Zebrafish Klf4 maintains the ionocyte progenitor population by regulating epidermal stem cell proliferation and lateral inhibition.

In the skin and gill epidermis of fish, ionocytes develop alongside keratinocytes and maintain body fluid ionic homeostasis that is essential for adaptation to environmental fluctuations. It is known that ionocyte progenitors in zebrafish embryos are specified from p63(+) epidermal stem cells through a patterning process involving DeltaC (Dlc)-Notch-mediated lateral inhibition, which selects scattered dlc(+) cells into the ionocyte progenitor fate. However, mechanisms by which the ionocyte progenitor population is modulated remain unclear. Kruppel-like factor 4 (Klf4) transcription factor was previously implicated in the terminal differentiation of mammalian skin epidermis and is known for its bifunctional regulation of cell proliferation in a tissue context-dependent manner. Here, we report novel roles for zebrafish Klf4 in the ventral ectoderm during embryonic skin development. We found that Klf4 was expressed in p63(+) epidermal stem cells of the ventral ectoderm from 90% epiboly onward. Knockdown or knockout of klf4 expression reduced the proliferation rate of p63(+) stem cells, resulting in decreased numbers of p63(+) stem cells, dlc(-)p63(+) keratinocyte progenitors and dlc(+) p63(+) ionocyte progenitor cells. These reductions subsequently led to diminished keratinocyte and ionocyte densities and resulted from upregulation of the well-known cell cycle regulators, p53 and cdkn1a/p21. Moreover, mutation analyses of the KLF motif in the dlc promoter, combined with VP16-klf4 or engrailed-klf4 mRNA overexpression analyses, showed that Klf4 can bind the dlc promoter and modulate lateral inhibition by directly repressing dlc expression. This idea was further supported by observing the lateral inhibition outcomes in klf4-overexpressing or knockdown embryos. Overall, our experiments delineate novel roles for zebrafish Klf4 in regulating the ionocyte progenitor population throughout early stem cell stage to initiation of terminal differentiation, which is dependent on Dlc-Notch-mediated lateral inhibition. Author summary The skin epidermis of terrestrial vertebrates is composed of a stratified epithelium, and requires kruppel-like factor 4 (KLF4) to establish a functional permeability barrier that protects animals from dehydration. In contrast, fish must tolerate variations in salinity and pH of the aquatic environment. As such, the fish skin epidermis is composed of keratinocytes and ionocytes, which transport ions and acid-base equivalents to maintain ionic and acid-base homeostasis of body fluids. In the present study, we used embryonic zebrafish as a model to investigate how Klf4 modulates the cell densities of matured ionocytes from early stem cell stages through the initiation of terminal differentiation by Dlc-Notch-mediated lateral inhibition. We showed that Klf4 promotes cell proliferation in epidermal stem cells, where it represses p53 expression and prevents cdkn1a/p21 induction. Additionally, Klf4 regulates the ionocyte progenitor population by directly repressing dlc expression and modulating lateral inhibition. Our findings describe novel roles for zebrafish Klf4 in ionocyte development and provide insights into the mechanism by which Klf4 regulates proliferation-differentiation balance in epidermal stem cells.