|Home | About | Journals | Submit | Contact Us | Français|
Cardiac specification models are widely utilized to provide insight into the expression and function of homologous genes and structures in humans. In Drosophila, contractions of the alary muscles control hemolymph inflow and support the cardiac tube, however embryonic development of these muscles remain largely understudied. We found that alary muscles in Drosophila embryos appear as segmental pairs, attaching dorsally at the seven-up (svp) expressing pericardial cells along the cardiac dorsal vessel, and laterally to the body wall. Normal patterning of alary muscles along the dorsal vessel was found to be a function of the Bithorax Complex genes abdominal-A (abd-A) and Ultrabithorax (Ubx) but not of the orphan nuclear receptor gene svp. Ectopic expression of either abd-A or Ubx resulted in an increase in the number of alary muscle pairs from seven to ten, and also produced a general elongation of the dorsal vessel. A single knockout of Ubx resulted in a reduced number of alary muscles, and double knockouts of both Ubx and abd-A prevented alary muscles from developing normally and from attaching to the dorsal vessel. These studies demonstrate an additional facet of muscle development that depends upon the Hox genes, and define for the first time mechanisms that impact development of this important subset of muscles.
The cardiac system in Drosophila is an excellent model system for the analysis of cardiac development in higher animals. The insect cardiac tube, or dorsal vessel, is comprised of a number of distinct cell types that arise through complex processes of cell specification in response to signals both from within the mesoderm and from the ectoderm (reviewed in Cripps and Olson, 2002; Tao and Schulz, 2007). The mature dorsal vessel is comprised several distinct cell types: muscular cardial cells, which mediate pumping of hemolymph (Bodmer and Frasch, 1998); pericardial cells, which play diverse roles in homeostasis, cardiac physiology, and formation of the adult circulatory system (Fujioka et al., 2005; Miller, 1950; Togel et al., 2008); and alary muscles, that are thought to support the dorsal vessel during locomotion, to help modulate the flow of hemolymph into the cardiac tube (Bate 1993; Rizki, 1978), and to serve as scaffolding for neurons at later developmental stages (Dulcis and Levine, 2003).
Within each of these components of the cardiac tube, there is also diversity along the anteroposterior (AP) axis. For example, hemolymph enters the muscular cardiac tube in a region termed the heart that spans approximately three posterior segments. Located in these heart segments are sets of specialized cells forming valve-like structures that express the seven-up (svp) orphan nuclear receptor gene (Molina and Cripps, 2001; Ponzielli et al., 2002). Hemolymph then is pumped anteriorly from the heart through the aorta, and ejected near the brain, prior to percolating back through the body cavity. A series of studies have demonstrated that the AP diversity in the cardiac tube arises from the actions of the homeotic selector genes, predominantly those of the Bithorax Complex (Lovato et al., 2002; Ponzielli et al., 2002; Lo et al., 2002; Perrin et al., 2004; Ryan et al., 2005).
While some of the factors that control patterning of many cell types in the dorsal mesoderm are understood, relatively little is known about the formation of alary muscles in Drosophila. The alary muscles are located symmetrically along the dorsal vessel in the mature embryo, attaching dorsally close to the cardiac tube and laterally to the body wall (Bate 1993). However a detailed description of their organization, and the identification of genes which impact their patterning, has yet to be presented. In this manuscript, we have studied the development of the alary muscles in wild-type and mutant embryos. We show that the alary muscles attach dorsally to the svp expressing pericardial cells adjacent to the cardiac tube, and laterally to one of two distinct locations on the body wall. In addition, we show that not all segments form alary muscles attaching to the cardiac tube. Importantly, genes of the Bithorax Complex, whose function is required for normal alary muscle development, control this process. These studies, for the first time, describe important characteristics of the alary muscles in Drosophila, and provide further insight into the roles of the Hox genes in muscle development.
One obstacle in the characterization of alary muscles has been the paucity of molecular markers that specifically identify these cells. Therefore, to determine the best method for observing the general physical structure of the alary muscles, wild-type Drosophila melanogaster embryos were stained with antibodies for targets prevalent in either cardiac or cardiac and skeletal muscle tissue.
