Disease Models & Mechanisms 6, 1057-1065 (2013) doi:10.1242/dmm.012138 REVIEW

Modeling human neurodevelopmental disorders in the Xenopus tadpole: from mechanisms to therapeutic targets

Kara G. Pratt1,* and Arseny S. Khakhalin2

The Xenopus tadpole model offers many advantages for studying the molecular, cellular and network mechanisms underlying neurodevelopmental disorders. Essentially every stage of normal neural circuit development, from axon outgrowth and guidance to activity-dependent homeostasis and refinement, has been studied in the frog tadpole, making it an ideal model to determine what happens when any of these stages are compromised. Recently, the tadpole model has been used to explore the mechanisms of epilepsy and autism, and there is mounting evidence to suggest that diseases of the nervous system involve deficits in the most fundamental aspects of nervous system function and development. In this Review, we provide an update on how tadpole models are being used to study three distinct types of neurodevelopmental disorders: diseases caused by exposure to environmental toxicants, epilepsy and seizure disorders, and autism. DMM

Introduction contralateral optic tectum (Gaze, 1958; Sperry, 1963), a midbrain Neurons have the amazing ability to self-assemble into highly structure that is homologous to the mammalian superior colliculus. organized circuits. These circuits give rise to our perceptions, The RGC axons form a highly organized topographic map within thoughts and emotions, and determine how we experience our their target structure, with neighboring RGCs making synapses world. Disorders in neural development, therefore, can often onto neighboring tectal neurons. This mirrors what is observed in compromise the quality of life. To date, there are no cures for many mammalian sensory circuits, including those within the prevalent neurodevelopmental disorders such as autism, epilepsy human nervous system. Furthermore, as in most mammalian and schizophrenia, and there are many gaps in what is known about excitatory synapses, RGC axons release glutamate, and tectal the underlying causes of these conditions. Animal models that allow neurons express AMPA and NMDA glutamate receptors (Wu et for a disorder to be studied at multiple levels, from molecules to al., 1996). Building on the contributions of the Xenopus model to behavior, can provide a more complete understanding of the the field of embryology, many aspects of Xenopus neural circuit associated gene locus and underlying mechanism(s), thereby development have been carefully studied, and meticulously promoting the design of novel approaches for treatment and described across the key developmental stages. For instance, it is prevention. well established that the axons of the RGCs reach the tectum at Disease Models & Mechanisms The Xenopus laevis tadpole possesses many qualities that make around developmental stage 39 [4-5 days post-fertilization (dpf)] it a powerful model to study disorders of the developing nervous (Holt, 1989; Dingwell et al., 2000), that the most dynamic phase of system. First and foremost, essentially every stage of normal neural circuit formation – both morphologically and functionally – occurs development, from neurogenesis and differentiation to axon between stage 44 and 47 (7-10 dpf), and that by stage 49 (~16-24 pathfinding, synapse maturation and circuit refinement, has been dpf) the circuit becomes more refined and stable (Sakaguchi et al., studied in detail in Xenopus tadpoles (Cline and Kelly, 2012; Sanes 1984; Cline et al., 1996b; Pratt and Aizenman, 2007). et al., 2012). Such a detailed understanding of normal Experimentally, Xenopus tadpoles pose many advantages. Because developmental processes is invaluable when seeking to determine of their transparency, and the extreme dorsal location of the brain, how they can malfunction. Compared with mammalian neural the axons and dendrites of single living neurons can be imaged in circuits, those of the tadpole are simpler, yet homologous in their vivo (Harris et al., 1987; Ruthazer et al., 2003; Cohen-Cory, 2007; Li basic organization. For example, in the tadpole retinotectal circuit et al., 2011; Dong and Aizenman, 2012) and, better yet, in awake (Fig. 1), retinal ganglion cells (RGCs) in the eye project their axons animals (Chen et al., 2012; Hossain et al., 2012). Time-lapse imaging to the brain, where they synapse onto tectal neurons in the of radial glia in the tectum has provided the first in vivo description of how neural activity affects the structure and function of these cells 1University of Wyoming, 1000 E University Avenue, Laramie, WY 82071, USA (Tremblay et al., 2009), and advances in morphometric software (Liu 2Brown University, 45 Prospect Street, Providence, RI 02912, USA et al., 2009; Chen et al., 2012) have allowed for an improved ability *Author for correspondence ([email protected]) to track and measure all processes in 3D across short intervals over © 2013. Published by The Company of Biologists Ltd long periods of time (Hossain et al., 2012). Furthermore, calcium This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution imaging in tadpoles can be carried out readily (Tao et al., 2001; Junek and reproduction in any medium provided that the original work is properly attributed. et al., 2010; Xu et al., 2011), including in vivo recordings from intact

