Periodic Reporting for period 2 - Heart2019 (Role of the cardiac neural crest cells in heart development and regeneration)
Período documentado: 2023-01-01 hasta 2023-12-31
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. 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 allowed 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 had 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.
In conclusion, this project revealed that CNC and CNC-derived Schwann cell precursors have regenerative potential for heart repair in 'lower' vertebrates and identified potential candidates related to these regenerative mechanisms. Additionally, the developed tools and datasets were used for several related projects. Finally, this grant was an outstanding experience that provided me with multidisciplinary collaborations and helped me prepare for the role of an independent scientist and leading my own research group.
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. 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 allowed 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 had 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.
In conclusion, this project revealed that CNC and CNC-derived Schwann cell precursors have regenerative potential for heart repair in 'lower' vertebrates and identified potential candidates related to these regenerative mechanisms. Additionally, the developed tools and datasets were used for several related projects. Finally, this grant was an outstanding experience that provided me with multidisciplinary collaborations and helped me prepare for the role of an independent scientist and leading my own research group.
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, 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. 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. This was an unexpected but important finding, and we decided to pursue this in more detail resulting in publication of this part of the dataset in a PNAS article (Stundl et al., 2023). Moreover, employing CM-DiI lineage tracing of CNC cells in lamprey, we made an unexpected observation of their contribution to the sympathetic ganglia. The sympathetic neurons arise from their precursors in close connection to the dorsal aorta, populate the extracardiac space, and then extend posteriorly into the trunk. Our work elucidating this process has been recently accepted for publication. 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. 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.
I put my additional effort into a very detailed characterization of CNC cells in developing and regenerating hearts at the transcriptional and molecular level. 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. 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, such as Sox10. The selected genes were validated by chromogenic in situ hybridization, and by hybridization chain reaction allowing analysis of up to three markers simultaneously. 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. Furthermore, I established and optimized protocols for cell isolations for single-cell RNA-sequencing in sturgeon.
The results of this grant were disseminated in several forms, such as scientific articles, popularization texts, presentations at scientific meetings, exhibition at European Researchers’ Night, and by press release.
I put my additional effort into a very detailed characterization of CNC cells in developing and regenerating hearts at the transcriptional and molecular level. 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. 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, such as Sox10. The selected genes were validated by chromogenic in situ hybridization, and by hybridization chain reaction allowing analysis of up to three markers simultaneously. 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. Furthermore, I established and optimized protocols for cell isolations for single-cell RNA-sequencing in sturgeon.
The results of this grant were disseminated in several forms, such as scientific articles, popularization texts, presentations at scientific meetings, exhibition at European Researchers’ Night, and by press release.
The most promising strategy for understanding common mechanisms of heart regeneration and the role of CNC 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. In addition to the discoveries mentioned above, we have identified a unique and unexpected neural crest morphogenesis in sturgeon and lamprey, 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.