Drosophila Stem Cell Niches: A Decade of Discovery
Suggests a Unified View of Stem Cell Regulation
Vicki P. Losick,1Lucy X. Morris,1Donald T. Fox,1and Allan Spradling1,*
1Howard Hughes Medical Institute Research Laboratories, Department of Embryology, Carnegie Institution for Science, 3520 San Martin
Drive, Baltimore, MD 21218, USA
The past decade ofresearch on Drosophila stem cells and niches has providedkey insights. Fly stem cells do
not occupy a special ‘‘state’’ based on novel ‘‘stem cell genes’’ but resemble transiently arrested tissue
progenitors. Moreover, individual stem cells and downstream progenitors are highly dynamic and dispens-
able, not tissue bulwarks. Niches, rather than fixed cell lineages, ensure tissue health by holding stem cells
and repressing cell differentiation inside, but not outside. We review the five best-understood adult
Drosophila stem cells and argue that the fundamental biology of stem cells and niches is conserved between
Drosophila and mice.
Stem cells and niches interest us because of their critical role in
maintaining adult tissues, as well as the insight they provide into
fundamental mechanisms of developmental biology. Any cell
that self-renews, whether via single asymmetric divisions or as
part of a larger group of cells coregulated over time, can rightly
be considered a stem cell. Analyzing the mechanisms that
specify self-renewal or differentiation within stem cell lineages
is simpler than deciphering tissue development as a whole.
Understanding how stem cell niches, spatially specific tissue
microenvironments, control stem cell differentiation potentially
illuminates how local conditions influence all tissue cells.
It has been slightly more than 10 years since a stem cell niche
was precisely documented using the Drosophila ovary (Xie and
Spradling, 2000). During this period, the study of fly stem cells
and niches has greatly expanded, and the knowledge gained
has begun to change our views of what stem cells are and how
they work (reviewed by Kirilly and Xie, 2007; Morrison and Spra-
dling, 2008; Pearson et al., 2009; Voog and Jones, 2010). In
particular, discovering that individual Drosophila stem cells
turn over regularly, compete for niche occupancy, and rapidly
differentiate when outside their normal milieu has focused
attention on the niche. Here we discuss recent studies of the
best-understood adult Drosophila stem cells to illuminate the
major mechanisms by which they operate. The role of spindle
orientation in stem cell biology is reviewed by Morin and Bel-
laı ¨che (2011) in this issue of Developmental Cell.
Drosophila stem cell niches were made accessible to study by
genetic tools that allow individual cells to be lineage labeled, and
enable gene function to be disrupted within marked cell clones.
Recently, mouse stem cell lineages have been similarly charac-
terized,especially inthetestis andsmallintestine.Whereasstem
cell divisions within small, simple Drosophila niches usually
produce asymmetric fate outcomes, daughter cells in mamma-
lian tissues often adopt fates stochastically. However, we argue
that these differences, like the obsolete distinction between
mosaic and regulative embryonic development, are not signifi-
cant and that the same basic mechanisms govern stem cells in
Drosophila and mice.
The Female Germline Stem Cell Niche: A Simple
All Drosophila niches studied to date function using two key
processes: adhesive interactions and asymmetric signaling.
Adhesive interactions between stem cells and niche cells
primarily determine niche size and occupancy (Figure 1A). In
addition, a sharply asymmetric signaling microenvironment
represses stem cell differentiation and promotes stem cell
adherence inside the niche but reduces adhesiveness and stim-
ulates development even one cell diameter outside (Figure 1A).
Little or no role has been found for intrinsic asymmetries during
stem cell division in programming differential cell fate outcomes.
As far as we know, Drosophila stem cell daughters are epigenet-
ically equivalent and diverge in fate based on the local signals
they receive inside versus outside the niche. However, only a
handful of adult stem cell types have been characterized to
date, and additional mechanisms may operate in other contexts,
such as developing neuroblasts (see Karlsson et al., 2010).
A prime example, the germline stem cell (GSC) niche located
at the tip of each Drosophila ovariole, maintains two to three
GSCs throughout pupal and adult life (Figure 1B). These simple
niches are established by three cooperating somatic cell types:
terminal filament cells, cap cells, and escort (or inner germarium
sheath) cells. Cap cells hold each GSC in place via adherens
junctions that prevent them from moving down the ovariole
(Song et al., 2002). The terminal filament produces Unpaired
(Upd) and related cytokines whose secretion activates Jak/Stat
signaling in cap and escort cells and stimulates Bmp ligand
production (Lo ´pez-Onieva et al., 2008; Wang et al., 2008a).
Bmp receptor activation in GSCs prevents transcription of the
master differentiation gene bag-of-marbles (bam). GSC divisions
are usually asymmetric in outcome not because the daughter
cells inherently differ, but because only one is able to remain in
the niche. Following an oriented GSC division, the proximal
daughter is usually well positioned to retain cap cell adhesion
and repression, while the distal daughter has little chance of
reentering the niche. Consequently, bam transcription is acti-
vated, and it differentiates into a cystoblast (CB), a cell that will
give rise to a new germline cyst and ultimately a new follicle.
Developmental Cell 21, July 19, 2011 ª2011 Elsevier Inc.
Because GSC maintenance and daughter differentiation are
governed by the local microenvironment, it is not surprising
that recent studies have revealed multiple, interlocking mecha-
nisms operating both inside and outside the niche that ensure
microenvironmental asymmetry. One of the most important
involves the extracellular matrix (ECM), which has long been
implicated in influencing stem cell activity in mammalian tissues.
The ECM surrounding the GSC niche differs from the ECM
downstream and actively limits the size of the repressive zone.
Dpp and Gbb, the Bmp ligands synthesized by cap cells, carry
not act over just a one-cell wide region without active restraint.
The type IV collagens Viking and Dcg-1 within the ECM can
bind Dpp and may limit its diffusion (Wang et al., 2008b), but
the heparin sulfate glycoprotein Dally plays a major role by
stabilizing Dpp and facilitating reception by germ cells (Guo
and Wang, 2009; Hayashi et al., 2009). Dally is abundant around
the niche but sparse away from the cap cells. Forcing high-level
dally expression in all escort cells blocks GSC differentiation,
suggesting that the low levels of Dally outside the niche are
required to downregulate Bmp reception in CBs. Dally and/or
other heparin sulfate glycoproteins probably require secondary
modifications to function because enzymes involved in this
process are also essential for niche function (Hayashi et al.,
2009). Thus, the range of the niche signal is limited by the ECM
and especially by the localized distribution of Dally.
