Wide Dispersion and Diversity of Clonally Related Inhibitory Interneurons

Abstract

The mammalian neocortex is composed of two major neuronal cell types with distinct origins: excitatory pyramidal neurons and inhibitory interneurons, generated in dorsal and ventral progenitor zones of the embryonic telencephalon, respectively. Thus, inhibitory neurons migrate relatively long distances to reach their destination in the developing forebrain. The role of lineage in the organization and circuitry of interneurons is still not well understood. Utilizing a combination of genetics, retroviral fate mapping, and lineage-specific retroviral barcode labeling, we find that clonally related interneurons can be widely dispersed while unrelated interneurons can be closely clustered. These data suggest that migratory mechanisms related to the clustering of interneurons occur largely independent of their clonal origin.

Figure 1. MGE Progenitors are Arranged in Radial Arrays of Clonally Related Cells

(A) 24 h after injection we observed a single radial glial cell and its progeny. Both the RG cell and progeny were positive for the proliferative cell marker Ki67. (B) Radial arrays of cells in the mouse MGE approximately 90 h after injection with a GFP-expressing retrovirus. Neurons are also observed migrating tangentially away from the clone (arrowhead). (C) Higher magnification view of the clone identifies 8 anatomically associated cells, one being a radial glial cell with an apical endfoot (arrowhead) attached to the ventricular surface. (D) Time-lapse imaging of a radial glial cell in the MGE shows an asymmetrical radial glial cell division at the ventricular surface that produces an IPC, and also a previously generated IPC dividing in the SVZ. (E) Retroviral GFP labeled MGE progenitors 48 h after viral infection. Non-radial glial progeny express bHLH transcription factors Mash1 (blue) and Olig2 (red) n= 134. (F) Time-lapse imaging of an E13.5 embryonic mouse organotypic slice shows a GFP+ IPC dividing to produce a pair of neuronal progeny. Scale bars: (A) 15µm, (B) 300µm, (D) 25µm, (E and F) 20µm.

Figure 2. Clustering of Sparsely Labeled Medial Ganglionic Eminence Progenitors

(A) E12.5 Nkx2.1-Cre;LSL-Tva mouse embryo showing the TVA+ region of the MGE progenitor zone where RCAS viral infection is permitted in green. (B–C`) P28 brain sections containing RCAS-GFP virus labeled clones stained with parvalbumin (white) (B and B`) or somatostatin (red) (C, and C`). (D) Pie chart showing the percentage of cells positive for parvalbumin (PV), somatostatin (SOM), or negative for both markers (Neither) from Nkx2.1-Cre;LSL-Tva injected brains (n= 4 brains, 701 cells). (E) Coronal section of P28 mouse brain injected with RCAS-EGFP virus at E12.5. Sparse EGFP+ neurons appear to cluster together into groups of two or more cells. (F) RCAS-EGFP labeled neurons, derived from infections of progenitors in the developing cortex with a, BrdU pulse given 24 h after infection (red), showing that EGFP+ neighboring cells often share the same BrdU labeling status. The upper box shows a pair of BrdU− migratory neurons (upper magnified panel), whereas the lower box shows a pair of BrdU+ migratory neurons (lower magnified panel). (G) E12.5 Nkx2.1-Cre;LSL-Tva mouse embryos injected with a mixture of RCAS-GFP and RCAS-mCherry viruses and harvested at P28. (H) Coronal sections of mice injected with mixed virus showing clusters of red, green and yellow cells. (I) Cumulative proportion of nearest neighbor distribution (NND) of same (black) and different (red) fluorophores (n=3 brains n=215 cells, p<0.01, Kolmogorov-Smirnov). Scale bars: (B) 50µm, (G) 100µm.

Figure 3. Lineage Analysis of Nkx2.1+ Progenitors Using Barcode Retroviral Library

(A) Schematic of the QmGFP-OL murine retroviral library. Each retrovirus expresses membrane GFP and contains a 24 bp barcode sequence. (B) EnvA pseudotyped retrovirus libraries were intraventricularly delivered into Nkx2.1-Cre;LSL-Tva embryos at E12.5; brains were harvested and analyzed at P28. (C) Stained neuron outlined for laser capture microdissection and catapulting. (D) Example gel of nested PCR products of dissected cells and GFP negative tissue sections used as controls. (E) Example barcode sequence alignment showing two four-cell clones with matching barcodes. (F) Three dimensional map of two four-cell clones shown in red and blue. Green spheres show cells which did not return a barcode sequence.

Figure 4. Interneuron Clones Consist of Widely Dispersed Cells of Diverse Subtypes

(A) Representative schematic of a four-cell clone containing three parvalbumin (PV) cells (white) and one somatostatin (SOM) cell (red) dispersed across thousands of microns in the brain. (B) Pie chart of forebrain regions which contain neurons that returned a barcode in Nkx2.1-Cre;LSL-Tva virus injected mice (n = 302 cells). (C) Bar graph showing the proportion of multi-cell clones composed of at least one cell type. S = somatostatin, P= parvalbumin, and N= negative for both markers (n=84 cells, 26 clones). (D) Box plot representing all of the nearest neighbor distances (NND) between pairs of cells in each clone. The marks in the boxes are the 1^st, 2^nd (median) and 3^rd quartile of the distances, with the dots representing the outliers. Superimposed red lines represent the mean distances in each group, and black bars represent the median distances (n = 2 brain hemispheres, 284 cells). (E) Cumulative (NND) of Nkx2.1-Cre;LSL-Tva brains (n = 284), with the gray area outlining 100 simulations of randomly distributed cells (complete spatial randomness). (F) Box plot representing NND of cortex cortical neurons. Sibling clones are significantly more widely distributed than unrelated cortical clones, and all neurons (both with and without tags) (multivs. single p < 0.01; multi vs. all p < 0.001, Kruskal-Wallis test) Scale bar: 50µm

Figure 5. Wide Dispersion and Diversity of Clonally Related Interneurons

Left: Coronal section of embryonic (E12.5) mouse brain illustrating distinct clonal lineages of MGE progenitors (red and green). Radial glia must divide asymmetrically to produce intermediate progenitors cells, which divide symmetrically to produce pairs of newborn interneurons. The newborn neurons must then migrate tangentially to reach their final destinations in the forebrain. Right: Postnatal day (P28) mouse brain illustrating clones composed of SOM and PV subtypes dispersed widely across different functional and structural regions of the cortex, striatum (not shown) and hippocampus.