Fig 2: Guidance receptors affect subcellular distribution and levels of WAVE/SCAR.Embryos are oriented with anterior to the left and dorsal up. (A) Embryos carrying the integrated, rescuing gfp::wve-1 (pjIs1) transgene were double-stained with antibodies to GFP (Abcam, ab6556, polyclonal) and AJM-1. Boxed regions are amplified and enhanced equally for contrast. Number of embryos that showed the represented phenotype are indicated. Dotted line outlines the basolateral region of the epidermal cells where the region is discernable. (B) Embryos carrying the rescuing integrated gfp::wve-1 transgene were double stained with mAb to GFP [49] and basolaterally localized UNC-70/beta spectrin [33]. Readings were taken across two cell junctions using the line tool in ImageJ for both the UNC-70 and the GFP signal. 5 readings were taken per cell and averaged and plotted in IPad Prism. SEM is shown. (C) Total levels of endogenous WVE-1 in whole worm lysates, or embryonic lysates (Materials and Methods) measured with a polyclonal antibody to WVE-1 [12]. Levels of WVE-1 normalized to tubulin and relative to WT are shown below the graph, based on the average of 4 blots from 3 sets of lysates. (D) Subcellular distribution of WVE-1 in fractionated lysates, measured using an antibody to endogenous WVE-1. Lysates were spun at increasing speeds and duration. Pellets were resuspended to match the volume of their partner Supernatant fraction. Equal volumes of each S and P fraction were loaded so that relative amounts of protein in the S vs. P fraction could be compared [34] (See Materials and Methods). 10 µl of each fraction were loaded. Numbers below each band represent the relative percentage of total protein found in each fraction. Numbers represent average of three blots (one set of lysates). S = supernatant, P = pellet. Graph shows average of total protein in S1 and P1 based on 3 blots. SEM is shown. Red rectangles indicate fractions with most significant changes compared to WT.

Fig 3: Identity and quantity of P2X7-EGFP expressing cell types in the CA1 region and comparison with P2X7 expression in wt mice.(A–F) Co-labeling of tg line 17 brain slices with anti-GFP antibody (ab6556, Abcam; A10262, Thermo Fisher Scientific) and antibodies for the indicated marker proteins (GFAP (MAB360, Millipore), S100ß (S2532, Sigma Aldrich)). Hippocampal CA1 regions are shown. Arrows indicate co-staining for S100ß and GFP. Cell nuclei were counterstained with DAPI (blue). PL, pyramidal cell layer; SR, stratum radiatum. Scale bar: 50 µm (G) Quantitative analysis of 10 ‘counting boxes’ (as shown in C–F) from five sections/mouse in each experiment. Bars represent mean ±SEM of three independent experiments/animals (total cell numbers in transgenic versus wt animals were: 14.4% vs. 12.2% Iba1 +cells, 10.4% vs. 11.0% Olig2 +cells, 7.3% vs. 8.4% NG2 +cells, 16.1 vs. 14.1% S100ß + cells). (H) Quantitative analysis of P2X7 protein reduction in conditional P2X7-/- mice (CNP-cre, Cx3cr1-cre). 75 µg cerebrum extracts (1% NP40) were analyzed by western blotting and infrared imaging with antibodies against P2X7 (Synaptic Systems) and fluorescent secondary antibodies (LI-COR 680RD dk anti-rb; LI-COR 800CW gt anti-ms). Data were normalized to P2X7 protein in wt animals. Bars represent mean ± SEM from 6 to 9 animals analyzed in three independent experiments. Significance between means was analyzed using two-tailed unpaired Student’s t-test and indicated as ****p<0.0001 compared to P2rx7fl/fl. For antibodies not specified in the legend see Key resources table.

