Induction of apoptosis and inhibition of cell growth by tbx5knockdown contribute to dysmorphogenesis in Zebrafish embryos

Lu Jenher, Tsai Tzuchun, Choo Sielin, Yeh Shuyu, Tang Renbing, Yang Anhang, Lee Hsinyu, Lu, Lu Jennkan

Description

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.

Sujets

• Zebrafish
• Mitocondria
• Apoptosis
• TBX5 (gene)
• Holt?Oram syndrome
• Cell cycle

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Luet al.Journal of Biomedical Science2011,18:73 http://www.jbiomedsci.com/content/18/1/73

R E S E A R C HOpen Access Induction of apoptosis and inhibition of cell growth bytbx5knockdown contribute to dysmorphogenesis in Zebrafish embryos 1,2*†1,2 54 1,22,3 4 Jenher Lu, Tzuchun Tsai, Sielin Choo , Shuyu Yeh , Renbing Tang, Anhang Yang, Hsinyu Leeand 4† Jennkan Lu

Abstract 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. Keywords:zebrafish, mitochondria, apoptosis,tbx5, HoltOram syndrome, cell cycle

1. Background Tbx5belongs to the Tbox family of transcription fac tors and is required for the embryonic development of the heart and forelimbs [1,2].Tbx5mutations in humans cause HoltOram syndrome (HOS), an autoso mal dominant condition characterized by a familial

* Correspondence: [e-mail] †Contributed equally 1 Department of Pediatrics and Pathology, Taipei Veterans General Hospital, Taipei, Taiwan Full list of author information is available at the end of the article

history of congenital heart defects and preaxial radial ray upperlimb defects [3,4]. The phenotypic manifesta tions oftbx5deficiency in different vertebrates are quite similar.Tbx5deficiency in zebrafish causes multiple organ defects during organogenesis, including a shor tened trunk, failure of cardiac looping formation, and hypogenesis or agenesis of the pectoral fins [5,6]. Either the type of mutation or the location of a mutation is predictive for the severity of heart or limb malforma tions in HoltOram syndrome patients. That is, there is

© 2011 Lu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.