Fig. 7. XOtx2 and XOtx5b are expressed in proliferating cells but do not stimulate proliferation. (A-F) A close-up of stage 41 retinal cryostat sections (10 µm) showing the ciliary marginal zone of embryos injected with BrdU and fixed 30 minutes later. Arrows in A-C and D-F point to the same cells. (A) A section stained for XOtx2 RNA shows labelled cells (arrows). (C) This section is stained with a BrdU antibody and is viewed under fluorescence. (B) Merged images of A and C (50% transparency). The same experiment is shown for XOtx5b in D-F. (G-H) The total number GFP-positive and BrdU+GFP-positive retinal cells were counted in five retinas lipofected with GFP and the vector alone (n=403), XOtx5b (n=355), EngR+XOtx5b (XOtx5bN+HD-EngR; n=317), VP16+XOtx5b (XOtx5b-VP16; n=384), VP16+XOtx2 (XOtx2-VP16; n=322) and XOTx2/5b (n=353), XOtx2 (n=418), or XOtx5b/2 (n=413). The percentage of BrdU-positive cells was calculated. None of the constructs tested gave an increase in proliferation of photoreceptor cells (G) or cells found in the outer edge of the inner nuclear layer (H). This analysis was done using a Student's t-test. n=the total number of cells counted. (I-R) XOtx2 modulates XOtx5b activity. (I-M) Embryos at stage 18 visualised under fluorescent light that have been injected on one side with the Xenopus opsin promoter fused to (I) GFP (Xop) DNA, ß-gal RNA (200 pg) and (J) 50 pg XOtx5b RNA (XOtx5b); (K) 25 pg XOtx5b and 25 pg XOtx2 (XOtx5b+XOtx2 1:1); (L) 17 pg XOtx5b and 33 pg XOtx2 (XOtx5b+XOtx2 1:2); (M) 50 pg XOtx2 (XOtx2). (N) Embryos from each set of injections [Xop (n=35); XOtx5b (n=37); XOtx5b+XOtx2 1:1 (n=28); XOtx5b+XOtx2 1:2 (n=29); XOtx2 (n=31)] were individually photographed under fluorescence and the fluorescence on both sides of the embryo was measured. The ratio of fluorescence on the injected versus uninjected side was calculated (relative GFP fluorescence) and an average for all the embryos from that set was made. Significant difference from the Xop-GFP control set using a Student's t-test was found for both the XOtx5b (P<0.001) and XOtx2 set (P<0.001). XOtx5b and XOtx2 together with Xop did not activate the Xop construct, suggesting that XOtx2 modulates the ability of XOtx5b to activate opsin. (O-Q) Sagittal section of stage 41 embryos lipofected with either GFP and pCS2⁺ vector (O), GFP, XOtx2 and XOtx5b (P), or GFP and XOtx5b (Q). Arrows point to an increase in bipolar cells in P and an increase in photoreceptors in Q. (R) Graph showing the percentage of GFP-positive retinal cells in retinas lipofected with GFP and the vector alone (n=1255); GFP, XOtx2 and XOtx5b (n=995); and GFP and XOtx5b (n=1188). Asterisks: P<0.01 as determined by a Student's t-test; n, number of GFP-positive retinal cells counted; GC, ganglion; Am, amacrine; BP, bipolar; Mü, Müller; H, horizontal, and PR, photoreceptor cells. Error bars represent s.e.m.

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