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1. Organism 2. Brain 3. Network 4. Cell 5. Synapse

OB FB

Glutamate OT

AMPA HB receptor

Cell NMDA 200 layer receptor μm

SC OT GABA GABA receptor

Whole-brain Cell imaging Behaviorimmunostaining Ca2+ imaging and reconstruction Electrophysiology a b DMM Normal c

a b Abnormal c

Fig. 1. The Xenopus tadpole as a research model, shown with key experimental techniques that are used to differentiate between normal and abnormal patterns of neural development. (1) Top: view of the animal at ca. 3 weeks post-fertilization. Several behavioral tests can be used to assess brain development: for example, wild-type animals usually swim along the sides of the container (represented by a circle; bottom), whereas animals with altered excitation/inhibition Disease Models & Mechanisms balance tend to circle in the middle of it. (2) Top: general view of the brain. OB, olfactory bulbs; OT, optic tectum; HB, hindbrain: SC, spinal cord; red, projections from the retina; green, tectal projections to the hindbrain; blue, descending projections to the spinal cord. An isolated brain provides an accessible in vitro preparation, and whole-brain immunostaining (bottom) can be used to quantify global alterations in brain biochemistry (an exaggerated staining for GABA is shown). (3) Top: horizontal section of the optic tectum (OT) and caudal forebrain (FB); at this level, Ca2+ imaging can be used to detect abnormal seizure-like patterns of activity (bottom). (4) At the neuron level, in vivo or ex vivo imaging allows assessment of cell morphology development. (5) At the synaptic level, electrophysiology offers a way to quantify maturation of synaptic and intrinsic properties of the cell through recordings of (a) evoked synaptic responses, (b) spiking in response to current injections and (c) spontaneous synaptic activity. The figure is inspired by experimental data published in the following papers: (Aizenman et al., 2002; Bestman et al., 2006; Ruthazer et al., 2006; Pratt and Aizenman, 2007; Hewapathirane et al., 2008; Bollmann and Engert, 2009; Hiramoto and Cline, 2009; Straka, 2010; Bell et al., 2011; Marshak et al., 2012; Miraucourt et al., 2012).

awake animals (Chen et al., 2012; Podgorski et al., 2012; Imaizumi (Pratt and Aizenman, 2007; Li et al., 2009; Pratt and Aizenman, 2009; et al., 2013). Expression of genes of interest can also be achieved in Straka and Simmers, 2012), synaptic maturation (Wu et al., 1996; vivo, using electroporation-based protocols (Haas et al., 2001; Haas Akerman and Cline, 2006; Aizenman and Cline, 2007; Deeg et al., et al., 2002; Bestman et al., 2006), or by mRNA injection into 2009; Khakhalin and Aizenman, 2012), synaptic plasticity (Engert appropriate cells of the early embryo (Demarque and Spitzer, 2010). et al., 2002; Mu and Poo, 2006; Pratt et al., 2008; Tsui et al., 2010) Because of the relatively high permeability of the tadpole blood-brain and cell intrinsic properties (Aizenman et al., 2003; Pratt and barrier, pharmacological manipulations of the nervous system are Aizenman, 2007; Winlove and Roberts, 2011). The behaviors usually achieved by simply adding the pharmacological agent to the controlled by corresponding neural circuits, including several types tadpole rearing solution. Electrophysiological techniques have been of escape behaviors (Roberts et al., 2000; Wassersug and Yamashita, successfully employed in Xenopus to quantify network connectivity 2002; Dong et al., 2009; Sillar and Robertson, 2009), orienting

