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Defects in cardiac valvulogenesis are a common cause of congenital heart disease, and the study of this process promises to provide mechanistic insights and lead to novel therapeutics. Normal valve development involves multiple signaling pathways, and recently roles have been identified for extracellular matrix components, including glycosaminoglycans. We therefore explored the role of the glycosaminoglycan chondroitin sulfate during zebrafish cardiac development. Beginning at 33 hours, there is a distinct zone of chondroitin sulfate expression in the atrioventricular (AV) boundary, in the cardiac jelly between the endocardium and myocardium. This expression is both spatially and temporally restricted, and is undetectable after 48 hours. Chemical as well as genetic inhibition of chondroitin synthesis results in AV canal defects, including loss of the atrioventricular constriction, blood regurgitation, and failure of circulation. Lack of chondroitin disrupts a marker of cell migration, results in a loss of myocardial and endothelial markers of valvulogenesis, and misregulates BMP expression, supporting an early role in AV canal development. In summary, we have defined a requirement for chondroitin sulfate expression in the normal patterning of the AV boundary, suggesting that this component of the cardiac jelly provides a necessary signal in this critical transition in vertebrate cardiogenesis.

SUMMARY

Developing myocardial cells respond to signals from the endocardial layer to form a network of trabeculae that characterize the ventricles of the vertebrate heart. Abnormal myocardial trabeculation results in specific cardiomyopathies in humans and yet trabecular development is poorly understood. We show that trabeculation requires Brg1, a chromatin remodeling protein, to repress ADAMTS1 expression in the endocardium that overlies the developing trabeculae. Repression of ADAMTS1, a secreted matrix metalloproteinase, allows the establishment of an extracellular environment in the cardiac jelly that supports trabecular growth. Later during embryogenesis, ADAMTS1 expression initiates in the endocardium to degrade the cardiac jelly and prevent excessive trabeculation. Thus, the composition of cardiac jelly essential for myocardial morphogenesis is dynamically controlled by ADAMTS1 and its chromatin-based transcriptional regulation. Modification of the intervening microenvironment provides a mechanism by which chromatin regulation within one tissue layer coordinates the morphogenesis of an adjacent layer.

A hallmark of heart-valve development is the swelling and deposition of extracellular matrix in the heart-valve region. Only myocardium overlying this region can signal to underlying endothelium and cause it to lose cell–cell contacts, delaminate, and invade the extracellular space abutting myocardium and endocardium to form endocardial cushions (EC) in a process known as epithelial to mesenchymal transformation (EMT). The heart-valve myocardium expresses bone morphogenetic protein-2 (Bmp2) coincident with development of valve mesenchyme. BMPs belong to the transforming growth factor beta superfamily (TGF-β) and play a wide variety of roles during development. We show that conditional ablation of Bmp2 in cardiac progenitors results in cell fate changes in which the heart-valve region adopts the identity of differentiated chamber myocardium. Moreover, Bmp2-deficient hearts fail to induce production and deposition of matrix at the heart-valve-forming region, resulting in the inability of the endothelium to swell and impairing the development of ECs. Furthermore, in collagen invasion assays, Bmp2 mutant endothelium is incapable of undergoing EMT, and addition of BMP2 protein to mutant heart explants rescues this phenotype. Our results demonstrate that Bmp2 is both necessary and sufficient to specify a field of cardiac progenitor cells as the heart-valve-inducing region amid developing atria and ventricles.

Rationale

Heart valves develop from precursor structures called cardiac cushions, an endothelial-lined cardiac jelly that resides in the inner side of the heart tube. The cushions are then invaded by cells from different sources, undergo a series of complicated and poorly understood remodeling processes, and give rise to valves. Disruption of the fibroblast growth factor (FGF) signaling axis impairs morphogenesis of the outflow tract (OFT). Yet, whether FGF signaling regulates OFT valve formation is unknown.

Objective

To study how OFT valve formation is regulated and how aberrant cell signaling causes valve defects.

