Diversification of habenular organization and asymmetries in teleosts: Insights from the Atlantic salmon and European eel.

Habenulae asymmetries are widespread across vertebrates and analyses in zebrafish, the reference model organism for this process, have provided insight into their molecular nature, their mechanisms of formation and their important roles in the integration of environmental and internal cues with a variety of organismal adaptive responses. However, the generality of the characteristics identified in this species remains an open question, even on a relatively short evolutionary scale, in teleosts. To address this question, we have characterized the broad organization of habenulae in the Atlantic salmon and quantified the asymmetries in each of the identified subdomains. Our results show that a highly conserved partitioning into a dorsal and a ventral component is retained in the Atlantic salmon and that asymmetries are mainly observed in the former as in zebrafish. A remarkable difference is that a prominent left-restricted pax6 positive nucleus is observed in the Atlantic salmon, but undetectable in zebrafish. This nucleus is not observed outside teleosts, and harbors a complex presence/absence pattern in this group, retaining its location and cytoarchitectonic organization in an elopomorph, the European eel. These findings suggest an ancient origin and high evolvability of this trait in the taxon. Taken together, our data raise novel questions about the variability of asymmetries across teleosts and their biological significance depending on ecological contexts.

Evolutionary origin of vertebrate OCT4/POU5 functions in supporting pluripotency.

The support of pluripotent cells over time is an essential feature of development. In eutherian embryos, pluripotency is maintained from naïve states in peri-implantation to primed pluripotency at gastrulation. To understand how these states emerged, we reconstruct the evolutionary trajectory of the Pou5 gene family, which contains the central pluripotency factor OCT4. By coupling evolutionary sequence analysis with functional studies in mouse embryonic stem cells, we find that the ability of POU5 proteins to support pluripotency originated in the gnathostome lineage, prior to the generation of two paralogues, Pou5f1 and Pou5f3 via gene duplication. In osteichthyans, retaining both genes, the paralogues differ in their support of naïve and primed pluripotency. The specialization of these duplicates enables the diversification of function in self-renewal and differentiation. By integrating sequence evolution, cell phenotypes, developmental contexts and structural modelling, we pinpoint OCT4 regions sufficient for naïve pluripotency and describe their adaptation over evolutionary time.

Developmental genoarchitectonics as a key tool to interpret the mature anatomy of the chondrichthyan hypothalamus according to the prosomeric model.

The hypothalamus is a key vertebrate brain region involved in survival and physiological functions. Understanding hypothalamic organization and evolution is important to deciphering many aspects of vertebrate biology. Recent comparative studies based on gene expression patterns have proposed the existence of hypothalamic histogenetic domains (paraventricular, TPa/PPa; subparaventricular, TSPa/PSPa; tuberal, Tu/RTu; perimamillary, PM/PRM; and mamillary, MM/RM), revealing conserved evolutionary trends. To shed light on the functional relevance of these histogenetic domains, this work aims to interpret the location of developed cell groups according to the prosomeric model in the hypothalamus of the catshark Scyliorhinus canicula, a representative of Chondrichthyans (the sister group of Osteichthyes, at the base of the gnathostome lineage). To this end, we review in detail the expression patterns of ScOtp, ScDlx2, and ScPitx2, as well as Pax6-immunoreactivity in embryos at stage 32, when the morphology of the adult catshark hypothalamus is already organized. We also propose homologies with mammals when possible. This study provides a comprehensive tool to better understand previous and novel data on hypothalamic development and evolution.

Evolutionary Transition in the Regulation of Vertebrate Pronephros Development: A New Role for Retinoic Acid.

