Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97772
DC FieldValueLanguage
dc.contributor許惠真zh_TW
dc.contributorHwei-Jan Hsuen_US
dc.contributor.author賴俊銘zh_TW
dc.contributor.authorChun-Ming Laien_US
dc.contributor.other生物科技學研究所zh_TW
dc.date2018zh_TW
dc.date.accessioned2019-03-22T06:05:10Z-
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dc.identifier.urihttp://hdl.handle.net/11455/97772-
dc.description.abstractStem cells reside in different types of supporting cells, or niches, which fine-tune stem cell maintenance and differentiation in response to physiological demand. However, little is known about how those niches are formed. Here, we have used Drosophila ovary as model to study how germline stem cell (GSC) niches are formed during development. Firstly, we have identified that Hedgehog (Hh) signaling establishes precursors for GSC niches by regulating cell-cell adhesion. Mechanistically, Hh produced from forming terminal filament (TF) signals posteriorly for somatic gonadal precursors (SGPs) to specify stromal intermingle cells (ICs), which contributes to both adult GSC maintenance niche-cap cells and differentiation niche- escort cells, by suppressing E-cadherin expression. We also identified Traffic Jam (an orthologue of a large Maf transcription factor in mammals) is a novel transcriptional target of Hh signaling to control cell adhesion by negatively regulating E-cadherin. Interestingly, the determinant of ICs differentiates into cap cells requires down-regulation of Hh signaling, thereby E-cadherin is able to be expressed in cap cells for GSC recruitment. Furthermore, we identified that Delta produced from TFs activates Notch signaling in adjacent ICs, and results in suppression of Hh signaling via Cul3HIB/SPOP-mediated Ci degradation. Our results demonstrate that spatio-temporal regulation of Hh and Notch signaling orchestrates GSC niche formation, and shed light on how organs utilize the signaling integration to carve out niches.en_US
dc.description.tableofcontentsTable of Content Abstract i Table of Content ii Abbreviations vii Introduction 1 Adult stem cells and their niches 1 The developing Drosophila ovary for studying stem cell niche formation 2 Cell-Cell interaction for stem cell niche formation 4 Hh signaling in organ development and stem cell niche formation 5 Notch signaling in stem cell niche formation 7 Results 9 Part I: Hedgehog signaling establishes precursors for GSC niches by regulating cell adhesion 9 Hh ligand produced from TFs signal to apical somatic cells and ICs 9 G-TRACE together with GAL80ts is reliable system to trace Hh-producing and –receiving cells lineages 10 Somatic Hh-producing cells contribute to GSC maintenance niche 11 Somatic Hh-receiving cells contribute to both GSC maintenance and differentiation niches 12 Disruption of somatic Hh signaling reduces the IC pool 13 Hh signaling maintains the IC pool for GSC niche formation 14 Hyperactivation of Hh signaling expands the IC pool, leading to over-populate differentiation niche 16 Hh signaling controls the cell fate decision between stromal ICs and epithelial basal cells 17 Hh signaling in ICs cell-autonomously promotes Tj expression but suppresses E-cadherin 20 Tj controls adhesion of somatic gonadal cells to adopt IC fate 22 Down-regulating shg or overexpressing tj in smo-knockdown somatic gonadal cells rescues soma-germline interaction and the IC pool 23 Hh signaling directly activates tj transcription through Ci-mediated canonical pathway 24 Discussion 27 Hh signaling in organogenesis 27 The autocrine-to-paracrine shift of Hh signaling in ovary development 28 Hh signaling in niche-stem cell formation and coordinated growth 29 Differential cell affinity in organ architecture 30 Part II: Notch signaling controls cap cell formation by suppressing Hh signaling 32 Notch signaling controls cap cell formation by preventing escort cell fate 32 Notch signaling sustains the adhesion of GSC-cap cells by suppressing Hh signaling 34 Knockdown ptc or overexpressing Ci-PKA is not sufficient to block Notch-mediated cap cell formation 34 Notch signaling suppresses Hh signaling for niche cap cell formation via Cul3HIB/SPOP-mediated Ci degradation 35 Discussion 37 Crosstalk between Hh and Notch signaling in GSC niche formation 37 The regulation of Hh signaling on Tj-mediated E-cadherin suppression 39 The regulation of Notch signaling in GSC niche formation 39 Future works 40 Summary 42 Future perspectives 43 Materials and Methods 44 Drosophila strains and culture 44 Developmental stage of larvae and pupae 45 GAL4-based cell lineage analysis 46 Genetic mosaic analysis 47 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) 48 Generation of Drosophila tj promoter constructs 49 Luciferase reporter assay 50 Western blot analysis 50 Chromatin immunoprecipitation (ChIP) 51 Immunostaining and fluorescence microscopy 53 Tables 56 Table 1: LacZ enhancer trap lines for examining gene transcriptional activity or marking specific cell types in developing and adult Drosophila ovary 56 Table 2: GAL4 lines for driving UAS transgene in specific cell types 57 Table 3: Mutant alleles combine with FRT site for generating GFP negative homozygous mutant cells 58 Table 4: UAS-RNAi lines for knocking down gene expression 59 Table 5: UAS transgenic lines for gene overexpression 60 Figures and Figure legends 61 Figure 1. Schematic of the ovary system and demonstration of Hh and Ptc antibodies specificity in wing discs. 