© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 3403-3415 doi:10.1242/dev.122226
RESEARCH ARTICLE
The receptor tyrosine kinase Pvr promotes tissue closure by
coordinating corpse removal and epidermal zippering
ABSTRACT
A leading cause of human birth defects is the incomplete fusion of
tissues, often manifested in the palate, heart or neural tube. To
investigate the molecular control of tissue fusion, embryonic dorsal
closure and pupal thorax closure in Drosophila are useful
experimental models. We find that Pvr mutants have defects in
dorsal midline closure with incomplete amnioserosa internalization
and epidermal zippering, as well as cardia bifida. These defects are
relatively mild in comparison to those seen with other signaling
mutants, such as in the JNK pathway, and we demonstrate that JNK
signaling is not perturbed by altering Pvr receptor tyrosine kinase
activity. Rather, modulation of Pvr levels in the ectoderm has an
impact on PIP3 membrane accumulation, consistent with a link to
PI3K signal transduction. Polarized PI3K activity influences
protrusive activity from the epidermal leading edge and the
protrusion area changes in accord with Pvr signaling intensity,
providing a possible mechanism to explain Pvr mutant phenotypes.
Tissue-specific rescue experiments indicate a partial requirement in
epithelial tissue, but confirm the essential role of Pvr in hemocytes for
embryonic survival. Taken together, we argue that inefficient removal
of the internalizing amnioserosa tissue by mutant hemocytes coupled
with impaired midline zippering of mutant epithelium creates a
situation in some embryos whereby dorsal midline closure is
incomplete. Based on these observations, we suggest that
efferocytosis (corpse clearance) could contribute to proper tissue
closure and thus might underlie some congenital birth defects.
KEY WORDS: Cell removal, Dorsal closure, Drosophila, Jun kinase,
PIP3, Pvr, Receptor tyrosine kinase, Morphometric analysis,
Thorax closure
INTRODUCTION
Tissue closure is an essential developmental process to seal a hole or
generate a contiguous tissue layer. Its common occurrence during
embryogenesis underscores the prevalence of birth defects in the
human population, such as cleft palate, spina bifida, ventral body
wall and cardiac septum defects, involving incomplete or improper
fusion (Brewer and Williams, 2004; Ray and Niswander, 2012).
Injury-induced tissue closure, or wound healing, is also essential
over a lifetime to repair damage and help repel infections (Martin
and Parkhurst, 2004).
1
Department of Microbiology and Molecular Genetics, School of Medicine,
2
University of Pittsburgh, Pittsburgh, PA 15219, USA. Center for Craniofacial and
Dental Genetics, Department of Oral Biology, School of Dental Medicine, University
of Pittsburgh, Pittsburgh, PA 15219, USA.
*Present address: Department of Molecular Genetics and Microbiology,
Duke University Medical Center, Durham, NC 27710, USA.
‡
Author for correspondence ([email protected])
Received 19 January 2015; Accepted 10 August 2015
Two genetically tractable models of tissue closure that have
contributed to our current understanding are dorsal closure and
thorax closure in Drosophila. Dorsal closure of the embryo
proceeds by the coordinated activities of a central squamous
epithelium, the amnioserosa (AS), and surrounding dorsal
epidermal epithelium (Kiehart et al., 2000). Cell contraction and
limited apoptosis of the AS, along with epidermal cell stretching
tightened by an actomyosin pursestring, together drive the
dorsalward movement of the epidermal leading edges (LEs)
(Blanchard et al., 2010; Fernández et al., 2007; Gorfinkiel et al.,
2009; Solon et al., 2009; Toyama et al., 2008). Dynamic LE cell
protrusions then facilitate zippering of the epithelium across the
midline concomitant with AS internalization and degeneration
(Jacinto et al., 2000, 2002; Kaltschmidt et al., 2002; Millard and
Martin, 2008). Although dorsal closure is robust to mechanical
perturbations due to distributed tension-based forces (Kiehart et al.,
2000; Rodriguez-Diaz et al., 2008), mutations in many signaling,
adhesion, cytoskeleton and trafficking proteins impact the speed
and efficiency of the process (Harden, 2002; Heisenberg, 2009).
Thorax closure occurs shortly after the Drosophila larva
pupariates to commence metamorphosis of the adult. A pair of
imaginal discs, which are epithelial precursors to the adult wing and
dorsal thorax (or notum), evert over the larval epidermis, displacing
it as they spread from lateral positions toward the midline (PastorPareja et al., 2004; Usui and Simpson, 2000). LE cells take on
a mesenchymal character and exhibit extensive actin-based
protrusions (Martin-Blanco et al., 2000). Within 8 h, midline
fusion bridges the contralateral cell sheets into a continuous
epithelium (Martin-Blanco et al., 2000; Usui and Simpson, 2000;
Zeitlinger and Bohmann, 1999), and further remodeling occurs to
form a mature epidermal barrier (Pastor-Pareja et al., 2004). Such
dynamic reorganization of epithelial margin cells to promote or
curtail motility highlights a similarity of Drosophila and vertebrate
tissue closure, in which mesenchyme function and signaling with
neighboring epithelia are major contributing factors (Heller et al.,
2014; Smith and Tallquist, 2010). One key difference is that in
Drosophila closure does not depend heavily on cell proliferation to
fuel tissue outgrowth across large distances (Bush and Jiang, 2012).
Despite this, the molecular pathways that orchestrate such largescale tissue fusions are generally well conserved, reflecting
fundamental strategies for animal morphogenesis.
Evidence from vertebrate studies indicates that Platelet-derived
growth factor receptor (Pdgfr) signaling organizes cell behaviors
that drive developmental closure processes in various organ systems
and organisms (Bush and Jiang, 2012). For instance, zebrafish
pdgfra directs morphogenesis of the craniofacial skeleton, and its
activity is sensitive to genetic and environmental perturbations
suspected in the etiology of mammalian clefting disorders (Eberhart
et al., 2008; McCarthy et al., 2013). Similarly, mutations in murine
Pdgfc ligand and Pdgfra receptor result in facial and palate clefts
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Rebecca A. Garlena1, Ashley L. Lennox1,*, Lewis R. Baker1, Trish E. Parsons2, Seth M. Weinberg2 and
Beth E. Stronach1,‡
RESEARCH ARTICLE
Development (2015) 142, 3403-3415 doi:10.1242/dev.122226
due to both reduced cell proliferation and inadequate migration of
neural crest derivatives (Ding et al., 2004; He and Soriano, 2013;
Smith and Tallquist, 2010). A human PDGFC noncoding
polymorphism has also been associated with cleft lip/palate (Choi
et al., 2009), signifying the importance of these growth factors and
cognate receptors for proper tissue closure. Although Pdgf
receptor tyrosine kinases signal through multiple molecular
pathways, namely the Mitogen-activated protein kinase (MAPK)
and phosphoinositide 3-kinase (PI3K) pathways (Heldin and
Westermark, 1999; Hoch and Soriano, 2003; Tallquist and
Kazlauskas, 2004), it is the loss of PI3K-mediated signaling
downstream of Pdgfrα that primarily accounts for such phenotypes
in the mouse and zebrafish mutants (Klinghoffer et al., 2002;
McCarthy et al., 2013; Pickett et al., 2008).
