Figure 3

Retinoid deficiency affects the olfactory neuronal lineage. (A-C) Treatment of HH14 stage chicken embryos with disulphiram (B) or AT-RA (C) beads impacts positively or negatively on OP mitotic (pH3-labeled) cells. (D,E) pH3 immunodetection in the OE of E12.5 wild-type and Raldh3^−/− mouse embryos, showing an increased number of labeled cells in the mutant. (F) Quantification of pH3+ (mitotic) or activated-caspase 3+ (apoptotic) cells in disulphiram or AT-RA-treated HH14 chicken OPs (n = 6 embryos/condition, expressed as percentages with respect to controls; *: p <0.05; **: p <0.01). (G) Quantification of pH3+ cells in the OE of E10.5 or E12.5 wild-type and Raldh3^−/− mouse embryos (n = 6 embryos/genotype; three sections of the OE were counted for each embryo; ***: p <0.001). (H) Examples of immunofluorescence detection of Ascl1 (top panels) or Neurog1 (bottom panels), after culturing E10.5 or E12.5 murine olfactory explants for 48 h in defined medium, in the presence (+Vit. A) or absence (−Vit. A) of vitamin A. The corresponding data are quantified in K,L. (I,J) Pax6 immunodetection in the OE of E12.5 wild-type and Raldh3^−/− embryos. (K,L) Quantitative analysis of neuronal progenitor populations in murine olfactory explants collected at E10.5 (left-side histograms) or E12.5 (right-side histograms), from wild-type (K) or Raldh3^−/−(L) mice, and cultured for 48 h in medium containing (+Vit. A, front row) or devoid of (−Vit. A, back row) vitamin A (n = 3 explants per stage and genotype; Additional file 3: Table S1 for statistics). Explants were analyzed by immunofluorescence using anti-Ascl1, -Neurog1, and/or TuJ1 antibodies. (M-P) TuJ1 immunodetection in the OE of E12.5 wild-type (M,O) and Raldh3^−/−(N,P) mice. (O,P) are higher magnifications of the distal OE, taken from specimens labeled with a different fluorochrome.