Sizzled-Tolloid Interactions Maintain Foregut Progenitors by Regulating Fibronectin-Dependent BMP Signaling

The liver, pancreas, and lungs are induced from endoderm progenitors by a series of dynamic growth factor signals from the mesoderm, but how the temporal-spatial activity of these signals is controlled is poorly understood. We have identified an extracellular regulatory loop required for robust bone morphogenetic protein (BMP) signaling in the Xenopus foregut. We show that BMP signaling is required to maintain foregut progenitors and induce expression of the secreted frizzled related protein Sizzled (Szl) and the extracellular metalloprotease Tolloid-like 1 (Tll1). Szl negatively regulates Tll activity to control deposition of a fibronectin (FN) matrix between the mesoderm and endoderm, which is required to maintain BMP signaling. Foregut-specific Szl depletion results in a loss of the FN matrix and failure to maintain robust pSmad1 levels, causing a loss of foregut gene expression and organ agenesis. These results have implications for BMP signaling in diverse contexts and the differentiation of foregut tissue from stem cells.     

Embryonic dorsal-ventral signaling: secreted frizzled-related proteins as inhibitors of tolloid proteinases

Here we report an unexpected role for the secreted Frizzled-related protein (sFRP) Sizzled/Ogon as an inhibitor of the extracellular proteolytic reaction that controls BMP signaling during Xenopus gastrulation. Microinjection experiments suggest that the Frizzled domain of Sizzled regulates the activity of Xolloid-related (Xlr), a metalloproteinase that degrades Chordin, through the following molecular pathway: Szl -| Xlr -| Chd -| BMP --> P-Smad1 --> Szl. In biochemical assays, the Xlr proteinase has similar affinities for its endogenous substrate Chordin and for its competitive inhibitor Sizzled, which is resistant to enzyme digestion. Extracellular levels of Sizzled and Chordin in the gastrula embryo and enzyme reaction constants were all in the 10(-8) M range, consistent with a physiological role in the regulation of dorsal-ventral patterning. Sizzled is also a natural inhibitor of BMP1, a Tolloid metalloproteinase of medical interest. Furthermore, mouse sFRP2 inhibited Xlr, suggesting a wider role for this molecular mechanism.     

Cardiac neural crest ablation alters Id2 gene expression in the developing heart

Id proteins are negative regulators of basic helix-loop-helix gene products and participate in many developmental processes. We have evaluated the expression of Id2 in the developing chick heart and found expression in the cardiac neural crest, secondary heart field, outflow tract, inflow tract, and anterior parasympathetic plexus. Cardiac neural crest ablation in the chick embryo, which causes structural defects of the cardiac outflow tract, results in a significant loss of Id2 expression in the outflow tract. Id2 is also expressed in Xenopus neural folds, branchial arches, cardiac outflow tract, inflow tract, and splanchnic mesoderm. Ablation of the premigratory neural crest in Xenopus embryos results in abnormal formation of the heart and a loss of Id2 expression in the heart and splanchnic mesoderm. This data suggests that the presence of neural crest is required for normal Id2 expression in both chick and Xenopus heart development and provides evidence that neural crest is involved in heart development in Xenopus embryos.     

The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field

The vertebrate heart arises from the fusion of bilateral regions of anterior mesoderm to form a linear heart tube. Recent studies in mouse and chick have demonstrated that a second cardiac progenitor population, known as the anterior or secondary heart field, is progressively added to the heart at the time of cardiac looping. While it is clear that this second field contributes to the myocardium, its precise boundaries, other lineages derived from this population, and its contributions to the postnatal heart remain unclear. In this study, we used regulatory elements from the mouse mef2c gene to direct the expression of Cre recombinase exclusively in the anterior heart field and its derivatives in transgenic mice. By crossing these mice, termed mef2c-AHF-Cre, to Cre-dependent lacZ reporter mice, we generated a fate map of the embryonic, fetal, and postnatal heart. These studies show that the endothelial and myocardial components of the outflow tract, right ventricle, and ventricular septum are derivatives of mef2c-AHF-Cre expressing cells within the anterior heart field and its derivatives. These studies also show that the atria, epicardium, coronary vessels, and the majority of outflow tract smooth muscle are not derived from this anterior heart field population. Furthermore, a transgene marker specific for the anterior heart field is expressed in the common ventricular chamber in mef2c mutant mice, suggesting that the cardiac looping defect in these mice is not due to a failure in anterior heart field addition to the heart. Finally, the Cre transgenic mice described here will be a crucial tool for conditional gene inactivation exclusively in the anterior heart field and its derivatives.     

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 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.     

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.     

