Protein Cell 2013, 4(9): 650–655

DOI 10.1007/s13238-013-3040-y Protein & Cell

MINI-REVIEW
Hedgehog in the Drosophila testis niche: what
does it do there?
Zhao Zhang, Chenyu Pan, Yun Zhao

State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China
 Correspondence: [email protected] (Y. Zhao), [email protected] (Z. Zhang)
Received May 10, 2013 Accepted June 2, 2013
Protein & Cell

ABSTRACT of niche-stem cell interaction have important implication for

regenerative medicine. In higher organisms, the regulatory
Stem cell niche is a specialized microenvironment crucial network underpinning niche-stem cell interaction is still not
to self-renewal. The testis in Drosophila contains two dif- well understood due to its intrinsic complexity. In this regard,
ferent types of stem cells, the germline stem cells and the the Drosophila testis offers a good system to address this is-
somatic cyst stem cells that are sustained by their respec- sue because it harbors one of the best characterized stem cell
tive niche signals, thus is a good system for studying niches, in which GSCs and somatic cyst stem cells (CySCs)
the interaction between the stem cells and their hosting co-exist (Fuller and Spradling, 2007). This system ensures that
niche. The JAK-STAT and BMP pathways are known to the number and the relative ratio of GSCs and CySCs are kept
play critical roles in the self-renewal of different kinds of steady from one individual fly to another, thus providing an ex-
stem cells, but the roles of several other pathways have cellent model for studying multiple signaling pathways, includ-
emerged recently in a complex signaling network in the ing those of the bone morphogenetic protein (BMP) and Janus
testis niche. Reports of independent observations from kinase-signal transducer and activator of transcription (JAK-
three research groups have uncovered an important role STAT) that controls this homeostasis (Issigonis and Matunis,
of Hedgehog (Hh) in the Drosophila testis niche. In this 2011). Here, we review several recent findings that generate
review, we summarize these recent findings and discuss insights into the role of Hh signaling in the Drosophila testis
the interplay between the Hh signaling mechanisms and niche. We also discuss several remaining questions for future
those of the JAK-STAT and BMP pathways. We also dis- investigation.
cuss directions for further investigation.
THE MECHANISM OF HH SIGNALING AND ITS
KEYWORDS Drosophila, testis, stem cell, Hedgehog, JAK- FUNCTION IN THE DROSOPHILA OVARY
STAT
The Hh pathway plays an important role in cell growth, pattern-
ing and stem cell maintenance during metazoan development
INTRODUCTION (Jiang and Hui, 2008). Hh ligands are morphogens whose con-
Stem cells are a type of undifferentiated cells that have unique centration gradient provides positional cues that dictate target
abilities to renew themselves and differentiate into specialized gene expression (Heemskerk and DiNardo, 1994; Tabata and
cell types while maintaining a stable population. Stem cells are Kornberg, 1994). In Drosophila, Hh binds its receptor Patched
critical during embryonic development, adult organ homeosta- (Ptc) (Ingham et al., 1991; Chen and Struhl, 1996) and co-
sis, and tissue repair. Germline stem cells (GSCs) are adult receptor iHog/Boi (Yao et al., 2006, 2010; Zheng et al., 2010)
stem cells responsible for production of gametes. The proper to relieve an inhibitory effect of Ptc on Smoothened (Smo), a
maintenance of GSCs depends on surrounding microenvi- signal transducer with seven transmembrane domains. Hh in-
ronment, or the niche, in which multiple signals instruct stem duces phosphorylation and stabilization of Smo at the plasma
cells to either renew themselves or differentiate (Li and Xie, membrane (Denef et al., 2000; Taipale et al., 2002; Jia et al.,
2005; Jones and Wagers, 2008). Advances in the knowledge 2004; Zhang et al., 2004; Apionishev et al., 2005), leading to

650 | September 2013 | Volume 4 | Issue 9 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2013
Hedgehog in Drosophila testis MINI-REVIEW

A B
Cyst cells

CySC
Testis

Hub cell
AG

ED GSC
Gonialblast

Figure 1. The structure of the reproductive system and stem cells in male Drosophila. (A) A cartoon of the reproductive system of a
male Drosophila, which harbors a pair of testes which produce sperms, and accessory glands (AG) which produce seminal fluid compo-
nents that mix with the sperm to protect and preserve them. Semen passes through ejaculatory duct (ED) during mating. (B) An enlarged
view of testis tip where GSCs and CySCs are arranged around the hub cells.