Methods and results

By employing mouse genetic manipulation, cell lineage tracing, ex vivo heart culture, and molecular biology approaches, we demonstrated that FGF signaling in the OFT myocardium upregulated Bmp4 expression, which then enhanced smooth muscle differentiation of neural crest cells (NCCs) in the cushion. FGF signaling also promoted OFT myocardial cell invasion to the cushion. Disrupting FGF signaling interrupted cushion remodeling with reduced NCCs differentiation into smooth muscle and less cardiomyocyte invasion, and resulted in malformed OFT valves.

Conclusions

The results demonstrate a novel mechanism by which the FGF-BMP signaling axis regulates formation of OFT valve primordia by controlling smooth muscle differentiation of cushion NCCs.

Endocardial cushions are precursors of mature atrioventricular (AV) valves. Their formation is induced by signaling molecules originating from the AV myocardium, including bone morphogenetic proteins (BMPs). Here, we hypothesized that BMP signaling plays an important role in the AV myocardium during the maturation of AV valves from the cushions. To test our hypothesis, we used a unique Cre/lox system to target the deletion of a floxed Alk3 allele, the type IA receptor for BMPs, to cardiac myocytes of the AV canal (AVC). Lineage analysis indicated that cardiac myocytes of the AVC contributed to the tricuspid mural and posterior leaflets, the mitral septal leaflet, and the atrial border of the annulus fibrosus. When Alk3 was deleted in these cells, defects were seen in the same leaflets, ie, the tricuspid mural leaflet and mitral septal leaflet were longer, the tricuspid posterior leaflet was displaced and adherent to the ventricular wall, and the annulus fibrosus was disrupted resulting in ventricular preexcitation. The defects seen in mice with AVC-targeted deletion of Alk3 provide strong support for a role of Alk3 in human congenital heart diseases, such as Ebstein’s anomaly. In conclusion, our mouse model demonstrated critical roles for Alk3 signaling in the AV myocardium during the development of AV valves and the annulus fibrosus.

The primitive heart tube is composed of an outer myocardial and an inner endocardial layer that will give rise to the cardiac valves and septa. Specification and differentiation of these two cell layers are among the earliest events in heart development, but the embryonic origins and genetic regulation of early endocardial development remain largely undefined. We have analyzed early endocardial development in the zebrafish using time-lapse confocal microscopy and show that the endocardium seems to originate from a region in the lateral plate mesoderm that will give rise to hematopoietic cells of the primitive myeloid lineage. Endocardial precursors appear to rapidly migrate to the site of heart tube formation, where they arrive prior to the bilateral myocardial primordia. Analysis of a newly discovered zebrafish Scl/Tal1 mutant showed an additional and previously undescribed role of this transcription factor during the development of the endocardium. In Scl/Tal1 mutant embryos, endocardial precursors are specified, but migration is severely defective and endocardial cells aggregate at the ventricular pole of the heart. We further show that the initial fusion of the bilateral myocardial precursor populations occurs independently of the endocardium and tal1 function. Our results suggest early separation of the two components of the primitive heart tube and imply Scl/Tal1 as an indispensable component of the molecular hierarchy that controls endocardium morphogenesis.

Author Summary

In its earliest functional form, the embryonic heart of all vertebrates is a simple linear tube consisting of two cell types. An outer muscular cell layer called the myocardium surrounds an inner vascular cell layer called the endocardium that connects the heart to the vascular system. The integration of both cell types is an important step during heart development, but the formation of the endocardial component of the heart tube is poorly understood. Here, we analyze the formation of the endocardium in the zebrafish embryo and show using time-lapse imaging that it is a highly dynamic structure. In addition, we have identified a zebrafish mutant with a specific defect during endocardial development. This defect is caused by a mutation in T cell acute leukemia 1, a gene that—when misexpressed—causes many cases of childhood leukemias. Here, we show an additional role for this gene during heart development. In mutant embryos, both endocardial and myocardial precursors are specified, but integration of both cell types does not occur properly due to a defective migration of the endocardial precursors. Given the many interactions that occur between the endocardium and the myocardium, our results will provide a more comprehensive understanding of heart development.

