ΔNp63 knockout mice reveal its indispensable role as a master regulator of epithelial development and differentiation

Abstract

SUMMARYThe transcription factor p63 is important in the development of the skin as p63-null mice exhibit striking defects in embryonic epidermal morphogenesis. Understanding the mechanisms that underlie this phenotype is complicated by the existence of multiple p63 isoforms, including TAp63 and DNp63. To investigate the role of DNp63 in epidermal morphogenesis we generated DNp63 knock-in mice in which the DNp63-specific exon is replaced by GFP. Homozygous DNp63 gfp/gfp animals exhibit severe developmental anomalies including truncated forelimbs and the absence of hind limbs, largely phenocopying existing knockouts in which all p63 isoforms are deleted. DNp63-null animals show a poorly developed stratified epidermis comprising isolated clusters of disorganized epithelial cells. Despite the failure to develop a mature stratified epidermis, the patches of DNp63-null keratinocytes are able to stratify and undergo a program of terminal differentiation. However, we observe premature expression of markers associated with terminal differentiation, which is unique to DNp63-null animals and not evident in the skin of mice lacking all p63 isoforms. We posit that the dysregulated and accelerated keratinocyte differentiation phenotype is driven by significant alterations in the expression of key components of the Notch signaling pathway, some of which are direct transcriptional targets of DNp63 as demonstrated by ChIP experiments. The analysis of DNp63 gfp/gfp knockout mice reaffirms the indispensable role of the DN isoform of p63 in epithelial biology and confirms that DNp63-null keratinocytes are capable of committing to an epidermal cell lineage, but are likely to suffer from diminished renewal capacity and an altered differentiation fate.

Notch signaling represses p63 expression in the developing surface ectoderm

“…7). Our model is consistent with the ability of p63 to directly regulate K14 expression (Romano et al, 2009), the absence of K14 expression in mice and hESCs lacking p63 isoforms (Mills et al, 1999;Romano et al, 2012;ShalomFeuerstein et al, 2011;Yang et al, 1999) and the expression of p63 as early as E9.5 in mouse embryos (Koster and Roop, 2004). Given the predominant expression of ΔNp63 in physiologically normal epidermal tissues and the requirement of ΔNp63 for epidermal development (Romano et al, 2009), ΔNp63 is likely to be the isoform expressed in the surface ectoderm.…”

“…In a similar fashion, p63 may negatively regulate Notch signaling pathway. In fact, mouse embryos lacking ΔNp63 display defects in Notch signaling, and ΔNp63 directly binds to the promoters of Notch1 (Nguyen et al, 2006) and Notch3 (Romano et al, 2012). Identification of the cell types that express Notch ligands and whether the downstream targets of Notch signaling, such as Rbp-Jk or Hes5, directly control p63 expression will be an avenue of future investigation.…”

“…Complete ablation of all p63 isoforms during mouse development leads to limb truncations, craniofacial malformations and the lack of an intact and functional epidermis (Mills et al, 1999;Yang et al, 1999). However, whether p63 controls epithelial progenitor self-renewal and/or lineage commitment to an epidermal fate remains controversial (Koster and Roop, 2004;Mills et al, 1999;Romano et al, 2012;Yang et al, 1999).…”

p53/p63/p73 in the Epidermis in Health and Disease

“…The p63 isoforms play distinct roles in the control of epidermal development; the DNp63 isoforms are much more abundant in the epidermis compared to TAp63, which is strongly expressed in basal epidermal keratinocytes and is markedly down-regulated in the spinous epidermal layer (Laurikkala et al 2006;Romano et al 2009Romano et al , 2012LeBoeuf et al 2010;Shalom-Feuerstein et al 2011). TAp63 is also expressed in response to stresses such as wound healing (Su et al 2009b).…”

“…TAp63 is also expressed in response to stresses such as wound healing (Su et al 2009b). DNp63 plays a major role in mediating the effects of p63 on epidermal development, whereas TAp63 keeps stem cells within the skin in quiescence to avoid early depletion of these cells and suppresses tumorigenesis in postnatal epidermis (Su et al 2009b;Romano et al 2012;Chakravarti et al 2014).…”