scRNA-seq analysis of OEs from UNO-treated male mice shows reduced quantities of newborn OSNs of musk-responsive subtypes on the closed side of the OE relative to the open. A. scRNA-seq datasets that were used to quantify newborn OSNs of musk-responsive subtypes were generated from the open and closed side of the OE of a mouse that was UNO-treated at P14 and sacrificed at P28 30. B. t-SNE plot representation of the scRNA-seq datasets corresponding to the open (left) and closed (right) sides of the OE, showing Omp (mature OSNs, top) and Gap43 (immature OSNs, bottom) expression. C. Quantification of individual (lines) and average (bars) percentages of the OE cell population represented by immature (Gap43+) OSNs of musk-responsive subtypes (left) or randomly chosen zone 2/3 subtypes (right) within the open and closed datasets. D. Identification of OSNs of the 4 musk-responsive subtypes that are more highly represented within the open dataset compared to the closed. Green arrows: Gap43+ OSNs. See also Supplementary Fig. 2.

Histological analyses confirm that olfactory deprivation reduces quantities of newborn OSNs of musk-responsive subtypes in male mice. A. Experimental timeline for the analysis of open-side biases in quantities of newborn OSNs of musk-responsive and control subtypes in male mice that were UNO treated at P14, weaned sex-separated at P21, EdU-labeled at P28, sacrificed at P35, and analyzed via OR-specific RNA-FISH and EdU staining. B-G. Representative images (left) and quantification (right) of newborn OSNs (OR+/EdU+) of subtypes Olfr235 (B), Olfr1440 (C), Olfr1431 (D), Olfr1437 (E), Olfr912 (F), or Olfr1463 (G) within sections of OEs from UNO-treated and EdU-labeled male mice that were exposed to themselves at the time of EdU labeling (♂ ® ♂). OR+/EdU+ cells (yellow arrows) are newborn OSNs of the indicated subtypes. Scale bars: 150 µm. Musk-responsive and control subtypes are labeled in purple and gray type, respectively. Each line represents a distinct mouse (n = 4–10 mice/OSN subtype).

Olfactory deprivation-induced reductions in quantities of newborn Olfr235 OSNs depend on exposure to male odors. A, B. Experimental timeline and schematic for the analysis of open-side biases in quantities of newborn OSNs of musk-responsive and control subtypes in male mice that were UNO treated at P14, weaned sex-separated or sex-combined at P21, EdU-labeled at P28, sacrificed at P35, and analyzed via OR-specific FISH and EdU staining. C, D. Representative images of OE sections stained for EdU and Olfr235 from UNO-treated female mice exposed to themselves (♀ ® ♀) (C) or to male mice (♀ ® ♂) and (D) at the time of EdU-labeling. OR+/EdU+ cells (yellow arrows) are newborn Olfr235 OSNs. Scale bars: 150 µm. E-I. Quantification (left) and UNO effect sizes (right) of newborn OSNs (OR+/EdU+) of subtypes Olfr235 (E), Olfr1440 (F), Olfr1431 (G), Olfr912 (H), and Olfr1463 (I) within OEs of UNO-treated male mice exposed to themselves (♂ ® ♂), female mice exposed to themselves (♀ ® ♀), or female mice exposed to male mice (♀ ® ♂) at the time of EdU labeling. Musk-responsive and control subtypes are labeled in purple and gray type, respectively. Each line or circle represents a distinct mouse (n = 4–10 mice per OSN subtype and condition). See also Supplementary Fig. 3.

UNO-induced changes in newborn Olfr1431 OSN quantities are intensified by exposure adult mice. A, B. Experimental timeline and schematic for the analysis of open-side biases in quantities of newborn OSNs of specific subtypes in male mice that were UNO treated at P14, weaned sex-separated (♂ ® ♂) or kept with parents (♂ ® adult ♀ + ♂) at P21, EdU-labeled at P28, sacrificed at P35, and analyzed via OR-specific FISH and EdU staining. C, D. Representative images of OE sections stained for EdU and Olfr1431 (C) or Olfr912 (D) from UNO-treated male mice exposed to their parents (♂ ® adult ♀ + ♂) at the time of EdU-labeling. OR+/EdU+ cells (yellow arrows) are newborn OSNs of the indicated subtypes. Scale bars: 150 µm. E, F. Quantification (left) and UNO effect sizes (right) of newborn OSNs (OR+/EdU+) of subtypes Olfr1431 (E) or Olfr912 (F) within OEs of UNO-treated male mice exposed to themselves (♂ ® ♂) or their parents (♂ ® adult ♀ + ♂) at the time of EdU labeling. Musk-responsive and control subtypes are labeled in purple and gray type, respectively. Each line or circle represents a distinct mouse (n = 5–10 mice per OSN subtype and condition). See also Supplementary Fig. 4.