Intercellular signaling involving escort cells helps maintain the
ECM’s asymmetry by reducing Dally production away from the
niche. When the transmembrane protease Stet, or its Egf sub-
strate ligands Spitz, Gurken, and Keren are mutated in germ
cells, or when Egfr or Map kinase components are disrupted in
escort cells, niche signals travel further than normal and block
CB differentiation (Schulz et al., 2002; Liu et al., 2010). Stet
mutants can be suppressed by mutating dally, indicating that
Egfr-mediated signaling in escort cells is required to limit Dally
secretion outside of the niche. Loss of Egfr signaling activity
causes escort cells to retract the cytoplasmic extensions that
normally separate cysts, and likely facilitate escort cell-germ
Several other pathways contribute to the sharp decrease in
Bmp reception a germ cell experiences upon exit from the niche.
The Bmp-induced ubiquitin E3 ligase Smurf mediates one such
process (Casanueva and Ferguson, 2004). The Ser/Thr-protein
kinase Fused, which also functions in hedgehog signaling, acts
cell autonomously in conjunction with Smurf to target the Dpp
receptor Tkv for degradation in CBs, and is essential for CB
differentiation (Narbonne-Reveau et al., 2006; Xia et al., 2010).
Fused binds Smurf and Tkv, and may directly phosphorylate
Tkv at serine 328. How this pathway is blocked in GSCs remains
unclear. Thus, the ability of germ cells to receive the repressive
Bmp signal is actively reduced outside the niche by multiple
A Translational Switch Specifies GSC Daughters as CBs
Theelaboratelyorchestrated downregulation ofBmpsignaling in
CBs exerts only one known direct action: the derepression of
bam transcription. Consistent with this simple mechanism,
Bam production is sufficient to induce GSC differentiation (Ohl-
stein and McKearin, 1997) through actions exerted primarily at
the translational level (Li et al., 2009; Shen et al., 2009)
(Figure 1C). In GSCs, differentiation-promoting mRNAs (most
of which remain unidentified) are repressed by regulatory com-
plexes containing the conserved germline proteins Nanos
(Nos) and Pumilio (Pum), whereas translation of E-cadherin
(E-cad) mRNA is high, supporting niche adhesion. Upon induc-
tion, Bam associates with the Benign gonial cell neoplasm
(Bgcn)and otherproteins tolower capcelladhesion andactivate
differentiation. The repressive Bam complex acts in part by
binding to the 30UTRs of E-cad and nanos mRNA. Reducing
E-Cad expression helps ensure that CBs do not reassociate
with cap cells (Jin et al., 2008), whereas lowering Nos levels
Bmp ligand (Dpp)
Figure 1. The Ovarian Germline Stem Cell Niche: A Well-Understood
Stem Cell Model System
(A) A generic stem cell niche (dashed line) containing one stem cell and
associated with a niche cell and a daughter cell. Two basic niche functions are
shown; occupancy (blue) is promoted by stimulating adherens junctions
between niche cells and cells within, but not outside, the niche. Fate regulation
(orange) is effected by repressing differentiation genes in cells inside, but not
outside, the niche.
(B) The Drosophila ovarian GSC niche contains one GSC; a daughter CB lies
just outside. The niche is generated by terminal filament (Tf), cap, and escort
cells. E-cad production is high in the GSC, supporting adhesion to the niche.
The master switch gene bam is repressed in GSCs by a Jak/Stat-to-Bmp
signaling cascade from the Tf, to the cap and escort cells. Bmp reception is
turned off in the CB, due to reduced signaling, reduced production of the Bmp
coreceptor Dally in response to Egfr signaling, and degradation of the Bmp
(C) Translational activation of CB differentiation. Differentiation-promoting
transcripts are repressed in the GSC via a Nos/Pum complex and by miRNAs.
Bam expression in CB represses E-Cad and Nos production, reducing
adhesion to the niche and activating mRNAs that induce differentiation.
Developmental Cell 21, July 19, 2011 ª2011 Elsevier Inc.
derepresses translation of differentiation genes. One mRNA
activated in CBs encodes another translational regulator, Brat.
Brat functions as a corepressor with Nos and Pum in the fly
embryo but probably acts with Pum independently of Nos to
mediate ovarian germ cell differentiation (Harris et al., 2011).
As CB differentiation commences, the translation of myc is
reduced to modulate cyst growth, while Mad translation (encod-
ing a SMAD protein) is also downregulated, further enforcing the
shutoff of Bmp signaling in CBs. Modeling these interactions
shows that the system as a whole can act as a robust, bistable
switch of GSC to CB fate specification (Kim et al., 2010; Harris
et al., 2011).
Multiple noncoding RNAs also modulate the fates of both
GSCs and CBs. Abolishing all miRNA activity by mutating genes
encoding key RISC components, including Dicer-I, Ago-1, or
Loquacious (Jin and Xie, 2007; Park et al., 2007; Yang et al.,
2007), causes GSCs to differentiate. One important miRNA
mediator is Mei-P26, a protein related to Brat whose level is
increased in early germ cells by the action of Vasa, which binds
to the mei-P26 30UTR and stimulates translation (Liu etal., 2009).
Both repressive miRNAs acting in GSCs such as bantam, and
differentiation-promoting miRNAs acting in CBs, are regulated
by Mei-P26 (Page et al., 2000; Neumu ¨ller et al., 2008; Yang
et al., 2009). Mei-P26 binds to Ago-1 and may influence the
RISC complex’s ability to process and degrade miRNA target
genes (Neumu ¨ller et al., 2008). A distinct miRNA, miR-184, stim-
ulates CB development by translationally repressing yet another
Bmp signaling component, the receptor Saxophone (Iovino
et al., 2009). A structurally distinct category of small RNAs called
piRNAs may also participate in the GSC-to-CB switch, because
they have been shown during embryonic development to target
relevant mRNAs, including nos (Rouget et al., 2010). Several
genes implicated in piRNA production and transposon regula-
tion, including Yb and piwi, act in somatic niche cells to maintain
normal signaling (Szakmary et al., 2009; Saito et al., 2010; Qi
et al., 2011).
