Genetics: Early Online, published on July 31, 2014 as 10.1534/genetics.114.166777
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8 Galactokinase is a novel modifier of calcineurin-induced cardiomyopathy in Drosophila
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15 Teresa E. Lee*, Lin Yu§, Matthew J. Wolf§†, and Howard A. Rockman*§†
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17 Departments of *Cell Biology, §Medicine, and †Molecular Genetics and Microbiology,
18 Duke University, Durham, NC, 27710
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Copyright 2014. 1
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3 Running title:
4 Galk modifies CanA cardiomyopathy in Drosophila
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6 Key words:
7 Galactokinase, Drosophila melanogaster, cardiomyopathy, calcineurin
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9 Corresponding author:
10 Howard A. Rockman, M.D.
11 Department of Medicine, Duke University Medical Center, DUMC 3104,
12 226 CARL Bldg., Research Dr., Durham, NC, 27710
13 E-mail: [email protected]
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2 1 ABSTRACT
2 Activated/uninhibited calcineurin is both necessary and sufficient to induce cardiac
3 hypertrophy, a condition that often leads to dilated cardiomyopathy, heart failure, and
4 sudden cardiac death. We expressed constitutively active calcineurin in the adult heart
5 of Drosophila melanogaster and identified enlarged cardiac chamber dimensions and
6 reduced cardiac contractility. In addition, expressing constitutively active calcineurin in
7 the fly heart using the Gal4/UAS system induced an increase in heart wall thickness.
8 We performed a targeted genetic screen for modifiers of calcineurin-induced cardiac
9 enlargement based on previous calcineurin studies in the fly and identified
10 galactokinase as a novel modifier of calcineurin-induced cardiomyopathy. Genomic
11 deficiencies spanning the galactokinase locus, transposable elements that disrupt
12 galactokinase, and cardiac-specific RNAi knockdown of galactokinase suppressed
13 constitutively active calcineurin-induced cardiomyopathy. In addition, in flies expressing
14 constitutively active calcineurin using the Gal4/UAS system, a transposable element in
15 galactokinase suppressed the increase in heart wall thickness. Lastly, genetic
16 disruption of galactokinase suppressed calcineurin-induced wing vein abnormalities.
17 Collectively, we generated a model for discovering novel modifiers of calcineurin-
18 induced cardiac enlargement in the fly and identified galactokinase as a previously
19 unknown regulator of calcineurin-induced cardiomyopathy in adult Drosophila.
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3 1 INTRODUCTION
2 Activated/uninhibited calcineurin is both necessary and sufficient to induce cardiac
3 hypertrophy (Molkentin et al. 1998; Wilkins and Molkentin 2002; van Berlo et al. 2013).
4 Transgenic mice expressing constitutively active calcineurin (CanAact) display cardiac
5 hypertrophy (Molkentin et al. 1998) and the genetic or pharmacological inhibition of
6 calcineurin suppresses agonist and pressure-overload induced cardiac hypertrophy
7 (Sussman et al. 1998; Taigen et al. 2000; Wilkins and Molkentin 2002; van Berlo et al.
8 2013). Prolonged cardiac hypertrophy is a known risk factor for dilated cardiomyopathy,
9 heart failure, and sudden death (LEVY et al. 1990; MESSERLI and KETELHUT 1991;
10 DRAZNER et al. 2004; GEORGE 2013; GROSSMAN and PAULUS 2013). In contrast, cardiac
11 hypertrophy stimulated by exercise is physiological, is not typically associated with
12 abnormal cardiac function, and does not stimulate calcineurin/NFAT signaling (WILKINS
13 et al. 2004), supporting the concept that calcineurin promotes pathological cardiac
14 hypertrophy.
15 Calcineurin acts as a calcium/calmodulin-dependent protein phosphatase that
16 consists of two subunits: a large CanA subunit (60kD), and a small CanB subunit
17 (19kD). In the mouse, there are three CanA genes (Ppp3ca, Ppp3cb, and Ppp3cc) and
18 two CanB genes (Ppp3r1 and Ppp3r2); in the fly, there are three CanA genes (CanA1,
19 CanA-14F, and PP2B-14D) and two CanB genes (CanB and CanB2) (NCBI Gene,
20 http://www.ncbi.nlm.nih.gov/gene). The large CanA subunit has phosphatase activity
21 and consists of several domains: the catalytic domain, which regulates protein
22 dephosphorylation (Klee et al. 1979), the CanB binding domain (Klee et al. 1988), the
23 calcium/calmodulin binding domain, and the autoinhibitory domain (Shibasaki et al.
4 1 2002). In the inactive state, the autoinhibitory domain inhibits the catalytic domain.
2 Binding of calcium/calmodulin activates calcineurin by alleviating this autoinhibition. A
3 constitutively active calcineurin (CanAact) is generated by eliminating the autoinhibitory
4 domain, and has been used to investigate calcineurin signaling (Molkentin et al. 1998;
5 Sullivan and Rubin 2002; Gajewski et al. 2003). Expression of constitutively active
act 6 calcineurin (CanA ) has been found to induce cardiac hypertrophy (MOLKENTIN et al.
7 1998), skeletal muscle hypertrophy (MUSARO et al. 1999; SEMSARIAN et al. 1999) and
8 slow twitch skeletal muscle specification (CHIN et al. 1998; WU et al. 2000). The smaller
9 subunit (CanB) is constitutively bound to CanA and is required for maintaining
10 calcineurin expression (WATANABE et al. 1995; KLEE et al. 1998; PARSONS et al. 2004).
11 Deficiency of CanB results in significant cardiomyopathy, including impaired
12 cardiomyocyte growth, impaired contractility, and lethality after birth (SCHAEFFER et al.
13 2009; MAILLET et al. 2010).
14 Calcineurin signaling in mammals involves calcineurin-dependent dephosphorylation
15 of nuclear factor of activated T cells (NFAT) transcription factors (Molkentin et al. 1998;
16 Okamura et al. 2000). In contrast, Drosophila do not have calcineurin-regulated
17 isoforms of NFAT and therefore use NFAT-independent pathways (Keyser et al. 2007).
18 Myocyte enhancer factor (Mef2) is a well-known NFAT-independent pathway that has
19 been implicated in calcineurin-mediated cardiac and skeletal muscle hypertrophy
20 (WILKINS and MOLKENTIN 2002; SAKUMA and YAMAGUCHI 2010). Cardiac calcineurin
21 expression has been shown to activate Mef2 reporter activity (PASSIER et al. 2000). The
22 expression of a dominant-negative Mef2 inhibited CanAact-induced cardiac enlargement
23 and overexpressing Mef2 caused cardiac chamber dilation (VAN OORT et al. 2006). In
5 1 skeletal muscle, calcineurin activates Mef2 with exercise (WU et al. 2001; SAKUMA et al.
2 2008). Mechanistically, calcineurin was found to co-immunoprecipitate with Mef2 and
3 induce activation of Mef2 through dephosphorylation (WU et al. 2001).
4 Previously, two independent screens have been conducted to identify modifiers of
5 calcineurin phenotypes in tissues other than the heart (Sullivan and Rubin 2002;
6 Gajewski et al. 2003). Sullivan and Rubin performed a dominant modifier screen in the
7 Drosophila eye and found five suppressor and four enhancer loci (Sullivan and Rubin
8 2002). Two modfier genes, CanB2 and sprouty were identified. However, modifier
9 genes within the seven other broadly mapped loci remained uncharacterized. Gajewski
10 et al found seven different deletion intervals that suppressed the lethal phenotype of
11 constitutively active calcineurin driven by the general mesodermal driver 24B (Gajewski
12 et al. 2003). CanB2 was determined to be a modifier and preliminary experiments
13 suggested that Mef2 might be the modifier for another interval. Importantly, only one
14 interval overlapped between these two studies on chromosome 3L, cytolocation 66F.
