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Disease Models & Mechanisms 4, 439-445 (2011) doi:10.1242/dmm.007351 PRIMER

Drosophila: a model for studying genetic and molecular aspects of and associated leukaemias

Michèle Crozatier1,* and Alain Vincent1,*

or short-term (ST) HSCs] (Wilson et al., haematopoietic stem cells (HSCs) give rise to a hierarchically organised 2008; Lo Celso et al., 2009; Xie et al., 2009). set of progenitors for erythroid, myeloid, lymphoid and lineages, Primed HSCs provide ongoing support for and are responsible for lifelong maintenance of the system. Dysregulation the daily normal regeneration of billions of of the haematopoietic differentiation programme is at the origin of numerous blood cells, whereas dormant HSCs that have pathologies, including leukaemias. With the discoveries that many transcriptional maximal, long-term reconstitution potential regulators and signalling pathways controlling development are are mobilised upon marrow injury conserved between humans and , the fruit fly has become (Wilson et al., 2008; Trumpp et al., 2010). Similarly to , Drosophila a good model for investigating the mechanisms underlying the generation of blood haematopoiesis occurs in two spatially and cell lineages and blood cell homeostasis. In this review article, we discuss how temporally distinct phases (Crozatier and

DMM genetic and molecular studies of Drosophila haematopoiesis can contribute to our Meister, 2007). The Drosophila life cycle understanding of the haematopoietic niche, as well as of the origin and/or comprises an embryonic stage, three larval progression of haematopoietic malignancies in humans. stages and metamorphosis, which gives rise to an adult. The first embryonic phase provides two types of circulating blood cells (haemocytes) – the plasmatocytes and the Transient and definitive adult . There is a rapid and crystal cells (see below) – both of which haematopoiesis in mammals and permanent perinatal shift of the entire subsist in the larva either in the haemolymph Drosophila to the bone marrow or attached to the inner epidermis in the case Vertebrate haematopoietic stem cells (HSCs) (Mikkola and Orkin, 2006; Dzierzak and of sessile haemocytes (Holz et al., 2003; Honti ensure continuous haematopoietic cell Speck, 2008). et al., 2010). The second phase of production throughout life. HSCs can be The decisions of HSCs to self-renew or haematopoiesis takes place in larvae, in a defined by their dual ability to self-renew, differentiate, and to become quiescent or specialised haematopoietic organ known as thereby maintaining their numbers, and to proliferate, are tightly controlled by the lymph gland (LG), which is situated along differentiate into all lineages of the blood integrating extrinsic cues provided by the the anterior part of the dorsal aorta (Lanot and immune system (Dykstra et al., 2007). niche. The concept of the niche was coined et al., 2001) (Fig. 1A). The LG contains Disease Models & Mechanisms Blood cells first appear in the mammalian more than 30 years ago to describe the haematopoietic progenitors (pro- conceptus, in the extraembryonic yolk sac, structural and regulatory microenvironment haemocytes) for three types of functional concomitant with the developing that sustains long-term renewal of bone haemocytes: first, the plasmatocytes, which vasculature. These early haematopoietic marrow HSCs (Schofield, 1978). Recent are -like cells involved in progenitors are, however, of more limited studies, including the successful detection of of apoptotic bodies and potency than adult HSCs. In mice, HSCs that HSCs in the bone marrow of live animals by pathogens, and, second, the crystal cells, confer complete, long-term, multilineage using real-time imaging technology, support which are required for melanisation, an haematopoietic repopulation of irradiated the view that there are two subpopulations insect-specific immune and wound-healing recipient mice first appear at embryonic day of HSCs: one that is quiescent [also referred response. These two haemocyte types are (E)10.5 in the aorta-gonad-mesonephros to as dormant, reserved or long-term (LT) released in the haemolymph upon dispersal (AGM) region of the embryo body and the HSCs], and the other that is more actively of the LG at the onset of metamorphosis. The , and they seed the fetal and the cycling [also referred to as primed, activated LG also gives rise to a third type of haemocyte, the lamellocytes, which are devoted to encapsulation of foreign bodies 1 Centre de Biologie du Développement, UMR 5547 CNRS/Université Toulouse III, 118 route de Narbonne 31062 that are too large to be phagocytosed. Toulouse cedex 9, France *Authors for correspondence ([email protected]; [email protected]) Lamellocytes do not differentiate in normal © 2011. Published by The Company of Biologists Ltd developmental conditions but only in This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share response to specific immune challenges such Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), which permits unrestricted non-commercial use, distribution and reproduction in any medium provided that the original work is properly cited and all further distributions as wasp parasitism (Rizki and Rizki, 1984; of the work or adaptation are subject to the same Creative Commons License terms. Lanot et al., 2001; Crozatier and Meister,

