InteractiveFly: GeneBrief

Adenosine deaminase-related growth factor A: Biological Overview | References

BIOLOGICAL OVERVIEW

Maintenance of a hematopoietic progenitor population requires extensive interaction with cells within a microenvironment or niche (see Hematopoetic progenitor maintenance in the Drosophila blood system). In the Drosophila hematopoietic organ, niche-derived Hedgehog signaling maintains the progenitor population. This study shows that the hematopoietic progenitors also require a signal mediated by Adenosine deaminase growth factor A (Adgf-A) arising from differentiating cells that regulates extracellular levels of adenosine. The adenosine signal opposes the effects of Hedgehog signaling within the hematopoietic progenitor cells and the magnitude of the adenosine signal is kept in check by the level of Adgf-A secreted from differentiating cells. These findings reveal signals arising from differentiating cells that are required for maintaining progenitor cell quiescence and that function with the niche-derived signal in maintaining the progenitor state. Similar homeostatic mechanisms are likely to be utilized in other systems that maintain relatively large numbers of progenitors that are not all in direct contact with the cells of the niche (Mondal, 2011).

The mammalian hematopoietic niche displays complex interactions between populations of HSCs and progenitors to maintain their numbers. The relative in vivo contributions of cues emanating from the microenvironment in regulating stem cell versus progenitor maintenance remains unclear. Several stem cell and progenitor populations demonstrate slow cell cycling and this property of 'quiescence' is critical for maintaining their integrity over a period of time (Mondal, 2011).

In vivo genetic analysis in Drosophila allows for the study of stem cell properties in their endogenous microenvironment (Losick, 2011). Drosophila blood cells, or hemocytes, develop within an organ called the lymph gland, where differentiating hemocytes, their progenitors, and the cells of the signaling microenvironment or niche, are found. Differentiated blood cells in Drosophila are all myeloid in nature and are located along the outer edge of the lymph gland, in a region termed the cortical zone (CZ. These arise from a group of progenitors located within an inner core of cells termed the medullary zone (MZ). The MZ cells are akin to the common myeloid progenitors (CMP) of the vertebrate hematopoietic system. They quiesce, lack differentiation markers, are multipotent, and give rise to all Drosophila blood lineage. MZ progenitors are maintained by a small group of cells, collectively termed the posterior signaling center (PSC), that function as a hematopoietic niche. Clonal analysis has suggested the existence of a niche-bound population of hematopoietic stem cells, although such cells have not yet been directly identified (Mondal, 2011).

The PSC cells express Hedgehog (Hh), which is required for the maintenance of the MZ progenitors. Cubitus interruptus (Ci) is a downstream effector of Hh signaling similar to vertebrate Gli proteins; it is maintained in its active Ci¹⁵⁵ form in the presence of Hh and degraded to the repressor Ci⁷⁵ form in the absence of Hh. PSC-derived Hh signaling causes MZ cells to exhibit high Ci¹⁵⁵ (Mondal, 2011).

Proliferation of circulating larval hemocytes is also regulated by Adenosine Deaminase Growth Factor-A (Adgf-A), which is similar to vertebrate adenosine deaminases (ADAs). Adgf-A is a secreted enzyme that converts extracellular adenosine into inosine by deamination. Two distinct adenosine deaminases, ADA1 and ADA2/CECR1, are found in humans. CECR1 is secreted by monocytes as they differentiate into macrophages (Zavialov, 2010b). In Drosophila, mutation of Adgf-A causes increased adenosine levels and increase in circulating blood cells (Dolezal, 2005; Zurovec, 2002; Mondal, 2011 and references therein).

Extracellular adenosine is sensed by the single Drosophila adenosine receptor (AdoR) that generates a mitogenic signal through the G protein/adenylate cyclase/cAMP-dependent Protein Kinase A (PKA) pathway (Dolezelova, 2007). A target of PKA is the transcription factor Ci, which also transduces the Hedgehog signal. This study explored the potential link between adenosine and Hedgehog signaling, both through PKA mediated regulation of Ci, and a model was proposed that the niche signal and the CZ signal interact to maintain the progenitor population in a quiescent and undifferentiated state within the MZ of the lymph gland (Mondal, 2011).

The first cells that express differentiation markers appear stereotypically at the peripheral edge of the lymph gland. These differentiating cells will eventually populate an entire peripheral compartment that will comprise the CZ. The timing of the first signs of differentiation matches closely with the onset of quiescence among the precursor population, eventually giving rise to the medullary zone (MZ) (Mondal, 2011).

The close temporal synchronization of CZ formation and the quiescence of MZ progenitors raised the intriguing possibility that the onset of differentiation might regulate the proliferation profile of the progenitors. To test this hypothesis, cell death was induced by expressing the pro-apoptotic proteins Hid and Reaper in the differentiating hemocytes, and the effect of their loss was assayed in the progenitor population. Loss of CZ cells was found to induce proliferation of the adjacent progenitor cells, which are normally quiescent at this stage (Mondal, 2011).

Candidate ligands in the lymph gland were knocked down by RNA interference (RNAi) and monitored for a loss of progenitor quiescence. This survey identified Pvf1 as a signaling molecule that is required for the maintenance of quiescence within the lymph gland. Expressing Pvf1^RNAi using Gal4 drivers specific to either niche (PSC) cells using Antp-gal4, progenitor cells using dome-gal4, or differentiating cells using Hml-gal4 showed that PSC-specific knockdown is sufficient to induce progenitor proliferation, whereas Pvf1 knockdown in progenitors or differentiating cells has no effect on the lymph gland. These results indicate that Pvf1 synthesized in the PSC is required for progenitor quiescence (Mondal, 2011).

To determine the site of Pvf1 function, its receptor Pvr was knocked down in the lymph gland using a similar approach. Interestingly, it was found that Pvr^RNAi expressed under the control of drivers specific to differentiating cells (Hml-gal4 and pxn-gal4) causes a loss of progenitor quiescence. The BrdU incorporating cells do not express differentiation markers. Thus, differentiation follows the proliferative event. Lymph glands are not similarly affected when Pvr function is downregulated in the progenitors themselves. These results indicate that Pvf1 originates in the niche and activates Pvr in maturing hemocytes, and that this signaling system is important for the quiescence of MZ progenitors. These results did not explain, though, how maturing cells might signal back to the progenitors causing them to maintain quiescence (Mondal, 2011).

