Fig 2: rCD72 induces molecular pathways consistent with tiling disruption and microglial immune response(A) Volcano plot showing protein abundance changes in microglia enriched from the mixed glia culture treated with Ctrl (vehicle 0.1% BSA) or 400ng/ml rCD72, N=6 each. Welch’s t-test was applied between Ctrl and rCD72, then p-values adjusted using BH method. Average fold-changes were calculated. Red dots denote downregulation and blue dots denote upregulation with rCD72 treatment (fold-change cutoff:1.5, adjusted p-value cutoff: 0.05). See also Table S6. (B) Enriched biological processes from significantly changing proteins. (C) Schematic depicting specific proteins related to enriched GO terms in (B) and their phosphorylation sites. The criterion for coloring was adjusted p-value <0.05 (no fold-change cutoff). The number of phosphorylation sites depicted on each protein is not necessarily the number of phosphorylation sites detected; however, any significant changes are shown. See Table S6. (D) Schematic depicting proteins within known CD72 signaling pathways and hypotheses about their roles in microglial tiling. Left of the black dotted line represents proteins at steady-state: CD72 is a coreceptor to an unknown microglial receptor that is analogous to the BCR in B cells. Right of the black dotted line: upon rCD72 treatment, LYN (kinase associated with the unknown receptor) phosphorylates the ITIM motif on CD72, recruiting SHP-1 (phosphatase) to downmodulate signaling by the unknown receptor, which could result in reduced ERK1/2 signaling, resulting in non-tiling. Coloring of proteins and phosphorylation sites on the right of the dotted line follows the same criterion as in (C).

Fig 3: CD72 overexpression in microglia recapitulates aspects of rCD72-mediated tiling disruption.(A) Workflow describing AAV-mediated overexpression of CD72 (or 3xFlag as control) in mixed glia, puromycin (puro) selection, microglia enrichment via magnetic activated cell sorting (MACS), then replating with uninfected non-microglia. Tiling analysis was performed 48 hours later. (B) Western blotting analysis on lysate from puro-selected and enriched CD72- or 3xFlag-overexpressing microglia stained for CD72 and β-actin as loading control. (C) Quantification of four technical replicates from western blot in (B). CD72 bands are normalized to β-actin band densities. Bars show mean ± SEM. *p<0.05 by Welch’s T-test. (D) Quantifications of tiling features. N=four images in each of five wells, 20 technical replicates. All p-values by Welch’s T-test are listed. (E) Representative images of microglia over-expressing 3xFlag or CD72.

Fig 4: An image-based screen identifies CD72 as a strong regulator of tiling(A) Tiling candidate list development. 241 CSPs were filtered by 1) StringDB functional annotations matching key words, 2) published bulk RNA sequencing dataset of significantly up- or downregulated (p-adjusted <0.05) microglial transcripts three days after distal middle cerebral artery occlusion stroke in mice. Known ligands to CSPs and controls for morphometric readouts were added. See Table S5. (B) Volcano plot of microglial transcripts three days after distal middle cerebral artery occlusion stroke in mice, with 26 significantly changing genes of corresponding CSPs (p-adjusted <0.05) highlighted in magenta dots. (C) Visual explanation of the six features quantified as the screen readout. Samples were stained for GFP (from Cx3cr1-GFP) to label microglia and DAPI. Cartoon yellow nuclei indicate that the overlap between GFP and DAPI signal was used for that quantification. See STAR Methods. (D) Data from NND, spatial regularity, and contacts of the Matrigel-embedded screen. N= Four images from each of three wells, 12 technical replicates. Bars show mean ± SEM. Two rounds of screening were performed, with rounds separated by vertical dotted lines. Molecules are shown in the order they were tested. X-axis text color represents category of molecule. Adjusted p-values (Benjamini-Hochberg (BH) method): *p<0.05 by Welch’s t-test between respective molecule and Ctrl (vehicle 0.1% BSA) within round. See also Figure S3A and B. (E) Summary heatmap of all features for every candidate in both rounds of the Matrigel-embedded screen. Color bar represents fold-change over respective Ctrl within round. X-axis text color represents the same as in (D). (F) Boxplots of six features showing differences between Ctrl and CD72 samples (data extracted from Matrigel-embedded screen). P values: *p<0.05, **CD72 (Matrigel-embedded screen). Red arrowheads indicate locations of upheld tiling in Ctrl and disrupted tiling in CD72 condition. Scale bar = 100μm.