Biochimica et Biophysica Acta 1812 (2011) 333–334

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Biochimica et Biophysica Acta

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Editorial Preface: Zebrafish Models of Neurology☆

Forming hypotheses about the Molecular Basis of Neurological benefitted primarily from biochemistry/ biophysics of protein Disease has traditionally been based on human genetics, biochemistry aggregation, cell culture work and mouse models; These paradigms and molecular biology, whereas testing these hypotheses requires us are challenging to implement in a fashion that tells us how disease in to turn to cell culture and animal models. Zebrafish have emerged as a one portion of the CNS spreads to another. Zebrafish are positioned tractable platform to complement and bridge these paradigms in brilliantly to fill this gap, not only because of its efficiency as a genetic many diseases, allowing integration of human genetics and cell and neuroscience model, but because zebrafish have a long history as biology within the in vivo setting. Perhaps it is in Neurology and a platform for assessing non-cell-autonomous effects of protein Neurodegeneration, however, where zebrafish have special promise perturbations in the in vivo setting. Thus it is common for zebrafish to make contributions. Four special advantages of zebrafish to biologists to ask if changing gene abundance or character in one group Neurology are worth highlighting here: efficient in vivo tests for of neurons is able to affect the fate and function of neighboring cells. genetic interactions, conserved cellular architecture of the CNS, the Creative and ambitious deployment of this ability will complement ability to create genetically mosaic animals, and tractable behaviour established tools to promise a deep understanding of how disease and electrophysiology to assess experimental interventions. spreads between cells. A tantalizing side-benefit is the ability to Linkages between Neurodegenerative diseases that were once deploy the fish in high-throughput screens of protein and small considered distinct are being recognized and celebrated as a path molecule effectors that represent therapeutic targets. forward to understanding the molecular basis of disease spread Finally, it is important to underline the cellular context of disease through the CNS. One example includes interactions between spreading during Neurolodegeneration. As disease spreads from cell notorious proteins in Alzheimer Disease (AD) and Prion Disease, to cell, the molecular basis of this spread is fundamentally influenced such that the Prion Protein may now be viewed as a receptor for Aβ by the cellular relationships. Importantly, The zebrafish has an oligomers. The abundance of such interactions between Neurological architecture of CNS cells that is conserved with that of humans. Diseases is growing, as protein interactome studies identify many Vasculature, blood-brain-barriers, and ventricles are all present from players in common, compelling the field to integrate an increasing list a young age, permitting tissue-level spread of molecules fundamental of genetic interactions into their models. The ability to efficiently to disease. Perhaps more importantly, the cells of the CNS develop in knockdown and overexpress gene products in vivo is propelling their normal environment, becoming polarized, interconnected and zebrafish to prominence at a rate commensurate with the complexity ensheathed in radial glia to form ion sinks. The cover image selected is of these genetic interactions. meant to highlight that this organization exists even in young On the other hand, various researchers have recently noted embryos. Certainly our understanding of hallmark synaptic dysfunc- intriguing parallels between prion diseases and the protein misfolding tion during Neurodegeneration must develop from paradigms where in other neurological diseases. In particular, questions are being raised synapses are formed and maintained with their typical glial partners as to the ability of misfolded proteins (including Tau and Aβ) to induce intact. So although zebrafish is not yet as advanced in its toolkit as the misfolding of normally folded protein, leading to a prion-like Drosophila, nor as tractable as cell culture, it has a relevant cellular domino effect and accumulation of protein dysfunction/aggregation architecture and genetic efficiency that make the paradigm a very that are hallmark disease pathologies. Consistent with such findings is attractive in vivo complement to studies in mouse models. the apparent spreading of disease pathology from a nucleation centre This issue of BBA: Molecular Basis of Disease has invited reviews through the CNS during clinical disease progression. Other celebrated from leading experts to consider the utility of zebrafish to Neurology. potential prion-like proteins include α-synuclein, polyglutamine These experts span from Neuroscientists who study fundamentals of proteins and SOD1 misfolding in ALS. zebrafish Biology, through to clinical Neurologists who recognize the Attention toward this prionoid nature of various neurological zebrafish as a relevant and potent paradigm to bring their hypotheses diseases is substantial, in large part because it identifies a novel to bedside in as timely a manner as possible. The first contributions therapeutic target: the mechanism of disease spreading. Unfortu- have sought to critically analyze previous work regarding particular nately, the field knows remarkably little about disease spreading, with Neurodegenerative diseases and how zebrafish have contributed to concepts in high-profile reviews centering on Tunneling Nanotubes this understanding. The subsequent contributions consider technical and exosome or synaptic release. Perhaps this fundamental lack of opportunities and challenges of using zebrafish genetics and beha- knowledge regarding disease spread through the CNS is the vioural outputs in Neurology, and the issue ends with the promise consequence of the tools available to neurologists and neuroscience provided by zebrafish to understand regeneration of the CNS for researchers. The mechanisms underlying AD, TSEs, and ALS have repair. I am grateful to each of these contributors, reviewers, and the ☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological BBA staff for their hardwork in communicating these important and Diseases. stimulating ideas.

0925-4439/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.bbadis.2010.11.004 334 Editorial

Allison is a neuroscientist with a special interest in research in the field of retina development and regeneration. His current research understanding processes of neurodegeneration and neural efforts focus on creating transgenic and mutant zebrafish to address hypotheses of regeneration. Towards this goal, Dr. Allison's research prion protein's normal function and to dissect the role of various prion protein integrates molecular biology, biochemistry, proteomics, domains. Other efforts use similar methods to examine the amyloid hypothesis of and electrophysiology to further develop zebrafish as a Alzheimer Disease. His Recent work is funded by a Recruitment Grant from PrioNet potent animal model. Particular emphasis of his research Canada and the Alberta Prion Research Institute and by a Young Investigators Award has been developing fish as effective models of neurode- from the Alzheimer Society of Canada. generative disease. He completed a Doctoral Program in 2004 with Professor Craig Hawryshyn at the University of Victoria, Canada, where he developed a unique model of neuronal degeneration and regeneration. This work was funded by fellowships from the Alzheimer Society of W. Ted Allison Canada and the Canadian Institute of Health Research University of Alberta, Centre for Prions and Protein Folding Diseases, Institute of Aging. Dr. Allison went on to complete an Departments of Biological Sciences & Medical Genetics, Edmonton, NSERC Post-Doctoral fellowship at the University of Michigan in the laboratory of Alberta, Canada T6G 2E9 Collegiate Professor Pamela Raymond. Dr. Allison's work at the University of Michigan created transgenic and mutant zebrafish has revealed genes important in the E-mail address: [email protected]. generation, positioning, and regeneration of neurons. Dr. Allison joined the Centre Tel.: +1 780 492 4430; fax: +1 780 492 9234. for Prions and Protein Folding Diseases as an Assistant Professor at the University of Alberta, Canada in 2008. His appointment is through the Department of Biological Sciences in the Faculty of Science, with a cross-appointment in the Department of 10 November 2010 Medical Genetics in the Faculty of Medicine and Dentistry. Dr. Allison is contributing to expanding the largest zebrafish facility in Western Canada and also maintains active Available online 27 November 2010 Biochimica et Biophysica Acta 1812 (2011) 335–345

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Biochimica et Biophysica Acta

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Review Zebrafish models for the functional genomics of neurogenetic disorders☆

Edor Kabashi a,b, Edna Brustein b, Nathalie Champagne a,b, Pierre Drapeau b,⁎ a Center for Excellence in Neuromics, CHUM Research Center and the Department of Medicine, Université de Montréal, Montréal, QC, Canada b Department of Pathology and Cell Biology and Groupe de recherche sur le système nerveux central, Université de Montréal, Montréal, QC, Canada article info abstract

Article history: In this review, we consider recent work using zebrafish to validate and study the functional consequences of Received 4 December 2009 mutations of human genes implicated in a broad range of degenerative and developmental disorders of the Accepted 22 September 2010 brain and spinal cord. Also we present technical considerations for those wishing to study their own genes of Available online 29 September 2010 interest by taking advantage of this easily manipulated and clinically relevant model organism. Zebrafish permit mutational analyses of genetic function (gain or loss of function) and the rapid validation of human Keywords: variants as pathological mutations. In particular, neural degeneration can be characterized at genetic, cellular, Zebrafish fi fi Human diseases functional, and behavioral levels. Zebra sh have been used to knock down or express mutations in zebra sh Neurogenetic diseases homologs of human genes and to directly express human genes bearing mutations related to Neurodegenerative disorders neurodegenerative disorders such as spinal muscular atrophy, ataxia, hereditary spastic paraplegia, Neurodevelopmental diseases amyotrophic lateral sclerosis (ALS), epilepsy, Huntington's disease, Parkinson's disease, fronto-temporal Animal models dementia, and Alzheimer's disease. More recently, we have been using zebrafish to validate mutations of Genomics synaptic genes discovered by large-scale genomic approaches in developmental disorders such as autism, Human genetics schizophrenia, and non-syndromic mental retardation. Advances in zebrafish genetics such as multigenic Trangenic animals analyses and chemical genetics now offer a unique potential for disease research. Thus, zebrafish hold much promise for advancing the functional genomics of human diseases, the understanding of the genetics and cell biology of degenerative and developmental disorders, and the discovery of therapeutics. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Apart from being a vertebrate with common organs and tissues such as a brain and spinal cord with conserved organization, the Zebrafish are used as a model for a wide variety of human diseases, attractiveness of zebrafish as a model lies in its biology and genetics. including cancer, cardiovascular disorders, angiogenesis, hemophilia, Zebrafish have large clutches of externally fertilized and transparent osteoporosis, diseases of muscle, kidney and liver, and, last but not the eggs, which develop rapidly and in synchrony, with neurogenesis least, disorders of the central nervous system. In recent years, starting around 10 h post-fertilization (hpf), synaptogenesis and the zebrafish have been used to study neurodegenerative disorders. first behaviors around 18 hpf and hatching around 52 hpf. Within Here we review zebrafish models of CNS diseases with an emphasis on 1 day of development, many pertinent features of the CNS appear and studies of degenerative neurological diseases. We also discuss our can be studied in relatively simple populations of identifiable neurons. recent efforts in extending these approaches to psychiatric disorders For example, the spinal cord is divided into 30 somites each of development. Although unlikely to yield accurate models for containing fewer than a dozen cell types that form relatively simple complex psychiatric disorders, genetic insights from studies of circuits. The rapid development of the zebrafish embryo allows for the zebrafish are pertinent in helping to identify relevant molecular and study of embryonic-lethal mutations as larvae can survive on their cellular mechanisms of human pathology and, at this level, promise yolk for up to a week, allowing studies of gene expression and insight to the development of therapeutics. In particular, the in vivo function throughout early developmental stages. biological validation of variants identified in human genetic and In addition to its advantageous biology, the second major genomic studies provides an important step in defining the advantage of the zebrafish as a model is the simplicity and pathological nature of mutations. effectiveness of manipulating gene expression for cell biological observations in living embryos with relevance to human pathology. The zebrafish genome is sequenced and, although not completely ☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological annotated, over 80% of gene structures are available and show a high Diseases. degree of synteny (nearest neighboring genes on the chromosomes) ⁎ Corresponding author. Department of Pathology and Cell Biology, Université de across vertebrate species as well as 50–80% homology with most Montréal, C.P. 6128, Succ. Centre-ville, Montréal, Québec H3C 3J7, Canada. Tel.: +1 514 343 7087; fax: +1 514 343 5755. human sequences. Homologs for most human genes can be identified E-mail address: [email protected] (P. Drapeau). in silico and, as described below, can be manipulated by gain- and loss-

0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2010.09.011 336 E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345 of-function approaches. However, the identification of a human Replacing targeted zebrafish genes with human genes can for homolog in zebrafish is complicated by the fact that a large number many genes be done as simply as by injection of human mRNA of genes are duplicated. This phenomenon is explained by whole simultaneously with the AMO as the latter is designed to specifically genome duplication that occurred during evolution [1]. The amino target the zebrafish and not the human sequence. We suggest using acid identity compared to the human gene is always higher for one of the zebrafish homologous cDNA if your gene of interest has a highly the two genes duplicated, indicating that the duplication appeared localized expression pattern like that of synaptic genes. Otherwise, before the species divergence. Therefore, when a gene is knocked human, mouse, or rat cDNA can be used and easily obtained down, it is important to consider that the duplicated gene can commercially. NCBI gives the link to cDNA clones for certain genes. compensate for the loss of function of the targeted homolog. Also, the The gene can be subcloned in expression vectors like pCS2 [4] or function could be partitioned between the two genes or simply lost for pcGlobin [5] to synthesize the 5′-capped mRNA that will be injected in one of the genes. Comparative sequence analysis allows identifying embryos. The pcGlobin vector has the advantage to have 5′UTR and 3′ the zebrafish homolog of a human gene. Three major genome UTR from Xenopus, and we also note that pcGlobin gives a better yield browsers, NCBI (www.ncbi.nlm.gov), Ensembl (www.ensembl.org) of mRNA. The cDNA can be tagged to MYC, flag, or HA epitopes to and UCSC (/genome.ucsc.edu/), can be use to search homologous gene follow the expression in fish. However, large DNA constructs such as sequences. In many cases, the homologous gene predictions can be plasmids often yield chimeric expression patterns in limited numbers found directly from either HomolGene (NCBI) or Orthologue Prediction of cells presumably as not all dividing progenitors retain the large (Ensembl). We have found that about half of the hundred or so human constructs. About half of the human genes we have tested permit synaptic genes on chromosome X (described later) have a predicted partial rescue of knockdown phenotypes, which then allows for fish ortholog, compared to ~30% in Drosophila and Caenorhabditis comparison between wild-type human mRNA and mRNA bearing elegans using the same databases. If the homologous gene is not disease-related mutations. Thus, in the space of a few weeks, each annotated or if the transcript prediction is not complete, a BLAST human variant can be validated in knockdown embryos once a clear search using nucleotide or protein sequence against the whole phenotype is recognized. Finally and as described below, recent genome assemblies (especially scaffolds based on BAC sequencing) transgenic technologies permit the creation of stable, targeted (cell- can allow the identification of homologous genes. TargetP and SignalP specific), and inducible lines for better spatially and temporally algorithms (www.cbs.dtu.dk/services/) can be used for signal peptide controlled and more detailed analyses of transgenic function. and cleavage site prediction. An additional phylogenetic analysis and multiple alignments of protein sequences (Clustal W) help to confirm the orthology. The syntenic regions can also be compared using Multi 2. Degenerative brain disorders Contig View (Ensembl). For additional information, the Sanger Institute (www.sanger.ac.uk) has excellent resources about zebrafish Degenerative disorders of the brain, including Alzheimer's, Par- sequence analysis. The sequence identity between zebrafish genes kinson's, and Huntington's diseases, are the first neurogenetic diseases and their human homologs is higher for ubiquitously expressed genes to have been studied using zebrafish [6,7]. Amyloid-beta plaques are than for highly specific genes such as some synaptic genes. The the strongest biological marker of Alzheimer's disease and the divergence is mainly found in domains important for membrane production of amyloid-beta is regulated by the presenilins 1 and 2 targeting as signal peptides and often in intracellular domains that (PS1, PS2). Surprisingly, wt zebrafish zPS1 was found to promote contain signaling motifs. Nonetheless, in general, the exon/intron aberrant amyloid-beta42 secretion when expressed in HEK 293 cells, boundaries and regulatory sequences as well as many other types of like the abnormal secretion associated with human PS1 mutations, and functional domains are evolutionarily conserved between the species. mutation of one residue in zPS1 abolished this activity [8]. Truncation As a simple starting point, the zebrafish homologs can be targeted for of zPS1 due to loss of exons 8 and 9 upon injection of splice acceptor knockdown by injection of selective anti-sense morpholino oligonu- AMOs had a dominant-negative effect by increasing PS1 expression [9] cleotides (AMOs) [2].ZFIN(zfin.org) has the principal database about and possibly the expression of other Alzheimer's-related genes [10]. AMO gene knockdown and phenotype. The parameters to consider in Zebrafish zPS2 is expressed at later stages and is regulated by zPS1 AMO design are the same as that for any oligonucleotide molecule: CG [11,12], but its role in amyloidogenesis in zebrafish is unclear. content, stem and loop structure, and the oligo length [3]. Gene-Tools Zebrafish possess two homologues of amyloid precursor protein Inc. (see www.gene-tools.com)offersanAMOdesignservicetothe (APP) with 63–66% homology to their human counterpart [13], and researchers purchasing their morpholinos. We recommend always their double knockdown causes a convergence–extension develop- checking if the AMO can bind to off-target sequences by doing a BLAST mental defect that is rescued by human APP mRNA but not by a against NCBI or Ensembl database. AMOs can be designed to target the Swedish mutation related to Alzheimer's disease [14]. The zebrafish ATG start site to block translation initiation or they can be used to block APPs possess a functional gamma-secretase complex to produce splice sites to produce truncated transcripts. The latter have the amyloid-beta [15] and inhibition of gamma-secretase [16] or knock- advantage of sparing maternal transcripts and permit RT-PCR amplifi- down of Pen-2 [17], another member of the presinilin complex, blocks cation and quantification of the gene knockdown, which is particularly Notch signaling to produce a severe neurogenic phenotype. Early useful when an antibody is unavailable. Further, the splice AMO can be studies also examined the tau protein in zebrafish as tau is present in used to mimic splicing defects, nonsense or truncation mutations human neurofibrillary tangles, mutations of tau are implicated in related to disease. AMOs resist degradation by ribonucleases and are dementia and tau may also contribute to Alzheimer's disease. The stable for several days, and in our hands when adequately controlled, zebrafish studies have shown that expression of human tau (driven they usually generate selective phenotypes (discussed below). Howev- transiently upon injection of a construct with a neural-specific variant er, AMOs must be injected intracellularly as they are more of a of the GATA2 promoter) results by 2 days in disruption of cytoskeletal pharmacological than a genetic tool, although they are more effective in structure, tau trafficking, and hyperphosphorylated fibrillar tau gene knockdown in zebrafish than interfering RNAs. Intracellular staining [18]. More recently, stable transgenic zebrafish expressing injection is feasible in the zebrafish embryo during the first 1–2hpfas mutated P301L human tau and a fluorescent reporter (driven pan- the blastomeres are open to the yolk prior to the 4th cell division. Free- neuronally from the HuC promoter) have permitted in vivo imaging of hand injection into the yolk near the blastomere border (Fig. 1) allows defective axonal growth, tau hyperphosphorylation, and the screening for the injection of a hundred eggs or so in 1–2 h, yielding a sufficient of novel therapeutic molecules [19]. These recent developments with number of treated embryos to permit statistical analyses of phenotypes zebrafish tau and amyloid open the possibility of further screens for and thus providing a genetic read-out often only 1 day later. novel therapeutics that, e.g., decrease the hyperphosphorylation of tau E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345 337

Fig. 1. Rescue of knockdown phenotypes by human mRNA allows for the validation of disease-related variants.

in HuC transgenics [19] or amyloid-beta load in amyloid transgenics aggregate formation and clearance, such as anti-prion compounds [20,21]. [35], or embryonic death, such as blockers of autophagy [37]. Research into Parkinson's disease has also used zebrafish as a Another CNS disorder that has been studied recently using model [22]. Zebrafish embryos treated with MPTP, a neurodegener- zebrafish is epilepsy [38] as clonus-like seizures are induced by ative chemical that reproduces some of the effects of idiopathic convulsant agents [39–42], which has permitted forward mutagenesis Parkinson's disease in mammalian models, demonstrated a loss of screens and the isolation of seizure-resistant mutants [43]. Zebrafish dopaminergic neurons, which could be rescued using the monoamine are now being used to screen for compounds that suppress seizures oxidase-B inhibitor deprenyl [23–25]. Half a dozen genes have been [44]. Furthermore, injection of mRNA with a mutation of the human identified in Parkinson's disease [26], and several of these have been PRICKLE1 gene implicated in epilepsy disrupts normal function when studied to date in zebrafish. For example, in zebrafish, the mRNA for overexpressed in zebrafish [45], demonstrating the usefulness of the ubiquitin processing gene UCH-L1 was detected by 1 day in the zebrafish for validating mutations of human genes causing brain ventral region of the midbrain and hindbrain and in the ventral disorders. diencephalon where it was co-expressed with markers of dopami- Our group has recently collaborated on the discovery of the nergic neurons [27]. The zebrafish DJ-1 protein has high (N80%) MEDNIK syndrome, a rare and severe autosomal recessive neurocu- homology with human and mouse DJ-1 (of unknown function) and is taneous disorder manifested by mental retardation, enteropathy, expressed throughout the body [28]. Knockdown of DJ-1 in the deafness, neuropathy, ichthyosis, and keratodermia that is often lethal zebrafish did not affect the number of dopaminergic neurons, but [46,47]. Affected individuals bear an A to G mutation in acceptor splice zebrafish embryos were more susceptible to oxidative stress and had site of exon 3 of the AP1S1 gene, which leads to a premature stop elevated SOD1 levels, while simultaneous knockdown of DJ-1 and p53 codon (Fig. 2A [47]). The AP1S1 gene encodes the small subunit σ1A of caused dopaminergic neuronal loss. Recently zebrafish Parkin, the first (AP-1) of four ubiquitous clathrin adaptor proteins [48–51]. another ubiquitin processing gene, was identified (62% homology Each one of the adaptor protein complexes is assembled from four with human PARKIN) and AMO abrogation of its activity leads to a subunits, and the σ subunit, the one affected in the MEDNIK significant decrease in the number of ascending dopaminergic syndrome, is part of the AP complex core and is suggested to neurons in the posterior tuberculum, which is homologous to the contribute to its stabilization. Also, together with the μ subunit, the σ substantia nigra in humans [29]. Screens for compounds promoting subunit is possibly involved in protein cargo selection [50,51].To (like MPTP) or preventing (such as caffeine) the Parkinsonian demonstrate that the mutation in the human AP1S1 gene indeed alters phenotype in zebrafish [30] should now be facilitated by the the biological function of this gene and underlies the MEDNIK availability of an enhancer trap line expressing GFP from the vesicular syndrome, we knocked down the function of the homologous Ap1s1 monoamine transporter 2 promoter [31]. gene in zebrafish. The zebrafish Ap1s1 protein shares 91% identity In zebrafish, the huntingtin (Htt) gene implicated in Huntington's with the human protein. To inhibit zebrafish Ap1s1 mRNA translation, disease has a predicted 70% identity with the human protein [32] and two types of AMOs were designed (Fig. 2A [47]). The first AMO its knockdown disrupts a number of features [33] and causes massive targeted the N-terminal, while the second AMO targeted the acceptor neuronal apoptosis due to reduced BDNF expression by 1 day of splice site of intron 2 (Fig. 2A), imitating the mutation found in development but not earlier [34]. It was recently observed that MEDNIK patients. Both AMOs caused similar, severe morphological injecting mRNA coding for the N-terminal fragment of Htt with and behavioral deficits. The 48 hpf KD larva was smaller and had different length polyQ repeats led to developmental abnormalities reduced pigmentation compared to the wild-type (WT) larva. Further, and apoptosis in the embryos as early as 1 day later [35]. Embryos blocking Ap1s1 translation caused disorganized skin formation in expressing a Q102 mRNA [35] or a Q56 plasmid [36] developed general, particularly affecting fin morphology (Fig. 2B; for further inclusions in the cytoplasm. These Huntington's disease models are information on the epithelial disorders, see Ref. [47]). In addition to being used to screen for novel therapeutics that could prevent the morphological deficits, the 48 hpf KD larva responded abnormally 338 E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345 to touch by coiling the tail instead of swimming away. The the start of the 3rd exon, rescued the phenotype (Fig. 2A). The latter compromised touch response of the KD larva could be attributed to result suggests that the predicted in frame protein, lacking only 3 a massive reduction (by half) in the spinal neuron population, amino acids (Fig. 2C), is functional and may explain the viability of the specifically due to a loss in the interneuron population. The specificity MEDNIK patients. However, the low expression level of the alterna- of the AMO effect was further confirmed both by Western blotting and tively spliced RNA (~10%) is insufficient to sustain normal develop- immunolabeling analysis (Fig. 2C and D). Most importantly, we could ment and function, further emphasizing the importance of AP1S1 in rescue the knockdown phenotype by co-injecting the AMO with normal development. Our in vivo zebrafish study of Ap1s1 function human AP1S1 wild-type mRNA (Fig. 2B and D), which is not targeted revealed its importance for appropriate neurogenesis and skin by the Ap1s1 AMO. In contrast, co-injection of AMO with mutated development. Accordingly, we could speculate that MEDNIK, a novel human AP1S1 mRNA missing exon 3 (Fig. 2A) failed to rescue the skin neurocutaneous syndrome, is caused by impaired development of and behavioral deficits, suggesting loss of function of this truncated various neural networks in the spinal cord and in the brain, explaining form of protein (Fig. 2C) and confirming the pathogenic nature of the the multifunctional human deficits such as ataxia, peripheral mutation found in MEDNIK patients. Further, co-injection of AMO and neuropathy, and mental retardation, which are concomitant with a an additional human AP1S1 mRNA isoform found in MEDNIK patients, perturbation in epithelial cell development in the skin and in the containing a cryptic splice acceptor site located 9 bp downstream of digestive system. We speculate that these generalized effects of AP1S1

Fig. 2. Evaluating a mutation found in human AP1S1 gene (MEDNIK syndrome) using zebrafish. (A) An illustration of the human AP1S1 gene to show the location of the mutation found in MEDNIK patients. An A to G mutation in the acceptor splice site of exon 3 resulted in skipping of this exon and led to a premature stop codon. The use of an alternative cryptic splice site located 9 bp downstream of the start of the 3rd exon resulted in mRNA lacking 9 bp. The location of the two different morpholinos targeting either ATG or exon 3 acceptor splice site of the zebrafish ap1s1 gene is indicated in green. The inset on the right depicts the clathrin adaptor protein 1 (AP-1) and its 4 subunits. It is the small subunit σ1 (red), which is mutated in MEDNIK patients. (B) Ap1s1 knockdown (KD) in zebrafish using morpholino oligo nucleotides targeting the N-terminal or the splice site (splice site, intron 2) resulted in a similar characteristic morphological phenotype (48 h post-fertilization), which is rescued by co-injection of the wild-type human AP1S1 (rescue +WT HmRNA) or the mRNA lacking 9 bp (rescue +−9 bp HmRNA), but not by the mutated human mRNA missing exon 3 (rescue +−exon3HmRNA). (C) Illustration of the predicated human AP1S1 proteins: normal protein containing 55AA (WT, black); truncated protein containing 19AA (red), the result of skipping exon 3; and a protein missing 3AA, a result of the alternative splicing (blue). Western blot analysis of Ap1s1 proteins from human (left) and zebrafish (right) to show that Ap1s1 protein is hardly expressed in MEDNIK patients and in ap1s1 knockdown (KD) zebrafish larva. To normalize the Western blot analysis, proteins extracted from WT, KD, and CTRL larvae were incubated with anti-actin. CTRL=control, WT=wild type. (D) Localization of Ap1s1 in skin cells of zebrafish wholemounts is illustrated using anti-Ap1s1 antibody immunofluorescence. Cell membrane (polygonal) and a well-defined perinuclear ring can be nicely observed in both normal (WT) and rescued larvae (rescue +WTHmRNA), whereas only a residual and diffuse staining could be observed in knockdown (KD) larvae (modified from Ref. [47]). E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345 339 mutation are due to a widespread deficit in vesicular transport and HSP patients [62]. Recently, SLC33A1 was identified as the gene protein sorting. responsible for SPG42 [63]. Knockdown of this gene in zebrafish embryos using AMO caused a curly-tail phenotype and defective axon 3. Degenerative spinal cord disorders outgrowth from the spinal cord [63]. Finally, knockdown of spastin (SPG4) caused widespread defects in neuronal connectivity and Zebrafish have also been proven to be particularly effective as a extensive CNS-specific apoptosis [64]. model for motor dysfunction [52]. Our group has worked extensively Spinal muscular atrophy (SMA) is an autosomally recessive motor on motor neuron diseases, including the most common of these neuron disorder caused by mutations in the survival motor neuron disorders, amyotrophic lateral sclerosis (ALS). The advantage of gene (SMN1). In a series of publications, Beattie's laboratory has studying motor neuron diseases in zebrafish consists in the rapid demonstrated that knockdown of the SMN1 in zebrafish embryos development of the spinal cord and allowing analysis of motor neuron (49% homology to human protein) causes motor axon outgrowth and branching patterns as early as 24 hpf. In addition, responses to touch pathfinding defects in early development [52,65]. Further, in a series and swimming can be monitored following hatching around 48 hpf of elegant rescue experiments, this group was able to show that a [53]. SOD1 (ALS1) is known to cause ALS in 10–20% of familial ALS conserved region in exon 7 of the SMN1 gene (QNQKE) is critical for cases mainly through an autosomal dominant toxic gain of function axonal outgrowth and that the plastin 3 (PLS3) may be important for and has 70% amino acid identity to the zebrafish homologue. axonal outgrowth since overexpression of plastin 3 rescued the Overexpression of mutant SOD1 in zebrafish leads to short motor phenotype caused by SMN1 knockdown [66,67]. Finally, transgenic axons with premature branching [54]. Interestingly, several other expression of homozygous deletion found in SMA patients that leads genes implicated in motoneuron degeneration show a similar to truncated SMN1 protein as well as knocking out the Smn1 gene in phenotype when tested in zebrafish. Alsin (ALS2) is the gene mutated zebrafish caused deficits in the neuromuscular junction formation and in juvenile ALS through autosomal recessive mode of action, which death at the larval stage [68]. leads in most cases to protein truncation. Corresponding to this, in zebrafish embryos, knockdown of Alsin (61% homology) by AMO 4. Developmental disorders causes shortening of motor axons and also loss of neurons in the spinal cord [55]. ELP3 was identified through association studies Although this developmental model is proving useful in the performed in ALS patients and in a Drosophila screen for genes study of late-onset degenerative diseases, zebrafish are just starting important for neuronal survival [56]. Knockdown of this gene (ELP3) to be used in the study of developmental brain diseases. Interesting in zebrafish caused increased branching and shortened motor axons speculations have been made on the potential of zebrafish for [56]. Recently, TDP-43 was found enriched in inclusion bodies from modeling developmental psychiatric disorders such as autism [69], spinal cord autopsy tissue obtained from ALS patients [57]. Moreover, but the lag in advancing these models is perhaps because the about 30 mutations have been identified in a considerable number of clinical phenotypes are so subtle and specifictohumanssuffering ALS patients, suggesting that this protein plays an important role in from disorders such as autism, schizophrenia, non-syndromic disease pathogenesis [58,59]. The zebrafish ztardbp gene product mental retardation, and others that zebrafish may appear as a (TDP-43) has 73% homology to the human protein. Knockdown of doubtful model. So far (and very recently), the only genes linked to TDP-43 (tardbp) in zebrafish embryos led to motor neuron branching schizophrenia to be studied in zebrafish using AMO methods are and motility deficits (Fig. 3). This AMO phenotype was rescued by co- DISC1 and NRG1 [70]. The receptor tyrosine kinase Met has been expressing human wild-type (WT) TDP-43 (which is not targeted by implicated in cerebellar development and autism, and knockdown the AMO) (Fig. 3). However, three ALS-related mutations of human of either Met alone or both of its Hgf ligands (two genes in TDP-43 identified in both in SALS and FALS cases (A315T, G348C, zebrafish) together using ATG or splice junction AMOs results in A382T) failed to rescue these phenotypes, indicating that these abnormal cerebellar and facial motoneuron development [71]. mutants are pathogenic (Fig. 3). A similar phenotype was observed However, neither the human homologs of these genes nor their when mutant (but not wt) TDP-43 was overexpressed, with the disease-related mutations have been tested in zebrafish. Our new G348C mutation being the most penetrant (Fig. 3). These results ongoing genomics approach, based on large-scale re-sequencing of indicate that TDP-43 mutations cause motor defects, suggesting that human synaptic genes in patients suffering from some of these both a loss as well as a gain of function may be involved in the disorders, is identifying mutations that can be validated, if not molecular mechanism of pathogenesis [125]. Finally, it is interesting exactly modeled, in zebrafish. to note that a number of groups generated ALS2 knockout mice yet As a part of our “Synapse to Disease” project, we are using a failed to observe motor deficits associated with motor neuron combination of AMO knockdown and gene overexpression to degeneration and concluded that ALS2 was not an important gene functionally validate novel synaptic gene mutations identified in for ALS [55,60]. However, the first three exons of ALS2 were left intact large cohorts of patients with autism, schizophrenia, or non- in these mice. We found that the motor phenotype in zebrafish upon syndromic mental retardation. Hundreds of synaptic genes were ALS2 KD could be partially rescued upon overexpression of the selected based on published studies and databases. Many genes were alternative transcript of the first three exons of ALS2, indicating that chosen because they are X-linked [72] as there are many more complete disruption could in fact be pathogenic in ALS [55]. This affected males than females in autism [73]. Other genes that were nicely illustrates the usefulness of zebrafish knockdown models for selected are those of the glutamate receptor complex because of their more complete disruption of gene function in some circumstances. association to neurodevelopmental diseases [74]. So far, the exonic Zebrafish have been also widely used to study the functional role regions of over 400 genes have been sequenced, and 15 de novo of gene mutations in another motor neuron disease, hereditary spastic mutations (present in the patients and not their parents) were paraplegia (HSP). Over 30 loci are known for this disorder, and at least genetically validated (manuscript submitted). We have identified 20 genes have been identified to cause spastic paraplegia [61]. Our deleterious rare de novo mutations in Shank3, IL1RAPL1, NRXN1, and group identified missense variants in the KIAA0196 gene at the SPG8 KIF17 genes, and all of these genes have orthologs in zebrafish. We locus and validated these mutations using the zebrafish model (87% have identified a de novo mutation in the Shank3 gene in a patient homology to the human protein) [62]. Knockdown of the zebrafish with autism [75] as Shank3 deletions or duplications were previously homolog of KIAA0196 gene caused a curly-tail phenotype coupled identified in patients with autism [76]. Shank3 encodes a scaffolding with shorter motor axons. This phenotype could be partially rescued protein found in excitatory synapses directly opposite to the pre- by the human WT KIAA0196 but not the two mutants identified in synaptic active zone. The Shank proteins link ionotropic and 340 E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345

Fig. 3. Motor phenotype of gain and loss of function of TDP-43 in zebrafish. (A) A schematic representation of TDP-43 protein showing the functional domains, localization of FLAG (3 kDa) and myc tags (12 kDa), and sites where the G348C mutation found in ALS patients was introduced by site-directed mutagenesis. (B) Locomotor phenotype. Zebrafish embryos develop a touch-evoked escape response as seen when embryos are injected with WT human TDP-43 RNA. Expression of TDP-43 G348C RNA causes a deficiency in the touch-evoked response. A similar phenotype is observed when the zf tdp-43 expression was knocked down using a specific AMO. This phenotype was rescued with expression of WT but not G348C RNA. (C) Motor axonal deficits. The behavioral (motor) phenotype was selectively associated with a shortening of the axon and premature branching in motor neurons (see G348C TDP-43 and tdp-43AMO) (modified from Kabashi et al., accepted at Hum. Mol. Genet.). metabotropic glutamate receptor complexes together and to the study. Mutations resulting in deletion of the TIR and the C-terminal cytoskeleton to regulate the structural organization of dentritic domains were previously identified in patients with non-syndromic spines. The splice mutation identified in the Shank3 gene would mental retardation [82,83], suggesting that those domains are result in a truncated protein lacking the Homer, Cortactin, and SAM important to the protein function. Two genes encode IL1RAPL1 in domains important for spine induction and dentritic targeting [77]. zebrafish (il1rapl1a and il1rapl1b) and share 69% and 75% identity, Zebrafish have two Shank3 homologous genes (shank3a and shank3b). respectively, with human protein. The divergence is mainly in the The identity to human protein is overall 65% and 62% for shank3a and signal peptide (il1rapl1a: 42%, b: 47%). Recently, a study using shank3b, respectively, but when we compare every individual zebrafish il1rapl1a showed that the mRNA is constantly expressed functional domain to the human one, the amino acids identity reach during early embryonic development, indicating that it is maternally 80% and is over 90% for the PDZ domain. The phenotype of Shank3 provided [84]. In this study, they also showed that knockdown of the knockdown zebrafish was not reported in the literature yet, and we other gene, il1rapl1b, using an ATG AMO caused a selective loss of observed major motility deficits that could be rescued by the rat synaptic endings in olfactory neurons and that il1rapl1b C terminus mRNA but not by some of the mutations [126]. and TIR domains regulate the synaptic vesicles accumulation and We also found de novo mutations in IL1RAPL1 in autistic patients morphological remodeling of axon terminals during synapse forma- [78]. IL1RAPL1 and 2 are plasma membrane proteins that belong to a tion [84]. These observations suggest that il1rapl1b may be essential class of the interleukin-1 receptor family characterized by a 150-aa C- for synapse formation and indicate how a loss-of-function IL1RAPL1 terminal domain that interacts with Neuronal Calcium Sensor-1 (NCS- mutant could affect brain neurodevelopment and its possible 1) [79,80]. A recent study from [81] used IL1RAPL1-deficient mouse to association to autism and non-syndromic mental retardation. To show that IL1RAPL1 controls inhibitory network during cerebellar determine more specifically the function of IL1RAPL1 in the context of development. In one patient, a de novo frameshift mutation in schizophrenia, we knocked down the expression of zebrafish il1rapl1a IL1RAPL1 causes a premature stop of the translation (I367fs) resulting with an ATG AMO and observed a severe phenotype with incomplete in a truncated protein, lacking the intracellular TIR domain and the C- development of the embryo, which could not be rescued by human terminal domain interacting with NCS-1 [78]. Moreover, a large mRNA. These preliminary (unpublished) results indicate that deletion of exons 3 to 7 of IL1RAPL1 in three brothers with autism IL1RAPL1 plays an important function during early embryonic and/or non-syndromic mental retardation was also identified in our development. Further work, such as with splice junction AMOs that E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345 341 spare maternal transcripts that are essential for early development, is 5.1. Targeted expression required in order to develop a pathogenic validation. Neurexins are predominantly pre-synaptic cell-adhesion mole- Most of the approaches taken to date to express foreign genes in cules. They can induce pre-synaptic differentiation by interacting with zebrafish are based on injection of mRNA for transient expression neuroligins. There are three neurexin genes (NRXN1, 2, and 3), each of throughout the embryo. This simple approach is valid for ubiqui- which encodes two major variants (alpha and beta). Only NRXN1 gene tously and early expressed genes such as AP1S1, SOD1, TARDBP and was shown to be disrupted using CNV analysis in autism [85,86]. More members of the ubiquination and beta-secretase complexes dis- recently, NRXN1 gene has been involved in CNV found in SCZ patients cussed above. However, mRNA injection risks expressing genes [87,88]. We have identified an insertion of 4 bp predicted to cause a ectopically, such as in the case of synaptic genes, and is usually only frameshift with a premature stop affecting the two major variants efficient at embryonic stages, prior to mRNA degradation that occurs alpha and beta of NRXN1 in SCZ patient. The protein is predicted to within a few days. The latter thus limits the time course for miss the transmembrane and intracellular domains. Two orthologs of phenotypic analysis, which is particularly problematic for studies of NRXN1 have been identified in zebrafish (Nrxn1a and 1b) with an neurogeneration in (often) adult-onset human diseases. More identity to the human protein over 70%. The greatest variability is specific expression patterns can be achieved by stable transgenesis found in the signal peptide (Nrxn1a, 27%; 1b 32%). An expression using the efficient Tol2 transposon system [97], but this is best suited analysis showed that all three Nrxn genes are expressed during for generating long-term models (rather than for preliminary or zebrafish embryonic development and some specific isoforms of large-scale screens) as it is a slower process that requires the raising Nrxn1a expressed at different stage of the development [89]. These of founder fish and their out-crossing to verify stable and specific results indicate the potential for developing zebrafish models of transgenic lines. A number of promoters are available for neural- neurexin mutations. However, the fact that there exist thousands of specific transgenic expression. For example, the α-tubulin promoter isoforms of Nrxn1 and the amino acids of the signal peptide are [98] was the first used to drive pan-neuronal expression and, as divergent could make the functional validation in zebrafish more described above, the HuC promoter expressed in post-mitotic challenging. neurons [19] and a neural-specific variant of the GATA2 promoter Kinesin 17 (KIF17) is a member of the kinesin superfamily [18] are also effective at early stages. More selective subsets of containing a motor domain for ATP hydrolysis. KIF17 binds to the neurons can be targeted using more restrictive promoters such as the scafolding Mint1 to transport the NMDA receptor subunit 2B (NR2B) dopaminergic-specific vesicular monoamine transporter 2 promoter to the dendrites along microtubules [90,91]. In addition, the K+ [31]. In the spinal cord, which is an important region for studies of channel Kv4.2, a major regulator of dendritic excitability, is also motor dysfunction and synaptic transmission, the developmentally transported to the dentrites by KIF17 [92]. In mice, overexpression of conserved transcriptional code [99] has permitted the generation of Kif17 enhances spatial and working memory [93]. The expression of transgenic lines for selective types of neurons. These include KIF17 was shown to be altered in a mouse models for Down syndrome motoneurons: HB9 [100], Isl-1 [101], commissural neurons: Evx1 (trisomy 21) leading to learning deficit [94]. We have identified a [102], mostly glycinergic neurons: Pax2.1 [103]; vGlyT2 [104], mostly nonsense mutation resulting in a protein that lacks the tail domain in glutamatergic neurons: Alx/Vsx2 [105], as well as sensory neurons: a SCZ patient. This domain was shown to interact with Mint1 Ngn1 [106] and oligodendrocytes: Nkx2.2a [107], olig2 [108] and following its phosphorylation by CaMKII [95]. One orthologous sox10 [109]. sequence was identified in zebrafish with an identity of 61% compare However, these stable transgenic models also have their limita- to the human amino acids. The divergence was mainly observed in the tions, and here, we consider three major ones that can be at least motifs important for cargo binding. The zebrafish KIF17 is widely partially avoided depending on necessity: ectopic expression, toxic expressed in the nervous system and retina [96]. By using low doses expression, and genetic background. Although promoter-based stable splicing AMO, this group demonstrates that KIF17 is essential for transgenic lines confer more selective expression patterns, rarely do vertebrate photoreceptor development and seems to have little effect they perfectly recapitulate the natural expression patterns as often on the global zebrafish development. At higher doses of either an ATG only subsets of enhancer elements are used in generating the or a splice junction AMO that micked the effect of the de novo transgenic constructs. Indeed, this is a limitation of the lack of a nonsense truncation mutation in schizophrenia, we observed a knock-in technology in zebrafish, requiring the integration of novel characteristic dose-dependent morphological trait with stunted, constructs in a wild-type background (considered below). However, curly embryos [127]. This validates that the nonsense mutation is a perfectly faithful expression patterns are not always essential as loss-of-function pathogenic effect. expression in incomplete or expanded subsets of neurons can be Together, these results with several zebrafish developmental useful to study. An approach that provides a more accurate expression genes indicate the potential usefulness of this model for validating pattern is the use of bacterial artificial chromosomes (BACs), such as brain disease mutations, but clearly each gene requires its own set of with the amyloid line mentioned above [21]. In principle (although carefully designed experiments. Whereas some human genes can be we are unaware of its practice with zebrafish), humanized models more difficult to study in zebrafish, with others, one can observe could be generated based on transgenesis with human BACs, as is faithful disease phenotypes that can be characterized in detail such as commonly done with mice. However, vertebrate genes can be for AP1S1 in MEDNIK and ALS2 in ALS and thus provide novel insights hundreds of kilobases in size and are not always completely contained to these disorders. We have observed some knockdown phenotypes in within single BACs. As an alternative, pufferfish (Takifugu rubripes) zebrafish that are not as obvious in knockout mice, which may reflect BACs can be used as the intergenic regions are much smaller, an a lower degree of compensation in zebrafish, perhaps because their approach we have taken in generating an Evx1 transgenic line [102], reproductive biology is based more on the quantity of offspring rather although even in this case less than 80% of the neurons labeled by a than necessarily on the quality of developmental compensation. selective antibody to evx1 also expressed GFP, indicating that not all evx1 cells transgenically express GFP. 5. Future directions A second issue in considering stable transgenic lines is toxicity, as many of the transgenes bearing disease-related mutations can be Several approaches have the potential of further advancing embryonic-lethal. A simple approach is to drive expression from a zebrafish models of human CNS disorders and making them uniquely heat shock promoter [110], although this may lose in generalized useful, in particular targeted expression, multigenic analysis and spatial expression what it gains in restricted temporal activation. A chemical genetics. powerful alternative is the use of combinatorial expression systems, 342 E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345 such as the cre-loxP system developed in mice and the yeast Gal4-UAS particularly as multiple conditions (e.g., different mutant alleles) binary system popular for invertebrate genetics, both of which have permit internal verification and comparison of the array results. A found applications in zebrafish [111,112]. Examples are the Evx1-Gal4 pertinent example is the recent microarray analysis [121] of three [102] and HuC-Gal4 [19] lines described earlier. The latter drives alleles of the neurogenic mindbomb mutant affecting Notch signaling transcription in both directions from the UAS in order to express two for which each allele affected the expression of hundreds of genes but constructs simultaneoulsy, such as a gene of interest (in this case less than 100 were commonly affected. Thus, zebrafish have a unique human P301L tau) and a reporter (dsRed) in the same cells. Another potential for rapidly analyzing multiple genes and dissecting complex technique for expressing more than one gene from the same construct genetic pathways. is to use the self-cleaving viral 2A peptide as a linker between the genes of interest [113]. 5.3. Chemical genetics A final concern is the creation of transgenic lines in a genetic background containing the endogenous gene as (in the absence of While zebrafish retain advantages for studying the genetics, knock-in technology) this results in the addition of another gene. One biology, and pathobiology of disease genes of interest, they also possibility that is rarely used is to create a transgenic in a knockout have the major advantage of being the only vertebrate model background. In principle, this can be done if mutants have been amenable to large chemical genetic screens [122]. Small chemical isolated in screens for related phenotypes or by TILLING (Targeted libraries of over 10,000 molecules, including approved drugs and Induced Local Lesions in Genomes) [114] available through a new randomly synthesized organic molecules of unknown function, have consortium (see https://webapps.fhcrc.org/science/tilling/). A new been screened in zebrafish models of cancer [123] and cardiovascular technology is the targeted lesioning of genes by zinc finger nucleases disorders [124], and some of these are in clinical testing. With the (ZFN) [115], although this is a complex and expensive technology that numerous models of brain diseases being developed in zebrafish, this at the moment is beyond the reach of small laboratories. Preliminary approach holds much potential for drug discovery. This is particularly success in targeting genes in zebrafish is encouraging, and the attractive in zebrafish as functional chemical genetic screens in vivo possibility to use homologous recombination, rather than non- with living embryos offer the potential of discovering therapeutics homologous end joining, to repair ZFN-induced double-strand breaks without prior knowledge or need for rational screening designs. Thus, may permit the development of a knock-in approach for zebrafish zebrafish are a unique model ranging from molecular genetic [116]. Much works remain to be done before this enticing possibility validation to drug discovery with important applications on the becomes practical, but nonetheless, a number of transgenic horizon for brain diseases, including many complex and untreatable approaches are now available for refined studies of gene expression disorders. and function in zebrafish, including human genes bearing disease- related mutations. References

5.2. Multigenic analysis [1] J.N. Volff, Genome evolution and biodiversity in teleost fish, Heredity 94 (2005) 280–294. [2] A. Nasevicius, S.C. Ekker, Effective targeted gene ‘knockdown’ in zebrafish, Nat. Although highly penetrant mutations of single genes are often Genet. 26 (2000) 216–220. implicated in CNS disorders, such as the majority considered in this [3] J.D. Moulton, Y.L. Yan, Using morpholinos to control gene expression, Curr. Protoc. Mol. Biol. (2008) Chapter 26, Unit 26 28. review, most diseases are multigenic in nature, requiring multiple [4] D.L. Turner, H. Weintraub, Expression of achaete-scute homolog 3 in Xenopus weakly penetrating mutations in predisposing genetic backgrounds in embryos converts ectodermal cells to a neural fate, Genes Dev. 8 (1994) order to produce the disease. Existing genetic models most easily 1434–1447. [5] H. Ro, K. Soun, E.J. Kim, M. Rhee, Novel vector systems optimized for injecting in address single gene mutations (or rather have focused on these vitro-synthesized mRNA into zebrafish embryos, Mol. Cells 17 (2004) 373–376. simple cases), but clearly, the future of these models lies in their [6] J.D. Best, W.K. Alderton, Zebrafish: an in vivo model for the study of neurological ability to provide insights to the nature of genetic interactions in diseases, Neuropsychiatr. Dis. Treat. 4 (2008) 567–576. fi disease. The zebrafish may prove to be particularly amenable to [7] P.W. Ingham, The power of the zebra sh for disease analysis, Hum. Mol. Genet. 18 (2009) R107–R112. multigenic analysis by a combination of the approaches described [8] U. Leimer, K. Lun, H. Romig, J. Walter, J. Grunberg, M. Brand, C. Haass, Zebrafish above. The use of two or even three AMOs is a simple solution once (Danio rerio) presenilin promotes aberrant amyloid beta-peptide production each AMO is validated. For example, in the context of Parkinson's and requires a critical aspartate residue for its function in amyloidogenesis, Biochemistry 38 (1999) 13602–13609. disease, simultaneous knockdown of DJ-1 and p53 was found to cause [9] S. Nornes, M. Newman, G. Verdile, S. Wells, C.L. Stoick-Cooper, B. Tucker, I. dopaminergic neuronal loss [28] and knockdown of both Hgf ligands Frederich-Sleptsova, R. Martins, M. Lardelli, Interference with splicing of was necessary to induce Met-related cerebellar defects [71]. Also, presenilin transcripts has potent dominant negative effects on presenilin – fi activity, Hum. Mol. Genet. 17 (2008) 402 412. photoactivatable caged AMOs can be used to release these at speci c [10] M. Newman, B. Tucker, S. Nornes, A. Ward, M. Lardelli, Altering presenilin gene time points [117]. This approach seems promising, but unfortunately, activity in zebrafish embryos causes changes in expression of genes with caged AMOs are not available commercially yet. Another major potential involvement in Alzheimer's disease pathogenesis, J. Alzheimers Dis. 16 (2009) 133–147. limitation of the AMO is the necessity to inject in embryo at 1 or 2 cells [11] C. Groth, S. Nornes, R. McCarty, R. Tamme, M. Lardelli, Identification of a second stage. An interesting alternative would be to use the vivo-AMOs that presenilin gene in zebrafish with similarity to the human Alzheimer's disease allow making gene knockdown and splice modification in adult gene presenilin2, Dev. Genes Evol. 212 (2002) 486–490. fi [12] S. Nornes, C. Groth, E. Camp, P. Ey, M. Lardelli, Developmental control of animals. The vivo-AMOs are modi ed AMO able to enter cells by presenilin1 expression, endoproteolysis, and interaction in zebrafish embryos, endocytosis. They could be conjugated with a dendrimeric octagua- Exp. Cell Res. 289 (2003) 124–132. nidine (Gene Tools) or a cell-penetrating peptide linked to phosphor- [13] A. Musa, H. Lehrach, V.A. Russo, Distinct expression patterns of two zebrafish odiamidate (PPMO) (AVI BioPharma Inc.). Vivo-AMOs were shown to homologues of the human APP gene during embryonic development, Dev. Genes Evol. 211 (2001) 563–567. be effective in mice [118,119], and they are currently tested in adult [14] P. Joshi, J.O. Liang, K. DiMonte, J. Sullivan, S.W. Pimplikar, Amyloid precursor zebrafish [3]. protein is required for convergent-extension movements during zebrafish – By performing AMO injections in mutants or transgenics, either development, Dev. Biol. 335 (2009) 1 11. [15] M. Newman, I.F. Musgrave, M. Lardelli, Alzheimer disease: amyloidogenesis, the stable or inducible, it should in principle be possible to readily test presenilins and animal models, Biochim. Biophys. Acta 1772 (2007) 285–297. several genes simultaneously or in different combinations, bestowing [16] A. Geling, H. Steiner, M. Willem, L. Bally-Cuif, C. Haass, A gamma-secretase zebrafish with a polyvalence that is difficult to envisage for other inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish, EMBO Rep. 3 (2002) 688–694. models such as mice. In the case of mutant and transgenic back- [17] W.A. Campbell, H. Yang, H. Zetterberg, S. Baulac, J.A. Sears, T. Liu, S.T. Wong, T.P. grounds, comparative microarray screening [120] becomes feasible, Zhong, W. Xia, Zebrafish lacking Alzheimer presenilin enhancer 2 (Pen-2) E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345 343

demonstrate excessive p53-dependent apoptosis and neuronal loss, J. Neuro- D.C. Slusarski, S.F. Berkovic, H.I. El-Shanti, A homozygous mutation in human chem. 96 (2006) 1423–1440. PRICKLE1 causes an autosomal-recessive progressive myoclonus epilepsy–ataxia [18] H.G. Tomasiewicz, D.B. Flaherty, J.P. Soria, J.G. Wood, Transgenic zebrafish model syndrome, Am. J. Hum. Genet. 83 (2008) 572–581. of neurodegeneration, J. Neurosci. Res. 70 (2002) 734–745. [46] T.G. Saba, A. Montpetit, A. Verner, P. Rioux, T.J. Hudson, R. Drouin, C.A. Drouin, An [19] D. Paquet, R. Bhat, A. Sydow, E.M. Mandelkow, S. Berg, S. Hellberg, J. Falting, M. atypical form of erythrokeratodermia variabilis maps to chromosome 7q22, Distel, R.W. Koster, B. Schmid, C. Haass, A zebrafish model of tauopathy allows in Hum. Genet. 116 (2005) 167–171. vivo imaging of neuronal cell death and drug evaluation, J. Clin. Invest. 119 [47] A. Montpetit, S. Cote, E. Brustein, C.A. Drouin, L. Lapointe, M. Boudreau, C. (2009) 1382–1395. Meloche, R. Drouin, T.J. Hudson, P. Drapeau, P. Cossette, Disruption of AP1S1, [20] J.A. Lee, G.J. Cole, Generation of transgenic zebrafish expressing green causing a novel neurocutaneous syndrome, perturbs development of the skin fluorescent protein under control of zebrafish amyloid precursor protein gene and spinal cord, PLoS Genet. 4 (2008) e1000296. regulatory elements, Zebrafish 4 (2007) 277–286. [48] J.S. Bonifacino, B.S. Glick, The mechanisms of vesicle budding and fusion, Cell 116 [21] L.A. Shakes, T.L. Malcolm, K.L. Allen, S. De, K.R. Harewood, P.K. Chatterjee, (2004) 153–166. Context dependent function of APPb enhancer identified using enhancer trap- [49] M.S. Robinson, Adaptable adaptors for coated vesicles, Trends Cell Biol. 14 containing BACs as transgenes in zebrafish, Nucleic Acids Res. 36 (2008) (2004) 167–174. 6237–6248. [50] M. Boehm, J.S. Bonifacino, Adaptins: the final recount, Mol. Biol. Cell 12 (2001) [22] L. Flinn, S. Bretaud, C. Lo, P.W. Ingham, O. Bandmann, Zebrafish as a new animal 2907–2920. model for movement disorders, J. Neurochem. 106 (2008) 1991–1997. [51] D.J. Owen, B.M. Collins, P.R. Evans, Adaptors for clathrin coats: structure and [23] S. Bretaud, S. Lee, S. Guo, Sensitivity of zebrafish to environmental toxins function, Annu. Rev. Cell Dev. Biol. 20 (2004) 153–191. implicated in Parkinson's disease, Neurotoxicol. Teratol. 26 (2004) 857–864. [52] C.E. Beattie, T.L. Carrel, M.L. McWhorter, Fishing for a mechanism: using zebrafish to [24] C.S. Lam, V. Korzh, U. Strahle, Zebrafish embryos are susceptible to the understand spinal muscular atrophy, J. Child Neurol. 22 (2007) 995–1003. dopaminergic neurotoxin MPTP, Eur. J. Neurosci. 21 (2005) 1758–1762. [53] P. Drapeau, L. Saint-Amant, R.R. Buss, M. Chong, J.R. McDearmid, E. Brustein, [25] E.T. McKinley, T.C. Baranowski, D.O. Blavo, C. Cato, T.N. Doan, A.L. Rubinstein, Development of the locomotor network in zebrafish, Prog. Neurobiol. 68 (2002) Neuroprotection of MPTP-induced toxicity in zebrafish dopaminergic neurons, 85–111. Brain Res. Mol. Brain Res. 141 (2005) 128–137. [54] R. Lemmens, A. Van Hoecke, N. Hersmus, V. Geelen, I. D'Hollander, V. Thijs, L. Van [26] A. Abeliovich, M. Flint Beal, Parkinsonism genes: culprits and clues, J. Den Bosch, P. Carmeliet, W. Robberecht, Overexpression of mutant superoxide Neurochem. 99 (2006) 1062–1072. dismutase 1 causes a motor axonopathy in the zebrafish, Hum. Mol. Genet. 16 [27] O.L. Son, H.T. Kim, M.H. Ji, K.W. Yoo, M. Rhee, C.H. Kim, Cloning and expression (2007) 2359–2365. analysis of a Parkinson's disease gene, uch-L1, and its promoter in zebrafish, [55] F. Gros-Louis, J. Kriz, E. Kabashi, J. McDearmid, S. Millecamps, M. Urushitani, L. Biochem. Biophys. Res. Commun. 312 (2003) 601–607. Lin, P. Dion, Q. Zhu, P. Drapeau, J.P. Julien, G.A. Rouleau, Als2 mRNA splicing [28] S. Bretaud, C. Allen, P.W. Ingham, O. Bandmann, p53-dependent neuronal cell variants detected in KO mice rescue severe motor dysfunction phenotype in death in a DJ-1-deficient zebrafish model of Parkinson's disease, J. Neurochem. Als2 knock-down zebrafish, Hum. Mol. Genet. 17 (2008) 2691–2702. 100 (2007) 1626–1635. [56] C.L. Simpson, R. Lemmens, K. Miskiewicz, W.J. Broom, V.K. Hansen, P.W. van Vught, [29] L. Flinn, H. Mortiboys, K. Volkmann, R.W. Koster, P.W. Ingham, O. Bandmann, J.E. Landers, P. Sapp, L. Van Den Bosch, J. Knight, B.M. Neale, M.R. Turner, J.H. Veldink, Complex I deficiency and dopaminergic neuronal cell loss in Parkin-deficient R.A. Ophoff, V.B. Tripathi, A. Beleza, M.N. Shah, P. Proitsi, A. Van Hoecke, P. Carmeliet, zebrafish (Danio rerio), Brain 132 (2009) 1613–1623. H.R. Horvitz, P.N. Leigh, C.E. Shaw, L.H. van den Berg, P.C. Sham, J.F. Powell, P. [30] W. Boehmler, J. Petko, M. Woll, C. Frey, B. Thisse, C. Thisse, V.A. Canfield, R. Verstreken, R.H. Brown Jr., W. Robberecht, A. Al-Chalabi, Variants of the elongator Levenson, Identification of zebrafish A2 adenosine receptors and expression in protein 3 (ELP3) gene are associated with motor neuron degeneration, Hum. Mol. developing embryos, Gene Expr. Patterns 9 (2009) 144–151. Genet. 18 (2009) 472–481. [31] L. Wen, W. Wei, W. Gu, P. Huang, X. Ren, Z. Zhang, Z. Zhu, S. Lin, B. Zhang, [57] M. Neumann, D.M. Sampathu, L.K. Kwong, A.C. Truax, M.C. Micsenyi, T.T. Chou, J. Visualization of monoaminergic neurons and neurotoxicity of MPTP in live Bruce, T. Schuck, M. Grossman, C.M. Clark, L.F. McCluskey, B.L. Miller, E. Masliah, transgenic zebrafish, Dev. Biol. 314 (2008) 84–92. I.R. Mackenzie, H. Feldman, W. Feiden, H.A. Kretzschmar, J.Q. Trojanowski, V.M. [32] C.A. Karlovich, R.M. John, L. Ramirez, D.Y. Stainier, R.M. Myers, Characterization Lee, Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyo- of the Huntington's disease (HD) gene homologue in the zebrafish Danio rerio, trophic lateral sclerosis, Science 314 (2006) 130–133. Gene 217 (1998) 117–125. [58] E. Kabashi, P.N. Valdmanis, P. Dion, D. Spiegelman, B.J. McConkey, C. Vande [33] A.L. Lumsden, T.L. Henshall, S. Dayan, M.T. Lardelli, R.I. Richards, Huntingtin- Velde, J.P. Bouchard, L. Lacomblez, K. Pochigaeva, F. Salachas, P.F. Pradat, W. deficient zebrafish exhibit defects in iron utilization and development, Hum. Camu, V. Meininger, N. Dupre, G.A. Rouleau, TARDBP mutations in individuals Mol. Genet. 16 (2007) 1905–1920. with sporadic and familial amyotrophic lateral sclerosis, Nat. Genet. 40 (2008) [34] H. Diekmann, O. Anichtchik, A. Fleming, M. Futter, P. Goldsmith, A. Roach, D.C. 572–574. Rubinsztein, Decreased BDNF levels are a major contributor to the embryonic [59] J. Sreedharan, I.P. Blair, V.B. Tripathi, X. Hu, C. Vance, B. Rogelj, S. Ackerley, J.C. phenotype of huntingtin knockdown zebrafish, J. Neurosci. 29 (2009) Durnall, K.L. Williams, E. Buratti, F. Baralle, J. de Belleroche, J.D. Mitchell, P.N. 1343–1349. Leigh, A. Al-Chalabi, C.C. Miller, G. Nicholson, C.E. Shaw, TDP-43 mutations in [35] N.W. Schiffer, S.A. Broadley, T. Hirschberger, P. Tavan, H.A. Kretzschmar, A. Giese, familial and sporadic amyotrophic lateral sclerosis, Science 319 (2008) C. Haass, F.U. Hartl, B. Schmid, Identification of anti-prion compounds as efficient 1668–1672. inhibitors of polyglutamine protein aggregation in a zebrafish model, J. Biol. [60] S. Hadano, S.C. Benn, S. Kakuta, A. Otomo, K. Sudo, R. Kunita, K. Suzuki- Chem. 282 (2007) 9195–9203. Utsunomiya, H. Mizumura, J.M. Shefner, G.A. Cox, Y. Iwakura, R.H. Brown Jr., J.E. [36] V.M. Miller, R.F. Nelson, C.M. Gouvion, A. Williams, E. Rodriguez-Lebron, S.Q. Ikeda, Mice deficient in the Rab5 guanine nucleotide exchange factor ALS2/alsin Harper, B.L. Davidson, M.R. Rebagliati, H.L. Paulson, CHIP suppresses polyglutamine exhibit age-dependent neurological deficits and altered endosome trafficking, aggregation and toxicity in vitro and in vivo, J. Neurosci. 25 (2005) 9152–9161. Hum. Mol. Genet. 15 (2006) 233–250. [37] A. Williams, S. Sarkar, P. Cuddon, E.K. Ttofi, S. Saiki, F.H. Siddiqi, L. Jahreiss, A. [61] S. Salinas, C. Proukakis, A. Crosby, T.T. Warner, Hereditary spastic paraplegia: Fleming, D. Pask, P. Goldsmith, C.J. O'Kane, R.A. Floto, D.C. Rubinsztein, Novel clinical features and pathogenetic mechanisms, Lancet Neurol. 7 (2008) targets for Huntington's disease in an mTOR-independent autophagy pathway, 1127–1138. Nat. Chem. Biol. 4 (2008) 295–305. [62] P.N. Valdmanis, I.A. Meijer, A. Reynolds, A. Lei, P. MacLeod, D. Schlesinger, M. [38] S.C. Baraban, Emerging epilepsy models: insights from mice, flies, worms and Zatz, E. Reid, P.A. Dion, P. Drapeau, G.A. Rouleau, Mutations in the KIAA0196 gene fish, Curr. Opin. Neurol. 20 (2007) 164–168. at the SPG8 locus cause hereditary spastic paraplegia, Am. J. Hum. Genet. 80 [39] J.A. Tiedeken, J.S. Ramsdell, DDT exposure of zebrafish embryos enhances seizure (2007) 152–161. susceptibility: relationship to fetal p,p′-DDE burden and domoic acid exposure of [63] P. Lin, J. Li, Q. Liu, F. Mao, R. Qiu, H. Hu, Y. Song, Y. Yang, G. Gao, C. Yan, W. Yang, C. California sea lions, Environ. Health Perspect. 117 (2009) 68–73. Shao, Y. Gong, A missense mutation in SLC33A1, which encodes the acetyl-CoA [40] S.C. Baraban, M.R. Taylor, P.A. Castro, H. Baier, Pentylenetetrazole induced transporter, causes autosomal-dominant spastic paraplegia (SPG42), Am. J. changes in zebrafish behavior, neural activity and c-fos expression, Neuroscience Hum. Genet. 83 (2008) 752–759. 131 (2005) 759–768. [64] J.D. Wood, J.A. Landers, M. Bingley, C.J. McDermott, V. Thomas-McArthur, L.J. [41] J.A. Tiedeken, J.S. Ramsdell, Embryonic exposure to domoic acid increases the Gleadall, P.J. Shaw, V.T. Cunliffe, The microtubule-severing protein Spastin is susceptibility of zebrafish larvae to the chemical convulsant pentylenetetrazole, essential for axon outgrowth in the zebrafish embryo, Hum. Mol. Genet. 15 Environ. Health Perspect. 115 (2007) 1547–1552. (2006) 2763–2771. [42] J.A. Tiedeken, J.S. Ramsdell, A.F. Ramsdell, Developmental toxicity of domoic acid [65] M.L. McWhorter, U.R. Monani, A.H. Burghes, C.E. Beattie, Knockdown of the in zebrafish (Danio rerio), Neurotoxicol. Teratol. 27 (2005) 711–717. survival motor neuron (Smn) protein in zebrafish causes defects in motor axon [43] S.C. Baraban, M.T. Dinday, P.A. Castro, S. Chege, S. Guyenet, M.R. Taylor, A large- outgrowth and pathfinding, J. Cell Biol. 162 (2003) 919–931. scale mutagenesis screen to identify seizure-resistant zebrafish, Epilepsia 48 [66] T.L. Carrel, M.L. McWhorter, E. Workman, H. Zhang, E.C. Wolstencroft, C. Lorson, (2007) 1151–1157. G.J. Bassell, A.H. Burghes, C.E. Beattie, Survival motor neuron function in motor [44] M.J. Winter, W.S. Redfern, A.J. Hayfield, S.F. Owen, J.P. Valentin, T.H. Hutchinson, axons is independent of functions required for small nuclear ribonucleoprotein Validation of a larval zebrafish locomotor assay for assessing the seizure liability biogenesis, J. Neurosci. 26 (2006) 11014–11022. of early-stage development drugs, J. Pharmacol. Toxicol. Methods 57 (2008) [67] G.E. Oprea, S. Krober, M.L. McWhorter, W. Rossoll, S. Muller, M. Krawczak, G.J. 176–187. Bassell, C.E. Beattie, B. Wirth, Plastin 3 is a protective modifier of autosomal [45] A.G. Bassuk, R.H. Wallace, A. Buhr, A.R. Buller, Z. Afawi, M. Shimojo, S. Miyata, S. recessive spinal muscular atrophy, Science 320 (2008) 524–527. Chen, P. Gonzalez-Alegre, H.L. Griesbach, S. Wu, M. Nashelsky, E.K. Vladar, D. [68] K.L. Boon, S. Xiao, M.L. McWhorter, T. Donn, E. Wolf-Saxon, M.T. Bohnsack, C.B. Antic, P.J. Ferguson, S. Cirak, T. Voit, M.P. Scott, J.D. Axelrod, C. Gurnett, A.S. Moens, C.E. Beattie, Zebrafish survival motor neuron mutants exhibit presyn- Daoud, S. Kivity, M.Y. Neufeld, A. Mazarib, R. Straussberg, S. Walid, A.D. Korczyn, aptic neuromuscular junction defects, Hum. Mol. Genet. 18 (2009) 3615–3625. 344 E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345

[69] V. Tropepe, H.L. Sive, Can zebrafish be used as a model to study the [87] G. Kirov, D. Gumus, W. Chen, N. Norton, L. Georgieva, M. Sari, M.C. O'Donovan, F. neurodevelopmental causes of autism? Genes Brain Behav. 2 (2003) 268–281. Erdogan, M.J. Owen, H.H. Ropers, R. Ullmann, Comparative genome hybridiza- [70]J.D.Wood,F.Bonath,S.Kumar,C.A.Ross,V.T.Cunliffe,Disrupted-in- tion suggests a role for NRXN1 and APBA2 in schizophrenia, Hum. Mol. Genet. 17 schizophrenia 1 and neuregulin 1 are required for the specification of (2008) 458–465. oligodendrocytes and neurones in the zebrafish brain, Hum. Mol. Genet. 18 [88] D. Rujescu, A. Ingason, S. Cichon, O.P. Pietilainen, M.R. Barnes, T. Toulopoulou, M. (2009) 391–404. Picchioni, E. Vassos, U. Ettinger, E. Bramon, R. Murray, M. Ruggeri, S. Tosato, C. [71] G.E. Elsen, L.Y. Choi, V.E. Prince, R.K. Ho, The autism susceptibility gene met Bonetto, S. Steinberg, E. Sigurdsson, T. Sigmundsson, H. Petursson, A. Gylfason, P.I. regulates zebrafish cerebellar development and facial motor neuron migration, Olason, G. Hardarsson, G.A. Jonsdottir, O. Gustafsson, R. Fossdal, I. Giegling, H.J. Dev. Biol. 335 (2009) 78–92. Moller, A.M. Hartmann, P. Hoffmann, C. Crombie, G. Fraser, N. Walker, J. Lonnqvist, J. [72] F. Laumonnier, P.C. Cuthbert, S.G. Grant, The role of neuronal complexes in Suvisaari, A. Tuulio-Henriksson, S. Djurovic, I. Melle, O.A. Andreassen, T. Hansen, T. human X-linked brain diseases, Am. J. Hum. Genet. 80 (2007) 205–220. Werge, L.A. Kiemeney, B. Franke, J. Veltman, J.E. Buizer-Voskamp, C. Sabatti, R.A. [73] D.H. Skuse, Imprinting, the X-chromosome, and the male brain: explaining sex Ophoff, M. Rietschel, M.M. Nothen, K. Stefansson, L. Peltonen, D. St Clair, H. differences in the liability to autism, Pediatr. Res. 47 (2000) 9–16. Stefansson, D.A. Collier, Disruption of the neurexin 1 gene is associated with [74] S.G. Grant, M.C. Marshall, K.L. Page, M.A. Cumiskey, J.D. Armstrong, Synapse schizophrenia, Hum. Mol. Genet. 18 (2009) 988–996. proteomics of multiprotein complexes: en route from genes to nervous system [89] A. Rissone, M. Monopoli, M. Beltrame, F. Bussolino, F. Cotelli, M. Arese, diseases, Hum. Mol. Genet. 14 (Spec No. 2) (2005) R225–R234. Comparative genome analysis of the neurexin gene family in Danio rerio: [75] J. Gauthier, D. Spiegelman, A. Piton, R.G. Lafreniere, S. Laurent, J. St-Onge, L. insights into their functions and evolution, Mol. Biol. Evol. 24 (2007) 236–252. Lapointe, F.F. Hamdan, P. Cossette, L. Mottron, E. Fombonne, R. Joober, C. [90] M. Setou, T. Nakagawa, D.H. Seog, N. Hirokawa, Kinesin superfamily motor Marineau, P. Drapeau, G.A. Rouleau, Novel de novo SHANK3 mutation in autistic protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport, patients, Am. J. Med. Genet. B Neuropsychiatr. Genet. 150B (2009) 421–424. Science (New York, N.Y.) 288 (2000) 1796–1802. [76] C.M. Durand, C. Betancur, T.M. Boeckers, J. Bockmann, P. Chaste, F. Fauchereau, G. [91] L. Guillaud, M. Setou, N. Hirokawa, KIF17 dynamics and regulation of NR2B Nygren, M. Rastam, I.C. Gillberg, H. Anckarsater, E. Sponheim, H. Goubran-Botros, trafficking in hippocampal neurons, J. Neurosci. 23 (2003) 131–140. R. Delorme, N. Chabane, M.C. Mouren-Simeoni, P. de Mas, E. Bieth, B. Roge, D. [92] P.J. Chu, J.F. Rivera, D.B. Arnold, A role for Kif17 in transport of Kv4.2, J. Biol. Heron, L. Burglen, C. Gillberg, M. Leboyer, T. Bourgeron, Mutations in the gene Chem. 281 (2006) 365–373. encoding the synaptic scaffolding protein SHANK3 are associated with autism [93] R.W. Wong, M. Setou, J. Teng, Y. Takei, N. Hirokawa, Overexpression of motor spectrum disorders, Nat. Genet. 39 (2007) 25–27. protein KIF17 enhances spatial and working memory in transgenic mice, Proc. [77] G. Roussignol, F. Ango, S. Romorini, J.C. Tu, C. Sala, P.F. Worley, J. Bockaert, L. Natl. Acad. Sci. U. S. A. 99 (2002) 14500–14505. Fagni, Shank expression is sufficient to induce functional dendritic spine [94] R. Roberson, L. Toso, D. Abebe, C.Y. Spong, Altered expression of KIF17, a synapses in aspiny neurons, J. Neurosci. 25 (2005) 3560–3570. kinesin motor protein associated with NR2B trafficking, may mediate learning [78] A. Piton, J.L. Michaud, H. Peng, S. Aradhya, J. Gauthier, L. Mottron, N. Champagne, deficits in a Down syndrome mouse model, Am. J. Obstet. Gynecol. 198 (2008) R.G. Lafreniere, F.F. Hamdan, R. Joober, E. Fombonne, C. Marineau, P. Cossette, M.P. 313 e311-314. Dube, P. Haghighi, P. Drapeau, P.A. Barker, S. Carbonetto, G.A. Rouleau, Mutations [95] L. Guillaud, R. Wong, N. Hirokawa, Disruption of KIF17-Mint1 interaction by in the calcium-related gene IL1RAPL1 are associated with autism, Hum. Mol. CaMKII-dependent phosphorylation: a molecular model of kinesin-cargo Genet. 17 (2008) 3965–3974. release, Nat. Cell Biol. 10 (2008) 19–29. [79] N. Bahi, G. Friocourt, A. Carrie, M.E. Graham, J.L. Weiss, P. Chafey, F. Fauchereau, [96] C. Insinna, N. Pathak, B. Perkins, I. Drummond, J.C. Besharse, The homodimeric R.D. Burgoyne, J. Chelly, IL1 receptor accessory protein like, a protein involved in kinesin, Kif17, is essential for vertebrate photoreceptor sensory outer segment X-linked mental retardation, interacts with Neuronal Calcium Sensor-1 and development, Dev. Biol. 316 (2008) 160–170. regulates exocytosis, Hum. Mol. Genet. 12 (2003) 1415–1425. [97] M.L. Suster, H. Kikuta, A. Urasaki, K. Asakawa, K. Kawakami, Transgenesis in [80] F. Gambino, A. Pavlowsky, A. Begle, J.L. Dupont, N. Bahi, R. Courjaret, R. Gardette, H. zebrafish with the tol2 transposon system, Methods Mol. Biol. 561 (2009) Hadjkacem, H. Skala, B. Poulain, J. Chelly, N. Vitale, Y. Humeau, IL1-receptor 41–63. accessory protein-like 1 (IL1RAPL1), a protein involved in cognitive functions, [98] D. Goldman, M. Hankin, Z. Li, X. Dai, J. Ding, Transgenic zebrafish for studying regulates N-type Ca2+-channel and neurite elongation, Proc. Natl. Acad. Sci. U. S. A. nervous system development and regeneration, Transgenic Res. 10 (2001) 104 (2007) 9063–9068. 21–33. [81] F. Gambino, M. Kneib, A. Pavlowsky, H. Skala, S. Heitz, N. Vitale, B. Poulain, M. [99] T.M. Jessell, Neuronal specification in the spinal cord: inductive signals and Khelfaoui, J. Chelly, P. Billuart, Y. Humeau, IL1RAPL1 controls inhibitory networks transcriptional codes, Nat. Rev. Genet. 1 (2000) 20–29. during cerebellar development in mice, Eur. J. Neurosci. 30 (2009) 1476–1486. [100] T. Nakano, M. Windrem, V. Zappavigna, S.A. Goldman, Identification of a [82] E. Tabolacci, M.G. Pomponi, R. Pietrobono, A. Terracciano, P. Chiurazzi, G. Neri, A conserved 125 base-pair Hb9 enhancer that specifies gene expression to spinal truncating mutation in the IL1RAPL1 gene is responsible for X-linked mental motor neurons, Dev. Biol. 283 (2005) 474–485. retardation in the MRX21 family, Am. J. Med. Genet. 140 (2006) 482–487. [101] S. Higashijima, Y. Hotta, H. Okamoto, Visualization of cranial motor neurons in [83] A. Carrie, L. Jun, T. Bienvenu, M.C. Vinet, N. McDonell, P. Couvert, R. Zemni, A. live transgenic zebrafish expressing green fluorescent protein under the control Cardona, G. Van Buggenhout, S. Frints, B. Hamel, C. Moraine, H.H. Ropers, T. of the islet-1 promoter/enhancer, J. Neurosci. 20 (2000) 206–218. Strom, G.R. Howell, A. Whittaker, M.T. Ross, A. Kahn, J.P. Fryns, C. Beldjord, P. [102] M.L. Suster, A. Kania, M. Liao, K. Asakawa, F. Charron, K. Kawakami, P. Drapeau, A Marynen, J. Chelly, A new member of the IL-1 receptor family highly expressed in novel conserved evx1 enhancer links spinal interneuron morphology and cis- hippocampus and involved in X-linked mental retardation, Nat. Genet. 23 regulation from fish to mammals, Dev. Biol. 325 (2009) 422–433. (1999) 25–31. [103]A.Picker,S.Scholpp,H.Bohli,H.Takeda,M.Brand,Anovelpositive [84] T. Yoshida, M. Mishina, Zebrafish orthologue of mental retardation protein transcriptional feedback loop in midbrain–hindbrain boundary development is IL1RAPL1 regulates presynaptic differentiation, Mol. Cell. Neurosci. 39 (2008) revealed through analysis of the zebrafish pax2.1 promoter in transgenic lines, 218–228. Development 129 (2002) 3227–3239. [85] P. Szatmari, A.D. Paterson, L. Zwaigenbaum, W. Roberts, J. Brian, X.Q. Liu, J.B. [104] D.L. McLean, J. Fan, S. Higashijima, M.E. Hale, J.R. Fetcho, A topographic map of Vincent, J.L. Skaug, A.P. Thompson, L. Senman, L. Feuk, C. Qian, S.E. Bryson, M.B. recruitment in spinal cord, Nature 446 (2007) 71–75. Jones, C.R. Marshall, S.W. Scherer, V.J. Vieland, C. Bartlett, L.V. Mangin, R. Goedken, A. [105] Y. Kimura, Y. Okamura, S. Higashijima, alx, a zebrafish homolog of Chx10, marks Segre, M.A. Pericak-Vance, M.L. Cuccaro, J.R. Gilbert, H.H. Wright, R.K. Abramson, C. ipsilateral descending excitatory interneurons that participate in the regulation Betancur, T. Bourgeron, C. Gillberg, M. Leboyer, J.D. Buxbaum, K.L. Davis, E. of spinal locomotor circuits, J. Neurosci. 26 (2006) 5684–5697. Hollander, J.M. Silverman, J. Hallmayer, L. Lotspeich, J.S. Sutcliffe, J.L. Haines, S.E. [106] P. Blader, C. Plessy, U. Strahle, Multiple regulatory elements with spatially and Folstein, J. Piven, T.H. Wassink, V. Sheffield, D.H. Geschwind, M. Bucan, W.T. Brown, temporally distinct activities control neurogenin1 expression in primary R.M. Cantor, J.N. Constantino, T.C. Gilliam, M. Herbert, C. Lajonchere, D.H. Ledbetter, neurons of the zebrafish embryo, Mech. Dev. 120 (2003) 211–218. C. Lese-Martin, J. Miller, S. Nelson, C.A. Samango-Sprouse, S. Spence, M. State, R.E. [107] A.N. Ng, T.A. de Jong-Curtain, D.J. Mawdsley, S.J. White, J. Shin, B. Appel, P.D. Tanzi, H. Coon, G. Dawson, B. Devlin, A. Estes, P. Flodman, L. Klei, W.M. McMahon, N. Dong, D.Y. Stainier, J.K. Heath, Formation of the digestive system in zebrafish: III. Minshew, J. Munson, E. Korvatska, P.M. Rodier, G.D. Schellenberg, M. Smith, M.A. Intestinal epithelium morphogenesis, Dev. Biol. 286 (2005) 114–135. Spence, C. Stodgell, P.G. Tepper, E.M. Wijsman, C.E. Yu, B. Roge, C. Mantoulan, K. [108] J. Shin, H.C. Park, J.M. Topczewska, D.J. Mawdsley, B. Appel, Neural cell fate analysis Wittemeyer, A. Poustka, B. Felder, S.M. Klauck, C. Schuster, F. Poustka, S. Bolte, S. in zebrafish using olig2 BAC transgenics, Methods Cell Sci. 25 (2003) 7–14. Feineis-Matthews, E. Herbrecht, G. Schmotzer, J. Tsiantis, K. Papanikolaou, E. [109] B.B. Kirby, N. Takada, A.J. Latimer, J. Shin, T.J. Carney, R.N. Kelsh, B. Appel, In vivo Maestrini, E. Bacchelli, F. Blasi, S. Carone, C. Toma, H. Van Engeland, M. de Jonge, C. time-lapse imaging shows dynamic oligodendrocyte progenitor behavior during Kemner, F. Koop, M. Langemeijer, C. Hijmans, W.G. Staal, G. Baird, P.F. Bolton, M.L. zebrafish development, Nat. Neurosci. 9 (2006) 1506–1511. Rutter, E. Weisblatt, J. Green, C. Aldred, J.A. Wilkinson, A. Pickles, A. Le Couteur, T. [110] T.A. Bayer, J.A. Campos-Ortega, A transgene containing lacZ is expressed in Berney, H. McConachie, A.J. Bailey, K. Francis, G. Honeyman, A. Hutchinson, J.R. Parr, primary sensory neurons in zebrafish, Development (Cambridge, England) 115 S. Wallace, A.P. Monaco, G. Barnby, K. Kobayashi, J.A. Lamb, I. Sousa, N. Sykes, E.H. (1992) 421–426. Cook, S.J. Guter, B.L. Leventhal, J. Salt, C. Lord, C. Corsello, V. Hus, D.E. Weeks, F. [111] M.E. Halpern, J. Rhee, M.G. Goll, C.M. Akitake, M. Parsons, S.D. Leach, Gal4/ Volkmar, M. Tauber, E. Fombonne, A. Shih, K.J. Meyer, Mapping autism risk loci UAS transgenic tools and their application to zebrafish, Zebrafish 5 (2008) using genetic linkage and chromosomal rearrangements, Nat. Genet. 39 (2007) 97–110. 319–328. [112] J. Dong, G.W. Stuart, Transgene manipulation in zebrafish by using recombi- [86] H.G. Kim, S. Kishikawa, A.W. Higgins, I.S. Seong, D.J. Donovan, Y. Shen, E. Lally, L.A. nases, Methods Cell Biol. 77 (2004) 363–379. Weiss, J. Najm, K. Kutsche, M. Descartes, L. Holt, S. Braddock, R. Troxell, L. Kaplan, F. [113] E. Provost, J. Rhee, S.D. Leach, Viral 2A peptides allow expression of multiple proteins Volkmar, A. Klin, K. Tsatsanis, D.J. Harris, I. Noens, D.L. Pauls, M.J. Daly, M.E. from a single ORF in transgenic zebrafish embryos, Genesis 45 (2007) 625–629. MacDonald, C.C. Morton, B.J. Quade, J.F. Gusella, Disruption of neurexin 1 associated [114] C.B. Moens, T.M. Donn, E.R. Wolf-Saxon, T.P. Ma, Reverse genetics in zebrafish by with autism spectrum disorder, Am. J. Hum. Genet. 82 (2008) 199–207. TILLING, Brief. Funct. Genomic Proteomic 7 (2008) 454–459. E. Kabashi et al. / Biochimica et Biophysica Acta 1812 (2011) 335–345 345

[115] J.E. Foley, J.R. Yeh, M.L. Maeder, D. Reyon, J.D. Sander, R.T. Peterson, J.K. [123] J.R. Yeh, K.M. Munson, K.E. Elagib, A.N. Goldfarb, D.A. Sweetser, R.T. Peterson, Joung, Rapid mutation of endogenous zebrafish genes using zinc finger Discovering chemical modifiers of oncogene-regulated hematopoietic differen- nucleases made by Oligomerized Pool ENgineering (OPEN), PLoS One 4 tiation, Nat. Chem. Biol. 5 (2009) 236–243. (2009) e4348. [124] A. Mukhopadhyay, R.T. Peterson, Deciphering arterial identity through gene [116] I.G. Woods, A.F. Schier, Targeted mutagenesis in zebrafish, Nat. Biotechnol. 26 expression, genetics, and chemical biology, Curr. Opin. Hematol. 15 (2008) (2008) 650–651. 221–227. [117] X. Ouyang, I.A. Shestopalov, S. Sinha, G. Zheng, C.L. Pitt, W.H. Li, A.J. Olson, J.K. [125] E. Kabashi, L. Lin, M.L. Tradewell, P.A. Dion, V. Bercier, P. Bourgouin, D. Rochefort, Chen, Versatile synthesis and rational design of caged morpholinos, J. Am. Chem. S.B. Hadj, H.D. Durham, C. Vande Velde, G.A. Rouleau, P. Drapeau, Gain and loss of Soc. 131 (2009) 13255–13269. function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in [118] S. Wu, J.M. Storey, K.B. Storey, Phosphoglycerate kinase 1 expression responds vivo, Human Mol. Gen. 19 (2010) 671–683. to freezing, anoxia, and dehydration stresses in the freeze tolerant wood frog, [126] J. Gauthier, N. Champagne, R.G. Lafrenière, L. Xiong, D. Spiegelman, E. Brustein, Rana sylvatica, J. Exp. Zool. A Ecol. Genet. Physiol. 311 (2009) 57–67. M. Lapointe, H. Peng, M. Côté, A. Noreau, F.F. Hamdan, A. Piton, A.M. Addington, [119] P.A. Morcos, Y. Li, S. Jiang, Vivo-morpholinos: a non-peptide transporter delivers J.L. Rapoport, L.E. DeLisi, M.-O. Krebs, R. Joober, F. Fathalli, F. Mouaffak, A.P. morpholinos into a wide array of mouse tissues, Biotechniques 45 (2008) Haghighi, C. Néri, M.-P. Dubé, M.E. Samuels, C. Marineau, E.A. Stone, P. Awadalla, 613–614 616, 618 passim. P.A. Barker, S. Carbonetto, P. Drapeau, G.A. Rouleau, and the S2D team, De novo [120] C.W. Sipe, M.S. Saha, The use of microarray technology in nonmammalian synaptic scaffolding protein SHANK3 mutations in patients ascertained with vertebrate systems, Methods Mol. Biol. 382 (2007) 1–16. schizophrenia, Proc. Natl. Acad. Sci. USA 107 (2010) 7863–7868. [121] A. Hegde, N.C. Qiu, X. Qiu, S.H. Ho, K.Q. Tay, J. George, F.S. Ng, K.R. Govindarajan, [127] J. Tarabeux, N. Champagne, E. Brustein, F.F. Hamdan, J. Gauthier, M. Lapointe, C. Z. Gong, S. Mathavan, Y.J. Jiang, Genomewide expression analysis in zebrafish Maios, A. Piton, D. Spiegelman, É. Henrion, S2D team, B. Mille, J.L. Rapoport, L.E. mind bomb alleles with pancreas defects of different severity identifies putative DeLisi, R. Joober, F. Fathalli, É. Fombonne, L. Mottron, N. Forget-Dubois, M. Boivin, Notch responsive genes, PLoS One 3 (2008) e1479. J.L. Michaud, R.G. Lafrenière, P. Drapeau, M.-O. Krebs, G.A. Rouleau, De Novo [122] L.I. Zon, R.T. Peterson, In vivo drug discovery in the zebrafish, Nat. Rev. Drug Truncating Mutation in KIF17 Associated with Schizophrenia, Biol. Psy. 68 Discov. 4 (2005) 35–44. (2010) 649–656. Biochimica et Biophysica Acta 1812 (2011) 346–352

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Review Zebrafish as a tool in Alzheimer's disease research☆

Morgan Newman a,1, Giuseppe Verdile b,1, Ralph N. Martins b, Michael Lardelli a,⁎ a Discipline of Genetics, School of Molecular and Biomedical Science, The University of Adelaide, SA 5005, Australia b Centre of Excellence for Alzheimer's Disease Research and Care, School of Exercise, Biomedical and Health Sciences, Edith Cowan University, Joondalup, WA 6027, Australia article info abstract

Article history: Alzheimer's disease is the most prevalent form of neurodegenerative disease. Despite many years of intensive Received 27 November 2009 research our understanding of the molecular events leading to this pathology is far from complete. No effective Accepted 21 September 2010 treatments have been defined and questions surround the validity and utility of existing animal models. The Available online 12 October 2010 zebrafish (and, in particular, its embryos) is a malleable and accessible model possessing a vertebrate neural structure and genome. Zebrafish genes orthologous to those mutated in human familial Alzheimer's disease have Keywords: been defined. Work in zebrafish has permitted discovery of unique characteristics of these genes that would have Zebrafish fi Alzheimer's disease been dif cult to observe with other models. In this brief review we give an overview of Alzheimer's disease and Presenilin transgenic animal models before examining the current contribution of zebrafish to this research area. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases. © 2010 Elsevier B.V. All rights reserved.