The anterior-posterior (AP) axis in chordates is regulated by a conserved set of genes and signaling pathways, including Hox genes and retinoic acid (RA), which play well-characterized roles in the organization of the chordate body plan. The intermediate mesoderm (IM), which gives rise to all vertebrate kidneys, is an example of a tissue that differentiates sequentially along this axis. Yet, the conservation of the spatiotemporal regulation of the IM across vertebrates remains poorly understood. In this study, we used a comparative developmental approach focusing on non-conventional model organisms, a chondrichthyan (catshark), a cyclostome (lamprey), and a cephalochordate (amphioxus), to assess the involvement of RA in the regulation of chordate and vertebrate pronephros formation. We report that the anterior expression boundary of early pronephric markers (Pax2 and Lim1), positioned at the level of somite 6 in amniotes, is conserved in the catshark and the lamprey. Furthermore, RA, driving the expression of Hox4 genes like in amniotes, regulates the anterior pronephros boundary in the catshark. We find no evidence for the involvement of this regulatory hierarchy in the AP positioning of the lamprey pronephros and the amphioxus pronephros homolog, Hatschek's nephridium. This suggests that despite the conservation of Pax2 and Lim1 expressions in chordate pronephros homologs, the responsiveness of the IM, and hence of pronephric genes, to RA- and Hox-dependent regulation is a gnathostome novelty.

Identifying Amygdala-Like Territories in Scyliorhinus canicula (Chondrichthyan): Evidence for a Pallial Amygdala.

To identify the putative amygdalar complex in cartilaginous fishes, our first step was to obtain evidence that supports the existence of a pallial amygdala in the catshark Scyliorhinus canicula, at present the prevailing chondrichthyan model in comparative neurobiology and developmental biology. To this end, we analyzed the organization of the lateral walls of the telencephalic hemispheres of adults, juveniles, and early prehatching embryos by immunohistochemistry against tyrosine hydroxylase (TH), somatostatin (SOM), Pax6, serotonin (5HT), substance P (SP), and Met-enkephalin (MetEnk), calbindin-28k (CB), and calretinin (CR), and by in situ hybridization against regulatory genes such as Tbr1, Lhx9, Emx1, and Dlx2. Our data were integrated with those available from the literature related to the secondary olfactory projections in this shark species. We have characterized two possible amygdalar territories. One, which may represent a ventropallial component, was identified by its chemical signature (moderate density of Pax6-ir cells, scarce TH-ir and SOM-ir cells, and absence of CR-ir and CB-ir cells) and gene expressions (Tbr1 and Lhx9 expressions in an Emx1 negative domain, as the ventral pallium of amniotes). It is perhaps comparable to the lateral amygdala of amphibians and the pallial amygdala of teleosts. The second was a territory related to the pallial-subpallial boundary with abundant Pax6-ir and CR-ir cells, and 5HT-ir, SP-ir, and MetEnk-ir fibers capping dorsally the area superficialis basalis. This olfactory-related region at the neighborhood of the pallial-subpallial boundary may represent a subpallial amygdala subdivision that possibly contains migrated cells of ventropallial origin.

When Bigger Is Better: 3D RNA Profiling of the Developing Head in the Catshark Scyliorhinus canicula.

We report the adaptation of RNA tomography, a technique allowing spatially resolved, genome-wide expression profiling, to a species occupying a key phylogenetic position in gnathostomes, the catshark Scyliorhinus canicula. We focused analysis on head explants at an embryonic stage, shortly following neural tube closure and of interest for a number of developmental processes, including early brain patterning, placode specification or the establishment of epithalamic asymmetry. As described in the zebrafish, we have sequenced RNAs extracted from serial sections along transverse, horizontal and sagittal planes, mapped the data onto a gene reference taking advantage of the high continuity genome recently released in the catshark, and projected read counts onto a digital model of the head obtained by confocal microscopy. This results in the generation of a genome-wide 3D atlas, containing expression data for most protein-coding genes in a digital model of the embryonic head. The digital profiles obtained for candidate forebrain regional markers along antero-posterior, dorso-ventral and left-right axes reproduce those obtained by in situ hybridization (ISH), with expected relative organizations. We also use spatial autocorrelation and correlation as measures to analyze these data and show that they provide adequate statistical tools to extract novel expression information from the model. These data and tools allow exhaustive searches of genes exhibiting any predefined expression characteristic, such a restriction to a territory of interest, thus providing a reference for comparative analyses across gnathostomes. This methodology appears best suited to species endowed with large embryo or organ sizes and opens novel perspectives to a wide range of evo-devo model organisms, traditionally counter-selected on size criterion.

