Abstract
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. Krüppel-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.
Highlights
Unlike terrestrial vertebrates, teleosts encounter and adapt to ionic, osmotic and acid-base fluctuations in aquatic environments
We showed that Kruppellike factor 4 (Klf4) promotes cell proliferation in epidermal stem cells, where it represses p53 expression and prevents cdkn1a/p21 induction
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
Summary
Teleosts encounter and adapt to ionic, osmotic and acid-base fluctuations in aquatic environments. To maintain body fluid ionic homeostasis, specialized ionocytes (previously called mitochondria-rich cells) develop predominantly in the skin of embryos and gills of adult fish. These cells regulate osmotic homeostasis through transepithelial ion-transport [1, 2]. Five types of ionocytes have been identified in the skin of zebrafish embryos, including H+-ATPase-rich (HR) cells, Na+, K+-ATPase-rich (NaR) cells, Na+-Clcotransporter-expressing (NCC) cells, SLC26-expressing cells, and K+-secreting (KS) cells [3] These ionocytes perform transepithelial H+ secretion/Na+ uptake/NH4+ excretion, Ca2+ uptake, Na+/Cl- uptake, Cl- uptake/HCO3- secretion, and K+ secretion, respectively, by utilizing various transporters located at the apical or basolateral cell surface [3,4,5,6]. Ionocyte progenitors transiently express ΔNp63 from the bud to the 14 somite stage, knockdown of ΔNp63 does not abolish proliferation or differentiation of ionocytes [7, 10], indicating that p63 is unlikely to be a master regulator of proliferation control in ionocyte progenitors
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