DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 3561
Development 137, 3561-3568 (2010) doi:10.1242/dev.053728
© 2010. Published by The Company of Biologists Ltd
Serpent, Suppressor of Hairless and U-shaped are crucial
regulators of hedgehog niche expression and prohemocyte
maintenance during Drosophila larval hematopoiesis
Yumiko Tokusumi*, Tsuyoshi Tokusumi*, Jessica Stoller-Conrad and Robert A. Schulz†
SUMMARY
The lymph gland is a specialized organ for hematopoiesis, utilized during larval development in Drosophila. This tissue is
composed of distinct cellular domains populated by blood cell progenitors (the medullary zone), niche cells that regulate the
choice between progenitor quiescence and hemocyte differentiation [the posterior signaling center (PSC)], and mature blood cells
of distinct lineages (the cortical zone). Cells of the PSC express the Hedgehog (Hh) signaling molecule, which instructs cells within
the neighboring medullary zone to maintain a hematopoietic precursor state while preventing hemocyte differentiation. As a
means to understand the regulatory mechanisms controlling Hh production, we characterized a PSC-active transcriptional
enhancer that drives hh expression in supportive niche cells. Our findings indicate that a combination of positive and negative
transcriptional inputs program the precise PSC expression of the instructive Hh signal. The GATA factor Serpent (Srp) is essential
for hh activation in niche cells, whereas the Suppressor of Hairless [Su(H)] and U-shaped (Ush) transcriptional regulators prevent
hh expression in blood cell progenitors and differentiated hemocytes. Furthermore, Srp function is required for the proper
differentiation of niche cells. Phenotypic analyses also indicated that the normal activity of all three transcriptional regulators is
essential for maintaining the progenitor population and preventing premature hemocyte differentiation. Together, these studies
provide mechanistic insights into hh transcriptional regulation in hematopoietic progenitor niche cells, and demonstrate the
requirement of the Srp, Su(H) and Ush proteins in the control of niche cell differentiation and blood cell precursor maintenance.
INTRODUCTION
Owing to highly productive research efforts in recent years,
Drosophila has emerged as a valuable organism for the discovery
and analysis of genes controlling hematopoiesis (Evans et al., 2003;
Sorrentino et al., 2005; Crozatier and Meister, 2007; Koch and
Radtke, 2007; Martinez-Agosto et al., 2007). As in vertebrate
animals, blood cell formation occurs in multiple spatiotemporal
waves during Drosophila development. Transplantation
experiments using single cells derived from blastoderm stage
embryos have indicated that blood cell precursors arise from two
distinct origins: cephalic mesoderm that generates embryonic
hemocytes and a thoracic mesodermal region that will form lymph
glands (Holz et al., 2003), the organ used for hematopoiesis during
larval development (Lebestky et al., 2000). Differentiated
hemocytes produced during both hematopoietic waves possess
characteristics comparable to those found in vertebrate myeloid
lineages (Meister and Langueux, 2003). Embryo-derived blood
cells include plasmatocytes and crystal cells. Plasmatocytes
constitute the major class, functioning as macrophage-like cells that
mediate phagocytosis of bacterial pathogens and apoptotic bodies.
Crystal cells make up the minor class, being involved in wound
healing and large foreign body encapsulation. Together, they
number ~700 cells by the end of embryogenesis and persist into
Department of Biological Sciences, University of Notre Dame, 147 Galvin Life
Sciences Hall, Notre Dame, IN 46556, USA.
*These authors contributed equally to this work
†
Author for correspondence ([email protected])
Accepted 1 September 2010
larval stages, when they undergo multiple rounds of mitosis,
generating 6000-8000 hemocytes by the end of larval development
(Sorrentino et al., 2007; Tokusumi et al., 2009b). A subpopulation
of these cells will take up residence as sessile hemocytes in
subepidermal locations within larvae, where they can be rapidly
converted into lamellocytes, a third hemocyte type that is produced
in response to wasp infestation (Markus et al., 2009; Honti et al.,
2010). Lamellocytes are large, flat, adhesive cells that are crucial
for the successful encapsulation of invasive species, such as the
parasitic wasp egg.
The lymph gland hematopoietic organ is formed near the end of
embryogenesis from two clusters of cells derived from anterior
cardiogenic mesoderm (Crozatier et al., 2004; Mandal et al., 2004).
About 20 pairs of hemangioblast-like cells give rise to three distinct
lineages that will form the lymph glands and anterior part of the
dorsal vessel. Notch (N) pathway signaling serves as the genetic
switch that differentially programs these progenitors towards cell
fates that generate the lymph glands (blood lineage), heart tube
(vascular lineage), or heart tube-associated pericardial cells
(nephrocytic lineage). An essential requirement has also been
proven for Tailup (Islet1) in lymph gland formation, in which it
functions as an early-acting regulator of serpent (srp), odd-skipped
and Hand hematopoietic transcription factor gene expression (Tao
et al., 2007).
