InteractiveFly: GeneBrief

Zinc finger protein RP-8: Biological Overview | References

Zfrp8 is essential for hematopoiesis in Drosophila. Zfrp8 (Zinc finger protein RP-8) is the Drosophila ortholog of the PDCD2 (programmed cell death 2) protein of unknown function, and is highly conserved in all eukaryotes. Zfrp8 mutants present a developmental delay, lethality during larval and pupal stages and hyperplasia of the hematopoietic organ, the lymph gland. This overgrowth results from an increase in proliferation of undifferentiated hemocytes throughout development and is accompanied by abnormal differentiation of hemocytes. Furthermore, the subcellular distribution of γ-Tubulin and Cyclin B is affected. Consistent with this, the phenotype of the lymph gland of Zfpr8 heterozygous mutants is dominantly enhanced by the l(1)dd4 gene encoding Dgrip91, which is involved in anchoring γ-Tubulin to the centrosome. The overgrowth phenotype is also enhanced by a mutation in Cdc27, which encodes a component of the anaphase-promoting complex (APC) that regulates the degradation of cyclins. No evidence for an apoptotic function of Zfrp8 was found. Based on the phenotype, genetic interactions and subcellular localization of Zfrp8, it is proposed that the protein is involved in the regulation of cell proliferation from embryonic stages onward, through the function of the centrosome, and regulates the level and localization of cell-cycle components. The overproliferation of cells in the lymph gland results in abnormal hemocyte differentiation (Minakhina, 2007).

The developmental mechanisms of human and Drosophila blood systems show remarkable parallels. In humans, several blood cell types with specific functions develop from the same pluripotent stem cells. In Drosophila, only a few specialized cell types exist, with functions similar to human cells. These are thought to originate from a common set of hematopoietic precursors. The development and specification of blood cells in humans and flies are controlled by conserved signaling pathways. Because of its relative simplicity, hematopoiesis in Drosophila is frequently used as a model to investigate the genetic control of hematopoiesis in flies and humans (Minakhina, 2007).

In Drosophila, mature hemocytes arise from two distinct sources: the mature larval circulating hemocytes derive from the embryonic head mesoderm, whereas the lymph gland hemocytes are normally released into circulation at the onset of metamorphosis and perdure into the adult stage. As in vertebrate blood and vascular systems, the Drosophila lymph gland hemocytes and heart cells derive from a common progenitor, called the hemangioblast or cardiogenic mesoderm, which further splits into the lymph gland and cardiogenic progenitors (Mandal, 2004; Minakhina, 2007).

Among the earliest requirements for the specification of blood progenitors in mammals and Drosophila are the highly conserved, GATA zinc-finger transcription factors. The Drosophila GATA-factor Pannier (Pnr) is required for early specification of the hemangioblast/cardiogenic mesoderm. Another GATA-factor, Serpent (Srp), plays a central role in committing mesodermal precursors to the hemocyte fate (Minakhina, 2007).

By the end of embryogenesis, the lymph gland is fully formed and contains mostly pro-hemocytes. The third instar larval lymph gland contains a pair of primary and several secondary lobes. Each primary lobe is subdivided into (1) the medullary zone, populated by slowly proliferating pro-hemocytes; (2) the cortical zone, containing differentiated hemocytes; and (3) the posterior signaling center (PSC), first defined as a small group of cells expressing the Notch ligand Serrate (Ser). Under the control of the EBF-homolog (early B-cell factor) collier (col; knot), PSCs function as a hematopoietic niche to maintain a population of blood cell precursors (Krzemien, 2007; Lebestky, 2003; Mandal, 2007). The blood cell precursors differentiate into three groups of hemocytes: plasmatocytes, crystal cells and lamellocytes. All three are released into the open circulating hemolymph during the onset of metamorphosis or as a part of an immune reaction (Holz, 2003; Jung, 2005). Differentiated plasmatocytes and crystal cells are found in both the cortical zone of the lymph gland and the larval hemolymph, but lamellocytes are rare (Minakhina, 2007).