1. Alzheimer's disease LDL and reduced HDL levels, hypertension, obesity and type II diabetes make important contributions to the risk of developing AD (reviewed by Alzheimer's disease (AD) is the most common form of neurodegen- Martins et al. [5]). Genome-wide association studies in humans have erative disease. Globally, the incidence of dementia was estimated in 2001 identified a number of possible additional loci associated with risk for to be 24.3 million with an estimated increase to 81.1 million in 2040 [1]. LOAD (reviewed by Rademakers and Rovelet-Lecrux [6]. In particular, two Clinically, AD is characterised by progressive memory loss and can include recent studies identified overlapping sets of candidates (see [7,8] and impairment of speech and motor ability, depression, delusions, hallucina- Table1). Individuals carrying the APOE ε4 allele also bear some risk of tions, aggressive behaviour and, ultimately, increasing dependence upon developing the early onset form of AD. Overall, EOAD accounts for only a others before death (recently reviewed by Voisin and Vellas [2]). The small percentage of AD cases but is the most severe form of the disease. major neuropathological hallmarks of the disease were first described by The majority of EOAD cases are familial forms of AD showing dominant Alois Alzheimer as “fibers” and “small military” foci (English translation, inheritance. [3]) Today these are commonly referred to as neurofibrillary tangles Familial AD (FAD) is typically associated with an early age of onset (NFTs) and amyloid plaques. Neurofibrillary tangles are hyperpho- as young as 25 years (reviewed by Verdile and Martins [20]). Most of sphorylated forms of the tau protein, while the major protein component our knowledge of the molecular events underlying the development of amyloid plaques is a small peptide referred to as amyloid beta (Aβ). of Alzheimer's disease comes from FAD since we can use genetic Other forms of lesion have also been found in AD brains (reviewed by analysis to identify the genes and proteins involved. Mutations at only Duyckaerts et al. [4]). three loci are known to cause FAD although other loci may yet be Alzheimer's disease can be classified broadly into two groups, late found (see review by Rademakers and Rovelet-Lecrux [6]). One locus onset AD (LOAD, occurring N65 years) and early onset AD (EOAD, hosts the Amyloid Precursor Protein (APP) from which Aβ is cleaved occurring b65 years). LOAD accounts for the vast majority of AD cases by the action of two “secretases,” β-secretase and γ-secretase (see (95%) and is associated with many risk factors. It is well established that Fig. 1). Mutations in APP alter these cleavages so that Aβ production is age and possession of the ε4alleleoftheAPOLIPOPROTEIN gene (APOE ε4) increased or more aggregation-prone forms of Aβ are formed are the major risk factors for most of the population (reviewed by Martins (reviewed by Verdile et al., [20]). The remaining two loci host the et al. [5]). However, other risk factors such as hormonal changes presenilin genes PSEN1 and PSEN2 with mutations in these genes associated with ageing, diet and lifestyle factors play very important accounting for 50–70% of EOFAD cases [21]. The presenilin proteins roles. Cardiovascular risk factors such as hypercholesterolemia, increased form the catalytic core of protein complexes with γ-secretase activity. Mutations in the presenilins also appear to alter the rate or form of Aβ production and many LOAD associated risk factors also regulate Aβ ☆ fi This article is part of a Special Issue entitled Zebra sh Models of Neurological levels in the brain. Therefore, it is not surprising that the currently Diseases. “ ⁎ Corresponding author. Tel.: +61 8 83033212; fax: +61 8 83034362. dominant hypothesis for the cause of FAD (and LOAD) is the amyloid E-mail address: [email protected] (M. Lardelli). hypothesis” that postulates that toxic oligomerisation of Aβ is the 1 Made equal contributions to this paper. initiating factor beginning a cascade of consequences such as synaptic

0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2010.09.012 M. Newman et al. / Biochimica et Biophysica Acta 1812 (2011) 346–352 347

directed at neutralising Aβ toxicity (i.e. PBT2) are addressing such issues (reviewed by Wisniewski, 2009 [29]). There are also therapies not directed at Aβ that show promise including the anti-histamine Latre- pirdine (formerly known as Dimebon™) which has completed phase IIIa trials and is currently in IIIb trials [30]. The mechanism(s) underlying the benefits of Latrepirdine is unclear but may include improving mitochon- drial activity. Another agent that has shown some promise is Rember™ (methylthionium chloride) which reduces tau aggregation (see http:// www.taurx.com/) although this drug was not seen to affect the histological or behavioural phenotypes of transgenic zebrafish expressing mutant human MAPT (see 2009 ICAD conference report at www. alzforum.org/new/detail.asp?id=2203). Methylthionium chloride is bet- ter known to those working with zebrafishasmethylenebluewhichis used to inhibit fungal growth and to stain the yellow pteridine-containing pigment cells of fish, xanthophores [31]. Recently, the combination of Dimebon (Latrepirdine) and methylene blue has also been shown to inhibit aggregation of TDP-43 [32], a protein component of ubiquitinated inclusions found in the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) (reviewed by Mackenzie [33]). The beneficial outcomes of these non-Aβ directed agents may provide further evidence for alternatives to the amyloid hypothesis, However, it is conceivable that these agents may be acting to ameliorate the consequences of Aβ toxicity e.g. by protecting neurons from insults such as aggregation of Aβ or other proteins. Fig. 1. The γ-secretase complex consists of four protein components including PSEN1 or PSEN2 that reside within lipid bilayers and cleave type 1 transmembrane domain proteins 2. Presenilin activity is involved in many aspects of AD pathology at a catalytic site dependent upon two aspartate residues (stars). APP is initially cleaved either by α-orβ-secretase to produce APPα or APPβ.Onlyβ-secretase cleavage permits Aβ peptide production after secondary cleavage by γ-secretase that also releases APP's The presenilins proteins are known to interact with both the APP and intracellular domain that can enter the nuclear to contribute to gene regulation. tau proteins [34–38] providing one possible link between Aβ production and changes in tau phosphorylation. Mice lacking expression of both Psen1 and Psen2 in forebrain tissue show a number of phenotypic effects dysfunction, inflammation and oxidative damage causing neuronal resembling Alzheimer's disease, i.e. neurodegeneration such as hyper- dysfunction, degeneration and death (see review in [22]). phosphorylation of tau, formation of intracellular tau inclusions, gliosis, The amyloid hypothesis is an attractively parsimonious explanation expansion of brain ventricles and neuronal death all in the absence of for AD pathogenesis and finds support from numerous phenomena any Aβ production [24]. Some characteristics of Alzheimer's disease, observed in EOFAD and LOAD brains. However, it fails to explain some such as increased formation of glia, may result from changes in observations in animal models and the outcomes of a number of human presenilin activity since γ-secretase complexes cleave not only APP clinical trials of drugs intended to ameliorate Aβ toxicity. For example, but also a large collection of other transmembrane proteins such as the majority of transgenic mice harbouring FAD-causing mutations NOTCH, E-cadherin, neuregulin-1 and syndecan (reviewed in Ref. [39]). leading to accumulation of human Aβ in their brains do not show Presenilins also function in a number of cellular processes, at least some dramatic neurodegeneration [23] (see the discussion in the 2008 paper of which appear independent of γ-secretase activity. For example, Kang by Chen et al. [24]). However, these transgenic mice do show cognitive et al. [40] showed that mouse Psen1 represents a second pathway for deficits in behavioural tests and show reduction in long-term phosphorylation of β-catenin independent of Axin. Presenilins also potentiation, most likely indicating loss in synaptic activity [25].To appear to act as Ca2+ leak channels in the endoplasmic reticulum [41]. obtain significant neurodegeneration or neuronal loss in transgenic Presenilins are also found in interphase kinetochores [42] (although mice requires expression of three or more mutations in AD-associated this observation has not been replicated) and changes in presenilin genes. Examples of mouse AD models used currently include “3xTg-AD” activity can cause aneuploidy ([43,44]) and dysregulation of cell cycle mice bearing mutant alleles of PS1, APP and MAPT [26] and “5xFAD” mice proteins [12,45]. Indeed, the activity in γ-secretase of at least some of bearing three APP and two PS1 mutant alleles [27]. However, the co- the proteins comprising these complexes does not appear to be their existence of three or more mutations in AD-associated genes is not a original function. Homologues of presenilins exist in plants and this situation that occurs in humans. A more physiologically relevant animal kingdom does not display γ-secretase activity [46]. model is required that more closely mirrors human AD neuropathology The great majority of the mutations causing FAD are found in PSEN1. including the observed neurodegeneration and neuronal loss (see the Currently over 180 different missense mutations are known (data on critical review by Buxbaum [28]). mutations are collated at Alzheimer Disease and Frontotemporal A number of therapeutic agents aimed at reducing Aβ production or Dementia Mutation Database, http://www.molgen.ua.ac.be/ADMuta- accumulation/aggregation have failed human clinical trials. These include tions/). There is also evidence that oligomeric forms of Aβ can feed back peptide mimetics that inhibit Aβ aggregation (i.e., Alzhemed™,) or agents onto the activity of γ-secretase ([47]). Thus it is possible that mutations that either inhibit γ-secretase activity (e.g. LY-411,575 and LY-450,139 ) that cause production of more Aβ, or more aggregation-prone forms of or target the production of the more neurotoxic Aβ42 (e.g. the NSAID, Aβ, may affect presenilin function and that changes in presenilin Flurizan™ and refer to www.alzforum.org regarding information on AD function may then affect tau phosphorylation, cell differentiation (e.g. drugs in clinical trials). This has raised questions over the validity of the via Notch signalling), calcium homeostasis, etc. amyloid hypothesis. However, issues surrounding penetration through the blood–brain barrier and the advanced state of neurodegeneration 3. Using zebrafish to study genes mutated in FAD when these drugs were given may explain their failure. Apparently successful therapeutics currently in phase II/III trials such as the Mice are the dominant model used for modelling of Alzheimer's disease immunotherapeutics Bapinezuimab™ and Solabezumab™ or agents pathology (reviewed in Ref. [48]) but non-mammalian organisms have also 348 M. Newman et al. / Biochimica et Biophysica Acta 1812 (2011) 346–352 enlightened us. Mutation screening in the nematode, Caenorhabditis elegans were able to do this. Also, their appb analysis utilized only one first showed the connection between the presenilins and Notch signalling morpholino so non-specific effects are not excluded). They observed [49,50]. Expression of human Aβ in C. elegans [51] and Drosophila [52] has defective convergent extension movements and reduced body length. been seen to cause amyloid deposits in muscle and neurodegeneration Interestingly, injection into zebrafish embryos of mRNA encoding respectively. Aβ toxicity can also be studied using yeast [53].Useofthese normal human APP was more effective at ameliorating the loss of organisms has opened up possibilities for screening drug libraries to find appb translation than an Alzheimer's mutant form (“APPswe” containing compounds blocking Aβ toxicity. the “Swedish” double mutation K595N with M596L [58]). This As a model organism, zebrafish presents many advantages for the demonstrates that zebrafish embryos can be exploited for analysis of study of human disease. The vertebrate zebrafish genome and anatomy differences in the activity of different mutant forms of APP [10]. show only ~420 million years (Myr) of divergence from the human Two papers examining transcriptional control of appb have been lineage [54] rather than the ~600 Myr of the ecdysozoans (Drosophila published [59,60]. Both emphasise the importance for expression of a and C. elegans, [55]). Therefore, most human genes have clearly regulatory element in the first intron of the gene. Approximately 14 identifiable orthologues in zebrafish (however, a genome duplication kb of DNA from the promoter region and first intron of the gene can early in the teleost (bony fish) lineage means that, in many cases, two largely recapitulate the embryonic transcription pattern of appb [59]. zebrafish genes together perform the function of an orthologue). The Attempts to model Aβ toxicity have, so far, proven difficult and no numerous and externally fertilized embryos of zebrafish are easily studies have yet been published. The existence of transgenic fish amenable to methods for manipulating gene and protein activity such as expressing human APPswe in neurons in zebrafishtheunderthecontrol injection of antisense oligonucleotides, mRNAs or transgenes and they of the promoter of the elavl3 gene (previously known as HuC)was can be arrayed in microtitre plates for screening of drug libraries. Cell reported at the 6th European Zebrafish Development and Genetics labelling and transplantation for tracking the behaviour and interactions Meeting in Rome in July 2009 (Hruscha, Teucke, Paquet, Haass and of living cells is also routine. A good indication of the potential utility of Schmid) but there is no data yet on any toxic effects. In our laboratories zebrafish for Alzheimer's disease research is the attempts to obtain we have followed a number of approaches to analysing Aβ toxicity. patent coverage of wide areas of the field (e.g. WO/2006/081539). Simple incubation of zebrafish embryos in media containing Aβ peptide Zebrafish possess genes orthologous to all the genes known to be appears to produce effects on neural development as monitored by involved in Alzheimer's disease (Table 1) The genes appa and appb [9] changes in the patterns of neurons observed in non-transgenic are duplicates of an ancestral teleost orthologue of human APP. The zebrafish. It can also induce cell and embryo death (Frederich-Sleptsova genes psen1 [11] and psen2 [16] are orthologues of human PSEN1 and et al. manuscript in preparation). We also attempted to generate a PSEN2, respectively. Our laboratory has also identified duplicates of an transgenic zebrafish model to facilitate screening for drugs suppressing ancestral teleost orthologue of the MAPT gene encoding tau protein, Aβ toxicity by expressing the 42 amino acid residue form of human Aβ mapta and maptb [56] (discussion of tau activities in zebrafish (Aβ42) in the melanophores (melanocytes) that constitute the zebra- including the MAPT-based model of neurodegeneration of Paquet et fish's dark surface stripes. The hope was to create a highly visible but al. 2009 [57] is to be found in an accompanying paper by Ed Burton). nevertheless viable phenotype in zebrafish larvae that could then be The body of research literature examining the endogenous zebrafish arrayed in microtitre dishes for exposure to various chemical com- appa, appb, psen1 and psen2 genes is still quite limited. Musa et al. [9] pounds. For this we used a DNA fragment from the promoter of the detected widespread and overlapping transcription of both appa and zebrafish gene mitfa (previously nacre) that can specifically drive appb by whole mount in situ transcript hybridisation (WISH) from mid- transcription in zebrafish melanophores [61]. This was coupled to DNA gastrulation. Later in development these two genes show differing coding for a secretory signal fused to Aβ42.Unfortunately,fish bearing patterns of transcription but, notably, at 24 hpf both are expressed in the this transgene only showed a disturbed pigmentation pattern at 16 developing forebrain (telencephalon) and ventral diencephalon. In months of age—too late for use in drug screening [81]. addition, appb is expressed in the ventral mesencephalon, a series of Analysis of presenilin function in zebrafish has tended to focus on nuclei in the hindbrain and in the spinal cord. Joshi et al. [10] recently psen1 due to the more frequent role played by mutations of its human examined the effects of reduction of appb function through use of orthologue, PSEN1 in AD. Zebrafish psen1 sequence and activity were antisense morpholino oligonucleotides blocking translation (while they first analysed by Leimer et al. in 1999 [11] who noted extensive also attempted to reduce appa mRNA translation it is not clear that they conservation of protein primary structure. Gene transcripts are

Table 1 Zebrafish genes orthologous to those involved in Alzheimer's disease.

Zebrafish orthologue Accession number Zebrafish references

Loci for dominant mutations in FAD Amyloid Precursor Protein (APP) appa NM_131564 [9,10] appb NM_152886 Presenilin 1 (PSEN1) psen1 NM_131024 [11–15] Presenilin 2 (PSEN2) psen2 NM_131514 [12,15,16]

Loci associated with risk for ADa Apolipoprotein E (APOE) apoeb NM_131098 Microtubule-Associated Protein Tau (MAPT) mapta N/A maptb N/A Clusterin (CLU) Clu NM_200802 Phosphatidylinositol Binding Clathrin Assembly Protein (PICALM) picalm NM_200927 Complement Component (3b/4b) Receptor 1 (CR1) Unknown

Loci for γ-secretase complex members Nicastrin (NCT) ncstn NM_001009556 [17] Anterior Pharynx Defective 1 Homolog A (APH1a) unknown Anterior Pharynx Defective 1 Homolog B (APH1b) aph1b NM_200115 [18] Presenilin Enhancer 2 (PSENEN) psenen NM_205576 [18,19]

a A large number of additional associated genes have been reported and are reviewed in Rademakers and Rovelet-Lecrux, 2009. M. Newman et al. / Biochimica et Biophysica Acta 1812 (2011) 346–352 349 apparently present in all cells at all developmental times examined. occur in cultured cells during successive passages and/or immortalisa- When zebrafish Psen1 protein expression was driven at high levels in tion. Also, cultured cells exist in a highly abnormal environment without cultured human HEK293 cells the zebrafish protein (as expected) the normal three-dimensional context of cellular contact and signalling. displaced human PSEN1 from γ-secretase complexes indicating This may explain, to some extent, differences that we have observed sufficient structural conservation to interact with other complex while analysing the function of presenilins in zebrafish embryos components. However, the C-terminal fragment (CTF) of zebrafish compared to what has been observed from mammalian cell culture. Psen1 generated by the endoproteolysis of the holoprotein (required to For example, when we overexpressed Psen2 protein in zebrafish activate γ-secretase activity [62])issufficiently different in size that it embryos at relatively high levels by mRNA injection we did not see a can be distinguished from the CTF of human PSEN1 on western blots. decrease in Psen1 levels [13] whereas transfection of constructs Also, zebrafish Psen1 possesses sufficient primary structural differences expressing presenilin proteins into cultured mammalian cells is from human PSEN1 that its γ-secretase cleavage of APP was abnormal known to reduce the levels of endogenous presenilin proteins [72]. leading to increased production of Aβ42 relative to production of the less A fertilized zebrafish egg represents a macroscopic single cell in aggregation-prone 40 amino acid residue form of Aβ (Aβ40). These which the activity of one or multiple endogenous genes/proteins can be authors also demonstrated that mutation of one of the two critical subtly manipulated to obtain a phenotypic or biochemical readout. We catalytic aspartate residues in zebrafish Psen1 could abolish its γ- attempted to model the molecular effects of mutations that alter human secretase activity. PSEN1 splicing to cause FAD by altering the splicing of zebrafish psen1 As expected from the expression of the psen1 transcript, we showed using injected morpholino antisense oligonucleotides. Mutations that Psen1 protein can be observed from the earliest stages of causing increased levels of a PSEN1 transcript isoform lacking exon development [14]. Interestingly, this did not appear to be the case for 8 or that cause loss of exon 9 can cause a form of FAD with a peculiar zebrafish psen2 that we identified in 2002 [16]. We detected the histology of plaques lacking neuritic cores and showing a “cotton wool” ubiquitous presence of psen2 transcripts at all stages but an antibody morphology [73,74]. Unfortunately, when morpholinos binding over raised against an oligopeptide corresponding to N-terminal fragment the splice acceptor sites of zebrafish exons cognate to human exon 8 or (NTF) sequence from Psen2 protein only revealed higher expression of exon 9 were injected we did not observe the desired exon loss from Psen2 after 6 hpf [16]. However, there is evidence from humans [63,64] transcripts. Instead we saw inclusion of cognate introns 7 or 8, and from our own unpublished work of alternative splicing and respectively, in a proportion of transcripts causing truncation of the phosphorylation of PSEN2 orthologous proteins so we presume that open reading frames after exons 7 or 8. Interestingly this produced very species of zebrafish Psen2 protein do, in fact, exist at the earliest strong dominant negative effects in the case of truncation after exon 7 developmental stages in this organism. sequence but not for truncation after exon 8 (which is very similar to a The essential role of presenilin activity in Notch signalling during normal Psen1 NTF). The dominant negative effects included cross- development and the resorption of dead embryos that occurs in mice inhibition of psen2 activity [14]. Injection of mRNAs encoding Psen1 has hindered the analysis of the roles of presenilin during vertebrate proteins truncated after exon 7 or exon 8 sequences produced similar development. Resorption is not a problem in zebrafish so we have been results ([14] and unpublished data). Tests with further morpholinos and keen to exploit morpholino antisense technology to block the activity of mRNAs have shown that transcripts producing dominant negative the zebrafish psen1 and psen2 genes. In mice, loss of Psen1 activity is truncated proteins are those that code for part or all of sequence cognate lethal during development and Psen1−/− embryos show a similar to human PSEN1 exons 6 and 7 but not exon 8. (Curiously one of two phenotype to those lacking mouse Notch1 activity [65,66].Incontrast, nonsense mutations in zebrafish psen1 currently under analysis in the mouse Psen2−/− embryos are viable with no obvious developmental laboratory of Dana Jongejan-Zivkovic falls in this region but there defects [67,68] (although adults do display haemorrhaging and appears to be no dominant negative effect probably due to nonsense histological changes in lung tissue [69]. However, mice lacking both mediated decay of the transcript before it can be translated into a Psen1 and Psen2 activity have a more severe phenotype and loss of γ- truncated protein. (Jongejan-Zivkovic personal communication). The secretase activity than when only Psen1 activity is absent [67]. When we discovery of potently dominant negative forms of truncated presenilin blocked psen1 translation in zebrafish we observed similarly irregular may be important since it has been suggested that a unifying feature of delineation of somites to that seen in Psen1−/− mouse embryos and the all the 180+ mutations in human PSEN1 may be that they are cyclic expression of the her1 gene that controls somite formation was hypomorphic with respect to γ-secretase activity [75]. It is possible disturbed in the presomitic mesoderm [13]. Interestingly, blockage of that the truncated forms of numerous proteins that are seen to translation of zebrafish psen2 transcripts also produces significant accumulate in the brains of LOAD individuals may represent a failure changes in embryo development including expansion of brain ventricles of RNA quality surveillance mechanisms such as nonsense mediated at 48 hpf and decreased trunk neural crest formation with increased decay [76]. This, combined with increased rates of aberrant splicing, etc. numbers of neurons at 24 hpf. The latter two defects at least can be seen in aged cells [77] may allow accumulation of dominant negative attributed to decreased Notch signalling indicating that Psen2 in truncated forms of presenilin in ageing brains that could suppress γ- zebrafish plays a more prominent role in Notch signalling than its secretase activity and thus contribute to LOAD. mammalian orthologue [15]. Indeed, in unpublished work, van Tijn et al. Azebrafish model of FAD or LOAD would be very useful for screening [70] have shown that zebrafish homozygous for null mutations in psen1 for drugs to prevent or ameliorate these diseases. However, even are, nevertheless, viable and fertile indicating that psen2 activity can embryo development in non-transgenic zebrafish can provide assays satisfy all essential psen1 functions, at least in a laboratory setting. for the actions of drugs relevant to Alzheimer's disease. The drug N-[N- The viable homozygous null psen1 mutant zebrafish of van Tijn et (3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT, al. [70] show decreased cell proliferation and de novo neurogenesis. [78]) is now commonly used to inhibit γ-secretase activity in zebrafish Such fish also present an opportunity for in vivo analysis of the effects [79]. Unfortunately, it is commonly and incorrectly assumed to be on psen1 function of mutations causing Alzheimer's disease [70,71]. blocking only Notch signalling which, while very important, is not Since presenilin proteins are ubiquitously expressed, simple ubiqui- the only influential signalling pathway facilitated by γ-secretase. For tous transgenic expression of mutated psen1 genes in fish lacking example, as noted above, Joshi et al. [10] showed that inhibition of appb endogenous psen1 activity can allow analysis of their function in vivo. activity causes aberrant convergence-extension movements although it The ability to analyse the function of genes involved in FAD in vivo is has not been shown that γ-secretase activity is required for this important since gene regulatory and functional data from cultured cells and treatment with DAPT [79,15] or simultaneous inhibition of and in vitro assays is sometimes not consistent with what occurs in the translation of both Psen1 and Psen2 by morpholino injection [15] whole organism. This may be due to genetic and epigenetic changes that does not produce this effect. We have previously shown that numbers of 350 M. Newman et al. / Biochimica et Biophysica Acta 1812 (2011) 346–352 a particular spinal cord cell type, the Dorsal Longitudinal Ascending [4] C. Duyckaerts, B. Delatour, M.C. Potier, Classification and basic pathology of Alzheimer disease, Acta Neuropathol. 118 (2009) 5–36. (DoLA) interneuron, appear dependent upon Psen2 but not Psen1 [5] I.J. Martins, E. Hone, J.K. Foster, S.I. Sunram-Lea, A. Gnjec, S.J. Fuller, D. Nolan, S.E. activity [14,15]. We used this as an assay for Psen2 activity to show that Gandy, R.N. Martins, Apolipoprotein E, cholesterol metabolism, diabetes, and the truncated Psen1 could suppress Psen2 activity [14]. Finally, in a convergence of risk factors for Alzheimer's disease and cardiovascular disease, fi Mol. Psychiatry 11 (2006) 721–736. publication from the Xia laboratory, Yang et al. [80] used zebra sh [6] R. Rademakers, A. Rovelet-Lecrux, Recent insights into the molecular genetics of embryos to observe the differential effects on development of drugs dementia, Trends Neurosci. 32 (2009) 451–461. inhibiting primarily the APP or NOTCH cleavage activities of γ-secretase. [7] J.C. Lambert, S. Heath, G. Even, D. Campion, K. Sleegers, M. Hiltunen, O. Combarros, Thus, zebrafish embryos can provide a valuable tool for discovery of D. Zelenika, M.J. Bullido, B. Tavernier, L. Letenneur, K. Bettens, C. Berr, F. Pasquier, N. Fievet, P. Barberger-Gateau, S. Engelborghs, P. De Deyn, I. Mateo, A. Franck, S. additional compounds that show differential inhibitory effects on γ- Helisalmi, E. Porcellini, O. Hanon, M.M. de Pancorbo, C. Lendon, C. Dufouil, C. secretase. The Xia laboratory has also used injection of different Jaillard, T. Leveillard, V. Alvarez, P. Bosco, M. Mancuso, F. Panza, B. Nacmias, P. combinations of morpholino antisense oligonucleotides to analyse Bossu, P. Piccardi, G. Annoni, D. Seripa, D. Galimberti, D. Hannequin, F. Licastro, H. Soininen, K. Ritchie, H. Blanche, J.F. Dartigues, C. Tzourio, I. Gut, C. Van the effects of inhibiting translation of additional protein components of Broeckhoven, A. Alperovitch, M. Lathrop, P. Amouyel, Genome-wide association γ-secretase complexes such as aph1b (orthologous to human APH1B) study identifies variants at CLU and CR1 associated with Alzheimer's disease, Nat. and psenen (orthologous to human PSENEN and previously named Genet. 41 (2009) 1047–1048. fi [8] D. Harold, R. Abraham, P. Hollingworth, R. Sims, A. Gerrish, M.L. Hamshere, J.S. PEN2). In this way they demonstrated that zebra sh Psenen inter- Pahwa, V. Moskvina, K. Dowzell, A. Williams, N. Jones, C. Thomas, A. Stretton, A.R. acts with NF-kappaB [19] and the p53-dependent apoptosis pathway Morgan, S. Lovestone, J. Powell, P. Proitsi, M.K. Lupton, C. Brayne, D.C. Rubinsztein, [18]. M. Gill, B. Lawlor, A. Lynch, K. Morgan, K.S. Brown, P.A. Passmore, D. Craig, B. McGuinness, S. Todd, C. Holmes, D. Mann, A.D. Smith, S. Love, P.G. Kehoe, J. Hardy, S. Mead, N. Fox, M. Rossor, J. Collinge, W. Maier, F. Jessen, B. Schurmann, H. van 4. Concluding remarks den Bussche, I. Heuser, J. Kornhuber, J. Wiltfang, M. Dichgans, L. Frolich, H. Hampel, M. Hull, D. Rujescu, A.M. Goate, J.S. Kauwe, C. Cruchaga, P. Nowotny, J.C. Morris, K. Mayo, K. Sleegers, K. Bettens, S. Engelborghs, P.P. De Deyn, C. Van Despite many years of intensive research uncertainty still surrounds Broeckhoven, G. Livingston, N.J. Bass, H. Gurling, A. McQuillin, R. Gwilliam, P. the fundamental mechanisms underlying AD pathology. There is Deloukas, A. Al-Chalabi, C.E. Shaw, M. Tsolaki, A.B. Singleton, R. Guerreiro, T.W. Muhleisen, M.M. Nothen, S. Moebus, K.H. Jockel, N. Klopp, H.E. Wichmann, M.M. considerable doubt about the relevance of existing transgenic mouse Carrasquillo, V.S. Pankratz, S.G. Younkin, P.A. Holmans, M. O'Donovan, M.J. Owen, models of the disease. Our lack of understanding has slowed the J. Williams, Genome-wide association study identifies variants at CLU and PICALM development of therapies to treat or prevent AD. The vertebrate genome associated with Alzheimer's disease, Nat. Genet. 41 (2009) 1088–1093. fi and neurobiology of zebrafish and our rapidly expanding ability to [9] A. Musa, H. Lehrach, V.A. Russo, Distinct expression patterns of two zebra sh homologues of the human APP gene during embryonic development, Dev. Genes manipulate gene activity in this organism offer alternative pathways in Evol. 211 (2001) 563–567. which to probe the mysteries of AD pathology. In particular, zebrafish [10] P. Joshi, J.O. Liang, K. Dimonte, J. Sullivan, S.W. Pimplikar, Amyloid precursor fi are well suited to biochemically-based approaches whereby gene protein is required for convergent-extension movements during Zebra sh development, Dev. Biol. 335 (2009) 1–11. activities are manipulated transiently and then the consequences [11] U. Leimer, K. Lun, H. Romig, J. Walter, J. Grunberg, M. Brand, C. Haass, Zebrafish examined in an otherwise normal cellular environment. As genome- (Danio rerio) presenilin promotes aberrant amyloid beta-peptide production and wide association studies reveal additional genes involved with requires a critical aspartate residue for its function in amyloidogenesis, Biochemistry 38 (1999) 13602–13609. development of AD pathology these biochemically-based approaches [12] M. Newman, B. Tucker, S. Nornes, A. Ward, M. Lardelli, Altering presenilin gene can rapidly contribute to our understanding of the relevance of these activity in zebrafish embryos causes changes in expression of genes with potential genes while reducing the chance of being misled by experimental involvement in Alzheimer's disease pathogenesis, J. Alzheimers Dis. 16 (2009) – fl 133 147. results from tissue culture systems that may not re ect in vivo reality. [13] S. Nornes, C. Groth, E. Camp, P. Ey, M. Lardelli, Developmental control of Presenilin1 expression, endoproteolysis, and interaction in zebrafish embryos, Exp. Cell Res. 289 (2003) 124–132. Acknowledgments [14] S. Nornes, M. Newman, G. Verdile, S. Wells, C.L. Stoick-Cooper, B. Tucker, I. Frederich-Sleptsova, R. Martins, M. Lardelli, Interference with splicing of This work was supported by a project grant from the National Presenilin transcripts has potent dominant negative effects on Presenilin activity, Hum. Mol. Genet. 17 (2008) 402–412. Health and Medical Research Council of Australia (453622), the [15] S. Nornes, M. Newman, S. Wells, G. Verdile, R.N. Martins, M. Lardelli, Independent Cancer Council of South Australia (S 24/04) and the School of and cooperative action of Psen2 with Psen1 in zebrafish embryos, Exp. Cell Res. Molecular and Biomedical Sciences of The University of Adelaide. 315 (2009) 2791–2801. fi Professor Martins is supported by grants from the McCusker [16] C. Groth, S. Nornes, R. McCarty, R. Tamme, M. Lardelli, Identi cation of a second presenilin gene in zebrafish with similarity to the human Alzheimer's disease Foundation for Alzheimer's Disease Research, Department of Veterans gene presenilin2, Dev. Genes Evol. 212 (2002) 486–490. Affairs, National Health and Medical Research Council (NHMRC), [17] S.-W. Chong, A. Amsterdam, H.-W. Ang, H. Yang, N. Hopkins, Y.-J. Jiang, Zebrafish Centre of Excellence for Alzheimer's disease Research and Care Nicastrin is required for mid- and hindbrain development, Dev. Biol. 331 (2009) 466. [18] W.A. Campbell, H. Yang, H. Zetterberg, S. Baulac, J.A. Sears, T. Liu, S.T. Wong, T.P. (RNM). Dr Verdile is generously supported by grants from Mr. Zhong, W. Xia, Zebrafish lacking Alzheimer presenilin enhancer 2 (Pen-2) Warren Milner (Milner English College, Perth, Western Australia) and demonstrate excessive p53-dependent apoptosis and neuronal loss, J. Neuro- – Ms Helen Sewell. The sources of funding listed above played no role in chem. 96 (2006) 1423 1440. [19] H. Zetterberg, W.A. Campbell, H.W. Yang, W. Xia, The cytosolic loop of the gamma- study design; in the collection, analysis and interpretation of data; in secretase component presenilin enhancer 2 protects zebrafish embryos from the writing of the report; or in the decision to submit the paper for apoptosis, J. Biol. Chem. 281 (2006) 11933–11939. publication. We are grateful to Prof. Christian Haass and others for [20] G. Verdile, R. Martins, Molecular Genetics of Alzheimer's Disease, in: D. Wildenauer (Ed.), Molecular Biology of Neuropsychiatric Disorders, Springer- valuable comments on the manuscript. The authors declare that they Verlag, Berlin, 2008, pp. 229–276. have no conflict of interest in this work. [21] N. Ercelen, S. Mercan, Alzheimer's disease and genes, Adv. Mol. Med. 1 (2005) 155–164. [22] G. Verdile, S. Fuller, C.S. Atwood, S.M. Laws, S.E. Gandy, R.N. Martins, The role of References beta amyloid in Alzheimer's disease: still a cause of everything or the only one who got caught? Pharmacol. Res. 50 (2004) 397–409. [1] C.P. Ferri, M. Prince, C. Brayne, H. Brodaty, L. Fratiglioni, M. Ganguli, K. Hall, K. [23] E. McGowan, F. Pickford, J. Kim, L. Onstead, J. Eriksen, C. Yu, L. Skipper, M.P. Murphy, J. Hasegawa, H. Hendrie, Y. Huang, A. Jorm, C. Mathers, P.R. Menezes, E. Rimmer, M. Beard, P. Das, K. Jansen, M. Delucia, W.L. Lin, G. Dolios, R. Wang, C.B. Eckman, D.W. Scazufca, Global prevalence of dementia: a Delphi consensus study, Lancet 366 Dickson, M. Hutton, J. Hardy, T. Golde, Abeta42 is essential for parenchymal and (2005) 2112–2117. vascular amyloid deposition in mice, Neuron 47 (2005) 191–199. [2] T. Voisin, B. Vellas, Diagnosis and treatment of patients with severe Alzheimer's [24] Q. Chen, A. Nakajima, S.H. Choi, X. Xiong, Y.P. Tang, Loss of presenilin function disease, Drugs Aging 26 (2009) 135–144. causes Alzheimer's disease-like neurodegeneration in the mouse, J. Neurosci. Res. [3] A. Alzheimer, R.A. Stelzmann, H.N. Schnitzlein, F.R. Murtagh, An English 86 (2008) 1615–1625. translation of Alzheimer's 1907 paper, “Uber eine eigenartige Erkankung der [25] D.S. Woodruff-Pak, Animal models of Alzheimer's disease: therapeutic implica- Hirnrinde”, Clin. Anat. 8 (1995) 429–431. tions, J. Alzheimers Dis. 15 (2008) 507–521. M. Newman et al. / Biochimica et Biophysica Acta 1812 (2011) 346–352 351

[26] S. Oddo, A. Caccamo, J.D. Shepherd, M.P. Murphy, T.E. Golde, R. Kayed, R. [54] V. Ravi, B. Venkatesh, Rapidly evolving fish genomes and teleost diversity, Curr. Metherate, M.P. Mattson, Y. Akbari, F.M. LaFerla, Triple-transgenic model of Opin. Genet Dev. 18 (2008) 544–550. Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic [55] K.J. Peterson, J.B. Lyons, K.S. Nowak, C.M. Takacs, M.J. Wargo, M.A. McPeek, dysfunction, Neuron 39 (2003) 409–421. Estimating metazoan divergence times with a molecular clock, Proc. Natl. Acad. [27] H. Oakley, S.L. Cole, S. Logan, E. Maus, P. Shao, J. Craft, A. Guillozet-Bongaarts, M. Sci. U. S. A. 101 (2004) 6536–6541. Ohno, J. Disterhoft, L. Van Eldik, R. Berry, R. Vassar, Intraneuronal beta-amyloid [56] M. Chen, R.N. Martins, M. Lardelli, Complex splicing and neural expression of aggregates, neurodegeneration, and neuron loss in transgenic mice with five duplicated tau genes in zebrafish embryos, J. Alzheimers Dis. 18 (2009) familial Alzheimer's disease mutations: potential factors in amyloid plaque 305–317. formation, J. Neurosci. 26 (2006) 10129–10140. [57] D. Paquet, R. Bhat, A. Sydow, E.M. Mandelkow, S. Berg, S. Hellberg, J. Falting, M. [28] J.N. Buxbaum, Animal models of human amyloidoses: are transgenic mice worth Distel, R.W. Koster, B. Schmid, C. Haass, A zebrafish model of tauopathy allows in the time and trouble? FEBS Lett. 583 (2009) 2663–2673. vivo imaging of neuronal cell death and drug evaluation, J. Clin. Invest. 119 (2009) [29] T. Wisniewski, AD vaccines: conclusions and future directions, CNS Neurol. 1382–1395. Disord. Drug Targets 8 (2009) 160–166. [58] M. Citron, T. Oltersdorf, C. Haass, L. McConlogue, A.Y. Hung, P. Seubert, C. Vigo- [30] R.S. Doody, S.I. Gavrilova, M. Sano, R.G. Thomas, P.S. Aisen, S.O. Bachurin, L. Seely, Pelfrey, I. Lieberburg, D.J. Selkoe, Mutation of the beta-amyloid precursor protein D. Hung, Effect of dimebon on cognition, activities of daily living, behaviour, and in familial Alzheimer's disease increases beta-protein production, Nature 360 global function in patients with mild-to-moderate Alzheimer's disease: a (1992) 672–674. randomised, double-blind, placebo-controlled study, Lancet 372 (2008) 207–215. [59] J.A. Lee, G.J. Cole, Generation of transgenic zebrafish expressing green fluorescent [31] S. Le Guyader, S. Jesuthasan, Analysis of xanthophore and pterinosome biogenesis protein under control of zebrafish amyloid precursor protein gene regulatory in zebrafish using methylene blue and pteridine autofluorescence, Pigment Cell elements, Zebrafish 4 (2007) 277–286. Res. 15 (2002) 27–31. [60] L.A. Shakes, T.L. Malcolm, K.L. Allen, S. De, K.R. Harewood, P.K. Chatterjee, Context [32] M. Yamashita, T. Nonaka, T. Arai, F. Kametani, V.L. Buchman, N. Ninkina, S.O. dependent function of APPb enhancer identified using enhancer trap-containing Bachurin, H. Akiyama, M. Goedert, M. Hasegawa, Methylene blue and dimebon BACs as transgenes in zebrafish, Nucleic Acids Res. 36 (2008) 6237–6248. inhibit aggregation of TDP-43 in cellular models, FEBS Lett. 583 (2009) 2419–2424. [61] R.I. Dorsky, D.W. Raible, R.T. Moon, Direct regulation of nacre, a zebrafish MITF [33] I.R. Mackenzie, The neuropathology of FTD associated With ALS, Alzheimer Dis. homolog required for pigment cell formation, by the Wnt pathway, Genes Dev. 14 Assoc. Disord. 21 (2007) S44–S49. (2000) 158–162. [34] G. Pigino, A. Pelsman, H. Mori, J. Busciglio, Presenilin-1 mutations reduce [62] T. Ratovitski, H.H. Slunt, G. Thinakaran, D.L. Price, S.S. Sisodia, D.R. Borchelt, cytoskeletal association, deregulate neurite growth, and potentiate neuronal Endoproteolytic processing and stabilization of wild-type and mutant presenilin, dystrophy and tau phosphorylation, J. Neurosci. 21 (2001) 834–842. J. Biol. Chem. 272 (1997) 24536–24541. [35] A. Takashima, M. Murayama, O. Murayama, T. Kohno, T. Honda, K. Yasutake, N. [63] G. Prihar, R.A. Fuldner, J. Perez-Tur, S. Lincoln, K. Duff, R. Crook, J. Hardy, C.A. Nihonmatsu, M. Mercken, H. Yamaguchi, S. Sugihara, B. Wolozin, Presenilin 1 Philips, C. Venter, C. Talbot, R.F. Clark, A. Goate, J. Li, H. Potter, E. Karran, G.W. associates with glycogen synthase kinase-3beta and its substrate tau, Proc. Natl. Roberts, M. Hutton, M.D. Adams, Structure and alternative splicing of the Acad. Sci. U. S. A. 95 (1998) 9637–9641. presenilin-2 gene, Neuroreport 7 (1996) 1680–1684. [36] G. Verdile, R.N. Martins, M. Duthie, E. Holmes, P.H. St George-Hyslop, P.E. Fraser, [64] J. Walter, A. Capell, J. Grunberg, B. Pesold, A. Schindzielorz, R. Prior, M.B. Podlisny, Inhibiting amyloid precursor protein C-terminal cleavage promotes an interaction P. Fraser, P.S. Hyslop, D.J. Selkoe, C. Haass, The Alzheimer's disease-associated with presenilin 1, J. Biol. Chem. 275 (2000) 20794–20798. presenilins are differentially phosphorylated proteins located predominantly [37] W. Xia, W.J. Ray, B.L. Ostaszewski, T. Rahmati, W.T. Kimberly, M.S. Wolfe, J. Zhang, within the endoplasmic reticulum, Mol. Med. 2 (1996) 673–691. A.M. Goate, D.J. Selkoe, Presenilin complexes with the C-terminal fragments of [65] J. Shen, R.T. Bronson, D.F. Chen, W. Xia, D.J. Selkoe, S. Tonegawa, Skeletal and CNS amyloid precursor protein at the sites of amyloid beta-protein generation, Proc. defects in Presenilin-1-deficient mice, Cell 89 (1997) 629–639. Natl. Acad. Sci. U. S. A. 97 (2000) 9299–9304. [66] P.C. Wong, H. Zheng, H. Chen, M.W. Becher, D.J. Sirinathsinghji, M.E. Trumbauer, H.Y. [38] W. Xia, J. Zhang, R. Perez, E.H. Koo, D.J. Selkoe, Interaction between amyloid precursor Chen, D.L. Price, L.H. Van der Ploeg, S.S. Sisodia, Presenilin 1 is required for Notch1 and protein and presenilins in mammalian cells: implications for the pathogenesis of DII1 expression in the paraxial mesoderm, Nature 387 (1997) 288–292. Alzheimer disease, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 8208–8213. [67] D.B. Donoviel, A.K. Hadjantonakis, M. Ikeda, H. Zheng, P.S. Hyslop, A. Bernstein, [39] J.V. McCarthy, C. Twomey, P. Wujek, Presenilin-dependent regulated intramembrane Mice lacking both presenilin genes exhibit early embryonic patterning defects, proteolysis and gamma-secretase activity, Cell. Mol. Life Sci. 66 (2009) 1534–1555. Genes Dev. 13 (1999) 2801–2810. [40] D.E. Kang, S. Soriano, X. Xia, C.G. Eberhart, B. De Strooper, H. Zheng, E.H. Koo, [68] H. Steiner, K. Duff, A. Capell, H. Romig, M.G. Grim, S. Lincoln, J. Hardy, X. Yu, M. Presenilin couples the paired phosphorylation of beta-catenin independent of axin: Picciano, K. Fechteler, M. Citron, R. Kopan, B. Pesold, S. Keck, M. Baader, T. Tomita, implications for beta-catenin activation in tumorigenesis, Cell 110 (2002) 751–762. T. Iwatsubo, R. Baumeister, C. Haass, A loss of function mutation of presenilin-2 [41] H. Tu, O. Nelson, A. Bezprozvanny, Z. Wang, S.F. Lee, Y.H. Hao, L. Serneels, B. De interferes with amyloid beta-peptide production and notch signaling, J. Biol. Strooper, G. Yu, I. Bezprozvanny, Presenilins form ER Ca2+ leak channels, a Chem. 274 (1999) 28669–28673. function disrupted by familial Alzheimer's disease-linked mutations, Cell 126 [69] A. Herreman, D. Hartmann, W. Annaert, P. Saftig, K. Craessaerts, L. Serneels, L. (2006) 981–993. Umans, V. Schrijvers, F. Checler, H. Vanderstichele, V. Baekelandt, R. Dressel, P. [42] J. Li, M. Xu, H. Zhou, J. Ma, H. Potter, Alzheimer presenilins in the nuclear Cupers, D. Huylebroeck, A. Zwijsen, F. Van Leuven, B. De Strooper, Presenilin 2 membrane, interphase kinetochores, and centrosomes suggest a role in deficiency causes a mild pulmonary phenotype and no changes in amyloid chromosome segregation, Cell 90 (1997) 917–927. precursor protein processing but enhances the embryonic lethal phenotype of [43] D.I. Boeras, A. Granic, J. Padmanabhan, N.C. Crespo, A.M. Rojiani, H. Potter, presenilin 1 deficiency, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 11872–11877. Alzheimer's presenilin 1 causes chromosome missegregation and aneuploidy, [70] P. van Tijn, J.T. Paridaen, C. van Rooijen, D. Zivkovic, Zebrafish models for familial Neurobiol. Aging 29 (2008) 319–328. Alzheimer's disease, 6th European Zebrafish Genetics and Development Meeting, [44] L.N. Geller, H. Potter, Chromosome missegregation and trisomy 21 mosaicism in 2009, Rome. Alzheimer's disease, Neurobiol. Dis. 6 (1999) 167–179. [71] D. Zivkovic, Investigator profile: an interview with Danica Zivkovic, Ph.D, [45] B. Malik, A. Currais, A. Andres, C. Towlson, D. Pitsi, A. Nunes, M. Niblock, J. Cooper, Zebrafish 4 (2007) 1–8. T. Hortobagyi, S. Soriano, Loss of neuronal cell cycle control as a mechanism of [72] G. Thinakaran, C.L. Harris, T. Ratovitski, F. Davenport, H.H. Slunt, D.L. Price, D.R. neurodegeneration in the presenilin-1 Alzheimer's disease brain, Cell Cycle 7 Borchelt, S.S. Sisodia, Evidence that levels of presenilins (PS1 and PS2) are (2008) 637–646. coordinately regulated by competition for limiting cellular factors, J. Biol. Chem. [46] A. Khandelwal, D. Chandu, C.M. Roe, R. Kopan, R.S. Quatrano, Moonlighting 272 (1997) 28415–28422. activity of presenilin in plants is independent of gamma-secretase and [73] D.M. Mann, A. Takeuchi, S. Sato, N.J. Cairns, P.L. Lantos, M.N. Rossor, M. Haltia, H. evolutionarily conserved, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 13337–13342. Kalimo, T. Iwatsubo, Cases of Alzheimer's disease due to deletion of exon 9 of [47] A. Sotthibundhu, A.M. Sykes, B. Fox, C.K. Underwood, W. Thangnipon, E.J. Coulson, the presenilin-1 gene show an unusual but characteristic beta-amyloid pathology Beta-amyloid(1–42) induces neuronal death through the p75 neurotrophin known as “cotton wool” plaques, Neuropathol. Appl. Neurobiol. 27 (2001) receptor, J. Neurosci. 28 (2008) 3941–3946. 189–196. [48] M. Newman, I.F. Musgrave, M. Lardelli, Alzheimer disease: amyloidogenesis, the [74] J.B. Kwok, G.M. Halliday, W.S. Brooks, G. Dolios, H. Laudon, O. Murayama, M. presenilins and animal models, Biochim. Biophys. Acta 1772 (2007) 285–297. Hallupp,R.F.Badenhop,J.Vickers,R.Wang,J.Naslund,A.Takashima,S.E.Gandy, [49] R. Baumeister, U. Leimer, I. Zweckbronner, C. Jakubek, J. Grunberg, C. Haass, P.R. Schofield, Presenilin-1 mutation L271V results in altered exon 8 splicing Human presenilin-1, but not familial Alzheimer's disease (FAD) mutants, facilitate and Alzheimer's disease with non-cored plaques and no neuritic dystrophy, Caenorhabditis elegans Notch signalling independently of proteolytic processing, J. Biol. Chem. 278 (2003) 6748–6754. Genes Funct. 1 (1997) 149–159. [75] M.S. Wolfe, When loss is gain: reduced presenilin proteolytic function leads to [50] X. Li, I. Greenwald, HOP-1, a Caenorhabditis elegans presenilin, appears to be increased Abeta42/Abeta40. Talking Point on the role of presenilin mutations in functionally redundant with SEL-12 presenilin and to facilitate LIN-12 and GLP-1 Alzheimer disease, EMBO Rep. 8 (2007) 136–140. signaling, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 12204–12209. [76] F.W. van Leeuwen, D.P. de Kleijn, H.H. van den Hurk, A. Neubauer, M.A. [51] C.D. Link, Expression of human beta-amyloid peptide in transgenic Caenorhabditis Sonnemans, J.A. Sluijs, S. Koycu, R.D. Ramdjielal, A. Salehi, G.J. Martens, F.G. elegans, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 9368–9372. Grosveld, J. Peter, H. Burbach, E.M. Hol, Frameshift mutants of beta amyloid [52] D.C. Crowther, K.J. Kinghorn, E. Miranda, R. Page, J.A. Curry, F.A. Duthie, D.C. Gubb, precursor protein and ubiquitin-B in Alzheimer's and Down patients, Science 279 D.A. Lomas, Intraneuronal Abeta, non-amyloid aggregates and neurodegeneration (1998) 242–247. in a Drosophila model of Alzheimer's disease, Neuroscience 132 (2005) 123–135. [77] E. Meshorer, H. Soreq, Pre-mRNA splicing modulations in senescence, Aging Cell 1 [53] P. Bharadwaj, L. Waddington, J. Varghese, I.G. Macreadie, A new method to (2002) 10–16. measure cellular toxicity of non-fibrillar and fibrillar Alzheimer's Abeta using [78] H.F. Dovey, V. John, J.P. Anderson, L.Z. Chen, P. de Saint Andrieu, L.Y. Fang, S.B. yeast, J. Alzheimers Dis. 13 (2008) 147–150. Freedman, B. Folmer, E. Goldbach, E.J. Holsztynska, K.L. Hu, K.L. Johnson-Wood, 352 M. Newman et al. / Biochimica et Biophysica Acta 1812 (2011) 346–352

S.L. Kennedy, D. Kholodenko, J.E. Knops, L.H. Latimer, M. Lee, Z. Liao, I.M. [79] A. Geling, H. Steiner, M. Willem, L. Bally-Cuif, C. Haass, A gamma-secretase Lieberburg,R.N.Motter,L.C.Mutter,J.Nietz,K.P.Quinn,K.L.Sacchi,P.A.Seubert, inhibitor blocks Notch signaling in vivo and causes a severe neurogenic G.M.Shopp,E.D.Thorsett,J.S.Tung,J.Wu,S.Yang,C.T.Yin,D.B.Schenk,P.C.May, phenotype in zebrafish, EMBO Rep. 3 (2002) 688–694. L.D. Altstiel, M.H. Bender, L.N. Boggs, T.C. Britton, J.C. Clemens, D.L. Czilli, D.K. [80] T. Yang, D. Arslanova, Y. Gu, C. Augelli-Szafran, W. Xia, Quantification of gamma- Dieckman-McGinty, J.J. Droste, K.S. Fuson, B.D. Gitter, P.A. Hyslop, E.M. secretase modulation differentiates inhibitor compound selectivity between two Johnstone, W.Y. Li, S.P. Little, T.E. Mabry, F.D. Miller, J.E. Audia, Functional substrates Notch and amyloid precursor protein, Mol. Brain 1 (2008) 15. gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain, [81] M. Newman, L. Wilson, E. Camp, G. Verdile, R.N. Martins, M. Lardelli, A zebrafish J. Neurochem. 76 (2001) 173–181. melanophore model of amyloidbeta toxicity, Zebrafish 7 (2010) 155–159. Biochimica et Biophysica Acta 1812 (2011) 353–363

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Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbadis

Review Zebrafish models of Tauopathy☆

Qing Bai a,b, Edward A. Burton a,b,c,d,e,f,⁎ a Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, USA b Department of Neurology, University of Pittsburgh School of Medicine, USA c Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, USA d Department of Neurology, VA Pittsburgh Healthcare System, USA e Geriatric Research, Education and Clinical Center, VA Pittsburgh Healthcare System, USA f Division of Movement Disorders, University of Pittsburgh Medical Center, USA article info abstract

Article history: Tauopathies are a group of incurable neurodegenerative diseases, in which loss of neurons is accompanied by Received 24 January 2010 intracellular deposition of fibrillar material composed of hyperphosphorylated forms of the microtubule- Accepted 8 September 2010 associated protein Tau. A zebrafish model of Tauopathy could complement existing murine models by providing a Available online 16 September 2010 platform for genetic and chemical screens, in order to identify novel therapeutic targets and compounds with disease-modifying potential. In addition, Tauopathy zebrafish would be useful for hypothesis-driven Keywords: Tau experiments, especially those exploiting the potential to deploy in vivo imaging modalities. Several Tauopathy considerations, including conservation of specialized neuronal and other cellular populations, and biochemical Progressive supranuclear palsy pathways implicated in disease pathogenesis, suggest that the zebrafish brain is an appropriate setting in which Zebrafish to model these complex disorders. Novel transgenic zebrafish lines expressing wild-type and mutant forms of Alzheimer's disease human Tau in CNS neurons have recently been reported. These studies show evidence that human Tau undergoes Transgenic disease-relevant changes in zebrafish neurons, including somato-dendritic relocalization, hyperphosphorylation Neurodegeneration and aggregation. In addition, preliminary evidence suggests that Tau transgene expression can precipitate neuronal dysfunction and death. These initial studies are encouraging that the zebrafish holds considerable promise as a model in which to study Tauopathies. Further studies are necessary to clarify the phenotypes of transgenic lines and to develop assays and models suitable for unbiased high-throughput screening approaches. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases. Published by Elsevier B.V.

1. Introduction briefly reviewing current knowledge and murine models of Tauo- pathy, we discuss the possible advantages of a zebrafish model and The microtubule-associated protein Tau (MAP-τ, ‘Tau’) undergoes whether a truly representative model encompassing key biochemical biochemical alterations, cellular redistribution, and deposition as events underlying Tauopathy can be recapitulated in the zebrafish insoluble intraneuronal fibrils (Fig. 1), in a variety of neurodegener- central nervous system. Finally, we review recent publications ative conditions that are collectively termed ‘Tauopathies’. Together, demonstrating initial proof of concept that Tauopathy zebrafish these diseases, which include Alzheimer's disease, progressive supra- models recapitulate core features of the human disorders. nuclear palsy and other conditions (Table 1), are an important cause of morbidity and mortality, with diverse clinical manifestations. No 2. The microtubule-associated protein Tau currently available treatments improve the prognosis of any of these relentlessly progressive diseases. Consequently, investigations aimed Neurons rely on fast axonal transport to shuttle organelles and at determining the underlying pathophysiology of Tauopathies, and macromolecules over long distances, allowing their physiological isolating novel therapeutic agents that prevent disease progression, distribution and turnover within axons and dendrites. Microtubules, are of great importance. In this review, we consider recent develop- which provide the tracks along which molecular motors rapidly ments concerning the possibility that a zebrafish Tauopathy model transport these diverse cargos, are composed of polymerized tubulin might be useful for therapeutic target and drug discovery in vivo. After monomers. Assembly of tubulin into microtubules is promoted by microtubule-associated proteins, the first of which to be identified was termed ‘Tau’ (τ was used to denote a factor essential for tubule ☆ fi This article is part of a Special Issue entitled Zebra sh Models of Neurological formation) [1,2]. Tau is expressed widely in neurons, where it is Diseases. enriched in the axonal compartment [3]. The microtubule-binding ⁎ Corresponding author. 7015 BST-3 3501 Fifth Avenue, Pittsburgh, PA 15217, USA. Tel.: +1 412 640 8480; fax: +1 412 648 8537. domain of Tau localizes to the C-terminal half of the protein [4,5] E-mail address: [email protected] (E.A. Burton). (Fig. 2). The N-terminal, or projection domain, contains a proline-rich

0925-4439/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.bbadis.2010.09.004 354 Q. Bai, E.A. Burton / Biochimica et Biophysica Acta 1812 (2011) 353–363

isoforms. In adult human brain, six isoforms are expressed, produced by alternative splicing of exons 2, 3 and 10 (exons 4A, 6 and 8 are not expressed in the CNS). Tau isoforms in the CNS contain either three or four copies of a tandem repeat containing tubulin-binding sequences, referred to as 3R- and 4R-Tau respectively (Fig. 2). Exon 10 encodes the second microtubule-binding repeat, such that inclusion of exon 10 in the mRNA results in translation of 4R-Tau, whereas exclusion of exon 10 results in a transcript encoding 3R-Tau [6,7]. Optional inclusion of exon 2, or exons 2 and 3, gives rise to N-terminal insertions of 29 (‘1N’)or58(‘2N’) amino acids respectively [8]. The six resulting CNS isoforms are shown in Fig. 2. Additional isoforms generated by incorporation of exon 4A (‘large Tau’) are expressed in the peripheral nervous system [9].

2.2. Functions of Tau

Association of Tau with tubulin promotes microtubule formation [1,2], and association of Tau with microtubules is thought to enhance their stability [10]. 4R-Tau binds to microtubules with greater avidity than 3R-Tau isoforms [4,11,12] (this is attributable to sequences in the first inter-repeat segment that are unique to 4R proteins, rather than to the additional copy of the microtubule-binding tandem repeat [13]), suggesting that expression of 4R-Tau may favor microtubule formation and stability. In healthy adult brain tissue, the 4R-/3R-Tau ratio is approximately 1, and disruption of this balance in the human brain may have serious consequences (see below) [14]. However, there are situations in which the 4R-/3R-Tau ratio may be altered physiologically. For example, during embryogenesis, Tau is exclusive- ly expressed in the brain as the 3R/0N isoform [8]. It has been argued Fig. 1. Neurofibrillary tangles in Alzheimer's disease. A: Neurofibrillary tangles in Alzheimer's disease prefrontal cortex are demonstrated using the Gallyas silver method that this might promote plasticity of neuronal processes during [127]. NFTs are seen as numerous argyrophilic (black) fibrillar intraneuronal inclusions development by reducing microtubule stability. Recent work has (arrows). Neuropil threads, axonal abnormalities also caused by accumulations of suggested that Tau interacts with motor proteins involved in axonal fibrillar Tau, are also seen (arrowheads). B: Neurofibrillary tangles in Alzheimer's transport, provoking different functional consequences for distinct disease hippocampus are demonstrated by immunohistochemistry, using an antibody types of motor molecules [15]. Kinesin, which transports cargo (AT8) that detects phospho-S202/T205-Tau. NFTs are seen as abundant immunoreac- tive (red) intraneuronal inclusions (arrows). Accumulations of phospho-Tau are also towards the distal end of the axon, tended to detach from the apparent in abnormal axonal terminals surrounding amyloid plaques (arrowheads). microtubule on encountering Tau, whereas the proximally-directed motor Dynein showed more directional reversal than detachment. These effects were apparent in the presence of 3R/0N Tau, and suggest region and multiple potential serine–threonine phosphorylation sites, that association of Tau with microtubules may modulate axonal and is thought to be involved in interactions with other cellular transport in addition to promoting microtubule stability. components.

2.3. Tau phosphorylation 2.1. The MAPT gene Tau undergoes post-translational changes, including physiological Tau is encoded by the MAPT gene, which is located on chromosome serine–threonine phosphorylation [16] and O-glycosylation [17],in 17 and contains 16 exons. Alternative splicing of the primary addition to tyrosine phosphorylation [18],SUMOylation[19] and transcript leads to a family of mRNAs, encoding different protein pathological nitration [20]. Of these changes, phosphorylation has

Table 1 Neurodegenerative diseases associated with prominent Tau pathology.

