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After the formation of the linear heart tube, it becomes divided into right and left components by the process of septation. Relatively late during this process, within the developing outflow tract, the initially mesenchymal outlet septum becomes muscularized as the result of myocardialization. Myocardialization is defined as the process in which existing cardiomyocytes migrate into flanking mesenchyme. Studies using genetically modified mice, as well as experimental approaches using in vitro models, demonstrate that Wnt and TGFβ signaling play an essential role in the regulation of myocardialization. They also show the significance of the interaction between cardiomyocytes, endocardial derived cells, neural crest cells, and the extracellular matrix. Interestingly, Wnt-mediated non-canonical planar cell polarity signaling was found to be a crucial regulator of myocardialization in the outlet septum and Wnt-mediated canonical β-catenin signaling is an essential regulator of the expansion of mesenchymal cells populating the outflow tract cushions.

This paper is dedicated to the memory of Dr. Adriana “Adri” Gittenberger-de Groot and in appreciation of her work in the field of developmental cardiovascular biology and the legacy that she has left behind. During her impressive career, Dr. Gittenberger-de Groot studied many aspects of heart development, including aspects of cardiac valve formation and disease and the role of the epicardium in the formation of the heart. In this contribution, we review some of the work on the role of epicardially-derived cells (EPDCs) in the development of the atrioventricular valves and their potential involvement in the pathogenesis of myxomatous valve disease (MVD). We provide an overview of critical events in the development of the atrioventricular junction, discuss the role of the epicardium in these events, and illustrate how interfering with molecular mechanisms that are involved in the epicardial-dependent formation of the atrioventricular junction leads to a number of abnormalities. These abnormalities include defects of the AV valves that resemble those observed in humans that suffer from MVD. The studies demonstrate the importance of the epicardium for the proper formation and maturation of the AV valves and show that the possibility of epicardial-associated developmental defects should be taken into consideration when determining the genetic origin and pathogenesis of MVD.

In this publication, dedicated to Professor Robert H. Anderson and his contributions to the field of cardiac development, anatomy, and congenital heart disease, we will review some of our earlier collaborative studies. The focus of this paper is on our work on the development of the atrioventricular mesenchymal complex, studies in which Professor Anderson has played a significant role. We will revisit a number of events relevant to atrial and atrioventricular septation and present new data on the development of the mesenchymal cap of the atrial septum, a component of the atrioventricular mesenchymal complex which, thus far, has received only moderate attention.

In August 2017, the Cardiovascular Developmental Biology Center (CDBC), together with the "Department of Regenerative Medicine and Cell Biology (RMCB) at the Medical University of South Carolina (MUSC), organized their 13th Annual CDBC Symposium. During this special event, which was organized in collaboration with The Anatomical Record, the unique and important contributions of Dr. Roger Markwald (known to all of us as Roger) to the field of cardiovascular research were celebrated. Fifteen leading investigators in the field presented their ideas and reported results of their studies to an audience that included many familiar faces from Roger's past and present. This group consisted of established investigators from around the world as well as young and upcoming scientists from local institutions. In their presentations, the platform speakers emphasized the significance of Roger's scientific contributions and advice to their professional development and career. In this Special Issue of The Anatomical Record, we assembled a collection of invited papers written by several attendees of the symposium. The issue also contains a number of articles written by colleagues who, for one reason or the other, were not able to attend the meeting, but expressed their desire to contribute to this special "festschrift" of The Anatomical Record in honor and recognition of Roger's amazing career. Anat Rec, 302:14-18, 2019. © 2018 Wiley Periodicals, Inc.

Primary cilia are small organelles projecting from the cell surface of many cell types. They play a crucial role in the regulation of various signaling pathway. In this study, we investigated the importance of cilia for heart development by conditionally deleting intraflagellar transport protein Ift88 using the col3.6-cre mouse. Analysis of col3.6;Ift88 offspring showed a wide spectrum of cardiovascular defects including double outlet right ventricle and atrioventricular septal defects. In addition, we found that in the majority of specimens the pulmonary veins did not properly connect to the developing left atrium. The abnormal connections found resemble those seen in patients with total anomalous pulmonary venous return. Analysis of mutant hearts at early stages of development revealed abnormal development of the dorsal mesocardium, a second heart field-derived structure at the venous pole intrinsically related to the development of the pulmonary veins. Data presented support a crucial role for primary cilia in outflow tract development and atrioventricular septation and their significance for the formation of the second heart field-derived tissues at the venous pole including the dorsal mesocardium. Furthermore, the results of this study indicate that proper formation of the dorsal mesocardium is critically important for the development of the pulmonary veins. Anat Rec, 302:136-145, 2019. © 2018 Wiley Periodicals, Inc.

Background Bicuspid aortic valve (BAV) disease is the most common congenital heart defect, affecting 0.5-1.2% of the population and causing significant morbidity and mortality. Only a few genes have been identified in pedigrees, and no single gene model explains BAV inheritance, thus supporting a complex genetic network of interacting genes. However, patients with rare syndromic diseases that stem from alterations in the structure and function of primary cilia ("ciliopathies") exhibit BAV as a frequent cardiovascular finding, suggesting primary cilia may factor broadly in disease etiology. Results Our data are the first to demonstrate that primary cilia are expressed on aortic valve mesenchymal cells during embryonic development and are lost as these cells differentiate into collagen-secreting fibroblastic-like cells. The function of primary cilia was tested by genetically ablating the critical ciliogenic gene Ift88. Loss of Ift88 resulted in abrogation of primary cilia and increased fibrogenic extracellular matrix (ECM) production. Consequentially, stratification of ECM boundaries normally present in the aortic valve were lost and a highly penetrant BAV phenotype was evident at birth. Conclusions Our data support cilia as a novel cellular mechanism for restraining ECM production during aortic valve development and broadly implicate these structures in the etiology of BAV disease in humans. Developmental Dynamics 246:625-634, 2017. © 2017 Wiley Periodicals, Inc.

