Crag Regulates Epithelial Architecture
and Polarized Deposition of Basement
Membrane Proteins in Drosophila
Natalie Denef,1Yu Chen,1Stephen D. Weeks,2Gail Barcelo,1and Trudi Schu ¨pbach1,*
1Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
2Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA
The polarized architecture of epithelia relies on an
interplay between the cytoskeleton, the trafficking
machinery, and cell-cell and cell-matrix adhesion.
Specifically, contact with the basement membrane
(BM), an extracellular matrix underlying the basal
side of epithelia, is important for cell polarity. How-
ever, little is known about how BM proteins them-
selves achieve a polarized distribution. In a genetic
screen in the Drosophila follicular epithelium, we
identified mutations in Crag, which encodes a con-
trafficking. Follicle cells mutant for Crag lose epithe-
lial integrity and frequently become invasive. The
loss of Crag leads to the anomalous accumulation
of BM components on both sides of epithelial cells
without directly affecting the distribution of apical
or basolateral membrane proteins. This defect is
not generally observed in mutants affecting epithelial
integrity. We propose that Crag plays a unique role in
organizing epithelial architecture by regulating the
polarized secretion of BM proteins.
Epithelia are organized as sheets of tightly adherent cells with
distinct apical-basal polarity (Muller and Bossinger, 2003). Intact
tissue architecture is vital for their function, and a loss of epithe-
lial organization is often associated with carcinoma progression
and tumor metastasis (Thiery, 2002). The polarized architecture
of epithelial cells is evident from the presence of distinct apical
and basolateral membrane domains that have different lipid
and protein compositions (Mostov et al., 2003; Muller and Bos-
singer, 2003). To establish and maintain these separate mem-
brane domains, newly synthesized and recycled proteins need
to be delivered to the correct location, a process that requires
the sorting of proteins into different apical and basolateral trans-
port vesicles, followed bytheir transport to, and fusion with, spe-
cific regions of the plasma membrane. Protein localization is
subsequently maintained through interactions with the underly-
ing cytoskeleton and other cortical protein complexes and
through intercellular junctions that inhibit diffusion between the
apical and basolateral membrane domains (Mostov et al.,
2003; Rodriguez-Boulan et al., 2005).
In order for apical and basolateral transport vesicles to reach
and fuse with the correct membrane region, initial cell surface
cells and with the extracellular matrix (ECM) (Nelson, 2003;
O’Brien et al., 2002). Of particular interest in this light is the base-
mentmembrane(BM),a specialized sheetofECMcontactingthe
basal side of epithelial tissues. The BM is composed primarily of
the secreted glycoproteins Laminin, Collagen IV, and Nidogen
and the heparan sulfate proteoglycan Perlecan. Interactions
between the BM and epithelial cells are mediated by a variety
of cell surface receptors including integrins and Dystroglycan
organisms and experimentsin culturedcells has indicated an im-
2003; O’Brien et al.,2002). Therefore, the accumulation of BM on
the basal side of the epithelium only is crucial to convey the posi-
tional information necessary to maintain a polarized architecture.
Even though substantial progress has been made in under-
standing the mechanisms that regulate the polarized trafficking
of basolateral membrane proteins (Mostov et al., 2003; Rodri-
guez-Boulan et al., 2005), very little is known about how basally
secreted proteins, such as BM components, are sorted and de-
livered specifically to the basal side of epithelial cells. Different
pathways for the polarized trafficking of basolateral membrane
and secreted proteins have been proposed, yet no components
specifically regulating the polarized secretion of BM proteins
have been identified (Boll et al., 1991; Caplan et al., 1987; Cohen
et al., 2001; De Almeida and Stow, 1991).
To study the establishment and maintenance of epithelial
architecture, we use the follicular epithelium (FE) in Drosophila
melanogaster, which forms a monolayer of somatic cells sur-
rounding the germline during oogenesis (Horne-Badovinac and
Bilder, 2005). The follicle cells (FC) that make up the FE display
a distinct apical-basal polarity, apparent in the presence of dif-
ferent membrane and cortical domains, apically localized adhe-
rens junctions, and the polarized organization of the cytoskele-
ton (Muller and Bossinger, 2003). As in other epithelia, the
basal side of the FE is in contact with the BM. The apical side,
however, contacts the germline rather than a lumen or the ex-
ternal environment. Cues on the basal, apical, and lateral sides
of the FC are required to establish and maintain a fully polarized
epithelial phenotype (Abdelilah-Seyfried et al., 2003; Bilder et al.,
354 Developmental Cell 14, 354–364, March 2008 ª2008 Elsevier Inc.
2000; Cox et al., 2001; Goode and Perrimon, 1997; Manfruelli
etal.,1996; Mullerand Bossinger, 2003;Tanentzapf etal.,2000).
To further elucidate the molecular mechanisms controlling ep-
ithelial organization, we took advantage of the stem-cell-derived
nature of the FE and performed a genetic mosaic screen to iden-
tify genes required for this process. We identified multiple alleles
of Crag (Calmodulin-binding protein related to a Rab3 GDP-GTP
epithelial characteristics and become motile and invasive. Strik-
Crag causes the aberrant accumulation of the BM components
Perlecan, Laminin, and Collagen IV on both sides of the epithe-
lium, a defect we did not generally observe in mutants disrupting
epithelial structure. We propose that Crag plays a unique role in
regulating epithelial architecture by specifically controlling the
polarized secretion of BM proteins. While it has been suggested
that BM components need specific factors to ensure their accu-
rate basal secretion, to our knowledge, Crag is the first protein
identified to be required for such a process.
CJ Mutant Follicle Cells Lose Epithelial Integrity
The Drosophila follicular epithelium (FE) forms a monolayer of
regularly shaped cuboidal cells with their apical sides facing the
developing germline (Figure 1A). To identify novel regulators of
epithelial architecture, we performed a genetic mosaic screen
identified bythe absence of GFP(Figure 1B).One complementa-
leles that cause defects in epithelial organization in follicle cell
(FC) clones. We named this complementation group CJ, after
a representative allele, CJ101. Instead of forming a regular epi-
thelial monolayer, CJ mutant FC accumulate in multiple layers
in which cells adopt round or irregular morphologies rather than
their normal cuboidal or columnar shapes (Figures 1A and 1B).
In addition, CJ mutant FC frequently become motile and invade
the germline cluster (Figure 1C). In a considerable number of
clones, defects appeared less severe, and a bilayer of cells with
epithelial characteristics could be observed (Figure 1D; 45% of
clones with abnormalities, n = 110). Interestingly, we noticed
that the bilayered, multilayered, and invasive regions regularly
comprised a mix of mutant and wild-type cells, suggesting that
mutant cells can cause a loss of epithelial structure in the
surrounding wild-type tissue (Figures 1B, 1C, 1E, and 1F). The
defects in epithelial architecture in CJ mutant clones were only
detected in FC at the poles of an egg chamber (e.g., arrow in
A differential response of FC reflecting their position within the
epithelium is commonly observed in mutants affecting epithelial
architecture (Abdelilah-Seyfried et al., 2003; Goode and Perri-
mon, 1997; Goode et al., 2005; Lee et al., 1997; Tanentzapf
et al., 2000).
Figure 1. CJ Mutant FC Lose Epithelial In-
(A) Wild-type egg chamber stained for F-actin
(red). The follicle cells (fc) have a regular morphol-
round the germline cells (gc). Apical (a) and basal
(b) sides are marked.
(B–D) Egg chambers containing CJ101 mutant fol-
licle cell clones marked by the absence of GFP
tant FC at the poles (arrow) are irregular in shape
and pile up in multiple layers. CJ101 mutant cells
on the lateral sides of the egg chamber (arrow-
head) are indistinguishable from surrounding
wild-type cells or cells in wild-type egg chambers.
(C) Stage 6 egg chamber in which FC have
invaded the germline cluster. Note that a mix of
mutant and wild-type cells is found within the
group of invading cells (arrow). (D) CJ101 mutant
cells at the posterior pole forming two layers main-
taining regular cell shape.
