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First published online 23 May 2007
doi: 10.1242/dev.003616
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Waksman Institute, Department of Molecular Biology and Biochemistry, Cancer Institute of New Jersey, Rutgers University, 190 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA.
* Author for correspondence (e-mail: steward{at}waksman.rutgers.edu)
Accepted 17 April 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-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 -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, we
propose 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.
Key words: Drosophila hematopoiesis, Lymph gland hyperplasia, Centrosome
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Hartenstein,
2006; Mandal et al.,
2004; Meister,
2004; Orkin,
2000).
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
(Holz et al., 2003;
Jung et al., 2005;
Lanot et al., 2001). 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
et al., 2004).
Among the earliest requirements for the specification of blood progenitors
in mammals and Drosophila are the highly conserved, GATA zinc-finger
transcription factors (Fujiwara et al.,
2004; Mandal et al.,
2004; Rehorn et al.,
1996). 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
(Mandal et al., 2004;
Rehorn et al., 1996).
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 - Flybase), PSCs
function as a hematopoietic niche to maintain a population of blood cell
precursors (Krzemien et al.,
2007; Lebestky et al.,
2003; Mandal et al.,
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
et al., 2003; Jung et al.,
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.
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)
(Daga et al., 1996;
Okuda et al., 2001).
Lamellocytes function in encapsulation. Their number is significantly
increased at the onset of metamorphosis and in response to infection
(Evans et al., 2003;
Lavine and Strand, 2002;
Meister, 2004;
Rizki and Rizki, 1992).
Differentiation of lamellocytes is connected to two major pathways - the
Drosophila Toll/NF-
B 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 (Luo et al.,
2002; Minakhina and Steward,
2006; Qiu et al.,
1998).
We identified a new gene, Zfrp8, 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.
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 -Tubulin anchoring. In the Zfrp8 mutant
lymph gland cells, both Cyclin B (CycB) and -Tubulin exhibit abnormal
subcellular distribution, suggesting that Zfrp8 plays an important role in
their regulation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T(2;3)B3, CyO; TM6B, Tb1 and GFP-marked balancers were used to identify mutant larvae. Homozygous and hemizygous mutant larvae were collected on egg-laying plates and incubated with additional yeast on standard cornmeal/molasses food at 23°C until they reached the third instar larval stage. Because the Zfrp8 larvae have a severe growth delay - they reach pupariaton 2-7 days after wild type - we collected the mutant larvae after their second molt and incubated for an additional 70 hours. The lymph glands of mutant animals were most similar to those of wild-type larvae grown 60 hours after the molt.
The C-terminally 3xHA-tagged (UAS-Zfrp8-HA) construct was made by PCR amplification of the Zfrp8 coding region and subsequent cloning into the Gateway vector pDONR4 (Life Technologies). The gene was transferred into pPWH (Gateway, Carnegie Institution) and transgenic flies were created following standard protocols. This transgene expressed under the control of the Hsp70-GAL4 driver was able to rescue Df(2R)SM206.
Zfrp8 genetic interactions
Gain-of-function mutations (M) in the hematopoietic/immunity genes
Toll, hop and Ras1 (Ras85D - Flybase) were crossed
into a heterozygous Zfrp8 loss-of-function background
(Zfrp8/+;M/+). The gain-of-function phenotypes including enlargement
of the lymph gland, melanotic tumor formation and lethality were monitored
(Asha et al., 2003;
Govind, 1996;
Luo et al., 2002;
Minakhina and Steward, 2006;
Qiu et al., 1998). Between 50
and 100 animals were examined for each genotype. Removal of one copy of
Zfrp8 did not affect any of the dominant phenotypes (see Table S1 in
the supplementary material).
The second approach was based on the mild haplo-insufficient phenotype of Zfrp8/+ larvae. The lymph gland of Df(2R)SM206/+ larvae is on average twice (2.2±0.5) as large as that of wild type (Fig. 3A,C). We found that the smallest gland of 50 Df(2R)SM206/+ lymph glands analyzed was larger than the largest wild-type gland. To test genes for their ability to modify the lymph gland phenotype, loss-of-function mutations in genes reported to function in fly hematopoiesis, cell-cycle genes and genes with an established function in programmed cell death, were crossed with Df(2R)SM206/+. From each cross, 30 transheterozygous larvae at wandering stage were analyzed; the area of 2D images of 6-12 lymph glands (one pair of primary and two pairs of secondary lobes) were measured, using Adobe PhotoShop and normalized to the size of the wild-type gland (see Table S1 in the supplementary material).
