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RESEARCH ARTICLE 5201
Development 138, 5201-5212 (2011) doi:10.1242/dev.069385
© 2011. Published by The Company of Biologists Ltd
Regulation of Drosophila glial cell proliferation by MerlinHippo signaling
B. V. V. G. Reddy and Kenneth D. Irvine*
SUMMARY
Glia perform diverse and essential roles in the nervous system, but the mechanisms that regulate glial cell numbers are not well
understood. Here, we identify and characterize a requirement for the Hippo pathway and its transcriptional co-activator Yorkie in
controlling Drosophila glial proliferation. We find that Yorkie is both necessary for normal glial cell numbers and, when activated,
sufficient to drive glial over-proliferation. Yorkie activity in glial cells is controlled by a Merlin-Hippo signaling pathway, whereas
the upstream Hippo pathway regulators Fat, Expanded, Crumbs and Lethal giant larvae have no detectable role. We extend
functional characterization of Merlin-Hippo signaling by showing that Merlin and Hippo can be physically linked by the Salvador
tumor suppressor. Yorkie promotes expression of the microRNA gene bantam in glia, and bantam promotes expression of Myc,
which is required for Yorkie and bantam-induced glial proliferation. Our results provide new insights into the control of glial
growth, and establish glia as a model for Merlin-specific Hippo signaling. Moreover, as several of the genes we studied have been
linked to human gliomas, our results suggest that this linkage could reflect their organization into a conserved pathway for the
control of glial cell proliferation.
INTRODUCTION
Neurons and glia are two distinct cell types that together form the
nervous system. Glial cells perform diverse essential functions to
support and protect neurons, and to guide and maintain their
connections (Freeman and Doherty, 2006; Barres, 2008). Despite
their fundamental importance, the mechanisms that control glial
cell numbers during development are not well understood.
Moreover, cancers associated with over-proliferation of glial cells
(gliomas) include the most common and deadly type of brain tumor
in adults (glioblastoma) and the most common solid tumors in
children. A number of different oncogenes and tumor suppressors
have been implicated in gliomas, including activation of receptor
tyrosine kinase pathways, activation of phosphoinositide 3 (PI3)
kinase signaling, activation of transforming growth factor- (TGF) signaling, elevation of Myc levels, expression of microRNAs
and loss of the tumor suppressor merlin (Ruttledge et al., 1994;
Herms et al., 1999; Maher et al., 2001; Lassman, 2004; Furnari et
al., 2007; Lau et al., 2008; Zheng et al., 2008; Abounader, 2009;
Godlewski et al., 2009; Silber et al., 2009). A better understanding
of the relationships among distinct genetic lesions associated with
gliomas could facilitate rational, targeted approaches to diagnosis
and treatment.
Human merlin is the product of the neurofibromatosis type 2
(neurofibromin 2, NF2) locus, a familial cancer syndrome in which
afflicted individuals develop tumors of the peripheral nervous
system, especially benign schwannomas and meningiomas, and
malignant mesotheliomas (Asthagiri et al., 2009). Merlin is a
member of the ezrin/radixin/moesin (ERM) protein family, which
Howard Hughes Medical Institute, Waksman Institute and Department of Molecular
Biology and Biochemistry, Rutgers The State University of New Jersey, Piscataway,
NJ 08854, USA.
*Author for correspondence ([email protected])
Accepted 3 October 2011
link the actin cytoskeleton to membrane proteins (Fievet et al.,
2007).
Gene-targeted
mutations
in
murine
merlin
(neurofibromatosis 2 – Mouse Genome Informatics), together with
experiments in cultured cells, have implicated it in a broad
spectrum of tumor biology, as knockout mice are prone to a range
of metastatic tumors, and merlin is required for contact inhibition
in cultured cells (McClatchey and Giovannini, 2005; Stamenkovic
and Yu, 2010). The action of merlin as a tumor suppressor has been
linked to several pathways and processes. One important link, first
identified in Drosophila, is to the Hippo signaling pathway
(Hamaratoglu et al., 2006).
Hippo signaling is a recently discovered pathway that controls
organ growth from Drosophila to humans (Fig. 1A) (Reddy and
Irvine, 2008; Pan, 2010; Zhao et al., 2010; Halder and Johnson,
2011). Hippo signaling is transduced through transcriptional coactivator proteins, known as Yap and Taz in mammals, and Yorkie
(Yki) in Drosophila, which regulate the expression of genes
important for growth, cell cycle progression and inhibition of
apoptosis (Oh and Irvine, 2010). Yki/Yap act in concert with DNAbinding partner proteins, which in Drosophila include Scalloped
(Sd), Homothorax (Hth) and Mad (Oh and Irvine, 2010; Oh and
Irvine, 2011). The transcriptional activity of Yki/Yap is negatively
regulated by the kinase Warts (Lats in mammals), which affects
Yki/Yap levels and localization. In Drosophila, the activity and
localization of Wts is regulated through multiple upstream branches
(Reddy and Irvine, 2008; Staley and Irvine, 2011). The two beststudied branches are Fat-Hippo signaling, which involves a
cadherin family protein called Fat, and Expanded-Hippo signaling,
which involves a FERM protein related to Merlin, called Expanded
(Ex). In Drosophila organs studied to date, Merlin has only modest
effects on Hippo signaling, apparently because of partial
redundancy with Ex (McCartney et al., 2000; Hamaratoglu et al.,
2006).
Studies in cultured mammalian cells, and more recently in mice,
support the existence of a merlin-hippo pathway in mammals, and
its importance to tumors associated with loss of merlin (Zhao et al.,
DEVELOPMENT
KEY WORDS: Drosophila, Hippo, Merlin, Glia
5202 RESEARCH ARTICLE
MATERIALS AND METHODS
Drosophila genetics
Expression of UAS lines in glia was achieved by crossing to repo-Gal4
UAS-mCD8:GFP flies (gift of J. Thomas, Salk Institute, San Diego, USA).
Expression of UAS lines in clones was achieved by crossing to hsFlp[122]; act>y+>gal-4 UAS-GFP (AyGal4). RNA interference (RNAi)
was induced using the following UAS-hairpin transgenes from the VDRC:
fat RNAi (9396), ex RNAi (22994), Mer RNAi (7161), Kibra RNAi
(100765), Myc RNAi (106066), mad RNAi (110517), yki RNAi (104523),
wts RNAi (106174), hpo RNAi (104169), sd RNAi (101497), lgl RNAi
(109604), crb RNAi (39177) and hth RNAi (108831). UAS-Dicer2 (Dietzl
et al., 2007) was combined with repo-Gal4 UAS-mCD8:GFP to obtain
efficient target gene knock down for all RNAi experiments. The
effectiveness of fat, ex, lgl, crb, yki, hpo, mad, sd and wts RNAi lines has
been described previously (Robinson et al., 2010; Oh and Irvine, 2011;
Rauskolb et al., 2011; Sun and Irvine, 2011). For over-expression
experiments, we used UAS-yki:V5, UAS-yki:V5S168A (Oh and Irvine, 2009),
UAS-bantam, UAS-Myc (gift of P. Gallant, Universität Würzburg,
Germany). To induce positively marked clones in glia, we crossed w repoGal4[4.3 ] UAS-mCD8:GFP repo-Flp5; FRT82B Tub-Gal80/TM2 (gift of
C. Klambt, Universität Münster, Germany) to wtsx1 FRT82B/TM6. As gene
expression reporters, we used GFP-ban sensor (Brennecke et al., 2003) and
th-lacZ.
Histology and imaging
Tissue was fixed and stained as described previously, using mouse antiRepo (1:400), rat anti-Elav [1:400, Developmental Studies Hybridoma
Bank (DSHB)], rabbit anti-Yki (1:400), guinea pig anti-dMyc (1:200, gift
of G. Morata, Universidad Autonoma de Madrid, Spain), guinea pig antiMerlin (gift of R. Fehon, University of Chicago, USA), goat anti-gal
(1:1000, Biogenesis), rabbit anti-Hth (1:400, gift of A. Salzberg, Israel
Institute of Technology, Haifa, Israel) and mouse anti-Diap1 (1:200, B.
Hay, California Institute of Technology, Pasadena, USA).
Ethynyl deoxyuridine (EdU) labeling and detection was performed using
Click-iT EdU Alexa Flour Imaging kits (Invitrogen) with Alexa-594 azide.
Dissected larvae were incubated with EdU (20 mM) at room temperature
for 10 minutes, washed with PBS and fixed with 4% paraformaldehyde in
PBS.
