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. Author manuscript; available in PMC: 2017 May 1.
Published in final edited form as: Dev Biol. 2016 Mar 13;413(1):16–25. doi: 10.1016/j.ydbio.2016.03.013

The canonical Wingless signaling pathway is required but not sufficient for inflow tract formation in the Drosophila melanogaster heart

Gloriana V Trujillo a,1, Dalea H Nodal a,1, Candice V Lovato a, Jill D Hendren a, Lynda A Helander a, TyAnna L Lovato a, Rolf Bodmer a,b, Richard M Cripps a,*
PMCID: PMC4834244  NIHMSID: NIHMS771181  PMID: 26983369

Abstract

The inflow tracts of the embryonic Drosophila cardiac tube, termed ostia, arise in its posterior three segments from cardiac cells that co-express the homeotic transcription factor Abdominal-A (abdA), the orphan nuclear receptor Seven-up (Svp), and the signaling molecule Wingless (Wg). To define the roles of these factors in inflow tract development, we assessed their function in inflow tract formation. We demonstrate, using several criteria, that abdA, svp, and wg are each critical for normal inflow tract formation. We further show that Wg acts in an autocrine manner to impact ostia fate, and that it mediates this effect at least partially through the canonical Wg signaling pathway. By contrast, neither wg expression nor Wg signaling are sufficient for inflow tract formation when expressed in anterior Svp cells that do not normally form inflow tracts in the embryo. Instead, ectopic abd-A expression throughout the cardiac tube is required for the formation of ectopic inflow tracts, indicating that autocrine Wg signaling must be supplemented by additional Hox-dependent factors to effect inflow tract formation. Taken together, these studies define important cellular and molecular events that contribute to cardiac inflow tract development in Drosophila. Given the broad conservation of the cardiac regulatory network through evolution, our studies provide insight into mechanisms of cardiac development in higher animals.

Keywords: Drosophila, heart, aorta, wingless, tinman, seven-up, abdominal-A, inflow tract, ostia

Introduction

Numerous distinct cell types contribute to the fully functional circulatory system of animals, and specific human diseases are known to arise from dysfunctions in, or malformations of, many of these cell types. Clearly, understanding the development and differentiation of individual components of the cardiac system will provide insight into disease mechanisms and possible therapeutic strategies. These studies are of particular significance since congenital heart disease is a common and debilitating birth defect (Christianson et al., 2006).

It is now becoming apparent that many instances of congenital cardiac disease arise from underlying genetic causes (reviewed in Pierpont et al., 2007; McCulley and Black, 2012) and de novo mutations (Zaidi et al 2013), therefore defining the developmental regulatory pathway for cardiac specification and differentiation is a critical component of understanding and treating such diseases. Much progress has been made in defining how individual genes control initial specification of the cardiac tissue, and in understanding how distinct developmental decisions are made within the cardiac lineage (for reviews see Cripps and Olson, 2002; McCulley and Black, 2012).

A striking finding from these studies is that the gene regulatory network for cardiac development shows significant conservation across vast evolutionary distance, since many of the genes that are important for heart specification in mammals are essential for cardiac specification in the fruit fly Drosophila melanogaster (Cripps and Olson, 2002; Tao and Schulz, 2007). Indeed, many regulatory genes known to be associated with human heart disease have direct orthologs in Drosophila that are required for normal fly cardiac development. These include NKX2.5/Tinman (Bodmer 1993; Azpiazu and Frasch, 1993; Schott et al., 1998); Myocyte enhancer factor-2 (MEF2; Lilly et al., 1995; Gonzalez et al., 2006); NOTCH1/Notch (Garg et al., 2005; Gajewski et al., 2000; Ward and Skeath, 2000); and GATA4/Pannier (Alvarez et al., 2003; Gajewski et al., 1999; Garg et al., 2003). Moreover, genetic screens in Drosophila have identified conserved pathways for cardiac development and maintenance (Yi et al 2006; Neely et al 2010). Such findings validate the utility of invertebrate systems for understanding human developmental mechanisms.

There is also conservation of the genetic pathways that control the formation of cardiac subtypes. In particular the cardiac inflow tracts share patterns of gene expression and gene function between Drosophila and vertebrates. In mammals, the atria correspond to the inflow tracts, and express the orphan nuclear receptor gene Chicken ovalbumin upstream promoter transcription factor-II (COUP-TFII) beginning at day 9 of embryonic development. Knockout of COUP-TFII function results in a loss of atrial cells in the developing mouse embryo (Pereira et al 1999), and it has now been established that COUP-TFII is a central determinant of atrial cell fate, lying at the top of a genetic hierarchy, and impacting the expression of several hundred genes (Wu et al 2013). Clearly, atrium formation is a complex process involving many regulatory steps, that might be readily dissected in a simpler model system.

The Drosophila cardiac system comprises the dorsal vessel, a linear tube located dorsally in the animal and running much of the length of the body. During normal circulation, hemolymph enters the tube in a posterior region termed the heart proper, through three pairs of specialized inflow tracts termed ostia. Upon contraction of the heart, hemolymph is then forced anteriorly through the part of the tube termed the aorta, and is expelled close to the brain (Figure 1A; Rizki, 1978).

Figure 1. Markers of inflow tract fate in the Drosophila embryonic heart.

Figure 1

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A. The embryonic dorsal vessel comprises a posterior heart region and an anterior aorta, each of which contain Tin cells and Svp cells. Heart fate is specified by abdA expression, and the part of the aorta that contains Svp cells is mostly specified by Ubx expression. Hemolymph enters the heart though three pairs of ostia (dashed arrows). Ostia are formed from Svp cells, and in the heart these cells become elongated. Image adapted from Bryantsev and Cripps (2009). B: abdominal-A protein (ABDA, Green) accumulates in the heart cells, but not the aorta. Arrows indicate the locations of ostia. MEF2 (Red) labels dorsal vessel nuclei and nuclei of adjacent skeletal muscles. C: Cardiac cells and a subset of pericardial cells accumulate the Tinman protein (Tin, Red) or they express svp visualized as βgal accumulation from a svp-lacZ enhancer trap (Green, arrows). A regular pattern is displayed, as four pairs of Tin myocardial cells alternate with two pairs of Svp cells. D: wingless (wg, Red, arrows) is expressed in the ostia which are the Svp expressing cells in the wild-type heart. E: CG8147 is expressed in the ostia. F: A wild-type dorsal vessel stained for the muscle structural protein Tropomyosin (Tm); note that the ostial cells (Arrows) are elongated compared to the adjacent myocardial Tin cells. G: Measurement of the axial ratios of myocardial Tin cells and Svp expressing ostia, underlining the differences in cell shapes. ****, p<0.0001, using Welch’s t-test. A-E: Stage 16 embryos, dorsal views. Bar, 20μm.

