碩士 / 國立臺灣大學 / 漁業科學研究所 / 95 / Previously we have isolated a KLF8 cDNA from zebrafish and it encodes 348 amino acids long polypeptide which sharing high (63%) amino acid sequence similarity with human KLF8. There are three tandem krüppel-type Cys2His2 zinc-finger motifs at the carboxyl terminus and a repressor motif (PVALS/T) located at the N-terminus of zebrafish KLF8. To explore the function of KLF8 on zebrafish development, we used KLF8 specific antisense morpholino oligomer (MO) to knock down the expression of KLF8. We observed defect in midbrain/hindbrain boundary formation in 24 hours post fertilization (hpf) morphant embryos. We also detected abnormal heart looping morphology with swollen pericardiac chamber, brain edema, small eyes, and malformed tail in 48hpf and 72hpf morphant embryos, respectively. In 96hpf morphant embryos,.we also found defects including lack of folds in intestinal epithelium and small intestinal lumen In addition, paraffin sectioning on 96hpf morphant embryos showed hypoplastic development of liver and exocrine pancreas, as well as the presence of cuboidal intestinal epithelium lacking of polarization. Therefore, the main goal of this thesis is to investigate function of KLF8 in zebrafish gut development. Results from DNA microarray revealed that up-regulation of p53 and Cyclin G1 expression in 72hpf morphant embryos. We also confirmed that △113p53 was the P53 isoform showing up-regulated expression in 72 hpf morphant embryos by Rapid amplification of cDNA ends. Real time Q-PCR was conducted to confirm some up-regulated genes in morphant embryos. Expression of △113p53 and Cyclin G1 were increased 7.76 and 2.88 fold respectively in 72hpf morphant embryos, but they were respectively increased 11.86 and 6.41 fold in 30hpf morphant embryos. Some p53 downstream genes including p21, Mdm2, Noxa and Puma were also up-regulated. Whole-mount in situ hybridization indicated p53 gene was abundantly expressed in the eyes, brain, spinal cord and gut in morphant embryos during 30 to 72 h of development. However no significant difference in p53 expression was detected between wild type and KLF8 morphant embryos at 96 hpf. We used acridine orange staining and found a significantly increased apoptosis in the eyes, brain and spinal cord in 25hpf morphant embryos. To explore how KLF8 affects p53 to regulate development of the digestive system, we co-injected KLF8-MO1 either with p53 splicing MO or with p53 ATG MO into 1-2 cells and examining their effects on foxa3 expressions in the liver and pancreas buds at 34hpf and on IFABP expression domain in the intestinal bulb at 72 hpf injected embryos. Approximately 16% 34hpf morphant embryos showing defects in liver and pancreas budding can be rescued by co-injection with p53 splicing MO but not with p53 ATG MO. However, approximately 34％ 72hpf morphant embryos that have been co-injected with p53 splicing MO showing increased IFABP expression domain in the intestinal bulb regions while no improvement of IFABP expression domain can be detected when co-injecting with p53 ATG MO. Whereas both foxa3 and IFABP expression domains in embryos that either have been injected with p53 splicing MO or p53 ATG MO alone were the same as wild type 34hpf and 72 hpf embryos. Overall, these results suggest that up-regulation of △113p53 caused by loss of KLF8 function may promote apoptosis in the fore gut and affect liver and pancreas budding as well as subsequent intestinal morphogenesis.
| Identifer | oai:union.ndltd.org:TW/095NTU05451014 |
| Date | January 2007 |
| Creators | Ming-Yuan Tsai, 蔡銘原 |
| Contributors | Wann-Nian Tzeng, 曾萬年 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 52 |
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