Stathmin-like 4是維持斑馬魚幼魚中腦內神經前驅細胞的關鍵因子

Abstract

細胞分裂與細胞分化往往需要精準分配彼此的比例來確保胚胎發育過程中適切的成長與神經特化。藉由鑽研斑馬魚腦部發育，我觀察到一個微管(microtubule)降解基因，stathmin-like 4 (stmn4)特別且專一地表現在背側中腦（dorsal midbrain）區域。而且該表現區域能與另一個泛神經分化代表基因，elavl3，交錯表現在發育中腦部，暗示stmn4於神經發育的角色。經由morpholino oligonucleotides (MOs)抑制部分蛋白質生成或是給予外加持續活化形式的Stmn4蛋白質，我發現不論是降低(knockdown)或是提高(overexpression)都導致神經分化訊號提早出現於背側中腦。同時，利用CRISPR/Cas9基因修改技術，我也製作出stmn4突變魚(mutant)。但是在其純合子（homozygous）突變幼魚裡卻只觀察到少於10%之前在降低表現幼魚(morphants)所看到的現象，這或許是因為stmn1b增加的補償作用來撫平stmn4的缺失。此外，在縮時攝影當中我發現到在morphants神經前驅細胞分裂與分化的差異。再者，morphants的細胞週期中明顯增長的G2相也能被我用外加的Cdc25a降回正常。最後，把Wnt訊號抑制更能將stmn4轉錄產物給剃除。這些結果都告訴我在神經龍骨時期（neural keel），Wnt訊息傳遞能藉由調控Stmn4在斑馬魚背側中腦的動態平衡來控制細胞週期中G2相長度，進而確保中腦免於過早進入神經分化。

A delicate balance between proliferating and differentiating signals is necessary to ensure proper growth and neuronal specification. By studying the developing zebrafish brain, we observed a specific and dynamic expression of a microtubule destabilizer gene, stathmin-like 4 (stmn4), in the dorsal midbrain region. The expression of stmn4 was mutually exclusive to a pan-neuronal marker, elavl3 that indicates its role in regulating neurogenesis. We showed the knockdown or overexpression of stmn4 resulted in premature neuronal differentiation in dorsal midbrain. We also generated stmn4 maternal-zygotic knockout zebrafish by the CRISPR/Cas9 system. Unexpectedly, only less than 10% of stmn4 mutants showed similar phenotypes observed in that of stmn4 morphants. It might be due to the complementation of the increased stmn1b expression observed in stmn4 mutants. In addition, time-lapse recordings revealed the changes in cellular proliferation and differentiation in stmn4 morphants. Stmn4 morphants displayed a longer G2 phase that could be rescued by Cdc25a. Furthermore, the inhibition of Wnt could reduce stmn4 transcripts. These results suggest that the Wnt-mediated Stmn4 homeostasis is crucial for preventing dorsal midbrain from premature differentiation via the G2 phase control during neural keel stage.