Introduction

The RNA-binding exon-junction complex (EJC) is an eukaryotic binding platform for many nuclear and cytoplasmic factors involved in mRNA processing like splicing, translation, localization, and non-sense decay (Tange et al. 2004; Palacios et al. 2004; Giorgi and Moore 2007; Le Hir and Seraphin 2008). The EJC consists of four core subunits; three of which (Magoh, RBM8A, and elF4A3) constitute the pre-EJC. The EJC components ensure tight control on spliced mRNA decisions by exerting general as well as specific binding properties (Roignant and Treisman 2010). Besides its association with the EJC, Magoh orthologous proteins play an ancient function in the control of mitotic spindle orientation, which is essential for regulating the balance between symmetric and asymmetric cell division (van der Weele et al. 2007). Localization of mRNAs by means of EJC-mediated cytoskeleton rearrangements generates cell asymmetry for germ cell specification and for the establishment of the antero-posterior (AP) and dorso-ventral (DV) axes (Micklem et al. 1997; Newmark et al. 1997; Li et al. 2000; Hachet and Ephrussi 2001; Wiens et al. 2006). The role of Magoh in the initial phase of axial patterning has been originally recognized in Drosophila, where the orthologous protein mago nashi controls the transport of oskar mRNA at the posterior pole, and the migration of the oocyte nucleus at the dorsal pole (Boswell et al. 1991; Newmark and Boswell 1994; Micklem et al. 1997; Hatchet and Ephrussi 2001). In Drosophila, mago nashi is also involved in photoreceptor differentiation by controlling the splicing patterns of MAPK mRNA (Roignant and Treisman 2010).

Functional studies of Magoh orthologs in vertebrates reveal a divergent scenario when compared with protostomes. In vertebrates, this EJC core factor is not required in axial patterning like in insects, but it has expanded its role in cell differentiation and renewal in the central nervous system, where Magoh is expressed in brain tissues during development (Silver et al. 2010; McMahon et al. 2016; Pilaz et al. 2016). In Xenopus, Magoh expression is restricted to neural tissues at neurula stage, and morpholino injection-mediated knockdown results in a significant reduction of neural crest cell (NCC)-derived pigment cells (Kenwrick et al. 2004; Haremaki et al. 2010). In human, Magoh is found within a 55-gene deletion on chromosome 1p32.3 that is associated with mental retardation and abnormalities in brain size (Brunetti-Pierri et al. 2008; Mulatinho et al. 2008; Silver et al. 2010; Viswanathan et al. 2018). Early lethality (E9.5) of Magoh knockout mice hinders the analysis of postnatal functions. However, the heterozygote phenotype shows microcephaly and melanocyte reduction due to defective mitosis of neural stem cells (NSC) and NCCs, respectively (Silver et al. 2010, 2013; Mao et al., 2016). In mammals, Magoh protein interacts with nuclear actin, and controls levels of cytoplasmic Lissencephaly 1 (Lis1), a microtubule-associated protein that is essential for mitotic spindle integrity. By modulating microtubule polarization, Magoh regulates the balance of asymmetric, proliferative, and neurogenic NSC divisions (Silver et al. 2010). Recently, Magoh has been associated with skipping of recursive splicing exons in the mouse brain, a mechanism thought to be present in all Deuterostomes (Blazquez et al. 2018). The creation of new splice forms may change protein structure and favor the fixation of new functions. It was suggested that the elaboration of additional transcripts was central to changes in size and organization during brain evolution (Kaas 2006; Holland and Short 2008; Bae et al. 2014).

Living members of the phylum Chordata belong to three groups, Cephalochordata (e.g., amphioxus), Tunicata (e.g., ascidians), and Craniata (e.g., vertebrates), which share, at least in some phases of their life cycle, the body plan organization and key anatomical features among which a dorsal neural tube and a notochord (Annona et al. 2015). The paucity of information on the evolutionary nature of EJC functions in chordates can be addressed in simple prototypical ascidian and cephalochordate embryos. Non-vertebrate chordates are crucially important for the comparative molecular approach as they provide evidence of synapomorphic features of the phylum. Here, we present an analysis of Magoh in the cephalochordate Branchiostoma lanceolatum and in the ascidian Ciona robusta.

