Drosophila Myogenesis Research Articles

Inscuteable and numb mediate asymmetric muscle progenitor cell divisions during Drosophila myogenesis.

Each larval hemisegment comprises approximately 30 uniquely specified somatic muscles. These derive from muscle founders that arise as distinct sibling pairs from the division of muscle progenitor cells. We have analyzed the progenitor cell divisions of three mesodermal lineages that generate muscle (and pericardial cell) founders. Our results show that Inscuteable and Numb proteins are localized as cortical crescents on opposite sides of dividing progenitor cells. Asymmetric segregation of Numb into one of the sibling myoblasts depends on inscuteable and is essential for the specification of distinct sibling cell fates. Loss of numb or inscuteable results in opposite cell fate transformations-both prevent sibling myoblasts from adopting distinct identities, resulting in duplicated or deleted mesodermal structures. Our results indicate that the muscle progenitor cell divisions are intrinsically asymmetric; moreover, the involvement of both inscuteable and numb/N suggests that the specification of the distinct cell fates of sibling myoblasts requires intrinsic and extrinsic cues.

The myogenic regulatory gene Mef2 is a direct target for transcriptional activation by Twist during Drosophila myogenesis.

MEF2 is a MADS-box transcription factor required for muscle development in Drosophila. Here, we show that the bHLH transcription factor Twist directly regulates Mef2 expression in adult somatic muscle precursor cells via a 175-bp enhancer located 2245 bp upstream of the transcriptional start site. Within this element, a single evolutionarily conserved E box is essential for enhancer activity. Twist protein can bind to this E box to activate Mef2 transcription, and ectopic expression of twist results in ectopic activation of the wild-type 175-bp enhancer. By use of a temperature-sensitive mutant of twist, we show that activation of Mef2 transcription via this enhancer by Twist is required for normal adult muscle development, and reduction in Twist function results in phenotypes similar to those observed previously in Mef2 mutant adults. The 175-bp enhancer is also active in the embryonic mesoderm, indicating that this enhancer functions at multiple times during development, and its function is dependent on the same conserved E box. In embryos, a reduction in Twist function also strongly reduced Mef2 expression. These findings define a novel transcriptional pathway required for skeletal muscle development and identify Twist as an essential and direct regulator of Mef2 expression in the somatic mesoderm.

Segregation of myogenic lineages in Drosophila requires numb.

Terminal divisions of myogenic lineages in the Drosophila embryo generate sibling myoblasts that found larval muscles or form precursors of adult muscles. Alternative fates adopted by sibling myoblasts are associated with distinct patterns of gene expression. Genes expressed in the progenitor cell are maintained in one sibling and repressed in the other. These differences depend on an asymmetric segregation of Numb between sibling cells. In numb mutants, muscle fates associated with repression are duplicated and alternative muscles are lost. If numb is overexpressed the reverse transformation occurs. Numb acts to block Notch-mediated repression of genes expressed in muscle progenitor cells. Thus asymmetric cell divisions are essential determinants of muscle fates during myogenesis in Drosophila

2 Drosophila Myogenesis and insights into the Role of nautilus

Several aspects of muscle development appear to be conserved between Drosophila and vertebrate organisms. Among these is the conservation of genes that are critical to the myogenic process, including transcription factors such as nautilus. From a simplistic point of view, Drosophila therefore seems to be a useful organism for the identification of molecules that are essential for myogenesis in both Drosophila and in other species. nautilus, the focal point of this review, appears to be involved in the specification and/or differentiation of a specific subset of muscle founder cells. As with several of its vertebrate and invertebrate counterparts, it is capable of inducing a myogenic program of differentiation reminiscent of that of somatic muscle precursors when expressed in other cell types. We therefore favor the model that nautilus implements the specific differentiation program of these founder cells, rather than their specification. Further analyses are necessary to establish the validity of this working hypothesis. Studies have revealed a critical role for Pax-3 in specifying a particular subset of myogenic cells, the progenitors of the limb muscles. These myogenic cells migrate from the somite into the periphery of the organism, where they differentiate. These myoblasts do not express MyoD or myf5 until they have arrived at their destination and begin the morphologic process of myogenesis (Bober et al., 1994; Goulding et al., 1994; Williams and Ordahl, 1994). They then begin to express these genes, possibly to put the myogenic plan into action. Thus, as with nautilus, MyoD and myf5 may be necessary for the manifestation of a muscle-specific commitment that has already occurred. By comparison with vertebrates, it was anticipated that the single Drosophila gene would serve the purpose of all four vertebrate genes. However, its restricted pattern of expression and apparent loss-of-function phenotype are inconsistent with this expectation. It remains to be determined whether nautilus functions in a manner similar to just one of the vertebrate genes. Since the myf5- and MyoD-expressing myoblasts are proliferative, the loss of one cell type appears to be compensated by proliferation of the remaining cell type. This apparent plasticity may obscure differences in mutant phenotype resulting from the loss of particular cells that express each of these genes. In Drosophila, by comparison, nautilus-expressing cells committed to the myogenic program undergo few, if any, additional cell divisions, and thus no other cells are available to compensate for the loss of nautilus. Therefore, the apparent differences between the Drosophila nautilus gene and its vertebrate counterparts may reflect, at least in part, differences in the developmental systems rather than differences in the function of the genes themselves.

