The development and differentiation of the body wall musculature in Drosophila are accompanied by changes in gene expression and cellular architecture. We isolated a Drosophila gene, termed rolling stone (rost), which, when mutated, specifically blocks the fusion of mononucleated cells to myotubes in the body wall musculature. β3 tubulin, which is an early marker for the onset of mesoderm differentiation, is still expressed in these cells. Gastrulation and mesoderm formation, as well as the development of the epidermis and of the central and peripheral nervous systems, appear quite normal in homozygous rolling stone embryos. Embryonic development stops shortly before hatching in a P-element-induced mutant, as well as in 16 EMS-induced alleles. In mutant embryos, other mesodermal derivatives such as the visceral mesoderm and the dorsal vessel, develop fairly normally and defects are restricted to the body wall musculature. Myoblasts remain as single mononucleated cells, which express muscle myosin, showing that the developmental program of gene expression proceeds. These myoblasts occur at positions corresponding to the locations of dorsal, ventral and pleural muscles, showing that the gene rolling stone is involved in cell fusion, a process that is independent of cell migration in these mutants. This genetic analysis has set the stage for a molecular analysis to clarify where the rolling stone action is manifested in the fusion process and thus gives insight into the complex regulating network controlling the differentiation of the body wall musculature.

The differentiation of body wall musclulature from the mesodermal germ layer is a highly conserved process in the animal kingdom. The development and differentiation of this germ layer are differentially regulated genetic processes.

A prerequisite for the formation of the mesoderm is the determination of the dorsoventral axis. For Drosophila, the existence of a cascade of maternally transcribed genes that drive the expression of the ventral morphogen dorsal has been shown. The dorsal gene product regulates the expression of the zygotic genes twist and snail which are essential for mesoderm formation (for review see St Johnston and Nüsslein-Volhard, 1992; Steward and Govind, 1993). In contrast to the early events of Drosophila mesoderm formation, much less is known about the differentiation of this germ layer. One of the earliest signs of mesoderm differentiation is the separation of the visceral and somatic mesoderm, which develop the gut musculature and the dorsal vessel, gonadal mesoderm, pharyngeal and body wall musculature, respectively. The larval body wall musculature of Drosophila consists of about 30 muscles per hemisegment (Crossley, 1978; Campos-Ortega and Hartenstein, 1985).

One morphological characteristic of the differentiation of the body wall musculature is the occurrence of mononucleated mesodermal cells that previously have undergone a determination as muscle founders (Bate, 1990). From these founder cells, muscle precursors, visible as bi- and trinucleated cells, develop in a specific pattern at the end of germband extension. During germband retraction, muscle development proceeds by the fusion of these precursor cells with fusion-competent mononucleated myoblasts to multinucleated myotubes. These myotubes are formed of 4-25 myoblasts (Bate, 1990).

In vertebrate cell culture systems, this fusion process can be induced by transfection of genes from the MyoD family, which consists of the four well-characterized genes MyoD, myogenin, Myf 5 and MRF 4 and code for transcriptional activators (for review see Olson, 1990). MyoD was first identified and cloned on the basis of its ability to convert non-muscle cells to a myogenic cell fate, and its specific expression in myoblasts (Davis et al., 1987; Weintraub et al., 1989). All members of this gene family encode transcription factors with a basic HLH domain which is necessary and sufficient for determining the myogenic potential in vitro (Olson, 1990). It has recently been shown that the inactivation of the myogenin gene in mice causes muscle deficiencies and neonatal death (Hasty et al., 1993; Nabeshima et al., 1993). Besides the muscle abnormalities, these mutant mice reveal skeletal defects.

The disruption of the MyoD or Myf-5 gene in mice has no effect on the development of the somatic musculature, presumably because of their functional redundancy in muscle morphogenesis (Braun et al., 1992; Rudnicki et al., 1992). Indeed, examination of mice carrying mutations in both of these genes revealed a complete absence of skeletal muscles. This suggests that either Myf-5 or MyoD is required for the determination or propagation of skeletal myoblasts (Rudnicki et al., 1993).

For Drosophila, the MyoD-related gene nautilus has been isolated using the conserved basic HLH domain as a probe (Michelson et al., 1990, Paterson et al., 1991). Nautilus is expressed in bi- and trinucleated mesodermal cells in every segment that can be followed into mature muscles and might be muscle precursor cells (Abmayr et al., 1992). Two other genes, the homeobox-related gene S59 (Dohrmann et al., 1990) and apterous (Bourgouin et al., 1992), a member of the LIM domain family, are specifically expressed during early fusion processes in a subset of muscle precursors. These genes most likely are involved in regulating cell fate of muscle precursors and events that determine the precise segmental pattern of the body wall musculature.

Besides transcriptional activators, we might expect that cell-cell adhesion molecules are involved in the development of myoblast to mature muscles, as has been made likely by the isolation of the vertebrate muscle-specific M-cadherin gene (Donalies et al., 1991). Expression of this member of the cadherin gene family has been shown to correlate with skeletal muscle development (Donalies et al., 1991, Moore and Walsh, 1993). For vertebrates, it has been proposed that neural cadherins also play a role in myoblast interactions (Knudsen et al., 1990a,b). Furthermore, it is likely that interaction between the nervous system and the somatic musculature is essential for proper development of somatic muscles. It is possible that some genes fulfil functions both in the development of the nervous system and during muscle development; for example, neurogenic mutants of Drosophila, like Notch and Delta, also show distortions in muscle differentiation (Corbin et al., 1991). In Drosophila, there are several other genes known to be important for the development of the somatic and visceral musculature, most of which encode for DNA-binding proteins (Azpiazu and Frasch, 1993; Dohrmann et al., 1990; Barad et al., 1988; Bodmer et al., 1990, Bodmer, 1993; Lai et al., 1993; Bourgouin et al., 1992).

To search for morphoregulatory molecules involved in Drosophila myogenesis, we chose a genetic approach to avoid a preselection of the kind of molecule involved. We reasoned that defects in the musculature will confer late embryonic lethality as the larvae would not be able to hatch. β3 tubulin was used to study of the differentiation of the mesoderm. It is expressed from the extended germband stage until shortly before hatching (Leiss et al., 1988). The β3 tubulin antibody stains mesodermal derivatives like somatic and visceral musculature and the dorsal vessel.

With the β3 tubulin antibody, we screened embryonic lethals for muscle defects (see Burchard et al., 1995) from a P-element screen of the second chromosome performed in parallel to the third chromosome (Cooley et al., 1988).