Fig 4: P2X7-EGFP expression in retina, sciatic nerves, spinal cord, and at the neuromuscular synapse.(A) EGFP exclusively co-localizes with microglia and endothelial cells in the adult mouse retina. Upper panel: Middle, retinal slice labeled for GFP (600 101 215, Rockland), Iba1 (marker for microglia/macrophages) and glutamine synthetase (marker for Müller glia). Left and right, retinal flat mounts scanned at the plane of the ganlion cell layer (GCL) and outer plexiform layer (OPL), respectively, to delineate microglia residing in these retinal layers. Astrocytes in the GCL were labeled with GFAP (G6171, Sigma-Aldrich). IPL, inner plexiform layer; INL, inner nuclear layer; ONL, outer nuclear layer. Lower panel: Co-staining of EGFP with neuronal marker PKCa (left) and glutamine synthetase (two right panels) at higher contrast and resolution to show absence of neuronal P2X7-EGFP. Cell nuclei were counterstained with Hoechst 33342 (blue) Scale bars: 20 µm. n = 2 individual line 61 in FVB/C57b/6 hybrid mice (B) Confocal images of GFP (ab6556, Abcam; A10262 or Thermo Fisher Scientific) co-immunostaining with antibodies against the indicated marker proteins in transgenic mice line 17 spinal cord slices (GFAP (MAB360, Millipore), S100ß (S2532, Sigma Aldrich)). Representative images were taken from the areas shown in the schematic overview. Arrows indicate co-staining for S100ß and GFP. Scale bar: 40 µm. Cell nuclei were counterstained with DAPI (blue). Representative images from n = 3 animals are shown. (C) Comparison of transgenic P2X7-EGFP fluorescence and endogenous P2X7 immunofluorescence (P2X7 antibody, Synaptic Systems) in teased sciatic nerve fibers of line 61 and wt mice, respectively. Representative images from at least 3 animals are shown. (D) Co-staining of P2X7-EGFP (A11122, Thermo Fischer Scientific, dilution 1:1000) in teased sciatic nerve fibers of line 46 with antibodies against axonal marker proteins demonstrates localization of the transgene at perinodal regions of Schwann cells. Scale bars: 50 µm. (E) Reconstructed 3-D images of the neuromuscular junction showing co-staining of P2X7-EGFP (ab6556, Abcam; or A10262, Thermo Fisher Scientific) with perisynaptic Schwann cells (S100ß (S2532, Sigma Aldrich)) as well as postsynaptic (a-Bungarotoxin, a-Bgt) and presynaptic (synaptophysin, Syn) marker proteins. The side view in the right panel shows no overlap between GFP and synaptophysin staining. Scale bars: 10 µm and 20 µm, respectively. Representative images from n = 3 animals are shown. For antibodies not specified in the legend see Key resources table.

Fig 5: Consequences of P2X7 overexpression under physiological and pathophysiological conditions.(A) Quantitative real-time PCR was performed in duplicates on samples from microglia isolated by immunomagnetic separation from control and postischemic (3 days post injury, 3 dpi) retinae. Bars represent mean ±SEM and include data from 3-4 animals/each genotype/condition. Significance between expression level in the untreated control eye of the respective genotype was analyzed using unpaired two-tailed Mann-Whitney-U-test and indicated as: *p<0.05. (B) Retinal slices labeled for the microglia/macrophage marker Iba1. Cell nuclei were counterstained with DAPI (red). Retinae were isolated from mice of which one eye had been subjected to transient ischemia. The untreated contralateral eye served as internal control. Dpi, days post-injury. Scale bars: 20 µm. Cell numbers of the inner retinal layers and microglia specifically were quantified in 2–5 central retinal slices per animal on basis of DAPI and Iba1 staining, respectively. Bars represent mean ±SEM and include data from 3 to 4 animals/genotype/condition. Note that data from transgenic mice were not significantly different in A and B. (C) Representative confocal images of coronal sections from posttraumatic GM at 5 dpi. Slices of the somatosensory grey matter (GM) from wt and transgenic animals stained for NeuN- (neurons) and Iba1- (microglia) positive cells are shown. White dotted lines indicate stab wounds; yellow dotted lines indicate NeuN-negative lesion areas. Insets show chosen borders between NeuN-positive and negative areas. Bar diagrams depict fractions of Iba1-positive and NeuN-negative areas in relation to DAPI-positive areas. Means of N = 2–3 animals (n = 6–7 sections per animal)±SEM are shown. Scale bar: 200 µm. One wt tissue broke and could not be analyzed. (D) Double immunostaining with GFP (ab6556, Abcam) and NeuN or GFAP (for astroglia (MAB360, Millipore)) shows no upregulation of P2X7-EGFP in these cell types within the penumbra of P2X7-EGFP and wt mice at 5 dpi. Note that immunofluorescence in the area immediately adjacent to the lesion core (~0–75 µm) is non-specific due to autofluorescence of cells within damaged tissue (inj), and this might obfuscate a potential P2X7-EGFP signal. Cell nuclei were counterstained with DAPI (blue). Scale bar: 100 µm. For antibodies not specified in the legend see Key resources table.