We have generated single cell transcriptomic atlases of vomeronasal organs (VNO) from juvenile and adult mice. Combined with spatial molecular imaging, we uncover a distinct, previously unidentified class of cells that express the vomeronasal receptors (VRs) and a population of canonical olfactory sensory neurons in the VNO. High-resolution trajectory and cluster analyses reveal the lineage relationship, spatial distribution of cell types, and a putative cascade of molecular events that specify the V1r, V2r, and OR lineages from a common stem cell population. The expression of vomeronasal and olfactory receptors follow power law distributions, but there is high variability in average expression levels between individual receptor and cell types. Substantial co-expression is found between receptors across clades, from different classes, and between olfactory and VRs, with nearly half from pairs located on the same chromosome. Interestingly, the expression of V2r, but not V1r, genes is associated with various transcription factors, suggesting distinct mechanisms of receptor choice associated with the two cell types. We identify association between transcription factors, surface axon guidance molecules, and individual VRs, thereby uncovering a molecular code that guides the specification of the vomeronasal circuitry. Our study provides a wealth of data on the development and organization of the accessory olfactory system at both cellular and molecular levels to enable a deeper understanding of vomeronasal system function.

Transcription often occurs in bursts as gene promoters switch stochastically between active and inactive states. Enhancers can dictate transcriptional activity in animal development through the modulation of burst frequency, duration, or amplitude. Previous studies observed that different enhancers can achieve a wide range of transcriptional outputs through the same strategies of bursting control. For example, in Berrocal et al., 2020, we showed that despite responding to different transcription factors, all even-skipped enhancers increase transcription by upregulating burst frequency and amplitude while burst duration remains largely constant. These shared bursting strategies suggest that a unified molecular mechanism constraints how enhancers modulate transcriptional output. Alternatively, different enhancers could have converged on the same bursting control strategy because of natural selection favoring one of these particular strategies. To distinguish between these two scenarios, we compared transcriptional bursting between endogenous and ectopic gene expression patterns. Because enhancers act under different regulatory inputs in ectopic patterns, dissimilar bursting control strategies between endogenous and ectopic patterns would suggest that enhancers adapted their bursting strategies to their trans-regulatory environment. Here, we generated ectopic even-skipped transcription patterns in fruit fly embryos and discovered that bursting strategies remain consistent in endogenous and ectopic even-skipped expression. These results provide evidence for a unified molecular mechanism shaping even-skipped bursting strategies and serve as a starting point to uncover the realm of strategies employed by other enhancers.