Download Gene Deletion Screen for Cardiomyopathy in Adult Drosophila

Gene Deletion Screen for Cardiomyopathy in Adult
Drosophila Identifies a New Notch Ligand
Il-Man Kim, Matthew J. Wolf, Howard A. Rockman
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Rationale: Drosophila has been recognized as a model to study human cardiac diseases.
Objective: Despite these findings, and the wealth of tools that are available to the fly community, forward genetic
screens for adult heart phenotypes have been rarely performed because of the difficulty in accurately measuring
cardiac function in adult Drosophila.
Methods and Results: Using optical coherence tomography to obtain real-time analysis of cardiac function in awake
Drosophila, we performed a genomic deficiency screen in adult flies. Based on multiple complementary
approaches, we identified CG31665 as a novel gene causing dilated cardiomyopathy. CG31665, which we name
weary (wry), has structural similarities to members of the Notch family. Using cell aggregation assays and
␥-secretase inhibitors we show that Wry is a novel Notch ligand that can mediate cellular adhesion with Notch
expressing cells and transactivates Notch to promote signaling and nuclear transcription. Importantly, Wry lacks
a DSL (Delta-Serrate-Lag) domain that is common feature to the other Drosophila Notch ligands. We further
show that Notch signaling is critically important for the maintenance of normal heart function of the adult fly.
Conclusions: In conclusion, we identify a previously unknown Notch ligand in Drosophila that when deleted causes
cardiomyopathy. Our study suggests that Notch signaling components may be a therapeutic target for dilated
cardiomyopathy. (Circ Res. 2010;106:1233-1243.)
Key Words: heart failure 䡲 cardiomyopathy gene 䡲 Drosophila, Notch signaling 䡲 optical coherence tomography
M
nity, forward genetic screens for adult heart phenotypes have
been rarely performed because of the difficulty in accurately
measuring cardiac function in adult Drosophila. Recently, we
developed a strategy to obtain rapid and accurate real-time
measurements of cardiac function in the awake adult fly by
optical coherence tomography (OCT).8 Using OCT, we performed a screen of adult flies that have genomic deficiencies
and identified a novel gene, CG31665 that has structural
similarities to members of the Notch family.
In Drosophila, Notch signaling is essential for cell differentiation and tissue development.21,22 The membrane-spanning
Notch receptor is stimulated by the ligands Delta (Dl) or Serrate
(Ser), which are present on the surface of neighboring cells.
After ligand binding, presenilin proteins mediate proteolytic
cleavage of the Notch Intracellular domain (NICD).21,23 Subsequently, the cleaved NICD undergoes nuclear translocation,
where it functions as a transcriptional coactivator for the Su(H)
(suppressor of Hairless) transcription factor, resulting in the
expression of specific target genes such as E(spl) (enhancer of
split). This activation is strictly controlled, and deregulation
causes extreme developmental defects.24 –27 The stringency of
the control system is provided by the general Notch antagonist
Hairless that assembles in a repressor complex on Notch target
ammalian animal models are useful to investigate the
molecular pathways and pathophysiology of cardiomyopathy. However, genetic screens in mammalian models are
complicated by time and effort required to identify the gene
responsible for disease-related phenotypes.1–3 As a model
system of human diseases, Drosophila has several advantages
including efficient genetic screening and mapping, a wellannotated genome, transposon insertion and molecularlydefined genomic deficiency stocks, and conserved signaling
pathways.4 –7 Additionally, the fruit fly has been recognized
as a model to study human cardiac diseases.6,8 The adult
Drosophila heart develops from the embryonic dorsal vessel
and shares similarities with vertebrate myocardium.9 –11 For
example, alterations in structural proteins such as troponin I,
tropomyosin, ␦-sarcoglycan, and dystrophin result in impaired Drosophila cardiac function.8,12–14 Moreover, mutations in ion channels including the potassium channel genes
KCNQ and HERG result in increased arrhythmia index and
shortened lifespan.15–17 Additionally, transcription factors
including Tin/Nkx2.5, Twist, Mef, and Hand have been
identified in Drosophila and have orthologs that are critical to
vertebrate heart development.6,18 –20 Despite these findings,
and the wealth of tools that are available to the fly commu-
Original received November 24, 2009; revision received February 15, 2010; accepted February 18, 2010.
From the Departments of Medicine (I.-M.K., M.J.W., H.A.R.), Cell Biology (H.A.R.), and Molecular Genetics (H.A.R.), Duke University Medical
Center, Durham, NC.
Correspondence to Howard A. Rockman, MD, Department of Medicine, Duke University Medical Center, DUMC 3104, 226 CARL Building, Research
Dr, Durham, NC 27710. E-mail [email protected]
© 2010 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.109.213785
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Non-standard Abbreviations and Acronyms
CSL
Dl
DN
E(spl)
H
N
NICD
OCT
RNAi
Ser
Su(H)
CBF1/suppressor of Hairless/Lag-1
Delta
dominant negative
enhancer of split
Hairless
Notch
Notch intracellular domain
optical coherence tomography
RNA interference
Serrate
suppressor of Hairless
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genes.21,22 Although Notch signaling is crucial in the development of many different Drosophila tissues including the
heart,22,28 –32 the role of Notch signaling in adult Drosophila
cardiac function is unknown. In mammals, Notch signaling
regulates many aspects of embryonic development, tissue homeostasis, and disease.33–36 In both humans and mice, mutations
in components of the Notch signaling pathway play a role in the
development of a number of congenital cardiovascular
diseases.33,37
Here, we identified a new gene that when deleted causes
dilated cardiomyopathy. The phenotype observed in deletion
mutants of CG31665 is consistent with a tired or fatigued
heart, and therefore we name the gene weary (wry). We show
that Wry functions as a Notch ligand, but is structurally
distinct from Dl and Ser. We further show that Notch
signaling is critically important for the maintenance of
normal heart function of the adult fly.
Methods
Molecular-defined genomic deficiency strains and transposon insertion stocks were obtained from the FlyBase repository at Bloomington, IL and PiggyBac insertion mutants were obtained from the
Exelixis repository at Harvard Medical School. Transgenic RNA
interference (RNAi) strains were obtained from the Vienna Drosophila RNAi Center.
Detailed descriptions of fly stocks, cardiac measurements in
adult Drosophila, quantitative RT-PCR, imaging wings, Drosophila cell culture and cell aggregation assays, ligand-dependent
luciferase assay, and statistical analysis are provided in the
expanded Methods section, available in the Online Data Supplement at http://circres.ahajournals.org.
