分析斑馬魚不同stathmin基因在胚胎中的表現

Abstract

在真核生物，微管在調節細胞生理訊息傳遞的過程中扮演很重要的角色，它能夠穩定細胞骨架以及影響神經發育。有許多不同的微管蛋白能夠調節微管的聚合，其中包含stathmin。stathmin蛋白是一種磷酸蛋白，它可調節微管的聚合，stathmin能夠和兩分子的α-和β-微管蛋白形成的二聚體結合，形成一個四聚體，因此stathmin能夠干擾微管的聚合化和去聚合化。藉由調節微管的聚合和去聚合，stathmin參與了許多不同的細胞訊息傳遞的過程，包括神經形成，細胞的分化和增生。在哺乳類的研究中指出stmn1廣泛的表現在細胞中，而stmn2，stmn3以及stmn4則主要表現在神經系統，在先前我們實驗室的研究，發現在斑馬魚中的一個stathmin蛋白，stathmin 2，在腦部發育中，扮演一個很重要的角色。目前已知斑馬魚中有7個不同的stathmin基因，為了瞭解stathmin在斑馬魚發育中扮演的角色，我選殖出斑馬魚stathmin蛋白的基因stmn 1a，stmn 2b和stmn 4l，發現它們和其他脊椎動物的stathmin蛋白有很高的相似度，並利用全標本包埋的原位雜交技術觀察斑馬魚stathmin蛋白在不同時間點，不同的斑馬魚stathmin蛋白的表現情形。在此研究中我觀察到，stmn 1a和stmn 2b從16小時就會表現在脊髓和三叉神經，但是stmn 4l要從18小時才會開始表現，而stmn 2b和stmn 4l主要會表現在中樞神經系統。此外，stmn 1a也會表現在眼睛，鰓弓以及胚胎的尾端。從24小時後，stmn 1a，stmn 2b和stmn 4l都會表現在下視丘，頭蓋，間腦以及端腦，在48小時後，stmn 2b和stmn 4l仍然會表現在中樞神經系統，不過stmn 1a則沒有表現在後腦。stathmin在中樞神經系統發育中的表現情形，顯示stathmin在斑馬魚中樞神經系統發育上，可能扮演一個很重要的角色。

Microtubules are well known to mediate many physiological processes in eukaryotic cell such as stable cell structure and axonal outgrowth. Microtubules can be regulated by different microtubule binding proteins to mediated their assembly and disassembly. Stathmin family proteins are phosphoprotein that regulates microtubules disassembly. Stathmin interacts with microtubules by binding to two moles of alpha and beta tubulin dimer to form a tetramer. Stathmins also interfere with microtubule polymerization by direct binding to microtubules. Via tight regulation of microtubule polymerization and depolymerization, stathmins are involved in different cellular processes, including neurogenesis, cell proliferation, differentiation and activities. In mammals, stmn 1 is ubiquitous expressed while other stathmin members, stmn 2, stmn 3, stmn 4 are mainly expressed in the nervous system. Previously, we have demonstrated that stmn 2, one of the zebrafish stathmin proteins, plays a vital role in zebrafish brain development. There are seven different stathmin genes in zebrafish. To understand the role of stathmin in zebrafish, I have cloned stmn 1a, stmn 2b and stmn 4l and demonstrated that they shared high identity with other vertebrate stathmin homologues. I analyzsed their temporal expression patterns using embryos at different stages by whole-mount in situ hybridization. I observed that stmn 1a and stmn 2b were expressed in spinal cord and trigeminal ganglia from 16 h post fertilization (hpf) but stmn 4l appeared from 18 hpf. stmn2b and stmn4l were mainly expressed in the central nervous system (CNS). Furthermore, the stmn1a transcripts were found in eyes, pharyngeal arches and tail bud. stmn 1a, stmn 2b and stmn4l were all expressed in hypothalamus, tegmentum, telencephalon and diencephalon after 24 hpf. After 48 hpf, stmn2b and stmn4l were still expressed in the CNS while stmn1a did not expressed in hindbrain. The dynamic expression of stathmin family genes in the developing central nervous system suggests a pivotal roles of those genes in regulating CNS development in zebrafish.