Conclusions
Consistent with their sequential appearances in embryonic retina, these results demonstrate sequential and surprisingly narrow developmental windows for integration/differentiation of cone and rod precursors following transplantation.
Methods
Rod and cone precursors were identified during development by their position in the outer retina and by immunostaining for markers of differentiation. Embryonic retinal cells from green fluorescent protein (GFP)(+) transgenic pigs at different developmental stages were transplanted into adult retinas and integration and differentiation was followed and quantified by immunostaining for markers of cone and rod differentiation.
Purpose
We followed cone and rod development in the pig and we correlated development with the potential for cone and rod precursor integration and differentiation following subretinal transplantation.
Results
Pig cones and rods are spatially segregated, allowing us to follow rod and cone development in situ. Gestation in the pig is 114 days. By embryonic day (E) 50, postmitotic cone progenitors had formed the outer two rows of the retina. These cone progenitors are marked by expression of Islet1 (ISL1) and Recoverin (RCVRN) (at this embryonic stage, RCVRN exclusively marks these cone precursors). By contrast, postmitotic neural retina leucine zipper (NRL)(+) rod precursors, located interior to the cone precursors, did not appear until E65. At E50, before NRL(+) rod precursors are evident, transplanted cells gave rise almost exclusively to cones. At, E57, transplanted cells gave rise to equal numbers of rods and cones, but by E65, transplanted cells gave rise almost exclusively to rods. Transplantation of cells at E85 or E105, as precursors initiate opsin expression, led to few integrated cells. Conclusions: Consistent with their sequential appearances in embryonic retina, these results demonstrate sequential and surprisingly narrow developmental windows for integration/differentiation of cone and rod precursors following transplantation.