capsulin與musculin在斑馬魚頭部肌肉發育的重要性

capsulin and musculin are required for zebrafish cranial muscle development

李耕琿(Gang-Hui Lee)

指導教授 ： 陳曜鴻

淡江大學/理學院/生命科學研究所碩士班/碩士(2009年)

https://doi.org/10.6846/TKU.2009.00874

摘要

Capsulin與Musculin為兩個具有basic helix I-loop-helix II(bHLH)結構之轉錄因子。為了深入研究capsulin與musculin如何參與頭部肌肉的發育，我們選用斑馬魚做為模式物種，首先，利用已知之斑馬魚capsulin胺基酸序列與其它脊椎動物進行比對，得知斑馬魚之capsulin與人類、牛、小鼠、大鼠與蟾蜍分別有75%、75%、76%、75%與71%之相似度，之後再透過反轉錄-聚合酶鏈鎖反應複殖出斑馬魚musculin序列並進行比對，得知斑馬魚之musculin與人類、牛、小鼠、大鼠與蟾蜍分別有63%、63%、63%、63%與62%之相似度。接著進行胚胎原位雜交，發現在受精後24小時的胚胎中capsulin mRNA 會表現在背側頭部後方近與卵黃交接處呈現四個點狀區塊，藉由dlx2a比對後發現capsulin表現在中胚層核心的位置上。隨後capsulin mRNA會開始分成兩排往頭部延伸表現，直到受精後三天左右就只剩在心臟部位有微弱的capsulin訊號表現；musculin的表現則區分為兩部分，在發育早期會表現在中胚層的位置上，至受精後36小時藉由myoD的比對發現其表現在顎間肌(im)的位置上，至晚期則會表現在全身骨骼肌上，因此得知兩者早期皆表現在中胚層上。之後我們使用反股寡核苷酸(morpholino)去抑制內生性capsulin與musculin的蛋白質轉譯，再藉由基因轉殖魚Tg(α-actin:RFP)及F59抗體染色觀察，發現注射過capsulin MO及musculin MO後，斑馬魚頭部肌肉完全消失的比率為52.9±3.7%及26.5±6.9%，因此我們認為斑馬魚頭部肌肉的發育需要capsulin與musculin。接著利用TUNEL assay觀察得知注射過capsulin MO及musculin MO後在後腦會有細胞凋亡的狀況，分別利用capsulin mRNA及musculin mRNA進行補救後細胞凋亡會有減緩的現象，再利用腹側中胚層核心專一性標記edn-1進行觀察，發現腹側中胚層核心不受影響，之後再利用肌肉發育相關因子myf5與myoD觀察，發現皆沒有表現，而當抑制myf5或myoD時，capsulin表現情形並無顯著變化，但晚期之musculin則受到影響，也正代表著capsulin與musculin位於中胚層核心調控myf5與myoD，而myf5與myoD也控制著晚期的musculin。另外我們也透過可定量式反轉錄聚合酶鏈鎖反應(Q-PCR)發現當capsulin或musculin被抑制時，musculin或capsulin表現量會有下降的狀況，所以接著利用musculin MO2+capsulin mRNA及capsulin MO1+musculin mRNA的實驗以補救表現量下降的狀況，亦發現頭部肌肉的缺失可被補救。綜合以上的實驗，我們推測早期capsulin與musculin在斑馬魚上會位於中胚層核心調控MRFs，晚期之musculin則可承接MRFs的訊號，進而控制整個頭部肌肉的生成。

關鍵字

斑馬魚 ； capsulin ； musculin ； morpholino ； 顏面肌肉發育

並列摘要

capsulin and musculin are two related transcription factors that controlled cranial muscles development in vertebrate, but their functions still unclear. Here, we used zebrafish as an animal model to study their roles during early developmental stage. After sequence comparison, we found that the zebrafish capsulin (musculin) polypeptide shares sequence identities of 75%(63%), 75%(63%), 76%(63%), 75%(63%) and 71%(62%) with the reported capsulin (musculin) of human, bovine, mice, mouse and Xenopus, respectively. Whole mount in situ hybridization experiments showed that capsulin was first detected at mesoderm of 20 hours post-fertilization (hpf) embryos. Comparing with expression pattern of dlx2a, we found that capsulin was expressed at the central parts of dlx2a of 24hpf and 30hpf embryos, indicating that capsulin was expressed at mesoderm core of cranial neural crest cells. musculin was expressed at mesoderm of early stage. At the later stage, musculin was expressed at intermandibularis (im) by comparing with the expression pattern of myoD. Using morpholino knockdown approach, Our data showed that knock capsulin and musculin down led to loss of all cranial muscles. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed that large amounts of cell apoptosis in the hindbrain region of capsulin-morphant and musculin-morphant, but ventral mesoderm core was not affected by capsulin MO and musculin MO. Molecular evidences showed that myf5 and myoD transcription were down-regulated at the precursor cells of head muscles in capsulin-morphant and musculin-morphant. musculin was down-regulated in myf5-morphant and myoD- morphant, whereas capsulin was not affected by myf5 MO and myoD MO. Taken together, we concluded that capsulin and musculin are required for zebrafish cranial muscles development.

並列關鍵字

Zebrafish ； capsulin ； musculin ； morpjolino ； cranial muscle development

參考文獻

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