Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster
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Evaluation Summary:
This paper presents a novel and potentially highly useful approach to monitor circadian rhythms in specific tissues. The elegant reporter that the authors have built has the potential to become an important tool for understanding how different body clocks respond to various inputs and genetic manipulations. The authors already apply it to show that different clocks appear to be responding differently to loss of signaling from a key circadian neuropeptide in Drosophila melanogaster. However, it is difficult to determine whether these results, as currently presented and analyzed, provide new insight into the relationship between brain and peripheral clocks. The work is of interest to the community of biologists studying biological rhythms.
(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
Circadian clocks are highly conserved transcriptional regulators that control ~24 hr oscillations in gene expression, physiological function, and behavior. Circadian clocks exist in almost every tissue and are thought to control tissue-specific gene expression and function, synchronized by the brain clock. Many disease states are associated with loss of circadian regulation. How and when circadian clocks fail during pathogenesis remains largely unknown because it is currently difficult to monitor tissue-specific clock function in intact organisms. Here, we developed a method to directly measure the transcriptional oscillation of distinct neuronal and peripheral clocks in live, intact Drosophila , which we term L ocally A ctivatable B io L uminescence, or LABL. Using this method, we observed that specific neuronal and peripheral clocks exhibit distinct transcriptional properties. Loss of the receptor for PDF, a circadian neurotransmitter critical for the function of the brain clock, disrupts circadian locomotor activity but not all tissue-specific circadian clocks. We found that, while peripheral clocks in non-neuronal tissues were less stable after the loss of PDF signaling, they continued to oscillate. We also demonstrate that distinct clocks exhibit differences in their loss of oscillatory amplitude or their change in period, depending on their anatomical location, mutation, or fly age. Our results demonstrate that LABL is an effective tool that allows rapid, affordable, and direct real-time monitoring of individual clocks in vivo.
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Author Response
Reviewer #3 (Public Review):
Here, Johnstone et al. developed novel tools to study endogenous and tissue-specific circadian clocks, which control gene expression oscillation over a 24-hour period. They find that these genetically encoded luciferase-based tools, which they call LABL (Locally Activatable BioLuminiscence). Other known techniques monitor downstream products of circadian clock gene activity (ie, neuronal calcium imaging) or utilize terminal assays such as qRT-PCR or require removal of organs for ex vivo monitoring. The authors show that their LABL technique faithfully mimics the oscillations of gene expression seen with other techniques for broad circadian expression drivers and for neuronally specific expression drivers but show different patterns for non-neuronal, so-called peripheral clocks. These …
Author Response
Reviewer #3 (Public Review):
Here, Johnstone et al. developed novel tools to study endogenous and tissue-specific circadian clocks, which control gene expression oscillation over a 24-hour period. They find that these genetically encoded luciferase-based tools, which they call LABL (Locally Activatable BioLuminiscence). Other known techniques monitor downstream products of circadian clock gene activity (ie, neuronal calcium imaging) or utilize terminal assays such as qRT-PCR or require removal of organs for ex vivo monitoring. The authors show that their LABL technique faithfully mimics the oscillations of gene expression seen with other techniques for broad circadian expression drivers and for neuronally specific expression drivers but show different patterns for non-neuronal, so-called peripheral clocks. These results suggest that the canonical hierarchy of central clocks regulating peripheral clocks may need closer re-examination.
The conclusions of this paper are mostly well supported but three specific aspects need to be clarified or tested.
- Figures 5A, 6B, and 6E are critical for the conclusions of this paper and from what this reviewer can tell, they support these conclusions but the overlay of mutant and wild type on the same graphs obscures both. This reviewer would suggest including split graphs with wild type and mutant alone for independent evaluation.
We agree with the reviewer that our well-intended attempt to compare two conditions ended up confusing both. We therefore show the wild-type graphs separately and earlier for clear demonstration that LABL can be used with drivers that target specific neuronal clusters.
