Non-canonical function of an Hif-1α splice variant contributes to the sustained flight of locusts

  1. Ding Ding
  2. Jie Zhang
  3. Baozhen Du
  4. Xuanzhao Wang
  5. Li Hou
  6. Siyuan Guo
  7. Bing Chen
  8. Le Kang

  • Curated by eLife

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    Evaluation Summary:

    The hypoxia inducible factor (Hif) pathway was defined based on its role in cellular adaptation to hypoxia. In this paper, the authors examine the function of the pathway under 'physiological' normoxia in highly aerobic locust flight muscle. They find that a muscle-specific variant, Hif-1alpha2, is induced extensively by flying. By integrating bioinformatic analyses, measurements of gene expression and regulation, metabolites as well as redox regulation and flight assays, it is shown that Hif-1alpha2 plays an important role in sustaining prolonged flight by promoting glucose oxidation and upregulating a reactive oxygen species quencher (DJ-1). This study demonstrates the physiological requirement for two Hif-1a variants in a highly aerobic tissue in migratory locusts, a species that is both physiologically fascinating and a major agricultural pest. The work will be of interest to colleagues studying the physiology of muscles and flight.

    (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 and Reviewer 3 agreed to share their name with the authors.)

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Abstract

The hypoxia inducible factor (Hif) pathway is functionally conserved across metazoans in modulating cellular adaptations to hypoxia. However, the functions of this pathway under aerobic physiological conditions are rarely investigated. Here, we show that Hif-1α2, a locust Hif-1α isoform, does not induce canonical hypoxic responses but functions as a specific regulator of locust flight, which is a completely aerobic physiological process. Two Hif-1α splice variants were identified in locusts, a ubiquitously expressed Hif-1α1 and a muscle-predominantly expressed Hif-1α2. Hif-1α1 that induces typical hypoxic responses upon hypoxia exposure remains inactive during flight. By contrast, the expression of Hif-1α2, which lacks C-terminal transactivation domain, is less sensitive to oxygen tension but induced extensively by flying. Hif-1α2 regulates physiological processes involved in glucose metabolism and antioxidation during flight and sustains flight endurance by maintaining redox homeostasis through upregulating the production of a reactive oxygen species (ROS) quencher, DJ-1. Overall, this study reveals a novel Hif-mediated mechanism underlying prolonged aerobic physiological activity.

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  1. Version published to 10.7554/elife.74554 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    This is a fascinating study that apparently began with an original observation (a Hif-1a splice variant heretofore unexamined in insect flight muscles) that sparked the sort of "can't miss" question that all scientists crave, where any outcome is interesting. In this case, what are two Hif-1a variants doing in a highly aerobic tissue in migratory locusts, a species that is both physiologically fascinating and a major agricultural pest? The authors undertook a well-designed and thorough experimental study that used a broad swath of methods to examine bioinformatic data, tissue- and age-specific gene and protein expression, downstream regulation of metabolic genes and metabolites, upstream regulation by PHD, redox regulation, and effects on speed and duration of locusts during prolonged flight. Numerous molecular manipulations were performed to make the study rigorous and results easy to interpret. Ultimately, by using this highly integrative approach, the study provides a compelling picture that the Hif-1a2 splice variant plays a previously undescribed function by regulating Dj-1, which is both an antioxidant and a regulator of other anti-oxidant genes, thereby limiting oxidative damage during prolonged aerobic activity and long migratory flights.

    The study and its presentation have many strengths. These include the clear formation of a series of testable hypotheses and critical experiments, progressing from each set of experimental results to the next hypotheses and experiments, and an interesting and nuanced discussion of the results that is well framed in prior findings in other species (including birds and humans) that are similar or different in their physiology and behavior. Ultimately it is an interesting and thought-provoking paper, and a valuable contribution to knowledge in areas that encompass oxygen-related regulatory biology, insect physiology, and animal flight.

    We greatly appreciate the reviewer’s invaluable and helpful comments.

