Anterior Heart Field Research Articles

307-OR: Delineating the Origin of Supraclavicular Brown Adipocytes

Brown adipose tissue (BAT) can use nutrients through non-shivering thermogenesis to generate heat. Thus, activation of BAT can be utilized to maintain body temperature and regulate metabolism. Healthy adult humans possess small amounts of BAT, with the most visible depot, the supraclavicular BAT (scBAT), located in the supraclavicular region of the neck. To understand how human scBAT functions, we previously identified BAT depots in the supraclavicular region of the mouse neck that anatomically resemble human scBAT. Similar to human scBAT, mouse scBAT contains a high density of mitochondria and expresses high levels of uncoupling protein 1. Mouse scBAT first appears in the late fetal stage in the ventral neck and continues to expand toward the lateral neck after birth. To identify the stem/progenitor cells that give rise to supraclavicular brown adipocytes, we performed a series of lineage tracing analyses. Unlike the well-studied mouse interscapular BAT depot, scBAT did not originate from the Myf5, Pax3, or Pax7-labeled progenitor cells that also give rise to skeletal muscle. Rather, a large portion of the supraclavicular brown adipocytes were labeled by Cre recombinase driven by the Mef2c anterior heart field promoter (AHF), which marks cells that give rise to the outflow tract and right ventricle of the heart. Mef2c-AHF+ cells isolated from the scBAT can spontaneously differentiate into highly thermogenic brown adipocytes in vitro, and the differentiation efficiency is further enhanced by addition of Bmp7 and PPARγ agonist, rosiglitazone. Together, our lineage tracing studies revealed that the scBAT is composed of heterogeneous brown adipocyte progenitor cells and that Mef2c-AHF-marked cardiac progenitor cells are one of the ancestors of these brown adipocytes. Understanding the trajectories of and molecular mechanisms by which these progenitor cells give rise to scBAT will provide new insights for developing a novel cell-based therapeutic tool for treating obesity and related metabolic complications. Disclosure Y.Ran: None. Z.Wang: None. M.G.Waterstraat: None. R.Caballero-juarez: None. M.Chen: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK116899)

Abstract 14797: Single Cell Multi-Modal Analyses Reveal Epigenomic and Transcriptomic Basis Underlying Cardiac Defects in Maternal Diabetes

Introduction: Birth defects can be caused by combinations of genetic and environmental influences. Large-scale DNA sequencing efforts reveal genetic influences, yet the environmental contributions have been mainly limited to association studies with little mechanistic insight. Hyperglycemia in pre-gestational diabetic (PGDM) mothers are among the most frequent environmental contributors to congenital defects, such as congenital heart defects and craniofacial anomalies. However, the cell types involved and underlying mechanisms by which PGDM affects these regions are unknown. Methods and Results: To determine how PGDM affects the epigenomic and transcriptomic state within individual cells during cardio-pharyngeal development, we utilized multi-modal single cell analyses with streptozotocin-induced mouse PGDM model. Analysis of E10.5 embryos showed that PGDM specifically affects the epigenomic state of two small subsets of cardiac and craniofacial progenitors. In PGDM derived embryos, a newly recognized subpopulation of anterior heart field progenitors expressing Alx3 acquires a more posterior-like identity in response to PGDM, based on gene expression and chromatin status. Similarly, a sub-population of neural crest-derived cells in the second pharyngeal arch, which contributes to craniofacial structures, also displayed patterning abnormalities. Analyses of differentially accessible chromatin regions revealed disrupted patterning was due to increased intrinsic retinoic acid signaling in affected cell types in PGDM. Conclusions: We showed the selective epigenomic vulnerability of a small subset of cells that help explain cardiac and craniofacial defects observed in PGDM despite all embryonic cells experiencing similar environmental exposure. We also showed that the selectivity was partly driven by a previously unrecognized dysregulation of retinoic acid signaling in PGDM. Discovery of the consequences of environmental factors on embryonic development using multi-modal single cell genomics will lead to potential gene-environment combinations that contribute to the complex and multifactorial diseases and preventive approaches to protect vulnerable cells.