If ovarian stem cells operate based on local environmental
signals, why do stem cell divisions usually produce asymmetric
cell fates? Asymmetry in GSC daughter cell fates is probably
an incidental by-product of the niche’s small size and physical
asymmetry. Adhesive cap cells are located only on the proximal
side, so distal GSC daughter cells are usually constrained to
leave. However, when both daughters do acquire cap cell
contact, they both become normal GSCs (Xie and Spradling,
2000), verifying that there are no important intrinsic differences
between them. Likewise, external germ cells can reenter the
niche and become GSCs (Kai and Spradling, 2004). GSCs do
contain a collection of aggregated organelles known as the
spectrosome or fusome that segregates asymmetrically in all
adult mitotic germ cell divisions in both sexes, and influences
microtubule organization and spindle orientation. The spectro-
some/fusome is held together by a-Spectrin and the Adducin-
like Hts protein (see Lighthouse et al., 2008). In spectrin or hts
mutant flies, the organelle is absent and its components
segregate symmetrically, but GSCs are not destabilized. In
contrast to its minimal role in the GSC, the spectrosome/fusome
is critically important for the downstream events of male and
female gametogenesis, including oocyte specification.
The Testis Stem Cell Niche: Increased Size
Malegametogenesis inDrosophila istypical ofdiversespeciesin
being supported during adulthood by GSCs. In the Drosophila
testis, a single large GSC niche, rather than 16–20 small niches
in the ovary that each head an ovariole, supplies germ cells for
sperm production (Figure 2A). This larger, more complex niche
offers many unique opportunities for understanding stem cell
biology. A round cluster of differentiated niche cells known as
thehubcontacts 7–12maleGSCs, arrayed aroundit likespokes.
The hub is anchored to the testis apex by integrins and the Lasp
protein (Lee et al., 2008; Tanentzapf et al., 2007). Two somatic
cyst progenitor cells (CySCs), a distinct type of stem cell,
surround each GSC, make their own contacts with the hub,
and are coregulated by the niche. GSCs and CySCs must be
coordinated because the GSC daughter, the gonialblast (GB),
acquires two CySC daughters that provide an external covering
as the GB divides synchronously to form a 16-cell cyst that will
give rise to 64 sperm. Although niche cells in both the male
and female derive from the embryonic gonadal mesoderm, the
Bmp ligand (Dpp/Gbb)
Figure 2. Regulation of Male GSC Niche and Dedifferentiation
(A) The Drosophila testis niche is shown with one GSC and one CySC;
adaughterGBliesjustoutside inassociation withacystcell.Nicheoccupancy
(blue) is promoted by Jak/Stat-stimulated E-Cad expression in the GSC. bam
is repressed in GSCs by a Jak/Stat to Bmp signaling cascade from the hub to
CySC. Bmp reception is turned off in the GB, due to reduced signaling and
Dally-like expression, which activates bam and currently unknown differenti-
(B) The growth of germline cysts downstream from the GSC (red) and its niche
is depicted for both Drosophila and mouse testis (details are uncertain in the
case of mice). Differentiation genes such as bam or ngn-3turn on in a slow and
variable manner in both species (dashed line), prior to the 16-cell stage,
leading to meiosis or commitment to mature (A1) spermatogonial fate. In rare
instances (dashed lines), cysts can break down and individual cyst cells can
reenter the niche and become GSCs, providing a stem cell reserve.
Developmental Cell 21, July 19, 2011 ª2011 Elsevier Inc.
time of niche formation differs between the sexes. Hub cells and
CySCs differentiate during embryonic stages, capture GSCs,
and begin functioning, whereas female niche cell precursors
remain undeveloped until late larval stages (Murray et al.,
2010). Thus, the testis provides an opportunity to study a niche
that must control and coordinate multiple stem cells and daugh-
ters using mechanisms that may be highly conserved.
Despite its greater complexity, the testis niche uses the same
general principles and many of the same genetic pathways that
operate in the ovary (Figure 2A). The niche comprises a one-cell
wide zone directly adjacent to the hub because only germ cells
directly contacting the hub via adherens junctions self-renew,
whereas daughters that move away begin cyst formation and
are wrapped by CySC daughters. Hub cells secrete Upd, which
occupancy and self-renewal. Upd signals received directly by
GSCs stimulate adhesiveness to the hub (Leatherman and Di-
nardo, 2008), and prevent CySCs from outcompeting GSCs for
niche contact (Issigonis et al., 2009; Sheng et al., 2009). Both
GSCs (Yamashita et al., 2003) and CySCs (Cheng et al., 2011)
orient their spindles to point away from the hub.
Upd signaling received by CySCs mediates the repressive
functions of the niche. Jak/Stat activation in CySCs autono-
mously represses differentiation by stimulating production of
gene products (Leatherman and Dinardo, 2008; Wang et al.,
2008a; Flaherty et al., 2010). Also in response to Upd, CySCs
nonautonomously maintain GSCs as stem cells by secreting
Gbb and Dpp, which activate Bmp reception (see Kirilly and
Xie, 2007; Wang et al., 2008a; Leatherman and Dinardo, 2010).
Bmp pathway activation in GSCs represses bam transcription
to prevent differentiation. Thus, both male and female GSC
differentiation is repressed by a Jak/Stat to Bmp signaling
cascade involving two distinct cell types.
The testis niche exhibits unique features, as well. Bmp activa-
tion does not block premature GSC differentiation exclusively by
repressing bam. GBs, unlike their CB counterparts, start dividing
before bam turns on (see Kirilly and Xie, 2007; Insco et al., 2009;
Sheng etal., 2009).Gbb playsa larger role in the male, while Dpp
is more potent in the female niche (Kawase et al., 2004). Finally,
GSC spindles are precisely oriented by an active mechanism
that is maintained by a checkpoint in males (see Morin and Bel-
laı ¨che, 2011, in this issue) but are more loosely controlled in
females (Deng and Lin, 1997; Morris and Spradling, 2011).
germ cells in the two sexes are maintained differently. The most
anterior escort cells surround GSCs in the female niche and
resemble CySCs both morphologically, and in their responses
to niche-generated Upd signals and germ cell-derived Egf
signals. However, they do not function as active stem cells but
remain in place as germ cells pass from one escort cell to the
next (Morris and Spradling, 2011). In contrast, CySCs not only
divide to produce cyst cells, but under some circumstances
CySC daughters can differentiate into hub cells (Voog et al.,
2008; Dinardo et al., 2011). Hub replenishment may help main-
tain niche function but has no known female analog.