15 Many pathways are conserved among mammalian and Drosophila cardiac
16 development (BODMER and VENKATESH 1998; CRIPPS and OLSON 2002; ZAFFRAN and
17 FRASCH 2002; ZAFFRAN et al. 2002). In fact, strategies based on fly genetics have been
18 used to identify genes that cause or modify cardiomyopathies (BIER and BODMER 2004;
19 WESSELLS and BODMER 2004; WOLF et al. 2006; YU et al. 2010). The Drosophila heart
20 can be efficiently monitored in real time in intact awake Drosophila using optical
21 coherence tomography (OCT) (WOLF et al. 2006). Importantly, Drosophila is well
22 adapted for genetic studies: it has a relatively short generation time, well developed
23 genetic manipulation methods, and well developed genetic resources, including
6 1 mutation stocks and vectors for making transgenics (KOHLER 1994; ST PIERRE et al.
2 2014). Therefore, we conducted studies using fly genetics to identify novel modifiers of
3 cardiac calcineurin.
4 We generated Drosophila that expressed constitutively active calcineurin (CanAact) in
5 the heart under the control of the cardiomyocyte-specific driver, tinC (tinC-CanAact), an
6 approach that is analogous to previous studies in transgenic mice (MOLKENTIN et al.
7 1998). tinC-CanAact flies had enlarged cardiac chamber dimensions and reduced
8 cardiac contractility. In addition, expressing CanAact in the heart with tinC-Gal4>UAS-
9 CanAact induced an increase in cardiac wall thickness. Based on the prior genetic
10 screens (Sullivan and Rubin 2002; Gajewski et al. 2003), we then designed a deficiency
11 screen targeting the overlapping region on chromosome 3L. Here we show that
12 deficiencies in a genetic region encoding galactokinase (Galk) rescued CanAact-induced
13 cardiac enlargement and decreased function. To test the hypothesis that Galk is a
14 novel modifier of calcineurin in the heart, two independent transposable element
15 insertions in Galk and cardiac-specific expression of RNAi directed against Galk were
16 studied. Genetic disruption of Galk rescued the cardiac enlargement phenotype in tinC-
17 CanAact flies and a transposable element insertion in Galk rescued tinC-Gal4>UAS-
18 CanAact-induced increase in cardiac wall thickness. In addition, genetic disruption of
19 Galk also suppressed an abnormal wing vein phenotype induced by the wing driver
20 e16E-Gal4>UAS-YCanAact. These findings suggest that galactokinase modifies
21 calcineurin-mediated cardiomyopathy in adult Drosophila.
7 1 MATERIALS AND METHODS
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3 Drosophila stocks
4 The following Drosophila stocks were obtained from the Bloomington Drosophila
5 Stock Center: w1118 (Flybase ID: FBst0003605), Df(3L)ED4413, Df(3L)ED4414,
6 Df(3L)ED4415, Df(3L)ED4416, Df(3L)ED4421, Df(3L)BSC130, Df(3L)BSC170,
7 Df(3L)BSC390, Df(2R)X1,Mef2X1/CyOAdhnB, sty∆5/TM3,P{35UZ}2, P{EP}CanB2EP774,
8 PBac{PB}Galkc03848, Mi{ET1}GalkMB10638, snaSco/SM6a, P{hsILMiT}2.4, P{en2.4-
9 Gal4}e16E (e16E-Gal4), P{GAL4-dpp.blk1}40C.6 (dpp-Gal4), P{Act5C-GAL4}25FO1
10 (Act5C-Gal4), P{GAL4-Mef2.R}3 (Mef2-Gal4). The double balancer line WR135 was
11 kindly provided by Dr. Robin Wharton. The P{tinC-Gal4} line was kindly provided by Dr.
12 Manfred Frasch (YIN and FRASCH 1998). The P{tinC-GFP} (tinC-GFP) line was
13 generated as previously described by inserting the 304 bp tinC genetic sequence into
14 the pGreen-H-Pelican vector (YIN and FRASCH 1998; BAROLO et al. 2000; YU et al. 2010)
15 and the construct injected at the Duke University Model Systems Genomics Facility.
16 The tinC-YCanAact, tinC-FCanAact, and UAS-YCanAact Drosophila lines were generated
17 at the Duke University Model Systems Genomics Facility by injecting the corresponding
18 constructs into Drosophila embryos.
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20 Calcineurin constructs
21 Constitutively active calcineurin was amplified from the calcineurin gene Pp2B-14D
22 as previously described using the primers: ATG TCT TCG AAT AAC CAG AGC AGC
23 AG (forward) and TCA GTT GCG TAT CAC CTC CTT GCG CA (reverse) (Sullivan and
8 1 Rubin 2002). Restriction enzyme sites and Flag-tagged CanAact (FCanAact) were
2 inserted by extending the N-terminal with primers carrying the corresponding
3 sequences. CanAact was amplified in topo vector (Invitrogen, Inc.) and inserted at the 3’
4 end to YFP in the pEYFP-C1 vector with appropriate restriction enzyme sites to form
5 YFP-tagged CanAact (YCanAact). Full length FCanAact or YCanAact PCR products were
6 subsequently cloned into the pCaSpeR5 Drosophila expression vector, inserting a tinC-
7 hsp70 promoter at the 5’ end as previously described (YIN et al. 1997; YU et al. 2010)
8 (Figure 1A). The catalytic activity of N-terminal YFP-tagged CanAact has been
9 confirmed in studies by a number of investigators, using well characterized NFAT
10 reporter assays (TOKOYODA et al. 2000; BURKARD et al. 2005), NFAT phosphorylation
11 assays (TOKOYODA et al. 2000), and NF-κB reporter assays (KANG et al. 2007). The
12 UAS-YCanAact construct was generated similarly by inserting YFP-tagged CanAact into
13 the pUAST vector following the UAS sequence.
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15 Optical coherence tomography (OCT) to measure cardiac function in adult
16 Drosophila
17 End-diastolic dimension and end-systolic dimension in adult Drosophila were
18 measured using OCT (Bioptigen, Inc. Durham, NC) as previously described (WOLF et al.
19 2006). Briefly, 7-10 days post-eclosion, female Drosophila were placed in GelWax
20 medium and allowed to awaken. M-mode images through the conical chamber were
21 collected for immobilized awake Drosophila. End-diastolic and end-systolic dimensions
22 (EDD and ESD, respectively) were measured in ImageJ, calibrated to a 125µm thick
23 glass slide. Fractional shortening (FS) is calculated as (End-diastolic dimension minus
9 1 end-systolic dimension/end-diastolic dimension) x 100% and used as a measure of
2 cardiac contractility. All end-systolic dimensions are supplied in Table S1.
