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Single-cell genomics of the mouse olfactory cortex reveals contrasts with neocortex and ancestral signatures of cell type evolution

Nature Neuroscience volume 28, pages 937–948 (2025)Cite this article

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Abstract

Understanding the molecular logic of cortical cell-type diversity can illuminate cortical circuit function and evolution. Here, we performed single-nucleus transcriptome and chromatin accessibility analyses to compare neurons across three- to six-layered cortical areas of adult mice and across tetrapod species. We found that, in contrast to the six-layered neocortex, glutamatergic neurons of the three-layered mouse olfactory (piriform) cortex displayed continuous rather than discrete variation in transcriptomic profiles. Subsets of piriform and neocortical glutamatergic cells with conserved transcriptomic profiles were distinguished by distinct, area-specific epigenetic states. Furthermore, we identified a prominent population of immature neurons in piriform cortex and observed that, in contrast to the neocortex, piriform cortex exhibited divergence between glutamatergic cells in laboratory versus wild-derived mice. Finally, we showed that piriform neurons displayed greater transcriptomic similarity to cortical neurons of turtles, lizards and salamanders than to those of the neocortex. In summary, despite over 200 million years of coevolution alongside the neocortex, olfactory cortex neurons retain molecular signatures of ancestral cortical identity.

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Fig. 1: Transcriptomic diversity of piriform cortex glutamatergic neurons.
Fig. 2: Transcriptomic diversity across the mouse three- to six-layered cortical continuum.
Fig. 3: Area-specific epigenetic states distinguish transcriptomically similar glutamatergic neurons across the adult mouse three- to six-layered cortical continuum.
Fig. 4: A potential link between immature neurons and the transcriptomic divergence of pyramidal cells between laboratory- and wild-derived mice in the posterior piriform cortex.
Fig. 5: Pir glutamatergic neurons display marked transcriptomic similarities to mature neurons of reptile and salamander.
Fig. 6: Salamander adult immature neurons display transcriptomic similarities to immature neurons of the Pir cortex.

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Data availability

Raw and processed sn-multiome-seq and RNA-seq data have been deposited and available in Gene Expression Omnibus under accession no. GSE239477. Seurat and Scanpy integrated objects are available upon request. All other data are included in the main paper or the supplementary materials. Source data are provided with this paper.

Code availability

The R and Python analysis scripts used for this paper are available at GitLab at https://gitlab.com/fleischmann-lab/papers/zeppilli-et-al-2023.

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Acknowledgements

We thank Z. Herbert and M. Berkeley from the Molecular Biology Core Facilities at the Dana-Farber Cancer Institute for sequencing services. We thank N. Eckart from 10x Genomics for excellent technical support. We thank K. Babcock, F. Mosti, D. Silver, R. Van Drunen, J. Ritt, S. Firestein, H. Berry, K. Franks, S. Pantalacci, G. Beslon, H. Wichterle, B. Mensh and T. Pievani for critical comments on the manuscript. We thank A. Pierre for software support and C. Bravo González-Blas for analysis support with SCENIC+. We thank N. Nisim for technical assistance with the wild-derived mice and the Brown and Weizmann animal facilities for animal care. Work in the A.F., R.S. and A.C. laboratories was supported by National Institutes of Health (NIH) grant NIDCD R01DC020478. Work in the A.F. laboratory was supported by grants from the NIH, NIDCD R01DC017437 to A.F., the Robert J. and Nancy D. Carney Institute for Brain Science to A.F. and the Carney Graduate Award in Brain Science to S.Z. Carney Institute computational resources used in this work were supported by the NIH Office of the Director grant S10OD025181. Work in the A.C. laboratory was supported by project AI4scMed, France 2030 ANR-22-PESN-0002. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Department of Neuroscience and Carney Institute for Brain Science, Brown University, Providence, RI, USA

