Embryonic even skipped–Dependent Muscle and Heart Cell Fates Are Required for Normal Adult Activity, Heart Function, and Lifespan
The Drosophila pair-rule gene even skipped (eve) is required for embryonic segmentation and later in specific cell lineages in both the nervous system and the mesoderm. We previously generated eve mesoderm-specific mutants by combining an eve null mutant with a rescuing transgene that includes the entire locus, but with the mesodermal enhancer removed. This allowed us to analyze in detail the defects that result from a precisely targeted elimination of mesodermal eve expression in the context of an otherwise normal embryo. Absence of mesodermal eve causes a highly selective loss of the entire eve-expressing lineage in this germ layer, including those progeny that do not continue to express eve, suggesting that mesodermal eve precursor specification is not implemented. Despite the resulting absence of a subset of muscles and pericardial cells, mesoderm-specific eve mutants survive to fertile adulthood, providing an opportunity to examine the effects of these developmental abnormalities on adult fitness and heart function. We find that in these mutants, flying ability, myocardial performance under normal and stressed conditions, and lifespan are severely reduced. These data imply a nonautonomous role of the affected pericardial cells and body wall muscles in developing and/or maintaining cardiac performance and possibly other functions contributing to normal lifespan. Given the similarities of molecular-genetic control between Drosophila and vertebrates, these findings suggest that peri/epicardial influences may well be important for proper myocardial function.
Although much effort has gone into defining developmental pathways, many of the mechanistic aspects of regulatory processes occurring during tissue specialization and organogenesis remain to be elucidated. The homeobox-containing gene even skipped (eve) is involved in several such aspects of tissue specialization. It was first identified in Drosophila as a segmentation gene.1 Later during development, eve is expressed in the nervous system and in dorsal muscle progenitors and pericardial cells.2 Regulatory elements for each of these aspects of the pattern were localized,3,4 and a transgenic copy of the eve gene was shown to fully supply eve function.3 Specific repressor domains of Eve were identified in cultured cell assays5,6 and subsequently shown to interact functionally with corepressors.7–9
eve is expressed in, and required for the formation of, a subset of dorsal muscle and pericardial (PC) cells.10–12 In the developing mesoderm, early progenitors in the cardiogenic region express eve in segmentally repeated clusters that later differentiate into eve-positive pericardial cells (EPCs) and dorsal acute muscle 1 (DA1).2 A transgene was generated that fully rescues the phenotype of eve null mutants in all other tissues, but gives no detectable expression or function in the mesoderm, and resulting embryos were shown to develop mesodermal-specific defects that include disruption of EPC and DA1 muscle formation.13
Although much progress has been made in recent years in understanding the specification of cell types in the embryonic dorsal vessel,14 contributions of PC cells to the formation and function of the larval and adult heart are unexplored.15 Here, we focus on 4 key aspects of that function. First, we find that eve is required for the correct specification of cell types that arise from the eve-expressing lineage. Second, we find that eve is required for formation not only of the lineage in which it is continuously expressed, but also of a second lineage in which its expression is normally transient. Third, we find that the repressor function of Eve is necessary and sufficient, in the context of its homeodomain, to provide full rescue, implying that Eve acts exclusively as a repressor. Fourth, in the absence of eve-dependent PC cells, whereas the myocardium forms normally, the larval and adult heart rate is reduced, and the susceptibility of the heart to fail is dramatically increased, suggesting that PC cells play a pivotal role in normal heart function.
Materials and Methods
Transgenic Lines, Constructs, and Antibodies
A genomic region spanning 15 kb (Figure 1A) is sufficient to completely rescue the phenotype of eve null mutants.3 An additional 0.6 kb at the 3′ end may increase expression at some chromosomal locations, but at the insertion sites used here, no differences are seen in the degree of rescue with or without this region. Details of transgenes and antibodies are provided in the online Data Supplement available at http://circres.ahajournals.org.
Analysis of Embryos
eve null backgrounds, either Df(2R)eve, Df(2R)eve/eve3, or eve3/eve3, all which gave indistinguishable results, were used in combination with mesodermal enhancer–deficient eve rescue transgenes and are referred to as eve “meso−” throughout the text. Embryos shown in Figure 1 are Df(2R)eve, whereas muscle preparations from larvae and adult are Df(2R)eve/eve3 or eve3/eve3.