Materials and methods

Synteny conservation analysis in chordates

The occurrence of synteny conservation was analyzed using the Genomicus (v97.01) (Louis et al. 2013), in addition to manual searches in cephalochordate (B. lanceolatum, Marletaz et al. 2018) and vase tunicate (C. robusta, JGI v2.0) genomes. In Fig. 1, we show conserved genes on both sides of the Magoh locus, and lineage-specific insertions of genes were discarded. In Supplementary Fig. S1, synteny conservation in tunicates was analyzed using data from the Aniseed web server (https://www.aniseed.cnrs.fr/aniseed/) except for C. robusta (Ensembl Genome Server, https://www.ensembl.org/). In Supplementary Table S1, the search for Magoh genes in selected craniate species was performed at the Ensembl Genome Server.

Fig. 1
figure 1

Schematic synteny representation of chordate Magoh genes. a The absence of syntenic conservation traces in non-vertebrate chordates (cephalochordates and tunicates) whereas high degree of conservation is highlighted in vertebrates. b A second functional ortholog is exclusively present in mammals, named MagohB. c A human Magoh pseudogene, named MAGOH2P, lies within an intron of the TMEM220 gene on chromosome 17. Chr, chromosome. d A second human Magoh pseudogene, MAGOH3P, is present on chromosome 14. Magoh is in blue, syntenic genes are in orange, unknown genes are in gray

Full size image

Animal sampling

Adult specimens of C. robusta were collected in the Fusaro Lake (Italy) by hand picking at low depth and transported in seawater tanks to the facilities of Stazione Zoologica Anton Dohrn Naples (SZN). Animals got acclimatized at ~20 °C for 2–3 days in open system tank setups and fed every day with a solution of marine microalgae concentrates (Shellfish Diet 1800™ Instant Algae®: 0.5 ml in 1 l of sea water). Subsequently, they were exposed to continuous lighting for few more days in order to accumulate mature gametes and to prevent gamete spawning. Adult specimens of B. lanceolatum were collected in the Gulf of Naples (Italy) by ship trawling at 10–15 m depth, and then kept in a seawater tank during transport up to the arrival at SZN laboratory facilities. Animals were quarantined upon observation and antibiotics treatment to prevent bacterial contamination for 20 days, and then introduced in tanks containing sandy substrate from the original sampling sites at ~17 °C with a 10/14 h day/night light cycle. Adult animals were fed every day with 200 ml of fresh microalgae mixture (Dunaliella tertiolecta, Isochrysis galbana, and Tetraselmis suecica).

Gamete sampling and collection of developmental stages

Ripe specimens of C. robusta were dissected at the base of the atrial siphon with a sterile blade in order to expose gonoducts. To avoid self-fertilization, only one kind of gamete was collected from each specimen. Sperm samples were collected from the spermiduct by using sterile glass Pasteur pipettes and pooled into pre-chilled vials in ice. Eggs were collected with sterile glass Pasteur pipettes and transferred in a 9-cm Petri dish filled with 0.22 μM Millipore filtered natural sea-water (MFSW) at 18 °C. Egg samples from different individuals were pooled in 9-cm Petri dishes allowing to expand the chorion and the follicle cells, which make the eggs floating and improve fertilization. Fertilization assays were carried out in 9-cm Petri dishes filled with 10 ml 0.22 μM MFSW at 18 °C, and different developmental stages of C. robusta were fixed overnight in 4% paraformaldehyde (PFA) in 3-(N-morpholino) propanesulfonic acid (MOPS) and further dehydrated to 70% ethanol (EtOH). Single ripe specimens of B. lanceolatum were induced to spawn by heat shock, as previously described (Fuentes et al. 2007). Eggs and sperms were collected with sterile plastic Pasteur pipettes and mixed in a 9-cm Petri dish filled with 0.22 μM MFSW at 19 °C to obtain fertilization (final sperm dilution 1:1000). Embryos were reared in MFSW at 19 °C in incubator. Desired developmental stages were fixed overnight at 4 °C in 4% PFA in a buffer containing 0.1 M MOPS, 0.5 M sodium chloride (NaCl), 2 mM magnesium sulfate (MgSO4), and 1 mM ethylene glycol tetraacetic acid (EGTA) pH 7.4 and further dehydrated to 70% EtOH. For both species, fixed developmental stages were then stored at − 20 °C for subsequent in situ hybridization or immunohistochemistry analyses.

