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First published online September 1, 2004
doi: 10.1242/10.1242/dev.01298

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The hedgehog pathway is a modulator of retina regeneration

Jason R. Spence^*, Mayur Madhavan^*, John D. Ewing, David K. Jones, Bret M. Lehman and Katia Del Rio-Tsonis

Department of Zoology, Miami University, Oxford, Ohio 45056, USA

View larger version (106K): [in a new window]   Fig. 1. Fgf2 induces retina regeneration in two distinct ways. (A) At E4 the retina is removed surgically leaving behind RPE and CB/CMZ. An Fgf2-soaked heparin bead (*) is then placed in the eye cup. (B) A heparin bead (not visible in this section) alone does not cause regeneration after 7 days (E11). (C) By E11, Fgf2 induces regeneration from the CB/CMZ (cr) and by the transdifferentiation of the RPE (td). (D-O) Histology of normally developing as well as regenerating retina at E7 (D-F), E9 (G-I), E11 (J-L) and E15 (M-O). D,G,J,M show normal development at each stage. Three days after retinectomy (E7) a transdifferentiated neuroepithelium (E) as well as a neuroepithelium generated from cells in the CB/CMZ (F) are present. At E9, the transdifferentiated neuroepithelium (H) as well as the neuroepithelium arising from the CB/CMZ (I) thicken and grow. (K,L) Seven days post-retinectomy, at E11, the various retinal layers become visible. By E15, regenerated retinas (N,O) are laminated and resemble an E11 developing retina (J). Scale bar: 100 µm (A,B); 500 µm (C); 100 µm (D-O). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; ne, neuroepithelium; l, lens; CB/CMZ, ciliary body/ciliary marginal zone; RPE, retinal pigmented epithelium.

View larger version (92K): [in a new window]   Fig. 2. Cells in the CB/CMZ are a source of regenerating retina. (A) Collagen type IX immunohistochemistry performed on an E4 operated eye 4 hours post-retinectomy shows the CB/CMZ region has not been removed. (B) An adjacent section shows that the CB/CMZ is rich in cells that express Pax6 (blue) and Chx10 (red). White arrowheads indicate progenitor cells that co-express Pax6 and Chx10. (C) Another adjacent section shows that within 4 hours of retinectomy, most cells in the CB/CMZ are proliferating and labeled with BrdU, suggesting that progenitor cells (like those shown in B) are proliferating. (D) The CB/CMZ was labeled with DiI (red) immediately after retinectomy. The collagen type IX (green) immunofluorescence confirms that the labeled region is the CB/CMZ. (E) At E5, 1 day after the CB/CMZ was labeled, DiI-labeled cells have migrated from the CB/CMZ and generated a neuroepithelium. (F) Three days after retinectomy this phenomenon is still apparent. The asterisk indicates the region where the CB/CMZ was originally labeled with DiI. c, cornea; CB/CMZ, ciliary body/ciliary marginal zone; l, lens; RPE, retina pigmented epithelium; pe, pigmented epithelium; cr, retina that regenerated from the CB/CMZ. The red arrowheads indicate the site at which the incision was made during the retinectomy. Scale bar in A is for A-C.

