Two Independent Functions of Collier/Early B Cell Factor in the Control of Drosophila Blood Cell Homeostasis

Blood cell production in the Drosophila hematopoietic organ, the lymph gland, is controlled by intrinsic factors and extrinsic signals. Initial analysis of Collier/Early B Cell Factor function in the lymph gland revealed the role of the Posterior Signaling Center (PSC) in mounting a dedicated cellular immune response to wasp parasitism. Further, premature blood cell differentiation when PSC specification or signaling was impaired, led to assigning the PSC a role equivalent to the vertebrate hematopoietic niche. We report here that Collier is expressed in a core population of lymph gland progenitors and cell autonomously maintains this population. The PSC contributes to lymph gland homeostasis by regulating blood cell differentiation, rather than by maintaining core progenitors. In addition to PSC signaling, switching off Collier expression in progenitors is required for efficient immune response to parasitism. Our data show that two independent sites of Collier/Early B Cell Factor expression, hematopoietic progenitors and the PSC, achieve control of hematopoiesis.


Introduction
Understanding how the balance between progenitor cell maintenance and differentiation is regulated in developing and adult tissues is a major area of investigation. The Drosophila larval hematopoietic organ, the lymph gland, is one established model to study the signalling mechanisms controlling this balance, under normal developmental and immunological challenging conditions [1][2][3][4][5][6][7]. Under normal developmental conditions, two types of blood cells (called hemocytes) differentiate in the lymph gland, the plasmatocytes endowed with phagocytic UAS-reaper and UAS-Dicer2 (Bloomington Stock Center). Larvae were raised, in uncrowded conditions from eggs collected for 8 or 12 hours, at 27°C except for RNAi interference experiments which were performed at 29°C. UAS-Dicer2 was co-expressed with RNAi constructs. Larvae were developmentally staged at 29°C as follows: early third larval instar (EL3): 66H after egg laying (AEL); mid third larval instar (ML3): 85H AEL, late third larval instar (LL3): 105H AEL.

Measurement of crystal cell, plasmatocyte and mitotic indexes
Optimized confocal sections were performed with Leica SP5 for 3D reconstructions. All quantifications were performed using the Volocity software and are representative of at least three independent experiments. Statistical analyses t test (Mann-Whitney nonparametric test) was performed using GraphPad Prism 5 software. Crystal cell and plasmatocyte indexes were displayed as the number of proPO-or Hnt-positive cells and the volume of P1 staining, per primary lobe relative to the lobe's volume. A minimum of 20 lymph glands were scored per genotype.

Wasp parasitism
Early third instar larvae were subjected to 1 H of egg-laying by wasps. The non-immune suppressive line of the wasp Leptopilina boulardi (NIS or G486 strain) was used [35]. For the lymph gland dispersal assay, larvae were dissected at the appropriate time, without prior fixation and lymph glands were analyzed for anterior lobe integrity. Anterior lobes showing no hemocyte dispersal were considered as intact anterior lobes. Un-parasitized or over-parasitized larvae were excluded from quantifications.

