The vertebrate peripheral nervous system (PNS) originates from neural crest and placodes. While its developmental origin is the object of intense studies, little is known concerning its evolutionary history. To address this question, we analyzed the formation of the larval tail PNS in the ascidian Ciona intestinalis. The tail PNS of Ciona is made of sensory neurons located within the epidermis midlines and extending processes in the overlying tunic median fin. We show that each midline corresponds to a single longitudinal row of epidermal cells and neurons sharing common progenitors. This simple organization is observed throughout the tail epidermis, which is made of only eight single-cell rows, each expressing a specific genetic program. We next demonstrate that the epidermal neurons are specified in two consecutive steps. During cleavage and gastrula stages, the dorsal and ventral midlines are independently induced by FGF9/16/20 and the BMP ligand ADMP, respectively. Subsequently, Delta/Notch–mediated lateral inhibition controls the number of neurons formed within these neurogenic regions. These results provide a comprehensive overview of PNS formation in ascidian and uncover surprising similarities between the fate maps and embryological mechanisms underlying formation of ascidian neurogenic epidermis midlines and the vertebrate median fin.
BackgroundThe past few years have seen a vast increase in the amount of genomic data available for a growing number of taxa, including sets of full length cDNA clones and cis-regulatory sequences. Large scale cross-species comparisons of protein function and cis-regulatory sequences may help to understand the emergence of specific traits during evolution.Principal FindingsTo facilitate such comparisons, we developed a Gateway compatible vector set, which can be used to systematically dissect cis-regulatory sequences, and overexpress wild type or tagged proteins in a variety of chordate systems. It was developed and first characterised in the embryos of the ascidian Ciona intestinalis, in which large scale analyses are easier to perform than in vertebrates, owing to the very efficient embryo electroporation protocol available in this organism. Its use was then extended to fish embryos and cultured mammalian cells.ConclusionThis versatile vector set opens the way to the mid- to large-scale comparative analyses of protein function and cis-regulatory sequences across chordate evolution. A complete user manual is provided as supplemental material.
This study indicates that a sequence-based dinucleotide signature, previously associated with nucleosome depletion and independent of transcription factor binding sites, contributes to the definition of a local cis-regulatory potential in two metazoa, Ciona intestinalis and Drosophila melanogaster.
After stimulation of the T cell receptor (TCR), the tyrosine residues 292 and 315 in interdomain B of the protein tyrosine kinase ZAP-70 become phosphorylated and plausibly function as docking sites for Cbl and Vav1, respectively. The two latter proteins have been suggested to serve as substrates for ZAP-70 and to fine-tune its function. To address the role of these residues in T cell development and in the function of primary T cells, we have generated mice that express ZAP-70 molecules with Tyr to Phe substitution at position 292 (Y292F) or 315 (Y315F). When analyzed in a sensitized TCR transgenic background, the ZAP-70 Y315F mutation reduced the rate of positive selection and delayed the occurrence of negative selection. Furthermore, this mutation unexpectedly affected the constitutive levels of the CD3-ζ p21 phosphoisoform. Conversely, the ZAP-70 Y292F mutation upregulated proximal events in TCR signaling and allowed more T cells to produce interleukin 2 and interferon γ in response to a given dose of antigen. The observation that ZAP-70 Y292F T cells have a slower rate of ligand-induced TCR downmodulation suggests that Y292 is likely involved in regulating the duration activated TCR reside at the cell surface. Furthermore, we showed that Y292 and Y315 are dispensable for the TCR-induced tyrosine phosphorylation of Cbl and Vav1, respectively. Therefore, other molecules present in the TCR signaling cassette act as additional adaptors for Cbl and Vav1. The present in vivo analyses extend previous data based on transformed T cell lines and suggest that residue Y292 plays a role in attenuation of TCR signaling, whereas residue Y315 enhances ZAP-70 function.
