The embryonic role of juvenile hormone in the firebrat, Thermobia domestica, reveals its function before its involvement in metamorphosis

  1. James W. Truman
  2. Lynn M. Riddiford
  3. Barbora Konopová
  4. Marcela Nouzova
  5. Fernando Noriega
  6. Michelle Herko

  • Curated by eLife

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    eLife assessment

    This important study presents findings regarding the role of Juvenile Hormone in development and cell differentiation in the ametabolous insect Thermobia domestica, providing an in-depth analysis of JH's roles in a member of this basally branching group. The evidence supporting the claims of the authors is convincing, drawing on a broad range of approaches and variety of experimental techniques. While the interpretation of this work in the wider context regarding its relevance for the evolution of metamorphosis is in some places overly speculative, the work will be of interest to evolutionary developmental biologists studying hormonal control of development and to entomologists studying the evolution of metamorphosis.

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Abstract

Juvenile hormone (JH) is a key regulator of insect metamorphosis. To understand its role before metamorphosis originated, we studied JH action in the ametabolous firebrat, Thermobia domestica . JH levels peak late in embryogenesis and are low through early juvenile stages. Chemical suppression of embryonic JH synthesis by 7-ethoxyprecocene blocks embryonic differentiation, but the latter is restored with exogenous JH. Premature exposure of younger embryos to JH suppresses growth and morphogenesis and the expression of morphogens, such as myoglianin. These embryos switch to premature differentiation as shown by muscle formation and synthesis of cuticle like that of later embryonic stages.

We hypothesize that this ancestral role of JH in supporting tissue differentiation was later exploited for the evolution of metamorphosis. In embryos, the temporal separation of morphogen signaling and JH secretion results in morphogenesis preceding differentiation. With the evolution of metamorphosis, embryonic morphogen systems were redeployed during juvenile growth for morphogenesis of imaginal primordia. JH was also redeployed, but it now occurred with morphogen signaling. This co-occurrence resulted in JH maintaining a juvenile quality to the bud, which the morphogens positive allometric growth. The disappearance of JH late in growth then allowed the unantagonized morphogens to drive the primordia into metamorphosis.

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  1. eLife

    eLife assessment

    This important study presents findings regarding the role of Juvenile Hormone in development and cell differentiation in the ametabolous insect Thermobia domestica, providing an in-depth analysis of JH's roles in a member of this basally branching group. The evidence supporting the claims of the authors is convincing, drawing on a broad range of approaches and variety of experimental techniques. While the interpretation of this work in the wider context regarding its relevance for the evolution of metamorphosis is in some places overly speculative, the work will be of interest to evolutionary developmental biologists studying hormonal control of development and to entomologists studying the evolution of metamorphosis.

  2. eLife

    Reviewer #1 (Public Review):

    Summary:
    This paper provides strong evidence for the roles of JH in an ametabolous insect species. In particular, it demonstrates that:
    • JH shifts embryogenesis from a growth mode to a differentiation mode and is responsible for terminal differentiation during embryogenesis. This, and other JH roles, are first suggested as correlations, based on the timing of JH peaks, but then experimentally demonstrated using JH antagonists and rescue thereof with JH mimic. This is a robust approach and the experimental results are very convincing.
    • JH redirects ecdysone-induced molting to direct formation of a more mature cuticle
    • Kr-h1 is downstream of JH in Thermobia, as it is in other insects, and is a likely mediator of many JH effects
    • The results support the proposed model that an ancestral role of JH in promoting and maintaining differentiation was coopted during insect radiations to drive the evolution of metamorphosis. However, alternate evolutionary scenarios should also be considered.

    Strengths:
    Overall, this is a beautiful, in-depth student. The paper is well-written and clear. The background places the work in a broad context and shows its importance in understanding fundamental questions about insect biology. The researchers are leaders in the field, and a strength of this manuscript is their use of a variety of different approaches (enzymatic assays, gene expression, agonists & antagonists, analysis of morphology using different types of microscopy and detection, and more) to attack their research questions. The experimental data is clearly presented and carefully executed with appropriate controls and attention to detail. The 'multi-pronged' approach provides support for the conclusions from different angles, strengthening conclusions. In sum, the data presented are convincing and the conclusions about experimental outcomes are well-justified based on the results obtained.

