跳到主要內容

臺灣博碩士論文加值系統

(3.229.142.104) 您好!臺灣時間:2021/07/27 08:13
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:江銘仁
研究生(外文):Min-Ren Chiang
論文名稱:探討線蟲中細胞遷移基因及時間和空間的調控機制
論文名稱(外文):Characterizing the Genetic Network that Regulates the Temporal and Spatial Cell Migration in C. elegans
指導教授:吳益群
口試委員:潘俊良陳昌熙許昭萍
口試日期:2015-07-15
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子與細胞生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:65
中文關鍵詞:線蟲細胞遷移遠頂細胞生殖腺
外文關鍵詞:C. eleganscell migrationdistal tip cellgonad
相關次數:
  • 被引用被引用:0
  • 點閱點閱:131
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
細胞遷移對於生物的生長和發育相當重要。在雌雄同體的線蟲中,生殖腺(gonad)的發育型態決定於位於生殖腺兩端遠頂細胞(distal tip cell)的遷移路徑,而開啟遠頂細胞進行向背遷移的訊息則是經由Netrin的受器(receptor) UNC-5所決定,另外unc-5的表現則是由上游的轉錄因子(transcription factor) DAF-12、LIN-29以及BLMP-1所調控,先前的研究指出LIN-29和DAF-12會促進unc-5的表現,而BLMP-1則會抑制unc-5表現。我們發現調控生理時間的基因lin-42能夠促進blmp-1及抑制lin-29在發育早期的表現(L2~early L3),來避免DTC提早進行轉彎。然而我們意外在blmp-1; daf-12的雙突變中觀察到包含正常、提早轉彎以及不轉彎的多樣(heterogeneous)突變性狀。為了探討多樣性狀的產生原因,我們利用轉錄報導基因(transcription reporter)以及單一分子螢光同位雜交法(smFISH)來觀察blmp-1是否會對lin-29及unc-5有未知的調控,結果得知blmp-1會在早期抑制lin-29的表現但是在晚期(mid L3~early L4)則是促進,此結果證實BLMP-1會經由促進lin-29進而調控unc-5的表現,而多樣性狀的產生可能是由於lin-29的不正常表現造成。我們同時建立了一個數學模擬程式來驗證lin-29及unc-5的表現形態對於性狀的影響,理論模擬的結果也顯示blmp-1;daf-12雙突變中多樣性的性狀是因為UNC-5蛋白的提早表現,以及表現較低很接近向背遷移時的閾值(threshold)所造成。我們也在特定時期調控上游lin-42的表現觀察到能提高lin-29及unc-5的轉錄,進而使UNC-5遠離閾值並降低不轉彎的性狀比例。然而我們發現在unc-5完全失去功能的突變中(null allele),後端(posterior)的生殖線仍然有大約30%的遠頂細胞是能正常進行向背遷移,但是當daf-12和lin-29同時突變時,全部的遠頂細胞都不會進行向背遷移。由這個結果我們推測除了UNC-5以外或許還有其他調控向背遷移的引導系統(guidance system),並且此引導系統的表現是受到DAF-12和LIN-29調控。我們利用DAF-12和LIN-29的DNA結合序列和基因功能性測試發現INA-1和MIG-6會與UNC-5共同調控遠頂細胞進行向背遷移。這項研究使遠頂細胞遷移的空間及時間調控機制更加完善,並且我們也利用基因調控機制解釋了在blmp-1; daf-12雙突變中多樣性狀產生的原因。

Cell migration plays an important role during animal development. In the C. elegans hermaphrodite, the shape of the gonad is determined by the migration pattern of two somatic distal tip cells (DTCs). UNC-5 is a netrin receptor that acts as a guidance cue to regulate the DTC dorsalward migration. The transcription factors DAF-12, LIN-29 and BLMP-1 act together to regulate unc-5 expression. Previous studies have shown that LIN-29 and DAF-12 suppress blmp-1 transcription and BLMP-1 negatively regulates unc-5 to control the normal timing of DTC migration. We found that LIN-42, C. elegans Period homolog, represses lin-29 transcription and activates blmp-1 transcription to prevent precocious DTC dorsal migration. Interestingly, blmp-1; daf-12 double mutants displayed a heterogeneous phenotype with a normal, precocious or retarded dorsalward turning. To investigate the causes of this heterogeneous phenotype, we used transcriptional reporter and single molecule fluorescence in situ hybridization (smFISH). We found earlier but lower lin-29 and unc-5 expression in blmp-1; daf-12 double mutants at the specific developmental stages, indicating that the early but low expression of lin-29 possibly propagates to unc-5. These data suggest that BLMP-1 may activate lin-29 expression at DTC turning stage and the heterogeneous phenotype may result from the abnormal expression of lin-29 in blmp-1; daf-12 mutants. We, therefore, developed a mathematical model to examine whether the earlier but lower lin-29 and unc-5 