Impairing one sensory modality enhances another by reconfiguring peptidergic signalling in Caenorhabditis elegans

  1. Giulio Valperga
  2. Mario de Bono

  • Curated by eLife

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    Evaluation Summary:

    This manuscript reports a surprising discovery in C. elegans: upon losing its normal sensory properties, the ADL nociceptive/pheromone sensing neuron relays and thereby enhances oxygen behavioral responses via neuropeptide signaling. This effect could be interpreted as cross-modal sensory plasticity or more general a cross-modulation between sensory circuits, a still open question that should be addressed in a revision. The study is relevant to scientists working on sensory neurobiology and neuronal plasticity.

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

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Abstract

Animals that lose one sensory modality often show augmented responses to other sensory inputs. The mechanisms underpinning this cross-modal plasticity are poorly understood. We probe such mechanisms by performing a forward genetic screen for mutants with enhanced O 2 perception in Caenorhabditis elegans . Multiple mutants exhibiting increased O 2 responsiveness concomitantly show defects in other sensory responses. One mutant, qui-1 , defective in a conserved NACHT/WD40 protein, abolishes pheromone-evoked Ca 2+ responses in the ADL pheromone-sensing neurons. At the same time, ADL responsiveness to pre-synaptic input from O 2 -sensing neurons is heightened in qui-1 , and other sensory defective mutants, resulting in enhanced neurosecretion although not increased Ca 2+ responses. Expressing qui-1 selectively in ADL rescues both the qui-1 ADL neurosecretory phenotype and enhanced escape from 21% O 2 . Profiling ADL neurons in qui-1 mutants highlights extensive changes in gene expression, notably of many neuropeptide receptors. We show that elevated ADL expression of the conserved neuropeptide receptor NPR-22 is necessary for enhanced ADL neurosecretion in qui-1 mutants, and is sufficient to confer increased ADL neurosecretion in control animals. Sensory loss can thus confer cross-modal plasticity by changing the peptidergic connectome.

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  1. Version published to 10.7554/elife.68040 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    In the present work Valperga and de Bono performed a forward genetic screen to identify candidate genes that would fulfill two criteria when mutant: 1) enhance an escape response to high ambient oxygen but 2) without modifications in the respective oxygen sensing neurons. They found that qui-1 mutants meet these criteria. qui-1 is known to act in the nociceptive neurons ASH and ADL (among others). The authors show that in qui-1 mutants ADL neurons are defective in normal chemo-sensation and upregulate neuropeptide secretion. This is associated with increased gene expression of neurosecretion components in ADL, among them two GPCR receptors (npr-22 and tkr-1); mutants in these receptors partially phenocopy the neurosecretion phenotype. The authors suggest an intriguing model in which ADL, upon loss of its normal sensory properties, relays peptidergic input from oxygen sensory circuits to peptidergic output towards yet unidentified downstream circuitry. This novel mechanism of sensory cross modality expands on on previous work on cross modality in C. elegans, where until now only one example been demonstrated, and where a different mechanisms than in the present study was described (Rabinowitch 2016). These findings could serve as generalizable models for other systems where cross-modal plasticity has been observed. Although many conclusions in this work are substantiated by cell specific rescue of qui-1 in ADL others are made based on correlated observations only. The study therefore would benefit from additional experiments that demonstrate a causal link between elevated neurosecretion in ADL and the associated changes in behavior. This could be achieved by ADL cell ablation experiments and specific interference with ADL neurosecretion.

    We thank the reviewer for this analysis of our work. We sought to address points raised in this summary using her/his suggestions.

    Reviewer #2 (Public Review):

    Loss of one sensory modality is often compensated with an increase in another sensory modality. Valperga and de Bono identify a possibly conserved mechanism that appears to heighten the worm's sensitivity to O2 while dampening other sensory responses. The mechanism that they discover suggests that increased neuropeptide secretion could be responsible for the overcompensation for a loss of a sense. The combined data based on forward genetic screening and behavioral analysis, imaging and genomics are convincing and interesting.

    1. I very much enjoyed reading a manuscript that uses 'good old' forward genetics to make an interesting discovery!
    1. The paper is well written and very easy to follow. The data quality and their display in the figures are very convincing, too.
    1. The proposed mechanism of using enhanced neuropeptide secretions for compensating the loss of one sensory modality with an increase of function of another is novel and could indeed be conserved.