Numbers of glial cells in eye discs were counted manually within the
Elav-staining region of eye discs. All the imaginal discs counted for each
genotype were staged based on the number of rows of Elav positive cells
(approximately ten rows) in eye discs. For brain measurements, stained
brains were mounted on slides using glass beads (60 mm diameter) as
spacers. Confocal images were taken under similar settings for the all
genotypes, and glia numbers were counted using Volocity software (Perkin
Elmer). Four to five animals were scored per genotype.
Co-immunoprecipitation
S2 cells were cultured with Schneider’s Drosophila medium (Invitrogen)
and 10% FBS (Sigma). For co-immunoprecipitations, transient
transfections were performed with equal amounts of DNA (0.5 mg per
construct) using Cellfectin (Invitrogen) in 6-well plates according to the
manufacturer’s protocol, using plasmids pAW-Gal4 (S. Blair, University of
Wisconsin, Madison, USA), pUAS-Flag:dMST, pAct-Ex:HA, pUAST
Merlin: HA, pMT-Sav:V5 and pMT SavDSarah:V5. Coimmunoprecipitation assays were performed according to published
protocols. In brief, cells were harvested 48 hours later in RIPA lysis buffer
(50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% NP40; 0.5% sodium
deoxycholate; 0.1% SDS; 1 mM EDTA) supplemented with protease
inhibitor cocktail (Roche). Cell lysates (500 mg for each sample) were
incubated with 10 ml anti-FLAG beads (Sigma) at 4°C for 2 hours followed
by five washes in RIPA buffer. Beads were then boiled in Laemmli sample
buffer at 100°C for five minutes and loaded onto SDS-PAGE gels.
Western-blotted proteins were visualized using HRP-conjugated mouse
anti-V5 (1:10,000, Invitrogen), HRP-conjugated mouse anti-Flag
(1:50,000, Sigma) and peroxidase-conjugated rat anti-HA (1:10,000, 3F10,
Roche).
RESULTS
Expression of Yki in glial cells
We recently described a role for Yki in controlling the proliferation
and differentiation of neuroepithelial cells in the Drosophila optic
lobe (Reddy et al., 2010). In the course of this study, we noticed
that Yki is also expressed in glial cells. This glial expression is
evident as a distinctive meshwork of Yki staining throughout the
central brain (Fig. 1C). The assignment of this Yki staining to glial
cells was confirmed by two observations. First, Yki expression
overlaps the glial-specific expression of the reversed polarity (repo)
gene (Xiong et al., 1994), as revealed by a repo-Gal4 line driving
the expression of a GFP transgene (UAS-mCD8:GFP) (Fig. 1C).
DEVELOPMENT
2007; Striedinger et al., 2008; Zhang et al., 2010; Zhao et al.,
2010). Despite this, the extent to which mammalian merlin
functions as a tumor suppressor through hippo signaling has
remained unclear, as merlin has been linked to diverse downstream
effectors (Rong et al., 2004; Maitra et al., 2006; Morrison et al.,
2007; Striedinger et al., 2008; Houshmandi et al., 2009; LopezLago et al., 2009; Li et al., 2010; Stamenkovic and Yu, 2010;
Zhang et al., 2010) and recent studies disagree about whether the
tumor suppressor function of merlin in liver is mediated through
Yap or through epidermal growth factor receptor (EGFR) signaling
(Benhamouche et al., 2010; Zhang et al., 2010).
The Drosophila central nervous system (CNS) comprises a
ventral nerve cord and two brain hemispheres. Recent studies have
begun to investigate Drosophila as a model for glial cell
proliferation and have shown that activation of some of the
signaling pathways implicated in human glioma, including EGFR,
TGF- and PI3 kinase, can also increase glial cell numbers in
Drosophila (Rangarajan et al., 2001; Klambt, 2009; Read et al.,
2009; Witte et al., 2009). The arrangement and morphology of glial
cells in the CNS complicates analysis of glial proliferation.
However, the developing Drosophila eye has also been used as a
model for glial cell development (Silies et al., 2010). Retinal glial
cells originate from the optic stalk, which connects the eye
imaginal disc to the brain, and then migrate into the eye disc as
photoreceptor cells differentiate (Fig. 1B). The arrangement and
accessibility of retinal glial cells and their migration into the eye
disc offers an accessible system to study glial cell proliferation and
migration.
Here, we analyze the contribution of the Hippo signaling
pathway to the control of glial cell proliferation in Drosophila,
using both the brain hemispheres and the eye disc as models. We
show that Merlin has a conserved role in controlling glial
proliferation from humans to Drosophila, and that its effects in
Drosophila can be accounted for by modulation of Hippo signaling.
Unlike previously examined tissues in Drosophila, in glial cells
Hippo signaling is controlled exclusively through a Merlin-Hippo
pathway and other upstream regulators have no detectable role. We
also establish that Yki activation is not only sufficient to promote
glial overgrowth, but also essential for normal glial growth during
nervous system development. Characterization of the downstream
regulatory pathways through which Yki acts reveals that Yki
functions with at least two different DNA-binding partners, and
regulates glial growth, at least in part, through the microRNA gene
bantam (ban), and that ban in turn acts in part through the
oncogene Myc. Our results define a regulatory pathway that
controls glial cell numbers in Drosophila, which includes genes
implicated in human gliomas, suggesting that the regulatory links
we identify and their importance to glial growth could be
conserved.
Development 138 (23)
Merlin-Hippo signaling in glia
RESEARCH ARTICLE 5203
Second, when RNAi-mediated downregulation of Yki was targeted
to glial cells, using repo-Gal4 to drive the expression of a yki
hairpin transgene (RNAi-yki), the meshwork staining of Yki in the
central brain was lost, whereas Yki expression in neuroepithelial
cells of the optic lobe was unaffected (Fig. 1D). These same
experiments identified Yki expression within glial cells of the
ventral nerve cord (VNC), eye disc and optic stalk (Fig. 1E-H).
Yki regulates glial cell proliferation
To investigate the role of Yki in glia, we took advantage of the
glial-specific depletion of Yki in repo-Gal4 RNAi-yki flies.
These animals can reach pupal stages, but never survive to
adulthood, indicating that the activity of Yki in glial cells is
essential for viability [yki mutants die as first instar larvae
(Huang et al., 2005)]. Examination of neural tissues revealed that
the brain and VNC are reduced in size and have fewer glial cells,
as identified by Repo antibody staining (Fig. 2A,B,D,E;
supplementary material Fig. S1A,B). We first quantified the
reduction in glial cells within the eye imaginal disc. In wild type,
the number of glial cells detected in the eye disc is correlated
with photoreceptor cell differentiation. In late third instar larvae
with ten rows of photoreceptor cells specified (as defined by
staining with the neuronal antigen Elav), wild-type eye discs had
on average 105 glial cells, whereas repo-Gal4 RNAi-yki eye
discs averaged 66 glial cells (Fig. 2D,E,H). Quantification also
confirmed a reduction in glial cell numbers within the brain and
even a decrease in total brain volume (Fig. 2I). These
observations establish that Yki is required for the normal
proliferation and/or survival of glial cells.
To investigate whether Yki is not only necessary for normal glial
growth, but also sufficient to drive over-proliferation of glial cells,
we used repo-Gal4 to express wild-type and activated forms of Yki
in glial cells. Expression of a wild-type transgene (UAS-yki:V5)
(Oh and Irvine, 2009) did not significantly affect glial cell numbers
(Fig. 2F). However, expression of a Yki isoform activated by
mutation of a key Wts phosphorylation site (UAS-ykiS168A:V5) (Oh
DEVELOPMENT
Fig. 1 Yki expression in glia. (A)Simplified schematic of components and regulatory connections within the Hippo signaling pathway.
(B)Schematic of an eye imaginal disc attached to the optic lobe of the brain. Glial nuclei are in red, photoreceptor neurons and their axonal
projections are in blue. A monolayer of glial cells surrounds the optic stalk and the glial cells from the optic stalk migrate into the eye disc with the
progression of morphogenic furrow. (C-H⬘) Yki expression (red) and glial cells marked by expression of GFP (green) from repo-Gal4 UAS-mCD8:GFP
transgenes in larval brain hemispheres (C,D), the posterior portion of the eye disc (E,G) and the VNC and medial brain hemispheres (F,H) of
Drosophila. In D,G and H, Yki was depleted from glial cells by expression of RNAi-yki and UAS-dcr2. C⬘-H⬘ show a single channel of the stain to the
left. The retinal glial staining of Yki appears to be relatively low compared with that in central brain glia, but this comparison is skewed by
substantial differences in morphology, and potentially cytoplasmic volumes, between these different glial cell types.
