Figure 4. Mapping of mDA territories in the adult mouse midbrain.

(A) A color-coded UMAP projection of the seven mDA territories and of the mostly-lesion (ML) clusters, with individual enriched genes plotted on smaller UMAPs. (B–D) Immunohistochemical staining with antibodies indicated in the ventral midbrain. (E) Fluorescent RNA in situ hybridization with Tacr3 probe combined with immunohistochemistry. Insets are shown as higher magnification on the right. (F) Schematic presentation of the localization of the seven mDA territories across the midbrain, with the most anterior section uppermost. SNc, Substantia Nigra pars compacta, SNcL, Substantia Nigra lateral, SNcV, Substantia Nigra ventral, SNcM, Substantia Nigra medial, VTAR, Ventral Tegmental Area rostral part, PBP, parabrachial pigmented nucleus, PN, paranigral nucleus, PIF, parainterfascicular nucleus, PAG, periaqueductal grey, RRF, retrorubral field, RLi, rostral linear nucleus, CLi, caudal linear nucleus of the raphe, IF, interfascicular nucleus, RN, red nucleus, IPN, interpeduncular nucleus, PN, pontine nucleus, fr, fasciculus retroflexus, ml, medial lemniscus. Scale bars = 200 μm.

Figure 4—figure supplement 1. Neighborhoods of Sox6 territory.

(A) Color-coded UMAP projections highlighting the four Sox6 neighborhoods. (B–F) Chromogenic in situ hybridization images from Allen Mouse Brain Atlas with the probes indicated, in situ sequencing for Aldh1a1, Th, and Ndnf in D and F. (G) Schematic model of the localization of Sox6 NH1-NH4, following the color code in A, with the most anterior section on the left. Scale bars = 500 µm.

Figure 4—figure supplement 2. Neighborhoods of Ebf1, Pdia5 and Otx2 territories.

(A–C) Neighborhoods of Ebf1 territory. Col24a1 was detected by in situ sequencing, dotted circle indicating area in PBP were Col24a1⁺ cells most enriched. Vip detected by fluorescent RNA in situ hybridization combined with TH immunohistochemistry. (D–F) Neighborhood 2 of Pdia5 territory showing enriched marker gene expression in the lateral SN. Images in (D) from Allen Mouse Brain Atlas. (F) Closeup images of SNcL with immunohistochemical staining with antibodies indicated, lateral side toward left. (G) Allen Mouse Brain Atlas images for Otx2 territory markers Cbln4 and Lpl, with the most anterior section upmost. (H) Allen Brain Atlas images of Otx2_NH1 marker Grp, most anterior section upmost. (I) In situ sequencing of Otx2_NH2 marker Csf2rb2, with more anterior section on top. (K) Color-coded UMAP projections of Ebf1, Pdia5 and Otx2 territories highlighting the neighborhoods. Scale bars are 200 µm in A, 100 µm in B and F, and 500 µm in D, G-I.

Figure 4—figure supplement 3. Neighborhoods of Gad2, Fbn2, and Pcsk6 territories.

(A–C) Neighborhoods of Gad2 territory. (A) Gad2 RNA in situ hybridization in Pitx3^EGFP/+ tissue to detect Th^low Gad2 neurons. Arrowheads point to some of the co-expressing cells. (C) In situ sequencing with Ebf2 to detect NH1 and Met to detect NH2, with the most anterior section upmost. (D–G) Neighborhoods of Fbn2 territory. (D, F) In situ sequencing of Fbn2, Rxfp1, and Dsg2. (G) Allen Brain Atlas images of Rxfp1, Col23a1, modified by adding lines and text to indicate boundaries of different VTA populations. Arrowheads point to areas where expression was detected. (H–I) Neighborhoods of Pcsk6 territory. Allen Brain Atlas Images of Pcsk6, Ano2, and Cpne2. In situ sequencing of Ccdc192, with arrowhead pointing to areas where expression was seen. (B, E, H) Color-coded UMAP projections with neighborhoods highlighted, accompanied by UMAPs showing marker gene expression. Scalebars are 100 µm in A and 500 µm in B, D-G, I.