Clonally Related Forebrain Interneurons Disperse Broadly across Both Functional Areas and Structural Boundaries

Abstract

The medial ganglionic eminence (MGE) gives rise to the majority of mouse forebrain interneurons. Here, we examine the lineage relationship among MGE-derived interneurons using a replication-defective retroviral library containing a highly diverse set of DNA barcodes. Recovering the barcodes from the mature progeny of infected progenitor cells enabled us to unambiguously determine their respective lineal relationship. We found that clonal dispersion occurs across large areas of the brain and is not restricted by anatomical divisions. As such, sibling interneurons can populate the cortex, hippocampus striatum, and globus pallidus. The majority of interneurons appeared to be generated from asymmetric divisions of MGE progenitor cells, followed by symmetric divisions within the subventricular zone. Altogether, our findings uncover that lineage relationships do not appear to determine interneuron allocation to particular regions. As such, it is likely that clonally related interneurons have considerable flexibility as to the particular forebrain circuits to which they can contribute.

Figure 1. Targeted infection of MGE progenitor cells with a retroviral library

A) Diagram summarizing the experimental design of this study. Orange depicts the area of Nkx2.1-cre expression. B) Experimental strategy. C) Overview images (black and white) and high-magnification images (green) of sections of embryonic mouse brains two to three days after injection of a retroviral library at E10.5 or E12.5. Radial clusters of cells expressing GFP (green, asterisks) were found in the VZ and SVZ of the MGE but not the CGE, LGE or cortex. Several cells with short processes arrayed along a radial process (white arrows). D) Three-dimensional reconstruction illustrating the distribution of GFP-expressing neurons with a barcode in the forebrain of a P16 mouse that was infected with a retroviral library at E10.5. E) Representative high-magnification images and their location within a 25 μm coronal brain section in the cortex (cyan tracing) and hippocampus (yellow tracing). Ctx, cortex; Hi, hippocampus; Scale bars, 500 μm; see also Fig. S1.

Figure 2. Dispersion of interneuron clones across forebrain structures

A) Quantification of single-cell and multi-cell clones identified by barcode analysis. (B) Histogram illustrating the probability in percent that barcoded neurons contribute to a clone of a particular size. (C) Categorization of clones according to their spread. Ctx, cortex; Hi, hippocampus; GP, globus pallidus; N = 3 brains. D) 3D-reconstructions of five clones (green and red dots) illustrating different modes of dispersion.

Figure 3. Dispersion of clonally related interneurons within brain structures

A) Pairwise distances of all multi-cell clones that were restricted to one brain structure are shown for three experiments (three analyzed brains). B) Histogram of pairwise distances for pooled data of three experiments. Bin-size, 500μm. C) Average distances between neurons within clones in the cortex, hippocampus and striatum. D) Dendrograms showing hierarchical relationships between clones of one brain structure grouped according to their Euclidian distances and labeled according to their lineal relationship. Numbers and colors indicate the lineal relationship between two or more cells based on barcode-sequencing. Colored lines below numbers mark isolated clonal clusters; colored dots mark split sibling neurons of the latter. E) Quantification of the distance between isolated clonal clusters. F) Nearest neighbor distance and average distance of multi-cell clones of one structure (clones) and all labeled neurons with a barcode (non-clones). See also Fig. S2, S4.

Figure 4. Spatially organized clonal units in the MGE

A) Diagram illustrating different kinds of cell divisions that have previously been described in the MGE of embryonic mice (Brown et al., 2011). B) High magnification images of groups of neurons (green) and their exact location depicted within low magnification overview images of coronal brain sections (black and white images). Rectangles indicate the location of high-magnification images; yellow circles and labels indicate the clonal relation between groups of neurons (i.e., monoclonal clusters of polyclonal cell groups). C) Representative examples of E10–12 and E10–13 MADM clones in the VZ/SVZ of the MGE. The total clone size was 11,1 ± 1,389 (N = 9) for E10–E12, and 21 ± 2,188 (N = 8) for E10–13, respectively. The majority of G2-X MADM clones (15/17) display unequal lineage trees with respect to the absolute number of labeled neurons in the two (red and green) sub-lineages. Scale bars, 500 μm (B); 50 μm (C); see also Fig. S5.

Figure 5. Distribution of transcriptionally silenced viral vectors

A) Schematic illustrating the strategy to recover silenced barcodes. Note: the spatial resolution of silenced barcodes is maintained only by anatomical structure and within the anterior-posterior axis. B) Quantification of single-cell and multi-cell clones identified by barcode analysis. C) The location of both GFP-positive and GFP-negative infected neurons is plotted relative to Bregma on the x-axis. Dots connected with a horizontal line represent the location of sibling neurons. Left, sibling cells that were dispersed either within the cortex (red), hippocampus (blue) or striatum (green); right, barcodes that represent sibling cells that were spread across structural boundaries. Relative clonal size (D) and spread both within and across brain structures (E). F) AD of multi-cell clones and single-cell clones in different brain structures.