Targeted deletion of Hand2 in cardiac neural crest-derived cells influences cardiac gene expression and outflow tract development

The basic helix-loop-helix DNA binding protein Hand2 has critical functions in cardiac development both in neural crest-derived and mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest has allowed us to genetically dissect Hand2-dependent defects specifically in outflow tract and cardiac cushion independent of Hand2 functions in mesoderm-derived structures. Targeted deletion of Hand2 in the neural crest results in misalignment of the aortic arch arteries and outflow tract, contributing to development of double outlet right ventricle (DORV) and ventricular septal defects (VSD). These neural crest-derived developmental anomalies are associated with altered expression of Hand2-target genes we have identified by gene profiling. A number of Hand2 direct target genes have been identified using ChIP and ChIP-on-chip analyses. We have identified and validated a number of genes related to cell migration, proliferation/cell cycle and intracellular signaling whose expression is affected by Hand2 deletion in the neural crest and which are associated with development of VSD and DORV. Our data suggest that Hand2 is a multifunctional DNA binding protein affecting expression of target genes associated with a number of functional interactions in neural crest-derived cells required for proper patterning of the outflow tract, generation of the appropriate number of neural crest-derived cells for elongation of the conotruncus and cardiac cushion organization. Our genetic model has made it possible to investigate the molecular genetics of neural crest contributions to outflow tract morphogenesis and cell differentiation.     

Myocardium at the base of the aorta and pulmonary trunk is prefigured in the outflow tract of the heart and in subdomains of the second heart field

Outflow tract myocardium in the mouse heart is derived from the anterior heart field, a subdomain of the second heart field. We have recently characterized a transgene (y96-Myf5-nlacZ-16), which is expressed in the inferior wall of the outflow tract and then predominantly in myocardium at the base of the pulmonary trunk. Transgene A17-Myf5-nlacZ-T55 is expressed in the developing heart in a complementary pattern to y96-Myf5-nlacZ-16, in the superior wall of the outflow tract at E10.5 and in myocardium at the base of the aorta at E14.5. At E9.5, the two transgenes are transcribed in different subdomains of the anterior heart field. A clonal analysis of cardiomyocytes in the outflow tract, at E10.5 and E14.5, provides insight into the behaviour of myocardial cells and their progenitors. At E14.5, most clones are located at the base of either the pulmonary trunk or the aorta, indicating that these derive from distinct myocardial domains. At E10.5, clones are observed in subdomains of the outflow tract. The distribution of small clones indicates proliferative differences, whereas regionalisation of large clones, that derive from an early myocardial progenitor cell, reflect coherent cell growth in the heart field as well as in the myocardium. Our results suggest that myocardial differences at the base of the great arteries are prefigured in distinct progenitor cell populations in the anterior heart field, with important implications for understanding the etiology of congenital heart defects affecting the arterial pole of the heart.     

The microRNA-processing enzyme Dicer is dispensable for somite segmentation but essential for limb bud positioning

Dicer is an enzyme that processes microRNAs (miRNAs) to their mature forms. As miRNAs were first discovered for their role in the control of developmental timing, we investigated their potential requirement in mouse somitogenesis, an event with precise temporal periodicity. To address the collective role of miRNAs in mesoderm development including somite formation, we used T (Brachyury)-Cre mouse line to inactivate Dicer in most cells of the mesoderm lineage. This Dicer mutant exhibits a reduced anterior–posterior axis. Somite number remains normal in mutant embryos up until the death of the embryos more than two days after Dicer inactivation. Consistent with this, the molecular machineries required for establishing segmentation, including clock and wave front, are not perturbed. However, somite size is reduced and later-formed somites are caudalized, coincident with increased cell death. Outside of the paraxial mesoderm and prior to apparent reduction of the axis in the mutant, the position of the hindlimb bud, a lateral plate mesoderm-derived structure, is posteriorly shifted and the timing of hindlimb bud initiation is delayed accordingly. We observed changes in the expression of genes critical for limb positioning, which include a shifted and delayed downregulation of Hand2 and Tbx3, and shifted and delayed upregulation of Gli3 in the prospective limb bud field. The 3′ UTRs of both Hand2 and Tbx3 harbor target sites for a seed sequence-sharing family of miRNAs mir-25/32/92/363/367. As an example of the family we show that mir-363, a miRNA with elevated expression in the prospective limb bud field, is capable of inhibiting Hand2/Tbx3 expression in vitro in a binding site-dependent manner. Together, our findings provide the first demonstration that in mouse embryonic mesoderm, while Dicer is dispensable for somite segmentation, it is essential for proper limb bud positioning.     

