Periodic Reporting for period 1 - GrowthDevStability (Characterization of the developmental mechanisms ensuring a robust symmetrical growth in the bilateral model organism Drosophila melanogaster)
Berichtszeitraum: 2020-06-01 bis 2022-05-31
Most animals, including humans, have one longitudinal or sagittal plane which divides the organism into two roughly mirror images, the left and right halves. For example, the surfaces of the two wings of the fruit fly Drosophila melanogaster, a privileged model organism, do not differ by more than 2% in wild-type animals. This feature is called Bilateral Symmetry and constitutes a key quality that defines mobility, predation and overall fitness. Even though many studies have focused on the autonomous mechanisms of organ size determination, less attention has been put on how organ growth is coordinated between the left and right halves in order to attain Bilateral Symmetry. The fact that symmetry is achieved even in the case of illness, starvation and injuries, suggests the occurrence of dedicated mechanisms that are able to compare the growth trajectories of each half and adjust when required.
The investigation on how Bilateral Symmetry is achieved has profound impacts, not only because it constitutes a fundamental question in Developmental Biology but also because it is directly linked to animal and human health and technology. The new concepts introduced by this investigation could help to better understand the pathophysiology of childhood growth disorders, as well as the growth disturbances observed in cancer survivors. It would also certainly have implications for large-scale regenerative research, in which a tight coordination of left and right halves should be attained.
The overarching objective of the proposed project was to find how and when organs asses their growth status in Drosophila, and which mechanisms are deployed to ensure fine size adjustment during normal animal development. In particular, my research was focused of the role of a signaling peptide that belongs to the superfamily of insulin/IGF/relaxin-like peptidic hormones and is called Drosophila insulin-like peptide 8 (Dilp8). I sought to characterize the source tissue that produces dilp8, how and when this production is induced, and what are the downstream events that dilp8 triggers for bilateral organ size adjustment.
After my two years of MSCA-funded research, the research team and I were able to define a cross-talk between Dilp8 and the growth-regulating hormone ecdysone that is key for developmental precision. This cross-talk has two functions: (i) it defines a critical time window after larval development during which wings adjust their size; (ii) it fine-tunes the levels of ecdysone signaling in the discs, which is crucial for their size adjustment.
The investigation on how Bilateral Symmetry is achieved has profound impacts, not only because it constitutes a fundamental question in Developmental Biology but also because it is directly linked to animal and human health and technology. The new concepts introduced by this investigation could help to better understand the pathophysiology of childhood growth disorders, as well as the growth disturbances observed in cancer survivors. It would also certainly have implications for large-scale regenerative research, in which a tight coordination of left and right halves should be attained.
The overarching objective of the proposed project was to find how and when organs asses their growth status in Drosophila, and which mechanisms are deployed to ensure fine size adjustment during normal animal development. In particular, my research was focused of the role of a signaling peptide that belongs to the superfamily of insulin/IGF/relaxin-like peptidic hormones and is called Drosophila insulin-like peptide 8 (Dilp8). I sought to characterize the source tissue that produces dilp8, how and when this production is induced, and what are the downstream events that dilp8 triggers for bilateral organ size adjustment.
After my two years of MSCA-funded research, the research team and I were able to define a cross-talk between Dilp8 and the growth-regulating hormone ecdysone that is key for developmental precision. This cross-talk has two functions: (i) it defines a critical time window after larval development during which wings adjust their size; (ii) it fine-tunes the levels of ecdysone signaling in the discs, which is crucial for their size adjustment.
Firstly, by measuring the volume of wing precursors and quantifying the left-right (L-R) variability, we found that wing size is adjusted through a major adjustment step in a Dilp8-dependent manner, at the beginning of pupal development.
In line with this finding, we observed that Dilp8 expression is sharply upregulated at the larva to pupa transition (WPP stage). Moreover, specific inhibition of dilp8 expression peak at WPP using a novel light-controlled tool (ShineGal4), allowed us to conclude that dilp8 at this stage is functionally required for maintaining low L-R wing size variability.
While very low or no expression of dilp8 was detected in wings, fat body, gut, brain and salivary glands, high dilp8 expression was detected in the epidermis Concordantly, inhibition of dilp8 expression in epidermal cells was sufficient to induce adult wing L-R variability. Therefore, the epidermis is the source of a burst of dilp8 expression at the WPP stage that triggers organ size adjustment.
The sharp expression of dilp8 in the WPP epidermis was indicative of a tight spatial and temporal transcriptional control. Ecdysone hormone titers increase gradually during the final larval stage and reach maximum levels at the WPP stage. Therefore, To test the possibility that dilp8 expression at WPP could rely on ecdysone, we silenced the expression of the ecdysone receptor (EcR) gene specifically in the epidermis, and observed a strong decrease in dilp8 expression at WPP. These results establish that a functional cross talk between ecdysone and Dilp8 takes place at WPP in the epidermis for the control of size adjustment.
To investigate whether Dilp8 also acts upstream of ecdysone for organ size adjustment, we compared the levels of circulating ecdysone in controls and dilp8 mutants at several timepoints around the WPP stage. We observed in dilp8 mutants a significant increase in circulating ecdysone at the WPP stage, followed by a sharper decrease afterwards. This data suggests that the key parameter for size adjustment is the level of ecdysone at the larva-to-pupa transition. 8 out of 9 EcR target genes were significantly upregulated in wings in the absence of Dilp8, indicating a clear effect on the intensity of EcR signaling in target tissues at WPP. Thus, we concluded that Dilp8 acts by ensuring proper ecdysone signaling in target tissues at the WPP stage.