    Sara Zeppilli, Robin Attey & Alexander Fleischmann

  2. Department of Biological Sciences, Columbia University, New York, NY, USA

    Alonso O. Gurrola & Maria A. Tosches

  3. Department of Computer Science, Center for Computational Molecular Biology, Brown University, Providence, RI, USA

    Pinar Demetci, Tuan M. Pham & Ritambhara Singh

  4. Department of Neurobiology, Harvard Medical School, Boston, MA, USA

    David H. Brann & Sandeep R. Datta

  5. Department of Brain Sciences, Weizmann Institute of Science, Rehovot, Israel

    Noga Zilkha & Tali Kimchi

  6. Inria Centre de Lyon, Villeurbanne, France

    Anton Crombach

  7. INSA-Lyon, CNRS, UCBL, LIRIS, UMR5205, Villeurbanne, France

    Anton Crombach

  8. INSA-Lyon, CITI, UR3720, Villeurbanne, France

    Anton Crombach

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Contributions

S.Z., A.C. and A.F. conceptualized the study with input from all authors. S.Z. conducted all experiments and supervised all computational analyses. S.Z., R.A. and A.C. performed the transcriptomic analysis. A.C. performed the epigenomic analysis. A.O.G. performed the cross-species transcriptomic analysis. P.D. performed the OT integration between datasets. T.M.P. performed the gene trajectory analysis. D.H.B. performed the immature neuron-related analysis. T.K. and N.Z. assisted with the wild-derived mice. S.Z., T.K., S.R.D., R.S., M.A.T., A.C. and A.F. provided supervision and scientific guidance. S.Z., A.C. and A.F. wrote the manuscript with input from all authors.

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Correspondence to Anton Crombach or Alexander Fleischmann.

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Extended data

Extended Data Fig. 1 Relative abundance, main cell types, and histological assessment across biological replicates and cortical areas of lab mice.

(a) Relative abundance of main cell types across biological replicates (donors) integrated from single-nucleus multiome sequencing (sn-multiome seq) experiments. From left to right: anterior piriform cortex (aPir) replicates, indicated by A; posterior piriform cortex (pPir) replicates, indicated by P; agranular insular cortex (AI) replicates, indicated by T; primary somatosensory cortex (SSp) replicates, indicated by N. Numbers correspond to the ID of mice. VLMC: vascular leptomeningeal cells; Micro: microglia; OPC_diff: differentiating oligodendrocytes; OPC: oligodendrocyte precursors; Oligo: oligodendrocytes; Astro: astrocytes; IN_MGE and IN_CGE: inhibitory neurons from medial and caudal ganglionic eminence, respectively; SL: semilunar cells; Pyr: pyramidal cells. (b) Post-hoc histological assessment of aPir and AI dissections from anterior coronal sections of adult lab mice ordered by ID mouse number. Asterisks indicate the microdissected area. Neurotrace counterstain in gray. (c) Same as in (b) but for pPir and SSp. (d) From left to right: UMAPs of aPir, pPir, AI, and SSp datasets color-coded by main cell types. (e) Gene expression levels of representative markers for each cell type across the four cortical areas, from left to right: aPir, pPir, AI, and SSp.

Extended Data Fig. 2 Quality control of single-nucleus multiome (RNA and ATAC) sequencing data of lab mice.

(a) Transcriptome quality control. Left: number of genes per nucleus quantified for each biological replicate. A indicates aPir replicates, P indicates pPir, T indicates AI, N indicates SSp. Numbers correspond to the ID of mice. Right: number of genes per nucleus identified for each main cell type. (b) Same as in (a) but for the fraction of mitochondrial content per nucleus. (c) Epigenome quality control for aPir biological replicates. From left to right: barcode rank plot, fragment size distribution, Transcription Start Site (TSS) enrichment and Fraction of Reads In Peaks (FRIP). (d) Same as in (c) but for pPir biological replicates. (e) Same as in (c) but for AI biological replicates. (f) Same as in (c) but for SSp biological replicates.

Extended Data Fig. 3 Piriform cortex-specific markers and histological validation.

