Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan.
PLoS One. 2011;6(6):e20974. doi: 10.1371/journal.pone.0020974. Epub 2011 Jun 8.
Mouse 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 FINDINGS: We 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/SIGNIFICANCE: This 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.
小鼠 Krüppel 样因子 4（Klf4）是一种含锌指的转录因子，对于结肠杯状细胞的终末分化是必需的。然而，使用 Klf4（-/-）小鼠或胃上皮细胞条件性缺失 Klf4 的小鼠进行的研究表明，Klf4 在上皮细胞增殖中的作用结果不同。我们使用斑马鱼作为模型生物，以进一步了解 Klf4 在肠道细胞增殖和分化中的作用。
方法/主要发现：我们通过反义形态发生素寡核苷酸敲低来表征哺乳动物 Klf4 同源物 klf4a 的功能。斑马鱼 Klf4a 与人类和小鼠 Klf4 具有高度的氨基酸相似性。系统发育分析将斑马鱼 Klf4a 与人类和小鼠 Klf4 一起分组在一个具有高自举值的分支中。在斑马鱼中，我们基于 BrdU 掺入、p-Histone 3 免疫染色和透射电子显微镜分析的结果表明，Klf4a 抑制肠道细胞增殖。在 80 和 102 小时受精后（hpf）klf4a 形态发生素的胚胎肠道中检测到 PepT1 表达减少。在 102 和 120 hpf klf4a 形态发生素的胚胎肠道中，鉴定到显著减少的粘蛋白蓝色染色的杯状细胞数量。用γ-分泌酶抑制剂处理的胚胎在肠道中显示出 Klf4a 表达增加，而用 Notch 细胞内结构域（NICD）mRNA 注射的胚胎则观察到 Klf4a 表达减少和杯状细胞数量减少。我们能够检测到在共同注射 klf4a 和 Notch1a NICD mRNA 的 102 hpf 胚胎中，杯状细胞数量的恢复。
结论/意义：本研究提供了体内证据，表明斑马鱼 Klf4a 对于抑制肠道细胞增殖是必需的。斑马鱼 Klf4a 对于杯状细胞的分化和肠上皮细胞的终末分化是必需的。此外，Notch 信号通路至少部分通过 Klf4a 调节斑马鱼肠道中杯状细胞的分化。
