# Characterization of the Drosophila Adult Hematopoietic System Reveals a Rare Cell Population With Differentiation and Proliferation Potential

Manon Boulet, Yoan Renaud, François Lapraz, Billel Benmimoun, Laurence

Vandel, Lucas Waltzer

► To cite this version:

Manon Boulet, Yoan Renaud, François Lapraz, Billel Benmimoun, Laurence Vandel, et al. Characterization of the Drosophila Adult Hematopoietic System Reveals a Rare Cell Population With Differentiation and Proliferation Potential. Frontiers in Cell and Developmental Biology, 2021, 9, pp.739357. 10.3389/fcell.2021.739357. hal-03429562

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# Characterization of the Drosophila Adult Hematopoietic System Reveals a Rare Cell Population With Differentiation and Proliferation Potential

OPEN ACCESS

**Edited by:**

Dominic C. Voon,
Kanazawa University, Japan

**Reviewed by:**

Tomomasa Yokomizo,
Kumamoto University, Japan
Jin Li Hua,
Northeast Forestry University, China

**\*Correspondence:**

Lucas Waltzer
lucas.waltzer@uca.fr

**†Present address:**

François Lapraz,
Université Côte d'Azur, Centre
National de la Recherche Scientifique,
Institut National de la Sante et de la
Recherche Medicale, Institut de
Biologie Valrose, Nice, France
Billel Benmimoun,
Institut Pasteur, Centre National de la
Recherche Scientifique, UMR 3738,
Paris, France

**Specialty section:**

This article was submitted to
Cell Growth and Division,
a section of the journal
Frontiers in Cell and Developmental
Biology

**Received:** 10 July 2021

**Accepted:** 22 September 2021

**Published:** 13 October 2021

**Citation:**

Boulet M, Renaud Y, Lapraz F, Benmimoun B, Vandel L and Waltzer L (2021) Characterization of the Drosophila Adult Hematopoietic System Reveals a Rare Cell Population With Differentiation and Proliferation Potential. *Front. Cell Dev. Biol.* 9:739357.
doi: 10.3389/fcell.2021.739357

Manon Boulet¹, Yoan Renaud¹, François Lapraz²†, Billel Benmimoun²†, Laurence Vandel¹ and Lucas Waltzer¹,²*

¹ Université Clermont Auvergne, Centre National de la Recherche Scientifique, Institut National de la Sante et de la Recherche Medicale, Institut Génétique Reproduction et Développement, Clermont-Ferrand, France, ² Centre de Biologie du Développement, Centre de Biologie Intégrative, Université de Toulouse, Centre National de la Recherche Scientifique, Université Paul Sabatier, Toulouse, France

While many studies have described Drosophila embryonic and larval blood cells, the hematopoietic system of the imago remains poorly characterized and conflicting data have been published concerning adult hematopoiesis. Using a combination of blood cell markers, we show that the adult hematopoietic system is essentially composed of a few distinct mature blood cell types. In addition, our transcriptomics results indicate that adult and larval blood cells have both common and specific features and it appears that adult hemocytes reactivate many genes expressed in embryonic blood cells. Interestingly, we identify a small set of blood cells that does not express differentiation markers but rather maintains the expression of the progenitor marker *domeMeso*. Yet, we show that these cells are derived from the posterior signaling center, a specialized population of cells present in the larval lymph gland, rather than from larval blood cell progenitors, and that their maintenance depends on the EBF transcription factor Collier. Furthermore, while these cells are normally quiescent, we find that some of them can differentiate and proliferate in response to bacterial infection. In sum, our results indicate that adult flies harbor a small population of specialized cells with limited hematopoietic potential and further support the idea that no substantial hematopoiesis takes place during adulthood.

**Keywords:** hematopoiesis, blood cell progenitor, Drosophila, imago, Collier

## INTRODUCTION

Several aspects of blood cell development and functions are conserved between mammals and Drosophila (Hartenstein, 2006; Banerjee et al., 2019). Hence, this insect has been extensively used as a simple genetic model organism to study the fundamental bases underlying hematopoiesis and blood cell functions (Letourneau et al., 2016; Boulet et al., 2018). Yet, while much effort has been devoted to the characterization of Drosophila blood cells at the embryonic and larval stages, the
adult stage remains less well characterized and conflicting data
have been published (see below). Thus, further work is essential
to gain a better understanding of the composition and dynamics
of the adult hematopoietic system.

As in vertebrates, Drosophila hematopoiesis occurs in
successive waves and stems from mesoderm-derived blood
cell progenitors (called prohemocytes) (Banerjee et al., 2019).
These progenitors can give rise to three main differentiated cell
types, collectively called hemocytes and related to vertebrate
myeloid cells: plasmatocytes, crystal cells, and lamellocytes (Gold
and Bruckner, 2015; Parsons and Foley, 2016). Plasmatocytes
are macrophages that form the bulk of the population; they
are essentially implicated in tissue remodeling and in the
cellular immune response, while crystal cells are implicated in
clotting and melanization (an insect-specific immune response).
Lamellocytes are normally barely present but their differentiation
is massively induced in the larvae in response to pathological
situations, such as infestation by parasitoid wasp eggs.

Plasmatocytes and crystal cells are first produced in the
embryo after a small group of prohemocytes emerges from the
early head mesoderm and differentiates (Tepass et al., 1994;
Lebestky et al., 2000; Bataille et al., 2005). These embryo-derived
hemocytes compose the larval peripheral blood cells, which
are observed either as free circulatory cells in the hemolymph
or attached under the cuticle (Holz et al., 2003; Makhijani
et al., 2011). Interestingly, differentiated larval plasmatocytes
can not only proliferate but also transdifferentiate into crystal
cells (Makhijani et al., 2011; Leitao and Sucena, 2015), and
it was proposed that the increase in peripheral blood cells
during larval development relies on self-renewing plasmatocytes
(Gold and Bruckner, 2015). Yet, undifferentiated blood cells
were also described among peripheral hemocytes (Sinenko et al.,
2010) and recent single-cell sequencing experiments suggest
that proliferative plasmatocytes retain a progenitor signature
(Cattenoz et al., 2020, 2021; Tattikota et al., 2020). In parallel, a
second wave of hematopoiesis takes place during the larval stages
in a specialized organ called the lymph gland (Lanot et al., 2001).
The lymph gland precursors derive from the embryonic lateral
mesoderm and develop in close association with the anterior part
of the dorsal vessel (Crozatier et al., 2004; Mandal et al., 2004).
In third instar larvae, the lymph gland is composed of three to
four pairs of lobes separated by pericardial cells (Lanot et al.,
2001; Rodrigues et al., 2021). The posterior lobes are essentially
composed of prohemocytes (Rodrigues et al., 2021), while the
anterior lobes contain blood cells progenitors, their differentiated
progenies (plasmatocytes and crystal cells), and a small cluster
of cells that form a niche, called the posterior signaling center
(PSC) (Jung et al., 2005; Letourneau et al., 2016). In normal
situations, lymph gland blood cells are released into circulation
during pupation when the lymph gland disperses (Honti et al.,
2010; Grigorian et al., 2011).

By comparison, the adult hematopoietic system of the fly
remains poorly characterized (Banerjee et al., 2019). Initial
examinations failed to reveal the presence of an hematopoietic
organ but showed that hemocytes are scattered throughout
the imago, mostly as sessile populations, and accumulate
along the heart in the abdomen (Elrod-Erickson et al., 2000;

Lanot et al., 2001), a general feature among insects (Yan and
Hillyer, 2020). In addition, it seemed that all the hemocytes
were non-dividing phagocytic cells whose number and
phagocytic activity decrease with age (Lanot et al., 2001;
Mackenzie et al., 2011; Woodcock et al. 2015). Besides,
transplantation experiments showed that both embryonic and
larval hematopoietic anlagen contribute to the adult blood cells
(Holz et al., 2003). Hence, the prevailing view was that the
adult hematopoietic system was solely composed of mature
plasmatocytes derived from the embryonic and larval stages,
and that no hematopoiesis took place during adulthood. Yet,
some data suggested a more contrasted situation. First, the
analysis of different markers revealed that crystal cells and
distinct subpopulations of plasmatocytes are present in the
adult (Kurucz et al., 2007; Clark et al., 2011). Second, a seminal
study by Ghosh et al. (2015) suggested that hematopoiesis also
occurs in the imago. Indeed, the authors proposed that blood cell
progenitors capable of differentiation persist in the adult and they
observed plasmatocyte proliferation in response to *Escherichia
coli* infection (Ghosh et al., 2015). However, these findings were
refuted by a subsequent publication (Sanchez Bosch et al., 2019),
in which the authors disproved the claim that the GATA factor
Serpent (SrP) is a marker of adult blood cell progenitors and
found no evidence for blood cell proliferation or differentiation.

Here, we set out to better characterize the Drosophila adult
hematopoietic system. First, we established the gene expression
profile of adult hemocytes and compared it to their larval
ascendant (larval peripheral hemocytes and lymph gland) to
define the common and specific features of these immune cells.
Next, we used a panel of hematopoietic markers to assess their
expression in the imago and to gain a better appreciation of
the adult blood cell landscape diversity and evolution with
age. Finally, we focused our analysis on a small population of
adult blood cells that does not express hemocyte differentiation
markers. We show that they originate from the PSC rather than
from larval blood cell progenitors and that they can proliferate
and differentiate in response to infection albeit unfrequently.
These PSC-derived cells most probably account for the previously
reported population of adult blood cell progenitor. Our findings
are discussed in view of the current controversies in the field.