How male GSCs are maintained by Bmp and possibly other
signals and how their differentiation outside the niche is
controlled are still poorly understood. Translational regulation
under the control of Nanos and Pumilio is important during the
embryonic development of male as well as female germ cells
(Asaoka-Taguchi et al., 1999). Yet, in Drosophila after the male
niche forms and spermatogenesis commences, Nanos and
Pumilio are no longer required, although they continue to be
expressed. Whether Bam and Bgcn control the translation of
stored mRNAs during the male differentiation program is
unknown. A threshold level of Bam is needed to terminate cysto-
cyte divisions (Insco et al., 2009), and minimal levels have to be
maintained while cysts are growing to prevent their premature
breakdown (Pek et al., 2009). Both the RNA-binding protein
How and miR-7, whose levels are controlled by Maelstrom,
can repress bam mRNA translation and are important for main-
taining Bam levels (Pek et al., 2009; Monk et al., 2010). However,
GB switch have yet to be described.
The male niche generates a spatially limited repressive micro-
environment; germ cells initiate the GB program shortly after
losing contact with the hub and by the time they have moved
one cell diameter away. As in females, the ECM helps control
the diffusion and presentation of Gbb and Dpp, but male GSC
maintenance requires the related protein Dally-like, instead of
Dally (Hayashi et al., 2009). Germline signaling via Stet-pro-
cessed Egf ligands activates Egfr/Map kinase signaling in cyst
cells that is required for them to extend membranes around
GBs and for their normal differentiation (Sarkar et al., 2007). It is
not clear if this involves ECM modification, however. Smurf
regulating Bmp signaling in males that it does in females. Males
do not require fused for fertility, but expressing constitutively
active Tkv using the bam promoter causes male sterility (Xia
et al.,2010). This same construct has no effect on female fertility,
presumably because of rapid Smurf/Fused-mediated Tkv turn-
over. Due to the absence of this pathway for downregulating
Bmp reception, the Bmp gradient may be shallower in the male
compared to the female niche region, which might explain why
bam turns on more slowly in males relative to the cyst divisions.
The tightly regulated spindle orientation characteristic of the
testis niche (Yamashita et al., 2003), which helps ensure divi-
sional asymmetry for both GSCs and CySCs, does not neces-
sarily contradict the idea that local environmental differences
specify daughter cell fates. Neither the spectrosome/fusome
nor any other GSC cellular component is known whose asym-
metric segregation is essential for stem cell maintenance or
GB specification. Indeed, as in the case of females, downstream
cyst cells can regain hub contact under a variety of circum-
stances, become GSCs, and function normally in the niche
microenvironment (Brawley and Matunis, 2004; Sheng et al.,
in the testis niche is needed to maintain its larger size and
complexity. Divisional orientation, supported by the spindle
checkpoint, may function primarily to preserve the regular inter-
digitated arrangement of GSCs and CySCs around the hub, not
to specify cell fates. Off-axis GSC divisions would reduce
GSC-CySC contact, which might perturb GSC regulation,
because repressive Bmp signals are transmitted via CySCs.
Altered organization of the stem cells around the hub might
also prevent GBs from acquiring the two CySC daughters
needed to found a new cyst.
Developmental Cell 21, July 19, 2011 ª2011 Elsevier Inc.
The Follicle Stem Cell Niche: Interaction at a Distance
The niche that supports production of the epithelial follicle cell
layer surrounding developing ovarian follicle differs in several
ways from the GSC niches. Instead of being associated with
a readily identified, differentiated cell population, the follicle
stem cell (FSC) niche and its single resident stem cell are not
morphologically distinctive and were only discovered through
lineage analysis (Margolis and Spradling, 1995). No genes have
thus far been found that are expressed differentially in the FSC
and its immediate daughter. Although fate specification in the
FSC lineage depends on Notch, Hedgehog (Hh), Wingless
(Wg), Jak/Stat, and other signals (see Huynh and St. Johnston,
2004; Kirilly and Xie, 2007; Nystul and Spradling, 2010), the
precise origin and timing of these signals have proved difficult
to pinpoint early in the lineage. Despite these differences, the
FSC niche supports cell production as efficiently and reliably
as the GSC niche throughout life.
The anatomy of the FSC niche and its lineage has been clari-
fied recently (Nystul and Spradling, 2007; Morris and Spradling,
2011). Posterior escort cells contact FSCs, and may organize
a cellular FSC niche on each lateral germarium surface. About
half the time, FSC daughters become cross-migrating cells
(Cmcs) that move to the opposite side of the germarium, where
they occasionally replace the resident stem cell. Successful
cross-migration and stem cell replacement are dependent on
a Delta-mediated Notch signal received by the migrating cell
from germ cells. The presence of Cmcs in variable locations, ex-
pressing genes indistinguishable from FSCs, has been a source
of confusion regarding FSC number. The situation provides one
of many examples where cellular behavior rather than gene
expression provides the only reliable means to identify stem
The genetic requirements of the FSC niche argue that it oper-
ates in a basically similar manner as other Drosophila niches.
E-Cad and b-Catenin are essential to niche function, suggesting
that adherens junctions tether FSCs in the niche (Song and Xie,
2002). Hypomorphic mutations of CycE that reduce kinase
activity cause preferential loss of FSCs, and this defect can be
suppressed by upregulating E-cad (Wang and Kalderon, 2009).
These results suggest that FSCs cycling below a minimum rate
detach from the niche. In vivo, however, even in the absence
of sufficient nutrition to make an egg, FSCs slow down but are
not lost (Drummond-Barbosa and Spradling, 2001). ECM com-
ponents such as Laminin A and integrins may also contribute
to FSC function (O’Reilly et al., 2008). However, the effects of
disrupting integrin functionin FSCs maybe secondary to general
changes in germarium structure that result from the lossof integ-
rin function in downstream follicle cells.
but FSCs require Hh, Wg, and Dpp signaling to be maintained
(reviewed in Kirilly and Xie, 2007). These signals may be sent
fromarelatively distantsource,the GSCniche, becauseterminal
filament and cap cells express pathway ligands much more
strongly than escort cells. FSC division responds to the level of
Hh, whose reception is mediated by Patched and the coreceptor
Boi (Hartman et al., 2010; Yan et al., 2010; Zheng et al., 2010).
