Induction of apoptosis and inhibition of cell growth by tbx5 knockdown contribute to dysmorphogenesis in Zebrafish embryos

Journal of Biomedical Science

Jennkan Lu¹  Hsinyu Lee⁴  Anhang Yang²  Renbing Tang²  Shuyu Yeh¹  Sielin Choo¹  Tzuchun Tsai³  Jenher Lu² 

[1] Laboratory of Molecular Biology, Institute of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan;School of Medicine, National Yang Ming University, Taipei, Taiwan;Department of Medical Research and Education, National Yang-Ming University Hospital, Yilan, Taiwan;Institute of Zoology, National Taiwan University, Taipei, Taiwan

关键词: cell cycle;    Holt-Oram syndrome;    tbx5;    apoptosis;    mitochondria;    zebrafish;   

Others  :  1146846
DOI  :  10.1186/1423-0127-18-73

 received in 2011-05-22, accepted in 2011-10-08,  发布年份 2011

【 摘 要 】

Background

The tbx5 mutation in human causes Holt-Oram syndrome, an autosomal dominant condition characterized by a familial history of congenital heart defects and preaxial radial upper-limb defects. We report aberrant apoptosis and dormant cell growth over head, heart, trunk, fin, and tail of zebrafish embryos with tbx5 deficiency correspond to the dysmorphogenesis of tbx5 morphants.

Methods

Wild-type zebrafish embryos at the 1-cell stage were injected with 4.3 nl of 19.4 ng of tbx5 morpholino or mismatch-tbx5-MO respectively in tbx5 morphants and mismatched control group. Semi-quantitative RT-PCR was used to for expression analysis of apoptosis and cell cycle-related genes. TUNEL and immunohistochemical assay showed the apoptosis spots within the local tissues. Ultra-structure of cardiac myocardium was examined by transmission electron microscope.

Results

Apoptosis-related genes (bad, bax, and bcl2), and cell cycle-related genes (cdk2, pcna, p27, and p57) showed remarkable increases in transcriptional level by RT-PCR. Using a TUNEL and immnuohistochemical assay, apoptosis was observed in the organs including the head, heart, pectoral fins, trunk, and tail of tbx5 knockdown embryos. Under transmission electron microscopic examination, mitochondria in cardiomyocytes became swollen and the myocardium was largely disorganized with a disarrayed appearance, compatible with reduced enhancement of myosin in the cardiac wall. The ATP level was reduced, and the ADP/ATP ratio as an apoptotic index significantly increased in the tbx5 deficient embryos.

Conclusion

Our study highlighted that tbx5 deficiency evoked apoptosis, distributed on multiple organs corresponding to dysmorphogenesis with the shortage of promising maturation, in tbx5 knockdown zebrafish embryos. We hypothesized that mesenchymal cell apoptosis associated with altered TBX5 level may subsequently interfered with organogenesis and contributed to dysmorphogenesis in tbx5 deficiency zebrafish embryos.

