指導教授：吳益群臺灣大學：分子與細胞生物學研究所蔣沆祥Jiang, Hang-ShiangHang-ShiangJiang2014-11-252018-07-062014-11-252018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261102計畫性細胞凋亡在生物體發育上扮演重要的角色。儘管執行計畫性細胞凋亡的基因已被發現且研究許多，這些基因如何在生物發育過程中被啟動與調控仍然有待發掘。我們發現在線蟲中，表皮生長因子〈LIN-3/EGF〉會以一種外來訊號的方式促進特定細胞的計畫性細胞凋亡。過少的EGF會導致計畫性細胞凋亡的減少；反之，過多的EGF會導致計畫性細胞凋亡的增加。這表示在生物體中，EGF必須保持在一個適當的濃度以確保適度的計畫性細胞凋亡進行。藉由遺傳分析，我們發現EGF會藉由上皮生長因子受器〈LET-23/EGFR〉活化下游Mitogen-Activated Protein Kinase〈MAPK〉路徑以及ETS轉錄因子〈LIN-1〉來促進線蟲中的BH3-only基因〈egl-1〉的轉錄並進一步引起特定細胞中的計畫性細胞凋亡。利用即時定量聚合酶連鎖反應和活體螢光觀測，我們發現在EGF的突變株中，egl-1的轉錄量會減少。更進一步地，利用凝膠遷移實驗〈EMSA〉以及異位表現基因的方式，我們證明了LIN-1可以直接結合到egl-1的啟動子上，而且這個結合對於表皮生長因子在活體中促進計畫性細胞凋亡來說是重要的。這些結果說明了表皮生長因子可以藉由提升egl-1的轉錄來促進特定計畫性細胞凋亡。 為了進一步找出在線蟲中參與計畫性細胞凋亡的調控因子，我們將grp-1突變株處理突變劑並找尋具有尾巴缺陷的突變株。GRP-1是一種ARF家族的GTP交換因子，當其和計畫性細胞凋亡相關基因同時產生突變時，突變株會展現尾巴缺陷的現象。藉由這個方式，我們找到了182個突變株，其中含有已知的計畫性細胞凋亡基因，如egl-1、ced-4、ced-3、ced-8和ced-11。除此之外，我們亦發現了PYR-1在線蟲中具有促進計畫性細胞凋亡的功能。這些結果顯示此基因掃描是具有其可行性。PYR-1是人類CAD (carbamoyl phosphate synthetase、aspartate transcarbamylase與dihydroorotase)的同源基因，參與在合成嘧啶的速率決定步驟。我們發現PYR-1影響到表皮細胞hyp8/9及分泌細胞excretory cell的姨細胞計畫性細胞凋亡，而且這個促進計畫性細胞凋亡的功能和其合成嘧啶的功能並不相關。除此之外，我們亦發現PYR-1是CED-3的酶作用物。因此，藉由這個基因掃描，我們找到了新的計畫性細胞凋亡相關基因並且初步了解其機制。Programmed cell death (PCD) is the physiological death of a cell mediated by an intracellular suicide program. Although key components of the PCD execution pathway have been identified, how PCD is regulated during development is poorly understood. We found that the epidermal growth factor (EGF)-like ligand LIN-3 acts as an extrinsic signal to promote the death of specific cells in Caenorhabditis elegans. The loss of LIN-3 or its receptor LET-23 reduced the death of these cells, while excess LIN-3 or LET-23 signaling resulted in an increase in cell deaths. Our molecular and genetic data support the model that the LIN-3 signal is transduced through LET-23 to activate the LET-60/RAS-MPK-1/ERK MAPK pathway and the downstream ETS domain-containing transcription factor LIN-1. LIN-1 binds to, and activates transcription of, the key pro-apoptotic gene egl-1, which leads to the death of specific cells. Our results provide the first evidence that EGF induces PCD at the whole organism level and reveal the molecular basis for the death-promoting function of LIN-3/EGF. In addition, the level of LIN-3/EGF signaling is important for the precise fine-tuning of the life-versus-death fate. Our data and the previous cell culture studies that EGF triggers apoptosis in some cell lines suggest that the EGF-mediated modulation of PCD is likely conserved in C. elegans and humans. To identify new genes involved in PCD in C. elegans, we conducted a genetic screen for mutants with a high penetrance of tail defects in the grp-1 background. grp-1 encodes a GTP exchange factor for ARFs and mutations defective in the activation, execution, or kinetics of PCD act synergistically with the grp-1 mutation to cause abnormal tail morphology. From this screen, we isolated 182 mutants. These mutants are classified into three subgroups, depending on their cell-corpse profiles: reduced/delayed, elevated and unchanged cell corpse numbers during