Temporal specificity and heterogeneity of Drosophila immune cells
2020; Springer Nature; Volume: 39; Issue: 12 Linguagem: Inglês
10.15252/embj.2020104486
ISSN1460-2075
AutoresPierre B. Cattenoz, Rosy Sakr, Alexia Pavlidaki, Claude Delaporte, Andrea Riba, Nacho Molina, Nivedita Hariharan, Tina Mukherjee, Angela Giangrande,
Tópico(s)Viral Infectious Diseases and Gene Expression in Insects
ResumoResource12 March 2020free access Temporal specificity and heterogeneity of Drosophila immune cells Pierre B Cattenoz Corresponding Author [email protected] orcid.org/0000-0001-5301-1975 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Rosy Sakr Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Alexia Pavlidaki Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Claude Delaporte Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Andrea Riba Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Nacho Molina orcid.org/0000-0003-0233-3055 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Nivedita Hariharan Institute for Stem Cell Science and Regenerative Medicine (inStem), Bangalore, India The University of Trans-disciplinary Health Sciences and Technology, Bangalore, India Search for more papers by this author Tina Mukherjee Institute for Stem Cell Science and Regenerative Medicine (inStem), Bangalore, India Search for more papers by this author Angela Giangrande Corresponding Author [email protected] orcid.org/0000-0001-6278-5120 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Pierre B Cattenoz Corresponding Author [email protected] orcid.org/0000-0001-5301-1975 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Rosy Sakr Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Alexia Pavlidaki Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Claude Delaporte Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Andrea Riba Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Nacho Molina orcid.org/0000-0003-0233-3055 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Nivedita Hariharan Institute for Stem Cell Science and Regenerative Medicine (inStem), Bangalore, India The University of Trans-disciplinary Health Sciences and Technology, Bangalore, India Search for more papers by this author Tina Mukherjee Institute for Stem Cell Science and Regenerative Medicine (inStem), Bangalore, India Search for more papers by this author Angela Giangrande Corresponding Author [email protected] orcid.org/0000-0001-6278-5120 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France Centre National de la Recherche Scientifique, UMR7104, Illkirch, France Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France Université de Strasbourg, Illkirch, France Search for more papers by this author Author Information Pierre B Cattenoz *,1,2,3,4, Rosy Sakr1,2,3,4,‡, Alexia Pavlidaki1,2,3,4,‡, Claude Delaporte1,2,3,4, Andrea Riba1,2,3,4, Nacho Molina1,2,3,4, Nivedita Hariharan5,6, Tina Mukherjee5 and Angela Giangrande *,1,2,3,4 1Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France 2Centre National de la Recherche Scientifique, UMR7104, Illkirch, France 3Institut National de la Santé et de la Recherche Médicale, U1258, Illkirch, France 4Université de Strasbourg, Illkirch, France 5Institute for Stem Cell Science and Regenerative Medicine (inStem), Bangalore, India 6The University of Trans-disciplinary Health Sciences and Technology, Bangalore, India ‡These authors contributed equally to this work *Corresponding author. Tel: +33 388653376; E-mail: [email protected] *Corresponding author. Tel: +33 388653381; E-mail: [email protected] EMBO J (2020)39:e104486https://doi.org/10.15252/embj.2020104486 See also: V Hartenstein (June 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Immune cells provide defense against non-self and have recently been shown to also play key roles in diverse processes such as development, metabolism, and tumor progression. The heterogeneity of Drosophila immune cells (hemocytes) remains an open question. Using bulk RNA sequencing, we find that the hemocytes display distinct features in the embryo, a closed and rapidly developing system, compared to the larva, which is exposed to environmental and metabolic challenges. Through single-cell RNA sequencing, we identify fourteen hemocyte clusters present in unchallenged larvae and associated with distinct processes, e.g., proliferation, phagocytosis, metabolic homeostasis, and