Single-cell RNA sequencing identifies novel cell types in Drosophila blood

Abstract

Drosophila has been extensively used to model the human blood-immune system, as both systems share many developmental and immune response mechanisms. However, while many human blood cell types have been identified, only three were found in flies: plasmatocytes, crystal cells and lamellocytes. To better understand the complexity of fly blood system, we used single-cell RNA sequencing technology to generate comprehensive gene expression profiles for Drosophila circulating blood cells. In addition to the known cell types, we identified two new Drosophila blood cell types: thanacytes and primocytes. Thanacytes, which express many stimulus response genes, are involved in distinct responses to different types of bacteria. Primocytes, which express cell fate commitment and signaling genes, appear to be involved in keeping stem cells in the circulating blood. Furthermore, our data revealed four novel plasmatocyte subtypes (Ppn⁺, CAH7⁺, Lsp⁺ and reservoir plasmatocytes), each with unique molecular identities and distinct predicted functions. We also identified cross-species markers from Drosophila hemocytes to human blood cells. Our analysis unveiled a more complex Drosophila blood system and broadened the scope of using Drosophila to model human blood system in development and disease.

Keywords: Blood; Drosophila; Plasmatocyte; Primocyte; Single-cell RNA-seq; Thanacyte.

Fig. 1.

Cell diversity in Drosophila blood cells delineated by single-cell transcriptomic analysis. A: Schematic representation of scRNA-seq workflow of blood cells from L3 stage larvae by 10x Chromium platform (10x Genomics, CA, USA). B: tSNE feature plot representing Drosophila blood scRNAseq data (left). Panel (right) shows cell numbers and percentage of cells/total for each tSNE cluster. C: Violin plots show expression of indicated genes across the different tSNE cell clusters: srp (pan-hemocyte) and representative marker genes for each of the five hemocyte clusters. X-axis: log scale normalized read count. Y: PM, plasmatocytes; CC, crystal cells; LM, lamellocytes; TH, thanacytes; PR, primocytes.

Fig. 2.

Characterization of plasmatocyte subtypes: Ppn⁺ PM, CAH7⁺ PM, Lsp⁺ PM and reservoir PM. A: tSNE plot of the plasmatocyte subclusters visualized by Seurat (R package). B: Heatmap representing the differentially expressed genes for each of the plasmatocyte subclusters. C: Single-cell trajectory for the four subtypes of plasmatocytes, reconstructed using Monocle. D – G: GO pathway analysis using the genes most differentially expressed in the Ppn⁺ PM (D), CAH7⁺ PM (E), Lsp⁺ PM (F) and reservoir PM (G) clusters. H: Feature plots showing the expression of marker genes specific to the Ppn⁺ PM, CAH7⁺ PM, Lsp⁺ PM and reservoir PM cell clusters. PM, plasmatocyte.

Fig. 3.

Labeling of Ppn⁺ and CAH7⁺ plasmatocytes by differentially expressed markers. A: Violin plot showing Ppn expression in plasmatocyte subclusters. Y-axis: log scale normalized read count. X-axis: 1: Ppn⁺ PM; 2: CAH7⁺ PM; 3: Lsp⁺ PM; 4: reservoir PM. B: Representing Apotome image of Ppn expression (green) in blood cells from Drosophila mCD8-GFP driven by Ppn-Gal4. Nuclear counterstain with DAPI (blue). Scale bar: 20 μm. C and E: Feature plots representing expression (level and distribution) in plasmatocytes for EcR (C) and ena (E). D and F: Representing Apotome images EcR (D; red) and Ena (F; red) protein expression in Drosophila blood cells, by immunofluorescence. Nuclear counterstain with DAPI (blue). Scale bars: 20 μm.

Fig. 4.

Identification of two novel cell types in Drosophila blood: thanacytes and primocytes. A: Feature plot representing beat-IIb gene expression in hemocytes (scRNA-seq data). Primocyte cell cluster bounded in blue dashed line. B: Violin plots showing the expression for beat-IIb, Z600 and Fas1 genes in Drosophila blood scRNA-seq data. C: Feature plot representing Ance-2 gene expression in hemocytes (scRNA-seq data). Thanacyte cell cluster bounded in blue dashed line. D: Violin plots for Ance-2, CG15506 and CG1648 expression in Drosophila blood scRNA-seq data. X-axis: PM, plasmatocytes; CC, crystal cells; LM, lamellocytes; TH, thanacytes; PR, primocytes. Y-axis: log scale normalized read count. E and F: GO pathway analysis of differentially expressed genes for primocyte (E) and thanacyte (F) cell clusters. Differentially expressed genes are shown in black, and enriched GO pathways are shown in green.

Fig. 5.

Labeling of thanacytes in Drosophila blood. A, B and E: tSNE feature plots of Drosophila blood scRNA-seq data. Thanacyte cell cluster bounded by blue dashed line. A: tSNE plot representing expression of Ance. B: tSNE plot representing expression of NimC1. E: tSNE plot representing expression of Tep4. C, D, F and G: Fluorescent images captured by Apotome microscopy. Scale bars: 20 μm. C: Ance protein expression (GFP; green) in Drosophila blood cells from Ance-GFP fly line. D: Ance protein expression (red) detected by antibody in Drosophila blood cells. F: Expression of Tep4 (green) in blood cells from a Drosophila Tep4-Gal4-driven UAS-mCD8-GFP line. G: NimC1 (P1), plasmatocyte marker, protein expression (red), and expression of Tep4 (green) in blood cells from Tep4-Gal4>UAS-mCD8-GFP Drosophila line. PM, indicates a GFP negative, NimC1 positive plasmatocyte; and, TH, indicates a GFP positive, NimC1 negative thanacyte.

Fig. 6.

Silencing of Tep4 expression in thanacytes led to distinct responses to different types of bacteria. A and B: Survival plots for control flies (w¹¹¹⁸: circle) and Tep4-IR flies (Tep4-Gal4>Tep4-IR: square) after intrathoracic injection with different types of bacteria. Whereas there is no difference observed between these two groups when injecting Escherichia coli (E. coli), Photorhabdus luminescens (P. luminsescens), Micrococcus luteus (M. luteus), or Staphylococcus aureus (S. aureus), injection of Photorhabdus asymbiotica (P. asymbiotica) (A) or Listeria monocytogenes (L. monocytogenes) (B) showed significant different survival curves. *P-value < 0.05. Graphs depict survival of 40 flies per experimental group, monitored for 60 h at 12-hour intervals.

Fig. 7.

Functional conservation between Drosophila and human blood cells. A: Summary of expression of Drosophila genes and their human homologs. B–D: tSNE feature plots of Drosophila blood scRNA-seq data. E–G: tSNE feature plots of human blood scRNA-seq data (peripheral blood mononuclear cells (Zheng et al., 2017)). B and E: Representing expression of Aldh (B), fly plasmatocyte cluster bounded by dashed blue line; and, its human homolog ALDH2 (E), CD14⁺ monocytes (CD14⁺ mono) and dendritic cells (DC) clusters indicated. C, D, F and G: Representing expression of CG30088 (C) and CG30090 (D), fly thanacytes cluster bounded by dashed blue line; and, their human homologs GZMB (F) and GZMH (G), respectively, natural killer cell and CD8+ (cytotoxic) T cell clusters indicated.