Results
Identification of a Haploinsufficient Region on
Chromosome 2L That Causes Dilated
Cardiomyopathy in Adult Drosophila
We used OCT to measure heart function in a screen of adult
Drosophila with molecularly-defined genomic deficiencies
along chromosome 2L. We identified a deletion mutant,
Df(2L)Exel7007, with the phenotype of dilated cardiomyopathy defined as enlarged end-diastolic and end-systolic dimensions, and impaired fractional shortening (Figure 1A and
1B). This mutant is haploinsufficient for a 193kb genomic
region corresponding to 22B1–22B5 on chromosome 2 and
covers 23 annotated genes.
To narrow down the candidate gene interval responsible for
the cardiomyopathic phenotype of Df(2L)Exel7007, we examined 3 overlapping deletion stocks. Df(2L)Exel8005 had a
dilated phenotype that was similar to Df(2L)Exel7007 and
allowed us to narrow the candidate interval to 16 genes (Figure
1C through 1E).
Because the majority of homozygous deletions are lethal,
deficiency stocks are maintained as heterozygotes in the
presence of a balancer chromosome. Because the cardiomyopathic phenotype in Df(2L)Exel7007 and Df(2L)Exel8005
could be theoretically attributed to the balancer chromosome,
we tested both deficiency strains in the context of w1118 to
remove the possible contribution of the balancer to cardiac
dysfunction. Both stocks retained the abnormal cardiac phenotype in the absence of balancer chromosomes (Online
Figure I, A through C), supporting our interpretation that a
gene deletion in Df(2L)Exel7007 is causative for a cardiomyopathic phenotype in the fly.
Transposon Insertions f00122 and 22697 Identify
CG31665 As a Cardiomyopathy Candidate Gene
To narrow the 130-Kb genomic region that included the 16
candidate genes, we examined heart function in seven transposon insertion lines within the genomic region. Of the seven
insertion lines screened, we identified 2 stocks (f00122 and
22697) that phenocopied the original deletion Df(2L)Exel7007
(Figure 1F and 1G). The remaining five insertion lines inserted
outside the CG31665 gene had a normal cardiac phenotype
(Figure 1F and 1G and data not shown).
Because both f00122 and 22697 are inserted in the gene
CG31665, we examined CG31665 gene expression by quantitative RT-PCR in Df(2L)Exel7007, Df(2L)Exel8005, f00122,
and 22697. All 4 stocks displayed a reduction in CG31665
expression (Figure 1H). Additionally, f00122 had normal expression levels of 2 neighboring genes (c-cup and CG31663),
whereas the 2 deficiency stocks showed reduced expression of
c-cup and CG31663 as expected (data not shown).
To validate that the piggyBac transposon insertion altered
cardiac function through its effect on CG31665, we excised the
piggyBac element from f00122 using precise excision.38 Precise
excision of the piggyBac element resulted in full restoration of
normal heart function (Online Figure I, E). These data suggest
that f00122 and 22697 interrupt the gene CG31665 and that
CG31665 is the gene responsible for the dilated cardiac phenotype in Df(2L)Exel7007 and Df(2L)Exel8005.
Transgenic Drosophila Harboring RNAi Directed
Against CG31665 Have Dilated Cardiomyopathy
We next obtained transgenic Drosophila strains that contain
UAS-RNAi targeted to genes within the candidate interval.
We crossed each stock with Gal4 driver lines using tinC,
actin or hsp70 promoters and evaluated heart function in the
F1 generation. To test whether knocking down CG31665
would result in a cardiomyopathic phenotype, we crossed
UAS-CG31665RNAi flies with the various Gal4 driver lines.
Actin or tinC promoter-induced CG31665 RNAi expression
resulted in abnormal cardiac function (Figure 2A and 2B).
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Figure 1. Screening of Exelixis deletions and transposon insertions identified CG31665 as a cardiomyopathy candidate gene. A,
Representative OCT M-mode images from indicated stocks. B, Summary data for M-mode images showing that Df(2L)Exel7007 has an
increased end systolic dimension and impaired fractional shortening. C, Representative OCT M-mode images from indicated flies. D,
Summary data for M-mode images. Df(2L)Exel8005 has dilated cardiac dimensions similar to Df(2L)Exel7007. E, Schematic diagram of
overlapping deletion stocks. Because Df(2L)Exel8005 also shows an abnormal phenotype, candidate interval is narrowed down to 16
genes. F, Representative OCT M-mode images from indicated stocks. G, Summary data for M-mode images. f00122 and 22697 display abnormal cardiac function. H, Representative quantitative real-time RT-PCR. f00122 and 22697 are inserted in the intron of
CG31665 gene as shown in cytological map of the CG31665 gene. The map is modified from GBrowse at FlyBase. CG31665 gene is
disrupted in 2 deficiency stocks as shown in E. Summary data show a significant reduction in CG31665 expression in all 4 stocks vs
w1118 controls. Data represent means⫾SE of OCT measurements (B, D, and G) and 3 independent experiments, each performed in
triplicate (H). A 125-␮m standard and 1 second scale bar is shown. *P⬍0.05 vs w1118.
Using quantitative real-time RT-PCR, we confirmed that
UAS-CG31665RNAi flies with actin-Gal4 had decreased
CG31665 expression compared to w1118 controls (Figure 2C).
Because the expression of the CG31665 RNAi transgene
was under the control of Gal4 drivers that are expressed early
during Drosophila development, we wanted to eliminate the
possibility that the abnormal heart function observed resulted
from altered gene expression during embryogenesis. To test
whether knocking down CG31665 gene expression postdevelopmentally causes dilated cardiomyopathy, we engineered
w;p{tub-Gal80ts};p{tinC⌬ 4-Gal4}/p{UAS-CG31665RNAi}
flies based on the temperature-sensitive Gal80 protein in the
context of the bipartite Gal4/UAS transgenic expression
system in Drosophila using the cardiac-specific driver,
tinC⌬4-Gal4.39 – 43 The Gal80ts protein is functional at 18°C
and binds to Gal4 thereby preventing Gal4 binding to a
UAS-CG31665RNAi transgene. However, at 26°C, the
Gal80ts/Gal4 complex dissociates and Gal4 then binds to the
UAS-CG31665RNAi to activate its transcription.39,41 A temperature shift from 18°C to 26°C resulted in the deterioration
of cardiac function that was corrected by a second temperature shift back to 18°C (Figure 2 D and 2E). These data
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Figure 2. Actin promoter–induced or cardiac-specific tinC promoter–induced CG31665RNAi expression resulted in dilated cardiomyopathy and cardiac-specific promoter-induced Wry expression rescues the abnormal phenotype of Df(2L)Exel7007. A,
Representative OCT M-mode images from indicated flies. B, Summary data for M-mode images. Actin- or tinC promoter–induced
CG31665RNAi expression caused abnormal cardiac function. C, CG31665RNAi flies with actin-Gal4 have decreased CG31665 expression. Representative quantitative real-time RT-PCR. Summary data show a significant reduction in CG31665 expression in
CG31665RNAi flies with actin-Gal4 vs w1118 controls. D, Representative OCT M-mode images from indicated flies. Flies were maintained at 18°C until eclosion. After flies were eclosed, the first set of flies was kept at 18°C and the second set of flies was kept at
26°C for 7 days. A third set of flies was kept at 26°C for 7 days and then switched back to 18°C for an additional 7 days. E, Summary
data for M-mode images. Temperature shift from 18°C to 26°C results in deterioration in cardiac function and a second temperature
shift back to 18°C restores cardiac function. F, Representative OCT M-mode images from indicated flies. G, Summary data for
M-mode images. The introduction of wry transgene with cardiac-specific promoter into the context of Df(2L)Exel7007 restored normal
cardiac function. Data represent means⫾SE of OCT measurements (B, E, and G) and 3 independent experiments, each performed in
triplicate (C). *P⬍0.05 vs controls.