Plasmatocytes, the predominant form of hemocytes in larvae, perform phagocytic functions and secrete extracellular matrix components and immune peptides similar to human white blood cells. Crystal cells are non-adhesive hemocytes responsible for melanization during wound healing and encapsulation of parasites. Crystal cell differentiation requires the cell-autonomous expression of the transcription factor Lozenge (Lz), homologous to the mammalian acute myeloid leukemia 1 protein (Aml1 or Runx1) (Minakhina, 2007).

Lamellocytes function in encapsulation. Their number is significantly increased at the onset of metamorphosis and in response to infection. Differentiation of lamellocytes is connected to two major pathways - the Drosophila Toll/NF-kappaB and the JAK/STAT - that regulate blood cells proliferation and activation during immune response. Constitutive activation of either pathway leads to overproliferation of circulating and lymph gland hemocytes, an increase in lamellocytes and activation of the cellular immune response (Minakhina, 2007).

A newly identified gene, Zfrp8, is essential for lymph gland growth and for the normal development of Drosophila larvae. Mutant larvae show hyperplasia of the hematopoietic organs. This phenotype is not linked to apoptosis but rather to an increase in cell proliferation. Mutant lymph glands also show a drastic increase in the number of lamellocytes (Minakhina, 2007).

These phenotypes are suppressed by mutations in the GATA factor gene pnr. Mutations in the two cell-cycle genes Cdc27 and l(1)dd4 [lethal (1) discs degenerate 4], have the opposite effect as they enhance the lymph gland overgrowth phenotype of Zfrp8/+. Cdc27 encodes a subunit of the APC complex, responsible for the turnover of cyclins, and l(1)dd4 encodes Dgrip91, a component of the centrosome involved in γ-Tubulin anchoring. In the Zfrp8 mutant lymph gland cells, both Cyclin B (CycB) and γ-Tubulin exhibit abnormal subcellular distribution, suggesting that Zfrp8 plays an important role in their regulation (Minakhina, 2007).

In the literature, the Zfrp8 vertebrate ortholog, PDCD2, is routinely referred to as an apoptotic gene solely because it was upregulated during steroid-induced programmed cell death in rat thymocytes. Subsequent studies, using different cells and assay conditions, found no connection between PDCD2 expression and programmed cell death (Chen, 2005; Fan, 2004; Kawakami, 1995; Steinemann, 2003; Minakhina, 2007).

It is unlikely that a reduction in cell death is the cause of the lymph gland overgrowth observed in Zfrp8 mutant larvae. Very few or no apoptotic cells are detected in wild-type larval lymph glands. This study found a statistically insignificant increase in the number of apoptotic cells in Zfrp8 mutants. No other evidence of change in programmed cell death in Zfrp8 mutant animals, no increase in apoptotic gene expression, no change in caspase cleavage and no genetic interaction of Zfrp8 with known apoptotic genes were found (Minakhina, 2007).

The results are consistent with an increase in cell division in Zfrp8 mutants throughout development. This conclusion is supported by the observation that Zfrp8 lymph glands are already twice the size of their normal counterparts in late-stage embryos, and that the number of cells in mitosis is about 30% higher in the mutant glands than in wild type (Minakhina, 2007).

Detailed analysis of Zfrp8 lymph glands shows that its phenotype is different from that of Drosophila hematopoietic/immunity mutants. Unlike hematopoietic/immunity mutants, the increase in lymph gland cell numbers is much larger than the increase in circulating hemocytes. Furthermore, the blood cell overproliferation in Zfrp8-null mutants is not accompanied by constitutive activation of immunity. Zfrp8 larvae show normal induction of immune peptide genes in response to bacterial challenge and normal wound clogging and wound melanization. That the requirements are different for Zfrp8 and known hematopoiesis and immunity genes is underlined by the absence of their genetic interaction (Minakhina, 2007).

In normal lymph glands, plasmatocytes are found mostly in the cortical region and very few lamellocytes are detected. The PSC is formed at the base of each primary lobe. The presence of additional PSCs in mutant lymph glands might indicate that additional primary lobes are formed by the large number of cells (Minakhina, 2007).

