Smim32 is expressed in the CLA, EP and TRN neurons.

(A) UMAP plot of single-cell transcriptomes of neurons from the anterior third of the cortex (7804 cells). Excitatory and inhibitory cell types are colored with red and blue tones, respectively. Each cluster is represented by a different color. A single cluster could be identified as excitatory neurons pertaining to the CLA based on the expression of previously identified markers.

(B,C) Cells shaded according to the normalized expression of (B) Nr4a2 and (C) Smim32 across neurons of the anterior cortex.

(D) Density plots of normalized expression levels of selected genes across clusters.

(E) UMAP plot of single-cell transcriptomes of neurons from the thalamus (7317 cells) with a similar color coding as in A.

(F,G) Cells shaded according to the normalized expression of (F) Pvalb and (G) Smim32 across neurons of the anterior cortex.

(H) Density plots of normalized expression levels of selected genes across clusters.

(I) Schematic of the localization of coronal sections shown in (L-Q). Anterior sections (K,M,O) comprise the CLA region, while posterior sections (L,N,P) include the TRN.

(J) Transcript topology (corresponding to the RefSeq NM_001378296.1) and genomic localization of Smim32. The region targeted by RNAscope ISH probes is highlighted in green.

(K) Illustrative image of CLA cells labeled by RNAscope ISH with probes for Smim32 (orange), Nr4a2 (magenta) and Pvalb (blue). Individual colored spots (mRNA puncta) correspond to mRNA transcripts. DAPI staining is shown in grey. Scale bar: 10 μm.

(L,M) Left, representative images of RNAscope ISH labeling of Smim32 transcripts in the (l) CLA/EP and in the (M) TRN. Scale bar: 250 μm. Right, cells expressing Smim32 across the entire hemisection, where dot color corresponds to the Smim32-expression level in each individual cell.

(N,O) Same as above, but showing Nr4a2 expression in the CLA/EP and in cortical layer 6 (L6). Scale bar: 250 μm.

(P-Q) Same as above, but showing Pvalb expression around the CLA/EP and in in the TRN. Scale bar: 250 μm.

(R,S) Correlation between expression levels of Smim32 and Nr4a2, and Smim32 and Pvalb, in cells from the (R) CLA/EP and cells from the (S) TRN. Each dot corresponds to one cell. All cells from both hemispheres, within the regions of interest (ROI, grey dotted line in (L,Q)), were included in the analysis. ρ value indicates Spearman’s rank correlation. CLA/EP, n = 6755 cells; TRN, n = 11139 cells.

(T,U) Illustrative map of cells co-expressing Smim32 and Nr4a2 (red) or Smim32 and Pvalb (purple grey) in the CLA/ EP, the TRN, and in L6. A cell was considered positive for a marker when a minimum of 20 mRNA puncta are attributed to this cell. The dotted lines highlight the ROIs used for quantification analyses shown in (V,W).

(V,W) Bar plots showing the percentages of cells positive for one or several markers. The analysis was done separately for each cell population.

Smim32 is co-expressed with known CLA markers.

(A) Correlation between expression levels of Smim32 and Lxn in cells from the CLA and EP. Each dot corresponds to one cell. Smim32 and Lxn: ρ value indicates Spearman’s rank correlation. n = 6429 cells.

(B) Illustrative plot of cells co-expressing Smim32 and Lxn (red) in the CLA and EP. Dotted lines highlight the ROIs used for quantification analyses shown in (C).

(C) Bar plots showing the percentage of cells positive for one or both markers in the CLA and EP regions. (D-R) Same as above, but with different markers of the CLA and surrounding cell populations. (D-F) Smim32 and Gnb4, n = 6429 cells. (G-I) Smim32 and Oprk1, n = 6780 pairs. (J-L) Smim32 and Car3, n = 6493 cells. (M-O) Smim32 and Ctgf, n = 7033 cells. (P-R) Smim32 and Rprm, n = 6683 cells.

Generation of mouse lines expressing Cre in Smim32-neurons.

(A) Schematic of the CRISPR-mediated insertion of IRES-Cre or IRES-FlpO after the Smim32 stop codon via homology direct repair (HDR) to generate the Smim32Cre or the Smim32Flpo alleles.

