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First published online 26 October 2005
doi: 10.1242/dev.02100

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Retinoic acid regulates the expression of dorsoventral topographic guidance molecules in the chick retina

¹ Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA
² Department of Biology, Oberlin College, 119 Woodland Street, Oberlin, OH 44074-1097, USA

View larger version (65K): [in a new window]   Fig. 1. RA activity assayed in DF-1 cells after co-transfection with RARE-lacZ and RCAS-DNhRAR. X-gal staining of DF-1 cells transfected with RARE-lacZ (RA-reporter) alone (A) or with RARE-lacZ and RCAS-DNhRAR (B).

View larger version (92K): [in a new window]   Fig. 2. Comparison in eye size and proliferation between RCAS-DNhRAR infected and uninfected eyes. (A) Two eyes from one E7 embryo, injected in the right eye with RCAS-DNhRAR at HH stage 17. Uninjected control eye on the left and the injected eye on the right. White arrowhead shows ventral fissure region of injected eye. (B,D) Dissociated cells from retina injected with RCAS-DNhRAR or (D) with RCAS-GFP, labeled with [3H] thymidine (dark grains) and 3c2 anti viral-gag antibody (red) and DAPI (blue). (C) Dissociated cell counts from 4 hour [3H] thymidine-labeled E7 retinae that were injected with RCAS-GFP control virus or RCAS-DNhRAR. The percentage of [3H] thymidine-labeled cells were counted among the infected, 3c2+ population (green bar and dark blue bars) as well as the uninfected, 3c2-population (red bars and light blue bars). The data for RCAS-DNhRAR are from nine infected retinae and the data for RCAS-GFP are from six infected retinae.

View larger version (86K): [in a new window]   Fig. 3. Expression of EphB2, EphB3, ephrin B2 and ephrin B1 in uninfected and RCAS-DNhRAR infected retinae. In situ hybridization carried out on flat-mounted E7 retina either uninjected or injected with RCAS-DNhRAR. Retinae shown with dorsal towards the top, ventral towards the bottom, anterior (nasal) towards the left and posterior (temporal) towards the right. In situ hybridization of uninjected E7 retina, with EphB2 (A), EphB3 (E), ephrin B2 (I) and ephrin B1 (M). Double in situ hybridization of RCAS-DNhRAR injected E7 retina with EphB2 and RCAS probes (B-D). Magnified view of the boxed area from B showing EphB2 expression (purple signal, C) and RCAS infection (blue signal, D). RCAS-DNhRAR injected E7 retina with double in situ hybridization for EphB3 and RCAS probes (F-H). Magnified view of the boxed area in F showing EphB3 expression (purple signal, G) and RCAS-infected area (blue signal, H). RCAS-DNhRAR-injected E7 retina with double in situ hybridization for ephrin B2 and RCAS probes (J-L). Magnified view of boxed area in J showing ephrin B2 expression (purple signal, K) and RCAS infected area (blue signal) (L). RCAS-DNhRAR injected E7 retina with double in situ hybridization for ephrin B1 (purple signal, N) and RCAS (blue signal, O). The white arrows indicate infected areas with subtle downregulation of ephrin B1 expression.

View larger version (66K): [in a new window]   Fig. 4. Expression of EphB2, EphB3, ephrin-B2 and ephrin-B1 in RCAS-DNcTRß2-infected retinae. Flat-mounted E7 retina oriented with dorsal towards the top, ventral towards the bottom, anterior (nasal) towards the left and posterior (temporal) towards the right, injected with RCAS-DNcTRß2 on which double in situ hybridization was carried out with the following probes: EphB2 (purple signal, A) and RCAS (blue signal, B), EphB3 (purple signal, C) and RCAS (blue signal, D), ephrin B2 (purple signal, E) and RCAS (blue signal, F) and ephrin B1 (purple signal, G) and RCAS (blue signal, H). X-gal staining of DF-1 cells transfected with DR4-lacZ (TH-reporter) alone (I) and with DR4-lacZ+ RCAS-DNcTRß2 (J).

