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.
