吳益群臺灣大學:分子與細胞生物學研究所葉怡君Yeh, Yi-ChunYi-ChunYeh2010-06-022018-07-062010-06-022018-07-062008U0001-2507200817400700http://ntur.lib.ntu.edu.tw//handle/246246/184703細胞遷移對生物的器官發育而言是一件重要的過程。線蟲的兩顆生殖腺遠端細胞的遷移過程，是研究細胞遷移的利器。遠端細胞的遷移過程有三個階段。線蟲孵出後，在第一齡末期進入第一階段，兩顆遠端細胞沿著腹部肌肉爬行。當線蟲成長到第三齡幼蟲中期時，遠端細胞轉九十度往背部行走，形成第二階段的遷移。到了第三齡幼蟲晚期，遠端細胞再度轉九十度，沿著背部肌肉往身體中央爬行，此時為第三階段。遠端細胞的遷移過程受到時間和空間上許多的因素調控。例如遠端細胞上的UNC-5細胞膜接收器和分布在腹部的UNC-6作用，控制遠端細胞往背部行走。三個調節發育時期的基因：dre-1、daf-12和lin-29，共同作用使蟲體的生殖腺在正常時期從第一階段的遷移路徑經第二階段再進入第三階段的初期。本實驗室先前分析認為DPY-24蛋白質藉由抑制unc-5的轉錄來避免第二階段爬行過早發生。在本實驗中，藉由分子遺傳技術，證實了一個新的dpy-24突變株，dpy-24(tk41)。dpy-24協同unc-5和unc-6在基因層次上調控遠端細胞在第三階段的方向選擇能力。證實了在DTC遷移過程中，unc-5和unc-6有調控第三階段方向性的能力。對於遠端細胞而言，DPY-24蛋白質只有在第一階段表現，在晚期則停止表現。本篇研究發現前述的調節發育時期的基因中，dre-1和daf-12參與了降低DPY-24晚期表現的機制。結果顯示了DPY-24受到嚴格的調控以規範進入第二階段爬行的時間點。Cell migration is critical for organogenesis during animal development. Patterned migration of gonadal distal tip cells (DTCs) in Caenorhabditis elegans is an ideal model for cell migration research. DTCs have three sequential migration phases. During phase I, DTCs migrate along the ventral body wall muscle in the L1 and L2 stages. At mid-L3, DTCs reorient and migrate dorsalward during phase II. In phase III, DTCs turn orthogonally and migrate along the dorsal body wall muscle toward midbody in the late L3 and L4 stages. Several factors have been identified and characterized that regulate DTC migration in temporally and spatially specific fashions. For example, UNC-5/Netrin receptor has been shown to act in DTCs to guide their phase II dorsal migration against the ventrally concentrated repellent of UNC-6/Netrin. Three heterochronic-related proteins, transcription factors LIN-29 and DAF-12 and F-box protein DRE-1, act redundantly to control the reflexion of the gonad in L3. Our lab has identified a zinc-finger-containing protein DPY-24 that functions to suppress the initiation of phase II migration in phase I by transcriptionally repressing unc-5. In this study, we have genetically and molecularly defined a new allele of dpy-24, tk41. In addition, we found that dpy-24 acts synergistically with unc-5 and unc-6 to regulate the phase III migration direction. This observation shows a previously unassigned function of unc-5 and unc-6 in the longitudinal DTC migration. Furthermore, DPY-24 protein is detected in phase I but not phase II or III in wild type; however, daf-12 and dre-1 double mutations block the down-regulation of DPY-24 in phase II and III. This result reveals a complex regulation of the DPY-24 level in specifying the timing of phase II migration.口試委員會審定書………………………………………………………………………i謝……………………………………………………………………………………...ii文摘要………………………………………………………………………………..iiibstract…………………………………………………………………………… ……ivntroduction……………………………………………………………………………...1aterials and Methods…………………………………………………………………14 C. elegans strains and culture methods…………………………………………...…14 Analysis of DTC migration phenotypes……………………………………………..15 Immunohistochemistry………………………………………………………………16 Image analysis……………………………………………………………………….16 Database searches and bioinformatics……………………………………………….16esults……………………………………………………………………… …………17 DTCs migration defect of dpy-24 mutants…………………………………………..17 Verification of a new dpy-24 allele…………………………………………………..18 DTCs migration defect of dpy-24;smg-2 mutants…………………………………...19 DTCs migration defect of unc-5 and unc-6 mutants………………………………...20 DTCs longitudinal migration defect of dpy-24; unc-5 and dpy-24; unc-6 mutants…20 transgene-dependent DTCs migration defect………………………………………..22 Temporal expression pattern of DPY-24 in DTCs……………………………….