expression cause heterogeneous phenotype in the double mutant. The results indicated the phenotypic variation in blmp-1; daf-12 mutants may result from the lower but noisy expression level of UNC-5 near the threshold of DTC dorsalward turning. Indeed, by manipulating the upstream lin-42 regulators to increase the lin-29 and unc-5 expression, we found the heterogeneous phenotype in blmp-1; daf-12 could be alleviated. The fact that about 30% of DTCs still undergo dorsalward migration in unc-5 null allele suggests that additional guidance system besides UNC-5 exists. No DTCs makes dorsalward migration in daf-12; lin-29 double mutants, therefore, the additional guidance system may be regulated by daf-12 and lin-29. Using the binding consensus sequences of DAF-12 and LIN-29 and a functional test, we identified MIG-6 and INA-1 as components of the guidance system that act in parallel with UNC-5 to direct DTC dorsalward migration. To sum up, our studies establish a comprehensive gene regulatory network consisting of temporal and spatial regulation for the DTC dorsalward migration in C. elegans, and provide the molecular bases for the heterogeneous phenotype observed in the mutants.

致謝 i
中文摘要 ii
Abstract iv
Table of Contents 1
Introduction 4
Materials and Methods 9
Strains 9
Transgenic worms 9
DNA constructs 10
RAN interference 10
Quantify the GFP reporter signal 11
Single molecule fluorescence in situ hybridization (smFISH) 11
Probes 12
Quantify the smFISH signal 12
Antibodies and immunostaining 12
Results 14
LIN-42 can activate blmp-1 expression without lin-29 during DTC phase I migration 14
LIN-42 may directly repress lin-29 transcription during DTC phase I migration 14
BLMP-1 can promote lin-29 expression during DTC dorsal turn 15
Lower lin-29 and unc-5 expression causes phenotypic heterogeneity 17
UNC-5 protein expression level correlates well with the timing of DTC dorsal turn 18
Upstream lin-42 gene fluctuation causes the no dorsal turn phenotype in blmp-1; daf-12 mutants 19
INA-1 and MIG-6 can function with UNC-5 to regulate DTC dorsal migration 20
Neither DAF-12 nor LIN-29 regulates the expression of mig-6s in DTCs 21
Discussions 23
The activation and repression of lin-29 by blmp-1 are both required for DTC dorsalward migration 23
DAF-12 may activate the transcription of lin-29 through the miRNA 24
In blmp-1; daf-12 mutants, the role UNC-5 expression on DTC dorsalward migration is not clear yet. 25
DAF-12 and LIN-29 may regulate the transcription of ina-1 and it probably affects the dorsalward migration of DTCs through mig-6 26
PAT-2 and PAT-3 may be required to work with UNC-5 to regulate DTCs dorsalward migration 28
There may exist a co-regulator that can switch the function of blmp-1 in different developmental stage 29
Figures 31
Figure 1. UNC-5/Netrin signaling pathway regulates DTCs dorsalward migration. 31
Figure 2. LIN-42 regulates blmp-1 and lin-29 in the opposite functions before the mid L3 stage. 33
Figure 3. blmp-1 activates the transcription of lin-29 to promote DTC dorsal turning after mid L3 stage. 34
Figure 4. lin-29 transcripts are reduced in blmp-1 and blmp-1; daf-12 double mutants after mid L3 in smFISH experiment. 37
Figure 5. Loss of blmp-1 results in reduced unc-5 transcription after mid L3 in the smFISH data. 38
