The myocardium utilizes a platelet-derived growth factor receptor alpha (Pdgfra)-phosphoinositide 3-kinase (PI3K) signaling cascade to steer toward the midline during zebrafish heart tube formation.

Coordinated cell movement is a fundamental process in organ formation. During heart development, bilateral myocardial precursors collectively move toward the midline (cardiac fusion) to form the primitive heart tube. Extrinsic influences such as the adjacent anterior endoderm are known to be required for cardiac fusion. We previously showed however, that the platelet-derived growth factor receptor alpha (Pdgfra) is also required for cardiac fusion (Bloomekatz et al., 2017). Nevertheless, an intrinsic mechanism that regulates myocardial movement has not been elucidated. Here, we show that the phosphoinositide 3-kinase (PI3K) intracellular signaling pathway has an essential intrinsic role in the myocardium directing movement toward the midline. In vivo imaging further reveals midline-oriented dynamic myocardial membrane protrusions that become unpolarized in PI3K-inhibited zebrafish embryos where myocardial movements are misdirected and slower. Moreover, we find that PI3K activity is dependent on and interacts with Pdgfra to regulate myocardial movement. Together our findings reveal an intrinsic myocardial steering mechanism that responds to extrinsic cues during the initiation of cardiac development.

The myocardium utilizes Pdgfra-PI3K signaling to steer towards the midline during heart tube formation.

Coordinated cell movement is a fundamental process in organ formation. During heart development, bilateral myocardial precursors collectively move towards the midline (cardiac fusion) to form the primitive heart tube. Along with extrinsic influences such as the adjacent anterior endoderm which are known to be required for cardiac fusion, we previously showed that the platelet-derived growth factor receptor alpha (Pdgfra) is also required. However, an intrinsic mechanism that regulates myocardial movement remains to be elucidated. Here, we uncover an essential intrinsic role in the myocardium for the phosphoinositide 3-kinase (PI3K) intracellular signaling pathway in directing myocardial movement towards the midline. In vivo imaging reveals that in PI3K-inhibited zebrafish embryos myocardial movements are misdirected and slower, while midline-oriented dynamic myocardial membrane protrusions become unpolarized. Moreover, PI3K activity is dependent on and genetically interacts with Pdgfra to regulate myocardial movement. Together our findings reveal an intrinsic myocardial steering mechanism that responds to extrinsic cues during the initiation of cardiac development.

Using Live Imaging to Examine Early Cardiac Development in Zebrafish.

Visualizing dynamic cellular behaviors using live imaging is critical to the study of cell movement and to the study of cellular and embryonic polarity. Similarly, live imaging can be vital to elucidating the pathology of genetic disorders and diseases. Model systems such as zebrafish, whose in vivo development is accessible to both the microscope and genetic manipulation, are particularly well-suited to the use of live imaging. Here we describe an overall approach to conducting live-imaging experiments with a specific emphasis on investigating cell movements during the early stages of heart development in zebrafish.

Human-gained heart enhancers are associated with species-specific cardiac attributes.

The heart, a vital organ which is first to develop, has adapted its size, structure and function in order to accommodate the circulatory demands for a broad range of animals. Although heart development is controlled by a relatively conserved network of transcriptional/chromatin regulators, how the human heart has evolved species-specific features to maintain adequate cardiac output and function remains to be defined. Here, we show through comparative epigenomic analysis the identification of enhancers and promoters that have gained activity in humans during cardiogenesis. These cis-regulatory elements (CREs) are associated with genes involved in heart development and function, and may account for species-specific differences between human and mouse hearts. Supporting these findings, genetic variants that are associated with human cardiac phenotypic/disease traits, particularly those differing between human and mouse, are enriched in human-gained CREs. During early stages of human cardiogenesis, these CREs are also gained within genomic loci of transcriptional regulators, potentially expanding their role in human heart development. In particular, we discovered that gained enhancers in the locus of the early human developmental regulator ZIC3 are selectively accessible within a subpopulation of mesoderm cells which exhibits cardiogenic potential, thus possibly extending the function of ZIC3 beyond its conserved left-right asymmetry role. Genetic deletion of these enhancers identified a human gained enhancer that was required for not only ZIC3 and early cardiac gene expression at the mesoderm stage but also cardiomyocyte differentiation. Overall, our results illuminate how human gained CREs may contribute to human-specific cardiac attributes, and provide insight into how transcriptional regulators may gain cardiac developmental roles through the evolutionary acquisition of enhancers.