The Tbx2 transcription factor is implicated in growth control based on its association with human cancers. In the heart, Tbx2 represses cardiac differentiation to mediate development of the atrioventricular canal (AVC). The zebrafish genome retains two tbx2 genes, and both are required for formation of the AVC. Here we show that both genes are also expressed earlier in the primitive heart tube, and we describe a previously unrecognized role for Tbx2 in promoting proliferation of presumptive myocardium at the heart tube stage. In contrast to single knockdowns, depletion of both gene products causes chamber defects, resulting in an expanded atrium and a smaller ventricle, associated with decreased proliferation of ventricular cardiomyocytes. The phenotype correlates with changes in the expression for known cardiac growth factors. Therefore, in zebrafish, two tbx2 genes are functionally redundant for regulating chamber development, while each gene is required independently for development of the AVC.

Hyaluronan is an extracellular matrix component implicated in expansion of the extracellular space, organization of supramolecular architecture, cell motility, proliferation, tumour metastases and wound healing. Hyaluronan is highly expressed in the developing heart but it is only a minor component of the mature heart. The loss of hyaluronan synthase-2 (Has2) results in embryonic lethality with a phenotype remarkably similar to that of the versican-deficient heart defect mouse. Has2-deficient embryos lack hyaluronan-containing cardiac jelly, and at embryonic day 9.5 show arrested development, with an apparent absence of the right ventricle and underdevelopment of the conustruncus segment, and pericardial effusion consistent with heart failure. Cardiac cushions are totally absent, and endocardial cell migration over collagen gels is not detectable in Has2-deficient atrioventricular (AV) canal explants. Endothelial to mesenchymal transformation is also defective in AV explants from Has2-null embryos. The normal phenotype is restored in AV canal explants from Has2-deficient embryos by co-culture with wild type AV canal explants, with conditioned media from wild type AV explants or with exogenous hyaluronan. These results provide evidence for a direct role for hyaluronan during endocardial cushion and AV canal morphogenesis.

While limb regeneration has been extensively studied in amphibians, little is known about the initial events in limb formation in metamorphosing anurans. The small secreted integrin ligand nephronectin (npnt) is necessary for development of the metanephros in mouse. Although expressed in many tissues, its role in other developmental processes is not well-studied. Here we show that a transgene insertion that disrupts this gene ablates forelimb formation in Xenopus tropicalis. Our results suggest a novel role for integrin signalling in limb development, and represent the first insertional phenotype to be cloned in amphibians.

Research Highlights

►The Xenopus tropicalis mutation xdm lacks all forelimb structures. ►Xdm results from a transgene integration in the nephronectin gene. ►Xdm tadpoles do not express tbx5 in the prospective forelimb field. ►Xdm is the first insertional mutation cloned in amphibians.

Cardiac malformations due to aberrant development of the atrioventricular (AV) valves are among the most common forms of congenital heart disease. At localized swellings of extracellular matrix known as the endocardial cushions, the endothelial lining of the heart undergoes an epithelial to mesenchymal transition (EMT) to form the mesenchymal progenitors of the AV valves. Further growth and differentiation of these mesenchymal precursors results in the formation of portions of the atrial and ventricular septae, and the generation of thin, pliable valves. Gata4, which encodes a zinc finger transcription factor, is expressed in the endothelium and mesenchyme of the AV valves. Using a Tie2-Cre transgene, we selectively inactivated Gata4 within endothelial-derived cells. Mutant endothelium failed to undergo EMT, resulting in hypocellular cushions. Mutant cushions had decreased levels of Erbb3, an EGF-family receptor essential for EMT in the atrioventricular cushions. In Gata4 mutant embryos, Erbb3 downregulation was associated with impaired activation of Erk, which is also required for EMT. Expression of a Gata4 mutant protein defective in interaction with Friend of Gata (FOG) cofactors rescued the EMT defect, but resulted in a decreased proliferation of mesenchyme and hypoplastic cushions that failed to septate the ventricular inlet. We demonstrate two novel functions of Gata4 in development of the AV valves. First, Gata4 functions as an upstream regulator of an Erbb3-Erk pathway necessary for EMT, and second, Gata4 acts to promote cushion mesenchyme growth and remodeling.

