brain research 1641 (2016) 111–121

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Review Evolution of the CNS myelin gene regulatory program

n Huiliang Li , William D. Richardson

Wolfson Institute for Biomedical Research, University College London, Gower Street, London WC1E 6BT, UK article info abstract

Article history: Accepted 5 October 2015 Myelin is a specialized subcellular structure that evolved uniquely in vertebrates. A Available online 22 October 2015 myelinated axon conducts action potentials many times faster than an unmyelinated Keywords: axon of the same diameter; for the same conduction speed, the unmyelinated axon Myelin would need a much larger diameter and volume than its myelinated counterpart. Hence myelin speeds information transfer and saves space, allowing the evolution of a powerful yet portable brain. Myelination in the (CNS) is controlled by a Olig1 gene regulatory program that features a number of master transcriptional regulators Olig2 including Olig1, Olig2 and Myrf. Olig family genes evolved from a single ancestral gene in MyRF non-chordates. Olig2, which executes multiple functions with regard to oligodendrocyte Evolution identity and development in vertebrates, might have evolved functional versatility fi Phylogeny through post-translational modi cation, especially phosphorylation, as illustrated by its evolutionarily conserved serine/threonine phospho-acceptor sites and its accumula- tion of serine residues during more recent stages of vertebrate evolution. Olig1, derived from a duplicated copy of Olig2 in early bony fish, is involved in oligodendrocyte development and is critical to in bony vertebrates, but is lost in birds. The origin of Myrf orthologs might be the result of DNA integration between an invading phage or bacterium and an early protist, producing a fusion protein capable of self- cleavage and DNA binding. Myrf seems to have adopted new functions in early vertebrates – initiation of the CNS myelination program as well as the maintenance of mature oligodendrocyte identity and myelin structure – by developing new ways to interact with DNA motifs specific to myelin genes. This article is part of a Special Issue entitled SI: Myelin Evolution. & 2015 Elsevier B.V. All rights reserved.

Contents

1. Introduction ...... 112 2. Oligodendrocyte lineage genes 1 and 2 (Olig1 and Olig2)...... 112 2.1. Phylogeny of Olig genes ...... 112

n Corresponding author. E-mail address: [email protected] (H. Li). http://dx.doi.org/10.1016/j.brainres.2015.10.013 0006-8993/& 2015 Elsevier B.V. All rights reserved. 112 brain research 1641 (2016) 111–121

3. Olig2 regulates oligodendrocyte development ...... 114 4. Phosphorylation contributes to Olig2's functional versatility ...... 115 5. Olig1 contributes to myelination and remyelination ...... 116 6. ...... 116 7. Overview ...... 118 Acknowledgments ...... 118 References ...... 118

1. Introduction 2. Oligodendrocyte lineage genes 1 and 2 (Olig1 and Olig2) Evolution of a genetic program specifying myelination of axons was a momentous event in the history of verte- 2.1. Phylogeny of Olig genes brates. By allowing a dramatic increase in the speed and efficiency of action potential propagation, myelin over- Olig genes, derived from a common ancestor, form a sub- – – came limits to the size of both brain and body as well as group of the basic helix loop helix (bHLH) family, which Olig1 Olig2 Olig3 Olig4 Bhlhb4 Bhlhb5. Olig1/2 increasing greatly the potential processing power of the includes , , , , and genes were first identified in mouse almost simultaneously brain-opening the way to the evolution of tetrapods, for by three groups searching for genes that regulate oligoden- example. Several genes encoding myelin structural com- drocyte development. Subsequently, Olig3, Olig4, Bhlhb4 and ponents are already present in the lancelet (a cephalo- Bhlhb5 were also identified. chordate) and/or lampreys (Agnatha) (Gould et al., 2005; Li Evidence of a common ancestor for Olig genes can be and Richardson, 2008; Smith et al., 2013; Werner, 2013). found in arthropods, nematodes and platyhelminthes. In the fi