suggest that postdevelopmental loss of expression of
CG31665 in adult fly heart results in inducible and reversible
dilated cardiomyopathy. Similar data were found using hsp70
promoter to induce CG31665 RNAi expression (Online
Figure II, A through C). We also examined other RNAi lines
in our candidate interval and show that cardiac induced RNAi
expression for these other genes has no effect on cardiac
function (Online Figure III). Lastly, we used the Gal80ts/Gal4
temperature-sensitive system for a UAS-RNAi line targeting
a neighboring gene (c-cup) and showed no change in cardiac
function with temperature shift (data not shown). These data
demonstrate that CG31665 is the causative gene responsible for the dilated cardiomyopathic phenotype of
Df(2L)Exel7007, and we named this gene weary (wry).
Expression of Recombinant Wry Restores Cardiac
Function in the Deletion Mutant Df(2L)Exel7007
To demonstrate that wry is directly responsible for the
cardiomyopathic phenotype, we examined the ability of
cardiac-specific expression of Wry to rescue the cardiac
abnormality in Df(2L)Exel7007. Expression of Wry in the
heart completely rescues the abnormal cardiac function of
Df(2L)Exel7007 supporting our conclusion that wry is the
causative gene (Figure 2F and 2G).
Disrupting Components of the Notch Signaling
Pathway Causes Dilated Cardiomyopathy
We performed bioinformatic analyses of Wry using
the Stockholm Bioinformatics Centre InParanoid
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Figure 3. Temperature-sensitive
dominant-negative Dl or dominantnegative Ser expression in adult fly
heart resulted in inducible and reversible dilated cardiomyopathy. A, Representative OCT M-mode images from
indicated flies. B, Summary data for
M-mode images. C, Representative OCT
M-mode images from indicated flies. D,
Summary data for M-mode images.
Temperature shift from 18°C to 26°C
results in deterioration in cardiac function and a second temperature shift
back to 18°C results in restoration of
cardiac function. Data represent
means⫾SE of OCT measurements.
*P⬍0.05 vs 18°C or 26°C to 18°C.
(http://inparanoid.sbc.su.se/cgi-bin/index.cgi), Ensembl (http://
www.ensembl.org), and PANTHER (http:// www.pantherdb.org)
databases to examine sequence homologies across multiple
species. Wry contains EGF repeats and a transmembrane
domain but does not possess a DSL (Dl-Ser-Lag) domain
(Online Figure IV), suggesting that it may be involved in
Notch signaling. To determine the transcriptional pattern of
wry expression, we performed quantitative RT-PCR analysis
and show that wry is expressed in all 4 life stages and
parallels the expression pattern of the known Notch ligand
Ser (Online Table I and Online Results). We next examined
the heart function in w;p{tub-Gal80ts};p{tinC⌬4-Gal4} flies
harboring p{UAS-Dl.DN(dominant-negative)} or UASSer.DN to determine whether Notch signaling is important for
post developmental cardiac function. Temperature shift from
18°C to 26°C resulted in deterioration in cardiac function and
a second temperature shift back to 18°C restored normal
function (Figure 3). We examined deficiency stocks for
Notch signaling components and observed that stocks with
deficiencies of Notch ligands or Su(H) had abnormal cardiac
function (Online Figure V, A and B). Additionally, we
observed that the cardiac-specific expression of RNAi directed against Notch receptor and ligands caused abnormal
cardiac function (Online Figure V, C and D and data not
shown). Because mutants in Notch signaling regulators often
have wing phenotypes,44,45 we next examined the wings of
Df(2L)Exel7007. Interestingly, Wry deficiency stocks had
normal appearing wings (Online Figure V, E). These data
support our hypothesis that disrupting Notch signaling in the
adult fly heart can result in an inducible and reversible dilated
cardiomyopathy.
Cardiac-Specific Expression of Notch Signaling
Components Rescues the Abnormal Phenotype
of Df(2L)Exel7007
Because wry appears to be the causative gene for the
phenotype of Df(2L)Exel7007, and may be involved in Notch
signaling, we tested whether specific components of the
Notch pathway can rescue Df(2L)Exel7007. The introduction
of a Notch transgene driven by a cardiac-specific promoter
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Figure 4. Cardiac-specific promoterinduced expression of Notch or Notch
ligands can rescue the abnormal phenotype of Df(2L)Exel7007. A, Representative OCT M-mode images from indicated flies. B, Summary data for
M-mode images. C, Representative OCT
M-mode images from indicated flies. D,
Summary data for M-mode images. E,
Representative OCT M-mode images
from indicated flies. F, Summary data for
M-mode images. The introduction of
Notch (N) or Dl or Ser transgene with
cardiac-specific promoter into the context of Df(2L)Exel7007 deletion restored
normal cardiac function. Data represent
means⫾SE of OCT measurements.
*P⬍0.05 vs controls.
into the context of Df(2L)Exel7007 deletion restored normal
cardiac function (Figure 4A and 4B). Additionally, cardiacspecific expression of Notch ligands rescued the abnormal
phenotype of Df(2L)Exel7007 (Figure 4C through 4F). Finally, cardiac-specific expression of Su(H), a positive regulator of Notch signaling, rescued the abnormal phenotype of
Df(2L)Exel7007, whereas the Notch antagonist Hairless, a
negative regulator of Notch signaling, did not restore normal
cardiac function (Figure 5A through 5D).
To demonstrate the specificity of Wry in restoring abnormal cardiac function, we show that transgenic flies harboring
either human ␦-sarcoglycan (Dsg) or fly Medea, a positive
regulator of BMP signaling,46 is unable to restore normal
cardiac function in Df(2L)Exel7007 (Online Figure VI).
