Central signalling pathways involved in the INSL5/RXFP4 axis

胰岛素样多肽5(INSL5)是松弛素家族多肽受体RXFP4的配体。我们之前确定INSL5是结肠和直肠肠内分泌细胞的激素产物，并与武田合作表明它在小鼠身上具有促食欲活性。为了研究INSL5/RXFP4轴的功能作用，我们建立了表达Insl5或Rxfp4的细胞用荧光记者标记的小鼠模型。使用我们的Rxfp4-CRE小鼠品系，我们鉴定了两个Rxfp4阳性细胞群：在结肠中表达TPH1的肠内分泌细胞群，可能代表肠嗜铬细胞群，以及在下丘脑外侧的限制性神经元群。在这个项目中，我们将研究INSL5/Rxfp4轴的性质和功能。该项目将与Giles Yeo(IMS)小组和Clemence Blouet(IMS)小组合作实施，Giles Yeo(IMS)小组以前拥有FACS纯化的POMC阳性神经元群体的单细胞RNAseq分析经验，Clemence Blouet小组(IMS)在通过应用例如细胞消融、DREADDS和神经元追踪技术来调查和操纵神经元群体方面拥有丰富的经验。我们的目标是：1.通过FACS纯化、单细胞RNAseq和LC-MS/MS多肽分析(IMS)鉴定Rxfp4标记的下丘脑神经元的多肽特征和转录图谱2.使用应用于原代神经元培养和下丘脑切片制备(IMS)的钙成像(+/-电生理)技术来表征Rxfp4阳性神经元的单细胞反应性。目的：探讨下丘脑Rxfp4神经元在控制摄食量中的作用。立体定向双侧下丘脑AAV注射将通过Cre依赖的caspase3表达来消融Rxfp4阳性细胞，或使用DREADDS激活它们。将检查小鼠的食物摄入量和食物偏好的变化(IMS/MedImmune)。建立适合血浆中INSL5定量的INSL5抗体，研究INSL5/Rxfp4轴在体内的病理生理调节。

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).