- Luminescence is a well-established, high-resolution real-time monitor; at the same time, my one concern is that luminescence via luciferase and feeding of luciferin substrate might be dependent on the host animal's feeding patterns. How do we know that the peaks and troughs of luminescence are not due to peaks and troughs of feeding and metabolism rather than peaks and troughs of circadian clock gene expression? Can the authors offer evidence to support the latter?
The reviewer makes a good point. We refer the reviewer to the section above on major concerns that we addressed above, point 4, “metabolism argument”.
- While the comparison of wild type to arrhythmic mutants is consistent with current data and seems to reflect faithful monitoring of tissue-specific circadian clock activity, the classic technique for demonstrating faithful monitoring of clock activity is to slow down or speed up the clock. The authors have themselves used this technique in previous publications, including using phosphosite-specific mutants of clock components and flies containing constitutively active or kinase-inactive regulators of clock activity. Another classic technique is to use short or long period mutants. Use of any of these types of mutants showing that they shift the luminescence rhythms generated by LABL would provide further evidence that LABL reflects endogenous, tissue-specific clock activity. Alternatively, monitoring the rhythm of a clock thought to be independent of central clock activity such as that in the antennae or Malpighian tubules and showing that this is not disrupted by central clock disruption would provide such support as well.
We thank the reviewer for this suggestion. We refer the reviewer to the major concerns that we addressed above, point 1, where we describe recording perS and perL mutants.
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Evaluation Summary:
This paper presents a novel and potentially highly useful approach to monitor circadian rhythms in specific tissues. The elegant reporter that the authors have built has the potential to become an important tool for understanding how different body clocks respond to various inputs and genetic manipulations. The authors already apply it to show that different clocks appear to be responding differently to loss of signaling from a key circadian neuropeptide in Drosophila melanogaster. However, it is difficult to determine whether these results, as currently presented and analyzed, provide new insight into the relationship between brain and peripheral clocks. The work is of interest to the community of biologists studying biological rhythms.
(This preprint has been reviewed by eLife. We include the public reviews from …
Evaluation Summary:
This paper presents a novel and potentially highly useful approach to monitor circadian rhythms in specific tissues. The elegant reporter that the authors have built has the potential to become an important tool for understanding how different body clocks respond to various inputs and genetic manipulations. The authors already apply it to show that different clocks appear to be responding differently to loss of signaling from a key circadian neuropeptide in Drosophila melanogaster. However, it is difficult to determine whether these results, as currently presented and analyzed, provide new insight into the relationship between brain and peripheral clocks. The work is of interest to the community of biologists studying biological rhythms.
(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):
Johnstone et al. developed an elegant luciferase-based construct (called LABL) to investigate in vivo the transcriptional dynamics controlled by the period promoter, one of the core circadian clock genes. Compared to previously generated reporter tools, the combination of LABL and the powerful UAS-GAL4 system provides an unmatched instrument to dissect with cell- and tissue-specific resolution the rhythmic transcriptional dynamics orchestrated by tissue-specific circadian clocks. Exploiting this strategy, the authors investigated rhythmic transcriptional dynamics in neuronal and peripheral clocks and their dependence from PDF signalling, a key neuropeptide for the functioning of Drosophila brain clock. They show that both neuronal and peripheral clocks exhibit distinct oscillatory properties and that they …
Reviewer #1 (Public Review):
Johnstone et al. developed an elegant luciferase-based construct (called LABL) to investigate in vivo the transcriptional dynamics controlled by the period promoter, one of the core circadian clock genes. Compared to previously generated reporter tools, the combination of LABL and the powerful UAS-GAL4 system provides an unmatched instrument to dissect with cell- and tissue-specific resolution the rhythmic transcriptional dynamics orchestrated by tissue-specific circadian clocks. Exploiting this strategy, the authors investigated rhythmic transcriptional dynamics in neuronal and peripheral clocks and their dependence from PDF signalling, a key neuropeptide for the functioning of Drosophila brain clock. They show that both neuronal and peripheral clocks exhibit distinct oscillatory properties and that they differentially behave upon loss of PDF signalling. Moreover, they uncover a peculiar ~60 h infradian rhythmic oscillation which is PDF signalling-dependent in neuronal clocks but not in most peripheral clocks. In sum, this manuscript provides an in-depth analysis of the oscillatory properties of neuronal and peripheral clocks in Drosophila, and whether they rely on PDF signalling to stabilize or sustain their oscillatory properties.