Disease Typical clinical presentations Typical Tau pathology Tau filaments Tau species Etiology

Alzheimer's disease (AD) Memory disorder, dysphasia, frontal Flame-shaped Paired helical 4R- and 3R-Tau Mostly unkown, few cases lobe cognitive–behavioral disorder, neurofibrillary tangles filaments, straight caused by mutations in APP, PS1 dementia filaments [123] or PS2 genes Progressive supranuclear palsy (PSP) Falls, rigidity, oculomotor disorder, Globose neurofibrillary Straight filaments Predominantly Unknown, linked to H1 cognitive–executive disorder tangles, tufted [124] 4R-Tau haplotype at MAPT locus astrocytes Corticobasal degeneration (CBD) Dystonia, myoclonus, apraxia, Ballooned neurons, Twisted ribbon Predominantly Unknown, linked to H1 cortical sensory loss, dementia pre-tangles, astrocytic filaments [125] 4R-Tau haplotype at MAPT locus plaques Pick's disease (PiD) Frontal lobe cognitive–behavioral Pick bodies Helical filaments; Predominantly Unknown disorder, dementia straight filaments 3R-Tau [126] Fronto-temporal dementia and Variable Parkinsonian motor Variable Variable Variable Mutations in MAPT gene parkinsonism linked to disorder and/or frontal lobe (see text) encoding Tau chromosome 17 (FTDP17) dementia Q. Bai, E.A. Burton / Biochimica et Biophysica Acta 1812 (2011) 353–363 355

Fig. 2. Isoforms of the microtubule-associated protein Tau. The schematic depicts the six Tau isoforms expressed in the adult human brain, labeled to the left of each protein. Positions of major protein domains are shown above the longest isoform. The N-terminal insertions encoded by exons 2 and 3, and the four tandem repeats (R1–4) in the microtubule-binding domains are indicated. Sites of known phosphorylation are shown above the longest isoform. The table to the right of the figure shows the number of amino acids, predicted molecular mass and electrophoretic mobility of each protein isoform.

been most extensively studied, since pathological hyperphosphorylation phosphorylates all of the residues found in Tauopathy specimens. is associated with Tauopathy [21]. Approximately 45 phosphorylation Furthermore, there are examples in vitro of cooperation between sites have been identified, which cluster in the proline rich C-terminal kinases; for example, cdk5-mediated phosphorylation of Tau pro- domains of the protein [22]. Phosphorylation of Tau, in particular at S262 motes further phosphorylation by GSK3β [37,38]. Consequently, it is and S356 within the microtubule-binding domain, promotes its thought that the transition from normal to hyperphosphorylated Tau detachment from microtubules, suggesting a potential mechanism in may be a hierarchical or sequential process dependent on phosphor- regulating microtubule stability and axonal transport [23,24]. In addition ylation by multiple different kinases. Several different protein to microtubule-binding affinity, phosphorylation may have other effects, phosphatases dephosphorylate Tau in vitro, including PP1, PP2A, such as modulating binding to the chaperone Pin1 [25] and possibly in PP2B and PP2C. Their specificities are overlapping and their roles in regulating putative signal transduction functions of Tau through vivo are incompletely understood. PP2A has emerged as a strong phosphorylation of SH3 domains [26]. candidate, since it appears to be the major brain-derived phosphatase Hyperphosphorylation of Tau is a characteristic feature in Tauopathy activity directed towards Tau in the rat brain [39] and is localized to specimens. No single phosphorylation site is specific for Tauopathy. microtubules (both through an association with Tau and also directly, Although some phospho-epitopes may be more readily detected in which may regulate its activity in vitro [40]). pathological specimens compared with non-Tauopathy material, most of the known phosphorylation sites have been demonstrated in normal biopsy material [16]. Hyperphosphorylation is a quantitative rather than 2.5. Tau deposits in sporadic neurodegenerative diseases qualitative phenomenon, defined by the degree to which Tau is phosphorylated at multiple sites. Phosphorylation is a dynamic process Tauopathies are characterized pathologically by the presence of and Tau is rapidly de-phosphorylated post mortem, accounting for the lack fibrillar deposits of hyperphosphorylated Tau. The histological of detectable phospho-epitopes in control autopsy specimens. It is unclear appearance and distribution of Tau deposits differs between diseases, whether a functional abnormality of phosphatase activity is present in as does the ultrastructural appearance of Tau filaments that compose Tauopathy samples, or whether the failure of de-phosphorylation in neurofibrillary tangles, Pick bodies and other inclusions (Table 1). Tauopathy autopsy material is attributable to changes in the properties of Formation of neurofibrillary tangles (NFTs) likely occurs through the hyperphosphorylated molecule as a phosphatase substrate. During oligomeric intermediates, and there is some evidence that these are normal development, Tau is present in a highly phosphorylated state the primary pathogenic species, rather than mature NFTs (see [27,28], suggesting that the balance of Tau phosphorylation and de- below). phosphorylation can change physiologically, without necessarily provok- The predominant Tau isoforms deposited in neurons differ between ing neurodegeneration. diseases. In Alzheimer's disease, SDS-PAGE analysis showed three major

2.4. Tau kinases

Table 2 Several cellular kinases are able to phosphorylate Tau in vitro or in Phylogenetic conservation of kinases implicated in tau physophorylation. cell culture (Table 2; reviewed in [22]). Many of these phosphorylate multiple residues of Tau, and individual residues can be phosphor- Tau Kinase Protein accession numbers % % identity consensus ylated by multiple different kinases, suggesting that Tau is a Human Zebrafish promiscuous kinase substrate. Consequently, it is difficult to be Glycogen synthase kinase NP_001139628 NP_571456 94.5 97.4 certain which kinase(s) are responsible for physiological or patho- 3β (GSK3β) logical phosphorylation of Tau in vivo. Kinases that have emerged as Cyclin-dependent kinase AAP35326 AAH85381 96.2 98.6 strong in vivo candidates include glycogen synthase kinase-3β 5 (cdk5) β – Mitogen activated protein NP_620407 BAB11813 90.8 93.5 (GSK3 ) [29 33] and cyclin-dependent protein kinase-5 (cdk5) kinase 1 (MAPK1, ERK1) [34,35]; both are expressed abundantly in neurons and associate Microtubule Affinity CAH72462 AAI55560 79.3 85.5 with microtubules (cdk5 is anchored to microtubules by Tau [36]). Regulating Kinase 1 However, it is likely that more than one kinase participates in the (MARK1) Casein kinase 1α (CK1α) NP_001883 NP_694483 99.3 99.3 events leading to hyperphosphorylation, since no single kinase 356 Q. Bai, E.A. Burton / Biochimica et Biophysica Acta 1812 (2011) 353–363

Tau immunoreactive bands of 68, 64 and 60 kDa present in detergent- First, dissociation between NFT formation and neuronal dysfunc- extracted material containing paired helical filaments [41].Afterde- tion or death was demonstrated in some of these models, suggesting phosphorylation of the same material, all six Tau isoforms were resolved that Tau hyper-phosphorylation, relocalization or oligomerization by electrophoresis, showing that hyperphosphorylation of Tau was may be more critical for the emergence of phenotypic abnormalities in responsible for the alteration in its electrophoretic mobility, and that AD these models than formation of NFTs. Abnormalities of hippocampal filaments are composed of all six Tau isoforms. Similar analyses of synaptic protein expression and physiology preceded the formation of specimens derived from other types of Tauopathy have revealed NFTs in mice expressing P301S mutant Tau [52]. Studies using a different results. In progressive supranuclear palsy and cortico-basal different model in which P301L Tau was expressed under an inducible degeneration, two major Tau bands of 68- and 64 kDa are derived from promoter showed that suppression of transgene expression after the hyperphosphorylation of 4R-Tau species [42,43]. Conversely, material onset of phenotypic abnormalities prevented neuronal loss and derived from Pick's disease brain shows two bands of 64- and 60-kDa, behavioral abnormalities but did not affect neurofibrillary tangle dephosphorylation of which reveals the presence of 3R-Tau species formation [53], and there was regional dissociation between NFT [43,44]. Interestingly, genetic data shows association between an formation and cell loss [54]. In addition, immunization of Tau/Aβ extended haplotype of markers across the MAPT locus, and PSP/CBD transgenic mice reduced soluble Tau in the brain but did not affect NFT [45]; recent data shows that this haplotype favors the splicing of exon formation — the animals showed phenotypic improvement, suggest- 10+ transcripts encoding 4R-Tau [46], suggesting that genetically- ing that neurobehavioral abnormalities were attributable to a soluble determined relative over-production of 4R-Tau might be one factor Tau species rather than NFT [55]. in the pathogenesis of these sporadic diseases. Second, Tau transgenic mice have provided a powerful means to test the interactions between different biochemical pathways impli- cated in neurodegeneration. For example, evidence of an in vivo 2.6. Hereditary fronto-temporal dementia and Parkinsonism linked to interaction between Tau and Aβ in the pathogenesis of Alzheimer's chromosome 17 disease was demonstrated by intracerebral inoculation of the Alzheimer's disease-associated amyloid peptide Aβ1–42, which Definitive evidence that the development of Tau pathology may be enhanced the formation of NFTs in a mouse P301L Tau-expressing mechanistically important in common sporadic Tauopathies comes model [56]. Furthermore, P301L Tau/APP double transgenic animals from the study of rare families, in which mutations within the MAPT showed exacerbation of NFT pathology in the presence of the APP gene are sufficient to cause a neurodegenerative disease with transgene, whereas Aβ plaque formation was unaffected by the over- prominent Tauopathy [14,47]. FTDP17 is inherited as an autosomal expression of mutant Tau, suggesting that β-amyloid formation or dominant trait and can manifest as a Parkinsonian movement another function of APP might be upstream of Tauopathy in this disorder or as a cognitive–behavioral disorder, resembling other model [57]. forms of fronto-temporal dementia (reviewed in [48]). To date, 39 Finally, murine models have provided powerful means to test different pathogenic MAPT mutations have been identified in FTDP17 hypotheses concerning experimental treatment approaches, and families. Mutations clustered around the 5′ boundary of intron 10 biochemical interventions aimed at understanding disease pathogen- alter the regulation of splicing, most frequently resulting in relative esis in vivo (reviewed in [58]). For example, administration of Li+,an over-production of exon 10+ transcripts and deposition of hyperpho- inhibitor of GSK3, reduced hyperphosphorylation of Tau in vivo and sphorylated 4R-Tau isoforms. Missense mutations within exon 10 also ameliorated some phenotypic abnormalities [31], and another kinase provoke a 4R-Tauopathy, by changing the biophysical properties of inhibitor targeting cdk5, GSK3 and MAPK1 delayed the onset of a 4R-Tau without altering the ratio of splice isoforms; these mutations motor deficit in Tau transgenic animals [59]. Other studies have also result in 4R-Tau deposition. Finally, mutations outside exon 10 deployed pharmacological interventions in order to clarify the are expressed in both 3R- and 4R-Tau, but may provoke selective potential therapeutic utility of interventions directed at restoring deposition of 3R- or 4R-, or both isoforms of Tau. A variety of loss of Tau microtubule-stabilizing function [60], enhancing clearance mechanisms has been proposed to account for the pathogenicity of of pathological Tau species from neurons by inhibiting refolding MAPT missense mutants, including promotion of Tau fibril formation, functions of the HSP90 complex to promote Tau degradation [61], and impairment of microtubule-binding and introduction or removal of curtailing a potentially pathogenic neuroinflammatory response by key phosphorylation sites [49]. Many of the mutants show multiple targeting microglial activation using immunosuppressive drugs [52]. functional abnormalities, such as alterations in 4R-/3R-splice ratio and Together, these observations show that experimental genetic reduced propensity to promote the stabilization of microtubules. manipulations, which mimic the mechanisms underlying FTDP17 in Although FTDP17 is an uncommon disease, the discovery of humans, provoke phenotypic changes relevant to human Tauopathy pathogenic MAPT mutations has provided important confirmation in transgenic animals. These models have been valuable for that primary abnormalities of Tau, including alterations in the relative hypothesis-driven experiments aiming to elucidate the pathogenesis abundance of 3R- and 4R-isoforms, can result in neurodegeneration of Tauopathy and for testing putative therapeutic approaches. and Tauopathy. Given the close similarity between FTDP17 and sporadic Tauopathies in some cases, it is reasonable to conclude that 4. Why would a zebrafish Tauopathy model be useful? abnormalities of cellular Tau metabolism may be central to neurode- generation in common sporadic Tauopathies. Consequently, study of The zebrafish is a small freshwater fish that has been used model systems in which Tauopathy is induced by molecular extensively in laboratory studies of vertebrate development. Fish manipulations that mimic the genetic mechanisms of FTDP17 might embryos develop externally allowing direct observation of embryo- yield important insights into pathophysiology and identify potential genesis. Many morphological and molecular parallels are shared treatment targets for common sporadic Tauopathies. between fish and other vertebrates allowing insights gained in the zebrafish model to be applied to other systems. Stemming from the 3. Transgenic mouse models of Tauopathy use of zebrafish in developmental studies, an extensive array of experimental methodologies has been developed. Straightforward A variety of different transgenic lines, over-expressing wild-type techniques are available for over-expression [62,63] or targeted or FTDP17 mutant human Tau, have been described (reviewed in knockdown of genes of interest [64], allowing the consequences of [50,51]). Several important themes that have emerged from studies of altered gene function to be determined readily in vivo.The these animals are briefly considered here. deployment of fluorescent reporters [65] allows direct observation Q. Bai, E.A. Burton / Biochimica et Biophysica Acta 1812 (2011) 353–363 357 of morphology or physiology of individual cells in vivo [66–68]. The to functional sub-specialization, suggesting that the zebrafish may be ability of zebrafish to breed regularly and produce sizeable clutches of a powerful tool in which to dissect multiple cellular roles of Tau. The embryos, and the practicable housing of significant numbers of two proteins encoded by the zebrafish genes have not yet been animals, has allowed development of techniques for large-scale detected, but both mapta and maptb mRNAs were expressed in the genetic [69–72] and chemical modifier [73–75] screens. These have developing CNS [97]. A complex pattern of alternative splicing of the provided numerous novel molecular insights into development, mapta and maptb transcripts suggests that, like human Tau, zebrafish through unbiased phenotype-driven approaches. More recently, it Tau isoforms with different numbers of microtubule-binding repeats has been demonstrated that intact zebrafish larvae provide an in vivo are expressed in the CNS, and larger forms of Tau are expressed in the neurobehavioral platform suitable for screens to find chemical peripheral nervous system. Interestingly, mapta gives rise to tran- modifiers of vertebrate behavior [76]. scripts encoding 4–6 microtubule-binding repeats, whereas maptb is It is possible that applying these types of analysis to zebrafish predominantly expressed as a 3-repeat isoform, raising the fascinat- models of neurodegenerative diseases would provide novel molecular ing possibility that conserved functions of mammalian 3R- and 4R-Tau insights into disease pathogenesis, and allow identification of are distributed between the two zebrafish genes [97]. potentially therapeutic compounds. In particular, screening studies carried out in the intact vertebrate CNS in vivo might uncover drug 6. Tools for the generation and analysis of zebrafish Tau models targets that are not present in cell culture models commonly used for discovery-driven approaches, such as pathogenic mechanisms that Generation of transgenic Tau zebrafish requires availability of are not cell-autonomous, or which are only expressed in end- appropriate methods for introducing exogenous DNA into the germ- differentiated neurons. Consequently, there has been significant line, and resources to direct MAPT transgene expression in the desired recent interest in the development of zebrafish models of Tauopathy. temporal and spatial pattern.

5. Is the zebrafish a suitable organism in which to study Tauopathy? 6.1. Techniques for establishment of transgenic lines

The proposed use of a small aquatic creature to study complex Transgenic zebrafish can be generated by micro-injecting linearized human neurobehavioral diseases naturally raises questions about plasmid DNA into the cytoplasm of one-cell stage embryos [100],but relevance — will it be possible to generate a truly representative this gives rise to inefficient genomic integration and significant model, with potential to yield important insights into the human mosaicism. Two technical advances, meganucleases and transposons, disorder? A variety of considerations suggest that the zebrafish may have substantially improved the efficiency by which foreign DNA can be be a good model system in which to study human neurological introduced into the zebrafish genome. (i) The meganuclease I-sce1 is an diseases. With respect to the human CNS, the zebrafish shows intron-encoded endonuclease from Saccharomyces cerevisiae [101], conservation of basic brain organization [77] and many key which cleaves DNA at an 18 bp sequence-specific recognition site not neuroanatomical [78,79] and neurochemical [80,81] pathways of found within the zebrafish genome. In meganuclease-mediated relevance to human disease; the zebrafish CNS contains microglia transgenesis, the transgene plasmid is designed so that the expression [68], cells with astrocytic properties [82,83], oligodendrocytes and cassette is flanked by I-sce1 sites, and is injected into zebrafish embryos myelin [84–87], and a blood–brain barrier [88], all of which have been with I-sce1 enzyme. Integration of a low transgene copy number usually implicated in the pathogenesis of neurological disease. These occurs at a single site, with substantially enhanced efficiency, and observations imply that the tissue environment in the zebrafish CNS reduced mosaicism, compared with naked DNA injection. This improves may present appropriate conditions in which to recapitulate patho- the rate of transmission of the transgene from the F0 to the F1 logical changes representative of the human disorders. In addition, generation [102] and results in simple Mendelian transmission of there is a striking degree of phylogenetic conservation of genes transgenes in subsequent crosses [103]. (ii) Type 2 transposons are implicated in the pathogenesis of neurodegenerative diseases, and a mobile DNA elements encoding a transposase, which mediates excision variety of other CNS functions [89–96]. This suggests that it will be of the transposon from the genome and its re-insertion at another possible to provoke neurodegeneration in zebrafish through con- location. The Tol2 transposon, found in the genome of medaka [104],has served biochemical pathways that are sufficiently close to those been engineered by deletion of the transposase gene to form a non- underlying human disorders that findings in the zebrafish model will autonomous mobile element, which can insert into the genome in the yield relevant insights into diseases mechanisms. It is noteworthy that presence of transposase supplied in trans [105]. In this approach to the zebrafish genome contains highly conserved orthologues of each transgenesis, the transgene expression cassette is flanked by transposon of the kinases implicated in Tau phosphorylation (Table 2 and Fig. 3), elements, and injected into zebrafish embryos with mRNA encoding which may be of particular relevance to Tauopathy in view of the transposase, resulting in highly efficient, usually multiple, single-copy proposed central role of hyperphosphorylation in pathogenesis. integration events. The technique has been used increasingly since its Similar considerations apply to other conserved pathways, and it introduction and subsequent refinement [62], because it is necessary to seems likely that findings in zebrafish models will be representative of inject and screen a relatively small number of embryos in order to the mechanisms underlying the pathogenesis of human Tauopathies identify stable transgenic lines. and will therefore be relevant to their understanding. 6.2. Promoter resources for generation of neurodegeneration models 5.1. Zebrafish paralogues of MAPT Most transgenic zebrafish lines have been constructed by expressing Recent work has identified two paralogues of MAPT in zebrafish, a cDNA under control of a characterized heterologous promoter annotated mapta and maptb [97]. Analysis of homology between fragment; cis-acting regulatory regions derived from zebrafish genes zebrafish and mammalian MAP-encoding genes, and examination of are usually employed for this purpose, because they may be associated synteny between zebrafish mapt and mammalian MAPT loci, strongly with more reliable transgene expression [106,107]. For the generation of suggest that the two zebrafish genes arose from duplication of an a Tauopathy model, desirable attributes for the promoter would include ancestral mapt locus. A genome duplication event is thought to have pan-neuronal activity and sufficient expression to provoke pathology. taken place during the evolution of teleosts [98]; consequently, there Three zebrafish promoter elements that drive transgene expression are dual zebrafish paralogues of many mammalian genes [92,99].Itis widely in CNS neurons have been described. (i) A neuronal enhancer possible that divergent evolution of the two mapt genes has given rise derived from the gata2 promoter was expressed at high levels in the 358 Q. Bai, E.A. Burton / Biochimica et Biophysica Acta 1812 (2011) 353–363

Fig. 3. Phylogenetic conservation of kinases implicated in Tau phosphorylation. The sequence figure shows the striking degree of amino acid conservation between human and zebrafish (A) GSK3β and (B) cdk5. Each panel shows the Human (upper) and zebrafish (lower) protein sequences, aligned using the ClustalW algorithm. Amino acids are shaded to show identical, conserved and non-similar residues. The catalytic domain of each kinase is indicated by underlining, and important functional sites labeled as indicated. developing CNS [108]. (ii) The zebrafish huc gene encodes an RNA ductive potential or preventing survival to sexual maturity. One binding protein, HuC/D, commonly used as an early neuronal marker possible solution would be deployment of a conditionally-expressing [109] — a 2.8 kb fragment of the proximal 5′ flanking region was system, for example the binary Gal4–UAS system previously sufficient to drive pan-neuronal expression in embryos [110]. (iii) The employed in Drosophila genetics. This system has been successfully eno2 gene, encoding the neuron-specific γ-enolase isoenzyme, is used in zebrafish, with recent modifications attempting to address expressed at low levels until 60–72 h post-fertilization, after which toxicity issues caused by the viral trans-activation domain usually high level expression persists into adulthood. The eno2 regulatory fused to Gal4, and inactivation of the UAS tandem repeats [112,113]. region is complex, containing an untranslated first exon, and an intronic Isolation of useful driver lines for generation of neurodegenerative CpG island that appears important for promoter activity. A 12 kb disease models is ongoing, and the optimal strategy for deployment of fragment of the promoter, including the first intron, was active in this technology is uncertain. Driver lines could be constructed by driving reporter gene expression in neurons, including many popula- expressing the Gal4-transactivator fusion protein using small pro- tions of relevance to disease, throughout the brain and spinal cord from moter fragments similar to the approach for generating cDNA 36–48 h post-fertilization through adulthood [111], and in the retina transgenic animals; this has been used successfully with the huC and visual pathways [103] (Fig. 4). The optimal promoter for promoter in a larval Tau model [114]. In addition, the highly efficient construction of Tauopathy models is unclear, and each of these genomic integration of transgenes using the Tol2 transposon has promoters has been deployed in studies of transgenic Tau zebrafish allowed the deployment of approaches in which random integration (see below). It is likely that further development of promoter resources of a gal4-expressing enhancer trap gives rise to robust stable and other approaches to generation of transgenic animals will yield expression of Gal4 under the endogenous regulatory elements of other useful reagents for this application. the gene of insertion [115]. Several such lines have been constructed and are currently being further evaluated. 6.3. Special considerations for expression of transgenes that provoke neurodegeneration in larvae 6.4. Assays for fully exploiting transgenic models

In order to fully exploit the potential of the zebrafish for high- The power of screening paradigms might be enhanced by throughput screening approaches, it may be necessary to drive Tau deployment of assays that exploit unique properties of the zebrafish expression at sufficiently high levels that disease pathogenesis occurs model. First, direct visualization of neuronal populations of interest in during larval stages of development, when zebrafish can be the zebrafish brain might allow non-invasive automated imaging to accommodated in 96-well plates. However, development of neuro- ascertain the morphology, integrity or viability of target cells as an degeneration at this early time point could provide strong selection assay end point. For example, the Tauopathies PSP and CBD are against establishment of transgenic lines, by compromising repro- associated with severe depletion of dopaminergic neurons causing a Q. Bai, E.A. Burton / Biochimica et Biophysica Acta 1812 (2011) 353–363 359

Fig. 4. The zebrafish eno2 promoter. The micrograph shows a single confocal plane through the head region of a Tg(eno2:egfp) zebrafish larva at 5 days post-fertilization, illustrating widespread neuronal expression of the eno2 promoter. Rostral is to left, dorsal up. GFP expression appears green; the section was counterstained using a blue nuclear marker to facilitate identification of anatomical landmarks. Key: Tel, telencephalon; TeO, optic tectum; Ce, cerebellum; MdO, medulla oblongata; SC, spinal cord; L, ocular lens; PRL, photoreceptor layer of retina; IPL, inner plexiform layer of retina; GCL, ganglion cell layer of retina; TG, trigeminal ganglion; OC, otic capsule; PLLG, posterior lateral line ganglion; LLN, axons of lateral line nerve.

Parkinsonian movement disorder that characterizes these condi- element [121]. In cultured cells, the fusion protein was phosphory- tions [116], and it would be of considerable interest to identify lated and associated with the cytoskeleton, similar to native Tau; in compounds that protect dopaminergic neurons from abnormal forms vitro studies showed that the fusion protein aggregated, validating its of Tau. Genetic labeling of dopamine neurons using fluorescent use in constructing an in vivo model of NFT formation. Zebrafish reporters is ongoing, and has proved to be complicated. The promoter embryos microinjected with the gata2:MAPT-egfp construct, showed regions of dopamine-neuron-specific genes are large and complex, mosaic neuronal expression of the fusion protein. Some neurons such that manageable fragments of the th [117,118] or slc6a3 [119] showed accumulation of fluorescent fibrillar structures resembling promoters have not shown dopaminergic neuron-specific activity in neurofibrillary tangles, in the cell body and proximal axon. The human vivo. Alternative approaches are being undertaken, including identi- Tau-GFP fusion protein was shown to be phosphorylated in the fication of conserved enhancers, using large genomic constructs likely zebrafish brain by western blot detection of a phospho-S396/S404 to contain more cis-acting elements, or adopting an enhancer trap epitope, and expression of glycogen synthase kinase-3 in the zebrafish approach to identify genomic integrants that recapitulate endogenous embryo and adult brain was demonstrated by western blot. This initial gene expression patterns. The latter approach has yielded a single study validated the use of a GFP fusion protein to monitor evolution of transgenic line in which an integration event in the vmat gene yielded tangle pathology in vivo, and suggested that human Tau is phosphor- zebrafish with GFP-expressing catecholaminergic neurons [67]. There ylated in the larval zebrafish, confirming the prediction that there is is a large literature on the development of promoter resources sufficient phylogenetic conservation of endogenous zebrafish kinases allowing genetic labeling of other specific neuronal populations using to modify the human protein. No stable lines were derived in this fluorescent reporters, which is outside the scope of the present study. review. Subsequently, stable transgenic zebrafish were constructed Second, neuronal dysfunction presumably precedes cell death in expressing human 4R/0N Tau [111], the most abundant Tau species Tauopathy, and cells in earlier stages of pathological evolution might deposited in progressive supranuclear palsy [43,44]. The transgene present a more tractable therapeutic target than those close to was expressed under transcriptional control of the eno2 promoter demise. It would be of considerable value to develop assays that [111]. The full phenotype of these transgenic fish has not yet been measure neuronal dysfunction, and which are sensitive to changes in reported, since the initial paper focused on characterization of the disease progression earlier in the evolution of pathology. Since genetic novel eno2 promoter. Abundant expression of human 4R/0N Tau was and chemical screening against a zebrafish Tauopathy model would found in the CNS, persisting into adulthood (Fig. 5A). In the adult be carried out in vivo, it might be possible to use automated motor brain, refractile Tau accumulations resembling neurofibrillary tangles behavioral assays as a surrogate marker of neuronal function. were found within neuronal cell bodies and proximal axons in CNS Consequently there is much current interest in establishing repro- regions of pathological relevance to PSP, including the optic tectum ducible and well-characterized behavioral assays for neurodegener- (Fig. 5B). Stable expression of the Tau transgene into adulthood in this ative phenotypes. A recent report showed that an automated motor model will allow analyses designed to test whether age-related behavioral test could be used to identify agents with neuropharma- pathology accumulates in these transgenic lines, similar to the cological properties in a chemical screen [76]. Assays of motor progressive changes seen in the human disease and recapitulated in function have been previously deployed in order to detect changes in some murine models, a potentially important step in validating the behavior caused by alterations in neurochemistry and dopamine cell model system. Furthermore, widespread neuronal expression of the integrity secondary to Parkinson's disease-associated toxins [81,120]. transgene may allow examination of factors involved in cellular It may be possible to use a similar approach to investigate changes in vulnerability to Tauopathy. behavior preceding demonstrable cell loss in a zebrafish Tauopathy More recently, exploitation of the Gal4–UAS system allowed model, in order to develop an assay to identify chemical modifiers of generation of a Tauopathy model showing a larval phenotype [114]. early pathological progression. This is potentially an important advance, since the development of phenotypic abnormalities at larval stages of development may allow 7. Current transgenic zebrafish models of Tauopathy high-throughput screening, as discussed above. Human 4R/2N-Tau, harboring a P301L mutation, was expressed from a novel bidirectional Three recent publications have described the first proof-of-principle UAS promoter, allowing simultaneous expression of a red fluorescent experiments showing that expression of human MAPT transgenes can reporter in Tau-expressing cells [114]. The high levels of mutant provoke relevant phenotypes in zebrafish. P301L Tau expression provoked by the huc:gal4-vp16 driver induced a In the first publication, a 4R-Tau-GFP fusion protein was transient motor phenotype during embryogenesis, likely caused by transiently over-expressed in zebrafish larvae, using a gata2 promoter peripheral motor axonal developmental abnormalities. At later time 360 Q. Bai, E.A. Burton / Biochimica et Biophysica Acta 1812 (2011) 353–363

solubility, fibril morphology and further clarify phosphorylation events. However, these data are encouraging that relevant pathways are sufficiently phylogenetically conserved between human and zebrafish to allow recapitulation of cellular processing events relating to Tau in the zebrafish model. The pathophysiological consequences of human Tau transgene expression in zebrafish CNS neurons are less clear. This is partly because a detailed phenotype has only been reported for a single transgenic Tau zebrafish line [114]. The motor axonal developmental abnormality, motor phenotype and cell death reported in this model have an uncertain relationship to the neuronal dysfunction and neurodegeneration that characterizes human Tauopathy, on account of the onset and subsequent resolution of abnormalities during early Fig. 5. Human Tau expression in Tg(eno2:MAPT)zebrafish neurons. A: A western blot was development, and the atypical anatomical distribution in the spinal made with protein lysate from wild-type zebrafish brain (lane 1), control post-mortem cord and motor neurons. One recurrent finding from the murine human cortex (lane 2) and Tg(eno2:MAPT) brain (lane 3). The blot was probed using an transgenic disease model literature is that phenotypes exhibited by β antibody to human Tau (upper panel) followed by an antibody to -actin (lower panel). cDNA transgenic animals are critically dependent on the promoter Abundant expression of human 4R-Tau is seen in transgenic zebrafish compared with the six isoforms in normal human brain [111].B:SectionsofTg(eno2:MAPT)zebrafish brain elements used to drive transgene expression. The temporal and were labeled using the antibody to human Tau used in the western blot experiment shown spatial expression pattern of the huC promoter used in this study in panel A and a histochemical reaction yielding a red product. The micrograph shows a directed the atypical pattern of phenotypic abnormalities. However, brainstem neuron with dense Tau immunoreactivity in the cell body and proximal axon, this may be unimportant for the purposes of drug and target discovery resembling neurofibrillary tangles shown in Fig. 1B [111]. relating to cellular mechanisms of Tau toxicity, provided that the molecular mechanisms underlying neuronal dysfunction and death are conserved between the model and the disorder. Further work, points, after recovery of motor function, enhanced cell death was including analysis of other zebrafish Tau lines constructed using observed in the spinal cord of transgenic animals. Abnormal Tau different promoter elements, will be necessary to clarify this point. conformers were detected using an antibody, MC1, which recognizes While detailed phenotypic analysis of the first cDNA transgenic Tau a discontinuous epitope present in Tauopathy tissue [122], as early as models is in progress, it is interesting to consider future directions in the 32 h post-fertilization; by 5 weeks of age, argyophilic material was generation of models and approaches for analysis. Use of models based detected in the spinal cord, but the animals were otherwise on over-expression of cDNA encoding human Tau would limit discovery phenotypically unremarkable at this time point. Subsequent loss of of novel molecular interventions to those targeting Tau modifications, transgene expression prevented the examination of later pathological Tau deposition or other abnormal effects exerted by Tau on neuronal changes. Tau phosphorylation was detected as early as 32 h post- function. However, there may be upstream targets related to transcrip- fertilization. Phospho-Tau specific antibodies labeled cells differen- tion and splicing that would not be represented in cDNA models. For tially; some phospho-epitopes, such as phospho-T231/S235 and example, changes in the cellular 3R-/4R-Tau ratio, caused by alteration phospho-S396/S404 were detected widely in Tau-expressing neurons, in the regulation of exon 10 splicing, underlie one form of FTDP17 and whereas others such as phospho-S422 and phospho-S202/T205 were may contribute to sporadic Tauopathies, where associated haplotypes initially present in only a small subset of Tau-expressing neurons. may alter 3R-/4R-Tau ratios [46]. It is not clear whether the human MAPT Between 2 and 7 days post-fertilization, however, expression of all of locus would be appropriately regulated in transgenic Tau zebrafish, these epitopes became widespread in Tau-expressing cells, suggesting allowing expression of physiological levels and patterns of alternatively that Tau had accumulated multiple phosphorylations in a sequential spliced products. Transgenic zebrafish lines harboring the entire MAPT progression of biochemical changes. Tau phosphorylation was gene would therefore be of considerable interest. Furthermore, since reduced by application of novel high-potency GSK3β inhibitors that zebrafish evolution may have resulted in the functions of 3R- and were designed to target the human enzyme, confirming that 4R-Tau isoforms becoming distributed between two different genes, an extensive homology between human and zebrafish kinases allows indication of whether alterations in the ratio of these forms can be small molecules to interact with orthologous targets from either pathogenic in zebrafish might be accomplished by morpholino species. This important study was the first detailed phenotypic knockdown experiments. description of a stable zebrafish Tauopathy model and showed proof Initial evidence shows that the zebrafish will most likely be a of principle that biochemical changes characteristic of human predictive platform for the discovery and assessment of Tau kinase Tauopathy can be recapitulated in larval zebrafish and modulated inhibitors, although arguably the greatest potential strength of the by chemical inhibitors. model might be in discovering new therapeutic targets. In order to gain novel insights from screening approaches, end points that are 8. Conclusions — what have we learned and what next? independent of presumptions about underlying mechanisms, such as neurobehavioral analyses or cell death assays, might be deployed. Examination of the first publications allows preliminary assessment Furthermore, experimental approaches that can be uniquely applied of the degree to which transgenic zebrafish models of Tauopathy seem in zebrafish larvae owing to their optical transparency, for example representative of human disorders. Evidence so far suggests that physiological imaging modalities relating to cellular calcium levels, processing of over-expressed human Tau in zebrafish CNS neurons have the potential to yield important insights from hypothesis- results in similar biochemical and pathological changes to those found in driven experiments that cannot be carried out using other available human Tauopathies. These changes include: aberrant localization of Tau models. to the somato-dendritic compartment; phosphorylation, with an In conclusion, the first reports show that construction of ordered (and possibly sequential) acquisition of phospho-epitopes; transgenic zebrafish expressing human Tau in CNS neurons is feasible adoption of abnormal conformations associated with Tauopathy; and and suggest that the resulting models will be relevant and useful. deposition as argyrophilic material. The availability of zebrafish lines Consequently, there is cause for cautious optimism that novel that express Tau in the brain through adulthood will allow more zebrafish Tauopathy models may provide important contributions to detailed biochemical studies of Tau in the zebrafish CNS to characterize ongoing efforts to address these common and devastating diseases. Q. Bai, E.A. Burton / Biochimica et Biophysica Acta 1812 (2011) 353–363 361

Acknowledgements microtubule binding and involves Ser-Pro and Thr-Pro motifs, FEBS Lett. 307 (1992) 199–205. [25] P.J. Lu, G. Wulf, X.Z. Zhou, P. Davies, K.P. Lu, The prolyl isomerase Pin1 restores We gratefully acknowledge research grant support from CurePSP — the function of Alzheimer-associated phosphorylated tau protein, Nature 399 The Society for Progressive Supranuclear Palsy (grants #441-05 and (1999) 784–788. [26] C.H. Reynolds, C.J. Garwood, S. Wray, C. Price, S. Kellie, T. Perera, M. Zvelebil, A. #468-08) and NINDS (NS058369). Yang, P.W. Sheppard, I.M. Varndell, D.P. Hanger, B.H. Anderton, Phosphorylation regulates tau interactions with Src homology 3 domains of phosphatidylinositol 3-kinase, phospholipase Cgamma1, Grb2, and Src family kinases, J. Biol. Chem. References 283 (2008) 18177–18186. [27] A. Kenessey, S.H. Yen, The extent of phosphorylation of fetal tau is comparable to [1] M.D. Weingarten, A.H. Lockwood, S.Y. Hwo, M.W. Kirschner, A protein factor that of PHF-tau from Alzheimer paired helical filaments, Brain Res. 629 (1993) essential for microtubule assembly, Proc. Natl. Acad. Sci. U. S. A. 72 (1975) 40–46. 1858–1862. [28] J.P. Brion, C. Smith, A.M. Couck, J.M. Gallo, B.H. Anderton, Developmental changes [2] G.B. Witman, D.W. Cleveland, M.D. Weingarten, M.W. Kirschner, Tubulin in tau phosphorylation: fetal tau is transiently phosphorylated in a manner requires tau for growth onto microtubule initiating sites, Proc. Natl. Acad. Sci. similar to paired helical filament-tau characteristic of Alzheimer's disease, J. U. S. A. 73 (1976) 4070–4074. Neurochem. 61 (1993) 2071–2080. [3] L.I. Binder, A. Frankfurter, L.I. Rebhun, The distribution of tau in the mammalian [29] M. Hong, D.C. Chen, P.S. Klein, V.M. Lee, Lithium reduces tau phosphorylation by central nervous system, J. Cell Biol. 101 (1985) 1371–1378. inhibition of glycogen synthase kinase-3, J. Biol. Chem. 272 (1997) [4] K.A. Butner, M.W. Kirschner, Tau protein binds to microtubules through a 25326–25332. flexible array of distributed weak sites, J. Cell Biol. 115 (1991) 717–730. [30] M. Hong, V.M. Lee, Insulin and insulin-like growth factor-1 regulate tau [5] N. Gustke, B. Trinczek, J. Biernat, E.M. Mandelkow, E. Mandelkow, Domains of tau phosphorylation in cultured human neurons, J. Biol. Chem. 272 (1997) protein and interactions with microtubules, Biochemistry 33 (1994) 9511–9522. 19547–19553. [6] M. Goedert, M.G. Spillantini, M.C. Potier, J. Ulrich, R.A. Crowther, Cloning and [31] W. Noble, E. Planel, C. Zehr, V. Olm, J. Meyerson, F. Suleman, K. Gaynor, L. Wang, J. sequencing of the cDNA encoding an isoform of microtubule-associated protein LaFrancois, B. Feinstein, M. Burns, P. Krishnamurthy, Y. Wen, R. Bhat, J. Lewis, D. tau containing four tandem repeats: differential expression of tau protein Dickson, K. Duff, Inhibition of glycogen synthase kinase-3 by lithium correlates mRNAs in human brain, EMBO J. 8 (1989) 393–399. with reduced tauopathy and degeneration in vivo, Proc. Natl. Acad. Sci. U. S. A. [7] M. Goedert, C.M. Wischik, R.A. Crowther, J.E. Walker, A. Klug, Cloning and 102 (2005) 6990–6995. sequencing of the cDNA encoding a core protein of the paired helical filament of [32] A. Caccamo, S. Oddo, L.X. Tran, F.M. LaFerla, Lithium reduces tau phosphorylation Alzheimer disease: identification as the microtubule-associated protein tau, but not A beta or working memory deficits in a transgenic model with both Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 4051–4055. plaques and tangles, Am. J. Pathol. 170 (2007) 1669–1675. [8] M. Goedert, M.G. Spillantini, R. Jakes, D. Rutherford, R.A. Crowther, Multiple [33] D. Terwel, D. Muyllaert, I. Dewachter, P. Borghgraef, S. Croes, H. Devijver, F. Van isoforms of human microtubule-associated protein tau: sequences and locali- Leuven, Amyloid activates GSK-3beta to aggravate neuronal tauopathy in bigenic zation in neurofibrillary tangles of Alzheimer's disease, Neuron 3 (1989) mice, Am. J. Pathol. 172 (2008) 786–798. 519–526. [34] W. Noble, V. Olm, K. Takata, E. Casey, O. Mary, J. Meyerson, K. Gaynor, J. [9] M. Goedert, M.G. Spillantini, R.A. Crowther, Cloning of a big tau microtubule- LaFrancois, L. Wang, T. Kondo, P. Davies, M. Burns, Veeranna, R. Nixon, D. associated protein characteristic of the peripheral nervous system, Proc. Natl. Dickson, Y. Matsuoka, M. Ahlijanian, L.F. Lau, K. Duff, Cdk5 is a key factor in tau Acad. Sci. U. S. A. 89 (1992) 1983–1987. aggregation and tangle formation in vivo, Neuron 38 (2003) 555–565. [10] D.N. Drechsel, A.A. Hyman, M.H. Cobb, M.W. Kirschner, Modulation of the [35] J.C. Cruz, H.C. Tseng, J.A. Goldman, H. Shih, L.H. Tsai, Aberrant Cdk5 activation by dynamic instability of tubulin assembly by the microtubule-associated protein p25 triggers pathological events leading to neurodegeneration and neurofibril- tau, Mol. Biol. Cell 3 (1992) 1141–1154. lary tangles, Neuron 40 (2003) 471–483. [11] M. Goedert, R. Jakes, Expression of separate isoforms of human tau protein: [36] K. Sobue, A. Agarwal-Mawal, W. Li, W. Sun, Y. Miura, H.K. Paudel, Interaction of correlation with the tau pattern in brain and effects on tubulin polymerization, neuronal Cdc2-like protein kinase with microtubule-associated protein tau, J. EMBO J. 9 (1990) 4225–4230. Biol. Chem. 275 (2000) 16673–16680. [12] D. Panda, J.C. Samuel, M. Massie, S.C. Feinstein, L. Wilson, Differential regulation [37] T. Li, C. Hawkes, H.Y. Qureshi, S. Kar, H.K. Paudel, Cyclin-dependent protein of microtubule dynamics by three- and four-repeat tau: implications for the kinase 5 primes microtubule-associated protein tau site-specifically for glycogen onset of neurodegenerative disease, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) synthase kinase 3beta, Biochemistry 45 (2006) 3134–3145. 9548–9553. [38] A. Sengupta, Q. Wu, I. Grundke-Iqbal, K. Iqbal, T.J. Singh, Potentiation of GSK-3- [13] B.L. Goode, S.C. Feinstein, Identification of a novel microtubule binding and catalyzed Alzheimer-like phosphorylation of human tau by cdk5, Mol. Cell. assembly domain in the developmentally regulated inter-repeat region of tau, J. Biochem. 167 (1997) 99–105. Cell Biol. 124 (1994) 769–782. [39] M. Goedert, R. Jakes, Z. Qi, J.H. Wang, P. Cohen, Protein phosphatase 2A is the [14] M. Hutton, C.L. Lendon, P. Rizzu, M. Baker, S. Froelich, H. Houlden, S. Pickering- major enzyme in brain that dephosphorylates tau protein phosphorylated by Brown, S. Chakraverty, A. Isaacs, A. Grover, J. Hackett, J. Adamson, S. Lincoln, D. proline-directed protein kinases or cyclic AMP-dependent protein kinase, J. Dickson, P. Davies, R.C. Petersen, M. Stevens, E. de Graaff, E. Wauters, J. van Baren, Neurochem. 65 (1995) 2804–2807. M. Hillebrand, M. Joosse, J.M. Kwon, P. Nowotny, P. Heutink, et al., Association of [40] E. Sontag, V. Nunbhakdi-Craig, G.S. Bloom, M.C. Mumby, A novel pool of protein missense and 5′-splice-site mutations in tau with the inherited dementia FTDP- phosphatase 2A is associated with microtubules and is regulated during the cell 17, Nature 393 (1998) 702–705. cycle, J. Cell Biol. 128 (1995) 1131–1144. [15] R. Dixit, J.L. Ross, Y.E. Goldman, E.L. Holzbaur, Differential regulation of dynein [41] M. Goedert, M.G. Spillantini, N.J. Cairns, R.A. Crowther, Tau proteins of Alzheimer and kinesin motor proteins by tau, Science 319 (2008) 1086–1089. paired helical filaments: abnormal phosphorylation of all six brain isoforms, [16] E.S. Matsuo, R.W. Shin, M.L. Billingsley, A. Van deVoorde, M. O'Connor, J.Q. Neuron 8 (1992) 159–168. Trojanowski, V.M. Lee, Biopsy-derived adult human brain tau is phosphorylated [42] N. Sergeant, J.P. David, D. Lefranc, P. Vermersch, A. Wattez, A. Delacourte, at many of the same sites as Alzheimer's disease paired helical filament tau, Different distribution of phosphorylated tau protein isoforms in Alzheimer's and Neuron 13 (1994) 989–1002. Pick's diseases, FEBS Lett. 412 (1997) 578–582. [17] F. Liu, K. Iqbal, I. Grundke-Iqbal, G.W. Hart, C.X. Gong, O-GlcNAcylation regulates [43] T. Arai, K. Ikeda, H. Akiyama, Y. Shikamoto, K. Tsuchiya, S. Yagishita, T. Beach, J. phosphorylation of tau: a mechanism involved in Alzheimer's disease, Proc. Natl. Rogers, C. Schwab, P.L. McGeer, Distinct isoforms of tau aggregated in neurons and Acad. Sci. U. S. A. 101 (2004) 10804–10809. glial cells in brains of patients with Pick's disease, corticobasal degeneration and [18] G. Lee, R. Thangavel, V.M. Sharma, J.M. Litersky, K. Bhaskar, S.M. Fang, L.H. Do, A. progressive supranuclear palsy, Acta Neuropathol. (Berl.) 101 (2001) 167–173. Andreadis, G. Van Hoesen, H. Ksiezak-Reding, Phosphorylation of tau by fyn: [44] N. Sergeant, A. Wattez, A. Delacourte, Neurofibrillary degeneration in progres- implications for Alzheimer's disease, J. Neurosci. 24 (2004) 2304–2312. sive supranuclear palsy and corticobasal degeneration: tau pathologies with [19] V. Dorval, P.E. Fraser, Small ubiquitin-like modifier (SUMO) modification of exclusively “exon 10” isoforms, J. Neurochem. 72 (1999) 1243–1249. natively unfolded proteins tau and alpha-synuclein, J. Biol. Chem. 281 (2006) [45] M. Baker, I. Litvan, H. Houlden, J. Adamson, D. Dickson, J. Perez-Tur, J. Hardy, T. 9919–9924. Lynch, E. Bigio, M. Hutton, Association of an extended haplotype in the tau gene [20] T. Horiguchi, K. Uryu, B.I. Giasson, H. Ischiropoulos, R. LightFoot, C. Bellmann, C. with progressive supranuclear palsy, Hum. Mol. Genet. 8 (1999) 711–715. Richter-Landsberg, V.M. Lee, J.Q. Trojanowski, Nitration of tau protein is linked to [46] T.M. Caffrey, C. Joachim, S. Paracchini, M.M. Esiri, R. Wade-Martins, Haplotype- neurodegeneration in tauopathies, Am. J. Pathol. 163 (2003) 1021–1031. specific expression of exon 10 at the human MAPT locus, Hum. Mol. Genet. 15 [21] M. Hasegawa, M. Morishima-Kawashima, K. Takio, M. Suzuki, K. Titani, Y. Ihara, (2006) 3529–3537. Protein sequence and mass spectrometric analyses of tau in the Alzheimer's [47] M.G. Spillantini, J.R. Murrell, M. Goedert, M.R. Farlow, A. Klug, B. Ghetti, Mutation disease brain, J. Biol. Chem. 267 (1992) 17047–17054. in the tau gene in familial multiple system tauopathy with presenile dementia, [22] D.P. Hanger, B.H. Anderton, W. Noble, Tau phosphorylation: the therapeutic Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 7737–7741. challenge for neurodegenerative disease, Trends Mol. Med. 15 (2009) 112–119. [48] J. van Swieten, M.G. Spillantini, Hereditary frontotemporal dementia caused by [23] J. Biernat, N. Gustke, G. Drewes, E.M. Mandelkow, E. Mandelkow, Phosphory- Tau gene mutations, Brain Pathol. 17 (2007) 63–73. lation of Ser262 strongly reduces binding of tau to microtubules: distinction [49] M. Hong, V. Zhukareva, V. Vogelsberg-Ragaglia, Z. Wszolek, L. Reed, B.I. Miller, between PHF-like immunoreactivity and microtubule binding, Neuron 11 D.H. Geschwind, T.D. Bird, D. McKeel, A. Goate, J.C. Morris, K.C. Wilhelmsen, G.D. (1993) 153–163. Schellenberg, J.Q. Trojanowski, V.M. Lee, Mutation-specificfunctionalim- [24] N. Gustke, B. Steiner, E.M. Mandelkow, J. Biernat, H.E. Meyer, M. Goedert, E. pairments in distinct tau isoforms of hereditary FTDP-17, Science 282 (1998) Mandelkow, The Alzheimer-like phosphorylation of tau protein reduces 1914–1917. 362 Q. Bai, E.A. Burton / Biochimica et Biophysica Acta 1812 (2011) 353–363

[50] V.M. Lee, T.K. Kenyon, J.Q. Trojanowski, Transgenic animal models of tauopa- [77] M.F. Wullimann, B. Rupp, H. Reichert, Neuroanatomy of the Zebrafish Brain, thies, Biochim. Biophys. Acta 1739 (2005) 251–259. Birkhauser-Verlag, Berlin, 1996. [51] F. Denk, R. Wade-Martins, Knock-out and transgenic mouse models of [78] T. Mueller, M.F. Wullimann, An evolutionary interpretation of teleostean tauopathies, Neurobiol. Aging 30 (2009) 1–13. forebrain anatomy, Brain Behav. Evol. 74 (2009) 30–42. [52] Y. Yoshiyama, M. Higuchi, B. Zhang, S.M. Huang, N. Iwata, T.C. Saido, J. Maeda, T. [79] E. Rink, M.F. Wullimann, Connections of the ventral telencephalon (subpallium) Suhara, J.Q. Trojanowski, V.M. Lee, Synapse loss and microglial activation in the zebrafish (Danio rerio), Brain Res. 1011 (2004) 206–220. precede tangles in a P301S tauopathy mouse model, Neuron 53 (2007) 337–351. [80] T. Mueller, P. Vernier, M.F. Wullimann, The adult central nervous cholinergic [53] K. Santacruz, J. Lewis, T. Spires, J. Paulson, L. Kotilinek, M. Ingelsson, A. system of a neurogenetic model animal, the zebrafish Danio rerio, Brain Res. Guimaraes, M. DeTure, M. Ramsden, E. McGowan, C. Forster, M. Yue, J. Orne, C. 1011 (2004) 156–169. Janus, A. Mariash, M. Kuskowski, B. Hyman, M. Hutton, K.H. Ashe, Tau [81] V. Sallinen, V. Torkko, M. Sundvik, I. Reenilä, D. Khrustalyov, J. Kaslin, P. Panula, suppression in a neurodegenerative mouse model improves memory function, MPTP and MPP+ target specific aminergic cell populations in larval zebrafish, J. Science 309 (2005) 476–481. Neurochem. 108 (3) (2008) 719–731. [54] T.L. Spires, J.D. Orne, K. SantaCruz, R. Pitstick, G.A. Carlson, K.H. Ashe, B.T. Hyman, [82] R.L. Bernardos, P.A. Raymond, GFAP transgenic zebrafish, Gene Expr. Patterns 6 Region-specific dissociation of neuronal loss and neurofibrillary pathology in a (2006) 1007–1013. mouse model of tauopathy, Am. J. Pathol. 168 (2006) 1598–1607. [83] K. Tomizawa, Y. Inoue, H. Nakayasu, A monoclonal antibody stains radial glia in [55] S. Oddo, V. Vasilevko, A. Caccamo, M. Kitazawa, D.H. Cribbs, F.M. LaFerla, the adult zebrafish (Danio rerio) CNS, J. Neurocytol. 29 (2000) 119–128. Reduction of soluble Abeta and tau, but not soluble Abeta alone, ameliorates [84] B.B. Kirby, N. Takada, A.J. Latimer, J. Shin, T.J. Carney, R.N. Kelsh, B. Appel, In vivo cognitive decline in transgenic mice with plaques and tangles, J. Biol. Chem. 281 time-lapse imaging shows dynamic oligodendrocyte progenitor behavior during (2006) 39413–39423. zebrafish development, Nat. Neurosci. 9 (2006) 1506–1511. [56] J. Gotz, F. Chen, J. van Dorpe, R.M. Nitsch, Formation of neurofibrillary tangles in [85] M. Yoshida, W.B. Macklin, Oligodendrocyte development and myelination in P301l tau transgenic mice induced by Abeta 42 fibrils, Science 293 (2001) GFP-transgenic zebrafish, J. Neurosci. Res. 81 (2005) 1–8. 1491–1495. [86] R.L. Avila, B.R. Tevlin, J.P. Lees, H. Inouye, D.A. Kirschner, Myelin structure and [57] J. Lewis, D.W. Dickson, W.L. Lin, L. Chisholm, A. Corral, G. Jones, S.H. Yen, N. composition in zebrafish, Neurochem. Res. 32 (2007) 197–209. Sahara, L. Skipper, D. Yager, C. Eckman, J. Hardy, M. Hutton, E. McGowan, [87] J. Schweitzer, T. Becker, M. Schachner, K.A. Nave, H. Werner, Evolution of myelin Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau proteolipid proteins: gene duplication in teleosts and expression pattern and APP, Science 293 (2001) 1487–1491. divergence, Mol. Cell. Neurosci. 31 (2006) 161–177. [58] K.R. Brunden, J.Q. Trojanowski, V.M. Lee, Advances in tau-focused drug discovery [88] J.Y. Jeong, H.B. Kwon, J.C. Ahn, D. Kang, S.H. Kwon, J.A. Park, K.W. Kim, Functional for Alzheimer's disease and related tauopathies, Nat. Rev. Drug Discov. 8 (2009) and developmental analysis of the blood – brain barrier in zebrafish, Brain Res. 783–793. Bull. 75 (2008) 619–628. [59] S. Le Corre, H.W. Klafki, N. Plesnila, G. Hubinger, A. Obermeier, H. Sahagun, B. [89] S. Bretaud, C. Allen, P.W. Ingham, O. Bandmann, p53-dependent neuronal cell Monse, P. Seneci, J. Lewis, J. Eriksen, C. Zehr, M. Yue, E. McGowan, D.W. Dickson, death in a DJ-1-deficient zebrafish model of Parkinson's disease, J. Neurochem. M. Hutton, H.M. Roder, An inhibitor of tau hyperphosphorylation prevents 100 (2007) 1626–1635. severe motor impairments in tau transgenic mice, Proc. Natl. Acad. Sci. U. S. A. [90] Q. Bai, S.J. Mullett, J.A. Garver, D.A. Hinkle, E.A. Burton, Zebrafish DJ-1 is 103 (2006) 9673–9678. evolutionarily conserved and expressed in dopaminergic neurons, Brain Res. [60] B. Zhang, A. Maiti, S. Shively, F. Lakhani, G. McDonald-Jones, J. Bruce, E.B. Lee, S.X. 1113 (2006) 33–44. Xie, S. Joyce, C. Li, P.M. Toleikis, V.M. Lee, J.Q. Trojanowski, Microtubule-binding [91] O. Anichtchik, H. Diekmann, A. Fleming, A. Roach, P. Goldsmith, D.C. Rubinsztein, drugs offset tau sequestration by stabilizing microtubules and reversing fast Loss of PINK1 function affects development and results in neurodegeneration in axonal transport deficits in a tauopathy model, Proc. Natl. Acad. Sci. U. S. A. 102 zebrafish, J. Neurosci. 28 (2008) 8199–8207. (2005) 227–231. [92] Z. Sun, A.D. Gitler, Discovery and characterization of three novel synuclein genes [61] C.A. Dickey, A. Kamal, K. Lundgren, N. Klosak, R.M. Bailey, J. Dunmore, P. Ash, S. in zebrafish, Dev. Dyn. 237 (2008) 2490–2495. Shoraka, J. Zlatkovic, C.B. Eckman, C. Patterson, D.W. Dickson, N.S. Nahman Jr., M. [93] L. Flinn, H. Mortiboys, K. Volkmann, R.W. Koster, P.W. Ingham, O. Bandmann, Hutton, F. Burrows, L. Petrucelli, The high-affinity HSP90 – CHIP complex Complex I deficiency and dopaminergic neuronal cell loss in parkin-deficient recognizes and selectively degrades phosphorylated tau client proteins, J. Clin. zebrafish (Danio rerio), Brain 132 (2009) 1613–1623. Invest. 117 (2007) 648–658. [94] W.A. Campbell, H. Yang, H. Zetterberg, S. Baulac, J.A. Sears, T. Liu, S.T. Wong, T.P. [62] K. Kawakami, Transgenesis and gene trap methods in zebrafish by using the Tol2 Zhong, W. Xia, Zebrafish lacking Alzheimer presenilin enhancer 2 (Pen-2) transposable element, Methods Cell Biol. 77 (2004) 201–222. demonstrate excessive p53-dependent apoptosis and neuronal loss, J. Neuro- [63] D. Soroldoni, B.M. Hogan, A.C. Oates, Simple and efficient transgenesis with chem. 96 (2006) 1423–1440. meganuclease constructs in zebrafish, Methods Mol. Biol. 546 (2009) 117–130. [95] C. Groth, S. Nornes, R. McCarty, R. Tamme, M. Lardelli, Identification of a second [64] A. Nasevicius, S.C. Ekker, Effective targeted gene ‘knockdown’ in zebrafish, Nat. presenilin gene in zebrafish with similarity to the human Alzheimer's disease Genet. 26 (2000) 216–220. gene presenilin2, Dev. Genes Evol. 212 (2002) 486–490. [65] K.G. Peters, P.S. Rao, B.S. Bell, L.A. Kindman, Green fluorescent fusion proteins: [96] U. Leimer, K. Lun, H. Romig, J. Walter, J. Grunberg, M. Brand, C. Haass, Zebrafish powerful tools for monitoring protein expression in live zebrafish embryos, Dev. (Danio rerio) presenilin promotes aberrant amyloid beta-peptide production Biol. 171 (1995) 252–257. and requires a critical aspartate residue for its function in amyloidogenesis, [66] D.L. McLean, J.R. Fetcho, Using imaging and genetics in zebrafish to study Biochemistry 38 (1999) 13602–13609. developing spinal circuits in vivo, Dev. Neurobiol. 68 (2008) 817–834. [97] M. Chen, R.N. Martins, M. Lardelli, Complex splicing and neural expression of [67] L. Wen, W. Wei, W. Gu, P. Huang, X. Ren, Z. Zhang, Z. Zhu, S. Lin, B. Zhang, duplicated tau genes in zebrafish embryos, J. Alzheimers Dis. 18 (2) (2009) 305–317. Visualization of monoaminergic neurons and neurotoxicity of MPTP in live [98] A. Amores, A. Force, Y.L. Yan, L. Joly, C. Amemiya, A. Fritz, R.K. Ho, J. Langeland, V. transgenic zebrafish, Dev. Biol. 314 (2008) 84–92. Prince, Y.L. Wang, M. Westerfield, M. Ekker, J.H. Postlethwait, Zebrafish hox [68] F. Peri, C. Nusslein-Volhard, Live imaging of neuronal degradation by microglia clusters and vertebrate genome evolution, Science 282 (1998) 1711–1714. reveals a role for v0-ATPase a1 in phagosomal fusion in vivo, Cell 133 (2008) [99] Y.C. Chen, M. Priyadarshini, P. Panula, Complementary developmental expression of 916–927. the two tyrosine hydroxylase transcripts in zebrafish, Histochem. Cell Biol. 132 (4) [69] W. Driever, L. Solnica-Krezel, A.F. Schier, S.C. Neuhauss, J. Malicki, D.L. Stemple, D.Y. (2009) 375–381. Stainier, F. Zwartkruis, S. Abdelilah, Z. Rangini, J. Belak, C. Boggs, A genetic screen [100] G.W. Stuart, J.V. McMurray, M. Westerfield, Replication, integration and stable for mutations affecting embryogenesis in zebrafish, Development 123 (1996) germ-line transmission of foreign sequences injected into early zebrafish 37–46. embryos, Development 103 (1988) 403–412. [70] L. Solnica-Krezel, A.F. Schier, W. Driever, Efficient recovery of ENU-induced [101] A. Jacquier, B. Dujon, An intron-encoded protein is active in a gene conversion mutations from the zebrafish germline, Genetics 136 (1994) 1401–1420. process that spreads an intron into a mitochondrial gene, Cell 41 (1985) [71] G. Golling, A. Amsterdam, Z. Sun, M. Antonelli, E. Maldonado, W. Chen, S. Burgess, 383–394. M. Haldi, K. Artzt, S. Farrington, S.Y. Lin, R.M. Nissen, N. Hopkins, Insertional [102] V. Thermes, C. Grabher, F. Ristoratore, F. Bourrat, A. Choulika, J. Wittbrodt, J.S. mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate Joly, I-SceI meganuclease mediates highly efficient transgenesis in fish, Mech. development, Nat. Genet. 31 (2002) 135–140. Dev. 118 (2002) 91–98. [72] A. Amsterdam, S. Burgess, G. Golling, W. Chen, Z. Sun, K. Townsend, S. Farrington, [103] Q. Bai, X. Wei, E.A. Burton, Expression of a 12-kb promoter element derived from M. Haldi, N. Hopkins, A large-scale insertional mutagenesis screen in zebrafish, the zebrafish enolase-2 gene in the zebrafish visual system, Neurosci. Lett. 449 Genes Dev. 13 (1999) 2713–2724. (2009) 252–257. [73] L.I. Zon, R.T. Peterson, In vivo drug discovery in the zebrafish, Nat. Rev. Drug [104] A. Koga, M. Suzuki, H. Inagaki, Y. Bessho, H. Hori, Transposable element in fish, Discov. 4 (2005) 35–44. Nature 383 (1996) 30. [74] H.M. Stern, R.D. Murphey, J.L. Shepard, J.F. Amatruda, C.T. Straub, K.L. Pfaff, G. [105] K. Kawakami, A. Shima, N. Kawakami, Identification of a functional transposase Weber, J.A. Tallarico, R.W. King, L.I. Zon, Small molecules that delay S phase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its suppress a zebrafish bmyb mutant, Nat. Chem. Biol. 1 (2005) 366–370. transposition in the zebrafish germ lineage, Proc. Natl. Acad. Sci. U. S. A. 97 [75] G. Molina, A. Vogt, A. Bakan, W. Dai, P. Queiroz de Oliveira, W. Znosko, T.E. (2000) 11403–11408. Smithgall, I. Bahar, J.S. Lazo, B.W. Day, M. Tsang, Zebrafish chemical screening [106] S. Higashijima, H. Okamoto, N. Ueno, Y. Hotta, G. Eguchi, High-frequency generation reveals an inhibitor of Dusp6 that expands cardiac cell lineages, Nat. Chem. Biol. of transgenic zebrafish which reliably express GFP in whole muscles or the whole 5 (2009) 680–687. body by using promoters of zebrafish origin, Dev. Biol. 192 (1997) 289–299. [76] J. Rihel, D.A. Prober, A. Arvanites, K. Lam, S. Zimmerman, S. Jang, S.J. Haggarty, D. [107] Q. Long, A. Meng, H. Wang, J.R. Jessen, M.J. Farrell, S. Lin, GATA-1 expression Kokel, L.L. Rubin, R.T. Peterson, A.F. Schier, Zebrafish behavioral profiling links pattern can be recapitulated in living transgenic zebrafish using GFP reporter drugs to biological targets and rest/wake regulation, Science 327 (2010) 348–351. gene, Development 124 (1997) 4105–4111. Q. Bai, E.A. Burton / Biochimica et Biophysica Acta 1812 (2011) 353–363 363

[108] A. Meng, H. Tang, B.A. Ong, M.J. Farrell, S. Lin, Promoter analysis in living [117] Y. Gao, P. Li, L. Li, Transgenic zebrafish that express tyrosine hydroxylase zebrafish embryos identifies a cis-acting motif required for neuronal expression promoter in inner retinal cells, Dev. Dyn. 233 (2005) 921–929. of GATA-2, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 6267–6272. [118] S. Meng, S. Ryu, B. Zhao, D.Q. Zhang, W. Driever, D.G. McMahon, Targeting retinal [109] C.H. Kim, E. Ueshima, O. Muraoka, H. Tanaka, S.Y. Yeo, T.L. Huh, N. Miki, Zebrafish dopaminergic neurons in tyrosine hydroxylase-driven green fluorescent protein elav/HuC homologue as a very early neuronal marker, Neurosci. Lett. 216 (1996) transgenic zebrafish, Mol. Vis. 14 (2008) 2475–2483. 109–112. [119] Q. Bai, E.A. Burton, Cis-acting elements responsible for dopaminergic neuron- [110] H.C. Park, C.H. Kim, Y.K. Bae, S.Y. Yeo, S.H. Kim, S.K. Hong, J. Shin, K.W. Yoo, M. specific expression of zebrafish slc6a3 (dopamine transporter) in vivo are Hibi, T. Hirano, N. Miki, A.B. Chitnis, T.L. Huh, Analysis of upstream elements in located remote from the transcriptional start site, Neuroscience 164 (2009) the HuC promoter leads to the establishment of transgenic zebrafish with 1138–1151. fluorescent neurons, Dev. Biol. 227 (2000) 279–293. [120] O.V. Anichtchik, J. Kaslin, N. Peitsaro, M. Scheinin, P. Panula, Neurochemical and [111] Q. Bai, J.A. Garver, N.A. Hukriede, E.A. Burton, Generation of a transgenic behavioural changes in zebrafish Danio rerio after systemic administration of 6- zebrafish model of Tauopathy using a novel promoter element derived from the hydroxydopamine and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine, J. zebrafish eno2 gene, Nucleic Acids Res. 35 (2007) 6501–6516. Neurochem. 88 (2004) 443–453. [112] K. Asakawa, M.L. Suster, K. Mizusawa, S. Nagayoshi, T. Kotani, A. Urasaki, Y. [121] H.G. Tomasiewicz, D.B. Flaherty, J.P. Soria, J.G. Wood, Transgenic zebrafish model Kishimoto, M. Hibi, K. Kawakami, Genetic dissection of neural circuits by Tol2 of neurodegeneration, J. Neurosci. Res. 70 (2002) 734–745. transposon-mediated Gal4 gene and enhancer trapping in zebrafish, Proc. Natl. [122] G.A. Jicha, R. Bowser, I.G. Kazam, P. Davies, Alz-50 and MC-1, a new monoclonal Acad. Sci. U. S. A. 105 (2008) 1255–1260. antibody raised to paired helical filaments, recognize conformational epitopes [113] M. Distel, M.F. Wullimann, R.W. Koster, Optimized Gal4 genetics for permanent on recombinant tau, J. Neurosci. Res. 48 (1997) 128–132. gene expression mapping in zebrafish, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) [123] M. Kidd, Paired helical filaments in electron microscopy of Alzheimer's disease, 13365–13370. Nature 197 (1963) 192–193. [114] D. Paquet, R. Bhat, A. Sydow, E.M. Mandelkow, S. Berg, S. Hellberg, J. Falting, M. [124] I. Tellez-Nagel, H.M. Wisniewski, Ultrastructure of neurofibrillary tangles in Distel, R.W. Koster, B. Schmid, C. Haass, A zebrafish model of tauopathy allows in Steele–Richardson–Olszewski syndrome, Arch. Neurol. 29 (1973) 324–327. vivo imaging of neuronal cell death and drug evaluation, J. Clin. Invest. 119 [125] H. Ksiezak-Reding, K. Morgan, L.A. Mattiace, P. Davies, W.K. Liu, S.H. Yen, K. (2009) 1382–1395. Weidenheim, D.W. Dickson, Ultrastructure and biochemical composition of [115] E.K. Scott, L. Mason, A.B. Arrenberg, L. Ziv, N.J. Gosse, T. Xiao, N.C. Chi, K. Asakawa, paired helical filaments in corticobasal degeneration, Am. J. Pathol. 145 (1994) K. Kawakami, H. Baier, Targeting neural circuitry in zebrafish using GAL4 1496–1508. enhancer trapping, Nat. Methods 4 (2007) 323–326. [126] D. Munoz-Garcia, S.K. Ludwin, Classic and generalized variants of Pick's disease: [116] D. Dickson, Sporadic tauopathies: Pick's disease, corticobasal degeneration, a clinicopathological, ultrastructural, and immunocytochemical comparative progressive supranuclear palsy and argyrophilic grain disease, in: M. Esiri, V.M. study, Ann. Neurol. 16 (1984) 467–480. Lee, J.Q. Trojanowski (Eds.), The Neuropathology of Dementia, Cambridge [127] F. Gallyas, Silver staining of Alzheimer's neurofibrillary changes by means of University Press, Cambridge, 2004, pp. 227–256. physical development, Acta Morphol. Acad. Sci. Hung. 19 (1971) 1–8. Biochimica et Biophysica Acta 1812 (2011) 364–380

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Review Investigating regeneration and functional integration of CNS neurons: Lessons from zebrafish genetics and other fish species☆

Valerie C. Fleisch a,b,⁎, Brittany Fraser b, W. Ted Allison a,b,c a Centre for Prions & Protein Folding Disease, University of Alberta, Edmonton, Alberta, T6G 2M8, Canada b Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2J9, Canada c Department of Medical Genetics, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada article info abstract

Article history: Zebrafish possess a robust, innate CNS regenerative ability. Combined with their genetic tractability and Received 6 January 2010 vertebrate CNS architecture, this ability makes zebrafish an attractive model to gain requisite knowledge for Received in revised form 5 October 2010 clinical CNS regeneration. In treatment of neurological disorders, one can envisage replacing lost neurons Accepted 21 October 2010 through stem cell therapy or through activation of latent stem cells in the CNS. Here we review the evidence Available online 28 October 2010 that radial glia are a major source of CNS stem cells in zebrafish and thus activation of radial glia is an attractive therapeutic target. We discuss the regenerative potential and the molecular mechanisms thereof, in Keywords: fi Zebrafish the zebra sh spinal cord, retina, optic nerve and higher brain centres. We evaluate various cell ablation Regeneration paradigms developed to induce regeneration, with particular emphasis on the need for (high throughput) Retina indicators that neuronal regeneration has restored sensory or motor function. We also examine the potential Optic nerve confound that regeneration imposes as the community develops zebrafish models of neurodegeneration. We Spinal cord conclude that zebrafish combine several characters that make them a potent resource for testing hypotheses Brain and discovering therapeutic targets in functional CNS regeneration. This article is part of a Special Issue Central nervous system entitled Zebrafish Models of Neurological Diseases. Stem cell © 2010 Elsevier B.V. All rights reserved. Glia

1. Introduction the highest degree of regenerative power, lack of genetic tractability as well as the absence of higher level brain centres are major drawbacks It had long been assumed that the mammalian central nervous for their use as animal models in CNS regeneration research. Among system (CNS) is incapable of regenerating neurons. However, today it vertebrates, a similarly high regenerative potential is found in is widely accepted that sources of adult neurogenesis do exist, and amphibians, such as the newt or Xenopus, and in fish. Newts are that these areas have the potential to underpin regeneration. Adult extremely difficult to maintain and breed under laboratory conditions, neurogenesis is localized to the dentate gyrus of the hippocampus as and genetic tools have only been developed recently. Xenopus and well as the lateral ventricles of the mammalian forebrain [1–3]. It has zebrafish are both popular animal models for CNS regeneration been shown that injury to the CNS (caused by ischemia, seizures, research, but this review will focus on the teleost model zebrafish. radiation or neurodegenerative disorders) triggers proliferation of Zebrafish are very potent in regenerating a variety of tissues. progenitor cells in the subgranular zone and the subventricular zone Traditionally, the zebrafish has been used as a model for studying the in mammalian animal models and humans. However, in most cases regeneration of the fins [6]. Since the retina is the most easily these newborn cells do not show long-term survival, fail to migrate or accessible part of the CNS, and morphology and function of the retina integrate properly into the neuronal network (for a review see [4]). are basically conserved among vertebrate species, zebrafish have also This lack of regenerative ability seems to have arisen in the course been extensively used for studying the regeneration of the retina and of evolution; most invertebrates (commonly used models are Hydra optic nerve (reviewed below). Moreover, zebrafish have also and planarians) as well as a number of anamniotic vertebrates, such as increasingly been used to study regeneration of other nervous tissues, amphibians and fish, are capable of regenerating whole body parts and such as brain nuclei and the spinal cord. In contrast to newts, zebrafish organs (for a review see [5]). Although invertebrates probably show are very easy and cost-effective to breed and produce high numbers of offspring [7]. Moreover, the abundance of genetic tools, including transgenic lines, knock-down (morpholino antisense technology [8]), ☆ fi This article is part of a Special Issue entitled Zebra sh Models of Neurological mutants and novel knock-out (zinc finger nuclease [9,10]) techniques Diseases. and microarrays, facilitate research aimed at identifying molecular ⁎ Corresponding author. Centre for Prions & Protein Folding Disease, University of Alberta, Edmonton, Alberta, T6G 2M8, Canada. Tel.: +1 780 492 1087. pathways underlying regenerative processes. Access to zebrafish E-mail address: valerie.fl[email protected] (V.C. Fleisch). embryos is easy, as larvae develop outside the mother, and

0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2010.10.012 V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380 365 transparency of larvae enables in vivo tracking of regenerating cells. columns of proliferating cells, termed “neurogenic clusters”, which Zebrafish also offer very powerful behavioral and electrophysiological express a similar set of stem cell markers as progenitor cells residing tools to assess functional integration of regenerated neurons/axons in the CMZ such as pax6, vsx1, notch-3 and n-cadherin [20–23]. Vihtelic [11,12]. et al. showed that PCNA (proliferating cell nuclear antigen, a marker Studying the exact mechanisms of how anamniotes such as for proliferation) positive cells can be detected as early as one day zebrafish utilize stem cells to replace neurons of the CNS is valuable after light-induced photoreceptor cell death in the INL (inner nuclear for helping us to understand why humans/mammals mostly lack this layer) in albino zebrafish. These progenitor cells migrate to the ONL regenerative ability and more importantly, will provide us with ideas (outer nuclear layer) within 96 h, where they differentiate into rod of how human CNS tissue could be stimulated to regenerate after and cone photoreceptors [24,25]. The identity of the cells comprising injury. This type of knowledge will be crucial for advancing stem cell these clusters was resolved only recently when several researchers therapeutics. As direct transplantation of stem cells has been proven found evidence that Müller glia are the major source of stem cells in inefficient for restoring missing/damaged tissues, future approaches the adult zebrafish retina. might involve switching existing dormant progenitor cells or even terminally differentiated cells back to a “stem cell mode”. 2.1.1. Retinal astrocytes (Müller glia cells) function as stem cells in the In order to address this scientific puzzle, a number of fundamental regenerating adult zebrafish retina questions immediately come to mind: What is the identity of the stem Studies in recent years have dramatically changed our view of cells giving rise to newborn neurons — are these cells the same ones astrocytes as “mere support cells” or “neuronal glue”. Astrocytes are across species and across different tissues? Is the limitation on CNS now viewed as highly important cells with a number of complex regeneration intrinsic to the neuron or do extrinsic signals control functions. Astrocytes are found in all tissues of the CNS (e.g. cerebellar regeneration (role of glia cell in inhibition versus promotion of Bergmann glia, retinal Müller glia) and have a number of established regeneration)? What are the molecular pathways underlying dedif- roles in metabolic support for neurons, K+ and neurotransmitter ferentiation of glial stem cells and specification of new neurons? Are uptake, synaptogenesis, angiogenesis and maintenance of the brain– regenerated neurons functionally integrated into the CNS and how is blood barrier (for a detailed recent review see [26]). Groundbreaking that achieved? studies of neuronal precursors in the CNS have elucidated the We are beginning to address these questions in zebrafish models existence of yet another type of glia cell [27,28]. These cells, exhibiting of CNS regeneration, and the present review aims to provide a neuronal stem cell features, are located in adult neurogenic regions summary of the (partial) answers that have arisen from these studies. (the adult SVZ, subventricular zone and SGZ, subgranular zone). They have been shown to give rise to newborn olfactory neurons [29–31] 2. Regeneration of various CNS tissues and granule neurons [32–34]. The brain is not the only CNS tissue containing a large number of Studies of neural regeneration in zebrafish have been carried out in astrocytes. Müller glia of the retina perform similar functions as other various CNS tissues. We review each in turn, dividing the topics into glial cells in the brain, including structural and metabolic support of retina, optic nerve, spinal cord, and brain nuclei. neurons, ion buffering, and neurotransmitter homeostasis. Studies in the chick retina [35,36] as well as experiments using 2.1. Retina transgenic zebrafish lines in combination with lineage tracing have revealed that, in addition to these basic support functions, Müller glia Ocular disorders involving degeneration of cells in the neural cells act as retinal stem cells after injury. Following photoreceptor cell retina are exceedingly common. Death of rod and/or cone photo- death, they undergo morphological as well as gene expression changes. receptors is found in retinal dystrophies, such as AMD (age-related Thummel et al. have shown that Müller glia cells dedifferentiate upon macular degeneration), Retinitis Pigmentosa, Usher Syndrome or injury as they lose expression of cell-type-specific markers such as GFAP Leber Congenital Amaurosis. Death of retinal ganglion cells is the (glial fibrillary acidic protein) and GS (glutamine synthetase) [37].Signs largest contributor to vision loss in glaucoma, a condition affecting the of dedifferentiation include re-expression of BLBP (brain lipid binding optic nerve. Cells lost from the retina cannot be replaced, as the protein), up-regulation of components of the Notch–Delta signalling mammalian retina does not have intrinsic repair processes, rendering pathway and redistribution of N-cadherin to the basolateral plasma the patient severely visually impaired or even completely blind. membrane [16]. In contrast to mammals, anamniotic vertebrates like zebrafish In succession, several groups used lineage tracing via transgenic have amazing regenerative capabilities, including the capacity to expression of GFP in Müller cells in combination with immunocytolo- regenerate any cell type in the retina. Cells are continuously added to gical stainings and BrdU labelling to provide evidence that Müller cells the zebrafish retina as it expands throughout the life of the fish. This not only respond to injury, but also contribute to the stem cells of the unique feature of teleost fish requires that sources of neurogenesis are retina and give rise to neuronal progenitors (Fig. 1). Fausett and maintained even in the adult. In the intact retina, progenitor cells have Goldman used the α1T transgenic line, driving transgene expression in a been classified as being localized to two distinct cellular compart- subpopulation of proliferative Müller glia cells after retinal injury [38]. ments [13,14]. The CMZ (ciliary marginal zone) gives rise to all retinal BrdU labelling experiments revealed a dramatic increase of BrdU+ cells cell types, whereas rod photoreceptors arise from a separate lineage of in the INL at 2 dpi (days post-injury), preceding a rise in BrdU+ cells in CMZ-derived Müller glia [13,15,16]. Neuronal progenitors of the CMZ the ONL at 3 dpi. Most of these proliferating cells were GFP+, had a express a number of markers such as n-cadherin, components of the Müller cell-like morphology and expressed the Müller cell markers Notch–Delta signalling pathway, rx1, vsx2/chx10 and pax6a [16]. GFAP and GS. BrdU labelling at a later time point, 2 dpi, revealed staining Proliferating Müller glia cells in the INL (inner nuclear layer) produce in the ONL and ganglion cell layer (GCL), suggesting migration of INL rod progenitors which subsequently migrate to the ONL (outer precursors. The progenitors that had presumably arisen from reactive nuclear layer), where they differentiate into mature rod photorecep- Müller glia cells started to express neuron-specific markers after 7 dpi, tors [15,17,18]. Cells within both of these compartments show and were localized to the INL, IPL and GCL. BrdU addition at 4 dpi and characteristic features of stem cells, including asymmetric division evaluation of cells at 60 and 180 dpi revealed cells expressing markers of and self-renewal. An additional population of stem cells is activated bipolar and amacrine cells, Müller glia and cone photoreceptors [39]. upon retinal injury and subsequently produces progenitors for Bernardos et al. used a gfap:GFP transgenic zebrafish line regenerating lost retinal cells (for a review see [16,19]). It has been harbouring the Müller cell-specific gfap promoter driving GFP shown that in fish, damage to the retina induces the formation of expression specifically in Müller glia cells [40]. Following light 366 V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380

Fig. 1. Radial glia throughout the CNS are a source of stem cells. Here we depict retinal Müller glia cells as an example of radial glial cells of the CNS. The retina of a 4 dpf zebrafish larva with its three nuclear layers (GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer) is shown as a reference for the location of retinal progenitors (A). Differentiated radial glia cells spanning the larval (and adult) zebrafish retina (B) become activated upon injury, dedifferentiate and start to proliferate (C). Neuronal progenitors of all retinal cell lineages arise from these activated cells (D), and migrate to specific retinal layers where they differentiate into neurons (E).

damage, the number of GFP+ Müller cells increased 3 fold up to 5 dpi, cells. Initially, researchers had speculated that apoptotic photorecep- indicating a proliferative response. Coinciding with a peak in GFP+ tors would provide a molecular signal to Müller glia cells, thereby cells, the expression of the progenitor cell markers pax6, pcna and crx activating dedifferentiation and proliferation. However, Fimbel et al. (a marker for photoreceptors progenitors) peaked between 1 and showed that damage to the GCL and INL, without substantial damage 5 dpi. The spatio-temporal distribution of pax6+ and crx+ cells was to photoreceptors, likewise causes Müller cells to re-enter the cell consistent with a transition from pax6 to crx expression in progenitors cycle and generate retinal progenitors [43]. Low doses of intravitreal as they migrated from the INL to the ONL. After 1 month, cones and ouabain injections caused GCL and INL death. At 1 day post-injury rods regenerated completely and Müller cell density fell back to (dpi), PCNA expression was seen in Müller glia cells, and by 5 dpi, its unlesioned levels. expression shifted to neuronal progenitors expressing the olig2:egfp Morris et al. specifically induced death of cone photoreceptors by transgene. After 60 dpi, 75% of ganglion and amacrine cells had using the pde6cw59 mutant zebrafish line, harbouring a mutation in the regenerated [43]. cone-specific phosphodiesterase gene. BrdU exposure 4 h before Various studies support Müller glia cells as one source, perhaps sacrifice and subsequent CAZ (carbonic anhydrase, a marker for even a major source, of progenitor cells in the zebrafish retina. Müller Müller glia cells) staining revealed a significant increase of BrdU+ cells glia cells are activated following retinal damage and subsequently in the INL, partly co-localizing with CAZ. These results suggest that at dedifferentiate, proliferate and give rise to different retinal cell types least a subset of Müller glia cells had re-entered the cell cycle [41]. (Fig. 1). As Müller glia cells are also found in the mammalian/human Studies using morpholino-mediated knock-down of PCNA [42] retina, understanding which molecular cues are required to turn a provided evidence that progenitor cell formation in zebrafish retina quiescent Müller glia cell into a retinal stem cell will be crucial for the after injury is dependent on proliferation of Müller glia cells. PCNA development of potential novel therapeutics which could be applied knock-down prior to light damage inhibited proliferation of Müller to trigger regeneration in the human retina. Although under normal glia cells and resulted in decreased levels of GS expression as well as physiological conditions, mammalian Müller glia cells do not re-enter Müller cell death. Importantly, rod photoreceptors and short and long the cell cycle in response to injury [44–46], it has been speculated that single cones could not be regenerated in morpholino-injected the human retina may have some regenerative potential. Several zebrafish larvae [42]. Assuming the effects of PCNA knock-down are studies have reported the presence of BrdU+ photoreceptors after primarily within the Müller glia, these data strongly supports the retina damage or after the addition of growth factors [47–51]. hypothesis that these cells are the source of retina stem cells. Moreover, the human retina may contain a compartment similar to Several researchers showed that damage to photoreceptors the zebrafish CMZ. In retinal explants, cells of the human retinal triggers a regenerative response through activation of Müller glia margin as well as the INL re-enter the cell cycle when EGF (epidermal V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380 367 growth factor) is added to the culture medium [52]. Thus the human growth factors such as l-plastin, CC chemokine, galectin family retina may harbour quiescent stem cells, which can be triggered to re- members, progranulin family members and midkines. In situ enter the cell cycle by the addition of growth factors. hybridization and immunohistological experiments of selected Unfortunately very little is known about the molecular pathways candidate genes showed that lgals9l1 (a member of the galectin leading to the cell-biological changes inducing the activation of Müller family) and progranulin-a were exclusively expressed in microglia, glia cells. In recent years, a number of microarray studies focussing on and lgals1l2 in microglia as well as Müller glia cell, confirming the temporal expression changes during retina regeneration in zebrafish specificity of the approach. have been conducted and have identified several candidate genes and Whereas Craig et al. had isolated Muller glia cell-derived pathways presumably implicated in the regeneration process. Data progenitor cells from the ONL, Qin et al. used the transgenic zebrafish collected in these studies will be presented in the next section of this reporter line Tg(gfap:GFP)mi2002 to directly isolate Müller glia cells review, and an overview of the molecular components that have been [57]. Gene expression profiles from intact and light-lesioned retinas at identified can be found in Table 1. 8, 16, 24 and 36 h post-treatment were compared. A set of 953 genes being differentially expressed was identified and subdivided into 3 2.1.2. Molecular pathways involved in retinal regeneration clusters, depending on their temporal expression patterns. One set of In order to identify candidate genes involved in the regenerative transcripts was up-regulated immediately after the lesion, and response in the zebrafish retina, a number of microarray-based included mainly genes implicated in translation and protein biosyn- studies have been performed. While one study used surgical removal thesis. A number of DNA replication and cell cycle genes were up- of a small piece of the retina [53], mostly light damage paradigms have regulated slightly delayed, reflecting re-entry of Müller cells into the been applied to cause retinal injury [54–57]. Microarray analysis cell cycle. Among the transcripts down-regulated after lesion were conducted on mRNA isolated from whole retina showed significant genes involved in chromatin assembly and ion homeostasis, consis- changes in expression of genes implicated in the cell cycle, cell tent with dedifferentiation of Müller glia cells. As expected, an up- proliferation, cell death, DNA replication, general metabolism and regulation of cell cycle genes and growth factors such as pcna axonal regeneration as expected [37,53,54,56]. Genes encoding the and pdgfa (platelet-derived growth factor) was observed as well. opsin proteins were decreased in expression, reflecting death of Interestingly, despite taking such a specific approach, Qin et al. photoreceptors, whereas cell cycle genes increased their expression, identified two candidate genes, hspd2 (heat shock 60-kDa protein 1) corresponding to progenitor proliferation [54]. As regeneration- and msp1 (monopolar spindle 1), which are not only essential for the specific responses are expected to occur in specific cell types (Müller regeneration of the retina, but likewise important for fin and heart glia cells and emanating progenitor cells) expression analysis of RNA tissue renewal in zebrafish [57]. extracted exclusively from those cells was expected to reveal novel Similar to hspd2 and msp1, the heparine-binding growth factors regeneration-specific candidate genes. Craig et al. examined the midkines a and b seem to play a significant role in regeneration of a expression changes in regenerating photoreceptor cells, by combining variety of tissues in the zebrafish (retina, heart and fin) [54,58,59]. light damage through continuous light of low intensity with laser- Photoreceptor apoptosis after light damage causes midkine expression capturing of ONL tissue containing photoreceptors and their progeni- pattern changes in the zebrafish retina [54]. In addition to their tors [55]. Samples collected at early time points after light damage developmental expression (midkine-a in progenitors, Müller cells and showed decreased expression of photoreceptor-specific genes (such horizontal cells and midkine-b in retinal ganglion and amacrine cells), as the opsins) and up-regulation of stress response genes, indicative both midkines are expressed in proliferating Müller glia cells and their of dying photoreceptors. In a second step, transcript changes at 24– progeny, and midkine-b is additionally found in HCs [54]. 48 h post-injury were examined, as they were thought to represent Other candidate genes identified in microarray experiments and changes in progenitor cells migrating to the ONL. Differentially hypothesized to be involved in the switch from differentiated Müller expressed genes found in this group included the transcription glia cell to proliferating retinal stem cell are stat3 (signal transducer factors sox11b, fos, jun and pax6a as well as genes encoding secreted and activator of transcription 3), ascl1a (ash1a) (achaete/scute

Table 1 List of molecular cues involved in retina regeneration in the zebrafish.