Neither lampreys nor hag sh, the other extant member of nematode Caenorhabditis elegans, the Olig homolog Hlh-17 is the Agnatha, are myelinated in their central or peripheral expressed at all development stages in the cephalic sheath nervous systems (CNS or PNS) (Bullock et al., 1984). This cells (considered to be glial cells) that ensheath four of the suggests that subsequent to the Agnatha, myelin evolved dopaminergic neurons (McMiller and Johnson, 2005) and through a process of “pick and mix” whereby pre-existing plays a role in dopamine signaling (Felton and Johnson, genes encoding proteins with favorable physico-chemical 2011). The Drosophila Olig homolog, Oli, is not expressed in properties were co-opted into a single myelin gene reg- glial lineage cells but in certain ventral sub- ulatory program that subsequently diverged into two types; Oli is responsible for regulating larval and adult closely-related programs in the central and peripheral locomotion and its loss of function can be partially compen- Olig2 nervous systems (in and Schwann cells, sated for by over-expression of chick (Oyallon et al., 2012). In the hemichordate Saccoglossus kowalevskii (acorn respectively). How this gene recruitment process occurred worm), the Olig homolog is first expressed in a cluster of is a fascinating question, the answer to which is likely to dorsal cells of the prosome base right after gastrulation; later illuminate evolutionary processes in general. A driving on, its expression expands across the entire proboscis ecto- force for the evolution of gene regulatory networks is derm and to other dorsal cells along the dorsal midline (Lowe thought to be alterations in the properties of key tran- et al., 2006). As it is still early days for research on non- fi scription factors (TFs) and their sequence-speci c binding chordate Olig genes, we have yet to form a clear picture of to DNA (Cheatle Jarvela and Hinman, 2015). Attention has their expression patterns, functions and evolutionary con- previously focused on genomic re-arrangements involving nections with their vertebrate descendants. Interestingly, in the insertion and deletion of cis-regulatory DNA sequence the genome of the cephalochordate Branchiostoma floridae motifs but recent studies suggest that mutations in the (lancelet) (http://genome.jgi-psf.org/Brafl1/Brafl1.home.html), coding regions of the TFs themselves-leading to altered there are at least two copies of Olig, one the precursor of protein expression, protein–protein interactions and/or Olig1/2/3/4 and the other the precursor of Bhlhb4/5, hinting at post-translational modifications-could have figured in a local chromosomal duplication resulting in the initial the evolution of gene regulatory networks more than divergence of Olig genes (Louis et al., 2012). fi previously imagined (Lynch et al., 2011; Reece-Hoyes As jawless sh are the only vertebrates without compact myelin, they are important phylogenetic tools for studying et al., 2013; Voordeckers et al., 2015). In this review we Olig gene evolution. The recent sea lamprey (Petromyzon focus on evolution of the myelin gene regulatory network marinus) genome project has identified about 26,000 protein- in oligodendrocytes, which involves a considerable num- coding genes including a group of genes for myelin compo- ber of TFs such as Olig1/2, Sox10, Nkx2.2, Nkx6.2, Mash1, nents (Smith et al., 2013). However, we cannot find any Olig Myrf,Sip1,Tcf7l2andsoon(Li et al., 2009; Zuchero and homolog in the current annotated gene set and performing a Barres, 2013; Huang et al., 2013; Emery and Lu, 2015). TBLASTN search with elephant shark (Callorhinchus milii) Olig2 Among these, Olig1/2, Sox10 and Myrf are considered the protein sequence (GenBank: XM_007906736) against Petromy- master regulators; here we concentrate on Olig1/2 and zon 7.0 genome sequence (www.ensembl.org/Petromyzon_ Myrf, since the role of Sox10 is reviewed separately in this marinus/) finds no match. The missing lamprey Olig might issue (see chapter by Wegner). simply reflect still-incomplete genome sequencing but, given brain research 1641 (2016) 111–121 113