Given the previously known role of Adgf-A in the control of hemocyte number in circulation (Dolezal, 2005), whether this protein plays a similar role in the lymph gland was investigated. Remarkably, downregulation of the secreted Adgf-A protein in the differentiating hemocytes of the CZ, achieved by expressing Adgf-A^RNAi under Hml-gal4 control, induces loss of quiescence of MZ progenitors, similar to that seen with loss of Pvr in the CZ. This suggests that Adgf-A may act as a signal originating from differentiating hemocytes that is required for maintaining progenitor quiescence. In support of this idea, while overexpression of Adgf-A in differentiating hemocytes alone does not affect normal zonation, it suppresses the induced progenitor proliferation caused by downregulation of Pvr. For loss of signaling molecules, it is the break in the signaling network necessary for reducing adenosine that causes continued proliferation and eventual differentiation. For rpr/hid the signaling cell itself has been removed, thereby causing a lack in a backward signal. Quantitative analysis of the data is consistent with a role for Adgf-A downstream of Pvr (Mondal, 2011).

The role of a niche signal is well established in many developmental systems that involve stem cell/progenitor populations. In the Drosophila lymph gland the niche expresses Hh and maintains a group of progenitor cells (Mandal, 2007). This current study establishes an additional mechanism, parallel to the niche signal that originates from differentiating cells, which also regulates quiescence of hematopoietic progenitors (Mondal, 2011).

The cells of the lymph gland proliferate at early stages, from embryo to mid second instar. At this stage, cells farthest from the PSC initiate differentiation and the rest enter a quiescent phase defining a MZ. In wild-type, the cells of the MZ remain quiescent and in progenitor form throughout the third instar, and this process requires a combination of the PSC and CZ signals. If either signal is removed, the progenitor population will eventually be lost due to differentiation. In many different genetic backgrounds, if quiescence is lost, the progenitor population initially continues to incorporate BrdU during the second instar without expressing any maturation markers. The differentiation phenotype, characterized by the expression of such markers, follows this abnormal proliferation. The net result is that whenever the progenitors accumulate BrdU (but not express any markers of differentiation) in the second instar, all cells of the lymph gland are differentiated and no MZ remains in the third instar. While the nature of the signal that triggers hemocyte differentiation is not known, withdrawal of Wingless may play a role in this process (Mondal, 2011).

Experimental analysis has demonstrated a novel role for Pvr in maturing hemocytes and its ligand, Pvf1, in the cells of the PSC. Pvf1 expression increases at a stage when the lymph gland is highly proliferative. At this critical window in development, Pvf1 originating from the PSC is transported to the differentiating hemocytes, binds to its receptor Pvr, and activates a STAT-dependent signaling cascade. At this stage, Pvf1 is sensed by all cells but it is only in the differentiating hemocytes that it activates Adgf-A in an AdoR/Pvr-dependent manner. This secreted factor Adgf-A is required for regulating extracellular adenosine levels. High adenosine would signal through AdoR and PKA to inactivate Ci and reduce the effects of the niche-derived Hedgehog signal leading to differentiation of the progenitor cells. The function of the Adgf-A signal is to reduce this adenosine signal and therefore reinforce the maintenance of progenitors by the Hedgehog signal. Thus, the Adgf-A and Hh signals work in the same direction but Adgf-A does so by negating a proliferative signal due to adenosine. In wild-type, equilibrium is reached through a signal that does not originate from the niche that opposes this proliferative process. The attractive step in this model is that the CZ and niche (in this case Hh-dependent) signals both impinge on common downstream elements allowing for control of the progenitor population relative to the niche and the differentiated cells. Most importantly, this is a mechanism for maintaining quiescence within a moderately large population of cells that is not in direct contact with a niche. By the time the three zone PSC/MZ/CZ system is set up in the late second instar all the cells of the MZ express high levels of E-cadherin, become quiescent and are maintained as progenitors and are capable of giving rise to all blood cell lineages. Under such circumstances, the interaction between a niche-derived signal and an equilibrium signal originating from differentiating cells can maintain homeostatic control of the progenitor population. Several vertebrate stem cell/progenitor scenarios such as during bone morphogenesis and hematopoiesis or in the Drosophila intestine have progenitors and differentiating cells in close proximity that could pose an opportunity for a similar niche and differentiating cell-derived signal interaction. In fact, evidence for such interactions have recently been provided for vertebrate skin cells (Mondal, 2011).

The role of small molecules such as adenosine has not yet been adequately addressed in vertebrate progenitor maintenance. A small molecule such as extracellular adenosine is unlikely to form a gradient over the population of cells and maintain such a gradient over a developmental time scale. It is much more likely that this system operates similar to the 'quorum sensing' mechanisms described for prokaryotes (Ng, 2009). A critical level of adenosine is required for proliferation and by expressing the Adgf-A signal this threshold amount is lowered, causing quiescence in the entire population (Mondal, 2011).

This study describes a developmental mechanism that is relevant to the generation of an optimal number of blood cells in the absence of any overt injury or infection. However, a system that utilizes such a mechanism to maintain a progenitor population could potentially sense a disruption upon induction of various metabolic stresses to cause differentiation of myeloid cells. Various mitochondrial and cellular stresses can cause an increase in extracellular adenosine (Fredholm, 2007), but whether they are relevant to this system remains to be studied. In the past, dual use has been observed of reactive oxygen species (ROS) as well as Hypoxia Inducible Factor-a (HIF-a) in both development and stress response of the Drosophila hematopoietic. Responses to injury have been described in the Drosophila intestine, and in satellite cells that respond during injury, a stress related signal could be the initiating factor that overrides a maintenance signal. Thus, the equilibrium generated through developmental interactions is disrupted to promote a cellular response to stress signals (Mondal, 2011).

