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. 2022 Aug 5;13:4571. doi: 10.1038/s41467-022-32230-2

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Reciprocal mating strategies used to generate MMc^low and MMc^pos offspring. b Number of MMc among brain cells from MMc^pos (n = 21) and MMc^low (n = 21) offspring isolated at E18.5 and identified by flow cytometry. c Same as (b) but for P8 (MMc^pos n = 15, MMc^low n = 20). d Representative contour plots used to identify MMc in fetal brain. e Number of fetal microglial cells, identified by flow cytometry in MMc^pos (n = 16) and MMc^low (n = 15) brains at E18.5 (p = 0.0366). f same as (e) but for P8 (MMc^pos n = 15, MMc^low n = 20). g Frequency of Ki-67 co-expression on fetal microglia, identified by flow cytometry at E18.5 (MMc^pos n = 9, MMc^low n = 12; p = 0.0007). h Volcano plot depicting comparative gene expression changes within fetal microglial cells isolated from MMc^pos and MMc^low mice at E18.5. Upregulated genes (n = 173) are marked in red, down-regulated genes (n = 29) in blue. Gene names are given when concordant expression was observed in two independent experiments; using transcriptome data from 2 to 3 biological replicates isolated each from 3 to 4 pooled litters. i Number of Vglut1⁺ presynaptic vesicles engulfed by microglial cells in the CA1 area of the hippocampus (HP) in MMc^pos (n = 22) and MMc^low (n = 33) offspring at P8 (p = 0.0048). j Representative photomicrograph of data shown in (i), microglial cells appear in yellow, engulfed Vglut1⁺ presynaptic vesicles as blue dots. Scale bar, 200 µm. k, l Same as (i), (j) but for Vglut2⁺ presynaptic vesicles (MMc^pos n = 9, MMc^low n = 15; p = 0.0693). m Number of dendritic intersections of co-cultured neurons and microglia isolated from MMc^pos embryos (N^posM^pos, gray) and neurons from MMc^pos embryos co-cultured with microglia from MMc^low embryos (N^posM^low, light blue). Data were generated in three independent experiments. n Representative photomicrographs of data shown in (m). Scale bar, 50 µm. o Number of branches per cell in co-culture conditions (N^posM^pos n = 30; N^posM^low n = 33, p = 0.0065). Significance was determined by a two-sided Mann–Whitney U test, *p < 0.05, **p < 0.01, ****p < 0.0001. Data are displayed as mean ± SEM and each circle denotes one pup (b, c, e–g, i, k) or neuron (n).