Single-cell transcriptome maps of myeloid blood cell lineages in Drosophila

Abstract

The Drosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and mature hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells, yet molecular underpinnings of the lymph gland hemocytes have been less investigated. Here, we use single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discover previously undescribed hemocyte types including adipohemocytes, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identify the developmental trajectory of hemocytes during normal development as well as the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. Finally, we establish similarities and differences between embryonically derived- and larval lymph gland hemocytes. Altogether, our study provides detailed insights into the hemocyte development and cellular immune responses at single-cell resolution.

Fig. 1. Major cell types identified in developing Drosophila lymph glands.

a A schematic diagram of the Drosophila lymph gland (left). Prohemocytes comprise the medullary zone at the inner core (blue) and give rise to mature hemocyte at the outermost area, called the cortical zone (red). Differentiating hemocytes in between the medullary zone and the cortical zone are termed the intermediate zone (yellow). The posterior signaling center (PSC) positions at the posterior end of the lymph gland (pink). Drosophila lymph glands (blue, DAPI) at three timepoints (72, 96, and 120 h AEL; After Egg Laying) (middle). Schematic workflow of sample preparation for scRNA-seq using Drop-seq (right). Scale bar, 30 μm. Lymph glands are demarcated by white dotted lines. b DAPI-positive cell counts of a single lymph gland lobe (n = 30 each for three timepoints). Red horizontal lines show median counts (397, 1392, and 4557 for 72, 96, and 120 h AEL, respectively). c A t-SNE plot showing the two-dimensional projection of eight major cell types identified in the scRNA-seq dataset (n = 22,645 in total; 72 h AEL, 2321; 96 h AEL, 9400; 120 h AEL, 10,924 cells). The count of each cell type is indicated in parentheses. Clusters are defined by following markers: Prohemocytes (PH: Tep4, Ance; 36.2%), Plasmatocytes (PM: Hml, Pxn, NimC1; 57.6%), Crystal cells (CC: lz, PPO1, PPO2; 1.3%), Lamellocytes (LM: atilla; 1.5%), the PSC (Antp, col; 0.9%), and Dorsal Vessel (DV: Mlc, Hand; 0.1%). Colors denote cell types. Dotted lines demarcate prohemocytes (blue) and plasmatocytes (red). d Two-dimensional projections of major cell types along developmental timepoints (left) and proportion of the cell types at each timepoint (right). Proportions of prohemocytes (blue) and plasmatocytes (red) are indicated. e Relative proportion (indicated as proportional ratio; top) and normalized cell counts (bottom) of each major cell type. Colors represent sampling timepoints.

Fig. 2. Subclustering analysis of hemocytes in the lymph gland.

a Subclusters of hemocytes—prohemocytes, plasmatocytes, lamellocyte, and crystal cells—are projected onto two-dimensional t-SNE plots. The numbers in the plots represent the subcluster number. b Dot plot presentation of significant gene sets in the 17 subclusters using Wilcoxon Rank-Sum test. 5 representative markers, srp, Tep4, Ance, Hml, and Pxn are indicated to the left column. Signature genes identified in this study are marked with subcluster markers. Dot color shows levels of average expression, and dot size represents the percentage of cells expressing the corresponding marker genes in each subcluster. Non-hematopoietic cell types are indicated (DV, dorsal vessel; RG, ring gland; Neurons). Cell count for each subcluster is listed in Supplementary Table 4. c Expressions of cell-cycle regulating genes in 17 subclusters. Dot color indicates average expression levels and dot size displays the percentage of cells with cell cycle controlling genes (Cdk1, CycD, and CycE for G1; stg, CycA, and CycB for G2; polo, aurB, and Det for M) in each subcluster.