Figure 6. The mRNA level of unc-5 cannot correlate with the DTC dorsalward migration in the smFISH data. 40
Figure 7. The protein level of unc-5 correlates well with the DTC dorsalward migration in the modeling data. 41
Figure 8. UNC-5 protein expression level correlates well with the time of dorsal turn. 44
Figure 9. Removing the residual expression of lin-42 at the lin-42-off state enhances the transcription of lin-29 and unc-5 and reduces no dorsal turning phenotype in blmp-1; daf-12 mutants. 45
Figure 10. UNC5 protein cannot be detected in DTCs of blmp-1; daf-12 mutants carrying the Punc-5::unc-5b::gfp transgene. 47
Figure 11. MIG-6 and integrin receptor can cooperate with UNC-5 guidance network to regulate the DTC dorsal migration. 49
Figure 12. A comprehensive genetic network used for the temporal and spatial control of DTC dorsalward migration. 50
Tables 51
Table 1. The regulation of LIN-42 on blmp-1 does not require LIN-29 51
Table 2. The regulation of LIN-42 on lin-29 does not require BLMP-1 52
Table 3. The genetic interaction of DTC dorsalward migration 53
Table 4. daf-12 knockout mutants have no DTC migration defect 54
Table 5. ina-1 and mig-6 cooperate with unc-5 to regulate DTC dorsal migration 55
Table 6. The expression of mig-6s is not regulated by DAF-12 and LIN-29 56
References 57
Supplementary materials 61
lin-29 smFISH probe 61
unc-5 smFISH probe 62



Abrahante, J.E., Miller, E.A., and Rougvie, A.E. (1998). Identification of heterochronic mutants in Caenorhabditis elegans. Temporal misexpression of a collagen::green fluorescent protein fusion gene. Genetics 149, 1335-1351.
Agawa, Y., Sarhan, M., Kageyama, Y., Akagi, K., Takai, M., Hashiyama, K., Wada, T., Handa, H., Iwamatsu, A., Hirose, S., et al. (2007). Drosophila Blimp-1 is a transient transcriptional repressor that controls timing of the ecdysone-induced developmental pathway. Molecular and cellular biology 27, 8739-8747.
Antebi, A., Yeh, W.H., Tait, D., Hedgecock, E.M., and Riddle, D.L. (2000). daf-12 encodes a nuclear receptor that regulates the dauer diapause and developmental age in C. elegans. Genes Dev 14, 1512-1527.
Baum, P.D., and Garriga, G. (1997). Neuronal migrations and axon fasciculation are disrupted in ina-1 integrin mutants. Neuron 19, 51-62.
Bethke, A., Fielenbach, N., Wang, Z., Mangelsdorf, D.J., and Antebi, A. (2009). Nuclear hormone receptor regulation of microRNAs controls developmental progression. Science 324, 95-98.
Bettinger, J.C., Lee, K., and Rougvie, A.E. (1996). Stage-specific accumulation of the terminal differentiation factor LIN-29 during Caenorhabditis elegans development. Development 122, 2517-2527.
Blelloch, R., and Kimble, J. (1999). Control of organ shape by a secreted metalloprotease in the nematode Caenorhabditis elegans. Nature 399, 586-590.
Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94.
Choi, P.J., Cai, L., Frieda, K., and Xie, X.S. (2008). A stochastic single-molecule event triggers phenotype switching of a bacterial cell. Science 322, 442-446.
Cram, E.J., Shang, H., and Schwarzbauer, J.E. (2006). A systematic RNA interference screen reveals a cell migration gene network in C. elegans. Journal of cell science 119, 4811-4818.
