1. Developmental Biology
  2. Immunology and Inflammation
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Eater cooperates with Multiplexin to drive the formation of hematopoietic compartments

  1. Gábor Csordás  Is a corresponding author
  2. Ferdinand Grawe
  3. Mirka Uhlirova  Is a corresponding author
  1. Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Germany
  2. Molecular Cell Biology, Institute I for Anatomy, University of Cologne Medical School, Germany
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Cite this article as: eLife 2020;9:e57297 doi: 10.7554/eLife.57297

Abstract

Blood development in multicellular organisms relies on specific tissue microenvironments that nurture hematopoietic precursors and promote their self-renewal, proliferation, and differentiation. The mechanisms driving blood cell homing and their interactions with hematopoietic microenvironments remain poorly understood. Here, we use the Drosophila melanogaster model to reveal a pivotal role for basement membrane composition in the formation of hematopoietic compartments. We demonstrate that by modulating extracellular matrix components, the fly blood cells known as hemocytes can be relocated to tissue surfaces where they function similarly to their natural hematopoietic environment. We establish that the Collagen XV/XVIII ortholog Multiplexin in the tissue-basement membranes and the phagocytosis receptor Eater on the hemocytes physically interact and are necessary and sufficient to induce immune cell-tissue association. These results highlight the cooperation of Multiplexin and Eater as an integral part of a homing mechanism that specifies and maintains hematopoietic sites in Drosophila.

Introduction

In the animal kingdom, the development and differentiation of immune cells is intimately linked to specific spatial compartments. These sites shelter developing immune precursors from external stimuli while providing signals to orchestrate their self-renewal, proliferation and differentiation. During mammalian embryonic development, the hematopoietic stem cells (HSCs) relocate to the fetal liver where they considerably expand in number, before they populate the spleen and the bone marrow in late embryonic stages (Gao et al., 2018). Postnatally, transplanted HSCs primarily seed the hematopoietic niches within the bone marrow by recognizing specific signals on the endothelial cells of the blood vessels, and undergoing trans-endothelial migration (Birbrair and Frenette, 2016). In the bone marrow, the HSCs attach to endosteal cells, which together form the endosteal HSC niche capable of renewing the entire blood cell pool throughout life (Birbrair and Frenette, 2016). Apart from various non-hematopoietic cells which guard HSC behavior, the extracellular matrix (ECM) has been recognized as an essential component of hematopoietic stem cell niches (Klamer and Voermans, 2014). The ECM provides a structured microenvironment to the niche and connects to HSCs through integrin-mediated adhesion (Gattazzo et al., 2014; Khurana et al., 2016). In turn, this mechanosensitive signal feeds back on the stem cells to regulate their proliferative capacity or differentiation (Choi and Harley, 2012; Lee et al., 2013). Although mounting evidence points to the requirement for the ECM in the homeostatic maintenance of the HSC population (Klamer and Voermans, 2014), it remains unexplored if ECM alters the behavior of immune precursors solely by conveying mechanical signals or whether there are receptor-ligand interactions that depend on the recognition of specific ECM components which trigger activation of discrete signaling pathways. Furthermore, it remains to be uncovered whether the presence of particular ECM proteins on niche surfaces alone can provoke the adhesion and expansion of the immune cells.

In recent years, the fruit fly Drosophila melanogaster emerged as an excellent model to study the dynamics of hematopoiesis (Banerjee et al., 2019). Similar to mammals, Drosophila immune cells, called hemocytes, are present from early embryonic stages, and reside in specific hematopoietic sites during development (Martinez-Agosto et al., 2007). In the larval stages, hemocytes form three hematopoietic tissues: the circulation, the lymph gland and the sessile hematopoietic pockets (Honti et al., 2014; Letourneau et al., 2016). The circulation comprises mostly macrophage-like cells (plasmatocytes) and crystal cells, which participate in the melanization of encapsulated foreign objects (e.g. parasitic wasp eggs) (Lanot et al., 2001). These capsules are largely formed by a third type of hemocytes, the lamellocytes, which are not present under homeostatic conditions, but rapidly differentiate upon immune challenge (Lanot et al., 2001). Unlike the freely moving cells in the circulation, the lymph gland is a compact multi-lobe hematopoietic organ on the anterior end of the dorsal vessel, where immune cell precursors differentiate into plasmatocytes and crystal cells (Jung, 2005; Krzemien et al., 2010). Importantly, the lymph gland-derived hemocytes enter the circulation only during pupariation or upon immune challenge such as parasitic attack (Krzemień et al., 2007; Sorrentino et al., 2002). The sessile hematopoietic pockets are located segmentally along the length of the larva in lateral and dorsal patches contained within epidermis and muscle tissue (Makhijani et al., 2011; Márkus et al., 2009). The sessile tissue is primarily composed of plasmatocytes, some of which undergo trans-differentiation into crystal cells (Leitão and Sucena, 2015). It has been demonstrated that the formation of sessile hematopoietic pockets is orchestrated by sensory neurons of the peripheral nervous system (PNS) that not only attract hemocytes but also support their survival and proliferation in situ by secreting Activin-β, a ligand of the TGF-β family (Makhijani et al., 2017; Makhijani et al., 2011). Furthermore, plasmatocytes require the cell-autonomous expression of Eater, a phagocytosis receptor of the Nimrod family (Kocks et al., 2005), to maintain their attachment to sessile pockets (Bretscher et al., 2015; Melcarne et al., 2019). The molecular counterpart of Eater on the body wall remains yet unknown. While these mechanisms anchor the immune cells to the epidermis, they do not isolate them, as there is a continuous exchange between circulating and sessile hemocytes (Honti et al., 2010; Lanot et al., 2001; Makhijani et al., 2011). Moreover, in response to various stress insults sessile hemocytes can be rapidly mobilized and enter circulation (Márkus et al., 2009; Vanha-Aho et al., 2015), while reestablishing the stereotypical pattern of hematopoietic pockets when the challenge ceases (Makhijani et al., 2011), highlighting the dynamic nature of the sessile hematopoietic compartment as well as a requirement for homing cues and adhesive surfaces.

Since the sessile hematopoietic pockets are formed in gaps between the larval epidermis and the body wall muscles (Makhijani et al., 2011), the immune cells are in intimate contact with the basement membranes covering these surfaces. In fact, hemocytes can relocate to other tissues with pathologically altered ECM composition and/or structure, such as imaginal discs or salivary glands with damaged basement membranes (Casas-Tintó et al., 2015; Hauling et al., 2014; Pastor-Pareja et al., 2008), tumors (Cordero et al., 2010; Kulshammer and Uhlirova, 2013; Pérez et al., 2017) or fibrotic adipose tissues (Zang et al., 2015). As a mechanism to cope with such insults and to participate in developmental tissue remodeling, hemocytes secrete ECM proteins, such as Laminins or Collagen IV (Bunt et al., 2010; Töpfer et al., 2019), as well as ECM processing enzymes or assembly factors (Martinek et al., 2008; Nelson et al., 1994). However, only little is known about the physical interaction between hemocytes and ECM under either pathological or homeostatic conditions.

Here, we show that the hemocyte-basement membrane interaction is crucial to the formation of the sessile hematopoietic pockets. We demonstrate that the sessile hemocytes require the interaction of the phagocytosis receptor Eater and the Collagen XV/XVIII ortholog Multiplexin in the epidermal basement membrane to maintain their association with the body wall. Importantly, by manipulating the basement membrane composition hemocytes can be redirected to other tissue surfaces, where they function similarly as they do in the sessile pockets.

Results

Increased Atf3 levels induce hemocyte attachment to the fat body

The basic leucine zipper (bZIP) domain protein, Activating transcription factor 3 (Atf3), has been defined as a stress-response gene pivotal to the maintenance of immune and metabolic homeostasis (Chakrabarti et al., 2014; Gilchrist et al., 2006; Jadhav and Zhang, 2017; Rynes et al., 2012; Zmuda et al., 2010), but has also been implicated in the regulation of cytoskeletal dynamics and vesicular trafficking (Boespflug et al., 2014; Donohoe et al., 2018; Yuan et al., 2013). We have reported previously that the fat body-specific overexpression of Atf3 under the C7-GAL4 driver (Koyama and Mirth, 2016) (hereafter abbreviated as C7>Atf3WT) induced a lean phenotype and decreased lipid droplet size (Rynes et al., 2012). Intriguingly, a closer examination of C7>Atf3WT larvae in which the fat body was visualized with a help of UAS-GFP and the hemocyte population with Hml:DsRed reporter (hereafter abbreviated as Hml:DsRed, C7>Atf3WT) revealed a massive attachment of hemocytes to the adipose tissue surface (Figure 1B, D and E), a phenotype not detected in control larvae (Figure 1A, C and E and Figure 1—figure supplement 1A). The fat body-associated hemocytes (hereafter referred to as FBAHs) present in Hml:DsRed, C7>Atf3WT larvae were densely packed and often formed contiguous monolayers over the entire length of the adipose tissue (Figure 1—figure supplement 1B). In contrast, the characteristic pattern of sessile hematopoietic pockets observed in controls was disrupted in Hml:DsRed, C7>Atf3WT larvae (Figure 1A and B), while the amount of circulating hemocytes was not significantly affected (Figure 1—figure supplement 1C). Importantly, we found that hemocytes also associated with clones of Atf3 overexpressing adipocytes induced by the heat shock FLPout (hsFLPout) technique (Figure 1F), suggesting that the attachment is an intrinsic property of the fat body cells with excessive Atf3 levels rather than a consequence of the systemic activation of blood cells. In support of this notion, neither the uptake of GFP-positive material originating from the fat body was observed in the hemocytes, nor signs of an encapsulation reaction, such as adhesion of lamellocytes or tissue melanization, were present in the case of C7>Atf3WT fat bodies (Figure 1—figure supplement 1D–1F).

Figure 1 with 1 supplement see all
Adipose tissue-specific Atf3 overexpression redirects hemocytes to the fat body surface.

(A–B) Sessile hemocytes present in control larvae as segmental stripes (A) are redirected to the surface of the fat body overexpressing Atf3 (B). Transgene and GFP expression was driven by the fat body-specific C7-GAL4 driver, while Hml:DsRed marks the hemocytes. The images are stitched from multiple Z-projections, where FBAHs are colored amber, all other hemocytes magenta and the fat body green. (C–D) In control larvae, no hemocytes are present on the fat body (C). Atf3 overexpression induces the attachment of hemocytes, which form large clusters on the fat body surface (D). Transgene and GFP expression was driven by the fat body-specific C7-GAL4 driver, while Hml:DsRed marks the hemocytes. (E) Quantification of fat body-attached hemocyte numbers upon Atf3 overexpression with the C7-GAL4 driver. Data points represent individual fat bodies. Unpaired nonparametric two-tailed Mann-Whitney test was used to calculate p-values. Error bars indicate SD, n = 15, ****p < 0.0001. (F) Clonal overexpression of Atf3 induced with the hsFLPout system causes selective hemocyte attachment (F', white, F'', amber) to the clonal adipocytes (marked with GFP). Hemocytes are identified based on strong Tubulin staining (F'), while the weak Tubulin staining in the fat body was pseudocolored magenta (F''). Fat bodies are outlined with dotted lines (C, D, F). The images are projections of multiple confocal sections. Scale bars: 100 μm (C, D, F). See also Figure 1—figure supplement 1 and Figure 1—source data 1.

Together these data indicate that the hemocytes either actively relocate from the sessile hematopoietic pockets or are redirected from the circulation to preferentially adhere to the fat body. Importantly, the interaction of hemocytes with Atf3 expressing adipocytes is not a result of anti-tissue immune response.

Redirected hemocytes proliferate and differentiate on the adipocyte surfaces and engage in immune response

The fact that the hemocytes favored the attachment to the C7>Atf3WT fat body at the expense of body wall sessile pockets indicated that the immune cells associated with Atf3 overexpressing adipocytes may form a de novo hematopoietic compartment reminiscent of the sessile tissue. To determine whether FBAHs would display features of the natural sessile hemocyte population, we focused on their morphological characteristics, mitotic activity, differentiation and ability to participate in the immune responses. Intriguingly, FBAHs, like sessile hemocytes (Lanot et al., 2001; Leitão and Sucena, 2015; Makhijani et al., 2011; Márkus et al., 2009), projected filopodia of varying lengths as well as lamellipodia (Figure 2A–B) and proliferated in situ as demonstrated by the Fly-FUCCI in vivo cell cycle reporter (Zielke et al., 2014) and immunostaining for a mitotic marker phospho-histone-H3 (pH3) (Figure 2C–D).

Fat body-associated hemocytes share features with sessile hemocytes.