Gene Classification Action Lesioning Reference paradigm

hspd1 (heat shock Mitochondrial protein chaperone involved Formation of retinal stem cells from Light damage [57] 60 kDa protein 1) in stress response dedifferentiating Müller cells mps1 (monopolar spindle 1) Kinase involved in mitotic checkpoint Proliferation of photoreceptor progenitors Light damage [57] regulation mdka (midkine-a) (secreted) heparin binding growth factor Not clear yet Light damage [54] mdkb (midkine-b) (secreted) heparin binding growth factor Not clear yet Light damage [54] ascl1a (ash1a) (achaete–scute Proneural gene Transition of Müller cell into proliferative stem cell Surgical excision [61] homolog 1a) notch3 Component of Notch–Delta signalling Progenitor proliferation Surgical excision [61] deltaA Component of Notch–Delta signalling Differentiation Surgical excision [61] (notch ligand) pax6b Transcription factor (neurogenesis, Hypothesized role in proliferation and migration Light damage [37] regeneration) of neuronal progenitor cells stat3 (signal transducer and Transcription factor Hypothesized to trigger Müller cell proliferation in the damaged Light damage [56,60] activator of transcription) retina (has been shown to do so in the undamaged retina) ngn1 (neurogenin 1) Transcription factor Hypothesized role in “re-differentiation” of Müller cells Light damage [37] olig2 (oligodendrocyte bHLH transcription factor Hypothesized role in maintaining Müller cells in a Light damage [37] transcription factor 2) dedifferentiated state lgals9l1-, 2 (galectin family) Secreted growth factor Up-regulated 24–48 h post-injury in microglia and Müller cells; Light damage [55] exact function unknown progranulin-a Secreted growth factor Up-regulated 24–48 h post-injury in microglia; exact function Light damage [55] unknown 368 V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380 homolog 1a) and members of the notch–delta family. Stat3 expression Although there is evidence that damage to photoreceptors and is increased in photoreceptors and Müller glia cells following light other cell types of the retina, trigger the regeneration response, the damage [56]. Interestingly, only a subset of Stat3+ Müller glia cells identity as well as the source of the actual “trigger signal” is not co-label with PCNA, which led Kassen et al. to hypothesize that Stat3 known. Fausett and Goldman speculated that the molecular signal might act as a molecular cue, triggering MCs to proliferate after injury. arises from cells near the actual lesion site, as 90% of Müller glia cells In a recent publication, Kassen et al. identified a potential upstream next to the lesion site are GFP+, whereas at sites far from lesion, regulator of Stat3, CNTF (ciliary neurotrophic factor). CNTF activates almost none are GFP+ [39]. Evidence in mouse models suggests that Stat3 to induce Müller cell proliferation in the undamaged retina [60]; photoreceptors signal damage to Müller glia through endothelin whether or not it plays a similar role in the injured retina remains to receptors [63]. be investigated. Finally, it is worth mentioning that Müller glia cells are not the Yurco and Cameron found up-regulation of members of the notch– only glial cell population in the zebrafish retina. Microglia are delta family, as well as ascl1a (ash1a) in their microarray analysis of phagocytic cells that secrete pro- or contra-inflammatory signals to differentially regulated genes after excision of a patch of adult either inhibit or promote neuronal repair and regeneration [64]. zebrafish retina. Importantly, ascl1a (ash1a) was up-regulated before Microglia hypertrophy at 24 h after light damage to photoreceptors components of Notch–Delta signalling showed increased expression. has been shown. Subsequently, they become polymorphic and They proposed a model in which Müller glia cells start to express migrate into the ONL [16,55]. Craig et al. found members of the ascl1a (ash1a) after injury and subsequently become divided into galectin and progranulin families up-regulated after light damage two sub-populations, a notch3-expressing population consisting of [55]. Progranulin-a, lgals1l1 as well as lgals1l2 are expressed in proliferating progenitors and a deltaA expressing population exiting microglia thus suggesting that microglia likely carry out an important the cell cycle and differentiating into neurons [61]. Morpholino role in retina regeneration and warrant further study. knock-down of ascl1a (ash1a) inhibits Müller glia cell activation and consequently regeneration [62]. 2.2. Optic nerve Although ngn1 (neurogenin-1) is expressed in proliferating progenitors and the transgene olig2:EGFP in dedifferentiated Müller Injuries of the optic nerve have a highly detrimental effect on a glia cells [37], nothing is yet known about their role in retina patient's life, severely impacting vision and ultimately leading to regeneration. complete blindness in a significant percentage of cases. Damage to the optic nerve can occur by either trauma (e.g. head trauma) or can be 2.1.3. Open questions caused by diseases leading to death of retinal ganglion cells and The unique capability to regenerate all retinal cell types, combined subsequent axon degeneration, such as general neurodegenerative with tractable genetics, makes zebrafish an ideal model for decipher- disorders (e.g. multiple sclerosis) or ocular disorders. The most ing the molecular pathways underlying regeneration. Understanding prominent of the latter is glaucoma in which increased intraocular zebrafish biology is valuable for developing future therapeutic pressure is thought to lead to optic nerve damage. Glaucoma is the interventions in human retinal disease. As evidence accumulates cause of 15–20% of vision loss worldwide and thus treatment and cure that the molecular components and stem cells implicated in retinal are major goals of public health research. regeneration might be similar to those involved in regeneration of Successful therapeutic interventions aim to halt nerve degenera- other tissues, retinal regeneration research will help us build our tion and ideally, trigger regeneration of retinal ganglion cells and most understanding of the general mechanisms of regenerative processes. importantly, axon regeneration. However, mammals (including Despite our progress a number of open questions remain. humans) are unable to regenerate an injured optic nerve under Differentiated Müller cells re-enter the cell cycle after injury to normal physiological circumstances. After injury, RGCs show only produce retinal progenitors; however, little is known about the transient sprouting and no long-distance regeneration. Compiling the extrinsic signals regulating lineage specification. In the undamaged findings of a great number of studies in rodent models, we can retina, Müller glia cell-derived progenitors in the INL primarily give conclude that failure of RGC axons to regenerate can be attributed to rise to rod photoreceptor progenitors; injury seems to induce a “fate two major reasons: switch” for Müller glia cells, turning them into progenitor cells for 1) Existence of a non-permissive/inhibitory environment and other retinal cell types. How this is achieved remains largely 2) Lack of growth-promoting molecules (in the environment or unknown. intrinsic to RGCs). Another unresolved question is if all or only a subset of Müller glia cells are multipotent and can give rise to retinal progenitors. pax6,a Experimental therapeutic strategies typically target one of these two gene associated with neurogenesis capacity and multipotency, is causes, either by trying to counteract inhibitors of axon re-growth or by expressed at low levels in all differentiated Müller cells in the activating the neuron-intrinsic capacity to regenerate (for a review see zebrafish retina; this may indicate that all Müller cells are able to [65]). Studies in several species have shown that macrophage activation dedifferentiate. Additionally, Fimbel et al. showed that the majority of (e.g. by injuring the lens or using pro-inflammatory agents) can MCs (90%) express PCNA at 3 dpi in the damaged retina [43]. promote (partial) axon regeneration [66–69]. More recently, research- Alternatively, there is compelling evidence indicating that it is only ers are trying to apply combinatorial treatments. Using a gene therapy a subset of MCs switching to the progenitor fate. Kassen et al. showed approach, Fischer et al. showed that activation of the RGC's intrinsic that Stat3 is expressed only in a subset of Müller glia cells during growth state while simultaneously overcoming inhibitory signals, regeneration [56]. Similarly, only a subset of BrdU+ cells in the INL co- significantly enhances axonal regeneration [70,71]. However, full localized with the Müller cell-specific marker CAZ upon light damage regeneration has not been achieved in any rodent animal models and in the study conducted by Morris et al. [41]. Thummel et al. observed it is not clear if regenerated axons are able to correctly pathfind and two distinct groups of MCs in the gfap:EGFP transgenic zebrafish line innervate targets in a precise fashion. The development of novel after damaging the zebrafish retina with intense light. While one therapeutic strategies certainly requires a thorough understanding of group of EGFP+ MCs maintained normal Müller cell morphology and the molecular basis of optic nerve degeneration and regeneration (or did not co-label with PCNA, another set of cells showed a reduced the lack thereof). amount of EGFP expression and did co-label with PCNA. The second Clearly, alternative vertebrate animal models with an intrinsic group likely represents MCs re-entering the cell cycle and dediffer- capacity for regeneration will help us decipher how cell-intrinsic and entiating [42]. micro-environmental inhibitory and permissive signals/factors drive V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380 369

RGC axon regeneration. Interestingly, the molecular and cellular switching RGCs to a regenerative state [78] or intravitreal injections reactions to optic nerve injury observed in mammalian models of exogenous CNTF [79].Inculturedgoldfish RGCs, CNTF in seem to be inversed relative to fish in many aspects. In zebrafish combination with two molecules secreted by optic nerve glia, we generally find an abundance of growth-promoting molecules. axogenesis factor-1 (AF-1) and AF-2, drives axon regeneration [80]. Inhibitory molecules, if expressed, usually exhibit a different pattern In zebrafish, CNTF is known to play roles in photoreceptor and/or timing of expression. This might partly be due to the fact that neuroprotection and induction of Müller glia cell proliferation in the the eye continues to grow throughout the lifespan and thus the visual retina of undamaged zebrafish [60]. Its implication in ON regeneration system is primed to continuously deal with newborn RGCs extending however has not been studied thus far. their axons to the optic tectum. It seems logical that signalling cues Another class of proteins acting as RGC-intrinsic outgrowth expressed during development will still be abundant in adulthood so molecules are the reggies (or flotillins). Morpholino-mediated they can be read by newborn RGCs. It has been clearly shown that combinatorial knock-down of the three zebrafish reggie orthologs after ON crush or lesioning, RGCs start to re-express molecules that 1a, 2a and 2b results in an overall reduction in RGC axon regeneration they also express during embryonic development. These include [81,82]. Similar to the situation in zebrafish, knocking down reggies in cytoskeletal proteins, membrane-bound recognition molecules of the mouse hippocampal and N2a cells inhibits neuronal differentiation immunoglobulin family (e.g. NCAM, L1 homologs) and GAP-43 and neurite extension in vitro [81]. These findings indicate that this (growth-associated protein 43) (for a review see [72–74]). GAP-43 class of scaffolding proteins is essential for ON regeneration, most is localized to growth cones and presynaptic terminals of developing likely by acting as microdomains for the assembly of multiprotein as well as regenerating axons [75]. Kaneda et al. examined the signalling complexes recruited during regeneration. expression patterns of GAP-43 and its phosphorylated isoform (which Members of the KLF (krüppel-like factor) family are clearly is known to be active at growth cones) in the regenerating zebrafish implicated in successful ON regeneration. They had been identified optic nerve and observed a biphasic increase of phospho-GAP-43. initially by microarray analysis of changes in gene expression during Interestingly, the two distinct phases (the first one a steep increase in ON injury in zebrafish. Veldman et al. had used laser micro-dissection expression after 7–14 days, the second one a long plateau increase to isolate RGCs from uninjured and 3-day post-injury zebrafish and after 30–50 days) coincided with the recovery of two visually- identified 347 up-regulated and 29 down-regulated genes. Out of six mediated behaviors (OMR and chasing behavior) [76]. genes chosen for further investigations using morpholino-mediated In the following section we will discuss recent studies aimed at knock-down, two out of those six, KLF6a and KLF7a, were necessary achieving an understanding of the molecular basics of optic nerve for robust RGC axon re-growth [83]. The same research group recently regeneration in the zebrafish (see Table 2 for a list of genes). also identified KLF4 as a key player responsible for the loss of axon re- growth capacity in adult mice [84]. Mice with a tissue-specific loss of 2.2.1. Molecular signals/pathways implicated in optic nerve regeneration KLF4 in RGC cells were subjected to ON crush and assessed for regeneration after 2 weeks. Strikingly, knock-out mice showed an 2.2.1.1. Growth-promoting signals. Regenerating RGCs need two types increase in the number of regenerated axons, however axons did not of “positive cues” enabling them to extend their axons to their regenerate fully. The authors propose that different members of the respective target regions: 1) molecular signals within the cell, KLF family act in concert to regulate the regenerative capacity of CNS switching (or maintaining) the cell to (in) a growth state, and 2) neurons in mice. permissive and growth-promoting signals in the environment of the 2.2.1.1.2. Growth-promoting molecules secreted from a permissive optic tract and tectum, allowing axon extension, pathfinding and environment. In addition to RGC-intrinsic molecular signals, cues from correct target innervations. the ON microenvironment play a significant role in axon outgrowth. 2.2.1.1.1. Intrinsic growth-promoting factors. Whereas zebrafish Glial cells are major players in axonal regeneration, as they secrete a continue to express a variety of genes needed for RGC development variety of growth-promoting molecules. Examples are axogenesis throughout their lifetime, mice often down-regulate their expression factors 1 and 2, which have been shown to activate the expression of prenatally. growth-related molecules via a purine-sensitive pathway in the In mice, several lines of evidence show that CNTF (ciliary goldfish [80]. Goldfish and zebrafish oligodendrocytes up-regulate the neurotrophic factor) acts as an RGC-intrinsic axon growth factor and expression of members of the immunoglobulin family, such as L1, P0 changes in CNTF expression levels during ON injury underlie RGC and Contactin1a, after ON lesion [72,85,86], suggesting roles in axon apoptosis. In untreated mice, CNTF receptor is lost from RGC axons regeneration and re-myelination. after optic nerve crush and glial cells (astrocytes and oligodendro- Interestingly, not only do glial cells seem to secrete permissive or cygtes) start to express CNTF shortly after lesioning. It is thought that inhibitory factors into the optic nerve microenvironment, but so do the loss of CNTF receptor on RGCs renders them insusceptible, leading photoreceptors. Matsukawa et al. isolated purpurin as a candidate to cell death, while the appearance of CNTF in glial cells promotes gene important in ON regeneration in goldfish by screening a cDNA scarring [77]. These processes can be stopped by inducing intraocular library for genes up-regulated during regeneration. The site of inflammation, which induces retinal astrocytes to release CNTF, purpurin expression was shown to be goldfish photoreceptors [87].

Table 2 Molecular cues implicated in successful optic nerve regeneration in the zebrafish.

Gene Classification Action Reference

reggie-1a, 2a, 2b Microdomain scaffolding protein (assembly Regulate axon regeneration, axon outgrowth and neuronal differentiation [81] of multiprotein complexes) CNTF (ciliary neurotrophic factor) Growth factor Drives axon regeneration in combination with axogenesis factors 1 and 2 [60,80] KLF 6a, 7a (krüppel-like factor) Transcription factors Thought to regulate axon regeneration [83,84] axogenesis factor 1, 2 Growth factors Activate expression of growth-related molecules [80] purpurin Retinol-binding protein Induces neurite outgrowth [87,88,109] netrin-1 Axon guidance molecule Pathfinding [89] Chondroitin sulfates Sulfated glycosaminoglycan Pathfinding [100] tenascin-R Repellent axon guidance molecule Pathfinding [104,105,110] ephrin-A Repellent axon guidance molecule Establishes retinotopy during regeneration [106–108] 370 V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380

Purpurin is a retinol-binding protein, and bound to retinol, induces et al. reported the expression of Sema3Aa in the retina, optic chiasm neurite outgrowth in retinal explant cultures. This outgrowth can be and optic tectum in zebrafish. However, it does not seem to be present inhibited by adding an RALDH2 (retinaldehyde dehydrogenase) during the time window when the axons extend [102]. Becker and inhibitor (disulfiram). This has led to the speculation that the action Becker state that neither of the two zebrafish 3A orthologs are of purpurin might be mediated by retinoic acid synthesis. Nagashima detectably expressed in the lesioned optic nerve tract in the adult et al. tested this hypothesis by looking at the expression levels of zebrafish (unpublished results reported in [73]). The lack of components of the retinoic acid synthesis pathway, such as RALDH2, expression of this repellent axon guidance molecule in zebrafish CYP26a1, CRABPs (cellular retinoic acid binding protein) and RARs might explain why axons are regenerated. (retinoic acid receptor). RALDH2, CRABPs and RARS were significantly While the lack of repellent axon guidance molecules in zebrafish up-regulated after optic nerve injury in goldfish, whereas CYP26a1 might help newly generated axons to extend, the expression of (an enzyme degrading retinoic acid) was down-regulated to about repellent guidance cues in specific patterns has been shown to 50% of normal levels. These results indicate that retinoic acid be essential for establishing correct pathfinding and retinotopy. signalling might play a key role during the first stage of optic nerve TenascinR is an extracellular matrix molecule acting as a repellent regeneration in fish [88]. guidance cue during development [103]. Areas of TenascinR expression The axon guidance molecule Netrin-1 is promoting RGC axon are also maintained in the adult zebrafish, bordering newly growing regeneration in goldfish. Netrin-1 expression is maintained until axons from the retinal periphery [104], indicating that it also is adulthood and receptors are up-regulated on newborn RGC axons implicated in guiding regenerating axons to their respective brain [89]. In contrast to zebrafish, its expression is developmentally down- targets. In mice, TenascinR similarly seems to act as a repellent guidance regulated in rats [89]. molecule. Outgrowth of embryonic and adult retinal ganglion cell axons from mouse retinal explants is significantly reduced on homogeneous 2.2.1.2. Inhibitors. Although initial sprouting of neurites is observed in substrates of TenascinR [105]. Becker et al. have observed an increase in mice after optic nerve injury, RGCs die shortly thereafter through the number of cells expressing TenascinR mRNA in the lesioned optic apoptosis, which is thought to be triggered by the activation of nerve and have hypothesized that the continued expression of the astrocytes initiating wound repair and scarring. Inhibitory signals can protein after an optic nerve crush may contribute to the failure of adult be roughly subdivided into myelin, glial scar and repellent axon retinal ganglion cells to regenerate their axons in vivo. guidance molecules. Another class of repellent cues involved in establishing retinotopy Myelin proteins are major inhibitors for axon regeneration in in the tectum are the Ephrin-As, acting via Eph receptors. Develop- mammals [90]. Data on how myelin affects axonal re-growth in fish is mental gradients of Ephrin-A2 and -A5 are retained in unlesioned and somewhat unclear. Whereas the role of myelin has not been studied in lesioned adult zebrafish [106], and blocking Eph receptors in goldfish zebrafish, findings from goldfish seem to argue that goldfish myelin is has a detrimental effect on establishing retinotopy during regenera- not a strong inhibitor [91–93]. The myelin-associated Nogo protein is tion [107]. Interestingly, while EphA3 and A5 (receptors) have been a major inhibitor of axon re-growth in the optic nerve and spinal cord found to be re-expressed in a gradient in the retina of goldfish [108], of mammals [94]. Knocking out Nogo A,B or C or inhibiting the Nogo their expression is not recapitulated during optic nerve regeneration receptor in mice abolishes this inhibitory effect and allows neurite in zebrafish [106]. extension into the ON lesion [95]. Although zebrafish do possess a Nogo A ortholog, the zebrafish isoform does lack the NogoA-specific 2.2.2. Open questions N-terminal domain that inhibits regeneration in mammals [96].A Data about molecular factors influencing optic nerve regeneration recent study examined the role of the second, C-terminal, inhibitory are accumulating and provide starting points for new therapeutic domain (Nogo-66) in zebrafish axon regeneration [97]. Binding of approaches. Recent gene therapy trials conducted in mouse models Nogo-66 to its receptor NgR has no inhibitory effect on zebrafish RGC have shown that combinatorial treatments can switch RGCs to a axon growth and in fact is growth promoting. growth state and lead to some extent of axon regeneration [111–113]. Another prominent source of inhibition in mammals is the glial Despite this progress, several open questions remain: Are the newly scar formed in response to optic never injury. A hallmark of the glial regenerated RGC axons going to innervate the appropriate pretectal scar is increased immunoreactivity for chondroitin sulfates, which has and tectal targets and re-establish a correct retinotopic map? been shown to inhibit axonal regeneration [98]. In an elegant Moreover, ultimately, will regeneration of RGC axons lead to recovery experiment, Charalambous et al. grafted chick neural tube stem cells of visual function? onto a lesioned rat optic nerve, and observed the induction of MMP Strikingly, the answer seems to be “yes” in zebrafish and goldfish. (matrix metalloproteinase)-2 and -14 in astrocytes of the optic nerve. RGC axons not only re-grow, but also find their way back to their This in turn led to the degradation of matrix chondroitin sulfate original targets, leading to full recovery of visual function [72,114– proteoglycans and allowed rat axons to migrate towards the thalamus 117]. Unfortunately, not much is known about the molecular path- and mid-brain [99]. Chondroitin sulfates are also present in the ways underlying restoration of vision, as research performed to date developing and adult non-retinorecipient pretectal brain nuclei of has mainly focussed on the first steps in ON regeneration, axon re- zebrafish. However, they are not found before or after an optic nerve growth and pathfinding. Integration of regenerated axons into crush in the optic nerve and tract in adult zebrafish [100]. Quite existing circuits is a prerequisite for restoration of vision in human different to the situation in rodents, where chondroitin sulfates seem patients. Therefore, a thorough understanding of these late-step to play a direct role in inhibition of axon regeneration, Becker and processes in regeneration on a molecular level is indispensable for Becker postulate that in zebrafish, chondroitin sulfates play a role in successful future therapeutic approaches. The zebrafish model system axon regeneration by acting as repellent axon guidance molecules has great potential for uncovering the molecular pathways underlying that help axons finding their correct path to their tectal targets [100]. network integration of regenerated optic nerve fibers. As the zebrafish retina continues to grow throughout the life of the fish, newborn RGCs are constantly added to the retina and their axons 2.3. Spinal cord need to find the correct path along the optic nerve. Developmentally regulated axon guidance molecules thus might remain in the ON Abundant research efforts to facilitate spinal cord regeneration in a microenvironment and play a key role in regeneration. clinical setting have focussed largely on rodent models, providing Semaphorin3a, a major repellent axon guidance cue, is up- valuable insights into the cellular processes required for functional regulated after axotomy of the rat ON for 28 days [101]. Calander recovery. Despite intense efforts, the limited ability of mammalian V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380 371 spinal cord neurons to generate new cells and form new connections function complement to tests in mammals, where L1 has been shown remains a limiting factor. Thus the goals of basic research, to allow to be up-regulated during regeneration in CNS nerve grafts and L1 translation into the clinical setting, include the enhancement of fusion protein application can promote functional recovery of regeneration by identifying interactions that alleviate these restric- locomotion [128,129]. Similarly, GAP-43 is up-regulated in zebrafish tions or activate latent cells. Anamniotic vertebrates exhibit substan- spinal cord regeneration [125], as in mammals [130–132]. tial spinal cord regenerative capacity and can thus provide insight into On the other hand, another cell-adhesion molecule, protein zero the events that underpin successful functional regeneration. Recent (P0, a component of the myelin sheath) shows substantive difference reviews and volumes compare the utility and contributions of between anamniotes with regenerative capacity when compared to anamniotic vertebrates to spinal cord regeneration [118,119]. mammals with limited spinal cord regenerative potential [85,133]: Clinical sources of spinal cord injury typically include trauma and This and difference to mammals allow studies in zebrafish to neurodegenerative diseases. Principal issues in clinical spinal cord contribute to understanding re-myelination following regeneration regeneration include: i) protection of surviving cells from secondary [133]. Recent work has examined another cell-adhesion molecule, degeneration following injury; ii) proliferation of new cells; iii) Contactin1a, which is up-regulated in both neurons and oligoden- growth of axons through/around scar; iv) connection of axons to drocytes during spinal cord lesion [86] and is discussed above in the original targets; and v) plasticity of the CNS to restore function despite section on optic nerve regeneration. imperfect pathfinding. Spinal cord regeneration from stem cells will Although many classes of neurons of the zebrafish regenerate require multimodal treatment to address neuron-intrinsic and following spinal cord lesion, some do not. One example of cells with neuron-extrinsic limitations. limited ability to regenerate their axons is the Mauthner cell Following spinal cord injury in mammals, the number of new [118,124]; a large and easily identifiable cell type that is amenable axons generated is very limited. In sum, it appears that the plasticity of to electrophysiological recording in larval zebrafish and controls spared axons is the principal substrate for the limited functional swimming [134–137]. Because other neurons consistently regenerate recovery that is achieved [120]. Thus, studies of enhanced and via the glial scar following lesion, Bhatt et al. have suggested that the directed axonal sprouting will be valuable to implementing treat- Mauthner cell has intrinsic properties (a surface protein or a ments. Regeneration of neurons will be especially important to component of its intracellular signalling cascade) that limit its human corticospinal lesions and restoration of voluntary motor regenerative capacity [138]. It is noteworthy that if such an intrinsic control, because neuronal sprouting is limited in these areas [120]. factor exists then it is presumably limiting regeneration through An understanding of neuronal differentiation and functional integra- detection of some property in the neuron's environment. tion is of high priority and a number of ongoing clinical trials using If this is the case, identification of molecules extrinsic to the stem cell therapy seek to address these issues [121–123]. Mauthner cell environment that interact with Mauthner-specific Zebrafish, with their capacity to regenerate neurons and their sensors is critically important. Although Mauthner cell regeneration is axons and regain function following experimentally induced spinal rare, it can be bolstered by altering intracellular signalling, including cord injury, offer promise in addressing several key questions. Beyond application of cAMP [138]. cAMP is known to affect neuronal the various advantages of the model system, studying functional sprouting and regeneration in other systems although which of the regeneration of the spinal cord in zebrafish is expected to be many pathways it may be modulating remains unclear. Mauthner cell especially fruitful. Drug and gene discovery in regard to spinal cord regeneration is an attractive target from a technical point-of-view, as regeneration are very practical in zebrafish because measurements of functional regeneration (reviewed below) can be assessed via trunk functional recovery can be automated in this highly active species movement [138] related to escape behavior, with the potential for (reviewed below). Unfortunately, their small size will not permit high-throughput screening in 96-well plates [139–141]. These large analysis of axon pathfinding to navigate large injuries. In the context cells can be recorded from or stimulated readily for electrophysiology of spinal injuries observed clinically, larger rat models may be [138]. considered too small. The size of an adult zebrafish is an order of Heterogeneous glial reactions that are expected to affect regene- magnitude less than the size of a typical human spinal cord lesion. ration of spinal cord axons in mammals also occur in zebrafish [125]. However the small size of the organism is an advantage in many Macrophages/microglia increase in numbers and phagocytose myelin regards, as in most model organisms, providing economy and debris, which may or may not be beneficial to axon regeneration generation time efficiency not practicable in larger animals. In sum, [142]. In mammals, such differences have been identified to underpin it appears that zebrafish will be invaluable to discovering targets for the permissive or restrictive properties of glia towards regeneration functional regeneration of the spinal cord, serving as a very effective [143]. compliment to murine and large-animal models. Studies of the zebrafish spinal cord have demonstrated robust 2.4. Brain nuclei regenerative capacity. Detailed research differentiates cell and injury types that influence the regenerative response and delineates the Clinical regeneration of neurons in higher brain centres is a molecules that underpin such differences. Regeneration of zebrafish laudable, oft-described goal of stem cell biology. Ambitious targets of adult spinal cord neurons from most of the spinal-projecting brain stem cell treatment include neurodegenerative diseases including nuclei is observed following spinal cord lesion [124]. Various nuclei Alzheimer Disease, Parkinson, and ALS [144–147], as well as stroke display different regenerative properties, including the molecular and traumatic brain injury. Indeed, proliferating cells may not only be pathways engaged, depending on the rostral–caudal position of the expected to replace lost neurons, but may also be able to support the lesion [125,126] and identifiable patterns within this heterogeneity survival of remaining cells. may be exploited to understand molecular pathways determining the Zebrafish are attracting considerable interest in understanding intrinsic ability to regenerate. Pathways underpinning regeneration cellular and molecular pathways directing radial glia to become stem seem to be primarily those previously noted for similar functions in cells, and directing CNS stem cells to replace lost neurons. Adult mammals and almost exclusively focus upon cell-adhesion proteins. neurogenesis is substantial in the zebrafish brain [148–156] and The immunoglobulin superfamily cell-adhesion protein L1.1 contri- includes brain centres beyond the restricted forebrain proliferation butes to spinal neuron growth, synapse formation and functional typically studied in mammalian models. Compared to mammals, recovery in zebrafish [127], as demonstrated by applying a morpho- zebrafish adult neurogenesis is located in multiple centres, but is lino directed against L1.1 to the spinal cord lesion. This effective gene fundamentally similar in process; subventricular proliferative zones knock-down technology is an economical and informative loss-of- give rise to nascent neurons that migrate radially to their differentiated 372 V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380 destination. There is a notable lack of published experimental paradigms example the optic nerve and spinal cord are accessible to surgical in which zebrafish brains are damaged, thus allowing the regenerative lesion, and the retina is accessible to neurotoxin delivery via response to be addressed. Nevertheless, these approaches are certainly intraocular injection, or to toxic light lesion in albino animals. We on the horizon [157], building on the substantial research in other fish review here methods that have been informative for regeneration models [158]. Efforts to define zebrafish adult stem cell properties will studies in the past, and look ahead to transgenic paradigms that are be very valuable in studies of functional regeneration. well suited to the zebrafish model and have the potential to allow cell Stem cells of the adult zebrafish brain have recently begun to be ablation/regeneration to be studied within a vast variety of neuronal defined, establishing zebrafish as a valuable model for adult brain cell types. We end with a commentary that assessing the function of stem cell biology. In the tegmentum, her5 (orthologue of mammalian regenerated neurons needs to become the benchmark of the field. Hes) cells meet the definitions of bona fide stem cells (including self- Thus physiological and behavioral measures of regeneration ought to renewal, slow proliferation and multipotency) and H/E(Spl) factors be a substantial component of assessing regeneration, and thus in the generally have a role in maintaining a neural progenitor state. These design of CNS regeneration paradigms. populations express markers of stem cells conserved with mamma- lian neural progenitors, including BLBP, GFAP, and Sox2. Presumptive 3.1. Advantages to refining methods neurons arising also express conserved markers, including asha1, her4, ngn1, and Delta [159,160]. A variety of lesioning paradigms have been employed for the induction of neuronal cell ablation with goals of learning more about 3. Lesioning paradigms regeneration of the zebrafish CNS. Techniques range in the specificity of lesioning (Fig. 2). Whereas surgical excisions of pieces of tissues are Animal models of neuronal regeneration necessarily depend upon not cell-specific, methods such as laser or light lesioning are potent to the manner in which the neurons (and neighbouring cells) are target specific layers of the CNS. These techniques have allowed damaged. Various paradigms have been developed in an opportunistic research groups to observe the regeneration process, but a pitfall is fashion, each with different goals and suite of advantages. In some that many cell types are ablated and thus questions concerning instances, experimentally induced damage may have a central goal of specific cell fate determination cannot be readily answered trying to replicate a disease process. Although a plethora of animal [23,24,161]. Cell ablation using a laser can also be used for targeting models of disease exist, few zebrafish models of disease have been only specific neuronal cells of interest in a tissue [162,163]. The use of brought to bear on regeneration. Instead, most work have focussed on chemical toxins, such as ouabain, injected into the eye also possesses a an experimental intervention (surgery, neurotoxin application, etc., certain level for controlled cell death. The concentration of the reviewed below) that strives for reproducibility. Traditionally such injected toxin dictates the extent of penetration of the toxin into the studies have been opportunistic in that they ablate accessible cells. For retinal layers, and therefore the amount of cell death [43,164]. Novel

Fig. 2. Multiple methods of neuronal ablation to study CNS regeneration in zebrafish. The method of cell ablation influences the complexity of regeneration and thus experimental design. The specificity of the neurons targeted (ablation of only one or some neuronal cell types versus ablation of an entire section of retinal tissue) is dependent on the technique: A) light/laser lesion, B) chemical ablation, C) surgical lesion or D) NTR transgenic ablation (see also Fig. 3). V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380 373 techniques employing transgenic-chemical ablation of neurons are paradigms in other tissues (e.g. surgical intervention in heart and tail proving to be the most specific means for targeted cell ablation [165]. fins). Regeneration paradigms can be refined through the standardiza- In studies of regeneration of the vertebrate spinal cord, researchers tion of the lesioning paradigms. Studies will become more comparable have attempted to simulate human spinal cord injury in an effort to between treatments or between research groups when protocols are discover potential therapies. Many investigations have employed performed with exacting rigor, eliminating some of the variables such teleost fish including adult zebrafish, cichlids and goldfish, and similar as the unwanted death of neighbouring neurons or the activation of surgical techniques have been used to lesion their spinal cords different cell death signalling pathways (i.e. apoptosis versus [124,170,179]. The vertebral column is exposed with a longitudinal necrosis) due to distinctive lesioning methods, both of which could incision, then is transected in the region of interest [124]. A similar have an impact on the regeneration of the neurons of interest technique to study spinal cord regeneration in mammals has been [166,167].Refinement of techniques also leads to simplifying the performed using adult rats and mice [169,172], and regeneration ability to visualize cell ablation and regeneration in vivo [165]. studies of Xenopus tadpoles have performed tail amputations to allow With each investigation of regeneration, the possibility of applying for comparisons of tail regeneration with spinal cord regeneration this knowledge towards therapeutics for the treatment of human [173]. neurodegenerative diseases gets closer to becoming a reality. In an investigation by Becker et al., axonal regeneration of the Characterization of the biological mechanisms and signalling path- zebrafish spinal cord was studied. After transection of the cord, re- ways involved in this process is crucial. Thus refined strategies for growth of projections from brain nuclei can be observed. Within six neuron damage/ablation, each with its particular advantages, are a weeks post lesion, most of the brain nuclei had axons that extended worthwhile consideration. past the transaction and into the distal spinal cord, indicative of regeneration [118]. 3.2. Methods of inducing neuronal death and damage 3.2.2. Laser lesioning 3.2.1. Surgical lesioning Laser lesioning has been applied to induce ablation of neural cells Surgical lesioning of CNS tissues has been performed on the optic in various tissues and organisms [23,161,180]. Regeneration of the nerve, spinal cord, brain and retinal neurons of many organisms including retina of teleost fish has been investigated by ablating photoreceptors teleost fish, mouse, rat and Xenopus [33,53,77,124,158,166–174]. with an argon laser. The argon laser promotes accurate placement of Optic nerve crush is a surgical lesioning approach that is routinely the lesions and can be used to ablate photoreceptor cells in the outer applied in studies using rat, mouse and teleost fish. The most common nuclear layer in the retina. In this procedure, the lens of the fish is surgical technique is to expose the optic nerve by gently pulling the surgically removed in order to gain access to the retina for focussing eyeball out of the socket then crushing it, taking care not to sever the the laser. The resulting lesions appear as bleached spots on the retina ophthalmic artery [77,100,106,166,167]. Optic nerve damage can [23,161]. easily be visualized and confirmed. In zebrafish, the optic nerve A particularly advantageous feature of laser lesioning is that it can usually has a whitish opaque colour, but upon damage, a translucent be employed on a cell-specific level to allow for regeneration studies stripe across the optic nerve becomes visible where the forceps had of subgroups of neurons. Moreover, it can be directed to ablation of made contact [106]. any part of the CNS. The targeted cells are labelled with an indicator Becker and Becker examined regeneration of the optic projection such as a fluorescent dye to allow for easy visualization of neurons. In in zebrafish after optic nerve crush to study the role of genes involved their study of the zebrafish hindbrain, Liu and Fetcho labelled cells in axonal growth, pathway selection, and target recognition [106]. The through retrograde uptake of the indicator CGD by injecting it into the axonal projection is retinotopic, meaning that it is formed from/ spinal cord of 4dpf fish [163]. After intense light exposure, the dye will composed of retinal ganglion cells projecting to the optic tectum have adverse affects, and thus cause cell death. The targeted neurons [175]. After lesioning, certain tectal areas were observed to be primary were identified by position and morphology, and subsequently targets for the regenerating axons in that there was an increased injured by exposing them to the maximum intensity of the laser. probability for functional integration of regenerated tissue. Becker Immediately after light treatment, fluorescence was observably and Becker found that the pattern of expression of the recognition decreased, and by 24 h after treatment it was no longer detectable molecules ephrin-A2 and ephrin-A5b correlated to the target areas, in confocal images, suggesting cell death [163]. A similar approach suggesting that these molecules may play important roles in was used on optic tectum in zebrafish larvae, utilizing effective functional regeneration of the optic nerve [106]. measures of visual function [181]. Moreover, Kimmel et al. utilized Although a major contributor to our understanding of nerve laser irradiation on trigeminal neurons to investigate if their input to regeneration, the relevance of optic nerve crush to human disease is the hindbrain Mauthner cell affects its dendrite outgrowth [182]. questionable. A significant advantage of this paradigm, however, These laser lesioning paradigms have not yet been used for includes the ability to trace pathfinding and target accuracy of the regeneration studies, but appear to be an attractive method for this regenerated axons (reviewed above). purpose. Retinal regeneration has been investigated by surgically removing Finally, it is noteworthy that femtosecond laser pulses have been portions of the retina or the retina in its entirety [176,177]. Mensinger employed to ablate specific cells in live zebrafish [183]. Although this and Powers investigated the effects of removing parts or the entire has not yet been used to ablate neuronal cells of zebrafish, it has been retina on regeneration in the teleost models goldfish and sunfish [178]. successful in studying neuronal regeneration in Caenorhabditis elegans While excision of the whole retina completely prevented regeneration, [184]. Developing this technology, and combining it with the optical leaving only 5% of retinal tissue intact led to restoration of functional transparency and transgenesis of zebrafish have substantial potential vision, as assayed by ERGs and dorsal light response (DLR). Surgical for specific ablation and neuronal regeneration. lesioning of the retina has also been used as a means to study gene expression profile changes during retinal regeneration [53]. 3.2.3. Light lesioning Retinal damage via surgery has been useful for observing neuronal Light-induced lesioning is a popular method of cell ablation regeneration, but is less powerful for studying only a specific subset of because it targets photoreceptor cells while leaving the remainder of retinal neurons because all neurons are regenerating at once [177].It the retina relatively unharmed, with less inflammatory response is noteworthy that this type of lesion leads to formation of a blastema compared to tissue injury [185]. This system has been shown to be during regeneration, and in this aspect may be comparable to lesion very effective for studies of the teleost retina, and is the most 374 V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380 commonly used lesioning paradigm for assessing regeneration in both One recently developed method of transgenic cell-specific ablation albino and pigmented fish [186–188]. Mechanisms of cell death in is nitroreductase-mediated conditional cell ablation [165]. Transgenic light lesion have been reviewed extensively elsewhere [187,189] and zebrafish express the Escherichia coli gene nfsB encoding the likely include both generation of reactive oxygen species and nitroreductase (NTR) enzyme. NTR is driven by a cell-specific accumulation of heat leading to photoreceptor death. promoter, resulting in its expression in the targeted neuron type. Fish are initially dark adapted for a particular period of time, These neurons will function normally until the application of a followed by the application of constant light, which induces prodrug, metronidazole (MTZ). MTZ binds to NTR and is electro- photoreceptor cell death through apoptosis [24,40,185,190]. The chemically reduced, converting it into a DNA cross-linking cytotoxin extent of retinal injury to the organism is affected by the fish's [165] (Fig. 3B). The toxic form of MTZ remains isolated in the neurons history of light exposure, the period, quality and intensity of light of interest and induces base transitions, transversion mutations, and exposure, and the organism's pigmentation and genetic background finally fragmentation of DNA [205]. This results in precise cell ablation [24,187,191]. Regeneration of photoreceptors following such lesions of the subset of neurons expressing NTR, while other neighbouring is substantial (please refer to the Retina section of this review). cells appear to be unaffected by the toxin [165].Ablationis discontinued upon the removal of the prodrug, allowing for 3.2.4. Chemical ablation regeneration to occur devoid of the influence of the toxin. The Another technique used primarily for the study of teleost retinal method has recently been used to ablate specific classes of bipolar regeneration is to expose the tissue of interest to toxic chemicals. cells in the zebrafish retina [204]. Based upon cell counts, the ablated Ouabain, a metabolic poison inhibiting the Na+/K+-ATPase, has bipolar cells reappear in the retina, presumably regenerating from a specifically been employed to induce degeneration of cells in the proliferating population. Another study has utilized a similar retina [43,164,192]. Depolarization of the plasma membrane triggers approach to ablate rod photoreceptors and demonstrated a differen- a cascade of enzymatic events, leading to cell death [193]. The extent tial proliferative response based on the abundance of rods ablated of cell death is proportional to the concentration of intravitreal toxin [241]. This study underlines the potential of the system, allowing [43,164,194]. High concentrations of ouabain have been shown to specific cell classes to regenerate among the complexity of cell types rapidly cause death to all retinal nuclear layers, while the use of lower in the retina. Certain questions remain to be tested, including whether concentrations can be useful in biasing ablation towards the inner adjacent cells are damaged or killed by the method, and whether the nuclear layers while leaving the photoreceptor layer relatively cell types regenerated are more or less diverse than those that are unscathed. Fimbel et al. took advantage of this element and ablated. The specificity of the system is tantalizing, as it ought to allow demonstrated that selective damage to cells of the INL by injection a simpler system to define molecular mechanisms during regenera- of low doses of ouabain could stimulate regeneration [43]. A zebrafish tion of a single cell type rather than many cell types. Ideally, via model for whole retina regeneration has been presented by Sherpa et comparison of regeneration of multiple classes, the community will al. who showed regeneration of RGCs after cytotoxin injection [194]. learn triggers of cell-type-specific regeneration and functional Intravitreal injection of 6-hydroxydopamine (6-OHDA) has been rewiring. used to ablate retinal dopaminergic cells in both goldfish and The methods used to date in zebrafish using nitroreductase cell zebrafish [195–199]. Almost 3 decades ago Negishi et al. had shown ablation also utilize the Gal4-VP16 and UAS combinatorial transgenic that loss of dopaminergic neurons in goldfish retinas triggered system [165,204]. This allows the exciting potential for combination increased proliferation of retinal progenitors [198]. Li et al. have with Gal4 enhancer trap lines [206–208] with many neuronal and used a behavioral test (escape resonse) to study the effect of loss of glial types now available for ablation in a variety of CNS tissues. dopaminergic neurons on visual sensitivity in the zebrafish [195]. Although the toxicity and transcriptional effects of VP16 itself need to be considered, this model system has enormous potential to 3.2.5. Transgenic expression of toxic proteins efficiently ablate single or multiple cell types to compare their Ablation of particular cells has been achieved by expression of regeneration and functional integration. disease genes or other toxic proteins in subsets of zebrafish photoreceptors [200,201]. This genetic ablation of cones versus rods 3.3. Regeneration has limited utility if function is not restored has led to important insights into retinal progenitor pools in the zebrafish retina [202]. In contrast to emerging transgenic methods The regeneration of a neuronal cell is an impressive process, but reviewed below, in which the toxicity is inducible and restricted to would be of limited utility if the cell does not functionally integrate the timeline of prodrug exposure, the continued fruitful nature of into the existing tissue system upon regeneration. This aspect is often these methods will be limited somewhat by the toxic protein being overlooked in regeneration studies. Neuronal regeneration in the abundant in the regenerated cell. Arguably, the early steps of cell zebrafish can easily be observed, but the functionality of the regeneration will be unperturbed in these models, and late-stage regenerated cells is often assumed but not experimentally tested. molecular signatures and functional measures of regeneration will Defining the molecular mechanisms that specify the regenerating need to consider whether the results are influenced by the toxic cell's fate will be an ongoing focus of the community, and will proteins used for ablation. certainly contribute to clinical specification of stem cells. On the other hand, specifying cell fates alone will not be useful if the cells are not 3.2.6. Transgenic conditional cell-specific ablation directed to reconnect into the neuronal network. Here we review Genetic engineering techniques have become very practical in the regeneration paradigms that can be coupled with informative and/or zebrafish, and with the advancement of these tools, mechanisms of efficient methods to assess the regenerated function using electro- development and regeneration have become readily observable. physiology and/or behavior. Recently, investigators have created conditional cell ablation techniques by combining genetic and chemical tools to target the degeneration of 3.3.1. Electrophysiology output particular cell types (Fig. 3) [165,203,204]. By specifically directing The ERG (electroretinogram) is a routinely used and extremely ablation of a particular neuronal cell class for a limited period of time, valuable tool for studying degeneration and dysfunction of retinal rather than causing simultaneous death to many types of neurons, as neurons in the zebrafish. This technique measures light-induced had been the standard procedure thus far, alterations and disruptions in changes of electrical activity in the eye [11,209,210]. The ERG is a relevant genetic pathways can be analyzed for their potential roles in method for probing outer retinal function and can be adapted to larval CNS regeneration. zebrafish and adult zebrafish, as well as multiple other organisms. In V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380 375

this non-invasive procedure, a recording electrode is placed onto the cornea of the zebrafish, and a response is induced by flashing light at specific intensities and durations [11]. The observed response is recorded by the instrumentation and interpreted into an ERG trace (Fig. 4) that has a form very simlar to the ERG of mammals. The vertebrate ERG output is expressed as three distinct waves (Fig. 4). An initial low-amplitude negative a-wave reflecting photo- receptor activity is followed by the large positive b-wave, mainly representing second-order (ON bipolar) neuron activity. The d-wave appears at light offset and is indicative of postsynaptic activity involved in the OFF response [11,211]. The ERG has been used in goldfish to assess functional recovery of retinal neurons after cytotoxic lesioning and surgical extirpation of the adult retina [178]. Recovery of the ERG a-wave up to 90% of control levels coincided perfectly with the regeneration of photo- receptors, and more importantly, also the b-wave amplitude recovered up to 50% in amplitude, demonstrating that regenerated photoreceptors made proper synaptic connections with second-order neurons [212]. The ERG has also been used to demonstrate functional integration during regeneration of a specific cone class in rainbow trout [191]. Restoration of function has also been established beyond the retina during cone photoreceptor regeneration in trout [213]. These recordings from the optic nerve where it enters the tectum have the advantage of demonstrating connectivity to higher brain centres, but require surgery and thus preclude documenting regeneration within the same individual. Electropysiological tools can also be employed for detecting electrical activity in the spinal cord of zebrafish. Patch-clamp electrodes are placed extracellularly on the zebrafish in order to record the locomotive response pattern [134,214].

3.3.2. Behavioral output Behavioral responses are another mode to assess functional integration of regenerated neurons into existing circuits. The loss of certain behaviors upon the ablation of neurons, as well as the recovery of those behaviors once the cells have regenerated, can be assessed by a number of well-established paradigms in the zebrafish. Typically, these have focussed on innate responses of fish to presented stimuli, rather than operant conditioning, thus simplifying the methods considerably. Indeed the simplicity of many of these assays should make them accessible to most any research group.

3.3.2.1. Regeneration of motor behaviors. Regeneration after spinal cord transection in adult zebrafish has been studied employing a simple swimming performance assay [215]. 1 and 3 months after lesioning, fish were transferred into a tunnel with water flowing at slow and fast speed to test for swimming endurance as a function of fiber re-growth [215]. Other examples of robust assays include the light-induced startle response used in high-throughput screening for motor deficits [139– 141]. This assays certainly could be used to assess replacement of functional connections during hindbrain or spinal cord regeneration. Escape responses in zebrafish larvae can also be induced with a light touch or puff of water directed at the trunk. This type of assay has

Fig. 3. Regeneration of a single neuronal class can be achieved following conditional cell-specific ablation. To reduce the heterogeneity of the regenerative resonse, one can reduce the complexity of the cell types that are ablated. This has recently been achieved using neuron-specific transgenic expression of the bacterial gene nitroreductase (nfsB, produces NTR protein), which kills cells upon application of the prodrug metronidazole (MTZ) [204]. A tissue-specific promoter fused to green fluorescent protein (GFP) drives expression of NTR in bipolar neurons of the retina (A). Cells expressing NTR develop normally until MTZ prodrug application, e.g. in a bath treatment (B). Upon application, MTZ is converted to toxin only in cells expressing NTR, leading to cross-linking of DNA and cell death (C,D). Removal of MTZ permits cell-specific regeneration, including expression of the fluorescent marker (E). This method can be combined efficiently with enhancer traps, allowing regeneration to be studied in a variety of cell populations (see text). 376 V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380

disease progression [224]. It is noteworthy that such models, where neuron death is a benchmark outcome, are anticipated to exhibit substantial regenerative response. Regeneration from intrinsic stem cells during degeneration will represent both confounds and opportunities to modelling neurodegenerative disease. In particular, longitudinal studies assessing cell loss by quantifying neuron popula- tions may be difficult if neurons are replaced at a rate similar to cell death. Perhaps such regeneration underpins the lack of obvious neuronal loss, despite neuropathological change in teleost models of light-induced photoreceptor death [225] or prion infectivity [226] (and other manuscripts in this volume). Instead, emphasis may be best placed on methods that assay cell death via expression of available cell death markers. Gene discovery approaches using zebrafish disease models may also need to consider regeneration in the way experiments are designed. For example proteomic, transcriptomic or metabolomic Fig. 4. CNS regeneration studies should emphasize functional integration of the studies of disease models will need to interpret changes in molecular regenerated cells. The electroretinogram (ERG) is an example of a non-invasive abundance in light of both degeneration and regeneration. One strategy technique that can be applied to measure neuronal function, thus allowing within- to address such issues may be to focus experiments on aged fish, e.g. individual examination of the regenerative process (e.g. measures before, during, and zebrafish more than two years old, where somatic growth and adult after cell ablation). Analysis of the ERG response typically focusses on the amplitude of the b-wave (bold, blue positive deflection) in response to light stimuli. The b-wave neurogenesis have slowed and the regenerative response is expected to primarily represents bipolar cell activity, and thus regeneration of this response can be be less robust. diagnostic for appropriate integration of photoreceptors and bipolar cells following Beyond experimental design, other fundamental processes in regeneration [191]. neurodegeneration may be different in an organism that is capable of replacing lost neurons. These may include the cellular and molecular been used to assess functional regeneration of the Mauthner cell in aspects of astrocyte reactions to injury. As reviewed above, popula- larval spinal cord [138]. tions of glia dedifferentiate to a progenitor cell state following zebrafish neuronal injury. This is in marked contrast to mammalian 3.3.2.2. Visually-evoked behaviors. Visually-evoked behaviors of zebra- systems and thus the molecular and cellular responses of glia, or at fish (and goldfish) can be easily stimulated and are readily accessible least a subset thereof, will be directed to a different biological process for measurement and genetic analysis of functional vision [12,216]. than one expects in a clinical setting. The optokinetic response (OKR) is a robust assay for visual Similarly, declines in adult neurogenesis have been associated function measuring zebrafish eye movements in response to a moving with onset of dementia in AD [227]. This has led to hypotheses that stimulus (grating) [217–220]. Ablation of cone photoreceptors for decreased neurogenesis is causal for learning deficits and cognitive example, would be expected to impair colour detection in zebrafish decline. Zebrafish represent an exciting opportunity to understand larvae. Consequently they would not be able to track the movements adult neurogenesis, however their continued robust generation of of coloured objects. Cone photoreceptors' regeneration would lead to new neurons into adulthood directly opposes the disease process recovery of functional colour vision and improved performance in the observed during clinical neurodegenerative decline. It will be of OKR assay could be observed. interest to examine if such neurogenesis is similarly affected in The OKR has been used to study retinal regeneration after ouabain various zebrafish models of human neurodegeneration. We expect injection in the goldfish [221]. Cytotoxin injection resulted in lack or that it will be important to develop methods that allow adult greatly reduced OKR performance. Regeneration of retinal cell layers neurogenesis to be slowed in these zebrafish disease models. resulted in partial recovery of several visually-evoked assays (ERG, A further complication to these disease models is that the disease- DRL and OKR), however visual sensitivity did not completely return to associated molecule being deployed in transgenic models is often control levels [221]. intimately linked to proliferation. One example is amyloid precursor In the optomotor response (OMR), motion in a particular direction is protein (APP), which is processed inappropriately in Alzheimer simulated and zebrafish will swim in the direction of the perceived Disease and overexpressed in various Alzheimer Disease animal motion [222,223]. Failure of zebrafish to respond to the OMR stimulus models. APP is enriched in neurogenic zones and acts to negatively can be attributed to distinct/different underlying causes, and thus the modulate neurogenesis via its intracellular domain [228,229] and OMR can be used for studying regeneration of various CNS tissues. promotes neuritogenesis through its extracellular domains [230– Swimming arrest will occur following lesion, and functional regene- 232]. Consistent with this, mouse models of Alzheimer Disease ration should be able to (at least partially) restore the optomotor integrating APP, Tau and Presenilin have reduced adult neurogenesis response. As the OMR is dependent not only on a functional retina, but [233–237]. Similarly, the prion protein (PrPC), often overexpressed in also on the correct propagation of visual stimuli to the fish optic tectum, animal models to allow cross-species infectivity and disease model- regeneration of the optic nerve can also be tested. Kaneda et al. observed ling, is also involved in modulating neurogenesis. PrPC is expressed in that the expression increase of the growth cone marker GAP-43 after adult neural precursors of mouse and positively modulates adult optic nerve injury temporally concurred with the recovery of two neurogenesis [238]. Overall, the substantial adult neurogenesis in visually-mediated behaviors (OMR and chasing behavior) [76]. zebrafish may be of great benefit to understanding the role for proteins like APP and PrP in neurogenesis in healthy brains. 4. Regeneration as a potential confound when Degeneration models based on overexpression of disease-associated studying neurodegeneration molecules may be especially prone to adult neural regeneration confounding data interpretations of described above. This will be International efforts are focussing on developing zebrafish as especially true when the products that recapitulate disease pheno- models of neurodegenerative disease, by identifying zebrafish types also inadvertently affect a proliferative response. mutants and deploying human versions of disease-associated genes We have briefly outlined potential confounds regarding interpreta- (see other papers in this volume). These efforts parallel those in mouse tion and experimental design that are worth considering as zebrafish and rat models, which have contributed much to understanding of models of neurodegeneration become available. Nevertheless, in V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380 377 each case, these confounds can be viewed as an opportunity to examine [15] P.R. Johns, Formation of photoreceptors in larval and adult goldfish, J. Neurosci. 2 (2) (1982) 178–198. the differences across taxa that permit the adult brain to host [16] P.A. Raymond, et al., Molecular characterization of retinal stem cells and their proliferation leading to replacement of new neurons. For example, niches in adult zebrafish, BMC Dev. Biol. 6 (2006) 36. adult neurogenesis can be viewed as a therapeutic target for Alzheimer [17] D. Julian, K. Ennis, J.I. Korenbrot, Birth and fate of proliferative cells in the inner nuclear layer of the mature fish retina, J. Comp. Neurol. 394 (3) (1998) 271–282. Disease treatments [239]. It is also noteworthy that stem cell therapy of [18] D.C. Otteson, A.R. D'Costa, P.F. Hitchcock, Putative stem cells and the lineage of degenerative diseases would need to be able to navigate these same rod photoreceptors in the mature retina of the goldfish, Dev. Biol. 232 (1) (2001) obstacles as well as be able to define mechanisms to limit proliferation 62–76. from becoming detrimental [146,240]. Thus it will be of great benefitto [19] P. Hitchcock, et al., Persistent and injury-induced neurogenesis in the vertebrate retina, Prog. Retin. Eye Res. 23 (2) (2004) 183–194. define mechanisms to limit growth signals from inducing death and to [20] P.F. Hitchcock, et al., Antibodies against Pax6 immunostain amacrine and limit proliferation from becoming detrimental itself through hyperpro- ganglion cells and neuronal progenitors, but not rod precursors, in the normal fi – liferation and cancer. Finally, in the same fashion as the retina, optic and regenerating retina of the gold sh, J. Neurobiol. 29 (3) (1996) 399 413. [21] E.M. Levine, et al., Restricted expression of a new paired-class homeobox gene in nerve and spinal cord efforts reviewed above, regeneration from normal and regenerating adult goldfish retina, J. Comp. Neurol. 348 (4) (1994) intrinsic stem cells in the brain centres of zebrafish will offer 596–606. opportunity to understand cellular and molecular events requisite to [22] S.A. Sullivan, et al., A goldfish Notch-3 homologue is expressed in neurogenic regions of embryonic, adult, and regenerating brain and retina, Dev. Genet. 20 replacing neurons lost during disease progression. (3) (1997) 208–223. [23] D.M. Wu, et al., Cones regenerate from retinal stem cells sequestered in the inner fi 5. Conclusion: zebrafish is a potent model of vertebrate nuclear layer of adult gold sh retina, Invest. Ophthalmol. Vis. Sci. 42 (9) (2001) 2115–2124. CNS regeneration [24] T.S. Vihtelic, D.R. Hyde, Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina, J. Neurobiol. 44 (3) (2000) – We have reviewed the intrinsic and impressive ability of zebrafish 289 307. [25] T.S. Vihtelic, et al., Retinal regional differences in photoreceptor cell death and to regenerate CNS cells and tissues. Combined with a substantial regeneration in light-lesioned albino zebrafish, Exp. Eye Res. 82 (4) (2006) repertoire of genetic tools and robust assays for functional integration 558–575. of regenerated cells, zebrafish are uniquely positioned to allow design [26] D.D. Wang, A. Bordey, The astrocyte odyssey, Prog. Neurobiol. 86 (4) (2008) 342–367. of sophisticated experiments to test hypotheses of CNS regeneration. [27] A. Bignami, et al., Localization of the glial fibrillary acidic protein in astrocytes by These approaches can be complemented by high-throughput in vivo immunofluorescence, Brain Res. 43 (2) (1972) 429–435. screens to explore for genetic and pharmacological modifiers of [28] L.F. Eng, et al., An acidic protein isolated from fibrous astrocytes, Brain Res. 28 (2) – fi (1971) 351 354. regeneration. Transgenic techniques and availability of these sh from [29] J. Altman, Autoradiographic and histological studies of postnatal neurogenesis. stock centres are allowing refined cell ablation techniques that will IV. Cell proliferation and migration in the anterior forebrain, with special herald cell-specific investigations of regeneration at both the reference to persisting neurogenesis in the olfactory bulb, J. Comp. Neurol. 137 – molecular and functional levels. Finally, we note that several (4) (1969) 433 457. [30] F. Doetsch, et al., Subventricular zone astrocytes are neural stem cells in the adult amenable paradigms are available to assess function of the regener- mammalian brain, Cell 97 (6) (1999) 703–716. ated CNS tissue, which ought be utilized to assess the integration of [31] M.B. Luskin, Restricted proliferation and migration of postnatally generated neurons into regenerated CNS circuits. neurons derived from the forebrain subventricular zone, Neuron 11 (1) (1993) 173–189. [32] J. Altman, G.D. Das, Autoradiographic and histological evidence of postnatal Acknowledgments hippocampal neurogenesis in rats, J. Comp. Neurol. 124 (3) (1965) 319–335. [33] H.A. Cameron, et al., Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat, Neuroscience 56 (2) (1993) 337–344. This work was funded by an NSERC Discovery Grant to WTA. VCF [34] M.S. Kaplan, J.W. Hinds, Neurogenesis in the adult rat: electron microscopic was supported by an SNF Fellowship and BF by an NSERC Scholarship. analysis of light radioautographs, Science 197 (4308) (1977) 1092–1094. [35] A.J. Fischer, T.A. Reh, Muller glia are a potential source of neural regeneration in the postnatal chicken retina, Nat. Neurosci. 4 (3) (2001) 247–252. References [36] A.J. Fischer, T.A. Reh, Potential of Muller glia to become neurogenic retinal progenitor cells, Glia 43 (1) (2003) 70–76. [1] C.G. Gross, Neurogenesis in the adult brain: death of a dogma, Nat. Rev. Neurosci. [37] R. Thummel, et al., Characterization of Muller glia and neuronal progenitors 1 (1) (2000) 67–73. during adult zebrafish retinal regeneration, Exp. Eye Res. 87 (5) (2008) 433–444. [2] D.C. Lie, et al., Neurogenesis in the adult brain: new strategies for central nervous [38] M.C. Senut, A. Gulati-Leekha, D. Goldman, An element in the alpha1-tubulin system diseases, Annu. Rev. Pharmacol. Toxicol. 44 (2004) 399–421. promoter is necessary for retinal expression during optic nerve regeneration [3] G. Kempermann, F.H. Gage, New nerve cells for the adult brain, Sci. Am. 280 (5) but not after eye injury in the adult zebrafish, J. Neurosci. 24 (35) (2004) (1999) 48–53. 7663–7673. [4] G.L. Ming, H. Song, Adult neurogenesis in the mammalian central nervous [39] B.V. Fausett, D. Goldman, A role for alpha1 tubulin-expressing Muller glia in system, Annu. Rev. Neurosci. 28 (2005) 223–250. regeneration of the injured zebrafish retina, J. Neurosci. 26 (23) (2006) [5] A. Sanchez Alvarado, P.A. Tsonis, Bridging the regeneration gap: genetic insights 6303–6313. from diverse animal models, Nat. Rev. Genet. 7 (11) (2006) 873–884. [40] R.L. Bernardos, et al., Late-stage neuronal progenitors in the retina are radial [6] S.L. Johnson, J.A. Weston, Temperature-sensitive mutations that cause stage- Muller glia that function as retinal stem cells, J. Neurosci. 27 (26) (2007) specific defects in zebrafish fin regeneration, Genetics 141 (4) (1995) 7028–7040. 1583–1595. [41] A.C. Morris, T. Scholz, J.M. Fadool, Rod progenitor cells in the mature zebrafish [7] R. Dahm, R. Geisler, Learning from small fry: the zebrafish as a genetic model retina, Adv. Exp. Med. Biol. 613 (2008) 361–368. organism for aquaculture fish species, Mar. Biotechnol. (N.Y.) 8 (4) (2006) [42] R. Thummel, et al., Inhibition of Muller glial cell division blocks regeneration of 329–345. the light-damaged zebrafish retina, Dev. Neurobiol. 68 (3) (2008) 392–408. [8] S.C. Ekker, Morphants: a new systematic vertebrate functional genomics [43] S.M. Fimbel, et al., Regeneration of inner retinal neurons after intravitreal approach, Yeast 17 (4) (2000) 302–306. injection of ouabain in zebrafish, J. Neurosci. 27 (7) (2007) 1712–1724. [9] Y. Doyon, et al., Heritable targeted gene disruption in zebrafish using designed [44] M.L. Chang, et al., Reactive changes of retinal astrocytes and Muller glial cells in zinc-finger nucleases, Nat. Biotechnol. 26 (6) (2008) 702–708. kainate-induced neuroexcitotoxicity, J. Anat. 210 (1) (2007) 54–65. [10] X. Meng, et al., Targeted gene inactivation in zebrafish using engineered zinc- [45] M.A. Dyer, C.L. Cepko, Control of Muller glial cell proliferation and activation finger nucleases, Nat. Biotechnol. 26 (6) (2008) 695–701. following retinal injury, Nat. Neurosci. 3 (9) (2000) 873–880. [11] Y.V. Makhankov, O. Rinner, S.C. Neuhauss, An inexpensive device for non- [46]X.Zhao,etal.,Growthfactor-responsive progenitors in the postnatal invasive electroretinography in small aquatic vertebrates, J. Neurosci. Meth. 135 mammalian retina, Dev. Dyn. 232 (2) (2005) 349–358. (1–2) (2004) 205–210. [47] J.L. Close, et al., Epidermal growth factor receptor expression regulates [12] V.C. Fleisch, S.C. Neuhauss, Visual behavior in zebrafish, Zebrafish 3 (2) (2006) proliferation in the postnatal rat retina, Glia 54 (2) (2006) 94–104. 191–201. [48] A.V. Das, et al., Neural stem cell properties of Muller glia in the mammalian retina: [13] P.R. Johns, Growth of the adult goldfish eye. III. Source of the new retinal cells, regulation by Notch and Wnt signaling, Dev. Biol. 299 (1) (2006) 283–302. J. Comp. Neurol. 176 (3) (1977) 343–357. [49] F. Osakada, et al., Wnt signaling promotes regeneration in the retina of adult [14] D.C. Otteson, P.F. Hitchcock, Stem cells in the teleost retina: persistent mammals, J. Neurosci. 27 (15) (2007) 4210–4219. neurogenesis and injury-induced regeneration, Vision Res. 43 (8) (2003) [50] J. Wan, et al., Preferential regeneration of photoreceptor from Muller glia after 927–936. retinal degeneration in adult rat, Vision Res. 48 (2) (2008) 223–234. 378 V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380

[51] J. Wan, et al., Sonic hedgehog promotes stem-cell potential of Muller glia in the [84] D.L. Moore, et al., KLF family members regulate intrinsic axon regeneration mammalian retina, Biochem. Biophys. Res. Commun. 363 (2) (2007) 347–354. ability, Science 326 (5950) (2009) 298–301. [52] B. Bhatia, et al., Distribution of Muller stem cells within the neural retina: [85] J. Schweitzer, et al., Expression of protein zero is increased in lesioned axon evidence for the existence of a ciliary margin-like zone in the adult human eye, pathways in the central nervous system of adult zebrafish, Glia 41 (3) (2003) Exp. Eye Res. 89 (3) (2009) 373–382. 301–317. [53] D.A. Cameron, et al., Gene expression profiles of intact and regenerating [86] J. Schweitzer, et al., Contactin1a expression is associated with oligodendrocyte zebrafish retina, Mol. Vis. 11 (2005) 775–791. differentiation and axonal regeneration in the central nervous system of [54] A.A. Calinescu, et al., Cellular expression of midkine-a and midkine-b during zebrafish, Mol. Cell. Neurosci. 35 (2) (2007) 194–207. retinal development and photoreceptor regeneration in zebrafish, J. Comp. [87] T. Matsukawa, et al., Role of purpurin as a retinol-binding protein in goldfish Neurol. 514 (1) (2009) 1–10. retina during the early stage of optic nerve regeneration: its priming action on [55] S. Craig, A.-A. Calinescu, P.F. Hitchcock, Identification of the molecular signatures neurite outgrowth, J. Neurosci. 24 (38) (2004) 8346–8353. integral to regenerating photoreceptors in the retina of the zebrafish, J. Ocul. Biol. [88] M. Nagashima, et al., Involvement of retinoic acid signaling in goldfish optic 1 (2008) 73–84. nerve regeneration, Neurochem. Int. 54 (3–4) (2009) 229–236. [56] S.C. Kassen, et al., Time course analysis of gene expression during light-induced [89] B. Petrausch, et al., Lesion-induced regulation of netrin receptors and photoreceptor cell death and regeneration in albino zebrafish, Dev. Neurobiol. 67 modification of netrin-1 expression in the retina of fish and grafted rats, Mol. (8) (2007) 1009–1031. Cell. Neurosci. 16 (4) (2000) 350–364. [57] Z. Qin, L.K. Barthel, P.A. Raymond, Genetic evidence for shared mechanisms of [90] J.W. Fawcett, Overcoming inhibition in the damaged spinal cord, J. Neurotrauma epimorphic regeneration in zebrafish, Proc. Natl Acad. Sci. USA 106 (23) (2009) 23 (3–4) (2006) 371–383. 9310–9315. [91] T. Sivron, M. Schwartz, Nonpermissive nature of fish optic nerves to axonal [58] C.L. Lien, et al., Gene expression analysis of zebrafish heart regeneration, PLoS growth is due to presence of myelin-associated growth inhibitors, Exp. Neurol. Biol. 4 (8) (2006) e260. 130 (2) (1994) 411–413. [59] M. Schebesta, et al., Transcriptional profiling of caudal fin regeneration in [92] M. Wanner, et al., Reevaluation of the growth-permissive substrate properties of zebrafish, Sci. World J. 6 (Suppl 1) (2006) 38–54. goldfish optic nerve myelin and myelin proteins, J. Neurosci. 15 (11) (1995) [60] S.C. Kassen, et al., CNTF induces photoreceptor neuroprotection and Muller glial 7500–7508. cell proliferation through two different signaling pathways in the adult zebrafish [93] M. Bastmeyer, et al., Growth of regenerating goldfish axons is inhibited by rat retina, Exp. Eye Res. 88 (6) (2009) 1051–1064. oligodendrocytes and CNS myelin but not but not by goldfish optic nerve tract [61] P. Yurco, D.A. Cameron, Cellular correlates of proneural and Notch–delta gene oligodendrocytelike cells and fish CNS myelin, J. Neurosci. 11 (3) (1991) expression in the regenerating zebrafish retina, Vis. Neurosci. 24 (3) (2007) 626–640. 437–443. [94] M.E. Schwab, Nogo and axon regeneration, Curr. Opin. Neurobiol. 14 (1) (2004) [62] B.V. Fausett, J.D. Gumerson, D. Goldman, The proneural basic helix–loop–helix gene 118–124. ascl1a is required for retina regeneration, J. Neurosci. 28 (5) (2008) 1109–1117. [95] Y. Su, et al., Axonal regeneration after optic nerve crush in Nogo-A/B/C knockout [63] A. Rattner, J. Nathans, The genomic response to retinal disease and injury: mice, Mol. Vis. 14 (2008) 268–273. evidence for endothelin signaling from photoreceptors to glia, J. Neurosci. 25 [96] H. Diekmann, et al., Analysis of the reticulon gene family demonstrates the (18) (2005) 4540–4549. absence of the neurite growth inhibitor Nogo-A in fish, Mol. Biol. Evol. 22 (8) [64] U.K. Hanisch, H. Kettenmann, Microglia: active sensor and versatile effector cells (2005) 1635–1648. in the normal and pathologic brain, Nat. Neurosci. 10 (11) (2007) 1387–1394. [97] H. Abdesselem, et al., No Nogo66- and NgR-mediated inhibition of regenerating [65] L. Benowitz, Y. Yin, Rewiring the injured CNS: lessons from the optic nerve, Exp. axons in the zebrafish optic nerve, J. Neurosci. 29 (49) (2009) 15489–15498. Neurol. 209 (2) (2008) 389–398. [98] J. Silver, J.H. Miller, Regeneration beyond the glial scar, Nat. Rev. Neurosci. 5 (2) [66] B. Lorber, M. Berry, A. Logan, Lens injury stimulates adult mouse retinal ganglion (2004) 146–156. cell axon regeneration via both macrophage- and lens-derived factors, Eur. J. [99] P. Charalambous, L.A. Hurst, S. Thanos, Engrafted chicken neural tube-derived Neurosci. 21 (7) (2005) 2029–2034. stem cells support the innate propensity for axonal regeneration within the rat [67] T. Okada, et al., Intravitreal macrophage activation enables cat retinal ganglion optic nerve, Invest. Ophthalmol. Vis. Sci. 49 (8) (2008) 3513–3524. cells to regenerate injured axons into the mature optic nerve, Exp. Neurol. 196 [100] C.G. Becker, T. Becker, Repellent guidance of regenerating optic axons by (1) (2005) 153–163. chondroitin sulfate glycosaminoglycans in zebrafish, J. Neurosci. 22 (3) (2002) [68] V. Pernet, A. Di Polo, Synergistic action of brain-derived neurotrophic factor and 842–853. lens injury promotes retinal ganglion cell survival, but leads to optic nerve [101] A. Shirvan, et al., Anti-semaphorin 3A antibodies rescue retinal ganglion cells dystrophy in vivo, Brain 129 (Pt 4) (2006) 1014–1026. from cell death following optic nerve axotomy, J. Biol. Chem. 277 (51) (2002) [69] Y. Yin, et al., Macrophage-derived factors stimulate optic nerve regeneration, 49799–49807. J. Neurosci. 23 (6) (2003) 2284–2293. [102] D.C. Callander, et al., Expression of multiple class three semaphorins in the retina and [70] D. Fischer, Z. He, L.I. Benowitz, Counteracting the Nogo receptor enhances optic along the path of zebrafish retinal axons, Dev. Dyn. 236 (10) (2007) 2918–2924. nerve regeneration if retinal ganglion cells are in an active growth state, [103] C.G. Becker, et al., Tenascin-R as a repellent guidance molecule for developing J. Neurosci. 24 (7) (2004) 1646–1651. optic axons in zebrafish, J. Neurosci. 23 (15) (2003) 6232–6237. [71] D. Fischer, et al., Switching mature retinal ganglion cells to a robust growth state [104] C.G. Becker, et al., Tenascin-R as a repellent guidance molecule for newly growing in vivo: gene expression and synergy with RhoA inactivation, J. Neurosci. 24 (40) and regenerating optic axons in adult zebrafish, Mol. Cell. Neurosci. 26 (3) (2004) (2004) 8726–8740. 376–389. [72] R.R. Bernhardt, Cellular and molecular bases of axonal regeneration in the fish [105] T. Becker, et al., Tenascin-R inhibits regrowth of optic fibers in vitro and persists central nervous system, Exp. Neurol. 157 (2) (1999) 223–240. in the optic nerve of mice after injury, Glia 29 (4) (2000) 330–346. [73] C.G. Becker, T. Becker, Growth and pathfinding of regenerating axons in the optic [106] C.G. Becker, R.L. Meyer, T. Becker, Gradients of ephrin-A2 and ephrin-A5b mRNA projection of adult fish, J. Neurosci. Res. 85 (12) (2007) 2793–2799. during retinotopic regeneration of the optic projection in adult zebrafish, [74] C.G. Becker, T. Becker, Adult zebrafish as a model for successful central nervous J. Comp. Neurol. 427 (3) (2000) 469–483. system regeneration, Restor. Neurol. Neurosci. 26 (2–3) (2008) 71–80. [107] J. Rodger, et al., EphA/ephrin-A interactions during optic nerve regeneration: [75] L.I. Benowitz, A. Routtenberg, GAP-43: an intrinsic determinant of neuronal restoration of topography and regulation of ephrin-A2 expression, Mol. Cell. development and plasticity, Trends Neurosci. 20 (2) (1997) 84–91. Neurosci. 25 (1) (2004) 56–68. [76] M. Kaneda, et al., Changes of phospho-growth-associated protein 43 (phospho- [108] C.E. King, et al., Transient up-regulation of retinal EphA3 and EphA5, but not GAP43) in the zebrafish retina after optic nerve injury: a long-term observation, ephrin-A2, coincides with re-establishment of a topographic map during optic Neurosci. Res. 61 (3) (2008) 281–288. nerve regeneration in goldfish, Exp. Neurol. 183 (2) (2003) 593–599. [77] J.A. Miotke, A.J. MacLennan, R.L. Meyer, Immunohistochemical localization of [109] M. Nagashima, et al., Purpurin is a key molecule for cell differentiation during the CNTFRalpha in adult mouse retina and optic nerve following intraorbital nerve early development of zebrafish retina, Brain Res. (2009). crush: evidence for the axonal loss of a trophic factor receptor after injury, [110] P. Pesheva, et al., The F3/11 cell adhesion molecule mediates the repulsion of J. Comp. Neurol. 500 (2) (2007) 384–400. neurons by the extracellular matrix glycoprotein J1-160/180, Neuron 10 (1) [78] A. Muller, T.G. Hauk, D. Fischer, Astrocyte-derived CNTF switches mature RGCs to (1993) 69–82. a regenerative state following inflammatory stimulation, Brain 130 (Pt 12) [111] K.S. Cho, D.F. Chen, Promoting optic nerve regeneration in adult mice with (2007) 3308–3320. pharmaceutical approach, Neurochem. Res. 33 (10) (2008) 2126–2133. [79] A. Muller, et al., Exogenous CNTF stimulates axon regeneration of retinal ganglion [112] K.S. Cho, et al., Re-establishing the regenerative potential of central nervous cells partially via endogenous CNTF, Mol. Cell. Neurosci. 41 (2) (2009) 233–246. system axons in postnatal mice, J. Cell Sci. 118 (Pt 5) (2005) 863–872. [80] B. Petrausch, et al., A purine-sensitive pathway regulates multiple genes [113] A.R. Harvey, M. Hellstrom, J. Rodger, Gene therapy and transplantation in the involved in axon regeneration in goldfish retinal ganglion cells, J. Neurosci. 20 retinofugal pathway, Prog. Brain Res. 175 (2009) 151–161. (21) (2000) 8031–8041. [114] M.P. Callahan, A.F. Mensinger, Restoration of visual function following optic [81] C. Munderloh, et al., Reggies/flotillins regulate retinal axon regeneration in the nerve regeneration in bluegill (Lepomis macrochirus)×pumpkinseed (Lepomis zebrafish optic nerve and differentiation of hippocampal and N2a neurons, gibbosus) hybrid sunfish, Vis. Neurosci. 24 (3) (2007) 309–317. J. Neurosci. 29 (20) (2009) 6607–6615. [115] R.A. Meyer, M.E. Zaruba, G.M. McKhann, Flow-cytometry of isolated cells from [82] C.A. Stuermer, The reggie/flotillin connection to growth, Trends Cell Biol. 20 (1) the brain, Anal. Quant. Cytol. Histol. 2 (1) (1980) 66–74. (2009) 6–13. [116] R.L. Meyer, Eye-in-water electrophysiological mapping of goldfish with and [83] M.B. Veldman, et al., Gene expression analysis of zebrafish retinal ganglion cells without tectal lesions, Exp. Neurol. 56 (1) (1977) 23–41. during optic nerve regeneration identifies KLF6a and KLF7a as important [117] D.P. Northmore, T. Masino, Recovery of vision in fish after optic nerve crush: a regulators of axon regeneration, Dev. Biol. 312 (2) (2007) 596–612. behavioral and electrophysiological study, Exp. Neurol. 84 (1) (1984) 109–125. V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380 379

