Somatic cell lineage is required for differentiation
and not maintenance of germline stem cells in
Jaclyn G. Y. Limaand Margaret T. Fullera,b,1
Departments ofaDevelopmental Biology andbGenetics, Stanford University School of Medicine, Stanford, CA 94305
Contributed by Margaret T. Fuller, September 23, 2012 (sent for review May 29, 2012)
Adult stem cells are believed to be maintained by a specialized
microenvironment, the niche, which provides short-range signals
that either instruct stem cells to self-renew or inhibit execution of
preprogrammed differentiation pathways. In Drosophila testes, so-
matic cyst stem cells (CySCs) and the apical hub form the niche for
neighboring germline stem cells (GSCs), with CySCs as the proposed
source of instructive self-renewal signals [Leatherman JL, Dinardo S
(2010) Nat Cell Biol 12(8):806–811]. In contrast to this model, we
show that early germ cells with GSC characteristics can be main-
tained over time after ablation of CySCs and their cyst cell progeny.
Without CySCs and cyst cells, early germ cells away from the hub
failed to initiate differentiation. Our results suggest that CySCs do
not have a necessary instructive role in specifying GSC self-renewal
and that the differentiated progeny of CySCs provide an environ-
ment necessary to trigger GSC differentiation. This work highlights
the complex interaction between different stem cell populations in
the same niche and how the state of one stem cell population can
influence the fate of the other.
replenish and repair specific tissues throughout the lifetime of an
organism (1, 2). Failureofa nichetomaintainitsappropriatestem
cell population may lead to degeneration, aging, or an inability to
repair tissue damage (3). Conversely, failure of a niche to properly
regulate differentiation versus proliferation may contribute to the
genesis of cancer in adult stem cell lineages (4). A comprehensive
understanding of how the local microenvironment of the stem cell
niche functions suggests strategies for expansion of adult stem
cell populations in vitro, facilitates design of artificial niches for
transplantation, and provides ideas for increasing maintenance
and functionality of endogenous adult stem cell populations used
for regenerative medicine.
The Drosophila testis stem cell niche, a key model for un-
(5–7), supports two distinct adult stem cell populations—germline
1A). GSCs and CySCs normally divide with oriented spindles to
produce daughters that remain next to the hub and self-renew and
daughters displaced away from the hub that initiate differentiation
(8, 9). GSCs give rise to gonialblasts (Gb) and CySCs give rise to
postmitotic cyst cells (10), a pair of which encapsulates each Gb to
form a cyst. The encapsulated Gb undergoes four rounds of syn-
chronous transit-amplifying (TA) divisions before entering meiosis
and terminal differentiation (Fig. 1A).
Both the apical hub and the CySCs influence the GSC state. A
cytokine-like signal from the hub activates the JAK-STAT sig-
naling pathway in both GSCs and CySCs (11, 12). Although JAK-
STAT signaling is required cell autonomously for CySC mainte-
nance, it is not necessary to retain GSCs in their stem cell state.
Rather, activity of Stat in the germline is essential for continued
attachment of GSCs to the hub and retains GSCs in their niche
(13). Several lines of evidence suggest that CySCs provide a niche
for maintenance of GSCs (13–15). Consistent with this model, it
he ability of a stem cell niche to maintain a population of stem
cells ensures the continued availability of adult stem cells to
has been proposed that self-renewal of GSCs is specified by in-
structive signal(s) from the CySCs, with a likely candidate being
TGF-β signaling (13).
hub in testes in which CySCs and cyst cells had been permanently
ablated. We further show that the progeny of GSC-like cells dis-
placed from the hub failed to initiate the TA program in the ab-
sence of CySCs and cyst cells, and instead continued to proliferate
as undifferentiated cells. Our findings suggest that CySCs do not
play a required instructive role in GSC self-renewal and that cyst
cells, the differentiated progeny of CySCs, are required for proper
onset of the germline differentiation.
Somatic CySCs and cyst cells in the testis were ablated by forced
expression of the apoptotic activator Grim (16) (Fig. 1 C–H).