The Spatiotemporal Pattern and Intensity of p53 Activation Dictates Phenotypic Diversity in p53-Driven Developmental Syndromes

Inappropriate activation of the p53 transcription factor contributes to numerous developmental syndromes characterized by distinct constellations of phenotypes. How p53 drives exquisitely specific sets of symptoms in diverse syndromes, however, remains enigmatic. Here, we deconvolute the basis of p53-driven developmental syndromes by leveraging an array of mouse strains to modulate the spatial expression pattern, temporal profile, and magnitude of p53 activation during embryogenesis. We demonstrate that inappropriate p53 activation in the neural crest, facial ectoderm, anterior heart field, and endothelium induces distinct spectra of phenotypes. Moreover, altering the timing and degree of p53 hyperactivation substantially affects the phenotypic outcomes. Phenotypes are associated with p53-driven cell-cycle arrest or apoptosis, depending on the cell type, with gene expression programs, rather than extent of mitochondrial priming, largely governing the specific response. Together, our findings provide a critical framework for decoding the role of p53 as a mediator of diverse developmental syndromes.

Wnt/\u03b2-catenin-mediated signaling re-activates proliferation of matured cardiomyocytes

BackgroundThe Wnt/β-catenin signaling pathway plays an important role in the development of second heart field (SHF Isl1+) that gives rise to the anterior heart field (AHF) cardiac progenitor cells (CPCs) for the formation of the right ventricle, outflow tract (OFT), and a portion of the inflow tract (IFT). During early cardiogenesis, these AHF CPCs reside within the pharyngeal mesoderm (PM) that provides a microenvironment for them to receive signals that direct their cell fates. Here, N-cadherin, which is weakly expressed by CPCs, plays a significant role by promoting the adhesion of CPCs within the AHF, regulating β-catenin levels in the cytoplasm to maintain high Wnt signaling and cardioproliferation while also preventing the premature differentiation of CPCs. On the contrary, strong expression of N-cadherin observed throughout matured myocardium is associated with downregulation of Wnt signaling due to β-catenin sequestration at the cell membrane, inhibiting cardioproliferation. As such, upregulation of Wnt signaling pathway to enhance cardiac tissue proliferation in mature cardiomyocytes can be explored as an interesting avenue for regenerative treatment to patients who have suffered from myocardial infarction.MethodsTo investigate if Wnt signaling is able to enhance cellular proliferation of matured cardiomyocytes, we treated cardiomyocytes isolated from adult mouse heart and both murine and human ES cell-derived matured cardiomyocytes with N-cadherin antibody or CHIR99021 GSK inhibitor in an attempt to increase levels of cytoplasmic β-catenin. Immunostaining, western blot, and quantitative PCR for cell proliferation markers, cell cycling markers, and Wnt signaling pathway markers were used to quantitate re-activation of cardioproliferation and Wnt signaling.ResultsN-cadherin antibody treatment releases sequestered β-catenin at N-cadherin-based adherens junction, resulting in an increased pool of cytoplasmic β-catenin, similar in effect to CHIR99021 GSK inhibitor treatment. Both treatments therefore upregulate Wnt signaling successfully and result in significant increases in matured cardiomyocyte proliferation.ConclusionAlthough both N-cadherin antibody and CHIR99021 treatment resulted in increased Wnt signaling and cardioproliferation, CHIR99021 was found to be the more effective treatment method for human ES cell-derived cardiomyocytes. Therefore, we propose that CHIR99021 could be a potential therapeutic option for myocardial infarction patients in need of regeneration of cardiac tissue.

Tbx1 represses Mef2c gene expression and is correlated with histone 3 deacetylation of the anterior heart field enhancer.

ABSTRACTThe TBX1 gene is haploinsufficient in 22q11.2 deletion syndrome (22q11.2DS), and genetic evidence from human patients and mouse models points to a major role of this gene in the pathogenesis of this syndrome. Tbx1 can activate and repress transcription, and previous work has shown that one of its functions is to negatively modulate cardiomyocyte differentiation. Tbx1 occupies the anterior heart field (AHF) enhancer of the Mef2c gene, which encodes a key cardiac differentiation transcription factor. Here, we show that increased dosage of Tbx1 correlates with downregulation of Mef2c expression and reduced acetylation of its AHF enhancer in cultured mouse myoblasts. Consistently, 22q11.2DS-derived and in vitro-differentiated human induced pluripotent stem cells (hiPSCs) expressed higher levels of MEF2C and showed increased AHF acetylation, compared with hiPSCs from a healthy donor. Most importantly, we show that in mouse embryos, loss of Tbx1 enhances the expression of the Mef2c-AHF-Cre transgene in a specific region of the splanchnic mesoderm, and in a dosage-dependent manner, providing an in vivo correlate of our cell culture data. These results indicate that Tbx1 regulates the Mef2c AHF enhancer by inducing histone deacetylation.