We found one mutant with a reduced set of somatic muscles (not enough muscles (nem), Burchard et al., 1995) and a second mutant, termed rolling stone (rost). Embryos homozygous for rost mutations are characterized by a high number of unfused myoblasts and only a few myotubes. Here, we describe the rost mutant phenotype in muscle differentiation and its relation to the differentiation of the epidermis and the nervous system. The genetic and cytological analysis suggest that rost function is specific for myoblast fusions.

Drosophila stocks

P-element-induced embryonic lethals were obtained from T. Orr-Weaver and A. Spradling. To screen for mutants with defects in muscle development, we stained 125 P-element mutant lines from the second and third chromosome, preselected for dying late in embryonic development, with the mesoderm-specific β3 tubulin antibody. Three of the P-element insertions seemed to have a muscle-specific defect. Furthermore, we stained mutants with chromosomal deficiencies covering 40% of the genome. However, most deletions revealed such severe distortions that the specificity of muscle phenotypes remained to be determined. In contrast, the P-element-induced mutant, rostP20, specifically revealed distortions in fusions from myoblasts to myotubes and was chosen for a detailed cytological and genetic analysis.

Genetics and EMS-mutagenesis

The P-element in the rostP20 mutant localises on the left arm of the second chromosome, determined by polytene in situ hybridization (data not shown). The rostP20 allele (l(2)neo14) was marked with bw and sp, and brought together by recombination with the previously marked chromosome of nemP8 (l(2)neo113, Burchard et al., 1995). Isogenic males of the phenotype cn bw sp were treated with ethyl methanesulfonate (Lewis and Bacher, 1968) and crossed to virgin females of the genotype b pr cn vgDbw sp/CyO. 10000 of the resulting cn bw sp/CyO males were tested for lethality of their offsprings by crossing with females of the genotype nemP8rostP20/CyO. Offspring containing a lethal mutation over the test chromosome were checked against nemP8/CyO and nemP20/CyO individually to distinguish between nem and rost alleles. A total of 16 EMS-induced rost alleles were obtained.

Reversion of the P-element insertion

The excision of the P-element of the mutant rostP20 should reverse its homozygous lethality to vitality, if indeed the P-element caused the mutation. This reversion experiment was performed by microinjection of the transposase-producing helper plasmid p25.7wc (Karess and Rubin, 1984) in the germ line of embryos with the genotype rostP20/CyO. The resulting flies were backcrossed to males or females of the line rostP20/CyO which contain the P-element insertion. Crossing of flies in which the P-element was excised by transposase results in homozygous viability in three independent lines recognizable by wild-type wings. Embryos stained with an antibody against β3 tubulin do not show any unfused myoblasts.

Antibody staining of embryos

Eggs laid by flies of the appropriate genetic constitution were collected on agar-apple juice plates. In order to obtain an age distribution that allowed visualization of different stages of muscle development, eggs were collected over a 24 hour period. Eggs were dechorionated, permeabilized and fixed essentially as described by Leiss et al. (1988). After washing and blocking in BBT (0.15% crystalline BSA, 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 40 mM MgCl2, 20 mM glucose, 50 mM sucrose, 0.1% Tween 20), the eggs were incubated overnight with a dilution of the appropriate antibody. For the analysis of the mesoderm formation, we used the twist antibody (Thisse et al., 1988; Leptin und Grunewald, 1990). For staining of mesodermal derivatives, the anti-β3 tubulin antibody and myosin antibody were used (Leiss et al., 1989; Kiehart und Feghali, 1986). The central and peripheral nervous system was stained with the monoclonal antibody mAb22C10 (Zipursky et al., 1984). The intersegmental and segmental neurons were visualized with the fasII antibody (mAb1D4, Grenningloh et al., 1991). The bound antibody was detected with a biotinylated secondary antibody and stained with the Vectastain ABC Elite-kit (VectorLabs) using diaminobenzidine as detection agent. Double stainings were performed as described by Lawrence et al. (1987). The stained embryos were embedded in Epon and photos taken under Nomarski optics with a Zeiss Axiophot microscope (Kodak, Ektachrome 25).

RNA in situ hybridization

In order to visualize muscle precursor cells in rost mutants, wholemount RNA hybridizations were performed with digoxigenin-labelled nautilus cDNA probes according to Tautz and Pfeiffle (1989). To identify homozygous mutant embryos at early developmental stages that had not yet shown severe morphogenetic alterations, we stained strains carrying the rost mutation balanced over CyO7.1. This balancer chromosome contained a hindgut/anal-pad β-gal fusion construct (Affolter et al., 1993). Embryos were hybridized simultaneously with a lacZ and a nautilus cDNA probe. Only those embryos that show no expression of the β-gal fusion construct are homozygous for the rost mutation.

Mesoderm formation is normal in rost mutants

Mesoderm formation depends on a cascade of maternally active genes as well as on two zygotically active genes twist and snail (Thisse et al., 1987; Bouley et al., 1987). It is possible to follow mesodermal cells during gastrulation using the twist antigen (Leptin and Grunewald, 1990). Thus, an antibody recognizing the twist antigen was used to characterize mesoderm formation in muscle mutants. Generally, we found no major difference in the pattern of twist expression in comparison to the wild type for the mutation described here (rolling stone, see below) nor for nem (not enough muscles, Burchardt et al., 1995) (data not shown). However, we cannot exclude that single cells, which may be essential for the initial formation of specific muscles, are missing (see below and Discussion).

The gene rolling stone is essential for myoblast fusion during embryonic muscle differentiation

As a marker to follow mesoderm differentation, P-elementinduced embryonic lethals were stained with the β3 tubulin antibody. The β3 tubulin is specifically expressed during embryonic mesoderm formation (Leiss et al., 1988), as well as during development of the adult musculature during metamorphosis (Bate et al., 1991). The β3 tubulin isotype is first detectable after mesoderm formation in the extended germband stage. It persists in splanchnopleura and somatopleura until the muscle differentation program has been completed (Leiss et al., 1988). Thus, the β3 tubulin isotype is a well-suited marker to follow the differentiation of the major mesodermal derivatives, e.g. somatic muscles, visceral muscles and dorsal vessel. In the wild-type, this β3 tubulin antigen is present in every single muscle of the body wall musculature (Fig. 2A). The somatic musculature is organized in ventral, pleural and dorsal groups. One of the analyzed P-element-induced mutations revealed a very specific distortion in myoblast fusion so that only a few myotubes were observed (Fig. 1). We named the corresponding wild-type gene rolling stone (rost) and the P-elementinduced allele rostP20. In homozygous rostP20 mutants, embryogenesis proceeds until shortly before hatching, when dorsal closure was visible already (Fig. 1D). In these embryos, the dorsal vessel was formed quite normally (Fig. 1D). Also the visceral musculature developed properly (Fig. 1B). However, major distortions were visible in the somatic mesoderm. In particular, free unfused myoblasts in the majority of mutant embryos at late stages of development characterize this mutant (Fig. 1C, E-G). Reflecting the variability of the P-element-induced phenotype, some embryos were observed showing far less unfused myoblasts and also a strongly reduced muscle pattern (Fig. 1H). The first distortions were detectable during germband retraction. The embryo shown in Fig. 1A, demonstrates this earliest visible fusion defect in the rostP20 mutant. Single myoblasts (white arrows) in the somatic mesoderm, shortly after separation from the visceral mesoderm become visible. This observation was proved by double stainings of rostP20/CyO7.1 embryos with antibodies against β3 tubulin and β-gal, which allow mutant and wild-type embryos to be distinguished by the expression of the reporter gene lacZ in the wild-type embryos. When embryogenesis proceeded, most myoblasts remained unfused (Fig. 1E-G). Nevertheless, the myoblasts were often arranged in specific groups in locations where in the wild-type ventral, pleural and dorsal groups of muscles were formed (Fig. 1G,E). Clearly, all muscles of the ventral (e.g. Fig. 1G), lateral (e.g. Fig. 1E,F) and dorsal group (e.g. Fig. 1D) can be affected. We find the same results in the analysis of the EMS-induced alleles (see below).

