RESEARCH ARTICLE 1493
Development 137, 1493-1503 (2010) doi:10.1242/dev.042804
© 2010. Published by The Company of Biologists Ltd
Drosophila VHL tumor-suppressor gene regulates epithelial
morphogenesis by promoting microtubule and aPKC stability
Serena Duchi1, Luca Fagnocchi1, Valeria Cavaliere1, Anita Hsouna2, Giuseppe Gargiulo1,† and Tien Hsu2,*,†
SUMMARY
Mutations in the human von Hippel-Lindau (VHL) genes are the cause of VHL disease, which displays multiple benign and malignant
tumors. The VHL gene has been shown to regulate angiogenic potential and glycolic metabolism via its E3 ubiquitin ligase function
against the alpha subunit of hypoxia-inducible factor (HIF). However, many other HIF-independent functions of VHL have been
identified and recent evidence indicates that the canonical function cannot fully explain the VHL mutant cell phenotypes. Many of
these functions have not been verified in genetically tractable systems. Using an established follicular epithelial model in
Drosophila, we show that the Drosophila VHL gene is involved in epithelial morphogenesis via stabilizing microtubule bundles and
aPKC. Microtubule defects in VHL mutants lead to mislocalization of aPKC and subsequent loss of epithelial integrity. Destabilizing
microtubules in ex vivo culture of wild-type egg chambers can also result in aPKC mislocalization and epithelial defects. Importantly,
paclitaxel-induced stabilization of microtubules can rescue the aPKC localization phenotype in Drosophila VHL mutant follicle cells.
The results establish a developmental function of the VHL gene that is relevant to its tumor-suppressor activity.
INTRODUCTION
Establishing and maintaining epithelial integrity is essential for
embryonic development, organogenesis and tissue remodeling. The
key characteristic of epithelial cells is asymmetrical specification of
membrane domains marked by domain-specific proteins (Knust and
Bossinger, 2002; Nelson, 2003; Tepass, 2002). The epithelial
morphogenic mechanism, although with some variations in different
epithelial tissues, is highly conserved from worm to mammal. The
crucial initial step in establishing epithelial polarity is the
specification of the apical domain, which is defined by the function
of a complex containing atypical PKC (aPKC), Bazooka (Baz;
mammalian and worm PAR-3) and PAR-6 (for a review, see Suzuki
and Ohno, 2006). The PAR complex is initially recruited by
activated Cdc42 to the apical domain. The three proteins were
originally thought to function as a complex; however, recent
evidence indicates that Baz might be required first to recruit the
aPKC–PAR-6 complex to the subapical domain juxtaposed to the
future adherens junction (AJ) (Harris and Peifer, 2007). The PAR
complex is required for the localization of another apical complex
containing Crumbs (Crb), Stardust (Std; mammalian Pals1) and
Discs lost (Dlt; mammalian Patj). The PAR- and Crb-containing
complexes occupy the apical-most region of the lateral membrane,
just apical to the AJs.
The apical complexes in turn restrict the localization of a third
complex comprising Scribble (Scrib; mammalian Scribble/Vartul),
Discs large (Dlg) and Lethal(2) giant larvae (Lgl) (Betschinger et al.,
1
Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, Via
Selmi 3, 40126 Bologna, Italy. 2Department of Pathology and Laboratory Medicine,
and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC
29425, USA.
*Present address: Boston University School of Medicine, Hem/Onc Section,
650 Albany Street, EBRC440, Boston, MA 02118, USA
†
Authors for correspondence ([email protected]; [email protected])
Accepted 23 February 2010
2005; Bilder et al., 2003) to the basolateral domain, while Lgl also
antagonizes the apical components and prevents their spreading to
the basolateral side (Yamanaka et al., 2003). The antagonistic action
of apical and basolateral complexes helps define the apicolateral loci
eventually occupied by AJs. It is not yet completely clear how the
initial localization of the apical complex is achieved.
The VHL tumor-suppressor gene mutations are the genetic cause
of the familial VHL disease. Germline mutations in VHL predispose
the patients to several benign and malignant tumors, including renal
cell carcinoma (RCC, kidney cancer), hemangioblastoma
(overgrowth of blood vessels in the retina and central nervous
system) and pheochromocytoma (tumors in the adrenal glands).
VHL protein has been shown to function as an E3 ubiquitin ligase.
Among its best-documented targets is the alpha subunit of the
hypoxia-inducible factor (HIF-). Therefore, the canonical tumorsuppressor function of VHL is modulation of the normal oxygensensing mechanism that regulates angiogenic response and
metabolic switch to glycolysis (Kaelin, 2008). However, how this
function correlates with the origin of epithelial tumors such as RCC
is unclear, although it is thought that HIF-independent mechanisms
might be involved (Frew and Krek, 2007).
VHL is evolutionarily conserved. In Drosophila, the VHL gene
has been implicated in tracheal tubule development and HIF-
regulation in the embryos based on biochemical and RNA
interference-mediated phenotypic studies (Adryan et al., 2000;
Arquier et al., 2006; Aso et al., 2000; Mortimer and Moberg, 2009).
In this report, we generated the first genomic Drosophila VHL
mutant and examined the function of VHL in epithelial
morphogenesis using a model epithelium – the follicle cells in the
egg chamber. We show that VHL regulates the proper localization
and stability of aPKC in the follicle cells and that this function is, at
least in part, mediated by the action of VHL on microtubule (MT)
stability. Without VHL function, MTs and aPKC are destabilized,
resulting in epithelial defects. These results establish a
developmental function of the VHL gene that is relevant to its tumorsuppressor activity.
DEVELOPMENT
KEY WORDS: Von Hippel-Lindau tumor-suppressor gene, Drosophila, Epithelial morphogenesis
1494 RESEARCH ARTICLE
Development 137 (9)
MATERIALS AND METHODS
Immunoprecipitation
Drosophila strains and genetics
Ovaries from 50-150 females were collected in 1⫻ PBS and rinsed twice
with 1⫻ PBS. Then 180 l protein extraction buffer (50 mM HEPES pH 7.6,
100 mM KCl, 1 mM EGTA, 1 mM MgCl2, 1% Triton-X 100, 10% glycerol)
and 20 l of 10⫻ protein inhibitor cocktail (Roche) were added. The ovaries
were homogenized manually on ice using an Eppendorf tissue grinder, then
sonicated on ice four times with 2-second pulses and 2-second intervals and
centrifuged at 6000 g for 20 minutes at 4°C. The supernatant was stored at
–20°C in 50 l aliquots and a small portion was used to perform a protein
quantification assay. 500 g of protein extracts were used for each
immunoprecipitation in a total volume of 500 l of protein extraction buffer.
