| Summary: | 博士 === 慈濟大學 === 醫學科學研究所 === 101 === Fibroblast growth factor (Fgf) signaling is important for embryonic development, including pattern formation, morphogenesis, and left-right asymmetry. It is involved in many cellular processes, including cell proliferation, differentiation, and apoptosis. Instead of 22 Fgf ligands and 4 Fgf receptors in mammalian, zebrafish has 27 Fgf ligands due to gene duplication. In this study, I investigated the functions of Fgf signaling in left-right (LR) pattern formation during early embryonic development and intestinal cell differentiation at late stages. Many organs in vertebrates are LR asymmetrical located. For example, liver is at the right side and stomach is at the left side in human. I used splicing blocking morpholinos (MOs) to specifically block the splicing of fgfr2b and fgfr2c variants, respectively. I found that the relative position of the liver and the pancreas was disrupted in fgfr2c morphants. Furthermore, the LR asymmetry of the heart became randomized. Expression pattern of the laterality controlling genes, spaw and pitx2c, was perturbed in the morphants. Lefty1 was not expressed in the posterior notochord, indicating that the molecular midline barrier was defective. It was also not expressed in the brain diencephalon. Kupffer’s vesicle (KV) size became smaller in fgfr2c morphants. Importantly, KV cilia were shorter in the morphants. The clustering of KV progenitors was disrupted in fgfr2c morphants. Furthermore, Fgf8 has synergistically effect with Fgfr2c in regulating laterality. Taking together, the fgfr2c isoform plays an important role in the LR asymmetry during zebrafish early development.
At late stage, I investigate the role of Fgf signaling in intestinal cell differentiation. There are four cell lineages derived from intestinal stem cells in the mammalian intestine: the non-secretory absorptive enterocytes, and the secretory cells, which include mucous-secreting goblet cells, regulatory peptide-secreting enteroendocrine cells, and antimicrobial peptide-secreting Paneth cells. Among them, only Paneth cells are located at bottom of crypt. Other cell types are distributed at crypt and villus. In zebrafish, crypt structure is not presented and Paneth cells have not been identified. Although Fgf signaling is important for cell proliferation and differentiation in various tissues, its role in intestinal differentiation is less well understood. Abnormal differentiation of goblet cells was observed when Fgf signaling was inhibited using SU5402 or in the Tg(hsp70l:dnfgfr1-EGFP) transgenic line after heat shock. I identified Fgfr2c as an important Fgf receptor in the intestine for cell differentiation. The number of goblet cells and enteroendocrine cells was reduced in fgfr2c morphants. In addition to secretory cells, enterocyte differentiation was also disrupted in fgfr2c morphants. Furthermore, proliferating cells were increased in the morphants. Interestingly, the loss of fgfr2c expression repressed secretory cell differentiation and increased cell proliferation in the mibta52b mutant in which secretory cells were overproduced due to defects for all the Notch signaling. In conclusion, Fgfr2c signaling derived from mesenchymal cells is important for regulating the differentiation of intestine epithelial cells by promoting cell cycle exit. These findings provide new evidences that Fgf signaling is required for the differentiation of intestinal cells in the zebrafish developing gut.
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