(E–H) Egg chambers containing CJ mutant follicle
cell clones marked by the absence of GFP (green)
and stained for aPKC (E), DE-Cadherin (F), FasII
(G), or Baz and Arm (H). (E) CJ101 mutant cells
show a loss of cortical apical staining of aPKC
([E0], red in [E]) when cells are multilayered (arrow)
but not when mutant cells have normal epithelial architecture (arrowhead). Wild-type (GFP-positive) cells are commonly found intermingled with mutant cells in
cases where severe multilayering is observed. Note that some mutant cells still accumulate high levels of cortical aPKC equivalent to apical levels in wild-type
cells. (F) DE-Cadherin levels ([D0], red in [D]) in multilayered CZ085 mutant FC are similar to the levels at the lateral plasma membrane below the junctions in wild-
type cells. Occasionally, high levels of DE-Cadherin staining can be seen (arrow). In clones at the lateral sides of the cyst, DE-Cadherin distribution appears iden-
tical to that inwild-typecells (arrowhead).(G) CJ101 mutant cells that have lost epithelial morphologyshow membrane staining of FasII ([G0], redin [G]) along their
entire cell circumference (arrow), whereas mutant cells with a normal morphology show a lateral restriction of FasII (arrowheads) as in wild-type cells. (H) CJ101
mutant clonesforming two layers, eachmaintaining the polarized distribution of theapicalcorticalprotein Baz ([H0],redin [H]) and the junctional protein Arm ([H00],
blue [pink in merge] in [H]). Scale bars in this and all subsequent figures represent 10 mm.
Crag Regulates Basement Membrane Deposition
Developmental Cell 14, 354–364, March 2008 ª2008 Elsevier Inc. 355
Epithelial Polarity in CJ Mutant Follicle Cells
Defects in epithelial organization, as observed in CJ mutant FC,
are typically associated with a loss of apical-basal polarity. To
assess epithelial polarity in CJ mutant cells, we analyzed the dis-
tribution of cortical and transmembrane proteins that normally
adopt asymmetric distributions along the apical-basal axis of
the epithelium. We examined the distribution of the apical
markers atypical Protein Kinase C (aPKC), Bazooka (Baz), Par-
6, and dPatj, the adherens junction components Armadillo
(Arm) and DE-Cadherin, and the basolateral proteins Discs large
(Dlg), Lethal giant larvae (Lgl), Fasciclin II, and b-spectrin. In mul-
tilayered CJ mutant clones, aPKC, Baz, Par6, and dPatj failed to
localize to the cortex (Figure 1E, arrow, and data not shown), in-
dicating a loss of apical membrane identity. Occasionally, corti-
cal foci accumulating apical markers were detected, possibly re-
flecting remnants of the apical membrane domain (Figure 1E). In
multilayered CJ mutant cells, Arm and DE-Cadherin were found
at the membrane at low levels, similar to those at the lateral
membrane below the junctions of wild-type cells (Figure 1F
and data not shown). Scattered foci accumulating Arm and
DE-Cadherin athigh levels, similar to those at the adherens junc-
tions of wild-type cells, were also observed (Figure 1F). Finally,
basolateral proteins were found to maintain their cortical locali-
in multilayered CJ mutant cells (Figure 1G, arrow, and data not
shown). Insummary, CJ mutantFC thathave completely lost ep-
ithelial integrity mislocalize apical, junctional, and basolateral
However, CJ mutant clones in which cells maintained their
normal epithelial morphology and monolayer organization dis-
played a wild-type distribution of apical, junctional, and basolat-
eral markers (Figures 1E–1G, arrowheads, and data not shown).
Similarly, in mutant clones adopting a bilayer organization, both
layers displayed discrete apical and basolateral membrane do-
mains without obvious defects in junctional integrity (Figure 1H).
proteins isonlyobservedincases whereepithelialarchitecture is
severely perturbed, the disruption of the apical and basolateral
membrane domains may not be the primary defect in CJ mutant
The Epithelial Defects in CJ Mutants Are Caused
by Mutations in the DENN Domain Protein Crag
We used a combination of meiotic recombination mapping and
complementation analysis with Duplications and Deficiencies
to map the lethal phenotype of CJ mutants and identified the ge-
nomic region around 7F7-8 as the region containing the lethal
point mutations in the coding region of a gene named Crag (for
Calmodulin-binding protein related to a Rab3 GDP/GTP ex-
change protein) (Xu et al., 1998) in which, prior to our study, to
our knowledge, no mutations had been isolated. The Crag locus
encodes two protein isoforms, Crag-PA and Crag-PB, of 1671
and 1644 amino acids, respectively (Figure 2A; Berkeley
Drosophila Genome Project). The strong alleles CragGG43,
CragCJ101, and CragCZ085contain premature stop codons at aa
161, 367, and 659, respectively. CragCB188, a weaker tempera-
ture-sensitive allele, contains a missense mutation in aa 469
changing a glycine residue to an aspartate. The early stop co-
dons in the CragGG43and CragCJ101alleles truncate the protein
before or within conserved protein domains (Figure 2A and see
below). Moreover, CragGG43, CragCJ101, and CragCZ085homozy-
gotes or hemizygotes die in early larval stages similarly to
CragCJ101/Df(1)KA14 transheterozygotes, providing evidence
that these alleles represent a strong or complete loss of Crag
function. A genomic rescue construct, including the Crag gene
and ?1.5 kb upstream and downstream sequences, as well as
HA-tagged Crag-PA and Crag-PB transgenes expressed using
the GAL4/UAS system, fully rescued the lethality and the pheno-
types in the FE. This confirms that the observed phenotypes are
caused by the absence of Crag function (Figures 2B and 2C).
Crag was originally identified as a Calmodulin-binding protein
(Xu et al., 1998) and is highly conserved in vertebrates and inver-
tebrates (Figure 2A) (Semova et al., 2003; GenBank). At their
Figure 2. Epithelial Defects in CJ Mutant FC Are Caused by Muta-
tions in Crag
(A) Schematic representation of the domain organization of the Crag protein
(top)andatabledepictingthepercentages ofsimilarity betweenconservedre-
gions in Crag, representative Crag homologs in different species, and the Dro-
sophila homolog of the DENN-containing protein Rab3-GEF (bottom). Crag
contains an amino-terminal DENN, uDENN, and dDENN domain (red, 1–3)
followed by a region responsible for Calmodulin binding (blue, 4, CaM) and
a conserved C-terminal region (green, 5, Cterm). The location of the nonsense
mutations found in the strong alleles CragGG43, CragCJ101, and CragCZ085and
the missense mutation present in the hypomorphic allele CragCB188are de-
picted with arrows below the protein (top). The percentages of similarity
were calculated using the EMBOSS global or local pairwise alignment tool.
H. sapiens Crag is human DENND4A (c-myc promoter-binding protein, Acc:
NP_005839), one of three Crag homologs in humans. D. rerio Crag is a zebra-
fish Crag homolog (Acc: CAK03709). C. elegans Crag is the protein F52C12.4.
Note that CaM and Cterm are not present in Rab3-GEF (n.p.).
(B) Ovariole of a CragCZ085homozygous female containing a Crag genomic
rescue construct stained for F-actin (red) and DNA (blue). The FE looks entirely
(C) Stage 6 egg chamber containing a CragCJ101mutant clone encompassing
the entire epithelium and expressing a HA-Crag transgene. No epithelial de-
fects are observed. The clone is marked by the absence of GFP (green), and
the egg chamber is stained for F-actin (red) and HA-Crag (blue).
Crag Regulates Basement Membrane Deposition
356 Developmental Cell 14, 354–364, March 2008 ª2008 Elsevier Inc.
amino termini, Crag proteins contain a DENN (for differentially
expressed in normal and neoplastic cells), uDENN (upstream of
DENN), and dDENN (downstream of DENN) domain (Levivier
Thefunctionof theDENNdomain isasyet unknown,butitspres-
notably to a number of Rab GTPases, has raised the possibility
(Levivier et al., 2001). The missense mutation in CragCB188in
a glycine residue that is conserved in nearly all DENN domains
suggests that this domain is critical for Crag function.
