Sumoylation is tumor-suppressive and confers proliferative quiescence to hematopoietic progenitors in Drosophila melanogaster larvae
How cell-intrinsic regulation of the cell cycle and the extrinsic influence of the niche converge to provide proliferative quiescence, safeguard tissue integrity, and provide avenues to stop stem cells from giving rise to tumors is a major challenge in gene therapy and tissue engineering. We explore this question in sumoylation-deficient mutants of Drosophila. In wild type third instar larval lymph glands, a group of hematopoietic stem/progenitor cells acquires quiescence; a multicellular niche supports their undifferentiated state. However, how proliferative quiescence is instilled in this population is not understood. We show that Ubc9 protein is nuclear in this population. Loss of the SUMO-activating E1 enzyme, Aos1/Uba2, the conjugating E2 enzyme, Ubc9, or the E3 SUMO ligase, PIAS, results in a failure of progenitors to quiesce; progenitors become hyperplastic, misdifferentiate, and develop into microtumors that eventually detach from the dorsal vessel. Significantly, dysplasia and lethality of Ubc9 mutants are rescued when Ubc9(wt) is provided specifically in the progenitor populations, but not when it is provided in the niche or in the differentiated cortex. While normal progenitors express high levels of the Drosophila cyclin-dependent kinase inhibitor p21 homolog, Dacapo, the corresponding overgrown mutant population exhibits a marked reduction in Dacapo. Forced expression of either Dacapo or human p21 in progenitors shrinks this population. The selective expression of either protein in mutant progenitor cells, but not in other hematopoietic populations, limits overgrowth, blocks tumorogenesis, and restores organ integrity. We discuss an essential and complex role for sumoylation in preserving the hematopoietic progenitor states for stress response and in the context of normal development of the fly.
Sumoylation is tumor-suppressive and confers
proliferative quiescence to hematopoietic progenitors
in Drosophila melanogaster larvae
Marta E. Kalamarz
, Indira Paddibhatla
, Christina Nadar
and Shubha Govind
Biology Department, The City College of the City University of New York, 138th Street and Convent Avenue, New York, NY 10031, USA
The Graduate Center of the City University of New York, 365 Fifth Avenue, New York, NY 10016, USA
*Author for correspondence (email@example.com)
Biology Open 1, 161–172
How cell-intrinsic regulation of the cell cycle and the extrinsic
influence of the niche converge to provide proliferative
quiescence, safeguard tissue integrity, and provide avenues
to stop stem cells from giving rise to tumors is a major
challenge in gene therapy and tissue engineering. We explore
this question in sumoylation-deficient mutants of Drosophila.
In wild type third instar larval lymph glands, a group of
hematopoietic stem/progenitor cells acquires quiescence; a
multicellular niche supports their undifferentiated state.
However, how proliferative quiescence is instilled in this
population is not understood. We show that Ubc9 protein is
nuclear in this population. Loss of the SUMO-activating E1
enzyme, Aos1/Uba2, the conjugating E2 enzyme, Ubc9, or the
E3 SUMO ligase, PIAS, results in a failure of progenitors to
quiesce; progenitors become hyperplastic, misdifferentiate,
and develop into microtumors that eventually detach from
the dorsal vessel. Significantly, dysplasia and lethality of Ubc9
mutants are rescued when Ubc9
is provided specifically in
the progenitor populations, but not when it is provided in the
niche or in the differentiated cortex. While normal
progenitors express high levels of the Drosophila cyclin-
dependent kinase inhibitor p21 homolog, Dacapo, the
corresponding overgrown mutant population exhibits a
marked reduction in Dacapo. Forced expression of either
Dacapo or human p21 in progenitors shrinks this population.
The selective expression of either protein in mutant
progenitor cells, but not in other hematopoietic populations,
limits overgrowth, blocks tumorogenesis, and restores organ
integrity. We discuss an essential and complex role for
sumoylation in preserving the hematopoietic progenitor states
for stress response and in the context of normal development
of the fly.
ß 2011. Published by The Company of Biologists Ltd. This is
an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial Share Alike
Key words: Dacapo, dysplasia, hematopoiesis, microtumor, niche,
organ integrity, p21, quiescence, stem cell, sumoylation, tumor
Tissue and organ regeneration in patients with lesions from
disease or surgery, or due to ageing, is a primary challenge in
biomedical research. Tissue engineering requires understanding
how normal tissues arise, develop, renew themselves, and
maintain their proliferative quiescence and homeostasis. Stem
cells provide proliferative quiescence and tissue integrity over
time (Morrison and Spradling, 2008). Proliferative quiescence is
characteristic property of some stem cells, which, as compared to
their more differentiated progenitors, undergo infrequent
divisions (Moore and Lyle, 2011). Loss of proliferative
quiescence in pre-malignant cells frequently accompanies the
development of cancer.
Mammalian cancers are composed of heterogeneous cell
populations that include few stem/stem-like cells and many more
differentiated cells with limited proliferative potential (Morrison
and Spradling, 2008; Wang, 2010). The growth and development
of a tumor depends on the complex interplay of both, the cell-
intrinsic mechanisms and the microenvironment. Tumors are
further characterized by dormancy or metastasis, and the nature of
these processes in relation to their origin remains largely unclear
(Morrison and Spradling, 2008; Wang, 2010). The mechanism of
proliferative quiescence in normal stem and related cancer cells is
not well understood (Moore and Lyle, 2011).
Drosophila has served as an excellent model system for cancer
research. One approach to studying cancer in flies is to screen the
genome for mutations in larval cells that promote tumorogenesis
and metastasis. In this approach, mutations are induced
selectively in specific tissues, where genetically affected
mutant cells form tumors in an otherwise wild type larval
body. The effects of a known or new oncogenic or tumor-
suppressive mutation can be studied in such mosaic animals
(Potter et al., 2000; Vidal and Cagan, 2006). In an ‘‘inverse
mosaic’’ approach, germline mutants that develop tumors with
high spatial and temporal specificity are studied by genetically
manipulating specific regions of the tumor, or its environment, by
expressing either the missing protein, or another protein,
suspected to play a role in tumor development (Manfruelli et
al., 1996; Qiu et al., 1998; Chiu et al., 2005). In either case,
mosaic animals can be created with fly or human proteins.
Research Article 161
In this study, we examined the origin of hematopoietic
microtumors in Ubc9 mutants of Drosophila (Chiu et al., 2005;
Huang et al., 2005). Microtumors are structures of at least
in projection area, consisting of at least 50 cells, and
aggregates are structures,10,000 mm
in projection area
(Kalamarz, 2010). Both classes of structures are found in more
than 80% of the Ubc9 mutants (Kalamarz, 2010). Microtumors
are composed mostly of blood cells (hemocytes), including
lamellocytes, and vary in the degree of melanization (Kalamarz,
2010). Ubc9 is the E2 SUMO-conjugating enzyme. Along with
the SUMO-activating E1 enzymes, Aos1 and Uba2, and the
SUMO E3 ligase, PIAS, Ubc9 participates in a highly-conserved
protein modification system (Mabb and Miyamoto, 2007;
Talamillo et al., 2008).
