Volume 92, Issue 1, Pages 31-44 (October 2016) Clonally Related GABAergic Interneurons Do Not Randomly Disperse but Frequently Form Local Clusters in the Forebrain  Khadeejah T. Sultan, Zhi Han, Xin-Jun Zhang, Anjin Xianyu, Zhizhong Li, Kun Huang, Song-Hai Shi  Neuron  Volume 92, Issue 1, Pages 31-44 (October 2016) DOI: 10.1016/j.neuron.2016.09.033 Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Interneuron Output of the MGE/PoA Progenitors in the Forebrain (A) Coronal sections of representative P21 Nkx2.1-Cre/Ai9-tdTomato brains showing tdTomato+ interneuron populations in the cortex (Ctx), hippocampus (Hip), and striatum (Str). Scale bar, 100 μm. (B and C) Quantification of the total number (B) and relative distribution (C) of tdTomato+ interneurons in the Ctx, Hip, Str, and globus pallidus (GP). Bars represent mean ± SD (n = 3 brains). (D) Three-dimensional stereological reconstructions of representative P21 brains that received intraventricular injection of low-titer RCAS retrovirus expressing EGFP at E12.5. Green, yellow, red, and white dots represent EGFP-labeled interneurons in the Ctx, Hip, Str, and GP, respectively. (E and F) Quantification of the total number (E) and relative distribution (F) of EGFP+ interneurons in the Ctx, Hip, Str, and GP labeled using in utero intraventricular RCAS-EGFP injection at E12.5. Data are presented as mean ± SD (n = 7 brains). See also Figures S1 and S2. Neuron 2016 92, 31-44DOI: (10.1016/j.neuron.2016.09.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 MGE/PoA-Derived Interneuron Clones in Mayer et al. Do Not Randomly Disperse in Different Forebrain Structures but Are Mostly Located in the Cortex or the Cortex and Hippocampus (A) Comparison of the fractions of clones located in the four brain structures (Ctx, Hip, Str, and GP) in the experimental data (red bars) with the predicted probabilities of clone distribution based on a random dispersion and the relative ratio of total MGE/PoA interneuron output to the four structures in P21 Nkx2.1-Cre/Ai9-tdTomato brains (black bars). Data are presented as mean ± SD (∗p < 0.05; ∗∗∗p < 0.001). (B) Comparison of the fractions of clones located in the four brain structures (Ctx, Hip, Str, and GP) in the experimental data (red bars) with the predicted probabilities of clone distribution based on a random dispersion and the relative ratio of the interneuron output of E12.5 dividing MGE/PoA VZ progenitor cells to the four structures labeled using in utero intraventricular RCAS-EGFP injection (black bars). Data are presented as mean ± SD (∗∗p < 0.01). (C) Quantification of the percentage of interneuron clones that are restricted to one brain structure (blue) or span more than one brain structure (green). Data are presented as mean ± SD (n = 3 brains). (D) Quantification of the percentage of interneuron clones restricted to the cortex, hippocampus, or striatum (n = 3 brains). (E) Quantification of the percentage of interneuron clones spanning different brain structures (n = 3 brains). Ctx, cortex; Hip, hippocampus; Str, striatum; GP, globus pallidus; OB, olfactory bulb. See also Figures S2 and S3 and Table S1. Neuron 2016 92, 31-44DOI: (10.1016/j.neuron.2016.09.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 MGE/PoA-Derived Interneuron Clones in Harwell et al. Are Mostly Restricted to the Cortex (A) Quantification of the percentage of clones that are either restricted to one brain structure (blue) or span more than one brain structure (green). (B) Quantification of the percentage of clones restricted to one brain structure (n = 2 hemispheres). (C) Quantification of the percentage of clones spanning more than one brain structure (n = 2 hemispheres). Ctx, cortex; Hip, hippocampus; Str, striatum. See also Table S2. Neuron 2016 92, 31-44DOI: (10.1016/j.neuron.2016.09.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 Interneuron Clones in Mayer et al. Reliably Form Local Clusters (A) Dendrograms of individual datasets containing multi-cell clones in the same brain structures according to their Euclidean distances. Numbers and colors indicate the lineal relationship between two or more cells based on the recovered barcodes. Colored lines below the numbers indicate spatially isolated clonal clusters. Colored dots mark a sibling neuron located away from the corresponding clonal cluster. Broken black lines indicate local clonal clusters occupying the same or nearby space. The superscript letters indicate the location of the same clone in different brain structures (C, cortex; H, hippocampus; O, olfactory bulb; C/O, cortex or olfactory bulb, uncertain based on the x-y-z spatial coordinates). (B) Quantification