Results
Low levels of col expression in lymph gland anterior lobes define a submedullar zone In wild type (wt) second larval instar lymph glands, both in situ hybridization and immunostaining indicated that all cells of the anterior lobes express low levels of col (Fig 1A, 1E and 1E'), in addition to high levels in the PSC and stochastic expression in the secondary lobes [10]. At this stage, primary lobe cells expressed dome-Gal4>mCD8-GFP (dome>GFP), a reporter insertion in domeless, which encodes the receptor of the JAK/STAT signalling pathway ( Fig  1E) [5,11]. In early third instar larvae, when hemocyte differentiation starts to form a cortical zone (CZ), both col mRNA and Col protein become restricted to the innermost part of the dome>GFP population of progenitors (Fig 1B and 1H), which displays a pseudo-epithelial structure [5]. In mid third instar larval lymph gland, col expression in the medullary zone (called col MZ hereafter) overlaps with the expression domain of the thioester-containing protein 4 (tep4) (Fig 1F and 1F') [36], which marks a sub-set of hemocyte progenitors in the medullary zone (Fig 1G and 1G'). In late third instar larvae, very low levels of col expression could be detected in medullary zone cells ( Fig 1D). In order to better characterize col MZ progenitors, we examined the cell division pattern in early and mid third instar larval lymph glands, by costaining for Col, dome>GFP and the mitotic cell marker H3P (phosphorylated histone H3). Active proliferation of dome>GFP progenitors in early third instar larvae was previously shown to contribute most of primary lobe growth [12,15]. In early third instar larval lymph gland, we found that the col MZ domain concentrates 88% of the H3P positive cells, while representing only 38% of the total volume of the medullary zone ( Fig 1I). This distribution showed that col MZ progenitors actively divide in early third instar larval lymph gland. In mid third instar larval lymph gland, 58% of H3P positive cells are localized in the col MZ domain ( Fig 1I). All together, these data revealed the existence of a core population of proliferating blood cell progenitors in the third instar larval lymph gland, which co-express col and tep4.

Col MZ prevents hematopoietic progenitors from differentiating
In order to address col function in the medullary zone, we examined progenitor and differentiated hemocyte populations when col expression was knocked down specifically in medullary zone cells, using RNA interference (dome>icol). In third instar larvae, we found that the population of dome>GFP cells decreased prematurely in dome>icol lymph glands (Fig 2A and 2B), with only a few GFP-positive cells persisting up to the end of larval development (Fig 2K and  2L). In parallel to the loss of GFP expression, col knock down in the medullary zone lead to the loss of the medullary zone epithelial-like structure (Fig 2C and 2D). Furthermore, the expression domain of two progenitor cell markers, latran (lat)/eye transformer (et) and tep4, was drastically reduced (Fig 2E-2H), supporting the conclusion that col MZ expression is required for progenitor identity.
To determine whether the loss of progenitor markers and medullary zone shaping when col is knocked down in the medullary zone impacted hemocyte differentiation, we stained dome>icol lymph glands for plasmatocyte and crystal cell markers, Cg25C-GFP [30] and proPO [11], respectively. In wild type third instar lymph gland, expression of these markers was restricted to the cortical zone ( Fig 2I). By contrast, when col was knocked down in the medullary zone, the innermost part of the anterior lobes already showed signs of differentiation at mid third larval instar (Fig 2J). Quantification of differentiated hemocytes in anterior lobes revealed a two-fold increase in both plasmatocyte and crystal cell numbers in early third larval instar when col expression was knocked down in the medullary zone compared to wild type In late third instar larval lymph glands, the bi-zonal organization of the anterior lobes was lost and differentiated hemocytes populated the entire anterior lobes, at the expense of progenitors (Fig 2K and 2L). However, no obvious differentiation defect could be associated in second instar larval lymph glands when col expression was knocked down in the medullary zone, indicating that other developmental signals are required for hemocyte differentiation (S1A, S1B and S1E-S1F' Fig). Whereas in control lymph glands, some lamellocytes (less than 10) differentiate in few lymph glands (18%, n = 49), most lymph glands lacking col in the medullary zone (75%, n = 52) develop lamellocytes as in control ( Fig  2M and 2N). Finally, there was no change in expression of either signaling (hhF4fGFP) (S2A and S2B Fig) or identity (Antp, Col) markers (S2C, S2D, S1G and S1H Figs) of the PSC in dome>icol lymph glands, indicating that hemocyte differentiation upon removal of col in the medullary zone is not a secondary consequence of a PSC identity defect. Together, these data revealed a previously unnoticed, cell-autonomous function of col in blood cell progenitors in preserving their identity throughout third instar.

