InteractiveFly: GeneBrief

blown fuse: Biological Overview | References


Gene name - blown fuse

Synonyms -

Cytological map position- 43E1-43E3

Function - cytoskeleton

Keywords - myoblast fusion

Symbol - blow

FlyBase ID: FBgn0004133

Genetic map position - 2R:3,467,990..3,474,548 [-]

Classification - Pleckstrin homology (PH) domain

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

Circular visceral muscles of Drosophila are binuclear syncytia arising from fusion of two different kinds of myoblasts: a circular visceral founder cell and one visceral fusion-competent myoblast. In contrast to fusion leading to the somatic body-wall musculature, myoblast fusion for the circular visceral muscles does not result in massive syncytia but instead in syncytia interconnected with multiple cytoplasmic bridges, which differentiate into large web-shaped muscles. These syncytial circular visceral muscles build a gut-enclosing network with the interwoven longitudinal visceral muscles. At the ultrastructural level, during circular visceral myoblast fusion and the first step of somatic myoblast fusion prefusion complexes and electron-dense plaques were not detectable which was surprising as these structures are characteristic for the second step of somatic myoblast fusion. Moreover, Blown fuse (Blow), a cytoplasmic protein essential for the second step of somatic myoblast fusion (Doberstein, 1997; Schröter, 2004), plays a different role in circular visceral myogenesis. Blow is known to be essential for progression beyond the prefusion complex in the somatic mesoderm; however, analysis of blow mutants established that it has a restricted role in stretching and outgrowth of the syncytia in the circular visceral muscles. Furthermore, in the visceral mesoderm, Blow is expressed in both the fusion-competent myoblasts and circular visceral founders, while expression in the somatic mesoderm is initially restricted to fusion-competent myoblasts. Different enhancer elements in the first intron of blow are responsible for this distinct expression pattern. Thus, a model for Blow is proposed in which this protein is involved in at least two clearly differing processes during Drosophila muscle formation, namely somatic myoblast fusion on the one hand and stretching and outgrowth of circular visceral muscles on the other (Schroter, 2006).

The ultrastructural analyses of the fusion mechanism of Drosophila circular visceral muscles indicates that fusion of the circular visceral muscles takes place without electron-dense vesicles or plaques and ends with incomplete membrane breakdown that is followed by the stretching of the syncytia around the gut. The start of this process resembles the observed contact structures between the somatic Fusion compentent myoblasts (FCMs) and founder cells during precursor formation which is different to the second fusion step during somatic myogenesis. However, there is also a clear difference in the ultrastructural features of fusing circular visceral muscles and the first somatic step of fusion: although cells first adhere in a very similar manner and fuse in both cases without a prefusion-complex or the formation of electron-dense plaques, the first somatic fusion proceeds to completion and results in a continuous syncytium, the precursor cell. Similarly, for vertebrate myogenesis, nascent myotubes containing about three nuclei are formed during the initial fusion steps. In contrast, the fusion of the visceral muscle founders (CVMs) is incomplete and leads to the formation of a web of interconnected strings, while no signs of string formation during the first somatic step of fusion were observed (Schroter, 2006).

Therefore, it is concluded that fusion of circular visceral muscles in Drosophila appears to be an unique process, sharing only similarities to somatic myogenesis in cell adhesion, but not in the presence of electron dense structures that precede fusion. This phenomenon is also reflected in the different use of the same proteins, e.g., Duf and Sns, in both mechanisms, namely somatic and visceral myoblast fusion (Schroter, 2006).

From an evolutionary view, however, both fusion mechanisms might have originated from an ancient general myoblast fusion mechanism, which is expected to be the case due to the molecular and ultrastructural similarities in cell adhesion, as well as the similarities in circular muscle stretching and somatic muscle attachment. The same ancient process might also be the evolutionary basis for vertebrate skeletal myogenesis but not for vertebrate smooth muscle formation. This is reflected in the expression of similar proteins, e.g., DOCK180, the vertebrate homolog to Mbc, and Hem2, a vertebrate Kette-homolog, although their involvement in vertebrate myoblast fusion remains to be confirmed (Schroter, 2006).