International audience; Non-syndromic mitral valve prolapse (MVP) is the most common heart valve disease affecting 2.4% of the population. Recent studies have identified genetic defects in primary cilia as causative to MVP, although the mechanism of their action is currently unknown. Using a series of gene inactivation approaches, we define a paracrine mechanism by which endocardially-expressed Desert Hedgehog (DHH) activates primary cilia signaling on neighboring valve interstitial cells. High-resolution imaging and functional assays show that DHH de-represses smoothened at the primary cilia, resulting in kinase activation of RAC1 through the RAC1-GEF, TIAM1. Activation of this non-canonical hedgehog pathway stimulates α-smooth actin organization and ECM remodeling. Genetic or pharmacological perturbation of this pathway results in enlarged valves that progress to a myxomatous phenotype, similar to valves seen in MVP patients. These data identify a potential molecular origin for MVP as well as establish a paracrine DHH-primary cilium cross-talk mechanism that is likely applicable across developmental tissue types.

Mitral valve prolapse (MVP) affects 1 in 40 people and is the most common indication for mitral valve surgery. MVP can cause arrhythmias, heart failure, and sudden cardiac death, and to date, the causes of this disease are poorly understood. We now demonstrate that defects in primary cilia genes and their regulated pathways can cause MVP in familial and sporadic nonsyndromic MVP cases. Our expression studies and genetic ablation experiments confirmed a role for primary cilia in regulating ECM deposition during cardiac development. Loss of primary cilia during development resulted in progressive myxomatous degeneration and profound mitral valve pathology in the adult setting. Analysis of a large family with inherited, autosomal dominant nonsyndromic MVP identified a deleterious missense mutation in a cilia gene, DZIP1 A mouse model harboring this variant confirmed the pathogenicity of this mutation and revealed impaired ciliogenesis during development, which progressed to adult myxomatous valve disease and functional MVP. Relevance of primary cilia in common forms of MVP was tested using pathway enrichment in a large population of patients with MVP and controls from previously generated genome-wide association studies (GWAS), which confirmed the involvement of primary cilia genes in MVP. Together, our studies establish a developmental basis for MVP through altered cilia-dependent regulation of ECM and suggest that defects in primary cilia genes can be causative to disease phenotype in some patients with MVP.

The mesothelium is an epithelial structure derived from the embryonic mesoderm. It plays an important role in the development of a number of different organs, including the heart, lungs, and intestines. In this publication, we discuss aspects of the development of the mesothelium, where mesothelial structures can be found, and review molecular and cellular characteristics associated with the mesothelium. Furthermore, we discuss the involvement of the mesothelium in a number of disease conditions, in particular in the pathogenesis of mesotheliomas with an emphasis on malignant pleural mesothelioma (MPM)—a primary cancer developing in the pleural cavity.

BACKGROUND The dorsal mesenchymal protrusion (DMP) is a second heart field (SHF) derived tissue involved in cardiac septation. Molecular mechanisms controlling SHF/DMP development include the Bone Morphogenetic Protein and Wnt/β-catenin signaling pathways. Reduced expression of components in these pathways leads to inhibition of proliferation of the SHF/DMP precursor population and failure of the DMP to develop. While the Sonic Hedgehog (Shh) pathway has also been demonstrated to be critically important for SHF/DMP development and atrioventricular septation, its role in the regulation of SHF proliferation is contentious. RESULTS Tissue-specific deletion of the Shh receptor Smoothened from the SHF resulted in compromised DMP formation and atrioventricular septal defects (AVSDs). Immunohistochemical analysis at critical stages of DMP development showed significant proliferation defect as well as reduction in levels of the Wnt/β-catenin pathway-intermediates β-catenin, Lef1, and Axin2. To determine whether the defects seen in the conditional Smoothened knock-out mouse could be attributed to reduced Wnt/β-catenin signaling, LiCl, a pharmacological activator of this Wnt/β-catenin pathway, was administered. This resulted in restoration of proliferation and partial rescue of the AVSD phenotype. CONCLUSIONS The data presented suggest that the Wnt/β-catenin pathway interact with the Shh pathway in the regulation of SHF/DMP-precursor proliferation and, hence, the development of the DMP.

