To shed light on the enigmatic origin of the vertebrate head, our study employs an integrated approach that combines single-cell transcriptomics, perturbations in signaling pathways, and cis-regulatory analysis in amphioxus. As a representative of a basal lineage within the chordate phylum, amphioxus retains many characteristics thought to have been present in the common chordate ancestor. Through cell type characterization, we identify the presence of prechordal plate-like, pre-migratory, and migratory neural crest-like cell populations in the developing amphioxus embryo. Functional analysis establishes conserved roles of the Nodal and Hedgehog signaling pathways in prechordal plate-like populations, and of the Wnt signaling pathway in neural crest-like populations' development. Furthermore, our trans-species transgenic experiments highlight similarities in the regulatory environments that drive neural crest-like and prechordal plate-like developmental programs in both vertebrates and amphioxus. Our findings provide evidence that the key features of vertebrate head development can be traced back to the common ancestor of all chordates.

• MeSH
• Biological Evolution * MeSH
• Neural Crest * metabolism   cytology   MeSH
• Head * embryology   MeSH
• Lancelets * genetics   embryology   MeSH
• Vertebrates * genetics   MeSH
• Hedgehog Proteins metabolism   genetics   MeSH
• Wnt Signaling Pathway genetics   MeSH
• Signal Transduction genetics   MeSH
• Gene Expression Regulation, Developmental * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Hedgehog Proteins MeSH

The Pax6 gene is essential for eye and brain development across various animal species. Here, we investigate the function of Pax6 in the development of the anterior central nervous system (CNS) of the invertebrate chordate amphioxus using CRISPR/Cas9-induced genome editing. Specifically, we examined Pax6 mutants featuring a 6 bp deletion encompassing two invariant amino acids in the conserved paired domain, hypothesized to impair Pax6 DNA-binding capacity and gene regulatory functions. Although this mutation did not result in gross morphological changes in amphioxus larvae, it demonstrated a reduced ability to activate Pax6-responsive reporter gene, suggesting a hypomorphic effect. Expression analysis in mutant larvae revealed changes in gene expression within the anterior CNS, supporting the conserved role of Pax6 gene in brain regionalization across chordates. Additionally, our findings lend support to the hypothesis of a zona limitans intrathalamica (ZLI)-like region in amphioxus, suggesting evolutionary continuity in brain patterning mechanisms. ZLI region, found in both hemichordates and vertebrates, functions as a key signaling center and serves as a restrictive boundary between major thalamic regions.

• Keywords
• amphioxus, brain, chordates, evolution, eye, genome editing, pax6,
• Publication type
• Journal Article MeSH