By the end of the third larval instar, each anterior lymph gland is
composed of three morphologically and molecularly distinct regions
(Jung et al., 2005). The posterior signaling center (PSC) is a cellular
domain formed during late embryogenesis due to the specification
function of the homeotic gene Antennapedia (Antp) (Mandal et al.,
2007) and the maintenance function of Collier (Col; Knot –
FlyBase), the Drosophila ortholog of the vertebrate transcription
DEVELOPMENT
KEY WORDS: Drosophila, Hedgehog, Hematopoiesis, Niche cells, Serpent, Suppressor of Hairless, U-shaped
3562 RESEARCH ARTICLE
we document crucial roles for the Srp, Su(H) and Ush
transcriptional regulators in the control of Hh niche expression and
verify their importance for normal blood cell homeostasis within
the Drosophila larval hematopoietic organ.
MATERIALS AND METHODS
Drosophila strains
We used the following lines in this study: ushVX22 and ushr24 (Cubadda et
al., 1997) (M. Haenlin); P85col-Gal4 and UAS-col (Crozatier et al., 2004;
Krzemien et al., 2007) (M. Crozatier); BcF6-GFP (Gajewski et al., 2007);
eater-DsRed and MSNF9mo-mCherry (Tokusumi et al., 2009a); tepIV-Gal4
(Drosophila Genetic Resource Center, Kyoto Stock Center, Japan) and
UAS-srp RNAi (two lines: GD12779, Vienna Drosophila RNAi Center; and
HM05094, Transgenic RNAi Resource Project at Harvard Medical
School). We also obtained several stocks from the Bloomington Stock
Center (Indiana University): w1118, N1N-ts1, Antp25, Antp17, Su(H)1,
Su(H)IB115, UAS-gapGFP, CyO Act-GFP, UAS-hh and UAS-dTCFDN.
Generation of transgenic Drosophila strains
Transgenic lines harboring hh DNA-GFP reporter fusions were generated as
described previously (Rubin and Spradling, 1982; Tokusumi et al., 2009b).
Briefly, we amplified hh genomic DNA fragments from a BAC clone using
PrimeStar DNA polymerase (Takara-Millus), followed by DNA subcloning
into the P-element vector pH Stinger (Barolo et al., 2000). For the analysis
of essential cis-regulatory elements, we introduced point mutations into trans
factor binding sites of the enhancer region with the QuikChange SiteDirected Mutagenesis Kit (Stratagene). The sequence of the PSC-active 1020
bp hhF4f enhancer DNA is given in Fig. S1 in the supplementary material.
The PSC-active hhF4l enhancer fragment corresponds to the 3⬘ 190 bp
interval of the larger DNA. Potential Antp, Srp, Su(H) (Brou et al., 1994;
Bailey and Posakony, 1995; Lecourtois and Schweisguth, 1995) and Col
(Dubois et al., 2007) binding sites are highlighted within the hhF4f sequence,
with the introduced nucleotide changes indicated. Sequence comparison of
hhF4f DNA from D. melanogaster with the corresponding DNAs of 11
additional Drosophila species showed that the putative EBF/Col binding site
was fully conserved in only six of 12 species (see Fig. S2 in the
supplementary material). Following germline transformation of hh DNAGFP reporter constructs, we established and analyzed at least ten independent
lines.
Immunostaining of Drosophila lymph glands
The following primary antibodies were used to determine protein
expression in lymph glands dissected from control or mutant animals:
rabbit anti-Hh (1:100; T. Kornberg) (Tabata and Kornberg, 1994), rabbit
anti-Ush (1:400) (Fossett et al., 2001), rabbit anti-Su(H) (1:1000; sc-25761,
Santa Cruz), mouse anti-Antp (1:100; 4C3, Developmental Studies
Hybridoma Bank), mouse anti-Ptc (1:200; Developmental Studies
Hybridoma Bank), and mouse anti-plasmatocyte (P1) antibody (1:100; I.
Ando) (Vilmos et al., 2004) . Lymph glands were dissected from staged
larvae, fixed with 4% paraformaldehyde in PBS for 1 hour at room
temperature, washed with PBS containing 0.1% Triton X-100 (PBST), and
blocked with 5% goat serum in PBST. We then added diluted primary
antibodies in PBST containing 5% goat serum, incubated overnight at 4°C,
washed with PBST several times, and then incubated with Alexa 488- or
555-conjugated secondary antibodies (1:500; Molecular Probes) in PBST
containing 5% goat serum for 1 hour at room temperature. After washing
with PBST, samples were mounted in 50% glycerol in PBS.
Immunostained samples were analyzed with a Zeiss Axioplan fluorescence
microscope or a Zeiss LSM710 laser-scanning confocal microscope.