(a) Left: gene expression levels of generic or subtype-specific markers for semilunar cells in the combined aPir and pPir datasets. Right: in situ hybridization images from the Allen Brain Atlas for some of the markers shown in the dotplot on the left, with the exception of the marker SATB1, which was validated using immunohistochemistry in Vglut1-CRE/INTACT-GFP transgenic mice. (b) Same as (a), but for generic or subtype-specific markers for pyramidal cells in the combined aPir and pPir datasets. (c) High magnifications in Pir layer 3 of immunohistochemical experiments using Vglut1-CRE/INTACT-GFP transgenic mice showing combinatorial expression of the specific marker for Pyr11, EBF2 (in magenta), with specific or generic markers for glutamatergic neurons (in cyan): EBF1 (Pyr10-11-specific); TBR1 and CUX1 (pan-excitatory/pyramidal neuron marker); MEIS1 (Vglut2-specific). Scale bar, 100 μm. (d) Same as (a), but for generic or subtype-specific markers for Vglut2 cells in the combined aPir and pPir datasets. (e) High magnifications in Pir layer 3 of immunohistochemical experiments using Vglut1-CRE/INTACT-GFP transgenic mice showing combinatorial expression of the specific marker for Vglut2 cells, PAPPA2 (in magenta), with generic markers for semilunar cells (RELN), INs (GABA), immature neurons (SOX11), and pyramidal cells (CUX1), or with markers highly specific to Vglut2 cells (MEIS1), or expressed in Pir layer 3 (BARHL1) (in cyan), to understand the identity of the uncharacterized Vglut2-expressing neuronal population. Scale bar, 100 μm. (f) Same as (a), but for generic or subtype-specific markers for immature neurons in the combined aPir and pPir datasets. (g) Same as (a), but for generic or subtype-specific markers for INs in the combined aPir and pPir datasets.

Extended Data Fig. 4 Integration of neurons from this study and a mouse single-cell reference atlas.

(a) Integration of neurons from this study and a mouse single-cell reference atlas17. UMAP of integrated neurons (n = 30,553). (b) UMAPs as shown in (a) with gene expression of generic markers for glutamatergic neurons (Slc17a7 and Slc17a6), inhibitory neurons (INs) (Gad2), and other established markers for neocortical projection neurons: Rorb (layer 4), Cux1 (layer 2/3), Ctip2 (layer 5), Fezf2 (layer 5), Foxp2 (layer 6), Nfia (layer 6). (c) Quantification of co-clustering between piriform, hippocampal formation and transition areas neurons from integration shown in (a). Neurons are grouped into main types per cortical area. Rectangles indicate co-clustering of neurons in the Seurat integrated clusters. Color represents the percentage of cells in an integrated cluster. L: layer; IT: intratelencephalic; NP: near projecting; CT: corticothalamic; Sub: subiculum; ProS: prosubiculum; PPP: para/post/pre subiculum; RHP: retrohippocampal region; DG: dentate gyrus; CA 1/2/3: hippocampal fields; IG: induseum griseum; FC: fasciola cinereal; AI: agranular insular; ENT: entorhinal (medial and lateral); TPE: Temporal association areas, Perirhinal area, Ectorhinal area. (d) Quantification of co-clustering between piriform glutamatergic neuron subtypes and hippocampal formation glutamatergic neurons from the integration shown in (a). Rectangles indicate co-clustering of neurons in the Seurat integrated clusters. Color represents the percentage of cells in an integrated cluster. Dentate gyrus (DG) glutamatergic neurons aligned with 13%, 31% and 7% of piriform subtypes Pyr7-8-9, respectively. CA 1 glutamatergic neurons aligned with 4% of piriform subtype Pyr7. DG: dentate gyrus; CA 1/2/3: hippocampal fields. (e) UMAPs of gene expression of transcription factors (TFs) highly enriched in piriform cortex compared to other cortical areas. (f) UMAPs of integrated neurons from this study and the mouse single-cell reference atlas (bottom) (Yao 2021), and visualization of only SSp neurons from the two studies (top: this study, bottom: single-cell reference atlas). (g) Quantification of co-clustering between SSp neurons of this study and of the single-cell reference atlas. Only SSp datasets were included in the integration to transfer projection neuron profile nomenclature to this study. SSp_yao indicates neurons from (Yao 2021), the rest corresponds to SSp clusters of this study. Dots indicate co-clustering of neurons in the Seurat integrated clusters. Size of the dots represents the percentage of cells in the integrated cluster. (h) Gene expression levels of Car3 in the SSp dataset. Car3 is expressed in SSp neurons co-clustering with piriform neurons in the immature neuron supertype. These Car3+ SSp neurons co-cluster with L4/5/6 intratelencephalic Car3+ neurons of the single-cell reference atlas. Given also the lack of DCX expression in SSp, we do not consider SSp neurons falling in the immature type as immature neurons. (i) Gene expression levels of the TF Rorb, which is highly enriched in the SL1 supertype (left), and of established TFs present in the SSp-specific supertypes (right). Pyr 14-15-16 of layers 4 and 5 are characterized by Rorb and Fezf2, Pyr17 is characterized by Foxp2, Bcl11b (Ctip2) and Nfia, corresponding to CT neurons of layers 6.

