Retroviral Lineage Analysis of the Vagal Neural Crest Reveals Multipotency Towards the Cardiac and Enteric Fates

Citation

Tang, Weiyi (2022) Retroviral Lineage Analysis of the Vagal Neural Crest Reveals Multipotency Towards the Cardiac and Enteric Fates. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/qakz-vm04. https://resolver.caltech.edu/CaltechTHESIS:04162022-233242577

Abstract

The neural crest is a migratory stem cell population that gives rise to the craniofacial skeleton, heart septa, pigment cells, and peripheral nervous system. Defects in neural crest development can lead to a broad range of congenital diseases, e.g., persistent truncus arteriosus, characterized by a mixture of oxygenated and deoxygenated blood, is related to the absence of the neural crest-derived outflow tract septum. Thus, a thorough understanding about neural crest migration, differentiation, and cell fate can shine lights on diagnosis and treatment of many congenital defects. A long-standing question is whether neural crest cells are composed of multipotent cells capable of giving rise to a wide range of cell types, or a mixture of fate-determined cells migrating to their destinations. Avian embryos resemble humans during neural crest development, but are more accessible to experimental manipulations than mammalian models, making them an ideal model to study the neural crest. Despite the abundance of information obtained from elegant experiments through interspecies grafting, the avian model lacks a direct tool to determine whether these cells are multipotent in vivo.

Here, we present a new clonal analysis tool that takes advantage of Replication Incompetent Avian retroviruses (RIAs). We validate the method in vitro and present the potential application in the chick embryo to test the multipotency of the trunk neural crest. Next, we perform RIA-mediated lineage tracing at a population level and uncover cardiomyocytes as a previously unknown cardiac neural crest derivative in both chicken and mouse. Furthermore, we utilize RIA-mediated clonal analysis to identify individual premigratory vagal neural crest cells as a multipotent stem cell that forms cell types in both the heart and the gut. We then confirm the results by single-cell photoconversion assay that further confirms that migrating neural crest cells are also multipotent. Time-lapse imaging shows that stochastic post-mitotic migration is a cellular mechanism underlying multipotency. Finally, molecular perturbation experiments show that CXCR4 and RET are essential guidance cues for migratory neural crest cells to enter the heart and the gut, respectively. Together, these results demonstrate the utility of using RIA viruses to tackle questions regarding the lineage, developmental potential, and migratory pathways followed by neural crest cells in avian embryos.

Thesis Files