View larger version (66K): [in a new window]   Fig. 3. Regeneration gives rise to all cells layers of the retina. Rows: de, normal retinal development; td, retinas that arose from transdifferention; cr, retinas that regenerated from the CB/CMZ. Immunohistochemistry using antibodies against Brn3a (ganglion cells) and Napa73 (NFL) are shown in the first column (A1-I1). Immunohistochemistry using antibodies against Pax6 (ganglion cells, amacrine cells and horizontal cells) and Chx10 (bipolar cells) are shown in column 2 (A2-I2). Immunohistochemistry using antibodies against visinin (photoreceptors) and Mitf (RPE) are shown in column 3 (A3-I3). Sections in column 4 (A4-I4) were stained for vimentin to detect Müller glia. Rows A, B and C show eyes at E7, D, E and F at E11 and G, H and I at E15. (Row A) At E7, during eye development, ganglion cells are starting to differentiate and an NFL is visible (A1), Pax6 is mostly expressed in the region where ganglion cells are present while Chx10 is expressed through a large part of the retina (A2). Photoreceptors have started to differentiate and Mitf expression in the RPE is noticeable (A3). At this stage Müller glia are also present (A4). (Row B) At E7 transdifferentiating retina shows some Napa73 staining but no Brn3a (B1). Pax6 is expressed throughout the retina but its expression is very prominent in the ganglion cells. There are low levels of Chx-10 at this stage (B2). There is no detectable Mitf expression in the retina that arises from transdifferentiation and visinin is expressed in the presumptive photoreceptor region. (B3). Vimentin expression is limited to Müller glias that span the retina (B4). (Row C) Retina that regenerates from the CB/CMZ shows a similar pattern of cell markers as the retina that arises from transdifferentiation. There is no Brn3a expression but there is some Napa73 staining (C1), and Pax6 is expressed throughout the retina, while there is a very low level of Chx10 expression (C2). However, visinin is not present and Mitf is expressed in the intact RPE (C3). Müller glia are present at this stage (C4). (Row D) By E11, developing retina has a clearly defined NFL, GCL (D1), INL (D2) and ONL (D3). Mitf continues to be expressed in the RPE (D3) and Müller glia are present through out the retina (D4). (Row E) Transdifferentiation results in all differentiated cell types by E11. Ganglion cells (E1), amacrine cells, bipolar cells, horizontal cells (E2), photoreceptors (E3) and Müller glia (E4) are present. The orientation of this layer, however, is flipped (E1-E4). (Row F) Regeneration from the CB/CMZ also results in production of all differentiated cell types by E11. The NFL, GCL (F1), INL (F2), and ONL (F3) have all the cells types seen in a normal retina. Here, the RPE is still intact and expresses Mitf (F3). Müller glia span the retina (F4). The orientation of this layer is similar to that of developing retina. These expression patterns continue to be maintained at E15 in the developing eye (G1-G4), the transdifferentiated retina (H1-H4) and the retina that regenerated from the CB/CMZ (I1-I4). Scale bar: 100 µm.

View larger version (70K): [in a new window]   Fig. 4. Shh and Ptc1 expression in E11 eyes. (A1-B3) Developing retina (de) at E11. (C1-D3) Retina regenerating from the CB/CMZ (cr). (E1-E3) Transdifferentiating retina (td) at E11. (A1) Expression of Shh mRNA during normal development is predominantly in the ganglion cells of the posterior retina. (A2) Shh protein expression in the posterior retina of the developing eye mirrors that of the mRNA expression. (A3) Ptc1 expression in the posterior retina of a developing eye is located through the INL and ONL. (B1) Shh mRNA expression in the anterior part of the developing eye includes the presumptive CMZ. (B2) Shh protein expression is similar to that of the mRNA expression. (B3) Expression of Ptc1 mRNA is in the anterior retina and ciliary region. (C1,D1) During CB/CMZ regeneration, Shh expression patterns basically remained unchanged, however the levels in the posterior region were low (arrowhead in C1). (C2,D2) Shh protein expression of retina regenerating from the CB/CMZ. (C3,D3). Ptc1 expression is present throughout part of the INL and ONL in the posterior (arrowhead in C3) as well as anterior part (arrowhead in D3) of the CB/CMZ regenerated retina. (E1) Transdifferentiating retina shows Shh mRNA expression in the ganglion cell layer (arrowhead). (E2) Shh protein expression is diffused through out the transdifferentiating retina but the expression is mainly in the ganglion cell layer (arrowhead). (E3) Ptc1 expression is predominantly in the INL of the transdifferentiated retina (arrowhead). F1, F2 and F3 are negative controls for Shh RNA, Shh Protein and Ptc1 RNA expression respectively. Arrowheads mark areas of expression.