Fig. 1.

Staining with β3 tubulin antibody shows fusion distortions of the somatic musculature in homozygous rostP20 embryos. (A) A stage 13 homozygous rostP20 embryo. The somatic musculature (sm) shows single unfused myoblasts (white arrows). (B) The visceral musculature (vm) of a homozygous stage 12 rostP20 embryo develops normally. (C) A mutant stage 14 embryo shows many unfused myoblasts (mb) in the dorsal musculature. (D) A dorsolateral view of a stage 15 embryo. Dorsal vessel (dv) is normal while characteristic unfused myoblasts (mb) are visible in the region of the dorsal musculature. (E) A lateral view of a stage 16 embryo illustrates the pleural musculature, which shows few myotubes (mt) and a great number of single unfused myoblasts (mb). The myoblasts are arranged at the location where in the wild-type pleural muscles are formed. (F) Higher magnification of E showing the ordered location of myoblasts (mb) preceding the formation of pleural muscles. (G) A stage 17 embryo shows unfused myoblasts (mb) in the pleural and ventral musculature. (H) This rare phenotype at stage 17 is characterized by a reduced number of muscles and only some unfused myoblasts are stained with the β3 tubulin antibody.

Fig. 1.

Staining with β3 tubulin antibody shows fusion distortions of the somatic musculature in homozygous rostP20 embryos. (A) A stage 13 homozygous rostP20 embryo. The somatic musculature (sm) shows single unfused myoblasts (white arrows). (B) The visceral musculature (vm) of a homozygous stage 12 rostP20 embryo develops normally. (C) A mutant stage 14 embryo shows many unfused myoblasts (mb) in the dorsal musculature. (D) A dorsolateral view of a stage 15 embryo. Dorsal vessel (dv) is normal while characteristic unfused myoblasts (mb) are visible in the region of the dorsal musculature. (E) A lateral view of a stage 16 embryo illustrates the pleural musculature, which shows few myotubes (mt) and a great number of single unfused myoblasts (mb). The myoblasts are arranged at the location where in the wild-type pleural muscles are formed. (F) Higher magnification of E showing the ordered location of myoblasts (mb) preceding the formation of pleural muscles. (G) A stage 17 embryo shows unfused myoblasts (mb) in the pleural and ventral musculature. (H) This rare phenotype at stage 17 is characterized by a reduced number of muscles and only some unfused myoblasts are stained with the β3 tubulin antibody.

Fig. 2.

Muscle phenotype of the EMS-induced allele rost15. (A) β3 tubulin expression in a stage 17 wild-type embryo is shown for comparison. vem, ventral muscles; pet 1-3, pleural external muscles 1-3; pit, pleural internal muscle. (B) This homozygous rost15 embryo represents a phenotype with a lot of myotubes and no significant proportion of unfused myotubes. The muscle pattern, however, is not complete and some of the muscles appear to be too short (white arrows) and disorganized. Identifiable myotubes of the ventral part of the musculature (vm) and the pleural muscles pet 1-3 are marked. (C) This homozygous rost15 embryo shows a lot of unfused myoblasts (mb) in the pleural musculature and some myotubes in the ventral musculature (vem). Often the pleural internal muscle (pit) has developed properly. (D) Dorsolateral view of a mutant stage 16 embryo reveals an intact dorsal vessel (dv) and a severe reduction of the dorsal musculature (dm).

Fig. 2.

Muscle phenotype of the EMS-induced allele rost15. (A) β3 tubulin expression in a stage 17 wild-type embryo is shown for comparison. vem, ventral muscles; pet 1-3, pleural external muscles 1-3; pit, pleural internal muscle. (B) This homozygous rost15 embryo represents a phenotype with a lot of myotubes and no significant proportion of unfused myotubes. The muscle pattern, however, is not complete and some of the muscles appear to be too short (white arrows) and disorganized. Identifiable myotubes of the ventral part of the musculature (vm) and the pleural muscles pet 1-3 are marked. (C) This homozygous rost15 embryo shows a lot of unfused myoblasts (mb) in the pleural musculature and some myotubes in the ventral musculature (vem). Often the pleural internal muscle (pit) has developed properly. (D) Dorsolateral view of a mutant stage 16 embryo reveals an intact dorsal vessel (dv) and a severe reduction of the dorsal musculature (dm).

EMS-induced rost alleles fall into one complementation group

The original mutant was induced by P-element mutagenesis. P-elements often integrate in upstream regions of a gene, thus the P-element does not necessarily destroy the function of the gene completely. Furthermore the P-element may cause mutations in two neighbouring genes. To clarify whether the rostP20 phenotype shows the complete phenotype of the gene, we performed an EMS mutagensis. We obtained 16 EMSinduced alleles to rostP20 (see Material and methods).

To decide if it indeed was a single gene, we performed a complementation analysis between all EMS-induced alleles, as well as with the rostP20 allele. The result showed that all mutations were alleles of a single gene. We found no interallelic complementation. The alleles reflect the rost phenotype in that the fusion process from myoblasts to myotubes is stopped, causing late embryonic lethality. Because of their role in this morphogenetic process, we sorted the rost alleles according to the degree of myotube formation (Table 1). As an example for the strongest class, the rost15 allele is shown in comparison to the wild type (Fig. 2A for the wild type, Fig. 2B-D for the rost15 allele). The dorsal vessel (Fig. 2D) and the visceral musculature developed normally in all alleles. This makes it very likely that the rost gene is essential for fusion of myoblasts to myotubes specifically and is not involved in the differentiation of the visceral musculature and the dorsal vessel. Fig. 2B and C demonstrates the variability of the rost phenotype in the EMS-induced alleles similar to the variabilty of the rostP20 mutant.