In Drosophila, receptor tyrosine kinase isoforms resembling
vertebrate Pdgfr, displaying extracellular Ig domains and a split
tyrosine kinase domain intracellularly, are encoded by the Pvr
(PDGF- and VEGF-receptor related) gene (Cho et al., 2002;
Duchek et al., 2001; Heino et al., 2001). Pvr knockdown has been
shown to impair thorax closure, suggesting an evolutionary
conservation of function in tissue closure; in this context, Jun
kinase (JNK) signaling is thought to mediate this effect (Ishimaru
et al., 2004). Numerous studies have shown that JNK signaling is a
key modulator of embryonic dorsal closure (Glise et al., 1995;
Riesgo-Escovar et al., 1996; Sluss et al., 1996), but curiously the
trigger for its activation in the embryo has been elusive. Together,
these observations spurred us to test directly the requirement for Pvr
and its three ligands, Pvf1-3 (Duchek et al., 2001), in dorsal closure
as upstream activators of JNK signaling. Here, we describe a role for
Pvr in embryonic dorsal closure and investigate the cause of closure
defects, the consequence for JNK signaling, and the cellular effects
on thorax closure. Moreover, we implicate Pvf1 as a relevant ligand
in these processes.
Pvr signaling is implicated in the process of thorax closure during
metamorphosis (Ishimaru et al., 2004). We hypothesized that Pvr
could play a role in embryonic dorsal closure as well. To test this, the
phenotypic consequences of embryonic lethal Pvr null mutations
were determined, paying specific attention to the dorsal aspect of the
secreted cuticle, revealing the fidelity of the closure process. Cuticle
preparations from Pvr zygotic or germline clone (GLC) mutant
embryos displayed mild dorsal midline defects centered around the
last segment to close (Fig. 1A-F). Phenotypes varied from wild type,
midline pinching and scabs, to dorsal holes with no more than four
segments unfused (Fig. 1G). Head involution defects were rare.
Although the cuticle phenotypes of Pvr mutant embryos were
relatively mild, they indicate an undescribed role for Pvr during
embryogenesis.
Pvr protein expression in the AS and ectoderm
The expression and requirement of Pvr in embryonic hemocytes
have been described (Brückner et al., 2004; Cho et al., 2002), but, in
addition, we detected Pvr protein in the AS and ectoderm, two
tissues orchestrating the closure process (Fig. 1H-K). Staining in the
AS developed during stage 10/11 when this tissue is flattened
between the extended germband and Pvr-expressing hemocytes are
migrating out of the head (Fig. 1H). During stage 12, Pvr was
detected in the ectoderm. Strong expression continued in these
tissues throughout the rest of embryogenesis (Fig. 1I,J). Upon
closure, dorsal midline cells remained enriched for Pvr (Fig. 1J).
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Fig. 1. Embryonic closure phenotypes and protein expression of Pvr.
(A-F) Larval cuticles. Wild type, with the segments indicated (A), is compared
with Pvr zygotic (B-D) or germline clone (GLC) (E,F) mutants displaying
midline scabs or holes (arrows). (G) Quantification of phenotypes in B-F
(n=124-310). (H-K″) Pvr immunostaining in wild-type embryos, without (H-J) or
with (K-K″) Fas3 colocalization. Germband extended (H), early dorsal closure
(I) and post-closure (J) embryos depict Pvr expression in hemocytes (HE),
amnioserosa (AS) and leading edge (LE) of the dorsal ectoderm (arrows, I,J).
(K) Confocal z-projection of dorsal quadrant from mid-closure embryo. Thin
lines demarcate the position of the x/z projection beneath (K′); single channels
are also shown. Dashed lines highlight Pvr localization at the interface between
LE and underlying AS. Note Pvr at lateral membranes of AS (black arrowhead).
Ectoderm has extensive colocalization of markers (open arrowhead). Planar
polarized enrichment of Pvr is seen in LE cells compared with Fas3
(K″, arrows). Lateral membrane colocalization is seen in the deeper section
(arrowhead). Scale bars: 50 μm in H-J; 10 μm in K-K″.
Surface (Fig. 1K,K″) and transverse (Fig. 1K′) views highlighted
Pvr along the lateral membranes of ectodermal epithelial cells,
similar to Fasciclin 3 (Fas3). Laterally localized Pvr was also
prominent in AS cells lacking Fas3. z-projection also revealed Pvr
enrichment at the adhesive interface between the ectodermal LE and
underlying peripheral AS cells (Fig. 1K,K′) (Wada et al., 2007).
Notably, single confocal sections of the LE showed marked planar
polarization of Pvr at the ‘free’ edge excluding Fas3 (Fig. 1K″),
where LE cells take on a mesenchymal-like phenotype with
extensive cellular projections (Bahri et al., 2010). In apical
surface sections, Pvr had a punctate appearance suggesting
receptor trafficking in endo/exocytic vesicles (Fig. 1K″).
Antibody specificity was confirmed by comparisons among wildtype, heterozygous and homozygous Pvr mutant embryos in which
DEVELOPMENT
RESULTS
Pvr is required for the completion of dorsal closure
the fluorescence intensity correlated directly with wild-type gene
copy number (supplementary material Fig. S1).
Late defects at the completion of dorsal closure in Pvr
mutant embryos
The cuticle defects in Pvr mutants suggested a requirement at late
stages; however, this could be secondary to delayed or inefficient
progression of closure. To determine the cause of the midline
defects, we evaluated molecular markers and dynamic parameters in
fixed and live embryos. Phalloidin staining for filamentous actin (Factin) or phosphotyrosine immunostaining in fixed embryos showed
no difference in the ability of Pvr mutants to fortify a prominent
actin pursestring at the LE or to assemble and maintain cellular
adherens junctions throughout the ectoderm and AS (Fig. 2A-D).
Time-lapse imaging of live embryos expressing a green fluorescent
protein (GFP)-tagged moesin actin-binding domain, moe::GFP
(Kiehart et al., 2000), revealed similar deployment of the
actomyosin cytoskeleton and dynamic AS behavior (Fig. 2E,F;
supplementary material Movie 1). In fact, measurements of the
width and height of the eye-shaped opening and the area of the
exposed AS in projected 2D images over time demonstrated that
closure of Pvr mutant embryos was nearly indistinguishable from
Development (2015) 142, 3403-3415 doi:10.1242/dev.122226
that of control heterozygotes (Fig. 2G,J; supplementary material
Fig. S2), arguing against inefficient closure as the basis for the late
defects. The velocity of the LEs, defined as dH/dt averaged over the
duration of closure, was 14±4 and 13±4 nm/s for Pvr heterozygous
and homozygous mutant embryos, respectively, similar to
published velocities (Hutson et al., 2003; Wells et al., 2014).
