Prolonged partner separation erodes nucleus accumbens transcriptional signatures of pair bonding in male prairie voles

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1. 

   Version published to 10.7554/elife.80517 on eLife

   Feb 28, 2023
2. 

   eLife

   Aug 23, 2022

   Author Response

   We thank the reviewers for their thoughtful and constructive comments which have helped us improve our manuscript. In our revised manuscript, we will respond to three main weaknesses:

   1. We will address the inconsistency in the experimental design across the behavior and the transcription experiments by repeating the behavior with an experimental timeline that more exactly matches that of the animals used in transcriptional studies;

   2. We will further validate and justify our use of TRAP and our focus on the NAc as the sole brain region of investigation;

   3. We will revise the language throughout the manuscript, especially in the discussion, to reduce anthropomorphizing of our results and interpretations. Below we have provided responses to specific concerns articulated by each reviewer.

   Reviewer #1 (Public Review):

   The …

   Author Response

   We thank the reviewers for their thoughtful and constructive comments which have helped us improve our manuscript. In our revised manuscript, we will respond to three main weaknesses:

   1. We will address the inconsistency in the experimental design across the behavior and the transcription experiments by repeating the behavior with an experimental timeline that more exactly matches that of the animals used in transcriptional studies;

   2. We will further validate and justify our use of TRAP and our focus on the NAc as the sole brain region of investigation;

   3. We will revise the language throughout the manuscript, especially in the discussion, to reduce anthropomorphizing of our results and interpretations. Below we have provided responses to specific concerns articulated by each reviewer.

   Reviewer #1 (Public Review):

   The monogamous vole provides unique opportunities to study the neural basis of pair bonding and this study exploits that opportunity in a novel way. Focusing on the nucleus accumbens, the authors conduct RNA-Seq to characterize the transcriptome in same-sex and opposite-sex pairs when bonded, when separated for a short time and when separated for a long time at which point the literature has in the past demonstrated the willingness to form a new bond. They determine that the transcriptome of pair bonding includes a preponderance of glial-associated gene changes and that it degrades with long-term separation. To the latter point, they then conduct a neuron enriching trap schema to find those genes subject to change specifically in neurons.

   The strength of the report is the clever experimental design, the unusual animal model, and the comparisons of same-sex and opposite-sex pairs and long-term and short-term separations.

   The weakness is that the behavioral changes observed are not what was expected based on prior work and are relatively modest, providing a disconnect between the outcome and the more dramatic transcriptional changes. A second weakness is the focus on the nucleus accumbens which is a brain region most closely associated with reward. While pair bonding may be rewarding, that component may be independent of the memory of a partner or the willingness to partner anew. Lastly, there is no clear connection between the identified transcriptome and either the formation or degradation of the pair bond.

   We thank the reviewer for noting the unique strengths of using prairie voles to investigate this specific question and for praising our experimental design, which compares opposite-sex and same-sex paired males at each time point to disentangle the effects of pair bonding from general social affiliation and isolation.

   Reviewers #1 and #3 noted the mismatch between the behavioral and transcriptional responses. Specifically, we found little evidence of bond dissolution following long term separation despite substantial erosion of the pair bond transcriptional signature. They further note that the experimental design employed to assess behavior and transcription differed, which may have contributed to the apparent mismatch. Importantly, our initial behavioral assessment as presented in Figure 1 of the manuscript had two strengths. It measured intra-animal changes in behavior over time and minimized the number of animals required. However, we agree with the reviewers, and we are currently repeating the behavior experiments to match the transcription experiments. Specifically, separated partners will be kept in separate colony rooms to ensure no possible access to partner-associated sensory cues (visual, auditory, olfactory), and we will use separate cohorts of animals for short- and long-term separation. This design avoids partner re-introduction during the short-term partner preference test. The results of this work will be informative regardless of outcome. If we observe a dissolution of pair bond behaviors, it indicates that re-exposure to a partner after a short, 48-hour separation has a powerful effect on bond duration following separation. If we do not observe any change in pair bond behaviors following separation, it would confirm that pair bond behaviors are more resistant to erosion than are transcriptional signatures of pair bonding.