Strengths:
The LABL construct is widely applicable in animal models with advanced genetic toolkits, and represent a major advancement in our investigation of cell- and tissue-specific dynamics underlying the rhythmic transcription driven by circadian clocks. This tool definitely represents an advancement compared to previously available reporter lines which did not allow tissue-specific or in vivo analyses. Moreover, especially the experiments dedicated to peripheral clocks provide interesting and novel insights, as they have been characterized to a less degree compared to the brain ones. Finally, the peculiar ~60 h infradian rhythmic oscillation identified represents, to my knowledge, a previously unidentified aspect of biological clocks, showing the LABL construct might provide further advancement in our understanding of molecular oscillations.Weaknesses:
Although the LABL construct could potentially provide a great tool for the interrogation of circadian clock properties (in animal models), I personally think the authors are stretching this aspect too much towards the biomedical application. In several parts of the paper, they envisaged an application in medical and pathophysiological contexts. Although in principle I agree this might represent a future application, currently a genetically-encoded reporter construct cannot be used for human studies (if we exclude in vitro studies i.e. cell culture and organoids), limiting the actual impact that the authors did not fail to emphasize. Thus, the authors should be more specifically focused on the short-term benefits (monitoring disease progression in animal models, in vitro studies, etc...). However, this should not diminish the benefits that such a tool could provide for studies in animal models. On the other hand, the authors do not provide insights on the nature of the ~60 h infradian rhythms identified. This is a very important element of the biological findings of the paper, yet the authors simply state that such detailed investigation goes beyond the scope of the study. I personally think that such novel and unusual oscillatory dynamics should have been better explained, theoretically, and perhaps also experimentally, to strengthen the biological advances provided by this study. Additionally, the authors fail to correctly place this phenomenon in the context of the existing literature, as variation of oscillatory dynamics in constant condition (i.e. switch from ~24 h to ~12 h oscillations in marine models kept in DD), in peripheral organs (i.e. ~12 h transcriptional oscillation in mouse liver) and upon clock manipulation have been already reported, although to my knowledge not of ~60 h. Finally, a substantial part of the experiments is dedicated to unravel the contribution of PDF signalling in driving neuronal and peripheral oscillations. Although this neuropeptide is widely considered as key to synchronize brain clocks, this is not known for the peripheral clocks explored in this study. PDF has been shown to coordinate peripheral clocks (i.e. prothoracic gland, Myers et al. 2003), but to my knowledge not in muscles, gut, fat body, all tissues where the expression of the PDFR has not been reported. All considered, it is not unexpected to me how, for most of the peripheral clocks investigated, PDF signalling is not critical. -
Reviewer #2 (Public Review):
Johnstone et al introduce a novel method to study circadian rhythms in a tissue-specific manner in Drosophila. The idea is to use an intersectional approach to express a luciferase reporter only in specific tissues, and then to monitor rhythmic luciferase activity in real time by feeding flies with luciferine. The authors convincingly demonstrate that their approach works, and are even able to record from a small number of brain circadian neurons. This should prove to be a potent approach to understanding how circadian clocks behave throughout the body of flies, in response to different environmental inputs or genetic manipulations. To further demonstrate that their approach will prove useful, the authors focus on flies that are missing PDF-receptor, which mediates communication between circadian pacemaker …
Reviewer #2 (Public Review):
Johnstone et al introduce a novel method to study circadian rhythms in a tissue-specific manner in Drosophila. The idea is to use an intersectional approach to express a luciferase reporter only in specific tissues, and then to monitor rhythmic luciferase activity in real time by feeding flies with luciferine. The authors convincingly demonstrate that their approach works, and are even able to record from a small number of brain circadian neurons. This should prove to be a potent approach to understanding how circadian clocks behave throughout the body of flies, in response to different environmental inputs or genetic manipulations. To further demonstrate that their approach will prove useful, the authors focus on flies that are missing PDF-receptor, which mediates communication between circadian pacemaker neurons expressing the neuropeptide PDF and other brain and body clocks. There, however, the message becomes murky. Results are not very consistent, and the authors do not place clearly their results in the context of previous work in flies, or in mammals. Also, there are concerns with the way the authors monitored the progressive loss of circadian rhythmicity, which is important to establish the impact of PDF-receptor signaling on various body clocks. Thus, while technically interesting, the advance of this paper is still limited because it does not yet provide clear new insight into the relationship between brain and body clocks.