Evx Expression, Pericardial Cell Counts, Cardiac and Lifespan Measurements, and Flight Assay
Details of assays for Evx expression, pericardial cell counts, heart rates, cardiac stress–induced failure rates, flight ability, and lifespan, are provided in the Data Supplement.
Mesoderm-Specific eve Mutants
Removing the mesodermal enhancer eme in the context of a rescue transgene removes all detectable mesodermal eve expression without affecting any other aspect of expression (Figure 1). When combined with a null mutation at the endogenous locus, this results in an eve “mesoderm negative” (eve meso−) mutant. A separate transgene containing eme upstream of a reporter gene (lacZ) can be used to faithfully mark the cells that would normally express eve. In the absence of mesodermal eve, the body wall muscles no longer show reporter gene expression (Figure 1F and 1G). In cells associated with the heart, however, the stable reporter protein persists until the formation of a seemingly normal heart tube (Figure 1G; supplemental Figure I). Except for the lack of eve expression (Figure 1B through 1G), other heart cell types, particularly the myocardial cells, appear to be present in their normal numbers and positions (Figure 1H and 1I; supplemental Figure I). In the absence of eve, the cells in which the reporter persists appear to be naïve and able to adopt other (cardiac) fates (Figure 1H and 1I), consistent with eve acting as an essential factor for specification of their lineage. Interestingly, the vertebrate eve homolog, Evx2, is also expressed in the heart, in an epicardial cell line, and in cells from primary epicardial explants (Figure 1J and 1K). Thus, eve/Evx may play a role in heart development in both insects and mammals.
Altered Expression of Identity Genes
Mesodermal eve expression normally occurs in founder cells that give rise to a subset of pericardial cells and to 2 muscles per hemisegment, DO2 and DA1 (Figure 2A).16–18 A cell within 1 eve-expressing cluster (cluster 2) initiates expression of Krüppel (Kr; Figure 2F). This Kr- and eve-expressing cell (progenitor 2) divides to yield 2 founder cells that express runt (Figure 2B), 1 of which is the founder of DO2 and continues to express runt, and the other of which is the EPC founder and turns off runt. The DO2 founder turns off eve shortly after runt is activated, whereas the EPC founder and the resulting EPCs continue to express eve. A cell within a second eve-expressing cluster (cluster 15) activates Kr (Figure 2F, outlined in white), then divides to yield the DA1 founder and a second cell that is fated to die. The DA1 muscle maintains eve expression.
Previous studies suggested that eve function is required for normal EPC differentiation12 and for the normal pattern of expression of ladybird.11,13 As a way to define the role of eve, we examined both runt and Kr expression in eve meso− embryos. In the absence of eve, both runt and Kr expression are either completely absent from or dramatically reduced in these muscle founder lineages (Figure 2). This strongly suggests that eve is required for these cells to adopt their normal fates. Thus, eve has either a direct or an indirect role (repression of a repressor) in activating Kr and runt. We distinguish between these possibilities below.
Muscle Defects in eve Meso− Larvae
To determine the extent to which eve function is required for normal muscle formation, we examined the musculature in eve meso− third instar larvae. The normal arrangement of dorsal muscles within each segment is clearly altered (Figure 3A through 3H). Both DA1 and DO2 are severely defective or aberrant, and other muscles in the vicinity exhibit alterations in their placement and size. A simple interpretation of the effects seen in a majority of segments is that DA1 is missing, and a single, large muscle occupies the normal positions of DO1 and DO2. The muscle occupying the normal position of DA2 is also enlarged. These enlarged muscles suggest that myoblasts that normally fuse with the DA1 and DO2 founders may instead fuse with other muscles nearby, or, alternatively, that in the absence of DA1 and DO2, attachment sites for adjacent muscles expand.
The DA1 and DO1 muscles are innervated in wild-type embryos by motorneurons that express eve.19–21 We used transgenes that express green fluorescent protein (GFP) in these neurons to label their axons in the muscle field.22 We found that in eve meso− embryos, one or both muscles in the normal positions of DA1/2 and DO1/2 are innervated by these eve-expressing neurons (Figure 3I through 3K). We also found that in eve meso− third instar larvae, both DA1/2 and DO1/2 are innervated (Figure 3C).