Over the past several years, significant effort has been directed at identifying the genes expressed in the dorsal vessel, and characterizing how they impact cardiac development and function. It is now known that the bulk of the cardiac tube arises from approximately ten trunk segments. In seven of these segments, the muscular component of the cardiac tube comprises alternating sets of cells that accumulate either the NK homeodomain transcription factor Tinman (Tin), or the single Drosophila COUP-TFII ortholog Seven-up (Svp). In each segment, there are four pairs of Tin cells, followed by two pairs of Svp cells (Bodmer and Frasch, 1999). Superimposed upon this segmental pattern is an axial component: the three most posterior segments of the cardiac tube accumulate the homeotic transcription factor abdominal-A (ABD-A), and the four segments anterior to this express the homeotic gene Ultrabithorax (Ubx) (Lovato et al., 2002; Lo et al., 2002; Ponzielli et al., 2002).

The expression of all of these regulatory genes is profoundly important for cardiac cell fate decisions: tin function is required for the differentiation of many aspects of the mature cardiac tube (Zaffran et al., 2006); abd-A is required and sufficient for specification of the posterior heart region (Lovato et al., 2002; Lo et al., 2002; Ponzielli et al., 2002); and Ubx is required for correct specification of Svp cells in the aorta, which is a region of the dorsal vessel that is narrower than the heart but that lacks inflow tracts in the embryo (Molina and Cripps, 2001; Perrin et al., 2004; Ryan et al., 2005). Importantly, the conjunction of svp and abd-A in the heart results in the formation of the ostia, or inflow tracts (Molina and Cripps, 2001; Lovato et al., 2002; Ponzielli et al., 2002), and svp is required for ostia formation in the embryo (Ponzielli et al., 2002). These studies expose a commonality between vertebrate and invertebrate mechanisms of inflow tract specification, since both COUP-TFII and svp are required in their respective organisms for inflow tract formation.

The specification and formation of ostia in the Drosophila heart probably arises from the combined action of a number of factors. Since abd-A is expressed in myocardial (Tin) cells of the cardiac tube, abd-A cannot be sufficient for ostium formation. Similarly, Svp cells are also found in the aorta, but the aorta does not contain ostia at the embryonic stage. In addition to – and in response to – svp and abd-A, the ostia cells of the heart also express the WNT signaling molecule Wingless, and Wg is not expressed by embryonic Svp cells in the aorta (Lo et al., 2002) (Figure 1D). Thus, one straightforward model is that ostia form in response to Wg accumulation, and svp and abd-A act simply to induce wg expression at the correct time and place.

Here, we have tested this and related models to define the cellular events that control inflow tract formation in the Drosophila heart. We generate criteria to define the formation of ostia, we confirm that svp and abd-A are required for ostia formation, and we show that wg is required for normal inflow tract development. We demonstrate that Wg produced by the ostia cells functions in an autocrine manner, and that the canonical Wg signaling pathway is required for normal inflow tract formation. Using ectopic expression studies, we find that expression of wg in the Svp cells of the aorta does not result in ostia formation, nor does direct activation of canonical Wg signaling in Svp cells of the aorta result in appearance of ectopic ostia, indicating that wg is required but not sufficient for the formation of these structures. By contrast, expression of abd-A in the Svp cells of the aorta can induce the appearance of some markers of ostia fate, and expression of abd-A throughout the cardiac tube induces ectopic ostium formation more robustly. Our studies therefore provide new insight into mechanisms of inflow tract formation, and underline the contributions of Hox factors and Wg signaling in this process. We anticipate that the relationships that we have uncovered will help to provide a framework for understanding the mechanisms of inflow tract formation in the mammalian cardiac system.

Materials and Methods

Drosophila stocks and crosses

All Drosophila symbols and genotypes are as described in Flybase (http://flybase.org). The following stocks and mutants were obtained from the Bloomington Drosophila Stock Center: UAS-pand∂N, UAS-wg, UAS-lacZ, 24B-Gal4, twi-Gal4, wgl–12. UAS-wg RNAi was from the Vienna Drosophila RNAi Center. UAS-abd-A was provided by Dr Alan Michelson (Harvard University; Michelson, 1994); svp1 was provided by Dr James Skeath (Washington University, St. Louis). To identify homozygous mutants from amongst lethal stocks, we used CyO, Cy elav-lacZ for wg mutants and TM3, Sb ftz-lacZ for svp mutants; both of these balancers were from Bloomington. We used morphology of the gut to identify abdA homozygous mutants.

P-element mediated germline transformation was carried out according to the methods of Rubin and Spradling (1982). Embryos of the genotype y w were injected with a mixture of the transforming plasmid and the transposase-encoding plasmid Delta2-3 (Robertson et al., 1988). G0 larvae were collected and adults crossed to y w for identification of transgenic animals in the G1 generation, based upon rescue of the white-eye phenotype. Standard genetic crosses were carried out to generate homozygous stocks for each insert.

Antibody staining and in situ hybridization

Antibody staining was carried out as described by Patel (1994). Antibodies used were rat anti-Tropomyosin (1:250; Peckham et al., 1993; AbCam Immunochemicals, MA); rabbit anti-MHC (1:500; Kiehart and Feghali, 1986); rabbit anti-MEF2 (1:1000; Lilly et al., 1995); mouse anti-Wg (1:10; Developmental Studies Hybridoma Bank, University of Iowa); rat anti-ABD-A (1:250; Macias et al., 1990); mouse anti-βGal (1:1000, Promega Corp.); and rabbit anti-Tin (1:500; Yin et al., 1997). Secondary antibodies were either: Biotinylated used at 1:1000, and detected using the Vectastain Elite kit (Vector Laboratories); or conjugated to Alexa fluors (Molecular Probes, Inc.), used at 1:2000, and detected by confocal microscopy on a Zeiss LSM780 system.

In situ hybridization was according to the methods of O’Neill and Bier (1994). A Digoxigenin-labeled wg probe was generated as described in Verzi et al. (2002), using the Roche RNA Labeling Kit (Roche). Incubation with anti-Digoxigenin (Roche) was extended to overnight, and hybridized probe was detected using NBT/BCIP. The lacZ probe was described in Ryan et al (2007). A probe for CG8147 was synthesized from the cDNA plasmid pOT2/SD27015, which was cleaved with PstI and probe synthesized using SP6 RNA polymerase. The plasmid was obtained from the Drosophila Genomics Resource Center (DGRC; https://dgrc.cgb.indiana.edu/).

Stained samples to be documented as whole-mounts were cleared in 80% (v/v) glycerol/1XPBS and viewed at 200X magnification for photography; samples to be filleted were dissected in 80% v/v glycerol/1XPBS and viewed at 630X magnification for photography. Samples at the higher magnification were photographed at multiple focal planes, and the resulting images were combined using Adobe Photoshop in order to generate a photomontage that provided a clear view of the structure of the dorsal vessel throughout its length.

DNA methods

Molecular cloning techniques were as described (Sambrook et al., 1998). To generate the plasmid used to create Gal4 transgenic lines, the Svp Cardiac Enhancer from the svp gene (Ryan et al., 2007) was cloned into pUASt (Brand and Perrimon, 1993). A verified clone was used for microinjection as described above.

