Notch Signaling Regulates Differentiation of Secretory Cell Lineage in Zebrafish Intestine.

• Main Authors: Jhuang-Wei Fu, 傅莊維
• Other Authors: Wen-Pin Wang
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/60544285531246578946

碩士 === 慈濟大學 === 分子生物暨人類遺傳學系碩士班 === 100 === In zebrafish, intestinal epithelium is composed of absorptive (enterocyte) and secretory (enteroendocrine and goblet cell) lineages. Previous data showed that activation of Notch signaling increased enterocyte number and, in contrast, reduction of Notch signaling promoted secretory cell number in vertebrates. In zebrafish, whether the Notch signaling regulates goblet cell differentiation is not clear. In this study, we used 2F11 antibody (for secretory cell) and WGA (wheat-germ agglutinin) staining (for goblet cell) to detect secretory cell differentiation. We found increased secretory cell (mainly enteroendocrine) number in mibta52b. Surprisingly, goblet cell number was reduced in this fish. rbpja MO-injected embryos were analyzed, and goblet cell number was greatly reduced in these morphants as expected. This result indicated that Notch signaling regulated goblet cell differentiation in zebrafish. Next, we used Hsp70:mibta52b transgenic fish for heat-shock (HS) at different stages and found the critical timing for secretory cell differentiation was 60 hpf. The differences between mib mutant and Hsp70:mibta52b in goblet cell differentiation might be due to insufficient induction of mib after HS. Further analysis using multiple HS to block Notch signaling continuously resulted in the reduction of goblet cells. Additionally, we screened the expression of Notch-related genes in larval intestine of Hsp70:mibta52b. We found deltaA, deltaD and atoh1b expression was increased after HS. However, goblet cell differentiation was not affected in deltaD homozygous mutant aei, which indicated that deltaD might not be the important Notch ligand for goblet cell differentiation. Furthermore, both deltaA mutants and deltaA morphants showed reduction of goblet cell differentiation. In atoh1b morphants, the goblet cell differentiation was reduced but the enteroendocrine cell differentiation was normal. The atoh1b MO inhibition of goblet cell differentiation was dosage-dependent. Since the increased goblet cell differentiation of Hsp70:mibta52b might be mediated by atoh1b, we injected the atoh1b MO into Hsp70:mibta52b embryos and HS at 60 hpf. We found that the induction of goblet cells in Hsp70:mibta52b was inhibited by atoh1b knockdown, but the increasing enteroendocrine cells were not affected. This study showed that Notch signaling regulated the differentiation of secretory cell lineage in zebrafish intestine. deltaA ligand and the downstream mediator atoh1b might be important for goblet cell differentiation.