DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 3367
Development 138, 3367-3376 (2011) doi:10.1242/dev.065797
© 2011. Published by The Company of Biologists Ltd
Live imaging of the Drosophila spermatogonial stem cell
niche reveals novel mechanisms regulating germline stem
cell output
X. Rebecca Sheng and Erika Matunis*
SUMMARY
Adult stem cells modulate their output by varying between symmetric and asymmetric divisions, but have rarely been observed in
living intact tissues. Germline stem cells (GSCs) in the Drosophila testis are anchored to somatic hub cells and were thought to
exclusively undergo oriented asymmetric divisions, producing one stem cell that remains hub-anchored and one daughter cell
displaced out of the stem cell-maintaining micro-environment (niche). We developed extended live imaging of the Drosophila
testis niche, allowing us to track individual germline cells. Surprisingly, new wild-type GSCs are generated in the niche during
steady-state tissue maintenance by a previously undetected event we term ‘symmetric renewal’, where interconnected GSCdaughter cell pairs swivel such that both cells contact the hub. We also captured GSCs undergoing direct differentiation by
detaching from the hub. Following starvation-induced GSC loss, GSC numbers are restored by symmetric renewals. Furthermore,
upon more severe (genetically induced) GSC loss, both symmetric renewal and de-differentiation (where interconnected
spermatogonia fragment into pairs while moving towards then establishing contact with the hub) occur simultaneously to
replenish the GSC pool. Thus, stereotypically oriented stem cell divisions are not always correlated with an asymmetric outcome in
cell fate, and changes in stem cell output are governed by altered signals in response to tissue requirements.
INTRODUCTION
Tissue maintenance relies on the output of adult stem cells that
divide to self-renew and give rise to differentiating progeny.
Residing in localized cellular micro-environments, or niches, stem
cells receive molecular signals that maintain their fate and regulate
their function (Morrison and Spradling, 2008). Although stem cells
are considered to be long-lived relative to their differentiating
progeny, they also undergo turnover (Doupe et al., 2010; Klein et
al., 2010; Lopez-Garcia et al., 2010; Snippert et al., 2010;
Wallenfang et al., 2006). Lost stem cells must be replaced to
sustain the tissue, as depletion of the stem cell pool could result in
tissue degeneration (Rossi et al., 2008). Contrary to previous
models, where stem cells are considered to have low turnover rates,
recent lineage tracing in the mouse testis, epidermis and intestine
indicate that the stem cell pool is fairly dynamic in actively cycling
tissues, and the fate of an individual stem cell cannot be reliably
predicted (Doupe et al., 2010; Klein et al., 2010; Lopez-Garcia et
al., 2010; Snippert et al., 2010). Formally, a stem cell can be
replenished either by the division of an existing stem cell to
produce two stem cells (symmetric division) or by the reversion of
a differentiating cell into a stem cell (de-differentiation) (Fuller and
Spradling, 2007; Morrison and Kimble, 2006). Both are thought to
occur in Drosophila and mammalian stem cell systems (Barroca et
al., 2009; Jiang et al., 2009; Nakagawa et al., 2007; Nakagawa et
al., 2010). However, direct visualization of these events in real time
in both normal and regenerating tissues has remained elusive.
Department of Cell Biology, 725 N. Wolfe Street, The Johns Hopkins University
School of Medicine, Baltimore, MD 21205, USA.
*Author for correspondence ([email protected])
Accepted 31 May 2011
Live imaging of stem cells in intact tissues has yielded
significant advances towards understanding stem cell function.
Tracking of marked undifferentiated spermatogonia in the intact
mouse testis revealed the putative niche at blood vessel branching
points (Yoshida et al., 2007), and live imaging of hematopoietic
stem and progenitor cells within the bone marrow identified
anatomical locations involved in proper stem cell function during
engraftment (Lo Celso et al., 2009; Xie et al., 2009). Therefore,
live imaging has tremendous potential to enhance our
understanding of stem cells, particularly when applied to tissues,
such as the Drosophila gonads, where stem cells can be
unambiguously distinguished from their differentiating daughters
(Fuller, 1998; Fuller and Spradling, 2007).
Drosophila spermatogenesis is initiated at the testis apex where
~10 germline stem cells (GSCs) are attached to a group of stromal
cells called the hub (Fig. 1A). GSCs always divide with their
mitotic spindles positioned perpendicularly with respect to the hub;
tethering of the older (mother) centrosome at the GSC-hub
interface ensures this. Thus, upon division, one GSC daughter
remains adherent to the hub, and the other is displaced away from
the hub, becoming a gonialblast (Hardy et al., 1979; Yamashita et
al., 2003; Yamashita et al., 2007; Yamashita et al., 2010).
Gonialblasts undergo four rounds of mitotic amplification with
incomplete cytokinesis to produce clusters of interconnected
spermatogonia that eventually form sperm (Fuller, 1998). This
niche also maintains ~20 somatic stem cells known as cyst stem
cells (CySCs) that flank GSCs and are also anchored to the hub
(Gonczy and DiNardo, 1996; Hardy et al., 1979). CySCs divide
asymmetrically to self-renew and produce cyst cells, two of which
envelop each gonialblast. As the gonialblast divides, its
accompanying cyst cells enlarge and sustain encystment. Both
GSCs and CySCs are maintained by Janus Kinase-Signal
DEVELOPMENT
KEY WORDS: Stem cell, Germline, Spermatogonia, Bag of marbles, Niche, De-differentiation, Migration, Live imaging, Symmetric renewal,
Regeneration
3368 RESEARCH ARTICLE
Development 138 (16)
Transducer and Activator of Transcription (Jak-STAT) signaling
initiated from the hub, which secretes the ligand Unpaired (Upd)
(Issigonis et al., 2009; Kiger et al., 2001; Leatherman and DiNardo,
2008; Tulina and Matunis, 2001). CySCs also contribute to the
niche, as Bone Morphogenetic Protein (BMP) signaling from
CySCs is required for GSC self-renewal. BMPs are insufficient to
generate ectopic GSCs, suggesting that additional signals function
in this niche (Kawase et al., 2004; Leatherman and DiNardo, 2010;
Schulz et al., 2004; Shivdasani and Ingham, 2003). Thus, the
Drosophila testis provides a genetically tractable system in which
to study mechanisms regulating stem cells in vivo.
