Central signalling pathways involved in the INSL5/RXFP4 axis

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Insulin-like peptide 5 (INSL5) is a ligand for the relaxin family peptide receptor RXFP4. We previously identified INSL5 as a hormonal product of colonic and rectal enteroendocrine cells, and in collaboration with Takeda showed that it has orexigenic activity in mice. To investigate the functional role of the INSL5/RXFP4 axis, we have generated mouse models in which cells expressing Insl5 or Rxfp4 were labelled with fluorescent reporters. Two Rxfp4-positive cells populations were identified using our Rxfp4-Cre mouse strain: an enteroendocrine cell population in the colon expressing Tph1 and likely representing an enterochromaffin cell population, and a restricted neuronal population in the lateral hypothalamus.In this project we will investigate the nature and functional roles of the INSL5/Rxfp4 axis. The project will be carried out in collaboration with the group of Giles Yeo (IMS), which has previous experience of single cell RNAseq analysis of FACS-purified POMC-positive neuronal populations, and with the group of Clemence Blouet (IMS) which has extensive experience of investigating and manipulating neuronal populations by application of e.g. cell ablation, DREADDs and neuronal tracing techniques. MedImmune Scientists will contribute to the in vivo characterisation of feeding behaviour in murine models and the development of INSL5 antibodies that will enable the measurement of INSL5 concentrations in plasma.Our aims are:1. To identify the peptidomic signature and transcriptomic profiles of Rxfp4-labelled hypothalamic neurons by FACS purification, single cell RNAseq and LC-MS/MS peptidomic analysis (IMS)2. To characterize the single cell responsiveness of Rxfp4 positive neurons using Ca2+ imaging (+/- electrophysiological) techniques applied to primary neuronal cultures and hypothalamic slice preparations (IMS).3. To establish the functional role of hypothalamic Rxfp4 neurons in the control of food intake. Stereotactic bilateral hypothalamic AAV injections will be used ablate Rxfp4 positive cells using Cre-dependent caspase3 expression, or to activate them using DREADDs. Mice will be examined for changes in food intake and food preference (IMS/MedImmune).4. To investigate the pathophysiological regulation of the INSL5/Rxfp4 axis in vivo by developing INSL5 antibodies suitable for INSL5 quantitation in plasma samples (MedImmune).