DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 1381
Development 139, 1381-1390 (2012) doi:10.1242/dev.070052
© 2012. Published by The Company of Biologists Ltd
The receptor tyrosine phosphatase Lar regulates adhesion
between Drosophila male germline stem cells and the niche
Shrividhya Srinivasan1, Anthony P. Mahowald2 and Margaret T. Fuller1,*
SUMMARY
The stem cell niche provides a supportive microenvironment to maintain adult stem cells in their undifferentiated state. Adhesion
between adult stem cells and niche cells or the local basement membrane ensures retention of stem cells in the niche
environment. Drosophila male germline stem cells (GSCs) attach to somatic hub cells, a component of their niche, through Ecadherin-mediated adherens junctions, and orient their centrosomes toward these localized junctional complexes to carry out
asymmetric divisions. Here we show that the transmembrane receptor tyrosine phosphatase Leukocyte-antigen-related-like (Lar),
which is best known for its function in axonal migration and synapse morphogenesis in the nervous system, helps maintain GSCs
at the hub by promoting E-cadherin-based adhesion between hub cells and GSCs. Lar is expressed in GSCs and early
spermatogonial cells and localizes to the hub-GSC interface. Loss of Lar function resulted in a reduced number of GSCs at the
hub. Lar function was required cell-autonomously in germ cells for proper localization of Adenomatous polyposis coli 2 and Ecadherin at the hub-GSC interface and for the proper orientation of centrosomes in GSCs. Ultrastructural analysis revealed that in
Lar mutants the adherens junctions between hub cells and GSCs lack the characteristic dense staining seen in wild-type controls.
Thus, the Lar receptor tyrosine phosphatase appears to polarize and retain GSCs through maintenance of localized E-cadherinbased adherens junctions.
INTRODUCTION
Many adult stem cells that maintain and repair tissues throughout
the life of an organism reside in a specialized microenvironment,
termed the stem cell niche, that helps retain stem cells in an
undifferentiated state via short-range signaling (Jones and Wagers,
2008; Morrison and Spradling, 2008). The ability of adult stem
cells to recognize and establish adhesion to the niche is crucial for
long-term maintenance of adult stem cells. The ability to
recapitulate and harness these mechanisms will be important for
the use of adult stem cells in regenerative medicine. Attachment of
stem cells to the niche also plays a role in orienting the axis of stem
cell divisions, allowing for either asymmetric divisions to give rise
to stem cells and differentiating daughter cells, or symmetric
divisions to expand the stem cell pool (Knoblich, 2008; Lin, 2008;
Morrison and Kimble, 2006). Cell adhesion molecules such as
cadherins and integrins have been identified as being upregulated
or important in adult stem cells in several systems (Ellis and
Tanentzapf, 2010; Raymond et al., 2009), underscoring the
importance of stem cell-niche or stem cell-extracellular matrix
attachments in maintaining niche structure, retaining stem cells in
the niche, and orienting stem cell divisions (Marthiens et al., 2010).
E-cadherin-based stem cell-niche adhesion has been shown to
play an important role in the maintenance of Drosophila germline
stem cells (GSCs) in their niche (Song et al., 2002; Voog et al.,
2008; Wang et al., 2006). Two populations of stem cells reside at
the apical tip of Drosophila testes: GSCs, which differentiate to
1
Department of Developmental Biology, Stanford University, School of Medicine,
Stanford, CA 94305, USA. 2Department of Molecular Genetics and Cell Biology,
University of Chicago, Chicago, IL 60637, USA.
*Author for correspondence ([email protected])
Accepted 16 January 2012
give rise to sperm; and somatic cyst stem cells (CySCs), which
constitute an important component of the GSC niche (Leatherman
and Dinardo, 2008; Leatherman and Dinardo, 2010) and give rise
to the cyst cells that envelop and ensure the proper differentiation
of germ cells (Kiger et al., 2000; Sarkar et al., 2007; Tran et al.,
2000). GSCs and CySCs are associated with, and organized
around, a tight cluster of postmitotic somatic cells called the hub
(Hardy et al., 1979). The hub contributes to the niche by secreting
the cytokine Unpaired (Upd; Outstretched – FlyBase), which
locally activates the Janus kinase-Signal transducer and activator
of transcription (JAK-STAT) pathway in the stem cells,
maintaining GSC attachment to the hub and preventing CySC
differentiation (Kiger et al., 2001; Leatherman and Dinardo, 2008;
Leatherman and Dinardo, 2010; Tulina and Matunis, 2001).
GSCs attach to the hub cells through adherens junctions, and
components of the adherens junctions, which include E-cadherin
(Shotgun – FlyBase) and Armadillo (Arm; fly -catenin), are
concentrated at the interface between GSCs and hub cells as well
as between adjacent hub cells (Yamashita et al., 2003). Marked
GSCs that lack E-cadherin function, induced by mitotic
recombination, fail to be maintained at the hub (Voog et al., 2008).
E-cadherin in GSCs might contribute to stem cell maintenance by
promoting GSC adhesion to the hub, so that GSCs continue to
receive Upd signals and are flanked by CySCs. The adherens
junctions between GSCs and hub cells also polarize GSCs by
recruiting one of the fly homologs of mammalian adenomatous
polyposis coli (APC), Apc2, to the cortex adjacent to the hub-GSC
interface in GSCs (Yamashita et al., 2003). The localized adherens
junctions and Apc2, in turn, maintain the stereotypic orientation of
the mother centrosome toward the hub, which sets up the strictly
oriented mitotic spindle and enables the normally asymmetric
outcome of GSC divisions (Inaba et al., 2010; Yamashita et al.,
2003; Yamashita et al., 2007). When a GSC divides, one of the
daughter cells inherits the adhesion with the hub and retains stem
DEVELOPMENT
KEY WORDS: Adult stem cells, Drosophila male germline, Receptor tyrosine phosphatase Lar, Stem cell-niche adhesion
1382 RESEARCH ARTICLE
MATERIALS AND METHODS
Fly husbandry and stocks
All stocks were grown on standard cornmeal/molasses/yeast/agar medium at
25°C unless otherwise stated. Strains are described in FlyBase
(http://flybase.org) or otherwise specified. y1w1 flies were used as wild-type
controls, except that Oregon R flies were used as wild-type controls for the
ultrastructural analysis. Flies used in this study include the following stocks
carrying Lar alleles: (1) w; Lar451, FRT40A/CyO, P{Ubi-GFP.S65T}, an
allele with an uncharacterized molecular lesion [gift from T. Clandinin
(Clandinin et al., 2001)]; (2) w; Lar2127, FRT40A/CyO, P{Ubi-GFP.S65T},
a loss-of-function allele with a nonsense mutation that truncates Lar protein
in the sixth fibronectin domain (Maurel-Zaffran et al., 2001); and (3)
Df(2L)E55, rdo1, hk1, LarE55, pr1/CyO, P{Ubi-GFP.S65T}, a deficiency that
deletes the 5⬘ portion of the Lar gene [Bloomington Drosophila Stock Center
(BDSC)]; and the following stocks carrying ena alleles: (1) w; FRTG13,
ena210/CyO and (2) w; FRTG13, ena23/CyO; both alleles harbor point
mutations in domains crucial for Ena function (Ahern-Djamali et al., 1998).