Fig. 3. In vivo validation of subclusters and markers in the lymph gland.

a Expression of GST-rich-marker genes, CG18547 and CG3397. mRNA transcripts of CG18547 (top; magenta) or CG3397 (bottom; magenta) are visualized at the outer demarcation of Tep4 (green; left). Magnified images: Tep4 and CG18547 or CG3397. b Expression of adipohemocyte-specific marker genes, Sirup or Lsd-2. A few Sirup-expressing (top; magenta) or Lsd-2 (bottom; magenta)-expressing cells are observed within the Hml⁺ (green) cortical zone. However, Sirup-positive or Lsd-2-positive hemocytes are devoid of Hml. Magnified images: Hml and Sirup or Lsd-2. c Lipid droplet-containing hemocytes in the lymph gland. When visualized with BODIPY (top; green) or Nile Red (bottom; magenta) lipid probes, a few hemocytes (Phalloidin, white) at the cortical zone hold a lipid droplet. Magnified images: actin structures (Phalloidin, white), a lipid droplet, and DAPI. d Plasmatocyte marker, vir-1 (green; vir-1^MiMiC) partially co-localizes with Pxn⁺ (top; magenta) or NimC1 (bottom; magenta). Magnified images: vir-1 and Pxn or NimC1. e PSC markers in the lymph gland. Antp⁺ PSC cells (magenta) co-localize with Ilp6 (green; Ilp6-gal4 UAS-mCD8::GFP), tau (green; tau^MiMiC), mthl7 (white; mthl7-gal4 UAS-GTRACE), or chrb (green; chrb^MiMiC). Ilp6-gal4 is expressed in other cell types including PH6 and crystal cells (Supplementary Fig. 2i). Magnified images: Antp and Ilp6, tau, mthl7, or chrb. f Crystal cell markers in the lymph gland. lz⁺ crystal cells (magenta) co-localized with Men (white; Men-gal4 UAS-GTRACE; left) or Numb (green; Numb::GFP; right). lz co-localizes with a subset of Men while all the Numb⁺ and lz⁺ overlap. Magnified images: lz and Men or Numb. g domeless-LexA generated in this study (magenta; domeless-LexA LexAop-mRFP-nls) partially co-localizes with Pxn (green). Magnified images: domeless and Pxn. White scale bar, 30 μm; yellow scale bar, 3 μm. Lymph gland is demarcated by white dotted line. Blue indicates DAPI. The dotted box in a–g indicates a magnified region. Each panel at the right displays magnified views.

Fig. 4. Pseudotime trajectory analysis of lymph gland hematopoiesis.

a A three-dimensional landscape of the lymph gland hematopoiesis trajectory using Monocle3 (n = 19,143). Non-hematopoietic cells were excluded. Black line indicates the trajectory. Colors indicate the six major cell types used. The inset shows the three ancestral PH subclusters, PH1, PH2, and PH3. b Trajectories re-drawn by developmental timepoints (top) and calculated pseudotime (bottom). Colors indicate the three real-timepoints (top) and pseudotime (bottom). c Relative densities of hemocytes segregated by three timepoints (top) and cell types (bottom) along pseudotime. PH1 and PH2 are separated from other PH subclusters for higher resolution. Colors in density plots correspond to pseudotime, as in b. d Nplp2-gal4 (dome^Meso-EBFP; Nplp2-gal4 UAS-EGFP; HmlΔ-dsRED) at 72, 96, or 120 h AEL lymph gland. Nplp2-gal4 (green) partially overlaps with dome^Meso (blue), Hml (magenta) or both. The proportion of Nplp2-gal4 expressing cells diminishes over development (36.08% at 72 h; 24.01% at 96 h; 17.81% at 120 h AEL). Graph indicates quantitations of the combinational proportions of dome^Meso+or dome^Meso−, Nplp2⁺ or Nplp2⁻, and Hml⁺ or Hml⁻ cells in one lymph gland lobe at 72 (n = 20), 96 (n = 27), or 120 (n = 22) h AEL. The bracket indicates the proportions of Nplp2-gal4. White scale bar, 30 μm. White dotted line demarcates the lymph gland. e Heatmap representation of the 35 signature genes identified in PH1, PH2, and the PSC (n = 77, 79, and 189 cells, respectively). The colored legend denotes the standardized level of the genes. f Four subgroups in PH1 and PH2 defined by the expression of Delta (Dl) and Notch (N). Colors show subgroups and shapes specify PH subclusters. X axis means Dl expression; Y axis, N expression. g Binary heatmap showing the activity of transcription factors in PH1, PH2, and the PSC predicted by SCENIC. Numbers in parentheses denote the count of downstream genes used to test the activity of transcription factors.

