E2F/Dp inactivation in fat body cells triggers systemic metabolic changes
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Evaluation Summary:
Zappia et al investigate the function of E2F transcriptional activity in the development of Drosophila, with the aim of understanding which targets the E2F/Dp transcription factors control to facilitate development. They characterize changes in gene expression by proteomic profiling, and characterize Dp loss phenotypes in muscle, fat body, and the whole body. They document low levels of glycolytic intermediates and circulating trehalose, which is traced to loss of Dp in the fat body. Strikingly, this phenotype and the resulting lethality during the pupal stage (metamorphosis) can be rescued by increasing dietary sugar. Hence the authors provides new insights in the function of E2F/Dp.
(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.)
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Abstract
The E2F transcription factors play a critical role in controlling cell fate. In Drosophila , the inactivation of E2F in either muscle or fat body results in lethality, suggesting an essential function for E2F in these tissues. However, the cellular and organismal consequences of inactivating E2F in these tissues are not fully understood. Here, we show that the E2F loss exerts both tissue-intrinsic and systemic effects. The proteomic profiling of E2F-deficient muscle and fat body revealed that E2F regulates carbohydrate metabolism, a conclusion further supported by metabolomic profiling. Intriguingly, animals with E2F-deficient fat body had a lower level of circulating trehalose and reduced storage of fat. Strikingly, a sugar supplement was sufficient to restore both trehalose and fat levels, and subsequently rescued animal lethality. Collectively, our data highlight the unexpected complexity of E2F mutant phenotype, which is a result of combining both tissue-specific and systemic changes that contribute to animal development.
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Author Response:
Reviewer #1:
Zappia et al investigate the function of E2F transcriptional activity in the development of Drosophila, with the aim of understanding which targets the E2F/Dp transcription factors control to facilitate development. They follow up two of their previous papers (PMID 29233476, 26823289) that showed that the critical functions of Dp for viability during development reside in the muscle and the fat body. They use Dp mutants, and tissue-targetted RNAi against Dp to deplete both activating and repressive E2F functions, focussing primarily on functions in larval muscle and fat body. They characterize changes in gene expression by proteomic profiling, bypassing the typical RNAseq experiments, and characterize Dp loss phenotypes in muscle, fat body, and the whole body. Their analysis revealed a consistent, …
Author Response:
Reviewer #1:
Zappia et al investigate the function of E2F transcriptional activity in the development of Drosophila, with the aim of understanding which targets the E2F/Dp transcription factors control to facilitate development. They follow up two of their previous papers (PMID 29233476, 26823289) that showed that the critical functions of Dp for viability during development reside in the muscle and the fat body. They use Dp mutants, and tissue-targetted RNAi against Dp to deplete both activating and repressive E2F functions, focussing primarily on functions in larval muscle and fat body. They characterize changes in gene expression by proteomic profiling, bypassing the typical RNAseq experiments, and characterize Dp loss phenotypes in muscle, fat body, and the whole body. Their analysis revealed a consistent, striking effect on carbohydrate metabolism gene products. Using metabolite profiling, they found that these effects extended to carbohydrate metabolism itself. Considering that most of the literature on E2F/Dp targets is focused on the cell cycle, this paper conveys a new discovery of considerable interest. The analysis is very good, and the data provided supports the authors' conclusions quite definitively. One interesting phenotype they show is low levels of glycolytic intermediates and circulating trehalose, which is traced to loss of Dp in the fat body. Strikingly, this phenotype and the resulting lethality during the pupal stage (metamorphosis) could be rescued by increasing dietary sugar. Overall the paper is quite interesting. It's main limitation in my opinion is a lack of mechanistic insight at the gene regulation level. This is due to the authors' choice to profile protein, rather than mRNA effects, and their omission of any DNA binding (chromatin profiling) experiments that could define direct E2F1/ or E2F2/Dp targets.