Clonal analysis has shown that Drosophila GSCs turn over with
an approximate half-life of 2 weeks, but are continually replaced
such that GSC number is relatively constant during adulthood
(Wallenfang et al., 2006). Replacement GSCs can be derived from
four- or eight-cell spermatogonial clusters that undergo dedifferentiation (Boyle et al., 2007; Cheng et al., 2008; Mair et al.,
2010; McLeod et al., 2010; Wallenfang, 2007; Wallenfang et al.,
2006). Whereas GSCs, gonialblasts or two-cell spermatogonial
clusters may also regenerate GSCs, these events have not been
detected thus far using fixed imaging. We developed long-term live
imaging conditions to directly observe GSC behavior during both
steady-state conditions and upon recovery from genetically induced
stem cell loss (referred to here as ‘regeneration’) in intact
Drosophila testes. Our data reveal that GSCs in the Drosophila
testis niche display various modes of stem cell renewal, which are
regulated in vivo by altered signals during regeneration.
MATERIALS AND METHODS
EdU incorporation
Dissected testes were incubated in Schneider’s media containing 10 mM
EdU (Invitrogen kit C10338) for 20 minutes. Immunostaining and
thymidine analog detection were performed as described previously
(Leatherman and DiNardo, 2008).
Extended live imaging
Testis culture conditions were adapted from other publications (Prasad et
al., 2007; Prasad and Montell, 2007). Testes were dissected into and rinsed
twice with Schneider’s insect medium (Gibco/Invitrogen 11720034, pH
adjusted to 7.0) supplemented with 15% FBS (Sigma F3018) and 0.5⫻
penicillin/streptomycin (Gibco/Invitrogen cat. 15140-122). Testes were
then rinsed twice with the same supplemented Schneider’s media with the
addition of MitoTracker Red CMXRos (M7512, Molecular Probes) at a
final concentration of 1 mM and 0.2 mg/ml insulin (Sigma I5500). Rinsed
testes were transferred with ~20 ml media onto a 60 mm coverslip, covered
with ~1 cm2 Teflon sheet (YSI Life Sciences #5793), and sealed with
Halocarbon oil 27 (Sigma H8773). Live images were acquired using either
an inverted Perkin Elmer UltraVIEW spinning-disk confocal, inverted
Leica SD4 spinning-disk confocal or upright Zeiss 710 NLO multiphoton
(JHU SOM Microscope Facility). Imaging parameters used are described
in Table 1. Up to eight different positions per slide were acquired using a
programmable xy stage. As the muscle sheath was intact, occasional
movies were not analyzed due to contractions. Furthermore, a range of
movie times were scored, as sample drift in the x, y or z planes occasionally
occurred and resulted in early termination of the movie as the sample
shifted away from the focal plane. The length of the movie and the type of
microsocpe used did not affect tissue viability.
Analysis of confocal images
Fixed testes were mounted in Vectashield and viewed with Zeiss LSM 5
Pascal. z-series of live testes were analyzed using the Pascal, Ultraview,
Slidebook, Zen, Bitplane’s Imaris or NIH ImageJ software.
Fly stocks and heat shock protocol
P{nos::eGFP-Moe::nos 3⬘UTR} flies were a gift from R. Lehmann
(Skirball Institute, NY, USA) (Sano et al., 2005). Hs-bam flies contain the
P[w+; Hsp70-bam+]18d transgene inserted on the X chromosome (Ohlstein
and McKearin, 1997). nanos-Gal4-VP16 flies (Van Doren et al., 1998)
(from E. Selva, University of Delaware, DE, USA) were crossed to UASGFP-Moesin Actin-binding domain flies (Bloor and Kiehart, 2001; Dutta
et al., 2002) (from D. Kiehart, Duke University, Durham, NC, USA) to
drive expression of GMA in germ cells. All other stocks were acquired
from the Bloomington Stock Center (Indiana). GSCs were depleted from
flies containing the Hs-bam transgene as described previously (Sheng et
al., 2009).
GSC cell cycle length estimation
As each testis contains on average 12.3 GSCs, we calculated the total
amount of time GSCs were imaged (12.3 GSC/testis ⫻ 197 hours) and
divided by the total number of GSC divisions observed during that time
(104) to derive a cell cycle time of 23.3 hours.
Mitotic index estimation
Live imaging of GSC division shows that mitosis takes between 40
minutes and 1 hour to complete. The % dividing GSCs is estimated by
dividing the length of mitosis by the length of the cell cycle (24 hours),
which is between 3% and 4%.
Comparing relative frequency of dividing germ cells
Immunostaining was performed as described (Matunis et al., 1997).
Primary antibodies: rabbit anti-Vasa at 1:200 (Santa Cruz); rabbit anti-GFP
at 1:10,000 (Torrey Pines Biolabs); mouse anti-1B1 at 1:25; and mouse
anti-Armadillo at 1:50 (all from Developmental Studies Hybridoma Bank,
University of Iowa). Alexa fluor-conjugated secondary IgG (H+L)
antibodies were diluted 1:200 for 405, and 1:400 for 488 conjugates.
Secondary antisera were: goat anti-rabbit 488 and goat anti-mouse 405
(Molecular Probes/Invitrogen). Nuclei were counterstained using 1 mg/ml
4⬘-6-diamidino-2-phenylindole (DAPI) (Roche Molecular Biochemical).
