Room temperature operable autonomously moving bio-microrobot powered by insect dorsal vessel tissue

Living muscle tissues and cells have been attracting attention as potential actuator candidates. In particular, insect dorsal vessel tissue (DVT) seems to be well suited for a bio-actuator since it is capable of contracting autonomously and the tissue itself and its cells are more environmentally robust under culturing conditions compared with mammalian tissues and cells. Here we demonstrate an autonomously moving polypod microrobot (PMR) powered by DVT excised from an inchworm. We fabricated a prototype of the PMR by assembling a whole DVT onto an inverted two-row micropillar array. The prototype moved autonomously at a velocity of 3.5 × 10(-2) μm/s, and the contracting force of the whole DVT was calculated as 20 μN. Based on the results obtained by the prototype, we then designed and fabricated an actual PMR. We were able to increase the velocity significantly for the actual PMR which could move autonomously at a velocity of 3.5 μm/s. These results indicate that insect DVT has sufficient potential as the driving force for a bio-microrobot that can be utilized in microspaces.     

Spontaneous contractions of dispersed vascular muscle in cell culture.

Dispersed vascular muscle cells from chick omphalomesenteric vessels maintained in primary cell culture contracted spontaneously. Six methods which produced contracting isolated muscle cells are described and compared. The combination of dispersion method and culture conditions to produce contracting muscle cells was more critical for vascular than for heart muscle. These findings of continuing pacemaker function demonstrate that functional integrity of isolated vascular muscle cells is possible to maintain. Further indication of the full functional state of the isolated vascular muscle cells was demonstrated by the sensitivity to norepinephrine at a physiological concentration (0.1 muM). Spontaneous contraction frequencies were similar to the range found in situ, and spontanious or norepinephrine-induced contractions had time courses corresponding to intact vessel contractions. This is the first report that isolated vascular muscle cells in primary cell culture retain functional characteristics found in situ and are suitable for pharmacological characterization of individual muscle cells.     

Electrical stimulation of cultured lepidopteran dorsal vessel tissue: an experiment for development of bioactuators

An insect dorsal vessel (DV) is well suited for a bioactuator since it is capable of contracting autonomously, and its tissue and cells are more environmentally robust under culturing conditions compared with mammalian tissue. In this study, electrical pulse stimulation was examined so as to regulate a bioactuator using the DV tissue. The DV tissue of a larva of Ctenoplusia agnate was assembled on a micropillar array, which was stimulated after culturing for about 3 wk. The contraction of the DV tissue was evaluated by image analysis to measure lateral displacements at the micropillar top. As a result, suitable stimulation conditions in a 35-mm petri dish were determined as: applied voltage of 10 V with 20-ms duration. Next, the time lag between the onset of electrical stimulus and the onset of mechanical contraction (electromechanical delay (EMD)) was estimated. A light-emitting diode (LED) was connected serially with the petri dish, and the LED flashed when electrical pulses were given. Movie images were analyzed in which electrical pulses made the DV tissue contract and the LED flashed virtually simultaneously; from these, the EMD was estimated as approximately 50 ms. These results suggest that the electrical pulse stimulation is capable of regulating the DV tissue, and the micropillar array is a useful biological tool to investigate physiological properties of muscle tissue.     

Isolation and maintenance-free culture of contractile myotubes from Manduca sexta embryos

Skeletal muscle tissue engineering has the potential to treat tissue loss and degenerative diseases. However, these systems are also applicable for a variety of devices where actuation is needed, such as microelectromechanical systems (MEMS) and robotics. Most current efforts to generate muscle bioactuators are focused on using mammalian cells, which require exacting conditions for survival and function. In contrast, invertebrate cells are more environmentally robust, metabolically adaptable and relatively autonomous. Our hypothesis is that the use of invertebrate muscle cells will obviate many of the limitations encountered when mammalian cells are used for bioactuation. We focus on the tobacco hornworm, Manduca sexta, due to its easy availability, large size and well-characterized muscle contractile properties. Using isolated embryonic cells, we have developed culture conditions to grow and characterize contractile M. sexta muscles. The insect hormone 20-hydroxyecdysone was used to induce differentiation in the system, resulting in cells that stained positive for myosin, contract spontaneously for the duration of the culture, and do not require media changes over periods of more than a month. These cells proliferate under normal conditions, but the application of juvenile hormone induced further proliferation and inhibited differentiation. Cellular metabolism under normal and low glucose conditions was compared for C2C12 mouse and M. sexta myoblast cells. While differentiated C2C12 cells consumed glucose and produced lactate over one week as expected, M. sexta muscle did not consume significant glucose, and lactate production exceeded mammalian muscle production on a per cell basis. Contractile properties were evaluated using index of movement analysis, which demonstrated the potential of these cells to perform mechanical work. The ability of cultured M. sexta muscle to continuously function at ambient conditions without medium replenishment, combined with the interesting metabolic properties, suggests that this cell source is a promising candidate for further investigation toward bioactuator applications.     

