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. 2017 Sep 28;28(11):3797–3815. doi: 10.1093/cercor/bhx241

Figure 8.

Figure 8.

Grin2b rescues the dendritic phenotype of Dlx1/2 CKO CINs. (a–d) Single confocal images of Dlx1/2f; GFP; CR-Cre (a, b) and Dlx1/2f; GFP; I12b-Cre (c, d) control (a, c) and mutant (b, d) mice at P30 showing the colocalization of GRIN2B (magenta) boutons onto GFP+ dendrite (arrowheads) at P30. (e) Quantification of GRIN2B+ boutons density on control (black and dark gray bars) and mutant (white and light gray bars) Dlx1/2f; GFP; CR-Cre (control: 0.67 ± 0.04 puncta/μm number, n = 23; mutant: 0.42 ± 0.03 puncta/μm, n = 24; P = 1.8e−005) or Dlx1/2f; GFP; I12b-Cre (control: 0.64 ± 0.06 puncta/μm number, n = 23; mutant: 0.37 ± 0.03 puncta/μm, n = 29; P = 2.4e−004) P30 mice and P8 Dlx1/2f; GFP; I12b-Cre (control: 0.76 ± 0.06 puncta/μm number, n = 25; mutant: 0.44 ± 0.03 puncta/μm, n = 29; P = 2.0e−005). ***P < 0.001 (t-test). Histograms show average ± SEM. n, number of cells. Scale bar (in b) a–d 10 μm. (f) Schematic diagram of the experimental assay. MGE from E13.5 Dlx1/2f control and mutants were dissected and dissociated. Cells, transfected with a mixture of CMV-Cre+Flex-GFP or CMV-hGRIN2B-T2a-Cre+Flex-GFP, were transplanted into cortices of P1 WT mice. Mice were analyzed 28 DAT (days after transplant). (g–i) Images and drawings of PV cells transfected with CMV-Cre+Flex-GFP (g, h) or CMV-hGRIN2B-T2a-Cre+Flex-GFP (i) from Dlx1/2f controls (g) or mutants (h, i). (j) Quantification of dendrite length of controls (black bar), mutants (white bar), and human GRIN2B rescued mutants (gray bar) 28 DAT (control: 2824.6 ± 237.3 μm, n = 10; mutant: 1550.1 ± 223.1 μm, n = 10; rescued: 2533.8 ± 163.2 P = 0.0005). ***P < 0.001, **P < 0.01 (ANOVA test, Tukey’s B post hoc). Histograms show average ± SEM. Scale bar (in g) G–I 100 μm. n, number of cells. (k) Diagram showing GE ChIP peaks in the Grin2b gene locus at E13.5 after H3K27Ac ChIP and at E13.5 and E16.5 after DLX2 ChIP. Three DLX2 peaks are identifiable at both ages (#1-3). (l) Luciferase reporter assay in P19 cells testing DLX2 activation of region #3 (orange box in k). Data are presented as fold change in activation with DLX2 over activation with GFP, and normalized to activation of the empty pGL4.23 control (pGL4.23: 1.0 ± 0.08, n = 3, same as control in Figure 5; Grin2b En3: 2.5 ± 0.20, n = 3, P = 0.01). Histograms show average ± SEM. *P < 0.05 (t-test).