A Drosophila model to screen Alport syndrome COL4A5 variants for their functional pathogenicity.

Alport syndrome is a hereditary chronic kidney disease, attributed to rare pathogenic variants in either of three collagen genes (COL4A3/4/5) with most localized in COL4A5. Trimeric type IV Collagen α3α4α5 is essential for the glomerular basement membrane that forms the kidney filtration barrier. A means to functionally assess the many candidate variants and determine pathogenicity is urgently needed. We used Drosophila, an established model for kidney disease, and identify Col4a1 as the functional homolog of human COL4A5 in the fly nephrocyte (equivalent of human podocyte). Fly nephrocytes deficient for Col4a1 showed an irregular and thickened basement membrane and significantly reduced nephrocyte filtration function. This phenotype was restored by expressing human reference (wildtype) COL4A5, but not by COL4A5 carrying any of three established pathogenic patient-derived variants. We then screened seven additional patient COL4A5 variants; their ClinVar classification was either likely pathogenic or of uncertain significance. The findings support pathogenicity for four of these variants; the three others were found benign. Thus, demonstrating the effectiveness of this Drosophila in vivo kidney platform in providing the urgently needed variant-level functional validation.

Promoting mitochondrial dynamics by inhibiting the PINK1-PRKN pathway to relieve diabetic nephropathy.

Diabetes is a metabolic disorder characterized by high blood glucose levels and is a leading cause of kidney disease. Diabetic nephropathy has been attributed to dysfunctional mitochondria. However, many questions remain about the exact mechanism. The structure, function and molecular pathways are highly conserved between mammalian podocytes and Drosophila nephrocytes; therefore, we used flies on a high-sucrose diet to model type 2 diabetic nephropathy. The nephrocytes from flies on a high-sucrose diet showed a significant functional decline and decreased cell size, associated with a shortened lifespan. Structurally, the nephrocyte filtration structure, known as the slit diaphragm, was disorganized. At the cellular level, we found altered mitochondrial dynamics and dysfunctional mitochondria. Regulating mitochondrial dynamics by either genetic modification of the Pink1-Park (mammalian PINK1-PRKN) pathway or treatment with BGP-15, mitigated the mitochondrial defects and nephrocyte functional decline. These findings support a role for Pink1-Park-mediated mitophagy and associated control of mitochondrial dynamics in diabetic nephropathy, and demonstrate that targeting this pathway might provide therapeutic benefits for type 2 diabetic nephropathy.

HIV-1 Nef acts in synergy with APOL1-G1 to induce nephrocyte cell death in a new Drosophila model of HIV-related kidney diseases.

Background: People carrying two APOL1 risk alleles (RA) G1 or G2 are at greater risk of developing HIV-associated nephropathy (HIVAN). Studies in transgenic mice showed that the expression of HIV-1 genes in podocytes, and nef in particular, led to HIVAN. However, it remains unclear whether APOL1-RA and HIV-1 Nef interact to induce podocyte cell death. Method: We generated transgenic (Tg) flies that express APOL1-G1 (derived from a child with HIVAN) and HIV-1 nef specifically in the nephrocytes, the fly equivalent of mammalian podocytes, and assessed their individual and combined effects on the nephrocyte filtration structure and function. Results: We found that HIV-1 Nef acts in synergy with APOL1-G1 resulting in nephrocyte structural and functional defects. Specifically, HIV-1 Nef itself can induce endoplasmic reticulum (ER) stress without affecting autophagy. Furthermore, Nef exacerbates the organelle acidification defects and autophagy reduction induced by APOL1-G1. The synergy between HIV-1 Nef and APOL1-G1 is built on their joint effects on elevating ER stress, triggering nephrocyte dysfunction and ultimately cell death. Conclusions: Using a new Drosophila model of HIV-1-related kidney diseases, we identified ER stress as the converging point for the synergy between HIV-1 Nef and APOL1-G1 in inducing nephrocyte cell death. Given the high relevance between Drosophila nephrocytes and human podocytes, this finding suggests ER stress as a new therapeutic target for HIV-1 and APOL1-associated nephropathies.

