Assembly and Exploration of a Single Cell Atlas of the Drosophila Larval Ventral Cord. Identification of Rare Cell Types

Abstract

Single‐cell RNA sequencing provides a new approach to an old problem: how to study cellular diversity in complex biological systems. This powerful tool has been instrumental in profiling different cell types and investigating, at the single‐cell level, cell states, functions, and responses. However, mining these data requires new analytical and statistical methods for high‐dimensional analyses that must be customized and adapted to specific goals. Here we present a custom multistage analysis pipeline which integrates modules contained in different R packages to ensure flexible, high‐quality RNA‐seq data analysis. We describe this workflow step by step, providing the codes, explaining the rationale for each function, and discussing the results and the limitations. We apply this pipeline to analyze different datasets of Drosophila larval ventral cords, identifying and describing rare cell types, such as astrocytes and neuroendocrine cells. This multistage analysis pipeline can be easily implemented by both novice and experienced scientists interested in neuronal and/or cellular diversity beyond the Drosophila model system. © 2021 US Government.

A single-cell transcriptomic atlas of complete insect nervous systems across multiple life stages

“…A major challenge is to combine genes, circuits, and behavior. Single-cell analyses have been performed in parts of the adult [18][19][20] or the larval nervous system at a single life stage [21,22]. However, a comprehensive transcriptomic atlas of the complete central nervous system from multiple samples and across multiple stages of larval life was previously not available.…”

Single‐Cell RNA Sequencing Analysis of the Drosophila Larval Ventral Cord

“…We apply this workflow to three separate samples and detail the technical challenges associated with successful application of scRNA-seq to studies on neuronal diversity. An accompanying article (Vicidomini, Nguyen, Choudhury, Brody, & Serpe, 2021) presents a custom multistage analysis pipeline that integrates modules contained in different R packages to ensure high-flexibility, high-quality RNA-seq data analysis. These protocols are developed for Drosophila larval ventral nerve cord, but could easily be adapted to other tissues and model organisms.…”

Systematic expression profiling of dprs and DIPs reveals cell surface codes in Drosophila larval peripheral neurons

“…Recent advances in single cell RNA sequencing (scRNAseq) provide a powerful, highthroughput approach to identify large scale gene expression patterns. Various Drosophila neural tissues have been analyzed by scRNAseq, including the adult brain (Davie et al, 2018), optic lobe (Konstantinides et al, 2018), adult VNC (Allen et al, 2020;Genovese et al, 2019), larval brain (Avalos et al, 2019), eye disc (Ariss et al, 2018) and the larval VNC (Nguyen et al, 2021;Vicidomini et al, 2021). Most studies report the transcriptome of large cell clusters of MNs, ganglion cells, neuroblasts, and glial cells due to the difficultly of matching single cell reads to a specific cell type and identity, impeding detailed analyses from scRNAseq data.…”

“…Because the GAL4 is inserted in coding regions, it should capture all regulatory mechanisms and faithfully report the expression of the corresponding endogenous mRNA (Nagarkar-Jaiswal et al, 2015a, 2015b. Thus, our dpr/DIP expression could serve as a map to identify individual MNs from a MN cluster in a larval VNC sample (Nguyen et al, 2021;Vicidomini et al, 2021). In addition to dprs and DIPs, other CSP subfamilies have been reported in several scRNAseq datasets, suggesting that expression maps of other subfamilies and even combinations of subfamilies can be utilized to refine cell types in datasets (Kurmangaliyev et al, 2020;Ma et al, 2021;Xie et al, 2021).…”