碩士 / 國立清華大學 / 生物資訊與結構生物研究所 / 95 / The genomic program for development operates primarily by the regulated expression of genes encoding transcription factors and components of cell signaling pathways. This program is executed by cis-regulatory DNAs (e.g., enhancers and silencers) that control gene expression and the trans-factors that bind on them. The regulatory inputs and functional outputs of developmental control genes constitute network-like architectures. Otx gene family contains pair type homeobox transcription factors that are highly conserved in all vertebrates. The common consider Otx gene has two roles, one is playing function during early embryogenesis and another is playing the crucial role in head development that depend on precisely regulated the temporal and spatial expression of Otx2.
To investigate the gene regulatory subnetwork center by Otx2. First, we knockdown the expression of Otx2 by Morpholino and then used the real time Q-PCR to screen the expression of 36 transcription factors in Otx2 morphant. From our Q-PCR data, we found Otx2 positively regulate five endoderm specific transcription factors Gata5, Foxa2, Gata6, Foxa3 and Sox17 at the 50% epiboly stage. At the segmentation stage, Otx2 regulated several transcription factors that expressed in the neuroectoderm. POU1, Brn1.2 and POU12 expressed overlap with Otx2 in the midbrain were been positively regulated, Six3a, Six3b, Foxh1, Sox4, Pax2a and Gbx2 expressed in the other brain region surrounding midbrain were been negatively regulated. All of them had specification function in the different brain regions. The interaction of Otx2 with them is for brain specification and regionalization.
Secondary, we compared genome sequence of Otx2 across several species to search the evolutionary conserved elements and construction of the regulatory elements driven GFP reporter constructs to study how the Otx2 has been regulated. Five of them are very highly conserved which we named them: Module 16, 27, 28, 29, and 36. Module 27, Module 28 and Module 29 are just immediate upstream of the 5’ transcription start site. In vivo promoter assay results suggest that Module 27 and Module 29 alone can drive GFP reporter gene in dorsal side as early as 5 hours post fertilization and anterior neuroectoderm at 75% epiboly to early somite stage. Module 29 also drives GFP later in the forebrain. Module 16 is 25 kb upstream of the Otx2 gene, which can regulate Module 27, and Module 29 expressed in the forebrain and midbrain. We are sure zebrafish Otx2 have two promoters and Module 16 can regulate both of them express in forebrain and midbrain. Search the transcription factor binding sites in the Module 29, we find 2 TCF binding sites 1 Foxh1 binding site and 1 SOX binding site. We are now performing mutation analysis to study the role of these transcription factor-binding sites.
| Identifer | oai:union.ndltd.org:TW/095NTHU5112033 |
| Date | January 2007 |
| Creators | Huang, Chang-Ben, 黃昶斌 |
| Contributors | 莊永仁 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 54 |
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