A functional cellular framework for sex and estrous cycle-dependent gene expression and behavior

Abstract

Sex hormones exert a profound influence on gendered behaviors. How individual sex hormone-responsive neuronal populations regulate diverse sex-typical behaviors is unclear. We performed orthogonal, genetically targeted sequencing of four estrogen receptor 1-expressing (Esr1⁺) populations and identified 1,415 genes expressed differentially between sexes or estrous states. Unique subsets of these genes were distributed across all 137 transcriptomically defined Esr1⁺ cell types, including estrous stage-specific ones, that comprise the four populations. We used differentially expressed genes labeling single Esr1⁺ cell types as entry points to functionally characterize two such cell types, BNSTpr^Tac1/Esr1 and VMHvl^Cckar/Esr1. We observed that these two cell types, but not the other Esr1⁺ cell types in these populations, are essential for sex recognition in males and mating in females, respectively. Furthermore, VMHvl^Cckar/Esr1 cell type projections are distinct from those of other VMHvl^Esr1 cell types. Together, projection and functional specialization of dimorphic cell types enables sex hormone-responsive populations to regulate diverse social behaviors.

Keywords: aggression; deep sequencing; estrous cycle; maternal behavior; mating; menstrual cycle; sex differences; social behaviors; synaptic plasticity; transcriptomically defined cell types.

Figure 1:. TRAPseq identification of sex differences in gene expression.

A. Schematic of TRAPseq workflow. B. Scatter plots of sDEGs in different Esr1⁺ populations. Dots represent DEGs with >1.5-fold change and FDR-adjusted p value < 0.05 (colored) or genes that did not meet both criteria (gray). Colored numbers show number of sDEGs upregulated in that condition and comparison for each region and (far right) for all regions combined. In total, TRAPseq identified 890 and 651 sDEGs in M v F_R and M v F_NR comparisons. v, versus; TPM, transcripts/million. n = 3/condition. C. ISH for sDEGs in coronal sections through regions enclosed within red dotted areas on Nissl-stained sections on left. ISHs confirm TRAPseq data showing higher expression of Pappa, Auts2, Criml, and Nripl in M and other genes upregulated in F_R or F_NR mice. Heatmap at far right shows scaled expression of DEGs that were visualized by ISH. Scale = mean z-scored expression of DEGs centered at zero for individual comparisons between conditions (sex or estrous state) and regions; analogous heatmaps provided for ISH studies in all figures. Coronal plane as well as dorsoventral and mediolateral orientations preserved for histological panels in all figures. n = 2/condition/probe. Red, mRNA; blue, DAPI. Scale bars = 100 μm. See also Fig. S1.

Figure 2:. TRAPseq identification of estrous stage-dependent differences in gene expression.

A. Scatter plots of eDEGs in different Esr1⁺ populations. Dots represent DEGs with >1.5-fold change and FDR-adjusted p value < 0.05 (colored) or genes that did not meet both criteria (gray). Colored numbers show number of eDEGs upregulated in that condition and comparison for each region and (far right) for all regions combined. In total, TRAPseq identified 770 eDEGs. n = 3. B. ISH for eDEGs. ISHs confirm TRAPseq data showing higher expression of Pgr15l in F_NR and other genes upregulated in F_R mice. n = 2/condition/probe. Scale bars = 100 μm. C. Heatmap of p values of individual DEGs for the relevant pairwise comparison (with darker green colors indicating more significant p values) illustrating that most DEGs are restricted to one Esr1⁺ population. D. Heatmap of p values of individual DEGs for the relevant pairwise comparison (with darker green colors indicating more significant p values) illustrating that most DEGs within an Esr1⁺ population are specific to one comparison between sexes or estrous states. See also Fig. S2.

Figure 3:. ASD-association and distribution of DEGs in vivo.

A. Heatmap of expression of ASD risk-conferring DEGs. Scale = z-scored expression of DEGs centered at zero for individual comparisons between conditions (sex or estrous state) and regions. See Fig. 1C for heatmap and ISH for Auts2, an ASD-risk conferring DEG. B. ISH for ASD-risk conferring DEGs confirm TRAPseq data showing upregulation of Tcf4, Ptchdl, Sox5, Rfx3, and Ube3a in males. n = 2/condition/probe. Scale bar = 100 μm. C. ISHs for DEGs show that they are distributed in a few (sparse) or most (dense) Esr1⁺ neurons in each region. Quantification of these ISHs revealed that ‘sparse’ and ‘dense’ DEGs were expressed in ≤10% and ≥70% of Esr1⁺ cells. n = 2/condition/probe. Scale bar = 100 μm. See also Fig. S3.

Figure 4:. Categorizing Esr1⁺ populations into cell types with snRNAseq.