The stocks (1) y1, w1, hs-FLP122; FRT40A, Ubi-GFP/CyO, (2) y1, w1, hsFLP122; FRT40A, arm-lacZ/CyO and (3) y1, w1, hs-FLP122; FRTG13, UbiGFP/CyO (BDSC) were paired with the above stocks for inducing mutant
clones or paired with FRT40A/CyO or FRTG13/CyO (BDSC) for inducing
wild-type control clones. nanos-GAL4-VP16 (NGVP16) (gift from R.
Lehmann, Skirball Institute, NY, USA) (Van Doren et al., 1998) and C587GAL4 (gift from S. Hou, NCI, Frederick, MD, USA) drivers were used to
express UAS-GFP--Tubulin in early germ cells and cyst cells, respectively.
To drive expression of UAS transgenes in wild-type or Lar mutant early
germ cells, sco/CyO; NGVP16 (BDSC) or Lar2127, FRT40A/CyO; NGVP16
virgin females were crossed to Lar451, FRT40A/CyO; UAS-DEFL #6-1. The
UAS-DEFL #6-1 line [containing E-cadherin-GFP downstream of UAS
sequences (Oda and Tsukita, 1999)] was acquired from the Drosophila
Genomics Research Center, Kyoto, Japan.
Immunofluorescence
Testes were dissected in PBS and fixed for 20 minutes in 4% formaldehyde
in PBS. To stain with antibodies, testes were permeabilized for 1 hour in
PBS with 0.3% Triton X-100 and 0.6% sodium deoxycholate at room
temperature, washed in PBST (0.1% Triton X-100 in PBS), blocked in
PBST with 3% BSA for 1 hour at room temperature or overnight at 4°C
and either incubated overnight at 4°C or for 2 hours at room temperature
in primary antibody diluted in PBST with 3% BSA. The testes were
washed in PBST, incubated in appropriate fluorochrome-conjugated
secondary antibodies raised in donkey (1:500; Jackson Labs) for 2 hours
at room temperature, again washed with PBST, and mounted in Vectashield
containing DAPI (Vector Laboratories).
Antibodies used in this study include those obtained from Drosophila
Studies Hybridoma Bank against the extracellular N-terminal domain of
Lar [mouse, 1:10 (Sun et al., 2000)], Arm [mouse, 1:10 (Riggleman et al.,
1990)], Spectrin [mouse, 1:10 (Dubreuil et al., 1987)], Enabled [mouse,
1:10 (Bashaw et al., 2000)], Fas III [mouse, 1:10 (Patel et al., 1987)] and
E-cadherin [rat, 1:10 (Oda et al., 1994)]. Other antibodies used include
those against -Tubulin (mouse, 1:50; Sigma), -galactosidase (mouse,
1:100; Sigma), Traffic jam [rat and guinea pig, 1:500; a gift from D. Godt
(Li et al., 2003)], Vasa (goat, 1:50-1:100; DC-13 from Santa Cruz Biotech),
green fluorescent protein (GFP) (rabbit, 1:500-1:1000, Invitrogen; sheep,
1:1000, AbD Serotec), Apc2 [rabbit, 1:5000; a gift from M. Bienz (Yu et
al., 1999)], Zinc finger homeodomain 1 (Zfh1) (rabbit, 1:5000; a gift from
R. Lehmann) and Stat92E (rabbit, 1:1000; a gift from E. Bach, NYU
School of Medicine, NY, USA) (Flaherty et al., 2010).
Images were captured using a Leica SP2 AOBS confocal laser-scanning
microscope and processed using Adobe Photoshop CS5 software. GSCs
were scored either as Vasa-positive cells adjacent to the hub (detected using
Arm or E-cadherin) and containing dot spectrosome (detected using
Spectrin) or as cells that are negative for Traffic jam staining and adjacent
to the hub.
Clonal analysis
Homozygous mutant clones in a heterozygous background were induced
using Flippase (FLP)-mediated mitotic recombination. y,w, hs-flp122;
FRT40A, Ubi-GFP virgin females were crossed to w; FRT40A or w; Lar451,
FRT40A/CyO or w; Lar2127, FRT40A/CyO males. Similarly, y,w, hs-flp122;
FRTG13, Ubi-GFP virgin females were crossed to w; FRTG13 or w; ena23,
FRTG13/CyO or w; ena210, FRTG13/CyO males to generate homozygous
ena mutant clones. To compare E-cadherin-GFP localization in GSCs that
were heterozygous and homozygous mutant for Lar function, hs-FLP122;
FRT40A, arm-lacZ/CyO; NGVP16 virgins were crossed to either w; Lar451,
FRT40A/CyO; UAS-DEFL #6-1 or w; Lar2127, FRT40A/CyO; UAS-DEFL
#6-1 males. The progeny were grown until the pupal stage at 25°C, then heat
shocked at 37°C for 2 hours on two consecutive days. Testes were dissected
from flies of appropriate genotypes on different days after the second day of
heat shock and examined for the presence of GFP-negative clones in germ
cells by fluorescence microscopy. Lar and ena mutant GSC clones were
identified by immunostaining with antibodies against GFP, Vasa and Arm.
CySC clones were identified with antibodies against GFP and Traffic jam.
E-cadherin-GFP expression in Lar mutant GSC clones was detected using
antibodies against GFP and counterstained with anti--galactosidase to
identify marked clones and with anti-E-cadherin and anti-Traffic jam to
identify GSCs. All clonal analyses were repeated independently at least three
times and the data are shown as an average of the independent experiments.
Analysis of Stat92E accumulation, Apc2 localization, centrosome orientation
and E-cadherin-GFP localization were performed in testes 5 days post-clone
induction, which is 3 days after eclosion of adult males.
