Zebrafish Krüppel-Like Factor 4a Represses Intestinal Cell Proliferation and Promotes Differentiation of Intestinal Cell Lineages

BackgroundMouse krüppel-like factor 4 (Klf4) is a zinc finger-containing transcription factor required for terminal differentiation of goblet cells in the colon. However, studies using either Klf4−/− mice or mice with conditionally deleted Klf4 in their gastric epithelia showed different results in the role of Klf4 in epithelial cell proliferation. We used zebrafish as a model organism to gain further understanding of the role of Klf4 in the intestinal cell proliferation and differentiation.
Methodology/Principal FindingsWe characterized the function of klf4a, a mammalian klf4 homologue by antisense morpholino oligomer knockdown. Zebrafish Klf4a shared high amino acid similarities with human and mouse Klf4. Phylogenetic analysis grouped zebrafish Klf4a together with both human and mouse Klf4 in a branch with high bootstrap value. In zebrafish, we demonstrate that Klf4a represses intestinal cell proliferation based on results of BrdU incorporation, p-Histone 3 immunostaining, and transmission electron microscopy analyses. Decreased PepT1 expression was detected in intestinal bulbs of 80- and 102-hours post fertilization (hpf) klf4a morphants. Significant reduction of alcian blue-stained goblet cell number was identified in intestines of 102- and 120-hpf klf4a morphants. Embryos treated with γ-secretase inhibitor showed increased klf4a expression in the intestine, while decreased klf4a expression and reduction in goblet cell number were observed in embryos injected with Notch intracellular domain (NICD) mRNA. We were able to detect recovery of goblet cell number in 102-hpf embryos that had been co-injected with both klf4a and Notch 1a NICD mRNA.
Conclusions/SignificanceThis study provides in vivo evidence showing that zebrafih Klf4a is essential for the repression of intestinal cell proliferation. Zebrafish Klf4a is required for the differentiation of goblet cells and the terminal differentiation of enterocytes. Moreover, the regulation of differentiation of goblet cells in zebrafish intestine by Notch signaling at least partially mediated through Klf4a.

研究背景 小鼠krüppel样因子4（Klf4）是一类含锌指结构的转录因子，对结肠杯状细胞的终末分化具有必需作用。然而，分别采用Klf4基因敲除（Klf4−/−）小鼠或胃上皮条件性敲除Klf4的小鼠开展的相关研究，在Klf4调控上皮细胞增殖的功能上得到了不一致的结果。本研究以斑马鱼为模式生物，旨在进一步阐明Klf4在肠道细胞增殖与分化中的作用。研究方法与主要结果 我们通过反义吗啉代寡核苷酸（antisense morpholino oligomer）敲低哺乳动物Klf4的同源基因klf4a，对其功能进行了系统表征。斑马鱼Klf4a与人类、小鼠Klf4的氨基酸序列具有高度同源性。系统发育分析显示，斑马鱼Klf4a与人类、小鼠Klf4共同聚类于一个bootstrap值较高的进化分支中。通过BrdU掺入实验、磷酸化组蛋白3（p-Histone 3）免疫染色以及透射电子显微镜分析，我们证实斑马鱼Klf4a可抑制肠道细胞增殖。在受精后80小时与102小时（hpf）的klf4a吗啉代敲低胚胎的肠球中，检测到PepT1的表达水平显著下调。在受精后102小时与120小时的klf4a敲低胚胎肠道内，阿尔辛蓝染色显示杯状细胞数量显著减少。经γ-分泌酶抑制剂处理的胚胎，其肠道内klf4a的表达水平升高；而注射Notch胞内域（Notch intracellular domain, NICD）mRNA的胚胎，则表现为klf4a表达下调且杯状细胞数量减少。我们还发现，同时注射klf4a与Notch 1a NICD mRNA的102小时胚胎中，杯状细胞数量得以恢复。研究结论与意义 本研究提供了体内实验证据，表明斑马鱼Klf4a对肠道细胞增殖的抑制作用不可或缺。斑马鱼Klf4a对于杯状细胞的分化以及肠上皮细胞的终末分化均为必需。此外，Notch信号通路对斑马鱼肠道杯状细胞分化的调控，至少部分是通过Klf4a介导实现的。