myoblast city

EFFECTS OF MUTATION part 1/2

Given the broad expression pattern of mbc, it was of interest to examine mbc mutant embryos for defects in other tissues. Mutant embryos exhibit a severe somatic muscle phenotype. By comparison, although the visceral musculature appears to be present, as evidenced by myosin-staining cells, obvious defects in midgut constriction and orientation are observed in ~25% of the embryos. However, these defects may be an indirect consequence of the lack of somatic muscles rather than a direct effect of the loss of Mbc in either the visceral mesoderm or the endoderm. The overall structure of the heart, which expresses Mbc late in development, appears to be normal (Erickson, 1997).

Although no epidermal defects had been reported in mbc mutant embryos, the early expression of mbc in the ectoderm, which persists in the epidermis into stage 14, led to a reexamination of mbc mutant embryos for epidermal defects. Visualization of the epidermis with an antibody to Fasciclin III, a cell surface glycoprotein, reveals defects in dorsal closure in ~80% of the mutant embryos. The extent of completion of dorsal closure varies from a relatively small opening surrounded by puckered misshapen cells to a very large opening. In the normal course of dorsal closure in a wild-type embryo, the epidermal cells elongate, and the epithelium stretches over the entire circumference of the embryo. In the early stages of dorsal closure in mbc mutant embryos, the cells along the leading edge of the epidermis appear to be normal. As dorsal closure nears completion, however, many cells along the leading edge cease to be elongated, adopt a rounded shape, and express Fasciclin III abnormally along their migrating edge (Erickson, 1997).

The cytoskeleton along the leading edge of the epidermis has been implicated in driving the process of dorsal closure. Fluorescently conjugated phalloidin, which binds filamentous actin, was used to examine mbc mutants for defects in cytoskeletal formation and organization. Both wild-type and mbc mutant embryos display some variability in the intensity and organization of staining. The signal in wild-type embryos is always stronger than that in mbc mutant embryos. While frequently more dramatic in cells along the migrating edge, this reduction in signal is also observed throughout the epidermis, consistent with the observed expression of mbc. In addition, it should be noted that ~20% of the mbc mutant embryos do not exhibit defects in dorsal closure. These embryos express relatively normal levels of filamentous actin and exhibit only mild cytoskeletal defects. In summary, this analysis suggests that there is a modest but reproducible reduction in cytoskeletal organization in the epidermis of mbc mutant embryos. Unfortunately, examination of the cytoskeletal structure in muscle cells was complicated by the dynamic nature of wild-type muscle cells, making rigorous comparisons with comparable muscle cells in mbc mutant embryos difficult (Erickson, 1997).

DOCK180, the apparent human homolog of mbc, may be involved in Crk-associated signal transduction from focal adhesions (Hasegawa, 1996). If mbc functions in a similar signal transduction pathway in Drosophila, it would be anticipated to be downstream of focal adhesions. Examination of putative focal adhesions in the epidermis of mbc mutant embryos was accomplished using a monoclonal antibody directed against phosphotyrosine. In contrast to the cytoskeletal defects [described above], comparison of phosphotyrosine staining patterns in the epidermis of wild-type embryos and homozygous mbc mutant embryos reveals no apparent differences during dorsal closure. This observation is consistent with the possibility that Mbc, like DOCK180, is downstream of phosphotyrosine-containing complexes in a signal transduction pathway that, in Drosophila, ultimately affects cell migration, dorsal closure, cytoskeletal organization, and myoblast fusion (Erickson, 1997).

Although mbc is quite highly expressed in the heart and the visceral musculature late in development, these tissues do not appear to be severely affected by the loss of mbc. The visceral musculature does appear to be somewhat defective, as evidenced by the absence of midgut constrictions in a low percentage of embryos, but the heart appears to be relatively normal. One interpretation of such behavior is that another gene, yet to be identified, serves a redundant role in these tissues. Another interpretation is that, while the level of expression observed in unfertilized eggs is quite low, adequate maternally derived Mbc protein may be available to embryos lacking zygotic expression of a functional protein. This may be particularly true for the pole cells, which express relatively high levels of Mbc early in development (Erickson, 1997).

