Sociosexual behavior requires both activating and repressive roles of Tfap2e/AP-2ε in vomeronasal sensory neurons
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Evaluation Summary:
This paper studies how pheromone-sensing neurons of the vomeronasal organ establish and maintain cell identity. A role for a particular transcription factor, AP-2e, is investigated using targeted gene knockout and rescue, and observations reveal an unexpected plasticity in the cell identity of mature sensory neurons. This paper should be of broad interest to neuroscientists and cell biologists studying mechanisms of cell development and differentiation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Neuronal identity dictates the position in an epithelium, and the ability to detect, process, and transmit specific signals to specified targets. Transcription factors (TFs) determine cellular identity via direct modulation of genetic transcription and recruiting chromatin modifiers. However, our understanding of the mechanisms that define neuronal identity and their magnitude remain a critical barrier to elucidate the etiology of congenital and neurodegenerative disorders. The rodent vomeronasal organ provides a unique system to examine in detail the molecular mechanisms underlying the differentiation and maturation of chemosensory neurons. Here, we demonstrated that the identity of postmitotic/maturing vomeronasal sensory neurons (VSNs), and vomeronasal-dependent behaviors can be reprogrammed through the rescue of Tfap2e/ AP-2ε expression in the Tfap2e Null mice, and partially reprogrammed by inducing ectopic Tfap2e expression in mature apical VSNs. We suggest that the TF Tfap2e can reprogram VSNs bypassing cellular plasticity restrictions, and that it directly controls the expression of batteries of vomeronasal genes.
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Evaluation Summary:
This paper studies how pheromone-sensing neurons of the vomeronasal organ establish and maintain cell identity. A role for a particular transcription factor, AP-2e, is investigated using targeted gene knockout and rescue, and observations reveal an unexpected plasticity in the cell identity of mature sensory neurons. This paper should be of broad interest to neuroscientists and cell biologists studying mechanisms of cell development and differentiation.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Lin et al investigate the role of AP-2e in vomeronasal sensory neurons through targeted gene deletion and rescue. They report that knockout of AP-2e reduces expression of basal markers, while induced expression can rescue basal identity. Moreover, forced expression of AP-2e in mature apical neurons causes them to express some basal markers. While it would be interesting if apical sensory neurons could be reprogrammed, additional evidence regarding shifts in receptor expression is needed before these conclusions can be made.
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Reviewer #2 (Public Review):
The manuscript by Forni and colleagues explores the fascinating question of cell fate plasticity by investigating the role of transcription factor AP-2epsilon at vomeronasal sensory neuron (VSN) specification. Building up upon their previous publication, this group defines the exact developmental timing at which VSNs adopt apical Vmn1R-specific expression programs or basal Vmn2R-specific programs, using scRNA-seq and elegant genetic manipulations. Having previously shown a role of AP-2epsilon in specifying the Vmn2R branch of VSN differentiation, they ask whether ectopically expressing AP-2epsilon in fully differentiated apical VSNs can transform them into basal OSNs (i.e. if they can force Vmn1R neurons to become Vmn2R neurons). Considering that the branching between the two VSN fates is a bona fide …
Reviewer #2 (Public Review):
The manuscript by Forni and colleagues explores the fascinating question of cell fate plasticity by investigating the role of transcription factor AP-2epsilon at vomeronasal sensory neuron (VSN) specification. Building up upon their previous publication, this group defines the exact developmental timing at which VSNs adopt apical Vmn1R-specific expression programs or basal Vmn2R-specific programs, using scRNA-seq and elegant genetic manipulations. Having previously shown a role of AP-2epsilon in specifying the Vmn2R branch of VSN differentiation, they ask whether ectopically expressing AP-2epsilon in fully differentiated apical VSNs can transform them into basal OSNs (i.e. if they can force Vmn1R neurons to become Vmn2R neurons). Considering that the branching between the two VSN fates is a bona fide developmental decision orchestrated by classical Notch-Delta fate decisions, these series of experiments are quite intriguing and of general value. To answer this question, the authors use Omp-Cre, a driver that is activated in apical VSNs (and in olfactory neurons) upon complete maturation. Strikingly they report that ectopic expression of AP-2epsilon results in ectopic expression of Vmn2Rs in the apical VSNs and ectopic expression of their signaling components (Gao). However, despite this apparent molecular transformation, there are no apparent behavioral changes in sociosexual behavioral tests. Finally, the authors perform CUT&RUN analysis in the VSNs with ectopic AP-2epsilon expression to decipher direct from indirect effects. In general, I find the experiments well designed, rigorously executed, and producing results of general interest (meaning beyond olfaction). The authors should expand this study with some experiments that will clarify why this molecular transformation does not alter sociosexual behaviors and perform a few control experiments for the CUT&RUN assays. They should also clarify if the Vmn1R genes and signaling components are still expressed in the transformed neurons.