   We have focused on the NAc because it is a critical hub that is engaged upon attachment formation and is implicated in loss processing. Specifically, studies have shown that blockade of neuromodulatory signaling (i.e. oxytocin and dopamine) in this region impairs bond formation and can lead to bond dissolution. Our group and others have demonstrated that plasticity within this region - in patterns of neuronal activity and in synaptic response to oxytocin - are associated with bond formation and maturation (1, 2). And literature on drugs of abuse has demonstrated an important role for the NAc in encoding of reward associations (3), which ultimately underlies partner preference. Additionally, in human neuroimaging studies, Prolonged Grief Disorder is associated with an enhanced signal in the NAc when viewing images of the lost loved one, suggesting that normal resolution of grief corresponds with a decrease in NAc activity elicited by reminders of the lost loved one (4). Thus, our focus on this region is well supported. Nonetheless, we recognize that the NAc does not act in a vacuum, and the efferent and afferent connectivity of different NAc cell types is well delineated, paving the way for future work (5, 6).

   Additionally, we agree with the reviewer that pair bonding behavior is multifaceted and comprised of several discrete behaviors that are not dissociable in the partner preference test. Partner-associated reward and partner memory may be independently encoded, and disruption of either process would manifest as a decrease or lack of partner preference. In our complete response to reviewers and revision of the manuscript, we will address this point more thoroughly. Finally, we interpret the reviewer’s last comment to be a request for functional manipulations to validate that the predicted transcriptional changes have a behavioral effect. This is beyond the scope of this manuscript but an active area of future research.

   Reviewer #2 (Public Review):

   The goal of this study is to understand the molecular mechanisms by which pair bonded animals recover following the loss of a partner.

   Strengths of this work include: (1) The organism - a novel model for studying pair bonding and loss; (2) The integrative nature of the study; it integrates behavior and brain gene expression RNASeq data and vTRAP; (3) The important and understudied question about how pair bonded animals recover from loss; (4) The thorough and careful analysis of highly multidimensional and complex datasets

   Weaknesses include: (1) the major comparison is between same vs opposite sex housed pairs. This design controls for social effects somewhat, but the two treatment groups differ not just with respect to whether or not they are pair bonded, but also in whether or not they had associated with a male or female. Differences between the treatments could reflect pair bonding, or perhaps something about the sex of the partner. It would be useful to have an additional control group, or data on the behavior of individuals within both types of pairs while they are co-housed. Were transcriptomic effects more detectable in pairs that were more bonded together behaviorally? That would suggest that the gene expression signatures really reflect something about the bond rather than other confounds, for example; (2) The vTRAP method is fancy but what is it really adding? (3) The authors interpret the transcriptomic differences as promoting the ability to form a new bond but there are probably other processes that are contributing to the differences in gene expression. Some of the differentially expressed genes could be involved in promoting a new pair bond, but there could also be a signature of the memory of the identity of the partner, the signature of the bond itself, etc. (4) Some of the interpretations go a little too far, especially in terms of anthropomorphism. The impact of the work includes further development of voles as an important model for studying social behavior and insights into the molecular processes important for recovering from the loss of a partner.

   We thank the reviewer for recognizing the strength of our study organism and experimental techniques as well as rigorous analyses to answer an important question about adapting to partner loss.

   Regarding the noted weaknesses:

   (1) We chose to compare opposite sex pair bonds to same sex affiliative relationships as this is the standard within the field, and we note that reviewers 1 and 3 found this to be a strength of our study design (7–11). Peer relationships in prairie voles are difficult to distinguish behaviorally from those of opposite-sex pairs (Fig 1) because both same and opposite-sex paired voles show selective preference for their pairmate and selective agression towards other voles (7). As such, the critical feature that makes pair bonding different is mating, which requires an opposite sex partner in voles, and our experiments are optimally designed to identify the longitudinal transcriptional changes that result from mating and cohabitating with an opposite-sex partner. In order to best match our two groups, only animals with a preference score >50% were included in the transcriptional experiment, ensuring that we were comparing animals with an affiliative preference for their partner - whether that individual was the same or opposite sex.

   We interpret the reviewers comment to be that they want us to compare opposite-sex-paired animals with and without bonds. This can be achieved two ways. First, we can compare to a promiscuous species, such as meadow voles, which will mate and cohabitate without forming bonds, but this is confounded by species differences in transcription that may exist independent of bonding. Second, we can compare bonded voles to the small subset that do not form bonds. While intriguing, this is experimentally challenging as only ~10-20% of males fail to form a bond when paired with a sexually receptive female (in the current study, 16% had a preference < 50% after two weeks of pairing, which is consistent with prior reports - (9–11)). Put simply, we would need to pair hundreds of voles to opportunistically collect a sufficient number of non-bonders for transcriptional assessment across our experimental conditions. While we hope to eventually be able to do such an experiment, litter sizes, consideration of animal welfare, and other constraints make this largely untenable at present.