Human archetypal pluripotent stem cells differentiate into trophoblast stem cells via endogenous BMP5/7 induction without transitioning through naive state.

Primary human trophoblast stem cells (TSCs) and TSCs derived from human pluripotent stem cells (hPSCs) can potentially model placental processes in vitro. Yet, the pluripotent states and factors involved in the differentiation of hPSCs to TSCs remain poorly understood. In this study, we demonstrate that the primed pluripotent state can generate TSCs by activating pathways such as Epidermal Growth Factor (EGF) and Wingless-related integration site (WNT), and by suppressing tumor growth factor beta (TGFß), histone deacetylases (HDAC), and Rho-associated protein kinase (ROCK) signaling pathways, all without the addition of exogenous Bone morphogenetic protein 4 (BMP4)-a condition we refer to as the TS condition. We characterized this process using temporal single-cell RNA sequencing to compare TS conditions with differentiation protocols involving BMP4 activation alone or BMP4 activation in conjunction with WNT inhibition. The TS condition consistently produced a stable, proliferative cell type that closely mimics first-trimester placental cytotrophoblasts, marked by the activation of endogenous retroviral genes and the absence of amnion expression. This was observed across multiple cell lines, including various primed induced pluripotent stem cell (iPSC) and embryonic stem cell (ESC) lines. Primed-derived TSCs can proliferate for over 30 passages and further specify into multinucleated syncytiotrophoblasts and extravillous trophoblast cells. Our research establishes that the differentiation of primed hPSCs to TSC under TS conditions triggers the induction of TMSB4X, BMP5/7, GATA3, and TFAP2A without progressing through a naive state. These findings propose that the primed hPSC state is part of a continuum of potency with the capacity to differentiate into TSCs through multiple routes.

Distinct Roles for COMPASS Core Subunits Set1, Trx, and Trr in the Epigenetic Regulation of Drosophila Heart Development.

Highly evolutionarily conserved multiprotein complexes termed Complex of Proteins Associated with Set1 (COMPASS) are required for histone 3 lysine 4 (H3K4) methylation. Drosophila Set1, Trx, and Trr form the core subunits of these complexes. We show that flies deficient in any of these three subunits demonstrated high lethality at eclosion (emergence of adult flies from their pupal cases) and significantly shortened lifespans for the adults that did emerge. Silencing Set1, trx, or trr in the heart led to a reduction in H3K4 monomethylation (H3K4me1) and dimethylation (H3K4me2), reflecting their distinct roles in H3K4 methylation. Furthermore, we studied the gene expression patterns regulated by Set1, Trx, and Trr. Each of the COMPASS core subunits controls the methylation of different sets of genes, with many metabolic pathways active early in development and throughout, while muscle and heart differentiation processes were methylated during later stages of development. Taken together, our findings demonstrate the roles of COMPASS series complex core subunits Set1, Trx, and Trr in regulating histone methylation during heart development and, given their implication in congenital heart diseases, inform research on heart disease.

A SNARE protective pool antagonizes APOL1 renal toxicity in Drosophila nephrocytes.

BACKGROUND: People of Sub-Saharan African ancestry are at higher risk of developing chronic kidney disease (CKD), attributed to the Apolipoprotein L1 (APOL1) gene risk alleles (RA) G1 and G2. The underlying mechanisms by which the APOL1-RA precipitate CKD remain elusive, hindering the development of potential treatments. RESULTS: Using a Drosophila genetic modifier screen, we found that SNARE proteins (Syx7, Ykt6, and Syb) play an important role in preventing APOL1 cytotoxicity. Reducing the expression of these SNARE proteins significantly increased APOL1 cytotoxicity in fly nephrocytes, the equivalent of mammalian podocytes, whereas overexpression of Syx7, Ykt6, or Syb attenuated their toxicity in nephrocytes. These SNARE proteins bound to APOL1-G0 with higher affinity than APOL1-G1/G2, and attenuated APOL1-G0 cytotoxicity to a greater extent than either APOL1-RA. CONCLUSIONS: Using a Drosophila screen, we identified SNARE proteins (Syx7, Ykt6, and Syb) as antagonists of APOL1-induced cytotoxicity by directly binding APOL1. These data uncovered a new potential protective role for certain SNARE proteins in the pathogenesis of APOL1-CKD and provide novel therapeutic targets for APOL1-associated nephropathies.

