Xenbase Image ID: 176850

Figure S3. Characterizing Molecular and Cellular Phenotypes in FHS Mutant Cranial Neural Crest Cells, Related to Figure 3. A) Confocal microscopy of immunofluorescence staining in WT and FHS MUT SRCAP CNCCs against canonical NCC markers NR2F1 (red), TFAP2A (orange), and p75 (green) with DAPI nuclear stain (blue). 10μm scale bars shown. (B) Fluorescence activated cell surface marker analysis of WT (WT-V5 lines #1-#3) and FHS MUT (FHS-V5 lines #1-#3) NCCs, with markers CD271 PE-Cy7, CD266 PE, CD105 PacificBlue, CD24 PerCP-Cy5, and CD10 APC. All are previously characterized markers for normal neural crest cells. (C) Enrichment of genes involved in calcium-dependent cell-cell adhesion (GO:0016339) with gene enrichment in red and associated gene names in blue. (D) Enrichment of migration genes enriched in HHSt11cluster 1 (Morrison et al., 2017) that are affected in FHS MUT SRCAP CNCCs with gene enrichment in red and associated gene names in blue. (E) Cell cycle analysis of WT (WT-V5 lines #1-#3) and FHS (FHS-V5 lines #3) NCCs for two distinct differentiations with cell cycle phases identified using Click-iT and FxCycle DNA staining with flow cytometry analysis. No significant differences detected by binomial regression. Confidence intervals obtained using quasi-binomial regression in glm. (F) CD24 flow cytometry analysis of WT and FHS MUT NCCs. CD24 by CD10 in one representative experiment, with three WT and three FHS MUT lines differentiated to CNCCs. (G) Fraction of CD24-low CNCCs compared between WT (orange) and FHS MUT (blue) over n=3 independent differentiations. Statistical analysis was analysis of covariance test with p=0.00003. (H) Imaging time course from migration assay (see Figure 3H) showing embryos selected for correct fluorescence at neurula stage (time=0 hours), migratory stage (time=5 hours) and tailbud stage (time=15 hours). 250μm scale bar shown. White line for each branchial arch and diagram on right shows number and patterning of branchial arches counted for quantifications. Image published in: Greenberg RS et al. (2019) Copyright © 2019. Image reproduced with permission of the Publisher, Elsevier B. V. Gene Synonyms Species Stage(s) Tissue nr2f1.L COUP transcription factor 1 X. laevis Sometime during NF stage 18 to NF stage 20 neural crest tfap2a.L ap2, AP2a, AP2alpha, tfap2a-a, tfap2a-b, tfap2alpha, XAP-2, xap2 X. laevis Sometime during NF stage 18 to NF stage 20 neural crest ngfr.L p75NTR, p75NTRa, p75NTRb X. laevis Sometime during NF stage 18 to NF stage 20 neural crest Image source: Published Larger Image Printer Friendly View

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