   Data on the behavior of individuals within both types of pairs while they are co-housed is already provided via results of a partner preference test performed after 2 weeks of co-housing and prior to re-housing or separation (Fig 2B and 3B). We find the reviewer’s suggestion of finding a relationship between the transcriptional signature and the pair bonding strength an interesting question, and we undertook a preliminary analysis examining whether animals with different pair bond strength aggregate on a PCA analysis of gene expression. There was no apparent relationship, although we are performing additional analyses such as exploratory factor analysis. The fact that we have not found a relationship between the baseline partner preference and the transcription in these initial analyses is perhaps unsurprising. First, bonding may require some threshold change in gene expression, with bond strength reflected in non-genomic information, such as synapse formation or strengthening, or axonal ensheathment. Second, we only performed transcriptional analyses on animals with a baseline partner preference >50%; we would not necessarily expect a dissociation given the uniformly strong bonds across these animals.

   (2) We feel that inclusion of TRAP adds substantially to this manuscript and to our understanding of the neuromolecular underpinnings of bonding and loss in the NAc. The value of this experiment is twofold. As noted by Reviewer 3, “the TRAP approach in prairie voles is novel and will provide a great resource to the research community.” The prairie vole community has just developed its first transgenic Cre lines, which could be paired with vTRAP to query bond-associated gene expression changes exclusively in Cre-expressing neurons (15). Second, we noticed a puzzle in our tissue-level data. The majority of cells in the NAc are neurons (16, 17), and the vast majority of pair bonding studies of this region have focused on neuronal phenotypes, but our transcriptional signatures were linked to changes in glial populations. Ultimately, changes in glia are likely to act via their interactions with neurons, and vTRAP enables us to query the neuronal transcriptional changes within our data. Supporting that this provides novel insights into our datasets, when we cluster transcripts based on their expression profiles following short and long-term separation, we predict different GO terms from the tissue level and neuronally-enriched gene sets. For instance, the GO terms resulting from cluster 2 for neuronal genes (Fig 4) includes “response to amphetamine” within the top 10 results, but the same cluster of genes from tissue level sequencing predicts this GO term as the 34th result.

   (3) We agree with the reviewer that adapting to partner loss is a multifaceted process that likely engages numerous biological and emotional systems in voles. The explanation we offer for the transcriptional changes during loss is based on previous work in the field and is one possible interpretation. We will expand on this point during revision of the manuscript.

   (4) We thank the reviewer for encouraging us to be objective with our interpretations. We will address this comment during revision of the manuscript.

   Finally, we thank the reviewer for recognizing the value of our study for not only the field of voles but the bereavement field more broadly.

   Reviewer #3 (Public Review):

   In this manuscript, the authors investigate the behavioral and brain transcriptional alterations associated with short- and long-term partner separation in the monogamous male prairie vole. Male prairie voles continue to show affiliative behavior after short- (2 days) and long-term (4-weeks) partner separation, with similar effects for same and opposite-sex pairs. However, the transcriptional signature in the nucleus accumbens exhibits marked alterations after long-term separation.

   Strengths:

   1. A key strength of this manuscript is its use of the monogamous prairie vole to investigate transcriptional alterations associated with pair bonding and subsequent pair separation. This sort of behavior cannot be investigated in typical rodent model systems (e.g., mice, rats), and the choice of using prairie voles allows for dissection of potential mechanisms of social bonding with relevance to partner loss in humans.

   1. Investigation of behavioral measures and transcriptional alterations at both short- and long-term time points after pairing and separation is a strength of the manuscript. These time points were selected based on previous studies in laboratory and wild prairie voles related to the time it takes to form a pair bond and for the male prairie vole to leave the nest after the loss of the female pair. The datasets generated will be of great use to the scientific community.

   1. The authors investigate the behavior and transcriptional profiles after same-sex as well as opposite-sex pairing. This is considered a thoughtful decision on the authors' part which allows them to tease apart the effects of same vs. opposite sex.

   1. The use of numerous behavioral measures to assess both affiliative and aggressive behaviors is a strength of the approach.

