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C. Gutierrez, M. M. McNally, M. V. Canto-Soler; RA4 and RMO270 Are Not Exclusive for Ganglion Cells During Retinal Development. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5943.
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The RA4 antibody recognizes a microtubule-associated protein expressed by long-axon projecting neurons in the chick, while RMO270, is a marker for neurofilaments expressed in migrating neuroblasts. In the retina, RA4 and RMO270 have long been considered exclusive to newly born migrating and differentiated ganglion cells (GCs). However in our experiments, observations suggested that GC progenitors may not be the only retinal progenitor cells (RPCs) expressing these markers. The purpose of this study is to determine whether RA4 and RMO270 are exclusive to newly born migrating GCs or if they label other RPC types as well.
Embryonic day (ED) 6 and 8 chick eyes were fixed in 4% para-formaldehyde, frozen and sectioned for immunohistochemical analysis. Double labeling fluorescent immunohistochemistry was used to analyze co-expression of RA4 and RMO270 with other transcription factors and markers specific for different cell types. Confocal microscopy was used to analyze the expression pattern of these markers in fundal and peripheral regions of the retina.
At ED6 RA4 showed a characteristic "radial" expression in the fundal region of the retina, demarcating the "neurogenic zone". At ED8, "radial" RA4+ cells were absent from the fundus but present in peripheral regions of the retina; in the fundus RA4 labeling was still observed in cells confined to the putative horizontal cell layer (HCL), and neural projections in the inner limiting membrane and the nerve fiber layer. At both stages of development all RA4+ cells were positive for RMO270. However we also observed a small population of cells positive for RMO270 and not for RA4. Interestingly, RMO270+/RA4- cells were evident at the leading edge of the neurogenic front, suggesting RMO270 expression may start earlier than RA4 in differentiating RPCs. In addition, double-labeling revealed these cells to also express markers characteristic of different retinal cell types. At ED6, RA4+/RMO270+ cells were also positive for transcription factors AP2-α, Lim1/2 and Prox1. In addition, a very small population of cells expressing Visinin was also positive for RA4 and RMO270. At ED8 RA4+/RMO270+ cells in the ganglion cell layer were co-labeled with Brn3a, a GC specific transcription factor, while those located in the putative HCL were positive for Prox1, Lim1/2, and AP2- α .
These observations suggest RA4 and RMO270 are not exclusive to GCs; rather, they appear to be transiently expressed in most, if not all, RPC types. Additional studies are underway to better characterize the spatial and temporal patterns of expression of RA4 and RMO270 in the developing retina.
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