In a first series of experiments, we utilized an antibody to Pericardin (anti-Prc), an extracellular matrix protein secreted by the pericardial cells (Chartier et al., 2002; Figure 1A and C), and anti-Tinman (anti-Tin), which marks the majority of cardial cells and a subset of pericardial cells (Bodmer, 1993; Azpiazu and Frasch, 1993; Figure 1B and C). Localization of these primary antibodies was visualized by immunofluorescence in whole-mount embryos. We found that, whereas it was possible to find preparations that showed Prc accumulation along the alary muscles, the signal in this region was faint and difficult to observe in all samples.
As an alternative option, we stained embryos with antibodies to muscle structural genes, such as anti-myosin heavy chain (anti-MHC; Kiehart and Feghali, 1986) or anti Tropomyosin (anti-Tm; Peckham et al., 1991). Localization of these antibodies was accomplished using immunohistochemistry. Stage 16 embryos were filleted and the dorsal vessel and associated tissues were viewed from the ventral side of the body wall. While more labor-intensive, this preparation gave richer structural images of alary muscles, presenting a much higher level of detail.
Using these preparations, we verified that there are seven pairs of alary muscles that attach medially and dorsally along the dorsal vessel (Figure 1D). These muscles were usually paired, although we occasionally observed a muscle attached in a slightly disordered manner, even in wild-type (arrowhead in Figure 1D). For the lateral attachment sites, the most anterior five alary muscle pairs extended past muscle DA2 and terminated at the cuticle slightly posterior to the dorsal insertion of skeletal muscle DT1 (Bate, 1993; Figure 1E, F). By contrast, the two most posterior alary muscle pairs extended further ventrally past DA2 and DA3, and inserted within the attachments of a cluster of muscles (Figure 1G). This pattern of alary muscles attachment was retained through the end of the larval stage (Figure 1F and G are preparations of third instar larvae). Clearly, the formation of the alary muscles is controlled by a complex series of processes that ensure that the correct number of fibers are generated and attached appropriately within the body.
In order to more directly characterize the dorsal attachment sites of the alary muscles, we studied the development of the alary cells relative to cardiac expression of svp. In wild-type embryos, svp is expressed continuously in seven groups of cardial cells along the AP axis (Bodmer and Frasch, 1998), and transiently in an equivalent number of pericardial cells (Lo and Frasch, 2001). To compare the arrangement of the alary muscles relative to the Svp cells, we double-stained embryos with antibodies to MHC (brown in Figure 2) and β-galactosidase expressed from a svp-lacZ fusion gene (black in Figure 2A-C; Ryan et al., 2005).
At stage 14, it was possible to identify all of the alary muscles as extensions running between the dorsal vessel and the lateral ectoderm (Figure 2A). The dorsal ends of the alary muscles consistently attached close to the Svp expressing pericardial cells. In particular, the alary muscles extended processes in either direction along the AP axis where they contacted the svp pericardial cells. This observation was also apparent at stage 16 (Figure 2B). At higher magnifications, details of the alary muscles could be clearly observed, including the multinucleate nature of the cells, and the processes that extend specifically towards the Svp pericardial cells. These studies identified the alary muscles as skeletal muscles (based upon their syncytial nature), and suggested a mechanism for how they might be oriented towards the dorsal vessel.
Given the close association of alary muscle development with the Svp pericardial cells, svp null embryos were evaluated for alary muscle patterning (Figure 2D and E). Despite the lack of differentiation of dorsal vessel cardial cells into Svp cells in these mutants, alary muscles still attached to the dorsal vessel and pericardial cells at the same locations (Figure 2D). While occasional patterning defects were observed (see asterisked fiber in Figure 2D), these were not significantly more prevalent than in wild-type animals (see for example Figure 1A).