Peptidergic control of the heart of the stick insect, Baculum extradentatum

The dorsal vessel of the Vietnamese stick insect, Baculum extradentatum, consists of a tubular heart and an aorta that extends anteriorly into the head. Alary muscles, associated with the heart, are anchored to the body wall with attachments to the dorsal diaphragm. Alary muscle contraction draws haemolymph into the heart through incurrent ostia. Excurrent ostia lie on the dorsal vessel in the last thoracic and in each of the first two abdominal segments. Muscle fibers are associated with these excurrent ostia. Crustacean cardioactive peptide (CCAP)- and proctolin-like immunoreactivity is present in axons of the segmental nerves that project to the dorsal vessel, and in processes extending over the heart and alary muscles. Proctolin-like immunoreactive processes are also localized to the valves of the incurrent ostia and to the excurrent ostia. Neither the link nerve neurons, nor the lateral cardiac neurons, stain positively for these peptides. Physiological assays reveal dose-dependent increases in heart beat frequency in response to CCAP and proctolin. Isolating the dorsal vessel from the ventral nerve cord led to a change in the pattern of heart contractions, from a tonic, stable heart beat, to one which was phasic. The tonic nature was restored by the application of CCAP.     

Electrically excitable normal rat kidney fibroblasts: A new model system for cell-semiconductor hybrids

In testing various designs of cell-semiconductor hybrids, the choice of a suitable type of electrically excitable cell is crucial. Here normal rat kidney (NRK) fibroblasts are presented as a cell line, easily maintained in culture, that may substitute for heart or nerve cells in many experiments. Like heart muscle cells, NRK fibroblasts form electrically coupled confluent cell layers, in which propagating action potentials are spontaneously generated. These, however, are not associated with mechanical disturbances. Here we compare heart muscle cells and NRK fibroblasts with respect to action potential waveform, morphology, and substrate adhesion profile, using the whole-cell variant of the patch-clamp technique, atomic force microscopy (AFM), and reflection interference contrast microscopy (RICM), respectively. Our results clearly demonstrate that NRK fibroblasts should provide a highly suitable test system for investigating the signal transfer between electrically excitable cells and extracellular detectors, available at a minimum cost and effort for the experimenters.     

Influence of different culture conditions on sarcoplasmic reticular calcium transport in isolated neonatal rat cardiomyocytes

This study investigates sarcoplasmic reticulum (SR) calcium-(Ca²⁺) transport ATPase (SERCA2a) and phospholamban (PLB) in cultured spontaneously contracting neonatal rat cardiomyocytes (CM) to ascertain the function of both SR proteins under various culture conditions. The two major SR proteins were readily detectable in cultured CM by immunofluorescent microscopy using specific anti-SERCA2 and anti-PLB antibodies. Double labeling technique revealed that PLB-positive CM also labeled with anti-SERCA2. Coexpression of SERCA2 and PLB in CM was supported by measurement of cell homogenate oxalate-supported Ca²⁺ uptake which was completely inhibited by thapsigargin and stimulated by protein kinase A-catalyzed phosphorylation. Under serum-free conditions, incubation of CM with the SERCA2a expression modulator 3,3,5-triiodo-L-thyronine (100 nM, 72 h) resulted in elevated Ca²⁺ uptake of +33%. Specific Ca²⁺ uptake activity was not altered if insulin was omitted from the serum-free culture medium but total SR Ca²⁺ transport activity was reduced under this culture condition. The results indicate that primary culture of spontaneously contracting neonatal rat CM can be employed as a useful model system for investigating both short- and long-term mechanisms determining the Ca²⁺ re-uptake function of the SR under defined culture conditions.     

Ostia, the inflow tracts of the Drosophila heart, develop from a genetically distinct subset of cardial cells.

