- 學位論文
myf5基因對斑馬魚頭部發育之功能分析
Roles of Myf5 During Craniofacial Developmnt in Zebrafish
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摘要
Myf5屬於肌肉發育調控因子，其為肌肉細胞命運決定所必需。然而過去對於Myf5的研究，主要著重在軀幹肌肉的發育；對於Myf5在頭部發育時扮演的角色，則很少探討。因此，首先利用whole mount in situ hybridization的方式，來觀察myf5和myoD在斑馬魚頭部肌肉表現的情形。結果顯示myf5表現於兩組斜肌(superior oblique & inferior oblique)、側直肌(lateral rectus)、胸骨舌骨肌( sternohyoideus)以及咽弧肌肉(pharyngeal arch muscles)的前驅細胞。相較於myf5表現於有限的肌肉前驅細胞，myod則在所有的頭部肌肉前驅細胞均會表現。抑制myf5轉譯的實驗則顯示，兩組斜肌、側直肌、胸骨舌骨肌以及所有的咽弧肌的形成需要myf5的功能；然而抑制myoD轉譯的實驗則顯示，四組直肌(superior rectus, inferior rectus, medial rectus & lateral rectus)以及腹側的咽弧肌的形成需要myod的功能。此外，在注射myf5-morpholino (MO)的胚胎中，共同注射myod mRNA，並無法拯救(rescue)抑制myf5後，頭部肌肉缺失之情形。而在注射myod-MO的胚胎中，共同注射myf5 mRNA，亦無法拯救抑制myod後，頭部肌肉缺失之情形。顯示myf5與myod在頭部軸側中胚層肌肉發育過程中並無互補(redundant)之能力，與其二者對軀幹部肌肉發育之調控具有差異。而且亦發現兩組斜肌由眼睛後方往眼睛前方遷移能力需要Myf5而非Myod。綜合上述實驗，證實Myf5與Myod在頭部肌肉發育過程中扮演各自獨立之功能，並根據實驗結果整合圖表，闡述Myf5與Myod如何參與調控斑馬魚頭部肌肉的生成。 另外，抑制myf5的轉譯，亦會導致頭形異常、頭部咽弧軟骨發育的缺失。並且在頭部dlx2、sox9與colo2a1表現之神經嵴細胞範圍會有減少現象。透過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。再進一步觀察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。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，表示頭部神經嵴細胞發育之影響應源自於後腦分節的缺失。再者，在注射myf5-MO的胚胎中，共同注射fgf3 mRNA，不但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基因藉由影響fgf3訊息傳遞路徑而調控後腦分節發育。myf5 在胚胎發育早期(shield stage)表現在non-axial mesoderm位置。實驗發現，Myf5的功能缺失，會造成dorsal organizer不正常延伸擴張，造成訊息傳遞混亂，進而影響隨之而後的後腦、神經嵴細胞與軟骨的發育。因此，我們研究結果指出，Myf5在頭部發育過程中，除扮演對肌肉特化之角色，更具有調控後腦分節、神經嵴細胞與咽弧軟骨發育的功能。
並列摘要
The myogenic regulatory factor Myf5 is well known as a fundamental molecule to trunk myogenesis. However, little is known about the role that myf5 plays in craniofacial development. We observed that zebrafish myf5 was detected in the primordia of the obliques, lateral rectus, sternohyoideus, and pharyngeal mesoderm cores. In contrast, myod transcripts were expressed in all head muscle precursors at later stages. Knockdown of myf5 revealed that Myf5 was required for the development of the obliques, lateral rectus, sternohyoideus, and all pharyngeal muscles, whereas knockdown of myod proved that Myod was required for the development of superior rectus, medial rectus, inferior rectus, lateral rectus, and the ventral pharyngeal muscles. myod mRNA did not rescue the loss of the cranial muscle caused by injecting myf5-morpholino, or vice versa, suggesting that the functions of Myf5 and Myod were not redundant in head paraxial mesoderm, a finding different from their functions in trunk myogenesis. Myf5, but not Myod, was required for the forward migration of myf5-positive oblique precursors. All evidences reveal that Myf5 and Myod function independently during cranial myogenesis. On the basis of the expression patterns of myf5 and myod, we propose a model to present how Myf5 and Myod are involved in head myogenesis of zebrafish. We show that Myf5 knockdown in zebrafish results in abnormal development of cranial cartilage. The expression of neural crests markers was dramatically reduced in the myf5 morphants. The TUNEL assay showed that apoptosis occurred significantly in the head of the myf5 morphants. Of interest, the pharyngeal arch defects found in the myf5 morphants were identical to those of the fgf3-morpholino(MO)-injected embryos, and the expression of fgf3 and its down-regulators erm and pea3 was greatly reduced in the myf5 morphants. 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, indicating that the defects in the crest and arch were attributable to the disordered hindbrain segmentation. 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 directs fgf3 signaling to mediate hindbrain development. We 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, which caused the defective hindbrain, crest, and cartilage development. Therefore, we propose that Myf5 plays roles in the development of the hindbrain boundary, cranial neural crest, and pharyngeal cartilage.
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