Ecdysone coordinates plastic growth with robust pattern in the developing wing
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Curated by eLife
Evaluation Summary:
This manuscript will be of broad interest for biologists, in particular developmental biologists and ecologists, as it addresses essential questions on the interaction between organisms and their environment. How organisms manage to maintain a stable phenotype (robustness) or how they adjust their phenotype (plasticity) in response to environmental variations is a major issue. In this article, the authors show that the hormone ecdysone is involved in Drosophila in the plasticity of wing size and the robustness of wing pattern.
(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
Animals develop in unpredictable, variable environments. In response to environmental change, some aspects of development adjust to generate plastic phenotypes. Other aspects of development, however, are buffered against environmental change to produce robust phenotypes. How organ development is coordinated to accommodate both plastic and robust developmental responses is poorly understood. Here, we demonstrate that the steroid hormone ecdysone coordinates both plasticity of organ size and robustness of organ pattern in the developing wings of the fruit fly Drosophila melanogaster . Using fed and starved larvae that lack prothoracic glands, which synthesize ecdysone, we show that nutrition regulates growth both via ecdysone and via an ecdysone-independent mechanism, while nutrition regulates patterning only via ecdysone. We then demonstrate that growth shows a graded response to ecdysone concentration, while patterning shows a threshold response. Collectively, these data support a model where nutritionally regulated ecdysone fluctuations confer plasticity by regulating disc growth in response to basal ecdysone levels and confer robustness by initiating patterning only once ecdysone peaks exceed a threshold concentration. This could represent a generalizable mechanism through which hormones coordinate plastic growth with robust patterning in the face of environmental change.
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Author Response
Reviewer #1 (Public Review):
The genetic destruction of the prothoracic gland by the cell death gene Grim is an accepted method. They need to indicate in this case, how long the prothoracic gland is detectable in the third instar after exposure to the elevated temperature to inactivate the GAL80. For instance, are any gland cells still functional at the time of critical weight? How does this affect the onset of Achaete progression which normally occurs beginning at 5 hr after ecdysis which they show is dependent on low levels of ecdysone? Do you get the same result if you place them at the elevated temperature 12 hr after ecdysis to the 2nd instar, a time when the ecdysteroid titer has already risen to cause the molt to the third instar?
These are all valid points, and future experiments should explore manipulating …
Author Response
Reviewer #1 (Public Review):
The genetic destruction of the prothoracic gland by the cell death gene Grim is an accepted method. They need to indicate in this case, how long the prothoracic gland is detectable in the third instar after exposure to the elevated temperature to inactivate the GAL80. For instance, are any gland cells still functional at the time of critical weight? How does this affect the onset of Achaete progression which normally occurs beginning at 5 hr after ecdysis which they show is dependent on low levels of ecdysone? Do you get the same result if you place them at the elevated temperature 12 hr after ecdysis to the 2nd instar, a time when the ecdysteroid titer has already risen to cause the molt to the third instar?
These are all valid points, and future experiments should explore manipulating larvae at earlier developmental times. We have not tried transferring L2 larvae to higher temperatures, but would be keen to explore this option. We would need to work out the timing of the ecdysteroid pulse in L2 larvae reared at 17oC first to make this work.
Our previous publication shows that some PG cells are still visible at 42 h after L3 ecdysis in PGX animals, although the whole ring gland is dramatically reduced in size (Herboso et al 2015 Supp Fig 3E,F). However, given the growth and patterning defects are similar to those found in previous studies where ecdysone signalling was inhibited in the wing disc (Mirth et al, 2009; Herboso et al 2015), and because these animals cannot pupariate, it is clear that the residual cells do not produce sufficient ecdysone to promote proper development. We believe the gland cannot produce normal ecdysone titres even early in the third instar because:
• Delays in patterning for Achaete and Senseless in the wing discs are similar to those we found when over expressing a dominant negative form of the ecdysone receptor specifically in the wing (Mirth et al 2009)
• The growth defects in the wing discs are similar to other manipulations that reduce ecdysone synthesis in the prothoracic gland, like expressing RNAi against smt3 (Herboso et al 2015)
• In response to Reviewer 3’s recommendation, we have analysed the ecdysone titres in fed PGX and control larvae and found that PGX produce significantly lower, albeit variable, 20E titres than controls (Figure 2 Supplement 1).
One concern with the paper is why the authors begin contrasting the effects of temperature on embryonic development and wing disc patterning in the Discussion on p25. This discussion seems irrelevant to the experimental manipulations done in this paper concerned with nutrition and hormone levels. Nothing was done relative to environmental temperature effects except to genetically kill the prothoracic gland, the source of ecdysone. I recommend omission of this section of the Discussion.
We were attempting to make a point about rates of patterning across developmental contexts, but obviously missed the mark. We have deleted this paragraph.
