The function of the IRM protein Kirre in sensory pathway formation

本资助申请旨在阐明IRM蛋白在果蝇视觉和嗅觉系统突触发生中的功能。IRM蛋白是免疫球蛋白超家族中一个保守的小细胞粘附蛋白家族。在果蝇中，该蛋白家族包括不规则Chiasm C/Roughest （rerc /Rst）、Kin-of-irre/ dumbfound （Kirre/Duf）及其相互作用的蛋白伙伴Sticks-and-Stones （SNS）和Hibris （Hbs）。它们与秀丽隐杆线虫的Syg-1和Syg-2有关，与哺乳动物的Neph1、Neph2、Neph3和Nephrin有关。我们将通过关注果蝇的单一神经元细胞类型来分析这些来自果蝇和小鼠的蛋白质在建立神经元连接中的作用。L4神经元是唯一的单极细胞在蝇片侵入邻近的盒。在近椎板层，它们与3个L2神经元和6个其他L4神经元形成突触。Kirre在这些侧枝之间的相互接触部位特异性表达，其缺失导致突触数量减少。在嗅觉系统中，果蝇的Kirre和Rst及其小鼠的同源基因也在嗅觉受体神经元的轴突末梢表达。利用突变分析和基于二元基因表达和翻转酶系统的各种方法，我们将操纵表达Kirre神经元及其相互作用的细胞伙伴的遗传构成，例如通过过表达IRM转基因或通过敲除使用UAS-RNAi表达的基因。细胞自主和非自主效应将通过使用遗传镶嵌分析和有丝分裂重组诱导Gal4抑制剂Gal80的损失来区分。对突触发生的影响将通过使用突触标记和在电子显微镜下直接检查来测试。突变体和转化体中连通性改变的行为后果将被用作敏感传感器，例如，通过在kirre-遗传背景上重新引入特定的kirre+细胞类型来恢复对特定气味的敏感性。

This grant application aims at the elucidation of IRM protein function in synaptogenesis, in both the visual and olfactory system of Drosophila. IRM proteins are a small conserved family of cell adhesion proteins of the immunoglobulin superfamily. In Drosophila this protein family comprises Irregular Chiasm C/Roughest (IrreC/Rst), Kin-of-irre/Dumbfounded (Kirre/Duf), and their interacting protein partners Sticks-and-Stones (SNS) and Hibris (Hbs). They are related to Syg-1 and Syg-2 in C. elegans and to Neph1, Neph2, Neph3 and Nephrin in mammals. We will analyze the role of these proteins from Drosophila and mouse in the establishment of neuronal connectivity by focusing on single neuronal cell types in the fly. L4 neurons are the only monopolar cells in the fly lamina that invade neighbouring cartridges. In the proximal lamina layer they form synapses with three L2 neurons and with six other L4 neurons. Kirre is specifically expressed at sites of mutual contact between these collaterals and its absence leads to reduced synapse numbers. In the olfactory system, Kirre and Rst in Drosophila and their orthologs in mouse are also expressed on axon terminals of olfactory receptor neurons. Using mutant analysis and various methods based on binary gene expression and flipase systems we will manipulate the genetic constitution of the Kirre expressing neurons and their interacting cellular partners, e. g. by overexpressing IRM transgenes or by knocking down genes expressed using UAS-RNAi. Cell autonomous and non-autonomous effects will be distinguished by the use of genetic mosaic analysis and the induced loss of Gal4 inhibitor Gal80 by mitotic recombination. Effects on synaptogenesis will be tested by use of synaptic markers and by direct inspection in the electron microscope. Behavioural consequences of altered connectivity in mutants and transformants will be used as sensitive sensors, e.g. rescue of sensitivity to specific odorants will be done by reintroducing cell type specifically kirre+ on kirre- genetic background.