The numbers of mitotic germ cells of a given stage (GSCs, gonialblasts,
two-, four- or eight-cell spermatogonia) were counted in both fixed and live
testes of genotypes P{nos::EGFP-moe::nos 3⬘UTR} and Hs-bam; nanosGal4; UAS-GMA/+, then divided by the total number of mitotic GSCs,
gonialblasts, two-, four- and eight-cell spermatogonia to obtain the percent
of total mitoses. In fixed tissues, cells were scored as being in mitosis if
they had high levels of GMA enrichment at the cell cortex and condensed
chromosomes. In live testes, cells were scored as having undergone mitosis
if they produced daughter cells (Fig. 1C-F, Fig. 2A-D; see Fig. S1A-D in
the supplementary material).
Table 1. Description of microscope parameters used for live imaging
Microscope
(number of movies)
Number of
z-sections/
timepoint
z-section
thickness (mm)
Interval length
(minutes)
Movie length
(minutes)
471 nm laser
exposure (ms)
568 nm laser
exposure (ms)
Binning
UltraView (23)
Leica SD4 (25)
Zeiss 710 NLO (5)
20-44
15-20
40
1.3-1.5
1.3-2
1
7-30
20-40
25
330-740
340-680
160-425
400-800
250-400
N/A
160-400
20-50
N/A
N/A
2⫻2
N/A
Scan speed,
averaging
N/A
N/A
Speed, 9;
average, 2
The various empirically determined specifications are listed for each microscope. We used the minimal laser power and exposure time to maintain specimen integrity during
image acquisition.
DEVELOPMENT
Immunostaining
Live imaging of fly GSCs
RESEARCH ARTICLE 3369
Fig. 1. Germ cells appear healthy and divide in live
imaged testes. (A)Schematic of the testis apex
depicting hub cells (blue), GSCs (green), mitotic spindle
(pink), centrosomes (white), spermatogonia and
gonialblasts (yellow), cyst stem cells (dark grey) and cyst
cells (light gray). (B)Graph comparing the relative
frequencies of dividing germ cells of a particular stage in
fixed and live imaged testes using the Chi-squared test.
(C-F)Frames and corresponding schematics showing
GSC division in a time-lapse imaged testis expressing
GMA in GSCs and spermatogonia (genotype: Hs-bam;
nanos-Gal4; UAS-GMA/+). (C)Before division occurs, the
GSC (arrowhead, green in schematic) has GMA enriched
at the stem cell-hub interface. (D)During mitosis, GMA
becomes highly enriched at the cell cortex, and later
marks the cleavage furrow during incomplete
cytokinesis. (E,F)A daughter gonialblast (arrow, yellow) is
formed (E) and remains displaced from the hub until the
end of the movie (F). Images are maximum intensity
projections of three 1.5mm optical sections. Illustrations
depicting the cells of interest are below their respective
panels. The hub is outlined. Scale bar: 10mm.
Flies were raised on standard molasses-based food, transferred to food
comprising 2.8% agar and 10% sucrose for 14 days, then returned to
standard food.
Statistical analysis
Averages were compared using two-tailed Student’s t-test assuming
unequal variances or ANOVA. All graphs were constructed and statistics
were confirmed using Graphpad’s Prism or R software. Comparison of live
imaged events between steady-state and regeneration was conducted
assuming that the probability of a given number of events occurred during
steady-state with a Poisson distribution (mean equal to the total number of
events observed during steady-state). Comparison of percentages was
conducted using Fisher Exact or Chi-Squared tests.
RESULTS
Live imaging is a valid method for analyzing
dividing germ cells in adult Drosophila testes
In order to observe dynamic cellular processes in real time within
a stem cell niche, we developed extended 4D (x, y, z and t) imaging
with the ability to resolve individual germline cells. We imaged live
testes expressing the Actin-binding domain of Moesin fused to
GFP (referred to as GMA) in the early germline (Dutta et al.,
2002), which reveals germ cell morphology in both live embryos
(Sano et al., 2005) and fixed testes (Sheng et al., 2009). In live
testes, this marker is enriched at germ cell cortices, at the GSC-hub
interface, and at cell-cell contacts within syncytial spermatogonia
(Fig. 1C-F) in a manner indistinguishable from that seen in fixed
testes (Sheng et al., 2009). Germ cells in mitosis also displayed
similar GMA localization to those found in fixed tissues (Fig. 1CF; see Fig. S1A-D in the supplementary material).
As previous live imaging of the Drosophila testis apex was on
shorter timescales (~2 hours) (Cheng et al., 2008), we measured
germ cell proliferation rates in our 27 movies of young wild-type
testes to determine whether our extended live imaging conditions
compromised cell viability. The length of our movies ranged
between 2.6 and 12 hours (average 7 hours), with z stacks
collected at 20-minute intervals. In our combined 197 hours of
imaging data, 104 GSC and 166 spermatogonial divisions were
observed (Fig. 1B-F; see Fig. S1A-D in the supplementary
material), indicating cells were viable and could proliferate given
our imaging conditions. We calculate the cell cycle length of a
single GSC to be ~24 hours during live imaging; this is
comparable with previous estimates of GSC cell cycle length
(Wallenfang et al., 2006) and mitotic index (Yamashita et al.,
2003). Consistent with this calculation, we never observed the
same GSC divide twice in our movies. The proportion of
dividing GSCs, gonialblasts and two- to eight-cell spermatogonia
was similar between fixed and live imaged testes (Fig. 1B), and
we did not observe significant changes in mitochondrial
morphology during imaging (see Fig. S6A,B in the
supplementary material), further indicating that imaging
conditions did not compromise cell viability or proliferation.