Digoxigenin-labeled riboprobe synthesis

For C. robusta, two oligonucleotides (Forward: 5′-GGAGTTTGAATTTCGACCAG-3′; Reverse: 5′-CTCTCATTGGGCTGCATTTC-3′) were designed to amplify a 358-bp Magoh fragment from ovary cDNA, upon the identification of the gene sequence in the C. robusta EST database (Satou et al. 2002). For B. lanceolatum two oligonucleotides (Forward: 5′-ATGGCTTCCAACGATTTCTATC-3′; Reverse: 5′-CTAGATGGGTTTAATCTTGAAG-3′) were designed to amplify a 441 bp Magoh fragment. For both species, PCR amplifications were performed in 50 μL containing 5 μL × 10 Roche PCR reaction buffer + Mg, 5 μL 10 mM Roche PCR Grade Nucleotide Mix (dNTPs), 5 μL each primer (10 mM), 5 μL × 10 BioLabs purified bovine serum albumine (BSA), 0.5 μL Roche Taq DNA polymerase, and 500 ng cDNA. PCR consisted of an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 60 s, and extension at 72 °C for 60 s, followed by a final extension at 72 °C for 10 min. PCR products were cloned into Dual TOPO-TA cloning Kit. For both species, digoxigenin (DIG)-labeled antisense and sense riboprobes for Magoh were synthesized using a template linearized with XhoI and transcribed using SP6 RNA polymerase for the antisense probe, or linearized with KpnI and transcribed using T7 RNA polymerase for the sense probe. Quantification of riboprobes was performed by DOT-BLOT analysis.

In situ hybridization and immunohistochemistry

In situ hybridization (ISH) experiments on different developmental stages of C. robusta were hand-performed as previously described (Christiaen et al. 2009). Pre-larval stages were manually dechorionated. ISH experiments were all performed in triplicate with consistent results. The sense riboprobe was tested in order to confirm signal specificity. For immunohistochemistry (IHC) analyses on developmental stages of C. robusta, two synthetic peptides, NH2-KIGSLVDVTDCKDSDG-COOH (amino acids (aa) 111–126) and NH2-AVLEELKRVIDDSEIMK-COOH (aa 63–79), corresponding to two predicted immunogenic Magoh epitopes, were used to immunize rabbits (PRIMM, Italy). Two specific anti-sera, C67 and D67, have been obtained and used for immunohistochemical studies as following: after rehydration in phosphate-buffered saline (PBS) 1X, pre-larval stages were manually dechorionated. Following, permeabilization was carried out in ice-cold acetone for 10 min at − 20 °C. Then, samples were incubated for 1 h in IB solution (PBS 1X, 2% BSA, 1% dimethyl sulfoxide (DMSO), 0.5% TritonX-100) with 10% sheep serum (SS), followed by overnight incubation in IB solution with 1% SS with primary antibody diluted 1:1000 at 4 °C. Hence, samples were washed (4 × 15 min, 1 × 1 h) in IB solution at room temperature, and then incubated overnight at 4 °C with 1:200 secondary anti-rabbit antibody HRP-conjugated in IB solution with 1% SS. Unbound antibody was washed away with IB solution (4 × 15 min, 1 × 1 h) and PBS 1X (1 × 15 m) at room temperature. Controls were run in parallel by using the corresponding pre-immune rabbit IgG at the same dilution. Staining was performed using DAB tablets.

