Compensatory engulfment and Müller glia reactivity in the absence of microglia

Abstract

Microglia are known for important phagocytic functions in the vertebrate retina. Reports also suggest that Müller glia have phagocytic capacity, though the relative levels and contexts in which this occurs remain to be thoroughly examined. Here, we investigate Müller glial engulfment of dying cells in the developing zebrafish retina in the presence and absence of microglia, using a genetic mutant in which microglia do not develop. We show that in normal conditions clearance of dying cells is dominated by microglia; however, Müller glia do have a limited clearance role. In retinas lacking intact microglial populations, we found a striking increase in the engulfment load assumed by the Müller glia, which displayed prominent cellular compartments containing apoptotic cells, several of which localized with the early phagosome/endosome marker Rab5. Consistent with increased engulfment, lysosomal staining was also increased in Müller glia in the absence of microglia. Increased engulfment load led to evidence of Müller glia reactivity including upregulation of gfap but did not trigger cell cycle re-entry by differentiated Müller glia. Our work provides important insight into the phagocytic capacity of Müller glia and the ability for compensatory functions and downstream effects. Therefore, effects of microglial deficiency or depletion on other glial cell types should be well-considered in experimental manipulations, in neurodegenerative disease, and in therapeutic approaches that target microglia. Our findings further justify future work to understand differential mechanisms and contexts of phagocytosis by glial cells in the central nervous system, and the significance of these mechanisms in health and disease.

Keywords: Müller glia; microglia; phagocytosis; reactivity; retina; zebrafish.

Figure 1:. Homozygous irf8 mutant zebrafish are deficient in retinal microglia, accumulate TUNEL+ cells, and have normal gross retinal organization

Representative images of 3 dpf irf8 wildtype (A, A’) and irf8 mutant (B-B”) zebrafish whole eyes stained for pan-leukocyte marker (L-plastin, magenta) and DAPI (blue). (B’) Enlarged region of dashed rectangle in B showing pyknotic nuclei observed in irf8 mutants. A’ and B” show single channel images of L-plastin staining. (C) Quantification of number of L-plastin+ cells per whole retina in irf8 wildtype, heterozygote, and mutant siblings. (D, D’, E, E’) Representative mages of whole eyes of irf8 wildtype and mutants stained with TUNEL (red) and DAPI (cyan) depicting TUNEL+ cell accumulation in mutants. D’ and E’ show single channel images of TUNEL staining. (F) Quantification of average number of TUNEL+ cells in irf8 wildtype, heterozygote, and mutant sibling retinas. (G) Quantification of average number of cleaved caspase 3 (CC3)+ cells in irf8 heterozygote, and mutant sibling retinas. (H-K) Representative images from retinal cryosections of irf8 wildtype and mutants, stained for (H-I) red/green cone photoreceptors (ZPR1, magenta) and bipolar cells (PKCα, green), or for (J-K) inner retinal neurons (HuC/D, magenta); DAPI staining in blue. Scale bars are as indicated; p values are shown for statistically significant differences between groups. Each dot in the graphs represents one eye that was quantified.

Figure 2:. Levels of engulfment by Müller glia and microglia during early retinal development

(A-A”) Image from whole eye (selected z projection) of wild-type fish stained for GS (magenta), TUNEL (green), and L-plastin (blue). Cells expressing both GS and L-plastin are visible (dotted outlines); one of these cells has engulfed a TUNEL+ cell (arrow). (B-B’) Image from whole eye (selected z projection, wildtype) showing GS+L-plastin- cell engulfing a TUNEL+ cell (arrow), which is considered a Müller cell engulfing a dying cell. (C) Phagocytic index (proportion of dying cells engulfed by the cell type) calculated for microglia and Müller glia in 3 dpf wild-type retinas, revealing phagocytic levels of the two cell types at this stage in retinal development.