Patterning the dorsal longitudinal flight muscles (DLM) of Drosophila: insights from the ablation of larval scaffolds.

The six Dorsal Longitudinal flight Muscles (DLMs) of Drosophila develop from three larval muscles that persist into metamorphosis and serve as scaffolds for the formation of the adult fibers. We have examined the effect of muscle scaffold ablation on the development of DLMs during metamorphosis. Using markers that are specific to muscle and myoblasts we show that in response to the ablation, myoblasts which would normally fuse with the larval muscle, fuse with each other instead, to generate the adult fibers in the appropriate regions of the thorax. The development of these de novo DLMs is delayed and is reflected in the delayed expression of erect wing, a transcription factor thought to control differentiation events associated with myoblast fusion. The newly arising muscles express the appropriate adult-specific Actin isoform (88F), indicating that they have the correct muscle identity. However, there are frequent errors in the number of muscle fibers generated. Ablation of the larval scaffolds for the DLMs has revealed an underlying potential of the DLM myoblasts to initiate de novo myogenesis in a manner that resembles the mode of formation of the Dorso-Ventral Muscles, DVMs, which are the other group of indirect flight muscles. Therefore, it appears that the use of larval scaffolds is a superimposition on a commonly used mechanism of myogenesis in Drosophila. Our results show that the role of the persistent larval muscles in muscle patterning involves the partitioning of DLM myoblasts, and in doing so, they regulate formation of the correct number of DLM fibers.

The function of type IV collagen during Drosophila muscle development

Type IV collagen forms a network that provides the major structural support for basement membranes. Basement membranes are specialized forms of extracellular matrix with important functions in development. One collagen gene (Dcg1) was characterized in Drosophila melanogaster and shown to encode a collagen chain related to vertebrate basement membrane type IV collagen chains. Therefore, to access the functional importance of type IV collagen during Drosophila myogenesis, we adopted two different approaches to decrease the Dcg1 gene expression in Drosophila embryos. We describe, here, that the decrease in Dcg1 gene expression causes, in particular, defective muscle attachments. These mutant phenotypes suggest that type IV collagen acts to stabilize cell-matrix interactions.

Myocyte-specific enhancer factor 2 acts cooperatively with a muscle activator region to regulate Drosophila tropomyosin gene muscle expression.

MEF2 (myocyte-specific enhancer factor 2) is a MADS box transcription factor that is thought to be a key regulator of myogenesis in vertebrates. Mutations in the Drosophila homologue of the mef2 gene indicate that it plays a key role in regulating myogenesis in Drosophila. We show here that the Drosophila tropomyosin I (TmI) gene is a target gene for mef2 regulation. The TmI gene contains a proximal and a distal muscle enhancer within the first intron of the gene. We show that both enhancers contain a MEF2 binding site and that a mutation in the MEF2 binding site of either enhancer significantly reduces reporter gene expression in embryonic, larval, and adult somatic body wall muscles of transgenic flies. We also show that a high level of proximal enhancer-directed reporter gene expression in somatic muscles requires the cooperative activity of MEF2 and a cis-acting muscle activator region located within the enhancer. Thus, mef2 null mutant embryos show a significant reduction but not an elimination of TmI expression in the body wall myoblasts and muscle fibers that are present. Surprisingly, there is little effect in these mutants on TmI expression in developing visceral muscles and dorsal vessel (heart), despite the fact that MEF2 is expressed in these muscles in wild-type embryos, indicating that TmI expression is regulated differently in these muscles. Taken together, our results show that mef2 is a positive regulator of tropomyosin gene transcription that is necessary but not sufficient for high level expression in somatic muscle of the embryo, larva, and adult.

