Abstract
The transcription factor p63 is a master regulator of ectoderm development. Although previous studies show that p63 triggers epidermal differentiation in vitro, the roles of p63 in developing embryos remain poorly understood. Here, we use zebrafish embryos to analyze in vivo how p63 regulates gene expression during development. We generate tp63-knock-out mutants that recapitulate human phenotypes and show down-regulated epidermal gene expression. Following p63-binding dynamics, we find two distinct functions clearly separated in space and time. During early development, p63 binds enhancers associated to neural genes, limiting Sox3 binding and reducing neural gene expression. Indeed, we show that p63 and Sox3 are co-expressed in the neural plate border. On the other hand, p63 acts as a pioneer factor by binding non-accessible chromatin at epidermal enhancers, promoting their opening and epidermal gene expression in later developmental stages. Therefore, our results suggest that p63 regulates cell fate decisions during vertebrate ectoderm specification.
Highlights
The transcription factor p63 is a master regulator of ectoderm development
The transcription factor (TF) p63 belongs to the p53 tumorsuppressor protein family and is a master regulator of ectoderm development that is known to play a key role during epidermal specification[1,2]
P63 is a likely candidate to act as a pioneer TF, which are TFs able to bind to non-accessible sites and open them by displacing nucleosomes, alone or assisted by other factors, in order to allow the binding of non-pioneer TFs and other proteins[25,26,27,28]
Summary
Deregulated epidermal expression in tp63−/− zebrafish mutant. In order to study the effects of the lack of p63 in vertebrate ectoderm development, we generated a tp[63] knockout zebrafish model using the CRISPR/Cas[9] genome editing system[33]. In tp63−/− embryos, in situ hybridization of tbx5a shows the formation of the mesodermal anlage that will generate the fin bud, but the apical ectodermal ridge (AER) is not formed and the appendage does not grow (Fig. 1b). They were able to grow until more than 40 hpf and pigment cells were less organized but normally visible, the epidermis stopped developing and protecting the animals. Analysis of enrichment of WT expression patterns showed an over-representation of genes expressed in the epidermis for the down-regulated genes and in mesoderm tissues for the up-regulated ones (Fig. 1f). This result suggests that p63 might be regulating gene expression in a tissueand stage-dependent manner through different BSs
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