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Reviewer #3 (Public Review):
Here, Johnstone et al. developed novel tools to study endogenous and tissue-specific circadian clocks, which control gene expression oscillation over a 24-hour period. They find that these genetically encoded luciferase-based tools, which they call LABL (Locally Activatable BioLuminiscence). Other known techniques monitor downstream products of circadian clock gene activity (ie, neuronal calcium imaging) or utilize terminal assays such as qRT-PCR or require removal of organs for ex vivo monitoring. The authors show that their LABL technique faithfully mimics the oscillations of gene expression seen with other techniques for broad circadian expression drivers and for neuronally specific expression drivers but show different patterns for non-neuronal, so-called peripheral clocks. These results suggest that the …
Reviewer #3 (Public Review):
Here, Johnstone et al. developed novel tools to study endogenous and tissue-specific circadian clocks, which control gene expression oscillation over a 24-hour period. They find that these genetically encoded luciferase-based tools, which they call LABL (Locally Activatable BioLuminiscence). Other known techniques monitor downstream products of circadian clock gene activity (ie, neuronal calcium imaging) or utilize terminal assays such as qRT-PCR or require removal of organs for ex vivo monitoring. The authors show that their LABL technique faithfully mimics the oscillations of gene expression seen with other techniques for broad circadian expression drivers and for neuronally specific expression drivers but show different patterns for non-neuronal, so-called peripheral clocks. These results suggest that the canonical hierarchy of central clocks regulating peripheral clocks may need closer re-examination.
The conclusions of this paper are mostly well supported but three specific aspects need to be clarified or tested.
Figures 5A, 6B, and 6E are critical for the conclusions of this paper and from what this reviewer can tell, they support these conclusions but the overlay of mutant and wild type on the same graphs obscures both. This reviewer would suggest including split graphs with wild type and mutant alone for independent evaluation.
Luminescence is a well-established, high-resolution real-time monitor; at the same time, my one concern is that luminescence via luciferase and feeding of luciferin substrate might be dependent on the host animal's feeding patterns. How do we know that the peaks and troughs of luminescence are not due to peaks and troughs of feeding and metabolism rather than peaks and troughs of circadian clock gene expression? Can the authors offer evidence to support the latter?
While the comparison of wild type to arrhythmic mutants is consistent with current data and seems to reflect faithful monitoring of tissue-specific circadian clock activity, the classic technique for demonstrating faithful monitoring of clock activity is to slow down or speed up the clock. The authors have themselves used this technique in previous publications, including using phosphosite-specific mutants of clock components and flies containing constitutively active or kinase-inactive regulators of clock activity. Another classic technique is to use short or long period mutants. Use of any of these types of mutants showing that they shift the luminescence rhythms generated by LABL would provide further evidence that LABL reflects endogenous, tissue-specific clock activity. Alternatively, monitoring the rhythm of a clock thought to be independent of central clock activity such as that in the antennae or Malpighian tubules and showing that this is not disrupted by central clock disruption would provide such support as well.
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