碩士 / 淡江大學 / 生命科學研究所碩士班 / 94 / The vertebrate skeleton consists of cartilage and bone. Cartilage originates from chondrocyte and bone from osteoblast and osteoclast. CBFA (core binding factor alpha subunit) belongs to the runt homology domain family, that promotes the osteoblast differentiation and bone formation in early development and also inhibits osteoblast differentiation during late development. This dual functions of the CBFA maintains the balance of the bone formation and resorption. cbfa-null mice displayed cranium, mandible, bone malformation and embryonic lethal (Komori et al, 1997). In order to extend our knowledge of skeleton development on aquatic animal, we studied the biological function of CBFA on zebrafish. We isolated the zebrafish cbfa coding region, which encodes a 204 amino-acids polypeptide. Comparing the deduced amino-acids sequences of zebrafish CBFA to human, rat, mice, chicken and fugu, we found that they shared 82%, 82%, 74%, 83% and 78%, respectively. Using Reverse Transcription-Polymerase Chain Reaction (RT-PCR), we found that endogenous cbfa expression can be detected from one cell stage to one month stage, suggesting cbfa is a maternal inheritance gene. Whole-mount in situ hybridization was also performed, and indicated that the cbfa transcripts were detected in the pharyngeal arch at 3-7dpf embryos. Using cbfa-morpholino following by Alcian blue staining, we found that the average length (L) and width (W) of morphants’ heads are 0.347±0.0379 mm and 0.266±0.018 mm, which are around 30% smaller than their wild type littermates (L:0.515±0.019 mm; W:0.323±0.073 mm). We also found that the CBFA-morphants displayed specific abnormalities, such as ceratobranchial missing, ceratohyal and Meckel’s cartilage deformation. The cbfa-MO-induced specific abnormalities are in a dosage-dependent manner. As the MO injection dosages increased (1.5 ng to 4.5 ng), the specific abnormalities rates increased (6.5±2.41 to 51.5±2.60%). cbfa mRNA were also injected in order to rescue CBFA-morphants. The abnormalities of CBFA-morphants were decreased in a dosage-dependent manner after rescued by cbfa mRNA. Finally, we used Dlx2 (all neural crest cells) and Sox9a (postmigration neural crest cells) riboprobes to carry out whole-mount in situ hybridization on CBFA-morphants. These results show that neural crest cell specification and migration was affected by cbfa. Base on these observations, we propose that cbfa gene is essential for neural crest cell specification into ceratobranchial, Meckel’s cartilage and ceratohyal pharyngeal arch cartilages development.
| Identifer | oai:union.ndltd.org:TW/094TKU05105006 |
| Date | January 2004 |
| Creators | Yung-Chang Lin, 林永蒼 |
| Contributors | 陳曜鴻 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 67 |
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