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Evaluation Summary:
This manuscript will be of broad interest for biologists, in particular developmental biologists and ecologists, as it addresses essential questions on the interaction between organisms and their environment. How organisms manage to maintain a stable phenotype (robustness) or how they adjust their phenotype (plasticity) in response to environmental variations is a major issue. In this article, the authors show that the hormone ecdysone is involved in Drosophila in the plasticity of wing size and the robustness of wing pattern.
(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):
This paper assesses the role of the molting hormone ecdysone in the coordination of growth and patterning in the Drosophila wing disc in response to food deprivation. Experimentally they use third instar-specific death of the prothoracic glands (PG), the source of ecdysone, to eliminate the hormone and the developmental time course of the appearance of two bristle markers, Achaete which normally begins progressing at about 5 hr after ecdysis to the third instar when the ecdysone titer just begins to increase and Senseless which only appears after the attainment of critical weight (a size that signals the ability to metamorphose without further feeding) after which small peaks of ecdysone occur. Then they assess the response of the experimentally manipulated larvae to 20-hydroxyecdysone (20E) in their food. …
Reviewer #1 (Public Review):
This paper assesses the role of the molting hormone ecdysone in the coordination of growth and patterning in the Drosophila wing disc in response to food deprivation. Experimentally they use third instar-specific death of the prothoracic glands (PG), the source of ecdysone, to eliminate the hormone and the developmental time course of the appearance of two bristle markers, Achaete which normally begins progressing at about 5 hr after ecdysis to the third instar when the ecdysone titer just begins to increase and Senseless which only appears after the attainment of critical weight (a size that signals the ability to metamorphose without further feeding) after which small peaks of ecdysone occur. Then they assess the response of the experimentally manipulated larvae to 20-hydroxyecdysone (20E) in their food. The experimental data are then modeled. Interestingly, the growth rates and Achaete patterning through developmental times are best fitted by a Gompertz function whereas the Senseless patterning over time shows a typical linear regression. Also, they assess the effects of manipulating the insulin-signaling pathway. They show that nutrition regulates disc growth by both ecdysone-dependent and ecdysone-independent means, but regulates patterning (i. e., differentiation) only by ecdysone levels. Thus, the growth response to the hormone is quite plastic whereas patterning requires a threshold level of ecdysone in order to begin. These two different responses to ecdysone and nutrition also seem to be typical of other holometabolous insects as evidenced by the published examples in Lepidoptera that they cite.
This group carefully stages animals and has accumulated a wealth of background information necessary for this type of study. They have wisely chosen two differentiation markers that begin at different times relative to the larva's physiological state with respect to nutrition and hormone levels, i.e. one that begins before critical weight when ecdysone titers are very low and one that begins after critical weight when the ecdysone titer rises in pulses. The data are well presented and generally support their conclusions.
The genetic destruction of the prothoracic gland by the cell death gene Grim is an accepted method. They need to indicate in this case, how long the prothoracic gland is detectable in the third instar after exposure to the elevated temperature to inactivate the GAL80. For instance, are any gland cells still functional at the time of critical weight? How does this affect the onset of Achaete progression which normally occurs beginning at 5 hr after ecdysis which they show is dependent on low levels of ecdysone? Do you get the same result if you place them at the elevated temperature 12 hr after ecdysis to the 2nd instar, a time when the ecdysteroid titer has already risen to cause the molt to the third instar?
One concern with the paper is why the authors begin contrasting the effects of temperature on embryonic development and wing disc patterning in the Discussion on p25. This discussion seems irrelevant to the experimental manipulations done in this paper concerned with nutrition and hormone levels. Nothing was done relative to environmental temperature effects except to genetically kill the prothoracic gland, the source of ecdysone. I recommend omission of this section of the Discussion.