To further support the hypothesis that somatic and visceral myoblast fusion are two closely related but clearly different molecular processes, somatic myogenesis relevant mutants from Drosophila were analyzed. No effect on circular visceral myoblast fusion was observed in blow or kette mutants but rather a phenotype affecting the muscle shaping and stretching. This is a further clear difference to somatic myoblast fusion, where Kette and Blow are required for the second step of fusion. Clearly, loss-of-function mutants for blow and kette exhibit different phenotypes in the somatic and visceral mesoderm. Therefore, it is proposed that these proteins are involved in different mechanisms during the development of both mesodermic tissues (Schroter, 2006).

During somatic myoblast fusion, Blow might be involved in transducing the cell-cell-adhesion signal in FCMs from Sns together with Kette and Crk. This would lead to progression beyond the prefusion complex, the formation of electron-dense plaques, and finally the fusion of the FCM with the syncytial precursor cell (Schroter, 2006).

A possible function for Blow during development of circular visceral muscles is the organization and rearrangement of actin together with the actin-regulating factor Kette. If this is true, then it might also explain the observed stretching phenotype. In other words, if actin could not reorganize, then cells would lose their ability to change their shape and thus to stretch and move along the gut for gaining the web-shaped morphology of the mature circular visceral muscles (Schroter, 2006).

Neither Blow mRNA nor protein is detectable before stage 10 in wild-type embryos; thus, it is highly unlikely that Blow mRNA or protein is maternally contributed. In contrast, wild-type embryos contain maternally contributed Kette. Therefore, it cannot be excluded that the maternal contribution of Kette plays a part in the fusion of circular visceral muscles as well as in the first somatic fusion step. For Blow, only the stretching defect was obseerved. Blow is clearly not involved in fusion of the circular visceral muscles (Schroter, 2006).

This study shows that Blow is involved in different processes that are essential for the proper formation of the musculature. Firstly, Blow is essential in somatic myoblast fusion for progression after the prefusion complex. Secondly, Blow is necessary for the stretching and outgrowth of the circular visceral muscles. In addition, Blow could also possibly play a role in the attachment of the somatic muscles at their epidermal destination; Blow was detected in the somatic muscle tips. These different functions of Blow are reflected by the separate enhancer elements guiding expression at specific times and in specific places. Thus, it is proposed that circular visceral myoblast fusion is independent of Blow, similar to the first somatic step of fusion. For the second step of fusion in the somatic mesoderm Blow is required in the FCMs and acts together in a cascade with Mbc, Crk, and the Kette/Sra-1/Abi-complex, which seems to be specific for somatic myogenesis. While in precursor cells Rolling pebbles (Rols) mediates signal transduction after cell-;cell recognition from Duf to the molecular components essential for maintenance of cell-cell contact and myoblast fusion, in FCMs, the component responsible for the initial signal transduction from Sns to this cascade is still unknown. Although the exact role of Blow in this signaling cascade also remains unclear, Blow acts upstream of the Kette/Sra-1/Abi-complex (Schroter, 2006).

During visceral myogenesis, Blow is expressed in both cell types, the FCMs and the circular visceral muscle founders. Therefore, it is proposed that Blow acts together with Kette to cause the stretching of the circular visceral muscles around the midgut. A similar process might later on in the somatic mesoderm be essential for the outgrowth and attachment of the somatic muscles, which is reflected by the concentration of Blow at the muscle ends in stage 16 embryos. It is further proposed that the molecular cascade for this process is similar to one essential for axonal pathfinding and neuronal outgrowth, since attachment phenotypes were observed in kette mutants as well as in other components of this mechanism, e.g., dock-mutants and mutant members of the Wasp-family (Schroter, 2006).

