Extended Data Fig. 5: Additional histology data to validate Trans-Seq data in the SC.

a-c, Validation of Trans-Seq marker gene expression from ESC1 using RNA-Scope in situ hybridization. Npnt (red) is not found in inhibitory neurons marked by Gad2-GFP (a). b, Higher magnification of the boxed area in a. Scale bars: (a, 250 µm; b, 50 µm), quantified in (c). n = 4 animals, per genotype. Data in this figure are presented as mean ± SEM. d-f, Immunohistochemistry using Npnt antibody onto SC excitatory-marking lines. d, Npnt antibody (red) co-localizes with Ntsr1-GN209-YFP (green) for wide-field excitatory neurons. e, f, By contrast, the Npnt antibody (red) does not co-localize with the Grp-KH288-YFP line (green) for narrow-field neurons (e) or the Rorb-YFP line (green) for stellate cells (f). Scale bars: (d-f, 250 µm). g-h, Validation of Trans-Seq marker gene expression from ESC2 using RNA-Scope in situ hybridization. (g) Cdh7 (red)/Slc17a6 (blue) double in situs do not label neurons in the Grp-KH288-YFP transgenic line for narrow-field excitatory neurons at higher magnification (h), n = 4 animals. i-j, Validation of Trans-Seq marker gene expression from ESC3 using RNA-Scope in situ hybridization. (i) Tac1 (red)/Slc17a6 (blue) double in situs do not label neurons in the Rorb-YFP knock-in line primarily for stellate excitatory neurons, at higher magnification (j), n = 4 animals. Red dotted circles in (b, h, j) indicate the absence of in situ signals with GFP staining. Scale bars (h-j, 50 µm). h, Validation of Trans-Seq top marker gene expression from ESC1 using RNA-Scope for in situ hybridization shows that Cbln2 RNA probes (red) are enriched in the Ntsr1-GN209-YFP transgenic line labeling wide-field excitatory neurons (green). Scale bar: 200 µm. n = 3 animals. i, Neuronal distribution of Ntsr1-GN209-YFP transgenic line, including wide-field neurons present in SO and a significant fraction of neurons in the SGI and other deep SC layers. The wide distribution of YFP-positive neurons beyond SO indicates that Ntsr1-GN209-YFP cannot be used to manipulate SO neurons within the SC selectively. Red and blue dotted lines mark the borders of the three zones (SGS, SO, and SGI) within the superficial SC. j, Only wide-field neurons in the SO are Npnt-positive (between red and blue dotted lines), but not the rest of Ntsr1-YFP positive neurons in the SC’s SGI and deep layers. These data further suggest that Npnt is a faithful molecular marker for these neurons within SO. n = 3 animals. Scale bars: (i, 2 mm; j, 500 µm).