Study of cranial neural crest cells positional identity significance and its influence on their plasticity

Cranial neural crest cells (CNCC) are a transient cell population that exhibits an anterior-posterior (AP) patterning reflective of where they originate in the neuroepithelium. However, contrary to most patterned tissues, this early positional information is lost upon delamination, with migratory CNCC adopting a uniform transcriptional signature which later re-diversifies as CNCC undergoes first commitment events allowing them to adapt to signals encountered during their migration towards the facial prominences. The mechanisms enabling CNCC to reprogram their positional identity are not fully understood. To study the molecular regulations of this process, we tested how an in vitro CNCC differentiation protocol recapitulates CNCC positional identity remodeling. By analyzing the expression of CNCC markers AP2α, Sox9, Twist1 and Pax7 we first determined that this in vitro system robustly generates CNCC that delaminate from the neurosphere and migrate extensively. We showed this protocol recapitulates CNCC AP patterning by analyzing the expression of positional markers Otx2 and Gbx2. Furthermore, we established this AP regionalization is set up progressively during neurospheres maturation and lost upon CNCC delamination and migration. Next, we assessed how changing the levels of FGF – a secreted signaling molecule described to have a posteriorizing effect on CNCC development – affects neurosphere AP patterning. While high levels of FGF inhibits CNCC formation, we showed that low FGF concentrations results in neurospheres with a more balanced AP patterning together with a robust production of CNCC. Lastly, we devised a new protocol to generate neurospheres by cell aggregation. This technique produces neurospheres of uniform size and AP patterning compared to neurospheres grown on cell culture plates. Altogether, our results show that our in vitro CNCC differentiation protocol represent a reliable tool to investigate the molecular mechanisms regulating CNCC positional identity remodeling during development.