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reflexes (Pronych et al., 1996; Simmons et al., 2004; Straka, 2010) the major targets of TH, however, is the brain, where disruption and social behaviors (Katz et al., 1981; Villinger and Waldman, 2012), of normal TH activity can lead to neurodevelopmental defects have been well described, and can be experimentally manipulated (Zoeller and Crofton, 2000). Given that it is unlikely that the more (Lum et al., 1982; Jamieson and Roberts, 2000; Wassersug and subtle defects associated with neurodevelopment (such as Yamashita, 2002; Simmons et al., 2004; Dong et al., 2009; Straka, incomplete synapse refinement for example) would disrupt the 2010). To sum up, these experimental approaches enable developing relatively gross changes associated with metamorphosis (such as neural circuits to be examined at the molecular, cellular and loss of the tail, emergence of limb buds and formation of lungs), behavioral levels – all in the same organism (Fig. 1). Moreover, disorders in nervous system development could go undetected. humans are genetically closer to Xenopus than to similar model Thus, it was necessary to develop a more sensitive molecular organisms, such as zebrafish, because teleosts (the ray-finned fishes) approach for the identification of neural-specific molecular are known to have divergent and highly specialized genomes markers that are associated with TH disruption. Experiments using (Postlethwait et al., 2004; Nakatani et al., 2007; Rash et al., 2012). quantitative reverse transcriptase PCR (qRT-PCR) revealed that Combined with the relatively low cost of housing, large number of the TH inhibitors methimazole and perchlorate alter neural TH embryos generated from one mating, and the ease of embryologic receptor expression in brain tissue of stage-54 tadpoles (Zhang et (De Robertis, 2006; Harland and Grainger, 2011; Pai et al., 2012) al., 2006). Furthermore, in a detailed study combining cDNA array and surgical (Constantine-Paton and Capranica, 1976; Filoni, 2009; analysis and qRT-PCR, perchlorate was found to significantly McKeown et al., 2013; Elliott et al., 2013) manipulations, these increase the expression of several neural mRNAs (Helbing et al., qualities render Xenopus an ideal model for neurodevelopmental 2007), including the mRNA for β-amyloid precursor , a research. protein whose improper processing has been highly implicated in In this Review, we first highlight how tadpoles have been used Alzheimer’s disease, and mRNAs that encode for myelin basic as a model for assaying the effects of environmental chemicals on protein and myelin proteolipid protein, both of which are major neurodevelopment, and how the model itself has evolved and been components of the myelin sheath that insulates axons and

