臺灣博碩士論文加值系統

脊髓內運動神元亞型在前後端體軸的分佈，主要是藉由視黃酸 (Retinoic acid, RA) 和纖維細胞生長因子 (Fibroblast growth factor, FGF)去引導中樞神經系統內的神經幹細胞分化。這些訊號分子會透過Hox基因的互相拮抗來確認每一種運動神經元在頭尾軸身份的建立，並調控每一種運動神經元亞型的特性、分佈及連結。
我們實驗室藉由胚胎幹細胞體外分化成運動神經元的方法，發現大部分3’Hox基因在受到RA作用8小時後都會立即進行轉錄，以Hoxa5基因的表現量最高。有趣的是，Hoxa5雖然在運動神經元前驅細胞轉錄，但是直到有絲分裂期後的運動神經元才會偵測到Hoxa5的蛋白。近年來的研究顯示，在Hox基因內區存在許多microRNA (miRNA)，其功能主要是藉由後轉錄調控來控制Hox基因的相互作用以確保體軸發育的一致性。因此，我們假設Hox蛋白質的延遲產生可能是藉由miRNA作用。
為了驗證此一假說，我們分別在體內和體外的運動神經元神經前驅細胞裡，以條件式基因剔除Dicer基因的方式研究神經前驅細胞失去miRNA之表型。我們的結果顯示當miRNA的生成受損後，會造成Hox蛋白的提早產生並呈現紊亂的表現量，當這些前驅細胞分化變為成熟的運動神經元時，會進一步造成Hox5和Hox8的邊界喪失。此一結果說明Hox基因轉錄與轉譯在時間上延遲機制可能是要讓miRNA過濾掉前驅細胞的轉錄雜訊，並控制Hox蛋白在體軸邊界可以清楚的區隔開來。
為了進一步鑑定出哪一個特殊的miRNA會參與訊號的過濾與邊界的確立，我
們利用數學模擬的方式找出Hox基因與miRNA的交互作用關係，預測的結果顯示兩種feed-forward loop (FFL)，可以解釋Dicer基因剔除後所造成 Hox蛋白質提早產生，與模糊的Hox5和Hox8邊界的現象。最後我們綜合理論計算和微陣列的結果，鑑定出單一miRNA – mir-27可能是會透過RA和Hoxc8的feed-forward loop來控制Hoxa5-Hoxc8邊界之確立。最後，我們藉由”功能獲得“與”功能喪失“的研究，證明mir-27是調節Hox蛋白質時間的延遲與空間分佈的主要調節者。我們的結果提供一個新的Hox基因與miRNA的迴路，來過濾轉錄雜訊並調控神經元亞型的身分和連結。

The rostrocaudal (RC) patterning of the developing spinal cord is controlled by extrinsic signals, such as retinoic acid (RA) and fibroblast growth factors(FGFs), that act on early neural progenitors within the nascent neural tube. The initial RC pattering leads to differential expression of Hox genes in postmitotic motor neurons (MNs) and specification of MN subtype identity and connectivity.
Through in vitro embryonic stem cell (ESC) derived motor neuron differentiation approach, we revealed that most of 3' Hox genes, particularly Hoxa5 transcription is induced within 8 hrs after the addition of RA, yet Hoxa5 protein is only detectable in post-mitotic MNs (~72 hrs after the addition of RA). In recent years, it has become clear that microRNA (miRNA) embedded within the Hox clusters is important to regulate Hox genetic network and to ensure axial identity. To address the mechanisms contributing to the timing lag between Hox transcription and translation and its physiological significance, we utilized conditional Dicer deletion in neural progenitors and revealed that miRNA biogenesis impairment leads to precocious and noisy expression of Hoxa5 protein, which later led to lousy Hox5-Hox8 boundary in vitro and in vivo.
Through exploring the Hox gene and miRNA network using in silico simulation, we predicted two feed-forward Hox-miRNA loops that might account for the precocious and noisy Hoxa5 expression, as well as the rambunctious boundary phenotype in Dicer mutants. Finally, we identified that mir-27 is a major regulator that coordinates the temporal delay and spatial collinaerity for Hox protein expression via gain- and loss-of-function studies. Our results provided a novel Hox-miRNA genetic circuit that controls timing of protein expression and confers robust individual neuron identity.