A Series of Mutations in the D-MEF2 Transcription Factor Reveal Multiple Functions in Larval and Adult Myogenesis in Drosophila

The D-mef2 gene encodes a MADS domain transcription factor expressed in differentiated muscles and their precursors in the Drosophila embryo. Embryos deficient for D-MEF2 protein due to a deletion of upstream transcriptional control sequences fail to form muscle, suggesting that the gene is required for muscle cell differentiation. To directly demonstrate a role for D-mef2 in embryonic myogenesis, we isolated gene mutants containing EMS-induced point mutations, characterized the effects of these mutations on D-MEF2 protein stability and nuclear localization, and analyzed the resulting muscle phenotypes. Our results show that in the somatic muscle lineage, D-mef2 is required for both the formation and patterning of body wall muscle. In the absence of somatic myogenesis, there is extensive apoptosis among the myoblast cell population. In contrast, in the cardiac muscle lineage, morphogenesis of the dorsal vessel occurs normally but the three myosin subunit genes are not expressed. Mutant embryos also exhibit an abnormal midgut morphology, which correlates with the absence of α PS2 integrin gene expression and muscle-specific enhancer function, suggesting that D-mef2 regulates the inflated locus which encodes this integrin subunit. D-MEF2 is also expressed in adepithelial cells and rare D-mef2 transheterozygous mutant adults fail to fly, consistent with defects observed in the indirect flight muscles. These results demonstrate that the D-mef2 gene has multiple functions in myogenesis and tissue morphogenesis during Drosophila development.

Expression of a MyoD family member prefigures muscle pattern in Drosophila embryos.

We have isolated a Drosophila gene that is expressed in a temporal and spatial pattern during embryogenesis, strongly suggesting an important role for this gene in the early development of muscle. This gene, which we have named nautilus (nau), encodes basic and helix-loop-helix domains that display striking sequence similarity to those of the vertebrate myogenic regulatory gene family. nau transcripts are initially localized to segmentally repeated clusters of mesodermal cells, a pattern that is reminiscent of the expression of the achaete-scute genes in the Drosophila peripheral nervous system. These early nau-positive cells are detected just prior to the first morphological evidence of muscle cell fusion and occupy similar positions as the later-appearing muscle precursors. Subsequently, nau transcripts are present in at least a subset of growing muscle precursors and mature muscle fibers that exhibit distinct segmental differences. These observations establish nau as the earliest known marker of myogenesis in Drosophila and indicate that this gene may be a key determinant of pattern formation in the embryonic mesoderm.

A Genetic Approach to the Study of Neurogenesis and Myogenesis

Extensive sequences of morphological differentiation have been described for Drosophila melanogaster neurogenesis and myogenesis. Neuroblasts and myoblasts become determined in the blastoderm or shortly thereafter. Each blast cell will then morphologically differentiate in vitro in sequences that closely resemble the events in vivo. Functional neurons, muscle cells and neuromuscular junctions form and these have normal biochemical and ultrastructural characteristics. A mutant hunt was conducted which was designed to generate mutations specific to neurogenesis or myogenesis. One mutation was recovered that apparently interfered only with myogenesis. The frequency of recovered mutations that affected cell differentiation was low and led to estimates that relatively few genes are required to preserve the structural integrity of cells during neurogenesis and myogenesis.

A 5-Bromodeoxyuridine-sensitive Interval during Drosophila Myogenesis

Drosophila myogenesis was monitored in vitro and the cells were treated with 5-bromodeoxyuridine (BrdU) or with thymidine at certain intervals. Muscle cells were scored for survival, contractility, and the uptake of thymidine and BrdU. Results indicated that the final S period for myoblasts takes place in vitro between 1.3 and 3.3 h following the initiation of gastrulation in the donor embryos. Treatment with 10(-4) M BrdU during this internal inhibited myogenesis, but later treatment did not. Thymidine reversed or prevented the BrdU effect if given before the final myoblast division, but not if given after wards. All results support the hypothesis that BrdU inhibits Drosophila myogenesis through its incorporation into DNA.

Ultrastructural differentiation during embryonic Drosophila myogenesis in vitro.

Cultures of embryonic Drosophila melanogaster cells were examined by electron microscopy and events in myogenesis were recorded. Thick and thin myofilaments, T-tubules and sarcoplasmic reticulum all appeared at about the same time, 10.5 hr. This was about 5 hr after the final division of myoblasts and about the time that muscle cells were elongating, aligning and fusing. Sarcoplasm typical of insect muscle was detected by 18.5 hr, as were myotendonal and tendocuticular junctions. Two populations of myocytes were detected, the cytoplasm of one more electron-dense than the other. The only previous report of myofibrilogenesis in invertebrate embryos had described novel mechanisms. In Drosophila embryonic material, however, the sequence of myofibrilogenesis resembled that in postembryonic insect or vertebrate material.