Protein & Cell

a conformational switch at the C-terminus of Smo (Zhao et al., recent paper reported that Hh regulates the expression of BMP
2007). This in turn promotes the dimerization of a downstream family ligands to maintain GSCs in Drosophila ovary, extending
kinase, Fused (Fu), causing it to be phosphorylated and there- the roles of Hh signaling in the GSC niche (Rojas-Rios et al.,
by activated, and leading to phosphorylation of kinesin Costal2 2012). As some similarities are shared by stem cells in differ-
(Cos2) (Therond et al., 1996; Nybakken et al., 2002; Ruel et ent systems (Decotto and Spradling, 2005; Losick et al., 2011),
al., 2007; Shi et al., 2011; Zhang et al., 2011; Zhou and Kal- these observations in the ovary suggested a possible role for
deron, 2011; Ranieri et al., 2012). The signal is further propa- Hh signaling in the testis system.
gated to the pathway transcription factor, Cubitus interruptus
(Ci), which is phosphorylated and converted into a truncated, DROSOPHILA TESTIS, A TALE OF TWO STEM
75-kDa Ci repressor (Ci75) in the absence of Hh. (Aza-Blanc CELLS
et al., 1997; Ohlmeyer and Kalderon, 1998; Methot and Basler,
1999). Hh signal stimulates the formation and activation of the Adult male Drosophila harbors a pair of testes that are a tube-
full-length, 155-kDa Ci activator (Ci155), which enters the nu- like coiled structure attaching to seminal vesicle (Fig. 1). At the
clear and turns on target gene expression. apex of the testis tube, 10–15 non-mitotic somatic cells form a
Previous studies in Drosophila ovary showed that Hh sign- tightly folded structure called the hub. About 10 GSCs, which
aling mainly functions in the somatic stem cells (Forbes et al., are round shaped, make direct contact with the hub cells to
1996a, 1996b; King et al., 2001; Zhang and Kalderon, 2001). form a rose-like structure. GSCs undergo asymmetric divi-
Hh protein is secreted by the terminal filament cells and cap sions, one of the daughter cells remains GSC, and the other
cells, which are present at the anterior tip of the germarium, to one (gonialblast) detaches itself from the hub cells and begins
regulate the self-renewal of follicle stem cells, a kind of somatic to differentiate. 16-cells spermatogonial clusters are formed
stem cell functions to differentiate into follicle cells to encapsu- after four mitotic cycles with incomplete cytokinesis. Spermato-
late passing cysts (Margolis and Spradling, 1995; Forbes et al., gonia differentiate into spermatocytes, which undergo meiosis
1996a; Zhang and Kalderon, 2001). Balanced Hh pathway ac- to finally form sperm. Two irregularly shaped CySCs enwrap
tivity is critical for normal egg chamber formation. Inactivation one GSC and make a narrow contact with the hub cells. Cy-
of Hh signaling reduces the number of follicle stem cells and SCs differentiate into cyst cells, which enwrap and accompany
stops egg chamber budding (Forbes et al., 1996a), while over- GSC daughter cells during spermatogenesis. Both of GSCs
activation of Hh signaling leads to over-proliferation of follicle and CySCs require niche for self-renewal. CySCs rely on the
stem cells, which accumulate between egg chambers (Forbes hub cells for niche signal, while the niches of GSCs are com-
et al., 1996a, 1996b; Zhang and Kalderon, 2001). In both kinds posed of signals from both the hub cells and CySCs (Voog et
of situation, GSCs could not develop properly to form mature al., 2008; Issigonis and Matunis, 2011).
eggs, suggesting a critical role of Hh signaling in female egg JAK-STAT signaling is critical to stem cell maintenance in
formation. The stem cell niche of ovary is defined by several the Drosophila testis (Kiger et al., 2001; Tulina and Matunis,
signaling pathways, including BMP, JAK-STAT, Wnt and Hh 2001). The ligand Unpaired (Upd) is produced by the hub cells
signaling. Using follicle stem cells as a model, it has been and received by GSCs and CySCs to activate the transcrip-
shown that Hh, JAK-STAT and Wnt signaling cooperate to tional factor STAT (Stat92E in Drosophila), which enters the
define an intersection of gradients of long-range niche signals nucleus to turn on different targets in GSCs and CySCs. STAT
which regulates the behavior of stem cells (Vied et al., 2012). A activation in CySCs leads to the expression of zfh-1 (Leather-

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2013 September 2013 | Volume 4 | Issue 9 | 651
 MINI-REVIEW Zhao Zhang et al.