Measurement of cardial cell dimensions

To determine the axial ratios of Svp cells and Tin cells in our experiments, samples were first stained for accumulation of either MHC or Tropomyosin. Individual stained embryos were then filleted using a fine tungsten needle, and the dorsal vessel was visualized using at 630x magnification. Images of the hearts were taken, often at several different focal planes to ensure that each cell was clearly visible. The maximum width and maximum length of each cardial cell was measured using ImageJ. Axial ratios were then calculated by dividing the length by the width to generate a unitless ratio. Averages of the axial ratios were calculated and used in the graphs. Welch’s t-tests were used to determine if different data sets differed significantly.

Results

Genetic markers of ostia fate in the Drosophila embryonic heart

As discussed in the introduction, the embryonic cardiac inflow tracts arise in the posterior region of the dorsal vessel termed the heart proper. This region is specified by the Hox gene abdA (Figure 1B), and ostia arise from cells in the heart that also express svp (Figure 1C) and wg (Figure 1D). We recently identified, from high throughput expression studies by Tomancek et al (2002), CG8147 as being expressed in the ostia cells of the dorsal vessel (Figure 1E). Early in development, CG8147 is expressed in cells including the trunk mesoderm and the anterior and posterior endoderm, but by stage 16 the expression is in the fat body and ostia cells in the heart. CG8147 encodes an alkaline phosphatase ortholog: to this point we have not investigated its role in heart development or function, however it is an effective marker of ostia fate.

Ostia cells are also characterized by their elongated shape as compared to neighboring Tin-positive myocardial cells (Figure 1F), and this can be quantified as a difference in axial ratios between the two cell types (Figure 1G). In summary, ostia cells express a unique set of regulatory factors and are distinguishable from non-ostia cells based upon morphological and gene expression criteria. To date, wg and CG8147 expression, plus ostia cell shape, are the only criteria that distinguish ostia cells from other myocardial cells.

abdA, svp, and wg mutants show reduction in ostia size

Based upon these new markers and criteria for ostia formation, we first wished to determine how loss of abdA or svp function would impact these markers. As previously reported by Ponzielli et al (2002), both abdA and svp are essential for the development of inflow tracts. We support and expand upon these studies, by analyzing CG8147 expression and ostia axial ratio in these mutants. For abdA, there was a reduction in the size of the heart lumen, and Svp cells in the heart did not elongate into the shape characteristic of ostia (Figure 2A, H). Moreover, there was a strong reduction or loss of CG8147 expression in the hearts of abdA mutants (Figure 2B).

Figure 2. Requirements for abdA, svp and wg in inflow tract development.

Figure 2

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Mutants or knockdowns for these three genes were assessed for ostia formation. A,B: In abd-A mutants there was a reduction in ostia cell size (ostia sets labeled 01-3) (A), and a loss of CG8147 expression (B). C, D: svp mutants also showed a reduction in ostia size (01-3)(C), and a loss of CG8147 expression in ostia (D). E: Expression of a wg-RNAi construct in the Svp cells caused a reduction in ostia size, albeit to a lesser extent that that observed in abdA or svp mutants. F: CG8147 expression in ostia was still observed in wg-RNAi hearts. G: Wingless temperature sensitive mutants (wgts) showed variable phenotypes, with some ostia cells showing reduced axial ratios, and other cells appearing more normal. H: Measurements of ostia cell axial ratio for the genotypes above. A Welch’s t-test was performed: *, p<0.05; **, p<0.01; ****, p<0.0001. Horizontal line indicates axial ratio of Tin cells in control animals. A–G: Stage 16 embryos, dorsal view. Some panels are montages of a single sample photographed at slightly different focal planes in order to clearly visualize the entire heart. A, C, E, G: Stained for the accumulation of the muscle structural proteins Myosin heavy-chain or Tropomyosin. B, D, F: CG8147 in situ hybridization stain. Bar, 20μm.

For svp, homozygous mutants formed a heart lumen, but there was once again no evidence of elongation of Svp cells into ostia (Figure 2C, H), and there was a complete loss of CG8147 expression in the heart (Figure 2D).

We next investigated the role of wg in ostia formation, by using a svp-Gal4 driver to express an RNAi targeting wg transcripts. Interestingly, the effects upon ostia formation of reducing Wg function in the heart were less clear cut than in mutants for abdA or svp, although a requirement for Wg in the development of the inflow tracts was still apparent. We used a svp-Gal4 driver to express an RNAi targeting wg transcripts, and observed a significant reduction in ostia axial ratio (Figure 2E, H), and a reduction (but not total loss) of CG8147 expression (Figure 2F). This result might reflect only a partial role for Wg in controlling inflow tract formation; alternatively, it is possible that there was incomplete knockdown of wg function by using the RNAi. To address this, we also reduced Wg function in the heart using a temperature-sensitive wg allele named wgts. We maintained mutant embryos at the permissive temperature until heart specification had occurred, and then shifted the embryos up to a restrictive temperature for the remainder of embryonic development. Embryos were stained for accumulation of Myosin heavy chain, and analyzed for ostia axial ratios. Once again, we observed an incomplete effect upon ostia formation: many Svp cells were reduced in size, however others were close to normal (Figure 2G). When we calculated axial ratios, we found that in the wgts mutants the axial ratio was significantly reduced (Figure 2H).

In summary, the regulatory genes abdA and svp are essential for the formation of inflow tracts, and wg has a partial but not complete requirement for the formation of these structures. Analysis of non-ostia Tin cells in the heart showed no significant differences between control and mutant lines, indicating that in the mutants and knockdowns there was not a global impact upon myocardial cell size (data not shown).

The Wg pathway signals in an autocrine fashion in the inflow tracts

In order to define how Wg signaling contributes to inflow tract formation, we next sought to determine if the signaling was autocrine or paracrine. We initially analyzed the expression of factors known to transduce the canonical Wg signaling pathway, to determine if they were differentially expressed in cells of the dorsal vessel. For the intracellular component Armadillo (Arm), we observed strong expression along the entire length of the dorsal vessel, with some enrichment on the luminal side of both heart and aorta cells (Figure 3A, B). For the Wg receptors Fz and Fz2, we were not able to detect significant accumulation of Fz in the dorsal vessel, although based upon results presented below, we nevertheless believe that there is low-level accumulation of Fz in the dorsal vessel that is below our levels of detection. For Fz2, we observed low levels of protein throughout the cardiac tube (Figure 3C,D). Together, these data indicated that most cardial cells of the dorsal vessel have the potential to respond to Wg signaling, but their expression patterns did not instruct us as to which cells might transduce the signal for ostia formation.

Figure 3. Wg signaling in the inflow tracts is autocrine.