Cardiac valve leaflets develop from rudimentary structures termed endocardial cushions. These pre-valve tissues arise from a complex interplay of signals between the myocardium and endocardium whereby secreted cues induce the endothelial cells to transform into migratory mesenchyme through an endothelial to mesenchymal transformation (EMT). Even though much is currently known regarding the initial EMT process, the mechanisms by which these undifferentiated cushion mesenchymal tissues are remodeled “post-EMT” into mature fibrous valve leaflets remains one of the major, unsolved questions in heart development. Expression analyses, presented in this report, demonstrate that periostin, a component of the extracellular matrix, is predominantly expressed in post-EMT valve tissues and their supporting apparatus from embryonic to adult life. Analyses of periostin gene targeted mice demonstrate that it is within these regions that significant defects are observed. Periostin null mice exhibit atrial septal defects, structural abnormalities of the AV valves and their supporting tensile apparatus, and aberrant differentiation of AV cushion mesenchyme. Rescue experiments further demonstrate that periostin functions as a hierarchical molecular switch that can promote the differentiation of mesenchymal cells into a fibroblastic lineage while repressing their transformation into other mesodermal cell lineages (e.g. myocytes). This is the first report of an extracellular matrix protein directly regulating post-EMT AV valve differentiation, a process foundational and indispensable for the morphogenesis of a cushion into a leaflet.

During early cardiac development, progenitors of the valves and septa of the heart are formed by an epithelial-mesenchymal cell transformation of endothelial cells of the atrioventricular (AV) canal. We have previously shown that this event is due to an interaction between the endothelium and products of the myocardium found within the extracellular matrix. The present study examines signal transduction mechanisms governing this differentiation of AV canal endothelium. Activators of protein kinase C (PKC), phorbol myristate acetate (PMA) and mezerein, both produced an incomplete phenotypic transformation of endothelial cells in an in vitro bioassay for transformation. On the other hand, inhibitors of PKC (H-7 and staurosporine) and tyrosine kinase (genistein) blocked cellular transformation in response to the native myocardium or a myocardially-conditioned medium. Intracellular free calcium concentration ([Ca2+]i) was measured in single endothelial cells by microscopic digital analysis of fura 2 fluorescence. Addition of a myocardial conditioned medium containing the transforming stimulus produced a specific increase in [Ca2+]i in "competent" AV canal, but not ventricular, endothelial cells. Epithelial-mesenchymal cell transformation was inhibited by pertussis toxin but not cholera toxin. These data lead to the hypothesis that signal transduction of this tissue interaction is mediated by a G protein and one or more kinase activities. In response to receptor activation, competent AV canal endothelial cells demonstrate an increase in [Ca2+]i. Together, the data provide direct evidence for a regional and temporal regulation of signal transduction processes which mediate a specific extracellular matrix-mediated tissue interaction in the embryo.

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The zebrafish is an ideal model organism for investigating the molecular mechanisms underlying cardiogenesis, due to the powerful combination of optical access to the embryonic heart and plentiful opportunities for genetic analysis. A continually increasing number of studies are uncovering mutations, morpholinos, and small molecules that cause striking cardiac defects and disrupt blood circulation in the zebrafish embryo. Such defects can result from a wide variety of origins including defects in the specification or differentiation of cardiac progenitor cells; errors in the morphogenesis of the heart tube, the cardiac chambers, or the atrioventricular canal or problems with establishing proper cardiac function. An extensive arsenal of techniques is available to distinguish between these possibilities and thereby decipher the roots of cardiac defects. In this chapter, we provide a guide to the experimental strategies that are particularly effective for the characterization of cardiac henotypes in the zebrafish embryo.