The extracts were pre-adsorbed with equilibrated 50 l of 50% protein A/G
agarose bead slurry (Pierce). The extracts were then incubated with ~3 g
of anti--tubulin or anti-GFP (either mouse or rabbit) before 50 l of
equilibrated protein A/G beads were added. Purified IgG was used as
control. The precipitated protein complexes were then subjected to western
blotting. Antibodies used in western blots were: mouse anti--tubulin
(1:250; see above), mouse anti--actin (1:2000; Sigma), rabbit anti-Bazooka
(1:5000; see above) and rabbit anti-VHL (1:10,000; see above).
Ex vivo culture of egg chambers
Ex vivo culture of egg chambers has been previously described (Prasad et
al., 2007). Briefly, ovaries were dissected at room temperature in PBS and
cultured at 25°C in oxygenated Schneider’s Complete Medium Cocktail
[Schneider’s Medium, heat-inactivated 15% fetal bovine serum (FBS), 0.6⫻
penicillin/streptomycin mix (Gibco/Invitrogen), 0.2 mg/ml fresh insulin, pH
6.95-7.0]. Paclitaxel and nocodazole (Sigma) were dissolved in DMSO and
used at indicated concentrations. After a 5-hour incubation at 25°C, the
ovaries were gently collected, washed and processed for immunostaining.
Immunohistochemistry
Ovaries were dissected at room temperature in PBS, pH 7.5, and fixed in 4%
paraformaldehyde or as specified (methanol was used for Crb staining).
Subsequent washes and incubations were performed in PBT (1⫻ PBS
containing 0.1% Triton X-100). Primary antibody incubation was performed
at 4°C overnight and antibodies were diluted in PBT containing 3% BSA.
Mouse monoclonal primary antibodies used were: -tubulin [1:25; E7,
Developmental Studies Hybridoma Bank (DSHB)], Arm (1:10; N2A71,
DHSB), Crb (1:5; Cq4, DSHB) and GFP (1:1000; sc-9996, Santa Cruz).
Primary rabbit polyclonal antibodies used were: cleaved caspase 3 (1:50;
Cell Signaling), phospho-histone H3 (1:200; Upstate), aPKC (1:200; C20,
Santa Cruz), HA (sc-805, Santa Cruz), Lgl (1:500) (Grifoni et al., 2004) and
Bazooka (1:2000) (Wodarz et al., 1999). Rabbit polyclonal anti-VHL
antibody was generated against full-length bacterially expressed His-tagged
protein. This antibody is not suitable for wholemount immunostaining of the
egg chambers. AlexaFluor 546-phalloidin and ToPro-3 are from
Invitrogen/Molecular Probes. AlexaFluor 488, 546 or 633-conjugated
secondary antibodies (Invitrogen/Molecular Probes) were used at 1:200
dilution and incubation performed for 2 hours at room temperature.
Propidium Iodide and DAPI are from Sigma. Egg chambers were mounted
in Fluoromount G (Electron Microscopy Sciences) and analyzed with the
TCS SL Leica confocal system or with an Olympus IX70 microscope
equipped with the FluoView 300 confocal capability. Digital images were
processed using the Photoshop software without biased manipulations.
RESULTS
Drosophila VHL mutant
The VHL mutation was generated using the homologous
recombination strategy (Rong et al., 2002). Homozygous VHL1
mutants are sluggish after hatching and die at the end of first instar
larval stage. Wild-type, heterozygous and homozygous first instar
larvae were hand-picked and subjected to genomic DNA PCR. The
homozygous mutant animals show complete loss of the wild-type
gene copy (Fig. 1A). The VHL1 allele, when paired with a deficiency
chromosome encompassing the VHL locus (at 47E), shows the same
late first instar lethality, suggesting that VHL1 is a null mutant. The
lethal phenotype can be rescued by expressing a wild-type VHL
cDNA under the control of the hsp70 promoter (data not shown).
Therefore, VHL gene truncation is the only major genetic defect in
the VHL1 allele.
Epithelial defects in VHL mutant follicle cells
The Drosophila follicle cells exhibit the typical epithelial polarity
exemplified by markers such as the apical PAR complex, AJ
components and the basolateral Lgl complex (Bilder et al., 2003;
Tanentzapf et al., 2000). Establishment of the epithelial polarity
begins soon after follicle cells diverge from the somatic stem cells
and encircle the germ cells (stage 1). The epithelium reaches
maturity at mid-oogenic stages (after stage 6 at ~30 hours of egg
chamber development; total developmental time ~70 hours), when
follicle cells cease to proliferate. To examine the phenotype in the
follicular epithelium, an adult tissue, we generated mosaic mitotic
mutant clones using the Flp/FRT system. Mutant clones are
identified by a lack of GFP expression. In an initial survey of
potential egg chamber phenotypes, prominent epithelial defects
were observed at stage 10 (about 50-55 hours into egg chamber
development; Fig. 1B-D). The most notable morphological
abnormalities were the piling-up of follicle cells (Fig. 1B,C, arrows)
and stretched follicular epithelium (brackets in Fig. 1C,D). The same
phenotypes were observed with the VHL deficiency Df(2R)en-A
(Fig. 1E,F). To quantify the phenotypes, 100 clones of various sizes
at stage 9-10 were analyzed. Clone sizes were categorized based on
the number of cells in an optical cross-section. As the penetrance in
single-cell clones is variable, probably because of phenotypic rescue
by the neighboring epithelial cells, we considered only clones larger
than two cells. Thirty-eight percent of clones showed stacking of
follicle cells (Fig. 1B, arrows) and the more severe, multilayering
phenotypes (more than three layers of cells; Fig. 1C, arrow); 42%
DEVELOPMENT
y1, w67c23 was used as the wild-type stock in this study. The Drosophila VHL1
mutant was generated by replacing the wild-type copy, via homologous
recombination (Rong and Golic, 2000), with a deletion that removes 81
codons encompassing the first two in-frame AUGs. The mutant stock is
maintained with the balancer chromosome CyO, Dfd-YFP that carries a YFP
reporter gene directed from the Dfd promoter (expressed in mandibles). The
Df(2R)en-A is a chromosomal deletion that uncovers the region 47D6-48F8
on the second chromosome, including the genomic VHL locus at 47E547E6. Follicle cell clones mutant for VHL were generated with site-directed
mitotic recombination using the Flp/FRT system (Xu and Rubin, 1993). The
mutant VHL alleles were crossed onto chromosome 2R carrying the yeast
recombination site FRTG13 at the base of the chromosome arm (at position
42B). The mitotic mutant clone was induced by crossing the VHL, FRTG13
fly with a transgenic strain carrying the wild-type FRTG13 chromosome (with
a ubiquitin promoter-driven GFP reporter gene, ubi-GFP) and the UAS-Flp
recombinase transgene under control of the T155-Gal4 driver. The genotype
of the VHL mutant mosaic female is (using VHL1 as an example): w67c23;
P{FRTwhs}G13, VHL1/P{FRTwhs}G13, P{ubi-GFP}; P{T155-Gal4},
P{UAS-Flp}/+. T155-Gal4 directs expression in all follicle cells throughout
oogenesis. FRT42D, aPKCk06403/CyO is a strong loss-of-function allele and
was a gift of W. M. Deng (Florida State University, USA) (Tian and Deng,
2008). The VHL full-length cDNA (Adryan et al., 2000) and the VHLYH
variant (single amino acid substitution mutation of tyrosine to histidine at
position 51, equivalent to human Y98) were cloned in the UAS-based
pGateway vector tagged with three HA epitopes at the 3⬘ end. These
expression vectors were used to transform the y w flies. Cy2-GAL4 was
kindly provided by T. Schüpbach (Princeton University, USA), and used for
UAS-driven expression in follicle cells. btl-GAL4 was used to drive the
expression of VHL in the tracheal and glial cells. Unless specified, all fly
stocks are from the Bloomington Stock Center.
