Fig. 1

The expression pattern of Nurr1 and Tle4 in the claustrum region distinguishes claustrum projection neurons from their cortical counterparts in layer 5 and 6. A Retrograde AAV-CAG-GFP was injected into the retrosplenial cortex (RSC), followed by tissue collection 14 days post injection. B An example tracer injection site in the retrosplenial cortex. C, E representative images of retrograde GFP labeling in the anterior claustrum (D, F), and representative images showing Nurr1 (D) and Tle4 (F) expression relative to GFP labeling. C–D are from the same slice, and E, F are from an adjacent slice of the same mouse. Dashed ellipses in (C–F) highlight GFP expression in the claustrum (CLA) without labeling the surrounding insula cortex (Ins) or striatum (str). G, H Representative images showing the degree of colocalization with Nurr1 (G) or Tle4 labelled cells (H). Single channel images are shown on the right of each panel. G’, H’ 3.5 × fold magnifications of areas in white boxes in (G, H), respectively, with single channel (left, middle) and merged (right) images. Orange arrowheads indicate examples of cell colocalization. I Venn diagrams showing the mean cell counts for Nurr⁺/GFP⁺ and Tle4⁺/GFP⁺ cells in the anterior CLA. Values throughout indicate mean ± standard deviation (n = 6 mice, 3 male and 3 female). J-L: The same as G-I but for the middle CLA. M–O The same as (G–I) for the posterior CLA. P Nurr1 and Tle4 colocalization in the CLA region was compared to layer 6b (Ctx L6b) of the primary motor cortex (MOp) and the somatosensory cortex (SSC). Q–S Representative images showing Nurr1 and Tle4 expression in the CLA region (Q), the MOp (R) and the SSC (S) (left, merged imaging channels; right, single channel images). Insets are 2.5 × fold magnifications of areas in white boxes. Red arrowheads indicate examples of cell colocalization. T–V Venn diagrams representing the mean number of cells expressing Nurr1 (magenta) and Tle4 (yellow), and colocalization between Nurr1 with Tle4 (n = 4 mice, 2 male and 2 female). The data in T are averaged across anterior, middle, and posterior CLA. Values in I, L, O, T, U, V represent mean ± standard deviation. Panels P–V: See Additional file 2: Table S1 for details on mouse sex