An in vivo RNAi screen uncovers the role of AdoR signaling and adenosine deaminase in controlling intestinal stem cell activity

Metabolites are increasingly appreciated for their roles as signaling molecules. To dissect the roles of metabolites, it is essential to understand their signaling pathways and their enzymatic regulations. From an RNA interference (RNAi) screen for regulators of intestinal stem cell (ISC) activity in the Drosophila midgut, this study identified adenosine receptor (AdoR) as a top candidate gene required for ISC proliferation. Ras/MAPK and Protein Kinase A (PKA) signaling act downstream of AdoR and Ras/MAPK mediates the major effect of AdoR on ISC proliferation. Extracellular adenosine, the ligand for AdoR, is a small metabolite that can be released by various cell types and degraded in the extracellular space by secreted adenosine deaminase. Interestingly, down-regulation of adenosine deaminase-related growth factor A (Adgf-A) from enterocytes is necessary for extracellular adenosine to activate AdoR and induce ISC overproliferation. As Adgf-A expression and its enzymatic activity decrease following tissue damage, this study provides important insights into how the enzymatic regulation of extracellular adenosine levels under tissue-damage conditions facilitates ISC proliferation (Xu, 2019).

The Drosophila midgut epithelium consists of multipotent intestinal stem cell (ISCs), their immediate progenies known as enteroblasts (EBs, which are progenitor cells primed for differentiation), and differentiated cells including enterocytes (ECs, which is the major cell type in number), and enteroendocrine cells (EEs). ISCs/EBs can adjust their proliferation and differentiation activities by deploying conserved core pathways such as JAK/Stat, Notch, Ras/MAPK, JNK, and Hippo. The dynamic responses of adult ISCs/EBs to different regenerative demands under physiological or pathological conditions depend on the machineries to detect microenvironment cues and modulate the activity of aforementioned core pathways, which have not been investigated in vivo systematically (Xu, 2019). To understand how ISCs/EBs sense their microenvironment, an RNAi screen was performed to identify receptor-coding genes that regulate ISC activity, among which Adenosine Receptor (AdoR) emerged as a top candidate required for ISC self-renewal and proliferation. Characterization of the AdoR-signaling pathway revealed the role of AdoR downstream pathways in regulating different aspects of ISC activity. Importantly, this study demonstrated that the mitogenic activity of the AdoR ligand, adenosine, is inhibited by adenosine deaminase-related growth factor A (Adgf-A) from ECs and that Adgf-A activity decreases following tissue damage. Altogether, this study demonstrates how an EC-derived metabolic enzyme modulates ISC activity by restricting extracellular adenosine (Xu, 2019).

An RNAi screen was performed for regulators of ISC activity and AdoR was identified as a gene required for Ras/MAPK and PKA signaling in the ISCs/EBs. Characterization of AdoR and its ligand revealed that, in the healthy midgut, EC-derived Adgf-A limits the bioavailability of extracellular adenosine and restricts AdoR signaling in ISCs/EBs to a baseline level that supports ISC maintenance. However, the damaged midgut lacks sufficient levels of Adgf-A to restrict extracellular adenosine, thus allowing the activation of AdoR and its downstream pathways to stimulate the regenerative activity of ISCs (Xu, 2019).

Purines not only are required for nucleic acid synthesis and the cellular energy supply, but also represent the most primitive and common extracellular chemical messengers. Extracellular adenosine acts on P1-type purinergic receptors, i.e., AdoRs. The effects of AdoR signaling on cell growth are context-dependent. For example, adenosine inhibits the growth of imaginal disk cells, and Adgf-A was initially identified as a growth factor that stimulates the proliferation of Drosophila imaginal disk and embryonic cells in vitro (Zurovec, 2002). In contrast, in both larval lymph gland (Mondal, 2011) and adult midgut, AdoR supports proliferation and differentiation in the stem/progenitor cells whereas AdgfA from a nonautonomous source suppresses AdoR activity. Despite the remarkably similar roles of AdoR in controlling behaviors of 2 different types of stem/progenitor cells, AdoR activation leads to hemapoietic progenitor exhaustion but ISC expansion. Furthermore, Ras/MAPK activity, rather than PKA (as in the hemapoietic progenitors), functions as a necessary and sufficient downstream component mediating AdoR-induced ISC overproliferation (Xu, 2019).

Identification of AdoR as an ISC regulator led to a dissection of the function of its downstream pathways, i.e., PKA and Ras/MAPK. Although earlier studies reported that EC-like differentiation in Caco2 colorectal cancer cells correlates with PKA activation and that pharmacological induction of cAMP/PKA suppresses the migration of mammalian intestinal or colorectal cancer cells, this study implicates PKA signaling in controlling ISC behaviors in vivo. This study found that PKA activation in ISCs/EBs induces ISC-EC differentiation and EB membrane elongation, whereas PKA activation in ECs nonautonomously stimulates ISC proliferation. PKA regulates cytoskeletal organizing proteins such as Rac, Cdc42, Rho, and PAK. Interestingly, PKA antagonizes Rac to induce morphological changes in neurons. A similar mechanism might explain how PKA affects EB morphology (Xu, 2019).

Ras/MAPK activity in the ISCs/EBs is responsive to a wide spectrum of inputs, including the EGFR pathway, the PDGF- and VEGF-receptor-related pathway, and cytosolic Ca2+ levels. This study confirmed AdoR as another upstream signal that can affect Ca2+ and Ras/MAPK activity. Since earlier studies suggested that GPCRs might affect intracellular Ca2+ levels, whereas high levels of cytosolic Ca2+ levels can induce Ras/MAPK activity in ISCs/EBs, it is likely that the detailed mechanism for AdoR to activate Ras/MAPK implicates the regulation of Ca2+ levels (Xu, 2019).

Following AdoR activation, both Ras/MAPK and PKA signaling are induced to facilitate ISC overproliferation and accelerated production of ECs, whereas the perdurance of PKA activity in a massive number of newly produced ECs has a synergistic effect with Ras/MAPK activity in ISCs/EBs in accelerating proliferation. Since human AdoRs are often highly expressed in carcinomas, a similar paradigm of PKA and Ras/MAPK synergy might fuel oncogenic growth in epithelial tissues (Xu, 2019).

Mammalian AdoRs and human ADA2 have been extensively studied in the hematopoietic and immune systems where ADA2 is produced by differentiating monocytes to stimulate T cell and macrophage proliferation (Hasco, 2008; Zavialov, 2010). Although mammalian AdoRs are expressed in human digestive epithelial cells, their functions remain elusive. Different groups have reported contradictory results suggesting either a protective or a pathological role of AdoR signaling during tissue damage in the mouse intestine, which could be due to the differences in mouse culture conditions, genetic backgrounds, damage models, or inflammation responses. Therefore, this study in Drosophila might help clarify the function of AdoR signaling in the digestive epithelium and in epithelial stem cells (Xu, 2019).

