Regulation ofDlx5andDlx6gene expression by p63 is involved in EEC and SHFM congenital limb defects

Abstract

The congenital malformation Split Hand-Foot Malformation (SHFM, or ectrodactyly) is characterized by a medial cleft of hands and feet, and missing central fingers. Five genetically distinct forms are known in humans; the most common (type-I) is linked to deletions of DSS1 and the distalless-related homeogenes DLX5 and DLX6. As Dlx5;Dlx6 double-knockout mice show a SHFM-like phenotype, the human orthologs are believed to be the disease genes. SHFM-IV and Ectrodactyly-Ectodermal dysplasia-Cleft lip (EEC) are caused by mutations in p63, an ectoderm-specific p53-related transcription factor. The similarity in the limb phenotype of different forms of SHFM may underlie the existence of a regulatory cascade involving the disease genes. Here, we show that p63 and Dlx proteins colocalize in the nuclei of the apical ectodermal ridge (AER). In homozygous p63-(null) and p63 EEC (R279H) mutant limbs, the AER fails to stratify and the expression of four Dlx genes is strongly reduced; interestingly, the p63 +/EEC and p63hindlimbs, which develop normally and have a normally stratified AER, show reduced Dlx gene expression. The p63 +/EEC mutation combined with an incomplete loss of Dlx5 and Dlx6 alleles leads to severe limb phenotypes, which are not observed in mice with either mutation alone. In vitro, ⌬Np63␣ induces transcription from the Dlx5 and Dlx6 promoters, an activity abolished by EEC and SHFM-IV mutations, but not by Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) mutations. ChIP analysis shows that p63 is directly associated with the Dlx5 and Dlx6 promoters. Thus, our data strongly implicate p63 and the Dlx5-Dlx6 locus in a pathway relevant in the aetio-pathogenesis of SHFM.

Split hand/foot malformation due to chromosome 7q aberrations(SHFM1): additional support for functional haploinsufficiency as the causative mechanism

“…15 This suggests DSS1 could also be involved in the pathogenesis of SHFM. However, studies in mice with double knockout of Dlx5 and Dlx6 showed typical ectrodactyly, 3,12 whereas Dss1 expression was normal in these mutant mice. 12,16 This suggests downregulation of DLX5 and DLX6 is sufficient to cause SHFM whereas DSS1 expression is normal.…”

“…In mice, Dlx transcription factors have been shown to have a key function in the development and morphogenesis of the limb skeleton. 3,12 Expression of Dlx5 and Dlx6 has been detected in the apical ectodermal ridge of the embryonic limb buds. Double knockout of Dlx5 and Dlx6 in the mouse leads to ectrodactyly.…”

“…This difference in dosage sensitivity of DLX5 and DLX6 suggests that humans are much more sensitive to disruptions in these genes than the mouse heterozygotes. 3,10,12 mice and humans has been shown for LMX1B, for which haploinsufficiency in humans causes nail patella syndrome (OMIM 161200), whereas only complete knockout mice exhibit such a phenotype. 13 The function of the DSS1 gene in limb formation, however, is less clear.…”

“…2,3,16 Second, the phenotype in SHFM4, due to p63 mutations, has been shown to be mediated through both Dlx5 and Dlx6. 3 In p63 À/À newborn mice, the hind limbs are absent, whereas the fore limbs are severely truncated in the distal segment. In these mice the expression of the Dlx5 and Dlx6 genes was significantly reduced.…”

“…2 Five types of non-syndromic SHFM that have been mapped to different loci can be distinguished genetically. 2,3 SHFM2 maps to Xq26 and SHFM5 to 2q24q31. Although the disease genes for SHFM2 and SHFM5 have not been identified yet, a function has been suggested for the HOXD genes in SHFM5.…”