斑馬魚FoxD5在體節形成中所扮演的角色

Abstract

在各胚層的發育中，Forkhead Box (Fox)轉錄因子皆扮演重要的命運決定角色。雖然已知斑馬魚中FoxD3在體節發育時，能維持myf5在體節的表現並藉此參與於肌肉的發育；但FoxD轉錄因子家族中的另一員，FoxD5，在體節發育時也會表現在presomitic mesoderm (PSM)前端生成中的體節處，而這個FoxD轉錄因子是否在體節發育的過程中扮演調控的角色目前仍不清楚。在本篇研究中，為了了解FoxD5在體節生成時的功能，利用專一抑制基因轉譯之Morpholino Oligonucletides (MO)對1-cell時期之斑馬魚胚胎進行顯微注射以抑制FoxD5的轉譯，並在體節發育已完成之24 hpf時、及體節仍在形成之14 hpf (約12-somite stage)時分別觀察FoxD5-MO注射個體 (FoxD5 morphants)，發現新生成之體節處有產生體節發育畸形的現象。利用全胚胎原位雜交實驗(Wholemount in situ Hybridization)，獲知於PSM呈現週期性表現之her1及deltaC基因，在FoxD5 morphants中週期性的表現並沒有受到影響；但是決定體節前後極性之mespa的表現下降，且mepsb的表現由條帶狀(stripe pattern)轉成”salt and pepper”之點狀表現；而參與體節表皮化之基因papc在體節S-II至S0之位置原本呈現3個條帶狀表現，但FoxD5 morphants卻增加為5至6個條帶狀之型態。這些結果顯示FoxD5並不影響體節之週期性形成，而是藉由調節體節生成之過程而參與在體節之發育。進一步觀察體節後期分化之肌肉發育，發現在FoxD5 morphants中雖然myf5在體節的表現下降卻由表現上升的myod所補償，因而使得myogenin的表現沒有發生變化，顯示FoxD5 morphants之肌肉發育沒有受到影響；而觀察抑制FoxD3的胚胎中，FoxD5在新生成體節中會有表現異位的表現，顯示FoxD3在PSM之功能可能由FoxD5來補充。另一方面，觀察過量表現FoxD5的胚胎，發現體節及肌肉發育沒有明顯異常，但體軸發育則產生嚴重畸形的性狀；根據上述觀察結果，推測FoxD5可能在體節及體軸發育上扮演不同角色：在體軸發育時，FoxD5則可能參與於胚胎軸向決定；而在體節中，FoxD5具有維持體節前後極性進而促使體節正常形成之功能。

In all germ layers, Forkhead Box (Fox) transcriptional factors play important roles in the determination of cell fates. During somitogenesis, zebrafish FoxD3 is known of maintaining the somitic expression of myf5, and subsequently regulates myogenesis. FoxD5, another gene of the FoxD family, is also expressed in forming somites of the anterior PSM. However, the regulatory function of FoxD5 in somitogenesis is not clear. To address this issue, we microinjected Morpholino Oligonucleotides (MO), which acts as a specific translational inhibitor, into 1-cell stage zebrafish embyos to knockdown the expression of FoxD5. Results showed that the newly forming somites of the FoxD5-MO-injected embryos were malformed both in the stage when the somitogenesis was completed (24-hpf) and in the stage when the somitogenesis was in progress (14-hpf). By whole-mount in situ hybridization, the cyclic patterns of her1 and deltaC in PSM were not altered in the FoxD5 morphants. Nevertheless, the anteroposterior polarity determinants mespa and mespb were both affected: the expression of mespa was downregulated and the expression of mespb was changed from the stripe pattern to the disrupted pattern called “salt and pepper”. In addition, the expression of papc, which is involved in the epithelializtion of somites, was increased from 3 bands of stripes at S-II, S-I, and S0 to 5-6 bands. These observations suggested that FoxD5 dose not take parts in the cyclic pre-pattern of the PSM, but involves in the process of somite formation. Furthermore, myogenesis was not affected in the FoxD5 morphants, owing to the results of myf5 downergulation in somites and myod upregulation due to the knockdown of myf5, and therefore resulted in myogenin normal expression. In addition, FoxD5 was upregulated in the FoxD3-knockdown embryos, suggesting that FoxD5 might compensate the functions of FoxD3 in the PSM. On the other hand, in spite of the severely distorted axis, the FoxD5-overexpressed embryos displayed neither somite nor muscle defects. Based on these results, we speculate that FoxD5 plays different roles in axis and somite formation. During somitogenesis, FoxD5 functions in the maintenance of the anteroposterior polarity of somites, and in turn plays roles in proper formation of somites.