Richard M. Cripps

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.

Pathogen Entrapment by Transglutaminase-A Conserved Early Innate Immune Mechanism

Clotting systems are required in almost all animals to prevent loss of body fluids after injury. Here, we show that despite the risks associated with its systemic activation, clotting is a hitherto little appreciated branch of the immune system. We compared clotting of human blood and insect hemolymph to study the best-conserved component of clotting systems, namely the Drosophila enzyme transglutaminase and its vertebrate homologue Factor XIIIa. Using labelled artificial substrates we observe that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Using both a human and a natural insect pathogen we provide functional proof for an immune function for transglutaminase (TG). Drosophila larvae with reduced TG levels show increased mortality after septic injury. The same larvae are also more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria while neither phagocytosis, phenoloxidase or—as previously shown—the Toll or imd pathway contribute to immunity. These results firmly establish the hemolymph/blood clot as an important effector of early innate immunity, which helps to prevent septic infections. These findings will help to guide further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions.

Drosophila MEF2 is a direct regulator of Actin57B transcription in cardiac, skeletal, and visceral muscle lineages

To identify regulatory events occurring during myogenesis, we characterized the transcriptional regulation of a Drosophila melanogaster actin gene, Actin 57B. Act57B transcription is first detected in visceral muscle precursors and is detectable in all embryonic muscles by the end of embryogenesis. Through deletion analysis we identified a 595 bp promoter element that was sufficient for high levels of expression in all three muscle lineages. This fragment contained a MEF2 binding site conserved between D. melanogaster and Drosophila virilis which bound MEF2 protein in embryo nuclear extracts. Mutation of the MEF2 site severely reduced promoter activity in embryos, and in Mef2 mutants Act57B expression was severely decreased, demonstrating MEF2 is an essential regulator of Act57B. We also showed that MEF2 likely acts synergistically with factors bound to additional sequences within the 595 bp element. These findings underline the importance of MEF2 in controlling differentiation in all muscle lineages. Our experiments reveal a novel regulatory mechanism for a structural gene where high levels of expression in all embryonic muscles is regulated through a single transcription factor binding site.

Cardiac gene regulatory networks in Drosophila

The Drosophila system has proven a powerful tool to help unlock the regulatory processes that occur during specification and differentiation of the embryonic heart. In this review, we focus upon a temporal analysis of the molecular events that result in heart formation in Drosophila, with a particular emphasis upon how genomic and other cutting-edge approaches are being brought to bear upon the subject. We anticipate that systems-level approaches will contribute greatly to our comprehension of heart development and disease in the animal kingdom.

Differential requirements for Myocyte Enhancer Factor-2 during adult myogenesis in Drosophila

Identifying the genetic program that leads to formation of functionally and morphologically distinct muscle fibers is one of the major challenges in developmental biology. In Drosophila, the Myocyte Enhancer Factor-2 (MEF2) transcription factor is important for all types of embryonic muscle differentiation. In this study we investigated the role of MEF2 at different stages of adult skeletal muscle formation, where a diverse group of specialized muscles arises. Through stage- and tissue-specific expression of Mef2 RNAi constructs, we demonstrate that MEF2 is critical at the early stages of adult myoblast fusion: mutant myoblasts are attracted normally to their founder cell targets, but are unable to fuse to form myotubes. Interestingly, ablation of Mef2 expression at later stages of development showed MEF2 to be more dispensable for structural gene expression: after myoblast fusion, Mef2 knockdown did not interrupt expression of major structural gene transcripts, and myofibrils were formed. However, the MEF2-depleted fibers showed impaired integrity and a lack of fibrillar organization. When Mef2 RNAi was induced in muscles following eclosion, we found no adverse effects of attenuating Mef2 function. We conclude that in the context of adult myogenesis, MEF2 remains an essential factor, participating in control of myoblast fusion, and myofibrillogenesis in developing myotubes. However, MEF2 does not show a major requirement in the maintenance of muscle structural gene expression. Our findings point to the importance of a diversity of regulatory factors that are required for the formation and function of the distinct muscle fibers found in animals.