Blood cells in normal Drosophila larvae circulate freely in the
hemolymph. Groups of blood cells are also present within the
hematopoietic organ, called lymph gland. The predominant cell
type is the macrophage-like plasmatocyte (Kurucz et al., 2007b),
which phagocytoses microbes and dead cells. The remaining
lineages are crystal cells and lamellocytes, both of which
facilitate melanization reactions (Kurucz et al., 2007a; Nam et
al., 2008). Large, adhesive lamellocytes differentiate in response
to parasitic wasp infection in both, circulation and the lymph
gland (Rizki and Rizki, 1992; Lanot et al., 2001; Sorrentino et al.,
The lymph gland originates in the embryo (Mandal et al.,
2004) and develops through larval stages (Lanot et al., 2001;
Holz et al., 2003). The lobes are arranged bilaterally and flank the
dorsal vessel in the anterior body segments (Shrestha and Gateff,
1982; Lanot et al., 2001; Qiu et al., 1998; Jung et al., 2005) (also
see Fig. 1A,B). By the first instar, anterior lobes form compact
cell clusters and by third instar they develop three zones (Jung et
Fig. 1. Aberrant gene expression in progenitors of Ubc9 lymph glands. Labeling: AL – anterior lobe(s), PL1 – first set of posterior lobes, PL2 – second set of posterior
lobes; asterisk – dorsal vessel (DV). (A) Lymph glands in second (L2) and third (L3) larval instars. Medullary zone (MZ, light green), cortical zone (CZ, dark green); the
niche (N, orange); unclassified cells (navy blue, P); pericardial cells (PC, light blue). Pairs of lobes aligned along the antero-posterior axis; PL1, PL2 consist of smaller
lobes (2–3 pairs each) distinguishable at L2, but forming a continuous lobe at L3. (B–E) Dome.GFP (green) in lymph glands of 4-day L2: Ubc9
6-day L3: Ubc9
(E). Lobes outlined in dotted marking (D,E). (F–G1) ZCL2897 (green) and Dome.DsRed (red) in wild type L3: AL (F–F’’),PL
(G–G’’). Dome.DsRed (F’,G’) and ZCL2897 (F’’,G’’) shown separately; overlap of expressions (F,F1,G,G1). Regions of F,G (white rectangles) shown magnified in
F1,G1, respectively. Yellow dotted markings outline the lobes (F–F’’,G–G’’). (H–K’) ZCL2897 (green) expression in Ubc9
(H, AL; I, PL1) and Ubc9
(J, AL; K, PL1) lymph glands; note: lobes in J and K are representative examples from different lymph glands. Increased ZCL2897 expression in Ubc9
overexposure and samples were therefore re-imaged after reducing detector gain (J’,K’). K–K’ is a fragment of S2L. Brightness and contrast were slightly modified in
panels F–G1 for clarity in merged images. Confocal sections (B–K’). Scale bars: 50 mm, except F1,G1 – 10 mm.
Hematopoietic microtumors in Ubc9 mutants of Drosophila 162
al., 2005; Mandal et al., 2007; Minakhina and Steward, 2010). A
small multicellular niche controls cell states in the other two
zones (Crozatier et al., 2004; Jung et al., 2005; Krzemien et al.,
2007; Mandal et al., 2007), which are located up to as many as 50
cell diameters away. Cells in medullary and cortical zone divide
actively until the third instar, when cells of the medullary zone
become proliferatively quiescent (Jung et al., 2005; Mandal et al.,
2007). The cell cycle mechanisms responsible for quiescence of
these multipotent hematopoietic stem cells and progenitors
remain largely unknown.
We show that Ubc9 microtumors derive from an initially
quiescent, heterogeneous, progenitor population of the medullary
zones of the anterior and posterior lobes. The largest microtumors
are likely derived from the highly enlarged posterior lobes, as
they abandon normal heterochronic development, and undergo
dysplasia, while still attached to the dorsal vessel, but then detach
from the dorsal vessel into the hemolymph as intact tumors.
Dysplastic growth is niche-independent. Other sumoylation
cascade enzymes, E1 subunits, and E3 ligase, PIAS, are also
needed for progenitor quiescence. Our studies suggest that the
cell cycle of this population is regulated, in part, by Dacapo/p21.
Of dozens of hematopoietic Drosophila mutants reported to date,
this is the first study where a clear cellular origin of microtumors
is defined. Changes in Ubc9 expression have been linked to
primary tumors in humans (Moschos et al., 2010). p21 is a known
drug target in cancer therapy. Its potential regulation via
sumoylation in Drosophila provides new insights into the
regulation of quiescence in an in vivo model system and into
the earliest steps in oncogenesis in humans.
Loss of Ubc9 affects gene expression, and size and integrity of
third instar lymph gland
Post-embryonic wild type lymph gland development is
heterochronic (Fig. 1A,B). From the onset of the third instar,
the posterior lobes of wild type lymph glands expand and
coalesce so that the initially distinct four to six pairs of cell
clusters form two sets of posterior lobes (Fig. 1A,B,D). The
growth of posterior lobes is developmentally synchronous in that
the first set expands earlier than the second set (Fig. 1B,D,
supplementary material Fig. S1A,C). We call them posterior
lobes, first set (PL1), and posterior lobes, second set (PL2)
Mutant Ubc9 lymph glands are variably overgrown and exhibit
aberrant differentiation of hemocytes (Chiu et al., 2005). Careful
analysis of scores of mutant glands revealed differential effects
on anterior versus posterior lobes (Fig. 1D,E, supplementary
material Fig. S1C,D). In many glands of 6–7-day third instar
larvae, the anterior lobes are completely absent or are partially
dispersed where peripheral cells in the cortex are lost to the
hemolymph (see below). In contrast, most posterior lobes are
severely overgrown and either remain tethered to the dorsal
vessel or detach (Fig. 1, supplementary material Fig. S1D, and
see below). Loss of posterior lobes coincides with the appearance
of large compact tumors in the hemolymph. This trend suggests
that the lymph gland itself may be the direct source of the
To examine whether Ubc9 has one primary function in normal
hematopoiesis and probe if all four defects (overgrowth,
misdifferentiation, lobe dispersal, and lobe detachment) are
triggered from an initial disruption of this primary function, we
compared the expression patterns of Dome.GFP, Hml.GFP,
and 76B.GFP in developing heterozygous and Ubc9 lymph
glands. We found no striking difference in late second or even
early third instar (day 4 after egg lay) animals (Fig. 1B,C,
supplementary material Fig. S1A,B). Most cells of the posterior
lobes do not express mature hemocyte markers, but express
Dome.GFP, when the Dome promoter is active (Fig. 1B,D)
(Jung et al., 2005; Krzemien et al., 2007). Dome encodes the
receptor for JAK-STAT signaling (Brown et al., 2001). At mid to
late third instar (day 5 to 6), all heterozygous anterior lobes
remain relatively small and structurally intact, while anterior
lobes of the mutant glands are either larger than control, or they
disperse. Mutant posterior lobes expand dramatically, but remain
largely intact (Fig. 1D,E, supplementary material Fig. S1C,D,
Fig. 4B,C,E,F). We found that the overgrown lobes themselves
are displaced and begin to detach from the dorsal vessel
(supplementary material Fig. S1D, Fig. S2J,L, Fig. 4E,F).
The expression of Dome.GFP in heterozygous lymph glands
remains high, while in mutant glands, it gradually decreases
during third instar and is virtually absent by late 6 day
(Fig. 1D,E). Loss of Dome.GFP expression in mutant lobes
does not result from increased apoptosis, as only less than 1% of
cells in the lobes of either genetic background are positive for
cleaved pro-caspase 3.
Dome.GFP expression is undetectable in circulating
hemocytes of both, control and mutant animals. Single
Dome.GFP cells in circulation or within microtumors are rare
(supplementary material Fig. S2A–D). Surprisingly, while
Dome.GFP is expressed weakly in the dorsal vessel of control
animals, it is highly upregulated after the onset of anterior lobe
dispersal in the mutant background (Fig. 1D,E, Fig. 5A,B;
asterisk). Together, these results suggest that a primary
hematopoietic effect of Ubc9 loss is on the cells of the
medullary zone. Additionally, Ubc9-dependent gene regulation
in the dorsal vessel coincides with loss of lobe integrity.
The expression of Hml.GFP is limited largely to the periphery
in all 6 day control anterior lobes and in approximately 10% (n51/
8) of the first set of posterior lobes (supplementary material
Fig. S1C). In all examined mutant anterior lobes and about 40%
53/8) of first posterior lobes, Hml.GFP cells are scattered
throughout the body of the lobe (supplementary material Fig. S1D).
The expanded posterior lobes of mutant glands contain more
Hml.GFP-expressing cells than the control posterior lobes
(supplementary material Fig. S1D). That both, Dome.GFP and
Hml.GFP expression becomes more pronounced in the first
posterior lobes of control glands at third instar, supports the notion
that this capacity to acquire zonation is heterochronic; it emerges
only after the anterior lobes have matured. Second, at third instar
Dome expression decreases in Ubc9 lymph gland and Hml
expression increases slightly in posterior lobes compared to
controls. These changes in the expression patterns occur
simultaneously with lymph gland overgrowth.