Flies carrying the temperature-sensitive Gal4 repressor, tub-
Gal80ts; the somatic-specific driver, c587Gal4; and UAS Grim
were grown to adulthood at 18 °C to avoid lethality during de-
velopment, then shifted to 30 °C to induce Grim expression.
Before the shift, immunofluorescence using antibodies against
the transcription factor Traffic Jam (Tj) showed densely stained
nuclei of CySCs flanking GSCs next to the hub (arrowheads),
cyst cells (arrows), and lighter stained nuclei in the apical hub
(Fig. 1B). By 1 d postshift to 30 °C, CySCs had been completely
ablated in most testes (80%, n = 84 testes) as marked by the
absence of Tj positive nuclei and presence of only Vasa-positive
cells next to the hub (Fig. 1 C and H). Because prolonged ex-
pression of Grim caused mortality, flies were shifted back to
permissive temperature after 1 d at 30 °C to allow the effects of
loss of CySCs to be followed over time.
Early Germ Cells Can Be Maintained After Ablation of CySCs and Cyst
Cells. Ablation of CySCs and cyst cells had profound but un-
expected effects on germ cell behavior. Consistent with previous
observations (13), 49% (n = 59) oftestesthat lackedCySCs or cyst
to the hub in the remaining 51% (Fig. 1 E and H). Similar pro-
portions of testes that lacked CySCs but retained germ cells were
also observed at 14 d (57%, n = 94 testes) and 21 d (49%, n = 81
testes) after pulse expression of Grim (Fig. 1H), suggesting that
early germ cells were stably maintained next to the hub in the ab-
senceofCySCsandcyst cells.Inwild-typetestes,GSCs aroundthe
hub were physically separated from one another by cytoplasmic
extensionsfromCySC cell bodies (Fig.1B).Incontrast, earlygerm
cells present next to the hub 7 d after ablation of CySCs and cyst
Author contributions: J.G.Y.L. designed research; J.G.Y.L. performed research; J.G.Y.L. and
M.T.F. analyzed data; and J.G.Y.L. and M.T.F. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| November 6, 2012
| vol. 109
| no. 45
cells were arranged in a tightly packed collar (Fig. 1E). In addition
to collar germ cells next to the hub, a second population of early
germ cells accumulated away from the hub over time in testes in
which CySCs and cyst cells had been ablated (Fig. 1 F and G).
Depletion of CySCs and cyst cells by RNAi knockdown of Stat in
these cells showed similar results as observed after ablation of
CySCs and cyst cells by pulse expression of Grim (Fig. S1 A–E).
Collar Germ Cells Maintained Next to the Hub in the Absence of CySCs
and Cyst Cells Had GSC Characteristics. Collar germcellsmaintained
next to the hub in the absence of CySCs and cyst cells retained
cytokine-like signal, Unpaired (Upd), that activates the JAK-
STAT signaling pathway in neighboring GSCs (11, 12). Like con-
trol GSCs (arrowheads, Fig. 2A), collar germ cells were Stat-pos-
itive (arrowheads, Fig. 2B), suggesting that they are responding
to the Upd signals from the hub. However, Stat protein was not
detected in the mass of germ cells away from the hub (Fig. 2B),
suggesting that these cells do not receive or respond to hub signals
hub. Similar results were observed after CySCs and cyst cells were
depleted by RNAi knockdown of Stat (Fig. S2A).
Collar germ cells maintained in the absence of CySCs and cyst
cellsalso displayedorientedcentrosomes(arrows, Fig.2 C and D),
a GSC characteristic that programs spindle orientation and the
asymmetric outcome of stem cell division (8, 17). When the du-
plicated centrosomes separate in the G2phase of the cell cycle in
wild-type Drosophila male GSCs, one normally remains near the
GSC–hub interface, whereas the other migrates to the opposite
collar germ cells with two centrosomes (n = 78) displayed normal
centrosome orientation, comparable to the 94% observed in con-
trol GSCs (n = 77) (Fig. 2D). Similar results were observed after
CySCs and cyst cells were depleted by RNAi knockdown of Stat in
these somatic cells (Fig. S2 C and D).