Wry Functions As a Notch Ligand
Because cardiac-specific expression of Notch ligands rescued
the abnormal phenotype of Wry deficiency stocks, we hypothesized that Wry acts as a functional Notch ligand. To test
this hypothesis, we conducted cell-aggregation assays with
cells expressing Notch, Dl, Ser, or Wry and examined the
ability of Notch to bind different ligands. Confocal images of
immunofluorescent cells containing Dl, Ser, or Wry indicate
that these proteins are each able to promote cellular adhesion
with Notch expressing cells as shown by the clustering of
ligand and receptor at sites of cellular contact (Figure 6A). Dl,
Ser, and Wry induced approximately 20% to 35% cell
aggregation compared to ⬍5% in Notch only containing cells
(Figure 6B). Next, we examined whether Wry activates
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Figure 5. Cardiac-specific promoterinduced expression of Su(H) can rescue the abnormal phenotype of
Df(2L)Exel7007. A, Representative OCT
M-mode images from indicated flies. B,
Summary data for M-mode images. C,
Representative OCT M-mode images
from indicated flies. D, Summary data
for M-mode images. The introduction of
Su(H) transgene (not Hairless [H] transgene) with cardiac-specific promoter into
the context of Df(2L)Exel7007 deletion
restored normal cardiac function. Data
represent means⫾SE of OCT measurements. *P⬍0.05 vs controls.
downstream nuclear transcription by performing liganddependent Notch-activity reporter assays using S2 cells. We
transfected S2N cells with the reporter construct, previously
shown to be responsive to transactivation by Dl.47 When S2N
cells were mixed with S2 cells overexpressing Wry, promoter
activity was transactivated by approximately 3-fold compared
with those mixed with untransfected cells (Figure 6C). As a
positive control, cells overexpressing Dl or Ser mixed with
S2N cells showed a 4 to 5 fold increase in promoter activity.
Importantly, Wry-mediated transactivation of Notch was
blocked by the ␥-secretase inhibitors, compound E and DAPT
(Figure 6C). Interestingly, it does not appear that Wry can
transactivate Notch signaling in mammalian C2C12 cells
(Online Figure VII and Online Results). Collectively, our
results suggest that Wry functions as a Notch ligand in
Drosophila to induce cleavage of Notch and effectively
activate transcriptional events in response to Notch signaling.
Cardiac-Specific Expression of Wry Rescues the
Abnormal Phenotype of SerRNAi Flies
Because in vitro cell adhesion assays (Figure 6) and in vivo
rescue studies (Figure 4C through 4F) suggest that Wry acts
as a Notch ligand, we tested whether Wry can rescue the
abnormal cardiac function in Ser loss-of-function mutants.
The cardiac-specific expression of a wry transgene into the
context of SerRNAi restored normal cardiac function. In
contrast, we show that the expression of Wry by a wingspecific vgMQ promoter did not rescue the wing morpholog-
ical abnormalities associated with SerRNAi flies, whereas Ser
restored both the cardiac and the wing phenotype in the
context of SerRNAi (Figure 7). These results suggest that
Wry functions as a novel Notch ligand in a somewhat
tissue-specific manner.
Discussion
In this study, we have identified wry as a gene associated with
dilated cardiomyopathy in adult Drosophila through the
examination of Drosophila mutants that have genomic haploinsufficiencies, transposon insertions, or cardiac-specific
RNAi expression. The protein encoded by wry has motifs that
include EGF repeats and a transmembrane domain consistent
with the Notch family of proteins but lacks a DSL domain.
Using cell aggregation assays and ␥-secretase inhibitors, we
show that Wry is a novel Notch ligand that can mediate
cellular adhesion with Notch expressing cells and transactivate Notch to promote signaling and nuclear transcription.
Notch signaling is important in directing the specification
of tissues including the heart in almost all developmental
stages in Drosophila.22,28,29,31,32 There are 2 types of Notch
ligands, which correspondingly activate distinct signaling
mechanisms.48 –50 Typical Notch ligands, which include Ser
and Dl in Drosophila and Jagged 1 to 2 and Dl-like 1, 3, and
4 in mammals, contain a DSL domain and transduce canonical Notch signaling pathway to the CSL (CBF1/suppressor
of Hairless/Lag-1)-NICD-Mastermind complex for the maintenance of stem or progenitor cells through transcriptional
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Figure 6. Wry can mediate cellular adhesion with Notchexpressing cells and transactivate Notch signaling pathway in
S2 cells. A, Panels 1 to 3 are representative of cell-aggregation
data from indicated cell mix. The cell mix used is labeled in each
panel and shows merged confocal images of green cell tracker–
labeled Notch cells and red cell tracker–labeled control (S2) or Dl
cells. Panels 4 to 6 show cell-aggregation assay of the Notch
binding potential of different ligands. The cell mix used is labeled in
each and shows merged cell tracker fluorescent images of Notch
cells (green) and immunofluorescence images of ligand cells (red).
Arrows represent the clustering of ligand and receptor at sites of
cellular contact. B, Graph shows the percentage of total Notchexpressing cells bound to cells that express different proteins or
control cells. C, Relative induction of luciferase activity. Control or
S2N cells were transfected with a reporter construct 2 X m3-Luc
and treated with vehicle only (DMSO) or Compound E or DAPT for
24 hours and then mixed with S2 cells expressing Dl, Ser, or Wry.
Activity was measured 4 hours after mixing. Activation of Notch by
S2Dl, S2Ser, or S2Wry cells resulted in a significant increase in
luciferase signal over controls, which were incubated with control
or S2N cells treated with ␥-secretase inhibitors. Data represent
means⫾SE of at least 4 independent experiments, each performed
in triplicate. *P⬍0.05 vs controls. Original magnification, ⫻150.
activation of Notch target genes such as E(spl) in Drosophila
and HES1 in mammals.35,51–53 In contrast, atypical Notch
ligands like DNER, F3/Contactin and NB-3 in mammals have
no DSL domain, and transduce noncanonical Notch signaling
to the CSL-NICD-Deltex complex for the differentiation of
progenitor cells through MAG transcriptional activation.48 –50
Notch signals are transduced to the canonical pathway (CSLNICD-Mastermind signaling cascade) or the noncanonical
pathway (CSL-NICD-Deltex signaling cascades) based on
the expression profile of Notch ligands, Notch receptors, and
Notch signaling modifiers.36 Interestingly, Wry does not have
a DSL domain although Wry does share EGF domain
homology with the 2 Drosophila Notch ligands.
