Deborah Yelon - US grants

The embryonic vertebrate heart has a simple anterior- posterior pattern: it is divided into two major chambers, an anterior ventricle and a posterior atrium. These two chambers are morphologically distinct; furthermore, the ventricular myocardium differs from the atrial myocardium by many molecular, histological, and physiological criteria. These intrinsic differences between the ventricle and the atrium are critical for proper cardiac function, and neonatal and adult cardiac conditions are often associated with chamber formation defects. Despite the functional importance of proper cardiac patterning, its genetic regulation is not yet understood. Through studies of cardiac chamber formation in the zebrafish embryo, we are identifying the genes that specify ventricular and atrial cell fates. In this proposal, we use zebrafish mutations to determine the roles of three key players in cardiac patterning: the transcription factor Hand2, the retinoic acid-synthesizing enzyme retinaldehyde dehydrogenase 2 (Raldh2), and an unidentified gene (heart of darkness (hod)). First, by creating fate maps for ventricular and atrial progenitors in wild-type and hand2 mutant zebrafish embryos, we will test models regarding the cellular function of Hand2 during myocardial differentiation and ventricle formation. Second, studies of raldh2 mutant embryos, including phenotypic characterization, fate mapping, and mosaic analysis, will test the hypothesis that retinoic acid signaling regulates atrial specification. Third, similar molecular and cellular analyses of the hod mutant phenotype will test the hypothesis that hod, like hand2, regulates early steps of myocardial differentiation and ventricle formation. Finally, we will clone the hod gene and begin the analysis of the hod gene product. Altogether, these experiments will provide a foundation for understanding the genetic pathways that regulate cardiac patterning in vertebrates.

DESCRIPTION (provided by applicant): The identification of genetic pathways controlling cardiac chamber formation is likely to illuminate the molecular mechanisms responsible for many cardiac structural birth defects. The cardiac homeodomain transcription factor gene Nkx2-5 is known to play a key role in cardiac development: Nkx2-5 is essential for normal chamber formation in mice, and mutations in human Nkx2-5 underlie familial cases of congenital heart disease. Despite the clear importance of Nkx2-5 in the cardiac regulatory hierarchy, the downstream components of the Nkx2-5 pathway and their contributions to cardiac chamber formation are not well understood. The PIs hypothesize that the essential effector genes for cardiac chamber formation are evolutionarily conserved components of the Nkx2-5 pathway. To test this hypothesis, they propose a two-year exploratory project that combines the expertise of the Yelon and Harvey laboratories for a comparative genetic analysis of Nkx2-5 function in zebrafish and mouse. Specifically, they will begin by characterizing the molecular anatomy of the zebrafish heart as a foundation for comparative analysis. Applying this knowledge, they will compare the loss-of-function phenotypes for the Nkx2-5 gene family in zebrafish and mouse. Then, taking advantage of advances in both mouse and zebrafish genomics, they will combine microarray analysis and comparative gene expression studies to identify genes downstream of Nkx2-5 in both species. Finally, with attention focused on conserved pathway components, they will utilize zebrafish reverse genetics to test their roles during cardiac chamber formation. Together, the proposed experiments will reveal conserved roles and components of the Nkx2-5 pathway. Furthermore, by capitalizing on the complementary advantages of two model organisms, the PIs will develop a new approach for functional annotation of cardiac gene expression profiles.

DESCRIPTION (provided by applicant): The embryonic vertebrate heart is composed of 2 major chambers, a ventricle and an atrium, each with a characteristic morphology that defines its functional capacity. Congenital heart defects are often associated with dysmorphic cardiac chambers, but the regulation of cardiac chamber morphogenesis is not well understood. Cardiac chamber morphogenesis can be divided into 2 major phases: heart tube assembly, in which bilateral precursor populations unite to form a tube, and chamber emergence, in which this simple cylinder transforms into a series of morphologically discrete chambers. The long-term goal of our research is to identify the cellular and molecular mechanisms that regulate these 2 phases of chamber morphogenesis. Using the zebrafish as a model organism, we can combine embryologic and genetic approaches, using high-resolution live imaging to determine how key genes influence cardiomyocyte behavior. Our preliminary time-lapse analyses of heart tube assembly suggest that this process is driven by multiple genes that collaborate to regulate regional differences in directed cardiomyocyte movements. Additionally, our initial morphometric analyses of chamber emergence suggest that this process is governed. by both intrinsic and extrinsic factors that control regional changes in cardiomyocyte morphology. Finally, our recent studies of novel mutations suggest that the slow fuse and change of heart genes are critical regulators of heart tube assembly and chamber emergence, respectively. Building on this foundation, we propose to test (1) how cardiomyocyte movements are regulated by a critical mass of cells, formation of polarized epithelia, interactions with endoderm and endocardium, and slow fuse function, and (2) how cardiomyocyte morphology is regulated by hemodynamics, sarcomere integrity, interactions with the endocardium, and change of heart function. Together, these studies will reveal essential regulatory mechanisms of cardiogenesis and also enrich our understanding of general paradigms for organ formation.