Collar germ cells also continued to proliferate long term in the
absence of CySCs and cyst cells. Immunofluorescence using the
mitotic marker phospho-histone H3 (PH3) showed that collar
germ cells next to the hub divided as individual cells (Fig. 2E) and
did so at a rate similar to GSCs in control testes; 2.8% (n = 12/
425) for collar germ cells compared with 2.6% (n = 13/497) for
control GSCs. Collar germ cells also had dot fusomes (arrows,
Fig. 2F), similar to wild-type GSCs and Gbs, and unlike the
branched fusomes typical of TA cells (Fig. 1A, arrows in Fig. 3A).
day(s) @ 30°C
21 day(s) @ 18°C
% of testes
cells. (A) Diagram of Drosophila spermatogenesis at the apical tip of the
testis. (Red) fusomes; (green) Bam protein expression. (B–G) Immunofluo-
rescence images of c587Gal4; UAS Grim; tubGal80tstestes stained with anti-
FasIII (white, hub), anti-Vasa (red, germ cells), and anti-Tj (green) nuclei
of hub, CySCs, and cyst cells). (B) Newly eclosed flies before shift to 30 °C.
(Arrowheads) CySCs; (arrows) cyst cells. (C) Flies shifted to 30 °C for 1 d. (D)
Flies shifted to 30 °C for 1 d and back to 18 °C for 7 d. (E) Flies shifted to 30 °C
for 1 d and back to 18 °C for 7 d. (F) Flies shifted to 30 °C for 1 d and back
to 18 °C for 14 d. (G) Flies shifted to 30 °C for 1 d and back to 18 °C for 21 d.
(H) Bar graph depicting phenotype distribution at different time points.
(Blue bar) Testes with CySCs and/or cyst cells (incomplete ablation); (red bar)
testes with early germ cells but lacked CySCs or cyst cells; (green bar) testes
lacking early germ cells, CySCs, and cyst cells. No significant difference in
phenotype distribution was observed among the 7-, 14-, and 21-d time
points. (Scale bar: B–G, 10 μm.)
Early germ cells can be maintained after ablation of CySCs and cyst
% of germ cells
next to hub
and cyst cells had GSC characteristics. Immunofluorescence images of testes
stained with anti-Vasa (red, germ cells) from (A) +; UAS Grim control flies and
(B, C, E, and F) c587Gal4; UAS Grim; tubGal80tsexperimental flies shifted to
30 °C for 1 d and back to 18 °C for 21 d. (A and B) Staining with anti-Stat
(green) and anti-FasIII (white, hub). (Arrowheads) Early germ cells next to the
hub; (*) Stat present at lower level in a Gb in control testes. (C) Staining with
anti-γ tubulin (green, centrosomes) and anti-E-cadherin (white, hub). (Arrows)
Oriented centrosomes in germ cell next to the hub. (D) Bar graph depicting
centrosome orientation in +; UAS Grim flies and c587Gal4; UAS Grim; tub-
Gal80tsflies. (Blue bar) Germ cells next to the hub that had two oriented
centrosomes; (red bar) germ cells next to the hub that had two misoriented
centrosomes. (E) Staining with anti-PH3 (green, mitotic marker) and anti-FasIII
(white, hub). (F) (Green) Anti-spectrin (fusome) and anti-FasIII (hub). (Arrows)
Dot fusomes in germ cells next to the hub. (Scale bar: A–C, E, and F; 10 μm.)