The medullary zone exhibits heterogeneity
To understand the effects of the Ubc9 mutation on cells of the
medullary zone, we simultaneously expressed Dome.DsRed
with ZCL2897 (a GFP protein trap) (Morin et al., 2001) in wild
type glands. ZCL2897 is expressed in cells of the medullary zone
of control animals (Fig. 1F,H) (Jung et al., 2005). Despite
substantial overlap in the expression of Dome.DsRed and
ZCL2897, there is significant heterogeneity in gene expression
Hematopoietic microtumors in Ubc9 mutants of Drosophila 163
(Fig. 1F–G1). At least three cell types are observed: those that
express both markers (Dome
, Fig. 1F1, cells with
yellow hue) and those that are strongly positive for one marker
, red cells, or Dome
, green cells,
Fig. 1F1,G1). Among the doubly-positive cells, there is no
apparent correlation in signal intensity of the two markers,
suggesting that the medullary zone population consists of distinct
We next monitored ZCL2897 expression in heterozygous and
Ubc9 third instar animals and found that, in contrast to
Dome.GFP, loss of Ubc9 activates ZCL2897 expression in
anterior and posterior lobes (Fig. 1H–K’). Unlike Dome.GFP,
high ZCL2897 expression is also found in mutant circulating
hemocytes, microtumors and overgrown lobes which are easily
spotted through the cuticle (supplementary material Fig. S2E–L).
Such overgrown, intact lobes, while still attached to the dorsal
vessel (Fig. 1E, supplementary material Fig. S2J,L; also see
Fig. 4E,F, Fig. 5B), correspond to the freely circulating
microtumors in size and shape (supplementary material
Fig. S2G,O; also see supplementary material Fig. S5B,H,
supplementary material Fig. S6C, supplementary material
Fig. S7H). This significant expansion of the ZCL2897
population suggests that Ubc9 restrains division, keeps
progenitors from entering an aberrant differentiation program,
and maintains organ integrity.
To test if ZCL2897 expression marks lamellocytes, we
examined relative expression of either MSNF9mo-mCherry
(MSNF9, supplementary material Fig. S3A–D), or Atilla
(supplementary material Fig. S3E,F) with ZCL2897. Both
methods revealed that while a significant number of mutant
ZCL2897-positive cells also express Atilla or MSNF9 (yellow
signal, supplementary material Fig. S3C,D,F; white arrowheads
in supplementary material Fig. S3G–R’), a number of ZCL2897
cells do not express either lamellocyte marker (supplementary
material Fig. S3G–R’, white arrows). We also identified rare
cells with low or absent ZCL2897 expression but positive for
MSNF9 (supplementary material Fig. S3O,O’, blue arrowheads)
or Atilla (M.K. and S.G., unpublished data). Thus, expansion of
ZCL2897 population in the mutant supports the idea that Ubc9
maintains proliferative quiescence in the progenitor population
and prevents their aberrant and lamellocyte differentiation.
Ubc9 affects cells of the transition zone
To probe the properties of the expanded population in mutant
glands with a Gal4 driver, whose expression is not downregulated
by the effects of the mutation, we examined the expression of the
76B.Gal4. This driver is expressed in few cells of the lymph
gland (Paddibhatla et al., 2010), although the identity of these cells
is not known. We found that at late third instar, many heterozygous
76B.GFP-expressing cells are located outside the Dome-MESO
boundary (i.e., they are negative for Dome-MESO; Fig. 2A, white
arrows) and do not express the Pro-PO (Fig. 2B–B1’’), Nim C
(Fig. 2C–C1’’), or MSNF9 (Fig. 2B–B1-), although rare
exceptions are observed (zoomed panels in Fig. 2A–C; yellow
arrows). Thus, 76B.GFP expression marks the cells that are
intermediate to the Dome-MESO-positive progenitors in the
medullary zone and the differentiated cells in the cortex.
Because most of the cells expressing 76B.GFP reside outside
the Dome-MESO boundary, interspersed in the cortex, and the
double positives with either Dome-MESO (Fig. 2A) or the Pro-PO/
Fig. 2. 76B-Gal4 characterization in
control and Ubc9 lymph glands.
(A–C10) L3 (6-day) Ubc9
lobes expressing 76B.GFP (green), co-
labeled with Dome-MESO (red; A–A20),
or Pro-Phenol Oxidase and misshapen
(PPO, red, and MSNF9, magenta,
respectively; B–B1-), or Nimrod C
(NimC, red; C–C10).
(D–E) 76B.GFP (green) expression in
(D) and Ubc9
(E) lymph glands. (F–G1-) L3 (6-day)
anterior lobes expressing
76B.GFP (green), labeled with Nimrod
C and misshapen (NimC, red and
MSNF9, magenta, respectively);
presented are lower (F–F1-) and upper
(G–G1-) optical sections of two lymph
glands. Split-channel images show green
(9), red (0) or magenta (-). Selected
regions (white rectangles) are shown as
high magnifications (panels labeled with
numbers 1–2). White arrows – cells
expressing singly 76B.GFP, and
yellow arrows – two markers; star–DV.
Confocal sections (A–G1-). Scale bars:
50 mm (A–A0,B,C,D,E,F,G) and 10 mm
Hematopoietic microtumors in Ubc9 mutants of Drosophila 164
Nim C (Fig. 2B,C) are rare, they most likely represent the
transitional precursors that are derived from the medullary zone
progenitors, but have not yet assumed a final differentiated
identity. The existence of this transition zone has been suggested in
recent studies (Krzemien et al., 2010).
Unlike Dome.GFP and Hml.GFP, 76B.GFP population is
significantly expanded in Ubc9 mutant glands (Fig. 2D,E). Some
mutant 76B.GFP cells are also positive for either MSNF9 or
Nim C (Fig. 2F–G1-; yellow arrows). [Ubc9 lymph glands have
very few crystal cells (Chiu et al., 2005) and these were therefore
not examined in mutant glands.] 76B.GFP expression is also
expanded in single cells in circulation or those in microtumors in
the hemolymph (supplementary material Fig. S2M–P). This
expanded expression of 76B.GFP parallels the expression
dynamics of ZCL2897 (supplementary material Fig. S2E–L) in
Ubc9 is expressed throughout the lymph glands
Ubc9 protein is ubiquitously expressed in the anterior and
posterior lobes of the control third instar animals, in both,
medulla (nuclear and cytoplasmic) and cortex (mainly nuclear;
supplementary material Fig. S4A–D’). In addition to the diffuse
nuclear signal (supplementary material Fig. S4B,B’,D,D’),
speckles are also present (supplementary material Fig. S4B,B’;
white arrows). Ubc9 is also expressed in the dorsal vessel
(supplementary material Fig. S4A,C,E,G; star). Ubc9
mutants exhibit significantly lower levels of the protein in the
entire organ (supplementary material Fig. S4E–H’). Both
hypomorphic alleles have been previously characterized
molecularly (Apionishev et al., 2001).
SUMO pathway components in hematopoiesis
If changes observed in Ubc9 mutant hematopoietic organ are due
to loss of sumoylation, then other enzymes of the sumoylation
cascade should be similarly required. To test this idea, we
examined larvae carrying loss-of-function mutations in E1
) and E3/PIAS (Su(var)2-10
E1 is an activating heterodimer of Aos1 and Uba2 subunits, while
PIAS, encoded by Su(var)2-10, serves as the E3 ligase. Like
Ubc9 glands, Aos1 and PIAS glands exhibit significant activation
of ZCL2897 (Fig. 3A–C). Mutants in each background produce
hematopoietic tumors (Fig. 3D,E) marked by increased
expression of ZCL2897. Numerous lamellocytes appear in
dispersing anterior lobes and in circulation (Fig. 3A–E and
M.K. and S.G., unpublished data).
To test if Dome.GFP expression is compromised by loss of
sumoylation enzymes, we performed knockdown of E1 subunits
via RNAi. Knock-down of either Aos1 or Uba2 led to significant
reduction of the Dome.GFP expression, lamellocyte
differentiation, anterior lobe dispersal (Fig. 3F–H), and
tumorogenesis (Fig. 3I,J). These observations parallel those for
Ubc9 mutants and demonstrate that sumoylation is a fundamental
mechanism through which cell division and differentiation of
hematopoietic progenitors is simultaneously regulated.
Ubc9 microtumors arise from progenitor hyperplasia of anterior
and posterior lobes
To more directly study the role of Ubc9 in the cell cycle, we
stained lymph glands in late third instar stage (day 6.5–7) for
phospho-histone H3 (Fig. 4A–F). At this stage, most control
animals pupariated or are about to pupariate; their lymph gland
lobes are relatively large and mitotically active (Fig. 4A–C). In
mutants, the anterior lobes are dispersed with only few cells
remaining (Fig. 4D, outline). The enlarged posterior lobes have
numerous mitotically-active cells; these lobes show signs of
detachment from the dorsal vessel (e.g., in Fig. 4E–F, only single
partially detached posterior lobes are visible). Lobes of both PL1
and PL2 are severely affected (Fig. 4E–F, merged confocal Z
Fig. 3. Sumoylation enzymes in larval
hematopoiesis. (A–E) ZCL2897 (green) expression in
wild type (A), Aos1
(B) and PIAS
glands and in tumors of Aos1
(D) and PIAS
(E) larvae. (F–H) Dome.GFP (green) in control
lymph gland (F, without RNAi constructs). Reduction
of Dome.GFP in lymph glands expressing
(G) and Dome.Uba2
(H). (I, J) Tumors form in animals expressing
(I) and Dome.Uba2
classes do not
produce tumors. Confocal sections (A–C,F,H,J) and
fluorescent microscopy (D,E,G,I). Scale bars: 50 mm.