mbc mutants have been identified as suppressors of a rac1 overexpression phenotype. To determine whether Rac and Cdc42 GTPases can disrupt eye development, transgenic flies were generated in which wild-type Rac1, Rac2, and Cdc42 GTPases were expressed in the developing eye under the control of the synthetic glass multimer reporter (GMR). Flies harboring a single copy of the rac1 transgene exhibit an externally rough eye, and retinal sections reveal a loss of pigment cells and a disruption of the normal ommatidial morphology, with occasional loss of photoreceptors. With two copies of the GMR-rac1 transgene, a complete disruption of normal eye structure is observed; a similar phenotype is observed in GMR-rac2 transgenic flies. The GMR-cdc42 transgenic flies exhibit externally rough eyes distinct from those seen in the rac1 and rac2 transgenics. Retinal sectioning reveals missing photoreceptors and a disruption of ommatidial morphology. Although the cdc42-induced eye phenotype somewhat resembles the GMR-rho1 phenotype, the cdc42 transgenics also exhibit an abnormal rhabdomere morphology. The postmitotic elongation event that establishes the depth of the retina was also examined. Overexpression of either rho1 or cdc42, but not rac1, disrupts the normal elongation of all retinal cells. Thus, each of these members of the Rho GTPase family, when overexpressed, induces distinct alterations of normal eye development (Nolan, 1998).

To identify specific components of a Rac1 signaling pathway in Drosophila, rac1 transgenic flies were used to screen for dominant mutations that specifically suppress the rac1-induced rough eye phenotype but not that caused by GMR-cdc42 or GMR-rho1. Chromosomal deficiences that cover either rac1 or rac2 each suppress the GMR-rac1 eye defect, confirming that the phenotype is sensitive to the levels of endogenous Rac activity and that Rac1 and Rac2 are normally expressed during eye development. To identify rac1-suppressing mutations, mutagenized wild-type males were mated with GMR-rac1 females and the resulting F₁ progeny were examined for suppression of the rough eye phenotype. A total of 23,000 F₁ flies were screened, and 36 dominantly suppressing mutations were identified. Three complementation groups were established on the basis of lethality, and a single complementation group of 11 alleles has been termed Suppressor of rac1 [Su(rac)1]. Each of the Su(rac)1 alleles dominantly suppresses the GMR-rac1-induced rough eye surface as well as the underlying retinal morphology, rescuing the percentage of normal appearing ommatidia from 3% in GMR-rac flies to 97% in GMR-rac1/Su(rac)1 flies. Each of these alleles also suppresses the GMR-rac2-mediated defect, although none suppresses a GMR-rho1 phenotype. These data suggest that Su(rac)1 encodes a specific component of a Rac-mediated signaling pathway (Nolan, 1998).

Because a specific requirement for Rac activity, but not that of Cdc42, has been demonstrated in the fusion of myoblasts during muscle development (Luo, 1994), the musculature of Su(rac)1 mutants was examined. Myoblast fusion is normally completed by stage 15; however, in stage 15 Su(rac)1 mutants, myoblasts are largely unfused. Meiotic mapping localized Su(rac)1 alleles to a chromosomal region similar to that of a previously reported gene, mbc, that is also associated with a loss-of-function myoblast fusion defect. Null alleles of mbc fail to complement the lethality and myoblast fusion phenotype of several alleles of Su(rac)1. Moreover, mbc alleles also suppress the GMR-rac1 phenotype. Together, these results indicate that Su(rac)1 is allelic to mbc. Although the role of Rac in myoblast fusion is unknown, these results suggest the Mbc mediates the activity of Rac in this morphogenetic process in which actin rearrangements have been implicated previously (Nolan, 1998).

Other phenotypes were examined that would be consistent with aberrant Rac signaling. Drosophila Rac1 has been implicated in axonal outgrowth (Luo, 1994), and mbc mutants exhibit a low penetrance defect in the fasciculation of axons of the ventral nerve cord neurons. Specifically, some mbc mutant embryos exhibit improper spacing between commissures and, in extreme cases, a lack of fasciculation of the longitudinal connectives, possibly because of abnormal migration of the central nervous system (CNS) neurons across the ventral surface. In support of a role for Mbc in cell migration is the recent observation that mutations in ced-5, the C. elegans homolog of mbc, result in defective migration of the distal tip cells of the gonad (Wu, 1998). Additionally, mutations in mig-2, a C. elegans gene encoding a Rac-like GTPase, also affect distal tip cell migration and axon outgrowth (Zipkin, 1997). Moreover, the mammalian Rac GTPase appears to regulate the motility of cultured fibroblasts. It is possible that a pathway mediated by both Rac and Mbc regulates neuronal migration and axon growth, and may explain the CNS defects observed in mbc mutant embryos (Nolan, 1998).