(A–B) Similar to sessile hemocytes in control larvae (B), FBAHs tightly cluster and form filopodia on the fat body surface (A). Images are depicting the fat body (A) and the epidermis (B). Transgene and GFP expression was driven by the fat body-specific C7-GAL4 driver (A). Hemocytes were stained with pan-hemocyte anti-Hemese antibody to reveal membrane morphology. Arrows indicate filopodia emanating from hemocytes, asterisks indicate nuclei of epidermal cells (B). (C–D) The ubiquitously expressed FUCCI cell cycle reporter (C) shows FBAHs in G1 (green), S (magenta) and G2/M (white, arrows) phases of the cell cycle. Mitotic FBAHs are highlighted by pH3 staining (D, arrowheads). Nuclei were pseudocolored (see Materials and methods) to indicate hemocytes (amber) or adipocytes (magenta) (C). (E–F) Crystal cells (arrowheads), and plasmatocyte-crystal cell intermediary hemocytes (arrows) are interspersed among plasmatocytes in FBAH clusters (E). Melanized crystal cells are attached to Atf3 overexpressing fat bodies (F, black cells). Transgene and GFP expression was driven by the fat body-specific C7-GAL4 driver. Crystal cells were identified by expression of the BcF6:GFP transgene (E) or melanization due to the presence of the Bc1 mutation (F). Plasmatocytes were revealed with staining against NimC1 (E'), while Hml:DsRed (E) or anti-Hemese immunostaining (F) was used to show all FBAHs. (G–I) FBAH numbers decline 48 hr after L. boulardi infection (H, I) compared to uninfected controls (G). Transgene and GFP expression was driven by the fat body-specific C7-GAL4 driver, while Hml:DsRed marks the hemocytes. Data points represent individual replicates. Unpaired nonparametric two-tailed Mann-Whitney test was used to calculate p-values. Error bars indicate SD, n ≥ 16, ****p < 0.0001 (I). (J–L) The lamellocyte differentiation 24 hr after parasitic infection (J, L) is not impaired in larvae expressing Atf3 in the fat body when compared to infected controls (J, K). Data points represent individual replicates, showing the percentage of lamellocytes (L1-positive cells) in all hemocytes (total DAPI count). Statistical significance was determined with two-tailed Student's t-test, error bars indicate SD, n = 6, n.s. = non significant (J). Images depict circulating immune cells bled from L. boulardi infected larvae. Phalloidin staining (K, L, green) labels all hemocytes, L1 staining (K', L', white) shows the lamellocytes. Transgene expression was driven by the fat body-specific C7-GAL4 driver. (M–P) Encapsulation (M) and melanization (O) of parasitic eggs are not hindered by Atf3 overexpression in the fat body (N, P). Lamellocytes surrounding the eggs 24 hr after infection were visualized with L1 staining (M, N). Brown coloration of the encapsulated eggs 48 hr following infestation indicates melanization (O, P). Nuclei were counterstained with DAPI (A–D, F, K–N). The images are projections of multiple confocal sections, fat bodies are outlined with dotted lines (C, D, G, H). Scale bars: 20 μm (A, B, E), 50 μm (C, D, F, M, N), 100 μm (G, H, K, L, O, P). See also Figure 2—source datas 1 and 2.

Staining against the plasmatocyte marker NimrodC1 (NimC1) (Kurucz et al., 2007a) and the crystal cell-specific BcF6:GFP reporter (Tokusumi et al., 2009) showed that like in the sessile tissue (Leitão and Sucena, 2015), the majority of the FBAHs were plasmatocytes, interspersed by a smaller number of crystal cells (Figure 2E). Surprisingly, a portion of the BcF6:GFP-positive crystal cells also stained for NimC1 and showed Hml:DsRed levels similar to plasmatocytes, suggesting that these cells are intermediate hemocytes in the process of plasmatocyte-crystal cell trans-differentiation (Figure 2E–E'). Moreover, we observed blackened crystal cells among FBAHs (Figure 2F) when combining the Hml:DsRed, C7>Atf3WT with the Bc1 mutation (Rizki et al., 1980). Since the runaway melanization cascade results in cell death (Lanot et al., 2001; Neyen et al., 2015), we speculate that their differentiation likely occurred on the adipocyte surface. Notably, both BcF6:GFP expressing and melanized crystal cells in the Bc1 background were mostly located within the FBAH clusters and were surrounded by plasmatocytes (Figure 2E–F).

Finally, to evaluate whether the FBAH population can participate in an immune response as previously reported for the sessile hemocytes (Márkus et al., 2009; Vanha-Aho et al., 2015), control and Hml:DsRed, C7>Atf3WT larvae were infected with Leptopilina boulardi parasitoid wasps. We found that the FBAHs detached from the Atf3 overexpressing adipose tissue 48 hr after parasitic infection (Figure 2G–I), and lamellocyte differentiation and capsule formation (Figure 2J, L, N and P) was not impaired to a noticeable extent when compared to control larvae (Figure 2J, K, M and O). Together, these data support the notion that hemocytes associated with the fat body in C7>Atf3WT larvae display a hematopoietic program reminiscent of sessile blood cells and engage in the innate immune defense. The C7>Atf3WT FBAH model thus represents a unique opportunity to dissect cellular and molecular mechanisms of hemocyte-tissue homing.

Basement membrane accumulation underlies hemocyte association to Atf3 overexpressing fat bodies

The Drosophila larval adipocytes are the primary source of the basement membrane components for most internal organs (Pastor-Pareja and Xu, 2011), including their own. A crucial step in the formation of basement membranes is the proper deposition and assembly of the ECM components which rely on an intricate cellular and membrane trafficking machinery. Hindering ECM protein secretion and release can lead to fibrotic accumulation (Shahab et al., 2015; Zang et al., 2015). Given the described role of Atf3 in cellular trafficking in epithelial cells (Donohoe et al., 2018), we asked whether the attachment of hemocytes to the Atf3 overexpressing fat body could be caused by changes in the structure or composition of the basement membrane. Indeed, the visualization of the Vkg::GFP reporter, a fusion protein of Collagen IVα2 and GFP (Morin et al., 2001), as well as immunostaining against Laminin and the Collagen XV/XVIII-type protein Multiplexin (Mp) revealed accumulation of ECM below the basement membrane in Atf3 overexpressing clonal adipocytes (Figure 3A–C). Transmission-electron microscopy (TEM) further showed that in contrast to the mostly smooth cell membranes of control adipocytes (Figure 3E), the C7>Atf3WT fat bodies formed deep pericellular folds containing electron-dense material (Figure 3F). These data suggest that the ECM proteins are trapped in pericellular spaces, which might attract hemocytes to the fat body surface.

Figure 3 with 2 supplements see all
Pericellular accumulation of basement membrane components underlies hemocytes attachment to Atf3 overexpressing fat bodies.

(A–D) Clonal Atf3 overexpression in the fat body leads to the enrichment of Collagen IV, Laminin and Mp on the surface of the adipocytes (A, C), and their accumulation in foci below the cell membrane (B, D). Clonal cells overexpressing Atf3 were induced with the hsFLPout technique, and are marked by the expression of myr-mRFP (A, B, magenta) or GFP (C, D, green). The expression of Collagen IV was visualized with the Vkg::GFP reporter, while Laminin and Mp expression was determined by immunostaining. The clonal adipocytes are indicated with cyan dots. The arrowhead points to hemocytes attached to the clonal cells (D). Images are single confocal planes taken at the adipose tissue surface (A, C) or ~5 μm below the tissue surface (B, D). (E–F) Transmission electron micrographs show that compared to controls (E), fat body-specific Atf3 overexpression causes the formation of cell membrane folds (F) and the entrapment of ECM material (arrowheads). The transgene expression was driven by the fat body-specific C7-GAL4 driver. Pericellular spaces between the cell membrane and the basement membrane are colored cyan. (G–K) Knockdown of Mp in Atf3 overexpressing fat bodies abolishes the attachment of hemocytes to the adipose tissue (H, J, K) when compared to Atf3 overexpression alone (G, I, K). Whole larval images are stitches of multiple Z-projections, where FBAHs are colored amber, all other hemocytes magenta (G, H). Transgene and GFP expression was driven by the fat body-specific C7-GAL4 driver, while Hml:DsRed marks the hemocytes. For whole larvae images, the localization of the DsRed signal was determined on every confocal section, and the hemocytes situated on the fat body surface were colored amber, the rest magenta (G, H). Data points represent individual replicates. Unpaired nonparametric two-tailed Mann-Whitney test was used to determine p-values, error bars indicate SD, n = 15, ****p < 0.0001 (K). (L–O) Knockdown of Mp in Atf3 overexpressing fat bodies restores the structure of the sessile hematopoietic pockets. Compared to controls (L, O), the overexpression of Atf3 in the adipose tissue disrupts the striped sessile hemocyte pattern (M, O). The pattern is restored following the simultaneous knockdown of Mp in the fat body (N, O). Transgene expression in the fat body was driven with the C7-GAL4 driver, while the Hml:DsRed reporter was used to determine hemocyte location. Images represent the stereotypical DsRed pattern generated from the alignment of five individual larvae (L–N) and were used for quantification (O). Data points represent the total fluorescence intensity of four regions encompassing the four posterior-most sessile bands, which were normalized to the mean of controls (represented as 1) and shown as fold change. One-way ANOVA multiple comparison with Tukey's correction was used to determine significance, error bars indicate SD, n = 5. **p = 0.0067, *p = 0.0165. Nuclei were counterstained with DAPI (C, D, I, J). The images are single confocal sections (A–D), or represent projections of multiple confocal sections (G–J), fat bodies are outlined with dotted lines (I, J). Scale bars: 20 μm (A–D), 5 μm (E, F), 100 μm (I, J). See also Figure 3—figure supplements 12 and Figure 3—source data 1 – 2.

To establish if there is a causal link between the accumulation of the basement membrane components and the attachment of hemocytes, we decided to interfere with the proper deposition of ECM proteins by silencing the matricellular chaperone SPARC (Shahab et al., 2015), and to reduce the amount of specific core ECM components in the C7>Atf3WT adipose tissue. As expected, inhibition of SPARC enhanced accumulation of ECM aggregates in C7>Atf3WT fat body cells which was associated with membrane blebbing (Figure 3—figure supplement 1A–1B). In contrast, the knockdown of Col4a1 (Collagen IVα1) and Trol (Perlecan) strongly suppressed fat body association of hemocytes, while silencing Vkg partially inhibited blood cell attachment (Figure 3—figure supplement 1C–1E and 1I) and silencing of Laminin-A, -B1 and -B2 (LanA, LanB1, LanB2) had no effect on the presence of FBAHs (Figure 3—figure supplement 1F-1I). It is important to note that similar to SPARC silencing, inhibition of Col4a1 and Trol caused marked alterations to fat body structure and impacted animal viability, as reported previously (Pastor-Pareja and Xu, 2011; Shahab et al., 2015). Surprisingly, downregulation of Mp manifested by a noticeable reduction of Mp levels (Figure 3—figure supplement 2A–2B), completely abolished hemocyte association to the Atf3 overexpressing fat body (Figure 3G-K and Figure 3—figure supplement 2C) without any adverse effect on tissue integrity, although the pericellular membrane folds with ECM material were still present (Figure 3—figure supplement 2D–2E). These results indicate that not the membrane folds, but their specific content is responsible for FBAH adhesion. To confirm this notion, we combined Mp knockdown with silencing of SPARC, which we found to exacerbate pericellular ECM accumulation (Figure 3—figure supplement 2F–2H). While the structure of C7>Atf3WTSPARCRNAiMpRNAi fat body remained disrupted and blebbing was still present (Figure 3—figure supplement 2G) the amount of FBAHs was significantly decreased compared to C7>Atf3WTSPARCRNAi adipose tissues (Figure 3—figure supplement 2H). Importantly, Mp knockdown in Atf3 overexpressing adipocytes resulted in restoration of the stereotypical pattern of the sessile hematopoietic pockets at the body wall (Figure 3L–O), supporting a notion that upon fat body-specific Atf3 overexpression hemocytes residing in the sessile pockets relocate and expand on the adipose tissue surface. These results demonstrate a functional link between ECM accumulation and hemocyte attachment to the fat body, and uncover Mp as a key component of this interaction.

Similar mechanisms drive sessile hematopoietic pocket formation and FBAH adhesion

Since FBAHs phenocopy sessile hemocytes in their morphology and behavior, we asked whether they share a common mechanism of tissue attachment. While little is known about the interaction that anchors the hemocytes to the epidermis, a loss of the plasmatocyte-specific phagocytosis receptor Eater (Kocks et al., 2005) was shown to completely abolish sessile pockets, increasing the number of circulating hemocytes (Bretscher et al., 2015; Melcarne et al., 2019). To address the requirement of Eater in FBAH attachment, we generated C7>Atf3WT larvae homozygous for the eater1 loss of function allele. Strikingly, these larvae lacked both sessile hematopoietic pockets and FBAHs (Figure 4A–E). To validate a specific requirement for Eater in the hemocytes, we first silenced its expression in C7>Atf3WT fat bodies, which did not interfere with hemocyte-fat body interaction (Figure 4F–G). Next, we generated larvae which aside from C7-GAL4 also carried the HmlΔ-GAL4 hemocyte-specific driver (Sinenko and Mathey-Prevot, 2004). While the expression of Atf3 in both the fat body and the hemocytes did not markedly impact hemocyte adhesion (Figure 4H), simultaneous Atf3 overexpression and Eater knockdown impaired FBAH formation (Figure 4I–J). These data define Eater as a hemocyte-specific adhesion molecule that acts both in the context of the sessile pockets and the hemocytes on the adipose tissue surface.

Figure 4 with 1 supplement see all
Mp and Eater control hemocyte attachment to the sessile hematopoietic pockets and on the surface of Atf3 overexpressing fat bodies.

