Generation of mice with inducible RasV12 expression in microglia
The double transgenic lines CAG-LSL-RasV12-IRES-EGFP; Cx3cr1CreER (Cx3cr1-RasV12 mice) and CAG-LSL-EGFP;Cx3cr1CreER (control mice) were used in this study. The chemokine receptor CX3CR1 is specifically expressed in monocytes and tissue-resident macrophages, including microglia ; hence, Cx3cr1 CreER knock-in mice expressed tamoxifen-inducible Cre recombinase in microglial cells. EGFP was also expressed after tamoxifen administration in both Cx3cr1-RasV12 mice and control mice, allowing for lineage tracing.
Control and Cx3cr1-RasV12 mice were born at the expected Mendelian ratio (Table 1). Tamoxifen was administered to control and Cx3cr1-RasV12 mice at postnatal day 14 (P14). Death of unknown causes was observed in Cx3cr1-RasV12 mice 9–10 days after tamoxifen injection. Since Cx3cr1 is also expressed in circulating monocytes [21, 22], we evaluated the presence of monocytes with Cre-mediated recombination in the peripheral blood by flow cytometry (Supplementary Fig. S1a). In Cx3cr1-RasV12 mice, EGFP expression was detected in approximately 1% of CD11b-positive cells (Supplementary Fig. S1b). Therefore, we could confidently conclude that EGFP-positive cells in the retina and brain were mostly microglia and that the representation of macrophages in this population is negligible.
Characterization of RasV12-expressing microglia in the retina and brain
We then evaluated the presence of RasV12-expressing microglia in the retina and the brain. Retinas harvested at day 7 were frozen-sectioned for histological examination. The number of EGFP-positive cells was considerably higher in retinas from Cx3cr1-RasV12 mice compared with control retinas (Fig. 1a, c). Moreover, the vast majority of EGFP-expressing cells also expressed Iba1, which is a marker of microglia and macrophages (Fig. 1b). We then examined the time course of EGFP expression at days 1, 3, 5, and 7 after tamoxifen administration. EGFP was expressed from day 3 in the retinas of control and Cx3cr1-RasV12 mice (Fig. 1e, arrowheads). EGFP-positive cells also expressed the microglia-specific marker TMEM119 (Fig. 1e) . EGFP+TMEM119+ cells accounted for nearly 90% of the total EGFP-positive cell population in control and Cx3cr1-RasV12 retinas at days 3, 5, and 7 after tamoxifen administration (Fig. 1F). The number of EGFP+TMEM119+ cells increased until day 7 in Cx3cr1-RasV12 retinas, whereas only a slight increase was observed in control retinas (Fig. 1e, g). Furthermore, immunohistochemical analysis revealed the presence of phosphorylated ERK1/2 in EGFP-positive microglia in Cx3cr1-RasV12 retinas (Fig. 1h, arrowheads), confirming Ras signaling pathway activation in these microglial cells.
In the brains of Cx3cr1-RasV12 mice, we observed prominent EGFP expression in the ventral orbital cortex (VO), lateral orbital cortex (LO), and ventral tenia tecta (VTT) at day 7 after tamoxifen administration (Supplementary Fig. Sa). Hematoxylin and eosin staining confirmed strong nuclear staining in these regions of the brain (Supplementary Fig. S2b). Similar to what we observed in the retina, EGFP expression was apparent from day 3 in the orbital cortex region of the brain (Supplementary Fig. S2c), and EGFP-positive cells also expressed Iba1 (Supplementary Fig. S2c, arrowheads d, e). The number of EGFP+Iba1+ cells continuously increased in the brain of Cx3cr1-RasV12 mice (Supplementary Fig. S2c, d). Moreover, ERK1/2 phosphorylation was detected in EGFP-positive cells in the brain of Cx3cr1-RasV12 mice (Supplementary Fig. S2f, arrowheads).
Migration and proliferation of RasV12-expressing microglia
Resting microglia are often found in the IPL and OPL of the retina . In control retinas, EGFP+TMEM119+ cells were primarily found in the IPL and OPL at day 7 after tamoxifen administration (Fig. 1e, 2a). In contrast, RasV12-expressing microglia were observed in the ganglion cell layer (GCL), ONL, and SR (Fig. 1e, 2a), suggesting microglial cell migration to the apical and basal sides of the retina.
At day 7 after tamoxifen administration, retinas from Cx3cr1-RasV12 mice contained a significantly higher number of EGFP+TMEM119+ cells compared with control retinas (Fig. 1g). Additionally, retinas from Cx3cr1-RasV12 mice contained a significantly higher number of proliferating microglial cells (Ki67+EGFP+; Fig. 2b, arrow heads). Ki67+EGFP+ cells comprised nearly 70% of the total EGFP-positive cell population in retinas from Cx3cr1-RasV12 mice (Fig. 2c). Although Ki67+EGFP+ cells were present in all retinal layers, notably high numbers were observed in the apical and basal regions (Fig. 2d).