Disease Models & Mechanisms 439 PRIMER Drosophila: a model for haematopoiesis

A Key Circulating haemocyte Sessile haemocyte

Aorta

LG T1 T2 T3 A1 A8 A2 A3 A4 A5 A6 A7

C CMP CLP B Crystal cells Plasmatocytes O2 Perivascular Lamellocytes niche ST-HSC Vasculature CZ ROS CAR ROS Upd3 Wg Hh JAK-STAT MZ JAK-STAT LT-HSC Wg Wg (Notch) (Upd3) Progenitors PSC N-cadherin (Ser) (Jagged) Osteoblastic dorsalAorta vessel niche BMPR1 Osteoblast Drosophila Bone DMM Mammals Fig. 1. Parallels in haematopoiesis between the Drosophila LG and mammalian bone marrow. (A) Schematic representation of a Drosophila third instar larva, illustrating the presence of circulating and sessile haemocytes of embryonic origin, and showing the position of the larval haematopoietic organ (the LG) along the dorsal aorta. T and A refer to thoracic and abdominal segments, respectively, and are numbered along the anterior-posterior axis. (B) The primary lobe of the LG consists of the PSC (blue), the MZ (yellow; containing haemocyte progenitors) and the CZ (red), in which differentiation of plasmatocytes and crystal cells takes place. Lamellocytes only differentiate in response to specific immune threats, at the expense of progenitors. There also exists a population of intermediate progenitors that have left the MZ (found within the orange region). Signalling pathways that are active in the PSC and MZ are indicated. Tightly controlled levels of ROS sensitise haemocyte progenitors to differentiation. Unpaired 3 (Upd3) is one activator of the JAK-STAT pathway, and Serrate (Ser) and Jagged are ligands of the Notch receptor. Their specific roles in the niche remain little understood. (C) Schematic representation of the quiescent LT-HSCs and primed (activated) ST- HSCs and their niches in mammalian bone marrow. Signalling pathways involved in the communication between HSCs and their niches are indicated. Common lymphoid and common myeloid precursors are indicated as CLP and CMP, respectively. Curved arrows indicate cell-autonomous effects within the PSC (B) or in LT-HTCs (C).

2007), stress conditions mediated by an identified by their expression of the Notch function was first proposed from work on increase of reactive oxygen species (ROS) and ligand Serrate (Ser) (Lebestky et al., 2003). A haematopoiesis (Schofield, 1978), but it mutant backgrounds in which their massive turning point in the use of Drosophila as a gained strong experimental and conceptual Disease Models & Mechanisms proliferation to the formation of model for haematopoiesis and haematopoietic support from studies in Drosophila germline ‘melanotic tumours’ that result from disorders was the discovery that the PSC plays stem cells (Xie and Spradling, 2000). Recent encapsulation of Drosophila tissue by a key function in controlling larval blood cell studies indicate that detailed characterisation lamellocytes (see below). homeostasis (Krzemien et al., 2007; Mandal of stem-cell–niche interactions will help to The mature Drosophila LG is composed of et al., 2007). Specifically, the PSC cells act in understand the pathology of human cancers a pair of anterior (primary) lobes, and several a non-cell-autonomous manner to maintain such as acute myeloid leukaemia (AML) (Box small posterior (secondary) lobes, which the activity of the Hedgehog (Hh) and JAK- 1). Despite intensive HSC studies in humans contain immature pro-haemocytes. The STAT signalling pathways in pro-haemocytes, and in vertebrate models, characterisation of primary lobes are organised into a medullary thereby preserving their multipotent the HSC niche has progressed slowly, mainly zone (MZ) composed of pro-haemocytes, a character. This key role of the PSC in owing to the complex anatomy of bone cortical zone (CZ) containing differentiated supporting Drosophila blood cell homeostasis marrow. Recent data have led to a model in haemocytes, and the so-called posterior revealed unanticipated parallels with the HSC which quiescent and primed stem cells signalling centre (PSC) (see below for details) niche in mammalian bone marrow (Krzemien interact with two different microenviron- (Lebestky et al., 2003; Jung et al., 2005) (Fig. et al., 2007; Mandal et al., 2007). ments: the endosteal surface covered by 1B). A pool of mitotically active intermediate immature osteoblasts (the ‘osteoblastic progenitors appears at the onset of haemocyte Drosophila as a model to study the niche’), which favours long-term quiescence, differentiation and accounts for the increasing haematopoietic niche and the sinusoidal vessels of the central bone number of differentiated haemocytes in the The concept that the niche can provide marrow (the ‘vascular niche’), which favours mature LG (Krzemien et al., 2010a; Krzemien structural and trophic support as well as proliferation of HSCs (Fig. 1C) (Trumpp et et al., 2010b). The PSC cells were initially appropriate signals to regulate al., 2010). The vessels and endosteal surfaces