(A–E) Eater loss abrogates the association of hemocytes both to the sessile pockets (A), and to Atf3 overexpressing fat bodies (B, D, E) compared to Atf3 overexpression alone (C, E). Transgene and GFP expression was driven by the fat-body-specific C7-GAL4 driver, while Hml:DsRed marks the hemocytes. Data points represent individual replicates. Nonparametric one-way Kruskal-Wallis test with Dunn’s multiple comparison was used to determine significance. Error bars indicate SD, n = 15. ****adjusted p < 0.0001 (E). (F–J) The knockdown of Eater in the Atf3 overexpressing fat body does not noticeably influence the association of hemocytes (G) compared to Atf3 overexpression alone (F). The combined expression of Atf3 in the fat body and the hemocytes does not markedly alter FBAH cluster formation (H), while simultaneous Eater knockdown suppresses hemocyte attachment (I, J), indicating hemocyte-specific requirement for Eater function. Transgene and GFP expression was driven either by the combination of the fat-body-specific C7-GAL4 driver and the hemocyte-specific HmlΔ-GAL4 (H–J), or with the C7-GAL4 alone (F, G), while Hml:DsRed marks the hemocytes. Data points represent individual replicates. Significance was determined by unpaired nonparametric two-tailed Mann-Whitney test, error bars represent SD, n = 5, **p = 0.0097 (J). (K–L) Knockdown of Mp in epidermal cells (L) disrupts the stereotypical banded sessile hemocyte pattern (K). Transgene and GFP expression was driven by the epidermis-specific a58-GAL4 driver, while Hml:DsRed marks the hemocytes. Images represent individual larvae (K, K', L, L'), or the stereotypical DsRed pattern generated from the alignment of five individual larvae (K'', L''). (M) Sessile hemocyte amounts significantly decrease following epidermal knockdown of Mp. Transgene expression was driven by the a58-GAL4 driver, while Hml:DsRed marks the hemocytes. Data points represent the total fluorescence intensity of four regions encompassing the four posterior-most sessile bands, which were normalized to the mean of controls (represented as 1) and shown as fold change. Statistical significance was determined with two-tailed student's t-test, error bars indicate SD, n = 5. ****p < 0.0001. (N–O) Compared to controls (N), the structure of both the dorsal stripe and lateral patches of the sessile hematopoietic tissue is disrupted upon knockdown of Mp in the epidermis (O). Note that hemocyte accumulation on the lateral side of larvae with epidermal-specific Mp knockdown is likely the consequence of decreased hemolymph flow due to the immobilization process (O’). Transgene expression was driven by the a58-GAL4 driver, while Hml:DsRed marks the hemocytes. Images depict the dorsal (N, O) and lateral (N’, O’) views of the A5-A6 larval segments from the same larvae. Tissues were counterstained with DAPI (C, D, F–I). Images are maximum projections of multiple confocal sections (C, D, F–I). Fat bodies are outlined with dotted lines (C, D, F–I). Scale bars: 100 μm (C, D, F–I). See also Figure 4—figure supplement 1 and Figure 4—source datas 13.

The notion of a common hemocyte adhesion mechanism prompted us to test if Mp is necessary for anchoring hemocytes to the sessile pocket as it is for FBAH formation. To this end, we silenced Mp with the a58-GAL4 (larval epidermis), mef2-GAL4 (body wall muscles) and elav-GAL4 (neurons) (Galko and Krasnow, 2004; Lin and Goodman, 1994; Ranganayakulu et al., 1996) drivers to account for the fact that the pockets reside between the larval epidermis and the body wall muscle layer, and their maintenance depends on the activity of peripheral neurons (Makhijani et al., 2011). While loss of Mp in the epidermal cells drastically disrupted the pattern of sessile tissue, resulting in a phenotype strikingly resembling that of eater1 (Figure 4K–O), Mp knockdown in the muscles and the neurons did not result in a similar dispersion of the sessile tissue (Figure 4—figure supplement 1A–1D). Interestingly, although effective in suppressing FBAHs on Atf3 overexpressing adipose tissues, knockdown of Col4a1 and Trol in the epidermis did not mimic the loss of sessile tissue structure inflicted by Mp knockdown (Figure 4—figure supplement 1E–1F). Furthermore, silencing Mp specifically in hemocytes had no visible impact on the integrity of the sessile hematopoietic pockets (Figure 4—figure supplement 1G–1H).

These results highlight a requirement for Eater and Mp to facilitate hemocyte-basement membrane interactions both in the case of the naturally occurring sessile hematopoietic pockets and the de novo hematopoietic compartment on Atf3 overexpressing fat bodies.

Immune cell-tissue attachment depends on the interaction of Multiplexin and Eater

The Drosophila Mp consists of an N-terminal Thrombospondin-like domain, followed by a Collagen triple helix domain, an NC1 trimerization domain and the C-terminal Endostatin-domain, all of which are present in its mammalian counterparts, Collagens XV and XVIII (Heljasvaara et al., 2017). Mp expression initiates during late embryonic development, mostly in the heart tube and the central nervous system, and is needed for motoaxonal pathfinding (Harpaz et al., 2013; Meyer and Moussian, 2009). These traits are distinct from the ubiquitous Col4a1 and Vkg, suggesting that Mp-containing basement membranes may have specific functions in cell adhesion and migration. Given the fact that hemocyte attachment to the epidermis as well as to C7>Atf3WT fat bodies was dependent on Mp, we asked whether its presence in the basement membrane may be sufficient to promote immune cell attachment. To this end, we generated a C-terminally GFP-tagged Mp transgene (UAS-Mp::GFP) and overexpressed it with the C7-GAL4 driver in the adipose tissue. Compared to control, Mp levels markedly increased in the basement membrane of C7>Mp::GFP fat body (Figure 5A–B) as determined by immunostaining with a Mp-specific antibody (Harpaz et al., 2013). However, GFP signal was restricted to the adipocyte cytoplasm (Figure 5—figure supplement 1A), suggesting that the C-terminal end of Mp undergoes proteolytic cleavage. Surprisingly, Mp::GFP overexpression in the fat body did not induce hemocyte adhesion (Figure 5A–B). Instead, it led to the complete dispersal of the sessile hematopoietic pockets, phenocopying homozygous eater1 mutants (Figure 5C–E, compare to Figure 4A). In addition, circulating hemocyte numbers in C7>Mp::GFP larvae significantly increased compared to controls, reaching similar levels as in eater1 mutants (Figure 5F). Immunoblots from the cell free hemolymph revealed a notable increase of the Mp protein in the circulation of C7>Mp::GFP larvae relative to controls (Figure 5G). These results indicate that while fat-body-produced Mp incorporates into the adipocyte ECM, it is also released into the hemolymph where it may interact with hemocytes and interfere with their binding to the basement membrane due to a saturation effect.

Figure 5 with 1 supplement see all
Fat body-wide Mp overexpression causes detachment of the sessile hemocytes.

(A–B) Similar to controls (A) fat bodies overexpressing Mp::GFP do not attract hemocytes (B), even though Mp integrates into the basement membrane of the adipose tissue (A'', B''). Transgene and GFP expression was driven by the fat-body-specific C7-GAL4 driver, while Hml:DsRed marks the hemocytes. The expression of Mp was determined with immunostaining. Fat bodies are outlined with dotted lines. Nuclei were counterstained with DAPI. Scale bars: 100 μm. (C–E) Fat body-specific Mp::GFP expression disrupts the segmentally organized sessile hematopoietic compartment (D) as observed in controls (C). Images represent individual larvae (C, C', D, D') or the stereotypical Hml:DsRed pattern generated from the alignment of five individual larvae (C'', D''). Data points represent the total fluorescence intensity of four regions encompassing the four posterior-most sessile bands, which were normalized to the mean of controls (represented as 1) and shown as fold change. Statistical significance was determined with two-tailed Student's t-test, error bars indicate SD, n = 5, ****p < 0.0001 (E). (F) Sessile hemocyte detachment following fat body-wide overexpression of Mp::GFP coincides with the elevation of circulating hemocyte numbers similar to eater deficiency. Data points represent individual replicates, which were normalized to control mean (represented as 1). Nonparametric one-way Kruskal-Wallis test with Dunn’s multiple comparison was used to determine significance, error bars indicate SD, n = 8, ***adjusted p = 0.0003, *adjusted p = 0.0175. (G) Mp levels increase in the circulation upon fat body-specific overexpression. Immunoblot against Mp shows multiple bands in cell-free hemolymph extracts, indicating extensive post-translational processing. Molecular weights (in kDa) are shown. Prophenoloxidase 1 (PPO1) served as a loading control. The expression of transgenes and GFP was driven with the fat body-specific C7-GAL4 driver, while hemocytes were recognized based on the expression of the Hml:DsRed reporter (A–G). See also Figure 5—figure supplement 1 and Figure 1—source datas 12.

To circumvent the hemolymph overload, we induced Mp::GFP expression only in clones, which resulted in local Mp accumulation over the targeted adipocytes and in their intercellular spaces (Figure 6A–E). Strikingly, clonal adipocytes overexpressing Mp::GFP were surrounded by hemocytes, which displayed previously established characteristics of FBAHs, namely tight clustering and projection of filopodia and lamellipodia, the ability to undergo mitosis on the tissue surface and the presence of crystal cells (Figure 6C–E). Moreover, Mp::GFP expression in the pouch region of the wing disc using the nubbin-Gal4, UAS-mCherry driver (nub>mCherry) promoted hemocyte adhesion to this specific domain (Figure 6F–G). Of note, this hemocyte epithelial tissue association was not provoked by apoptosis of imaginal cells (Figure 6—figure supplement 1A–1B). Together these results demonstrate that Mp is not only necessary but also sufficient to facilitate hemocyte attachment to tissue surfaces.

Figure 6 with 1 supplement see all
The interaction of Mp and Eater underlies hemocyte attachment to tissue surfaces.

(A–B) In contrast to expression in the whole adipose tissue, clonal overexpression of Mp::GFP in the fat body attracts hemocytes and causes local incorporation of Mp into adipocyte basement membrane, (B) compared to controls (A). Heat-shock induced FLPout clones were distinguished based on their expression of GFP. Clonal adipocytes are indicated with cyan dots. Nuclei were pseudocolored amber to indicate FBAH nuclei, and magenta to show adipocyte nuclei. (C–D) Hemocytes attached to the surface of Mp::GFP overexpressing adipocyte clones (C, D) cluster tightly together and extend filopodia (C', arrows), and some undergo cell division (D’). Immunostaining against Hemese visualizes hemocytes (C, D, amber, C', white), phospho-histone H3 staining shows mitotic nuclei (D', white, indicated with arrows). (E) Crystal cells (indicated with arrows) are present on the surface of Mp::GFP overexpressing adipocyte clones (E). Immunostaining against Hemese was used to visualize hemocytes, and melanized crystal cells can be identified due to the presence of the Bc1 mutation (E', black cells). (F–G) While in control wing discs no hemocytes can be observed on the basal side of the wing pouch (F), overexpression of Mp::GFP in this domain using the nub-GAL4, UAS-mCherry driver (F, G, cyan, outlined with cyan dotted lines) is sufficient to cause hemocyte attachment (G). The hemocytes were visualized with immunostaining against Hemese. Images represent projections of multiple confocal sections from the basal side of the wing disc. (H) Mp::FLAG co-precipitates with Eater::GFP from Drosophila S2 cells lysates. The GFP-tagged Eater served as the bait (IP:GFP). Eater and Mp proteins were detected with the anti-GFP and anti-FLAG tag-specific antibodies. The lower panel shows input extracts with α-Tubulin serving as a loading control. (I) Adipocyte-specific overexpression of Atf3 redirects hemocytes (orange) to the fat body surface (green) from the sessile hematopoietic pockets (purple), where they proliferate (cells with two nuclei) and trans-differentiate into crystal cells (cyan) and can detach from upon immune challenge, similar to their natural hematopoietic environment. The presence of Mp in the basement membrane promotes hemocyte attachment both in the sessile compartment and on the fat body surface upon Atf3 or clonal Mp expression through its interaction with the phagocytosis receptor Eater (right panels). Tissues were counterstained with DAPI (A–G). Images are projections of multiple confocal sections (A–G). Fat bodies (A, B, E) or wing discs (F) are outlined with white dotted lines. Scale bars: 100 μm (A, B), 20 μm (C–E), 50 μm (F, G). See also Figure 6—figure supplement 1.

The necessity of Eater and Mp to secure the immune cells to the basement membrane and the results from Mp overexpression experiments prompted us to test if the two proteins might physically interact. To this end, we performed co-immunoprecipitation experiments with tagged Eater::GFP and Mp::FLAG proteins in S2 cells, and found that the two proteins indeed co-precipitated (Figure 6H). While we cannot exclude that the binding might be indirect, these results present an argument that Mp acts as an interacting partner of Eater on the basement membranes of C7>Atf3WT fat bodies, and on the epidermal surface covering the sessile hematopoietic pockets (Figure 6I).