MATERIALS AND METHODS

Fly Strains

The following strains were used in this study: *w*¹¹¹⁸ (BL3605),
*srpD-GAL4* (Waltzer et al., 2002; Crozatier et al., 2004),
*srpHemo-H2A-3xmCherry* (Gyoergy et al., 2018) (BL78361),
*HmlΔ-GAL4* (Sinenko and Mathey-Prevot, 2004) (BL30141
and BL30140), *crq-GAL4* (Olofsson and Page, 2005) (BL25041),
*lz-GAL4* (Lebestky et al., 2000) (BL6313), *sn-GAL4* (Avet-
Rochex et al., 2010; Zanet et al., 2012), *dot-GAL4* (Kimbrell
et al., 2002) (BL6902), *dome*PG125-*GAL4* (Bourbon et al., 2002),
*domeMeso-GAL4* (Oyallon et al., 2016), *tep4-GAL4* (Avet-Rochex
et al., 2010) (DGRC #105442), *Ance*MiMiC-*GFP* (BL59829),
*pcol85-GAL4* (Crozatier et al., 2004), *col*(GMR13A11)-*GAL4*
(BL49248), *Antp-GAL4* (Mandal et al., 2007), *Ubx(M3)-GAL4*
(de Navas et al., 2006), *Pxn-GAL4* (Stramer et al., 2005), *Pxn-RedStinger* (Rodrigues et al., 2021), *BcF2-GFP* (Gajewski et al., 2007), *BcF6-mCherry* (Tokusumi et al., 2009a), *msnF9-mCherry*, *msnF9-GFP* (Tokusumi et al., 2009b), *Cg-GAL4* (Asha et al., 2003) (BL7011), *Cg25C-GFP* (Sorrentino et al., 2007), *UAS-RNAi col* (Baumgardt et al., 2007), *kn<sup>col1</sup>* and *kn<sup>col1</sup>P{col5-cDNA}* (Crozatier and Vincent, 1999), *UAS-Bax* (Gaumer et al., 2000), *hop<sup>Tum-1</sup>* (BL8492), *UAS-2xEGFP* (BL6874), *UAS-2xEYFP* (BL6660) *UAS-RedStinger* (BL8546), *UAS-mCD8GFP* (BL5138), *UAS-RNAi srp* (VDRC109521), *tubP-Gal80<sup>ts</sup>* (BL7018), *G-TRACE* (*UAS-RedStinger*, *UAS-FLP*, *Ubi-p63E(FRT.STOP)Stinger*) (BL28281), *UAS-FUCCI* (*UAS-GFP.E2f1.1-230*, *UAS-mRFP1.NLS.CycB.1-266*) (BL55110).

Drosophila stocks and crosses were maintained on standard fly medium (75 g/l organic corn flour, 28 g/l dry yeast, 40 g/l sucrose, 8 g/l agar, 10 ml/l Moldex 20%). All crosses and collections were performed at 25°C with the exception of experiments involving tub-GAL80ts, which were performed at 18°C before transferring the progenies to 29°C as indicated in the result section.

## Adult Blood Cells Preparations

To bleed adult flies, aged-matched mated individuals were anesthetized, washed in 70% ethanol and air dried before cutting the last abdominal segment with a clean scalpel. Then a fine glass needle was inserted in the anterior part of the thorax and PBS was perfused under air pressure. Flushed hemocytes were collected in a 12-well plate (Nunc) containing 600 µl PBS and a round glass coverslip. In general, six drops of PBS (~60 µl) were collected per fly and five flies were bled in a single well. The plates were centrifuged at 1000 rpm for 2 min before adding 300 µl of 16% formaldehyde for 30 min. For in situ observation of blood cells in adult abdomen, flies were dissected essentially as described in Ghosh et al. (2015). Briefly: flies were anesthetized, stuck on their dorsal side in a paraffin-coated plastic dish and dissected in PBS. The wings and most of the thorax were discarded and the ventral abdomen was incised on both sides with thin scissors before delicately removing the gonads and the gut. The dissected abdomens were fixed in 4% formaldehyde for 30 min. Samples were then processed as described below for immunofluorescence and/or confocal imaging.

## Immunostainings and *in situ* Hybridizations

Immunostainings and RNA *in situ* hybridizations were essentially performed as described in Miller et al. (2017). The following antibodies were used: mouse anti-P1/NimC1 (Kurucz et al., 2007), mouse anti-Col (Krzemien et al., 2007), mouse anti-β integrin/Myospheroid (CF6.G11, DSHB), mouse anti-Antp (8C11, DSHB), rabbit anti-GFP (Torrey Pines), anti-DIG coupled to alkaline phosphatase (Roche) and Alexa-fluor labeled secondary antibodies (Molecular Probes). Nuclei were stained with DAPI and the actin cytoskeleton with fluorescent labeled phalloidin (Molecular Probes). The slides were mounted in Vectashield (Vector Laboratories) and observed under a confocal microscope (Zeiss LSM800 or Leica SP5). Confocal images are displayed as maximum intensity projection of Z-stacks.

## Bacterial and Wasp Infections

*Escherichia coli* (DH5α strain) were grown overnight in LB broth and pelleted at 4000 rpm for 15 min. A fine tungsten needle was dipped in the bacterial pellet and inserted into the dorsal abdomen or under the wing hinge of anesthetized 1-week-old females. A heat-sterilized tungsten needle was used to prick the flies that were used as non-infected control. Three groups of 10 flies were used in each experiment. The flies were then reared for 16 h (FUCCI experiments) to 96 h (G-TRACE experiments) and dissected or bled as described above. For wasp infestations, late second instar larvae were subjected to parasitism by ~10 *Leptopilina boulardi* (strain G486) females for 2 h, as described previously (Benmimoun et al., 2015). Successful infestation was assessed by looking for melanotic nodule 48 h later. The corresponding larvae were transferred to fresh vial and the surviving adult flies were bled 5 days after emergence.

## EdU Incorporation Assays

For EdU incorporation assays, 2-day-old *domeMeso-GAL4*, *UAS-EYFP* females were grown on 2 ml of fly food medium supplemented with 0.4 mM EdU (5-ethynyl-2'-deoxyuridine) for 5 days before infection with *E. coli* as described above. After 24 h on medium containing fresh EdU, infected and control flies were bled and EdU incorporation in hemocytes was assessed using Click-IT Plus EdU kit with AlexaFluor 555 dye according to the manufacturer's instructions (Molecular Probes #C10638).

## RNA-Sequencing

For RNA-seq experiments, wandering third instar larvae or 4- to 5-day-old adult *w*¹¹¹⁸ females were used to prepare independent biological triplicates. Adult blood cells were retrieved by perfusing the flies with PBS as described above and directly collected in a 1.5 ml Eppendorf tube on ice. Peripheral larval blood cells were collected by delicately peeling the dorsal cuticle with forceps and dripping the larva in a 5 µl drop of PBS on parafilm. Upon microscopic examination, we found that >90% of the cells retrieved with this protocol were hemocytes (*Pxn-RedStinger*⁺), with no visible contamination by fat body cells. The anterior lobes of the larval lymph glands were dissected in PBS. Blood cells and lymph glands were transferred to 1.5 ml Eppendorf tubes, pelleted by centrifugation at 1000 rpm for 2 min and processed for RNA extraction using Arcturus PicoPure RNA kit (ThermoFisher #KIT0204). For each replicate, ~100 adult flies and 20 larvae were used. RNA samples were run on Agilent Bioanalyzer to assess sample quality and concentration. Samples were converted to cDNA using Nugen's Ovation RNA-Seq System (Catalog # 7102-A01). Libraries were generated using Kapa Biosystems library preparation kit (#KK8201) and multiplexed libraries were sequenced on a 1x75 flow cell on the HiSeq2000 device (Illumina). Reads were filtered and trimmed to remove adapter-derived or low-quality bases using Trimmomatic and checked again with FASTQC. Illumina reads were aligned to Drosophila reference genome (dm6 Ensembl release 70) with Hisat2. Read counts were
generated for each annotated gene using HTSeq-Count. RPKM (Reads Per Kilobase of exon per Megabase of library size) values were calculated using Cufflinks. Reads normalization, variance estimation and pair-wise differential expression analysis with multiple testing correction was conducted using the R Bioconductor DESeq2 package. Heatmaps and hierarchical clustering were generated with “pheatmap” R package. Gene ontology enrichment analyses were performed using Genomatix. The RNA-seq data were deposited on GEO under the accession number GSE174107.

## Real-Time Quantitative Polymerase Chain Reaction

For RT-quantitative polymerase chain reaction (qPCR), RNA samples were prepared from adult or larval bleeds and larval lymph glands (dissected as described above) using RNeasy kit (Qiagen) with an additional on-column DNAse treatment with RNase-Free DNase Set (Qiagen). Reverse transcription was performed with SuperScript IV Reverse Transcriptase (ThermoFisher) according to manufacturer's instruction on 100 ng of RNA and using a mix (1:1) of random primers (Invitrogen) and oligo dT (Promega). qPCRs were performed with SsoFast EvaGreen (Biorad) on a LightCycler 480 Instrument II (Roche Life Science). The sequences of the primers used to assess the expression of the different genes are provided in **Supplementary Table 1**. qPCR data were analyzed with ΔΔCt method and gene expressions were normalized to *rp49* (synonym: *RpL32*). All experiments were performed using biological triplicates.

# RESULTS

## Adult Blood Cells Exhibit a Distinct Gene Expression Profile as Compared to Larval Blood Cells

As a first step to characterize Drosophila adult hematopoietic system, we used a perfusion protocol similar to the one used to bleed mosquitoes (Muller et al., 1999) in order to retrieve adult blood cells and define their gene expression program (see the section "Materials and Methods"). We found that ~99% of the cells collected with this technique in 5-day-old flies expressed the pan-hemocyte marker *srp* as revealed by using the *srpD-GAL4* driver (Waltzer et al., 2002) or the *srpHemo-H2A::3mCherry* reporter (Gyoergy et al., 2018; **Figures 1A,C,E**), indicating that this approach allows to obtain clean blood cell preparations. Of note, when using young adults (24-48 h after eclosion), the bleeds were contaminated with remnants of the larval fat body (**Figure 1B**, arrow), which is known to dissociate into large individual adipose cells during metamorphosis (Nelliot et al., 2006) and gets cleared in the first 2 days of adulthood (Aguila et al., 2007). Using aged flies (35-day-old), we found that ~4.6% of the cells did not express *srp* and we recovered fewer hemocytes (**Figures 1D-F**), which is consistent with previous reports showing that blood cell number decreases

with aging (Mackenzie et al., 2011; Woodcock et al., 2015; Sanchez Bosch et al., 2019).