(B-E) Left, epifluorescence images of knockin mice crossed with mice carrying a Cre-dependent or a Flippase-dependent reporter allele R26tdT. Representative coronal sections at the level of the CLA and TRN. Right, maps of tdT relative expression levels within the represented section. Each dot represents a cell.

(F) Schematic of the linearized BAC transgene use to generate the different Tg(Smim32-Cre) lines. A detailed description of the transgene can be found in the Materials and Methods section.

(G-J) Left, epifluorescence images of BAC transgenic mice (G,H: line 61Irod, I,J: line 62Irod) crossed with mice carrying a Cre-dependent reporter allele R26tdT. Representative coronal sections at the level of the CLA and TRN. Right, maps of tdT relative expression within the represented section.

Characterization of the expression of reporter genes in adult mice.

(A) Schematics showing the regions (according to the Allen Brain Reference Atlases – Adult Mouse, atlas.brain-map.org) that were analyzed to compare expression levels of reporter genes in the knockin and in the BAC transgenic lines. Red dotted lines highlight the ROIs used for quantitative analyses shown in (F-H). br: distance from bregma in mm.

(B-D) RNAscope ISH labeling of Smim32 (white) and tdT (red) mRNA on a coronal section in the anterior CLA of a Smim32Cre/wt;R26tdT/wt mouse. The white square highlights the region magnified in (E). Scale bar: 200 μm.

(E) CLA cells co-expressing Smim32 and tdT. Scale bar: 50 μm.

(F-H) Bar plots showing the percentage of cells co-expressing Smim32 with the Cre-inducible reporter allele R26tdT. A cell was considered as positive for a marker when a minimum of 20 mRNA puncta were attributed to this cell. Numbers in the bar plots indicate the number of cells analyzed in the corresponding region and antero-posterior position.

Expression of transgenes during development

(A-C) Expression of the Cre-dependent reporter gene R26-tdT in brain sections of (A) Smim32-Cre, (B) Tg(Smim32-Cre)61Irod and (C) Tg(Smim32-Cre)62Irod mice carrying the R26tdT allele, from P10 to P200. Only brain regions with persistent expression in adult Smim32-Cre mice are shown here. A vertical grey bar indicates the first observation of tdT-positive cells within the structure. The heatmap shows relative gene expression levels in sections from P10, normalized across all regions and all mouse lines.

(D) Schematics of the regions of interest (blue) where tdT expression was quantified.

Examples of Cre-dependent reporters used with Smim32 BAC transgenics.

(A,B) Tg(Smim32-Cre)61Irod mouse expressing ChR2 and EYFP after injection of a Cre-dependent viral vector with ChR2 and EYFP in the CLA. DAB IHC staining of YFP in a coronal section. The black square indicates the region magnified in (B). Scale bars: 200 um (A) and 100 um (B).

(C,D) Coronal section of a Tg(Smim32-Cre)61Irod; R26HA-hM3Dq/wt mouse, where the HA-hM3Dq tag was labeled by IHC (green). The white square highlights the region magnified in (D). Scale bars: 200 um (C) and 100 um (D).

(E-H) Confocal image of a pyramidal neuron in the CLA periphery of a Tg(Smim32-Cre)62Irod; R26HA-hM3Dq/tdT, DAPI staining of the nuclei (blue), (F) NR4A2 immunolabelling (magenta), (G) HA immunolabelling (green) and (H) tdT endogenous fluorescence (red). Scale bar: 15 um.

(I,J) Expression of the Cre-inducible diphtheria toxin A in Smim32-positive cells of the CLA. Tg(Smim32-Cre)61Irod mice were crossed with NSEDTA/wt (neuron-specific enolase 2) mice, and the persistence of Smim32-positive cells in the CLA and EP was evaluated by RNAscope ISH labeling. Coronal sections of a Tg(Smim32-Cre)61Irod; NSEDTA/wt and a control NSEDTA/wt mouse were labeled with probes against Smim32 (yellow), Nr4a2 (magenta) and the cortical layer 6 marker Rprm (light blue).

(K-N) Higher magnification of the CLA region, with all three channels (K,L) and only the Smim32 channel (M,N).