International audience; Mitral valve prolapse (MVP) is a common cardiac valve disease that affects nearly 1 in 40 individuals. It can manifest as mitral regurgitation and is the leading indication for mitral valve surgery. Despite a clear heritable component, the genetic aetiology leading to non-syndromic MVP has remained elusive. Four affected individuals from a large multigenerational family segregating non-syndromic MVP underwent capture sequencing of the linked interval on chromosome 11. We report a missense mutation in the DCHS1 gene, the human homologue of the Drosophila cell polarity gene dachsous (ds), that segregates with MVP in the family. Morpholino knockdown of the zebrafish homologue dachsous1b resulted in a cardiac atrioventricular canal defect that could be rescued by wild-type human DCHS1, but not by DCHS1 messenger RNA with the familial mutation. Further genetic studies identified two additional families in which a second deleterious DCHS1 mutation segregates with MVP. Both DCHS1 mutations reduce protein stability as demonstrated in zebrafish, cultured cells and, notably, in mitral valve interstitial cells (MVICs) obtained during mitral valve repair surgery of a proband. Dchs1(+/-) mice had prolapse of thickened mitral leaflets, which could be traced back to developmental errors in valve morphogenesis. DCHS1 deficiency in MVP patient MVICs, as well as in Dchs1(+/-) mouse MVICs, result in altered migration and cellular patterning, supporting these processes as aetiological underpinnings for the disease. Understanding the role of DCHS1 in mitral valve development and MVP pathogenesis holds potential for therapeutic insights for this very common disease.

Background: Our previous study on mouse mutants with the ureteric bud (UB) epithelium-specific Dicer deletion (Dicer UB mutants) demonstrated the significance of UB epithelium-derived miRNAs in UB development. Results: Our whole-genome transcriptional profiling showed that the Dicer mutant UB epithelium abnormally retained transcriptional features of the early UB epithelium and failed to express many genes associated with collecting duct differentiation. Furthermore, we identified a temporal expression pattern of early UB genes during UB epithelium development in which gene expression was detected at early developmental stages and became undetectable by embryonic day 14.5. In contrast, expression of early UB genes persisted at later stages in the Dicer mutant UB epithelium and increased at early stages. Our bioinformatic analysis of the abnormally persistently expressed early genes in the Dicer mutant UB epithelium showed significant enrichment of the let-7 family miRNA targets. We further identified a temporal expression pattern of let-7 miRNAs in the UB epithelium that is anti-parallel to that of some early UB genes during kidney development. Conclusions: We propose a model in which the let-7 family miRNAs silence the expression of a subset of early genes in the UB epithelium at later developmental stages to promote collecting duct differentiation. Developmental Dynamics 244:444–456, 2015. © 2014 Wiley Periodicals, Inc.

Congenital heart malformations are the most common type of defects found at birth. About 1% of infants are born with one or more heart defect on a yearly basis. Congenital Heart Disease (CHD) causes more deaths in the first year of life than any other congenital abnormality, and each year, nearly twice as many children die in the United States from CHD as from all forms of childhood cancers combined. Atrioventricular septal defects (AVSD) are congenital heart malformations affecting approximately 1 in 2000 live births. Babies born with an AVSD often require surgical intervention shortly after birth. However, even after successful surgery, these individuals typically have to deal with lifelong complications with the most common being a leaky mitral valve. In recent years the understanding of the molecular etiology and morphological mechanisms associated with the pathogenesis of AVSDs has significantly changed. Specifically, these studies have linked abnormal development of the Dorsal Mesenchymal Protrusion (DMP), a Second Heart Field-derived structure, to the development of this congenital defect. In this review we will be discuss some of the latest insights into the role of the DMP in the normal formation of the atrioventricular septal complex and in the pathogenesis of AVSDs.

Partitioning of the four-chambered heart requires the proper formation, interaction and fusion of several mesenchymal tissues derived from different precursor populations that together form the atrioventricular mesenchymal complex. This includes the major endocardial cushions and the mesenchymal cap of the septum primum, which are of endocardial origin, and the dorsal mesenchymal protrusion (DMP), which is derived from the Second Heart Field. Failure of these structures to develop and/or fully mature results in atrial septal defects (ASDs) and atrioventricular septal defects (AVSD). AVSDs are congenital malformations in which the atria are permitted to communicate due to defective septation between the inferior margin of the septum primum and the atrial surface of the common atrioventricular valve. The clinical presentation of AVSDs is variable and depends on both the size and/or type of defect; less severe defects may be asymptomatic while the most severe defect, if untreated, results in infantile heart failure. For many years, maldevelopment of the endocardial cushions was thought to be the sole etiology of AVSDs. More recent work, however, has demonstrated that perturbation of DMP development also results in AVSD. Here, we discuss in detail the formation of the DMP, its contribution to cardiac septation and describe the morphological features as well as potential etiologies of ASDs and AVSDs.

The importance of the epicardium for myocardial and valvuloseptal development has been well established; perturbation of epicardial development results in cardiac abnormalities, including thinning of the ventricular myocardial wall and malformations of the atrioventricular valvuloseptal complex. To determine the spatiotemporal contribution of epicardially derived cells to the developing fibroblast population in the heart, we have used a mWt1/IRES/GFP-Cre mouse to trace the fate of EPDCs from embryonic day (ED)10 until birth. EPDCs begin to populate the compact ventricular myocardium around ED12. The migration of epicardially derived fibroblasts toward the interface between compact and trabecular myocardium is completed around ED14. Remarkably, epicardially derived fibroblasts do not migrate into the trabecular myocardium until after ED17. Migration of EPDCs into the atrioventricular cushion mesenchyme commences around ED12. As development progresses, the number of EPDCs increases significantly, specifically in the leaflets which derive from the lateral atrioventricular cushions. In these developing leaflets the epicardially derived fibroblasts eventually largely replace the endocardially derived cells. Importantly, the contribution of EPDCs to the leaflets derived from the major AV cushions is very limited. The differential contribution of EPDCs to the various leaflets of the atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the pathogenesis of abnormalities that preferentially affect individual components of this region of the heart. The notion that there is a significant difference in the contribution of epicardially and endocardially derived cells to the individual leaflets of the atrioventricular valves has also important pragmatic consequences for the use of endocardial and epicardial cre-mouse models in studies of heart development. (C) 2012 Elsevier Inc. All rights reserved