The homeobox gene tinman and the nuclear receptor gene seven-up are expressed in mutually exclusive dorsal vessel cells in Drosophila, however, the physiological reason for this distinction is not known. We demonstrate that tin and svp-lacZ expression persists through the larval stage to the adult stage in the same pattern of cells expressing these genes in the embryo. In the larva, six pairs of Svp-expressing cells form muscular ostia, which permit hemolymph to enter the heart for circulation, however, more anterior Svp-expressing cells form the wall of the dorsal vessel. During pupation, the adult heart forms from a chimera of larval and imaginal muscle fibers. The portion of the dorsal vessel containing the larval ostia is histolyzed and the anterior Svp-expressing cells metamorphose into imaginal ostia. This is the first demonstration that the significant molecular diversity of cardial cells identified in the embryonic heart correlates with the formation of physiologically and functionally distinct muscle cells in the animal. Furthermore, our experiments define the cellular changes that occur as the larval heart is remodeled into an imaginal structure in an important model organism.     

Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation

This work describes the step-by-step development of a novel, serum-free, in vitro cell culture system resulting in the formation of robust, contracting, multinucleate myotubes from dissociated skeletal muscle cells obtained from the hind limbs of fetal rats. This defined system consisted of a serum-free medium formulation developed by the systematic addition of different growth factors as well as a nonbiological cell growth promoting substrate, N-1[3-(trimethoxysilyl) propyl] diethylenetriamine. Each growth factor in the medium was experimentally evaluated for its effect on myotube formation. The resulting myotubes were evaluated immunocytochemically using embryonic skeletal muscle, specifically the myosin heavy chain antibody. Based upon this analysis, we propose a new skeletal muscle differentiation protocol that reflects the roles of the various growth factors which promote robust myotube formation. Further observation noted that the proposed skeletal muscle differentiation technique also supported muscle–nerve coculture. Immunocytochemical evidence of nerve–muscle coculture has also been documented. Applications for this novel culture system include biocompatibility and skeletal muscle differentiation studies, understanding myopathies, neuromuscular disorders, and skeletal muscle tissue engineering.     

Engineered heart tissue graft derived from somatic cell nuclear transferred embryonic stem cells improve myocardial performance in infarcted rat heart

The concept of regenerating diseased myocardium by implanting engineered heart tissue (EHT) is intriguing. Yet it was limited by immune rejection and difficulties to be generated at a size with contractile properties. Somatic cell nuclear transfer is proposed as a practical strategy for generating autologous histocompatible stem (nuclear transferred embryonic stem [NT‐ES]) cells to treat diseases. Nevertheless, it is controversial as NT‐ES cells may pose risks in their therapeutic application. EHT from NT‐ES cell‐derived cardiomyocytes was generated through a series of improved techniques in a self‐made mould to keep the EHTs from contraction and provide static stretch simultaneously. After 7 days of static and mechanical stretching, respectively, the EHTs were implanted to the infarcted rat heart. Four weeks after transplantation, the suitability of EHT in heart muscle repair after myocardial infarction was evaluated by histological examination, echocardiography and multielectrode array measurement. The results showed that large (thickness/diameter, 2–4 mm/10 mm) spontaneously contracting EHTs was generated successfully. The EHTs, which were derived from NT‐ES cells, inte grated and electrically coupled to host myocardium and exerted beneficial effects on the left ventricular function of infarcted rat heart. No teratoma formation was observed in the rat heart implanted with EHTs for 4 weeks. NT‐ES cells can be used as a source of seeding cells for cardiac tissue engineering. Large contractile EHT grafts can be constructed in vitro with the ability to survive after implantation and improve myocardial performance of infarcted rat hearts.     