    We are grateful to the reviewer for the encouraging review of our work.

    Reviewer #3 (Public Review):

    The work by Valperga and de Bono aims to uncover molecular components of cross-modal plasticity, a system-wide form of neuronal remodeling that responds to sensory loss by altering the performance of remaining sensory modalities. The study focuses on the interplay between oxygen-sensing and pheromone detection in C. elegans. The data presented are mostly convincing and revealing. However, the message and the overall context within which the findings are framed are problematic.

    The authors rightly assert that the molecular processes underlying cross-modal plasticity are not fully understood. However, they emphasize that the important challenge is to reveal genetic lesions that result in sensory loss and drive cross-modal plasticity. I find this to be over-specific and imprecise. There are many possible causes for sensory loss, some are genetic, some are non-genetic (e.g., certain diseases and injuries). In any case, the causes for sensory loss are usually independent of the processes that give rise to cross-modal plasticity. The genetics behind cross-modal plasticity enables the response to sensory loss, it does not cause the sensory loss. Genetic lesions to genes involved in cross-modal plasticity disrupt cross-modal plasticity, they don't induce it. Curiously, the authors sought to find single genes whose removal is simultaneously associated with both the loss of a sensory modality and the enhancement of another. This was done using a forward genetic screen for C. elegans mutants displaying enhanced oxygen sensation.

    We thank our reviewer for her/his thoughtful comments. We have revised our introduction to take account of her/his comments, and to remove the misleading statements s/he highlights.

    The analysis was further complicated by the fact that the screen was performed on strains whose oxygen sensitivity is already modified due to dysregulated activity in the RMG hub-and-spoke neural circuit, which integrates diverse sensory signals to control locomotion. Mutagenesis was performed on either the N2 strain, exhibiting RMG suppression, and thus decreased oxygen sensitivity, or flp-21 mutants, displaying excessive RMG activation, and increased oxygen sensitivity.

    We chose two genetic backgrounds for our mutant screens that attenuate the output of the RMG hub interneurons. Both backgrounds include a gain-of-function allele of the neuropeptide receptor NPR-1 that inhibits RMG output. The NPR-1 receptor has multiple peptide ligands, so in the second screen we reduced NPR-1 inhibitory signalling by deleting one these ligands, FLP-21. Neither of the two strains we used, N2 or flp-21, show appreciable O2 responses on food, and do not aggregate or accumulate on thicker parts of the food lawn, facilitating our screen (See Figure 1B).

    The screen yielded a gene, qui-1, whose dysfunction led to enhanced oxygen sensing (it is unclear if this is in the N2 or flp-21 background). The authors found that increased neuropeptide release from the pheromone-sensing neuron ADL underlies the increase in oxygen sensitivity. Furthermore, the qui-1 mutation was shown to diminish ADL pheromone responses. Therefore, a very particular genetic coupling between loss of pheromone sensation and enhanced oxygen sensitivity was revealed.

    We have indicated the parental origin of the qui-1 mutant in the revised manuscript.

    To generalize this finding, several additional mutant genes (not from the screen) were examined, including genes from the BBS family as well as wrt-6 and fig-1. They too displayed enhanced oxygen sensing linked to increased ADL neuropeptide secretion. However, their effects on ADL pheromone sensation were not reported. The main conclusion I draw from these findings is that the ADL neurons are able to modulate oxygen sensitivity by relaying information about oxygen levels from the RMG circuit to locomotor circuits via neuropeptide secretion. It is not at all clear that loss of pheromone sensation in the qui-1 case is the cause for increased neuropeptide release, or whether it is just one out of the many outcomes of mutating this gene. A much cleaner and more revealing experiment could have been, for example, to examine worms lacking the functional pheromone receptor OCR-2 in ADL. In fact, unlike qui-1 mutants who showed diminished oxygen responses in ADL, previous work from the de Bono group (Fenk and de Bono 2017) demonstrated that ADL O2 response are normal in ocr-2 mutants, indicating a profound difference between loss of pheromone sensitivity due to receptor dysfunction (ocr-2) and the unknown and broad effects of qui-1.