    Weaknesses:
    This paper provides more detail than is likely needed for readers outside the field but also provides sufficient depth for those in the field. This is both a strength and a weakness. I would suggest the authors shorten some aspects of their text to make it more accessible to a broader audience. In particular, the discussion is very long and accompanied by two model figures. The discussion could be tightened up and much of the text used for a separate review article (perhaps along with Figure 11) that would bring more attention to the proposed evolution of JH roles.

  3. eLife

    Reviewer #2 (Public Review):

    The authors have studied in detail the embryogenesis of the ametabolan insect Thermobia domestica. They have also measured the levels of the two most important hormones in insect development: juvenile hormone (JH) and ecdysteroids. The work then focuses on JH, whose occurrence concentrates in the final part (between 70 and 100%) of embryo development. Then, the authors used a precocene compound (7-ethoxyprecocene, or 7EP) to destroy the JH producing tissues in the embryo of the firebrat T. domestica, which allowed to unveil that this hormone is critically involved in the last steps of embryogenesis. The 7EP-treated embryos failed to resorb the extraembryonic fluid and did not hatch. More detailed observations showed that processes like the maturational growth of the eye, the lengthening of the foregut and posterior displacement of the midgut, and the detachment of the E2 cuticle, were impaired after the 7EP treatment. Importantly, a treatment with a JH mimic subsequent to the 7EP treatment restored the correct maturation of both the eye and the gut. It is worth noting that the timing of JH mimic application was essential for correcting the defects triggered by the treatment with 7EP.

    This is a relevant result in itself since the role of JH in insect embryogenesis is a controversial topic. It seems to have an important role in hemimetabolan embryogenesis, but not so much in holometabolans. Intriguingly, it appears important for hatching, an observation made in hemimetabolan and in holometabolan embryos. Knowing that this role was already present in ametabolans is relevant from an evolutionary point of view, and knowing exactly why embryos do not hatch in the absence of JH, is relevant from the point of view of developmental biology.

    Then, the authors describe a series of experiments applying the JH mimic in early embryogenesis, before the natural peak of JH occurs, and its effects on embryo development. Observations were made under different doses of JHm, and under different temporal windows of treatment. Higher doses triggered more severe effects, as expected, and different windows of application produced different effects. The most used combination was 1 ng JHm applied 1.5 days AEL, checking the effects 3 days later. Of note, 1.5 days AEL is about 15% embryonic development, whereas the natural peak of JH occurs around 85% embryonic development. In general, the ectopic application of JHm triggered a diversity of effects, generally leading to an arrest of development. Intriguingly, however, a number of embryos treated with 1 ng of JHm at 1.5 days AEL showed a precocious formation of myofibrils in the longitudinal muscles. Also, a number of embryos treated in the same way showed enhanced chitin deposition in the E1 procuticle and showed an advancement of at least a day in the deposition of the E2 cuticle.

    While the experiments and observations are done with great care and are very exhaustive, I am not sure that the results reveal genuine JH functions. The effects triggered by a significant pulse of ectopic JHm when the embryo is 15% of the development will depend on the context: the transcriptome existing at that time, especially the cocktail of transcription factors. This explains why different application times produce different effects. This also explains why the timing of JHm application was essential for correcting the effects of 7EP treatment. In this reasoning, we must consider that the context at 85% development, when the JH peaks in natural conditions and plays its genuine functions, must be very different from the context at 15% development, when the JHm was applied in most of the experiments. In summary, I believe that the observations after the application of JHm reveal effects of the ectopic JHm, but not necessarily functions of the JH. If so, then the subsequent inferences made from the premise that these ectopic treatments with JHm revealed JH functions are uncertain and should be interpreted with caution.

    Those inferences affect not only the "JH and the progressive nature of embryonic molts" section, but also, the "Modifications in JH function during the evolution of hemimetabolous and holometabolous life histories" section, and the entire "Discussion". In addition to inferences built on uncertain functions, the sections mentioned, especially the Discussion, I think suffer from too many poorly justified speculations. I love speculation in science, it is necessary and fruitful. But it must be practiced within limits of reasonableness, especially when expressed in a formal journal.

    Finally, In the section "Modifications in JH function during the evolution of hemimetabolous and holometabolous life", it is not clear the bridge that connects the observations on the embryo of Thermobia and the evolution of modified life cycles, hemimetabolan and holometabolan.