Dominant suppressor of Df(2R)SM206/+ lymph gland overgrowth, pnrMD237, was also tested for its ability to suppress the phenotype of Df(2R)SM206 homozygous larvae. We found that pnrMD237 not only dominantly suppresses the lymph gland phenotype, but also improves the survival of mutants through the larval stages (Fig. 3B,F and see Table S1 in the supplementary material).
Because the apical caspase Dronc (also known as Nedd2-like caspase) is
known to function in the induced apoptosis of blood cells
(Chew et al., 2004), homozygous
and hemizygous combinations of dronc and Zfrp8
(Zfrp8M-1-1/Df(2R)206; dronc2/+ and
Zfrp8M-1-1/+;
dronc2/dronc51) were tested. No
modifications of the Zfrp8M-1-1/Df(2R)206
dronc2/dronc51 phenotype were
observed.
Immunostaining
For antibody staining, third instar larvae were dissected in
phosphate-buffered saline (PBS) and lymph glands with adjacent pericardial
cells, brain and discs were immediately transferred to a glass slide. Excess
liquid was removed with tissue paper and the samples air dried for 30 seconds
and then fixed at 80°C for 1 minute. The slides were kept at -70°C for
several days until staining. The samples were additionally fixed for 40
minutes in 4% paraformaldehyde in PBST (PBS, 0.1% Tween 20) and washed several
times in PBST. Antibody staining was performed essentially as described
(Jung et al., 2005;
Minakhina and Steward, 2006;
Oegema et al., 1999). Between
20 and 40 larvae of each genotype were stained with each antibody.
Rat anti-Ser antibodies were obtained from Dr C. Irvine (Rutgers
University, Piscataway, NJ). Anti-Hemese monoclonal antibody (H2), antibodies
specific for lamellocytes (L1) and for plasmatocytes (P1) were obtained from
Dr I. Ando (Biological Research Center, Szeged, Hungary) and used at 1:500
dilution. Rabbit polyclonal anti-H3P antibody (Upstate, Lake Placid, NY,
1:1000), rabbit anti-HA (Sigma, St Louis, MO, 1:1000), mouse anti-HA (Roche,
1:100) and mouse anti--Tubulin (Sigma, St Louis, MO, 1:100) were used.
Mouse monoclonal anti-Cyclin B (F2F4) and anti-Cyclin A (A12) antibodies
developed by Drs C. Lehner (University of Bayreuth, Bayreuth, Germany) and P.
O'Farrell (University of California, San Francisco, CA) were obtained from the
Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA) and
used 1:120. Rabbit anti-Dgrip84 and anti-Dgrip91 were kindly provided by Dr M.
Moritz (UCSF, San Francisco, CA), and anti-CP309 by Drs D. Ducat and Y. Zheng
(Carnegie Institution of Washington/HHMI, Baltimore, MD) and used 1:1000.
Rabbit anti-active Caspase 3 (BD Biosciences, San Jose, CA) and anti-Srp were
obtained from Dr K. Hoshizaki (University of Nevada, Las Vegas, NV) and used
at 1:1500.
Secondary goat anti-mouse Cy3, goat anti-rat Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa Fluor 488 donkey anti-rabbit antibodies (Molecular Probes, Eugene, OR) were used at 1:500. DNA was stained using Hoechst 33258 (Molecular Probes, Eugene, OR). Samples were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and examined with a Zeiss Axioplan-2 microscope. Hematopoietic nodule images were captured using a Leica DM IRBE laser scanning confocal microscope. The images were analyzed with Image Pro Plus and Leica Microsystems software and further processed using Adobe PhotoShop.
Analysis of cell cycle in lymph glands
To determine the S-phase index, BrdU incorporation was performed as
previously described (Jung et al.,
2005). Immediately upon fixation, lymph glands were stained with
mouse monoclonal anti-BrdU antibody (Becton Dickinson, Franklin Lakes, NJ,
1:100 dilution). The number of positive nuclei per field was counted, and the
ratio of BrdU-positive nuclei to the total number of nuclei was calculated.
This ratio was averaged over at least ten fields from different lobes of lymph
glands of five larvae (more than 2000 cells counted for each genotype).
To count the number of centrosomes, we stained cells with anti-CP309,
anti-Dgrip91 and anti-Dgrip84 antibodies
(Kawaguchi and Zheng, 2004;
Oegema et al., 1999). We
captured a series of confocal sections through one layer of lymph gland cells
(about 5 µm) at x63 magnification, and counted the number of cells
with one or two centrosomes using 3D projection. Cells in at least five
independent fields were counted for each marker. All three markers gave
similar results.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B pathway genes and pursued it because of its
hematopoietic phenotype. Zfrp8 encodes a conserved protein, called
PDCD2 in mammals, that has not been functionally characterized so far. Like
the mammalian gene, Zfrp8 is transcribed at low levels and is
ubiquitously expressed in embryos and the majority of tissues
(Mihola et al., 2007;
Rebhan et al., 1997) (data not
shown).