[118] C.G. Becker, T. Becker, Model Organisms in Spinal Cord Regeneration, Wiley-VCH [153] G.K.H. Zupanc, K. Hinsch, F.H. Gage, Proliferation, migration, neuronal differen- Verlag GmbH, 2007. tiation, and long-term survival of new cells in the adult zebrafish brain, J. Comp. [119] P. Chapouton, R. Jagasia, L. Bally-Cuif, Adult neurogenesis in non-mammalian Neurol. 488 (3) (2005) 290–319. vertebrates, Bioessays 29 (8) (2007) 745–757. [154] K. Hinsch, G.K.H. Zupanc, Isolation, cultivation, and differentiation of neural stem [120] A. Blesch, M.H. Tuszynski, Spinal cord injury: plasticity, regeneration and the cells from adult fish brain, J. Neurosci. Meth. 158 (1) (2006) 75–88. challenge of translational drug development, Trends Neurosci. 32 (1) (2009) [155] K. Hinsch, G.K.H. Zupanc, Generation and long-term persistence of new neurons 41–47. in the adult zebrafish brain: a quantitative analysis, Neuroscience 146 (2) (2007) [121] A.F. Cristante, et al., Stem cells in the treatment of chronic spinal cord injury: 679–696. evaluation of somatosensitive evoked potentials in 39 patients, Spinal Cord 47 (10) [156] B. Adolf, et al., Conserved and acquired features of adult neurogenesis in the (2009) 733–738. zebrafish telencephalon, Dev. Biol. 295 (1) (2006) 278–293. [122] G.W.J. Hawryluk, et al., Protection and repair of the injured spinal cord: a review [157] V. Kroehne, J. Kaslin, M. Brand, Proliferation and cell fates during regeneration of of completed, ongoing, and planned clinical trials for acute spinal cord injury, the adult zebrafish brain, Mech. Dev. 126 (2009) S291–S292. Neurosurg. Focus 25 (5) (2008). [158] G.K. Zupanc, et al., Generation, long-term persistence, and neuronal differenti- [123] J.W. Rowland, et al., Current status of acute spinal cord injury pathophysiology ation of cells with nuclear aberrations in the adult zebrafish brain, Neuroscience and emerging therapies: promise on the horizon, Neurosurg. Focus 25 (5) 159 (4) (2009) 1338–1348. (2008). [159] P. Chapouton, et al., her5 expression reveals a pool of neural stem cells in the [124] T. Becker, et al., Axonal regrowth after spinal cord transection in adult zebrafish, adult zebrafish midbrain, Development 133 (21) (2006) 4293–4303. J. Comp. Neurol. 377 (4) (1997) 577–595. [160] C. Stigloher, et al., Identification of Neural Progenitor Pools by E(Spl) Factors in [125] T. Becker, et al., Readiness of zebrafish brain neurons to regenerate a spinal axon the Embryonic and Adult Brain, 2008. correlates with differential expression of specific cell recognition molecules, [161] J.E. Braisted, T.F. Essman, P.A. Raymond, Selective regeneration of photoreceptors J. Neurosci. 18 (15) (1998) 5789–5803. in goldfish retina, Development 120 (9) (1994) 2409–2419. [126] T. Becker, et al., Differences in the regenerative response of neuronal cell [162] S.G. He, R.H. Masland, Retinal direction selectivity after targeted laser ablation of populations and indications for plasticity in intraspinal neurons after spinal cord starburst amacrine cells, Nature 389 (6649) (1997) 378–382. transection in adult zebrafish, Mol. Cell. Neurosci. 30 (2) (2005) 265–278. [163] K.S. Liu, J.R. Fetcho, Laser ablations reveal functional relationships of segmental [127] C.G. Becker, et al., L1.1 is involved in spinal cord regeneration in adult zebrafish, hindbrain neurons in zebrafish, Neuron 23 (2) (1999) 325–335. J. Neurosci. 24 (36) (2004) 7837–7842. [164] W. Maier, H. Wolburg, Regeneration of the goldfish retina after exposure to [128] J. Haspel, M. Grumet, The L1CAM extracellular region: a multi-domain protein with different doses of ouabain, Cell Tissue Res. 202 (1) (1979) 99–118. modular and cooperative binding modes, Front. Biosci. 8 (2003) S1210–S1225. [165] S. Curado, D.Y.R. Stainier, R.M. Anderson, Nitroreductase-mediated cell/tissue [129] C. Roonprapunt, et al., Soluble cell adhesion molecule L1-fc promotes locomotor ablation in zebrafish: a spatially and temporally controlled ablation method with recovery in rats after spinal cord injury, J. Neurotrauma 20 (9) (2003) 871–882. applications in developmental and regeneration studies, Nat. Protoc. 3 (6) [130] C.M. Booth, M.C. Brown, Expression of GAP-43 mRNA in mouse spinal cord (2008) 948–954. following unilateral peripheral nerve damage: is there a contralateral effect? Eur. [166] N.C. Gellrich, et al., Quantification of histological changes after calibrated J. Neurosci. 5 (12) (1993) 1663–1676. crush of the intraorbital optic nerve in rats, Br. J. Ophthalmol. 86 (2) (2002) [131] H. Linda, et al., Expression of GAP-43 mRNA in the adult mammalian spinal cord 233–237. under normal conditions and after different types of lesions, with special [167] L. Sarikcioglu, N. Demir, A. Demirtop, A standardized method to create optic reference to motoneurons, Exp. Brain Res. 91 (2) (1992) 284–295. nerve crush: Yasargil aneurysm clip, Exp. Eye Res. 84 (2) (2007) 373–377. [132] A.B. Schmitt, et al., GAP-43 (B-50) and C-Jun are up-regulated in axotomized [168] G. Alonso, A. Privat, Neuropeptide Y-producing neurons of the arcuate nucleus neurons of Clarke's nucleus after spinal cord injury in the adult rat, Neurobiol. regenerate axons after surgical deafferentation of the mediobasal hypothalamus, Dis. 6 (2) (1999) 122–130. J. Neurosci. Res. 34 (5) (1993) 510–522. [133] G. Jeserich, K. Klempahn, M. Pfeiffer, Features and functions of oligodendrocytes [169] E.J. Bradbury, et al., Chondroitinase ABC promotes functional recovery after and myelin proteins of lower vertebrate species, J. Mol. Neurosci. 35 (1) (2008) spinal cord injury, Nature 416 (6881) (2002) 636–640. 117–126. [170] S.J. Zottoli, M.M. Freemer, Recovery of C-starts, equilibrium and targeted feeding [134] P. Drapeau, et al., In vivo recording from identifiable neurons of the locomotor after whole spinal cord crush in the adult goldfish Carassius auratus, J. Exp. Biol. network in the developing zebrafish, J. Neurosci. Meth. 88 (1) (1999) 1–13. 206 (17) (2003) 3015–3029. [135] J.I.P. Prugh, C.B. Kimmel, W.K. Metcalfe, Non-invasive recording of the Mauthner [171] M. Schwartz, Optic nerve crush: protection and regeneration, Brain Res. Bull. 62 neuron action potential in larval zebrafish, J. Exp. Biol. 101 (DEC 1982) 83–92. (6) (2004) 467–471. [136] M. Chong, P. Drapeau, Interaction between hindbrain and spinal networks [172] M. Bilgen, Magnetic resonance microscopy of spinal cord injury in mouse using a during the development of locomotion in zebrafish, Dev. Neurobiol. 67 (7) miniaturized implantable RF coil, J. Neurosci. Meth. 159 (1) (2007) 93–97. (2007) 933–947. [173] J.M.W. Slack, G. Lin, Y. Chen, The Xenopus tadpole: a new model for regeneration [137] J.R. Fetcho, The utility of zebrafish for studies of the comparative biology of research, Cell. Mol. Life Sci. 65 (1) (2008) 54–63. motor systems, J. Exp. Zool. B Mol. Dev. Evol. 308B (5) (2007) 550–562. [174] G.K. Zupanc, Adult neurogenesis and neuronal regeneration in the brain of [138] D.H. Bhatt, et al., Cyclic AMP-induced repair of zebrafish spinal circuits, Science teleost fish, J. Physiol. Paris 102 (4–6) (2008) 357–373. 305 (5681) (2004) 254–258. [175] R.L. Meyer, Mapping the normal and regenerating retinotectal projection of [139] D. Kokel, R.T. Peterson, Chemobehavioural phenomics and behaviour-based goldfish with autoradiographic methods, J. Comp. Neurol. 189 (2) (1980) psychiatric drug discovery in the zebrafish, Brief. Funct. Genomic. Proteomic. 7 273–289. (6) (2008) 483–490. [176] D.A. Cameron, S.S. Easter, Cone photoreceptor regeneration in adult fish retina — [140] L.K. Mathew, et al., Unraveling tissue regeneration pathways using chemical phenotypic determination and mosaic pattern formation, J. Neurosci. 15 (3) genetics, J. Biol. Chem. 282 (48) (2007) 35202–35210. (1995) 2255–2271. [141] R.T. Peterson, et al., Use of non-mammalian alternative models for neurotox- [177] P.F. Hitchcock, et al., Local regeneration in the retina of the goldfish, J. Neurobiol. icological study, Neurotoxicology 29 (3) (2008) 546–555. 23 (2) (1992) 187–203. [142] T. Becker, C.G. Becker, Regenerating descending axons preferentially reroute to [178] A.F. Mensinger, M.K. Powers, Visual function in regenerating teleost retina the gray matter in the presence of a general macrophage/microglial reaction following surgical lesioning, Vis. Neurosci. 24 (3) (2007) 299–307. caudal to a spinal transection in adult zebrafish, J. Comp. Neurol. 433 (1) (2001) [179] G. Fridberg, et al., Regeneration of caudal neurosecretory system in cichlid 131–147. teleost Tilapia mossambica, J. Exp. Zool. 162 (3) (1966) 311–366. [143] J. Frisen, et al., Adhesive/repulsive properties in the injured spinal cord: relation [180] D.S. Hughes, R.R. Schade, M. Ontell, Ablation of the fetal mouse spinal cord — the to myelin phagocytosis by invading macrophages, Exp. Neurol. 129 (2) (1994) effect on soleus muscle, Dev. Dyn. 193 (2) (1992) 164–174. 183–193. [181] T. Roeser, H. Baier, Visuomotor behaviors in larval zebrafish after GFP-guided [144] S.U. Kim, J. de Vellis, Stem cell-based cell therapy in neurological diseases: a laser ablation of the optic tectum, J. Neurosci. 23 (9) (2003) 3726–3734. review, J. Neurosci. Res. 87 (10) (2009) 2183–2200. [182] C.B. Kimmel, K. Hatta, W.K. Metcalfe, Early axonal contacts during development [145] K. Sugaya, et al., Practical issues in stem cell therapy for Alzheimer's disease, of an identified dendrite in the brain of the zebrafish, Neuron 4 (4) (1990) Curr. Alzheimer Res. 4 (4) (2007) 370–377. 535–545. [146] P. Taupin, Adult neurogenesis, neural stem cells and Alzheimer's Disease: [183] V. Kohli, A.Y. Elezzabi, Laser surgery of zebrafish (Danio rerio) embryos using developments, limitations, problems and promises, Curr. Alzheimer Res. 6 (6) femtosecond laser pulses: optimal parameters for exogenous material delivery, (2009) 461–470. and the laser's effect on short- and long-term development, BMC Biotechnol. [147] M. Valenzuela, et al., Neural stem cell therapy for neuropsychiatric disorders, 8 (2008) 7. Acta Neuropsychiatr. 19 (1) (2007) 11–26. [184] M.F. Yanik, et al., Neurosurgery: functional regeneration after laser axotomy, [148] H. Grandel, et al., Neural stem cells and neurogenesis in the adult zebrafish brain: Nature 432 (7019) (2004) 822. origin, proliferation dynamics, migration and cell fate, Dev. Biol. 295 (1) (2006) [185] S. Shahinfar, D.P. Edward, M.O.M. Tso, A pathological study of photoreceptor cell 263–277. death in retinal photic injury, Curr. Eye Res. 10 (1) (1991) 47–59. [149] J. Kaslin, et al., Stem cells in the adult zebrafish cerebellum: initiation and [186] L.R. Marotte, J. Wyedvorak, R.F. Mark, Retinotectal reorganization in goldfish. maintenance of a novel stem cell niche, J. Neurosci. 29 (19) (2009) 6142–6153. 2. Effects of partial tectal ablation and constant light on the retina, Neuroscience [150] M.F. Wulliman, Neuroanatomy of Zebrafish Brain, Birkhauser Boston, 1996. 4 (6) (1979) 803–810. [151] G.K.H. Zupanc, Neurogenesis and neuronal regeneration in the adult fish brain, J. [187] D.T. Organisciak, B.S. Winkler, Retinal light damage — practical and theoretical Comp. Physiol. Neuroethol. Sens. Neural Behav. Physiol. 192 (6) (2006) 649–670. considerations, Prog. Retin. Eye Res. 13 (1) (1994) 1–29. [152] G.K.H. Zupanc, Towards brain repair: insights from teleost fish, Semin. Cell Dev. [188] J.S. Penn, Effects of continous light on the retina of a fish, notemigonus- Biol. 20 (6) (2009) 683–690. crysoleucas, J. Comp. Neurol. 238 (1) (1985) 121–127. 380 V.C. Fleisch et al. / Biochimica et Biophysica Acta 1812 (2011) 364–380

[189] A. Wenzel, et al., Molecular mechanisms of light-induced photoreceptor [216] S.C.F. Neuhauss, Behavioral genetic approaches to visual system development apoptosis and neuroprotection for retinal degeneration, Prog. Retin. Eye Res. and function in zebrafish, J. Neurobiol. 54 (1) (2003) 148–160. 24 (2) (2005) 275–306. [217] S.E. Brockerhoff, Measuring the optokinetic response of zebrafish larvae, Nat. [190] A.S. Abler, et al., Photic injury triggers apoptosis of photoreceptor cells, Res. Protoc. 1 (5) (2006) 2448–2451. Commun. Mol. Pathol. Pharmacol. 92 (2) (1996) 177–189. [218] S.S. Easter, G.N. Nicola, The development of eye movements in the zebrafish [191] W.T. Allison, et al., Degeneration and regeneration of ultraviolet cone photo- (Danio rerio), Dev. Psychobiol. 31 (4) (1997) 267–276. receptors during development in rainbow trout, J. Comp. Neurol. 499 (5) (2006) [219] O. Rinner, J.M. Rick, S.C.F. Neuhauss, Contrast sensitivity, spatial and temporal 702–715. tuning of the larval zebrafish optokinetic response, Invest. Ophthalmol. Vis. Sci. [192] P.A. Raymond, M.J. Reifler, P.K. Rivlin, Regeneration of goldfish retina: rod 46 (1) (2005) 137–142. precursors are a likely source of regenerated cells, J. Neurobiol. 19 (5) (1988) [220] Y.Y. Huang, S.C. Neuhauss, The optokinetic response in zebrafish and its 431–463. applications, Front. Biosci. 13 (2008) 1899–1916. [193] R.C. Valente, et al., Mechanisms of ouabain toxicity, FASEB J. 17 (10) (2003) [221] A.E. Lindsey, M.K. Powers, Visual behavior of adult goldfish with regenerating 1700–1702. retina, Vis. Neurosci. 24 (3) (2007) 247–255. [194] T. Sherpa, et al., Ganglion cell regeneration following whole-retina destruction in [222] S.C.F. Neuhauss, et al., Genetic disorders of vision revealed by a behavioral screen zebrafish, Dev. Neurobiol. 68 (2) (2008) 166–181. of 400 essential loci in zebrafish, J. Neurosci. 19 (19) (1999) 8603–8615. [195] L. Li, J.E. Dowling, Effects of dopamine depletion on visual sensitivity of zebrafish, [223] M.B. Orger, H. Baier, Channeling of red and green cone inputs to the zebrafish J. Neurosci. 20 (5) (2000) 1893–1903. optomotor response, Vis. Neurosci. 22 (3) (2005) 275–281. [196] Z.S. Lin, S. Yazulla, Depletion of retinal dopamine does not affect the ERG b-wave [224] A.L. Phinney, et al., Mouse models of Alzheimer's disease: the long and increment threshold function in goldfish in vivo, Vis. Neurosci. 11 (4) (1994) filamentous road, Neurol. Res. 25 (6) (2003) 590–600. 695–702. [225] W.T. Allison, et al., Photic history modifies susceptibility to retinal damage in [197] Z.S. Lin, S. Yazulla, Depletion of retinal dopamine increases brightness perception albino trout, Vis. Neurosci. 23 (1) (2006) 25–34. in goldfish, Vis. Neurosci. 11 (4) (1994) 683–693. [226] E. Salta, et al., Evaluation of the possible transmission of BSE and scrapie to [198] K. Negishi, T. Teranishi, S. Kato, New dopaminergic and indoleamine-accumu- gilthead sea bream (Sparus aurata), PLoS ONE 4 (7) (2009) e6175. lating cells in the growth zone of goldfish retinas after neurotoxic destruction, [227] R.J.E. Armstrong, R.A. Barker, Neurodegeneration: a failure of neuroregenera- Science 216 (4547) (1982) 747–749. tion? Lancet 358 (9288) (2001) 1174–1176. [199] K. Negishi, T. Teranishi, S. Kato, Growth zone of the juvenile goldfish retina [228] Q.H. Ma, et al., A TAG1-APP signalling pathway through Fe65 negatively revealed by fluorescent flat mounts, J. Neurosci. Res. 7 (3) (1982) 321–330. modulates neurogenesis, Nat. Cell Biol. 10 (3) (2008) 283–294. [200] A.C. Morris, et al., Cone survival despite rod degeneration in XOPS-mCFP [229] P. Porayette, et al., Differential processing of amyloid-beta precursor protein transgenic zebrafish, Invest. Ophthalmol. Vis. Sci. 46 (12) (2005) 4762–4771. directs human embryonic stem cell proliferation and differentiation into [201] G. Stearns, et al., A mutation in the cone-specific pde6 gene causes rapid cone neuronal precursor cells, J. Biol. Chem. 284 (35) (2009) 23806–23817. photoreceptor degeneration in zebrafish, J. Neurosci. 27 (50) (2007) 13866–13874. [230] Y. Chen, C. Dong, A beta 40 promotes neuronal cell fate in neural progenitor cells, [202] A.C. Morris, et al., Genetic dissection reveals two separate pathways for rod and Cell Death Differ. 16 (3) (2009) 386–394. cone regeneration in the teleost retina, Dev. Neurobiol. 68 (5) (2008) 605–619. [231] M. Gralle, S.T. Ferreira, Structure and functions of the human amyloid precursor [203] J.M. Davison, et al., Transactivation from Gal4-VP16 transgenic insertions for tissue- protein: the whole is more than the sum of its parts, Prog. Neurobiol. 82 (1) specific cell labeling and ablation in zebrafish, Dev. Biol. 304 (2) (2007) 811–824. (2007) 11–32. [204] X.F. Zhao, S. Ellingsen, A. Fjose, Labelling and targeted ablation of specific bipolar [232] E.A. Milward, et al., The amyloid protein precursor of Alzheimer disease is a cell types in the zebrafish retina, BMC Neurosci. 10 (2009). mediator of the effects of nerve growth factor on neurite outgrowth, Neuron 9 (1) [205] H. Pisharath, Validation of nitroreductase, a prodrug-activating enzyme, mediated (1992) 129–137. celldeathinembryoniczebrafish (Danio rerio), Comp. Med. 57 (3) (2007) 241–246. [233] S.H. Choi, et al., Non-cell-autonomous effects of presenilin 1 variants on [206] K. Asakawa, et al., Genetic dissection of neural circuits by Tol2 transposon- enrichment-mediated hippocampal progenitor cell proliferation and differenti- mediated Gal4 gene and enhancer trapping in zebrafish, Proc. Natl Acad. Sci. USA ation, Neuron 59 (4) (2008) 568–580. 105 (4) (2008) 1255–1260. [234] F.V. Ermini, et al., Neurogenesis and alterations of neural stem cells in mouse [207] E.K. Scott, H. Baier, The cellular architecture of the larval zebrafish tectum, as models of cerebral amyloidosis, Am. J. Pathol. 172 (6) (2008) 1520–1528. revealed by gal4 enhancer trap lines, Front. Neural Circuits 3 (2009) 13. [235] J.J. Rodriguez, V.C. Jones, A. Verkhratsky, Impaired cell proliferation in the [208] E.K. Scott, et al., Targeting neural circuitry in zebrafish using GAL4 enhancer subventricular zone in an Alzheimer's disease model, NeuroReport 20 (10) trapping, Nat. Methods 4 (4) (2007) 323–326. (2009) 907–912. [209] S. Saszik, J. Bilotta, C.M. Givin, ERG assessment of zebrafish retinal development, [236] P.H. Wen, et al., Overexpression of wild type but not an FAD mutant presenilin-1 Vis. Neurosci. 16 (5) (1999) 881–888. promotes neurogenesis in the hippocampus of adult mice, Neurobiol. Dis. 10 (1) [210] M.W. Seeliger, et al., Genetic separation of rod and cone-driven activity in RPE65 (2002) 8–19. knockout mice identifies the rods as the source of vision in a form of lebers [237] C. Zhang, et al., Long-lasting impairment in hippocampal neurogenesis congenital amaurosis, Invest. Ophthalmol. Vis. Sci. 43 (2002) 3682. associated with amyloid deposition in a knock-in mouse model of familial [211] J.E. Dowling, Functional and pharmacological organization of the retina, Arch. Alzheimer's disease, Exp. Neurol. 204 (1) (2007) 77–87. Neurol. 44 (11) (1987) 1208-1208. [238] A.D. Steele, et al., Prion protein (PrPc) positively regulates neural precursor [212] A.F. Mensinger, M.K. Powers, Visual function in regenerating teleost retina proliferation during developmental and adult mammalian neurogenesis, Proc. following cytotoxic lesioning, Vis. Neurosci. 16 (2) (1999) 241–251. Natl Acad. Sci. USA 103 (9) (2006) 3416–3421. [213] H.I. Browman, C.W. Hawryshyn, The developmental trajectory of ultraviolet [239] A. Abdipranoto, et al., The role of neurogenesis in neurodegenerative diseases photosensitivity in rainbow trout is altered by thyroxine, Vision Res. 34 (11) and its implications for therapeutic development, CNS Neurol. Disord. Drug (1994) 1397–1406. Targets 7 (2) (2008) 187–210. [214] J.P. Gabriel, et al., Locomotor pattern in the adult zebrafish spinal cord in vitro, [240] B. Waldau, A.K. Shetty, Behavior of neural stem cells in the Alzheimer brain, Cell. J. Neurophysiol. 99 (1) (2008) 37–48. Mol. Life Sci. 65 (15) (2008) 2372–2384. [215] W. van Raamsdonk, et al., Long term effects of spinal cord transection in [241] J.E. Montgomery, et al., A novel model of retinal ablation demonstrates that the zebrafish: swimming performances, and metabolic properties of the neuromus- extent of rod cell death regulates the origin of the regenerated zebrafish rod cular system, Acta Histochem. 100 (2) (1998) 117–131. photoreceptors, J. Comp. Neurol. 518 (6) (2010) 800–814. Biochimica et Biophysica Acta 1812 (2011) 381–389

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Biochimica et Biophysica Acta

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Review Behavioural assessments of neurotoxic effects and neurodegeneration in zebrafish☆

Keith B. Tierney ⁎

Department of Biological Sciences, University of Alberta, Z 718 Biological Sciences Building, Edmonton, Alberta, Canada T6G 2E9 article info abstract

Article history: Altered neurological function will generally be behaviourally apparent. Many of the behavioural models Received 2 December 2009 pioneered in mammalian models are portable to zebrafish. Tests are available to capture alterations in basic Received in revised form 27 September 2010 motor function, changes associated with exteroceptive and interoceptive sensory cues, and alterations in Accepted 21 October 2010 learning and memory performance. Excepting some endpoints involving learning, behavioural tests can be Available online 28 October 2010 carried out at 4 days post fertilization. Given larvae can be reared quickly and in large numbers, and that software solutions are readily available from multiple vendors to automatically test behavioural responses in Keywords: fi Zebrafish 96 larvae simultaneously, zebra sh are a potent and rapid model for screening neurological impairments. Behaviour Coupling current and emerging behavioural endpoints with molecular techniques will permit and accelerate Activity the determination of the mechanisms behind neurotoxicity and degeneration, as well as provide numerous Sensorimotor means to test remedial drugs and other therapies. The emphasis of this review is to highlight unexplored/ Learning endpoints underutilized behavioural assays for future studies. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases. © 2010 Elsevier B.V. All rights reserved.

1. Introduction through injury and developmental abnormalities, can cause altered behaviours. Malformed limbs or other morphologically-based condi- 1.1. The importance of behavioural endpoints tions may be associated with behaviours not apparent in normal individuals. There are also a suite of developmental issues that are In animals, and arguably all life throughout the biological related to neurological function, however, they are not related to kingdoms, behaviours are ways to integrate into the biotic and abiotic neurotoxins or neurodegeneration. Cerebral palsy, for instance, can environment. Behaviours are the actions and reactions taken to affect gross and/or fine motor control and have very apparent internal and external cues that are intended to place organisms in a behavioural abnormalities [1]. Additionally, in healthy animals, preferable position with respect to fitness. Conditions and situations internal, physiological processes can also modify neuron function that cause deviant or impaired behaviours therefore have clear and these can result in behaviour phenotypes. For the purposes of this survival implications. The inclusion of a behavioural assessment endpoint review, only behavioural alterations arising though neurological in studies of disease and neurotoxin exposure, especially those involving pathways and directly involving neurotoxin exposure or progressive non-lethal impairments, will help address whether sub-organismal neurological pathologies will be given consideration. changes will affect survival. However, there is another reason to include Behavioural alterations arising from neurotoxin exposure and behavioural endpoints—they can be used to rapidly and effectively detect neurodegeneration come about through very different means, even changes of molecular to system level origins. though the behavioural “phenotype” (observable manifestation) may In vertebrates, all behaviours are achieved through nervous appear similar in some instances. Neurotoxic agents affect the control. However, not all behavioural modifications are of neurolog- functionality of “normal” neurons and may be reversible and of ical origin. Non-neurological alterations can also affect behaviour— limited duration. In contrast, neurodegeneration collectively refers to some musculoskeletal issues, for example, such as those arising a group of typically irreversible processes that work at the genetic to system level, all of which result in the loss of neurons and/or their functionality. Mechanistically, neurotoxic agents act in one of two Abbreviations: AChE, acetylcholinesterase; Anti-AChE, anticholinesterase; CPP, fi conditioned place preference; CS, conditioned stimulus; dpf, days post fertilization; ways: in general, non-speci c ways (e.g. polar or non-polar narcosis OKR, optokinetic response; OMR, optomotor response; OP, organophosphorus agent; [2]); or through affecting genetic and/or protein receptors. Either OSN, olfactory sensory neuron; PEA, phenylethyl alcohol; PTZ, pentylenetetrazole; UCS, mode of action can cause changes at molecular, cellular, system and unconditioned stimulus levels beyond. It must be noted that the first mode of action could ☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological include disruption of cells in addition to neurons. With neurodegen- Diseases. ⁎ Tel.: +1 780 492 5339. eration, mechanisms of action include protein misfolding and E-mail address: [email protected]. conformational disorders (proteopathies) and/or astrogliosis, i.e. an

0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2010.10.011 382 K.B. Tierney / Biochimica et Biophysica Acta 1812 (2011) 381–389 increase in astrocytes due to neuron death. For each mechanism of neuron (or neural tissue) impairment, it should be possible to isolate an individual behavioural phenotype for screening purposes. All behaviours involve motion, and so given the appropriate methods, all are quantifiable. The evolution of software solutions in the last decade or so, including solutions from EthoVision (www.noldus.com), LocoScan (www.cleversysinc.com), Loligo (www.loligosystems.com), and Video- Track (www.viewpoint.fr) has greatly facilitated the accurate assessment of subtle and directed movements. However, unlike the movement of mammalian models, fish move in three dimensions. In studies of fish motion, movement in the vertical plane is typically ignored or minimized (i.e. by keeping the tank water shallow) [3–5], however, in at least one case, a proprietary protocol was developed to include it [6]. Nevertheless, with a model such as a zebrafish, the best solution may be to minimize Fig. 1. Behavioural responses can be evoked by internal and external signals and/or be water depth, to perhaps twice body length. This depth does not appear to altered by impaired neurological condition. With zebrafish, assessment methodologies result in overt stress and most fish settle down (acclimate) within 30 min exist to test neurological performance in absence of signals through to the ability of the (personal observation). Current technology permits the rapid, and brain to retain and integrate sensory signals of diverse origins, which makes zebrafish a potentially repeated, non-lethal testing of numerous fish (e.g. 4+ adults powerful model for testing compounds that affect neurons over brief to life-spanning timeframes. CPP=conditioned place preference. to 96 larvae) [7,8]. Additionally, a method has been constructed to calculate very subtle differences in the swimming mechanics of individual fish [4]. Caveats exist in behavioural assessments. Organisms are designed ability, and/or impaired olfactory sensory neuron (OSN) function, and/ with systems that provide resilience and can act to retain higher level or cognitive ability. responses [9]. Also, factors that affect responses at lower organisational Implicit in neurodegeneration and neurotoxin exposure is that levels, e.g. protein level, may not necessarily have an obvious behavioural organism condition, and so likely behavioural responses, will change phenotype. Behavioural studies clearly need a mechanistically-based with time. With neurotoxins, there are four temporal phases to rationale for including a motion-based endpoint. describe their effects: direct, secondary, tertiary and quaternary effects. The first two phases deal with the presence and actions of an 1.2. The relevance of zebrafish models agent within the organism, and the latter two, without. Direct effects are those local or systemic effects that occur over the short run and Ten years ago, zebrafish were identified as an up and coming model would typically be the “intended” effects, such as the actions of an for genetic disorders and developmental biology in humans [10].Since anticholinesterase (anti-AChE) drug. Secondary effects consist of this time, the zebrafish genome has been fully sequenced and many adaptations made over longer periods to restore equilibrium from genes of high mammalian homology have been identified. Five years drug actions, such as upregulation of acetylcholinesterase (AChE) ago, zebrafish were proposed as an untapped behaviour-genetic model expression. Tertiary effects are those that follow from the discontin- organism [11]. There are now high-throughput behavioural zebrafish- uation of administration of an agent that has become physically based screens able to detect specific neurological alterations/patholo- depended, i.e. involve withdrawal and stress. Over extended periods, gies, some of which take advantage of protocols similar in function and tertiary effects may give way to quaternary effects, such as purpose to those used on rodents (e.g. olfactory-based endpoints). malnutrition. The majority of behavioural assays focus on direct and Furthermore, zebrafish are more amenable to manipulation and secondary effects, however, there are studies of tertiary effects, e.g. behavioural testing during early development. Their transparent larvae withdrawal from cocaine [16]. facilitate localization of proteins and the use of morpholino manipula- In the following, a diverse array of endpoints using apparatus from tion. For these reasons, and given that rodents are more expensive and simple, one chambered tests, to complex multi-chamber, decision- require greater growth periods than zebrafish, the adoption of zebrafish based challenges are discussed (Table 1). The majority of the models continues in typically rodent dominated fields. This review is behavioural endpoints can be conducted on zebrafish 3–4 days post intended to communicate the current use of behavioural endpoints in fertilization [17,18], excepting learning/memory-based endpoints. zebrafish, and how protocols used with other animals may be adapted. Numerous behavioural assessment avenues remain unexplored. 2.1. Basic motor response endpoints

2. Types of behavioural endpoints Compounds that modulate neuron firing rate have potential to affect locomotory activity. In fishes, locomotory activity endpoints As with most animals, fishes exhibit intricate behaviours that include swimming speed [19], distance covered [14], and turning rate depend on locomotion and may be the result of complex decisions (angular velocity) [20]. Changes in zebrafish activity have been noted [12]. Behavioural responses must be viewed in a three-part hierarchical in studies investigating the mechanisms of addiction to ampheta- manner: (1) basic motor responses, which underlie (2) sensorimotor mines [21], cocaine [16], ethanol [11,22], and nicotine [23], as well as responses, that facilitate and/or integrate with (3) learning and memory the effects of pesticides [24,25]. (Fig. 1). Although the first “behavioural level,” arguably does not involve Whether any given drug/neurotoxin increases or decreases true behaviour since it does not necessarily include sensory responses, activity is related to concentration/dose and time. For example, many studies have included locomotory (activity) changes as a exposure to a low concentrations of D-amphetamine caused hyper- behavioural endpoint [13–15]. While this endpoint may appear activity while higher concentrations caused hypoactivity [21]. simplistic, it does not mean it is not meaningful, in fact, quite the Cholinesterase inhibiting drugs/pesticides are well known to have opposite: all behaviours are predicated on the ability to move. similar effects [26–29]. Clearly, agonizing stimulatory receptors or Furthermore, in tests of higher-level behaviours, such as those involving inhibiting neurotransmitter degradation will potentially lead to learning, it may be difficult to rule out potential neurological issues of activity increases in the short run that are not sustainable in the the lower founding “levels.” For example, if feeding response decreased long. In fact, persistent stimulation may result in excitotoxicity and in fish exposed to a dissolved neurotoxin such as an organophosphorus neuron apoptosis. Behavioural manifestations of neurotoxin exposure (OP) insecticide, the effect could be the result of impaired locomotory may not appear until later in life. Additionally, some activity changes K.B. Tierney / Biochimica et Biophysica Acta 1812 (2011) 381–389 383

Table 1 Behavioural test endpoints and their associated stimuli available to fishes, including zebrafish. Some endpoints that are underutilized or hold promise in toxicity or degenerative studies are in bold.

Apparatus Stimulus Stim. type Type of test References

Open arena None None Activity levelA [14,16,20–22,24,25,30] Space Vis Searching behaviourA [22,23,70,90] Food, dead Vis; Olf Appetitive behaviourA [37] Food, alive Vis; Olf Prey captureA [40,42,43] Tap Aud Startle responseA [7] Glass bead Aud; Vis Startle responseA [58] Predator Vis Alarm responseA [39,40] Predator scent Olf Alarm responseB [68–70] Simulated conspecific Social AggressivenessB [11,22] Confined tube, or flume Water flow Motion UcritB [83,84] Social Vis; Motion SchoolingB [58,59] Moving background (striped drum) Simulated object Vis OMRA [47,49,50] Vis OKRA [51,52] ⁎ B Counter-current olfactometer Odours Vis; Olf Attraction; avoidance [15,74,75] ⁎ B Y-maze Odours Olf Attraction [76–79] T-maze Preferred location Vis CPPB [93] Food Chemo CPPB [97] Location Drug CPPB [97,98] Two-chamber tank Shade Vis CPPB [22] Electric field Pain Aversive CPAB [16] Colour Vis CPPB [99] Three-chamber, gated Confinement Vis; Stress CPP, CPAB [100] Three-chamber, separated Social Vis SchoolingB [22]

Abbreviations: Aud, auditory; CPA, conditioned place aversion; CPP, conditioned place preference; OKR, optokinetic response; Olf, olfactory; OMR, optomotor response; Ucrit, critical swimming speed; Vis, visual. Note: Zebrafish age may affect whether specific endpoints are appropriate, as the endpoints may depend on the development of swimming ability, sensory responses, and memory. In general, assays requiring swimming or sensory-evoked swimming are available as soon as 48 post hatch (~4 dpf; endpoints marked A) [37,106]. Swimming intensive and memory based endpoints have mostly been conducted on adults (approx. ≥3 months), however many of these assays could likely be conducted on juveniles (endpoints marked B). An exception: OMR relies on immobilizing fish, and so fish cannot beN7 dpf [51]. A caveat: olfactory-based alarm responses may be most robust and least variable at 50 dpf [73]. ⁎ Odours include food, conspecific, pheromones, predator, and synthetic compounds. may only be evident after the removal of the drug, i.e. during endpoints clearly have application, but they may be more of an withdrawal [16]. umbrella for other neurological impairments isolatable through Zebrafish activity has proven a viable endpoint for detecting sense-specific tests. neurological impairments received during development. For example, embryonic zebrafish exposed to chlorpyrifos, an organophosphorus 2.2. Sensorimotor endpoints insecticide and ubiquitous environmental pollutant, had reduced swimming activity 6+ days later [24]. If persisting neurological Fishes have a suite of exteroceptive senses analogous to those of impairment or neurodegeneration are not readily apparent, a drug mammals. These include vision (photoreceptor-based), audition can be used to evoke activity. Altered neurology will be apparent in a (mechanoreceptor-), olfaction, gustatory (both chemosensor-), balance lower response threshold to the drug. The convulsant (seizure and somatosensory, which includes touch (mechanoreceptor-), pres- inducing drug) pentylenetetrazole (PTZ) has been used in recent sure, temperature (thermoreceptor-) and pain (nociceptor-). Fishes also studies in zebrafish [30], including high throughput 24 and 96 well posses senses that do not have mammalian analogs, e.g. magnetorecep- plate assays [3,31,32], for just such a purpose. Specifically, zebrafish tion. Furthermore, some of the analogous senses, e.g. gustation, are embryos injected with domoic acid (DA), a neurotoxin of diatom morphologically dissimilar—fishes, including zebrafish, have solitary origin, reduced the concentration of PTZ required to evoke stage I and chemosensory cells (SCCs), which are externally-located taste buds II seizure activity [33]. Activity endpoints can also be used to detect [36,37]. And unlike mammals, sound and motion are not only perceived neurodegeneration. Specifically, to model the effects of Parkinson by the ear and vestibular (inertial detection) system, but by an disease, neurotoxic drugs can be injected directly into specific brain additional system, the lateral line. In this system, neuromast cells regions (using microinjection), although this has historically not structurally similar to inner ear mechanoreceptor “hair” cells, detect carried out on fish [34]. A recent study demonstrated that unilateral changes in water pressure and motion relative to themselves. These injection of the drugs 1,2,3,6-tetrahydropyridine (MPTP) and 6- cells can also detect sounds and provide information regarding hydroxydopamine (6-OHDA), two neurotoxins with specific targets, acceleration and velocity [38]. In the below, known and potential reduced the speed and distance zebrafish travelled [14]. Microinjec- sensorimotor endpoints will be discussed. tion techniques make the zebrafish model portable to a variety of The senses enable reflexive, innate responses to “unconditioned neural ablation studies. stimuli” (UCS), as well as learned responses to “conditioned stimuli” A consideration for activity-based sensory endpoints is that they (CS). Both UCS and CS can be used to test sensorimotor responses, and may not actually be stimulus-free. When a fish or other animal is can be positive (attractive) or aversive. UCS evoked behavioural introduced into a new environment, such as a test tank, they will responses stimuli have been included in this section, but their likely begin searching and forming a cognitive map of their new modification is included in the proceeding section on learning and surroundings [35]. This process will draw upon one or more senses memory. Positive UCS or CS stimuli may arrive via various senses but and involve synaptic plasticity (discussed below). Even with elimi- uniformly enable the organism to place itself in an actual or perceived nation of searching behaviour, perhaps accomplished through tank improved fitness position that otherwise offers some benefitor acclimation, activity changes may be based on input from the reward; example stimuli include food, cover, mate call or odour. interoceptive senses, i.e. the perception of internal movement and/ Aversive stimuli do the opposite; examples stimuli include electric or pain (aspects of the somatosensory system). Activity-based shocks, abrupt and severe changes in lighting, temperature, sound, 384 K.B. Tierney / Biochimica et Biophysica Acta 1812 (2011) 381–389

(unpleasant) odours, or other sensory assaults causing discomfort. A test of optomotor response (OMR) was pioneered over 80 years Stimuli can also be neutral, i.e. perceptible but without an associated ago (see [45]) and has been frequently used on a variety of animals behaviour [8]. For behavioural endpoints, both CS and UCS stimuli are since, including mice [46] and zebrafish [47]. The test is one of routinely used in behavioural studies in fishes, including those with tracking: animals reflexively keep pace with a rotating a striped or and without conditioning. “grated” drum. At a high enough rotation speed, the lines will blur and Changes in sensory responses can be due to impaired sensory fish will cease to follow. The parameter of interest is the point at perception or due to impaired motor performance. Separating which the animal ceases to track. Excitatory and inhibitory drugs perceptual vs. motor impairment in sensory studies may involve should increase and decrease this point, respectively, and neurode- using an additional endpoint, such as cell or tissue level assessment of generation will obviously affect it. Another way to conduct the test is neuron function. Another challenge is that most behaviours are to hold the drum speed constant but vary the illumination [48]. This multisensory, and isolating specific impairments may be difficult or method was capable of resolving optomotor differences between impossible. Nevertheless, behavioural endpoints exist to evoke strains. A different version of the test replaces the uniform stripes responses through each of the senses. with one large stripe. The large stripe represents a threatening object, and so fish will swim away from it. This test proved capable of 2.2.1. Visual endpoints detecting retinal degeneration in a “night blind” mutant [49]. Unless experiments are conducted under darkness or with visually A variation of the OMR assay includes using competing visual occluded fish, all behavioural endpoints arguably have some visual grating [50]. Specifically, a background reference grating is kept at a component. In fish, vision specific endpoints include predator constant speed in one direction while a test grating of variable speed (simulated or actual) avoidance, prey capture, optomotor and is moved in the other. Fish will move in the direction of the more optokinetic responses. All of these methods have clearly defined strongly perceived cue. Three scenarios are possible: the fish will track endpoints and involve coordination, except the last, which uses the reference cue when the test cue is of lower contrast, or they will immobilized fish. track the test cue when it is of higher contrast, or when there is One of the most basic assays draws upon phototaxis, i.e. light insufficient contrast between the two, they will stop moving. The null seeking behaviour. Zebrafish are active during the day, and so should motion point provides an index of visual guided response, with acuity spend time in light unless they perceive a risky (predation-prone) inversely related to contrast. This test identified performance condition. When given a choice between a lighted or shaded area in an differences from a mutation in a vesicular glutamate transporter. open arena, zebrafish did indeed spend less time under shade (~40%) The OMR has been adapted to isolate eye tracking ability in static [22]. Ethanol exposure caused a concentration-dependent decline in larval zebrafish [51,52]. In this “optokinetic response” (OKR) assay, the proportion of time spent under shade. With exposures to ethanol, larvae are immobilized in methylcellulose gel, placed in the same zebrafish sheltering was reduced to ~10–15%, i.e. exposure evoked visual scenario as adults (a circular arena with a rotating striped risky behaviour. Such a robust and easily quantifiable behavioural perimeter), and smooth tracking and rapid saccades with stripe contrast could be exploited to test other neurological impairments. sweep are recorded. Impaired altered visual performance will be Alarm (antipredator) behaviours are often used in fish research, evident in decreased saccadic movement [52–54]. The output of eye although they are usually investigated using olfactory cues. Neverthe- velocity in degrees/s can be determined using computer software (a less, visual cues can be used to evoke alarm [39,40].Specifically, protocol is available [55]). A consideration is that fish cannot be N7 presenting fish with a large shape modelled after a predator or an dpf because gel immobilization becomes problematic [51]. The OKR actual predatory fish, such as in a neighbouring tank, can evoke test is functionally equivalent to the finger tracking-based field dashing, freezing and shelter seeking. Fast start responses (s-starts) sobriety test, where the degree at which eye motion switches from have been shown to increase in response to the presentation of a smooth tracking to saccadic movement is indicative of intoxication. simulated predator (painted falcon tube), and be sensitive to the effects Zebrafish are a schooling species that exhibits territoriality [56,57]. of ethanol exposure [22]. A recent paper suggested that alarm response Although schooling and aggressive behaviour are not solely driven by could be a very valuable tool for mechanistically understanding the vision–they can rely on sensory input from the octavolateralis (sound/ roles of signalling molecules, specific receptors and neurons in neural motion), etc.–they are predicated on it. The propensity of a small group circuitry [41]. One of the advantages to aversive responses is that they of zebrafish that was physically but not visually isolated from a larger are easy to score either manually or through video analysis. However, school to swim near the larger group, i.e. their “social preference,” was in some stocks of lab reared fish, innate responses may have been lost. inhibited in a concentration-specificmannerbyethanol[22]. School In this case, conditioning would need to be carried out. cohesion, apparent in “nearest neighbour distance” (NND), may also be Visually guided prey capture (as opposed to chemosensory-guided) used as a sensitive indicator of intoxication [58] and genetic-based has been used in a variety of fish species, although not typically on such a differences [59]. Aggressive behaviour (agonistic behaviour) consists of small one. Nevertheless, visually guided appetitive behaviours can be alternating and/or coincident fin displays and attack behaviours. carried out even in 7-day-old zebrafish using paramecia [42].Otherprey Displays consist of erection of dorsal, pectoral or anal fins, and/or items such as daphnia, hyalella and brine shrimp have been used in slapping of the caudal fin; attack behaviours include biting motions and other species, and there is no reason why they cannot also be used with directed swimming at the attacker [22]. An inclined mirror can be used zebrafish. Assay endpoints include time to initiate attack (latency), to simulate a conspecific, and assess aggression [11,22].Inan number of attacked or captured prey, capture efficiency, and time to “aggressiveness test,” ethanol exposure was correlated with increased cessation [40,42,43]. For example, a recent study on striped bass noted time near the mirror and aggressive displays [22]. Automatic video that time to prey capture was increased 6 days following exposure to an analysis of fin motions obviously poses a challenge, but changes in speed anti-AChE pesticide, the OP diazinon [44]. Another used zebrafish larvae and distance near the mirror would be easy to determine. with laser ablated premotor neurons in the retinotectum and reticulospinal neurons to isolate neural circuitry associated with visually 2.2.2. Olfactory endpoints directed prey capture [42]. Tracking software currently uses contrast In fish, olfaction is so important to so many behaviours that it cannot (i.e. a dark object, the animal, against a light background) to track as be done without (reviewed in [60]). This is the reason numerous studies many as ten objects in one open arena (EthoVision; www.noldus.com). have focused on its impairment through exposure to a variety of However, prey may not be easily discernable, and their removal from an neurotoxic contaminants (reviewed in [61]). In humans, olfaction is not arena may not yet be easily quantifiable. A high throughput version of so indispensable, however, its loss does correlate with neurodegener- this assays remains for development. ative diseases such as Parkinson's and Alzheimer's [62]. The use of K.B. Tierney / Biochimica et Biophysica Acta 1812 (2011) 381–389 385 zebrafish olfactory-based behavioural endpoints of neurodegeneration For example, the antibiotic streptomycin can negatively affect fish remains largely unexplored. A recent publication suggests olfactory behaviour [80]. For these reasons, hair cell alteration has mammalian endpoints may also be viable in prion research [63]. parallel (reviewed in [17]). In fishes, odorants and odorant classes are associated with specific An auditory-evoked startle response has recently been adapted behavioural responses, which facilitate isolating specificneuronal from rodents to zebrafish [7]. In the study, adult zebrafish were placed impairments. Odorants include amino acids, bile salts and pheromones, individually in an eight tank array and video was recorded from which evoke feeding and antipredator, social (assumed), alarm and overhead. The stimulus consisted of an automated, mechanical “tap” mating-based responses, respectively [60]. Amino acids associated with to the tank bottom. Fish swimming distance was determined over the food, such as L-alanine [64], evoked appetitive behaviours such attraction following 5 s, and the taps were repeated every minute for a total of (directed movement up a concentration gradient) [65].Amixtureoften ten times. This session was then subsequently repeated. This design is amino acids, including L-alanine, evoked increases in turning rate and particularly interesting because its neurological assessment is two- activity level at 4 dpf [37], suggesting the possibility of using amino acid dimensional: it tests an innate, behavioural reflex (which includes responses in high throughput assays. Bile salts remain untested as integration of sound and space, and depends on physiological behavioural modulators in zebrafish, but given their efficacy at evoking condition), as well as the ability to adapt (habituate) rapidly to a OSN responses [66], they may be of use in olfactory-based assays. stimulus. In their study, they found that both endpoints could show Pheromone-based behavioural endpoints of mating have been restricted changes due to larval exposure to chlorpyrifos. However, it was to other species (e.g. F-type prostaglandins evoke substrate probing in arguably the ability to adapt that highlighted the biggest neurological salmonids [67]), however, the behaviours evoked by alarm pheromone alteration. The difference in startle response magnitude was greater are well studied. and more significant with repetition. The purpose of this high Alarm pheromone is anxiogenic and makes for a very reliable throughput auditory-based test was to assess the persisting effects endpoint since it is an innate reflex. Alarm responses involve a set of of chlorpyrifos exposure, however there is no reason this assay could stereotypical behaviours, which include dashing, freezing, jumping and not be used to screen/assess a variety of neurological conditions/ erratic movements [68–70]. All the actions are “fight or flight” in nature, pathologies. Furthermore, given their findings regarding habituation, and are associated with increases in stress hormone concentrations [70]. studies including other stimuli (such as odorants), could benefit from Many fish studies have included alarm response as an endpoint of including such a parameter. olfactory neuron function [61]. Numerous neurotoxic agents, including anti-AChE pesticides, alter alarm response, possibly through impairing 2.2.4. Interoceptive sensory endpoints both peripheral (OSNs) and central neurons [61,71]. Alarm behaviour Fish rely on balance and experience pain. However, unlike endpoints are highly sensitive, with some OPs impairing responses with mammals, equilibrioception (balance) is accomplished through the exposures to just 1 ppb [28,71]. Studies wishing to test the functionality octavolateralis system, which includes neurons that perceive sound of the hypothalamic-pituitary-interrenal (HPI) axis (analogous to the and motion. Additionally, fish do not rely upon their “limbs” (i.e. fins) hypothalamic-pituitary-adrenal (HPA) axis in mammals) may benefit for balance. In fact, removing two or more fins has frequently been from the inclusion of an alarm assay [41]. used as a non-invasive, low impact means of identifying fish [81,82]. A recent study found that hypoxanthine 3-N-oxide (H3NO) Nevertheless, neurotoxins affecting the vestibular portion (i.e. inertial evoked alarm response in zebrafish [72]. Whether this compound is sense) of the inner ear, or neurodegeneration in general, may alarm pheromone or a component thereof, this discovery has great conceivably affect balance, and so may impair rheotactic ability. potential to improve the standardization of alarm response testing, Performance deficiencies could be gauged through measuring the since a molecularly based behavioural concentration-response curve time to current orientation. can now be constructed. The previous alarm response testing methods Decades of research on fish swimming performance has relied on involved the use of a skin extract or filtrate derived from the skin of mild electrical shock to evoke swimming [83,84]. Detecting altered conspecifics. These methods suffer by not being able to standardize neurological performance through nociceptor-based response is not the unknown(s) pheromone, however some did endeavour to do so common in fish, but it does show promise, particularly with agents/ by measuring total protein [6]. In zebrafish, the alarm response may conditions which modify anxiety. A surprising result of a recent study be most robust and least variable 50 dpf [73]. was that cocaine did not alter a behavioural avoidance of a mild One of the simplest tests for olfactory-evoked motion involves the electrical field [16]. Given that establishing a local electric field in an use of an avoidance trough or “counter current olfactometer.” In this open arena is not difficult, and that fish will instinctively avoid it, this device, water/odour sources flow from either end of rectangular method has promise for screening neurotoxins with anaesthetic trough and exit in the center without mixing appreciably. In this way, effects. there are effectively two odour zones in the tank, and fish can choose to spend time in either one. As long as a fish is swimming back and 2.3. Learning and memory based endpoints forth prior to odorant introduction (i.e. has not preferred an end before stimulus introduction), both attractive and aversive responses In mammals, the hippocampus is involved with cognitive can be determined [15,74,75]. This test is not unlike a Y-maze, where plasticity. Zebrafish lack a hippocampus, but they do have a odours flow down two arms to a null or mixing zone. Y-maze can be developmentally similar region, the lateral pallium, that appears problematic in some cases because fish may elect to make no decision, functionally equivalent [85]. Reference memory (i.e. acquired mem- i.e. not explore the arms. Nevertheless, both Y-mazes and avoidance ories) and working memory (i.e. the attentional component of short- troughs have been useful in fish studies of the attractive or aversive term memory), and their interplay (i.e. cognitive plasticity) can be qualities of amino acids, pheromones and neurotoxic contaminants tested in a variety of ways, including both classical (Pavlovian) and [76–79]. operant (instrumental) conditioning. Both conditioning methods evoke response(s) using a pre-existing behaviour (evoked by either 2.2.3. Auditory endpoints CS or UCS), but for classical conditioning, a neutral stimulus is converted Even though the anatomy of the zebrafish ear is dissimilar to that into a positive CS (through their repeated pairing), and in operant of mammals (i.e. no cochlea), the neuron hair cell structure is highly conditioning consequences are used to reinforce or extinguish a CS. In the conserved. Additionally, the lateral line cells are structurally and below, endpoints are discussed for (1) attention, for (2) searching functionally the same as ear hair cells. Both ear and later line cells can (map building) behaviour, for (3) the acquisition of responses to be disturbed by compounds of human origin (i.e. “ototoxic” agents). neutral stimuli (classical conditioning), as well as for (4) the 386 K.B. Tierney / Biochimica et Biophysica Acta 1812 (2011) 381–389 reinforcement or extinguishment of positive and negative stimuli zebrafish study using a net as a freight stimulus showed that the (operant conditioning). latency to discover an escape portal decreased significantly over six training sessions [92]. 2.3.1. Attentional endpoints Behaviours generally result from the integration of multiple 2.3.3. Classical conditioning endpoints signals. What is important is the ability to discern important signals Zebrafish studies have demonstrated that both visual [93] and amidst background noise. Neurological filtering in vertebrates has olfactory [8] cues unassociated with behavioural response(s) can be been referred to as the “cocktail party effect,” coined to describe the converted to behavioural response cues. For example, over a few ability of a person to hear a signal such as their name spoken in a room (b10) trials, zebrafish were able to visibly discriminate and select full of chatting people [86]. Implicit in “selective attention” is that coloured arms of a T-maze where they would receive a food reward distracting signals be ignored. One assessment methodology used in [93]. As for odorant-based learning, a recent study demonstrated that non-human primates involves coupling a positive stimulus, such as by pairing PEA (a neutral stimulus) with food odours (L-alanine and L- food, with the occurrence of another stimulus, such as a particular valine) over a number of training sessions, appetitive swimming visual signal, and varying delay between the two [87]. Distractability behaviour (turning rate) could be evoked by the addition of PEA alone can be assessed though inserting irrelevant stimuli in the delay (i.e. it was converted to a CS) [8]. Classical conditioning endpoints period. Since filtering draws upon reference memory and working may be increasingly adopted since conditioning can be achieved memory, it is a useful correlate of neurological function. While rapidly. For example, just one pairing of a neutral stimulus (red light) numerous paradigms exist to test selective attention-based tests in with alarm pheromone (UCS) evoked significant conditioning in a rodents and primates [87,88], no obvious correlate currently exist for related species (fathead minnow) [94]. These data suggest that the zebrafish. However, a functionally similar test might be carried out by strength of the innate response determines the rate of learning. challenging zebrafish with competing cues of fitness relevance. For In many species of vertebrates, shaping is used to evoke a desired example, fish could be exposed to a food odorant, which would behaviour. This method consists of reinforcing successive steps initiate appetitive behavioural response, then interrupted with an towards a target behaviour. This method of reference memory alarm cue, which should evoke anti-predator behaviour. The degree creation may be unreasonable for fish; however autoshaping may and rate at which the appetitive behaviour is replaced and anti- be a viable option. Autoshaping was developed in pigeons and predator responses exhibited (i.e. evokes attention) could prove consisted of the pairing of a visual cue (light) with food. Initially the meaningful endpoints of sensorimotor function and cognition. To pigeons will respond (peck) to the UCS, but over time will respond to explicitly test memory function, the UCS food and/or predator cues the CS alone [95]. There is no reason why this model would not also could be replaced with CS cues, such as behaviourally neutral amino work in fish. Zebrafish will swim into odour sources and sometimes acids or PEA. An attentional endpoint could be especially useful in “bite” at water inflows including food odour (personal observation). testing cholinergic system functionality, since working memory is With repeated trials, they could be expected to bite at a water flow dependent on it [89], and since many drugs agonize or antagonize source alone. cholinergic receptors and neurodegenerative diseases such as Alzheimer's impair it. 2.3.4. Operant conditioning endpoints A variety of methods using open arena to multi-chambered arenas 2.3.2. Searching (spatial) endpoints exist to test stimulus reinforcement or extinguishment. Open arenas Introduction to new surroundings will stimulate searching behav- have been used for tests involving food odour and predator scent. In iour and map building [35]. The formation of a new cognitive map is the first, an appetitive response to food flake was enhanced by pairing another form of neural plasticity and therefore a possible performance it with amino acids food odours [8]. In the second, a study of a closely metric for neurological impairment. Numerous studies have already related species (fathead minnow) found that it took 6 to 8 days and validated that searching behaviour in an open arena is a meaningful two to four days to visually and olfactorily acquire a predator, endpoint in zebrafish [22,70,90]. Searching behaviour was recently used presumably by the pairing of a UCS (alarm pheromone) with visual in combination with two anxiogenic factors, alarm pheromone or cues and scent, respectively [39]. Visual and odorant cues clearly have caffeine, and two axiolytic factors, ethanol and fluoxetine (i.e. the SSRI potential for assessing cognition, and the differential time course Prozac), as an endpoint in “a novel tank test” [70]. Release into a new could provide a valuable diagnostic tool. tank evoked a bottom seeking in control fish, which was followed by A common method of operant conditioning involves presenting an subsequent upper tank exploration. With alarm pheromone and animal with one or more choices between arena arms or areas, and caffeine exposure, the number of upper tank explorations was reduced, based on the decision, either rewarding the animal with food or and the time taken to explore (latency) increased. With exposure to punishing it with pain or stress. A popular paradigm is “conditioned ethanol or fluoxetine, the reverse pattern was noted: transitions to the place preference” (CPP) or sometimes just “place preference” (PP), in upper tank increased, and the latency to upper tank searching was which animals are rewarded with a drug after selecting a particular greatly diminished. Nicotine had the opposite effect, fish were not as location [96]. The reinforcing stimulus needs to be a positive inclined to dive to the tank bottom [23]. psychoactive stimulant, such as cocaine [97] or amphetamine [98]. Spatial memory is mediated through the lateral pallium. Studies A version of CPP is “conditioned place avoidance” (CPA), which is have found that lesioning the functionally equivalent region in rats where the conditioned area was initially avoided [99]. For example, by (hippocampus), abolished searching behaviour [91]. Given the ability “addicting” zebrafish to D-amphetamine, preference for a side of the to focally lesion larval zebrafish brain tissue and neurons using laser tank with two large freight inducing black dots could be evoked [99]. ablation [42], studies of searching hold promise for cognitive In general, CPP and CPA are good models for testing the neurological endpoints. A tried and true test of spatial memory in rats is the bases for addiction, but they remain largely unadopted but available Morris water navigation task, which is essentially map building under in toxicity or degenerative studies. stress and with visual cue reference. The rate of learning of the At least two methods exist to test vision-based learning in fishes, platform location can be gauged by the latency to platform discovery the T-maze and the three chambered arena. Both methods share some and distance covered in successive trials. Learning under stress in fish similarities to the radial arm maze (RAM), frequently used in could be accomplished in a variety of means, such as by the ability to mammalian models. In the T-maze, fish are introduced into the long find an escape portal (to another potion of a test tank), under the arm (base of the T) and they will generally swim to one or both of the threat of predation, evoked by visual or olfactory stimuli. One sort arms. However, as many as 5% fish may not move from the base K.B. Tierney / Biochimica et Biophysica Acta 1812 (2011) 381–389 387 arm unless provoked [97]. The intended outcome is for a fish to favour Acknowledgments one arm based on the desirability of the settings (“spatial preference”) [97], or a reward coupled to a visual cue [93]. In tests using the three The author is grateful to W. Ted Allison for motivation in doing this chambered arena, fish are introduced to a center area that is flanked work, and for grants to KBT from the Canadian Wildlife Federation on either side by gated chambers [100]. To generate avoidance (CWF) and the University of Alberta. behaviour of one of the side chamber, fish are penalized (using fi con nement) for making repeated selections of one tank side (in References general, to condition an animal to avoid an area based on a visual cue, nociceptor and stress based methods are used [101]). The three [1] R.N. Boyd, M.E. Morris, H.K. Graham, Management of upper limb dysfunction in chamber confinement test demonstrated that cholinergic agonization children with cerebral palsy: a systematic review, Eur. J. Neurol. 8 (2001) 150–166. can improve the number of correct decisions [100]. [2] D.W. Roberts, J.F. Costello, Mechanisms of action for general and polar narcosis: a In all operant models discussed above, a conditioned CS will lose difference in dimension, QSAR Comb. Sci. 22 (2003) 226–233. its behavioural stimulatory efficacy as its repeated presentation goes [3] S.C. Baraban, M.R. Taylor, P.A. Castro, H. Baier, Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression, Neuroscience unpaired with the initial pre-existing, positive stimulus [8]. As with 131 (2005) 759–768. acquisitional endpoints, the rate of change in the reinforced decision [4] E. Fontaine, D. Lentink, S. Kranenbarg, U.K. Muller, J.L. van Leeuwen, A.H. Barr, J.W. can provide a correlate of neurological function [102]. Burdick, Automated visual tracking for studying the ontogeny of zebrafish swimming, J. Exp. Biol. 211 (2008) 1305–1316. [5] C.S. Lam, V. Korzh, U. Strahle, Zebrafish embryos are susceptible to the 3. Problems associated with behavioural endpoints dopaminergic neurotoxin MPTP, Eur. J. Neurosci. 21 (2005) 1758–1762. [6] J.F. Sandahl, D.H. Baldwin, J.J. Jenkins, N.L. Scholz, A sensory system at the interface between urban stormwater runoff and salmon survival, Environ. Sci. Variation in responses/traits/parameters increases with organisa- Technol. 41 (2007) 2998–3004. tional level, and so behaviours are obviously the most variable of [7] D. Eddins, D. Cerutti, P. Williams, E. Linney, E.D. Levin, Zebrafish provide a organismal responses. Additionally, in fish, as other animals, their sensitive model of persisting neurobehavioral effects of developmental chlorpyrifos exposure: Comparison with nicotine and pilocarpine effects and state will affect memory recall and motivation, and they are not relationship to dopamine deficits, Neurotoxicology and Teratology In Press, without personality. Furthermore, with aquatic species, special Corrected Proof (2009). consideration must be made for the water source. Municipal water [8] O.R. Braubach, H.-D. Wood, S. Gadbois, A. Fine, R.P. Croll, Olfactory conditioning fi – is often rife with neurotoxic contaminants, which may vary in in the zebra sh (Danio rerio), Behav. Brain Res. 198 (2009) 190 198. [9] K.B. Tierney, C.J. Kennedy, Chapter 6: Background Toxicology, in: P.J. Walsh, S.L. concentration considerably even over brief time periods. Water Smith, L.E. Fleming, H. Solo-Gabriele, W.H. Gerwick (Eds.), Oceans and Human needs to be carbon filtered and tested to remove/account for any Health: Risks and Remedies from the Seas, vol. 1, Elsevier Science Publishers, – confounding chemical effects. New York, 2008, pp. 101 120. [10] L.I. Zon, Zebrafish: A new model for human disease, Genome Res. 9 (1999) An appreciable amount of individual variation can be accounted 99–100. for by classifying behavioural phenotypes, such as low or high activity [11] R. Gerlai, Zebra fish: an uncharted behavior genetic model, Behav. Genet. 33 subgroups. Doing so will increase the power of the test; however, not (2003) 461–468. [12] V.A. Braithwaite, Cognitive ability in fish, in: K.A. Sloman, R.W. Wilson, S. all traits that appear obviously related to behaviour may be so. In Balshine (Eds.), Behaviour and physiology of fish, vol. 24, Academic Elsevier juvenile brook trout, for example, swimming performance ability was Press, San Diego, 2006, pp. 1–37. not associated with actual (conative) swimming activity [103]. Some [13] W. Boehmler, T. Carr, C. Thisse, B. Thisse, V.A. Canfield, R. Levenson, D4 Dopamine receptor genes of zebrafish and effects of the antipsychotic clozapine temporal issues can be exploited; food responsiveness and willing- on larval swimming behaviour, Genes Brain Behav. 6 (2007) 155–166. ness to take risks, for example, are enhanced with brief periods of food [14] O.V. Anichtchik, J. Kaslin, N. Peitsaro, M. Scheinin, P. Panula, Neurochemical and deprivation [65,103]. Social status can also affect stress level which behavioural changes in zebrafish Danio rerio after systemic administration of 6-hydroxydopamine and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine, J. will in turn modulate activity and stimuli responses [103]. If social Neurochem. 88 (2004) 443–453. endpoint was to be used, accounting for dominance behaviour in fish [15] K.B. Tierney, C.R. Singh, P.S. Ross, C.J. Kennedy, Relating olfactory neurotoxicity to may present a challenge. Careful observation and selective removal of altered olfactory-mediated behaviors in rainbow trout exposed to three – individuals of stronger resource utilization (e.g. food consumption) currently-used pesticides, Aquat. Toxicol. 81 (2007) 55 64. [16] M.A. López-Patiño, L. Yu, H. Cabral, I.V. Zhdanova, Anxiogenic effects of cocaine could help resolve variation. withdrawal in zebrafish, Physiol. Behav. 93 (2008) 160–171. [17] T.T. Whitfield, P. Mburu, R.E. Hardisty-Hughes, S.D.M. Brown, Models of fi 4. Conclusions congenital deafness: mouse and zebra sh, Drug Discov. Today: Disease Models 2 (2005) 85–92. [18] L. Li, Zebrafish mutants: behavioral genetic studies of visual system defects, Dev. Vertebrates have been proposed as the best models for elucidating Dyn. 221 (2001) 365–372. mechanisms of neurodegeneration [104]. Among vertebrate models, [19] S.L. Beauvais, S.B. Jones, J.T. Parris, S.K. Brewer, E.E. Little, Cholinergic and fi fi behavioral neurotoxicity of carbaryl and cadmium to larval rainbow trout zebra sh are nding ever increasing application. In studies of drugs, (Oncorhynchus mykiss), Ecotoxicol. Environ. Saf. 49 (2001) 84–90. zebrafish make for excellent models, since delivery can be achieved in [20] S.L. Beauvais, S.B. Jones, S.K. Brewer, E.E. Little, Physiological measures of neurotoxicity many cases by simple addition to the tank water. Furthermore, of diazinon and malathion to larval rainbow trout (Oncorhynchus mykiss)andtheir correlation with behavioral measures, Environ. Toxicol. Chem. 19 (2000) 1875–1880. microinjection techniques are now available to deliver agents directly [21] T.D. Irons, R.C. MacPhail, D.L. Hunter, S. Padilla, Acute neuroactive drug to specific cells/tissues, even within larval fish [105]. This review has exposures alter locomotor activity in larval zebrafish, Neurotoxicol. Teratol. 32 highlighted behavioural endpoints available to zebrafish that probe (2010) 84–90. [22] R. Gerlai, M. Lahav, S. Guo, A. Rosenthal, Drinks like a fish: zebra fish (Danio rerio) basic neurological function, that test behaviours predicated on the as a behavior genetic model to study alcohol effects, Pharmacol. Biochem. Behav. function of diverse types of sensory neurons, and that investigate 67 (2000) 773–782. cognitive performance. Some behavioural endpoints test multiple [23] E.D. Levin, Z. Bencan, D.T. Cerutti, Anxiolytic effects of nicotine in zebrafish, – neurologically-based abilities; this review is intended to provide a Physiol. Behav. 90 (2007) 54 58. [24] E.D. Levin, H.A. Swain, S. Donerly, E. Linney, Developmental chlorpyrifos effects template by which to link and/or apportion specific physiological, on hatchling zebrafish swimming behavior, Neurotoxicol. Teratol. 26 (2004) perceptual and cognitive impairments. Most methodologies described 719–723. herein have very tight parallel with mammalian models. Furthermore, [25] C.E.W. Steinberg, R. Lorenz, O.H. Spieser, Effects of atrazine on swimming behavior of zebrafish, Brachydanio rerio, Water Res. 29 (1995) 981–985. many behavioural tests, especially those using high throughput [26] K.N. Baer, K. Olivier, C.N. Pope, Influence of temperature and dissolved oxygen on screens, remain in evolution. The future holds promise for the the acute toxicity of profenofos to fathead minnows (Pimephales promelas), Drug development of a suite of standardized behavioural assays that can Chem. Toxicol. 25 (2002) 231–245. [27] S. Bretaud, P. Saglio, J.-P. Toutant, Effets du carbofuran sur l'activité de be used to determine the mechanisms by which neurotoxic effects l'acétylcholinestérase cérébrale et sur l'activité de nage chez Carassius auratus and neurodegenerative diseases develop and progress. (Cyprinidae), Cybium 25 (2001) 33–40. 388 K.B. Tierney / Biochimica et Biophysica Acta 1812 (2011) 381–389

[28] J.F. Sandahl, D.H. Baldwin, J.J. Jenkins, N.L. Scholz, Comparative thresholds for [59] K.B. Tierney, D.A. Paterson, C.J. Kennedy, The influence of maternal condition on acetylcholinesterase inhibition and behavioral impairment in coho salmon offspring performance in sockeye salmon (Oncorhynchus nerka Walbaum), J. Fish exposed to chlorpyrifos, Environ. Toxicol. Chem. 24 (2005) 136–145. Biol. 75 (2009) 1244–1257. [29] S.K. Brewer, E.E. Little, A.J. DeLonay, S.L. Beauvais, S.B. Jones, M.R. Ellersieck, [60] H. Hino, N.G. Miles, H. Bandoh, H. Ueda, Molecular biological research on Behavioral dysfunctions correlate to altered physiology in rainbow trout olfactory chemoreception in fishes, J. Fish Biol. 75 (2009) 945–959. (Oncorynchus mykiss) exposed to cholinesterase-inhibiting chemicals, Arch. [61] K.B. Tierney, D.H. Baldwin, T.J. Hara, P.S. Ross, N.L. Scholz, C.J. Kennedy, Review: Environ. Contam. Toxicol. 40 (2001) 70–76. olfactory toxicity in fishes, Aquat. Toxicol. (2009) 1–25, doi:10.1016/j. [30] K.B. Tierney, X. Ren, Z. Alyasha'e, B. Zielinski, Towards a mechanistic aquatox.2009.09.019. understanding of food odor driven motion using zebrafish (Danio rerio), 15th [62] C. Hawkes, Olfaction in neurodegenerative disorder, Mov. Disord. 18 (2003) International Symposium on Olfaction and Taste, vol. 33, Oxford Univ Press, San 364–372. Francisco, CA, 2008, pp. S156–S157. [63] C.E. Le Pichon, M.T. Valley, M. Polymenidou, A.T. Chesler, B.T. Sagdullaev, A. [31] M.J. Winter, W.S. Redfern, A.J. Hayfield, S.F. Owen, J.-P. Valentin, T.H. Hutchinson, Aguzzi, S. Firestein, Olfactory behavior and physiology are disrupted in prion Validation of a larval zebrafish locomotor assay for assessing the seizure liability protein knockout mice, Nat. Neurosci. 12 (2009) 60–69. of early-stage development drugs, Journal of Pharmacological and Toxicological [64] W.E.S. Carr, J.C. Netherton III, R.A. Gleeson, C.D. Derby, Stimulants of feeding Methods 57, 176–187. behavior in fish: analyses of tissues of diverse marine organisms, Biol. Bull. 190 [32] S.C. Baraban, M.T. Dinday, P.A. Castro, S. Chege, S. Guyenet, M.R. Taylor, A large- (1996) 149–160. scale mutagenesis screen to identify seizure-resistant zebrafish, Epilepsia 48 [65] C.W. Steele, D.W. Owens, A.D. Scarfe, Attraction of zebrafish, Brachydanio rerio,to (2007) 1151–1157. alanine and its suppression by copper, J. Fish Biol. 36 (1990) 341–352. [33] J.A. Tiedeken, J.S. Ramsdell, Embryonic exposure to domoic acid increases the [66] W.C. Michel, D.S. Derbidge, Evidence of distinct amino acid and bile salt susceptibility of zebrafish larvae to the chemical convulsant pentylenetetrazole, receptors in the olfactory system of the zebrafish, Danio rerio, Brain Res. 764 Environ. Health Perspect. 115 (2007) 1547–1552. (1997) 179–187. [34] M.F. Beal, Experimental models of Parkinson's disease, Nat. Rev. Neurosci. 2 [67] F. Laberge, T.J. Hara, Behavioural and electrophysiological responses to F-prostaglan- (2001) 325–334. dins, putative spawning pheromones, in three salmonid fishes, J. Fish Biol. 62 (2003) [35] W.J. Bell, Searching behaviour: the behavioural ecology of finding resources, 206–221. Chapman and Hall, Cambridge, 1991. [68] K. von Frisch, Über einen Schreckstoff der Fischhaut und seine biologische [36] K. Kotrschal, W.-D. Krautgartner, A. Hansen, Ontogeny of the solitary Bedeutung, Z. Vgl. Physiol. 29 (1941) 46–145. chemosensory cells in the zebrafish, Danio rerio, Chem. Senses 22 (1997) [69] N. Speedie, R. Gerlai, Alarm substance induced behavioral responses in zebrafish 111–118. (Danio rerio), Behav. Brain Res. 188 (2008) 168–177. [37] S.M. Lindsay, R.G. Vogt, Behavioral responses of newly hatched zebrafish (Danio rerio) [70] R.J. Egan, C.L. Bergner, P.C. Hart, J.M. Cachat, P.R. Canavello, M.F. Elegante, S.I. to amino acid chemostimulants, Chem. Senses 29 (2004) 93–100. Elkhayat, B.K. Bartels, A.K. Tien, D.H. Tien, S. Mohnot, E. Beeson, E. Glasgow, H. [38] C.B. Braun, S. Coombs, R.R. Fay, What is the nature of multisensory interaction Amri, Z. Zukowska, A.V. Kalueff, Understanding behavioral and physiological between octavolateralis sub-systems? Brain Behav. Evol. 59 (2002) 162–176. phenotypes of stress and anxiety in zebrafish, Behav. Brain Res. 205 (2009) [39] G.E. Brown, D.P. Chivers, R.J.F. Smith, Differential learning rates of chemical 38–44. versus visual cues of a northern pike by fathead minnows in a natural habitat, [71] N.L. Scholz, N.K. Truelove, B.L. French, B.A. Berejikian, T.P. Quinn, E. Casillas, T.K. Environ. Biol. Fish. 49 (1997) 89–96. Collier, Diazinon disrupts antipredator and homing behaviors in chinook salmon [40] V.N. Mikheev, J. Wanzenbock, A.F. Pasternak, Effects of predator-induced visual (Oncorhynchus tshawytscha), Can. J. Fish. Aquat. Sci. 57 (2000) 1911–1918. and olfactory cues on 0+ perch (Perca fluviatilis L.) foraging behaviour, Ecol. [72] K.V. Parra, J.C. Adrian Jr., R. Gerlai, The synthetic substance hypoxanthine 3-N-oxide Freshw. Fish 15 (2006) 111–117. elicits alarm reactions in zebrafish (Danio rerio), Behav. Brain Res. 205 (2009) [41] S.J. Jesuthasan, A.S. Mathuru, The alarm response in zebrafish: Innate fear in a 336–341. vertebrate genetic model, J. Neurogenet. 22 (2008) 211–228. [73] S.R. Blechinger, R.C. Kusch, K. Haugo, C. Matz, D.P. Chivers, P.H. Krone, Brief [42] E. Gahtan, P. Tanger, H. Baier, Visual prey capture in larval zebrafish is controlled embryonic cadmium exposure induces a stress response and cell death in the by identified reticulospinal neurons downstream of the tectum, J. Neurosci. 25 developing olfactory system followed by long-term olfactory deficits in juvenile (2005) 9294–9303. zebrafish, Toxicol. Appl. Pharmacol. 224 (2007) 72–80. [43] J. Savitz, L. Bardygula-Nonn, Behavioral interactions between coho (Oncorhynchus [74] E.E. Little, S.E. Finger, Swimming behavior as an indicator of sublethal toxicity in kisutch) and chinook salmon (Oncorhynchus tshawytscha)andpreyfish species, Ecol. fish, Environ. Toxicol. Chem. 9 (1990) 13–19. Freshw. Fish 6 (1997) 190–195. [75] J.A. Hansen, D.F. Woodward, E.E. Little, A.J. Delonay, H.L. Bergman, Behavioral [44] K.M. Gaworecki, A.P. Roberts, N. Ellis, A.D. Sowers, S.J. Klaine, Biochemical and avoidance: possible mechanism for explaining abundance and distribution of behavioral effects of diazinon exposure in hybrid striped bass, Environ. Toxicol. trout species in a metal-impacted river, Environ. Toxicol. Chem. 18 (1999) Chem. 28 (2009) 105–112. 313–317. [45] C. Schlieper, Farbensinn der tiere und optomotorische reaktionen, J. Comp. [76] Y. Ishida, H. Kobayashi, Avoidance behavior of carp to pesticides and decrease of Physiol. A Neuroethol. Sens. Neural Behav. Physiol. 6 (1927) 453–472. the avoidance threshold by addition of sodium lauryl sulfate, Fish. Sci. Tokyo 61 [46] J. Abdeljalil, M. Hamid, O. Abdel-mouttalib, R. Stéphane, R. Raymond, A. Johan, S. (1995) 441–446. José, C. Pierre, P. Serge, The optomotor response: a robust first-line visual [77] J.D. Morgan, G.A. Vigers, A.P. Farrell, D.M. Janz, J.F. Manville, Acute avoidance screening method for mice, Vis. Res. 45 (2005) 1439–1446. reactions and behavioral responses of juvenile rainbow trout (Oncorhynchus [47] A. Krauss, C. Neumeyer, Wavelength dependence of the optomotor response in mykiss) to Garlon 4® Garlon 3A® and Vision® herbicides, Environ. Toxicol. zebrafish (Danio rerio), Vis. Res. 43 (2003) 1275–1284. Chem. 10 (1991) 73–79. [48] K. Ijiri, Vestibular and visual contribution to fish behavior under microgravity, [78] H. Yambe, M. Shindo, F. Yamazaki, A releaser pheromone that attracts males in Adv. Space Res. 25 (2000) 1997–2006. the urine of mature female masu salmon, J. Fish Biol. 55 (1999) 158–171. [49] L. Li, J.E. Dowling, A dominant form of inherited retinal degeneration caused by a [79] T.J. Hara, Feeding behaviour in some teleosts is triggered by single amino acids non-photoreceptor cell-specific mutation, Proc. Natl Acad. Sci. USA 94 (1997) primarily through olfaction, J. Fish Biol. 68 (2006) 810–825. 11645–11650. [80] J.M. Gardiner, J. Atema, Sharks need the lateral line to locate odor sources: [50] M.C. Smear, H.W. Tao, W. Staub, M.B. Orger, N.J. Gosse, Y. Liu, K. Takahashi, M.-m. rheotaxis and eddy chemotaxis, J. Exp. Biol. 210 (2007) 1925–1934. Poo, H. Baier, Vesicular glutamate transport at a central synapse limits the acuity [81] I.K. Birtwell, R. Fink, D. Brand, R. Alexander, C.D. McAllister, Survival of pink of visual perception in zebrafish, Neuron 53 (2007) 65–77. salmon (Oncorhynchus gorbuscha) fry to adulthood following a 10-day exposure [51] S.E. Brockerhoff, J.B. Hurley, U. Janssen-Bienhold, S.C. Neuhauss, W. Driever, J.E. to the aromatic hydrocarbon water-soluble fraction of crude oil and release to Dowling, A behavioral screen for isolating zebrafish mutants with visual system the Pacific Ocean, Can. J. Fish. Aquat. Sci. 56 (1999) 2087–2098. defects, Proc. Natl Acad. Sci. USA 92 (1995) 10545–10549. [82] K.B. Tierney, E. Stockner, C.J. Kennedy, Changes in immunological characteristics [52] O. Rinner, J.M. Rick, S.C.F. Neuhauss, Contrast sensitivity, spatial and temporal and disease resistance of juvenile coho salmon (Oncorhynchus kisutch Walbaum) tuning of the larval zebrafish optokinetic response, Investig. Ophthalmol. Vis. Sci. in response to dehydroabietic acid exposure under varying thermal conditions, 46 (2005) 137–142. Water Qual. Res. J. Can. 39 (2004) 176–184. [53] F.M. Richards, W.K. Alderton, G.M. Kimber, Z. Liu, I. Strang, W.S. Redfern, J.P. [83] C.H. Hammer, Fatigue and exercise tests with fish, Comp. Biochem. Physiol. A Valentin, M.J. Winter, T.H. Hutchinson, Validation of the use of zebrafish larvae in Physiol. 112 (1995) 1–20. visual safety assessment, J. Pharmacol. Toxicol. Meth. 58 (2008) 50–58. [84] K.B. Tierney, M. Casselman, S. Takeda, A.P. Farrell, C.J. Kennedy, The relationship [54] T.P. Barros, W.K. Alderton, H.M. Reynolds, A.G. Roach, S. Berghmans, Zebrafish: between cholinesterase inhibition and two types of swimming performance in an emerging technology for in vivo pharmacological assessment to identify chlorpyrifos-exposed coho salmon (Oncorhynchus kisutch), Environ. Toxicol. potential safety liabilities in early drug discovery, Br. J. Pharmacol. 154 (2008) Chem. 26 (2007) 998–1004. 1400–1413. [85] F. Rodríguez, J.C. López, J.P. Vargas, C. Broglio, Y. Gómez, C. Salas, Spatial memory [55] C. Hodel, S.C.F. Neuhauss, Computer-based analysis of the optokinetic response and hippocampal pallium through vertebrate evolution: insights from reptiles in zebrafish larvae, Cold Spring Harbor Protocols 2008 (2008)8 pdb.prot4961-. and teleost fish, Brain Res. Bull. 57 (2002) 499–503. [56] R. Spence, G. Gerlach, C. Lawrence, C. Smith, The behaviour and ecology of the [86] E.C. Cherry, Some experiments on the recognition of speech, with one and with zebrafish, Danio rerio, Biol. Rev. 83 (2008) 13–34. two ears, J. Acoust. Soc. Am. 25 (1953) 975–979. [57] C. Brown, K.N. Laland, Social learning in fishes: a review, Fish Fish. 4 (2003) [87] M.A. Prendergast, Assessment of distractability in non-human primates 280–288. performing a delayed matching task, in: J.J. Buccafusco (Ed.), Methods of [58] C.A. Dlugos, R.A. Rabin, Ethanol effects on three strains of zebrafish: model Behavior Analysis in Neuroscience, CRC Press, Boca Raton, 2001, pp. 123–140. system for genetic investigations, Pharmacol. Biochem. Behav. 74 (2003) [88] P.J. Bushnell, B.J. Strupp, Assessing attention in rodents, in: J.J. Buccafusco (Ed.), 471–480. Methods of Behavior Analysis in Neuroscience, CRC Press, Boca Raton, 2009. K.B. Tierney / Biochimica et Biophysica Acta 1812 (2011) 381–389 389

