Cardiac neural crest is necessary for normal addition of the myocardium to the arterial pole from the secondary heart field

In cardiac neural-crest-ablated embryos, the secondary heart field fails to add myocardial cells to the outflow tract and elongation of the tube is deficient. Since that study, we have shown that the secondary heart field provides both myocardium and smooth muscle to the arterial pole. The present study was undertaken to determine whether addition of both cell types is disrupted after neural crest ablation. Marking experiments confirm that the myocardial component fails to be added to the outflow tract after neural crest ablation. The cells destined to go into the outflow myocardium fail to migrate and are left at the junction of the outflow myocardium with the nascent smooth muscle at the base of the arterial pole. In contrast, the vascular smooth muscle component is added to the arterial pole normally after neural crest ablation. When the myocardium is not added to the outflow tract, the point where the outflow joins the pharynx does not move caudally as it normally should, the aortic sac is smaller and fails to elongate resulting in abnormal connections of the outflow tract with the caudal aortic arch arteries.     

Second heart field and the development of the outflow tract in human embryonic heart

The second heart field (SHF) is indicated to contribute to the embryonic heart development. However, less knowledge is available about SHF development of human embryo due to the difficulty of collecting embryos. In this study, serial sections of human embryos from Carnegie stage 10 (CS10) to CS16 were stained with antibodies against Islet‐1 (Isl‐1), Nkx2.5, GATA4, myosin heavy chain (MHC) and α‐smooth muscle actin (α‐SMA) to observe spatiotemporal distribution of SHF and its contribution to the development of the arterial pole of cardiac tube. Our findings suggest that during CS10 to CS12, SHF of the human embryo is composed of the bilateral pharyngeal mesenchyme, the central mesenchyme of the branchial arch and splanchnic mesoderm of the pericardial cavity dorsal wall. With development, SHF translocates and consists of ventral pharyngeal mesenchyme and dorsal wall of the pericardial cavity. Hence, the SHF of human embryo shows a dynamic spatiotemporal distribution pattern. The formation of the Isl‐1 positive condense cell prongs provides an explanation for the saddle structure formation at the distal pole of the outflow tract. In human embryo, the Isl‐1 positive cells of SHF may contribute to the formation of myocardial outflow tract (OFT) and the septum during different development stages.     

Ablation of the secondary heart field leads to tetralogy of Fallot and pulmonary atresia

Recent studies in chick and mouse embryos have identified a previously unrecognized secondary heart field (SHF), located in the ventral midline splanchnic mesenchyme, which provides additional myocardial cells to the outflow tract as the heart tube lengthens during cardiac looping. In order to further delineate the contribution of this secondary myocardium to outflow development, we labeled the right SHF of Hamburger-Hamilton (HH) stage 14 chick embryos via microinjection of DiI/rhodamine and followed the fluorescently labeled cells over a 96-h time period. These experiments confirmed the movement of the SHF into the outflow and its spiraling migration distally, with the right side of the SHF contributing to the left side of the outflow. In contrast, when the right SHF was labeled at HH18, the fluorescence was limited to the caudal wall of the lengthening aortic sac. We then injected a combination of DiI and neutral red dye, and ablated the SHF in HH14 or 18 chick embryos. Embryos were allowed to develop until day 9, and harvested for assessment of outflow alignment. Of the embryos ablated at HH14, 76% demonstrated cardiac defects including overriding aorta and pulmonary atresia, while none of the sham-operated controls were affected. In addition, the more severely affected embryos demonstrated coronary artery anomalies. The embryos ablated at HH18 also manifested coronary artery anomalies but maintained normal outflow alignment. Therefore, the myocardium added to the outflow by the SHF at earlier stages is required for the elongation and appropriate alignment of the outflow tract. However, at later stages, the SHF contributes to the smooth muscle component of the outflow vessels above the pulmonary and aortic valves which is important for the development of the coronary artery stems. This work suggests a role for the SHF in a subset of congenital heart defects that have overriding aorta and coronary artery anomalies, such as tetralogy of Fallot and double outlet right ventricle.     