Collar germ cells maintained next to the hub in the absence of CySCs
| www.pnas.org/cgi/doi/10.1073/pnas.1215516109Lim and Fuller
Without CySCs or Cyst Cells, Mass Germ Cells Away From the Hub
Failed to Properly Enter the TA Program. Although CySCs and cyst
cells were not required for maintenance of GSC-like cells next to
cell daughters execute finite rounds of mitotic division before en-
tering terminal differentiation. Control TA spermatogonia divided
with incomplete cytokinesis and remained interconnected by
fusome, a membranous structure that normally forms a branched
network within each cyst (Fig. 1A, arrows in Fig. 3A). In contrast,
most mass germ cells had dot (arrowheads, Fig. 3B) or dumbbell
shaped fusomes (arrows, Fig. 3B), reminiscent of GSCs or
gonialblasts. TA cells within a cyst also undergo mitosis in syn-
chrony; in control testes, 26% of early germ cell divisions away
from the hub occurred as single cells, 25% as 2-cell clusters and
E). However, 90% of mass germ cell divisions occurred as single
divided as single cells, they did so at a lower rate compared with
GSCs; 0.72% (39/5393) for mass germ cells compared with 2.8%
and 2.6% for collar germ cells and control GSCs respectively.
Similar results were observed after CySCs and cyst cells were de-
pleted by RNAi knockdown of Stat (Fig. S2 B and F).
TA spermatogonial cells turn on expression of bag-of-marbles
cell stage (Fig. 1A) (20). In control testes, expression of GFP from
a Bam-GFP fusion protein (21) was detected in 4-, 8-, and 16-cell
spermatogonial cysts, but not in GSCs or Gbs (Fig. 4A). However,
mass germ cells that accumulated in testes depleted of CySCs and
cyst cells showed no detectable expression of Bam-GFP (Fig. 4B).
Immunofluorescence using the antibodies against phosphoSMAD
and spermatogonia in control testes (Fig. 4C).
Our observation that GSCs can be maintained long term in the
Drosophila testis without CySCs indicates that CySCs are not
required for GSC self-renewal as previously proposed (13). GSCs
may have an intrinsic capacity to undergo prolonged self-renewal
without external environmental stimuli, as has been suggested for
mouse ES cells (22). It is also possible that GSC maintenance
could be specified by other unidentified signal(s) from the hub.
Our finding that cyst cell function is required for early germ cells
to properly enter the TA program suggests that a key role of these
somatic support cells is to promote GSC differentiation, likely by
active differentiation inputs and/or by down-regulation of the re-
proper cyst cell function for germ cells to initiate differentiation is
its downstream effector Raf in somatic cells in the testis is required
for male germ cells to enter the TA program (23, 24). The TGF-β
pathwayhas beenshownto be requiredcell-autonomouslyfor GSC
maintenance (25–27). The high levels of pSMAD observed in
mass germ cells suggest that the somatic support cells may nor-
mally function in either physically restricting TGF-β signaling
from reaching germ cells away from the hub or in providing cues
that down-regulate the ability of germ cells to respond to TGF-β
also observed in ectopic GSC-like cells depleted of Stat (13), even
though somatic cells were still present and seemed to function
normally in promoting germ cell differentiation.
cells had been ablated could be attributed to the high levels of
TGF-β signaling in mass germ cells. Consistent with this observa-
tion, TGF-β signals from niche cells repress the expression of Bam
in GSCs in the female germline (7). Unlike the female germline
however, function of Bam and deactivation of TGF-β signaling is
not necessary for male GSCs to enter the TA program (19, 27),
to initiate differentiation. Our finding that cyst cells are required
for germ cells to enter the TA divisions raises the possibility that
cyst cells may be the source of such factors.
CySCs may normally provide an environment that allows GSC
maintenance because CySCs are kept in a state in which they are
unable to induce germ cell differentiation. We posit that Chinmo
and the transcriptional repressor Zfh1, which function cell au-
tonomously to maintain CySC identity, may also act to block ex-
pression of cyst cell programs that instruct germ cells to initiate
differentiation. This model accounts for the results that forced
overexpression of either Zfh1 or Chinmo in the cyst cell lineage
resulted in continued proliferation of undifferentiated early germ
cells (14, 15).
the ablation of the somatic cell lineage. The absence of germ cells
in 49% of testes depleted of CySCs and cyst cells may be due to
leaky expression of the c587Gal4 driver in the germline, compro-
mised hub function or both. Low GFP expression had been occa-
from 20 testes), suggesting that the driver may have low activity in
the germline. Ablation of somatic cells may also have negative
consequences on the hub, because hub cells are derived from the
same somatic lineage (28). Thus, even though the hub is physically
present in testes in which CySCs and cyst cells had been ablated, it
may not be fully functional in some testes and therefore fail to
promote germ cell attachment to the hub.