Col MZ and the PSC independently regulate distinct facets of lymph gland homeostasis
Enhanced hemocyte differentiation was previously reported in both col mutant larvae [11] and hh ts mutants [13], and interpreted as a consequence of PSC loss or of impaired hh expression in PSC cells, respectively. Enhanced hemocyte differentiation reported here upon col depletion in the medullary zone raised the question of whether col MZ expression was controlled by Hh issued from the PSC. The GATA factor Serpent (Srp) controls hh expression in the PSC [31]. We therefore suppressed hh expression in PSC cells by downregulating srp using the PSC driver pcol-Gal4 (col>isrp). In agreement with previous reports [13,31], we confirmed that loss of hh expression in the PSC, visualized by the hh transcriptional reporter hhF4fGFP [31] (S3A and S3B Fig), caused a significant increase in crystal cell and plasmatocyte differentiation compared to wild type, confirming that the PSC regulates the rate of hemocyte differentiation ( Fig  3A-3F). However, increased hemocyte differentiation remained restricted to the periphery of the lymph gland, unlike in lymph glands lacking col in the medullary zone (compare Figs 3B with 2J and 2L). We then looked at expression of both col in the medullary zone (Fig 3G and  3H) and domeMESOGFP, a marker of JAK/STAT positive-progenitors, (Fig 3I-3K) and found that neither was lost in third instar col>isrp lymph glands. Altogether, our data indicate that col expression in the medullary zone and its function in those cells are independent of hh expressed by PSC cells.
To further investigate a possible regulation of col MZ expression by the PSC, we genetically modified the PSC size. PSC cell number was reduced from an average of 40 to less than 5 cells per lobe by antagonizing Wg signaling in the PSC, through expression of a dominant-negative form of TCF/pangolin(pan) (col>TCF DN ) (S3E Fig) [20,21,26]. Quantification of cell differentiation in this genetic context revealed a reproducible, mild reduction of crystal cell number in anterior lobes (Fig 3L-3N). In contrast, neither col MZ (Fig 3O and 3P) nor domeMESOGFP expression (Fig 3Q-3S) was detectably modified. In a reciprocal experiment, we overexpressed the proto-oncogene Myc in PSC cells (col>Myc), which leads to strong increase of both the PSC cell number and the pool of domeMESO-positive medullary zone progenitors in anterior lobes, at the expense of differentiated hemocytes [20]. We found that col MZ expression was not positive and -negative progenitors in early (EL3) and mid (ML3) third instar larvae. In EL3 LGs, 88 ± 8% are within the dome>GFP/Col co-expression domain (red), the other cells (green) are dome>GFP positive. In ML3 LGs, 58 ± 6% are within the Col expressing domain (red). Vertical bars correspond to SD. Scale bars: 40 μm.
doi:10.1371/journal.pone.0148978.g001 noticeably modified in this background (S3C and S3D Fig). Thus, altering PSC cell number, and thereby overall PSC signaling, does not significantly impact col MZ expression. From these data, we conclude that col MZ expression is independent of the PSC, and that col expressed in the medullary zone and in the PSC control blood cell homeostasis in the lymph gland, by regulating progenitor maintenance and hemocyte differentiation, respectively.
Switching off col expression in the medullary zone upon parasitism is independent of the PSC Wasp parasitism provokes a phase of active proliferation in early third instar larval lymph glands, followed by massive lamellocyte differentiation, at the expense of progenitor maintenance [11,12]. We found that col MZ expression was specifically lost 6H post-parasitism, while col remained strongly expressed in PSC cells (Fig 4A and 4B) [10,28]. This observation raised the possibility that silencing col MZ activity was a pre-requisite for massive lamellocyte differentiation. To test this possibility, we artificially forced col expression in the medullary zone (dome>col), in larvae that were wasp parasitized at early third instar. While dispersal of lymph glands and release of lamellocytes into circulation was robustly observed in wild type larvae 24H post-parasitism, it was only observed in 40% of dome>col lymph glands parasitized larvae ( Fig 4C). Furthermore, only few differentiated lamellocytes were observed at the cortex of dome>col lymph gland anterior lobes which did not disperse 24H post-parasitism (Fig 4D and  4E). We conclude that down-regulation of col expression in the medullary zone following wasp infestation is a pre-requisite for efficient differentiation of lamellocytes in the lymph gland and their release into the hemolymph upon lymph gland dispersal.
The question remained as to whether col expression in the medullary zone endowed progenitor cells with the ability to differentiate into lamellocytes. To address this question, we subjected to wasp parasitism larvae in which col MZ was downregulated from second instar larval stage (dome>icol), and analyzed lymph glands 24H post-parasitism. We found that col knock down in the medullary zone lead to lymph gland dispersal, similar to wild type ( Fig 4C). Staining with anti-αPS4, a lamellocyte-specific marker [11], confirmed that lamellocytes differentiated massively in the lymph gland as in wild type (Fig 4D and 4F). Thus, col MZ expression is not required for cell fate specification of blood cell progenitors into lamellocytes upon parasitism.
Our previous studies identified the PSC as essential for lymph gland responsiveness to parasitism [10,11]. Indeed, massive differentiation of lamellocytes did not occur in col mutant lymph glands where the PSC was not specified [10]. Neither did it occur in wild type larvae where PSC cells were ablated by targeted expression of reaper (rpr), a cell-death inducer [11]. Our finding that col MZ was downregulated in response to parasitism thus opened the possibility that this regulation was dependent on PSC function. To test this hypothesis, we eliminated PSC cells (col>rpr) in larvae (S4A and S4B Fig), which were then subjected to parasitism before examining lymph glands. Although lymph glands lacking a PSC rarely dispersed 24H post-parasitism (Fig 4I), col MZ was notably downregulated 6H post-parasitism in col>rpr lymph glands, similar to wild type (Fig 4G and 4H). Thus, the down regulation of col MZ expression in blood cell progenitors upon parasitism does not require the PSC. Moreover, these data indicate that progenitor cells respond directly to immune cues.