Wild-type GSCs divide with stereotypical spindle
orientation and generally result in asymmetric
cell fate
GSCs in young flies are reported to always divide with spindles
oriented perpendicular to the hub to produce one GSC still attached
to the hub and one differentiating daughter cell displaced away from
the hub, thus accomplishing asymmetric division (Cheng et al., 2008;
Yamashita et al., 2003). We tracked all 104 GSC divisions until the
end of each movie to determine the outcome of the division. With
the exception of one case (see Fig. S2 in the supplementary material),
every GSC that divided during live imaging (103/104) initially
displaced a daughter cell away from the hub, confirming that this
stereotypical GSC behavior occurs reliably in our live imaging
conditions (Fig. 2A-D). Furthermore, daughter cells remained
displaced from the hub in 95 out of 104 divisions for the remainder
of the movie (see Movie 1, Fig. S7 and Table S2 in the
supplementary material). Thus, asymmetric division is the
predominant outcome following GSC divisions in vivo (Cheng et al.,
2008; Yamashita et al., 2003) and during live imaging.
An oriented GSC division can produce two GSCs,
resulting in symmetric renewal
While tracking wild-type GSCs, we were surprised to see seven
GSC-daughter pairs swivel such that the daughter cell initially
distal to the hub changed its position and gained direct contact
DEVELOPMENT
Protein starvation protocol
3370 RESEARCH ARTICLE
Development 138 (16)
Fig. 2. GSC division can result in the production of
a GSC and GB (asymmetric division), two GSCs
(symmetric renewal) or the loss of a GSC
(symmetric differentiation). (A-L)Time-lapse images
taken from a movie of P{nos::EGFP-moe::nos 3⬘UTR}
testis. (A-D)Example of a GSC division that results in a
daughter cell (arrow) remaining displaced away from
the hub until the end of the movie. Arrowhead
indicates daughter cell. (E-H)GSC-daughter pairs
(arrowhead, arrow) can swivel such that the initially
displaced daughter contacts and is maintained at the
hub. (I-L)GSC-daughter pairs (arrowhead, arrow) can
detach from the hub and remain morphologically
indistinguishable from two-cell spermatogonial clusters
for the remainder of the movie. Illustrations
representing the cells of interest are below their
respective panels with colors corresponding to their
function as suggested by their anatomical location:
green, GSC; yellow, daughter cell. Asterisks indicate the
hub. Scale bar: 10mm.
An oriented GSC division can produce a two-cell
spermatogonial cluster, resulting in symmetric
differentiation
Previous clonal analysis in fixed testes demonstrated that GSC
loss occurs in wild-type testes upon aging (Wallenfang et al.,
2006). Using live imaging, we detected 16 cases of GSC loss in
young wild-type testes, each characterized by a GSC-daughter
pair progressively losing hub contact over ~20-40 minutes time
(Fig. 2I-L; see Movie 1, Fig. S7 and Table S2 in the
supplementary material). GSC loss occurred at random times
between 20 and 250 minutes (average 120 minutes) after the
beginning of imaging, suggesting that the loss is not due to
disruption of tissue integrity caused by imaging. Interestingly, we
never observed GSCs without interconnected daughters losing
hub contact, suggesting that GSC loss does not occur in the late
G2 or M phases of the cell cycle (see Fig. S3 and Table S1 in the
supplementary material). GSC-daughter pairs that detached from
the hub stayed interconnected and appeared morphologically
healthy for the remainder of the imaging time (from 2 to 9 hours),
suggesting that differentiation rather than death occurred
following loss. Consistent with this hypothesis, STAT92E was not
detected in pairs of germ cells detached from the hub (n44
testes), and apoptotic GSCs have never been detected in fixed
wild-type testes (Boyle et al., 2007; Brawley and Matunis, 2004;
Issigonis et al., 2009; Sheng et al., 2009). Together, these data
suggest that stem cell loss during steady-state is via symmetric
differentiation characterized by loss of adhesion of a
mother/daughter pair followed by differentiation of both cells.
EdU pulse-labeling confirms that symmetrical GSC
events observed in live imaging also occur in vivo
We next sought to confirm that symmetric renewal of GSCs occurs
in vivo, and is not an artifact of live imaging conditions. As
symmetric renewal is distinguished by the movement of a daughter
cell into the niche while attached to a GSC, we looked for such pairs
of cells in fixed tissues by incubating testes briefly (20 minutes) in
media containing the thymidine analog EdU prior to fixation. Similar
to wild-type female Drosophila GSCs (Carpenter, 1981; de Cuevas
DEVELOPMENT
with the hub (Fig. 2E-H; see Movie 1, Fig. S7 and Table S2 in
the supplementary material). This event has never been reported
to occur in this system, and would not have been identified in
fixed images. In all seven cases, both daughter cells were
maintained next to the hub for the remainder of the movie (2-5
hours), suggesting that GSCs in the testis can produce two stem
cells from one division. We refer to this phenomenon here as
‘symmetric renewal’. To determine whether both daughters
acquire (or retain) GSC character, we analyzed the expression of
the well-characterized GSC marker STAT92E (Boyle et al.,
2007; Chen et al., 2002; Issigonis et al., 2009; Johansen et al.,
2003; Leatherman and DiNardo, 2008; Read et al., 2004; Tulina
and Matunis, 2001). STAT92E was detected in all GSCs (n556)
in fixed young wild-type testes (not shown). Thus, although GSC
divisions are stereotypically oriented, daughter cells displaced
away from the niche occasionally enter the niche and become
stem cells. As we do not detect single gonialblasts or any type
of spermatogonial cluster reverting into GSCs in 197 hours of
imaging, our data strongly support the hypothesis that symmetric
renewal of existing GSCs is the primary mode for GSC
regeneration in young testes.