VHL regulates epithelial morphogenesis
RESEARCH ARTICLE 1495
Fig. 1. Drosophila VHL mutant follicle cells exhibit epithelial
defects. (A)VHL1 mutant allele contains a deletion removing the first two
in-frame AUG codons. PCR primers (black line fragments) for
distinguishing wild-type and VHL1 genomic segments are shown with
expected PCR fragment sizes. Genomic DNAs from first-instar larvae of
the indicated genotypes were subject to PCR amplification using the VHLspecific primers indicated on the left or rp49-specific primers as control.
(B-D)VHL1 mutant clones were generated as described in Materials and
Methods. Relevant cell types are marked in B for reference herein. FC,
follicle cells; NC, nurse cells; O, oocyte. The apical surface of the follicle
cells is in contact with the germ cell complex (nurse cells and the oocyte).
Mutant cells are identified by a lack of GFP expression (dotted lines or
brackets). Egg chambers were stained for DNA (ToPro-3 in B and DAPI in
C,D). The mutant epithelia show stacking of cells (arrows in B), piling-up
(more than three layers of cells; arrow in C) and stretching (brackets in
C,D). (E,F)Mutant clones of Df(2R)en-A encompassing the VHL locus
were generated as above. Egg chambers were stained for DNA
(Propidium Iodide). Stacking of epithelial cells is seen (arrows).
(G)Quantification of cell numbers in mutant clones compared with the
sister wild-type cell clones. Twenty-five clones are presented with
increasing sizes. There are no significant size differences between mutant
and wild-type clones. Scale bars: 20m.
showed flattening/stretching of epithelium (Fig. 1C,D, brackets);
and the remaining 20% showed swelling (see below). We also
determined that VHL loss-of-function mutation did not affect cell
Loss of VHL in follicle cell clones affects cell
polarity
Multilayering is a typical terminal phenotype for polarity defects in
epithelial cells. In homozygous VHL1 clones, the aPKC protein was
mislocalized or greatly diminished from the apical membrane
domain, which abuts the germ cell complex. This defect occurred
early in oogenesis in 82% (n28) of the stage 3-4 clones (~10-24
hours of egg chamber development; stage 4 as an example in Fig.
2A). At later stages (>stage 8; >40 hours of egg chamber
development) in homozygous VHL1 clones, mislocalization of
aPKC was more pronounced, showing either cytoplasmic
localization or absence in 96% (n27) of stage 10 clones or 83%
(n29) of stage 8-9 clones (Fig. 2B,C). Interestingly, in many
mutant cells [61% (n28) of stage 8-9 clones], aPKC was detected
in the perinuclear region (Fig. 2C⬙, sharp arrows), suggesting a
defect in translocation of the protein. We next analyzed the
distribution of the aPKC partner Bazooka (Baz). Baz localization
was largely normal in VHL mutant cells at the same stage 3-4, with
only a slight decrease in the apical domain and some spreading to
the lateral domain (50%, n28; Fig. 2D). The overall levels
diminished significantly later in 83% (n29) of stage 9-10 clones
(Fig. 2E). Perinuclear accumulation was also detected in 61%
(n28) of stage 10 clones (Fig. 2E). The time difference in
localization defects of these two proteins indicates that Baz
mislocalization at later stage is probably an indirect effect
downstream of mislocalized aPKC.
The PAR complex has been shown to be a master regulator of
epithelial polarity (Suzuki and Ohno, 2006). We therefore
examined representative markers of different apicolateral
membrane domains. The subapical region (SAR) can be visualized
by Crumbs (Crb), a transmembrane protein that interacts with
aPKC and is required for the establishment of this domain (Tepass
et al., 1990; Wodarz et al., 1995). It has been shown that
phosphorylation of Crb by aPKC in Drosophila embryonic
blastodermal cells is necessary for Crb stabilization in the apical
domain (Sotillos et al., 2004). In homozygous VHL1 clones at stage
4, apical localization of Crb was only slightly reduced (but not
absent; Fig. 2F,F⬘). Co-staining of Crb and aPKC at stage 4 showed
that in the VHL1 clone, aPKC is undetectable while Crb is fading
but still present in the apical domain (see Fig. S2A in the
supplementary material). Later at stage 8, Crb was mislocalized to
the basal domain and formed large aggregates in 89% (n28) of
stage 8-10 clones (Fig. 2G,G⬘, arrows).
AJs are the mature epithelial cell landmark and can be visualized
by the Drosophila -catenin encoded by the armadillo (arm) locus
(Peifer, 1993; Tanentzapf et al., 2000). Arm distribution was normal
DEVELOPMENT
proliferation or viability. As mitotic recombination in a
heterozygous progenitor cell (VHL1/ubi-GFP) generates one VHL
homozygote (no GFP) and one wild type (two copies of GFP), the
numbers of high GFP-expressing cells and GFP-negative cells
should be equal if the mutant cells do not exhibit an altered rate of
proliferation or viability. We calculated the ratio of the cell number
within the VHL1 mutant clones versus the cell number within the
sister wild-type clones (number of clones examined25) and
obtained a mean value of 0.96 (± 0.16 s.d.). Importantly, the ratio
between the cell number in mutant and wild-type sister clones did
not change as a function of the clone size (Fig. 1G). Furthermore,
staining for mitosis and apoptosis markers phospho-histone H3 and
cleaved caspase 3, respectively, also showed that VHL mutation does
not affect cell proliferation or caspase-mediated apoptosis (see Fig.
S1 in the supplementary material).
1496 RESEARCH ARTICLE
Development 137 (9)
at stage 4 (Fig. 2H,H⬘). Co-staining of aPKC and Arm confirmed
that, in VHL mutant cells at stage 4, apical aPKC is greatly
diminished but Arm appears normal (see Fig. S2B in the
supplementary material). Arm distribution became abnormal in VHL
mutant cells after stage 6 of oogenesis. As shown in Fig. 2I,I⬘, loss
of VHL caused a pronounced alteration of the epithelial monolayer
(swelling as an example) and Arm overaccumulated to the apical
and basolateral membrane domains in 89% (n27) of stage 8-10
clones (Fig. 2I⬘, arrows). The pattern of Arm spreading is consistent
with the notion that apical and SAR polarity proteins are crucial for
the formation and restriction of AJ at the apicolateral domain of the
epithelial cells.
