Fig. 6: Activity partially controls the later phase of in vitro maturation.

a Schematic of loss of function experiments in the presence of TTX. b Differential accessibility between DIV28 and DIV28 + TTX conditions. Each dot represents a single ATAC-seq peak. Peaks in orange gain accessibility and peaks in purple lose accessibility in the absence of activity (FDR < 0.05, as calculated by DiffBind). c Top two motifs enriched in peaks that lose (top) or gain (bottom) accessibility in the absence of activity. HOMER outputs show the de novo motif (top) and the best-matched known transcription factor motif (bottom) along with p value and prevalence. d Heatmaps show ATAC-seq reads at 1674 chromatin regions that lose accessibility in the absence of activity. Each panel spans ±1 kb from the center of the peak. e Lineplots showing cumulative accessibility at top 10k chromatin regions that gain accessibility between DIV0 and DIV7 (left) and between DIV7 and DIV28 (right). f, g In vitro expression of genes up- or downregulated in the absence of activity. ***p value <1e–5 calculated by two-tailed t-tests; n = 108 down and 89 up genes. h Correlation between maturation and activity-dependent gene expression changes. Each dot on the plot represents a gene, the x-axis plots the change in expression over time, and y-axis plots the change in expression in TTX condition compared to no TTX at DIV28. Genes colored in shades of blue are up or downregulated at those ages with p < 0.001 and fold change >2 (EdgeR, Fig. 4d). The percent of the colored genes in each quadrant with log₂ fold change > 0 or 1 is also reported. i Principal component analysis of in vitro chromatin accessibility (left) and expression data (right). Each dot represents combined data from 2-3 replicates. j Scoring of Thy1-P2A-SUN1-GFP reporter and SPP1 protein in cultured motor neurons in the presence or absence of activity, as detected by immunostaining. Error bars show SEM; n = 3 independent biological replicates shown as black dots. For box plots, center line is the median, the interquartile range is 25–75th percentile, and outliers are eliminated; p values from two-tailed t-tests. Source data are provided as a Source Data file.