Time-lapse imaging did however suggest faltering of zipping in
some embryos, which was appreciated by projecting time series data
as 2D images (Fig. 2H,I). Consistent with this, quantification of the
change in length over time between the anterior canthus and
cephalic dorsal ridge (dB) demonstrated shorter mean lengths in the
mutants (Fig. 2I,J). We also noted that the plane of the zipping
canthi was typically deeper than the rest of the LE (Fig. 2E,F),
a pattern observed in other mutants (Nowotarski et al., 2014) but
of unknown cause. Finally, cell adhesion between the AS and
epidermis was typically maintained as closure proceeded, although
in some specimens the LE pursestring was less uniform and tissues
detached as final segments approached the midline, presumably
yielding those cuticles with a hole or scab.
Another terminal phenotype in Pvr mutants was a split dorsal
vessel, or cardia bifida (Chartier et al., 2002), observed in 6% of
heterozygotes (n=16) versus 65% of homozygous mutants (n=37).
Fig. 2. Dorsal closure progression in Pvr mutants. (A-D) F-actin or phosphotyrosine distribution in mid- and late-stage heterozygous (A,C) or homozygous
(B,D) Pvr mutant embryos. LE actin (arrowhead) accumulates normally in the mutant (B). At late stages, note the midline (bracketed) defects and unapposed
cardiac cells (cc) in mutants (B′,D) compared with controls (A′,C). Cardiac cells (arrowhead), which normally trail the LE (arrow, C), were exposed in unclosed Pvr5
mutants (D). (E-I) moe::GFP localization in live embryos. LE pursestring (arrowhead, E,F) is less uniform and zipping canthi are in different focal planes in Pvr
mutants (arrows, E,F). (G) Single image from time-lapse movie of dorsal closure illustrating the values quantified in J (see Materials and methods).
(H,I) Cumulative projections of 20 frames from time-lapse movies of Pvr heterozygote (H) or GLC mutant (I). Contours show 5-min intervals. Control embryo has
closed by t100, but the mutant has stalled. (J) Scatter plot comparing change in length of the indicated parameters after 60 min; red bars show mean value. t0 was
set when AS height (H ) equaled 85±4 μm. dB was significantly different between heterozygous control and Pvr mutants. *P<0.05 (unpaired t-test). n=5 control,
n=7 mutant embryos. Scale bars: 20 μm, except 50 μm in G.
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Single rows of cardiac cells remained adherent with their lateral
neighbors, but failed to coalesce across the midline to form the heart
tube (Fig. 2B′,D and Fig. 3). The position and extent of the unfused
dorsal vessel corresponded precisely with the overlying epidermal
hole, but, even in embryos that appeared closed, incomplete vessel
apposition was sometimes evident in deeper focal planes (Fig. 3B).
Incidentally, we have encountered dorsal open embryos of unrelated
genotypes without cardia bifida (supplementary material Fig. S3A),
implying that closure of the different tissues is not absolutely
interdependent, a conclusion corroborated by a recent study (Haack
et al., 2014). Thus, the cardia bifida phenotype described here might
reflect a direct requirement for Pvr and is likely to underlie a
reported malfunction in heart pumping mechanics in late-stage Pvr
mutant embryos (Wu and Sato, 2008).
Often, Pvr mutants displayed cells and/or debris within the
unclosed hole (Fig. 3C) or outside the epidermis (supplementary
material Fig. S3B). In wild-type embryos, concurrent with dorsal
vessel and epidermal closure, the AS becomes internalized,
degenerates, and disappears rapidly through hemocyte
phagocytosis (Reed et al., 2004; Rugendorff et al., 1994).
Presumably, the AS has to get out of the way before converging
tissues meet at the midline, but inefficient hemocyte phagocytosis
might result in corpses or debris obstructing the fusing tissues.
Hemocytes also secrete extracellular matrix proteins required for
embryonic morphogenesis (Bunt et al., 2010; Olofsson and Page,
2005). Together, impaired hemocyte survival and migration, two
characteristic phenotypes of Pvr mutants (Brückner et al., 2004;
Cho et al., 2002), could contribute to both decreased removal of cell
corpses and compromised integrity of tissues fusing at the midline.
Pvr function in multiple tissues accounts for embryonic
viability and completion of closure
To determine the tissue requirements for Pvr in proper dorsal
closure, tissue-specific rescue experiments were conducted using
various Gal4 drivers to induce wild-type receptor in a Pvr mutant
background. Rescue was assessed by quantifying the lethal fraction
of embryos and the penetrance of cuticle phenotypes compared with
controls without either the rescue transgene or the driver. Both
Development (2015) 142, 3403-3415 doi:10.1242/dev.122226
control crosses resulted in an embryonic lethal fraction of onequarter, the expected Mendelian frequency for homozygous
recessive Pvr mutants (Fig. 4A). Expression of Pvr in epithelial
tissues, such as the ectoderm, AS and endoderm, with 69B-G4
significantly reduced embryonic lethality. By contrast, restricted
expression of Pvr in the AS using LP1-G4 provided no rescue, and
the majority of cuticles had obvious dorsal defects (Fig. 4A). These
results suggest that embryonic survival requires Pvr expression in
both dorsal ectoderm and AS, or in the ectoderm alone. We
confirmed transgene expression and the effects on different tissues
by immunostaining for Pvr and Fas3, which showed that transgenic
Pvr protein exceeded the levels of endogenous protein (Fig. 4B-E).
Interestingly, we found that high-level expression of Pvr in the AS
(with LP1-G4 or 69B-G4) led to abnormal upregulation of Fas3
(Fig. 4C; supplementary material Fig. S4). Coincidentally, remnants
of AS tissue were left behind at the midline after closure
(supplementary material Fig. S4), suggesting that Pvr could
modulate the cell adhesion machinery.
With the crq-G4 driver directing Pvr expression in the hemocytes
as early as stage 11, the fraction of lethal embryos was dramatically
reduced and the hemocyte survival and migration phenotypes were
rescued (Fig. 4A,E; supplementary material Fig. S5). Despite this,
Fas3 staining revealed remaining dorsal midline defects, including
lack of epidermal fusion and cardia bifida, as compared with
similarly staged heterozygous embryos (Fig. 4E). Therefore, the
embryonic lethality of Pvr mutants is largely attributable to
impaired hemocyte functions; however, complementation of Pvr
in epithelial derivatives excluding the hemocytes (using 69B-G4)
promoted survival to a small degree, with demonstrable rescue of
the midline closure defects. Altogether, we conclude that there is a
dual tissue requirement for endogenous Pvr in the hemocytes and
ectoderm to allow embryonic survival and proper tissue closure.