8-Oxoguanine DNA glycosylase 1 (ogg1) maintains the function of cardiac progenitor cells during heart formation in zebrafish

Genomic damage may devastate the potential of progenitor cells and consequently impair early organogenesis. We found that ogg1, a key enzyme initiating the base-excision repair, was enriched in the embryonic heart in zebrafish. So far, little is known about DNA repair in cardiogenesis. Here, we addressed the critical role of ogg1 in cardiogenesis for the first time. ogg1 mainly expressed in the anterior lateral plate mesoderm (ALPM), the primary heart tube, and subsequently the embryonic myocardium by in situ hybridisation. Loss of ogg1 resulted in severe cardiac morphogenesis and functional abnormalities, including the short heart length, arrhythmia, decreased cardiomyocytes and nkx2.5⁺ cardiac progenitor cells. Moreover, the increased apoptosis and repressed proliferation of progenitor cells caused by ogg1 deficiency might contribute to the heart phenotype. The microarray analysis showed that the expression of genes involved in embryonic heart tube morphogenesis and heart structure were significantly changed due to the lack of ogg1. Among those, foxh1 is an important partner of ogg1 in the cardiac development in response to DNA damage. Our work demonstrates the requirement of ogg1 in cardiac progenitors and heart development in zebrafish. These findings may be helpful for understanding the aetiology of congenital cardiac deficits.     

DM-GRASP/ALCAM/CD166 is required for cardiac morphogenesis and maintenance of cardiac identity in first heart field derived cells

Vertebrate heart development requires specification of cardiac precursor cells, migration of cardiac progenitors as well as coordinated cell movements during looping and septation. DM-GRASP/ALCAM/CD166 is a member of the neuronal immunoglobulin domain superfamily of cell adhesion molecules and was recently suggested to be a target gene of non-canonical Wnt signalling. Loss of DM-GRASP function did not affect specification of cardiac progenitor cells. Later during development, expression of cardiac marker genes in the first heart field of Xenopus laevis such as Tbx20 and TnIc was reduced, whereas expression of the second heart field marker genes Isl-1 and BMP-4 was unaffected. Furthermore, loss of DM-GRASP function resulted in defective cell adhesion and cardiac morphogenesis. Additionally, expression of DM-GRASP can rescue the phenotype that results from the loss of non-canonical Wnt11-R signalling suggesting that DM-GRASP and non-canonical Wnt signalling are functionally coupled during cardiac development.     

Reassessment of Isl1 and Nkx2-5 cardiac fate maps using a Gata4-based reporter of Cre activity

Isl1 and Nkx2-5-expressing cardiovascular progenitors play pivotal roles in cardiogenesis. Previously reported Cre-based fate-mapping studies showed that Isl1 progenitors contribute predominantly to the derivatives of the second heart field, and Nkx2-5 progenitors contributed mainly to the cardiomyocyte lineage. However, partial recombination of Cre reporter genes can complicate interpretation of Cre fate-mapping experiments. We found that a Gata4-based Cre-activated reporter was recombined by Isl1^Cre and Nkx2-5^Cre in a substantially broader domain than previously reported using standard Cre-activated reporters. The expanded Isl1 and Nkx2-5 cardiac fate maps were remarkably similar, and included extensive contributions to cardiomyocyte, endocardial, and smooth muscle lineages in all four cardiac chambers. These data indicate that Isl1 is expressed in progenitors of both primary and secondary heart fields, and that Nkx2-5 is expressed in progenitors of cardiac endothelium and smooth muscle, in addition to cardiomyocytes. These results have important implications for our understanding of cardiac lineage diversification in vivo, and for the interpretation of Cre-based fate maps.     

