碩士 / 國立臺灣海洋大學 / 生物科技研究所 / 94 / Abstract
We have previously cloned a zebrafish KLF4a cDNA, which encodes 396 amino acids. Amino acid sequence comparison showed that zebrafish KLF4a shares 65.8％ sequence similarity with mammalian KLF4. KLF4a is a meternal mRNA, it was expressed in the eyes, otic vesicle, midbrain-hindbrain boundary (MHB), ventricular zone at 24 hpf (hour post fertilization), and was expressed in the liver, exocrine pancreas, intestine between 48 to 96 hours of development. Previous results from antisense morpholino oligomer knock down experiments using KLF4a-MO1 showed that there were no foregut looping and epithelial folding in the intestine of KLF4a 72 hpf morphant. Decrease in expression levels or domains of IFABP、LFABP, and Trypsin, which are involved in digestive tract metabolism were also detected in embryos that have been injected with KLF4a-MO1.
The purpose of this thesis is to investigate the function of KLF4a in the digestive tract development in detail. We designed another antisense morpholino oligomer, KLF4a-MO3, and compared morphant phenotype and rate when respectively injected with either KLF4a-MO3 (10 ng) or coinjected KLF4a-MO1 & MO3(4 ng each). A KLF4a dominant-negative plasmid (150 pg) that can compete with endogenous KLF4a for binding site on target gene promoters, and KLF4a 5mm-MO3 (10ng) were used as controls. Results revealed malformation of midbrain-hindbrain boundary and reduction in midbrain ventricles in 24 hpf embryos that have been injected with either KLF4a-MO3 orco-injected KLF4a-MO1&MO3. We also observed the absence of foregut looping and epithelial folding in the intestine of 72-96 hpf embryos that have been injected with either KLF4a-MO3 orco-injected KLF4a-MO1&MO3. These morphant phenotypes were similar to those observed previously when injecting with KLF4a-MO1. While phenotype of embryos that have been injected with either KLF4a 5mm-MO3 or KLF4a dominant-negative plasmid were the same as wild-type. Whole-mount in situ hybridization was further conducted to study the effects on expressions of several digestive tract marker genes in KLF4a morphant embryos. We detected that expression levels of IFABP and Trypsin were not apparently altered, but their expression domains were decreased, and the expression level and domain of LFABP were reduced, while the expression level and domain of insulin were not affected in 72 hpf embryos that have been respectively injected with either KLF4a-MO3, KLF4a-MO1&MO3, or KLF4a dominant- negative plasmid. However, expressions of these four marker genes in KLF4a 5mm-MO3 72 hpf morphant were the same as in wild-type embryos. Therefore, we suggest that KLF4a may be involved in zebrafish liver development. In addition, these results partly showed the specificity of KLF4a-MO3 used in this study. In order to further confirm the specificity of KLF4a-MO3, we also prepared KLF4a polyclonal antibody. Results of western blot analyses revealed that KLF4a polyclonal antibody can not recognize endogenous KLF4a protein in 72 hpf embryos; perhaps due to the amount of endogenous klf4a protein is low. However, it can recognize overexpressed KLF4a protein isolated from 72 hpf embryos that have been injected with 50pg pcDNA-KLF4a plasmid. Furthermore, level of overexpressed KLF4a protein can be reduced when embryos coinjecting different amount of KLF4a-MO3 with pcDNA-KLF4a plasmid. These results further confirm the specificity of KLF4a-MO3 used in this study. Moreover, we also investigated effects on expression of transcription factors that are involved in GI tract development in KLF4a morphant embryos. We observed that the expression domains in the liver, intestine, and exocrine pancreas of HNF4α、HNF1β and GATA6 were affected in KLF4a morphant embryos. In addition, paraffin sectioning and HE staining analyses showed a decrease in the intestinal lumen, no epithelial folding, and hypoplasia of liver and exocrine pancreas in 96 hpf KLF4a morphant embryos. Overall, our results demonstrated that KLF4a plays important roles in the development of intestine, liver, and exocrine pancreas.
| Identifer | oai:union.ndltd.org:TW/094NTOU5111018 |
| Date | January 2006 |
| Creators | Chein-Tso Chan, 詹健佐 |
| Contributors | Sheng-Ping L. Hwang, 黃聲蘋 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 110 |
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