Anton L. Bryantsev

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.

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.

Hsp27 is persistently expressed in zebrafish skeletal and cardiac muscle tissues but dispensable for their morphogenesis

Constitutive expression of Hsp27 has been demonstrated in vertebrate embryos, especially in developing skeletal and cardiac muscle. Results of several previous studies have indicated that Hsp27 could play a role in the development of these tissues. For example, inhibition of Hsp27 expression has been reported to cause defective development of mammalian myoblasts in vitro and frog embryos in vivo. In contrast, transgenic mice lacking Hsp27 develop normally. Here, we examined the distribution of Hsp27 protein in developing and adult zebrafish and effects of suppressing Hsp27 expression using phosphorodiamidate morpholino oligonucleotides (PMO) on zebrafish development. Consistent with our previous analysis of hsp27 messenger RNA expression, we detected the protein Hsp27 in cardiac, smooth, and skeletal muscle of both embryonic and adult zebrafish. However, embryos lacking detectable Hsp27 after injection of antisense hsp27 PMO exhibited comparable heart beat rates to that of control embryos and cardiac morphology was indistinguishable in the presence or absence of Hsp27. Loss of Hsp27 also had no effect on the structure of the skeletal muscle myotomes in the developing embryo. Finally, embryos injected with antisense hsp27 and scrambled control PMO displayed equal motility. We conclude that Hsp27 is dispensable for zebrafish morphogenesis but could play a role in long-term maintenance of heart and muscle tissues.

Extradenticle and Homothorax Control Adult Muscle Fiber Identity in Drosophila

Here we identify a key role for the homeodomain proteins Extradenticle (Exd) and Homothorax (Hth) in the specification of muscle fiber fate in Drosophila. exd and hth are expressed in the fibrillar indirect flight muscles but not in tubular jump muscles, and manipulating exd or hth expression converts one muscle type into the other. In the flight muscles, exd and hth are genetically upstream of another muscle identity gene, salm, and are direct transcriptional regulators of the signature flight muscle structural gene, Actin88F. Exd and Hth also impact muscle identity in other somatic muscles of the body by cooperating with Hox factors. Because mammalian orthologs of exd and hth also contribute to muscle gene regulation, our studies suggest that an evolutionarily conserved genetic pathway determines muscle fiber differentiation.

Cardiac expression of the Drosophila Transglutaminase (CG7356) gene is directly controlled by myocyte enhancer factor-2.

The myocyte enhancer factor‐2 (MEF2) family of transcription factors plays key roles in the activation of muscle structural genes. In Drosophila, MEF2 accumulates at high levels in the embryonic muscles, where it activates target genes throughout the mesoderm. Here, we identify the Transglutaminase gene (Tg; CG7356) as a direct transcriptional target of MEF2 in the cardiac musculature. Tg is expressed in cells forming the inflow tracts of the dorsal vessel, and we identify the enhancer responsible for this expression. The enhancer contains three binding sites for MEF2, and can be activated by MEF2 in tissue culture and in vivo. Moreover, loss of MEF2 function, or removal of the MEF2 binding sites from the enhancer, results in loss of Tg expression. These studies identify a new MEF2 target in the cardiac musculature. These studies provide a possible mechanism for the activation of transglutaminase genes. Furthermore, given the relevance of transglutaminase genes to human disease, these studies provide a possible mechanism for their activation. Developmental Dynamics 237:2090–2099, 2008.

Analysis of Skeletal Muscle Development in Drosophila

The Drosophila system has been invaluable in providing important insights into mesoderm specification, muscle specification, myoblast fusion, muscle differentiation, and myofibril assembly. Here, we present a series of Drosophila protocols that enable the researcher to visualize muscle precursors and differentiated muscles, at all stages of development. In doing so, we also highlight the variety of techniques that are used to create these findings. These protocols are directly used for the Drosophila system, and are provided with explanatory detail to enable the researcher to apply them to other systems.

Arrest is a regulator of fiber-specific alternative splicing in the indirect flight muscles of Drosophila

The RNA-binding protein Arrest occupies a novel intranuclear domain and directs flight muscle–specific patterns of alternative splicing in flies.

Purification of cardiac cells from Drosophila embryos

Recent genome-level innovations have enabled the identification of the entire cadre of genes that are expressed in specific tissues at particular developmental times. However, to be informative as to how individual cell types develop, this process relies upon the successful and efficient purification of cells for a particular tissue. Here, we describe a method to isolate cardiac cells from Drosophila embryos. We generated transgenic embryos in which a cardiac-specific enhancer of the Sulphonylurea receptor (Sur) gene drove expression of the green fluorescent protein (GFP) gene. Homogenized embryos were subjected to fluorescence activated cell sorting (FACS), resulting in approximately 50,000 cardiac cells purified. The prevalence of cardiac cells in the purified population was high, based upon a significant enrichment for cardiac-specific marker genes, including Sur and Toll. This enrichment also enabled the identification of cardiac-specific alternatively spliced isoforms of the Zasp66 gene. In the future, this approach can be used to describe the cardiac transcriptome of Drosophila at distinct stages of embryonic development.

The Drosophila Z-disc protein Z(210) is an adult muscle isoform of Zasp52, which is required for normal myofibril organization in indirect flight muscles

Background: Several components of Drosophila flight muscles are not characterized. Results: The Z disc protein Z(210) is an adult isoform of Zasp52, and Zasp52 is required for sarcomere structure in adult muscles. Conclusion: Flight muscles utilize a novel Zasp52 isoform. Significance: Drosophila Zasp52 is homologous to human ZASP, which is a disease gene. The Z-disc is a critical anchoring point for thin filaments as they slide during muscle contraction. Therefore, identifying components of the Z-disc is critical for fully comprehending how myofibrils assemble and function. In the adult Drosophila musculature, the fibrillar indirect flight muscles accumulate a >200 kDa Z-disc protein termed Z(210), the identity of which has to date been unknown. Here, we use mass spectrometry and gene specific knockdown studies, to identify Z(210) as an adult isoform of the Z-disc protein Zasp52. The Zasp52 primary transcript is extensively alternatively spliced, and we describe its splicing pattern in the flight muscles, identifying a new Zasp52 isoform, which is the one recognized by the Z(210) antibody. We also demonstrate that Zasp52 is required for the association of α-actinin with the flight muscle Z-disc, and for normal sarcomere structure. These studies expand our knowledge of Zasp isoforms and their functions in muscle. Given the role of Zasp proteins in mammalian muscle development and disease, our results have relevance to mammalian muscle biology.