In carcinomas, ADA2 is focally and frequently deleted, based on copy number analysis. Deleterious ADA2 mutations have been identified in colorectal cancers in The Cancer Genome Atlas (TCGA) and Catalogue of Somatic Mutations in Cancer projects. Moreover, ADA2 expression is significantly down-regulated in colorectal cancers, according to microarray studies and RNA-seq datasets from TCGA. Further, anti-ADA2 stainings were detected in the normal digestive epithelium but not in colorectal cancers. Therefore, the down-regulation of ADA2 in colorectal carcinomas has been observed at DNA, RNA, and protein levels. Unfortunately, ADA2 cannot be studied in a mouse model because of a rodent-specific gene loss event during evolution. Moreover, murine developmental and physiological programs have adapted to the loss of ADA2, as transgenic expression of human ADA2 in mice results in abnormal development and embryonic/neonatal lethality. Therefore, these findings describe a striking case in which flies are uniquely suited for understanding the function and regulation of an important disease-related gene (Xu, 2019).

Expression of Drosophila Adenosine Deaminase in immune cells during inflammatory response

Extra-cellular adenosine is an important regulator of inflammatory responses. It is generated from released ATP by a cascade of ectoenzymes and degraded by adenosine deaminase (ADA). There are two types of enzymes with ADA activity: ADA1 and ADGF/ADA2. ADA2 activity originates from macrophages and dendritic cells and is associated with inflammatory responses in humans and rats. Drosophila possesses a family of six ADGF proteins with ADGF-A being the main regulator of extra-cellular adenosine during larval stages. This study present the generation of a GFP reporter for ADGF-A expression by a precise replacement of the ADGF-A coding sequence with GFP using homologous recombination. The reporter is specifically expressed in aggregating hemocytes (Drosophila immune cells) forming melanotic capsules; a characteristic of inflammatory response. The vital reporter thus confirms ADA expression in sites of inflammation in vivo and demonstrates that the requirement for ADA activity during inflammatory response is evolutionary conserved from insects to vertebrates. These results also suggest that ADA activity is achieved specifically within sites of inflammation by an uncharacterized post-transcriptional regulation based mechanism. Utilizing various mutants that induce melanotic capsule formation and also a real immune challenge provided by parasitic wasps, it was shown that the acute expression of the ADGF-A protein is not driven by one specific signaling cascade but is rather associated with the behavior of immune cells during the general inflammatory response. Connecting the exclusive expression of ADGF-A within sites of inflammation, as presented in this study, with the release of energy stores when the ADGF-A activity is absent, suggests that extra-cellular adenosine may function as a signal for energy allocation during immune response and that ADGF-A/ADA2 expression in such sites of inflammation may regulate this role (Novakova, 2011).

The adenosine deaminase activity of ADGF-A is an important regulator of extra-cellular adenosine in Drosophila larvae. Therefore a vital GFP reporter system was made which would allow observation of dynamic changes in the ADGF-A expression in vivo (Novakova, 2011).

This work demonstrates that it was possible to use the ‘ends-in’ based method of homologous recombination to specifically and precisely replace a target gene sequence with that of a reporter (or other heterologous sequence). This approach was used to precisely exchange the entire coding-sequence of the ADGF-A gene with that of the dGFP reporter, leaving intact all the regulatory sequences, including the whole 5' and 3' UTRs, of the locus. This allowed faithful reproduction of the natural expression pattern of ADGF-A in a manner offering maximal fidelity, especially when compared with more conventional methods of transgenic reporter fly generation that rely on random sites of genomic integration (Novakova, 2011).

Molecular and phenotypic characterizations of recombination events, coupled with the developmental and the hemocyte-specific expression profiling clearly demonstrated the successful creation of the reporter. Furthermore that it faithfully recapitulates the expression pattern of the endogenous ADGF-A. Although newer, and arguably less laborious, strategies to target genes or to tag proteins utilizing novel recombineering strategies are now becoming available, it is important to note that the reporter does represents the first faithful reporter of ADGF-A expression to be published. It is anticipated that this will be of use to the wider Drosophila community, especially in light of the absence of anti-ADGF-A antiserum (Novakova, 2011).

Although reporter-derived AGFP mRNA was present in all developmental stages (and in similar levels to the endogenous ADGF-A transcripts), no GFP protein expression was detected at any developmental stage under normal growth conditions. This may be because the expression levels were too low to be detectable using the available methodology. Additionally in the case of heterozygous animals, the reporter was only expressed from one of the two alleles, thus lowering the potentially detectable expression to ~50% compared to normal endogenous ADGF-A expression. However, the detectable GFP fluorescence only at the sites of hemocyte aggregation in both AGFP homozygous and heterozygous animals suggests that heterozygosity of the reporter was not the reason. Also, a destabilized version of GFP was used that is reported to undergo degradation within a couple hours. While this allowed observation of the important dynamic changes in ADGF-A expression, it probably also lowered the overall reporter signal and as a consequence its sensitivity, especially when compared with more stable GFP variants. Therefore it cannot be concluded that there is no expression of ADGF-A/AGFP at the protein level under normal growth conditions but rather if there is any expression, it is certainly very low (Novakova, 2011).

In agreement with the previously reported expression of ADGF-A mRNA in the hematopoietic organ, this study found that the ADGF-A/AGFP mRNA was quite abundant in hemocytes under normal physiological conditions; typified by circulating and sessile non-activated macrophage-like cells called plasmatocytes. Nevertheless, GFP fluorescence was rarely observed in these cells. However it was found that GFP expression was induced in hemocytes during the formation of melanotic capsules, a type of immune response typical of insects. The purpose of melanization is to either isolate and destroy larger foreign bodies that are too big to be phagocytosed or its involvement in the healing of larger wounds. Melanotic capsule formation can be considered a form of inflammatory response since it involves the recruitment and adherence of immune cells (including the macrophage-like plasmatocytes and specialized insect cells called lamellocytes) to large invading objects, as required. Furthermore, both plasmatocytes and lamellocytes expressed the AGFP reporter as they became adhesive and especially in the site of melanotic capsule formation, i.e. the site of inflammation (Novakova, 2011).