Evolution of lbx spinal cord expression and function.

Ladybird homeobox (Lbx) transcription factors have crucial functions in muscle and nervous system development in many animals. Amniotes have two Lbx genes, but only Lbx1 is expressed in spinal cord. In contrast, teleosts have three lbx genes and we show here that zebrafish lbx1a, lbx1b, and lbx2 are expressed by distinct spinal cell types, and that lbx1a is expressed in dI4, dI5, and dI6 interneurons, as in amniotes. Our data examining lbx expression in Scyliorhinus canicula and Xenopus tropicalis suggest that the spinal interneuron expression of zebrafish lbx1a is ancestral, whereas lbx1b has acquired a new expression pattern in spinal cord progenitor cells. lbx2 spinal expression was probably acquired in the ray-finned lineage, as this gene is not expressed in the spinal cords of either amniotes or S. canicula. We also show that the spinal function of zebrafish lbx1a is conserved with mouse Lbx1. In zebrafish lbx1a mutants, there is a reduction in the number of inhibitory spinal interneurons and an increase in the number of excitatory spinal interneurons, similar to mouse Lbx1 mutants. Interestingly, the number of inhibitory spinal interneurons is also reduced in lbx1b mutants, although in this case the number of excitatory interneurons is not increased. lbx1a;lbx1b double mutants have a similar spinal interneuron phenotype to lbx1a single mutants. Taken together these data suggest that lbx1b and lbx1a may be required in succession for correct specification of dI4 and dI6 spinal interneurons, although only lbx1a is required for suppression of excitatory fates in these cells.

Loss of Active Neurogenesis in the Adult Shark Retina.

Neurogenesis is the process by which progenitor cells generate new neurons. As development progresses neurogenesis becomes restricted to discrete neurogenic niches, where it persists during postnatal life. The retina of teleost fishes is thought to proliferate and produce new cells throughout life. Whether this capacity may be an ancestral characteristic of gnathostome vertebrates is completely unknown. Cartilaginous fishes occupy a key phylogenetic position to infer ancestral states fixed prior to the gnathostome radiation. Previous work from our group revealed that the juvenile retina of the catshark Scyliorhinus canicula, a cartilaginous fish, shows active proliferation and neurogenesis. Here, we compared the morphology and proliferative status of the retina in catshark juveniles and adults. Histological and immunohistochemical analyses revealed an important reduction in the size of the peripheral retina (where progenitor cells are mainly located), a decrease in the thickness of the inner nuclear layer (INL), an increase in the thickness of the inner plexiform layer and a decrease in the cell density in the INL and in the ganglion cell layer in adults. Contrary to what has been reported in teleost fish, mitotic activity in the catshark retina was virtually absent after sexual maturation. Based on these results, we carried out RNA-Sequencing (RNA-Seq) analyses comparing the retinal transcriptome of juveniles and adults, which revealed a statistically significant decrease in the expression of many genes involved in cell proliferation and neurogenesis in adult catsharks. Our RNA-Seq data provides an excellent resource to identify new signaling pathways controlling neurogenesis in the vertebrate retina.

Differential expression of five prosomatostatin genes in the central nervous system of the catshark Scyliorhinus canicula.

Five prosomatostatin genes (PSST1, PSST2, PSST3, PSST5, and PSST6) have been recently identified in elasmobranchs (Tostivint et al., General and Comparative Endocrinology, 2019, 279, 139-147). In order to gain insight into the contribution of each somatostatin to specific nervous systems circuits and behaviors in this important jawed vertebrate group, we studied the distribution of neurons expressing PSST mRNAs in the central nervous system (CNS) of Scyliorhinus canicula using in situ hybridization. Additionally, we combined in situ hybridization with tyrosine hydroxylase (TH) immunochemistry for better characterization of PSST1 and PSST6 expressing populations. We observed differential expression of PSST1 and PSST6, which are the most widely expressed PSST transcripts, in cell populations of many CNS regions, including the pallium, subpallium, hypothalamus, diencephalon, optic tectum, midbrain tegmentum, and rhombencephalon. Interestingly, numerous small pallial neurons express PSST1 and PSST6, although in different populations judging from the colocalization of TH immunoreactivity and PSST6 expression but not with PSST1. We observed expression of PSST1 in cerebrospinal fluid-contacting (CSF-c) neurons of the hypothalamic paraventricular organ and the central canal of the spinal cord. Unlike PSST1 and PSST6, PSST2, and PSST3 are only expressed in cells of the hypothalamus and in some hindbrain lateral reticular neurons, and PSST5 in cells of the region of the entopeduncular nucleus. Comparative data of brain expression of PSST genes indicate that the somatostatinergic system of sharks is the most complex reported in any fish.