Figure 3

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A–D: Expression of Wg signaling pathway components in the stage 16 dorsal vessel. A: Armadillo (Arm, Red) is detected throughout the dorsal vessel. B: Merge of MEF-2 accumulation (Green) and Armadillo accumulation (Red). C: Frizzled 2 (Fz2, Red) expression is weak but detectable in the aorta and heart. D: Merge of MEF-2 accumulation (Green) and Fz2 accumulation (Red). E: Schematic of 6THlacZ. Pangolin (Pan)/dTCF and Helper sites are fused to a lacZ gene to report the activation of the Wg signaling pathway. F: 6THlacZ embryos were stained using in situ hybridization for lacZ expression. Expression was observed in the ostia and not in the Tin expressing cells that surround them, indicating that the wg expression in the ostia cells results in autocrine signaling. Bar, 20μm.

To gain more direct insight into the target cells for Wg signaling in the heart, we analyzed expression of a Wg reporter termed 6TH-lacZ, in which several Pan binding sites are fused to a minimal promoter-lacZ construct (Figure 3E; Chang et al 2008). Since Pan activation is the downstream effect of canonical Wg signaling (Brunner et al 1997; van de Wetering et al 1997), lacZ expression labels cardiac cells receiving and transducing the Wg signal. Importantly, activity of this reporter does not indicate expression of pan, but instead its activation by the Wg signaling pathway. When we stained the 6TH-lacZ embryos with a lacZ antisense riboprobe, we observed strong and highly specific lacZ expression in the ostia cells. There was no lacZ expression in the neighboring Tin-positive myocardial cells (Figure 3F).

These data indicate that whereas multiple cardial cells might be able to respond to Wg signaling based upon expression of Wg signaling pathway components, the ostia cells transduce the signal into Pan-dependent gene expression. Overall, we conclude that the Wg signal most likely functions in an autocrine manner to influence inflow tract development.

The canonical Wg signaling pathway is partially required for ostia development

Having demonstrated that the canonical Wg signaling pathway is activated in the ostia cells, we next wanted to determine whether this pathway is required for inflow tract development. To achieve this, we inactivated the pathway either by expression in the ostia cells of a dominant negative isoform of Pan, or by knocking down expression of Wg receptors. In parallel, we used 6TH-lacZ expression in the ostia as an indication of the efficacy of interfering with the pathway. 6TH-lacZ expression in control animals was normal (Figure 4A), as was expression of CG8147 (Figure 4B).

Figure 4. The canonical Wg signaling pathway is partially required for inflow tract development.

Figure 4

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A: 6THlacZ expression in the ostia of control embryos. B: CG8147 expression in the ostia of control embryos. C, D: Expression in the Svp cells of a dominant-negative isoform of Pan resulted in a loss of expression in the ostia for both lacZ (C) and CG8147 (D). Knockdown of Fz resulted in loss of expression for 6THlacZ (E), but not for CG8147 (F). Knockdown of Fz2 resulted in a loss of expression for 6THlacZ (G) and CG8147 (H). A–H: Stage 16 embryos, dorsal views. I: Quantification of the ostia cell axial ratio measured from Tropomyosin stained embryos. Horizontal line indicates axial ratio of Tin cells in control animals. Welch’s t-test was used to generate statistics. *, p<0.05. Bar, 20μm

When we expressed panDeltaN in the Svp cells, we observed a loss of 6TH-lacZ expression, indicating that the canonical pathway had been inhibited (Figure 4C). In addition, we observed a failure of CG8147 expression (Figure 4D). When the svp>panDeltaN animals were stained for accumulation of Tm, the ostia cell axial ratio was significantly reduced (Figure 4I).

When we knocked down expression of Fz or Fz2 in the ostia, there was also a partial loss of ostia cell fate. Knockdown of expression of either receptor caused a loss of 6TH-lacZ expression (Figure 4E, G), a reduction in CG8147 expression (Figure 4F, H), and a significantly reduced axial ratio compared to controls (Figure 4I). Once again, analysis of non-ostia Tin cells showed no significant differences between control and mutant lines, indicating that in the mutants and knockdowns there was not a global impact upon myocardial cell size (data not shown).

We can make a number of conclusions from these observations. Firstly, Fz and Fz2 are each necessary to fully transduce the autocrine Wg signal in the ostia. Secondly, the Wg pathway is critical for full CG8147 expression, where the partial expression of CG8147 in the fz knockdown could result from residual fz and fz2 expression in this knockdown. Thirdly, CG8147 expression is a more sensitive read-out of Wg signaling than is 6TH-lacZ. Overall, it is clear that Wg signaling in the ostia cells is necessary for their normal development into inflow tracts.

Wg signaling is not sufficient for ostia development

Having demonstrated a requirement for Wg signaling for complete ostia formation, we next sought to determine what genes are sufficient to form cells characteristic of inflow tracts. Inflow tracts do not form in the embryonic/larval aorta even though there are Svp cells in the aorta (Molina and Cripps, 2001). Therefore expression of svp itself, although essential for ostia formation, is not sufficient for ostia development. Aorta Svp cells do not express wg (Lo et al 2002), nor do they express abd-A (Lovato et al 2002; Lo et al 2002; Ponzielli et al 2002), thus expression of either of these factors might promote ostia formation.

To determine if Wg signaling could act upon Svp cell fate to promote ostia formation, we expressed wg, or activated the Wg signaling pathway, in the aorta Svp cells. We once again monitored ostia fate in experimental embryos by analyzing 6TH-lacZ expression, CG8147 expression, and cell axial ratios in experimental samples, that were compared to controls (shown in Figure 5 A–C).

Figure 5. Wg signaling is not sufficient for inflow tract development.

Figure 5

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A, D, G: lacZ in situ hybridization stain. B, E, H: CG8147 in situ hybridization stain. C, F, I: Tropomyosin (Tm) antibody stains. A: Control 6THlacZ line shows lacZ expression in the Svp cells of the ostia (arrows) but not in the aorta. B: Control embryo showing CG8147 expression in ostia cells. C: Control embryo stained for Tropomyosin (Tm). D–I: When members of the Wg signaling pathway were expressed in all Svp cells, there was ectopic expression of the 6THlacZ reporter in the aorta, but there was not ectopic expression of CG8147 in the aorta, nor were there morphological changes to the Svp cells in the aorta. Arrows indicate ostia cells in the heart, and arrowheads indicate Svp cells in the aorta. D–F: Ectopic expression of wg resulted in expansion of 6THlacZ expression into the aorta cells (D), but no expansion of CG8147 expression (E) and no ectopic ostia formation (F). Note that Svp cells in the aorta (arrowheads) have similar axial ratios to the neighboring myocardial Tin cells when Wg is expressed in the Svp cells. G–I: Expression of armS10 in the Svp cells of the aorta also resulted in expansion of 6THlacZ expression into the aorta cells (G), but no expansion of CG8147 expression (H) and no ectopic ostia formation (I). J: Graph showing Svp cell axial ratios measured from Tm stained embryos of the indicated genotypes. Welch’s t-test was used to generate statistics. **, p<0.01. NS, not significant. A–I: Stage 16 embryos, dorsal view. WT, wild-type. Bar, 20μm

