Fig. 2

Spatial pattern of Nr2f2 and Tle4 expression enables localization of claustrum projection neurons. A1–A2 Representative images of the anterior claustrum showing retrograde GFP labeling (A1) and Nr2f2 expression (A2) following AAV injection into the retrosplenial cortex (Retro-RSC). Dashed ellipses highlight GFP expression in the claustrum (CLA) without labeling the surrounding insula cortex (Ins) or striatum (str). B–D Representative images showing colocalization of GFP labeling with Nr2f2 in the anterior (B), middle (C) and posterior (D) CLA (left, merged imaging channels; right, single channel images). (B’) 2.3 × fold magnifications of area in the white boxes in (B) with single channel (top, middle) and merged (bottom) images. Orange arrowheads indicate examples of cell colocalization. E–G Venn diagrams representing the mean number of cells expressing Nr2f2 (red) and GFP (cyan), and colocalization of GFP with Nr2f2 in the anterior (E), middle (F), and posterior (G) CLA. Values indicate mean ± standard deviation from n = 6 mice (3 male and 3 female). H1, H2 Two representative images of the same coronal plane showing lack of colocalization between Nr2f2 and Tle4 in the CLA. H2 is a magnified image of the dashed box in (H1). For H2, merged imaging channels are on the left and single channel images are on the right. Inset (bottom-left) is 2.5 × fold magnifications of area in the white box. I Venn diagram representing the mean number of cells expressing Nr2f2 (red) and Tle4 (yellow), and the colocalization of Nr2f2 with Tle4, averaged across anterior, middle and posterior CLA (n = 6 mice, 3 male and 3 female). J1–J3 Example images of the CLA region showing polygons outlining the boundaries of GFP cells and neuropil (cyan, J1), Nr2f2 concentric expression (red, J2), and absence of Tle4 expression (grey, J3). K1, K2 Overlay of polygons from (J1–J3). Bottom-left insets show spatial registration of GFP outline with Nr2f2 outline (K1), and GFP outline with Tle4 outline (K2) (see Materials and Methods for details). Pink represents the topographic overlap of GFP/Nr2f2 expression, grey represents the region of low Tle4 expression. L Cumulative spatial overlap of GFP⁺/Nr2f2⁺ labeling (left), and the spatial location of GFP labeling within the region of relative Tle4 absence (GFP⁺/Tle4⁻, right), in the anterior (top), middle (middle) and posterior (bottom) CLA across 6 mice. Values in E, F, G, I represent mean ± standard deviation