We appreciate the reviewer’s comment. Based on previously published chromatin profiling data for E2F/Dp and Rbf in thoracic muscles (Zappia et al 2019, Cell Reports 26, 702–719) we discovered that both Dp and Rbf are enriched upstream the transcription start site of both cell cycle genes and metabolic genes (Figure 5 in Zappia et al 2019, Cell Reports 26, 702–719). Thus, our data is consistent with the idea that the E2F/Rbf is binding to the canonical target genes in addition to a new set of target genes encoding proteins involved in carbohydrate metabolism. We think that E2F takes on a new role, and rather than being re-targeted away from cell cycle genes. We agree that the mechanistic insight would be relevant to further explore.
Reviewer #2:
The study sets out to answer what are the tissue specific mechanisms in fat and muscle regulated by the transcription factor E2F are central to organismal function. The study also tries to address which of these roles of E2F are cell intrinsic and which of these mechanisms are systemic. The authors look into the mechanisms of E2F/Dp through knockdown experiments in both the fat body* (see weakness) and muscle of drosophila. They identify that muscle E2F contributes to fat body development but fat body KD of E2F does not affect muscle function. To then dissect the cause of adult lethality in flies, the authors proteomic and metabolomic profiling of fat and muscle to gain insights. While in the muscle, the cause seems to be an as of yet undetermined systemic change , the authors do conclude that adult lethality in fat body specific Dp knockdown is the result of decrease trehalose in the hemolymph and defects in lipid production in these flies. The authors then test this model by presenting fat body specific Dp knockdown flies with high sugar diet and showing adult survival is rescued. This study concurs with and adds to the emerging idea from human studies that E2F/Dp is critical for more than just its role in the cell-cycle and functions as a metabolic regulator in a tissue-specific manner. This study will be of interest to scientists studying inter-organ communication between muscle and fat.
The conclusions of this paper are partially supported by data. The weaknesses can be mitigated by specific experiments and will likely bolster conclusions.
- This study relies heavily on the tissue specificity of the Gal4 drivers to study fat-muscle communication by E2F. The authors have convincingly confirmed that the cg-Gal4 driver is never turned on in the muscle and vice versa for Dmef2-Gal4. However, the cg-Gal4 driver itself is capable of turning on expression in the fat body cells and is also highly expressed in hemocytes (macrophage-like cells in flies). In fact, cg-Gal4 is used in numerous studies e.g.:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4125153/ to study the hemocytes and fat in combination. Hence, it is difficult to assess what contribution hemocytes provide to the conclusions for fat-muscle communication. To mitigate this, the authors could test whether Lpp-Gal4>Dp-RNAi (Lpp-Gal4 drives expression exclusively in fat body in all stages) or use ppl-Gal4 (which is expressed in the fat, gut, and brain) but is a weaker driver than cg. It would be good if they could replicate their findings in a subset of experiments performed in Figure 1-4.
This is indeed an important point. We apologize for previously not including this information. Reference is now on page 7.
Another fat body driver, specifically expressed in fat body and not in hemocytes, as cg-GAL4, was tested in previous work (Guarner et al Dev Cell 2017). The driver FB-GAL4 (FBti0013267), and more specifically the stock yw; P{w[+mW.hs]=GawB}FB P{w[+m*] UAS-GFP 1010T2}#2; P{w[+mC]=tubP-GAL80[ts]}2, was used to induce the loss of Dp in fat body in a time-controlled manner using tubGAL80ts. The phenotype induced in larval fat body of FB>DpRNAi,gal80TS recapitulates findings related to DNA damage response characterized in both Dp -/- and CG>Dp- RNAi (see Figure 5A-B, Guarner et al Dev Cell 2017). The activation of DNA damage response upon the loss of Dp was thoroughly studied in Guarner et al Dev Cell 2017. The appearance of binucleates in cg>DpRNAi is presumably the result of the abnormal transcription of multiple G2/M regulators in cells that have been able to repair DNA damage and to resume S-phase (see discussion in Guarner et al Dev Cell 2017). More details regarding the fully characterized DNA damage response phenotype were added on page 6 & 7 of manuscript.
Additionally, r4-GAL4 was also used to drive Dp-RNAi specifically to fat body. But since this driver is weaker than cg-GAL4, the occurrence of binucleated cells in r4>DpRNAi fat body was mild (see Figure R1 below).