During early embryogenesis, the formation of the cardiac atrioventricular canal (AVC) facilitates the transition of the heart from a linear tube into a chambered organ. However, the genetic pathways underlying this developmental process are poorly understood. The T-box transcription factor Tbx20 is expressed predominantly in the AVC of early heart tube. It was shown that Tbx20 activates Nmyc1 and suppresses Tbx2 expression to promote proliferation and specification of the atrial and ventricular chambers, yet it is not known if Tbx20 is involved in early AVC development. Here, we report that mice lacking Tbx20 in the AVC myocardium fail to form the AVC constriction, and the endocardial epithelial-mesenchymal transition (EMT) is severely perturbed. Tbx20 maintains expression of a variety of genes, including Bmp2, Tbx3 and Hand1 in the AVC myocardium. Intriguingly, we found Bmp2 downstream genes involved in the EMT initiation are also downregulated. In addition, re-expression of Bmp2 in the AVC myocardium substantially rescues the EMT defects resulting from the lack of Tbx20, suggesting Bmp2 is one of the key downstream targets of Tbx20 in AVC development. Our data support a complex signaling network with Tbx20 suppressing Tbx2 in the AVC myocardium but also indirectly promoting Tbx2 expression through Bmp2. The spatiotemporal expression of Tbx2 in the AVC appears to be balanced between these two opposing signals. Overall, our study provides genetic evidence that Tbx20 has essential roles in regulating AVC development that coordinate early cardiac chamber formation.

The development of the valves and septa of the heart depends on the formation and remodeling of endocardial cushions. Here, we report that the alternative splicing regulator muscleblind-like 1 (MBNL1) exhibits a regionally restricted pattern of expression in canal region endocardium and ventricular myocardium during endocardial cushion development in chicken. Knockdown of MBNL1 in atrioventricular explants leads to a transforming growth factor β-dependent increase in epithelial-mesenchymal transition (EMT) of endocardial cells. This reveals a novel role for MBNL1 during embryonic development, and represents the first evidence that an alternative splicing regulator is a key player in endocardial cushion development.

Complete atrioventricular canal (CAVC), also referred to as complete atrioventricular septal defect, is characterised by an ostium primum atrial septal defect, a common atrioventricular valve and a variable deficiency of the ventricular septum inflow. CAVC is an uncommon congenital heart disease, accounting for about 3% of cardiac malformations. Atrioventricular canal occurs in two out of every 10,000 live births. Both sexes are equally affected and a striking association with Down syndrome was found. Depending on the morphology of the superior leaflet of the common atrioventricular valve, 3 types of CAVC have been delineated (type A, B and C, according to Rastelli's classification). CAVC results in a significant interatrial and interventricular systemic-to-pulmonary shunt, thus inducing right ventricular pressure and volume overload and pulmonary hypertension. It becomes symptomatic in infancy due to congestive heart failure and failure to thrive. Diagnosis of CAVC might be suspected from electrocardiographic and chest X-ray findings. Echocardiography confirms it and gives anatomical details. Over time, pulmonary hypertension becomes irreversible, thus precluding the surgical therapy. This is the reason why cardiac catheterisation is not mandatory in infants (less than 6 months) but is indicated in older patients if irreversible pulmonary hypertension is suspected. Medical treatment (digitalis, diuretics, vasodilators) plays a role only as a bridge toward surgery, usually performed between the 3rd and 6th month of life.