The DENN domain in Crag is followed by a region that medi-
ates Calmodulin binding in vitro (Xu et al., 1998). To ask if Crag
also binds Calmodulin in vivo in the FC, we performed coimmu-
noprecipitations between HA-Crag and Calmodulin and found
that Crag can pull down Calmodulin and vice versa in a Ca2+-
dependent manner (Figure S1 in the Supplemental Data avail-
able with this article online). At its carboxy terminus, Crag
contains a domain that is conserved among Crag homologs
but is not found in other proteins.
Crag Localizes to the Plasma Membrane
and Endosomal Compartments
a role for Crag in regulating protein trafficking. To ask whether
Crag protein distribution is consistent with such a role, we raised
antibodies against Crag and, in addition, expressed tagged
formed in the germarium and maintains expression in the entire
epithelium throughout its development (Figures 3A and 3B and
data not shown). The specificity of Crag protein expression
was validated by the absence of staining in Crag mutant clones
Strikingly, in wild-type FC Crag protein accumulates at the
plasma membrane and is found in punctate structures through-
out the cytoplasm (Figure 3C). In early stages of oogenesis, Crag
is present along the apical and lateral cell cortex (Figure 3A). In
later stages, a bias to the lateral cortex becomes apparent,
although Crag staining at or just below the apical surface can
be observed (Figure 3B and data not shown). In all stages, Crag
is clearly absent from the basal cortex (Figures 3A and 3B).
Overexpressed HA-tagged transgenes show a comparable
Figure 3. Crag Expression Pattern and Subcellular Distribution
(A and B) Wild-type egg chambersstained with an antibody against the carboxy-terminal half of Crag (red). (A) Germarium and early stages of oogenesis showing
Crag expression in all FC and FC precursors. Crag is also present in the germline. Note the absence of Crag staining at the basal cortex (asterisk). (B) Cross-
section through the FE of a stage 10 egg chamber shows that Crag accumulates at the lateral cortex. Low levels of Crag protein can also be observed at the
apical (a) cortex, but no Crag is present at the basal (b) cortex.
(C)OpticalsectionthroughtheFEofastage 9eggchamberwithaCragCJ101mutantcloneshowing accumulation ofCragatthecortexandinintracellularpunctae
in wild-type cells ([C0], red in [C]). The specificity of the Crag antibody is evident from the absence of staining in the mutant clone (marked by the absence of GFP,
(Dand E)OverexpressedHA-Crag localizesinthecytoplasmand accumulates atthecortexandinpunctate structuressimilarly toendogenousCrag.(E)HA-Crag
is enriched at the lateral cortex.
(F–I) Optical sections through the FE of stage 8 or 9 egg chambers expressing HA-Crag (red) and GFP-Rab7 (G), GFP-Rab11 (H), GFP-Rab5 (I), or HA-Crag alone
(F). No significant colocalization is observed between HA-Crag and the Golgi protein Lva ([F], green) or between HA-Crag and Rab7-GFP ([G], green). Substantial
overlap is seen between HA-Crag and GFP-Rab11 ([H], green) and between HA-Crag and GFP-Rab5 ([I], green).
(J and K) Optical section through the FE of wild-type stage 9 egg chambers stained for endogenous Crag (red) and Rab11 ([J], green) or Rab5 ([K], green). A clear
overlap is observed between Crag- and Rab11-positive intracellular vesicles ([J], arrowheads). Colocalization between Crag and Rab5 protein is most obvious at
the plasma membrane ([K], arrowheads).
Crag Regulates Basement Membrane Deposition
Developmental Cell 14, 354–364, March 2008 ª2008 Elsevier Inc. 357
(apico)-lateral cortical enrichment and punctate staining in the
cytoplasm (Figures 3D and 3E).
To analyze the nature of the punctate cytoplasmic Crag stain-
ing, we performed colocalization studies with markers for vari-
ous membrane compartments. We did not detect significant co-
localization between HA-Crag and the Golgi marker Lava Lamp
(Lva; Sisson et al., 2000) or the late endosomal marker GFP-
Rab7 (Entchev et al., 2000) (Figures 3F and 3G). We did, how-
ever, observe a substantial overlap between HA-Crag and
GFP-Rab11, which localizes to the recycling endosome (Emery
et al., 2005) (Figure 3H). We also observed colocalization with
the early endosomal marker GFP-Rab5 (Wucherpfennig et al.,
2003) (Figure 3I). To ensure that the observed overlap between
HA-Crag and GFP-Rab5 and GFP-Rab11 was not an artifact
of protein overexpression, we stained for endogenous Crag,
Rab5, and Rab11 proteins and found that Crag colocalizes
strongly with Rab11 in intracellular punctae (Figure 3J). Overlap
between Rab5 and Crag was predominantly observed in punc-
tae at the cell cortex (Figure 3K) but was less compelling in cyto-
Taken together, the presence of Crag protein at the cell cortex
and in endosomal compartments is consistent with a putative
role in trafficking.
Crag Mutant Follicle Cells Show Specific Defects
in the Polarized Distribution of BM Components
The polarized sorting and delivery of newly synthesized and en-
docytosed membrane proteins are crucial for epithelial architec-
ture (Mostov et al., 2003; Rodriguez-Boulan et al., 2005). As
other DENN-domain-containing proteins have been implicated
in the regulation of trafficking and Crag localization is consistent
with such a role, we asked whether the epithelial defects in Crag
mutantFCwere duetoafailureinpolarized trafficking.Wethere-
fore analyzed the distribution of a number of transmembrane
proteins that are normally present on specific membrane do-
mains in the epithelium. We focused ouranalysis on Crag mutant
cells that maintained a normal morphology and monolayer orga-
nization to avoid secondary effects due to a loss of epithelial
structure. We found that the distributions of the apical receptor
Notch, the adherens junction protein DE-Cadherin, and the ba-
solateral adhesion molecules Fasciclin II, Fasciclin III, and Neu-
roglian were indistinguishable in wild-type and Crag mutant FC
that maintained a normal morphology (Figures 4A–4C and data
not shown; n > 20 for each). Since we did not observe obvious
defects in thepolarized distribution of apical, junctional, orbaso-
lateral transmembrane proteins, Crag most likely does not
directly regulate the polarized trafficking of these proteins.
and the BM in controlling epithelial architecture (Li et al., 2003;
(Pcan), a secreted heparan sulfate proteoglycan and major com-
ponent of the BM (Yurchenco et al., 2004). In wild-type egg
chambers Pcan accumulates exclusively on the basal side of
the FE (Figure 4D) (Schneider et al., 2006). Strikingly, Crag
mutant FC deposit Pcan on both their basal and apical sides
(Figures 4E–4G; 100% of stage 6–9 egg chambers with clones,
n > 100). This aberrant localization was seen even in very small
clones, at the poles as well as on the sides of the egg chamber
(Figures 4E–4G). Occasionally, wild-type cells adjacent to cells
lacking Crag accumulated Pcan on their apical side, presumably
due to local diffusion of Pcan secreted by the mutant cells
(Figure 4G, arrow). Abnormal apical Pcan staining was observed
with several Crag alleles, was fully suppressed upon expression
of a genomic rescue construct (0% of stage 6–9 egg chambers
showed apical Pcan, n > 100), and was partially rescued when
a HA-tagged Crag transgene was expressed in cells mutant for
Crag (64% of egg chambers with clones showed no apical
Pcan, 36% showed residual apical Pcan in a fraction of mutant
cells, n= 50) (Figure 4H and data not shown). These data confirm
that the presence of the BM component Pcan on both sides of
the epithelium is due to the absence of Crag activity.
To examine whether the aberrant apical localization of Pcan in
alyzed the distribution of two other major BM components, the
secreted glycoproteins Laminin (Lam) and Collagen IV (Coll IV).