Clonal derivation of a rat muscle cell strain that forms contraction-competent myotubes

Summary A muscle cell strain capable of forming contracting myotubes was isolated from an established rat embryo cell line. The myogenic cells, termed rat myoblast omega or RMo cells, have a diploid complement of chromosomes (n=42). In the presence of mitogen-containing growth medium, RMo cells proliferated with a cell generation time of about 12 hours. In mitogen-depleted medium, RMo cells withdrew from the cell cycle and formed myotubes that spontaneously contracted. Differentiated RMo cells produced creatine kinase isozymes in a ratio characteristic of skeletal muscle cells. RMo cells were easy to cultivate. Cells proliferated and differentiated equally well on gelatin-coated or noncoated culture dishes, at clonal or mass culture densities, and in all basal media tested. In most experiments, growth medium consisted of horse serum-containing medium supplemented with either chicken embryo extract or FGF activity; cells proliferated equally well in medium containing unsupplemented calf serum. RMo cells differentiated if growth medium was not replenished regularly. Alternatively, differentiation was induceable by incubation in mitogen-depleted medium consisting of basal medium supplemented either with 10⁻⁶ M insulin, 0.5% serum, or 50% conditioned growth medium. RMo cells were competently transformed with cloned exogenous genes. Because it forms functional myofibrils, the RMo cell line constitutes a useful model system for studying the cell biology and biochemistry of proteins involved in contractile apparatus assembly and muscle disease. This work was supported by NIH research grant GM34432 and Research Career Development Award AG00334. Editor's Statement This report documents the characterization of a differentiating rat cell line that does not show the karyotypic shift toward polyploidy usually observed in rodent cell lines. Investigators already are finding this line valuable in studies of regulation of growth and differentiation.     

Do rat cardiac myocytes release ATP on contraction?

ATP is released by numerous cell types in response to mechanical strain. It then acts as a paracrine or autocrine signaling molecule, inducing a variety of biological responses. In this work, we addressed the question whether mechanical force acting on the membranes of contracting cardiomyocytes during periodic longitudinal shortening can stimulate the release of ATP. Electrically stimulated isolated adult rat cardiomyocytes as well as spontaneously contracting mouse cardiomyocytes derived from embryonic stem (ES) cells were assayed for ATP release with the use of luciferase and a sensitive charge-coupled device camera. Sensitivity of soluble luciferase in the supernatant of cardiomyocytes was 100 nM ATP, which is 10-fold below the EC₅₀ values for most purinergic receptors expressed in the heart (1.5–20 µM). Light intensities were not different between resting or contracting adult rat cardiomyocytes. Similar results were obtained with ES-cell-derived contracting mouse cardiomyocytes. ATP release was measurable only from obviously damaged or permeabilized cells. To increase selectivity and sensitivity of ATP detection we have targeted a recombinant luciferase to the sarcolemmal membrane using a wheat germ agglutinin-IgG linker. Contraction of labeled adult rat cardiomyocytes was not associated with measurable bioluminescence. However, when human umbilical vein endothelial cells were targeted with membrane-bound luciferase, shear stress-induced ATP release could be clearly detected, demonstrating the sensitivity of the detection method. In the present study, we did not detect ATP release from contracting cardiomyocytes on the single cell level, despite adequate sensitivity of the detection system. Thus deformation of the contracting cardiomyocyte is not a key stimulus for the release of cellular ATP. cardiomyocytes; luciferase     

Hormonal regulation of rat granulosa cell deoxyribonucleic acid synthesis: effects of estrogens

Although it is widely accepted that estrogens exert a major trophic effect on follicular growth, their mechanism of action has not been established. We examined the effect of estrogen treatment in vivo or in vitro on DNA synthesis in rat granulosa cells cultured under defined conditions (DMEM:F12, collagen-coated plastic wells). Treatment with diethylstilbestrol (DES) in vivo (silastic implants containing 5 mg DES) for at least 2 days was required to observe a significant stimulation of 3H-thymidine incorporation by insulin (1 microgram/ml) in culture. Rat thecal/interstitial cells (TI) were isolated from DES-treated rats and cultured under the same conditions as granulosa cells. Conditioned media from TI cells stimulated DNA synthesis in granulosa cell cultures (as much as twofold). This effect was markedly amplified by estradiol treatment (1 microgram/ml) of the TI cell cultures. Addition of estradiol to granulosa cell cultures enhanced the effect of conditioned medium from nontreated TI cells. Conditioned medium from estradiol-treated TI cells stimulated DNA synthesis in granulosa cells from both DES-treated and nontreated rats. Estradiol had no effect when added directly to purified granulosa cell cultures but stimulated 3H-thymidine incorporation in crude preparations of ovarian cells. The stimulatory effects of TI cell-conditioned medium and insulin were reflected in the final cell densities achieved after 9 days in culture. We conclude that the mitogenic actions of estrogens in the ovary involve sensitization of granulosa cells to locally present mitogens like insulin and a TI cell-derived growth factor.     