View larger version (91K): [in a new window]   Fig. 5. Expression of EphB2, EphB3, ephrin B2 and ephrin B1 in RCAS-Cyp26A1-infected retinae. Flat-mounted E7 retina oriented with dorsal towards the top and ventral towards the bottom, anterior (nasal) towards the left and posterior (temporal) towards the right, injected with RCAS-Cyp26A1 on which double in situ hybridization was carried out with the following probes: ephrin B2 (purple signal, A) and RCAS (blue signal, E), ephrin-B1 (purple signal, B) and RCAS (blue signal, F), EphB2 (purple signal, C) and RCAS (blue signal, G) and EphB3 (purple signal, D) and RCAS (blue signal, H). Black arrows indicate areas of loss of expression of ephrin B2 (A) and RCAS infection (E). White arrowheads point to areas of RCAS infection overlapping with ephrin B1 (F), EphB2 (G) and EphB3 (H). In situ hybridization of uninjected E7 retinae with ephrin B2 (I), ephrin B1 (J) and Cyp26A1 (K). White arrows indicate ventral expression borders of ephrin B2 (I) and ephrin B1 (J) and expression domain of Cyp26A1 (K). X-gal staining of DF-1 cells transfected with RARE-lacZ (RA-reporter) alone (L), with RARE-lacZ+ RCAS-DNhRAR (M) and with RARE-lacZ+ RCAS-Cyp26A1 (N).

View larger version (75K): [in a new window]   Fig. 6. Expression of RALDH1, RALDH3 and Cyp26A1 on retinae infected with RCAS-DNhRAR. Flat-mounted E7 retina oriented with dorsal towards the top, ventral towards the bottom, anterior (nasal) towards the left and posterior (temporal) towards the right, injected with RCAS-DNhRAR, on which double in situ hybridization was carried out with the following probes: RALDH1 (purple signal, A,D,G) and RCAS (blue signal, D), RALDH3 (purple signal, B,E,H) and RCAS (blue signal, E) and Cyp26A1 (purple signal, C,F,I,L) and RCAS (blue signal, F,L). White arrowhead (A) indicates area of upregulation of RALDH1 within its normal domain of expression. Black arrowhead (B) indicates upregulation of RALDH3 within its normal domain of expression. Black arrowheads indicate RCAS-infected regions within the normal domain of expression of RALDH1 (D) and of RALDH3 (E) and black arrows indicate RCAS-infected regions outside the normal domain of expression of RALDH1 (D) and of RALDH3 (E). (G,J) Magnified views of boxed region in A showing upregulated RALDH1 expression (G) and RCAS infection (J); (H,K) magnified views of boxed region in B showing upregulated RALDH3 expression (H) and RCAS infection (K); (I,L) magnified views of C and F showing loss of Cyp26A1 expression (I) and RCAS infection (L). Uninjected E7 retinae showing RALDH1 (M) and RALDH3 (N) expression domains.

View larger version (69K): [in a new window]   Fig. 7. In situ hybridization with Vax and Tbx5 on retinae infected with RCAS-DNhRAR and in situ hybridization with RALDH1 and RALDH3 on RCAS-Vax infected retinae. Flat-mounted E7 retina oriented with dorsal towards the top and ventral towards the bottom, anterior (nasal) towards the left and posterior (temporal) towards the right, injected with RCAS-DNhRAR, on which in situ hybridization was carried out with the following probes: Vax (purple signal, A) and Tbx5 (purple signal, C). The RCAS infection (brown) was detected by immunohistochemistry using 3c2 anti viral-Gag antibody (B,D). (E-H) E7 retina oriented as above, injected with RCAS-Vax on which double in situ hybridization was carried out with RALDH3 (purple signal, E,G) and RCAS (blue signal) (F,H). Magnified views of E and F are shown in G and H, respectively. (I,J) E7 retina also injected with RCAS-Vax on which in situ hybridization was carried out with RALDH1 (lack of purple signal, I) and RCAS (blue signal, J).

View larger version (26K): [in a new window]   Fig. 8. Models describing the two possible modes of regulation of expression of EphB2, EphB3 and ephrin B2 by RA, Vax and Tbx5. Model 1: Vax and Tbx5 function in the dorsal and ventral retina to regulate the expression of ephrin B2, EphB2 and EphB3 exclusively through the RA synthesizing enzymes, RALDH1 and RALDH3. Model 2: Vax and Tbx5 function independently of RA to regulate expression of EphB2, EphB3 and ephrin B2. According to this model, both Tbx5-dependent and RA-dependent positive inputs are required to express ephrin B2 in the dorsal retina. The enhancer regions of EphB2 and EphB3 would require positive inputs from both Vax and RA for their expression in the ventral retina.