In contrast to wild-type epithelia where Lam and Coll IV are pres-
ent only basally (Gutzeit et al., 1991; Lunstrum et al., 1988), both
proteins were found on the basal as well as the apical side of fol-
licular epithelia with Crag mutant clones (Figures 4I–4K; 87% of
egg chambers with clones showed apical Lam, n = 93; 99% or
100% showed apical Coll IV as visualized by antibody staining
[n = 75] or by Coll IVa2 [=Vkg]-GFP expression [n = 60], respec-
tively). As for Pcan, this defect was observed even when only
a few cells lacked Crag activity (e.g., Figure 4K) and was seen
both at the poles and on the sides of the egg chamber. However,
differences between the distribution of Pcan on the one hand
and that of Lam and Coll IV on the other hand were apparent.
First, we noticed a greater nonautonomy in aberrant Lam and
Coll IV localization, where both proteins were often observed
on the apical side of wild-type cells several cell diameters
away from a clone (Figure 4J and data not shown). Second,
whereas strong apical Pcan staining was typically observed on
all cells within a Crag mutant clone, not all cells in a clone accu-
mulated Lam or Coll IV on their apical side (Figure 4I). Both
observations may be explained by a stronger binding of Pcan
to surface receptors on the apical side of the FC allowing itto ac-
cumulate strongly upon secretion and preventing it from diffus-
ing over longer distances.
To determine whether the anomalous apical deposition of BM
proteins in Crag mutant cells was a specific feature of the FE, or
was a more general defect seen in other epithelia, we examined
Pcan distribution in embryos lacking both maternal and zygotic
Crag expression. Consistent with a more general role for Crag
in regulating the accurate deposition of BM proteins in epithelia,
we found that in late stages of embryogenesis Pcan accumu-
lated aberrantly on the apical side of the epidermis in Crag
mutants (Figure S2).
troglycan and the bPSintegrin subunit. Both receptors are pres-
ent at low levels at the apical plasma membrane of wild-type FC
in addition to their basal localization (Deng et al., 2003; Fernan-
of bPSwere detected in the absence of Crag (data not shown).
Conversely, Dystroglycan appeared to accumulate at higher
levels at the apical plasma membrane of Crag mutant
FC (100% of egg chambers with clones, n = 48) (Figure 4L).
However, this increase in apical localization was not always
Crag Regulates Basement Membrane Deposition
358 Developmental Cell 14, 354–364, March 2008 ª2008 Elsevier Inc.
cell-autonomous and is therefore likely to represent a secondary
effect through stabilization of low levels of apical protein upon
binding to BM proteins.
Taken together, our results demonstrate that Crag is required
to ensure the accumulation of BM components exclusively on
the basal side of epithelia, possibly by regulating their accurate
polarized trafficking. Because this defect is observed in mutant
cells irrespective of their morphology, it is not a consequence
of the loss of epithelial structure but may instead be the primary
tion in Crag mutant FC.
Mislocalization of BM Components Is Not Generally
Observed in Mutants Affecting Epithelial Organization
We next wanted to address whether the aberrant accumulation
of BM proteins on the apical side of the FE is a defect caused
specifically by the absence of functional Crag protein or whether
it is more generally observed in the absence of proteins that
Disrupt the Polarized Distribution of BM
chamber containing CragCJ101clones, marked by
the absence of GFP (green) and stained for Notch
(A), DE-Cadherin (B), or FasIII (C) (red in merged
images [top panels], grayscale in bottom panels).
(A) Notch accumulates at the apical plasma mem-
brane and in vesicles in control (green) and mutant
membrane and at higher levels at the adherens
junctions. No differences were observed between
control and mutant FC. (C) The localization of
FasIII to the (baso)lateral membrane is unaltered
in the absence of Crag.
(D–H) A control egg chamber (D), egg chambers
with CragCJ101clones (E–G), or an egg chamber
with CragCJ101clones coexpressing HA-Crag (H)
stained for Pcan (red in merged images, grayscale
bers without clones, Pcan is seen in intracellular
punctae in the FC and accumulates exclusively
on the basal side of the epithelium. (E and E0) In
Crag mutant clones, Pcan accumulates strongly
on the apical and basal sides of the FE (arrow), in
contrast to the surrounding wild-type cells (arrow-
head) where Pcan is present solely on the basal
side. Note that the mutant cells accumulating
Pcan apically still have a normal morphology and
that apical Pcan is observed at the anterior pole
as well as on the side of the egg chamber. (F)
Pcan accumulates apically of a CragCJ101mutant
clone at the posterior pole of an egg chamber.
Note also that the epithelium is bilayered and
that Pcan is present between the two layers. (G)
Even very small clones show anomalous apical lo-
calization of Pcan. Apical Pcan can also be seen
in wild-type cells adjacent to the clone (arrow;
boundaries of the clone are marked with dashed
lines). (H) No apical accumulation of Pcan is
detected in a Crag mutant clone when HA-Crag
is coexpressed. The boundary of the clone is
marked with dashed lines in the bottom panel.
No difference in Pcan staining is observed be-
tween cells inside or outside the clone.
(I–K) Cross-sections through egg chambers with
CragCJ101clones stained for Lam ([I], red in top
panel, grayscale in bottom panel), Coll IV ([K],
red), or coexpressing Coll IVa2(Vkg)-GFP ([J],
green). Clones are marked by the absence of nu-
4. Crag MutantFC Specifically
clear and cytoplasmic GFP (green). (I) Lam accumulates strongly on the apical side of CragCJ101FC (arrows). Note however that not all cells within the clone
show apical Lam staining (arrowheads). (K) Even a small CragCJ101clone exhibits apical accumulation of Coll IV. (J) Vkg-GFP accumulates on the apical side
of CragCJ101FC (arrowhead) and of adjacent wild-type cells (arrow).
(L) An egg chamber with CragCJ01clones stained for Dystroglycan (Dg, red). Dg accumulates at the basal and apical surface in the absence of Crag. (L0and L00)
Higher magnification of two regions at the boundary between wild-type and mutant cells depicted in (J). Dg levels are increased in mutant cells and in wild-type
cells adjacent to the mutant clones (arrows; boundaries of the clones are marked with a dashed line).
Crag Regulates Basement Membrane Deposition
Developmental Cell 14, 354–364, March 2008 ª2008 Elsevier Inc. 359
ization of Pcan in FC lacking components of three major classes
of polarity regulators: (1) the Baz/Par-6 complex, (2) the Crumbs
(Crb) complex, and (3) the Dlg group (Muller and Bossinger,
2003). Mutual antagonistic interactions between proteins of
these different classes have been shown to lead to the formation
of separate apical and basolateral membrane domains (Bilder
et al., 2003; Tanentzapf and Tepass, 2003). As for Crag mutant
clones, werestricted ouranalysis to mutantcellsthat maintained
a regular monolayer organization to avoid secondary effects
once epithelial integrity was compromised. In FC mutant for
the null allele par6D226, Pcan was never found to accumulate
on the apical side (n = 25; Figure 5A). Similarly, in crb11A2or
dlgm52mutantFC Pcanwasnot foundapically(n=25and n=26,
respectively; Figures 5B and 5C). Hence, mistargeting of BM
components to the apical side of the FE does not appear to be
a general defect observed in mutants affecting epithelial archi-
tecture but is instead specifically caused by the absence of
Crag function. Crag therefore appears to act in a unique and
novel pathway, distinct from the Baz, Crb, and Dlg groups of
proteins, to regulate epithelial organization.
The Presence of Pcan on the Apical Side of Crag
Mutant Cells Does Not Depend on Transcytosis
from the Basal Side
Different mechanisms could lead to the presence of BM compo-
nents on the apical side of the epithelium in Crag mutant cells.