   1. The authors use many biostatistical approaches (e.g., RRHO, WGCNA, Enrichr) to probe the transcriptomics data. These approaches allow the authors to move beyond simply assessing transcriptional profiles separately, but to look for patterns that are similar or different across datasets.

   1. The authors use rigorous statistical methods to assess behavioral measures.

   1. The TRAP approach in prairie voles is novel and will provide a great resource to the research community.

   Weaknesses:

   1. The methods state that prairie voles were treated differently in the behavioral and transcriptomics studies. Specifically, for the separation in the behavioral studies, prairie voles were separated by sight, but not necessarily by the smell from partners (i.e., partners were kept ~1 foot apart). However, prairie voles in the transcriptomics studies were separated by both sight and smell (i.e., partners were sacrificed after separation). Thus, it is possible that the lack of degradation of pair bond-related behavior after long-term separation might be due to these prairie voles being able to smell their partners after separation. This is considered a moderate flaw in the design of the studies which limits the integration of results between behavior and transcriptomics. This might be why the authors do not see a strong behavioral degradation of pair bond-related behavior after long-term separation but do see a strong transcriptional signature.

   1. While RRHO is helpful to assess overall patterns of transcriptional signatures across datasets, its utility for determining the exact transcripts is limited. This is because of how RRHO determines the overlapping transcripts for its Venn diagram feature (by taking the point where the p-value is most significant and taking the list to the outside corner of that quadrant).

   1. TRAP expression was verified in only one animal. Thus, the approach has not been appropriately confirmed.

   We thank the reviewer for their thoughtful comments on the innovative strengths and advantages of our manuscript.

   Regarding the noted weaknesses:

   (1) Please see our response to Reviewer #1, who shares your concerns.

   (2) We agree that RRHO is particularly useful for assessment of overall patterns. We interpret the Reviewer’s comment to mean that when extracting the overlapping gene lists from an RRHO quadrant for downstream analyses, we should filter that list for genes whose differential expression passes a nominal p-value cutoff to reduce the amount of biologically insignificant conclusions we are drawing from the RRHO data. Our initial analyses used just such a threshold-based approach by identifying GO terms via differentially expressed genes of the combined pair bond (Figure 2) using both p-value and log2Fold cutoffs. This analysis revealed a number of terms associated with glial cell proliferation, differentiation, and function (Fig 2H). Such processes occur over a time frame of days to weeks, with different phases of differentiation characterized by different gene expression profiles. To explore this further, we used the genes in the UU and DD RRHO quadrants without implementing a p-value cutoff to see if additional genes associated with these GO-identified pathways may be showing subtle but consistent directional changes (Fig 3). Importantly, we only use the overlapping RRHO gene lists to determine how previously defined biological processes via DEG-predicted GO terms change across conditions; we are not using the RRHO gene lists to generate new GO terms. This allowed us to look for patterns within the identified pathways that may give insight into how transcription might be affecting gliogenesis. This analysis was similarly suggested to us from other experienced users of RRHO plots (see Acknowledgements). There are also several published studies that use RRHO UU and DD quadrant overlap (18–22).

   (3) Most labs rarely confirm Cre-dependence of vectors in more than one or two animals as the results, including those shown in Fig S9A, are typically definitive (i.e. no expression in the absence of Cre, expression in the presence of Cre). In addition to the images shown in figure S9A, we used fluorescent guided dissection to harvest tissue/mRNA, serving as an additional visual confirmation of RPL10-GFP expression in the animals used to generate Figure 4. Since submission, we have also confirmed that this vector also expresses in rats when Cre-recombinase is present. However, prior to resubmission, we will perform additional surgeries to confirm that TRAP is only expressed in the presence of Cre-recombinase.

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   10. A. J. Grippo, B. S. Cushing, C. S. Carter, Depression-like behavior and stressor-induced neuroendocrine activation in female prairie voles exposed to chronic social isolation. Psychosomatic Medicine. 69, 149–157 (2007).
       

   11. A. J. Grippo, D. Gerena, J. Huang, N. Kumar, M. Shah, R. Ughreja, C. Sue Carter, Social isolation induces behavioral and neuroendocrine disturbances relevant to depression in female and male prairie voles. Psychoneuroendocrinology (2007), doi:10.1016/j.psyneuen.2007.07.004.
       

