Figure 4

Retinoic acid is required for sustained olfactory neurogenesis. (A,B) Pax6 immunochemical detection on sections of the OE of E17.5 wild-type (A) and Raldh3^−/−(B) embryos. Insets show details of the basal region of the epithelium (boxed) at a higher magnification. Asterisks point to dark stained areas probably due to artifactual trapping of the immunochemical reaction product. (C,D) TuJ1 immunofluorescence on sections of the OE of E17.5 wild-type (C) and Raldh3^−/−(D) embryos. While numerous TuJ1+ neurons are seen along the recess of the ethmoturbinate cavity in the mutant (bracket in D), neurons are markedly depleted in more distal regions of the epithelium (compare insets in C and D). (E-I) Detection of fast- and slow-proliferating cells in E17.5 OE after IdU/BrdU injections at E15.5/E16.5 (wild-type: E,F; Raldh3^−/−: G,H). (E,G) BrdU labeling (green). (F,H) Merged labeling, IdU+/BrdU- cells appearing orange to yellow. Insets show higher magnifications of selected regions. (I) Quantification of IdU+ and BrdU+ cells (n = 3 animals/genotype; *: p <0.05). (J-M) Analysis of neuronal regeneration in E17.5 olfactory explants (wild-type: J,L; Raldh3^−/−: K,M) cultured for 48 h in neurobasal medium containing vitamin A, with BrdU added during the first 24 h. (J,K) Explants at the start of culture. (L,M) Explants at 48 h. TuJ1 (green), BrdU (red), DAPI (blue). sc: sustentacular cells; bc: basal cells.