View larger version (36K): [in a new window]   Fig. 5. Manipulation of Shh levels. (A) Ectopic expression of Shh using Rcas-Shh. (A1) Rcas-Shh/Fgf2-treated eyes show an increase in the expression of Shh mRNA (arrowhead). (A2) Shh sense probe. (A3) Ectopic Shh expression upregulates Ptc1 levels as indicated by the arrowhead. (A4) Ptc1 sense probe. (A5) Ectopic Shh expression via a retroviral vector corresponds to an increase in Shh protein (arrowheads). (A6) AMV3C2 staining shows the presence of Rcas virus coat protein in Rcas-Shh-treated eyes (arrowheads). (B) Real-time PCR analysis of the relative Ptc1 levels in Fgf2, RCAS-Shh/Fgf2, and 100 µM KAAD/Fgf2-treated eyes show that Rcas-Shh treatment causes and significant increase in Ptc1 (P<0.01) levels while KAAD/Fgf2 treatment reduces Ptc1 levels (P<0.05). * statistically significant. Error bars are s.e.m.

View larger version (93K): [in a new window]   Fig. 6. Effect of Shh on regeneration. Inhibition of the Shh pathway using KAAD increases the domain of transdifferentiation (A-C,J-L,S-U) compared to eyes that received Fgf2 alone (D-F,M-O,V-X). However, overexpression of Shh using Rcas-Shh decreases transdifferentiation (G-I,P-R,Y,Z). (A-I) E7. (A) At E7, KAAD/Fgf2-treated eyes show regeneration from the CB/CMZ (cr) as well the transdifferentiation of RPE (td). (B) A close up view of CB/CMZ from an E7 KAAD/Fgf2-treated eye. (C) A close up view of transdifferentiation from an E7 KAAD/Fgf2-treated eye. (D) Regenerating eye that has been treated with Fgf2 only. (E) A close up view of CB/CMZ regenerated retina. (F) Close up view of transdifferentiated retina. (G) Regenerating eye that has been treated with Rcas-Shh/Fgf2 shows almost no transdifferentiation. (H) CB/CMZ regenerated retina, where Shh has been overexpressed does not look different from that of E7 Fgf2-treated regenerating neuroepithelium (E). (I) Occasionally, there are small areas of transdifferentiation in the regenerating eyes that received Rcas/Shh/Fgf2. The neuroepithelium at this stage is similar to that of an Fgf2-treated transdifferentiating retina (F). (J-R) E11. (J) At E11, KAAD/Fgf2-treated eyes show an increased domain of transdifferentiation when compared to Fgf2-treated eyes of the same stage (M). (P) At this stage, several eyes that overexpress Shh show some transdifferentiation. KAAD/Fgf2 and Fgf2-treated eyes show similar organization having a GCL, IPL and INL (K,L,N,O); whereas Rcas-Shh/Fgf2-treated eyes seem to be more differentiated with a visible OPL and ONL (Q,R). (S-Z) E15. (S) At E15, most of the retina in KAAD/Fgf2-treated eyes results from transdifferentiation. However, there exist small regions that contain CB/CMZ regenerated retina. (V) Eyes treated with Fgf2 show both transdifferentiated retina and CB/CMZ regenerated retina. (Y) Rcas-Shh/Fgf2-treated eyes do not show any transdifferentiated retina and all the retina present is produced by regeneration from the CB/CMZ. All the retinas at this stage show similar levels of spatial organization (T,U,W,X,Z). Scale bars: 500 µm (A,D,G,J,M,S); 100 µm and is applicable to B,C,E,F,H,I,K,L,N,O,Q,R,T,U,W,X,Z; in P is 500 µm and applies to Y; 500 µm (V). An asterisk marks an Fgf2-coated heparin bead.