Eve Functions as a Repressor in the Mesoderm
The changes in gene expression observed in eve meso− embryos (Figure 2) suggest that eve acts, directly or indirectly, as an activator of Kr and runt. Previous analyses of eve function suggested that it acts as a repressor of transcription.8,22–24 If this is true in the mesoderm, then at least 1 intermediary gene that is repressed by eve normally represses runt and Kr. To study the domain requirements of eve, we expressed transgenes in the eve meso− background that contained the eve mesodermal enhancer driving expression of modified Eve proteins. With the wild-type Eve coding region, the mesodermal defect is completely rescued (supplemental Figure IIA and IIB). In contrast, when the Eve homeodomain (HD)-containing region alone is so expressed, a very limited degree of rescue is observed (supplemental Figure IIC and IID). Importantly, when the heterologous Engrailed repressor domain is added to the HD construct, full rescuing ability is restored (supplemental Figure IIE), implying that eve acts exclusively as a repressor in the mesoderm.
We also examined the ability of Eve to act as a direct repressor in the mesoderm by targeting it to a reporter transgene using the Gal4 DNA binding domain. When a Gal4-Eve fusion protein containing both of the repressor domains of Eve is combined with an eve lineage–specific Gal4-UAS–containing reporter, the reporter is strongly repressed (supplemental Figure IIF through IIH).
eve Meso− Larvae Have Fewer Pericardial Cells
eve meso− embryos develop into viable adults, providing an opportunity to examine the role of PC cells in larval and adult heart function. The absence of mesodermal eve does not noticeably affect the assembly of the myocardial cells at the dorsal midline, which will give rise to the contractile part of the heart tube (data not shown). To examine the contribution of EPCs to larval heart development, eve meso− larvae were dissected and PC cells were counted. Wild-type and wild-type eve-rescued larvae show 7 to 8 PC cells per segement, and eve meso− heterozygotes show 6 (Figure 4A and 4D). In contrast, 2 independent eve meso− lines displayed a marked reduction, with an average of only 3 to 4 PC cells per segment (Figure 4B through 4D). Thus, eve is required to produce the normal complement of larval PC cells.
Functional Heart Defects
Because eve meso− animals are missing half or more of their PC cells, we examined the effect on heart function in pupae and adults. We found that neither wild-type eve-rescued pupae nor those heterozygous for eve and carrying one copy of a meso− rescue transgene exhibited heart rates that differed significantly from controls (Figure 4E). However, all 3 meso− lines, which carry independent transgene insertions, exhibited a significant reduction in heart rate (30% to 50%, Figure 4E).
We examined adults of the same genotypes to assess whether defective functions persist through partial remodeling of the heart during metamorphosis.25 The wild-type heart rate is 2.9 beats/second (Hz) in 1-week-old adults and 2.6 Hz in 3-week-old adults (Figure 5A).26 Compared with wild type, both the eve null background rescued by a wild-type rescue transgene and the eve meso− heterozygotes (J49/CyO) exhibit a reduced heart rate at both ages, probably attributable to a genetic background effect inherent to these eve rescue lines that serve as controls. eve meso− flies, however, develop a dramatically lower heart rate with age, and one of the meso− lines also shows a severely reduced heart rate at an early age (Figure 5A).
Heart function can also be assayed by quantifying stress tolerance, using an external current to briefly pace the heart to about twice the normal rate, then charting the percentage that undergo either fibrillation or cardiac arrest26 (termed heart failure). In wild-type flies, the ability of the heart to withstand such stress is highly age-dependent, with stress-induced failure rates increasing dramatically (2- to 3-fold) between 1 and 5 weeks of age.26,27 Because eve meso− flies seldom reach 5 weeks of age, we examined flies at 1 and 3 weeks of age. Neither heterozygous eve meso− nor eve rescued flies differed from wild type (Figure 5B). In contrast, one eve meso− line showed a significantly increased failure rate at 1 week of age, whereas the other showed a disastrously high failure rate at 3 weeks of age (Figure 5B). Although dorsal somatic muscle defects might also conceivably affect heart function, these results suggest that the reduction in PC cell number causes a slowed heartbeat and reduces cardiac stress resistance.
Effects on Lifespan
To further assess the importance of fully functional heart and muscle activity and potentially of other results of mesodermal eve expression (see Figure 5C, and “Other functional defects” in the Data Supplement), we examined the life spans of eve meso− flies. Such flies display a significantly reduced mean and maximal lifespan (Figure 5D), suggesting that the presence of mesodermal Eve is required not only for normal activity levels but also for a normal lifespan.