humoral response. Finally, we characterize the changes occurring in the hemocyte clusters upon wasp infestation, which triggers the differentiation of a novel hemocyte type, the lamellocyte. This first molecular atlas of hemocytes provides insights and paves the way to study the biology of the Drosophila immune cells in physiological and pathological conditions. Synopsis Single cell transcriptome analyses of control and challenged Drosophila hemocytes identify immune cell populations associated with distinct biological processes. The transcriptome of the fly immune cells undergoes a metabolic switch during development. A single-cell atlas of fly immune cells in control and wasp-infested larvae. Fourteen immune cell populations are identified that are associated with distinct cellular processes. Two additional populations appear upon immune challenge. Introduction The innate immune response has been the object of intense investigation in Drosophila melanogaster, as this model shows mechanisms that are conserved throughout evolution, from pattern recognition molecules to immune molecular cascades (Akira et al, 2006; Kleino & Silverman, 2014). Given the importance of innate immunity in a variety of physiological and pathological processes including tumor progression (Ratheesh et al, 2015), the current challenge is to characterize immune cell heterogeneity and identify specific hemocyte populations. This is the aim of the present work. Three classes of hemocytes have so far been identified as follows: the plasmatocytes, the crystal cells, and the lamellocytes (Honti et al, 2014). The plasmatocytes are the most abundant cell type and are responsible for the main functions of the hemocytes: phagocytosis, secretion of extracellular matrix proteins (ECM), signaling molecules, and antimicrobial peptides (AMPs; Yasothornsrikul et al, 1997; Basset et al, 2000; Sears et al, 2003; Ferrandon et al, 2004; Baer et al, 2010; Gold & Bruckner, 2015). The crystal cells account for less than 5% of the total hemocyte population, with distinctive crystals inside them that are composed of prophenoloxidases (PPO; Rizki & Rizki, 1959). These enzymes are released in large quantity upon wounding and constitute a key component for the melanization process (Rizki & Rizki, 1959). The lamellocytes are flat and large cells that only appear upon challenge. They are considered activated immune cells (Gold & Bruckner, 2015) that arise through plasmatocyte trans-differentiation or from a mitotic-dedicated precursor (Anderl et al, 2016). In the embryo, the hemocytes contribute to the clearance of apoptotic cells and the deposition of ECM-related molecules including Peroxidasin (Pxn) and Viking (Vkg; Nelson et al, 1994; Yasothornsrikul et al, 1997). By the larval stage, the organism interacts with the external environment and responds to metabolic and oxidative stress as well as to infection- or injury-related stimuli. The hemocytes must therefore adapt to these new, highly demanding, settings. In addition, while during embryogenesis, the hemocytes are highly motile and patrol the whole organism, during the larval life a large fraction of them, called resident hemocytes, colonize segmentally repeated epidermal-muscular pockets in which cell proliferation is enhanced (Makhijani et al, 2011). Upon wounding, septic infection, or infestation by parasitic wasps, the resident hemocytes are mobilized and enter in circulation to reach the site of the immune challenge (Owusu-Ansah & Banerjee, 2009; Dragojlovic-Munther & Martinez-Agosto, 2012). Thus, hemocyte localization adapts to homeostatic and challenged conditions. We here characterize the transcriptional changes occurring during development and the different types of hemocytes present in the larva. Comparing the bulk RNA sequencing data allows us to define stage-specific features: In the embryo, hemocytes contribute to the shaping of the tissues and are glycolytic, whereas in the larva, hemocytes show a strong phagocytic potential and a metabolic switch toward internalization of glucose and lipid and toward beta oxidation. The single-cell RNA sequencing (scRNA-seq) assay allows us to identify fourteen clusters of larval plasmatocytes and to assign specific molecular and cellular features, including nutrient storage, proliferative potential, antimicrobial peptide production, and phagocytosis. Finally, as a first