RESEARCH ARTICLE 1395
Development 137, 1395-1404 (2010) doi:10.1242/dev.045450
© 2010. Published by The Company of Biologists Ltd
Non-cell-autonomous control of denticle diversity in the
Drosophila embryo
Stacie A. Dilks and Stephen DiNardo*
SUMMARY
Certain Drosophila embryonic epidermal cells construct actin-based protrusions, called denticles, which exhibit stereotyped,
column-specific differences in size, density and hook orientation. This precise denticle pattern is conserved throughout all
drosophilids yet studied, and screening for mutations that affect this pattern has been used to identify genes involved in
development and signaling. However, how column-specific differences are specified and the mechanism(s) involved have remained
elusive. Here, we show that the transcription factor Stripe is required for multiple aspects of this column-specific denticle pattern,
including denticle hook orientation. The induction of stripe expression in certain denticle field cells appears to be the primary
mechanism by which developmental pathways assign denticle hook orientation. Furthermore, we show that the cytoskeletal linker
protein Short stop (Shot) functions both cell-autonomously and non-autonomously to specify denticle hook orientation via
interaction with the microtubule cytoskeleton. We propose that stripe mediates its effect on hook orientation, in part, via
upregulation of shot.
INTRODUCTION
During development, groups of equivalent cells differentiate to form
a highly patterned body plan through the use of signals secreted by
organizer regions. The process of differentiation therefore requires
a cell to have the capacity to receive a signal, and to then translate
that signal into some cellular response, such as a change in genetic
program, adhesion properties or morphogenetic movements.
Historically, developmental biology has focused primarily on the
signal cascades that are required for differentiation, rather than on
the vital next step of translating a developmental signal into a
cellular event. However, the gap between developmental signaling
cues and cell biological responses is being bridged, and certain
model systems have emerged in recent years that can link organizing
signals to cellular morphogenesis.
One such system is the ventral epidermis of the Drosophila
embryo. In late embryogenesis, these epidermal cells adopt one of
two cell fates: either they remain apically smooth or they produce an
elaborately shaped actin-based protrusion called a denticle. Later,
these latter cells secrete a hardened cuticle that conforms to the
shape of the plasma membrane, molding the actin-based protrusions
in the cuticle (Hillman and Lesnik, 1970; Martinez-Arias and Bate,
1993; Payre, 2004). Importantly, denticle characteristics differ from
column to column, giving rise to a highly reproducible pattern that
has served as the basis for several screens to identify patterning
genes (Nüsslein-Volhard and Wieschaus, 1980).
It is not known how the details of this intricate pattern first arise,
nor how they are achieved by cytoskeletal components. Although
cytoskeletal coordination has been studied extensively in migrating
and cultured cells, less is known about cytoskeletal coordination in
Department of Cell and Developmental Biology, University of Pennsylvania School of
Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
*Author for correspondence ([email protected])
Accepted 4 February 2010
epithelia in vivo. In fact, many epithelia produce actin-based
protrusions that display elaborate shapes, such as vertebrate
stereocilia and Drosophila sensory bristles and ventral denticles.
However, how cytoskeletal networks are influenced to create these
shapes is not well understood.
The denticle field within most abdominal parasegments consists
of seven columns of epidermal cells that can each produce several
denticles. Although the posterior-most two columns of cells produce
small, non-descript denticles, cell columns 1-5 produce denticles
that are uniquely patterned from column to column. These cell
columns differ from one another in the size, shape, number and hook
orientation of the denticles they produce, as well as in the expression
of certain cell fate markers and signaling components (LohsSchardin et al., 1979; Martinez-Arias and Bate, 1993). Of note, cell
columns 1 and 4 are the only columns that produce denticles that
hook towards the anterior (see Fig. 1A). This anterior hooking is
unique, in the wild-type denticle field as well as in situations in
which the field is artificially expanded (O’Keefe, 1997; Szüts,
1997). This prompted us to examine what it is that is unique about
columns 1 and 4 that allows them to produce denticles that have a
reversed hook orientation. To date, no specific cell fate determinant
has been found to be common to these two cell columns that might
provide a clue to this process.
As the epidermal cells form denticles apically, a subset of these
epidermal cells (called tendon cells) forms junctions with myotubes
at their basal surface. This process is known as epidermal-muscle
attachment (EMA). Upon muscle contraction, mechanical
information is communicated from the EMA junction to the larval
cuticle, which allows for efficient larval crawling. Interestingly,
reversed hook orientation (towards the anterior) is found adjacent to
muscle attachment sites, suggesting that the tendon cells might play
some role in anterior hooking (see Fig. 2A). The early growth
response (EGR)-like transcription factor Stripe is necessary for the
specification and maturation of tendon cells, the recruitment of
myotubes and the stabilization of EMA junctions (Becker et al.,
1997; Frommer et al., 1996; Strumpf and Volk, 1998; Subramanian
DEVELOPMENT
KEY WORDS: Drosophila, Denticle, Short stop, Spectraplakin, Stripe
1396 RESEARCH ARTICLE
embryos were processed for immunofluorescence using a high
concentration-short fixation protocol, in which dechorionated embryos
were rocked in vials containing a 1:1 mixture of heptane and 37%
formaldehyde for 5-7 minutes (Teodoro and O’Farrell, 2003). Embryos
were then pipetted onto a glass slide, picked up on double-sided sticky tape
fixed to a cover slip, placed in a watch glass and covered with PBS. To
preserve phalloidin binding to F-actin, a needle was used to poke the
embryos out of their vitelline membranes. Fixation for anti-Shot and antiArmadillo (Arm) involved a short heating step in which dechorionated
embryos were immersed in a hot Triton X-100 (0.03%)-NaCl (0.4%)
solution (E-Wash) and then quickly cooled by addition of excess chilled EWash. Vitelline membranes were removed by shaking the embryos
vigorously in a 1:1 mixture of heptane and methanol. This fixation
procedure appears to highlight the localization of proteins such as Arm at
the zonula adherens at the expense of other cellular pools (Müller et al.,
1999).