In fetal valve maturation the mechanisms by which the relatively homogeneous proteoglycan-rich extracellular matrix (ECM) of endocardial cushions is replaced by a specialized and stratified ECM found in mature valves are not understood. Therefore, we reasoned that uncovering proteases critical for ‘remodeling’ the proteoglycan rich (extracellular matrix) ECM may elucidate novel mechanisms of valve development. We have determined that mice deficient in ADAMTS5, (A Disintegrin-like And Metalloprotease domain with ThromboSpondin-type 1 motifs) which we demonstrated is expressed predominantly by valvular endocardium during cardiac valve maturation, exhibited enlarged valves. ADAMTS5 deficient valves displayed a reduction in cleavage of its substrate versican, a critical cardiac proteoglycan. In vivo reduction of versican, in Adamts5−/− mice, achieved through Vcan heterozygosity, substantially rescued the valve anomalies. An increase in BMP2 immunolocalization, Sox9 expression and mesenchymal cell proliferation were observed in Adamts5−/− valve mesenchyme and correlated with expansion of the spongiosa (proteoglycan-rich) region in Adamts5−/− valve cusps. Furthermore, these data suggest that ECM remodeling via ADAMTS5 is required for endocardial to mesenchymal signaling in late fetal valve development. Although adult Adamts5−/− mice are viable they do not recover from developmental valve anomalies and have myxomatous cardiac valves with 100% penetrance. Since the accumulation of proteoglycans is a hallmark of myxomatous valve disease, based on these data we hypothesize that a lack of versican cleavage during fetal valve development may be a potential etiology of adult myxomatous valve disease.

SummaryDNA methylation is a major epigenetic mechanism for gene silencing. Whereas methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here, we show that either knockout or catalytic inactivation of the DNA repair enzyme thymine DNA glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific developmentally and hormonally regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.PaperClip

Aims Human congenital heart disease linked to mutations in the homeobox transcription factor, NKX2-5, is characterized by cardiac anomalies, including atrial and ventricular septal defects as well as conduction and occasional defects in contractility. In the mouse, homozygous germline deletion of Nkx2-5 gene results in death around E10.5. It is, however, not established whether Nkx2-5 is necessary for cardiac development beyond this embryonic stage. Because human NKX2-5 mutations are related to septum secundum type atrial septal defects (ASD), we hypothesized that Nkx2-5 deficiency during the processes of septum secundum formation may cause cardiac anomalies; thus, we analysed mice with tamoxifen-inducible Nkx2-5 ablation beginning at E12.5 when the septum secundum starts to develop. Methods and results Using tamoxifen-inducible Nkx2-5 gene-targeted mice, this study demonstrates that Nkx2-5 ablation beginning at E12.5 results in embryonic death by E17.5. Analysis of mutant embryos at E16.5 shows arrhythmias, contraction defects, and cardiac malformations, including ASD. Quantitative measurements using serial section histology and three-dimensional reconstruction demonstrate growth retardation of the septum secundum and enlarged foramen ovale in Nkx2-5-ablated embryos. Functional cardiac defects may be attributed to abnormal expression of transcripts critical for conduction and contraction, including cardiac voltage-gated Na+ channel pore-forming α-subunit (Nav1.5-α), gap junction protein connexin40, cardiac myosin light chain kinase, and sarcolipin within 4 days after tamoxifen injection. Conclusion Nkx2-5 is necessary for survival after the mid-embryonic stage for cardiac function and formation by regulating the expression of its downstream target genes.

Myxoid degeneration of the cardiac valves is a common feature in a heterogeneous group of disorders that includes Marfan syndrome and isolated valvular diseases. Mitral valve prolapse is the most common outcome of these and remains one of the most common indications for valvular surgery. While the etiology of the disease is unknown, recent genetic studies have demonstrated that an X-linked form of familial cardiac valvular dystrophy can be attributed to mutations in the Filamin-A gene. Since these inheritable mutations are present from conception, we hypothesize that filamin-A mutations present at the time of valve morphogenesis lead to dysfunction that progresses postnatally to clinically relevant disease. Therefore, by carefully evaluating genetic factors (such as filamin-A) that play a substantial role in MVP, we can elucidate relevant developmental pathways that contribute to its pathogenesis. In order to understand how developmental expression of a mutant protein can lead to valve disease, the spatio-temporal distribution of filamin-A during cardiac morphogenesis must first be characterized. Although previously thought of as a ubiquitously expressed gene, we demonstrate that filamin-A is robustly expressed in non-myocyte cells throughout cardiac morphogenesis including epicardial and endocardial cells, and mesenchymal cells derived by EMT from these two epithelia, as well as mesenchyme of neural crest origin. In postnatal hearts, expression of filamin-A is significantly decreased in the atrioventricular and outflow tract valve leaflets and their suspensory apparatus. Characterization of the temporal and spatial expression pattern of filamin-A during cardiac morphogenesis is a crucial first step in our understanding of how mutations in filamin-A result in clinically relevant valve disease.