Fig. 3. GSCs undergoing symmetric renewal are detected in fixed
tissues. (A-C)Testes of genotype P{nos::EGFP-moe::nos 3⬘UTR} were
incubated in media containing EdU for 20 minutes, fixed and then
visualized for EdU (red) and immunostained for GMA (green), Armadillo
and 1B1 (blue). (A)Two labeled GSCs (solid arrowheads) both attached to
labeled daughter cells (arrows). (B)A labeled GSC-daughter pair with
both cells attached to the hub (solid arrowheads) and fusome (open
arrowhead; inset shows the blue channel) between the cells. No single
daughter cells were nearby, suggesting that abscission had not occurred
prior to fixation. (C)An EdU-labeled GSC-daughter pair (arrows) displaced
from the hub with a long GMA-rich protrusion still attached at the hub
(open arrowhead; inset shows the green channel). (D)A GSC-daughter
pair (solid arrowheads) with both cells at the hub in Cnn HK21/mfs3
trans-heterozygotes immunostained with the germ cell marker Vasa
(green) instead of GFP. Scale bar: 10mm. (E)The number of GSCs is not
significantly different between heterozygous controls and transheterozygous or homozygous centrosomin mutants (nnumber of
testes). (F)The percentage of EdU labeled GSCs with daughters adjacent
to the hub was significantly increased in centrosomin mutants, including
the heterozygous HK21 allele (nnumber of GSCs). (G)Schematic of
starvation and feeding protocol performed on P{nos::EGFP-moe::nos
3⬘UTR} flies; broken lines represent time spent on 10% sucrose food and
unbroken lines represent time spent on standard molasses food.
(H)Starvation causes a modest but significant decrease in the number of
GSCs compared with age-matched controls, followed by recovery upon
feeding (nnumber of testes). (I)The percentage of GSC-daughter pairs
with both cells at the hub increases after starvation and after 1 day of
feeding, but returns to baseline after 5 days of feeding (nnumber of
GSCs). Error bars represent s.d. Statistical analysis of GSC number
performed using ANOVA with Dunn’s and Tukey’s multiple comparison
tests (*P<0.05). Statistical analysis comparing percentages performed
using Chi-squared test.
RESEARCH ARTICLE 3371
and Spradling, 1998), male GSCs and their daughter cells undergo
S-phase while interconnected (Fig. 3A,B), and every EdU-positive
GSC (n361) was connected to a daughter. We then assessed the
position of EdU-positive GSC-daughter pairs with respect to the hub.
Ninety-five percent of EdU-positive GSCs were attached to
daughters displaced away from the hub (Fig. 3A). However, 5% of
EdU-positive GSC-daughter pairs were positioned such that both
cells contacted the hub (Fig. 3B). These cells were morphologically
similar to symmetrically renewing GSCs observed during live
imaging (Fig. 2G,H), as both cells were enriched for GMA at the
hub-germ cell interface. It is unlikely that these EdU-positive cells
are two adjacent GSCs that coincidentally both labeled and both
completed abscission of their daughter cells prematurely, as we did
not detect single EdU-positive gonialblasts nearby (n374
gonialblasts). Furthermore, pulse-chase BrdU/EdU labeling
experiments (see Fig. S4 in the supplementary material) and fusome
and Anillin morphology (see Fig. S3 in the supplementary material)
revealed that GSC-daughter abscission occurs in G2, many hours
after S phase (see Table S1 in the supplementary material). Finally,
a spherical fusome was detected between the two cells, and EdU
label intensity was similar within each pair, as expected if both cells
were interconnected and entered S phase simultaneously (Fig. 3B,
inset). As we did not detect movement of two-cell spermatogonial
clusters into the niche during our live imaging, we do not believe
these pairs of cells represent de-differentiating spermatogonia.
Instead, they represent symmetrically renewing GSCs, which are in
vivo products within this stem cell niche.
We next looked for signs of symmetric differentiation in fixed
EdU-labeled testes. GSC-daughter pairs that have recently detached
from the hub are indistinguishable from two-cell spermatogonia in
fixed tissues. However, we infrequently saw examples (three cases
in over 300 GSCs) of EdU-positive GSC-daughter pairs with fine
protrusions projecting between adjacent GSCs to contact the hub
(Fig. 3C). As these are fixed images, we cannot determine whether
the pairs of cells are moving towards or away from the hub.
However, in our live imaging, we saw only the latter. Thus, these
examples most probably represent GSC-daughter pairs that are
detaching from the hub in unperturbed wild-type tissues, resulting
in symmetric differentiation.
Genetic and environmental factors affect
symmetric GSC renewal frequency
Having devised an assay for identifying symmetric renewals in
fixed tissue, we wanted to confirm that it behaved as expected in a
genetic background known to increase the frequency of GSC
renewal. Thus, we analyzed centrosomin (cnn) homozygous and
trans-heterozygous mutants, where randomization of the division
plane is thought to yield increased numbers of GSCs (Inaba et al.,
2010; Yamashita et al., 2003). As expected, whereas the overall
EdU labeling frequency remained similar to controls (~30%), the
percentage of GSC-daughter pairs where both cells touched the hub
increased significantly from 5% in heterozygous controls to over
15% in the mutants (Fig. 3D,F), consistent with the finding that
GSCs lacking Cnn can place daughter cells into the niche due to
disruption of spindle orientation (Yamashita et al., 2003). In
contrast to previously published findings, we did not see a
significant increase in the number of GSCs per testis compared
with sibling controls (Fig. 3E), suggesting that factors other than
division orientation regulate overall GSC number.
We next asked whether changes in GSC division outcome could
be detected in physiologically relevant circumstances such as poor
nutrition. Protein starvation increases the rate of GSC loss in the
DEVELOPMENT
Live imaging of fly GSCs
Fig. 4. Live imaging shows de-differentiating spermatogonia
moving to make contact with the niche and also undergoing
symmetric renewal. (A-F)Time-lapse images taken from movie of Hsbam; Nanos-Gal4; UAS-GMA testes depleted of GSCs and allowed to
recover for 36 hours before imaging. Numbered cells and hub (dotted
circle) are depicted in illustrations. (A)Two pairs of germ cells (1,2) and
(3,4), with GMA enrichment between the syncytial cells, are initially not
in contact with the hub. (B)Pair (1,2) moves first such that both cells
make large hub contacts, (C) followed by pair (3,4). Cells 1 and 3 are
contacting the hub, but out of the focal plane. (D)Cell 4, which had
attached to the hub, divides to produce daughter 4a. (E)4a initially has
a very little hub contact; (F) this gradually increases. This is an example
of a division where the daughter cell is not displaced from the hub.