Table 1.

Classification of rost alleles according to the degree of myotube formation

Classification of rost alleles according to the degree of myotube formation
Classification of rost alleles according to the degree of myotube formation

The six alleles (see Table 1) stronger than rostP20 indicate that this phenotype very likely represents the null situation. Furthermore, we proved numerous chromosomal deletions but none of them uncovered the rost gene. Cloning and sequence analysis of the strongest alleles will finally clarify the null phenotype.

Expression of the nautilus gene is independent of the rost gene

One key step in the process of muscle formation is the segregation of muscle precursors at the stage of germband retraction. Nautilus, the Drosophila homologue of the vertebrate myogenic transcription factor MyoD is expressed in a subset of muscle precursors (Michelson et al., 1990; Paterson et al., 1991). To test whether the rost gene is essential in this early myogenic cells, we looked for the presence of muscle precursor cells in rost mutants by analysing the expression pattern of nautilus in rost mutants (Fig. 3), assuming that the rost phenotype might be a result of the absence of muscle precursors. During germband retraction, when nautilus expression begins, the rost phenotype is not recognizable by β3 tubulin staining, but homozygous rost/rost embryos can be identified by lack of the balancer marker (see Material and Methods). The nautilus expression in rostP20 homozygous mutants (Fig. 3A,C,E,G) is shown in comparison to wild-type embryos (Fig. 3B,D,F,H). In a lateral view no difference is visible at stage 10, when nautilus is first expressed in single cells per hemisegment (Fig. 3A,B). Shortly later, more cells express nautilus (Fig. 3C,D). No differences are observed in dorsal view. In the wild type (Fig. 3F) and in rostP20 mutants (Fig. 3E), precursors of the ventral muscles are clearly visible. In stage 13/14, wild-type and mutant embryos reveal an identical nautilus expression pattern, as far as is detectable with this method.

Fig. 3.

Nautilus is expressed in muscle precursors in homozygous rostP20 embryos. Wild-type embryos were stained with a nautilus cDNA probe. rostP20/CyO7.1 (see Material and methods) embryos were stained simultaneously with a nautilus cDNA and a lacZ probe to distinguish between homozygous rostP20 embryos and embryos containing the balancer chromosome. (A,C,E,G) Homozygous rostP20 and (B,D,F,H) wild-type embryos. Embryos for B and D were taken from a staining of wild-type embryos, all others are from a rostP20/CyO7.1 offspring. The lacZ reporter gene expression in the hindgut/anal-pad-precursor is shown in F and H (arrowhead). B and D show a lateral view of wild-type embryos (stage 10 and 11) selected by the double staining. Nautilus is expressed in the lateral muscle precursors (A) and shortly later in the medial precursors as well (C). Compared to wild-type embryos (B,D) at similiar stages and orientation there is no obvious difference in the expression pattern. E shows a mutant embryo (stage 10) with a normal level of nautilus expression in all precursors (arrows). A wild-type embryo of the same stage is shown in F. Later on (stage 13) there are no differences in the nautilus expression pattern between homozygous rostP20 (G) and wild-type (H) embryos.

Fig. 3.

Nautilus is expressed in muscle precursors in homozygous rostP20 embryos. Wild-type embryos were stained with a nautilus cDNA probe. rostP20/CyO7.1 (see Material and methods) embryos were stained simultaneously with a nautilus cDNA and a lacZ probe to distinguish between homozygous rostP20 embryos and embryos containing the balancer chromosome. (A,C,E,G) Homozygous rostP20 and (B,D,F,H) wild-type embryos. Embryos for B and D were taken from a staining of wild-type embryos, all others are from a rostP20/CyO7.1 offspring. The lacZ reporter gene expression in the hindgut/anal-pad-precursor is shown in F and H (arrowhead). B and D show a lateral view of wild-type embryos (stage 10 and 11) selected by the double staining. Nautilus is expressed in the lateral muscle precursors (A) and shortly later in the medial precursors as well (C). Compared to wild-type embryos (B,D) at similiar stages and orientation there is no obvious difference in the expression pattern. E shows a mutant embryo (stage 10) with a normal level of nautilus expression in all precursors (arrows). A wild-type embryo of the same stage is shown in F. Later on (stage 13) there are no differences in the nautilus expression pattern between homozygous rostP20 (G) and wild-type (H) embryos.

Myosin heavy chain is present in unfused myoblasts of rost mutants

The analysis of several rost alleles revealed that the majority of myoblasts remain as mononucleated cells. These mesodermal cells turn off twist expression and start the muscle differentiation program as is evident by the expression of the characteristic β3 tubulin isotype. We addressed the question of whether these cells, during differentiation, express the genetic repertoire of muscle-specific proteins, like myosin, despite their failure to build myotubes. With an antibody recognizing myosin heavy chain (Kiehart and Feghali, 1986) that stains the complete set of fused muscles, we analyzed several EMSinduced alleles as well as the original P-element-mediated mutant. As a representative example, we show the results for the rost5 allele, which exhibits a medium mutant phenotype that is very similar to the original P-element mutant. Although in wild-type embryos single myoblasts did not express myosin, unfused myoblasts in the rost allele showed expression of this protein (Fig. 4). This myosin expression in unfused myoblasts shows that these cells complete their developmental program independently of the myotube formation.

Fig. 4.

Lateral view of a homozygous stage 16 rost5 mutant. Expression of muscle myosin in unfused myoblasts and myotubes is obvious.

Fig. 4.

Lateral view of a homozygous stage 16 rost5 mutant. Expression of muscle myosin in unfused myoblasts and myotubes is obvious.

Ectodermal derivatives, epidermis, and central and peripheral nervous system develop normally in rost mutants

The rost mutation causes failure of myoblast fusions. This defect may be due to the missing rost gene product in myoblasts themselves. However, it cannot be ruled out that distortions in the epidermis, for example in the muscle attachment sites, or in the development of the nervous system, caused the muscle phenotype. No defect was detected in cuticular preparations of rost mutants (data not shown). For the analysis of the nervous system, we performed double staining experiments. The peripheral and central nervous system were stained with the monoclonal antibody mAb22C10 (Zipursky et al., 1984) and the mesoderm with the β3 tubulin antibody (Leiss et al., 1988). For the wild-type, staining of the nervous system is shown in Fig. 5A and C in dark brown and muscles are shown in light brown. In homozygous rostP20 mutants, many unfused β3-positive myoblasts (light brown) were observed. In these mutant embryos, the morphology of the central and peripheral nervous system was quite normal (Fig. 5B,D), though we cannot exclude that single cells were missing.

