Fig. 1: MEF2C is expressed by microglia throughout development, and loss of MEF2C leads to phenotypic changes.
a, Experimental schematic. Fetal and postnatal brain cortical RNA sequencing data were previously published27. In parallel, brain cortical samples were fixed and stained for MEF2C, CTIP and IBA1 across developmental time points. b, MEF2C RNA levels are greater in isolated microglia than in bulk cortex at both fetal (n = 19 microglia and 10 bulk cortex) and postnatal (PN; n = 18 microglia and 5 bulk cortex) developmental stages. c, Representative confocal images from the cortex of human brain tissue at gestational week (GW) 11 demonstrating colocalization of MEF2C with IBA1+ microglia and CTIP+ cortical neurons. Left, MEF2C colocalization with IBA+ microglia across developmental stages (fetal stages: GW11, 13 and 17; postnatal stage: 17 years (YR)). d, Quantification of MEF2C+ cells demonstrates more colocalization in IBA1+ microglia than in CTIP+ cortical neurons in both fetal and postnatal human brain. Fetal microglia also demonstrate higher MEF2C intensity than fetal cortical neurons. Microglia and cortical neurons in postnatal brains demonstrate equivalent levels of MEF2C intensity (n = 1 fetal (GW 17) and 2 postnatal (17 and 20 YR) samples; data points represent technical replicates, the mean measurement from five to ten fields per section from three sections per sample); AU, arbitrary units. e, Schematic of the CRISPR–Cas9 strategy for MEF2C haploinsufficiency (MHS) and KO human iPS cell lines. f, Quantification of western blots showing a dose-dependent reduction in MEF2C protein in MHS and KO iPS cell lines differentiated into microglia (iMGs; n = 3 independent iPS cell lines per genotype). g, Representative confocal images demonstrating genotype-dependent knockdown of MEF2C protein levels in iMGs (replicated across all lines; n = 3 independent iPS cell lines per genotype). h, High-throughput image segmentation of iMG morphology for all genotypes. i, High-throughput image analysis identifying MEF2C dose-dependent reductions in microglia ramification length and number of branch points per cell body cluster in MHS and KO iMGs (n = 7 control (Ctl), 10 MHS and 9 KO iMG biological samples derived from independent iPS cell lines from separate differentiation batches); scale bars, 10 μm in c, g and h. Data in b, d, f and i are presented as mean ± s.e.m. Data points in i represent the mean measurement for all technical replicates from one independent line in a differentiation batch. Statistical analyses in b, d, f and i were performed using one-way analysis of variance (ANOVA) with P values adjusted for multiple comparisons by Tukey’s method. Graphics in a and e were created using BioRender.com.
