How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here we mutate an invertebrate orphan relaxin receptor gene, the Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3), and find body asymmetries similar to those found in insulin-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, we map this Lgr3 activity to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.

How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here, we mutated an invertebrate orphan relaxin receptor, the Drosophila Lgr3, and found body asymmetries similar to those found in insulin/relaxin-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNAi against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By fluorescently-tagging the endogenous Lgr3 protein and performing CNSspecific RNAi, we find that Lgr3 is expressed and required in a novel subset of CNS neurons to transmit the peripheral tissue stress signal, Dilp8, to the neuroendocrine centers controlling developmental timing. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations. Main textHow different organs in the body sense growth perturbations in distant tissues to coordinate their size and differentiation status during development is poorly understood 1,2 . We have previously discovered a hormone in Drosophila, the insulin/relaxin-like peptide Dilp8, which ensures organ and body size coordination 3 . In developing larvae, Dilp8 is produced and secreted from abnormally-growing imaginal discs. Its activity transiently delays the onset of metamorphosis by inhibiting the biosynthesis of the major insect molting hormone ecdysone by the prothoracic gland, a part of a compound endocrine peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/017053 doi: bioRxiv preprint first posted online Mar. 25, 2015; peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/017053 doi: bioRxiv preprint first posted online Mar. 25, 2015; peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/017053 doi: bioRxiv preprint first posted online Mar. 25, 2015; peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/017053 doi: bioRxiv preprint first posted online Mar. 25, 2015; peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/017053 doi: bioRxiv preprint first posted online Mar. 25, 2015; peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/017053 doi: bioRxiv preprint first posted online Mar. 25, 2015; peer-reviewed) is the ...

Innate behaviors consist of a succession of genetically-hardwired motor and physiological subprograms that can be coupled to drastic morphogenetic changes. How these integrative responses are orchestrated is not completely understood. Here, we provide insight into these mechanisms by studying pupariation, a multi-step innate behavior of Drosophila larvae that is critical for survival during metamorphosis. We find that the steroid-hormone ecdysone triggers parallel pupariation neuromotor and morphogenetic subprograms, which include the induction of the relaxin-peptide hormone, Dilp8, in the epidermis. Dilp8 acts on six Lgr3-positive thoracic interneurons to couple both subprograms in time and to instruct neuromotor subprogram switching during behavior. Our work reveals that interorgan feedback gates progression between subunits of an innate behavior and points to an ancestral neuromodulatory function of relaxin signaling.

Age-related macular degeneration (AMD) is among the main pathologies leading to blindness in adults and has currently no cure or effective treatment. Selective apoptosis of retina pigment epithelial (RPE) cells results in the progressive loss of photoreceptor neurons, with the consequent gradual vision loss. Oxidative stress plays an important role in this process. We have previously determined that activation of RXRs protects rat photoreceptor neurons from oxidative stress-induced apoptosis. In this study we investigated whether RXR ligands prevented apoptosis in an RPE cell line, D407 cells, exposed to hydrogen peroxide (H2O2). H2O2 induced apoptosis of D407 cells, promoting p65NFκB nuclear translocation, increasing Bax mRNA expression, activating caspase-3 and altering cell morphology. We show, for the first time, that HX630, a RXR pan-agonist, protected D407 cells from H2O2-induced apoptosis, preventing p65NFκB nuclear translocation, increasing Bclxl and PPARγ mRNA levels and simultaneously decreasing Bax mRNA levels and caspase-3 activation. Pretreatment with a RXR antagonist blocked HX630 protection. LG100754, which binds RXRs but only activates heterodimers and is an antagonist of RXR homodimers, also had a protective effect. In addition, only agonists known to bind to RXR/PPARγ were protective. As a whole, our results suggest that RXR activation protects RPE cells from oxidative stress-induced apoptosis and this protection might involve signaling through a heterodimeric receptor, such as RXR/PPARγ. These data also imply that RXR agonists might provide potential pharmacological tools for treating retina degenerative diseases.

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Müller glial cells (MGC) are stem cells in the retina. Although their regenerative capacity is very low in mammals, the use of MGC as stem cells to regenerate photoreceptors (PHRs) during retina degenerations, such as in retinitis pigmentosa, is being intensely studied. Changes affecting PHRs in diseased retinas have been thoroughly investigated; however, whether MGC are also affected is still unclear. We here investigated whether MGC in retinal degeneration 1 ( rd1 ) mouse, an animal model of retinitis pigmentosa, have impaired stem cell properties or structure. rd1 MGC showed an altered morphology, both in culture and in the whole retina. Using mixed neuron-glial cultures obtained from newborn mice retinas, we determined that proliferation was significantly lower in rd1 than in wild type (wt) MGC. Levels of stem cell markers, such as Nestin and Sox2 , were also markedly reduced in rd1 MGC compared to wt MGC in neuron-glial cultures and in retina cryosections, even before the onset of PHR degeneration. We then investigated whether neuron-glial crosstalk was involved in these changes. Noteworthy, Nestin expression was restored in rd1 MGC in co-culture with wt neurons. Conversely, Nestin expression decreased in wt MGC in co-culture with rd1 neurons, as occurred in rd1 MGC in rd1 neuron-glial mixed cultures. These results imply that MGC proliferation and stem cell markers are reduced in rd1 retinas and might be restored by their interaction with “healthy” PHRs, suggesting that alterations in rd1 PHRs lead to a disruption in neuron-glial crosstalk affecting the regenerative potential of MGC.

Innate behaviors consist of a succession of genetically-hardwired motor and physiological subprograms that can be coupled to drastic morphogenetic changes. How these integrative responses are orchestrated is not completely understood. Here, we provide insight into these mechanisms by studying pupariation, a multi-step innate behavior of fly larvae that is critical for survival during metamorphosis. We find that the steroid-hormone ecdysone triggers parallel pupariation neuromotor and morphogenetic subprograms, which include the induction of the relaxin-peptide hormone, Dilp8, in the epidermis. Dilp8 acts on six Lgr3-positive thoracic interneurons to couple both subprograms in time and to instruct neuromotor subprogram switching during behavior. Our work reveals that interorgan feedback gates progression between subunits of an innate behavior and points to an ancestral neuromodulatory function of relaxin signaling.