Discussion

The hematopoietic microenvironments are essential for immune cell development and hematopoietic homeostasis by providing molecular cues and physical interactions that control HSC and progenitor cell localization, maintenance and differentiation. In contrast, emerging evidence suggest that niche alterations can drive premature hematopoietic aging and malignancies (Ho et al., 2019; Walkley et al., 2007a; Walkley et al., 2007b). Fundamental principles of hematopoiesis and hematopoietic niche formation show similarities across phyla (Martinez-Agosto et al., 2007). In this study, we employed the Drosophila melanogaster model to gain a mechanistic understanding of how hematopoietic microenvironments arise. We show that Atf3 overexpression in adipocytes promotes the formation of a de novo hematopoietic compartment on the adipose tissue surface at the expense of the naturally occurring sessile hematopoietic cell pool (Figure 6I). The fat body-associated hemocytes showed round/spherical morphology, and projected short filopodia and lamellipodia, which are characteristic to the sessile hemocytes (Lanot et al., 2001, Figure 2B) and hemocytes adhering to Laminin-coated substrates (Sampson and Williams, 2012), but are distinct from the elongated phenotype of the migratory embryonic macrophages and the pupal hemocytes assisting tissue remodeling (Evans and Wood, 2011; Sampson et al., 2013). The characteristic morphology and the absence of tissue debris-phagocytosis and encapsulation response suggest that FBAHs are in a naïve homeostatic state, not engaged in immune response. Moreover, similar to unchallenged sessile hemocytes, FBAHs proliferate and differentiate in situ on the adipose tissue surface. Although hemocytes also proliferate in the circulation, the division rate of those in the sessile pockets was shown to be higher due to the stimulating effects of Activin-β secreted by peripheral sensory neurons (Makhijani et al., 2017; Makhijani et al., 2011). Since the fat body lacks the innervation of the epidermal cells, the proliferation of FBAHs may be part of a hemocyte-autonomous developmental program, which is supported by the physical adhesion to the tissue and cell-cell contacts between attached hemocytes. Such contacts appear instrumental in the trans-differentiation of plasmatocytes to crystal cells in the sessile pockets, which requires Serrate-Notch juxtacrine signaling among the clustered cells (Leitão and Sucena, 2015). Similarly, FBAH clusters contained fully and partially differentiated crystal cells intermingled with the hemocytes, which suggests that plasmatocyte-crystal cell trans-differentiation is cluster-dependent and does not require signals secreted by peripheral neurons.

Importantly, the simultaneous appearance of the FBAHs and a decline of the sessile hemocytes did not significantly impact the amount of circulating blood cells. This may indicate that FBAHs are dynamic, entering and leaving the circulation at a comparable rate to sessile hemocytes (Honti et al., 2010; Makhijani et al., 2011; Welman et al., 2010). The dynamic nature of FBAHs is further underlined by the fact that they detached after parasitic wasp infestation, mimicking the described behavior of the sessile population (Márkus et al., 2009; Vanha-Aho et al., 2015).

The analysis of FBAHs provided us with an avenue to explore the mechanistic underpinnings of hemocyte-tissue interactions, and highlighted the importance of basement membrane proteins in the formation of Drosophila hematopoietic tissues. We identified Mp, the Drosophila ortholog of Collagens XV/XVIII as a necessary ECM protein for hemocyte attachment to tissue surfaces. We show that Mp loss not only blocked hemocyte attachment to the Atf3 overexpressing fat body, but also restored the integrity of the sessile pockets, while its epidermal knockdown caused sessile tissue disintegration. It is important to note that the formation of FBAHs, but not the sessile hematopoietic pockets, was abolished also by inhibiting Col4a1 and Trol. While these ECM components may directly facilitate hemocyte attachment, it is more likely that they are required to form the general lattice structure of the basement membrane to which Mp anchors. Supporting this argument, when ECM accumulation in the fat body was exacerbated by inhibiting SPARC, simultaneous Mp loss abolished FBAH clusters without restoring the fat body integrity. Thus, unlike the ubiquitously present ECM components Collagen IV, Laminins or Trol (Pastor-Pareja and Xu, 2011) the expression of Mp is restricted to tissues where it locally incorporates into the basement membrane to facilitate cell adhesion (Harpaz et al., 2013; Meyer and Moussian, 2009).

Importantly, we show that the clonal gain of Mp induced localized FBAH clusters very similar to those found on Atf3-expressing adipocytes, demonstrating that Mp is not only required but also sufficient to promote hemocyte adhesion. Furthermore, Mp expression in the wing pouch also attracted hemocytes to the wing disc surface, which suggests that this phenomenon is not restricted to the adipose tissue. Surprisingly, Mp overexpression in the entire fat body did not attract hemocytes. Instead, it resulted in the complete loss of the sessile population. We propose that the increased Mp levels in the hemolymph compete with the basement membrane-incorporated protein for the binding of its receptor on the hemocytes preventing their association with the epidermal or fat body ECMs. These results also suggest that both the tissue specificity and the levels of endogenous Mp expression are tightly regulated. In the context of the sessile hematopoietic pockets, this could mean that low amounts of Mp in the epidermal basement membrane are sufficient to anchor hemocytes through a specific and strong interaction between Mp and its receptor on the hemocytes.

The de novo hematopoietic tissue model and the biochemical assay from S2 cells imply that this hemocyte-specific binding partner is the scavenger receptor Eater. Although the phagocytic properties of Eater have been extensively characterized (Chung and Kocks, 2011; Kocks et al., 2005; Melcarne et al., 2019), its requirement for the formation of hematopoietic pockets was only recently established (Bretscher et al., 2015; Melcarne et al., 2019). We find that Eater is not only essential to maintain the sessile hemocytes but also needed for FBAH formation, further underlining the idea of common hemocyte attachment mechanisms (Figure 6I).

Interestingly, the mammalian counterpart of Mp, Collagen XV is restricted to the cardiac and skeletal muscles (Hägg et al., 1997) while its other ortholog Collagen XVIII is ubiquitously expressed throughout development (Miosge et al., 2003). Both ColXV and ColXVIII undergo a multitude of posttranslational modifications, which include the addition of chondroitin- or heparan-sulfate side-chains (Dong et al., 2003), and extensive protein cleavage which produces Endostatin, an anti-angiogenic peptide that is widely studied because of its link to cell migration, proliferation and tumorigenesis (O'Reilly et al., 1997; Walia et al., 2015). Similarly, Mp was found to be modified by Glycosaminoglycan (GAG) chains, and the overexpression of its N- and C-terminal domains had distinct effects on the motoaxon guidance (Meyer and Moussian, 2009; Momota et al., 2011). The hemocyte-specific counterpart of Mp, Eater, on the other hand, is a member of the Nimrod superfamily of transmembrane phagocytosis receptors that contain multiple EGF-like domains (Kocks et al., 2005; Somogyi et al., 2008). While Nimrod family members have no direct orthologs in mammals, multiple EGF-like domain-containing transmembrane proteins such as MEGF10 have been associated with cell adhesion and migration (Suzuki and Nakayama, 2007). Since Mp is extensively processed upon its secretion to the extracellular space, it is tempting to speculate that its interaction with Eater may be due to particular posttranslational modifications, such as chondroitin sulfate addition or proteolytic cleavage (Meyer and Moussian, 2009; Momota et al., 2011), which sets it apart from the ubiquitously present ECM proteins in Drosophila.

Materials and methods

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (Drosophila melanogaster)w1118BDSCRRID:BDSC_3605
Strain, strain background (Drosophila melanogaster)w; C7-GAL4Rynes et al., 2012
Strain, strain background (Drosophila melanogaster)w; UAS-Atf3WTSekyrova et al., 2010
Strain, strain background (Drosophila melanogaster)w; Hml:DsRedMakhijani et al., 2011
Strain, strain background (Drosophila melanogaster)w; hsFLP, act>y+>GAL4, UAS-GFPSekyrova et al., 2010
Strain, strain background (Drosophila melanogaster)w; hsFLP, act>y+>GAL4, UAS-myr.mRFPSekyrova et al., 2010
Strain, strain background (Drosophila melanogaster)w;; Ubi-GFP.E2f11-230, Ubi-mRFP1.NLS.CycB1-266Zielke et al., 2014RRID:BDSC_55124
Strain, strain background (Drosophila melanogaster)w;; BcF6:GFPTokusumi et al., 2009
Strain, strain background (Drosophila melanogaster)w; Bc1Rizki et al., 1980
Strain, strain background (Drosophila melanogaster)w; P[PTT-un1]vkgG454Morin et al., 2001DGRC 11069
Strain, strain background (Drosophila melanogaster)w; MpRNAiVDRCv38189
Strain, strain background (Drosophila melanogaster)w; MpRNAi IIVDRCv35431
Strain, strain background (Drosophila melanogaster)w; Cg25CRNAiVDRCv28369
Strain, strain background (Drosophila melanogaster)w, trolRNAiVDRCv22642
Strain, strain background (Drosophila melanogaster)w; vkgRNAiVDRCv16986
Strain, strain background (Drosophila melanogaster)w; LanARNAiVDRCv18873
Strain, strain background (Drosophila melanogaster)w; LanB1RNAiVDRCv23119
Strain, strain background (Drosophila melanogaster)w; LanB2RNAiVDRCv42559
Strain, strain background (Drosophila melanogaster)w; SPARCRNAiVDRCv16677
Strain, strain background (Drosophila melanogaster)w;; a58-GAL4Galko and Krasnow, 2004
Strain, strain background (Drosophila melanogaster)w;; mef2-GAL4Ranganayakulu et al., 1996RRID:BDSC_27390
Strain, strain background (Drosophila melanogaster)w, elav-GAL4Lin and Goodman, 1994RRID:BDRSC_458
Strain, strain background (Drosophila melanogaster)eater1Bretscher et al., 2015RRID:BDSC_68388
Strain, strain background (Drosophila melanogaster)w; HmlΔ-GAL4Sinenko and Mathey-Prevot, 2004RRID:BDSC_30139
Strain, strain background (Drosophila melanogaster)w; nub-GAL4, UAS-mCherryBDSCRRID:BDSC_63148
Strain, strain background (Drosophila melanogaster)w;; UAS-eaterRNAiVDRCv4301
Strain, strain background (Drosophila melanogaster)w;; UAS-Mp::GFPThis study
Cell line (Drosophila melanogaster)Schneider 2 (S2) cellsDrosophila Genomic Resource CenterRRID:CVCL_Z992
Recombinant DNA reagentpENTR4-dualThermo FisherA10465
Recombinant DNA reagentpTWGDGRC1076
Recombinant DNA reagentpTWFDGRC1116
Recombinant DNA reagentpTWG-MpThis studyUsed to generate UAS-Mp::GFP Drosophila line
Transfected construct (Drosophila melanogaster)pTWF-MpThis studyUsed to transfect S2 cells
Transfected construct (Drosophila melanogaster)pTWG-EaterThis studyUsed to transfect S2 cells
Transfected construct (Drosophila melanogaster)pWA-GAL4Oda and Tsukita, 1999Used to transfect S2 cells
AntibodyAnti-Hemese (mouse monoclonal)Kurucz et al., 2003 (I. Ando)H2IF (1:100)
AntibodyAnti-L1 (mouse monoclonal)Kurucz et al., 2007b (I. Ando)H10IF (1:100)
AntibodyAnti-NimrodC1 (mouse monoclonal)Kurucz et al., 2007a (I. Ando)N1+N47IF (1:100)
AntibodyAnti-Laminin (rabbit polyclonal)Abcamab47651, RRID:AB_880659IF (1:500)
AntibodyAnti-alpha-Tubulin (mouse monoclonal)DSHBAA4.3, RRID:AB_579593IF (1:200), WB (1:1000)
AntibodyAnti-phospho-histone H3 (rabbit polyclonal)Cell SignalingCat# 9701, RRID:AB_331535IF (1:500)
AntibodyAnti-cleaved-Dcp-1 (rabbit polyclonal)Cell SignalingCat# 9578, RRID:AB_2721060IF (1:500)
AntibodyAnti-Endostatin (rat polyclonal)Harpaz et al., 2013 (T. Volk)IF (1:200), WB (1:1000)
AntibodyAnti-FLAG M2 (mouse monoclonal)Sigma-AldrichF1804, RRID:AB_439685WB (1:1000)
AntibodyAnti-GFP (rabbit polyclonal)AcrisTP401, RRID:AB_2313770WB (1:5000)
AntibodyAnti-PPO1 (rabbit polyclonal)Jiang et al., 1997 (M. Kanost)WB (1:750)
AntibodyAnti-mouse IgG Cy3 (donkey polyclonal)Jackson Immuno Research715-165-1511IF (1:500)
AntibodyAnti-mouse IgG Cy5 (donkey polyclonal)Jackson Immuno Research715-175-1510IF (1:500)
AntibodyAnti-rabbit IgG Cy3 (donkey polyclonal)Jackson Immuno Research711-165-152IF (1:500)
AntibodyAnti-rabbit IgG Cy5 (donkey polyclonal)Jackson Immuno Research711-175-152IF (1:500)
AntibodyAnti-rat IgG Cy5 (donkey polyclonal)Jackson Immuno Research712-175-153IF (1:500)
AntibodyAnti-mouse IgG HRP (donkey polyclonal)Jackson Immuno Research715-035-150WB (1:5000)
AntibodyAnti-rabbit IgG HRP (donkey polyclonal)Jackson Immuno Research711-035-152WB (1:5000)
AntibodyAnti-rat IgG HRP (donkey polyclonal)Jackson Immuno Research712-035-153WB (1:5000)
Chemical compound, drug4′,6-Diamidine-2′-phenylindole (DAPI)Carl Roth GmBH.6335,11 mg/ml
Chemical compound, drugN-PhenylthioureaSigma-AldrichP76290.01% w/V
Software, algorithmFIJISchindelin et al., 2012http://fiji.sc, RRID:SCR_003070
Software, algorithmGraphPad Prism 6GraphPadRRID:SCR_002798
Software, algorithmPhotoshop CS5.5Adobe Systems, IncRRID:SCR_014199
Software, algorithmFluoView FV-10ASWOlympusRRID:SCR_014215
Software, algorithmcellSens standard v1.11OlympusRRID:SCR_014551
Software, algorithmCellProfilerKamentsky et al., 2011RRID:SCR_007358
Other reagentPhalloidin-Alexa 488Molecular ProbesA12379Used for F-actin staining
Other reagentDabco-MowiolSigma-AldrichD27802,81381Mounting medium
Other reagentTransIT-Insect ReagentMirusMIR6100Tranfection reagent
Other reagentGFP-trap beadsChromotekgtma-20, RRID:AB_2631406GFP trap beads for co-immunoprecipitation
Commercial assay or kitGateway LR Clonase IIThermo Fisher11791–020Gateway clonase for entry-destination (LR) recombination

Drosophila husbandry

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Flies were kept at 25°C on a diet consisting of 0.8% (wt/vol) agar, 8% cornmeal, 1% soy meal, 1.8% dry yeast, 8% malt extract, and 2.2% sugar beet syrup supplemented with 0.625% propionic acid and 0.15% Methylparaben (Sigma-Aldrich). In standard crosses, 10 virgins were crossed with five males. The crosses were flipped daily, and the larvae were analyzed 6 days after egg laying (AEL). Driver lines crossed to w1118 served as controls.