    We thanks the reviewer for this important suggestion. We have sought to test our model with a functional experiment that selectively disrupts sensory input into the ADL neurons. To achieve this, we decided to knock down a protein required for intraflagellar transport, OSM-6, rather than the OCR-2 TRP channel subunit. OCR-2 mediates not only pheromone responses in ADL, but also O2-escape behavior (de Bono et al., 2002). This may reflect a broader role for OCR-2 in ADL than sensory transduction. Disrupting OSM-6 truncates sensory cilia and severely compromises many chemosensory responses, but only weakly reduces aggregation and O2 responses.

    To target OSM-6 degradation specifically to the ADL neurons we knocked in DNA encoding an Auxin Inducible Degron (AID) into the osm-6 locus, and expressed TIR1 in ADL to achieve cell-specificity. TIR1 is required for AID. We have added the new data to Figure 4F–G and Figure 4 – figure supplement 2. We show that expressing TIR1 in ADL disrupts OSM-6::AID function both in the presence and absence of Auxin. This agrees with recent work that tested the efficiency and specificity of the AID system (Hills-Muckey et al., 2021). A partial OSM-6::AID reduction in ADL recapitulates many of the phenotypes of qui-1 mutants, including increased neurosecretion from ADL, heightened ADL responses to O2 inputs and a small but significant enhancement of the O2-escape response. We think these new data support our interpretation that a change in ADL’s sensory properties leads to heightened response of ADL neurons to O2 inputs, a phenotype observed in qui-1 and multiple other sensory defective mutants and a hallmark of cross-modal plasticity. However, the effects of knocking down osm-6 on ADL function also appear to be complex, as the stronger osm-6 knockdown achieved by adding auxin to the osm-6::AID knockin animals expressing TIR1 in ADL, unexpectedly gives weaker phenotypes than when auxin is absent.

    In fact, it would be interesting if the authors could explain or speculate how qui-1 eliminates ADL O2 responses, and how neuropeptide signaling from the RMG circuit via the NPR-22 neuropeptide receptor bypasses this lack of response and drives enhanced neuropeptide secretion in ADL, as they report.

    We can only speculate why O2-evoked responses in ADL disappear in qui-1 mutants. One possibility is that ADL becomes less excitable due to the reconfigured gene expression associated with loss of qui-1 in ADL. This model would predict that selectively knocking down qui-1 in ADL would confer the same Ca2+ response phenotype. Blocking ADL neurosecretion with TeTx in qui-1 mutants would test if the increased ADL neurosecretion we describe feeds back to reduce the O2-evoked Ca2+ response in ADL. An alternative hypothesis is that the effect of disrupting qui-1 is non-cell-autonomous, altering excitatory or inhibitory input to ADL from other qui-1 expressing neurons. We have not tested if neurosecretion from other qui-1-expressing neurons is altered in qui-1 mutants.

    Strikingly, while disrupting qui-1 leads to loss of a measurable O2-evoked Ca2+ response in ADL, these neurons display elevated O2-evoked neurosecretion in qui-1 mutants. This implies that some O2-evoked Ca2+ responses are retained in ADL’s axons in qui-1 mutants. It also suggests that other second messengers upregulate neurosecretion. Elevating cAMP, for example, can promote dense-core vesicle release more efficiently than increasing Ca2+ levels (Costa et al., 2017). Altered G-protein coupled receptor signalling could lead to elevated cAMP levels and increased neurosecretion in qui-1 mutants. It is worth noting that in N2 controls, ADL does not display O2-evoked neurosecretion despite showing measurable Ca2+ responses.

    The work includes a transcriptomic analysis comparing ADL-specific gene expression between wild type and the qui-1 mutant. Unlike other experiments in the study, in which the specific effects of mutations were confirmed through rescue experiments and the use of additional alleles, thus eliminating potential confounds with background mutations, the transcriptomic experiment did not apply such controls. Therefore, it is hard to conclude whether the reported changes in transcription are due solely to the qui-1 mutation or to other unrelated genetic modifications in the mutant strain.

    We worried about unspecific effects of background mutations both on the ADL transcriptome and on other qui-1 related phenotypes. We regret we did not explicitly address this point in our initial submission. To remove background mutations, mutants isolated in our screen, including qui-1, were backcrossed with the N2 laboratory strain a minimum of four times. These qui-1 animals were further crossed into a 5 times outcrossed line that expresses the fluorescent protein mKate specifically in ADL, to generate the strains from which we sorted ADL neurons by FACS. Mutant and transgenic strains were outcrossed using the N2 laboratory strain. We explain this in the Methods section of the revised manuscript.

