臺灣博碩士論文加值系統

果蠅胚胎的前後軸向的形成，在卵發生時期就已決定。在卵發育中期，bcd與osk mRNA的不對稱的分布提供了一個主要的機制，其中osk mRNA在卵中的坐落位置決定未來胚胎腹部與生殖前驅細胞的形成。當參與osk mRNA定位的基因發生突變時，會使得胚胎腹部與生殖前驅細胞無法正確的形成，造成胚胎腹部腹節缺失的posterior group突變性狀。
Smad是TGFβ訊息路徑中不同的Smad蛋白質所共用的轉錄因子。人類的SIF，Smad Interacting Factor，與Smad有蛋白質-蛋白質間的結合。果蠅的Smad結合蛋白，dSIF，在生殖細胞進行的基因重組分析搜尋中，因為呈現腹部腹節缺失的posterior group突變胚胎性狀而被篩選研究。dSIF與人類SIF在N端形成演化上相當保守的區域。在dSIF突變缺失的基因背景下，卵殼呈現腹部化現象，這與Dpp (Decapentaplegic，果蠅的TGFβ同源基因蛋白) 的受體基因突變時腹部化的卵殼性狀相吻合；由於dSIF突變可以抑制因為Dpp過度表現所呈現的胚胎背部化的性狀，這顯示dSIF與Dpp路徑有相互作用的關係。
在dSIF突變的卵巢中，osk mRNA及Osk蛋白的表現量皆呈現下降或消失的性狀，但在定位上卻是正常的 ; Staufen(Stuf)和Gurken (Grk，果蠅的TGFα)蛋白質的表現量亦呈現下降或消失的性狀。這些現象與Dpp受體基因突變時，會產生母源產物無法運送到卵細胞的性狀相類似。因此，在dSIF突變中母源產物的普遍性而非專一性的減少及dSIF蛋白於護理細胞與卵細胞交接處如守門員似的分布加上其分子上的特性，我們推測dSIF很可能參與運送母源產物的機制。

Body axis of Drosophila embryos is determined through the asymmetry localization of maternal components during oogenesis. In the mid-oogenesis, two key components, bicoid (bcd) mRNA and oskar (osk) mRNA, distribute anteriorly and posteriorly of the oocyte, respectively. Mutations of genes resulted in osk mRNA mislocalization produce the "posterior group" embryonic phenotype, which was previously defined as the lost of abdomen and pole cells.
Smad is the common mediator of TGFβ signaling pathway. In human, the binding between SIF (Smad interacting factor) and Smad implicate the possible function of SIF in TGFβ signaling. In this study, the Drosophila homologue of SIF, dSIF, is identified because of its maternal-effect posterior group defect. The eggs derived from dSIF mutant female exhibit ventralized eggshell phenotype similar to that are laid by mother carrying decapentaplegic (dpp, Drosophila TGFβ homologue) receptor mutant. Furthermore, dorsalized embryonic phenotype caused by dpp and thick vein (tkv) overexpression could be suppressed by dSIF mutant. It is suggested that dSIF is a new component acting in Dpp signaling.
During oogenesis, dSIF is essential for the formation of the proper level of Osk protein and mRNA; nevertheless, their localizations are not affected in mutant. The expression level of Staufen (Stau) and Gurken (Grk) also decreased. In dpp receptor mutants, saxophone (sax) and tkv, their mutant ovaries exhibited maternal-component transport defect phenotype. Based on the general but not specific decrease of maternal components and the nearby oocyte-nurse cell-junction gatekeeper-like localization of dSIF protein in egg chamber, we propose that dSIF may participate in the transportation mechanism of maternal components from nurse cells to oocyte.

Abstract 1
中文摘要 2
Table of Content 3
List of Tables 6
List of Figures 7
Introduction 9
Genetics dissecting of Drosophila embryogenesis10
Overview of Drosophila Oogenesis 11
Anterior-posterior axis determination of oocyte12
Dorsal-ventral axis determination of oocyte 14
Cytoplasmic streaming during oogenesis 15
Directed Transport 15
Rapid Transport 15
Nurse Cell Cytoplasmic Actin Bundle Assembly 16
TGFβ signaling 17
The role of TGFβ signaling during oogenesis 19
dSIF , a novel gene in Dpp signaling pathway20
Materials and Methods 22
Fly stocks and maintenance 22
The autosomal FLP-DFS technique 22
Genetics screen of P-transposon induced zygotic lethal with specific maternal effect phenotype on the second chromosome 23
Germ-line clone production 23
Cuticle preparation 24
In situ hybridization 24
For embryo 24
For ovary 26
Immuno-antibody staining 28
For embryo 28
For ovary 28
X-gal staining for ovary 29
Single fly PCR 29
Mis-expression experiment 30
Plasmid rescue 30
Inverse PCR 31
Antibody preparation 32
Genomic rescued construct and UAS rescued construct 32
Bioinformatics analysis 33
For the P-transposon induced homozygous lethal mutations 33
For the detail analysis of dSIF 33
Isolation of plasmid DNA 34
Result 35
Molecular and maternal function characterizations of P-transposon-induced zygotic lethal mutants 35
Identification of a novel posterior group gene, dSIF 36
The posterior phenotype and the lethality of dSIFb53 result from the P-element insertion 37
dSIF belongs to a novel protein family, which may involve in TGFβ signaling 39
The lethal phase of dSIF zygotic mutant 42
The mRNA expression pattern of dSIF in Drosophila ovary and embryo 43
β-glycosidase activity of dSIFb53 44
The protein distribution pattern of dSIF in Drosophila ovary 44
The dSIF protein expression is disrupted in dSIF mutant egg chamber 45
dSIF suppresses the overexpressed dpp embryonic phenotype 45
dSIF suppresses the overexpressed tkv embryonic phenotype 46
dSIF is epistatic to dpp weak allele 47
dSIFb53 is not soma-required during oogenesis 48
dSIF is required for the posterior localization of Osk protein and osk mRNA 48
dSIF is required for the posterior localization of Staufen protein 49
Ovarian dpp expression pattern is not changed in dSIF mutation 50
dSIF is required for the expression of Gurken protein 50
dSIF changes the expression domain of EGFR target gene, kekkon-151
Discussion53
dSIFb53 is a weak hypomorph allele54
dSIF encodes a cytosolic-nuclear bizonal protein54
dSIF is a new component of Dpp/TGFβ signaling pathway55
The organization of microtubule is not affected in dSIF mutant ovary56
dSIF is not involved in a general translation activation machinery in the oocyte57
dSIF is involved in the formation of posterior-assembly-complex through the transportation of maternal materials57
The role of dSIF in dorsal-anterior follicle cell patterning58
Acknowledgements60
Reference61

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