that adult liver tissue is the source of the sequenced DNA, it Olig is also possible that the locus in the lamprey somatic Olig Pre-chordates genome has been lost due to rearrangement at embryonic stages. It is known that during embryonic devel- Ancestor of Ancestor of opment the lamprey undergoes programmed genome rear- Invertebrates Olig1/2/3/4 Bhlhb4/5 Chordates rangement, which can lead to the removal of certain repetitive sequences, entire or individual Jawless fish genes, resulting in deletion of 20% of germline DNA from Cartilaginous fish somatic cells (Smith et al., 2009, 2012). Although the mechan- Bony fish ism and effect of such genomic rearrangement are not Olig4 Olig2 Olig3 Bhlhb4 Olig1 Bhlhb5 Amphibians understood, it seems to be a shared property of the jawless fish because hagfish (Myxine glutinosa) are believed to undergo Reptiles similar large-scale rearrangements, based on the fact that the Birds genome size of their somatic tissue is smaller than that of germline tissue (Kubota et al., 1997, 2001; Goto et al., 1998). Mammals We have searched another relatively new genome database derived from testis of Japanese lamprey (Lethenteron japoni- cum) using TBLASTN with Olig2 protein sequence of the Olig elephant shark against the draft genome assembly LetJap1.0 Pre-chordates (http://jlampreygenome.imcb.a-star.edu.sg/blast/) and have identified DNA fragments encoding partial orthologs of Bhlhb4 small-scale genome duplication and Bhlhb5 and two Olig2/3/4 orthologs – one full length and Olig1/2/3/4 Bhlhb4/5 one a small portion. These data imply that at least 4 copies of Invertebrates Olig family genes have been deleted in lamprey somatic cells. Chordates On the other hand, lamprey genome analysis suggests that two rounds of WGD & local genome duplication two-rounds of whole-genome duplication might have Olig2 fi Olig2L Bhlhb5 occurred in a common ancestor of jawless sh (Agnatha) Cartilaginous and gnathostomes, meaning that all Olig genes except for fish Olig1 would have already been in existence when lampreys Olig3 first appeared. We cannot be certain about this until the lamprey germline genome project has been completed. It Olig3L Bhlhb4 would also be of interest to sequence the hagfish genome. Cartilaginous fish (family Chondrichthyes) are the most further divergence ancient living species to possess myelin. We are able to Olig2 retrieve sequences of Olig2, Olig2-like, Olig3, Olig3-like and Olig1 Bhlhb5 Bony Bhlhb5 from the elephant shark genome database (http:// fish esharkgenome.imcb.a-star.edu.sg) and Olig2, Olig3 and Bhlhb4 Olig3 from the little skate (Leucoraja erinacea) database. It is inter- esting to note that the elephant shark has two Olig2 homologs Olig4 Bhlhb4 just 28 kb apart in one scaffold (scaffold 170; Venkatesh et al., 2014), which is similar to the way that Olig1 and Olig2 are Fig. 1 – Phylogeny of Olig genes. (A) Olig genes were evolved located in a synteny block in bony vertebrates, lending from a common invertebrate ancestor gene which was support to our previous hypothesis that Olig1 might have duplicated in an early chordate to provide the ancestral genes evolved from a local duplication at an ancestral Olig2 locus (Li of Olig1/2/3/4 and Bhlhb4/5. Although we are unsure how and Richardson, 2008). Given that the elephant shark has two many Olig paralogs there are in jawless fish (Agnatha), pre- copies of Olig3 but no Olig4, it is likely that one of the Olig3 Olig1 (Olig2-like), Olig2, Olig3, pre-Olig4 (Olig3-like), Bhlhb4 and copies has diverged to become Olig4 in higher species. There- Bhlhb5 all exist in cartilaginous fish. Olig4 is subsequently lost fore, since their divergence from the Chondrichthyes, jawed from all Amniota and Olig1 from Aves. (B) Hypothetical model vertebrates have possessed all of the Olig family genes with of Olig gene evolution. A local chromosomal duplication in the two exceptions: the loss of Olig4 in amniotes (including birds, early chordate resulted in the production of ancestors of Olig1/ reptiles and mammals) and the curious absence of Olig1 2/3/4 and Bhlhb4/5. After two rounds of whole genome from birds. duplication during early vertebrate evolution, accompanied by Taking all this into account we have updated our view of gene loss, the Olig1/2/3/4 gene ancestor gave rise to Olig2, Olig3 Olig gene evolution (Li and Richardson, 2008)(Fig. 1). We now and Olig4 while the Bhlhb4/5 gene ancestor gave rise to Bhlhb4 hypothesize the following sequence of events: (1) a single and Bhlhb5. Meanwhile, a local duplication around the Olig2 founder Olig gene was present in non-chordates, (2) a local locus produced a synteny block containing two Olig2 genes chromosomal duplication in an invertebrate chordate gener- and one of these subsequently underwent recombination ated two copies of Olig, (3) following two rounds of whole with another distantly related bHLH family gene to produce genome duplication and subsequent massive gene loss in a Olig1 (adapted and updated from Li and Richardson, 2008) common ancestor of jawless fish and jawed vertebrates 114 brain research 1641 (2016) 111–121