RESULTS
Characterization of a transcriptional enhancer
controlling hh expression in hematopoietic niche
cells
As Hh produced by the PSC is a crucial signaling molecule
regulating the maintenance of hemocyte precursors within the
medullary zone (Mandal et al., 2007), we were interested to elucidate
DEVELOPMENT
factor early B-cell factor (Crozatier et al., 2004). PSC cells
selectively express the Hedgehog (Hh) and Serrate (Ser) signaling
molecules and extend numerous thin filopodia into the neighboring
medullary zone (Lebestky et al., 2003; Krzemien et al., 2007;
Mandal et al., 2007). This latter lymph gland domain is populated by
undifferentiated and slowly proliferating blood cell progenitors
(Mandal et al., 2007). Prohemocytes within the medullary zone
express the Hh receptor Patched (Ptc) and the Hh pathway
transcriptional effector Cubitus interruptus (Ci). Medullary zone cells
also express components of the Jak/Stat signaling pathway. By
contrast, the third lymph gland domain – the cortical zone – solely
contains differentiating and mature hemocytes, such as plasmatocytes
and crystal cells. Upon wasp parasitization, or in certain altered
genetic backgrounds, lamellocytes will also appear in the cortical
zone as a third type of differentiated hemocyte (Lanot et al., 2001;
Sorrentino et al., 2002; Sorrentino et al., 2007; Gao et al., 2009;
Markus et al., 2009; Tokusumi et al., 2009b).
Two independent studies have provided compelling data to
support the contention that the PSC functions as a hematopoietic
progenitor niche within the lymph gland, with this cellular domain
being essential for maintaining normal hemocyte homeostasis
(Krzemien et al., 2007; Mandal et al., 2007). These investigations
showed that communication between the PSC and prohemocytes
present in the medullary zone is crucial for the preservation of the
progenitor population and to prevent these cells from becoming
abnormally programmed to differentiate into mature hemocytes.
Seminal findings from these studies can be summarized as follows
(Crozatier and Meister, 2007): Col expression must be restricted to
the PSC by the localized expression of Ser; Hh must be expressed
selectively in the PSC, coupled with the non-autonomous activation
of the Hh signaling pathway in prohemocytes of the medullary
zone; and the PSC triggers activation of the Jak/Stat pathway
within cells of the medullary zone. With the perturbation of any of
these molecular events, the precursor population of the medullary
zone is lost owing to the premature differentiation of hemocytes,
which swell the cortical zone. Although the exact interrelationship
of Ser, Hh and Jak/Stat signaling within the lymph gland is
currently unknown, the cytoplasmic extensions emanating from
PSC cells might facilitate instructive signaling between these niche
cells and hematopoietic progenitors present in the medullary zone
(Krzemien et al., 2007; Mandal et al., 2007). A more recent study
showed that components of the Wingless (Wg) signaling pathway
are expressed in the stem-like prohemocytes to reciprocally
regulate the proliferation and maintenance of cells within the
supportive PSC niche (Sinenko et al., 2009). The cellular
organization and molecular signaling of the Drosophila lymph
gland are remarkably similar to those of the hematopoietic stem
cell niches of vertebrate animals, including several mammals
(Gering and Patient, 2005; Yamashita et al., 2005; Martinez-Agosto
et al., 2007; Orkin and Zon, 2008).
These findings have documented a complex role for distinct
signaling pathways and a sophisticated organization of nonequivalent cell types within the hematopoietic progenitor-niche
microenvironment of the Drosophila lymph gland. To gain
mechanistic insights into the genetic control of one of these
pathways, we pursued the transcriptional enhancer controlling the
PSC-specific expression of the essential Hh signaling molecule. We
rationalized that through a detailed analysis of the positive and
negative inputs influencing this PSC-active regulatory module,
enlightening information could be gained on those genes required
for the precise expression of Hh in niche cells and on their role in
the maintenance of the hematopoietic progenitor population. Here,
Development 137 (21)
Regulators of hedgehog niche expression
RESEARCH ARTICLE 3563
the genetic mechanisms controlling hh cell-specific expression in the
hematopoietic niche. Towards this goal, we localized the
transcriptional enhancer controlling hh expression in the PSC.
Initially, we scanned seven 3-kb DNA intervals, spanning 21 kb of
hh upstream and intragenic sequence, for regulatory DNAs that
could drive GFP reporter expression selectively in the PSC domain
of the lymph gland. The overlapping hhF4 and hhF5 DNAs, derived
from hh intron 1, possessed the ability to direct GFP expression
exclusively in the PSC (Fig. 1A). The smaller, 1.0 kb hhF4f interval
likewise directed GFP expression in niche cells, indicating that it
harbored a regulatory module sufficient for hh transcription in the
PSC (Fig. 1A,B). Double-labeling experiments verified the fidelity
of the transcriptional enhancer, as the regulatory DNA was active in
PSC cells that also expressed Antp (Fig. 1C) and Hh (Fig. 1D).