Extended Data Fig. 5 Epigenetic divergence of transcriptome-based supertypes across mouse cortical areas.

(a) UMAP of multiome ATAC data colored by aPir, pPir, AI, and SSp datasets. Neurons are integrated using Harmony. (b) UMAP as in (a) colored by the corresponding transcriptome-based supertype. For interneurons areas mix, while for glutamatergic neurons piriform separates from SSp, while AI overlaps with both (see (a)). (c) UMAP as in (a) colored by epigenome-based leiden clusters. (d) Mapping of transcriptome-based clusters (RNA, supertypes) to epigenome-based (ATAC, leiden) clusters. For glutamatergic neurons, multiple area-specific epigenome-based clusters correspond to a single supertype. Adjusted Rand Indices (ARIs) quantify the cluster overlap: for all neurons= 0.43; for INs= 0.88; for glutamatergic neurons= 0.37. (e) High-quality e-regulons are selected for downstream analysis based on the correlation between AUC (Area Under the Curve) scores for target genes and target CREs (Cis Regulatory Elements). A correlation cut-off of 0.4 and a minimum number of target genes of 10 are used. (f) Upset plot of the intersection of target genes for the e-regulon Rorb(+), which is shared across aPir, pPir, AI and SSp. Vertical bars show the number of target genes in the corresponding intersection of the matrix below. Horizontal bars show the total number of target genes for each cortical area. Of note, main text states 9% overlap between aPir and SSp target genes. That overlap is computed with a Jaccard similarity index and equivalent to taking in the upset plot the (relative) size of a combination of intersections. (g) Upset plot of the intersection of target CREs for the e-regulon Rorb(+). See (f) for details. (h) From left to right, average log-normalized expression of target genes (TGs) of Rorb identified in aPir, pPir, AI, and SSp. TGs of a given area are shown across areas (even if they are regulated by other TFs). (i) Difference of gene expression between target genes identified in aPir and SSp for the e-regulon Rorb(+) remains within a 2-fold change. The average log- normalization expression (y-axis) is only used to spread data points. (j) From left to right, average log-normalized expression of a random set of genes (n = 500) for aPir, pPir, AI, and SSp, similar to (h). Expression patterns are qualitatively equivalent as in (h). (k) Difference of gene expression between set of random genes in aPir and SSp datasets remains within a 2-fold change and indicates that Rorb TGs in (i) behave as a set of random genes.

Extended Data Fig. 6 Cell type discreteness and TF coexpression and repression analyses.

(a) Kernel-density estimate plots showing cluster distance per cortical area computed on integrated clusters composed of glutamatergic neurons from all areas using highly variable transcription factors. Integration was performed between our data (aPir, pPir, AI, SSp) and cortical areas from Yao et al.17: SSp (as internal control to compare with our SSp dataset), primary motor cortex (MOp), visual cortex (VIS), and dentate gyrus (DG). (b) Top 500 positively (left) and negatively (right) correlated TF pairs projected from one cortical area (source, rows) to another (target, columns). Distances are normalized to be 1.0 when source is target. Lower distance indicates lower correlation match. TF-TF correlations were computed from log-normalized expression matrices for each area (see Supplementary Fig. 3c). (c) Repressive (top row) and activating (middle row) TF-TF interactions identified per cortical area e-GRN (aPir, pPir, SSp). AI was excluded for lack of laminar information. Repression and activation were determined using SCENIC + , which considered (anti)correlations for each e-regulon as computed between the TF, its target genes and its target CREs. Results in main text are based on an (anti)correlation value of 0.4 (red line) and minimum number of target genes 10. Only TFs expressed in at least 20% of neurons in a given cortical layer are considered. Gray area indicates bounds of pattern stability. (d) Activating and repressing ATAC regions recovered from leave-10%-out datasets. Each dot represents the ratio of regions in the leave-10%-out dataset over the original e-GRN, for both activating and repressing regions. Dots are colored by cortical area. To indicate the trend per cortical area, linear regressions (solid lines) with +/- bootstrapped 95% confidence intervals (shaded area) are shown. As a reference, the dashed black line is the equal ratio of recovery for activating and repressing regions. (e) Gene expression levels of predicted piriform repressors shown in Fig. 3i in aPir (top) and pPir (bottom). Neurons are labeled by cortical layer (SL and Pyr cells), neuron type (INs and Vglut2 cells), or “Unknown” if layer information was unavailable.