Dysregulation of TBX1 dosage in the anterior heart field results in congenital heart disease resembling the 22q11.2 duplication syndrome.

Non-allelic homologous recombination events on chromosome 22q11.2 during meiosis can result in either the deletion (22q11.2DS) or duplication (22q11.2DupS) syndrome. Although the spectrum and frequency of congenital heart disease (CHD) are known for 22q11.2DS, there is less known for 22q11.2DupS. We now evaluated cardiac phenotypes in 235 subjects with 22q11.2DupS including 102 subjects we collected and 133 subjects that were previously reported as a confirmation and found 25% have CHD, mostly affecting the cardiac outflow tract (OFT). Previous studies have shown that global loss or gain of function (LOF; GOF) of mouse Tbx1, encoding a T-box transcription factor mapping to the region of synteny to 22q11.2, results in similar OFT defects. To further evaluate Tbx1 function in the progenitor cells forming the cardiac OFT, termed the anterior heart field, Tbx1 was overexpressed using the Mef2c-AHF-Cre driver (Tbx1 GOF). Here we found that all resulting conditional GOF embryos had a persistent truncus arteriosus (PTA), similar to what was previously reported for conditional Tbx1 LOF mutant embryos. To understand the basis for the PTA in the conditional GOF embryos, we found that proliferation in the Mef2c-AHF-Cre lineage cells before migrating to the heart, was reduced and critical genes were oppositely changed in this tissue in Tbx1 GOF embryos versus conditional LOF embryos. These results suggest that a major function of TBX1 in the AHF is to maintain the normal balance of expression of key cardiac developmental genes required to form the aorta and pulmonary trunk, which is disrupted in 22q11.2DS and 22q11.2DupS.

Abstract 240: Asb2-dependent Proteolysis of the Cytoskeleton Directs Cardiac Development and Disease

Congenital heart diseases (CHDs) account for 25% of birth defects and are major risk factors for adult cardiovascular problems. Partial disease penetrance is seen even in autosomal dominant disorders and genotype/phenotype correlations remain a clinical challenge; thus, the need to understand different regulators of cardiac formation. The Ubiquitin-Proteasome System (UPS) is important in controlling protein turnover during organ development but its role in the mammalian heart remains ambiguous. We have identified a specificity subunit of ubiquitin-mediated proteolysis (Asb2) as being specific for the cardiac myogenic lineage. Asb2 was previously shown to regulate hematopoietic and skeletal muscle cell differentiation through targeting filamin proteins (FlnA, B and C), actin-binding proteins important for cytoskeleton stabilization. In our present study, we show that Asb2 is markedly enriched in myocardial progenitor cells and cardiomyocytes. To investigate the role of Asb2 and UPS dependent proteolysis in heart development, we generated two cardiac-specific murine knockouts (KOs): Nkx Cre .Asb2 -/- and Mef2c Cre .Asb2 -/- (deleting Asb2 in early cardiomyocyte progenitors and anterior heart field progenitors, respectively). Both KOs are embryonic lethal with pericardial edema. We used tissue clarifying and confocal microscopy to define the morphological defects of the Asb2 null heart. Moreover, we found that FlnA is overexpressed in the hearts of these mice and its deletion therein partially rescues their lethality. In addition, using transcriptomic analysis on Asb2-null e9.5 hearts, we identified novel potential Asb2 targets in the heart. Finally, to understand the role of Asb2 in the differentiation and function of human cardiomyocytes, we used CRISPR/Cas9 genome editing technique to generate Asb2-null human induced pluripotent stem cells. Collectively, our study provides a novel mechanistic understanding of the role of the UPS proteasome in cardiac development, myocardial function, and disease pathogenesis. Given recent interests in both the UPS and the cytoskeleton as therapeutic targets, our study provides an innovative platform for the development of pharmacotherapy for cardiac disease.