誌謝----------------- i
中文摘要--------- iii
英文摘要----------- v
目錄--------------- vi
圖目錄------------ vii
第一章 緒論------ 1
第二章 材料與方法-------------------------------- 5
第三章結果 ----------------------------------------------- 18
3.1 Hox蛋白質在運動神經元前驅細胞會延遲產生
3.2在體內和體外運動神經元前驅細胞剔除Dicer基因會造成Hoxa5蛋白質提早產生且呈現擾亂
3.3當胚胎的Dicer剔除會造成脊髓的Hoxa5/Hoxc8邊界位移和喪失清晰的邊界
3.4時間與空間的模擬Hox與miRNA的交互作用
3.5藉由mir-27的調控Hoxa5的表現
3.6 mir-27在脊髓表現的分析
3.7 mir-27與Hoxa5 3’UTR作用位置的分析
3.8 mir-27的生物功能分析
第四章討論------------------------------------ 27
4.1 miRNA過濾Hox基因的轉錄雜訊並調節Hox蛋白質的延遲效應以決定細胞的身份
4.2 在胚胎中的Hox – miRNA迴路會提升後端抑制前端的機制
第五章參考文獻------------------------- 32
圖表------------------------------------- 40
附圖------------------------------------------ 60

Abranches, E., Guedes, A.M., Moravec, M., Maamar, H., Svoboda, P., Raj, A., and Henrique, D. (2014). Stochastic NANOG fluctuations allow mouse embryonic stem cells to explore pluripotency. Development 141, 2770-2779.
 Alexander, T., Nolte, C., and Krumlauf, R. (2009). Hox genes and segmentation of the hindbrain and axial skeleton. Annu Rev Cell Dev Biol 25, 431-456.
 Asli, N.S., and Kessel, M. (2010). Spatiotemporally restricted regulation of generic motor neuron programs by miR-196-mediated repression of Hoxb8. Dev Biol 344, 857-868.
 Balaskas, N., Ribeiro, A., Panovska, J., Dessaud, E., Sasai, N., Page, K.M., Briscoe, J., and Ribes, V. (2012). Gene regulatory logic for reading the Sonic Hedgehog signaling gradient in the vertebrate neural tube. Cell 148, 273-284.
 Brend, T., Gilthorpe, J., Summerbell, D., and Rigby, P.W. (2003). Multiple levels of transcriptional and post-transcriptional regulation are required to define the domain of Hoxb4 expression. Development 130, 2717-2728.
 Chang, H.H., Hemberg, M., Barahona, M., Ingber, D.E., and Huang, S. (2008). Transcriptome-wide noise controls lineage choice in mammalian progenitor cells. Nature 453, 544-547.
 Chen, J.-A., Huang, Y.-P., Mazzoni, E.O., Tan, G.C., Zavadil, J., and Wichterle, H. (2011a). Mir-17-3p Controls Spinal Neural Progenitor Patterning by Regulating Olig2/Irx3 Cross-Repressive Loop. Neuron 69.
 Chen, J.A., Chu, S.T., and Amaya, E. (2007). Maintenance of motor neuron progenitors in Xenopus requires a novel localized cyclin. EMBO Rep 8, 287-292.
 Chen, J.A., Huang, Y.P., Mazzoni, E.O., Tan, G.C., Zavadil, J., and Wichterle, H.
(2011b). Mir-17-3p controls spinal neural progenitor patterning by regulating Olig2/Irx3 cross-repressive loop. Neuron 69, 721-735.
 Chen, J.A., and Wichterle, H. (2012). Apoptosis of limb innervating motor neurons and erosion of motor pool identity upon lineage specific dicer inactivation. Frontiers in neuroscience 6, 69.