In Drosophila follicle cells, as in other epithelial tissues,
antagonistic interaction between the PAR complex and the
basolateral Lgl complex is necessary for the establishment and
maintenance of epithelial polarity (Bilder et al., 2003; Hutterer et
al., 2004; Tanentzapf and Tepass, 2003). Lgl can compete with
Baz/PAR-3 for binding to aPKC-PAR-6 and destabilize the PAR
complex (Yamanaka et al., 2003). Conversely, aPKC
phosphorylates Lgl, resulting in its dissociation from the
membrane domain (Betschinger et al., 2005). In wild-type
epithelial cells, Lgl is located in the lateral membrane domain with
enrichment toward the basal side. At stage 4 in the homozygous
VHL1 clones, Lgl was slightly reduced but present on the
basolateral domain (Fig. 2J,J⬘), but greatly reduced from the
membrane domain in 90% (n29) of stage 8-10 clones (Fig.
2K,K⬘). Interestingly, it has been shown that a functional but
mislocalized aPKC-PAR-6 complex could still phosphorylate Lgl
in the cytoplasm (Hutterer et al., 2004). It is therefore probable that
the mislocalized aPKC in VHL mutant cells can phosphorylate Lgl
and prevent its membrane localization.
We have previously shown that RNA interference-mediated
knock-down of VHL resulted in defects in tracheal development
during embryogenesis (Adryan et al., 2000). Homozygous VHL
‘escaper’ mutant flies (~10% of adult progenies from heterozygous
cross) can be recovered by re-expression of the VHL cDNA
sequence from the breathless (btl) gene promoter, which is specific
for the trachea and glial cells. As btl is not expressed in the follicle
cells, we reasoned that the rescued adult VHL1 homozygous females
should exhibit severe epithelial defects in the follicle cells and serve
as a confirmation of the VHL clonal phenotypes. We first confirmed
that overexpression of VHL from the btl promoter in wild-type
females did not cause epithelial defects (Fig. 3A). Conversely, the
VHL homozygous females rescued by btl>VHL were sterile, in
which 70% of the ovarioles contained only degenerated egg
chambers. In the rest, we observed very severe epithelial defects in
their egg chambers, including prominent expansion of the
epithelium into the germ cell proper (Fig. 3B,C, arrows). In other
cases, the entire epithelium became multilayered as shown in the
optical cross-section through the center of the egg chamber in Fig.
3D. The invading follicle cells showed a loss of apical specification
as Arm was expressed evenly throughout the cell peripheries (Fig.
3B,C, arrows). In the severely multilayered epithelium (Fig. 3D),
DEVELOPMENT
Fig. 2. Altered epithelial marker expression in VHL mutant cells.
Egg chambers containing VHL1 clones (no GFP) were dissected and
stained for indicated epithelial markers (red). (A,A⬘) A stage-4 egg
chamber showing significantly reduced aPKC in mutant cells (single
cells are marked by empty arrows and multi-cell clones are marked by
the dotted line) compared with the neighboring normal cells. (B,B⬘) A
stage-10A egg chamber showing a mutant clone (dotted line) with no
aPKC expression (empty arrow). (C-C⬙) A stage-9 egg chamber showing
cytoplasmic accumulation (C⬘) and perinuclear accumulation (sharp
arrows in C⬙) of aPKC. Dotted lines mark the mutant clones. (D,D⬘) A
stage-4 egg chamber. The VHL1 mutant clone (dotted line) shows some
lateral spreading (arrow) but retains the proper apical localization of
Baz. (E,E⬘) A stage-10A egg chamber. VHL1 mutant clones (dotted lines)
either lose Baz expression (empty arrows) or show perinuclear
localization of Baz (sharp arrows in ⬘). (F,F⬘) A stage-4 egg chamber.
The VHL1 mutant clone (dotted line) shows slightly reduced apical Crb.
(G,G⬘) A stage-8 egg chamber. The VHL1 mutant clone (dotted line)
shows basally localized Crb and disrupted epithelium (arrows). (H,H⬘) A
stage-4 egg chamber. The VHL1 mutant clones (dotted lines) show
normal distribution of Arm. (I,I⬘) A stage-9 egg chamber. The VHL1
mutant clone (dotted line) shows apical and lateral overaccumulation of
Arm and swelling of the epithelium (arrows). (J,J⬘) A stage-4 egg
chamber. The VHL1 mutant clones (dotted lines) show slightly reduced
Lgl (empty arrows). (K,K⬘) A stage-10B egg chamber. The VHL1 mutant
clone (dashed line) shows greatly diminished expression of Lgl (empty
arrow). Scale bars: 20m.
VHL regulates epithelial morphogenesis
RESEARCH ARTICLE 1497
Fig. 3. Homozygous VHL1 egg chambers show severe epithelial
defects. Lethality of homozygous VHL1 is rescued by btl-driven VHL
transgene. (A)A control egg chamber from wild-type females
expressing VHL from the btl promoter. (B-F)Ovaries from surviving VHL1
homozygous females expressing btl>VHL. Egg chambers were
processed for staining for Arm and aPKC (A-D) or -tubulin and aPKC
(E,F) and ToPro-3. (B,C)A stage-7 and a stage-8 egg chamber,
respectively, showing piling-up of follicle cells and expansion of the
epithelium into the germ cell proper (arrows). (D)A stage-10B egg
chamber. The entire follicular epithelium is multilayered. The nurse cell
complex has collapsed (bracket). (E,F)Two examples of rescued VHL
mutant egg chambers showing greatly diminished MTs and aPKC. All
images were confocal optical cross-sections through the center of the
egg chambers. Scale bars: 20m.
Arm overaccumulated in the apical domain in the inner layer facing
the germline complex and spread throughout the lateral membrane
domains in the stacking layers. We also observed a collapsed nurse
cell complex (Fig. 3D, bracket), which, in the wild-type egg
chamber, occupies the anterior half of the egg chamber at stage 10.
This might be the result of germline cytoskeletal defects or the result
of structural defects in the squamous follicle cells that cover the
nurse cell complex. In all egg chambers examined, aPKC was
mislocalized (cytoplasmic localization or spreading throughout the
lateral membrane) and greatly reduced.
