In mice, discrete odors can selectively promote the neurogenesis of sensory neuron subtypes that they stimulate

Reviewer #1 (Public Review):

Summary:

Olfactory sensory neurons (OSNs) in the olfactory epithelium detect myriads of environmental odors that signal essential cues for survival. OSNs are born throughout life and thus represent one of the few neurons that undergo life-long neurogenesis. Until recently, it was assumed that OSN neurogenesis is strictly stochastic with respect to subtype (i.e. the receptor the OSN chooses to express).

However, a recent study showed that olfactory deprivation via naris occlusion selectively reduced birthrates of only a fraction of OSN subtypes and indicated that these subtypes appear to have a special capacity to undergo changes in birthrates in accordance with the level of olfactory stimulation. These previous findings raised the interesting question of what type of stimulation influences neurogenesis, since naris occlusion does not only reduce the exposure to potentially thousands of odors but also to more generalized mechanical stimuli via preventing airflow.

In this study, the authors set out to identify the stimuli that are required to promote the neurogenesis of specific OSN subtypes. Specifically, they aim to test the hypothesis that discrete odorants selectively stimulate the same OSN subtypes whose birthrates are affected. This would imply a highly specific mechanism in which exposure to certain odors can "amplify" OSN subtypes responsive to those odors suggesting that OE neurogenesis serves, in part, an adaptive function.

To address this question, the authors focused on a family of OSN subtypes that had previously been identified to respond to musk-related odors and that exhibit higher transcript levels in the olfactory epithelium of mice exposed to males compared to mice isolated from males. First, the authors confirm via a previously established cell birth dating assay in unilateral naris occluded mice that this increase in transcript levels actually reflects a stimulus-dependent birthrate acceleration of this OSN subtype family. In a series of experiments using the same assay, they show that one specific subtype of this OSN family exhibits increased birthrates in response to juvenile male exposure while a different subtype shows increased birthrates to adult mouse exposure. In the core experiment of the study, they finally exposed naris occluded mice to a discrete odor (muscone) to test if this odor specifically accelerates the birth rates of OSN types that are responsive to this odor. This experiment reveals a complex relationship between birth rate acceleration and odor concentrations showing that some muscone concentrations affect birth rates of some members of this family and do not affect two unrelated OSN subtypes.

Strengths:

The scientific question is valid and opens an interesting direction. The previously established cell birth dating assay in naris occluded mice is well performed and accompanied by several control experiments addressing potential other interpretations of the data.

Weaknesses:

(1) The main research question of this study was to test if discrete odors specifically accelerate the birth rate of OSN subtypes they stimulate, i.e. does muscone only accelerate the birth rate of OSNs that express muscone-responsive ORs, or vice versa is the birthrate of muscone-responsive OSNs only accelerated by odors they respond to?

This question is only addressed in Figure 5 of the manuscript and the results only partially support the above claim. The authors test one specific odor (muscone) and find that this odor (only at certain concentrations) accelerates the birth rate of some musk-responsive OSN subtypes, but not two other unrelated control OSN subtypes. This does not at all show that musk-responsive OSN subtypes are only affected by odors that stimulate them and that muscone only affects the birthrate of musk-responsive OSNs, since first, only the odor muscone was tested and second, only two other OSN subtypes were tested as controls, that, importantly, are shown to be generally stimulus-independent OSN subtypes (see Figure 2 and S2).

As a minimum the authors should have a) tested if additional odors that do not activate the three musk-responsive subtypes affect their birthrate b) choose 2-3 additional control subtypes that are known to be stimulus-dependent (from their own 2020 study) and test if muscone affects their birthrates.

(2) The finding that Olfr1440 expressing OSNs do not show any increase in UNO effect size under any muscone concentration (Figure 5D, no significance in line graph for UNO effect sizes, middle) seems to contradict the main claim of this study that certain odors specifically increase birthrates of OSN subtypes they stimulate. It was shown in several studies that olfr1440 is seemingly the most sensitive OR for muscone, yet, in this study, muscone does not further increase birthrates of OSNs expressing olfr1440. The effect size on birthrate under muscone exposure is the same as without muscone exposure (0%).

In contrast, the supposedly second most sensitive muscone-responsive OR olfr235 shows a significant increase in UNO effect size between no muscone exposure (0%) and 0.1% as well as 1% muscone.

