Targeted disruption of the aldh1a3 (Raldh3) gene. (A) Structure of the targeting vector (pHR3.3) and partial restriction map (not to scale) of the aldh1a3 locus before (WT allele, +) and after homologous recombination (L3 allele) and Cre-mediated recombination (L2 and L–alleles). Black boxes (7–9) stand for exons. The location of restriction sites (E5, EcoRV; H, HindIII; N, NsiI) and of the 3′ external probe is indicated. On the bottom part of the diagram, the sizes of the restriction fragments obtained for each allele are in kilobases. Arrowhead flags represent loxP sites. Arrows indicate the location of primers 1–3 used for PCR genotyping. (B Left) Southern blot analysis of HindIII-digested genomic DNA from ES cell clones with the indicated aldh1a3 genotype, using the 3′ probe. (Right) PCR analysis of tail genomic DNA from mice with the indicated aldh1a3 genotype. The identities of the different alleles are indicated on the right. (C) Western blot analysis of head proteins (100 μg) from E13.5 fetuses with the indicated aldh1a3 genotype, using an anti-Raldh3 polyclonal Ab. Excision of exons 8 and 9 resulted in a null allele, as confirmed by the absence of Raldh3 (R3) in E13.5 aldh1a3^L–/L– fetuses. The upper band is a nonspecific signal (NS), showing similar amounts of protein on each lane.

Effect of Raldh3 inactivation on RA synthesis at E11.5. (A and D) In situ hybridization with a digoxigenin-labeled Raldh3 antisense probe. (B, C, E, and F) Distribution of β-galactosidase activity driven by the RARE-hsp68-lacZ transgene (18). Raldh3 inactivation suppresses RA synthesis in the ventral retina, ventral cornea, nasal epithelium, and nasolacrimal groove. The disappearance of the RA responsiveness in the mesenchyme adjacent to the ventral retina and to the nasal epithelium may reflect a low level of Raldh3 expression, not detected by in situ hybridization. Note that the areas responsive to RA in the mutant dorsal retina and cornea express Raldh1 at this developmental stage (21, 36). C, cornea; D, dorsal retina; L, lens; M, mesenchyme; N, nasal cavity; NG, nasolacrimal groove; O, optic nerve; V, ventral retina. [Scale bar = 150 μm (A–C) and 100 μm (D–F).]

Raldh3 inactivation results in ocular and nasal defects. (A–H) Frontal histological sections through WT and aldh1a3^KO heads at E14.5 (A and C) and E18.5 (B and D–H). (I and J) Views of the internal surface of the nasal cavities at E18.5. C, cornea; CH, choana; D, dorsal retina; DN, dorsal nasal concha; E1–E3, ethmoturbinates; E2/E3, fused second and third ethmoturbinates; F, persistent nasal fin; L, lens; MS, maxillary sinus; N, nasal cavity; ND, nasolacrimal duct (which drains tears from the conjunctiva of the eye to the nasal cavity); NS, nasal septum; O, optic nerve; P, nasopharynx; R, retrolenticular membrane (persistent primary vitreous body); V, ventral retina; VN, ventral nasal concha. The asterisk in D denotes the absence of the HG. [Scale bar = 150 μm (A and C), 300 μm (B, D, G, and H), and 500 μm (E and F).]

CA, which is caused by the persistence of the nasal fin, is prevented by maternal administration of RA. (A–D) Frontal histological sections of WT and aldh1a3^KO embryos through the posterior portion of the nasal cavities at E11.5. In the WT embryo, the nasal cavity is large and the nasal fin has undergone a cleavage process, giving rise to the oropharyngeal membrane. In the aldh1a3^KO mutant, the cavity is narrow and the fin persists. (E–G) External lateral views of E12.5 heads. (H–J) Frontal histological sections at the level of the vomeronasal organ at E12.5. The embryos in G and J were from a mother treated with RA by oral gavage between E9 and E11. This RA treatment permits opening of the choana into the oral cavity and allows invagination of the nasolacrimal groove ectoderm but at this stage does not fully restore the growth of the ventral retina. (K) WT and aldh1a3^KO mice (1 mo old) born from a mother given dietary nonteratogenic doses of RA from E8.5 to E14.5. (L–O) Frontal histological sections through heads of mice shown in K. The nasal defects and HG agenesis caused by aldh1a3 knockout are prevented. Note, however, that a normal shape of the ethmoturbinates is not totally restored. Additionally, examination of serial sections shows that the opening of the nasal cavity into the nasopharynx is reduced in length (0.2 mm instead of 1 mm in WT). B, brain; CH, choana; D, dorsal retina; E, ethmoturbinate; F, nasal fin; L, lens; M, molar; MS, maxillary sinus; N, nasal cavity; ND, nasolacrimal duct; NG, nasolacrimal groove; NL, nasolateral process; NM, nasomedial process; NS, nasal septum; O, oropharyngeal cavity; OM, oropharyngeal membrane; P, nasopharynx; R, retina; S, secondary palate; T, tongue; V, ventral retina; VO, vomeronasal organ; *, blind choanal pit. [Scale bar = 150 μm (A and C), 50 μm (B and D), 300 μm (H–J), and 1.2 mm (L–O).]

Persistence of Fgf8 expression in the nasal fin of aldh1a3^KO mutants. (A–D, F–I, and K–N) External views of whole-mount in situ hybridizations of the nasal region of WT and aldh1a3^KO embryos from E10 to E11 seen from the front with digoxigenin-labeled Raldh3 and Fgf8 antisense probes. (E, J, and O) In situ hybridization with digoxigenin-labeled Raldh3 or Eya2 antisense probes on frontal histological sections through the posterior portion of the nasal cavities at E11. Embryos in C, H, and M are identical to D, I, and N, respectively, but their heads were slightly tilted to better visualize the ventral portion of the nasal fin. Note that expression of Fgf8 in E10 and E10.5 aldh1a3^KO embryos was indistinguishable from that in WT. At E11, the nasal fin no longer expresses Fgf8 in WT embryos, even though it is still present as demonstrated by Eya2 expression. F, nasal fin; L, nasolateral process; M, nasomedial process; N, nasal cavity. [Scale bar = 80 μm (E, J, and O).]