The steroid-hormone ecdysone coordinates parallel pupariation neuromotor and morphogenetic subprograms via epidermis-to-neuron Dilp8-Lgr3 signal induction

Funding Information: We thank Drs. Carlos Ribeiro, Christen Mirth, Elio Sucena, Filip Port, Frank Schnorrer, Julien Colombani, Maria Dominguez, Maria Luisa Vasconcelos, Pierre Leopold, Simon Bullock, Rita Teodoro, Gerald Rubin, Melissa Harrison, Kate O’Connor-Giles, Jill Wildonger, Mariana Melani, Pablo Wappner, and Christian Wegener for fly stocks and reagents. We thank Ryohei Yagi and Konrad Basler for the LHV2 plasmid and Brain McCabe for the mhc-Gateway destination plasmid. We thank Carlos Ribeiro and Dennis Goldschmidt for help in designing and constructing one of the pupariation arenas and Mariana Melani, Pablo Wappner, Arash Bashirullah, and Filip Port for sharing resources and unpublished data. We thank Arash Bashirullah, Fillip Port, and Carlos Ribeiro for discussions and/or comments on the manuscript, and Jim Truman for discussions on Fraenkel’s pupariation factors. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. Work in the Integrative Biomedicine Laboratory was supported by the European Commission FP7 (PCIG13-GA-2013-618847), by the FCT (IF/00022/2012; Congento LISBOA-01-0145-FEDER-022170, cofinanced by FCT/Lisboa2020; UID/Multi/04462/2019; PTDC/BEXBCM/1370/2014; PTDC/MED-NEU/30753/2017; PTDC/BIA-BID/31071/2017; FCT SFRH/BPD/94112/ 2013; SFRH/BD/94931/2013), the MIT Portugal Program (MIT-EXPL/BIO/0097/2017), and FAPESP (16/09659-3, 16/10342-4, and 17/17904-0). AG is a CONICET researcher, YV holds a CONICET postdoctoral fellowship and FPS and MJD hold a PhD fellowship from CONICET. Work in the Garelli lab was supported by ANPCyT (Agencia Nacional para la Promoción de la Ciencia y la Tecnología, PICT 2014-2900 and PICT 2017-0254) and CONICET (PIP11220150100182CO). Publisher Copyright: © 2021, The Author(s). Copyright: Copyright 2021 Elsevier B.V., All rights reserved.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.publishersversionpublishe

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Progression through pupariation behaviors requires dilp8-Lgr3 signaling between the cuticle epidermis and thoracic CNS neurons

Higher dipterans undergo metamorphosis within a puparium, a protective capsule made up of the reshaped and hardened cuticle of the last larval instar. Puparium formation (pupariation) in Drosophila starts when wandering stage larvae reduce their locomotion and initiate increasingly strong whole body contractions that together with the internalization of the three anteriormost body segments remodel the body and cuticle, reducing their length/width ratio from ~5 to ~3. This behavior lasts 5-10 min and culminates with the extrusion of a proteinaceous mix (glue) produced by the salivary glands. The animal then slowly moves forward in a caterpillar-like fashion, traveling about half its length for ~1 min to its final pupariation site. We call this behavior glue-spreading behavior (GSB), as it helps to spread the glue over the ventral part of the animal, promoting its attachment to the underlying substrate. Following GSB, the final shape of the puparium is set, even though weak and periodic contractions occur over the next 40-50 min (post-GSB), the operculum becomes defined, and the cuticle (i.e., the future puparium) starts to gradually sclerotize and tan. Here, we show that proper progression through three pupariation behaviors (pre-GSB, GSB, and postGSB) requires the Dilp8-Lgr3 pathway, a relaxin-like pathway that has been previously implicated in controlling the timing of pupariation in animals carrying aberrantly growing imaginal discs. During pupariation, however, Dilp8-Lgr3 signaling is spatiallyand temporally-distinct: a strong, epidermis-derived dilp8 expression peak that starts at the pre-GSB phase of pupariation appears to signal via Lgr3 in a novel subpopulation of thoracic CNS neurons. While this signaling ensures partial progression through the preGSB phase, and total progression through GSB and post-GSB phases, it is not required for sclerotization and tanning. These results demonstrate a new transient epidermis to neuron signaling event that facilitates progression through the cascade of pupariationassociated behaviors.Fil: Garelli, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidade Nova de Lisboa; PortugalFil: Heredia, Fabiana. Universidade Nova de Lisboa; PortugalFil: Volonté, Yanel Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidade Nova de Lisboa; PortugalFil: Pereirinha, Joana. Universidade Nova de Lisboa; PortugalFil: Casimiro, Andrea. Universidade Nova de Lisboa; PortugalFil: Viegas, Filipe. Universidade Nova de Lisboa; PortugalFil: Belém, Claudia. Universidade Nova de Lisboa; PortugalFil: Tanaka, Kohtaro. Instituto Gulbenkian de Ciências; PortugalFil: Cardoso, Gisela. Universidade Nova de Lisboa; Portugal. Universidade de Sao Paulo; BrasilFil: Macedo, Andre. Universidade Nova de Lisboa; PortugalFil: Leal, Ana. Universidade Nova de Lisboa; PortugalFil: Kotowicz, Malwina. Universidade Nova de Lisboa; PortugalFil: Prado Spalm, Facundo Heber. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Mendes, César. Universidade Nova de Lisboa; PortugalFil: Gontijo, Alisson M.. Universidade Nova de Lisboa; PortugalInsect Hormones 2019KolymbariGreciaBashirullah, ArashLeopold, Pierr