Unveiling Complexity and Multipotentiality of Early Heart Fields.

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Cardiac Morphogenesis: Crowding and Tension Resolved through Social Distancing.

Organ maturation entails the reshaping of simple tissues into more complex structures critical for function. In a recent issue of Nature, Priya et al. show how tension heterogeneity between developing cardiomyocytes can coordinate the cell behaviors that remodel the architecture of the cardiac chamber wall.

Using Zebrafish to Analyze the Genetic and Environmental Etiologies of Congenital Heart Defects.

Congenital heart defects (CHDs) are among the most common human birth defects. However, the etiology of a large proportion of CHDs remains undefined. Studies identifying the molecular and cellular mechanisms that underlie cardiac development have been critical to elucidating the origin of CHDs. Building upon this knowledge to understand the pathogenesis of CHDs requires examining how genetic or environmental stress changes normal cardiac development. Due to strong molecular conservation to humans and unique technical advantages, studies using zebrafish have elucidated both fundamental principles of cardiac development and have been used to create cardiac disease models. In this chapter we examine the unique toolset available to zebrafish researchers and how those tools are used to interrogate the genetic and environmental contributions to CHDs.

Canonical Wnt5b Signaling Directs Outlying Nkx2.5+ Mesoderm into Pacemaker Cardiomyocytes.

Pacemaker cardiomyocytes that create the sinoatrial node are essential for the initiation and maintenance of proper heart rhythm. However, illuminating developmental cues that direct their differentiation has remained particularly challenging due to the unclear cellular origins of these specialized cardiomyocytes. By discovering the origins of pacemaker cardiomyocytes, we reveal an evolutionarily conserved Wnt signaling mechanism that coordinates gene regulatory changes directing mesoderm cell fate decisions, which lead to the differentiation of pacemaker cardiomyocytes. We show that in zebrafish, pacemaker cardiomyocytes derive from a subset of Nkx2.5+ mesoderm that responds to canonical Wnt5b signaling to initiate the cardiac pacemaker program, including activation of pacemaker cell differentiation transcription factors Isl1 and Tbx18 and silencing of Nkx2.5. Moreover, applying these developmental findings to human pluripotent stem cells (hPSCs) notably results in the creation of hPSC-pacemaker cardiomyocytes, which successfully pace three-dimensional bioprinted hPSC-cardiomyocytes, thus providing potential strategies for biological cardiac pacemaker therapy.

Platelet-derived growth factor (PDGF) signaling directs cardiomyocyte movement toward the midline during heart tube assembly.

Communication between neighboring tissues plays a central role in guiding organ morphogenesis. During heart tube assembly, interactions with the adjacent endoderm control the medial movement of cardiomyocytes, a process referred to as cardiac fusion. However, the molecular underpinnings of this endodermal-myocardial relationship remain unclear. Here, we show an essential role for platelet-derived growth factor receptor alpha (Pdgfra) in directing cardiac fusion. Mutation of pdgfra disrupts heart tube assembly in both zebrafish and mouse. Timelapse analysis of individual cardiomyocyte trajectories reveals misdirected cells in zebrafish pdgfra mutants, suggesting that PDGF signaling steers cardiomyocytes toward the midline during cardiac fusion. Intriguingly, the ligand pdgfaa is expressed in the endoderm medial to the pdgfra-expressing myocardial precursors. Ectopic expression of pdgfaa interferes with cardiac fusion, consistent with an instructive role for PDGF signaling. Together, these data uncover a novel mechanism through which endodermal-myocardial communication can guide the cell movements that initiate cardiac morphogenesis.

Myocardial plasticity: cardiac development, regeneration and disease.

The adult mammalian heart is unable to recover from myocardial cell loss due to cardiac ischemia and infarction because terminally differentiated cardiomyocytes proliferate at a low rate. However, cardiomyocytes in other vertebrate animal models such as zebrafish, axolotls, newts and mammalian mouse neonates are capable of de-differentiating in order to promote cardiomyocyte proliferation and subsequent cardiac regeneration after injury. Although de-differentiation may occur in adult mammalian cardiomyocytes, it is typically associated with diseased hearts and pathologic remodeling rather than repair and regeneration. Here, we review recent studies of cardiac development, regeneration and disease that highlight how changes in myocardial identity (plasticity) is regulated and impacts adaptive and maladaptive cardiac responses.