    1. Something that is present in a supplementary figure but not discussed in the text is a taxonomic consideration of the presence of Hif-1a splice variants in other insects. Are these unique to locusts or Orthoptera, or are they general to all insects? There are, for example, four Hif-1a splice variants in Drosophila, so the authors should discuss what is known and unknown in this realm.

    The reviewer raises a good point. We performed taxonomic analysis of Hif-1α splice variants across taxa based on transcriptome data and documentary reports (Figure 1-figure supplement 2). Hif-1α in the locust species generates two transcripts, i.e., the full-length Hif-1α1 and the short Hif-1α2 that lacks the domain C-TAD. We analyzed the transcriptome data of Deracantha onos (Orthoptera), Grylloblatta bifratrilecta (Grylloblattodea) and Periplaneta americana (Blattodea). Only in D. onos did we find Hif-1α transcript variants with structure similar to those in the locust. Previous reports also showed that the C-TAD domain as well as its inhibitor FIH are absent in Hif-1α at the genomic level in some complete metamorphosis insects, including wasps (Hymenoptera), fruit flies (Diptera), moths and butterflies (Lepidoptera). Thus, the C-TAD-lacked Hif-1α transcripts seem to commonly exist in different insect taxa. However, different from the Orthoptera species, the C-TAD-lacked transcripts of Hif-1α in other taxa are not generated from alternative splicing.

    We have added the Hif-1α splice variants of D. onos to Figure 1-figure supplement 2, and submitted these transcripts to NCBI with gene accession number ON137898 and ON137899. A statement of this issue have been added in Results as follows:

    “Evolutionary analysis revealed that such Hif-1α splice form also exists in other Orthoptera (Accession no. ON137898 and ON137899 for Deracantha onos), some birds (e.g., XP_025006307.1 for Gallus gallus and XP_013038471.1 for Anser cygnoides domesticus), and human (NP_851397.1). Additionally, the TADs of Hif-α have varied distributions amongst insects. In incomplete metamorphosis insects and beetles the Hif-α protein possesses two TADs (N-TAD and C-TAD), but in flies and moths the C-TAD and its inhibitor FIH are completely missing at the genomic level. Therefore, C-TAD-lacking Hif-1α transcripts, with distinct origins, seem to commonly exist in different insect taxa (Figure 1-figure supplement 2).” (Line 108-116)

    Meanwhile, we have given discussion as follows:

    “Alternative splicing may be a source of functional innovation for Hif-α in locusts. In this study, we found that Hif-1α in locust species generates two transcripts, i.e., the full-length Hif-1α1 and the short Hif-1α2 that lacks the C-TAD domain. The C-TAD of Hif-α is under strong selective pressure in invertebrates; it first appears in non-bilaterians (Nematostella vectensis) and has a varied distribution amongst invertebrates (Graham and Presnell, 2017). This domain and its inhibitor FIH are completely absent at the genomic level in some newly emerged insect species, including wasps (Hymenoptera), true flies (Diptera), moths and butterflies (Lepidoptera), all of which are outstanding flyers (Graham and Presnell, 2017). Genetic variations in the Hif pathway can affect the tracheal volume and flight performance of lowland butterfly populations under well oxygenate environment (Marden et al., 2013). This evidence combined with our findings, implies that the emergence of C-TAD-lacking Hif-1α transcripts is likely to be a substrate for flight adaptation in some insect species.” (Line 366-377)

    1. The most prominent unanswered question from a mechanistic standpoint is "what causes the Hif-1a2 variant to have unique upstream and downstream regulation?". Age and tissue specific expression of Hif-1a2 implies that the locust Hif-1a gene may have promoters that differently affect alternative splicing during development, and in an oxygen sensitive fashion in mature flight muscle. The paper states that lack of regulation of genes that inhibit mitochondria suggests that Hif-1a2 transcription factor activity is altered by absence of the C-TAD. Figure 6F is a compact summary of the functional differences, but a more complex supplementary figure showing a hypothesis that summarizes both the upstream and downstream regulatory details would help readers form a mechanistic understanding. The text could do this by elaborating a bit more on the ideas in lines 288-290.