As suggested by the reviewer, Lpp-GAL4 was used to knock down the expression of Dp specifically in fat body. All animals Lpp>DpRNAi died at pupa stage. New viability data were included in Figure 1-figure supplement 1. Also, larval fat body were dissected and stained with phalloidin and DAPI to visualize overall tissue structure. Binucleated cells were present in Lpp>DpRNAi fat body but not in the control Lpp>mCherry-RNAi (Figure 2-figure supplement 1B). These results were added to manuscript on page 7.
Furthermore, Dp expression was knockdowned using a hemocyte-specific driver, hml-GAL4. No defects were detected in animal viability (data not shown).
Thus, overall, we conclude that hemocytes do not seem to contribute to the formation of binucleated-cells in cg>Dp-RNAi fat body.
Finally, since no major phenotype was found in muscles when E2F was inactivated in fat body (please see point 3 for more details), we consider that the inactivation E2F in both fat body and hemocytes did not alter the overall muscle morphology. Thus, exploring the contribution of cg>Dp-RNAi hemocytes in muscles would not be very informative.
- The authors perform a proteomics analysis on both fat body and muscle of control or the respective tissue specific knockdown of Dp. However, the authors denote technical limitations to procuring enough third instar larval muscle to perform proteomics and instead use thoracic muscles of the pharate pupa. While the technical limitations are understandable, this does raise a concern of comparing fat body and muscle proteomics at two distinct stages of fly development and likely contributes to differences seen in the proteomics data. This may impact the conclusions of this paper. It would be important to note this caveat of not being able to compare across these different developmental stage datasets.
We appreciate the suggestion of the reviewer. This caveat was noted and included in the manuscript. Please see page 11.
- The authors show that the E2F signaling in the muscle controls whether binucleate fat body nuclei appear. In other words, is the endocycling process in fat body affected if muscle E2F function is impaired. However, they conclude that imparing E2F function in fat does not affect muscle. While muscle organization seems fine, it does appear that nuclear levels of Dp are higher in muscles during fat specific knock-down of Dp (Figure 1A, column 2 row 3, for cg>Dp-RNAi). Also there is an increase in muscle area when fat body E2F function is impaired. This change is also reflected in the quantification of DLM area in Figure 1B. But the authors don't say much about elevated Dp levels in muscle or increased DLM area of Fat specific Dp KD. Would the authors not expect Dp staining in muscle to be normal and similar to mCherry-RNAi control in Cg>dpRNAi? The authors could consider discussing and contextualizing this as opposed to making a broad statement regarding muscle function all being normal. Perhaps muscle function may be different, perhaps better when E2F function in fat is impaired.
The overall muscle structure was examined in animals staged at third instar larva (Figure 1A-B). No defects were detected in muscle size between cg>Dp-RNAi animals and controls. In addition, the expression of Dp was not altered in cg>Dp-RNAi muscles compared to control muscles. The best developmental stage to compare the muscle structure between Mef2>Dp-RNAi and cg>Dp-RNAi animals is actually third instar larva, prior to their lethality at pupa stage (Figure 1- figure supplement 1).
Based on the reviewer’s comment, we set up a new experiment to further analyze the phenotype at pharate stage. However, when we repeated this experiment, we did not recover cg>Dp-RNAi pharate, even though 2/3 of Mef2>Dp-RNAi animals survived up to late pupal stage. We think that this is likely due to the change in fly food provider. Since most cg>DpRNAi animals die at early pupal stage (>75% animals, Figure 1-figure supplement 1), pharate is not a good representative developmental stage to examine phenotypes. Therefore, panels were removed.
Text was revised accordingly (page 6).
- In lines 376-380, the authors make the argument that muscle-specific knockdown can impair the ability of the fat body to regulate storage, but evidence for this is not robust. While the authors refer to a decrease in lipid droplet size in figure S4E this is not a statistically significant decrease. In order to make this case, the authors would want to consider performing a triglyceride (TAG) assay, which is routinely performed in flies.
Our conclusions were revised and adjusted to match our data. The paragraph was reworded to highlight the outcome of the triglyceride assay, which was previously done. We realized the reference to Figure 6H that shows the triglyceride (TAG) assay was missing on page 17. Please see page 17 and page 21 of discussion.