Fig. 5.

The central and peripheral nervous system in homozygous rostP20 embryos. The embryos were stained with the β3 tubulin antibody (Leiss et al., 1988) to detect the musculature and with mAb22C10 (Zipursky et al., 1984) to stain the central and peripheral nervous system. (A)A lateral view of a wild-type embryo showing the pleural musculature (light-brown staining) and dorsal hair sensilla (dh) and the lateral chordotonal organs (lch5) of the peripheral nervous system (dark-brown staining). (B) Lateral view of a homozygous rostP20 mutant reveals no defects in the dorsal hair sensilla (dh) and the lateral chordotonal organs (Ich 5). (C) The ventral cord (vc) of a wild-type central nervous system and the ventral musculature (vem) are visible. (D) The ventral cord (vc) of a mutant embryo shows no obvious defects. Unfused myoblasts and some myotubes of the ventral musculature (vem) are stained with the β3 tubulin antibody

Fig. 5.

The central and peripheral nervous system in homozygous rostP20 embryos. The embryos were stained with the β3 tubulin antibody (Leiss et al., 1988) to detect the musculature and with mAb22C10 (Zipursky et al., 1984) to stain the central and peripheral nervous system. (A)A lateral view of a wild-type embryo showing the pleural musculature (light-brown staining) and dorsal hair sensilla (dh) and the lateral chordotonal organs (lch5) of the peripheral nervous system (dark-brown staining). (B) Lateral view of a homozygous rostP20 mutant reveals no defects in the dorsal hair sensilla (dh) and the lateral chordotonal organs (Ich 5). (C) The ventral cord (vc) of a wild-type central nervous system and the ventral musculature (vem) are visible. (D) The ventral cord (vc) of a mutant embryo shows no obvious defects. Unfused myoblasts and some myotubes of the ventral musculature (vem) are stained with the β3 tubulin antibody

In addition, we were interested in the pattern of motoneurons in the rost mutant. Previously, it was shown that innervation is not an essential requirement for the early stages of myogenesis (Bate, 1990; Broadie and Bate, 1993). The initial myoblast fusions begin with the onset of germband retraction (Bate, 1990), yet pioneering of the motor nerves does not start before completion of germband retraction. Nevertheless, the motor nerves are closely associated with the developing myotubes for many hours prior to the establishment of the mature muscle pattern (Johansen et al., 1993; Broadie and Bate, 1993) so that this association might be essential for the completion of fusions of the myoblasts.

To examine the pattern of motoneurons in the rost mutant, we performed double stainings of motoneurons and muscles in homozygous rost embryos. The results for the EMS-induced allele rost5 are shown in Fig. 6. With the fas II antibody (a gift from G. Helt and C. S. Goodman, Grenningloh et al., 1991) we visualized all intersegmental (ISN) and segmental (SN) neurons (Grenningloh et al., 1991; Seeger et al., 1993; Van Vactor et al., 1993;) in parallel to the staining of mesodermal cells with the anti-β3 antibody (Fig. 6, black: fas II, brown: β3 tubulin). In mid embryonic stages (stage 14), we found an obviously normal pattern (Fig. 6A) and guiding of motoneurons in the absence of multinucleated myotubes, which can be well observed at a higher magnification (Fig. 6B). In later stages of development (stage 16), the pattern of motoneurons in rost mutants was strongly changed (not shown), which is presumably the result of the missing muscles.

Fig. 6.

Motoneuron development in homozygous rost5 mutants. Double staining of motoneurons (shown in black) and musculature (shown in brown) in homozygous rost embryos visualized with the fas II antibody (Grenningloh et al., 1991) and β3 tubulin antibody (Leiss et al., 1988). Intersegmental (ISN) and segmental neurons (SN) are marked. (A) This homozygous rost5 embryo at stage 14 of embryogenesis shows a normal segmentally arranged motoneuron pattern. (B) Higher magnification of the embryo shown in A clearly reveals independance of motoneuron outgrowth and muscle fusions.

Fig. 6.

Motoneuron development in homozygous rost5 mutants. Double staining of motoneurons (shown in black) and musculature (shown in brown) in homozygous rost embryos visualized with the fas II antibody (Grenningloh et al., 1991) and β3 tubulin antibody (Leiss et al., 1988). Intersegmental (ISN) and segmental neurons (SN) are marked. (A) This homozygous rost5 embryo at stage 14 of embryogenesis shows a normal segmentally arranged motoneuron pattern. (B) Higher magnification of the embryo shown in A clearly reveals independance of motoneuron outgrowth and muscle fusions.

The somatic musculature develops from mesodermal stem cells that are determined as myoblasts. During muscle development, myoblasts fuse with each other to form multinucleated myofibers (see Introduction). Muscle fibers insert into apodemes and it has been suggested that this connection induces a regulatory cascade leading to β1 tubulin expression (Buttgereit, 1993). Previous analysis of deletion mutants of the X-chromosome revealed that deletion of a number of loci disturbs the muscle pattern (Drysdale et al., 1993); furthermore, a P-element mutation has been detected, leading to a strongly reduced number of myotubes (Burchard et al., 1995). Here,we analyze the gene rolling stone (rost) which is essential for the fusion of myoblasts to myotubes. In mutant embryos, only a few myotubes are formed. The majority of myoblasts, however, do not fuse or only fuse partially, as is visualized by β3 tubulin staining. However, the program of gene expression proceeds, as is evident from the decrease of twist expression in the wild-type, activation of β3 tubulin and myosin heavy chain expression, the last being a typical gene product of mature muscle fibers in wild-type embryos (Kiehart and Feghali, 1986). In rost mutants, however, myosin is detected also in the unfused myoblasts. This shows that the fusion process is dispensable for the expression of this protein. The rost mutant phenotype may be a consequence of defects in the mesoderm itself or of signals derived from ectodermal derivatives such as the nervous system or the apodemes (muscle attachment sites). In homozygous rost mutants, mesoderm formation is quite normal. However, we cannot exclude that some mesodermal cells are missing. In addition, the morphology of the CNS and PNS, including the motoneurons, seems normal in early stages of development. In later stages, the motoneuron pattern shows strong morphological abberations in a rost mutant allele. In the absence of most of the somatic muscles, this modification could be a secondary effect driven by the dramatic defects in the mesoderm. We conclude that the correct outgrowth and guidance of motoneurons is independent of the fusion of myoblasts to myotubes during muscle formation. Broadie and Bate (1993) could not detect significant differences between muscle development in the presence or absence of motorneuron innervation, showing that innervation is not a prerequisite for muscle formation. Due to the limited resolution of antibody stainings, we cannot exclude disorganization of a small population of nerve cells. Our investigation of the mesoderm formation and of the morphology of the nervous system allows us to state that the gross organization is not disturbed. This is in contrast to the previously characterized gene nem, which is essential for the formation of the complete set of somatic muscles, as well as for the development of the pentascolopidial cells of the peripheral nervous system (Burchard et al., 1995). In rost mutants, as well as in nem mutants, the visceral mesoderm, the dorsal vessel and the pharynx musculature develop properly. This parallels the early separation of regulatory pathways in these tissues for the β3 tubulin gene (Gasch et al., 1989, Hinz et al., 1992).