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Reviewer #2 (Public Review):
Alves et al investigate hormone-dependent control of developmental plasticity. While some traits exhibit robustness (ie. Low variability) in the face of changing environmental conditions, others exhibit plasticity (ie. High variability). The authors focus on how the ecdysone steroid hormone links nutritional status in the larval stages of D. melanogaster development to robust and plastic traits in the wing. Prior work from this group and others demonstrated that nutritional status controls production of ecdysone, a key hormone signal that coordinates developmental timing in insects. In addition, ecdysone has been shown to promote wing growth during the larval stages of development, and it determines the timing of wing patterning. However, whereas wing growth (and hence wing size) is a plastic trait (e.g. …
Reviewer #2 (Public Review):
Alves et al investigate hormone-dependent control of developmental plasticity. While some traits exhibit robustness (ie. Low variability) in the face of changing environmental conditions, others exhibit plasticity (ie. High variability). The authors focus on how the ecdysone steroid hormone links nutritional status in the larval stages of D. melanogaster development to robust and plastic traits in the wing. Prior work from this group and others demonstrated that nutritional status controls production of ecdysone, a key hormone signal that coordinates developmental timing in insects. In addition, ecdysone has been shown to promote wing growth during the larval stages of development, and it determines the timing of wing patterning. However, whereas wing growth (and hence wing size) is a plastic trait (e.g. restricted nutrition leads to smaller wings), wing patterning is a robust trait (e.g. small and large wings have the same constituent parts). It is unclear how ecdysone might coordinate both growth plasticity and patterning robustness. The authors combine several existing methodologies to support their conclusions. These include two types of precise measurement at successive stages of larval development (wing disc size, and spatiotemporal expression of the sensory organ patterning genes Achaete and Senseless); while conceptually straightforward, these dissections and measurements are challenging to execute. They manipulate systemic ecdysone levels by genetically ablating the prothoracic gland in a developmentally regulated manner. They control nutritional status by culturing larvae in normal food or starvation conditions. Lastly, they add defined amounts of ecdysone directly to the food to control systemic hormone levels. The combination of these approaches allows them to test three stated hypotheses: (1) ecdysone's effects on growth and patterning occur at different times. (2) ecdysone's effects on growth and patterning are interdependent. (3) ecdysone regulates growth and patterning independently.
The authors' highly quantitative approaches confirm the role for ecdysone in controlling wing disc growth and patterning, and they support a role of ecdysone in independently regulating wing growth and patterning (Hypothesis 3). Importantly, these quantitative approaches extend understanding by convincingly demonstrating that nutrition regulates disc growth in both an ecdysone-dependent and an ecdysone-independent manner - supplementing starved larvae with ecdysone only partially rescues disc growth. They also demonstrate that nutrition regulates disc patterning only through ecdysone - supplementing starved larvae with ecdysone fully rescues disc patterning. Lastly, they propose that robustness of wing patterning is due to a threshold response to ecdysone, whereas plasticity of wing size is due to a graded response to ecdysone. While the data supporting these last conclusions are provocative, they are not decisive. In particular, additional data are needed to strengthen the conclusion that disc size is controlled by a graded response to ecdysone. A final weakness of the paper is that the authors have not provided a causal link between the proposed graded response to ecdysone and plasticity of wing growth.
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Reviewer #3 (Public Review):
Strengths:
The authors investigated the role of ecdysone in the plasticity of wing disc size and the robustness of wing disc patterning.
The authors use the genetic tools available in the model species Drosophila melanogaster to ablate the prothoracic glands that produce ecdysone. They also manipulate ecdysone level by adding it to the food or manipulating the activity of the insulin pathway in the prothoracic glands where it regulates ecdysone synthesis.
The authors measure ecdysone level in control or starved larvae with normal or ablated prothoracic glands fed with different concentration of ecdysone. They show that starvation increase ecdysone level in both genotypes and that supplementing the food with ecdysone is efficient in increasing ecdysone level in larvae.
They use normal or poor medium to …
Reviewer #3 (Public Review):
Strengths:
The authors investigated the role of ecdysone in the plasticity of wing disc size and the robustness of wing disc patterning.
The authors use the genetic tools available in the model species Drosophila melanogaster to ablate the prothoracic glands that produce ecdysone. They also manipulate ecdysone level by adding it to the food or manipulating the activity of the insulin pathway in the prothoracic glands where it regulates ecdysone synthesis.
The authors measure ecdysone level in control or starved larvae with normal or ablated prothoracic glands fed with different concentration of ecdysone. They show that starvation increase ecdysone level in both genotypes and that supplementing the food with ecdysone is efficient in increasing ecdysone level in larvae.
They use normal or poor medium to analyse the role of nutrition on wing growth and patterning.
They follow size of wing imaginal discs and the progression of the patterning of these discs using neurogenic proteins (Achaete, Senseless) expressed in the developing peripheral nervous system (sensory bristles).
They show that nutrition regulate growth by ecdysone and another mechanism and that nutrition regulates pattering only via ecdysone.
Interestingly, growth shows a linear response to ecdysone level, whereas patterning shows a threshold response.
The linear response explains how nutrition induced fluctuation of ecdysone concentration leads to size plasticity and induce a robust patterning by initiating it only when a particular ecdysone level is reached.
The manuscript is very well written. The authors make three conflicting hypothesis in the introduction that they test experimentally. The experiments are rigorously designed and performed with appropriate controls. Their interpretation is justified. The methods used such as the rigorous staging of the patterning or the manipulation of ecdysone level by different means will be useful to other researchers. This article will have a strong impact as it illustrates how a single hormone coordinates the plasticity of size and the robustness of patterning of an organ.
I do not see weaknesses in this article.
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