Figure 3. Histological validation of molecularly distinct mitral and tufted cell types.

(A) Combined average (avg) raw expression level of the top n differentially expressed (DE) genes for each mitral cell type (M1 n=14, M2 n=11, M3 n=10), overlaid onto the subclustering UMAP space (shown in Figure 2D). DE genes were selected if their log fold change was greater than 4 (see Materials and methods for details). M1-specific genes: Kcng1, Lhx5, Sertm1, Gabra2, Doc2b, Cntn6, Olfr1259, Nrp2, C1ql1, Ebf1, Baiap3, Adgrl2, Dsc2, Chrna5; M2-specific genes: Piezo2, Vgll2, Zfp114, Nts, Ros1, Samsn1, Grid2, Smpx, Itga4, Itga9, Sema6d; M3-specific genes: Cadps2, Calca, Fst, Ets1, Ednra, Cdkn1c, Mustn1, Smoc2, Cnr1, Ccno. (B) Violin plots showing maximum raw expression value of selected mitral cell type-specific DE genes across mitral and tufted cell clusters for further validation with smFISH. (C) Combined average (avg) raw expression level of the top n DE genes for each tufted cell type (T1 n=9, ET1 n=7, ET2 n=9, ET4 n=6), overlaid onto the subclustering UMAP space (shown in Figure 2D). T1-specific genes: Barhl2, Sgcg, Vdr, Olfr111, Olfr110, Cacna1g, Fam84b, Kcna10, Tspan10; ET1-specific genes: Coch, Wnt5b, Rorb, Chst9, Tpbgl, Clcf1, Rxfp1; ET2-specific genes: Lhx1, Ebf3, Trp73, Edn1, Ebf2, Nr2f2, Uncx, Psrc1, Dsp; ET4-specific genes: Ly6g6e, Foxo1, Siah3, Galnt12, Itga8, Ets2, Grik4. (D) Violin plots showing maximum raw expression value of selected tufted cell type-specific DE genes across mitral and tufted cell clusters for further validation with smFISH. (E, F) Schematic representations of the smFISH images for validating projection neuron type-specific marker genes upon rAAVretro-CAG-H1B-GFP injection into PCx and AON. The schemes depict the laminar location visualized in the histological images from a coronal section of the ipsilateral hemisphere to the injection site. EPL: external plexiform layer; MCL: mitral cell layer; GL: glomerular layer. (G–I) smFISH showing combinatorial expression of mitral cell type-specific marker genes for M1, M2 and M3 cells in the mitral cell layer. High magnifications (top right) show co-labeling of viral GFP with the in situ mRNA probe. (G and G’). The M3 markers Cadps2 and Calca are co-expressed in subpopulations of cells in the mitral cell layer, indicated by the yellow/magenta arrowheads. (H and H’) The M3 marker Cadps2 and M1 marker Kcng1 are expressed in distinct subpopulations of cells in the mitral cell layer, indicated by the magenta and yellow arrowheads respectively. (I and I’) The M3 marker Cadps2 and M2 marker Vgll2 are mutually exclusive in subpopulations of cells in the mitral cell layer, indicated by the yellow and magenta arrowheads respectively. For additional histological analysis and quantification of co-expression see Figure 3—figure supplement 1. (J–L) smFISH images showing combinatorial expression patterns of tufted cell type-specific marker genes for validating T1, ET1, ET2 and ET4 clusters as distinct projection neuron types in the external plexiform and glomerular layers. High magnifications (top right) show co-labeling of viral GFP with the in situ mRNA probe. As described for the mitral cell types, yellow or magenta arrowheads show non-overlapping patterns (K, K’: T1–ET1 and L, L’: ET1–ET4), and yellow/magenta arrowheads show co-expression patterns (J, J’: T1–T1). For additional histological analysis and quantification of co-expression see Figure 3—figure supplement 1. DAPI counterstain in blue. Scale bars, 50 μm and 10 μm (high magnifications).

Figure 3—figure supplement 1. Histological analysis of DE genes for distinct mitral cell types.