Here, we demonstrate that ADAMTS9, a highly conserved versican-degrading protease, is required for correct cardiovascular development and adult homeostasis. Analysis of Adamts9+/LacZ heterozygous adult mice revealed anomalies in the aortic wall, valvulosinus and valve leaflets. Abnormal myocardial projections and `spongy' myocardium consistent with non-compaction of the left ventricle were also found in Adamts9+/LacZ mice. During development, Adamts9 was expressed in derivatives of the Secondary Heart Field, vascular smooth muscle cells (VSMC) in the arterial wall, mesenchymal cells of the valves, and non-myocardial cells of the ventricular myocardium but expression also continued in the adult heart and ascending aorta. Thus, the adult cardiovascular anomalies found in Adamts9+/LacZ hearts could result from subtle developmental alterations in extracellular matrix remodeling or defects in adult homeostasis. The valvular and aortic anomalies of Adamts9+/LacZ hearts were associated with accumulation of versican and a decrease in cleaved versican relative to WT littermates. These data suggest a potentially important role for ADAMTS9 cleavage of versican, or other, as yet undefined substrates in development and allostasis of cardiovascular extracellular matrix. In addition these studies identify ADAMTS9 as a potential candidate gene for congenital cardiac anomalies. Mouse models of ADAMTS9 deficiency may be useful to study myxomatous valve degeneration.

The development of the embryonic heart is dependent upon the generation and incorporation of different mesenchymal subpopulations that derive from intra- and extra-cardiac sources, including the endocardium, epicardium, neural crest, and second heart field. Each of these populations plays a crucial role in cardiovascular development, in particular in the formation of the valvuloseptal apparatus. Notwithstanding shared mechanisms by which these cells are generated, their fate and function differ profoundly by their originating source. While most of our early insights into the origin and fate of the cardiac mesenchyme has come from experimental studies in avian model systems, recent advances in transgenic mouse technology has enhanced our ability to study these cell populations in the mammalian heart. In this article, we will review the current understanding of the role of cardiac mesenchyme in cardiac morphogenesis and discuss several new paradigms based on recent studies in the mouse.

Aortic aneurysm and dissection is a common phenotype, accounting for 1% to 2% of all deaths in industrialized countries and ≈50 000 deaths annually in the United States.1 In contrast to abdominal aortic aneurysm, thoracic aortic aneurysm, particularly in the ascending segment, commonly occurs in young individuals in the absence of identifiable environmental risk factors. Marfan syndrome (MFS) is the most common syndromic presentation of ascending aortic aneurysm, but other syndromes such as vascular Ehlers-Danlos syndrome and Loeys-Dietz syndrome (LDS) also have ascending aortic aneurysms and the associated cardiovascular risk of aortic dissection and rupture. Familial segregation of the risk for ascending aortic aneurysm can also occur in the absence of associated systemic findings of a connective tissue abnormality in patients with familial thoracic aortic aneurysm and dissection (FTAAD) or bicuspid aortic valve with ascending aortic aneurysm (BAV/AscAA). The knowledge gained through basic and clinical research focused on MFS has improved and will continue to improve the care of patients with these related conditions. Recent paradigm-shifting discoveries about the molecular pathogenesis of MFS have highlighted the need for a focused research agenda to solidify the gains of the past 30 years and set the stage for future advances in MFS and related conditions. In April 2007, the National Heart, Lung, and Blood Institute (NHLBI) and the National Marfan Foundation convened a working group on research in MFS and related disorders to foster a multidisciplinary discussion. The working group, which included experts in cardiovascular disease, developmental biology, genetics and genomics, and proteomics, was charged with identifying opportunities and barriers to advancing the research agenda and developing recommendations to the NHLBI in the context of the Institute’s strategic plan (http://apps.nhlbi.nih.gov/strategicplan/). MFS is a systemic disorder of connective tissue caused by heterozygous mutations in the gene ( FBN1 ) that encodes the extracellular matrix …

The development of the venous pole of the heart is undoubtedly one of the most complex remodeling events during the formation of the 4-chambered heart. It involves the creation of two separate atrial chambers, the development of the atrial/atrioventricular septal complex, the processes that lead to the incorporation of the pulmonary venous return to the left atrium, and the incorporation of the caval veins and coronary sinus into the right side of the heart. In many of these processes, the dorsal mesenchymal protrusion, a derivative of the second heart field, plays a crucial role.

In recent years, insights into the role of the epicardium in cardiac development have significantly changed. An important contribution to this increasing knowledge comes from the availability of mouse models that facilitate the study of the fate of the epicardial cell lineage and that allow epicardial-specific manipulation of expression of genes involved in regulation of epicardial cell behavior. In this contribution we will discuss our growing understanding of the role of the epicardium and epicardially derived cells in the formation of the atrioventricular valve leaflets. We will illustrate how epicardially derived cells specifically contribute to the development of the leaflets that derive from the lateral atrioventricular cushions, and we will discuss the role of Bmp signaling, through the Bmp receptor BmpR1A/Alk3, in the regulation of the preferentially migration of EPDCs into the parietal AV valve leaflets.