Secondary heart field contributes myocardium and smooth muscle to the arterial pole of the developing heart

The arterial pole of the heart is the region where the ventricular myocardium continues as the vascular smooth muscle tunics of the aorta and pulmonary trunk. It has been shown that the arterial pole myocardium derives from the secondary heart field and the smooth muscle tunic of the aorta and pulmonary trunk derives from neural crest. However, this neural crest-derived smooth muscle does not extend to the arterial pole myocardium leaving a region at the base of the aorta and pulmonary trunk that is invested by vascular smooth muscle of unknown origin. Using tissue marking and vascular smooth muscle markers, we show that the secondary heart field, in addition to providing myocardium to the cardiac outflow tract, also generates prospective smooth muscle that forms the proximal walls of the aorta and pulmonary trunk. As a result, there are two seams in the arterial pole: first, the myocardial junction with secondary heart field-derived smooth muscle; second, the secondary heart field-derived smooth muscle with the neural crest-derived smooth muscle. Both of these seams are points where aortic dissection frequently occurs in Marfan's and other syndromes.     

Pulmonary endoderm,second heart field and the morphogenesis of distal outflow tract in mouse embryonic heart

The second heart field (SHF), foregut endoderm and sonic hedgehog (SHH) signaling pathway are all reported to associate with normal morphogenesis and septation of outflow tract (OFT). However, the morphological relationships of the development of foregut endoderm and expression of SHH signaling pathway members with the development of surrounding SHF and OFT are seldom described. In this study, serial sections of mouse embryos from ED9 to ED13 (midgestation) were stained with a series of marker antibodies for specifically highlighting SHF (Isl‐1), endoderm (Foxa2), basement membrane (Laminin), myocardium (MHC) and smooth muscle (α‐SMA) respectively, or SHH receptors antibodies including patched1 (Ptc1), patched2 (Ptc2) and smoothened, to observe the spatiotemporal relationship between them and their contributions to OFT morphogenesis. Our results demonstrated that the development of an Isl‐1 positive field in the splanchnic mesoderm ventral to foregut, a subset of SHF, is closely coupled with pulmonary endoderm or tracheal groove, the Isl‐1 positive cells surrounding pulmonary endoderm are distributed in a special cone‐shaped pattern and take part in the formation of the lateral walls of the intrapericardial aorta and pulmonary trunk and the transient aortic‐pulmonary septum, and Ptc1 and Ptc2 are exclusively expressed in pulmonary endoderm during this Isl‐l positive field development, suggesting special roles played in inducing the Isl‐l positive field formation by pulmonary endoderm. It is indicated that pulmonary endoderm plays a role in the development and specification of SHF in midgestation, and that pulmonary endoderm‐associated Isl‐l positive field is involved in patterning the morphogenesis and septation of the intrapericardial arterial trunks.     

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 Hoxb1^Cre, the contribution of Hoxa1-enhIII-Cre and Hoxa3^Cre-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.     

Misexpression of noggin leads to septal defects in the outflow tract of the chick heart.

BMP-2 and BMP-4 are known to be involved in the early events which specify the cardiac lineage. Their later patterns of expression in the developing mouse and chick heart, in the myocardium overlying the atrioventricular canal (AV) and outflow tract (OFT) cushions, also suggest that they may play a role in valvoseptal development. In this study, we have used a recombinant retrovirus expressing noggin to inhibit the function of BMP-2/4 in the developing chick heart. This procedure resulted in abnormal development of the OFT and the ventricular septum. A spectrum of abnormalities was seen ranging from common arterial trunk to double outlet right ventricle. In hearts infected with noggin virus, where the neural crest cells have been labelled, the results show that BMP-2/4 function is required for the migration of neural crest cells into the developing OFT to form the aortopulmonary septum. Prior to septation, misexpression of noggin also leads to a decrease in the number of proliferating mesenchymal cells within the proximal cushions of the outflow tract. These results suggest that BMP-2/4 function may mediate several key events during cardiac development.     