Ultrastructural analysis of adherens junctions in GSCs
Testes were dissected and incubated in fixative (2% glutaraldehyde in 0.1
M cacodylate buffer at pH 7.2) at room temperature for 15 minutes, then
transferred to ice in this solution for up to 1 hour. Testes were postfixed
with 2% OsO4 for 2 hours at 4°C, then transferred to 1% uranyl acetate
overnight at 4°C. Tissues were dehydrated in ethanol and embedded in
Epon resin. Serial sections (80 nm) were picked up on formvar-covered
wire loops, transferred to slot grids and stained with uranyl acetate and lead
citrate. Sections were viewed with a Technai F30 electron microscope at
300 kV and images recorded on a Gatan digital camera. The membrane
interface for each hub-GSC combination was scanned over multiple
sections to identify the presence of adherens junctions.
DEVELOPMENT
cell fate. The daughter displaced from the hub becomes a
gonialblast and initiates the differentiation program, embarking on
four rounds of transit-amplification divisions with incomplete
cytokinesis, then switching to spermatocyte fate, meiosis and
spermatid differentiation (Davies and Fuller, 2008). The gonialblast
and the transit-amplifying spermatogonial cells have the capacity
to dedifferentiate to GSC fate and reoccupy the niche (Brawley and
Matunis, 2004; Cheng et al., 2008; Sheng et al., 2009), although
the mechanism by which dedifferentiating germ cells recognize and
form attachment to the niche is not known.
Here we discover a new player in the mechanism that maintains
GSCs in their niche in Drosophila testes: the receptor tyrosine
phosphatase Leukocyte-antigen-related-like (Lar), which is best
known for its role in axonal migration, target selection and synapse
formation in the nervous system (Baker and Macagno, 2010;
Chagnon et al., 2004; Johnson and Van Vactor, 2003; Sethi et al.,
2010). Drosophila Lar is a transmembrane type IIA receptor
protein tyrosine phosphatase that contains N-terminal
immunoglobulin-like domains and membrane-proximal fibronectin
type III repeats in its extracellular region, a single-pass
transmembrane domain and two tandem repeats of protein tyrosine
phosphatase domains in its cytoplasmic region (Chagnon et al.,
2004). Lar expression is upregulated in a microarray analysis of
testes with elevated numbers of early germ and cyst cells (D. L.
Jones, E. L. Davies and M.T.F., unpublished results). Our data
suggest that Lar, expressed in GSCs and early germ cells, maintains
GSCs at the hub by promoting E-cadherin-based adhesion between
hub cells and GSCs.
Development 139 (8)
RESULTS
Lar is expressed in early germ cells and localizes
to the hub-GSC interface
Immunofluorescence analysis using antibodies against Lar revealed
expression of Lar protein at the apical tip of adult (Fig. 1A,A⬘) and
third instar larval (data not shown) wild-type testes. Examination
of testes in which early germ cells were marked by the expression
of GFP-tagged -Tubulin revealed anti-Lar immunofluorescence
in GSCs, where it localized to the membrane at the interface
between the hub and GSCs (arrowheads, Fig. 1A,A⬘,B⬘). Lar was
also detected in two- and four-cell spermatogonial cysts, with only
a weak signal in eight-cell spermatogonial cysts, where it localized
to the membrane between mitotic sister germ cells of a cyst
(arrows, Fig. 1A,A⬘). Lar signal was below detection in CySCs
(arrowheads, Fig. 1C⬘), somatic cyst cells, and in germ cells in later
stages of differentiation.
Lar helps maintain GSCs at the hub
Adult escapers of a heteroallelic combination of Lar alleles,
Lar451/Lar2127, survived for 2-3 days post-eclosion. Examination of
testes isolated from newly eclosed Lar451/Lar2127 males by phasecontrast microscopy revealed a normal progression of germ cell
differentiation, similar to that seen in wild-type testes (data not
shown), suggesting that Lar function is not necessary for germ cell
differentiation. Although reproductive tracts from Lar mutant
males had motile sperm, Lar451/Lar2127 males were sterile. Lar
Fig. 1. Lar is expressed in early germ cells and localizes to the
hub-GSC interface. (A,A⬘) Apical tip of adult wild-type Drosophila
testes, with early germ cells marked by expression of GFP--Tubulin
(green) under control of NGVP16, immunostained with anti-Lar (red, A;
white, A⬘), co-stained with anti-GFP (green) and DAPI (blue).
Arrowheads indicate Lar localized to the hub-GSC interface in GSCs.
Arrows indicate Lar at the membrane between sister germ cells in twoand four-cell spermatogonial cysts. (B,B⬘) High-magnification view of
GSCs marked by expression of GFP--Tubulin (green) under control of
NGVP16, immunostained for Lar (red, B; white, B⬘). Arrowheads
indicate Lar localization at the hub-GSC interface. (C,C⬘) Cyst cells
marked by the expression of GFP--Tubulin (green) under the control
of C587-GAL4, immunostained for Lar (red, C; white, C⬘). Arrowheads
indicate the absence of Lar localization at the interface between hub
and CySCs. The star indicates the Hub. Scale bars: 25 m.
RESEARCH ARTICLE 1383
staining was not detected at the apical tip in testes isolated from 0to 1-day-old Lar451/Lar2127 adult males (Fig. 2B,B⬘) or from larval
testes (data not shown), suggesting that the antibodies used
specifically detected Lar protein in wild-type testes (Fig. 2A,A⬘)
and that the allelic combination is a protein null.
Examination of Lar mutant adult or larval testes immunostained
with antibodies against Vasa to mark germ cells, Zfh1 to mark early
cyst cell nuclei and Arm to mark the hub, revealed a defect in GSC
maintenance. The mutant testes had fewer GSCs adjacent to the
hub compared with wild type (Fig. 2C,D; data not shown for larval
testes). In addition, although in wild-type testes Zfh1-positive
nuclei of cyst cells were found one GSC diameter away from the
hub (arrows, Fig. 2C⬘) (Leatherman and Dinardo, 2008), in Lar
mutant testes Zfh1-positive cyst cells were positioned adjacent to
the hub in regions where GSCs were lacking (arrows, Fig. 2D⬘).
