Fig 1: Low density isolated cell analysis of intermediate epiblast region identifies cell autonomous specification of neural crest in culture.(A) Schematic showing HH stage 8 and EGK stage XII embryos used for low density isolated cell analysis and the workflow. Cells were dissociated from Stage 8 embryo marked by Pax7 expressing neural fold region (boxes sections within the red region) (positive control), plated, fixed and immunostained for Pax7/Sox9. Stage XII embryo explants marked as 1 (intermediate) and 2 (medial) regions were dissociated and cultured at very low density (10–20 cells/cm2) on a thin layer of collagen gel under non-inducing conditions for 30h. (B) Isolated cells in the low density culture were immunostained for Pax7/Sox9 expression from neural fold region, intermediate region (section 1) at time 0 (immediately after plating), intermediate region (section 1) used as no primary control, and from sections 1 and 2. Intermediate region (section 1) had the highest Pax7 positive cells (38%) with 8% Pax+ cells in medial region (n = 4). (C) Explants from EGK St. XII epiblast were dissected, dissociated and cultured for 25h as described in (A) in presence or absence of Wnt-ligand inhibitor (WIF-1) or β-catenin inhibitor (XAV939) and processed for Pax7 immunofluorescence. Six panels from 4 individual embryo explants show distant cells cultured in isolation express Pax7 under control and WIF-1 treated conditions, while no Pax7 expression was seen under XAV939 treatment. (D) Average cell counts for Pax7 positive cells from the three different conditions, untreated control, WIF-1-treated and XAV939-treated (n = 3), and (E) Total DAPI positive cells under these conditions (n = 3). Error bars represent SEM from three separate embryo. Cell counts are provided in supplemental Table 1.