Retinitis pigmentosa (RP) is a type of inherited retinal degenerative disease, which leads to blindness. The primary pathological event of this disease is the death of rods because of genetic mutations. The S334ter-line-3 rat is a transgenic model developed to express a rhodopsin mutation similar to that found in RP. In this study, the rod's death triggered are organization of the cone mosaic into an orderly array of rings. Four observations were relevant to understand this reorganization. First, rods died in hot spots, which progressively increased as circular waves, leaving rod-less zones behind. Second, rings of cones formed around these zones. Third, remodeled Müller glia processes enveloped cones and filled the center of their rings. Zonula occludens-1 located between the photoreceptor inner segments and the apical processes of Müller cells showed in the rings. Fourth, these rings were formed before the onset of cone cell deaths and were maintained until late stages of RP. From these observations,we hypothesize that cone-Müller-cell interactions mediate and maintain the rings. A test of this hypothesis can be performed by injecting DL-a-aminoadipic acid (AAA), which is known to disrupt Müller cell metabolism. A single intravitreal injection of AAA at P50 disrupted the rings of cones 3 days after the injection. These findings indicate that the rearrangement of cones in rings is modulated by Müller cells in RP. Thus, if the relationship between photoreceptors and Müller glia is better understood, the latter could potentially be manipulated for effective neuroprotection or the restoration of normal cone arrays.

We have recently described the surviving cones and Müller-glia process remodeling in retinitis pigmentosa (RP) and shown that rod degeneration triggers the reorganization of the cone mosaic into an orderly array of rings. Within these rings, remodeled Müller-glia processes envelope cones. Here, we report the spatiotemporal pattern of healthy rods, their relationship with dying rods and the way that rod death stimulates the modification of cone spatial-distribution patterns and Müller-glia processes in the S334ter-line-3 rat, a transgenic model expressing a rhodopsin mutation that causes RP. The spatial patterns of rods, cones, microglial and Müller cells were labeled by immunocytochemistry with cell-type-specific markers at various stages of deveopment in rat whole-mount retinas. Spatial patterns of dying cells were examined by TUNEL staining. The S334ter rod mosaic began to develop small holes around postnatal day 10. These hot-spots of cell death progressively increased in size, leaving larger rod-less holes behind. The holes were temporarily occupied by active microglial cells, before being replaced by remodeled Müller-cell processes. Our data suggest that the hot spots of rod death create holes in the rod mosaic early in retinal degeneration and that the resulting pattern triggers the modification of the spatial-distribution patterns of cones and glia cells.

In retinitis pigmentosa (RP), the death of cones normally follows some time after the degeneration of rods. Recently, surviving cones in RP have been studied and reported in detail. These cones undergo extensive remodeling in their morphology. Here we report an extension of the remodeling study to consider possible modifications of spatial-distribution patterns. For this purpose we used S334ter-line-3 transgenic rats, a transgenic model developed to express a rhodopsin mutation causing RP. In this study, retinas were collected at postnatal (P) days P5–30, 90, 180, and P600. We then immunostained the retinas to examine the morphology and distribution of cones and to quantify the total cone numbers. Our results indicate that cones undergo extensive changes in their spatial distribution to give rise to a mosaic comprising an orderly array of rings. These rings first begin to appear at P15 at random regions of the retina and become ubiquitous throughout the entire tissue by P90. Such distribution pattern loses its clarity by P180 and mostly disappears at P600, at which time the cones are almost all dead. In contrast, the numbers of cones in RP and normal conditions do not show significant differences at stages as late as P180. Therefore, rings do not form by cell death at their centers, but by cone migration. We discuss its possible mechanisms and suggest a role for hot spots of rod death and the remodeling of Müller cell process into zones of low density of photoreceptors.

These findings confirm that TIMP-1 induced M-cone mosaics in S334ter-line-3 to gain homogeneity without reaching the degree of regularity seen in normal retinal mosaics. Even if TIMP-1 fails to promote regularity, the effects of this drug on homogeneity appear to be so dramatic that TIMP-1 may be a potential therapeutic agent. TIMP-1 improves sampling of the visual field simply by causing homogeneity.

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Due to the limited specificity of 'classical' AT1 cell markers such as AQP5, PDPN, HOPX, and AGER, there is a need for identifying new AT1 cell markers and tools for characterizing AT1 cell phenotype. Recently, through transcriptome profiling or single-cell-RNA sequencing, respectively, we have identified and characterized two new AT1 cell markers, GRAMD2 and GPRC5A. We further generated a new transgenic mouse line, Gramd2CreERT2, and tested AT1 cell specificity of this strain. METHODS: We used CRISPR/Cas-assisted genetargeting to knockin creERT2 and nuclear GFP (nGFP) into the stop codon of the endogenous Gramd2 gene. Because GFP protein could not be detected, we crossed the GD2CE (Gramd2 promoter-driving creERT2) mouse with the Cre-reporter mouse strain mTmG (membrane-targeted tandem dimer Tomato (mT) prior to Cremediated excision and membrane-targeted GFP (mG) after excision) to generate GD2CE;mTmG mice to evaluate lineage labeling by GD2CE by Western blotting, and double labeling immunofluorescence (IF) of tissues and isolated cells for GFP, AT1, AT2 and airway markers.RESULTS: By western blot, GFP is exclusively detected in the lung but not in other organs, indicating lung specificity. In lung sections, GFP colocalized with AT1 cell markers AQP5 or PDPN, but not with AT2 cell marker proSPC, indicating that in distal lung, GD2CE;mTmG lineage labels AT1 cells. However, the GFP positive signal was not detected in all AQP5 or PDPN positive AT1 cells, suggesting either incomplete recombination or heterogeneity of Gramd2 gene expression among AT1 cells. In the distal airway, very few GFP positive cells were detected and co-stained with CC10 (1.91 ± 0.36% of CC10 + cells were GFP and CC10 double-positive). We further quantified recombination efficiency by performing IF in cytospins of crude alveolar epithelial cell preparations. In cytospins, GFP colocalized with AQP5 (52.83 ± 3.42% of AQP5 + cells were GFP and AQP5 double-positive) and rarely with proSPC (0.56 ± 0.56% of SPC + cells were GFP and SPC double-positive), confirming AT1 cell labeling in alveolar epithelium. Furthermore, we successfully utilized GD2CE;mTmG mice to sort GFP+ AT1 cells of high purity using a GFP antibody. 98.57 ± 0.79% of GFP+ cells were AQP5 + , and 99.72 ± 0.19% of GFP+ cells were GPRC5A + . CONCLUSIONS: GD2CE mice label AT1 cells in distal lung with high specificity, with minimal labeling of airway cells. GD2CE mice are a novel tool for study of AT1 cell biology and will be useful for both isolation of AT1 cells and in vivo studies.

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