Loss-of-function mutations reveal that the Drosophila nautilus gene is not essential for embryonic myogenesis or viability.

nautilus (nau), the single Drosophila member of the bHLH-containing myogenic regulatory family of genes, is expressed in a subset of muscle precursors and differentiated fibers. It is capable of inducing muscle-specific transcription as well as myogenic transformation, and plays a role in the differentiation of a subset of muscle precursors into mature muscle fibers. In previous studies, the nau zygotic loss-of-function phenotype was determined using genetic deficiencies in which the gene is deleted. We note that this genetic loss-of-function phenotype differs from the loss-of-function phenotype determined using RNA interference (L. Misquitta and B. M. Paterson, 1999, Proc. Natl. Acad. Sci. USA 96, 1451-1456). The present study re-examines this loss-of-function phenotype using EMS-induced mutations that specifically alter the nau gene, and extends the genetic analysis to include the loss of both maternal and zygotic nau function. In brief, embryos lacking nau both maternally and zygotically are missing a distinct subset of muscle fibers, consistent with its apparent expression in a subset of muscle fibers. The muscle loss is tolerated, however, such that the loss of nau both maternally and zygotically does not result in lethality at any stage of development.

Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion.

The Drosophila sticks-and-stones (sns) locus was identified on the basis of its mutant phenotype, the complete absence of body wall muscles and corresponding presence of unfused myoblasts. The genetic location of the mutation responsible for this apparent defect in myoblast fusion was determined by recombination and deficiency mapping, and the corresponding wild-type gene was isolated in a molecular walk. Identification of the SNS coding sequence revealed a putative member of the immunoglobulin superfamily (IgSF) of cell adhesion molecules. As anticipated from this homology, SNS is enriched at the membrane and clusters at discrete sites, coincident with the occurrence of myoblast fusion. Both the sns transcript and the encoded protein are expressed in precursors of the somatic and visceral musculature of the embryo. Within the presumptive somatic musculature, SNS expression is restricted to the putative fusion-competent cells and is not detected in unfused founder cells. Thus, SNS represents the first known marker for this subgroup of myoblasts, and provides an opportunity to identify pathways specifying this cell type as well as transcriptional regulators of fusion-specific genes. To these ends, we demonstrate that the presence of SNS-expressing cells is absolutely dependent on Notch, and that expression of SNS does not require the myogenic regulatory protein MEF2.

Identification of a Drosophila homologue to vertebrate Crk by interaction with MBC.

The vertebrate adapter protein termed Crk was initially identified from the chicken CT10 retrovirus on the basis of its transforming activity (Mayer et al., 1988. Nature 332, 272-275). We have identified a Drosophila protein with homology to vertebrate Crk, termed dCRK, by interaction with the protein encoded by the Drosophila myoblast city (mbc) gene. The dCRK protein has extensive homology to the both the Crk-II form of vertebrate Crk and the Crk-related protein CRKL, and includes one SH2 domain followed by two SH3 domains. A single protein of approx. 37kDa is detected in extracts from embryos, and Northern analysis revealed a single transcript of 1.3kb. The dCrk mRNA is abundant throughout embryogenesis, declines during the larval stages, and reappears during pupation. In addition, maternally-provided transcripts have been detected. During embryogenesis, the spatial distribution of this transcript is relatively broad and appears to include all germ layers. Finally, dCrk is located on the fourth chromosome, approximately at cytological position 101F-102A.

A role for nautilus in the differentiation of muscle precursors.