PDCD2/Zfrp8 is present in eukaryotes from yeast to humans. The mammalian
proteins are more than 80% identical, and the identity between the fly and the
human orthologs is 
40%, distributed along the entire protein (see Fig. S1
in the supplementary material). The C-terminal half of PDCD2/Zfrp8 proteins is
called the PDCD2(C) domain (Chen et al.,
2005). It is present only in PDCD2/Zfrp8 orthologs and in one
predicted protein in vertebrates and flies. It has not been functionally
characterized and might not represent a bone fide domain.
The zinc-finger domain, named MYND because of the conservation of its
general structure in the myeloid tumor gene 8 (also known as MTG8, ETO and
RUNX1T1), the Drosophila proteins Nervy and in Deaf1
(Gross and McGinnis, 1996), is
also present in a number of other transcriptional regulators including MTGR1,
NUDR and BS69 (also known as CBFA2T2, DEAF1 and ZMYND11, respectively). In
these proteins, MYND fosters protein-protein interaction and recruits
co-repressors (Ansieau and Leutz,
2002; Ibanez et al.,
2004; Jensik et al.,
2004; Lutterbach et al.,
1998). The sequence adjacent to the MYND domain (see Fig. S1 in
the supplementary material, underlined) has been shown to interact with Host
cell factor 1 (also known as HCF-1 and HCFC1), an important regulator of cell
cycle (Mahajan and Wilson,
2000; Scarr and Sharp,
2002). We find that Drosophila Hcf and Zfrp8 also
interact in vitro in a manner similar to the human and worm orthologs (results
not shown).
Zfrp8 is essential for fly hematopoiesis
To investigate the in vivo function of Zfrp8, we used two
previously described deficiencies, Df(2R)SM206 and
Df(2R)SM183 (Minakhina et al.,
2003) and the new EMS allele, Zfrp8M-1-1. The
deficiencies were originally obtained by mobilizing a P-element located at the
5' end of the Zfrp8 protein-coding region. Whereas the larger
deletion, Df(2R)SM183, abolishes expression of the two genes
tamo and Zfrp8, the smaller deletion, Df(2R)SM206,
does not affect the expression of the neighboring tamo gene
(Minakhina et al., 2003). The
EMS allele, Zfrp8M-1-1, was isolated as a lethal over both
deficiencies. More than 90% of Zfrp8M-1-1 hemizygous and
homozygous late third instar larvae produce melanotic masses that are seen as
black spots outlining the shape of the lymph glands in pupae (see Fig. S2B,D
in the supplementary material). In Zfrp8M-1-1, the
absolutely conserved glutamic acid 296 is changed to a lysine (E296K; see Fig.
S1 in the supplementary material).
Homozygotes for the Zfrp8-null allele, Df(2R)SM206, show
a severe growth and developmental delay and most animals die in larval stages.
Few animals survive until pupariation and are smaller than their heterozygous
siblings. About 2% of homozygous Df(2R)SM206 escapers survive until
adulthood, but have poor viability and female fertility.
Zfrp8M-1-1 homozygous and hemizygous larvae show 5%
adult survival.
All Zfrp8 larvae have an enormous overgrowth of the hematopoietic organs, the lymph glands. The mutant lymph glands are at least 25 times the size of the organ of wild-type third instar larvae. In developmentally delayed Zfrp8 animals, it could grow to up to 70 times the normal size (Fig. 1A,B, and see Fig. S2A,B in the supplementary material). Other organs were often reduced in size proportional to the size of the larvae. Zfrp8 was also found to have a mild haplo-insufficient phenotype; the lymph glands of Df(2R)SM206/+ larvae were about twice (2.2±0.5) the size of the average wild-type gland (Fig. 3, and see Table S1 in the supplementary material).
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). Up
to 90% of mutant animals expressing the transgene survived and were fertile.
The size of their lymph glands was about two to three times the size of that
of wild type (2.8±1.0). It is possible that the lymph gland phenotype
would be rescued to wild-type size if the transgene were expressed at higher
levels specifically in the gland. Alternatively, the growth of the gland could
be controlled extrinsically.