(3) The authors introduce their choice to study this particular family of OSN subtypes with first, the previous finding that transcripts for one of these musk-responsive subtypes (olfr235) are downregulated in mice that are deprived of male odors. Second, musk-related odors are found in the urine of different species. This gives the misleading impression that it is known that musk-related odors are indeed excreted into male mouse urine at certain concentrations. This should be stated more clearly in the introduction (or cited, if indeed data exist that show musk-related odors in male mouse urine) because this would be a very important point from an ethological and mechanistic point of view.

In addition, this would also be important information to assess if the chosen muscone concentrations fall at all into the natural range.

Related: If these are male-specific cues, it is interesting that changes in OR transcripts (Figure 1) can already be seen at the age of P28 where other male-specific cues are just starting to get expressed. This should be discussed.

(4) Figure 5: Under muscone exposure the number of newborn neurons on the closed sides fluctuates considerably. This doesn't seem to be the case in other experiments and raises some concerns about how reliable the naris occlusion works for strong exposure to monomolecular odors or what other potential mechanisms are at play.

(5) In contrast to all other musk-responsive OSN types, the number of newborn OSNs expressing olfr1437 increases on the closed side of the OE relative to the open in UNO-treated male mice (Figure 1). This seems to contradict the presented theory and also does not align with the bulk RNAseq data (Figure S1).

(6) The authors hypothesize in relation to the accelerated birthrate of musk-responsive OSN subtypes that "the acceleration of the birthrates of specific OSN subtypes could selectively enhance sensitivity to odors detected by those subtypes by increasing their representation within the OE". However, for two other OSN subtypes that detect male-specific odors, they hypothesize the opposite "By contrast, Olfr912 (Or8b48) and Olfr1295 (Or4k45), which detect the male-specific non-musk odors 2-sec-butyl-4,5-dihydrothiazole (SBT) and (methylthio)methanethiol (MTMT), respectively, exhibited lower representation and/or transcript levels in mice exposed to male odors, possibly reflecting reduced survival due to overstimulation."

Without any further explanation, it is hard to comprehend why exposure to male-derived odors should, on one hand, accelerate birthrates in some OSN subtypes to potentially increase sensitivity to male odors, but on the other hand, lower transcript levels and does not accelerate birth rates of other OSN subtypes due to overstimulation.

Reviewer #2 (Public Review):

In their paper entitled "In mice, discrete odors can selectively promote the neurogenesis of sensory neuron subtypes that they stimulate" Hossain et al. address lifelong neurogenesis in the mouse main olfactory epithelium. The authors hypothesize that specific odorants act as neurogenic stimuli that selectively promote biased OR gene choice (and thus olfactory sensory neuron (OSN) identity). Hossain et al. employ RNA-seq and scRNA-seq analyses for subtype-specific OSN birthdating. The authors find that exposure to male and musk odors accelerates the birthrates of the respective responsive OSNs. Therefore, Hossain et al. suggest that odor experience promotes selective neurogenesis and, accordingly, OSN neurogenesis may act as a mechanism for long-term olfactory adaptation.

The authors follow a clear experimental logic, based on sensory deprivation by unilateral naris occlusion, EdU labeling of newborn neurons, and histological analysis via OR-specific RNA-FISH. The results reveal robust effects of deprivation on newborn OSN identity. However, the major weakness of the approach is that the results could, in (possibly large) parts, depend on "downregulation" of OR subtype-specific neurogenesis, rather than (only) "upregulation" based on odor exposure. While, in Figure 6, the authors show that the observed effects are, in part, mediated by odor stimulation, it remains unclear whether deprivation plays an "active" role as well. Moreover, as shown in Figure 1C, unilateral naris occlusion has both positive and negative effects in a random subtype sample.

Another weakness is that the authors build their model (Figure 8), specifically the concept of selectivity, on a receptor-ligand pair (Olfr912 that has been shown to respond, among other odors, to the male-specific non-musk odors 2-sec-butyl-4,5-dihydrothiazole (SBT)) that would require at least some independent experimental corroboration. At least, a control experiment that uses SBT instead of muscone exposure should be performed. In this context, it is somewhat concerning that some results, which appear counterintuitive (e.g., lower representation and/or transcript levels of Olfr912 and Olfr1295 in mice exposed to male odors) are brushed off as "reflecting reduced survival due to overstimulation." The notion of "reduced survival" could be tested by, for example, a caspase3 assay.
Important analyses that need to be done to better be able to interpret the findings are to present (i) the OR+/EdU+ population of olfactory sensory neurons not just as a count per hemisection, but rather as the ratio of OR+/EdU+ cells among all EdU+ cells; and (ii) to the ratio of EdU+ cells among all nuclei (UNO versus open naris). This way, data would be normalized to (i) the overall rate of neurogenesis and (ii) any broad deprivation-dependent epithelial degeneration.