In the Drosophila embryo, nautilus is expressed in a subset of muscle precursors and differentiated fibers and is capable of inducing muscle-specific transcription, as well as myogenic transformation. In this study, we examine the consequences of nautilus loss-of-function on the development of the somatic musculature. Genetic and molecular characterization of two overlapping deficiencies, Df(3R)nau-9 and Df(3R)nau-11a4, revealed that both of these deficiencies remove the nautilus gene without affecting a common lethal complementation group. Individuals transheterozygous for these deficiencies survive to adulthood, indicating that nautilus is not an essential gene. These embryos are, however, missing a subset of muscle fibers, providing evidence that (1) some muscle loss can be tolerated throughout larval development and (2) nautilus does play a role in muscle development. Examination of muscle precursors in these embryos revealed that nautilus is not required for the formation of muscle precursors, but rather plays a role in their differentiation into mature muscle fibers. Thus, we suggest that nautilus functions in a subset of muscle precursors to implement their specific differentiation programs.

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.

Drosophila myoblast city encodes a conserved protein that is essential for myoblast fusion, dorsal closure, and cytoskeletal organization.

The Drosophila myoblast city (mbc) locus was previously identified on the basis of a defect in myoblast fusion (Rushton et al., 1995. Development [Camb.]. 121:1979-1988). We describe herein the isolation and characterization of the mbc gene. The mbc transcript and its encoded protein are expressed in a broad range of tissues, including somatic myoblasts, cardial cells, and visceral mesoderm. It is also expressed in the pole cells and in ectodermally derived tissues, including the epidermis. Consistent with this latter expression, mbc mutant embryos exhibit defects in dorsal closure and cytoskeletal organization in the migrating epidermis. Both the mesodermal and ectodermal defects are reminiscent of those induced by altered forms of Drac1 and suggest that mbc may function in the same pathway. MBC bears striking homology to human DOCK180, which interacts with the SH2-SH3 adapter protein Crk and may play a role in signal transduction from focal adhesions. Taken together, these results suggest the possibility that MBC is an intermediate in a signal transduction pathway from the rho/rac family of GTPases to events in the cytoskeleton and that this pathway may be used during myoblast fusion and dorsal closure.

Ectopic expression of MEF2 in the epidermis induces epidermal expression of muscle genes and abnormal muscle development in Drosophila.

Myocyte-specific enhancer-binding factor 2 (MEF2) is a myogenic regulatory factor in vertebrates and Drosophila. Whereas the role of MEF2 in regulating vertebrate myogenesis and muscle genes has been extensively studied, little is known of the role of MEF2 in regulating Drosophila myogenesis. We have shown in a recent analysis of the regulation of the Drosophila Tropomyosin I (TmI) gene in transgenic flies that MEF2 is a positive regulator of TmI expression in the somatic body-wall muscles of embryos, larvae, and adults. To understand further the role of MEF2 in myogenesis and test the role of MEF2 in regulating TmI expression, we have used the yeast GAL4/UAS system to generate embryos in which MEF2 is ectopically expressed in tissues where it is not normally expressed or embryos in which MEF2 is overexpressed in the mesoderm and muscles. We observe that ectopic expression of MEF2 in the epidermis and the ventral midline cells in embryos activates the expression of TmI and other muscle genes in these tissues and that this activation is stage-dependent suggesting a requirement for additional factors. Furthermore, ectopic expression of MEF2 in the epidermis results in a decrease in the expression of signaling molecules in the epidermis and a failure of the embryo to properly form body-wall muscles. These results indicate that MEF2 can function out of context in the epidermis to induce the expression of muscle genes and interfere with a requirement for the epidermis in muscle development. We also find that the level of MEF2 in the mesoderm and/or muscles in embryos is critical to body-wall muscle formation; however, no effect is observed on the development of the visceral muscle or dorsal vessel.

