Characterization of YFP+ cells in the retina of CD11c-eYFP transgenic mice: the effects of breeding out the Crb1^rd8 mutation

Abstract

Purpose: The normal CNS parenchyma is traditionally thought to be devoid of professional antigen presenting cells (APCs); however, a population of putative dendritic cells (DCs) expressing yellow fluorescent protein (YFP) are present in the neural retina of C57Bl/6N CD11c-eYFP mice. We have previously shown that these mice carry the Crb1^rd8 mutation, which results in retinal dystrophic lesions. We hypothesized that the accumulation of YFP+ putative DCs within the retina may be the result of the pathology associated with the Crb1^rd8 mutation.

Methods: C57Bl/6N CD11c-eYFP mice were outcrossed with C57Bl/6J mice to create Crb1^rd8/wt offspring; heterozygous offspring were then intercrossed to generate CD11c-eYFP Crb1^wt/wt mice. At 6 weeks of age, CD11c-eYFP Crb1^wt/wt mice (n=50) underwent clinical fundus examination in brightfield and fluorescent modes. At 8 weeks of age, eyes were collected from perfusion-fixed mice (n=5) and retinal wholemounts were processed for confocal microscopy. Single cell suspensions were prepared from pooled retinas (n=10 mice), stained with an antibody panel for the identification of APC surface markers and subsequently characterized by flow cytometry.

Results: In vivo examination of the eyes of CD11c-eYFP Crb1^wt/wt mice revealed normal appearance of the fundus in brightfield mode, with no retinal lesions observed. However, fluorescent fundus imaging demonstrated the continued presence of YFP+ cells in the retina. Quantitative analysis of retinal wholemounts by confocal microscopy revealed a density of 94±3.3 (mean ± SEM) YFP+ cells/mm². These YFP+ cells, which were predominantly distributed within the plexiform and nerve fiber-ganglion cell layers of the retina, co-expressed the microglial markers CD11b, F4/80 and Iba-1, whereas few YFP+ cells expressed the APC markers CD11c, CD80, CD86, CD103, CD8, DEC205, 33D1 and MHC class II.

Conclusions: The detection of YFP+ cells in the retina of CD11c-eYFP Crb1^wt/wt mice indicates that the presence of these cells is not wholly a consequence of the Crb1^rd8 mutation. Retinal YFP+ cells displayed an immunophenotype that is consistent with microglia, and not DCs, suggesting that the YFP+ cells in the retina of CD11c-eYFP Crb1^wt/wt mice represent a subset of microglia.