3 Several types of controls were used and compared for our study: w1118 (Flybase ID:
4 FBst0003605; EDD= 54.10 ± 2.95 microns; FS= 98.79 ± 1.21%), w1118 used to create
5 transgenics from the Duke University Model Systems Genetics Facility (Flybase ID:
6 FBst0006326; EDD= 41.53 ± 5.08 microns; FS= 99.76 ± 1.21%), tinC-Gal4 (EDD=
7 51.59 ± 4.14 microns; FS= 96.70 ± 1.53%), tinC-Gal4 heterozygous with w1118 (EDD=
8 48.34 ± 2.34 microns; FS= 98.92 ± 0.74%), tinC-GFP (EDD= 52.88 ± 5.85 microns; FS=
9 98.24 ± 1.76%) and tinC-GFP heterozygous with w1118 (EDD= 56.34 ± 4.32 microns;
10 FS= 98.78 ± 0.58%). We performed a one-way ANOVA comparing OCT measurements
11 between all control groups and show that none were significantly different from each
12 other (Figure S1A).
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14 Histology
15 Histology was performed according to standard paraffin embedding and hematoxylin
16 and eosin staining procedures for the fly (ASHBURNER 1989). Briefly, 3-5 days post-
17 eclosion, Drosophila were washed with 70% EtOH before fixing in 10% buffered
18 formalin at 4˚ overnight. This fixes the hearts at their most relaxed state, end-diastole.
19 To assess this, OCT images were measured and compared between alive w1118 end-
20 diastolic dimension (46.37 ± 4.91 microns, N=9) and the size of the heart after EtOH
21 fixation (50.19 ± 6.09 microns, N=9), P=N.S., student’s t test. The next day, Drosophila
22 were dehydrated starting with a PBS wash and subsequent alcohol gradient into xylene.
23 Drosophila were then incubated overnight at 60˚ under vacuum in liquid paraffin wax.
10 1 Samples were then positioned appropriately in molds and allowed to harden. 8 µm
2 consecutive sections were cut using a microtome and adhered to poly-L-lysine-coated
3 glass slides. The slides were subsequently rehydrated into water and stained with
4 hematoxylin and eosin. The slides were brought back through the alcohol gradient and
5 xylene and mounted in Cytoseal XYL mounting medium (Thermo Fisher Scientific Inc.).
6 Images were subsequently quantified in ImageJ, calibrated with a hemocytometer grid
7 measuring 50µm. Locating the position along the cardiac tube for measurement was
8 determined as previously described by examining the portion of the sections where an
9 en face section of the cardiac tube was visible and measuring three sections (24µm)
10 posterior to this section (YU et al. 2010). Wall thickness was calculated as the average
11 of dorsal, ventral, left, and right walls, excluding the longitudinal dorsal muscle
12 underlying the ventral side of the conical chamber.
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14 Minos excision
15 The Minos insertion in Galk, Mi{ET1}GalkMB10638, was excised precisely according to
16 standard transposable element excision procedures (ARCA et al. 1997; METAXAKIS et al.
17 2005). Briefly, the Mi{ET1}GalkMB10638 males were crossed to virgin females containing
18 the Minos transposase snaSco/SM6a, P{w+mC=hsILMiT}2.4. After three days, adult flies
19 were removed, and the embryos were heat shocked in a 37˚C water bath 1 hour for 4
20 consecutive days. Male progeny were selected for the presence of the Minos insertion
21 and the Minos transposase according to eye color (the Minos element expresses GFP,
22 and Minos transposase expresses a red eye color from mini-white in the insertion) and
23 crossed to the double balancer fly stock WR135 (Sp/CyO;TM2/MKRS). Male progeny
11 1 were selected for the absence of GFP and mini-white, indicating a successful excision,
2 and crossed again to WR135 virgin females. Progeny harboring excisions were
3 crossed to each other to make a homozygous stock. These stocks were assayed for
4 the presence of a precise excision using primers sequencing through the affected
5 genomic region.
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7 qRT-PCR
8 Drosophila were collected 3-5 days after eclosion and 5 whole flies were collected
9 or 10-15 fly hearts were dissected for each group. RNA was extracted using RNA-Bee
10 RNA isolation reagent (Amsbio) according to the manufacturer’s protocol. SuperScript
11 II (Invitrogen) reverse transcription according to the manufacturer’s protocol was
12 followed by real-time PCR analysis using a predesigned taqman probe for Galk (probe:
13 Dm01801608_g1, Applied Biosystems, Inc.).
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15 Tissue-specific phenotypes
16 For wing vein phenotypes, Drosophila from the respective crosses were collected at
17 3-5 days after eclosion. The wings were detached using forceps and the ventral surface
18 was placed face down on GelWax plates. The wings were examined for abnormalities
19 under a dissection microscope at 40x or imaged under a Leica M165FC Fluorescence
20 stereo microscope equipped with a Leica DFC310FX camera at 50x. A range of wing
21 vein abnormalities were observed, which were divided into normal, abnormalities of the
22 posterior crossvein (PCV), or abnormalities of both the PCV and the longitudinal vein 5
23 (L5). The number of wings under each category of wing vein phenotype was counted
12 1 for each group and the percentage of total wings counted was calculated. Statistical
2 significance to detect for a rescue of the abnormal wing vein phenotype (pooling the two
3 different types of wing vein abnormalities) with the Df(3L)ED4416 deficiency or
4 Mi{ET1}GalkMB10638 insertion was determined using Fisher’s exact test. Tissue-specific
5 expression of CanAact with dpp-Gal4, Act5C-Gal4, and Mef2-Gal4 was analyzed
6 similarly by counting the number of progeny with the respective phenotypes according
7 to the crosses and genotypes described in the figure legend.
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9 Statistical analysis
10 All data was analyzed in GraphPad Prism 6 software. Student’s t test was
11 performed for single comparisons and one-way ANOVA and post-hoc analysis with
12 Bonferroni correction was conducted for group comparisons. Fisher’s exact test was
13 employed to determine the rescue of the proportion of flies displaying wing or lethality
14 phenotypes.
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13 1
2 RESULTS
3 Expression of constitutively active calcineurin (CanAact) in the adult fly heart
4 caused cardiac enlargement
5 To identify genetic modifiers of cardiac calcineurin, we generated two types of
6 sensitized Drosophila lines expressing CanAact in the heart: 1) CanAact under the direct
7 control of the tinC promoter, a 304 bp intronic region within the tinman gene that
8 controls cardiac-specific gene expression with a minimal hsp70 promoter (tinC-CanAact)
act 9 (YIN et al. 1997; LO and FRASCH 2001; WOLF et al. 2006). tinC-CanA transgenic lines
10 were generated under the direct control of the cardiac-specific tinC genomic fragment in
11 the context of an hsp70 minimal promoter to harbor either a flag- or YFP (yellow
12 fluorescent protein)- tagged CanAact (FCanAact or YCanAact respectively, Figure 1A). 2)
13 CanAact under control of the UAS promoter (UAS-CanAact) where cardiac-specific
14 CanAact was expressed in Drosophila using the Gal4/UAS system (tinC-Gal4>UAS-
15 CanAact) (Brand and Perrimon 1993).
16 We performed measurements of cardiac chamber dimensions during complete
17 relaxation (end-diastole) and full contraction (end-systole) using OCT (Wolf et al. 2006);
18 fractional shortening was calculated as described in methods and used as a
19 measurement of cardiac contractility. In agreement with previous studies, w1118 control
20 Drosophila displayed close to 100% fractional shortening (WOLF et al. 2006) (Figure 1B,
21 S1A). Drosophila expressing either FCanAact or YCanAact displayed enlarged end-
22 diastolic dimension and reduced fractional shortening (Figure 1B, Figure S1B). In order
23 to account for possible positional effects of the transgene, end-diastolic dimensions in
14 1 several different lines were tested. tinC-YCanAact or tinC-FCanAact insertions on the
2 first, second, and third chromosomes all induced enlarged end-diastolic dimensions,
3 suggesting that the transgene is causing cardiac enlargement and the phenotype is not
4 due to disruption of an endogenous gene from transgenic insertion (Figure S1C).