Next, we established the gene expression profile of adult hemocytes collected from 5-day-old flies by RNA sequencing. For comparison, we also established the transcriptome of third instar larval peripheral hemocytes (i.e., embryo-derived hemocytes) and lymph gland hemocytes. All experiments were performed in biological triplicates and at least 14 million mapped reads per sample. We found that 6802 genes were expressed with a RPKM > 1 in all three samples of adult hemocytes (corresponding to 39% of the genes in Drosophila reference genome dm6) (**Supplementary Table 2**). Similarly, larval peripheral blood cells and lymph gland hemocytes expressed 6282 and 6220 genes (**Supplementary Tables 3, 4**), respectively. As shown in **Figure 2A**, adult hemocytes (AD), lymph gland (LG) and larval peripheral hemocytes (PH) shared the expression of 5351 genes, i.e., between 79 and 86% of their respective transcriptome. Among the 268 genes annotated in Flybase (FB_2021_02) as being expressed in blood cells (i.e., hemocytes, plasmatocytes, crystal cells, lamellocytes or prohemocytes, **Supplementary Table 5**), 216 were retrieved in our adult hemocyte data set (2.1-fold over-enrichment, *p*-value < 5.5E-45), 197 in the lymph gland (2.1-fold over-enrichment, *p*-value < 3.5E-37), and 198 in peripheral hemocytes (2.0-fold over-enrichment, *p*-value < 3.4E-37), with 188 being common to all three samples (**Supplementary Figure 1**). Thus, most hemocyte markers expressed in larval blood cells are also expressed in the adult. Notably, 11 of these hemocyte markers (*Col4a1/Cg25C*, *Ppn*, *Nplp-2*, *Pxn*, *Fer2LHC*, *Fer1HCH*, *PPO2*, *PPO1*, *crq*, *Sr-Cl* and *Glt*) were among the 60-most strongly expressed genes in adult blood cells (**Supplementary Table 2**). In contrast, *gcm* and *gcm2*, which were shown to be expressed in embryonic but not in larval blood cells (Avet-Rochex et al., 2010; Bazzi et al., 2018; Ramond et al., 2020), were not detected in adult (or larval) hemocyte RNA-seq samples (**Supplementary Tables 2-4**).

Despite expressing many genes in common, adult and larval hemocytes also exhibit distinct features. Indeed, principal component analysis showed that 77% of gene expression variance among samples was accounted for by stage differences (adult *versus* larva) (**Figure 2B**). Furthermore, differential gene expression analysis with DESeq2 revealed that 1074 genes were differentially expressed between adult blood cells and both larval peripheral hemocytes and lymph glands (adjusted *p*-value < 0.01 and fold change > 2), with 864 genes up-regulated and 210 down-regulated in adult hemocytes (**Figures 2C,D** and **Supplementary Table 6**). For comparison, 330 genes were differentially expressed between larval peripheral hemocytes and both adult blood cells and lymph glands (151 up-regulated, 179 down-regulated), and 218 genes were differentially expressed between lymph glands and both adult and larval peripheral hemocytes (99 up-regulated, 119 down-regulated). For instance, consistent with previous immunostaining results (Kurucz et al., 2007), we observed that *Hemese* (*He*) expression was down-regulated in adult hemocytes as compared to larval ones (**Supplementary Table 6**). In addition, to validate these results, we performed RT-qPCR on several genes that were differentially expressed between adult and larval blood
<figure><img src="image_3.png" /><figcaption>**FIGURE 1** | Recovery of Drosophila blood cell by bleeding. (A–D) Confocal images of cells recovered from *srpD-GAL4*, *UAS-mCherry* (*A*, *srpD-GAL4*) or *srpHemo-His2A-RFP* (B–D, *srpHemo*) adult females. Cells were counterstained with phalloidin (green) and DAPI (blue). The age of the flies is indicated below each panel. Scale bar: 200 µm. The arrow in panel (B) indicates a fat body cell surrounded by hemocytes. (E) Quantifications of the proportion of cells (DAPI⁺) expressing *srpHemo-His2A-RFP* according to fly age. Means and standard deviations of five independent experiments are shown. A total of 2316, 2351, and 1516 cells were counted in 2-day-, 5-day-, and 35-day-old flies, respectively. (F) Quantifications of the absolute number of cells recovered per fly at different ages. Means and standard deviations from eight independent experiments are represented. Kruskal–Wallis test was used for statistical analysis. n.s., non-significant (*p* > 0.05); \*\* *p* < 0.01.</figcaption></figure>

cells using independent RNA samples. Thereby, we confirmed that *hsp23*, *Tig*, *mthl6* and *CG15115* were downregulated in adult hemocytes while *ninaD*, *Fhos*, *Tps1*, *Cyp12a4*, *Ald* and *Men* were up-regulated (**Figure 2E**). Furthermore, reporter lines for *NimC4* and *Mp* also showed that these two genes were expressed in adult but not in larval hemocytes (**Figures 2F,G**).

Gene Ontology enrichment analyses underlined the up-regulation of genes implicated in small molecule metabolism processes, including ATP metabolism (as seen with the overexpression of several enzymes involved in glycolysis, TCA cycle, or oxidative phosphorylation) and a down-regulation of genes regulating chromatin organization in adult hemocytes (**Table 1**). Along the same line, Gene Ontology analysis considering the 1000 most strongly expressed genes in larval, lymph gland or adult blood cells revealed shared features between them, with for instance a strong over-representation for genes implicated in translation, vesicle-mediated transport or immune response, but also confirmed the singular enrichment for ATP metabolic processes among highly expressed genes in adult hemocytes (**Table 2**).

Surprisingly, one of the top-enriched genes in adult hemocytes is *NimC4*, which was recently shown to be highly enriched in stage 16 embryonic hemocytes as compared to larval descendants (i.e., larval peripheral hemocytes) in a comparative transcriptomic analysis (Cattenoz et al., 2020). Moreover, reminiscent of the situation that we observed, embryonic and their larval progenies were found to have distinct metabolic gene signature. We thus reanalyzed the RNA-seq data from Cattenoz et al. (2020) using the same settings as for our transcriptomes. Thereby, we established a list of 4892 differentially expressed genes between embryonic and peripheral hemocytes (adjusted *p*-value < 0.01 and fold change > 2; **Supplementary Table 7**) that we compared with our results. Strikingly, 49% (422/864) of the genes up-regulated in adult *versus* larval hemocytes are also over-expressed in embryonic blood cells as compared to larval blood cells (2.9-fold over-enrichment, *p* < 4.8E-113) (**Figure 2H**). Similarly, 46% (97/210) of the genes down-regulated in adult *versus* larval hemocytes are also repressed in

embryonic blood cells (4.2-fold over-enrichment, *p* < 1.3E-38) (**Figure 2H**). The common over-expressed genes in embryonic and adult hemocytes *versus* larval ones were implicated in small molecule/ATP metabolic processes, while down-regulated genes were involved in cell cycle (**Table 3**). Hence, many genes and processes differentially regulated between adult and larval hemocytes are similarly regulated between embryonic and larval hemocytes.

In sum, these data show that adult hemocytes share a significant portion of their transcriptome with larval blood cells but also exhibit distinct features, some of which appear to be shared with embryonic hemocytes.

## Characterization of the Adult Hematopoietic System Landscape

To better define the cellular composition of the adult hematopoietic system, we then analyzed the expression of a series of well-characterized reporters classically used to study embryonic and larval blood cell types (see the section "Materials and Methods").

First, we assessed the expression of plasmatocyte and crystal cell differentiation markers. In 5-day-old flies, the plasmatocyte reporters for *Peroxidasin* (*Pxn*), *Collagen 25C* (*Cg25C*, also known as *Collagen type IV alpha1*), *Hemolectin* (*Hml*), and *croquemort* (*crq*) were expressed in ~95%, 90%, 65%, and 25% of the blood cells, respectively, while those for the crystal cell markers *Black cells* (*Bc*, also known as *Prophenoloxidase 1*, *PPO1*) and *lozenge* (*lz*) were expressed in 8 to 12% of the cells (**Figures 3A–E,G** and **Supplementary Figures 2A–F**). The proportion of hemocytes expressing these reporters was similar in 35-day-old flies, except for a slight but significant decline in the proportion of *Pxn*⁺ cells (**Figure 3G**). The analysis of double transgenic lines revealed that (almost) all the cells expressing *Hml* co-expressed *srp* and *Pxn* (**Figures 3H–H", K–K", Q**). Besides, ~90% of the *crq*⁺ cells were *Hml*⁺ (**Figures 3O–O", Q**). Among the *Bc*⁺ cells, 100% expressed *srp* and *Pxn*, ~90% *Cg25C* and 60% *Hml* (**Figures 3I–I", L–L", N–N", P–P", Q**). Of note, it was previously observed
<figure><img src="image_4.png" /></figure>

**FIGURE 2** | Analysis of adult blood cell gene expression profile. (A) Venn diagrams showing the number of genes expressed (RPKM > 1 in all three biological replicates) in adult hemocytes (AD), larval peripheral hemocytes (PH) and larval lymph glands (LG) as determined by RNA-seq on w<sup>115B</sup> females. (B) Principal component analysis of the RNA-seq profiles of adult, peripheral and lymph gland biological triplicates. (C) Venn diagrams showing the overlaps between differentially expressed genes in pairwise comparisons (adjusted *p*-value < 0.01 and fold change > 2). Upper panels: up-regulated genes. Lower panels: down-regulated genes. The number of up- or down-regulated genes in each condition is indicated in brackets and the number of genes commonly deregulated is indicated in the Venn diagram intersection. (D) Heat map of the genes differentially expressed between adult versus larval hemocytes. The genes tested in panels **E** to **G** are indicated. (E) The expression level of the indicated genes was determined by RT-qPCR on adult hemocytes, larval peripheral hemocytes and larval lymph glands. Expression levels were normalized to *rp49*. Means and SEM from three independent experiments are represented. (F,G) Confocal images showing GFP expression in adult and larval hemocytes from *Mp-GFP* (F) or *NimC4-GFP* (G) reporter lines. Nuclei were stained with DAPI (blue). For adult and larval peripheral hemocytes (upper and middle panels), the samples were stained with phalloidin (red). Lower panels show a lymph gland anterior lobe. Scale bar: 50 µm. (H) Venn diagrams showing the overlaps of up- or down-regulated genes between adult versus larval hemocytes (AD, blue) or embryonic versus larval hemocytes (Emb, orange).

that the “plasmatocyte” markers *Cg25C*, *Pxn* and *Hml* are also expressed in larval crystal cells (Jung et al., 2005). We did not observe significant shifts in the repartition of these populations between 5- and 35-day-old flies, indicating that the overall composition of the adult hematopoietic system remains stable during aging (Figure 3Q).