(Dehal and Boore, 2005), the two divergent Olig precursors pMN domain after MN production is complete, preserving the gave rise to Olig2/3/4 and Bhlhb4/5 respectively, (4) a local progenitor cell properties of pMN for later production of genome duplication at the Olig2 locus formed a synteny block oligodendrocyte precursors (OPs). Olig2 is down-regulated containing two copies of Olig2, (5) in early bony fish, one Olig2 rapidly in migrating post-mitotic MN precursors and is absent locus recombined with a distant relative (another bHLH gene), from mature MNs; forced expression of Olig2 in MN precur- undergoing domain insertion, rearrangement and/or reshuf- sors prevents their differentiation into functional MNs (Lee fling (Atchley and Fitch, 1997; Morgenstern and Atchley, 1999) et al., 2005). At the end of MN production, pMN progenitors to form Olig1, and (6) subsequently Olig4 was lost from switch fate and start to generate migratory OPs that express amniotes and Olig1 from birds. platelet-derived growth factor receptors (alpha subunit, Despite having a common origin, Olig genes display Pdgfra) and transcription factor Sox10. Olig2 is critical to OP diverse functions. Bhlhb4, which is expressed in the pancreas specification in the spinal cord and, in Olig2-KO mice, Pdgfra/ and brain (Bramblett et al., 2002), marks the diencephalic– Sox10-positive cells are completely absent (Lu et al., 2002; mesencephalic boundary and is also required for the matura- Takebayashi et al., 2002; Zhou and Anderson, 2002). Olig2 also tion of retinal rod bipolar cells (Bramblett et al., 2004). Bhlhb5, plays an important role in the later events of OP differentia- which can be detected in the CNS and in sensory organs such tion and oligodendrocyte maturation in the spinal cord, as the eye, hair follicle, cochlea of the developing inner ear because conditional knockout (cKO) of Olig2 in OPs (using and nasal epithelium (Brunelli et al., 2003), is critical for the CNP-Cre) represses OP differentiation and results in hypo- specification of bipolar and amacrine neurons in the retina myelination, while cKO of Olig2 in immature oligodendro- (Feng et al., 2006) and is necessary for the survival of a subset cytes (using PLP-CreER) facilitates oligodendrocyte maturation of inhibitory interneurons that control pruritic (itch) circuits and accelerates myelination (Mei et al., 2013). In addition, in the dorsal horn (Ross et al., 2010). Olig3 is expressed in both Olig2's function in oligodendrocyte development in the spinal dorsal and ventral precursor domains of the developing cord of vertebrates is evolutionarily conserved. For instance, spinal cord but is only required for the patterning of dorsal knockdown of Olig2 with morpholino antisense oligonucleo- spinal cord (Muller et al., 2005; Liu et al., 2014). Olig3 is also tides in zebrafish spinal cord prevents development of both expressed in the dorsal hindbrain where it plays an essential MNs and oligodendrocytes, suggesting that Olig2 is charged role in the development of brainstem nuclei and rhombic-lip- with the same task in teleost oligodendrocyte development derived neurons (Liu et al., 2008; Storm et al., 2009). The above as in mammals (Park et al., 2002). roles of Olig genes in the development of neural progenitors It is unclear whether a similar neuron-to-oligodendrocyte and neurons presumably evolved prior to their relatively new fate switch takes place in the ventral VZ of the brain. In the functions in controlling myelination. To understand how developing forebrain, Olig2 starts to be expressed at E9.5, myelination comes about, we need to start by looking into mainly in the thalamus (ventral diencephalon). Later on at the two major regulators of myelination – Olig1 and Olig2. E12.5, Olig2 is highly expressed in the medial ganglionic eminence (MGE) and the anterior entopeduncular area (AEP) and weakly expressed in the lateral ganglionic eminence 3. Olig2 regulates oligodendrocyte development (LGE) (Takebayashi et al., 2000; Woodruff et al., 2001). Olig2- positive precursor cells in the MGE and LGE can give rise to The majority (Z80%) of oligodendrocyte lineage cells in the GABAergic interneurons, which migrate throughout the perinatal spinal cord are descended from a ventrally-located developing forebrain and into the cerebral cortex, and choli- precursor domain of the embryonic ventricular zone, the nergic projection neurons, which remain in the ventral pMN domain (for “progenitors of motor neurons”) telencephalon (Furusho et al., 2006; Miyoshi et al., 2007; Ono (Richardson et al., 2006; Tripathi et al., 2011). Prior to neural et al., 2008). Loss of Olig2 results in a 40% decrease in the tube closure, Olig2 mRNA is expressed in the pMN domain as number of cholinergic neurons in the basal forebrain while well as the neighboring p3 and p2 domains that abut pMN on there is no change in the number of GABAergic interneurons its ventral and dorsal sides, respectively (Lu et al., 2000; (Furusho et al., 2006; Ono et al., 2008). In the forebrain, Takebayashi et al., 2000; Zhou et al., 2000). From about oligodendrocyte specification begins at E13.5 as indicated by embryonic day 9.5 (E9.5) in the mouse, Olig2 expression is the expression of OP marker Pdgfra at the ventral boundary of confined to pMN, possibly via a microRNA-mediated mRNA the MGE (Tekki-Kessaris et al., 2001). Unlike the spinal cord, silencing mechanism in the adjacent domains (Chen et al., Sox10/Pdgfra-expressing OPs are still present in the forebrain 2011). Induced by the gradient of a signaling molecule Shh of Olig2 null mice although in dramatically reduced numbers (sonic hedgehog) secreted from the notochord and floor plate, compared to wild type controls (Lu et al., 2002). Conditionally Olig2 defines pMN by repressing Irx3 (for p2 identity) and deleting Olig2 in embryonic neural stem cells (NSCs), using a Nkx2.2 (for p3 identity) (Marquardt and Pfaff, 2001; Novitch Cre transgene driven either by the human GFAP promoter et al., 2001); in Olig2 knockout (KO) mice the pMN domain is (expressed in all NSCs in mice) or by the Emx1 promoter lost and the p2 domain expands ventrally into what would (restricted to cortical NSCs), has no obvious effect on OP normally be pMN territory (Lu et al., 2002; Takebayashi et al., specification but can inhibit further differentiation of OPs into 2002; Zhou and Anderson, 2002). Olig2 knockout (KO) mice oligodendrocytes, leading to decreased myelin gene expres- therefore lack spinal motor neurons and the animals die at sion and hypo-myelination (Yue et al., 2006). Another study birth (Lu et al., 2002; Takebayashi et al., 2002; Zhou and found that when Olig2 is conditionally deleted in cortical OPs Anderson, 2002). However, the role of Olig2 extends beyond using an OP-specific NG2-Cre, they change fate and transform MN specification because it continues to be expressed in the into GFAPþ (Zhu et al., 2012). brain research 1641 (2016) 111–121 115