We found that the organization and location of the Zfpr8 mutant
lymph glands were similar to those of wild-type larvae. The relative
proportions of primary and secondary lobes (marked I and II in the figures)
were preserved and all lobes were overgrown
(Fig. 1A,B,
Fig. 2 and see Fig. S2A,B in
the supplementary material). Anti-Hemese (He) antibody
(Kurucz et al., 2003)
recognized the plasma membrane-associated protein specific for pro-hemocytes
and hemocytes in both wild-type and mutant organs
(Fig. 1E,F). Anti-Srp antibody
(Sam et al., 1996) staining
showed that Zfpr8 lymph glands are already twice as large as
wild-type glands by stage 16 to 17. The number of Srp-positive cells in
wild-type organs varied from 19 to 25, whereas Df(2R)SM206 homozygous
organs contained 35-50 cells (Fig.
1C,D). During the 5 days of larval growth and development, the
size difference between wild-type and mutant lymph glands increased
exponentially, and in third instar mutant larvae the glands virtually filled
the mid-anterior part of the larva.
The Zfrp8 phenotype is completely different from that observed in
mutants of genes functioning in the immune response. In mutants with
constitutively induced immunity, such as in Toll or JAK/STAT
(hop/Stat92E) gain-of-function mutants, the number of circulating
hemocytes is markedly increased (Luo et
al., 2002; Minakhina and
Steward, 2006; Qiu et al.,
1998). By contrast, in Zfrp8 larvae, the number of cells
in the lymph glands was dramatically increased, but the number of circulating
hemocytes was only about six times higher than in the wild type
(Fig. 1A,B,G).
Hemocyte differentiation in Zfrp8 mutants
We further defined the role of Zfrp8 in hemocyte differentiation
by staining mutant and wild-type lymph glands with antibodies recognizing
specific markers. These antibodies were for plasmatocytes (P1); lamellocytes
(L1); crystal cells (anti-Lozenge); and specific for the PSC (anti-Ser)
(Lebestky et al., 2003).
Despite the increase in the number of the plasmatocytes, their proportion in
the mutant lymph glands was similar to that in wild type
(Fig. 2C,D). Crystal cells,
positive for anti-Lozenge staining (Jung
et al., 2005), were also increased proportionally to overgrowth
and were distributed similar to those in wild type (data not shown). By
contrast, the number of L1-positive pro-lamellocytes or lamellocytes was
drastically increased in the mutant (Fig.
2F). In wild-type lymph glands and hemolymph, lamellocytes are
rare (<0.5% of hemocytes, Fig.
2E) (Holz et al.,
2003; Jung et al.,
2005; Sorrentino et al.,
2002), but in mutant glands the percentage of L1-positive cells
varied from 5% to 60% and they were not restricted to the cortical zone of the
primary lymph gland lobes, but were found in the presumptive medullary zone
and in secondary lobes. The number of circulating lamellocytes varied from
very few to several hundred per larva (>10% hemocytes). The increase in the
number of lamellocytes reflects reprogramming of pro-hemocytes, similar to
what is observed in response to parasitization or during metamorphosis
(Krzemien et al., 2007).
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The size and appearance of the PSC was similar in Zfrp8 and
wild-type late third instar larvae. In the wild-type, small islands of
Ser-positive hemocytes were located at the posterior of each primary lobe
(Fig. 2A,B)
(Jung et al., 2005). More than
half of the mutant organs analyzed had three to five Ser-positive islands
(Fig. 2B), but the number of
positive cells was not massively increased. The number of Ser-positive islands
were proportional to the size of the gland, suggesting that the increase in
hemocytes results in the formation of the additional PSCs, possibly by
splitting the islands during growth.
Zfrp8 genetically interacts with pannier, which encodes a GATA factor essential for lymph gland development
To investigate connections between Zfrp8 and genes known to
function in Drosophila hematopoiesis, we took advantage of the
dominant lymph gland phenotype observed in Df(2R)SM206/+ larvae, and
looked for dominant modification of this phenotype in transheterozygous larvae
(see Material and methods; Fig.
3A,C). We also investigated whether loss of one copy of
Zfrp8 modified the phenotypes caused by specific gain-of-function
mutations. Mutations in 14 genes were tested (for full results see Table S1 in
the supplementary material). Although most mutations did not modify the
Df(2R)SM206/+ lymph gland phenotype, two alleles of pnr,
pnrVX6 and pnrMD237, dominantly suppressed
the lymph gland overgrowth of Df(2R)SM206/+
(Fig. 3E). Moreover, in
Df(2R)SM206/Df(2R)SM206; pnrMD237/+ larvae, the lymph
glands were significantly smaller than in Df(2R)SM206/Df(2R)SM206;
+/+ larvae (Fig. 3B,F).
Pnr functions in the specification of the hemangioblasts,
mesodermal cells giving rise to both cardiac and lymph gland cells
(Mandal et al., 2004). Zfrp8
might function with Pnr, a transcription factor, in controlling the early
establishment of the lymph gland. The interaction of Zfrp8 with
pnr is not restricted to embryonic development because the dose
reduction of pnr not only suppresses the lymph gland phenotype, but
also increases the larval survival rate of Zfrp8 mutants, suggesting
that the two proteins also function together at other stages of development
and in other tissues.