APOL1-G2 accelerates nephrocyte cell death by inhibiting the autophagy pathway.

People of African ancestry who carry the APOL1 risk alleles G1 or G2 are at high risk of developing kidney diseases through not fully understood mechanisms that impair the function of podocytes. It is also not clear whether the APOL1-G1 and APOL1-G2 risk alleles affect these cells through similar mechanisms. Previously, we have developed transgenic Drosophila melanogaster lines expressing either the human APOL1 reference allele (G0) or APOL1-G1 specifically in nephrocytes, the cells homologous to mammalian podocytes. We have found that nephrocytes that expressed the APOL1-G1 risk allele display accelerated cell death, in a manner similar to that of cultured human podocytes and APOL1 transgenic mouse models. Here, to compare how the APOL1-G1 and APOL1-G2 risk alleles affect the structure and function of nephrocytes in vivo, we generated nephrocyte-specific transgenic flies that either expressed the APOL1-G2 or both G1 and G2 (G1G2) risk alleles on the same allele. We found that APOL1-G2- and APOL1-G1G2-expressing nephrocytes developed more severe changes in autophagic pathways, acidification of organelles and the structure of the slit diaphragm, compared to G1-expressing nephrocytes, leading to their premature death. We conclude that both risk alleles affect similar key cell trafficking pathways, leading to reduced autophagy and suggesting new therapeutic targets to prevent APOL1 kidney diseases.

Single-cell profiling of the developing embryonic heart in Drosophila.

Drosophila is an important model for studying heart development and disease. Yet, single-cell transcriptomic data of its developing heart have not been performed. Here, we report single-cell profiling of the entire fly heart using ∼3000 Hand-GFP embryos collected at five consecutive developmental stages, ranging from bilateral migrating rows of cardiac progenitors to a fused heart tube. The data revealed six distinct cardiac cell types in the embryonic fly heart: cardioblasts, both Svp+ and Tin+ subtypes; and five types of pericardial cell (PC) that can be distinguished by four key transcription factors (Eve, Odd, Ct and Tin) and include the newly described end of the line PC. Notably, the embryonic fly heart combines transcriptional signatures of the mammalian first and second heart fields. Using unique markers for each heart cell type, we defined their number and location during heart development to build a comprehensive 3D cell map. These data provide a resource to track the expression of any gene in the developing fly heart, which can serve as a reference to study genetic perturbations and cardiac diseases.

The Roles of Histone Lysine Methyltransferases in Heart Development and Disease.

Epigenetic marks regulate the transcriptomic landscape by facilitating the structural packing and unwinding of the genome, which is tightly folded inside the nucleus. Lysine-specific histone methylation is one such mark. It plays crucial roles during development, including in cell fate decisions, in tissue patterning, and in regulating cellular metabolic processes. It has also been associated with varying human developmental disorders. Heart disease has been linked to deregulated histone lysine methylation, and lysine-specific methyltransferases (KMTs) are overrepresented, i.e., more numerous than expected by chance, among the genes with variants associated with congenital heart disease. This review outlines the available evidence to support a role for individual KMTs in heart development and/or disease, including genetic associations in patients and supporting cell culture and animal model studies. It concludes with new advances in the field and new opportunities for treatment.

H3K36 Di-Methylation Marks, Mediated by Ash1 in Complex with Caf1-55 and MRG15, Are Required during Drosophila Heart Development.

Methyltransferases regulate transcriptome dynamics during development and aging, as well as in disease. Various methyltransferases have been linked to heart disease, through disrupted expression and activity, and genetic variants associated with congenital heart disease. However, in vivo functional data for many of the methyltransferases in the context of the heart are limited. Here, we used the Drosophila model system to investigate different histone 3 lysine 36 (H3K36) methyltransferases for their role in heart development. The data show that Drosophila Ash1 is the functional homolog of human ASH1L in the heart. Both Ash1 and Set2 H3K36 methyltransferases are required for heart structure and function during development. Furthermore, Ash1-mediated H3K36 methylation (H3K36me2) is essential for healthy heart function, which depends on both Ash1-complex components, Caf1-55 and MRG15, together. These findings provide in vivo functional data for Ash1 and its complex, and Set2, in the context of H3K36 methylation in the heart, and support a role for their mammalian homologs, ASH1L with RBBP4 and MORF4L1, and SETD2, during heart development and disease.