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   Read the original source
3. 

   eLife

   Aug 23, 2022

   Evaluation Summary:

   This work will be of interest to behavioral neuroscientists with a focus on social behavior. The interrogation of the transcriptional signature of pair-bonding, in both short and long-term, is unique and made possible with the use of the monogamous vole. That there is a "degrading" of the transcriptome of pair bonding following separation is evident but there is a gap in understanding how the gene expression changes relate to behavior.

   (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. Reviewer #1 agreed to share their name with the authors.)

   Read the original source
4. 

   eLife

   Aug 23, 2022

   Reviewer #1 (Public Review):

   The monogamous vole provides unique opportunities to study the neural basis of pair bonding and this study exploits that opportunity in a novel way. Focusing on the nucleus accumbens, the authors conduct RNA-Seq to characterize the transcriptome in same-sex and opposite-sex pairs when bonded, when separated for a short time and when separated for a long time at which point the literature has in the past demonstrated the willingness to form a new bond. They determine that the transcriptome of pair bonding includes a preponderance of glial-associated gene changes and that it degrades with long-term separation. To the latter point, they then conduct a neuron enriching trap schema to find those genes subject to change specifically in neurons.

   The strength of the report is the clever experimental design, the …

   Reviewer #1 (Public Review):

   The monogamous vole provides unique opportunities to study the neural basis of pair bonding and this study exploits that opportunity in a novel way. Focusing on the nucleus accumbens, the authors conduct RNA-Seq to characterize the transcriptome in same-sex and opposite-sex pairs when bonded, when separated for a short time and when separated for a long time at which point the literature has in the past demonstrated the willingness to form a new bond. They determine that the transcriptome of pair bonding includes a preponderance of glial-associated gene changes and that it degrades with long-term separation. To the latter point, they then conduct a neuron enriching trap schema to find those genes subject to change specifically in neurons.

   The strength of the report is the clever experimental design, the unusual animal model, and the comparisons of same-sex and opposite-sex pairs and long-term and short-term separations.

   The weakness is that the behavioral changes observed are not what was expected based on prior work and are relatively modest, providing a disconnect between the outcome and the more dramatic transcriptional changes. A second weakness is the focus on the nucleus accumbens which is a brain region most closely associated with reward. While pair bonding may be rewarding, that component may be independent of the memory of a partner or the willingness to partner anew. Lastly, there is no clear connection between the identified transcriptome and either the formation or degradation of the pair bond.

   Read the original source
5. 

   eLife

   Aug 23, 2022

   Reviewer #2 (Public Review):

   The goal of this study is to understand the molecular mechanisms by which pair bonded animals recover following the loss of a partner.

   Strengths of this work include: (1) The organism - a novel model for studying pair bonding and loss; (2) The integrative nature of the study; it integrates behavior and brain gene expression RNASeq data and vTRAP; (3) The important and understudied question about how pair bonded animals recover from loss; (4) The thorough and careful analysis of highly multidimensional and complex datasets

   Weaknesses include: (1) the major comparison is between same vs opposite sex housed pairs. This design controls for social effects somewhat, but the two treatment groups differ not just with respect to whether or not they are pair bonded, but also in whether or not they had associated with …

   Reviewer #2 (Public Review):

   The goal of this study is to understand the molecular mechanisms by which pair bonded animals recover following the loss of a partner.

   Strengths of this work include: (1) The organism - a novel model for studying pair bonding and loss; (2) The integrative nature of the study; it integrates behavior and brain gene expression RNASeq data and vTRAP; (3) The important and understudied question about how pair bonded animals recover from loss; (4) The thorough and careful analysis of highly multidimensional and complex datasets

   Weaknesses include: (1) the major comparison is between same vs opposite sex housed pairs. This design controls for social effects somewhat, but the two treatment groups differ not just with respect to whether or not they are pair bonded, but also in whether or not they had associated with a male or female. Differences between the treatments could reflect pair bonding, or perhaps something about the sex of the partner. It would be useful to have an additional control group, or data on the behavior of individuals within both types of pairs while they are co-housed. Were transcriptomic effects more detectable in pairs that were more bonded together behaviorally? That would suggest that the gene expression signatures really reflect something about the bond rather than other confounds, for example; (2) The vTRAP method is fancy but what is it really adding? (3) The authors interpret the transcriptomic differences as promoting the ability to form a new bond but there are probably other processes that are contributing to the differences in gene expression. Some of the differentially expressed genes could be involved in promoting a new pair bond, but there could also be a signature of the memory of the identity of the partner, the signature of the bond itself, etc. (4) Some of the interpretations go a little too far, especially in terms of anthropomorphism.

