斑馬魚Stathmin家族基因在胚發育時期之基因表現分析

Characterization and Expression Analysis of Stathmin Family Genes during Embryogenesis in Zebrafish, Danio reri

Stathmin家族基因因其細胞微管去組裝之功能而可整合不同細胞內訊息傳遞之路徑，然而我們對其在胚胎發育過程中所扮演之角色卻不甚明瞭，因此我們由斑馬魚中選殖出包括stathmin 1b（stmn1b），stathmin-like 2a（stmn2a），stathmin-like 3（stmn3）及stathmin-like 4（stmn4）等四種不同stathmin基因並分析其特性。此四種stathmin基因均帶有與哺乳動物相似且高度保守的類stathmin及可被磷酸化之區域。利用反轉錄聚合酶鏈式反應（RT-PCR）和全固定原位雜合反應分析（whole-mount in situ hybridization analysis，WISH）我們發現此四種斑馬魚stathmins主要表現在中樞神經系統，但其表現彼此間有時間及空間上之歧異，這也顯示了這些stathmin在斑馬魚發育過程中之神經發育可能扮演某些角色。在以嗎啉基寡核苷酸（morpholino oligonucleotide, MO）弱化stahmin基因之斑馬魚胚中我們觀察到下列表徵：頭部變小，腦室偏大，腦水腫及中後腦間隔變窄。同時我們亦以islet1基因WISH的方法觀察神經之發育，也發現到islet1之表現在stmn2a MO處理之胚中明顯降低。這些缺陷無法在非對應性的stmn2a MO注射的胚中發現，更重要的其缺陷可為同時與stmn2a MO注射之stmn2a mRNA所救回。總之這些結果證實了stmn2a在斑馬魚腦部之發育扮演關鍵之角色。

Stathmin family genes are known to mediate microtubule disassembly that makes them potential regulators for integrating diverse intracellular signaling pathways. To investigate the roles of stathmins in zebrafish (Danio rerio) development, we cloned and characterized four zebrafish stathmin genes, including stathmin 1b (stmn1b), stathmin-like 2a (stmn2a), stathmin-like 3 (stmn3), and stathmin-like 4 (stmn4). All four stathmins contains a conserved stathmin-like domain with consensus phosphorylation domains and share high homology with their vertebral counterparts. RT-PCR and whole-mount in situ hybridization analyses revealed that zebrafish stathmins are mainly expressed in the central nervous system with divergent temporal and spatial expressions. This suggests roles of stathmins in neuronal regulation during development in zebrafish. By knocking down stmn2a we observed smaller brain, enlarge brain ventricle, brain edema and narrowed midbrain and hindbrain boundary. We also confirmed the observed phenotypes by using whole-mount ISH against islet1 and found that islet1 expression was reduced in stmn2a MO-injected embryos. In addition, these brain defects were specifically due to the loss of stmn2a because they could not be induced by a mis-match stmn2a MO and could be rescued by co-injecting stmn2a mRNA. Collectively, these results suggest a pivotal role of stmn2a in zebrafish brain development.