Images are maximum intensity projections of three 1.5mm optical
sections. The hub is indicated by an asterisk. Scale bar: 10mm.
Drosophila ovary and testis (Hsu and Drummond-Barbosa, 2009;
Mair et al., 2010; McLeod et al., 2010). We confirmed that protein
starvation reduces the number of GSCs compared with age-matched
controls (8.6±1.8 versus 10.5±2.3, P<0.05, Fig. 3G,H). This effect is
fully reversed by returning starved flies to standard food (Fig. 3G,H)
(McLeod et al., 2010). As an increase in de-differentiation or
misoriented centrosomes was not previously detected during GSC
recovery (McLeod et al., 2010), we hypothesized that symmetric
renewals were responsible for the modest increase in GSC number.
Therefore, we determined the percentage of EdU-positive GSCdaughter pairs with both cells contacting the hub; this was
significantly higher both after starvation (14%) and after 1 day of
feeding (21%) compared with fully fed age-matched controls (3%),
suggesting that nutrition levels affect the frequency of symmetric
renewals (Fig. 3I). This increase in symmetric renewal was transient
and returned to baseline after 5 days of feeding (6.5%). Together, our
data show that the frequency of symmetric renewals responds to
environmental and genetic perturbations.
Development 138 (16)
Live imaging directly reveals spermatogonial
fragmentation and movement into the niche
during regeneration
In order to better understand regeneration in vivo, we made 26
movies (254 hours) of testes undergoing de-differentiation.
Although de-differentiation occurs in wild-type testes, the
probability of observing it in young flies is very low (Cheng et al.,
2008). To capture de-differentiation events during live imaging, we
used flies that had first been genetically depleted of GSCs and were
in the process of recovering their stem cells. During recovery, high
levels of spermatogonial dedifferentiation replenish lost GSCs
(Brawley and Matunis, 2004; Sheng et al., 2009). In our assay, heat
shock-induced ectopic expression of the differentiation factor Bagof-Marbles (Bam) reduces the number of GSCs to less than one per
testis, but testes regain wild-type numbers of GSCs after 4-5 days
of recovery at the permissive temperature (Sheng et al., 2009). As
our previous work suggested that de-differentiating spermatogonia
were undergoing cellular rearrangements between 12 and 36 hours
post heat-shocks (Sheng et al., 2009), we focused on these
timepoints.
Fixed images suggested that spermatogonia, normally thought to
be immobile, can acquire motility and move to make hub contact
during dedifferentiation (Sheng et al., 2009). Our movies validated
this hypothesis, as we saw 25 cases where spermatogonia initially
not contacting the hub move in and established hub contact (Fig.
4A-C; see Fig. S7, Table S2 and Movie 2 in the supplementary
material). Following contact, which occurred despite the presence
of cyst stem cells occupying the positions directly next to the hub
(see Fig. S6 in the supplementary material) (Sheng et al., 2009),
GMA became enriched at the hub-GSC interface (Fig. 4B).
Spermatogonia of most developmental stages were able to revert;
we saw two gonialblasts, 14 two-cell, four four-cell and one eightcell cluster establish hub contact during our movies. Before
initiation of movement, the proximal edges of the reverting
spermatogonia were between 2 and 11 mm (average 4 mm) away
from the edge of the hub. Cells that moved to contact the hub were
not always those closest to the hub (Fig. 4A-C, cells 3 and 4),
indicating that proximity to the hub does not necessarily correlate
with reversion. Our previous work showed that some
spermatogonia had protrusions on their cell surface during dedifferentiation (Sheng et al., 2009), and we observed similar
protrusions on live spermatogonia (see Movie 2 in the
supplementary material). However, protrusions only occasionally
correlated with cell movement towards the hub, indicating they
may reflect other cellular events such as changes in encystment by
somatic cells. Live imaging has now directly demonstrated the
ability of spermatogonia to move into the niche, gain hub contact
and then resume divisions (Fig. 4D-F and Movie 3 in the
supplementary material), thereby reverting to a less differentiated
cell.
Live imaging also allowed us to visualize fragmentation of
spermatogonial clusters, a process inferred to occur specifically
during dedifferentiation (Brawley and Matunis, 2004; Cheng et al.,
2008; Kai and Spradling, 2004; Sheng et al., 2009). In live wildtype testes, cytoplasmic stretching occurs only between the GSC
and daughter gonialblast during abscission (see Fig. S1G-I in the
supplementary material), and germ cells within a cluster of
interconnected spermatogonia always divide synchronously (see
Fig. S1A-D in the supplementary material). In testes undergoing
dedifferentiation, we observed stretching of the cytoplasm between
interconnected spermatogonia followed by mitoses of resulting
smaller clusters at different timepoints. For example, two pairs of
DEVELOPMENT
3372 RESEARCH ARTICLE
Live imaging of fly GSCs
RESEARCH ARTICLE 3373
cells arise from a stretched four-cell spermatogonial cluster; one of
the resulting pairs divided over 2 hours before the other, further
indicating they were no longer interconnected (see Fig. S5 in the
supplementary material). In 254 hours of imaging, we saw two
four-cell, one eight-cell and one 16-cell cluster fragment into twocell clusters, and one eight-cell fragment into two four-cell clusters.
As we observed only five clear examples, fragmentation is either
rare or our methods are not optimized for detecting it. Nonetheless,
live imaging clearly shows spermatogonial clusters near the hub
fragmenting into smaller clusters while entering the niche –
hallmarks of de-differentiation that had never been directly
visualized.
