Extended Data Fig. 4: Excess Igfbp2 in ACM inhibits neurite outgrowth.
a. Expression of IGF family members in cortical cell types (data from Zhang et al., 2014). b,c. Addition of Igfbp2 protein to WT ACM inhibits WT neurite outgrowth, which is reduced by adding IGF1. Addition of CPE protein to WT ACM does not inhibit WT neurite outgrowth. b. Example images of WT neurons cultured for 48 hours, conditions as marked (image merge of MAP2 + Tau). c. Quantification of total neurite outgrowth. Example experiment shown, repeated 2 times with same result, number of neurons: control alone=49, control ACM = 50, CPE alone=36, CPE ACM = 46, Igfbp2 alone=44, Igfbp2 ACM = 48, Igfbp2 ACM + Igf1 = 39. d. smFISH against Igfbp2 mRNA in the P7 visual cortex in Aldh1l1-GFP mice to mark astrocytes, combined with probe for OPCs (Cspg4). See Fig. 4g for quantification. N = 3 WT mice. e. Example images from Fig. 4d prior to processing and analysis. Neurons immunostained with MAP2 (dendrites, green) and tau (axon, red). f. Relative frequency distribution plot of total neurite outgrowth length, pooled data from 3 experiments, same data as Fig. 4e. g. Adding the IgG control antibody to WT ACM does not alter neurite outgrowth. Example experiment shown, repeated twice with same result. Number of neurons: control alone=216, control ACM = 333, Igfbp2-Ab alone=257, Igfbp2-Ab ACM = 267, IgG con-Ab alone=277, IgG con-Ab ACM = 266. Violin plots (c,g), dashed line marks median, dotted lines 25th and 75th percentile. Statistics by Kruskal-Wallis one-way ANOVA on ranks with Dunn’s test for multiple comparisons, p values compared to control alone condition (c).
