Tbx20 regulation of cardiac cell proliferation and lineage specialization during embryonic and fetal development in vivo

TBX20 gain-of-function mutations in humans are associated with congenital heart malformations and myocardial defects. However the effects of increased Tbx20 function during cardiac chamber development and maturation have not been reported previously. CAG-CAT-Tbx20 transgenic mice were generated for Cre-dependent induction of Tbx20 in myocardial lineages in the developing heart. βMHCCre-mediated overexpression of Tbx20 in fetal ventricular cardiomyocytes results in increased thickness of compact myocardium, induction of cardiomyocyte proliferation, and increased expression of Bmp10 and pSmad1/5/8 at embryonic day (E) 14.5. βMHCCre-mediated Tbx20 overexpression also leads to increased expression of cardiac conduction system (CCS) genes Tbx5, Cx40, and Cx43 throughout the ventricular myocardium. In contrast, Nkx2.5Cre mediated overexpression of Tbx20 in the embryonic heart results in reduced cardiomyocyte proliferation, increased expression of a cell cycle inhibitor, p21(CIP1), and decreased expression of Tbx2, Tbx5, and N-myc1 at E9.5, concomitant with decreased phospho-ERK1/2 expression. Together, these analyses demonstrate that Tbx20 differentially regulates cell proliferation and cardiac lineage specification in embryonic versus fetal cardiomyocytes. Induction of pSmad1/5/8 at E14.5 and inhibition of dpERK expression at E9.5 are consistent with selective Tbx20 regulation of these pathways in association with stage-specific effects on cardiomyocyte proliferation. Together, these in vivo data support distinct functions for Tbx20 in regulation of cardiomyocyte lineage maturation and cell proliferation at embryonic and fetal stages of heart development.     

Tbx1基因功能下调影响斑马鱼Tbx20和Tbx2表达

目的采用吗啡啉修饰反义寡核苷酸显微注射方法下调斑马鱼Tbx1基因表达,研究斑马鱼Tbx1基因功能下调对其他两个T盒基因Tbx20和Tbx2表达的影响。方法采用吗啡啉修饰的反义寡核苷酸显微注射方法抑制斑马鱼Tbx1基因表达,分别将2.5、5、8、10 ng吗啡啉反义寡核苷酸在斑马鱼0-4细胞期注入胚胎,并构建Tbx20,骨形成蛋白2b（Bmp2b）和Tbx2反义RNA探针,进行整体原位杂交,观察Tbx1基因下调对Tbx20、Bmp2b及Tbx2表达的影响。结果Tbx1吗啡啉寡核苷酸显微注射组胚胎表现出鳃弓、耳囊、心血管系统和胸腺的发育异常。Tbx1基因下调导致Tbx20的表达出现改变,Tbx20在心脏的表达与对照组相比明显下调,神经元的表达范围明显缩小;Tbx1基因功能下调会导致Bmp2b在心脏和咽囊的表达减低,Bmp2b在后部咽囊的表达较前部咽囊减低得更为明显;Tbx1基因功能下调胚胎,Tbx2在鳃弓的表达模式发生改变,48 hpf,Tbx2在鳃弓的表达出现从后向前逐渐减低,鳃弓的表达范围较对照组明显缩小。结论Tbx1在发育过程中,会对其他T盒基因,如Tbx20和Tbx2具有激活或抑制的调控作用。Tbx1对Tbx20的作用可能是通过影响Bmp2b的途径,继发地影响Tbx20的表达。Tbx1基因功能下调,会改变Tbx2在鳃弓的表达模式。     

Tbx18通过下调EMT信号分子参与调控心脏发育

转录因子Tbx18(Tbx18)在小鼠胚胎心外膜上皮细胞表达并调控心外膜上皮细胞向心系细胞分化.上皮间充质转化(EMT)过程是器官发育和形成的重要机制.为阐述Tbx18通过调控下游EMT关键信号分子参与心外膜上皮细胞分化和心脏发育,本研究运用Tbx18-Cre/Rosa26R-EYFP双杂合基因敲入小鼠和免疫荧光共聚焦,证实Tbx18+心系细胞和EMT关键信号分子Snail1、Smad、Slug、Twist在发育后期胚鼠心外膜和心外膜下间充质发生共聚焦.同时还发现,Tbx18在胚鼠不同发育阶段的表达模式和Tbx18+心系细胞内上述EMT关键信号分子的表达模式相似.Tbx18和EMT关键信号分子在发育心脏存在相似的时空表达模式,因此,它们之间可能存在相互调控作用.运用Tbx18突变技术揭示了Tbx18突变型胚鼠心脏EMT关键信号分子表达水平均较野生型显著下调,直接证实了上述4个EMT信号分子是Tbx18的可能靶点.理解Tbx18参与心脏发育的下游靶点有助于改善成年心脏损伤后的再生修复.