Figure 1.

Transcriptional Profiling of Early NC

(A) Schematic representation of Citrine⁺ NC cells (green) and Citrine⁻ non-NC cells (gray) in early chicken embryos. Red dashed line indicates dissected cranial regions.

(B) In vivo NC-specific activity of FoxD3 enhancer, NC1, at 5-6ss and 8-10ss.

(C) Number of differentially expressed (enriched and depleted) genes in 5-6ss and 8-10ss NC compared to their respective non-NC controls and between stages.

(D and E) Volcano plots showing enriched genes (LogFoldChange >1, base mean >50) in 5-6ss (D) and 8-10ss (E) compared to corresponding non-NC cells; magenta, transcription factors; green, cell-surface molecules; yellow, signaling molecules; blue, differentiation genes. Differential expression was determined using DESeq2 with a negative binomial model, p-values calculated using Wald test, corrected for multiple testing using the Benjamin-Hochberg method (padj).

(F) GO terms associated with NC-enriched genes. Fold enrichments were obtained using statistical overrepresentation test, p-values calculated with binomial distributions and Bonferroni correction for multiple hypothesis testing.

(G) Co-expression clusters of highly correlated genes identified by WGCNA; representative genes are shown. Two replicates per stage for NC and non-NC cells are shown on the x axis.

(H) Cellular co-localization of cluster-iii genes Zfhx4, TFAP2B, Pax7, and Sox10 at 6ss (top panel) and 8ss (bottom panel), detected by HCR.

(I) Co-localization of cluster-iv genes Otogl, Arnt2, and Col9a3 at 8ss.

(J) Co-localization of cluster-vii genes Lmo4, Mef2C, and Col2a1 at 10ss.