Interferon Regulatory Factor 6 (IRF6) is a transcription factor that, in mammals, is required for the differentiation of skin, breast epithelium, and oral epithelium. However, the transcriptional targets that mediate these effects are currently unknown. In zebrafish and frog embryos Irf6 is necessary for differentiation of the embryonic superficial epithelium, or periderm. Here we use microarrays to identify genes that are expressed in the zebrafish periderm and whose expression is inhibited by a dominant-negative variant of Irf6 (dnIrf6). These methods identify Grhl3, an ancient regulator of the epidermal permeability barrier, as acting downstream of Irf6. In human keratinocytes, IRF6 binds conserved elements near the GHRL3 promoter. We show that one of these elements has enhancer activity in human keratinocytes and zebrafish periderm, suggesting that Irf6 directly stimulates Grhl3 expression in these tissues. Simultaneous inhibition of grhl1 and grhl3 disrupts periderm differentiation in zebrafish, and, intriguingly, forced grhl3 expression restores periderm markers in both zebrafish injected with dnIrf6 and frog embryos depleted of Irf6. Finally, in Irf6 deficient mouse embryos, Grhl3 expression in the periderm and oral epithelium is virtually absent. These results indicate that Grhl3 is a key effector of Irf6 in periderm differentiation.

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An epidermis surrounds all vertebrates, forming a water barrier between the external environment and the internal space of the organism. In the zebrafish, the embryonic epidermis consists of an outer enveloping layer (EVL) and an inner basal layer that have distinct embryonic origins. Differentiation of the EVL requires the maternal effect gene poky/ikk1 in EVL cells prior to establishment of the basal layer. This requirement is transient and maternal Ikk1 is sufficient to allow establishment of the EVL and formation of normal skin in adults. Similar to the requirement for Ikk1 in mouse epidermis, EVL cells in poky mutants fail to exit the cell cycle or express specific markers of differentiation. In spite of the similarity in phenotype, the molecular requirement for Ikk1 is different between mouse and zebrafish. Unlike the mouse, EVL differentiation requires functioning Poky/Ikk1 kinase activity but does not require the HLH domain. Previous work suggested that the EVL was a transient embryonic structure, and that maturation of the epidermis required replacement of the EVL with cells from the basal layer. We show here that the EVL is not lost during embryogenesis but persists to larval stages. Our results show that while the requirement for poky/ikk1 is conserved, the differences in molecular activity indicate that diversification of an epithelial differentiation program has allowed at least two developmental modes of establishing a multilayered epidermis in vertebrates.

Centrosomes consist of a pair of cylindrical centrioles oriented perpendicularly to each other and function as the main microtubule organizing center of the cell. Centrosomes also play roles in intracellular trafficking, cell polarity, organization of the mitotic spindle, and organization of primary cilia. The two centrioles within a centrosome each self-duplicate once during the cell cycle, but are asymmetric in their maturation state. This results in two differently aged centrosomes, an older mother, and a younger daughter, which are then separated upon cell division so that each new cell inherits one centrosome. Centrosome inheritance, therefore, is asymmetric and has been shown to influence cell fate in Drosophila neuroblasts and male germline stem cells (Yamashita and Fuller, 2008). A recent study of mouse cortex neuroepithelia found that inheritance of the mother centrosome correlated with progenitor fate, while inheritance of the daughter the centrosome segregated with differentiated progeny (Wang et al., 2009). To address the question of whether centrosome inheritance influences cell fate in vivo, we have generated a zebrafish transgenic line, ß actin:Kaede-Centrin, that takes advantage of the photoconvertable Kaede protein fused to the centrosomal protein Centrin, to label differently aged centrosomes. Importantly, we found that Centrin protein stably binds to centrosomes, thus photoconverted Kaede-Centrin protein will remain steadily bound so that a centrosome labeled with Kaede-Centrin can be followed through several cell cycles. Currently we are using the transgenic line to study the role of asymmetric centrosome inheritance in cell fate decisions in the retina.Epidermal differentiation in the zebrafish begins with formation of the enveloping layer (EVL). Rapid differentiation of the EVL is required to protect the developing embryo from the hypotonic environment. The EVL differentiates directly from the external cells of the blastoderm. These cells dramatically slow their cell cycle at the onset of zygotic transcription, form a permeability barrier and express differentiation markers, including many homologs of genes expressed in differentiating mammalian epidermis. The EVL differentiates prior to formation of the p63 positive epidermal basal layer raising questions as to the degree of conservation of pathways regulating differentiation of the EVL and mammalian epidermis. Zebrafish poky mutants have defective EVL differentiation and the blastoderm clears and lyses as control embryos are initiating gastrulation. poky encodes the zebrafish homolog of mammalian IKK1/IKKa, a kinase required for epidermal differentiation in the mouse. To examine the extent of molecular conservation in the pathways that regulate epidermal differentiation between zebrafish and mammals we have examined the expression and activities of additional zebrafish homologs of mammalian epidermal differentiation genes. Among these we have identified the zebrafish homolog of Ripk4, another kinase required for epidermal differentiation i...