Skeletal Mineralization in Association with Type X Collagen Expression Is an Ancestral Feature for Jawed Vertebrates.

In order to characterize the molecular bases of mineralizing cell evolution, we targeted type X collagen, a nonfibrillar network forming collagen encoded by the Col10a1 gene. It is involved in the process of endochondral ossification in ray-finned fishes and tetrapods (Osteichthyes), but until now unknown in cartilaginous fishes (Chondrichthyes). We show that holocephalans and elasmobranchs have respectively five and six tandemly duplicated Col10a1 gene copies that display conserved genomic synteny with osteichthyan Col10a1 genes. All Col10a1 genes in the catshark Scyliorhinus canicula are expressed in ameloblasts and/or odontoblasts of teeth and scales, during the stages of extracellular matrix protein secretion and mineralization. Only one duplicate is expressed in the endoskeletal (vertebral) mineralizing tissues. We also show that the expression of type X collagen is present in teeth of two osteichthyans, the zebrafish Danio rerio and the western clawed frog Xenopus tropicalis, indicating an ancestral jawed vertebrate involvement of type X collagen in odontode formation. Our findings push the origin of Col10a1 gene prior to the divergence of osteichthyans and chondrichthyans, and demonstrate its ancestral association with mineralization of both the odontode skeleton and the endoskeleton.

Revisiting the evolution of the somatostatin family: Already five genes in the gnathostome ancestor.

The somatostatin (SST) family members are a group of neuropeptides that are best known for their role in the regulation of growth, development and metabolism. The occurrence of six paralogous SST genes named SST1, SST2, SST3, SST4, SST5 and SST6 has been reported in vertebrates. It has been proposed that SST1, SST2 and SST5 arose in 2R from a common ancestral gene. SST3 and SST6 would have been subsequently generated by tandem duplications of the SST1 and SST2 genes respectively, at the base of the actinopterygian lineage. SST4 is thought to have appeared more recently from SST1, in teleost-specific 3R. In order to gain more insights into the SST gene family in vertebrates, we sought to identify which paralogs of this family are present in cartilaginous fish. For this purpose, we first searched the recently available genome and transcriptome databases from the catshark Scyliorhinus canicula. In a previous study, three S. canicula SST genes, called at that time SSTa, SSTb and SSTc, were identified and proposed to correspond to SST1, SST5 and SST2 respectively. In the present work, two additional SST genes, called SSTd and SSTe, were found in S. canicula plus two other chondrichtyan species, elephant shark (Callorhinchus milii) and whale shark (Rhincodon typus). Phylogeny and synteny analyses were then carried out in order to reveal the evolutionary relationships of SSTd and SSTe with other vertbrates SSTs. We showed that SSTd and SSTe correspond to SST2 and SST3 respectively, while SSTc corresponds to SST6 and not to SST2 as initially proposed. Our investigations in other vertebrate species also led us to find that the so-called SST2 gene in chicken, lungfish, sturgeons and teleosts actually corresponds to SST6. Conversely, the so-called SST6 gene in actinopterygians corresponds to SST2. Taken together, our results suggest that: i) SST3 and SST6 were already present in the gnathostome ancestor, much earlier than previously thought; ii) SST6 was also present in the tetrapod ancestor and still occurs in living birds; with this respect, it is likely that SST6 was independently lost several times during evolution: in amphibians, squamates and mammals; iii) SST2, SST3 and SST5 were probably lost in euteleosts, sarcopterygians and tetrapods, respectively.