5 Cardiac enlargement persisted in tinC-CanAact flies compared to controls over 5 weeks
6 of age (Figure S1D).
7 Histological analyses confirmed that the tinC-CanAact fly hearts were significantly
8 larger than controls (Figure 1C). The heart wall was significantly thicker in tinC-
9 Gal4>UAS-CanAact but not tinC-CanAact flies (Figure S1E-F). This distinction may be
10 due to augmented CanAact expression with tinC-Gal4>UAS-CanAact.
11 Immunofluorescence staining and confocal microscopy confirmed the cardiac-specific
12 expression of tinC-CanAact (Figure S1G). To determine whether the enlargement in
13 heart size was due to increase in number of cells or increase in cell size, we examined
14 the number of nuclei in tinC-CanAact and tinC-GFP control hearts. Confocal microscopy
15 of tinC-CanAact hearts expressing cardiac-specific nuclear localized RFP had the same
16 number of cardiomyocytes compared to controls, indicating that the phenotype was not
17 the result of abnormalities in cardiomyocyte number (Figure S1H).
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19 A region on chromosome 3L harbored novel modifiers of tinC-CanAact-mediated
20 cardiac enlargement
21 To test the feasibility of finding a modifier using tinC-CanAact as the sensitized line,
22 we examined the effect of genetic deficiency of the known downstream regulator of
15 1 calcineurin, Mef2 (VAN OORT et al. 2006). A deficiency in the known calcineurin modifier
2 Mef2 rescued tinC-CanAact-induced cardiac enlargement (Figure S2).
3 Previous screens identified that a region located at cytolocation 66F rescued 24B-
4 CanAact-induced lethality (Gajewski et al. 2003) and corresponded to a suppressor and
5 enhancer region for GMR-CanAact-induced rough eye (Sullivan and Rubin 2002).
6 Therefore, we reasoned that a deficiency screen covering this region would likely
7 identify a modifier of CanAact-induced cardiomyopathy. Genomic deficiency lines
8 surrounding this region were initially examined for the ability to suppress tinC-CanAact-
9 mediated cardiac enlargement (Figure 2A). Two lines, Df(3L)ED4416 and
10 Df(3L)BSC130, suppressed tinC-CanAact-induced cardiac enlargement (Figure 2B-C,
11 S3A). Excluding the regions in which the deficiencies did not rescue the phenotype, we
12 narrowed the region down to 80 kb that is predicted to encode 13 genes (Figure 2A). Of
13 note, Df(3L)ED4421 spans the suppressor region, but exerts a cardiac enlargement
14 phenotype on its own, indicating that genes within this large deficiency induce cardiac
15 dilation independent of CanAact (Figure S3A). For this reason, the Df(3L)ED4421
16 deficiency was not considered when determining the suppressor region.
17 We validated the suppressor region corresponding to Df(3L)ED4416 and
18 Df(3L)BSC130 by evaluating the cardiac phenotype in tinC-Gal4>UAS-CanAact
19 Drosophila in the context of these deficiencies. tinC-Gal4>UAS-CanAact flies displayed
20 cardiac enlargement similar to that of tinC-CanAact flies (Figure 3). Genetic disruption of
21 the known calcineurin modifiers Mef2, sty, or CanB2 rescued the fractional shortening of
22 tinC-Gal4>UAS-CanAact flies (Figure 3, S3B), demonstrating the fidelity of using a
23 sensitized Drosophila line to find modifiers of calcineurin. Cardiac enlargement (end-
16 1 diastolic dimension) of tinC-Gal4>UAS-CanAact hearts was rescued by Df(3L)ED4416
2 and Df(3L)BSC130 (Figure 3). These results suggest that Df(3L)ED4416 and
3 Df(3L)BSC130 harbor modifiers for CanAact-induced cardiac enlargement.
4
5 Identification of galactokinase as a novel modifier of calcineurin-induced cardiac
6 enlargement
7 The refined suppressor region contained a total of 13 genes (Figure 2A). Two genes,
8 galactokinase and arginine kinase, have high expression in the adult fly heart
9 (flyatlas.org) (Chintapalli et al. 2007; Robinson et al. 2013). In order to determine
10 whether Galk is a modifier of the tinC-CanAact cardiac phenotype, we tested two
11 Drosophila lines that had transposable element insertions in Galk and decrease Galk
12 transcript expression: PBac{PB}Galkc03848 and Mi{ET1}GalkMB10638. In the context of
13 tinC-CanAact, PBac{PB}Galkc03848 and Mi{ET1}GalkMB10638 rescued the tinC-CanAact-
14 induced decrease in cardiac contractility, and a precise excision of the
15 Mi{ET1}GalkMB10638 insertion, Mi{ET1}Galkrev, reverted this rescue (Figure 4A-B, S3C).
16 Additionally, cardiac-specific expression of RNAi directed against Galk also rescued
17 tinC-CanAact-induced cardiac enlargement (Figure 4C-D). Galk expression levels were
18 reduced in the transposable element insertion and Galk RNAi-expressing flies (Figure
19 4E-G). To test whether the tinC-Gal4>UAS-YCanAact-induced hypertrophy would be
20 rescued, we examined whether Mi{ET1}GalkMB10638 would prevent the CanAact-induced
21 increase in cardiac wall thickness. Flies expressing tinC-Gal4>UAS-YCanAact show
22 rescue of the increase in wall thickness (Figure 4H). Additionally, tinC-CanAact flies had
23 reduced lifespans compared to control, which was rescued in the context of
17 1 Mi{ET1}GalkMB10638 (Figure S4). These results support the hypothesis that Galk is a
2 modifier of calcineurin in the heart.
3 We then tested whether Mi{ET1}GalkMB10638 would rescue a non calcineurin-mediated
4 cardiomyopathy of the troponin I mutant hdp2 that shows a flight muscle abnormality
MB10638 5 and cardiac dilation (BEALL and FYRBERG 1991; WOLF et al. 2006). Mi{ET1}Galk
6 did not rescue the cardiac dilation phenotype of heterozygous hdp2 flies (Figure S5),
7 indicating that Galk modification is specific to CanAact-induced cardiomyopathy.
8 We also tested Argk as a possible modifier for calcineurin. A Drosophila line with an
9 insertion in Argk, PBac{WH}Argkf05525, had normal cardiac function and did not rescue
10 tinC-CanAact-induced cardiac enlargement (Figure S6A-C).
11 In addition, the dorsocross genes (Doc1, Doc2, and Doc3) are homologous to
12 mammalian T-box genes and known to regulate embryonic cardiac development (Reim
13 and Frasch 2005). Accordingly, we examined the effects of these candidate genes on
14 the tinC-CanAact cardiac enlargement phenotype using OCT. Doc1, Doc2, and Doc3
15 RNAi lines were obtained and crossed into tinC-Gal4, tinC-CanAact flies to test for
16 rescue of the CanAact phenotype. Genetic disruption of the Doc genes with RNAi did
17 not rescue tinC-CanAact-induced cardiac enlargement (Figure S6D). Interestingly,
18 several cardiac-specific RNAi lines directed against the Doc genes caused an enlarged
19 cardiac phenotype on their own (Figure S6D).