Figure 3:. Increased engulfment activity of Müller glia in the absence of microglia

(A-A’) Selected z projection from irf8+/+ whole eye (microglia sufficient) zebrafish stained for TUNEL (magenta), L-plastin (yellow), and GS (green). TUNEL+ cell engulfment by microglia (L-plastin+ cells, yellow arrows) and Müller glia (GS+ L-plastin- cells with radial orientation, white arrow) is apparent. (B) Selected z projection from irf8−/− whole eye (microglia deficient sibling) showing increased TUNEL+ cell association with Müller glia (GS+ with radial orientation); many of these appear to be engulfed. Also see Movie 1. (C) Quantification of average number of TUNEL+ engulfed by Müller glia per whole retina, in the presence (irf8 +/+,+/−) and absence (irf8 −/−) of microglia. (D) Calculation of the engulfment index of Müller glia (proportion of TUNEL+ cells engulfed by Müller glia) in the three genotypes. The engulfment index of Müller glia is significantly increased in microglia deficient retinas. Scale bars are as indicated; p values are shown for statistically significant differences between groups. Each dot in the graphs represents one eye that was quantified. (E) Selected z projection of gfap:GFP Müller glia (green) in irf8−/− whole mounted retina containing compartments with cleaved caspase 3 (CC3, magenta) signal (arrows). (E’) Enlarged view of the region outlined in E, showing a single z plane in the xy field, with orthogonal views of the z stack region. (F-H) Images of retinal cryosections from 4 dpf irf8 +/+ (F) or irf8 −/− (G,H) zebrafish showing increased TUNEL+ (green) engulfment by Müller glia (GS+ with radial orientation, magenta) in the absence of microglia. (F, inset) Tangentially oriented GS+ cell engulfing TUNEL+ cell in irf8 wild-type retina, likely a microglial cell. (G,H) TUNEL+ cells are apparently engulfed by Müller glia in peripheral and central retina, but many unengulfed TUNEL+ cells are also seen in irf8 mutants, within the CMZ as well as the differentiated retina. Accumulated apoptotic cells are also apparent in the irf8−/− brain (G).

Figure 4.. Engulfed apoptotic cells visualized in Müller glia with membrane tagged reporter.

(A-B’) Images acquired at 3 dpf from whole eyes of live, anesthetized transient transgenics visualizing individual Müller glia (through mosaic expression of the TP1:mcherry-CAAX transgene, magenta) with engulfed apoptotic cells (Acridine Orange, AO, green). Asterisk indicates location of the Müller glia soma, arrows represent enclosed AO+ compartments. Also see 3D renderings in Movies 3 and 4. (A-A’) A single Müller cell with one AO+ compartment. A” shows enlarged area outlined in A’ with a single z plane and orthogonal views of the z stack region. (B-B’) A single Müller cell with multiple AO+ compartments, indicating engulfment of multiple apoptotic cells. Arrowhead indicates an AO+ cell that is touching this Müller cell but is not enclosed in a compartment (see 3D rendering in Movie 4). B” shows enlarged area outlined in B’ with a single z plane and orthogonal views of the z stack region.

Figure 5:. Visualization of Müller glia engulfment of dying cells in retinal cryosections

High magnification (60X) images from 3–4 dpf retinal cryosections. (A-B’) Images from sections stained for GS (magenta), TUNEL (green), and DAPI (blue) in each genotype. (A) Müller glia in wild-type retinas showing typical radial orientation and morphology with cell body (asterisk) in the inner nuclear layer (INL) and radial processes reaching from the ganglion cell layer to the outer nuclear layer. (B-B’) In the absence of microglia, Müller glia were often hypertrophic and observed forming pockets, or compartments, that excluded the GS label and often had TUNEL+DAPI+ label inside (arrows). In addition, pyknotic nuclei (strong condensed DAPI signal, blue) were often visible within Müller glia (GS, green) compartments (C) in irf8 mutants. (D-D’) Phagocytosis in microglia-deficient retinas apparently occurring in various regions of the INL by multiple Müller glia (GS, magenta) highlighted by white arrows, showing engulfment of apoptotic cells, visualized by TUNEL (green) or pyknotic nuclei (DAPI). Asterisks indicate putative location of the Müller glia cell body. The yellow arrow indicates a region of strong TUNEL signal with diffuse DAPI signal, suggesting the compartment contains degraded material. (E-E’) Müller glia in irf8 mutants with apparent engulfment of multiple apoptotic cells (TUNEL, green and pyknotic nuclei, arrows); Müller glia outlined by white dotted line.

Figure 6:. Rab5 staining of Müller glia in retinal cryosections.