Exposure to muscone modulates deprivation-induced reductions in quantities of newborn OSNs of musk-responsive subtypes. A. Experimental timeline for the analysis of open-side biases in quantities of newborn OSNs of specific subtypes in female mice that were UNO treated at P14, weaned sex-separated at P21, exposed to varying concentrations of muscone starting at P21, EdU-labeled at P28, sacrificed at P35, and analyzed via OR-specific FISH and EdU staining. B. Representative image of an OE section stained for EdU and Olfr235 from a UNO-treated female mouse exposed to muscone (♀ ® 0.1% muscone) at the time of EdU-labeling. OR+/EdU+ cells (yellow arrows) are newborn Olfr235 OSNs. Scale bar: 150 µm. C–E. Quantification of newborn OSNs (OR+/EdU+) (left) and UNO effect sizes for newborn (middle) and total (right) OSNs of subtypes Olfr235 (C), Olfr1440 (D), or Olfr1431 (E) within OEs of UNO-treated female mice exposed to 0, 0.1, 1, or 10% muscone at the time of EdU labeling. Musk-responsive subtypes are labeled in purple type. Each line or circle represents a distinct mouse (n = 5–10 mice per OSN subtype and condition). See also Supplementary Fig. 5.

Exposure of non-occluded female mice to male odors or muscone induces elevated quantities of newborn OSNs of musk-responsive subtypes. A. Experimental timeline for the quantification of newborn OSNs of specific subtypes in non-occluded mice that were weaned sex-separated, sex-combined, or exposed to muscone starting at P21, EdU-labeled at P28, sacrificed at P35, and analyzed via OR-specific FISH and EdU staining. B. Representative images (left) of OE sections stained for Olfr235 mRNA and EdU, and quantification (right) of newborn (OR+/EdU+) Olfr235 OSNs within OEs from non-occluded females exposed to themselves (♀ ® ♀), males exposed to themselves (♂ ® ♂), females exposed to males (♀ ® ♂), or females exposed to muscone (♀ ® 0.1% muscone) at the time of EdU-labeling. C, D. Representative images (left) of OE sections stained for EdU and Olfr1440 (C) or Olfr1431 (D) mRNAs, and quantification (right) of newborn (OR+/EdU+) Olfr1440 (C) and Olfr1431 (D) OSNs within OEs of non-occluded females exposed to themselves (♀ ® ♀) or to muscone (♀ ® 0.1% muscone) at the time of EdU-labeling. E. Quantification of newborn (OR+/EdU+) Olfr912 OSNs within OEs from non-occluded females exposed to themselves (♀ ® ♀), males exposed to themselves (♂ ® ♂), females exposed to males (♀ ® ♂), or females exposed to muscone (♀ ® 0.1% muscone) at the time of EdU-labeling. OR+/EdU+ cells (yellow arrows) are newborn OSNs of the indicated subtype. Scale bars: 50 µm. Musk-responsive and control subtypes are labeled in purple and gray type, respectively. Each circle represents a distinct mouse (n = 4–10 mice per OSN subtype and condition).