A similar phenotype has been observed as a consequence of overexpression of a mutated GTPase Drac1 in the mesoderm (Luo et al., 1994). The role of this protein in the muscleforming process in wild-type development remains to be determined. In some deletion mutants described by Drysdale et al. (1993), unfused myoblasts were also observed. Interestingly, one of the EMS-induced nem alleles (nem22) also shows fusion distortions (Burchard et al., 1995). In this case, myoblasts still aggregate, which is in contrast to rost mutants. Other mutants are known to be essential for the development of the visceral musculature but have only moderate effects on the somatic musculature (Bodmer et al., 1993; Azpiazu and Frasch, 1993). In tinman mutants, the muscle pattern is somewhat disorganized and the muscle number is usually lower than normal. In mutants for bagpipe, a gene required for the specification of the visceral mesoderm, the somatic musculature appears normal (Azpiazu and Frasch, 1993).

There are two classes of cell types that are essential for the formation of the somatic musculature. First, there is a class of founder cells that precedes muscle formation. Second, there is a class of fusion competent cells that fuse with the founder cells to form muscle precursors and muscles (Bate, 1990). A possible function of the rost gene could be to define the founder cells or to convert premature founder cells to fusion-competent founder cells. Failure of rost expression may have the consequence that the founder cells cannot be specified and the fusion process is disturbed.

So far a few genes that are expressed in muscle precursor cells have been isolated and their gene products localized in situ, for example nautilus, the MyoD homologue of Drosophila (see Introduction). We started to analyse the presence of muscle precursors in rost mutants by the detection of nautilus expression with in situ hybridizations.

We found an expression of nautilus in the rolling stone mutant from the extended germband stage onwards. In comparison to the wild type, all nautilus-expressing precursors are visible in the rost mutant. This result indicates that the fusion defect in the rost mutant is not caused by the absence of muscle precursors. The early muscle differentiation, characterized by the formation of muscle precursors is not disturbed in rost embryos as far as nautilus expression indicates. The high number of β3 tubulin- and myosin-expressing cells, which are not fusion competent, indicates that the cell specificity of myogenic cells is determined. In conclusion we found that the precursors and the cells to fuse with them are present in rost embryos. Furthermore the expression of nautilus in the rost mutant implicates that nautilus is independent of rost function.

Many morphogenetic events like cell adhesion, cell migration, cell fusion, shaping, attachment to apodemes, innervation and expression of muscle-specific proteins are essential for muscle development. Furthermore, individual muscles gain their identity under control of homeotic gene products (Hooper, 1986). The homeobox-containing gene S59 may also fall into this class, as this gene is active in specific muscle precursors (Dohrmann et al., 1990). Besides transcription factors, proteins mediating cell-cell adhesion certainly play a role in these fusion processes. Because myogenesis involves the fusion of mononucleated myoblasts to multinucleated myotubes, the recognition and adhesion steps are believed to be important for this highly specific process. A number of adhesion molecules such as N-CAM (e.g. Knudsen et al., 1990a,b) and integrins seems to be important in myoblast fusion. Molecules like integrins are essential for muscle attachment to apodemes as is evident in the mutant lethal (1) myospheroid, which eliminates the PSβ subunit (Newman and Wright, 1981; MacKrell et al., 1988; Leptin et al., 1989). Recently, a cadherin isotype was identified (Donalies et al., 1991; Moore and Walsh, 1993) that plays a role in myogenesis.

We have started the cloning and molecular analysis of the rost gene, which in combination with the molecular analysis of the EMS-induced alleles should elucidate the function of this gene. Determination of the spatiotemporal pattern of expression will indicate whether the rost gene encodes a functionally important molecule of the muscle cells themselves or whether the rost gene is transcribed in another tissue but influences muscle development.

We thank Ruth Hyland for excellent technical assistence, Detlev Buttgereit for stimulating discussions and Heike Sauer for her secretary work. We acknowledge D. Kiehard for providing the anti-myosin antibody, L. Zipursky for providing mAb22C10, G. Helt and C. Goodman for the fas II antibody. We thank also Marcos A. Gonzales Gaitan for help with whole-mount in situ hybrization, Alan Michelson for providing the nautilus cDNA probe and Markus Affolter and Uwe Walldorf for the CyO7.1 balancer strain. We are very grateful to Günther Korge for help with the interpretation of polytene chromosome in situ hybridizations. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Re628/7-1/7-2) and the Fonds der Chemischen Industrie to R. R.-P.