To determine if the alary muscles were still extending towards the Svp pericardial cells in svp mutants, we studied alary muscle attachment in homozygous mutants for a svp enhancer trap line, svp3. In these mutants, the Svp pericardial cells can still be visualized based upon residual β-galactosidase accumulation (black in Figure 2E); nevertheless, the alary muscles were still observed to orient towards these marked pericardial cells (Figure 2E). These data indicated that alary muscle patterning along the dorsal vessel is not dictated by the presence or absence of svp expression. One interpretation of these data is that there is some form of molecular signature in the Svp pericardial cells, presumably produced independently of svp function, that is required for normal alary muscle attachment. A more simple explanation is that the alary muscles attach to the dorsal vessel in the location immediately above where they are specified, and that this location just happens to be in the vicinity of the Svp pericardial cells.
Since the alary muscles attaching to the dorsal vessel only formed in a subset of embryonic segments, and given the role of Hox genes in controlling AP pattern in the cardiac tube in Drosophila, we next investigated a potential influence of the Hox genes upon the patterning and development of the alary muscles. Presence or absence of Hox expression in the alary muscles was evaluated using wild-type embryos immunohistochemically stained for the products of the two Hox genes abd-A and Ubx. The presence of Hox gene products is indicated by a stained black nucleus. Absence of Hox gene expression is signified by a clear or unstained muscle. Representative images of stained alary muscles are shown in Figure 3 A–D.
We found that Ubx and abd-A expression in the alary muscles was generally consistent with the known pattern of segmental expression of these genes. The nuclei of the three most anterior alary pairs were positive for Ubx expression and negative for abdA. Posterior to the three pairs, the expression pattern in the nuclei of the alary muscles switched to positive for abd-A and negative for Ubx (Figure 3 A–D). These studies suggested that individual Hox genes might be responsible for the development of different subsets of alary muscles. A summary of our descriptive studies of the alary muscles is presented in Figure 3E.
Based on our findings of Hox gene expression in the nuclei of the alary muscles, we sought to determine whether patterning changes in the alary muscles resulted from knockout or over-expression for each of Ubx and abd-A.
Loss of function experiments provided evidence that the patterning of alary muscles was under the direct control of Hox genes (Figure 4). When compared to the wild-type (Figure 4A), mutants unable to express Ubx failed to develop the 2–3 most anterior alary pairs, consistent with those muscles which we had identified as accumulating Ubx protein in wild-type (Figure 4B). By contrast, animals that were unable to produce abd-A still formed seven pairs of alary muscles along the cardiac tube (Figure 4C). This apparently wild-type phenotype presumably arises from the ability of Ubx to specify alary muscles in posterior segments in the absence of abd-A function. Double knockout mutants for both Ubx and abd-A produced individuals with a drastically reduced number of alary muscles. Of the few muscles that did develop, all were randomly positioned and consisted of no true alary muscle pairs (Figure 4D).
To complement the loss-of-function data, we also ectopically expressed either Ubx or abd-A throughout the mesoderm (Figure 5). In both cases, this expansion of Hox gene expression produced embryos with elongated cardiac tubes and an increase in the number of normally developed alary muscle pairs (Figure 5). For ectopic expression of Ubx, we observed 9.75 +/− 0.16 (n=8) muscles on each side of the dorsal vessel (Figure 5A), significantly more than the seven observed in wild-type. Similarly, ectopic abd-A produced on average 9.50 +/− 0.23 (n=12) alary muscles on each side of the dorsal vessel (Figure 5B). These supernumary muscles were usually paired, and in many cases the most anterior pair of muscles was angled acutely to ensure contact with the dorsal vessel (arrowheads in Figure 5). This latter observation probably arises from the fact that the ventrolateral attachment site on the cuticle is still located in an anterior segment, whereas even under conditions of Hox gene over-expression the dorsal vessel ultimately shortens to occupy slightly more posterior sections.
Consistent with the requirements for Ubx or abd-A (in the absence of Ubx) to specify alary muscles in loss-of-function assays, both Ubx and abd-A were equally effective at generating supernumerary alary muscles. Taken together, these studies identify the Hox genes as being critical to alary muscle development, and further expand the cadre of tissues types which are subject to Hox gene influence.