Two recent papers report that the PSCs are essential for maintaining the undifferentiated hemocyte population in the medullary zone and that they control lamellocyte differentiation during parasitic infection (Krzemien, 2007; Mandal, 2007). Lack of the transcription factor collier, essential for PSC maintenance, leads to a decrease in the pro-hemocyte population and abolishes lamellocyte differentiation. Loss of Zfrp8 leads to the opposite phenotype - an increase in pro-hemocyte proliferation, beginning during embryogenesis, and an increased number of cells acquiring the lamellocyte fate. Expansion of the PSCs alone does not account for this phenotype. Ectopic expression of the homeotic gene Antennapedia results in expansion of the PSCs, and a concomitant increase of the medullar zone, but not the gland overgrowth (Mandal, 2007). Therefore, it is unlikely that Zfrp8 is directly involved in the establishment of PSCs (Minakhina, 2007).

The results link the Zfrp8 overgrowth phenotype to a defect in normal cell proliferation. In mutant lymph glands, the cell-cycle markers γ-Tubulin and CycB are misregulated. Zfrp8 genetically interacts with at least two genes functioning in the cell cycle, Cdc27 encoding a subunit of the anaphase-promoting complex (APC), and l(1)dd4 encoding the Drosophila gamma-ring protein Dgrip91 (Minakhina, 2007).

Dgrip91 and γ-Tubulin are components of the γ-TuRC microtubule-nucleating complex anchored to centrosomes. Beyond the conventional role in microtubule organization, centrosomes also serve as a scaffold for anchoring a number of cell-cycle regulators. For instance, centrosome-association of Cdc27 and CycB proteins plays an important role in CycB activation, degradation and entrance into mitosis (Minakhina, 2007).

The link between the phenotypes described above and Zfrp8 function became clear when it was discovered that a proportion of Zfrp8 protein localizes adjacent to the centrosome in wild-type tissue. This subcellular localization is consistent with a function of Zfrp8 in centrosome organization and in the anchoring of proteins such as γ-Tubulin and CycB to this organelle (Minakhina, 2007).

Zfrp8 might also affect the expression of bona fide cell-cycle regulators. The protein contains a zinc-finger domain, MYND, present in a number of transcriptional regulators, that fosters protein-protein interactions and recruits co-repressors. PDCD2/Zfrp8 is known to interact with the HCF-1 transcriptional regulator, which suggests that PDCD2/Zfrp8 might be involved in regulating the cell cycle at the transcriptional level (Minakhina, 2007).

Zfrp8 might have a dual function, through its association with the centrosome and as a transcriptional regulator of the cell cycle. Several transcriptional regulators have been found to localize to the centrosome, but their centrosomal function has not been documented (Minakhina, 2007).

Zfrp8 function is essential for the control of cell proliferation already in the embryo. With this being the case, it functions upstream from most of the conserved signaling pathways involved in fly hematopoiesis and immunity. Because of the similarity of the protein in flies and vertebrates, it is possible that PDCD2 has a similar function in vertebrate hematopoiesis (Minakhina, 2007).

Hematopoietic stem cells in Drosophila

The Drosophila lymph gland, the source of adult hemocytes, flanks the dorsal vessel and is established by mid-embryogenesis. During larval stages, a pool of pluripotent hemocyte precursors differentiate into hemocytes that are released into circulation upon metamorphosis or in respond to immune challenge. This process is controlled by the posterior signaling center (PSC), which is reminiscent of the vertebrate hematopoietic stem cell niche. Using lineage analysis, bona fide hematopoietic stem cells (HSCs) were identified in the lymph glands of embryos and young larvae, which give rise to a hematopoietic lineage. These lymph glands also contain pluripotent precursor cells that undergo a limited number of mitotic divisions and differentiate. It was further found that the conserved factor Zfrp8/PDCD2 (Minakhina, 2007) is essential for the maintenance of the HSCs, but dispensable for their daughter cells, the pluripotent precursors. Zfrp8/PDCD2 is likely to have similar functions in hematopoietic stem cell maintenance in vertebrates (Minakhina, 2010).

