臺灣博碩士論文加值系統

glycogen synthase kinase 3β (GSK3β)是一多功能的serine/threonine蛋白激酶，在生物體內許多器官組織中都會表現。GSK3β參與許多複雜的生理反應，如：細胞存活、發育型態、致癌過程等。基於以上的特性，在活體內研究GSK3β如何參與單一調控機制的實驗進行不易，因此GSK3β在心臟胚胎發育過程及對心臟功能上的影響機制仍未明。在本實驗中，我們使用綠螢光標定心臟的斑馬魚轉殖品系來輔助心臟形態功能實驗觀察的進行。為了有效且專一的抑制GSK3β表現，我們在胚胎一細胞時期注射GSK3β antisense morpholino oligonucleotide（gsk3β-MO）來抑制GSK3β蛋白質生成。在注射gsk3β-MO的胚胎中發現：授精後24小時（24 hours postfertilization；24 hpf），心臟先驅細胞已在中軸位置癒合並形成心管。然而過了36 hpf，管狀心臟卻仍無法完成正確地進行左移（jogging）與彎曲（looping）等左右非對稱發育過程（cardiac left-right asymmetry）。授精後第四天（4 days postfertilization；4 dpf）之後，可觀察到細長的條狀心臟，且心跳速率變慢、心臟收縮力減弱，甚至有圍心腔異常腫大的情形。注射gsk3β-MO導致心臟發育異常，且損害的程度和比例會隨著gsk3β-MO劑量提高而增加；同時注射gsk3β-MO和GSK3β mRNA則可達到救援（rescue）的效果：心臟異常的比例由嚴重異常43％，輕微異常53.5％降為嚴重異常31.8％，輕微異常12.1％。在原位雜交（in situ hybridization）實驗中，得知注射gsk3β-MO胚胎的cardiac myosin light chain（cmlc2）mRNA表現正常； atrial natriuretic factor（ANF）mRNA表現量大增；和bone morphogenetic protein（bmp4）mRNA有異位性表現且表現量大增，顯示心臟左右非對稱發育受到干擾。此外，已知GSK3β在Wnt/β-catenin pathway會與axin、adenomatous polyposis coli （APC）形成複合體，而在注射axin1-MO胚胎中也可觀察到心臟左右非對稱發育異常的情形。因此，經由實驗結果推測GSK3β調控斑馬魚胚胎心臟發育過程中的左右非對稱發育；而其所參與的調控路徑為Wnt/β-catenin pathway。

Glycogen synthase kinase 3β (GSK3β) encodes a multifunctional serine/threonine protein kinase, which is ubiquitously expressed in organisms. Although GSK3β is known to play roles in many biological processes, including cell survival, tumorigenesis and developmental patterning, it remains unclear that the function of GSK3β in cardiogenesis in vivo. We used a GFP-tagged heart transgenic zebrafish to address this question. In order to specifically inhibit GSK3β, gsk3β antisense morpholino oligonucleotide (gsk3β-MO) was injected into one-celled embryos to block the translation of the gsk3β mRNA. In gsk3β-MO -injected embryos, we found that heart precursor cells lined up at the midline at 24 hpf, failed to complete the heart positioning, and then stretched slowly to a thin, ‘string-like’ shape about 96 hpf. In addition, in this morphants, the heart rate was slower, the contraction was weaker, and pericardial edema was commonly observed. Knowdown of GSK3β resulted in a severe disruption of early (jogging) and late (looping) aspects of cardiac left-right asymmetry. The degree of cardiac defects due to GSK3β attenuation was dose-dependent. But these defectives could be rescued by injecting synthetic gsk3β mRNA. Consistent with the morphological change of heart, the expression of bmp4, a heart-asymmetry marker and a target of Wnt/β-catenin signaling pathway, was upregulated in GSK3β morphants: the asymmetry of heart was completely disrupted. Interestingly, we found that cardiac defects happened in the gsk3β-MO-injected embryos were similar to those observed in axin1 morphants. Therefore, our findings strongly suggest that GSK3β plays a role in L/R-biased heart positioning through Wnt/β-catenin pathway during zebrafish cardiogenesis.