 Cohen, M., Page, K.M., Perez-Carrasco, R., Barnes, C.P., and Briscoe, J. (2014). A theoretical framework for the regulation of Shh morphogen-controlled gene expression. Development 141, 3868-3878.
 Coulombe, Y., Lemieux, M., Moreau, J., Aubin, J., Joksimovic, M., Berube-Simard, F.A., Tabaries, S., Boucherat, O., Guillou, F., Larochelle, C., et al. (2010). Multiple promoters and alternative splicing: Hoxa5 transcriptional complexity in the mouse embryo. PLoS One 5, e10600.
 Dasen, J.S. (2013). Long noncoding RNAs in development: solidifying the Lncs to Hox gene regulation. Cell reports 5, 1-2.
 Dasen, J.S., De Camilli, A., Wang, B., Tucker, P.W., and Jessell, T.M. (2008). Hox repertoires for motor neuron diversity and connectivity gated by a single accessory factor, FoxP1. Cell 134, 304-316.
 Dasen, J.S., and Jessell, T.M. (2009). Hox networks and the origins of motor neuron diversity. Curr Top Dev Biol 88, 169-200.
 Dasen, J.S., Liu, J.P., and Jessell, T.M. (2003). Motor neuron columnar fate imposed by sequential phases of Hox-c activity. Nature 425, 926-933.
 Dasen, J.S., Tice, B.C., Brenner-Morton, S., and Jessell, T.M. (2005). A Hox regulatory network establishes motor neuron pool identity and target-muscle connectivity. Cell 123, 477-491.
 Dessaud, E., Yang, L.L., Hill, K., Cox, B., Ulloa, F., Ribeiro, A., Mynett, A., Novitch, B.G., and Briscoe, J. (2007). Interpretation of the sonic hedgehog
morphogen gradient by a temporal adaptation mechanism. Nature 450, 717-720.
 Ebert, M.S., Neilson, J.R., and Sharp, P.A. (2007). MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods 4, 721-726.
 Ebert, M.S., and Sharp, P.A. (2012). Roles for microRNAs in conferring robustness to biological processes. Cell 149, 515-524.
 Gerard, M., Zakany, J., and Duboule, D. (1997). Interspecies exchange of a Hoxd enhancer in vivo induces premature transcription and anterior shift of the sacrum. Dev Biol 190, 32-40.
 Harfe, B.D., McManus, M.T., Mansfield, J.H., Hornstein, E., and Tabin, C.J. (2005). The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb. Proc Natl Acad Sci U S A 102, 10898-10903.
 Hayashi, S., Tenzen, T., and McMahon, A.P. (2003). Maternal inheritance of Cre activity in a Sox2Cre deleter strain. Genesis 37, 51-53.
 Hornstein, E., Mansfield, J.H., Yekta, S., Hu, J.K., Harfe, B.D., McManus, M.T., Baskerville, S., Bartel, D.P., and Tabin, C.J. (2005). The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development. Nature 438, 671-674.
 Hornstein, E., and Shomron, N. (2006). Canalization of development by microRNAs. Nat Genet 38 Suppl, S20-24.
 Inui, M., Martello, G., and Piccolo, S. (2010). MicroRNA control of signal transduction. Nature reviews Molecular cell biology 11, 252-263.
 Johnston, R.J., Jr., and Desplan, C. (2010). Stochastic mechanisms of cell fate specification that yield random or robust outcomes. Annu Rev Cell Dev Biol 26, 689-719.
 Joksimovic, M., Jeannotte, L., and Tuggle, C.K. (2005). Dynamic expression of murine HOXA5 protein in the central nervous system. Gene expression patterns :
GEP 5, 792-800.
 Juan, A.H., and Ruddle, F.H. (2003). Enhancer timing of Hox gene expression: deletion of the endogenous Hoxc8 early enhancer. Development 130, 4823-4834.
 Jung, H., Lacombe, J., Mazzoni, E.O., Liem, K.F., Jr., Grinstein, J., Mahony, S., Mukhopadhyay, D., Gifford, D.K., Young, R.A., Anderson, K.V., et al. (2010). Global control of motor neuron topography mediated by the repressive actions of a single hox gene. Neuron 67, 781-796.