Quantitative assessment revealed that wild-type adult testes had
an average of 8.06±1.58 GSCs per testis, whereas testes isolated
from Lar451/Lar2127 mutant adults averaged 3.35±1.52 GSCs per
testis (Fig. 2E). A similar reduction in GSC numbers was also
observed in Lar451/Lar2127 mutant larval testes (5±1.5 GSCs per testis
in Lar mutants compared with 9.1±1.2 GSCs per testis in wild type;
data not shown), suggesting that fewer GSCs are maintained adjacent
to the hub. Testes isolated from Lar2127/Df(2L)E55 adult escapers
also averaged 3.14±1.29 GSCs per testis (Fig. 2E). Interestingly,
adult testes from Lar mutant alleles heterozygous with wild type
(Lar451/+ or Lar2127/+) also showed a slight, but statistically
significant, reduction in GSC numbers compared with wild-type
controls (Fig. 2E), suggesting that Lar function to maintain GSCs
might be dose dependent. The number of Zfh1-positive cyst cell
nuclei within one cell diameter of the hub was slightly reduced in
testes isolated from Lar451/Lar2127 mutant adults (13±3.1 CySCs per
testis; n52 testes) compared with wild type (16.95±3.4 CySCs per
testis; n60 testes) (P<0.0001, two-tailed Student’s t-test). Since Lar
protein expression was not detected in CySCs (Fig. 1C), the slight
reduction in the number of Zfh1-positive cells in Lar mutants might
be due to indirect effects on the cyst cells.
Lar functions cell-autonomously to maintain GSCs
Clonal analysis revealed that Lar function is required cellautonomously for GSC maintenance, but not for germline
differentiation. Negatively marked homozygous Lar mutant clones
were generated for two different Lar alleles using the FLP-FRTbased mitotic recombination system in a Lar heterozygous
background. Lar protein was not detected in GSCs homozygous for
either the Lar451 or the Lar2127 allele by immunostaining 5 days postclone induction (PCI) (supplementary material Fig. S1A,B),
suggesting that both alleles of Lar are protein null. By day 3 PCI,
only 46.5% of testes carrying the Lar451 allele and 38% of testes
carrying the Lar2127 allele had at least one marked homozygous
mutant GSC compared with 85.6% testes in the wild-type control
(Fig. 2F). Under the same conditions of clone induction, at day 3 PCI
germ cells in later stages of differentiation that were homozygous
mutant for either of the two Lar alleles (GFP negative) were present
at levels similar to the wild-type controls (Fig. 2G), suggesting that
the low percentage of testes with Lar mutant GSCs was not due to a
defect in clone induction. GFP-negative spermatocytes and round or
elongated spermatids were present 7 days PCI (supplementary
material Fig. S1C-H), suggesting that, at least until the spermatid
stage, Lar function might not be required for germ cell
differentiation. The percentage of testes having marked Lar451 or
Lar2127 mutant GSCs steadily decreased to ~20% by day 15 PCI,
suggesting that Lar functions cell-autonomously to maintain GSCs.
DEVELOPMENT
Stem cell-niche adhesion by Lar
1384 RESEARCH ARTICLE
Development 139 (8)
Fig. 2. Lar functions cell-autonomously to maintain
GSCs at the hub. (A-B⬘) Apical tips of 0- to 1-day-old
adult Drosophila testes stained with antibodies against Lar
(green in A,B; white in A⬘,B⬘), co-stained for E-cadherin
(red) to mark the hub and with DAPI (magenta) to mark
DNA in (A) wild type and (B) Lar451/Lar2127 mutant. GSCs
are outlined. Arrows indicate that Lar is expressed and
localizes to the hub-GSC interface in wild type (A⬘), but
not in the mutant (B⬘). (C-D⬘) Wild-type (C,C⬘) and
Lar451/Lar2127 mutant (D,D⬘) adult testes immunostained
with anti-Vasa (red) to label germ cells, anti-Arm
(magenta) to mark the hub and (C⬘,D⬘, arrows) anti-Zfh1
(green) to label early cyst cell nuclei. (E) Quantitation of the
number of GSCs adjacent to the hub per testis, reported
as average ± s.d. N, the number of testes scored for each
genotype. P-values of Student’s t-test (two-tailed) are
shown. (F-H) Germline clonal analysis of the Lar mutant
alleles Lar451 (red) and Lar2127 (green) compared with
FRT40A controls (blue). The reduction in the number of
GFP-negative Lar mutant GSCs maintained (F), the
decrease in Lar mutant spermatocyte cysts and spermatids
(G) and the maintenance of CySCs (H) scored at different
time points post-clone induction (PCI). Numbers next to
the data points indicate the number of testes scored for
each genotype at the different time points. The same
numbers of testes were scored for F and H. Data are
average ± s.d. Scale bars: 25 m.
loss-of-function alleles ena23 and ena210 were generated using the
FLP-FRT system and scored for maintenance at different time
points PCI. At 2 days PCI, 83.5% of testes carrying the ena23 allele
and 90% of testes carrying the ena210 allele had at least one marked
homozygous mutant GSC, comparable to wild-type controls (100%
testes) (Fig. 3C). The percentage of testes with ena210 homozygous
mutant GSC clones dropped from 90% at day 2 PCI to 56% at day
3 and to 45.5% by day 15 PCI, as compared with 100% (day 3) to
95.2% (day 15) in wild-type controls. The percentage of testes
containing marked GSC clones homozygous mutant for ena23 did
not drop significantly over time, remaining at 73.5% at day 15 PCI.
Thus, unlike marked Lar mutant GSC clones, which were steadily
lost over time (Fig. 2F), ena mutant GSCs, after an initial loss for
one of the alleles examined, were maintained, suggesting that ena
might not be the sole or key downstream target of Lar for the
maintenance of GSCs at the hub.
Lar function is not required for Stat92E
accumulation in GSCs
Immunofluorescence staining for expression of Stat92E, an
indicator of JAK-STAT signaling activity (Chen et al., 2002),
revealed that Lar was not required for the accumulation of Stat92E
protein in the GSCs that remained next to the hub. In wild-type
testes, Stat92E is expressed in GSCs, CySCs and, to a lesser extent,
in some gonialblasts (Fig. 4A,A⬘). The GSCs remaining at the hub
in Lar451/Lar2127 adult testes had Stat92E protein in the cytoplasm
and the nucleus (Fig. 4B,B⬘). Stat92E protein was also detected in
DEVELOPMENT
By day 15 PCI, similar to the reduction in the percentage of testes
with marked Lar mutant GSC clones, the percentage of testes with
negatively marked clones of spermatocytes and later stages of germ
cell differentiation also dropped to ~20%, likely reflecting the
corresponding loss of marked GSCs (Fig. 2G). Consistent with the
inability to detect Lar protein by immunostaining in CySCs and cyst
cells, Lar mutant CySC clones were maintained at similar levels as
wild-type control clones (Fig. 2H), indicating that Lar function is not
required cell-autonomously for CySC maintenance.