Clones overexpressing specific transgene were generated with the FLPout technique utilizing heat shock-induced FLP expression which removes an FRT-flanked stop cassette separating a constitutive Act5C promoter from the GAL4 coding sequence (hsFLP, Act5C>y+>GAL4). Clonal cells were marked by the expression of UAS-GFP or UAS-myr-mRFP. To generate adipocyte clones, first instar larvae (24 hr AEL) were heat-shocked in a 37°C water bath for 20 min, and were afterwards kept at 25°C until processing on day six AEL.

Drosophila melanogaster lines

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The following Drosophila melanogaster strains were used: w1118 (BDSC; RRID:BDSC_3605), w; Hml:DsRed (Makhijani et al., 2011), w; C7-GAL4 (Rynes et al., 2012), w; C7-GAL4, UAS-Atf3WT (Rynes et al., 2012), w; C7-GAL4, UAS-GFP, Hml:DsRed (recombined in this study), w; C7-GAL4, UAS-GFP, Hml:DsRed, UAS-Atf3WT (recombined in this study), w; nub-GAL4, UAS-mCherry (BL 63148, Bloomington Drosophila Stock Center), w; hsFLP, Act5C>y+>GAL4, UAS-GFP (Sekyrova et al., 2010), w; hsFLP, Act5C>y+>GAL4, UAS-GFP, UAS-Atf3WT (Donohoe et al., 2018), w; hsFLP, Act5C>y+>GAL4, UAS-myr-mRFP, UAS-Atf3WT (recombined in this study), w;; Ubi-GFP.E2f11-230, Ubi-mRFP1.NLS.CycB1-266 (Zielke et al., 2014), w;; BcF6:GFP (Tokusumi et al., 2009), w; Bc1 (Rizki et al., 1980), w; P[PTT-un1]vkgG454 (vkg::GFP, Morin et al., 2001), w; UAS-MpRNAi (v38189, VDRC), w; UAS-MpRNAi II (v35431, VDRC), w; UAS-Col4a1RNAi (v28369, VDRC), w, UAS-trolRNAi (v22642, VDRC), w; UAS-vkgRNAi (v16986, VDRC), w; UAS-LanARNAi (v18873, VDRC), w; UAS-LanB1RNAi (v23119, VDRC), w; UAS-LanB2RNAi (v42559, VDRC), w; UAS-SPARCRNAi (v16677, VDRC), w; UAS-MpRNAi, UAS-SPARCRNAi (recombined in this study), w; Hml:DsRed; a58-GAL4, UAS-GFP (Galko and Krasnow, 2004, recombined in this study), w; Hml:DsRed; mef2-GAL4, UAS-GFP (Ranganayakulu et al., 1996, recombined in this study), w, elav-GAL4; Hml:DsRed, UAS-GFP (Lin and Goodman, 1994, recombined in this study), eater1 (Bretscher et al., 2015), w; C7-GAL4, UAS-GFP, Hml:DsRed, UAS-atf3WT; eater1 (recombined in this study), w; HmlΔ-GAL4, UAS-GFP (Sinenko and Mathey-Prevot, 2004), w;; UAS-eaterRNAi (v4301, VDRC), w;; UAS-Mp::GFP (this study).

Whole larval imaging

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Third instar larvae were collected and washed in PBS and placed in glass dissection dishes filled with PBS on ice for 15 min for immobilization. Imaging was performed with an Olympus SZX-16 microscope fitted with a DP72 camera. GFP and RFP images were captured with the cellSens standard v1.11 software (Olympus, RRID:SCR_014551).

Stereotypical sessile tissue patterns were generated from larvae imaged under identical conditions. One larva was selected as a reference and every other image was aligned to match the reference in Adobe Photoshop CS5.5 (Adobe Systems, Inc, RRID:SCR_014199) using the Puppet warp tool. Aligned images were projected in Fiji v1.52i (Schindelin et al., 2012, RRID:SCR_003070) with ‘Average intensity’ projection, using the ‘Fire’ lookup table to enhance visualization.

For confocal imaging of whole fixed larvae, third instar wandering larvae were washed in 70% ethanol and were then injected with 4% paraformaldehyde with a sharpened glass injection capillary. The fixed larvae were immediately placed on a glass slide with a double-sided tape and imaged with an Olympus FV-1000 confocal microscope, with an UPlanSApo 10x (NA0.40) objective. Images were taken using the multi-area module of the FluoView FV-10ASW (Olympus, RRID:SCR_014215) software. Overlapping images were stitched in Fiji v1.52i and all single Z-planes were exported for both GFP (fat body) and DsRed (hemocytes) channels. Each individual confocal section was examined for GFP expression (fat body) and Hml:DsRed expression (hemocyte). The DsRed channel was locally recolored on each section when it was determined to overlap with the GFP signal to amber using Adobe Photoshop CS5.5, then all Z-planes were maximum projected. Sessile- and lymph gland hemocytes are shown in magenta.

Tissue dissection and immunostaining

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Fat body dissection from third instar larvae was performed in PBS by opening the posterior of the larvae and inverting the carcass. After removal of the intestine, larvae were fixed in 4% paraformaldehyde for 1.5 hr. Larval epidermal fillet samples were prepared according to Brent et al., 2009, fixed in 4% paraformaldehyde, followed by the gentle removal of most body wall muscles. Tissues were blocked in 0.5% BSA (A3059, Sigma-Aldrich) supplemented with 0.1% TritonX-100 (T8787, Sigma-Aldrich) in PBS. Wing disc dissection was performed as described in Donohoe et al., 2018. Primary antibody staining was performed overnight at 4°C on a nutating mixer with anti-α-Tubulin (mouse, 1:200, DSHB, AA4.3, RRID:AB_579593), anti-Hemese (mouse, 1:100, Kurucz et al., 2003), anti-L1 (mouse, 1:100, Kurucz et al., 2007b), anti-NimC1 (mouse, 1:100, Kurucz et al., 2007a), anti-Laminin (rabbit, 1:500, Abcam, ab47651, RRID:AB_880659), anti-phospho-histone H3 (rabbit, 1:500, Cell Signaling, Cat# 9701, RRID:AB_331535), anti-Endostatin (rat, 1:200, Harpaz et al., 2013) and anti-cleaved-Dcp-1 (rabbit, 1:500, Cell Signaling, Cat# 9578, RRID:AB_2721060) antibodies diluted in the blocking solution. After washing, the samples were incubated with the corresponding Cy3- or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch) for 1.5 hr at room temperature and counterstained with DAPI (1 μg/ml, 6335.1, Carl Roth GmbH) to visualize nuclei. The fat bodies were dissected from the carcass and mounted in Dabco-Mowiol (Sigma-Aldrich). Tissues were imaged on an Olympus FV-1000 confocal microscope with UPlanSApo 10x (NA0.40), UPlanSApo 20x (NA0.75), UPlanSApo-O 20x (NA0.85), UPlanFLN-O 40x (NA1.30) and UPlanSApo-O 60x (NA1.35) objectives using the FluoView FV-10ASW software. All fat body images represent Z-projections (unless otherwise indicated), which were generated with Fiji v1.52i.

For the pseudocoloring of hemocyte and adipocyte nuclei, confocal Z-stacks of the DAPI channel were divided into surface sections (~0–8 μm from the surface) and deeper sections (>8 μm from the surface), which were projected and exported separately using Fiji v1.52i. The surface projection containing the hemocyte nuclei was then colored amber, while the deeper projection with the adipocyte nuclei was colored magenta. The two projections were merged in Adobe Photoshop CS5.5.

Leptopilina boulardi infection

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Flies were reared on the following diet: 8% cornmeal, 1% agar, 4% yeast, 5% saccharose and 0.16% methylparaben. Fifty early third instar larvae were placed in an infection chamber with 100 female L. boulardi wasps for 15 min (Bajgar et al., 2015). After the removal of the wasps, the larvae were kept at 25°C for 24 or 48 hr before bleeding or dissection.

Hemocyte isolation and staining

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Third instar larvae were bled into 1x PBS containing 0.01% n-phenylthiourea (31056, Sigma-Aldrich) on 12 spot glass slides (HM-101, Hendley-Essex). Allowing hemocytes to adhere for 45 min, samples were then fixed in acetone. Blocking was performed with 0.5% BSA in 1x PBS for 30 min. Hemocytes were stained with anti-Hemese (mouse, 1:100, Kurucz et al., 2003) or anti-L1 (mouse, 1:100, Kurucz et al., 2007b) antibodies and anti-mouse-Cy3 conjugated secondary antibodies (1:500, Jackson Immunoresearch). Nuclei were counterstained with DAPI. Samples were mounted on glass slides in Dabco-Mowiol 4–88 and imaged on an Olympus FV-1000 confocal microscope with UPlanSApo 10x (NA0.40) and UPlanSApo-O 20x (NA0.85) objectives.

Transmission electron microscopy

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Fat bodies were fixed in 2.5% glutaraldehyde diluted in 100 mM phosphate buffer (PB), washed in 100 mM PB and postfixed in 2% osmium tetroxide in PB for 1 hr on ice. Contrasting was performed with 2% uranyl acetate, after which the samples were dehydrated in ethanol and embedded in acetone-resolved araldite. Electron microscopy was performed with an EM 109 (Zeiss) microscope.

Cloning of Eater and Mp expression plasmids and generation of UAS-Mp::GFP Drosophila line

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To express C-terminally GFP- or FLAG-tagged Eater and Multiplexin proteins under UAS control, eater and Mp cDNA was cloned into pENTR4-dual vector between BamHI and NotI sites, and subsequently recombined using LR Clonase II (11791–020, Life Technologies) into pTWG (Mp and Eater) and pTWF (Mp) vectors, respectively (T. Murphy, Drosophila Genomic Resource Center). The UAS-Mp::GFP transgenic flies were obtained by standard P-element-mediated germline transformation of pTWG-Mp plasmid into w1118 Drosophila embryos (Genetics Fly Facility, The University of Cambridge, UK). Multiple transformants were recovered and tested, all showing comparable Mp::GFP expression.

S2 cell culture and cell lysis

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Schneider 2 (S2) cells (Drosophila Genomic Resource Center, RRID:CVCL_Z992) were cultured at 25°C in Shields and Sang M3 insect medium (S8398-1L, Sigma-Aldrich) containing 8% fetal bovine serum (Gibco, Life Technologies) without antibiotics. There are no verified reports of Mycoplasma infection in S2 cells (Cherbas and Gong, 2014). S2 cells were only used to express transgenic proteins for biochemical experiments. All functional data were obtained from in vivo studies in a Drosophila melanogaster model. Cells were transfected using TransIT Insect transfection reagent (MIR 6100, Mirus). Expression of UAS-driven cDNAs was induced by co-transfection with a pWA-GAL4 plasmid expressing GAL4 under an actin5C promoter. Cells were lysed 36 hr after transfection in lysis buffer containing 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1% Triton X-100, 0.01% Igepal, and protease inhibitors (Roche Applied Science). Protein concentration was quantified using Bradford reagent (K015.1, Roth GmBH) according to manufacturer’s instructions.

Co-immunoprecipitation and immunoblotting

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For each sample, 1 mg of S2 cell protein lysate was incubated with GFP-Trap beads (gtma-20, Chromotek, RRID:AB_2631406) overnight. Following five washes with Lysis buffer, bound proteins were eluted with Glycine-HCl on 37°C, followed by neutralization. Proteins resolved on 10% SDS-PAGE were detected by immunoblotting with anti-Flag M2 (mouse, 1:1000, F1804, Sigma Aldrich, RRID:AB_262044), anti-GFP (rabbit, 1:5000, TP401, Acris, RRID:AB_2313770) and anti-α-Tubulin (mouse, 1:1000, DSHB, AA4.3, RRID:AB_579593) antibodies, followed by incubation with corresponding HRP-conjugated secondary antibodies (Jackson Immuno Research). Chemiluminescent signal was captured using ImageQuant LAS4000 reader (GE Healthcare, RRID:SCR_014246).

Hemolymph was collected from twenty third instar larvae per replicate in 100 μL PBS. After centrifugation, 5000 rpm for 10 min, the supernatant was precipitated with 100 μL of ice cold acetone for 1 hr at −20°C. Afterwards, the proteins were pelleted and resuspended in 50 μL in Lysis buffer (see above), and equal volumes were resolved on 10% SDS-PAGE. Immunoblotting was carried out with anti-Endostatin (rat, 1:1000, Harpaz et al., 2013) and anti-PPO1 (rabbit, 1:750, Jiang et al., 1997) antibodies.

