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CoordinGrowthDev Report Summary

Project ID: 657685
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - CoordinGrowthDev (Characterization of the mechanism of inter-organ communication coordinating tissue growth and developmental timing)

Reporting period: 2016-03-01 to 2018-02-28

Summary of the context and overall objectives of the project

Over the recent years, research in developmental biology has stumbled upon the problem of how development ensures that organisms achieve appropriate body and organ size. The coordination of organ growth with the developmental program is essential for the emergence of adults with proper body size and proportions. These species-specific features condition many traits of adult life such as fitness and survival. Studies on fruit flies have allowed setting up the conceptual frame for this critical research. Our current understanding is that tissue growth and the developmental program are synchronized by feedback control mechanisms that block the transition to the next developmental period until all tissues have completed growth. Yet, we still lack a coherent picture of how these feedbacks operate at the organism level from a mechanistic point of view. Recent work in Drosophila has started unraveling the nature of the signals that couple tissue growth with developmental progression. The relaxin-like peptide Dilp8 is key in this coupling, raising the possibility that peptides with similar roles may exist in vertebrates.

Dilp8 is secreted by growing, regenerating and tumorous tissues. It acts remotely on steroid hormone production to inhibit the transition to the pupal stage and to delay development. In addition, the negative impact of Dilp8 on tissue growth suggests that it could act as a synchronization factor among growing tissues. The current project aimed at characterizing the molecular mechanisms by which Dilp8 coordinates development of the different body parts. To do so, we have identified and characterized the receptor for Dilp8, the relaxin family receptor Lgr3. In addition, we functionally identified two Lgr3-positive neurons in each brain lobe that are required for Dilp8 functions. Reducing Lgr3 levels in these neurons results in adult flies exhibiting increased fluctuating bilateral symmetry, therefore recapitulating the phenotype of dilp8 mutants. Our work revealed a novel Dilp8/Lgr3 neuronal circuitry involved in a feedback mechanism that ensures coordination between organ growth and developmental transitions and prevents developmental variability. Finally, ongoing work focuses on the role of Dilp8 in inter-organ coordination of tissue growth, with an emphasis on the molecular mechanism linking organ growth status with Dilp8 upregulation and the systemic response.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The objectives were to identify the receptor for Dilp8, to identify the target tissue mediating Dilp8 action on developmental timing, and finally to characterize the role of Dilp8 in the coordination of tissue growth.

We conducted a functional genetic screen that led us to the identification of the relaxin family receptor Lgr3 as the receptor for Dilp8. This objective was achieved by combining the LexA/LexO system, used to overexpress Dilp8 in imaginal discs, with the Gal4/UAS system, used to knock-down a collection of membrane receptors in candidate target tissues. Only the downregulation of Lgr3 in neuronal cells was able to rescue the developmental delay induced by Dilp8 overexpression.

In order to precisely identify the Dilp8-responsive neurons, we investigated lgr3 expression pattern with lgr3-Gal4 tools. By conducting intersection experiments, we functionally identified a single pair of bilateral Lgr3-positive neurons mediating Dilp8 function, referred to as growth coordinating Lgr3 (GCL) neurons. Interestingly, GCL neurons physically interact with PTTH neurons, which are known to project their axons directly onto the prothoracic gland (PG) and control ecdysone production. These results unravel the mechanism by which growing tissues indirectly regulate the production of steroid hormone.

Apart from its effect on developmental timing, the overexpression of Dilp8 in imaginal discs reduces their growth rate. We found that silencing lgr3 in GCL neurons, but not in the wing discs, rescued disc growth inhibition caused by Dilp8 overexpression. This suggests that Dilp8/Lgr3 signaling in GCL neurons could modulate the growth of peripheral tissues in a systemic manner through the control of ecdysone production. Moreover, analysis of fluctuating asymmetry revealed that Dilp8/Lgr3 signaling in the two GCL neurons is required for the maintenance of bilateral symmetry and the control of developmental variability.

This work was published in Current Biology, and an article written in French for a non-specialized audience was published in Médecine/Sciences. This work was also presented at the 24th European Drosophila Research Conference, at the 30th Annual French Drosophila Conference and at the 5th Drosophila growth and regeneration meeting.

In addition, we are currently addressing the role of Dilp8 in the inter-organ coordination (IOC) of growth. When growth of one imaginal disc domain is perturbed, other domains and other discs slow down their growth, maintaining proper inter-disc and intra-disc proportions. We show that Dilp8 is required for IOC. Our work also reveals that the stress-response transcription factor Xrp1 is a key player in IOC upstream of dilp8. These observations indicate that Xrp1 and Dilp8 constitute a new independent regulatory module that ensures growth coordination during development.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Body proportions are key characteristics that distinguish one animal species from another. Dilp8 is the humoral signal secreted by undergrowing, regenerating or tumorous tissues to inhibit the production of steroid hormone and therefore to delay metamorphosis. The identification of the receptor for Dilp8, Lgr3, and the two Lgr3-positive bilateral neurons that mediate regulation of ecdysone greatly enhance our understanding of how organ-intrinsic size modulates whole-body physiology. Interestingly, the Dilp8-Lgr3-PG circuitry shares some characteristics with the hypothalamic-pituitary axis in humans in which the hypothalamus integrates and relays information about the state of the body to the pituitary gland through synaptic communication with other neurons or the release of hormones. The observation that mammalian relaxin family peptides are expressed in the hypothalamus opens the possibility that similar relaxin dependent mechanisms monitoring the growth status of peripheral tissues are at play in humans. In line with this, reduced body mass, stress induced by heavy surgery, inflammatory diseases, and dietary restrictions have all been associated with a delay in puberty. We therefore propose that the Dilp8-Lgr3 axis represents an ancient surveillance mechanism ensuring developmental stability that may be conserved in higher animals.

Finally, our ongoing work on the molecular mechanism of coordination of tissue growth constitute an important step in the field of systemic growth control by showing how inter-organ communication contributes to final body proportions. Our results establish Dilp8 as a master regulator of the systemic response to a local growth perturbation, being the key signal for both the modulation of developmental timing and the coordination of growth between remote organs. After unraveling signaling events downstream of Dilp8 (receptor and target tissue), our current work identifies a new upstream regulator of Dilp8, the stress-response factor Xrp1, which couples organ growth status with the systemic response.

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