Fig 1.
snRNA-seq atlas of the juvenile mouse primary whisker somatosensory cortex (wS1).
(A) Schematic of the mouse whisker somatosensory system, including the facial whisker pad and the whisker somatosensory cortex (wS1). wS1 contains a somatotopic map of the whisker pad in which individual whiskers are represented by neural activity within barrel columns of the cortex. (B) Experimental design and developmental timeline for snRNA-seq profiling of one reference (control) dataset at P22 and three experimental conditions: an earlier time during development (P12) and following two different whisker deprivation paradigms at P22. RWD, row-whisker deprivation. AWD, all-whisker deprivation. (C) General experimental and computational workflow for snRNA-seq profiling and subsequent confirmatory studies. Created in BioRender. Shekhar, K. (2025) https://BioRender.com/6wszljc. (D) Representation of cortical neuron diversity explored in this study highlighting the three taxonomic levels: classes, subclasses, and types. Created in BioRender. Shekhar, K. (2025) https://BioRender.com/kxs5b3n. (E) Tracksplot showing marker genes (rows) for each neuronal subclass (columns). Data was aggregated from 81,462 nuclei across all four conditions and each subclass was subsampled to the size of the smallest subclass for plotting purposes. (F) Relative frequencies of neuronal subclasses are highly consistent across biological replicates and experimental conditions. The highest variance is seen for L6b glutamatergic neurons, whose frequency ranges from 0.1% to 1% of all neurons. Each biological replicate contains cells derived from three mice (see Materials and methods, S1 Data).
Fig 2.
Gene expression changes between P12 and P22 are enriched in neurodevelopmental processes.
(A) Overview of analyses to classify genes based on subclass variability and temporal differential expression. P12 contains two biological replicates, and P22 contains four biological replicates. Each replicate contains cells from three mice. (B) Scatter plot of subclass variability (SV) scores and temporal differential expression (tDE) scores of genes in glutamatergic (left) and GABAergic neurons (right). Scores along each axis are capped at the value of 2 (S1 Data). (C) Same as B for glutamatergic neurons with four gene categories highlighted. Subpanels (clockwise, starting from top left) correspond to cell adhesion molecules (CAMs), transcription factors (TFs), housekeeping genes (HKs), and ion channels (ICs). Boxes on the right of each panel list the odds ratio (OR) and adjusted p-value (Padj) for the enrichment of the corresponding gene category in each quadrant (Fisher’s exact test). Gray values indicate neither enrichment nor depletion, while red and green indicate depletion and enrichment, respectively (see Materials and methods, S1 Data for details). (D) Q1 is enriched in gene ontology (GO) programs associated with neurodevelopment, while Q3 is enriched in general housekeeping processes (S1 Data). (E) Stacked violin plot of example genes (rows) from Q1 with the highest tDE score in L2/3. Columns correspond to subclasses at P12 and P22, violins represent the expression distribution, and color represents median expression. Genes are colored according to the functional categories as in panel C (S1 Data). (F) FISH for tDE genes. Representative images (top) from an ‘across-row’ (see Materials and methods) section in wS1. Cortical layers 2/3 and 4 are indicated on the sections. Quantification (bottom) of mean intensity in nuclei revealed strong temporal regulation of gene expression in L2/3 consistent with what was measured with snRNA-seq (see Fig 2E). Mann–Whitney test, ****p < 0.0001. L2/3_Col19a1: n = 1,390, 1,042 nuclei, L4_Col19a1: n = 1,028, 813 nuclei, L2/3_Sorcs3: n = 1,455, 1,102, L4_Sorcs3: n = 1,021, 813 L2/3_Gabrg3: n = 1,433, 1,311, L4_Gabrg3: n = 1,026, 812. Three mice per condition, two slices per mouse (S1 Data).
Fig 3.
L2/3 pyramidal cell type composition and gene expression are selectively regulated during development.
(A) Schematic for transferring P22 cell type labels (reference data) to P12 cells to facilitate cell type-level comparisons. (B) Within glutamatergic neurons (~80% of all neurons), all cell types except for L2/3_A and L2/3_B have approximately the same relative frequency between P12 and P22. Pearson correlation coefficient between the relative frequencies is indicated on top (S1 Data). (C) Scatter plot of PC1 vs. PC2 for L2/3 neurons at P12 (top row) and P22 (bottom row). Within each row, the leftmost panel highlights cells colored by their type-identity (L2/3_A, L2/3_B, and L2/3_C). In the remaining three panels within each row, cells are colored based on their aggregate expression levels of markers for each type (see Materials and methods). Between P12 and P22, L2/3_A markers decrease in expression and increase in specificity, while L2/3_B markers increase in expression during development, driving cell type identity maturation (S1 Data). (D) Representative FISH images showing labeling of cell type markers Cdh13, Trpc6, and Chrm2 at P12 and P22 within wS1 L2/3. Arrowheads indicate putative Cdh13-expressing interneurons (See S5A Fig). (E) Summary plots based on overlay of all images of L2/3 visualizing expression of Cdh13, Trpc6, and Chrm2 at P12 and P22. Circles represent individual excitatory cells within L2/3, colored based on their expression of one or more marker genes (color code as in panel E). To the right of each summary plot is the kernel density estimate for each type along the pial-ventricular axis. Cdh13+ cells dominate in upper L2/3 at P12, whereas Trpc6+ cells are more abundant at P22. N = 10–12 slices from 3 mice per time point. (F) Quantification of the fraction of excitatory (Slc17a7+) L2/3 cells expressing one or more markers Cdh13, Trpc6, and Chrm2 at P12 and P22. N = 10–12 slices from 3 mice per time point (S1 Data).