5204 RESEARCH ARTICLE
Development 138 (23)
and Irvine, 2009) induced a substantial increase in glial cells, and
a corresponding increase in the size of the VNC and brain
hemispheres (Fig. 2C,G,I; supplementary material Fig. S1C). The
increase in glial cell numbers was hard to discern by Repo staining
in cross sections through the VNC and central brain, presumably
owing to the dispersed nature of glial cell nuclei. However, it was
clearly evident in projections through the optic stalk, which
becomes much thicker and contains many more glial cells (Fig.
2G), and also by the increased number of glial cell nuclei in optical
sections through the outer cortex of the brain hemispheres (Fig.
2C). Using image-analysis software, we were able to confirm an
increase in total glial cell numbers throughout the brain, as well as
an increase in total brain volume (Fig. 2I). Labeling for cells in S
phase with EdU confirmed that expression of activated-Yki is
associated with increased retinal glial cell proliferation (Fig. 2J,K).
Under similar conditions, EdU labeling of central brain glia was
observed only rarely, both in wild type and in animals expressing
activated-Yki (supplementary material Fig. S1D,E). We surmise
that at late third instar most non-retinal glial cells are insensitive to
activated-Yki, and that the measurable increase in glial cells within
the central brain reflects the cumulative effect of Yki activation
throughout development.
DEVELOPMENT
Fig. 2 Influence of Yki on glial growth. (A-C⬘) Drosophila brain lobes, at the same magnification, stained for glial nuclei with anti-Repo (red)
from larvae expressing repo-Gal4 UAS-dcr2 UAS-mCD8:GFP (green) alone (A, control) or with UAS-RNAi-yki (B) or UAS Yki:V5S168A (C). (D-G⬘) Third
instar eye discs, stained for Repo (red) and Elav (blue), from larvae expressing repo-Gal4 UAS-mCD8:GFP (green) alone (D, control) or with UASRNAi-yki (E), UAS-Yki:V5 (F) or UAS-Yki:V5S168A (G). (H)Histogram showing average number of glial nuclei per eye disc in larvae expressing the
indicated UAS-RNAi or over-expression transgenes, under repo-Gal4 control. (I)Histograms showing average number of glial nuclei per brain
hemisphere (blue, left) or average brain hemisphere volume (green, right, normalized to the wild-type average) in larvae expressing UAS-RNAi-yki or
UAS-Yki:V5S168A, as indicated, under repo-Gal4 control. (J-K⬘) Third instar eye discs, stained for Repo (red) and EdU (cyan), from larvae expressing
repo-Gal4 alone (J, control) or with UAS-Yki:V5S168A (K). A⬘-G⬘,J⬘,K⬘ show a single channel of the stain to the left. Error bars represent s.d.
Merlin-Hippo signaling in glia
RESEARCH ARTICLE 5205
A Merlin-Hippo pathway regulates glial cell
numbers through Yki
Yki is the transcriptional effector of the Hippo signaling pathway
(Oh and Irvine, 2010). One of the upstream regulators of the Hippo
pathway is Merlin, which was first identified as a tumor suppressor
gene in humans through its influence on the proliferation of
peripheral glial cells (Gusella et al., 1996). RNAi-mediated
depletion of Merlin in glial cells substantially increased glial cell
numbers in both the optic stalk and the brain cortex (Fig. 2H, Fig.
3A; supplementary material Fig. S2A). Expression of a dominantnegative form of Merlin also increased glial cell numbers
(supplementary material Fig. S2E).
To investigate whether this influence of Merlin on glial cell
numbers in Drosophila is consistent with an influence on Hippo
signaling, we examined the consequences of depletion of other
Hippo pathway components. RNAi of the Yki kinase Warts, or
the Warts kinase Hippo (Hpo), substantially increased glial cell
numbers (Fig. 2H, Fig. 3C,D; supplementary material Fig.
S2C,D). Kibra was recently identified as a protein that interacts
with Merlin and modulates Hippo pathway activity (Genevet et
al., 2010; Ling et al., 2010; Yu et al., 2010); depletion of Kibra
also increased glial cell numbers (Fig. 2H, Fig. 3E). These
observations, together with the consequences of expression of
activated-Yki, indicate that depletion of Merlin and inactivation
of the Hippo pathway result in similar glial overgrowth
phenotypes.
Two additional experiments were performed to test directly the
hypothesis that Merlin controls glial growth through Yki. First, we
examined the subcellular localization of Yki, which is normally
predominantly cytoplasmic, but which accumulates in the nucleus
when upstream tumor suppressors in the Hippo pathway are mutant
(Dong et al., 2007; Oh and Irvine, 2008). Depletion of Merlin, or
mutation of wts, within clones of glial cells elevated nuclear
localization of Yki in comparison with neighboring wild-type cells
DEVELOPMENT
Fig. 3 Influence of Hippo signaling on glial growth. (A-F⬘) Drosophila third instar eye discs, stained for Repo (red) and Elav (blue), from larvae
expressing repo-Gal4 UAS-dcr2 UAS-mCD8:GFP (green) and UAS-RNAi-Mer (A), UAS-RNAi-Mer UAS-RNAi-yki (B), UAS-RNAi-hpo (C), UAS-RNAi-wts
(D), UAS-RNAi-kibra (E) or UAS-RNAi-fat (F). (G,G⬘) Posterior eye disc and optic stalk, stained for Mer (red) and Repo (blue), from larvae expressing repoGal4 UAS-mCD8:GFP (green). (H)Western blots, probed with anti-Flag, anti-V5 and anti-HA, on samples from S2 cells co-transfected to express
Hpo:Flag (all lanes), Sav:V5 (lane 4), Sav-Sarah:V5 (C-terminal truncation lacking SARAH domain, lane 5), Ex:HA (lanes 2-5) or Merlin:HA (lanes 1, 3-5).
The top three panels show the input (cell lysate), the bottom three panels show the blots on material precipitated by anti-Flag beads. (I-J⬘) Posterior eye
disc and optic stalk from animals with Flp-out clones expressing UAS-RNAi-Mer UAS-dcr2 (I) or MARCM clones mutant for wtsX1 (J) marked by
expression of UAS-GFP (green) and stained to reveal expression of Yki (red) and Repo (blue). White arrows point to elevated nuclear Yki within the
clone, gray arrows point to low nuclear Yki in wild-type glial cells. A⬘-G⬘,I⬘,J⬘ show a single channel of the stain to the left.
(Fig. 3I,J). Thus, Merlin and Wts regulate Yki localization in glia.
The functional significance of this effect on Yki localization was
established by epistasis tests, which revealed that depletion of Yki
suppressed the increase in glial cell numbers associated with
depletion of Merlin (Fig. 2H, Fig. 3B; supplementary material Fig.
S2B). Thus, the Merlin overgrowth phenotype in Drosophila glia
is yki-dependent.
Genetic studies of Hippo signaling in Drosophila have
identified Fat-dependent and Ex-dependent pathways as major
regulators of Hippo signaling in other tissues (Reddy and Irvine,
2008; Staley and Irvine, 2011). However, in earlier studies, we
noted that expression of Fat and Ex in the brain appeared to be
restricted to neuroepithelial cells, whereas Merlin is expressed
throughout the brain (Reddy et al., 2010). Confirmation that this
ubiquitous expression of Merlin includes glial cells was provided
by staining eye discs for both Merlin expression and glial cell
markers (Fig. 3G). To confirm that Fat and Ex do not influence
glial growth, their expression was downregulated in glial cells
using RNAi lines that generate strong phenotypes in imaginal
discs. RNAi of fat or ex in glial cells did not increase glial cell
numbers (Fig. 2H, Fig. 3F; supplementary material Fig. S2G). As
an additional test, we examined animals mutant for null alleles of
fat or ex. These survive beyond late third instar, but have fewer
photoreceptor cells (Feng and Irvine, 2007; Pellock et al., 2007)
and we observed concordant decreases in numbers of glial cells
in the eye disc and a lack of glial overgrowth (supplementary
material Fig. S2H,I). We also examined two more recently
identified regulators of Hpo signaling, crumbs (crb) and lethal
giant larvae [lgl; l(2)gl – FlyBase] (Chen et al., 2010; Grzeschik
et al., 2010; Ling et al., 2010; Robinson et al., 2010; Sun and
Irvine, 2011). Depletion of their expression had no detectable
effect on glial cell numbers (supplementary material Fig. S2J,K).
Although we can not exclude the possibility that null alleles of
crb or lgl might affect glial cells, these RNAi lines phenocopy
strong mutant alleles in imaginal discs (Robinson et al., 2010;
Sun and Irvine, 2011), and crb is known to act through ex, which,
as noted above, has no role in glia. Thus, our observations,
together with the strong effect of Merlin depletion on glial cell
numbers, imply that in Drosophila glial cells Hippo signaling is
regulated exclusively by a Merlin-dependent pathway.