These genetic studies, in which we failed to detect an interaction of Zfrp8 with genes functioning in hemocyte differentiation, suggest that Zfrp8 regulates proliferation of blood cells prior to differentiation.
Role of Zfrp8 in the regulation of cell proliferation
The overgrowth phenotype suggests that Zfrp8 is essential for
controlling cell death of hematopoietic precursor cells or, alternatively, the
number of cell divisions. To investigate these possibilities we performed
genetic interaction assays with mutants in 14 genes with an established
function in programmed cell death or involved in the cell cycle (see Table S1
in the supplementary material). Mutations in two genes involved in cell-cycle
regulation, Cdc27 (Cdc27L7123) and
l(1)dd4 (l(1)dd41 and
l(1)dd42), showed dominant enhancement of the
Df(2R)SM206/+ lymph gland phenotype, at least doubling the size of
the gland. The resultant (Df(2R)SM206/+;
Cdc27L7123/+ and
l(1)dd42/+;Df(2R)SM206/+) lymph glands were five times the
size of wild-type lymph glands (Fig.
3G,H, and see Table S1 in the supplementary material). This
demonstrates that the Df(2R)SM206/+ lymph gland size is sensitive to
the dosage of genes involved in the cell cycle and links Zfrp8 itself
to the cell cycle.
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). In mutant glands, the proportion of cells in S phase was
about 1.3 (25±2%) times greater than in wild type (20±2%). As in
wild type, BrdU-positive cells were found in all lobes. We next evaluated the mitotic index (mitotic cells/total cells) in wild-type and mutant glands by staining of histone H3 phosphorylated at serine 10 (H3P), which is specific to mitosis (Fig. 4C-E). The average mitotic index in the entire lymph glands increased from 6±1% in wild type to an average of 8.5±1% in Df(2R)SM206/Df(2R)SM206 and Zfrp8M-1-1/Zfrp8M-1-1. Overall, the mutant tissue showed a mild increase in cell proliferation, but the continuous growth increase over a period of several days converted the slightly increased gland observed in embryos into the massively overgrown lymph gland observed in third instar larvae.
In Zfrp8 mutants, the increase in mitotic cells was concentrated in secondary lymph gland lobes (II) that usually contain less differentiated hemocytes. Such areas had greater than 10% of cells in mitosis, indicating actively growing regions (Fig. 4D,E arrows). Primary lobes (I) usually had fewer mitotic cells.
In Drosophila, accumulation of Cyclins A and B (CycA and CycB) is
detected in cells from late S/G2 to prophase and anaphase, respectively
(Huang and Raff, 1999;
Parry and O'Farrell, 2001).
Our staining experiments showed that CycA and CycB are abundant in wild-type
brain and discs (Fig. 5). CycA
was also detected in the lymph glands, whereas CycB was virtually absent from
the glands (Fig. 5A,C,D,G,H).
In mutant lymph glands, CycB was dramatically increased. The protein was
detected in the cytoplasm of more than 50% of cells and was particularly
abundant in 25% (Fig.
5B,E,F). However, in mutant brains, the CycB level was comparable
to that in wild type. The CycA level and distribution were similar in mutant
and wild-type glands and brains (Fig.
5G-J).
The apparent high levels of CycB in many cells of mutant glands raised the
question of whether it reflects an increase in the number of cells in G2. We
visualized centrosomes using antibodies to -Tubulin and two other
components of the -Tubulin ring complex (-TuRC),
Drosophila gamma-ring proteins (GRIPs) Dgrip84 (Grip84 - Flybase) and
Dgrip91 (Fig. 6 and see Fig. S3
in the supplementary material) (Colombie et
al., 2006; Oegema et al.,
1999). We also stained the centrosome protein CP309, which is
required for microtubule nucleation
(Kawaguchi and Zheng, 2004).
The number of centrosomes is indicative of G1 (one centrosome) and G2 (two).
We counted cells containing one and two foci of Dgrip84, Dgrip91 or CP309 in
lymph gland lobes and found that the ratio of cells before and after
chromosome duplication was about 1:1 in wild-type and mutant glands,
suggesting that the ratio of G1/G2 cells in mutants and wild type is the
same.