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longevity following repeated expansion expression levels and activity in the PSC Box 1. Understanding the (Fleming et al., 2008). Parathyroid hormone hematopoietic niche supports (Krzemien et al., 2007) (Fig. 1B). How Hh, studies of cancer (PTH) signalling in osteoblasts leads to an Wg, Notch and other signalling outputs are increased number of HSCs, probably integrated in PSC cells to control JAK-STAT Seminal studies of acute myeloid leukaemia mediated through high expression levels of signalling activity in pro-haemocytes and to (AML), an aggressive malignancy of immature the Notch ligand Jagged1 in osteoblasts control blood homeostasis is now being haematopoietic cells in the bone marrow, (Calvi et al., 2003). Conversely, signalling by addressed in several laboratories. Our recent suggested that AML ‘leukaemic stem cells’ (LSCs) arose from HSCs (Bonnet and Dick, 1997; bone morphogenic proteins (BMPs) studies of the LG have uncovered a Reya et al., 2001). This led to the cancer stem controls the number of HSCs by regulating previously unidentified mode by which JAK- cell (CSC) hypothesis that proposed that many the number of osteoblastic cells (Zhang et STAT signalling is regulated, involving a human cancers are organised in cellular al., 2003). Finally, CXC- ligand 12 non-signalling dominant-negative form of a hierarchies, similar to normal tissues. At the (CXCL12) produced by the reticular cells receptor (Makki et al., 2010). hierarchy apex are multipotent, largely [CXCL12-abundant reticular (CAR) cells] Ongoing characterisation and genetic quiescent, long-lived CSCs with self-renewal surrounding the sinusoidal endothelial cells manipulation of PSC function in controlling capacity that sustain disease (Bonnet and Dick, 1997). Investigation of a large number of is required for maintaining the primed HSC Drosophila haematopoiesis are thus expected human AML samples has recently shown that pool. The interconnections between Wnt, to provide clues as to how the mammalian haematopoietic progenitors, rather than HSCs, PTH, CXCL12 and Notch signalling, bone marrow niche controls the balance could be the origin of LSCs. The most however, remain unclear. between HSC self-renewal and differentia- important aspect is the ability of LSCs to self- Despite the tremendous amount of tion. renew, a crucial property of stem cells (Goardon information gathered from these et al., 2011; Majeti and Weissman, 2011). Mouse investigations, our knowledge about the ROS levels in regulating xenograft models of acute lymphoblastic niche and its function remains fragmentary. haematopoiesis: a Drosophila case leukaemia (ALL) recently showed that