The opportunities and challenges of using Drosophila to model human cardiac diseases.

The Drosophila heart tube seems simple, yet it has notable anatomic complexity and contains highly specialized structures. In fact, the development of the fly heart tube much resembles that of the earliest stages of mammalian heart development, and the molecular-genetic mechanisms driving these processes are highly conserved between flies and humans. Combined with the fly's unmatched genetic tools and a wide variety of techniques to assay both structure and function in the living fly heart, these attributes have made Drosophila a valuable model system for studying human heart development and disease. This perspective focuses on the functional and physiological similarities between fly and human hearts. Further, it discusses current limitations in using the fly, as well as promising prospects to expand the capabilities of Drosophila as a research model for studying human cardiac diseases.

SARS-CoV-2 Nsp6 damages Drosophila heart and mouse cardiomyocytes through MGA/MAX complex-mediated increased glycolysis.

SARS-CoV-2 infection causes COVID-19, a severe acute respiratory disease associated with cardiovascular complications including long-term outcomes. The presence of virus in cardiac tissue of patients with COVID-19 suggests this is a direct, rather than secondary, effect of infection. Here, by expressing individual SARS-CoV-2 proteins in the Drosophila heart, we demonstrate interaction of virus Nsp6 with host proteins of the MGA/MAX complex (MGA, PCGF6 and TFDP1). Complementing transcriptomic data from the fly heart reveal that this interaction blocks the antagonistic MGA/MAX complex, which shifts the balance towards MYC/MAX and activates glycolysis-with similar findings in mouse cardiomyocytes. Further, the Nsp6-induced glycolysis disrupts cardiac mitochondrial function, known to increase reactive oxygen species (ROS) in heart failure; this could explain COVID-19-associated cardiac pathology. Inhibiting the glycolysis pathway by 2-deoxy-D-glucose (2DG) treatment attenuates the Nsp6-induced cardiac phenotype in flies and mice. These findings point to glycolysis as a potential pharmacological target for treating COVID-19-associated heart failure.

Lpt, trr, and Hcf regulate histone mono- and dimethylation that are essential for Drosophila heart development.

Mammalian KMT2C, KMT2D, and HCFC1 are expressed during heart development and have been associated with congenital heart disease, but their roles in heart development remain elusive. We found that the Drosophila Lpt and trr genes encode the N-terminal and C-terminal homologs, respectively, of mammalian KMT2C or KMT2D. Lpt and trr mutant embryos showed reduced cardiac progenitor cells. Silencing of Lpt, trr, or both simultaneously in the heart led to similar abnormal cardiac morphology, tissue fibrosis, and cardiac functional defects. Like KMT2D, Lpt and trr were found to modulate histone H3K4 mono- and dimethylation, but not trimethylation. Investigation of downstream genes regulated by mouse KMT2D in the heart showed that their fly homologs are similarly regulated by Lpt or trr in the fly heart, suggesting that Lpt and trr regulate an evolutionarily conserved transcriptional network for heart development. Moreover, we showed that cardiac silencing of Hcf, the fly homolog of mammalian HCFC1, leads to heart defects similar to those observed in Lpt and trr silencing, as well as reduced H3K4 monomethylation. Our findings suggest that Lpt and trr function together to execute the conserved function of mammalian KMT2C and KMT2D in histone H3 lysine K4 mono- and dimethylation required for heart development. Possibly aided by Hcf, which we show plays a related role in H3K4 methylation during fly heart development.

Using Drosophila Nephrocytes to Understand the Formation and Maintenance of the Podocyte Slit Diaphragm.