characterization of the immune response at the single-cell level, we assess the transcriptional changes induced by infestation by the parasitic wasp Leptopilina boulardi, one of the most studied pathways linked to cellular immunity. The wasp lays eggs in the Drosophila larva and triggers hemocyte proliferation as well as lamellocyte differentiation (Markus et al, 2009), with subsequent encapsulation of the wasp egg and its death through the increased levels of reactive oxygen species (ROS). The scRNA-seq assay identifies two lamellocyte populations, a mature one with a strong glycolytic signature, and a population that expresses both lamellocyte and plasmatocyte features, likely originating through trans-differentiation (Anderl et al, 2016). The response to wasp infestation involves the embryonic hemocytes that differentiate from the procephalic mesoderm (1st wave of hematopoiesis; Tepass et al, 1994), as well as the hemocytes that originate from the lymph gland, the site of the 2nd hematopoietic wave. While in not-infested (NI) conditions, the lymph gland histolyses and releases hemocytes in circulation during the pupal life, upon wasp infestation (WI), it undergoes precocious histolysis so that both lymph gland and embryonic-derived hemocytes populate the larva (Letourneau et al, 2016; Bazzi et al, 2018; Banerjee et al, 2019). Our single-cell RNA sequencing assay identifies the same number of plasmatocyte clusters as that observed in normal conditions, strongly suggesting that the plasmatocytes from the first and second hematopoietic waves share the same features. In sum, this work characterizes the transcriptional changes occurring during hemocyte development and the hemocyte populations present in the Drosophila larva. It also provides the molecular signature and the initial characterization of the larval hemocyte repertoire as well as numerous novel markers in NI and in WI conditions. These first bulk and single-cell RNA-seq data pave the way to understand the role of the immune system in development and physiology. Results Comparing the bulk transcriptomes from embryonic (E16) and larval (WL) hemocytes In the embryo, insulated from most immune challenges by the eggshell, the hemocytes main functions are developmental. They clear the organism from apoptotic bodies issued from organogenesis and secrete extracellular components. In the larva, the hemocytes display new properties to respond to the microorganism-rich environment in which they grow. To identify the changes occurring in the hemocytes during development, we compared the hemocytes’ transcriptomes from mature, stage 16 (E16) embryos and from third-instar wandering larvae (WL). The comparison shows 3,396 genes significantly up-regulated in E16 and 1,593 up-regulated in WL hemocytes (Fig 1A, data in Dataset EV1). Most plasmatocyte markers such as Hemese (He; Kurucz et al, 2003), Singed (Sn; Zanet et al, 2009), Eater (Kocks et al, 2005), Hemolectin (Hml; Goto et al, 2001), Serpent (Srp; Shlyakhover et al, 2018), Nimrod C1 (NimC1, also called P1; Kurucz et al, 2007), Croquemort (Crq; Franc et al, 1996), and Pxn (Nelson et al, 1994) are strongly expressed at both stages but enriched in WL hemocytes (Fig 1C). Crystal cell markers are also present in the transcriptome: Pebbled (Peb) and Lozenge (Lz) are detected at relatively low levels, in agreement with the small number of crystal cells in the E16 and WL hemolymph (Rizki & Rizki, 1959). The crystal cell-specific markers PPO1 and PPO2, on the other hand, are among the genes expressed at the highest levels, highlighting their key function and the sharp specialization of the crystal cells (Binggeli et al, 2014). Figure 1. Hemocytes display distinct properties at E16 and WL stage A. Transcriptome comparison of hemocytes from stage 16 (E16) embryos and wandering 3rd-instar larvae (WL). The x-axis is the average gene expression levels (n = 3), and the y-axis is the log2 fold change WL/E16. P-values are indicated with the color code. B. Gene Ontology (GO) term enrichment analysis in E16 (green) and WL (red) hemocytes. The fold enrichments for a subset of significant GO terms are displayed; the number of genes and the P-value of the GO term enrichment are indicated in brackets. C–E. Scatter plots as in (A) highlighting in black subsets of known genes expressed in hemocytes (C) or genes associated with the GO term extracellular matrix (D) and phagocytosis (E). F, G. Phagocytosis assay on E16 (F) and WL hemocytes (G) srp(hemo)-moesin-RFP. The beads (in green) are phagocytosed by the hemocytes (in red). The WL hemocytes show greater phagocytic capacity compared to the embryonic ones after 5 min of exposure. Full stacks are displayed, and the scale bars represent 20 μm. Data information: Related to Appendix Figs S1 and S2, and Datasets EV1 and EV4. Download figure Download PowerPoint Surprisingly, most lamellocyte markers such as myospheroid (Mys or L4; Irving et al, 2005), Misshapen (Msn; Braun et al, 1997), Cher (or L5; Rus et al, 2006), and Atilla (or L1; Honti et al, 2009) were also detected at significant levels in the hemocytes from both stages. This suggests that they are expressed at basal levels in normal hemocytes and are strongly induced in lamellocytes and/or that few lamellocytes are present in basal conditions. At last, Gcm is involved in hemocyte development in the early embryo (stages 8–10; Bernardoni et al, 1997) and is no longer expressed by E16 (Bazzi et al, 2018). Accordingly, Gcm transcripts are barely detected in E16 and WL transcriptomes (levels < 40 normalized read count). Overall, these data prove the efficiency of the experimental design to purify hemocytes. Embryonic hemocytes express ECM components We next carried out a GO term enrichment analysis on the genes up-regulated in either population (|log2 fold change WL/E16| > 1, adjusted P-value < 0.01; Dataset EV1). The E16 hemocytes display a striking enrichment for gene coding for extracellular matrix components (ECM; Fig 1B and D). Out of 162 gene coding for ECM proteins, 138 are enriched in E16 hemocytes. To confirm the expression pattern of the ECM genes, we compared these data with two in situ hybridization databases (Berkeley Drosophila Genome Project (Hammonds et al, 2013; Tomancak et al, 2002, 2007) and Fly-FISH (Lecuyer et al, 2007; Wilk et al, 2016); Appendix Fig S1D and E). Most genes for which we could find data are specifically expressed in hemocytes in the embryo (Appendix Fig S1D and E). The expression/secretion of few specific ECM compounds by the hemocytes during embryonic development was previously described. The integrins alphaPS1 (Mew) and Mys as well as the integrin ligand Tiggrin (Tig) are secreted by the hemocytes at the level of muscle insertion to stabilize strong attachment between the cells (Fogerty et al, 1994; Bunch et al, 1998). The laminins LanA, LanB1, LanB2, and Wb are secreted by the hemocytes for them to migrate efficiently throughout the embryo (Sanchez-Sanchez et al, 2017). Pxn and the collagen Vkg and Col4a1 secretion by the hemocytes are essential for the condensation of the ventral nerve cord (Olofsson & Page, 2005). Finally, SPARC is produced by the hemocytes and is necessary for basal lamina assembly (Martinek et al, 2008). These 11 compounds are expressed at extremely high levels in the embryo and remain highly expressed in the larva (Fig 1D), suggesting that the role of these specific genes is preserved throughout development. Among the remaining ECM genes enriched in E16, we distinguished a large group of ECM compounds described as constituent of the cuticle: 23 Tweedles (Twdl), 56 Cuticular Proteins (Cpr and Ccp), nine Larval Cuticle Proteins (Lcp), and nine Mucins (Muc; Fig 1D, annotated in Dataset EV1). This calls for a role of the embryonic hemocytes in cuticle deposition. We also identified 21 ECM genes strongly up-regulated in the embryo (log2FC < −3, P-value < 0.01, annotated in Dataset EV1). These include the two heart-specific ECM compounds Pericardin (Prc) and Lonely heart (Loh;Maroy et al, 1988; Chavez et al, 2000; Charles, 2010), Thrombospondin (Tsp), which interacts with the integrins Mew, Mys, and If at the tendon-muscle attachment sites (Chanana et al, 2007) and Shifted (Shf) that modulate Hedgehog diffusion (Gorfinkiel et al, 2005; not exhaustive list). This strongly calls for additional embryo-specific pathways for the deposition of the ECM, in which future studies will elucidate. Larval hemocytes express specific scavenger receptors The GO terms enriched in WL compared to E16 hemocytes highlight phagocytosis and, to a lower extent, signaling pathways involved in the immune response (JNK and Wnt; Fig 1B and E, and Dataset EV1). Among the genes involved in phagocytosis, a large panel is coding for transmembrane phagocytic