Melanotic capsule formation were induced in larvae by four different ways. First, in the adgf-a mutant, it was induced by a disintegration of endogenous larval tissue, the fat body. Second, in the cactus mutant, it was induced by constitutive activation of Toll signaling leading to lamellocytes differentiation and the spontaneous aggregation of both plasmatocytes and lamellocytes with consequent melanotic capsule formation. Third, in the hop^Tum mutant, hyperactivation of the JAK/STAT signalling cascade led to melanotic capsule formation. Last, melanotic capsule formation was induced by a genuine immune reaction to an egg deposited by a parasitic species of wasp. This last approach demonstrated that the observed AGFP reporter expression did not occur as a consequence of the genetic manipulations used in the other three cases, but rather was indicative of a bona fide, in vivo immune response. In all four cases the AGFP reporter was expressed only in the adherent hemocytes of the melanotic capsule, thus indicating that the ADGF-A protein expression is tightly associated with hemocyte function during the immune reaction regardless of the inducing factor and signaling cascades involved (Novakova, 2011).

Ni significant increases were seen in AGFP mRNA levels in hemocytes isolated from larvae forming melanotic capsules (and expressing abundantly detectable GFP fluorescence), when compared to hemocytes from AGFP/+ larvae with no melanotic capsules (and exhibiting a lack of GFP fluorescence). In addition, the ADGF-A/AGFP mRNA was quite abundant in these unchallenged wild-type hemocytes, corresponding to ~10% of the mRNA levels of the ribosomal house-keeping protein gene, Rp49. These results suggest the possibility of a post-transcriptional regulative mechanism of ADGF-A/AGFP protein expression. It should be stressed that both 5' and 3' UTRs of the ADGF-A mRNA were preserved in the AGFP reporter mRNA; only the coding sequence was replaced. Therefore, the potential for adgf-a gene UTR-mediated post-transcriptional regulation of the GFP reporter sequence exists. Since adenosine is readily transported across the plasma membrane by nucleoside transporters, an intriguing possibility may be provided by the potential for a riboswitch mechanism, similar to that present in prokaryotic adenosine deaminase based regulation. Riboswitches have mostly been described as mechanisms of bacterial regulation that enable rapid responses to environmental stimuli. They typically act via the binding of specific ligands (e.g. purine molecules) to riboswitch regulatory elements in 5' UTR of mRNA's, thus prompting or inhibiting their translation. It is therefore possible that when hemocytes find themselves in environments of high concentrations of extra-cellular adenosine, they take the adenosine up and it then binds to a riboswitch within the 5' UTR of the ADGF-A mRNA, that subsequently activates the translation of the ADGF-A protein. Accordingly, increases were observed in the expression of the AGFP reporter system, when isolated but non-adhered hemocytes were challenged by a dose of exogenous adenosine. However it was not possible to obtain quantifiable data due to the inherently weak AGFP signal in this system. Furthermore, it is also possible that the activation or adherence of hemocytes also play a role in this regulation that could not yet be modeled. Nevertheless, it will be very important to further explore the mechanism of this post-transcriptional regulation evidently at work in in this system and investigate whether a similar mechanism is also operating in mammalian systems (Novakova, 2011).

There are at least two ways ADA2 can influence the inflammatory response. Firstly, by its catalytic-independent signaling function and secondly, via regulating adenosine levels through its adenosine deaminase enzymatic activity. It is not known if Drosophila ADGF-A exerts a signaling function similar to human ADA2. This function might be an evolutionary adaptation in vertebrates since the signaling function is associated with adaptive immunity (Zavialov, 2010b) and includes cells that are not present in the innate immunity of Drosophila. Interestingly, insect wounds do not undergo the same burst of cellular proliferation and differentiation that characterizes mammalian wounds healing. However, ADGF-A does shares all the protein domains, including the putative receptor binding domain, with human ADA2 (Zavialov, 2010a) and thus the potential signaling role of ADGF proteins in insects should to be addressed (Novakova, 2011).

The role of ADGF/ADA2 in the site of inflammation is certainly linked to its catalytic activity (the conversion of extra-cellular adenosine to inosine). There are at least two important roles of extra-cellular adenosine during inflammatory response; to mitigate the severity of the potentially harmful response by its anti-inflammatory role and to readjust the energy 'supply-to-demand' ratio by stimulating additional blood flow and glucose release from stores (Novakova, 2011).

Changes in the relative amounts of ATP and adenosine form the core of inflammatory response regulation, and act through the purinergic receptors (Bours, 2006). The release of ATP into the extra-cellular space acts a potently pro-inflammatory, 'danger-associated molecular pattern' (DAMP) signal. Such inflammatory processes are associated with a significant increase in the expression of ecto-5'nucleotidase that acts to rapidly convert ATP into adenosine (Antonioli, 2008). Furthermore, in vertebrates, adenosine itself is a strongly anti-inflammatory molecule (Bours, 2006). and acts later to down-regulate inflammation. Therefore during acute inflammation, increased extra-cellular adenosine is rapidly metabolized to inosine by adenosine deaminase. Indeed, a close correlation can be observed between inflammation and local increases in adenosine deaminase activity (Valdes, 2003; Conlon, 2004). The results in Drosophila confirm this correlation in vivo and suggest that adenosine plays a similar role during the inflammatory response of insects as in vertebrates (Novakova, 2011).