[89] M.I. Miranda, G. Ferreira, L. Ramírez-Lugo, F. Bermúdez-Rattoni, Role of [98] J. Ninkovic, A. Folchert, Y.V. Makhankov, S.C.F. Neuhauss, I. Sillaber, U. Straehle, L. cholinergic system on the construction of memories: taste memory encoding, Bally-Cuif, Genetic identification of AChE as a positive modulator of addiction to Neurobiol. Learn. Mem. 80 (2003) 211–222. the psychostimulant D-amphetamine in zebrafish, J. Neurobiol. 66 (2006) [90] R. Blaser, R. Gerlai, Behavioral phenotyping in zebrafish: comparison of three 463–475. behavioral quantification methods, Behav. Res. Meth. 38 (2006) 456–469. [99] J. Ninkovic, L. Bally-Cuif, The zebrafish as a model system for assessing the [91] J. O'Keefe, L. Nadel, The hippocampus as a cognitive map, Oxford University reinforcing properties of drugs of abuse, Methods 39 (2006) 262–274. Press, Oxford, 1978. [100] E. Levin, J. Limpuangthip, T. Rachakonda, M. Peterson, Timing of nicotine effects [92] D. Arthur, E. Levin, Spatial and non-spatial visual discrimination learning in on learning in zebrafish, Psychopharmacology 184 (2006) 547–552. zebrafish (Danio rerio), Anim. Cogn. 4 (2001) 125–131. [101] J. Garcia, P.S. Lasiter, F. Bermudez-Rattoni, D.A. Deems, A general theory of [93] R.M. Colwill, M.P. Raymond, L. Ferreira, H. Escudero, Visual discrimination aversion learning, Ann. NY Acad. Sci. 443 (1985) 8–21. learning in zebrafish (Danio rerio), Behav. Process. 70 (2005) 19–31. [102] T.E. Orr, P.A. Walters, R.L. Elkins, Fundamentals, methodologies, and uses of taste [94] W.K. Yunker, D.E. Wein, B.D. Wisenden, Conditioned alarm behavior in fathead aversion learning, in: Buccafusco (Ed.), Methods of Behavior Analysis in minnows (Pimephales promelas) resulting from association of chemical alarm Neuroscience, CRC Press, Boca Raton, 2001, pp. 51–70. pheromone with a nonbiological visual stimulus, J. Chem. Ecol. 25 (1999) [103] M. Farwell, R.L. McLaughlin, Alternative foraging tactics and risk taking in brook 2677–2686. charr (Salvelinus fontinalis), Behav. Ecol. 20 (2009) 913–921. [95] C.M. Locurto, H.S. Terrace, J. Gibbon, Autoshaping and conditioning theory, in: C.M. [104] J.D. Cooper, C. Russell, H.M. Mitchison, Progress towards understanding disease Locurto, H.S. Terrace, J. Gibbon (Eds.), Autoshaping and Conditioning Theory, mechanisms in small vertebrate models of neuronal ceroid lipofuscinosis, Academic Press, New York, 1981, p. 313. Biochim Biophys Acta (BBA), Mol. Basis Dis. 1762 (2006) 873–889. [96] J.R. James, R. Young, J.A. Rosecrans, Conditioned place preference: an approach to [105] C. Cianciolo Cosentino, B.L. Roman, I.A. Drummond, N.A. Hukriede, Intravenous evaluating positive and negative drug-induced stimuli, in: J.J. Buccafusco (Ed.), microinjections of zebrafish larvae to study acute kidney injury, J. Vis. Exp. 42 Methods of Behavior Analysis in Neuroscience, CRC Press, Boca Raton, 2001, pp. 81–90. (2010) e2079. [97] T. Darland, J.E. Dowling, Behavioral screening for cocaine sensitivity in [106] J.S.S. Easter, G.N. Nicola, The development of vision in the zebrafish (Danio rerio), mutagenized zebrafish, Proc. Natl Acad. Sci. USA 98 (2001) 11691–11696. Dev. Biol. 180 (1996) 646–663. Biochimica et Biophysica Acta 1812 (2011) 390–401

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Review Aberrant forebrain signaling during early development underlies the generation of holoprosencephaly and coloboma☆

Patricia A. Gongal a,1, Curtis R. French a, Andrew J. Waskiewicz a,b,c,⁎ a Department of Biological Sciences, University of Alberta, Edmonton, Canada T6G 2E9 b Centre for Neuroscience, University of Alberta, Edmonton, Canada T6G 2E9 c Women & Children's Health Research Institute, University of Alberta, Edmonton, Canada T6G 2E9 article info abstract

Article history: In this review, we highlight recent literature concerning the signaling mechanisms underlying the develop- Received 1 December 2009 ment of two neural birth defects, holoprosencephaly and coloboma. Holoprosencephaly, the most common Accepted 8 September 2010 forebrain defect, occurs when the cerebral hemispheres fail to separate and is typically associated with Available online 16 September 2010 mispatterning of embryonic midline tissue. Coloboma results when the choroid fissure in the eye fails to close. It is clear that Sonic hedgehog (Shh) signaling regulates both forebrain and eye development, with defects in Keywords: Shh, or components of the Shh signaling cascade leading to the generation of both birth defects. In addition, Holoprosencephaly Coloboma other intercellular signaling pathways are known factors in the incidence of holoprosencephaly and Sonic hedgehog coloboma. This review will outline recent advances in our understanding of forebrain and eye embryonic Gdf6 pattern formation, with a focus on zebrafish studies of Shh and retinoic acid pathways. Given the clear overlap Retinoic acid in the mechanisms that generate both diseases, we propose that holoprosencephaly and coloboma can Retina represent mild and severe aspects of single phenotypic spectrum resulting from aberrant forebrain development. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases. © 2010 Elsevier B.V. All rights reserved.

1. Introduction to holoprosencephaly and coloboma 2. Part I: Holoprosencephaly

As the vertebrate embryo develops, the ventral forebrain and retina Holoprosencephaly (HPE) is the most common forebrain birth are subject to similar extracellular cues that drive their development. A defect in humans [1] and encompasses a broad range of craniofacial combination of human genetic, animal model, and cell biochemical and neural defects. Traditionally, HPE has been defined as the studies has shed light on the signaling pathways that are essential for failure of the forebrain to divide into two distinct cerebral hemi- patterning this region of the embryo. Within the forebrain, researchers spheres during development. In the most profound cases, the fetus have identified deficiencies in Nodal, Shh, and retinoic acid (RA) displays cyclopia and the complete absence of lobar divisions in the pathways, as causes of the ventral forebrain birth defect holoprosen- cerebrum [2–7]. The HPE spectrum also includes ethmocephaly, cephaly. In the retina, mutations in components of the Shh, RA, and where the fetus displays a proboscis (an abnormal tubular nose-like BMP signaling pathways prevent proper closure of the choroid fissure, structure) and closely-set eyes, and cebocephaly, where the fetus and result in coloboma. The overlap in genetic causes for these two has closely-set eyes and single nostril [8].Moresubtledefectshave distinct birth defects implies that they may be more accurately also been classified as falling within the HPE spectrum, including considered as components of a common phenotypic spectrum. This median cleft lip, absence of the olfactory bulb, agenesis of the review will define the recent advances in our understanding of corpus callosum, and a single, centrally located maxillary incisor Holoprosencephaly (Part I) and Coloboma (Part II), and propose the [2,8]. While HPE is rare in live births (1/16,000), approximately 1/ commonality of their causality. 250 conceptuses are thought to be affected by HPE [1,9]. This large difference in frequency likely reflects an incompatibility of

☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological severe HPE with life, and a high rate of miscarriage of affected Diseases. embryos. ⁎ Corresponding author. CW405, Biological Sciences Building, Biological Sciences HPE can be caused by both genetic abnormalities and exposure to Department, University of Alberta, Edmonton, Alberta, Canada T6G 2E9. Tel.: +1 780 teratogens, including alcohol [10,11] and retinoic acid [12,13]. 492 4403; fax: +1 780 492 9234. Approximately one-quarter of cases are due to detectable genetic E-mail address: [email protected] (A.J. Waskiewicz). 1 Present address: Laboratoire de Génétique Moléculaire du Développement, U784 anomalies [5]. Mapping chromosomal deletions in affected patients INSERM, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris cedex 05, France. facilitated the identification of the first genes linked to HPE [14–20],

0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2010.09.005 P.A. Gongal et al. / Biochimica et Biophysica Acta 1812 (2011) 390–401 391 and later work has demonstrated a high frequency of microdeletions signaling in the formation of the vertebrate prechordal plate. and point mutations in these genes [21]. Disruption of this tissue results in the fusion of the eye fields and Research in zebrafish over the last decade has yielded many deletion of ventral forebrain tissue, a phenotype that strongly insights into the normal function of HPE genes, and how their resembles the most severe cases of HPE [32–36]. The prechordal disruption can result in human disease. The eight genes linked to plate is the anterior-most derivative of Spemann's organizer (the HPE have been shown to play important roles in three major shield in zebrafish), which, during gastrulation, acts as a signaling developmental signaling pathways: Nodal, Sonic hedgehog, and center to induce dorsal and anterior structures. The shield is specified retinoic acid (RA). The forkhead box transcription factor FOXH1 (also very early in zebrafish development (the 128 cell-stage) and arises known as FAST1 and schmalspur in zebrafish) [22] and EGF-CFC family from the cells of the dorsal margin. The transcription factor β-catenin protein CRIPTO (also known as TDGF1 in mammals and one-eyed is stabilized in the nucleus of prospective shield cells, and when pinhead in zebrafish) [23] are components of the Nodal pathway. The zygotic transcription initiates, activates transcription of the Nodal signaling ligand SONIC HEDGEHOG (SHH), transmembrane receptor ligand and morphogen squint [37]. Within the shield, levels of Nodal PATCHED (PTC) and transmembrane sterol-sensing protein DIS- signaling are asymmetric. The most vegetal cells are subject to the PATCHED-1 (DISP1) are core members of the Sonic hedgehog signaling highest levels of Nodal signaling, and become the prechordal plate, pathway, while the transcription factor SINE OCULIS HOMEOBOX-3 while cells positioned nearer the animal pole, exposed to lower levels (SIX3) directly regulates shh expression [24–27]. The atypical home- of Nodal signaling, become notochord [38]. The prechordal plate, after odomain transcription factor TG-INTERACTING FACTOR (TGIF) regulates the cell movements of gastrulation, comes to underlie the ventral retinoic acid signaling during development [28–30], while ZINC forebrain, while the notochord underlies the more posterior neural FINGER OF CEREBELLUM (ZIC) transcription factors have recently tube. been proposed as a regulator of Nodal, Sonic hedgehog, and retinoic acid signaling in the forebrain [31]. There is significant cross- 2.2. Nodal signaling and HPE regulation among these three pathways, which has important implications for early brain development. This cross-regulation is an Nodal is a member of the TGF-β superfamily of signaling proteins. important consideration in interpreting phenotypes associated with Members of this protein superfamily exist as dimers, and activate an alteration of gene function within the three pathways, such as in type I and type II transmembrane serine/threonine kinase receptors. HPE patients (see Fig. 1 for an anatomical overview of forebrain Nodals bind the type I receptor protein ALK4 or the complex of ALK7 signaling relevant to HPE). (type I) and ActRIIB (type II) [39]. The HPE gene CRIPTO/oep physically interacts with the ligand–receptor complex, and is required for Nodal 2.1. Specification of the prechordal plate signal transduction, positioning this protein as a Nodal co-receptor [40–43]. Ligand binding results in the phosphorylation of the type I The prechordal plate is the anterior-most axial mesoderm, and a receptors, which in turn phosphorylate the transcription factor critical ventral patterning center that underlies the developing Smad2. Phosphorylated Smad2 is imported into the nucleus, where forebrain. Research in zebrafish has delineated a clear role for Nodal it binds Smad4 and other cofactors, including bonnie and clyde (bon; mixer) and the HPE gene FOXH1 (schmalspur) [44,45]. The Smad complex then activates the transcription of target genes, such as the prechordal-plate-specific transcription factor goosecoid (gsc) [46] and the morphogen shha [47]. Forward genetic screens in zebrafish have identified mutations in the Nodal pathway components linked to HPE: the co-receptor Cripto/ oep, and the Smad2 cofactor FOXH1/sur [48,49]. Loss of function of these genes causes the loss or reduction of the shield and the elimination of the prechordal plate, causing severe forebrain deficits within the HPE spectrum, including cyclopia.

2.3. Cripto/one-eyed pinhead in HPE

oep transcript is maternally deposited in 1-cell zebrafish embryos, and embryos lacking both maternal and zygotic oep (MZoep) have a much more severe phenotype than zygotic mutants. MZoep mutants fail to develop a morphologically distinguishable shield, and lack all shield-specific gene expression. As a consequence, embryos lack most mesodermal cell types, including the prechordal plate and notochord. This in turn causes a failure to specify ventral neurectoderm, and embryos display a cyclopic phenotype [40]. In zygotic oep mutants that retain maternal sources of this protein, shield-specific gene expression is initiated normally, but fails to be maintained. The notochord is specified correctly, and expresses shha, suggesting that low levels of Nodal signaling are sufficient for notochord development. However, zygotic oep mutants lack pre- Fig. 1. Ventral forebrain patterning is regulated by signaling between the pectoral plate and overlying neural tube. Holoprosencephaly (HPE) is caused by a complex set of chordal plate, and shha is not expressed in forebrain regions, molecular genetic signaling pathways. Retinoic acid (RA, gradient shown in red) plays a indicating that the specification of prechordal plate is particularly key role in this process and is synthesized in the posterior paraxial mesoderm by sensitive to a reduction in Nodal signaling [36]. Aldh1a2 and degraded anteriorly by Cyp26a1. Tgif, which causes HPE, regulates Two human patients with mutations in CRIPTO have been expression of both cyp26a1 and aldh1a2. Within the prechordal plate (PCP, blue), described [23]. One patient displays several HPE-like microforms, signaling among Zic, Nodal (Cyclops or Cyc) and Sonic hedgehog (Shh) creates a signaling center underlying the ventral diencephalon. Shh signaling activates both ptc1 including a hypoplastic corpus callosum. This patient has a hetero- and nkx2 genes. zygous P125L mutation in the CFC domain of the oep orthologue 392 P.A. Gongal et al. / Biochimica et Biophysica Acta 1812 (2011) 390–401

TDGF1, which is required for the protein to bind type I TGF-β ventral neuronal identities, and the correct separation of the eye field receptors. While wild type TDGF1 will rescue the zebrafish oep mutant into two distinct domains [55]. phenotype, a P125L mutant version does not, suggesting that this The mechanism of Shh signal transduction has been the subject of protein is nonfunctional, and the malformations in this patient may be intense research. The protein undergoes extensive post-translational due to TDGF1 haploinsufficiency. A second patient displays semi-lobar modifications, including autocatalytic cleavage and cholesterylation of HPE, and a V159M mutation in CRIPTO, which is in a domain thought the N-terminus, which becomes the mature ligand [56]. Subsequently, to mediate the protein's association with the cell membrane [50]. a palmitate moiety is added to the N-terminus of the ligand by the However, this mutant version of the protein can still rescue an oep enzyme known as Hedgehog acyltransferase or Skinny hedgehog [57]. mutant, suggesting that the basis of HPE in this case may involve The N-terminal signaling ligand processed with both lipid modifica- additional genetic or environmental factors. Indeed, within most tions is denoted as ShhNp. Cleavage and lipid modification is required patient families, the penetrance of HPE is highly variable. It has been for secretion outside the cell. A series of proteins, including Dispatched widely speculated that interaction between multiple genetic defi- and You/Scube2 are required for its export. ShhNp then binds to ciencies or environmental insults are responsible for this variability, transmembrane proteins (Interference hedgehog; IHOG and Brother and underlie most cases of HPE [4–6]. of IHOG; BOI) that facilitate its interaction with the Patched receptor [55]. In the absence of ligand, Ptc inhibits the function of Smoothened 2.4. The transcription factor Foxh1 and HPE (Smo). Upon ligand binding, this inhibition is relieved; Smo becomes activated, and induces a signaling cascade that leads to the regulation FOXH1 is a Smad-binding protein which targets activated Smad of the localization or cleavage of Gli transcription factors. In Drosophila, complexes to specificDNAsequences[51].Itisrequiredfor a single Gli protein, named Cubitus interruptus (Ci), is present. Upon transduction of a subset of Nodal signals, and is partially redundant ligand binding, the cleavage of Ci into a repressor isoform is inhibited, with the paired-like homeobox transcription factor Bon/Mixer [52]. and the activator isoform transcriptionally upregulates target genes Zebrafish lacking maternal and zygotic Sur (MZsur) have reduced, but [58]. not eliminated, shield-specific gene expression. MZsur embryos also Sixty-four unique mutations in the SHH gene have been linked to lacking bon have abolished shield-specific gene expression, indicating holoprosencephaly [59]. These include frameshift and nonsense that these genes are partially redundant. Foxh1 mouse mutants mutations, missense mutations in functional domains or uncharacter- entirely lack specification of the node, prechordal plate, and ized but conserved domains, or changes in residues important for notochord, and display a severely hypoplastic forebrain [53]. processing, such as the post-translational addition of palmitate and Similarly, MZsur zebrafish mutants also show the elimination of the cholesterol moieties. Two orthologues of SHH exist in zebrafish, shha prechordal plate, although notochord-specific gene expression is still and shhb (formerly known as tiggy-winkle hedgehog). This complicates present. Interestingly, notochord-specific genes are activated even in analysis of mutants for these two genes, as each can compensate for the absence of both bon and sur, suggesting that these two are not the the other's function. Mutants lacking shha have a mild phenotype, only Smad cofactors that can transduce Nodal signal to specify including abnormal fin and somite patterning, and axonal pathfinding notochord in zebrafish. Besides its role as a Nodal signal transducer, defects, while knockdown of shhb alone causes no detectable Sur is also required for the maintenance of cyclops and squint phenotype [60–62]. Because of redundancy, shh loss of function has expression during gastrulation [44], however, expression of neither mostly been accomplished in zebrafish by mutations in or pharma- cyclops nor squint can rescue prechordal plate specification, cyclopia, cological inhibition of Smoothened (Smo) [63].Thecompound or other phenotypes in MZsur embryos. Therefore, it is likely that cyclopamine directly binds and inhibits Smo signal transduction these defects mostly result from an inability to transduce Nodal [64]. Unlike the Shh ligand itself, smo does not appear to be duplicated signals within the axial mesoderm. in the zebrafish genome, and smo mutants show a severe phenotype, A number of mutations in FOXH1 have been identified in HPE although not as severe as cyclopamine-treated embryos. Smo is patients [22], including those that disrupt the forkhead DNA binding present maternally, and maternal transcript is likely to allow for some domain and Smad-interaction domain. The activity of these mutant early Shh signal transduction, which cyclopamine would block. smo versions has been assessed by measuring the activity of RNA to rescue mutants have a suite of defects, including synopthalmia and improper zebrafish sur mutants. These studies demonstrate that mutations in motor neuron specification in the spinal cord. Moreover, mutants the Smad-interacting domain eliminate protein activity, while show a dorsalized forebrain and a complete lack of hypothalamic variants in the forkhead domain reduce protein activity by approx- tissue. smo mutants also have a lack of anterior pituitary cell fates, and imately 20% [22]. instead form an ectopic lens at this site. Interestingly, an optic stalk Overall, in live births, the occurrence of Nodal pathway mutations is marker, pax2a, is ectopically expressed in the forebrain, suggesting rare [22]. Work in animal models suggests that this is likely due to the that hedgehog signals play a key role in distinguishing forebrain from essential nature of this signal pathway during the very early stages of eye identities [63]. development. Indeed, in patients suffering from recurrent miscarriage, a The HPE gene PATCHED is the receptor for Shh, and is in fact a very high proportion of embryos lost in the first trimester show severe negative regulator of the pathway. Unliganded Ptc represses Smo, and neural and midline defects [54]. Embryos with severe loss of Nodal ligand binding prevents Ptc repression activity. Therefore, loss of Ptc function are likely not viable, and it is plausible that Nodal pathway function results in constitutive activation of the Shh pathway [65–67]. mutations contribute to a proportion of early pregnancy losses. Zebrafish have two ptc homologues, and mutants for both ptc1 and ptc2 have a strong upregulation of hedgehog signaling, concomitant 2.5. Sonic hedgehog signaling and HPE with a ventralization of the neural tube and mispatterning of the somites [68]. Conversely, gain of Ptc function results in constitutive Defects in the Sonic hedgehog pathway are the most frequent repression of the pathway, and also results in phenotypes within the cause of HPE [18], and this pathway has long been known to be spectrum of HPE, including midline defects, such as malformation of essential for forebrain patterning. An essential role for Nodal signaling the corpus callosum [66,69]. is the initiation of shh transcription in the prechordal plate and Several PTC mutations have been linked to HPE. Two of these are in notochord [38]. Shh secreted from the axial mesoderm initiates shh the extracellular domain in a position thought to interact with the transcription in the floor plate, the most ventral and medial cells of the SHH ligand. Two others are located within intracellular domains of the neural tube. A graded hedgehog signal emanating from both the axial protein, and it is hypothesized that these domains might be important mesoderm and floor plate is required for diencephalon specification, for PTC's interaction with SMO [70]. It has been proposed that these P.A. Gongal et al. / Biochimica et Biophysica Acta 1812 (2011) 390–401 393 mutations represent a gain of PTC inhibitory function of the SHH contribution to zebrafish neural or eye patterning has yet to be pathway, as HPE phenotypes are well-known to be linked to SHH loss established. However, mutations in mouse and human Cyp1b1 are of function. However, it is possible that a gain of SHH function could linked to the development of juvenile-onset glaucoma, suggesting an cause HPE-like phenotypes as well, since in zebrafish, a loss of Ptc1 important role in eye development [86–88]. and consequential increase in signaling results in narrow-set eyes and The best characterized role of RA during development is in the moderate forebrain patterning defects [68]. hindbrain, where it acts as a potent posteriorizing signal on neural The function of the HPE gene DISPATCHED has also been modeled tissue. High levels of RA signaling are required for specification of in zebrafish, where there is a single described orthologue [71]. caudalhindbrainfates[82,83,89–91]. During gastrulation, the Although disp1 expression is broad, it is enriched in regions of high forebrain is insulated from the posteriorizing influence of RA through Shh signaling: the ventral forebrain, notochord, and floor plate. In the activity of cyp26 (cytochrome P450, subfamily XXVI) genes, which Drosophila, disp has been shown to be required for export of processed hydroxylate RA, a modification thought to target the molecule for Shh ligand [72]. Suggesting that this function is conserved, disp loss- degradation [92]. cyp26a1 is the earliest expressed cyp26, and is of-function in mice and zebrafish causes phenotypes typical of Shh expressed in the anterior neurectoderm, initiating during gastrulation deficiency [73–76].Zebrafish embryos with mutations in disp1 [93]. Slightly later, cyp26b1 and cyp26c1 transcription is initiated in a (chameleon) have phenotypes typical of a reduction in Shh signaling: subset of telencephalic cells (cyp26c1) and the diencephalon (cyp26b1 strongly reduced expression of shh target genes, and a dorsalized and cyp26c1) [94,95], where they limit RA activity. Both cyp26b1 and neural tube. Further supporting the model that disp regulates Shh cyp26c1 are also expressed in dynamic, rhombomere-specific patterns export, overexpressing shh RNA in disp1 zebrafish mutants causes an in the hindbrain, where they are essential for proper anterior– attenuated transcriptional response compared to shh overexpression posterior patterning [94–97]. Together, RA synthesis and degradation in wild type embryos [76]. genes set up a highly dynamic pattern of RA levels in the developing CNS. 2.6. The transcription factor Six3 and HPE Abnormal RA levels have severe phenotypic consequences, particularly for the forebrain. Exposure to exogenous RA during Zebrafish models of the function of the HPE gene SIX3 are development causes severe craniofacial defects, eye patterning complicated by the presence of three SIX3-related genes, six3a, anomalies, and ectopic forebrain expression of posterior CNS genes six3b, and six7, which are all expressed in the prechordal plate during [11,98,99]. RA levels become abnormally high when Cyp26 activity is gastrulation, and then in the anterior neurectoderm during somito- reduced, which has been modeled in mice, chick, and zebrafish. genesis [77]. Interestingly, however, six3b/six7 loss of function Overall, embryos with reduced Cyp26 function display defects in indicates that Six3 proteins are required in the neurectoderm to patterning of the diencephalon, eye, hindbrain and neural tube repress Nodal target gene expression, which contributes to the proper closure defects [30,96,100–102]. Further, without Cyp26 activity, establishment of left/right asymmetry [77]. Complete loss of six3 gene embryos are highly sensitive to environmental changes in retinoids, function may reveal additional roles for six3 regulation of Nodal and the entire neural tube takes on posterior hindbrain identity upon signaling. exposure to doses of retinal that cause no defects in wild type Zebrafish rescue studies indicate that the vast majority of HPE- embryos [96]. associated SIX3 mutations act as hypomorphs [78]. Work in mice High levels of retinoic acid have recently been linked to DiGeorge suggests that defects in a direct interaction between SIX3 and the Shh syndrome (also known as velo-cardio-facial syndrome), a suite of signaling pathway underlie the development of HPE [24]. Geng and multiple congenital defects, including craniofacial abnormalities such colleagues generated mice with a HPE mutant version of Six3 as cleft palate [103,104]. A critical region mapped in patients with knocked-in to the endogenous Six3 locus. While heterozygous mutant chromosomal deletions encompasses the gene tbx1, which regulates mice display HPE-like phenotypes at a low frequency, removing one expression of all three cyp26 genes during somitogenesis [102]. Taken copy of Shh results in severe cyclopia and loss of ventral diencephalic together with the large body of evidence that increased developmen- gene expression. Further, mice heterozygous for the mutant Six3 and tal RA levels can cause forebrain patterning defects, it is plausible that with one copy of Shh deleted have a loss of both Shh and Six3 changes in RA signaling underlie some of the phenotypes observed in expression in the ventral diencephalon, indicating that these two DiGeorge syndrome. genes form a critical positive feedback loop. The regulation of Shh by RA deficiency also causes severe developmental defects. Loss of RA Six3 is direct, and the Six3-binding site in the Shh forebrain enhancer has been modeled in vitamin A-deficient quail and swine [105–107], is mutated in a patient with semi-lobar HPE [79], indicating the mouse and zebrafish mutants for RA-synthesizing genes importance of the genetic interaction between these two proteins for [82,83,90,108], and chick and zebrafish by application of pharmaco- forebrain development. logical inhibitors [89]. Although defects observed vary slightly depending on the organism, common phenotypes include the 2.7. Retinoic acid signaling and HPE formation of a single telencephalic vesicle, mispatterning of the diencephalon, failure to specify the caudal hindbrain, and eye Retinoic acid has a critical role in early neural patterning. This small, abnormalities. Thus, both too much and too little RA cause defects diffusible molecule is synthesized from vitamin A (retinol) precursors in similar tissues, and many of these phenotypes are reminiscent of through a series of enzymatic reactions. The retinol dehydrogenase HPE. Interestingly, similar defects are observed in a gain or loss of RA, Rdh10 catalyzes the conversion of retinol to retinaldehyde during such as the mispatterning of the diencephalon and failure of the development [80], and aldehyde dehydrogenases (raldh or aldh1a neural tube to close. gene family) convert retinal to retinoic acid [81]. There are two characterized aldh1a genes in zebrafish: aldh1a2, which is expressed at 2.8. The transcription factor TGIF and HPE high levels in paraxial mesoderm and in the dorsal retina [82,83], and aldh1a3, which is expressed in the ventral retina flanking the optic Until recently, little was known about the role of the HPE gene TGIF stalk [84]. Recently, an additional enzyme, cyp1b1, has been demon- (5′-TG-3′ interacting factor) in forebrain development. Work in cell strated to synthesize RA in multiple tissues during chick development culture systems demonstrates that TGIF can transcriptionally regulate [85]. This gene is expressed in a much more restricted domain in both retinoid and TGF-β signaling. Tgif can bind Smad2 and Smad3, zebrafish than chick, and is present in the anterior telencephalon and and represses the transcription of a TGF-β-dependent reporter [109], eye, beginning mid-somitogenesis (Gongal et al., submitted). Its and mutant versions of TGIF that occur in human patients do not show 394 P.A. Gongal et al. / Biochimica et Biophysica Acta 1812 (2011) 390–401 this same repression activity [110]. As alterations of Nodal signaling multiple points in the pathway, including transcription of the ligand, are well-known to cause forebrain defects in animal models, and response to the signal, and post-translational processing. The Nodal is a TGF-β pathway, it was widely assumed that misregulation mechanisms of these interactions are unclear, and identifying the of Nodal function explained the incidence of HPE in human patients precise molecular bases that underlie RA-Shh interactions are key deficient in TGIF [5,7,110–113]. However, there has been a notable topics for future work. absence of evidence from animal models for this hypothesis. Interpreting the effects of changes in shh expression can be In fact, TGIF was originally identified because of its involvement challenging, as transcription of this gene is regulated by an with retinoid signaling. This atypical homeodomain transcription autoregulatory loop. When Shh signaling is reduced, shh transcription factor was discovered in a search for proteins that bind to a retinoid X is upregulated [118], so both upregulation and downregulation of the response element (RXRE) [29]. Bartholin and colleagues provide pathway may be correlated with an increase in shh transcription. further evidence that TGIF can regulate retinoid signaling [28]. They Nevertheless, shh expression is critical for activity of the pathway, and convincingly show that TGIF binds RXRs (most robustly RXRα and RA regulates signaling at this level. RXRγ, and RXRβ to a lesser extent), and that TGIF can form a complex Aldh1a2 mouse mutants have normal Shh transcription (and both with RXR and its nuclear receptor binding partners, including protein levels, detected by immunohistochemistry) in the axial RARs and PPARs (peroxisome proliferator activated receptor). Using a mesoderm during early stages of forebrain patterning. Slightly later reporter assay, Tgif can repress RA-dependent transcription under the in mouse development (by the 16 somite stage), however, RA does control of a DR5 element. However, this element is activated by RXR regulate the transcription of Shh itself. This regulation is tissue- expression, regardless of the presence or absence of ligand. Further, it specific, however, as in a Aldh1a2 mutant, Shh is decreased in some isn't clear whether this repression activity is specific for RXR or tissues (ventral diencephalon), and increased in others (infundibu- generally acts on other nuclear receptors. Therefore, the in vivo lum) [108]. The functional consequences of this loss of transcript are relevance of Tgif's function in this assay remains to be established. unclear, but occur too late to explain early loss of Shh target gene Four independent groups have generated Tgif-null mice [28,114– expression in aldh1a2 mutants. 116]. None of these mutants has neural patterning phenotypes. Mice In contrast, transcriptional regulation of shh by RA seems to be have multiple copies of Tgif-like genes, including Tgif2, Tgif-like-on- more important in bird development. In vitamin A-deficient quail, shh the-X and Tgif-like-on-the-Y, whose functions are poorly understood, expression is reduced in prechordal plate at early stages [106]. and which may compensate for the loss of Tgif activity in these Further, locally applying RAR and RXR inhibitors in the chick forebrain mutants. However, depending on genetic background, heterozygous abolishes shh expression. Treated embryos display cyclopia and loss of and homozygous mutants do have an increased rate of exencephaly forebrain tissues [119]. This forebrain phenotype is more severe than when treated with RA [28,116], suggesting that Tgif plays a role in the an Aldh1a2 mouse mutant, perhaps because receptor inhibition causes regulation of RA signaling. a more profound loss retinoid activity. Aldh1a2 and Aldh1a3 are A fifth group interested in Tgif function noted that some patients partially redundant in the mouse forebrain patterning, but double had a nonsense mutation that left the first repressor domain and mutants cannot be analyzed (without maternal supplementation with homeodomain intact, but eliminated the second and third repressor RA), due to very early lethality caused by cardiac defects. domains. Kuang and colleagues generated a mouse with an equivalent A second method of RA regulation of Shh signaling has recently mutation by deleting Tgif's third exon. Interestingly, mice with this been revealed in mouse. Aldh1a2 mutant mice have a reduction in Shh mutation do show neural patterning defects, although these are target gene expression, such as Gli3 and Olig2, while no change in Shh strain-specific [117]. TgifΔexon3 mice show severe hypoplasia of the transcription or protein levels is detectable [108]. Importantly, Ribes forebrain and exencephaly. Shh protein levels are reduced, and the and colleagues demonstrated that RA deficiency causes a failure to ventral forebrain marker Nkx2.2 has reduced expression. Because of appropriately respond to the Shh signal [120]. Although the these phenotypes, Kuang and colleagues propose that this version of mechanism of this interaction is not yet understood, treating Tgif may function as a dominant negative. However, defects in this Aldh1a2 mutants in vitro with Shh ligand does not result in high mutant are still highly dependent on genetic background, which levels of Shh target gene expression, as does treating wild type suggests that there are unknown and essential genetic modifiers that embryos. These results could reflect a role for RA in regulating the exist in this strain. activity of Shh protein activity, localization, processing, degradation, In contrast to mice, zebrafish have only two tgif family genes, tgif or RA signaling could be required for the transcription or function of and tgif2, and therefore genetic redundancy may be less of an issue for a Shh pathway component. Future experiments will reveal the myriad functional analyses. Studies in zebrafish have revealed that Tgif is an of mechanisms used in regulating Shh activity, such as modulation of essential regulator of retinoic acid metabolism gene expression [30]. palmitoylation [121,122]. Tgif is required for the initiation of both cyp26a1 and aldh1a2, indicating that this gene regulates both RA degradation and synthesis. 2.10. Zic genes and the coordinated regulation of Shh and RA signaling tgif morphants have reduced diencephalic gene expression, a phenotype that resembles that of cyp26a1 mutants, suggesting that While cross-talk between the Shh and RA pathways is likely essential excess RA in the forebrain is responsible for this mispatterning. These to coordinate their activities during neural patterning, these two results suggest a molecular basis for Tgif-dependent holoprosence- pathways are also connected by common regulators. A transcription phaly; embryos lacking TGIF function may be highly sensitive to factor that regulates both the RA and Shh pathways has been identified: teratogenesis from environmental fluctuations in vitamin A [30]. zic1. The precise effect of a single factor transcriptionally regulating components of multiple pathways is not well understood. However, it is 2.9. Genetic interactions between Sonic hedgehog and retinoic acid plausible that this method of regulation functions to maintain a precise signaling in the forebrain balance between the activities of the two pathways. zic1 (whose related gene zic2 hasbeenlinkedtoHPE)hasbeen While deleterious for the forebrain during gastrulation, RA is in linked to disruptions to both the retinoic acid and Shh signaling fact required for proper forebrain development at later stages. One of pathways [31]. zic1 morphants show a loss of Shh target gene expression its important roles is facilitating Sonic hedgehog signaling. The genetic (ptc1), concomitant with a mild reduction in shha expression and a interactions between Shh and RA signaling are complex, and are quite strong reduction in shhb expression. zic1 loss-of-function results in different between species, as well as between different tissues and higher levels of RA signaling in the eye and forebrain mid-somitogen- developmental stages (see below). RA regulates Shh signaling at esis. zic1 morphants have an expansion of optic stalk in the eye and P.A. Gongal et al. / Biochimica et Biophysica Acta 1812 (2011) 390–401 395 forebrain, a tissue that strongly expresses the RA-synthesizing genes 3.1. Sonic hedgehog signaling is required for closure of the optic fissure aldh1a3 and cyp1b1, which might cause the high RA levels observed in the eye and forebrain. Overexpressing cyp26a1 makes some zic1 To date, only one family has been identified with coloboma due to morphant phenotypes more severe, suggesting that RA deficiency, mutation in the sonic hedgehog (shh) gene itself [135]. It is likely that rather than excess, contributes to these phenotypes. Indeed, RA levels in null mutations, or even strong hypomorphic mutations in the Shh the somites are reduced, as assayed by RARE-yfp reporter expression. gene are incompatible with life as Hedgehog signaling is required Nevertheless, although the detailed mechanisms underlying Zic1 forebrain patterning and development. Thus, most knowledge function are not yet clear, its overall function is clearly to regulate concerning Shh and its role in ocular coloboma comes from both RA and Shh signaling, which may help facilitate the coordinated experiments in animal models. Both the zebrafish and mouse have actions of these two pathways during neural development. proven to be useful models for the study of the eye, and have provided Zebrafish zic2 has also been shown to play a key role in regulating valuable insight as to the role of Shh and its downstream target genes forebrain development. Morphant embryos that lack zic2a display a in the development of coloboma. pronounced reduction in arx and dlx2 gene expression, indicating a In zebrafish, like most other vertebrates, there are two tissues that defect in specification of the prethalamic region of the forebrain [123]. act as sources of Sonic hedgehog ligand that are relevant for the Though the changes in ptc1 gene expression are mild, it is possible development of the eye: first, the floorplate in the ventral midline of that other zic genes are compensating for a loss of zic2a [124]. Recent the diencephalon, and second, the neural retina itself. It is the former research on zic2a has shown that it plays an essential role in that is responsible for eye patterning and closure of the optic fissure regulating proper formation of the eye. Indeed many of the genes [138], while Shh signaling derived from within the retina is required discussed in this section function at the same developmental stage to later in development for the timely cell cycle exit and differentiation regulate formation of the eye. Given the striking similarities between of neural lineages [139]. forebrain and eye patterning mechanisms, the second component of this review will deal with eye formation birth defects, most notably 3.2. Sonic hedgehog regulates genes required for eye patterning colobomata, and the known signaling pathways that regulate ocular morphogenesis. The zebrafish eyecup is partitioned early in development, estab- lishing the retina, optic stalk, and retinal pigmented epithelium into molecularly distinct domains. Midline diencephalic Sonic hedgehog is 3. Part II: Coloboma required for this partition by maintaining the expression boundary of spatially restricted transcription factors [138]. Loss of shh in zebrafish During early development, the vertebrate eye contains a transient leads to severe coloboma, and the loss of ventral-anterior homeobox 1 ventral fissure that is essential for the entry of mesenchyme cells into and 2 (vax1 and vax2), which delineate the optic stalk and ventral the retina, where they will give rise to blood vesicles that will nourish neural retina regions [140]. Inactivation of both vax genes in zebrafish the eye throughout the life of the organism [125]. Failure of the optic results in severe coloboma [140], supporting the model that shh acts fissure to close results in coloboma, an important cause of visual genetically upstream of vax genes to regulate closure of the optic impairment worldwide. Depending on the size and location, this fissure (Fig. 2). congenital malformation can lead to blind spots, lazy eye, and reduced In addition to regulating vax transcription factors, Shh is required visual acuity in patients [126]. for the proper expression of members of the paired box family of Coloboma can affect numerous tissues in the posterior eye including transcription factors, with pax2 and pax6 being important for eye the choroid, and optic nerve, as well as tissues of the anterior eye such as development and closure of the optic fissure. Shh is required for the iris, cornea, and lens [127]. Ocular colobomata have been observed expression of pax2, while negatively regulating the levels of pax6 as an independent defect, or as a component of syndromes that often [138,141]. Mutations in both genes have been mapped in patients include other ocular abnormalities as well as craniofacial and neuro- with coloboma or colobomataneous syndromes; mutations in pax2 logical phenotypes [128]. These include microphthalmia [127,129,130], are linked to renal-coloboma syndromes [142,143] while mutations in cataracts [131], and CHARGE syndrome (coloboma, heart defects, pax6 tend to be found in patients with coloboma of the optic nerve choanal atresia, retarded growth, and genital and ear anomalies) (along with other retinal phenotypes including aniridia). pax2 [132]. Colobomata can be sporadic, or heritable with autosomal expression is restricted to proximal eye structures such as the optic dominant, recessive, and X-linked inheritance cases being reported. stalk and fissure, while pax6 is restricted to more distal structures. The The vertebrate eye is patterned along proximal–distal axis early in expression of pax2 in cells surrounding the choroid fissure requires development. The distal eye cells give rise to neural retina and retinal Shh, and overexpression of shh leads to pax2 expression in more distal pigmented epithelium, while more proximal cells give rise to the optic structures such as the neural retina [138]. Conversely, loss of Shh stalk and choroid. Although transient, the choroid fissure acts as an signaling results in reduced expression of pax6 neural retina. Thus, it entryway into the eye, allowing vascularization to occur. Before the appears that control of pax gene expression via shh is required for the onset of cellular differentiation in the retina, the fissure begins to close proper partition of early eye to define and maintain the partition of using a contact-dependent dissolution of the basal lamina at the optic stalk and neural retina. It is clear from studies involving both Pax contacting neuroepithelial on each side of the optic fissure [133]. and Vax proteins, that early embryonic patterning of the eye into Thus, genetic abnormalities or environmental toxins that disrupt this distinct optic stalk and neural retinal lineages is essential for process, likely present as coloboma. subsequent fusion of the optic fissure. A number of genetic lesions and chromosomal rearrangements Recent evidence from zebrafish model studies suggests that the have been identified that lead to coloboma in human patients, many regulation of pax genes via Sonic hedgehog signaling is indirect, and of which involve the loss of Sonic hedgehog signaling or one of its involves the regulation of other factors that modulate pax gene downstream target genes [134,135]. As aberrant Shh signaling is one expression. One such factor, Zinc finger protein of the cerebellum 2a of the leading causes of holoprosencephaly, it is important to note (Zic2a), has been shown to negatively regulate the transcriptional that many individuals suffering from mild forms of HPE output of Sonic hedgehog signaling, while itself being transcription- also display coloboma. In addition, defects in the RA [136,137],and ally regulated by Hedgehog signaling cascades [124]. Loss of zic2a Bmp [127] signaling pathways have been implicated in the results in severe coloboma [124,144,145] likely due to mispatterning generation of coloboma or syndromes involving colobomataneous of the optic stalk and retina, as optic stalk markers are ectopically phenotypes. expressed in the retina in Zic2a-depleted embryos [124]. 396 P.A. Gongal et al. / Biochimica et Biophysica Acta 1812 (2011) 390–401

tion of the retinoblastoma gene (Rb), leading to abnormal cell cycle progression and increased proliferation [147]. Work in mice demon- strates that accurate regulation of the cell cycle acts upstream of Sonic Hedgehog and causes abnormalities in the eye. Loss of function studies of involving the cell cycle regulator E2F4 result in coloboma, and aberrant expression of Sonic Hedgehog ligand at the ventral midline [148]. E2F4 is a transcription factor downstream of the Rb gene, further linking Rb cell cycle control to the development of coloboma. In addition, Shh effector genes are abnormally expressed upon loss of E2F4, with Pax2 expression extending ectopically to the distal optic cup, and reduced Pax6 expression in the ventral optic cup [148].

3.3. Retinoic acid plays a key role in closure of the optic fissure

Retinoic acid has long been implicated in the development of the eye in humans and animal models. Like Sonic hedgehog, mutations in components of the core RA pathway are rarely found in human patients with coloboma due to the wide array of functions retinoic acid performs in the developing embryo. However mutations in Retinol Binding Protein (rbp) have been described in a sibling-pair that presented with iris coloboma [149]. In both siblings, no serum Rbp was detected, and patients had approximately 17% of normal retinol levels. In addition to iris coloboma, these patients also suffered from night blindness but were otherwise healthy. Furthermore, there is evidence that suggests that in some populations, vitamin A-deficient (VAD) diets are correlated with the incidence of coloboma [136], reiterating the link between aberrant retinoid levels to the develop- ment of coloboma. Animal models have yielded major insights into the role of retinoic Fig. 2. Sonic hedgehog signaling is required for eye patterning the eye along the medio- acid during eye development. For example, mice that receive lateral axis. A complex transcriptional interaction between Sonic hedgehog (Shh) and teratogenic doses of retinoic acid while pregnant give birth to pups Zic pathways specifies the identity of the midline of the ventral diencephalon. The Shh with eye defects, including coloboma [150,151]. In zebrafish, local fi diffusible ligand is required for speci cation of ventral eye structures, including the delivery of RA to the eye via implanted RA soaked beads not only optic stalk and ventral neural retina. Shh is required for expression of vax2 and pax2 fi transcription factors, both of which are expressed in the optic stalk and ventral retina. results in coloboma, but can also induce ectopic ssure formation This signaling cascade is opposed in the dorsal retina through the action of Bmp ligands, anywhere in the retina [152]. Furthermore, failure to close the optic such as Gdf6. Bmp signaling cascades are required for the expression of dorsal retinal fissure in RA treated mice correlates with changes in gene expression fi speci c transcription factors such as Tbx5 and Tbx2. Loss of Shh signaling, or any of its along the proximal–distal axis of the developing eye [151] in a downstream signaling components can lead to coloboma. Loss of Bmp signaling in the dorsal retina can also lead to coloboma, as well as ectopic fissure formation in the dorsal manner comparable to that seen with a loss of Sonic hedgehog retina. signaling. Epistasis analysis indicates that sonic hedgehog itself is downregulated by teratogenic doses of RA [153], although this relationship was only tested in tissues other than the floorplate (the Although numerous candidate genes have been identified down- critical source of Shh ligand for optic fissure closure). Nevertheless, stream of Shh for the development of coloboma, there is little data this demonstrates that RA can act upstream of Shh in a living concerning the mechanism by which fusion of the optic fissure is organism. prevented in shh mutants. In wild type animals, fusion is facilitated by Although teratogenic doses of RA clearly regulate Shh signaling contact between the epithelial tissue on either side of the optic fissure and cause coloboma, little work has been done involving manipula- leading to a degradation of the basal lamina. Inhibition of Shh tion of RA at physiologically relevant levels. In most studies, embryos signaling and the consequential mispatterning along the proximal– are incubated in a concentration of RA that far exceeds what an distal axis of the eyecup have been shown in some cases to physically embryo would naturally be exposed to during development. In the prevent contact between epithelium on either side of the optic fissure. developing embryo, RA levels are higher in the ventral retina than in For example, in the zebrafish blowout mutant where there is a the dorsal retina, thus creating a gradient of retinoic acid signaling mutation in ptc1, (a gene that functions as a Shh receptor and negative across the dorsal–ventral axis of the eye [154,155]. This gradient regulator of Shh signaling; see Section I of this review), ectopic cannot be mimicked by simply exposing the whole embryo to RA. expression of optic stalk markers such as pax2, is observed, and the Thus, loss of function studies are required to determine if endogenous stalk grows ectopically into the retina, physically preventing contact RA is required for closure of the optic fissure. These studies have been between the epithelial tissue on both sides of the optic fissure [146]. fewer in number, and the role of endogenous RA in fissure closure Conversely, when pax2 is lost in zebrafish, contact between the appears to differ among different model systems. It has been noted, epithelial tissues on either side of the optic fissure still occurs, yet for example, that treatment of developing mice with citral, a there is no dissolution of the basal lamina and the fissure persists. competitive inhibitor of aldehyde dehydrogenases [156–158] caused Thus, pax2 must be required for fissure closure subsequent to contact a delay in (although did not prevent) the closure of the optic fissure between the two sides of the fissure. [151]. Vitamin A depletion in mice also leads to coloboma that occurs It has recently been demonstrated that cell cycle regulation may with a reduction in ventral retinal cell number and a loss of cell also play a key role in regulation of optic fissure closure, and is linked adhesion molecules surrounding the optic fissure [159]. This to Shh signaling. For example, mouse humpty dumpty mutants display phenotype may be due to abnormal morphogenesis of the early severe coloboma and cell cycle defects. This transgenic mouse eyecup, as opposed to later patterning events, as when aldehyde contains a mutation in Phactr4, which results in hyperphosphoryla- dehydrogenase genes are knocked out in mice, the dorsal eyecup P.A. Gongal et al. / Biochimica et Biophysica Acta 1812 (2011) 390–401 397 invaginates, but the ventral eyecup completely fails to form [160]. constitute antagonizing signaling centers required for patterning the Similarly, in zebrafish, treatment of embryos with citral results in the eye along the dorsal–ventral axis and closure of the optic fissure. failure of the ventral eye to form, while the dorsal half remains In many model systems, Bmp4 and Gdf6 have been proposed to relatively normal [154]. Knockout of the dorsal-specific aldh1a2 does function redundantly. As loss of gdf6a results in coloboma and has not result in coloboma, although it is likely that compensation by the been shown to regulate retinal expression of bmp4 [162], it is likely ventral-specific aldh1a3, for which there is currently no mutant that Gdf6a also plays a role in the formation of coloboma in embryos zebrafish strain available. Thus it appears in both mice and zebrafish, lacking Smoothened function. Surprisingly, loss of Smoothened does endogenous RA may affect closure of the optic fissure not because of not result in any change of expression retinoic acid synthesizing genes improper patterning of ocular tissues, but due to its requirement for such as Aldh1a2 or Aldh1a3 [165]. Gdf6 has been shown to regulate the earlier morphological events leading to morphologically normal eyes. expression of both of these genes, demonstrating that although Bmp4 and Gdf6a act redundantly for some developmental processes, at least some independent functions for each molecule are required for 3.4. Bmp signaling and coloboma closing the optic fissure.

Mutations or chromosomal aberrations involving members of the 3.5. Coloboma may be linked to overall growth of the eye bone morphogenetic protein (Bmp) family have also been described in human patients with coloboma. Asai-Coakwell and colleagues Mutations in human BMP4 and GDF6 result in both coloboma and demonstrated that a deletion encompassing GROWTH AND DIFFER- microphthalmia, indicating that closure of the optic fissure may be ENTIATION FACTOR 6 (GDF6/BMP13) is likely responsible for bilateral genetically linked with the overall growth of the eye in utero.A retinal coloboma and unilateral iris coloboma [127]. Further studies number of other genes have been reported to cause both micro- revealed that mutations in the prodomain of GDF6 account for 1.6% of phthalmia and coloboma, including transcription factors such as retinal colobomata in the human population [161]. This mutation orthodenticle homeobox 2 (otx2) and Sex determining region Y-box 2 results in reduced Gdf6 protein secretion from the cell, which is (sox2). Whole gene deletions in Otx2 tend to result in severe mechanistically consistent with Gdf6's non-cell autonomous effects microphthalmia, while milder mutations tend to result in either [162,163]. Mutations or chromosomal aberrations involving the unilateral or bilateral coloboma [167–169], suggesting that the level of closely-related bmp4 can also cause coloboma in human patients gene activity influences both growth of the eye and closure of the [164]. optic fissure. Although required for differentiation of the retinal Animal models have confirmed the association of gdf6 and bmp4 pigmented epithelium [170,171], otx2 expression is also observed in with coloboma, and have provided insights as to the mechanism by the early proliferating cells of the eyecup [172], and could thus affect which each gene can affect closure of the optic fissure. Both genes are growth of the eye before it is required for specification of RPE. expressed in the retina during early stages of development, and However to date, there is no data that sheds light on the mechanism knockdown of gdf6a (one of two gdf6 paralogues in the teleost by which otx2 influences both growth of the eye and closure of the genome) in zebrafish results in retinal coloboma [127,162]. Similarly optic fissure. to exogenous RA application, loss of Gdf6a signaling in zebrafish leads Mutations in the SOX2 gene have also been identified in human to ectopic fissure formation [162,163]. Studies of axial patterning patients [173]. Similar to Otx2, the severity of loss of function in Sox2 molecules expressed in the retina indicate that gdf6a is responsible for correlates with the severity of disease, whereby mutations causing limiting ventral eye marker expression, as loss of this protein results complete loss of function result in severe microphthalmia, while in expansion of vax2 expression to include both the ventral and dorsal milder variants tend to manifest as coloboma [167,174]. Although it is retina. This may explain the ectopic fissure in the dorsal eye, which is not known whether Sox2 is directly involved in closure of the optic respecified to a ventral fate in the absence of Gdf6a. In addition, Gdf6a fissure, the product of this gene is required for the proliferation of is required for proper establishment of RA signaling in the eye. For early retinal progenitor cells [174], reiterating the potential genetic example, the dorsal-specific aldh1a2 is not expressed in embryos link between eye growth and closure of the optic fissure. lacking Gdf6a, while the ventral-specific aldh1a3 is upregulated [162]. While details regarding the mechanism by which bmp4 can cause 4. Conclusions coloboma are not as well understood, it has been noted that bmp4 expression overlaps with both sonic hedgehog and patched1 expres- The genetic pathways that influence the formation of HPE and sion. Further suggesting a genetic interaction between BMPs and coloboma, as outlined in this review, have considerable overlap. Sonic hedgehog signaling is that an increased phenotypic severity is Holoprosencephaly and colobomata represent birth defects that can observed in human patients with low-penetrance mutations of both arise from aberrant Sonic hedgehog signaling. Shh is required in the bmp4 and shh than with either single mutation [164]. Supporting this forebrain for the formation of ventral neural structures and division of model, studies in mice have implicated Bmp4 as essential for the early eye field into two distinct hemispheres. Interference with Hedgehog induced coloboma. In a conditional knockout of the this signaling pathway, either through genetic mutation or via Smoothened gene, which is required to transduce the Hedgehog environmental factors can thus cause holoprosencephaly. Forebrain signal, Bmp4 expression is strongly expanded throughout the ventral Shh signaling is also needed for early eye patterning, a process that is retina [165].AsBmp4 expression is normally confined to the dorsal required for closure of the optic fissure. Given the overlap in retina, the authors speculate that coloboma in these embryos may be mutational etiology, holoprosencephaly and coloboma are likely to caused by ectopic BMP signaling. In addition, the expression of ventral represent severe and mild ends of the same phenotypic spectrum. retinal and optic stalk markers, including Vax2 and Pax2, show Retinoic Acid signaling pathway can also play a role in the reduced expression in Smoothened-null embryos, however only at development of HPE and coloboma. RA is required for proper developmental time points after ectopic expression of Bmp4 is forebrain morphogenesis and influences Shh signaling through a yet observed. Thus, loss of ventral retinal and optic stalk gene expression, unknown mechanism. In the eye, RA is required for morphological and the resulting coloboma in embryos suggest that the lack of events required for proper eye morphogenesis, and possibly for Hedgehog signaling results in the inability to limit Bmp4 signaling. patterning eye tissues. Nodal signaling is also important, as it is Furthermore, overexpression of Bmp4 in the developing optic vesicles required for the initiation of shh expression in the ventral forebrain. In can inhibit Hedgehog expression itself [166], supporting a model the eye, Bmp signaling is required for eye patterning, and thus closure whereby localized expression of Sonic hedgehog and Bmp ligands of the optic fissure, possibly through an antagonistic signaling 398 P.A. Gongal et al. / Biochimica et Biophysica Acta 1812 (2011) 390–401 relationship with Shh. It is thus clear that alteration of genetic spectrum of the sonic hedgehog gene in holoprosencephaly: SHH mutations fi fl cause a signi cant proportion of autosomal dominant holoprosencephaly, Hum. pathways that subsequently in uence Shh signaling, in addition to the Mol. Genet. 8 (1999) 2479–2488. Shh pathway itself, can result in the generation of HPE or coloboma. [26] E. Roessler, Y. Ma, M.V. Ouspenskaia, F. Lacbawan, C. Bendavid, C. Dubourg, P.A. Beachy, M. Muenke, Truncating loss-of-function mutations of DISP1 contribute to holoprosencephaly-like microform features in humans, Hum. Genet. 125 References (2009) 393–400. [27] L.G. Shaffer, A. Theisen, B.A. Bejjani, B.C. Ballif, A.S. Aylsworth, C. Lim, M. [1] E. Matsunaga, K. Shiota, Holoprosencephaly in human embryos: epidemiologic McDonald, J.W. Ellison, D. Kostiner, S. Saitta, T. Shaikh, The discovery of studies of 150 cases, Teratology 16 (1977) 261–272. microdeletion syndromes in the post-genomic era: review of the methodology [2] M.M. Cohen Jr., Problems in the definition of holoprosencephaly, Am. J. Med. and characterization of a new 1q41q42 microdeletion syndrome, Genet. Med. 9 Genet. 103 (2001) 183–187. (2007) 607–616. [3] W. DeMyer, W. Zeman, C. Palmer, The face predicts the brain: diagnostic [28] L. Bartholin, S.E. Powers, T.A. Melhuish, S. Lasse, M. Weinstein, D. Wotton, TGIF significance of median facial anomalies for holoprosencephaly (arhinencephaly), inhibits retinoid signaling, Mol. Cell. Biol. 26 (2006) 990–1001. Pediatrics 34 (1964) 256–263. [29] E. Bertolino, B. Reimund, D. Wildt-Perinic, R.G. Clerc, A novel homeobox protein [4] M. Hayhurst, S.K. McConnell, Mouse models of holoprosencephaly, Curr. Opin. which recognizes a TGT core and functionally interferes with a retinoid- Neurol. 16 (2003) 135–141. responsive motif, J. Biol. Chem. 270 (1995) 31178–31188. [5] M. Muenke, P.A. Beachy, Genetics of ventral forebrain development and [30] P.A. Gongal, A.J. Waskiewicz, Zebrafish model of holoprosencephaly demon- holoprosencephaly, Curr. Opin. Genet. Dev. 10 (2000) 262–269. strates a key role for TGIF in regulating retinoic acid metabolism, Hum. Mol. [6] M. Muenke, M.M.J. Cohen, Genetic approaches to understanding brain Genet. 17 (2008) 525–538. development: holoprosencephaly as a model, Men. Retard. Dev. Disabil. Res. [31] D. Maurus, W.A. Harris, Zic-associated holoprosencephaly: zebrafish Zic1 Rev. 6 (2000) 15–21. controls midline formation and forebrain patterning by regulating Nodal, [7] D. Wallis, M. Muenke, Mutations in holoprosencephaly, Hum. Mutat. 16 (2000) Hedgehog, and retinoic acid signaling, Genes Dev. 23 (2009) 1461–1473. 99–108. [32] M. Brand, C.P. Heisenberg, R.M. Warga, F. Pelegri, R.O. Karlstrom, D. Beuchle, A. [8] K. Shiota, S. Yamada, M. Komada, M. Ishibashi, Embryogenesis of holoprosence- Picker, Y.J. Jiang, M. Furutani-Seiki, F.J. van Eeden, M. Granato, P. Haffter, M. phaly, Am. J. Med. Genet. 143A (2007) 3079–3087. Hammerschmidt, D.A. Kane, R.N. Kelsh, M.C. Mullins, J. Odenthal, C. Nusslein- [9] C.L. Olsen, J.P. Hughes, L.G. Youngblood, M. Sharpe-Stimac, Epidemiology Volhard, Mutations affecting development of the midline and general body of holoprosencephaly and phenotypic characteristics of affected children: shape during zebrafish embryogenesis, Development 123 (1996) 129–142. New York State, 1984–1989, Am. J. Med. Genet. 73 (1997) 217–226. [33] B. Feldman, M.A. Gates, E.S. Egan, S.T. Dougan, G. Rennebeck, H.I. Sirotkin, A.F. [10] D. Higashiyama, H. Saitsu, M. Komada, T. Takigawa, M. Ishibashi, K. Shiota, Schier, W.S. Talbot, Zebrafish organizer development and germ-layer formation Sequential developmental changes in holoprosencephalic mouse embryos require nodal-related signals, Nature 395 (1998) 181–185. exposed to ethanol during the gastrulation period, Birth Defects Res. A Clin. [34] K. Hatta, C.B. Kimmel, R.K. Ho, Charline Walker, The cyclops mutation blocks Mol. Teratol. 79 (2007) 513–523. specification of the floor plate of the zebrafish central nervous system, Nature [11] K.K. Sulik, D.B. Dehart, J.M. Rogers, N. Chernoff, Teratogenicity of low doses of all- 350 (1991) 339–341. trans retinoic acid in presomite mouse embryos, Teratology 51 (1995) 398–403. [35] M. Nomura, E. Li, Smad2 role in mesoderm formation, left–right patterning and [12] M.M. Cohen Jr., K.K. Sulik, Perspectives on holoprosencephaly: Part II. Central craniofacial development, Nature 393 (1998) 786–790. nervous system, craniofacial anatomy, syndrome commentary, diagnostic ap- [36] A.F. Schier, S.C. Neuhauss, K.A. Helde, W.S. Talbot, W. Driever, The one-eyed proach, and experimental studies, J. Craniofac. Genet. Dev. Biol. 12 (1992) 196–244. pinhead gene functions in mesoderm and endoderm formation in zebrafish and [13] E.J. Lammer, D.T. Chen, R.M. Hoar, N.D. Agnish, P.J. Benke, J.T. Braun, C.J. Curry, interacts with no tail, Development 124 (1997) 327–342. P.M. Fernhoff, A.W. Grix Jr., I.T. Lott, et al., Retinoic acid embryopathy, N. Engl. J. [37] A.F. Schier, W.S. Talbot, Molecular genetics of axis formation in zebrafish, Annu. Med. 313 (1985) 837–841. Rev. Genet. 39 (2005) 561–613. [14] S. Brown, P. Jackey, J. Lien, D. Warburton, A small deletion in the putative ‘critical [38] K. Grittsman, W.S. Talbot, A. Schier, Nodal signalling patterns the organizer, region’ in chromosome 13q32 in a fetus with isolated holoprosencephaly, Am. J. Development 127 (2000) 921–932. Hum. Genet. 57 (1995) 606. [39] A. Mehra, J.L. Wrana, TGF-beta and the Smad signal transduction pathway, [15] F. Gurrieri, B.J. Trask, G. van den Engh, C.M. Krauss, A. Schinzel, M.J. Pettenati, D. Biochem. Cell Biol. 80 (2002) 605–622. Schindler, J. Dietz-Band, G. Vergnaud, S.W. Scherer, et al., Physical mapping of [40] K. Gritsman, J. Zhang, S. Cheng, E. Heckscher, W.S. Talbot, A.F. Schier, The EGF-CFC the holoprosencephaly critical region on chromosome 7q36, Nat. Genet. 3 protein one-eyed pinhead is essential for nodal signaling, Cell 97 (1999) 121–132. (1993) 247–251. [41] E. Reissmann, H. Jornvall, A. Blokzijl, O. Andersson, C. Chang, G. Minchiotti, M.G. [16] M. Muenke, L.J. Bone, H.F. Mitchell, I. Hart, K. Walton, K. Hall-Johnson, E.F. Ippel, J. Persico, C.F. Ibanez, A.H. Brivanlou, The orphan receptor ALK7 and the Activin Dietz-Band, K. Kvaloy, C.M. Fan, et al., Physical mapping of the holoprosence- receptor ALK4 mediate signaling by Nodal proteins during vertebrate develop- phaly critical region in 21q22.3, exclusion of SIM2 as a candidate gene for ment, Genes Dev. 15 (2001) 2010–2022. holoprosencephaly, and mapping of SIM2 to a region of chromosome 21 [42] C. Yeo, M. Whitman, Nodal signals to Smads through Cripto-dependent and important for Down syndrome, Am. J. Hum. Genet. 57 (1995) 1074–1079. Cripto-independent mechanisms, Mol. Cell 7 (2001) 949–957. [17] J. Overhauser, H.F. Mitchell, E.H. Zackai, D.B. Tick, K. Rojas, M. Muenke, Physical [43] J. Zhang, W.S. Talbot, A.F. Schier, Positional cloning identifies zebrafish one-eyed mapping of the holoprosencephaly critical region in 18p11.3, Am. J. Hum. Genet. pinhead as a permissive EGF-related ligand required during gastrulation, Cell 92 57 (1995) 1080–1085. (1998) 241–251. [18] E. Roessler, E. Belloni, K. Gaudenz, P. Jay, P. Berta, S.W. Scherer, L.C. Tsui, M. Muenke, [44] H.M. Pogoda, L. Solnica-Krezel, W. Driever, D. Meyer, The zebrafish forkhead Mutations in the human Sonic Hedgehog gene cause holoprosencephaly, transcription factor FoxH1/Fast1 is a modulator of nodal signaling required for Nat. Genet. 14 (1996) 357–360. organizer formation, Curr. Biol. 10 (2000) 1041–1049. [19] E. Roessler, E. Belloni, K. Gaudenz, F. Vargas, S.W. Scherer, L.C. Tsui, M. Muenke, [45] H.I. Sirotkin, M.A. Gates, P.D. Kelly, A.F. Schier, W.S. Talbot, Fast1 is required for the Mutations in the C-terminal domain of Sonic Hedgehog cause holoprosencephaly, development of dorsal axial structures in zebrafish, Curr. Biol. 10 (2000) 1051–1054. Hum. Mol. Genet. 6 (1997) 1847–1853. [46] K. Gritsman, W.S. Talbot, A.F. Schier, Nodal signaling patterns the organizer, [20] U. Schell, J. Wienberg, A. Kohler, P. Bray-Ward, D.E. Ward, W.G. Wilson, W.P. Development 127 (2000) 921–932. Allen, R.R. Lebel, J.R. Sawyer, P.L. Campbell, D.J. Aughton, H.H. Punnett, E.J. [47] F. Muller, S. Albert, P. Blader, N. Fischer, M. Hallonet, U. Strahle, Direct action of Lammer, F.T. Kao, D.C. Ward, M. Muenke, Molecular characterization of the nodal-related signal cyclops in induction of sonic hedgehog in the ventral breakpoints in patients with holoprosencephaly and definition of the HPE2 midline of the CNS, Development 127 (2000) 3889–3897. critical region 2p21, Hum. Mol. Genet. 5 (1996) 223–229. [48] M. Brand, C.-P. Heisenberg, R.M. Warga, F. Pelegri, R.O. Karlstrom, D. Beuchle, A. [21] C. Bendavid, C. Dubourg, I. Gicquel, L. Pasquier, P. Saugier-Veber, M.R. Durou, S. Picker, Y.-J. Jiang, M. Furutani-Seiki, F.J.M.v. Eeden, M. Granato, P. Haffter, M. Jaillard, T. Frebourg, B.R. Haddad, C. Henry, S. Odent, V. David, Molecular Hammerschmidt, D.A. Kane, R.N. Kelsh, M.C. Mullins, J.r. Odenthal, C. Nusslein- evaluation of foetuses with holoprosencephaly shows high incidence of Volhard, Mutations affecting development of the midline and general body microdeletions in the HPE genes, Hum. Genet. 119 (2006) 1–8. shape during zebrafish embryogenesis, Development 123 (1996). [22] E. Roessler, M.V. Ouspenskaia, J.D. Karkera, J.I. Velez, A. Kantipong, F. Lacbawan, [49] A.F. Schier, S.C. Neuhauss, M. Harvey, J. Malicki, L. Solnica-Krezel, D.Y. Stainier, F. P. Bowers, J.W. Belmont, J.A. Towbin, E. Goldmuntz, B. Feldman, M. Muenke, Zwartkruis, S. Abdelilah, D.L. Stemple, Z. Rangini, H. Yang, W. Driever, Mutations Reduced NODAL signaling strength via mutation of several pathway members affecting the development of the embryonic zebrafish brain, Development 123 including FOXH1 is linked to human heart defects and holoprosencephaly, Am. J. (1996) 165–178. Hum. Genet. 83 (2008) 18–29. [50] G. Minchiotti, S. Parisi, G. Liguori, M. Signore, G. Lania, E.D. Adamson, C.T. Lago, M.G. [23] J.M. de la Cruz, R.N. Bamford, R.D. Burdine, E. Roessler, A.J. Barkovich, D. Donnai, Persico, Membrane-anchorage of Cripto protein by glycosylphosphatidylinositol and A.F. Schier, M. Muenke, A loss-of-function mutation in the CFC domain of TDGF1 its distribution during early mouse development, Mech. Dev. 90 (2000) 133–142. is associated with human forebrain defects, Hum. Genet. 110 (2002) 422–428. [51] M. Whitman, Nodal signaling in early vertebrate embryos: themes and [24] X. Geng, C. Speirs, O. Lagutin, A. Inbal, W. Liu, L. Solnica-Krezel, Y. Jeong, D.J. variations, Dev. Cell 1 (2001) 605–617. Epstein, G. Oliver, Haploinsufficiency of Six3 fails to activate Sonic hedgehog [52] P.S. Kunwar, S. Zimmerman, J.T. Bennett, Y. Chen, M. Whitman, A.F. Schier, expression in the ventral forebrain and causes holoprosencephaly, Dev. Cell 15 Mixer/Bon and FoxH1/Sur have overlapping and divergent roles in Nodal (2008) 236–247. signaling and mesendoderm induction, Development 130 (2003) 5589–5599. [25] L. Nanni, J.E. Ming, M. Bocian, K. Steinhaus, D.W. Bianchi, C. Die-Smulders, A. [53] P.A. Hoodless, M. Pye, C. Chazaud, E. Labbe, L. Attisano, J. Rossant, J.L. Wrana, Giannotti, K. Imaizumi, K.L. Jones, M.D. Campo, R.A. Martin, P. Meinecke, M.E. FoxH1 (Fast) functions to specify the anterior primitive streak in the mouse, Pierpont, N.H. Robin, I.D. Young, E. Roessler, M. Muenke, The mutational Genes Dev. 15 (2001) 1257–1271. P.A. Gongal et al. / Biochimica et Biophysica Acta 1812 (2011) 390–401 399