In addition to the defects in myoblast fusion and CNS development, Su(rac)1 alleles exhibit a dorsal closure defect similar to that reported previously for mbc mutants and embryos expressing dominant-negative rac1 (Harden, 1995). During dorsal closure, two symmetric epithelial monolayers coordinately migrate from their lateral position to fuse along the dorsal midline. The row of cells along the dorsal apical edge, known as the leading up edge (LE) cells, elongate first and remain morphologically distinct from the more ventral cells until the two sheets have nearly met at the midline. Recently, a Rac-mediated signaling pathway that regulates this process has been elucidated. Rac1 appears to activate the c-Jun amino (N)-terminal kinase (JNK) pathway, which leads to decapentaplegic (dpp) expression in the LE cells of the dorsal epidermis, and several JNK pathway mutants associated with reduced dpp expression exhibit similar dorsal closure defects, including hemipterous (hep; Jun kinase kinase), basket (Jun kinase), Djun, and kayak (c-Fos). To determine whether Mbc mediates the activity of Rac in the activation of JNK during dorsal closure, the expression of DPP mRNA was examined in mbc mutant embryos. In wild-type embryos, dpp is expressed predominantly in the visceral mesoderm and the LE of the dorsal epidermis. In mbc mutant embryos, 50% of which exhibit dorsal closure defects, dpp is expressed at normal levels in the majority of embryos but appears to be mildly reduced specifically in the leading edge cells of some of these embryos. This is in contrast to hep mutant embryos, in which dpp expression in leading edge cells is clearly absent. This result suggests that Mbc is not absolutely required for JNK pathway activation and may play a distinct role in dorsal closure. However, the possiblity cannot be excluded that Mbc contributes to the activation of JNK in the leading edge cells, but the effects of its absence are masked by a redundant function of Cdc42, which is also capable of activation of JNK in the leading edge cells (Nolan, 1998 and references).

The mammalian protein DOCK180, has been demonstrated to interact directly with Rac, but it is unlikely to act as a RacGEF, that is, it is unlikely that DOCK180 functions directly as a Rac activator. There are two models that most simply explain the role of Mbc in dorsal closure. Possibly, Mbc is required for Rac activation in the leading edge cells during dorsal closure, but some functional redundancy for JNK regulation, which takes place in mbc mutants, is provided by Cdc42. In this scenario, Mbc is still required for Rac-dependent cytoskeletal changes, reflecting a more stringent requirement for Rac activity in regulating cell morphology than in regulating transcription. Consistent with such a possibility, it is found that only a small fraction of mbc mutant embryos that exhibit a dorsal closure defect exhibit any detectable reduction in dpp expression. Although this result suggests a lesser role for Mbc in JNK activation than in cytoskeletal regulation, it is observed that overexpression of DOCK180 leads to activation of JNK in transfected mammalian cells, suggesting that Mbc can potentially play a role in activating JNK in vivo. Alternatively, two separate pools of Rac, with different subcellular localizations, may be utilized for distinct biological processes. In this scenario, Mbc promotes activation of a pool of Rac that regulates reorganization of the actin cytoskeleton but does not substantially affect the pool of Rac required for JNK activation. Consistent with this model, it is found that DOCK180 colocalizes with Rac in membrane ruffles, raising the possibility that Rac, and perhaps other GTPases, can regulate distinct biological processes within a single cell by virtue of subcellularly localized activation (Nolan, 1998).