(O) Proportion of Smim32-positive cells within the CLA and EP (among all cells in the ROI, dotted lines in (I,J)). Tg(Smim32-Cre)61Irod; NSEDTA/wt, n=1 mouse, n= 8 hemisections; NSEDTA/wt, n=1 mouse, n= 8 hemisections. **** p<0.001, two-way RM ANOVA, Fisher’s LSD test.

(P,Q) Coronal section of a Tg(Smim32-Cre)61Irod; TIGREOptopatch3/wt mouse, with endogenous eYFP fluorescence. DAPI staining of nuclei (blue). The white square highlights the region magnified in (N). Scale bars: 200 μm (M) and 100 μm (N).

(R,S) Coronal section of a Tg(Smim32-Cre)62Irod; TIGREGCaMPf6/wt mouse, where GCaMPf6 is revealed by IHC labelling of GFP. DAPI staining of nuclei (blue). Scale bar: 200 μm.

Primers for genotyping.

List of transgenic mouse lines.

SRA run accessions for single-cells RNAseq analysis.

List of RNAscope probes

List of antibodies used for IHC labeling.

Statistical data.

Smim32 is expressed in excitatory neurons in the CLA and in inhibitory neurons in the TRN.

(A,B) Correlation between expression levels of Smim32 and Slc17a6, and Smim32 and Gad1, in cells from the (A) CLA/EP and from the (B) TRN. Each dot corresponds to one cell. All cells from both hemispheres, within the regions of interest (ROI, dotted line in (C,D)), were included in the analysis. ρ value indicates Spearman’s rank correlation. CLA/EP, n = 6272 cells; TRN, n = 11101 cells.

(C,D) Illustrative map of cells co-expressing Smim32 with Slc17a6 (red) or Smim32 with Pvalb (purple grey) in the CLA/ EP and in the TRN. A cell was considered positive for a marker when a minimum of 20 mRNA puncta were attributed to this cell. The dotted lines highlight the ROIs used for quantification analyses shown in (E,F).

(E,F) Bar plots showing the percentages of cells positive for one or several markers. The analysis was done separately for each cell population.

Proportion of CLA neuronal population expressing Cre driven reporter.

(A) Quantification of tdT expressing cells in Nr4a2-positive neuronal population in Smim32Cre/wt; R26tdT/wt mice. Bar plots show the percentage of Nr4a2-positive cells from the CLA and EP that co-express tdT. Numbers in the bar plots indicate the number of cells analyzed in the corresponding region and anteroposterior position. n= 1 Smim32Cre/wt; R26tdT/wt mouse.

(B-C) Same as above, but for Tg(Smim32-Cre)61Irod and Tg(Smim32-Cre)62Irod mice. n= 2 Tg(Smim32-Cre)61Irod mice, n=1 Tg(Smim32-Cre)62Irod mouse.

Smim32 expression in the kidney

(A) Schematic of a mouse kidney with the cutting plane of the section shown in (B) and (C).

(B) Reporter expression in the kidney of a Tg62(Smim32-Cre) mouse.

(C) Zoom on the papilla, where arrays of reporter-expressing cells can be observed, likely forming the walls of distal tubules.

(D) Re-analysis of mouse collecting duct scRNAseq data from Chen et al40. Statistics were computed for cell types (rows of the matrix) and genes of interest (columns of the matrix). For each gene, mean expression values (mean TPM) were divided by the maximum observed mean expression across all samples to produce the scaled mean TPM value (max mean TPMs: Smim32=0.216, Atp6v1b1=353, Atp6v1g3=3088, Trpc7=2.06, Nr4a2=8.35). On the right, the dark areas of the pie charts represent the proportion of cells expressing more than 1 TPM of Smim32 for each cell type.

Representative images of Cre-driven tdT expression in Smim32 transgenic mice

(A) Schematics of the regions of interest within which tdT expression was quantified.

(B-D) Representative epifluorescence images of tdT expression in Smim32-Cre, Tg(Smim32-Cre)61Irod and Tg(Smim32-Cre)62Irod mice carrying the Cre-dependent R26-tdT allele. Left panels, overview of the section stained with DAPI (blue) and endogenous tdT fluorescence (white). Scale bar: 1 mm. The white square highlights the region magnified in the right panels. White arrowheads point towards tdT expressing cells in regions where expression is very sparse.