臺灣博碩士論文加值系統

(1)探討Plexin A、Semaphorin 2a、MICAL-like、Eph和Ephrin在果蠅卵子發育過程中扮演的角色
果蠅卵子發育過程是研究細胞增殖，分化，形態發生和集體細胞遷移的有效模型系統。過程中需要多個信號傳導途徑的協調，細胞連接和細胞骨架的重排。有研究觀測到Plexin A（PlexA）、Semaphorin 2a（Sema2a）、MICAL-like、Eph以及Ephrin基因在果蠅卵巢中有高度或中度的表現，Semaphorin / Plexin，ephrin / Eph除了在神經系統發育過程扮演引導軸突的功能外，這些配體/受體還調控上皮細胞遷移和分離。因此我們認為Plexin A，Semaphorin 2a，MICAL-like，Eph或Ephrin可能參與在果蠅卵子發育的過程。我使用UAS-GAL4系統和RNAi果蠅株來將濾泡細胞前體（follicle cell precursors），與卵室（Egg chamber）前端及後端的濾泡細胞中PlexA，Sema2a，MICAL-like，Eph或Ephrin的基因表現減量。當減少PlexA基因表現時，會減弱莖細胞（stalk cells）內插（intercalation）的能力。然而減少Sema2a，MICAL-like、Eph或Ephrin的基因表現後，濾泡細胞的增殖，莖細胞和極細胞（polar cells）的形成，伸展細胞（stretched cells）的形態轉變和邊界細胞（border cells）的遷移並不會受到影響，這表明這些基因活性在濾泡細胞形態發生過程中是不必要的。進一步的減少生殖細胞（germline cells）中的PlexA，Sema2a，MICAL-like，Eph或Ephrin的基因表現量，但並沒有觀察到特徵的改變。綜合上述，結果顯示在卵子發生過程中PlexA會調節莖（stalk）的形成，但Sema2a、MICAL-like、Eph、Ephrin是不必要的。

(2)探討Cpsf5或Cpsf6是否在果蠅卵子發育過程中與訊息傳遞路徑相互作用
Hippo信息傳遞路徑是保守的，其透過調節細胞增殖和凋亡來控制器官大小。在果蠅中，Hippo路徑主要的組成是激酶Hippo（Hpo），Warts（Wts）和轉錄輔助因子Yorkie（Yki）。雖然許多Hippo路徑的組成已經確定了，但是我們仍然對Hippo路徑所調控的機制沒有完整理解。最近的一項研究藉由利用免疫共沉澱法，提出了與Hippo路徑蛋白質有蛋白質相互接觸作用的網路（PPIN）。根據Hippo-PPIN，先前我們發現了與Yki相互作用的蛋白Cpsf5和Cpsf6，在果蠅卵子發育過程的濾泡細胞中起重要作用。在濾泡細胞中降低了Cpsf5或Cpsf6的基因表現後，會減弱邊界細胞遷移，增加極細胞的數量，促進增殖並延遲了從有絲分裂到核內再複製（endocycle）的轉變。而許多信息傳遞路徑已經被證明能夠調節濾泡細胞增殖、分化和邊界細胞遷移，例如Bmp，Wnt，Notch和JAK-STAT路徑。在本論文中，我分析了四個報告基因的果蠅株： Bmp路徑的Daughters against dpp-lacZ（dad-lacZ）， Wnt路徑的Frizzled3-RFP（Frz3-RFP）， Notch路徑的Notch response element-GFP（NRE-GFP）和JAK-STAT路徑的STAT-GFP。降低了Cpsf5或Cpsf6的基因表現量，並不影響這些報告基因的強度或形態模式，結果顯示Cpsf5或Cpsf6不透過Bmp，Wnt，Notch和JAK-STAT路徑來調控濾泡細胞增殖、分化和邊界細胞遷移。

(1)Investigating the role of Plexin A, Semaphorin 2a, MICAL-like, Eph and Ephrin during Drosophila oogenesis

