轉錄因子Six1在斑馬魚頭部及顏部肌肉發育的功能

Abstract

six1基因屬於sine oculis/six家族的一員，其在型態發生、器官生成及細胞分化上扮演重要角色。於果蠅的研究中已發現，six家族、eyeless (pax)家族、eyes absent (eya)家族與dachshund (dach)家族會形成一個協同調節網絡以促進複眼生成。而於six1基因剔除鼠中，Six1缺失會影響軀幹部的肌肉發育，這指出Six1對於軀幹部肌肉發育是必須的，然而其對於頭部肌肉所扮演的角色仍是未知。為進一步了解six1於頭部肌肉發育上的功能，我們選定斑馬魚作為模式動物以探討。因此，我首先利用whole mount in situ hybridization的方式，來觀察six1在斑馬魚頭部肌肉的表現情形。結果顯示six1可於所有的頭部肌肉前趨細胞表現，抑制six1轉譯的實驗則顯示，胸骨舌骨肌(sternohyoideus)、中直肌(medial rectus)、下直肌(inferior rectus)及所有咽弧肌之形成需要six1的功能；並且這些肌肉的myod與myogenin表現亦需要six1的功能。此外，外源性myod mRNA的添加，可以回復生成six1基因抑制劑(six1-MO)所造成缺失的肌肉，這顯示six1可會透過myod而調控頭部肌肉發育。而且我也觀察到pax3基因抑制劑所造成的頭部肌肉缺失與six1基因抑制劑相同，但外源性的six1, myod或myf5 mRNA皆無法回復生成pax3基因抑制劑所造成的肌肉缺失，因此推論six1與pax3不是處於同一基因網絡以調控頭部肌肉發育。除此之外，於注射six1或pax3基因抑制劑的胚胎中，皆可觀察到側鳍(fin bud)與後軸下肌(posterior hypaxial muscle)會缺失。故综合以上實驗結果，在斑馬魚，six1於頭部肌肉發育扮演重要的角色。

six1 gene belongs to the sine oculis/six gene family that plays important roles in morphogenesis, organogenesis, and cell differentiation. Studies in Drosophila have revealed that six family, eyeless (pax family), eyes absent (eya family) and dachshund (dach family) form a synergistic regulatory network to trigger compound eye organogenesis. six1 knockout mice showed that knockdown of six1 causes extensive muscle hypoplasia which affects most of the trunk muscles, indicating that six1 is required for trunk myogenesis. However, the function of six1 in craniofacial myogenesis remains unknown. To further understand the role of six1 in the development of head muscles, we used zebrafish as a model organism. In this study, I used whole mount in situ hybridization to observe the expression pattern of six1 in the head of zebrafish. Results showed that six1 was expressed in all head-muscle progenitor cells. Knockdown of six1 proved that Six1 was required for the development of sternohyoideus, medial rectus, inferior rectus, and all pharyngeal arch muscles. Six1 was also required for the expression of myod and myogenin in these muscles. Exogenous myod mRNA can rescue the loss of the cranial muscles caused by six1-morpholino(MO), indicating myod may be a target gene of Six1 to regulate craniofacial myogenesis. Moreover, we found that the defect of craniofacial muscles caused by pax3-MO phenocopied that of six1-MO. But exogenous six1, myod or myf5 mRNA did not rescue the loss of the cranial muscles caused by injecting pax3-MO, suggesting six1 and pax3 could not function in the same genetic network to regulate development of head muscles. In addition, we also observed that fin bud and posterior hypaxial muscles disappeared in six1 and pax3 morphants . Taken together, our results revealed that six1 played an important role in craniofacial myogenesis.