Stimulation-dependent changes in newborn OSN quantities are observed immediately after neurogenesis and stable thereafter, consistent with a mechanism involving altered birthrate. A. Experimental timeline for analysis of the time-dependence of open-side biases in quantities of newborn OSNs of specific subtypes in mice that were UNO treated at P14, weaned sex-separated at P21, exposed to muscone (subset of mice) starting at P21, EdU-labeled at P28, sacrificed at P32 (4 d post-EdU) or P35 (7 d post-EdU), and analyzed via OR-specific FISH and EdU staining. B. Representative image of an OE section stained for EdU and Olfr235 from UNO-treated male mice exposed to themselves (♂ ® ♂) at the time of EdU-labeling and sacrificed 4 d post-EdU. OR+/EdU+ cells (yellow arrows) are newborn Olfr235 OSNs. Scale bar: 150 µm. C, D. Quantification of (left) and UNO effect sizes for (right) newborn Olfr235 OSNs (OR+/EdU+) within OEs of UNO-treated males exposed to themselves (♂ ® ♂) (C) or females exposed to muscone (♀ ® 0.1% muscone) (D) at the time of EdU labeling. Each line or circle represents a distinct mouse (n = 4–7 mice per OSN subtype and condition). E. Experimental timeline for analysis of the time-dependence of muscone exposure-induced increases in quantities of newborn OSNs of specific subtypes non-occluded mice that were weaned sex-separated at P21, exposed to muscone (subset of mice) starting at P21, EdU-labeled at P28, sacrificed at P32 (4 d post-EdU) or P35 (7 d post-EdU), and analyzed via OR-specific FISH and EdU staining. F. Representative images of OE sections stained for Olfr235 mRNA and EdU, from non-occluded females exposed to themselves (♀ ® ♀; left) or to muscone (♀ ® 0.1% muscone; right) at the time of EdU-labeling. Scale bars: 50 µm. G–I. Quantification of newborn (OR+/EdU+) Olfr235 (G), Olfr1440 (H), and Olfr1431 (I) OSNs within OEs of non-occluded females exposed to themselves (♀ ® ♀; black circles) or to muscone (♀ ® 0.1% muscone; purple circles) at the time of EdU-labeling. Musk-responsive subtypes are labeled in purple type. Each circle represents a distinct mouse (n = 4–10 mice per OSN subtype and condition). See also Supplementary Fig. 6.

Model for the selective acceleration of the birthrates of specific OSN subtypes by discrete odors that stimulate them. A fraction of OSN subtypes (e.g., Olfr235), upon stimulation by discrete odors (e.g., muscone), undergo accelerated rates of neurogenesis. Other subtypes (e.g., Olfr912) do not exhibit altered rates of neurogenesis upon stimulation by discrete odors that stimulate them (e.g., SBT). A hypothetical mechanism involves selective stimulation-dependent signaling by mature OSNs of specific subtypes to neural progenitors.

Identification of OSN subtypes that are candidates for undergoing sex-specific- and/or musk odor-accelerated neurogenesis. A. Experimental schematic for the identification of OSN subtypes whose representation depends on exposure to sex-specific odors. Datasets used in this study were generated from mice that were housed either sex-separated or sex-combined from weaning (P21) until the time of sacrifice (6 months), followed by OEs dissection and analysis via bulk RNA-seq 17,65. B. OSN subtypes previously identified as responsive to sex-specific odors and/or musk-like odors. SBT, 2-sec-butyl-4,5-dihydrothiazole; MTMT, (methylthio)methanethiol; †, 17,65; ‡, 32; #, 34; §, 35; ¶, 60.

scRNA-seq analysis of OEs from UNO-treated male mice shows reduced quantities of newborn OSNs of musk-responsive and/or male-biased subtypes on the closed side of the OE relative to the open. A. (top) t-SNE plot representation of scRNA-seq datasets corresponding to the open (left) and closed (right) sides of the OE, showing Gap43 (immature OSNs) expression 30. (bottom) Identification of immature OSNs of the 5 known musk-responsive subtypes (Olfr235, Olfr1440, Olfr1431, Olfr1434, Olfr1437) within the open and closed datasets. B. Quantification of individual (lines) and average (bars) percentages of the OE cell population represented by immature (Gap43+) OSNs of musk-responsive and/or male-biased subtypes (left) or known stimulation-independent subtypes (right) within the open and closed datasets. Related to Fig. 1.

Olfactory deprivation-induced reductions in quantities of newborn Olfr235 OSNs depend on exposure to male odors. A–E. Quantification (left) and UNO effect sizes (right) of total OSNs (OR+) of subtypes Olfr235 (A), Olfr1440 (B), Olfr1431 (C), Olfr912 (D), and Olfr1463 (E) within OEs of UNO-treated male mice exposed to themselves (♂ ® ♂), female mice exposed to themselves (♀ ® ♀), or female mice exposed to male mice (♀ ® ♂) at the time of EdU labeling. Musk-responsive and control subtypes are labeled in purple and gray type, respectively. Each line or circle represents a distinct mouse (n = 4–10 mice per OSN subtype and condition). Related to Fig. 3.

