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First published online 5 October 2005
doi: 10.1242/dev.02031

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Retinoic acid-dependent eye morphogenesis is orchestrated by neural crest cells

Nicolas Matt^*, Valérie Dupé^*, Jean-Marie Garnier, Christine Dennefeld, Pierre Chambon, Manuel Mark and Norbert B. Ghyselinck

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut Clinique de la Souris (ICS), CNRS/INSERM/ULP, Collège de France, BP10142, 67404 Illkirch Cedex, CU de Strasbourg, France

View larger version (45K): [in a new window]   Fig. 1. Targeted disruption of the Aldh1a1 gene encoding RALDH1. (A) Structure of the targeting vector and partial restriction map of the Aldh1a1 locus before [wild-type (WT) allele, +] and after homologous recombination (L3 allele) and Cre-mediated recombination (L2 and L-alleles). Black boxes (labeled 7-9) stand for exons. The location of restriction sites (B, BamHI; H, HpaI; S, SalI) and of the 3' external probe is indicated. Arrowhead flags represent loxP sites. Arrows indicate the location of primers 1 and 2 used for PCR genotyping. Sizes of the restriction fragments obtained for each allele are shown below and are in kilobases (kb). (B) Southern blot analysis of HpaI-digested genomic DNA from ES cell clones with the indicated Aldh1a1 genotype, using the 3' probe. (C) PCR analysis of tail genomic DNA from mice with the indicated Aldh1a1 genotype. The identities of the different alleles are indicated on the right. (D) Western blot analysis of liver proteins (50 µg) from adult mice with the indicated Aldh1a1 genotype, using anti-RALDH1 (upper panel) and anti-RALDH3 (lower panel) polyclonal antibodies, showing the absence of RALDH1 (R1) and a normal amount of RALDH3 (R3) in Aldh1a1L–/L– mice.

View larger version (105K): [in a new window]   Fig. 2. Expression patterns of RALDH1, RALDH2 and RALDH3 during eye development. In situ hybridization using digoxigenin-labeled Aldh1a1 (A,D,G), Aldh1a2 (B,E,H) and Aldh1a3 (C,F,I) antisense riboprobes at E10.5 (A-C), E11.5 (D-F) and E13.5 (G-I). Arrowheads (C,F) indicate the peripheral portion of the dorsal retina. c, presumptive corneal ectoderm; d, dorsal retina; le, lens; m, periocular mesenchyme; mu, muscle; o, optic nerve; rpe, retinal pigment epithelium; v, ventral retina. Scale bar in I: 40 µm for A-C; 50 µm for D-F; 80 µm for G-I.

View larger version (103K): [in a new window]   Fig. 3. Effects of RALDH1 and RALDH3 inactivation, alone or in combination, on the RA-dependent activity of a reporter transgene. Distribution of ß-galactosidase activity driven by the RARE-lacZ transgene in wild-type (A,E,I), Aldh1a1-null (B,F,J), Aldh1a3-null (C,G,K) and Aldh1a1/3-null (D,H,L) mutants at E10.5 (A-D), E11.5 (E-H) and E13.5 (I-L). Red arrowheads (E,H) indicate dorsal and ventral eyelid grooves. Black arrowhead (J) indicates the most peripheral portion of the dorsal retina where RARE-lacZ activity is not abolished in Aldh1a1-null fetuses. The faint staining observed at E10.5 in the retina of Aldh1a1/3-null mutants (D) reflects a residual activity of the ß-galactosidase synthesized at an earlier stage, as no lacZ mRNA can be detected using in situ hybridization. Note also that the ß-galactosidase activity detected at E13.5 in the ventral retina of the Aldh1a3-null fetus (K) results from expression of Aldh1a1. c, presumptive corneal ectoderm; d, dorsal retina; le, lens; m, periocular mesenchyme; o, optic nerve; rpe, retinal pigment epithelium; v, ventral retina. Scale bar in L: 40 µm for A-C; 50 µm for D-F; 80 µm for G-I.

View larger version (96K): [in a new window]   Fig. 4. Ablation of RALDH3 alters development of the ventral periocular mesenchyme. (A,D) Frontal histological sections through the head of E12.5 wild-type (A) and Aldh1a3-null (D) fetuses. (B,E) Distribution of apoptotic cells (assessed by TUNEL assays) in E10.5 wild-type (B) and Aldh1a3-null (E) fetuses. (C,F) Whole mount in situ hybridization with a digoxigenin-labeled Eya2 antisense riboprobe on E11.5 wild-type (C) and Aldh1a3-null (F) fetuses. Note that the histological sections illustrated have been selected to provide the best match between mutant and wild-type embryos. Arrows indicate the optic nerve exit point; red brackets indicate the periocular mesenchyme. d, dorsal retina; e, eye cup; f, forebrain; g5, ganglion of the Vth nerve; le, lens or lens placode; v, ventral retina. Scale bar in D: 80 µm for A,D; 25 µm for B,E.

