- 學位論文
斑馬魚肌肉調控因子MYF5的轉錄調控與生物特性
Transcriptional regulation and biological Function of Zebrafish Myogenic Regulatory Factor, MYF5
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摘要
Myf5是一種basic helix-loop-helix轉錄因子，負責控制胚胎發育時期肌肉組織的分化，myf-5的表現具有組織專一性與時期專一性的特性。在哺乳類與魚類中對於myf-5基因的調控機制至今尚不清楚。利用顯微注射方法研究myf-5 promoter的結果顯示，在myf-5上游-82/-62序列為一重要的cis-acting element，對myf-5基因的開啟有很大的影響 (Chen et al., 2003)，但是到底是何種轉錄因子結合在我們發現的調控區上，目前不得而知。本文利用yeast one hybrid system找出與斑馬魚myf-5基因上游調控序列-82/-62 casette結合的轉錄因子- Forkhead box d3 (Foxd3)。in vitro transactivation assay證明Foxd3能促進斑馬魚myf5啟動子活化。在注射foxd3-morpholino (MO)的胚胎中，發現myf5只侷限在presomitic mesoderm表現，在體節與其鄰近的adaxial cells則無法偵測到。Foxd3對myf5的調控是有時間性的: 在受精後12-18小時的過程中，Foxd3負責維持Myf5在體節與其鄰近的adaxial cells的表現。此外，myod的表現則未受到影響。在注射pax3-MO的胚胎中，發現foxd3的表現幾乎完全消失，而myf5的表現情況則與注射foxd3-morpholino (MO)的胚胎類似。在同時注射pax3-MO與foxd3 mRNA的胚胎中，發現myf5在體節與其鄰近adaxial cells的表現能被拯救(rescue)。我們推論在胚發育的過程中，Pax3控制Foxd3表現，之後Foxd3再調控Myf5的表現，進而促進肌肉的發育。由於Foxd3之前主要被認為是調控神經脊細胞發育的轉錄因子，因此本篇文章是第一篇證明Foxd3參與myf5的調控並在體節發育過程中扮演重要角色。 另ㄧ方面，抑制myf5的轉譯，亦會導致原腸化異常、體軸與頭部畸形 (Chen and Tsai 2002)。本實驗發現myf5 在胚胎發育早期(shield stage)表現在non-axial mesoderm位置。注射myf5-MO，抑制myf5的功能，會造成原腸化時期dorsal organizer不正常延伸擴張。並且在Myf5缺失之胚胎個體中，後腦krox20與 pax6基因表現亦受到嚴重影響，後腦神經的發育也受到嚴重影響。此外頭部dlx2、sox9與colo2a1表現之神經嵴細胞範圍會有減少現象，晚期則出現頭部形態異常、頭部咽弧軟骨發育缺失。TUNEL assay實驗證實，在Myf5缺失之胚胎個體中，頭部區域會出現嚴重細胞凋亡現象。進一步觀察Myf5缺失胚胎，發現頭部軟骨的缺失型態與胚胎個體注射fgf3-MO之結果相似。因此，在Myf5缺失之胚胎個體中，偵測fgf3與下游訊息影響基因erm與pea3之表現，均出現減少現象。在同時注射myf5-MO與fgf3 mRNA的胚胎中，發現可拯救後腦分節的缺失，減少角鰓骨(ceratobranchial)完全缺失的比例，更明顯減弱頭部區域細胞凋亡現象。這些證據顯示，myf5在原腸化過程扮演重要角色，Myf5的缺失造成後腦fgf3訊息傳遞缺失，干擾後腦分節發育，進而影響之後的神經嵴細胞與軟骨的發育。因此，我們研究結果指出，Myf5除了參與肌肉發育(myogenesis)以外，在原腸化時期、後腦、神經脊細胞與軟骨發育都扮演重要角色。
並列摘要
Myf5, one of the basic helix-loop-helix transcription factors, controls muscle differentiation and is expressed in somites during early embryogenesis. However, the transcription factors bound to the cis-elements of myf5 are poorly understood. In this study, we used the yeast onehybrid assay and found that Forkhead box d3 (Foxd3) interacted specifically with the -82/-62 cassette, a key element directing somite-specific expression of myf5. The dual-luciferase assay revealed that the expression of Foxd3 potently transactivated the myf5 promoter. Knocking down foxd3 with morpholino oligonucleotide (MO) resulted in a dramatic down-regulation of myf5 in somites and adaxial cells but not in the presomitic mesoderm. On the other hand, myod expression remained unchanged in foxd3 morphants. Foxd3 mediation of myf5 expression is stagedependent, maintaining myf5 expression in the somites and adaxial cells during the 7- to 18-somite stage. Furthermore, in the pax3 morphant, the expression of foxd3 was down-regulated greatly and the expression of myf5 was similar to that of the foxd3 morphant. Co-injection of foxd3 mRNA and pax3-MO1 greatly restored the expression of myf5 in the somites and adaxial cells, suggesting that pax3 induces foxd3 expression, which then induces the expression of myf5. This report is the first study to show that Foxd3, a well-known regulator in neural crest development, is also involved in myf5 regulation. To understand whether myf5 plays other roles than myogenesis during embryogenesis. We first observed that myf5 was expressed in the non-axial mesoderm at the shield stage. Knockdown of Myf5 resulted in abnormal expansion and disorder of the dorsal organizer. We proved that the segmentation of the hindbrain were affected severely in the myf5 morphants due to either lost or defective expression of krox20 and pax6,. The expression of neural crests markers was dramatically reduced in the myf5 morphants; five-day-old myf5 morphants had serious chondrodysplasia in craniofacial cartilage. The TUNEL assay showed that apoptosis occurred significantly in the head of the myf5 morphants. These findings suggest that the reduction of head size and the absence of pharyngeal cartilage formation induced by myf5 inactivation were due primarily to apoptosis. Of interest, the pharyngeal arch defects found in the myf5 morphants were identical to those of the fgf3-MO-injected embryos, and the expression of fgf3 and its down-regulators erm and pea3 was greatly reduced in the myf5 morphants. The fgf3 transcripts also were reduced in the myf5 morphants, but co-injection of fgf3 mRNA and myf5-MO1 into the embryos rescued the hindbrain patterning and the ceratobranchial cartilage defects; the apoptotic signals were also reduced. This evidence suggests that, myf5 is involved in axial and non-axial mesoderm interaction during gastrulation. The disrupted hindbrain segmentation affects the fgf3 signaling, thus causing the CNC to undergo apoptosis. Myf5 is necessary for dorsal organizer patterning, hindbrain segmentation, CNC survival, and cranial cartilage formation.
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