Zfh-1
al., 2012; Zhang et al., 2013). The revealed roles of Hh signal-
JAK-STAT STAT Self-renewal ing in the testis extend the conserved regulation of Hh signal-
activation Chinmo
ing in stem cell biology.

Upd
gbb HH IS ONLY RECEIVED BY CYSCS TO REGULATE
dpp CySC ITS SELF-RENEWAL, NOT GSCS

JAK-STAT
Both GSCs and CySCs make direct contact with hub cells,
Hub activation Hh proteins derived by hub cells are specifically received by
STAT
adhesion CySCs. The components of Hh signaling, Ptc, Smo and Ci are
gbb
BMP detected in CySCs (Michel et al., 2012; Amoyel et al., 2013;
dpp Mad Self-renewal
activation Zhang et al., 2013). By using lacZ enhancer trap flies or GFP
reporter flies, the transcriptions of ptc and ci are shown to ex-
Hub cells GSC
ist in CySCs (Michel et al., 2012; Amoyel et al., 2013; Zhang
et al., 2013). Ptc expression responds to different Hh signal-
Figure 2. JAK-STAT signaling and BMP signaling in
ing activity in a similar way with Hh cascade in the wing disc,
the regulation of stem cells in Drosophila testis. JAK-
suggesting that Hh signaling in CySCs is functional (Zhang et
STAT ligand Upd is secreted by hub cells and received by
Protein & Cell