【 授权许可】

   
2011 Lu et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

【 参考文献 】

• [1]Bruneau BG, Nemer G, Schmitt JP, Charron F, Robitaille L, Caron S, Conner DA, Gessler M, Nemer M, Seidman CE, Seidman JG: A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 2001, 106:709-721.
• [2]Koshiba-Takeuchi K, Takeuchi JK, Matsumoto K, Momose T, Uno K, Hoepker V, Ogura K, Takahashi N, Nakamura H, Yasuda K, Ogura T: Tbx5 and the retinotectum projection. Science 2000, 287:134-137.
• [3]Basson CT, Bachinsky DR, Lin RC, Levi T, Elkins JA, Soults J, Grayzel D, Kroumpouzou E, Traill TA, Leblanc-Straceski J, Renault B, Kucherlapati R, Seidman JG, Seidman CE: Mutations in human TBX5 [corrected] cause limb and cardiac malformation in Holt-Oram syndrome. Nat Genet 1997, 15:30-35.
• [4]Li QY, Newbury-Ecob RA, Terrett JA, Wilson DI, Curtis AR, Yi CH, Gebuhr T, Bullen PJ, Robson SC, Strachan T, Bonnet D, Lyonnet S, Young ID, Raeburn JA, Buckler AJ, Law DJ, Brook JD: Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family. Nat Genet 1997, 15:21-29.
• [5]Garrity DM, Childs S, Fishman MC: The heartstrings mutation in zebrafish causes heart/fin Tbx5 deficiency syndrome. Development 2002, 129:4635-4645.
• [6]Lu JH, Lu JK, Choo SL, Li YC, Yeh HW, Shiue JF, Yeh VC: Cascade effect of cardiac myogenesis gene expression during cardiac looping in tbx5 knockdown zebrafish embryos. J Biomed Sci 2008, 15:779-787.
• [7]Camarata T, Krcmery J, Snyder D, Park S, Topczewski J, Simon HG: Pdlim7 (LMP4) regulation of Tbx5 specifies zebrafish heart atrio-ventricular boundary and valve formation. Dev Biol 2010, 337:233-245.
• [8]Garg V, Kathiriya IS, Barnes R, Schluterman MK, King IN, Butler CA, Rothrock CR, Eapen RS, Hirayama-Yamada K, Joo K, Matsuoka R, Cohen JC, Srivastava D: GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5. Nature 2003, 424:443-447.
• [9]Hiroi Y, Kudoh S, Monzen K, Ikeda Y, Yazaki Y, Nagai R, Komuro I: Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyocyte differentiation. Nat Genet 2001, 28:276-280.
• [10]Boogerd CJ, Dooijes D, Ilgun A, Mathijssen IB, Hordijk R, van de Laar IM, Rump P, Veenstra-Knol HE, Moorman AF, Barnett P, Postma AV: Functional analysis of novel TBX5 T-box mutations associated with Holt-Oram syndrome. Cardiovasc Res 2010, 88:130-139.
• [11]Goetz SC, Brown DD, Conlon FL: TBX5 is required for embryonic cardiac cell cycle progression. Development 2006, 133:2575-2584.
• [12]Pyati UJ, Look AT, Hammerschmidt M: Zebrafish as a powerful vertebrate model system for in vivo studies of cell death. Semin Cancer Biol 2007, 17:154-165.
• [13]Poelmann RE, Gittenberger-de Groot AC: Apoptosis as an instrument in cardiovascular development. Birth Defects Res C Embryo Today 2005, 75:305-313.
• [14]Bursch W, Oberhammer F, Schulte-Hermann R: Cell death by apoptosis and its protective role against disease. Trends Pharmacol Sci 1992, 13:245-251.
• [15]Cole LK, Ross LS: Apoptosis in the developing zebrafish embryo. Dev Biol 2001, 240:123-142.
• [16]Uchida D, Yamashita M, Kitano T, Iguchi T: Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile zebrafish. J Exp Biol 2002, 205:711-718.
• [17]Molin DG, DeRuiter MC, Wisse LJ, Azhar M, Doetschman T, Poelmann RE, Gittenberger-de Groot AC: Altered apoptosis pattern during pharyngeal arch artery remodelling is associated with aortic arch malformations in Tgfbeta2 knock-out mice. Cardiovasc Res 2002, 56:312-322.
• [18]Fischer U, Schulze-Osthoff K: Apoptosis-based therapies and drug targets. Cell Death Differ 2005, 12(Suppl 1):942-961.
• [19]Gustafsson AB, Gottlieb RA: Heart mitochondria: gates of life and death. Cardiovasc Res 2008, 77:334-343.
• [20]Shoubridge EA, Wai T: Mitochondrial DNA and the mammalian oocyte. Curr Top Dev Biol 2007, 77:87-111.
• [21]Van Blerkom J: Mitochondria in human oogenesis and preimplantation embryogenesis: engines of metabolism, ionic regulation and developmental competence. Reproduction 2004, 128:269-280.
• [22]Pradelli LA, Beneteau M, Ricci JE: Mitochondrial control of caspase-dependent and -independent cell death. Cell Mol Life Sci 2010, 67:1589-1597.
• [23]Ricci JE, Munoz-Pinedo C, Fitzgerald P, Bailly-Maitre B, Perkins GA, Yadava N, Scheffler IE, Ellisman MH, Green DR: Disruption of mitochondrial function during apoptosis is mediated by caspase cleavage of the p75 subunit of complex I of the electron transport chain. Cell 2004, 117:773-786.
• [24]Waterhouse NJ, Goldstein JC, von Ahsen O, Schuler M, Newmeyer DD, Green DR: Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process. J Cell Biol 2001, 153:319-328.
• [25]Bradbury DA, Simmons TD, Slater KJ, Crouch SP: Measurement of the ADP:ATP ratio in human leukaemic cell lines can be used as an indicator of cell viability, necrosis and apoptosis. J Immunol Methods 2000, 240:79-92.
• [26]Lemasters JJ, Qian T, Bradham CA, Brenner DA, Cascio WE, Trost LC, Nishimura Y, Nieminen AL, Herman B: Mitochondrial dysfunction in the pathogenesis of necrotic and apoptotic cell death. J Bioenerg Biomembr 1999, 31:305-319.
• [27]Gustafsson AB, Gottlieb RA: Mechanisms of apoptosis in the heart. J Clin Immunol 2003, 23:447-459.
• [28]Halestrap AP: Calcium, mitochondria and reperfusion injury: a pore way to die. Biochem Soc Trans 2006, 34:232-237.
• [29]Mahyar-Roemer M, Katsen A, Mestres P, Roemer K: Resveratrol induces colon tumor cell apoptosis independently of p53 and precede by epithelial differentiation, mitochondrial proliferation and membrane potential collapse. Int J Cancer 2001, 94:615-622.
• [30]He ML, Chen Y, Peng Y, Jin D, Du D, Wu J, Lu P, Lin MC, Kung HF: Induction of apoptosis and inhibition of cell growth by developmental regulator hTBX5. Biochem Biophys Res Commun 2002, 297:185-192.