DMM refined over the years. We then describe a recently designed tadpole facilitates appropriate action potential conduction. The effects that model of epileptic seizures that has already led to the finding of a these perchlorate-induced increases in mRNAs could have on the built-in protective mechanism that is activated in response to a developing tadpole brain, however, remain unknown. seizure. Finally, we present a new and exciting tadpole model to Advances in both imaging and electrophysiological approaches study autism. have enabled the tadpole to become a powerful in vivo model for investigating, at a high resolution, how environmental chemicals Characterizing the effects of environmental toxicants can affect developing neurons. For instance, a study using the Rana on development in a tadpole model pipiens tadpole has shown that chronically exposing tectal neurons During development, neural circuits can be particularly sensitive to low, sub-micromolar levels of lead decreases both RGC axon to chemicals in the environment. In humans, for example, doses arbor area and branchtip number. The same group showed that of methylmercury that are neurotoxic to the embryonic central acute lead exposure weakens synaptic transmission between RGC nervous system (CNS) have no effect on the maternal CNS axons and tectal dendrites (Cline et al., 1996a). More recently, the (Castoldi et al., 2001). Similarly, exposure of the developing nervous Xenopus tadpole was used to characterize the effects of sub-lethal system of a rat pup to lead (a heavy metal) often results in concentrations of methylisothiazolinone (MIT; a biocide commonly encephalopathy, whereas the mature rat brain remains unaffected used in several cosmetics, including shampoo) on many aspects of when exposed to the same amount of lead (Holtzman and Hsu, nervous system function (Spawn and Aizenman, 2012). For Disease Models & Mechanisms 1976). Thus, chemicals in the environment that are deemed to be example, overall visual system function was tested using protocols innocuous to the adult CNS can be harmful to a developing brain. designed to characterize tectum-dependent and thalamus- Having been used for decades by researchers in academia as dependent visual behaviors (Dong et al., 2009). MIT-exposed well as the US government’s Environmental Protection Agency tadpoles displayed deficits in only the tectum-dependent visual (EPA) to assay toxic and teratogenic effects of environmental behavior, suggesting a malfunction in retinotectal synaptic chemicals, the Xenopus tadpole is not new to the field of transmission. Although no differences were observed in synaptic embryotoxicology (Dumpert and Zietz, 1984; Degitz et al., 2003; transmission between RGC inputs and tectal neurons in the MIT- Richards and Cole, 2006; Berg et al., 2009). The tadpole has served treated tadpoles, the pattern of the recurrent tecto-tectal as a workhorse for these studies mostly because their connectivity – which is activated by RGC inputs – (Fig. 1C) was metamorphosis from tadpole to frog depends entirely on thyroid altered in a way that suggests lack of circuit refinement. At the hormone (TH) (Damjanovski et al., 2000), and, in turn, one of the single-neuron level, no differences in intrinsic excitability and most prevalent environmental contaminants are the TH inhibitors, synaptic strengths were observed in MIT-treated tadpoles (Fig. 1D). a major class of endocrine disruptors. In the tadpole, if TH action In summary, the deficits in tectum-dependent visual behavior and is inhibited, metamorphosis stalls, whereas exposure to TH in pre- unrefined tecto-tectal connectivity, combined with the absence of metamorphic tadpoles induces precocious metamorphosis noticeable changes in intrinsic or synaptic properties, suggest that (Helbing et al., 2007). Because the progression of metamorphosis chronic MIT exposure causes problems at the circuit level and not is well described and obvious, alterations can be readily identified. at the single-cell level. For neurotoxicology research, this study Hence, this became a convenient way to test many classes of exemplifies how chronic exposure to concentrations of a chemical chemicals for their ability to disrupt TH activity (Gutleb et al., 2000; with no noticeable effects on either survival or morphology can Tietge et al., 2005; Cheng et al., 2011; Lorenz et al., 2011). One of still compromise a developing neural circuit. Overall, this study

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demonstrates the level of detail at which neurons and neural circuits Tadpole models for the study of autism spectrum can be assayed using the Xenopus tadpole, and, more specifically, disorders how a deficit in a behavior can be tracked down and studied at the Autism spectrum disorders (ASD) are paradoxical: the syndromes circuit and single-neuron level. within this group present with a highly recognizable set of symptoms, yet they can be caused by a diverse array of genetic A tadpole model for epileptic seizure abnormalities and environmental insults, such as prenatal infection, In addition to environmental toxins, developing circuits are hormonal exposure and teratogens (Newschaffer et al., 2007; particularly susceptible to epileptic activity. A protocol to reliably Abrahams and Geschwind, 2008). Mutations in more than 40 genes induce controlled seizures in the Xenopus tadpole has been have been shown to increase susceptibility to ASD, yet none of these developed (Hewapathirane et al., 2008) and has already led to a mutations are completely penetrant, i.e. cause ASD with 100% fundamental insight into how endogenous polyamines can play a probability (Lichtenstein et al., 2010; Neale et al., 2012). neuroprotective role in response to an epileptic seizure (Bell et al., Furthermore, although the defining symptoms of ASD, such as 2011). deficits in language, social interactions and personal interests are The development of the tadpole model for studying epileptic manifested at the highest cognitive levels, the etiology of ASD has seizures began with a detailed characterization of the ability of been linked to abnormalities in surprisingly fundamental aspects several different classes of chemoconvulsants to reliably induce of nervous system functioning and development. This includes seizures in stage-47 tadpoles. Several different classes of known defects in synaptic plasticity (Krey and Dolmetsch, 2007; Markram convulsants were tested: GABA-receptor antagonists et al., 2008; Bhakar et al., 2012), inhibition/excitation balance (Perry [pentylenetetrazole (PTZ), picrotoxin and bicuculline], glutamate et al., 2007; Markram and Markram, 2010; Marín, 2012), receptor agonists (kainate), muscarinic receptor agonists microcircuitry organization (Geschwind, 2009) and neuron-glia (pilocarpine) and potassium channel inhibitors (4-aminopyridine) interactions (Abrahams and Geschwind, 2008), as well as long- (Hewapathirane et al., 2008). All of these convulsants seemed to range underconnectivity and local overconnectivity, as a