Next, we tested whether lamellocytes could be retrieved in the adult. Intriguingly, we found that *msnF9* reporter, whose activity is induced in lamellocytes in the larvae (Tokusumi et al., 2009b), was expressed in ~92% of the adult hemocytes (Supplementary Figure 2G). However, we did not observe any cell with high actin content and/or expressing the β-integrin Myospheroid
<table><caption>**TABLE 1** | Main gene ontology terms enriched in genes over- (up AD) or under- (down AD) expressed in adult as compared to larval hemocytes.</caption><thead><tr><th>GO-term</th><th>Up AD</th><th>Down AD</th></tr></thead><tbody><tr><td>Small molecule metabolic process</td><td>5.06E-42 (n = 158)</td><td></td></tr><tr><td>ATP metabolic process</td><td>1.66E-39 (n = 63)</td><td></td></tr><tr><td>Inner mitochondrial membrane protein</td><td>3.50E-30 (n = 52)</td><td></td></tr><tr><td>Oxidoreductase activity</td><td>7.74E-29 (n = 113)</td><td></td></tr><tr><td>Nucleosome</td><td></td><td>6.27E-53 (n = 45)</td></tr><tr><td>Chromatin organization</td><td></td><td>5.46E-29 (n = 51)</td></tr></tbody></table>

p-Values and gene numbers are indicated for each category.

<table><caption>**TABLE 2** | Main gene ontology terms among the 1000 most strongly expressed genes in adult hemocytes (AD), peripheral hemocytes (PH) and lymph glands (LG).</caption><thead><tr><th>GO-term</th><th>AD top 1000</th><th>PH top 1000</th><th>LG top 1000</th></tr></thead><tbody><tr><td>Cytoplasmic translation</td><td>1.50E-71 (n = 89)</td><td>2.33E-70 (n = 88)</td><td>3.53E-77 (n = 93)</td></tr><tr><td><strong>Generation of precursor metabolites</strong></td><td><strong>6.91E-42 (n = 83)</strong></td><td>1.20E-04 (n = 30)</td><td>3.67E-04 (n = 24)</td></tr><tr><td><strong>ATP metabolic process</strong></td><td><strong>1.61E-41 (n = 156)</strong></td><td>1.89E-06 (n = 28)</td><td>8.22E-07 (n = 27)</td></tr><tr><td>Structural constituent of ribosome</td><td>2.79E-31 (n = 80)</td><td>6.00E-28 (n = 75)</td><td>4.48E-31 (n = 79)</td></tr><tr><td><strong>Oxidoreductase activity</strong></td><td><strong>4.19E-17 (n = 105)</strong></td><td>2.77E-04 (n = 67)</td><td>1.29E-03 (n = 64)</td></tr><tr><td>Vesicle-mediated transport</td><td>2.44E-14 (n = 86)</td><td>2.13E-18 (n = 94)</td><td>6.25E-12 (n = 81)</td></tr><tr><td>Phagocytosis</td><td>8.43E-09 (n = 18)</td><td>7.54E-09 (n = 18)</td><td>2.52E-04 (n = 12)</td></tr><tr><td>Immune system process</td><td>1.48E-05 (n = 56)</td><td>2.72E-07 (n = 61)</td><td>2.17E-07 (n = 62)</td></tr></tbody></table>

p-Values and gene numbers are indicated for each category. Categories particularly enriched in adult hemocytes are highlighted in bold.

(Mys), two characteristic features of lamellocytes (Irving et al., 2005; Supplementary Figures 3C,I), suggesting that lamellocytes are not present and that the msnF9 reporter is constitutively active in adult blood cells. Since lamellocytes are absent in healthy larvae but massively induced in response to infection by eggs of the parasitoid wasp *Leptopilina boulardi* (Lanot et al., 2001), we assessed their presence in adult flies that survived wasp infestation. Still, β-integrin and phalloidin staining did not reveal the presence of lamellocytes in infected adults, while they were readily recovered in bleeds from infected larvae (Supplementary Figures 3B,D,H,J). Furthermore, when using flies carrying the gain-of-function *HopT* Tum-1, which exhibit constitutive activation of the JAK kinase Hopscotch leading to lamellocyte production in the larvae (Luo et al., 1995), we did not observe lamellocytes in the adult either (Supplementary Figures 3E,F,K,L). Thus, it appears that lamellocytes do not persist during adulthood.

Finally, to reveal the presence of putative blood cell progenitors, we made use of *tepIV-GAL4*, *Ance-GFP*, *domePG125-GAL4* and *domeMeso-GAL4*, all of which have been shown to label larval prohemocytes in the lymph gland (Krzemien et al., 2007; Avet-Rochex et al., 2010; Louradour et al., 2017; Cho

<table><caption>**TABLE 3** | Main gene ontology terms enriched in genes up- or down-regulated in adult (AD) and embryonic (Emb) hemocytes as compared to larval hemocytes.</caption><thead><tr><th>GO-term</th><th>Up AD and Emb</th><th>Down AD and Emb</th></tr></thead><tbody><tr><td>Small molecule metabolic process</td><td>2.48E-24 (n = 83)</td><td></td></tr><tr><td>ATP metabolic process</td><td>4.21E-22 (n = 34)</td><td></td></tr><tr><td>Respiratory chain complex</td><td>3.07E-21 (n = 26)</td><td></td></tr><tr><td>Oxidoreductase activity</td><td>1.37E-15 (n = 58)</td><td></td></tr><tr><td>Cell cycle</td><td></td><td>9.73E-8 (n = 20)</td></tr></tbody></table>

p-Values and gene numbers are indicated for each category.

et al., 2020; Rodrigues et al., 2021) or in peripheral blood cells (Fu et al., 2020). While we did not detect any expression of *tepIV-GAL4*, *Ance-GFP* or *domePG125-GAL4* in adult hemocytes (Supplementary Figures 2H-J), we found that *domeMeso-GAL4* was expressed in ~4% of cells in both 5- and 35-day-old flies (Figures 3F,G). *domeMeso* was expressed in cells exhibiting a characteristic fusiform shape, with one or two filopodial extension, but not in round hemocytes (Figures 3F,J-J”,M-M”). Importantly, none of the fusiform cells expressed the blood cell differentiation markers tested above (*Pxn*, *Cg25C*, *Hml*, *crq*, *Bc*, *lz* and *msn*), but they expressed *srp*, albeit at lower level than the other blood cells (Figures 3H-P” and Supplementary Figures 4A-H). Also, immunostaining against the plasmatocyte marker P1/NimC1 as well as *in situ* hybridization against *Pxn* or against the crystal cell marker *Bc/PPO1* confirmed that *domeMeso*/fusiform cells did not express these blood cell differentiation markers (Supplementary Figures 4I-K), strongly suggesting that they could be blood cell progenitors.

In sum, our data indicate that the adult hematopoietic system is essentially composed of plasmatocytes (~85%) and crystal cells (8 to 10%) and could harbor a small population of undifferentiated cells (~4%). Furthermore, as schematized in Figure 3R, adult blood cells can be classified into different subcategories (depending on the expression of *Pxn*, *Hml*, *crq*, *Bc* and *domeMeso*) whose proportions are relatively stable between young and old flies.