The following antibodies (and dilutions) were used for 2 hours at room
temperature or overnight at 4°C: guinea pig anti-Stripe and rat anti-Stripe A
(1:500, gifts from Talila Volk), mouse anti-Shot [1:10, Developmental
Studies Hybridoma Bank (DSHB)], mouse anti-phosphorylated tyrosine
(1:500, Molecular Probes), rat anti-Filamin [N- and C-terminal, 1:4000, a
gift from Lynn Cooley (Sokol and Cooley, 2003)], rabbit anti-Arm (1:200,
a gift from Eric Wieschaus, Princeton University), rat anti-DE-cadherin
(Shotgun – FlyBase) (1:20, DSHB), mouse anti-Coracle (1:500, DSHB) or
mouse anti-b-tubulin (1:10, DSHB). Secondary antibodies, used at 1:400 for
1 hour, were conjugated to Alexa Fluor (Molecular Probes) or Cy3 or Cy5
dyes (Jackson Labs). Alexa 488-, Alexa 568- or Rhodamine-coupled
phalloidin was used at 1:200 (Molecular Probes). Stained embryos were
mounted in Prolong Gold (Molecular Probes) and images were obtained
using structured illumination (Zeiss Apotome) and assembled in Adobe
Photoshop.
Temperature-shift experiments
Embryos were collected for 2 hours at room temperature, aged at 18°C for
the indicated time, and then shifted to 29°C. The length of time at 18°C was
determined by trial and error such that a denticle hooking phenotype was
observed without cell fate change or cell column loss. For cuticle
preparations, embryos were aged at 29°C until just prior to hatching (~20-21
hours after egg laying), genotyped and processed. For immunofluorescence,
embryos were aged at 29°C until stage 15-16, genotyped, then fixed and
processed. For Egfrts experiments, embryos containing a heteroallelic
combination of Egfrtsla over the amorphic Egfrf24 allele were used.
Quantification
We analyzed the null mutations for sr and mys, sr155 (FBal0032779) and
mys1 (FBal0012678), as well as the strong sr hypomorphs sr3999
(FBal0009498) and sr461 (FBal0032784). Deficiencies uncovering shot were
Df(2R)Exel7128 (FBab0038035) and Df(2R)383BSC (FBab0045182); the
deficiency uncovering stripe was Df(3)DG4 (FBab0002521). All stocks
were balanced over TM6 Hu Ubi-GFP, CyO Kr-Gal4 UAS-GFP, or FM7 B
Kr-Gal4 UAS-GFP for cuticle analysis. ptc-Gal4, en-Gal4, hhts2, UASdicer2 and the deficiencies uncovering shot were from the Bloomington
Stock Center; Egfrtsla (FBal0083481) was from Kevin Moses (Rodrigues et
al., 2005) and Egfr24 (FBal0003552) was from Trudi Schüpbach (Clifford
and Schüpbach, 1989); UAS-EFhand-GAS2-GFP, UAS-srB, UAS-srA and
UAS-dsshot were from Talila Volk (Dorfman et al., 2002; Volohonsky et al.,
2007); UAS-spastin was from Kai Zinn (Sherwood et al., 2004); and UASshotL(A) was from Peter Kolodziej (Lee and Kolodziej, 2002a).
To quantify denticle hook orientation, cuticles were prepared and
visualized using standard phase-contrast microscopy. Each scorable
denticle in the column of interest was manually tallied as either hooking
towards the anterior or posterior, or as being ambiguous/unhooked. To
quantify denticle density, we stained embryos with phalloidin to label the
actin-based protrusions and for phosphorylated tyrosine to label cell
outlines. We then manually tallied the number of protrusions per cell in
each column. Whenever possible, segments A3-A7 were scored in each
embryo. Data were gathered from multiple segments of multiple animals
and compiled in Excel (Microsoft). For all genotypes, more than 100
individual denticles from at least five embryos were scored for
quantification.
To quantify stripe-expressing nuclei (as in Fig. 1), embryos were
sorted for genotype, stained with anti-Stripe and visualized by
immunofluorescence. stripe-expressing nuclei were counted manually, and
the ratio of stripe-expressing nuclei in column 2 versus column 5 was
calculated and compared between genotypes. As a control, the ratio of
stripe-expressing nuclei in column 5 versus the smooth field was also
calculated and compared between genotypes.
Cuticle preparation and immunohistochemistry
shot dsRNA
Embryos were collected on apple agar plates, aged, and either processed to
visualize cuticle pattern by phase-contrast microscopy (van der Meer, 1977)
or fixed and processed for immunofluorescence. In most cases, embryos
were genotyped using a fluorescence stereomicroscope. In most cases,
Females expressing both UAS-shot-dsRNA and UAS-dicer2 (to strengthen
the RNAi phenotype) were crossed to males expressing either Act5C-Gal4
or ptc-Gal4. As UAS-shot-dsRNA is on the X chromosome, half of the
progeny should express shot-dsRNA in the ectopic pattern. Although we
MATERIALS AND METHODS
Fly strains
DEVELOPMENT
et al., 2003; Yarnitzky et al., 1997). Here, we describe a novel
function of stripe in the tendon cells: assigning anterior denticle
hooking to neighboring cells.
Although Stripe is presumed to have many targets, relatively few
have been studied in detail. One such target is the spectraplakin short
stop (shot). Spectraplakins are extremely large cytoskeletal proteins
with characteristics of both the spectrin and plakin families, and are
generally thought to function by linking cytoskeletal filaments to one
another or to the membrane (Roper et al., 2002). shot, the only known
Drosophila spectraplakin, contains at least four transcriptional start
sites and exhibits extensive alternative splicing, producing a highly
modular protein with diverse functions (Gregory and Brown, 1998;
Lee et al., 2003; Lee et al., 2000; Roper and Brown, 2003; van Vactor
et al., 1993). MACF and BPAG1 (dystonin), vertebrate homologs of
Shot, are some of the few proteins capable of binding actin filaments
and microtubules simultaneously (Karakesisoglou et al., 2000; Lee
and Kolodziej, 2002a; Leung et al., 1999). Shot can also associate with
junctional proteins, integrins and E-cadherin (Gregory and Brown,
1998; Lee et al., 2003; Lee and Kolodziej, 2002b; Roper and Brown,
2003; Subramanian et al., 2003).