Pvr signals independently of the JNK pathway during dorsal
closure
Constitutively active Pvr is reportedly sufficient to induce JNK
target gene expression in the embryo (Ishimaru et al., 2004),
predicting that Pvr mutations might diminish JNK signaling,
DEVELOPMENT
Fig. 3. Cardia bifida in Pvr mutants. (A-C) Single confocal
sections of epidermis or dorsal vessel showing the indicated
fluorescent markers in live embryos. Late-stage heterozygotes
(A,A′) have closed (midline bracketed) and cardiac cells have
coalesced underneath in an aligned double row (arrowheads).
Pvr mutants have mild (dorsal vessel only, B,B′) or moderate
(C,C′) ectodermal hole and split vessel phenotypes. Scale bar:
20 μm.
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Development (2015) 142, 3403-3415 doi:10.1242/dev.122226
causing dorsal closure defects. Yet our observations revealed that
the phenotype of Pvr mutants was comparatively mild. Thus, we
tested directly whether Pvr activity is necessary or sufficient for
induction of JNK signaling by systematically evaluating a
transcriptional reporter for JNK pathway activity in Pvr mutant
and overexpression contexts. In control embryos, the puc-lacZ
reporter was prominently expressed in the most dorsal epidermal
cells surrounding the AS tissue (Fig. 5A). As positive controls,
dorsal overexpression of wild-type or kinase-dead versions of the
JNK kinase kinase Slpr led to ectopic induction or inhibition of
puc-lacZ, respectively (Fig. 5B,C,H). By contrast, puc-lacZ
expression was unaltered in embryos similarly expressing wildtype or constitutively active Pvr (λPvr) (Fig. 5D,E,H), and in
embryos with Pvr expression or activity depleted by RNA
interference (RNAi), dominant-negative inhibition, or GLC
mutation (Fig. 5F-H; supplementary material Fig. S6). Collectively,
these results demonstrate that Pvr signaling does not directly
regulate JNK signaling in the embryonic dorsal ectoderm.
To reconcile these results with previous studies, we considered
the effects of excessive Pvr activation over a prolonged period of
Fig. 5. JNK signaling is unaffected by changes in Pvr activity.
(A-G) Immunostaining of mid-stage dorsal closure embryos for Fas3 (or Pvr)
and β-gal protein from the JNK transcriptional reporter puc-lacZ at the LE
(arrows). pnr-G4 directs transgene expression in the dorsal ectoderm. Slpr
overexpression induced ectopic puc-lacZ (B), whereas kinase-dead Slpr
blocked it (C). Overexpression of wild-type (D) or constitutively active (E) Pvr
had no effect on puc-lacZ. Similarly, reporter expression was normal upon
depletion of Pvr by RNAi (F) or in GLC mutants (G), as confirmed by anti-Pvr
co-immunostaining. Scale bars: 50 μm. (H) Quantification of β-gal-positive
nuclei, n≥4 embryos. Bars show mean±s.d. *P<0.05 (one-way ANOVA, with
Bonferroni post-hoc test) for comparison with ‘no Tg’ control.
λPvr overexpression. In late-stage embryos specifically, we counted
the same or slightly increased numbers of puc-lacZ-positive cells,
but they were disorganized, reflecting a striking alteration of cell and
embryo morphology including induction of Matrix metalloprotease
1 (Mmp1) expression (supplementary material Fig. S7). Under
these circumstances, the apparent increase in JNK target gene
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Fig. 4. Complementation of Pvr in embryonic hemocytes rescues
lethality. (A) Quantification of embryonic lethal fraction (left) and cuticle
phenotypes (right) from tissue-specific rescue crosses. The lethal fraction was
significantly reduced by expression of Pvr in ectodermal derivatives (69B-G4)
or hemocytes (crq-G4). Bars show mean±s.d. *P<0.0001 (Chi-square test).
n=269-394. Line indicates the expected fraction (0.25) for homozygous
recessive embryonic lethal mutation. (B-E) Pvr immunostaining detects
endogenous and overexpressed protein in merged and single-channel images
(embryo anterior). Fas3 is shown in merged and righthand panels (embryo
posterior). Arrows point to the dorsal midline, which is closed in similarly staged
control (B) and 69B-G4 (D) embryos, but open in LP1-G4 (C) and crq-G4 (E)
embryos. Scale bar: 20 μm.
expression is likely to have resulted from effects secondary to
epithelial disorganization, as described previously for imaginal
epithelial tissues (Rosin et al., 2004).
Pvr is necessary and sufficient for maximal PI3K pathway
signaling
Given the distinct embryonic phenotypes and lack of impact on JNK
signaling during dorsal closure, we considered whether Pvr, like its
mammalian counterparts, could regulate PI3K signaling, using a
fluorescent reporter to monitor production of phosphatidylinositol
(3,4,5)-trisphosphate (PIP3). tGPH encodes GFP fused to a PIP3binding PH domain, which relocates to the plasma membrane in
response to PIP3 production, reflecting a bias in the opposing
enzymatic activities of PI3K and the phosphatase Pten (Britton
et al., 2002). During dorsal closure, PIP3 distribution is polarized at
the LE interface, driving actin-based protrusive activity to facilitate
midline zippering (Pickering et al., 2013). We confirmed this
localization and quantified tGPH fluorescence at the LE junctions
versus lateral membranes in live wild-type or Pvr mutant embryos.
Mutants had significantly reduced (by 36%) fluorescence intensity
relative to the control, although the overall profile of junctional
enrichment was preserved (Fig. 6A-D).
Next, we expressed λPvr in segmental stripes of cells using the
en-Gal4 driver and measured tGPH fluorescence. In these embryos,
the reporter was strongly recruited to the membranes of cells in
the expressing stripes, while the adjacent non-expressing stripes
showed a profile equivalent to that of control embryos (Fig. 6E-G;
supplementary material Fig. S8). Quantification revealed a two-fold
Development (2015) 142, 3403-3415 doi:10.1242/dev.122226
increase in fluorescence at the LE junctions upon λPvr expression
(Fig. 6H). In keeping with the role of elevated PIP3 in regulating
dynamic protrusive activity, large protrusions were observed to
emanate from LE and AS cells with λPvr overexpression (Fig. 6I,J;
supplementary material Fig. S8A-D). The total area and maximum
length of LE projections from cells co-expressing λPvr and GFPlabeled actin were significantly enhanced relative to wild type
(Fig. 6K,L). Collectively, these data suggest that Pvr function is
necessary for maximal PIP3 enrichment at the membrane and
sufficient for protrusions, but is not necessary for the polarized
accumulation of PIP3 at the LE junctions.
These results point to a signaling mechanism linking Pvr to PI3K
in the epidermis. To test this model further, we expressed wild-type
PI3K in Pvr mutant embryos using the 69B-Gal4 driver. Although
there was no effect on embryonic lethality, boosting PI3K levels
increased the fraction of wild-type cuticles by 10% relative to the
control, comparable to expression of PvrWT (supplementary material
Fig. S8E). Moreover, adult progeny from these crosses revealed
dominant genetic suppression of a PI3K-induced phenotype by
heterozygosity of Pvr (supplementary material Fig. S8F). Thus,
mechanistically, the influence of Pvr on the PI3K pathway could
account in part for the ectodermal requirement during closure.
