Figure 2. Characterization of discrete MSN subtypes.

A) Hierarchical clustering of the highest pairwise gene correlations in MSNs shows two strongly anticorrelated clusters of genes which include known MSN subtype markers for D1- and D2-MSNs.

B) rPCA of MSNs, Cells (rows) are ordered by their projection onto PC1 and genes (columns) by their positive (left) or negative (right) contribution to PC1. This identifies three molecularly distinct populations, assigned to D1-MSN (red), D2-MSN (green) and D1/2 hybrid MSN (yellow). Bar on the left shows the experimental origin of cells.

C) The distribution of single MSNs projected onto the D1-D2 scores. The D1 and D2 score is calculated by summing the scaled expression values of the genes shown in Fig 2B. D1- and D2-MSNs form distinct peaks, and the novel MSNs are distributed between the 2 peaks.

D) Biplot of D1- and D2-MSN cells by their expression of D1 genes (y-axis) and of D2 genes (x-axis). Scaled expression of Drd1 is shown by the color of the cell points.

E) Sagittal brain section of a D2-GFP/D1-TdTom double reporter mouse showing tdTom-GFP double positive novel MSNs in both nucleus accumbens and dorsal striatum.

F) Confocal imaging of striatal slices of D2-GFP/D1-TdTom double reporter mice demonstrating the existence of D1/2 hybrid MSNs in striatum.

G) rPCA of MSNs in D1 part of Figure 2D using all expressed genes, cells (rows) are ordered by their projection onto PC1 and genes (columns) by their positive (left) or negative (right) contribution to PC1. This identifies two molecularly distinct populations, assigned to Major (Foxp1) D1-MSN (red), and Pcdh8-MSN (yellow).

H) Biplot of major D1- and Pcdh8-MSN cells by their expression of D1 genes (y-axis) and of Pcdh8 genes (x-axis). Scaled expression of Penk is shown by the color of the cell points.

I) The distribution of MSNs projected onto Pcdh8-Foxp1 scores. Major-D1 and Tacr1-MSNs form distinct peaks

J) Box plots showing specific Pcdh8 and Tacr1 expression in Tacr1-MSNs compared to other cell types in MSN.

K) rPCA of MSNs in D2 part of Figure 2D, cells (rows) are ordered by their projection onto PC1 and genes (columns) by their positive (left) or negative (right) contribution to PC1. This identifies two molecularly distinct populations, assigned to D2-MSN (green), and Htr7-MSN (light green).

L) Biplot of D2- and Htr7-MSN cells by their expression of Htr7- genes (y-axis) and of Synpr- genes (x-axis). Scaled expression of Tac1 is shown by the color of the cell points.

M) The distribution of MSNs projected onto Htr7-Synpr scores. Major D2- and Htr7-MSNs form distinct peaks

N) Box plots showing specific Htr7 and Agtr1a expression in Htr7-MSNs compared to other cell types in MSN.