Figure 1.

Transcriptome Characterization of foxd3-Expressing NC

(A) Zebrafish embryo stages examined in this study (hpf – hours post fertilization). 75% epiboly—a gastrulation stage during which embryonic shield and hypoblast are formed. 1–2 and 5–6 somite stages—induced and specified premigratory neural crest (NC), respectively. 14–16ss – migratory NC. Foxd3 expression is labeled in green.

(B) Experimental pipeline for obtaining foxd3-expressing cells and performing single-cell RNA-seq (scRNA-seq). The genetrap zebrafish line, Gt(foxd3-citrine)^ct110, expressing foxd3-Citrine fusion is outcrossed to wild-type resulting in fluorescent signal in endogenous foxd3+ cells, enabling their isolation by FACS. 5–6ss Citrine-positive NC cells are collected into individual wells of the 96-well plate and processed for smartseq2-based scRNA-seq. Total of 94 cells was sorted with two empty, External RNA Controls Consortium (ERCC)-only wells left as controls.

(C) Heatmaps illustrating the hierarchical clustering of foxd3+ single cells at 75% epiboly (200 cells) and 5–6ss (93 cells) and showing transcriptional levels (depicted in Log2 RPKM) of selected NC and stem cell genes. NC cells that express negligent levels of bona fide NC specifiers (zic2b, tfap2a, sox10, twist1b, ets1, or pax3a) but high levels of lratb, cxcr4b, and ved, as well as other markers of stemness (snai1a, vent, vox, and cx43.4), possibly representing pluripotent non-specified NC progenitors maintained in premigratory NC (boxed and labeled in red).

(D) tSNE plots for selected stem cell (sox2, pou2f1, vent) and NC genes (snai1a, sox5, tfap2a, sox10) indicate individual epiblast, and NC cells do not reveal cell subpopulations. Clustering of 5–6ss NC cells identifies a small group of cells that appear to be pluripotent non-specified NC progenitors. See Figure S1 for scRNA-seq quality control (QC) and more details.