Reduced dosage of β-catenin provides significant rescue of cardiac outflow tract anomalies in a Tbx1 conditional null mouse model of 22q11.2 deletion syndrome

The 22q11.2 deletion syndrome (22q11.2DS; velo-cardio-facial syndrome; DiGeorge syndrome) is a congenital anomaly disorder in which haploinsufficiency of TBX1, encoding a T-box transcription factor, is the major candidate for cardiac outflow tract (OFT) malformations. Inactivation of Tbx1 in the anterior heart field (AHF) mesoderm in the mouse results in premature expression of pro-differentiation genes and a persistent truncus arteriosus (PTA) in which septation does not form between the aorta and pulmonary trunk. Canonical Wnt/β-catenin has major roles in cardiac OFT development that may act upstream of Tbx1. Consistent with an antagonistic relationship, we found the opposite gene expression changes occurred in the AHF in β-catenin loss of function embryos compared to Tbx1 loss of function embryos, providing an opportunity to test for genetic rescue. When both alleles of Tbx1 and one allele of β-catenin were inactivated in the Mef2c-AHF-Cre domain, 61% of them (n = 34) showed partial or complete rescue of the PTA defect. Upregulated genes that were oppositely changed in expression in individual mutant embryos were normalized in significantly rescued embryos. Further, β-catenin was increased in expression when Tbx1 was inactivated, suggesting that there may be a negative feedback loop between canonical Wnt and Tbx1 in the AHF to allow the formation of the OFT. We suggest that alteration of this balance may contribute to variable expressivity in 22q11.2DS.

Mechanism responsible for D-transposition of the great arteries: Is this part of the spectrum of right isomerism?

D-transposition of the great arteries (TGA) is one of the most common conotruncal heart defects at birth and is characterized by a discordant ventriculoarterial connection with a concordant atrioventricular connection. The morphological etiology of TGA is an inverted or arrested rotation of the heart outflow tract (OFT, conotruncus), by which the aorta is transposed in the right ventral direction to the pulmonary trunk. The rotational defect of the OFT is thought to be attributed to hypoplasia of the subpulmonic conus, which originates from the left anterior heart field (AHF) residing in the mesodermal core of the first and second pharyngeal arches. AHF, especially on the left, at the early looped heart stage (corresponding to Carnegie stage 10-11 in the human embryo) is one of the regions responsible for the impediment that causes TGA morphology. In human or experimentally produced right isomerism, malposition of the great arteries including D-TGA is frequently associated. Mutations in genes involving left-right (L-R) asymmetry, such as NODAL, ACTRIIB and downstream target FOXH1, have been found in patients with right isomerism as well as in isolated TGA. The downstream pathways of Nodal-Foxh1 play a critical role not only in L-R determination in the lateral plate mesoderm but also in myocardial specification and differentiation in the AHF, suggesting that TGA is a phenotype in heterotaxia as well as the primary developmental defect of the AHF.

Generation of iPSC from cardiac and tail-tip fibroblasts derived from a second heart field reporter mouse

Mef2c Anterior Heart Field (AHF) enhancer is activated during embryonic heart development and it is expressed in multipotent cardiovascular progenitors (CVP) giving rise to endothelial and myocardial components of the outflow tract, right ventricle and ventricular septum. Here we have generated iPSC from transgenic Mef2c-AHF-Cre x Ai6(RCLZsGreen) mice. These iPSC will provide a novel tool to investigate the AHF-CVP and their cell progeny.

Sequential Binding of MEIS1 and NKX2-5 on the Popdc2 Gene: A Mechanism for Spatiotemporal Regulation of Enhancers during Cardiogenesis.

SummaryThe homeobox transcription factors NKX2-5 and MEIS1 are essential for vertebrate heart development and normal physiology of the adult heart. We show that, during cardiac differentiation, the two transcription factors have partially overlapping expression patterns, with the result that as cardiac progenitors from the anterior heart field differentiate and migrate into the cardiac outflow tract, they sequentially experience high levels of MEIS1 and then increasing levels of NKX2-5. Using the Popdc2 gene as an example, we also show that a significant proportion of target genes for NKX2-5 contain a binding motif recognized by NKX2-5, which overlaps with a binding site for MEIS1. Binding of the two factors to such overlapping sites is mutually exclusive, and this provides a simple regulatory mechanism for spatial and temporal synchronization of a common pool of targets between NKX2-5 and MEIS1.