receptors involved in pathogen recognition, such as the Nimrod family (Eater (Kocks et al, 2005), NimC1 (Kurucz et al, 2007) and NimC2), several scavenger receptors (Sr-CI and Sr-CIV (Lazzaro et al, 2004), He, Peste (Cuttell et al, 2008; Hashimoto et al, 2009) as well as the integrins Scab (alpha-PS3) and Integrin beta-nu (Itgbn; Nonaka et al, 2013). Noteworthy, the E16 embryonic hemocytes are specifically enriched for NimC4 (also called Simu), a receptor of the Nimrod family that is involved in the phagocytosis of apoptotic bodies (Fig 1E; Kurant et al, 2008; Roddie et al, 2019). The WL hemocytes are also enriched for opsonins. These secreted molecules bind to the pathogens and promote their phagocytosis by the macrophages. Tep1 and Tep4 (Dostalova et al, 2017; Haller et al, 2018) are among the genes expressed at the highest levels in WL hemocytes, and Tep1 presents the strongest enrichment. Most of the genes involved in phagosome formation are also enriched at this stage: Arp3, Rac1, Rac2, SCAR, WASP, Chic, and Cdc42 (Pearson et al, 2003). Finally, genes involved in phagosome maturation (Rab14) and phagolysosome formation (Vps39) are enriched as well (Fig 1E; Garg & Wu, 2014; Jiang et al, 2014). The scavenger receptors and the opsonins cover a large panel of pathogens (for review, see Melcarne et al, 2019), indicating an overall switch for hemocytes’ function from apoptotic body scavenging and cuticle production at embryonic stages to pathogen scavenging at the WL stage. Since it was previously shown that the hemocytes present in the embryo are able of phagocytosis (Vlisidou et al, 2009; Tan et al, 2014), we compared the phagocytic capacity of E16 and WL hemocytes upon exposing them to fluorescent beads. The results clearly show that the larval hemocytes phagocytose faster and more than the embryonic ones (Fig 1F and G). In sum, the transcriptome analysis reveals a change in the function of the hemocytes during development, from building the ECM and the cuticle to adopting a defense profile against immune challenges. Metabolic shift between embryonic and larval hemocytes The properties of the immune cells are directly dependent on their metabolic state, which is constrained by their micro-environment (reviewed in Sieow et al, 2018). We hypothesized that the hemocytes display distinct metabolic states according to the nutritional environment present in the two developmental stages, as embryos are closed systems, whereas larvae have been feeding for most of their life. To address this hypothesis, we analyzed the expression profiles of the energy metabolic pathways in E16 and WL hemocytes. The transcriptome data comparison reveals that the larval hemocytes are most likely internalizing and metabolizing lipids through the beta oxidation pathway to generate acetyl CoA and drive the TCA cycle (Appendix Fig S2A–C). This notion is supported by the up-regulation of genes encoding lipid-scavenging receptors, and the down-regulation of genes is involved in lipid biosynthetic (TAG) pathway (Appendix Fig S2A and B). The down-regulation of genes involved in glycolysis, mainly phosphofructokinase and pyruvate dehydrogenase (Appendix Fig S2B), implies that the larval hemocytes do not rely on this process to drive the TCA cycle. The transcriptional down-regulation of gluconeogenic genes (phosphoenolpyruvate carboxykinase and fructose 1, six bisphosphatase) suggests the absence of gluconeogenesis in these cells. However, a significant up-regulation of the Glut1 sugar transporter suggests active uptake of glucose by the larval hemocytes. The down-regulation of glycolytic genes downstream of G6P and up-regulation of genes of the pentose phosphate pathway (PPP) imply that the internalized glucose could be potentially used to generate pentose sugars for ribonucleotide synthesis and redox homeostasis through the generation of NADPH. Corroborating this observation is also the strong up-regulation of redox homeostatic enzymes (Appendix Fig S2A). In contrast to the larval hemocytes, the E16 hemocytes are glycolytic and rely less on oxidative metabolism (Appendix Fig S2B). This is supported by the strong up-regulation of a key glycolytic enzyme, lactate dehydrogenase, which is essential for the conversion of pyruvate to lactate. Furthermore, these cells likely metabolize lipids at a lower level, as enzymes of the beta oxidation pathway are transcriptionally