Cell quantification and statistical analysis

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Fat body associated hemocytes were quantified based on Z-projection images taken with Olympus FV-1000 confocal microscope fitted with UPlanSApo 20x (NA0.75) objective. The area imaged for comparisons is depicted in Figure 1—figure supplement 1B. The area selected is devoid of hemocytes in wild-type larvae, and lacks non-adipose tissue, such as the gonad precursors or the tracheal branches found at the posterior end. Quantification was performed manually using the CellCounter plugin in Fiji v1.52. For quantification of circulating hemocytes images were acquired using the UPlanSApo 10x (NA0.40) objective. One representative image per larva was processed for cell counting. DAPI positive nuclei were counted with CellProfiler (Kamentsky et al., 2011, RRID:SCR_007358), using a customized pipeline. Lamellocyte percentage was determined by comparing the number of L1-positive cells to all counted DAPI-positive nuclei.

For the quantification of sessile hemocytes, whole larval images aligned to a reference (see ‘Whole larval imaging’ section) were analyzed. These images were loaded in Fiji v1.52i as hyperstacks, containing both control larvae and the genotypes of interest. Using the Rectangle tool, an area was selected where no hemocytes were present, where the average pixel intensity was measured for each control larva and averaged across larvae to establish background levels. Using the Rectangle tool, selections corresponding to single sessile stripes in the A4, A5, A6 and A7 segments were made on control larvae, and the average intensity of the Hml:DsRed signal was measured for the relevant genotypes within this selection. From every measurement the previously established background values were subtracted, then for each larva the four corrected intensity values were added together.

Statistical analysis and plotting were carried out with Prism 6 (GraphPad, RRID:SCR_002798), using one-way ANOVA assuming unequal variances with multiple comparisons or unpaired Student's t-test. For comparison of cell counts, one-way Kruskal-Wallis test with Dunn's multiple comparison or nonparametric two-tailed Mann-Whitney test were used. For fat-body-associated hemocytes, every data point represents the number of attached hemocytes within the quantified area, while for circulating hemocytes and sessile hemocyte quantifications, values were normalized to control mean (represented as 1) and shown on the graphs as fold change (F.C.). All experiments represent at least two independent temporal replicates.

Sample-size criteria was estimated post hoc with G*Power 3.1 (Faul et al., 2009). All significantly different datasets exceeded 0.99 Power (1-beta) with respective sample sizes, means and standard deviations.

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Decision letter

  1. Jiwon Shim
    Reviewing Editor; Hanyang University, Republic of Korea
  2. Utpal Banerjee
    Senior Editor; University of California, Los Angeles, United States
  3. Dan Hultmark
    Reviewer; Umeå University, Sweden

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Drosophila hemocytes come in and out of hematopoietic microenvironments within which they proliferate or differentiate. Despite its importance in hematopoiesis, the molecular mechanism underlying hemocyte-microenvironment interaction remains poorly understood. Here, Uhlirova and colleagues identify critical roles for basement membrane proteins, eater and Multiplexin, in the formation of hematopoietic compartments, providing fundamental insights into how hematopoietic niches arise and maintain.

Decision letter after peer review:

Thank you for submitting your article "Eater cooperates with Multiplexin to drive the formation of hematopoietic compartments" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Utpal Banerjee as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Dan Hultmark (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv (if it is not already there) along with this decision letter and a formal designation that the manuscript is "in revision at eLife". Please let us know if you would like to pursue this option. (If your work is more suitable for medRxiv, you will need to post the preprint yourself, as the mechanisms for us to do so are still in development.)

Summary:

In this manuscript, Uhlirova and colleagues have reported an interesting molecular mechanism underlying the hemocyte recruitment to its environments, which bears a clinical potential to human immune cell biology. The authors first focused on a novel phenomenon that overexpression of Atf3 recruits hemocytes to the fat body while altering patterns of hematopoietic pockets in segmentally repeated epidermal sites. The authors went on to understand a molecular basis for the redirection of hemocytes and discovered that Collagen XV/XVIII type protein Multiplexin (Mp) is required for the Atf3-mediated hemocyte homing dependent on the Eater expression in hemocytes. Given that overexpression of Mp in the fat body disrupts the overall pattern of hematopoietic pockets and that Mp is detected in the hemocyte-free hemolymph, the authors raised a valid hypothesis that Mp can be deposited to the hemolymph, which consequently promotes the systemic hemocyte homing to the sessile pockets.

Essential revisions:

As you will find below, all of the reviewers found the study interesting and agreed on the significance of your work. At the same time, a number of critical criticisms were raised, which require additional data analyses and new experiments. The full comments of the reviewers are attached to provide further details.

1) Identify division and differentiation rates of hemocytes in the Atf3 or Mp expression background.

- As indicated in the comments below (reviewer 1 and reviewer 2), it will be critical to show the redirection of hemocytes to a new tissue would recapitulate hematopoiesis in the hematopoietic pocket.

2) Improve figure images and provide details in figure legends.

- There are multiple concerns about this issue from three reviewers. Please find the comments below for details.

- Of note, it will be important to adequately address reviewer 3's Essential revisions 3 and 4 where the reviewer raised concerns about images showing the fat body associated hemocytes.

3) Provide quantitation of results.

- All the reviewers asked for proper quantitations of data for clarity. Please find the details below.

Reviewer #1:

In this manuscript, Uhlirova and colleagues have reported a critical role for basement membrane proteins in the recruitment of hemocytes in the fat body. The authors first focused on a novel phenomenon that overexpression of Atf3 recruits hemocytes to the fat body while altering patterns of hematopoietic pockets in segmentally repeated epidermal sites. Interestingly, hemocytes attached to the fat body undergo mitosis similar to the ones found in hematopoietic pockets and exhibit normal morphologies. Moreover, hemocytes associated with the fat body relocate to the circulation upon wasp infestation, indicating that these hemocytes display a normal hematopoietic program reminiscent of sessile hemocytes. The authors further went on to understand a molecular basis for the attachment and discovered that Collagen XV/XVIII type protein Multiplexin (Mp) is required for the Atf3-mediated hemocyte homing which is dependent on the Eater expression in hemocytes. Supporting these findings, the authors validated a physical interaction between Mp and eater by biochemical experiments. Given that overexpression of Mp in the fat body disrupts the overall pattern of hematopoietic pockets and that Mp is detected in the hemocyte-free hemolymph, the authors raised a valid hypothesis that Mp can be deposited to the hemolymph critical for the systemic hemocyte homing to the sessile pockets.

This study provides a fundamental insight into molecular mechanisms underlying the hematopoietic environment essential for immune cell development and its function and prompts future studies linking the ECM landscape with the hematopoietic pocket formation and hemocyte development.

Essential revisions:

1) The precise quantitation of data and presenting proper controls will enhance the clarity of the manuscript.

1.1) Figure 1C-D': In Figure 1E, the authors quantified FBAH in imaged fat bodies. However, the number of fat body cells per one image could be variable depending on the size of the fat body, dissecting methods or imaging and so on. For example, the mean value shown in Figure 1E is different from Figure 2I even though these data represent the same genotype. It will be important to deliberate on the precise quantitation method for the FBAH phenotype as it is one of the most important findings described in the manuscript.

1.2) The control shown in Figure 1E is identical to the one shown in Figure 2I and Figure 3—figure supplement 2E. It will be essential to ALWAYS repeat relative controls side by side with associating experiments.

1.3) Figure 2J-K', the number of lamellocytes and the percentage of melanotic capsule formation need to be quantified.

1.4) In the same vein, Figure 3—figure supplement 1C-K' requires proper quantitation with enough biological replicates (n) to draw a conclusion.

1.5) Figure 1A-B, Figure 3G-H, Figure 4A,G,H, Figure 4—figure supplement 1A-F, Figure 5C-D: authors have shown the relocation of sessile hemocytes. Again, it is one of the main phenotypes shown in the paper; however, none of these are quantitated. The patterns of sessile hemocytes are quite variable even amongst controls. Therefore, authors need to quantify the phenotype with their own measure and with enough sample size.

1.6) In Figure 4D-E': though it has been shown in the previous study that eater is essential for the hemocyte homing and that eater is primarily expressed in hemocytes, C7-gal4, hml-gal4 (dual gal4) is a different genetic background and can cross-react. Therefore, additional controls (C7-gal4, hml-gal4/+, C7-gal4, hml-gal4; eater RNAi) are required at least to be represented as quantitation data.

2) It is not clear whether the overall hemocyte number (or hemocyte development) is changed upon Atf3_WT or Mp::GFP expression, which may, in turn, modify the circulating or sessile hemocyte population.

2.1) Figure 2C: To claim whether the epidermal sites are more supportive of the proliferation of hemocytes than those in the fat body, the ratio of mitotic cells needs to be quantified as shown in Makhijani et al., 2017. And without showing the number of total hemocytes, it is hard to understand which site is more prominent in the proliferation and differentiation of hemocytes.

2.2) The total number of hemocytes together with circulating hemocytes needs to be shown in Atf3_wt or Mp::GFP expression (Figure 1—figure supplement 1C, Figure 5E). Moreover, the number of circulating and total hemocytes in Atf3_WT; MpRNAi rescue background should be indicated.

3) In Figure 2E-E', it is not clear whether crystal cells express high levels of NimC1 in the images as NimC1-positive membranes are anyway juxtaposed. It will be great to have a bleeding of FABH or high mag of crystal cells with better resolution.

4) Though authors have shown that FBAHs are relocated to the circulation upon wasp parasitism, these data are not sufficient to conclude that the fat body FBAH follows the identical hematopoietic program to sessile blood cells. It is possible that FBAHs relocation is less frequent than regular sessile hemocytes found in the epidermis/neuron due to the tight association. It will be important to show whether FBAHs can be reattached after physical disturbances as shown in Makhijani et al., 2011.

Reviewer #2:

This very well written and interesting manuscript addresses the clinically relevant question of what molecules drive immune cells' interaction with environments that promote their proliferation and differentiation. The authors use the basic system of Drosophila and provide convincing evidence that an ECM component, Multiplexin, is necessary for the recruitment of Drosophila immune cells (hemocytes) to the previously identified hematopoietic pockets and for their recruitment to a new position on the fat body where they are can divide and differentiate as they do in the native environment. They define the phagocytosis receptor Eater, which has previously been found to be crucial for hemocyte localization to the hematopoietic pockets, as the partner in mediating hemocyte binding to Multiplexin in both locations. I think this work is exciting and mostly well conducted and deserves to be published in ELife, with some alteration of the text to reflect that some of the current conclusions require a modest amount of new data and analysis.

1) The paper in places implies that Multiplexin is capable of redirecting hemocytes to a new location in which they can function as in the hematopoietic pocket. This finding would be extremely exciting if true. But though the authors show relocalization to the fat body in the experiment in which they induce clones of Mp (Figure 5G-H), they do not assess the division and differentiation capacity of the hemocytes there. Thus Atf3 expression may induce other changes in the adipocytes than just Mp expression to allow Fat Body associated hemocytes (FBAH) to divide and differentiate there (as seen in Figure 2C-F); Mp may thus be required for hemocyte adhesion yet not sufficient to induce all niche dependent functions. To address sufficiency for proliferation the authors could do a pH3 staining upon the induction of clones in strains they already have (those used in Figure 5G-H). To address sufficiency for differentiation, they could put one copy of Bc1 into that background.

Alternatively they should alter the paper to make clearer that they have not explicitly investigated if or shown that the ECM component, Mp, drives hematopoiesis in the Title, the Abstract, the Introduction, the Discussion section (specifically "indistinguishable").

2) They conduct experiments to assess the importance of Mp for attachment to the endogenous sessile niches. Their conclusion (subsection “Similar mechanisms drive sessile hematopoietic pocket formation and FBAH adhesion”) that the knockdown of Mp by RNAi produces a phenotype strikingly resembling that seen in the eater1 mutant (and thus that Mp is required for all attachment to endogenous hematopoetic environments) doesn't seem supported by the comparison of Figure panels 4A' to 4H'. While the dorsal patches seem to be gone in the Mp knockdown, it appears as if there is still a repeated hemocyte pattern on the lateral sides in the larvae shown. Having a close up of the lateral and dorsal niches for these genotypes instead of the GFP channel (whose relevance as a marker of the driver pattern is also not explained in the figure legends) would clarify this issue. It is clear there is a strongly reduced attachment in the absence of Mp, but if this the same in all the different endogenous hematopoetic locations is important to determine. Given that Mp has been reported to be expressed in the heart tube, next to where the dorsal patches are, and not to my knowledge near the PNS, which flanks the lateral ones, would fit with there being a variable effect in the different regions. This Figure has no quantitation, which also weakens this conclusion. If they do not have the data in hand to make the new figure/ do the quantitation, then they should soften the conclusion that this is the same as the eater1 phenotype and address textually that the effect on the lateral patches has not been rigorously assessed.

Reviewer #3:

I believe this is a great paper, with exciting science, but I find it unnecessarily difficult to read and to assess critically. I was easily distracted while reading it and I may have missed important points.

Essential revisions:

1) Conceptually, it would be helpful to give the reasons for choosing to overexpress Atf3 in the fat body. I would also like to see more background information about this transcription factor in the Introduction.

2) I also miss a discussion about the possible physiological role of the system described here. Is Atf3 also involved in defining hemocyte docking sites elsewhere? Or should I regard Atf3 overexpression as an artificial but useful experimental system for the study of hematopoiesis?