    The extensive outcrossing make us confident that the large majority of differentially regulated genes between wild type and qui-1 samples in ADL are due to the absence of qui-1. Supporting this, both mutations in neuropeptide receptors identified by our profiling, npr-22 and tkr-1, suppress ADL’s elevated neurosecretion. Nevertheless, we have added a note to explicitly bring up the concern raised by our reviewers, that some transcriptional differences could be the result of background mutations.

    Overall, except for where mentioned, the data presented are solid and consistent. However, the conclusion that the study reveals a molecular pathway for cross-modal plasticity is less convincing. The chain of events does not include some form of sensory loss, leading to subsequent, independent neural plasticity, as expected for cross-modal plasticity. Rather, a very broad genetic switch is described that can simultaneously change receptor abundance and neuropeptide release. Thus, an equally interesting and more coherent framing of the data could be that the study uncovered a genetic regulator, yet to be fully characterized, of oxygen-dependent behavior in a non-oxygen sensing neuron, adding to previous literature on neural circuit cross-talk.

    We are grateful to the reviewer for her/his thorough and critical analysis of our work, which has prompted us to perform additional experiments and helped us revise our manuscript. These additional data clarify our final interpretation of the data regarding cross-modal plasticity.

  3. eLife

    Evaluation Summary:

    This manuscript reports a surprising discovery in C. elegans: upon losing its normal sensory properties, the ADL nociceptive/pheromone sensing neuron relays and thereby enhances oxygen behavioral responses via neuropeptide signaling. This effect could be interpreted as cross-modal sensory plasticity or more general a cross-modulation between sensory circuits, a still open question that should be addressed in a revision. The study is relevant to scientists working on sensory neurobiology and neuronal plasticity.

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

  4. eLife

    Reviewer #1 (Public Review):

    In the present work Valperga and de Bono performed a forward genetic screen to identify candidate genes that would fulfill two criteria when mutant: 1) enhance an escape response to high ambient oxygen but 2) without modifications in the respective oxygen sensing neurons. They found that qui-1 mutants meet these criteria. qui-1 is known to act in the nociceptive neurons ASH and ADL (among others). The authors show that in qui-1 mutants ADL neurons are defective in normal chemo-sensation and upregulate neuropeptide secretion. This is associated with increased gene expression of neurosecretion components in ADL, among them two GPCR receptors (npr-22 and tkr-1); mutants in these receptors partially phenocopy the neurosecretion phenotype. The authors suggest an intriguing model in which ADL, upon loss of its normal sensory properties, relays peptidergic input from oxygen sensory circuits to peptidergic output towards yet unidentified downstream circuitry. This novel mechanism of sensory cross modality expands on on previous work on cross modality in C. elegans, where until now only one example been demonstrated, and where a different mechanisms than in the present study was described (Rabinowitch 2016). These findings could serve as generalizable models for other systems where cross-modal plasticity has been observed. Although many conclusions in this work are substantiated by cell specific rescue of qui-1 in ADL others are made based on correlated observations only. The study therefore would benefit from additional experiments that demonstrate a causal link between elevated neurosecretion in ADL and the associated changes in behavior. This could be achieved by ADL cell ablation experiments and specific interference with ADL neurosecretion.

  5. eLife

    Reviewer #2 (Public Review):

    Loss of one sensory modality is often compensated with an increase in another sensory modality. Valperga and de Bono identify a possibly conserved mechanism that appears to heighten the worm's sensitivity to O2 while dampening other sensory responses. The mechanism that they discover suggests that increased neuropeptide secretion could be responsible for the overcompensation for a loss of a sense. The combined data based on forward genetic screening and behavioral analysis, imaging and genomics are convincing and interesting.

    1. I very much enjoyed reading a manuscript that uses 'good old' forward genetics to make an interesting discovery!

    2. The paper is well written and very easy to follow. The data quality and their display in the figures are very convincing, too.

    3. The proposed mechanism of using enhanced neuropeptide secretions for compensating the loss of one sensory modality with an increase of function of another is novel and could indeed be conserved.

  6. eLife

    Reviewer #3 (Public Review):

    The work by Valperga and de Bono aims to uncover molecular components of cross-modal plasticity, a system-wide form of neuronal remodeling that responds to sensory loss by altering the performance of remaining sensory modalities. The study focuses on the interplay between oxygen-sensing and pheromone detection in C. elegans. The data presented are mostly convincing and revealing. However, the message and the overall context within which the findings are framed are problematic.