down-regulated compared to the larval hemocytes. Generation of single-cell RNA-seq datasets from NI and WI larvae The Drosophila larva contains plasmatocytes and crystal cells that are resident or in circulation. Upon wasp infestation, the lamellocytes are produced from precursors (Anderl et al, 2016) or by plasmatocyte trans-differentiation (Stofanko et al, 2010). To obtain a comprehensive repertoire of the hemocyte populations present in the larva, we generated two single-cell libraries on the hemocytes from not-infested WL (NI dataset) and from WL infested by the parasitoid wasp L. boulardi (WI dataset). The NI cells comprise the resident and the circulating embryonic-derived hemocytes, and the WI cells include in addition the hemocytes released from the lymph gland. The hemocytes were collected from pools of 20 female larvae. The libraries were produced using the Chromium single-cell 3′mRNA-seq protocol (10 × Genomics). The NI library contains 7,606 cells (mean read per cell = 37,288; median genes per cell = 959) and the WI library 8,058 cells (mean read per cell = 32,365; median genes per cell = 1,250). The libraries were merged to cluster the hemocytes presenting similar expression profiles using the Seurat toolkit (Butler et al, 2018; Stuart et al, 2019; Appendix Fig S3A). Subclustering was then applied to refine the grouping of the cells leading to the identification of 16 clusters of hemocytes (Appendix Fig S3A′ and C–D″). The identity of each cluster was assigned using the list of known markers for the crystal cells (Lz, Peb, PPO1, PPO2), for the lamellocytes (Mys, Msn, Cher, Atilla, ItgaPS4, PPO3), and for the plasmatocytes (Sn, Pxn, Hml, Eater, NimC1, Crq, He, Srp; Appendix Fig S3B). Of note, the single-cell data show that the lamellocyte markers Mys, Msn, Cher, and Atilla detected in the bulk RNA-seq on WL and E16 are expressed in the plasmatocytes and enriched in the lamellocytes (Appendix Fig S3B). We identified 13 clusters of plasmatocytes and one cluster of crystal cells in both the NI and the WI larvae and two clusters of lamellocytes specifically found in the WI larvae. The name of each cluster corresponds to the name of one of the main markers or to specific biological features (Fig 2A and B). Importantly, all cells analyzed in these datasets present known hemocyte markers, which indicate a high purity of the samples. Figure 2. Fourteen hemocyte populations can be distinguished in WL by single-cell RNA-seq UMAP projection representing the 14 clusters of cells identified in the hemocyte pools from OregonR WL (NI dataset). Number of cells and proportion of each cluster in the NI dataset. GO term enrichment analysis for each cluster. The x-axis is the GO term enrichment, the color gradient (black to light blue) indicates the P-value, and the number of genes and the GO term category (CC: cellular compound, BP: biological process, MF: molecular function) are indicated between brackets. Top 5 markers of each cluster. The expression levels are represented by the gradient of purple levels) and the percentage of cells with the size of the dots. Data information: Related to Appendix Figs S3 and S4, Datasets EV2 and EV3. Download figure Download PowerPoint Characterization of the transcriptomic profile in normal conditions Following the identification of the clusters, our first aim was to characterize the properties of the clusters in the NI dataset. Thus, we carried out GO term analyses on the genes enriched in each of them (Dataset EV2, Fig 2C) using DAVID (Huang da et al, 2009). In addition, to estimate whether the clusters are enriched/specifically localized in the circulating or in the resident compartments, we performed qPCR assays on hemocytes from either compartment to measure the expression levels of the strong markers of the different clusters (Fig 3A). Figure 3. Localization of the NI hemocyte clusters Identification of the position (circulating/resident) of the clusters within the larva by qPCR. The left panel indicates the distribution of each marker across all clusters (as in Fig 2D), and the right panel indicates the log2 of the ratio between the expression level in the circulating versus the resident compartment. Positive values indicate an enrichment in the circulating compartment and negative values in the resident compartment (n = 5, mean ± S
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