al., 2013). Clonal assay showed that CySCs loss of smo are
both CySCs and GSCs. STAT activation in CySCs leads to
unable to self-renew and begin to differentiate into Eya positive
expression of zfh-1 and chinmo to regulate the self-renewal
daughter cells, while CySCs loss of ptc over-proliferate (Michel
of CySCs, while activated STAT in GSCs only regulates the
et al., 2012; Amoyel et al., 2013; Zhang et al., 2013). Those
hub adhesion of GSCs. Hub cells and CySCs derived BMP
ligands, gbb and dpp, are received by GSCs which is critical
results indicate that a proper activity of Hh signaling is critical
for the GSC self-renewal. for CySC maintenance. Manipulation of Hh signaling in GSCs
by RNAi or clonal assay has no effect on GSC number, which
is consistent with the absent of Hh signaling components in
man and Dinardo, 2008) and chinmo (Flaherty et al., 2010), GSCs (Michel et al., 2012; Amoyel et al., 2013; Zhang et al.,
two factors sufficient for CySC self-renewal. JAK-STAT signal- 2013). In summary, the independent findings of three groups
ing does not primarily regulate self-renewal of GSCs, but gov- lead to a consistent conclusion that Hh signaling regulates
erns niche adhesion (Leatherman and Dinardo, 2010; Singh et CySC number and maintenance in a cell-autonomous manner.
al., 2010). GSC self-renewal requires BMP signaling activation
during which Mothers against dpp (Mad) is phosphorylated THE RELATIONSHIP BETWEEN HH SIGNALING
and translocates to the nucleus to repress transcription of the AND JAK-STAT SIGNALING
differentiation factor bag of marbles (bam) (Affolter and Basler,
2007). Two BMP ligands, Decapentaplegic (Dpp) and Glass In CySCs, JAK-STAT signaling is the primary pathway to regu-
bottom boat (Gbb) are secreted from the hub cells, as well as late self-renewal through targeting on zfh-1 (Leatherman and
CySCs with activated JAK-STAT signaling (Shivdasani and In- Dinardo, 2008) and chinmo (Flaherty et al., 2010). What is
gham, 2003; Kawase et al., 2004; Schulz et al., 2004) (Fig. 2). the relationship between the emerging Hh signaling and JAK-
STAT signaling? Two groups (Amoyel et al., 2013; Zhang et al.,
2013) investigated it with different approaches and got a similar
HH SIGNALING PATHWAY, A NEW PLAYER IN THE
conclusion that Hh signaling and JAK-STAT signaling work in
DROSOPHILA TESTIS
parallel to regulate CySC self-renewal, with minor differences.
Although a Hh enhancer trap fly named hhp30 (hh-lacZ) was Amoyel et al. (2013) found that the readouts of JAK-STAT and
observed a specific and strong lacZ expression in the hub cells Hh signaling are not affected by each other. MARCM assay
17 years ago (Forbes et al., 1996a), the knowledge of Hh sign- showed that ectopic activation of the JAK-STAT signaling can-
aling in the testis is still poor. Until very recently, three groups not rescue CySCs lacking smo. Hh activation is not sufficient
reported their independent findings to draw a picture of a func- for the maintenance of CySCs loss of Stat92E or chinmo,
tional and critical role of Hh signaling in the testis (Michel et though the CySCs differentiation is delayed. They finally ar-
al., 2012; Amoyel et al., 2013; Zhang et al., 2013). In a fashion gued that Hh signaling and JAK-STAT signaling may target on
similar with the role of Hh signaling in the ovary, Hh ligands are different factors to regulate the self-renewal of CySCs (Fig. 3).
secreted by somatic cells in both ovary and testis system (cap Zhang et al. (2013) showed that Stat92E activity is not altered
cells in the ovary and hub cells in the testis). Although GSCs by different Hh signaling activity. Genetic interaction experi-
tightly attach to the Hh secreting cells (hub cells) in the testis, ments were performed with Stat92E temperature sensitive (ts)
Hh ligands are not received by GSCs. Hh signaling regulates and UAS-Smo mutants. Activated Smo rescues the CySCs
the self-renewal of somatic stem cells (follicle stem cells in the number by loss of Stat92E, while overexpression of Smo domi-
ovary and CySCs in the testis). Hh signaling contributes to the nant negative form in Stat92Ets background causes a complete
GSC niche through regulating BMP signaling (Rojas-Rios et loss of CySCs number. On the other hand, Stat92E overex-

652 | September 2013 | Volume 4 | Issue 9 © Higher Education Press and Springer-Verlag Berlin Heidelberg 2013
Hedgehog in Drosophila testis MINI-REVIEW

X deron, 2002; Jia et al., 2004), the extent of pathway activity is

Ptc
Ptc

SSm
not even, especially in testis. Further investigation is needed to

m
oo
test the relationship between the degree of Hh pathway activity
Ci
Self-renewal and niche function.

STAT SUMMARY AND PERSPECTIVES

GSC niche?
Hh CySC The emerging role of Hh signaling in testis draws a beautiful
image of how different signaling pathways work together to
maintain the homeostasis of stem cell niche. Hub cells derived
Hh ligands are received by cyst cells to primarily regulate the
self-renewal of CySCs, and a secondary effect of Hh signal-
Mad Self-renewal
ing on BMP signaling in GSCs is mediated by CySCs. One
Hub cells GSC source of Hh signaling contributes to the regulation of two dif-
ferent populations of stem cells, implying a novel niche-stem
cell interaction. Desert hedgehog (Dhh), which is one of the
Figure 3. The emerging role of Hh signaling in the testis.
three Hh proteins in mammal, is specifically expressed in the
Hh ligands derived from hub cells are received by CySCs.