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21 Tissue-specific suppression of CanAact-induced phenotypes in non-cardiac
22 tissues with genetic deficiency of Galk
18 1 Next, we used a Gal4/UAS system for driving CanAact with non-cardiac tissue
2 drivers. The expression of CanAact under control of the engrailed wing driver e16E-Gal4
3 caused an abnormality in the posterior crossveins (PCV) and the longitudinal wing vein
4 L5 (Figure 5). Importantly, Df(3L)ED4416 significantly rescued the e16E-Gal4>UAS-
5 YCanAact-induced abnormal wing vein phenotype. The percentage of flies that had
6 normal wing veins significantly increased from 11% to 44%, and the percentage of flies
7 with abnormal wing vein phenotypes decreased from 89% to 56% with a genetic
8 deficiency encompassing Galk (Figure 5). To further investigate the involvement of
9 Galk in wing vein abnormality, the effect of disruption of Galk with a transposable
10 element insertion was examined in e16E-Gal4>UAS-YCanAact wings. Disruption of
11 Galk with Mi{ET1}GalkMB10638 in e16E-Gal4>UAS-YCanAact flies also rescued the wing
12 vein phenotypes, albeit at a lesser rate compared to the deficiency, with the percentage
13 of normal wing vein flies increasing from 5% to 31%, and the percentage of abnormal
14 winged flies decreasing from 95% to 69% in the context of Mi{ET1}GalkMB10638 (Figure 5,
15 lower right panel).
16 Expression of CanAact under control of the ubiquitous driver, Act5C-Gal4, caused
17 larval lethality (Figure 6A), while expression under the ectodermal driver, dpp-Gal4,
18 caused a complete inability of the wing to expand (Figure 6B), and expression under the
19 muscle driver, Mef2-Gal4, caused lethality at a pupal stage (Figure 6C). In contrast to
20 the observed rescue of the abnormal wing vein phenotype, Df(3L)ED4416 was not
21 sufficient to rescue the wing or lethality phenotypes driven by Act5C-Gal4, dpp-Gal4, or
22 Mef2-Gal4, suggesting that deficiency of Galk suppresses CanAact induced phenotypes
23 in a tissue-specific manner. We also examined the ability of Mi{ET1}GalkMB10638 to
19 1 suppress the Mef2-Gal4>UAS-YCanAact lethality phenotype, and show that
2 Mi{ET1}GalkMB10638 was not able to rescue the Mef2-Gal4>UAS-YCanAact- induced
3 lethality (Figure 6C), further indicating that Galk may not modify Mef2-Gal4>UAS-
4 YCanAact-induced lethality.
5
20 1 DISCUSSION
2 Calcineurin is a known mediator of cardiac hypertrophy in mammalian hearts and
3 understanding the signals that regulate calcineurin has the potential to alter cardiac
4 pathology. Here we show that 1) CanAact in the Drosophila heart induced cardiac
5 enlargement and reduced cardiac contractility; 2) Galk disruption suppressed the
6 CanAact-mediated cardiac enlargement, increase in wall thickness, and posterior wing
7 vein phenotypes; and 3) Galk regulation of CanAact-induced phenotypes was tissue
8 specific. Using the resources that are available in fly genetics, the ability to phenotype
9 in vivo cardiac chamber sizes, and the unique fact that flies lack calcineurin-regulated
10 NFAT, we identified a potential new regulator of calcineurin in the Drosophila heart. We
11 also observed that CanAact induced sustained cardiac enlargement in Drosophila during
12 aging and reduced life span. Furthermore, known regulators of calcineurin signaling,
13 including Mef2, rescued the observed cardiac abnormalities, supporting our hypothesis
14 that these approaches can identify modifiers of calcineurin-mediated cardiac
15 abnormalities.
16 Two prior screens in the fly scored changes in eye morphology or lethality caused by
17 activated calcineurin and identified several genomic regions that harbor potential
18 modifiers. Four enhancer regions and five suppressor regions were identified in a
19 mutagenesis screen in the fly eye (Sullivan and Rubin 2002); whereas seven
20 suppressor regions were discovered in a deficiency screen for lethality (Gajewski et al.
21 2003). These studies identified cytological regions in the fly genome and, based on
22 these important findings, we focused our attention on a region that was common to both
23 studies but lacked identification of the candidate modifier of calcineurin. Using
21 1 molecularly-defined genomic deficiencies, transposable element insertions, precise
2 excisions, and transgenic RNAi, we identified that galactokinase was a candidate
3 modifier of calcineurin-mediated cardiac abnormalities in the fly. Galactokinase may
4 function as either an enhancer of calcineurin signaling or in a pathway downstream of
5 calcineurin activation. Interestingly, previous studies have not implicated an interaction
6 between calcineurin and galactokinase.
7 Our results demonstrate that deficiency of Mef2 suppressed cardiac-CanAact-induced
8 cardiac enlargement. Since Drosophila do not express calcineurin-regulated NFAT, this
9 implies that Mef2 functions independent of NFAT in our system. In agreement with our
10 results, studies in skeletal muscle and C2C12 myogenic cells have shown that
11 calcineurin binds to Mef2, leading to hypophosphorylation and enhanced transcription
12 activity (Wu et al. 2000; Wu et al. 2001). However, studies in Jurkat T-lymphocytes
13 suggest that calcineurin-regulated Mef2 activation requires recruitment of NFATc2
14 (Blaeser et al. 2000; Youn et al. 2000). In the mouse heart, the major pathway appears
15 to be NFAT since expressing dominant negative Mef2 only rescued cardiac dilation and
16 not hypertrophy (VAN OORT et al. 2006). In the same mouse study, overexpressing Mef2
17 induced a cardiac dilation phenotype without hypertrophy, suggesting that Mef2 mainly
18 contributes to the dilation phenotype induced by calcineurin. Interestingly, flies
19 expressing tinC-Gal4>UAS-Mef2 had significantly increased end-diastolic dimensions
20 (Figure S7), similar to the tinC-CanAact phenotype, although the flies overexpressing
21 Mef2 did not display reduced fractional shortening, suggesting that Mef2 is necessary
22 but not sufficient for calcineurin-induced reduction in contractility. These results suggest
23 that an NFAT-independent pathway through Mef2 in Drosophila may regulate cardiac
22 1 enlargement, and that additional factors outside of Mef2 may be involved in controlling
2 cardiac function.
3 Several potential genes were excluded as modifiers from our original candidate
4 region. The Doc genes are known to regulate Drosophila cardiac development (REIM
5 and FRASCH 2005), and we showed that knocking down Doc expression with RNAi
6 caused cardiac enlargement, and did not rescue tinC-CanAact. This implies that Doc
7 genes are important for cardiac development but we do not have evidence for them
8 regulating calcineurin. Argk has high expression in the adult Drosophila heart. Argk is
9 an enzyme that transfers the phosphate on ATP to arginine, creating an energy rich
10 buffer for maintaining ATP concentration (Newsholme et al. 1978). Although it is
11 conceivable that Argk functions to provide sufficient energy for the fly myocardium, our
12 results showed that disrupting Argk with a transposable element did not produce a
13 phenotype and did not rescue calcineurin-induced cardiac enlargement. These results
14 suggest that Argk does not regulate calcineurin-induced cardiac enlargement.
15 However, we note that the gene expression was decreased by only 30% in the
16 heterozygous PBac{PB}Argkf05255 line used (Figure S6C) and it is possible that the lack
17 of rescue was due to incomplete knock-down of gene expression.