Whole mount in situ hybridizations on different developmental stages of B. lanceolatum were performed essentially as previously described (Annona et al. 2017), with the following modifications. After rehydration, embryos were digested with 7.5 μL of Proteinase K for 5–30 min depending on the size and stage of development (Yu and Holland 2009). Pre-hybridization was conducted at 50–65 °C with gentle shaking for at least 3 h. Hybridization was performed overnight at 60 °C with 50–200 ng of DIG-labeled probes. The embryos were blocked in 10% PBT (in phosphate-buffered saline—Tween20 buffer) for at least 3 h at room temperature, and then incubated in pre-absorbed 1:1500 anti-DIG-alkaline phosphatase at 4 °C overnight. After stopping the staining reaction, all developmental stages of cephalochordate and tunicate species were mounted in 80–100% glycerol and imaged using a Zeiss Axio Imager M1 microscope equipped with Axiocam digital camera.

Results

Synteny conservation in vertebrates and gene duplication events in mammals

Genomic databases show that Magoh is an ancient eukaryotic gene that maintained an highly conserved structure and is widely present as a single copy gene. The alignment of Magoh proteins, moreover, revealed a high degree of sequence identity (data not show), and this did not allow us to perform an informative phylogenetic analysis. Gene order on a chromosome of evolutionary distant species is often a symptom of a well-established and conserved expression pattern in ontogenetic pathways (Kuraku and Meyer 2012). For this reason we looked at the syntenic conservation of the Magoh gene locus being interested in finding an explanation for the duality of neural/non-neural expression patterns observed in evolution. Strikingly, we did not find any kind of conservation in neighboring genes in the genomes of metazoans, including non-vertebrate chordates, i.e., one cephalochordate and six tunicate species (Fig. 1a; Supplementary Fig. S1), despite the fact that at least the amphioxus genome is considered very much static and hardly changed respect to the chordate’s ancestor genome, one example of which is the evolutionary conserved Hox cluster (Pascual-Anaya et al. 2008, 2012, 2013). Conversely, traces of syntenic conservation are detected in the genomes of teleosts (Magoh - Cpt2), while Magoh is nestled in a conserved block of genes in amphibians, birds, and mammals (Fig. 1a). On the other hand, mammals showed one additional Magoh gene, called MAGOHB (Fig. 1b). In human, there are also two pseudogenes, named MAGOH2P and MAGOH3P. The first one, MAGOH2P, probably originated from a retrotranscription event being a single exon gene (Fig. 1c) (Singh et al. 2013). Considering that multiple copies of Magoh are only present in mammals, and not in all vertebrates, we deduced that gene duplication events presumably took place at the stem of vertebrates, therefore excluding the genesis of Magoh genes by whole genome duplication events (WGD) (Ohno 1970) (Supplementary Table S1). Gene loss may also have played an important role in shaping the Magoh gene repertoire in different vertebrate lineages.