embryogenesis. By SNP mapping and complementation tests, 22 mutations isolated were localized in known ced genes. This result showed that this screen is feasible to identify genes important for PCD. In addition, we have isolated pyr-1, encoding C. elegans CAD (carbamoyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase). In mammals, CAD has been reported to control the rate-limiting step during pyrimidine biosynthesis. We found that pyr-1 promotes the PCD of the aunt cells of hyp8/9 and excretory cell and this cell death-promoting function is independent of its CAD activity. Moreover, we showed that PYR-1 is a substrate of CED-3 in vitro. Therefore, we identified a previously unassigned pro-apoptotic function of PYR-1.摘要 i ABSTRACT iii TABLE OF CONTENTS v Chapter 1. LIN-3/EGF Promotes the Programmed Cell Death of Specific Cells in C. elegans by Transcriptional Activation of the Pro-apoptotic Gene egl-1 1 INTRODUCTION 3 MATERIALS AND METHODS 7 General Methods and Strains 7 RNA interference (RNAi) 7 Transgenic animals 8 Heat shock treatment 9 Molecular biology 10 Cell death assays 11 Quantitative real-time reverse transcriptase (RT)-PCR 12 Quantification of Pegl-1::gfp intensity in ABprpppapp 12 Prediction of potential LIN-1 binding sites 13 Electrophoretic mobility shift assay (EMSA) 14 Statistical analysis 15 RESULTS 17 lin-3 and let-23 promote specific PCDs in C. elegans 17 Overexpression of lin-3 cause ectopic cell deaths 20 let-23 is expressed in dying cells, including ABpl/rpppapp 21 LIN-3 can act as an extrinsic signal to promote PCD 22 lin-3 signaling up-regulates egl-1 transcription 23 LET-23 transduces the cell death-promoting signal via the LET-60-MPK-1 pathway 25 LET-23 transduces the cell death-promoting signal via the transcription factor LIN-1, but not LIN-31 26 LIN-1 activates egl-1 transcription by direct binding to the egl-1 promoter 27 DISCUSSION 31 LIN-3 signaling promotes PCD through LET-60-MPK-1 signaling and LIN-1 to activate egl-1 transcription 31 An appropriate level of extrinsic LIN-3 signaling is important for the precise fine-tuning of the life-versus-death fate of cells 36 The LET-60-MPK-1 pathway promotes germline and developmental cell deaths 38 Pathological and physiological roles of EGFR in promoting cell death 39 REFERENCES 41 FIGURES AND TABLES 53 Figure 1. lin-3 and let-23 promote specific PCDs in C. elegans 54 Figure 2. Overexpression of lin-3 induces ectopic gland cell death(s) through let-23 and the core PCD pathway 57 Figure 3. let-23 is expressed in dying cells, whereas lin-3 acts in a cell-nonautonomous manner to promote PCD 60 Figure 4. lin-3 promotes egl-1 transcription 64 Figure 5. lin-3, lin-1 and the LET-60-MPK-1 pathway act in the same pathway to promote PCD 65 Figure 6. lin-3 promotes PCD through transcriptional activation of egl-1 by LIN-1 67 APPENDIX 71 Appendix 1. Loss of lin-3 causes the disappearance of ABpl/rpppapp corpse(s) 71 Appendix 2. The lin-3(e1417) mutation does not affect the duration of the first 13 cell corpses derived from the AB lineage 73 Appendix 3. Analysis of extra surviving cells in the pharyngeal