However, lowering extra-cellular adenosine levels may have an important function beyond the site of inflammation. Extra-cellular adenosine is traditionally regarded as a local signal due to its usually rapid metabolism; therefore only exerting localized effects on cells/tissues surrounding its site of production. However, studies in the rat model suggest it can act over longer distances in a hormone-like manner (Cortes, 2009). In this rat model, lower-limb ischemia causes the muscular accumulation of both extra-cellular adenosine and inosine that upon reperfusion are rapidly released into the circulating blood. It is these plasma nucleosides that then promote hepatic glucose release and eventual hyperglycemia, via the activation of A3 adenosine receptor on hepatocytes. It was recently demonstrated that extra-cellular adenosine can also act as an anti-insulin hormone stimulating a release of glucose from stores in the Drosophila model (Zuberova, 2010). Therefore a connection between the work presented here (demonstrating the quite exclusive expression of ADGF-A in sites of inflammation) and a previous study (showing the hyperglycemic effect caused by the deficiency of ADGF-A associated with increased extra-cellular adenosine (Zuberova, 2010) could lead to a reappraisal of the roles of extra-cellular adenosine and its regulation by ADGF-A/ADA2. Accordingly, damaged tissues and sites of inflammation generating significant amounts of extra-cellular adenosine, may serve as sites of hormone production that alert an organism towards the appropriate allocation of energy reserves towards mounting a necessary immune reaction. Such a mechanism would need to be under tight control as not to precipitate harmful hyperglycemia and eventually uncontrolled loss of energy reserves. Therefore, once the stimulus for the inflammation is under control, marked by the presence of sufficient activated immune cells at the inflammation site, the signal for energy release should be suppressed. This could explain why ADGF-A protein is only expressed in fully adhered immune cells at site of inflammation i.e., to dampen this important but potentially dangerous signal. The deficiency of ADGF-A protein that causes hyperglycemia and progressive loss of energy reserves in flies (Zuberova, 2010) illustrates the potential importance of this regulatory circuit. The ability of extra-cellular adenosine to stimulate glucose release over longer distances (Cortes, 2009) and the expression of ADA2 in sites of inflammation (Valdes, 2003; Conlon, 2004) suggest that similar roles of adenosine and ADA2 in energy allocation could be applicable to mammalian systems (Novakova, 2011).

To better understand the immunomodulatory roles of ATP and adenosine it is important to monitor dynamic changes in the expression of their receptors and the enzymes regulating their in vivo concentrations. This is especially applicable in a time when increasing attention is being paid to the role of adenosine system in inflammation and its involvement in, for example, the pathophysiology of inflammatory bowel diseases. This work demonstrates that Drosophila could serve as a valuable and convenient model for visualization of such dynamic changes an in vivo context (Novakova, 2011).

This work reports the creation of a functional in vivo expression reporter for ADGF-A using precise gene replacement homologous recombination procedures. This work confirms the expression of ADGF/ADA2 enzymes in the site of inflammation by showing that ADGF-A expression occurs specifically in the adhered immune cells of such sites in Drosophila. This supports the view that the inflammatory response and its regulation are evolutionary ancient and Drosophila and mammalian systems share common mechanistic features. This model also suggests that the expression of adenosine deaminase during inflammation response might be regulated at post-transcriptional level, therefore potentially providing a means of rapid responding regulation. Previously uncharacterised observations of ADGF-A expression highlight its potential regulatory role in energy allocation stimulated by extra-cellular adenosine (Novakova, 2011).

A role for adenosine deaminase in Drosophila larval development

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. This study has established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organThe hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as has been speculated for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals (Dolezal, 2005).

This study has established an ADA deficiency model in Drosophila in order to study the effects of altered adenosine levels in vivo. A loss-of-function mutation was produced in the ADGF-A gene, which produces a product (ADGF-A) with ADA activity. When homozygous, the mutation causes abnormal hemocyte development, leading to melanotic tumor formation (Rizki, 1957), as well as fat-body disintegration associated with death during the larval stage or delayed transition to the pupal stage of development. In agreement a previous study using cells cultured in vitro (Zurovec, 2002), this study has shown that ADA enzymatic activity is essential for ADGF-A function in vivo, when this function is assayed by testing for rescue of the mutant phenotype. Just as increased levels of both ADA substrates, adenosine and deoxyadenosine, are found in blood of SCID patients, adgf-a mutant larvae also have elevated levels of adenosine and deoxyadenosine, indicating that the mutant phenotype is caused by disturbance in the turnover of these nucleosides (Dolezal, 2005).

Expression of ADGF-A only in the lymph glands is sufficient to fully rescue the mutant phenotype, indicating that the hemocytes within the lymph glands play a major role in regulation of adenosine levels in the hemolymph. A similar regulatory role has also been attributed to blood cells in humans. This suggests a function for ADGF-A within the lymph gland. However, ADGF-A behaves as a soluble growth factor and could be released from the lymph gland to activate targets elsewhere in the larval body. The results show that ADGF-A functions by limiting the level of extracellular adenosine, and in this way the protein could have a systemic function even if it were restricted to its tissue of origin. Although tests did not exclude a role for ADGF-A in circulating hemocytes (which constitute a separate lineage from the lymph gland hemocytes, it was shown that expression of ADGF-A in circulating hemocytes is not required for rescue of the adgf-a mutant phenotype, since e33C-Gal4/UAS-ADGF-A - which expresses ADGF-A in the lymph gland but not in circulating hemocytes - fully rescued the phenotype (Dolezal, 2005).

Late third-instar larvae homozygous for the adgf-a mutation contain, on average, seven times more hemocytes in circulation than wild-type larvae, and most of these cells show strong adhesive properties compared to normal larval plasmatocytes, which remain rounded after settling down on the substrate. Although these cells share other characteristics with plasmatocytes, they are normally not seen in circulation until they are released from the lymph glands at the onset of metamorphosis under the regulation of ecdysone to serve as phagocytes for histolysing tissues during metamorphosis—thus, they are referred to as pupal macrophages. In agreement with the presence of these cells in circulation, at least the first lobes of the lymph glands are usually completely dispersed in late third-instar mutant larvae. This indication of precocious metamorphic changes in the mutant is further supported by the finding that hemocytes aggregate in a segmental pattern in early rather than late third instar, and that the hemocytes lose expression of Hemolectin in late third-instar larvae rather than at the onset of metamorphosis (Dolezal, 2005).

Recent studies show that the Toll signaling pathway, which is already known to be involved in the control of innate immunity of both Drosophila and mammals, may also be involved in the control of hemocyte differentiation in the Drosophila larva. Constitutive activation of Toll signaling leads to developmental arrest and hematopoietic defects associated with melanotic tumor formation, similar to the phenotype of the adgf-a mutant. The current work also shows that forced expression of the ADGF-A gene can rescue the effects of overactive Toll signaling, suggesting that ADGF-A might function downstream of Toll signaling to control its effects. This conclusion is consistent with the existence of a putative binding site for Dorsal (one of two known effectors of Toll signaling) in the ADGF-A promoter. It will be important to explore this connection further, since recent studies suggest an interaction between adenosine signaling and the NF-kappaB signaling pathway, which is the mammalian counterpart of the Toll pathway (Dolezal, 2005).