Magoh expression in Ciona robusta

We wished to analyze the expression patterns of the Magoh gene in embryos of the model ascidian C. robusta using various developmental stages from unfertilized eggs to larvae (see “Materials and methods”). In the ascidian, mRNA encoding the EJC protein Magoh is maternally supplied, with a significant amount of transcript distributed throughout the unfertilized egg cytoplasm (Fig. 2a). Upon fertilization, the transcript is polarized in a small subcortical area at the vegetal pole (Fig. 2b). Following the first cell division, Magoh mRNA distribution in the vegetal hemisphere spreads toward the animal pole (Fig. 2c–e). At the 4- and 8-cell stages, the in situ hybridization signal is localized to all blastomeres (Fig. 2f–h). At the 16-cell stage, Magoh mRNA staining is detected broadly all through the embryo, absent only from the B5.2 cells (Fig. 2i). At the 32-cell stage, zygotic expression is unequally segregated to the animal (ventral) hemisphere of the embryo, where it labels most epidermal precursor cells (Fig. 2j, k). From the 44/64- to 110-cell stages, Magoh expression occurs diffusely in all blastomeres except for the endodermal precursor cells (Fig. 2l, m). Then, in situ hybridization on late gastrula/neural plate and neurula stage embryos shows restriction of Magoh transcript distribution in embryonic mesenchyme cells and at the tip of the notochord cell mass (Fig. 2n–q). At tailbud stage, Magoh transcript is detected also in the sensory vesicle (Fig. 2r, s). Finally, tadpole larvae show detectable Magoh expression in palps and in a large domain including sensory vesicle, neck, visceral ganglion, and posterior trunk neural tube (Fig. 2t).

Fig. 2
figure 2

Developmental expression of Magoh in Ciona robusta. a mRNA distribution in the unfertilized egg. Polar body (asterisk) marks the animal (ventral) pole at the upper side. b mRNA segregation at the vegetal pole in the fertilized egg. c-e Expression expanded half-way toward the animal pole at 2-cell stage (c frontal view, d vegetal view, e lateral view). f-h 4- and 8-cell stage. i Diffuse expression at 16-cell stage except for the B5.2 cell pair. j, k Expression on the vegetal side at 32-cell stage (j, vegetal view, k lateral view, vegetal to left). l, m Expression in all but the endodermal precursor cells at 44/64- and 110-cell stages (vegetal views). n, o Expression in the muscle cell progenitors at late gastrula/neural plate stage (n vegetal view, o lateral view, vegetal to left). p, q In situ staining in mesenchymal cells at neurula stage (p vegetal view, q lateral view, vegetal to left). r, s Expression in sensory vesicle, mesenchyme, and posterior notochord at tailbud stage (r vegetal view, s lateral view, vegetal to left). t Early tadpole larvae showing expression in palps, sensory vesicle, neck, visceral ganglion, and posterior trunk neural tube. EN endoderm, ME mesenchyme, NO notochord, NT neural tube, OC ocellus, OT Otolith, P palps, SV sensory vesicle, VG visceral ganglion

Full size image

Magoh immunoreactivity in Ciona robusta

To examine the expression pattern of C. robusta Magoh protein throughout embryogenesis, we used two anti-sera in whole mount on most developmental stages used for ISH and on juveniles. Immunoreactivity staining pattern was highly reminiscent of mRNA. Maternally positioned Magoh protein shows a diffuse distribution in the cytoplasm before fertilization (Fig. 3a). Following sperm entry, immunoreactivity is significantly restricted to the vegetal pole (Fig. 3b). During the ooplasmic segregation that follows egg fertilization, immunochemical staining shows a perinuclear signal around the female nucleus, in correspondence with the future posterior pole (Fig. 3c). Prior any sign of cell division, the perinuclear domain splits presumably because of ongoing mitosis (Fig. 3d). Then, the two signals separate and are positioned at the vegetal hemisphere (Fig. 3e). At the 2-cell stage, Magoh protein distribution is polarized to the vegetal pole to less extent than the mRNA pattern. In addition, a stripe of high protein signal at the vegetal pole suggests the future plan of the second cell division (Fig. 3f). Then, protein localization from the 4-cell to tailbud stage is highly reminiscent of mRNA distribution (Fig. 3g–q). In addition, a transient signal is found at the 110-cell stage in endoderm progenitor cells A7.2 that will give rise to the esophagus (Hirano and Nishida 1997, 2000) (Fig. 3k). At larval stage, Magoh protein is observed in two axon bundles elongating from the sensory vesicle into the anterior spinal cord (Fig. 3r), and at the edge of siphon primordia, which are ectodermal evaginations just anterior (Fig. 3r) and posterior (Fig. 3r′) to the sensory vesicle. After metamorphosis, Magoh immunolocalization occurs in the esophagus of stage 4 juveniles (Fig. 3s), and in particular in a group of ciliated epithelial cells abutting where gonads will form (Okada and Yamamoto 1999; Yamamoto and Okada 1999).