Synthetic lethal interaction between the JNK pathway and
Pvf1
Although our observations demonstrate that Pvr does not directly
signal through JNK in embryonic dorsal closure, published studies
do provide evidence that a Pvr ligand, Pvf1, and JNK signaling
Fig. 6. Pvr is required for maximal PIP3 accumulation at the LE. (A,B,E,F) Confocal z-projections of the LE of mid-dorsal closure stage live embryos
expressing the PIP3 reporter tGPH. Controls with two (A) or one (E) copy of tGPH compared with Pvr mutant (B) or constitutively active Pvr expression in
segmental stripes with en-G4 (red bracket) versus control stripe (blue bracket). (C,G) Mean tGPH fluorescence along 5 μm spanning the LE cell junction enriched
for PIP3 or lateral membrane control (yellow arrows, A). Error bars represent s.e.m. n=24-28 junctions, 6-7 embryos (C); n=60 junctions, 15 embryos (G). Line
color (G) matches bracketed stripes in F. (D,H) Fold change in maximum tGPH fluorescence between control and experimental genotypes, normalized to
corresponding averaged lateral membrane values. Error bars show s.e.m. (I,J) en-G4 driving GFP-labeled Actin to reveal LE protrusions (outlines, arrows),
without (I) or with (J) λPvr co-expression. (K,L) Quantification of LE protrusion area and length, respectively. Mean±s.d. n=4 stripes, two time points, 6-7 embryos.
***P<0.0001 (unpaired t-test). Scale bars: 10 μm.
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Development (2015) 142, 3403-3415 doi:10.1242/dev.122226
Table 1. Synthetic lethal genetic interaction between Pvf1 and JNK
pathway mutants
Test cross
% Eclosion of mutant
male adult* (n ‡)
% Embryonic
lethality§ (n ¶)
hep1/FM6 × +/Y
Pvf1EP1624/FM7 × +/Y
hep1 Pvf1EP1624/FM7 × +/Y
slprBS06/FM7 × +/Y
slprBS06 Pvf1EP1624/FM7 × +/Y
100 (161)
79 (170)
1 (262)
11 (537)
0 (>100)
1 (240)
5 (550)
28 (314)
4 (881)
27 (1891)
*Relative to FM/Y; 100% expected if fully viable.
‡
Total number of male progeny.
§
25% expected if fully lethal.
¶
Total number of embryos.
participate in some of the same developmental processes. For
example, Pvf1 null mutants are adult viable, but mutant males show
morphological defects of the external genitalia (Macias et al., 2004),
a phenotype seen in slprBS06 zygotic mutant males, in which JNK
signaling is impaired (Polaski et al., 2006). Pvf1/Pvr and JNK
signaling are also required for wound healing, regulating distinct
aspects of the process (Brock et al., 2012; Wu et al., 2009).
To test whether Pvf1 might also participate in embryonic dorsal
closure along with Pvr and JNK, we generated slpr, Pvf1 double
mutants and discovered a synthetic lethal genetic interaction (Table 1)
resulting in fully penetrant embryonic lethality and failure of dorsal
closure, comparable to slpr GLC mutants (Fig. 7). The lethal phase
and phenotype of a maternal effect viable JNKK [hemipterous (hep) –
FlyBase] allele, hep1, alone or with Pvf1, were also compared,
revealing similar synthetic lethality (Table 1, Fig. 7A). Given
possible redundancy among Pvf ligands, the slprBS06 mutation was
combined with alleles of Pvf2 and Pvf3. Although the lethality
associated with Pvf2 was partially suppressed by simultaneous loss of
slpr in the double mutants, neither of the ligands enhanced the
lethality or cuticle phenotype of the slpr mutant, in contrast to Pvf1
(Fig. 7A). These data thereby support the idea that Pvf1 is a candidate
for the activation of embryonic Pvr and that this activity, in
connection with JNK signaling, is required for proper dorsal closure.
Notum morphology reveals opposing effects of Pvr and JNK
signaling on thorax closure
We showed previously that modulating the levels of JNK signaling
during thorax closure leads to concomitant changes in midline
notum morphology in the adult (Garlena et al., 2010). Underlying
these morphological changes is the participation of JNK in
regulating cell adhesion, shape, polarity and motility during
spreading of the imaginal cell sheets toward the midline (MartinBlanco et al., 2000; Pastor-Pareja et al., 2004; Zeitlinger and
Bohmann, 1999). Ishimaru et al. (2004) described thorax closure
defects arising from knockdown of Pvr and, based on genetic
interactions and reduced puc-lacZ expression, proposed that the
phenotypes were due to loss of JNK signaling. Since we found no
direct dependence of JNK signaling on Pvr in the embryo, we reevaluated their relationship in thorax closure using a geometric
morphometric strategy with adult phenotypes (Fig. 8) and cell
biological observations of pupal development (Figs 9 and 10).
Geometric morphometrics is well suited to account for shape
variation among individuals in a population, separating or
clustering groups with statistical power. To investigate how notum
shape correlates with signaling intensity, we systematically
perturbed slpr-dependent JNK signaling, or Pvr signaling through
knockdown or overexpression constructs within the pnr-Gal4
domain, encompassing a broad region spanning the dorsal
midline. Altered signaling of these two pathways resulted in
reproducible phenotypes with some superficial similarities
(Fig. 8A, compare b-e with g-j). After digitizing landmarks on
images of specimens of different genotypes (supplementary material
Fig. S9A, Table S1), the resulting coordinates were characterized
using principal components analysis (PCA) to quantify the major
modes of shape variation. The two pathways were first analyzed
individually (supplementary material Fig. S9B,C).
Analysis of all genotypes together revealed an overlap of group
clusters, with the notable outcome that increased signaling in one
pathway phenocopied reduced signaling in the other (Fig. 8B),
suggesting a reciprocal relationship in thorax closure. For instance,
SlprWT and Pvf1RNAi individuals grouped together on the negative
side of the PC1 axis, reflecting shape variation characterized by
longer, narrower notum morphology, as depicted beneath in the
wireframe diagram (Fig. 8B). PvrRNAi also distributed on this side
but separated from the former groups and controls by differences in
PC2, where lateral landmarks were wider and the scutellum was
notably reduced. Substantial overlap of groups along the positive
PC1 axis correlated with widening of all landmarks and a slight
anterior shift in the scutellum. These shapes arose from reduced
JNK activity using dominant interfering proteins (SlprAAA, SlprSH3)
or from elevated Pvr signaling (Fig. 8B). Together, PC1 and PC2
described nearly 71% of the total shape variation.
In light of these results, we next asked which cell behaviors induced
during the pupal stages might account for the morphological
phenotypes in adults. Several drivers were used to express GFP and
experimental constructs in particular tissue regions. The pnr-Gal4
Fig. 7. Specificity of synthetic lethal interaction between Pvf1 and the JNK pathway. (A) Quantification of percent embryonic lethality of single versus double
mutants. (B-D′) Darkfield images of cuticles from control (B) and mutant embryos with large dorsal holes (arrows, C-D′). D and D′ show two examples of the same
genotype.