The Isl1/Ldb1 Complex Orchestrates Genome-wide Chromatin Organization to Instruct Differentiation of Multipotent Cardiac Progenitors

Cardiac stem/progenitor cells hold great potential for regenerative therapies; however, the mechanisms regulating their expansion and differentiation remain insufficiently defined. Here we show that Ldb1 is a central regulator of genome organization in cardiac progenitor cells, which is crucial for cardiac lineage differentiation and heart development. We demonstrate that Ldb1 binds to the key regulator of cardiac progenitors, Isl1, and protects it from degradation. Furthermore, the Isl1/Ldb1 complex promotes long-range enhancer-promoter interactions at the loci of the core cardiac transcription factors Mef2c and Hand2. Chromosome conformation capture followed by sequencing identified specific Ldb1-mediated interactions of the Isl1/Ldb1 responsive Mef2c anterior heart field enhancer with genes that play key roles in cardiac progenitor cell function and cardiovascular development. Importantly, the expression of these genes was downregulated upon Ldb1 depletion and Isl1/Ldb1 haplodeficiency. In conclusion, the Isl1/Ldb1 complex orchestrates a network forheart-specific transcriptional regulation and coordination in three-dimensional space during cardiogenesis.

Impaired development of left anterior heart field by ectopic retinoic acid causes transposition of the great arteries.

BackgroundTransposition of the great arteries is one of the most commonly diagnosed conotruncal heart defects at birth, but its etiology is largely unknown. The anterior heart field (AHF) that resides in the anterior pharyngeal arches contributes to conotruncal development, during which heart progenitors that originated from the left and right AHF migrate to form distinct conotruncal regions. The aim of this study is to identify abnormal AHF development that causes the morphology of transposition of the great arteries.Methods and ResultsWe placed a retinoic acid–soaked bead on the left or the right or on both sides of the AHF of stage 12 to 14 chick embryos and examined the conotruncal heart defect at stage 34. Transposition of the great arteries was diagnosed at high incidence in embryos for which a retinoic acid–soaked bead had been placed in the left AHF at stage 12. Fluorescent dye tracing showed that AHF exposed to retinoic acid failed to contribute to conotruncus development. FGF8 and Isl1 expression were downregulated in retinoic acid–exposed AHF, and differentiation and expansion of cardiomyocytes were suppressed in cultured AHF in medium supplemented with retinoic acid.ConclusionsThe left AHF at the early looped heart stage, corresponding to Carnegie stages 10 to 11 (28 to 29 days after fertilization) in human embryos, is the region of the impediment that causes the morphology of transposition of the great arteries.

Functional cardiomyocytes derived from Isl1 cardiac progenitors via Bmp4 stimulation.

As heart failure due to myocardial infarction remains a leading cause of morbidity worldwide, cell-based cardiac regenerative therapy using cardiac progenitor cells (CPCs) could provide a potential treatment for the repair of injured myocardium. As adult CPCs may have limitations regarding tissue accessibility and proliferative ability, CPCs derived from embryonic stem cells (ESCs) could serve as an unlimited source of cells with high proliferative ability. As one of the CPCs that can be derived from embryonic stem cells, Isl1 expressing cardiac progenitor cells (Isl1-CPCs) may serve as a valuable source of cells for cardiac repair due to their high cardiac differentiation potential and authentic cardiac origin. In order to generate an unlimited number of Isl1-CPCs, we used a previously established an ESC line that allows for isolation of Isl1-CPCs by green fluorescent protein (GFP) expression that is directed by the mef2c gene, specifically expressed in the Isl1 domain of the anterior heart field. To improve the efficiency of cardiac differentiation of Isl1-CPCs, we studied the role of Bmp4 in cardiogenesis of Isl1-CPCs. We show an inductive role of Bmp directly on cardiac progenitors and its enhancement on early cardiac differentiation of CPCs. Upon induction of Bmp4 to Isl1-CPCs during differentiation, the cTnT+ cardiomyocyte population was enhanced 2.8±0.4 fold for Bmp4 treated CPC cultures compared to that detected for vehicle treated cultures. Both Bmp4 treated and untreated cardiomyocytes exhibit proper electrophysiological and calcium signaling properties. In addition, we observed a significant increase in Tbx5 and Tbx20 expression in differentiation cultures treated with Bmp4 compared to the untreated control, suggesting a link between Bmp4 and Tbx genes which may contribute to the enhanced cardiac differentiation in Bmp4 treated cultures. Collectively these findings suggest a cardiomyogenic role for Bmp4 directly on a pure population of Isl1 expressing cardiac progenitors, which could lead to enhancement of cardiac differentiation and engraftment, holding a significant therapeutic value for cardiac repair in the future.