[54] H.J. Carp, Recurrent miscarriage: genetic factors and assessment of the embryo, acid signalling in the zebrafish embryo is necessary during pre-segmentation Isr. Med. Assoc. J. 10 (2008) 229–231. stages to pattern the anterior–posterior axis of the CNS and to induce a pectoral [55] P.W. Ingham, M. Placzek, Orchestrating ontogenesis: variations on a theme by fin bud, Development 129 (2002) 2851–2865. sonic hedgehog, Nat. Rev. Genet. 7 (2006) 841–850. [84] S. Pittlik, S. Domingues, A. Meyer, G. Begemann, Expression of zebrafish aldh1a3 [56] S.F. Farzan, S. Singh, N.S. Schilling, D.J. Robbins, The adventures of sonic (raldh3) and absence of aldh1a1 in teleosts, Gene Expr. Patterns 8 (2008) 141–147. hedgehog in development and repair. III. Hedgehog processing and biological [85] D. Chambers, L. Wilson, M. Maden, A. Lumsden, RALDH-independent generation activity, Am. J. Physiol. Gastrointest. Liver Physiol. 294 (2008) G844–849. of retinoic acid during vertebrate embryogenesis by CYP1B1, Development 134 [57] Z. Chamoun, R.K. Mann, D. Nellen, D.P. von Kessler, M. Bellotto, P.A. Beachy, K. (2007) 1369–1383. Basler, Skinny hedgehog, an acyltransferase required for palmitoylation and [86] R.T. Libby, R.S. Smith, O.V. Savinova, A. Zabaleta, J.E. Martin, F.J. Gonzalez, S.W. activity of the hedgehog signal, Science 293 (2001) 2080–2084. John, Modification of ocular defects in mouse developmental glaucoma models [58] P. Aza-Blanc, T.B. Kornberg, Ci: a complex transducer of the hedgehog signal, by tyrosinase, Science 299 (2003) 1578–1581. Trends Genet. 15 (1999) 458–462. [87] I. Stoilov, A.N. Akarsu, I. Alozie, A. Child, M. Barsoum-Homsy, M.E. Turacli, M. Or, [59] E. Roessler, K.B. El-Jaick, C. Dubourg, J.I. Velez, B.D. Solomon, D.E. Pineda-Alvarez, R.A. Lewis, N. Ozdemir, G. Brice, S.G. Aktan, L. Chevrette, M. Coca-Prados, M. F. Lacbawan, N. Zhou, M. Ouspenskaia, A. Paulussen, H.J. Smeets, U. Hehr, C. Sarfarazi, Sequence analysis and homology modeling suggest that primary Bendavid, S. Bale, S. Odent, V. David, M. Muenke, The mutational spectrum of congenital glaucoma on 2p21 results from mutations disrupting either the hinge holoprosencephaly-associated changes within the SHH gene in humans predicts region or the conserved core structures of cytochrome P4501B1, Am. J. Hum. loss-of-function through either key structural alterations of the ligand or its Genet. 62 (1998) 573–584. altered synthesis, Hum. Mutat. (2009). [88] I. Stoilov, A.N. Akarsu, M. Sarfarazi, Identification of three different truncating [60] X. Feng, E.G. Adiarte, S.H. Devoto, Hedgehog acts directly on the zebrafish mutations in cytochrome P4501B1 (CYP1B1) as the principal cause of primary dermomyotome to promote myogenic differentiation, Dev. Biol. 300 (2006) congenital glaucoma (Buphthalmos) in families linked to the GLC3A locus on 736–746. chromosome 2p21, Hum. Mol. Genet. 6 (1997) 641–647. [61] A. Nasevicius, S.C. Ekker, Effective targeted gene ‘knockdown’ in zebrafish, Nat. [89] L. Maves, C.B. Kimmel, Dynamic and sequential patterning of the zebrafish Genet. 26 (2000) 216–220. posterior hindbrain by retinoic acid, Dev. Biol. 285 (2005) 593–605. [62] H.E. Schauerte, F.J. van Eeden, C. Fricke, J. Odenthal, U. Strahle, P. Haffter, Sonic [90] K. Niederreither, J. Vermot, N. Messaddeq, B. Schuhbaur, P. Chambon, P. Dolle, hedgehog is not required for the induction of medial floor plate cells in the Embryonic retinoic acid synthesis is essential for heart morphogenesis in the zebrafish, Development 125 (1998) 2983–2993. mouse, Development 128 (2001) 1019–1031. [63] Z.M. Varga, A. Amores, K.E. Lewis, Y.L. Yan, J.H. Postlethwait, J.S. Eisen, M. [91] K. Niederreither, J. Vermot, B. Schuhbaur, P. Chambon, P. Dolle, Retinoic acid Westerfield, Zebrafish smoothened functions in ventral neural tube specification synthesis and hindbrain patterning in the mouse embryo, Development 127 and axon tract formation, Development 128 (2001) 3497–3509. (2000) 75–85. [64] J.K. Chen, J. Taipale, M.K. Cooper, P.A. Beachy, Inhibition of Hedgehog signaling by [92] R.J. White, T.F. Schilling, How degrading: Cyp26s in hindbrain development, Dev. direct binding of cyclopamine to Smoothened, Genes Dev. 16 (2002) 2743–2748. Dyn. 237 (2008) 2775–2790. [65] Y. Chen, G. Struhl, Dual roles for patched in sequestering and transducing [93] T. Kudoh, S.W. Wilson, I.B. Dawid, Distinct roles for Fgf, Wnt and retinoic acid in Hedgehog, Cell 87 (1996) 553–563. posteriorizing the neural ectoderm, Development 129 (2002) 4335–4346. [66] L.V. Goodrich, D. Jung, K.M. Higgins, M.P. Scott, Overexpression of ptc1 inhibits [94] X. Gu, F. Xu, X. Wang, X. Gao, Q. Zhao, Molecular cloning and expression of a induction of Shh target genes and prevents normal patterning in the neural tube, novel CYP26 gene (cyp26d1) during zebrafish early development, Gene Expr. Dev. Biol. 211 (1999) 323–334. Patterns 5 (2005) 733–739. [67] P.W. Ingham, A.M. Taylor, Y. Nakano, Role of the Drosophila patched gene in [95] Q. Zhao, B. Dobbs-McAuliffe, E. Linney, Expression of cyp26b1 during zebrafish positional signalling, Nature 353 (1991) 184–187. early development, Gene Expr. Patterns 5 (2005) 363–369. [68] M.J. Koudijs, M.J. den Broeder, E. Groot, F.J. van Eeden, Genetic analysis of the two [96] R.E. Hernandez, A.P. Putzke, J.P. Myers, L. Margaretha, C.B. Moens, Cyp26 zebrafish patched homologues identifies novel roles for the hedgehog signaling enzymes generate the retinoic acid response pattern necessary for hindbrain pathway, BMC Dev. Biol. 8 (2008) 15. development, Development 134 (2007) 177–187. [69] J.L. Mullor, I. Guerrero, A gain-of-function mutant of patched dissects different [97] R.J. White, Q. Nie, A.D. Lander, T.F. Schilling, Complex regulation of cyp26a1 responses to the hedgehog gradient, Dev. Biol. 228 (2000) 211–224. creates a robust retinoic acid gradient in the zebrafish embryo, PLoS Biol. 5 [70] J.E. Ming, M.E. Kaupas, E. Roessler, H.G. Brunner, M. Golabi, M. Tekin, R.F. (2007) e304. Stratton, E. Sujansky, S.J. Bale, M. Muenke, Mutations in PATCHED-1, the receptor [98] S.Q. Cohlan, Congenital anomalies in the rat produced by excessive intake of for SONIC HEDGEHOG, are associated with holoprosencephaly, Hum. Genet. 110 vitamin A during pregnancy, Pediatrics 13 (1954) 556–567. (2002) 297–301. [99] Y.L. Yan, T. Jowett, J.H. Postlethwait, Ectopic expression of hoxb2 after retinoic [71] F.J. van Eeden, M. Granato, U. Schach, M. Brand, M. Furutani-Seiki, P. Haffter, M. acid treatment or mRNA injection: disruption of hindbrain and craniofacial Hammerschmidt, C.P. Heisenberg, Y.J. Jiang, D.A. Kane, R.N. Kelsh, M.C. Mullins, J. morphogenesis in zebrafish embryos, Dev. Dyn. 213 (1998) 370–385. Odenthal, R.M. Warga, M.L. Allende, E.S. Weinberg, C. Nusslein-Volhard, [100] S. Abu-Abed, P. Dolle, D. Metzger, B. Beckett, P. Chambon, M. Petkovich, The Mutations affecting somite formation and patterning in the zebrafish, Danio retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain rerio, Development 123 (1996) 153–164. patterning, vertebral identity, and development of posterior structures, Genes [72] R. Burke, D. Nellen, M. Bellotto, E. Hafen, K.A. Senti, B.J. Dickson, K. Basler, Dev. 15 (2001) 226–240. Dispatched, a novel sterol-sensing domain protein dedicated to the release of [101] Y. Emoto, H. Wada, H. Okamoto, A. Kudo, Y. Imai, Retinoic acid-metabolizing cholesterol-modified hedgehog from signaling cells, Cell 99 (1999) 803–815. enzyme Cyp26a1 is essential for determining territories of hindbrain and spinal [73] T. Caspary, M.J. Garcia-Garcia, D. Huangfu, J.T. Eggenschwiler, M.R. Wyler, A.S. cord in zebrafish, Dev. Biol. 278 (2005) 415–427. Rakeman, H.L. Alcorn, K.V. Anderson, Mouse Dispatched homolog1 is required for [102] C. Roberts, S. Ivins, A.C. Cook, A. Baldini, P.J. Scambler, Cyp26 genes a1, b1 and c1 are long-range, but not juxtacrine, Hh signaling, Curr. Biol. 12 (2002) 1628–1632. down-regulated in Tbx1 null mice and inhibition of Cyp26 enzyme function produces [74] T. Kawakami, T. Kawcak, Y.J. Li, W. Zhang, Y. Hu, P.T. Chuang, Mouse dispatched a phenocopy of DiGeorge syndrome in the chick, Hum. Mol. Genet. 15 (2006) mutants fail to distribute hedgehog proteins and are defective in hedgehog 3394–3410. signaling, Development 129 (2002) 5753–5765. [103] V.S. Aggarwal, B.E. Morrow, Genetic modifiers of the physical malformations in [75] Y. Ma, A. Erkner, R. Gong, S. Yao, J. Taipale, K. Basler, P.A. Beachy, Hedgehog- velo-cardio-facial syndrome/DiGeorge syndrome, Dev. Disabil. Res. Rev. 14 mediated patterning of the mammalian embryo requires transporter-like (2008) 19–25. function of dispatched, Cell 111 (2002) 63–75. [104] S.C. Butts, The facial phenotype of the velo-cardio-facial syndrome, Int. J. Pediatr. [76] Y. Nakano, H.R. Kim, A. Kawakami, S. Roy, A.F. Schier, P.W. Ingham, Inactivation Otorhinolaryngol. 73 (2009) 343–350. of dispatched 1 by the chameleon mutation disrupts Hedgehog signalling in the [105] F. Hale, Pigs born without eyeballs, J. Heredity 24 (1933) 105–106. zebrafish embryo, Dev. Biol. 269 (2004) 381–392. [106] A. Halilagic, M.H. Zile, M. Studer, A novel role for retinoids in patterning the avian [77] A. Inbal, S.H. Kim, J. Shin, L. Solnica-Krezel, Six3 represses nodal activity to forebrain during presomite stages, Development 130 (2003) 2039–2050. establish early brain asymmetry in zebrafish, Neuron 55 (2007) 407–415. [107] M. Maden, E. Gale, I. Kostetskii, M. Zile, Vitamin A-deficient quail embryos have [78] S. Domene, E. Roessler, K.B. El-Jaick, M. Snir, J.L. Brown, J.I. Velez, S. Bale, F. half a hindbrain and other neural defects, Curr. Biol. 6 (1996) 417–426. Lacbawan, M. Muenke, B. Feldman, Mutations in the human SIX3 gene in [108] V. Ribes, Z. Wang, P. Dolle, K. Niederreither, Retinaldehyde dehydrogenase 2 holoprosencephaly are loss of function, Hum. Mol. Genet. 17 (2008) 3919–3928. (RALDH2)-mediated retinoic acid synthesis regulates early mouse embryonic [79] Y. Jeong, F.C. Leskow, K. El-Jaick, E. Roessler, M. Muenke, A. Yocum, C. Dubourg, X. forebrain development by controlling FGF and sonic hedgehog signaling, Li, X. Geng, G. Oliver, D.J. Epstein, Regulation of a remote Shh forebrain enhancer Development 133 (2006) 351–361. by the Six3 homeoprotein, Nat. Genet. 40 (2008) 1348–1353. [109] D. Wotton, R.S. Lo, S. Lee, J. Massague, A Smad transcriptional corepressor, Cell 97 [80] L.L. Sandell, B.W. Sanderson, G. Moiseyev, T. Johnson, A. Mushegian, K. Young, J.P. (1999) 29–39. Rey, J.X. Ma, K. Staehling-Hampton, P.A. Trainor, RDH10 is essential for synthesis [110] K.W. Gripp, D. Wotton, M.C. Edwards, E. Roessler, L. Ades, P. Meinecke, A. of embryonic retinoic acid and is required for limb, craniofacial, and organ Richieri-Costa, E.H. Zackai, J. Massague, M. Muenke, S.J. Elledge, Mutations in development, Genes Dev. 21 (2007) 1113–1124. TGIF cause holoprosencephaly and link NODAL signalling to human neural axis [81] M. Maden, Retinoid signalling in the development of the central nervous system, determination, Nat. Genet. 25 (2000) 205–208. Nat. Rev. Neurosci. 3 (2002) 843–853. [111] M.M. Cohen Jr., K. Shiota, Teratogenesis of holoprosencephaly, Am. J. Med. Genet. [82] G. Begemann, T.F. Schilling, G.J. Rauch, R. Geisler, P.W. Ingham, The zebrafish 109 (2002) 1–15. neckless mutation reveals a requirement for raldh2 in mesodermal signals that [112] C. Dubourg, L. Lazaro, L. Pasquier, C. Bendavid, M. Blayau, F. Le Duff, M.R. Durou, S. pattern the hindbrain, Development 128 (2001) 3081–3094. Odent, V. David, Molecular screening of SHH, ZIC2, SIX3, and TGIF genes in [83] H. Grandel, K. Lun, G.J. Rauch, M. Rhinn, T. Piotrowski, C. Houart, P. Sordino, A.M. patients with features of holoprosencephaly spectrum: mutation review and Kuchler, S. Schulte-Merker, R. Geisler, N. Holder, S.W. Wilson, M. Brand, Retinoic genotype–phenotype correlations, Hum. Mutat. 24 (2004) 43–51. 400 P.A. Gongal et al. / Biochimica et Biophysica Acta 1812 (2011) 390–401

[113] E. Roessler, M. Muenke, Holoprosencephaly: a paradigm for the complex ophthalmologic delineation of the renal-coloboma syndrome, Ophthalmic genetics of brain development, J. Inherit. Metab. Dis. 21 (1998) 481–497. Genet. 24 (2003) 191–202. [114] J.Z. Jin, S. Gu, P. McKinney, J. Ding, Expression and functional analysis of Tgif [143] A. Taranta, A. Palma, V. De Luca, A. Romanzo, L. Massella, F. Emma, L. Dello Strologo, during mouse midline development, Dev. Dyn. 235 (2006) 547–553. Renal-coloboma syndrome: a single nucleotide deletion in the PAX2 gene at Exon [115] L. Mar, P.A. Hoodless, Embryonic fibroblasts from mice lacking Tgif were 8 is associated with a highly variable phenotype, Clin. Nephrol. 67 (2007) 1–4. defective in cell cycling, Mol. Cell. Biol. 26 (2006) 4302–4310. [144] P. Elms, P. Siggers, D. Napper, A. Greenfield, R. Arkell, Zic2 is required for neural [116] J. Shen, C.A. Walsh, Targeted disruption of Tgif, the mouse ortholog of a human crest formation and hindbrain patterning during mouse development, Dev. Biol. holoprosencephaly gene, does not result in holoprosencephaly in mice, Mol. Cell. 264 (2003) 391–406. Biol. 25 (2005) 3639–3647. [145] E. Herrera, L. Brown, J. Aruga, R.A. Rachel, G. Dolen, K. Mikoshiba, S. Brown, C.A. [117] C. Kuang, Y. Xiao, L. Yang, Q. Chen, Z. Wang, S.J. Conway, Y. Chen, Intragenic Mason, Zic2 patterns binocular vision by specifying the uncrossed retinal deletion of Tgif causes defects in brain development, Hum. Mol. Genet. 15 (2006) projection, Cell 114 (2003) 545–557. 3508–3519. [146] J. Lee, J.R. Willer, G.B. Willer, K. Smith, R.G. Gregg, J.M. Gross, Zebrafish blowout [118] J.J. Sanz-Ezquerro, C. Tickle, Autoregulation of Shh expression and Shh induction provides genetic evidence for Patched1-mediated negative regulation of of cell death suggest a mechanism for modulating polarising activity during Hedgehog signaling within the proximal optic vesicle of the vertebrate eye, chick limb development, Development 127 (2000) 4811–4823. Dev. Biol. 319 (2008) 10–22. [119] R.A. Schneider, D. Hu, J.L. Rubenstein, M. Maden, J.A. Helms, Local retinoid [147] T.H. Kim, J. Goodman, K.V. Anderson, L. Niswander, Phactr4 regulates neural tube signaling coordinates forebrain and facial morphogenesis by maintaining FGF8 and optic fissure closure by controlling PP1-, Rb-, and E2F1-regulated cell-cycle and SHH, Development 128 (2001) 2755–2767. progression, Dev. Cell 13 (2007) 87–102. [120] V. Ribes, I. Le Roux, M. Rhinn, B. Schuhbaur, P. Dolle, Early mouse caudal [148] V.A. Ruzhynsky, M. Furimsky, D.S. Park, V.A. Wallace, R.S. Slack, E2F4 is required development relies on crosstalk between retinoic acid, Shh and Fgf signalling for early eye patterning, Dev. Neurosci. 31 (2009) 238–246. pathways, Development 136 (2009) 665–676. [149] M.W. Seeliger, H.K. Biesalski, B. Wissinger, H. Gollnick, S. Gielen, J. Frank, S. Beck, [121] M.H. Chen, Y.J. Li, T. Kawakami, S.M. Xu, P.T. Chuang, Palmitoylation is required E. Zrenner, Phenotype in retinol deficiency due to a hereditary defect in retinol for the production of a soluble multimeric Hedgehog protein complex and long- binding protein synthesis, Invest. Ophthalmol. Vis. Sci. 40 (1999) 3–11. range signaling in vertebrates, Genes Dev. 18 (2004) 641–659. [150] H. Ozeki, S. Shirai, Developmental eye abnormalities in mouse fetuses induced by [122] J. Feng, B. White, O.V. Tyurina, B. Guner, T. Larson, H.Y. Lee, R.O. Karlstrom, J.D. retinoic acid, Jpn. J. Ophthalmol. 42 (1998) 162–167. Kohtz, Synergistic and antagonistic roles of the Sonic hedgehog N- and C- [151] D.L. Stull, K.C. Wikler, Retinoid-dependent gene expression regulates early terminal lipids, Development 131 (2004) 4357–4370. morphological events in the development of the murine retina, J. Comp. Neurol. [123] N.A. Sanek, Y. Grinblat, A novel role for zebrafish zic2a during forebrain 417 (2000) 289–298. development, Dev. Biol. 317 (2008) 325–335. [152] G.A. Hyatt, E.A. Schmitt, N. Marsh-Armstrong, P. McCaffery, U.C. Drager, J.E. [124] N.A. Sanek, A.A. Taylor, M.K. Nyholm, Y. Grinblat, Zebrafish zic2a patterns the Dowling, Retinoic acid establishes ventral retinal characteristics, Development forebrain through modulation of Hedgehog-activated gene expression, Devel- 122 (1996) 195–204. opment 136 (2009) 3791–3800. [153] J.A. Helms, C.H. Kim, D. Hu, R. Minkoff, C. Thaller, G. Eichele, Sonic hedgehog [125] Y.R. Barishak, Embryology of the eye and its adnexae, Dev. Ophthalmol. 24 participates in craniofacial morphogenesis and is down-regulated by teratogenic (1992) 1–142. doses of retinoic acid, Dev. Biol. 187 (1997) 25–35. [126] D. Taylor, Developmental abnormalities of the optic nerve and chiasm, Eye 21 [154] N. Marsh-Armstrong, P. McCaffery, W. Gilbert, J.E. Dowling, U.C. Drager, Retinoic (2007) 1271–1284. acid is necessary for development of the ventral retina in zebrafish, Proc. Natl. [127] M. Asai-Coakwell, C.R. French, K.M. Berry, M. Ye, R. Koss, M. Somerville, R. Acad. Sci. U. S. A. 91 (1994) 7286–7290. Mueller, V. van Heyningen, A.J. Waskiewicz, O.J. Lehmann, GDF6, a novel locus [155]A.Perz-Edwards,N.L.Hardison,E.Linney,Retinoicacid-mediatedgene for a spectrum of ocular developmental anomalies, Am. J. Hum. Genet. 80 (2007) expression in transgenic reporter zebrafish, Dev. Biol. 229 (2001) 89–101. 306–315. [156] M.J. Connor, Oxidation of retinol to retinoic acid as a requirement for biological [128] C.Y. Gregory-Evans, M.J. Williams, S. Halford, K. Gregory-Evans, Ocular activity in mouse epidermis, Cancer Res. 48 (1988) 7038–7040. coloboma: a reassessment in the age of molecular neuroscience, J. Med. Genet. [157] M.J. Connor, M.H. Smit, Terminal-group oxidation of retinol by mouse epidermis. 41 (2004) 881–891. Inhibition in vitro and in vivo, Biochem. J. 244 (1987) 489–492. [129] E. Ferda Percin, L.A. Ploder, J.J. Yu, K. Arici, D.J. Horsford, A. Rutherford, B. Bapat, [158] T.J. Schuh, B.L. Hall, J.C. Kraft, M.L. Privalsky, D. Kimelman, v-erbA and citral D.W. Cox, A.M. Duncan, V.I. Kalnins, A. Kocak-Altintas, J.C. Sowden, E. Traboulsi, reduce the teratogenic effects of all-trans retinoic acid and retinol, respectively, M. Sarfarazi, R.R. McInnes, Human microphthalmia associated with mutations in in Xenopus embryogenesis, Development 119 (1993) 785–798. the retinal homeobox gene CHX10, Nat. Genet. 25 (2000) 397–401. [159] A.W. See, M. Clagett-Dame, The temporal requirement for vitamin A in the [130] D. Morrison, D. FitzPatrick, I. Hanson, K. Williamson, V. van Heyningen, B. Fleck, I. developing eye: mechanism of action in optic fissure closure and new roles for Jones, J. Chalmers, H. Campbell, National study of microphthalmia, anophthal- the vitamin in regulating cell proliferation and adhesion in the embryonic retina, mia, and coloboma (MAC) in Scotland: investigation of genetic aetiology, J. Med. Dev. Biol. 325 (2009) 94–105. Genet. 39 (2002) 16–22. [160] A. Molotkov, N. Molotkova, G. Duester, Retinoic acid guides eye morphogenetic [131] R.V. Jamieson, R. Perveen, B. Kerr, M. Carette, J. Yardley, E. Heon, M.G. Wirth, V. movements via paracrine signaling but is unnecessary for retinal dorsoventral van Heyningen, D. Donnai, F. Munier, G.C. Black, Domain disruption and patterning, Development 133 (2006) 1901–1910. mutation of the bZIP transcription factor, MAF, associated with cataract, ocular [161] M. Asai-Coakwell, C.R. French, M. Ye, K. Garcha, K. Bigot, A.G. Perera, K. anterior segment dysgenesis and coloboma, Hum. Mol. Genet. 11 (2002) 33–42. Staehling-Hampton, S.C. Mema, B. Chanda, A. Mushegian, S. Bamforth, M.R. [132] L.E. Vissers, C.M. van Ravenswaaij, R. Admiraal, J.A. Hurst, B.B. de Vries, I.M. Doschak, G. Li, M.B. Dobbs, P.F. Giampietro, B.P. Brooks, P. Vijayalakshmi, Y. Janssen, W.A. van der Vliet, E.H. Huys, P.J. de Jong, B.C. Hamel, E.F. Schoenmakers, Sauve, M. Abitbol, P. Sundaresan, V. van Heyningen, O. Pourquie, T.M. H.G. Brunner, J.A. Veltman, A.G. van Kessel, Mutations in a new member of the Underhill, A.J. Waskiewicz, O.J. Lehmann, Incomplete penetrance and pheno- chromodomain gene family cause CHARGE syndrome, Nat. Genet. 36 (2004) typic variability characterize Gdf6-attributable oculo-skeletal phenotypes, 955–957. Hum. Mol. Genet. 18 (2009) 1110–1121. [133] M. Torres, E. Gomez-Pardo, P. Gruss, Pax2 contributes to inner ear patterning and [162] C.R. French, T. Erickson, D.V. French, D.B. Pilgrim, A.J. Waskiewicz, Gdf6a is optic nerve trajectory, Development 122 (1996) 3381–3391. required for the initiation of dorsal–ventral retinal patterning and lens [134] M.R. Eccles, L.A. Schimmenti, Renal-coloboma syndrome: a multi-system development, Dev. Biol. 333 (2009) 37–47. developmental disorder caused by PAX2 mutations, Clin. Genet. 56 (1999) 1–9. [163] N.J. Gosse, H. Baier, An essential role for Radar (Gdf6a) in inducing dorsal fate in [135] L.A. Schimmenti, J. de la Cruz, R.A. Lewis, J.D. Karkera, G.S. Manligas, E. Roessler, the zebrafish retina, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 2236–2241. M. Muenke, Novel mutation in sonic hedgehog in non-syndromic colobomatous [164] P. Bakrania, M. Efthymiou, J.C. Klein, A. Salt, D.J. Bunyan, A. Wyatt, C.P. Ponting, A. microphthalmia, Am. J. Med. Genet. 116A (2003) 215–221. Martin, S. Williams, V. Lindley, J. Gilmore, M. Restori, A.G. Robson, M.M. Neveu, G.E. [136] S.J. Hornby, S.J. Ward, C.E. Gilbert, L. Dandona, A. Foster, R.B. Jones, Holder, J.R. Collin, D.O. Robinson, P. Farndon, H. Johansen-Berg, D. Gerrelli, N.K. Environmental risk factors in congenital malformations of the eye, Ann. Trop. Ragge, Mutations in BMP4 cause eye, brain, and digit developmental anomalies: Paediatr. 22 (2002) 67–77. overlap between the BMP4 and hedgehog signaling pathways, Am. J. Hum. Genet. [137] J.G. Wilson, C.B. Roth, J. Warkany, An analysis of the syndrome of malformations 82 (2008) 304–319. induced by maternal vitamin A deficiency. Effects of restoration of vitamin A at [165] L. Zhao, H. Saitsu, X. Sun, K. Shiota, M. Ishibashi, Sonic hedgehog is involved in various times during gestation, Am. J. Anat. 92 (1953) 189–217. formation of the ventral optic cup by limiting Bmp4 expression to the dorsal [138] R. Macdonald, K.A. Barth, Q. Xu, N. Holder, I. Mikkola, S.W. Wilson, Midline domain, Mech. Dev. (2009). signalling is required for Pax gene regulation and patterning of the eyes, [166] Y. Ohkubo, C. Chiang, J.L. Rubenstein, Coordinate regulation and synergistic Development 121 (1995) 3267–3278. actions of BMP4, SHH and FGF8 in the rostral prosencephalon regulate [139] C.J. Neumann, C. Nuesslein-Volhard, Patterning of the zebrafish retina by a wave morphogenesis of the telencephalic and optic vesicles, Neuroscience 111 of sonic hedgehog activity, Science 289 (2000) 2137–2139. (2002) 1–17. [140] M. Take-uchi, J.D. Clarke, S.W. Wilson, Hedgehog signalling maintains the optic [167] A.M. Hever, K.A. Williamson, V. van Heyningen, Developmental malformations of stalk-retinal interface through the regulation of Vax gene activity, Development the eye: the role of PAX6, SOX2 and OTX2, Clin. Genet. 69 (2006) 459–470. 130 (2003) 955–968. [168] N.K. Ragge, A.G. Brown, C.M. Poloschek, B. Lorenz, R.A. Henderson, M.P. Clarke, [141] S.C. Ekker, A.R. Ungar, P. Greenstein, D.P. von Kessler, J.A. Porter, R.T. Moon, P.A. I. Russell-Eggitt, A. Fielder, D. Gerrelli, J.P. Martinez-Barbera, P. Ruddle, J. Beachy, Patterning activities of vertebrate hedgehog proteins in the developing Hurst, J.R. Collin, A. Salt, S.T. Cooper, P.J. Thompson, S.M. Sisodiya, K.A. eye and brain, Curr. Biol. 5 (1995) 944–955. Williamson,D.R.Fitzpatrick,V.vanHeyningen, I.M. Hanson, Heterozygous [142] L.A. Schimmenti, G.S. Manligas, P.A. Sieving, Optic nerve dysplasia and renal mutations of OTX2 cause severe ocular malformations, Am. J. Hum. Genet. 76 insufficiency in a family with a novel PAX2 mutation, Arg115X: further (2005) 1008–1022. P.A. Gongal et al. / Biochimica et Biophysica Acta 1812 (2011) 390–401 401

[169] A. Wyatt, P. Bakrania, D.J. Bunyan, R.J. Osborne, J.A. Crolla, A. Salt, C. Ayuso, R. [172] C.R. French, T. Erickson, D. Callander, K.M. Berry, R. Koss, D.W. Hagey, J. Stout, K. Newbury-Ecob, Y. Abou-Rayyah, J.R. Collin, D. Robinson, N. Ragge, Novel Wuennenberg-Stapleton, J. Ngai, C.B. Moens, A.J. Waskiewicz, Pbx home- heterozygous OTX2 mutations and whole gene deletions in anophthalmia, odomain proteins pattern both the zebrafish retina and tectum, BMC Dev. Biol. microphthalmia and coloboma, Hum. Mutat. 29 (2008) E278–283. 7 (2007) 85. [170] N. Fujimura, M.M. Taketo, M. Mori, V. Korinek, Z. Kozmik, Spatial and temporal [173] P. Wang, X. Liang, J. Yi, Q. Zhang, Novel SOX2 mutation associated regulation of Wnt/beta-catenin signaling is essential for development of the with ocular coloboma in a Chinese family, Arch. Ophthalmol. 126 (2008) retinal pigment epithelium, Dev. Biol. 334 (2009) 31–45. 709–713. [171] J.R. Martinez-Morales, V. Dolez, I. Rodrigo, R. Zaccarini, L. Leconte, P. Bovolenta, S. [174] O.V. Taranova, S.T. Magness, B.M. Fagan, Y. Wu, N. Surzenko, S.R. Hutton, L.H. Saule, OTX2 activates the molecular network underlying retina pigment Pevny, SOX2 is a dose-dependent regulator of retinal neural progenitor epithelium differentiation, J. Biol. Chem. 278 (2003) 21721–21731. competence, Genes Dev. 20 (2006) 1187–1202. Biochimica et Biophysica Acta 1812 (2011) 402–414

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Biochimica et Biophysica Acta

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Review Fish models in prion biology: Underwater issues☆

Edward Málaga-Trillo a,⁎,1, Evgenia Salta b,1, Antonio Figueras c, Cynthia Panagiotidis d, Theodoros Sklaviadis b,⁎ a Department of Biology, University of Konstanz, 78457 Konstanz, Germany b Department of Pharmacology, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece c Instituto de Investigaciones Marinas, CSIC, 36208, Vigo, Spain d Centre for Research and Technology-Hellas, Institute of Agrobiotechnology, 57001, Thessaloniki, Greece article info abstract

Article history: Transmissible spongiform encephalopathies (TSEs), otherwise known as prion disorders, are fatal diseases Received 8 December 2009 causing neurodegeneration in a wide range of mammalian hosts, including humans. The causative agents – Received in revised form 11 September 2010 prions – are thought to be composed of a rogue isoform of the endogenous prion protein (PrP). Beyond these Accepted 21 September 2010 and other basic concepts, fundamental questions in prion biology remain unanswered, such as the Available online 7 October 2010 physiological function of PrP, the molecular mechanisms underlying prion pathogenesis, and the origin of prions. To date, the occurrence of TSEs in lower vertebrates like fish and birds has received only limited Keywords: fi fi Prion protein attention, despite the fact that these animals possess bona de PrPs. Recent ndings, however, have brought Transmissible spongiform encephalopathy fish before the footlights of prion research. Fish models are beginning to provide useful insights into the roles Neurodegeneration of PrP in health and disease, as well as the potential risk of prion transmission between fish and mammals. Fish Although still in its infancy, the use of fish models in TSE research could significantly improve our basic Zebrafish understanding of prion diseases, and also help anticipate risks to public health. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Alzheimer's disease (AD) and Parkinson's disease (PD), prion diseases have received special attention because of their infectious nature and 1.1. Prion disorders the associated risk of epidemics. In fact, prion diseases gained considerable notoriety in the 1990s Prion diseases or TSEs are a group of rare but fatal neurological because of the massive outbreak of mad cow disease (bovine spongi- disorders that affect humans and animals. Whether sporadic, inherited form encephalopathy, BSE) in Europe and the negative impact it had on or acquired, these illnesses generally correlate with the accumulation of public health and the food industry. TSEs are, however, not a recent misfolded PrP in the brain and the appearance of widespread phenomenon. Their history dates back to the 1700s when scrapie, a neurodegeneration after long incubation times [1]. The classical related neurological condition among sheep and goats, was first histopathological landmarks of TSEs are spongiform vacuolation, described in England [3]. Other TSEs in animals include chronic wasting neuronal loss and astrocytic gliosis, whereas the main clinical disease (CWD) in deer and elk, transmissible mink encephalopathy and manifestations in humans include progressive dementia, cerebellar feline spongiform encephalopathy (FSE) [4]. The most common TSEs in ataxia and myoclonus [2]. Interestingly, although such symptoms are humans, Creutzfeldt–Jacob disease (CJD) and Gerstmann Sträussler also observed in more common neurodegenerative disorders like Scheinker syndrome (GSS), were discovered much later (1920s and 1930s, respectively [5]), and it was not until the 1950s that the study of kuru, another human prion disease, set off a chain of revolutionary Abbreviations: TSEs, transmissible spongiform encephalopathies; PrP, prion protein; BSE, bovine spongiform encephalopathy; CJD, Creutzfeldt–Jakob disease; discoveries that would redefine the meaning of “pathogen” and push a GPI, glycosylphospatidylinositol; MBM, meat and bone meal; p.i., post inoculation; i.c., biological dogma against the ropes. intracerebrally; PK, proteinase K; GI, gastrointestinal ☆ fi This article is part of a Special Issue entitled Zebra sh Models of Neurological 1.2. The nature of the infectious TSE agent Diseases. ⁎ Corresponding authors. E. Málaga-Trillo is to be contacted at Department of Biology, University of Konstanz, Universitätstrasse 10, 78457, Konstanz, Germany. Tel.: Kuru was originally described as a neurological epidemic of the Fore +49 7531 884581; fax: +49 7531 884863. T. Sklaviadis, Department of Pharmacology, linguistic group in Papua New Guinea [2]. For some time, the disease School of Pharmacy, Aristotle University of Thessaloniki (AUTH), AUTH Campus, 54214 was believed to be infectious, and possibly transmitted during Thessaloniki, Greece. Tel.: +30 2310 997615; fax: +30 2310 997720. cannibalistic rituals. Nevertheless, failure to transmit kuru to laboratory E-mail addresses: [email protected] (E. Málaga-Trillo), [email protected] (T. Sklaviadis). animals led to the erroneous assumption that it had a genetic basis. 1 Equally contributing authors. Towards 1959, remarkable similarities between kuru, CJD and scrapie

0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2010.09.013 E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414 403 were recognized at the neuropathological, clinical and epidemiological abnormalities, their only clear “phenotype” is their resistance to prion levels [6,7]. Since scrapie had been already shown to be transmissible infection [36]. It is probably not exaggerated to say that as many PrP [8], it became apparent that kuru and CJD were also of infectious functions have been proposed as methods have been employed to etiology. Indeed, by 1968, both human diseases had been successfully address this question. Thus, the list of putative PrP roles in vitro and in transmitted to chimpanzees [9,10]. Despite these advances, the nature vivo includes activities as diverse as cytoprotection from apoptosis [37] of the scrapie agent continued to be the subject of intense debate for and oxidative stress [38], olfactory behavior [39], copper metabolism many years. Because TSEs exhibit strain variation and long incubation [40], neurogenesis [41], neurite outgrowth and neuronal survival [42], periods, it was argued that the agent could be a slow-virus [11]. lymphocyte activation [43], hematopoietic stem cell self-renewal [44], However, direct proof for this “viral hypothesis” has been difficult to synaptic function [45], activation of signal transduction [46],and obtain [12]. Instead, it has been shown that the transmissible agent is binding to cell adhesion molecules [47]. Unfortunately, it is not clear resistant to formalin (a chemical that inactivates viruses) and to various how many of these findings may be of actual physiological relevance. In other treatments that destroy nucleic acids, but not to proteolytic addition, it seems unlikely that a single molecular function of PrP could digestion [13–15]. These and other developments finally led to the account for such diverse biological roles. enunciation of the “protein-only hypothesis” by Stanley Prusiner The clear correlation between PrP misfolding and neurodegenera- (1982), according to which TSEs are caused by prions, small protein- tion may suggest that PrPSc is the direct cause of prion disease. aceous particles capable of replicating in the absence of nucleic acids Nevertheless, solid experimental evidence indicates that prions [14,16]. cannot induce neuronal damage in the absence of PrPC. For instance, The protein-only hypothesis has not remained uncontested. While PrP knockout mice are resistant to scrapie inoculation [48], even if some continue to advocate the existence of an unidentified scrapie they carry PrPC-expressing neurografts [49], or if PrPC depletion is agent [17–19], others see the notion of an infectious, self-replicating induced postnatally [50]. Notably, transgenic mice expressing only a protein as a clear violation of Crick's central dogma of molecular biology GPI-anchorless version of PrP (thus unable to attach to the plasma [20], which excludes the flow of biological information from protein to membrane) can efficiently replicate prions without developing prion protein. Notwithstanding the controversy, Prusiner's theory reasonably disease [51]. Altogether, these studies demonstrate that prion accounts for the complex etiology of TSEs, the physicochemical neurotoxicity is not triggered by PrPSc itself, but by an activity of properties of the agent, and the existence of scrapie strains [3].Today, PrPC at the cell surface. Accordingly, it has been proposed that prion it is widely accepted that prions are largely – if not entirely – composed replication might cause PrPC to either lose a neuroprotective role, gain of PrPSc, an abnormally folded isoform of the cellular prion protein a neurotoxic one, or subvert a neuroprotective role into a neurotoxic (PrPC) [21]. The molecular mechanism by which normal PrPC “converts” one [52]. Therefore, a thorough characterization of PrPC function will into infectious PrPSc is, however, poorly understood. It has been likely be instrumental in ascertaining the molecular basis of prion- proposed that PrPSc can associate with PrPC and induce its conversion induced neurodegeneration. into more PrPSc, triggering a chain reaction [22].Alternatively,PrPSc and PrPC may coexist in equilibrium until mutation or sporadic events favor 1.4. Open challenges in prion biology and pathogenesis the formation of oligomeric PrPSc seeds, which undergo repeated aggregation and fragmentation cycles [23]. In either case, the concept of The past five decades have seen remarkable achievements that PrP conversion is central to the protein-only hypothesis because it shaped our current understanding of TSEs. These include the explains the ability of prions to replicate and propagate disease. transmissibility of kuru and CJD to laboratory animals, the isolation of the scrapie agent, the development of the protein-only hypothesis, 1.3. The prion protein the identification of PrP and its isoforms, and the study of prion propagation in genetically modified mice. Yet, very little has been In 1982, PrPSc was identified as the major constituent of infective learned about basic aspects of prion pathogenesis, such as the fractions purified from hamster brain homogenates [24]. Subsequent mechanisms of PrP misfolding and prion replication, the physiological characterization revealed that the pathogenic protein was host-encoded function of PrP, or the cellular pathways through which prions induce and not the product of a viral gene, as it had been assumed previously neurodegeneration. To this day, prion disorders remain incurable, and [25,26]. It was also established that PrPSc is posttranslationally derived neither early diagnostic methods nor anti-prion drugs have success- from PrPC [27]. Thus, although the two isoforms differ greatly in their fully been developed. Of special concern is the possibility that animal spatial conformation [28], they have the same amino acid sequence and prion diseases may be transmitted to humans via the food chain. In are encoded by the same single-copy gene, Prnp [29]. Under normal fact, experimental evidence indicates that transmission of BSE to conditions, PrPC is a glycoprotein tethered to the outer plasma humans was the likeliest cause for the appearance of a new variant membrane by a glycosylphosphatidylinositol (GPI) anchor. Its unique form of CJD (vCJD) [53,54]. These findings illustrate the need to ensure molecular structure, studied by nuclear magnetic resonance (NMR) and food safety, not only through efficient quality control but also by crystallographic techniques, can be roughly divided into two halves: a experimentally assessing the risk of prion transmission across a wide flexible N-terminal domain rich in repetitive motifs, and a C-terminal range of animal hosts. globular domain containing a characteristic array of three α-helices and So far, only beef and lamb have been identified as potential sources of two β-sheets. At the center of the polypeptide chain, a short dietary prion infections. Other major animal food sources like fish and hydrophobic stretch connects the two major domains [30,31].In poultry appear to have remained free of prion contamination. However, contrast, attempts to resolve the molecular structure of PrPSc have not it is not clear whether these organisms are susceptible to prion been successful. pathogenesis because TSE research has been carried out predominantly Almost 30 years after the discovery of PrP, there is still no consensus in mice and other mammals. Therefore, the study of prion biology in about what its physiological role might be. Since PrP has no obvious non-mammals, particularly in fish, is of great relevance to food safety. similarities to known protein families, functional affinities cannot be Fish constitute a vital food resource for humans and animals worldwide inferred from amino acid sequence comparisons. Neither can organ- or (Table 1), with direct impact on important economic activities like tissue-specific functions be hypothesized because PrP is widely fishing and aquaculture. expressed in most adult and embryonic cell types [32–35].Astonish- In addition, several fish species have become valuable tools in ingly, the generation and analysis of PrP knockout mice failed to provide biomedical research. One of them, the zebrafish (Danio rerio), is an conclusive information about the physiological role of PrP, as these excellent model to study vertebrate development and the biology of animals develop and behave rather normally. Aside from a few subtle human disease. It has many advantages as a genetic system, such as its 404 E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414

Table 1 divergent regions of the protein, respectively. Remarkably, and despite Commercially important fish species worldwide. having undergone substantial sequence variation, the globular domain Source: Food and Agriculture Organization of the United Nations, Fisheries and retained its structural fold throughout evolution [67,69].Theseobserva- Aquaculture Department (http://www.fao.org/fishery/en). tions strongly suggest that the globular domain carries out an important Common Scientific Main producers Main consumers function, which was already present in the last common ancestor of all name name vertebrates. Whether prion behavior was already an intrinsic attribute of Salmon Salmo salar Chile, Norway, Scotland, USA, Japan, Europe, such primitive PrPs, or whether it appeared late in evolution remains to Canada North America be clarified. Carp Cyprinus carpio Central Europe, China Austria, Germany, Hungary, Poland So far, TSEs have only been reported in mammals. This is not Tilapia Oreochromis Africa, China, Papua New Guinea, USA, China surprising, since prion biology is a field primarily focused on the study of niloticus Philippines, Thailand, Indonesia human diseases. Nevertheless, if PrP is intrinsically able to form niloticus infectious prions, then its presence in non-mammals implies that Tuna Thunnus Japan, Australia Japan, Australia these animals would – at least in theory – be susceptible to some form of albacores fi Catfish Ictalutus USA, China USA, China prion disease. Evaluating this scenario is particularly dif cult without punctatus basic knowledge about PrP conversion and prion replication in non- Trout Oncorhynchus Europe, USA, Chile Europe, USA, mammalian species. Moreover, it is not even clear what the eventual mykiss Japan manifestations of TSEs in fish or birds might be. Clearly, extensive Seabream Sparus aurata Mediterranean countries Europe Sea bass Dicentrarchus Mediterranean countries Europe experimentation will be required to establish whether these organisms labrax can acquire and transmit prion disease.

2.2. Prion transmission and the species barrier large offspring size and short generation time [55]. Furthermore, zebrafish embryos are transparent and develop externally, making Prion transmission across different species is limited by the so-called them amenable to detailed cellular analyses and genetic manipula- species barrier. Following primary inoculation between two species, tions. Moreover, the zebrafish is rapidly emerging as a valuable tool in only some animals develop disease, with long incubation periods that clinical and pharmaceutical research aimed at drug discovery and may reach the normal life span of the species. This phenomenon is validation [56,57]. clearly illustrated by the resistance to TSE observed in rabbits, even after In this article, we review current knowledge about the occurrence of intracranial inoculation with TSE agents of sheep, mouse and human PrPs, prions, and TSEs among vertebrates, focusing on recent advances origin [70]. It has been suggested that the unique structural character- using fish models to characterize the physiological function of PrP and istics of rabbit PrP make it less prone to pathogenic conversion [70,71]. the possible risk of prion transmission between fish and mammals. Typically, species barriers become attenuated after serial passaging of a prion strain, resulting in shortened incubation times and the consequent 2. Prion disease: a mammalian attribute? adaptation of the host to the specific strain [72]. At the molecular level, the efficiency of interspecies prion transmission appears to depend on 2.1. PrP and prion-like “behavior” the degree of sequence and structural identity between the PrPs of the two species involved [73]. For instance, wild type mice do not become Besides PrP, a handful of mammalian proteins appear to be able to infected with hamster prions, whereas transgenic mice overexpressing replicate in a prion-like manner. Among them are the molecular culprits hamster PrP do [74]. However, BSE can effectively be transmitted to a of known neurodegenerative disorders, such as amyloid-β (Alzheimer's wide range of species and maintain its pathogenic characteristics, even disease), α-synuclein (Parkinson's disease), tau (Tauopathies) and when passaged through an intermediate species carrying a different PrP amyloid A (AA amyloidosis) [58]. Such observations are undoubtedly coding gene [75]. intriguing, yet it still needs to be established whether the rogue versions It has been proposed that every species harbors a number of possible of these proteins are indeed infectious and, like bona fide prions, capable PrPSc conformational variants, with a thermodynamically “preferred” of spreading disease between individuals or even across species. In conformation (strain) being the decisive factor for interspecies fungi, at least six different proteins exhibit prion-like behavior, as transmission. According to this “conformational selection model” [76], judged by their ability to propagate phenotypic traits between cells asignificant overlap between the preferred PrPSc configurations of two without the need for nucleic acids [59]. However, these proteins have species is likely to result in a relatively easy transmission of prions neither structural nor functional similarities to PrP, or to one another. between them [77]. On the contrary, two species not sharing common Thus, while the study of fungal prions has provided valuable information PrPSc structures would have to overcome a considerable barrier to about the molecular basis of prion conversion, it is not clear to what transmission, and pathogenesis would not ensue without modification extent this knowledge may apply to mammalian prion disease [60]. of the strain type. Therefore, evaluation of the prion transmission from In addition to mammals, PrP homologues have also been identified in one species to another should also be based on the structural identity birds [61],reptiles[62], amphibians [63] and fish [64],butnotin between the host and donor PrP molecules and not exclusively on their invertebrates. Comparison of amino acid sequences indicates that PrPs amino acid sequence [78]. have evolved differently in each vertebrate class. For instance, although The route of infection is another parameter affecting the magnitude mammalian and avian sequences are strongly conserved, with similarity of species barriers. Most inoculation studies rely on the use of scores of ~90% in each group, conservation between the two groups is intracranial injection because “natural” per os infection is usually less rather low (~30% similarity) [65,66]. Likewise, PrP sequences among effective [79,80]. Notably, even after homologous oral transmission of bony fish are relatively well conserved (50–60% similarity), but exhibit BSE to cattle, animals may remain presymptomatic for 28 months, only 20–25% similarity to mammalian sequences [67]. PrPs also exhibit despite the evident PrPSc deposition in their brains [81]. Although the considerable variation in size, ranging from ~260 amino acids in relative importance of the aforementioned factors has been extensively mammals and birds to ~600 amino acids in fish. Despite these large studied, the outcome of an intra-species prion transmission cannot be differences in length and sequence, all vertebrate PrPs share the same predicted in advance. It is noteworthy, that two different TSE strains that protein domains (Fig. 1) [67,68]. The degree of conservation varies can effectively infect one species may present completely modified significantly along the polypeptide, with the short hydrophobic stretch transmissibility potentials when transmitted to a second species. Thus, it and the repetitive domain being the most conserved and the most has been proposed that a more suitable term to describe these E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414 405

Fig. 1. Fish and mammalian prion proteins. Upper panel: Fish possess two prion proteins, PrP-1 and PrP-2. Despite differences in sequence and length, fish and mammalian PrPs share the same protein domains (drawn at scale). Lower panel: Recent studies indicate that a common involvement of vertebrate PrPs in cell–cell communication. In cultured cells, PrPs elicit cell–cell contact formation. In the zebrafish embryo, PrP-1 and PrP-2 are deployed during gastrulation and neural development, respectively. L = leader peptide, R = repetitive domains, H = hydrophobic stretch, G = globular domain; orange triangles indicate the ends of the mature polypeptides. PrP localization at cell contacts is depicted in red.

phenomena would be “transmission barriers” instead of “species [64]. Subsequent cloning and sequencing of PrP genes in salmon, trout, barriers” [76]. carp, stickleback, sea bream and zebrafish [67,68,85,86] revealed that Recent data have led to the reconsideration of many parameters that bony fish possess two PrP orthologs, PrP-1 and PrP-2, which probably were thought to be indicative of a species barrier, such as the lack of arose during a fish-specific genome duplication [87]. Northern and clinical symptoms. Interestingly, cases of subclinical infection in Western Blot analyses indicated that the fish proteins are expressed at mammals displaying neuropathological and biochemical abnormalities, particularly high levels in adult fish brains, as well as in muscle, skin, often including PrPSc accumulation, have been reported [82]. Thus, heart and gills [67]. Although quite variable in size and sequence, PrP- prion-inoculated animals harboring high infectivity levels, and even 1 and PrP-2 present characteristic features of mammalian PrPs: an N- abnormal PrP aggregates in their brains, may under certain circum- terminal signal peptide, conserved N-glycosylation sites, two cysteine stances remain asymptomatic carriers throughout their lifetime. residues that form a disulphide bond, a central hydrophobic stretch, Notably, subclinical disease has been reported even after homologous and the C-terminal signal for the attachment of a GPI-anchor (Fig. 1). transmission, for instance, in studies using the oral route of infection and An intriguing feature of fish PrPs is the presence of a highly conserved low dosage inoculum [79,83,84]. motif of 13 amino acids in length between the repetitive region and the hydrophobic stretch [67]; the structural or functional importance 3. Fish prion proteins of this motif is not evident from its sequence. The repetitive domains of fish PrPs are easily recognizable but at the 3.1. Duplicated PrPs in bony fish same time clearly different from those of tetrapod PrPs. For example, while a constant number of nearly identical octarepeats is typical for PrP genes from land vertebrates have typically been cloned using mammals (or hexarepeats in birds), in fish the number and length of conventional procedures such as cDNA hybridization and PCR degenerate repeats vary from species to species. Nevertheless, all PrP screening. In contrast, fish PrP genes could not be isolated with repeats can be considered variations of two basic types of repeats (A and these methods due to their large DNA sequence divergence. The first B), which evolved differentially in every vertebrate class [67].The fish PrP homologue was identified in Fugu rubripes by searching sequence of the globular domain also has diverged considerably genome databases for ORFs encoding key structural features of PrPs between fish and mammals, owing to high rates of amino acid 406 E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414 substitutions, insertions and deletions. Notably, these changes predom- activity. Furthermore, the ability of mouse PrP to partially revert the inantly affect residues outside key structural motifs, and are not PrP-1 phenotype strongly suggests that this activity of PrP is expected to affect the globular fold [67]. conserved between fish and mammals [92]. It is therefore surprising Apart from PrP-1 and PrP-2, other genes partially resembling PrP that the loss of such an important function would not be evident in PrP have been reported in fish. Originally described as PrP-like and Shadoo knockout mice, especially since PrP is expressed at high levels in the [88,89], they are also known as PrP-1-rel and PrP-2-rel because of their mouse embryo. A plausible explanation for this discrepancy is that the genomic location, directly downstream of PrP-1 and PrP-2, respec- mouse knockout phenotype may become masked by genetic tively [67]. The encoded products are GPI-anchored polypeptides of compensation or developmental plasticity [96]. only 150–180 amino acids in length, expressed in brain tissues, and The relative simplicity of the zebrafish gastrula makes the PrP-1 containing the characteristic PrP hydrophobic stretch. Further phenotype an excellent choice to analyze conserved mechanisms of similarities to PrPs include a β-strand motif in PrP-rel-1 identical to PrP function at the molecular and cellular levels. Detailed character- the first β-strand of PrP, and the presence of degenerated B- and A- ization of PrP-1 knockdown embryos revealed that the developmental like repeats in PrP-rel-2. This situation is reminiscent to that of Prnp arrest was caused by the loss of tissue cohesion and the consequent and its genomic neighbor Doppel. Thus, PrP-rels, like Doppel, may failure to carry out epiboly, an early morphogenetic cell movement. have arisen from the tandem duplication of an ancestral PrP gene, and Because epiboly relies on the control of cell–cell adhesion, this finding then diverged as a result of high substitution rates and the differential led to the discovery that PrP-1 regulates the stability of E-cadherin/ß- loss of motifs. Interestingly, although PrP-rels are predicted to be catenin adhesive complexes at the plasma membrane [92]. Ongoing unstructured proteins, their mammalian homologue Shadoo has biochemical work indicates that the mechanism behind this effect is recently received attention because it appears to influence biological the ability of PrP-1 to control the levels and membrane localization of and pathogenic activities of PrP in vivo [90,91]. ß-catenin as well as the levels and phosphorylation state of Src- related kinases (Sempou and Málaga-Trillo, in preparation). Related 3.2. Zebrafish PrP-1 and PrP-2 unpublished in vivo pharmacological data suggest that PrP-1 acts to prevent the degradation of some of these proteins. The characterization of PrP homologues in the zebrafish opened the These results are in agreement with long proposed roles of PrP as a exciting possibility to study prion biology in a new model organism. Like signal transduction molecule [46]. Concretely, the experiments with other bony fish, the zebrafish has two prion protein orthologs, PrP-1 and zebrafish gastrulae indicate that a complex signaling cascade is PrP-2, mapped to chromosomes 10 and 25, respectively [67].Genomic initiated by the interaction between PrP-1 molecules across opposite studies revealed that the two genes originated from the duplication of a cell membranes. Accordingly, in vitro experiments show that fish, large chromosomal block that corresponds to those containing the Prnp amphibian, avian and mammalian PrPs share the intrinsic ability to gene in mammals. That both gene copies are fully functional has been engage in such homophilic trans-interactions, promoting the forma- biochemically confirmed in fish and mammalian cells [92,93].Inthese tion of cell–cell contacts, and triggering intracellular signaling via Src- studies, it was shown that the encoded polypeptides become properly related kinases [92]. Of special interest within the scope of this review glycosylated and attached to the plasma membrane via a GPI-anchor. As is the finding of heterologous interactions between zebrafish and is often the case with duplicated genes [94], PrP-1 and PrP-2 are thought mammalian PrPs [92], which raise the question of whether PrP to have retained complementary subsets of ancestral PrP functions. conversion and TSE transmission can take place across such distant Accordingly, zebrafish PrPs have evolved distinct spatiotemporal species. patterns of embryonic expression: while PrP-1 is ubiquitously expressed Altogether, the combined work in zebrafish embryos and at high levels in very early stages of embryogenesis (blastula and mammalian cells indicate that PrPs play crucial roles in cell–cell gastrula stages), PrP-2 is strongly upregulated at later stages in communication (Fig. 1). These data are in line with the notion that PrP developing neurons [92]. At later larval stages and into adulthood, provides protective signals required for neuronal growth and/or both genes continue to be expressed in the CNS, although PrP-2 at much survival [97,98]. Understanding the precise nature of such signals will higher levels than PrP-1. The differences between the expression be crucial to uncover the early cellular mechanisms of prion patterns of PrP-1 and PrP-2 are due to the divergent evolution of DNA pathogenesis in the mammalian brain. transcriptional regulatory sequences upstream of each gene (Barth and Málaga-Trillo, unpublished data). Interestingly, the expression of 4. Risk of prion disease in fish zebrafish PrPs (particularly PrP-2) in the developing nervous system bears striking similarities to that of PrP in the mouse embryo [32].It 4.1. TSEs and the food chain remains to be determined if, like PrP-1,mammalianPrP plays a role during blastula and early gastrula stages. The appearance of BSE in Europe has been causally linked to the common practice of feeding cattle with meat and bone meal (MBM) of 3.3. PrP loss of function phenotypes in zebrafish embryos bovine origin [99]. Approximately 200,000 BSE cases were reported between 1985 and 2000, and it has been estimated that roughly three One of the many experimental advantages of the zebrafish is the million infected animals could have entered the human food chain possibility to knockdown the expression of any given gene using before developing a clinical disease [81]. Due to its low cost and high morpholino antisense oligonucleotides. When microinjected into nutritional value, MBM has become an important ingredient in the early fish embryos, morpholinos rapidly bind to their target mRNAs diet of non-ruminant livestock and farmed fish. Hence, there is the and block their translation [95]. Recently, this method was success- theoretical risk that farmed animals unintentionally fed with prion- fully applied to study PrP loss of function in the zebrafish. In contrast contaminated MBM might develop TSEs or serve as asymptomatic to the absence of clear phenotypes in PrP knockout mice, knockdown carriers of a disease [100]. Since BSE was initially reported, the of zebrafish PrPs revealed that these genes play essential roles during European Commission (EC) has implemented a comprehensive set of distinct phases of embryonic development. Specifically, PrP-1 knock- TSE risk-reducing measures to protect humans from BSE, as well as to down causes lethal embryonic arrest during gastrulation, whereas control and eventually eradicate prion diseases in animals. Among PrP-2 knockdown severely impairs brain development. Notably, the these, a total EU-wide feed ban on the use of rendered animal proteins PrP-1 phenotype can be rescued by PrP-1 or PrP-2 overexpression, in the nutrition of all animals was imposed, with minor exceptions indicating that although the two proteins are expressed in different (i.e. use of fish feeds in the diets of non-ruminants like pigs and developmental contexts, they share essentially the same biological chicken). E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414 407

Experimental demonstration that TSEs can propagate in a new makes it rather difficult to exclude such a possibility. Regarding the host without typical pathognomonic brain lesions, clinical disease or convertibility potential of fish PrPs, it has been suggested that the fatality, has led to the conclusion that prion-infected individuals could extended type A-repeat domain in sea bream PrP-1 and other fish PrPs go undetected within an exposed population [100]. This implies that could confer on these proteins the ability to self-polymerize and farmed animals may carry the disease but not develop it until they are aggregate [85]. In addition, it has been noted that motifs similar to slaughtered. Human consumption of meat from these animals would, those observed in fish PrPs can also be identified in unrelated proteins in turn, give rise to repeated infection cycles long before clinical signs such as the piscine annexin 11 and the mammalian lectin L-29 [77]. can be recognized. Moreover, the relatively recent discovery of Notably, both these proteins display self-aggregation properties, and unconventional TSE phenotypes, such as atypical scrapie in sheep annexin 11 is, like PrP, a neuro-specific membrane protein. and goats [101,102], and bovine amyloidotic spongiform encepha- lopathy (BASE) in cattle [103], usually displaying decreased or altered 4.3. Transmission studies in commercially important fish species PrPSc resistance to proteolytic treatment and less pronounced clinical signs, has further made it difficult to define prion diseases in absolute At present, only a few studies have explored the experimental terms and thus, to implement effective rules to eliminate them. transmission of TSEs to teleost fish. The first attempts in this direction involved the oral and parenteral inoculation of two commercially 4.2. Risk of prion transmission to fish important fish species, rainbow trout and turbot, with the 139A mouse-adapted scrapie strain [105]. Although the fish displayed no According to the aforementioned arguments, fish, poultry and pigs clinico-histopathological signs during the three month-experimental represent potential candidate hosts for prion infection. In order to assess period, a mouse bioassay revealed that they carried residual the risk of prion transmission to fish, five main parameters should be infectivity. Specifically, mice intracranially inoculated with trout considered: 1. the potential use of prion-contaminated MBM as a intestinal extracts one day after oral challenge were positive for nutritional supplement in aquaculture, 2. the consumption of infected brain PrPSc deposition approximately 200 days post inoculation (p.i.), farmed fish by humans, 3. the use of feeds or other products (i.e. gelatin, despite the absence of clinical symptoms. Similar results were milk replacers) derived from TSE-affected fish for mammalian or piscine obtained when mice were intracerebrally (i.c.) inoculated with spleen nutrition, 4. the use of fishmeals cross-contaminated with MBM in extracts from trouts and turbots, 15 days after parenteral inoculation mammalian diets, and 5. the escape of infected fish, or the release of of the fish with scrapie material. Finally, brain tissue from parenterally infected waste from aquaculture facilities into the marine environment. inoculated turbots 15 and 90 days after challenge was also able to These scenarios are depicted in Fig. 2. elicit PrPSc accumulation in the brains of recipient mice, without With fish mariculture turning into a very important and rich causing clinical disease. protein source for humans, consumers may become concerned about Recently, we reported data on the oral transmission of BSE and the possibility of farmed fish developing prion disease, or serving as scrapie to gilthead sea bream (Sparus aurata) [106]. Interestingly, at passive carriers of prion infectivity. All farmed fish receive commercial two years p.i., a number of fish that had been force-fed BSE- or feeds containing 40–55% protein, and since some of it is likely to be of scrapie-infected brain homogenates developed abnormal plaque-like animal origin, the possibility of feed contamination with mammalian deposits in their brains. Specifically, the brains of two out of five fish prions cannot be excluded. Interestingly, in the late 1980s, total fish inoculated with scrapie developed signs of abnormal protein feed production in the UK was in the order of 75,000 tons per year, aggregation at 24 months p.i. These aggregates were positively with the use of about 3750 tons of MBM in fish feed [104]. stained with polyclonal antibodies raised against fish PrPs, but However, how realistic is the thesis that fish prion proteins may showed no proteinase K (PK)-resistance or Congo red birefringence misfold and form abnormal aggregates? The striking conservation of (Fig. 3A). The brains of the BSE-challenged fish, however, displayed a structural motifs between mammalian and piscine PrP molecules much more striking picture, having already developed the first signs of abnormal deposition at eight months p.i. A general progression in size, PK-resistance and morphological features was observed there- after, resulting in an impressive number of aggregates in all the brain regions examined at 24 months p.i. Three out of five fish sacrificed at this time point showed 500–800 deposits per brain section each, 70– 85% of which were PK-resistant and had a mean diameter of 30 μm. These aggregates were PAS-positive, congophilic and birefringent in polarized light, indicating an amyloid or amyloid-like fibrillar structure (Fig. 3B). In contrast to the TSE-challenged individuals, no signs of abnormal aggregation or any other lesions were observed in the brains of the control fish “challenged” with bovine or ovine brain homogenates prepared from healthy animals. Altogether, the devel- opment of abnormal brain deposits in BSE-challenged sea bream constitutes an unprecedented histopathology in fish.

4.4. Neuropathology in TSE-challenged sea bream

The lesions observed in the brains of the BSE-infected sea bream share some remarkable similarities with those typically seen in prion- affected mammalian brains. For instance, the deposits in fish brains were only detectable where neuronal parenchyma was present, a feature greatly resembling the localization of prion amyloid plaques in mammals [107]. The most extensively affected regions included the cerebellum (which displayed a mammal-like deposition motif [108]), Fig. 2. Prion diseases and the food chain. Potential scenarios for the transmission of fi prions between mammals and fish are illustrated. Arrows indicate the direction of the lateral nucleus of the ventral telencephalic area (a sh counterpart possible transmission events. to the basal nucleus of Meynert in mammals [109]), the lateral 408 E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414

Fig. 3. Experimental transmission of prions between mammals and fish. Inoculation of fish with ovine (A), bovine (B) and mouse (C) prions and their various outcomes. The photographs on the upper right corner illustrate the development of abnormal amyloid deposition in the optic tectum of BSE-challenged sea bream, using the periodic acid Schiff (PAS) staining reaction. The right image (scale bar=10 μm) is a zoom-in of the lesion indicated in the left image (scale bar =100 μm). i.c., intracerebrally. telencephalic pallium (homologue to the mammalian hippocampus carriers of the infection without ever developing a clinical disease. For [110]), and finally the thalamus and diencephalon. Notably, no example, the absence of clinical symptoms has been reported in mice spongiosis was observed in any of the brain regions examined. However, following the primary transmission of sheep and hamster scrapie. cases of TSE subtypes that develop little or no vacuolation have also been However, sequential passage of infectious material did result in the reported for human prion diseases [111]. Interestingly, the neurode- development of clinical disease [82]. Interestingly, differential generation observed in BSE-challenged sea bream affected only susceptibility to specific pathogens is also known among piscine neurites, as opposed to the “classically defined” degeneration of species. For instance, closely related fish species may exhibit neuronal somata. Based on the morphological progression of the differential resistance to viral infections, as illustrated by the abnormal deposits and the level of neurite involvement, aggregates development of clinical disease in sea bass but not in sea bream were classified into two main categories. “Pre-mature” deposits, infected with nodavirus [112,113]. On the other hand, the absence of observed mostly at earlier time points and consisting mainly of clinical symptoms in BSE-challenged sea bream could be related to the dystrophic neurites that have lost their coherence, appear to mark remarkable ability of teleost fish to continuously produce new initial stages of neurodegeneration. “Mature” deposits, observed neurons throughout their lifetime. It is well known that adult fish 24 months p.i., seem to result from the complete deconstruction of can regenerate destroyed retinal tissue, optic nerve axons, and fibers, leading to the development of a more homogenous and degenerated brain stem neurites, resulting in functional recovery of flocculated extracellular material, with increased PrP-immunoreactiv- these structures [114,115]. Given the fact that farmed fish remain in ity, congophilia and birefringence in polarized light. The two types of aquaculture facilities up to 20 months before reaching the required aggregates in the brains of BSE-challenged sea bream could be commercial weight, the lack of clinical disease in infected animals interpreted as different stages of pathogenesis, with the former may become a crucial issue of unanticipated relevance for public temporally preceding the latter. For scrapie-challenged sea bream, the health. distribution and morphology of the observed neuropathology could not be evaluated and classified as mentioned earlier, due to the limited 4.6. Differential susceptibility of fish species to prion infection number of fish developing abnormal deposits in this group. In a parallel study, we inoculated European sea bass (Dicentrarchus 4.5. Absence of “clinical symptoms” in fish: presymptomatic carrier labrax) with the same infectious material, namely BSE and scrapie. state? Interestingly, in contrast to the sea bream, none of the challenged individuals showed any clinical symptoms or histopathological Visual examination of challenged fish throughout the post evidence of abnormal deposition at any point throughout the time inoculation period revealed no signs of altered swimming behavior course of the study. Sea bass and sea bream are both Perciformes, the or eating habits. However, previous studies have shown that species largest class of teleosts. Although they belong to different families, once thought to be resistant to certain TSE strains can be life-long Sparidae (sea bream) and Moronidae (sea bass), the two species are E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414 409 quite closely related at the phylogenetic and anatomical levels. Since the GI tract may serve as a potential portal for exogenous PrPSc,dueto the amino acid sequence of sea bass PrP is not available, it is not clear the high PrP mRNA levels detected in the posterior intestine of zebrafish how it might differ from that of sea bream PrP. However, the two larvae [68]. In mammals, studies have shown that the pathogenic agent proteins display different reactivities against anti-fish PrP antibodies. may invade the CNS directly, by exploiting the extended autonomous Conceivably, such differences could confer on the sea bass PrP intestinal innervation [120]. In particular, it has been proposed that BSE resistance to the PrPSc in the inoculum, and thus, reinforce the neuroinvasion may take place via the parasympathetic nerve fibers of “transmission barrier” in these experiments. the vagus nerve into the brain stem of orally-challenged cattle [81].Such Another factor possibly involved in the different reaction of the a route of dispersion requires the adequate expression of endogenous two species to prion inoculation might be the ease with which PrPC within the peripheral nervous system [121]. In the sea bream, exogenous PrPSc can cross the intestinal barrier and reach the CNS immunohistochemical examination of intestinal tissue revealed three [116]. In teleosts, like in other vertebrates, oral pathogens are initially main regions of intense PrP-immunopositivity, namely the serosa, the confronted by activated immune cells on the enteric wall. Macro- myenteric plexus (corresponding to Auerbach's plexus in mammals) phages, whose efficiency contributes to the resistance against and the submucous plexus (a homologue to mammalian Meissner's pathogens, play an important role in this process. While in sea bass, plexus). Both the myenteric and the submucous plexus contain many enteric phagocytes seem to become extensively activated upon parasympathetic nerves and ganglia, one of which is a nucleus of the microbial attack [117], it has been reported that the intestinal mucosa vagus nerve running to the medulla oblongata in the brain stem of of the sea bream may be relatively permissive to infection by certain mammals and teleosts. The hypothesis that the infectious agent might bacteria [118]. use the enteric nervous system to invade the fish CNS is supported by Although plausible, these hypotheses do not entirely explain the the progressive changes observed in the regional distribution of differential sensitivity of the two species to prion infection, since cases abnormal deposits in sea bream brain [81]. Initially, the region affected of pathogenesis in which the clinically affected species is the sea bass is the posterior part of the brain, with the brain stem displaying the most and not the sea bream are also known [112]. It is noteworthy, severe lesion profile. Subsequently, anterior anatomical regions show however, that the different susceptibility of the two species can be signs of abnormal aggregation as well, although posterior brain areas regarded as an internal control in our transmission experiments. continue to be affected. Interestingly, it has been proposed that direct Specifically, the fact that despite the close relationship between the neuroinvasion of the infectious agent following per os challenge might two species, only sea bream were affected, strengthens the hypothesis be related to the absence of clinical symptoms and the reduced attack that the observed neuropathology represents a novel piscine rate of the transmitted disease [122], an observation that is in agreement amyloidosis triggered by mammalian prions. Thus, transmissibility with the asymptomatic amyloidosis developed by BSE-challenged sea of prions to fish exhibits species specificity, a common feature of TSEs. bream.

4.7. Possible routes of neuroinvasion: tissues harboring infectivity 5. Future directions

Immunohistochemical examination of peripheral tissues from TSE- 5.1. Monitoring for infectious neurodegenerative disease in farmed fish inoculated sea bream including spleen and intestine revealed no signs of abnormal protein accumulation at any of the time points tested. The rapid worldwide expansion of fish farming calls for the Therefore, it is likely that the transit of prions from the fish implementation of efficient measures to ensure animal health and gastrointestinal (GI) tract to the CNS might have occurred long before food safety. The fact that pathogens can spread considerably more easily the first time point of sacrifice, perhaps only a few hours or days after through flowing water than between land animals is one of the main oral challenge, leaving no trace of infectivity in the peripheral tissues difficulties when dealing with aquatic diseases. Over the last years, the tested thereafter. Transmission studies in rainbow trout and turbot have aquaculture industry has been faced with an increasing number of shown that specific binding of exogenous PrPSc (139A scrapie strain) can infectious diseases affecting both farmed and feral populations. be detected in the intestinal mucosa for up to seven days p.i., despite the Surveillance and monitoring programs have therefore been developed absence of active uptake by the epithelial cells [105,119]. in many countries, in an attempt to prevent and control the emergence Preliminary data from a more recent study demonstrate the of new fish pathologies [123]. The list of notifiable diseases varies importance of residual infectivity, suggesting that after oral challenge depending on the specific needs of each region. In Europe, all countries with the ME7 mouse-adapted scrapie, prions can cross the intestinal are asked to comply with the Council Directive (2006/88/EC) on health wall of the sea bream and be delivered from the peritoneal cavity into requirements for aquaculture animals. Nevertheless, it would be the spleen (Figueras et al., unpublished data). More specifically, sensible to enrich the list of large-scale spot checks in fish farms by inoculated fish were sacrificed at regular time points, and various including the histological examination of fish brains, in order to detect tissues, including spleen and intestine, were used to intracranially possible signs of feed-induced or even spontaneous PrPSc amyloidosis. inoculate mice (Fig. 3C). The TSE-challenged fish never showed As described earlier, BSE-infected sea bream developed the first signs of histopathological or clinical symptoms throughout the 18 month- abnormal aggregation already at eight months p.i., a time period that lies experimental period. However, serial passage to mice of tissues from within the grow-out phase of fish breeding. Moreover, the fact that fish sacrificed one week p.i. induced vacuolation in the brains of some these animals remained asymptomatic throughout the experimental of the mice, 300 days after the intracranial inoculation. Concretely, period raises the possibility that affected fish may generally go spongiosis without clinical disease was detected in five out of 19 undetected before ending up on our plates. recipient mice inoculated with scrapie-infected fish spleens, and in 12 out of 17 individuals challenged with scrapie-infected fish intestines. 5.2. Further transmission studies in commercially important fish species These findings suggest, on one hand, that the TSE agent might have been able to reach the fish brain in a remarkably short period of time, Gilthead sea bream and European sea bass are the two most either after peripheral propagation or even by direct neuroinvasion. important farmed fish in the Mediterranean Sea, with the main On the other hand, this study emphasizes the need for a thorough producers being Greece, Turkey, Spain, France and Italy. In 2004, the examination of peripheral tissues in asymptomatic individuals annual production levels of both species reached 160,000 tons following prion inoculation. worldwide. Therefore, assessing the risk of TSE transmission to these There is no doubt that the intestine represents the main entry point fish is an issue of major importance for public health. On a global scale, for oral pathogens. In zebrafish, for example, it has been proposed that the practice of aquaculture is experiencing steady growth rates, driven 410 E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414 by the increasing demand for aquatic food products. According to fish would pose to humans. On the other hand, addressing the cellular projections by the Food and Agriculture Organization of the United and molecular aspects of prion transmission to fish requires the use of Nations (FAO), the global production of farmed fish will have to reach established model organisms. The zebrafish is best fitted for this 80 million tons by 2050, in order to maintain the current level of fish purpose, not only because of its well-characterized neuroanatomy consumption per capita. Amazingly, not all countries have introduced and neural development, but also because of the availability of useful laws to regulate the use of rendered mammalian proteins for fish research tools, such as mutant strains, GFP-transgenic lines, specific nutrition and, in some cases, those that have, have incurred in minor and antibodies, cell lines and a fully sequenced genome. Additionally, major violations [124]. Hence, the likelihood that contaminated MBMs zebrafish larvae are optimally suited for the study of neurological might have been inadvertently fed to farmed fish is considerably high. In diseases because they display quantifiable neuropathological and this regard, the amyloidosis observed in the brains of BSE-fed sea bream behavioral phenotypes similar to those observed in humans [125].It demonstrates that certain fish species can develop neuropathology would be particularly interesting to know whether scrapie or CJD following ingestion of prion-contaminated meals. Therefore, further prions can be efficiently inoculated to transgenic zebrafish expressing transmission studies with other commercially important fish species, mouse or human PrPs. Similarly, expression of pathogenic PrP including salmon (Salmo salar), trout (Oncorhynchus mykiss), and carp mutants in wildtype and transgenic fish could be of great value in (Cyprinus carpio) should be conducted in order to evaluate the elucidating the genetic basis of prion disease. Engineering of these likelihood of acquiring TSEs upon consumption of infected feeds animals, currently underway, will provide an ideal tool to systemat- (Fig. 4). In addition, primary TSE transmission studies in piscine species ically analyze prion replication in fishbrains,aswellasthe that are considered future candidates for mariculture would be development of PrP aggregates, brain lesions, and possibly clinical advisable, especially those closely related to sea bream, such as the disease. Another fascinating perspective is the possibility to study the common dentex (Dentex dentex, Sparidae)andtheredporgy(Pagrus effect of exogenously added prions on zebrafish embryos. Such pagrus, Sparidae). experiments could significantly facilitate the analysis of PrPC and PrPSc behavior in vivo, thus contributing valuable insights into the 5.3. Zebrafish as a model for prion pathobiology relationship between prion conversion and PrP dysfunction. Alto- gether, transmission experiments in zebrafish could help to effective- Clearly, transmission experiments in commercially important ly evaluate the susceptibility of fish to TSEs, as well as the mechanistic species are necessary to directly assess the risk that TSE-infected link between PrP function and prion pathogenesis.