Fig. 3
figure 3

Distribution of Magoh protein in Ciona robusta developmental stages. a Maternal protein in the unfertilized egg. b Segregation of immunoreactivity following fertilization (animal pole at the upper side). c-e Perinuclear signal during mitosis (animal pole at the upper side; asterisk indicates polar body). f At 2-cell stage, Magoh protein is preferentially distributed at the vegetal pole. Arrows point to expression in the second cleavage plane. g, h Immunoreactivity pattern at 4- and 8-cell stage (g animal view, h lateral view). i-k Protein expression in all but the endodermal precursor cells at 32-cell and 110-cell stage (i, k vegetal view, j lateral view). l, m Immunoreactive signal in mesodermal cells at gastrula stage (l vegetal view, m lateral view). n, o Magoh localization in mesenchymal and muscle progenitor cells and posterior notochord of neural plate and neurula stages (vegetal view). p, q Protein localization in sensory vesicle, neural tube, muscle and notochord at tailbud stage (p lateral view, q ventral view). r, r′ Protein signal in axon bundles elongating from the sensory vesicle to the anterior nerve cord (arrowheads), and in primordial siphons (arrows) at larval stage. s Immunostaining in esophageal cells in a juvenile. E esophagus, EN endoderm, M muscle, N neck, NC nerve cord, NT neural tube, NO notochord, OC ocellus, OT otolith, ST stomach, SV sensory vesicle

Full size image

Magoh expression in Branchiostoma lanceolatum

Magoh is a maternal gene in amphioxus as shown by its expression in unfertilized eggs (Fig. 4a) and persists after fertilization when it is possible to observe the fertilization membrane (Fig. 4b). During the first phases of embryonic cleavage, Magoh is equally distributed in the two, four, and following blastomeres (Fig. 4c–f). During gastrulation (Fig. 4g, h) and neurulation (Fig. 4i, j), Magoh is extensively diffused in the whole embryo except for epidermis. Few hours of development later (pre-mouth larval stage), it becomes clearly confined to the ventral part from the rostral to the most posterior regions (Fig. 4k). Here, Magoh is strongly expressed in the region of the embryo where the mouth opening will soon be formed (white arrow), along the forming gut until the tailbud, and a faint expression persists in the brain vesicle (black arrow), that is the most rostral structure of the nervous system, in connection with the nerve cord (Fig. 4k). At larval stage, the expression becomes more faint but still detectable in the pharyngeal area (white arrow) and in trunk mesoderm (Fig. 4l). Overall, Magoh expression pattern in amphioxus during embryonic development is therefore ventral, except for the brain vesicle.

Fig. 4
figure 4

Developmental expression of Magoh in Branchiostoma lanceolatum. a-j Diffuse expression from unfertilized egg to late neurula (h anterior to left; i anterior to left, dorsal view; j anterior to left and dorsal to top, lateral view). k In pre-mouth larva, Magoh expression is detected in the brain vesicle (black arrow), pharyngeal area (white arrow) and ventral trunk. i At larval stage, faint mRNA labeling occurs in pharyngeal area (white arrow) and trunk (k, i anterior to left, dorsal to top, lateral view). EP epidermis, FM fertilization membrane

Full size image

Discussion

The ancestry of the Magoh gene in organismal evolution can be traced back to yeast, suggesting that this EJC component serves basic cellular functions. This is reflected in a striking degree of sequence conservation, as noticeable when plotting the phylogenetic tree of Magoh proteins onto classical eukaryotic taxonomy. Gene order organization in the chromosomal region encompassing the Magoh locus reveals syntenic conservation in all tetrapod genomes, suggesting a conserved cis-regulatory control depending on the chromosomal context. However, a complex pattern of species-specific gene loss and duplication events makes complex our understanding of the evolutionary history of Magoh in the mammalian lineage. One hypothesis could be that soon after the initial vertebrate WGD events, multiple Magoh paralogs were lost in ancestral vertebrates before the split between jawless and jawed vertebrates, and thus, only one Magoh is retained in most of the vertebrate lineages. On the other hand, the mammalian Magoh appeared to experience additional duplications and gave rise to multiple copies; however, some duplicated copies were subsequently lost in some lineages.