Eldar, A., and Elowitz, M.B. (2010). Functional roles for noise in genetic circuits. Nature 467, 167-173.
Fielenbach, N., Guardavaccaro, D., Neubert, K., Chan, T., Li, D., Feng, Q., Hutter, H., Pagano, M., and Antebi, A. (2007). DRE-1: an evolutionarily conserved F box protein that regulates C. elegans developmental age. Developmental cell 12, 443-455.
Finney, M., and Ruvkun, G. (1990). The unc-86 gene product couples cell lineage and cell identity in C. elegans. Cell 63, 895-905.
Gerisch, B., and Antebi, A. (2004). Hormonal signals produced by DAF-9/cytochrome P450 regulate C. elegans dauer diapause in response to environmental cues. Development 131, 1765-1776.
Gerisch, B., Weitzel, C., Kober-Eisermann, C., Rottiers, V., and Antebi, A. (2001). A hormonal signaling pathway influencing C. elegans metabolism, reproductive development, and life span. Developmental cell 1, 841-851.
Hedgecock, E.M., Culotti, J.G., and Hall, D.H. (1990). The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans. Neuron 4, 61-85.
Hochbaum, D., Zhang, Y., Stuckenholz, C., Labhart, P., Alexiadis, V., Martin, R., Knolker, H.J., and Fisher, A.L. (2011). DAF-12 regulates a connected network of genes to ensure robust developmental decisions. PLoS genetics 7, e1002179.
Horn, M., Geisen, C., Cermak, L., Becker, B., Nakamura, S., Klein, C., Pagano, M., and Antebi, A. (2014). DRE-1/FBXO11-dependent degradation of BLMP-1/BLIMP-1 governs C. elegans developmental timing and maturation. Developmental cell 28, 697-710.
Huang, T.F., Cho, C.Y., Cheng, Y.T., Huang, J.W., Wu, Y.Z., Yeh, A.Y., Nishiwaki, K., Chang, S.C., and Wu, Y.C. (2014). BLMP-1/Blimp-1 regulates the spatiotemporal cell migration pattern in C. elegans. PLoS genetics 10, e1004428.
Itoh, B., Hirose, T., Takata, N., Nishiwaki, K., Koga, M., Ohshima, Y., and Okada, M. (2005). SRC-1, a non-receptor type of protein tyrosine kinase, controls the direction of cell and growth cone migration in C. elegans. Development 132, 5161-5172.
Jafari, G., Burghoorn, J., Kawano, T., Mathew, M., Morck, C., Axang, C., Ailion, M., Thomas, J.H., Culotti, J.G., Swoboda, P., et al. (2010). Genetics of extracellular matrix remodeling during organ growth using the Caenorhabditis elegans pharynx model. Genetics 186, 969-982.
Jeon, M., Gardner, H.F., Miller, E.A., Deshler, J., and Rougvie, A.E. (1999). Similarity of the C. elegans developmental timing protein LIN-42 to circadian rhythm proteins. Science 286, 1141-1146.
Ji, N., Middelkoop, T.C., Mentink, R.A., Betist, M.C., Tonegawa, S., Mooijman, D., Korswagen, H.C., and van Oudenaarden, A. (2013). Feedback control of gene expression variability in the Caenorhabditis elegans Wnt pathway. Cell 155, 869-880.
Ji, N., and van Oudenaarden, A. (2012). Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos. WormBook : the online review of C elegans biology, 1-16.
Jia, K., Albert, P.S., and Riddle, D.L. (2002). DAF-9, a cytochrome P450 regulating C. elegans larval development and adult longevity. Development 129, 221-231.
Kamath, R.S., Martinez-Campos, M., Zipperlen, P., Fraser, A.G., and Ahringer, J. (2001). Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome biology 2, RESEARCH0002.
Kawano, T., Zheng, H., Merz, D.C., Kohara, Y., Tamai, K.K., Nishiwaki, K., and Culotti, J.G. (2009). C. elegans mig-6 encodes papilin isoforms that affect distinct aspects of DTC migration, and interacts genetically with mig-17 and collagen IV. Development 136, 1433-1442.