    We’re glad to follow the reviewer’s suggestion and added a supplementary figure to present a mechanistic hypothesis (Figure 6-figure supplement 2). We added discussion on this issue:

    “The regulatory mechanism underlying the spatiotemporal expression of Hif-1α2 remains elusive. Alternative splicing is one of the main sources of spatiotemporally specific mRNA expression and proteomic diversity in eukaryotes. The diverse expression of alternatively spliced mRNA isoforms is usually attributed to alternative promoters or regulatory splicing factors (Fu and Ares, 2014; Russcher et al., 2007). Alternative promoters can produce a wide variety of transcripts at transcription initiation sites or even affect the splicing patterns of downstream exons (Zavolan et al., 2003). The regulatory splicing factors with cell-type–specific expression can bind specifically to enhancers or silencers of a premature mRNA to promote or repress splicing (Fu and Ares, 2014). Therefore, alternative usage of promoters or regulatory splicing factors could contribute to the age and tissue-specific expression of the locust Hif-1α transcripts (Figure 6-figure supplement 2). However, detailed mechanism requires further elucidation.”(Line 404-414)

    The downstream regulatory mechanism was discussed in Line 285-294.

    1. In the conclusion, the authors should perhaps be more explicit about the hypothesis that Hif-1a2, which is expressed in normoxia and more so at low oxygen tension, provides continuously variable expression of anti-oxidant genes so that protection is in place before the damage occurs. This is different from the way Hif-1a1 is typically activated only at very low oxygen tension, which in a highly active tissue may provide protective effects too late to prevent oxidative damage. Thinking in this way may stimulate experiments across time courses and/or graded oxygen tension that provide additional insight and further refine thinking about canonical versus non-canonical function of Hif gene variants. Such a discussion may be a springboard for pondering why all species don't do this. Or is it possible that they do, and this study is only the first glimpse?

    We appreciate the reviewer for providing the thoughtful insight for discussion. Following the reviewer’s suggestion, we have given a discussion on this topic in Discussion as follows:

    “The two Hif-1α splices may coordinate their roles in long-lasting flight tasks. In locusts, the canonical role of Hif pathway is modulated by Hif-1α1, which regulates metabolic reprogramming and possibly controls tracheal growth under low oxygen tension. However, the abundant tracheal system of the locust flight muscle may keep the intracellular oxygen tension above the low level that triggers Hif-1α1 stability. Meanwhile, the relatively easy task of flying with weight support on a flight mill in the present study may render the role of Hif-1α1 in flight muscle undetectable. Nevertheless, when it comes to highly active tissue, Hif-1α1 may provide protective effects too late to prevent oxidative damage. Instead, Hif-1α2, which is expressed in normoxia and has a graded activity with decreasing oxygen, provides continuously variable expression of antioxidant genes so that protection is in place before the damage occurs. This is different from the way Hif-1α1 is typically activated only at very low oxygen tension. As shown in Figure 1-figure supplement 2, the similar transcript form of locust Hif-1α2 also exists in some other insect species and birds. Therefore, the Hif-1α2-mediated protective mechanism is possibly applicable to other flying animals, with the locust in this study as the first glimpse.”(Line 390-403)

    1. On a related note, the discussion may benefit by considering other findings regarding oxidative damage caused by flight in insects that differ in their flight physiology, behavior and life history. (https://academic.oup.com/biomedgerontology/article/61/2/136/542463; https://www.science.org/doi/abs/10.1126/science.aah4634; https://journals.biologists.com/jeb/article/221/6/jeb171009/246/Enzyme-polymorphism-oxygen-and-injury-a-lipidomic. Have these species independently evolved different mechanisms, or might this new discovery be part of a suite of mechanisms for oxygen-related physiological and protective mechanisms in insect flight muscles?