Boi production in anterior cells sequesters Hh away from the
FSCs, thereby limiting FSC proliferation (Hartman et al., 2010).
gene expression differences between the FSC and its daughters
might bedue to the shallow ligand gradients expected fromsuch
Dynamic Stem Cells Undergo Replacement
and Compete for Niche Occupancy
from the study of Drosophila stem cells is their regular turnover
and replacement. Initially, the observed turnover of marked
stem cells was assumed to result from cell loss (Margolis and
Spradling, 1995). Eventually, the realization that the departing
cells were being replaced provided proof for the stem cell niche
(Xie and Spradling, 2000). Three of the four well-studied
Drosophila stem cell types that reside in niches undergo regular
replacement by other competent progenitors. In the ovarian
GSC niche, half of the stem cells are replaced every 2–3 weeks
by daughters of neighboring stem cells (Xie and Spradling,
2000). In the testis, GSCs in flies more than 2 weeks old are
replaced even more frequently, mostly by cells derived from
the breakdown of two-, four- and eight-cell cysts (Brawley and
Matunis, 2004; Cheng et al., 2008 Sheng et al., 2009)
(Figure 2B). FSCs are replaced every 2–3 weeks by FSC daugh-
Spradling, 2007). The net result is that very few of the initial stem
cells that populate a tissue’s niches remain throughout adult life.
Stem cell replacement by the daughters of other nearby stem
cells causes the size and shape of marked stem cell clones to
continue changing long after the time required for a cell to transit
the entire lineage. The movement, growth, and extinction of cell
stem cell replacement represent a distinct process of dynamic
logical significance remains little explored.
Replacement potentially places each stem cell in competition
with every other stem cell that lies within reach of its replace-
ment-competent daughters. However, it has yet to be decisively
demonstrated that competition between stem cell genotypes
serves a biological purpose. Which of two wild-type GSCs or
FSCs gets replaced appears to be random (Xie and Spradling,
2000; Nystul and Spradling, 2007). Equivalently, one could say
that the two GSCs (or FSCs) undergo ‘‘neutral competition.’’
However, if one stem cell is wild-type and one is mutant, then
competition might maintain stem cell quality by ensuring that
weaker cells are expelled (Nystul and Spradling, 2007; Jin
et al., 2008; Johnston, 2009; Rhiner et al., 2009). GSCs with
reduced levels of E-Cad (Jin et al., 2008) or with reduced Myc
expression (Rhiner et al., 2009) are lost preferentially. A potential
disadvantage of maintaining stem cell quality by cell competition
ability to replace wild-type stem cells. This is exactly what was
observed when E-Cad or Myc levels were increased above
normal. Whether competition occurs in wild-type tissues be-
tween spontaneously arising genotypes and whether such
changes impact aging and cancer development remain to be
Chromatin Regulation of Stem Cells and Differentiation
Proper chromatin organization is critically important for stem
cells to maintain their full differentiation potential despite on-
going proliferation and environmental fluctuations. Epigenetic
states must remain flexible enough to support differentiation
Developmental Cell 21, July 19, 2011 ª2011 Elsevier Inc.
along multiple pathways but maintain sufficient programming to
interpret rather general tissue signals so as to produce appro-
priate cell types. The chromatin requirements for repressing
bam transcription in GSCs provide a relevant focus for under-
standing stem cell chromatin programming. Iswi, a component
of the NURF nucleosome remodeling complex, is specifically
needed (Xi and Xie, 2005). Steroid hormone signaling can modu-
late this requirement (Ables and Drummond-Barbosa, 2010).
action between SMADs and the lamin-like nuclear membrane
protein Otefin (Jiang et al., 2008). Otefin mutant ovaries lose
GSCs, suggesting that bam cannot be repressed sufficiently
without this interaction. Otefin has been proposed to tether
bam to the nuclear periphery, but moving SMAD-bound bam
to the nuclear margin by an independent mechanism did not
rescue Otefin mutants (Sui and Yang, 2011). NURF is also
required in male GSCs (Cherry and Matunis, 2010).
contribute to stem cell maintenance. The histone lysine methyl-
transferase Setdb1, which generates repressive histone H3
lysine 9 methylation, is required to maintain GSCs, and its
expression relative to the related methyltransferase Su(var)3-9
changes during early germ cell development (Clough et al.,
2007; Yoon et al., 2008). Scrawny, an H2B Ubiquitin protease
that acts autonomously within stem cells, likely dampens
premature gene activation to help maintain GSCs, FSCs, and
intestinal stem cells (ISCs) (Buszczak et al., 2009). Niche cells
also require chromatin-modifying proteins. The histone deme-
thylase Lsd1 functions within escort cells to limit niche size in
the ovary (Eliazer et al., 2011). The piRNA pathway in niche
chromatin factors that
cells may not just suppress transposon damage but may also
actively program chromatin and gene activity (Yin and Lin,
2007). Enhanced GSC loss caused by piRNA pathway muta-
tions in niche cells can be partially suppressed by mutating
corto, which encodes a chromatin-repressor protein (Smuld-
ers-Srinivasan et al., 2010). It is still challenging to identify the
direct targets of all these chromatin-modifying proteins and to
distinguish chromatin regulation that is unique to stem cell
function from general requirements for differentiation or gene
Chromatin also plays critical but incompletely understood
roles during lineage differentiation downstream from the stem
cell. The FSC lineage shows particular promise as a system for
systematically analyzing the chromatin changes involved in
stem cell differentiation. An assay based on Gal4/UAS variega-
tion allows changes in epigenetic stability to be measured at
each step in the main body cell lineage and to be dissected
genetically (Skora and Spradling, 2010). Consistent with gain-
of-function assays in embryos, epigenetic instability in the FSC
lineage is very high in early progenitors but stabilizes 80-fold
to be involved in modulating FSC chromatin have been identified
genetically. The nucleosome remodeling protein Dom, rather
than Iswi, functions in this tissue (Xi and Xie, 2005). Mutating
Psc and Su(z)2, Polycomb group genes that are involved in inter-
preting spatial patterning, disrupts differentiation downstream
from the FSC (Li et al., 2010).