DMM leukaemic proliferation in bone marrow alters New approaches are necessary to determine The functional difference between the the stromal microenvironment and creates how the integration of different signalling osteoblastic and vascular niches in mouse ‘malignant niches’ that out-compete native pathways in the niche maintains HSCs. Many bone marrow might be related to oxygen HSCs. Normal HSCs engaged by the malignant transcriptional regulators and signalling availability: oxygen levels are probably higher niche exhibit abnormal behaviour, and AML pathways involved in haematopoiesis are in the vascular niche, whereas the LT-HSC LSCs that home to and engraft within the conserved between humans and Drosophila osteoblastic niche is hypoxic (Parmar et al., osteoblastic niche are protected from (Table 1 and Fig. 1B,C). Therefore, the 2007; Simsek et al., 2010). A low-oxygen -induced . These findings raise many questions about the nature Drosophila PSC, which controls the differ- microenvironment could be essential for the of the dysfunction of stem and/or progenitor entiation of pro-haemocytes in a non-cell- maintenance of HSCs and for their cells and their niche in cancer, as well as related autonomous manner, represents an protection from detrimental ROS questions about therapy selection and/or interesting, genetically tractable model with accumulation (Jang and Sharkis, 2007), intensification (Colmone et al., 2008). which to study signal integration and whereas a high-oxygen microenvironment crosstalk during blood cell development. could favour proliferation (for a review, see are intimately entwined within trabecular PSC cells are specified in the embryo by Eliasson and Jonsson, 2010). Notably, the bone and might play a coordinated and the expression of two transcription factors, forkhead O (FoxO) transcription factors play interrelated role, rather than having binary the homeotic protein Antennapedia (Antp) essential roles in preserving quiescence and Disease Models & Mechanisms functions. and Collier (Col), the Drosophila orthologue enhancing survival of HSCs in the bone Genetic, transgenic and molecular of early B-cell factors (EBFs) (Crozatier et al., marrow through regulating the response to studies in mice that made use of promoters 2004; Mandal et al., 2007). In the absence of physiological oxidative stress (Tothova et al., and inducible systems that are specific to the Col activity, the PSC is not specified and 2007), although how this regulation is exerted osteoblastic lineage have uncovered several larval pro-haemocytes are not maintained, remains poorly understood. The distinct signalling pathways that are involved in the and differentiate prematurely. Mouse EBF2 metabolism of HSCs [and probably also communication between HSCs and their was recently shown to act in osteoblastic cells leukaemic stem cells (Box 1)], which utilise niche(s) (Table 1 and Fig. 1C). In brief, and to regulate the maintenance of HSCs, in glycolysis instead of mitochondrial oxidative dormant HSCs and osteoblasts bind via part by modulating Wnt signalling phosphorylation, does indeed reflect their homotypic N-cadherin interactions. (Kieslinger et al., 2010). This finding is location in a hypoxic niche (Simsek et al., Expression of angiopoietin-1 and particularly interesting given that Wingless 2010). Notably, quiescent leukaemic cells by osteoblasts contributes to (Wg; the Drosophila orthologue of Wnt) that reside in the hypoxic endosteum are both the adhesion of HSCs to the signalling has been shown to control both the resistant to radio- and chemotherapy osteoblastic niche and to HSC quiescence number of PSC cells and the maintenance of (Ishikawa et al., 2007; Richardson, 2011). (Arai et al., 2004; Nilsson et al., 2005). pro-haemocytes (Sinenko et al., 2009). PSC The link between ROS levels and HSC Enforced expression of the Wnt inhibitor cells also express the morphogen Hh, and Hh survival therefore contributes to Dickkopf1 in osteoblasts showed that Wnt signalling is required to maintain haemocyte understanding the connection between ROS pathway activation in the niche is required homeostasis in the LG (Mandal et al., 2007). levels in cancer stem cells (Box 1) and the to limit HSC proliferation and thereby to Finally, Ser-mediated Notch signalling from resistance of tumours to radiation (Diehn et preserve HSC self-renewal capacity and the PSC is required to maintain normal Col al., 2009).