region in the ced-3(n2427) seneitized background 75 Appendix 4. lin-3 requires let-23 and the core PCD pathway to incerase numbers of embryonic cell corpses 77 Appendix 5. Mutants defective in the LET-60-MPK-1 pathway have reduced numbers of cell corpses 79 Appendix 6. The PI3K pathway and PLC genes are not involved in embryonic PCD 81 Appendix 7. lin-1 mutants, but not lin-31 mutants, have reduced numbers of cell corpses 83 Chapter 2. Identify New Genes Involved in Programmed Cell Death in C. elegans 85 INTRODUCTION 87 MATERIALS AND METHODS 89 General Methods and Strains 89 Transgenic animals 89 Molecular biology 90 Cell death assays 90 Mutagenesis screen 91 Statistical analysis 92 RESULTS 93 Defects in the activation, execution, or kinetics of PCD cause the tail defect in the grp-1 background 93 Extra hyp8/9 results in the tail defect 94 Identify new genes involved in PCD by a genetic screen in the grp-1 background 96 45 mutants with reduced or delayed cell corpse profiles are isolated. 97 16 mutants with increased numbers of cell corpses are isolated 97 99 mutants with normal cell corpse numbers are isolated 98 pyr-1 is isolated as a new PCD gene 99 DISCUSSION 103 Extra hyp8/9 causes the tail defect in the grp-1; ced-3 mutants 103 A genetic screen in the grp-1 sensitized background can isolate PCD-defective mutations 104 Mutants with abnormal tail morphogenesis independent of PCD can also be isolated from this genetic screen 105 REFERENCES 107 FIGURES AND TABLES 111 Figure 1. A model illustrates how the grp-1 and ced-3 mutations may act synergistically to result in extra neurons 111 Figure 2. The grp-1(gm350); ced-3(n717) double mutant displays abnormal tail morphology 113 Figure 3. The grp-1; ced-3 double mutant has ventral ridge(s) posterior to the anus 115 Figure 4. The grp-1; ced-3 mutant has extra hyp8/9 and PHsh 118 Figure 5. Extra hyp8/9 is sufficient to cause the tail defect 119 Figure 6. The proposed lineages in worms of the indicated genotypes 121 Figure 7. The flowchart and brief description of a genetic screen to isolate mutants with tail defects in the grp-1 sensitized background 123 Figure 8. PYR-1 can be cleaved by CED-3 in vitro and is important in the PCD of the aunt cells of hyp8/9 and excretory cell 125 Figure 9. The expression pattern of pyr-1 127 Table 1. Defects in the induction, execution, or kinetics of PCD cause tail defects in the grp-1 background. 129 Table 2. Mutants with known ced genes (22 mutants) 131 Table 3. Mutants with reduced or delayed cell corpse profiles (45 mutants) 133 Table 4. Mutants with increased numbers of cell corpses (16 mutants) 135 Table 5. Mutants with unchanged numbers of cell corpses (grp-1-dependent, 39 mutants) 137 Table 6. Mutants whose tail defects are grp-1-independent (24 mutants). 139 Table 7. Unanalyzed mutants (36 mutants) 141 APPENDIX 143 Appendix 1. A proposed lineage in the mutants with both tail defects and increased numbers of cell corpses. 1432954687 bytesapplication/pdf論文公開時間：2019/07/29論文使用權限：同意無償授權表皮生長因子計畫性細胞凋亡線蟲[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261102/1/ntu-103-D94b43001-1.pdf