 Kalir, S., Mangan, S., and Alon, U. (2005). A coherent feed-forward loop with a SUM input function prolongs flagella expression in Escherichia coli. Molecular systems biology 1, 2005 0006.
 Kondo, T., and Duboule, D. (1999). Breaking colinearity in the mouse HoxD complex. Cell 97, 407-417.
 Kondrashov, N., Pusic, A., Stumpf, C.R., Shimizu, K., Hsieh, A.C., Xue, S., Ishijima, J., Shiroishi, T., and Barna, M. (2011). Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning. Cell 145, 383-397.
 Lacombe, J., Hanley, O., Jung, H., Philippidou, P., Surmeli, G., Grinstein, J., and Dasen, J.S. (2013). Genetic and functional modularity of Hox activities in the specification of limb-innervating motor neurons. PLoS Genet 9, e1003184.
 Liu, J.P. (2006). The function of growth/differentiation factor 11 (Gdf11) in rostrocaudal patterning of the developing spinal cord. Development 133, 2865-2874.
 Liu, J.P., Laufer, E., and Jessell, T.M. (2001). Assigning the positional identity of spinal motor neurons: rostrocaudal patterning of Hox-c expression by FGFs, Gdf11, and retinoids. Neuron 32, 997-1012.
 Luxenhofer, G., Helmbrecht, M.S., Langhoff, J., Giusti, S.A., Refojo, D., and Huber, A.B. (2014). MicroRNA-9 promotes the switch from early-born to
late-born motor neuron populations by regulating Onecut transcription factor expression. Dev Biol 386, 358-370.
 Mahony, S., Mazzoni, E.O., McCuine, S., Young, R.A., Wichterle, H., and Gifford, D.K. (2011). Ligand-dependent dynamics of retinoic acid receptor binding during early neurogenesis. Genome Biol 12, R2.
 Mallo, M., and Alonso, C.R. (2013). The regulation of Hox gene expression during animal development. Development 140, 3951-3963.
 Mansfield, J.H., and McGlinn, E. (2012). Evolution, expression, and developmental function of Hox-embedded miRNAs. Curr Top Dev Biol 99, 31-57.
 Mazzoni, E.O., Mahony, S., Iacovino, M., Morrison, C.A., Mountoufaris, G., Closser, M., Whyte, W.A., Young, R.A., Kyba, M., Gifford, D.K., et al. (2011). Embryonic stem cell-based mapping of developmental transcriptional programs. Nat Methods 8, 1056-1058.
 Mazzoni, E.O., Mahony, S., Peljto, M., Patel, T., Thornton, S.R., McCuine, S., Reeder, C., Boyer, L.A., Young, R.A., Gifford, D.K., et al. (2013). Saltatory remodeling of Hox chromatin in response to rostrocaudal patterning signals. Nat Neurosci 16, 1191-1198.
 McGlinn, E., Yekta, S., Mansfield, J.H., Soutschek, J., Bartel, D.P., and Tabin, C.J. (2009). In ovo application of antagomiRs indicates a role for miR-196 in patterning the chick axial skeleton through Hox gene regulation. Proc Natl Acad Sci U S A 106, 18610-18615.
 Otaegi, G., Pollock, A., Hong, J., and Sun, T. (2011). MicroRNA miR-9 modifies motor neuron columns by a tuning regulation of FoxP1 levels in developing spinal cords. J Neurosci 31, 809-818.
 Pearson, J.C., Lemons, D., and McGinnis, W. (2005). Modulating Hox gene
functions during animal body patterning. Nat Rev Genet 6, 893-904.
 Peljto, M., Dasen, J.S., Mazzoni, E.O., Jessell, T.M., and Wichterle, H. (2010). Functional diversity of ESC-derived motor neuron subtypes revealed through intraspinal transplantation. Cell Stem Cell 7, 355-366.
 Philippidou, P., and Dasen, J.S. (2013). Hox genes: choreographers in neural development, architects of circuit organization. Neuron 80, 12-34.