3) In general, the figure legends give little or no information about the experiments shown. Instead they merely describe what conclusions I am expected draw, something that is anyway already explained in the text. There are many examples:

a) In Figure 1, the reader has to guess how the different colors are generated. I assume that the green color comes from a UAS-GFP construct, driven by the C7 fat body-specific driver. An Hml:DsRed construct is apparently also involved. It should give red fluorescence to plasmatocytes. Apparently, that this has been converted to the "magenta" color, here labeled "Hemocyte" (I comment the "amber"-colored hemocytes below). Similar difficulties complicate interpretations in other figures too.

b) The units on y-axes are sometimes unclear. Does "Number of FBAHs" (Figure 1, Figure 2, and Figure 3) refer to the number per unit area as defined in Figure 1—figure supplement 1? And what is "Circulating hemocytes (F.C.)" in Figure 1, Figure 1—figure supplement 1 and Figure 5?

c) If "Hemese" (in Figure 2 and Figure 5) refers to antibody staining, for general visualization of hemocytes, then say so.

d) What is "myr-RFP" (Figure 3A and B), and what does it signify?

I will stop giving examples here, but my progress through the manuscript was slowed down dramatically by the fact that I constantly had to make guesses about what I could see in the figures.

4) How was the "amber"-coloring of hemocytes generated in Figure 1B (and elsewhere)? The text in the Materials and methods section seems to indicate that these cells were simply enhanced in this way via Photoshopping. That does give a striking effect, but is not valid evidence that these cells are attached to the fat body.

5) On a similar note, the intracellular vacuoles in Figure 3F are colored "cyan", presumably by Photoshop, to generate the impression that they are contiguous with the similarly colored "pericellular" space under the basement membrane. This is a potentially dangerous way to fool the eye. Do the authors have evidence that these systems are contiguous, or that they are related in other ways? Is there evidence that the associated electron-dense material consists of extracellular matrix proteins?

6) As shown here, several extracellular matrix proteins are enriched over Atf3-expressing cells. Does that mean that the basement membrane is thickened? Zooming in on Figure 3F I get an impression that this may be the case.

7) I find no information about how the Atf3 clones were generated for Figure 1F, and very rudimentary information about the clones in Figure 3 and Figure 5.

8) The magnification and resolution is not sufficient to show the filopodia and lamellipodia in Figure 2A, 2B, Figure 5G and 5H, even when I print out these figures over entire pages. They are even difficult to see when I zoom them in on the computer.

9) Has the C7 driver ever been described? The authors refer to a previous article from the same lab (Rynes et al., 2012), where I only find a reference back to a paper by Grönke et al., (2003). However, C7 is not mentioned there. How specific is the C7 driver, and why is it used?

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Eater cooperates with Multiplexin to drive the formation of hematopoietic compartments" for further consideration by eLife. Your revised article has been evaluated by Utpal Banerjee (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:

Your revised manuscript was evaluated by original reviewers (reviewer #1 and reviewer #2) and an additional reviewer (reviewer #4). All the reviewers agreed that now the manuscript is greatly strengthened and is ready for publication after incorporating a few minor changes as suggested by reviewer #2 and #4. Reviewer #4 recommended providing additional statistical information in Materials and methods section and applying generalized linear modeling for count data.

Reviewer #1:

With an extensive revision, the authors significantly improved the manuscript and satisfactorily addressed all my concerns. Additional data and quantitation looked convincing, and detailed descriptions of the Materials and methods section and Figure legends enhanced its readability.

Reviewer #2:

The authors have fully addressed my major concerns. I believe the work is greatly strengthened and now makes a convincing case that Mp is capable of inducing the hemocyte proliferation and differentiation in the new fat body location. The quantitation of the Mp knockdown showing the effect on sessile hemocytes has also been conducted, fully anchoring their conclusions on Mp importance for hemocyte localization at endogenous sites of hematopoiesis.

I look forward to seeing this exciting work online soon.

Reviewer #4:

This manuscript provides novel insight into the interaction of Drosophila blood cells, the hemocytes, with other tissues during the formation of sessile hemocytes clusters. The manuscript provides evidence on a key role of Multiplexin in the formation of these clusters, which, as this manuscript and previous work shows, are important sites for hemocyte proliferation and probably also differentiation. The reviewers in the first round of assessment have pointed out valid concerns regarding the manuscript and, in my opinion, the authors have done a good job addressing these concerns. I have only few remaining points/questions for the authors.

On some occasions, the number of animals tested seem quite low, especially when Drosophila material is not exactly scarce. However, I don't see this as a decisive problem in this study, one reason being that differences between controls and experimental crosses are in general quite clear. Also, multiple experiments conducted back each other up. However, could authors provide more detailed information on how experiments were replicated? Specifically, were the animals studied from the progeny of one cross/the same patch of crosses or were experiments replicated temporally? This could be clarified in the Materials and methods section since it helps in assessing the reproducibility of the results.

https://doi.org/10.7554/eLife.57297.sa1

Author response

Reviewer #1:

In this manuscript, Uhlirova and colleagues have reported a critical role for basement membrane proteins in the recruitment of hemocytes in the fat body. The authors first focused on a novel phenomenon that overexpression of Atf3 recruits hemocytes to the fat body while altering patterns of hematopoietic pockets in segmentally repeated epidermal sites. Interestingly, hemocytes attached to the fat body undergo mitosis similar to the ones found in hematopoietic pockets and exhibit normal morphologies. Moreover, hemocytes associated with the fat body relocate to the circulation upon wasp infestation, indicating that these hemocytes display a normal hematopoietic program reminiscent of sessile hemocytes. The authors further went on to understand a molecular basis for the attachment and discovered that Collagen XV/XVIII type protein Multiplexin (Mp) is required for the Atf3-mediated hemocyte homing which is dependent on the Eater expression in hemocytes. Supporting these findings, the authors validated a physical interaction between Mp and eater by biochemical experiments. Given that overexpression of Mp in the fat body disrupts the overall pattern of hematopoietic pockets and that Mp is detected in the hemocyte-free hemolymph, the authors raised a valid hypothesis that Mp can be deposited to the hemolymph critical for the systemic hemocyte homing to the sessile pockets.

This study provides a fundamental insight into molecular mechanisms underlying the hematopoietic environment essential for immune cell development and its function and prompts future studies linking the ECM landscape with the hematopoietic pocket formation and hemocyte development.

Essential revisions:

1) The precise quantitation of data and presenting proper controls will enhance the clarity of the manuscript.

1.1) Figure 1C-D': In Figure 1E, the authors quantified FBAH in imaged fat bodies. However, the number of fat body cells per one image could be variable depending on the size of the fat body, dissecting methods or imaging and so on. For example, the mean value shown in Figure 1E is different from Figure 2I even though these data represent the same genotype. It will be important to deliberate on the precise quantitation method for the FBAH phenotype as it is one of the most important findings described in the manuscript.

We expanded the Materials and methods section to accurately describe how the quantifications were performed. The area of the fat body assessed throughout the study and across the different genotypes was specifically selected because of its flat surface and absence of additional tissues that can associate with this organ (such as salivary gland in the anterior part or the gonads or tracheae at the posterior end). Furthermore, while structural variances could indeed be observed in response to manipulating various genes, such as SPARC or vkg, the overall size of the adipose tissue was not noticeably affected by co-expression of additional transgenes with UAS-Atf3WT.

Regarding the differences in FBAHs numbers in Figure 1E and Figure 2I, we consider different food composition as an important and likely source of variability in the number of FBAHs between the two datasets mentioned. The wasp infection experiments were performed in the lab of Dr. Tomas Dolezal (University of South Bohemia, Czech Republic). This means that flies/larvae were reared on different fly food than the larvae throughout the manuscript. The numbers therefore should be interpreted within the context of the particular experiment. We now describe in detail the experimental setup for the wasp infestation experiments in the Materials and methods section including the fly food composition.

1.2) The control shown in Figure 1E is identical to the one shown in Figure 2I and Figure 3—figure supplement 2E. It will be essential to ALWAYS repeat relative controls side by side with associating experiments.

The control numbers (indicated in gray) were repeated because these genotypes were dissected and quantified as parts of the same experiments to allow comparison across genotypes. To align with the order of the data presentation, the data had to be “artificially” split. We now provide additional data with separate controls for each experiment shown. In the case of Figure 3K and Figure 3—figure supplement 1I we indicate in the supporting datasets that the results belong to the same experiment.

1.3) Figure 2J-K', the number of lamellocytes and the percentage of melanotic capsule formation need to be quantified.

We now include the quantification of lamellocytes (Figure 2J). The capsule formation rate cannot be assessed as the wasp infestation experiments were performed in the lab of Dr. Tomas Dolezal (University of South Bohemia, Czech Republic) that is currently not accessible to us due to the travel ban for the University of Cologne employees due to COVID-19 pandemic. We would like to note that the data shown in Figure 2J-O was only included to indicate that lamellocyte differentiation and encapsulation is not noticeably affected in C7>Atf3WT larvae, and we did not intend to draw conclusions about the efficiency of the immune response. We adjusted the wording of the manuscript to indicate this.

1.4) In the same vein, Figure 3—figure supplement 1C-K' requires proper quantitation with enough biological replicates (n) to draw a conclusion.

We performed the quantifications which are now included in Figure 3—figure supplement 1I and reflect on the data in the text.

1.5) Figure 1A-B, Figure 3G-H, Figure 4A,G,H, Figure 4—figure supplement 1A-F, Figure 5C-D: authors have shown the relocation of sessile hemocytes. Again, it is one of the main phenotypes shown in the paper; however, none of these are quantitated. The patterns of sessile hemocytes are quite variable even amongst controls. Therefore, authors need to quantify the phenotype with their own measure and with enough sample size.

We devised a quantification method of the sessile hemocyte patterns based on image analysis. The description of the quantification method is now included in the Materials and methods section. The results are included in the relevant figures. For data shown in Figure 4—figure supplement 1A-F, we adapted relevant statements in the Results section.

1.6) In Figure 4D-E': though it has been shown in the previous study that eater is essential for the hemocyte homing and that eater is primarily expressed in hemocytes, C7-gal4, hml-gal4 (dual gal4) is a different genetic background and can cross-react. Therefore, additional controls (C7-gal4, hml-gal4/+, C7-gal4, hml-gal4; eater RNAi) are required at least to be represented as quantitation data.

We performed quantifications comparing C7>Hml>Atf3WT and C7>Hml>Atf3WTeaterRNAi and included the results in the manuscript and in Figure 4H-J.

2) It is not clear whether the overall hemocyte number (or hemocyte development) is changed upon Atf3_WT or Mp::GFP expression, which may, in turn, modify the circulating or sessile hemocyte population.

2.1) Figure 2C: To claim whether the epidermal sites are more supportive of the proliferation of hemocytes than those in the fat body, the ratio of mitotic cells needs to be quantified as shown in Makhijani et al., 2017. And without showing the number of total hemocytes, it is hard to understand which site is more prominent in the proliferation and differentiation of hemocytes.

The manuscript does not state that “the epidermal sites are more supportive of the proliferation of hemocytes than those in the fat body”, only that the epidermally associated hemocytes proliferate more than freely circulating ones, citing the data and conclusions from Makhijani et al., 2017. Our intention was to determine the behavior of hemocytes on the fat body surface, and the phenomena of hemocyte proliferation and plasmatocyte-crystal cell trans-differentiation are indicative of an unchallenged, homeostatic hemocyte developmental program.

Furthermore, we indicate in the Discussion section that differences likely exist between FBAHs and sessile hemocytes, but the mechanistic basis of their attachment is conserved.

2.2) The total number of hemocytes together with circulating hemocytes needs to be shown in Atf3_wt or Mp::GFP expression (Figure 1—figure supplement 1C, Figure 5E). Moreover, the number of circulating and total hemocytes in Atf3_WT; MpRNAi rescue background should be indicated.

The experiments combining Atf3WT and MpRNAi expression in the fat body were aimed to show that on one hand the hemocyte attachment to the fat body is dependent on Mp, and on the other hand, when hemocytes cannot attach to the adipose tissue the pattern of the sessile hematopoietic compartment is restored, for which we now include additional data in Figure 3L-O. We would also like to note that the comparison of total hemocyte numbers using the methods from Petraki et al., 2015 may not reflect the accurate values, as those are optimized for circulating and sessile cells, and not hemocytes attached to the internal organs.

3) In Figure 2E-E', it is not clear whether crystal cells express high levels of NimC1 in the images as NimC1-positive membranes are anyway juxtaposed. It will be great to have a bleeding of FABH or high mag of crystal cells with better resolution.

We exchanged the panels in Figure 2E-E’ to better show the colocalization of the Bc:GFP signal and the NimC1 staining on the intermediate hemocytes.

4) Though authors have shown that FBAHs are relocated to the circulation upon wasp parasitism, these data are not sufficient to conclude that the fat body FBAH follows the identical hematopoietic program to sessile blood cells. It is possible that FBAHs relocation is less frequent than regular sessile hemocytes found in the epidermis/neuron due to the tight association. It will be important to show whether FBAHs can be reattached after physical disturbances as shown in Makhijani et al., 2011.

We did not intend to suggest that FBAHs and sessile hemocytes behave “identically”, rather that tissue/fat bodyattached hemocytes seem to behave in a similar fashion. To reflect this more accurately, we changed the wording of the manuscript. On a second note, similarly to Essential revisions 2.2, the physical mobilization assay described in Makhijani et al., 2011 was specifically created to release sessile hemocytes which adhere to the epidermis and are sensitive to external force (e.g. brush strokes, vortexing). FBAHs on the other hand are on the surface of the adipose tissue, which is floating in the hemolymph, and are therefore much less prone to these insults.