When we expressed wg in the aorta Svp cells by crossing svp-Gal4 with UAS-wg, there was ectopic activation of the Wg signaling pathway based upon robust 6TH-lacZ expression in the four sets of aorta Svp cells (Figure 5D). However, there was no expansion of CG8147 expression into the aorta (Figure 5E), nor was there a detectable increase in Svp cell axial ratio compared to controls (Figure 5F, J). We complemented these results by expressing in the Svp cells an activated form of Arm termed ArmS10, that constitutively activates the canonical Wg pathway. Once again, there was clear evidence of pathway activation in the aorta Svp cells (Figure 5G). Although there was no expansion of CG8147 expression into the aorta (Figure 5H), there was a small but significant increase in Svp cell axial ratio compared to controls (Figure 5I, J). We conclude from these experiments that activation of the canonical Wg pathway in Svp cells is not sufficient to direct the formation of complete ostia, but there might be an intermediate fate whereby ostia cell size can increase slightly without expression of CG8147.

abd-A promotes ectopic ostia formation

Since abd-A promotes the specification of the posterior heart that includes the inflow tracts, we determined the degree to which expression of abd-A in the aorta could promote inflow tract fate. Prior studies had indicated that expression of abd-A throughout the cardiac tube could promote ectopic heart and inflow tract fate, based upon ectopic expression of wg and alterations in the sizes of aorta Svp cells (Lovato et al 2002; Ponzielli et al 2002; Lo et al 2002; Perrin et al 2004; Ryan et al 2005). However, these studies did not evaluate CG8147 expression nor Svp cell axial ratios, nor did they assess the effects of ectopic abd-A expression in just the aorta Svp cells.

Therefore, we used svp-Gal4 to express abd-A in the aorta Svp cells, and monitored wg and CG8147 expression, and Svp cell axial ratios. Compared to controls (Figure 6A–C), we observed ectopic wg expression and ectopic CG8147 expression in the four sets of aorta Svp cells of experimental animals (Figure 6D, E). In addition, there was a significant increase in the axial ratios of the aorta Svp cells (Figure 6F, J), although the increased axial ratio was not as great as that observed for wild-type ostia (Figure 6J).

Figure 6. abd-A promotes ectopic ostia formation more effectively when all cardiac cells are transformed.

Figure 6

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Embryos were stained for wg transcripts (A, D, G), CG8147 transcripts (B, E, H), and Tropomyosin accumulation (C, F, I). Arrows indicate ostia in the heart region, and arrowheads indicate Svp cells within the aorta. D–F: When abd-A was expressed in all the cardiac Svp cells there was an expansion of wg (D) and CG8147 (E) expression into the aorta. F: The MHC stain showed small increases in the size of the Svp cells in the aorta. G–I: Using 24B-Gal4+twist-Gal4 as drivers, there was expanded expression of wg (G) and CG8147 (H) into the Svp cells of the aorta, and there was formation of ectopic ostia based upon accumulation of Tm (I). J: Graph reporting the Svp cell axial ratio measured from Tm stained embryos. Welch’s t-test was used. ***, p<0.001; ****, p<0.0001. A–I: Stage 16 embryos, dorsal views. A, B, D, E, G, H: Bar, 20μm. C, F, I: Bar, 15 μm.

In parallel, we also expressed abd-A throughout the cardiac tube and assessed the effects upon aorta Svp cell fate. Note that since abd-A promotes Svp cell fate in the cardiac tube, there are ten sets of Svp cells in these animals (Perrin et al 2004; Ryan et al 2005). Again, we observed ectopic wg and CG8147 expression in these animals (Figure 6G, H), and a further increase in the sizes of the Svp cells (Figure 6 I, J).

We conclude from these observations that although svp and wg are required for normal ostia formation, these factors either alone or in combination cannot program inflow tract fate. Instead, they function in collaboration with abd-A to promote the changes in morphology and gene expression that are characteristic of the inflow tracts.

Discussion

Cardiac development comprises both the initial specification of heart-forming cells, as well as the specification and differentiation of distinct cell types within the cardiac tissue. Understanding the genes and molecular mechanisms that contribute to cell type specification is critical to understanding disease mechanisms that impact specific cardiac cell sub-types.

Here, we have used the Drosophila system to define the mechanisms that contribute to formation of the cardiac inflow tracts, termed ostia. We find that a number of distinct regulatory molecules are required for normal inflow tract development, and it is interesting to note evolutionary conservation in this process: just as svp is required for inflow tract formation in Drosophila, a mammalian ortholog termed COUP-TFII is expressed in the developing atria and is required for proper atrium formation in mice (Pereira et al., 1999; Petit et al., 2007; Wu et al 2013).

Moreover, roles in heart development for vertebrate homeotic genes have recently been identified: cardiac defects are observed in mouse knockouts of Hoxa1 (Makki and Capecchi, 2012), and combined deletion of both HoxA and HoxB complexes results in cardiac looping defects (Soshnikova et al 2013). These studies parallel the demonstration by ourselves and others that homeotic genes pattern the cardiac tube (Lovato et al 2002; Lo et al 2002; Ponzielli et al 2002), and our demonstration in this paper that abdA is required for morphogenesis of the inflow tract.

A role for WNT molecules, the mammalian orthologs of Wg, in cardiac development is also becoming more apparent. Whereas WNTs can act to suppress cardiogenesis (Marvin et al., 2001; Schneider and Mercola, 2001), this activity follows an earlier requirement for WNT signaling in cardiac specification (Naito et al., 2006; reviewed in Cohen et al., 2008). These latter findings mirror the early requirement for wg in cardiac specification in Drosophila (Wu et al., 1995), but do not address a later role in the formation of specific cell types within the developing vertebrate heart. Along these lines, Hurlstone et al. (2003) found that components of the canonical Wnt signaling pathway act in zebrafish to mediate cardiac valve development. Whether molecular mechanisms of valve development and of ostia development are conserved remains to be determined. The recent studies of Wu et al (2012) also identified WNT2 expression as being higher in the atria versus ventricles, and reduced in COUP-TFII knockout hearts. In addition, WNT2-expressing cells were shown to contribute to the developing mouse inflow tract (Peng et al 2013). These data support a conserved role for Wg/WNT proteins in controlling the development of the cardiac inflow tracts.

While our studies demonstrate the importance of normal Wg signaling to ostia formation, there are still details of ostia specification and differentiation that remain to be resolved. A recent study described enriched expression of a Wg paralog, DWnt4, in the ostia cells (Tauc et al 2012), and it is likely that this factor also contributes to Wg signaling in the ostia and potentially to ostia formation. Moreover, since canonical Wg signaling is not sufficient for ostia specification, factors in addition to Wg signaling are necessary for ostia formation, and our studies place these yet-to-be defined factors genetically downstream of abd-A. Clearly, the formation of the Drosophila inflow tract arises from a complex genetic pathway. Based upon the demonstrated commonalities in inflow tract development between Drosophila and mammals, dissection of this process in a model system such as Drosophila is likely to provide an important framework for understanding inflow tract formation in higher animals.