Different aspects of the male GSC program appear to be
controlled by multiple inputs from the local microenvironment at
% of types
4- / 8- cell
Control (n=53) Exp (n=59)
to properly enter the TA program. Immunofluorescence images of testes
stained with anti-Vasa (red, germ cells) from (A, C) +; UAS Grim control flies
and (B and D) c587Gal4; UAS Grim; tubGal80tsexperimental flies shifted to
30 °C for 1 d and back to 18 °C for 21 d. (A and B) (Green) Anti-spectrin
(fusome) and anti-FasIII (hub). (A) (Arrows) Branched fusomes in TA sper-
matogonia. (B) (Arrowheads) Dot fusomes; (arrows) dumbbell-shaped
fusomes. (C and D) Staining with anti-PH3 (green, mitotic marker) and anti-
FasIII (white, hub). (Arrow) Synchronous spermatogonial division; (arrow-
head) single cell division. (E) Bar graph depicting types of divisions of early
germ cells away from the hub. (Blue bar) Dividing single cell; (red bar) di-
viding two-cell cysts; (green bar) dividing four- and/or eight-cell cyst. (Scale
bar: A–D; 10 μm.)
Without CySCs or cyst cells, mass germ cells away from the hub failed
Lim and Fuller PNAS
| November 6, 2012
| vol. 109
| no. 45
the testis apical tip, indicating a complex niche. In addition to
influences exerted by CySCs, two key characteristics of GSCs
appear to be governed directly by activation of the JAK-STAT
signaling pathway in GSCs by signals from the hub: attachment
to the hub and orientation of centrosomes with respect to the
GSC–hub interface, resulting in oriented stem cell division.
The function of Stat has been shown to be cell-autonomously
required for continued attachment of GSCs to the hub (13).
Orientation of centrosomes toward the hub in GSCs is also
dependent on action of the JAK-STAT signaling pathway;
centrosomes became misoriented in germ cells next to the hub
soon after Stat was depleted in all cells by shifting stattsmutant
flies to restrictive temperature (13). Our finding that the collar
germ cells maintained in absence of CySCs have oriented
centrosomes indicates that the centrosome orientation program
characteristic of GSCs is likely an effect of Stat activation in the
GSCs by Upd signals from the hub rather than an indirect
consequence of signals from the CySCs.
Although the model that niches serve primarily to promote
stem cell self-renewal may be true for many adult stem cell sys-
tems (7, 29, 30), our results indicate that components of the
microenvironment may play key roles that can trigger stem cell
differentiation. Indeed, as we come to understand the workings of
more complex niches in mammalian systems, we may find com-
binations of these two opposing influences, with some areas of the
niche promoting self-renewal of stem cells, whereas neighboring
regions drive differentiation of stem cell daughters. A balance
between these competing regulatory machineries may serve as an
important defense against stem cell overproliferation or loss.
Likewise, loss or subversion of the ability of local stromal cells to
promote differentiation may contribute to abnormal proliferation
of undifferentiated cells in tumors.
Materials and Methods
Fly Husbandry and Stocks. Flieswereraisedonstandardcornmealmolassesagar
medium. Fly strains used in this study are c587Gal4 (S. Hou, National Cancer In-
104055), eyaA3 Gal4 (S. Dinardo, University of Pennsylvania, Philadelphia), UAS
Grim (D. Bennett, University of Liverpool, Liverpool, UK), UAS stat92E RNAi
(Vienna Drosophila Resource Center [VDRC] 106980), and tubGal80ts(Bloo-
mington Drosophila Stock Center 7018). Bam expression was analyzed using
a transgenic BamGFP protein fusion reporter (D. McKearin, Chevy Chase, MD).