The functions of a few transcription factors have been shown to
be vital for the generation and maintenance of the PSC cell
population within the lymph gland: Antp specifies the PSC (Mandal
et al., 2007), whereas Col maintains the PSC population (Crozatier
et al., 2004). We monitored the activity of the hhF4f-GFP transgene
in mutant backgrounds that altered the functions of these
hematopoietic regulators. In Antp loss-of-function embryos, the PSC
was lost and a corresponding absence of hhF4f-GFP expression was
observed (Fig. 1E). By contrast, forced expression of Col during
lymph gland development resulted in an overproduction of PSC
cells, with the supernumerary cells being labeled with the GFP
reporter driven by the hh enhancer (Fig. 1F). Wg signaling also
positively regulates the proliferation and maintenance of these niche
cells (Sinenko et al., 2009). Consistent with this observation, we
failed to detect hhF4f-GFP-positive cells in lymph glands expressing
a dominant-negative version of TCF (Pangolin – FlyBase), a
downstream effector of Wg signaling (Fig. 1G). Together, these
genetic studies confirm the fidelity of the hh regulatory module for
hematopoietic niche cells.
glands isolated from transgenic larvae. Through these analyses, we
were able to further delimit the PSC regulatory module to the 190
bp hhF4l interval of hh intron 1 (Fig. 2A). A sequence comparison
of this region of the Drosophila melanogaster genome with
corresponding DNA intervals of 11 other sequenced Drosophila
species revealed the precise conservation of four putative
transcription factor binding sites: a possible Antp recognition
element, two GATA sequences that could bind Srp, and a potential
recognition site for the Su(H) protein (Fig. 2B; see Fig. S1 in the
supplementary material). As mutation of the putative Antp binding
site within the 1.0 kb hhF4f DNA (hhF4f mAntp construct) and 190
bp hhF4l DNA (hhF4l mAntp construct) did not affect hh enhancer
function (Fig. 2A), we concluded that this sequence was not a
direct target of Antp activation. Thus, we pursued Srp and Su(H)
as potential transcriptional regulators of the hh enhancer.
A role for the hematopoietic factor Srp in the regulation of hh
PSC expression was implicated by the finding that mutation of both
conserved GATA recognition sequences (hhF4f mGATA construct)
led to a complete loss of PSC enhancer activity (Fig. 2C). More
direct evidence for Srp involvement in hh niche expression was
found when RNAi knockdown of srp function resulted in a
complete loss of hh enhancer activity (Fig. 2D) and an absence of
Hh protein (Fig. 2E), as derived from expression of the endogenous
gene, from niche cells of the PSC. RNAi knockdown of srp also
resulted in defects in niche cell differentiation, based on the
absence of filopodial extensions (Fig. 2G). As an important control
for these experiments, we showed that PSC cell nuclei present in
lymph glands dissected from P85col>UAS-srp RNAi animals failed
to accumulate Srp protein at detectable levels (see Fig. S3 in the
supplementary material). In summary, the enhancer mutagenesis
and srp loss-of-function data demonstrate that this hematopoietic
GATA factor serves as an activator of hh expression in the PSC
niche.
Srp is required for hh expression and cellular
differentiation in the PSC
Six additional 5⬘- or 3⬘-truncated versions of the PSC-active hhF4f
DNA were generated and tested for their ability to drive GFP
reporter expression in hematopoietic niche cells within lymph
Su(H) prevents hh expression in blood cell
precursors within the medullary zone
As noted above, the minimal hh PSC enhancer present in the 190
bp hhF4l DNA contains an evolutionarily conserved GTGGGAA
element, which is a potential recognition sequence for the
DEVELOPMENT
Fig. 1. Localization and characterization of a Drosophila
hh transcriptional enhancer that is active in the PSC.
(A)Location and posterior signaling center (PSC) activity of
overlapping hh-GFP enhancer test DNAs. (B)hhF4f-GFP
transgene activity in the PSC. (C)Co-expression of hhF4f-GFP
and Antp in the PSC. (D)Co-expression of hhF4f-GFP and Hh
in the PSC. (E)Absence of hhF4f-GFP expression (arrowhead)
in lymph glands dissected from Antp mutant larvae.
(F)Supernumerary hhF4f-GFP-positive PSC cells in Col gainof-function larvae. (G)Absence of hhF4f-GFP expression
(arrowhead) in lymph glands dissected from larvae
expressing a dominant-negative form of TCF. The PSCspecific Gal4 driver P85col was used to express UAS-col or
UAS-TCFDN in niche cells (F,G). Scale bars: 20m.
3564 RESEARCH ARTICLE
Development 137 (21)
transcriptional repressor Su(H). Using an antibody directed against
Su(H), we demonstrated that this protein is expressed in lymph
glands, with highest accumulation in hematopoietic progenitors
within the medullary zone and a lower accumulation in
differentiated hemocytes in the cortical zone (Fig. 3A). A doublelabeling experiment to detect Su(H) and GFP driven by the hh PSC
enhancer also showed a low, but detectable, presence of Su(H) in
niche cells (Fig. 3B).