Function of the heart begins long before its formation is complete. Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification. However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established. Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish. To address whether myocardial function is required for EC formation, we examined silent heart (sih−/−) embryos, which lack a heartbeat due to mutation of cardiac troponin T (tnnt2), and observed that atrioventricular (AV) ECs do not form. Likewise, we determined that cushion formation is blocked in cardiofunk (cfk−/−) embryos, which exhibit cardiac dilation and no early blood flow. In order to further analyze the heart defects in cfk−/− embryos, we positionally cloned cfk and show that it encodes a novel sarcomeric actin expressed in the embryonic myocardium. The Cfks11 variant exhibits a change in a universally conserved residue (R177H). We show that in yeast this mutation negatively affects actin polymerization. Because the lack of cushion formation in sih- and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development. Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development. Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease.

Cardiac anomolies can result from very early defects in heart development. In zebrafish, such defects have been shown to be caused by a new gene called cardiofunk

Vascular endothelial growth factor-receptors (VEGF-Rs) are pivotal regulators of vascular development, but a specific role for these receptors in the formation of heart valves has not been identified. We took advantage of small molecule inhibitors of VEGF-R signaling and showed that blocking VEGF-R signaling with receptor selective tyrosine kinase inhibitors, PTK 787 and AAC 787, from 17–21 hours post fertilization (hpf) in zebrafish embryos resulted in a functional and structural defect in cardiac valve development. Regurgitation of blood between the two chambers of the heart, as well as a loss of cell-restricted expression of the valve differentiation markers notch 1b and bone morphogenetic protein-4 (bmp-4), was readily apparent in treated embryos. In addition, microangiography revealed a loss of a definitve atrioventricular constriction in treated embryos. Taken together, these data demonstrate a novel function for VEGF-Rs in the endocardial endothelium of the developing cardiac valve.

The epithelial–mesenchymal interactions required for kidney organogenesis are disrupted in mice lacking the integrin α8β1. None of this integrin's known ligands, however, appears to account for this phenotype. To identify a more relevant ligand, a soluble integrin α8β1 heterodimer fused to alkaline phosphatase (AP) has been used to probe blots and cDNA libraries. In newborn mouse kidney extracts, α8β1-AP detects a novel ligand of 70–90 kD. This protein, named nephronectin, is an extracellular matrix protein with five EGF-like repeats, a mucin region containing a RGD sequence, and a COOH-terminal MAM domain. Integrin α8β1 and several additional RGD-binding integrins bind nephronectin. Nephronectin mRNA is expressed in the ureteric bud epithelium, whereas α8β1 is expressed in the metanephric mesenchyme. Nephronectin is localized in the extracellular matrix in the same distribution as the ligand detected by α8β1-AP and forms a complex with α8β1 in vivo. Thus, these results strongly suggest that nephronectin is a relevant ligand mediating α8β1 function in the kidney. Nephronectin is expressed at numerous sites outside the kidney, so it may also have wider roles in development. The approaches used here should be generally useful for characterizing the interactions of novel extracellular matrix proteins identified through genomic sequencing projects.

NDRG4 is a novel member of the NDRG family (N-myc downstream-regulated gene). The roles of NDRG4 in development have not previously been evaluated. We show that, during zebrafish embryonic development, ndrg4 is expressed exclusively in the embryonic heart, the central nervous system (CNS) and the sensory system. Knockdown ndrg4 in zebrafish embryos causes a marked reduction in proliferative myocytes and results in hypoplastic hearts. This growth defect is associated with cardiac phenotypes in morphogenesis and function, including abnormal heart looping, inefficient circulation and weak contractility. We reveal that ndrg4 is required for restricting the expression of versican and bmp4 to the developing atrioventricular canal. This constellation of ndrg4 cardiac defects phenocopies those seen in mutant hearts of heartstrings (hst), the tbx5 loss-of-function mutants in zebrafish. We further show that ndrg4 expression is significantly decreased in hearts with reduced tbx5 activities. Conversely, increased expression of tbx5 that is due to tbx20 knockdown leads to an increase in ndrg4 expression. Together, our studies reveal an essential role of ndrg4 in regulating proliferation and growth of cardiomyocytes, suggesting that ndrg4 may function downstream of tbx5 during heart development and growth.