Hematopoietic microtumors in Ubc9 mutants of Drosophila 165
sections) and the number of phospo-histone H3-positive cells
ranges between 200–800 per posterior lobe set, compared to 30–
80 phospho-histone H3-positive cells in the corresponding
To clarify the identity of mitotic cells and examine their
relation to Dome.GFP expression, we stained anterior lobes of
slightly younger early 6-day lymph glands (where Dome.GFP is
still detectable) and visualized differentiated plasmatocytes (anti-
Nimrod C antibody) or lamellocytes (anti-Atilla antibody) with
anti-phospho-histone H3 antibody. Most of the Dome.GFP cells
in control glands are phospho-histone H3-negative, confirming
proliferative quiescence of this cell population (Fig. 4G,G1,I,I1,
arrowheads indicate mitotic cells). Not surprisingly, markers for
mitosis and Nimrod C rarely colocalized in cells of either
genotype (Fig. 4G–H1, arrow). None of the lamellocytes were in
division (Fig. 4I–J1). Notably however, loss of Dome.GFP
precedes increase in proliferation, as phospho-histone H3
staining is observed in regions of mutant lobes with low
Dome.GFP signal, but only rarely among the Dome.GFP-
positive cells (Fig. 4H,H1,J,J1).
Collectively, these observations strongly suggest that the solid
compact large Ubc9 microtumors result primarily from the
excessive mitoses in the lymph gland lobes. The expanded lobes
are severed from the dorsal vessel to become free-floating
microtumors. Some small tumors and aggregates are likely
derived from clusters of cells dispersed from the anterior lobes.
These conclusions are supported by the following: (1) Extensive
mitoses and overgrowth in the anterior and posterior mutant lobes
of 6 to 7 day old organs and their partial dispersal (Figs 1, 3, 4, 5,
supplementary material Figs S1, S2). (2) Massive overgrowth of
the remaining posterior lobes with enhanced expression of
ZCL2897 (Figs 1K, supplementary material Fig. S2J,L,
supplementary material Fig. S3D,F) or 76B (Fig. 2E,
supplementary material Fig. S2O,P) in the lobes and
Fig. 4. Overproliferation of immature
cells in Ubc9 lymph gland.
(A–F) Phosphorylated histone H3 (PH3,
white) in 6.5–7-day L3 Ubc9
PL1, PL2 (A,B,C, respectively) and
AL (D; remaining cells
outlined), PL1, PL2 (E,F, respectively).
Star marks DV. Optical Z-sections
merged (A–C,E–F). Mutant PL1 and
PL2 (E,F) are partially detached from
the DV and misaligned; lobe orientation
(top – anterior, bottom – posterior) is
reverse of the DV. (G–J1) Dome.GFP
(green), PH3 (white; arrowheads) and
Nimrod C (red, G–H1), or Atilla (red, I–
J1) in 6-day AL in Ubc9
(G,G1,I,I1) and Ubc9
(H,H1,J,J1) animals. PH3/Nimrod C
localization in the same cell (G1, arrow).
Regions indicated in G,H,I,J magnified
in G1,H1,I1,J1, respectively. Confocal
sections (A–J1). Scale bars: 50 mm.
Hematopoietic microtumors in Ubc9 mutants of Drosophila 166
microtumors. (3) The morphologies of overgrown ZCL2897
lobes match those of the microtumors in the
hemolymph. (4) The time of microtumor appearance in the
hemolymph correlates with observed detachment of the
overgrown lobes from the dorsal vessel.
Ubc9 function is essential in hematopoietic progenitors
To delineate the spatio-temporal requirement of Ubc9 in
restraining division and differentiation of hematopoietic
progenitors, we provided wild type Ubc9 protein to these
populations via Dome-Gal4 and 76B-Gal4. The experimental
rescue class (Ubc9; Dome.Ubc9
) animals exhibit
simultaneous and remarkable amelioration from the differential
effects of the mutation on the anterior and posterior lobes: (1)
The normal temporal and spatial regulation of the Dome
promoter is restored in both anterior and posterior lobes and
cells of the dorsal vessel (Fig. 5A–C). (2) The normal course of
lobe development is restored, i.e., not only are the rescued
posterior lobes comparable in size to control posterior lobes, they
remain tethered to the dorsal vessel. (3) Even though the cortical
zone of some rescue class glands shows differentiating
lamellocytes, the overall proportions of the medullary and
cortical zones return to normal. Overexpression of
reduces the number of Dome.GFP cells very
slightly (Fig. 5D). (4) A stark reduction in tumorogenesis is noted
as reduction in the proportion of animals carrying free
microtumors (supplementary material Fig. S5D; microtumor
penetrance from 75% to 11%) or aggregates (clusters of 15–50
cells; supplementary material Fig. S5E; from 82% to 23%). Other
non-hematopoietic defects, i.e., delay in the onset of pupariation
and adult lethality, are also rescued. These rescued adults carry
no visible microtumors.
Significantly, like Dome.Ubc9
Ubc9 defects. Since its expression is high in mutant cells
(Fig. 2D,E), it is possible to visualize the remedial effects of
as it shrinks the GFP-positive cell population,
restores coherent lymph gland lobes (I.P. and S.G., unpublished
data), prevents posterior lobe detachment, and reduces the tumor
burden (supplementary material Fig. S5F–I).
In contrast to the full rescue with the Dome.Ubc9
transgenes, we found that large microtumors
persisted with Collagen.Ubc9
material Fig. S6A–D; Cg-Gal4 is expressed in the lymph gland
cortical zone, circulating hemocytes, and fat body) (Asha et al.,
2003). All together, these observations are consistent with the
interpretation that even though Ubc9 influences all hematopoietic
compartments and the integrity of the lymph gland, the primary
function of the protein is to maintain quiescence in hematopoietic
progenitors. Sumoylation appears to serve a critical tumor-
suppressive function by regulating the gene expression and the
cell cycle of hematopoietic progenitors of the third instar larval
Ubc9 hyperplasia is niche-independent
To examine the requirement for Ubc9 in the niche, we compared
niche morphology and size, and the membranous projections
emanating from the niche into the medullary zone (Krzemien et
al., 2007; Mandal et al., 2007) in heterozygous and mutant
glands. We found no significant difference in the niche size,
measured either as the number of cells expressing Antennapedia
protein (supplementary material Fig. S7A–C, 5 day old animals)
or Antp.GFP (supplementary material Fig. S7D–F, 6 day old
animals). There was no difference in the niche projections, which
were sparse in both backgrounds (supplementary material
Fig. 5. Dome.Ubc9
restores Ubc9 lymph gland size and
Dome.GFP expression. (A–D) Dome.GFP in Ubc9
(D). Asterisk (DV); dotted line outlines the lobes
(A–D). Confocal sections (A–D). Scale bars: 50 m m.
Hematopoietic microtumors in Ubc9 mutants of Drosophila 167
Fig. S7D,E). Cells of the dorsal vessel immediately adjacent to
the niche express Antp (by both criteria), although we found no
difference in its expression between heterozygous and mutant
glands (supplementary material Fig. S7A,B,D,E; asterisks). An
occasional population of Antp.GFP cells is found in the
posterior lobes of the mutant or in microtumors (M.K. and
S.G., unpublished data).
To link Ubc9 function in the niche to overproliferation, we
progeny. These rescue class
larvae did not experience relief from hematopoietic defects
(supplementary material Fig. S7G–K) and died during pupal
stages, just like their mutant siblings. Overexpression of Ubc9
in the niche (Antp-Gal4) did not modify the niche or lobe
morphology, nor did it induce lamellocytes (M.K. and S.G.,
unpublished data). Likewise, mutants were not rescued when
wild type protein was supplied in the niche by Collier-Gal4
(M.K. and S.G, unpublished data) (Crozatier et al., 2004). These
observations demonstrate that progenitor hyperplasia in mutants
is niche-independent and that its function is autonomous with
respect to the progenitor pool.