Merlin and Ex are both FERM domain proteins, and both are
thought to inhibit Yki activity principally by promoting Hippo
activity. Consistent with a recent report (Yu et al., 2010), we
observed that Ex, but not Merlin, could be directly co-precipitated
with Hpo when expressed together in cultured Drosophila S2 cells
(Fig. 3H). Although Merlin does not bind Hpo directly, both Merlin
and Hpo have been reported to be able to bind to the scaffolding
protein Salvador (Harvey et al., 2003; Pantalacci et al., 2003; Udan
et al., 2003; Wu et al., 2003; Formstecher et al., 2005; Yu et al.,
2010). To investigate whether Salvador might thus be able to serve
as a bridge linking Hpo to Merlin, we assayed for Salvadordependent co-precipitation of Merlin with Hpo in S2 cells. Indeed,
when all three proteins were co-expressed, Merlin and Hpo were
efficiently co-precipitated, and this depended upon the presence of
the Hpo-binding region of Salvador (the SARAH domain) (Fig.
3H). Thus, both Ex and Mer can form complexes with Hpo, but
Mer-Hpo complexes are Sav-dependent. Although the role of
Salvador has previously been suggested as being to promote
phosphorylation of Wts by Hpo (Harvey et al., 2003; Pantalacci et
al., 2003; Udan et al., 2003; Wu et al., 2003), our observations
suggest that Sav might also have a distinct role in facilitating MerHippo signaling.
Development 138 (23)
Fig. 4 Requirements for DNA-binding partners of Yki in glial
growth. (A-F⬘) Drosophila third instar eye discs, stained for Repo (red)
and Elav (blue), from larvae expressing repo-Gal4 UAS-dcr2 UASmCD8:GFP (green) and UAS-RNAi-sd (A), UAS-RNAi-mad (B), UASRNAi-hth (C), UAS-RNAi-sd UAS-Yki:V5S168A (D), UAS-RNAi-mad UASYki:V5S168A (E) or UAS-RNAi-hth UAS-Yki:V5S168A (F). A⬘-F⬘ show a
single channel of the stain to the left.
Both Sd and Mad are required for Yki-promoted
glial growth
Yki functions as a transcriptional co-activator protein and regulates
the expression of downstream target genes in conjunction with
DNA-binding partners. Three distinct Yki partners have been
identified in Drosophila: Sd, Hth and Mad (for a review, see Staley
and Irvine, 2011). Within imaginal discs, Sd is normally only
required for growth of the wing, but it is also required more
broadly for overgrowths associated with Yki over-expression (Wu
et al., 2008; Zhang et al., 2008). A requirement for Hth in Ykidependent growth has been identified in anterior eye disc cells,
which do not require Sd (Peng et al., 2009). Mad is required
broadly for growth in many Drosophila tissues (Affolter and
Basler, 2007). This requirement for Mad reflects its role as the
transcriptional effector of Dpp signaling and had been suggested to
stem from its ability to repress expression of the transcriptional
repressor protein Brinker (Affolter and Basler, 2007). However,
recent studies indicated that Mad can also promote growth in
imaginal discs in conjunction with Yki, at least in part by
promoting transcription of ban (Oh and Irvine, 2011).
The requirements for each of these DNA-binding Yki partners
in glial cell proliferation was assessed by RNAi-mediated
depletion. These studies identified requirements for both Sd and
Mad, but not Hth, in glial cell proliferation (Fig. 4A-C, Fig. 2H).
The lack of effect of Hth was not due to ineffective RNAi, because
antibody staining confirmed that hth RNAi could deplete Hth
protein (supplementary material Fig. S3A). Similar to yki RNAi,
DEVELOPMENT
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RESEARCH ARTICLE 5207
Fig. 5. Regulation of th and ban in glia. (A,A⬘) Posterior eye disc
and optic stalk from Drosophila larva with Flp-out clones expression
UAS-Yki:V5S168A, marked by expression of UAS-GFP (green), and
stained to reveal expression of th-lacZ (red) and Repo (blue). White
arrow points to elevated th-lacZ within the clone, gray arrow points to
low expression in wild-type glial cells. (B-C⬘) Larva with Flp-out clones,
marked by expression of GFP (green) and stained to reveal expression of
Diap1 (red) and Repo (blue) and expressing either UAS-sd-RNAi (B, gray
arrow points to lower Diap1 within the clone) or UAS-mad-RNAi (C,
white arrow points to normal Diap1 within the clone). (D,D⬘) Wild-type
optic stalk stained for Repo (blue), Armadillo (red) and GFP-ban sensor
(green). White arrows point to elevated GFP-ban within axons, gray
arrows point to low GFP-ban in glial cells. (E-I⬘) Larva with Flp-out
clones, marked by expression of Dicer (red), and stained to reveal
expression of GFP-ban sensor (green) and Repo (blue) and expressing
UAS-Yki:V5S168A (E, white arrow points to lower GFP-ban within the
clone, gray arrow points to GFP-ban expression in wild-type glial cells),
UAS-yki RNAi (F), UAS-mad RNAi (G), UAS-mad RNAi and UASYki:V5S168A (H) or UAS-sd RNAi (I). Clones are outlined by yellow
dashed lines. A⬘-C⬘,E⬘-I⬘ show a single channel of the stain to the left.
D⬘ shows GFP-ban and Arm channels, as indicated.
depletion of sd or mad in glial cells reduced glial cell numbers (Fig.
4A,B, Fig. 2H). Moreover, depletion of Sd or Mad suppressed the
ability of activated-Yki to promote glial growth, whereas depletion
of Hth did not (Fig. 2H, Fig. 4D-F).
The observation that both Sd and Mad are required for Ykipromoted glial growth indicates that, in glia, both of these DNAbinding partners are required, possibly to regulate distinct sets of
downstream genes. One gene identified as a direct target of Yki-Sd
regulation in imaginal discs, and not Yki-Mad regulation, is thread
(th), which encodes Diap1 (Wu et al., 2008; Zhang et al., 2008; Oh
and Irvine, 2011). Expression of a th-lacZ reporter was upregulated
by expression of YkiS168A:V5 in glial cells (Fig. 5A), consistent
with the inference that Sd is a Yki partner in glia, with th as one of
its targets. Moreover, depletion of sd, but not depletion of mad,
reduced Diap1 expression in glial cells (Fig. 5B,C).
Another key downstream target of Yki in imaginal discs is the
microRNA (miRNA) gene bantam (ban) (Brennecke et al., 2003;
Nolo et al., 2006; Thompson and Cohen, 2006). In imaginal discs,
Yki can promote ban expression in conjunction with either Sd, Hth
or Mad, acting through distinct enhancers (Zhang et al., 2008; Peng
et al., 2009; Oh and Irvine, 2011). ban expression can be detected
using a GFP-ban sensor, which inversely reports ban expression
by virtue of ban target sites in the 3⬘ UTR of a GFP-expressing
transgene (Brennecke et al., 2003). The GFP-ban sensor is
expressed at high levels in retinal axons within the optic stalk, but
is barely detectable within glial cells, which implies that ban is
expressed in glial cells (Fig. 5D). A further reduction of the ban
sensor in glial cells could be induced by expression of activatedYki (Fig. 5E), whereas yki RNAi upregulated ban sensor
expression (Fig. 5F). Thus, ban is regulated downstream of Yki in
glial cells. Investigation of requirements for Sd and Mad in glial
cells revealed that mad RNAi elevated ban sensor expression (Fig.
5G), and was epistatic to Yki activation for ban regulation (Fig.
5H). Conversely, sd RNAi had no detectable effect on ban sensor
expression in glia (Fig. 5I). Thus, ban is apparently regulated in
glial cells through a Yki-Mad transcription factor complex, and not
a Yki-Sd complex.
DEVELOPMENT
Merlin-Hippo signaling in glia
5208 RESEARCH ARTICLE
Development 138 (23)
Regulation of glial growth by Yki through ban
and Myc
To assess the influence of ban on glial growth, we took the
advantage of transgenes that drive expression of ban under UASGal4 control. Over-expression of ban in glial cells, under repoGal4 control, induced substantial over-proliferation of both retinal
and central brain glia (Fig. 6B,K), even greater than that induced
by activated-Yki. Conversely, ban mutant animals, which survive
to the third larval instar, have greatly reduced numbers of glial cells
(Fig. 6A,L). Thus, ban is a crucial growth regulator in glial cells.
ban-induced glial overgrowth is not blocked by depletion of yki
(Fig. 6C). Moreover, expression of activated-Yki could not reverse
the reduction in glial cell numbers associated with mutation of ban,
nor it could rescue the reduced growth of ban mutants, even using
a variety of Gal4 drivers (repo-Gal4, Fig. 6M; tub-Gal4 or actinGal4, not shown). These observations suggest that ban is the key
downstream target of Yki for the promotion of glial cell
proliferation.