Protein & Cell

Activated Hh signaling regulates the self-renewal of CySCs, testis but not the ovary (Bitgood et al., 1996). Dhh knockout
which is independent of STAT activity. BMP signaling in the mice are sterile (Bitgood et al., 1996) and somatic Leydig cells
GSCs are regulated by Hh activity in the CySCs, thus dual are severely defective (Yao et al., 2002). Despite the inves-
roles of Hh signaling in the testis niche are revealed. tigation that Dhh signaling is critical for a normal function of
testis, the inside mechanism of spermatogenesis is still poorly
understood due to the complex of niche-stem cell interactions
pression partially rescues CySC loss induced by smo RNAi. In in mammal. The knowledge of niche-stem cell regulations in fly
summary, they indicated that Hh signaling might share similar testis may provide us some clues for better understanding of
self-renewal factors with JAK-STAT signaling (Fig. 3). The di-
mammalian spermatogenesis. However, the new findings also
versity of conclusions comes from different approaches used,
raise many important questions that need to be addressed in
indicating the complexity of interactions between signaling
the future.
pathways than expected.
Why the major members of Hh signaling are expressed
in cyst cells? Although ptc, smo and ci are found to express
THE CONTRIBUTION OF HH SIGNALING TO GSC in cyst cells including CySCs, the inside mechanism of this
NICHE specific distribution is not clear. According to the experience
CySCs play another role to function as a part of GSC niche, in wing discs, the expression pattern of ptc is defined by Ci
probably through regulating BMPs expression (Kawase et al., activity, it seems reasonable that ptc is not expressed in GSCs
2004; Leatherman and Dinardo, 2008, 2010). Hh signaling due to lack of Ci activity. Engrailed (En) directly represses
regulates the self-renewal of CySCs, raising a possibility that ci expression in posterior compartment cells of wing discs
Hh signaling in CySCs contributes to niche function. All of the (Schwartz et al., 1995), providing a possibility of the absence
three groups observed that loss of Hh signaling in CySCs or of Ci in GSCs. There are other two possibilities. First, there is
whole testis results in the reduced GSC number (Michel et al., a cyst specific protein turns on the expression of ci or smo in
2012; Amoyel et al., 2013; Zhang et al., 2013). Based on the CySCs. Second, there is a germline specific protein represses
observations that Hh signaling is not received by GSCs, the the transcription of ci or smo in GSCs. Further whole genome
GSC loss is likely a secondary effect of Hh signaling in CySCs. RNAi screening may give some answers to those questions.
Different views were pointed out on the niche function of Hh Are there other targets of Ci to regulate the self-renewal of
signaling. Amoyel et al. (2013) found that overexpression of Ci CySCs? The finding of Amoyel et al. (2013) raises some hints
active form (Ciact) cannot rescue GSC loss in Stat92Ets testes. that, besides zfh-1 and chinmo, there may exist other targets of
They conducted that Hh signaling does not contribute to the Ci in CySCs. This unknown Ci targeting factor likely regulates
GSC niche. Zhang et al. (2013) showed that BMP signaling in CySCs self-renewal. Ci/Gli family transcriptional factors bind to
GSCs is affected by Hh signaling activity from CySCs. They a specific DNA motif (GACCACCCA) (Kinzler and Vogelstein,
also found that activated Hh signaling in CySCs reduce GSC 1990) in the regulation area of target genes, which may help
number, probability through cell competition. Hh signaling us to better identify those unknown factors in the future. Hh is
thus plays dual roles in testis niche. Facing those two different a morphogen to turn on different target genes in a concentra-
conclusions, people may want to compare their experiment de- tion dependent manner, such as dpp, ptc and en in wing discs
tails. They used different approaches to activate Hh signaling, (Jiang and Hui, 2008). A predictable Hh dosage from hub cells
one is Ciact, another one is SmoSD123. Though both of them are to surrounding cells may also result in different Ci targets ex-
shown to activate Hh signaling in wing discs (Price and Kal- pression. Further investigation is needed to give us a better

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2013 September 2013 | Volume 4 | Issue 9 | 653
 MINI-REVIEW Zhao Zhang et al.

understanding of morphogen in stem cell niche. a repressor. Cell 89, 1043–1053.