ISCs and Their Niche
The Drosophila posterior midgut contains 800–1000 active ISCs
that replenish the abundant enterocytes (ECs) and rare enter-
oendocrine (ee) cells of the gut every 1–2 weeks (Figure 3A).
The abundance, simple lineage, and high activity of ISCs make
the fly midgut an exceptionally attractive system for studying
stem cell regulation (reviewed in Casali and Batlle, 2009; Karpo-
wicz et al., 2010; Hou, 2010; Zhao and Xi, 2010). However, it is
still unclear if ISCs are located in niches similar to those
described for reproductive tissue stem cells. ISCs are arrayed
irregularly across the basement membrane of the midgut, sur-
rounded on all sides by ECs, and associated with no special
cell or morphological structure that might organize a niche.
Muscle layers located beneath the basement membrane en-
sheath the gut with circular and longitudinal fibers. Wg is ex-
pressed in some intestinal muscles and was proposed to
generate an ISC niche (Lin et al., 2008). However, Wg signal
reception in ISCs may not be required for stem cell maintenance
but, rather, to tune their proliferation rate. Clones expressing
a constitutively active b-catenin are hyperplasic but do not block
differentiation (Lee et al., 2009).
ISC niches might be fixed but cryptic, like FSC niches, or they
might form around each ISC anywhere on the basement
membrane. There is reason to suspect that ISCs might be able
to generate their own niche. CySCs can contribute to their niche
because they are able to generate replacement hub cells (Voog
et al., 2008). During development ISC precursors within midgut
‘‘imaginal islands’’ (Jiang and Edgar, 2009) are maintained by
one or two island cells that differentiate early (Mathur et al.,
2010). Developing progenitors produce Egfr ligands that act as
their own mitogens (Jiang and Edgar, 2009). Recently, Paneth
cells, one of the cell types produced by mouse ISCs, have
UpdVn, Spi, Krn
Figure 3. The Responsive Drosophila Intestinal Stem Cell Lineage
(A) Diagram showing the Drosophila ISC in its location on the basement
membrane, near a recent daughter enteroblast (EB), several mature enter-
ocytes (EC, blue), and rare enteroendocrine cells (ee, green).
(B)TheleveloftheNotchligand Delta intheISCactswiththeJak/Statpathway
to specify EC versus ee fates.
(C) Stress, infection, and/or damage to the intestinal epithelium trigger
signaling through Hpo/Wts, Jnk, Jak/Stat, and Egfr/Ras/Mapk pathways to
modulate the ISC’s proliferative response.
Developmental Cell 21, July 19, 2011 ª2011 Elsevier Inc.
been clearly shown to play key roles in the niche within the intes-
tinal crypt (Sato et al., 2011) (Figure 4A).
ISCs also play an active role in specifying the fate of their
daughters. The choice between an EC or ee cell fate outcome
depends on Notch signaling between the stem cell and its
daughter, the enteroblast (EB), shortly after division. An ISC
that expresses high levels of the Notch ligand Delta will signal
strongly and induce EC differentiation, whereas ISCs with low
Delta expression will trigger differentiation toward an ee fate
(Ohlstein and Spradling, 2007; Bardin et al., 2010; Takashima
et al., 2011) (Figure 3B). The direct targets of Notch signaling
that mediate differentiation and fate choice are poorly under-
stood. In addition, the Jak/Stat pathway coordinates with Notch
to regulate EB differentiation. Stat is expressed in ISCs and EBs
and is required for differentiation, even when Notch signaling is
activated (Beebe et al., 2010).
Stem Cells Respond to Environmental Cues
Drosophila stem cells do not just function autonomously in their
niche but are exquisitely sensitive to conditions that impinge on
the anticipated need for cell production (reviewed in Drummond-
Barbosa, 2008). For example, when nutrients are readily avail-
able and environmental conditions for embryo development
are favorable, GSC niches support 4-fold higher levels of stem
cell activity, and the ovary produces many more eggs per unit
time. ISCs also respond to nutrition, stress, and infection. In
several tissues, stem cell activity declines with age (Boyle
etal.,2007;Panetal.,2007; reviewedin Wangand Jones, 2011).
Dietary conditions directly impact stem cell division rate both
through the insulin signaling pathway and by alternative routes
(Hsu et al., 2008; LaFever et al., 2010; Amcheslavsky et al.,
2009; Biteau et al., 2010; McLeod et al., 2010; Ueishi et al.,
2009). For example, in the ovary both InR and Tor activity are
required to prevent GSCs from arresting in G2 of the cell cycle.
Drosophila insulin-like peptides (Dilps) not only bind to receptors
expressed on GSCs but also modulate how cap cells interact
with GSCs (Hsu and Drummond-Barbosa, 2009) and respond
to Notch signaling (Hsu and Drummond-Barbosa, 2011). Even
under laboratory conditions, where food is relatively plentiful,
a significant fraction of GSCs pause in the G2 phase of the cell
cycle. In addition to InR and Tor-mediated signals, arrest and
restart in G2 may require degradation of CycA, a process that
can affect GSC maintenance (Chen et al., 2009). Release from
arrest can be recapitulated by the addition of insulin to germaria
developing in vitro (Morris and Spradling, 2011).
The intestinal epithelium, unlike the germline, is in constant
contact with the external environment due to the ingestion of
nutrients. Even in the absence of stress, the gut hosts a commu-
nity of commensal and symbiotic microorganisms that interact
extensively with ECs and participate in setting the basal level
of cell turnover (Buchon et al., 2009). Therefore, it is not
surprising that ISCs are highly responsive to external influences.
Indeed, much has been learned about how they are regulated
when the intestine is exposed to pathogens or toxic agents
that can damage the gut epithelium (Apidianakis et al., 2009;
Biteau et al., 2008; Buchon et al., 2009, 2010; Chatterjee and
Ip, 2009; Cronin et al., 2009; Karpowicz et al., 2010; Jiang
et al., 2009, 2011; Ren et al., 2010; Shaw et al., 2010; Staley
and Irvine, 2010).