Our results suggest that Wry regulates Notch signaling in a
DSL domain-independent manner which has not been previously recognized to occur in Drosophila. Interestingly, the
identification of Notch signaling in normal adult Drosophila
cardiac function has not been previously described. Our
results show that the cardiac-specific expression of Notch
ligands can rescue the abnormal cardiac phenotype observed
in Df(2L)Exel7007, supporting the concept that Wry functions as in the Notch pathway. Interestingly, Df(2L)Exel7007
does not have abnormal wing vein morphology, a phenotype
that is associated with deficiency mutants for Notch ligands.44,45,54 In addition, Df(2L)Exel7007 does not possess
the characteristic abnormalities in external sensory organs
including bristle morphology defects (data not shown) that
have previously been identified in mutations of Notch regulators.55,56 These results are consistent with a recent study that
used genome-wide RNAi screens of Notch signaling and
showed no apparent abnormalities in wing vein morphology
or external sensory organs in flies that expressed UASCG31665RNAi under the control of a pnr (notum specific)Gal4 or MS1096 (wing-specific)-Gal4 driver.30
In general, an identification of abnormalities that cause
dilated cardiomyopathies in adult Drosophila requires a
distinction between mutations that alter heart formation from
postdevelopmental effects on cardiac function. During larval
stages, the Drosophila cardiovascular system is composed of
a dorsal vessel containing 52 pairs of cardiomyocytes that
display both endothelial and muscle cell characteristics. The
cardiomyocytes form a tubular structure flanked by pericardial cells.9,10 Abnormal cardiac function in adult Drosophila
can result from defects in embryonic dorsal vessel development mimicking congenital abnormalities20 and abnormalities
arising in postdevelopment. To address this issue we used a
temperature sensitive Gal80ts system to drive expression of
UAS-WryRNAi, Dl.DN, and Ser.DN in the fully developed
adult fly heart. The transgene expression in tinC-expressing
cells could have effects on cardiomyocytes in an autonomous
manner, or could affect neighboring cells to influence heart
function in a nonautonomous manner. Indeed, nonautonomous influence of the epi-/pericardial cells on the function of
the fly heart has been postulated.57,58 However, in both cases,
our results demonstrate that the inhibition and subsequent
restoration of Notch signaling results in the induction and
reversal of abnormal cardiac function, respectively. Thus, the
regulation of Notch signaling appears to be an important
component in the maintenance of normal cardiac function in
the adult fly. In mammals, Notch signaling is involved in
many aspects of development and disease.33–36 Although
human mutations in Notch signaling and mutant mouse
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Figure 7. Cardiac-specific promoterinduced expression of Wry rescues
the abnormal cardiac phenotype of
SerRNAi flies. A, Representative OCT
M-mode images from indicated flies. B,
Summary data for M-mode images. Data
represent means⫾SE of OCT measurements. *P⬍0.05 vs controls. C, Ser lossof-function mutants had abnormal wing
vein morphology shown as arrows. Only
Ser is able to rescue the abnormal wing
phenotype induced by Ser knockdown.
N values are numbers of flies having a
wing phenotype divided by numbers of
total counted flies.
models of Notch signaling have already been implicated in
congenital heart disease,33,37 the involvement of Notch signaling in adult cardiac disease remains unclear. Our study
suggests that gene orthologs involved in Notch signaling may
be important in pathogenesis of mammalian dilated cardiomyopathy and that Notch signaling components may be a
therapeutic target for dilated cardiomyopathy.
Acknowledgments
We thank Weili Zou for technical assistance, Michelle Casad for
generating Drosophila reagents, and Teresa Lee for cloning.
Sources of Funding
This work was supported by NIH grants HL083065 (to H.A.R.) and
K08 HL085072 (to M.J.W.), American Heart Association National
Innovative Research Grant 0970391N (to M.J.W.), American Heart
1242
Circulation Research
April 16, 2010
Association Postdoctoral Fellowship 0825499E (to I.M.K.), and the
GlaxoSmithKline Research and Education Foundation for Cardiovascular Disease (to M.J.W.).
Disclosures
None.
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Novelty and Significance
What Is Known?
●
●
The Notch signaling pathway is important in many different tissues at
all developmental stages in Drosophila.
Whether Notch signaling is important to maintain normal heart
function in adult Drosophila is not known.
What New Information Does This Article Contribute?
●
●
●
This study identifies a new gene that causes dilated cardiomyopathy
in adult Drosophila.
This gene, which we name weary (wry), functions as a novel Notch
ligand to maintain normal heart function.
Disrupting Notch signaling in the adult fly heart causes dilated
cardiomyopathy.
A genomic-deficiency screen along chromosome 2L was performed to identify genes that cause dilated cardiomyopathy in
awake adult Drosophila. This search identified CG31665 as a
cardiomyopathy candidate gene, which we name weary (wry).
The Wry has EGF repeat motifs and a transmembrane domain
similar to that found in Notch ligands, but it is structurally
distinct from Delta and Serrate because Wry lacks a DSL
(Delta-Ser-Lag) domain common to other Notch ligands. Despite
the absence of a DSL domain, Wry can mediate cellular
adhesion with Notch expressing cells, and transactivate Notch
signaling in Drosophila S2 cells like the 2 other known Notch
ligands. Wry has not been previously described, nor has it
been shown in the fly that a Notch ligand can regulate Notch
signaling in a DSL domain-independent manner. Additionally,
deficiencies in Wry do not possess the characteristic abnormalities in external sensory organs and wings that have been
identified in mutations of Notch regulators. Based on the
findings, this newly identified Notch ligand may play a
fundamental role in maintaining normal cardiac function, and
gene orthologs involved in Notch signaling may be important
in the pathogenesis of human dilated cardiomyopathy.
Gene Deletion Screen for Cardiomyopathy in Adult Drosophila Identifies a New Notch
Ligand
Il-Man Kim, Matthew J. Wolf and Howard A. Rockman
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Circ Res. 2010;106:1233-1243; originally published online March 4, 2010;
doi: 10.1161/CIRCRESAHA.109.213785
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2010 American Heart Association, Inc. All rights reserved.
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CIRCRESAHA/2009/213785/R1
Supplemental Materials:
Detailed Methods
Fly stocks. Molecular-defined genomic deficiency strains and transposon insertion stocks were
obtained from the FlyBase repository at Bloomington, IL and PiggyBac insertion mutants were
obtained from the Exelixis repository at Harvard Medical School. Transgenic RNAi strains were
obtained from the Vienna Drosophila RNAi Center. w[*]; P{tubP-GAL80[ts]}10; TM2/TM6B, Tb[1]
was obtained from the Bloomington Drosophila Stock Center 1. Transgenic p{UAS-N}, p{UASSerrate}, p{UAS-Delta}, p{UAS-Su(H)}, and p{UAS-H} fly lines were kindly provided by Dr.