## Abdominal Hematopoietic Hubs Have a Similar Composition as Adult Bleeds

Since adult bleeds may not accurately reflect the composition of the adult hematopoietic system, we also assessed the expression of some of the above reporters in the dorsal abdomen. As shown in Figures 4A-G,A'-G' *srp*, *Pxn*, *Cg25C*, *Hml*, *crq*, *Bc*, or *domeMeso* expression was observed as dispersed cells in the abdomen, especially in segments A1 to A3 along the heart, in the so-called “hematopoietic hubs.” *srp* expression was also prominent in pericardial cells (Figure 4A, arrows). In the hematopoietic hubs, *Hml* cells represented 65% of the hemocytes as judged by co-labeling with *srp* or *Pxn* and no *Hml* *srp* or cells *Hml* *Pxn* were observed (Figures 4H-H”,K-K”,P-P”). Crystal cells (*Bc*) and putative prohemocytes (*domeMeso*) represented ~10% and ~3% of the hemocytes, respectively (Figures 4I-I”,J-J”,P-P”). 90% of the crystal cells expressed *Cg25C* and 55% of them expressed *Hml* (Figures 4M-M”,O-O”,P-P”). All *domeMeso* cells expressed *srp* at low levels and none expressed
<figure><img src="image_5.png" /></figure>

**FIGURE 3** | Analysis of blood cell transgenic markers in adult bleeds. (A–F) Confocal views showing representative bleeds from 5-day-old flies carrying the following transgenes: *Pxn-RedStinger* (**A**, *Pxn-RFP*), *Cg25C-GFP* (**B**), *HmlΔ-GAL4*,*UAS-2xEYFP* (**C**, *HmlΔ>GFP*), *crq-GAL4*,*UAS-2xEYFP* (**D**, *crq>GFP*), *Bcf2-GFP* (**E**, *Bc-GFP*), *domeMeso-GAL4*,*UAS-2xEYFP* (**F**, *domeMeso>GFP*). Cells were counterstained with phalloidin (**A**: green, **B–F**: red) and DAPI (blue). Scale bar 200 µm. (G) Quantifications of blood cell proportions expressing the indicated transgenes in 5- or 35-day-old flies. Means and standard deviations from four to six independent experiments are represented. From 200 to 600 DAPI+ cells were scored in each experiment. \*\*p < 0.01 (Student's t-test). (H–P) High magnification views showing the different types of blood cells observed in bleeds from 5-day-old flies carrying the following combination of transgenes: *srpHemo-His2A-RFP*, *HmlΔ-GAL4*,*UAS-2xEYFP* (**H**), *srpHemo-His2A-RFP*, *Bcf2-GFP* (**I**), *srpHemo-His2A-RFP*, *domeMeso-GAL4*,*UAS-2xEYFP* (**J**), *Pxn-RedStinger*, *HmlΔ-GAL4*,*UAS-2xEYFP* (**K**), *Pxn-RedStinger*, *Bcf2-GFP* (**L**), *Pxn-RedStinger*, *domeMeso-GAL4*,*UAS-2xEYFP* (**M**), *HmlΔ-GAL4*,*UAS-2xEYFP*, *Bcf6-mCherry* (**N**), *HmlΔ-GAL4*,*UAS-2xEYFP*, *crq-RedStinger* (**O**), *Cg25C-GFP*, *Bcf6-mCherry* (**P**). GFP and RFP signals are nuclear; cells were counterstained with DAPI (blue) and phalloidin (green). The top three panels show round cells expressing or not RFP and/or GFP; the bottom panels (**) show fusiform cells together with their GFP/RFP expression status. Scale bar 20 µm. (Q) Quantifications of the proportion of cells expressing the indicated combination of markers in 5- or 35-day-old flies. Mean values from at least three independent experiments are presented. No significant difference (Student's t-test *p* < 0.05) was observed in the proportion of GFP+RFP+, GFP+RFP- or GFP-RFP+ blood cell populations between 5- and 35-day-old flies. The total numbers of GFP+ and/or RFP+ cells scored are indicated at the top of each column. (R) Schematic representation of the adult hematopoietic landscape. The proportion of cells expressing each marker is indicated.

*Pxn* (Figures 4J–J”, L–L”, P–P”). Similar observations were made when analyzing the expression of these markers in 35-day-old flies and we did not observe significant differences in the proportion of each blood cell populations between 5-day- and 35-day-old flies (Figure 4P). These results are consistent with our observations on adult bleeds and indicate that the adult hematopoietic system is composed of different subpopulations of plasmatocytes and crystal cells as well as a few putative blood cell progenitors.

Hemolectin Is Expressed in a
Sub-Population of Adult Hemocytes

The *Hml* driver has been used in several instances to manipulate
gene expression in adult blood cells and/or test the function of
these cells, notably by generating “hemoless” adults through the
expression of an apoptosis inducer in blood cells (Charroux and
Royet, 2009; Defaye et al., 2009; Ayyaz et al., 2015; Ghosh et al.,
2015; Woodcock et al., 2015; Sanchez Bosch et al., 2019). Yet,
our results suggest that this driver is not active in every adult

hemocyte but rather labels a specific subpopulation. To confirm
this hypothesis, we expressed the apoptotic inducer Bax under
the control of *Hml* either during the whole development or only
from day 1 of adulthood by using a *tubGAL-80ts* transgene to
restrict its expression temporally, and we assessed the presence
of hemocytes in 10-day-old adult flies. *Hml*-driven expression
of Bax specifically during adulthood caused an almost total
ablation of *Hml*<sup>+</sup> blood cell population as compared to control
flies (Figures 5A–A”*,C–C”*), but *Pxn*<sup>+</sup> cells were still present
in adult bleeds and abdominal preparations (Figures 5B–B”*,D–
D”*). When Bax was expressed from embryogenesis onward,
we observed an efficient ablation of *Hml*<sup>+</sup> cells and very few
*Pxn*<sup>+</sup> blood cells in adult abdominal hubs but, in contrast
with the normal situation (Figures 5E–E”*,G–G”*), we retrieved
many *Pxn*<sup>-</sup> cells when these individuals were bled (Figures 5F–
F”*,H–H”*). While some of these *Pxn*<sup>-</sup> cells were fusiform,
as in the control, most had the typical round morphology of
differentiated hemocytes, suggesting that the chronic expression
of Bax in *Hml*<sup>+</sup> cells leads to the repression of *Pxn*. Consistent
with this hypothesis, immunostaining against P1/NimC1 showed
<figure><img src="image_6.png" /></figure>

**FIGURE 4** | Analysis of blood cell transgenic markers in the adult abdomen. (A–F) Confocal views of the abdominal segments A1 to A4 of 5-day-old females carrying the following transgenes: *srpHemo-His2A-RFP* (*A*), *Pxn-RedStinger* (*B*), *Cg25C-GFP* (*C*), *HmlΔ-GAL4*, *UAS-2xEYFP* (*D*), *crq-GAL4*, *UAS-2xEYFP* (*E*), *Bcf2-GFP* (*F*), *domeMeso-GAL4*, *UAS-2xEYFP* (*G*). Cells were counterstained with phalloidin (*A*,*B*: green; *C*–*G*: red) and DAPI (blue). Arrows in panel (*A*) indicate pericardial cells expressing *srpHemo*. The lower panels show only the transgene expression. Scale bar 200 µm. (**H–O**) High magnification views of hemocytes in the abdominal hematopoietic hubs (heart region in A1 or A2 segments) of 5-day-old females carrying the following transgenes: *srpHemo-His2A-RFP*, *HmlΔ-GAL4*, *UAS-2xEYFP* (*H*), *srpHemo-His2A-RFP*, *Bcf2-GFP* (*I*), *srpHemo-His2A-RFP*, *domeMeso-GAL4*, *UAS-2xEYFP* (*J*), *Pxn-RedStinger*, *HmlΔ-GAL4*, *UAS-2xEYFP* (*K*), *Pxn-RedStinger*, *domeMeso-GAL4*, *UAS-2xEYFP* (*L*), *HmlΔ-GAL4*, *UAS-2xEYFP*, *Bcf6-mCherry* (*M*), *HmlΔ-GAL4*, *UAS-2xEYFP*, *crq-RedStinger* (*N*), *Cg25C-GFP*, *Bcf6-mCherry* (*O*). The lower panels show only the red (*H''-O''*) or the green (*H'''-O''') channel. Nuclei were stained with DAPI (blue). Scale bar 50 µm. (**P**) Quantifications of the proportion of cells expressing the indicated combination of markers in 5- or 35-day-old flies. Mean values from at least three independent experiments are presented. No significant difference (Student's *t*-test *p* < 0.05) was observed in the proportion of GFP⁺RFP⁺, GFP⁺RFP⁻ or GFP⁻RFP⁺ blood cell populations between 5- and 35-day-old flies. The total numbers of GFP⁺ and/or RFP⁺ cells scored are indicated at the top of each column.

<figure><img src="image_7.png" /></figure>

**FIGURE 5** | Adult blood cells are still present following the ablation of Hml⁺ hemocytes. Confocal views of adult bleeds (*A*,*B*,*E*,*F*,*I*,*J*) and abdominal hubs (*C*,*D*,*G*,*H*,*K*,*L*) from tub-GAL80ts, *Pxn-RedStinger*, *HmlΔ-GAL4*, *UAS-2xEYFP* (*A*,*C*,*E*,*G*,*I*,*K*) and tub-GAL80ts, *Pxn-RedStinger*, *HmlΔ-GAL4*, *UAS-2xEYFP*, *UAS-Bax* (*B*,*D*,*F*,*H*,*J*,*L*) flies expressing (or not) Bax in Hml⁺ cells only during adulthood (*A*–*D*) or throughout their life (*E*–*L*). Cells were stained with phalloidin (*A*,*B*,*E*,*F*: red; *C*,*D*,*G*,*H*,*K*,*L*: white; *I*,*J*: no phalloidin) and nuclei with DAPI (blue). (*I*–*L*): immunostaining against P1/NimC1 (red). Lower panels show high magnification views of hemocytes. (*A'''-L''') Merged blue, red and green channels, (*A''''-L''''*): red channel only, (*A'''''-L''''*): green channel only. Upper panels: scale bar 200 µm; lower panels: scale bar 20 µm.

that many cells expressing this plasmatocyte marker were still present in adult bleeds and abdominal preparations following the constitutive expression of Bax in *Hml⁺* cells (Figures 5J–J''',L''–L''''

and Supplementary Figure 5). These results show that killing *Hml⁺* blood cells is not sufficient to generate “hemoless” flies and might alter the gene expression program of the remaining blood
<figure><img src="image_8.png" /><figcaption>**FIGURE 6** | Fusiform cells are derived from the lymph gland posterior signaling center. (A–F,L–W) Cell lineage analyses of adult blood cells origin. Confocal views showing the different types of fusiform (A,D,G,J–L,O,R,U) or round (B,C,E,F,H,I,M,N,P,Q,S,T,V,W) cells recovered in adult flies expressing the G-TRACE reporter transgenes under the control of the indicated drivers. Present and past expression of the drivers are revealed by nuclear RFP (red, A'–F' and L'–W') and nuclear GFP (green, A''–F'' and L''–W''), respectively. (G–H) Expression of Col (green) in adult blood cells as revealed by immunostaining. (I–K) Confocal views of adult bleeds (I,J) and abdominal hematopoietic hub (K) from *Pxn-RedStinger*, *pcol85-GAL4*,*UAS-mCD8-GFP* flies. (A–W) Scale bar 20 µm. Cells were counterstained with phalloidin (A–H,L–W: red; I–K: white) and DAPI (blue).</figcaption></figure>

cells. These data also indicate that during adulthood *Hml<sup>+</sup>Pxn<sup>+</sup>* and *Hml<sup>-</sup>Pxn<sup>+</sup>* cells form two distinct populations and that fusiform cells are not derived from *Hml<sup>+</sup>* cells.