Collectively, these and our previous results indicate that epidermal Dilp8 acts on ecdysone accumulation and modulates the level and timing of EcR signaling in peripheral tissues for disc size adjustment.
The results described in this section where made publicly available in the Open-access repository Biorxiv under the title "Dilp8 controls a time window for tissue size adjustment in Drosophila" (doi: https://doi.org/10.1101/2020.11.09.375063(öffnet in neuem Fenster)). The dissemination of this publication was encouraged by active sharing through the Pierre Leopold laboratory Tweeter account (@LeopoldLab). The publication is currently under editorial revision at the Open-access journal Nature Communications and will be duly disseminated once accepted.
In line with this finding, we observed that Dilp8 expression is sharply upregulated at the larva to pupa transition (WPP stage). Moreover, specific inhibition of dilp8 expression peak at WPP using a novel light-controlled tool (ShineGal4), allowed us to conclude that dilp8 at this stage is functionally required for maintaining low L-R wing size variability.
While very low or no expression of dilp8 was detected in wings, fat body, gut, brain and salivary glands, high dilp8 expression was detected in the epidermis Concordantly, inhibition of dilp8 expression in epidermal cells was sufficient to induce adult wing L-R variability. Therefore, the epidermis is the source of a burst of dilp8 expression at the WPP stage that triggers organ size adjustment.
The sharp expression of dilp8 in the WPP epidermis was indicative of a tight spatial and temporal transcriptional control. Ecdysone hormone titers increase gradually during the final larval stage and reach maximum levels at the WPP stage. Therefore, To test the possibility that dilp8 expression at WPP could rely on ecdysone, we silenced the expression of the ecdysone receptor (EcR) gene specifically in the epidermis, and observed a strong decrease in dilp8 expression at WPP. These results establish that a functional cross talk between ecdysone and Dilp8 takes place at WPP in the epidermis for the control of size adjustment.
To investigate whether Dilp8 also acts upstream of ecdysone for organ size adjustment, we compared the levels of circulating ecdysone in controls and dilp8 mutants at several timepoints around the WPP stage. We observed in dilp8 mutants a significant increase in circulating ecdysone at the WPP stage, followed by a sharper decrease afterwards. This data suggests that the key parameter for size adjustment is the level of ecdysone at the larva-to-pupa transition. 8 out of 9 EcR target genes were significantly upregulated in wings in the absence of Dilp8, indicating a clear effect on the intensity of EcR signaling in target tissues at WPP. Thus, we concluded that Dilp8 acts by ensuring proper ecdysone signaling in target tissues at the WPP stage.
Collectively, these and our previous results indicate that epidermal Dilp8 acts on ecdysone accumulation and modulates the level and timing of EcR signaling in peripheral tissues for disc size adjustment.
The results described in this section where made publicly available in the Open-access repository Biorxiv under the title "Dilp8 controls a time window for tissue size adjustment in Drosophila" (doi: https://doi.org/10.1101/2020.11.09.375063(öffnet in neuem Fenster)). The dissemination of this publication was encouraged by active sharing through the Pierre Leopold laboratory Tweeter account (@LeopoldLab). The publication is currently under editorial revision at the Open-access journal Nature Communications and will be duly disseminated once accepted.
At the time of the initiation of this MSCA fellowship, the mechanism by which the Dilp8 hormone controls developmental stability in Drosophila remained unknown. Two distinct hypotheses could account for such control. Continuous feedbacks taking place during the growth phase could maintain organs on a growth trajectory leading to an appropriate final size. Alternatively, developing organs could randomly deviate from a standard growth trajectory up to a time window in development where the extent of the deviation is evaluated and a correction is made.
As indicated in the results sections above, the work during this MSCA fellowship let us conclude that Dilp8 mode of action is compatible to the second model, by regulating the emergence of the time window for correction. Notably, this time window is relatively short when compared to the total time of Drosophila development (7 hs vs 5 days), indicating that a significant correction of L-R tissue size variation occurs rather abruptly, instead of gradually. A similar short time window for correction has been found in another model organism, the Zebrafish, suggesting that our findings could reflect a general strategy for size adjustment in animals. Furthermore, the conclusions obtained might have direct relevance for the mammalian system, where an analogous systemic control of limb symmetrical growth has been established.
In conclusion, the general concept of a stepwise regulation of bilateral symmetry found in this project could potentially be relevant for the clinic, in the treatment of malformations arising during human development as the result of pathogenic environmental conditions or malfunction of the physiological buffering of developmental errors.
As indicated in the results sections above, the work during this MSCA fellowship let us conclude that Dilp8 mode of action is compatible to the second model, by regulating the emergence of the time window for correction. Notably, this time window is relatively short when compared to the total time of Drosophila development (7 hs vs 5 days), indicating that a significant correction of L-R tissue size variation occurs rather abruptly, instead of gradually. A similar short time window for correction has been found in another model organism, the Zebrafish, suggesting that our findings could reflect a general strategy for size adjustment in animals. Furthermore, the conclusions obtained might have direct relevance for the mammalian system, where an analogous systemic control of limb symmetrical growth has been established.
In conclusion, the general concept of a stepwise regulation of bilateral symmetry found in this project could potentially be relevant for the clinic, in the treatment of malformations arising during human development as the result of pathogenic environmental conditions or malfunction of the physiological buffering of developmental errors.