Spyros Artavanis-Tsakonas 2. For UAS transgene expression, we used several Gal4 driver
lines. The flies express Gal4 by the cardiomyocyte-specific constitutive tinC promoter3-5 or the
ubiquitous actin 5c promoter 6 or the heat-shock inducible hsp70 promoter 7 or the wing- specific
vgMQ promoter8. Transgenic flies harboring p{UAS-Wry} were engineered as previously
described 7, 9, 10. Briefly, the cDNA encoding wry was obtained by RT-PCR of w1118 total RNA,
sequenced, and subcloned into pUAST. All fly stocks were maintained on yeast paste nutrient
media (yeast, cornmeal, molasses, and agar with tegosept) and kept at either 18oC or 26oC
according to standard procedures.
Cardiac measurements in adult Drosophila. Cardiac function in adult Drosophila was
measured using a custom built OCT microscopy system (Bioptigen, Inc) as previously described
7
. Briefly, adult Drosophila at 7 days post eclosion were briefly subjected to CO2, gently placed
on a soft gel support, and allowed to fully awaken based on body movement. Multiple OCT mmodes of heart function from awake Drosophila were recorded along with a 125 micron
standard. All images were processed using ImageJ software using Bioptigen software. M-mode
images were used to measure cardiac parameters determined from 3 consecutive heart beats.
Cardiac chamber dimensions were measured from the edge of the superior and inferior walls
during mid-diastole for EDD (end-diastolic dimension) and mid-systole for ESD (end-systolic
dimension). Fractional shortening (FS) was calculated as [(EDD - ESD)/EDD] × 100 and
represents a surrogate measurement of contractility. All OCT imaging was conducted at room
temperature.
QRT-PCR. Thirty flies of 7 days age were individually collected from each group and embryos,
larvae, pupae, and dissected adult tissues were collected from w1118 to define the transcriptional
expression pattern of wry and ser. Total RNA was prepared using RNA-Bee (Tel-Test "B," Inc.)
and treated with RNase-free DNaseI to remove genomic DNA. cDNA was synthesized using
invitrogen SuperScript II reverse transcriptase. Applied Biosystems Taqman Gene expression
assays were used to perform quantitative (real time) RT-PCR (fly wry, Dm01808177_m1, fly ser,
Dm02151542_m1, and fly ribosomal protein L32, Dm02151827_g1 for endogenous control).
The following reaction components were used for each probe: 2 uL cDNA, 12.5 ul 2X TaqMan
Universal PCRMaster Mix (Applied Biosystems), 1.25 ul probe, and 9.25 ul water in a 25 µL
total volume. Reactions were amplified and analyzed in triplicate using an ABI PRISM® 7000
Sequence Detection System. PCR reaction conditions were as follows: Step 1: 50°C for 2
minutes, Step 2: 95°C for 10 minutes, Step 3: 40 cycles of 95°C for 15 seconds followed by
60°C for 1 minute. Expression relative to ribosomal protein L32 was calculated using 2-∆∆Ct and
levels were normalized to baseline.
Imaging wings. Adult wings were removed and placed on a cover slide. Images were taken on
a Zeiss Microscope with a x5 objective using PXCam ARC software and assembled using
Adobe Photoshop.
1
CIRCRESAHA/2009/213785/R1
Drosophila cell culture and cell aggregation assays. Drosophila S2 cells were maintained at
25°C in standard Schneider M3 medium (Invitrogen) supplemented with 10% fetal bovine
serum. S2N cells and S2Dl cells were described previously 11, 12 and were kindly provided by Dr.
Spyros Artavanis-Tsakonas. For Ser and Wry expression, S2 cells were transiently transfected
with pMT/Myc-Ser (S2Ser) or pUAST/HA-Wry and ubiquitin-Gal4 (S2Wry). All transfections
were performed using Cellfectin reagent (Invitrogen). Cell aggregation assays were performed
as described previously 11, 13. Briefly, S2N cells were induced with 1 mM CuSO4. In parallel,
separate cultures of S2, S2Dl, S2Ser and S2Wry cells were induced. After induction, S2N cells
were incubated with green cell tracker (Invitrogen) and S2 or S2Dl cells were incubated with red
cell tracker (Invitrogen) as described previously 14. And then cells were mixed and aggregated
with gentle rotation before transferring the cell suspension to confocal dishes coated in collagen
(Roche) for confocal micrographs. For immunostaining, S2N cells were incubated with green
cell tracker and mixed with S2Dl, S2Ser, or S2Wry cells. And then, cells were incubated in
Rabbit anti-Dl (1:50, Santa Cruz) or anti-Myc (1:1000, Sigma) or anti-HA (1:1000, Sigma)
polyclonal antibody, followed by incubation with Texas-red-conjugated anti-rabbit (1:500,
Invitrogen) secondary antibody. Confocal images were obtained as described previously 15.
Ligand-dependent luciferase assay. Ligand-dependent Notch luciferase assays in S2 cells
were performed as described previously 12. Briefly, S2N cells were transfected with 2 X m3-Luc
construct and induced with 1 mM CuSO4. In parallel, separate cultures of S2, S2Dl, S2Ser and
S2Wry cells were induced and 0.5 ml of the cultures were mixed with 0.5 ml of transfected S2N
cells and rocked at room temperature. Samples of 100 l were taken at 0h and 4 h, and
luciferase signals were developed using a Luc-Screen assay (Applied Biosystems) and
measured using a NOVOstar plate reader (BMG) as described previously 16. To quantify the fold
induction after ligand activation, values at 0 h were subtracted from values at 4 h to correct for
basal expression, then the corrected values at 4 h for S2Dl, S2Ser or S2Wry cells were divided
by the corrected values at 4 h for S2 cells. To examine the effect of γ-secretase inhibitors, S2N
cells were pretreated with 100nM of Compound E (Alexis Biochemicals) or DAPT (Sigma) or
vehicle DMSO alone before mixing with ligand-expressing cells as described previously 17, 18.
Luciferase assays in C2C12 cells were performed as described previously19. Briefly, C2C12
cells were transfected with a reporter construct pGa981-6 using FuGENE6 reagents (Roche
Molecular Biochemicals) . Fourty-eight hours after transfection, cells were co-cultured with
ligand-expressing HEK293 cells. Luciferase signals were measured as above.
Statistical Analysis. Data are expressed as mean±SE. Statistical significance was determined
using one-way ANOVA with Bonferroni correction for multiple comparisons or Student’s
unpaired t-tests. A P value of < 0.05 was considered statistically significant.
