Distinct roles for Distal-less genes Dlx3 and Dlx5 in regulating ectodermal development in Xenopus

In vertebrates, there are six or more copies of genes related to the Drosophila pattern formation homeodomain gene Distal-less. Among this family, Dlx3 and Dlx5 share extensive sequence homology and have similar, but distinctive, expression patterns, suggesting that these two factors may have substantially redundant developmental functions. Here we show that at the earliest phases of embryogenesis in Xenopus, there are significant differences between Dlx3 and Dlx5 expression and that this correlates with different functions in the restriction of neural crest and neural plate boundaries, respectively.     

Phosphorylation of murine homeodomain protein Dlx3 by protein kinase C

The Dlx3 homeodomain gene is expressed in terminally differentiated murine epidermal cells. As demonstrated for differentiation-specific granular markers, Dlx3 is activated in primary mouse keratinocytes cultured in vitro by increasing the level of the extracellular Ca(2+). This activation is mediated through a protein kinase C-dependent (PKC) pathway. In this study, we investigated whether PKC can modulate the activity of murine Dlx3 protein. Using in vitro kinase assays, we show that PKC enzymes phosphorylate the Dlx3 protein. Using keratinocyte nuclear extracts for the kinase reaction, we determined that Dlx3 protein is phosphorylated, and the phosphorylation is inhibited by the PKC-specific inhibitor GF109203X, suggesting that Dlx3 is phosphorylated by PKC in vivo. Of the PKC isoforms present in the epidermis, we tested alpha, delta, epsilon and zeta. Dlx3 is primarily phosphorylated by PKC alpha. By deletion and mutational analysis, we show that the serine residue S(138), located in the homeodomain of Dlx3 protein, was specifically phosphorylated by PKC. The phosphorylation of purified Dlx3 proteins by PKC partially inhibited formation of complexes between Dlx3 protein and DNA. These results suggest that Dlx3 protein can be directly phosphorylated by PKC and this affects the DNA binding activity of Dlx3.     

Ectopic expression of the Dlx genes induces glutamic acid decarboxylase and Dlx expression.

The expression of the Dlx homeobox genes is closely associated with neurons that express gamma-aminobutyric acid (GABA) in the embryonic rostral forebrain. To test whether the Dlx genes are sufficient to induce some aspects of the phenotype of GABAergic neurons, we adapted the electroporation method to ectopically express DLX proteins in slice cultures of the mouse embryonic cerebral cortex. This approach showed that ectopic expression of Dlx2 and Dlx5 induced the expression of glutamic acid decarboxylases (GADs), the enzymes that synthesize GABA. We also used this method to show cross-regulation between different Dlx family members. We find that Dlx2 can induce Dlx5 expression, and that Dlx1, Dlx2 and Dlx5 can induce expression from a Dlx5/6-lacZ enhancer/"reporter construct.     

Fgf-dependent otic induction requires competence provided by Foxi1 and Dlx3b

Background  

The inner ear arises from a specialized set of cells, the otic placode, that forms at the lateral edge of the neural plate adjacent to the hindbrain. Previous studies indicated that fibroblast growth factors (Fgfs) are required for otic induction; in zebrafish, loss of both Fgf3 and Fgf8 results in total ablation of otic tissue. Furthermore, gain-of-function studies suggested that Fgf signaling is not only necessary but also sufficient for otic induction, although the amount of induced ectopic otic tissue reported after misexpression of fgf3 or fgf8 varies among different studies. We previously suggested that Foxi1 and Dlx3b may provide competence to form the ear because loss of both foxi1 and dlx3b results in ablation of all otic tissue even in the presence of a fully functional Fgf signaling pathway.     

Dlx5 and Dlx6 homeobox genes are required for specification of the mammalian vestibular apparatus

The mammalian inner ear is a complex organ that develops from a surface ectoderm into distinct auditory and vestibular components. Congenital malformation of these two components resulting from single or multiple gene defects is a common clinical occurrence and is observed in patients with split hand/split foot malformation, a malformation which is phenocopied by Dlx5/6 null mice. Analysis of mice lacking Dlx5 and Dlx6 homeobox genes identified their restricted and combined expression in the otic epithelium as a crucial regulator of vestibular cell fates. Otic induction initiates without incident in Dlx5/6(-/-) embryos, but dorsal otic derivatives including the semicircular ducts, utricle, saccule, and endolymphatic duct fail to form. Dlx5 and Dlx6 seem to influence vestibular cell fates by restricting Pax2 and activating Gbx2 and Bmp4 expression domains. Given their proximity to the disease locus and the observed phenotype in Dlx5/6 null mice, Dlx5/6 are likely candidates to mediate the inner ear defects observed in patients with split hand/split foot malformation.