臺灣博碩士論文加值系統

背景與目標: 初級纖毛在中樞神經系統結構形成中，扮演類似天線傳遞訊息的角色，刺蝟因子為其訊號傳遞中之一條路徑。Gli2 是初級纖毛上傳遞訊號的重要蛋白，主要作用在初級纖毛的頂端。先前的文獻指出，初級纖毛頂端的蛋白對於纖毛生成過程中扮演重要的角色。另外，初級纖毛的長短也會去調控細胞週期，當初級纖毛較長時會使細胞延遲進入G1/S 期。然而，Gli2如何去調控初級纖毛之生成並進一步影響細胞週期仍有待探究。

假設:我們假設 Gli2藉由調控初級纖毛生成進而改變細胞週期。

材料與方法: 我們以CRISPR/Cas9方法建立Gli2剔除之NIH3T3細胞株，接著用免疫組織染色法觀察初級纖毛的生成。此外我們利用流式細胞儀檢測剔除Gli2後細胞週期之變化。

結果:我們發現將Gli2 蛋白剔除後的細胞會有較長的初級纖毛，並延遲進入G1/S 期。另外，我們觀察到Gli2調控初級纖毛長度與細胞的自噬路徑有關，在Gli2剔除的細胞株中自噬小體表現的LC3-II蛋白量較多，且其中心粒蛋白OFD1的表現量下降。

結論: Gli2 蛋白會經由調控細胞自噬小體及中心粒蛋白OFD1，進而影響初級纖毛長度且進一步改變細胞週期之分布。

Background: The central nervous system arises from the neural tube, consisting of neural stem cells, which give rise to neurons and glia cells. Interestingly, many neural stem cells contain the primary cilium, a microtubule-based organelle projecting from the plasma membrane. Primary cilia are critical in numerous functions ranging from mechanosensation, proliferation, and differentiation. Importantly, primary cilia participate in patterning of the central nervous system by functioning as cellular antennae for transmitting molecular signals, such as Sonic Hedgehog (SHH) signaling. Gli2, a fundamental player in the SHH signaling, is known for regulating cell cycle progression. Interestingly, Gli2 is also involved in cell-cycle re-entry from G0 in many cell types, including neural progenitors. However, unlike Gli2-dependent cell cycle progression, how Gli2 regulates cell cycle re-entry is not fully elucidated.

Hypothesis: We hypothesize that Gli2 could regulate cell cycle re-entry mediated by primary cilia.

Materials and Methods: We generated a Gli2-knockout cell line by CRISPR/Cas9 technology in NIH3T3 cells in which the functions of SHH players on the primary cilium were broadly studied. The primary cilia were labeled by using immunostaining and then observed them with confocal microscopy. In addition, we utilized flow cytometry to survey cell cycle progression.

Results: We validated that cells depleted of Gli2 possess longer primary cilia; meanwhile, we monitored a delay for cell-cycle re-entry in Gli2-knockout cells by flow cytometry. In addition, we found that Gli2 can induce more autophagy expression and downregulate OFD1 (oral-facial-digital syndrome1) protein level. Conversely, blocking the autophagic flux with the autophagy inhibitor, 3-methyladenine (3-MA), these phenotypes can be rescued.

Conclusion: Gli2 can affect cell cycle re-entry through regulating primary cilia length. We discovered that the autophagy-dependent OFD1 removal is the key pathway for Gli2-dependent ciliary length control.

Contents...................................... i
Acknowledgements.............................. iii
摘要.......................................... v
Abstract...................................... vi
Table of Illustrations........................ viii

Chapter 1: Introduction 1
1.1 A brief history and importance
of the primary cilium......................... 1
1.2 The structure of primary cilium........... 2
1.3 The function of primary cilium
in SHH signaling pathway...................... 2
1.4 Gli2 and Holoprosencephaly type 9......... 4
1.5 The correlation of ciliary length
and cell cycle progression.................... 5
1.6 The relationship of autophagy
and ciliogenesis.............................. 6
1.7 Overview of CRISPR/Cas9 system............ 7

Chapter 2: Material and Methods 9
2.1 Plasmids.................................. 9
2.2 Cell culture and transfection............. 10
2.3 Live imaging.............................. 10
2.4 CRISPR/Cas9 technology.................... 11
2.5 Western Blot.............................. 15
2.6 Luciferase reporter assay................. 17
2.7 Immunocytochemistry....................... 18
2.8 Flow cytometry............................ 20
2.9 Microscopy................................ 21

