Poky/Ikk1/Ikka promotes Ripk4 function in zebrafish epidermal differentiation

Abstract

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...