Figure 5.

Aldh1a2-independent RA signaling during PS-like differentiation

(A) Reporter expression after 5 days of PS-like differentiation of 2KI Aldh1a2^−/− mESCs transgenic for an RA signaling reporter (DR5-RARE-Scarlet). Bar: 100 μm.

(B) Scheme of the experimental principle to monitor the impact of perturbing RA signaling on the PS-like differentiation of Aldh1a2^−/− mESCs.

(C and D) Sox1-GFP reporter expression in Aldh1a2^−/− cells after PS-like differentiation. (C) (black: control, orange: AGN, red: vitamin A and quantification). (D) (n = 3 independent experiments; ^∗∗, p < .01; two-sided unpaired t test; data represented as mean ± SD).

(E) Scheme of an RA-responsive transcriptional reporter relying on three RA-responsive elements (RARE), each consisting of three RAR binding sites (DR, direct repeats; cDR, composite direct repeat; min CMV, minimal CMV promoter; BGH pA, bovine growth hormone poly A).

(F and G) Reporter expression after 5 days of PS-like differentiation of 2KI wild-type (Aldh1a2^+/+) (F) or 2KI Aldh1a2^−/− (G) mESCs transgenic for the cDR RA signaling reporter. Bar: 100 μm.

(H) Sox1-GFP reporter expression after PS-like differentiation of 2KI cells (gray) or Rbp1^−/−Stra6^−/− cells without (blue) or with (red) additional vitamin A.

(I) Quantification of (H) data (n = 3 independent experiments; ^∗, p < .05; ^∗∗∗, p < .001; two-sided unpaired t test; data represented as mean ± SD).

(J) Sox1-GFP and cDR-RARE-Scarlet reporter expression after PS-like differentiation of 2KI mESCs, 2KI mESCs transgenic for the cDR RA signaling reporter (Aldh1a2^+/+), Aldh1a2^−/−, or Aldh1a2^−/−Rbp1^−/−Stra6^−/− mESCs. Dotted lines: gates fixed according to the non-transgenic mESCs negative control. See also Figure S5.