View larger version (98K): [in a new window]   Fig. 5. Absence of RA signaling in the periocular mesenchyme results in severe ocular defects. (A-E) External views of the ocular region of E14.5 fetuses (genotypes as indicated). (F-L) Frontal histological sections through heads of E14.5 (F-J) and E18.5 (K,L) fetuses. (M,N) Distribution of ß-galactosidase activity driven by the R26R transgene in E10.5 embryos bearing the Wnt1-Cre transgene either in a wild-type genetic background (M, WTNCC+/+ lacZ), or in a genetic background containing loxP-flanked Rarb and Rarg genes (N, Rarb/gNCC–/– lacZ). In both WTNCC+/+ lacZ and Rarb/gNCC–/– lacZ embryos, the ß-galactosidase staining is identical, indicating that ablation of RARß and RAR does not alter the migration of neural crest cells (NCCs) in the periocular mesenchyme. Asterisks (I,J,L) indicate undifferentiated mesenchyme replacing the eyelids and cornea; dotted lines (F-J) indicate the equatorial plane of the lens; arrowhead (L) points to the coloboma of the retina. a, anterior chamber; c, cornea; d, dorsal retina; e, eyelid; i, iris; j, conjunctival sac; le, lens; m, periocular mesenchyme; o, optic nerve; rpe, retinal pigment epithelium; r, retrolenticular membrane; s, sclera; v, ventral retina. Scale bar in N: 120 µm for F-J; 250 µm for K,L; 25 µm for M,N.

View larger version (89K): [in a new window]   Fig. 6. Cell proliferation in the periocular mesenchyme is not altered upon ablation of RALDH1 and RALDH3, or of RARß and RAR. (A-C) Distribution of proliferating cells (assessed by Ki-67 expression) in E11.5 wild-type (A), Aldh1a1/3-null (B) and Rarb/gNCC–/– lacZ (C) fetuses. The histological sections illustrated have been selected to provide the best match between mutant and wild-type embryos. d, dorsal retina; le, lens; m, periocular mesenchyme; o, optic nerve; v, ventral retina. Scale bar: 50 µm.

View larger version (124K): [in a new window]   Fig. 7. Ablation of RALDH1 and RALDH3 impairs programmed cell death and differentiation in the periocular mesenchyme. (A,B,D,E) Distribution of apoptotic cells (assessed by TUNEL assays) in E11.5 wild-type (A,B) and Aldh1a1/3-null (D,E) fetuses. (C,F,G-N) In situ hybridization with digoxigenin-labeled Eya2 (C,F), Foxc1 (G,K), Pitx2 (H,L) Fgfr2 (I,M) and Pax6 (J,N) antisense riboprobes on frontal sections through the eye of E11.5 wild-type (C,G,H,I,J) and Aldh1a1/3-null (F,K,L,M,N) fetuses. Note that the histological sections illustrated have been selected to provide the best match between mutant and wild-type embryos. Red brackets indicate the periocular mesenchyme. d, dorsal retina; le, lens; o, optic nerve; rpe, retinal pigment epithelium; v, ventral retina. Scale bar: 50 µm.

View larger version (75K): [in a new window]   Fig. 8. Ablation of Rarb and Rarg in neural crest cells impairs apoptosis and gene expression in the periocular mesenchyme. (A-D) Distribution of apoptotic cells (assessed by TUNEL assays) in E11.5 wild-type (A,B) and Rarb/gNCC–/– lacZ (C,D) fetuses. (E-H) In situ hybridizations with digoxigenin-labeled Eya2 (E,G) and Foxc1 (G,H) antisense riboprobes on frontal sections through the eye of E11.5 wild-type (E,F) and Rarb/gNCC–/– lacZ (G,H) fetuses. The histological sections illustrated have been selected to provide the best match between mutant and wild-type embryos. (A-D) Sections are counterstained with methyl green. Red brackets indicate the periocular mesenchyme. d, dorsal retina; le, lens; o, optic nerve; rpe, retinal pigment epithelium; v, ventral retina. Scale bar: 50 µm.

View larger version (96K): [in a new window]   Fig. 9. RA is not required for establishing the dorsoventral axis of the retina. (A-D) Whole mount in situ hybridization of E10.5 wild-type (A,C) and Aldh1a1/3-null (B,D) fetuses using digoxigenin-labeled antisense riboprobes for Tbx5 (A,B) and Vax2 (C,D). (E,F) In situ hybridization with a digoxigenin-labeled Pax2 (E,F) antisense riboprobe on frontal sections through the eye of E11.5 wild-type (E) and Aldh1a1/3-null (F) fetuses. (G,H) Distribution of apoptotic cells (assessed by TUNEL assays) in E10.5 wild-type (G) and Aldh1a1/3-null (H) fetuses. d, dorsal retina; le, lens; rpe, retinal pigment epithelium; v, ventral retina. Scale bar: 50 µm.

View larger version (68K): [in a new window]   Fig. 10. RA-dependent eye morphogenesis is orchestrated by the NCC-derived periocular mesenchyme. RALDH1 (red) and RALDH3 (orange), expressed in the retina, the retinal-pigmented epithelium, the lens and the corneal ectoderm, are the only RA-synthesizing enzymes required in the eye region from E10.5 onwards. The RA synthesized in these locations (yellow arrows) diffuses towards the periocular mesenchyme (POM, blue), where it determines eye morphogenesis through the activation of RXR/RARß and RXR/RAR heterodimers. The latter may control (1) the Eya2-dependent apoptosis involved in POM remodeling; (2) the expression of Foxc1 and Pitx2 transcription factors that are required for the eye anterior segment morphogenesis; and (3) the production of an unknown signal (question mark) necessary for the growth of the ventral retina. Asterisk indicates the RA-depleted zone expressing the catabolizing enzymes CYP26A1 and CYP26C1. This drawing is representative of the E11.5 condition.