N-cadherin prevents the premature differentiation of anterior heart field progenitors in the pharyngeal mesodermal microenvironment

The cardiac progenitor cells (CPCs) in the anterior heart field (AHF) are located in the pharyngeal mesoderm (PM), where they expand, migrate and eventually differentiate into major cell types found in the heart, including cardiomyocytes. The mechanisms by which these progenitors are able to expand within the PM microenvironment without premature differentiation remain largely unknown. Through in silico data mining, genetic loss-of-function studies, and in vivo genetic rescue studies, we identified N-cadherin and interaction with canonical Wnt signals as a critical component of the microenvironment that facilitates the expansion of AHF-CPCs in the PM. CPCs in N-cadherin mutant embryos were observed to be less proliferative and undergo premature differentiation in the PM. Notably, the phenotype of N-cadherin deficiency could be partially rescued by activating Wnt signaling, suggesting a delicate functional interaction between the adhesion role of N-cadherin and Wnt signaling in the early PM microenvironment. This study suggests a new mechanism for the early renewal of AHF progenitors where N-cadherin provides additional adhesion for progenitor cells in the PM, thereby allowing Wnt paracrine signals to expand the cells without premature differentiation.

Hox genes define distinct progenitor sub-domains within the second heart field

Much of the heart, including the atria, right ventricle and outflow tract (OFT) is derived from a progenitor cell population termed the second heart field (SHF) that contributes progressively to the embryonic heart during cardiac looping. Several studies have revealed anterior-posterior patterning of the SHF, since the anterior region (anterior heart field) contributes to right ventricular and OFT myocardium whereas the posterior region gives rise to the atria. We have previously shown that Retinoic Acid (RA) signal participates to this patterning. We now show that Hoxb1, Hoxa1, and Hoxa3, as downstream RA targets, are expressed in distinct sub-domains within the SHF. Our genetic lineage tracing analysis revealed that Hoxb1, Hoxa1 and Hoxa3-expressing cardiac progenitor cells contribute to both atria and the inferior wall of the OFT, which subsequently gives rise to myocardium at the base of pulmonary trunk. By contrast to Hoxb1Cre, the contribution of Hoxa1-enhIII-Cre and Hoxa3Cre-labeled cells is restricted to the distal regions of the OFT suggesting that proximo-distal patterning of the OFT is related to SHF sub-domains characterized by combinatorial Hox genes expression. Manipulation of RA signaling pathways showed that RA is required for the correct deployment of Hox-expressing SHF cells. This report provides new insights into the regulatory gene network in SHF cells contributing to the atria and sub-pulmonary myocardium.

BMP-mediated inhibition of FGF signaling promotes cardiomyocyte differentiation of anterior heart field progenitors

The anterior heart field (AHF) encompasses a niche in which mesoderm-derived cardiac progenitors maintain their multipotent and undifferentiated nature in response to signals from surrounding tissues. Here, we investigate the signaling mechanism that promotes the shift from proliferating cardiac progenitors to differentiating cardiomyocytes in chick embryos. Genomic and systems biology approaches, as well as perturbations of signaling molecules, in vitro and in vivo, reveal tight crosstalk between the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) signaling pathways within the AHF niche: BMP4 promotes myofibrillar gene expression and cardiomyocyte contraction by blocking FGF signaling. Furthermore, inhibition of the FGF-ERK pathway is both sufficient and necessary for these processes, suggesting that FGF signaling blocks premature differentiation of cardiac progenitors in the AHF. We further revealed that BMP4 induced a set of neural crest-related genes, including MSX1. Overexpression of Msx1 was sufficient to repress FGF gene expression and cell proliferation, thereby promoting cardiomyocyte differentiation. Finally, we show that BMP-induced cardiomyocyte differentiation is diminished following cranial neural crest ablation, underscoring the key roles of these cells in the regulation of AHF cell differentiation. Hence, BMP and FGF signaling pathways act via inter- and intra-regulatory loops in multiple tissues, to coordinate the balance between proliferation and differentiation of cardiac progenitors.