RasV12-expressing microglia exhibited ameboid morphology with a large cell body and short pseudopodia (Fig. 2b), which is typically observed in activated microglia [25, 26]. Consistently, Ccl2, Ccl3, Cxcl1, Cxcl5, Il1b, Il6, and Tnf were significantly upregulated in Cx3cr1-RasV12 retinas (Fig. 2e), further supporting microglial activation (Nakagawa and Chiba, 2014; Franco and Fernández-Suárez, 2015). In the brain, although a significant number of EGFP-positive cells expressed Ki67 (Supplementary Fig. S3a, b), Ki67-positive cells comprised less than 30% of the total EGFP-positive cell population (Supplementary Fig. S3c).
RasV12 expression in microglia promotes photoreceptor degeneration
Histological analysis indicated the loss of photoreceptors in Cx3cr1-RasV12 retinas (Fig. 3a, arrowheads). Immunohistochemical staining revealed that microglial cells were localized close to the degenerating regions of the ONL (Fig. 3b). Moreover, differential interference contrast (DIC) imaging showed a reduction in the thickness of the outer layer of Cx3cr1-RasV12 retinas (Fig. 3b, c). We also performed Annexin V staining to measure apoptosis  and found an increased number of apoptotic cells in Cx3cr1-RasV12 retinas. Importantly, the number of apoptotic cells was higher in CD73-positive rod cells than other retinal cells (Fig. 3d, e). In Cx3cr1-RasV12 retinas, we also observed upregulation of Edn2 and Fgf2 (Fig. 3f), which are commonly induced upon photoreceptor damage .
Retinal degeneration is often accompanied by reactive gliosis, which is characterized by strong GFAP expression . Consistently, GFAP was upregulated in Cx3cr1-RasV12 retinas (Fig. 3g, h), and microglia were localized adjacent to the GFAP-positive MGCs (Fig. 3f, enlarged panels). We found no differences in the numbers of RBPMS-positive retinal ganglion cells (Supplementary Fig. S4a), AP-2α-positive amacrine cells (Supplementary Fig. S4b), Chx10-positive bipolar cells (Supplementary Fig. S4c), and Calbindin-positive horizontal cells (Supplementary Fig. S4d) in RasV12-expressing and control retinas, suggesting that survival and maintenance of these cell types was not affected by RasV12 expression in microglia.
RasV12-expressing microglia in the ONL, but not in the INL, mediates rod photoreceptor phagocytosis
Confocal microscopy revealed that RasV12-expressing microglia engulfed recoverin-positive rod photoreceptors in the damaged ONL regions (Fig. 4a); hence, we measured phagocytosis related molecules in Cx3cr1-RasV12 retinas. In Cx3cr1-RasV12 mice, EGFP-positive cells strongly expressed CD68 (Fig. 4b, arrow heads), which is a lysosomal protein highly expressed in activated microglia [7, 31]. Interestingly, the number of CD68-expressing cells was higher in the ONL and SR than in the inner layers of the retina (Fig. 4c). Furthermore, population of the EGFP+CD68+TMEM119+ cells in EGFP and Tmem119 double positive cells was considerably higher in the ONL and SR than in other retinal regions (Fig. 4d). Microglial iNOS expression was demonstrated in rd8 mice[28, 32], and an optic nerve injury mouse model . Immunohistochemical staining revealed strong iNOS expression in EGFP-positive microglial cells in Cx3cr1-RasV12 retinas (Fig. 4e). EGFP+iNOS+ cells were also observed in the inner retinal layers (Fig. 4e, f); nevertheless, this double-positive cell population was higher in the ONL and SR than in other regions of the retina (Fig. 4g). Furthermore, the phagocytosis-related genes C3 and Cd68 were upregulated in Cx3cr1-RasV12 retinas (Fig. 4h).
The behavior of RasV12-expressing microglia differs in the IPL and ONL
We next examined the behavior of RasV12-expressing microglia in different regions of the retina. We prepared retinal explants from Cx3cr1-RasV12 mice and labeled the nuclei of rod photoreceptors with Hoechst33342. Time-lapse imaging revealed that RasV12-expressing microglia in the ONL engulfed photoreceptors by extending their pseudopodia. Moreover, multiple nuclei were observed in individual microglial cells, suggesting rod photoreceptor engulfment by microglia (Fig. 5, upper panel, Supporting information movie 1). However, we found no evidence of cells engulfment by RasV12-expressing microglia in the IPL (Fig. 5, lower panel, Supporting information movie 2).