Misexpression of nautilus induces myogenesis in cardioblasts and alters the pattern of somatic muscle fibers.

nautilus (nau), one member of the myogenic regulatory family of bHLH-encoding genes, is expressed in a subset of muscle precursors and differentiated fibers in the Drosophila embryo. To elucidate the role of nautilus in myogenesis, we have misexpressed it using the GAL4-targeted system. We find that ectopic expression results in lethality throughout Drosophila development. We analyzed the effects of embryonic expression in mesodermal tissues that include the cardioblasts of the dorsal vessel as well most, if not all, of the presumptive somatic muscle precursors. Immunohistochemical staining for muscle MHC revealed abnormalities that include an absence of cardial cells, coincident with the appearance of novel muscle fibers adjacent to the dorsal vessel. Moreover, many cardioblasts express increased levels of muscle-specific genes such as myosin, actin 57B, and Mlp60A, a protein that is restricted to the somatic, visceral, and pharyngeal muscles. These data suggest that the missing cardial cells have been transformed into cells with properties similar to those of the somatic muscles. In addition, ubiquitous expression of nautilus in somatic muscle cells of these embryos resulted in muscle pattern defects. Specifically, muscles that do not normally express nautilus were frequently absent, and novel fibers were observed in positions reminiscent of nau-expressing muscles. These data imply that nautilus can alter the developmental program of muscle precursors. In summary, we suggest that nautilus induces myogenic differentiation in vivo when ectopically expressed in developing cardioblasts and may affect the myogenic differentiation program of specific muscle fibers.

Mutations in a novel gene, myoblast city, provide evidence in support of the founder cell hypothesis for Drosophila muscle development.

We have used mutations in the newly identified gene myoblast city to investigate the founder cell hypothesis of muscle development in Drosophila melanogaster. In embryos mutant for myoblast city the fusion of myoblasts into multinucleate muscles is virtually abolished. Nevertheless, a subset of the myoblasts develop specific muscle-like characteristics, including gene expression appropriate to particular muscles, migration to the appropriate part of the segment, correct position and orientation, and contact by motor neurons. We suggest that this subset of myoblasts represents the proposed muscle founder cells and we draw an analogy between these founder cells and the muscle pioneers described for grasshopper muscle development.

Embryonic development of the larval body wall musculature of Drosophila melanogaster.

The somatic, or body wall, muscles of the larva of Drosophila melanogaster are composed of an elaborate pattern of segmentally repeating fibers that form during embryogenesis. The primordia of these muscles progress from morphologically indistinct mesodermal cells to multinucleate syncytia with unique characteristics that include shape, size, location and attachment to the epidermis. Although relatively little is known about the development of the musculature and the mechanisms by which this elaborate pattern is achieved, recent progress has begun to reveal key players in this process.

Drosophila MEF2, a transcription factor that is essential for myogenesis.

mef2 encodes the only apparent Drosophila homolog of the vertebrate myocyte-specific enhancer factor 2 (MEF2). We show herein that the Drosophila MEF2 protein is expressed throughout the mesoderm following gastrulation. Later in embryogenesis, its expression is maintained in precursors and differentiated cells of the somatic and visceral musculature, as well as the heart. We have characterized genetic deficiencies and EMS-induced point mutations that result in complete loss of MEF2 protein in homozygous mutant embryos. These embryos exhibit a dramatic absence of myosin heavy chain (MHC)-expressing myoblasts and lack differentiated muscle fibers. Examination of earlier events of muscle development indicates that the specification and early differentiation of somatic muscle precursors are not affected because even-skipped-, nautilus-, and beta 3-tubulin-expressing myoblasts are present. However, these partially differentiated cells are unable to undergo further differentiation to form muscle fibers in the absence of mef2. The later aspects of differentiation of the visceral mesoderm and the heart are also disrupted in mef2 mutant embryos, although the specification and early development of these tissues appear unaffected. Midgut morphogenesis is disrupted in the mutant embryos, presumably as a consequence of abnormal development of the visceral mesoderm. In the heart, the cardial cells do not express MHC. These results indicate that MEF2 is required for later aspects of differentiation of the three major types of musculature, which include body wall muscles, gut musculature, and the heart, in the Drosophila embryo.

D-mef2: a Drosophila mesoderm-specific MADS box-containing gene with a biphasic expression profile during embryogenesis.