The development of a properly functioning 4-chambered heart relies on the correct formation of the septal structures that separate the atrial and ventricular chambers. Perturbation of this septation process results in a spectrum of cardiac malformations involving the atrial and ventricular septal structures. Atrioventricular septal defects (AVSDs) form a class of congenital heart defects that are characterized by the presence of a primary atrial septal defect, a common atrioventricular junction, and frequently also a ventricular septal defect. While AVSD were historically considered to result from failure of the endocardial atrioventricular cushions to properly develop and fuse, more recent studies have determined that inhibition of the development of the dorsal mesenchymal protrusion (DMP), a derivative of the second heart field, can lead to AVSDs as well. In this chapter, we review what is currently known about the molecular mechanisms and pathways that are involved in DMP development and the pathogenesis of AVSD.

To expand our insight into cardiac development, a comparative DNA microarray analysis was performed using tissues from the atrioventricular junction (AVJ) and ventricular chambers of mouse hearts at embryonic day (ED) 10.5–11.0. This comparison revealed differential expression of approximately 200 genes, including cartilage link protein 1 (Crtl1). Crtl1 stabilizes the interaction between hyaluronan (HA) and versican, two extracellular matrix components essential for cardiac development. Immunohistochemical studies showed that, initially, Crtl1, versican, and HA are co-expressed in the endocardial lining of the heart, and in the endocardially derived mesenchyme of the AVJ and outflow tract (OFT). At later stages, this co-expression becomes restricted to discrete populations of endocardially derived mesenchyme. Histological analysis of the Crtl1-deficient mouse revealed a spectrum of cardiac malformations, including AV septal and myocardial defects, while expression studies showed a significant reduction in versican levels. Subsequent analysis of the hdf mouse, which carries an insertional mutation in the versican gene (CSPG2), demonstrated that haploinsufficient versican mice display septal defects resembling those seen in Crtl1−/− embryos, suggesting that reduced versican expression may contribute to a subset of the cardiac abnormalities observed in the Crtl1−/− mouse. Combined, these findings establish an important role for Crtl1 in heart development.

As part of a large-scale noninvasive fetal ultrasound screen to recover ethylnitrosourea (ENU)-induced mutations causing congenital heart defects in mice, we established a high-throughput ultrasound scanning strategy for interrogating fetal mice in utero utilizing three orthogonal imaging planes defined by the fetus’ vertebral column and body axes, structures readily seen by ultrasound. This contrasts with the difficulty of acquiring clinical ultrasound imaging planes which are defined by the fetal heart. By use of the three orthogonal imaging planes for two-dimensional (2D) imaging together with color flow, spectral Doppler, and M-mode imaging, all of the major elements of the heart can be evaluated. In this manner, 10,091 ENU-mutagenized mouse fetuses were ultrasound scanned between embryonic days 12.5 and 19.5, with 324 fetuses found to die prenatally and 425 exhibiting cardiovascular defects. Further analysis by necropsy and histology showed heart defects that included conotruncal anomalies, obstructive lesions, and shunt lesions as well as other complex heart diseases. Ultrasound imaging also identified craniofacial/head defects and body wall closure defects, which necropsy revealed as encephalocele, holoprosencephaly, omphalocele, or gastroschisis. Genome scanning mapped one ENU-induced mutation associated with persistence truncus arteriosus and holoprosencephaly to mouse chromosome 2, while another mutation associated with cardiac defects and omphalocele was mapped to mouse chromosome 17. These studies show the efficacy of this novel ultrasound scanning strategy for noninvasive ultrasound phenotyping to facilitate the recovery of ENU-induced mutations causing congenital heart defects and other extracardiac anomalies.

In humans, genetic disorders affecting post-squalene cholesterol biosynthesis result in a variety of dysmorphology syndromes. One key feature of all of these is the presence of mental retardation and another is the lack of a robust genotype-phenotype correlation. Knockout mice defective in the 3beta hydroxysterol Delta7 reductase (Dhcr7), a model for the most common of such disorders in humans, the Smith-Lemli-Opitz syndrome, all die within 24 h of birth. The cause of this postnatal mortality in these mice has not been fully established. In the present study, we tested the hypothesis that CNS dysfunction was a major cause of this lethality and investigated whether transgenic expression of normal human DHCR7 in neuronal tissues could rescue this neonatal lethality. Transgenic mice, expressing DHCR7 driven by murine nestin promoter, were bred onto Dhcr7 knock-out (Dhcr7(-1-)) background and resulted in a partial rescue of neonatal lethality in 11 of 91 (12%) of transgene-positive Dhcr7(-1-) pups. Despite biochemical analyses that showed continued profound cholesterol deficiency in brain, rescued animals survived between 3 and 17 days. Thus, one important conclusion to be drawn is that defects in CNS in Dhcr7 knockout mice may contribute to the early lethality. Another conclusion is that even small and subtle changes in the brain sterol metabolism were sufficient to enable rescue. These data also provide important clues as to the cause of the variable expressivity seen in SLOS.

Signaling by means of vascular endothelial cell growth factor (VEGF) and its receptors (VEGFRs) is required for cardiovascular development. To examine how VEGF/VEGFR receptor signaling affects early endocardial cell behavior, embryonic quail hearts were subjected to elevated VEGF165 levels (five- to nine-somite stage). Primitive embryonic hearts microinjected with recombinant human (rh)VEGF165 exhibit several distinct malformations compared with hearts in untreated embryos: the endocardial tube is malformed with tortuous cords and folds surrounded by a diminished cardiac jelly space, and the lumens of affected hearts are conspicuously reduced. Furthermore, the embryonic heart fails to loop properly. Inhibition of bending is accompanied by an apparent failure of the dorsal mesocardium to atrophy—an event thought to be necessary for heart bending. Instead of atrophy, VEGF-treated mesocardia exhibit a marked increased in the number of resident endothelial cells. Collectively, the data suggest that the abnormally robust mesocardia in VEGF-treated hearts impede the mechanical deformation required for normal heart bending. We conclude that the excessive VEGF signaling culminates in a physical or biomechanical mechanism that acts over a wide, tissue-level, length scale to cause a severe developmental defect—failure of heart bending. Developmental Dynamics 235:10–18, 2006. © 2005 Wiley-Liss, Inc.