18 A deficiency encompassing Galk suppressed the CanAact-induced phenotypes in
19 heart and posterior wing. However, this deficiency did not suppress phenotypes driven
20 by ubiquitous Act5C-Gal4, ectodermal dpp-Gal4, or mesodermal Mef2-Gal4 drivers.
21 These observations could be explained by the tissue-specific context in which
22 calcineurin was expressed. The expression of each driver occurs in different tissues.
23 Although e16E-Gal4 and dpp-Gal4 expression of CanAact induced wing phenotypes,
23 1 e16E-Gal4 drives expression in the posterior wing while dpp-Gal4 drives expression
2 anterior to the anterior-posterior boundary during imaginal disc development at the third
3 instar larva stage (BROWER 1986; DE CELIS 1997). Many signaling factors are
4 differentially expressed during wing development in these two separate compartments
5 to guide correct patterning. For example, engrailed and hedgehog are expressed
6 posteriorly, guiding formation of the posterior wing veins (BROWER 1986; LEE et al.
7 1992) while dpp and the EGFR inhibitor knot are expressed specifically in the anterior
8 wing imaginal disc (DE CELIS 1997; MOHLER et al. 2000). It is possible that Galk
9 modification of calcineurin signaling is regulated by these differentially expressed
10 factors. In addition, Act5C-Gal4 and Mef2-Gal4 driving CanAact induced lethality that
11 was not suppressed by deficiency of Galk. These drivers induce transcript expression
12 in multiple tissues during early embryonic development (BURN et al. 1989; BOUR et al.
13 1995). One explanation for the findings is that the signals at an early stage of
14 development involve pathways that are not modulated by Galk.
15 We examined the survival of tinC-CanAact Drosophila and in the context of the
16 insertion Mi{ET1}GalkMB10638. Of note, in mice, cardiac-specific expression of CanAact
17 also induced premature sudden death (MOLKENTIN et al. 1998). Flies heterozygously
18 expressing tinC-CanAact had a significantly reduced life span compared to the control
19 group. This reduction was suppressed in the context of one copy of Mi{ET1}GalkMB10638
20 (Figure S4), further suggesting that deficiency in Galk suppresses multiple cardiac-
21 calcineurin-induced phenotypes. However, a caveat to the interpretation of this
22 experiment is the possibility of genetic background confounding the beneficial effects
23 seen by disruption of Galk.
24 1 Galactokinase belongs to the GHMP ATP-dependent kinase family (named after the
2 four representative kinases in this family: galactokinase, homoserine kinase,
3 mevalonate kinase, and phosphomevalonate kinase) (Holden et al. 2004). In the fly,
4 galactokinase phosphorylates galactose and N-acetyl-galactosamine (GalNAc), allowing
5 further utilization in either metabolism (energy production) or glycosylation (protein
6 modification) (Holden et al. 2004). These pathways potentially lead to cardiac
7 enlargement: either galactokinase promotes a higher level of phosphorylated galactose,
8 galactose-1-p, leading to a diseased state, or downstream reactions involving UDP-
9 galactose incorporation into glycosylated proteins promotes cardiac enlargement, or
10 both mechanisms may be required. A previous screen examining Drosophila cardiac
11 development discovered that mutations in HMG-CoA reductase (the rate-limiting
12 enzyme in the mevalonate pathway) induces a cardiac phenotype by
13 geranylgeranylation of the G protein Gγ1, suggesting a pathway by which post-
14 translational modifications can alter the fly heart (Yi et al. 2006).
15 In additional experiments, we show that a transposable element insertion in galectin
16 (a galactoside-binding lectin) suppressed the tinC-CanAact cardiac phenotype (Figure
17 S8A-B). Mammalian Galectin3 has been shown to bind to Galk-regulated N-
18 acetyllactosamine side chains on EGFR, preventing endocytosis and enhancing
19 signaling of isolated mouse mammary cells (Partridge et al. 2004). Driving activated
20 EGFR in the Drosophila heart has been shown to induce a cardiac hypertrophy
21 phenotype (Yu et al. 2013) and sprouty, a regulator of EGFR signaling, has been shown
22 to modify calcineurin-induced rough eye (Sullivan and Rubin 2002). Although
23 speculation, one potential mechanism by which galactokinase functions to modulate
25 1 calcineurin-induced cardiac enlargement is by influencing co-translational glycosylation
2 of EGFR or another yet unidentified cell surface protein that is bound by galectin (Figure
3 S8C).
4 A yeast homologue of Galk, Gal3p, has been found to act as a transcriptional
5 activator by interacting with Gal80p (Zenke et al. 1996); transcriptional regulation is
6 activated with the binding of galactose and ATP to Gal3p. This suggests the possibility
7 that Galk may act as a modifier of transcription for known pathways including Mef2.
8 Whether Galk functions as part of the transcriptional machinery remains to be
9 determined.
10 In conclusion, we have developed a model for screening for novel modifiers of
11 constitutively active calcineurin in the Drosophila heart and identified galactokinase as a
12 novel modifier of constitutively active calcineurin-induced cardiomyopathy, shortened
13 life span, and wing vein abnormality in adult Drosophila. These findings have set up a
14 system for delineating the pathways involved in CanAact-induced cardiomyopathy in
15 Drosophila.
16
17
18 ACKNOWLEDGMENTS
19 This work was supported by grants from the National Institutes of Health. HL083065
20 (H.A.R.) and HL116581 (M.J.W.), and an American Heart Association Predoctoral
21 Award 11PRE7100004 (T.E.L.)
26 1
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13
31 1 FIGURES AND FIGURE LEGENDS
2 Figure 1
A CanA Catalytic B M I CanAact tinC YFP/Flag Catalytic B M stop
B w1118 80 *** 100 ** 80 125µm 60 tinC-YCanAact/+ 60 40
Fractional 40
0.8sec 20 Shortening (%) 20 End-diastolic
Dimension (microns) 0 0 w1118 tinC- w1118 tinC- YCanAact/+ YCanAact/+ C tinC-GFP 2.0 * 200
) ** 2 1.5 150
1.0 100 tinC-YCanAact 50
Lumen Area Lumen 0.5 (1000 microns
0 Perimeter (microns) 0
50 microns tinC-GFP tinC- tinC-GFP tinC- 3 YCanAact YCanAact 4 Figure 1. tinC-CanAact flies display a cardiac enlargement phenotype. 5 (A) Domain structure of calcineurin, tinC-YFP-tagged CanAact (tinC-YCanAact), and 6 tinC-Flag-tagged CanAact (tinC-FCanAact). CanAact= constitutively active calcineurin. B= 7 CanB binding domain. M= Calmodulin binding domain. I= autoinhibitory domain. tinC 8 is composed of a 304 bp genomic DNA element driving transgene expression in all cells 9 constituting the heart tube as previously described (YIN et al. 1997; LO and FRASCH 10 2001; WOLF et al. 2006). (B) Representative OCT m-mode images and summary data 11 for end-diastolic dimension and fractional shortening of w1118 control (N=16) and 12 heterozygous tinC-YCanAact from cross with w1118 (N=19) flies. CanAact flies show 13 enlarged end-diastolic dimensions and reduced fractional shortening. (C) 14 Representative transverse paraffin sections of homozygous tinC-GFP control (N=11) 15 and tinC-YCanAact (N=9) fly hearts are shown (Blue arrows indicate the fly heart). 16 Summary data for lumen area and perimeter are shown in microns. The tinC-YCanAact 17 fly displayed a significantly enlarged cardiac lumen area and perimeter. (Student’s t 18 test, *P<0.05, **P<0.01, ***P<0.001 compared to either w1118 or tinC-GFP control. Data 19 represent mean ±SEM.)