Disease Models & Mechanisms 441 PRIMER Drosophila: a model for haematopoiesis

Table 1. Conserved proteins with important roles in haematopoiesis Drosophila Mammalian function function Transcription factors and cofactorsa Srp (GATA) Specification of haemocytes; progenitor maintenance (Lebestky et Maintenance of HSCs; lineage commitment; regulation of al., 2000; Lebestky et al., 2003; Tokusumi et al., 2010) differentiation (Orkin and Zon, 2008; Braun and Woollard, 2009) Lz (RUNX) Specification of haemocytes (Lebestky et al., 2000; Lebestky et al., Maintenance of HSCs; lineage commitment; regulation of 2003) differentiation (Wang et al., 2010) Ush (FOG) Specification of haemocytes; differentiation of lamellocytes Lineage commitment; regulation of differentiation (Tsiftsoglou (Lebestky et al., 2000; Sorrentino et al., 2007) et al., 2009) Col (EBF) Maintenance of PSC cells (Crozatier et al., 2004; Krzemien et al., B-cell differentiation; maintenance of the niche (Lin and 2007) Grosschedl, 1995; Kieslinger et al., 2010) Antp (HOX) Formation of the PSC (Jung et al., 2005) HSC proliferation and self-renewal; ; B (Argiropoulos and Humphries, 2007) Signalling pathwaysa Notch Differentiation of crystal cells (Lebestky et al., 2003) Maintenance of HSCs; haematopoietic regeneration; regulation of differentiation (Weber and Calvi, 2010; Renstrom et al., 2010) Hedgehog Maintenance of pro-haemocytes (Mandal et al., 2007) Lineage commitment; regulation of differentiation (Hofmann et al., 2009; Campbell et al., 2008; Gao et al., 2009) Wingless (WNT) Proliferation of PSC cells; maintenance of pro-haemocytes; Maintenance and proliferation of HSCs; regulation of B- and T- differentiation of crystal cells (Sinenko et al., 2009) cell differentiation (Grigoryan et al., 2008) JAK-STAT Maintenance of pro-haemocytes; differentiation of HSC renewal during regeneration; lineage commitment (Chung DMM lamellocytes (Krzemien et al., 2007; Harrison et al., 1995; Luo et al., 2006) et al., 1995) ROS/FoxO Maintenance of pro-haemocytes (Owusu-Ansah and Banerjee, Maintenance of HSCs; differentiation of erythroid cells (Tothova 2009) et al., 2007) Pvf (PDGF/VEGF) Differentiation of plasmatocytes (Jung et al., 2005) Maintenance of HSCs (VEGF) (Gerber and Ferrara, 2003) aMammalian genetic or functional homologues are shown in parentheses.

Owusu-Hansa and Banerjee (Owusu- (Sinenko et al., 2009; Makki et al., 2010). This fusion product (see below), is found in ~12% Ansah and Banerjee, 2009) established the would provide a more integrated view of how of AML patients. Under normal conditions, role of ROS in the control of Drosophila haematopoiesis is controlled in different AML1, a RUNX-domain protein, acts as a haematopoiesis, revealing that the environmental conditions. tissue-specific transcriptional activator at accumulation of ROS in the LG is tightly multiple steps during haematopoiesis, controlled during development. This report Using Drosophila to identify new whereas ETO (also known as RUNX1T1 and showed that ROS levels in pro-haemocytes oncogene partners MTG8) functions as a transcriptional are an intrinsic factor that sensitises these Drosophila can provide a valuable in vivo repressor. The commonly accepted view is Disease Models & Mechanisms progenitors to differentiation. Furthermore, screening platform for modifiers, enhancers that the AML1-ETO fusion product acts as they found that there is a tight correlation or suppressors of human oncogenic protein a constitutive repressor of AML1 protein between FoxO overactivation and the function, as illustrated by studies of AML. function and inhibits myeloid differentiation downregulation of Polycomb (an epigenetic The pathogenesis of AML is considered to while promoting proliferation of multilineage ) in cells with high ROS levels; this is involve multistep genetic alterations in progenitors. Interestingly, the Drosophila in agreement with a previous report that several genes that predispose or promote RUNX protein Lozenge is required for the showed that repressing the activity of a leukaemias [for a recent WHO differentiation of crystal cells. Two Polycomb protein provoked abnormal classification, see Dohner et al. (Dohner et independent groups expressed human lamellocyte differentiation (Remillieux- al., 2010)]. Analyses of chromosomal AML1-ETO in Drosophila haemocyte Leschelle et al., 2002). Drosophila translocations in the bone marrow of AML precursors and showed that this results in haematopoiesis is thus an excellent model in patients have shown that several increased proliferation of circulating which to study the tight developmental transcription factors that are crucial for progenitors, the formation of ‘melanotic control of ROS levels that might play a key haematopoiesis also play important roles in tumours’ or ‘pseudotumours’ and prevention role in the transition from HSCs to myeloid leukaemogenesis. These include AML1, of crystal cell differentiation. Both groups progenitors in mammals (Tothova et al., LMO2, SALL, MLL and SCl/Tal1, all of then performed genetic screens to identify 2007). Similar studies might also be useful for which are proteins with clearly identified modifiers of these blood phenotypes, which dissecting how signalling via ROS is Drosophila orthologues (FlyBase; could potentially be modulators of the connected to other signalling pathways, such http://flybase.org/). human leukaemogenic protein. One group as the Wg and JAK-STAT pathways required The t(8;21) (q22;q22) chromosomal revealed that in components of the for pro-haemocyte maintenance in the LG translocation, which creates the AML1-ETO Wnt4-Frizzled and Ca2+ signalling pathways