Abmayr
,
S. M.
,
Michelson
,
A. M.
,
Corbin
,
V.
,
Young
,
M. W.
and
Maniatis
,
T.
(
1992
).
nautilus, a Drosophila member of the myogenic regulatory gene family
. In
Neuromucular Development and Disease
(ed.
A. M.
Kelly
and
H. M.
Blau
).
New York
:
Raven Press, Ltd
.
Affolter
,
M.
,
Walldorf
,
U.
,
Kloter
,
U.
,
Schier
,
A. F.
and
Gehring
,
W. J.
(
1993
).
Regional repression of a Drosophila POU box gene in the endoderm involves inductive interactions between germ layers
.
Development
117
,
1199
1210
.
Azpiazu
,
N.
and
Frasch
,
M.
(
1993
).
Tinman and bagpipe: two homeo box genes that determine cell fate in the dorsal mesoderm of Drosophila
.
Genes Dev
.
7
,
1325
1340
.
Barad
,
M.
,
Jack
,
T.
,
Chadwick
,
R.
and
McGinnis
,
W.
(
1988
).
A novel, tissue-specific, Drosophila homeobox gene
.
EMBO J
.
7
,
2151
2161
.
Bate
,
M.
(
1990
).
The embryonic development of larval muscles in Drosophila
.
Development
110
,
791
804
.
Bate
,
M.
,
Rushton
,
E.
and
Currie
,
D. A.
(
1991
).
Cells with persistent twist expression are the embryonic precursors of adult muscles in Drosophila
.
Development
113
,
79
89
.
Bodmer
,
R.
,
Jan
,
L. Y.
and
Jan
,
Y. N.
(
1990
).
A new homeobox-containing gene, msh-2, is transiently expressed early during mesoderm formation in Drosophila
.
Development
110
,
661
669
.
Bodmer
,
R.
(
1993
).
The gene tinman is required for specification of the heart and visceral muscles in Drosophila
.
Development
118
,
719
729
.
Bouley
,
J. L.
,
Dennefeld
,
C.
and
Alberga
,
A.
(
1987
).
The Drosophila developmental gene snail encodes a protein with nucleic acid binding fingers
.
Nature
330
,
395
398
.
Bourgouin
,
C.
,
Lundgren
,
S. E.
and
Thomas
,
J. B.
(
1992
).
apterous is a Drosophila LIM domain gene required for the development of a subset of embryonic muscles
.
Neuron
9
,
549
561
.
Braun
,
T.
,
Rudnicki
,
M. A.
,
Arnold
,
H. H.
and
Jaenisch
,
R.
(
1992
).
Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death
.
Cell
71
,
369
382
.
Broadie
,
K.
and
Bate
,
M.
(
1993
).
Muscle development is independent of innervation during Drosophila embryogenesis
.
Development
119
,
533
543
Burchard
,
S.
,
Paululat
,
A.
,
Hinz
,
U.
and
Renkawitz-Pohl
,
R.
(
1995
).
The mutant not enough muscles (nem) reveals reduction of the Drosophila embryonic muscle pattern
.
J. Cell Sci
.
108
,
1443
1454
.
Buttgereit
,
D.
(
1993
).
Redundant enhancer elements guide β1 tubulin expression in apodemes during Drosophila embryogenesis
.
J. Cell Sci
.
105
,
721
727
.
Campos-Ortega
,
J. A.
and
Hartenstein
,
V.
(
1985
).
The Embryonic Development of Drosophila melanogaster. Berlin and Heidelberg: Springer Verlag
.
Cohen
,
B.
,
McGuffin
,
E.
,
Pfeifle
,
C.
,
Segal
,
D.
and
Cohen
,
S. M.
(
1992
).
apterous, a gene required for imaginal disc development in Drosophila encodes a member of the LIM family of developmental regulatory proteins
.
Genes Dev
.
6
,
715
729
.
Cooley
,
L.
,
Berg
,
C.
and
Spradling
,
A.
(
1988
).
Controlling P-element insertional mutagenesis
.
Trends Genet
.
4
,
254
258
.
Corbin
,
V.
,
Michelson
,
A. M.
,
Abmayr
,
S. M.
,
Neel
,
V.
,
Alcamo
,
E.
,
Maniatis
,
T.
and
Young
,
M. W.
(
1991
).
A role for the Drosophila neurogenic genes in mesoderm differentiation
.
Cell
67
,
311
323
.
Crossley
,
A. C.
(
1978
).
The morphology and development of the Drosophila muscular system
. In
The Genetics and Development of Drosophila
vol.
2b (eds Ashburner, M. and Wright, T
.), pp
499
560
.
New York
:
Academic press
.
Davis
,
R. L.
,
Weintraub
,
H.
and
Lassar
,
A. B.
(
1987
).
Expression of a single transfected cDNA converts fibroblasts to myoblasts
.
Cell
51
,
987
1000
.
Dohrmann
,
C.
,
Azpiazu
,
N.
and
Frasch
,
M.
(
1990
).
A new Drosophila homeo box gene is expressed in mesodermal precursor cells of distinct muscles during embryogenesis
.
Genes Dev
.
4
,
2098
2111
.
Donalies
,
M.
,
Cramer
,
M.
,
Ringwald
,
M.
and
Starzinski-Powitz
,
A.
(
1991
).
Expression of M-cadherin, a member of the cadherin multigene family, correlates differentiation of skeletal muscle cells
.
Proc. Natl. Acad. Sci. USA
88
,
8024
8028
.
Drysdale
,
R.
,
Rushton
,
E.
and
Bate
,
M.
(
1993
).
Genes required for embryonic muscle development in Drosophila melanogaster
.
Roux’s Arch. Dev. Biol
.
202
,
279
295
.
Gasch
,
A.
,
Hinz
,
U.
and
Renkawitz-Pohl
,
R.
(
1989
).
Intron and upstream sequences regulate expression of the Drosophila β3 tubulin gene in the visceral and somatic musculature, respectively
.
Proc. Natl. Acad. Sci. USA
86
,
3215
3218
.
Grenningloh
,
G.
,
Rehm
,
E. J.
and
Goodman
,
C. S.
(
1991
).
Genetic analysis of growth cone guidance in Drosophila: fasciclin II functions as a neuronal recognition molecule
.
Cell
67
,
45
57
.
Hasty
,
P.
Bradley
,
A.
,
Morris
,
J. H.
,
Edmondson
,
D. G.
,
Venuti
,
J. M.
,
Olson
,
E.
and
Klein
,
W. H.
(
1993
).
Muscle deficiency and neonatal death in mice with a target mutation in the myogenin gene
.
Nature
364
,
501
506
.
Hinz
,
U.
,
Wolk
,
A.
and
Renkawitz-Pohl
,
R.
(
1992
).
Ultrabithorax is a regulator of β3 tubulin expression in the Drosophila visceral mesoderm
.
Development
116
,
543
554
.
Hooper
,
J. E.
(
1986
).
Homeotic gene function in the muscles of Drosophila larvae
.
EMBO J
.
5
,
2321
2329
.
Johansen
,
J.
,
Halpern
,
M. E.
and
Keshishian
,
H.
(
1993
).
Axonal guidance and the development of muscle fiber-specific innervation in Drosophila embryos
.
J. Neurosci
.
9
,
4318
4332
.
Karess