DEVELOPMENT
Fig. 6. Role of ban and Myc in Glial growth. (A,A⬘) Third instar eye disc, stained for Repo (red) and Elav (blue), from ban⌬1 mutant Drosophila
larva. (B-H⬘) Third instar eye discs, stained for Repo (red) and Elav (blue), from larvae expressing repo-Gal4 UAS-dcr2 UAS-mCD8:GFP (green) and
UAS-ban (B), UAS-ban UAS-RNAi-yki (C), UAS-RNAi-Myc (D), UAS Yki:V5S168A UAS-RNAi-Myc (E), UAS-ban UAS-RNAi-Myc (F), UAS-Myc (G) or UASMyc UAS-Yki:V5S168A (H). (I-J⬘) Posterior eye disc and optic stalk from larvae with Flp-out clones expressing UAS Yki:V5S168A (I) or UAS-ban (J)
marked by expression of UAS-GFP (green), and stained to reveal expression of Myc (red) and Repo (blue). (K-P⬘) Brain lobes, stained for glial nuclei
with anti-Repo (red) from larvae expressing repo-Gal4 UAS-dcr2 UAS-mCD8:GFP (green) and UAS-ban (K), ban⌬1 mutant (L), ban⌬1; UASYki:V5S168A (M), UAS-RNAi-Myc (N), UAS-Myc (O) or UAS-Myc UAS-Yki:V5S168A (P). Note the moderate (K) and extreme (P) thickening of the cortex
(highlighted by arrows), including multiple cell layers, induced by ban (K) or activated Yki and Myc (P). A⬘-P⬘ show a single channel of the stain to
the left.
Increased expression of the Myc oncogene has been correlated
with gliomas in humans (Herms et al., 1999). Myc has also recently
been identified as a direct target of Yki in imaginal discs (Neto-Silva
et al., 2010), and we observed a modest upregulation of Myc when
activated-Yki was expressed (Fig. 6I). ban has also been reported to
upregulate Myc protein levels in wing discs, acting through
downregulation of the ubiquitin ligase Mei-P26 (Herranz et al.,
2010). Myc antibody staining revealed that ban could also upregulate
Myc levels in glial cells (Fig. 6J). To assess the functional
significance of this upregulation, we depleted Myc from glial cells
using RNAi. Loss of Myc severely reduced glial cell numbers in
otherwise wild-type animals (Fig. 6D,N), and also suppressed the
glial overgrowth phenotypes of both activated-Yki and ban (Fig.
6E,F). Thus, Yki- and ban-induced glial growth are Myc-dependent.
On its own, Myc over-expression induces only a very mild glial
overgrowth phenotype (Fig. 2H, Fig. 6G,O) (Read et al., 2009), but
co-expression of Myc and Yki resulted in a much stronger glial overproliferation phenotype than either gene alone (Fig. 6H,P), and was
associated with substantial EdU-labeling of cortex glia even at late
third instar, whereas expression of Myc or activated-Yki alone did
not have this effect (supplementary material Fig. S1). Thus, Myc is
a downstream target of Yki in glia, but Myc also acts synergistically
with other Yki targets to induce glial cell proliferation.
DISCUSSION
Regulation of glial growth by a Merlin-Hippo
signaling pathway
Merlin was first identified as the product of a human tumor
suppressor gene, NF2, loss of which in peripheral glial cells results
in benign tumors (Gusella et al., 1996). Merlin has also been
identified as an inhibitor of gliomas (Lau et al., 2008). Our
observations indicate that the role of Merlin as a negative regulator
of glial cell proliferation is conserved from humans to Drosophila
and, thus, that Drosophila can serve as a model for understanding
Merlin-dependent regulation of glial growth.
Studies in Drosophila imaginal discs first linked Merlin to Hippo
signaling (Hamaratoglu et al., 2006), and Merlin was subsequently
linked to Hippo signaling in mammalian cells (Striedinger et al.,
2008; Zhang et al., 2010), including its role in meningioma
(Striedinger et al., 2008). However, the tumor suppressor activity
of Merlin has also been linked to other downstream effectors in
mammals, including Erb2, Src, ras, rac, TORC1 (CRTC1 – Human
Gene Nomenclature Database) and CRL4 (IL17RB – Human Gene
Nomenclature Database) (Tikoo et al., 1994; Shaw et al., 2001;
Curto et al., 2007; Morrison et al., 2007; Houshmandi et al., 2009;
Lopez-Lago et al., 2009; Benhamouche et al., 2010; Li et al.,
2010), creating some uncertainty regarding the general importance
of the linkage of Merlin to Hippo in growth control. We found that
depletion of Merlin, depletion of other tumor suppressors in the
Hippo pathway, or expression of an activated form of Yki, all result
in similar glial overgrowth phenotypes. Moreover, depletion of
Merlin increased nuclear localization of Yki, and depletion of Yki
suppressed the overgrowth phenotype of Merlin. Together, these
observations clearly establish that the glial overgrowth phenotype
associated with Merlin depletion in Drosophila is mediated through
the Hippo signaling pathway.
A noteworthy feature of Hippo signaling in Drosophila glial
cells is that Merlin appears to be uniquely required as an upstream
regulator of Hippo signaling, as the Fat-dependent, Ex-dependent
and Lgl-dependent branches have no detectable role. Glia might,
thus, provide an ideal model for mechanistic investigations of the
Merlin branch of Hippo signaling. Fat-Hippo signaling employs Fat
RESEARCH ARTICLE 5209
as a transmembrane receptor and Dachsous as its transmembrane
ligand (Bennett and Harvey, 2006; Cho et al., 2006; Silva et al.,
2006; Willecke et al., 2006; Rogulja et al., 2008; Feng and Irvine,
2009), whereas Ex-Hippo signaling appears to employ Crumbs as
a transmembrane receptor and ligand (Chen et al., 2010; Ling et al.,
2010; Robinson et al., 2010). By contrast, Drosophila
transmembrane proteins that mediate extracellular signaling and
interact with Merlin have not yet been identified. Distinct
mechanisms might also be involved in signal transduction
downstream of Merlin. Although there is evidence that Ex and
Merlin can both influence Hippo activity, Ex, but not Mer, can
directly associate with Hpo. Conversely, Merlin, but not Ex, can
interact directly with Salvador, and Merlin, Salvador and Hippo can
form a trimeric complex. Moreover, the kibra loss-of-function
phenotype is weaker than expanded in imaginal discs, but
comparable to Merlin (Baumgartner et al., 2010; Genevet et al.,
2010; Yu et al., 2010), and we found that depletion of kibra also
has a significant effect on glial cell proliferation. Kibra is highly
expressed in mammalian brain, and alleles of KIBRA (WWC1 –
Human Gene Nomenclature Database) have been linked to human
memory performance (Papassotiropoulos et al., 2006). The role of
kibra in regulating glial cell numbers in Drosophila thus raise the
possibility that the influence of KIBRA on human memory might
reflect a role in glial cells.
Finally, we note that although Hippo signaling has been
investigated in several different organs in Drosophila, including
imaginal discs, ovarian follicle cells, neuroepithelial cells and
intestinal cells, these all involve roles in epithelial cells, in which
upstream regulators of the pathway (e.g. Fat, Ex, Mer) all have a
distinctive localization near adherens junctions. The identification
of a requirement for Hippo signaling in glia is the first time in
Drosophila that a role for the pathway has been identified in nonepithelial cells. Indeed, in previous studies we found that Hippo
signaling influences proliferation of neuroepithelial cells, but other
neuronal cell types, including neuroblasts, ganglion mother cells
and neurons, are insensitive to Yki (Reddy et al., 2010).
Identification of genes that promote glial growth
Considerable attention has been paid to genes for which mutation
or inappropriate activation can cause over-proliferation of glial
cells, resulting in glial tumors. However, less is known about the
mechanisms required for normal glial growth. Through loss-offunction studies, we identified several genes essential for normal
glial cell numbers, including yki, sd, ban, mad and myc. The
requirement for yki, mad and sd, together with epistasis studies,
identifies a requirement for active Yki in glial growth. This in turn
implies that downregulation of Hippo signaling is important for
normal glial growth. Understanding how this is achieved will
provide further insights into the regulation of glial cell numbers.