中文摘要------------------------------------------------------1
英文摘要------------------------------------------------------3
前言------------------------------------------------------------5
材料與方法--------------------------------------------------11
結果-----------------------------------------------------------15
討論-----------------------------------------------------------24
參考文獻-----------------------------------------------------31
圖表-----------------------------------------------------------39
附錄-----------------------------------------------------------57

Alexander J and Stainier DY. 1999. A molecular pathway leading to endoderm formation in zebrafish. Curr Biol. 9(20):1147-57.

Ali A, Hoeflich KP, Woodgett JR. 2001. Glycogen synthase kinase-3: properties, functions, and regulation. Chem Rev. 101(8):2527-40.

Badorff C, Ruetten H, Mueller S, Stahmer M, Gehring D, Jung F, Ihling C, Zeiher AM, Dimmeler S. 2002. Fas receptor signaling inhibits glycogen synthase kinase 3ß and induces cardiac hypertrophy following pressure overload. J Clin Invest. 109: 373–381.

Basson CT, Huang T, Lin RC, Bachinsky DR, Weremowicz S, Vaglio A, Bruzzone R, Quadrelli R, Lerone M, Romeo G, Silengo M, Pereira A, Krieger J, Mesquita SF, Kamisago M, Morton CC, Pierpont ME, Muller CW, Seidman JG, Seidman CE. 1999. Different TBX5 interactions in heart and limb defined by Holt-Oram syndrome mutations. Proc Natl Acad Sci U S A. 96(6):2919-24.

Bruneau BG, Nemer G, Schmitt JP, Charron F, Robitaille L, Caron S, Conner DA, Gessler M, Nemer M, Seidman CE, Seidman JG. 2001. A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell. 106(6):709-21.

Brown NA and Wolpert L. 1990. The development of handedness in left/right asymmetry. Development 109:1-9.

Chen JN, van Eeden FJ, Warren KS, Chin A, Nusslein-Volhard C, Haffter P, Fishman MC. 1997. Left-right pattern of cardiac BMP4 may drive asymmetry of the heart in zebrafish. Development. 124(21):4373-82.

Chin AJ, Chen JN, Weinberg ES. 1997. Bone morphogenetic protein-4 expression characterizes inductive boundaries in organs of developing zebrafish. Development Genes and Evolution. 207, 107-114.

Chin AJ, Tsang M, Weinberg ES. 2000. Heart and gut chiralities are controlled independently from initial heart position in the developing zebrafish. Dev Biol. 227(2):403-21.

Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. 1995. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature. 378: 785–789.

Danos MC, Yost HJ. 1995. Linkage of cardiac left-right asymmetry and dorsal-anterior development in Xenopus. Development 121:1467-74.

Danos MC, Yost HJ. 1996. Role of notochord in specification of cardiac left-right orientation in zebrafish and Xenopus. Dev. Biol. 177:96-103.

Driever W, Solnica-Krezel L, Schier AF, Neuhauss SC, Malicki J, Stemple DL, Stainier DY, Zwartkruis F, Abdelilah S, Rangini Z, Belak J, Boggs C. 1996. A genetic screen for mutations affecting embryogenesis in zebrafish. Development. 123:37-46.

Fishman MC, Chien KR. 1997. Fashioning the vertebrate heart: earliest embryonic decisions. Development. 124(11):2099-117.

Frame S, Cohen P, Biondi RM. 2001. A common phosphate binding site explains the unique substrate specificity of GSK3 and its inactivation by phosphorylation. Mol Cell. 7(6):1321-7.

Frame S, Cohen P. 2001. GSK3 takes centre stage more than 20 years after its discovery. Biochem J. 359: 1–16.

Garrity DM, Childs S, Fishman MC. 2002. The heartstrings mutation in zebrafish causes heart/fin Tbx5 deficiency syndrome. Development. 129(19):4635-45.

Grimes CA, Jope RS. 2001. The multifaceted roles of glycogen synthase kinase 3ß in cellular signaling. Prog Neurobiol. 65: 391–426.