Multiple isoforms of Shot are present in the Drosophila
embryonic epidermis and these are distinctly regulated. Full-length
and other long Shot isoforms are ubiquitously expressed in the
epidermis from an early embryonic stage and are required to
maintain epithelial integrity (Roper and Brown, 2003). However, a
shorter isoform of Shot is upregulated in response to stripe
specifically in the tendon cells (Becker et al., 1997; Roper and
Brown, 2003). In these cells, Shot acts downstream of stripe, and is
speculated to link the actin and microtubule cytoskeletons,
preventing the tendon cell from rupturing upon muscle contraction,
although alternative models remain possible (Bottenberg, 2009; Lee
et al., 2007; Subramanian et al., 2003).
In this work, we link developmental patterning signals to the
morphogenetic event of denticle shaping via stripe and shot. First,
we demonstrate that stripe is required, non-cell-autonomously, for
the unique anterior hook orientation observed in the column 1 and
4 denticles. Secondly, our data strongly suggest that stripe
specifies anterior denticle hooking, in part via upregulation of shot.
The involvement of shot reveals novel roles for spectraplakins and
the microtubule cytoskeleton in the shaping of actin-based
protrusions.
Development 137 (8)
Control of denticle diversity
RESEARCH ARTICLE 1397
could not unambiguously genotype these embryos, roughly half (18 out of
46) of the embryos produced from this cross exhibited a pattern that deviated
from the wild type and we assumed these to be the mutant class and scored
these embryos for hook orientation as described above. In embryos stained
in parallel, column-selective knockdown of Shot was observed (see Fig. 6).
RESULTS
hedgehog and spitz are required for anterior
denticle hooking
Once the denticle field is established, short-range signals subdivide
the field into progressively smaller territories (Alexandre et al., 1999).
It has been observed that the orientation of denticle hooks reverses
across certain territorial boundaries, specifically at the boundaries
between columns 1 and 2 and columns 4 and 5 (Alexandre et al.,
1999). As we (and others) had previously shown that the Hedgehog
(Hh)/Smoothened and Spitz (Spi)/Egfr pathways have specific effects
across these respective boundaries (Hatini and DiNardo, 2001), we
tested whether these signals were involved in denticle hooking. We
bypassed earlier requirements for Hedgehog and Egfr by using
temperature-sensitive alleles (hhts2, Egfrtsla) (Ma et al., 1993;
Rodrigues et al., 2005). We raised embryos at the permissive
temperature until the actin-based protrusions began to form, then we
shifted to the restrictive temperature and scored denticle hook
orientation after cuticle deposition.
Cell columns 1 and 4 are the only columns that produce denticles
that hook towards the anterior (Fig. 1A). Anterior hook orientation
is consistent in wild type, with ~95% of column 1 and 4 denticles
hooking towards the anterior and only ~5% failing to hook (Fig. 1G,
see Materials and methods for scoring criteria). When we inactivated
Egfr, we found that 70% of column 4 denticles exhibited incorrect
hook orientation (Fig. 1C,G), whereas all other columns were
unchanged. Similarly, when we inactivated hh, 57% of column 1
denticles failed to hook towards the anterior (Fig. 1E,G), whereas all
other columns remained wild-type. These hooking phenotypes were
not due to changes in denticle field specification, as all denticle
columns were present. From these data, we conclude that the hh and
Egfr pathways are required for anterior denticle hooking in cell
columns 1 and 4, respectively.
stripe is required for anterior denticle hook
orientation
We next sought to identify a target(s) of these signaling pathways
involved in denticle hook orientation. We and others have
previously characterized a target gene common to both the Hh and
Egfr pathways: that of the transcription factor stripe (Hatini and
DiNardo, 2001). stripe is expressed in columns 2 and 5 in the
prospective denticle field and in one additional column in the
smooth field (Fig. 1B; Fig. 2A). Each column of stripe expression
is dependent on distinct signals: Hh signaling is required in
column 2 and Spi/Egfr signaling is required in column 5 (Hatini
and DiNardo, 2001; Piepenburg et al., 2000; Wiellette and
McGinnis, 1999).
First, we verified that Stripe protein was selectively depleted from
the proper columns in embryos compromised for Hh or Egfr
pathway activation. In Egfrts embryos, Stripe protein was lost
selectively from cell column 5 and retained in column 2 (Fig. 1D)
(Hatini and DiNardo, 2001). Although hhts embryos maintained
Stripe expression in some column 2 cells (Fig. 1F), their number was
significantly reduced from 11.5 to 6.2 Stripe-positive nuclei per
column. The effect was selective for column 2, as column 5
remained unchanged compared with controls (14.5 per column; see
Materials and methods).
To determine whether stripe is required for denticle hook
orientation, we examined stripe mutant embryos. Compared with
wild type, mutant embryos exhibited a dramatic decrease in anterior
hooking in both cell columns 1 and 4 (Fig. 2C). In stripe mutant
embryos, 88% of column 1 denticles and 63% of column 4 denticles
exhibited a loss of anterior hooking (Fig. 2E).
These hooking defects did not appear to be associated with
additional defects in planar cell polarity. In stripe mutant embryos,
as in wild type, the denticles were arranged in columns (Fig. 2B,C)
(Dickinson, 1997). Also, the actin-based protrusions remained
positioned along the posterior edges of cells and exhibited posterior
outgrowth (Fig. 2F,G) (Price et al., 2006; Walters et al., 2006). As
stripe mutant embryos exhibited these characteristics of polarity, we
conclude that the primary defect in stripe mutants is a loss of anterior
denticle hooking orientation, rather than a defect in tissue polarity.