Homeotic selector genes control the patterning of seven-up expressing cells in the Drosophila dorsal vessel

The linear cardiac tube of Drosophila, the dorsal vessel, is an important model organ for the study of cardiac specification and patterning in vertebrates. In Drosophila, the Hox segmentation gene abdominal-A (abd-A) is required for the specification of a functionally distinct heart region at the posterior of the dorsal vessel, from which blood is pumped anteriorly through a tube termed the aorta. Since we have previously shown that the posterior part of the aorta is specified during embryogenesis to form the adult heart during metamorphosis, we determined if the embryonic aorta is also patterned by the function of Hox segmentation genes. Using gain- and loss-of-function experiments, we demonstrate that the three Hox genes expressed in the posterior aorta and heart are sufficient to confer heart or posterior aorta fate throughout the dorsal vessel. Additionally, we demonstrate that Ultrabithorax and abd-A, but not Antennapedia, function to control cell number in the dorsal vessel. These studies add robustness to the model that homeotic selector genes pattern the Drosophila dorsal vessel, and further extend our understanding of how the cardiac tube is patterned in animal models.

Myocyte enhancer factor 2 and chorion factor 2 collaborate in activation of the myogenic program in Drosophila.

ABSTRACT The process of myogenesis requires the coordinated activation of many structural genes whose products are required for myofibril assembly, function, and regulation. Although numerous reports have documented the importance of the myogenic regulator myocyte enhancer factor 2 (MEF2) in muscle differentiation, the interaction of MEF2 with cofactors is critical to the realization of muscle fate. We identify here a genomic region required for full MEF2-mediated activation of actin gene expression in Drosophila, and we identify the zinc finger transcriptional regulator chorion factor 2 (CF2) as a factor functioning alongside MEF2 via this region. Furthermore, although both MEF2 and CF2 can individually activate actin gene expression, we demonstrate that these two factors collaborate in regulating the Actin57B target gene in vitro and in vivo. More globally, MEF2 and CF2 synergistically activate the enhancers of a number of muscle-specific genes, and loss of CF2 function in vivo results in reductions in the levels of several muscle structural gene transcripts. These findings validate a general importance of CF2 alongside MEF2 as a critical regulator of the myogenic program, identify a new regulator functioning with MEF2 to control cell fate, and provide insight into the network of regulatory events that shape the developing musculature.

Thin, a Trim32 ortholog, is essential for myofibril stability and is required for the integrity of the costamere in Drosophila

Myofibril stability is required for normal muscle function and maintenance. Mutations that disrupt myofibril stability result in individuals who develop progressive muscle wasting, or muscular dystrophy, and premature mortality. Here we present our investigations of the Drosophila l(2)thin [l(2)tn] mutant. The “thin” phenotype exhibits features of the human muscular disease phenotype in that tn mutant larvae show progressive muscular degeneration. Loss-of-function and rescue experiments determined that l(2)tn is allelic to the tn locus [previously annotated as both CG15105 and another b-box affiliate (abba)]. tn encodes a TRIM (tripartite motif) containing protein highly expressed in skeletal muscle and is orthologous to the human limb-girdle muscular dystrophy type 2H disease gene Trim32. Thin protein is localized at the Z-disk in muscle, but l(2)tn mutants showed no genetic interaction with mutants affecting the Z-line–associated protein muscle LIM protein 84B. l(2)tn, along with loss-of-function mutants generated for tn, showed no relative mislocalization of the Z-disk proteins α-Actinin and muscle LIM protein 84B. In contrast, tn mutants had significant disorganization of the costameric orthologs β-integrin, Spectrin, Talin, and Vinculin, and we present the initial description for the costamere, a key muscle stability complex, in Drosophila. Our studies demonstrate that myofibrils progressively unbundle in flies that lack Thin function through progressive costamere breakdown. Due to the high conservation of these structures in animals, we demonstrate a previously unknown role for TRIM32 proteins in myofibril stability.

Bithorax Complex genes control alary muscle patterning along the cardiac tube of Drosophila

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 10, and also produced a general elongation of the dorsal vessel. A single knockout of Ubx resulted in a reduced number of alary muscles. 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.

Characterization of muscle actin genes in Drosophila virilis reveals significant molecular complexity in skeletal muscle types.

Actin is a ubiquitous and highly conserved eukaryotic protein required for cell motility and locomotion. In this manuscript, we characterize the four muscle actin genes of the insect Drosophila virilis and demonstrate strong similarities between the D. virilis genes and their homologues in Drosophila melanogaster; intron locations are conserved, and there are few amino acid differences between homologues. We also found strong conservation in temporal expression patterns of the muscle actin genes – the homologues of the D. melanogaster genes Act57B and Act87E are expressed throughout the life cycle, whereas the other two D. virilis genes, homologous to Act79B and Act88F are specific to pupal and adult stages. In situ hybridization revealed that each D. virilis gene is expressed in a unique pattern in the muscles of the thorax and abdomen. These muscle‐specific patterns of actin isoforms suggest a greater physiological diversity for the adult muscles of insects than has been appreciated to date from their categorization into fibrillar, tubular (non‐fibrillar) and supercontractile muscle types.