Various stimuli that damage ECs stimulate the production and
release of Jak/Stat ligands (Upd2 and Upd3) and Egfr ligands
(Vn, Spi, and Krn). In fact, the EC seems to be the major source
of these stimulatory cytokines, because genetically inhibiting
these factors in ECs can block accelerated stem cell division
under conditions of stress. Clonal or tissue-wide activation of
Jak/Statand/or Egfrpathways inISCs mimics theeffect oftissue
injury by accelerating ISC division, whereas impairing either
pathway blocks the ability of ISC to respond. Thereisalso cross-
talk between the Jak/Stat and Egfr pathways. Blocking Ras
signaling in clones expressing Upd reduces ISC proliferation,
and likewise, loss of Stat can reduce clone size when cell
division is stimulated with the Egfr ligand Vn (Jiang et al.,
2011). Depending on the type and severity of injury, cytokine
production by ECs can also be induced by the Jnk and Hippo/
Warts pathways leading to further modulation of the ISC division
rate (Figure 3C).
Figure 4. The Mouse ISC Niche
(A) Diagram of a cross section of a mouse intestinal crypt showing stem cells
(ISC, green) and Paneth cells (blue) (adapted from Snippert et al., 2010).
Oriented ISC division generates competition to maximize ISC-Paneth cell
contacts between the ISC daughters and neighboring stem cells. One stem
cell is forced out of the niche by ‘‘neutral competition’’ and differentiates.
(B) Diagram showing similarity between the Drosophila GSC niche (top) and
the mouse ISC niche (bottom). Stem cells in both niches are retained by
GSC division, the distal daughter usually loses the competition for niche
contact, exits, and differentiates, thereby generating asymmetric daughter cell
fates. In the larger mouse niche, one of the recently divided ISC daughters or
another adjacent ISC loses the competition for niche contact, exits the niche,
and differentiates, generating symmetric or asymmetric cell fates. The prac-
tical effect of both systems for cell production is similar or identical.
Developmental Cell 21, July 19, 2011 ª2011 Elsevier Inc.
Do Mouse and Drosophila Stem Cells and Niches Use
Similar Regulatory Strategies?
The basic mechanisms of metazoan development and gene
regulation arose prior to the divergence of the major phyla and
have been conserved in evolution. However, mammalian tissues
are much larger than Drosophila tissues, contain many more
stem cells, live much longer, and contend with potentially
different challenges resulting from the organisms’ different
ecological lifestyles. Studies of mammalian stem cells have
been intensively pursued due to their medical importance, and
have spanned a wide range of species and tissues (reviewed in
Barker et al., 2010). Despite this, the technical difficulty of
mapping mammalian stem cells and niches at the level of indi-
vidual cells in vivo has made direct comparisons with Drosophila
difficult (see Morrison and Spradling, 2008). Recently, geneti-
cally controlled lineage tracing in the mouse epidermis, testis,
small intestine, and several other tissues has started to over-
come this problem (Clayton et al., 2007; Yoshida et al., 2007;
Barker et al., 2007; Hsu et al., 2011). Moreover, a stem cell niche
in themurinesmallintestinehasnowbeencharacterized indetail
(Sato et al., 2011). Whether these findings reveal fundamental
differences in the operation of mammalian and Drosophila
stem cell lineages and niches is a subject of current interest
(Nakagawa et al., 2010; Snippert et al., 2010; Klein et al., 2010).
The Mouse Testis and Stem Cell Replacement
The mouse testis houses thousands of spermatogonial stem
cells (here called GSCs) along the basal layer of the seminiferous
tubules that can be assayed by lineage marking or transplanta-
tion (reviewed in Oatley and Brinster, 2008; Yoshida, 2010).
Mouse spermatogonia, like those in Drosophila and many other
species, develop from single cells (As) into germline cysts inter-
connected by intercellular bridges via synchronous divisions
(Figure 2B). This suggests that the single cells represent stem
cells and GBs, whereas the interconnected cells form a develop-
mental sequence. Clusters of up to 16 or 32 cells appear mor-
phologically ‘‘undifferentiated,’’ whereas cells in larger cysts
are thought to have initiated a differentiation process leading
cia and Russell, 2001).
Whether discrete niches support mouse GSCs as in
Drosophila remains controversial. Somatic Sertoli cells contact
Ascells and clustered spermatogonia and may serve niche-like
functions possibly analogous to the roles played by Drosophila
somatic cyst cells or escort cells. GSCs require the Tgf-b family
member Gdnf produced by Sertoli cells and received by
germline Gfra1/cRet-1 receptors to maintain self-renewal. The
conserved Nanos2 gene is preferentially expressed from Ascells
to smallcysts, and is essential for GSC maintenance (Sada etal.,
2009). Germ cell production is thought to respond to nutritional
conditions via a pathway involving mTorc1 and the transcription
factor Plzf, which feeds back on Gdnf reception (Hobbs et al.,
2010). Despite the evolutionary conservation revealed by these
shared features, the existence of a murine male GSC niche
remains unclear. No cellular structure analogous to the
Drosophila hub appears to be present. Instead, regions of the
basal layer near interstitial cells and blood vessels may provide
niche function (Yoshida et al., 2007; but see Oatley et al.,
2011). Knockdown experiments failed to detect a requirement
for either Stat3 or E-Cad in mouse GSCs assayed by transplan-
tation (Kanatsu-Shinohara et al., 2008; Oatley et al., 2010). Until
more is learned about the existence and nature of a male niche,
it remains difficult to compare this aspect of mouse and
Drosophila stem cell biology.
However, one very striking similarity has been documented
between cell replenishment in Drosophila and mouse testes.
This is the dynamic character of the early germ cells (Figure 2B).
Mouse cysts can lose their interconnections and form new As
cells (Nakagawa et al., 2010). Cyst breakdown also takes place
in Drosophila, especially in older animals, and the released
germ cells can reenter the niche and function as GSCs (Brawley
and Matunis, 2004; Cheng et al., 2008; Sheng et al., 2009).