**domeMeso<sup>+</sup> “Undifferentiated Hemocytes” Are Not Derived From Larval Blood Cell Progenitors but From the Lymph Gland Posterior Signaling Center**

A previous report showed that a small fraction of cells expressing Srp did not express the blood cell differentiation markers P1/NimC1 (for plasmatocytes) or Hindsight (for crystal cells), suggesting that they could be hematopoietic progenitors (Ghosh et al., 2015). In addition, these cells were proposed to derive from the posterior lobes of the larval lymph gland based on lineage tracing experiments using the *col-GAL4* driver *GMR13A11*. While *col-GAL4*-derived cells from the larval lymph gland were shown to contribute to adult plasmatocytes and crystal cells (Ghosh et al., 2015), it remains to demonstrate that these cells can proliferate and/or differentiate during adulthood and thus constitute a genuine adult hematopoietic progenitor population. Our results above strongly suggested that fusiform/domeMeso<sup>+</sup> cells could correspond to this putative hematopoietic progenitor population. To test this hypothesis, we first assessed their origin. Using the G-TRACE cell lineage tracing

technique (Evans et al., 2009) with either *GMR13A11* or the classically used *pcol85 col-GAL4* line, we found that all fusiform cells belonged to the *col* lineage-traced cells (**Figures 6A,D**), indicating that they correspond to the previously described adult blood cell progenitors. Interestingly, we found that these cells maintain *col* driver expression (**Figures 6A–A”,D–D”*), while we only observed past/lineage-traced expression in round hemocytes (**Figures 6B–B”,E–E”*). This result was confirmed by immunostaining, which revealed that Col was expressed in fusiform/domeMeso<sup>+</sup> cells, but not in other adult blood cells (**Figures 6G–G”,H–H”* and **Supplementary Figures 6A,B**). Furthermore, in accordance with the above characterization of domeMeso<sup>+</sup> cells, *col<sup>+</sup>* cells were *Pxn<sup>-</sup>* both in adult bleeds and in abdominal hematopoietic hubs (**Figures 6J–J”,K–K”*).

These results are thus consistent with the hypothesis that *col<sup>+</sup>* progenitors could give rise to differentiated hemocytes. Importantly though, the *col-GAL4* lines *pcol85* and *GMR13A11* are not only expressed in blood cell progenitors present in the lymph gland posterior lobes but also in the PSC (Benmimoun et al., 2012; Ghosh et al., 2015; Rodrigues et al., 2021). Moreover, recent single-cell RNA-seq analyses revealed that PSC-like cells expressing *col* and *Antp* are present in circulating larval blood cells (Cattenoz et al., 2021). Hence, these putative adult hematopoietic progenitors could have diverse origins. First, to test whether they were derived from the
<figure><img src="image_9.png" /><figcaption>**FIGURE 7** | Fusiform cells can differentiate and proliferate. (A–C) Confocal views of blood cells from *domeMeso-GAL4*, *UAS-2xEYFP* (*A*, *ctr*), *col*⁻/⁻, *domeMeso-GAL4*, *UAS-2xEYFP* (*B*, *col*⁻/⁻) and *domeMeso-GAL4*, *UAS-2xEYFP*, *UAS-RNAi col* (*C*, *RNAi col*) adult flies. Blood cells were stained with phalloidin (red) and DAPI (blue). Scale bar 200 µm. (D) Quantifications of the proportion of *domeMeso*⁺ hemocytes. Means and standard deviations of at least three independent experiments are presented. A total of 2612, 2350, and 4271 hemocytes were scored in control (*ctr*), *col*⁻/⁻, and *RNAi col* flies, respectively.
***p < 0.001 (Student's t-test). (E–J) Blood cells from *tub-GAL80ts*, *domeMeso-GAL4*, *UAS2x-EYFP* adult flies expressing (G–J) or not (*E*,*F*) *col* RNAi only during adulthood. P1/NimC1 expression (red/white in lower panels) was revealed by immunostaining. Nuclei were stained with DAPI (blue). Scale bar: 20 µm. (K) Quantifications of the proportion of *domeMeso*high/fusiform hemocytes (green) and *domeMeso*low/round hemocytes in *tub-GAL80ts*, *domeMeso-GAL4*, *UAS2x-EYFP* adult flies expressing (*RNAi col*) or not (*ctr*) *col* RNAi during adulthood. Means and standard deviations of at least three independent experiments are presented. A total of 2892 and 4489 hemocytes were scored in *ctr* and *RNAi col* flies, respectively. ***p < 0.001 (Student's t-test). (L–Q) Blood cells from *tub-GAL80ts*, *domeMeso-GAL4*, *G-TRACE* flies raised at permissive temperature (L,M) or switched from restrictive to permissive temperature 24 h after adult emergence (N–Q). *domeMeso* live expression is revealed by nuclear RFP (red) and its lineage-traced expression by nuclear GFP (green). P1/NimC1 expression (red)</figcaption></figure>

(Continued)
<caption>**FIGURE 7** | (Continued)</caption>

was revealed by immunostaining. Nuclei were stained with DAPI. Scale bar: 20 µm. *ctr*, control non-infected flies; *inf*, flies infected with *E. coli*. (R) Quantifications of the proportion of *domeMeso*<sup>+</sup> cells expressing NimC1 in *tub-GAL80ts*, *domeMeso-GAL4*, *G-TRACE* flies raised at permissive temperature only during adulthood and infected (*inf*) or not (*ctr*) with *E. coli*. Means and standard deviations of at least seven independent experiments are presented. A total of 369 (*ctr*) and 449 (*inf*) *domeMeso*<sup>+</sup> cells were scored. ***p* < 0.001 (Student's t-test). (S-Y) Representative images of the cell cycle status observed in fusiform or round hemocytes from *srpD-GAL4*, *UAS-FUCCI* control (S-U) or *E. coli*-infected (V-Y) adult flies. Cells in G1 phase express the nuclear GFP only, cells in G2/M express both nuclear GFP and RFP, cells in S phase express the nuclear RFP only. Cell morphology was visualized by phalloidin staining (green). Nuclei were stained with DAPI (blue). Scale bar 20 µm. (Z) Quantifications of the proportion of fusiform or round hemocytes in the different phase of the cell cycle in control (*ctr*) or in *E. coli*-infected (*inf*) *srpP-FUCCI* adult flies. Mean values from at least six independent experiments are presented. A total of 646 (*ctr*) and 515 (*inf*) fusiform hemocytes as well as 842 (*ctr*) and 1336 (*inf*) round hemocytes were scored.

embryo/peripheral hemocytes or from the lymph gland, we
performed lineage-tracing experiments with *sn-GAL4*, which
is specifically expressed in embryo-derived hemocytes (Avet-
Rochex et al., 2010), and *dot-GAL4*, which is specifically expressed
in lymph gland-derived cells (Honti et al., 2010). In line with
the idea that adult blood cells are derived from both waves of
hematopoiesis (Holz et al., 2003; Sanchez Bosch et al., 2019),
we observed lineage-traced expression in round hemocytes with
both drivers (**Figures 6M–M”,P–P”**). In contrast, fusiform/Col+
cells were lineage-traced with *dot-GAL4* (*n* = 73/95) but
none (*n* = 0/96) with *sn-GAL4* (**Figures 6L–L”,O–O”** and
**Supplementary Figures 6C,D**), demonstrating that they are
solely derived from the lymph gland. Next, we asked whether
fusiform cells are derived from the lymph gland posterior lobes or
the PSC using, respectively, *Ubx(M3)-GAL4*, which is expressed
only in the posterior lobes (Rodrigues et al., 2021), and *Antp-
GAL4*, which is expressed in the PSC but not in the posterior
lobes (Evans et al., 2009; Rodrigues et al., 2021). Importantly, we
found that fusiform cells were lineage-traced with *Antp-GAL4*
(*n* = 29/38), while *Ubx(M3)-GAL4* activity was traced in round
hemocytes but not in fusiform cells (*n* = 0/83) (**Figures 6R–
W,R’-W’,R”-W”**). Furthermore, immunostaining revealed that
the PSC marker Antp is specifically expressed in *domeMeso*<sup>+</sup>
blood cells (**Supplementary Figures 6E,F**). In addition, as shown
for PSC cells in the larval lymph gland (Tokusumi et al.,
2010), the knock-down of *srp* in *domeMeso*<sup>+</sup> cells abrogated
the formation of filopodial extensions typical of these cells
(**Supplementary Figures 6G,H**). In sum, these results show
that fusiform/col<sup>+</sup>/domeMeso<sup>+</sup> cells are derived from the PSC
rather than from undifferentiated blood cells originating from the
lymph gland posterior lobes.

