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First published online June 8, 2005
doi: 10.1242/10.1242/dev.01882

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Retinal neurons regulate proliferation of postnatal progenitors and Müller glia in the rat retina via TGFß signaling

Neurobiology and Behavior Program, Department of Biological Structure, 357420 Health Sciences Center, University of Washington, School of Medicine, Seattle, WA 98195, USA

View larger version (57K): [in a new window]   Fig. 1. Retinal progenitor proliferation declines between P4 and P12. Rats were injected every 3 hours for 9 hours with BrdU on the postnatal day indicated. At P4 (A), BrdU-positive cells can be found centrally and peripherally in both the INL and ONL (inset). By P6 (B), few or no cells incorporate BrdU in the central retina; (B, inset), however, BrdU-positive cells can be found in the peripheral regions (asterisks). At P8 (C), BrdU incorporation is restricted to the periphery. By P10 (D), some BrdU labeling can be found at the margin (arrow). At P12 (E), there is no BrdU labeling within the retina (arrow marks the retinal margin). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 200 µm.

View larger version (71K): [in a new window]   Fig. 2. Soluble signal from retinal cells inhibits postnatal proliferation. (A) Experimental protocol (see text). Cells were cultured in the presence of 5 million retinal cells for 12 hours, BrdU was added for the final 2 hours of progenitor cell culture (B,D) and the final 6 hours of glial cell culture (C,E). (B-E) Example fields of nestin+ (red) BrdU-labeled (green) progenitor cells (B,D), and CRALBP+ (red) BrdU-labeled (green) Müller glia (C,E), under control conditions (no added cells: B,C) or with the addition of 5 million dissociated retinal cells (D,E). (F) Quantification of BrdU+/nestin+ cells shows a significant reduction in proliferation with 5 million added cells (15±2.2%) compared with control (24.9±2.4%). (G) Quantification of BrdU+/CRALBP+ cells shows significant inhibition of glial proliferation with addition of 5 million cells (23.0±2.8%) compared with control treated cells (44.3±2.3%). *P<0.02, pairwise comparison (Student's t-test).

View larger version (21K): [in a new window]   Fig. 3. Inhibition of TGFß signaling restores proliferation in addback conditions. (A) Quantification of nestin+/BrdU+ cells shows restoration of proliferation to near control levels (white bar; 24.9±2.4%) in the presence of TGFßRII-Fc and 5 million retinal cells (dark gray bar; 24.4±1.8%) compared with 5 million cells alone (light gray bar; 15±2.2%). Neither ActivinRII-Fc nor BMPRIB-Fc had a significant effect on proliferation (black bars). (B) Quantification of CRALBP+/BrdU+ cells shows TGFßRII-Fc restores proliferation of CRALBP+ Müller glial cells in the presence of 5 million retinal cells (dark gray bar; 41.7±3.9%), compared with 5 million cells alone (light gray bar; 23±2.8%), neither ActivinRII-Fc, nor BMPRIB-Fc affected glial proliferation in this assay (black bars). Error bars=s.e.m. *P<0.02, **P<.005 pairwise comparisons (Student's t-test).

View larger version (75K): [in a new window]   Fig. 4. TGFß ligands and receptors are expressed in P4 rat retina. (A-F) Immunolocalization of TGFßRI (red, A) and II (red, B) in nestin-positive (green, C,F) processes of progenitor cells in the central retina. Arrowheads indicate double-labeled cells. (G) Quantitative RT-PCR results show TGFß2 is expressed 80-fold higher than TGFß3 and eightfold higher than TGFß1 in P4 retinal tissue. (H-J) TGFß2 (red) is expressed by ß3 tubulin-positive (green) ganglion and amacrine cells (arrowheads). (K) RT-PCR for TGFßs and receptors P4 retinal mRNA. Scale bar: 50 µm.

View larger version (97K): [in a new window]   Fig. 5. TGFß ligands and receptors are expressed in P10 rat retina. (A-F) Immunolocalization of TGFßRI (red, A) and II (red, D) glast-positive (C,F green) cell bodies (arrowheads) and processes (arrows) in central retina. (G-I) TGFß2 (red) is expressed by ß3 tubulin-positive (green) neurons of the inner retina (arrowheads). (J) RT-PCR analysis shows that transcripts for TGFß ligands 1 and 3, as well as for receptors I and II, are expressed in P10 rat retinas. (K) Quantitative RT-PCR showing TGFß2 is expressed 12-fold higher than TGFß1 at P10. Scale bar: 50 µm.