of the percentage of local clusters (i.e., the lowest hierarchical branch in the dendrogram) that are clonally related in the experimental dataset (red bars) compared with the percentage estimated from random permutations (i.e., reshuffling) of the clonal identity in the same dendrograms repeated for 100 rounds (black bars). Data are shown as the total percentage in the whole dataset as well as mean ± SD (n = 3 brains) (∗∗∗p < 0.001). (C) Quantification of the percentage of clones that form local clusters in the experimental dataset (red bars) compared with the percentage estimated from random permutations of the clonal identity in the same dendrograms repeated for 100 rounds (black bars). Data are shown as the total percentage in the whole dataset as well as mean ± SD (n = 3 brains) (∗∗∗p < 0.001). (D) Histograms showing the frequency of local clonal clustering estimated from random permutations of clones. Red arrows indicate the number of local clonal clusters observed in each of the three experimental datasets. See also Figure S5. Neuron 2016 92, 31-44DOI: (10.1016/j.neuron.2016.09.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Interneuron Clones in Mayer et al. Are Spatially Segregated in the Brain (A) Quantification of the average intra-clonal and inter-clonal Euclidean distances for individual clones in each experimental brain dataset. The “inter-clonal” and “intra-clonal” distances for single cells represent the average distance between individual clones and all single barcoded cells as a “clone.” Each black dot represents the average distance for an individual clone. Red lines represent mean ± SD (∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; n.s., not significant). (B) Quantification of the average inter-clonal and intra-clonal distances for all clones and single cells. Data are presented as mean ± SD (n = 3 brains; ∗∗∗∗p < 0.0001; n.s., not significant). (C) Histograms of inter-clonal and intra-clonal distances for clones and single cells in all experimental brains (n = 3 brains; chi-square test, ∗∗∗∗p < 0.0001). See also Figures S5 and S6. Neuron 2016 92, 31-44DOI: (10.1016/j.neuron.2016.09.033) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Interneuron Clones in Harwell et al. Exhibit a Clustering Feature in Spatial Distribution (A) Dendrograms of the left and right hemispheres of the single barcoded experimental dataset according to the Euclidean distances of multi-cell clones in the same brain structure. Numbers and colors indicate the lineal relationship between two or more cells based on the recovered barcodes. Colored lines below the numbers indicate spatially isolated clonal clusters. Colored dots mark a sibling neuron located away from the corresponding clonal cluster. Broken black lines indicate local clonal clusters occupying the same or nearby space. (B) Quantification of the percentage of local clusters (i.e., the lowest hierarchical branch in the dendrogram) that are clonally related in the experimental dataset (red bars) compared with the percentage estimated from random permutations of the clonal identity in the same dendrograms repeated for 100 rounds (black bars). Data are shown as the total percentage in the whole dataset as well as mean ± SD (n = 2 hemispheres). (C) Quantification of the percentage of clones that form local clusters in the experimental dataset (red bars) compared with the percentage estimated from random permutations of the clonal identity in the same dendrograms for 100 rounds (black bars). Data are shown as the total percentage in the whole dataset as well as mean ± SD (n = 2 hemispheres). (D) Histograms showing the frequency of local clonal clustering estimated from random permutations of clones. Red arrows indicate the number of local clonal clusters observed in the experimental dataset. (E) Quantification of the average intra-clonal and inter-clonal Euclidean distances for individual clones in each experimental dataset. The “inter-clonal” and “intra-clonal” distances for single cells represent the average distance between individual clones and all single barcoded cells as a “clone.” Each black dot represents the average distance for an individual clone. Red lines represent mean ± SD (∗p < 0.05; ∗∗∗p < 0.001; n.s., not significant). (F) Quantification of the average inter-clonal and intra-clonal distances for all clones and single cells. Data are presented as mean ± SD (n = 2 hemispheres; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; n.s., not significant). See also Figure S7 and Table S2. Neuron 2016 92, 31-44DOI: (10.1016/j.neuron.2016.09.033) Copyright © 2016 Elsevier Inc. Terms and Conditions