Newly synthesized BM proteins could be targeted aberrantly to
the apical side in the absence of Crag activity. Alternatively,
BM components could be transported from the basal to the
apical side through transcytosis in mutant cells. It was shown
that the latter mechanism is responsible for the formation
of a small apical BM cap over anterior polar cells at stage 8 of
oogenesis (Medioni and Noselli, 2005). To distinguish between
these two scenarios, we generated FC clones lacking both
Crag and Pcan (referred to as pcan?Crag?clones). pcan?and
pcan?Crag?clones lose the punctate intracellular Pcan staining
observed in wild-type cells (Figures 6A and 6B). This punctate
staining may therefore reflect Pcan in the biosynthetic pathway.
However, small or medium sized pcan?and pcan?Crag?clones
maintain basal Pcan localization, either due to the diffusion of
Pcan synthesized and secreted by neighboring wild-type cells
or to a low turnover of Pcan protein once assembled in the
BM (Figures 6A and 6B). In contrast to Crag mutant cells,
pcan?Crag?FC never show Pcan staining on their apical side
(n > 50; compare Figures 6B and 6C). Because Pcan is still pres-
ent on the basal side of these clones, this suggests that apical
Pcan in Crag mutant cells does not originate from transcytosed
basal material but rather from newly synthesized protein.
FC lacking both Pcan and Crag were identical to Crag mutant
FC, but differed from pcan mutant clones. This was apparent
from their general morphology and organization and from the lo-
calization of Laminin and Dystroglycan (Figures 6D and 6E and
data not shown). Whereas Laminin localization is not obviously
affected in pcan mutant clones (n > 50; and Schneider et al.
), ectopic apical Laminin can readily be observed in
pcan?Crag?clones, as in Crag mutant FC (93% of egg cham-
bers with pcan?Crag?clones showed apical Laminin, n = 84)
(Figure 6D). Similarly, Dystroglycan is found at increased levels
at the apical surface in pcan?Crag?clones (Figure 6E). These re-
sults indicate that the presence of Pcan on the apical side of the
FC is not a prerequisite for the presence of apical Laminin and
Dystroglycan and the disruption of epithelial architecture in
Crag mutant cells. The presence of Laminin and/or other BM
components may suffice to induce the epithelial defects
observed in Crag mutant cells.
The maintenance of epithelial architecture relies on the polarized
trafficking of apical and basolateral proteins to their respective
membrane domains (Mostov et al., 2003; Rodriguez-Boulan
et al., 2005). Whereas considerable progress has been made in
elucidating the molecular mechanisms regulating the polarized
trafficking of basolateral transmembrane proteins, little is known
about how basolaterally secreted proteins, such as components
of the BM, obtain their polarized distribution. Here, we report
a functional characterization of the evolutionarily conserved pro-
tein Crag. We show that Crag mutant FC lose epithelial integrity
and become invasive. We propose that Crag plays a unique and
regulating the polarized secretion of BM components.
First, we have shown that Crag mutant cells lose the polarized
distribution of the BM components Pcan, Lam, and Coll IV.
Instead of being present exclusively on the basal side of the
epithelium, these BM proteins were found to accumulate both
Figure 5. Mislocalization of Pcan Is Not a General Defect in FC
Mutant for Regulators of Epithelial Organization
(A–C) Egg chambers containing par6D226(A), crb11A2(B), or dlgm52(C) mutant
accumulates exclusively on the basal side of par6, crb, or dlg mutant cells that
maintain a regular monolayer organization (arrowheads) or have formed multi-
ple layers of irregularly shaped cells (arrows). Aberrant apical Pcan was never
Crag Regulates Basement Membrane Deposition
360 Developmental Cell 14, 354–364, March 2008 ª2008 Elsevier Inc.
basally and apically in the absence of Crag. This was true for all
mutant clones analyzed, regardless of their position within the
epithelium and even in the absence of any other discernible phe-
notypes. This contrasts with apical, junctional, and basolateral
failure to restrict the accumulation of BM components to the
basal side of the FE is therefore likely the primary defect in
Crag mutant cells. Second, the mistargeting of BM components
in Crag mutant cells appears to be a specific consequence of the
loss of Crag function and not a general defect observed in the
absence of other proteins known to regulate epithelial organiza-
tion. FC mutant for par-6, crb, or dlg and thus lacking the activity
of any one of three major polarity complexes show no alterations
in the accumulation of BM proteins. Crag may therefore be the
first factor identified to specifically control the accurate deposi-
tion of BM on the basal side of epithelial cells.
Based on the well-established role of the BM in regulating
epithelial organization, we propose that the presence of BM
components on both sides of the FE is responsible for the loss
of epithelial integrity in the absence of Crag. Studies in both
model organisms and cultured cells have pointed to a central
role for the BM in regulating epithelial polarity and tissue organi-
zation (Li et al., 2003; Miner and Yurchenco, 2004; Yurchenco
Figure 6. The Accumulation of Pcan on the
Apical Side of Crag Mutant FC Originates
from Newly Synthesized Protein
(A–C) Egg chambers with pcannull(A), pcannull
CragCJ101(B), or CragCJ101(C) clones marked by
the absence of GFP (green) and stained for Pcan
(red). Higher magnifications of the boxed areas in
(A)–(C) are depicted in (A00)–(C00). (A) Whereas
Pcan can be detected in intracellular punctae in
cellular Pcan staining. In contrast, Pcan is present
on the basal side of the FE in control and pcannull
cells. (B) Pcan staining in pcannullCragCJ101FC
looks similar to that in pcannullclones. Note that
even though basal Pcan can readily be observed,
no apical accumulation is visible. (C) Conversely,
a striking apical Pcan localization is observed in
(D and E) Egg chambers with pcannullCragCJ101
clones marked by the absence of GFP (green)
and stained for Lam ([D], red) or Dystroglycan
([E], red). (D) Apical Lam staining is observed in
pcannullCragCJ101clones similarly to CragCJ101
clones. (E) Dystroglycan accumulates strongly at
the apical surface in pcannullCragCJ101clones.
et al., 2004). In particular, contact with
the BM has been shown to direct the ori-
entation of the apical-basal axis of epi-
thelia, with the apical pole forming op-
posite to the side contacting the BM
(O’Brien etal.,2002).Exposure of theapi-
cal surface of cultured epithelial cysts or
monolayers to either collagen or laminin
triggers a reversal of polarity (Chambard
et al., 1981; Hall et al., 1982; O’Brien
et al., 2001; Schwimmer and Ojakian, 1995; Wang et al., 1990;
Yu et al., 2005; Zuk and Matlin, 1996). Polarity reversal is com-
pleted only after the BM present on the original basal side of
the epithelium has been degraded, and it is preceded by a loss
of apical membrane identity, a dispersal of basolateral proteins,
and the formation of multiple cell layers (Schwimmer and Oja-
kian, 1995; Wang et al., 1990; Zuk and Matlin, 1996). This artifi-
cially induced situation shows noticeable similarities with the be-
havior of Crag mutant FC where BM components are found in
contact with both sides of the epithelium. Cells form multiple
layers, the cortical localization of apical proteins is lost, and ba-
solateral proteins are found along the entire cell surface. Thus,
the presence of a BM on both sides of the FE in the absence
of Crag can account for the observed phenotypes. Even though
the significance of the BM in organizing epithelial architecture
has been well established, our results underscore the impor-
tance of maintaining a polarized deposition of BM proteins on
the basal side of epithelial cells.
Interestingly, epithelial disorganization isnotrestricted to Crag
mutant cells but is also seen in neighboring wild-type cells. This
nonautonomy can be explained by the observation that Pcan,
Lam, and Coll IV were found on the apical side of wild-type cells
neighboring mutant cells, presumably due to their diffusion
within the extracellular space.