DISCUSSION
Live imaging of the Drosophila germline stem cell niche has
directly demonstrated many aspects of GSC behavior that were
impossible to observe in fixed tissues. Asymmetrically oriented
divisions do not necessarily determine asymmetric cell fate, but can
occasionally result in the production of two GSCs. This is the
primary mechanism by which GSCs are replenished in healthy
tissues to compensate for GSC loss. As GSC-daughter pairs are
Fig. 5. The outcomes of GSC division are significantly altered
between steady-state and regeneration. (A)Graph showing the
119 events observed during steady-state and 102 events during
regeneration categorized as asymmetric division, symmetric renewal,
loss or reversion. The total number of events is derived from the
number of observed GSC mitoses (regardless of division orientation)
plus the number of cases of spermatogonial reversion, symmetric
renewal and symmetric differentiation that occurred without an
observed GSC division. GSCs that undergo mitosis and produce
daughter cells that remained displaced away from the hub until the end
of imaging were scored as asymmetric division. GSC-daughter pairs
that swivel such that the daughter cell makes and maintains contact
with the hub throughout the remaining imaging period (this includes
cases where a GSC divided parallel or 45° to the hub) were scored as
symmetric renewals. GSC-daughter pairs detaching from the hub and
remaining detached throughout the subsequent imaging period were
scored as symmetric differentiation (loss). Gonialblasts, two-, four-,
eight- or 16-cell spermatogonial clusters moving from not being in
direct contact to being attached to the hub were scored as reversions.
Chi-squared analysis reveals the proportions of the events to be
significantly different. (B)Schematic showing relative frequency of
events during steady-state and regeneration.
adjacent to the hub and are enriched in the maintenance factor
STAT92E (Boyle et al., 2007; Issigonis et al., 2009; Leatherman
and DiNardo, 2008), the process of symmetric renewal is probably
distinct from de-differentiation of spermatogonia (which are non
hub-adherent and express the differentiation factor Bam). The
frequency of symmetric renewal increases during GSC recovery
after protein starvation, and during GSC regeneration after
genetically induced stem cell depletion. In the latter case, where the
rate of GSC regeneration is higher, GSCs are concurrently derived
from de-differentiating spermatogonia, a process characterized by
movement, fragmentation and adhesion to the hub by
spermatogonial cells. Together, these data demonstrate that lost
DEVELOPMENT
The proportions of misoriented GSC divisions, GSC
reversions and symmetric renewals are
significantly higher during regeneration
GSCs in the ovary are able to divide parallel to niche-generating
cells after stem cell turnover (Xie and Spradling, 2000), and it has
been proposed that male GSCs could also divide with a mitotic
spindle parallel to the hub during GSC regeneration (Fuller and
Spradling, 2007; Spradling, 2008). Therefore, we wanted to
determine whether testes undergoing regeneration produce stem
cells that repopulate the niche via symmetric renewals derived from
divisions with a misoriented spindle. Surprisingly, even in testes
undergoing regeneration, 91% (58/64) of germ cell mitoses at the
hub produced a daughter cell that was initially displaced from the
hub, indicating that misoriented divisions are not the primary
method to generate additional stem cells during regeneration (see
Fig. S7 in the supplementary material). However, the proportion of
misoriented hub-contacting germ cell divisions in regenerating
testes is slightly higher than wild type (6/64 versus 1/104, P≤0.05;
see Movie 3 in the supplementary material), consistent with
previous findings that GSCs derived from de-differentiation have
higher incidence of misoriented centrosomes and division planes
(Cheng et al., 2008).
The stem cell output of tissues in steady-state versus tissues
undergoing regeneration is probably different in order to fulfill the
cellular requirements of each situation. We compared the relative
frequencies of events resulting in asymmetric division, symmetric
renewal, symmetric differentiation and reversion during steadystate (total events119) and regeneration (total events102). During
regeneration, we saw 47 cases of asymmetric outcome, 22 cases of
symmetric renewals, 25 cases of spermatogonial reversion and 8
cases of loss (see Fig. S7, Table S2 in the supplementary material).
Although the relative frequency of germ cell loss was not
significantly changed during regeneration, the relative frequency
of asymmetric division decreased by about one-half (Fig. 5; see
Table S2 in the supplementary material). Furthermore, both
symmetric renewal and reversion increased over threefold (Fig. 5;
see Table S2 in the supplementary material). Thus, symmetric
renewal and reversion are regulated processes, and the overall
status of the tissue can direct stem cell behavior via changes in cell
signaling.
GSCs can be regenerated by multiple mechanisms, some or all of
which may be similar to events occurring in other stem cell
systems.
As changes in stem cell output are observed during regeneration,
signaling from support cells or from systemic factors may underlie
these effects. Niche-generating cells, transit amplifying daughter
cells or even differentiated daughter cells may potentially signal to
stem cells and modulate their division output. In the Drosophila
testis, GSC maintenance depends on Jak-STAT signaling initiated
from the hub, but it is not known whether this same pathway
regulates division outcome. As STAT-null GSCs are rapidly lost
from the niche (Brawley and Matunis, 2004; Kiger et al., 2001;
Leatherman and DiNardo, 2010; Tulina and Matunis, 2001), low
levels of Jak-STAT signaling due to fluctuations in gene expression
may be sufficient to cause GSC loss. In support of this hypothesis,
three out of 556 GSCs examined for STAT92E expression had low
levels of this protein. However, the mRNA expression pattern of
the Jak-STAT pathway ligand Upd is unchanged during dedifferentiation (Sheng et al., 2009), suggesting that genes other than
Upd may affect symmetric renewals. BMP signaling, which is
required for GSC maintenance (Kawase et al., 2004; Leatherman
and DiNardo, 2010; Schulz et al., 2004; Shivdasani and Ingham,
2003), is a good candidate. Combining live imaging with genetic
tools for monitoring levels of signaling pathway activation in the
Drosophila testis will provide a powerful platform for
understanding how cell signaling affects the outcome of stem cell
divisions in real time.
