Discovery and assessment of conserved Pax6 target genes and enhancers

Abstract

The characterization of transcriptional networks (TNs) is essential for understanding complex biological phenomena such as development, disease, and evolution. In this study, we have designed and implemented a procedure that combines in silico target screens with zebrafish and mouse validation, in order to identify cis-elements and genes directly regulated by Pax6. We chose Pax6 as the paradigm because of its crucial roles in organogenesis and human disease. We identified over 600 putative Pax6 binding sites and more than 200 predicted direct target genes, conserved in evolution from zebrafish to human and to mouse. This was accomplished using hidden Markov models (HMMs) generated from experimentally validated Pax6 binding sites. A small sample of genes, expressed in the neural lineage, was chosen from the predictions for RNA in situ validation using zebrafish and mouse models. Validation of DNA binding to some predicted cis-elements was also carried out using chromatin immunoprecipitation (ChIP) and zebrafish reporter transgenic studies. The results show that this combined procedure is a highly efficient tool to investigate the architecture of TNs and constitutes a useful complementary resource to ChIP and expression data sets because of its inherent spatiotemporal independence. We have identified several novel direct targets, including some putative disease genes, among them Foxp2; these will allow further dissection of Pax6 function in development and disease.

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

“…Nuclear protein gene Mab21l1 (Yamada et al, 2003) and the secreted frizzled-related protein gene Sfrp2 (Wawersik et al, 1999) are also downregulated in the Sey mutant. In a zebrafish study of pax6 targets (Coutinho et al, 2011) a number of genes are downregulated upon morpholino antisense oligonucleotide treatment of embryos and studies on specific differentiated tissues in the mouse, e.g. the lens, provide a more global analysis of later Pax6 targets (Wolf et al, 2009).…”

The zebrafish eye—a paradigm for investigating human ocular genetics

“…32 Interestingly, morpholino-induced knockdown of pax6a or simultaneous knockdown of pax6a and pax6b results in a similar but more severe phenotype; morphants present with reduced body size and neural tube girth, morphologically abnormal brain development, and microphthalmia. 33 TALENs-mediated knockdown of pax6a and pax6b simultaneously, by targeting conserved sequences between these paralogs, resulted in eye phenotypes including microphthalmia, closely recapitulating the ocular morphant phenotype described. 34 The strategy employed not only confirms the efficiency of TALENsmediated gene disruption, but also emphasises the ability to induce bi-allelic and paralogous genome editing, which is important when investigating genes that are duplicated in the zebrafish genome.…”

Prdm12 specifies V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes in Xenopus

“…To determine whether Pax6 binds this enhancer, ChIP-qPCR was performed on caudal neural tube tissues from E11.5 embryos using an anti-Pax6 antibody (Coutinho et al, 2011) and primers that span the identified potential Pax6 binding sites. This region was found to be significantly enriched compared with a negative control region (Kirrel2) (Borromeo et al, 2014) (supplementary material Fig.…”