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.     

Up-regulation of natriuretic peptides in the ventricle of Csx/Nkx2-5 transgenic mice

A cardiac homeobox-containing gene Csx/Nkx2-5, which is essential for cardiac development, is abundantly expressed in the adult heart as well as in the heart primordia. Targeted disruption of this gene results in embryonic lethality due to abnormal heart morphogenesis. To elucidate the role of Csx/Nkx2-5 in the adult heart, we generated transgenic mice which overexpress human Csx/Nkx2-5. The transgene was expressed abundantly in the heart and the skeletal muscle. mRNA levels of several cardiac genes including natriuretic peptides, CARP, MLC2v, and endogenous Csx/Nkx2-5 were increased in the ventricle of the transgenic mice. Electron microscopic analysis revealed that the ventricular myocardium of the transgenic mice had many secretory granules, which disappeared after administration of vasopressin. These results suggest that Csx/Nkx2-5 regulates many cardiac genes and induces formation of secretory granules in the adult ventricle.     

单纯性先天性心脏病中TBX5基因表达异常的机制

为探讨人类单纯性先天性心脏病患者中TBX5基因表达下调的可能原因, 应用变性高效液相色谱(DHPLC)方法检测100例单纯性先天性心脏病患者中TBX5基因上游1 200 bp调控区的突变情况; 应用甲基化敏感性限制性内切酶(MS-RE)法检测50例单纯性先天性心脏病患者和5例非先天性心脏病患者心肌组织TBX5基因启动子区两个CpG岛(转录起始点上游-49~-188 bp和-247~-464 bp处)的甲基化情况; 应用P-match软件预测小鼠Tbx5基因上游转录因子Nkx2-5的结合位点, 构建Nkx2-5表达载体转染小鼠H9C2(2-1)心肌细胞, RT-PCR及Western blotting检测Tbx5基因表达, 凝胶阻滞实验(EMSA)验证Nkx2-5和Tbx5基因的作用。结果在100例单纯性先天性心脏病患者中, 未检测到TBX5基因上游1 200 bp调控区突变; 非先天性心脏病患者和单纯性先天性心脏病患者在两个CpG岛存在相同的甲基化; 小鼠Tbx5基因转录起始点上游-312~-315 bp可能存在Nkx2-5的结合位点, 转染Nkx2-5表达载体后Tbx5基因在mRNA及蛋白质水平均有表达增高趋势, Nkx2-5在体外可以与Tbx5基因上游-312~-315 bp序列相结合。以上结果提示TBX5基因调控区突变和两个CpG岛的甲基化不是单纯性先天性心脏病患者心肌组织中TBX5基因表达下调的原因, TBX5基因表达下调可能由于NKX2-5的表达异常引起。     

Cre-mediated excision of Fgf8 in the Tbx1 expression domain reveals a critical role for Fgf8 in cardiovascular development in the mouse

Tbx1 has been implicated as a candidate gene responsible for defective pharyngeal arch remodeling in DiGeorge/Velocardiofacial syndrome. Tbx1(+/-) mice mimic aspects of the DiGeorge phenotype with variable penetrance, and null mice display severe pharyngeal hypoplasia. Here, we identify enhancer elements in the Tbx1 gene that are conserved through evolution and mediate tissue-specific expression. We describe the generation of transgenic mice that utilize these enhancer elements to direct Cre recombinase expression in endogenous Tbx1 expression domains. We use these Tbx1-Cre mice to fate map Tbx1-expressing precursors and identify broad regions of mesoderm, including early cardiac mesoderm, which are derived from Tbx1-expressing cells. We test the hypothesis that fibroblast growth factor 8 (Fgf8) functions downstream of Tbx1 by performing tissue-specific inactivation of Fgf8 using Tbx1-Cre mice. Resulting newborn mice display DiGeorge-like congenital cardiovascular defects that involve the outflow tract of the heart. Vascular smooth muscle differentiation in the great vessels is disrupted. This data is consistent with a model in which Tbx1 induces Fgf8 expression in the pharyngeal endoderm, which is subsequently required for normal cardiovascular morphogenesis and smooth muscle differentiation in the aorta and pulmonary artery.     

