轉錄因子CBFA (core binding factor alpha)在斑馬魚軟骨發育過程中之功能探討

脊椎動物的骨骼主要是由軟骨母細胞組成的軟骨及硬骨母細胞與蝕骨細胞組成的硬骨所構成。cbfa（core binding factor alpha）為runt同源轉錄因子家族，在胚胎發育早期會促進硬骨母細胞分化及硬骨形成，晚期則會抑制硬骨母細胞分化藉以維持骨骼發育的平衡。在小鼠研究中，cbfa基因剔除的小鼠其顱骨、顏面骨骼及全身硬骨缺失且在出生隨即死亡，在胚胎早期發育不易觀察且耗時。我們利用斑馬魚胚胎透明易於觀察與胚胎數量多等特性進行研究。在本篇論文中，我們複殖出斑馬魚cbfa的基因序列可轉譯成204個胺基酸。經序列比對後得知斑馬魚CBFA胺基酸序列與人類、大鼠、小鼠、雞、河豚的相同性分別為82 %、82 %、74 %、83 %與78 %。並利用反轉錄-聚合酶鏈鎖反應偵測斑馬魚內生性cbfa的表現時期，發現自受精後1細胞時期到1個月的胚胎皆可偵測到cbfa的基因表現，推測應為母系遺傳。接著利用胚胎原位雜交法觀察cbfa mRNA在胚胎早期發育的時空分佈，發現在受精後3到7天會表現在咽弧（pharyngeal arch）。為了更進一步為了確定cbfa在骨骼發育中的功能，我們使用胚胎抑制劑（morpholino）去抑制內生性CBFA的蛋白質轉譯，在每個胚胎注射入1.5~4.5 ng，3天後觀察到畸形率為6.5±2.41~51.5±2.60 %。由外觀發現此畸形為頭部尺寸變小，野生種長為0.515±0.019 mm、寬為0.323±0.073 mm，cbfa被knock down後之胚胎長為0.347±0.037 mm、寬為0.266±0.018 mm，平均大約縮小30 %。接著利用阿爾襄藍染色法特異性的染出軟骨，結果發現畸型是由於角鰓骨（ceratobranchial）缺失、密克爾氏軟骨（Meckel’s cartilage）及角舌軟骨（ceratohyal）變形等咽弧軟骨發育有缺失而造成頭部尺寸變小。最後利用顯微注射打入cbfa mRNA去補回胚胎抑制劑所造成的缺陷，發現咽弧軟骨發育缺失的比例顯著地降低，代表CBFA的功能確實與角鰓骨、密克爾氏軟骨及角舌軟骨等骨骼發育有關而其他軟骨並不受影響。此外，我們也利用所有神經嵴細胞移動的標記Dlx2，及神經嵴細胞移動後的標記Sox9a，將CBFA-morphant進行胚胎原位雜交法，推測cbfa會調控顱神經嵴細胞的特化及移動最後形成咽弧。綜合以上的實驗，我們推測cbfa與斑馬魚神經嵴細胞特化及移動成為角鰓骨、密克爾氏軟骨及角舌軟骨等下顎軟骨的發育有關，這些研究結果能讓我們更了解cbfa在水生脊椎動物胚胎早期發育中所扮演的角色。

關鍵字

斑馬魚； cbfa ；軟骨；發育

並列摘要

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.

並列關鍵字

zebrafish ； cbfa ； cartilage ； development

參考文獻

1. Ducy, P. and G. Karsenty, Genetic control of cell differentiation in the skeleton. Curr Opin Cell Biol, 1998. 10(5): p. 614-9.

2. Lian, J.B., et al., Regulatory controls for osteoblast growth and differentiation: role of Runx/Cbfa/AML factors. Crit Rev Eukaryot Gene Expr, 2004. 14(1-2): p. 1-41.

4. Kimmel, C.B., C.T. Miller, and C.B. Moens, Specification and morphogenesis of the zebrafish larval head skeleton. Dev Biol, 2001. 233(2): p. 239-57.

5. Langille, R.M. and B.K. Hall, Role of the neural crest in development of the cartilaginous cranial and visceral skeleton of the medaka, Oryzias latipes (Teleostei). Anat Embryol (Berl), 1988. 177(4): p. 297-305.

6. Le Douarin, N.M., The Neural Crest. 1982.

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轉錄因子CBFA (core binding factor alpha)在斑馬魚軟骨發育過程中之功能探討

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