A primary culture system of adult rat heart cells for the study of toxicologic agents

Summary Tricyclic antidepressants (TCAs) are currently used in the treatment of mental depression and nocturnal enuresis. Clinically, these drugs are useful; however, cardiotoxicity can occur even with therapeutic dosages. For example, TCAs are known to alter myocardial function, induce arrhythmias, and produce heart block in individuals with a normal cardiovascular history. The present study was undertaken to establish a culture system of spontaneously contracting adult primary myocardial cells for toxicologic testing and to examine their contractility, morphology, and lactate dehydrogenase release (LDH) after treatment with one of the most cardiotoxic TCAs, amitriptyline. Primary myocardial cell cultures were obtained from approximately 60- to 90-day-old Sprague-Dawley rats. After the cells had been grown in culture for 11 days, they were treated with amitriptyline (1 × 10⁻³, 1 × 10⁻⁴, and 1 × 10⁻⁵ M) for 2 to 24 h. The highest concentration of amitriptyline (1 × 10⁻³ M) completely destroyed the cardiac muscle cells. In addition to moderate and severe vacuole, granule, and pseudopodia formation, all contractile activity was inhibited as early as 2 h after exposure to the intermediate concentration of 1 × 10⁻⁴ M amitriptyline. Significant LDH release did not occur until 8 h after treatment with this intermediate concentration. Even though there was no significant LDH release at all 3 time points tested, there was a 50% decrease in beating activity (154±9 to 77±5 beats/min) and initiation of vacuole formation by 2 h with the lowest concentration of amitriptyline (1 × 10⁻⁵ M). This study presents a new apparatus for the isolation of adult cardiac myocytes for the establishment of primary cell cultures for toxicologic testing. Furthermore, these data demonstrate that amitriptyline induces a concentration- and time-dependent cardiotoxic profile in a model of spontaneously contracting adult cardiac muscle cells in culture.     

Hypoxia-induced pulmonary artery adventitial remodeling and neovascularization: contribution of progenitor cells

Information is rapidly emerging regarding the important role of the arterial vasa vasorum in a variety of systemic vascular diseases. In addition, increasing evidence suggests that progenitor cells of bone marrow (BM) origin may contribute to postnatal neovascularization and/or vascular wall thickening that is characteristic in some forms of systemic vascular disease. Little is known regarding postnatal vasa formation and the role of BM-derived progenitor cells in the setting of pulmonary hypertension (PH). We sought to determine the effects of chronic hypoxia on the density of vasa vasorum in the pulmonary artery and to evaluate if BM-derived progenitor cells contribute to the increased vessel wall mass in a bovine model of hypoxia-induced PH. Quantitative morphometric analyses of lung tissue from normoxic and hypoxic calves revealed that hypoxia results in a dramatic expansion of the pulmonary artery adventitial vasa vasorum. Flow cytometric analysis demonstrated that cells expressing the transmembrane tyrosine kinase receptor for stem cell factor, c-kit, are mobilized from the BM in the circulation in response to hypoxia. Immunohistochemistry revealed an increase in the expression of c-kit+ cells together with vascular endothelial growth factor, fibronectin, and thrombin in the hypoxia-induced remodeled pulmonary artery vessel wall. Circulating mononuclear cells isolated from neonatal calves exposed to hypoxia were found to differentiate into endothelial and smooth muscle cell phenotypes depending on culture conditions. From these observations, we suggest that the vasa vasorum and circulating progenitor cells could be involved in vessel wall thickening in the setting of hypoxia-induced PH.     

A non-contact suspension culture approach to the culture of osteogenic cells derived from a CD49elow subpopulation of human bone marrow-derived cells

We demonstrate that adult human bone marrow (BM) contains a population of mesenchymal stromal cells (MSCs) that can be expanded in non-adherent, cytokine-dependent, suspension culture conditions for at least 42 days. The cells generated during suspension culture lacked detectable levels of gene expression associated with differentiated mesenchymal cell types, including bone, muscle and fat, suggesting that suspension culture maintains MSCs in an uncommitted state. However, when these undifferentiated cells were taken out of suspension culture and placed in adherent osteogenic conditions, osteogenic genes were upregulated and morphologically identifiable bone matrix was elaborated. Flow cytometric analysis of uncultured, density gradient-separated human BM revealed that colony forming unit-fibroblast (CFU-F) and CFU-osteoblast (CFU-O) activity was associated with a CD45(-) CD49e(low) phenotype. Importantly, suspension-grown MSCs, capable of CFU-F and CFU-O development, maintained the CD45(-)CD49e(low) phenotype whereas MSCs directly cultured under adherent conditions rapidly upregulated CD49e expression and were associated with a CD45(-)CD49e(high) phenotype. Tracking the CD49e(low) expression under suspension culture conditions provides a mechanism to isolate an expanding suspension-grown MSC population with osteogenic potential. This could provide a potential strategy to isolate populations of MSCs, with functional osteogenic capacity, in a scalable and controllable culture system for therapeutic applications.     