Fig. 5. Expression of PH1 in the lymph gland.

a STAT92E^Act (green) and Tep4⁺ (magenta) or Antp⁺ (white) cells are mutually exclusive (Tep4-gal4 UAS-mCherry; STAT92E::edGFP). Magnified images: STAT92E^Act and Tep4⁺ cells near Antp⁺ PSC. b STAT92E^Act (green) do not co-localize with cells expressing high (PSC) or low (PHs) levels of collier (magenta). Magnified images: STAT92E^Act and col⁺ cells near the PSC. c. The number of STAT92E^Act cells (green) increases during lymph gland development (72 (n = 24), 96 (n = 17), and 120 h (n = 20) AEL). Antp⁺ (magenta) and STAT92E^Act are separable at all timepoints. Graphs represent quantitation of the number (left) or the proportion (right) of STAT92E^Act cells in one lymph gland lobe. d. Genetic ablation of the PSC (pCol85-gal4; STAT92E::edGFP UAS-Hid, Rpr) attenuates STAT92E^Act (green) expression (top). Graph indicates quantitations of the number of STAT92E^Act cells in one lymph gland lobe (bottom, Mann–Whitney test, ***P < 0.0001). pCol85-gal4/+ (n = 13) or pCol85-gal4 UAS-Hid, Rpr (n = 19). e Dl⁺ cells (magenta) are localized adjacent to the PSC (Antp, green). Magnified images: Dl⁺ and Antp⁺ cells. Cyan dotted lines demarcate Dl⁺ cells. f Dl⁺ cells (magenta) co-localize with STAT92E^Act (green). Magnified view: Dl⁺ and STAT92E^Act cells (right). A few Dl⁺ cells that do not express STAT92E::edGFP are indicated (arrowhead). g Lineage tracing of Dl⁺ cells (green, traced; magenta, real-time; blue, Antp). Delta^BL45136-gal4 UAS-GTRACE covers the entire lymph gland. Arrowheads represent Dl⁺ cells next to the Antp⁺ Dl⁺ PSC. h Model of the lymph gland hematopoiesis. PH1 cells are adjacent to the PSC. PH1 and PSC or PH2 are mutually exclusive. There are multiple states of prohemocytes including GST-rich and intermediary PHs/PMs. Plasmatocytes represent a heterogeneous cell population including adipohemocytes and late plasmatocytes, PM3-4. Colors indicate subclusters. Putative subclusters in the medullary zone (MZ), the cortical zone (CZ), or the intermediate zone (IZ) are described (right). Subcluster-specific markers verified in this study are listed (right). Red asterisks indicate 120 h AEL-specific subclusters. White scale bar, 30 μm; yellow scale bar, 3 μm. White dotted line demarcates the lymph gland. The dotted box (a–b, e–g) indicates a magnified region. Each panel at the right displays magnified views. Median value is represented in graphs (c, d).

Fig. 6. Comparison of normal and wasp infested lymph glands.

a Lymph gland (Antp-gal4 UAS-GTRACE; Antp, magenta) remains at 24 h post infestation (PI) (DAPI, blue). Hemocytes disintegrate by 48 h PI. Graph indicates the number of DAPI⁺ cells at 24 h PI. Normal (n = 51), 24 h PI (n = 44). Mann-Whitney test, **P = 0.0011. b UMAP projections of major cell types defined in normal (top, ‘n’) and wasp infested (bottom, ‘i’) lymph glands at 96 h AEL (thus, 24 h PI). c Relative proportion (top) or normalized cell counts (bottom) of major cell types in normal (blue) and wasp infested (red) lymph glands. d Two-dimensional projections of subclustered cells in normal (left) and wasp infested (right) lymph glands. e Normalized cell counts (top) or proportional ratio (bottom) of subclustered cells in normal (blue) or wasp infested (red) lymph glands. f. Wasp infestation reduces STAT92E^Act (top; green) or Dl⁺ (bottom; magenta) PH1 populations. Graphs represent quantitation of the number of STAT92E ^Act (top) or the proportion of Dl⁺ (bottom) cells after infestation (24 h PI). Normal (n = 27) or 24 h PI (n = 18) for Top and Normal (n = 10), or 24 h PI (n = 13). Mann–Whitney test, ***P < 0.0001. g Top 20 genes in iLM1 or iLM2. Color bar indicates the level of scaled gene expression. h Dot plot expresses the level of marker genes in normal (blue) or wasp infested (red) lymph glands (top) using Wilcoxon Rank-Sum test. Scaled expressions of cell-cycle regulating genes in subclusters upon wasp infestation (bottom). Dot color: average expression levels. Dot size: percentage of cells expressing cell cycle genes in a subcluster. Cell count for each subcluster is listed in Supplementary Table 5. i PSC cell numbers do not change upon wasp infestation by 24 h PI (Antp, magenta; Traced, white; Antp-gal4 UAS-GTRACE). Graph indicates the number of Antp⁺ cells. Normal (n = 19) or infested (n = 17). Mann–Whitney test, n.s.: not significant. White scale bar, 30 μm; Yellow scale bar, 3 μm. Lymph glands are demarcated by white dotted lines. Median value is shown in graphs. Colors in b and d indicate each cell type. Graphs indicate the number of cells in one lymph gland lobe.