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Drosophila tional effector of the pathway, STAT92E fly. This opens the prospect of manipulating Advantages of as a (STAT92ENC ) also promotes and/or signalling activities model for haematopoiesis and leukaemias tumorigenesis (Ekas et al., 2010). Together in vivo, simultaneously in HSCs and niche with the key role of JAK-STAT signalling in cells, and decrypting their reciprocal • Drosophila is amenable to genetic and the maintenance of pro-haemocytes in the communication under physiological and transgenic manipulations. Homologous LG (Makki et al., 2010), these tumorous pathological conditions. Large-scale screens recombination and targeted RNAi technologies phenotypes could be particularly relevant to for melanotic-tumour suppressor genes have are routinely used, both in vivo and in cell culture assays. Large collections of mutants and the study of human leukaemias, because already been performed, and new genes and double-stranded RNA (dsRNA) libraries are mutations and translocations of JAK genes gene networks controlling blood cell available. are associated with a variety of homeostasis have been identified (Minakhina • Drosophila has a very short generation time haematopoietic malignancies (Bromberg et and Steward, 2006; Stofanko et al., 2008; and is easy to maintain in large populations al., 1999; Vainchenker et al., 2008; Mullighan Avet-Rochex et al., 2010; Bina et al., 2010; that are convenient for large-scale genetic et al., 2009). For example, an activating Minakhina and Steward, 2010). On the basis screens as well as transcriptome, proteome and mutation in the human tyrosine kinase JAK2 of these pioneering experiments, and the interactome analyses. (JAK2V617F) is responsible for various forms recent demonstration that Drosophila can be • The genomic sequences of 12 Drosophila species and of several Diptera of medical of myeloid leukaemia. Mouse models have used to identify the core regulatory network interest (mosquitoes) are available. This allows a confirmed that this mutation causes myelo- that contributes to the development of AML high precision of sequence comparison of proliferative neoplasms and has differential (Osman et al., 2009; Sinenko et al., 2010), it coding and regulatory regions between these effects on HSCs and myeloid progenitor cells is expected that more genetic screens species and mutants, or of variants within a (Levine et al., 2005; Mullighan et al., 2009; performed in flies will provide novel entries given species. Mullally et al., 2010). It is therefore possible to the study of acute and chronic leukaemias The problem of functional redundancy that is • that the constitutive activity of conserved often encountered when studying human in mammals.