Highlights.

  • The Drosophila heart has inflow tracts, termed ostia, that express Wg and CG8147, and that are elongated relative to neighbouring cardial cells

  • Ostia formation depends upon autocrine Wingless signaling

  • The canonical Wg signaling pathway is necessary but not sufficient for ostia formation

  • Wg signaling also requires Hox gene expression for ostia to form

Acknowledgments

We are very grateful to the individuals who provided critical reagents for this work: Drs Manfred Frasch, Daniel Kiehart, Alan Michelson and James Skeath. The project was funded by HL080545 from the NIH/NHLBI and 14GRNT20490250 from the American Heart Association Southwest Affiliate, awarded to RMC; and by HL054732 from the NIH/NHLBI awarded to RB. GVT was supported by ASERT/IRACDA grant K12 GM088021; DN was supported by IMSD grant R25 GM060201. We acknowledge technical support from the Department of Biology’s Molecular Biology Facility, supported by P20 GM103452 from the Institute Development Award (IDeA) Program of NIGMS.

Footnotes

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References

  1. Akasaka T, Klinedinst S, Ocorr K, Bustamante EL, Kim SK, Bodmer R. The ATP-sensitive potassium (KATP) channel-encoded dSUR gene is required for Drosophila heart function and is regulated by Tinman. Proc Natl Acad Sci USA. 2006;103:11999–12004. doi: 10.1073/pnas.0603098103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alvarez AD, Shi WS, Wilson BA, Skeath JB. pannier and pointedP2 act sequentially to regulate Drosophila development. Development. 2003;130:3015–3026. doi: 10.1242/dev.00488. [DOI] [PubMed] [Google Scholar]
  3. Azpiazu N, Frasch M. tinman and bagpipe: Two homeobox genes that determine cell fates in the dorsal mesoderm of Drosophila. Genes and Development. 1993;7:1325–1340. doi: 10.1101/gad.7.7b.1325. [DOI] [PubMed] [Google Scholar]
  4. Bejsovec A, Arias AM. Roles of Wingless in pattering the larval epidermis of Drosophila. Development. 1991;113:471–485. doi: 10.1242/dev.113.2.471. [DOI] [PubMed] [Google Scholar]
  5. Bodmer R. The gene tinman is required for specification of the heart and visceral muscles in Drosophila. Development. 1993;118:719–729. doi: 10.1242/dev.118.3.719. [DOI] [PubMed] [Google Scholar]
  6. Bodmer R, Frasch M. Genetic determination of Drosophila heart development. In: Harvey RP, Rosenthal N, editors. Heart Development. Academic Press; San Diego, CA: 1999. [Google Scholar]
  7. Brand T. Heart development: molecular insights into cardiac specification and early morphogenesis. Developmental Biology. 2003;258:1–19. doi: 10.1016/s0012-1606(03)00112-x. [DOI] [PubMed] [Google Scholar]
  8. Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118:401–415. doi: 10.1242/dev.118.2.401. [DOI] [PubMed] [Google Scholar]
  9. Brunner E, Peter O, Schweizer L, Basler K. pangolin encodes a Lef-1 homologue that acts downstream of Armadillo to transduce the Wingless signal in Drosophila. Nature. 1997;385:829–833. doi: 10.1038/385829a0. [DOI] [PubMed] [Google Scholar]
  10. Bryantsev AL, Cripps RM. Cardiac gene regulatory networks in Drosophila. Biochim Biophys Acta: Gene Reg Mech. 2009;1789:343–353. doi: 10.1016/j.bbagrm.2008.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cadigan KM, Fish MP, Rulifson EJ, Nusse R. Wingless repression of Drosophila frizzled2 expression shapes the wingless morphogen gradient in the wing. Cell. 1998;93:767–777. doi: 10.1016/s0092-8674(00)81438-5. [DOI] [PubMed] [Google Scholar]
  12. Chang MV, Chang JL, Gangopadhyay A, Sherarer A, Cadigan KM. Activation of Wingles targets requires bipartite recognition of DNA by TCF. Curr Biol. 2008;18:1877–1881. doi: 10.1016/j.cub.2008.10.047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Christianson A, Howson CP, Modell B. Global report on birth defects: the hidden toll of dying and disabled children. White Plains, NY: March of Dimes Birth Defects Foundation; 2006. [Google Scholar]
  14. Cohen ED, Tian Y, Morrisey EE. Wnt signaling: an essential regulator of cardiovascular differentiation, morphogenesis and progenitor self-renewal. Development. 2008;135:789–798. doi: 10.1242/dev.016865. [DOI] [PubMed] [Google Scholar]
  15. Combs MD, Yutzey KE. Heart valve development: regulatory networks in development and disease. Circ Res. 2009;105:408–421. doi: 10.1161/CIRCRESAHA.109.201566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cripps RM, Olson EN. Control of cardiac development by an evolutionarily conserved transcriptional network. Developmental Biology. 2002;246:14–28. doi: 10.1006/dbio.2002.0666. [DOI] [PubMed] [Google Scholar]
  17. Garg V, Kathiriyra IS, Barnes R, Schluterman MK, King IN, Butler CA, Rothrock CR, Eapen RS, Hirayama-Yamada K, Joo K, Matsuoka R, Cohen JC, Srivastava D. GATA4 mutations cause human congenital heart defects and reveal interactions with TBX5. Nature. 2003;424:443–447. doi: 10.1038/nature01827. [DOI] [PubMed] [Google Scholar]
  18. Garg V, Kathiriya IS, Barnes R, Schluterman MK, King IN, Butler CA, Rothrock CR, Eapen RS, Hirayama-Yamada K, Joo K, Matsuoka R, Cohen JC, Srivastava D. Mutations in NOTCH1 cause aortic valve disease. Nature. 2005;437:270–274. doi: 10.1038/nature03940. [DOI] [PubMed] [Google Scholar]
  19. Gajewski K, Fossett N, Molkentin JD, Schulz RA. The zinc finger proteins Pannier and GATA4 function as cardiogenic factors in Drosophila. Development. 1999;126:5679–5688. doi: 10.1242/dev.126.24.5679. [DOI] [PubMed] [Google Scholar]
  20. Gajewski K, Choi CY, Kim Y, Schulz RA. Genetically distinct cardial cells within the Drosophila dorsal vessel. Genesis. 2000;28:36–43. doi: 10.1002/1526-968x(200009)28:1<36::aid-gene50>3.0.co;2-4. [DOI] [PubMed] [Google Scholar]