Extended Data Fig. 7 Adult piriform cortex immature neurons and the transcriptomic divergence of pyramidal cells between lab and wild mice and between human individuals.

(a) Left: distribution of piriform cortex mature and immature neurons in reanalyzed MERFISH data (Zhang et al.25). Right: cumulative distribution along the anterior–posterior axis of piriform cortex of all pyramidal cells, immature neurons, and pyramidal cells from Yao 2023 aligning with subtype Pyr7 (Yao et al.64; Zhang et al.25). (b) Classification accuracy of support vector machine (SVM) trained on combined RNA and ATAC data of the pPir dataset. Each neuron type is distinguished. (c) Same as (b) but applied to subtypes. Each neuron subtype is distinguished. (d) Conservation scores for aPir, pPir, and SSp. Scores are median probabilities of transcriptomic similarity between lab and wild datasets, quantified by considering all mature neurons, or neurons of a given cell type (SL, Pyr, Vglut2, and IN). A conservation score of 0.5 indicates perfect mixing between lab and wild datasets. 95% confidence intervals (CIs) for each neuron type are reported in brackets. (e) Statistical significance of pairwise differences in conservation score between cortices reported per neuron type (see also (d)). P-values are calculated using two-sided Mann–Whitney U-Tests and Bonferroni-corrected. P-values in red are smaller than α0.01(Bonf.)=8.34e-4, p values in yellow are (only) smaller than α0.05(Bonf.)=4.167e-3. (f) Similarity (as cosine distance) between lab and wild neuron types upon integrating lab and wild pPir datasets using scVI. Pyramidal cells were on average less similar to each other than the other neuron types. (g) Percentage of lab pPir neighbors in scVI integration when computing nearest neighbor graphs from 5 to 100. Data shown as mean +/- bootstrapped 95% CIs, for 7861 aligned (other Pyr cells) and 184 misaligned cells (wild-specific Pyr cells). Alignment of cells between lab and wild datasets is taken from the OT integration shown in (c). (h) Classification accuracy of SVM trained on combined lab and wild pPir data. Neuron types are distinguished with 98.8% accuracy. (i) Immunohistochemistry using lab (left) and wild-derived mice (right) of markers for piriform neuronal populations (in cyan), namely RELN for SL, CUX1 for Pyr, GABA for INs, co-stained with DCX (in magenta), a canonical marker for immature neurons. Inset white boxes with higher magnification show coexpression of CUX1 with DCX in both lab and wild-derived mice. Scale bar, 100 μm. (j) Conservation scores per neuron type for adult human piriform (PIR) and primary somatosensory cortex (S1C) computed using published adult human whole-brain sn-RNA-seq data (Siletti et al.41). Scores are computed per pair of three human donors. Abbreviated donor IDs: h18 for H.18.30.001, h19.1 for h19.30.001, and h19.2 for H19.30.002. Within violin plots, black circles mark medians and black bars indicate 95% CIs. Bonferroni-corrected significance thresholds were α0.01(Bonf.)=1.59e-4 and α0.05(Bonf.)=7.94e-4. IT: intratelencephalic; NP: near projecting (see Supplementary Table 1 for sample size, see (l) for statistical significance). (k) Conservation scores as in (d), but for human piriform (left) and primary somatosensory cortex (right). Scores are median probabilities of transcriptomic similarity per pair of donors. (l) Statistical significance of pairwise differences in conservation score as shown in (e), but between donors. P-values are calculated using two-sided Mann–Whitney U-Tests and reported per neuron type.

