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First published online 12 April 2006
doi: 10.1242/dev.02328

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Retinoic acid guides eye morphogenetic movements via paracrine signaling but is unnecessary for retinal dorsoventral patterning

Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.

View larger version (71K): [in a new window]   Fig. 1. Morphogenetic movement defects in developing mouse eyes deficient for RA synthesis. (A-D) Hematoxylin and eosin staining of frontal sections through the eye of E14.5 wild-type (WT), Raldh1-/- (R1-/-), Raldh3-/- (R3-/-) and Raldh1-/-;Raldh3-/- double null (R1;R3-/-) embryos. The double-arrow indicates thickening of the neural retina in R3-/- and R1;R3-/- embryos; notice the lack of a vitreous body and the presence of a retrolenticular membrane in these mutants. (E-H) Expression of RARE-lacZ (an RA-reporter transgene) in whole-mount E10.5 wild-type and mutant embryos. Note the near complete loss of RARE-lacZ expression in eyes of R1;R3-/- embryos. Olfactory pit staining is completely dependent upon Raldh3, but forebrain staining is not dependent upon Raldh1 or Raldh3. c, cornea; D, dorsal; e, eye; el, eyelid; f, forebrain; olf, olfactory pit; pm, perioptic mesenchyme; rlm, retrolenticular membrane; V, ventral; vb, vitreous body.

View larger version (97K): [in a new window]   Fig. 2. Rescue of Raldh1-/-;Raldh3-/- eye morphogenetic movements by maternal dietary RA supplementation. (A-F) Raldh1-/- and Raldh1-/-;Raldh3-/- littermates were subjected to maternal dietary RA supplementation from E8.5 until the point of analysis, then analyzed by hematoxylin-eosin staining of frontal sections through the eye of E14.5 embryos (A,B), or RARE-lacZ expression in frontal sections of E11.5 (C,D) and E12.5 (E,F) eyes. (G-K) Whole-mount in situ hybridization to detect the expression of Raldh1, Raldh2 and Raldh3 in E10.5 whole-mount eyes (G-I), and Crabp1 and Crabp2 in E11.5 eyes following frontal sectioning (J,K). c, cornea; D, dorsal; el, eyelid; nr, neural retina; nr/vb, neural retina/vitreous body junction; olf, olfactory pit; pm, perioptic mesenchyme; rpe, retinal pigment epithelium; V, ventral; vb, vitreous body.

View larger version (61K): [in a new window]   Fig. 3. Raldh1-mediated RA signaling to perioptic mesenchyme. (A,B) RARE-lacZ expression in frontal sections of E11.5 wild-type (A) and Raldh3-/- (B) eyes. D, dorsal; pm, perioptic mesenchyme; V, ventral.

View larger version (69K): [in a new window]   Fig. 4. Loss of RA curtails apoptosis in perioptic mesenchyme. (A,B) Detection of phosphohistone 3 (H3P) in frontal sections of E11.5 Raldh1-/- (A) and Raldh1-/-;Raldh3-/- (B) eyes reveals no difference in cell proliferation. (C,D) TUNEL assay in frontal sections of E11.5 Raldh1-/- (C) and Raldh1-/-;Raldh3-/- (D) eyes reveals mesenchymal regions of apoptosis that are lost in the double mutant (arrows). D, dorsal; V, ventral.

View larger version (79K): [in a new window]   Fig. 5. Raldh1 and Raldh3 are not required to establish or maintain dorsoventral patterning of the retina. All panels consist of frontal sections of eyes at either E10.5 (A-D) or E14.5 (E-H). (A) Tbx5 mRNA in a wild-type embryo. (B) Vax2 mRNA in a wild-type embryo. (C) Double in situ hybridization showing both Tbx5 and Vax2 mRNA in a Raldh1-/-;Raldh3-/- embryo. (D) Vax2 mRNA in limited-rescue Raldh1-/-;Raldh2-/-;Raldh3-/- triple mutant embryo. (E-H) Detection of ephrin B2 mRNA (E,F) and Ephb2 mRNA (G,H) in wild-type and Raldh1-/-;Raldh3-/- embryos reveals that the double mutant still expresses both genes at relatively normal levels in their correct dorsoventral positions. D, dorsal; V, ventral.

View larger version (85K): [in a new window]   Fig. 6. Severe ventral optic cup invagination defects in embryos lacking RA synthesis. All embryos (except that shown in E) were subjected to maternal dietary RA supplementation from E6.75-E8.5, then returned to a normal diet until the point of analysis (limited-rescue). (A-D) Raldh2-/- and Raldh2-/-;Raldh3-/- littermates were analyzed for Raldh1 expression at E10.5 (frontal sections; A,B), and for RARE-lacZ expression at E11.5 (frontal sections; C,D); asterisks mark the region failing to undergo ventral invagination. (E) RARE-lacZ expression in unrescued E10.5 Raldh1-/-;Raldh3-/- embryo. (F,G) RARE-lacZ expression in limited-rescue E10.5 Raldh1-/-;Raldh2-/-;Raldh3-/- triple mutant embryo; note the complete lack of staining in the eye compared with the double mutant (E), the lack of olfactory staining (confirming the Raldh3-/- genotype), and the reduced trunk staining and forelimb bud growth (confirming the Raldh2-/- genotype). (H) Frontal section through a triple mutant optic vesicle (from embryo in F), demonstrating a complete loss of RARE-lacZ expression and a failure of ventral invagination (asterisk), but persistence of dorsal invagination in the absence of RA activity. D, dorsal; e, eye; f, forebrain; fl, forelimb bud; le, lens; olf, olfactory pit; V, ventral.

View larger version (129K): [in a new window]   Fig. 7. Sources of RA and essential target tissues for RA signaling during optic cup formation. (A-E) RARE-lacZ expression in unrescued Raldh1-/-;Raldh3-/- embryos. (F-K) Raldh2 expression in wild-type embryos. (L-P) RARE-lacZ expression in limited-rescue Raldh1-/-;Raldh2-/- embryos. (Q-U) Raldh3 expression in wild-type embryos. (V-X) RARE-lacZ expression in limited-rescue Raldh2-/-;Raldh3-/- embryos. (Y) Raldh1 expression in a wild-type embryo. In all panels, dorsal is at the top and ventral at the bottom, and sections are frontal except for J, which is a horizontal section that has temporal at the top and nasal at the bottom. Embryo stages are indicated on each panel. D, dorsal; f, forebrain; le, lens; nr, neural retina; ov, optic vesicle; pm, perioptic mesenchyme; res, limited rescue (maternal RA from E6.75-E8.5); rpe, retinal pigment epithelium; se, surface ectoderm; tm, temporal mesenchyme; V, ventral.

View larger version (16K): [in a new window]   Fig. 8. Model for RA action during eye development. The two phases of RA signaling during eye development, both of which occur through the cell-nonautonomous actions of RALDH genes that generate paracrine RA signals affecting the morphogenetic movements of neighboring cells. See text for a more complete description.