Crag Regulates Basement Membrane Deposition
Developmental Cell 14, 354–364, March 2008 ª2008 Elsevier Inc. 361
How could the absence of Crag protein lead to the aberrant
deposition of BM components on the apical side of epithelial
cells? Previous work in tissue culture has suggested that epithe-
lial cells secrete BM proteins predominantly from their basal side
process mediating this polarized secretion (Boll et al., 1991;
Caplan et al., 1987; Natori et al., 1992; Unemori et al., 1990). Fur-
thermore, it has been suggested that different trafficking routes
for basally secreted and basolateral transmembrane proteins
exist (Boll et al., 1991; Caplan et al., 1987; Cohen et al., 2001;
De Almeida and Stow, 1991). It is thus conceivable that Crag
plays a role in specifically controlling the polarized secretion of
gested by the presence of a conserved DENN domain at its
amino terminus. DENN domains are found in a large number of
proteins in nearly all eukaryotes. The precise function of the
DENN domain remains to be elucidated, but its presence in
a number of proteins involved in endocytosis or exocytosis,
most notably in a number of Rab interactors, led to the hypoth-
esis that the DENN domain regulates membrane trafficking (Al-
laire et al., 2006; Clague and Lorenzo, 2005; Falbel et al., 2003;
Levivier et al., 2001; Miyoshi and Takai, 2004). Moreover, the
localization of Crag to the cell cortex and to recycling and early
endosome membranes is consistent with a role in membrane
We showed that apical Pcan in Crag mutant cells originates
from newly synthesized protein and not from transcytosed basal
protein because Crag pcan double mutant clones that maintain
basal Pcan do not show apical Pcan. Assuming that in wild-
type FC newly synthesized BM components are secreted exclu-
sively on the basal side, Crag could act by ensuring one or more
steps during their polarized secretion. Crag may facilitate the
sorting of newly synthesized BM components. In the absence
of Crag, inaccurate sorting would lead to the packaging of BM
proteins in vesicles destined for both apical and basolateral
secretion. The colocalization of Crag protein with the recycling
endosome (RE) markerRab11 may point toa rolefor Craginpro-
tein sorting, as the RE has been shown to be a hub for the sorting
of both newly synthesized and endocytosed apical and basolat-
eral proteins (Ang et al., 2004; Rodriguez-Boulan et al., 2005). Al-
ternatively, Crag may act at a later step and direct the polarized
transport, targeting, or fusion of vesicles containing BM com-
ponents specifically with the basal membrane. Interestingly,
since Crag is absent from the basal cortex in FC, Crag would
play a repulsive role, inhibiting the targeting or fusion of BM pro-
tein-containing vesicles with apical and lateral membranes. In
the absence of Crag, transport vesicles would be allowed to
fuse with basolateral as well as apical membranes, leading to
the observed apolar distribution of Lam, Coll IV and Pcan.
Even though evidence from previous work points toward the
active sorting and polarized secretion of BM proteins (Boll
et al., 1991; Caplan et al., 1987; Natori et al., 1992; Unemori
et al., 1990), the possibility remains that BM components are se-
creted both apically and basally in wild-type FC but stabilized
only on the basal side, or removed preferentially apically, for
instance through degradation by localized matrix metallopro-
teases. While these scenarios require additional assumptions,
Crag could mediate either of these processes and regulate the
basal accumulation of BM proteins more indirectly, yet
In summary, we have uncovered a crucial and, to our knowl-
edge, novel role for the conserved protein Crag in the regulation
of epithelial architecture of the Drosophila FE. Crag appears to
specifically affect the polarized trafficking of BM components.
Even though the existence of different trafficking routes for
basally secreted and basolateral transmembrane proteins has
specific component required to ensure the polarized accumula-
tion of BM on the basal side of epithelial cells. It therefore serves
lecular analysis. It will be interesting to elucidate the molecular
machinery through which Crag regulates the polarized deposi-
tion of BM components.
Fly Stocks and Genetics
Control flies used were y w FRT19A or OreR. Deficiency and Duplication lines
and P element lines used for mapping (Bellen et al., 2004) were obtained from
Bloomington. The mutant lines dlgm52FRT101 (Woods and Bryant, 1991),
blich, 2001) were gifts from J. Zallen. pcannullFRT101 (Schneider et al., 2006)
was a gift from S. Baumgartner. The transgenic lines UAS-GFP-Rab5
(Wucherpfennig et al., 2003), UAS-GFP-Rab7 (Entchev et al., 2000), and
UAS-GFP-Rab11 (Emery et al., 2005) were gifts from M. Gonzalez-Gaitan.
GR1-Gal4 was used for overexpression experiments. The Vkg-GFP line
FlyTrap. Follicle cell clones were generated using the FRT/UAS-Flp/GAL4 sys-
tem (Duffy et al., 1998).The genotypes of femalesused for the analysescan be
found in the Supplemental Data.
Males of the genotype y w FRT19A were mutagenized and balanced as de-
scribed by Chen and Schupbach (2006). Females from each lethal line were
crossed to males of the genotype Ubi-GFP FRT19A/Y; e22c-Gal4, UAS-Flp/
CyO. Female offspring of the genotype * FRT19A/Ubi-GFP FRT19A; e22c-
Gal4, UAS-Flp/+ were dissected, fixed, and stained with Phalloidin and
Hoechst. Ovaries were mounted in Aquapolymount and scored for epithelial
defects using an Axiophot epifluorescence microscope. In 2 separate screens
a total of 12 Crag alleles were isolated.
Mapping of Crag Mutations
We used recombination with visible recessive markers to map the lethal muta-
tion in Crag mutants to the region between cut (7B4) and vermillion (9F11). The
lethal phenotype was rescued by duplications uncovering genomic regions
7A8; 8A5 and 7D; 8B3-D7 (Dp(1;2) sn[+]72d and Dp(1;Y)850, respectively)
allowing us to use the Deficiencies Df(1)KA14 and Df(1)RA2 to further map
the mutation between 7F1 and 8A4. This also allowed us to confirm that all
Crag alleles isolated were allelic. Subsequent recombination mapping with P
element insertions placed the lethal mutation between BG02451 (7F4) and
were balanced over an FM7c, Kr > GFP balancer chromosome and genomic
DNA from ?10 to 15 non-GFP-expressing Crag?/Y first instar larvae was iso-
lated using a method described in http://www.fruitfly.org/about/methods/
inverse.pcr.html. PCR products covering the Crag gene region were se-
quenced, and sequences were compared with those of FRT19A control prod-
ucts.Foreachallele,PCR productsofatleasttwoindependent genomic preps
Ovaries were dissected in PBS, fixed in PBS with 4% paraformaldehyde for 20
min at room temperature, and stained as described (Ashburner, 1989).
Schneider’s medium was used instead of PBS for dissections and fixations
Crag Regulates Basement Membrane Deposition
362 Developmental Cell 14, 354–364, March 2008 ª2008 Elsevier Inc.
in the case of Crag and HA-Crag stainings. Ovaries, mounted in Aquapoly-
mount, were visualized using a Zeiss laser scanning confocal microscope
(lsm510). Primary antibodies used were rat polyclonal CragC (this study,
1:200), rabbit aPKC (1:1000, Santa Cruz), mouse Arm (N2 7A1, 1:50, DSHB),
rabbit Baz (1:500 [Wodarz et al., 1999]), rat DE-Cadherin (DCAD2, 1:20,
DSHB), mouse CollagenIV (6G7, 1:10 [Murray et al., 1995]) rabbit DG-cyto
(1:1000 [Deng et al., 2003]), mouse FasII (1D4, 1:1000 conc., DSHB), mouse
FasIII (7G10, 1:10, DSHB), mouse HA (F-7, 1:1000, Santa Cruz), rat HA
(3F10, 1:50, Roche), rabbit Laminin (1:1000 [Fessler et al., 1987]), rabbit Lam-
inin-A (293, 1:20 [Gutzeit et al., 1991]), mouse Notch (C17.9C6, 1:100, DSHB),
rabbit Pcan (1:1000 [Friedrich et al., 2000]), rabbit Rab5 (1:50 [Wucherpfennig
et al., 2003]), and mouse Rab11 (1:20, BD Biosciences). Secondary antibodies
were AlexaFluor488, 568, 647 conjugated (Molecular probes) and used at
1:1000. Phalloidin conjugates and Hoechst were from Molecular Probes.