In Drosophila, the seven pairs of embryonic alary muscles attach to Svp pericardial cells along the dorsal vessel as it migrates dorsally towards its final location. The alary muscles persist throughout larval development, playing what are thought to be important roles in stabilizing the location of the heart in the body cavity. In addition, modified alary muscles are also found in the adult (Molina and Cripps, 2001), and there is evidence from some insects that contraction of these adult muscles is concordant with heart beating (Dulcis and Levine, 2003). These published data suggest important functions for the alary muscles throughout the life cycle.
Attachment of the alary muscles to the cardiac tube occurs in the vicinity of the Svp pericardial cells. Our data clearly identify processes emanating from the alary muscles towards the pericardial cells. It is reasonable to propose that the Svp pericardial cells produce or present some molecule(s) to which the alary muscles attach, although the nature of this molecule has yet to be defined. This suggestion is consistent with the observations of Curtis et al. (1999), who noted that in the developing pupa the pericardial cells and alary muscles are connected by significant amounts of connective tissue. In addition, the expression of this unknown molecule must be independent of svp function, since in svp mutants the patterning of the alary muscles appears largely normal. The cardiac tube expresses several secreted molecules which are known to function in cell attraction (Tao and Schulz, 2007), however an enrichment for any of these in the Svp pericardial cells has not been reported.
Our data also demonstrate that normal alary muscle patterning is under the direct control of the Hox genes Ubx and abd-A. This finding is consistent with previous research on the role of the Bithorax Complex in patterning of cardiac and skeletal muscle within the mesoderm (Lovato et al. 2002, Michelson 1994; Lo et al., 2002; Ponzielli et al., 2002), and the more general function of the Hox genes in controlling AP diversity. We note that the domains of Hox gene function in alary muscles bear a closer resemblance to their expression in developing skeletal muscles rather than Hox gene expression in the cardiac tube (compare the conclusions of Michelson (1994) with those of Lovato et al. (2002), Lo et al. (2002), and Ponzielli et al. (2002)). This observation is consistent with the conclusion that the alary muscles are skeletal muscle derivatives based upon their multinucleate nature.
Previous research further illustrates the requirement of the Hox genes abd-A and Ubx in the normal development and patterning of Svp cardial and pericardial cells (Perrin et al., 2004; Ryan et al, 2005). These studies also conducted experiments whereby both abd-A and Ubx were manipulated in knockout as well as over-expression conditions, and the nature of our results are in general consistent with previous findings for the cardiac Svp cells: over-expression of abd-A and Ubx produced three additional sets of Svp expressing cardial cells; and knockout conditions produced either no change in Svp cardial cell number (for abd-A mutants), loss of anterior Svp cardial cells (for Ubx mutants), or almost no sets of Svp cardial cells (for Ubx abd-A mutants).
Orthologs of Drosophila Hox genes are detected in the developing human heart, as well as being widely expressed in the neighboring viscera such as the lungs, spleen, liver, pancreas and epidermis (Hwang et al. 2006). In other vertebrates, Hox genes have been generally (although not specifically) implicated in cardiac development (reviewed in Lo and Frasch, 2003). A recent genome-scale study of skeletal muscle development also established an AP pattern of Hox gene expression in the developing embryonic skeletal myoblasts (Biressi et al., 2007). Together, our studies, and those cited here, further support a general role for Hox gene patterning of muscle derivatives broadly across the Animal Kingdom.
What are the embryonic origins of the alary muscles? As indicated previously, this question is difficult to answer absent a specific marker for the alary muscles early in embryonic development, and it must be noted that our analyses are therefore by necessity end-point assays carried out at stage 16. Nevertheless given the syncytial nature of the alary muscles, they likely arise from specific founders cells specified at particular locations in the somatic mesoderm. Furthermore, since there is only one alary muscle which arises in each hemisegment, it can be proposed that the founder for this muscle arises from an asymmetrical cell division. Experiments were carried out to test this hypothesis using mutants for sanpodo and numb, which are genes in the asymmetric cell division pathway (reviewed in Roegiers and Jan, 2004). While sanpodo mutants produced individuals with a partial loss of alary muscles, the numb mutants were so disrupted at the level of the whole organism that we were unable to discern any sign of the forming alary muscles if present. This issue might be addressed in the future via analysis of alary muscle precursor formation in these mutants, once suitable markers become available. Alternatively, a strategy for following cell lineages in founder cells might prove useful.