Discussion
Here, we characterize a novel core population of blood cell progenitors in the Drosophila lymph gland that expresses col, where it plays an essential, cell-autonomous function in preventing hematopoietic progenitors from differentiating. The same conclusion was reported recently by another team, using similar experiments [37]. Maintenance of progenitors was previously assigned to col expression in the PSC [11,13]. Here, we further redefine the PSC function in normal developmental conditions, which is to regulate the rate of blood cell differentiation, and we confirm its requirement for lamellocytes production and lymph gland dispersal upon wasp parasitism. We further show that switching off col expression in the medullary zone is also required for lymph gland response to parasitism, independently from PSC activity. Thus, two independent Col expression sites in the lymph gland, in core hematopoietic progenitors and the PSC, control Drosophila larval hematopoiesis under normal developmental  col expression in the medullary zone defines a sub-population of lymph gland proliferating progenitors Our data confirm that, in addition to the PSC and posterior lobes, col is expressed in a sub-population of progenitors in the medullary zone [10,37]. Hence, the medullary zone population is heterogeneous, as it contains both col-positive and -negative hematopoietic progenitor cells. In addition, we show that progenitors expressing Col actively proliferate and could contribute to the extensive lymph gland growth that occurs in early third instar larvae, while outer medullary zone, col-negative cells could correspond to intermediate progenitors primed to differentiate and contributing to hemocyte differentiation along lymph gland maturation. The persistence of progenitors expressing col in anterior lobes up to metamorphosis raises the question of their fate beyond larval development. We have previously shown that col was expressed in a fraction of cells in posterior lymph gland lobes that do not show signs of hemocyte differentiation in normal developmental conditions [10], and we proposed that posterior lobes could be reservoirs of precursors for adult hemocytes [38], one possibility recently supported by lineage tracing experiments [39]. Our finding of a pool of col-expressing progenitors in the medullary zone raises the question of the respective contributions of these and posterior lobe cells to adult hematopoiesis.
We previously showed that forced col expression in the entire medullary zone prevented hemocyte differentiation [11]. This study, in addition to data presented by Benmimoun et al. (2015), indicating that col expression in the medullary zone maintains progenitors in third instar lymph glands, shows that the restriction of col expression to a core medullary zone progenitors that occurs in early third instar larvae allows Col negative progenitors to progress towards differentiation. In addition, our study shows that forcing col expression in progenitors prevents them from differentiating massively into lamellocytes in response to parasitism. This finding disagrees with the conclusion raised by Benmimoun et al. (2015) that Col overexpression does not affect lamellocyte differentiation in response to wasp parasitism although the extent of differentiation was not quantified. While varying levels of ectopic col expression in progenitors could possibly explain this discrepancy, the notable defect of lymph glands dispersal upon parasitism strengthens the conclusion that removal of col expression in the medullary zone is required for massive lamellocyte differentiation. Various genes and signalling pathways have been shown to regulate progenitor cell maintenance versus differentiation in the medullary zone [2,13,16,18,19,21,23,25,31,[40][41][42][43][44][45][46][47][48][49]. Integrating the regulation of col expression in the medullary zone into regulatory networks controlling blood cell homeostasis is an important line of future investigation.