DEVELOPMENT
Fig. 1. hedgehog and spitz are required
for anterior denticle hooking. (A-F) Faceon view of Drosophila cuticle preparations
(A,C,E) and staining with anti-Stripe
(magenta) and phalloidin (green) (B,D,F).
Circles indicate denticles with incorrect hook
orientation. (A,B) Egfrts/+ embryo. Note the
anterior hooking specifically in columns 1
and 4 (arrows). (C,D) Egfrts embryo,
upshifted at 9 hours. Note the loss of Stripe
in column 5. (E,F) hhts2 embryo, upshifted at
10 hours. Note the reduction of Stripe in
column 2 (see text). Anterior is to the left in
all figures. (G) Quantification of hook
orientation in wild type (hhts2/+), hhts2 and
Egfrts. **, P<0.01. Scale bar: 10 mm in
A,C,E; 20 mm in B,D,F.
1398 RESEARCH ARTICLE
Development 137 (8)
Previous work has suggested that the cuticle pattern arises as a
result of local signaling interfaces within the epidermis
(Alexandre et al., 1999). As stripe is induced asymmetrically
across these interfaces via hh and spi, we propose that signaling
interfaces could influence denticle diversity by initiating stripe
expression in the neighboring cell (Fig. 2A) (Hatini and DiNardo,
2001).
stripe is required non-cell-autonomously for
anterior denticle hook orientation
To test directly whether non-autonomous stripe expression is
sufficient for anterior hooking, we attempted to rescue the column
1 hooking defect in the stripe mutant by adding back stripe to
column 2 using the patched (ptc)-Gal4 driver. ptc is highly
expressed in column 2 (and more weakly to the posterior), but not
in column 1 (Fig. 2H). Restoring stripe expression using ptc-Gal4
indeed restored proper anterior hooking in cell column 1 (Fig. 2D,I,
compare with 2C). We conclude that the requirement for stripe is
strictly non-autonomous, and that the expression of stripe in cell
column 2 is necessary and sufficient for the anterior hooking of cell
column 1. Currently, no Gal4 drivers exist that can restore stripe
selectively to column 5 cells, but based on our Egfrts data, we
presume that column 5 expression of stripe similarly acts nonautonomously on column 4 hooking.
stripe specifies denticle density
Previously, cells of all columns were thought to produce two to three
denticles each (Gergen and Wieschaus, 1985; Price et al., 2006;
Szabad et al., 1979), but it was recently appreciated that denticle
density varies across the field (Price et al., 2006). We found that cells
can produce anywhere from a single denticle to as many as five
denticles per cell (see Fig. S1 in the supplementary material). The
stripe-expressing columns averaged the fewest denticles per cell (1.2),
whereas column 1 cells averaged 2.4 denticles per cell (see Fig. S1 in
the supplementary material; see Materials and methods). However,
embryos null for stripe showed an increased denticle density for both
columns 2 and 5 without affecting the other cell columns (see Fig. S1
in the supplementary material). Reciprocally, ectopic expression of
stripe using engrailed (en)-Gal4, which is highly expressed in denticle
column 1, significantly reduced average denticle density from 2.4 to
1.3 denticles per cell. These data indicate that stripe function plays a
key role in specifying denticle density.
Muscle attachment is not required for denticle
hook orientation
Since stripe is necessary for muscle attachment, we wondered
whether the tension exerted by body wall muscles on column 2 and
5 cells was responsible for the anterior denticle hooking observed in
neighboring cells. To test this, we examined myospheroid (mys)
DEVELOPMENT
Fig. 2. stripe is required for denticle hook
orientation. (A) Schematic of one parasegment of
ventral epidermis in cross-section, showing nuclear
accumulation of Stripe (purple) in cells corresponding to
denticle columns 2 and 5, and in one smooth field cell
column (S). Note that denticle hook orientation reverses
at stripe-expressing sites. (B) Wild-type cuticle
preparation. Note the anterior hooking specifically in
columns 1 and 4 (arrows). (C) sr3999 cuticle. Hooking
defects are observed in both column 1 and 4 (circles).
(D) ptc::sr; sr3999 cuticle. Note the restoration of anterior
hooking in column 1 (arrow). (E) Quantification of
denticle hook orientation in column 1 and column 4 in
sr3999/+, sr3999 and sr155/Df(3)DG4 (the deficiency
uncovering stripe) embryos. (F,G) Denticles arising from
column 4 cells of wild-type (F; sr3999/+) or stripe mutant
(G; sr3999) embryos showing phalloidin staining (green),
with anti-phospho-tyrosine (magenta) to label cell
outlines. Note that the actin-based protrusions arise from
the posterior of the cell and elongate towards column 5
cells. (H) ptc::sr; sr3999 embryo. Note the restoration of
column 2 Stripe expression (arrow). (I) Quantification of
column 1 hook orientation in sr3999/+, sr3999 and ptc::sr;
sr3999 embryos. **, P<0.01. Scale bar: 10 mm in B,C,D; 4
mm in F,G; 20 mm in H.
Control of denticle diversity
RESEARCH ARTICLE 1399
Fig. 4. shot is required for anterior denticle hooking. (A) Df/+
cuticle. (B) Df(2R)383BSC/Df(2R)Exel7128 (deficiencies uncovering
shot) cuticle. Hooking defects are indicated by circles.
(C) Df(2R)383BSC/Df(2R)Exel7128 cuticle. Note the disorganization and
hooking defects in all columns. (D) Quantification of hook orientation in
Df/+ and Df/Df embryos. **, P<0.01. (E) Act5c::dsshot cuticle, exhibiting
global disorganization and hooking defects. Scale bar: 10 mm.
mutant embryos, which lack b-integrin, an essential component of
EMA junctions required for late tendon cell maturation (MartinBermudo, 2000).
