Sequencing of Pax6 Loci from the Elephant Shark Reveals a Family of Pax6 Genes in Vertebrate Genomes, Forged by Ancient Duplications and Divergences

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.

Functional characteristics of novel pancreatic Pax6 regulatory elements

“…In contrast to the downstream region of PAX6 , which has a significant role in disease aetiology of aniridia patients, the upstream region towards RCN1 is less well studied; it includes the only E-200 element and E-55 cluster, implicating this region as a regulatory ‘desert’ even though it is known to contain a number of evolutionarily conserved sites (23,24). As cis -regulatory elements are key to modulation of gene expression through transcription factor binding and are increasingly linked to complex disease phenotypes, we set out to identify and characterize novel PAX6 regulatory elements specific to pancreatic expression, owing to its roles in pancreatic development and T2D.…”

Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients

“…Rather, there is another possibility that a second pax6 -related gene, pax6.2, may have redundant function in Xenopus embryos and may partially rescue pax6 mutations. It was recently discovered that non-mammalian vertebrates all appear to have this second pax6 -like gene and that the gene has been lost in birds and mammals (Ravi et al, 2013). X. tropicalis Pax6.2 (XM_004912020) consists of 281 amino acids and has 85% amino-acid identity with the region of amino acids 137–424 of X. tropicalis Pax6.…”

“…We have already used CRISPR-mediated gene targeting to mutate the pax6.2 and are currently raising animals to evaluate the contribution of this gene to eye development. It has been shown that in zebrafish, an antisense morpholino oligonucleotide directed against pax6.2 does reduce eye size in wild-type embryos (Ravi et al, 2013). Our analysis of expression of pax6.2 is consistent with a role in retinal phenotype in the pax6 mutant because, as noted earlier, expression is restricted to the dorsal retina (which persists in the mutant), but also we note that pax6.2 is not expressed in the lens, and that the lens is completely absent in the pax6 mutant.…”

SBE6: a novel long-range enhancer involved in driving sonic hedgehog expression in neural progenitor cells

“…The amplicon was cloned directly into an hsp68-LacZ vector containing a P4-P1r entry cassette [28]. Transgenic mice were generated by microinjection into mouse oocytes, and the analysis of transgenic lines was carried out as previously described [28]. Two independently derived E11.5 SBE6.1-LacZ embryos were independently analysed; one a transient insertion, the second from a stable line.…”