Department of Ophthalmology and Visual Sciences, University of Louisville Health Sciences Center, Louisville, Kentucky.
Invest Ophthalmol Vis Sci. 2014 Jan 15;55(1):301-9. doi: 10.1167/iovs.13-12600.
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.
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.
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.
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.
我们观察了猪眼的视锥和视杆发育，并将其与视锥和视杆前体细胞在视网膜下移植后的整合和分化潜能相关联。
通过在外视网膜中的位置和对分化标志物的免疫染色，在发育过程中鉴定视杆和视锥前体细胞。从不同发育阶段的 GFP(+)转基因猪胚胎视网膜中分离出胚胎视网膜细胞，移植到成年视网膜中，并通过免疫染色对分化标志物进行检测，以跟踪和量化整合和分化情况。
猪的视锥和视杆是空间分离的，这使我们能够在原位观察视杆和视锥的发育。猪的妊娠期为 114 天。在胚胎第 50 天（E）时，有丝分裂后视锥祖细胞已经形成了视网膜的外两层。这些视锥祖细胞通过表达 Islet1（ISL1）和 Recoverin（RCVRN）来标记（在这个胚胎阶段，RCVRN 专门标记这些视锥前体细胞）。相比之下，位于视锥祖细胞内部的有丝分裂后神经视网膜亮氨酸拉链（NRL）（+）视杆前体细胞直到 E65 才出现。在 E50 时，在 NRL（+）视杆前体细胞出现之前，移植细胞几乎只产生视锥。在 E57 时，移植细胞产生等量的视杆和视锥，但在 E65 时，移植细胞几乎只产生视杆。在 E85 或 E105 时移植细胞，即前体细胞开始表达视蛋白时，导致整合细胞数量很少。
与它们在胚胎视网膜中的顺序出现一致，这些结果表明，在视锥和视杆前体细胞移植后的整合/分化过程中，存在顺序和令人惊讶的狭窄发育窗口。