Molecular ontogeny of the stomach in the catshark Scyliorhinus canicula.

The origin of extracellular digestion in metazoans was accompanied by structural and physiological alterations of the gut. These adaptations culminated in the differentiation of a novel digestive structure in jawed vertebrates, the stomach. Specific endoderm/mesenchyme signalling is required for stomach differentiation, involving the growth and transcription factors: 1) Shh and Bmp4, required for stomach outgrowth; 2) Barx1, Sfrps and Sox2, required for gastric epithelium development and 3) Cdx1 and Cdx2, involved in intestinal versus gastric identity. Thus, modulation of endoderm/mesenchyme signalling emerges as a plausible mechanism linked to the origin of the stomach. In order to gain insight into the ancient mechanisms capable of generating this structure in jawed vertebrates, we characterised the development of the gut in the catshark Scyliorhinus canicula. As chondrichthyans, these animals retained plesiomorphic features of jawed vertebrates, including a well-differentiated stomach. We identified a clear molecular regionalization of their embryonic gut, characterised by the expression of barx1 and sox2 in the prospective stomach region and expression of cdx1 and cdx2 in the prospective intestine. Furthermore, we show that gastric gland development occurs close to hatching, accompanied by the onset of gastric proton pump activity. Our findings favour a scenario in which the developmental mechanisms involved in the origin of the stomach were present in the common ancestor of chondrichthyans and osteichthyans.

The Chemosensory Receptor Repertoire of a True Shark Is Dominated by a Single Olfactory Receptor Family.

Throughout the animal kingdom chemical senses are one of the primary means by which organisms make sense of their environment. To achieve perception of complex chemosensory stimuli large repertoires of olfactory and gustatory receptors are employed in bony vertebrates, which are characterized by high evolutionary dynamics in receptor repertoire size and composition. However, little is known about their evolution in earlier diverging vertebrates such as cartilaginous fish, which include sharks, skates, rays, and chimeras. Recently, the olfactory repertoire of a chimera, elephant shark, was found to be curiously reduced in odorant receptor number. Elephant sharks rely heavily on electroreception to localize prey; thus, it is unclear how representative their chemosensory receptor repertoire sizes would be for cartilaginous fishes in general. Here, we have mined the genome of a true shark, Scyliorhinus canicula (catshark) for olfactory and gustatory receptors, and have performed a thorough phylogenetic study to shed light on the evolution of chemosensory receptors in cartilaginous fish. We report the presence of several gustatory receptors of the TAS1R family in catshark and elephant shark, whereas TAS2R receptors are absent. The catshark olfactory repertoire is dominated by V2R receptors, with 5-8 receptors in the other three families (OR, ORA, TAAR). Species-specific expansions are mostly limited to the V2R family. Overall, the catshark chemosensory receptor repertoires are generally similar in size to those of elephant shark, if somewhat larger, showing similar evolutionary tendencies across over 400 Myr of separate evolution between catshark and elephant shark.

Characterization of Gonadotropin-Releasing Hormone (GnRH) Genes From Cartilaginous Fish: Evolutionary Perspectives.