View larger version (41K): [in a new window]   Fig. 7. Shh maintains the RPE phenotype and its actions in the RPE and the CB/CMZ depend on Fgfr signaling. (A) Developing eyes were treated with Fgf2 at E4, or Rcas-Shh and Fgf2 at E3.5 and E4, respectively, and analyzed at E6. Ectopic Shh was sufficient to inhibit transdifferentiation in developing eyes. B, Fgf2 bead; t, transdifferentiated retina. Arrowhead indicates developing retina. (B) Ffgr signaling activity was measured 4 hours after treatment by immunohistochemistry using a phospho-ERK antibody in retinectomized eyes with: no Fgf2, Fgf2, Fgf2/KAAD or Fgf2/Rcas-Shh. Fgf2 activates the Fgfr signaling pathway, whereas ectopic Shh inhibits this activity. Scale bar: 50 µm. (C) Retinectomies were performed at E4 and eyes were treated with Fgf2±PD 173074, or with Rcas-Shh±PD 173074, and analyzed 3 da ys later. Ectopic Shh stimulates regeneration from the CB/CMZ, as does Fgf2. Both of these activities are significantly inhibited when PD 173074, a potent and specific Fgfr inhibitor, is used.

View larger version (54K): [in a new window]   Fig. 8. Effects of Shh on organization of the regenerating retina. Eyes that were treated with KAAD/Fgf2 (A,D,G,J), Fgf2 only (B,E,H,K) or Rcas-Shh/Fgf2 (C,F,I) were collected at E11 (A-F) or E15 (G-K) and were subjected to immunohistochemical analysis. Brn3a and Napa-73 immunoreactivity in E11 KAAD/Fgf2-treated eyes suggests that there are more ganglion cells and nerve fibers in both the CB/CMZ regeneration (A1) and retina that arises from transdifferentiation of the RPE (D1) when compared to the Fgf2-treated controls in (B1) and (E1) respectively. When eyes were treated with Rcas-Shh/Fgf2, regeneration from the CB/CMZ (C1) and from transdifferentiation (F1) seem to have a reduced NFL and number of ganglion cells. There seem to be no difference in the expression patterns of Pax6 and Chx10 in KAAD/Fgf2 (A2,D2) or Rcas-Shh /Fgf2 (C2,F2) when compared with the Fgf2-treated controls (B2,E2, respectively). There are no major differences in the expression of visinin and Mitf in the CB/CMZ regenerate when comparing KAAD/Fgf2-treated eyes (A3) with Fgf2-treated eyes (B3) and Rcas-Shh/Fgf2-treated (C3) eyes. There is also no difference in the expression of visinin in retina that regenerated by transdifferentiation, when we compare KAAD/Fgf2-treated (D3) with Fgf2 (E3) and Rcas-Shh/Fgf2-treated (F3) eyes. Similar patterns are observed at E15. Expression patterns of Pax6 and Chx10 are comparable among KAAD/Fgf2-treated (G2 and J2), Fgf2 only (H2 and K2) and Rcas-Shh/Fgf2-treated (I2) eyes. At E15, visinin is present while Mitf is absent in all transdifferentiated retina that arises in KAAD/Fgf2-treated eyes (J3) and control (K3) eyes. Scale bar: 50 µm.

View larger version (18K): [in a new window]   Fig. 9. Cellular effects of Shh manipulations. (A) KAAD/Fgf2-treated eyes show a significantly increased number of Brn3a-positive ganglion cells compared with Fgf2-treated eyes (P<0.001). This corresponds to a 23.09% increase in the number of ganglion cells. Rcas-Shh/Fgf2-treated eyes have a significantly lower number of ganglion cells (P<0.001) than Fgf2-treated controls, corresponding to a 42.98% decrease in the number of ganglion cells. (B) Rcas-Shh/Fgf2-treated eyes show increased cell death throughout the retina when compared with Fgf2 (P<0.001) and KAAD/Fgf2-treated eyes. There is no significant difference between KAAD/Fgf2 eyes and Fgf2-treated eyes. (C) Rcas-Shh/Fgf2-treated eyes also show increased cell death in the presumptive ganglion cell layer, compared with Fgf2-treated controls P<0.001). There is no statistically significant difference in cell death between KAAD/Fgf2-treated eyes and Fgf2-treated eyes. * denotes statistical significance. Error bars shown are s.e.m.

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