Delamination of neural crest cells requires transient and reversible Wnt inhibition mediated by Dact1/2. Gallus gallus

Neural crest cells are a transient embryonic population, hence are neither present after bith and nor are they readily accesible for analysis. Therefore, little is known about the genetic networks that regulate NC especification, delamitation and migration from the dorsal neural tube to their final destination along the embryo. Here we have developed a novel resource for lineage tracing and for the isolation of neural crest cells in the chick embryo, enabling us to perform a genome-wide expression screen in neural crest progenitors versus neuroepithelial cells. Overall design: Plasmid DNA encoding the pBRE-tk-EGFP reporter construct and the pCAGGS-IRES-H2B-RFP construct were coelectroporated into HH10/11 chick embryos and the NT was dissected out of the embryos 24 h later (HH10/11+24hpe). Nearly 30 electroporated embryos were pooled, and a single cell suspension was obtained. GFP and RFP fluorescent cells were sorted using a MoFlo flow cytometer (DakoCytomation, Fort Collins, Colorado, USA). At least 100,000 RFP+ cells and 20,000 GFP+ cells were obtained per pool, and total RNA was extracted from the resulting cell sorted populations, 90% of RFP+/GFP+ cells or 90% of RFP+/GFP- cells, using a standard Trizol (Promega) protocol.