    The authors rightly assert that the molecular processes underlying cross-modal plasticity are not fully understood. However, they emphasize that the important challenge is to reveal genetic lesions that result in sensory loss and drive cross-modal plasticity. I find this to be over-specific and imprecise. There are many possible causes for sensory loss, some are genetic, some are non-genetic (e.g., certain diseases and injuries). In any case, the causes for sensory loss are usually independent of the processes that give rise to cross-modal plasticity. The genetics behind cross-modal plasticity enables the response to sensory loss, it does not cause the sensory loss. Genetic lesions to genes involved in cross-modal plasticity disrupt cross-modal plasticity, they don't induce it. Curiously, the authors sought to find single genes whose removal is simultaneously associated with both the loss of a sensory modality and the enhancement of another. This was done using a forward genetic screen for C. elegans mutants displaying enhanced oxygen sensation.

    The analysis was further complicated by the fact that the screen was performed on strains whose oxygen sensitivity is already modified due to dysregulated activity in the RMG hub-and-spoke neural circuit, which integrates diverse sensory signals to control locomotion. Mutagenesis was performed on either the N2 strain, exhibiting RMG suppression, and thus decreased oxygen sensitivity, or flp-21 mutants, displaying excessive RMG activation, and increased oxygen sensitivity.

    The screen yielded a gene, qui-1, whose dysfunction led to enhanced oxygen sensing (it is unclear if this is in the N2 or flp-21 background). The authors found that increased neuropeptide release from the pheromone-sensing neuron ADL underlies the increase in oxygen sensitivity. Furthermore, the qui-1 mutation was shown to diminish ADL pheromone responses. Therefore, a very particular genetic coupling between loss of pheromone sensation and enhanced oxygen sensitivity was revealed.

    To generalize this finding, several additional mutant genes (not from the screen) were examined, including genes from the BBS family as well as wrt-6 and fig-1. They too displayed enhanced oxygen sensing linked to increased ADL neuropeptide secretion. However, their effects on ADL pheromone sensation were not reported. The main conclusion I draw from these findings is that the ADL neurons are able to modulate oxygen sensitivity by relaying information about oxygen levels from the RMG circuit to locomotor circuits via neuropeptide secretion. It is not at all clear that loss of pheromone sensation in the qui-1 case is the cause for increased neuropeptide release, or whether it is just one out of the many outcomes of mutating this gene. A much cleaner and more revealing experiment could have been, for example, to examine worms lacking the functional pheromone receptor OCR-2 in ADL. In fact, unlike qui-1 mutants who showed diminished oxygen responses in ADL, previous work from the de Bono group (Fenk and de Bono 2017) demonstrated that ADL O2 response are normal in ocr-2 mutants, indicating a profound difference between loss of pheromone sensitivity due to receptor dysfunction (ocr-2) and the unknown and broad effects of qui-1.

    In fact, it would be interesting if the authors could explain or speculate how qui-1 eliminates ADL O2 responses, and how neuropeptide signaling from the RMG circuit via the NPR-22 neuropeptide receptor bypasses this lack of response and drives enhanced neuropeptide secretion in ADL, as they report.

    The work includes a transcriptomic analysis comparing ADL-specific gene expression between wild type and the qui-1 mutant. Unlike other experiments in the study, in which the specific effects of mutations were confirmed through rescue experiments and the use of additional alleles, thus eliminating potential confounds with background mutations, the transcriptomic experiment did not apply such controls. Therefore, it is hard to conclude whether the reported changes in transcription are due solely to the qui-1 mutation or to other unrelated genetic modifications in the mutant strain.

    Overall, except for where mentioned, the data presented are solid and consistent. However, the conclusion that the study reveals a molecular pathway for cross-modal plasticity is less convincing. The chain of events does not include some form of sensory loss, leading to subsequent, independent neural plasticity, as expected for cross-modal plasticity. Rather, a very broad genetic switch is described that can simultaneously change receptor abundance and neuropeptide release. Thus, an equally interesting and more coherent framing of the data could be that the study uncovered a genetic regulator, yet to be fully characterized, of oxygen-dependent behavior in a non-oxygen sensing neuron, adding to previous literature on neural circuit cross-talk.

  7. Version published to 10.1101/2021.03.11.435052v1 on bioRxiv