Apical ectodermal ridge (AER) is an ectodermal structure essential for induction, patterning and outgrowth of limbs. Fgf8 is the representative molecule produced in AER, and is indispensable for limb bud outgrowth. Meanwhile, Jag2 is also highly expressed in AER and suppresses Fgf8 production in AER through Notch signaling pathway. However, transcriptional regulation of these essential molecules in AER has been unrevealed. Transcription factor p63 is highly expressed in AER, and p63 null mouse neonates present severely truncated limbs. Multiple transcript variants of p63 have been identified, and previous studies indicate that these phenotypes in p63 null mice are mainly caused by hypoplasia of AER; however, the underlying molecular mechanisms and specific roles of p63 transcript variants are not fully understood. The present study aimed to reveal specific expressions and roles of each p63 transcript variant in limb development.
FACS analysis of limb bud cells obtained from mouse E11.5 embryos showed that both dNp63 and TAp63 were expressed much more abundantly in AER cells than in non-AER cells. In AER-specific p63 knockout mice, forelimb autopods were truncated, and hindlimb zeugopods and autopods were hypoplastic. Expressions of Fgf8 and Jag2 were significantly decreased in limb buds of E11.5 AER-specific p63 knockout embryos. When we introduced dNp63 and TAp63 into mouse ES cells, Fgf8 and Jag2 were upregulated, respectively. Luciferase assay using B16 cells showed that dNp63 mainly enhanced promoter activity of Fgf8, while TAp63 strongly enhanced that of Jag2. Chromatin immunoprecipitation assay using mouse ES cells confirmed binding of p63 protein to the responsive elements identified in Fgf8 and Jag2 promoters.
Considering that Fgf8 is essential for maintenance of AER and that Jag2/Notch signaling negatively regulates growth and function of AER, dNp63 and TAp63 may regulate limb development in different ways through transcriptional induction of different target genes in AER.