Figure 4
From: Heterogeneous fates of simultaneously-born neurons in the cortical ventricular zone
Isochronic early AP-born neuron diversity emerges during differentiation. (a) Heterogeneity of E12 to E15-born apical progenitors, 1 day(d)- and 4d-old neurons (N1d and N4d), and adult neurons from superficial and deep layers (from ref.2,19). Within-type heterogeneity is quantified by comparing variance explained by the first principal component (PC1) using top 80 variable genes in actual vs. randomly permuted data, when ratio = 1, the PC1 variance obtained with actual data is similar to that obtained with permuted data (i.e. similar to that obtained by chance—see Methods and ref.20; n = 80 cells per cross-validation, n = 100 cross-validations per condition, Two-way repeated measures ANOVA of E12-E13 vs. E14-E15, Mann–Whitney test of DL vs. SL). (b) Molecular clustering of 1d-old neurons. Left: tSNE plot. Right: Clusters with pie charts representing the distribution of cells per day of birth in each cluster. Color code indicates day of birth. Bottom right, heatmap of the top differentially expressed genes per cluster. (c) Left: Top 89 most variable genes at E12 and E13 normalized expression in 1d-old neurons born at E12, E13, E14 and E15. The most variable genes in early-born neurons display higher levels of expression in late-born neurons (Two-way ANOVA of E12-E13 vs. E14-E15 expression values). Right: Expression of late-born neuron markers in 1d-old neurons born at E12, E13, E14 and E15. A fraction of early-born neurons expresses late-born markers (highlighted in blue), as shown by the pie charts (bottom right). The expression threshold was established for each marker as the mean expression value for late-born cells in clusters 4 and 5 (dashed horizontal line). The pie charts represent only cells with expression > 0 RPM (to avoid bias due to drop-out events). (d) Micrographs showing E13.5-born 2d-old neurons (i.e. at E15.5) expressing CUX1 protein (white arrowheads). (e) Summary of the findings. Illustration is from L. Le Trung. Data are from refs2,19 (a–c). Scale bars: 30 µm (d, low mag), 15 µm (d, high mag). AP Apical Progenitor; d days after injection; E Embryonic day; N Neuron; n.s. not significant; *P < 0.05.