  21. Gonzalez P, Garcia-Castro M, Reguero JR, Batalla A, Ordonez AG, Palop RL, Lozano I, Montes M, Alvarez V, Coto E. The pro279leu variant in the transcription factor MEF2A is associated with myocardial infarction. (Letter) J Med Genet. 2006;43:167–169. doi: 10.1136/jmg.2005.035071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Grumbling G, Strelets V The FlyBase Consortium. Flybase: anatomical data, images, and queries. Nucleic Acids Res. 2006;34:D484–D488. doi: 10.1093/nar/gkj068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hendren JD, Shah AP, Arguelles AM, Cripps RM. Cardiac expression of the Drosophila Sulphonylurea receptor gene is regulated by an intron enhancer dependent upon the NK homeodomain transcription factor Tinman. Mech Dev. 2007;124:416–426. doi: 10.1016/j.mod.2007.03.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hurlstone AF, Haramis AP, Wienholds E, Begthel H, Korving J, VanEeden F, Cuppen E, Zivkovic D, Plasterk RH, Clevers H. The Wnt/beta-catenin pathway regulates cardiac valve formation. Nature. 2003;425:633–637. doi: 10.1038/nature02028. [DOI] [PubMed] [Google Scholar]
  25. Kiehart DP, Feghali R. Cytoplasmic myosin from Drosophila melanogaster. J Cell Biol. 1986;103:1517–1525. doi: 10.1083/jcb.103.4.1517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lilly B, Zhao B, Ranganayakulu G, Paterson BM, Schulz RA, Olson EN. Requirement of MADS domain transcription factor D-MEF2 for muscle formation in Drosophila. Science. 1995;267:688–93. doi: 10.1126/science.7839146. [DOI] [PubMed] [Google Scholar]
  27. Lo P, Frasch M. A role for the COUP-TF-related gene seven-up in the diversification of cardioblast identites in the dorsal vessel of Drosophila. Mechanisms of Development. 2001;104:49–60. doi: 10.1016/s0925-4773(01)00361-6. [DOI] [PubMed] [Google Scholar]
  28. Lo P, Skeath J, Gajewski K, Schulz RA, Frasch M. Homeotic genes autonomously specify the anteroposterior subdivision of the Drosophila dorsal vessel into aorta and heart. Developmental Biology. 2002;251:307–319. doi: 10.1006/dbio.2002.0839. [DOI] [PubMed] [Google Scholar]
  29. Lo PCH, Frasch M. Establishing A-P polarity in the embryonic heart tube: a conserved function of Hox genes in Drosophila and vertebrates ? Trends Cardiovasc Med. 2003;13:182–187. doi: 10.1016/s1050-1738(03)00074-4. [DOI] [PubMed] [Google Scholar]
  30. Lovato TL, Nguyen TP, Molina MR, Cripps RM. The Hox gene abdominal-A specifies heart cell fate in the Drosophila dorsal vessel. Development. 2002;129:5019–5027. doi: 10.1242/dev.129.21.5019. [DOI] [PubMed] [Google Scholar]
  31. Macias A, Casanova J, Morata G. Expression and regulation of the abd-A gene of Drosophila. Development. 1990;110:1197–1207. doi: 10.1242/dev.110.4.1197. [DOI] [PubMed] [Google Scholar]
  32. Makki N, Capecchi MR. Cardiovascular defects in a mouse model of HOXA1 syndrome. Hum Mol Genet. 2012;21:26–31. doi: 10.1093/hmg/ddr434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Marvin MJ, Di Rocco G, Gardiner A, Bush SM, Lassar AB. Inhibition of Wnt activity induces heart formation from posterior mesoderm. Genes Dev. 2001;15:316–327. doi: 10.1101/gad.855501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McCulley DJ, Black BL. Transcription factor pathways and congenital heart disease. Curr Topics Dev Biol. 2012;100:253–277. doi: 10.1016/B978-0-12-387786-4.00008-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Michelson AM. Muscle pattern diversification in Drosophila is determined by the autonomous function of homeotic genes in the embryonic mesoderm. Development. 1994;120:755–768. doi: 10.1242/dev.120.4.755. [DOI] [PubMed] [Google Scholar]
  36. Molina MR, Cripps RM. Ostia, the inflow tracts of the Drosophila heart, develop from a genetically distinct subset of cardial cells. Mech Dev. 2001;109:51–59. doi: 10.1016/s0925-4773(01)00509-3. [DOI] [PubMed] [Google Scholar]
  37. Naito AT, Shiojima I, Akazawa H, Hidaka K, Morisaki T, Kikuchi A, Komuro I. Developmental stage-specific biphasic roles of Wnt/beta-catenin signaling in cardiomyogenesis and hematopoiesis. Proc Natl Acad Sci USA. 2006;103:19812–19817. doi: 10.1073/pnas.0605768103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nasonkin I, Alikasifoglu A, Ambrose C, Cahill P, Cheng M, Sarniak A, Egan M, Thomas PM. A novel sulfonylurea receptor family member expressed in the embryonic Drosophila dorsal vessel and tracheal system. J Biol Chem. 1999;274:29420–29425. doi: 10.1074/jbc.274.41.29420. [DOI] [PubMed] [Google Scholar]
  39. O’Neill JW, Bier E. Double-label in situ hybridization using biotin and digoxigenin-tagged RNA probes. BioTechniques. 1994;17:870–875. [PubMed] [Google Scholar]
  40. Patel N. Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes. In: Fyrberg EA, Golstein LSB, editors. Methods in Cell Biology. Vol. 44. Academic Press; Boston, MA: 1994. pp. 445–487. [DOI] [PubMed] [Google Scholar]
  41. Peckham M, Cripps RM, White DCS, Bullard B. Mechanics and protein content of insect flight muscles. J Exp Biol. 1992;168:57–76. [Google Scholar]
  42. Peng T, Tian Y, Boogerd CJ, Lu MM, KAdzik RS, Stewart KM, Evans SM, Morrisey EE. Coordination of heart and lung co-development by a multipotent cardiopulmonary pregenitor. Nature. 2013;500:589–592. doi: 10.1038/nature12358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pereira FA, Qiu Y, Zhou G, Tsai MJ, Tsai SY. The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development. Genes Dev. 1999;13:1037–1049. doi: 10.1101/gad.13.8.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Perrin L, Monier B, Ponzielli R, Astier M, Semeriva M. Drosophila cardiac tube organogenesis requires multiple phases of Hox activity. Developmental Biology. 2004;272:419–431. doi: 10.1016/j.ydbio.2004.04.036. [DOI] [PubMed] [Google Scholar]
  45. Petit FG, Jamin SP, Kurihara I, Behringer RR, DeMayo FJ, Tsai M-J, Tsai SY. Deletion of the orphan nuclear receptor COUP-TFII in uterus leads to placental deficiency. Proc Nat Acad Sci. 2007;104:6293–6298. doi: 10.1073/pnas.0702039104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pierpont ME, Basson CT, Benson DW, Jr, Gelb BD, Giglia TM, Goldmuntz E, McGee G, Sable CA, Srivastava D, Webb CL. Genetic basis for congenital heart defects: current knowledge: a scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: endorsed by the American Academy of Pediatrics. Circulation. 2007;115:3015–3038. doi: 10.1161/CIRCULATIONAHA.106.183056. [DOI] [PubMed] [Google Scholar]