One of the most remarkable features of the adgf-a mutant phenotype is the disintegration of the fat body in third-instar larvae, another indication of precocious metamorphic changes since the disintegration normally occurs much later, during pupal life. Furthermore, study of this mutant provides strong evidence that the fat body disintegration is promoted by the action of hemocytes. Fat body disintegration was significantly suppressed when the hemocyte number was reduced using the l(3)hem¹ mutation, and fully blocked by the croquemort (crq) mutation which affects a CD36-related receptor (Croquemort) expressed on macrophages and required in phagocytosis of apoptotic cells. Human CD36 is a scavenger receptor which, in combination with the macrophage vitronectin receptor and thrombospondin, binds apoptotic cells. A similar role of Croquemort for removing histolysing tissues during Drosophila metamorphosis has not yet been tested, but seems likely since the crq mutant used in this study (crq^KG01679) is lethal in pupae (Dolezal, 2005).

The idea that hemocytes are involved in fat body dissociation in Drosophila is further supported by work on the flesh fly Sarcophaga. Natori's group showed that proteinase cathepsin B was released from pupal hemocytes when they interacted with the fat body, and that this enzyme digested the basement membrane of the fat body, causing the tissue to dissociate (Yano, 1995). It was also shown that the interaction of hemocytes with the fat body is mediated by a 120-kDa membrane protein localized specifically on pupal hemocytes (Hori, 2000). This protein was suggested to be a scavenger receptor, but it does not seem to be homologous to Drosophila Croquemort. Published evidence is consistent with the idea that more than one scavenger receptor is involved in this process (Dolezal, 2005).

The precocious metamorphic changes that appear to occur in response to elevated adenosine in the adgf-a mutant larvae lead to the suggestion that adenosine may act as a regulatory signal for these processes during normal development. One possibility is that adenosine acts as a downstream effector of ecdysone-regulated prepupal changes, and that the increase in adenosine concentration is mediated by ecdysone-induced down-regulation of ADGF-A expression. This is supported by the presence of multiple sites for ecdysone-inducible transcription regulators in the ADGF-A promoter. Adenosine could serve as a signal for macrophage differentiation, and the lack of adenosine deaminase activity due to the adgf-a mutation could cause precocious differentiation of these cells in mutant larvae. Direct tests are being carried out of the idea that the differentiation of hemocytes in mutant larvae is caused by elevated adenosine. If confirmed, this effect would have general significance, since in ADA-deficient mice, inflammatory changes in the lungs include an accumulation of activated alveolar macrophages, and this could also be mediated by elevated adenosine (Dolezal, 2005).

The elevated adenosine in the adgf-a mutant larvae leads to precocious changes (hemocyte differentiation and fat body disintegration) resembling those normally occurring at the time of metamorphosis, but it also is associated with an apparently opposite effect, in that it causes a significant delay in progress through the third larval instar and a decrease in the frequency of successful pupariation (formation of the puparium from the larval cuticle), which is one of the earliest steps in metamorphosis. It is concluded that the mutation has additional effects on the hormonal regulation of development (Dolezal, 2005).

One possible explanation for the developmental delay and failure to pupariate is that the adgf-a mutation affects the production or release of ecdysteroid hormones from the major endocrine organ of the Drosophila larva - the ring gland. This is supported by the fact that pupariation rate and survival of the adgf-a mutant can be significantly improved by expression of transgenic ADGF-A in the ring gland and salivary glands. It is suggested that this somehow interferes with the regulation of hormone release. Other mutants with hormonal dysregulation show delayed larval development and failure to pupariate. Presumably the elevated adenosine in the adgf-a mutant blocks the production or release of ecdysone from the ring gland by an unknown mechanism. This idea is supported by the finding that both pupariation rate and survival of the adgf-a mutant can also be improved by feeding the mutant larvae with 20E in the diet. Thus it is clear that the adgf-a mutant is arrested in development due to an effect of the mutation on hormone production from the ring gland (Dolezal, 2005).

The arrest of development in the adgf-a mutants was significantly suppressed by loss of the adenosine receptor caused by the adoR mutation: larvae simply homozygous for adgf-a pupated after two or more days, whereas larvae also homozygous for adoR pupated within 1 d after their heterozygous siblings. Therefore, adenosine signaling through the AdoR must play a role in the developmental arrest of the adgf-a mutant, and this is most likely mediated by signaling to the ring gland, where AdoR is expressed. The mutation in AdoR does not block macrophage differentiation and fat-body disintegration, so this effect must involve another, as yet uncharacterized mechanism independent of AdoR signaling. Work using adenosine-receptor deficient mammalian cells also suggested the existence of a novel, undefined adenosine signaling mechanism. However, the role of elevated deoxyadenosine in these effects cannot be excluded. Drosophila, now with the advantage of the well-characterized adgf-a mutant, could serve as an ideal model system in which to investigate this mechanism (Dolezal, 2005).

In previous work using cells cultured in vitro, it was shown that, as in mammals, adenosine can block proliferation and/or survival of some Drosophila cell types (Zurovec, 2002). The present work established a Drosophila model to study altered levels of adenosine and deoxyadenosine in vivo, and it was shown that loss of ADGF-A function causes an increase of these nucleosides in larval hemolymph. Although the adgf-a mutation leads to larval or pupal death, this study has shown that this is not due to the adenosine or deoxyadenosine simply blocking cellular proliferation or survival, as the experiments in vitro would suggest. Rather, this mutation leads to an increase in number of hemocytes at the end of larval development due to the differentiation and release of hemocytes from the lymph glands. Hemocytes also differentiate and are released from the lymph glands during systemic infection. Together with the result suggesting an interaction between Toll signaling and ADGF-A, this leads to the hypothesis that adenosine controls hemocyte differentiation in response to infection, and that it signals through the adenosine receptor to postpone the next developmental step, metamorphosis. This would be consistent with the role of adenosine as a 'stress hormone' in mammals. A similar process of hemocyte differentiation and release from the lymph glands normally takes place at the onset of metamorphosis, when pupal macrophages remove histolyzing tissues. The ADGF-A promoter contains consensus binding sites for effectors of both Toll and ecdysone signaling. This raises the possibility that adenosine plays a role in the control of metamorphosis as well as in the response to stress (Dolezal, 2005).

Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila by depleting extracellular adenosine

A protein family in Drosophila is described containing six adenosine deaminase-related growth factors (ADGFs) that are homologous to a mitogenic growth factor discovered in conditioned medium from cells of a different fly species, Sarcophaga. Closely related proteins have been identified in other animals, and a human homolog is implicated in the genetic disease Cat-Eye Syndrome. The two most abundantly expressed ADGFs in Drosophila larvae are ADGF-A, which is strongly expressed in the gut and lymph glands, and ADGF-D, which is mainly expressed in the fat body and brain. Recombinant ADGF-A and ADGF-D are active adenosine deaminases (ADAs), and they cause polarization and serum-independent proliferation of imaginal disk and embryonic cells in vitro. The enzymatic activity of these proteins is required for their mitogenic function, making them unique among growth factors. A culture medium prepared without adenosine, or depleted of adenosine by using bovine ADA, also stimulates proliferation of imaginal disk cells, and addition of adenosine to this medium inhibits proliferation. Thus ADGFs secreted in vivo may control tissue growth by modulating the level of extracellular adenosine (Zurovec, 2002; full text of article).

Search PubMed for articles about Drosophila Adgf-A

Alcada-Morais, S., Goncalves, N., Moreno-Juan, V., Andres, B., Ferreira, S., Marques, J. M., Magalhaes, J., Rocha, J. M. M., Xu, X., Partidario, M., Cunha, R. A., Lopez-Bendito, G. and Rodrigues, R. J. (2021). Adenosine A2A Receptors Contribute to the Radial Migration of Cortical Projection Neurons through the Regulation of Neuronal Polarization and Axon Formation. Cereb Cortex. PubMed ID: 34184030

Antonioli, L., et al. (2008). Pharmacological modulation of adenosine system: novel options for treatment of inflammatory bowel diseases. Inflamm. Bowel Dis. 14(4): 566-574. PubMed ID: 18022872

Bours, M. J., et al. (2006). Adenosine 5'-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation. Pharmacol Ther. 112(2): 358-404. PubMed ID: 16784779

Conlon, B. A. and Law, W. R. (2004). Macrophages are a source of extra-cellular adenosine deaminase-2 during inflammatory responses. Clin. Exp. Immunol. 138(1): 14-20. PubMed ID: 15373900

Cortés, D., et al. (2009). Evidence that endogenous inosine and adenosine-mediated hyperglycaemia during ischaemia-reperfusion through A3 adenosine receptors. Auton. Autacoid Pharmacol. 29(4): 157-164. PubMed ID: 19740086

Dolezal, T. et al. (2005). A role for adenosine deaminase in Drosophila larval development. PLoS Biol. 3: e201. PubMed ID: 15907156

Dolezelova, E., et al. (2007). A Drosophila adenosine receptor activates cAMP and calcium signaling. Insect Biochem. Mol. Biol. 37: 318-329. PubMed ID: 17368195

Fredholm, B. B. (2007). Adenosine, an endogenous distress signal, modulates tissue damage and repair. Cell Death Differ. 14: 1315-1323. PubMed ID: 17396131

Hori, S., Kobayashi, A., Natori, S. (2000). A novel hemocyte-specific membrane protein of Sarcophaga (flesh fly). Eur. J. Biochem. 267: 5397-5403. PubMed ID: 10951197

Losick, V. P., et al. (2011). Drosophila stem cell niches: A decade of discovery suggests a unified view of stem cell regulation. Dev. Cell 21: 159-171. PubMed ID: 21763616

Mandal, L., et al. (2007). A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors. Nature 446(7133): 320-4. PubMed ID: 17361183

Mondal, B. C., et al. (2011). Interaction between differentiating cell- and niche-derived signals in hematopoietic progenitor maintenance. Cell 147(7): 1589-600. PubMed ID: 22196733

Ng, W. L. and Bassler, B. L. (2009). Bacterial quorum-sensing network architectures. Annu. Rev. Genet. 43: 197-222. PubMed ID: 19686078

Novakova, M. and Dolezal, T. (2011). Expression of Drosophila adenosine deaminase in immune cells during inflammatory response. PLoS ONE 6(3): e17741. PubMed ID: 21412432

Rizki, M. T. M. (1957). Tumor formation in relation to metamorphosis in Drosophila melanogaster. J. Morphol. 100: 459-472

Valdés, L., et al. (2003). Tuberculous pleural effusions. Eur. J. Intern. Med. 14(2): 77-88. PubMed ID: 12719023

Xu, C., Franklin, B., Tang, H. W., Regimbald-Dumas, Y., Hu, Y., Ramos, J., Bosch, J. A., Villalta, C., He, X. and Perrimon, N. (2020). An in vivo RNAi screen uncovers the role of AdoR signaling and adenosine deaminase in controlling intestinal stem cell activity. Proc Natl Acad Sci U S A 117(1): 464-471. PubMed ID: 31852821

Yano, T., Takahashi, N., Kurata, S. and Natori, S. (1995). Regulation of the expression of cathepsin B in Sarcophaga peregrina (flesh fly) at the translational level during metamorphosis. Eur. J. Biochem. 234: 39-43. PubMed ID: 8529666

Zavialov, A. V., et al. (2010a). Structural basis for the growth factor activity of human adenosine deaminase ADA2. J. Biol. Chem. 285(16): 12367-12377. PubMed ID: 20147294

Zavialov, A. V., et al. (2010b). Human adenosine deaminase 2 induces differentiation of monocytes into macrophages and stimulates proliferation of T helper cells and macrophages. J. Leukoc. Biol. 88(2): 279-90. PubMed ID: 20453107

Zuberova, M., et al. (2010). Increased extra-cellular adenosine in Drosophila that are deficient in adenosine deaminase activates a release of energy stores leading to wasting and death. Dis. Model Mech. 3(11-12): 773-784. PubMed ID: 20940317

Zurovec, M., et al. (2002). Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila by depleting extracellular adenosine. Proc. Natl. Acad. Sci. 99: 4403-4408. PubMed ID: 11904370

date revised: 2 January 2023

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