2
CIRCRESAHA/2009/213785/R1
Supplemental Results
Wry is expressed from embryonic stages through adulthood
To determine the transcriptional pattern of wry expression, we performed QRT-PCR analysis in
four life stages of w1118; embryo, larva, pupa, and adult. Wry was expressed in all four stages
and highly expressed in 16-24 hour embryo, pupa, and adult head. The expression of wry in
dissected adult heart was detectable but low compared to other tissues such as muscles
(Online Table I). We also examined the transcriptional pattern of ser expression using QRTPCR. The Notch ligand Ser was expressed in all four life stages, highly enriched in pupa, and at
a low level in the adult heart which paralleled the expression of wry (Online Table I). These
Notch ligands appear to play an important role in maintaining the normal cardiac function in the
adult fly despite the relatively low expression in the heart.
Wry can not transactivate Notch signaling in mammalian cells
The fact that Wry acts as a functional Notch ligand in flies prompted us to examine whether Wry
activates transcription downstream of Notch signaling in mammalian cells. To assess the ligand
activity of Wry, we performed luciferase reporter assays using C2C12 mouse myoblast cell lines
that endogenously express Notch and its downstream signaling molecule20. We transfected
C2C12 cells with a reporter construct pGa981-6 carrying RBP-J binding sites that has been
shown to be transactivated by NICD and suppressed by a dominant-negative form of RBP-J19.
When C2C12 cells were co-cultured with HEK 293 cells overexpressing Wry, promoter activity
was not transactivated compared with those mixed with control cells. As a positive control, cells
overexpressing DNER (a mammalian Notch ligand) mixed with C2C12 cells showed a 7-fold
increase in promoter activity compared to control cells (Online Figure VII). These results
suggest that Wry may not function as a Notch ligand in mammalian cells.
3
CIRCRESAHA/2009/213785/R1
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Eiraku M, Tohgo A, Ono K, Kaneko M, Fujishima K, Hirano T, Kengaku M. DNER acts
as a neuron-specific Notch ligand during Bergmann glial development. Nat Neurosci.
2005;8:873-880.
Shawber C, Nofziger D, Hsieh JJ, Lindsell C, Bogler O, Hayward D, Weinmaster G.
Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway.
Development. 1996;122:3765-3773.
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Supplemental Figure Legends
Online Figure I. Both the Df(2L)Exel7007 and Df(2L)Exel8005 display the abnormal
cardiac phenotype with absence of balancer chromosomes and precise excision of
piggyBac element from f00122 restores normal cardiac function. (A) Breeding strategy to
separate effects of balancer vs. deletion chromosome. (B) Representative OCT M-mode images
of F1 flies from the cross in A. A 125 micron standard and 1 second scale bar is shown. (C)
Summary data for M-mode images. Both the Df(2L)Exel7007/+ and Df(2L)Exel8005/+ have
dilated cardiomyopathy phenotypes. Each graph represents the summary data for OCT
measurements and is expressed as the mean +/- SE (n=15, 20, and 11 as shown in the order).
(D) Breeding strategy to excise the piggyBac element from f00122. The excision of the
piggyBac element from f00122 is based on the introduction of the piggyBac transposase into the
germ line. Viable offsprings that have the piggyBac element excised are identified by eye color
change. (E) Summary data for M-mode images from w1118, f00122, and precise excision line
Ex(f00122). Ex(f00122) restores normal cardiac function. Each graph represents the summary
data for OCT measurements and is expressed as the mean +/- SE (n=15,18, and 15 as shown
in the order). * : p<0.05 vs CyO/+ or w1118or EX(f00122).
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Online Figure II. Heat shock-induced CG31665 RNAi expression in adult fly resulted in
dilated cardiomyopathy. (A) Representative OCT M-mode images from Hsp70-Gal4;UASCG31665 RNAi with either no heat shock or heat shock and UAS-CG31665 RNAi flies with heat
shock. We subjected 3 day old flies to daily 1h heat shocks at 370C for 3 days and examined
cardiac function at 7 days post eclosion. A 125 micron standard and 1 second scale bar is
shown. (B) Summary data for M-mode images. Heat shock-induced CG31665 RNAi expression
caused abnormal cardiac function. Each graph represents the summary data for OCT
measurements and is expressed as the mean +/- SE (n=8, 18, and 13 as shown in the order).
(C) Hsp70-Gal4;UAS-CG31665 RNAi flies with heat shock have decreased CG31665
expression. Representative quantitative real-time RT-PCR of either no heat shock or heat shock
in UAS-CG31665 RNAi flies with hsp70-Gal4. Summary data show a significant reduction in
CG31665 expression in heat shock vs. no heat shock controls. Data represent mean ± SE of at
least three independent experiments, each performed in triplicates. *: p<0.05 vs. controls.
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Online Figure III. Cardiac-specific tinC promoter-induced RNAi expression for other
candidate genes displayed normal cardiac function. (A) Representative OCT M-mode
images of F1 flies from the cross in UAS-Dicer2;tinc-Gal4 and UAS-RNAi for candidate genes.
The numbers indicate Vienna RNAi stock numbers for each gene. A 125 micron standard and 1
second scale bar is shown. (B) Summary data for M-mode images. Cardiac induced RNAi
expression for other candidate genes caused normal cardiac phenotypes. Each graph
represents the summary data for OCT measurements and is expressed as the mean +/- SE
(n=15, 11, 12, 10, 13, 13, 13, 10, 12, 15, 9, 10, 11 and 10 as shown in the order).