3409
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
pattern evolved from expression in the proximal part of the larval wing
discs that prefigure the adult notum and in the larval dorsal epidermis
upon which the disc cells migrate toward the midline, to a broad dorsal
stripe overlapping the midline of the scutum and entire scutellum
(Fig. 9A,B). The domain of a second driver, C15-Gal4, was restricted
more proximally in the wing discs and delimited the margins of the
fusing tissues in pupae, making it an excellent marker of thorax closure
(Fig. 9C). Confocal imaging further showed GFP expression in ∼2-8
cells at the leading front of the pupal disc during closure, but not in the
underlying larval epithelium, in contrast to pnr-Gal4 (Fig. 9, compare
B with D). LE cells, distinguished by slightly larger nuclei, were
strikingly enriched for Pvr protein, above the uniform levels present in
the rest of the disc epithelium and larval epidermis (Fig. 9D,D″).
Although the narrower expression domain of C15-Gal4 facilitated
the best view of the leading front during closure, the ultimate effects
on adult morphology were mild and limited to the scutellum (not
shown). A third reagent, He-Gal4, was used to drive expression of
constructs in larval hemocytes that pervade the tissues during closure
(Fig. 9E), analogous to our use of crq-Gal4 in the embryo.
The most striking tissue-level phenotypes during thorax closure
were the effects of Pvr signaling on the integrity of the larval
3410
epidermis specific to pnr-Gal4 (Fig. 10B,C) and the accumulation
of F-actin and changes in cell morphology at the tissue margins
visualized clearly with C15-Gal4 (Fig. 10G,H). Altering Pvr
activity in the pnr domain led to dissolution of the larval epithelium
and fragility of the specimens during dissection (Fig. 10B,C),
phenotypes rarely encountered with perturbation of JNK signaling
(Fig. 10D,E). With Pvr knockdown, larval cells rounded up and
nuclei condensed, reminiscent of apoptosis (Fig. 10C). These cells
persisted at the midline without being engulfed by hemocytes,
which are normally present during closure as seen with HeGal4>GFP (Fig. 10K-O). As such, the adult notum possessed
occasional midline scabs (Fig. 8Aj), recalling the phenotype of Pvr
mutant embryos. In the C15 domain, Pvr-depleted margin cells also
rounded up, lost contact and dispersed, consistent with blunted actin
accumulation and protrusions (Fig. 10H). Although some pyknotic
nuclei and dissolution of the larval epidermis also resulted from
elevated Pvr in the pnr domain, the phenotype was distinct in that
the bright round larval cells seen in Pvr-depleted pupae were not
evident (Fig. 10B,C). Some larval cells had rounded up but the
central tissue appeared highly disorganized, less adherent and more
mesenchymal, with cells of different shapes, possibly contributing
to impaired disc migration due to the compromised larval epithelial
substrate. C15-Gal4 expression of PvrWT revealed intense F-actin
staining in the imaginal cells, which was especially dramatic at the
apical surface compared with the control (Fig. 10G), and the disc
margin appeared broader and less tightly constrained compared with
the other genotypes (Fig. 10G). Hemocytes expressing Pvr were
clearly present and capable of corpse engulfment, but were sparse
with PvrRNAi (Fig. 10L,M).
The effects of altering JNK signaling with all three drivers were
subtle in comparison to Pvr (Fig. 10D,E,I,J,N,O). Slpr
overexpression moderately enhanced F-actin accumulation,
whereas Slpr inhibition reduced it (Fig. 10I,J), but JNK signaling
appeared to have much less impact on the larval epithelium. Thus,
the thorax morphologies induced by reciprocal changes in both
pathways cannot be explained simply by their effects on F-actin
alone, or the LE in isolation, but must also take into account effects
on the intervening larval tissue, the interface between them, and
possibly the phagocytic hemocytes, which are also thought to be
part of the pnr expression domain (Minakhina et al., 2011).
DISCUSSION
Here, we show evidence of a role for Pvf1/Pvr in tissue closure,
acting independently of JNK signaling, but with an influence on the
PI3K pathway. Foremost, in the embryo, Pvr is required in both
hemocytes and ectoderm to support embryo viability and proper
midline zippering. We envision that impaired hemocyte migration
and function in Pvr mutants (Brückner et al., 2004) slows AS
internalization and removal. This occurs to a varying extent, likely
contributing to the low penetrance and variable expressivity of the
Pvr midline phenotypes. Indeed, Brückner et al. (2004) predicted
that many phenotypes found in Pvr mutants might be indirect
consequences of hemocyte dysfunction. This has certainly been
borne out in independent studies of central nervous system
development (Olofsson and Page, 2005; Sears et al., 2003),
malpighian tubule morphogenesis (Bunt et al., 2010), and now
dorsal closure. Yet, sole rescue of hemocyte function did not
eliminate midline phenotypes compared with similarly staged
control embryos, and a significant degree of rescue was achieved by
Pvr expression in ectodermal derivatives. In fact, PIP3 accumulation
at the ectodermal LE is altered in Pvr loss- and gain-of-function
contexts, denoting input to the PI3K pathway. In this light, the
DEVELOPMENT
Fig. 8. JNK and Pvr signaling during thorax closure cause reciprocal
changes in adult notum morphology. (A) Examples of notum morphology of
control groups (a,f ) or adults with pnr-G4-directed transgene expression (b-e,
g-j). Yellow arrowheads indicate the dorsal midline, red arrowheads point to the
scutellum. (B) Principal components analysis (PCA) of group shapes from
expression of the indicated transgenes driven by pnr-G4. Individuals are
represented graphically as points. Group differences are depicted as
deformations of landmark positions in a wireframe diagram (black)
corresponding to position along the adjacent PC axis, as compared with the
mean shape from all groups (gray).
Development (2015) 142, 3403-3415 doi:10.1242/dev.122226
RESEARCH ARTICLE
Development (2015) 142, 3403-3415 doi:10.1242/dev.122226
planar polarized distribution of Pvr at the LE is noteworthy and
could differentially impact PI3K signaling components. The effects
of Pvr on the magnitude of membrane-associated PIP3 appear
distinct, however, from that of Bazooka and Pten phosphatase,
which are required to polarize PIP3 distribution in LE cells
(Pickering et al., 2013). Altogether, the cumulative defects in
hemocyte function, slowing removal of the AS in Pvr mutants,
coupled with submaximal PIP3-dependent protrusive activity of the
epidermal LE, result in compromised midline zippering, leaving
debris-filled dorsal holes and causing cardia bifida. This multitissue requirement for Pvr in facilitating dorsal closure recalls the
process of wound healing in which contraction of wound bed
mesenchyme cooperates with wound edge filopodial extensions to
facilitate re-epithelialization of embryonic wounds (Redd et al.,
2004).
