Fig. 5. Effects of VP16 or Engrailed fusion constructs of XOtx5b and XOtx2 on retinal cell fate decisions. (A,B) Cryostat sections (10 µm) of retinas lipofected with (A) GFP and the pCS2⁺ vector or (B) XOtx5bN+HD-VP16 and GFP then stained with an anti-cone photoreceptor antibody (calbindin in red). Arrowheads indicate cells that fluoresce with GFP alone (green) and arrows indicate cells expressing both GFP and calbindin (yellow). (C) Graph showing an increase in the distribution of GFP-positive photoreceptor cells in retinas colipofected with XOtx5bN+HD-VP16 and GFP (n=344) when compared to GFP and pCS2⁺ (n=493). Values were calculated as in Fig. 4D,E; P0.04. In C,D,G and J asterisks indicate P values as calculated using a Student's t-test; n=the total number of retinal cells counted; error bars, s.e.m. (D) Graph showing the types of photoreceptor cells produced in retinas co-lipofected with GFP and pCS2⁺ (n=184) or VP16-XOtx5bHD and GFP (n=267). Cells positive for GFP (unlabelled) or GFP and calbindin (cones) were counted in one retina and the percentage of each determined. Percentages from both cones and unlabeled cells were averaged in all retinas counted. The error bar indicates s.e.m. Even though a larger number of photoreceptors were produced in XOtx5bN+HD-VP16 lipofected retinas (C), cones and unlabeled cell numbers were similar to each other in retinas lipofected with either XOtx5bN+HD-VP16 or the control construct. (E) Cryostat section (10 µm) of a retina co-lipofected with GFP and XOtx2N+HD-EngR, which shows a decreased number of bipolar cells. White lines (also in F-H) mark the inner and outer plexiform layers. (F) GFP and XOtx5bN+HD-EngR co-lipofected retina (10 µm cryostat section) showing a distinct decrease of fluorescently labelled photoreceptor cells. (G) The percentage of GFP-positive photoreceptor cells in the XOtx5bN+HD-EngR+GFP (n=651) co-lipofected retinas is decreased while the percentage of GFP-positive bipolar cells is decreased in the XOtx2N+HD-EngR+GFP (n=474) co-lipofected retinas when compared to controls (GFP and pCS2⁺, n=692). This is a representative experiment. Lipofection of each construct with the control was repeated in two separate experiments, resulting in identical population shifts as shown [XOtx2N+HD-EngR+GFP (n=1692); XOtx5bN+HD-EngR+GFP (n=2121)]. P0.05, (H-I) Sagittal section of stage 41 retinas that have been co-lipofected with GFP and either (H) XOtx5bN+HD-VP16 or (I) XOtx2N+HD-VP16. In both, there are a large number of labelled photoreceptor cells. Scale bar: 30 µm. (J) The percentage of each retinal cell type, which is GFP-positive, in retinas lipofected with XOtx5bN+HD-VP16 (n=342) or XOtx2N+HD-VP16 (n=294) and GFP. GFP and the expression vector alone acted as the control (n=535). Nearly all of the GFP-positive cells are photoreceptors in the experimental lipofected retinas. The asterisk denotes significant differences between the control and each of the constructs lipofected (P0.02); GC, ganglion cells; Am, amacrine cells; BP, bipolar cells; Mü, Müller cells; H, horizontal cells; PR, photoreceptor cells. (K) A schematic of the constructs used in the above lipofections. HD, homeodomain. The numbers above the bars indicate the amino acid residues.

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