Drosophila oogenesis is an efficient model system for studying cell proliferation, differentiation, morphogenesis and collective cell migration. Coordination of multiple signaling pathways, rearrangement of cell junctions and cytoskeletons are required for the oogenesis processes. Interestingly, the mRNA levels of Plexin A, Semaphorin 2a, MICAL-like, Eph and Ephrin are high or moderate in the ovary of Drosophila. Semaphorin/Plexin, ephrin/ Eph guide axons along the right direction during the development of the nervous system. Additionally, these ligands/receptors regulate migration and segregation of epithelial cells. Therefore, we suspected that Plexin A, Semaphorin 2a, MICAL-like, Eph and Ephrin may play roles during oogenesis. I used UAS-GAL4 system and RNAi lines to knock down PlexA, Sema2a, MICAL-like, Eph or Ephrin in follicle cell precursors, anterior and posterior follicle cells. Knockdown of PlexA attenuated intercalation of stalk cells. However, knockdown of Sema2a, MICAL-like, Eph or Ephrin did not affect follicle cell proliferation, stalk cell and polar cell formation, stretched cell morphogenesis and border cell migration, suggesting that these genes are dispensable during follicle cell morphogenesis. I further knocked down PlexA, Sema2a, MICAL-like, Eph and Ephrin in germline cells and no phenotype was observed. Together, my results suggest that PlexA regulate stalk formation, while Sema2a, MICAL-like, Eph and Ephrin are dispensable during oogenesis.

(2)To investigate whether Cpsf5 and Cpsf6 interact with the Bmp, Wnt, Notch and JAK-STAT pathways during oogenesis
The Hippo signaling pathway is conserved and controls organ size through regulating cell proliferation and apoptosis. In Drosophila, key components of the Hippo pathway are Hippo (Hpo), Warts (Wts) kinases and a transcriptional coactivator Yorkie (Yki). Many components of Hippo pathway have been identified. Nonetheless, our understanding about the regulatory mechanism of the Hippo pathway remains incomplete. A recent study has revealed protein-protein physical interaction network (PPIN) of the Hippo pathway by using Co-Immunoprecipitation. Based on Hippo-PPIN, we previously found that Yki-interacting proteins Cpsf5 and Cpsf6 play important roles in follicle cells during oogenesis. Knockdown Cpsf5 or Cpsf6 in follicle cells attenuated border cell migration, increased the number of polar cells, promoted proliferation and delayed transition from mitosis to endocycle. Many signaling pathways have been demonstrated to regulate follicle cell proliferation, differentiation, and border cell migration, such as the Bmp, Wnt, Notch and JAK-STAT pathways. Here, I analyzed four reporter lines: Daughters against dpp-lacZ (dad-lacZ) for the Bmp pathway, Frizzled3-RFP (Frz3-RFP) for the Wnt pathway, Notch response element-GFP (NRE-GFP) for the Notch pathway, and STAT-GFP for the JAK-STAT pathway. Knockdown of Cpsf5 or Cpsf6 did not affect the intensity or pattern of these reporter lines, indicating that Cpsf5 or Cpsf6 does not regulate follicle cell proliferation, differentiation and border cell migration through the Bmp, Wnt, Notch and JAK-STAT pathway.

摘要...............................................(i)
Abstract..........................................(iii)
Table of Contents.................................(v)

Chapter 1: Investigating the role of Plexin A, Semaphorin 2a, MICAL-like, Eph and Ephrin during Drosophila oogenesis

1. Introduction...................................(1)
-Drosophila oogenesis
-Axon guidance cues
-The roles of axon guidance molecules in non-neuronal cells
2. Materials and methods...........................................(7)
-Fly stocks
-Antibodies and Reagents
-RNAi experiments
-Immunostaining and fluorescence microscopy
-Statistics
3. Results........................................(9)
4. Discussion.....................................(11)
5. References.....................................(13)
6. Figures and Legends...........................................(16)

Chapter 2: To investigate whether Cpsf5 and Cpsf6 interact with the Bmp, Wnt, Notch and JAK-STAT pathways during oogenesis

1. Introduction...................................(38)
-The Hippo pathway
-Cpsf5 and Cpsf6 physically interact with Yki
-Previous studies related to Cpsf5 and Cpsf6
-The Bmp pathway
-The Wnt pathway
-The Notch pathway
-The JAK-STAT pathway
2. Materials and methods...........................................(43)
-Fly stocks
-Antibodies and Reagents
-RNAi experiments
-Immunostaining and fluorescence microscopy
3. Results........................................(44)
4. Discussion.....................................(46)
5. References.....................................(47)
6. Figures and Legends...........................................(50)

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