DMM produce a common type of behavioral seizure in tadpoles. The consequence of altered axon guidance and dendritic arborization behavior commences with intermittent bouts of rapid swimming, (Rinaldi et al., 2008; Geschwind, 2009). These features suggest that followed by immobility, deviations from the normal head-down tail- ASD represents a uniquely human response to a broad class of up posture, and lateral movements of the head, followed ultimately developmental dysregulations (Peça and Feng, 2012) and, therefore, by full-blown seizure behavior – C-shaped contractions evoked by the mechanisms of ASD are likely to be successfully addressed in abnormal unilateral axial muscle contractions that are so strong animal models not necessarily capable of expressing most that they result in the entire tadpole displaying a stereotypical ‘C’ behavioral and cognitive symptoms of ASD (Patterson, 2011). These shape. Because all of the different classes of convulsant induced animal models would include mammals, but also fish (Tropepe and the same type of seizure, it was concluded that this is a ‘true’ seizure Sive, 2003; Kabashi et al., 2011), birds (Panaitof, 2012), insects rather than the effects of a particular drug on motor function. The (Gatto and Broadie, 2011) and amphibians. GABA receptor antagonist PTZ was determined to be the optimal With this in mind, a successful experimental approach in chemoconvulsant for the tadpole seizure model because it reliably Xenopus would entail looking directly at the changes caused by induces the stereotypical C-shape contractions at doses that are known ASD-associated developmental perturbations at the cellular neither lethal nor toxic, and in vivo field potential recordings in and network levels. One of the unique benefits of the tadpole is the optic tectum revealed robust epileptiform activity, i.e. high- the ease at which gene expression can be altered in individual amplitude spiking, in response to PTZ application. This neurons, and the convenience of registering the consequences of Disease Models & Mechanisms epileptiform activity can be blocked completely by administration these perturbations in vivo, allowing a way to differentiate between of the anti-epileptic drug valproate. An advantage of this model is cell-autonomous and network-level effects of ASD-associated that immobilization of the tadpole for electrophysiology or imaging mutations. As a good example, when a wild-type human MeCP2 experiments can be achieved using reversible paralytics or agar gene [a mutation in this gene causes Rett syndrome in humans, immersion, thereby allowing seizures to be studied in the absence and is strongly comorbid with ASD (Samaco and Neul, 2011)] was of anesthetic agents (Hewapathirane et al., 2008). overexpressed in Xenopus tectal neurons in vivo, these neurons In a recent study by Bell et al. (Bell et al., 2011) involving a were found to develop fewer, albeit longer, dendrites compared protocol consisting of two consecutive PTZ-induced seizures and with normal tectal cells (Marshak et al., 2012). Hence, in Xenopus, a combination of behavioral, electrophysiological and as in humans and rodents, variations in MeCP2 activity cause pharmacological experiments, it was shown that the first initial redistribution between close- and long-range network PTZ-induced seizure in a tadpole increases the production of connections, which is one of the landmark circuit abnormalities polyamines [an observation that had also been reported in a rodent in ASD (Geschwind, 2009). This work also illustrates that key seizure model (Hayashi et al., 1993)]. Elevated polyamine levels were transcription regulators are sufficiently conserved between found to boost the production and release of the inhibitory Xenopus and humans (Amir et al., 1999), allowing the human transmitter GABA, which rendered tadpoles less prone to future MeCP2 gene to interact (Marshak et al., 2012) with native Xenopus seizures. Similarly, exposing tadpoles to enhanced visual stimulation pathways (Stancheva et al., 2003). led to increased GABA levels in the tectum, providing another Although not every gene linked to ASD in humans (Abrahams compelling example of how GABA can function in a homeostatic and Geschwind, 2008; Neale et al., 2012) has been identified and manner in response to abnormally high levels of circuit activity studied in Xenopus thus far (Hellsten et al., 2010), a large proportion (Miraucourt et al., 2012). have been; see Table 1 for a list. Among all ASD-related genetic