   The impact of the work includes further development of voles as an important model for studying social behavior and insights into the molecular processes important for recovering from the loss of a partner.

   Read the original source
6. 

   eLife

   Aug 23, 2022

   Reviewer #3 (Public Review):

   In this manuscript, the authors investigate the behavioral and brain transcriptional alterations associated with short- and long-term partner separation in the monogamous male prairie vole. Male prairie voles continue to show affiliative behavior after short- (2 days) and long-term (4-weeks) partner separation, with similar effects for same and opposite-sex pairs. However, the transcriptional signature in the nucleus accumbens exhibits marked alterations after long-term separation.

   Strengths:

   1. A key strength of this manuscript is its use of the monogamous prairie vole to investigate transcriptional alterations associated with pair bonding and subsequent pair separation. This sort of behavior cannot be investigated in typical rodent model systems (e.g., mice, rats), and the choice of using prairie voles allows …

   Reviewer #3 (Public Review):

   In this manuscript, the authors investigate the behavioral and brain transcriptional alterations associated with short- and long-term partner separation in the monogamous male prairie vole. Male prairie voles continue to show affiliative behavior after short- (2 days) and long-term (4-weeks) partner separation, with similar effects for same and opposite-sex pairs. However, the transcriptional signature in the nucleus accumbens exhibits marked alterations after long-term separation.

   Strengths:

   1. A key strength of this manuscript is its use of the monogamous prairie vole to investigate transcriptional alterations associated with pair bonding and subsequent pair separation. This sort of behavior cannot be investigated in typical rodent model systems (e.g., mice, rats), and the choice of using prairie voles allows for dissection of potential mechanisms of social bonding with relevance to partner loss in humans.
   2. Investigation of behavioral measures and transcriptional alterations at both short- and long-term time points after pairing and separation is a strength of the manuscript. These time points were selected based on previous studies in laboratory and wild prairie voles related to the time it takes to form a pair bond and for the male prairie vole to leave the nest after the loss of the female pair. The datasets generated will be of great use to the scientific community.
   3. The authors investigate the behavior and transcriptional profiles after same-sex as well as opposite-sex pairing. This is considered a thoughtful decision on the authors' part which allows them to tease apart the effects of same vs. opposite sex.
   4. The use of numerous behavioral measures to assess both affiliative and aggressive behaviors is a strength of the approach.
   5. The authors use many biostatistical approaches (e.g., RRHO, WGCNA, Enrichr) to probe the transcriptomics data. These approaches allow the authors to move beyond simply assessing transcriptional profiles separately, but to look for patterns that are similar or different across datasets.
   6. The authors use rigorous statistical methods to assess behavioral measures.
   7. The TRAP approach in prairie voles is novel and will provide a great resource to the research community.

   Weaknesses:

   1. The methods state that prairie voles were treated differently in the behavioral and transcriptomics studies. Specifically, for the separation in the behavioral studies, prairie voles were separated by sight, but not necessarily by the smell from partners (i.e., partners were kept ~1 foot apart). However, prairie voles in the transcriptomics studies were separated by both sight and smell (i.e., partners were sacrificed after separation). Thus, it is possible that the lack of degradation of pair bond-related behavior after long-term separation might be due to these prairie voles being able to smell their partners after separation. This is considered a moderate flaw in the design of the studies which limits the integration of results between behavior and transcriptomics. This might be why the authors do not see a strong behavioral degradation of pair bond-related behavior after long-term separation but do see a strong transcriptional signature.
   2. While RRHO is helpful to assess overall patterns of transcriptional signatures across datasets, its utility for determining the exact transcripts is limited. This is because of how RRHO determines the overlapping transcripts for its Venn diagram feature (by taking the point where the p-value is most significant and taking the list to the outside corner of that quadrant).
   3. TRAP expression was verified in only one animal. Thus, the approach has not been appropriately confirmed.

   Read the original source
7. 

   Version published to 10.1101/2021.07.14.452355v2 on bioRxiv

   May 20, 2022
8. 

   Version published to 10.1101/2021.07.14.452355v1 on bioRxiv

   Jul 14, 2021