    Thanks for providing these informative documents. We have added a discussion on this issue:

    “Additionally, oxidative damage caused by flight in insects differs in their flight physiology, behavior and life history. A sustained flight throughout life can cause a higher mortality rate to Drosophila (Magwere et al., 2006). Flight activity of honey bees directly leads to increased oxidative damage, which in turn detrimentally affects their flight performance and foraging ability (Margotta et al., 2018). Insects have evolved a series of adaptive strategies to cope with intermittent and migratory flight-induced oxidative stress. Glanville Fritillary butterflies carrying Sdhd M allele are associated with the activated Hif signalling, reduced metabolic rate, and larger tracheal volume in larvae, and these associations contribute to less oxidative injury in flight muscle and better flight performance during intermittent flight in adults (Marden et al., 2021; Pekny et al., 2018). Nectar feeding hawkmoths use their antioxidant stores during migratory flight and through PPP to produce an antioxidant potential to recover from oxidative damage during rest (Levin et al., 2017). While, the utilization of PPP was reported to be positively correlated with the activation of Hif pathway (Sadiku and Walmsley, 2019; Tokuda et al., 2019). Therefore, at the molecular level, the Hif pathway likely plays a central role in regulating redox homeostasis during insect flight.”(Line 321-335)

  3. eLife

    Evaluation Summary:

    The hypoxia inducible factor (Hif) pathway was defined based on its role in cellular adaptation to hypoxia. In this paper, the authors examine the function of the pathway under 'physiological' normoxia in highly aerobic locust flight muscle. They find that a muscle-specific variant, Hif-1alpha2, is induced extensively by flying. By integrating bioinformatic analyses, measurements of gene expression and regulation, metabolites as well as redox regulation and flight assays, it is shown that Hif-1alpha2 plays an important role in sustaining prolonged flight by promoting glucose oxidation and upregulating a reactive oxygen species quencher (DJ-1). This study demonstrates the physiological requirement for two Hif-1a variants in a highly aerobic tissue in migratory locusts, a species that is both physiologically fascinating and a major agricultural pest. The work will be of interest to colleagues studying the physiology of muscles and flight.

    (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 and Reviewer 3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    This is a fascinating study that apparently began with an original observation (a Hif-1a splice variant heretofore unexamined in insect flight muscles) that sparked the sort of "can't miss" question that all scientists crave, where any outcome is interesting. In this case, what are two Hif-1a variants doing in a highly aerobic tissue in migratory locusts, a species that is both physiologically fascinating and a major agricultural pest? The authors undertook a well-designed and thorough experimental study that used a broad swath of methods to examine bioinformatic data, tissue- and age-specific gene and protein expression, downstream regulation of metabolic genes and metabolites, upstream regulation by PHD, redox regulation, and effects on speed and duration of locusts during prolonged flight. Numerous molecular manipulations were performed to make the study rigorous and results easy to interpret. Ultimately, by using this highly integrative approach, the study provides a compelling picture that the Hif-1a2 splice variant plays a previously undescribed function by regulating Dj-1, which is both an antioxidant and a regulator of other anti-oxidant genes, thereby limiting oxidative damage during prolonged aerobic activity and long migratory flights.

    The study and its presentation have many strengths. These include the clear formation of a series of testable hypotheses and critical experiments, progressing from each set of experimental results to the next hypotheses and experiments, and an interesting and nuanced discussion of the results that is well framed in prior findings in other species (including birds and humans) that are similar or different in their physiology and behavior. Ultimately it is an interesting and thought-provoking paper, and a valuable contribution to knowledge in areas that encompass oxygen-related regulatory biology, insect physiology, and animal flight.

    Something that is present in a supplementary figure but not discussed in the text is a taxonomic consideration of the presence of Hif-1a splice variants in other insects. Are these unique to locusts or Orthoptera, or are they general to all insects? There are, for example, four Hif-1a splice variants in Drosophila, so the authors should discuss what is known and unknown in this realm.

    The most prominent unanswered question from a mechanistic standpoint is "what causes the Hif-1a2 variant to have unique upstream and downstream regulation?". Age and tissue specific expression of Hif-1a2 implies that the locust Hif-1a gene may have promoters that differently affect alternative splicing during development, and in an oxygen sensitive fashion in mature flight muscle. The paper states that lack of regulation of genes that inhibit mitochondria suggests that Hif-1a2 transcription factor activity is altered by absence of the C-TAD. Figure 6F is a compact summary of the functional differences, but a more complex supplementary figure showing a hypothesis that summarizes both the upstream and downstream regulatory details would help readers form a mechanistic understanding. The text could do this by elaborating a bit more on the ideas in lines 288-290.

