周子賓臺灣大學：分子與細胞生物學研究所陳崇豪Chen, Chung-HaoChung-HaoChen2007-11-252018-07-062007-11-252018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/49936形態決定素(morphogen)是具有訊息傳遞能力的蛋白質，在發育組織中各有其獨特的濃度梯度。藉此，接受不等訊息強度的細胞群會開啟特定的下游基因表現，進而引導了細胞命運的正確決定與發育過程的進行。近年來的研究顯示，位於細胞膜上的巨型多醣蛋白HSPGs對於形態決定素Hedgehog(Hh), Wingless(Wg), Decapentaplegics(Dpp)在果蠅翅碟上的移動與訊息傳遞皆扮演重要的角色。我們先前的研究指出一個新發現的節向決定基因Rotini(Rti)能決定HSPGs的表現，同時也影響到形態決定素的行為。我們更發現Rti座落於高基氏體，且能夠直接與一個可能的v-SNARE, dVti1交互作用。這些資訊顯示Rti應是一個參與於蛋白質運輸的新成員，並且由Rti調控的輸送過程可能決定了HSPGs合成的正確發生，因此引發了後續對於形態決定素的廣泛影響。 此外，合成HSPGs的核心蛋白Dally-like(Dlp)，在翅碟上具有一特殊的分布: Dlp在翅碟腹背交界處表現量極低，而此處正是Wg分泌的所在。我們進一步證明了Wg的訊息傳遞對於Dlp的抑制作用不僅是必須性的，且足以在Wing pouch及notum上被異位引發。由於Dlp的表現能夠擴大Wg在翅碟上的分布，因此形態決定素Wg可以藉由其訊息傳遞路徑對Dlp產生抑制作用，以此來回饋調控本身的散佈範圍。這些結果顯示形態決定素與多醣蛋白HSPGs之間的調控關係是互為因果的。Morphogens are signaling proteins which form their own specific gradients over the developing tissues and subsequently pattern the field by activating different sets of genes at distinct signaling intensities. In recent years, macromolecules heparan sulfate proteoglycans (HSPGs) which locate on the cell surface were found to be essential for modulating the movement and distribution of multiple morphogens such as Hedgehog (Hh), Wingless (Wg) and Decapentaplegics (Dpp) in Drosophila wing imaginal discs. In previous studies, we identified a novel segment polarity gene, rotini (rti), which determines the distribution of these essential morphogens and is required for the normal expression of functional HSPGs. Additionally, we found Rti is a Golgi-resided protein and capable to physically interact with a putative V-SNARE, dVti1, that is a core member in the process of vesicle traffickings. Therefore, we suggest that Rti is a novel component in the trafficking pathway and the Rti-mediated transportation is necessary for the biosynthesis of functional HSPGs, which in turn influences the HSPGs-controlled morphogen behaviors on the cell surface. Moreover, we found one glypican of HSPGs core proteins, Dally-like (Dlp), is almost absent within cells along the D-V boundary of wing discs, and this pattern is roughly complementary to Wg expression. Our analysis showed that high level of Wg signaling is not only necessary but also sufficient to repress Dlp expression in the wing pouch and notum region in wing discs. As the Dlp-composed HSPGs are potent to modulate Wg distribution, we suggest that Wg can restrict its own spreading range via its own signaling. It also indicates that the regulations on HSPGs and morphogens are interdependent.Table of content List of figures…………………………………………………………………..…7 Introduction…………………………………………………………………….…9 I. Positional information guided by secreted morphogens………...9 II. Formation of morphogen gradient…………………………………..10 1. Morphogen transportation………………………………………….…11 a. Planar transcytosis……………………………………………………11 b. Argosomes……………………………………………………………..12 c. Cytoneme……………………………………………………………....13 d. Restricted diffusion…………………………………………………...13 2. Enhanced degradation………………………….…………………...…14 3. Receptor/ligand interaction…………………………………………...15 4. Lipid modification……………………………………………………...16 5. HSPGs modulation……………………………………………………..17 a. The biosynthesis of HSPGs…………………………………………...17 b. Functional HSPGs and morphogen patterning……………….…….19 III. Hedgehog and Wingless signal transduction in wing discs……20 1. Hedgehog pathway……………………………………………………...21 2. Wingless pathway……………………………………………………….23 IV. Rotini, a novel segment polarity gene involved in HSPGs expression………………………………………………………………….24 1. Identification of rotini locus…………………………………………….24 2. Rotini is a Golgi resided protein……………………………………….25 3. rotini mutant impairs Hh expression and signaling………………...25 4. HSPGs decreased within rotini mutant clones……………………....26 V. Vesicle trafficking in morphogen distribution……………………26 1. Vesicle budding and fusion……………………………………………….27 2. Trafficking mutants can interfere with morphogen patterning………..28 Materials and Methods………………………………………………………30 1. Fly stocks and maintenance………………………………………….....30 2. The autosomal FLP-DFS technique…………………………………...30 3. Heat shock treatment……………………………………………………31 4. GAL4-UAS system………………………………………………………..31 5. Generation of recombinant mutant clones in somatic tissues……..31 6. Generation of flip-out gain of function clones in somatic tissues…32 7. Fluorescence antibody staining of imaginal discs…………………...32 8. GST pull-down…………………………………………….……………..34 9. Membrane Stripping…………………………………………………….36 10. Cloning constructs…………………………………………………..36 11. micro-ingestion and transgenic fly…………………………………38 Result……………………… ……………………………………………………...39 I. Rotini, a Golgi-resided human Gpp34 homologue, interacts with a putative v-SNARE dVti1 and determines the HSPGs expression and morphogen distribution……………………………..39 1. Overexpression of Rti disrupts Drosophila wing and eye development………………………………………………………………..39 2. Abnormal Rti expression alters Hh, Wg and Dpp distribution in wing discs…………………………………………………………………..41 3. Rti, a Golgi-resided protein, interacts and colocalizes with a putative Drosophila v-SNARE dVti1……………………………………………..43 4. dVti1 genetically interacts with Rti in adult tissue development….46 II. The expression of Dally-like, a HSPGs core protein, is negatively regulated by Wingless signaling to modulate morphogen Wingless distribution in Drosophila wing discs……………………47 1. Dlp expression is roughly complementary to the Wingless expression stripe in wing discs………………………………………………………..47 2. Wg signaling is necessary for the negative regulation of Dlp along the D-V boundary…………………………………………………………48 3. Ectopic Wg signaling is sufficient to repress Dlp expression in wing pouch and Notum region………………………………………………...50 4. Wg spreading range increases in the Wg signaling decreasing zone where Dlp up-regulated……………………………………………..…...51 5. The Wg-mediated repression of Dlp does not act through the extracellular enzyme Notum…………………………………………….51 6. Wg signaling may crosstalk with Hh signaling by Dlp……………...53 Discussion……………………………………………………………………...…54 1. Defective HSPGs alter Hh, Wg and Dpp morphogen generally……………………………………………………………………55 2. The role of Rti in the trafficking pathway…………………………….57 3. May trafficking mutants alter HSPGs expression? …………………59 4. The regulation of HSPGs by morphogen signalings……………..….61 Acknowledgement……………………………………………………………..63 Reference………………………………………………………………………....64 Figures……………………………………………………………………………..731799312 bytesapplication/pdfen-US高基氏體多醣蛋白形態決定素GolgiHSPGsmorphogenotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49936/1/ntu-94-R92b43001-1.pdf