Fig. 5. Sparse-induced knockout of the Tgfb1 gene in individual adult microglia supports an autocrine mechanism of microglial TGF-β ligand production and signaling regulation.

A Mouse model was used to induce Tgfb1 KO in mosaic sparse individual microglia and an experimental timeline depicting a titrated dose of TAM. B–D Representative images showing IBA1, TMEM119, and YFP expression and co-localization in B control tissue at 2 weeks post TAM, C sparse iKO tissue at 2 weeks showing loss of TMEM119 expression in sparse individual microglia, and D absence of TMEM119−/IBA1+ parenchyma microglia in the sparse Tgfb1 iKO brain at 8 weeks post TAM. The yellow dotted outline in C highlights singular microglia showing loss of homeostatic TMEM119 expression. White arrows highlight YFP+ cells showing no loss of homeostatic TMEM119 expression. Note that at this low dosage of TAM, the recombination of individual floxed alleles (R26-YFP reporter or the floxed Tgfb1 gene) occurs independently of each other, therefore YFP+ cells could not track a sparse Tgfb1 KO microglia, consistent with our recent study³². E, F Quantification of percentages of cell populations for E YFP+, F TMEM− YFP+, and G TMEM− cells out of total IBA1+ cells at 2 and 8 weeks post sparse TAM administration (for E–G, n = 6, 4, and 9 for each group presented, *p = 0.0195 and 0.0123 for (F) and ****p < 0.0001, E–G analyzed by one way ANOVA, two-sided, Tukey’s multiple comparison). H, I Quantification of TMEM119 expression at H 2 weeks post (n = 12, 12, and 9 for each group, ****p < 0.0001, one-way ANOVA, two-sided, Tukey’s multiple comparisons) and I 8 weeks post (n = 16 and 15 for each group, not significant) low dose TAM administration from individual TMEM+ cells, TMEM− cells, and YFP+ cells. J–K Detailed morphological analysis of individual microglia in sparse iKO mice at 2 weeks post TAM characterizing J the total process length and K the total terminal end number of individual TMEM119+ or TMEM119− microglia (n = 8 for each group for J and K, ****p < 0.0001, I–K analyzed by Student’s t-test, two-sided). Animals pooled from different cohorts of TAM treatment. Mean ± SE. Scale bar = 100 µm. A Created with BioRender.com and released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a source data file.