The 10th annual Weinstein Conference was held May 13–16, 2004, at Leeuwenhorst Conference Center in the Netherlands outside of Leiden. The Weinstein meeting, founded by Dr. Constance Weinstein and subsequently named in her honor by the attendees, is one of the leading annual gatherings of researchers studying heart development. Basic research scientists and research clinicians were brought together from around the world to share their latest data and concepts of heart development. This year the meeting was sponsored by the Leiden University Medical Center in collaboration with the Medical University of South Carolina in Charleston and was attended by approximately 270 participants. The meeting began Friday evening with a welcoming reception followed by the keynote lecture by Margaret E. Buckingham of the Pasteur Institute. Dr. Buckingham’s lecture entitled, “Programming Cells to Form Muscle: The Cellular Contribution to Cardiogenesis,” reviewed her recently published work on lineage analysis of the anterior heart field in the mouse by a retrospective clonal analysis approach that takes advantage of an infrequent and spontaneous mitotic recombination of an inactivated nlaacZ reporter into an active nlacZ reporter in mouse (Meilhac et al., 2004). The analysis demonstrated that two lineages arise in the heart fields from a common progenitor. These two lineages then contribute unequally to specific regions of the developing heart. The left ventricle and the outflow tract seem to derive from a single lineage, whereas other regions derive from both. During questions and discussion, Roger Markwald proposed that the two lineages are derived at earlier stages from the primitive lateral heart fields and then are sorted into the separate lineages. The observation that some chambers derive from more than one lineage is consistent with earlier concepts that suggested the primitive heart forms initially in a segmental manner, but the mature chambers of the heart result from cellular interactions and remodeling of these primitive segments (Mjaatvedt et al., 1999). The cellular and molecular interactions leading to this specification of precardiac lineages will no doubt be a hot topic of future research in this area. SESSIONS

The cGATA-6 gene is flanked by an enhancer that selectively marks the atrioventricular conduction system (AVCS) in transgenic mice. This enhancer reads anterior/posterior and medial/lateral positional information very early in the cardiogenic program and remains active in progressively more restricted regions of primary myocardium leading up to the emergence of a histologically distinct AVCS. We undertook to parse this enhancer to resolve how the respective AVCS-specific transcription program is regulated at the molecular level. We determined that this AVCS enhancer includes a 102 bp module that is sufficient to restrict expression to primary nonchamber myocardium. This offers a novel tool to analyze the early molecular delineation of primary and chamber myocardium, which subsequently give rise to components of the central and peripheral conduction system, respectively. Furthermore, we show that this 102 bp module in turn contains a nested 47 bp core module that has the potential to direct expression specifically to the AVCS domain of primary myocardium, albeit with low efficiency. Accordingly, we show that a GATA site and a GC-rich site in the 102 bp region bolster the activity of the nested 47 bp AVCS core region even within the context of the parental 1,478 bp enhancer. These are the first functional elements to be reported for a cardiac conduction system-specific control region.

Periostin, a member of the fasciclin gene family, acts as a cell adhesion molecule through binding to cell surface integrins. Periostin expression has previously been shown to increase substantially following transforming growth factor beta (TGF-beta) and bone morphogenetic protein stimulation. As these molecules are indispensable for cardiac development, we sought to clone the chicken ortholog of periostin and evaluate its spatiotemporal expression pattern during heart morphogenesis. We show by Northern analysis, whole mount and section in situ hybridization experiments that periostin is predominantly expressed in the developing endothelium of the ventricular trabeculae as well as in the endothelium and mesenchyme of the outflow tract and atrioventricular endocardial cushions. Cardiac expression continues into fetal development where periostin is seen predominantly in the valve leaflets and supporting chordae tendinae.

After its initial formation the epicardium forms the outermost cell layer of the heart. As a result of an epithelial-to-mesenchymal transformation (EMT) individual cells delaminate from this primitive epicardial epithelium and migrate into the subepicardial space (Perez-Pomares et al., Dev Dyn 1997; 210:96-105; Histochem J 1998a;30:627-634). Several studies have demonstrated that these epicardially derived cells (EPDCs) subsequently invade myocardial and valvuloseptal tissues (Mikawa and Fischman, Proc Natl Acad Sci USA 1992;89:9504-9508; Mikawa and Gourdie, Dev Biol 1996;174:221-232; Dettman et al., Dev Biol 1998;193:169-181; Gittenberger de Groot et al., Circ Res 1998;82:1043-1052; Manner, Anat Rec 1999;255:212-226; Perez-Pomares et al., Dev. Biol. 2002b;247:307-326). A subset of EPDCs continue to differentiate in a variety of different cell types (including coronary endothelium, coronary smooth muscle cells (CoSMCs), interstitial fibroblasts, and atrioventricular cushion mesenchymal cells), whereas other EPDCs remain in a more or less undifferentiated state. Based on its specific characteristics, we consider the EPDC as the ultimate 'cardiac stem cell'. In this review we briefly summarize what is known about events that relate to EPDC development and differentiation while at the same time identifying some of the directions where EPDC-related research might lead us in the near future.