DMM genes owing to vertebrate genome components of the JAK-STAT pathway can ACKNOWLEDGEMENTS duplications is minimised in Drosophila. be used in RNAi screens to identify in vivo We thank Caroline Monod and Cédric Polesello for • Drosophila models of leukaemia created by suppressors of JAK-STAT activity. Such their comments on the manuscript. Work in the expressing human oncogenic or abnormal suppressors might have conserved functions authors’ laboratory is supported by CNRS, University Toulouse III, Ministère de la Recherche (ANR proteins can be used to search for new genetic in restricting acute lymphoblastic leukaemia « programme blanc »), ARC (Association pour la regulators. (ALL) or AML progression in humans and Recherche sur le Cancer), AFM (Association Française could provide potential targets for contre les Myopathies) and FRM (Fondation pour la Recherche Médicale). increased AML1-ETO activity (Sinenko et al., therapeutic intervention. 2010). The other group identified eight COMPETING INTERESTS The authors declare that they do not have any suppressor candidates, including Calpain B Conclusions competing or financial interests. (CalpB), a Ca2+-dependent protease (Osman For the past 30 years, Drosophila et al., 2009). Functional analysis of CalpB melanogaster has been a central model for REFERENCES revealed that it is required to stabilize AML1- understanding the genetic and molecular Arai, F., Hirao, A., Ohmura, M., Sato, H., Matsuoka, S., Takubo, K., Ito, K., Koh, G. Y. and Suda, T. (2004). ETO in the nucleus. Interestingly, CalpB was bases of animal development. During this Tie2/angiopoietin-1 signaling regulates hematopoietic also found to interact with AML1-ETO in period, new genetic and genomic resources stem cell quiescence in the bone marrow niche. Cell human cells (Osman et al., 2009), opening up and technologies have been developed that 118, 149-161. Disease Models & Mechanisms the possibility that it might have a role in allow tissue-specific knockdown of gene Argiropoulos, B. and Humphries, R. K. (2007). Hox genes in hematopoiesis and leukemogenesis. regulating leukaemogenesis. This functional expression, gene replacement and site- Oncogene 26, 6766-6776. study provides proof of principle that specific integration of modified and/or Avet-Rochex, A., Boyer, K., Polesello, C., Gobert, V., expression of human oncogenic factors in exogenous genes in the Drosophila genome. Osman, D., Roch, F., Auge, B., Zanet, J., Haenlin, M. and Waltzer, L. (2010). An in vivo RNA interference Drosophila haemocytes can help to identify The recent description of Drosophila screen identifies gene networks controlling core regulatory networks whose haematopoietic stem-like cells and their Drosophila melanogaster blood cell homeostasis. BMC dysregulation in humans contributes to the niche – and the accumulating evidence that Dev. Biol. 10, 65. development of leukaemias. many of the basic pathways and processes are Bina, S., Wright, V. M., Fisher, K. H., Milo, M. and Zeidler, M. P. (2010). Transcriptional targets of As noted above, AML1-ETO expression in also present in vertebrates – establishes Drosophila JAK/STAT pathway signalling as effectors Drosophila provokes an increased Drosophila as a model for studying the of haematopoietic tumour formation. EMBO Rep. 11, proliferation of haemocyte precursors and genetic aspects of haematopoiesis and 201-207. Bonnet, D. and Dick, J. E. (1997). Human acute myeloid the formation of melanotic tumours or haematopoietic disorders. leukemia is organized as a hierarchy that originates pseudotumours (Osman et al., 2009; Sinenko A new repressible binary expression from a primitive hematopoietic cell. Nat. Med. 3, 730- et al., 2010). Such tumours were previously system for transgene expression, the Q 737. observed in a gain-of-function mutation system, has recently been developed (Potter Braun, T. and Woollard, A. (2009). RUNX factors in development: lessons from invertebrate model affecting the kinase domain of the Hopscotch et al., 2010). The combination of the Q systems. Blood Cells Mol. Dis. 43, 43-48. kinase (Hoptuml mutation), the Drosophila system and the previously established GAL4 Bromberg, J. F., Wrzeszczynska, M. H., Devgan, G., JAK-STAT kinase (Harrison et al., 1995; Luo inducible system will allow the development Zhao, Y., Pestell, R. G., Albanese, C. and Darnell, J. E., Jr (1999). Stat3 as an oncogene. Cell 98, 295-303. et al., 1995). Furthermore, misexpression of of strategies for manipulating transgene Calvi, L. M., Adams, G. B., Weibrecht, K. W., Weber, J. a dominant-active form of the transcrip- expression in different cell types in the same M., Olson, D. P., Knight, M. C., Martin, R. P.,

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