DESCRIPTION (provided by applicant): Formation of the outflow tract (OFT) is an essential aspect of cardiogenesis: the dimensions, orientation, and subdivision of the OFT are crucial for effective transport of blood from the heart to the periphery. OFT development initiates with the assembly of a small myocardial tube, which subsequently provides a vital foundation for OFT remodeling. Given the importance of establishing the OFT myocardium, the embryonic origins of OFT cardiomyocytes (CMs) have been of great interest. A series of studies in mouse and chick embryos have illuminated two major sources of cardiac progenitor cells, termed the first heart field (FHF) and the second heart field (SHF). Notably, the initial foundation of the OFT is built by SHF-derived CMs that are appended to the arterial pole of the heart. Although several signaling pathways have been implicated in regulating SHF differentiation, little is known about which genes function downstream of these key signals to execute OFT assembly or how the multiple relevant pathways interact to set the dimensions of the OFT. Here, we exploit the utility of the zebrafish as a model organism in order to identify novel regulators of OFT formation. Preliminary studies suggest that the zebrafish OFT, like the amniote OFT, is constructed from a population of SHF-derived CMs. Furthermore, in zebrafish, as in amniotes, Fgf signaling is required to promote the production of OFT CMs. However, it is unclear which genes act downstream of Fgf signaling to recruit the appropriate number of CMs into the OFT. Our preliminary data reveal an interesting set of genes - cell adhesion molecule 4 (cadm4), cadm3, and cadm2a - that are repressed by Fgf signaling and play essential roles in restricting the formation of OFT myocardium. These data suggest an intriguing model in which Fgf signaling drives the recruitment of OFT CMs by limiting the expression of cadm genes and thereby altering critical extracellular interactions of SHF-derived progenitor cells. In this proposal, we will test this model in detail by establishing the origins of the zebrafish OFT, deciphering the mechanisms of Cadm function, and integrating the Fgf-Cadm pathway into the context of the multiple influences that converge to define the size of the OFT. In Aim 1, we will employ fate mapping, time-lapse tracking, and assays for the timing of myocardial differentiation to determine whether the zebrafish OFT myocardium is derived from a SHF equivalent. In Aim 2, we will use loss-of-function, gain-of-function, structure-function, and biochemical analyses to test if Cadms mediate extracellular interactions that inhibit recruitment of OFT CMs. In Aim 3, we will identify signals that counterbalance the impact of the Fgf-Cadm pathway on OFT size, focusing on the roles played by Notch, Bmp, and retinoic acid signaling in limiting the dimensions of the zebrafish OFT. Together, these experiments are likely to reveal new mediators of OFT CM recruitment, to uncover a novel mechanism for regulating OFT size through modulation of extracellular interactions, and to shed light on the network of pathways that collaborate to insure an appropriate myocardial foundation for the embryonic OFT. PUBLIC HEALTH RELEVANCE: Cardiac defects are found in as many as 1 in 100 live births and 1 in 10 still births and frequently include problems with the formation of the cardiac outflow tract. Outflow tract development initiates with the assembly of a small tube of muscle, the precise dimensions of which are essential for its subsequent remodeling into a mature structure. Therefore, a better comprehension of the mechanisms controlling the initial investment of muscle into the outflow tract is likely to illuminate the causes of cardiac birth defects and may also suggest strategies for directing multipotent cells to become cardiac muscle.

