Genomic Resources and Annotations for a Colonial Ascidian, the Light-Bulb Sea Squirt Clavelina lepadiformis.

Ascidian embryos have been studied since the birth of experimental embryology at the end of the 19th century. They represent textbook examples of mosaic development characterized by a fast development with very few cells and invariant cleavage patterns and lineages. Ascidians belong to tunicates, the vertebrate sister group, and their study is essential to shed light on the emergence of vertebrates. Importantly, deciphering developmental gene regulatory networks has been carried out mostly in two of the three ascidian orders, Phlebobranchia and Stolidobranchia. To infer ancestral developmental programs in ascidians, it is thus essential to carry out molecular embryology in the third ascidian order, the Aplousobranchia. Here, we present genomic resources for the colonial aplousobranch Clavelina lepadiformis: a transcriptome produced from various embryonic stages, and an annotated genome. The assembly consists of 184 contigs making a total of 233.6 Mb with a N50 of 8.5 Mb and a L50 of 11. The 32,318 predicted genes capture 96.3% of BUSCO orthologs. We further show that these resources are suitable to study developmental gene expression and regulation in a comparative framework within ascidians. Additionally, they will prove valuable for evolutionary and ecological studies.

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.

Highly distinct genetic programs for peripheral nervous system formation in chordates.

BACKGROUND: Vertebrates develop their peripheral nervous system (PNS) from transient unique embryonic structures, the neural crest, and the ectodermal placodes that are located at the border of the forming central nervous system. By contrast, in the invertebrate chordates, amphioxus and ascidians, a large part of the PNS originates at the opposite of the embryo, in the ventral ectoderm. In both groups, a biphasic mechanism regulates ventral PNS formation: high BMP levels specify a neurogenic territory within which glutamatergic epidermal sensory neuron formation is controlled by the Notch pathway. Given these similarities and the phylogenetic relationships within chordates, it is likely that ventral PNS is an ancestral feature in chordates and that it has been lost in vertebrates. RESULTS: In order to get insights into the molecular control of ventral PNS formation and to test the hypothesis of their homology and potential contribution to the emergence of vertebrate PNS, we undertook a close comparison of ventral PNS formation in the ascidian Phallusia mammillata and the amphioxus Branchiostoma lanceolatum. Using timed RNA-seq series, we identified novel markers of the ventral PNS during different phases of its development in both species. By extensively determining the expression of paralogous and orthologous genes, we observed that only a minority of genes have a shared expression in the ventral PNS. However, a large fraction of ventral PNS orthologous genes are expressed in the dorsally forming PNS of vertebrates. CONCLUSIONS: Our work has significantly increased the molecular characterization of ventral PNS formation in invertebrate chordates. The low observed conservation of gene expression in the ventral PNS suggests that the amphioxus and ascidian ventral PNS are either not homologous, or alternatively extensive drift has occurred in their regulatory mechanisms following a long period (600 My) of separate evolution and accelerated evolution in the ascidian lineage. The homology to genes expressed in the dorsally forming PNS of vertebrates suggests that ancestral sensory neurons gene networks have been redeployed in vertebrates.

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.

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.

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.

Prosomeric organization of the hypothalamus in an elasmobranch, the catshark Scyliorhinus canicula.

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 α-acetylated-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.

The ancestral role of nodal signalling in breaking L/R symmetry in the vertebrate forebrain.

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.

Mutation in the human homeobox gene NKX5-3 causes an oculo-auricular syndrome.

Several dysmorphic syndromes affect the development of both the eye and the ear, but only a few are restricted to the eye and the external ear. We describe a developmental defect affecting the eye and the external ear in three members of a consanguineous family. This syndrome is characterized by ophthalmic anomalies (microcornea, microphthalmia, anterior-segment dysgenesis, cataract, coloboma of various parts of the eye, abnormalities of the retinal pigment epithelium, and rod-cone dystrophy) and a particular cleft ear lobule. Linkage analysis and mutation screening revealed in the first exon of the NKX5-3 gene a homozygous 26 nucleotide deletion, generating a truncating protein that lacked the complete homeodomain. Morpholino knockdown expression of the zebrafish nkx5-3 induced microphthalmia and disorganization of the developing retina, thus confirming that this gene represents an additional member implicated in axial patterning of the retina.

Eight previously unidentified mutations found in the OA1 ocular albinism gene.

BACKGROUND: Ocular albinism type 1 (OA1) is an X-linked ocular disorder characterized by a severe reduction in visual acuity, nystagmus, hypopigmentation of the retinal pigmented epithelium, foveal hypoplasia, macromelanosomes in pigmented skin and eye cells, and misrouting of the optical tracts. This disease is primarily caused by mutations in the OA1 gene. METHODS: The ophthalmologic phenotype of the patients and their family members was characterized. We screened for mutations in the OA1 gene by direct sequencing of the nine PCR-amplified exons, and for genomic deletions by PCR-amplification of large DNA fragments. RESULTS: We sequenced the nine exons of the OA1 gene in 72 individuals and found ten different mutations in seven unrelated families and three sporadic cases. The ten mutations include an amino acid substitution and a premature stop codon previously reported by our team, and eight previously unidentified mutations: three amino acid substitutions, a duplication, a deletion, an insertion and two splice-site mutations. The use of a novel Taq polymerase enabled us to amplify large genomic fragments covering the OA1 gene. and to detect very likely six distinct large deletions. Furthermore, we were able to confirm that there was no deletion in twenty one patients where no mutation had been found. CONCLUSION: The identified mutations affect highly conserved amino acids, cause frameshifts or alternative splicing, thus affecting folding of the OA1 G protein coupled receptor, interactions of OA1 with its G protein and/or binding with its ligand.