Lineage tracing and transplantation experiments suggest that
cyst breakdown is a source of replacement stem cells in mice
as well (Nakagawa et al., 2007). Long-term lineage studies indi-
cate that cysts break down at an easily detectable rate (Klein
et al., 2010). Shortly before, during, or after cyst breakdown,
germ cells turn off differentiation-associated genes such as
ngn3, and induce self-renewal-associated genes such as
nanos2 and Gfra1. This might explain why some Ascells and
short cysts express ngn3, whereas some longer cysts express
genes associated with self-renewal (Suzuki et al., 2009; Naka-
gawa et al., 2010). Despite the existence of cyst breakdown
and dedifferentiation, most mouse cysts probably do not expe-
rience such events or else cyst cell numbers would not usually
correspond to powers of two.
The heterogeneity of gene expression within cysts and the
observation that small cysts or even single Ascells can begin
to differentiate directly as A1cells (Nakagawa et al., 2010) have
called into question for some the view of early cysts as a funda-
mental development sequence. However, the evidence that
fragmented cysts can start to differentiate is insufficient to
compel such a conclusion. Drosophila female cysts with half
the normal number of cells can still form fertile gametes, and
even cysts with just a few cells, such as those produced by hts
mutants, support extensive differentiation (but not fertility).
Consequently, a conservative interpretation would be that in
both Drosophila and mice, cysts usually differentiate without
interruption along the canonical pathway, whereas a few break
down and supply replacement stem cells. As part of the break-
down process, some single cyst cells and small cyst fragments
with discordant gene expression are generated that develop
extensively but eventually arrest and die.
The Mouse ISC Niche
Recently, the mammalian small intestine has emerged as one of
the best-characterized mammalian stem cell niches (reviewed in
Barker et al., 2010) (Figure 4A). Paneth cells, an ISC-derived cell
type with antimicrobial function, are found at the base of the
crypt interdigitated between about 14 ISCs, where they help
generate and maintain an ISC niche (Sato et al., 2011). Paneth
cells adhere to ISCs and express Egf, Tgfa, Wnt3, and Dll4,
factors that are needed to maintain ISC activity in culture (Sato
et al., 2009). Niche signals probably induce ISCs to express
Lgr5, a Wnt-inducible orphan G protein-coupled receptor that
is dispensable for ISC function, but which has been widely
used as an ISC marker (Barker et al., 2007; Barker and Clevers,
2010). After ISC daughters lose contact with Paneth cells, they
to divide and differentiate while moving up the crypt wall.
Developmental Cell 21, July 19, 2011 ª2011 Elsevier Inc.
The similarities between the mouse intestinal niche and those
studied previously in the fly are striking. The ISC niche depends
on a specific cell, the Paneth cell, to which ISCs attach and
that sends signals that repress differentiation, exactly as in
Drosophila niches. Reducing the number of Paneth cells in a
crypt reduces the number of ISCs, but those ISCs that are left
are still attached to the remaining Paneth cells (Sato et al.,
2011), exactly as when cap cell or hub cell number is decreased
(Kitadate and Kobayashi, 2010; Song et al., 2007). When crypts
are lineage labeled with a single Lgr5+ cell, some become fully
labeled after several months (Barker et al., 2007), indicating
that ISCs at the crypt base are regularly replaced by other ISCs
or their daughters. Indeed, competition for Paneth cell contact
has been proposed to underlie ISC maintenance (Sato et al.,
2011) (Figure 4A), just as competition for cap cell contact plays
a key role for Drosophila GSC maintenance (Jin et al., 2008).
Interestingly, the fates of ISC daughters following division are
usually symmetric, rather than asymmetric as in the case of the
well-studied Drosophila niches (Snippert et al., 2010). Following
an oriented division (Quyn et al., 2010), the recently divided ISCs
and their nearby neighbors undergo ‘‘neutral competition,’’
probably to maximize ISC-Paneth cell contact, and soon one
cell is ejected from the niche (Figures 4A and 4B, arrows). There-
fore, the net effect of ISC division, just as in the Drosophila
niches, is to produce a cell that renews the crypt stem cell
pool and a cell that differentiates. This system of orientated divi-
sion followed by neutral competition likely helps, in some way
not currently understood, to maintain (1) ISC and Paneth cell
number and position, (2) the ability to orient subsequent ISC divi-
sions, and (3) the ability to direct exiting cells upward.
Although ‘‘neutral competition’’ might seem different from
asymmetric division, this is not the case when the daughters of
stem cell division are intrinsically equal. Which equivalent cell
remains and which exits the niche after each stem cell division
will have little if any functional consequence as long as robust
niche mechanisms guarantee that both events remain coupled.
Inthisrespect, stem cell biology appears to belike other aspects
of embryonic and tissue development. Patterning in these
systems depends on cellular interactions, but not lineage; repro-
ducible lineages usually result from small systems with few cells
and many constraints that just generate reproducible intercel-
has provided new insights into how cell production is maintained
in adult tissues. Well-characterized stem cells reside in highly
asymmetric microenvironments capable of holding stem cells in
tiation. The number of niches and their size are tightly regulated,
perhaps because of their potential to support excessive cell
production. Consequently, niches are associated with robust,
self-reinforcing mechanisms to ensure that on average, half the
daughters of stemcell division emerge fromthe niche into condi-
tions conducive to differentiation. The methods used to reinforce
functional asymmetry are diverse and will provide insights into
the even greater mechanistic sophistication throughout meta-
zoan tissues. Another decade analyzing Drosophila niches will
be even more informative than the last.
In contrast, close examination has scraped some of the mis-
placed luster off stem cells themselves. So far, only extrinsic
mechanisms of cell fate specification have been uncovered,
and unique biological mechanisms shared among different
stem cells have not been identified. The master controls of cell
of the system, rather than in a special cell population. Early
progenitors play a previously unappreciated role, based on their
able niches, and regain stem cell function. At present one can
only speculate that the entire system of niches, reversible differ-
entiation, competition, and replacement evolved to maintain
order among potentially unruly somatic cell populations as
they deal with growth, stress, aging, mutation, and disease.
While a few details will differ, the basic strategies described
here appear to be used by stem cells throughout diverse organ-
isms, including Drosophila and mice. Thus, further study of stem
cell biology promises new insights into the most fundamental
mechanisms that govern the metazoan genome.
V.P.L. is a fellow of the Jane Coffin Childs Memorial Fund; D.T.F., L.X.M, and
A.S. are supported by HHMI.
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