Fig. 7. Lymph gland hematopoiesis following wasp infestation.

a Three-dimensional pseudotime trajectory of subclustered cells from the normal lymph gland dataset. Colors indicate cell types. b Expression patterns of 14 marker genes– kn (col), IM18, Ance, Tep4, Hml, Pxn, eater, vkg, NimC1, Pvr, atilla, Dbi, ItgalphaPS4, and CG1208–in two-dimensional (UMAP 1 and 3) pseudotime trajectory. Color bar denotes the log₁₀-scaled level of gene expression. Gray color indicates no expression. c A three-dimensional trajectory landscape of major cell types under wasp infestation (left), and additional representation of trajectory over calculated pseudotime using Monocle3 (right). Box indicates the cells used for subtrajectory analysis in d. Colors in legends show pseudotime (right). d Subtrajectory analysis of four subclusters—iPH4, iPM1, iLM1, and iLM2—detected in the trajectory to iLM (left). Two different waves, arrow 1/2 (iPH4/NimC1⁻ iPM1, thus, intermediate cells) and arrow 3 (NimC1⁺ iPM1), advance towards iLM with distinct gene modules (right). Shared gene modules between iPH or iPM with iLM are indicated in boxes. Colored scale represents the z-transformed mean expression level of gene modules. e Lamellocytes differentiate from intermediate iPHs (Nplp2-gal4 UAS-GTRACE) or iPMs (Hml-gal4 UAS-GTRACE) upon wasp infestation. Nplp2⁺ iPHs (green, traced; blue, DAPI; top) or Hml⁺ iPMs (green, traced; blue, DAPI; bottom) express atilla (magenta) upon wasp infestation. Insets indicate magnified views of atilla⁺ cells. Cyan dotted lines within insets demarcate traced iLMs. f iLM and iCC lineages are separable. iLMs emerge independently of iCCs (lz-gal4^DBD, Pxn-gal4^AD UAS-GTRACE). lz-gal4^DBD, Pxn-gal4^AD UAS-GTRACE trace the majority of lz-expressing crystal cells (lz, magenta; traced, white). However, atilla⁺ iLMs are lz^DBD/Pxn^AD-negative (atilla, cyan; traced, white). White scale bar, 30 μm; yellow scale bar, 3 μm. Lymph glands are demarcated by white dotted lines. Magnified images are shown on the right of each panel.

Fig. 8. Transcriptome-wide comparisons between embryonic and larval lymph gland hemocytes in Drosophila.

a Two-dimensional projections of hemocytes in the lymph gland (top) and circulation (bottom) at 96 and 120 h AEL. b Combined projection (top) and proportions of major cell types (bottom) in the lymph gland (yellow) and in circulation (cyan). Inset (top) shows the ratio of major cell types between the lymph gland (L) and in circulation (C). c Dot plot of marker genes highly enriched in a lineage-specific or cell type-specific manner. The colors show the origin of the datasets (yellow, lymph gland; cyan, circulation). Significant marker genes with FDR-adjusted P < 0.001 were outlined.