Enrichment of Neural Crest Cells by Antibody Labeling and Flow Cytometry for Single‐Cell Transcriptomics in a Lizard

Neural crest cells (NCCs) are a key component of the vertebrate body plan and contribute to a variety of different traits. Recent advances in single-cell transcriptomics (scRNA-seq) have significantly improved our understanding of NCC biology. However, their dynamic migratory behavior and spatiotemporal heterogeneity in the developing embryo pose significant challenges for their identification and isolation. Consequently, most studies of NCCs have been confined to model organisms with established transgenic tools or established methods for in ovo manipulation. To overcome this limitation, we present a novel approach that combines antibody labeling with fluorescence activated cell sorting to enrich for NCCs and we demonstrate the approach in the common wall lizard (Podarcis muralis). Through microscopy, reverse transcription quantitative polymerase chain reaction and single-cell RNA sequencing, we show that the method enriches for NCCs as efficiently as methods relying on transgenic animals. Using this technique, we successfully characterize transcriptional profiles of NCCs in wall lizard embryos. We anticipate that this method can be applied to a wide range of vertebrates that lack transgenic tools, enabling deeper insights into the diverse roles of neural crest cells in development and evolution.