After the development of the linear heart tube, additional myocardium is formed leading to the muscular mantle around the caval and pulmonary veins and the muscular septa in the embryonic heart. Here, we report the results of our in vivo and in vitro studies of this late myocardium-generating process in the mouse. By using an immunohistochemical approach, we determined that myocardium formation starts around embryonic day 12 in the dorsal mesocardium. In subsequent stages of development, the process extends downstream into the intracardiac mesenchymal tissues of the atrioventricular canal and outflow tract and upstream into the extracardiac mediastinal mesenchyme embedding the pulmonary and caval veins. Given the spatiotemporal pattern of myocardium formation, we applied a three-dimensional in vitro explant culture assay to investigate the myocardium-generating potential of the different cardiac compartments. We determined that this potential is stage- and mesenchyme-dependent. This latter finding suggests an important role for mesenchyme in myocardium formation after the development of the linear heart tube.

A series of recent studies strongly suggests that the myocardium of the cardiac distal outflow tract (d-OFT) does not derive from the original precardiac mesoderm but, instead, differentiates from a so-called anterior heart field. Similar findings were also reported for the endocardium of the d-OFT. However, very little information is available on the origin of the epicardium of the OFT. To address this issue, we have performed a study in which we have combined experimental in vivo and in vitro techniques (construction of proepicardial chimeras, proepicardial ablation, OFT insertion of eggshell membrane pieces, and culture on collagen gels) with molecular characterization techniques to determine this origin and define the properties of d-OFT epicardium compared with proepicardially derived epicardium. Our results demonstrate that the coelomic/pericardial epithelium in the vicinity of the aortic sac (and not the proepicardium) is the origin of d-OFT epicardium. This "pericardially" derived epicardium and the proepicardially derived epicardial tissues differ in their morphologic appearance, gene-expression profile, and in their ability to undergo epithelial-to-mesenchymal transformation. We conclude that the heterogeneity in the epicardial cell population of the OFT could be a factor in the complex developmental remodeling events at the arterial pole of the heart.

The dorsal mesenchymal protrusion (DMP) is a second heart field (SHF) derived tissue involved in cardiac septation. Molecular mechanisms controlling SHF/DMP development include the Bone Morphogenetic Protein and Wnt/β-catenin signaling pathways. Reduced expression of components in these pathways leads to inhibition of proliferation of the SHF/DMP precursor population and failure of the DMP to develop. While the Sonic Hedgehog (Shh) pathway has also been demonstrated to be critically important for SHF/DMP development and atrioventricular septation, its role in the regulation of SHF proliferation is contentious.Tissue-specific deletion of the Shh receptor Smoothened from the SHF resulted in compromised DMP formation and atrioventricular septal defects (AVSDs). Immunohistochemical analysis at critical stages of DMP development showed significant proliferation defect as well as reduction in levels of the Wnt/β-catenin pathway-intermediates β-catenin, Lef1, and Axin2. To determine whether the defects seen in the conditional Smoothened knock-out mouse could be attributed to reduced Wnt/β-catenin signaling, LiCl, a pharmacological activator of this Wnt/β-catenin pathway, was administered. This resulted in restoration of proliferation and partial rescue of the AVSD phenotype.The data presented suggest that the Wnt/β-catenin pathway interact with the Shh pathway in the regulation of SHF/DMP-precursor proliferation and, hence, the development of the DMP.

Epicardially derived cells (EPDCs) delaminate from the primitive epicardium through an epithelial-to-mesenchymal transformation (EMT). After this transformation, a subpopulation of cells progressively invades myocardial and valvuloseptal tissues. The first aim of the study was to determine the tissue-specific distribution of two molecules that are thought to play a crucial function in the interaction between EPDCs and other cardiac tissues, namely the Wilms' Tumor transcription factor (WT1) and retinaldehyde-dehydrogenase2 (RALDH2). This study was performed in normal avian and in quail-to-chick chimeric embryos. It was found that EPDCs that maintain the expression of WT1 and RALDH2 initially populate the subepicardial space and subsequently invade the ventricular myocardium. As EPDCs differentiate into the smooth muscle and endothelial cell lineage of the coronary vessels, the expression of WT1 and RALDH2 becomes downregulated. This process is accompanied by the upregulation of lineage-specific markers. We also observed EPDCs that continued to express WT1 (but very little RALDH2) which did not contribute to the formation of the coronary system. A subset of these cells eventually migrates into the atrioventricular (AV) cushions, at which point they no longer express WT1. The WT1/RALDH2-negative EPDCs in the AV cushions do, however, express the smooth muscle cell marker caldesmon. The second aim of this study was to determine the impact of abnormal epicardial growth on cardiac development. Experimental delay of epicardial growth distorted normal epicardial development, reduced the number of invasive WT1/RALDH2-positive EPDCs, and provoked anomalies in the coronary vessels, the ventricular myocardium, and the AV cushions. We suggest that the proper development of ventricular myocardium is dependent on the invasion of undifferentiated, WT1-positive, retinoic acid-synthesizing EPDCs. Furthermore, we propose that an interaction between EPDCs and endocardial (derived) cells is imperative for correct development of the AV cushions.