Given this demonstration of Su(H) expression in lymph glands,
we mutated the conserved GTGGGAA element within the context
of the 1.0 kb hhF4f PSC enhancer [hhF4f mSu(H) construct; Fig.
2A] and generated transgenic flies harboring this enhancer test
DNA. Mutation of the prospective Su(H) binding site resulted in
the de novo expression of the GFP reporter in prohemocytes of the
medullary zone (Fig. 3C). Furthermore, in a Su(H) loss-of-function
mutant background, we observed an expansion of hh PSC
transcriptional enhancer activity to cells of the medullary zone (Fig.
3F) and ectopic expression of Hh protein in the same cells (Fig.
3G). In the same Su(H) mutant, Antp expression was not expanded,
indicating the maintenance of a normal PSC cell population in this
genetic background (Fig. 3H). It has been previously reported that
Su(H) can function as a repressor of gene expression in an Nindependent manner (Koelzer and Klein, 2003; Koelzer and Klein,
2006). We tested the activity of the hh-GFP transgene in lymph
glands obtained from Nts mutant larvae and determined that the
PSC-active enhancer functioned normally in this altered genetic
background (data not shown). These results demonstrated that
Su(H) serves as a crucial negative regulator of hh transcription (in
an N-independent mode), preventing both endogenous and
transgenic hh expression in blood cell progenitors of the medullary
zone.
Srp and Su(H) are required for prohemocyte
maintenance and the control of hemocyte
differentiation in the lymph gland
Previous studies have shown that the disruption of Hh signaling
from the PSC niche to hematopoietic precursor cells within the
medullary zone results in a loss of the progenitor population and in
enhanced differentiation of plasmatocytes and crystal cells
throughout the lymph gland (Mandal et al., 2007). Since RNAi
knockdown of srp resulted in an absence of Hh in the PSC, we
investigated the effect of Srp perturbation on prohemocyte and
hemocyte homeostasis. The Hh receptor Ptc serves as a marker of
hemocyte precursors within the medullary zone (Mandal et al.,
2007) (Fig. 4A) and we observed a strong reduction in the number
of Ptc-positive cells in the induced srp mutant background (Fig.
4B). Also, in srp mutant lymph glands, a large increase in the
number of differentiated plasmatocytes (Fig. 4E) and crystal cells
(Fig. 4H) was observed based on the use of the P1 antibody and
BcF6-GFP markers. Additionally, we determined the circulating
hemocyte concentration in control versus P85col>UAS-srp RNAi
third instar larvae and observed a 67% increase in blood cell
number due to perturbation of srp function in niche cells (see Fig.
S4 in the supplementary material). Thus, the knockdown of srp
function in lymph glands, and the resulting loss of Hh expression
in the PSC, culminated in a blood cell phenotype identical to that
seen in hh mutant lymph glands, with a loss of the progenitor
population and copious production of differentiated plasmatocytes
and crystal cells.
Comparable analyses were undertaken with lymph glands
obtained from Su(H) loss-of-function mutants, which showed the
abnormal expansion of Hh expression to progenitor cells within the
medullary zone. As with the disruption of srp function, Su(H)
mutant tissues exhibited a loss of Ptc-positive prohemocytes (Fig.
DEVELOPMENT
Fig. 2. Srp function is required for hh PSC expression
and niche cell differentiation. (A)Detailed mapping of
the regulatory elements required for hh enhancer activity
in the PSC. Asterisk, expanded GFP expression is observed.
(B) Sequence comparison (100 bp) of the crucial cisregulatory element cluster present in the PSC-active hhF4l
DNA of D. melanogaster with the corresponding hh DNAs
of 11 additional Drosophila species. (C)Mutation of two
evolutionarily conserved GATA sequences in the hhF4fmGATA DNA results in a complete loss of PSC enhancer
activity (arrowhead). (D,E)RNAi knockdown of srp function
results in a loss of hh enhancer activity (arrowhead in D)
and in the absence of Hh protein (arrowhead in E) from
PSC niche cells. The lymph gland is outlined. (F)Filopodial
extensions (arrow) are observed in niche cells of a control
lymph gland. (G)RNAi knockdown of srp function results
in defects in niche cell differentiation, based on the
absence of filopodial extensions (arrowhead). The UAS-srp
RNAi strain GD12779 was used in these srp knockdown
experiments, expressed under the control of the PSCspecific Gal4 driver P85col (D,E,G). Scale bars: 20m.
Fig. 3. Su(H) is a repressor of hh expression in hemocyte
precursors within the medullary zone. (A,B)Su(H) is strongly
expressed in prohemocytes present in the medullary zone (MZ), and
weakly expressed in differentiated hemocytes and hhF4f-GFP-positive
niche cells. (C-E)Mutation of the evolutionarily conserved Su(H)
recognition sequence in the hhF4fmSu(H)-GFP DNA results in the de
novo appearance of the GFP reporter in prohemocytes in the MZ, as
compared with eater-DsRed expression in plasmatocytes in the cortical
zone (CZ). (F,G)Expansion of hh PSC enhancer activity, and ectopic
expression of Hh protein, in medullary zone cells in an Su(H) loss-offunction mutant background. (H)In the same Su(H) mutant
background, a normal PSC cell population is maintained, as Antp
protein expression is not expanded. Scale bar: 20m.