Drosophila blood cell development occurs in two phases. In the first, 'primitive' phase, hemocytes develop from the early embryo head mesoderm and supply the pool of circulating blood cells. The second phase gives rise to adult hemocytes, produced in a small organ, the lymph gland. The larval lymph gland and the differentiation of hemocytes have been studied using a range of cell-specific markers. The primary, largest lobe of the larval lymph gland is sub-divided into the posterior signaling center (PSC), the medullary zone (MZ) and the cortical zone (CZ) (see illustration in Jung, 2005). The MZ has been thought to contain a relatively uniform population of pluripotent prohemocytes (PH), sometimes called stem-like cells. These cells migrate into the CZ as they differentiate into plasmatocytes (PM), crystal cells (CC) and lamellocytes (LM). The homeostasis between prohemocytes and differentiated blood cells is maintained by the PSC (Minakhina, 2010 and references therein).

To investigate the existence of stem cells in the Drosophila lymph gland, clones were induced in embryos and first instar larvae, using the MARCM technique combined with UAS-GFP reporters. This technique results in marking a single cell and its progeny, and revealed that in wild-type lymph glands both persistent and transient clones are induced, indicating the presence of hematopoietic stem cells (Minakhina, 2010).

Because stem cells usually represent only a small fraction of the cells in an organ, they are difficult to identify and study. The MARCM technique was chosen because it marks cells undergoing mitosis, such as stem cells, which are particularly active in young animals. Clones were produced at four embryonic stages, 2-6 hours, 6-12 hours, 12-18 hours and 18-24 hours, and in first instar larvae, by exposing animals to 38^oC for one hour to activate the heat-inducible FLP-recombinase. 2-6 hour embryos contain about nine precursor cells that will form one lymph gland lobe and the cardioblasts. In 6-12 hour embryos, a lymph gland lobe contains about 12 cells, and at stage 16, 20-25 cells. By late first instar larval stage cells have undergone, on average, one additional division. In the absence of infection, hemocytes remain in the lymph gland until metamorphosis when they are released into circulation. This aspect of hemocyte development allowed wild-type and Zfrp8 clones to be followed from the embryo to the third instar larval stage by noting the distribution of marked cells in the lymph glands. As expected, wild-type and mutant PSC clones were obtained with similar frequency (about 6%-9%) after induction between 6-18 hours. They had comparable phenotypes and did not mix with non-PSC hemocytes (Minakhina, 2010).

Except for the cells in the PSC, the hemocyte precursors within the embryonic lymph glands appear identical and were therefore expected to have similar lineage potential, and to produce clones of comparable size and appearance. However, a large variety of non-PSC clones were recovered that were subdivided into four types according to their size, shape and location. Type 1 are large clones encompassing 10-30% of all of the lymph gland cells that form cohesive clusters. They occupy a large part of the medulla and extend into the cortex, where they scatter into secondary small clusters. All ten type 1 clones that were stained with the PSC marker Antp contained cells, probably the founder cells, and were in immediate contact with the PSC. The frequency of type 1 clones remained about the same (18-29%) independent of when they were induced during embryogenesis. But their frequency was strongly reduced when the clones were induced in first instar larvae (Minakhina, 2010).

Type 1 clones showed the characteristics of 'persistent; clones that are expected when the clone is induced in HSCs or their precursors (primordial cells). Founder cells in these clones were in contact with the PSC hematopoietic niche, they could self-renew and were pluripotent, meaning that they could differentiate into plasmatocytes, crystal cells and probably lamellocytes (there are too few lamellocytes in a normal lymph gland to establish this positively). By contrast, type 3 and 4 clones clearly arose from cells that have no self-renewal properties, cells that divide, migrate into the cortex, and differentiate. Because these cells are gradually removed from the medulla, type 3 and 4 clones are considered to be 'transient'. The types of clones obtained are consistent with the existence of stem cells that can self-renew and replenish the population of pluripotent hemocyte precursors, while their daughter cells divide several times and commit to differentiation. The four types of clones also indicate that the hematopoietic lineage contains at least three developmental stages in addition to the stem cells. All persistent and most transient clones consisted of one or several contiguous patches and scattered cells, indicating that cell mixing was prevalent, especially when cells moved into the cortex (Minakhina, 2010).