To ablate CySC and cyst cells, virgin female c587Gal4; Sco/Cyo; tubGal80ts
flies were crossed to w/Y; UAS Grim males and the progeny were grown
to eclosion at 18 °C. Newly eclosed (0–1 d old) males were shifted to 30 °C for
to w/Y; UAS Grim males to generate control flies; the progeny were grown as
described previously. Either c587 Gal4; Sco or Cyo/UAS Grim; tubGal80tsex-
perimental flies, or yw; +/UAS Grim control flies were analyzed at indicated
time points by whole mount immunofluorescence and confocal microscopy.
To induce RNAi of Stat in CySCs and cyst cells, w; tj Gal4; tubGa80tswere
crossed to w/Y; UAS stat92E RNAi (VDRC 106980) and the progeny was
grown to eclosion at 18 °C. Newly eclosed males were shifted to 30 °C. In
parallel, virgin female yw flies were crossed to w/Y; UAS stat92E RNAi males
and were grown as described previously to generate control flies. Either w;
tj Gal4/UAS stat92E RNAi; tub-Gal80ts/+ experimental, or y w; UAS stat92E
RNAi/+ control flies were analyzed at indicated time points by whole mount
immunofluorescence and confocal microscopy.
Whole Mount Immunofluorescence. Testes were dissected in 1× PBS and fixed
with 4% (vol/vol) formaldehyde diluted in 1× PBS for 20 min at room
temperature. Testes were then permeabilized for 2 h in 1× PBS with 0.6%
(vol/vol) Triton-X 100 and 0.6% (wt/vol) sodium deoxycholate at room
temperature, then washed once with 1× PBS with 0.1% (vol/vol) Triton-X
100 (PBST). Primary and secondary antibody incubations were performed in
1× PBST with 3% (wt/vol) BSA; overnight at 4 °C for primary and 2 h in the
dark at room temperature for secondary. Testes were washed with 1× PBST
three times at room temperature after each antibody incubation and
mounted in Vectashield mounting media with DAPI (Vector Labs). Primary
antibodies used were guinea pig anti-Tj (a gift from D. Godt, Toronto;
1:5,000); mouse anti-Fasciclin III (7G10; Developmental Studies Hybridoma
Bank [DSHB]; 1:10); mouse anti-DE-cadherin (DCAD2; DSHB; 1:40); mouse
anti-Alpha Spectrin (3A9; DSHB; 1:5); goat anti-Vasa (dC-13; Santa Cruz
Biotechnology; 1:100); rabbit anti-GFP (Invitrogen; 1:3,000); rabbit anti-STAT92E
(a gift from D. Montell, Baltimore; 1:1,000); rabbit anti-STAT92E (a gift from
E. Bach, New York; 1:1,000); rabbit anti-PhosphoHistone3 Thr3 (Upstate Bio-
(DSHB; 1:10); and rabbit anti-pSMAD (gift from E. Laufer, New York; 1:1,000).
DyLight conjugated donkey secondary antibodies were used at 1:500
(Jackson ImmunoResearch Laboratories). Images were taken using a Leica
SP2 AOBS Confocal Laser Scanning microscope and processed with Adobe
Photoshop CS4. Early germ cells were defined to be Vasa-positive cells that
were <10 μm in diameter and GSCs were scored as Vasa-positive cells in
contact with the hub. For centrosome orientation counts, only GSCs that
contained two centrosomes were scored in the analysis. Centrosomes were
scored as oriented if at least one of them was at the GSC-hub interface. For
the purpose of determining the mitotic index of mass germ cells, Z-stack
images were first taken using the Leica TCS SP5 system. The images were
then compiled into 3D renditions and the percentage of PH3 (green) cells
that were both Vasa- (red) and DAPI- (blue) positive were measured using
Volocity 3D Image Analysis Software.
ACKNOWLEDGMENTS. This work was supported by grants from the Starr
Stanford Graduate Fellowship and the Genentech Graduate Fellowship (to
J.G.Y.L.) and National Institutes of Health Grant R01GM080501 (to M.T.F.).
The support of the Reed-Hodgson Professorship in Human Biology (M.T.F.)
made part of this work possible.
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Lim and FullerPNAS
| November 6, 2012
| vol. 109
| no. 45