Combining single-cell RNA-sequencing with a molecular atlas unveils new markers for C. elegans neuron classes

Abstract

Single-cell RNA-sequencing (scRNA-seq) of the Caenorhabditis elegans (C. elegans) nervous system offers the unique opportunity to obtain a partial expression profile for each neuron within a known connectome. Building on recent scRNA-seq data [1] and on a molecular atlas describing the expression pattern of ~800 genes at the single cell resolution [2], we designed an iterative clustering analysis aiming to match each cell-cluster to the ~100 anatomically defined neuron classes of C. elegans. This heuristic approach successfully assigned 58 clusters to their corresponding neuron class. Another 11 clusters grouped neuron classes sharing close molecular signatures and 7 clusters were not assigned. Based on these 76 molecular profiles, we designed 15 new neuron class-specific promoters validated in vivo. Amongst them, 10 represent the only specific promoter reported to this day, expanding the list of neurons amenable to genetic manipulations. Finally, we observed a differential expression of functionally relevant genes between sensory-, inter-, and motor neurons in C. elegans, suggesting the mode of functional diversification may vary accordingly to the neuronal modalities.Recent progresses in molecular profiling using single-cell RNA-sequencing (scRNAseq) allowed exploring neuronal diversity at the molecular level in human and mouse brain [4][5][6]. In comparison, the nervous system of Caenorhabditis elegans (C. elegans) adult hermaphrodite has a simple and well-described structure composed of only 302 neurons. Despite its anatomical simplicity, the neuronal diversity of C. elegans encompasses 118 neuron classes identified by the examination of their complete diagram of connectivity as revealed by serial sections and electron microscopy [7][8][9]. Based on expression patterns at the single-cell resolution, the 118 anatomically defined neuron classes in the adult hermaphrodite would correspond to 118 or more unique molecular identities [2, 10]. However, the transcriptional profiles are only known for a few neuron classes purified by FACS-sorting [11][12][13][14].The exquisitely described small circuits composed by few neurons in C. elegans allow exploring how cell-to-cell communication can shape behaviour. The function of several C. elegans neuron classes has been defined using laser ablation, genetic and optogenetic methods [15, 16]. These later methods, however, rely on reliable promoters driving transgene expression in a single neuron class to be characterized. Currently, cell-specific promoters are described for only 29 neuron classes. This current lack of cell-specific promoters has limited the optogenetic and chemogenetic analysis of the neuronal functions to a fraction of the C. elegans nervous system.