The visceral musculature of the Drosophila midgut consists of an inner layer of circular and an outer layer of longitudinal muscles. The circular muscles are organized as binucleate syncytia that persist through metamorphosis. At stage 11, prior to the onset of the fusion processes, two classes of myoblasts are detected within the visceral trunk mesoderm. One class expresses the founder-cell marker rP298-LacZ in a one- to two-cells-wide strip along the ventralmost part of the visceral mesoderm, whereas the adjacent two to three cell rows are characterised by the expression of Sticks and stones (SNS). During the process of cell fusion at stage 12, SNS expression decreases within the newly formed syncytia that spread out dorsally over the midgut. At both margins of the visceral band several cells remain unfused and continue to express SNS. Additional rP298-LacZ-expressing cells arise from the posterior tip of the mesoderm, migrate anteriorly and eventually fuse with the remaining SNS-expressing cells, generating the longitudinal muscles. Thus, although previous studies have proposed a separate primordium for the longitudinal musculature located at the posteriormost part of the mesoderm anlage, cell lineage analyses as well as morphological observations reveal that a second population of cells originates from the trunk mesoderm. Mutations of genes that are involved in somatic myoblast fusion, such as sns, dumbfounded ( (duf or kirre) or myoblast city (mbc), also cause severe defects within the visceral musculature. The circular muscles are highly unorganized while the longitudinal muscles are almost absent. Thus the fusion process seems to be essential for a proper visceral myogenesis. These results provide strong evidence that the founder-cell hypothesis also applies to visceral myogenesis, employing the same genetic components as are used in the somatic myoblast fusion processes (Klapper, 2002).

The gene mbc, a homolog of human DOCK180, is expressed in all somatic myoblasts during the fusion processes. In embryos mutant for mbc, syncytia within somatic muscles are almost absent, presumably due to defects in the rearrangement of the cytoskeleton during preceding myoblast fusion. Mbc is also expressed in the visceral mesoderm from stage 12 onward. Embryos mutant for mbc not only exhibit defects in the formation of midgut constrictions but also show severe abnormalities in the formation of visceral muscles. At stage 12 the visceral band is randomly interrupted and the elongated FAS III-expressing cells seem to be disoriented. During further development, parts of the visceral band either stretch out in the dorsoventral direction, as in the wild type, or form disarranged patches. The status of the founder cells in an mbc mutant background was analyzed by expressing rP298-LacZ in the strain mbc^C1. At stage 14 the number of rP298-LacZ-expressing cells within the remaining population of myoblasts appears to be reduced and large gaps within the visceral band are visible that are not present in the wild type. Many globular cells are still observed at the margins of the visceral band that appear to be unfused myoblasts. Antibody staining reveals a prolonged expression of SNS within a corresponding subpopulation of myoblasts. As described for duf and sns mutant embryos, no myoblast fusions were detectable. While the circular musculature shows severe defects and is apparently reduced, the longitudinal musculature is completely absent at the end of embryogenesis (Klapper, 2002).

By using the GAL4/UAS transplantation system for cell lineage analyses of the mesoderm anlage, syncytia were detected not only within somatic muscles but also in the visceral musculature. Since the visceral muscles have classically been described as mononuclear, this surprising observation led an examination of syncytia formation within this tissue. Evidence is provided that the circular visceral muscles of the midgut are likewise organized as syncytia. The first signs of GFP expression (driven by daGAL4) within these muscles were observed in embryos at stage 15. Since there is a considerable delay of about 2-4 h between the activation of the UAS-GFP construct and the formation of the fluorescent product, it is assumed that the formation of syncytia begins at stage 12. This is consistent with the observation of the first fusion processes within the visceral band. Using GFP expression as an in vivo marker, individual syncytia could be followed throughout development. In contrast to longitudinal muscle fibers, which have been found to contain up to six nuclei, the circular muscles of hindgut as well as midgut always comprise two nuclei each. Thus, fusion processes within this tissue stop after the formation of binucleate minimal syncytia that each cover one-half of the gut tube. This is a curious finding since in other muscle types of Drosophila many nuclei share a common cytoplasm to generate a large structural and functional unit (Klapper, 2002).

Following labelled syncytia through all stages of development, it has been shown that the visceral musculature is not replaced by a newly formed imaginal tissue but persists through metamorphosis. Since the visceral musculature plays a crucial role for the proper formation of the midgut during embryonic development, the persisting visceral musculature might again serve as a template for remodelling of the gut tube during metamorphosis (Klapper, 2002).