To confirm that EMA junctions are not formed in mys embryos, we
stained mys mutant embryos with antibodies specific to each of the
two stripe isoforms (Volohonsky et al., 2007). The early isoform,
stripe-B, is induced via hh and spi (Hatini and DiNardo, 2001;
Volohonsky et al., 2007). Stripe-B initiates tendon cell differentiation
and attracts approaching myotubes (Volohonsky et al., 2007). When
muscles attach, stripe gene expression switches to the late isoform,
shot is required for denticle hook orientation
As Stripe is a transcription factor, we wondered whether any known
Stripe-induced genes might function in denticle hooking. One such
target, shot, was of interest owing to its ability to organize cytoskeletal
networks. To test its involvement in denticle hook orientation, we
examined embryos deficient for shot. The shot3 allele has been
suggested to be amorphic with respect to axon extension, although a
small percentage of characterized axons exhibit a normal trajectory
(Lee et al., 2000). However, in the epidermis, we detected residual
Shot protein in tendon cells from shot3 embryos (see Fig. S2 in the
supplementary material) (Roper and Brown, 2003). Thus, to
definitively deplete Shot, we produced embryos containing a
heteroallelic combination of mapped deletions that uncover the shot
DEVELOPMENT
Fig. 3. Muscle attachment is not required for denticle hook
orientation. (A) mys1 cuticle, exhibiting proper hook orientation
(compare with Fig. 1A). (B) Quantification of denticle hook orientation
in Tubulin::stripe (Tub::sr) Drosophila embryos. (C-C⬙) Wild-type (mys/+)
stage 16 embryo, showing Stripe-B (SrB, magenta; single channel in C⬘)
and Stripe-A (SrA, green; single channel in C⬙). Both Stripe isoforms are
localized to epidermal-muscle attachment (EMA) cells. (D-D⬙) mys1
embryo, showing Stripe-B (magenta; single channel in D⬘) and Stripe-A
(green; single channel in D⬙). Note the loss of Stripe-A. (E-E⬙) Tub::srB,
showing Stripe-B (green; single channel in E⬘) and Stripe-A (magenta;
single channel in E⬙). Although the Stripe-B pattern is drastically altered,
Stripe-A remains as in wild type. Scale bar: 10 mm in A; 20 mm in C-E⬙.
stripe-A (Nabel-Rosen et al., 1999; Volohonsky et al., 2007).
Formation of proper EMA junctions is required for this switch, so the
presence of Stripe-A within a cell indicates stably attached muscles
(Becker et al., 1997). When we stained mys embryos with isoformspecific antibodies, we found that Stripe-B levels appeared
comparable to those of wild type, but mys mutant embryos lacked
detectable Stripe-A (Fig. 3D, compare with 3C). Therefore, we
conclude that mys embryos lack stable EMA junctions, as previously
reported (Martin-Bermudo, 2000). Although mys embryos fail to
attach muscles, the denticle hooking pattern of mys embryos was
indistinguishable from that of wild type (Fig. 3A). These data suggest
that neither muscle tension nor complete EMA specification is
required for anterior denticle hook orientation.
Furthermore, whereas broad ectopic expression of ectopic StripeB using Tubulin (Tub)-Gal4 led to hooking defects in both column
1 and column 4 (Fig. 3B), it did little to alter the location of muscle
attachment sites or Stripe-A expression (Fig. 3E) (Becker et al.,
1997). The lack of correlation between denticle phenotype and EMA
specification indicates that stripe functions in denticle hooking
independently of its role in muscle attachment.
1400 RESEARCH ARTICLE
Development 137 (8)
locus (see Fig. S2 in the supplementary material). We could not detect
Shot protein in the tendon cells of these embryos (see Fig. S2 in the
supplementary material). Such embryos exhibited a range of denticle
phenotypes, from general shape and organization defects (Fig. 4C) to
a more specific hooking defect in which 62% of column 1 denticles
and 75% of column 4 denticles exhibited incorrect hook orientation
(Fig. 4B,D), which is reminiscent of the stripe mutant phenotype.
As shot is not the only gene uncovered by the heteroallelic
deficiency, we wished to confirm that the phenotype observed was in
fact due to the loss of Shot. We expressed shot double-stranded (ds)
RNA using the general driver Actin (Act5c)-Gal4. In these embryos,
we observed strong phenotypes that were very reminiscent of the
deficiency phenotype, including defects in epithelial organization and
hook orientation (Fig. 4E). We conclude that shot has a general role in
epithelial organization (Roper and Brown, 2003) and acts in hooking
orientation as well. The variable expressivity of the shot phenotype
has been observed previously (Gregory and Brown, 1998; Lee et al.,
2000) and suggests the existence of other proteins partially redundant
with shot. Consistent with this, restoring the stripe-dependent isoform
of shot [shotL(A)-GFP] (Lee and Kolodziej, 2002a) to column 2 was
not sufficient to rescue the hooking defects of stripe mutants (see Fig.
S3 in the supplementary material). This suggests the existence of other
Stripe targets relevant to denticle hook orientation.
Shot is enriched in denticles and across certain
cell interfaces
To further understand the denticle phenotypes, we reinvestigated the
accumulation of Shot protein within denticle field cells, focusing
apically, where the actin-based protrusions arise. As previously
shown, in stage 14-15 embryos, Shot was detectable in all epidermal
cells and enriched in tendon cells (Fig. 5A) (Gregory and Brown,
1998; Roper and Brown, 2003; Strumpf and Volk, 1998). We also
observed Shot puncta that colocalized with Filamin (Cheerio –
FlyBase), an actin-associated protein that is enriched in protrusions
(Fig. 5A).