8
Online Figure IV
A
Delta
Serrate
Wry
Delta
Serrate
Wry
Delta
Serrate
Wry
Delta
Serrate
Wry
Delta
Serrate
Wry
Delta
Serrate
Wry
Delta
Serrate
Wry
B
KGFTNPIQFPFSFSWPGTFSLIVEAWHDTNNSGNARTNKLLIQRLLVQQVLEVSSEWKTN
---VGAIVLPFTFRWTKSFTLILQALDMYNTS--YPDAERLIEETSYSGVILPSPEWKTL
DAMLAQQPTASKLNIVDLYPLKIEDIQPIIRNDNDELIKQRTVFTQHENAEEYKQNLMPN
*
KSESQYTSLEYDFRVTCDLNYYGSGCAKFCRPRDD--------SFGHSTCSETGEIICLT
DHIGRNARITYRVRVQCAVTYYNTTCTTFCRPRDD--------QFGHYACGSEGQKLCLN
EVEMMVDKEVIEMKDKIQINQEKFGSSTTVKPTTSRPHQGTTADFTGKLLADQDDLITEI
*
*
GWQGDYCHIPKCAKGCE--HGHCDKPNQCVCQLGWKGALCNECVLEPNCIHGTCN-KPWT
GWQGVNCEEAICKAGCDPVHGKCDRPGECECRPGWRGPLCNECMVYPGCKHGSCNGSAWK
ESKFQLHETTTLRPRVTTVQHPGNTFIDRRVKKSFEFPMQHRASDVMAMPHIDFANTKFQ
*
167
223
240
-------------------------GYQCECPIGYSGPNCDLQLDNCSPNPCINGGSCQP
TTTRKMAKPSGLPCSGHGSCEMSDVGTFCKCHVGHTGTFCEHNLNECSPNPCRNGGICLD
-----------------------MEEYECQCCSGFAGPHCEE-IDACNPSPCTNNGICVD
* * * * *
* * ** * * *
SG--------KCICPAGFSGTRCETNIDDCLGHQCENGGTCID----MVNQYRCQCVPGF
GDG-----DFTCECMSGWTGKRCSERATGCYAGQCQNGGTCMPGAPDKALQPHCRCAPGW
LSQGHEGNSYQCLCPYGYAGKNCQYESDPCNPAECMNGGSCVG----NSTHFRCDCAPGF
* * * * *
*
* *** *
* * **
HGTHCSSKVDLCLIRPCANGGTCLNLNNDYQCTCRAGFTGKDCSVDIDECSSGPCHNGGT
TGLFCAEAIDQCRGQPCHNGGTCESGAGWFRCVCAQGFSGPDCRINVNECSPQPCQGGAT
TGPLCQHSLNECESSPCVHG-ICVDQEDGFRCFCQPGFAGELCNFEYNECESNPCQNGGE
* *
*
** * *
* * ** * *
**
** *
CMNRVNSFECVCANGFRGKQCDEES----------------YDSVTFDAHQYGATTQARA
CIDGIGGYSCICPPGRHGLRCEILLSDPKSACQNASNTISPYTALNRSQNWLDIALTGRT
CIDHIGSYECRCTKGFSGNRCQIKVD-------------------FCANKPCPEGHRCID
*
* * * * *
435
815
753
219
275
300
276
335
360
483
870
809
543
930
868
587
990
909
CIRCRESAHA/2009/213785/R1
Online Figure IV. Wry does not have a DSL domain. (A) Amino acid alignments in Delta,
Serrate, and Wry. * represents amino acid identity in three proteins. The DSL (Delta-SerrateLag) domain is underlined and characters in red colors represent the consensus sequence.
There are some homologies in EGF repeats shown as yellow boxes. (B) Schematic
representation of domain architecture in Delta, Serrate, and Wry. While two Notch ligands have
a DSL domain, Wry does not have a DSL domain. Additionally, Serrate has a VWC (von
willebrand factor type C) domain.
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Online Figure V. Disrupting Notch signaling components caused dilated cardiomyopathy
and Wry deficiency stocks have no wing phenotypes. (A) Representative OCT M-mode
images from w1118, Serrate Df, Delta Df, and Su(H)Df. A 125 micron standard and 1 second
scale bar is shown. (B) Summary data for M-mode images. Df(3R)Exel6208, Df(3R)BSC475,
and Df(2L)BSC299 displayed abnormal cadiac function. Each graph represents the summary
data for OCT measurements and is expressed as the mean +/- SE (n=15, 14, 19, and 23 as
shown in the order). (C) Representative OCT M-mode images from w1118 and UAS-N
RNAi/tincΔ4-Gal4. A 125 micron standard and 1 second scale bar is shown. (D) Summary data
for M-mode images. Cardiac-specific promoter-induced N RNAi expression caused abnormal
cardiac function. Each graph represents the summary data for OCT measurements and is
expressed as the mean +/- SE (n=15 and 18 as shown in the order). (E) While Delta loss-offunction mutants have thickening of wing vein shown as arrows, Wry deficiency stocks have
normal wing morphology. *: p<0.05 vs. w1118.
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Online Figure VI. Cardiac-specific promoter-induced human Dsg or Medea expression
can not rescue the abnormal phenotype of Df(2L)Exel7007. (A) Representative OCT Mmode images from flies harboring UAS-Dsg and tincΔ4-Gal4 driver, flies harboring
Df(2L)Exel7007 and UAS-Dsg, and flies harboring Df(2L)Exel7007, UAS-Dsg and tincΔ4-Gal4
driver. A 125 micron standard and 1 second scale bar is shown. (B) Summary data for M-mode
images. The introduction of Dsg transgene with cardiac-specific promoter into the context of
Df(2L)Exel7007 deletion did not restore normal cardiac function. Each graph represents the
summary data for OCT measurements and is expressed as the mean +/- SE (n=8, 7, and 9 as
shown in the order). (C) Representative OCT M-mode images from flies harboring UAS-Medea
and tincΔ4-Gal4 driver, flies harboring Df(2L)Exel7007 and UAS-Medea, and flies harboring
Df(2L)Exel7007, UAS-Medea and tincΔ4-Gal4 driver. A 125 micron standard and 1 second
scale bar is shown. (B) Summary data for M-mode images. The introduction of Medea
transgene with cardiac-specific promoter into the context of Df(2L)Exel7007 deletion did not
restore normal cardiac function. Each graph represents the summary data for OCT
measurements and is expressed as the mean +/- SE (n=11, 7, and 9 as shown in the order). *:
p<0.05 vs. controls.
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Online Figure VII. Wry can not transactivate Notch signaling pathway in mammalian cells.
Relative induction of luciferase activity. C2C12 cells were transfected with a reporter construct
pGa981-6 carrying RBP-J binding sites and then co-cultured with HEK 293 cells expressing
DNER (a mammalian Notch ligand), Wry, or a control vector (CMV-empty). Activity was
measured 48 h after start of co-culture. Data represent mean ± SE of at least three independent
experiments, each performed in triplicate. *p<0.05 vs. controls.
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Online Table I . Transcriptional expression patterns of wry and ser in wild-type flies at
four life stages.
wry
ser
Embryo
0-8h
8-16h
16-24h
1 ± 0.03
176.3 ± 18.6
347 ± 66.7
17.9 ± 4.7
86.4 ± 10.3
17.8 ± 2.3
Larva
25.1 ± 6.8
5.6 ± 2.2
Pupa
352 ± 90.3
626.9 ± 30.4
Adult
Head part
Thorax part
Abdomen part
Heart
Leg Muscle
Wing
470 ± 102.6
155.3 ± 41.3
2.9 ± 0.7
0.3 ± 0.06
61.7 ± 12.5
5.8 ± 1.7
5.1 ± 1.2
1.3 ± 0.9
7.2 ± 1
0.8 ± 0.3
30.5 ± 4.4
2.7 ± 0.3
Representative quantitative real-time RT-PCR. Total RNA and cDNA were prepared from
embryos in indicated time points after birth, larvae, pupae, and dissected adult tissues of w1118.
Summary data show relative gene expression of wry and ser at all four stages (expressed as
fold-change compared to the wry expression level at 0-8h embryo). Data represent mean ± SE
of at least three independent experiments, each performed in triplicate.
13