 Raj, A., Peskin, C.S., Tranchina, D., Vargas, D.Y., and Tyagi, S. (2006). Stochastic mRNA synthesis in mammalian cells. PLoS Biol 4, e309.
 Rinn, J.L., Kertesz, M., Wang, J.K., Squazzo, S.L., Xu, X., Brugmann, S.A., Goodnough, L.H., Helms, J.A., Farnham, P.J., Segal, E., et al. (2007). Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129, 1311-1323.
 Rosenfeld, N., and Alon, U. (2003). Response Delays and the Structure of Transcription Networks. Journal of Molecular Biology 329, 645-654.
 Sanchez, A., Choubey, S., and Kondev, J. (2013). Regulation of noise in gene expression. Annual review of biophysics 42, 469-491.
 Schilling, T.F., Nie, Q., and Lander, A.D. (2012). Dynamics and precision in retinoic acid morphogen gradients. Curr Opin Genet Dev 22, 562-569.
 Schmiedel, J.M., Klemm, S.L., Zheng, Y., Sahay, A., Bluthgen, N., Marks, D.S., and van Oudenaarden, A. (2015). Gene expression. MicroRNA control of protein expression noise. Science 348, 128-132.
 Takashima, Y., Era, T., Nakao, K., Kondo, S., Kasuga, M., Smith, A.G., and Nishikawa, S. (2007). Neuroepithelial cells supply an initial transient wave of MSC differentiation. Cell 129, 1377-1388.
 Thomsen, S., Azzam, G., Kaschula, R., Williams, L.S., and Alonso, C.R. (2010). Developmental RNA processing of 3'UTRs in Hox mRNAs as a
context-dependent mechanism modulating visibility to microRNAs. Development 137, 2951-2960.
 Trapnell, C., Cacchiarelli, D., Grimsby, J., Pokharel, P., Li, S., Morse, M., Lennon, N.J., Livak, K.J., Mikkelsen, T.S., and Rinn, J.L. (2014). The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat Biotechnol 32, 381-386.
 Tschopp, P., and Duboule, D. (2011). A genetic approach to the transcriptional regulation of Hox gene clusters. Annual review of genetics 45, 145-166.
 Tung, Y.T., Lu, Y.L., Peng, K.C., Yen, Y.P., Chang, M., Li, J., Jung, H., Thams, S., Huang, Y.P., Hung, J.H., et al. (2015). Mir-17 approximately 92 Governs Motor Neuron Subtype Survival by Mediating Nuclear PTEN. Cell reports.
 Vidigal, J.A., and Ventura, A. (2015). The biological functions of miRNAs: lessons from in vivo studies. Trends in cell biology 25, 137-147.
 Wichterle, H., Lieberam, I., Porter, J.A., and Jessell, T.M. (2002). Directed differentiation of embryonic stem cells into motor neurons. Cell 110, 385-397.
 Woltering, J.M., and Durston, A.J. (2008). MiR-10 represses HoxB1a and HoxB3a in zebrafish. PLoS One 3, e1396.
 Xue, S., Tian, S., Fujii, K., Kladwang, W., Das, R., and Barna, M. (2015). RNA regulons in Hox 5' UTRs confer ribosome specificity to gene regulation. Nature 517, 33-38.
 Yekta, S., Shih, I.H., and Bartel, D.P. (2004). MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594-596.
 Yekta, S., Tabin, C.J., and Bartel, D.P. (2008). MicroRNAs in the Hox network: an apparent link to posterior prevalence. Nat Rev Genet 9, 789-796.
 Zakany, J., Gerard, M., Favier, B., and Duboule, D. (1997). Deletion of a HoxD enhancer induces transcriptional heterochrony leading to transposition of the
sacrum. EMBO J 16, 4393-4402.
 Zhang, L., Radtke, K., Zheng, L., Cai, A.Q., Schilling, T.F., and Nie, Q. (2012). Noise drives sharpening of gene expression boundaries in the zebrafish hindbrain. Molecular systems biology 8, 613.