Fig 4.
Comparative transcriptomic analysis of wS1 and V1.
(A) Heatmap highlighting the overlap of tDE genes between all pairs of wS1 subclasses (P12 vs. P22; rows) and V1 subclasses (P14 vs. P21; columns). The left and right panels correspond to downregulated and upregulated genes. Values correspond to Bonferroni-corrected -log10(Padj) values from a hypergeometric test of overlap of tDE genes. The background set for these tests was the set of all the genes regulated in any subclass. Except for L4, subclasses with fewer than 10 tDE genes (S2C and S7A Figs) showed little to no overlap. The value Padj = 0.05 is highlighted on the scalebar (right). (B) Top 5 GO terms from shared downregulated and upregulated genes in V1 and wS1 in corresponding subclasses from panel A. See S7C Fig for a full list (S1 Data). (C) Examples of shared genes that are temporally downregulated (left) and upregulated (right) in L2/3 neurons between V1 and wS1 (S1 Data). (D) Scatter plots showing highly similar relative frequencies between matched cell types (see S7D Fig) across V1 and wS1 (S1 Data).
Fig 5.
Full-face 10-day all-whisker deprivation (10d AWD) does not influence cell type maturation.
(A) All whisker deprivation is conducted for 10 days, from P12 to P22 (10-d AWD). All whiskers on both sides of the face are plucked, then checked every other day and plucked if there is regrowth. Controls are sham mice, which are anesthetized the same amount of time as 10d AWD but not plucked. (B) Glutamatergic neuronal types have approximately the same relative frequency in P22 10d AWD (y-axis) and normal P22 (x-axis) (S1 Data). (C) PC1 vs. PC2 scatter plot for L2/3 neurons at P22 10d AWD (top) and normal P22 (bottom), highlighting the location of types and the type-specific marker scores. Representation as in Fig 3C. Scores are similar between the two datasets (see also S8D Fig) (S1 Data). (D) L2/3 markers genes, as in S4C Fig, are shown as a function of cells’ position along PC1 comparing patterns between P22 with normal experience and P22 10d AWD (S1 Data).
Fig 6.
Brief row whisker deprivation (1d RWD) upregulates activity-dependent gene expression programs in deprived columns.
(A) Schematic representation of the 1d row-whisker deprivation (RWD) manipulation and ‘across-row’ S1 section in which all barrel columns are identifiable. A representative image of DAPI labeling in ‘across-row’ S1 section. (B) Dotplots of genes upregulated by 1d RWD in glutamatergic subclasses (panels). Within each panel, rows indicate condition and columns indicate genes. The size of each circle corresponds to the % of cells with nonzero expression, and the color indicates average expression level (S1 Data). (C) Representative wide-field images of wS1. Barrels and barrel fields are indicated with light gray rectangles and labeled. Boxes indicate the locations of ROIs used for analysis in G. (D) Npas4 intensity inside Slc17a7+ L2/3 excitatory cells is increased after 1d RWD. Violin plots show median (dashed line) and quartiles (dotted lines) across individual cells. Mann–Whitney test, p < 0.0001 ****, n = 1,852, 1,699 cells, respectively, from 5 mice per group (S1 Data). (E) Junb intensity inside Slc17a7+ L2/3 cells is increased after 1d RWD. Mann–Whitney test, p < 0.0001 ****, n = 1,864, 1,703 cells, respectively, from 5 mice per group (S1 Data). (F) Btg2 intensity inside Slc17a7+ L2/3 cells is increased after 1d RWD. Mann–Whitney test, p < 0.0001 ****, n = 1,836, 1,706 cells respectively, from 5 mice per group (S1 Data). (G) L2/3 C and D columns were compared to examine whether gene expression changes are specific to the deprived (D) column. Pseudo-colored outlines of Npas4, Junb, and Btg2 expressing cells. Each plot is an overlay of 5 images of L2/3 S1 from 3 mice. Only Slc17a7+ cells are shown. (H) Quantification of column-specific gene expression changes in 1d RWD. Plotted values are the difference between the fraction of Npas4/Junb/Btg2 expressing cells among all excitatory (Slc17a7+) cells in the L2/3 D column and the corresponding fraction in the L2/3 C column. Symbols represent individual sham or 1d RWD mice. For each condition, mean and SEM (error bars) are shown. Two-way RM-ANOVA, Whisker Experience: F(1,8) = 11.41, **p = 0.0097 with Šidák correction for multiple comparisons, Npas4: **p = 0.002. JunB: n.s., p = 0.20. Btg2: *p = 0.014 (S1 Data).