DESCRIPTION (provided by applicant): Formation of the outflow tract (OFT) is an essential aspect of cardiogenesis: the dimensions, orientation, and subdivision of the OFT are crucial for effective transport of blood from the heart to the periphery. OFT development initiates with the assembly of a small myocardial tube, which subsequently provides a vital foundation for OFT remodeling. Given the importance of establishing the OFT myocardium, the embryonic origins of OFT cardiomyocytes (CMs) have been of great interest. A series of studies in mouse and chick embryos have illuminated two major sources of cardiac progenitor cells, termed the first heart field (FHF) and the second heart field (SHF). Notably, the initial foundation of the OFT is built by SHF-derived CMs that are appended to the arterial pole of the heart. Although several signaling pathways have been implicated in regulating SHF differentiation, little is known about which genes function downstream of these key signals to execute OFT assembly or how the multiple relevant pathways interact to set the dimensions of the OFT. Here, we exploit the utility of the zebrafish as a model organism in order to identify novel regulators of OFT formation. Preliminary studies suggest that the zebrafish OFT, like the amniote OFT, is constructed from a population of SHF-derived CMs. Furthermore, in zebrafish, as in amniotes, Fgf signaling is required to promote the production of OFT CMs. However, it is unclear which genes act downstream of Fgf signaling to recruit the appropriate number of CMs into the OFT. Our preliminary data reveal an interesting set of genes - cell adhesion molecule 4 (cadm4), cadm3, and cadm2a - that are repressed by Fgf signaling and play essential roles in restricting the formation of OFT myocardium. These data suggest an intriguing model in which Fgf signaling drives the recruitment of OFT CMs by limiting the expression of cadm genes and thereby altering critical extracellular interactions of SHF-derived progenitor cells. In this proposal, we will test this model in detail by establishing the origins of the zebrafish OFT, deciphering the mechanisms of Cadm function, and integrating the Fgf-Cadm pathway into the context of the multiple influences that converge to define the size of the OFT. In Aim 1, we will employ fate mapping, time-lapse tracking, and assays for the timing of myocardial differentiation to determine whether the zebrafish OFT myocardium is derived from a SHF equivalent. In Aim 2, we will use loss-of-function, gain-of-function, structure-function, and biochemical analyses to test if Cadms mediate extracellular interactions that inhibit recruitment of OFT CMs. In Aim 3, we will identify signals that counterbalance the impact of the Fgf-Cadm pathway on OFT size, focusing on the roles played by Notch, Bmp, and retinoic acid signaling in limiting the dimensions of the zebrafish OFT. Together, these experiments are likely to reveal new mediators of OFT CM recruitment, to uncover a novel mechanism for regulating OFT size through modulation of extracellular interactions, and to shed light on the network of pathways that collaborate to insure an appropriate myocardial foundation for the embryonic OFT.

PROJECT SUMMARY Organogenesis relies upon the carefully coordinated regulation of collective cell movement, in which a group of cells operate as a cohesive entity, coordinating their individual trajectories to reach a common destination. Cardiogenesis, for example, employs collective cell movement during multiple phases of morphogenesis, including the assembly of the heart tube, the protrusion of trabeculae, and the construction of septae. Despite the importance of these morphogenetic processes, we do not yet understand the molecular mechanisms that govern collective cell behavior in the developing heart. In particular, the cues that control the timing and routes of cardiac cell movement remain largely mysterious. Here, we aim to decipher the genetic pathways that control collective cell movement during heart tube assembly in the zebrafish embryo. To build the heart tube, bilateral groups of cardiomyocytes move toward the midline and merge through a process called cardiac fusion. Our prior studies have suggested a model in which interactions between the myocardium, endoderm, and extracellular matrix (ECM) act to facilitate cardiac fusion. However, the elucidation of these tissue-level interactions has not answered key open questions regarding the molecular mechanisms that drive cell behavior. Notably, we do not yet know which signals dictate the direction of cardiomyocyte trajectories or which cues control the rate of cardiomyocyte mobility. It is therefore exciting that we will investigate two novel regulators of cardiomyocyte movement ? the platelet-derived growth factor receptor Pdgfra and the transmembrane protein Tmem2 ? that are poised to address these unresolved issues. First, to test the hypothesis that medially-located PDGF ligands activate Pdgfra in cardiomyocytes and thereby control the direction of cardiomyocyte movement, we will (a) employ time-lapse analysis to pinpoint the impact of pdgfra on myocardial cell behavior, (b) use tissue-specific transgenes to determine where pdgfra acts to influence cardiac fusion, (c) test whether PDGF ligands act as directional cues for myocardial movement, (d) identify effector pathways acting downstream of Pdgfra in this context, and (e) evaluate whether Pdgfra plays a comparable role during cardiac fusion in mouse. Second, to test the hypothesis that the Tmem2 ectodomain facilitates an efficient rate of myocardial motility through modulation of the ECM, we will (a) employ time-lapse analysis to determine the influence of tmem2 on myocardial cell behavior, (b) determine whether tmem2 has a non-autonomous effect on myocardial movement, (c) test whether Tmem2 regulates cardiac fusion by modulating the ECM, and (d) utilize structure-function and proteomic analyses to identify which domains of Tmem2 are required for its function and which proteins interact with these domains. Together, these studies will reveal essential mechanisms of heart tube assembly, uncover new paradigms for the regulation of collective cardiac cell movement, shed light on the origins of congenital heart disease, and facilitate future tissue engineering approaches for cardiac repair.