The number of centrosomes and appearance of Dgrip91, Dgrip84 and CP309 were
similar in mutant and wild-type lymph gland cells, but the key -TuRC
component, -Tubulin, showed abnormal distribution in Zfrp8
mutants (Fig. 6A-D, and see
Fig. S3 in the supplementary material). Multiple -Tubulin foci that did
not overlap with other centrosomal markers were observed. The phenotype
suggests that Zfrp8 is involved in -Tubulin recruitment to centrosomes
(Fig. 6C,D, and see Fig. S3 in
the supplementary material). This phenotype is consistent with our genetic
finding that the lymph gland overgrowth of Zfpr8 is dominantly
enhanced by the mutation in l(1)dd4, which encodes Dgrip91 involved
in -Tubulin centrosome anchoring.
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-Tubulin and Dgrip84 foci at centrosomes
(Fig. 6E,F). We also observed
similar localization of human PDCD2 in ML-1 cultured cells (data not shown).
These results suggest that Zfrp8 might function with the centrosomes
and participate in anchoring proteins to this organelle. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Subsequent studies, using different cells and assay conditions, found no
connection between PDCD2 expression and programmed cell death
(Chen et al., 2005;
Fan et al., 2004;
Kawakami et al., 1995;
Steinemann et al., 2003).
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
(Chiu and Govind, 2002;
Jung et al., 2005). We found a
statistically insignificant increase in the number of apoptotic cells in
Zfrp8 mutants (see Fig. S4 in the supplementary material). No other
evidence of change in programmed cell death in Zfrp8 mutant animals,
no increase in apoptotic gene expression, no change in caspase cleavage (data
not shown) and no genetic interaction of Zfrp8 with known apoptotic
genes (and see Table S1 in the supplementary material) were found.
Our 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.
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 (see Fig. S2E in the supplementary material) and normal wound clogging and wound melanization (not shown). That the requirements are different for Zfrp8 and known hematopoiesis and immunity genes is underlined by the absence of their genetic interaction (and see Table S1 in the supplementary material).
|
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 et al., 2007;
Mandal et al., 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 et al., 2007).
Therefore, it is unlikely that Zfrp8 is directly involved in the
establishment of PSCs.
Zfrp8 and the cell cycle
Our results link the Zfrp8 overgrowth phenotype to a defect in
normal cell proliferation. In mutant lymph glands, the cell-cycle markers
-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
(Barbosa et al., 2000;
Oegema et al., 1999).
Dgrip91 and -Tubulin are components of the -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
(Debec and Montmory, 1992;
Jackman et al., 2003;
Kramer et al., 2004;
Wakefield et al., 2000).
The link between the phenotypes described above and Zfrp8 function
became clear when we 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 -Tubulin and CycB to this organelle.
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 (Ansieau and Leutz,
2002; Ibanez et al.,
2004; Jensik et al.,
2004; Lutterbach et al.,
1998). 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.
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
(Andersen et al., 2003;
Doxsey et al., 2005;
Hsu and White, 1998).
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
(Hartenstein, 2006;
Evans et al., 2003).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/13/2387/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Andersen, J. S., Wilkinson, C. J., Mayor, T., Mortensen, P., Nigg, E. A. and Mann, M. (2003). Proteomic characterization of the human centrosome by protein correlation profiling. Nature 426,570 -574.[CrossRef][Medline]
Ansieau, S. and Leutz, A. (2002). The conserved
Mynd domain of BS69 binds cellular and oncoviral proteins through a common
PXLXP motif. J. Biol. Chem.
277,4906
-4910.
Asha, H., Nagy, I., Kovacs, G., Stetson, D., Ando, I. and
Dearolf, C. R. (2003). Analysis of Ras-induced
overproliferation in Drosophila hemocytes. Genetics
163,203
-215.
Barbosa, V., Yamamoto, R. R., Henderson, D. S. and Glover, D.
M. (2000). Mutation of a Drosophila gamma tubulin ring
complex subunit encoded by discs degenerate-4 differentially disrupts
centrosomal protein localization. Genes Dev.
14,3126
-3139.
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.[CrossRef][Medline]
Chew, S. K., Akdemir, F., Chen, P., Lu, W. J., Mills, K., Daish, T., Kumar, S., Rodriguez, A. and Abrams, J. M. (2004). The apical caspase dronc governs programmed and unprogrammed cell death in Drosophila. Dev. Cell 7,897 -907.[CrossRef][Medline]
Chiu, H. and Govind, S. (2002). Natural infection of D. melanogaster by virulent parasitic wasps induces apoptotic depletion of hematopoietic precursors. Cell Death Differ. 9,1379 -1381.[CrossRef][Medline]
Colombie, N., Verollet, C., Sampaio, P., Moisand, A., Sunkel,
C., Bourbon, H. M., Wright, M. and Raynaud-Messina, B.
(2006). The Drosophila gamma-tubulin small complex subunit
Dgrip84 is required for structural and functional integrity of the spindle
apparatus. Mol. Biol. Cell
17,272
-282.
