Institute of Cellular and Organismic Biology (ICOB), Academia Sinica, Taipei, Taiwan, Republic of China.
Department of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan, Republic of China.
PLoS Genet. 2019 Apr 1;15(4):e1008058. doi: 10.1371/journal.pgen.1008058. eCollection 2019 Apr.
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.
在鱼类的皮肤和鳃表皮中，离子细胞与角质形成细胞一起发育，维持体液离子的动态平衡，这对于适应环境波动至关重要。已知斑马鱼胚胎中的离子细胞祖先是通过涉及 DeltaC（Dlc）-Notch 介导的侧向抑制的模式形成过程从 p63+表皮干细胞中特化而来的，该过程将分散的 dlc+细胞选择进入离子细胞祖细胞命运。然而，离子细胞祖细胞群体如何被调节的机制尚不清楚。Krüppel 样因子 4（Klf4）转录因子先前被牵连到哺乳动物皮肤表皮的终末分化中，并且以其在组织背景依赖性方式对细胞增殖的双功能调节而闻名。在这里，我们报道了斑马鱼 Klf4 在胚胎皮肤发育过程中对腹侧外胚层的新作用。我们发现 Klf4 在 90% 胚环延伸后从腹侧外胚层的 p63+表皮干细胞中表达。klf4 表达的敲低或敲除降低了 p63+干细胞的增殖率，导致 p63+干细胞、dlc-p63+角蛋白前体细胞和 dlc+p63+离子细胞祖细胞的数量减少。这些减少随后导致角蛋白细胞和离子细胞密度降低，这是由于众所周知的细胞周期调节剂 p53 和 cdkn1a/p21 的上调所致。此外，对 dlc 启动子中的 KLF 基序进行突变分析，结合 VP16-klf4 或 engrailed-klf4 mRNA 过表达分析，表明 Klf4 可以结合 dlc 启动子并通过直接抑制 dlc 表达来调节侧向抑制。这一观点进一步得到了在 klf4 过表达或敲低胚胎中观察到的侧向抑制结果的支持。总体而言，我们的实验描绘了斑马鱼 Klf4 在调节整个早期干细胞阶段到终末分化开始的离子细胞祖细胞群体中的新作用，这依赖于 Dlc-Notch 介导的侧向抑制。