Although it was heavily enriched within the tendon cells, as
expected, Shot exhibited an additional component not previously
appreciated. Apically, at stage 15, using anti-Armadillo (Arm) to
label adherens junctions, we observed Shot protein not only in
column 2 cells, but also along the posterior of column 1 cells,
leading to its apparent accumulation along both sides of the column
1-2 cell interface (Fig. 5B). We observed identical accumulation at
the posterior of column 4 cells (Fig. 5B). Artifacts of imaging or cell
architecture were ruled out, as expression of lacZ in the cytoplasm
of column 2 cells was imaged solely within those cells (see Fig. S4A
in the supplementary material). Thus, Shot specifically accumulated
along the posterior interface within column 1 and 4 cells, abutting
the Stripe-expressing column 2 and 5 cells. Interestingly, these are
the same two interfaces along which denticles reverse their hook
orientation, suggesting a correlation between the two events.
In older embryos, Shot protein became anteriorly enriched in
column 2 and 5 cells. Although cuticle deposition prevents the
visualization of cell outline markers at this stage, Shot was enriched
anterior to tendon cell nuclei (Fig. 5C), which themselves are centrally
positioned within the cells (see Fig. S4B,C in the supplementary
material). This anterior bias was specific to the denticle field cells, as
Shot accumulated on either side of nuclei within smooth field cells
(Fig. 5C). As MACF can localize, stabilize and bundle microtubules,
DEVELOPMENT
Fig. 5. shot is enriched in denticles and
across certain cell interfaces. (A-A⵮) Stage
15 wild-type Drosophila embryo, showing
colocalization of Shot (green; single channel
in A⬘) with Filamin (magenta; single channel
in A⬙), a marker of actin-based protrusions.
Colocalization was detectable from the onset
of actin-based protrusion formation. (A⵮)
Magnification of the boxed area in A.
(B-B⵮) Stage 16 wild-type embryo, showing
Shot (magenta; single channel in B⬘) and
Arm (green; single channel in B⬙). White line
marks the anterior (left) boundary of tendon
cells. Note the Shot accumulation on the
anterior side (left) of the cell boundary. (B⵮)
Magnification of the boxed area in B.
(C-C⵮) Stage 17 wild-type embryo, showing
Shot (green; single channel in C⬘) and Stripe
(magenta; single channel in C⬙). Note the
Shot protein enrichment anterior to tendon
cell nuclei in the denticle field, but not in the
smooth field (arrow). (C⵮) Magnification of
the boxed area in C. Scale bar: 20 mm,
except 5 mm in A⵮,B⵮,C⵮.
Control of denticle diversity
RESEARCH ARTICLE 1401
it is possible that this enrichment reflects a specialized cytoskeletal
complex at the anterior interface of column 2 and 5 cells (Leung et al.,
1999; Roper et al., 2002; Sanchez-Soriano et al., 2009).
shot functions non-cell-autonomously in denticle
hooking
Although Shot is upregulated in the tendon cells, Shot is also present
at lower levels throughout the epidermis and accumulates within the
denticle itself (Fig. 5A) (Gregory and Brown, 1998; Roper and
Brown, 2003), in addition to along the 1-2 and 4-5 boundaries. To
assess which pool(s) of Shot is required for denticle hooking
orientation, we asked whether interfering with Shot function in cell
column 2 could cause a non-autonomous phenotype in column 1.
First, we expressed shot dsRNA under the control of ptc-Gal4,
which significantly reduced Shot protein levels in cell column 2
(Fig. 6A). We also interfered with Shot using a dominant-negative
construct, UAS-shot-EGG, which contains its EF-hand domain and
microtubule-binding GAS2 domain (Fig. 6H, boxed region) fused
to GFP (Gregory and Brown, 1998; Subramanian et al., 2003). UASshot-EGG localizes to microtubules in tendon cells and presumably
functions by blocking the interactions of all Shot isoforms with
microtubules (Subramanian et al., 2003).
Remarkably, both ptc::dsshot and ptc::shot-EGG embryos
displayed non-autonomous denticle hook orientation defects in
column 1 (Fig. 6B,D,F,G). In addition, embryos depleted for Shot
also exhibited gaps among column 1 denticles (Fig. 6C; see
Materials and methods). Taken together, the stripe-dependent
upregulation and accumulation of Shot across the column 1-2 (and
4-5) boundaries is suggestive of the non-autonomous requirement
for Shot in denticle hook orientation. Furthermore, these results are
consistent with a role for stripe-dependent shot expression in nonautonomous anterior hooking.
We turned next to the ubiquitously expressed pool of Shot, which
accumulates along the actin-based protrusions and at the posterior
boundary of column 1, and asked whether Shot might also play an
intrinsic role in denticle shaping. To test this, we expressed UASshot-EGG under the control of en-Gal4, which is strongly expressed
in cell column 1. In these embryos, ~65% of column 1 denticles
exhibited a defect in anterior hooking and appeared poorly shaped
and aligned (Fig. 6E,G). Thus, we conclude that Shot is intrinsically
required for denticles to hook properly, and we hypothesize that this
reflects a role for the ubiquitously expressed pool of Shot. Since the
dominant-negative construct, which is thought to block the
interaction of Shot with the microtubule cytoskeleton, generated
DEVELOPMENT
Fig. 6. shot is required non-cellautonomously. (A-A⬙) ptc::dsshot Drosophila
embryo, showing Stripe (magenta; single
channel in A⬘) and Shot (green; single channel in
A⬙). Note the strong reduction of Shot in column
2, but not column 5 (compare with Fig. 5C). Ptc
also drives expression in the smooth field, and is
likely to account for the slight decrease of Shot
in the ‘S’ column. (B) ptc::dsshot cuticle, showing
hooking defects in column 1 (circles).
(C) ptc::dsshot cuticle, showing hooking defects
and a gap in column 1. (D) Quantification of
column 1 hook orientation in ptc::dsshot.
‘Affected’ refers to those embryos assumed to
carry the dsRNA transgene, whereas ‘unaffected’
refers to presumed sibling controls (see Materials
and methods). (E) en::shot-EGG cuticle, showing
hooking defects in column 1 (circles).