A requirement for Mad, together with its upstream regulator
Thickveins (Tkv), in promoting retinal glial cell proliferation was
described previously by Rangarajan et al. (Rangarajan et al., 2001).
Our studies of glial cells, together with recent work in imaginal
discs (Oh and Irvine, 2011), emphasize that in mediating the
growth-regulating activity of Hippo signaling, Yki utilizes multiple
DNA-binding partners (i.e. Mad and Sd) in the same cells at the
same time to regulate distinct downstream target genes required for
tissue growth.
Although Yki activity influenced glial cell numbers throughout
the nervous system, direct analysis of cell proliferation by EdU
labeling revealed that retinal glia were more sensitive to Yki
activation at late third instar than central brain glia, and significant
DEVELOPMENT
Merlin-Hippo signaling in glia
induction of central brain glial cell proliferation was only observed
when Yki activation was combined with Myc over-expression.
Further studies will be required to define the basis for this
differential sensitivity, but the implication that the proliferative
response to Yki is modulated by developmental stage and/or glial
cell type has important implications for diseases associated with
both excess and deficits of glial cells.
A regulatory pathway for glial growth
Our studies in Drosophila delineate functional relationships among
genes involved in the control of glial cell proliferation. Mammalian
homologs of Merlin, Yki and Myc have been implicated in glioma
(Herms et al., 1999; Uppal and Coatesworth, 2003; Lau et al., 2008;
Striedinger et al., 2008). Although a mammalian homolog of ban has
not been described, other miRNAs have also been linked to glioma
(Silber et al., 2009). Our observations imply that these genes can be
placed into a pathway, in which Merlin, through Hippo signaling,
regulates Yki, Yki regulates ban, and ban regulates Myc. However,
as expression of Myc alone did not lead to substantial overgrowth of
glia, Yki and ban must also have other downstream targets important
for the promotion of glial cell proliferation. Moreover, our
observations indicate that a Yki-Sd complex is also required for glial
growth. In addition to the well characterized downstream target
Diap1, Yki-Sd complexes in glial cells might regulate Myc directly,
as suggested by studies in imaginal discs (Neto-Silva et al., 2010),
and might regulate cell cycle genes in conjunction with E2F1 (E2f –
FlyBase) (Nicolay et al., 2011).
The influence of activated-Yki on a ban-GFP sensor, together
with the observations that yki is not required for ban-mediated
overgrowth, whereas ban is required for Yki-mediated overgrowth,
position ban downstream of Yki. This is consistent with studies of
Hippo signaling in imaginal discs, in which ban has also been
identified as a target of Yki for growth regulation (Nolo et al.,
2006; Thompson and Cohen, 2006). The placement of Myc
downstream of Yki and ban is supported by the observation that
Myc levels can be increased by expression of ban or activated-Yki,
and by genetic tests that indicate that Myc is required for Yki- and
ban-promoted glial overgrowth. A mechanism by which ban can
regulate Myc levels, involving downregulation of a ubiquitin ligase
that negatively regulates Myc, was identified recently in imaginal
discs (Herranz et al., 2010), and might also function in glial cells.
Myc has been reported to downregulate Yki expression in imaginal
discs (Neto-Silva et al., 2010) and, although we have not
investigated whether a similar negative-feedback loop exists in
glial cells, the synergistic enhancement of glial cell proliferation
observed when Yki and Myc were co-expressed is consistent with
this possibility, as the expression of both genes under heterologous
promoters could bypass negative regulation of Yki by Myc.
The Myc proto-oncogene is de-regulated or amplified in several
human cancers, including gliomas (Herms et al., 1999; Herms et
al., 2000; Pelengaris et al., 2002). The sensitivity of Yki/baninduced overgrowth to reduced Myc levels parallels studies of
glioma models involving other signaling pathways. For example,
Myc is upregulated by EGFR, and is limiting for EGFR-PI3Kinduced glial cell overgrowth in a Drosophila glioma model (Read
et al., 2009), and p53 and Pten-driven glioma in mouse models is
also Myc dependent (Zheng et al., 2008). Considering the evidence
linking Merlin and Yap to glial growth in mammals (Gusella et al.,
1996; Lau et al., 2008; Striedinger et al., 2008), and the
identification of Myc as a downstream target of Yap in cultured
cells (Dong et al., 2007), it is likely that Yap could also influence
glial growth in mammals, in part, through regulation of Myc.
Development 138 (23)
Acknowledgements
We thank S. Blair, R. Fehon, B. Hay, C. Klambt, P. Gallant, G. Morata, A.
Salzberg, J. Thomas, the Developmental Studies Hybridoma Bank and the
Bloomington stock center for antibodies and Drosophila stocks.
Funding
This research was supported by the Howard Hughes Medical Institute.
Deposited in PMC for release after 6 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.069385/-/DC1
References
Abounader, R. (2009). Interactions between PTEN and receptor tyrosine kinase
pathways and their implications for glioma therapy. Expert Rev. Anticancer
Ther. 9, 235-245.
Affolter, M. and Basler, K. (2007). The Decapentaplegic morphogen gradient:
from pattern formation to growth regulation. Nat. Rev. Genet. 8, 663-674.
Asthagiri, A. R., Parry, D. M., Butman, J. A., Kim, H. J., Tsilou, E. T.,
Zhuang, Z. and Lonser, R. R. (2009). Neurofibromatosis type 2. Lancet 373,
1974-1986.
Barres, B. A. (2008). The mystery and magic of glia: a perspective on their roles
in health and disease. Neuron 60, 430-440.
Baumgartner, R., Pörnbacher, I., Buser, N., Hafen, E. and Stocker, H. (2010).
The WW domain protein Kibra acts upstream of Hippo in Drosophila. Dev. Cell
18, 309-316.
Benhamouche, S., Curto, M., Saotome, I., Gladden, A. B., Liu, C. H.,
Giovannini, M. and McClatchey, A. I. (2010). Nf2/Merlin controls progenitor
homeostasis and tumorigenesis in the liver. Genes Dev. 24, 1718-1730.
Bennett, F. C. and Harvey, K. F. (2006). Fat cadherin modulates organ size in
Drosophila via the Salvador/Warts/Hippo signaling pathway. Curr. Biol. 16,
2101-2110.
Brennecke, J., Hipfner, D. R., Stark, A., Russell, R. B. and Cohen, S. M.
(2003). bantam encodes a developmentally regulated microRNA that controls
cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell
113, 25-36.
Chen, C. L., Gajewski, K. M., Hamaratoglu, F., Bossuyt, W., SansoresGarcia, L., Tao, C. and Halder, G. (2010). The apical-basal cell polarity
determinant Crumbs regulates Hippo signaling in Drosophila. Proc. Natl. Acad.
Sci. USA 107, 15810-15815.
Cho, E., Feng, Y., Rauskolb, C., Maitra, S., Fehon, R. and Irvine, K. D.
(2006). Delineation of a Fat tumor suppressor pathway. Nat. Genet. 38, 11421150.
Curto, M., Cole, B. K., Lallemand, D., Liu, C. H. and McClatchey, A. I.
(2007). Contact-dependent inhibition of EGFR signaling by Nf2/Merlin. J. Cell
Biol. 177, 893-903.
Dietzl, G., Chen, D., Schnorrer, F., Su, K. C., Barinova, Y., Fellner, M.,
Gasser, B., Kinsey, K., Oppel, S., Scheiblauer, S. et al. (2007). A genomewide transgenic RNAi library for conditional gene inactivation in Drosophila.
Nature 448, 151-156.
Dong, J., Feldmann, G., Huang, J., Wu, S., Zhang, N., Comerford, S. A.,
Gayyed, M. F., Anders, R. A., Maitra, A. and Pan, D. (2007). Elucidation of
a universal size-control mechanism in Drosophila and mammals. Cell 130,
1120-1133.
Feng, Y. and Irvine, K. D. (2007). Fat and expanded act in parallel to regulate
growth through warts. Proc. Natl. Acad. Sci. USA 104, 20362-20367.
Feng, Y. and Irvine, K. D. (2009). Processing and phosphorylation of the Fat
receptor. Proc. Natl. Acad. Sci. USA 106, 11989-11994.
Fievet, B., Louvard, D. and Arpin, M. (2007). ERM proteins in epithelial cell
organization and functions. Biochim. Biophys. Acta 1773, 653-660.
Formstecher, E., Aresta, S., Collura, V., Hamburger, A., Meil, A., Trehin, A.,
Reverdy, C., Betin, V., Maire, S., Brun, C. et al. (2005). Protein interaction
mapping: a Drosophila case study. Genome Res. 15, 376-384.