A-D. Violin plots classifying Esr1⁺ cell types (rows) by virtue of expression of sex hormone receptors, major neuronal neurotransmitter type (excitatory and inhibitory), and enriched genes in the BNSTpr (A), MeA (B), POA (C), and VMHvl (D). CPM, counts per million. n = 10,392 (BNSTpr), 15,929 (MeA), 17,784 (POA), and 5671 (VMHvl) Esr1+ neurons. See also Fig. S4 and Tables S5, S6.

Figure 5:. Distributive representation of DEGs across Esr1⁺ cell types.

A. UMAP representation of cell types in Esr1⁺ populations, with numbers corresponding to those in Fig. 4. B. UMAP representation of Esr1⁺ cell types by sex or estrous state shows that most of them are proportionally equivalently distributed. C. UMAP representation of excitatory and inhibitory cell types. D. UMAP representation shows that each cell type expresses ≥20% of DEGs in the respective Esr1⁺ population. E. UMAP representation with scaled distribution of DEGs. Individual cell types express both sDEGs and eDEGs, with different cell types expressing proportionally more DEGs upregulated in a given condition (sex or estrous stage). Gray cell types (scaled value = “0”) express equivalent proportion of DEGs upregulated in both conditions being compared. See also Fig. S5 and Table S2.

Figure 6:. The BNSTpr^Tac1/Esr1 cell type, among all BNSTpr^Esr1 cell types, is essential for sex recognition, mating, and aggression in males.

A. Violin plots of a subset of DEGs in the BNSTpr^Tac1/Esr1 cell type. Table S7 lists all DEGs in this cell type. B. Salt and pepper distribution of Tac1 mRNA in Esr1⁺ BNSTpr neurons. Scale bar = 100 μm. C. Schematic of intersectional chemogenetic strategy to inhibit BNSTpr^Tac1/Esr1 (top) or BNSTpr^Tac1−/Esr1 cell types. D. Inhibition of the BNSTpr^Tac1/Esr1 cell type, but not BNSTpr^Tac1−/Esr1 cell types, abolishes male preference for female urine. E. Inhibition of the BNSTpr^Tac1/Esr1 cell type reduces the probability of resident males initiating mating toward receptive females as well as the number of mount or intromission events/test. F. Inhibition of the BNSTpr^Tac1/Esr1 cell type reduces the probability of resident males attacking intruder males as well as the number of attacks/test. G. Inhibition of BNSTpr^Tac1−/Esr1 cell types does not alter mating of resident males with receptive females. H. Inhibition of BNSTpr^Tac1−/Esr1 cell types does not alter aggression of resident males toward intruder males. Mean ± SEM. n = 2 (B), 8 (D-H). *p < 0.05, **p < 0.01, ***p < 0.001. See also Fig. S6.

Figure 7:. The VMHvl^Cckar/Esr1 and VMHvl^{Cckar−/Esr1} cell types are required for female mating and maternal aggression, respectively.

A. Violin plot showing FR-specific expression of Cckar in the VMHvl^Cckar/Esr1 cell type (#20). B. Representative eDEGs significantly enriched in VMHvl^Cckar/Esr1 cell type compared to VMHvl^{Cckar−/Esr1} cell types. C. Cckar expression is restricted to the ventrolateral component of the Esr1⁺ VMHvl population, in agreement with previous work (Hashikawa et al., 2017; Xu et al., 2012). Scale bar = 100 μm. D. Schematic of intersectional chemogenetic strategy to inhibit VMHvl^Cckar/Esr1 (top) or VMHvl^{Cckar−/Esr1} (bottom) cell types. E. Inhibition of VMHvl^Cckar/Esr1 cell type in Fr mice significantly reduces lordosis, receptivity index and increases rejection behavior. Lordosis quotient, (lordosis events/# of mounts); Receptivity index (# of intromissions/# of mounts). F. Inhibition of the VMHvl^{Cckar−/Esr1}, but not VMHvl^Cckar/Esr1, cell types abrogates maternal aggression. G. Inhibition of VMHvl^{Cckar−/Esr1} cell types in F_R mice does not disable mating behavior. H. Schematic of strategy to label presynaptic termini of VMHvl^Cckar/Esr1 and Cckar⁻ neurons. I. Higher density of mCherry⁺ termini of VMHvl^Cckar/Esr1 neurons in AVPV of F_R compared to F_NR mice. For all findings related to mCherry⁺ termini in this and subsequent Figures, density of termini is normalized to the number of mCherry⁺ soma in the VMHvl as described previously (Inoue et al., 2019). J. No change in density of mCherry⁺ termini of VMHvl^Cckar− neurons in AVPV between F_R and F_NR mice. Mean ± SEM. n = 2 (B), 6 (F), ≥7 (G), 8 (H), ≥7 (J), and 6 (K). *p < 0.05. See also Fig. S7 and Table S7.