Wild-type cone photoreceptors persist despite neighboring mutant cone degeneration

Abstract

In many retinal diseases, the malfunction that results in photoreceptor loss occurs only in either rods or cones, but degeneration can progress from the affected cell type to its healthy neighbors. Specifically, in human and mouse models of Retinitis Pigmentosa the loss of rods results in the death of neighboring healthy cones. Significantly less is known about cone-initiated degenerations and their affect on neighboring cells. Sometimes rods remain normal after cone death, whereas other patients experience a loss of scotopic vision over time. The affect of cone death on neighboring cones is unknown. The zebrafish is a cone-rich animal model in which the potential for dying cones to kill neighboring healthy cones can be evaluated. We previously reported that the zebrafish cone phosphodiesterase mutant (pde6c(w59)) displays a rapid death of cones soon after their formation and a subsequent loss of rods in the central retina. In this study we examine morphological changes associated with cone death in vivo in pde6c(w59) fish. We then use blastulae transplantations to create chimeric fish with a photoreceptor layer of mixed wild-type (WT) and pde6c(w59) cones. We find that the death of inoperative cones does not cause neighboring WT cone loss. The survival of WT cones is independent of transplant size and location within the retina. Furthermore, transplanted WT cones persist at least several weeks after the initial death of dysfunctional mutant cones. Our results suggest a potential for the therapeutic transplantation of healthy cones into an environment of damaged cones.

Figure 1.

Morphological changes in individual cones undergoing death in the pde6c ^w59 mutant. Wild-type and mutant embryos were injected with TαCP:MCFP at the one-cell stage. At 4 dpf, mosaic patches of MCFP-positive cones were imaged once an hour for 7 h. One cell in each genotype was pseudocolored throughout the time course of the recording to facilitate its identification. Scale bar, 10 μm.

Figure 2.

Death of the cone population occurs in two stages in pde6c ^w59. A, B, Eyes from wild-type (A) and pde6c ^w59 (B) Tg(TαCP:MCFP) fish were removed at the indicated ages and inverted onto a slide to allow imaging of the cones in the central retina at the back of the eye. Scale bar, 50 μm. C, Cell counts show a rapid decline in cone number in pde6c ^w59 between 4 and 5 dpf followed by a slow loss over the next several days. Percentages are pde6c ^w59 over wild-type cell counts at each time point, and error bars are the SEM.

Figure 3.

Mutant eyes have an increased density of TUNEL-positive cells. A, Eyes from wild-type and pde6c ^w59 Tg(TαCP:MCFP) fish were removed at the indicated ages and immunolabeled with TUNEL to show late stage apoptotic cells. Scale bar, 50 μm. B, Higher magnification images show that most MCFP-positive cells are not TUNEL positive. Scale bar is 50 μm. C, The number of TUNEL-positive cells per eye is increased in mutant eyes as compared to wild-type siblings. Error bars are the SEM.

Figure 4.

There is no cone to cone bystander effect in the pde6c ^w59 mutant. Blastulae stage transplants were performed to create chimeric fish containing a mixed photoreceptor layer. A–D, All cones are shown in gray scale. Donor cones express GFP (bright cytoplasmic fill), host cones express MCFP (outer segment fluorescence, visible in 7 dpf images). Genotypes are listed as donor → host. Transplant size is determined at 3 dpf, and compared to images at 6–9 dpf. Scale bar, 50 μm. Counts below the images show a dot for each transplanted cone within the associated image above. A, Mutant cells die in a mutant host. B, Mutant cells die in a wild-type host. C, Wild-type cells are viable in a wild-type host. D, Wild-type cells are viable in a mutant host. E, The viability of multiple transplants such as those in A–D was graphed as the percentage of cells from the 3 dpf counts that remained after 6 dpf. Error bars are the SEM, and n = the number of fish. F, G, Higher magnification images of Tg(TαCP:MCFP) donors in a pde6c ^w59 host show some shortening of WT cones in the central retina. Host cones (mutant) are not fluorescent. Scale bar, 10 μm.

Figure 5.

Location and size does not influence the viability of wild-type donors in the mutant hosts. A, Images from wild-type donors in pde6c ^w59 hosts were divided into the peripheral, intermediate, and central retina (see diagram). The viability of transplants in each region is shown as a percentage of cells from the 3 dpf image that remained after 6 dpf. There was not a difference between regions. Error bars are the SEM, and n = the number transplant patches. B, C, The size of the transplant does not influence wild-type viability. B, Higher magnification images from Tg(TαCP:GFP) donors in pde6c ^w59 Tg(TαCP:MCFP) host showing the continued presence of isolated individual donor cells at 3 and 6 dpf (arrows). Scale bar, 10 μm. C, Three-dimensional reconstructions of the image stacks were used to count the cones within isolated patches of wild-type transplant in either wild-type or pde6c ^w59 hosts. The viability of these transplants is graphed as a function of the number of cells in the patch at 3 dpf. No differences are seen between wild-type donors in the two host environments.

Figure 6.

Transplanted wild-type cones are still present at 21 dpf. Chimeric fish were grown to 21 dpf, cryosectioned, and imaged. A, Low-magnification images show the retinal layers with the decreased size of the ONL in the mutant retina. Cones from wild-type donors expressing Tg(TαCP:GFP) are shown in green. Scale bar, 50 μm. B, High-magnification images show the morphology of the wild-type donor cones in gray scale. Wild-type cones in the mutant retina are shorter, presumably due to collapse of the ONL. Scale bar, 10 μm.