Contrasting roles of the PSC under normal and parasitism conditions
Modifying the PSC cell number and/or gene expression program showed that the PSC, while not required to maintain col MZ progenitors, regulates hemocyte differentiation. Reducing srp expression, thereby hh production, in PSC cells favors crystal cell and plasmatocyte differentiation (Fig 3) [13,16]. Conversely, increasing levels of PSC signals by genetically increasing cell numbers reduce hemocyte differentiation [20,21,23,24,26,37]. Paradoxically, reducing drastically PSC cell number can also result in decreased crystal cell differentiation without affecting blood cell progenitor maintenance (Fig 3). Reduced crystal cell number with no progenitor maintenance defect was also reported in PSC-ablated lymph glands [37]. This data supports our conclusion that the PSC regulate hemocyte differentiation in the lymph gland. How can one reconcile this apparent paradox? We hypothesize that hemocyte homeostasis integrates positive and negative differentiation signals issued from the PSC. Altering one signal at a time could thus affect PSC cell number and/or signaling ability, in the same or opposite ways. One example is reducing col expression in the PSC specifically in larvae, which both increases PSC cell proliferation and decreases hh signalling with no or little consequence on hemocyte differentiation [20]. How can the PSC regulate blood cell differentiation independently of progenitor maintenance? Many cells in third instar larval lymph glands show neither high levels of medullary zone gene expression nor expression of hemocyte differentiation genes, suggesting that they most likely represent an heterogeneous cell population encompassing intermediate, faterestricted progenitors and precursors committed to differentiate [15,16,42,50]. One attractive hypothesis is that PSC signaling regulates the differentiation of this overlooked pool of intermediate lymph gland cells, a subject of future investigation.
The PSC plays an essential role in mounting a cellular immune response to wasp parasitism (this work and [10,11,27,37]). Our present data further establish that silencing col expression in the medullary zone is also essential for this response, independently from PSC activity ( Fig  4). Thus, our study establishes, for the first time, that several signals, PSC-relayed and PSCindependent, must be integrated by medullary zone cells for an effective immune response, pointing to a more integrated process than previously thought for fighting wasp parasitism. A previous study showed that switching off JAK/STAT signalling in medullary zone cells was necessary for massive lamellocyte production upon wasp parasitism [34], suggesting crosstalks between JAK/STAT signalling and the regulation of col expression in the medullary zone.

Conservation of Col/EBF functions during hematopoiesis
Four mammalian Col orthologs, EBF1-4 have been characterized [51]. EBF1, initially characterized for its role in immature B-Cell progenitors [52], regulates differentiation, stage-specific signaling, proliferation, and survival of B cells [53]. Mouse EBF2 was later shown to be expressed in immature osteoblasts, one component of the endosteal HSC niche in the adult bone marrow [54]. Thus, different EBFs control different aspects of vertebrate hematopoiesis and immune cell differentiation. The different roles of Col in the Drosophila lymph gland provide a framework for investigating the evolution of COE (Collier/EBF) hematopoietic functions throughout the animal kingdom.