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Evaluation Summary:
Zappia et al investigate the function of E2F transcriptional activity in the development of Drosophila, with the aim of understanding which targets the E2F/Dp transcription factors control to facilitate development. They characterize changes in gene expression by proteomic profiling, and characterize Dp loss phenotypes in muscle, fat body, and the whole body. They document low levels of glycolytic intermediates and circulating trehalose, which is traced to loss of Dp in the fat body. Strikingly, this phenotype and the resulting lethality during the pupal stage (metamorphosis) can be rescued by increasing dietary sugar. Hence the authors provides new insights in the function of E2F/Dp.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private …
Evaluation Summary:
Zappia et al investigate the function of E2F transcriptional activity in the development of Drosophila, with the aim of understanding which targets the E2F/Dp transcription factors control to facilitate development. They characterize changes in gene expression by proteomic profiling, and characterize Dp loss phenotypes in muscle, fat body, and the whole body. They document low levels of glycolytic intermediates and circulating trehalose, which is traced to loss of Dp in the fat body. Strikingly, this phenotype and the resulting lethality during the pupal stage (metamorphosis) can be rescued by increasing dietary sugar. Hence the authors provides new insights in the function of E2F/Dp.
(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.)
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Reviewer #1 (Public Review):
Zappia et al investigate the function of E2F transcriptional activity in the development of Drosophila, with the aim of understanding which targets the E2F/Dp transcription factors control to facilitate development. They follow up two of their previous papers (PMID 29233476, 26823289) that showed that the critical functions of Dp for viability during development reside in the muscle and the fat body. They use Dp mutants, and tissue-targetted RNAi against Dp to deplete both activating and repressive E2F functions, focussing primarily on functions in larval muscle and fat body. They characterize changes in gene expression by proteomic profiling, bypassing the typical RNAseq experiments, and characterize Dp loss phenotypes in muscle, fat body, and the whole body. Their analysis revealed a consistent, striking …
Reviewer #1 (Public Review):
Zappia et al investigate the function of E2F transcriptional activity in the development of Drosophila, with the aim of understanding which targets the E2F/Dp transcription factors control to facilitate development. They follow up two of their previous papers (PMID 29233476, 26823289) that showed that the critical functions of Dp for viability during development reside in the muscle and the fat body. They use Dp mutants, and tissue-targetted RNAi against Dp to deplete both activating and repressive E2F functions, focussing primarily on functions in larval muscle and fat body. They characterize changes in gene expression by proteomic profiling, bypassing the typical RNAseq experiments, and characterize Dp loss phenotypes in muscle, fat body, and the whole body. Their analysis revealed a consistent, striking effect on carbohydrate metabolism gene products. Using metabolite profiling, they found that these effects extended to carbohydrate metabolism itself. Considering that most of the literature on E2F/Dp targets is focused on the cell cycle, this paper conveys a new discovery of considerable interest. The analysis is very good, and the data provided supports the authors' conclusions quite definitively. One interesting phenotype they show is low levels of glycolytic intermediates and circulating trehalose, which is traced to loss of Dp in the fat body. Strikingly, this phenotype and the resulting lethality during the pupal stage (metamorphosis) could be rescued by increasing dietary sugar. Overall the paper is quite interesting. It's main limitation in my opinion is a lack of mechanistic insight at the gene regulation level. This is due to the authors' choice to profile protein, rather than mRNA effects, and their omission of any DNA binding (chromatin profiling) experiments that could define direct E2F1/ or E2F2/Dp targets.
