Department of Pediatrics and Pathology, Taipei Veterans General Hospital, Taipei, Taiwan.
J Biomed Sci. 2011 Oct 8;18(1):73. doi: 10.1186/1423-0127-18-73.
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.
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.
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.
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.
人类中的 tbx5 突变导致 Holt-Oram 综合征，这是一种常染色体显性遗传病，其特征是家族性先天性心脏病和前轴上肢缺陷。我们报道了 tbx5 缺陷的斑马鱼胚胎头部、心脏、躯干、鳍和尾部存在异常凋亡和休眠细胞生长，这与 tbx5 突变体的发育不良相对应。
将 1 细胞期野生型斑马鱼胚胎分别用 4.3 nl 的 19.4 ng tbx5 嵌合体或错配 tbx5-MO 注射到 tbx5 嵌合体和错配对照组中。半定量 RT-PCR 用于分析凋亡和细胞周期相关基因的表达。TUNEL 和免疫组织化学检测显示局部组织内的凋亡点。透射电子显微镜检查心肌超微结构。
RT-PCR 显示凋亡相关基因（bad、bax 和 bcl2）和细胞周期相关基因（cdk2、pcna、p27 和 p57）转录水平显著增加。使用 TUNEL 和免疫组化检测，在 tbx5 敲低胚胎的头部、心脏、胸鳍、躯干和尾部等器官中观察到凋亡。透射电镜检查显示，心肌细胞中的线粒体肿胀，心肌大部分排列紊乱，肌球蛋白在心脏壁中的增强减少。tbx5 缺陷胚胎中的 ATP 水平降低，ADP/ATP 比值作为凋亡指数显著增加。
我们的研究表明，tbx5 缺乏导致凋亡，分布在多个器官，与缺乏成熟的发育不良相对应，在 tbx5 敲低的斑马鱼胚胎中。我们假设与 TBX5 水平改变相关的间充质细胞凋亡可能随后干扰器官发生，并导致 tbx5 缺陷斑马鱼胚胎的发育不良。
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