The Pathways in Biological Sciences (PiBS) Training Program at UCSD is a new T32 Training Program evolved from the successful 40+ year Cell and Molecular Genetics (CMG) Training Program. PiBS will provide an enhanced practices pathway to produce leaders in diverse biology careers, including academic and industrial research, education, writing, consulting, and policy. Trainees will be a select subset of 30 UCSD Biological Sciences PhD students, conducting research in a variety of pressing problems in foundational and translational biology by the use of mechanistic molecular approaches. Students will be invited to become PiBS Trainees at the end of their first year, soon after choosing a thesis advisor, for a two year PiBS-supported period, followed by maintained Trainee status for the remainder of their PhD. PiBS will instill and amplify six core competencies needed for success, including critical thinking, knowledge acquisition, experimental ability with emphasis on rigor, reproducibility and quantitation, effective communication, leadership tools including team building, networking, and collaborative problem solving, and career development. To this end, the PiBS program will conduct a variety of Trainee-specific activities: yearly one-on-one meetings with the PiBS Director, BGGN290 - a class for in-depth analysis and critique of invited seminar speakers, a twice-yearly public colloquium of Trainee research presentations, a yearly Trainee-organized Symposium of invited leaders from a chosen field, a scientific writing workshop, a path-to-career workshop, career networking guidance, an inclusive mentoring workshop, a white-board ?jam? to enhance clear low tech exposition of science, and ?One Book-One Program?- an annual group discussion of a mutually chosen book. The PiBS Directors actively participate in selection and stewardship of Training faculty who can serve as PhD mentors for PiBS Trainees. This will include training of PiBS faculty in dedicated and inclusive mentoring and ongoing assessment of their effectiveness. The PiBS mission includes oversight mechanisms to evaluate the success and effectiveness of the PiBS program with particular emphasis on evaluating our PiBS training faculty, to ensure our vision of involvement, inclusion, best-practices mentoring, rigorous scientific approaches, and effective impartation of the core competencies required for Trainee success. PiBS Trainee outcomes will be clearly documented, continuously curated, and fully available to Trainees and the Division of Biological Sciences through web- based resources to best self-assess our progress and to inform future Trainees about the most impactful choices for their individual career goals. The PiBS mission is deeply dedicated to maximizing the diversity of the Trainee pool to provide opportunities to the broadest pool of talented students; we strive to create high cultural diversity in the Trainee experience, both to immediately foster distinct and creative viewpoints in the graduate environment and to eventually help create a fully diverse mentoring and participant base as the leadership of biological science and biology-oriented careers in the future.

PROJECT SUMMARY Organogenesis requires the execution of interwoven patterning processes that sculpt the distinct functional components of an organ with exquisite specificity. In the context of the embryonic heart, specific territories within each cardiac chamber take on unique attributes: for example, the pacemaker cells that reside within the atrial inflow tract (IFT) have particular conductive properties that are integral to their role in initiating the heartbeat. Cardiac pacemaking activity must be confined to a discrete region of the heart in order to avoid arrhythmia, but we do not yet fully understand the genetic pathways that define the dimensions of the IFT. How are an appropriate number of specialized cardiomyocytes established at the IFT? Prior studies have shown that IFT progenitor cells inhabit discrete outlying regions of the anterior lateral plate mesoderm (ALPM). Moreover, we have demonstrated that canonical Wnt signaling is active in these outlying regions and that the ligand Wnt5b acts to drive IFT differentiation. Thus, Wnt signaling plays a key role in promoting IFT development, but we do not yet understand how Wnt pathway activity is restricted to the edges of the ALPM. Here, we propose to utilize the suite of genetic and embryological approaches available in the zebrafish in order to identify essential patterning mechanisms that constrain IFT dimensions. Importantly, our preliminary studies suggest that the number of IFT cardiomyocytes is constrained through a two-phase process, with distinct signaling pathways operating at successive developmental stages. First, in the early embryo, we propose that Hedgehog (Hh) signaling restricts the allocation of progenitor cells into the IFT lineage. Later, in the ALPM, we propose that Fgf signaling reinforces constraints on the number of IFT cardiomyocytes by restricting the distribution of Wnt signaling. Together, our preliminary data highlight previously unappreciated roles for both Hh and Fgf signaling and suggest a novel model for the molecular mechanisms that restrict the size of the IFT. To test this model, we will employ loss- and gain-of-function analysis, fate mapping, and mosaic analysis in order to (1) determine whether Hedgehog signaling constrains specification of IFT progenitor cells and (2) ascertain whether Fgf signaling constrains differentiation of IFT cardiomyocytes. In addition, our model predicts that IFT progenitor cells possess distinct molecular characteristics prior to their overt differentiation into IFT cardiomyocytes. To test this, we will (3) define the developmental path of IFT progenitors by integrating spatial and transcriptomic data, thereby revealing how the signaling pathways that specify the IFT lineage set the stage for differentiation of the IFT myocardium. Taken together, our proposed studies will provide novel insight into the network of signaling pathways that control IFT dimensions, thereby illuminating new paradigms for the regulation of cardiac patterning. Moreover, our work has the potential to shed light on the developmental origins of congenital cardiac conduction disorders and may also facilitate future innovations in regenerative medicine.