    In the conclusion, the authors should perhaps be more explicit about the hypothesis that Hif-1a2, which is expressed in normoxia and more so at low oxygen tension, provides continuously variable expression of anti-oxidant genes so that protection is in place before the damage occurs. This is different from the way Hif-1a1 is typically activated only at very low oxygen tension, which in a highly active tissue may provide protective effects too late to prevent oxidative damage. Thinking in this way may stimulate experiments across time courses and/or graded oxygen tension that provide additional insight and further refine thinking about canonical versus non-canonical function of Hif gene variants. Such a discussion may be a springboard for pondering why all species don't do this. Or is it possible that they do, and this study is only the first glimpse?

    On a related note, the discussion may benefit by considering other findings regarding oxidative damage caused by flight in insects that differ in their flight physiology, behavior and life history. (https://academic.oup.com/biomedgerontology/article/61/2/136/542463; https://www.science.org/doi/abs/10.1126/science.aah4634; https://journals.biologists.com/jeb/article/221/6/jeb171009/246/Enzyme-polymorphism-oxygen-and-injury-a-lipidomic). Have these species independently evolved different mechanisms, or might this new discovery be part of a suite of mechanisms for oxygen-related physiological and protective mechanisms in insect flight muscles?

  5. eLife

    Reviewer #2 (Public Review):

    I am impressed by the comprehensiveness of the work. A caveat is that I am not a molecular biologist and so my expertise in this area is lacking, particularly with regards to the lab-based methodology. My only primary concern (or confusion) with the paper is the link between Hif-1alpha2, which is shown to be induced by flying, and the upregulation of glucose oxidation facilitating prolonged flight. The comparative physiology greats, August Krogh and Torkel Weis-Fogh, showed many years ago that locust flight was only initially powered by carbohydrate catabolism, and that sustained locust flight is powered by lipid catabolism. I believe more recent work has confirmed this finding. Thus, I am unconvinced, that the upregulation of glucose oxidation is likely to facilitate prolonged flight. In my mind, I could imagine that it may rather help to augment the power output of initial flight, when locust metabolic requirements are greatest as they must power the requirements of ascent. Thereafter, I think locusts switch to lipid catabolism, and generally utilise thermal currents and tail winds to migrate. I could be convinced otherwise. Perhaps this just needs to be better clarified in the paper.

  6. eLife

    Reviewer #3 (Public Review):

    The functions of hypoxia-inducible factor (Hif) in cellular adaptations to hypoxia have been widely investigated; however, the roles of Hif in aerobic conditions have drawn less attention. In this study, the authors identified the expression of Hif-1a2, a splice variant of Hif-1a, in locusts flight muscles and found that Hif-1a2 sustains prolonged flight behavior of flight muscles independent of oxygen concentrations. Hif-1a2 activates DJ-1, an oxidative stress sensor, to maintain the redox homeostasis in flight muscles. Long-term flight is generally energetically demanding and unfavorable to tissues and animals and these findings elucidate the mechanisms underlying how flight muscle cells alleviate oxidative damages through unconventional roles of Hif-1a2.

    The authors have clearly demonstrated from the expression of Hif-1a variants in locusts to the direct target of Hif-1a2 in flight muscles with appropriate methods and experiments. It is well supported by a series of data that Hif-1a2 is specific to flight muscles and is required for prolonged flight. Moreover, the transcriptome analysis indicated differentially expressed genes in Hif-1a2 RNAi genetic background including glucose metabolism and DJ-1. Despite the Hif-1a2-dependent expression, genes involved in glucose metabolism are generally dispensable for long flight behaviors. However, loss of Hif-1a2 elevates the level of ROS identical to the phenotype led by DJ-1 knock-down. Further, the authors probed that Hif-1a2 directly binds to DJ-1 as a downstream for the long flight. Overall, experiments are well executed and analyzed, and the claims are sufficiently supported by the data.

  7. Version published to 10.1101/2021.11.10.468104v1 on bioRxiv