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Exogenous growth factors induce the production of ganglion cells at the retinal margin

Department of Biological Structure, University of Washington, Seattle, WA 98195, USA

View larger version (93K): [in a new window]   Fig. 1. The proliferation and accumulation of CMZ cells is stimulated by insulin, but not by FGF2. (A-D) Vertical sections of the temporal retinal margin that have been labeled for BrdU immunoreactivity. Sections were obtained from eyes that were treated with (A) saline, (B) insulin alone, (C) FGF2 alone, and (D) insulin and FGF2. (E) Histogram demonstrating that the number of BrdU-labeled cells added to the nasal and temporal peripheral edge of the retina is stimulated by insulin, but is not further stimulated by co-application of FGF2. (F-T) Vertical sections of the retinal margin were double labeled with antibodies to Pax6 (green) and Chx10 (red). Tissues were obtained from eyes that received two consecutive daily injections of (F-H) saline or (I-T) insulin alone. Tissues were obtained (F-N) 24 hours or (O-T) 10 days after the final injection. Small arrows indicate cells that are double-labeled for Pax6 and Chx10, arrowheads indicate cells labeled for Pax6 or Chx10 alone, and the large arrows indicate the retinal margin, with the pars plana to the right and more central regions of the retina the left. The boxed areas in K and Q are enlarged in L-N and R-T, respectively. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars: in D, 50 µm in A-D; in Q, 50 µm in F-K and O-Q.

View larger version (58K): [in a new window]   Fig. 2. Markers of ganglion cells are expressed in the CMZ after treatment with insulin alone and insulin with FGF2. (A-E) Vertical sections of (A) central retina and (B-E) the retinal margin that have been labeled for neurofilament immunoreactivity. Sections were obtained from eyes that were treated with three consecutive daily injections of (A,B) saline, (C) insulin alone or (D,E) insulin and FGF2. Retinas were processed for immunocytochemistry (A-D) 24 hours after the final injection or (E) 4 days after the final injection. Arrows indicate the CMZ, with the ciliary body to the right and central regions of the retina to the left. Scale bar: 50 µm in A-E. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; NFL, nerve fiber layer. (F) Histogram demonstrating that the number of neurofilament-positive cells in the CMZ is increased by injections of insulin alone and insulin and FGF2, but not by FGF2 alone.

View larger version (117K): [in a new window]   Fig. 3. Neurofilament-expressing cells in the CMZ are newly generated and express markers of differentiated neurons. (D-F) Vertical sections of (A-C,G-I,M-O) the retinal margin and (D-F,J-L) the central retina were labeled with antibodies to (A; red) the 160 kDa subunit of neurofilament, (D,G,J,M; red) 145 kDa subunit of neurofilament, (B; green) BrdU, (E,H; green) Hu, and (K,N; green) Islet1. C,F,I,L,O are merged images. Tissues were obtained from eyes that were treated with three consecutive daily injections of insulin and FGF2. The small arrows indicate double-labeled cells, and the large arrowhead in J-L indicates a neurofilament-positive ganglion cell that does not express Islet1. Scale bars: in C, 50 µm in A-C; in O, 50 µm in D-O. NF, neurofilament; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

View larger version (41K): [in a new window]   Fig. 4. Insulin and FGF2 induce the production of Islet1-expressing cells in the ganglion cell layer at the retinal margin. (A) Histogram demonstrating that the number of BrdU/Islet1-labeled cells in the inner nuclear layer at the retinal margin of insulin-treated eyes is increased between 1 and 4 days after the final injection. (B) Histogram demonstrating that the number of BrdU/Islet1-labeled cells in the ganglion cell layer is stimulated by insulin and FGF2. (C-E) Vertical sections of the retinal margin that were labeled for BrdU (green) and Islet1 (red). Tissues were obtained from eyes 4 days after the final dose of three consecutive daily injections of insulin and FGF2. Scale bar: in E, 50 µm in C-E. INL, inner nuclear layer; GCL, ganglion cell layer.

View larger version (77K): [in a new window]   Fig. 5. Insulin and FGF2 induce the production of Brn3.0-expressing ganglion cells at the retinal margin. Vertical sections of (A-C) central retina and (D-I) the retinal margin were double-labeled for (A,C,D,F; green) neurofilament and (B,C,E,F; red) Brn3.0 or (G,I; green) BrdU and (H,I; red) Brn3.0. Tissues were obtained from eyes that received three consecutive daily injections of (D-F) insulin alone or (A-C,G-I) insulin and FGF2. The large arrow indicates the retinal margin, with the ciliary body to the right and central regions of the retina to the left. The small arrows in A-C indicate a displaced ganglion cell that expresses neurofilament but not Brn3.0, and the small arrows in G-I indicate double labeled cells. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars: in F, 50 µm in D-F: in I, 50 µm in A-C,G-I.

View larger version (67K): [in a new window]   Fig. 6. Insulin and FGF2 induce the production of neurofilament-expressing ganglion cells at the retinal margin. Whole-mount preparations of the retinal margin were obtained from eyes 4 days after three consecutive daily injections of insulin and FGF2. Tissues were labeled with antibodies to (A,C,D,F; green) BrdU and (B,C,E,F; red) neurofilament. Large arrows indicate the retinal margin and small arrows indicate double-labeled cells. Scale bar: 50 µm.

View larger version (91K): [in a new window]   Fig. 7. Cath5 is expressed at the retinal margin of eyes treated with insulin and FGF2. Vertical sections of the retinal margin were labeled for Cath5 transcripts. In situ hybridization was used to detect Cath5 mRNA in tissues treated with (B) insulin alone or (A,C) insulin and FGF2. Tissues were processed for in situ hybridization 24 hours after the final dose of three consecutive daily injections of growth factor. Large arrows in B,C indicate the retinal margin and the small arrows indicate Cath5-expressing cells. The boxed area in C is increased 2.5-fold below to better demonstrate Cath5-expressing cells. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: 50 µm. (D) Histogram demonstrating the numbers of Cath5-expressing cells observed at the retinal margin of eyes treated with insulin alone, FGF2 alone, and a combination of insulin and FGF2.

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