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Table 1. Genes that are linked to ASD in humans, and that have been identified and studied inXenopus Human gene Gene function Association with ASD Studies in Xenopus MeCP2 Transcription modulator Linked to Rett syndrome (Samaco and Expression (Stancheva et al., 2003); effects on cell Neul, 2011) morphology (Marshak et al., 2012). See the text for details. FMR1 (aka Regulator of and mRNA Linked to Fragile X syndrome (Spencer et Genetics (Lim et al., 2005; Huot et al., 2012); FMRP), FXR1, shuttling al., 2006; Abrahams and Geschwind, expression (Blonden et al., 2005); effects on FXR2 2008; Levenga et al., 2010; Guo et al., development (Huot et al., 2005; Gessert et al., 2011; Bhakar et al., 2012) 2010; Huot et al., 2012). See the text for details. NLGN Neuroligins (synaptic adhesion ) (Jamain et al., 2003) Effects on cell morphology development (Chen et al., 2010). See the text for details. NRX Neurexins (synaptic adhesion proteins) (Feng et al., 2006) Effects on cell morphology development (Chen et al., 2010). See the text for details. MEF2 Transcription factor Linked to Fragile X syndrome (Tsai et al., Effects on cell functional and morphological 2012) maturation (Chen et al., 2012) Shank Synaptic scaffolding proteins (Abrahams and Geschwind, 2008; Peça and Expression pattern (Gessert et al., 2011) Feng, 2012) WNT-2 Signaling protein, developmental (Wassink et al., 2001) Expression pattern, effects on development regulator (Landesman and Sokol, 1997)

CACNA1C CaV1.2 channel gene (voltage-dependent Linked to Timothy syndrome (Krey and Expression pattern (Lewis et al., 2009) ion channel) Dolmetsch, 2007) IR Insulin receptor (Chiu and Cline, 2010; Stern, 2011) Effects on cell function and morphology (Chiu et al., 2008) DMM PER1 Circadian gene (member of a Period (Nicholas et al., 2007) Expression regulation (Zhuang et al., 2000) family) GRIK2 Kainate glutamate receptor (ionotropic (Jamain et al., 2002) Channel properties (Ishimaru et al., 1996) receptor, aka GluR6, GluK2) PTEN Cell cycle regulator, tumor suppressor Linked to Cowden syndrome (Butler et al., Effects on development (Ueno et al., 2006) gene 2005)

conditions, the one that is most researched in a Xenopus model is tectal neurons by either overexpression of mutant forms of mouse Fragile X syndrome, which is linked to the deactivation of a single Nlg-1, competitive saturation of native Nlg-1 extracellular domains gene, FMR1 (Abrahams and Geschwind, 2008; Levenga et al., 2010; with soluble neurexin motifs, or a knock-down of native Nlg-1 with Bhakar et al., 2012). The tadpole can be easily employed for studying morpholinos, the dendritic tree maturation profile for these associated network pathology via single-gene manipulation in the neurons was altered. Specifically, the tectal neurons failed to developing embryo. It has already been shown that manipulation establish new synapses with RGC axons, demonstrated higher of the fmr1 gene in the developing Xenopus embryo disrupts proper motility of dendritic filopodia, and had a simplified and immature Disease Models & Mechanisms somite formation (Huot et al., 2012), and it will be interesting to dendritic arbor morphology overall (Chen et al., 2010). Moreover, observe how altered FMR1 expression affects developing neural by comparing effects of these perturbations on the dynamics of circuits in humans. The Fragile-X-related genes are well described dendritic arbor development, the authors managed to convincingly in Xenopus (both X. tropicalis and X. laevis), with frogs having fewer reconstruct the pattern of protein interactions occurring during homologs within the gene family (fmrp and fxr1p) (Lim et al., 2005) filopodia stabilization and synapse formation. This work provides than do humans (FMRP, FXR1P and FXR2P), as well as fewer another example of how in situ time-lapse imaging in a live tadpole isoforms per gene (Huot et al., 2005). Even so, it has been brain (Bestman et al., 2012; Hossain et al., 2012) can be combined demonstrated that antibodies to human FMRP and FXRP proteins with genetic manipulations (Bestman et al., 2006; Bestman and are effective against Xenopus proteins (Blonden et al., 2005; Huot Cline, 2008; Liu and Haas, 2011) and electrophysiology (Pratt and et al., 2005), and that expression of human FMRP or FXRP rescues Aizenman, 2007) to dissect the functional role of target synaptic fmrp and fxr1p knockdown phenotypes in Xenopus, respectively proteins and their influence on network formation (Chen and Haas, (Huot et al., 2005; Gessert et al., 2010). 2011). Neuroligins and neurexins (synaptic adhesion proteins), together Finally, Xenopus modeling can help us to probe the link between with Shank and PSD-95 scaffolding proteins, make up another ASD and immune activation in the brain (Deverman and Patterson, functional group of proteins that share high degrees of homology 2009). Although increases in glia activation and levels of pro- between Xenopus and mammals (Ichtchenko et al., 1996; Chen et inflammatory cytokines have been observed in individuals with al., 2010; Gessert et al., 2011), have mutations in corresponding ASD, it is unclear whether these phenomena contribute to the genes linked to ASD in human patients (Jamain et al., 2003; Feng cause, or are a consequence of the disorder (Cohly and Panja, 2005). et al., 2006; Marín, 2012), and have been successfully studied in When tadpoles were chronically exposed to interleukins (IL-1β, IL- Xenopus. When neuroligin-1 function was disrupted in Xenopus 6) or tumor necrosis factor (TNFα), tectal neurons were