VHL functions via stabilizing MTs
Drosophila aPKC can control epithelial polarity during
epithelialization of embryonic blastoderm by regulating MT
organization (Harris and Peifer, 2007). In human cells, VHL has
been shown to interact with the PAR-3–PAR-6–aPKC complex
(Schermer et al., 2006), which in turn regulates MT–cortex
interactions and coordinates growth of cortical MTs. Conversely,
because MTs are a major transport route for epithelial markers
(Musch, 2004; Rodriguez-Boulan et al., 2005), the disruption of MT
might indirectly affect aPKC localization. To distinguish between
these two possibilities, we first analyzed MT distribution in aPKC
mutant clones. Homozygous aPKC mutant clones, genetically
marked by the absence of GFP and verified by lack of aPKC
antibody staining, showed disorganized MT structure but not
diminished MTs (Fig. 4E,F). Even at later stages (>stage 8) when
Drosophila VHL mutant clones showed consistent loss of MTs,
aPKC mutant cells exhibited a disorganized MT network but no loss
of MTs (Fig. 4G). Closer examination showed that instead of
organized cortical bundling (Fig. 4G⵮), the aPKC mutant cells
exhibited a dotted pattern of MT structure (Fig. 4E⬘,G⬙, empty
arrowheads), indicating a truncated MT structure. Other aspects of
the VHL mutant phenotypes, such as piling up (Fig. 4E, arrows) and
stretching or gaps in epithelium (Fig. 4F,H, brackets), were
observed. Therefore, the aPKC mutant exhibits some, but not all, of
the phenotypes in the VHL1 mutant.
We next examined whether disruption of MTs can directly
influence aPKC localization. Wild-type egg chambers were
dissected and cultured ex vivo in the presence of DMSO (control),
paclitaxel (MT stabilizing agent) or nocodazole (MT destabilizing
agent). After a 5-hour incubation, stage 8-9 egg chambers were
examined for expression of aPKC and -tubulin. Egg chambers
treated with DMSO (Fig. 5A) showed normal organization of the
MT network (Fig. 5A⬘) and normal epithelial structure (Fig. 5A⬙),
with apically enriched aPKC (Fig. 5A). Paclitaxel increased the
levels of MT bundles throughout the egg chamber (Fig. 5B⬘),
DEVELOPMENT
MT defects
It has been shown that human VHL can promote MT stability
against MT destabilizing agents colcemid and nocodazole
(Hergovich et al., 2003; Lolkema et al., 2004), although the
physiological role of this activity has not been elucidated. We
therefore examined whether MT organization is affected in
Drosophila VHL mutant follicle cells. In many organ-associated
epithelial cells such as the follicle cells, MTs are not centrosomeanchored, but are instead organized in cortical bundles parallel to the
apicobasal axis, with the plus-end localized basally (Bartolini and
Gundersen, 2006). In follicle cells, an apical meshwork of MTs also
exists. As shown in Fig. 4, VHL1 mutant cells already displayed
either disorganization or loss of MTs in pre-stage 6 clones (Fig. 4AB⬘), in step with loss of apical aPKC (Fig. 4A⬙,B⬙). Loss of MTs
along the lateral cortex became more obvious in later egg chambers
(Fig. 4C). The MT defects were observed in all VHL1 clones. It has
been shown that unbundled MTs are lost during fixation with
standard immunostaining protocol (e.g. 4% paraformaldehyde) but
can be preserved and detected using more extensive fixation/crosslinking conditions (e.g. 8% paraformaldehyde) (Theurkauf, 1994).
Surprisingly, even with 8% paraformaldehyde fixation, VHL mutant
cells still exhibited loss of MTs (Fig. 4D,D⬘), suggesting that either
the stability of MTs in VHL1 mutant cells was severely compromised
or the plus-end polymerization became defective. In all VHL1
homozygous egg chambers from btl>VHL-rescued mutant females,
MT was barely detectable (Fig. 3E,F).
1498 RESEARCH ARTICLE
Development 137 (9)
especially in the nurse cells (yellow arrow). Follicle cells were
somewhat thickened but maintained the monolayer characteristics
(Fig. 5B⬙). aPKC localization was also normal (Fig. 5B). By
contrast, nocodazole treatment greatly diminished MTs throughout
the epithelium (Fig. 5C⬘,D⬘,E⬘) and caused stacking (Fig. 5C⬙,E⬙,
sharp arrows). Concomitantly, aPKC was either diminished or
mislocalized, not only in cells that showed epithelial defects (Fig.
5C, sharp arrows), but also in morphologically normal epithelium
(Fig. 5C, arrows; Fig. 5D). Actin filaments showed some
disorganization but were largely intact (Fig. 5E), indicating that
nocodazole treatment for 5 hours does not cause general
disintegration of the follicular epithelium. In nocodazole-treated
samples, follicle cells without disintegration of epithelial structure
were shown to demonstrate that aPKC and MT disruption is the
direct result of nocodazole treatment, not downstream indirect
effects from epithelial destruction. Our crucial observation is that
there is 90% (n20) correlation between MT disruption and aPKC
reduction. We therefore conclude that MT destabilization can
directly cause aPKC mislocalization similar to that observed in the
VHL mutant in vivo. It is interesting to note that these defects
occurred in stage 9-10 egg chambers within 5 hours of nocodazole
treatment without cell division (follicle cell division ceased after
stage 6). This suggests that maintenance of epithelial integrity is a
dynamic and continuous process, for which MTs are crucially
important.
To further demonstrate that VHL function in follicle cells is
mediated by MT organization, we examined whether stabilizing
MTs can rescue VHL phenotypes. As shown in Fig. 5F,G, ex vivo
cultured egg chambers containing VHL1 mutant clones (Fig. 5F⬙,G⬙,
dashed lines) were treated with either solvent (DMSO) or paclitaxel.
Homozygous VHL1 clones from stage 9 egg chambers without drug
treatment showed that MTs are compromised in the mutant cells,
with significant loss along the lateral cortex (Fig. 5F⬘), consistent
with the in vivo VHL phenotype. In 100% (n20) of VHL1 clones,
aPKC was almost completely lost from the apical domain (Fig. 5F).
By contrast, in 80% of the homozygous VHL1 clones (n20) from
egg chambers treated with paclitaxel, MTs recovered and their
correct distribution was restored (Fig. 5G⬘). Importantly, within
these rescued clones, the correct apical localization of aPKC was
invariably (100%) re-established (Fig. 5G,G⵮, arrows).
Because aPKC can also regulate MT organization, we tested
whether paclitaxel can also rescue the MT phenotype in aPKC
mutant cells. Interestingly, only 45% of the aPKC mutant clones
(n20) could be rescued under the same treatment condition as for
the VHL1 clones described above (Fig. H,I). This indicates that
aPKC regulates overlapping as well as different aspects of MT
organization than VHL. Taken together, the above results
demonstrate that the epithelial function of VHL is mainly mediated
via stabilizing MTs, although we cannot exclude the possibility that
VHL can also influence the stability of aPKC as 45% of aPKC
mutant clones can be similarly rescued and 20% of the VHL mutants
cannot be rescued by paclitaxel treatment.
VHL regulates MTs and aPKC stability
The functional relationship among VHL, aPKC and MTs was further
tested in cultured Drosophila S2 cells and in ovaries expressing the
VHL full-length sequence or VHL RNA interference construct
(VHLi) under heat-shock promoter control. Protein extracts were
DEVELOPMENT
Fig. 4. MT defects in VHL1 mutant cells.