spinal white matter; phosphorylation of the triple serine 4. Phosphorylation contributes to Olig2's motif is required for neural progenitor proliferation according functional versatility to neurosphere culture but is not needed for OP specification and oligodendrocyte differentiation (Sun et al., 2011). The fi How can Olig2 play multiple roles in NSC speci cation and triple serine motif of Olig2 is also phosphorylated in - oligodendrocyte development? Unlike other neurogenic bHLH positive human , repressing the p53-mediated apop- TFs such as Mash1 and Ngn2 that are transiently expressed totic pathway (Sun et al., 2011; Meijer et al., 2014). In addition, and function at restricted times during development, Olig2 Olig2 with phosphorylated triple serine motif binds to a has sustained expression throughout development and into transcriptionally active chromatin domain, regulating target adulthood in all stages of oligodendrocyte lineage cells. (Meijer et al., 2014). We have provided fi Therefore, it is likely that post-translational modi cation is evidence that Olig2 controls the MN-OP fate switch by the key to Olig2's functional versatility. Post-translational reversible phosphorylation on S147, a conserved protein modification can greatly change the properties of the amino kinase A (PKA) target site; Olig2-S147 is phosphorylated acids and the proteins that contain them in response to during ventral spinal cord patterning and MN generation developmental need and evolutionary pressure, which might but de-phosphorylated at the onset of OL specification (Li be essential to the evolution of organismal complexity et al., 2011). In vivo evidence acquired from Olig2-S147A (Prabakaran et al., 2012). mutant mice indicates that phosphorylation at this site is One common post-translational modification is protein required for the ventral patterning and MN generation, while phosphorylation on serine, threonine and tyrosine resi- data from in ovo electroporation of chick embryos and P19 dues. We have found that TFs in general tend to possess embryonal carcinoma cells (which resemble NSCs) show that more serine residues relative to other classes of protein, de-phosphorylation at S147 favors OP fate specification. based on comparison of 542 TFs and 15,861 non-TFs in a Furthermore, S147 phosphorylation causes Olig2 to switch mouse UniProtKB/Swiss-Prot database (Jiang and Li, unpub- its preferred binding partner from itself (or Olig1) to Ngn2, lished). Olig2 in particular is a serine-rich protein contain- and this regulated exchange of co-factors is required for and ing50serineresiduesaswellas14threoninesand triggers the MN-OP fate switch (Li et al., 2011). In addition, S30 3 tyrosines out of 323 amino acids, showing great potential of Olig2 has been found to be a target of protein kinase B (e.g. for phosphorylation. We chose GPS3.0 (Group-based Pre- Akt) and S30 phosphorylation triggers translocation of Olig2 diction System, version 3, http://gps.biocuckoo.org), a from the nucleus to the cytoplasm (Setoguchi and Kondo, newly updated bioinformatics software program to predict 2004). In vitro NSC culture shows that Akt, possibly by potential protein phosphorylation sites given that the phosphorylating S30, facilitates Olig2's exclusion from the previous version GPS2.0 performed large-scale prediction nucleus and stimulates NSCs to differentiate into astrocytes on more than 13,000 mammalian protein phosphorylation in the presence of ciliary neuronotrophic factor (CNTF) sites with remarkable accuracy (Xue et al., 2008). By setting (Setoguchi and Kondo, 2004). a high cut-off threshold (with a 2% false positive rate for Seen from an evolutionary perspective, of all these known serine/threonine kinases and a 4% false positive rate for Olig2 phosphorylation sites, the triple serine motif, S/T box, tyrosinekinases),wecalledupallofthemurineOlig2 S30 and S147, only S147 is conserved among vertebrates and serine/threonine/tyrosine sites that can be phosphorylated their invertebrate forerunners, which is not surprising con- by at least one protein kinase. This computer prediction is sidering the fact that Olig2 S147 phosphorylation is indis- backed up by our experimental data, which indicate that pensible to motor neuron generation in mouse spinal cord. By Olig2 over-expressed in COS-7 cells can be phosphorylated analogy, Oli might also be required for motor neuron genera- at multiple sites and that the phosphorylation can be tion in the ventral nerve cord of Drosophila and phosphoryla- reversed by alkaline phosphatase treatment (Li et al., tion might also be involved (Oyallon et al., 2012). However, 2011). Mass spectrometry has identified phosphorylation flies have no oligodendrocytes or compact myelin and pre- on serine 10 (S10), S13, S14, S81 and S263 and threonine 43 sumably do not need the dephosphorylated form of Olig2- (T43) of mouse Olig2 in neural stem cells or COS-7 cells (Sun S147 to direct NSCs to a glial fate. Nonetheless, it would be et al., 2011) although, due to limitations to the sensitivity of interesting to find out if Oli is phosphorylated/dephosphory- mass spectrometry, it is impossible to identify all phos- lated during Drosophila nerve cord development. The triple phorylation sites on Olig2 in a few experiments. serine motif of Olig2 is conserved in bony vertebrates but not Murine Olig2 contains a cluster of 12 contiguous serine/ in elephant sharks, suggesting that cartilaginous fish do not threonine residues from S77 to S88, i.e. an S/T box, which is a need extensive OP proliferation to generate a large number of substrate of casein kinase II (CK2) (Huillard et al., 2010). Data oligodendrocytes as in mammals. In contrast, the S/T box and obtained from neurosphere culture suggest that S/T box S30 are only present in mammals, although phosphorylation phosphorylation by CK2 serves to regulate neural progenitor at those two sites has yet to be confirmed in vivo, leaving us proliferation and oligodendrocyte production (Huillard et al., wondering if their phosphorylation could lead to some Olig2 2010). Apart from the S/T box, mouse Olig2 has a triple serine activities that are specific to mammals. Close examination of motif comprising S10, S13 and S14, and the phosphorylation mouse Olig2 protein reveals that S/T142 and T151 of the bHLH state of this triple serine motif changes during development, domain are conserved in ancestral Olig genes and S320 at the affecting the proliferation of neural progenitors. The triple Olig2 C-terminus is conserved in jawed vertebrates. These serine motif is phosphorylated during OP proliferation in conserved phosphorylation sites might be pointers to key embryonic spinal cord but dephosphorylated in postnatal mechanisms of CNS development and would be worth 116 brain research 1641 (2016) 111–121

