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

Regulation of gene expression is arguably the main mechanism underlying the phenotypic diversity of tissues within and between species. Here we assembled an extensive transcriptomic dataset covering 8 tissues across 20 bilaterian species and performed analyses using a symmetric phylogeny that allowed the combined and parallel investigation of gene expression evolution between vertebrates and insects. We specifically focused on widely conserved ancestral genes, identifying strong cores of pan-bilaterian tissue-specific genes and even larger groups that diverged to define vertebrate and insect tissues. Systematic inferences of tissue-specificity gains and losses show that nearly half of all ancestral genes have been recruited into tissue-specific transcriptomes. This occurred during both ancient and, especially, recent bilaterian evolution, with several gains being associated with the emergence of unique phenotypes (for example, novel cell types). Such pervasive evolution of tissue specificity was linked to gene duplication coupled with expression specialization of one of the copies, revealing an unappreciated prolonged effect of whole-genome duplications on recent vertebrate evolution.

• MeSH
• Phylogeny MeSH
• Insecta * genetics   MeSH
• Evolution, Molecular * MeSH
• Vertebrates * genetics   MeSH
• Organ Specificity MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• 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

Several bioinformatic tools have been developed for genome-wide identification of orthologous and paralogous genes. However, no corresponding tool allows the detection of exon homology relationships. Here, we present ExOrthist, a fully reproducible Nextflow-based software enabling inference of exon homologs and orthogroups, visualization of evolution of exon-intron structures, and assessment of conservation of alternative splicing patterns. ExOrthist evaluates exon sequence conservation and considers the surrounding exon-intron context to derive genome-wide multi-species exon homologies at any evolutionary distance. We demonstrate its use in different evolutionary scenarios: whole genome duplication in frogs and convergence of Nova-regulated splicing networks ( https://github.com/biocorecrg/ExOrthist ).

• Keywords
• Alternative splicing, Intron-exon structures, Orthology, Paralogy,
• MeSH
• Alternative Splicing MeSH
• Exons * MeSH
• Genome MeSH
• Introns MeSH
• Conserved Sequence MeSH
• Humans MeSH
• Evolution, Molecular * MeSH
• Mice MeSH
• Software * MeSH
• Computational Biology * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The evolution of the vertebrate eye remains so far unresolved. Amphioxus frontal eye pigment cells and photoreceptors were proposed to be homologous to vertebrate photoreceptors and retinal pigmented epithelium, based on ultrastructural morphology and gene expression analysis in B. floridae. Here, we present comparative molecular data using two additional amphioxus species, a closely related B. lanceolatum, and the most divergent A. lucayanum. Taking advantage of a unique set of specific antibodies we characterized photoreceptors and putative interneurons of the frontal eye and investigated its neuronal circuitry. Our results corroborate generally conserved molecular fingerprint among cephalochordate species. Furthermore, we performed pharmacological perturbations and found that the Notch signaling pathway, a key regulator of retina development in vertebrates, is required for correct ratios among frontal eye cell types. In summary, our study provides a valuable insight into cell-type relationships in chordate visual organs and strengthens the previously proposed homology between amphioxus frontal eye and vertebrate eyes.

• Keywords
• Notch signaling, chordates, eye evolution, gene expression, interneurons, light detection, photoreceptors, vision,
• Publication type
• Journal Article MeSH