A subpopulation of embryonic microglia respond to maternal stress and influence nearby neural progenitors

•Embryonic hypothalamic microglia are heterogeneous•A subpopulation of embryonic reside adjacent to NSCs•Maternal cold stress impacts signaling nearby NSCs in males•Exposure gestational leads disrupted social behaviors adulthood The interplay between neurons and as they integrate stressors regulate homeostasis is growing interest. We asked if the hypothalamus were likewise responsive and, so, whether their precocious activation perturbs neural stem cell (NSC) programs. performed single-cell transcriptomics define heterogeneity identified four microglial subsets, including a that was stress. Stress exposure elevated CCL3 CCL4 secretion, but only male brains, ex vivo treatment altered proliferation differentiation. Concomitantly, decreased PVN oxytocin embryos, which reversed by depletion. Adult offspring exposed displayed behaviors, dependent, males. 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Here, profiled isolated wild-type embryos scRNAseq. one direct contact responded observed secretion C-C motif chemokine ligand 3 (CCL3) 4 (CCL4), directly NSC culture. At cellular level, (PVN), phenotype blocked Finally, had lasting effects, since changed elimination again, Together, these data suggest perturbations manner. some react change properties might interfere neurodevelopment. To test this, employed scRNAseq examine dynamics E15.5, period terminating gliogenesis being initiated (Aslanpour 2020Aslanpour Schuurmans Neurog2 Acts Classical Proneural Gene Ventromedial Hypothalamus Is Required Phase Neurogenesis.J. 2020; 40: 3549-3563Crossref (5) Marsters 2016Marsters C.M. H.F. Aslanpour Klenin Wilkinson Pittman Q.J. Oligodendrocyte Ascl1.Neural Dev. 11: 20Crossref (17) assess microglia, pregnant dams chose physiologically relevant model plays prominent thermoregulation (Bachtell 2003Bachtell R.K. Tsivkovskaia N.O. 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By gating CD11bhigh CD45+ (CD45low-high; Figure S1B), collected both CD45low CD45high border-associated (BAMs), reasoned would helpful comparison purposes. Enriched processed droplet-based 10× Genomics platform (v3 chemistry), previously described (Stratton 2019Stratton Sinha Shin W. Labit Chu T.H. Shah P.T. Midha Biernaskie Droplet Barcoding-Based Single Cell Transcriptomics Mammalian Tissues.J. Vis. Exp. https://doi.org/10.3791/58709Crossref combined total 2,623 sequenced (wild-type stress) 15 S1). Despite purification, proportion (?19%) contaminating (e.g., NSCs, newly born glia, endothelial pericytes, etc.) S1D), so our initial clustering strategies excluded analyses 1B Monocytes comprised ?2.4% retained final allow comparisons definitive subsets 1B). significant enrichment mitochondrial genes stressed each clusters, exception S1M). driven either adaptation death (O’Flanagan 2019O’Flanagan C.H. Campbell K.R. A.W. Kabeer Lim J.L.P. Biele Eirew Lai McPherson Kong al.Dissociation solid tumor tissues protease RNA-seq minimizes conserved collagenase-associated responses.Genome Biol. 20: 210Crossref (61) Picard McEwen, 2018Picard McEwen B.S. Psychological mitochondria: systemic review.Psychosom. Med. 2: 141-153Crossref (79) scored KEGG-curated apoptosis gene set found no any clusters S1N), challenge. Unsupervised integrated (anchored stressed) revealed 1B), highly correlated (E14) neonatal (P4, P5), not (P30, P100) aged, states captured Hammond colleagues S2) Both broadly represented across 1C). state-specific regulatory network case hypoxia-inducible factor 1-alpha (Hif1a, Table S1), implicated susceptibility cell-cycle based scoring strategy Tirosh (Tirosh 2016Tirosh Izar Prakadan S.M. Wadsworth 2nd, M.H. Treacy Trombetta J.J. Rotem Rodman Murphy al.Dissecting multicellular ecosystem metastatic melanoma RNA-seq.Science. 352: 189-196Crossref (1652) demonstrated Cluster uniquely G2/M, while Clusters 2 primarily contained G1 S, 1 G1, S 1D). velocity predicts source point different 1E). Canonical genes, Fc receptor-like (Fcrls, 1F), purinergic P2y, G-protein coupled, 12 (P2ry12, 1G), triggering expressed (Trem2, 1H), complement C1q subcomponent subunit (C1qa, 1I), C-X3-C (Cx3cr1, 1J), colony stimulating (Csf1r, 1K) all clusters. transcripts apolipoprotein E (Apoe, 1L) hexosaminidase ? (Hexb, 1M) regards ubiquity Fcrls (90% microglia), Trem2 (91% C1qa (98% microglia) evenly distributed 1–4 1F F?, 1H 1H?, 1I 1I?), whereas P2ry12 (79% absent substantial 1G 1G?). Notably, Cx3cr1, Csf1r, Apoe, Hexb numerous 1J–1M). monocyte/macrophage population (Ccr2, 1N); interferon induced transmembrane protein (Ifitm3, 1O); placenta 8 (Plac8, 1P); histocompatibility 2, class II antigen A, alpha (H2-Aa, 1Q), rarely 1N–1Q). same time, exhibited own profiles. membrane-spanning 4-domains, subfamily member 7 (Ms4a7) showed 6.3-fold 1R), secreted phosphoprotein (Spp1) 9.2-fold enriched 1S), baculoviral repeat-containing 5 (Birc5) 6.1-fold 1T), 119 (Tmem119) 1.6-fold 1U). demonstrate share core program, brain. further fluorescent situ hybridization (FISH) generated atlas. first confirmed monocyte cluster 2A). addition Ccr2, Ifitm3, Plac8, H2-Aa, several upregulated almost exclusively group S100 calcium binding A6 (S100a6) cytochrome b-245 chain (Cybb; Furthermore, Ccr2+ did co-label marker 2A??) localized outside lining outer pial surface 2A?–2A??), consistent lineage. most transcriptionally similar cluster. Ms4a7, platelet (Pf4) coagulating XIII (F13a1; 2B). FISH Ms4a7 labeled inside 2B? 2B?). Ms4a7+ residing co-labeled 2B??), defines intermediate acquiring it invades proper. Considering shared overlapping Ifitm3; 1O) spatial distribution 2A??–2B??) macrophages, primed quickly respond capacity) foreign insults blood-brain barrier (BBB) transcriptomic places status proliferating 4, respectively. Spp1, fatty acid-binding (Fabp5), lipoprotein lipase (Lp1; 2C). Unexpectedly, tile instead restricted region third ventricle 2C?), smaller resided near edge supraoptic