(A-D⬘) Ovaries containing VHL1 clones (no GFP) were
dissected and egg chambers stained for -tubulin and
aPKC (A-B⬙) or -tubulin and nuclei (ToPro-3; C-D⬘). The
ovary in D was fixed with 8% formaldehyde. All others
were with 4% formaldehyde. (A-A⬙) A stage-4 egg
chamber showing VHL1 clones (dotted lines) with
mislocalized and diminished MTs (A⬘). aPKC is reduced
in these mutant cells (A⬙). (B-B⬙) A stage-5 egg chamber
showing a stretched epithelium (bracket). A VHL1 clone
(dotted lines) shows diminished MTs and aPKC (empty
arrows) compared with normal cells (arrows). In
stretched epithelial cells (bracket), residual MTs occupy
very thin cytoplasmic space, thus appearing condensed
and stained strongly. (C,C⬘) A stage-10A egg chamber.
A VHL1 clone (dotted line) shows loss of MTs along the
lateral cortex. (D,D⬘) A stage-7 egg chamber. Even with
8% formaldehyde fixation, MTs are still lost in the VHL1
clone (dotted line). (E-H⬘) Ovaries containing aPKC
clones were dissected and egg chambers stained for tubulin, GFP and DNA (ToPro-3 in E) or aPKC (F-H).
(E,E⬘) A stage-4 egg chamber. MTs are not diminished in
the mutant cells (dotted line) but show a dotted pattern
(empty arrowheads). Arrows point to mutant regions
that show the piling-up phenotype. (F,F⬘) A stage-6 egg
chamber containing two aPKC clones (dotted lines),
which show abnormal cell shape or stretched
epithelium (brackets). MTs are not diminished in either
case. (G-G⵮) A stage-9 egg chamber containing 2 aPKC
clones (dotted lines). The aPKC mutant clones show
disorganized and truncated (dotted lines) MT pattern
(empty arrowhead) compared with the normal bundles
(G⵮). (H,H⬘) A stage-9 egg chamber showing a gap in
the epithelium (brackets). Scale bars: 20m.
VHL regulates epithelial morphogenesis
RESEARCH ARTICLE 1499
subjected to western blotting. Overexpression of full-length VHL
increased the VHL protein level modestly and expression of VHLi
significantly reduced VHL protein levels in both S2 cells (Fig. 6A)
and in ovaries (Fig. 6B). In S2 cells, overexpression of VHL
increased the levels of aPKC and PAR-6, whereas knockdown of
VHL greatly reduced their levels (Fig. 6A). However, the effects of
altered VHL levels on the levels of aPKC and PAR-6 were present
but less pronounced in vivo (Fig. 6B). Also interestingly, although
VHL knockdown did not alter the total cellular tubulin level in S2
cells, in ovaries, VHL knockdown greatly reduced tubulin levels,
consistent with the immunostaining results (Fig. 4). Perhaps in cell
culture, the MTs are less prone to dynamic instability and therefore
more stable. This is particularly true when compared with follicular
epithelium, in which the epithelial sheet movement at stage 9,
accompanied by rapid growth into a columnar shape, requires
extensive remodeling and turnover of cytoskeletons.
We next examined whether VHL can interact with tubulin and
aPKC in vivo. For this purpose, we used transgenic flies expressing
aPKC-GFP fusion protein, which has been shown to be fully
functional (Tian and Deng, 2008). Ovarian extracts expressing
aPKC-GFP fusion proteins were processed for immunoprecipitation.
As shown in Fig. 6C, aPKC-GFP pulled down its known partner
DEVELOPMENT
Fig. 5. MT instability leads to aPKC localization and
epithelial defects. (A-E⬙) y w (representing wild type),
(F-G⵮) VHL1 clone-containing or (H-I⵮) aPKC mutant clonecontaining egg chambers were cultured ex vivo and
treated with DMSO (A,F), paclitaxel (3.2M for B and
6.4M for G-I) or nocodazole (20M, C-E). Five hours
after drug treatment, the egg chambers were processed
for staining for -tubulin, nuclei (ToPro-3) and either aPKC
or actin (Phalloidin) as indicated. (A-A⬙) A stage-9 egg
chamber treated with DMSO showing normal epithelium.
(B-B⬙) A paclitaxel-treated stage-10A egg chamber
showing increased level of MT bundles, especially in the
germ cells (yellow arrow). aPKC expression is normal (B).
There is some thickening of the follicle cells but the
epithelium maintains the monolayer morphology (B⬙).
(C-C⬙) A nocodazole-treated stage-10A egg chamber
showing diminished aPKC and MTs either in a region
showing stacking of follicle cells (sharp arrows) or in a
morphologically normal region of the epithelium (arrows).
(D-D⬙) A nocodazole-treated stage-9 egg chamber
showing diminished aPKC (D) and MTs (D⬘) in the entire
epithelium (also see merged view in D⬙). (E-E⬙) A
nocodazole-treated stage-8 egg chamber. MTs are
diminished (E⬘) and the epithelium shows multilayered
phenotypes (sharp arrows). Actin filament staining shows
that although the epithelial structure is disrupted, the drug
dosage used does not cause general disintegration of the
follicle cells. (F-F⵮) Enlarged view of a DMSO-treated stage9 egg chamber containing a VHL1 clone (dashed lines;
identified as lack of GFP in F⬙). MTs are diminished along
the lateral cortex of the mutant cells (inset) and aPKC is
also diminished (F,F⵮). (G-G⵮) Enlarged view of a paclitaxeltreated stage-9 egg chamber containing a VHL1 clone
(dashed lines; lack of GFP in G⬙). The MT bundles are
restored in the mutant cells and aPKC regains the apical
localization (arrows). The percentage in G⵮ indicates that
80% of the VHL1 mutant clones (n20) are rescued by the
treatment. (H-I⵮) Egg chambers containing aPKC mutant
clones (dotted lines; lack of aPKC in H and I and lack of
GFP in H⬙ and I⬙) were treated with paclitaxel as above. The
treatment restores MT bundles (arrow in H⬘) in 45% of the
clones (H⬙). The rest still exhibit disorganized and dotted
MT pattern (inset in I⬘). Scale bars: 20m.
1500 RESEARCH ARTICLE
Development 137 (9)
Fig. 6. VHL interacts with MTs and aPKC. Head-to-head VHL cDNA
duplex (dVHLi) or full-length VHL (dVHL) were cloned into the pCaSpe-hs
vector and transfected into Schneider (S2) cells or used to generate
transgenic flies. (A)Lysates from S2 cells transfected with control plasmid
or vectors expressing full-length VHL or VHL duplex were westernblotted for indicated proteins. (B)Ovaries from females carrying hs-VHL
(two lines) or VHLi were dissected and protein lysates processed for
western blotting with the indicated antibodies. (C)Protein lysates from
ovaries carrying Cy2-Gal4/UAS-aPKC-GFP were processed for
immunoprecipitation (IP) and probed with the antibodies indicated on
the left. Input denotes western blots of total protein lysates without IP.