Loss of Ubc9 is linked to reduction of Dacapo levels
Protein interaction data suggested direct association of Ubc9 with
Drosophila CDK inhibitor Dacapo (Dap) (Stanyon et al., 2004).
To test if Dap levels are affected in Ubc9 cells, we stained lymph
glands with anti-Dap antibody (described in de Nooij et al.) (de
Nooij et al., 1996). In control glands, levels of Dap protein differ:
cytoplasmic Dap is somewhat higher in the compact region of the
medullary zone (dotted lines Fig. 6A,A’), than in the cytoplasm
of Dome.GFP-negative cells. This correlation is maintained in
Ubc9 glands, where cytoplasmic Dap signal is significantly
reduced in cells with lower Dome.GFP signal and loss of the
compact architecture (Fig. 6A–B’). The overall correlation
between high Dome.GFP and high Dap signals suggests that
sumoylation maintains quiescence by controlling cell cycle exit
by sustaining high levels of Dacapo. While in both, heterozygous
Fig. 6. Lymph gland cells respond to
cell cycle inhibitors Dacapo/p21 and
human p21 rescues Ubc9
tumorogenesis. (A–B’) Dacapo (red)
and Dome.GFP (green) expression in
(A,A’) and Ubc9
(B,B’) lymph glands. Indicated regions
(A,A’) are magnified in the insets.
(A’,B’) show Dacapo staining only.
(C–F) Lymph glands with Dome.GFP
(C); Dome.Dap, GFP (D); Dome.p21,
GFP (E) expression; nuclei labeled in
white. Absolute cell numbers in
Dome.GFP; Dome.Dap, GFP;
Dome.p21, GFP (F; average 6 SE,
n>5 animals per genotype); p values
relative to control are shown on the
graph (for Dome.GFP populations with
green highlight, for remaining GFP
cells with blue highlight). Dotted lines
outline MZ (white) and CZ (yellow).
(G–L) 76B.GFP (green) in Ubc9
AL (G), PL1 (H); Ubc9
AL (I), PL1
, 76B.p21, GFP AL
(K), PL1 (L). Outlines: compact tissue
(white line), lobe edges (yellow).
Confocal sections (A–L). Brightness of
images in A–B’ was slightly increased
for clarity without modifying the result.
Scale bars: 50 mm.
Hematopoietic microtumors in Ubc9 mutants of Drosophila 168
and mutant glands, Dacapo levels are lower in cells outside the
medulla, in both backgrounds Dap protein is clearly detected
Expression of human p21 relieves Ubc9 overproliferation
Dacapo shares structural and functional similarity with vertebrate
cyclin/cyclin-dependent kinase (CDK) inhibitors, p21/p27 (de
Nooij and Hariharan, 1995; de Nooij et al., 1996; Lane et al.,
1996). Like overexpression of Ubc9
, both Dome.Dap and
Dome.p21 lead to reduction of the progenitor population
(Fig. 6C–F). The effect of Dome.p21 is stronger than that of
Dome.Dap [n520606384 cells in control (average 6 SD, per
both anterior lobes), to n515666449 in Dome.Dap, and
n59526301 in Dome.p21 (Fig. 6C–F)].
If the primary function of sumoylation is to maintain
quiescence in progenitors, expression of p21 in this population
may be sufficient to partially restore lymph gland homeostasis.
To test this hypothesis, we created Dome.p21; Ubc9 animals.
, Dome.p21 resulted in only temporary
and weak rescue (supplementary material Fig. S8A) presumably
because in Dome.p21; Ubc9 glands, Dome.GFP levels
continue to remain low (M.K. and S.G., unpublished data).
In contrast to Dome.p21, both, 76B.Dap and 76B.p21
prevent overgrowth of the progenitor population in mutant
glands, restoring their normal compact morphology. There is a
decline in the 76B.GFP-positive cells (Fig. 6K,L), the lobes do
not disperse or dislocate, and microtumor penetrance is
significantly reduced (Fig. 6K,L, supplementary material
Fig. S8B). However, when p21 was provided in cells of the
cortical zone and circulating hemocytes (with SrpHemo, Hemese,
Hml,orCg), we found no evidence of tumor rescue. Thus,
downregulation of Dap expression in Ubc9 mutant lymph gland
progenitors and Ubc9 rescue with 76B.Dap/p21 confirm the
tumor-suppressive function of Ubc9 in the hematopoietic
progenitors and suggest that cell cycle inhibition is likely
maintained through sumoylation.
Mammalian cancer stem cells, characterized in many cancer
types, persist for a long time, and like their putative parental
cells, remain proliferatively quiescent. This phenotype is thought
to make them resistant to chemotherapy. Whether quiescence
plays a role in cancer stem cell biology and how these cells retain
proliferative quiescence, despite transitioning into a diseased
state, is not clearly understood (reviewed in Moore and Lyle,
2011). Our studies here provide an important avenue to
investigate the regulatory cell cycle mechanisms of normal and
quiescent cancer cells at the earliest stage of cancer development.
Tumorogenesis results from failure to quiesce, dysplasia of
heterogeneous progenitors, and dispersal and detachment
In a quest to identify the source of microtumors in Ubc9 mutants,
we discovered that even though Ubc9 protein is ubiquitously
expressed, it plays a specific and essential, niche-independent
function in maintaining proliferative quiescence within
progenitors of the medullary and transition zones. Reduction of
sumoylation via knockdown of any of the other core enzymes of
the pathway also leads to progenitor dysplasia and
tumorogenesis. Once detached from the dorsal vessel, the
microtumors float in the hemolymph (Fig. 1, supplementary
material Figs S1, S2, Fig. 3, supplementary material Fig. S5).
The progenitor population that serves as the source of
microtumors is heterogeneous with respect to Dome.GFP and
ZCL2897 expression. One of the earliest detectable effects of the
mutation is on the differential expression of Dome.GFP and
ZCL2897 or 76B.GFP in the expanding population (Fig. 1,
supplementary material Fig. S2, Figs 3, 7). The onset of the
effects of Ubc9 mutation coincides with the period when the
progenitors in the medulla of the anterior lobes undergoes
proliferative restraint (Jung et al., 2005). At the same time, cells
of the posterior lobes lag behind; they continue to divide and
follow a defined heterochronic developmental pattern
(Fig. 1A,B,D, supplementary material Fig. S1A,C). It is
somewhat surprising that even though the Ubc9 mutation has
differential effects on cells of the anterior versus posterior lobes,
the overproliferation defects in both are largely rescued by
ectopic expression of p21/Dap. This observation suggests a
fundamental role for the enzyme in inhibiting cell cycle
progression and conferring quiescence to progenitors. Since the
decline in Dome.GFP expression precedes overproliferation in
mutant lobes (Fig. 4) and each defect can be rescued by the
expression of wild type Ubc9, it is possible that Dome.GFP
expression marks the quiescent cell state. The inability of p21 or
Dap to restore normal Dome.GFP expression attests to the
notion that the sequential series of events, even at the earliest
stages of tumorogenesis, can be genetically teased out in vivo.
While the changes in cell identities in mutant lobes are
complex, the discovery of heterogeneity in the medullary zone
populations of anterior and first posterior lobes is consistent with
recent reports that this population has distinct fate-restricted cell
populations (Krzemien et al., 2010; Minakhina and Steward,
Fig. 7. Sumoylation controls proliferation of progenitor cells along with
Dacapo. Hematopoietic progenitors express high level of either one or both,
ZCL2897 (green) and Dome.GFP (red). Some of these cells and cells in
transition zone express 76B-Gal4 (cyan). At third instar stage, progenitors enter
quiescence. Heterogeneity and absence of mature marker expression suggest
similarity to mammalian transit amplifying cells (Morrison and Spradling,
2008; Shaker and Rubin, 2010) or Drosophila testis progenitors (Shivdasani
and Ingham, 2003). We propose that sumoylation regulates multiple events
including maintenance of high levels of Dacapo protein in these cells. In the
absence of sumoylation enzymes Aos1/Uba2, Ubc9, and PIAS, these cells fail
to quiesce and progress into G2/M phase (cells with purple outline) and
misdifferentiate (dark green cells); dysplasia and tumorogenesis follow.
Hematopoietic microtumors in Ubc9 mutants of Drosophila 169
2010). Our results suggest that lymph gland progenitors are
similar to mammalian transit amplifying cells (Morrison and
Spradling, 2008; Shaker and Rubin, 2010) or those in the
Drosophila testis (Shivdasani and Ingham, 2003), that have
limited proliferative capacity and possess a restricted
differentiation potential relative to their multipotent stem cells.