32 1 Figure 2 A Chromosome 3L 8800k 8900k 9000k 9100k 9200k Gene Span
Deficiency Span Df(3L)ED4416 Df(3L)BSC130 Df(3L)ED4414 Df(3L)BSC170 Df(3L)ED4415 Df(3L)BSC390 Df(3L)ED4413 Df(3L)ED4421
8980k 9000k 9020k 9040k CG5644 CG5068 CG5087 CG5194 Doc1 CG5144 Galk CG5280 Doc3 Doc2 Argk CG13314 smg ** B ** * 80
60
40
20 End-diastolic
Dimension (microns) 0 C ** ** 100 **
80
60
40 Fractional 20 Shortening (%) 0 act tinC-YCanA - + + + + + + + + + Deficiency - -
(3L)BSC130 (3L)BSC17(3L)0 BSC390 2 Df(3L)ED4416Df Df(3L)ED4421Df(3L)ED4414Df(3L)ED4415Df(3L)ED4413Df Df 3 Figure 2. Molecularly-defined deficiencies Df(3L)ED4416 and Df(3L)BSC130 4 rescued calcineurin-mediated abnormalities in adult flies. 5 (A) Genetic map of deficiency stocks tested (adapted from Gbrowse, 6 http://flybase.org/cgi-bin/gbrowse/dmel). Dotted lines indicate the suppressor region. 7 Genes within the suppressor region are shown in the magnified view below. (Green 8 bars= rescuing deficiencies. Red bars= non-rescuing deficiencies. Black bar= 9 deficiency that causes cardiomyopathy on its own.) (B-C) Summary data for (B) end- 10 diastolic dimension and (C) fractional shortening of w1118 control (first column) tinC- 11 YCanAact alone and in the context of molecularly-defined genomic deficiencies. All 12 deficiencies were tested in the heterozygous states. (N=17-51 for each group.) Two 13 deficiency lines rescued the tinC-YCanAact phenotype, narrowing down the original 14 suppressor region to the region in between the dotted lines in (A). Note that 15 Df(3L)ED4421 covers the deficiency region but was dilated on its own without CanAact 16 expression (Figure S3), and was not considered in defining the deficiency region. (One- 17 way ANOVA with Bonferroni correction. *P<0.05, **P<0.0001. Data represent mean 18 ±SEM.) 19
33 1 2 Figure 3 A * * *** *** * * * 80
60
40
End-diastolic 20
Dimension (microns) 0
B *** *** ** ** *** 100 *** ***
80
60
40 Fractional
Shortening (%) 20
0 tinC-Gal4 - - + + + + + + + act UAS-YCanA - + - + + + + + +
Genetic Disruption - - -- X1 5 EP774 sty
(3L)BSC130 Df(3L)ED4416Df 3 Df(2R)X1,Mef2 P{EP}CanB2 4 Figure 3. tinC-Gal4>UAS-CanAact displayed cardiac enlargement that was 5 rescued by tinC-CanAact-rescuing deficiencies and diminished contractility 6 phenotype that was rescued by known calcineurin modifiers. 7 (A-B) Summary data for (A) end-diastolic dimension and (B) fractional shortening of 8 w1118 controls (N=9), tinC-Gal4>UAS-YCanAact (N=32) alone, heterozygous tinC-Gal4 9 (N=7) and tinC-Gal4>UAS-YCanAact in the presence of Mef2 (N=11), sty (N=12), CanB2 10 (N=23), Df(3L)ED4416 (N=18), and Df(3L)BSC130 (N=13). All transgenes and 11 mutations were heterozygous. Known modifiers of calcineurin signaling, Mef2, sty, and 12 CanB2, rescued tinC-Gal4>UAS-YCanAact cardiac contractility (sty rescued fractional 13 shortening but not end-diastolic dimension). In addition, the tinC-YCanAact-rescuing 14 deficiencies Df(3L)ED4416 and Df(3L)BSC130 also rescued tinC-Gal4>UAS-YCanAact 15 cardiac enlargement (Df(3L)BSC130 rescued end-diastolic dimension but not fractional 16 shortening). (*P<0.05, **P<0.01, ***P<0.0001. One-way ANOVA with Bonferroni 17 correction. Data represent mean ±SEM.)
34 1 Figure 4 *** A B * * 100 * ** ** ** 80 80
60 60
40 40 Fractional
End-diastolic 20 20 Shortening (%)
Dimension (microns) 0 0 - + + + + - + + + + tinC-YCanAact - - --+ - - --+ PBac{PB}Galkc03848 - - --+ - - --+ Mi{ET1}GalkMB10638 - - -- + - - -- + Mi{ET1}Galkrev
*** C *** ** * ** D *** * ** 100 80
60
40 50 Fractional 20 Shortening (%) End-diastolic
Dimension (microns) 0 0 - +- - - + - +- - - + tinC-YCanAact -- + - ++ -- + - ++ UAS-Galk RNAi - -- + + + - -- + + + tinC-Gal4
E F G * * ** ** * 1.0 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.5 0.4 * 0.2 0.2 (Fold/Control) (Fold/Control) (Fold/Control) Galactokinase Galactokinase Galactokinase 0.0 0.0 0.0 act act 1118 1118 tinC-YCanA c03848 w MB10638 w rev
MB10638 tinC-Gal4/+ tinC-YCanA
PBac{PB}GalkMi{ET1}Galk Mi{ET1}Galk Mi{ET1}Galk tinC-Gal4>Galk RNAi
H tinC-Gal4/+ tinC-Gal4>>UAS-YCanAact tinC-Gal4>UASYCanAact Mi{ET1}Galk
50 microns 10 **
5
(microns) 0 + + + tinC-Gal4 Wall thickness - ++ UAS-YCanAact - - + Mi{ET1}Galk
2 3 Figure 4. Genetic disruption of Galk rescued tinC-CanAact-induced reduction in 4 cardiac contractility and tinC-Gal4>UAS-CanAact- induced hypertrophy. 5 (A-B) Summary data for (A) end-diastolic dimension and (B) fractional shortening of 6 flies expressing w1118 control, tinC-YCanAact, the transposable elements
35 1 PBac{PB}Galkc03848, Mi{ET1}GalkMB10638, or precise excision of Mi{ET1}GalkMB10638 2 (Mi{ET1}Galkrev), in the context of tinC-YCanAact. The two transposable elements in 3 Galk rescued tinC-YCanAact -mediated cardiac contractility (fractional shortening), while 4 a precise excision reverted the rescue. (A: *P<0.05, **P<0.01; B: *P<0.05, **P<0.001, 5 ***P<0.0001 compared to tinC-YCanAact or Mi{ET1}GalkMB10638 as indicated with an over 6 bar. N=14-27 for each group. One-way ANOVA with Bonferroni correction for multiple 7 comparisons, all data represent mean ±SEM.) (C-D) Summary data for (C) end- 8 diastolic dimension and (D) fractional shortening of w1118 control, CanAact only, UAS- 9 Galk no driver (heterozygous with w1118), tinC-Gal4 driver only (heterozygous with 10 w1118), Galk RNAi only flies, and flies expressing tinC-YCanAact in the context of cardiac 11 Galk RNAi. RNAi to Galk significantly rescued tinC-YCanAact -mediated cardiac 12 enlargement. (*P<0.01, **P<0.0001 compared to tinC-YCanAact. N=7-20 for each 13 group. One-way ANOVA with Bonferroni correction for multiple comparisons, all data 14 represent mean ±SEM.) (E) qRT-PCR for Galk expression of w1118 control, 15 homozygous PBac{PB}Galkc03848, or homozygous Mi{ET1}GalkMB10638. Galk expression 16 is downregulated by transposable element insertions. (*P<0.001 compared to w1118 17 control. N=3 in each group. One-way ANOVA with Bonferroni correction for multiple 18 comparisons, all data represent mean ±SEM.) (F) qRT-PCR of Galk expression in w1118 19 control flies, flies heterozygous for tinC-YCanAact alone, in the context of heterozygous 20 Mi{ET1}GalkMB10638, or a precise excision of Mi{ET1}GalkMB10638, Mi{ET1}Galkrev. 