A single-cell transcriptome atlas of the adult muscle precursors uncovers early events in fiber-type divergence inDrosophila

Abstract

In Drosophila, the wing disc-associated adult muscle precursors (AMPs) give rise to the fibrillar indirect flight muscles (IFM) and the tubular direct flight muscles (DFM). To understand early transcriptional events underlying this muscle diversification, we performed single cell RNA-sequencing experiments and built a cell atlas of AMPs associated with third instar larval wing disc. Our analysis identified distinct transcriptional signatures for IFM and DFM precursors that underlie the molecular basis of their divergence. The atlas further revealed various states of differentiation of AMPs, thus illustrating previously unappreciated spatial and temporal heterogeneity among them. We identified and validated novel markers for both IFM and DFM precursors at various states of differentiation by immunofluorescence and genetic tracing experiments. Finally, we performed a systematic genetic screen using a panel of markers from the reference cell atlas as an entry point and found a novel gene, Ama, which is functionally important in muscle development. Thus, our work provides a framework of leveraging scRNA-seq for gene discovery and therefore, this strategy can be applied to other scRNA-seq datasets.

Single-cell transcriptomics of theDrosophilawing disc reveals instructive epithelium-to-myoblast interactions

“…The few tracheal cells were identified by high expression of the genes waterproof (wat) and tracheal-prostasin (tpr) (Jaspers et al, 2014;Drees et al, 2019), and the hemocytes were distinguished by high expression levels of Hemese (He) and regucalcin (Kurucz et al, 2003;Klebes et al, 2005) ( Figure 1E, Figure 1 -figure supplement 1). We observed a similar set of marker genes as other recently published wing disc scRNAseq analyses (Deng et al, 2019;Bageritz et al, 2019;Zappia et al, 2019). Altogether, we recovered profiles for 19,885 adult muscle precursors (AMPs), 7,104 wing disc epithelial cells, 15 tracheal cells, and 12 hemocytes.…”

“…Some of these identified genes were expressed at higher levels within certain subsets of indirect AMP, such as the transcription factors h and rotund (rn) that jointly mark a subset of indirect AMPs (Figure 3B and Figure 3 -figure supplement 3). Using a rn-Gal4 line, we confirmed the expression of rn in a subset of AMPs nearest to the stalk of the wing disc ( Figure 3 -figure supplement 3), which largely overlaps with the pattern identified for the gene h via antibody staining (Zappia et al, 2019). Potentially, h and rn may separate indirect AMPs into subsets that contribute to different muscle fibers, although such a role has not been described.…”

“…We found that one cluster was characterized by high levels of ct and low levels of vg ( Figure 3B, D-F Classification of direct and indirect AMP cells based on vg and ct expression enabled us to identify additional genes that are differentially expressed between the two canonical AMP cell types ( Figure 3B and Materials and Methods). These genes include Zn finger homeodomain 1 (zfh1), hairy (h), and naked cuticle (nkd) for indirect AMPs and Amalgam (Ama) and wing blister (wb) for direct AMPs, which were also identified in a recent single-cell study on the AMPs (Zappia et al, 2019). Some of these identified genes were expressed at higher levels within certain subsets of indirect AMP, such as the transcription factors h and rotund (rn) that jointly mark a subset of indirect AMPs (Figure 3B and Figure 3 -figure supplement 3).…”

“…Single-cell transcriptomics provides a powerful paradigm for mapping the cellular composition of developing organs (Schier, 2020), including vertebrate appendages (Fabre et al, 2018;Cao et al, 2019;Feregrino et al, 2019), and has been successfully utilized for characterizing the Drosophila wing disc at single snapshots during development (Deng et al, 2019;Bageritz et al, 2019;Zappia et al, 2019). Beyond cataloging cell types, transcriptomescale analysis of single cells opens the way for a comprehensive evaluation of how interactions between cells may facilitate development (Satija et al, 2015;Karaiskos et al, 2017;Deng et al, 2019;Bageritz et al, 2019).…”