BMP-mediated inhibition of FGF signaling promotes cardiomyocyte differentiation of anterior heart field progenitors

The anterior heart field (AHF) encompasses a niche in which mesoderm-derived cardiac progenitors maintain their multipotent and undifferentiated nature in response to signals from surrounding tissues. Here, we investigate the signaling mechanism that promotes the shift from proliferating cardiac progenitors to differentiating cardiomyocytes in chick embryos. Genomic and systems biology approaches, as well as perturbations of signaling molecules, in vitro and in vivo, reveal tight crosstalk between the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) signaling pathways within the AHF niche: BMP4 promotes myofibrillar gene expression and cardiomyocyte contraction by blocking FGF signaling. Furthermore, inhibition of the FGF-ERK pathway is both sufficient and necessary for these processes, suggesting that FGF signaling blocks premature differentiation of cardiac progenitors in the AHF. We further revealed that BMP4 induced a set of neural crest-related genes, including MSX1. Overexpression of Msx1 was sufficient to repress FGF gene expression and cell proliferation, thereby promoting cardiomyocyte differentiation. Finally, we show that BMP-induced cardiomyocyte differentiation is diminished following cranial neural crest ablation, underscoring the key roles of these cells in the regulation of AHF cell differentiation. Hence, BMP and FGF signaling pathways act via inter- and intra-regulatory loops in multiple tissues, to coordinate the balance between proliferation and differentiation of cardiac progenitors.

Human Cardiac Stem Cells

The view of the heart as a static organ implies that myocyte death and formation play a negligible role in cardiac homeostasis. Although stem cells have been unexpectedly identified in several organs, including the brain, kidney, lung, and skeletal muscle, the search for a cardiac stem cell (CSC) has been perceived as a futile effort, given the acknowledged lack of regenerative potential of the myocardium. Nevertheless, in the past several years, the demonstration of myocyte renewal in the normal and diseased heart has revealed a new, dynamic, and lively picture of this organ. Components of the cell cycle machinery and markers of cell replication have been detected in cardiomyocytes. The demonstration that karyokinesis and cytokinesis involve cells expressing contractile proteins has provided evidence that cardiomyocyte division occurs in the adult heart.1 More recently, pulse-chase assays with thymidine analogs,2 lineage tracing protocols,3 and 14C birth dating of cells4 have shown the existence of myocyte turnover, a process that has been found to differ in magnitude according to the methods used for its documentation and quantification. Article see p 2559 Over the years, the heart has provided us with the evidence that solves the critical problem of the origin of cardiomyocytes. At this moment in time, paraphrasing Eugenio Montale, we could say that the human heart is “on the verge of betraying [its] final secret offering the opportunity to uncover…the still point of the world, the link that won't hold, the thread to untangle that will finally lead to the heart of a truth.”5 Newly generated myocytes may derive from division of preexisting parenchymal cells or from activation and differentiation of resident CSCs. Discriminating between these two possibilities is not an easy task. Fate-mapping strategies, which are commonly used to track the origin of cells and …

The role of secondary heart field in cardiac development

Although de la Cruz and colleagues showed as early as 1977 that the outflow tract was added after the heart tube formed, the source of these secondarily added cells was not identified for nearly 25 years. In 2001, three pivotal publications described a secondary or anterior heart field that contributed to the developing outflow tract. This review details the history of the heart field, the discovery and continuing elucidation of the secondarily adding myocardial cells, and how the different populations identified in 2001 are related to the more recent lineage tracing studies that defined the first and second myocardial heart fields/lineages. Much recent work has focused on secondary heart field progenitors that give rise to the myocardium and smooth muscle at the definitive arterial pole. These progenitors are the last to be added to the arterial pole and are particularly susceptible to abnormal development, leading to conotruncal malformations in children. The major signaling pathways (Wnt, BMP, FGF8, Notch, and Shh) that control various aspects of secondary heart field progenitor behavior are discussed.