Clonal analysis and dynamic imaging identify multipotency of individual Gallus gallus caudal hindbrain neural crest cells toward cardiac and enteric fates

Neural crest stem cells arising from caudal hindbrain (often called cardiac and posterior vagal neural crest) migrate long distances to form cell types as diverse as heart muscle and enteric ganglia, abnormalities of which lead to common congenital birth defects. Here, we explore whether individual caudal hindbrain neural crest precursors are multipotent or predetermined toward these particular fates and destinations. To this end, we perform lineage tracing of chick neural crest cells at single-cell resolution using two complementary approaches: retrovirally mediated multiplex clonal analysis and single-cell photoconversion. Both methods show that the majority of these neural crest precursors are multipotent with many clones producing mesenchymal as well as neuronal derivatives. Time-lapse imaging demonstrates that sister cells can migrate in distinct directions, suggesting stochasticity in choice of migration path. Perturbation experiments further identify guidance cues acting on cells in the pharyngeal junction that can influence this choice; loss of CXCR4 signaling results in failure to migrate to the heart but no influence on migration toward the foregut, whereas loss of RET signaling does the opposite. Taken together, the results suggest that environmental influences rather than intrinsic information govern cell fate choice of multipotent caudal hindbrain neural crest cells. Neural crest stem cells formed from the caudal hindbrain migrate long distances to the heart and gut, but how cell fate is determined is unclear. Here, the authors use multiplex clonal analysis and single-cell photoconversion lineage tracing to show environmental not intrinsic factors affect the cell fate of multipotent caudal hindbrain cells in the chick.