臺灣博碩士論文加值系統

Stathmin為一小分子磷酸蛋白，具調節微管去聚合能力，以整合細胞增殖及分化相關之訊息傳遞。在哺乳動物中，stathmin分布於細胞質，為一廣泛表現於各類細胞的磷酸蛋白，而SCG10、SCLIP和RB3/RB3’/RB3”等其他同為stathmin family的基因則只表現在神經系統且嵌合於細胞膜上。 為研究stathmin在斑馬魚胚發育所扮演之角色，我們自斑馬魚胚胎中選殖STMN1、STMN2及STMN4，以RT-PCR及原位雜合分析(Whole-mount in situ hybridization)其基因表現，結果顯示stathmin主要表現在神經系統，但與哺乳動物不同的是，zSTMN1和zSTMN2主要表現在腦和眼，zSTMN4則廣泛表現在各種組織；在胚胎發育的過程中，zSTMN2持續在發育各階段表現，zSTMN1和zSTMN4則在受精後16小時才開始有表現。大致上，三個基因都在受精後16小時開始表現在spinal cord neurons 和otic capsules，但zSTMN1在受精後36小時以後的表現都侷限在腦部，zSTMN2在受精後24小時以後的表現主要在腦部及spinal cord neurons，zSTMN4的表現量較弱且幾乎整個胚體都有表現。當以morpholino oligos抑制zSTMN1、zSTMN2及zSTMN4之轉譯時，亦造成斑馬魚胚不同程度的缺陷及死亡，顯示這三個stathmin family genes在斑馬魚胚發育過程中可能扮演不同之角色。

Stathmin (STMN), a small phosphoprotein, integrates diverse intracellular signaling pathways involving the controls of cell proliferation, differentiation and activities by destablizing microtubules. Stathmin is the genetic element of a family, including stathmin (STMN1), SCG10 (STMN2), SCLIP10 (STMN3) and RB3 (STMN4) with its splicing variants RB3’ (STMN4’) and RB3” (STMN4”). In mammals, STMN1 is a ubiquitous and cytosolic protein, whereas other members of the stathmin family, STMN2 (SCG10), STMN3, STMN4/STMN4’/STMN4”, are exclusively expressed in the nervous system and attached to cell membrane. To understand the roles of stathmins in zebrafish development, we have cloned zebrafish orthologs of STMN1, 2 and 4 from embryos and showed that they shares high amino acid identities and characteristic motifs with other vertebrates. In the present study, we showed that zSTMN1 and zSTMN2 were expressed mainly in adult brain and eye, while zSTMN4 distributed more widely among tissues. During embryonic development, the expression of STMN2 was continuative throughout embryonic stages, while STMN1 and STMN4 were turned on at 12~16 hour post fertilization (hpf). Whole-mount in situ hybridization showed that all three stathmins were expressed in the spinal cord neurons and otic capsules at 16hpf, the expressions were extended to central nervous system (CNS) at about 18 hpf and retina at 48hpf, respectively. Moreover, localization of STMN1 was restricted to CNS after 36 hpf. In contrast, STMN2 and STMN4 were localized in both spinal cord neurons and CNS. Knocking down stathmins by morpholino oligos resulted in differential defects and embryonic death. These results suggest that different stathmins may play differential roles during development in zebrafish.

CONTENTS…………………………………………………………………………Ⅰ
LIST OF TABLE AND FIGURES…………………………………………………Ⅱ
中文摘要……………………………………………………………………………Ⅲ
ABSTRACT…………………………………………………………………………Ⅳ
INTRODUCTION……………………………………………………………………1
MATERIALS AND METHODS……………………………………………………6
Fish maintenance and embryo collection…………………………………………6
In silica cloning and sequence analysis……………………………………………6
RT-PCR analysis……………………………………………………………………7
Whole-mount in situ hybridization…………………………………………………8
Morpholino oligos injection………………………………………………………8
Embryo observations and photography……………………………………………9
RESULTS……………………………………………………………………………10
Sequence and phylogenetic analysis of stathmin family proteins…………………10
Expression pattern of zebrafish stahtmin family genes……………………………12
Knockdown of stathmin family by morpholino oligonucleotides…………………13
DISCUSSION………………………………………………………………………16
REFERENCE……………………………………………………………………22

TABLE………………………………………………………………………………32

FIGURES……………………………………………………………………………33

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