Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice

Abstract

Epidermal development requires the transcription factor p63, as p63À/À mice are born dead, without skin. The gene expresses two proteins, one with an amino-terminal transactivation domain (TAp63) and one without (DNp63), although their relative contribution to epidermal development is unknown. To address this issue, we reintroduced TAp63a and/or DNp63a under the K5 promoter into p63À/À mice by in vivo genetic complementation. Whereas p63À/À and p63À/À;TA mice showed extremely rare patches of poorly differentiated keratinocytes, p63À/À;DN mice showed significant epidermal basal layer formation. Double TAp63a/ DNp63a complementation showed greater patches of differentiated skin; at the ultrastructural level, there was clear reformation of a distinct basal membrane and hemidesmosomes. At the molecular level, DNp63 regulated expression of genes characteristic of the basal layer (K14), interacting (by Chip, luc assay) with the third p53 consensus site. Conversely, TAp63 transcribed the upper layer's genes (Ets-1, K1, transglutaminases, involucrin). Therefore, the two p63 isoforms appear to play distinct cooperative roles in epidermal formation.

p63 in epithelial development

“…These data [27,29] are therefore somewhat in contrast with the results from Roop's group [24,31], who showed that, in vivo siRNA for TAp63 produced a dramatic phenotype, with failure to organize pluristratified epithelia but no significant effect was observed on the basal layer. However, in our model [27] we investigated the role in early development using a genetically pure model, with the total absence of one isoform, the elegant in vivo study by Roop using siRNA addressed a slightly different question, representing a model of isoform unbalance.…”

“…Indeed, a detailed analysis of the expression of K5 and loricrin in the epidermis of E16 wt and p63 -/-mice, revealed that cells remaining on the surface of the p63 embryos are committed to an epidermal lineage that progress through differentiation [26]. We have obtained similar evidences using a different approach, we complemented p63-deficient mice by expressing TAp63 and Np63 transgenes under the control of the K5 promoter [27]. Hence, by crossing TAp63 or Np63 epidermal transgenic mice into p63-/-mice, we generated mice expressing one single isoform to identify the relative contribution of each isoform to the development of the epidermis and in thymic epithelial cells [27; 28].…”

“…Although we were unable to obtain a full reversion of the phenotype of the null mice, the study showed that transgenic mice complemented with Np63 (p63 -/-; N) developed a significant basal layer, while transgenic mice complemented with TAp63 (p63 -/-;TA) did not differ from p63 -/-mice. Double complemented mice exhibited a greater degree of re-epithelialization with expression of keratins of the basal layer (K5 and K14), and differentiation-specific markers such as K1 and loricrin but complete cornification remained impaired [27]. These data suggest that, although Np63 plays a pivotal role in development, any contribution by TAp63 is only concomitant or subsequent to the function of Np63 .…”

“…In addition to TAp63, Np63 also showed transcriptional activity which is due to the presence of a C-terminal TA domain see Fig.1C. Analysis of target genes induced by either TAp63 or Np63 in Saos2 cells showed that TAp63α mainly regulates terminal differentiation markers such as K1, K10, profilaggrin and involucrin, while Np63α induces the early differentiation marker K14 [27]. These data suggested that Np63 is important for maintaining the proliferative potential of the basal layer, whereas TAp63 contributes by acting synergistically and/or subsequently to Np63 to allow differentiation [27].…”

“…Analysis of target genes induced by either TAp63 or Np63 in Saos2 cells showed that TAp63α mainly regulates terminal differentiation markers such as K1, K10, profilaggrin and involucrin, while Np63α induces the early differentiation marker K14 [27]. These data suggested that Np63 is important for maintaining the proliferative potential of the basal layer, whereas TAp63 contributes by acting synergistically and/or subsequently to Np63 to allow differentiation [27]. Furthermore, these results are consistent with a study based on RNA interference directed against p63 isoforms, in which the authors demonstrated that Np63 isoforms are the main mediators of p63 effects and that TAp63 isoforms contribute to later stages of differentiation in mature keratinocytes.…”