exploring. In addition, the number of serine residues in Olig2 Traces of Olig1 can be found in elephant sharks. However, orthologs increases gradually along the evolutionary path, all avian genome databases (including chicken, turkey, bud- from 28 in elephant shark to 50 in mouse, raising the gerigar, medium ground finch and zebra finch genomes) lack possibility that by increasing phosphorylation sites, Olig2 any Olig1 homolog. Moreover, no bird Olig1 gene sequences might have gained functional versatility to provide a genetic can be found in NCBI Genbank, contrasting with 21 listed bird basis for species to adapt under selective pressure. Olig2 gene sequences. Taken together, we infer that Aves do not have Olig1. Olig1 is likely to be one of many genes lost in birds. In fact, there are genomic clues pointing to a massive 5. Olig1 contributes to myelination and gene loss in birds: the genome size of Aves is smaller than remyelination that of other tetrapod classes (Szarski, 1976; Tiersch and Wachtel, 1991); the number of protein coding genes (paralogs) In the spinal cord, Olig1 mRNA is first detected in the p3 and in a Gallus gene family is normally lower than that in its pMN ventral progenitor domains and becomes confined to mammalian or reptile counterpart (Hughes and Friedman, the pMN domain by E10.5 (Chen et al., 2011). However, Olig1 2008; Zhang et al., 2014); and the gene number of avian protein can only be detected after E18.5 (Fu et al., 2009). Loss genomes is 70% of that of the (Zhang of Olig1 does not impair MN development in the spinal cord, et al., 2014). This large-scale loss of DNA segments might but leads to an about 30% increase in the number of adult have happened after birds split from other reptiles around cortical interneurons (Silbereis et al., 2014). Evidence from 100 million years ago and, as a result, birds might have taken three independently generated Olig1 KO mouse lines (Lu a minimalist approach to adaptation by undergoing func- et al., 2002; Paes de Faria et al., 2014) suggests that Olig1 tional compensation and innovation in paralogous gene has no effect on oligodendrocyte development in the spinal copies (Zhang et al., 2014). Given that Olig1 is involved in cord, apart from causing a slight delay in OP differentiation. oligodendrocyte function in teleosts and mammals, it would fi In stark contrast to this are data from another Olig1 KO line be intriguing to nd out how birds tackle myelination and (Xin et al., 2005), in which myelination in the spinal cord is remyelination without a contribution from Olig1. completely blocked and the animals die around the third postnatal week. The Olig1 KO of Xin et al. (2005) was derived 6. Myelin regulatory factor from the Olig1 KO of Lu et al. (2002) by removing the Pgk-neo cassette from the Olig1 null allele in the latter. Xin et al. (2005) Myelin regulatory factor (Myrf or Mrf) is also known as GM98 invoked the presence of the Pgk-neo cassette in the Lu et al. (gene mode 98) in mouse and C11ORF9 (chromosome 11 open (2002) line to explain the difference between the two KO lines, reading frame 9) in human. Unlike other master regulators of speculating that the Pgk promoter might up-regulate the oligodendrocyte development such as Olig1/2 and Sox10, Olig2 adjacent gene, thereby compensating for the loss of which are expressed at all stages of the oligodendrocyte Olig1. However, a later study (Samanta et al., 2007) demon- lineage, Myrf is exclusively expressed in differentiated oligo- strated that Olig2 gene expression is not altered in the Olig1 dendrocytes in the mouse CNS. Myrf does not start to be KO of Lu et al. (2002). In respect of oligodendrocyte develop- expressed until OP differentiation has been initiated. Myrf is ment in the brain, our own Olig1 KO models (Paes de Faria critical to oligodendrocyte differentiation and is necessary to et al., 2014) exhibit no abnormal phenotypes, while the Olig1 drive the CNS myelin transcriptional program during devel- KO of Lu et al. (2002) displays a subtle delay in oligodendro- opment (Emery et al., 2009). Moreover, Myrf is necessary for fi cyte maturation in the spinal cord and impaired OP speci ca- oligodendrocyte generation in the adult CNS and conditional tion and oligodendrocyte differentiation in the corpus deletion of Myrf in adult OPs can prevent new oligodendro- callosum (Dai et al., 2015). Such differences in the phenotypes cytes from being generated, leading to defects in motor of Olig1 null lines might be attributed to different genetic learning (McKenzie et al., 2014). Myrf is also required for backgrounds, different nutritional and/or environmental con- maintaining the identity of mature oligodendrocytes and ditions or unintended genetic alterations at the Olig1/2 locus myelin structure; cKO of Myrf in mature oligodendrocytes during removal of the Pgk-neo cassette. Notwithstanding (e.g. using Plp-CreER or Sox10-CreER) causes dramatic down- these uncertainties, Olig1 is believed to play a role in regulation of myelin protein expression and the breakdown myelination in some contexts. For instance, Olig1 can physi- of myelin sheaths (Koenning et al., 2012; McKenzie et al., cally interact with Sox10 to promote myelin basic protein (Mbp) 2014). In addition, Myrf can interact physically with Sox10 to transcription via evolutionarily conserved DNA motifs in the control myelin gene expression directly (Hornig et al., 2013). Mbp promoter (Li et al., 2007). Moreover, Olig1 is critical to Current data on Myrf in non-mammals is scanty, but we have remyelination following lysolecithin- or cuprizone-induced detected Myrf expression in oligodendrocytes of adult zebra- demyelination (Arnett et al., 2004) and is necessary for fish (Li and Richardson, data not shown). transplanted neural progenitor cells to differentiate and Myrf contains a DNA-binding domain, an intramolecular remyelinate in virus-induced demyelination (Whitman chaperone auto-processing (ICA) domain and a transmembrane et al., 2012). In addition, at two weeks postnatal, Olig1 domain (Bujalka et al., 2013; Li et al., 2013)(Fig. 2A) and belongs translocates from the cell nucleus to the cytoplasm, but to a class of membrane-bound TFs that are translated as returns to the nucleus during early remyelination, suggesting transmembrane proteins but subsequently undergo proteolytic that the subcellular location of Olig1 might have an impact on processing, releasing a soluble TF that translocates to the myelination and remyelination (Arnett et al., 2004). nucleus to participate in transcriptional regulation. brain research 1641 (2016) 111–121 117