The neuropeptide gonadotropin-releasing hormone (GnRH) plays an important role in the control of reproductive functions. Vertebrates possess multiple GnRH forms that are classified into three main groups, namely GnRH1, GnRH2, and GnRH3. In order to gain more insights into the GnRH gene family in vertebrates, we sought to identify which paralogs of this family are present in cartilaginous fish. For this purpose, we searched the genomes and/or transcriptomes of three representative species of this group, the small-spotted catshark, Scyliorhinus canicula, the whale shark, Rhincodon typus and the elephant shark Callorhinchus milii. In each species, we report the identification of three GnRH genes. In catshark and whale shark, phylogenetic and synteny analysis showed that these three genes correspond to GnRH1, GnRH2, and GnRH3. In both species, GnRH1 was found to encode a novel form of GnRH whose primary structure was determined as follows: QHWSFDLRPG. In elephant shark, the three genes correspond to GnRH1a and GnRH1b, two copies of the GnRH1 gene, plus GnRH2. 3D structure prediction of the chondrichthyan GnRH-associated peptides (GAPs) revealed that catshark GAP1, GAP2, and elephant shark GAP2 peptides exhibit a helix-loop-helix (HLH) structure. This structure observed for many osteichthyan GAP1 and GAP2, may convey GAP biological activity. This HLH structure could not be observed for elephant shark GAP1a and GAP1b. As for all other GAP3 described so far, no typical 3D HLH structure was observed for catshark nor whale shark GAP3. RT-PCR analysis revealed that GnRH1, GnRH2, and GnRH3 genes are differentially expressed in the catshark brain. GnRH1 mRNA appeared predominant in the diencephalon while GnRH2 and GnRH3 mRNAs seemed to be most abundant in the mesencephalon and telencephalon, respectively. Taken together, our results show that the GnRH gene repertoire of the vertebrate ancestor was entirely conserved in the chondrichthyan lineage but that the GnRH3 gene was probably lost in holocephali. They also suggest that the three GnRH neuronal systems previously described in the brain of bony vertebrates are also present in cartilaginous fish.

The nanos1 gene was duplicated in early Vertebrates and the two paralogs show different gonadal expression profiles in a shark.

Nanos are RNA-binding proteins playing crucial roles in germ cell development and maintenance. Based on phylogenetic and synteny analyses, this study reveals that nanos1 gene has undergone multiple duplications and gene copies losses in Vertebrates. Chondrichthyan species display two nanos1 genes (named nanos1A/1B), which were both retrieved in some Osteichthyes at basal positions in Sarcopterygii and Actinopterygii lineages. In contrast, Teleosts have lost nanos1A but duplicated nanos1B leading to the emergence of two ohnologs (nanos1Ba/1Bb), whereas Tetrapods have lost nanos1B gene. The two successive nanos gene duplications may result from the second and third whole genome duplication events at the basis of Vertebrates and Teleosts respectively. The expression profiles of nanos1A and nanos1B paralogs were characterized in the dogfish, Scyliorhinus canicula. Nanos1A was strongly expressed in brain and also localized in all germ cell types in the polarized testis. In contrast, nanos1B was detected in testis with the highest expression in the germinative zone. In addition, Nanos1B protein was predominantly located in the nuclei of male germinal cells. In the ovary, both paralogs were detected in germinal and somatic cells. Our study opens new perspectives concerning the complex evolution of nanos1 paralogs and their potential distinct roles in Vertebrates gonads.

The Shark Basal Hypothalamus: Molecular Prosomeric Subdivisions and Evolutionary Trends.

The hypothalamus is a key integrative center of the vertebrate brain. To better understand its ancestral morphological organization and evolution, we previously analyzed the segmental organization of alar subdivisions in the catshark Scyliorhinus canicula, a cartilaginous fish and thus a basal representative of gnathostomes (jawed vertebrates). With the same aim, we deepen here in the segmental organization of the catshark basal hypothalamus by revisiting previous data on ScOtp, ScDlx2/5, ScNkx2.1, ScShh expression and Shh immunoreactivity jointly with new data on ScLhx5, ScEmx2, ScLmx1b, ScPitx2, ScPitx3a, ScFoxa1, ScFoxa2 and ScNeurog2 expression and proliferating cell nuclear antigen (PCNA) immunoreactivity. Our study reveals a complex genoarchitecture for chondrichthyan basal hypothalamus on which a total of 21 microdomains were identified. Six belong to the basal acroterminal region, the rostral-most point of the basal neural tube; seven are described in the tuberal region (Tu/RTu); four in the perimamillar region (PM/PRM) and four in the mamillar one (MM/RM). Interestingly, the same set of genes does not necessarily describe the same microdomains in mice, which in part contributes to explain how forebrain diversity is achieved. This study stresses the importance of analyzing data from basal vertebrates to better understand forebrain diversity and hypothalamic evolution.

Neurogenetic asymmetries in the catshark developing habenulae: mechanistic and evolutionary implications.