Our observation that both symmetric renewal and GSC loss
occur when the GSC is attached to a daughter cell suggests that
there may be a cell cycle-specific gene expression profile that
primes the cells for these events to occur during S or early G2 in
the cell cycle. We speculate that the abscission accompanying
symmetric renewal is similar to that occurring in GSC-GB pairs,
another G2 event. Cell cycle regulation, which is characterized
by a short G1 phase and relatively long S phase, maintains
pluripotency in many types of cultured stem cells (Singh and
Dalton, 2009). As GSCs in the Drosophila testis have short G1
phases (see Table S1 in the supplementary material), and
Drosophila GSCs require distinct cell cycle regulators (Wang
and Lin, 2005), investigation of cell cycle regulation of
Drosophila GSC division outcome may be informative.
We showed that GSCs in both centrosomin mutants and
starved wild-type flies have increased frequencies of symmetric
renewal, but surprisingly, there is no corresponding rise in GSC
numbers. These results suggest that increased symmetric renewal
is counterbalanced by increased GSC loss. Cnn mutant GSC are
reported to have abnormal cell morphology and often appear to
be detaching from the hub, suggesting an overall maintenance
defect (Yamashita et al., 2007). During starvation, lowered
insulin signaling results in GSCs loss, and this effect can be
rescued by overactivation of insulin signaling (McLeod et al.,
2010; Ueishi et al., 2009). Our results indicate that symmetric
renewals of GSCs undergoing oriented divisions are the source
of new GSCs. Starved flies initially have low insulin signaling,
but when returned to normal food for a day have higher insulin
signaling (McLeod et al., 2010). However, we find that both
timepoints exhibited increased symmetric renewals, leading us
to believe that activation of insulin signaling does not directly
modulate division outcome. Perhaps during starvation, lowered
insulin signaling causes GSC loss, which in turn triggers a
compensatory increase in symmetric renewal. However,
symmetric renewals are not able to fully compensate for the loss,
Development 138 (16)
yielding an overall decrease in GSC number. When flies are refed and insulin signaling returns to normal, GSCs are no longer
rapidly lost, and the same rate of symmetric renewal is now able
to increase overall GSC number. Together, our results suggest
that the behavior of stem cells within the niche is much more
dynamic than previously expected, and indicate that GSC
number is controlled by the relative rates of symmetric renewal
versus loss, not by the orientation of the division plane.
Why do the majority of Drosophila GSCs undergo asymmetric
division if symmetric renewal plus symmetric differentiation
produces the same output? As GSCs and CySCs function
together within the niche during spermatogenesis, robust division
orientation of both populations may enable differentiating
germline cells to be generated at a rate that matches cyst cell
production. Asymmetric divisions may also prevent clonal
expansion of stem cells harboring harmful mutations within the
niche, which can compete for niche occupancy (Issigonis et al.,
2009; Johnston, 2009). However, clonal expansion may not
always be harmful; mammalian niches regularly progress
towards mono-clonality with stem cells exhibiting neutral drift
dynamics (Klein et al., 2010; Lopez-Garcia et al., 2010; Snippert
et al., 2010). Perhaps symmetrically renewing divisions are not
detrimental to mammalian systems because mammalian niches
are not as constrained spatially, and mammalian stem cells are
often motile (Morrison and Spradling, 2008; Nakagawa et al.,
2010; Yoshida et al., 2007). So far, asymmetric division in
Drosophila testes correlates with optimal GSC function, as it
becomes less robust with aging (Cheng et al., 2008). Whether
symmetric divisions increase during aging has not been
examined, but it might occur because GSCs are thought to be
lost more frequently due to decreased maintenance cues (Boyle
et al., 2007; Wallenfang, 2007). Interestingly, depleting
STAT92E from GSCs displaces them from the hub, yet they are
not lost from the tissue. Instead, they associate with BMPproducing CySCs, which probably promote GSC renewal.
However, GSC division orientation is now randomized
(Leatherman and DiNardo, 2010); suggesting that their output is
composed of symmetric renewals and symmetric differentiation.
Furthermore, APC2 mutants that affect centrosome position and
E-cadherin mutants that have misoriented divisions still have
wild-type GSC numbers (Inaba et al., 2010). Together, these
observations suggest that the Drosophila testis stem cell niche
does not require invariant asymmetric GSC division outcomes.
As mammalian stem cells are thought to undergo symmetric
renewal in combination with stochastic differentiation, rather
than strict asymmetric divisions, GSCs in Drosophila may share
more aspects of stem cell behavior with mammalian systems
than has been previously assumed. We observe wild-type GSCs
losing niche attachment and directly differentiating, which is
consistent with reports that subsets of undifferentiated
spermatogonia in the mouse testes can directly differentiate
(Nakagawa et al., 2010; Yoshida et al., 2006). Although Fig. 2EL shows a lost GSC being replaced by a neighboring GSC
undergoing symmetric renewal, this was our only example where
these events are coupled together. Thus, stem cell loss and
symmetric renewal may occur stochastically in Drosophila
GSCs, as in the mouse testis (Klein et al., 2010). We also show
that differentiating spermatogonia revert into GSCs, which is
consistent with findings that differentiating spermatogonia can
contribute to the stem cell pool during reconstitution of
spermatogenesis in the mouse testes (Barroca et al., 2009;
Nakagawa et al., 2007; Nakagawa et al., 2010). Therefore, our
DEVELOPMENT
3374 RESEARCH ARTICLE
system provides an ideal platform for determining regulators of
stem cell loss and replacement in vivo that may also be
conserved in mammalian tissues.
Acknowledgements
We gratefully thank our colleagues who have supplied us with suggestions,
stocks and technical assistance. We thank Dr Margaret de Cuevas for editing,
Dr Geraldine Seydoux for providing comments on the manuscript, Drs Mohit
Prasad and Denise Montell for imaging advice, Dr Melanie Issigonis for
STAT29E immunostaining, and the Johns Hopkins University SOM Microscope
Facility for technical assistance. This work was supported by NIH grants
HD052937 and HD040307 (E.L.M.). Deposited in PMC for release after 12
months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.065797/-/DC1
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