Heparan sulphate proteoglycans such as glypicans are essential modulators of intercellular communication during embryogenesis. In Xenopus laevis embryos, the temporal and spatial distribution of Glypican 4 (Gpc4) transcripts during gastrulation and neurulation suggests functions in early development of the central nervous system. We have functionally analysed the role of Xenopus Gpc4 by using antisense morpholino oligonucleotides and show that Gpc4 is part of the signalling network that patterns the forebrain. Depletion of GPC4 protein results in a pleiotropic phenotype affecting both primary axis formation and early patterning of the anterior central nervous system. Molecular analysis shows that posterior axis elongation during gastrulation is affected in GPC4-depleted embryos, whereas head and neural induction are apparently normal. During neurulation, loss of GPC4 disrupts expression of dorsal forebrain genes, such as Emx2, whereas genes marking the ventral forebrain and posterior central nervous system continue to be expressed. This loss of GPC4 activity also causes apoptosis of forebrain progenitors during neural tube closure. Biochemical studies establish that GPC4 binds FGF2 and modulates FGF signal transduction. Inhibition of FGF signal transduction, by adding the chemical SU5402 to embryos from neural plate stages onwards,phenocopies the loss of gene expression and apoptosis in the forebrain. We propose that GPC4 regulates dorsoventral forebrain patterning by positive modulation of FGF signalling.
In chordates, neural induction is the first step of a complex developmental process through which ectodermal cells acquire a neural identity. In ascidians, FGF-mediated neural induction occurs at the 32-cell stage in two blastomere pairs, precursors respectively of anterior and posterior neural tissue. We combined molecular embryology and cis-regulatory analysis to unveil in the ascidian Ciona intestinalis the remarkably simple proximal genetic network that controls posterior neural fate acquisition downstream of FGF. We report that the combined action of two direct FGF targets, the TGFβ factor Nodal, acting via Smad- and Fox-binding sites, and the transcription factor Otx suffices to trigger ascidian posterior neural tissue formation. Moreover, we found that this strategy is conserved in the distantly related ascidian Phallusia mammillata, in spite of extreme sequence divergence in the cis-regulatory sequences involved. Our results thus highlight that the modes of gene regulatory network evolution differ with the evolutionary scale considered. Within ascidians, developmental regulatory networks are remarkably robust to genome sequence divergence. Between ascidians and vertebrates, major fate determinants, such as Otx and Nodal, can be co-opted into different networks. Comparative developmental studies in ascidians with divergent genomes will thus uncover shared ascidian strategies, and contribute to a better understanding of the diversity of developmental strategies within chordates.
Ascidians with very similar embryos but highly divergent genomes are thought to have undergone extensive developmental system drift. We compared, in four species (Ciona and Phallusia for Phlebobranchia, Molgula and Halocynthia for Stolidobranchia), gene expression and gene regulation for a network of six transcription factors regulating peripheral nervous system (PNS) formation in Ciona. All genes, but one in Molgula, were expressed in the PNS with some differences correlating with phylogenetic distance. Cross-species transgenesis indicated strong levels of conservation, except in Molgula, in gene regulation despite lack of sequence conservation of the enhancers. Developmental system drift in ascidians is thus higher for gene regulation than for gene expression and is impacted not only by phylogenetic distance, but also in a clade-specific manner and unevenly within a network. Finally, considering that Molgula is divergent in our analyses, this suggests deep conservation of developmental mechanisms in ascidians after 390 My of separate evolution.
The tail ascidian larval peripheral nervous system is made up of epidermal sensory neurons distributed more or less regularly in ventral and dorsal midlines. Their formation occurs in two-steps: the ventral and dorsal midlines are induced as neurogenic territories by Fgf9/16/20 and Admp respectively. The Delta2/Notch interaction then controls the number of neurons that form. The genetic machinery acting between the inductive processes taking place before gastrulation and neuron specification at tailbud stages are largely unknown. The analysis of seven transcription factors expressed in the forming midlines revealed an unexpected complexity and dynamic of gene expression. Their systematic overexpression confirmed that these genes do not interact following a linear cascade of activation. However, the integration of our data revealed the distinct key roles of the two upstream factors Msxb and Nkx-C that are the earliest expressed genes and the only ones able to induce neurogenic midline and ESN formation. Our data suggest that Msxb would be the primary midline gene integrating inputs from the ventral and dorsal inducers and launching a pan-midline transcriptional program. Nkx-C would be involved in tail tip specification, in maintenance of the pan-midline network and in a posterior to anterior wave controlling differentiation.
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