At species-specific times in embryonic development, the pro-epicardial organ appears as an outcropping of the mesothelial body wall, near the sinus venosus–liver region. The pro-epicardial vesicles attach to the myocardium, flatten, and join to form the epicardium. The epicardium shows epithelial–mesenchymal transformation: cells detach from the epithelium, fill the subepicardial space, and invade the heart tube. Epicardium-derived cells migrate as far as the core of the endocardial cushions, which differentiate into the atrioventricular valve leaflets. In the cardiac wall, other epicardium-derived cells differentiate into interstitial fibroblasts and adventitial and smooth muscle cells of the coronary arteries.

Using neural crest tracings in mouse embryos (Wnt1-Cre-lacZ), we studied the patterning of cardiac neural crest cells during development. Participation of neural crest cells in the formation of the vascular media could not be excluded, although epicardium-derived cells have hitherto been considered responsible for formation of the coronary arterial smooth muscle cells. The endothelial cells of the coronary network derive mostly from the endothelium of the sinus venosus–liver region by vasculogenesis and angiogenesis. However, an epicardium-derived cell origin of some endothelial cells cannot be ruled out. The coronary vasculature is closely related to the differentiating Purkinje network, but isolated epicardium-derived cells are also associated with Purkinje cells. After ablating the pro-epicardial organ in quail embryos, we found severe malformations in the myocardial architecture, leading to the hypothesis that epicardium-derived cells give instructive signals to the myocardium for proper differentiation of the compact and the trabeculated compartments. (Tex Heart Inst J 2002;29:255–61)

The directionality of local blood flow in the zebrafish embryonic heart is essential for proper heart valve formation.

Heart valve anomalies are some of the most common congenital heart defects, yet neither the genetic nor the epigenetic forces guiding heart valve development are well understood. When functioning normally, mature heart valves prevent intracardiac retrograde blood flow; before valves develop, there is considerable regurgitation, resulting in reversing (or oscillatory) flows between the atrium and ventricle. As reversing flows are particularly strong stimuli to endothelial cells in culture, an attractive hypothesis is that heart valves form as a developmental response to retrograde blood flows through the maturing heart. Here, we exploit the relationship between oscillatory flow and heart rate to manipulate the amount of retrograde flow in the atrioventricular (AV) canal before and during valvulogenesis, and find that this leads to arrested valve growth. Using this manipulation, we determined that klf2a is normally expressed in the valve precursors in response to reversing flows, and is dramatically reduced by treatments that decrease such flows. Experimentally knocking down the expression of this shear-responsive gene with morpholine antisense oligonucleotides (MOs) results in dysfunctional valves. Thus, klf2a expression appears to be necessary for normal valve formation. This, together with its dependence on intracardiac hemodynamic forces, makes klf2a expression an early and reliable indicator of proper valve development. Together, these results demonstrate a critical role for reversing flows during valvulogenesis and show how relatively subtle perturbations of normal hemodynamic patterns can lead to both major alterations in gene expression and severe valve dysgenesis.

Author Summary

The growth and development of vertebrates are critically dependent on efficient cardiac output to drive blood circulation. An essential step of heart development is the formation of heart valves, whose leaflets are made through a complex set of cellular rearrangements of endothelial cells. Endothelial cells experience high flow forces as blood circulates. Moreover, heart valves and associated structures can be malformed when flow forces are abnormal, suggesting that these flow forces are in fact required for proper valve formation. Whether it is the force of the blood flow, its directionality (forward or reverse), or both that are important is not clear. We studied the interplay during valve development between key genes known to be involved in the process and epigenetic influences such as flow forces. Using zebrafish, whose optical clarity allows analyzing blood flow patterns at high resolution, we identified the presence of reversing flows specifically at the level of valve precursors. By manipulating blood flow patterns, we show that reversing flows are essential for valve morphogenesis. Specifically, we show that the expression of the gene klf2a depends on the presence of reversing flows and is required for valve development. We predict that by influencing levels of klf2a, reversing flows constitute an important stimulus controlling the appropriate biological responses of endothelial cells during valve formation.