A known substrate of the phosphatase activity of Lar, Enabled
(Ena), localizes to the hub-GSC interface. However, Ena is not
absolutely required for GSC maintenance. Drosophila Ena, a
member of the Ena/VASP family of filamentous actin (F-actin)
regulators, physically and genetically interacts with Lar and serves
as substrate for Lar in vitro (Wills et al., 1999). Immunostaining
with anti-Ena in wild-type testes revealed localization of Ena to the
hub-GSC interface (arrows, Fig. 3A⬘), similar to Lar localization.
Examination of Ena localization in Lar451/Lar2127 mutant GSCs
revealed that Ena failed to localize at the hub-GSC interface in
about half the GSCs (arrowhead, Fig. 3B⬘). Of the 145
Lar451/Lar2127 mutant GSCs scored, 51.7% had Ena distribution
throughout the GSC cortex, as compared with 18.7% in wild-type
GSCs (171 wild-type GSCs were scored).
To test the possibility that Lar might act through Ena in GSCs,
with failure of Ena localization at the hub-GSC interface a possible
cause of GSC loss, the maintenance of GSCs upon loss of ena
function was determined. GSCs homozygous mutant for the ena
RESEARCH ARTICLE 1385
Fig. 3. Ena function is not absolutely required for GSC
maintenance. (A-B⬘) 0- to 1-day-old wild-type (A,A⬘) and Lar451/Lar2127
mutant (B,B⬘) adult Drosophila testes immunostained with anti-Ena
(green, A,B; white A⬘,B⬘), co-stained with anti-Vasa (red) to mark germ
cells and anti-E-cadherin (magenta) to mark the hub. Arrows (A⬘,B⬘)
indicate Ena localization to the hub-GSC interface. Arrowhead (B⬘)
indicates an example where Ena failed to localize at the hub-GSC
interface in a Lar451/Lar2127 mutant GSC. The star indicates the Hub.
(C) Maintenance of GFP-negative ena23 (red) and ena210 (green) mutant
GSCs scored at different time points PCI compared with FRTG13
controls (blue). Numbers next to the data points indicate the number of
testes scored for each genotype at the different time points. Data are
average ± s.d. Scale bars: 25 m.
Fig. 4. Lar function is not required for Stat92E accumulation in
GSCs. (A-B⬘) 0- to 1-day-old wild-type (A,A⬘) and Lar451/Lar2127 mutant
(B,B⬘) adult Drosophila testes immunostained with anti-Stat92E (red,
A,B; white, A⬘,B⬘) and anti-Vasa (green) antibodies. (A,B) GSCs (arrows)
and CySCs (arrowheads) express Stat92E. (B⬘) A germ cell (long arrow)
displaced from the hub expresses Stat92E. (C-D⬙) Expression of Stat92E
(red, C,D; white, C⬘,D⬘) in homozygous Lar451 (arrows, C-C⬙) and
Lar2127 (arrows, D-D⬙) mutant GSC clones 5 days PCI as compared with
heterozygous control GSCs (arrowheads). Homozygous mutant GSCs
(green outline in C⬙,D⬙) are detected by the absence of anti-GFP
immunostaining (green, C,D; white C⬙,D⬙). The star indicates the Hub.
Scale bars: 25 m.
Vasa-positive cells displaced away from the hub in Lar451/Lar2127
testes (long arrow, Fig. 4B⬘), suggesting that the loss of GSCs was
not due to loss of JAK-STAT signaling in Lar mutants. Similarly,
Stat92E levels in Lar451 or Lar2127 homozygous mutant GSCs were
similar to those in the neighboring heterozygous GSCs (Fig. 4C,D).
Relative levels of Stat92E protein in CySCs versus GSCs were
similar in Lar mutants and wild-type controls (arrowheads, Fig.
4A,B). Loss of GSCs in Lar mutants was unlikely to be due to
changes in JAK-STAT-mediated competition by CySCs as
previously described (Issigonis et al., 2009), where loss of
Suppressor of cytokine signaling at 36E function, a negative
regulator of the JAK-STAT pathway, resulted in upregulation of
Stat92E in CySCs relative to neighboring GSCs, causing CySCs to
outcompete GSCs for attachment to the hub.
adjacent to the hub in 0- to 1-day-old Lar451/Lar2127 testes, the
Apc2 detected was distributed around the GSC cortex, rather than
localized to the hub-GSC interface (Fig. 5B,B⬘,E). By contrast,
only 21.2% of GSCs in wild-type testes had anti-Apc2 signal
distributed around the GSC cortex (Fig. 5E). Furthermore, 76.6%
of homozygous Lar451 and 57% of homozygous Lar2127 mutant
GSCs at 5 days PCI also exhibited Apc2 distributed over the GSC
cortex (Fig. 5C,D,F) compared with 27.3% and 23.8% of the
neighboring heterozygous control GSCs from the same testes,
respectively (Fig. 5F). These observations suggest that Lar function
might cell-autonomously contribute to the correct localization of
Apc2 to the hub-GSC interface.
Mislocalization of Apc2 in Lar mutant GSCs remaining at the
hub might lead to an increased frequency of misoriented
centrosomes in those cells. GSCs with two centrosomes in Lar
mutant testes (Fig. 5G, supplementary material Fig. S2A,B) or in
homozygous Lar mutant GSC clones induced in heterozygous
animals (Fig. 5H) showed an increased percentage of misoriented
centrosomes compared with their controls. In 0- to 1-day-old
Lar451/Lar2127 adults, neither of the centrosomes was adjacent to
the hub-GSC interface in 37.8% of the total GSCs with two
centrosomes, compared with 11.9% of GSCs in wild-type controls
(Fig. 5G). Similarly, in homozygous Lar451 and Lar2127 mutant
Localization of Apc2 to the hub-GSC interface and
proper orientation of centrosomes in GSCs require
Lar function
The GSCs remaining at the hub in Lar mutant testes tended to
exhibit abnormal localization of Apc2 protein. Drosophila Apc2 is
enriched at the cortical region of GSCs adjacent to the hub-GSC
interface (Fig. 5A,A⬘) (Yamashita et al., 2003). Immunostaining
with anti-Apc2 revealed that, in 57.5% of the GSCs that remained
DEVELOPMENT
Stem cell-niche adhesion by Lar
1386 RESEARCH ARTICLE
Development 139 (8)
Fig. 5. Apc2 localization and centrosome orientation
are altered in Lar mutant GSCs. (A-B⬘) Apc2 (white, A,B;
red, A⬘,B⬘) localization in wild-type (A,A⬘) and Lar451/Lar2127
mutant (B,B⬘) GSCs from 0- to 1-day-old adult Drosophila
testes. Anti-Vasa (green, A⬘,B⬘) marks germ cells.
