Periodic Reporting for period 1 - Heart2019 (Role of the cardiac neural crest cells in heart development and regeneration) Reporting period: 2021-01-01 to 2022-12-31 Summary of the context and overall objectives of the project The neural crest is a key stem cell population characterized by its multipotency, migratory behavior, and broad ability to differentiate into many derivatives, such as the craniofacial skeleton, melanocytes, and various parts of the cardiovascular system. The cardiac neural crest (CNC) is one of the most unique neural crest subpopulations that contributes to the proper development of the parts of the heart including the outflow tract, coronary vessels, valves, and cardiomyocytes. Classical and genetic ablation studies in chick and zebrafish embryos showed that removal and dysregulation of CNC development led to a broad range of heart defects and diseases, including persistent truncus arteriosus, cardiomyopathies and heart failure. Heart defects are the most common congenital birth defects in newborns, and defects in septation of the heart and outflow tract are among the most common defects of the general population requiring surgical repair. For example, Jacobsen syndrome is often associated with ventricular septal defects caused by a mutation in Ets-1 gene which is known to be associated with CNC development. In addition, the loss of Ets-1 also negatively influences the expression of the pan-neural crest marker Sox10 leading to defects in neural crest migration. The CNC also has a key role in the repair and healing of damaged heart muscle in fish. Recently, it has been shown that zebrafish CNC cells may represent a population of quiescent stem cells that differentiate to cardiomyocytes after myocardium injury by redeploying a neural crest developmental gene regulatory program. Based on our current knowledge, only a limited number of vertebrates are capable of scar-free regeneration of myocardium with newly differentiating cardiomyocytes entirely replacing the injured tissue. Although the data from zebrafish demonstrate that CNC cells proliferate and differentiate into new cardiomyocytes upon injury, not much is known about their role in the heart development and repair in basal vertebrates. Thus, using a multiorganismal approach allows us to discover fundamental mechanisms driving heart regeneration across vertebrates and better understand the potential of applying this knowledge to heal the human heart after a heart attack and during heart damage.The project has four main objectives as follows:1. To examine the fate of CNC and identify its developmental potential in lamprey, sturgeon, and salamander.2. To determine the developmental potential of CNC to differentiate into cardiomyocytes.3. To define whether CNC cells represent a source of “stem cells” for heart regeneration.4. To determine whether the mechanism by which CNC cells contribute to heart regeneration is common between basal and jawed vertebrates. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The migratory pathways and derivatives formed by neural crest are regionalized according to their axial level of origin, such that cells from a given axial level give rise to a characteristic array of progeny and follow distinct pathways. Thus, as the first part of this project (Specific Aim 1: To examine the fate of CNC), I focused on the identification of when (at which developmental stage) and where (at which axial level which may vary across vertebrates) CNC arises, migrate and what is the fate of this cell population. For this purpose, I primarily used focal injections of CM-DiI into the premigratory CNC cells at different axial levels (WPD1.1) and I identified significant differences in the fate of CNC between different species, e.g. in contrast to zebrafish, lamprey CNC does not contribute to the ventricle. From the CM-DiI labeling experiments, I also discovered a unique developmental ability of trunk neural crest cells to form bone, which has important implications for our understanding of neural crest evolution. These results will be published soon. Besides CM-DiI injections, I also used different fate mapping techniques, such as using replication-incompetent viruses that allow us to stain the entire cell progeny (WPD1.2). Unfortunately, this technique did not work in our model organisms. Given that CNC cells give rise to a portion of cardiomyocytes in zebrafish and chick, I have analyzed the ability of CNC in our models to differentiate into the cardiomyocytes by performing hybridization chain reaction and immunohistochemistry on CM-DiI injected samples. Simultaneously, I also asked whether the progeny of these cells might contribute to heart regeneration and for this purpose I established procedures for heart resections in our model organisms (WPD1.4). I put my additional effort into a very detailed characterization of CNC cells in developing and regenerating hearts at the transcriptional and molecular level (Specific Aim 2: Transcriptional profiling). For that reason, I collected injured ventricles at different time points, performed transcriptional profiling, and analyzed the RNA-sequencing results, including the comparison of these datasets with data from uninjured ventricles and our zebrafish datasets (WPD2.1). This approach helped us to determine transcription factors and signaling molecules that are enriched in injured hearts including the neural crest stem cell transcription factors, Sox10. The selected genes were validated by chromogenic in situ hybridization, and by hybridization chain reaction allowing analysis of up to three markers simultaneously (WPD2.2). I identified that Ccn2a, one of the most interesting genes associated with heart regeneration, is co-expressed with Sox10. Interestingly, I also identified similar expression patterns of these genes in other tissues after injury. For the functional validation of candidate genes, we optimized protocols for CRISPR/Cas9 mutagenesis, which led to a publication (WPD2.3; WPD 3.1). Furthermore, I established and optimized protocols for cell isolations for single-cell RNA-sequencing in sturgeon (WPD2.4). Importantly, this allows us to obtain a detailed characterization of CNC cells in heart development and repair. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The most promising strategy for understanding common mechanisms of heart regeneration and the role of cardiac neural crest in this process is to focus on basal vertebrates such as lamprey or sturgeon. To date, no developmental data describe cardiac neural crest cells in lamprey or sturgeon. Even though Covid pandemic has negatively impacted major aspects of this project, I have made significant progress in all main objectives. In addition to the discoveries mentioned above, I have identified a unique and unexpected neural crest morphogenesis in sturgeon, which has a significant value for understanding neural crest evolution in vertebrates. Although the scientific outcomes from this project are in the area of basic research and have an impact on the scientific community, the results of neural crest regenerative potential may contribute to knowledge that helps promote heart regeneration after injury. This project is still ongoing and promises exciting results, and we expect to have a much better understanding of the neural crest roles in the development and repair of vertebrate tissues such as heart. Cardiac neural crest contribution into the heart of lamprey (A), sturgeon (B), and salamander (C).