4C) and the overproduction of differentiated plasmatocytes (Fig.
4F). By contrast, an increase was not observed in the crystal cell
population in Su(H) mutant lymph glands (Fig. 4I), probably
because this gene is required for lozenge activation and crystal cell
differentiation (Duvic et al., 2002; Lebestky et al., 2003). Together,
these findings demonstrated that Srp and Su(H) are required for
prohemocyte maintenance and for the regulation of hemocyte
differentiation in the lymph gland.
Ush is a negative regulator of hh expression in
blood cell precursors and differentiated
hemocytes
As all lymph gland cells express Srp (Jung et al., 2005; Sorrentino
et al., 2007), the question arose as to the mechanism by which this
hematopoietic GATA factor activates hh selectively in the PSC and
not in cells throughout the remainder of the lymph gland. hh
repression by Su(H) is certainly part of the answer to this question
RESEARCH ARTICLE 3565
Fig. 4. Srp and Su(H) loss of function disrupt blood cell
homeostasis in the lymph gland. (A)Ptc expression marks the PSC in
lymph glands dissected from a wild-type Drosophila larva. (B,C)srp
RNAi knockdown and Su(H) loss of function result in a strong reduction
in Ptc-positive cells in the lymph glands. (D)P1 antibody detects
plasmatocytes located in the cortical zone of a lymph gland dissected
from a wild-type larva. (E,F)srp RNAi knockdown and Su(H) loss of
function result in differentiated plasmatocytes throughout the mutant
lymph glands. (G)BcF6-GFP expression serves as a marker for crystal
cells in lymph glands dissected from a wild-type larva. (H)srp RNAi
knockdown results in differentiated crystal cells throughout the lymph
gland. (I)Reduced numbers of BcF6-GFP-positive crystal cells are
observed in Su(H) mutant lymph glands. The UAS-srp RNAi strain
GD12779 was used in these srp knockdown experiments, expressed
under the control of the PSC-specific Gal4 driver P85col (B,E,H). Scale
bar: 20m.
of regulatory control. In addition, we considered the possibility that
the transcriptional regulator Ush functions with Srp in precisely
regulating hh expression. Previous reports demonstrated that the
SrpNC-Ush (SrpNC is an Srp isoform with N- and C-terminal zinc
fingers) combination functions as a negative regulator of crystal
cell lineage specification (Fossett et al., 2003; Muratoglu et al.,
2007).
To address a potential role for Ush in hh regulation, we assayed
its expression in lymph glands. Ush protein was expressed in most
lymph gland cells, with the exception of the hhF4f-GFP-positive
PSC cells (Fig. 5A). Forcing the expression of Ush in PSC cells
resulted in a loss of hh PSC enhancer activity, while maintaining the
Antp-positive niche cell population (Fig. 5B). Expressing Ush in the
PSC also led to defects in niche cell differentiation, based on a lack
of filopodia formation, processes that normally extend from PSC
cells (Fig. 5C). Conversely, in lymph glands dissected from ush lossof-function animals, an expansion of hhF4f-GFP transgene activity
DEVELOPMENT
Regulators of hedgehog niche expression
Fig. 5. Ush is a negative regulator of hh expression in the lymph
gland. (A)Ush is expressed in most cells of the lymph gland, with the
exception of hhF4f-GFP-positive cells of the PSC. (B)Forced expression
of Ush in niche cells using P85col results in a lack of expression of the
hhF4f-GFP transgene, while maintaining the Antp-positive PSC
population. (C)Forced expression of Ush in niche cells using P85col
results in defects in niche cell differentiation, based on the reduction of
filopodial extensions (arrowhead). (D,G)Expanded expression of the
hhF4f-GFP transgene in lymph glands dissected from ush loss-offunction Drosophila larvae. (E)Normal PSC cell number and Antp
expression in lymph glands isolated from a ush loss-of-function larva.
(F)Ectopic expression of Hh protein in a lymph gland dissected from a
ush loss-of-function larva. (H)Expanded expression of the hhF4f-GFP
transgene and supernumerary lamellocyte production in lymph glands
isolated from a ush loss-of-function larva. Scale bars: 20m.
(Fig. 5D,G,H) and Hh protein accumulation (Fig. 5F,G) were
observed, implicating Ush as an additional and crucial negative
regulator of hh transcription in this hematopoietic organ. A reduction
in ush function also culminated in a robust production of
lamellocytes in the lymph glands (Fig. 5H) owing to its proven
function as a positive regulator of prohemocyte maintenance and as
a negative regulator of lamellocyte differentiation (Sorrentino et al.,
2007; Gao et al., 2009).