All four types of clones were observed in wild-type glands, independently of when the clones were induced, suggesting that already at the earliest embryonic stage the lymph gland cells have different developmental potentials, and that all cell types persist at least through the first larval instar. These observations suggest that some of the primordial cells do not form stem cells but undergo differentiation similar to what is observed in the ovary, where some prestem cells fail to form stem cells and instead undergo differentiation. The proportion of type 1 clones was significantly lower in first instar larvae than in early embyos, indicating that the number of stem cells stays relatively constant while their daughter cells multiply. Stem cells are likely to be present still in later larval stages, but they would be difficult to detect because of their relatively low numbers and because their mitotic activity may be reduced. Furthermore, if clones were induced in second and third instar larvae, the short time between the induction of the clones and their analysis would not be sufficient to see a clear difference beween persistent and transient clones (Minakhina, 2010).

The results show that embryonic and first instar larval lymph glands contained HSCs (type 1 clones), transient pluripotent progenitors (type 2 and 3 clones), and cells with limited mitotic potential (type 4). The presence of HSCs in wild-type glands was further validated by the fact that these cells were lost in the absence of Zfrp8 (Minakhina, 2010).

Zfrp8 (Minakhina, 2007), also called PDCD2, is highly conserved from flies to humans, and its molecular and physiological function is generally not well understood. Loss of Zfrp8 causes a unique phenotype in Drosophila. The lymph gland is enlarged already in mid-embryogenesis and by the late third instar larval stage, the lymph gland size is increased 10 to 50 times, accompanied by lamellocyte overproliferation (Minakhina, 2010).

To study the function of Zfrp8 throughout hematopoiesis, GFP-labeled homozygous mutant Zfrp8 clones were induced in Zfrp8 heterozygous animals. Analysis of the Zfrp8 mutant lymph gland clones showed that their occurrence differed remarkably from that of wild type. The most striking result was that no type 1 (HSC) clones were detected. The percentage of type 2 clones was reduced, whereas that of type 3 and 4 clones was increased, especially when induced in young embryos. The percentage of mosaic animals with no lymph gland clones was double that of wild type (Minakhina, 2010).

In spite of this shift, the phenotypes of type 2, 3 and 4 clones were indistinguishable from that of wild type. Lack of Zfrp8 did not result in hemocyte or lamellocyte overproliferation within the clone. The pluripotency of Zfrp8 mutant prohemocytes was the same as that of wild-type cells, indicating that Zfrp8 is not required in cells that give rise to transient clones. A similar result was found in other tissues where the clonal loss of Zfrp8 resulted in cells that looked indistinguishable from their wild-type neighbors. Cell proliferation, viability or differentiation was not affected (Minakhina, 2010).

The absence of persistent clones, the decrease of animals with clones in the lymph gland, the increase of type 3 and 4 clones in young animals, and the absence of a phenotype within the clones, all suggest that Zfrp8 is required specifically in stem cells. Stem cells lacking Zfrp8 loose their ability to self-renew and instead behave like more mature prohemocytes (Minakhina, 2010).

To ascertain whether the Zfrp8 mutant phenotype was consistent with the loss of HSCs, mutant lymph gland growth and hemocyte differentiation were examined during several stages of larval development. Peroxidasin (Pxn) is an early cortex marker expressed in cells committed to differentiation (see Jung, 2005). As in wild type, in second instar mutant glands Pxn-negative cells were detected in the medulla and positive cells in the cortex, indicating that these Zfrp8 mutant glands contain hemocyte precursors and prohemocytes. But in early third instar mutant larvae, all lymph gland cells had become Pxn-positive, indicating that all hemocyte precursor cells, normally present in the medulla, had matured. The absence of hemocyte precursors is consistent with the finding that HSCs, which would replenish this hemocyte population throughout development, were missing in Zfrp8 mutant lymph glands. Thus, the lack of Zfrp8 explains the absence of HSCs and the subsequent loss of the medulla. Larvae without a PSC also lack medulla. The overlap of these two phenotypes is consistent with the PSC controlling the development of the HSCs. Conversely, the massive Zfrp8 mutant hemocyte overgrowth was not seen in animals without a PSC, which indicates the existence of an additional signal, possibly also originating in the PSC, that controls hemocyte proliferation and differentiation (Minakhina, 2010).