Background

The heart forms from a linear tube that is subject to complex remodeling during embryonic development. Hallmarks of this remodeling are the looping of the heart tube and the regionalization into chamber and non-chamber myocardium. Cardiomyocytes in the future chamber myocardium acquire different cellular and physiological characteristics through activation of a chamber-specific genetic program, which is in part mediated by T-box genes.

Methodology/Principal Finding

We characterize two new zebrafish T-box transcription factors, tbx3b and tbx2a, and analyze their role during the development of the atrioventricular canal. Loss- and gain-of-function analyses demonstrate that tbx3b and tbx2a are necessary to repress the chamber-genetic program in the non-chamber myocardium. We also show that tbx3b and tbx2a are required to control cell proliferation in the atrioventricular canal and that misregulation of cell proliferation in the heart tube influences looping. Furthermore, we characterize the heart phenotype of a novel Tbx3 mutation in mice and show that both the control of cell proliferation and the repression of chamber-specific genetic program in the non-chamber myocardium are conserved roles of Tbx3 in this species.

Conclusions/Significance

Taken together, our results uncover an evolutionarily conserved role of Tbx2/3 transcription factors during remodeling of the heart myocardium and highlight the importance of controlling cell proliferation as a driving force of morphogenesis.

Malformations of the cardiovascular system are the most common type of birth defect in humans, frequently affecting the formation of valves and septa. During heart valve and septa formation, cells from the atrio-ventricular canal (AVC) and outflow tract (OFT) regions of the heart undergo an epithelial-to-mesenchymal transformation (EMT) and invade the underlying extracellular matrix to give rise to endocardial cushions. Subsequent maturation of newly formed mesenchyme cells leads to thin stress-resistant leaflets. TWIST1 is a basic helix-loop-helix transcription factor expressed in newly formed mesenchyme cells of the AVC and OFT that has been shown to play roles in cell survival, cell proliferation and differentiation. However, the downstream targets of TWIST1 during heart valve formation remain unclear. To identify genes important for heart valve development downstream of TWIST1, we performed global gene expression profiling of AVC, OFT, atria and ventricles of the embryonic day 10.5 mouse heart by tag-sequencing (Tag-seq). Using this resource we identified a novel set of 939 genes, including 123 regulators of transcription, enriched in the valve forming regions of the heart. We compared these genes to a Tag-seq library from the Twist1 null developing valves revealing significant gene expression changes. These changes were consistent with a role of TWIST1 in controlling differentiation of mesenchymal cells following their transformation from endothelium in the mouse. To study the role of TWIST1 at the DNA level we performed chromatin immunoprecipitation and identified novel direct targets of TWIST1 in the developing heart valves. Our findings support a role for TWIST1 in the differentiation of AVC mesenchyme post-EMT in the mouse, and suggest that TWIST1 can exert its function by direct DNA binding to activate valve specific gene expression.

Development of the mammalian heart is mediated by complex interactions between myocardial, endocardial, and neural crest-derived cells. Studies in Drosophila have shown that the Slit-Robo signaling pathway controls cardiac cell shape changes and lumen formation of the heart tube. Here, we demonstrate by in situ hybridization that multiple Slit ligands and Robo receptors are expressed in the developing mouse heart. Slit3 is the predominant ligand transcribed in the early mouse heart and is expressed in the ventral wall of the linear heart tube and subsequently in chamber but not in atrioventricular canal myocardium. Furthermore, we identify that the homeobox gene Nkx2-5 is required for early ventral restriction of Slit3 and that the T-box transcription factor Tbx2 mediates repression of Slit3 in nonchamber myocardium. Our results suggest that patterned Slit-Robo signaling may contribute to the control of oriented cell growth during chamber morphogenesis of the mammalian heart.