Analysis of the establishment of epithalamic asymmetry in two non-conventional model organisms, a cartilaginous fish and a lamprey, has suggested that an essential role of Nodal signalling, likely to be ancestral in vertebrates, may have been largely lost in zebrafish. In order to decipher the cellular mechanisms underlying this divergence, we have characterised neurogenetic asymmetries during habenular development in the catshark Scyliorhinus canicula and addressed the mechanism involved in this process. As in zebrafish, neuronal differentiation starts earlier on the left side in the catshark habenulae, suggesting the conservation of a temporal regulation of neurogenesis. At later stages, marked, Alk4/5/7 dependent, size asymmetries having no clear counterparts in zebrafish also develop in neural progenitor territories, with a larger size of the proliferative, pseudostratified neuroepithelium, in the right habenula relative to the left one, but a higher cell number on the left of a more lateral, later formed population of neural progenitors. These data show that mechanisms resulting in an asymmetric, preferential maintenance of neural progenitors act both in the left and the right habenulae, on different cell populations. Such mechanisms may provide a substrate for quantitative variations accounting for the variability in size and laterality of habenular asymmetries across vertebrates.

Fin-fold development in paddlefish and catshark and implications for the evolution of the autopod.

The evolutionary origin of the autopod involved a loss of the fin-fold and associated dermal skeleton with a concomitant elaboration of the distal endoskeleton to form a wrist and digits. Developmental studies, primarily from teleosts and amniotes, suggest a model for appendage evolution in which a delay in the AER-to-fin-fold conversion fuelled endoskeletal expansion by prolonging the function of AER-mediated regulatory networks. Here, we characterize aspects of paired fin development in the paddlefish Polyodon spathula (a non-teleost actinopterygian) and catshark Scyliorhinus canicula (chondrichthyan) to explore aspects of this model in a broader phylogenetic context. Our data demonstrate that in basal gnathostomes, the autopod marker HoxA13 co-localizes with the dermoskeleton component And1 to mark the position of the fin-fold, supporting recent work demonstrating a role for HoxA13 in zebrafish fin ray development. Additionally, we show that in paddlefish, the proximal fin and fin-fold mesenchyme share a common mesodermal origin, and that components of the Shh/LIM/Gremlin/Fgf transcriptional network critical to limb bud outgrowth and patterning are expressed in the fin-fold with a profile similar to that of tetrapods. Together these data draw contrast with hypotheses of AER heterochrony and suggest that limb-specific morphologies arose through evolutionary changes in the differentiation outcome of conserved early distal patterning compartments.

A Developmental Study of the Cerebellar Nucleus in the Catshark, a Basal Gnathostome.

The output of the cerebellar cortex is mainly released via cerebellar nuclei which vary in number and complexity among gnathostomes, extant vertebrates with a cerebellum. Cartilaginous fishes, a basal gnathostome lineage, show a conspicuous, well-organized cerebellar nucleus, unlike ray-finned fishes. To gain insight into the evolution and development of the cerebellar nucleus, we analyzed in the shark Scyliorhinus canicula (a chondrichthyan model species) the developmental expression of several genes coding for transcription factors (ScLhx5,ScLhx9,ScTbr1, and ScEn2) and the distribution of the protein calbindin, since all appear to be involved in cerebellar nuclei patterning in other gnathostomes. Three regions (subventricular, medial or central, and lateral or superficial) became recognizable in the cerebellar nucleus of this shark during development. Present genoarchitectonic and neurochemical data in embryos provide insight into the origin of the cerebellar nucleus in chondrichthyans and support a tripartite mediolateral organization of the cerebellar nucleus, as previously described in adult sharks. Furthermore, the expression pattern of ScLhx5,ScLhx9, and ScTbr1 in this shark, together with that of markers of proliferation, migration, and early differentiation of neurons, is compatible with the hypothesis that, as in mammals, different subsets of cerebellar nucleus neurons are originated from progenitors of 2 different sources: the ventricular zone of the cerebellar plate and the rhombic lip. We also present suggestive evidence that Lhx9 expression is involved in cerebellar nuclei patterning early on in gnathostome evolution, rather than representing an evolutionary innovation of the dentate nucleus in mammals, as previously hypothesized.