  47. Ponzielli R, Astier M, Chartier A, Therond A, Semeriva M. Heart tube patterning in Drosophila requires integration of axial and segmental information provided by the Bithorax Complex genes and Hedgehog signaling. Development. 2002;129:4509–4521. doi: 10.1242/dev.129.19.4509. [DOI] [PubMed] [Google Scholar]
  48. Rizki TM. The circulatory system and associated cells and tissues. In: Ashburner M, Wright TRF, editors. The Genetics and Biology of Drosophila. 2b. Academic Press; New York: 1978. pp. 397–452. [Google Scholar]
  49. Robertson HM, Preston CR, Phillis RW, Johnson-Schlitz DM, BWK, Engels WR. A stable genomic source of P element transposase in Drosophila melanogaster. Genetics. 1988;118:461–470. doi: 10.1093/genetics/118.3.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rubin GM, Spradling GM. Genetic-transformation of Drosophila with transposable element vectors. Science. 1982;218:348–353. doi: 10.1126/science.6289436. [DOI] [PubMed] [Google Scholar]
  51. Ryan KM, Hoshizaki DK, Cripps RM. Homeotic selector genes control the patterning of seven-up cells in the Drosophila dorsal vessel. Mech Dev. 2005;122:1023–1033. doi: 10.1016/j.mod.2005.04.007. [DOI] [PubMed] [Google Scholar]
  52. Ryan KM, Hendren JD, Helander LA, Cripps RM. The NK homeodomain transcription factor Tinman is a direct activator of seven-up in the Drosophila dorsal vessel. Dev Biol. 2007;302:694–702. doi: 10.1016/j.ydbio.2006.10.025. [DOI] [PubMed] [Google Scholar]
  53. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning A Laboratory Manual. Cold Spring Harbor; New York: 1998. [Google Scholar]
  54. Schneider VA, Mercola M. Wnt antagonism initiates cardiogenesis in Xenopus laevis. Genes Dev. 2001;15:304–315. doi: 10.1101/gad.855601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Schott JJ, Benson DW, Basson CT, Pease W, Silberbach GM, Moak JP, Maron BJ, Seidman CE, Seidman JG. Congenital heart disease caused by mutations in the transcription factor Nkx2-5. Science. 1998;281:108–111. doi: 10.1126/science.281.5373.108. [DOI] [PubMed] [Google Scholar]
  56. Sharma Y, Cheung U, Larsen EW, Eberl DF. Drosophila Pkd2 Is Haploid-insufficient for Mediating Optimal Smooth Muscle Contractility. Genesis. 2002;34:115–118. [Google Scholar]
  57. Soshnikova N, Dewaele R, Janvier P, Krumlauf R, Duboule D. Duplications of hox gene clusters and the emergence of vertebrates. Dev Biol. 2013;378:194–199. doi: 10.1016/j.ydbio.2013.03.004. [DOI] [PubMed] [Google Scholar]
  58. Tao Y, Schulz RA. Heart development in Drosophila. Semin Cell Dev Biol. 2007;18:3–15. doi: 10.1016/j.semcdb.2006.12.001. [DOI] [PubMed] [Google Scholar]
  59. Tauc HM, Mann T, Werner K, Pandur P. A role for Drosophila Wnt-4 in heart development. Genesis. 2012;50:466–481. doi: 10.1002/dvg.22021. [DOI] [PubMed] [Google Scholar]
  60. Tomancak P, Beaton A, Weiszmann R, Kwan E, Shu S, Lewis SE, Richards S, Ashburner M, Hartenstein V, Celniker SE, Rubin GM. Systematic determination of patterns of gene expression during Drosophila embryogenesis. Genome Biol. 2002;3:1–14. doi: 10.1186/gb-2002-3-12-research0088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Van der Wetering M, Cavallo R, Dooijes D, van Beest M, van Es J, Loureiro J, Ypma D, Hursh D, Jones T, Bejsovec A, Peifer M, Mortin M, Clevers H. Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF. Cell. 1997;88:789–799. doi: 10.1016/s0092-8674(00)81925-x. [DOI] [PubMed] [Google Scholar]
  62. Verzi MP, Anderson JP, Dodou E, Kelly KK, Greene SB, North BJ, Cripps RM, Black BL. N-Twist, an evolutionarily conserved bHLH protein expressed in the developing CNS, functions as a transcriptional inhibitor. Developmental Biology. 2002;249:174–190. doi: 10.1006/dbio.2002.0753. [DOI] [PubMed] [Google Scholar]
  63. Ward EJ, Skeath JB. Characterization of a novel subset of cardiac cells and their progenitors in the Drosophila embryo. Development. 2000;127(22):4959–4969. doi: 10.1242/dev.127.22.4959. [DOI] [PubMed] [Google Scholar]
  64. Wu X, Golden K, Bodmer R. Heart development in Drosophila requires the segment polarity gene wingless. Dev Biol. 1995;169:619–628. doi: 10.1006/dbio.1995.1174. [DOI] [PubMed] [Google Scholar]
  65. Wu SP1, Cheng CM, Lanz RB, Wang T, Respress JL, Ather S, Chen W, Tsai SJ, Wehrens XH, Tsai MJ, Tsai SY. Atrial identity is determined by a COUP-TFII regulatory network. Dev Cell. 2013;25:417–426. doi: 10.1016/j.devcel.2013.04.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Yin ZZ, Xu XL, Frasch M. Regulation of the Twist target gene tinman by modular cis-regulatory elements during early mesoderm. Development. 1997;124:4971–4982. doi: 10.1242/dev.124.24.4971. [DOI] [PubMed] [Google Scholar]
  67. Zaffran S, Reim I, Qian L, Lo PC, Bodmer R, Frasch M. Cardioblast-intrinsic Tinman activity controls proper diversification and differentiation of myocardial cells in Drosophila. Development. 2006;133:4073–4083. doi: 10.1242/dev.02586. [DOI] [PubMed] [Google Scholar]
  68. Zaidi S, Choi M, Wakimoto H, Ma L, Jiang J, Overton JD, Romano-Adesman A, Bjornson RD, Breitbart RE, Brown KK, Carriero NJ, Cheung YH, Deanfield J, DePalma S, Fakhro KA, Glessner J, Hakonarson H, Italia MJ, Kaltman JR, Kaski J, Kim R, Kline JK, Lee T, Leipzig J, Lopez A, Mane SM, Mitchell LE, Newburger JW, Parfenov M, Pe’er I, Porter G, Roberts AE, Sachidanandam R, Sanders SJ, Seiden HS, State MW, Subramanian S, Tikhonova IR, Wang W, Warburton D, White PS, Williams IA, Zhao H, Seidman JG, Brueckner M, Chung WK, Gelb BD, Goldmuntz E, Seidman CE, Lifton RP. De novo mutations in histone-modifying genes in congenital heart defects. Nature. 2013;498:220–223. doi: 10.1038/nature12141. [DOI] [PMC free article] [PubMed] [Google Scholar]

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