We have identified a mesoderm-specific Drosophila gene, designated D-mef2. The encoded protein contains the MADS- and MEF2-specific domains, which are characteristic of the myocyte-specific enhancer factor 2 (MEF2) family of transcription factors. D-mef2 RNA is first detectable in the presumptive mesoderm at late cellular blastoderm stage and is expressed in all mesoderm after invagination. Following the dorsal migration of the mesodermal layer, D-mef2 expression becomes restricted to the primordia for visceral muscle and the heart. In the second phase, D-mef2 expression is first distinct in heart precursors and then becomes prominent sequentially in visceral and somatic muscles. twi activity is required for D-mef2 expression, while sna function may be needed for the maintenance of D-mef2 expression but not its initiation. D-mef expression is not dependent on the function of tin, and embryos that are deficient for the mesodermal gene DFR1 also show normal initiation of D-mef2 expression at blastoderm. These results suggest that D-mef2 could have a function in early mesoderm differentiation and may be required for subsequent cell fate specifications within the somatic and visceral/heart mesodermal layers.

A role for the Drosophila neurogenic genes in mesoderm differentiation.

The neurogenic genes of Drosophila have long been known to regulate cell fate decisions in the developing ectoderm. In this paper we show that these genes also control mesoderm development. Embryonic cells that express the muscle-specific gene nautilus are overproduced in each of seven neurogenic mutants (Notch, Delta, Enhancer of split, big brain, mastermind, neuralized, and almondex), at the apparent expense of neighboring, nonexpressing mesodermal cells. The mesodermal defect does not appear to be a simple consequence of associated neural hypertrophy, suggesting that the neurogenic genes may function similarly and independently in establishing cell fates in both ectoderm and mesoderm. Altered patterns of beta 3-tubulin and myosin heavy chain gene expression in the mutants indicate a role for the neurogenic genes in development of most visceral and somatic muscles. We propose that the signal produced by the neurogenic genes is a general one, effective in both ectoderm and mesoderm.

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.

Transcriptionally active immediate-early protein of pseudorabies virus binds to specific sites on class II gene promoters.

In the presence of partially purified pseudorabies virus immediate-early protein, multiple sites of DNase I protection were observed on the adenovirus major late and human hsp 70 promoters. Southwestern (DNA-protein blot) analysis demonstrated that the immediate-early protein bound directly to the sequences contained in these sites. These sequences share only limited homology, differ in their affinities for the immediate-early protein, and are located at different positions on these two promoters. In addition, the site-specific binding of a temperature-sensitive immediate-early protein was eliminated by the same heat treatment which eliminates its transcriptional activating function, whereas the binding of the wild-type protein was unaffected by heat treatment. Thus, site-specific binding requires a functionally active immediate-early protein. Furthermore, immediate-early-protein-dependent in vitro transcription from the major late promoter was preferentially inhibited by oligonucleotides which are homologous to the high-affinity binding sites on the major late or hsp 70 promoters. These observations suggest that transcriptional stimulation by the immediate-early protein involves binding to cis-acting elements.

Transcriptional regulation by the immediate early protein of pseudorabies virus during in vitro nucleosome assembly.

An in vivo transcriptional activator, the immediate early protein (IE) of pseudorabies virus, potentiates the activity of the major late promoter in a reconstituted chromatin assembly system where the assembly of preinitiation complexes is in competition with the assembly of promoter sequences within nucleosomes. IE function requires the simultaneous action of TFIID and results in the formation of stable preinitiation complexes within nucleosome-assembled templates. IE is unable to reverse nucleosome-mediated repression, once established, or to further increase the activity of previously activated templates. These results indicate that IE stimulates TFIID binding to promoter sequences, effectively competing with nucleosomes, during chromatin reconstitution. The specific implications for IE function in vivo and the general implications for cellular gene regulation are discussed.

Identification of a functional mammalian spliceosome containing unspliced pre-mRNA.