Functional Requirements in All eve-Expressing Mesodermal Lineages
By constructing a mesoderm-specific eve mutant, we established that eve is required not only in the eve-expressing lineages in which it is maintained during terminal differentiation (the eve-expressing pericardial cells and the DA1 muscle), but also in the lineage in which it is expressed in the progenitor but turned off in the muscle founder cell and the resulting DO2 muscle. Thus, both DA1 and DO2 require mesodermal eve to be specified, and without this specification, the pattern and size control of some remaining muscles are compromised (Figures 2 and 3⇑). Importantly for heart function (discussed below), in the absence of mesodermal eve expression, the majority of the large larval PC cells are missing (Figure 4).
It is intriguing that even though ectopic Eve expression can interfere with the DO2 fate,11 and eve is normally turned off as runt is activated in the lineage (Figure 2A), eve function is nonetheless required for DO2 formation, apparently because of a requirement in the progenitor before the lineage divisions.
Eve Protein Function in the Mesoderm
When normal eve expression in the mesoderm is replaced by expression of the eve HD (with repressor domains deleted), a similar but less severe muscle deficiency is observed compared with the complete absence of mesodermal eve (supplemental Figure II). In particular, a muscle in the DO2 position is usually formed, whereas DA1 is still absent. Additionally, there is occasionally an extra muscle ventral to DO2, as if the DO2 founder was duplicated. Importantly, however, when the heterologous Engrailed repressor domain is added to the HD construct, full rescuing ability is restored (supplemental Figure II). This suggests that Eve functions in the mesoderm primarily or exclusively as a repressor, and in turn that eve acts indirectly to activate Kr and runt in the mesoderm. Good candidates for intermediary repressors are ladybird and the muscle identity gene msh.11,13
Eve and PC Cells Influence Larval and Adult Heart Function
A reduced number of larval PC cells (and dorsal somatic muscles) caused by a lack of mesodermal eve expression results in severely compromised heart function and is likely to contribute to a shortened lifespan. A less drastic effect on cardiac performance and lifespan is observed when manipulating insulin signaling exclusively in the heart.27 The functional role of pericardial cells in insect hearts is not well understood,15 but they may contribute to heart function by secreting hormones or by gathering such peptides from circulating hemolymph and “presenting” them to the myocardium. It has been suggested that pericardial cells may function as nephrocytes,15 and at this point we cannot rule out that a potential accumulation of toxic agents, as a consequence of fewer pericardial cells, contributes to the observed phenotypes.
As in insects, the developmental and functional interactions between the vertebrate epicardium and the myocardium are not well understood. Recent studies have suggested that the loss of epicardial function results in impaired growth of the myocardium at mid-gestation.28–30 The epicardium is thought to be a source of signals and secreted factors that affect myocardial proliferation and differentiation, as well as influencing formation of the conduction system.31 Even though it cannot yet be decided whether the mammalian epicardium has a developmental program in common with a fly’s pericardial cells, they both depend on GATA factors for formation.30,32 In addition, the Evx2 homolog of eve is indeed expressed in the mammalian heart, including in epicardial tissue (Figure 1J and 1K). Our findings are consistent with pericardial cells in Drosophila functioning as a source of signals that affect the myocardium. Possibly because the myocardium, which is maintained by proliferation in vertebrates, does not proliferate in flies after it is developmentally specified,25 pericardial deficiency does not appear to result in morphological heart defects (supplemental Figure I). Rather, defects manifest themselves as functional deficits. This provides an opportunity to study the influence of these heart-associated cell types on cardiac physiology in the absence of myocardial defects. Epicardial lineages in vertebrates may contribute analogously to normal cardiac physiology and performance.
M.Z. is a recipient of a fellowship from the Spanish Ministry of Science and Education. P.R-L. is funded by the National Institutes of Health (National Heart, Lung, and Blood Institute). J.B.J. received support for this work from the National Institutes of Health and the National Science Foundation (award 0416760), and R.B. from the National Heart, Lung, and Blood Institute and the National Institute on Aging of the National Institutes of Health. We thank Manfred Frasch, Corey Goodman, Steve Small, the Developmental Studies Hybridoma Bank at the University of Iowa, and the Bloomington Stock Center for reagents. We thank Jian Zhou and Erin Fitzgerald for excellent technical assistance.
↵*Both authors contributed equally to this study.
Original received December 9, 2004; revision received September 29, 2005; accepted October 10, 2005.
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