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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.

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 Funded by the Ministerio de Economía Industria y Competitividad (to EC; grant number BFU-2017-89861-P) and Xunta de Galicia Predoctoral Fellowship (to IH-N; grant number ED 481 A 2018 216). Both grants were partially financed by the European Social Fund SI

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 (ESCs), we found 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. This specialization of these duplicates enables the diversification of function in self-renewal and differentiation. By integrating sequence evolution, ESC phenotypes, developmental contexts and structural modelling, we pinpoint OCT4 regions sufficient for naïve pluripotency and describe their adaptation over evolutionary time.

Analysis of the establishment of epithalamic asymmetry in two non-conventional model organisms, a cartilaginous fsh and a lamprey, has suggested that an essential role of Nodal signalling, likely to be ancestral in vertebrates, may have been largely lost in zebrafsh. 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 zebrafsh, neuronal diferentiation 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 zebrafsh also develop in neural progenitor territories, with a larger size of the proliferative, pseudostratifed 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 diferent cell populations. Such mechanisms may provide a substrate for quantitative variations accounting for the variability in size and laterality of habenular asymmetries across vertebrates. The work was funded by CNRS, Université Pierre et Marie Curie, grant N° ANR-16-CE13-0013-02 to S.M., and grants from the Spanish Dirección General de Investigación-FEDER (BFU2014-58631-P), Xunta de Galicia-FEDER (ED341D R2016/032) to E.C. R.L. and M.L. were supported was supported by CNRS and Région Occitanie doctoral fellowships SI

The hypothalamus has been a central topic in neuroanatomy because of its important physiological functions, but its mature organization remains elusive. Deciphering its embryonic and adult organization is crucial in an evolutionary approach of the organization of the vertebrate forebrain. Here we studied the molecular organization of the hypothalamus and neighboring telencephalic domains in a cartilaginous fish, the catshark, Scyliorhinus canicula, focusing on ScFoxg1a, ScShh, ScNkx2.1, ScDlx2/5, ScOtp, and ScTbr1 expression profiles and on the identification αacetylated-tubulin-immunoreactive (ir), TH-ir, 5-HT-ir, and GFAP-ir structures by means of immunohistochemistry. Analysis of the results within the updated prosomeric model framework support the existence of alar and basal histogenetic compartments in the hypothalamus similar to those described in the mouse, suggesting the ancestrality of these subdivisions in jawed vertebrates. These data provide new insights into hypothalamic organization in cartilaginous fishes and highlight the generality of key features of the prosomeric model in jawed vertebrates. This work was supported by grants from the Spanish Dirección General de Investigación-FEDER (BFU2010- 15816), the Xunta de Galicia (10PXIB200051PR, CN 2012/237), European Community-Research Infrastructure Action under the FP7 “Capacities” Specific Programme (ASSEMBLE 227799), the Région Centre, Région Bretagne (EVOVERT grant number 049755; PEPTISAN project), National Research Agency (grant ANR-09-BLAN-026201), CNRS, Université d’Orléans and Université Pierre et Marie Curie. GNSD would like to thank Spanish SEPE for its funding support SI

International audience; Left-right asymmetries in the epithalamic region of the brain are widespread across vertebrates, but their magnitude and laterality varies among species. Whether these differences reflect independent origins of forebrain asymmetries or taxa-specific diversifications of an ancient vertebrate feature remains unknown. Here we show that the catshark Scyliorhinus canicula and the lampreys Petromyzon marinus and Lampetra planeri exhibit conserved molecular asymmetries between the left and right developing habenulae. Long-term pharmacological treatments in these species show that nodal signalling is essential to their generation, rather than their directionality as in teleosts. Moreover, in contrast to zebrafish, habenular left-right differences are observed in the absence of overt asymmetry of the adjacent pineal field. These data support an ancient origin of epithalamic asymmetry, and suggest that a nodal-dependent asymmetry programme operated in the forebrain of ancestral vertebrates before evolving into a variable trait in bony fish.