Fig. 4: ZBTB18 regulates axon guidance genes and the mammalian-specific Robo1 enhancer.
From: Adaptive evolution of gene regulatory networks in mammalian neocortex
a, Coronal sections from PD 0 brain showing ZBTB18 expression and GFP-labelled corpus callosum axons after IUE at PCD 15.5 with Neurod1–Cre and CALNL–Gfp (left) or Neurod1–Zbtb18 (right) plasmids into Zbtb18fl/fl mice. Enlarged panels show GFP images in greyscale. b, Top five Gene Ontology terms for genes that are downregulated (red) or upregulated (blue) in the Zbtb18−/− (KO) and mouse compared with Zbtb18+/− (control) at PCD 14.5 (upper bar plot) and in Neurod6–Cre; Zbtb18 cKO compared with control at PD 0 (lower bar plot). c, Genes encoding axon guidance molecules and upregulated and downregulated in the Zbtb18 KO mouse compared with Zbtb18+/− (control). d, Line graphs showing the H3K27ac peaks from IT neurons, ET neurons and ZBTB18-HA ChIP–seq peaks associated with mouse Robo1. e, Luciferase reporter activity with Robo1-E1 enhancer and ZBTB18. An unpaired two-tailed t-test was performed to detect differences between the control and experimental conditions. The graph represents mean ± s.e.m. **P = 0.00672 (n = 3). f, ISH shows Robo1, Robo2 and Robo3 expressions in the Zbtb18 −/− mouse in the neocortical plate. g–i, Representative images of the IUE with pCag–Robo1 allow Gfp-expressing co-electroporated upper-layer neurons to project GFP-positive axons to and across the corpus callosum (arrows in the inset) analysed at PD 0 (g) and PD 21 (h). Top, number of axons crossing at the midline on the contralateral side of the IUE at PD 21 (i). Bottom, number of cells electroporated on the ipsilateral side (i). An unpaired two-tailed t-test was performed to detect differences between the control and experimental conditions. The graph represents mean ± s.e.m. *P = 0.030. For each IUE experiment, n = 6 (control) and n = 3 (Robo1) animals were analysed at PD 0; n = 3 (control) and n = 3 (Robo1) animals were analysed at PD 21. For RNA-seq, n = 3 per condition. The ISH data shown are representative of the data generated from multiple sections (n = 3 animals). Scale bars, 500 μm (a,g,h), 150 μm (f), 250 μm (g,h (inset)).
