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Supplementary Figure 1: Generation and Characterization of a Conditional Lhx6 Allele

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Supplementary Figure 1: Generation and Characterization of a Conditional Lhx6 Allele Supplementary Figure 1: Generation and characterization of a conditional Lhx6 allele (A) Schematic representation of the strategy used to generate the Lhx6fl allele by homologous recombination. A1 shows the targeted region of the wild-type Lhx6 locus. Exons 1a, 1b (generated by alternative splicing), 2 and 3 as well as the probes used for Southern blot analysis are indicated. H, HindIII. A2 shows the targeting construct including exons 1b, 2 and 3 flanked by loxP sites and PGK-Neo positive selection cassette flanked by FRT sites. The targeted Lhx6 allele following homologous recombination and removal of the PGK-Neo cassette (by FlpE-induced recombination of the FRT sites) is shown in A3 and A4, respectively. A5, following CRE-mediated recombination exons 1b, 2 and 3 are deleted. (B) Southern Blot analysis of HindIII digested genomic DNA from wild type (Lhx6+/+) and Lhx6fl/+ animals hybridized with 5’ and 3’ homology probes verifying the generation of the Lhx6fl allele. (C,D) Cryosections from the cortex of P18 control Nkx2.1-Cre;Ai14;Lhx6+/fl (Ctrl; C) or mutant Nkx2.1-Cre;Ai14;Lhx6fl/- (Mut; D) littermates were immunostained with a polyclonal Lhx6 antibody. The Lhx6fl allele was recombined with the Nkx2.1::Cre transgene and recombination was followed using the Cre-dependent Ai14 (tdT) reporter. Lhx6 expression (blue) is undetectable in MGE CIs (red) in Lhx6fl/- mice. (E) Quantification of Lhx6+ MGE-derived CIs in Ctrl and Mut mice. (F-J) Quantification of the co-localization of MGE and CGE markers in Ctrl and Mut mice. Note the dramatic reduction of MGE specific CI markers and the small increase of CGE-specific markers. Data expressed as mean ± SEM. Statistical significance evaluated using Student’s t-test, * denotes p < 0.05. Supplementary Figure 2: Quantification of CI subtypes in the motor cortex of Lhx6fl/+ (Ctrl) and Lhx6fl/- (Mut) mice. The data in panels A-F are from the animals used for Fig. 1. Specifically, A, B, C, D, E and F correspond respectively to panels G, L, M, I, J and K of 1 Figure 1. Note that the changes observed in motor cortex are similar to those observed in the somatosensory cortex. Supplementary Figure 3: Characterisation of Pv+ CIs in Lhx6 mutants. (A-D) Coronal sections from the cortex of P18 Nkx2.1-Cre;Ai14;Lhx6fl/- (A) and Htr3a-Cre;Ai14;Lhx6-/- (C) mice immunostained for Pv (green). CIs derived from the MGE (A) or CGE (C) are tdT+ (red). In panels B and D, which correspond to A and C respectively, the lineage tracer is shown in white. (E-H) Coronal sections from P18 VGat-Cre;Lhx6Fl/- mice double immunostained for Pv (green) and Lhx6 (red in E) and Sox6 (red in G). In panels F and H, which correspond to E and G respectively, the Lhx6 and Sox6 signal is shown in white. Yellow arrowheads indicate Pv-expressing cells. Supplementary Figure 4: No changes in proliferation or migration of CI precursors in Lhx6 mutant embryos. (A,B,E,F) Coronal brain sections from E14.5 wild-type (A,E) and Lhx6 mutant (B,F) mouse embryos immunostained for pH3. Sections shown in A and B are from the MGE level while sections shown in E and F are from the CGE level. (C,D,G,H) The set of sections shown in A,B,E and F imaged for EdU. (I) DAPI staining of 3 different sections across the rostro-caudal axis of an E16.5 wt mouse brain, representing the areas analysed for Sp8 immunostaining. Boxes indicate the two areas (A and B) along the latero- medial axis used for Sp8+ CI quantification shown in L. (J,K) Sp8+ migrating interneurons (green) in cortical sections of the A2 brain area (I) from wild-type (J) and Lhx6 mutant (K) E16.5 embryos. (L) Quantification of Sp8+ CIs along different regions of Lhx6+/+ (black) and Lhx6-/- (red) E16.5 mouse brains. Supplementary Figure 5: Global transcriptomic analysis of Lhx6 Mutants. (A) - MA plot summarizing the results of the differential expression analysis between Lhx6+/- and Lhx6- /- forebrains. Significantly upregulated genes are shown in red (977 genes) while significantly down-regulated genes are shown in blue (730 genes). Significance was set as a false 2 discovery rate of <= 0.05. (B) - Hierarchical clustering of Lhx6+/- and Lhx6-/- forebrain samples. Clusters were generated using the 25 most significantly upregulated genes in dissociated hippocampal cultures upon treatment with bicucullin(Yu