(A) Schematic representation of the smFISH images for validating selected mitral cell type-specific marker genes upon rAAVretro-CAG-H2B-GFP injection into PCx and AON. The scheme depicts the laminar location visualized in the histological images from a coronal section of the ipsilateral hemisphere to the injection site. MCL: mitral cell layer. (B–D) smFISH showing co-expression of M1-specific marker genes with viral GFP. Labeled nuclei are indicated by the magenta/green arrowheads. (E) smFISH showing co-expression of two M1-specific marker genes. Labeled nuclei are indicated by the yellow/magenta arrowheads. (J) Same as (E) but for M2-specific marker gene. (N) Same as (E) but for M3-specific marker gene. (F, G) Same as (B–D) but for M2-specific marker gene. (H, I) In situ hybridization images from the Allen Brain Atlas showing additional M2-specific DE genes. (K - M) Same as (B–D) but for M3-specific marker gene. (O–Q) smFISH images showing mutually exclusive expression of mitral cell type-specific marker genes for M1, M2, and M3. Yellow and magenta arrowheads show mutually exclusive expression of M1 versus M2 (O–P), and M1 versus M3 (Q) marker genes. DAPI counterstain in blue. Scale bars, 50 μm. (R) Combinatorial quantification of mitral cell type-specific smFISH probes. Each dot is color-coded by the mitral cell type membership between cells of the same clusters or represented in grey for cells across different clusters. Each dot represents the fraction of co-expression between two cluster-specific probes.

Figure 3—figure supplement 2. Histological analysis of DE genes for distinct tufted cell types.

(A) Schematic of the smFISH images for validating selected tufted cell type-specific marker genes upon rAAVretro-CAG-H2B-GFP injection into PCx and AON. The scheme depicts the laminar location visualized in the histological images from a coronal section of the ipsilateral hemisphere to the injection site. GL=glomerular layer; EPL=external plexiform layer. (B) smFISH showing co-expression of two T1-specific marker genes. Labeled nuclei are indicated by the yellow/magenta arrowheads. High magnification (left) shows clear co-labeling of the two mRNA probes Barhl2 and Olfr110. (C) In situ hybridization images from the Allen Brain Atlas showing one additional T1-specific DE gene. (D) Same as (B) but for ET1-specific marker gene. (E) In situ hybridization images from the Allen Brain Atlas showing additional ET1-specific DE genes. (F) Same as (B) but for ET2-specific marker gene. (G) In situ hybridization images from the Allen Brain Atlas showing additional ET2-specific DE genes. (H) Same as (B) but for ET4-specific marker gene. (I–J–K) smFISH images showing mutually exclusive expression of tufted cell type-specific marker genes for T1, ET1, ET2, and ET4. Yellow and magenta arrowheads show mutually exclusive expression of T1 versus ET4 (I), ET2 versusET4 (J), and ET1 versus ET4 (K) marker genes. DAPI counterstain in blue. Scale bars, 50 μm. (L) Combinatorial quantification of tufted cell type-specific smFISH probes. Each dot is color-coded by tufted cell type membership between cells of the same clusters or represented in grey for cells across different clusters. Each dot represents the fraction of the co-expression between the two cluster-specific probes visualized next to the dot.

Figure 3—figure supplement 3. Excitability-related, cell adhesion-related, and pan-OB projection neuron DE genes.

(A) Violin plots showing maximum raw expression values of genes encoding for channels as key candidates for controlling the differential excitability of different MC and TC types. Kcng1 gene is known to encode for a voltage-gated potassium channel that forms heterotetrameric channels with the ubiquitously expressed delayed rectifying Kv2.1 potassium channel (Kcnb1). (B) Violin plots showing maximum raw expression values of genes encoding for cell adhesion and axon guidance molecules known to control the formation and maintenance of neuronal connectivity that were differentially expressed in MC and TC types. (C) Violin plots showing maximum raw expression values of pan-external tufted cell marker genes (Gabbr2, Shisa6, Pip5k1b), pan-tufted cell marker genes (Zbtb20, Astn2), and pan-mitral cell marker genes (Fam19a2, Tnfaip8).