Till now, we know that hub cells secrete at least three kinds Bitgood, M.J., Shen, L., and McMahon, A.P. (1996). Sertoli cell signal-
of ligands: Upd for JAK-STAT signaling, Gbb/Dpp for BMP ing by Desert hedgehog regulates the male germline. Curr Biol 6,
signaling and Hh for Hh signaling. How can the hub cells ex- 298–304.
press exact amount of different ligands? The proper amount Chen, Y., and Struhl, G. (1996). Dual roles for patched in sequestering
of different ligands is critical for individual signaling activities. and transducing Hedgehog. Cell 87, 553–563.
There are several possible approaches for maintaining the Decotto, E., and Spradling, A.C. (2005). The Drosophila ovarian and
testis niche homeostasis. One is that hub cells secrete a pro- testis stem cell niches: similar somatic stem cells and signals. Dev
Cell 9, 501–510.
grammed amount of diverse ligands during different develop-
Denef, N., Neubuser, D., Perez, L., and Cohen, S.M. (2000). Hedge-
mental stage, and the interaction between different signaling
hog induces opposite changes in turnover and subcellular localiza-
pathways buffers the abnormal of niche environment. Conclu-
tion of patched and smoothened. Cell 102, 521–531.
sion from Zhang et al. (2013) that Hh signaling plays dual roles
Flaherty, M.S., Salis, P., Evans, C.J., Ekas, L.A., Marouf, A., Zavadil, J.,
in testis niche is an example to show how a single signaling
Banerjee, U., and Bach, E.A. (2010). chinmo is a functional effector
buffers the whole niche. Another possible approach is that hub
of the JAK/STAT pathway that regulates eye development, tumor
cells could sense the demands of different ligands from CySCs
formation, and stem cell self-renewal in Drosophila. Dev Cell 18,
and GSCs to make a corresponding responds. Further study 556–568.
on Hh signaling in testis should therefore not only provide in- Forbes, A.J., Lin, H., Ingham, P.W., and Spradling, A.C. (1996a).
Protein & Cell

sight into how Hh signaling functions in testis niche but also hedgehog is required for the proliferation and specification of ovar-
shed light on how different signaling pathways are connected ian somatic cells prior to egg chamber formation in Drosophila.
in niche-stem cell biology. Development 122, 1125–1135.
Forbes, A.J., Spradling, A.C., Ingham, P.W., and Lin, H. (1996b). The
ACKNOWLEDGEMENTS role of segment polarity genes during early oogenesis in Drosophi-
We apologize to colleagues whose works were not cited owing to la. Development 122, 3283–3294.
space limitation. This work was supported by grants from the National Fuller, M.T., and Spradling, A.C. (2007). Male and female Drosophila
Basic Research Program (973 Program) (Nos. 2010CB912101 and germline stem cells: two versions of immortality. Science 316,
2011CB943902), the National Natural Science Foundation of China 402–404.
(Grant No. 31171414). Heemskerk, J., and DiNardo, S. (1994). Drosophila hedgehog acts as
a morphogen in cellular patterning. Cell 76, 449–460.
ABBREVIATIONS Ingham, P.W., Taylor, A.M., and Nakano, Y. (1991). Role of the Dros-
ophila patched gene in positional signalling. Nature 353, 184–187.
BMP, bone morphogenetic protein; Ci, Cubitus interruptus; Ci155, 155- Issigonis, M., and Matunis, E. (2011). SnapShot: stem cell niches of
kDa Ci activator; Ci75, 75 kDa Ci repressor; Ciact, Ci active form; Cos2, the Drosophila testis and ovary. Cell 145, 994–994.
Costal2; CySC, cyst stem cell; Dhh, Desert hedgehog; En, Engrailed; Jia, J., Tong, C., Wang, B., Luo, L., and Jiang, J. (2004). Hedgehog
Fu, Fused; Gbb, Glass bottom boat; GSC, germline stem cell; Hh, signalling activity of Smoothened requires phosphorylation by pro-
Hedgehog; JAK-STAT, Janus kinase-signal transducer and activator of tein kinase A and casein kinase I. Nature 432, 1045–1050.
transcription; Mad, Mothers against dpp; Ptc, Patched; Smo, Smooth- Jiang, J., and Hui, C.C. (2008). Hedgehog signaling in development
ened and cancer. Dev Cell 15, 801–812.
Jones, D.L., and Wagers, A.J. (2008). No place like home: anatomy
COMPLIANCE WITH ETHICS GUIDELINES and function of the stem cell niche. Nature reviews. Mol Cell Biol 9,
Zhao Zhang, Chenyu Pan, and Yun Zhao declare that they have no 11–21.
conflict of interest. Kawase, E., Wong, M.D., Ding, B.C., and Xie, T. (2004). Gbb/Bmp
signaling is essential for maintaining germline stem cells and for
repressing bam transcription in the Drosophila testis. Development
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