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Reviewer #3 (Public Review):
Animals are able to detect an almost unlimited number of odorants or pheromones in the environment for their survival and reproduction. This extraordinary capacity is achieved by hundreds to thousands of olfactory receptors from different receptor families expressed in olfactory sensory neurons. Accordingly, the olfactory sensory neurons can be classified into distinct neuronal subpopulations based on the expressed receptors. How the distinct neuronal subpopulations are generated and maintained is still a mystery. Here, Lin et al. utilized the vomeronasal system, a specialized olfactory subsystem in rodents, as a model to answer this important question. The vomeronasal epithelium are composed of two main types of vomeronasal sensory neurons (VSNs), apical and basal VSNs, which express V1Rs and V2Rs, …
Reviewer #3 (Public Review):
Animals are able to detect an almost unlimited number of odorants or pheromones in the environment for their survival and reproduction. This extraordinary capacity is achieved by hundreds to thousands of olfactory receptors from different receptor families expressed in olfactory sensory neurons. Accordingly, the olfactory sensory neurons can be classified into distinct neuronal subpopulations based on the expressed receptors. How the distinct neuronal subpopulations are generated and maintained is still a mystery. Here, Lin et al. utilized the vomeronasal system, a specialized olfactory subsystem in rodents, as a model to answer this important question. The vomeronasal epithelium are composed of two main types of vomeronasal sensory neurons (VSNs), apical and basal VSNs, which express V1Rs and V2Rs, respectively. In their previous studies (Lin et al., Developmental Biology, 2018), the authors find that a transcription factor, AP-2e, is specifically expressed in maturing and mature V2R-expressing basal VSNs. They also show that deletion of AP-2e results in a reduced number of basal VSNs. In the current study, they further ask if AP-2e functions as a master regulator that is able to fully reprogram differentiated neurons or a terminal selector that maintains the fate of basal VSNs. They generated a knock-in mouse line to ectopically express AP-2e in apical VSNs, and investigated the transcriptomic changes in apical VSNs. By combining histological imaging and single-cell RNA sequencing (scRNA-seq) analysis, they find that ectopic expression of AP-2e in apical VSNs only induces expression of some basal VSN-specific genes but is unable to fully transform apical VSNs into basal VSNs. Thus, they conclude that AP-2e likely acts as a terminal selector for the identity of basal VSN subpopulations by activating some basally enriched genes while suppressing apically enriched genes. They further show that detection of conspecific odorants is impaired in mice with AP-2e ectopic expression.
The conclusions of this paper are mostly supported by data, but some key aspects of gene expression analysis need to be strengthened.
Strengths:
The authors provide genetic tools to rescue or ectopically express AP-2e, which enables precise manipulation of AP-2e in vivo. In addition, the scRNA-seq results nicely reconstruct the process of apical-basal VSN differentiation dichotomy and correlate well with the histological results.Weaknesses:
Although the present data mainly support the authors' hypothesis, a few aspects of data analysis are required to reinforce their conclusions. The changes of V1Rs and V2Rs after ectopic AP-2e expression are lacking. Direct histological evidence for basally enriched genes in apical VSNs is also needed. Further, more detailed analyses on VSN activation challenged by conspecific odorants could consolidate the molecular and behavioral findings. -
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