Constructs and Antibody Production
To generate the Crag genomic rescue construct, BAC clone RP98-40O10 was
To generate pUASp-HA-CragA and pUASp-HA-CragB, the coding regions
and 30UTRs of Crag-PA and Crag-PB were cloned into pUASp in-frame with
an N-terminal triple HA tag. For Crag-PB we used EST clone AT30747. Be-
cause no full-length cDNA encoding Crag-PA was available, we constructed
full-length Crag-PA by exchanging a 1 kb DraIII/FspI fragment from AT30747
with that of LD37492, which contains the Crag-PA-specific exon 10. For anti-
body production a fragment encoding aa 919–1666 of Crag-PA was cloned
into pGEX-4T-1. GST-CragC was purified on Ni-NTA agarose (Novagen) and
Glutathione-Sepharose (GE Healthcare) and injected into rats to generate rat
polyclonal antibodies (Pocono Rabbit Farm and Laboratory Inc.).
Supplemental Data include two figures and Supplemental Experimental Pro-
cedures and are available at http://www.developmentalcell.com/cgi/content/
We thank S. Baumgartner, L. and J. Fessler, M. Gonzalez-Gaitan, H. Gutzeit,
H. Ruohola-Baker, A. Wodarz, J. Zallen, the Developmental Studies Hybrid-
oma Bank, and the Bloomington stock center for providing flies and anti-
bodies; J. Goodhouse for advice with confocal microscopy; and members of
the Schu ¨pbach and Wieschaus labs for feedback and advice. We also thank
S. De Renzis, C. Denef, F. Ulrich, Y. Yan, and J. Zallen for helpful comments
on this or previous versions of the manuscript. This work was supported by
the Howard Hughes Medical Institute and US Public Health Service Grants
PO1 CA41086 and RO1 GM077620. ND was supported by a Long-Term Fel-
lowship from the Human Frontier Science Program during part of this work.
Received: November 6, 2006
Revised: September 13, 2007
Accepted: December 18, 2007
Published: March 10, 2008
Abdelilah-Seyfried, S., Cox, D.N., and Jan, Y.N. (2003). Bazooka is a permis-
sive factor for the invasive behavior of discs large tumor cells in Drosophila
ovarian follicular epithelia. Development 130, 1927–1935.
Allaire, P.D., Ritter, B., Thomas, S., Burman, J.L., Denisov, A.Y., Legendre-
Guillemin, V., Harper, S.Q., Davidson, B.L., Gehring, K., and McPherson,
P.S. (2006). Connecdenn, a novel DENN domain-containing protein of neuro-
nal clathrin-coated vesicles functioning in synaptic vesicle endocytosis. J.
Neurosci. 26, 13202–13212.
Ang, A.L., Taguchi, T., Francis, S., Folsch, H., Murrells, L.J., Pypaert, M.,
Warren, G., and Mellman, I. (2004). Recycling endosomes can serve as inter-
mediates during transport from the Golgi to the plasma membrane of MDCK
cells. J. Cell Biol. 167, 531–543.
Ashburner, M. (1989). A Laboratory Manual (New York: Cold Spring Harbor
Bellen, H.J., Levis, R.W., Liao, G., He, Y., Carlson, J.W., Tsang, G., Evans-
Holm, M., Hiesinger, P.R., Schulze, K.L., Rubin, G.M., et al. (2004). The
BDGP gene disruption project: single transposon insertions associated with
40% of Drosophila genes. Genetics 167, 761–781.
Bilder, D., Li, M., and Perrimon, N. (2000). Cooperative regulation of cell polar-
ity and growth by Drosophila tumor suppressors. Science 289, 113–116.
Bilder, D., Schober, M., and Perrimon, N. (2003). Integrated activity of PDZ
protein complexes regulates epithelial polarity. Nat. Cell Biol. 5, 53–58.
Boll, W., Partin, J.S., Katz, A.I., Caplan, M.J., and Jamieson, J.D. (1991).
Distinct pathways for basolateral targeting of membrane and secretory pro-
teins in polarized epithelial cells. Proc. Natl. Acad. Sci. USA 88, 8592–8596.
Buszczak, M., Paterno, S., Lighthouse, D., Bachman, J., Planck, J., Owen, S.,
Skora, A.D., Nystul, T.G., Ohlstein, B., Allen, A., et al. (2007). The carnegie pro-
Caplan, M.J., Stow, J.L., Newman, A.P., Madri, J., Anderson, H.C., Farquhar,
sorting of secreted proteins. Nature 329, 632–635.
on the orientation of epithelial cell polarity: follicle formation from isolated
thyroid cells and from preformed monolayers. J. Cell Biol. 91, 157–166.
Chen, Y., and Schupbach, T. (2006). The role of brinker in eggshell patterning.
Mech. Dev. 123, 395–406.
Clague, M.J., and Lorenzo, O. (2005). The myotubularin family of lipid phos-
phatases. Traffic 6, 1063–1069.
Cohen, D., Musch, A., and Rodriguez-Boulan, E. (2001). Selective control of
basolateral membrane protein polarity by cdc42. Traffic 2, 556–564.
protein kinase C are required to regulate oocyte differentiation in the Drosoph-
ila ovary. Proc. Natl. Acad. Sci. USA 98, 14475–14480.
De Almeida, J.B., and Stow, J.L. (1991). Disruption of microtubules alters
polarity of basement membrane proteoglycan secretion in epithelial cells.
Am. J. Physiol. 261, C691–C700.
Deng, W.M., Schneider, M., Frock, R., Castillejo-Lopez, C., Gaman, E.A.,
Baumgartner, S., and Ruohola-Baker, H. (2003). Dystroglycan is required for
polarizing the epithelial cells and the oocyte in Drosophila. Development
Duffy, J.B., Harrison, D.A.,and Perrimon, N. (1998). Identifying locirequired for
follicular patterning using directed mosaics. Development 125, 2263–2271.
Emery, G., Hutterer, A., Berdnik, D., Mayer, B., Wirtz-Peitz, F., Gaitan, M.G.,
and Knoblich, J.A. (2005). Asymmetric Rab 11endosomes regulate delta recy-
clingand specify cellfate inthe Drosophila nervous system. Cell122,763–773.
Entchev, E.V., Schwabedissen, A., and Gonzalez-Gaitan, M. (2000). Gradient
formation of the TGF-beta homolog Dpp. Cell 103, 981–991.
Falbel, T.G., Koch, L.M., Nadeau, J.A., Segui-Simarro, J.M., Sack, F.D., and
Bednarek, S.Y. (2003). SCD1 is required for cytokinesis and polarized cell
expansion in Arabidopsis thaliana. Development 130, 4011–4024.
Fernandez-Minan, A., Martin-Bermudo, M.D., and Gonzalez-Reyes, A. (2007).
Integrin signaling regulates spindle orientation in Drosophila to preserve the
follicular-epithelium monolayer. Curr. Biol. 17, 683–688.
Fessler, L.I., Campbell, A.G., Duncan, K.G., and Fessler, J.H. (1987). Drosoph-
ila laminin: characterization and localization. J. Cell Biol. 105, 2383–2391.
domain V of Drosophila melanogaster. Sequence, recombinant analysis and
tissue expression. Eur. J. Biochem. 267, 3149–3159.
Goode, S., and Perrimon, N. (1997). Inhibition of patterned cell shape change
and cell invasion by Discs large during Drosophila oogenesis. Genes Dev. 11,
Crag Regulates Basement Membrane Deposition
Developmental Cell 14, 354–364, March 2008 ª2008 Elsevier Inc. 363
Goode, S., Wei, J., and Kishore, S. (2005). Novel spatiotemporal patterns of Download full-text
epithelial tumor invasion in Drosophila discs large egg chambers. Dev. Dyn.
Gutzeit, H.O., Eberhardt, W., and Gratwohl, E. (1991). Laminin and basement
membrane-associated microfilaments in wild-type and mutant Drosophila
ovarian follicles. J. Cell Sci. 100, 781–788.