et al., 2015). Log2 fold- change of each gene in each sample was calculated relative to the mean expression of that gene across all samples. Clustering was performed on the euclidean distance between the Log2 values. Genes upregulated upon bicucullin treatment are similarly upregulated upon deletion of Lhx6. (C) Table showing a list of Gene Ontology terms that were significantly (FDR<0.01) enriched in the genes differentially expressed upon deletion of Lhx6 in forebrains. Supplementary Figure 6: Nuclear c-fos expression increases in hM3D(Gq) expressing CIs following CNO administration. (A,B,D,E) Example confocal micrographs of coronal sections from mice electroporated in utero with a mixture of pCAGGS-hM3D(Gq)-IRES- RFP and pCAGGS-IRES-GFP plasmids and immunostained for GFP (green), RFP (red) and c-fos (white). Sections are from untreated (A) or CNO (D) treated animals. Panels B and E are identical to those in A and D respectively, but show only the c-fos signal (white). (C,F) Cumulative histogram of normalized c-fos intensity in nuclei from cells that are either negative (black) or positive (red) for RFP (hM3D(Gq)). Quantifications are from either untreated (C) or CNO (F) treated animals. Table S1: Summary of statistical analyses References Yu, H., Su, Y., Shin, J., Zhong, C., Guo, J.U., Weng, Y.L., Gao, F., Geschwind, D.H., Coppola, G., Ming, G.L., and Song, H. (2015). Tet3 regulates synaptic transmission and homeostatic plasticity via DNA oxidation and repair. Nat Neurosci 18, 836-843. 3 Fate Mapping (Figures 1,2, S1 and S2) Average SEM Average SEM N cells N cells Test Figure Cell Type Control Control Mutant Mutant Control Mutant p Used Panel MGE CI SSC Student's 1G (% Control) 100 7 65 5 5855 3907 0.02 t test CGE CI SSC (% Student's 1I Control) 100 7 185 21 984 1787 0.01 t test pan CI SSC (% Student's 1H Control) 100 5 97 1 6001 6198 0.6 t test Reelin+ CGE+ 1J SSC Student's (cells/mm2) 49.9 5.7 84.3 7 220 357 0.02 t test VIP+ CGE+ SSC Student's 1K (cells/mm2) 43 5 41.2 2.7 146 153 0.7 t test Pv+ MGE- SSC Student's 1L (cells/mm2) 29.7 0.4 69.2 3.8 80 208 0.009 t test Sst+ MGE- SSC Student's 1M (cells/mm2) 28 2.5 24.3 2.6 77 73 0.4 t test P2 MGE CI (% Student's 2I Control) 100 0.4 100.3 3.9 4048 5395 0.9 t test P2 CGE CI (% Student's 2I Control) 100 2.4 88.6 7 1009 773 0.2 t test P7 MGE CI (% Student's 2I Control) 100 5.5 71.2 3.8 5272 2878 0.01 t test P7 CGE CI (% Student's 2I Control) 100 9.1 153.8 3.4 1974 3670 0.02 t test P18 MGE CI Student's 2I (% Control) 100 6.7 65.3 4.7 23164 14257 0.02 t test P18 CGE CI (% Student's 2I Control) 100 3.2 176.3 13 3382 6318 0.02 t test TUNEL+MGE+ Student's 2K (% MGE+) 3.1 0.4 7.6 1.3 12712 5436 0.07 t test TUNEL+CGE+ Student's 2L (% CGE+) 2 0.1 0.7 0.1 3468 9943 0.0006 t test MGE CI MC Student's S2A (% Control) 100 11 65 5 6369 4269 0.07 t test CGE CI MC (% Student's S2D Control) 100 6 172 11 891 1693 0.002 t test Reelin+ CGE+ S2E MC Student's (cells/mm2) 44.7 3.5 94.9 7.1 191 448 0.009 t test VIP+ CGE+ MC Student's S2F (cells/mm2) 52.8 5.3 59.4 2.8 125 164 0.5 t test Pv+ MGE- MC Student's S2B (cells/mm2) 36 4.1 82.8 5.3 114 259 0.003 t test Sst+ MGE- MC Student's S2C (cells/mm2) 32.6 8.1 29.1 3 108 99 0.7 t test Lhx6+MGE+ (% Student's S1E MGE+) 89.3 4.9 0.5 0.3 1242 605 0.003 t test Pv+MGE+ (% Student's S1F MGE+) 54.2 1.7 4.7 0.5 5415 3389 0.001 t test Sst+MGE+ (% Student's S1G MGE+) 29 1.1 0.3 0.2 5749 3225 0.001 t test Reelin+MGE+ Student's S1H (% MGE+) 22.9 1 8 1.9 5504 3038 0.005 t test VIP+MGE+ (% Student's S1I MGE+) 0 0 5.1 0.4 5238 3383 0.005 t test Sp8+MGE+ (% Student's S1J MGE+) 0 0 5.8 1 4748 3084 0.03 t test Grafting GAD67:GFP CIs (Figure 3K) Individual Average SEM Control Mutant N animals n Cells n Cells Control Host Host Host Control Control Mutant Experiment 1 (% Control Average) 100 13 198 4 4933 2441 Experiment 2 (% Control Average) 100 9 184 5 16927 6223 Experiment 3 (% Control) 100 157 1 5434 8554 Average Average SEM Mutant p Test Used Mutant Host Host (% Control) Student's t test single sample (mean 181 13 0.02 100) Grafting hM3D(GQ) expressing CI (Figure 4J) Average SEM Average SEM Vehicle Vehicle CNO CNO p test RFP+ (% GFP+) Student's t test (paired 47 3 61 3 0.01 samples) Individual Fraction Fraction N Cells N Cells Experiments Vehicle CNO Vehicle CNO Experiment 1 RFP+ (% GFP+) 53.63 65.76 330 444 Experiment 2 RFP+ (% GFP+) 45.34 62.34 397 308 Experiment 3 RFP+ (% GFP+) 41.67 54.86 228 175 Material and Methods.
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  • Supporting Information
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