DISCUSSION
The Drosophila lymph gland has emerged as an informative tissue
for the study of cellular organization and intercellular signaling
within a hematopoietic progenitor-niche cell microenvironment.
Development 137 (21)
Four signaling pathways have been identified thus far that control
blood cell homeostasis within this larval hematopoietic organ: Hh,
Jak/Stat, N and Wg (Crozatier et al., 2004; Krzemien et al., 2007;
Mandal et al., 2007; Sinenko et al., 2009). Our current studies have
focused on the regulation of Hh expression in the PSC owing to the
vital role of the Hh signal sent from the niche cells to the hemocyte
precursors, which maintains this progenitor population while
preventing premature and precocious blood cell differentiation. The
utilization of this strategy has allowed us to gain mechanistic
insights into the mode of hh transcriptional control in the PSC and
the crucial functions of its positive and negative regulators in
prohemocyte maintenance.
Through detailed molecular and gene expression analyses we
have identified the PSC-active transcriptional enhancer within hh
intron 1 and delimited its location to a minimal 190 bp region. The
hh enhancer-GFP transgene faithfully recapitulates the niche cell
expression of Hh derived from the endogenous gene, as doublelabeling experiments with the GFP marker and Antp or Hh show a
clear co-expression in PSC domain cells. Appropriately, GFP
expression is not detected in Antp loss-of-function or TCFDN
genetic backgrounds, which culminate in an absence of niche cells
from the lymph gland. The hematopoietic GATA factor Srp serves
as a positive activator of hh PSC expression, as mutation of two
evolutionarily conserved GATA elements in the enhancer abrogates
its function and Srp functional knockdown via srp RNAi results in
hh enhancer-GFP transgene inactivity and the absence of Hh
protein expression. An additional intriguing phenotype was
observed in lymph glands expressing the srp RNAi transgene, that
being a strong reduction in the number of filopodial extensions
emerging from cells of the PSC. This phenotype suggests a
functional role for Srp in the correct differentiation of niche cells,
via a requirement for normal Hh presentation from these cells
and/or the transcriptional regulation by Srp of additional genes
needed for the formation of filopodia.
As Srp accumulates in all cells of the lymph gland (Jung et al.,
2005; Sorrentino et al., 2007), a question arose as to how hh
expression is restricted to cells of the PSC. This paradox could be
explained by a mechanism in which hh expression is also under
some means of negative transcriptional control in non-PSC cells of
the lymph gland. This possibility proved to be correct, with our
analyses identifying two negative regulators of hh lymph gland
expression. The first is Su(H). Mutation of the evolutionarily
conserved GTGGGAA element, a predicted recognition sequence
for this transcriptional repressor, resulted in an expanded activity
of the hh PSC enhancer-GFP transgene; that is, we observed the de
novo appearance of GFP in prohemocytes of the medullary zone.
Likewise, ectopic medullary zone expression of the wild-type PSC
enhancer-GFP transgene and of Hh protein was seen in lymph
glands mutant for Su(H). These findings, coupled with the
detection of Su(H) in blood cell progenitors, strongly implicate this
factor as a transcriptional repressor of the hh PSC enhancer,
restricting its expression to niche cells.
Additional studies identified Ush as a second negative regulator
of hh expression. Ush is expressed in most cells of the lymph
gland, with the exception of those cells resident within the PSC
domain. Previous research demonstrated that ush expression in the
lymph gland is under the positive control of both Srp (Muratoglu
et al., 2006) and the Jak/Stat signaling pathway (Gao et al., 2009).
Why Ush protein fails to be expressed in the PSC remains to be
determined. Forced expression of ush in niche cells resulted in
inactivation of the hh PSC enhancer and reduced the formation of
filopodia. We hypothesize that Ush might be forming an inhibitory
DEVELOPMENT
3566 RESEARCH ARTICLE
Fig. 6. Model of the positive and negative inputs controlling hh
expression in hematopoietic progenitor niche cells. Srp is a
transcriptional activator of hh expression in niche cells of the Drosophila
PSC. Such cells are specified or maintained via the functions of Antp,
Col and TCF. Su(H) serves as a transcriptional repressor of hh expression
in prohemocytes located in the medullary zone. Ush functions as a
negative regulator of hh expression in blood cell progenitors of the
medullary zone and in differentiated hemocytes located in the cortical
zone, possibly through its interaction with Srp and the formation of a
repressive SrpNC-Ush complex. The positive regulation of ush
expression by Srp and dStat (Stat92E – FlyBase) has been described
previously (Muratoglu et al., 2006; Gao et al., 2009).
complex with the SrpNC protein, changing Srp from a positive
transcriptional activator to a negative regulator of hh lymph gland
expression. Such a mechanism has been demonstrated previously
in the negative regulation by Ush of crystal cell lineage
commitment (Fossett et al., 2003; Muratoglu et al., 2007). The
expansion of wild-type hh enhancer-GFP transgene and Hh protein
expression to prohemocytes within the medullary zone and to
differentiated hemocytes within the cortical zone in lymph glands
mutant for ush is also supportive of Ush functioning as a negative
regulator of hh expression.