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Disease Models & Mechanisms Thus, as a vertebrate model, X. tropicalis offers all the advantages Chen,S. X.,Tari,P. K.,She,K.andHaas,K.(2010). Neurexin-neuroligin cell adhesion of the X. laevis model without the experimental complications that complexes contribute to synaptotropic dendritogenesis via growth stabilization are inherent to a polyploid genome. Hence, the X. tropicalis tadpole mechanisms in vivo. Neuron 67, 967-983. Chen, S. X., Cherry, A., Tari, P. K., Podgorski, K., Kwong, Y. K. and Haas, K. (2012). has been predicted to be the animal model of the future (Amaya The transcription factor MEF2 directs developmental visually driven functional and et al., 1998). structural metaplasticity. Cell 151, 41-55. In conclusion, the Xenopus tadpole has been and continues to Cheng,Y.,Cui,Y.,Chen,H. M.andXie,W. P.(2011). Thyroid disruption effects of environmental level perfluorooctane sulfonates (PFOS) in Xenopus laevis. be a powerful and prolific model to study, at many levels, both Ecotoxicology 20, 2069-2078. normal and abnormal neural development. The ability to study Chiu, S. L. and Cline, H. T. (2010). Insulin receptor signaling in the development of developing neural circuits at these different levels and with high neuronal structure and function. Neural Dev. 5, 7. resolution allows the tadpole to contribute greatly to the Chiu, S. L., Chen, C. M. and Cline, H. T. (2008). Insulin receptor signaling regulates synapse number, dendritic plasticity, and circuit function in vivo. Neuron 58, 708- identification of currently unknown targets to treat 719. neurodevelopmental disorders in humans. Cline, H. T. and Kelly, D. (2012). Xenopus as an experimental system for developmental neuroscience: introduction to a special issue. Dev. Neurobiol. 72, 463- ACKNOWLEDGEMENTS 464. We thank Carlos Aizenman, Harold Bergman, Jane Sullivan, Lulu Tsai and Ryan Cline, H. T., Witte, S. and Jones, K. W. (1996a). Low lead levels stunt neuronal growth Maloney for helpful comments and suggestions. in a reversible manner. Proc. Natl. Acad. Sci. USA 93, 9915-9920. COMPETING INTERESTS Cline, H. T., Wu, G. Y. and Malinow, R. (1996b). In vivo development of neuronal The authors declare that they do not have any competing or financial interests. structure and function. Cold Spring Harb. Symp. Quant. Biol. 61, 95-104. Coen, L., Le Blay, K., Rowe, I. and Demeneix, B. A. (2007). Caspase-9 regulates FUNDING apoptosis/proliferation balance during metamorphic brain remodeling in Xenopus. K.G.P. is supported by a grant from the National Institute of General Medical Proc. Natl. Acad. Sci. USA 104, 8502-8507. Sciences (P30 GM103398) from the National Institutes of Health. A.S.K. is funded Cohen-Cory, S. (2007). Imaging retinotectal synaptic connectivity. CSH Protoc 2007, by NIH R01 EY019578-03 grant (PI: Carlos Aizenman). pdb prot4782.

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