Baz, but not tubulin (left panels), while VHL was coimmunoprecipitated by either -tubulin or aPKC-GFP (right panels).
Interestingly, VHL interactions with MTs and aPKC are probably
not within the same complex as the majority of aPKC does not
associate with microtubules (aPKC-GFP does not bring down
tubulin). These results are consistent with a direct regulatory
function of VHL on MTs and suggest an additional VHL function in
directly regulating aPKC stability.
To test more specifically the significance of reduced aPKC
protein level in VHL mutant cells, we examined whether
overexpression of aPKC can rescue the VHL knockdown phenotype.
UAS-aPKC-GFP; hs-VHLi flies were crossed with flies from the
Cy2-Gal4 line, which expresses the Gal4 transgene in all the follicle
cells covering the oocyte from stage 8 (Queenan et al., 1997). Heatshock-induced VHL knockdown was activated by heat-treatment of
flies at 37°C for 1 hour per day for 4 days. After 12 hours at 29°C
from the fourth heat-shock treatment (29° promotes the expression
aPKC-GFP transgene), ovaries from females carrying hs-VHLi and
UAS-aPKC-GFP or hs-VHLi alone (both with Cy2-Gal4) were
dissected and analyzed. The piling up phenotype was tabulated from
stage 4 to 9. When quantifying the extent of rescue by exogenously
expressed aPKC-GFP, only cells expressing GFP were examined.
That is, an egg chamber containing pile-up follicle cells in the nonGFP-expressing region was not counted; these pile-up follicle cells
cannot be regarded as ‘not rescued’ as they do not express aPKCGFP. In 50% of stage 4 and 5 hs-VHLi egg chambers analyzed
(n30), follicular epithelium lost the coherent single-layered shape
after heat-shock treatments and endogenous aPKC was either
reduced or absent in the affected epithelium (see Fig. S3 in the
supplementary material). The same percentage of mutants was
obtained for the hs-VHLi flies with UAS-aPKC-GFP expression
(n30) in stage 4-5 egg chambers. This is expected as the Cy2-Gal4
driver is only expressed in the follicular epithelium after stage 8.
A disease-related VHL mutant defective in MT
stabilization cannot rescue the aPKC localization
phenotype
A few disease-causing mutations in human VHL have been shown to
ameliorate the ability of VHL to promote MT stability in cultured
cells (Hergovich et al., 2003). One of these amino acid substitutions
affects a highly conserved tyrosine residue (Y98 to histidine). We
generated the equivalent mutation in Drosophila VHL (Y51 to H in
the Drosophila counterpart) as a hemagglutinin fusion protein. The
wild-type VHL-HA transgene (VHLwt-HA) is functional as it can
rescue VHL1 phenotypes in 71% of the VHL mutant clones (n24),
whereas the Y-to-H mutant (VHLYH-HA) cannot in 100% of the
clones examined (n26; see Fig. S4 in the supplementary material).
Anti-HA staining in VHLwt-HA-expressing egg chambers showed
punctate cytoplasmic localization of the wild-type VHLwt-HA fusion
protein (see Fig. S5A in the supplementary material). There was
substantial co-localization of VHLwt-HA and the cortical MT
bundles along the lateral membrane, in agreement with the
immunoprecipitation result, although not all VHL punctae were
associated with MTs (see Fig. S5A in the supplementary material).
Consistent with the property of the human Y98H mutant protein,
Drosophila VHLYH-HA retained, at least partly, its ability to colocalize with the MT (see Fig. S5B in the supplementary material).
Importantly, the expression levels of the wild-type and YH mutant
VHL proteins were the same in the follicle cells, measured by ImageJ
software analysis of confocal images of two-cell clusters from five
different same-stage egg chambers of each strain. VHL also showed
localization along the MT bundles in cultured S2 cells (see Fig. S5B
in the supplementary material). The effects of the wild-type and YH
mutant VHL on MT stability were further tested ex vivo. As shown
before, nocodazole treatment on a normal egg chamber disrupted the
MT organization and reduced the overall levels of MT bundles
(compare Fig. 7A,B). Overexpression of VHLwt-HA counteracted the
destructive effects of nocodazole (compare Fig. 7B-D). By contrast,
the VHLYH-HA mutant had only limited effect (compare Fig. 7E,F).
The MTs in these follicle cells contained only a mesh in the apical
region. These observations are quantified in Fig. 7G. Interestingly,
without intact MT bundles, the VHLYH protein itself became
mislocalized. It did not colocalize with the disorganized MT and
became more nuclear (Fig. 7F, insets). This indicates that cytoplasmic
distribution of the VHL protein is itself MT-dependent.
DISCUSSION
The role of VHL and MTs in epithelial
morphogenesis
In this report, we show that Drosophila VHL is important for
establishing and maintaining epithelial integrity via its regulation of
MT and aPKC stability. We observed MT disruption and epithelial
DEVELOPMENT
This set of early eggs serves as a convenient internal control.
Conversely, 60% of stage 9 egg chambers analyzed (n36) from hsVHLi females displayed a multilayered phenotype, whereas 43% of
stage 9 egg chambers from UAS-aPKC-GFP-expressing flies (n36)
displayed the same phenotype. The extent of rescue, although
notable, is not statistically significant (based on Pearson’s Chisquared test, P0.2357; see Fig. S3 in the supplementary material).
The partial rescue might be because of an insufficient expression
level of aPKC-GFP. This is not probable, however, as the same
Cy2-Gal4-driven aPKC-GFP can rescue an aPKC mutant (data not
shown). More plausible is that the direct stabilizing (versus proper
localization) action of VHL on aPKC plays an appreciable but minor
role in epithelial morphogenesis.
VHL regulates epithelial morphogenesis
RESEARCH ARTICLE 1501
phenotypes early in oogenesis. This indicates that MT bundles in
developing epithelial cells are crucial for epithelial development and
are under pressure from dynamic instability. Without stabilizing
activity provided by VHL, MTs are disorganized and ultimately
disintegrate, resulting in loss of epithelial integrity. We show that
disrupted MTs interfere with proper localization of aPKC, which in
turn leads to mislocalization of downstream epithelial markers and
epithelial defects. Our ex vivo experiment also demonstrates that
epithelial defects can occur within a short time (relative to the entire
oogenesis time frame) after destabilizing MTs in non-proliferating
epithelial cells. This indicates that the maintenance of epithelial
integrity is a dynamic and continuous process even in a stable
epithelium, for which MTs are crucially important. Previous studies
using RNA interference-mediated knockdown demonstrated a
morphogenic role of VHL in trachea development (Adryan et al.,
2000; Mortimer and Moberg, 2009). The tracheal phenotypes appear
to be the result of elevated cell motility and ectopic chemotactic
signaling. Therefore, the tracheal function of VHL might be
mediated via different VHL targets in a tissue-specific context.