Coordinating cardiomyocyte interactions to direct ventricular chamber morphogenesis.

Many organs are composed of complex tissue walls that are structurally organized to optimize organ function. In particular, the ventricular myocardial wall of the heart comprises an outer compact layer that concentrically encircles the ridge-like inner trabecular layer. Although disruption in the morphogenesis of this myocardial wall can lead to various forms of congenital heart disease and non-compaction cardiomyopathies, it remains unclear how embryonic cardiomyocytes assemble to form ventricular wall layers of appropriate spatial dimensions and myocardial mass. Here we use advanced genetic and imaging tools in zebrafish to reveal an interplay between myocardial Notch and Erbb2 signalling that directs the spatial allocation of myocardial cells to their proper morphological positions in the ventricular wall. Although previous studies have shown that endocardial Notch signalling non-cell-autonomously promotes myocardial trabeculation through Erbb2 and bone morphogenetic protein (BMP) signalling, we discover that distinct ventricular cardiomyocyte clusters exhibit myocardial Notch activity that cell-autonomously inhibits Erbb2 signalling and prevents cardiomyocyte sprouting and trabeculation. Myocardial-specific Notch inactivation leads to ventricles of reduced size and increased wall thickness because of excessive trabeculae, whereas widespread myocardial Notch activity results in ventricles of increased size with a single-cell-thick wall but no trabeculae. Notably, this myocardial Notch signalling is activated non-cell-autonomously by neighbouring Erbb2-activated cardiomyocytes that sprout and form nascent trabeculae. Thus, these findings support an interactive cellular feedback process that guides the assembly of cardiomyocytes to morphologically create the ventricular myocardial wall and more broadly provide insight into the cellular dynamics of how diverse cell lineages organize to create form.

The tumor suppressor PTEN and the PDK1 kinase regulate formation of the columnar neural epithelium.

Epithelial morphogenesis and stability are essential for normal development and organ homeostasis. The mouse neural plate is a cuboidal epithelium that remodels into a columnar pseudostratified epithelium over the course of 24 hr. Here we show that the transition to a columnar epithelium fails in mutant embryos that lack the tumor suppressor PTEN, although proliferation, patterning and apical-basal polarity markers are normal in the mutants. The Pten phenotype is mimicked by constitutive activation of PI3 kinase and is rescued by the removal of PDK1 (PDPK1), but does not depend on the downstream kinases AKT and mTORC1. High resolution imaging shows that PTEN is required for stabilization of planar cell packing in the neural plate and for the formation of stable apical-basal microtubule arrays. The data suggest that appropriate levels of membrane-associated PDPK1 are required for stabilization of apical junctions, which promotes cell elongation, during epithelial morphogenesis.

tal1 Regulates the formation of intercellular junctions and the maintenance of identity in the endocardium.

The endocardium forms the inner lining of the heart tube, where it enables blood flow and also interacts with the myocardium during the formation of valves and trabeculae. Although a number of studies have identified regulators in the morphogenesis of the myocardium, relatively little is known about the molecules that control endocardial morphogenesis. Prior work has implicated the bHLH transcription factor Tal1 in endocardial tube formation: in zebrafish embryos lacking Tal1, endocardial cells form a disorganized mass within the ventricle and do not populate the atrium. Through blastomere transplantation, we find that tal1 plays a cell-autonomous role in regulating endocardial extension, suggesting that Tal1 activity influences the behavior of individual endocardial cells. The defects in endocardial behavior in tal1-deficient embryos originate during the earliest steps of endocardial morphogenesis: tal1-deficient endocardial cells fail to generate a cohesive monolayer at the midline and instead pack tightly together into a multi-layered aggregate. Moreover, the tight junction protein ZO-1 is mislocalized in the tal1-deficient endocardium, indicating a defect in intercellular junction formation. In addition, we find that the tal1-deficient endocardium fails to maintain its identity; over time, a progressively increasing number of tal1-deficient endocardial cells initiate myocardial gene expression. However, the onset of defects in intercellular junction formation precedes the onset of ectopic myocardial gene expression in the tal1-deficient endocardium. We therefore propose a model in which Tal1 has distinct roles in regulating the formation of endocardial intercellular junctions and maintaining endocardial identity.