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Extended Data Fig. 8 Transcriptomically-defined cell types across cortical areas of wild-derived mice.

(a) Relative abundance of main cell types across biological replicates (donors) integrated from single-nucleus RNA (sn-RNA-seq) and multiome (sn-multiome seq) sequencing experiments. Replicates IDs 1 and 2 derive from sn-multiome seq experiments, replicates IDs 3, 4, 5 and 6 derive from sn-RNA-seq experiments. From left to right: aPir replicates, indicated by A; pPir replicates, indicated by P; SSp replicates, indicated by N. Numbers correspond to the ID of the donor mouse. W: wild. (b) Post-hoc histological assessment of aPir, pPir, and SSp dissections from anterior and posterior coronal sections of adult wild-derived mice ordered by ID mouse number. Asterisks indicate the microdissected area. Neurotrace counterstain in gray. (c) Left: number of genes per nucleus quantified for biological replicates. A indicates aPir replicates, P indicates pPir, N indicates SSp. Numbers correspond to the ID of mice. Right: number of genes per nucleus identified for main cell types. (d) Same as in (c) but for the fraction of mitochondrial content per nucleus. (e) From left to right: UMAPs of aPir, pPir, and SSp datasets color-coded by main cell types. (f) Gene expression levels of representative markers for each cell type across the three cortical areas, from left to right: aPir, pPir, and SSp. (g) Optimal transport (OT) alignment of main cell types between lab and wild datasets for each cortical area, from left to right: aPir, pPir, and SSp. Color and size of dots indicate the probability of alignment.

Extended Data Fig. 9 Quantification of co-clustering between cortical glutamatergic neurons of mice, reptiles, and salamander grouped by areas and LISI scores.

(a) Broad quantification of co-clustering in the integrated clusters between glutamatergic neurons from mouse and non-mammalian cortical areas, highlighting greater transcriptomic similarity of piriform glutamatergic neurons to those of non-mammals than to those of the neocortex. Pir: piriform; NCx: neocortex; DCtx, LCtx: dorsal, lateral cortex; aDVR: anterior dorsal ventricular ridge; dDP, LP, dVP: deep dorsal, lateral, deep ventral pallium. Rectangles indicate co-clustering of neurons (rows) in the integrated clusters (columns). Color of the rectangle represents the percentage of neurons in the integrated cluster. (b) Distribution of LISI scores across neuronal clusters of the datasets Tosches et al.45 (turtle and lizard), Hain et al.43 (lizard), and Woych et al.42 (salamander). A mean value close to 4 indicates a cell type that is well-mixed with neurons from other species, while a value close to 0 indicates a cell type mixed only with neurons from the same species. Box-and-whisker plots show min to max, center (median), 25th and 75th percentile box bounds, and whiskers extending to 1.5 * interquartile range (see Supplementary Table 1 for sample size).

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Extended Data Fig. 10 Cross-species gene module score and TF coexpression analyses.

(a) Expression enrichment of gene modules Pyr-like (top, blue) and SL-like (bottom, orange) in this study (left), in the salamander dataset (right), and in Yao., 2021 (middle). NTS: neurotensin neurons. A positive score indicates that the set of genes in the module are expressed in a particular cluster more highly compared to the average expression across all clusters of the dataset (see Supplementary Table 1 for sample size). (b) UMAPs of SL-like (top) and Pyr-like (bottom) gene module scores across datasets shown in (a), from left to right: this study, Yao et al.17, and Woych et a.l42. Color bar indicates the enrichment score of a module.

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Supplementary text: abbreviations, Additional Methods, Supplementary Figs. 1–6 and Supplementary Tables 1–3.

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Statistical source data for Figs. 4k and 5e and Extended Data Figs. 7f, 9b and 10a.

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Zeppilli, S., Gurrola, A.O., Demetci, P. et al. Single-cell genomics of the mouse olfactory cortex reveals contrasts with neocortex and ancestral signatures of cell type evolution. Nat Neurosci 28, 937–948 (2025). https://doi.org/10.1038/s41593-025-01924-3

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