Freeman, M. R. and Doherty, J. (2006). Glial cell biology in Drosophila and
vertebrates. Trends Neurosci. 29, 82-90.
Furnari, F. B., Fenton, T., Bachoo, R. M., Mukasa, A., Stommel, J. M., Stegh,
A., Hahn, W. C., Ligon, K. L., Louis, D. N., Brennan, C. et al. (2007).
Malignant astrocytic glioma: genetics, biology, and paths to treatment. Genes
Dev. 21, 2683-2710.
Genevet, A., Wehr, M. C., Brain, R., Thompson, B. J. and Tapon, N. (2010).
Kibra is a regulator of the Salvador/Warts/Hippo signaling network. Dev. Cell
18, 300-308.
Godlewski, J., Newton, H. B., Chiocca, E. A. and Lawler, S. E. (2009).
MicroRNAs and glioblastoma; the stem cell connection. Cell Death Differ. 17,
221-228.
DEVELOPMENT
5210 RESEARCH ARTICLE
Grzeschik, N. A., Parsons, L. M., Allott, M. L., Harvey, K. F. and Richardson,
H. E. (2010). Lgl, aPKC, and Crumbs regulate the Salvador/Warts/Hippo
pathway through two distinct mechanisms. Curr. Biol. 20, 573-581.
Gusella, J. F., Ramesh, V., MacCollin, M. and Jacoby, L. B. (1996).
Neurofibromatosis 2, loss of merlin’s protective spell. Curr. Opin. Genet. Dev.
6, 87-92.
Halder, G. and Johnson, R. L. (2011). Hippo signaling: growth control and
beyond. Development 138, 9-22.
Hamaratoglu, F., Willecke, M., Kango-Singh, M., Nolo, R., Hyun, E., Tao, C.,
Jafar-Nejad, H. and Halder, G. (2006). The tumour-suppressor genes
NF2/Merlin and Expanded act through Hippo signalling to regulate cell
proliferation and apoptosis. Nat. Cell Biol. 8, 27-36.
Harvey, K. F., Pfleger, C. M. and Hariharan, I. K. (2003). The Drosophila Mst
ortholog, hippo, restricts growth and cell proliferation and promotes
apoptosis. Cell 114, 457-467.
Herms, J., Neidt, I., Luscher, B., Sommer, A., Schurmann, P., Schroder, T.,
Bergmann, M., Wilken, B., Probst-Cousin, S., Hernaiz-Driever, P. et al.
(2000). C-MYC expression in medulloblastoma and its prognostic value. Int. J.
Cancer 89, 395-402.
Herms, J. W., von Loewenich, F. D., Behnke, J., Markakis, E. and
Kretzschmar, H. A. (1999). c-myc oncogene family expression in glioblastoma
and survival. Surg. Neurol. 51, 536-542.
Herranz, H., Hong, X., Perez, L., Ferreira, A., Olivieri, D., Cohen, S. M. and
Milan, M. (2010). The miRNA machinery targets Mei-P26 and regulates Myc
protein levels in the Drosophila wing. EMBO J. 29, 1688-1698.
Houshmandi, S. S., Emnett, R. J., Giovannini, M. and Gutmann, D. H.
(2009). The neurofibromatosis 2 protein, merlin, regulates glial cell growth in
an ErbB2- and Src-dependent manner. Mol. Cell. Biol. 29, 1472-1486.
Huang, J., Wu, S., Barrera, J., Matthews, K. and Pan, D. (2005). The Hippo
signaling pathway coordinately regulates cell proliferation and apoptosis by
inactivating Yorkie, the Drosophila Homolog of YAP. Cell 122, 421-434.
Klambt, C. (2009). Modes and regulation of glial migration in vertebrates and
invertebrates. Nat. Rev. Neurosci. 10, 769-779.
Lassman, A. B. (2004). Molecular biology of gliomas. Curr. Neurol. Neurosci.
Rep. 4, 228-233.
Lau, Y. K., Murray, L. B., Houshmandi, S. S., Xu, Y., Gutmann, D. H. and Yu,
Q. (2008). Merlin is a potent inhibitor of glioma growth. Cancer Res. 68,
5733-5742.
Li, W., You, L., Cooper, J., Schiavon, G., Pepe-Caprio, A., Zhou, L., Ishii, R.,
Giovannini, M., Hanemann, C. O., Long, S. B. et al. (2010). Merlin/NF2
suppresses tumorigenesis by inhibiting the E3 ubiquitin ligase CRL4(DCAF1) in
the nucleus. Cell 140, 477-490.
Ling, C., Zheng, Y., Yin, F., Yu, J., Huang, J., Hong, Y., Wu, S. and Pan, D.
(2010). The apical transmembrane protein Crumbs functions as a tumor
suppressor that regulates Hippo signaling by binding to Expanded. Proc. Natl.
Acad. Sci. USA 107, 10532-10537.
Lopez-Lago, M. A., Okada, T., Murillo, M. M., Socci, N. and Giancotti, F. G.
(2009). Loss of the tumor suppressor gene NF2, encoding merlin,
constitutively activates integrin-dependent mTORC1 signaling. Mol. Cell. Biol.
29, 4235-4249.
Maher, E. A., Furnari, F. B., Bachoo, R. M., Rowitch, D. H., Louis, D. N.,
Cavenee, W. K. and DePinho, R. A. (2001). Malignant glioma: genetics and
biology of a grave matter. Genes Dev. 15, 1311-1333.
Maitra, S., Kulikauskas, R. M., Gavilan, H. and Fehon, R. G. (2006). The
tumor suppressors Merlin and expanded function cooperatively to modulate
receptor endocytosis and signaling. Curr. Biol. 16, 702-709.
McCartney, B. M., Kulikauskas, R. M., LaJeunesse, D. R. and Fehon, R. G.
(2000). The neurofibromatosis-2 homologue, Merlin, and the tumor
suppressor expanded function together in Drosophila to regulate cell
proliferation and differentiation. Development 127, 1315-1324.
McClatchey, A. I. and Giovannini, M. (2005). Membrane organization and
tumorigenesis-the NF2 tumor suppressor, Merlin. Genes Dev. 19, 2265-2277.
Morrison, H., Sperka, T., Manent, J., Giovannini, M., Ponta, H. and
Herrlich, P. (2007). Merlin/neurofibromatosis type 2 suppresses growth by
inhibiting the activation of Ras and Rac. Cancer Res. 67, 520-527.
Neto-Silva, R. M., de Beco, S. and Johnston, L. A. (2010). Evidence for a
growth-stabilizing regulatory feedback mechanism between Myc and Yorkie,
the Drosophila homolog of Yap. Dev. Cell 19, 507-520.
Nicolay, B. N., Bayarmagnai, B., Islam, A. B., Lopez-Bigas, N. and Frolov,
M. V. (2011). Cooperation between dE2F1 and Yki/Sd defines a distinct
transcriptional program necessary to bypass cell cycle exit. Genes Dev. 25,
323-335.
Nolo, R., Morrison, C. M., Tao, C., Zhang, X. and Halder, G. (2006). The
bantam microRNA is a target of the hippo tumor-suppressor pathway. Curr.
Biol. 16, 1895-1904.
Oh, H. and Irvine, K. D. (2008). In vivo regulation of Yorkie phosphorylation
and localization. Development 135, 1081-1088.
Oh, H. and Irvine, K. D. (2009). In vivo analysis of Yorkie phosphorylation sites.
Oncogene 28, 1916-1927.
RESEARCH ARTICLE 5211
Oh, H. and Irvine, K. D. (2010). Yorkie: the final destination of Hippo signaling.
Trends Cell Biol. 20, 410-417.
Oh, H. and Irvine, K. D. (2011). Cooperative regulation of growth by Yorkie
and Mad through bantam. Dev. Cell 20, 109-122.
Pan, D. (2010). The hippo signaling pathway in development and cancer. Dev.
Cell 19, 491-505.
Pantalacci, S., Tapon, N. and Leopold, P. (2003). The Salvador partner Hippo
promotes apoptosis and cell-cycle exit in Drosophila. Nat. Cell Biol. 5, 921927.
Papassotiropoulos, A., Stephan, D. A., Huentelman, M. J., Hoerndli, F. J.,
Craig, D. W., Pearson, J. V., Huynh, K.-D., Brunner, F., Corneveaux, J.,
Osborne, D. et al. (2006). Common Kibra alleles are associated with human
memory performance. Science 314, 475-478.
Pelengaris, S., Khan, M. and Evan, G. (2002). c-MYC: more than just a matter
of life and death. Nat. Rev. Cancer 2, 764-776.