基因谱系示踪技术揭示小鼠Tbx18+心脏祖细胞向心系细胞分化的潜能

心脏祖细胞(cardiac progenitor cells,CPCs)的研究对阐明先天性心脏病的机制及治疗心血管疾病具有重要意义.哺乳动物的心脏组织由多种不同CPCs分化形成.转录因子Tbx18在发育中的心外膜中表达,对心脏的发育形成起重要的调节作用.为了在组织及活体细胞水平检测和阐明Tbx18+CPC的分化潜能,应用Cre-LoxP系统建立Tbx18+CPCs基因命运谱系示踪模型:Tbx18-Cre/Rosa26R-EYFP和Tbx18-Cre/Rosa26R-LacZ双杂合基因敲入小鼠.该双杂合基因敲入小鼠通过Cre的表达能有效地示踪Tbx18+细胞在胚胎和成年小鼠中的分化命运.Tbx18-Cre/Rosa26R-EYFP双杂合小鼠心脏能非常容易地利用流式细胞分选系统(FACS)分离出YFP+细胞,也可在倒置共聚焦显微镜下观察.应用X-gal染色分析其表达模式,揭示Tbx18命运谱系参与心房肌、室间隔、心室肌、冠状动脉、瓣膜等的形成.应用免疫荧光技术初步揭示Tbx18+CPCs向心脏肌钙蛋白T(cTNT)阳性心肌细胞和平滑肌肌球蛋白重链11(MYH11)阳性血管平滑肌细胞分化的潜能.心脏是一个由多种肌肉和非肌肉组织细胞构成的复杂器官.推测Tbx18可能在心脏祖细胞向肌源性细胞分化的信号通路中起重要调节作用.在上述研究中应用基因谱系示踪技术,验证Tbx18可作为一类CPCs的标志,为更深入揭示心脏祖细胞向心系细胞的分化潜能打下基础.     

Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

About two-thirds of human congenital heart disease involves second heart field-derived structures. Histone-modifying enzymes, histone deacetylases (HDACs), regulate the epigenome; however, their functions within the second heart field remain elusive. Here we demonstrate that histone deacetylase 3 (HDAC3) orchestrates epigenetic silencing of Tgf-β1, a causative factor in congenital heart disease pathogenesis, in a deacetylase-independent manner to regulate development of second heart field-derived structures. In murine embryos lacking HDAC3 in the second heart field, increased TGF-β1 bioavailability is associated with ascending aortic dilatation, outflow tract malrotation, overriding aorta, double outlet right ventricle, aberrant semilunar valve development, bicuspid aortic valve, ventricular septal defects, and embryonic lethality. Activation of TGF-β signaling causes aberrant endothelial-to-mesenchymal transition and altered extracellular matrix homeostasis in HDAC3-null outflow tracts and semilunar valves, and pharmacological inhibition of TGF-β rescues these defects. HDAC3 recruits components of the PRC2 complex, methyltransferase EZH2, EED, and SUZ12, to the NCOR complex to enrich trimethylation of Lys-27 on histone H3 at the Tgf-β1 regulatory region and thereby maintains epigenetic silencing of Tgf-β1 specifically within the second heart field-derived mesenchyme. Wild-type HDAC3 or catalytically inactive HDAC3 expression rescues aberrant endothelial-to-mesenchymal transition and epigenetic silencing of Tgf-β1 in HDAC3-null outflow tracts and semilunar valves. These findings reveal that epigenetic dysregulation within the second heart field is a predisposing factor for congenital heart disease.     

Comparative gene expression analysis and fate mapping studies suggest an early segregation of cardiogenic lineages in Xenopus laevis

Retrospective clonal analysis in mice suggested that the vertebrate heart develops from two sources of cells called first and second lineages, respectively. Cells of the first lineage enter the linear heart tube and initiate terminal differentiation earlier than cells of the second lineage. It is thought that both heart lineages arise from a common progenitor cell population prior to the cardiac crescent stage (E7.5 of mouse development). The timing of segregation of different lineages as well as the molecular mechanisms underlying this process is not yet known. Furthermore, gene expression data for those lineages are very limited. Here we provide the first comparative study of cardiac marker gene expression during Xenopus laevis embryogenesis complemented by single cell RT-PCR analysis. In addition we provide fate mapping data of cardiac progenitor cells at different stages of development. Our analysis indicates an early segregation of cardiac lineages and a fairly complex heterogeneity of gene expression in the cardiac progenitor cells. Furthermore, this study sets a reference for all further studies analyzing cardiac development in X. laevis.     

Identification and biological characterization of chicken embryonic cardiac progenitor cells

Objectives: Many kinds of cardiac progenitor cell populations have been identified, including c‐kit⁺, Nkx2.5⁺s and GATA4⁺ cells. However, these progenitors have limited ability to differentiate into different cardiac cell types. Recently, a new kind of cardiac progenitor cell named the multipotent Isl1⁺ cardiovascular progenitor (MICPs) has been identified, which also expresses Nkx2.5, GATA4, CD34 and Flk1. Materials and methods: In this study, we have isolated and characterized MICPs from chicken embryonic heart tissues using immunofluorescence and PCR. Results: Results shown that they express markers of cardiac progenitor cells, with high clonality. They have the ability to self‐renew and can give rise to three types of heart cell in vitro. Conclusions: Myocytes, smooth muscle cells and endothelial cells. Our work provides evidence for a developmental paradigm of the heart, that endothelial and muscle lineage diversification arises from multipotent cardiac progenitor cells. Existence of these cells provides a new opportunity for myocardial injury repair.     