Haq S, Choukroun G, Kang ZB, Ranu H, Matsui T, Rosenzweig A, Molkentin JD, Alessandrini A, Woodgett J, Hajjar R, Michael A, Force T. 2000. Glycogen synthase kinase-3ß is a negative regulator of cardiomyocyte hypertrophy. J Cell Biol. 151: 117–130.
Haq S, Choukroun G, Kang ZB, Ranu H, Matsui T, Rosenzweig A, Molkentin JD, Alessandrini A, Woodgett J, Hajjar R, Michael A, Force T. 2000. Glycogen synthase kinase-3ß is a negative regulator of cardiomyocyte hypertrophy. J Cell Biol. 151: 117–130.

Hardt SE and Sadoshima J. 2002. Glycogen synthase kinase-3beta: a novel regulator of cardiac hypertrophy and development. Circ Res. 90(10):1055-63.

Hashimoto H, Rebagliati M, Ahmad N, Muraoka O, Kurokawa T, Hibi M, Suzuki T. 2004. The Cerberus/Dan-family protein Charon is a negative regulator of Nodal signaling during left-right patterning in zebrafish. Development. 131(8):1741-53.

He X, Saint-Jeannet JP, Woodgett JR, Varmus HE, Dawid IB. 1995. Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos. Nature. 374(6523):617-22.

Hedgepeth CM, Conrad LJ, Zhang J, Huang HC, Lee VM, Klein PS. 1997. Activation of the Wnt signaling pathway: a molecular mechanism for lithium action. Dev Biol. 185(1):82-91.

Heisenberg CP, Houart C, Take-Uchi M, Rauch GJ, Young N, Coutinho P, Masai I, Caneparo L, Concha ML, Geisler R, Dale TC, Wilson SW, Stemple DL. 2001. A mutation in the Gsk3-binding domain of zebrafish Masterblind/Axin1 leads to a fate transformation of telencephalon and eyes to diencephalon. Genes Dev. 15(11):1427-34.

Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O, Woodgett JR. 2000. Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation. Nature. 406(6791):86-90.

Huang CJ, Tu CT, Hsiao CD, Hsieh FJ, Tsai HJ. 2003. Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish. Dev Dyn. 228(1):30-40.

Hunter JJ, Chien KR. 1999. Signaling pathways for cardiac hypertrophy and failure. N Engl J Med. 341: 1276–1283.

Hurlstone AF, Haramis AP, Wienholds E, Begthel H, Korving J, Van Eeden F, Cuppen E, Zivkovic D, Plasterk RH, Clevers H. 2003. The Wnt/beta-catenin pathway regulates cardiac valve formation. Nature. 425(6958):633-7.

Hyatt, B. A., Lohr, J. L. and Yost, H. J. 1996. Initiation of vertebrate leftright axis formation by maternal Vg1. Nature. 384:62-5.

Isaac, A., Sargent, M. G. and Cooke, J. 1997. Control of vertebrate left-right asymmetry by a snail-related zinc finger gene. Science. 275:1301-4.

Klein PS, Melton DA. 1996. A molecular mechanism for the effect of lithium on development. Proc Natl Acad Sci U S A. 93(16):8455-9.

Lawson ND, Weinstein BM. 2002. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol. 248(2):307-18.

Levin M, Johnson RL, Stern CD, Kuehn M and Tabin C. 1995. A molecular pathway determining left-right asymmetry in chick embryogenesis. Cell. 82:803-14.

Liberatore CM, Searcy-Schrick RD, Yutzey KE. 2000. Ventricular expression of tbx5 inhibits normal heart chamber development. Dev Biol. 2223(1):169-80.

Logan CY, Nusse R. 2004. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol. 20:781-810.

Martinez SE, Beavo JA, Hol WG. 2002. Two-Billion-Year-Old Molecular Switches that Bind Cyclic Nucleotides. Mol Interv. 2(5):317-23.

Michael A, Haq S, Chen X, Hsich E, Cui L, Walters B, Shao Z, Bhattacharya K, Kilter H, Huggins G, Andreucci M, Periasamy M, Solomon RN, Liao R, Patten R, Molkentin JD, Force T. 2004. Glycogen synthase kinase-3beta regulates growth, calcium homeostasis, and diastolic function in the heart. Biol Chem. 279(20):21383-93.