Bringing these results together, a model can be proposed for the
regulatory events that culminate in the precise expression of the
vital Hh signaling molecule in niche cells (Fig. 6). Srp is a direct
transcriptional activator of hh in the lymph gland and Hh protein
is detected in niche cells due to this activity. hh expression is
inhibited in prohemocytes of the medullary zone by Su(H) action,
while a repressive SrpNC-Ush transcriptional complex prevents
Srp from activating hh expression in prohemocytes and in
differentiated hemocytes of the medullary zone and cortical zone.
Together, these positive and negative modes of regulation would
allow for the niche cell-specific expression of Hh and facilitate the
localized presentation of this crucial signaling molecule to
neighboring hematopoietic progenitors.
The identification of Srp and Su(H) as key regulators of Hh
expression in the larval hematopoietic organ prompted an
investigation into the functional requirement of these proteins in
the control of blood cell homeostasis. Since Srp knockdown by
RNAi leads to an absence of the crucial Hh signal, it was not
surprising to find that normal Srp function is required for
prohemocyte maintenance and the control of hemocyte
differentiation within the lymph gland; that is, we observed a
RESEARCH ARTICLE 3567
severe reduction of Ptc-positive hematopoietic progenitors and a
strong increase in differentiated plasmatocytes and crystal cells in
srp mutant tissue.
Likewise, Ptc-positive prohemocytes were lost and large numbers
of plasmatocytes were prematurely formed in Su(H) mutant lymph
glands. This disruption of prohemocyte maintenance occurred even
though Hh protein expression was expanded throughout the
medullary zone. This raised the question as to why expanded Hh
protein and possible Hh pathway activation did not increase the
progenitor population in Su(H) mutant lymph glands, instead of the
observed loss of prohemocytes and appearance of differentiated
plasmatocytes. One explanation might be that the PSC niche is not
expanded in Su(H) mutant lymph glands (Fig. 3H) and Hh might
only function in promoting blood cell precursor maintenance within
the context of the highly ordered progenitor-niche microenvironment.
It has been hypothesized that the filopodial extensions that emanate
from differentiated niche cells are crucial for Hh signal transduction
from the PSC to progenitor cells of the medullary zone (Krzemien et
al., 2007; Mandal et al., 2007). The possibility exists that ectopic Hh
protein, which is not produced or presented by niche cells, is unable
to positively regulate prohemocyte homeostasis. An experimental
result consistent with this hypothesis is that expression of UAS-hh
under the control of the medullary zone-specific tepIV-Gal4 driver
failed to expand the blood cell progenitor population (data not
shown). A second possibility is that the Hh pathway transcriptional
effector Ci might require the co-function of Su(H) in its control of
prohemocyte maintenance. This model would predict that, in the
absence of Su(H) function, Hh signaling would be less (or non)
effective in controlling the genetic and cellular events needed for the
maintenance of the prohemocyte state. Third, Su(H) might regulate
additional target genes, the expression (or repression) of which is
crucial for normal blood cell precursor maintenance and the
prevention of premature hemocyte differentiation. Finally, we cannot
rule out the possibility that the expression of ectopic Hh in medullary
zone cells, in the context of the adverse effects of Su(H) loss of
function in these cells, culminates in the disruption of normal Hh
pathway signaling due to an unforeseen dominant-negative effect.
In summary, these findings add significantly to our knowledge
of hematopoietic transcription factors that function to control stemlike progenitor maintenance and blood cell differentiation in the
lymph gland. An additional conclusion from these studies is that
the hh enhancer-GFP transgene can serve as a beneficial reagent to
identify and characterize genes and physiological conditions that
control the cellular organization of the hematopoietic progenitorniche cell microenvironment. RNAi-based genetic screens could be
undertaken using this high-precision marker to determine signaling
pathways and/or environmental stress conditions that might alter
niche cell number and function, leading to an alteration in
hematopoietic progenitor maintenance coupled with the robust
production of differentiated blood cells. Much remains to be
determined about the regulated control of these critical
hematopoietic changes and their likely relevance to hematopoietic
stem cell-niche interactions in mammals.
Acknowledgements
We thank I. Ando, M. Crozatier, M. Haenlin, T. Kornberg and the various stock
centers for antibodies and Drosophila strains; and T. Vargo-Gogola and the
Indiana University Medical School, South Bend, for use of their Zeiss LSM710
laser confocal microscope. This work was supported by grants to R.A.S. from the
National Institutes of Health (HL071540) and the Notre Dame Initiative in Adult
Stem Cell Research. This research was facilitated through the use of the Notre
Dame Integrated Imaging Facility. Deposited in PMC for release after 12 months.
DEVELOPMENT
Regulators of hedgehog niche expression
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.053728/-/DC1
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