(F) ptc::shot-EGG cuticle, showing hooking
defects in column 1 (circles). Some hooking
defects are also observed in column 4 and this is
likely to be due to Ptc driving shot-EGG in
column 4 cells. (G) Quantification of column 1
hook orientation in en::shot-EGG and ptc::shotEGG. **, P<0.01. (H) Structure of Shot. The
boxed regions are included in the shot-EGG
construct. Scale bar: 20 mm in A-A⬙; 10 mm in
B,C,E,F.
1402 RESEARCH ARTICLE
Development 137 (8)
hooking defects when expressed in either column 2 or column 1
cells, we next tested whether microtubules acted in either, or both,
of the non-autonomous and intrinsic facets of denticle hooking.
Shot functions with the microtubule cytoskeleton
As globally disrupting microtubules would be likely to cause a
severe phenotype, we disrupted microtubules in a column-specific
manner by ectopically expressing the microtubule-severing protein
Spastin (Sherwood et al., 2004; Trotta et al., 2004).
First, we expressed UAS-spastin under the control of en-Gal4,
severely depleting microtubules specifically in En-expressing cells,
including column 1 (Fig. 7A). This correlated with a loss of column
1 denticle hooking, with ~60% of column 1 denticles failing to hook
(Fig. 7B,E). These data are consistent with the observation that latestage protrusions contain microtubules at their base (Price et al., 2006).
Next, we expressed UAS-spastin under control of ptc-Gal4,
depleting microtubules in column 2 (Fig. 7C). ptc::spastin mutant
cuticles exhibited a non-autonomous denticle orientation phenotype
in column 1 (Fig. 7D), with ~44% of column 1 denticles affected
(Fig. 7E). Therefore, we conclude that microtubules are required
both intrinsically for denticle hooking and in the non-autonomous
specification of anterior hooking. Furthermore, these data are
consistent with our hypothesis that Shot acts via the microtubule
cytoskeleton to properly orient denticle hooks.
Stripe links patterning signals to denticle
diversity
stripe is a genetic target of both the Hh and Egfr pathways, and is
regulated by Hh (and Wingless) directly via distinct cis-regulatory
elements (Hatini and DiNardo, 2001; Piepenburg et al., 2000). Thus,
our data suggest that the signals responsible for the elaborate
denticle pattern (Alexandre et al., 1999) act by determining stripe
expression in the appropriate cell columns.
Our work significantly advances the current understanding of
how pattern diversity is produced within the cuticle. The allocation
of cells into the smooth and denticle fields occurs when competitive
signaling interactions between the Wingless and Egfr pathways
result in restricted expression of the transcription factor Shavenbaby
(Svb; Ovo – FlyBase) (Mevel-Ninio et al., 1991; O’Keefe et al.,
1997; Payre et al., 1999; Szüts et al., 1997). Svb then regulates target
genes that participate in actin filament, extracellular matrix and
membrane morphogenesis to construct denticles (Chanut-Delalande
et al., 2006). However, whereas svb is sufficient for denticle
production, stripe is required for denticle diversity by regulating
Fig. 7. shot functions with the microtubule cytoskeleton.
(A) en:spastin Drosophila embryo, showing phalloidin (magenta; to
label actin) and b-tubulin (green) staining. Note the depletion of
microtubules in column 1. (B) en-Gal4; UAS-spastin cuticle. Note the
column 1 hooking defect. (C) ptc-Gal4; UAS-spastin embryo, showing
phalloidin and b-tubulin staining. Note the depletion of microtubules in
column 2. (D) ptc-Gal4; UAS-spastin. Note the column 1 hooking
defect. It appeared that column 2 was also affected in these embryos,
but this was difficult to score as column 2 exhibits only weak hooks.
(E) Quantification of column 1 hook orientation in en-Gal4; UAS-spastin
and ptc-Gal4; UAS-spastin embryos. **, P<0.01. (F) Model of stripedependent denticle hook orientation (see text). Attachments to muscle
and exoskeleton are not depicted. Scale bar: 10 mm in B,D; 20 mm
in A,C.
target genes that modulate denticle morphogenesis. How the Stripe
targets interact with, and modify, the output of Svb targets in specific
denticle columns will be interesting to explore.
Taking our data together with previously published work, stripe has
at least three functions in this epidermis: the specification of the
muscle attachment sites (Frommer et al., 1996; Volk and
VijayRaghavan, 1994; Volohonsky et al., 2007), the reversal of
DEVELOPMENT
DISCUSSION
Screening for mutations that affect the cuticle pattern has been a
powerful approach to identify genes involved in developmental
decisions (Luschnig et al., 2004; Mayer and Nüsslein-Volhard,
1988; Nüsslein-Volhard and Wieschaus, 1980). Some years ago, it
became clear that the column-specific differences in denticle shape
and hook orientation occurred as a result of differential activation
of the signaling pathways that pattern the epidermis (Alexandre et
al., 1999). However, since then, no downstream targets have been
identified that selectively affect column-specific denticle diversity,
and the mechanisms involved have remained elusive. Here, we
show that stripe integrates signaling information and positional
cues to specify denticle density and anterior hook orientation.
Furthermore, we show that stripe governs hook orientation, in part
via the spectraplakin shot. Thus, the stripe-shot circuit has the
potential to link the unpatterned blastoderm to the fully patterned
cuticle.
denticle hooking orientation, and the determination of denticle density.
At least some of these functions are separable, as we show that muscle
attachment is not required for hook orientation. However, the ventral
denticle pattern is conserved throughout all the drosophilids studied
so far, which suggests that these three biological characteristics are
functionally coupled (McGregor et al., 2007). This, in turn, suggests
an explanation for why select denticle columns are anteriorly hooked.