Disheveled mediated planar cell polarity signaling is required in the second heart field lineage for outflow tract morphogenesis

Disheveled (Dvl) is a key regulator of both the canonical Wnt and the planar cell polarity (PCP) pathway. Previous genetic studies in mice indicated that outflow tract (OFT) formation requires Dvl1 and 2, but it was unclear which pathway was involved and whether Dvl1/2-mediated signaling was required in the second heart field (SHF) or the cardiac neural crest (CNC) lineage, both of which are critical for OFT development. In this study, we used Dvl1/2 null mice and a set of Dvl2 BAC transgenes that function in a pathway-specific fashion to demonstrate that Dvl1/2-mediated PCP signaling is essential for OFT formation. Lineage-specific gene-ablation further indicated that Dvl1/2 function is dispensable in the CNC, but required in the SHF for OFT lengthening to promote cardiac looping. Mutating the core PCP gene Vangl2 and non-canonical Wnt gene Wnt5a recapitulated the OFT morphogenesis defects observed in Dvl1/2 mutants. Consistent with genetic interaction studies suggesting that Wnt5a signals through the PCP pathway, Dvl1/2 and Wnt5a mutants display aberrant cell packing and defective actin polymerization and filopodia formation specifically in SHF cells in the caudal splanchnic mesoderm (SpM), where Wnt5a and Dvl2 are co-expressed specifically. Our results reveal a critical role of PCP signaling in the SHF during early OFT lengthening and cardiac looping and suggest that a Wnt5a→ Dvl PCP signaling cascade may regulate actin polymerization and protrusive cell behavior in the caudal SpM to promote SHF deployment, OFT lengthening and cardiac looping.     

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.     

A fate map of Tbx1 expressing cells reveals heterogeneity in the second cardiac field

Tbx1 is required for the expansion of second heart field (SHF) cardiac progenitors destined to the outflow tract of the heart. Loss of Tbx1 causes heart defects in humans and mice. We report a novel Tbx1(Cre) knock-in allele that we use to fate map Tbx1-expressing cells during development in conjunction with a reporter and 3D image reconstruction. Tbx1 descendants constitute a mesodermal cell population that surrounds the primitive pharynx and approaches the arterial pole of the heart from lateral and posterior, but not anterior directions. These cells populate most of the outflow tract with the exception of the anterior portion, thus identifying a population of the SHF of distinct origin. Both myocardial and underlying endocardial layers were labeled, suggesting a common origin of these cell types. Finally, we show that Tbx1(Cre)-positive and Tbx1(Cre)-negative cell descendants occupy discrete domains in the outflow tract throughout development.     

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.     

Pitx2 promotes development of splanchnic mesoderm-derived branchiomeric muscle

Recent experiments, showing that both cranial paraxial and splanchnic mesoderm contribute to branchiomeric muscle and cardiac outflow tract (OFT) myocardium, revealed unexpected complexity in development of these muscle groups. The Pitx2 homeobox gene functions in both cranial paraxial mesoderm, to regulate eye muscle, and in splanchnic mesoderm to regulate OFT development. Here, we investigated Pitx2 in branchiomeric muscle. Pitx2 was expressed in branchial arch core mesoderm and both Pitx2 null and Pitx2 hypomorphic embryos had defective branchiomeric muscle. Lineage tracing with a Pitx2cre allele indicated that Pitx2 mutant descendents moved into the first branchial arch. However, markers of both undifferentiated core mesoderm and specified branchiomeric muscle were absent. Moreover, lineage tracing with a Myf5cre allele indicated that branchiomeric muscle specification and differentiation were defective in Pitx2 mutants. Conditional inactivation in mice and manipulation of Pitx2 expression in chick mandible cultures revealed an autonomous function in expansion and survival of branchial arch mesoderm.     

Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart

Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell-, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0-Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells-and under the right conditions-differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.     