The podocyte slit diaphragm (SD) is an essential component of the glomerular filtration barrier and its disruption is a common cause of proteinuria and many types of kidney disease. Therefore, better understanding of the pathways and proteins that play key roles in SD formation and maintenance has been of great interest. Podocyte and SD biology have been mainly studied using mouse and other vertebrate models. However, vertebrates are limited by inherent properties and technically challenging in vivo access to the podocytes. Drosophila is a relatively new alternative model system but it has already made great strides. Past the initial obvious differences, mammalian podocytes and fly nephrocytes are remarkably similar at the genetic, molecular and functional levels. This review discusses SD formation and maintenance, and their dependence on cell polarity, the cytoskeleton, and endo- and exocytosis, as learned from studies in fly nephrocytes and mammalian podocytes. In addition, it reflects on the remaining gaps in our knowledge, the physiological implications for glomerular diseases and how we can leverage the advantages Drosophila has to offer to further our understanding.

Understanding Individual SARS-CoV-2 Proteins for Targeted Drug Development against COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic, responsible for millions of deaths globally. Even with effective vaccines, SARS-CoV-2 will likely maintain a hold in the human population through gaps in efficacy, percent vaccinated, and arising new strains. Therefore, understanding how SARS-CoV-2 causes widespread tissue damage and the development of targeted pharmacological treatments will be critical in fighting this virus and preparing for future outbreaks. Herein, we summarize the progress made thus far by using in vitro or in vivo models to investigate individual SARS-CoV-2 proteins and their pathogenic mechanisms. We have grouped the SARS-CoV-2 proteins into three categories: host entry, self-acting, and host interacting. This review focuses on the self-acting and host-interacting SARS-CoV-2 proteins and summarizes current knowledge on how these proteins promote virus replication and disrupt host systems, as well as drugs that target the virus and virus interacting host proteins. Encouragingly, many of these drugs are currently in clinical trials for the treatment of COVID-19. Future coronavirus outbreaks will most likely be caused by new virus strains that evade vaccine protection through mutations in entry proteins. Therefore, study of individual self-acting and host-interacting SARS-CoV-2 proteins for targeted therapeutic interventions is not only essential for fighting COVID-19 but also valuable against future coronavirus outbreaks.

Drosophila, a powerful model to study virus-host interactions and pathogenicity in the fight against SARS-CoV-2.

The COVID-19 pandemic is having a tremendous impact on humanity. Although COVID-19 vaccines are showing promising results, they are not 100% effective and resistant mutant SARS-CoV-2 strains are on the rise. To successfully fight against SARS-CoV-2 and prepare for future coronavirus outbreaks, it is essential to understand SARS-CoV-2 protein functions, their host interactions, and how these processes convey pathogenicity at host tissue, organ and systemic levels. In vitro models are valuable but lack the physiological context of a whole organism. Current animal models for SARS-CoV-2 research are exclusively mammals, with the intrinsic limitations of long reproduction times, few progeny, ethical concerns and high maintenance costs. These limitations make them unsuitable for rapid functional investigations of virus proteins as well as genetic and pharmacological screens. Remarkably, 90% of the SARS-CoV-2 virus-host interacting proteins are conserved between Drosophila and humans. As a well-established model system for studying human diseases, the fruit fly offers a highly complementary alternative to current mammalian models for SARS-CoV-2 research, from investigating virus protein function to developing targeted drugs. Herein, we review Drosophila's track record in studying human viruses and discuss the advantages and limitations of using fruit flies for SARS-CoV-2 research. We also review studies that already used Drosophila to investigate SARS-CoV-2 protein pathogenicity and their damaging effects in COVID-19 relevant tissues, as well as studies in which the fly was used as an efficient whole animal drug testing platform for targeted therapeutics against SARS-CoV-2 proteins or their host interacting pathways.

Slit diaphragm maintenance requires dynamic clathrin-mediated endocytosis facilitated by AP-2, Lap, Aux and Hsc70-4 in nephrocytes.

