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Figures
- Fig. 1.
RA localization in Raldh2–/– embryos carrying the RARE-lacZ RA-reporter gene. (A-F) Whole-mount staining for expression of RARE-lacZ was performed on Raldh2–/– (A,C,E) and wild-type (B,D,F) embryos at E8.75 (A,B), E9.5 (C,D) and E10.5 (E,F). (G,H) Frontal sections of Raldh2–/– (G) and wild-type (H) embryos at E8.75 show a large difference in RARE-lacZ expression in the eye field. (I,J) The correspondence between Raldh2 expression and RA generation in the eye field and trunk of E8.25 wild-type embryos is demonstrated by RARE-lacZ expression (I) and detection of Raldh2 mRNA by whole-mount in situ hybridization (J). (K-M) Raldh2 mRNA detection is also shown in wild-type embryos at E8.75 (K), E9.5 (L) and E10.5 (M), where it is seen that the eye signal is now gone, but is replaced by nearby maxillary expression. e, eye field surface ectoderm; f, forebrain; h, hindbrain; m, midbrain; mx, maxillary process; ol, olfactory pit; op, optic vesicle.
- Fig. 2.
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.
- Fig. 3.
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).
- Fig. 4.
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.
- Fig. 5.
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.
- Fig. 6.
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.
- Fig. 7.
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.
- Fig. 8.
Early expression of novel RA-generating activities in the neural tube and heart. Staining for RARE-lacZ expression is shown for Raldh2–/– and wild-type embryos treated with only two doses of RA at E6.75 and E7.25 (A-H) or three doses of RA at E6.75, E7.25 and E7.75 (I-L). For the E7.25 rescue: (A) E8.5 embryo shown 30 hours after the last dose; (B) E9.25 embryo shown 48 hours after the last dose; dorsal view of rescued Raldh2–/– embryo at E8.5 (C) and wild-type embryo (D). (E-G) Frontal sections through the hindbrain at E9.25; (H) transverse section through the spinal cord of an E9.25 rescued embryo. For the E7.75 rescue: (I) E9.25 embryo shown 36 hours after the last dose and (J) E10.25 embryo shown 60 hours after the last dose; also shown are higher magnification views at E9.25 of the heart for rescued Raldh2–/– embryo (K) and wild-type embryo (L). a, atrium, c, conotruncus; fp, floorplate; h, hindbrain; he, heart; ot, otic vesicle; r4/5, rhombomere 4/5 boundary; s, somite; sc, spinal cord; sv, sinus venosa; v, ventricle; vi, ventral interneurons.
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