Endothelial neuropilin disruption in mice causes DiGeorge syndrome-like malformations via mechanisms distinct to those caused by loss of Tbx1

The spectrum of human congenital malformations known as DiGeorge syndrome (DGS) is replicated in mice by mutation of Tbx1. Vegfa has been proposed as a modifier of DGS, based in part on the occurrence of comparable phenotypes in Tbx1 and Vegfa mutant mice. Many additional genes have been shown to cause DGS-like phenotypes in mice when mutated; these generally intersect in some manner with Tbx1, and therefore impact the same developmental processes in which Tbx1 itself is involved. In this study, using Tie2Cre, we show that endothelial-specific mutation of the gene encoding the VEGFA coreceptor neuropilin-1 (Nrp1) also replicates the most prominent terminal phenotypes that typify DGS. However, the developmental etiologies of these defects are fundamentally different from those caused by absence of TBX1. In Tie2Cre/Nrp1 mutants, initial pharyngeal organization is normal but subsequent pharyngeal organ growth is impaired, second heart field differentiation is normal but cardiac outflow tract cushion organization is distorted, neural crest cell migration is normal, and palatal mesenchyme proliferation is impaired with no change in apoptosis. Our results demonstrate that impairment of VEGF-dependent endothelial pathways leads to a spectrum of DiGeorge syndrome-type malformations, through processes that are distinguishable from those controlled by Tbx1.     

An Nkx2-5/Bmp2/Smad1 negative feedback loop controls heart progenitor specification and proliferation

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.     

Tbx5 specifies the left/right ventricles and ventricular septum position during cardiogenesis

Extensive misexpression studies were carried out to explore the roles played by Tbx5, the expression of which is excluded from the right ventricle (RV) during cardiogenesis. When Tbx5 was misexpressed ubiquitously, ventricular septum was not formed, resulting in a single ventricle. In such heart, left ventricle (LV)-specific ANF gene was induced. In search of the putative RV factor(s), we have found that chick Tbx20 is expressed in the RV, showing a complementary fashion to Tbx5. In the Tbx5-misexpressed heart, this gene was repressed. When misexpression was spatially partial, leaving small Tbx5-negative area in the right ventricle, ventricular septum was shifted rightwards, resulting in a small RV with an enlarged LV. Focal expression induced an ectopic boundary of Tbx5-positive and -negative regions in the right ventricle, at which an additional septum was formed. Similar results were obtained from the transient transgenic mice. In such hearts, expression patterns of dHAND and eHAND were changed with definitive cardiac abnormalities. Furthermore, we report that human ANF promoter is synergistically activated by Tbx5, Nkx2.5 and GATA4. This activation was abrogated by Tbx20, implicating the pivotal roles of interactions among these heart-specific factors. Taken together, our data indicate that Tbx5 specifies the identity of LV through tight interactions among several heart-specific factors, and highlight the essential roles of Tbx5 in cardiac development.     

Efficient Cre-mediated deletion in cardiac progenitor cells conferred by a 3'UTR-ires-Cre allele of the homeobox gene Nkx2-5

Conditional gene targeting and transgenic strategies utilizing Cre recombinase have been successfully applied to the analysis of development in mouse embryos. To create a conditional system applicable to heart progenitor cells, a Cre recombinase gene linked at its 5' end to an internal ribosome entry site (IRES) was inserted into the 3' untranslated region of the cardiac homeobox gene Nkx2-5 using gene targeting. Nkx2-5IRESCre mice were fully viable as homozygotes. We evaluated the efficacy of Cre-mediated deletion by crossing Nkx2-5IRESCre mice with the Cre-dependent R26R and Z/AP reporter strains. Efficient deletion was observed in the cardiac crescent and heart tube in both strains. However, the Z/AP locus showed transient resistance to deletion in caudal heart progenitors. Such resistance was not evident at the R26R locus, suggesting that Cre-mediated deletion in myocardium may be locus-dependent. From cardiac crescent stages, deletion was seen not only in myocardium, but also endocardium, dorsal mesocardium and pericardial mesoderm. The Cre domain apparently includes cells dorsal to the heart that have been shown to constitute a secondary heart field, contributing myocardium to the outflow tract. Other sites of Nkx2-5 expression, including pharyngeal endoderm and its derivatives, branchial arch epithelium, stomach, spleen, pancreas and liver, also showed efficient deletion. Our data suggest that the Nkx2-5IRESCre strain will be useful for genetic dissection of the multiple tiers of lineage allocation to the forming heart as well as of molecular interactions within the heart fields and heart tube.