Pellock, B. J., Buff, E., White, K. and Hariharan, I. K. (2007). The Drosophila
tumor suppressors Expanded and Merlin differentially regulate cell cycle exit,
apoptosis, and Wingless signaling. Dev. Biol. 304, 102-115.
Peng, H. W., Slattery, M. and Mann, R. S. (2009). Transcription factor choice
in the Hippo signaling pathway: homothorax and yorkie regulation of the
microRNA bantam in the progenitor domain of the Drosophila eye imaginal
disc. Genes Dev. 23, 2307-2319.
Rangarajan, R., Courvoisier, H. and Gaul, U. (2001). Dpp and Hedgehog
mediate neuron-glia interactions in Drosophila eye development by promoting
the proliferation and motility of subretinal glia. Mech. Dev. 108, 93-103.
Rauskolb, C., Pan, G., Reddy, V., Oh, H. and Irvine, K. D. (2011). Zyxin links
Fat signaling to the Hippo pathway. PLoS Biol. 9, e1000624.
Read, R. D., Cavenee, W. K., Furnari, F. B. and Thomas, J. B. (2009). A
Drosophila model for EGFR-Ras and PI3K-dependent human glioma. PLoS
Genet. 5, e1000374.
Reddy, B. V. and Irvine, K. D. (2008). The Fat and Warts signaling pathways:
new insights into their regulation, mechanism and conservation. Development
135, 2827-2838.
Reddy, B. V., Rauskolb, C. and Irvine, K. D. (2010). Influence of fat-hippo and
notch signaling on the proliferation and differentiation of Drosophila optic
neuroepithelia. Development 137, 2397-2408.
Robinson, B. S., Huang, J., Hong, Y. and Moberg, K. H. (2010). Crumbs
regulates Salvador/Warts/Hippo signaling in Drosophila via the FERM-domain
protein Expanded. Curr. Biol. 20, 582-590.
Rogulja, D., Rauskolb, C. and Irvine, K. D. (2008). Morphogen control of
wing growth through the Fat signaling pathway. Dev. Cell 15, 309-321.
Rong, R., Tang, X., Gutmann, D. H. and Ye, K. (2004). Neurofibromatosis 2
(NF2) tumor suppressor merlin inhibits phosphatidylinositol 3-kinase through
binding to PIKE-L. Proc. Natl. Acad. Sci. USA 101, 18200-18205.
Ruttledge, M. H., Sarrazin, J., Rangaratnam, S., Phelan, C. M., Twist, E.,
Merel, P., Delattre, O., Thomas, G., Nordenskjold, M., Collins, V. P. et al.
(1994). Evidence for the complete inactivation of the NF2 gene in the majority
of sporadic meningiomas. Nat. Genet. 6, 180-184.
Shaw, R. J., Paez, J. G., Curto, M., Yaktine, A., Pruitt, W. M., Saotome, I.,
O’Bryan, J. P., Gupta, V., Ratner, N., Der, C. J. et al. (2001). The Nf2 tumor
suppressor, merlin, functions in Rac-dependent signaling. Dev. Cell 1, 63-72.
Silber, J., James, C. D. and Hodgson, J. G. (2009). microRNAs in gliomas: small
regulators of a big problem. Neuromolecular Med. 11, 208-222.
Silies, M., Yuva-Aydemir, Y., Franzdottir, S. R. and Klambt, C. (2010). The
eye imaginal disc as a model to study the coordination of neuronal and glial
development. Fly (Austin) 4, 71-79.
Silva, E., Tsatskis, Y., Gardano, L., Tapon, N. and McNeill, H. (2006). The
tumor-suppressor gene fat controls tissue growth upstream of expanded in the
hippo signaling pathway. Curr. Biol. 16, 2081-2089.
Staley, B. K. and Irvine, K. D. (2011). Hippo signaling in Drosophila: recent
advances and insights. Dev. Dyn. doi: 10.1002/dvdy.22723.
Stamenkovic, I. and Yu, Q. (2010). Merlin, a “Magic” Linker between
Extracellular Cues and Intracellular Signaling Pathways that Regulate Cell
Motility, Proliferation, and Survival. Curr. Protein Pept. Sci. 11, 471-484.
Striedinger, K., VandenBerg, S. R., Baia, G. S., McDermott, M. W.,
Gutmann, D. H. and Lal, A. (2008). The neurofibromatosis 2 tumor
suppressor gene product, merlin, regulates human meningioma cell growth by
signaling through YAP. Neoplasia 10, 1204-1212.
Sun, G. and Irvine, K. D. (2011). Regulation of Hippo signaling by Jun kinase
signaling during compensatory cell proliferation and regeneration, and in
neoplastic tumors. Dev. Biol. 350, 139-151.
Thompson, B. J. and Cohen, S. M. (2006). The Hippo pathway regulates the
bantam microRNA to control cell proliferation and apoptosis in Drosophila.
Cell 126, 767-774.
Tikoo, A., Varga, M., Ramesh, V., Gusella, J. and Maruta, H. (1994). An antiRas function of neurofibromatosis type 2 gene product (NF2/Merlin). J. Biol.
Chem. 269, 23387-23390.
Udan, R. S., Kango-Singh, M., Nolo, R., Tao, C. and Halder, G. (2003). Hippo
promotes proliferation arrest and apoptosis in the Salvador/Warts pathway.
Nat. Cell Biol. 5, 914-920.
DEVELOPMENT
Merlin-Hippo signaling in glia
Uppal, S. and Coatesworth, A. P. (2003). Neurofibromatosis type 2. Int. J. Clin.
Pract. 57, 698-703.
Willecke, M., Hamaratoglu, F., Kango-Singh, M., Udan, R., Chen, C. L., Tao,
C., Zhang, X. and Halder, G. (2006). The fat cadherin acts through the hippo
tumor-suppressor pathway to regulate tissue size. Curr. Biol. 16, 2090-2100.
Witte, H. T., Jeibmann, A., Klambt, C. and Paulus, W. (2009). Modeling
glioma growth and invasion in Drosophila melanogaster. Neoplasia 11, 882888.
Wu, S., Huang, J., Dong, J. and Pan, D. (2003). hippo encodes a Ste-20 family
protein kinase that restricts cell proliferation and promotes apoptosis in
conjunction with salvador and warts. Cell 114, 445-456.
Wu, S., Liu, Y., Zheng, Y., Dong, J. and Pan, D. (2008). The TEAD/TEF family
protein Scalloped mediates transcriptional output of the Hippo growthregulatory pathway. Dev. Cell 14, 388-398.
Xiong, W. C., Okano, H., Patel, N. H., Blendy, J. A. and Montell, C. (1994).
repo encodes a glial-specific homeo domain protein required in the Drosophila
nervous system. Genes Dev. 8, 981-994.
Yu, J., Zheng, Y., Dong, J., Klusza, S., Deng, W. M. and Pan, D. (2010). Kibra
functions as a tumor suppressor protein that regulates Hippo signaling in
conjunction with Merlin and Expanded. Dev. Cell 18, 288-299.
Development 138 (23)
Zhang, L., Ren, F., Zhang, Q., Chen, Y., Wang, B. and Jiang, J. (2008). The
TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in
organ size control. Dev. Cell 14, 377-387.
Zhang, N., Bai, H., David, K. K., Dong, J., Zheng, Y., Cai, J., Giovannini, M.,
Liu, P., Anders, R. A. and Pan, D. (2010). The Merlin/NF2 tumor suppressor
functions through the YAP oncoprotein to regulate tissue homeostasis in
mammals. Dev. Cell 19, 27-38.
Zhao, B., Wei, X., Li, W., Udan, R. S., Yang, Q., Kim, J., Xie, J., Ikenoue, T.,
Yu, J., Li, L. et al. (2007). Inactivation of YAP oncoprotein by the Hippo
pathway is involved in cell contact inhibition and tissue growth control. Genes
Dev. 21, 2747-2761.
Zhao, B., Li, L., Lei, Q. and Guan, K. L. (2010). The Hippo-YAP pathway in
organ size control and tumorigenesis: an updated version. Genes Dev. 24,
862-874.
Zheng, H., Ying, H., Yan, H., Kimmelman, A. C., Hiller, D. J., Chen, A. J.,
Perry, S. R., Tonon, G., Chu, G. C., Ding, Z. et al. (2008). Pten and p53
converge on c-Myc to control differentiation, self-renewal, and transformation
of normal and neoplastic stem cells in glioblastoma. Cold Spring Harb. Symp.
Quant. Biol. 73, 427-437.
DEVELOPMENT
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