The anterior heart-forming field: voyage to the arterial pole of the heart

Studies of vertebrate heart development have identified key genes and signalling molecules involved in the formation of a myocardial tube from paired heart-forming fields in splanchnic mesoderm. The posterior region of the paired heart-forming fields subsequently contributes myocardial precursor cells to the inflow region or venous pole of the heart. Recently, a population of myocardial precursor cells in chick and mouse embryos has been identified in pharyngeal mesoderm anterior to the early heart tube. This anterior heart-forming field gives rise to myocardium of the outflow region or arterial pole of the heart. The amniote heart is therefore derived from two myocardial precursor cell populations, which appear to be regulated by distinct genetic programmes. Discovery of the anterior heart-forming field has important implications for the interpretation of cardiac defects in mouse mutants and for the study of human congenital heart disease.     

Role of myocardial hypoxia in the remodeling of the embryonic avian cardiac outflow tract

The embryonic cardiac outflow myocardium originates from a secondary heart-forming field to connect the developing ventricles with the aortic sac. The outflow tract (OFT) subsequently undergoes complex remodeling in the transition of the embryo to a dual circulation. In avians, elimination of OFT cardiomyocytes by apoptosis (stages 25-32) precedes coronary vasculogenesis and is necessary for the shortening of the OFT and the posterior rotation of the aorta. We hypothesized that regional myocardial hypoxia triggers OFT remodeling. We used immunohistochemical detection of the nitroimidazole EF5, administered by intravascular infusion in ovo, as an indicator of relative tissue oxygen concentrations. EF5 binding was increased in the OFT myocardium relative to other myocardium during these stages (25-32) of OFT remodeling. The intensity of EF5 binding paralleled the prevalence of apoptosis in the OFT myocardium, which are first detected at stage 25, maximal at stage 30, and diminished by stage 32. Evidence of coincident hypoxia-dependent responses included the expression of the vascular endothelial growth factor (VEGF) receptor 2 by the OFT myocardium, the predominant expression of VEGF122 (diffusible) isoform in the OFT, and the recruitment of QH1-positive pro-endothelial cells to the OFT and vasculogenesis. Exposure of embryos to hyperoxia (95% O(2)/5% CO(2)) during this developmental window reduced the prevalence of cardiomyocyte apoptosis and attenuated the shortening and rotation of the OFT, resulting in double-outlet right ventricle morphology, similar to that observed when apoptosis is directly inhibited. These results suggest that regional myocardial hypoxia triggers cardiomyocyte apoptosis and remodeling of the OFT in the transition to a dual circulation, and that VEGF autocrine/paracrine signaling may regulate these processes.     

Smad signaling in the neural crest regulates cardiac outflow tract remodeling through cell autonomous and non-cell autonomous effects

Neural crest cells (NCCs) are indispensable for the development of the cardiac outflow tract (OFT). Here, we show that mice lacking Smad4 in NCCs have persistent truncus arteriosus (PTA), severe OFT cushion hypoplasia, defective OFT elongation, and mispositioning of the OFT. Cardiac NCCs lacking Smad4 have increased apoptosis, apparently due to decreased Msx1/2 expression. This contributes to the reduction of NCCs in the OFT. Unexpectedly, mutants have MF20-expressing cardiomyocytes in the splanchnic mesoderm within the second heart field (SHF). This may result from abnormal differentiation or defective recruitment of differentiating SHF cells into OFT. Alterations in Bmp4, Sema3C, and PlexinA2 signals in the mutant OFT, SHF, and NCCs, disrupt the communications among different cell populations. Such disruptions can further affect the recruitment of NCCs into the OFT mesenchyme, causing severe OFT cushion hypoplasia and OFT septation failure. Furthermore, these NCCs have drastically reduced levels of Ids and MT1-MMP, affecting the positioning and remodeling of the OFT. Thus, Smad-signaling in cardiac NCCs has cell autonomous effects on their survival and non-cell autonomous effects on coordinating the movement of multiple cell lineages in the positioning and the remodeling of the OFT.