Frs2alpha-deficiency in cardiac progenitors disrupts a subset of FGF signals required for outflow tract morphogenesis

The cardiac outflow tract (OFT) is a developmentally complex structure derived from multiple lineages and is often defective in human congenital anomalies. Although emerging evidence shows that fibroblast growth factor (FGF) is essential for OFT development, the downstream pathways mediating FGF signaling in cardiac progenitors remain poorly understood. Here, we report that FRS2alpha (FRS2), an adaptor protein that links FGF receptor kinases to multiple signaling pathways, mediates crucial aspects of FGF-dependent OFT development in mouse. Ablation of Frs2alpha in mesodermal OFT progenitor cells that originate in the second heart field (SHF) affects their expansion into the OFT myocardium, resulting in OFT misalignment and hypoplasia. Moreover, Frs2alpha mutants have defective endothelial-to-mesenchymal transition and neural crest cell recruitment into the OFT cushions, resulting in OFT septation defects. These results provide new insight into the signaling molecules downstream of FGF receptor tyrosine kinases in cardiac progenitors.     

Combined effects of octopamine and filling pressure on the isolated heart of the lobster,Panulirus japonicus

Summary The effects of octopamine (OA) on the isolated and pumping heart of Panulirus japonicus were investigated. Under low filling pressure, OA (10^-7-10^-5 M) decreased the heart rate and enhanced the first systolic contraction (FSC) while reducing the second systolic contraction (SSC). The decrease in heart rate was transient whereas the enhancement of the FSC continued for 10 min or more after washout of OA. Pressure and OA synergistically intensified the FSC and reduced the SSC. The effect of OA on heart rate changed from a deceleration to an acceleration as the pressure was raised. Octopamine decreased both the firing frequency and the burst rate of the small cardiac neurons in a dose-dependent manner. In contrast, the amine often induced a slow ramp potential in the large cardiac neurons and increased their burst rate. Octopamine had no effect on the excitatory junction potentials evoked in cardiac muscle cells. Contraction and closure of the posterior cardioarterial valve were induced by OA, resulting in rebound internal heart pressure and stretching of the heart. The stretch augmented the heart rate and the FSC. The inhibition of small cardiac neurons by OA was related to the reduction of the heart rate and the SSC. The activation of large cardiac neurons and of cardiac and valve muscles by OA augmented the heart rate and the FSC. These multiple actions on the perfused heart lead to abolition of the SSC. After abolition of the SSC, increases of pressure can rapidly raise the heart rate.Abbreviations EJP excitatory junction potential - FLP first large potential - FSC first systolic contraction - OA octopamine - SLP second large potential - SSC second systolic contraction     

Combinatorial signaling in the heart orchestrates cardiac induction, lineage specification and chamber formation

The complexity of mammalian cardiogenesis is compounded, as the heart must function in the embryo whilst it is still being formed. Great advances have been made recently as additional cardiac progenitor cell populations have been identified. The induction and maintenance of these progenitors, and their deployment to the developing heart relies on combinatorial molecular signalling, a feature also of cardiac chamber formation. Many forms of congenital heart disease in humans are likely to arise from defects in the early stages of heart development; therefore it is important to understand the molecular pathways that underlie some of the key events that shape the heart during the early stages of it development.     

Pitx2 regulates cardiac left-right asymmetry by patterning second cardiac lineage-derived myocardium

Current models of left-right asymmetry hold that an early asymmetric signal is generated at the node and transduced to lateral plate mesoderm in a linear signal transduction cascade through the function of the Nodal signaling molecule. The Pitx2 homeobox gene functions at the final stages of this cascade to direct asymmetric morphogenesis of selected organs including the heart. We previously showed that Pitx2 regulated an asymmetric pathway that was independent of cardiac looping suggesting a second asymmetric cardiac pathway. It has been proposed that in the cardiac outflow tract Pitx2 functions in both cardiac neural crest, as a target of canonical Wnt-signaling, and in the mesoderm-derived cardiac second lineage. We used fate mapping, conditional loss of function, and chimera analysis in mice to investigate the role of Pitx2 in outflow tract morphogenesis. Our findings reveal that Pitx2 is dispensable in the cardiac neural crest but functions in second lineage myocardium revealing that this cardiac progenitor field is patterned asymmetrically.