Re-evaluation of JNK and Pvr signaling in thorax closure
provided several new insights. First, the application of geometric
morphometrics provided a powerful method to objectively quantify
morphological features that can often be subtle or vary among
individuals. Our analyses revealed evidence for an inverse
relationship between pathway activity and notum shape.
Upregulation of JNK signaling phenocopied downregulation of
Pvf1/Pvr activity and vice versa, which is incompatible with a direct
role for Pvr in JNK activation as proposed (Ishimaru et al., 2004).
Instead, the implication is that the pathways act in opposition or in a
balanced manner to regulate cellular machinery driving the closure
process. Second, our evidence supports the notion that Pvr regulates
PI3K signaling at this later stage. We found that Pvr dominantly
suppressed a PI3K overexpression phenotype consistent with
positive regulation of signaling by Pvr. Also, the induction of
cuticle melanization by Pvr overexpression and reduced
pigmentation with RNAi might connect to PI3K. Cuticle
pigmentation relies on catecholamine derivatives, the enzymatic
production of which is sensitive to nutrient status through the
Insulin receptor/PI3K/Akt signaling axis (Shakhmantsir et al.,
2014; Zitserman et al., 2012). The strength of Akt activity towards
its targets, Tor and FoxO, correlates directly with the intensity of
pigmentation, and our data suggest that this cascade is also
responsive to Pvr activity. Pvr activation of PI3K signaling could
therefore explain the inverse phenotypic effects with JNK described
here, as antagonistic crosstalk between Akt/Tor and JNK signaling
is believed to regulate inverse cell fates, such as survival or
apoptosis (Eijkelenboom and Burgering, 2013; Puig and Mattila,
2011). It is likely, though, that the function of Pvr in thorax closure
is multifaceted, providing trophic signals for the larval epidermis
and hemocytes, and morphogenetic signals in the imaginal
epithelium and LE, similar to its roles in the embryo. Indeed,
there is a striking parallel between disruption of tissue in the pupae
upon Pvr knockdown – as characterized by diminished actin, loss of
structured cell shape, tissue break up, limited corpse clearance and
defective midline fusion – and the embryonic tissue and cuticle
phenotypes.
3411
DEVELOPMENT
Fig. 9. Pvr expression at the LE of spreading imaginal
discs during thorax closure. (A,C) GFP expression with
pnr-G4 (A) or C15-G4 (C) drivers in progressively older live
pupae reveals apposition and fusion of notum tissue at the
midline (arrows) during thorax closure. Boxes indicate
regions shown as dissected, fixed tissues in panels beneath.
(B-B‴) pnr drives expression in discs (small packed nuclei) in
a broad proximal domain and in the intervening larval
epidermal tissue (large nuclei). DAPI staining shows nuclei
(blue). F-actin is enriched at the LE margins (arrowheads).
(D-D‴) GFP expression in the C15 domain is restricted to a
narrow region of the disc margins. Endogenous Pvr protein is
enriched in mesenchymal-like marginal cells (arrowheads).
(E-E‴) For comparison, the He-G4 driver directs GFP
expression in hemocytes infiltrating the tissues. Actin
protrusions are prominent, knitting together disc margins.
Scale bars: 50 μm.
RESEARCH ARTICLE
Development (2015) 142, 3403-3415 doi:10.1242/dev.122226
Fig. 10. Differential effects of Pvr on tissues during thorax closure. (A-O) Confocal images of fixed pupal tissues during thorax closure with the indicated
markers. (A-E) pnr-G4-directed GFP expression without (A) or with (B-E) transgene co-expression. Altering Pvr levels has a dramatic effect on centrally
positioned larval cells. The grayscale DAPI channel (beneath) highlights pyknotic nuclei (B,C) versus the control (A) or slpr-expressing (D,E) samples.
(F-J) Single apical and basal sections of pupal wing discs expressing GFP and transgenes with C15-G4. The midline is toward the right in merged and singlechannel images (beneath). F-actin at the leading front (arrows) is highly enriched upon Pvr overexpression (G), and blunted by Pvr knockdown (H) or Slpr
inhibition (J). Slpr overexpression has a mild enhancing effect on actin levels (I). Arrowheads identify round stray cells depleted for Pvr and leaving the leading
edge (H), whereas cells with increased Pvr are flat and spreading (G). (K-O) Confocal projection of circulating hemocytes labeled by He-G4-directed GFP
expression 12 μm below the notum epithelium, which has closed at the midline (arrowheads). Internalized larval cells with pyknotic nuclei are seen engulfed
inside hemocytes (arrows). PvrRNAi depletes hemocyte numbers. Scale bars: 50 μm.
3412
provides added value especially for organisms with rapid embryonic
development, just like maintaining a streamlined factory assembly line.
MATERIALS AND METHODS
Fly stocks
UAS-Pvf1 (McDonald et al., 2003); UAS-PvrWT, UAS-λPvr, P{EP}
Pvf1EP1624 (Duchek et al., 2001); UAS-PvrΔC (UAS-PvrDN), Pvr1, Pvr5
(Brückner et al., 2004); UAS-slprWT, UAS-slprKD, UAS-slprAAA, UASslprSH3 (Garlena et al., 2010); slprBS06 (Polaski et al., 2006); hep1 (Glise
et al., 1995); slprHMS00742 (TRiP) (Ni et al., 2008); PvrKK101575 (VDRC)
(Dietzl et al., 2007); PvrNIG.8222R, Pvf1NIG.7103R (NIG-Fly); UAS-mCherry.
CAAX, UAS-srcEGFP, UAS-Act5C::GFP, PBac{PB}Pvrc02195, P{XP}
Pvf2d02444, PBac{PB}Pvf2c06947, Mi{MIC}Pvf3MI04168, En-Gal4e16E,
UAS-PI3K92E.Exel (BDSC); pucE69 (also known as puc-lacZ) (Ring and
Martinez Arias, 1993); pnr-Gal4MD237 (Calleja et al., 1996); C15-Gal4
(also known as P{GMR40B01-Gal4}attP2) (Pfeiffer et al., 2008); crq-Gal4
(Olofsson and Page, 2005); 69B-Gal4 (Brand and Perrimon, 1993); LP1Gal4 (also known as cald-Gal4; LP1 is also known as Orct2 – FlyBase)
(Herranz et al., 2006); He-Gal4, UAS-GFP.nls (Zettervall et al., 2004);
P{His2Av-mRFP}, P{sGMCA} (also known as H2Av::RFP, moe::GFP)
(Kiehart et al., 2000); tGPH (Britton et al., 2002). Pvf1 recombinants with
hep and slpr were verified by complementation and PCR to detect the P{EP}
insertion. w1118 was our experimental control.
Clonal analysis
hs-Flp; ovoD1, FRT40 males were mated with heterozygous Pvr1, FRT40
females. Larvae were heat shocked for 2 h at 37°C on two consecutive days
to produce female GLCs. Paternal rescue was 50%.