Chapter 3: Results 22
3.1 Knockdown or overexpress Gli2 protein
affected primary cilia formation.............. 22
3.2 The Gli2-/- cells were established
by utilizing CRISPR/Cas9 technology........... 26
3.3 Depletion of Gli2 induced
longer primary cilia.......................... 36
3.4 Primary cilia mediate the delay of
cell cycle re-entry in Gli2 knockout cells.... 45
3.5 Gli2 knockout enhanced the length of
primary cilia through autophagy-mediated
OFD1 removal.................................. 54

Chapter 4: Discussion 63
4.1 Possible mechanisms for ciliary length........................................ 64
4.2 The possible link between Gli2 and autophagy................................. 65
4.3 The role of OFD1 in ciliary length control........................ 66
4.4 Role of Gli2 in cell cycle regulation..... 67

References 68


Table of Illustrations
Table 1 Commercial plasmids................... 9
Table 2 Reagents used in CRISPR/Cas 9 technology.................................... 14
Table 3 Primary antibodies used in Western Blot............................... 16
Table 4 Secondary antibodies used in Western Blot............................... 16
Table 5 Materials reagents used in Western Blot............................... 16
Table 6 Primary antibodies in immunostaining............................. 19
Table 7 Secondary antibodies in immunostaining............................. 19
Table 8 Reagents used in cell cycle analysis...................................... 20
Table 9 The blastn results of potential cells............................... 27

Figure 1 The effect of Gli2 knockdown in the primary cilia formation....................... 24
Figure 2 Gli2-/- cells were generated by using CRISPR/Cas9 technology.................. 29
Figure 3 Depletion of Gli2 induces longer primary cilia.......................... 38
Figure 4 Primary cilia are required for the delay of cell cycle reentry in Gli2 knockout cells........................... 48
Figure 5 Gli2-/- cells enhanced the length of primary cilia through
autophagy-mediated OFD1 removal............... 57

Bertolacinia, C.D.P. et al. Clinical findings in patients with GLI2 mutations – phenotypic variability. Clinical Genetics 81, 70–75 (2012).

Chuang, P.-T. & McMahon, A.P. Vertebrate Hedgehog signalling modulated by induction of a Hedgehog-binding protein. Nature 397, 617-621 (1999).

Doudna, J.A. & Charpentier, E. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).

Feather, S.A., Winyard, P.J.D., Dodd, S. & A. S. Woolf2. Oral-facial-digital syndrome type 1 is another dominant polycystic kidney disease: clinical, radiological and histopathological features of a new kindred. Nephrol Dial Transplant 12, 1354-1361 (1997).

Gibbons, IR. The relationship between the fine structure and direction of beat in gill cilia of a lamellibranch mollusc. J Biophys Biochem Cytol. 11, 179–205 (1961).

Gabriel, E. et al. CPAP promotes timely cilium disassembly to maintain neural progenitor pool. The EMBO Journal 35, 803-819 (2016).


Goto, H., Inoko, A. & Inagaki, M. Cell cycle progression by the repression of primary cilia formation in proliferating cells. Cellular and Molecular Life Sciences 70, 3893–3905 (2013).

Heyne, G.W. et al. Gli2 gene-environment interactions contribute to the etiological complexity of holoprosencephaly: evidence from a mouse model. Disease Models & Mechanisms 9, 1307-1315 (2016).

Hsiao, Y.-C., Tuz, K. & Ferland, R.J. Trafficking in and to the primary cilium. Cilia 1:4 (2012).

Izawa, I., Goto, H., Kasahara, K. & Inagaki, M. Current topics of functional links between primary cilia and cell cycle.Cilia 4:12 (2015).

Jimenez-Sanchez, M. et al. The Hedgehog signalling pathway regulates autophagy. Nature communication 3:1200 (2012).

Kobayashi, T. & Dynlacht, B.D. Regulating the transition from centriole to basal body. J. Cell Biol. 193, .435-444 (2011).

Kim, S. & Tsiokas, L. Cilia and cell cycle re-entry: More than a coincidence. Cell Cycle 10, 2683-2690 (2011).

Kim, S. et al. Nde1-mediated inhibition of ciliogenesis affects cell cycle re-entry. Nature Cell Biology 13, 351-360 (2011).

Keeling, J., Tsiokas, L. & Maskey, D. Cellular Mechanisms of Ciliary Length Control. Cells 5(1):6 (2016).

Kim, S., Lee, K., Choi, J.-H., Ringstad, N. & Dynlacht, B.D. Nek2 activation of Kif24 ensures cilium disassembly during the cell cycle. Nature communication 6:8087 (2015).