Hall, H.G., Farson, D.A., and Bissell, M.J. (1982).Lumen formation byepithelial
cell lines in response to collagen overlay: a morphogenetic model in culture.
Proc. Natl. Acad. Sci. USA 79, 4672–4676.
Horne-Badovinac, S., and Bilder, D. (2005). Mass transit: epithelial morpho-
genesis in the Drosophila egg chamber. Dev. Dyn. 232, 559–574.
Lee, J.K., Brandin, E., Branton, D., and Goldstein, L.S. (1997). alpha-Spectrin
is required for ovarian follicle monolayer integrity in Drosophila melanogaster.
Development 124, 353–362.
I. (2001). uDENN, DENN, and dDENN: indissociable domains in Rab and MAP
kinases signaling pathways. Biochem. Biophys. Res. Commun. 287, 688–695.
Li, S., Edgar, D., Fassler, R., Wadsworth, W., and Yurchenco, P.D. (2003). The
role of laminin in embryonic cell polarization and tissue organization. Dev. Cell
Lunstrum, G.P., Bachinger, H.P., Fessler, L.I., Duncan, K.G., Nelson, R.E., and
Fessler,J.H.(1988). DrosophilabasementmembraneprocollagenIV.I. Protein
characterization and distribution. J. Biol. Chem. 263, 18318–18327.
Manfruelli, P., Arquier, N., Hanratty, W.P., and Semeriva, M. (1996). The tumor
suppressor gene, lethal(2)giant larvae (1(2)g1), is required for cell shape
change of epithelial cells during Drosophila development. Development 122,
Medioni, C., and Noselli, S. (2005). Dynamics of the basement membrane in
invasive epithelial clusters in Drosophila. Development 132, 3069–3077.
Miner, J.H., and Yurchenco, P.D. (2004). Laminin functions in tissue morpho-
genesis. Annu. Rev. Cell Dev. Biol. 20, 255–284.
Miyoshi, J., and Takai, Y. (2004). Dual role of DENN/MADD (Rab3GEP) in
neurotransmission and neuroprotection. Trends Mol. Med. 10, 476–480.
Mostov, K., Su, T., and ter Beest, M. (2003). Polarized epithelial membrane
traffic: conservation and plasticity. Nat. Cell Biol. 5, 287–293.
Muller, H.A., and Bossinger, O. (2003). Molecular networks controlling epithe-
lial cell polarity in development. Mech. Dev. 120, 1231–1256.
Murray, M.A., Fessler, L.I., and Palka, J. (1995). Changing distributions of ex-
tracellular matrix components during early wing morphogenesis in Drosophila.
Dev. Biol. 168, 150–165.
Natori, Y., O’Meara, Y.M., Manning, E.C., Minto, A.W., Levine, J.S., Weise,
W.J., and Salant, D.J. (1992). Production and polarized secretion of basement
membrane components by glomerular epithelial cells. Am. J. Physiol. 262,
Nelson, W.J. (2003). Adaptation of core mechanisms to generate cell polarity.
Nature 422, 766–774.
O’Brien, L.E., Jou, T.S., Pollack, A.L., Zhang, Q., Hansen, S.H., Yurchenco, P.,
and Mostov, K.E. (2001). Rac1 orientates epithelial apical polarity through
effects on basolateral laminin assembly. Nat. Cell Biol. 3, 831–838.
O’Brien, L.E., Zegers,M.M., and Mostov, K.E. (2002). Opinion: building epithe-
lial architecture: insights from three-dimensional culture models. Nat. Rev.
Mol. Cell Biol. 3, 531–537.
Petronczki, M., and Knoblich, J.A. (2001). DmPAR-6 directs epithelial polarity
and asymmetric cell division of neuroblasts in Drosophila. Nat. Cell Biol. 3,
Rodriguez-Boulan, E., Kreitzer, G., and Musch, A. (2005). Organization of
vesicular trafficking in epithelia. Nat. Rev. Mol. Cell Biol. 6, 233–247.
Schneider, M., Khalil, A.A., Poulton, J., Castillejo-Lopez, C., Egger-Adam, D.,
Wodarz, A., Deng, W.M., and Baumgartner, S. (2006). Perlecan and Dystrogly-
can act at the basal side of the Drosophila follicular epithelium to maintain
epithelial organization. Development 133, 3805–3815.
Schwimmer, R., and Ojakian, G.K. (1995). The alpha 2 beta 1 integrin regulates
collagen-mediated MDCK epithelial membrane remodeling and tubule forma-
tion. J. Cell Sci. 108, 2487–2498.
Semova, N., Kapanadze, B., Corcoran, M., Kutsenko, A., Baranova, A., and
Semov, A. (2003). Molecular cloning, structural analysis, and expression of
a human IRLB, MYC promoter-binding protein: new DENN domain-containing
protein family emerges. Genomics 82, 343–354.
Sisson, J.C., Field, C., Ventura, R., Royou, A., and Sullivan, W. (2000). Lava
lamp, a novel peripheral golgi protein, is required for Drosophila melanogaster
cellularization. J. Cell Biol. 151, 905–918.
Tanentzapf, G.,and Tepass,U.(2003).Interactionsbetweenthecrumbs,lethal
giant larvae and bazooka pathways in epithelial polarization. Nat. Cell Biol. 5,
Tanentzapf, G., Smith, C., McGlade, J., and Tepass, U. (2000). Apical, lateral,
and basal polarization cues contribute to the development of the follicular
epithelium during Drosophila oogenesis. J. Cell Biol. 151, 891–904.
Thiery, J.P. (2002). Epithelial-mesenchymal transitions in tumour progression.
Nat. Rev. Cancer 2, 442–454.
Unemori, E.N., Bouhana, K.S., and Werb, Z. (1990). Vectorial secretion of ex-
tracellular matrix proteins, matrix-degrading proteinases, and tissue inhibitor
of metalloproteinases by endothelial cells. J. Biol. Chem. 265, 445–451.
Wang, A.Z., Ojakian, G.K., and Nelson, W.J. (1990). Steps in the morphogen-
esis of a polarized epithelium. II. Disassembly and assembly of plasma mem-
(MDCK) cysts. J. Cell Sci. 95, 153–165.
an apical cue for Inscuteable localization in Drosophila neuroblasts. Nature
Woods, D.F., and Bryant, P.J. (1991). The discs-large tumor suppressor gene
of Drosophila encodes a guanylate kinase homolog localized at septate junc-
tions. Cell 66, 451–464.
Wucherpfennig, T., Wilsch-Brauninger, M., and Gonzalez-Gaitan, M. (2003).
Role of Drosophila Rab5 during endosomal trafficking at the synapse and
evoked neurotransmitter release. J. Cell Biol. 161, 609–624.
Xu, X.Z., Wes, P.D., Chen, H., Li, H.S., Yu, M., Morgan, S., Liu, Y., and Montell,
C.(1998). Retinal targets for calmodulin include proteins implicated insynaptic
transmission. J. Biol. Chem. 273, 31297–31307.
Yu, W., Datta, A., Leroy, P., O’Brien, L.E., Mak, G., Jou, T.S., Matlin, K.S.,
Mostov, K.E., and Zegers, M.M. (2005). Beta1-integrin orients epithelial polar-
ity via Rac1 and laminin. Mol. Biol. Cell 16, 433–445.
Yurchenco, P.D., Amenta, P.S., and Patton, B.L. (2004). Basement membrane
assembly, stability and activities observed through a developmental lens.
Matrix Biol. 22, 521–538.
Zuk, A., and Matlin, K.S. (1996). Apical beta 1 integrin in polarized MDCK cells
mediates tubulocyst formation in response to type I collagen overlay. J. Cell
Sci. 109, 1875–1889.
Crag Regulates Basement Membrane Deposition
364 Developmental Cell 14, 354–364, March 2008 ª2008 Elsevier Inc.