DEVELOPMENT
While it is commonly appreciated that the failure to remove
apoptotic cells during tissue repair or homeostasis can result in
pathology, as debris and leaky corpses trigger the immune
response and inflammation (Franc, 2002; Hochreiter-Hufford and
Ravichandran, 2013), the consequences for proper developmental
tissue closure might be underappreciated as a possible contribution to
embryonic clefting defects. Intriguingly, murine JNK pathway
mutants with increased apoptosis and neural tube closure defects
have been described (Chi et al., 2005; Sabapathy et al., 1999), but to
what extent the removal of dead cells contributes to the phenotype, if at
all, is not clear. Moreover, programmed cell death is a normal part of
other tissue closure events in craniofacial development and
palatogenesis (Ray and Niswander, 2012). Thus, it will be important
to ascertain the consequences not only of regulated apoptosis, but also
of the fate of dying cells in cleft mutants. The characterization of Pvr
mutants, impaired in hemocyte and ectodermal function, provides an
opportunity to explore this phenomenon further. It will be interesting
to determine whether the prolonged presence of debris or corpses
simply poses a physical barrier to tissue closure, triggers secondary
signals detrimental to further development, or whether there is a
temporal window in which a series of developmental events must
proceed without disruption to subsequent steps. Closely coupling
programmed cell death and phagocytosis appears to be a conserved
mechanism to ensure expeditious corpse removal while maintaining
immune tolerance during tissue homeostasis and infection (Stefater
et al., 2011). Likewise, efficient coupling during organogenesis
RESEARCH ARTICLE
Tissue staining
Embryo immunostaining used standard methods (Rothwell and Sullivan,
2000). F-actin staining was similar but excludes methanol in favor of hand
devitellinization. Staining reagents were: rat anti-Pvr 1:500 (Sears et al.,
2003), rat anti-Pvr 1:500 (Rosin et al., 2004), mouse anti-Fas3 1:40 (7G10,
DSHB), presorbed rabbit anti-β-gal 1:1500 (Cappel, 55976), mouse
anti-phosphotyrosine 1:1000 (4G10, EMD Millipore, 05-321), Texas Redphalloidin 1:500 (Life Technologies), and Vectashield with DAPI mounting
media (Vector Labs). Balancer chromosomes carrying lacZ, GFP or RFP
markers were used to distinguish genotypes in embryo populations.
Preparation of larval cuticles has been described (Baril et al., 2009).
Pupal dissections
GFP-expressing pupae, aged 4.5-6 h after puparium formation, were poked
with fine-tipped forceps in the posterior pupal case, releasing pressure. The
posterior third of the case was peeled away and gut and internal tissues were
removed. Remaining tissue was fixed overnight at 4°C in 4% formaldehyde
diluted in PBT (1× PBS pH 7, 0.1% Triton X-100), followed by three
10 min washes in PBS. Samples were dissected further under GFP
fluorescence by pushing tissue away from the casing. Samples were then
prepared for phalloidin or immunostaining and imaging.
Development (2015) 142, 3403-3415 doi:10.1242/dev.122226
Resource Center (VDRC), Bloomington Drosophila Stock Center (BDSC) and
National Institute of Genetics (NIG-Fly) for stocks and reagents, to Developmental
Studies Hybridoma Bank (DSHB) for antibodies, and to F. Homa and G. Campbell
for use of their microscopes.
Competing interests
The authors declare no competing or financial interests.
Author contributions
B.E.S. conceived the experiments. R.A.G., A.L.L. and B.E.S. performed the
experiments and analyzed the results. L.R.B. obtained images and performed
landmarking and data conversion for the morphometric analysis. T.E.P. and S.M.W.
analyzed the morphometric data and contributed to presentation and description of
the data. B.E.S. wrote the manuscript.
Funding
This work was supported by funding from the National Institutes of Health/Institute
for Child Health and Human Development (NIH/NICHD) [grant number HD045836]
to B.E.S., along with start-up funding from the University of Pittsburgh School of
Medicine. L.R.B. was supported by a Brackenridge Research Fellowship from
the University of Pittsburgh Honors College. Deposited in PMC for release after
12 months.
Supplementary material
Adult flies were glued on a slide, ventral side down. Digital images of the
notum were taken and the program tpsDIG2 (http://life.bio.sunysb.edu/
morph/) was used to collect 14 landmarks and five semi-landmarks covering
features of the scutum, scutellum and scuto-scutellar suture (supplementary
material Fig. S9A). The resulting x,y coordinate positions were aligned
using tpsRelw v1.49 (http://life.bio.sunysb.edu/morph/), in which the raw
landmarks are translated, rotated and scaled to remove any variation not due
to shape. Semi-landmarks were slid along a path tangent to the curve at that
semi-landmark to minimize the Procrustes distance between each target and
reference specimen. The resulting shape coordinates were imported into
MorphoJ v1.05f (Klingenberg, 2011) for statistical analysis using PCA to
quantify the major modes of shape variation and to test for differences
among the groups. Shape variation and group differences were then
visualized graphically and as deformations in wireframe diagrams.
Microscopy
Images of adult flies and pupae were obtained with NIS-Elements software
using a DS-Fi1 digital camera and SMZ1500 stereomicroscope (all Nikon).
Embryonic cuticles were imaged with a Leica DMI4000B inverted
microscope and DFC480 camera. Stained embryos and pupal tissues were
imaged using an Olympus FV1000 Fluoview confocal system on an IX81
compound inverted microscope and processed with Adobe Photoshop.
Preparation and time-lapse imaging of embryos was as previously described
(Evans et al., 2010) with the following details. moe::GFP embryos were
imaged with a 20× objective for up to 2 h. Every 5 min, z-series of four to
eight slices (1.5 μm step size) were captured. A 40× objective was used for
5-min movies with 30-s intervals to visualize cell protrusions and
contractility. Files were imported into ImageJ (NIH), assembled into
movies, cropped, and adjusted for brightness and contrast. Height (h, H) and
width (W) were measured as straight-line lengths (Fig. 2). AS area (A) was
defined using the Freehand selection tool followed by a Fit ellipse function.
An arbitrary value (B) was defined as the midline distance between the
anterior canthus and cephalic dorsal ridge. Live tGPH-expressing stage 14
embryos were imaged at 40× using identical settings for all genotypes.
Projected z-stacks of four slices were quantified in ImageJ with a Profile plot
of gray values (Pickering et al., 2013). Fold change was calculated by
dividing maximum mean pixel intensity for control and experimental
genotypes by mean lateral membrane intensity to control for different
background intensities. puc-lacZ quantification in the embryo has been
described (Stronach et al., 2014). Graphing and statistical tests were
performed with Prism GraphPad. Details are provided in the figure legends.
Acknowledgements
We are grateful to members of the fly community, D. Montell, K. Bruckner, P. Rorth,
P. Garrity, B. Shilo, G. Campbell and B. McCartney, and the Vienna Drosophila
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.122226/-/DC1
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