With an appropriate immune or developmental cue, Drosophila
hematopoietic progenitors may re-enter the cell cycle to produce
What is the physiological significance of retaining some cells
in quiescence at this stage in larval life? One possibility is that
mitotic exit shelters progenitors from precocious development
and provides a mechanism that determines the number of times
they must divide before they differentiate. Additionally, a reserve
of progenitors, ready to divide and differentiate rapidly guards
larvae against natural enemies such as parasitic wasps that attack
them at this stage of the life cycle (Sorrentino et al., 2002). This
tactic parallels mitotic exit of hematopoietic stem cells (HSCs) in
mice about three weeks after birth, or in humans, at about four
years of age, when they become adult HSCs. The dormant adult
HSCs are activated as the organism recovers from injury
(Trumpp et al., 2010).
This similarity in strategies between flies and humans in
normal hematopoiesis is further reinforced even when the process
becomes aberrant. Like in dUbc9 mutants, uncontrolled
proliferation of progenitors in human leukemias can occur
independently of the signals from the niche (supplementary
material Fig. S7) (Passegue et al., 2003). It is intriguing that
Antp, a niche marker, is also expressed in the dorsal vessel
(supplementary material Fig. S7). Furthermore, Dome.GFP
expression, undetectable in normal cells, is strongly activated
in mutant cells of the dorsal vessel (Figs 1, 5). Thus, it is possible
that cues from the cells of the dorsal vessel influence the state of
the hematopoietic progenitors and integrity of the lobes.
Conversely, the status of the progenitors themselves may
determine the association of the lobes to the dorsal vessel.
Further analysis of Ubc9 mutants will clarify the role of the
microenvironment in supporting progenitor quiescence and
maintaining tissue integrity.
Dacapo/p21 contributes to progenitor quiescence
A key mechanism by which sumoylation maintains proliferative
quiescence in larval hematopoiesis is cell cycle regulation
through Dacapo/p21. In the embryo, Dap/p21 binds to cyclin
E/Cdk2 complexes to block the G1/S transition in cell cycle
(Lane et al., 1996). Furthermore, the human p21 protein can
block mitosis in the Drosophila eye (Tseng and Hariharan, 2002).
This function of Dap/p21 in larval hematopoiesis is similar to the
roles of p27
(Fero et al., 1996) or p21
(Cheng et al.,
2000) in enforcing HSC quiescence.
We found that Dap is expressed in Dome .GFP progenitors in
wild type and mutant glands, and is reduced shortly after
Dome.GFP is downregulated in mutant glands (Fig. 6).
Overexpression of Dap/p21 in these cells leads to decrease in
progenitor number. It is noteworthy that dap mutants do not
exhibit apparent tumorous overgrowth (M.K. and S.G.,
unpublished data) (de Nooij et al., 1996; Lane et al., 1996), a
trait that is similar to young p21 null mice (Adnane et al., 2000;
Martin-Caballero et al., 2001). However, with age, or in the
presence of other mutations (e.g., oncogenic Ras), p21 null mice
are prone to developing tumors (Adnane et al., 2000; Martin-
Caballero et al., 2001; Jackson et al., 2002). It is therefore very
likely that tumorogenesis in Ubc9 mutants is supported not only
by loss of Dap/p21 but also by the activation of other oncogenic
and pro-inflammatory proteins (Fig. 7).
The mechanism by which Ubc9 controls Dap protein levels is
not known. dap transcription has been studied in embryonic
development where it regulates mitotic exit (de Nooij et al., 1996;
Lane et al., 1996; Liu et al., 2002). High dap transcript levels in
stage 16 embryonic central and peripheral nervous system, or in
differentiating postmitotic cells of a developing eye disc,
correlate with exit from mitosis (de Nooij et al., 1996; Lane et
al., 1996; Liu et al., 2002). These observations suggest that
regulation of dap transcription is coupled with mitotic exit, and it
is therefore possible that its transcription in the lymph gland
progenitors is similarly synchronized. Microarray experiments of
whole Ubc9 larvae compared to their heterozygous siblings
indicate dap transcript downregulation (S.G., unpublished data).
An intriguing possibility is that Dacapo itself, or another protein
in complex with Dap, is a sumoylation target. In high throughput
yeast two-hybrid assay, Dap was found to physically interact with
Ubc9 (Stanyon et al., 2004). Future experiments including
biochemical analyses of Dap and interacting proteins are
required to test this idea.
Unscrambling Ubc9 functions in cancer and inflammation
The causal relationship between cancer and inflammation is now
widely accepted, even though the mechanisms that establish and
sustain this relationship remain unresolved (Karin and Greten,
2005; Mantovani et al., 2008). Drosophila Toll-Dorsal pathway
not only manages immunity, but also governs hematopoietic
development (Qiu et al., 1998; Govind, 2008). Ubc9 microtumor
development requires Rel/NF-kappa B family transcription
factors Dorsal and Dif (Chiu et al., 2005; Huang et al., 2005).
Aberrant activation of NF-kappa B signaling in Ubc9 mutants
resembles hematopoieitic malignancies in vertebrates that arise
due to ectopic germline or somatic disruption of the pathway
(Courtois and Gilmore, 2006).
We recently discovered that sumoylation provides a
homeostatic mechanism to restrain systemic inflammation in
the fly larva, where it keeps the Toll/Dorsal-dependent immune
response in check. Ubc9 controls the ‘‘set point’’ by maintaining
normal levels of IkB/Cactus protein in immune tissues
(Paddibhatla et al., 2010). The Ubc9 cancer-inflammation
model offers novel opportunities to examine the dynamics of
tumor growth, its relationship to metastasis, and the links
between cancer and inflammation. Ubc9 tumors are sensitive to
aspirin (I.P. and S.G., unpublished results). This model is well-
suited for identifying and testing drugs that target highly-
conserved biochemical mechanisms, such as sumoylation, which
oversee self-renewal pathways in progenitor populations.
Material and Methods
Fly strains and culture
The following lines were obtained: y w; Ubc9
and y w; Ubc9
(Dr S. Tanda) (Chiu et al., 2005); w
(17744) and yw;Smt3
GFP (10419; Drosophila Bloomington Stock Center); PIAS alleles Su(var)2-10
CyO, act-GFP and Su(var)2-10
/CyO, act-GFP (Dr G. Karpen) (Hari et al., 2001);
MSNF9mo-mCherry from Dr R. Schultz (Tokusumi et al., 2009), Dome-MESO
(Hombria et al., 2005; Krzemien et al., 2007). ZCL2897 was obtained from Yale
GFP Protein Trap Collection (Morin et al., 2001).
UAS lines: UAS-Aos1
(Vienna Stock Center) and UAS-Uba2
Harvard Medical School); UAS-Ubc9
(Dr S. Tanda) (Apionishev et al., 2001);
Hematopoietic microtumors in Ubc9 mutants of Drosophila 170
UAS-p21 and UAS-Dap (Dr I. Hariharan) (Tseng and Hariharan, 2002); UAS-
mCD8-GFP (5137), UAS-DsRed (6280) and UAS-myr-mRFP (7119) from
Drosophila Bloomington Stock Center.
Gal4 lines: Domeless-Gal4 (Bourbon et al., 2002) and Collier-Gal4 (Krzemien
et al., 2007) from Dr M. Crozatier, Antennapedia-Gal4 (Dr S. Minakhina)
(Emerald and Cohen, 2004), HemolectinD-Gal4 (Sinenko and Mathey-Prevot,
2004) and Collagen-Gal4 (7011; Bloomington Stock Center) (Asha et al., 2003);
Hemese-Gal4 (Dr I. Ando) (Zettervall et al., 2004) and Serpent-Gal4 (Bruckner et
al., 2004) are expressed only in some lymph gland cells and circulating hemocytes;
76B-Gal4 (Harrison et al., 1995).
The ZCL2897, UAS and Gal4 transgenes were integrated into mutant
backgrounds by standard crosses. ZCL2897 and Dome.myr-mRFP combination
was lethal in the Ubc9
Drosophila cultures were maintained on standard media. Six- or twelve-hour
egglays were cultured at 23.5
Comparison of mutant and heterozygote was done on the same day, where
available. Heterozygotes pupate at day 6; some of the Ubc9 mutants remain in L3
at day 8.