Are there mammalian versions of the alary muscles that we see in Drosophila? While in some cases it is difficult to assign directly homologous structures between insects and mammals, there are a number of ligaments known to stabilize the location of the heart in mammals. These include in particular the ligaments which attach the outer pericardial layer to the diaphragm and spinal column, as well as the sternopericardiac ligaments which connect the pericardium to the sternum (Standring, 2005). Thus, since the requirement for structures to stabilize the heart within the body cavity appears to be conserved, the molecular mechanisms responsible for their development might also bear some resemblances to each other.
Drosophila stocks were maintained at 25°C on Carpenter’s medium (Carpenter, 1950) in plastic bottles or vials. The strain w1118 was used as the non-mutant control. Knockout stocks were: TM1/Ubx109 for abd-A plus Ubx; TM1/Ubx9.22 for Ubx; TM1/abdAMX1 for abd-A; TM3/svp1 and TM3/svp3 for svp. These stocks were obtained from the Bloomington Drosophila Stock Center, except the svp1 stock which was from James Skeath (Washington University, St Louis, MO). Expression assays were carried out utilizing the GAL4-UAS system (Brand and Perrimon, 1993): UAS-Ubx and the 24B-Gal4 driver line were obtained from the Bloomington Stock Center. UAS-abd-A was obtained from Alan Michelson (Harvard University, MA). The SCE svp-lacZ strain was described in Ryan et al. (2007).
Antibody staining of embryos was performed in accordance with Patel (1994). Larval fillets were prepared according to Molina and Cripps (2001). The primary antibodies and concentrations were: Mouse anti-Pericardine at 1:4 (Chartier et al., 2002), rabbit anti-Tinman at 1:250 (Yin et al., 1997), mouse anti-UBX at 1:50 (White and Wilcox, 1984), mouse anti-abd-A at 1:000 (Macias et al., 1990), mouse anti-myosin heavy-chain (MHC) at 1:500 (Kiehart and Feghali, 1986), and rat anti-Tropomyosin (Abcam Inc, Cambridge, MA) at 1:1000. Biotinylated secondary antibodies were anti-mouse and anti-rat, both used at concentrations of 1:1000 (Vector Laboratories, Burlingame, CA). Antibody detection was performed using the Vectastatin Elite kit (Vector Laboratories, Burlingame, CA) and DAB staining kit (Vector Laboratories, Burlingame, CA). Fluorescent staining was generated using the anti-mouse or anti-rabbit secondary antibodies Alexa-488 or Alexa-568 conjugated, at concentrations of 1:2000 (Molecular Probes, Seattle, WA). Non-fluorescent double-stains were carried out by staining embryos sequentially with antibodies and developing the first antibody using DAB staining reagent with NiCl2 added; followed by the second antibody detected with DAB staining solution lacking NiCl2.
Microscopy was performed on an Olympus BX51 microscope using DIC or fluorescent optics. Stained embryos used for dissection were initially submerged in 100 μL of 80% (v/v) glycerol/1XPBS, and dissected as described in Lovato et al. (2002). Filleted preparations were viewed at 600x magnification using an oil immersion lens and DIC optics. Representative samples were photographed using 35mm slide film, and the resulting slides were scanned and assembled using Adobe Photoshop. For the filleted preparations, the figures presented here are representative montages of individual photographs taken at slightly different focal planes in order to fully document the dorsal vessel and alary muscles.
We wish to extend our gratitude to the organizations and individuals who have generously provided reagents for our study: Danielle Gratecos, Manfred Frasch, Alan Michelson, James Skeath, Daniel Kiehart. We thank the Bloomington Drosophila Stock Center for providing fly stocks. The funding for this study was provided by NIH grant number HL080545 to RMC. EML is supported by the NIGMS/IMSD grant number GM080201. DT was supported by the NIGMS/PREP training number R25 GM075149. We acknowledge technical support from the Department of Biology’s Molecular Biology Facility, supported by NIH grant number 1P20RR18754 from the Institute Development Award (IDeA) Program of the National Center for Research Resources.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.