Drosophila grh mutant embryos have slack and fragile cuticles, as well as “grainy” and discontinuous head skeletons. Null mutations are lethal, as the embryos fail to develop past the embryonic/larval transition point due to their extremely fragile epidermal barriers. These phenotypes clearly point to defects in the formation of chitin-based cuticular structures in grh mutant embryos. Which encodes dopa decarboxylase, an enzyme required to generate the reactive quinone molecules used to cross-link chitin fibers and proteins in the Drosophila cuticle. Furthermore, grh embryos are permeable to exogenously applied dyes, and the removal of GRH from imaginal disc cells results in reduced expression of at least two cell-adhesion genes. These findings suggest that the paracellular integrity of the epithelial barrier underlying the cuticle becomes compromised in Drosophila grh mutants. In addition to the developmental functions of GRH in Drosophila, it is also necessary for the proper expression of several cuticular-barrier genes that are activated during the regenerative process following epidermal wounding. GRH family proteins are also important for epidermal-barrier formation in the distantly related invertebrate C. elegans. RNAi targeted against Ce-Grh-1 results in embryos with a fragile and puckered hypodermis – a similar phenotype to that seen in Drosophila. Ce-Grh-1 binds the same palindromic consensus DNA sequences as Drosophila GRH, and the Ddc gene in C. elegans has GRH binding sites upstream of its promoter. Strikingly, conservation of GRH family transcription factor function extends to vertebrates as well, despite vast differences in the structural components of epidermal barriers between and within protostome and deuterostome animals. In Xenopus laevis, expression of a dominant negative form of XGRHL1 leads to a malformed epidermis, partly due to lowered expression of keratin. While the DNA-binding specificity of GRH family proteins has been conserved between protostome and deuterostome animals, the downstream effectors of GRH-like proteins in distantly related species do not appear to be homologous, but instead carry out analogous functions suited to the specific barrier being generated or GDC-0879 905281-76-7 regenerated after wounding. For instance, the epidermal defects in Grhl3-deficient mice correlate with reduced levels of transglutaminase 1 transcription, as well as reduced transcription for many genes that are structural barrier components of differentiated corneocytes. Transglutaminase 1 is an enzyme necessary for the cross-linking of keratin and other proteins in the mammalian epidermis, and it plays an analogous role to that of dopa decarboxylase in the Drosophila cuticle. In sum, there exists a high-level functional conservation of GRH proteins as regulators of epidermal integrity and wound healing in both protostome and deuterostome animals, despite the significant structural differences in barrier composition across the animal kingdom. This functional conservation is reminiscent of other cases in which high-level transcription factor function has been conserved over great evolutionary time despite the drift of specific downstream effectors. Since the function of GRH-like proteins in epidermal-barrier formation and wound healing appears well conserved in triploblastic animals.