Kim, H.S., Kitano, Y., Mori, M., Takano, T., Harbaugh, T.E., Mizutani, K., Yanagimoto, H., Miwa, S., Ihara, S., Kubota, Y., et al. (2014). The novel secreted factor MIG-18 acts with MIG-17/ADAMTS to control cell migration in Caenorhabditis elegans. Genetics 196, 471-479.
Kimble, J., and Hirsh, D. (1979). The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans. Developmental biology 70, 396-417.
Margadant, C., Monsuur, H.N., Norman, J.C., and Sonnenberg, A. (2011). Mechanisms of integrin activation and trafficking. Current opinion in cell biology 23, 607-614.
Meighan, C.M., and Schwarzbauer, J.E. (2007). Control of C. elegans hermaphrodite gonad size and shape by vab-3/Pax6-mediated regulation of integrin receptors. Genes Dev 21, 1615-1620.
Meighan, C.M., and Schwarzbauer, J.E. (2014). alpha Integrin cytoplasmic tails have tissue-specific roles during C. elegans development. The International journal of developmental biology 58, 325-333.
Mello, C., and Fire, A. (1995). DNA transformation. Methods in cell biology 48, 451-482.
Niu, W., Lu, Z.J., Zhong, M., Sarov, M., Murray, J.I., Brdlik, C.M., Janette, J., Chen, C., Alves, P., Preston, E., et al. (2011). Diverse transcription factor binding features revealed by genome-wide ChIP-seq in C. elegans. Genome research 21, 245-254.
Powell, D.R., Hernandez-Lagunas, L., LaMonica, K., and Artinger, K.B. (2013). Prdm1a directly activates foxd3 and tfap2a during zebrafish neural crest specification. Development 140, 3445-3455.
Raj, A., Rifkin, S.A., Andersen, E., and van Oudenaarden, A. (2010). Variability in gene expression underlies incomplete penetrance. Nature 463, 913-918.
Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden, A., and Tyagi, S. (2008). Imaging individual mRNA molecules using multiple singly labeled probes. Nature methods 5, 877-879.
Reddien, P.W., and Horvitz, H.R. (2000). CED-2/CrkII and CED-10/Rac control phagocytosis and cell migration in Caenorhabditis elegans. Nature cell biology 2, 131-136.
Reinhart, B.J., Slack, F.J., Basson, M., Pasquinelli, A.E., Bettinger, J.C., Rougvie, A.E., Horvitz, H.R., and Ruvkun, G. (2000). The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature 403, 901-906.
Schmid, R.S., Shelton, S., Stanco, A., Yokota, Y., Kreidberg, J.A., and Anton, E.S. (2004). alpha3beta1 integrin modulates neuronal migration and placement during early stages of cerebral cortical development. Development 131, 6023-6031.
Su, M., Merz, D.C., Killeen, M.T., Zhou, Y., Zheng, H., Kramer, J.M., Hedgecock, E.M., and Culotti, J.G. (2000). Regulation of the UNC-5 netrin receptor initiates the first reorientation of migrating distal tip cells in Caenorhabditis elegans. Development 127, 585-594.
Tennessen, J.M., Gardner, H.F., Volk, M.L., and Rougvie, A.E. (2006). Novel heterochronic functions of the Caenorhabditis elegans period-related protein LIN-42. Developmental biology 289, 30-43.
Wong, M.C., and Schwarzbauer, J.E. (2012). Gonad morphogenesis and distal tip cell migration in the Caenorhabditis elegans hermaphrodite. Wiley interdisciplinary reviews Developmental biology 1, 519-531.
Yang, Y., Lee, W.S., Tang, X., and Wadsworth, W.G. (2014). Extracellular matrix regulates UNC-6 (netrin) axon guidance by controlling the direction of intracellular UNC-40 (DCC) outgrowth activity. PloS one 9, e97258.


QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top