Katoh, Y. & Katoh, M. Hedgehog Target Genes: Mechanisms of Carcinogenesis Induced by Aberrant Hedgehog Signaling Activation. Current Molecular Medicine 9, 873-886 (2009).

Kramann, R. et al. Pharmacological GLI2 inhibition prevents myofibroblast cell-cycle progression and reduces kidney fibrosis. The Journal of Clinical Investigation 125(8), 2935-2951 (2015).

Lin, Y.T. et al. YAP regulates neuronal differentiation through Sonic hedgehog signaling pathway. Experimental cell research 318, 1877-1888 (2012).

Lopes, C.A.M. et al. Centriolar satellites are assembly points for proteins implicated in human ciliopathies, including oral-facial-digital syndrome 1. Journal of Cell Science 24, 600-612 (2011).

Matise, M.P. & Joyner, A.L. Gli genes in development and cancer. Oncogene 18, 7852 -7859 (1999).


Maskey, D. et al. Cell cycle-dependent ubiquitylation and destruction of NDE1 by CDK5-FBW7 regulates ciliary length. The EMBO Journal 34(19), 2424-2440 (2015).

MI, F. et al. Oral-facial-digital type I protein is required for primary cilia formation and left-right axis specification. Nature genetics 38(1), 112-117 (2006).

Mill, P. et al. Sonic hedgehog-dependent activation of Gli2 is essential for embryonic hair follicle development. Genes and Development 2003, 282–294 (2002).

Pampliega, O. et al. Functional interaction between autophagy and ciliogenesis. Nature 502, 194-200 (2013).

Pugacheva, E.N., Jablonski, S.A., Hartman, T.R., Henske, E.P. & Golemis, E.A. HEF1-dependent Aurora A activation induces disassembly of the primary cilium. Cell 129(7), 1351–1363 (2007).

Reiter, J.F., Blacque, O.E. & Leroux, M.R. The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization. EMBO reports 13(7), 608-618 (2012).

Roessler, E. et al. Loss-of-function mutations in the human GLI2 gene are associated with pituitary anomalies and holoprosencephaly-like features. PNAS 100, 13424-13429 (2003).

Satir, P. Landmarks in cilia research from Leeuwenhoek to us. Cell motility and the cytoskeleton 32, 90-94 (1995).
Spassky, N. et al. Primary Cilia are required for cerebellar development and Shh-dependent expansion of progenitor pool. Developmental Biology 317, 246–259 (2008).

Sasaki, H., Nishizaki, Y., Hui, C.-c., Nakafuku, M. & Kondoh, H. Regulation of Gli2 and Gli3 activities by an amino-terminal repression domain: implication of Gli2 and Gli3 as primary mediators of Shh signaling. Development 126, 3915-3924 (1999).

Song, G. et al. CRISPR/Cas9: A powerful tool for crop genome editing. The Crop Journal 4, 75 – 82 (2016).

Tran, P.V., Sharma, M., Li, X. & Calvet, J.P. Developmental Signaling: Does It Bridge the Gap Between Cilia Dysfunction and Renal Cystogenesis? Birth Defects Res C Embryo Today 02, 159–173 (2014).

Tang, Z. et al. Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites. Nature 502(7470), 254-257 (2013).

Tang, Z., Zhu, M. & Zhong, Q. Self-eating to remove cilia roadblock. Autophagy 10:2, 379-381 (2014).

Tang, X. et al. Inhibition of Hedgehog signaling pathway impedes cancer cell proliferation by promotion of autophagy. European Journal of Cell Biology 94, 223-233 (2015).


Vogel, T.W., Carter, C.S., Abode-Iyamah, K., Z., Q. & Robinson, S. The role of primary cilia in the pathophysiology of neural tube defects. Neurosurgical
Focus. Cell biology international 33, 333-339 (2005).

Varjosalo, M. & Taipale, J. Hedgehog: functions and mechanisms. Gene and development 22(18), 2454-2472 (2008).

Verhey, K.J., Dishinger, J. & Kee, H.L. Kinesin Motors and Primary Cilia. Biochem Soc Trans. 39(5), 1120-1125 (2011).

Wheatley, D.N. Landmarks in the first hundred years of primary (9+0) cilium research. Cell biology international 29, 333-339 (2005).

Xu, Y., An, Y., Wang, X., Zha, W. & Li, X. Inhibition of the Hedgehog pathway induces autophagy in pancreatic ductal adenocarcinoma cells. Oncology Reports 31, 707-712 (2014).