Gene expression in the eye of Raldh2^–/– embryos. Whole-mount in situ hybridization was performed in Raldh2^–/– and wild-type embryos to examine expression of Raldh1 at E9.5 (A,B), Tbx5 at E9.5 (C,D), Raldh3 at E8.75 (E,F) and Pax2 at E9.5 (G,H). dr, dorsal retina; e, eye field surface ectoderm; vr, ventral retina.

Clearance of maternally administered RA in rescued Raldh2^–/– embryos carrying RARE-lacZ. The time course of RARE-lacZ induction was examined in Raldh2^–/– and wild-type embryos following RA administration at 12-hour intervals from E6.75-E8.25. β-galactosidase activity is shown at E8.5, 6 hours after the last RA dose (A,F); E8.75, 12 hours after the last RA dose (B,G); and E9.25, 24 hours after the last RA dose (C,H). For comparison, RARE-lacZ expression in untreated embryos of the same stages can be seen in Fig. 1. Expression of Cyp26A1 during RA clearance was monitored in Raldh2^–/– and wild-type embryos by whole-mount in situ hybridization at E8.75, 12 hours after the last RA dose (D,I); and E9.25, 24 hours after the last RA dose (E,J).

Endogenous RA localization in conditionally rescued Raldh2^–/– embryos carrying RARE-lacZ. Staining for expression of RARE-lacZ was performed on Raldh2^–/– and wild-type embryos treated with RA every 12 hours from E6.75-E8.25, then examined at E9.5 (A,B) or E10.5 (C,D). f, forebrain; f/m, forebrain/midbrain junction; h, hindbrain; he, heart; m, midbrain; mx, maxillary process; n, nasolacrimal groove; ol, olfactory pit; op, optic vesicle; sc, spinal cord; u, urinary tract; *, truncated forelimb bud.

Novel RA-generating activity in neural tissues. Shown are vibratome sections of E10.5 Raldh2^–/– and wild-type embryos treated with RA to E8.25 showing localization of RARE-lacZ expression. (A,B) Transverse sections of the posterior spinal cord; the lower level of β-galactosidase activity observed in the intermediate region (vi) of the wild-type relative to the corresponding region of the mutant may be due to sequestration of the substrate (X-gal) by the high level of β-galactosidase present dorsally and in the somites, thus reducing the amount of substrate able to diffuse into the intermediate region during staining of whole-mount embryos; alternatively, the mutant may have higher expression of the novel enzyme. (C-H) Frontal sections of forebrain (C,D); frontal sections of midbrain (E,F); and oblique transverse sections through the hindbrain just rostral to the occipital somites (G,H). drg, dorsal root ganglia; f, forebrain; f/m, forebrain/midbrain junction; fp, floorplate; m, midbrain; r, rhombomere; s, somite; vi, ventral interneurons.

Induction of Raldh1 in dorsal retina of Raldh2^–/– embryos by conditional RA rescue. (A,B) RARE-lacZ expression in the eye of E10.5 Raldh2^–/– embryos treated with RA to E8.25 is shown (A) as a whole-mount stained for only 2 hours and (B) as a frontal section to depict the high level of expression in dorsal retina. (C,D) Combined detection of Raldh1+Raldh2 mRNAs by whole-mount in situ hybridization at E9.75, using both probes simultaneously, for Raldh2^–/– (C) and wild-type (D) embryos treated with RA to E8.25; this shows that the rescued mutant, which has very low Raldh2 mRNA detection relative to wild type, now expresses Raldh1 in the dorsal retina. dr, dorsal retina; ol, olfactory pit; rpe, retinal pigment epithelium.

RARE-lacZ and Raldh3 expression in the eye, olfactory pit and ureteric bud of conditionally rescued Raldh2^–/– embryos. (A) RARE-lacZ staining in the head of an E10.5 Raldh2^–/– embryo treated with RA to E8.25 is shown in a sagittal section. (B,C) Combined detection of Raldh3+Raldh2 mRNAs by whole-mount in situ hybridization at E9.5 using both probes simultaneously for E9.5 Raldh2^–/– (B) and wild-type embryos treated with RA to E8.25 (C); the rescued mutant expresses Raldh3 in the optic vesicle and olfactory pit. (D,E) Higher magnification views of Raldh3 mRNA detection in the head of conditionally rescued Raldh2^–/– embryos at E9.5 (D) and E10.5 (E). (F-H) Analysis of posterior urinary tract of E10.5 Raldh2^–/– embryos treated with RA to E8.25 shows expression of RARE-lacZ in whole-mount and frontal section, respectively (F,G), plus expression of Raldh3 mRNA in whole-mount (H). f, forebrain; m, midbrain; ol, olfactory pit; op, optic vesicle; rpe, retinal pigment epithelium; vr, ventral retina; u, urinary tract.