Since myoblast fusions were discovered within the visceral musculature, whether these syncytia are also formed by Duf-expressing founder and Sns-expressing fusion-competent cells was examined. duf can be detected in muscle precursors as long as they incorporate further fusion-competent cells into the syncytia. The expression of Duf and Sns within the visceral mesoderm suggests a function of specifying founder and fusion-competent cells similar to that observed for the somatic musculature. To clarify whether these genes indeed play a functional role in visceral myoblast fusion, the phenotypes of sns and duf mutant embryos was examined with respect to syncytia formation within the developing visceral musculature. In wild type embryos the ventrally located cells of the visceral band exhibit all characteristics of muscle founder cells, in that they express Duf, become elongated and fuse specifically with Sns-expressing myoblasts. Without duf function no signs of visceral muscle fusion are detectable. It is therefore proposed that duf is the key component in the visceral muscle founder cells. This observation is in agreement with the postulated role of Duf as an attractant for fusion-competent cells in the somatic musculature (Klapper, 2002).

These results indicate that loss of duf function leads to a complete loss of fusion even though the two populations of founders and fusion-competent myoblasts are already aligned within the visceral band at stage 11. Therefore, in the visceral mesoderm, Duf expression seems not only to attract the fusion-competent cells but also to play a crucial role in the fusion process itself. Probably the gaps within the visceral band of duf mutant embryos may indicate an early Duf adhesion function necessary for a proper anterior-posterior alignment of the visceral myoblasts. Furthermore, despite initial cell alignment at stage 11, this contact between founder cells and fusion-competent myoblasts is lost during further development, so that additional gaps appear between these cell layers. In sns mutant embryos, again, no myoblast fusions were detectable. The phenotype closely resembles that of duf mutant embryos, since the two populations of founders and fusion-competent cells seem to reject one another. However, the large gaps within the visceral band of duf embryos were never observed in sns mutants. Whereas in the anterior part of the midgut severe defects of the visceral musculature are obvious at later stages of embryogenesis, the posterior part looks quite normal. This regional difference is somewhat difficult to explain, since at earlier stages no such regional distinctions were observed. It is thought that either in the posterior visceral band sns function can be mimicked or bypassed later on by another spatially restricted gene product, or that even unfused myoblasts can form a seemingly normal musculature in the posterior part. The stronger phenotype at later stages in duf mutant embryos may indicate that the founder cells play a more crucial role in visceral myogenesis than the fusion-competent cells. In somatic myogenesis, founder and fusion-competent cells play specific roles in the recognition process, while in mbc mutants the fundamental capability to fuse is lost in both cell types. Because here intracellular components of the fusion apparatus are affected, fusion-competent cells still aggregate around founders, but the tight membrane junctions are not formed and fusion does not occur. As is consistent with the somatic phenotype, in visceral myogenesis, fusion is totally blocked although founder and fusion-competent cells are in direct contact and still express Sns and Duf (Klapper, 2002).

Taken together, these results provide evidence that the founder-cell hypothesis also applies to visceral myogenesis employing the same genetic components as used in the somatic myoblast fusion processes. Thus, the specification of myoblasts as either founder or fusion-competent cells might be a fundamental step preceding syncytia formation (Klapper, 2002).

Previous analyses have provided evidence that the primordium for the longitudinal midgut musculature is located at the posteriormost tip of the mesoderm anlage. In byn mutant embryos the hindgut as well as the longitudinal musculature are absent. The longitudinal musculature can be rescued, if byn is ectopically expressed exclusively within the posterior tip of the mesoderm anlage, leading to the conclusion that the entire anlage of the longitudinal musculature is located within this posterior region. However, after transplantation within the central region of the trunk, mesoderm labelled longitudinal muscles were frequently obtained. Taking into account that the founder-cell hypothesis is also valid for this tissue, this seeming contradiction can now be solved. The founder cells of the longitudinal musculature that comprise the genetic information for the specific tissue identity arise from the posterior tip of the mesoderm anlage, while the fusion-competent cells originate from the entire trunk region as indicated by confocal analysis. During the fusion processes it is possible to distinguish between two different events of syncytia formation. (1) At stage 12 all the ventrally located founder cells simultaneously become extended dorsally. Each of them fuse with a single SNS-expressing fusion-competent cell, eventually differentiating into a binucleate circular muscle. (2) The remaining SNS-expressing cells migrate to both margins of the visceral band and successively fuse with the longitudinal founder cells that invade from the posterior tip of the mesoderm. It is thus reasonable that in byn mutant embryos the entire longitudinal musculature is missing, since the fusion-competent cells alone are not capable of differentiating longitudinal muscles (Klapper, 2002).

continued: see Effects of Mutation part 2/2 |

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