Fig. 4. Future scenarios for transmission studies. Examples of primary and secondary inoculation studies required for the evaluation of prion transmissibility between piscine and mammalian species. Tg = transgenic, wt = wildtype, ZF = zebrafish, boPrP = bovine PrP, ovPrP = ovine PrP, huPrP = human PrP, moPrP = mouse PrP. E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414 411

5.4. Assessing transmissibility and convertibility 5.5. Dissecting the physiological roles of PrP in the zebrafish

Issues of prion transmissibility between fish and mammals need to The molecular basis of prion disease is closely associated with the be given full consideration because of the possibility that fish fed with structural and functional properties of PrP. Therefore, deep under- prion-contaminated MBM may further transmit TSEs through the standing of prion pathogenesis in fish and mammals cannot be food chain. To evaluate this scenario, secondary transmission reached without basic knowledge about the physiological roles of PrP. experiments between BSE-affected fish brains and mice carrying the The knockdown phenotypes described in zebrafish constitute the first bovine Prnp gene are currently underway. Further studies in experimental evidence that the lack of PrP can cause dramatic transgenic fish will also help assess the risk of potential transmission abnormalities in a whole organism [92]. In addition, the finding that of piscine amyloidoses to mammals or to other fish species (Fig. 4). PrPs regulate cell–cell communication via known cellular pathways For instance, the use of transgenic zebrafish carrying the bovine Prnp, provides an ideal opportunity to establish how PrP may exert its or mice carrying the gene coding for fish PrP on a PrP knockout proposed neuroprotective role (Fig. 5). It is noteworthy that some of background would answer a plethora of questions concerning not the molecules affected in PrP-1 knockdown embryos (Src-kinases, E- only host-related pathogenic determinants but also the specific cadherin, β-catenin, etc.) are not only essential for early zebrafish conditions required for certain intra-species transmissions. development but also play important roles in neuronal function and The discovery of residual prion infectivity in fish intestines and during neurodegeneration [96]. Therefore, it will be crucial to further spleens is also a matter of great concern, as it suggests that even fish characterize the signaling pathways under the control of PrP-1 using species resistant to prion infection might harbor infectivity shortly biochemical, cell biological, proteomic and imaging tools. Such after ingestion of contaminated meals, thus being able to transmit the experiments are currently being carried out in fish embryos and disease to consumers. Similar studies should be undertaken employ- embryonic stem cells. Ideally, this basic knowledge should be ing a variety of fish species and TSE strains, including atypical scrapie integrated into the design and interpretation of studies addressing and BSE, to assess residual infectivity levels not only in spleen and the function of PrP in mammalian cells, as well the experimental intestine, but also in blood and other “high risk” tissues (Fig. 4). transmission of prion disease between fish and mammals. Furthermore, bioassays should also include “humanized” mice in Characterization of the PrP-2 knockdown phenotype is currently in order to directly assess the risk of transmission to humans. progress. Preliminary results show that PrP-2 is required for the Another basic question that requires further clarification is the proliferation and differentiation of different groups of embryonic convertibility potential of fish PrPs. Ongoing experiments using the neurons (Málaga-Trillo and Luncz, unpublished data). A similar sensitive protein misfolding cyclic amplification (PMCA) method phenotype has been reported for PrP knockout mice [41], although [126,127] may reveal whether piscine PrPs have the intrinsic ability to the defect does not impair neural development to the extent seen in misfold and aggregate. In addition, the use of TSE-infected cell lines the zebrafish [92]. Because PrP-2 also shares the ability to elicit cell– could become an important tool to investigate possible molecular cell adhesion and intracellular signaling, it is likely to regulate neural interactions between mammalian PrPSc and fish PrPC, leading to the development via the same molecular pathways controlled by PrP-1 in conversion of piscine PrPs. Aside from transmissibility and convert- the early gastrula. Thus, morphological and molecular analyses of the ibility issues, the composition of the deposits found in the brains of the PrP-2 phenotype may provide valuable mechanistic insights into the BSE-challenged sea bream deserves further examination. Concretely, roles of PrP during neuronal differentiation, axon growth, and the identification of other aggregated molecules in brain deposits synaptogenesis. Integrating the emerging zebrafish data within the could help define the properties of these novel amyloid plaques, and vast amount of information available from mammalian cells and perhaps their mechanistic connection to neurodegeneration. For knockout mice promises to become a lengthy but rewarding task. instance, previous data on the composition of amyloid deposits present in degenerating neuronal tissue have suggested that the 6. Concluding remarks components of such aggregates may represent the outcome of the degeneration process rather than the cause of neuronal cell death For many years, prion researchers have relied heavily on the use of [128]. Thus, the study of novel proteinaceous deposits may contribute mammalian models as experimental systems. This strategy, and new leads to the debate about the causal link between neurotoxicity particularly the development of genetically modified mice, has led to and protein oligomerization. outstanding discoveries in the field. At the same time, the

Fig. 5. Exploring the roles of PrP in health and disease. It has been proposed that neuronal death results from failure by PrP to provide an unknown neuroprotective function. It remains to be clarified how the various cellular and molecular processes influenced by PrP in vivo may converge into such a neuroprotective role. 412 E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414 experimental difficulties associated with the study of TSEs have [24] D.C. Bolton, M.P. McKinley, S.B. Prusiner, Identification of a protein that purifies with the scrapie prion, Science 218 (1982) 1309–1311. exposed the need for new animal models in prion biology. Recently, [25] B. Oesch, D. Westaway, M. Walchli, M.P. McKinley, S.B. Kent, R. Aebersold, R.A. researchers have explored the utility of fish to address questions Barry, P. Tempst, D.B. Teplow, L.E. Hood, et al., A cellular gene encodes scrapie PrP concerning the physiological roles of PrP and the risk of TSEs in these 27–30 protein, Cell 40 (1985) 735–746. [26] B. Chesebro, R. Race, K. Wehrly, J. Nishio, M. Bloom, D. Lechner, S. Bergstrom, K. organisms. An interesting picture has emerged, suggesting that Robbins, L. Mayer, J.M. Keith, et al., Identification of scrapie prion protein-specific transmission of prions could potentially take place between com- mRNA in scrapie-infected and uninfected brain, Nature 315 (1985) 331–333. mercially important fish and mammals, although the implications of [27] D.R. Borchelt, M. Scott, A. Taraboulos, N. Stahl, S.B. Prusiner, Scrapie and cellular these findings for human and animal health remain to be clarified. prion proteins differ in their kinetics of synthesis and topology in cultured cells, J. Cell Biol. 110 (1990) 743–752. Much remains to be done in order to confirm or rule out the existence [28] K.M. Pan, M. Baldwin, J. Nguyen, M. Gasset, A. Serban, D. Groth, I. Mehlhorn, Z. of bona fide prions in fish. Nevertheless, the use of fish models in TSE Huang, R.J. Fletterick, F.E. Cohen, et al., Conversion of alpha-helices into beta- research will likely uncover novel aspects of prion transmission across sheets features in the formation of the scrapie prion proteins, Proc. Natl Acad. Sci. USA 90 (1993) 10962–10966. a wide range of hosts, relevant to human health. In addition, the [29] K. Basler, B. Oesch, M. Scott, D. Westaway, M. Walchli, D.F. Groth, M.P. McKinley, finding that fish and mammalian PrPs share important roles in cell– S.B. Prusiner, C. Weissmann, Scrapie and cellular PrP isoforms are encoded by the cell communication might help elucidate the molecular mechanisms same chromosomal gene, Cell 46 (1986) 417–428. [30] R. Riek, S. Hornemann, G. Wider, R. Glockshuber, K. Wuthrich, NMR by which prions induce neuronal death, setting the stage for the characterization of the full-length recombinant murine prion protein, mPrP identification of new anti-prion therapeutic targets. (23–231), FEBS Lett. 413 (1997) 282–288. [31] K.J. Knaus, M. Morillas, W. Swietnicki, M. Malone, W.K. Surewicz, V.C. Yee, Crystal structure of the human prion protein reveals a mechanism for oligomerization, Acknowledgements Nat. Struct. Biol. 8 (2001) 770–774. [32] J. Manson, J.D. West, V. Thomson, P. McBride, M.H. Kaufman, J. Hope, The prion protein gene: a role in mouse embryogenesis? Development 115 (1992) 117–122. This work is supported by a DFG grant (MA 2525/2-1) and University [33] M.J. Ford, L.J. Burton, R.J. Morris, S.M. Hall, Selective expression of prion protein of Konstanz funds awarded to EMT. ES, TS and AF are funded by EU in peripheral tissues of the adult mouse, Neuroscience 113 (2002) 177–192. grants (QLK5-2002-00866 for ES, TS, AF and FOOD-CT-2004-506579 for [34] W. Goldmann, G. O'Neill, F. Cheung, F. Charleson, P. Ford, N. Hunter, PrP (prion) gene expression in sheep may be modulated by alternative polyadenylation of ES, TS). ES is a scholar of the Greek States Scholarships Foundation (IKY), its messenger RNA, J. Gen. Virol. 80 (Pt 8) (1999) 2275–2283. and the sea bream and sea bass transmission experiments were part of [35] D.A. Harris, P. Lele, W.D. Snider, Localization of the mRNA for a chicken prion her doctoral thesis. AF would like to thank Raquel Aranguren and Beatriz protein by in situ hybridization, Proc. Natl Acad. Sci. USA 90 (1993) 4309–4313. [36] A.D. Steele, S. Lindquist, A. Aguzzi, The prion protein knockout mouse: a Novoa for contributing to the secondary transmission study of ME7- phenotype under challenge, Prion 1 (2007) 83–93. infected sea bream tissues to mice. [37] C. Kuwahara, A.M. Takeuchi, T. Nishimura, K. Haraguchi, A. Kubosaki, Y. Matsumoto, K. Saeki, T. Yokoyama, S. Itohara, T. Onodera, Prions prevent neuronal cell-line death, Nature 400 (1999) 225–226. References [38] D.R. Brown, W.J. Schulz-Schaeffer, B. Schmidt, H.A. Kretzschmar, Prion protein- deficient cells show altered response to oxidative stress due to decreased SOD-1 [1] S.B. Prusiner, The prion diseases, Brain Pathol. 8 (1998) 499–513. activity, Exp. Neurol. 146 (1997) 104–112. [2] J. Collinge, Prion diseases of humans and animals: their causes and molecular [39] C.E. Le Pichon, M.T. Valley, M. Polymenidou, A.T. Chesler, B.T. Sagdullaev, A. basis, Annu. Rev. Neurosci. 24 (2001) 519–550. Aguzzi, S. Firestein, Olfactory behavior and physiology are disrupted in prion [3] C.M. Poser, Notes on the history of the prion diseases, Part I Clin. Neurol. protein knockout mice, Nat. Neurosci. 12 (2009) 60–69. Neurosurg. 104 (2002) 1–9. [40] D.R. Brown, K. Qin, J.W. Herms, A. Madlung, J. Manson, R. Strome, P.E. Fraser, T. [4] A. Aguzzi, F. Baumann, J. Bremer, The prion's elusive reason for being, Annu. Rev. Kruck, A. von Bohlen, W. Schulz-Schaeffer, A. Giese, D. Westaway, H. Neurosci. 31 (2008) 439–477. Kretzschmar, The cellular prion protein binds copper in vivo, Nature 390 [5] C.M. Poser, Notes on the history of the prion diseases, Part II Clin. Neurol. (1997) 684–687. Neurosurg. 104 (2002) 77–86. [41] A.D. Steele, J.G. Emsley, P.H. Ozdinler, S. Lindquist, J.D. Macklis, Prion protein [6] W.J. Hadlow, Scrapie and kuru, Lancet 2 (1959) 289–290. (PrPc) positively regulates neural precursor proliferation during developmental [7] I. Klatzo, D.C. Gajdusek, V. Zigas, Pathology of kuru, Lab. Invest. 8 (1959) 799–847. and adult mammalian neurogenesis, Proc. Natl Acad. Sci. USA 103 (2006) [8] J. Cuillé, P.L. Chelle, La maladie dite tremblante du mouton est-elle inoculable? 3416–3421. CR Acad. Sci. 203 (1936) 1552–1554. [42] S. Chen, A. Mange, L. Dong, S. Lehmann, M. Schachner, Prion protein as trans- [9] D.C. Gajdusek, C.J. Gibbs, M. Alpers, Experimental transmission of a kuru-like interacting partner for neurons is involved in neurite outgrowth and neuronal syndrome to chimpanzees, Nature 209 (1966) 794–796. survival, Mol. Cell. Neurosci. 22 (2003) 227–233. [10] C.J. Gibbs Jr., D.C. Gajdusek, D.M. Asher, M.P. Alpers, E. Beck, P.M. Daniel, W.B. [43] N.R. Cashman, R. Loertscher, J. Nalbantoglu, I. Shaw, R.J. Kascsak, D.C. Bolton, P.E. Matthews, Creutzfeldt–Jakob disease (spongiform encephalopathy): transmis- Bendheim, Cellular isoform of the scrapie agent protein participates in sion to the chimpanzee, Science 161 (1968) 388–389. lymphocyte activation, Cell 61 (1990) 185–192. [11] H. Diringer, Transmissible spongiform encephalopathies (TSE) virus-induced [44] C.C. Zhang, A.D. Steele, S. Lindquist, H.F. Lodish, Prion protein is expressed on amyloidoses of the central nervous system (CNS), Eur. J. Epidemiol. 7 (1991) long-term repopulating hematopoietic stem cells and is important for their self- 562–566. renewal, Proc. Natl Acad. Sci. USA 103 (2006) 2184–2189. [12] B. Chesebro, Introduction to the transmissible spongiform encephalopathies or [45] J. Collinge, M.A. Whittington, K.C. Sidle, C.J. Smith, M.S. Palmer, A.R. Clarke, J.G. prion diseases, Br. Med. Bull. 66 (2003) 1–20. Jefferys, Prion protein is necessary for normal synaptic function, Nature 370 [13] I.H. Pattison, Resistance of the scrapie agent to formalin, J. Comp. Pathol. 75 (1994) 295–297. (1965) 159–164. [46] S. Mouillet-Richard, M. Ermonval, C. Chebassier, J.L. Laplanche, S. Lehmann, J.M. [14] S.B. Prusiner, Novel proteinaceous infectious particles cause scrapie, Science 216 Launay, O. Kellermann, Signal transduction through prion protein, Science 289 (1982) 136–144. (2000) 1925–1928. [15] T. Alper, W.A. Cramp, D.A. Haig, M.C. Clarke, Does the agent of scrapie replicate [47] G. Schmitt-Ulms, G. Legname, M.A. Baldwin, H.L. Ball, N. Bradon, P.J. Bosque, K.L. without nucleic acid? Nature 214 (1967) 764–766. Crossin, G.M. Edelman, S.J. DeArmond, F.E. Cohen, S.B. Prusiner, Binding of neural [16] J.S. Griffith, Self-replication and scrapie, Nature 215 (1967) 1043–1044. cell adhesion molecules (N-CAMs) to the cellular prion protein, J. Mol. Biol. 314 [17] B. Chesebro, B. Caughey, Scrapie agent replication without the prion protein? (2001) 1209–1225. Curr. Biol. 3 (1993) 696–698. [48] H. Bueler, A. Aguzzi, A. Sailer, R.A. Greiner, P. Autenried, M. Aguet, C. Weissmann, [18] C.I. Lasmezas, J.P. Deslys, O. Robain, A. Jaegly, V. Beringue, J.M. Peyrin, J.G. Mice devoid of PrP are resistant to scrapie, Cell 73 (1993) 1339–1347. Fournier, J.J. Hauw, J. Rossier, D. Dormont, Transmission of the BSE agent to mice [49] S. Brandner, S. Isenmann, A. Raeber, M. Fischer, A. Sailer, Y. Kobayashi, S. Marino, in the absence of detectable abnormal prion protein, Science 275 (1997) C. Weissmann, A. Aguzzi, Normal host prion protein necessary for scrapie- 402–405. induced neurotoxicity, Nature 379 (1996) 339–343. [19] L. Manuelidis, Z.X. Yu, N. Barquero, B. Mullins, Cells infected with scrapie and [50] G. Mallucci, A. Dickinson, J. Linehan, P.C. Klohn, S. Brandner, J. Collinge, Depleting Creutzfeldt–Jakob disease agents produce intracellular 25-nm virus-like parti- neuronal PrP in prion infection prevents disease and reverses spongiosis, Science cles, Proc. Natl Acad. Sci. USA 104 (2007) 1965–1970. 302 (2003) 871–874. [20] M.E. Keyes, The prion challenge to the ‘central dogma’ of molecular biology, [51] B. Chesebro, M. Trifilo, R. Race, K. Meade-White, C. Teng, R. LaCasse, L. Raymond, 1965–1991, Stud. Hist. Philos. Biol. Biomed. Sci. 30 (1999) 1–19. C. Favara, G. Baron, S. Priola, B. Caughey, E. Masliah, M. Oldstone, Anchorless [21] C. Weissmann, The state of the prion, Nat. Rev. Microbiol. 2 (2004) 861–871. prion protein results in infectious amyloid disease without clinical scrapie, [22] S.B. Prusiner, Molecular biology of prion diseases, Science 252 (1991) 1515–1522. Science 308 (2005) 1435–1439. [23] J.T. Jarrett, P.T. Lansbury Jr., Seeding “one-dimensional crystallization” of [52] L. Westergard, H.M. Christensen, D.A. Harris, The cellular prion protein (PrP(C)): amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie? Cell 73 its physiological function and role in disease, Biochim. Biophys. Acta 1772 (1993) 1055–1058. (2007) 629–644. E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414 413

[53] C.I. Lasmezas, J.P. Deslys, R. Demaimay, K.T. Adjou, F. Lamoury, D. Dormont, O. [85] L. Favre-Krey, M. Theodoridou, E. Boukouvala, C.H. Panagiotidis, A.I. Papado- Robain, J. Ironside, J.J. Hauw, BSE transmission to macaques, Nature 381 (1996) poulos, T. Sklaviadis, G. Krey, Molecular characterization of a cDNA from the 743–744. gilthead sea bream (Sparus aurata) encoding a fish prion protein, Comp. [54] J. Collinge, K.C. Sidle, J. Meads, J. Ironside, A.F. Hill, Molecular analysis of prion Biochem. Physiol. B Biochem. Mol. Biol. 147 (2007) 566–573. strain variation and the aetiology of ‘new variant’ CJD, Nature 383 (1996) [86] B. Oidtmann, D. Simon, N. Holtkamp, R. Hoffmann, M. Baier, Identification of cDNAs 685–690. from Japanese pufferfish (Fugu rubripes) and Atlantic salmon (Salmo salar)coding [55] G. Streisinger, C. Walker, N. Dower, D. Knauber, F. Singer, Production of clones of for homologues to tetrapod prion proteins, FEBS Lett. 538 (2003) 96–100. homozygous diploid zebra fish (Brachydanio rerio), Nature 291 (1981) 293–296. [87] J.H. Postlethwait, Y.L. Yan, M.A. Gates, S. Horne, A. Amores, A. Brownlie, A. [56] T.P. Barros, W.K. Alderton, H.M. Reynolds, A.G. Roach, S. Berghmans, Zebrafish: Donovan, E.S. Egan, A. Force, Z. Gong, C. Goutel, A. Fritz, R. Kelsh, E. Knapik, E. an emerging technology for in vivo pharmacological assessment to identify Liao, B. Paw, D. Ransom, A. Singer, M. Thomson, T.S. Abduljabbar, P. Yelick, D. potential safety liabilities in early drug discovery, Br. J. Pharmacol. 154 (2008) Beier, J.S. Joly, D. Larhammar, F. Rosa, M. Westerfield, L.I. Zon, S.L. Johnson, W.S. 1400–1413. Talbot, Vertebrate genome evolution and the zebrafish gene map, Nat. Genet. 18 [57] C. Chakraborty, C.H. Hsu, Z.H. Wen, C.S. Lin, G. Agoramoorthy, Zebrafish: a (1998) 345–349. complete animal model for in vivo drug discovery and development, Curr. Drug [88] M. Premzl, L. Sangiorgio, B. Strumbo, J.A. Marshall Graves, T. Simonic, J.E. Gready, Metab. 10 (2009) 116–124. Shadoo, a new protein highly conserved from fish to mammals and with [58] A. Aguzzi, Cell biology: beyond the prion principle, Nature 459 (2009) 924–925. similarity to prion protein, Gene 314 (2003) 89–102. [59] R.B. Wickner, H.K. Edskes, F. Shewmaker, T. Nakayashiki, Prions of fungi: [89] T. Suzuki, T. Kurokawa, H. Hashimoto, M. Sugiyama, cDNA sequence and tissue inherited structures and biological roles, Nat. Rev. Microbiol. 5 (2007) 611–618. expression of Fugu rubripes prion protein-like: a candidate for the teleost [60] M.F. Tuite, Yeast prions and their prion-forming domain, Cell 100 (2000) orthologue of tetrapod PrPs, Biochem. Biophys. Res. Commun. 294 (2002) 912–917. 289–292. [90] R. Young, B. Passet, M. Vilotte, E.P. Cribiu, V. Beringue, F. Le Provost, H. Laude, J.L. [61] J.M. Gabriel, B. Oesch, H. Kretzschmar, M. Scott, S.B. Prusiner, Molecular cloning Vilotte, The prion or the related Shadoo protein is required for early mouse of a candidate chicken prion protein, Proc. Natl Acad. Sci. USA 89 (1992) embryogenesis, FEBS Lett. 583 (2009) 3296–3300. 9097–9101. [91] J.C. Watts, B. Drisaldi, V. Ng, J. Yang, B. Strome, P. Horne, M.S. Sy, L. Yoong, R. [62] T. Simonic, S. Duga, B. Strumbo, R. Asselta, F. Ceciliani, S. Ronchi, cDNA cloning of Young, P. Mastrangelo, C. Bergeron, P.E. Fraser, G.A. Carlson, H.T. Mount, G. turtle prion protein, FEBS Lett. 469 (2000) 33–38. Schmitt-Ulms, D. Westaway, The CNS glycoprotein Shadoo has PrP(C)-like [63] B. Strumbo, S. Ronchi, L.C. Bolis, T. Simonic, Molecular cloning of the cDNA coding protective properties and displays reduced levels in prion infections, EMBO J. 26 for Xenopus laevis prion protein, FEBS Lett. 508 (2001) 170–174. (2007) 4038–4050. [64] E. Rivera-Milla, C.A. Stuermer, E. Malaga-Trillo, An evolutionary basis for scrapie [92] E. Málaga-Trillo, G.P. Solis, Y. Schrock, C. Geiss, L. Luncz, V. Thomanetz, C.A. disease: identification of a fish prion mRNA, Trends Genet. 19 (2003) 72–75. Stuermer, Regulation of embryonic cell adhesion by the prion protein, PLoS Biol. [65] F. Wopfner, G. Weidenhofer, R. Schneider, A. von Brunn, S. Gilch, T.F. Schwarz, T. 7 (2009) e1000055. Werner, H.M. Schatzl, Analysis of 27 mammalian and 9 avian PrPs reveals high [93] M. Miesbauer, T. Bamme, C. Riemer, B. Oidtmann, K.F. Winklhofer, M. Baier, J. conservation of flexible regions of the prion protein, J. Mol. Biol. 289 (1999) Tatzelt, Prion protein-related proteins from zebrafish are complex glycosylated 1163–1178. and contain a glycosylphosphatidylinositol anchor, Biochem. Biophys. Res. [66] T. van Rheede, M.M. Smolenaars, O. Madsen, W.W. de Jong, Molecular evolution Commun. 341 (2006) 218–224. of the mammalian prion protein, Mol. Biol. Evol. 20 (2003) 111–121. [94] A. Force, M. Lynch, F.B. Pickett, A. Amores, Y.L. Yan, J. Postlethwait, Preservation [67] E. Rivera-Milla, B. Oidtmann, C.H. Panagiotidis, M. Baier, T. Sklaviadis, R. of duplicate genes by complementary, degenerative mutations, Genetics 151 Hoffmann, Y. Zhou, G.P. Solis, C.A. Stuermer, E. Malaga-Trillo, Disparate evolution (1999) 1531–1545. of prion protein domains and the distinct origin of Doppel- and prion-related loci [95] A. Nasevicius, S.C. Ekker, Effective targeted gene ‘knockdown’ in zebrafish, Nat. revealed by fish-to-mammal comparisons, FASEB J. 20 (2006) 317–319. Genet. 26 (2000) 216–220. [68] E. Cotto, M. Andre, J. Forgue, H.J. Fleury, P.J. Babin, Molecular characterization, [96] E. Malaga-Trillo, E. Sempou, PrPs: Proteins with a purpose (lessons from the phylogenetic relationships, and developmental expression patterns of prion zebrafish), Prion 3 (2009) 129–133. genes in zebrafish (Danio rerio), FEBS J. 102 (3) (2005) 651–655. [97] R. Chiesa, D.A. Harris, Fishing for prion protein function, PLoS Biol. 7 (2009) e75. [69] L. Calzolai, D.A. Lysek, D.R. Perez, P. Guntert, K. Wuthrich, Prion protein NMR [98] M.C. Sorgato, C. Peggion, A. Bertoli, Is indeed the prion protein an Harlequin structures of chickens, turtles, and frogs, Proc. Natl Acad. Sci. USA 102 (3) (2005) servant of “many” masters? Prion 3 (4) (2009) 202–205. 651–655. [99] J.W. Wilesmith, G.A. Wells, M.P. Cranwell, J.B. Ryan, Bovine spongiform [70] I. Vorberg, M.H. Groschup, E. Pfaff, S.A. Priola, Multiple amino acid residues encephalopathy: epidemiological studies, Vet. Rec. 123 (1988) 638–644. within the rabbit prion protein inhibit formation of its abnormal isoform, J. Virol. [100] D. Matthews, B.C. Cooke, The potential for transmissible spongiform encepha- 77 (2003) 2003–2009. lopathies in non-ruminant livestock and fish, Rev. Sci. Tech. 22 (2003) 283–296. [71] Y. Wen, J. Li, W. Yao, M. Xiong, J. Hong, Y. Peng, G. Xiao, D. Lin, Unique structural [101] S.L. Benestad, J.N. Arsac, W. Goldmann, M. Noremark, Atypical/Nor98 scrapie: characteristics of the rabbit prion protein, J. Biol. Chem. 285 (41) (2010) properties of the agent, genetics, and epidemiology, Vet. Res. 39 (2008) 19. 31682–31693. [102] T. Baron, A.G. Biacabe, J.N. Arsac, S. Benestad, M.H. Groschup, Atypical [72] V. Beringue, J.L. Vilotte, H. Laude, Prion agent diversity and species barrier, Vet. transmissible spongiform encephalopathies (TSEs) in ruminants, Vaccine 25 Res. 39 (2008) 47. (2007) 5625–5630. [73] A.F. Hill, J. Collinge, Species-barrier-independent prion replication in apparently [103] C. Casalone, G. Zanusso, P. Acutis, S. Ferrari, L. Capucci, F. Tagliavini, S. Monaco, M. resistant species, APMIS 110 (2002) 44–53. Caramelli, Identification of a second bovine amyloidotic spongiform encepha- [74] S.B. Prusiner, M. Scott, D. Foster, K.M. Pan, D. Groth, C. Mirenda, M. Torchia, S.L. Yang, lopathy: molecular similarities with sporadic Creutzfeldt–Jakob disease, Proc. D. Serban, G.A. Carlson, et al., Transgenetic studies implicate interactions between Natl Acad. Sci. USA 101 (2004) 3065–3070. homologous PrP isoforms in scrapie prion replication, Cell 63 (1990) 673–686. [104] G.A. Wells, A.C. Scott, C.T. Johnson, R.F. Gunning, R.D. Hancock, M. Jeffrey, M. [75] M.M. Simmons, J. Spiropoulos, S.A. Hawkins, S.J. Bellworthy, S.C. Tongue, Dawson, R. Bradley, A novel progressive spongiform encephalopathy in cattle, Approaches to investigating transmission of spongiform encephalopathies in Vet. Rec. 121 (1987) 419–420. domestic animals using BSE as an example, Vet. Res. 39 (2008) 34. [105] L. Ingrosso, B. Novoa, A.Z. Valle, F. Cardone, R. Aranguren, M. Sbriccoli, S. [76] J. Collinge, A.R. Clarke, A general model of prion strains and their pathogenicity, Bevivino, M. Iriti, Q. Liu, V. Vetrugno, M. Lu, F. Faoro, S. Ciappellano, A. Figueras, Science 318 (2007) 930–936. M. Pocchiari, Scrapie infectivity is quickly cleared in tissues of orally-infected [77] B. Sweeting, M.Q. Khan, A. Chakrabartty, E.F. Pai, Structural factors underlying farmed fish, BMC Vet. Res. 2 (2006) 21. the species barrier and susceptibility to infection in prion disease, Biochem. Cell [106] E. Salta, C. Panagiotidis, K. Teliousis, S. Petrakis, E. Eleftheriadis, F. Arapoglou, N. Biol. 88 (2010) 195–202. Grigoriadis, A. Nicolaou, E. Kaldrymidou, G. Krey, T. Sklaviadis, Evaluation of the [78] Y. Wang, B. Hu, Y. Liu, H. Zhang, Y. Song, New insight into the species barrier of possible transmission of BSE and scrapie to gilthead sea bream (Sparus aurata), mammalian prions obtained from structure-based conservation analysis, PLoS ONE 4 (2009) e6175. Intervirology 51 (2008) 210–216. [107] H. Budka, Histopathology and immunohistochemistry of human transmissible [79] A.M. Thackray, M.A. Klein, R. Bujdoso, Subclinical prion disease induced by oral spongiform encephalopathies (TSEs), Arch. Virol. Suppl. (2000) 135–142. inoculation, J. Virol. 77 (2003) 7991–7998. [108] U. Unterberger, T. Voigtlander, H. Budka, Pathogenesis of prion diseases, Acta [80] R.H. Kimberlin, C.A. Walker, Pathogenesis of mouse scrapie: effect of route of Neuropathol. 109 (2005) 32–48. inoculation on infectivity titres and dose–response curves, J. Comp. Pathol. 88 [109] W. Butler, Comparative Vertebrate Neuroanatomy: Evolution and Adaptation, (1978) 39–47. Wiley-IEEE, 2005. [81] C. Hoffmann, U. Ziegler, A. Buschmann, A. Weber, L. Kupfer, A. Oelschlegel, B. [110] C. Salas, C. Broglio, E. Duran, A. Gomez, F.M. Ocana, F. Jimenez-Moya, F. Hammerschmidt, M.H. Groschup, Prions spread via the autonomic nervous Rodriguez, Neuropsychology of learning and memory in teleost fish, Zebrafish 3 system from the gut to the central nervous system in cattle incubating bovine (2006) 157–171. spongiform encephalopathy, J. Gen. Virol. 88 (2007) 1048–1055. [111] G. Almer, J.A. Hainfellner, T. Brucke, K. Jellinger, R. Kleinert, G. Bayer, O. Windl, H. [82] A.F. Hill, J. Collinge, Subclinical prion infection in humans and animals, Br. Med. A. Kretzschmar, A. Hill, K. Sidle, J. Collinge, H. Budka, Fatal familial insomnia: a Bull. 66 (2003) 161–170. new Austrian family, Brain 122 (Pt 1) (1999) 5–16. [83] A.M. Thackray, M.A. Klein, A. Aguzzi, R. Bujdoso, Chronic subclinical prion disease [112] J. Castri, R. Thiery, J. Jeffroy, P. de Kinkelin, J.C. Raymond, Sea bream Sparus induced by low-dose inoculum, J. Virol. 76 (2002) 2510–2517. aurata, an asymptomatic contagious fish host for nodavirus, Dis. Aquat. Organ. [84] S.J. Collins, V. Lewis, M.W. Brazier, A.F. Hill, V.A. Lawson, G.M. Klug, C.L. Masters, 47 (2001) 33–38. Extended period of asymptomatic prion disease after low dose inoculation: [113] R. Aranguren, C. Tafalla, B. Novoa, A. Figueras, Experimental transmission of assessment of detection methods and implications for infection control, encephalopathy and retinopathy induced by nodavirus to sea bream, Sparus Neurobiol. Dis. 20 (2005) 336–346. aurata L., using different infection models, J. Fish Dis. 25 (2002) 317–324. 414 E. Málaga-Trillo et al. / Biochimica et Biophysica Acta 1812 (2011) 402–414

[114] C.G. Becker, T. Becker, Adult zebrafish as a model for successful central nervous [122] J.C. Espinosa, M. Morales, J. Castilla, M. Rogers, J.M. Torres, Progression of prion system regeneration, Restor. Neurol. Neurosci. 26 (2008) 71–80. infectivity in asymptomatic cattle after oral bovine spongiform encephalopathy [115] G.K.H. Zupanc, Towards brain repair: insights from teleost fish, Semin. Cell Dev. challenge, J. Gen. Virol. 88 (2007) 1379–1383. Biol. 20 (2009) 683–690. [123] T. Hastein, A. Hellstrom, G. Jonsson, N.J. Olesen, E.R. Parnanen, Surveillance of fish [116] Y. Ano, A. Sakudo, H. Nakayama, T. Onodera, Uptake and dynamics of infectious diseases in the Nordic countries, Acta Vet. Scand. Suppl. 94 (2001) 43–50. prion protein in the intestine, Protein Pept. Lett. 16 (2009) 247–255. [124] Opinion of the Scientific Panel on Biological Hazards on a request from the [117] A.D. Vale, The professional phagocytes of sea bass (Dicentrarchus labrax L.): European Parliament on the assessment of the health risks of feeding of cytochemical characterization of neutrophils and macrophages in the normal ruminants with fishmeal in relation to the risk of TSE, EFSA J. 443 (2007) 1–26. and inflamed peritoneal cavity, Fish & Shelfish Immunology 13 (2002) 183–198. [125] J.D. Best, W.K. Alderton, Zebrafish: an in vivo model for the study of neurological [118] M. Chabrillon, Interactions of fish pathogenic Vibrio species with the mucous diseases, Neuropsychiatr. Dis. Treat. 4 (2008) 567–576. surfaces of gilthead seabream (Sparus aurata), Recent Research Developments in [126] N. Fernandez-Borges, J. de Castro, J. Castilla, In vitro studies of the transmission Microbiology, 2003, pp. 41–53. barrier, Prion 3 (2009) 220–223. [119] A.Z. Dalla Valle, M. Iriti, F. Faoro, C. Berti, S. Ciappellano, In vivo prion protein [127] G.P. Saborio, B. Permanne, C. Soto, Sensitive detection of pathological prion intestinal uptake in fish, APMIS 116 (2008) 173–180. protein by cyclic amplification of protein misfolding, Nature 411 (2001) [120] M. Beekes, P.A. McBride, The spread of prions through the body in naturally 810–813. acquired transmissible spongiform encephalopathies, FEBS J. 274 (2007) 588–605. [128] R.A. Armstrong, P.L. Lantos, N.J. Cairns, What determines the molecular [121] A. Aguzzi, M. Heikenwalder, G. Miele, Progress and problems in the biology, composition of abnormal protein aggregates in neurodegenerative disease? diagnostics, and therapeutics of prion diseases, J. Clin. Invest. 114 (2004) 153–160. Neuropathology 28 (2008) 351–365. Biochimica et Biophysica Acta 1812 (2011) 415–422

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Biochimica et Biophysica Acta

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Review Microdomain-forming proteins and the role of the reggies/flotillins during axon regeneration in zebrafish☆

Claudia A.O. Stuermer ⁎

University of Konstanz, Department of Biology, 78465 Konstanz, Germany article info abstract

Article history: The two proteins reggie-1 and reggie-2 (flotillins) were identified in axon-regenerating neurons in the central Received 15 September 2010 nervous system and shown to be essential for neurite growth and regeneration in fish and mammals. Reggies/ Received in revised form 30 November 2010 flotillins are microdomain scaffolding proteins sharing biochemical properties with lipid raft molecules, form Accepted 2 December 2010 clusters at the cytoplasmic face of the plasma membrane and interact with signaling molecules in a cell type Available online 13 December 2010 specific manner. In this review, reggie microdomains, lipid rafts, related scaffolding proteins and caveolin— which, however, are responsible for their own microdomains and functions—are introduced. Moreover, the Keywords: Axon regeneration function of the reggies in axon growth is demonstrated: neurons fail to extend axons after reggie knockdown. Microdomain Furthermore, our current concept of the molecular mechanism underlying reggie function is presented: the Reggie/flotillin association of glycosyl-phophatidyl inositol (GPJ)-anchored surface proteins with reggie microdomains elicits Recruitment signals which activate src tyrosine and mitogen-activated protein kinases, as well as small guanosine 5′- Targeted delivery triphosphate-hydrolyzing enzymes. This leads to the mobilization of intracellular vesicles and to the recruitment of bulk membrane and specific cargo proteins, such as cadherin, to specific sites of the plasma membrane such as the growth cone of elongating axons. Thus, reggies regulate the targeted delivery of cargo— a process which is required for process extension and growth. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases. © 2010 Elsevier B.V. All rights reserved.

1. Introduction mechanisms, will be reviewed in this article. But before reggies are considered in the context of axon growth, their properties as The zebrafish and goldfish visual pathway has served as a model constituents of “lipid rafts,” or better microdomains will be discussed system for the exploration of rules governing over the development of and compared to other microdomains and microdomain-forming the retinotopic projection of retinal ganglion cell (RGC) axons onto proteins in zebrafish. Prior to this, however, the reasons why axons in the optic tectum [1,2]. The restoration of the retinotopic map by the CNS of fish regenerate—as opposed to mammals which do not— regenerating RGC axons after optic nerve lesion has also been will be considered in brief. intensively analyzed in this system. Because of its remarkable fi regeneration-supporting properties, the sh optic nerve is, moreover, 2. Axon regeneration in the CNS and the identification of the suited to search for factors underlying successful regeneration of reggie proteins injured CNS fiber tracts per se. It is expected that insights into fi conditions allowing successful regrowth in sh might provide One reason for successful regeneration in the CNS is the growth- valuable clues for stimulation of axon regeneration in the mammalian permissive nature of the glial cell environment in the fish optic nerve CNS where regeneration does not spontaneously occur to any [5,6]. The mammalian CNS, by contrast, presents several growth fi signi cant extent. Experiments along these lines have led to the inhibiting molecules on glial cells and in the extracellular matrix discovery of two proteins, reggie-1 and reggie-2 [3], which turned out which block axon growth and prevent regeneration [7]. The second to be essential for growth and regeneration [4], hence their name reg- reason for the success of axon regeneration is also dependent on the gie. Evidence for a role of the reggies in axon growth and regeneration, special neuron-intrinsic properties of nerve cells in fish [8]. Retinal together with our current understanding of the underlying molecular ganglion cells (RGCs), for instance, promptly upregulate growth- associated proteins after optic nerve lesion which enables the neurons to regrow their axons and to provide them with cytoskeletal proteins, cell adhesion molecules, the necessary receptors for guidance cues ☆ This article is part of a Special Issue entitled Zebrafish Models of Neurological and, of course, bulk membrane material [4,8,9]. This ability to switch Diseases. ⁎ Tel.: +49 7531 882236. on expression of genes which were active during development E-mail address: [email protected]. but downregulated thereafter is by far less developed in mammals

0925-4439/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2010.12.004 416 C.A.O. Stuermer / Biochimica et Biophysica Acta 1812 (2011) 415–422 although in principle possible [10 and see below]. More than a dozen many intracellular vesicles [9,18,19], including the Rab11 recycling growth-associated proteins in RGCs have been identified at the compartment. Consistent with their identity as microdomain proteins, molecular level, and these were recently complemented by a reggies were independently identified as components of the protein proteomics approach in mammalian cortical neurons [11]. fraction known as DRMs (detergent resistant membranes). The DRM The two proteins discovered in this context, reggie-1 and reggie-2 contains molecules that are insoluble in non-ionic detergents (such as [3,11], are 45% identical to one another and have a molecular weight Triton X-100) and that are regarded as “lipid raft” components. Together of 48 kD. Reggies were also identified in mammals [12,13] where they with other DRM proteins, the reggies “float” after sucrose density occur in basically all cells examined so far. In the rat retina, they are centrifugation [12], and hence, they were named flotillins [12]. Reggie-1 upregulated in exactly the 3% of RGCs that, as demonstrated by corresponds to flotillin-2, and reggie-2 to flotillin-1. Reggies/flotillins Richardson et al. [10], can regenerate their axon in a growth- are often used as markers for the lipid raft fraction in biochemical work. permissive environment. We prefer the term “reggie microdomain” (instead of lipid raft) because However, it was not immediately clear how reggies regulate reggies assemble by oligomerization into clusters of ≤100 nm [9,20] and regeneration. Unlike cell adhesion molecules which are also upregu- are, therefore, visible at the light microscopic level by immunofluores- lated during axon regeneration [14,15] and mediate the interaction of cence analysis [13,20,21] with specific antibodies. The microdomains the elongating growth cone with the environment, the reggies were appear as dots or puncta at the plasma membrane of cells and along found to reside at the cytoplasmic face of the plasma membrane. They axons, growth cones, filopodia and as densely packed rows of dots, for are anchored to the membrane by myristoyl and palmitoyl residues instance, at cell–cell contacts [21,22] and in the T cell cap [20,23].This [16,17] and through stretches of hydrophobic amino acids in their “pearl on a string-like” arrangement in T cell caps and at cell contacts N-terminal head domain (Fig. 1). It was also observed that the reggies do (Fig. 2) is formed by a reduction of the distance between individual not only occur at the plasma membrane but also in association with microdomains [22].

Fig. 1. The microdomain scaffolding proteins and their domains. Reggie-1 and reggie-2 (flotillin) possess an N-terminal head domain (light blue), with two hydrophobic regions (yellowish) and acylation sites (red) for membrane attachment. The tail domain (gray) has strikingly many EA repeats for α-helical coiled-coil formation and oligomerization. The other microdomain scaffolding proteins share some degree of sequence homology with reggie and among each other and also form oligomers. C.A.O. Stuermer / Biochimica et Biophysica Acta 1812 (2011) 415–422 417

Fig. 2. Distribution and function of reggie microdomains in T cells and neurons. A) In T cells, reggie-1 (and -2)—visualized here by a reggie-1 specific antibody (red)—is clustered at one pole of the cell, the preformed reggie cap which represents a platform for the association of specific GPI-anchored proteins, such as PrP and Thy-1 and signaling. B) Reggie microdomains cluster at cell–cell contact sites (arrowheads) by reducing the distance between individual microdomains (red, immunofluorescence with anti-reggie-1 antibodies). C) The growth cone from a zebrafish RGC axon shows the distribution of reggie microdomains (puncta) at its central region as well as in its filopodia. D) Individual zebrafish RGC neuron and its axon immunostained with a reggie-1 specific antibody and showing the punctate microdomains along the axon. E) Hippocampal neurons differentiate in vitro (E, control siRNA) and extend neurites whereas their counterparts fail to produce neurites after exposure to reggie-1 specific siRNAs (F). G) Hippocampal neurons often produce club-shaped endings where a growth cone should have been, after perturbation of reggie-1 expression. Scale bars: 20 μm.

3. Microdomains and lipid rafts membrane interaction and stretches of amino acids (tail domain) which promote oligomerization [33]. These protein oligomers, when labeled Microdomains, caveolae and lipid rafts represent functional units by antibodies or tags, make microdomains visible at the light in cellular membranes whose existence depends on mainly saturated microscopic level and amenable to direct analysis of distribution and sphingolipids and cholesterol in a liquid ordered phase and function. segregated from unsaturated lipids in the liquid disordered phase Lipid rafts and microdomains confer inhomogeneities to the [24,25]. Rafts range in size from a few nanometer encompassing by plasma membrane and allow the formation of membrane compart- estimate six GPI-anchored proteins (nanoclusters) [26] to several ments [25] which are used by specific membrane proteins for cluster hundred nanometers (known as “clustered rafts”) [25] are dynamic formation, protein–protein interactions and signaling [34] as well as and generally short-lived. They can be stabilized by the actin for the recruitment and targeted delivery of membrane proteins to cytoskeleton [27] which influences their distribution, half life and specific sites [9]. GPI-anchored proteins are typical cell-surface size. The existence of lipid rafts has been heavily disputed [28] residents of rafts and microdomains whereas proteins with acyl particularly since they often escape microscopic observation due to residues (palmitoylation, myristoylation and others) typically reside their small size [26]. Recent results now demonstrate the phase at the cytoplasmic face of the microdomain plasma membrane. separation of sphingolipids and cholesterol in giant plasma mem- Certain transmembrane proteins also acquire a preference for the lipid brane vesicles of vertebrate cells which support again the raft concept raft and microdomain environment, in particular certain growth [29]. factor and transmitter receptors [25]. Microdomains—in contrast to rafts—depend on specific scaffolding While being intensively discussed among cell biologist and experts proteins that have an affinity for the lipid raft environment and share in lipidomics, rafts and microdomains do not play such a central role many raft properties. A family of microdomain proteins with a in research on zebrafish development or disease. Few exceptions are characteristic membrane interaction domain at their N-terminal the microdomains scaffolded by the proteins caveolin (i.e. caveolae), “head” domain consists of stomatin, prohibitin, flotillin/reggie, podocin podocin and reggie which will be described next and whose function and erlin [9,30–32]. The scaffolding proteins (Fig. 1) possess a region for in zebrafish embryos and/or adult fish will be discussed subsequently. 418 C.A.O. Stuermer / Biochimica et Biophysica Acta 1812 (2011) 415–422

4. Microdomains and caveolae cholesterol levels has not been reported nor is clear how caveolin- dependent receptor-ligand interaction and signal transduction may Among the most intensively studied microdomains are caveolae, change. The enzymes involved in cholesterol synthesis [43] are 100 nm flask-shaped plasma membrane invaginations, whose forma- expressed in zebrafish and in the embryo as maternal messages at tion depends on caveolin [35]. Each of the other scaffolding proteins, the one and two cell stage predicting that cholesterol biosynthesis is reggie/flotillin, stomatin, podocin, prohibitin and erlin, forms its own crucial already in the early blastocytes. Altogether, it appears that microdomain without producing membrane indentations or other caveolin-1 and caveolae control the differentiation of specificcell morphologically conspicuous structures. types in the embryo, yet the underlying mechanism is not well There was a debate as to whether reggies represent additional understood. constituents of caveolae which would imply that they serve as a second scaffold of the caveolar invaginations [12].Reggieproteins, however, are present in basically every cell analyzed so far, whereas 5.2. Caveolin-3 caveolin is not. Caveolin, not reggie, is absolutely required for caveola formation. Moreover, caveolae are absent from neurons and Zebrafish also expresses a homolog of mammalian caveolin-3 lymphocytes [13,20,21,36] which do have reggie microdomains. (cav-3) which is a muscle-specific isoform in mammals and fish Even in cells with caveolae, such as astrocytes, caveolae and reggie [44,45]. Cav-3 in zebrafish was found in the first differentiating proteins occupy non-overlapping regions of the plasma membrane muscle precursor cells and later in skeletal and cardiac muscle fibers [21], so that reggies are clearly not constituents of caveolae. In fact, where expression correlated with the presence of caveolae. Cav-3, we are not aware of any report on microdomains with mixed however, also appeared in pectoral fin, facial muscle and notochord protein scaffolds. [44]. Downregulation of cav-3 expression caused severe muscle The microdomain scaffolding proteins are evolutionarily highly abnormalities and uncoordinated movement and increased the conserved [32,37] suggesting important functions across kingdoms of number of muscle pioneer-like cells adjacent to the notochord. The life. Accordingly, inspection of databases shows the presence of signaling pathways which depend on cav-3 and caveolae and steer homologs of caveolin, stomatin, prohibitin, reggie/flotillin, podocin muscle development have not been described and are to this end and erlin in zebrafish. Aside from their sequence, however, not much unknown. is known on their function. We found no report on zebrafish stomatin, prohibitin and erlin. But work was published on caveolin-1 and caveolin-3 in the early embryo, on podocin during kidney formation, 6. Zebrafish podocin and on reggie-1 and reggie-2 during axon regeneration. The reggies will be considered in greater detail because they were discovered in Podocin is expressed by podocytes that are necessary for the the fish CNS and functionally characterized in the context of axon blood filtration barrier in the kidney glomerulus [46,47].Podocytes regeneration [3,4]. Moreover, results from fish together with insights form the slit diaphragm, a specialized cell–cell adhesion complex at from various cell types and species has led to a new concept the podocyte foot processes surrounding glomerular blood vessels. explaining reggie functions [9] but before its presentation published Slit diaphragm formation depends on the cell adhesion proteins (of data on zebrafish caveolin-1, caveolin-3 and podocin will be the immunoglobulin superfamily) nephrin and Neph as well as summarized. podocin [48].Muchasinhighervertebrates,zebrafish nephrin and podocin are specifically expressed in pronephric podocytes and 5. Zebrafish caveolin and caveolae required for the development of pronephric podocyte cell struc- tures [49]. Morpholino-mediated knockdown of podocin (or nephrin) In mammals, caveolin and caveolae have been implicated in many resulted in loss of a slit diaphragm at 72 and 96 hpf and failure to form functions such as endocytosis, transcytosis, signaling, trafficking of normal podocyte foot processes. Absence of normal podocin (or cholesterol (for review see reference 38) so that it came as a surprise nephrin) expression resulted in defects in glomerular filtration and that the caveolin-1 knockout mice had at first sight no apparent aberrant passage of high molecular filtrate [49,50]. The exact cellular phenotype. But mice turned out to suffer from pulmonary defects, and molecular mechanism of podocin function during slit diaphragm particularly under the challenge of exercise, and to lack the ability to formation is not known. However, human podocin mutations C- appropriately adapt their blood pressure [39]. terminal to the membrane association (head) domain resulted in loss of podocin at the cell membrane and aberrant accumulation in ER 5.1. Caveolin-1 secretory pathway vesicles [51]. Podocin mutants also resulted in failed delivery of nephrin to the slit diaphragm thus indicating a role of In zebrafish, caveolin-1 and caveolae were shown to exist during podocin in nephrin trafficking [48]. the development in neuromasts and in notochord cells (and other structures) [40] which exhibited caveolae in abundance. Morpho- lino-mediated knockdown to assess the potential role of caveolin in 7. Reggie microdomains embryogenesis showed that the formation of neuromasts was perturbed and the notochord disrupted [40]. Another publication Reggie microdomains are defined by hetero-oligomeric clusters of reports additional defects in the eye and in somites at 12 h post reggie-1 and reggie-2. To this end, it has not been explicitly examined fertilization (hpf) as well as disruption of the actin cytoskeleton whether reggie-1 and reggie-2 can perform separate functions. [41]. Later defects emerged in the vascular endothelium [41] and in However, it has become clear that reggie-2 undergoes proteasomal the smaller size of the embryos [40]. In addition, the lateral line degradation when reggie-1 is downregulated (53), for example after developed abnormally most likely because of the defects in siRNA and morpholino-mediated interference with reggie-1 expres- neuromasts and notochord [40,41]. The embryos might suffer from sion levels in neurons, epithelial cells and zebrafish embryo. It is other problems as, for instance, reduced cholesterol levels of the unclear why reggie-2 is so tightly regulated in dependence of reggie-1 plasma membrane since caveolin has been proposed to function as a nor has it been thoroughly analyzed whether reggie-1 and reggie-2 carrier for cholesterol [42] and cholesterol is needed for the can subserve separate functions in other cell types. Therefore, we do existence of lipid rafts and microdomains [25]. Whether morpho- not distinguish between reggie-1 and -2 in the text when they were lino-treated fish develop severe abnormalities due to imbalanced not addressed individually. C.A.O. Stuermer / Biochimica et Biophysica Acta 1812 (2011) 415–422 419

7.1. Association of the GPI-anchored prion protein and Thy-1 with reggie It is known that the small GTPases regulate actin dynamics and vesicle microdomains trafficking [4,60–62]. Vesicles carrying reggie were observed to shuttle between the plasma membrane and more distant intracellular sites [61] Reggie-1 and -2 were isolated from larval goldfish by co- including the recycling compartment. This indicates that reggie immunoprecipitation with an antibody against goldfish Thy-1 [3]. microdomains at vesicle membranes participate in the signaling This is in line with the fact that Thy-1, as a GPI-anchored protein, required for vesicle mobilization, trafficking and targeting. These events associates preferentially with reggie microdomains [20,21,52,53] not require the activity of small GTPases that, in turn, have a preference for only in fish but also in all vertebrate cells studied so far. Moreover, rafts [9,58,63]. Thy-1 is upregulated in fish RGCs after nerve lesion like reggie [53] The reggie/flotillin-binding protein CAP/ponsin and its relatives and co-localizes with reggie in microdomains in growth cones, along ArgBP2 and vinexin were implied in the interaction with the actin axons, at cell–cell contact sites and in the T cell cap [9]. In the cytoskeleton [56] in which reggie/flotillin is also involved [64] and uninjured fish visual system, Thy-1 as well as reggie, is colocalized in with substrate and cell adhesion proteins, such as integrins and the few axons from new RGCs that are constantly added to the fish cadherins. Together, these findings suggest that reggie activates a retina [3]. Thus, Thy-1 seems to cocluster preferentially with reggie pathway for polarized delivery of cargo [9]. The type of cargo depends microdomains in vitro and in vivo. This applies also to the GPI- on the cell type and includes, for example, transporters (such as anchored cellular prion protein [PrPc; 20] and the zebrafish homologs Glut4) [55], cell adhesion molecules (such as cadherins, integrins) PrP-1 and PrP-2 [54]. That this association of PrP and Thy-1 with [54,56], receptors, ion channels and many other proteins that need to reggie-1 and -2 is functionally important was demonstrated first in T be targeted to specific sites of cells [9]. The sites are “marked” by the cells as will be described below. Further insights into the role of the clustering of reggie/flotillin microdomains and co-clusters of GPI- reggies came from studies on insulin stimulation of adipocytes (fat anchored proteins, which provides external signals and specificity cells) [55,56] and on cell–cell contact formation in various types of (Fig. 3). Reggies/flotillins at membranes of intracellular vesicles cells [9]. [18,61] might represent similar signaling centers for clustering of signaling molecules to promote vesicle transport, sorting and targeted 7.2. Reggie and PrP signaling in T cells delivery of cargo such as cadherin (Fig. 3) [9].

In T cells, reggie microdomains are pre-clustered at one pole of the cell, representing ‘the preformed reggie cap’ [23] where the T cell 7.4. Reggies, PrP and cadherin recruitment receptor complex and the signaling molecules associated with T cell activation coalesce upon stimulation [20]. The preformed reggie cap is Reggies are clustered at contact sites between cells [20,21] and so important for T cell activation. A dominant-negative reggie construct is PrP. PrP–PrP trans-interactions in contacts between mammalian disrupts the cap and interferes with the formation of the immuno- logical synapse [19]. In T cells, the activation of GPI-anchored proteins by antibody cross-linking is sufficient for TCR complex assembly in the cap [34]. This applies, for instance, to Thy-1 and PrP, whose activation leads to their selective association with the reggie cap [20,21]. When clustered in the reggie cap, PrP elicits the phosphorylation of the MAP (mitogen-activated protein) kinase ERK1/2 and evokes a distinct Ca2+ signal leading to the recruitment of CD3 [20,57], the major T cell receptor component, to the membrane in the cap. Yet, CD3 recruitment is not sufficient for the full activation of the T cell receptor complex [20], which would require cross-linking of CD3 or stimulation by antigen. T cell receptor recruitment involves the communication with actin dynamics through src tyrosine kinases (fyn, lck, src), Rho-GTPases and the signaling molecule Vav [19]. The conclusion from these results is that the microdomains consisting of reggie oligomers serve as platforms for PrP cluster formation, signaling, actin rearrangement and the recruitment of CD3 (from internal stores) to the cap [9]. The involvement of reggie in the recruitment of a membrane protein (Glut4) to specific sites of the plasma membrane has previously been observed in adipocytes [55,56] as is discussed next.

7.3. Reggie-associated signaling and the recruitment of Glut4 in adipocytes

In adipocytes (fat cells) flotillin-1/reggie-2 interact with the c-cbl- associated adaptor protein (CAP) and via CAP with a signaling complex that activates the small guanosine 5′-triphosphate-hydrolyzing enzyme (GTPase) TC10 [55] in lipid rafts (reggie/flotillin microdomains). This Fig. 3. Recruitment and targeted delivery of membrane and membrane proteins results in the translocation of the glucose transporter Glut4 from a regulated by reggie. PrP is typically associated with reggie microdomains and clustered specific vesicular storage compartment to the plasma membrane at contact sites between cells (cell 1 with cell 2). PrP trans-interaction in reggie [55,56]. This step requires the exocyst and the (Ras-related) GTPase microdomains leads to signal transduction including the CAP/reggie-associated RalA. TC10 and RalA associate with vesicles, such as the recycling signaling complex which activates the small GTPase TC10. This leads to the mobilization of cargo (cadherin)-loaded vesicles which also have reggie microdomains, through compartment and (transport) vesicles en route to the plasma TC10 and RalA. Vesicles are transported (along cytoskeletal elements) to the plasma — membrane [58,59] which also carry reggie in agreement with the membrane and fuse resulting in membrane growth and cadherin delivery at cell contact role of the reggie proteins in activation of TC10 and other small GTPases. sites. 420 C.A.O. Stuermer / Biochimica et Biophysica Acta 1812 (2011) 415–422 epithelial and neuronal cells or zebrafish embryonic blastocytes cause was applied to the regenerating axons behind the first transection and PrP clustering and co-clustering with reggies [9,54]. PrP clusters in morpholino application site in the optic nerve which allows to reggie microdomains apparently regulate cell contact formation in quantify the regenerating axons by the second label in their parent zebrafish embryos as well as in mammalian cells (Fig. 3) as well as RGCs. This approach showed a 70% reduction of RGCs with regenerating downstream actin rearrangement [54]. In zebrafish embryos, PrP-1 axons in the optic nerve showing that reggie morpholinos block axon accumulates selectively at contact sites between cells at the blastula regeneration and, in other words, suggest that reggies are necessary for and gastrula stage. PrP clustering results in signaling. This signal is axon growth and regeneration. necessary for the PrP-dependent recruitment of epithelial (E)- This conclusion was supported by individual neurons from the cadherin from the internal vesicle pool to the contacts between cells mouse hippocampus in culture in which reggie expression levels were in zebrafish as well as in various mammalian cells [9,54]. In zebrafish, downregulated by reggie-specific siRNAs [4]. These neurons failed to PrP downregulation by morpholinos has dramatic effects: the cells differentiate and did not form axons or failed to elongate them. Axons lose contact and the embryo dies from its failure to proceed through often showed immobile club-shaped endings where a growth cone gastrulation [54]. Search for the underlying mechanism showed that should have been and did not grow (Fig. 2). Other neurons had shorter E-cadherin was retained in intracellular recycling vesicles instead of or no processes (Fig. 2), were abnormally large and malformed and emerging at the cell-surface where E-cadherin should be engaged in produced conspicuous bulges instead of axons and dendrites. The homophilic adhesion. elongation process was apparently blocked because, as we believe, How GPI-anchored proteins such as PrP communicate with reggie membrane proteins or membrane and proteins from the internal for the recruitment of cadherins and other membrane proteins vesicle pool were not appropriately supplied to the prospectively remains to be solved. How proteins with lipid anchors confined to growing tips [9]. Thus, the mechanism of membrane and protein one leaflet of the lipid bilayer are capable of signal transduction recruitment which is necessary for process growth is controlled by without associated transmembrane proteins is also unsolved and reggies. Therefore, reggies clearly regulate regeneration and axon debated in many reports and reviews dealing with lipid rafts and their growth. function [25]. Recent results from a biophysical simulation approach, Such phenotypes indicate a disturbance in the communication however, suggest that cluster formation of GPI-anchored proteins with the cytoskeleton, which was examined in N2a neuroblastoma with lipid anchors in the outer leaflet of the plasma membrane is cells. Reggie mis- and downregulation perturbed the activation levels crucial and efficient in influencing the adjacent cytoplasmic leaflet, of the Rho-GTPases Rac, Rho and cdc42 which changed the activation which offers another cluster of proteins (such as the reggie of the downstream effectors WASP, Arp2/3, cortactin and cofilin and microdomain) with their own lipid anchors—the myristoyl and of focal adhesion kinase (FAK), p38 and ras [4,62]. This is compatible palmitoyl residues in reggie (Matthias Weiss, pers. commun.). with the view that the reggie proteins can affect and modify actin Moreover, sphingolipids with lipid chains longer than the width of dynamics [64], in connection with the recruitment of membrane and one leaflet are enriched in rafts and microdomains. It is thus proteins (or simply membrane building blocks) from the constitutive conceivable that co-clusters of proteins in the outer and inner leaflet secretory pathway and recycling compartment to the growing tips [9]. of the plasma membrane can transduce signals into the cell, activating src tyrosine kinases that are also lipid-anchored and able to interact 9. An explanation of reggie functions with reggie (such as fyn) [65]. Thus, reggies appear to function as platforms necessary for the Taken together, it seems that reggies are crucial for the assembly of specific cell-surface proteins, for signal transduction and recruitment of membrane and specific membrane proteins (or activation of small GTPases to regulate actin dynamics. Specificity and membrane building blocks) to specific regions or domains of the the trigger for signaling appear to come from the cell-surface associates cell (Fig. 3). Neurons with elongating and regenerating axons, where of reggie/flotillin microdomains, which are cell type dependent (insulin reggies are enriched at growing tips, seem to suffer badly when receptor in adipocytes, PrP (Thy-1) in T cells and PrP (Thy-1) at cell reggies are missing since blockage of reggie function impairs the contact sites in the embryo and cells in vitro) and domain specific(the delivery of material for growth. Membrane proteins are recruited cap in T cells, contact sites). The cell-surface-derived signals often (or from vesicular pools (recycling compartment, transport vesicles, always) target GTPases and actin dynamics, conceivably to activate and Golgi-associated vesicles) and moved along cytoskeletal elements, recruit specific vesicles from the intracellular pool such as Glut4 and and this requires the small GTPases of the Rho- and Ras-family and E-cadherin. their effectors. Reggie-associated signal transduction and membrane Thus, consistent with finding reggie-1 and -2 in basically all cell protein recruitment appear to be activated by surface molecules with types and species as distant as flies, fish and mammals [53], their specificaffinities for the reggie microdomain environment such as function seems to be of general importance for the communication GPI-anchored proteins and certain receptor types. The surface between cells and for membrane protein sorting, trafficking and proteins can be activated by ligands, binding partners (PrP–PrP delivery. How does this relate to axon growth? trans-interaction) or cross-linking antibodies mimicking ligands which activate signaling molecules located in reggie microdomains: 8. Reggies regulate axon regeneration src tyrosine kinases, small GTPases, cbl and interacting adaptor complexes. Signaling provokes the recruitment of vesicles and To examine whether and how reggies might regulate axon thereby the targeted delivery of membrane building blocks and regeneration and growth, we applied morpholinos in a piece of proteins for axon growth, adhesion, navigation as well as for contact gelfoam to the transected right nerve of adult zebrafish and a control formation between cells and for the establishment of specific morpholino to the left transected nerve [4] following a procedure by membrane domains such as the leading edge, growth cone, its Becker et al. [66]. The morpholinos are being taken up by the severed lamellipodia and filopodia. This implies a role of the reggies in the axons and transported retrogradely to the RGCs of origin. The recruitment of specific vesicles and certain membrane proteins from morpholinos are labeled by a fluorescent tag (lissamine) thus vesicles and stores (transporters, receptors, ion channels and allowing identification of morpholino-laden RGCs in retina whole adhesion molecules) [9]. mounts. Miniexplants of retinae of treated fish send out axons. The This role of the reggies in the mediation of the recruitment of quantification of axons extending from the explants showed a 45% proteins/membrane from intracellular pools requires their existence in decrease from morpholino-treated RGCs compared to control. A all cells of vertebrates and invertebrates and explains why they are so second experiment gave even more striking effects: a fluorescent dye important for axon growth. Upregulation of reggie expression is a C.A.O. Stuermer / Biochimica et Biophysica Acta 1812 (2011) 415–422 421 prerequisite for axon growth/regeneration not only in fish but also in [15] C.A.O. Stuermer, C.A. Leppert, Molecular determinants of retinal axon pathfinding fi fi in sh, in: N.A. Ingoglia, M. Murray (Eds.), Axonal Regeneration in the Central the mammalian CNS [4,13].In sh, all RGCs express reggie at high levels Nervous System, Marcel Dekker Inc., New York, 2000. and regenerate their axons. In the mammalian retina, only 3% of the [16] I.C. Morrow, S. Rea, S. Martin, I.A. Prior, R. Prohaska, J.F. Hancock, D.E. James, R.G. neurons regenerate their axons and express reggie. When new methods Parton, Flotillin-1/reggie-2 traffics to surface raft domains via a novel Golgi- independent pathway. Identification of a novel membrane targeting domain and a are applied to stimulate the remaining neurons to upregulate reggie, role for palmitoylation, J. Biol. Chem. 277 (2002) 48834–48841. axon regeneration might be successful to an extent that would allow [17] C. Neumann-Giesen, I. Fernow, M. Amaddii, R. Tikkanen, Role of EGF-induced recovery of function in mammals. First results in this direction are tyrosine phosphorylation of reggie-1/flotillin-2 in cell spreading and signaling to – encouraging (unpublished results: Jan Koch, Paul Lingor, Matthias Bähr the actin cytoskeleton, J. Cell Sci. 120 (2007) 395 406. [18] J.F. Dermine, S. Duclos, J. Garin, F. St-Louis, S. Rea, R.G. Parton, M. Desjardins, [Göttingen] in collaboration with Gonzalo Solis and C.A.O. Stuermer Flotillin-1-enriched lipid raft domains accumulate on maturing phagosomes, [Konstanz]). In light of such results it is tempting to speculate that J. Biol. Chem. 276 (2001) 18507–18512. upregulation of reggie in injured neurons after spinal cord lesion might [19] M. Langhorst, A. Reuter, G. Luxenhofer, E.M. Boneberg, D.F. Legler, H. Plattner, C.A.O. Stuermer, Preformed reggie/flotillin caps: stable priming platforms for macrodomain promote axon regrowth and improve the fatal conditions after lesion. assembly in T cells, FASEB J. 20 (2006) 711–713. Upregulation of reggie and the ensuing growth response might provide [20] C.A.O. Stuermer, M. Langhorst, M. Wiechers, D.F. Legler, S. Hannbeck von c neurons with the necessary “power” to resist and counteract effects Hanwehr, A.H. Guse, H. Plattner, PrP capping in T cells promotes its association with the lipid raft proteins reggie-1 and reggie-2 and leads to signal transduction, from neurodegenerative diseases. FASEB J. 2 (2004) 1–27. This unexpected role of the reggies predicts on the one hand [21] C.A.O. Stuermer, D.M. Lang, F. Kirsch, M.F. Wiechers, S.-O. Deininger, H. Plattner, related functions of other microdomain proteins and requires on the Glycosylphosphatidyl inositol-anchored proteins and fyn kinase assemble in fi fi noncaveolar plasma membrane microdomains de ned by reggie-1 and -2, Mol. other hand identi cation of all players in the reggie-dependent Biol. Cell 12 (2001) 3031–3045. signaling pathway. Moreover, whether reggies act as molecular [22] C.A.O. Stuermer, H. Plattner, The “lipid raft”/microdomain proteins reggie-1 and shuttles between plasma membrane and intracellular vesicle pools reggie-2 (flotillins) are scaffolds for protein interaction and signaling, Biochem. Soc. Symp. 72 (2005) 109–118. needs to be addressed experimentally. The most crucial issue in the [23] L. Rajendran, M. Masilamani, S. Solomon, R. Tikkanen, C.A.O. Stuermer, Asymmetric context of axonal regeneration is the identification of the factors that localization of flotillins/reggies in preassembled platforms confers inherent polarity control reggie upregulation in neurons during axon outgrowth and to hematopoietic cells, Proc. Natl Acad. Sci. USA 100 (2003) 8241–8246. regeneration in fish and in mammals so to stimulate neurons to [24] K. Simons, E. Ikonen, Functional rafts in cell membranes, Nature 387 (1997) 569–572. produce new processes upon injury and resist degeneration. [25] K. Simons, W.L. Vaz, Model systems, lipid rafts, and cell membranes, Annu. Rev. Biophys. Biomol. Struct. 33 (2004) 269–295. [26] D. Goswami, K. Gowrishankar, S. Bilgrami, S. Ghosh, R. Raghupathy, R. Chadda, R. Acknowledgment Vishwakarma, M. Rao, S. Mayor, Nanoclusters of GPI-anchored proteins are formed by cortical actin-driven activity, Cell 135 (20098) 1085–1097. [27] P.F. Lenne, L. Wawrezinieck, F. Conchonaud, O. Wurtz, A. Boned, X.-J. Guo, H. Our work is supported by the Deutsche Forschungsgemeinschaft Rigneault, H.T. He, D. Marguet, Dynamic molecular confinement in the plasma and Fonds der Chemischen Industrie. I want to thank Ulrike Binkle, membrane by microdomains and the cytoskeleton network, EMBO J. 25 (2006) Marianne Wiechers and Vsevolod Bodrikov for the figures and all 3245–3256. [28] J.F. Hancock, Lipid rafts: contentious only from simplistic standpoints, Nat. Rev. co-workers for the experiments and results in our reggie research. Mol. Cell Biol. 7 (2006) 456–462. [29] H.-J. Kaiser, D. Lingwood, I. Levental, J.L. Sampaio, L. Kalvodova, L. Rajendran, K. Simons, Order of lipid phases in model and plasma membranes, Proc. Natl Acad. References Sci. USA 106 (2009) 16645–16650. [30] D.T. Browman, M.B. Hoegg, S.M. Robbins, The SPFH domain-containing proteins: [1] R.M. Gaze, The Formation of Nerve Connections, Academic Press, London, 1970. more than lipid raft markers, Trends Cell Biol. 17 (2007) 394–402. [2] C.A.O. Stuermer, Retinotopic organization of the developing retinotectal projec- [31] I.C. Morrow, R.G. Parton, Flotillins and the PHB domain protein family: rafts, tion in the zebrafish embryo, J. Neurosci. 8 (1988) 4513–4530. worms and anaesthetics, Traffic 6 (2005) 725–740. [3] T. Schulte, K.A. Paschke, U. Laessing, F. Lottspeich, C.A.O. Stuermer, Reggie-1 and [32] E. Rivera-Milla, C.A.O. Stuermer, E. Malaga-Trillo, Ancient origin of reggie reggie-2, two cell surface proteins expressed by retinal ganglion cells during axon (flotillin) and reggie-like proteins and convergent evolution of SPFH domain, regeneration, Development 124 (1997) 577–587. Cell. Mol. Life Sci. 63 (2006) 343–357. [4] C. Munderloh, G.P. Solis, F. Jaeger, M. Wiechers, C.A.O. Stuermer, Reggies/flotillins [33] G.P. Solis, M. Hoegg, C. Munderloh, E. Rivera-Milla, C.A.O. Stuermer, Rules for regulate retinal axon regeneration in the zebrafish optic nerve and signal reggie oligomerization and stabilization, Biochem. J. 403 (2007) 313–322. transduction in N2a cells, J. Neurosci. 29 (2009) 6607–6615. [34] K. Simons, R. Ehehalt, Cholesterol, lipid rafts, and disease, J. Clin. Invest. 110 (2002) [5] H.B Abdesselem, A. Shypitsyna, C.A.O. Stuermer, No Nogo66-mediated inhibition 597–603. of regenerating axons in the zebrafish optic nerve, J. Neurosci. 29 (2009) [35] K.G. Rothberg, J.E. Heuser, W.C. Donzell, Y.S. Ying, J.R. Glemney, R.G. Anderson, 15489–15498. Caveolin, a protein component of caveolae membrane coats, Cell 68 (1992) [6] H. Diekmann, M. Klinger, T. Oertle, M. Pogoda, D. Heinz, M.E. Schwab, C.A.O. 673–682. Stuermer, Evidence for the absence of the neurite growth inhibitory protein [36] A.M. Fra, E. Williamson, K. Simons, R.G. Parton, De novo formation of caveolae in Nogo-A in fish, J. Mol. Evol. 22 (2005) 1635–1648. lymphocytes by expression of VIP21–caveolin, Proc. Natl Acad. Sci. USA 92 (1995) [7] I.C. Maier, M.E. Schwab, Sprouting, regeneration and circuit formation in the 8655–8659. injured spinal cord: factors and activity, Phil. Trans. R. Soc. B Biol. Sci. 361 (2006) [37] M.F. Langhorst, A. Reuter, C.A.O. Stuermer, Scaffolding microdomains and beyond 1611–1634. —the function of reggie/flotillin proteins, Mol. Cell. Life Sci. 62 (2005) 2228–2240. [8] C.A.O. Stuermer, M. Bastmeyer, M. Bähr, G. Strobel, K. Paschke, Trying to [38] M. Kirkham, S.J. Nixon, M.T. Howes, L. Abi-Rached, D.E. Wakeham, M. Hanzal- understand axonal regeneration in the CNS of fish, J. Neurobiol. 23 (1992) Bayer, C. Ferguson, M.M. Hill, M. Fernandez-Rojo, D.A. Brown, J.F. Hancock, F.M. 537–550. Brodsky, R.G. Parton, Evolutionary analysis and molecular dissection of caveola [9] C.A.O. Stuermer, The reggie/flotillin connection to growth, Trends Cell Biol. biogenesis, J. Cell Sci. 121 (2008) 2075–2086. (2009), doi:10.1016/j.tcb2009.10.003. [39] M. Drab, P. Verkade, M. Elger, M. Kasper, M. Lohn, B. Lauterbach, J. Menne, C. [10] P.M. Richardson, U.M. McGuinness, A.J. Aguayo, Axons from CNS neurons Lindschau, F. Mende, F.C. Luft, Loss of caveolae, vascular dysfunction, and pulmonary regenerate into PNS grafts, Nature 284 (1980) 264–265. defects in caveolin-1 gene-disrupted mice, Science 293 (2001) 2449–2452. [11] A.M. Taylor, N.C. Berchtold, V.M. Perreau, C.H. Tu, N.L. Jeon, C.W. Cotman, Axonal [40] S.J. Nixon, A. Carter, J. Wegner, C. Ferguson, M. Floetenmeyer, J. Riches, B. Key, M. mRNA in uninjured and regenerating cortical mammalian axons, J. Neurosci. 29 Westerfield, R.G. Parton, Caveolin-1 is required for lateral line neuromast and (2009) 4697–4707. notochord development, J. Cell Sci. 120 (2007) 2151–2161. [12] P.E. Bickel, P.E. Scherer, J.E. Schnitzer, P. Oh, M.P. Lisanti, H.F. Lodish, Flotillin and [41] P.-K. Fang, K.R. Solomon, L. Zhuang, Q. Maosong, M. McKee, M.R. Freeman, P.C. epidermal surface antigen define a new family of caveolae-associated integral Yelick, Caveolin-1alpha and -1beta perform nonredundant roles in early membrane proteins, J. Biol. Chem. 272 (1997) 13793–13802. vertebrate development, Am. J. Pathol. 169 (2006) 2209–2222. [13] D.M. Lang, S. Lommel, M. Jung, R. Ankerhold, B. Petrausch, U. Laessing, M.F. [42] M. Murata, J. Peranen, R. Schreiner, F. Wieland, T.V. Kurzchalia, K. Simons, VIP21/ Wiechers, H. Plattner, C.A.O. Stuermer, Identification of reggie-1 and reggie-2 as caveolin is a cholesterol-binding protein, Proc. Natl Acad. Sci. USA 92 (1995) plasmamembrane-associated proteins which cocluster with activated GPI- 10339–10343. anchored cell adhesion molecules in non-caveolar micropatches in neurons, J. [43] J.L. Thorpe, M. Doitsidou, S.-Y. Ho, E. Raz, S.A. Farber, Germ cell migration in Neurobiol. 37 (1998) 502–523. zebrafish is dependent on HMGCoA reductase activity and prenylation, Dev. Cell 6 [14] K.A. Paschke, F. Lottspeich, C.A.O. Stuermer, Neurolin, a cell surface glycoprotein (2004) 295–302. on growing retinal axons in the goldfish visual system, is reexpressed during [44] S.J. Nixon, J. Wegner, C. Ferguson, P.-F. Méry, J.F. Hancock, P.D. Currie, B. Key, M. retinal axonal regeneration, J. Cell Biol. 117 (1992) 863–875. Westerfield, R.G. Parton, Zebrafish as a model for caveolin-associated muscle 422 C.A.O. Stuermer / Biochimica et Biophysica Acta 1812 (2011) 415–422

disease; caveolin-3 is required for myofibril organization and muscle cell [56] N. Kioka, K. Ueda, T. Amachi, Vinexin, CAP/ponsin, ArgBP2: a novel adaptor patterning, Hum. Mol. Genet. 14 (2005) 1727–1743. protein family regulating cytoskeletal organization and signal transduction, Cell [45] Z. Tang, P.E. Scherer, T. Okamoto, K. Song, C. Chu, D.S. Kohtz, I. Nishimoto, H.F. Lodish, Struct. Funct. 27 (2002) 1–7. M.P. Lisanti, Molecular cloning of caveolin-3, a novel member of the caveolin gene [57] V.N.B. Das, A. Dujeancourt, M.I. Toulouze, T. Galli, P. Roux, A. Dautry-Varsat, A. family expressed predominantly in muscle, J. Biol. Chem. 271 (1996) 2255–2261. Alcover, Activation-induced polarized recycling targets T cell antigen receptors to [46] T. Benzing, Signaling at the slit diaphragm, J. Am. Soc. Nephrol. 15 (2004) 1382–1391. the immunological synapse: involvement of the SNARE complexes, Immunity 20 [47] S. Roselli, O. Gribouval, N. Boute, M. Sich, F. Benessy, T. Attie, M.C. Gubler, C. (2004) 577–588. Antignac, Podocin localizes in the kidney to the slit diaphragm area, Am. J. Pathol. [58] L. Chang, S.-H. Chiang, A.R. Saltiel, TC10alpha is required for insulin-stimulated 160 (2002) 131–139. glucose uptake in adipocytes, Endocrinology 148 (2007) 27–33. [48] T.B. Huber, M. Simons, B. Hartleben, L. Sernetz, M. Schmidts, E. Gundlach, M.A. [59] L.A. Feig, Ral-GTPases: approaching their 15 minutes of fame, Trends Cell Biol. 13 Saleem, G. Walz, T. Benzing, Molecular basis of the functional podocin–nephrin (2003) 419–425. complex: mutations in the NPHS2 gene disrupt nephrin targeting to lipid raft [60] G. Lalli, A. Hall, Ral GTPases regulate neurite branching through GAP-43 and the microdomains, Hum. Mol. Genet. 12 (2004) 3397–3405. exocyst complex, J. Cell Biol. 171 (2005) 657–669. [49] A.G. Kramer-Zucker, S. Wiessner, A.M. Jensen, I.A. Drummond, Organization of the [61] M. Langhorst, A. Reuter, M. Wiechers, S. Hannbeck, H. Plattner, C.A.O. Stuermer, pronephric filtration apparatus in zebrafish requires Nephrin, Podocin and the Trafficking and association with PrPc of the microdomain proteins reggie-1 and FERM domain protein Mosaic eyes, Dev. Biol. 285 (2005) 316–329. reggie-2 (flotillins), Eur. J. Cell Biol. 87 (2008) 211–226. [50] I.A. Drummond, Kidney development and disease in the zebrafish, Am. Soc. [62] M.F. Langhorst, C. Munderloh, S. Mueller, F. Jaeger, S. Hartmann, G. Luxenhofer, M. Nephrol. 16 (2005) 299–304. Wiechers, C.A.O. Stuermer, Reggies/flotillins regulate Rho GTPase signalling [51] S. Roselli, I. Moutkine, O. Gribouval, A. Benmerah, C. Antignac, Plasma membrane during axon outgrowth and axon regeneration, Eur. J. Cell Biol. 87 (2008) targeting of podocin through the classical exocytic pathway: effect of NPHS2 921–931. mutations, Traffic 5 (2004) 37–44. [63] X.-W. Chen, M. Inoue, S.C. Hsu, A.R. Saltiel, RalA-exocyst-dependent recycling [52] S.O. Deininger, L. Rajendran, F. Lottspeich, M. Przybylski, H. llges, C.A.O. Stuermer, endosome trafficking is required for the completion of cytokinesis, J. Biol. Chem. A. Reuter, Identification of teleost Thy-1 and association with the microdomain/ 281 (2006) 38609–38616. lipid raft reggie proteins in regenerating CNS axons, Mol. Cell. Neurosci. 22 (2003) [64] M.F. Langhorst, S. Hannbeck von Hanwehr, G.P. Solis, H. Plattner, C.A.O. Stuermer, 544–554. Linking membrane microdomains to the cytoskeleton: regulation of the lateral [53] A. Reuter, E. Malaga-Trillo, U. Binkle, E. Rivera-Milla, R. Beltre, Y. Zhou, M. mobility of reggie-1/flotillin-2 by interaction with actin, FEBS Lett. 581 (2007) Bastmeyer, C.A.O. Stuermer, Evolutionary analysis and expression of teleost Thy- 4697–4703. 1, Zebrafish 1 (2004) 191–201. [65] J. Liu, S.M. DeYoung, M. Zhang, L.H.S.A.R. Dold, The stomatin/prohibitin/flotillin/ [54] E. Malaga-Trillo, G.P. Solis, V. Tomanetz, C. Geis, L. Luncz, C.A.O. Stuermer, HflK/C domain of flotillin-1 contains distinct sequences that direct plasma Regulation of embryonic cell adhesion by the prion protein, PLoS Biol. 7 (2009) membrane localization and protein interactions in 3T3-L1 adipocytes, J. Biol. e1000055. Chem. 280 (2005) 16125–16134. [55] C.A. Baumann, V. Ribon, M. Kanzaki, D.C. Thurmond, S. Mora, S. Shigematsu, P.E. [66] C.G. Becker, B.C. Lieberoth, F. Morellini, J. Feldner, T. Becker, M. Schachner, L1.1 is Bickel, J.E. Pessing, A.R. Saltiel, CAP defines a second signalling pathway required involved in spinal cord regeneration in adult zebrafish, J. Neurosci. 24 (2004) for insulin-stimulated glucose transport, Nature 407 (2000) 202–207. 7837–7842.