FIGURE 6. Visualization and subclustering of mWAKE⁺ neurons in the SCN.

(a-c) Representative images of mWake expression in the SCN are shown. (a) Anti-V5 IF staining (green) with DAPI in mWake^(V5/+) mice. (b) Chromogenic RNAscope ISH for mWake mRNA (red) with hematoxylin counterstain (purple) in wild-type (WT) mice. (c) Endogenous tdTomato fluorescence (red) in mWake^(Cre/+) mice with DAPI. In (a-c), the dashed line outlines the SCN. The asterisks in (c) indicate the tdTomato⁺ processes in the dorsal SCN. The scale bar in (a) is equivalent to 500 μm, and applies across the row. (d) UMAP plot of mWake^SCN neurons reveals five subgroups with key gene markers: cluster 0 (Vip, Grp, red, n=160 cells); cluster 1 (Rorb, Penk, yellow, n=140 cells); cluster 2 (Grp, Pcp4, green, n=119 cells); cluster 3 (Vip, Penk, blue, n=89 cells); cluster 4 (Vip, Prok2, Nms, purple, n=33 cells). (e) Violin plot depicting the expression of key molecular markers for each of the 5 clusters. Each row depicts the expression of a single gene, with expression level on the y-axis, and the percentage of the cells expressing at that level forming the width. (f-s) Multiplex fluorescent RNAscope in situ hybridization from mWake^(Cre/+) SCN showing relative distribution of markers. (f-i) Representative images using Vip (f), tdTomato (g), Prok2 (h) probes, as well as merged image with DAPI (i) are shown. (j) Higher magnification image of the area denoted by the box in (i). Dashed circles highlight Vip⁺, tdTomato⁺, Prok2⁺ cells from cluster 4. (k-n) Representative images using Vip (k), tdTomato (l), Rorb (m) probes, as well as merged image with DAPI (n) are shown. (o) Higher magnification image of the area denoted by the box in (n). Dashed circles highlight Vip⁻, tdTomato⁺, Rorb⁺ cells from cluster 1. (p-s) Representative images using Grp (p), tdTomato (q), Rorb (r) probes, as well as merged image with DAPI (s) are shown. The scale bars in (i), (n), and (s) denote 100 μm, and the scale bars in (j) and (o) denote 25 μm.