(C-D⬙) Localization of Apc2 (white, C,D; red, C⬘,D⬘) in
homozygous Lar451 (arrows, C-C⬙) and Lar2127 (arrows, DD⬙) mutant GSC clones 5 days PCI as compared with
neighboring heterozygous control GSCs (arrowheads).
Homozygous mutant GSCs (green outline, C⬙,D⬙) are
detected by the absence of anti-GFP immunostaining
(green, C⬘,D⬘; white C⬙,D⬙). Orange solid lines indicate
cortical sites in GSCs with prominent Apc2 localization. The
star indicates the Hub. (E,F) Quantitation of GSCs with Apc2
localization to the hub-GSC interface versus GSCs with
Apc2 localization distributed more broadly over the GSC
cortex in (E) GSCs from wild-type versus Lar451/Lar2127
mutant testes and (F) heterozygous and homozygous
FRT40A, Lar451 and Lar2127 GSCs. (G,H) Quantitation of
GSCs with oriented versus misoriented centrosomes in
GSCs with two centrosomes (G) from wild-type versus
Lar451/Lar2127 mutant testes and (H) in heterozygous and
homozygous FRT40A, Lar451 and Lar2127 GSCs. N, number
of GSCs scored for each genotype. P-values of Fisher’s exact
test (two-tailed) are shown. Scale bars: 25 m.
Defective adherens junctions between hub cells
and GSCs in Lar mutants
The hub-GSC interface, where Lar localizes, is enriched in adherens
junctions (Yamashita et al., 2003), raising the possibility that Lar
affects hub-GSC adhesion. Ultrastructural analysis revealed
abnormal adherens junctions between GSCs and hub cells in Lar
mutant testes. Adherens junctions between GSCs and the hub, as
analyzed for eight GSCs from three wild-type testes by
transmission electron microscopy, appeared as electron-dense
structures between the opposing cell membranes (Fig. 6A). By
contrast, of the nine Lar mutant GSCs from five testes analyzed,
none had strong electron-dense adherens junctions between hub and
GSCs (Fig. 6B). Instead, in regions where the plasma membranes
between hub cell and an adjacent GSC were closely opposed, the
intervening material displayed only weakly electron-dense staining
compared with the wild type (compare magnified images 1, 2 and 3
in Fig. 6A with 4, 5 and 6 in 6B), suggesting that adherens junctions
between hub and GSCs in Lar mutant testes might be weak
compared with those in wild-type controls.
Interestingly, the membranes between adjacent hub cells also
exhibited weakly electron-dense staining in Lar mutant testes as
compared with wild-type controls (data not shown). The localization
of E-cadherin (supplementary material Fig. S3B) and Arm (Fig. 2D⬘)
between hub cells of Lar451/Lar2127 mutant testes appeared normal.
However, Fas III staining, which is localized to the membrane
between hub cells in wild-type testes (supplementary material Fig.
S3A,A⬘), was reduced in Lar451/Lar2127 mutant testes (supplementary
material Fig. S3B,B⬘), suggesting a possible role for Lar in the hub
in addition to its germ cell-autonomous roles.
Retention of GFP-tagged E-cadherin at the hubGSC interface is affected in Lar mutant GSCs
The reduction in the number of GSCs retained at the hub, the
mislocalization of Apc2 around the GSC cortex and the abnormal
appearance of adherens junctions by ultrastructural analysis in Lar
loss-of-function mutants suggest that Lar might help maintain hubGSC adhesion, possibly through effects on E-cadherin, a major
structural component of adherens junctions (Harris and Tepass,
2010) that is present at the membranes between hub and GSCs in
wild-type testes (arrow, Fig. 6C⬘) (Yamashita et al., 2003).
Although anti-E-cadherin staining revealed localization at the hubGSC interface in some GSCs in 0- to 1-day-old Lar451/Lar2127
mutant testes, similar to in wild type (arrow, Fig. 6D⬘), levels of Ecadherin staining were lower or missing altogether in other Lar
mutant GSCs (arrowhead, Fig. 6D⬘).
As E-cadherin present on GSC membranes is difficult to
distinguish from that present on the adjacent hub cell membranes
when the two are juxtaposed at the hub-GSC interface, the
DEVELOPMENT
GSCs at 5 days PCI, 52% and 48.8% of homozygous mutant GSCs
exhibited misoriented centrosomes, respectively, as compared with
14.4% and 12% in neighboring heterozygous GSCs (Fig. 5H).
Stem cell-niche adhesion by Lar
RESEARCH ARTICLE 1387
Fig. 6. Abnormal adherens junctions between hub cells and GSCs
in Lar mutants. (A,B) Electron micrographs show the interface
between hub cells and GSCs in (A) wild-type and (B) Lar451/Lar2127
mutant Drosophila testes. The dashed blue line lies adjacent to the
membrane between a hub cell on the left and a GSC on the right. Red
boxes enclose adherens junctions in wild type (1-3) and possible
adherens junctions in Lar mutants (4-6). Magnified images of the areas
enclosed by the red boxes are shown beneath. (C-D⬘) 0- to 1-day-old
wild-type (C,C⬘) and Lar451/Lar2127 mutant (D,D⬘) adult testes
immunostained with anti-E-cadherin (green, C,D; white C⬘,D⬘), costained with anti-Vasa (red) to mark germ cells. Arrows (C⬘,D⬘) indicate
that E-cadherin is present at the hub-GSC interface. Arrowhead
indicates a Lar451/Lar2127 mutant GSC that lacks E-cadherin at the hubGSC interface. Scale bars: 500 nm in A,B; 25 m in C-D⬘.