The Gal4/UAS system (Brand and Perrimon, 1993) was used. Dome.Ubc9
rescue: y w UAS-Ubc9
, UAS-mCD8-GFP/CyO y
and y w Dome-Gal4/
flies were crossed; simultaneous cross: y w/Y; Ubc9
and y w Dome-Gal4/FM7c; Ubc9
heterozygote Dome-Gal4/y w; Ubc9
, UAS-mCD8-GFP/CyO y
, mutant Dome-
Gal4/y w; Ubc9
, rescue Dome-Gal4/y w UAS-Ubc9
and overexpression Dome-Gal4/y w UAS-
, UAS-mCD8-GFP/CyO y
classes were scored.
Remaining rescues were similarly designed: UAS-Ubc9
p21-carrying flies in Ubc9
background were crossed to those carrying
selected Gal4 (Dome, 76B, Hemolectin, Collagen, Hemese, Serpent, Antp) and
allele; UAS-GFP or UAS-mCD8-GFP transgenes were carried
by either one, or both parents. The F1 Ubc9
mutant combination, rescue,
overexpression and heterozygous control were scored.
Tumor penetrance in larvae was scored after dissection (at a magnification of
200 x), except in 76B.Ubc9
and 76B.p21 rescue experiments where tumors
were scored in intact animals. Since tumor penetrance is inherently variable, for
well-controlled conditions and comparable results, all control, mutant, rescue and
overexpression animals were grown and scored simultaneously under the same
conditions. All experiments were performed in duplicate or triplicate.
Standard antibody staining protocol was used (described in Paddibhatla et al.)
(Paddibhatla et al., 2010). Antibodies used: rabbit anti-phospho-histone H3 (1:200,
Molecular Probes), mouse anti-P1/Nimrod C1 and mouse anti-L1/Atilla (1:10)
(Vilmos et al., 2004; Kurucz et al., 2007a), mouse anti-Prophenol Oxidase (1:10,
Dr T. Trenczek, University of Giessen), rabbit anti-Ubc9 (1:1500, received from
Dr R. Tanguay) (Joanisse et al., 1998), anti-Antennapedia 8C11 (1:20,
Developmental Studies Hybridoma Bank, The University of Iowa), mouse anti-
Dacapo (1:4, received from Dr I. Hariharan (de Nooij and Hariharan, 1995), mouse
anti-beta-galactosidase 40-1a (1:10, Developmental Studies Hybridoma Bank, The
University of Iowa). Fluorescently-labeled secondary antibodies (Molecular
Probes and Jackson Immunological), Phalloidin (Invitrogen) and nuclear dye
Hoechst 33258 (Molecular Probes) were used.
Image acquisition and processing
Whole larvae were imaged in Leica stereomicroscope. Images of dissected and
stained tissues were acquired in a Zeiss Laser Scanning Confocal or Zeiss
Axioscope 2 Plus Fluorescence microscopes, and formatted in Zeiss LSM5 and
AxioVision LE 4.5 software, respectively. Figures were assembled in Adobe
Photoshop CS5. Cell counts were performed using Volocity software (Perkin
Elmer). Nuclear staining is represented in the figures in blue, unless stated
otherwise. Slight adjustments of brightness and contrast were applied equally to
images of both, control and mutant, where applicable, and are explicitly stated in
corresponding figure legend. None of these modifications affect or modify the
result in a significant way.
We are grateful to our colleagues for stocks and reagents and to
members of the Govind Lab for feedback. We thank J. Uribe, C.
Chand, R. Rajwani, and Z. Papadopol for help with experiments and
D. Fimiarz for help with confocal imaging, image processing and
data retrieval. We thank the TRiP facility at Harvard Medical School
for RNAi lines. This work was supported in part by funds from
NSF (1121817), USDA (NRI/USDA CSREES 2006-03817 and
2009-35302-05277), NIH NIGMS S06 GM08168, G12-RR03060,
P50-GM68762), and PSC-CUNY.
Adnane, J., Jackson, R. J., Nicosia, S. V., Cantor, A. B., Pledger, W. J. and Sebti,
S. M. (2000). Loss of p21WAF1/CIP1 accelerates Ras oncogenesis in a transgenic/
knockout mammary cancer model. Oncogen e 19, 5338-5347.
Apionishev, S., Malhotra, D., Raghavachari, S., Tanda, S. and Rasooly, R. S. (2001).
The Drosophila UBC9 homologue lesswright mediates the disjunction of homologues
in meiosis I. Genes Cells 6, 215-224.
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,
Bourbon, H. M., Gonzy-Treboul, G., Peronnet, F., Alin, M. F., Ardourel, C.,
Benassayag, C., Cribbs, D., Deutsch, J., Ferrer, P., Haenlin, M. et al. (2002). A P-
insertion screen identifying novel X-linked essential genes in Drosophila. Mech. Dev.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering
cell fates and generating dominant phenotypes. Development 118, 401-415.
Brown, S., Hu, N. and Hombria, J. C. (2001). Identification of the first invertebrate
interleukin JAK/STAT receptor, the Drosophila gene domeless. Curr. Biol. 11, 1700-
Bruckner, K., Kockel, L., Duchek, P., Luque, C. M., Rorth, P. and Perrimon, N.
(2004). The PDGF/VEGF receptor controls blood cell survival in Drosophila. Dev.
Cell 7, 73-84.
Cheng, T., Rodrigues, N., Shen, H., Yang, Y., Dombkowski, D., Sykes, M. and
Scadden, D. T. (2000). Hematopoietic stem cell quiescence maintained by p21cip1/
waf1. Science 287, 1804-1808.
Chiu, H., Ring, B. C., Sorrentino, R. P., Kalamarz, M., Garza, D. and Govind, S.
(2005). dUbc9 negatively regulates the Toll-NF-kappa B pathways in larval
hematopoiesis and drosomycin activation in Drosophila. Dev. Biol. 288, 60-72.
Courtois, G. and Gilmore, T. D. (2006). Mutations in the NF-kappaB signaling
pathway: implications for human disease. Oncogene 25, 6831-6843.
Crozatier, M., Ubeda, J. M., Vincent, A. and Meister, M. (2004). Cellular immune
response to parasitization in Drosophila requires the EBF orthologue collier. PLoS
Biol. 2, e196.
de Nooij, J. C. and Hariharan, I. K. (1995). Uncoupling cell fate determination from
patterned cell division in the Drosophila eye. Science 270, 983-985.
de Nooij, J. C., Letendre, M. A. and Hariharan, I. K. (1996). A cyclin-dependent
kinase inhibitor, Dacapo, is necessary for timely exit from the cell cycle during
Drosophila embryogenesis. Cell 87, 1237-1247.
Emerald, B. S. and Cohen, S. M. (2004). Spatial and temporal regulation of the
homeotic selector gene Antennapedia is required for the establishment of leg identity
in Drosophila. Dev. Biol. 267, 462-472.
Fero, M. L., Rivkin, M., Tasch, M., Porter, P., Carow, C. E., Firpo, E., Polyak, K.,
Tsai, L. H., Broudy, V., Perlmutter, R. M. et al. (1996). A syndrome of multiorgan
hyperplasia with features of gigantism, tumorigenesis, and female sterility in
p27(Kip1)-deficient mice. Cell 85, 733-744.
Govind, S. (2008). Innate immunity in Drosophila: pathogens and pathways. Insect Sci.
Hari, K. L., Cook, K. R. and Karpen, G. H. (2001). The Drosophila Su(var)2-10 locus
regulates chromosome structure and function and encodes a member of the PIAS
protein family. Genes Dev. 15, 1334-1348.
Harrison, D. A., Binari, R., Nahreini, T. S., Gilman, M. and Perrimon, N. (1995).
Activation of a Drosophila Janus kinase (JAK) causes hematopoietic neoplasia and
developmental defects. EMBO J. 14, 2857-2865.
Holz, A., Bossinger, B., Strasser, T., Janning, W. and Klapper, R. (2003). The two
origins of hemocytes in Drosophila. Development 130, 4955-4962.
Hombria, J. C., Brown, S., Hader, S. and Zeidler, M. P. (2005). Characterisation of
Upd2, a Drosophila JAK/STAT pathway ligand. Dev. Biol. 288, 420-433.
Huang, L., Ohsako, S. and Tanda, S. (2005). The lesswright mutation activates Rel-
related proteins, leading to overproduction of larval hemocytes in Drosophila
melanogaster. Dev. Biol. 280, 407-420.
Jackson, R. J., Adnane, J., Coppola, D., Cantor, A., Sebti, S. M. and Pledger, W. J.