Morisco C, Zebrowski D, Condorelli G, Tsichlis P, Vatner SF, Sadoshima J. 2000. The Akt-glycogen synthase kinase 3ß pathway regulates transcription of atrial natriuretic factor induced by ß-adrenergic receptor stimulation in cardiac myocytes. J Biol Chem. 275: 14466–14475.
Meno C, Saijoh Y, Fujii H, Ikeda M, Yokoyama T, Yokoyama M, Toyoda Y and Hamada H. 1996. Left-right asymmetric expression of the TGF beta-family member lefty in mouse embryos. Nature. 381:151-5.

Morisco C, Seta K, Hardt SE, Lee Y, Vatner SF, Sadoshima J. 2001. Glycogen synthase kinase 3ß regulates GATA4 in cardiac myocytes. J Biol Chem. 276: 28586–28597.
Nasevicius A, Ekker SC. 2000. Effective targeted gene ''knockdown'' in zebrafish. Nat Genet. 26(2):216-20.
Olson EN, Molkentin JD. 1999. Prevention of cardiac hypertrophy by calcineurin inhibition: hope or hype? Circ Res. 84: 623–632.
Pelster B and Burggren WW. 1996. Disruption of hemoglobin oxygen transport does not impact oxygen-dependent physiological processes in developing embryos of zebra fish (Danio rerio). Circ Res. 79(2):358-62.
Plyte SE, Hughes K, Nikolakaki E, Pulverer BJ, Woodgett JR. 1992. Glycogen synthase kinase-3: functions in oncogenesis and development. Biochim Biophys Acta. 1114: 147–162.
Ryves WJ, Harwood AJ. 2001. Lithium inhibits glycogen synthase kinase-3 by competition for magnesium. Biochem Biophys Res Commun. 280(3):720-5.
Sadoshima J, Izumo S. 1997. The cellular and molecular response of cardiac myocytes to mechanical stress. Annu Rev Physiol. 59: 551–571.
Schneider VA, Mercola M. 2001. Wnt antagonism initiates cardiogenesis in Xenopus laevis. Genes Dev. 15(3):304-15.
Stainier DY. 2001. Zebrafish genetics and vertebrate heart formation. Nat Rev Genet. 2(1):39-48.
Sugden PH. 1999. Signaling in myocardial hypertrophy: life after calcineurin? Circ Res. 84: 633–646.
Stambolic V, Ruel L, Woodgett JR. 1996. Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr Biol. 6: 1664–1668.

Szeto DP, Griffin KJ, Kimelman D. 2002. HrT is required for cardiovascular development in zebrafish. Development. 129(21):5093-101.

Tsuda T, Philp N, Zile MH and Linask KK. 1996. Left-right asymmetric localization of flectin in the extracellular matrix during heart looping. Dev. Biol. 173:39-50.

Tzahor E, Lassar AB. 2001. Wnt signals from the neural tube block ectopic cardiogenesis. Genes Dev. 15(3):255-60.

Wagman R, Yorke E, Ford E, Giraud P, Mageras G, Minsky B, Rosenzweig K. 2003. Respiratory gating for liver tumors: use in dose escalation. Int J Radiat Oncol Biol Phys. 55(3):659-68.

Willert K, Shibamoto S, Nusse R. 1999. Wnt-induced dephosphorylation of axin releases beta-catenin from the axin complex. Genes Dev. 13(14):1768-73.

Woodgett JR. 1990. Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J. 9:243–248.

Woodgett JR. 1991. A common denominator linking glycogen metabolism, nuclear oncogenes and development. Trends Biochem Sci. 16(5):177-81.

Woodgett JR. 2001. Judging a protein by more than its name: GSK-3. Sci STKE. 100: RE12.

Yamamoto H, Kishida S, Kishida M, Ikeda S, Takada S, Kikuchi A. 1999. Phosphorylation of axin, a Wnt signal negative regulator, by glycogen synthase kinase-3beta regulates its stability. J Biol Chem. 274(16):10681-4.