As muscle attachments are required for larval movement, perhaps a
reversal in hook orientation at the attachment site provides for more
efficient traction. Similarly, the denticles produced by tendon cells,
although fewer, are often more robust and might be better able to
transmit tension. We suggest that stripe could integrate patterning
information to correlate muscle attachment with denticle number and
shape, the latter via cytoskeletal remodeling or modulation of cuticle
secretion components. It should be possible to design biophysical
approaches to test these inferences.
Spectraplakins are required for denticle hooking
One Stripe target that we implicated is the spectraplakin, shot. We
found that Shot acts intrinsically in denticle hooking, but is also
important for the non-autonomous influence that one cell column
exerts on another. The phenotypic similarities between stripe and its
target shot strongly suggest that shot acts as part of a nonautonomous, stripe-controlled circuit, rather than in some parallel
pathway. In turn, this suggests that the smaller, stripe-dependent
Shot isoform functions in the non-autonomous circuit. In addition,
although we found that muscle tension is not required, the
involvement of Shot suggests that the non-autonomous signal from
tendon cells to the responding cells might still be mechanical in
nature. To our knowledge, this is the first work to demonstrate a
functional role for either spectraplakins or microtubules in the
shaping of actin-based protrusions.
shot is required intrinsically
Although Shot is required intrinsically for columns 1 and 4 to
construct properly hooked denticles, there are two pools of Shot to
consider here. Thus, we cannot differentiate whether Shot is required
intrinsically to respond to stripe-dependent cues or for a separate
structural purpose. Of course, these possibilities are not mutually
exclusive, and it seems likely that Shot is intrinsically required at
multiple steps.
Shot, actin filaments and microtubules are all found within the
protrusion (Price et al., 2006). Since Shot has the ability to bind
both actin and microtubules simultaneously (Karakesisoglou et al.,
2000; Lee and Kolodziej, 2002a; Leung et al., 1999), the intrinsic
role of Shot might be to physically link these cytoskeletal networks
within the protrusion itself (Fig. 7F). In addition, it was recently
demonstrated that MACF possesses actin-induced ATPase activity,
which is hypothesized to move microtubules along actin filaments
(Wu et al., 2008). Thus, Shot could function within the protrusion
by maintaining physical tension between the cytoskeletal
networks.
Conversely, as Shot is also enriched at the posterior cortex of the
responding cells, this pool of Shot sits in close proximity to the
denticles it influences (Fig. 7F). Thus, it is tempting to speculate that
this pool of Shot is required to respond to the hooking cues given by
the tendon cell. One unresolved issue is how Shot in column 1
becomes enriched along the 1-2 boundary. As Shot protein fails to
localize to the posterior cortex of columns 1 and 4 in the absence of
stripe (see Fig. S5 in the supplementary material), it is tempting to
speculate that stripe-dependent Shot stabilizes intrinsic Shot at select
column boundaries (Fig. 7F).
RESEARCH ARTICLE 1403
Shot is required non-cell-autonomously
Although we demonstrate that Shot is required in tendon cells for
denticles in the adjacent column to hook properly (Fig. 6), the
specific molecular requirement for Shot in the tendon cell remains
to be determined.
The column 1-2 and 4-5 interfaces are enriched for a number of
cytoskeletal components (Walters et al., 2006; Simone and DiNardo,
2010), and late stage embryos show dramatic enrichment of Shot
protein at these interfaces (Fig. 5C). stripe does not control the
special nature of these interfaces (R. Simone and S.D., unpublished
observation), and raises the wider question of what initially
differentiates these interfaces from other column boundaries. We
speculate that the same signaling cues that initiate stripe expression
(Hh and Spi) might also be responsible for the specialization of the
1-2 and 4-5 interfaces, and serve to coordinate cytoskeletal
enrichment, muscle attachment and reversal of denticle hook
orientation. As previously mentioned, we speculate that stripedependent Shot could be required to stabilize this enrichment along
the 1-2 and 4-5 column boundaries.
Notably, in response to Stripe, a specialized microtubule array is
formed within the tendon cell that runs from the basal to the apical cell
surface (Becker et al., 1997; Gregory and Brown, 1998; Subramanian
et al., 2003). Shot localizes along this array in two places – the basal
cell surface, where the EMA junction will form, and the apical portion
of the cell, where the microtubule array links to the cortical actin
cytoskeleton (Gregory and Brown, 1998; Volk and VijayRaghavan,
1994). We do not know whether the apical-basal microtubule array is
responsible for the localization of Shot, or whether this array is
required for denticle hooking, although this would be consistent with
the requirement we observed for microtubules.
Summary
It appears that the non-autonomous stripe-shot circuit culminates in
the localization of Shot protein across the boundaries where denticle
hooks reverse. As spectraplakins can stabilize, localize and bundle
microtubule arrays, as well as create specialized membrane domains
via membrane protein clustering (Leung et al., 1999; Roper et al.,
2002; Sanchez-Soriano et al., 2009), a likely hypothesis is that Shot
organizes a specialized microtubule array or other cytoskeletal
complex at these interfaces.
As denticle hooks manifest themselves at the level of the cuticle
and not in bending of the actin filaments themselves (S.A.D. and
S.D., unpublished observation) (Dickinson, 1997; Price et al., 2006),
we speculate that this complex could control polarized secretion,
extracellular matrix deposition or membrane-matrix attachment.
Alternatively, as we also discovered a role for microtubules in
denticle hooking, perhaps Shot remodels the cytoskeleton via
membrane-microtubule attachment, as it does in tracheal cells (Lee
and Kolodziej, 2002b).
Acknowledgements
We thank the fly community, especially Talila Volk, and the Bloomington Stock
Center for stocks and antibodies; Talila Volk and Nick Brown for their insightful
input; the Ghabrial and DiNardo laboratories for discussions; and Mitra Eghbal
for work on denticle density. This work was supported by an NIH Training
Program in Genetics T32GM008216 to S.A.D. and NIH RO1 GM45747 to S.D.
Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.045450/-/DC1
DEVELOPMENT
Control of denticle diversity
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1404 RESEARCH ARTICLE