Ventricle and outflow tract of the African lungfish Protopterus dolloi

We report a morphologic study of the heart ventricle and outflow tract of the African lungfish Protopterus dolloi. The ventricle is saccular and appears attached to the anterior pericardial wall by a thick tendon. An incomplete septum divides the ventricle into two chambers. Both the free ventricular wall and the incomplete ventricular septum are entirely trabeculated. Only a thin rim of myocardium separates the trabecular system from the subepicardial space. The outflow tract consists of proximal, middle, and distal portions, separated by two flexures, proximal and distal. The proximal outflow tract portion is endowed with a layer of compact, well-vascularized myocardium. This portion is homologous to the conus arteriosus observed in the heart of most vertebrates. The middle and distal outflow tract portions are arterial-like, thus being homologous to the bulbus arteriosus. However, the separation between the muscular and arterial portions of the outflow tract is not complete in the lungfish. A thin layer of myocardium covers the arterial tissue, and a thin layer of elastic tissue underlies the conus myocardium. Two unequal ridges composed of loose connective tissue, the spiral and bulbar folds, run the length of the outflow tract. They form an incomplete division of the outflow tract, but fuse at the distal end. The two folds are covered by endocardium and contain collagen, elastin, and fibroblast-like cells. They appear to be homologous to the dextro-dorsal and sinistro-ventral ridges observed during the development of the avian and mammalian heart. Two to three rows of vestigial arterial-like valves appear in the dorsal and ventral aspects of the conus. These valves are unlikely to have a functional role. The possible functional significance of the "gubernaculum cordis," the thick tendon extending between the anterior ventricular surface and the pericardium, is discussed.     

Mesoderm progenitor cells of common origin contribute to the head musculature and the cardiac outflow tract

During early embryogenesis, heart and skeletal muscle progenitor cells are thought to derive from distinct regions of the mesoderm (i.e. the lateral plate mesoderm and paraxial mesoderm, respectively). In the present study, we have employed both in vitro and in vivo experimental systems in the avian embryo to explore how mesoderm progenitors in the head differentiate into both heart and skeletal muscles. Using fate-mapping studies, gene expression analyses, and manipulation of signaling pathways in the chick embryo, we demonstrate that cells from the cranial paraxial mesoderm contribute to both myocardial and endocardial cell populations within the cardiac outflow tract. We further show that Bmp signaling affects the specification of mesoderm cells in the head: application of Bmp4, both in vitro and in vivo, induces cardiac differentiation in the cranial paraxial mesoderm and blocks the differentiation of skeletal muscle precursors in these cells. Our results demonstrate that cells within the cranial paraxial mesoderm play a vital role in cardiogenesis, as a new source of cardiac progenitors that populate the cardiac outflow tract in vivo. A deeper understanding of mesodermal lineage specification in the vertebrate head is expected to provide insights into the normal, as well as pathological, aspects of heart and craniofacial development.     

Dynamic control of head mesoderm patterning

The embryonic head mesoderm gives rise to cranial muscle and contributes to the skull and heart. Prior to differentiation, the tissue is regionalised by the means of molecular markers. We show that this pattern is established in three discrete phases, all depending on extrinsic cues. Assaying for direct and first-wave indirect responses, we found that the process is controlled by dynamic combinatorial as well as antagonistic action of retinoic acid (RA), Bmp and Fgf signalling. In phase 1, the initial anteroposterior (a-p) subdivision of the head mesoderm is laid down in response to falling RA levels and activation of Fgf signalling. In phase 2, Bmp and Fgf signalling reinforce the a-p boundary and refine anterior marker gene expression. In phase 3, spreading Fgf signalling drives the a-p expansion of MyoR and Tbx1 expression along the pharynx, with RA limiting the expansion of MyoR. This establishes the mature head mesoderm pattern with markers distinguishing between the prospective extra-ocular and jaw skeletal muscles, the branchiomeric muscles and the cells for the outflow tract of the heart.     

Sonic hedgehog maintains proliferation in secondary heart field progenitors and is required for normal arterial pole formation