Daga, A., Karlovich, C. A., Dumstrei, K. and Banerjee, U.
(1996). Patterning of cells in the Drosophila eye by Lozenge,
which shares homologous domains with AML1. Genes Dev.
10,1194
-1205.
Debec, A. and Montmory, C. (1992). Cyclin B is associated with centrosomes in Drosophila mitotic cells. Biol. Cell 75,121 -126.[CrossRef][Medline]
Doxsey, S., Zimmerman, W. and Mikule, K. (2005). Centrosome control of the cell cycle. Trends Cell Biol. 15,303 -311.[CrossRef][Medline]
Evans, C. J., Hartenstein, V. and Banerjee, U. (2003). Thicker than blood: conserved mechanisms in Drosophila and vertebrate hematopoiesis. Dev. Cell 5, 673-690.[CrossRef][Medline]
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.[CrossRef][Medline]
Fujiwara, Y., Chang, A. N., Williams, A. M. and Orkin, S. H.
(2004). Functional overlap of GATA-1 and GATA-2 in primitive
hematopoietic development. Blood
103,583
-585.
Govind, S. (1996). Rel signalling pathway and the melanotic tumour phenotype of Drosophila. Biochem. Soc. Trans. 24,39 -44.[Medline]
Gross, C. T. and McGinnis, W. (1996). DEAF-1, a novel protein that binds an essential region in a Deformed response element. EMBO J. 15,1961 -1970.[Medline]
Hartenstein, V. (2006). Blood cells and blood cell development in the animal kingdom. Annu. Rev. Cell Dev. Biol. 22,677 -712.[CrossRef][Medline]
Holz, A., Bossinger, B., Strasser, T., Janning, W. and Klapper,
R. (2003). The two origins of hemocytes in Drosophila.
Development 130,4955
-4962.
Hsu, L. C. and White, R. L. (1998). BRCA1 is
associated with the centrosome during mitosis. Proc. Natl. Acad.
Sci. USA 95,12983
-12988.
Huang, J. and Raff, J. W. (1999). The disappearance of cyclin B at the end of mitosis is regulated spatially in Drosophila cells. EMBO J. 18,2184 -2195.[CrossRef][Medline]
Ibanez, V., Sharma, A., Buonamici, S., Verma, A., Kalakonda, S.,
Wang, J., Kadkol, S. and Saunthararajah, Y. (2004). AML1-ETO
decreases ETO-2 (MTG16) interactions with nuclear receptor corepressor, an
effect that impairs granulocyte differentiation. Cancer
Res. 64,4547
-4554.
Jackman, M., Lindon, C., Nigg, E. A. and Pines, J. (2003). Active cyclin B1-Cdk1 first appears on centrosomes in prophase. Nat. Cell Biol. 5, 143-148.[CrossRef][Medline]
Jensik, P. J., Huggenvik, J. I. and Collard, M. W.
(2004). Identification of a nuclear export signal and protein
interaction domains in deformed epidermal autoregulatory factor-1 (DEAF-1).
J. Biol. Chem. 279,32692
-32699.
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.
Kawaguchi, S. and Zheng, Y. (2004).
Characterization of a Drosophila centrosome protein CP309 that shares homology
with Kendrin and CG-NAP. Mol. Biol. Cell
15, 37-45.
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.[Medline]
Kramer, A., Mailand, N., Lukas, C., Syljuasen, R. G., Wilkinson, C. J., Nigg, E. A., Bartek, J. and Lukas, J. (2004). Centrosome-associated Chk1 prevents premature activation of cyclin-B-Cdk1 kinase. Nat. Cell Biol. 6, 884-891.[CrossRef][Medline]
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.[CrossRef][Medline]
Kurucz, E., Zettervall, C. J., Sinka, R., Vilmos, P., Pivarcsi,
A., Ekengren, S. Hegedus, Z., Ando, I. and Hultmark, D.
(2003). Hemese, a hemocyte-specific transmembrane protein,
affects the cellular immune response in Drosophila. Proc. Natl.
Acad. Sci. USA 100,2622
-2627.
Lanot, R., Zachary, D., Holder, F. and Meister, M. (2001). Postembryonic hematopoiesis in Drosophila. Dev. Biol. 230,243 -257.[CrossRef][Medline]
Lavine, M. D. and Strand, M. R. (2002). Insect hemocytes and their role in immunity. Insect Biochem. Mol. Biol. 32,1295 -1309.[CrossRef][Medline]
Lebestky, T., Jung, S. H. and Banerjee, U.
(2003). A Serrate-expressing signaling center controls Drosophila
hematopoiesis. Genes Dev.
17,348
-353.