View larger version (30K): [in a new window]   Fig. 6. TGFß treatment reduces proliferation in P4 retina. P4 retinas were cultured as whole explants for 24 hours with BrdU. (A,B) BrdU labeling of control (A) and TGFß2-treated (B) explants. Fewer BrdU-positive cells are present in TGFß2-treated P4 explants. (C) P4 explants were dissociated after 24 hours of culture and processed for immunohistochemistry. TGFß1 (gray bar) and 2 (dark gray bar) reduced proliferation to 51.3% (±6.3) and 52.4% (±1.2) of control (white bar), respectively. No significant difference in BrdU+ cell numbers was observed in TGFß3 treated explants (black bar). (D) Dose-response curve indicates 13.4% (±1.7) of cells are BrdU-positive in control treated explants, compared to 8.4% (±1.6) at 0.5 ng/ml TGFß2, 7.4% (±0.7) at 5 ng/ml, and 6.4% (±0.4) at 50 ng/ml. *P<0.01, **P<0.005, pairwise comparison (Student's t-test).

View larger version (37K): [in a new window]   Fig. 7. Inhibition of TGFß signaling increases proliferation in P6 retinal explants. (A,B) BrdU labeling of P6 explants cultured as whole, floating retinas for 24 hours with BrdU, in control media, including mouse IGG (A) or in the presence of monoclonal antibody against TGFß ligands 1, 2 and 3 (B). Treatment with anti-TGFß antibody resulted in a 170.8% (±18.9) increase in the percentage of cells that were BrdU+, compared with control. *P<0.01, pairwise comparison (Student's t-test).

View larger version (63K): [in a new window]   Fig. 8. Inhibition of TGFßRI signaling increases proliferation in the central P6 retina. Postnatal day 5.5 rat pups were given intraocular injections of either DMSO (control) or 40 nanomoles SB431542, small molecule inhibitor of the TGFßRI kinase domain. Pups were injected with BrdU 12 hours later, at P6. (A) A section located at the level of the optic nerve (ON) shows few cells are BrdU+ (green) after intraocular injection with DMSO. (B) SB431542-treated retinas contain numerous BrdU-labeled (green) cells in the region of the optic nerve (ON). (C) Quantification of the number of BrdU+ cells in sections of the retina adjacent to the optic nerve show DMSO-treated control animals (n=6) average 105 (±27) BrdU+ cells/mm2 central retina, whereas SB-431542 treated animals (n=5) average 240 (±40) BrdU+ cells/mm2 central retina. **P<0.01. Scale bar: 100 µm.

View larger version (109K): [in a new window]   Fig. 9. Inhibition of TGFß signaling in vivo enhances Müller glial proliferation in response to EGF at P10. Postnatal day 10 rat pups received intraocular injections of either PBS/BSA (A-C,G), anti-TGFß cocktail (5 µg mouse-anti-TGFß and 1.25 µg TGFßRII-fc), 250 ngEGF(H), or a combination of EGF and anti-TGFß cocktail (D-F,I). Intraocular injections were followed by BrdU injections. (A-C) In PBS/BSA-injected animals, only an occasional BrdU (green) labeled cell was found. (D-F) In the anti-TGFß/EGF-treated animals, BrdU (green) and CRALBP (red) staining shows multiple double-positive cells (arrowheads) in both the inner and outer nuclear layers of the retina. (G-I) Low magnification views of control injected retina (G), EGF injected retina (H) or EGF + anti-TGFß injected retina (I). (J), Quantification of in vivo results, from counts of CRALBP+/BrdU+ cells present in central retinal sections: control, 12 (±6) cells/mm2 retina; anti-TGFß, 11.1 (±4) cells/mm2; EGF, 333.5 (±92) cells/mm2; EGF + anti-TGFß, 630 (±81) cells/mm2; *P<0.05, **P<0.005, pairwise comparison (Student's t-test). Scale bar: 50 µm.

View larger version (96K): [in a new window]   Fig. 10. p27kip1 expression is disrupted in vivo with inhibition of TGFß signaling. Sections shown represent typical sections from the central-most region of the retina from animals receiving intraocular injections of either PBS/BSA, 250 ng EGF or 250 ng EGF + anti-TGFß cocktail. (A-C) PBS/BSA injected animals show distinct p27kip1 (red, A) labeling of CRALBP-positive (green, C) cell bodies in the inner nuclear layer (arrows). (D-F) In EGF-treated animals, p27kip1 staining (red, D) is still present in CRALBP+ (green, F) Müller glia, although slightly reduced, compared with control-treated animals. (G-I) p27kip1 (red, G) labeling is largely absent from the CRALBP+ (green, I) Müller glia after treatment with EGF and anti-TGFß cocktail combined. Scale bar: 50 µm.