Olig2 transcriptional regulation (Bujalka et al., 2013; Li et al., 2013). TSM S/T bHLH Similarly, the MrfA of Dictyostelium is first affixed to the ER membrane in a trimer fashion via the C-terminal transmem- S10 S30 S85 S147 323 S13 Akt S87 PKA brane and ICA domains, then undergoes constitutive self- S14 CKII cleavage, releasing the N-terminal fragment, which remains in the cytoplasm of growing cells but accumulates in the Fig. 2 – Phosphorylation sites of of murine Olig2 (GenBank: nuclei of anterior-like cells and pre-stalk cells (Senoo et al., NP_058663). Functionally confirmed phosphorylation 2012). regions and sites in Olig2 are shown. Akt, CKII and PKA are Apart from the activities of DNA binding and ICA domains, confirmed kinases that can phosphorylate Olig2 at specific the C-terminus of Myrf is thought to be involved in cellular sites. bHLH: basic helix–loop–helix domain; TSM: triple secretion, which is supported by the observation that all serine motif; S/T: serine and threonine rich domain. known Myrf orthologs are initially located in the ER mem- brane and by the finding that pqn-47 of nematodes is required for regulated secretion of cuticle components or The DNA binding domain of Myrf is homologous to the hormones during larval molting cycles (Russel et al., 2011). Ndt80 (non-dityrosine 80) of yeast. This Ndt80 DNA binding Taking into account the fact that, during myelination, oligo- domain is conserved with a similar crystal structure among dendrocytes transport huge amounts of myelin component a range of eukaryotic organisms from protists and fungi to proteins, cholesterol and membrane lipids through the secre- mammals (Montano et al., 2002; Xu et al., 1995). Yeast tory pathway (Anitei and Pfeiffer, 2006), it is possible that Ndt80 can bind to a so-called midsporulation element Myrf and pqn-47 might play a similar secretory role despite (MSE) with the consensus sequence CRCAA [where R¼A being phylogenetically distant. In vitro assays with oligoden- or G; (Chu et al., 1998)]. However, it is not possible for a drocyte cell lines show that Myrf's N-terminal transcription fi chimeric protein comprising the human Myrf DNA binding factor fragment is not suf cient to promote oligodendrocyte domain to repair defects in yeast caused by Ndt80 inactiva- maturation compared to full-length Myrf, suggesting that the tion (Fingerman et al., 2004). A different view argues that C-terminus of Myrf is also important to oligodendrocyte Myrf and its orthologs are wrongly annotated as homo- maturation, possibly by controlling the ER secretory pathway logous to yeast Ndt80 because they have no genuine (Li et al., 2013). Interestingly, Myrf can bind to Sox10, not nuclear localization signal (NLS), illustrated by the fact that through its transcriptional domain but through the mem- brane bound C-terminal domain (Hornig et al., 2013), its ortholog in C. elegans, pqn-47 (prion-like glutamine/ although it is still unclear whether this phenomenon is asparagine rich protein 47), is not localized in the nucleus peculiar to Myrf and Sox10 or whether it underlies a common at any developmental stage (Russel et al., 2011). Never- mechanism for the secretion of myelin related proteins. theless, a DNA motif (CTGGYAC, Y¼C or T) that is different Given the fact that yeast Ndt80 has no ICA domain and from the MSE sequence has been identified and proven to that those ICA domain-containing proteins in phages and be able to bind to murine Myrf, leading to activation of prokaryotes such as Bdellovibrio bacteriovorus have no Ndt80 myelin gene expression (Bujalka et al., 2013). In addition, domain, while protists including choanoflagellates and Dic- MrfA, the Myrf ortholog in Dictyostelium (slime mold), can tyostelium have Myrf orthologs containing both an ICA bind to a 39-mer CA-rich motif (Senoo et al., 2012). domain and an Ndt80-like DNA binding domain, a likely Unlike other membrane-bound TFs that require the ubi- scenario for the emergence of Myrf's ancestor during evolu- quitin/proteasome-dependent system or regulated intra- tion is that an invasion event of a phage or bacterium into an membrane proteolysis for cleavage (Hoppe et al., 2000; early protist resulted in the phage/bacterial DNA integrating Wang et al., 1994), Myrf can self-process its proteolytic into the protist genome, producing a fusion protein com- activation through the ICA domain. To date, only two verte- posed of an ICA domain and a DNA binding domain (Roberts, brate membrane-bound TFs-Myrf and its paralog Myrfl (mye- 2013). In addition, most Metazoa possess two Myrf homologs, – lin regulatory factor-like) have proven to have self-cleavage hinting at early genome duplication in eukaryotes. Our Myrf ability. The ICA domain of Myrf harks back to endosialidases, phylogenetic tree illustrates that two Myrf homologs evolved the tailspike proteins of bacteriophages, which are essential simultaneously in invertebrates and that subsequently, in for bacteriophages to infect bacteria (Stummeyer et al., 2005). vertebrates, the two Myrf homologs diverged to form two The ICA domain causes the tailspike protein to form trimers, separate gene branches, Myrf and Myrfl (Fig. 2B). According to triggering auto-proteolytic cleavage and ending in the release a mouse CNS RNA-seq database, Myrfl is not expressed in the of the mature protein (Schulz et al., 2010). During phage CNS (Zhang et al., 2014). It is curious that no extra Myrf fi infection of bacteria, the tailspike trimers rst attach to the homologs have appeared in vertebrates-considering the two- surface of the host, and then undergo ICA domain-mediated round genome duplication. However, they might first have self-cleavage, producing an active endosialidase to break been generated then deleted in the following massive gene down the host's cell wall (Schwarzer et al., 2007). In the loss. As with Olig genes, one Myrf homolog can be found in endoplasmic reticulum (ER) of mouse oligodendrocytes, the Japanese lamprey testis genome, but no Myrf homolog membrane-bound Myrf also forms trimers via a leucine can be retrieved at the moment in the database of sea zipper in the ICA domain; after self-cleavage, the detached lamprey genome derived from somatic tissue. Myrf might N-terminal trimer of Myrf containing the DNA-binding have gained the function of regulating myelin gene expres- domain translocates from ER into the nucleus to execute sion in early vertebrates; it would be worth looking into how 118 brain research 1641 (2016) 111–121