(2002). Loss of the cell cycle inhibitors p21(Cip1) and p27(Kip1) enhances
tumorigenesis in knockout mouse models. Oncogene 21, 8486-8497.
Joanisse, D. R., Inaguma, Y. and Tanguay, R. M. (1998). Cloning and developmental
expression of a nuclear ubiquitin-conjugating enzyme (DmUbc9) that interacts with
small heat shock proteins in Drosophila melanogaster. Biochem. Biophys. Res.
Commun. 244, 102-109.
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.
Kalamarz, M. (2010). Origin and development of hematopoietic tumors in sumoylation
mutants of Drosophila melanogaster. PhD dissertation, pp. 112, Department of
Biology, The Graduate Center of The City University of New York, USA.
Karin, M. and Greten, F. R. (2005). NF-kappaB: linking inflammation and immunity
to cancer development and progression. Nat. Rev. Immunol. 5, 749-759.
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.
Hematopoietic microtumors in Ubc9 mutants of Drosophila 171
Krzemien, J., Oyallon, J., Crozatier, M. and Vincent, A. (2010). Hematopoietic
progenitors and hemocyte lineages in the Drosophila lymph gland. Dev. Biol. 346,
Kurucz, E., Vaczi, B., Markus, R., Laurinyecz, B., Vilmos, P., Zsamboki, J., Csorba,
K., Gateff, E., Hultmark, D. and Ando, I. (2007a). Definition of Drosophila
hemocyte subsets by cell-type specific antigens. Acta Biol. Hung. 58 Suppl., 95-111.
Kurucz, E., Markus, R., Zsamboki, J., Folkl-Medzihradszky, K., Darula, Z.,
Vilmos, P., Udvardy, A., Krausz, I., Lukacsovich, T., Gateff, E. et al. (2007b).
Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila
plasmatocytes. Curr. Biol. 17, 649-654.
Lane, M. E., Sauer, K., Wallace, K., Jan, Y. N., Lehner, C. F. and Vaessin, H.
(1996). Dacapo, a cyclin-dependent kinase inhibitor, stops cell proliferation during
Drosophila development. Cell 87, 1225-1235.
Lanot, R., Zachary, D., Holder, F. and Meister, M. (2001). Postembryonic
hematopoiesis in Drosophila. Dev. Biol. 230, 243-257.
Liu, T. H., Li, L. and Vaessin, H. (2002). Transcription of the Drosophila CKI gene
dacapo is regulated by a modular array of cis-regulatory sequences. Mech. Dev. 112,
Mabb, A. M. and Miyamoto, S. (2007). SUMO and NF-kappaB ties. Cell Mol. Life Sci.
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.
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.
Manfruelli, P., Arquier, N., Hanratty, W. P. and Semeriva, M. (1996). The tumor
suppressor gene, lethal(2)giant larvae (1(2)g1), is required for cell shape change of
epithelial cells during Drosophila development. Development 122, 2283-2294.
Mantovani, A., Allavena, P., Sica, A. and Balkwill, F. (2008). Cancer-related
inflammation. Nature 454, 436-444.
Martin-Caballero, J., Flores, J. M., Garcia-Palencia, P. and Serrano, M. (2001).
Tumor susceptibility of p21(Waf1/Cip1)-deficient mice. Cancer Res. 61, 6234-6238.
Minakhina, S. and Steward, R. (2010). Hematopoietic stem cells in Drosophila.
Development 137, 27-31.
Moore, N. and Lyle, S. (2011). Quiescent, slow-cycling stem cell populations in
cancer: a review of the evidence and discussion of significance. J. Oncol. 2011, pii:
Morin, X., Daneman, R., Zavortink, M. and Chia, W. (2001). A protein trap strategy
to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila.
Proc. Natl. Acad. Sci. USA 98, 15050-15055.
Morrison, S. J. and Spradling, A. C. (2008). Stem cells and niches: mechanisms that
promote stem cell maintenance throughout life. Cell
Moschos, S. J., Jukic, D. M., Athanassiou, C., Bhargava, R., Dacic, S., Wang, X.,
Kuan, S. F., Fayewicz, S. L., Galambos, C., Acquafondata, M. et al. (2010).
Expression analysis of Ubc9, the single small ubiquitin-like modifier (SUMO) E2
conjugating enzyme, in normal and malignant tissues. Hum. Pathol. 41, 1286-1298.
Nam, H. J., Jang, I. H., Asano, T. and Lee, W. J. (2008). Involvement of pro-
phenoloxidase 3 in lamellocyte-mediated spontaneous melanization in Drosophila.
Mol. Cells 26, 606-610.
Paddibhatla, I., Lee, M. J., Kalamarz, M. E., Ferrarese, R. and Govind, S. (2010).
Role for sumoylation in systemic inflammation and immune homeostasis in
Drosophila larvae. PLoS Pathog, 6, e1001234.
Passegue, E., Jamieson, C. H. M., Ailles, L. E. and Weissman, I. L. (2003). Normal
and leukemic hematopoiesis: are leukemias a stem cell disorder or a reacquisition of
stem cell characteristics? Proc. Natl. Acad. Sci. USA 100, 11842-11849.
Potter, C. J., Turenchalk, G. S. and Xu, T. (2000). Drosophila in cancer research. An
expanding role. Trends Genet. 16, 33-39.
Qiu, P., Pan, P. C. and Govind, S. (1998). A role for the Drosophila toll/cactus pathway
in larval hematopoiesis. Development 125, 1909-1920.
Rizki, T. M. and Rizki, R. M. (1992). Lamellocyte differentiation in drosophila larvae
parasitized by leptopilina. Dev. Comp. Immunol. 16, 103-110.
Shaker, A. and Rubin, D. C. (2010). Intestinal stem cells and epithelial-mesenchymal
interactions in the crypt and stem cell niche. Transl. Res. 156, 180-187.
Shivdasani, A. A. and Ingham, P. W. (2003). Regulation of stem cell maintenance and
transit amplifying cell proliferation by TGF-beta signaling in Drosophila spermato-
genesis. Curr. Biol. 13, 2065-2072.
Shrestha, R. and Gateff, E. (1982). Ultrastructure and cyto-chemistry of the cell-types
in the larval hematopoietic organs and hemolymph of Drosophila-Melanogaster. Dev.
Growth Differ. 24, 65-82.
Sinenko, S. A. and Mathey-Prevot, B. (2004). Increased expression of Drosophila
tetraspanin, Tsp68C, suppresses the abnormal proliferation of ytr-deficient and Ras/
Raf-activated hemocytes. Oncogene 23, 9120-9128.
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.
Stanyon, C. A., Liu, G., Mangiola, B. A., Patel, N., Giot, L., Kuang, B., Zhang, H.,
Zhong, J. and Finley, R. L., Jr (2004). A Drosophila protein-interaction map
centered on cell-cycle regulators. Genome Biol. 5, R96.
Talamillo, A., Sanchez, J. and Barrio, R. (2008). Functional analysis of the
SUMOylation pathway in Drosophila. Biochem. Soc. Trans. 36, 868-873.
Tokusumi, T., Shoue, D. A., Tokusumi, Y., Stoller, J. R. and Schulz, R. A. (2009).
New hemocyte-specific enhancer-reporter transgenes for the analysis of hematopoi-
esis in Drosophila. Genesis 47, 771-774.
Trumpp, A., Essers, M. and Wilson, A. (2010). Awakening dormant haematopoietic
stem cells. Nat. Rev. Immunol. 10
Tseng, A. S. and Hariharan, I. K. (2002). An overexpression screen in Drosophila for
genes that restrict growth or cell-cycle progression in the developing eye. Genetics
Vidal, M. and Cagan, R. L. (2006). Drosophila models for cancer research. Curr. Opin.
Genet. Dev. 16, 10-16.
Vilmos, P., Nagy, I., Kurucz, E., Hultmark, D., Gateff, E. and Ando, I. (2004). A
rapid rosetting method for separation of hemocyte sub-populations of Drosophila
melanogaster. Dev. Comp. Immunol. 28, 555-563.
Wang, J. C. (2010). Good cells gone bad: the cellular origins of cancer. Trends Mol.
Med. 16, 145-151.
Zettervall, C. J., Anderl, I., Williams, M. J., Palmer, R., Kurucz, E., Ando, I. and
Hultmark, D. (2004). A directed screen for genes involved in Drosophila blood cell
activation. Proc. Natl. Acad. Sci. USA 101, 14,192-14,197.
Hematopoietic microtumors in Ubc9 mutants of Drosophila 172