Finally, the paper will benefit from improved data presentation and adequate statistical testing. Images in Figures 2 - 7, showing both EdU labeling of newborn neurons and OR-specific RNA-FISH, are hard to interpret. Moreover, t-tests should not be employed when data is not normally distributed (as is the case for most of their samples).

Reviewer #3 (Public Review):

Summary:

Neurogenesis in the mammalian olfactory epithelium persists throughout the life of the animal. The process replaces damaged or dying olfactory sensory neurons. It has been tacitly that replacement of the OR subtypes is stochastic, although anecdotal evidence has suggested that this may not be the case. In this study, Santoro and colleagues systematically test this hypothesis by answering three questions: is there enrichment of specific OR subtypes associated with neurogenesis? Is the enrichment dependent on sensory stimulus? Is the enrichment the result of differential generation of the OR type or from differential cell death regulated by neural activity? The authors provide some solid evidence indicating that musk odor stimulus selectively promotes the OR types expressing the musk receptors. The evidence argues against a random selection of ORs in the regenerating neurons.

Strengths:

The strength of the study is a thorough and systematic investigation of the expression of multiple musk receptors with unilateral naris occlusion or under different stimulus conditions. The controls are properly performed. This study is the first to formulate the selective promotion hypothesis and the first systematic investigation to test it. The bulk of the study uses in situ hybridization and immunofluorescent staining to estimate the number of OR types. These results convincingly demonstrate the increased expression of musk receptors in response to male odor or muscone stimulation.

Weaknesses:

A major weakness of the current study is the single-cell RNASeq result. The authors use this piece of data as a broad survey of receptor expression in response to unilateral nasal occlusion. However, several issues with this data raise serious concerns about the quality of the experiment and the conclusions. First, the proportion of OSNs, including both the immature and mature types, constitutes only a small fraction of the total cells. In previous studies of the OSNs using the scRNASeq approach, OSNs constitute the largest cell population. It is curious why this is the case. Second, the authors did not annotate the cell types, making it difficult to assess the potential cause of this discrepancy. Third, given the small number of OSNs, it is surprising to have multiple musk receptors detected in the open side of the olfactory epithelium whereas almost none in the closed side. Since each OR type only constitutes ~0.1% of OSNs on average, the number of detected musk receptors is too high to be consistent with our current understanding and the rest of the data in the manuscript. Finally, unlike the other experiments, the authors did not describe any method details, nor was there any description of quality controls associated with the experiment. The concerns over the scRNASeq data do not diminish the value of the data presented in the bulk of the study but could be used for further analysis.

A weakness of the experiment assessing musk receptor expression is that the authors do not distinguish immature from mature OSNs. Immature OSNs express multiple receptor types before they commit to the expression of a single type. The experiments do not reveal whether mature OSNs maintain an elevated expression level of musk receptors.

There are also two conceptual issues that are of concern. The first is the concept of selective neurogenesis. The data show an increased expression of musk receptors in response to male odor stimulation. The authors argue that this indicates selective neurogenesis of the musk receptor types. However, it is not clear what the distinction is between elevated receptor expression and a commitment to a specific fate at an early stage of development. As immature OSNs express multiple receptors, a likely scenario is that some newly differentiated immature OSNs have elevated expression of not only the musk receptors but also other receptors. The current experiments do not distinguish the two alternatives. Moreover, as pointed out above, it is not clear whether mature OSNs maintain the increased expression. Although a scRNASeq experiment can clarify it, the authors, unfortunately, did not perform an in-depth analysis to determine at which point of neurogenesis the cells commit to a specific musk receptor type. The quality of the scRNASeq data unfortunately also does not lend confidence for this type of analysis.

A second conceptual issue, the idea of homeostasis in regeneration, which the authors presented in the Introduction, needs clarification. In its current form, it is confusing. It could mean that a maintenance of the distribution of receptor types, or it could mean the proper replacement of a specific OR type upon the loss of this type. The authors seem to refer to the latter and should define it properly.