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doi: 10.1242/10.1242/dev.00114

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The chicken RaxL gene plays a role in the initiation of photoreceptor differentiation

Department of Genetics, Harvard Medical School, Howard Hughes Medical Institute, 200 Longwood Avenue, Boston, MA 02115, USA

View larger version (66K): [in a new window]   Fig. 1. The amino acid sequence alignment of chick RaxL (cRaxL) and human RAX2 (hRAX2) (A). Identical amino acid residues between these two proteins are indicated by asterisks. Gaps required for optimal alignment are represented by dashes. The homeodomain is underlined. (B-Q) Whole-mount in situ hybridization of Rax (B-I) and RaxL (J-Q) on Hamburger-Hamilton stage 11 (B,C,J,K), stage 12 (D,E,L,M), stage 14 (F,G,N,O), and stage 15 (H,I,P,Q) chick embryos. (B,D,J,L) Dorsal views of the embryos; (C,E,K,M) ventral views of the same embryos. (F,H,N,P) Lateral views; (G,I,O,P) Magnified frontal or ventral views. The arrowheads and arrows indicate the retina and ventral diencephalon, respectively.

View larger version (121K): [in a new window]   Fig. 2. In situ hybridization of Rax (A-D,I-L) and RaxL (E-H,M-P) on retinal sections of E5 (A,E), E6 (B,F), E7 (C,D,G,H), E9 (I,M), E11 (J,N), E14 (K,O), and E19 (L,P) chick embryos. (C,G) Sections from the peripheral region; (D,H) sections from the central region of the E7 retina. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. In B and F, arrows indicate Rax and RaxL non-expressing domains, respectively. In H, strong expression of RaxL in developing photoreceptor cells is indicated by an arrow and the weak expression zone in INL is indicated by an arrowhead.

View larger version (84K): [in a new window]   Fig. 3. Flat-mount in situ hybridization of E7.5 chick retinas electroporated with EnRaxLC (A-F), EnRaxC (G-L), EnRaxLHD (M,N), and EnIrx (O,P) viral constructs. Endogenous visinin (A-C,G-I,M-P) and Rxrg transcripts (D-F,J-L) are represented by the purple stain and exogenous EnRaxC, EnRaxLC, EnRaxLHD and EnIrx by the green-blue stain. The sections of the retina samples in B,E,H,K are shown in C,F,I,L, respectively. Scale bars: in A, 2 mm for all the flat-mount images; in C, 30 µm for C,F,I,L.

View larger version (115K): [in a new window]   Fig. 4. Flat-mount in situ hybridization of E9 chick retinas electroporated with EnRaxLC viral construct. The signals of endogenous Brn3a (A-C), Chx10 (D-F) and Pax6 (G-I) transcripts are shown in purple and EnRaxLC viral transcript is shown in green-blue. The sections of the retina samples in B,E,H are shown in C,F,I, respectively. Scale bars: in A is 2 mm for A,B,D,E,G,H; in F, 30 µm for C,F,I. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer.

View larger version (24K): [in a new window]   Fig. 5. CAT activities were assayed in COS cells transiently transfected with the RET1-CAT reporter construct (A) together with Rax or RaxL expression vectors (B) or with combinations of different ratios of expression vectors as indicated (C). The CAT activity from each transfection was normalized for internal transfection efficiency (see Materials and Methods). The relative CAT activity was normalized to the value from empty vector (B) or from RaxL transfection alone (C). The data presents the average value of triplicates. EnRax* represents EnRaxC, EnRaxLC or EnRaxLHD indicated in C.

View larger version (134K): [in a new window]   Fig. 6. Flat-mount in situ hybridization of E7.5 chick retinas electroporated with EnRaxLHD alone (A-C), EnRaxLHD plus RaxL (D-G), EnRaxLHD plus Rax (H-K) or EnRaxLHD plus mouse Crx (L-N) viral constructs. The sections of the retina samples in F,J are shown in G,K, respectively. The signals of endogenous visinin transcripts are shown in purple, EnRaxLHD viral transcript is shown in green-blue, and 3C2 mAb, which stains all viral infected patches, is shown in brown. The arrows in D,E indicate the visinin expression is partially rescued by RaxL. Scale bars: in A, 2 mm for A-F,H-N; in G, 30 mm for G,K.

View larger version (65K): [in a new window]   Fig. 7. Western blot with anti-visinin mAb showing a 24 kDa visinin protein in E5.5, 6.5, 7.5 and 8.5 chick retinal extracts (A, lanes 1 to 4). The photoreceptors in the ONL of E7 (B) and E18 (C) retina are stained with visinin mAb. The examples of FACS analyses of RCAS- (D) and EnRaxLHD- (E) infected retina are shown, with the upper-right quadrants representing the visinin and p27 (virus infected) double-positive population; and the upper- and lower-right two quadrants representing all viral-infected cells (p27 positive). The average percentages of double-positive cells among all viral infected population are indicated in the F. n indicates the retina sample number. P values are based on the Student's t-test of two-tailed distribution as follows: *, P<0.005; **, P=0.152; ***, P=0.226. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.

View larger version (80K): [in a new window]   Fig. 8. Phospho-Histone H3 staining (A,B) and TUNEL analysis on E7.5 retinal (C-F) and brain (G,H) sections infected with EnRaxLHD virus (A,B,E-H) or control EnIrx virus (C,D). The phospho-Histone H3 or TUNEL-positive cells are shown in yellow or green, viral-infected cells are shown in red and nuclei stained with DAPI are in blue. The red and green merged images are presented in A,C,E,G. The same fields with red and blue merged images are presented in B,D,F,H, respectively.