Luo, H., Rose, P. E., Roberts, T. M. and Dearolf, C. R. (2002). The Hopscotch Jak kinase requires the Raf pathway to promote blood cell activation and differentiation in Drosophila. Mol. Genet. Genomics 267, 57-63.[CrossRef][Medline]
Lutterbach, B., Sun, D., Schuetz, J. and Hiebert, S. W.
(1998). The MYND motif is required for repression of basal
transcription from the multidrug resistance 1 promoter by the t(8;21) fusion
protein. Mol. Cell. Biol.
18,3604
-3611.
Mahajan, S. S. and Wilson, A. C. (2000).
Mutations in host cell factor 1 separate its role in cell proliferation from
recruitment of VP16 and LZIP. Mol. Cell. Biol.
20,919
-928.
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.[CrossRef][Medline]
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.[CrossRef][Medline]
Meister, M. (2004). Blood cells of Drosophila: cell lineages and role in host defence. Curr. Opin. Immunol. 16,10 -15.[CrossRef][Medline]
Mihola, O., Forejt, J. and Trachtulec, Z. (2007). Conserved alternative and antisense transcripts at the programmed cell death 2 locus. BMC Genomics 8, 20.[CrossRef][Medline]
Minakhina, S. and Steward, R. (2006). Melanotic
mutants in Drosophila: pathways and phenotypes.
Genetics 174,253
-263.
Minakhina, S., Yang, J. and Steward, R. (2003). Tamo selectively modulates nuclear import in Drosophila. Genes Cells 8,299 -310.[Abstract]
Oegema, K., Wiese, C., Martin, O. C., Milligan, R. A., Iwamatsu,
A., Mitchison, T. J. and Zheng, Y. (1999). Characterization
of two related Drosophila gamma-tubulin complexes that differ in their ability
to nucleate microtubules. J. Cell Biol.
144,721
-733.
Okuda, T., Nishimura, M., Nakao, M. and Fujita, Y. (2001). RUNX1/AML1: a central player in hematopoiesis. Int. J. Hematol. 74,252 -257.[Medline]
Orkin, S. H. (2000). Diversification of haematopoietic stem cells to specific lineages. Nat. Rev. Genet. 1,57 -64.[Medline]
Owens, G. P., Hahn, W. E. and Cohen, J. J.
(1991). Identification of mRNAs associated with programmed cell
death in immature thymocytes. Mol. Cell. Biol.
11,4177
-4188.
Parry, D. H. and O'Farrell, P. H. (2001). The schedule of destruction of three mitotic cyclins can dictate the timing of events during exit from mitosis. Curr. Biol. 11,671 -683.[CrossRef][Medline]
Qiu, P., Pan, P. C. and Govind, S. (1998). A role for the Drosophila Toll/Cactus pathway in larval hematopoiesis. Development 125,1909 -1920.[Abstract]
Rebhan, M., Chalifa-Caspi, V., Prilusky, J. and Lancet, D. (1997). GeneCards: Encyclopedia for Genes, Proteins and Diseases. Rehovot, Israel: Weizmann Institute of Science, Bioinformatics Unit and Genome Center. http://www.genecards.org/.
Rehorn, K. P., Thelen, H., Michelson, A. M. and Reuter, R. (1996). A molecular aspect of hematopoiesis and endoderm development common to vertebrates and Drosophila. Development 122,4023 -4031.[Abstract]
Rizki, T. M. and Rizki, R. M. (1992). Lamellocyte differentiation in Drosophila larvae parasitized by Leptopilina. Dev. Comp. Immunol. 16,103 -110.[CrossRef][Medline]
Sam, S., Leise, W. and Hoshizaki, D. K. (1996). The serpent gene is necessary for progression through the early stages of fat-body development. Mech. Dev. 60,197 -205.[CrossRef][Medline]
Scarr, R. B. and Sharp, P. A. (2002). PDCD2 is a negative regulator of HCF-1 (C1). Oncogene 21,5245 -5254.[CrossRef][Medline]
Sorrentino, R. P., Carton, Y. and Govind, S. (2002). Cellular immune response to parasite infection in the Drosophila lymph gland is developmentally regulated. Dev. Biol. 243,65 -80.[CrossRef][Medline]
Steinemann, D., Gesk, S., Zhang, Y., Harder, L., Pilarsky, C., Hinzmann, B., Martin-Subero, J. I., Calasanz, M. J., Mungall, A., Rosenthal, A. 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.[CrossRef][Medline]
Wakefield, J. G., Huang, J. Y. and Raff, J. W. (2000). Centrosomes have a role in regulating the destruction of cyclin B in early Drosophila embryos. Curr. Biol. 10,1367 -1370.[CrossRef][Medline]
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