,
R. E.
and
Rubin
,
G. M.
(
1984
).
Analysis of P transposable element functions in Drosophila
.
Cell
38
,
135
146
.
Kiehart
,
D. P.
and
Feghali
,
R.
(
1986
).
Cytoplasmic myosin from Drosophila melanogaster
.
J. Cell Biol
.
103
,
1517
1525
.
Knudsen
,
K. D.
,
Myers
,
L.
and
McElwee
,
S. A.
(
1990a
).
A role for the Ca2+- dependent adhesion molecule, N-cadherin, in myoblast interaction during myogenesis
.
Exp. Cell. Res
.
188
,
175
184
.
Knudsen
,
K. A.
,
McElwee
,
S. A.
and
Myers
,
L.
(
1990b
).
A role for the neural adhesion molecule, N-CAM, in myoblast interaction during myogenesis
.
Dev. Biol
.
138
,
159
168
.
Lai
,
Z.-C.
,
Rushton
,
E.
,
Bate
,
M.
and
Rubin
,
G. M.
(
1993
).
Loss of function of the Drosophila zfh-1 gene results in abnormal development of mesodermal derived tissues
.
Proc. Natl. Acad. Sci. USA
90
,
4122
4126
.
Lawrence
,
P. A.
,
Johnston
,
P.
,
MacDonald
,
P.
and
Struhl
,
G.
(
1987
).
Borders of parasegments in Drosophila embryos are delimited by the fushi tarazu and even-skipped genes
.
Nature
328
,
440
442
.
Leiss
,
D.
,
Hinz
,
U.
,
Gasch
,
A.
,
Mertz
,
R.
and
Renkawitz-Pohl
,
R.
(
1988
).
β3 tubulin expression characterizes the differentiating mesodermal germ layer during Drosophila embryogenesis
.
Development
104
,
525
531
.
Leptin
,
M.
,
Bogaert
,
T.
,
Lehmann
,
R.
and
Wilcox
,
M.
(
1989
).
The function of PS integrins during Drosophila embryogenesis
.
Cell
56
,
401
408
.
Leptin
,
M.
and
Grunewald
,
B.
(
1990
).
Cell shape changes during gastrulation in Drosophila
.
Development
110
,
73
84
.
Lewis
,
E. B.
and
Bacher
,
F.
(
1968
).
Methods of feeding ethyl methane sulfonate (EMS) to Drosophila males
.
Drosophila Inf. Service
43
,
193
.
Luo
,
L.
,
Liao
,
Y. J.
,
Jan
,
L. Y.
and
Jan
,
Y. N.
(
1994
).
Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion
.
Genes Dev
.
8
,
1787
1802
.
MacKrell
,
A. J.
,
Blumberg
,
B.
,
Haynes
,
S. R.
and
Fessler
,
J. H.
(
1988
).
The lethal myospheroid gene of Drosophila encodes a membrane protein homologous to vertebrate integrine β subunits
.
Proc. Natl. Acad. Sci. USA
85
,
2633
2637
.
Michelson
,
A. M.
,
Abmayr
,
S. M.
,
Bate
,
M.
,
Martinez-Arias
,
A.
and
Maniatis
,
T.
(
1990
).
Expression of a MyoD family member prefigures muscle pattern in Drosophila embryos
.
Genes Dev
.
4
,
2086
2097
.
Moore
,
R.
and
Walsh
,
F. S.
(
1993
).
The cell adhesion molecule M-cadherin is specifically expressed in developing and regenerating, but not denervated skeletal muscle
.
Development
117
,
1409
1420
.
Nabeshima
,
Y.
,
Hanaoka
,
K.
,
Hayasaka
,
M.
,
Esumi
,
E.
,
Li
,
S.
,
Nonaka
,
I.
and
Nabeshima
,
Y.
(
1993
)
Myogenin gene disruption results in perinatal lethality because of severe muscle defects
.
Nature
364
,
532
535
.
Newman
,
S. M.
and
Wright
,
T. R.
(
1981
).
Histological and ultrastructural analysis of developmental defects produced by the mutant lethal(1)myospheroid in Drosophila melanogaster
.
Dev. Biol
.
86
,
393
402
.
Olson
,
E. N.
(
1990
).
MyoD family: a paradigm for development?
Genes Dev
.
4
,
1454
1461
.
Paterson
,
B. M.
,
Walldorf
,
U.
,
Eldridge
,
J.
,
Dübendorfer
,
A.
,
Frasch
,
M.
and
Gehring
,
W. J.
(
1991
).
The Drosophila homologue of vertebrate myogenic-determination genes encodes a transiently expressed nuclear protein marking primary myogenic cells
.
Proc. Natl. Acad. Sci. USA
88
,
3782
3786
.
Rudnicki
,
M. A.
,
Braun
,
T.
,
Hinuma
,
S.
and
Jaenisch
,
R.
(
1992
).
Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development
.
Cell
71
,
383
390
.
Rudnicki
,
M. A.
,
Schnegelsberg
,
P. N. J.
,
Stead
,
R. H.
,
Braun
,
T.
,
Arnold
,
H.-H.
and
Jaenisch
,
R.
(
1993
).
MyoD or Myf-5 is required for the formation of sceletal muscle
.
Cell
75
,
1351
1359
.
Seeger
,
M.
,
Tear
,
G.
,
Ferres-Marco
,
D.
and
Goodman
,
C. S.
(
1993
).
Mutations affecting growth cone guidance in Drosophila: Genes necessary for guidance toward or away from the midline
.
Neuron
10
,
409
426
.
St. Johnston
,
R. D.
and
Nüsslein-Volhard
,
C.
(
1992
).
The origin of pattern and polarity in the Drosophila embryo
.
Cell
68
,
1
20
.
Steward
,
R.
and
Govind
,
S.
(
1993
).
Dorsal-ventral polarity in the Drosophila embryo
.
Current Opinion in Genetics and Development
3
,
556
561
.
Tautz
,
D.
and
Pfeiffle
,
C.
(
1989
).
A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback
.
Chromosoma
98
,
81
85
.
Thisse
,
B.
,
El Messal
,
M.
and
Perrin-Schmitt
,
F.
(
1987
).
The twist gene: isolation of a zygotic gene necessary for the establishment of dorso-ventral pattern
.
Nucl. Acids Res
.
15
,
3439
3453
.
Thisse
,
B.
,
Stoetzel
,
C.
,
Gorostiza-Thisse
,
C.
and
Perrin-Schmitt
,
F.
(
1988
).
Sequence of the twist gene and nuclear localization in endomesodermal cells of early Drosophila embryos
.
EMBO J
.
7
,
2175
2183
.
Van Vactor
,
D.
,
Sink
,
H.
,
Fambrough
,
D.
,
Tsoo
,
R.
and
Goodman
,
C. S.
(
1993
).
Genes that control neuromuscular specificity in Drosophila
.
Cell
73
,
1137
1153
.
Weintraub
,
H.
,
Tapscott
,
S. J.
,
Davis
,
R. L.
,
Thayer
,
M. J.
,
Adam
,
M. A.
,
Lassar
,
A. B.
and
Miller
,
A. D.
(
1989
).
Activation of muscle-specific genes in pigment, nerve, fat, liver and fibroblast cell lines by forced expression of MyoD
.
Proc. Natl. Acad. Sci. USA
86
,
5434
5438
.
Zipursky
,
S. L.
,
Venkatesh
,
T. R.
,
Teplow
,
D. B.
and
Benzer
,
S.
(
1984
).
Neuronal development in the Drosophila retina: monoclonal antibodies as molecular probes
.
Cell
36
,
15
26
.