UNO-induced changes in quantities of newborn OSNs of specific subtypes as a function of the ages of mice within the odor environment. A–C. Quantification (left) and UNO effect sizes (right) of newborn OSNs (OR+/EdU+) of subtypes Olfr235 (A), Olfr1440 (B) or Olfr1437 (C) within OEs of UNO-treated male mice exposed to themselves (♂ ® ♂) or their parents (♂ ® adult ♀ + ♂) at the time of EdU labeling. Musk-responsive subtypes are labeled in purple type. Each line or circle represents a distinct mouse (n = 3–9 mice per OSN subtype and condition). Related to Fig. 4.

Effects of exposure to muscone on UNO-induced changes in the quantities of total and newborn OSNs of specific subtypes. A–C. Quantification of total OSNs (OR+) of subtypes Olfr235 (A), Olfr1440 (B), or Olfr1431 (C) within OEs of UNO-treated female mice exposed to 0, 0.1, 1, or 10% muscone at the time of EdU labeling. D–G. Quantification of (left) or UNO effect sizes for (right) newborn OSNs (OR+/EdU+) (D, F) or total OSNs (OR+) (E, G) of subtypes Olfr912 (D, E) or Olfr1463 (F, G) within OEs of UNO-treated female mice exposed to varying concentrations of muscone at the time of EdU labeling. Musk-responsive and control subtypes are labeled in purple and gray type, respectively. Each line or circle represents a distinct mouse (n = 5–10 mice per OSN subtype and condition). Related to Fig. 5.

Stimulation-dependent changes in newborn OSN quantities are observed immediately after neurogenesis and stable thereafter, consistent with a mechanism involving altered birthrate. A, B, D–G. Quantification of (left) and UNO effect sizes for (right) newborn Olfr1440 (A), Olfr1431 (B), Olfr912 (D, F), Olfr1463 (E), and Olfr235 (G) OSNs (OR+/EdU+) within OEs of UNO-treated males exposed to themselves (♂ ® ♂) (A, B, D, E), females exposed to muscone (♀ ® 0.1% muscone) (F), or females exposed to themselves (♀ ® ♀) (G) at the time of EdU labeling. C. Representative image of an OE section stained for EdU and Olfr235 mRNA from UNO-treated female mice exposed to muscone (♀ ® 0.1% muscone) at the time of EdU-labeling and sacrificed 4 d post-EdU. OR+/EdU+ cells (yellow arrows) are newborn Olfr235 OSNs. Scale bar: 150 µm. H. Quantification of newborn (OR+/EdU+) Olfr912 OSNs within OEs of non-occluded females exposed to themselves (♀ ® ♀; black circles) or to muscone (♀ ® 0.1% muscone; purple circles) at the time of EdU-labeling. Musk-responsive and control subtypes are labeled in purple and gray type, respectively. Each line or circle represents a distinct mouse (n = 2–10 mice per OSN subtype and condition). Related to Fig. 7.

Muscone exposure-dependent increases in quantities of newborn OSNs of musk-responsive subtypes persist into adulthood. A. Experimental timeline for the quantification of newborn OSNs of specific subtypes in non-occluded adult mice that were weaned sex-separated with or without muscone exposure starting at P21, EdU-labeled at P56–58, sacrificed at P65, and analyzed via OR-specific FISH and EdU staining. B–E. Representative images (left) of OE sections stained for EdU and Olfr235 (B), Olfr1440 (C), Olfr1431 (D), or Olfr912 (E) mRNAs, and quantification (right) of newborn (OR+/EdU+) Olfr235 (B), Olfr1440 (C), Olfr1431 (D), or Olfr912 (E) OSNs within OEs of non-occluded females exposed to themselves (♀ ® ♀) or to muscone (♀ ® 0.1% muscone) at the time of EdU-labeling. OR+/EdU+ cells (yellow arrows) are newborn Olfr235 OSNs. Scale bars: 150 µm. Musk-responsive and control subtypes are labeled in purple and gray type, respectively. Each circle represents a distinct mouse (n = 5–7 mice per OSN subtype and condition).

Summary of animals used.

Summary of FISH probes used.