…..23 DRE-1 and DAF-12 act together to regulate DPY-24 expression level after phase I.25iscussion………………………………………………………………………………27 DTCs migration defect of unc-5, unc-6, and dpy-24 mutants……………………….27 DTCs longitudinal migration defect of dpy-24; unc-5 and dpy-24; unc-6 mutants…28 lag-2::gfp transgenes caused a DTC migration defect………………………………29 Temporal expression pattern of DPY-24 in DTCs…………………………………...30 The regulation of post-translation of DPY-24……………………………………….31eference……………………………………………………………………………….35 igures……………………………………………………………………… ……... …..I Figure 1. DTC migration pattern of N2 hermaphrodite……………………………….I Figure 2. The predicted protein structures of wild-type and mutant DPY-24……...…II Figure 3. DTC migration pattern of dpy-24 mutants………………………………...III Figure 4. DTC failed in phase II migration in unc-5(e53)………………………..…IV Figure 5. DPY-24 is expressed in DTC during phase I migration and diminished thereafter………………………………………………………………………………..V Figure 6. DPY-24 is diminished after phase I in dre-1(dh99)………………………VI Figure 7. DPY-24 is diminished after phase I in daf-12(rh61rh411)……………….VII Figure 8. DPY-24 is weakly expressed in DTC during phase III migration in dre-1(dh99);daf-12(rh61rh411)………………………………………………………VIII Figure 9. Percentage of ectopic DPY-24 expression in DTC during phase III migration and the ratio is normalized to phase I……………………………………...IX Figure 10. The model of the complex regulations of DPY-24 level and the dpy-24- involved-regulations of DTC migration……………………………………………….Xables………………………………………………………………………………. …XI Table 1. DTCs migration defect of dpy-24 and dpy-24;smg-2……………………...XI Table 2. The allele characterization of tk41………………………………………...XII Table 3. tk41 and tp5 belong to the same complementation group………………...XIII Table 4. DTCs migration defect of unc-5, unc-6 and unc-40……………………...XIV Table 5. DTC longitudinal migration defect of dpy-24;unc-5 mutants…………….XV Table 6. DTC longitudinal migration defect of dpy-24;unc-6 mutants………...….XVI Table 7. Transgenic-dependent DTC migration defect…………………………...XVII Table 8. dpy-24 promoter is turned on from phase I to III……………………....XVIII Table 9. 3''UTR is not sufficient for DPY-24 downregulation…………………….XIX Table 10. Coding region is sufficient for DPY-24 downregulation………………..XX Table 11. dpy-24 promoter maybe weaker in phase II and III than I………………XXIupplementary Figures……………………………………………………………....XXII Figure S1. The amino acids alignment of DPY-24 homologes………………...…XXII Figure S2. The protein structure of wild type and mutant UNC-5……………....XXIII Figure S3. The protein structure and domain function of UNC-6…………….…XXIV Figure S4. The predicted cleavage sites of DPY-24……………………………....XXV Figure S5. The predicted miRNAs which interact with dpy-24 3’UTR………....XXVIupplementary Table..……………………………………………………………. XXVII Table S1. The strains containing GFP-tagging DTCs…………………………..XXVIIapplication/pdf1601554 bytesapplication/pdfen-US細胞遷移遠端細胞unc-5dpy-24cell migrationdistal tip cell (DTC)http://ntur.lib.ntu.edu.tw/bitstream/246246/184703/1/ntu-97-R95b43009-1.pdf