localization of E-cadherin in GSCs was examined by expressing
UAS-E-cadherin-GFP specifically in the germ cells under the
control of the NGVP16 driver. E-cadherin-GFP expression by the
UAS-GAL4 system in Lar mutant GSCs failed to rescue the loss
of GSCs in Lar451/Lar2127 mutant testes. In addition, E-cadherin
failed to localize at the membrane in Lar mutant GSCs. When Ecadherin-GFP was expressed in wild-type (Fig. 7A,A⬘) or Lar
heterozygous (Fig. 7B,B⬘) GSCs using the NGVP16 driver, the
GFP-tagged protein localized to the hub-GSC interface,
consistent with the localization of adherens junctions between the
hub and GSCs (Inaba et al., 2010; Yamashita et al., 2003). By
contrast, E-cadherin-GFP did not localize to the hub-GSC
interface in the GSCs that remained next to the hub in Lar mutant
testes. Of the 87 GSCs analyzed from 27 Lar mutant testes, only
six GSCs exhibited cortical expression of E-cadherin-GFP. Of
these, three GSCs exhibited E-cadherin-GFP localization to the
hub-GSC interface. Strikingly, in the remaining 81 Lar mutant
GSCs no E-cadherin-GFP expression was detected (Fig. 7C,C⬘)
on either the GSC plasma membrane or in the cytoplasm. This
lack of E-cadherin-GFP in Lar mutant GSCs was not due to an
inability of NGVP16 to drive expression, as UAS-EGFP was
expressed under the control of NGVP16 in Lar mutant GSCs
(data not shown). Further, E-cadherin-GFP expression and
localization were affected in Lar451 or Lar2127 homozygous
DISCUSSION
This work identifies a role for the transmembrane receptor tyrosine
phosphatase Lar, acting cell-autonomously to maintain attachment
of Drosophila male GSCs to the hub. Lar function appears to
promote the maintenance of robust adherens junctions between
GSCs and hub cells and to localize and/or retain E-cadherin at the
hub-GSC interface. Consistent with the recently demonstrated
requirement for E-cadherin to polarize GSCs by localizing Apc2 at
the hub-GSC interface and to establish centrosome orientation in
GSCs (Inaba et al., 2010), Apc2 was often mislocalized around the
GSC cortex and centrosomes were often misoriented in Lar mutant
GSCs.
Lar may function in parallel with other cell signaling pathways
that are important for maintaining attachment of GSCs to the hub.
Activation of the JAK-STAT pathway in Drosophila male GSCs
maintains GSCs at the hub (Leatherman and Dinardo, 2010).
However, Stat92E protein levels appeared normal in Lar mutant
GSCs, suggesting that Lar function is not required for activation of
the JAK-STAT pathway. The Rap1 GTPase/Rap1 guanine
nucleotide exchange factor (Rap-GEF) signaling pathway also
regulates hub-GSC adhesion (Wang et al., 2006). Like Lar mutants,
Rap-GEF mutants have impaired adherens junctions at the hubGSC interface resulting in GSC loss. However, Rap-GEF function
is required in hub cells, whereas Lar functions in GSCs to promote
hub-GSC adhesion. Interestingly, expression of E-cadherin-GFP in
either hub cells or GSCs of Rap-GEF mutants resulted in wild-type
numbers of GSCs and restored E-cadherin localization at the hubGSC interface (Wang et al., 2006), whereas expression of Ecadherin-GFP in Lar mutant GSCs did not rescue the loss of GSCs,
suggesting that the Rap-GEF and Lar signaling pathways might use
different mechanisms to build and/or maintain adherens junctions
between the hub cells and GSCs.
The ability of some GSCs to persist next to the hub in Lar
mutant testes might be due to partial redundancy between Lar and
other tyrosine phosphatases such as the type IIA family receptor
tyrosine phosphatase Ptp69D, which has overlapping functions
with Drosophila Lar in the central nervous system and the visual
cortex and shares common signaling mechanisms (Clandinin et al.,
2001; Desai et al., 1997; Garrity et al., 1999; Krueger et al., 1996;
Maurel-Zaffran et al., 2001). Alternatively, weak hub-GSC
adhesion in Lar mutant testes might enable CySCs to compete for
attachment to the hub, displacing some, but not all, GSCs from the
hub. CySCs normally have smaller regions of contact with the hub
than do GSCs (Hardy et al., 1979) but can outcompete GSCs from
the hub when provided with an advantage. For example,
DEVELOPMENT
mutant GSC clones 5 days PCI, as compared with heterozygous
GSC counterparts (Fig. 7D,E), suggesting a cell-autonomous
requirement of Lar for E-cadherin localization to the hub-GSC
interface. In 60% of Lar451 and 63.8% of Lar2127 homozygous
mutant GSCs scored, very little or no E-cadherin-GFP was
detected at the hub-GSC interface, as compared with 14.2% and
18.8% of the neighboring heterozygous GSCs, respectively (Fig.
7F). In contrast to Lar451/Lar2127 mutant GSCs, where no Ecadherin-GFP expression was detected (arrowheads, Fig. 7C⬘),
some Lar451 or Lar2127 homozygous mutant GSC clones had low
levels of E-cadherin-GFP present at the hub-GSC interface
(arrow, Fig. 7D⬘). The Lar451/Lar2127 mutant adult GSCs lacked
Lar function since early development, whereas the homozygous
mutant GSC clones lacked Lar function for only 5 days,
suggesting a progressive loss of E-cadherin-GFP from the hubGSC interface in the mutant GSC clones.
1388 RESEARCH ARTICLE
Development 139 (8)
overexpression of components of the integrin-based adhesion
system in CySCs resulted in displacement of GSCs from the hub
by CySCs (Issigonis et al., 2009).
In wild-type testes, Lar localizes to the hub-GSC interface,
which is the region of cell cortex where localized adherens
junctions anchor GSCs to their niche (Yamashita et al., 2003).
Adherens junctions are formed by extended clustering of
transmembrane cadherin proteins that form homotypic interactions
with cadherins on opposing cell membranes (Harris and Tepass,
2010). The highly conserved cytoplasmic tail of E-cadherin acts as
an anchor for -catenin and p120-catenin and indirectly for catenin through its interaction with -catenin (Pokutta and Weis,
2007). Lar also localizes to adherens junctions in epithelial cells
(Aicher et al., 1997) and in neuronal synapses that are enriched in
cadherin-catenin complexes (Dunah et al., 2005). Lar physically
associates with the cadherin-catenin complex in cultured cells
(Aicher et al., 1997; Kypta et al., 1996; Muller et al., 1999) and
with N-cadherin in Drosophila embryos (Prakash et al., 2009).
Adherens junctions are associated with underlying arrays of
cortical F-actin (Hirokawa et al., 1983), organized by the high local
concentration of -catenin dimers (Drees et al., 2005; Weis and
Nelson, 2006). F-actin filaments, in turn, regulate the stability and
strength of adherens junctions (Chu et al., 2004; Ehrlich et al.,
2002; Hansen et al., 2002; Jamora and Fuchs, 2002; Vaezi et al.,
2002). Biochemical and genetic analyses of Lar indicate a role in
regulating the actin cytoskeleton. Loss of Lar function in
Drosophila oocytes results in defects in follicle formation, egg
elongation and anterior-posterior polarity that are correlated with
defects in actin filament organization (Bateman et al., 2001;
Frydman and Spradling, 2001). Lar might help to maintain hubGSC adhesion by interacting with and modulating the function of
regulators of F-actin. Drosophila Lar and its homologs physically
and genetically interact with Ena, a member of the Ena/VASP
family of actin regulators (Biswas et al., 2002; Wills et al., 1999).
