Comparative analysis of ventral sensory neuron specification mechanisms in invertebrate chordates: insights into the origin and the evolution of the vertebrate peripheral nervous system. – VentralPNS

The 2-step mode of vPNS formation had been described in reference invertebrate chordates: ventral ectoderm is induced as a neurogenic territory by BMP signaling, then the number of cells that become neurons is regulated by Notch signaling. Using this starting point, we aimed at deciphering and comparing the gene regulatory networks regulating vPNS formation in other species, several ascidian species and the European amphioxus Branchiostome lanceolatum. First, using in situ hybridization we determined whether already known vPNS genes were expressed in the vPNS of other species. Next, by combining functional approaches (pharmacological treatments, classical embryology, CRISPR/Cas9), RNA-seq and phylogenetic analyses, we identified novel vPNS markers, and, for some of them, determined their function. Finally, by focusing on cis-regulatory DNA, we examined whether conserved gene expression patterns were regulated through similar mechanisms.

A number of othologous genes are expressed in the vPNS of several invertebrate chordates, and, for a small number that we have tested, they share similar regulation and function, in agreement with the proposed vPNS homology. However, the gene networks are very different following 600 millions years of separate evolution. Interestingly, the majority of the vPNS genes have their orthologs expressed in the PNS of vertebrates. This suggests that the vPNS gene network, active in the ventral part of the embryo at the base of chordates, has been recruited dorsally and has contributed to the emergence of the vertebrate PNS.

Our 'invertebrate' point of view on peripheral nervous system evolution has brought original hypotheses that couldn't have come from a 'vertebrate-centric' vision.
Our study in invertebrate chordates has increased the molecular description of vPNS formation. Interestingly, a majority of the orthologs of the vPNS genes are expressed in the dorsal PNS of vertebrates. In contrast, for the hypothetical vPNS GRNs of amphioxus and ascidian having few genes in common, we propose that the vPNS is ancestral in chordates and that extensive developmental system drift has occurred since the ancient separation of the two lineages.
Future studies focusing on functional experiments in a number of species would be essential to extend our comparative analysis of these GRNs.

Coulcher JF, Roure A, Chowdhury R, Robert M, Lescat L, Bouin A, Carvajal Cadavid J, Nishida H and Darras S (2020). Conservation of peripheral nervous system formation mechanisms in divergent ascidian embryos. eLife 9, e59157.
Comparison of vPNS gene expression and regulation in several ascidian species.

Darras S (2021). En masse DNA Electroporation for in vivo Transcriptional Assay in Ascidian Embryos. Bio-protocol 11(18): e4160.
Protocol for performing electroporation-based transgenesis in several ascidian species.

Chowdhury R, Roure A, le Pétillon Y, Mayeur H, Daric V and Darras S (2022). Highly distinct genetic programs for peripheral nervous system formation in chordates. BMC Biol 20, 1–25.
Comparison of the vPNS gene networks between amphioxus and ascidian.

While vertebrate peripheral nervous system (PNS) exclusively arises from dorsal structures (neural crest and placodes), a large part of invertebrate chordate (ascidians and amphioxus) PNS originates from the ventral ectoderm. BMP signals induce a neurogenic field from which sensory neurons are selected by Notch signaling. This ventral PNS (vPNS) likely corresponds to an ancestral chordate structure that may have been lost in vertebrates or shifted dorsally to form placodes/neural crest, possibly via co-option of ancestral specification mechanisms. Our project aims at exploring functionally the ventral PNS using both groups of invertebrate chordates as references. We will perform a thorough side-by-side comparison to 1) identify vPNS genes, 2) define their hierarchy and transcriptional regulation, 3) determine whether their mode of regulation is conserved and 4) probe the existence of vertebrate counterparts for either a vPNS territory, vPNS neurons or vPNS gene regulatory networks.