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First published online 19 October 2005
doi: 10.1242/dev.02096

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Retinal ganglion cell-derived sonic hedgehog locally controls proliferation and the timing of RGC development in the embryonic mouse retina

¹ Molecular Medicine Program, Ottawa Health Research Institute and University of Ottawa Eye Institute, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada
² Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada

View larger version (126K): [in a new window]   Fig. 1. Spatial and temporal expression pattern of Shh, Gli1 and RGC differentiation. In situ hybridization for Shh and Gli1 mRNA and immunohistochemistry for Brn3b staining on serial sections of the mouse eye at E12 (A-C) and E13 (D-F). (A-C) Shh expression is initiated behind the leading edge of RGC differentiation, as marked by Brn3b staining (lines in B) at E12. (C) Gli1 expression is localized to the neuroblast layer overlying the Shh-expression domain. (D-F) By E13, the Shh and Gli expression domains have expanded across the retina, but still lag behind the leading edge of RGC differentiation (lines in E). Scale bar: 200 µm.

View larger version (103K): [in a new window]   Fig. 2. Shh inactivation in the peripheral retina is associated with increased cell cycle exit and depletion of the RPC pool. (A-L) In situ hybridization on wild-type and -Cre;Shh–/c retinas at E14 and P0. (A-D) Gli and (E-H) cyclin D1 expression are downregulated in the peripheral retina of mutant mice at both ages (between arrowheads in B,D,F,H). (I-K) In situ hybridization for Rax expression to mark the neuroblast layer, which by P0 is reduced in thickness in the peripheral retina of the mutant mice (between the arrowheads in L). (M) The number of S-phase cells is reduced in the peripheral retina of the mutant mice at E14. (N) The number of birthdated cells is increased in the peripheral retinas of mutant mice. RPC were labelled at E13 with BrdU and the number of heavily labelled nuclei was counted in the central versus peripheral retina at P0. *P<0.005. Scale bar: 100 µm.

View larger version (94K): [in a new window]   Fig. 3. Shh inactivation in the peripheral retina is associated with increased RGC development. (A-D) Retinas from wild-type and -Cre;Shh–/c mice at E17 stained for BrdU and Brn3b. RPC were labelled in vivo with BrdU at E16 and harvested 24 hours later. B and D are high magnification views of the neuroblast layers in A and B, respectively. Arrows in D indicate examples of double-labelled cells. The layer of Brn3b+ RGCs is thickened and there is an increase in double-labelled cells in the -Cre;Shh–/c retina (C,D). (E) Quantification of the number of Brn3b+ cells in a 300 µm region in the central and peripheral regions of wild-type and mutant retinas at P0. (F,G) Birthdating analysis in the embryonic -Cre;Shh–/c retina. RPC were labelled in vivo with BrdU at E13 and the retinas were harvested at P0. The number of Brn3b+ cells with intense BrdU-labelling (F) and the proportion of Brn3b+ cells among the heavily BrdU-labelled cohort (G) were quantified in 300 µm regions of the central and peripheral retinas in wild-type and mutant mice. *P<0.005. Scale bar: 100 µm in A,C; 50 µm in B,D.

View larger version (73K): [in a new window]   Fig. 4. Hh signalling inhibits RGC development in vitro. (A-C) Brn3b staining in E12 retinal explants cultured for 48 hours under control conditions (A) or in the presence of recombinant Shh-N (B) or anti-Hh (C). The most dramatic change in Brn3b staining is observed in the peripheral region (closer to the lens) of the retinal explants. (D) Co-localization of silver grains marking [3H]thymidine labelled cells and Brn3b staining. RPC were labelled with [3H]thymidine for 4 hours, washed and cultured for an additional 48 hours. Arrows indicate double-labelled cells, arrowheads indicate Brn3b+ [3H]thymidine– cells. (E) Dissociated cell scoring to quantify the proportion of RGCs among the [3H]thymidine+ cohort in E12 retinal explants cultured for 48 hours under the indicated treatment conditions. Treatment with isotype-matched anti-LFA antibodies (1E6) was no different than control and is not shown. n3 for each condition. (F) Proportion of S-phase, Brn3b+ and the non-RGC component of the Tuj1+ population (determined by subtracting the number of Brn3b+ from the number of Tuj1+ cells) in E12 explants cultured for 48 hours under the indicated conditions and analyzed by dissociated cell scoring. *P<0.005, ** P<0.005. Scale bars: 100 µm in A-C; 25 µm in D.

View larger version (88K): [in a new window]   Fig. 5. Shh inactivation affects differentiation and the development of late-born cell types. Retinal sections from wild-type (A,C,G,I,K,M,O) and -Cre;Shh–/c mice (B,D,H,J,L,N,P) were stained at P0 for syntaxin (A,B) to identify amacrine and horizontal cells; at P6 for cone-arrestin (C,D) to identify cone photoreceptors; at P9 for Hoechst (G,H) to mark nuclei and for PKC (I,J) to identify rod bipolar cells; and at P6 for Hoechst (K,L), rhodopsin (M,N) to identify rod photoreceptor cells and CRALBP (O,P) to identify Müller glia. Red arrows in B indicate the line of differentiating horizontal cells in the outer plexiform layer in the periphery of the -Cre;Shh–/c retina. Horizontal cells are not differentiated in the control retina at this stage (A). The intensity of cone arrestin staining is greater in the periphery of the -Cre;Shh–/c retina compared with wild-type littermates (C,D). (E) Quantification of retinal cell types in the central and peripheral regions of the wild-type and mutant retinas at P1 (rods), P6 (cones, Müller glia) and P9 (bipolar cells). (F) The distribution of cells between the different retinal layers in a 100 µm region of the central and peripheral retinas of wild-type and mutant mice at P6. (G-J) Bipolar cells are reduced in number and disorganized in degenerating periperal retinas of -Cre;Shh–/c mice. **P<0.05, *P<0.005. (K-P) Degenerative changes take place in the periphery of the -Cre;Shh–/c retina, as indicated by the rosettes (arrows in L), gaps in rhodopsin staining (arrows in N) and infiltration of Müller glial cell bodies into the ONL (arrows in P). Scale bar: 100 µm.

View larger version (67K): [in a new window]   Fig. 6. Ectopic Hh pathway activation restores cell cycle gene expression and bipolar cell development in retinal explants from -Cre;Shh–/c mice. Retinal explants from wild-type and -Cre;Shh–/c mice at P1 were cultured for 2 (A-L) or 9 (M-X) days and processed for in situ hybridization to detect mRNA for Gli (A-D), cyclin D1 (E-H) and Chx10 (I-L) or stained for Hoechst to reveal nuclei and anti-PKC (M-P), anti-CRALBP (Q-T) and anti-syntaxin (U-X) antibodies. The peripheral region of the explants is shown in these panels. Scale bar: 100 µm.

View larger version (100K): [in a new window]   Fig. 7. Shh signalling modulates Hes gene expression in RPC. (A,B) In situ hybridization for Hes1 mRNA in the retina of wild-type (A) and -Cre;Shh–/c (B) mice at P0. Hes1 expression is reduced in the peripheral retina of the -Cre;Shh–/c (between the red arrows). (C) Semi-quantitative RT-PCR analysis of gene expression in control E18 retinas and in E18 retinal explants cultured for 1 and 3 days under the indicated conditions. Scale bar: 100 µm in A,B.