The Sonic hedgehog (Shh)-null mouse was initially described as a phenotypic mimic of Tetralogy of Fallot with pulmonary atresia (Washington Smoak, I., Byrd, N.A., Abu-Issa, R., Goddeeris, M.M., Anderson, R., Morris, J., Yamamura, K., Klingensmith, J., and Meyers, E.N. 2005. Sonic hedgehog is required for cardiac outflow tract and neural crest cell development. Dev. Biol. 283, 357–372.); however, subsequent reports describe only a single outflow tract, leaving the phenotype and its developmental mechanism unclear. We hypothesized that the phenotype that occurs in response to Shh knockdown is pulmonary atresia and is directly related to the abnormal development of the secondary heart field. We found that Shh was expressed by the pharyngeal endoderm adjacent to the secondary heart field and that its receptor Ptc2 was expressed in a gradient in the secondary heart field, with the most robust expression in the caudal secondary heart field, closest to the Shh expression. In vitro culture of secondary heart field with the hedgehog inhibitor cyclopamine significantly reduced proliferation. In ovo, cyclopamine treatment before the secondary heart field adds to the outflow tract reduced proliferation only in the caudal secondary heart field, which coincided with the region of high Ptc2 expression. After outflow tract septation should occur, embryos treated with cyclopamine exhibited pulmonary atresia, pulmonary stenosis, and persistent truncus arteriosus. In hearts with pulmonary atresia, cardiac neural crest-derived cells, which form the outflow tract septum, migrated into the outflow tract and formed a septum. However, this septum divided the outflow tract into two unequal sized vessels and effectively closed off the pulmonary outlet. These experiments show that Shh is necessary for secondary heart field proliferation, which is required for normal pulmonary trunk formation, and that embryos with pulmonary atresia have an outflow tract septum.     

Cx43基因敲除鼠胚心脏近端流出道组织中Galpha13信号通路基因的差异表达

目的研究Cx43基因剔除(Cx43KO)小鼠胚胎心脏近端流出道组织中基因表达谱的改变,筛选可能导致Cx43KO小鼠流出道梗阻的相关基因。方法以胎龄(embryonic day,ED)14.5天的Cx43KO和野生型(Cx43WT)鼠胚心脏近端流出道部分为研究对象,分别提取总RNA,逆转录成cDNA;并在体外转录为cRNA,同时进行生物素标记及片段化;再与Affymetrix-4302.0基因芯片进行杂交。杂交信号经扫描后,应用相关生物信息软件分析基因表达情况。结果与Cx43WT组相比,Cx43KO组中表达上调2倍以上的基因共有287个,表达下调2倍以上的基因有199个。其中表达差异的基因参与转录调控、细胞周期等主要生理过程。进一步筛查表达差异1.5倍以上的基因发现,Galpha13信号通路上的多个基因在Cx43KO组有明显变化。结论利用基因芯片技术初步筛选出与Cx43KO鼠胚心脏近端流出道发育有关的多个基因,其中Galpha13信号通路上的相关基因可能与Cx43KO小鼠流出道梗阻的发生有关。     

DNA methylation abnormalities in congenital heart disease

Congenital heart defects represent the most common malformation at birth, occurring also in ∼50% of individuals with Down syndrome. Congenital heart defects are thought to have multifactorial etiology, but the main causes are largely unknown. We have explored the global methylation profile of fetal heart DNA in comparison to blood DNA from control subjects: an absolute correlation with the type of tissue was detected. Pathway analysis revealed a significant enrichment of differential methylation at genes related to muscle contraction and cardiomyopathies in the developing heart DNA. We have also searched for abnormal methylation profiles on developing heart-tissue DNA of syndromic and non-syndromic congenital heart defects. On average, 3 regions with aberrant methylation were detected per sample and 18 regions were found differentially methylated between groups. Several epimutations were detected in candidate genes involved in growth regulation, apoptosis and folate pathway. A likely pathogenic hypermethylation of several intragenic sites at the MSX1 gene, involved in outflow tract morphogenesis, was found in a fetus with isolated heart malformation. In addition, hypermethylation of the GATA4 gene was present in fetuses with Down syndrome with or without congenital heart defects, as well as in fetuses with isolated heart malformations. Expression deregulation of the abnormally methylated genes was detected. Our data indicate that epigenetic alterations of relevant genes are present in developing heart DNA in fetuses with both isolated and syndromic heart malformations. These epimutations likely contribute to the pathogenesis of the malformation by cis-acting effects on gene expression.     

Comparison of the four mouse fasciclin-containing genes expression patterns during valvuloseptal morphogenesis

All four mammalian fasciclin-containing genes are expressed in the adult valves and are localized in partially overlapping and reciprocal patterns during cardiovascular development. Spatiotemporal comparison of the fasciclin-containing secreted adhesion genes, TGFbeta induced clone H3 (betaigH3) and periostin, revealed that they are co-localized within the outflow tract endocardial cushions, but that betaigH3 expression is restricted to the septal cushions within the atrioventricular canal. Conversely, the fasciclin-containing transmembrane gene, stabilin-1, is predominately expressed in the endocardial layer overlaying the cushions and lining the developing heart. However, expression of the fasciclin-containing transmembrane gene, stabilin-2 is only present in the post-natal mature valve endothelial cells. These data illustrate for the first time that the primitive endocardial cushions dynamically express multiple fasciclin-containing adhesion molecules as they undergo the key steps of seeding, proliferation, differentiation, fusion, mesenchymal condensation and remodeling during mouse heart development.