21 (*P<0.05, **P<0.001 compared to heterozygous tinC-YCanAact/Mi{ET1}GalkMB10638. 22 N=8 in each group. One-way ANOVA with Bonferroni correction for multiple 23 comparisons, all data represent mean ±SEM.) (G) Galk expression for tinC-Gal4 24 heterozygous with w1118 (driver only) and heterozygous UAS-Galk RNAi knockdown 25 with a tinC-Gal4 driver (Galk RNAi). Galk expression is downregulated by transposable 26 element insertions. N=3 for each group. Student’s t-test *P<0.001 Galk RNAi vs. tinC- 27 Gal4 driver only. (H) Representative hematoxylin and eosin stained histological 28 sections and quantification of wall thickness for heterozygous tinC-Gal4 (from cross with 29 w1118, N=8), tinC-Gal4>UAS-YCanAact (N=13), or tinC-Gal4>UAS-YCanAact in the 30 context of Mi{ET1}GalkMB10638 (N=6). tinC-Gal4>UAS-YCanAact caused a cardiac 31 hypertrophy phenotype that was rescued by Mi{ET1}GalkMB10638. Blue arrowheads point 32 to the heart. *P<0.05, one-way ANOVA with Bonferroni correction, all data represent 33 mean ±SEM. (All transgenes were heterozygous unless otherwise noted.) 34
36 1 Figure 5 CanAact-induced wing vein phenotype Percent progeny from e16E-Gal4 x UAS-YCanAact/CyO;Df(3L)ED4416/MKRS or e16E-Gal4 x UAS-YCanAact/CyO;Mi{ET1}Galk/MKRS e16E-Gal4 driver only normal wing veins abnormal PCV abnormal PCV and L5
100 11% 80 1mm 24% 44% 60 40 50% act 65% e16E-Gal4>UAS-YCanA 20 * 6%
normal Percent total (%) 0
1mm No deficiencyDf(3L)ED4416 5% abnormal 100 posterior 80 2323%% 31% crossvein PCV 60 (PCV) 31% 40 72% 1mm * 20 38% abnormal Percent total (%) 0 PCV and longitudinal L5 vein 5 (L5) No Minos 1mm Mi{ET1}Galk 2 e16E-Gal4>UAS-YCanAact 3 Figure 5. The deficiency Df(3L)ED4416 and Minos insertion in Galk 4 Mi{ET1}GalkMB10638 rescued e16E-Gal4>UAS-YCanAact -induced wing vein loss. 5 Wing vein phenotypes of heterozygous e16E-Gal4 driver only control or e16E- 6 Gal4>UAS-YCanAact flies: normal, abnormality of the posterior cross vein (PCV) or 7 abnormality of both PCV and longitudinal vein 5 (L5). Progeny were counted from the 8 cross e16E-Gal4 x UAS-YCanAact/CyO;Df(3L)ED4416/MKRS or e16E-Gal4 x UAS- 9 YCanAact/CyO;Mi{ET1}GalkMB10638/MKRS. The graph represents percentage of all 10 heterozygous e16E-Gal4>UAS-YCanAact flies counted with normal or abnormal wings in 11 the context of heterozygous MKRS balancer (no deficiency), Df(3L)ED4416, or 12 Mi{ET1}GalkMB10638. Df(3L)ED4416 partially rescues the e16E-Gal4>UAS-YCanAact- 13 induced wing vein loss phenotype (No deficiency N=46, Df(3L)ED4416 N=54). 14 Mi{ET1}GalkMB10638 also partially rescued the e16E-Gal4>UAS-YCanAact -induced wing 15 vein loss phenotype (No Minos N=129, Mi{ET1}GalkMB10638 N=242). (Arrowheads point 16 to the shortened abnormal PCV or L5, respectively. Significant rescue of the wing vein 17 abnormality with Df(3L)ED4416 and Mi{ET1}GalkMB10638 as determined by Fisher’s exact 18 test *P<0.0001.) 19
37 1 Figure 6 A CanAact-induced lethality C
Recovered progeny from CanAact-induced lethality Act5C-Gal4/CyO x UAS-YCanAact/CyO;Df(3L)ED4416/TM2 Recovered progeny from Genotype No deficiency Df(3L)ED4416 Mef2-Gal4 x UAS-YCanAact/CyO;Df{3L}ED4416/MKRS
No UAS-YCanAact expression 130 194 Genotype No deficiency Df(3L)ED4416
act Act5C-Gal4>UAS-YCanA 0 0 (No rescue) No UAS-YCanAact expression 52 49 B Mef2-Gal4>UAS-YCanAact 0 0 (No rescue) CanAact-induced wing phenotype w1118 dpp-Gal4>UAS-YCanAact
Recovered progeny from Mef2-Gal4 x UAS-YCanAact/CyO;Mi{ET1}GalkMB10638/MKRS
Genotype No Minos Mi{ET1}GalkMB10638 Percent abnormal winged progeny from dpp-Gal4/TM6B x UAS-YCanAact/CyO;Df(3L)ED4416/TM2 No UAS-YCanAact expression 104 107
act Genotype No deficiency Df(3L)ED4416 Mef2-Gal4>UAS-YCanA 0 0 (No rescue) No UAS-YCanAact expression 0% (N=0/20) 0% (N=0/14) dpp-Gal4>UAS-YCanAact 72.22% (N=8/11) 69.59% (N=16/23) 2 (No rescue) 3 Figure 6. The deficiency Df(3L)ED4416 did not rescue Act5C-Gal4>UAS-YCanAact- 4 induced lethality, dpp-Gal4>UAS-YCanAact -induced unexpanded wing, or Mef2- 5 Gal4>UAS-YCanAact-induced pupal lethality. 6 (A) Progeny number from the cross Act5C-Gal4/CyO x UAS- 7 YCanAact/CyO;Df(3L)ED4416/TM2. Driving UAS-YCanAact with an actin (Act5C-Gal4) 8 driver resulted in a lethal phenotype, which was not rescued by Df(3L)ED4416. (B) 9 Percent of total abnormal-winged progeny from the cross dpp-Gal4/TM6B x UAS- 10 YCanAact/CyO;Df(3L)ED4416/TM2. Expressing CanAact with a dpp driver resulted in a 11 shriveled abnormal wing phenotype. This was not rescued by Df(3L)ED4416. (C) Total 12 progeny from the cross Mef2-Gal4 x UAS-YCanAact/CyO;Df(3L)ED4416/MKRS or Mef2- 13 Gal4 x UAS-YCanAact/CyO;Mi{ET1}GalkMB10638/MKRS. Expressing CanAact with a Mef2- 14 driver resulted in a pupal lethal phenotype that was not rescued by Df(3L)ED4416 or 15 Mi{ET1}GalkMB10638. Data was analyzed using Fisher’s exact test.
38