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

The core molecular mechanisms of dorsal organizer formation during gastrulation are highly conserved within the chordate lineage. One of the key characteristics is that Nodal signaling is required for the organizer-specific gene expression. This feature appears to be ancestral, as evidenced by the presence in the most basally divergent chordate amphioxus. To provide a better understanding of the evolution of organizer-specific gene regulation in chordates, we analyzed the cis-regulatory sequence of amphioxus Chordin in the context of the vertebrate embryo. First, we generated stable zebrafish transgenic lines, and by using light-sheet fluorescent microscopy, characterized in detail the expression pattern of GFP driven by the cis-regulatory sequences of amphioxus Chordin. Next, we performed a 5'deletion analysis and identified an enhancer sufficient to drive the expression of the reporter gene into a chordate dorsal organizer. Finally, we found that the identified enhancer element strongly depends on Nodal signaling, which is consistent with the well-established role of this pathway in the regulation of the expression of dorsal organizer-specific genes across chordates. The enhancer identified in our study may represent a suitable simple system to study the interplay of the evolutionarily conserved regulatory mechanisms operating during early chordate development.

• Keywords
• Amphioxus, Chordate evolution, Chordin, Enhancer, Gene regulation, Light-sheet microscopy, Nodal, Organizer of gastrulation,
• MeSH
• Zebrafish genetics   metabolism   MeSH
• Gene Expression MeSH
• Lancelets * genetics   metabolism   MeSH
• Transforming Growth Factor beta metabolism   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
• chordin MeSH Browser
• Transforming Growth Factor beta MeSH

Deciphering the mechanisms of axis formation in amphioxus is a key step to understanding the evolution of chordate body plan. The current view is that Nodal signaling is the only factor promoting the dorsal axis specification in the amphioxus, whereas Wnt/β-catenin signaling plays no role in this process. Here, we re-examined the role of Wnt/βcatenin signaling in the dorsal/ventral patterning of amphioxus embryo. We demonstrated that the spatial activity of Wnt/β-catenin signaling is located in presumptive dorsal cells from cleavage to gastrula stage, and provided functional evidence that Wnt/β-catenin signaling is necessary for the specification of dorsal cell fate in a stage-dependent manner. Microinjection of Wnt8 and Wnt11 mRNA induced ectopic dorsal axis in neurulae and larvae. Finally, we demonstrated that Nodal and Wnt/β-catenin signaling cooperate to promote the dorsal-specific gene expression in amphioxus gastrula. Our study reveals high evolutionary conservation of dorsal organizer formation in the chordate lineage.

• Keywords
• axial patterning, body plan, branchiostoma floridae, branchiostoma lanceolatum, developmental biology, evolution, evolutionary biology, wnt/β-catenin signaling,
• MeSH
• beta Catenin metabolism   MeSH
• Biological Evolution MeSH
• HEK293 Cells MeSH
• Lancelets embryology   metabolism   MeSH
• Humans MeSH
• Goosecoid Protein metabolism   MeSH
• Nodal Protein metabolism   MeSH
• Smad2 Protein metabolism   MeSH
• Wnt Signaling Pathway * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• beta Catenin MeSH
• Goosecoid Protein MeSH
• Nodal Protein MeSH
• Smad2 Protein MeSH

The midbrain-hindbrain boundary (MHB) is one of the key organizing centers of the vertebrate central nervous system (CNS). Its patterning is governed by a well-described gene regulatory network (GRN) involving several transcription factors, namely, pax, gbx, en, and otx, together with signaling molecules of the Wnt and Fgf families. Here, we describe the onset of these markers in Oryzias latipes (medaka) early brain development in comparison to previously known zebrafish expression patterns. Moreover, we show for the first time that vox, a member of the vent gene family, is expressed in the developing neural tube similarly to CNS markers. Overexpression of vox leads to profound changes in the gene expression patterns of individual components of MHB-specific GRN, most notably of fgf8, a crucial organizer molecule of MHB. Our data suggest that genes from the vent family, in addition to their crucial role in body axis formation, may play a role in regionalization of vertebrate CNS.

• Keywords
• Gene regulatory network, Heat shock element, Midbrain-hindbrain boundary, fgf8, medaka, vox,
• MeSH
• Embryo, Nonmammalian metabolism   MeSH
• Gene Regulatory Networks MeSH
• Homeodomain Proteins genetics   metabolism   MeSH
• Mesencephalon embryology   metabolism   MeSH
• Oryzias embryology   genetics   MeSH
• Rhombencephalon embryology   metabolism   MeSH
• Fish Proteins genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Homeodomain Proteins MeSH
• Fish Proteins MeSH