This study has found evidence for a Drosophila hematopoietic lineage established by a stem cell and, further, that the identity of the HCS is dependent on the function of Zfrp8. It is possible that the Zfrp8 human homolog, the PDCD2 protein, has a similar function. PDCD2 is more highly expressed in a CD34⁺ bone marrow fraction, enriched in HSCs, than in a sample of total bone marrow cells. Consistent with this observation, transcriptional profiling of mouse embryonic, neural and hematopoietic stem cells showed an enrichment of PDCD2 mRNA in all three stem cells (Ramalho-Santos, 2002; Minakhina, 2010).

Search PubMed for articles about Drosophila Zfrp8

Chen, Q., Qian, K. and Yan, C. (2005). Cloning of cDNAs with PDCD2(C) domain and their expressions during apoptosis of HEK293T cells. Mol. Cell. Biochem. 280: 185-191. PubMed Citation: 16311922

Fan, C. W., Chan, C. C., Chao, C. C., Fan, H. A., Sheu, D. L. and Chan, E. C. (2004). Expression patterns of cell cycle and apoptosis-related genes in a multidrug-resistant human colon carcinoma cell line. Scand. J. Gastroenterol. 39: 464-469. PubMed Citation: 15180185

Holz, A., Bossinger, B., Strasser, T., Janning, W. and Klapper, R. (2003). The two origins of hemocytes in Drosophila. Development 130: 4955-4962. PubMed Citation: 12930778

Jung S. H., Evans C. J., Uemura C. and Banerjee U. (2005). The Drosophila lymph gland as a developmental model of hematopoiesis. Development 132: 2521-2533. PubMed Citation: 15857916

Kawakami, T., Furukawa, Y., Sudo, K., Saito, H., Takami, S., Takahashi, E. and Nakamura, Y. (1995). Isolation and mapping of a human gene (PDCD2) that is highly homologous to Rp8, a rat gene associated with programmed cell death. Cytogenet. Cell Genet. 71: 41-43. PubMed Citation: 7606924

Krzemien, J., Dubois, L., Makki, R., Meister, M., Vincent, A. and Crozatier, M. (2007). Control of blood cell homeostasis in Drosophila larvae by the posterior signalling centre. Nature 446: 325-328. PubMed Citation: 17361184

Lebestky, T., Jung, S. H. and Banerjee, U. (2003). A Serrate-expressing signaling center controls Drosophila hematopoiesis. Genes Dev. 17: 348-353. PubMed Citation: 12569125

Mandal, L., Banerjee, U. and Hartenstein, V. (2004). Evidence for a fruit fly hemangioblast and similarities between lymph-gland hematopoiesis in fruit fly and mammal aorta-gonadal-mesonephros mesoderm. Nat. Genet. 36: 1019-1023. PubMed Citation: 15286786

Mandal, L., Martinez-Agosto, J. A., Evans, C. J., Hartenstein, V. and Banerjee, U. (2007). A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors. Nature 446: 320-324. PubMed Citation: 17361183

Minakhina, S., Druzhinina, M., and Steward R. (2007). Zfrp8, the Drosophila ortholog of PDCD2, functions in lymph gland development and controls cell proliferation. Development 134: 2387-2396. PubMed Citation: 17522156

Minakhina, S. and Steward, R. (2010). Hematopoietic stem cells in Drosophila. Development 137(1): 27-31. PubMed Citation: 20023157

Ramalho-Santos M., Yoon S., Matsuzaki Y., Mulligan R. C. and Melton D. A. (2002). 'Stemness': transcriptional profiling of embryonic and adult stem cells. Science 298: 597-600. PubMed Citation: 12228720

Steinemann, D., et al. (2003). Identification of candidate tumor-suppressor genes in 6q27 by combined deletion mapping and electronic expression profiling in lymphoid neoplasms. Genes Chromosomes Cancer 37: 421-426. PubMed Citation: 12800155

date revised: 20 March 2010

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