Reviewer #2:

This very well written and interesting manuscript addresses the clinically relevant question of what molecules drive immune cells' interaction with environments that promote their proliferation and differentiation. The authors use the basic system of Drosophila and provide convincing evidence that an ECM component, Multiplexin, is necessary for the recruitment of Drosophila immune cells (hemocytes) to the previously identified hematopoietic pockets and for their recruitment to a new position on the fat body where they are can divide and differentiate as they do in the native environment. They define the phagocytosis receptor Eater, which has previously been found to be crucial for hemocyte localization to the hematopoietic pockets, as the partner in mediating hemocyte binding to Multiplexin in both locations. I think this work is exciting and mostly well conducted and deserves to be published in ELife, with some alteration of the text to reflect that some of the current conclusions require a modest amount of new data and analysis.

1) The paper in places implies that Multiplexin is capable of redirecting hemocytes to a new location in which they can function as in the hematopoietic pocket. This finding would be extremely exciting if true. But though the authors show relocalization to the fat body in the experiment in which they induce clones of Mp (Figure 5G-H), they do not assess the division and differentiation capacity of the hemocytes there. Thus Atf3 expression may induce other changes in the adipocytes than just Mp expression to allow Fat Body associated hemocytes (FBAH) to divide and differentiate there (as seen in Figure 2C-F); Mp may thus be required for hemocyte adhesion yet not sufficient to induce all niche dependent functions. To address sufficiency for proliferation the authors could do a pH3 staining upon the induction of clones in strains they already have (those used in Figure 5G-H). To address sufficiency for differentiation, they could put one copy of Bc1 into that background.

Alternatively they should alter the paper to make clearer that they have not explicitly investigated if or shown that the ECM component, Mp, drives hematopoiesis in the Title, the Abstract, the Introduction, the Discussion section (specifically "indistinguishable").

We now include experiments demonstrating the cell proliferation as well the presence of crystal cells among hemocytes attached to Mp::GFP overexpressing fat body clones (Figure 6C-E). We would like to add that while hemocyte division does take place in the clusters attached to the Mp::GFP clonal cells, the amount of attached hemocytes and the size of the clones vary among experimental replicates making the quantification of these phenomena problematic.

Furthermore, we include new data showing that Mp::GFP overexpression in the wing imaginal disc, also causes the specific attachment of hemocytes, further strengthening the evidence for the crucial role of Mp to facilitate hemocyte-tissue association (Figure 6F-G). Additionally, we reworded the cited conclusions to more appropriately describe the phenotypes observed.

2) They conduct experiments to assess the importance of Mp for attachment to the endogenous sessile niches. Their conclusion (subsection “Similar mechanisms drive sessile hematopoietic pocket formation and FBAH adhesion”) that the knockdown of Mp by RNAi produces a phenotype strikingly resembling that seen in the eater1 mutant (and thus that Mp is required for all attachment to endogenous hematopoetic environments) doesn't seem supported by the comparison of Figure panels 4A' to 4H'. While the dorsal patches seem to be gone in the Mp knockdown, it appears as if there is still a repeated hemocyte pattern on the lateral sides in the larvae shown. Having a close up of the lateral and dorsal niches for these genotypes instead of the GFP channel (whose relevance as a marker of the driver pattern is also not explained in the figure legends) would clarify this issue. It is clear there is a strongly reduced attachment in the absence of Mp, but if this the same in all the different endogenous hematopoetic locations is important to determine. Given that Mp has been reported to be expressed in the heart tube, next to where the dorsal patches are, and not to my knowledge near the PNS, which flanks the lateral ones, would fit with there being a variable effect in the different regions. This Figure has no quantitation, which also weakens this conclusion. If they do not have the data in hand to make the new figure/ do the quantitation, then they should soften the conclusion that this is the same as the eater1 phenotype and address textually that the effect on the lateral patches has not been rigorously assessed.

The experiments showing the disruption of the sessile compartment are now complemented with additional images, showing closeups of the dorsal and lateral patches (Figure 4N-O). Additionally, to provide better representation for the sessile tissue pattern, we now include quantifications for the relevant genotypes, and compare the stereotypical sessile tissue pattern with larvae where the compartment is disrupted (Figure 4K-M). We would like to note that on the overview images, it is possible to interpret the hemocytes as “repeated hemocyte pattern”, but in reality, this is due to the immobilization (cooling the larvae) and the settling of the circulating hemocytes. Since the contractions of the dorsal vessel diminish in low temperature, the cells in the circulation can accumulate between the muscle and epidermal layers, creating the impression of a repeated pattern (Figure 4N-O).

Reviewer #3:

I believe this is a great paper, with exciting science, but I find it unnecessarily difficult to read and to assess critically. I was easily distracted while reading it and I may have missed important points.

Essential revisions:

1) Conceptually, it would be helpful to give the reasons for choosing to overexpress Atf3 in the fat body. I would also like to see more background information about this transcription factor in the Introduction.

See below.

2) I also miss a discussion about the possible physiological role of the system described here. Is Atf3 also involved in defining hemocyte docking sites elsewhere? Or should I regard Atf3 overexpression as an artificial but useful experimental system for the study of hematopoiesis?

The use of Atf3 was based on our previous observation of the hemocyte attachment to the fat body. The overexpression of Atf3 in the fat body at high levels is indeed artificial, and our goal was to exploit this as a platform to understand the fundamental mechanisms underlying hemocyte attachment to Drosophila tissues in an accessible manner. As the reviewer suggested, we now include additional background about Atf3 in the leading paragraph of the Result section.

3) In general, the figure legends give little or no information about the experiments shown. Instead they merely describe what conclusions I am expected draw, something that is anyway already explained in the text. There are many examples:

a) In Figure 1, the reader has to guess how the different colors are generated. I assume that the green color comes from a UAS-GFP construct, driven by the C7 fat body-specific driver. An Hml:DsRed construct is apparently also involved. It should give red fluorescence to plasmatocytes. Apparently, that this has been converted to the "magenta" color, here labeled "Hemocyte" (I comment the "amber"-colored hemocytes below). Similar difficulties complicate interpretations in other figures too.

b) The units on y-axes are sometimes unclear. Does "Number of FBAHs" (Figure 1, Figure 2, and Figure 3) refer to the number per unit area as defined in Figure 1—figure supplement 1? And what is "Circulating hemocytes (F.C.)" in Figure 1, Figure 1—figure supplement 1 and Figure 5?

c) If "Hemese" (in Figure 2 and Figure 5) refers to antibody staining, for general visualization of hemocytes, then say so.

d) What is "myr-RFP" (Figure 3A and B), and what does it signify?

I will stop giving examples here, but my progress through the manuscript was slowed down dramatically by the fact that I constantly had to make guesses about what I could see in the figures.

We expanded the Figure legends and the Materials and methods section considerably to facilitate reading, including the specific suggestions of the reviewer.

4) How was the "amber"-coloring of hemocytes generated in Figure 1B (and elsewhere)? The text in the Materials and methods section seems to indicate that these cells were simply enhanced in this way via Photoshopping. That does give a striking effect, but is not valid evidence that these cells are attached to the fat body.

We added the description to the Materials and methods section. Briefly, larvae were fixed with the injection of 4% paraformaldehyde, immobilized on microscope slides and image with confocal microscope. Each individual confocal section was examined for GFP expression (fat body) and Hml:DsRed expression (hemocyte). The DsRed channel was locally recolored on each section when it was determined to overlap with the GFP signal. The attachment of the hemocytes is also demonstrated throughout the study on dissected fat bodies (e.g. Figure1—figure supplement 1).

5) On a similar note, the intracellular vacuoles in Figure 3F are colored "cyan", presumably by Photoshop, to generate the impression that they are contiguous with the similarly colored "pericellular" space under the basement membrane. This is a potentially dangerous way to fool the eye. Do the authors have evidence that these systems are contiguous, or that they are related in other ways? Is there evidence that the associated electron-dense material consists of extracellular matrix proteins?

We based our assessment on two previous publications, which used similar indication for the pericellular spaces, and concluded that the trapped material consists of ECM proteins. The representative Figure from Zang et al., 2015, shows the same type of distinction between pericellular spaces and cytoplasm/organelles, and points out the electron dense material as ECM protein aggregates.

6) As shown here, several extracellular matrix proteins are enriched over Atf3-expressing cells. Does that mean that the basement membrane is thickened? Zooming in on Figure 3F I get an impression that this may be the case.

We include a closeup image Author response image 1 to compare the two genotypes. According to our measurements in both genotypes the average thickness of the BM is 65-85 nms, which also corresponds to data published in Dai et al., 2017 (Figure S1B). What can create the impression of thicker BM is the proximity of the cytoplasm in C7>Atf3WT fat bodies. In controls the pericellular space between the cell membrane and the BM is considerably wider.

Author response image 1
Transmission electron micrographs of control (left) and Atf3 overexpressing (right) adipocytes.

Transgene and GFP expression was driven by the fat bodyspecific C7-GAL4 driver. The average thickness of the BM is 65-85 nms in both genotypes. Basement membranes are indicated with arrowheads Scale bars: 2 μm.

7) I find no information about how the Atf3 clones were generated for Figure 1F, and very rudimentary information about the clones in Figure 3 and Figure 5.

We generated the clones using the hsFLPout system. We now include a description in the Materials and methods section and add description into respective Figure legends.

8) The magnification and resolution is not sufficient to show the filopodia and lamellipodia in Figure 2A, 2B, Figure 5G and 5H, even when I print out these figures over entire pages. They are even difficult to see when I zoom them in on the computer.

We now include higher magnification images of these samples with the filopodia and lamellipodia indicated.

9) Has the C7 driver ever been described? The authors refer to a previous article from the same lab (Rynes et al., 2012), where I only find a reference back to a paper by Grönke et al., (2003). However, C7 is not mentioned there. How specific is the C7 driver, and why is it used?

The C7-GAL4 is a strong fat body-specific driver expressed in the fat body cells from early larval stages. A detailed description of the driver can be found in Koyama and Mirth, 2016, which we now refer to in the manuscript.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Reviewer #4:

This manuscript provides novel insight into the interaction of Drosophila blood cells, the hemocytes, with other tissues during the formation of sessile hemocytes clusters. The manuscript provides evidence on a key role of Multiplexin in the formation of these clusters, which, as this manuscript and previous work shows, are important sites for hemocyte proliferation and probably also differentiation. The reviewers in the first round of assessment have pointed out valid concerns regarding the manuscript and, in my opinion, the authors have done a good job addressing these concerns. I have only few remaining points/questions for the authors.

On some occasions, the number of animals tested seem quite low, especially when Drosophila material is not exactly scarce. However, I don't see this as a decisive problem in this study, one reason being that differences between controls and experimental crosses are in general quite clear. Also, multiple experiments conducted back each other up. However, could authors provide more detailed information on how experiments were replicated? Specifically, were the animals studied from the progeny of one cross/the same patch of crosses or were experiments replicated temporally? This could be clarified in the Materials and methods section since it helps in assessing the reproducibility of the results.

All experiments represent at least two independent temporal replicates. We now include the sentence in Materials and methods section.

https://doi.org/10.7554/eLife.57297.sa2

Article and author information

Author details

  1. Gábor Csordás

    Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
    Contribution
    Conceptualization, Resources, Data curation, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
    For correspondence
    cgabor@uni-koeln.de
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6871-6839
  2. Ferdinand Grawe

    1. Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
    2. Molecular Cell Biology, Institute I for Anatomy, University of Cologne Medical School, Cologne, Germany
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  3. Mirka Uhlirova

    Institute for Genetics and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Writing - original draft, Project administration, Writing - review and editing
    For correspondence
    mirka.uhlirova@uni-koeln.de
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5735-8287

Funding

Deutsche Forschungsgemeinschaft (UH 243/3-1)

  • Mirka Uhlirova

Deutsche Forschungsgemeinschaft (EXC 2030 - 390661388)

  • Mirka Uhlirova

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Talila Volk, Michael R Kanost, Bruno Lemaitre, István Andó, the Bloomington Drosophila Stock Center supported by NIH grant NIH P40OD018537 (BDSC, Bloomington, IN, USA), the Drosophila Genomics Resource Center supported by NIH grant 2P40OD010949 (DGRC, Bloomington, IN, USA), the Vienna Drosophila Resource Center (VDRC, Vienna, Austria), and the Developmental Studies Hybridoma Bank (DSHB, Iowa City, IA, USA) for fly stocks, plasmids, cell line, and antibodies. We are grateful to Tomáš Doležal and Pavla Nedbalová for access to and sharing experience with the parasitoid wasp infection model at the University of South Bohemia (Ceske Budejovice, Czech Republic). We also thank Marek Jindra (Biology Center CAS, Czech Republic) and the Bioscience Imaging and Histology Unit of the Institute of Entomology (Biology Center CAS, Czech Republic) for microscope access. We are grateful to Steffen Erkelenz for advice on cloning and immunoprecipitation experiments, Nils Teuscher and Tina Bresser for excellent technical assistance, and the entire Uhlirova laboratory for discussion. This work was funded by UH 243/3–1 project to M.U and under Germany's Excellence Strategy – CECAD, EXC 2030 – 390661388 both from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation).

Senior Editor

  1. Utpal Banerjee, University of California, Los Angeles, United States

Reviewing Editor

  1. Jiwon Shim, Hanyang University, Republic of Korea

Reviewer

  1. Dan Hultmark, Umeå University, Sweden

Publication history

  1. Received: March 26, 2020
  2. Accepted: September 18, 2020
  3. Version of Record published: October 7, 2020 (version 1)

Copyright

© 2020, Csordás et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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