Influence of baculovirus-host cell interactions on complex N-linked glycosylation of a recombinant human protein

The conditions required for mammalian-type complex N-linked glycosylation of human proteins produced in insect cells with the baculovirus expression vector system were investigated. Marked alterations to N-linked glycosylation of human placental secreted alkaline phosphatase (SEAP) were observed with different baculovirus species, insect cell lines, and cell culture media. When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was used to produce SEAP in Trichoplusia ni (Tn-4h) cells cultured in serum-free medium, structural analyses indicated <1% hybrid and no complex oligosaccharides attached to SEAP, a typical result with the baculovirus expression vector system. However, when fetal bovine serum was added to the culture medium, 48 +/- 4% of the oligosaccharides were hybrid or complex (but asialylated) glycans. When a recombinant T. ni nucleopolyhedrovirus (TnSNPV) was similarly used to express SEAP in Tn-4h cells cultured in serum-containing medium, only 24 +/- 3% of the glycans contained terminal N-acetylglucosamine and/or galactose residues. In contrast, SEAP produced in Sf9 cells grown in serum-containing medium with AcMNPV contained <1% hybrid oligosaccharides and no complex oligosaccharides. The results illustrate that baculovirus type, host cell type, and the growth medium all have a strong influence on the glycosylation pathway in insect cells, resulting in significant alterations in structures and relative abundance of N-linked glycoforms. Although the addition of sialic acid residues to the SEAP glycans was not detected, possible approaches to obtain sialylated glycans are discussed.     

Differentiation of skeletal muscle and integration of myotubes with silicon microstructures using serum-free medium and a synthetic silane substrate

This protocol describes a cell culture model to study the differentiation of fetal rat skeletal muscle cells. The model uses serum-free medium, a nonbiological substrate N-1[3(trimethoxysilyl)propyl] diethylenetriamine (DETA) and fabricated microcantilevers to promote the differentiation of dissociated rat myocytes into robust myotubes. In this protocol, we also describe how to characterize the myotubes on the basis of morphology, immunocytochemistry and electrophysiology. Here, four major techniques are employed: fabrication of cantilevers, surface modification of the glass and cantilever substrates with a DETA SAM, a serum-free medium and refined culture techniques. This culture system has potential applications in biocompatibility studies, bioartificial muscle engineering, skeletal muscle differentiation studies and for better understanding of myopathies and neuromuscular disorders. The model can be established in 26-33 d.     

The Hox gene abdominal-A specifies heart cell fate in the Drosophila dorsal vessel

The Drosophila melanogaster dorsal vessel is a linear organ that pumps blood through the body. Blood enters the dorsal vessel in a posterior chamber termed the heart, and is pumped in an anterior direction through a region of the dorsal vessel termed the aorta. Although the genes that specify dorsal vessel cell fate are well understood, there is still much to be learned concerning how cell fate in this linear tube is determined in an anteroposterior manner, either in Drosophila or in any other animal. We demonstrate that the formation of a morphologically and molecularly distinct heart depends crucially upon the homeotic segmentation gene abdominal-A (abd-A). abd-A expression in the dorsal vessel was detected only in the heart, and overexpression of abd-A induced heart fate in the aorta in a cell-autonomous manner. Mutation of abd-A resulted in a loss of heart-specific markers. We also demonstrate that abd-A and sevenup co-expression in cardial cells defined the location of ostia, or inflow tracts. Other genes of the Bithorax Complex do not appear to participate in heart specification, although high level expression of Ultrabithorax is capable of inducing a partial heart fate in the aorta. These findings for the first time demonstrate a specific involvement for Hox genes in patterning the muscular circulatory system, and suggest a mechanism of broad relevance for animal heart patterning.