Alternatively, regulation of MT stabilization might also be the
underlying mechanism. We favor a separate, tissue-specific function
for VHL as the tracheal defects in VHL knockdown can be relieved
by decreased expression of breathless, which encodes the
chemotactic signaling receptor in the trachea. The two VHL
functions, however, are not necessarily mutually exclusive. These
different organ systems might in the future serve as a model for
testing whether the various functions assigned to VHL are tissuespecific and context-dependent.
Human VHL has been shown to translocate aPKC to MTs,
thereby influencing MT reorganization (Schermer et al., 2006). In
this study, we show that the aPKC mutant can affect MT
organization but not stability, whereas VHL can influence both.
Conversely, we show that disruption of MTs alone can result in
aPKC mislocalization resembling that observed in VHL mutant
cells. Importantly, paclitaxel-induced MT stabilization can rescue
aPKC localization in VHL mutant follicle cells. We therefore
conclude that a major function of VHL in the follicular epithelium is
regulation of MT stability. Loss of MTs leads to aPKC
mislocalization and degradation. Conversely, our results also
indicate that part of the VHL epithelial functions might be mediated
by its direct effect on aPKC stability, as exogenously expressed
aPKC-GFP fusion protein can partially rescue (not statistically
significant) the VHL mutant phenotype. Indeed, we also demonstrate
that VHL can co-immunoprecipitate with tubulin or aPKC, and that,
at least in S2 cells, aPKC levels can be affected by VHL levels
without affecting tubulin. Taken together, it appears that the
epithelial function of VHL is mediated through stabilization of MT,
with an auxiliary role in directly stabilizing aPKC.
It has been suggested that VHL interacts with MTs via the
kinesin 2 family of motors (Lolkema et al., 2007). Future studies
using our epithelial system should also address this issue in vivo.
Also interestingly, we show that the YH mutant can associate with
MT but has little MT-stabilizing activity. This suggests that the YH
mutant might be defective in recruiting other proteins, possibly
including aPKC, that are important for regulating MT functions. In
light of the role of Drosophila VHL in regulating MT stability, a
function presumably important for all cells, it is curious that the
tissue-specific btl-driven VHL expression can rescue the
homozygous lethality of VHL1. We have shown that tracheal
defects are the major embryonic phenotype observed in VHL
mutant (Adryan et al., 2000) (data not shown). In the course of
attempting to rescue the tracheal phenotype with btl-driven VHL
DEVELOPMENT
Fig. 7. A VHL disease-related mutant
defective in MT stabilization cannot
restore MT stability against
nocodazole. Control flies (Cy2-Gal4) or
flies expressing VHLwt-HA or VHLYH-HA
were conditioned at 25°C with live yeast
for 1 day and incubated at 29°C for 5
days to induce transgene expression
before the ovaries were dissected and
egg chambers cultured ex vivo. Cultured
egg chambers were treated with DMSO
(A,C,E) or nocodazole (B,D,F) for 5
hours and processed for staining for tubulin, HA and nuclei (ToPro-3). Apical
sides of the follicle cells are marked in
the insets (ap). Nocodazole treatment
significantly reduced MTs in control egg
chambers (compare B with A). VHLwt-HA
can restore the stability of MTs in the
presence of nocodazole (compare D
with C and B), whereas VHLYH-HA
cannot (compare F with E).
(G)Quantification of MT phenotype.
Statistical analysis is based on Pearson’s
Chi-squared test, in which P<0.01 is
considered significant. Scale bars:
20m.
(A.H. and T.H., unpublished), we noted the appearance of rescued
homozygous adults. This indicates that the MT stabilizing function
of VHL is not required in all tissues. It is possible that although
VHL can enhance MT stability, by itself it is not an essential factor
for MT polymerization. As such, some tissues might be less
dependent on VHL levels. In the follicular environment, MT
rearrangement, including depolymerization and repolymerization,
is crucial when the entire epithelial sheet moves over the germ cell
complex while the cells grow increasingly columnar. MT
stabilization facilitated by VHL might be of particular importance
during this process.
Tumor-suppressor function of VHL
The best-documented function of VHL is its E3 ubiquitin ligase
activity that targets the alpha subunit of the HIF transcription factor.
This activity provides an elegant mechanistic explanation for the
hypervascularity of many of the VHL tumors and for a potential
contributor to the metabolic switch to glycolysis, as HIF can
upregulate pro-angiogenic factors such as vascular-endothelial
growth factor and components in the glucose metabolic pathway.
However, recent evidence has suggested that VHL is a
multifunctional protein. It can function as a regulator of matrix
deposition (Ohh et al., 1998), integrin assembly (Esteban-Barragan
et al., 2002), endocytosis (Champion et al., 2008; Hsu et al., 2006),
kinase activity (Yang et al., 2007), senescence (Young et al., 2008),
protein stabilities (Chitalia et al., 2008; Roe and Youn, 2006) and
tight junction formation (Calzada et al., 2006; Harten et al., 2009),
among many others (Frew and Krek, 2007). Whether tight junction
disassembly in VHL mutant cells is HIF-dependent is still
unresolved; however, other – HIF-independent – functions appear
to facilitate protein stability or activity instead of destabilizing them
as a ubiquitin ligase. Such chaperon/adaptor function has also been
implicated in promoting stability of MTs (Hergovich et al., 2003;
Lolkema et al., 2004). The MT-stabilizing function, although
potentially highly significant, has so far only been linked to cilium
biogenesis and mitotic spindle orientation in cultured RCC and renal
tubule cells (Esteban et al., 2006; Lolkema et al., 2008; Lutz and
Burk, 2006; Schermer et al., 2006; Thoma et al., 2009). The
physiological and developmental significance of this function has
not been elucidated in vivo. Indeed, it is unclear how loss of many
of these HIF-independent functions contributes to VHL tumor
formation because of a lack of tractable genetic models.
One crucial element in tumorigenesis is the breakdown of
epithelial integrity that ultimately leads to epithelial-tomesenchymal transition. This report provides the first demonstration
of a potential tumor-suppressor function for VHL in regulating
epithelial morphogenesis via its role in promoting MT stability.
Future studies should exploit further this genetic system for
elucidating how a myriad of disease-related VHL point mutations
might differentially influence such function.
Acknowledgements
We thank W. M. Deng (Florida State University), D. Grifoni (University of
Bologna), T. Schüpbach (Princeton University) and A. Wodarz (Georg-AugustUniversity Göttingen) for generous gifts of antibodies and fly stocks. K.
Lavenburg contributed to the initial cloning of the Drosophila VHL genomic
sequence for subsequent mutagenesis. The work was supported by grants
from the National Institutes of Health (PO1CA78582 and RO1CA109860) to
T.H., a grant from the University of Bologna (RFO 2007) to G.G. and V.C. and a
Marco Polo Fellowship from the University of Bologna to S.D. Deposited in
PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Development 137 (9)
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.042804/-/DC1
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DEVELOPMENT
VHL regulates epithelial morphogenesis