**domeMeso<sup>+</sup> Cells Can Differentiate and Proliferate**

Since *col* is required for PSC development (Crozatier et al., 2004), we tested its role for fusiform cell development. In contrast with control flies, no *domeMeso*<sup>+</sup> cells (or fusiform cells) were present in *col*<sup>-/-</sup> adults (**Figures 7A,B,D**). In addition, RNAi-mediated knock-down of *col* specifically in *domeMeso*<sup>+</sup> cells was sufficient to ablate this lineage (**Figures 7C,D**). To test whether the maintenance of *col* expression in *domeMeso*<sup>+</sup> cells is required in the adult, we used a *tub-GAL80*<sup>ts</sup> transgene to restrict *col* knock-down to adulthood. Following the inhibition of *col* expression for 48 h in adult flies almost no *domeMeso*<sup>+</sup>/fusiform cells were retrieved, yet we observed a few cells that maintained

*domeMeso* expression at lower levels and adopted a round
shape similar to differentiated hemocytes (**Figures 7E–K**).
Interestingly, in contrast with *domMeso*<sup>high</sup>/fusiform cells, these
*domeMeso*<sup>low</sup> cells expressed the plasmatocyte marker P1/NimC1
(**Figures 7H–J**). Similarly, *in situ* hybridization revealed that
unlike their normal counterparts, these cells also expressed the
differentiation marker *Pxn* (**Supplementary Figure 7**). These
results show that *col* controls cell-autonomously *domeMeso*<sup>+</sup>
cells development and suggest that its expression in these cells
is continuously required to prevent their differentiation.

In light of the above results, we used the G-TRACE system to test whether *domeMeso*<sup>+</sup> cells naturally differentiate during adulthood. Because *domeMeso-GAL4* is expressed in blood cell progenitors in the larval lymph gland (Louradour et al., 2017; Rodrigues et al., 2021), we used a *tubGAL80*<sup>ts</sup> transgene to temporally restrict its activity. When *domeMeso-GAL4* activity was left unrestricted (i.e., when flies were raised at 29°C from embryonic to adult stages), we observed lineage-traced expression of *domeMeso* in round cells (which expressed the plasmatocyte marker P1/NimC1) as well as live (and traced) expression in fusiform cells (**Figures 7L–L”,M–M”). When *dome-GAL4* activity was restricted to the adult (i.e., when flies were raised at 18°C until adulthood and then switched at 29°C for up to 2 weeks), *domeMeso*<sup>+</sup>*G-TRACE* expression was observed in fusiform cells but not in other hemocytes, except for one cell from one out of seven experiments (**Figures 7N–N”,O–O”,R and Supplementary Table 8**). Strikingly though, we found that upon infection of adult flies by *E. coli*, in addition to the fusiform cells, some round/NimC1<sup>+</sup> cells were recurrently lineage-traced with *domeMeso* (**Figures 7P–P”,Q–Q”,R, and Supplementary Table 8**). Thus, while *domeMeso*<sup>+</sup> cells do not usually differentiate during adulthood under normal conditions, they can give rise to plasmatocytes in response to an immune challenge.

Finally, it was proposed that infection of adult flies with *E. coli* can induce *Hml*<sup>+</sup> cells proliferation at low frequency (Ghosh et al., 2015), but these results were refuted by Sanchez Bosch et al. (2019), who brought several lines of evidence that *Hml*<sup>+</sup> cells do not proliferate even in response to infection (Sanchez Bosch et al., 2019). Yet, *Hml* is not expressed in all blood cells and notably not in *domeMeso*<sup>+</sup>/fusiform cells. We thus reassessed the cell cycle status of adult blood cells using *srpD-GAL4*, which is expressed in all the hemocytes, together with the two-color FUCCI cell cycle indicator (Zielke et al., 2014). We found that 97% of the round hemocytes were in G1 phase and 3% in G2/M but none in S phase, while 73% of the fusiform cells were in G1,
27% in G2/M (Figures 7S–S”, T–T”, U–U”, Z and Supplementary Table 9). Of note except for one fusiform cell in one experiment out of six, we did not observe cells in S phase. These results strongly suggest that blood cell do not proliferate in uninfected adults. Following infection with *E. coli*, the proportion of round or fusiform cells in G1 or G2/M remained similar (Figures 7V–V”, W–W”, Y–Y”, Z), but in contrast with round hemocytes, a few fusiform cells (2.1%) reproducibly entered S phase (Figures 7X–X”, Z and Supplementary Table 9). A similar proportion of fusiform cells (1.5%) entered S phase upon infection of 35-day-old flies (Supplementary Table 9), suggesting that they maintain their proliferating potential during aging. In addition, when we used EdU incorporation to assess *domeMeso*<sup>+</sup> cell proliferation, a few *domeMeso*<sup>+</sup> cells (n = 8/370) incorporated EdU following infection with *E. coli*, while none were observed in control flies (n = 0/225) (Supplementary Figure 8). These results thus indicate that *domeMeso*<sup>+</sup>/fusiform cells can proliferate, albeit at low frequency, in response to an immune challenge.

DISCUSSION

In mammals, the life-long production of the different blood
cell types relies on the presence of long-term hematopoietic
stem and progenitor cells, which are specified in the embryo
and reside in specific niches from where they can be mobilized
to respond to the needs of the organism (Cool and Forsberg,
2019). While several aspects of hematopoiesis are conserved from
mammals to Drosophila (Banerjee et al., 2019), the presence of a
similar long-term blood cell progenitor population in Drosophila
remains elusive and the overall composition of the adult
hematopoietic system is still a matter of debate. In particular, two
successive publications came to different conclusions concerning
the presence of hematopoietic progenitors and the mere existence
of hematopoiesis in adult flies (Ghosh et al., 2015; Sanchez
Bosch et al., 2019). Overall, our data indicate that the Drosophila
adult hematopoietic system is essentially composed of different
subpopulations of mature blood cells, including PSC-derived cells
which seem to have some limited hematopoietic capacity, and
concur with the hypothesis that no significant hematopoiesis
takes place during adulthood.

Our results show that the bleeding protocol that we used
ensures a swift and representative collection of the blood cell
types present in the adult with minimal contamination from
other tissues. This simple protocol is thus suitable to define
the adult hemocyte gene repertoire without applying tissue
dissociation and cell-purification protocols, which might alter
gene expression. Thereby, we could show that adult hemocytes
share an important part of their transcriptome with their
larval parents, but also exhibit some clearly distinct features.
Notably several blood cell markers are differentially expressed
between adult and larval hemocytes. For instance, *NimC4* and
*drpr*, which work together for apoptotic cell clearance in the
embryo (Kurant et al., 2008), or *Fer1HCH* and *Fer2LHC*, which
encode the two components of the iron transporter ferritin and
control larval blood cell differentiation (Yoon et al., 2017), are
strongly up-regulated in adult hemocytes. Moreover, our findings

highlight an unexpected degree of similarities between adult and
embryonic blood cells as compared to larval blood cells. Indeed,
the comparison of our results with the recently described embryo
*versus* larval blood cell gene repertoire (Cattenoz et al., 2020)
revealed that almost half of the genes differentially expressed
in adult *versus* larval hemocytes are similarly regulated in
embryonic *versus* larval blood cells. This is particularly striking
for adult up-regulated genes as 422 (out of 864) of them are
also more strongly expressed in embryonic hemocytes, indicating
that adult blood cells reactivate a large number of embryonic
blood cell markers. Among those, many are involved in energy
metabolism. Actually embryo-derived hemocytes undergo a shift
from glycolysis during embryogenesis to lipid β-oxidation during
the larval stages (Cattenoz et al., 2020) and our results strongly
argue that a second metabolic shift occurs during adulthood. The
important role of energy metabolism regulation in Drosophila
blood cells is underscored by recent publications showing that
fatty acid β-oxidation is required for blood cell progenitor
differentiation in the larval lymph gland (Tiwari et al., 2020) and
that infection leads to changes in blood cell metabolism which are
important for an effective immune response (Bajgar et al., 2015;
Krejcova et al., 2019). Prominent changes in energy metabolism
have been observed also in mammalian blood cells in response
to development, aging, infection or cancer (Rashkovan and
Ferrando, 2019; Faas and de Vos, 2020; Nakamura-Ishizu et al.,
2020). Getting further insights into the pathways controlling
these shifts will be thus of great interest. More generally, the
differences between embryonic, larval and adult blood cell gene
expression programs likely reflect distinct biological functions
which warrant further investigation.

Our analysis of different blood cell differentiation markers
indicate that the adult hematopoietic system comprises different
subtypes of plasmatocytes as well as ~10% of crystal cells, but no
lamelocytes. These finding are consistent with previous reports
(Lanot et al., 2001; Kurucz et al., 2007; Ghosh et al., 2015;
Sanchez Bosch et al., 2019), and underscore the heterogeneity
of the plasmatocyte/crystal cell populations, which can be
subdivided according to the expression of *Hml*, *crq* and, to a
lesser extent, *Cg25C*. It will be interesting to analyze whether
these subpopulations have distinct functions for instance in the
immune response or the removal of apoptotic cells, as recently
shown for embryonic macrophage subpopulations (Coates et al.,
2021). The proportion of these subpopulations was similar
between young and aged flies, suggesting that they do not
represent differentiation intermediates inherited from pupal
hemocytes. Along that line, our cell ablation experiments argue
against the idea that all adult hemocytes are derived from *Hml*<sup>+</sup>
cells or that adult *Hml*<sup>+</sup>*Pxn*<sup>+</sup> cells differentiate into *Hml*<sup>-</sup>*Pxn*<sup>+</sup>
cells during adulthood. Besides, our observations are in line with
previous reports showing that adult blood cells do not proliferate
(Lanot et al., 2001; Sanchez Bosch et al., 2019) and that their
number decreases with aging (Mackenzie et al., 2011; Woodcock
et al., 2015; Sanchez Bosch et al., 2019). Therefore, it seems
that the adult hematopoietic system is essentially composed of
mature blood cells.