61 Figure 2. Expression patterns of Hh, Ptc, and ptc>lacZ during ovarian development. 63 Figure 3. G-TRACE is reliable cell lineage tracing system under GAL4-GAL80ts control. 65 Figure 4. Gonadal hh-GAL4 and ptc-GAL4-expressing cells contribute to adult GSC niches. 67 Figure 5. Somatic Hh signaling controls soma-germline interaction and IC pool as well as GSC recruitment. 69 Figure 6. Disruption of somatic Hh signaling results in ovary morphogenesis failure. 71 Figure 7. Somatic Hh signaling is essential for GSC maintenance and differentiation niche formation. 72 Figure 8. Overexpression of hh promotes IC formation and accompany with over-populated PGCs. 74 Figure 9. Overexpression of hh results in an expanded population of escort cells. 75 Figure 10. Somatic Hh signaling controls the cell fate decision between stromal ICs and epithelial basal cells. 77 Figure 11. Suppression of somatic Hh signaling does not decrease Egfr signaling and induce cell death. 79 Figure 12. Somatic Hh signaling directly controls Tj and E-cadherin expression in ICs. 80 Figure 13. Piwi is dispensable for soma-germline interaction that controlled by Hh signaling. 82 Figure 14. Tj modulates E-cadherin expression for somatic cells to intermingle with PGCs and adopt IC cell fate. 83 Figure 15. Overexpression of tj in somatic gonadal precursors increases PGCs. 84 Figure 16. Overexpression of tj in smo-knockdown SGPs rescues soma-germline interaction as well as adult GSCs and their niches. 85 Figure 17. Tj acts downstream of Hh signaling to control IC formation. 87 Figure 18. Hh signaling activates tj transcription via Ci. 88 Figure 19. Hh signaling is down-regulated in forming cap cells and absent from adult cap cells. 90 Figure 20. Notch signaling is required for cap cell formation and down-regulation of Hh signaling in cap cells. 91 Figure 21. Notch mutant does not induce cap cell apoptosis. 93 Figure 22. Notch signaling sustains E-cadherin expression by suppressing Hh signaling. 95 Figure 23. Overexpressed a Cul3HIB/SPOP resistant form of Ci blocks cap cell formation. 96 Figure 24. Hyperactivation of Hh signaling is sufficient to decrease Notch-mediated cap cell formation. 98 Figure 25. Model of Hh and Notch signaling in the formation of Drosophila ovarian GSC niches. 99 Reference: 100 Appendix 111 Overexpression of hh promotes cap cell to terminal filament cell transformation by non-canonical Hh pathway 111 Overexpression of hh facilitates transformed terminal filament cell formation by promoting Dl expression 113 Discussion 116 The competition of niches for GSC maintenance or differentiation 116 Crosstalk between non-canonical Hh pathway and Dl-Notch signaling in GSC niche formation 117 Appendix Figure A1. Overexpression of hh promotes cap cell to TF cell conversion via non-canonical Hh signaling. 118 Appendix Figure A2. E-cadherin is dispensable for hh-overexpressing mediated cap cell to TFC conversion. 119 Appendix Figure A3. Notch signaling is up-regulated in the transformed TFCs. 120 Appendix Figure A4. Knockdown Notch suppresses hh-overexpressing-mediated transformed TFC formation and restores GSCs. 121 Appendix Figure A5. Overexpression of hh promotes Dl expression in transformed TFCs. 122 Appendix Figure A6. Dl genetically interact with hh to facilitate transformed TFC formation. 123 Appendix Figure A7. Possible mechanism of overexpression of hh drives cap cell to TF cell conversion. 124 Publication 125 Honor 126zh_TW
dc.language.isoen_USzh_TW
dc.rights同意授權瀏覽/列印電子全文服務,2021-08-20起公開。zh_TW
dc.subject生殖幹細胞zh_TW
dc.subject微環境zh_TW
dc.subject細胞表面黏著特性zh_TW
dc.subjectGermline Stem Cellen_US
dc.subjectNicheen_US
dc.subjectCell adhesionen_US
dc.titleHedgehog 訊息對卵巢生殖幹細胞微環境(niche)形成所扮演的角色zh_TW
dc.titleRoles of Hedgehog Signaling for Ovarian Germline Stem Cell Niche Formationen_US
dc.typethesis and dissertationen_US
dc.date.paperformatopenaccess2021-08-20zh_TW
dc.date.openaccess2021-08-20-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypethesis and dissertation-
item.cerifentitytypePublications-
item.fulltextwith fulltext-
item.languageiso639-1en_US-
item.grantfulltextrestricted-
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