Drosophila Ena and its mammalian homologs localize to adherens
junctions and have been implicated in the formation and
strengthening of adherens junctions in several cell types (Baum and
Perrimon, 2001; Grevengoed et al., 2001; Kris et al., 2008; Scott
et al., 2006; Vasioukhin et al., 2000). However, although Ena
localized to the hub-GSC interface, where adherens junctions are
present, its function was not absolutely required for GSC
maintenance, suggesting that other F-actin regulators in addition to
Ena function to maintain hub-GSC adhesion.
Lar protein localized to the hub-GSC interface might instead, or
in addition, regulate the tyrosine phosphorylation state of
components of the adherens junctions to maintain strong adhesion
between hub cells and GSCs. Regulation of the tyrosine
phosphorylation of components of adherens junctions plays an
important role in modulating the adhesive state of cells (Daniel and
Reynolds, 1997; Lampugnani et al., 1997; Roura et al., 1999; Wang
and Hartenstein, 2006). Tyrosine phosphorylation of E-cadherin in
epithelial cells induces loss of cell-cell contacts and the endocytosis
DEVELOPMENT
Fig. 7. Retention of E-cadherin-GFP at the hub-GSC
interface is affected in Lar mutant GSCs. (A-C⬘) Ecadherin-GFP expressed in GSCs and early germ cells using the
NGVP16 driver detected with anti-GFP (green, A-C; white, A⬘C⬘) in 0- to 1-day-old wild-type (A,A⬘), Lar451/+ (B,B⬘) or
Lar451/Lar2127 (C,C⬘) mutant adult Drosophila testes. Vasa (red)
marks germ cells and Arm (magenta) marks the hub.
Arrowheads (C,C⬘) mark the interface between hub and a
Lar451/Lar2127 mutant GSC where E-cadherin-GFP fails to
localize. (D-E⬙) Localization of E-cadherin-GFP (green, D,E;
white, D⬘,E⬘) in homozygous Lar451 mutant (arrows and white
outline, D-D⬙) and Lar2127 mutant (arrows and white outline,
E-E⬙) GSC clones 5 days PCI as compared with heterozygous
control GSCs (arrowheads and red outline, D⬙,E⬙).
Homozygous mutant GSCs are detected by the absence of
anti--galactosidase immunostaining (red, D,E; white, D⬙,E⬙).
(D⬘,E⬘) There is reduced or absent E-cadherin-GFP at the hubGSC interface in homozygous mutant GSCs (arrows), whereas
localization of E-cadherin-GFP is seen at the hub-GSC
interface in heterozygous GSCs (arrowheads). The star
indicates the Hub. (F) Quantitation of GSCs with E-cadherinGFP expression and localization at the hub-GSC interface
versus GSCs with little or no E-cadherin-GFP expression as
detected in heterozygous and homozygous Lar451 and Lar2127
GSCs from clonal analysis. N, number of GSCs scored for each
genotype. P-values of Fisher’s exact test (two-tailed) are
shown. Scale bars: 25 m.
of E-cadherin (Fujita et al., 2002; Potter et al., 2005). A possible
role of Lar is to maintain adherens junctions by dephosphorylating
E-cadherin. Alternatively, Lar might target E-cadherin to the
membrane to build adherens junctions, as has been shown for the
mammalian homolog of Lar in cultured hippocampal neurons,
where it promotes the accumulation of cadherin-catenin complexes
at the synapse to enhance cell adhesion (Dunah et al., 2005).
Alternatively, or in addition, Lar might regulate tyrosine
phosphorylation of the catenins associated with E-cadherin at the
hub-GSC interface. Tyrosine phosphorylation of -catenin leads to
loss of cadherin–-catenin interaction and to internalization of Ecadherin, reducing the strength of adherens junctions (Lilien and
Balsamo, 2005). Mammalian Lar has been shown to
dephosphorylate -catenin in vitro (Dunah et al., 2005; Kypta et
al., 1996; Muller et al., 1999), suggesting that in vivo Lar might
promote cell adhesion by regulating the phosphorylation of catenin.
In addition to GSCs, Lar protein was also detected in two-, fourand eight-cell transit-amplifying spermatogonial cysts, which have
the ability to dedifferentiate and reoccupy the hub to replace lost
GSCs (Brawley and Matunis, 2004; Cheng et al., 2008; Sheng et
al., 2009). Under conditions that promote dedifferentiation,
spermatogonial cells send out dynamic, actin-rich, thin protrusions,
suggesting acquisition of motility (Sheng et al., 2009). An
intriguing possibility is that Lar might facilitate the ability of
dedifferentiating spermatogonial cells to reorganize their actin
cytoskeleton to recognize and build adherens junctions with the
hub, similar to the role of Lar in the nervous system, where it
promotes axonal migration, possibly by facilitating reorganization
of the actin cytoskeleton (Chagnon et al., 2004). One of the ligands
of Lar identified in the nervous system, the heparan sulfate
proteoglycan Dally-like (Dlp), is expressed by hub cells and helps
maintain GSCs in their undifferentiated state (Hayashi et al., 2009).
At Drosophila neuromuscular junctions, Dlp interacts with and
inhibits the phosphatase activity of Lar to regulate active zone
morphology and function at synapses (Johnson et al., 2006).
Similarly, in dedifferentiating spermatogonial cells, interaction of
Lar in GSCs with Dlp on hub cells might inhibit cell motility and
promote the formation of adherens junctions between hub cells and
the dedifferentiating germ cells.
Acknowledgements
We thank the Bloomington Drosophila Stock Center and Drosophila Genomics
Research Center for fly stocks; the Drosophila Studies Hybridoma Bank for
providing antibodies; Thomas Clandinin, Dorothea Godt, Ruth Lehmann,
Mariann Bienz, Steven Hou and Erika Bach for generously providing reagents;
Thomas Clandinin and members of the Fuller laboratory for helpful discussions
and advice; and Leanne Jones and Erin Davies for sharing microarray data in
which Lar expression was identified in early germ and cyst cells.
Funding
This work was supported by the National Institutes of Health [National
Research Service Award (NRSA) Postdoctoral Fellowship F32 GM083351 to
S.S., NIH R01 GM080501 to M.T.F.]. M.T.F. gratefully acknowledges the ReedHodgson Professorship in Human Biology, which partially supported this work.
Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.070052/-/DC1
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