BACKGROUND: The Slit diaphragm (SD) is the key filtration structure in human glomerular kidney that is affected in many types of renal diseases. SD proteins are known to undergo endocytosis and recycling to maintain the integrity of the filtration structure. However, the key components of this pathway remain unclear. METHODS: Using the Drosophila nephrocyte as a genetic screen platform, we screened most genes involved in endocytosis and cell trafficking, and identified the key components of the cell trafficking pathway required for SD protein endocytosis and recycling. RESULTS: We discovered that the SD protein endocytosis and recycling pathway contains clathrin, dynamin, AP-2 complex, like-AP180 (Lap), auxilin and Hsc70-4 (the endocytosis part) followed by Rab11 and the exocyst complex (the recycling part). Disrupting any component in this pathway led to disrupted SD on the cell surface and intracellular accumulation of mislocalized SD proteins. We also showed the first in vivo evidence of trapped SD proteins in clathrin-coated pits at the plasma membrane when this pathway is disrupted. CONCLUSIONS: All genes in this SD protein endocytosis and recycling pathway, as well as SD proteins themselves, are highly conserved from flies to humans. Thus, our results suggest that the SD proteins in human kidney undergo the same endocytosis and recycling pathway to maintain the filtration structure, and mutations in any genes in this pathway could lead to abnormal SD and renal diseases.

Characterization of SARS-CoV-2 proteins reveals Orf6 pathogenicity, subcellular localization, host interactions and attenuation by Selinexor.

BACKGROUND: SARS-CoV-2 causes COVID-19 which has a widely diverse disease profile. The mechanisms underlying its pathogenicity remain unclear. We set out to identify the SARS-CoV-2 pathogenic proteins that through host interactions cause the cellular damages underlying COVID-19 symptomatology. METHODS: We examined each of the individual SARS-CoV-2 proteins for their cytotoxicity in HEK 293 T cells and their subcellular localization in COS-7 cells. We also used Mass-Spec Affinity purification to identify the host proteins interacting with SARS-CoV-2 Orf6 protein and tested a drug that could inhibit a specific Orf6 and host protein interaction. RESULTS: We found that Orf6, Nsp6 and Orf7a induced the highest toxicity when over-expressed in human 293 T cells. All three proteins showed membrane localization in COS-7 cells. We focused on Orf6, which was most cytotoxic and localized to the endoplasmic reticulum, autophagosome and lysosomal membranes. Proteomics revealed Orf6 interacts with nucleopore proteins (RAE1, XPO1, RANBP2 and nucleoporins). Treatment with Selinexor, an FDA-approved inhibitor for XPO1, attenuated Orf6-induced cellular toxicity in human 293 T cells. CONCLUSIONS: Our study revealed Orf6 as a highly pathogenic protein from the SARS-CoV-2 genome, identified its key host interacting proteins, and Selinexor as a drug candidate for directly targeting Orf6 host protein interaction that leads to cytotoxicity.

Functional analysis of SARS-CoV-2 proteins in Drosophila identifies Orf6-induced pathogenic effects with Selinexor as an effective treatment.

BACKGROUND: SARS-CoV-2 causes COVID-19 with a widely diverse disease profile that affects many different tissues. The mechanisms underlying its pathogenicity in host organisms remain unclear. Animal models for studying the pathogenicity of SARS-CoV-2 proteins are lacking. METHODS: Using bioinformatic analysis, we found that 90% of the virus-host interactions involve human proteins conserved in Drosophila. Therefore, we generated a series of transgenic fly lines for individual SARS-CoV-2 genes, and used the Gal4-UAS system to express these viral genes in Drosophila to study their pathogenicity. RESULTS: We found that the ubiquitous expression of Orf6, Nsp6 or Orf7a in Drosophila led to reduced viability and tissue defects, including reduced trachea branching as well as muscle deficits resulting in a "held-up" wing phenotype and poor climbing ability. Furthermore, muscles in these flies showed dramatically reduced mitochondria. Since Orf6 was found to interact with nucleopore proteins XPO1, we tested Selinexor, a drug that inhibits XPO1, and found that it could attenuate the Orf6-induced lethality and tissue-specific phenotypes observed in flies. CONCLUSIONS: Our study established Drosophila as a model for studying the function of SARS-CoV2 genes, identified Orf6 as a highly pathogenic protein in various tissues, and demonstrated the potential of Selinexor for inhibiting Orf6 toxicity using an in vivo animal model system.

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

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.