A DBD/Ndt80 TMD 393-540 768-786 7. Overview

ICA Myrf SBD 1138 The invention of myelin was a major event that changed the 587-647 960-1049 course of evolution. Based on developmental clues, we had B previously hypothesized that myelin forming cells evolved from so-called “motor glia” that had the same developmental Vertebrate origin as motor neurons (Richardson et al., 1997; Li and Myrf orthologs Richardson, 2008). A new gene regulatory network was required to shape the motor glia into today's oligodendro- cytes; in this regard, cis-regulatory elements were integrated into a group of genes encoding myelin-forming proteins, Vertebrate while transcription factors simultaneously adopted new Myrfl orthologs functions by undergoing post-transcriptional modification to permit interactions with novel transcriptional co-factors and cis-regulatory elements. This would have led to the expres- sion of myelin genes in a spatially and temporally controlled fi fi Invertebrate manner. As jawless sh and cartilaginous sh occupy key Myrf orthologs nodes in myelin evolution, future research on myelin evolu- tion would benefit from studying the expression patterns and molecular functions of master myelin regulators Olig1/2, Emergence of Sox10 and Myrf in the lamprey and hagfish as well as in ICA/TMD cartilaginous fish such as dogfish, elephant shark or Emergence of Ntd80 little skate.

Fig. 3 – Phylogeny of Myrf. (A) Structure of murine Myrf protein (GenBank: XP_006526992.1). DBD: DNA binding domain; ICA, intramolecular chaperone auto-processing; Acknowledgments TMD: transmembrane domain; SBD, Sox10 binding domain. (B) Myrf phylogenetic tree. Myrf homolog/ortholog Work in the authors' laboratories was supported by the UK sequences were downloaded from NCBI (www.ncbi.nlm.nih. Biotechnology and Biological Sciences Research Council (BB/ gov) and Ensembl (www.ensembl.org/Petromyzon_marinus/ J006602/1 and BB/L003236/1), the Medical Research Council ). MEGA6/ClustalW was used to draw the rooted (G0800575), the Wellcome Trust (WT100269MA) and the phylogenetic tree (http://www.megasoftware.net/mega. European Research Council (ERC “Ideas” Program 293544). php). Myrf orthologs might result from an invasion event of a phage or bacterium into an early protist resulting in the references phage/bacterial DNA (encoding ICA domain) integrating into the protist genome (encoding DBD/Ndt80p domain). In invertebrates, two Myrf homologs evolved simultaneously; Anitei, M., Pfeiffer, S.E., 2006. Myelin biogenesis: sorting out in vertebrates, these two Myrf homologs diverged to form protein trafficking. Curr. Biol. 16, R418–R421. Myrf and Myrfl branches. Hs: Homo sapiens;Mm:Mus Arnett,H.A.,Fancy,S.P.,Alberta,J.A.,Zhao,C.,Plant,S.R.,Kaing,S., musculus; Rn: Rattus norvegicus (brown rat); Gg: Gallus gallus Raine, C.S., Rowitch, D.H., Franklin, R.J., Stiles, C.D., 2004. bHLH transcription factor Olig1 is required to repair demyelinated (chicken); Ac: Anolis carolinensis (Carolina anole, a lizard); Xt: lesions in the CNS. Science 306, 2111–2115. Xenopus tropicalis (clawed frog); Dr: Danio rerio (zebrafish); Atchley, W.R., Fitch, W.M., 1997. A natural classification of the Cm: Callorhinchus milii (elephant shark or chimera); Ci: Ciona basic helix–loop–helix class of transcription factors. Proc. intestinalis (sea squirt, an ascidian) Cf: Camponotus floridanus Natl. Acad. Sci. USA 94, 5172–5176. (Florida carpenter ant); Sc: Saccharomyces cerevisiae (brewers' Bramblett, D.E., Copeland, N.G., Jenkins, N.A., Tsai, M.J., 2002. yeast); Ce: Caenorhabditis elegans (a nematode worm); Df: BHLHB4 is a bHLH transcriptional regulator in pancreas and Dictyostelium fasciculatum (slime mold); Mb: Monosiga brain that marks the dimesencephalic boundary. Genomics 79, 402–412. brevicollis (a choanoflagellate, close relative of metazoans); Bramblett, D.E., Pennesi, M.E., Wu, S.M., Tsai, M.J., 2004. The Bb: Bdellovibrio bacteriovorus (a motile gram-negative transcription factor Bhlhb4 is required for rod bipolar cell bacterium that invades and parasitizes other bacteria); maturation. Neuron 43, 779–793. CWSAFP: cell wall surface anchor family protein; DTFP: Brunelli, S., Innocenzi, A., Cossu, G., 2003. Bhlhb5 is expressed in distal tail fiber protein. the CNS and sensory organs during mouse embryonic devel- opment. Gene Expr. Patterns 3, 755–759. Bujalka, H., Koenning, M., Jackson, S., Perreau, V.M., Pope, B., Hay, Myrf functions in fish oligodendrocytes and whether inverte- C.M., Mitew, S., Hill, A.F., Lu, Q.R., Wegner, M., Srinivasan, R., brate or mammalian Myrfl orthologs (full-length, N-terminal, Svaren, J., Willingham, M., Barres, B.A., Emery, B., 2013. MYRF is a membrane-associated transcription factor that autopro- or C-terminal) can rescue a null mutation of Myrf in fish teolytically cleaves to directly activate myelin genes. PLoS (Fig. 3). Biol. 11, e1001625. brain research 1641 (2016) 111–121 119

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