  4. eLife

    Reviewer #3 (Public Review):

    Summary:
    In this manuscript, the authors use inhibitors and mimetics of juvenile hormone (JH) to demonstrate that JH has a key role in late embryonic development in Thermobia, specifically in gut and eye development but also resorption of the extraembryonic fluid and hatching. They then exogenously apply JH early in development (when it is not normally present) to examine the biological effects of JH at these stages. This causes a plethora of defects including developmental arrest, deposition of chitin, limb development, and enhanced muscle differentiation. The authors interpret these early effects on development as JH being important for the shift from morphogenetic growth to differentiation - a role that they speculate may have facilitated the evolution of metamorphosis (hemi- and holo-metaboly). This paper will be of interest to insect evo-devo researchers, particularly those with interests in the evolution of metamorphosis.

    Strengths:
    The experiments are generally conducted very well with appropriate controls and the authors have included a very detailed analysis of the phenotypes.
    The manuscript significantly advances our understanding of Thermobia development and the role of JH in Thermobia development.
    The authors interpret this data to present some hypotheses regarding the role of JH in the evolution of metamorphosis, some aspects of which can be addressed by future studies.

    Weaknesses:
    The results are based on using inhibitors and mimetics of JH and there was no attempt to discern immediate effects of JH from downstream effects. The authors show, for instance, that the transcription of myoglianin is responsive to JH levels, it would have been interesting to see if any of the phenotypic effects are due to myoglianin upregulation/suppression (using RNAi for example). These kinds of experiments will be necessary to fully work out if and how the JH regulatory network has been co-opted into metamorphosis.

    The results generally support the authors' conclusions. However, the discussion contains a lot of speculation and some far-reaching conclusions are made about the role of JH and how it became co-opted into controlling metamorphosis. There are some interesting hypotheses presented and the author's speculations are consistent with the data presented. However, it is difficult to make evolutionary inferences from a single data point as although Thermobia is a basally branching insect, the lineage giving rise to Thermobia diverged from the lineages giving rise to the holo- and hemimetabolous insects approx.. 400 mya and it is possible that the effects of JH seen in Thermobia reflect lineage-specific effects rather than the 'ancestral state'. The authors ignore the possibility that there has been substantial rewiring of the networks that are JH responsive across these 400 my. I would encourage the authors to temper some of the discussion of these hypotheses and include some of the limitations of their inferences regarding the role of JH in the evolution of metamorphosis in their discussion.

  5. Arcadia Science

    In this manuscript, the authors examined the role of juvenile hormone (JH) in the development of an ametabolous insect, the firebrat Thermobia domestica. JH is produced during the post-embryonic development of hemimetabolous and holometabolous insects and is necessary for metamorphosis. The authors suppress JH production and precociously induce JH signaling through exogenous application of synthetic JH mimics to understand the role of JH in Thermobia development.

    The authors demonstrate that JH is necessary for Thermobia embryos to undergo proper differentiation – embryos treated with 7EP, which inhibits the production of endogenous JH, demonstrate delayed development of various body features, such as the ommatidia.

    Conversely, embryos treated precociously with exogenous JHm (juvenile hormone mimic) showed developmental defects consistent with premature differentiation, such as the development of myofibrils earlier than in vehicle-treated embyros.

    Overall, the authors provide multiple lines of evidence for their findings, from measuring hormone concentrations through chemical assays, evaluating gene expression using qPCR, and analyzing embryonic morphology using stains and brightfield classification of phenotypes. The authors propose that JH was coopted from an ancestral state, in which it regulated the morphogenesis to differentiation switch during embryonic development, to its derived state in hemi- and holometabolous insects, where it performs an analogous role in the maturation of nymph- and larval-stage insects towards adulthood.

  6. Arcadia Science

    reated embryos that completed katatrepsis also showed the prematureproduction of myofibrils (Fig. 8D) and they underwent substantial growth.

    It's cool to see that, concurrent with degradation of limb morphogenesis, you could simultaneously observe differentiation.

  8. Arcadia Science

    The latter two events, though, were restored to7EP-treated embryos by application of JHm shortly after dorsal closure.

    It's surprising how quickly the JHm + 7EP treated embryos recovered the extraembryonic fluid reabsorption, given that the amount of gene expression rescue by JHm in Fig. 3B seemed to be more limited.

  9. Arcadia Science

    (A) Titer of juvenile hormone III (JH-III) during thelast 60% of embryogenesis and the first eight days of juvenile life

    To improve clarity, the arrows in panel A could also have dotted lines extended across panels B and C; currently, a casual glance could lead to the misinterpretation that they might be marking some feature of the JH-III molecule.

  12. Version 1 published on bioRxiv