Figure 2

Retinoic acid promotes specification of trunk NCCs.

(A) RT-qPCR analysis of cranial (ETS1) and trunk (PHOX2B) NCC markers from H1 and WTC differentiated via Dalton protocol. (B) Schematic detailing the timing of retinoic acid (RA) treatment during NCC differentiation. (C) RT-qPCR analysis for cranial (ETS1) and trunk (PHOX2B) markers from H1 cells differentiated with RA added starting at each day indicated, demonstrating that RA addition at Day 3 or 4 resulted in maximal upregulation of trunk (PHOX2B) and suppression of cranial (ETS1) markers. (D) NCC treated with or without RA were stained for PHOX2B and DAPI. (E) Percentage of PHOX2B⁺ cells (PHOX2B⁺ /DAPI) was quantified using ImageJ. (F,G) NCC differentiated without or with RA (@ day 3) were electroporated with a constitutive expressing mCherry plasmid and a SOX10 E1-GFP reporter activated specifically in trunk NCC. After 72 hours, cells were analyzed for GFP expression via immunofluorescence (F) and quantified using ImageJ (GFP⁺/mCherry⁺) (G) *p < 0.05, **p < 0.01. Scale bars represent 50 μm.