In this study of developmental expression patterns, Magoh mRNA and protein are supplied maternally and are found distributed in the entire ooplasm in both cephalochordate and tunicate unfertilized eggs. Conversely, patterns are very different between the two species during early cleavage stages. While Magoh transcript remains widely distributed in all cells until neurula stage in amphioxus, ascidian mRNA and protein become initially restricted toward the vegetal pole (future dorsal), likely co-localizing with myoplasm, and then move to the future posterior pole following ooplasmic segregation. In 2-cell stage Ciona embryos, Magoh immunostaining in a vegetal stripe corresponding to the prospective plane of the second blastomere division suggests an association between Magoh activity and cell cleavage mechanisms. Subcellular restriction of the Magoh ortholog since first cell divisions supports a conserved role in the polarization of the oocyte cytoskeleton during the initial establishment phase of anterior-posterior axis patterning of tunicates. Here, restriction of Magoh activity toward the future dorsal side of the tunicate embryo is in contrast with the expected dorsal-ventral inversion of anatomical topographies at the transition from protostomes to deuterostomes (Arendt and Nübler-Jung 1994, 1997; Holley et al. 1995; De Robertis and Sasai 1996). To be clarified, this apparent discrepancy will need to be the subject of functional approaches. Notably, lack of subcellular Magoh mRNA segregation during early development in cephalochordates is possibly due to a secondary change of the regulatory context.

In non-vertebrate chordates, Magoh mRNA and protein are observed in muscle/mesenchyme and neural precursors. In Ciona, the mesodermal pattern changes at 64-cell stage, when separation of muscle and mesenchyme differentiation takes place. From this stage, Magoh expression persists only in lineages giving rise to the mesenchyme (Hirano and Nishida 1997). Magoh expression in mesoderm and endoderm precursors of ascidian and amphioxus larvae is in line with data from insects to man. In human, Magoh is transcribed in gastric epithelial progenitor cells but with no apparent function in differentiation (Micklem et al. 1997; Newmark et al. 1997; Mills et al. 2002). However, there are preliminary indications that the genetic program initiating ventral mesoderm formation in amphioxus may not be involved in the specification of the ventral mesoderm as a whole (Holland and Holland 2007). Moreover, the presence of Magoh expression in eye precursor cells in vertebrates (Pozzoli et al. 2004) and the absence of that in ascidians may reflect differences in the mechanisms for eye development between the two groups of chordates.

Among chordates, craniates have large, elaborate brains with diverse peripheral sensory systems. The 1000-fold increase of the cerebral cortex in the lineage leading to humans is thought to be the result of changes in proliferation, death, and/or differentiation of cells in pre-established compartments (Rakic 1995; Hill and Walsh 2005; Kriegstein et al. 2006). Here, the expression of Magoh in neural cells of non-vertebrate chordates could be related to high levels of mRNA splicing events. Alternatively, our findings may raise the hypothesis that Magoh was recruited in neural cell renewal and differentiation in the last common ancestor of chordates. In this context, recruitment of Magoh expression in the central nervous system, along with the occurrence of lineage-specific events of gene duplication, are perhaps better understood for the role in the creation of alternative splice forms to generate a much larger number of proteins available for mediating new functions in relation to brain size and complexity (Blazquez et al. 2018). Understanding the evolutionary path connecting Magoh function to neural stem cells regulation might illuminate the evolution of the central nervous system in chordates and the etiology of brain pathologies like microcephaly and depression (Segman et al. 2005).