Ghosh et al. (2015) observed some *Srp*<sup>+</sup> blood cells that
did not express the differentiation markers *NimC1* or *Hnt* and
were lineage-traced with *col-GAL4*. Although the differentiation and proliferative potentials of these cells during adulthood were not assessed, the authors concluded that blood cell progenitors derived from the lymph gland posterior lobes persist during adulthood. Our results contradict this model. Indeed, while we also observed the presence of a small proportion of *srp*⁺ cells that do not express hematopoietic differentiation markers and are lineage-traced as originating from the lymph gland, we demonstrated that these cells actually derive from the PSC rather than from the posterior lobes. Actually, they exhibit a characteristic fusiform shape with filopodial extensions reminiscent of PSC cells (Krzemien et al., 2007; Mandal et al., 2007), and they express Col, which is required for PSC development but also for their maintenance in the adult. Moreover, although these cells express *domeMeso*, they do not express other prohemocytes markers (*tep4*, *Ance* or *dome-GAL4*), and we did not find evidence that they differentiate during adulthood in normal conditions. Nonetheless these cells hold some hematopoietic potential. Notably, upon *E. coli* infection, some of them can enter S phase and can differentiate into plasmatocytes as evidenced by NimC1 induction and morphological changes. Along the same line, infection of larvae with *E. coli* was shown to induce PSC cells proliferation and to affect their morphology (Khadilkar et al., 2017). However, given the low number of PSC-derived cells and the limited response we observed in infected adults, it is unlikely that *de novo* production of mature blood cells is their main function. In the lymph gland, the PSC forms a highly specialized cluster of cells that does not contribute to the general pool of blood cells but controls their fate, in particular in response to immune challenges (Crozatier et al., 2004; Benmimoun et al., 2015). It is tempting to speculate that these PSC-derived cells may have similar functions in the adult. As the knock-down of *col* resulted in the ablation of the PSC-derived cells and the resulting adults were viable and did not show any obvious phenotype, it would be interesting to challenge these flies with different pathogens to try to shed light on the physiological role of these cells.

In sum, our results strongly suggest that the Drosophila adult hematopoietic system does not harbor a true blood cell progenitor population with significant proliferation and differentiation potential. Still, different types of mature, potentially specialized, blood cells are present during adulthood. Along that line it will be interesting to decipher the function of the PSC-derived population, as these cells have been shown to control the cellular immune response to some specific immune challenges in the larva and may play a similar role in the adult. Finally, it is expected that applying single cell RNA-sequencing approaches to the adult hematopoietic system will bring a better description of its constituents and could reveal some of its functional features.

## DATA AVAILABILITY STATEMENT

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and

accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/geo/, GSE174107.

## AUTHOR CONTRIBUTIONS

MB, FL, BB, and LW performed the experiments. YR performed bioinformatics analyses. MB, FL, YR, LV, and LW analyzed the data. MB, FL, YR, BB, LV, and LW designed the study. LW wrote the manuscript. All authors read, and approved the submitted version.

## FUNDING

This work was supported by grants from the Agence Nationale de la Recherche (ANR 13-BSV3-08 and 17-CE12-0030-03), i-Site CAP20-25 and the Indo-French Centre for the Promotion of Advanced Research (IFCPAR/CEFIPRA 5503-1) to LW. MB was supported by Ph.D. fellowships from the Université Clermont-Auvergne and the Fondation pour la Recherche Médicale (FRM).

## ACKNOWLEDGMENTS

We thank the Toulouse RIO and Clermont-Ferrand CLIC platforms for assistance with confocal imaging. We thank all our former colleagues for valuable discussions and the Drosophila community, the Drosophila Bloomington Stock Center and the Developmental Study Hybridoma Bank (DSHB) for fly stocks and antibodies.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2021.739357/full#supplementary-material

**Supplementary Figure 1** | Venn diagrams showing the number of genes expressed (RPKM > 1 in all three biological replicates) in adult hemocytes (AD), in larval peripheral hemocytes (PH) and in larval lymph glands (LG) as determined by RNA-seq on w¹¹¹⁸ females, in comparison with those annotated as hemocyte markers according to Flybase.

**Supplementary Figure 2** | Confocal views showing representative bleeds from 5-day-old flies carrying the following transgenes: *Cg-GAL4*, *UAS-RedStinger* (*A*, *Cg25C>RFP*), *HmlΔ-GAL4*, *UAS-RedStinger* (*B*, *HmlΔ>RFP*), *crq-RFP* (*C*), *Bcf6-mCherry* (*D*, *Bcf-RFP*), *lz-GAL4*, *UAS-RedStinger* (*E*, *lz>RFP*), *pxn-GAL4*, *UAS-2xEYFP* (*F*, *pxn>GFP*), *msnF9-GFP* (*G*, *msn-GFP*), *Ance-GFP* (*H*), *tep4-GAL4*, *UAS-2xEYFP* (*I*, *tep4>GFP*), *domePG125-GAL4*, *UAS-2xEYFP* (*J*, *dome>GFP*). Cells were counterstained with phalloidin (*A–E*: green, *F–J*: red) and DAPI (blue). Scale bar 200 µm. The percentage of cells expressing each transgene is indicated in the bottom right corner of each panel.

**Supplementary Figure 3** | Confocal views of larval (*A*,*B*,*E*,*G*,*H*,*K*) and adult (*C*,*D*,*F*,*I*,*J*,*L*) blood cells from control (*A*,*C*,*G*,*I*) or *L. boulardi*-infected (*B*,*D*,*H*,*J*) w¹¹¹⁸ flies or from *HopTum-1* flies (*E*,*F*,*K*,*L*). (*A–F*) Blood cells morphology was revealed with phalloidin staining (red). (*G–L*) Blood cells were stained with phalloidin (red) and anti-β-integrin (green). (*A–L*) Nuclei were stained with DAPI (blue). Scale bar 20 µm.

**Supplementary Figure 4** | **(A–H)** Confocal views of hemocytes from adult flies carrying the following transgenes: *srpHemo-His2A-RFP* (*A*), *Pxn-RedStinger* (*B*),
Cg25C-GFP (C), HmlΔ-GAL4, UAS2xEYFP (D), crq-GAL4, UAS-RedStinger (E), Bcf6-mCherry (F), Iz-GAL4, UAS-RedStinger (G), msnF9-mCherry (H). Cells were counterstained with phalloidin (green) and DAPI (blue). Lower panels display the red (A,B,E-H) or green (C,D) channel only. (I-K) Confocal views of hemocytes from *domeMeso*-GAL4, UAS-2xEYFP adult flies following immunostaining against P1/NimC1 (I, red) or *in situ* hybridization against *Pxn* (J, red) or *PPO1/Bc* (K, red). Cell nuclei were stained with DAPI (blue). The lower panels display the red channel only. (A-K) Scale bar 20 µm. Left panels display round hemocytes and right panels show fusiform/*domeMeso*⁺ cells.

**Supplementary Figure 5** | Confocal views of abdominal hematopoietic hubs from tub-GAL80ts, *Pxn-RFP*, HmlΔ-GAL4, UAS-2xEYFP control (A) and tub-GAL80ts, *Pxn-RFP*, HmlΔ-GAL4, UAS-2xEYFP, UAS-Bax (B) flies raised at 29°C. Cells were stained with anti-P1/NimC1 (white) and DAPI (blue). (A',B'): green (GFP) and red (RFP) channels. (A'',B''): white channel (P1/NimC1). Scale bar 20 µm.

**Supplementary Figure 6** | (A–F) Confocal views of Col (A–D) or Antp (E,F) expression in fusiform (A,C,E) or round (B,D,F) hemocytes from *domeMeso*-GAL4, UAS-2xEYFP (A,B,E,F) or dot-GAL4, G-TRACE (C,D) adult flies. (C,D) G-traced (past) activity of dot-GAL4 is visualized by nuclear GFP expression. No present expression (nuclear RFP) was observed. (G,H) Confocal views of *domeMeso*-GAL4, UAS-2xEYFP cells in control (G, ctr) and UAS-RNAi *srp* (H, RNAi *srp*) adult flies. The knock-down of *srp* by RNAi prevents filopodia extension by *domeMeso*⁺ cells. Cells were stained with DAPI (blue) and phalloidin (red). Scale bar: 20 µm.

**Supplementary Figure 7** | Blood cells from tub-GAL80ts, *domeMeso*-GAL4, UAS2x-EYFP adult flies expressing (C,D) or not (A,B) col RNAi only during adulthood. *Pxn* expression (red) was visualized by *in situ* hybridization and EYFP (green) expression by immunostaining. Nuclei were stained with Topro3 (blue). The lower panels display the red channel only. Scale bar 20 µm. Left panels (A,C) show *domeMeso*⁺ cells and right panels (B,D) show *domeMeso*⁻ cells.

**Supplementary Figure 8** | (A–C) Confocal views of *domeMeso*-GAL4, UAS2x-EYFP blood cells from control (A) or *E. coli*-infected (B,C) adult flies fed with EdU. *In vivo* EdU incorporation was visualized in red. Nuclei were stained with DAPI. Scale bar 20 µm. The lower panels show only the green (A'-C') or the red (A''-C'') channel.

**Supplementary Table 1** | List of primers used for qPCR.

**Supplementary Table 2** | List of genes expressed in adult hemocytes (RPKM > 1 in all three samples).

**Supplementary Table 3** | List of genes expressed in larval peripheral hemocytes (RPKM > 1 in all three samples).

**Supplementary Table 4** | List of genes expressed in larval lymph glands (RPKM > 1 in all three samples).

**Supplementary Table 5** | List of hemocytes markers on Flybase (expression: hemocytes, plasmatocytes, crystal cells, lamellocytes or prohemocytes).

**Supplementary Table 6** | List of differentially expressed genes between adult, larval peripheral and lymph gland blood cells (fold change > 2, adjusted *p*-value < 0.01).

**Supplementary Table 7** | List of differentially expressed genes between embryonic and larval peripheral hemocytes (fold change > 2, adjusted *p*-value < 0.01) (from EMBL-EBI data set: E-MTAB-8702).

**Supplementary Table 8** | Quantifications of *domeMeso*⁺ cells differentiating into NimC1⁺ hemocytes in control or in *E. coli*-infected adult flies (*tubGAL80ts*, *domeMeso*⁺>*G-TRACE*).

**Supplementary Table 9** | Quantifications of round or fusiform hemocytes in G1, S or G2/M phase of the cell cycle in control or in *E. coli*-infected adult flies (*srp*>'*FUCCI*).

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