Functional 60S spliceosomes were assembled under conditions that block the first step of the mRNA splicing reaction. This block was imposed by carrying out the splicing reaction in nuclear extracts preincubated in 2.5 mM EDTA. Preparative amounts of the spliceosomes were isolated by gel filtration chromatography and shown to be functional by in vitro complementation assays. The unspliced pre-mRNA in the complex was converted to spliced products when incubated in cytoplasmic S100 extracts or in heat-treated or micrococcal nuclease-treated nuclear extracts. The latter result, in conjunction with the size of the complex, suggests that the spliceosome contains all of the small nuclear ribonucleoproteins (snRNPs) required for both steps of the splicing reaction. Biochemical characterization of the 5' cleavage reaction revealed that ATP and MgCl2 are required for this step in the splicing pathway. The presence of U1 snRNP in the blocked complex was demonstrated by quantitative immunoprecipitation of this complex by an anti-U1 snRNP monoclonal antibody.

The pseudorabies immediate early protein stimulates in vitro transcription by facilitating TFIID: promoter interactions.

The pseudorabies virus immediate early (IE) protein, partially purified from infected HeLa cells, stimulated transcription initiation by RNA polymerase II and associated factors in HeLa nuclear extracts. This stimulation was maximal at low template concentrations, where the basal level of transcription was also low. In an analysis of the limitations on transcription under these conditions, it was found that transcription could be increased drastically not only by IE addition but also by (1) the addition of nonpromoter-containing DNA, which titrated nonspecific DNA-binding proteins in the crude nuclear extract, and (2) preincubation of the template with either the nuclear extract (in the absence of Mg2+) or with the TATA box-binding factor, TFIID. These results suggest that in the absence of IE, nonspecific DNA-binding proteins competed with TFIID for binding to the promoter, thus making TFIID: promoter interactions limiting for transcription. The stimulation of transcription effected by IE was essentially the same as that observed following preassociation of TFIID with the template or by titration of nonspecific DNA-binding proteins. Moreover, the presence of IE under the latter conditions did not stimulate transcription further. These observations strongly suggest that all of these manipulations affected the same limiting step and, thus, that IE accentuated the rate or extent of formation of a preinitiation complex involving the TATA factor, rather than subsequent initiation or elongation steps.

In vitro stimulation of specific RNA polymerase II-mediated transcription by the pseudorabies virus immediate early protein.

Nuclear extracts from human cells infected with pseudorabies virus (PRV) exhibited higher levels of accurate transcription of RNA polymerase II genes than did control extracts from mock-infected cells. Stimulation was maximal at low DNA concentrations and was not gene-specific. It was heat sensitive in extracts from cells infected with a virus containing a temperature sensitive mutation in the immediate early (IE) gene. The stimulatory activity copurified from the IE protein and was also heat sensitive when purified with cells infected with tsG, further indicating that the IE protein was responsible for this stimulation. These results thus demonstrate an in vitro system that mimics, at least in part, the in vivo stimulatory action of the PRV IE protein. They further imply that the IE protein acts not by increasing the amounts of cellular transcription factors, but rather by directly or indirectly altering their activities.

Nucleosome repeat structure is present in native salivary chromosomes of Drosophila melanogaster.

The regularly repeating periodic nucleosome organization is clearly resolved in the chromatin of the isolated salivary chromosomes of Drosophila melanogaster. A new microsurgical procedure of isolation in buffer A of Hewish and Burgoyne (1973, Biochem. Biophys. Res. Commun., 52:504-510) yielded native Drosophila salivary chromosomes. These chromosomes were then swollen and spread by a modified Miller procedure, stained or shadowed, and examined in the electron microscope. Individual nucleoprotein fibers were resolved with regularly repeated nucleosomes of approximately 10 nm diameter. Micrococcal nuclease digestion of isolated salivary nuclei gave a family of DNA fragments characteristic of nucleosomes for total chromatin, 5S gene, and simple satellite (rho = 1.688 g/cm3) sequences.