Retinal cryosections from gfap:GFP (green) irf8+/+ and irf8−/− siblings stained for Rab5-GTPase (magenta) taken at 60X magnification. (A-A”) Shows a z projection of a Müller glial cell in irf8 +/+ retina without observable engulfment activity, asterisk indicates location of the cell body. Rab5 signal is diffuse in the cytoplasm at the soma with occasional punctal staining. (B-B”) A z projection of a Müller glial cell in irf8−/− mutant with a cellular compartment excluding the cytoplasmic GFP reporter signal. Rab 5 signal localizes around this compartment. (C) Enlarged region indicated by the box in B”, showing only Rab5 signal, in a single z plane (optical section). (D) The same optical section as shown in C, but merged with the gfap:GFP reporter, with orthogonal views provided showing that Rab5 signal localizes around the compartment.

Figure 7:. Lysosomal staining provides additional evidence of compensatory phagocytic activity by Müller glia in the absence of microglia

Retinal cryosections from irf8 +/+ and irf8 −/− siblings stained for GS (magenta) and LAMP1 (lysosomal label, yellow). Images show consecutive z-planes from 1 micron spaced confocal z-stacks (optical sections). White arrows point to LAMP1+ puncta/compartments localizing to GS+ Müller glia cells. Also see 3D renderings in Movies 4 and 5. (A-A”’) LAMP1 staining is visible in Müller glia in wild-type retinas, consistent with some phagocytic activity in the normal state (white arrows). (B-B”’) Increased staining of lysosomes in Müller glia is seen in microglia deficient retinas (arrows). LAMP1+ puncta are more frequently observed and often seen adjacent to apparent compartments where GS staining is excluded (green arrows), which are likely phagosomes. C-E. Orthogonal views provided for specific cells of interest to confirm LAMP1 staining is within Müller glia. C. Pertains to the Müller glia cell body outlined in A”’ in irf8 +/+. D and E. Pertain to the Müller glia cell bodies outlined in B”’ in irf8 −/−. F. Quantification of the fraction of Müller glia area that is LAMP1+ from images of retinal cryosections from each genotype.

Figure 8:. Evidence of Müller glia reactivity in the absence of microglia, as a result of increased phagocytic load

(A) RT-qPCR was used to measure relative expression levels of gfap in individual pairs of eyes from 3–4 dpf microglia sufficient (irf8 +/+,+/−) and deficient (irf8 −/−) siblings. The graph shows fold change (FC) of gfap expression calculated relative to the microglia sufficient group, using two calibrator genes. (B) Results from repeat of experiment represented in A, but additionally inhibiting phagocytosis (L-SOP treatment) in half of the embryos of each genotype. When phagocytosis is inhibited, gfap expression is no longer increased in microglia-deficient retinas, indicating that reactivity is a downstream effect of Müller cell phagocytosis. Each dot in the graph represents one pair of eyes from a single embryo. (C,D) Images of GFAP (red) protein expression, using immunostaining of retinal cryosections, in the presence (irf8 +/+) and absence (irf8 −/−) of microglia. (E) Comparison of intensity measurement of GFAP protein levels from the two groups represented in C-D. Each dot represents average intensity calculated from 2 retinal cryosections of each genotype (4 samples per genotype). (F-G’) Examples of images of retinal cryosections from microglia sufficient (irf8 +/−) and deficient (irf8 −/−) fish (5 dpf) also expressing the gfap:nGFP (green) transgene and stained for DAPI (blue). (H) Quantification of the average number of cells expressing gfap:nGFP in each genotype. (I) A subset of H, cells with bright GFP expression only. Statistically significant differences are indicated (**p<0.01, *p<0.04)

Figure 9:. No evidence of cell cycle re-entry by differentiated Müller glia in microglia deficient retinas

(A-B”’) Images of 4 dpf retinal cryosections after EdU immersion from 2 to 4 dpf, stained for EdU (magenta), GS (green), L-plastin (red), and DAPI (blue). (A-A”’) Representative images from microglia sufficient wild-type siblings. (B-B”’) Representative images from microglia deficient irf8 mutant siblings. (C) Quantification of average number of EdU+ Müller glia (defined as GS+L-plastin-) cells per retina in each genotype. (D) Counts of average number of EdU+ cells per retina within the central retinal region (*p<0.03) in each genotype. EdU counts are broken down by retinal layer in Supplemental Figure 3. (E-F) Fold change expression of progenitor cell marker, ascl1a (E), and cell cycle marker, Id2a (F) in pairs of eyes from each genotype measured by RT-qPCR.