Evaluating roles of KLF4a in the differentiation of intestinal cell during embryonic development

• Main Authors: I-Chen Li, 李怡禛
• Other Authors: Sheng-Ping L. Hwang
• Format: Others
• Language: en_US
• Online Access: http://ndltd.ncl.edu.tw/handle/48554437467045615278

碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 95 === Abstract zebrafish KLF4a cDNA encoding a zinc finger transcription factor was cloned previously and the amino acid sequence shares 65.8 % similarity with mammalian KLF4. Previous study has shown that one can effectively inhibit KLF4a expression in zebrafish embryos by co-injecting 4 ng each of KLF4a specific antisense morpholino oligomers including KLF4a-MO and KLF4a-MO3. As a result, malformation of midbrain-hindbrain boundary, reduction in the area of midbrain ventricle, absence of intestinal epithelial folding, and reduction in the intestinal lumen were detected in KLF4a morphant embryos at different developmental stages. In order to investigate possible function of KLF4a on the differentiation of different intestinal cell types, whole-mount in situ hybridization on agr2, which is expressed in most of secretory cells including goblet cells, and alcian blue staining that can stain acidic mucins within goblet cells were used to compare goblet cell numbers in KLF4a-MO1 & KLF4a-MO3 co-injected and wild type embryos. The number of alcian blue stained goblet cells in 102 hpf KLF4a morphants was less than wild-type embryos while the number of alcian blue staining cells in 120 hpf and 144 hpf KLF4a morphants are similar to wild-type embryos. However the mature alcian blue stained goblet cells with larger size and darker blue color in mucous granules were less in 102 hpf, 120 hpf, and 144 hpf KLF4a morphants as compared with respective wild type embryos. These findings indicate that KLF4a may regulate terminal differentiation of goblet cell. The observation that sparse distribution of glucagon-positive cells in the intestine of 120 hpf KLF4a morphant also indicates the terminal differentiation of enteroendocrine cell was also regulated by KLF4a. Furthermore decreased alkaline phosphatase activity and IFABP expression were detected in respective 75 hpf KLF4a morphant embryos. This result indicates KLF4a may mediate the differentiation of enterocytes. Delta/Notch signaling plays a crucial role in secretory fate decision of zebrafish intestinal epithelium. Expression levels of Notch3/5, Notch 4/6, and Delta D were similar in 75 hpf KLF4a morphant embryos as compared with wild type. In contrast, the expression of KLF4a was higher in embryos which Notch signaling was inhibited by DAPT, a γ-secretase inhibitor. It indicates that KLF4a was negatively regulated by Notch signaling. In order to investigate possible function of KLF4a on intestinal cell proliferation, immunohistochemistry using anti Phospho-Histone H3 antibody was used to detect those intestinal cells in the M phase of cell cycle. Results showed the presence of similar numbers of Phospho-Histone H3 stained intestinal cells in both 74 hpf KLF4a-MO1 & KLF4a-MO3 co-injected and wild type embryos. Overall, our results demonstrate that KLF4a may modulate cell differentiation of enterocyte. KLF4a may regulate the terminal differentiation of two secretory cells including goblet and enteroendocrine cells and this activity is negatively regulated by Notch signaling. Whereas KLF4a may not regulate cell proliferation in the intestine of zebrafish embryos.