-
Reviewer #2 (Public Review):
The study sets out to answer what are the tissue specific mechanisms in fat and muscle regulated by the transcription factor E2F are central to organismal function. The study also tries to address which of these roles of E2F are cell intrinsic and which of these mechanisms are systemic. The authors look into the mechanisms of E2F/Dp through knockdown experiments in both the fat body* (see weakness) and muscle of drosophila. They identify that muscle E2F contributes to fat body development but fat body KD of E2F does not affect muscle function. To then dissect the cause of adult lethality in flies, the authors proteomic and metabolomic profiling of fat and muscle to gain insights. While in the muscle, the cause seems to be an as of yet undetermined systemic change , the authors do conclude that adult …
Reviewer #2 (Public Review):
The study sets out to answer what are the tissue specific mechanisms in fat and muscle regulated by the transcription factor E2F are central to organismal function. The study also tries to address which of these roles of E2F are cell intrinsic and which of these mechanisms are systemic. The authors look into the mechanisms of E2F/Dp through knockdown experiments in both the fat body* (see weakness) and muscle of drosophila. They identify that muscle E2F contributes to fat body development but fat body KD of E2F does not affect muscle function. To then dissect the cause of adult lethality in flies, the authors proteomic and metabolomic profiling of fat and muscle to gain insights. While in the muscle, the cause seems to be an as of yet undetermined systemic change , the authors do conclude that adult lethality in fat body specific Dp knockdown is the result of decrease trehalose in the hemolymph and defects in lipid production in these flies. The authors then test this model by presenting fat body specific Dp knockdown flies with high sugar diet and showing adult survival is rescued. This study concurs with and adds to the emerging idea from human studies that E2F/Dp is critical for more than just its role in the cell-cycle and functions as a metabolic regulator in a tissue-specific manner. This study will be of interest to scientists studying inter-organ communication between muscle and fat.
The conclusions of this paper are partially supported by data. The weaknesses can be mitigated by specific experiments and will likely bolster conclusions.
This study relies heavily on the tissue specificity of the Gal4 drivers to study fat-muscle communication by E2F. The authors have convincingly confirmed that the cg-Gal4 driver is never turned on in the muscle and vice versa for Dmef2-Gal4. However, the cg-Gal4 driver itself is capable of turning on expression in the fat body cells and is also highly expressed in hemocytes (macrophage-like cells in flies). In fact, cg-Gal4 is used in numerous studies e.g.:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4125153/ to study the hemocytes and fat in combination. Hence, it is difficult to assess what contribution hemocytes provide to the conclusions for fat-muscle communication. To mitigate this, the authors could test whether Lpp-Gal4>Dp-RNAi (Lpp-Gal4 drives expression exclusively in fat body in all stages) or use ppl-Gal4 (which is expressed in the fat, gut, and brain) but is a weaker driver than cg. It would be good if they could replicate their findings in a subset of experiments performed in Figure 1-4.
The authors perform a proteomics analysis on both fat body and muscle of control or the respective tissue specific knockdown of Dp. However, the authors denote technical limitations to procuring enough third instar larval muscle to perform proteomics and instead use thoracic muscles of the pharate pupa. While the technical limitations are understandable, this does raise a concern of comparing fat body and muscle proteomics at two distinct stages of fly development and likely contributes to differences seen in the proteomics data. This may impact the conclusions of this paper. It would be important to note this caveat of not being able to compare across these different developmental stage datasets.
The authors show that the E2F signaling in the muscle controls whether binucleate fat body nuclei appear. In other words, is the endocycling process in fat body affected if muscle E2F function is impaired. However, they conclude that imparing E2F function in fat does not affect muscle. While muscle organization seems fine, it does appear that nuclear levels of Dp are higher in muscles during fat specific knock-down of Dp (Figure 1A, column 2 row 3, for cg>Dp-RNAi). Also there is an increase in muscle area when fat body E2F function is impaired. This change is also reflected in the quantification of DLM area in Figure 1B. But the authors don't say much about elevated Dp levels in muscle or increased DLM area of Fat specific Dp KD. Would the authors not expect Dp staining in muscle to be normal and similar to mCherry-RNAi control in Cg>dpRNAi? The authors could consider discussing and contextualizing this as opposed to making a broad statement regarding muscle function all being normal. Perhaps muscle function may be different, perhaps better when E2F function in fat is impaired.
In lines 376-380, the authors make the argument that muscle-specific knockdown can impair the ability of the fat body to regulate storage, but evidence for this is not robust. While the authors refer to a decrease in lipid droplet size in figure S4E this is not a statistically significant decrease. In order to make this case, the authors would want to consider performing a triglyceride (TAG) assay, which is routinely performed in flies.
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