Quantitative semi-automated analysis of morphogenesis with single-cell resolution in complex embryos.

A quantitative understanding of tissue morphogenesis requires description of the movements of individual cells in space and over time. In transparent embryos, such as C. elegans, fluorescently labeled nuclei can be imaged in three-dimensional time-lapse (4D) movies and automatically tracked through early cleavage divisions up to ~350 nuclei. A similar analysis of later stages of C. elegans development has been challenging owing to the increased error rates of automated tracking of large numbers of densely packed nuclei. We present Nucleitracker4D, a freely available software solution for tracking nuclei in complex embryos that integrates automated tracking of nuclei in local searches with manual curation. Using these methods, we have been able to track >99% of all nuclei generated in the C. elegans embryo. Our analysis reveals that ventral enclosure of the epidermis is accompanied by complex coordinated migration of the neuronal substrate. We can efficiently track large numbers of migrating nuclei in 4D movies of zebrafish cardiac morphogenesis, suggesting that this approach is generally useful in situations in which the number, packing or dynamics of nuclei present challenges for automated tracking.

Pten regulates collective cell migration during specification of the anterior-posterior axis of the mouse embryo.

Pten, the potent tumor suppressor, is a lipid phosphatase that is best known as a regulator of cell proliferation and cell survival. Here we show that mouse embryos that lack Pten have a striking set of morphogenetic defects, including the failure to correctly specify the anterior-posterior body axis, that are not caused by changes in proliferation or cell death. The majority of Pten null embryos express markers of the primitive streak at ectopic locations around the embryonic circumference, rather than at a single site at the posterior of the embryo. Epiblast-specific deletion shows that Pten is not required in the cells of the primitive streak; instead, Pten is required for normal migration of cells of the Anterior Visceral Endoderm (AVE), an extraembryonic organizer that controls the position of the streak. Cells of the wild-type AVE migrate within the visceral endoderm epithelium from the distal tip of the embryo to a position adjacent to the extraembryonic region. In all Pten null mutants, AVE cells move a reduced distance and disperse in random directions, instead of moving as a coordinated group to the anterior of the embryo. Aberrant AVE migration is associated with the formation of ectopic F-actin foci, which indicates that absence of Pten disrupts the actin-based migration of these cells. After the initiation of gastrulation, embryos that lack Pten in the epiblast show defects in the migration of mesoderm and/or endoderm. The findings suggest that Pten has an essential and general role in the control of mammalian collective cell migration.

The RA domain of Ste50 adaptor protein is required for delivery of Ste11 to the plasma membrane in the filamentous growth signaling pathway of the yeast Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, pheromone response requires Ste5 scaffold protein, which ensures efficient G-protein-dependent recruitment of mitogen-activated protein kinase (MAPK) cascade components Ste11 (MAPK kinase kinase), Ste7 (MAPK kinase), and Fus3 (MAPK) to the plasma membrane for activation by Ste20 protein kinase. Ste20, which phosphorylates Ste11 to initiate signaling, is activated by binding to Cdc42 GTPase (membrane anchored via its C-terminal geranylgeranylation). Less clear is how activated and membrane-localized Ste20 contacts Ste11 to trigger invasive growth signaling, which also requires Ste7 and the MAPK Kss1, but not Ste5. Ste50 protein associates constitutively via an N-terminal sterile-alpha motif domain with Ste11, and this interaction is required for optimal invasive growth and hyperosmotic stress (high-osmolarity glycerol [HOG]) signaling but has a lesser role in pheromone response. We show that a conserved C-terminal, so-called "Ras association" (RA) domain in Ste50 is also essential for invasive growth and HOG signaling in vivo. In vitro the Ste50 RA domain is not able to associate with Ras2, but it does associate with Cdc42 and binds to a different face than does Ste20. RA domain function can be replaced by the nine C-terminal, plasma membrane-targeting residues (KKSKKCAIL) of Cdc42, and membrane-targeted Ste50 also suppresses the signaling deficiency of cdc42 alleles specifically defective in invasive growth. Thus, Ste50 serves as an adaptor to tether Ste11 to the plasma membrane and can do so via association with Cdc42, thereby permitting the encounter of Ste11 with activated Ste20.