Abstract
Pax6 is a developmental control gene essential for eye development throughout the animal kingdom. In addition, Pax6 plays key roles in other parts of the CNS, olfactory system, and pancreas. In mammals a single Pax6 gene encoding multiple isoforms delivers these pleiotropic functions. Here we provide evidence that the genomes of many other vertebrate species contain multiple Pax6 loci. We sequenced Pax6-containing BACs from the cartilaginous elephant shark (Callorhinchus milii) and found two distinct Pax6 loci. Pax6.1 is highly similar to mammalian Pax6, while Pax6.2 encodes a paired-less Pax6. Using synteny relationships, we identify homologs of this novel paired-less Pax6.2 gene in lizard and in frog, as well as in zebrafish and in other teleosts. In zebrafish two full-length Pax6 duplicates were known previously, originating from the fish-specific genome duplication (FSGD) and expressed in divergent patterns due to paralog-specific loss of cis-elements. We show that teleosts other than zebrafish also maintain duplicate full-length Pax6 loci, but differences in gene and regulatory domain structure suggest that these Pax6 paralogs originate from a more ancient duplication event and are hence renamed as Pax6.3. Sequence comparisons between mammalian and elephant shark Pax6.1 loci highlight the presence of short- and long-range conserved noncoding elements (CNEs). Functional analysis demonstrates the ancient role of long-range enhancers for Pax6 transcription. We show that the paired-less Pax6.2 ortholog in zebrafish is expressed specifically in the developing retina. Transgenic analysis of elephant shark and zebrafish Pax6.2 CNEs with homology to the mouse NRE/Pα internal promoter revealed highly specific retinal expression. Finally, morpholino depletion of zebrafish Pax6.2 resulted in a “small eye” phenotype, supporting a role in retinal development. In summary, our study reveals that the pleiotropic functions of Pax6 in vertebrates are served by a divergent family of Pax6 genes, forged by ancient duplication events and by independent, lineage-specific gene losses.
Highlights
Development is critically dependent on a core set of developmental regulator genes, most of which are highly conserved across metazoans and carry out pleiotropic functions as part of multiple gene regulatory networks
Different origins of the Pax6 duplicates of zebrafish versus acanthopterygian teleosts To identify duplicate Pax6 genes in teleost fishes other than zebrafish, we searched the genomes of four other teleost fish species for which genomic sequences were available in the Ensembl database: medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), green spotted pufferfish (Tetraodon nigroviridis) and fugu (Takifugu rubripes)
Comparison of cis-regulatory activity in intron 7 of Pax6 To assess how the presence or absence of particular conserved noncoding elements (CNEs) correlates with functional cis-regulatory activity we focused on intron 7 of the Pax6 gene
Summary
Development is critically dependent on a core set of developmental regulator genes, most of which are highly conserved across metazoans and carry out pleiotropic functions as part of multiple gene regulatory networks. In addition Pax plays important roles in development and maintenance of the endocrine pancreas, the olfactory system and the central nervous system (CNS) where it is required for multiple cellular processes including maintenance of the neuronal progenitor pool at early developmental stages, and neurogenesis at later stages [10]. It is required for cell migration and axon guidance in parts of the brain (reviewed in [11,12]). In humans heterozygous disruption of the gene gives rise to the congenital eye Author Summary
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