Taken together it has been shown that during Drosophila embryogensis at least two different modes of myogenesis take place: (1) the two-stepped process of somatic myoblast fusion, requiring Blow and the actin-regulating factor Kette for fusion of the precursor cell with additional FCMs and (2) the incomplete fusion of circular visceral founder-cells with one FCM each, requiring Blow and Kette for developing cell morphology after fusion. While process (1) is a very suitable model for vertebrate myogenesis, further studies would be required to see whether process (2) is found in other systems apart from Drosophila. Therefore, it remains to be clarified in which way longitudinal visceral myoblasts of Drosophila fuse and whether this process is similar to either of the two proposed in this study or whether it represents a third way of myogenesis (Schroter, 2006).

kette and blown fuse interact genetically during the second fusion step of myogenesis in Drosophila

Drosophila myoblast fusion proceeds in two steps. The first step gives rise to small syncytia, the muscle precursor cells, which then recruit further fusion competent myoblasts to reach the final muscle size. Kette has been identified as an essential component for myoblast fusion. In kette mutants, founder cells and fusion-competent myoblasts are determined correctly and overcome the very first fusion. But then, at the precursor cell stage, fusion is interrupted. At the ultrastructural level, fusion is characterized by cell-cell recognition, alignment, formation of prefusion complexes, electron dense plaques and membrane breakdown. In kette mutants, electron dense plaques of aberrant length accumulate and fusion is interrupted owing to a complete failure of membrane breakdown. Furthermore, kette interacts genetically with blown fuse (blow) which encodes a novel cytoplasmic protein and is to be required to proceed from prefusion complexes to the formation of the electron dense plaques. Interestingly, a surplus of Kette can replace Blow function during myogenesis. A model is proposed in which Dumbfounded/Sticks and stones-dependent cell adhesion is mediated over Rolling Pebbles, Myoblast city, Crk, Blown fuse and Kette, and thus induces membrane fusion (Schröter, 2004).

kette mutants exhibit a strong myoblast fusion phenotype. Null alleles of kette show numerous unfused myoblasts, while in hypomorphic alleles the fusion phenotype is less severe but defects in muscle attachment become obvious. These fusion defects are due to the intrinsic function of Kette in the myogenic mesoderm (Schröter, 2004).

Furthermore, it has been shown that founder cells and fusion-competent myoblasts are correctly determined in kette mutants and muscle precursor cells are properly formed during the first myoblast fusion step. Electron microscopic analysis of kette mutants revealed that the second myoblast fusion step is interrupted during formation of the electron-dense plaques and thus kette mutants stop development shortly after blow but before sns15 mutants (Schröter, 2004).

Myoblast fusion requires intensive membrane rearrangements and thus an active modulation of the F-actin cytoskeleton, e.g., as seen during endocytosis. In support of the idea that muscle development depends on a F-actin dynamics, myofibre atrophy is observed in the Wave1 knockout mice. The Wave regulator Kette has been shown to be required for myoblast fusion in the fly embryo. Previous biochemical analyses showed that Kette fulfils a dual role in the regulation of the actin cytoskeleton. In one case, Kette promotes F-actin formation at the cell-membranes via Wasp; in the other, Kette inhibits Scar/Wave function in the cytosol (Schröter, 2004).

In addition to a role of Kette during myoblast fusion, high expression of Kette is found at the growing tips of mature myotubes. These structures are rich in F-actin and, like growth cones, migrate towards the muscle-attachment sites (Schröter, 2004).

Within the two-step model of myoblast fusion, kette can be placed relative to other components of the fusion process. The initial recognition between founder cells and fusion-competent myoblasts is mediated by the Ig-domain proteins Duf/Kirre and Rst in the founder cell. The extracellular domain of Duf/Kirre interacts with Sns, another member of the immunoglobulin superfamily, which is expressed in fusion competent myoblasts. This interaction may signal into both cell types and thus initiate the first fusion step that leads to the formation of precursor cells (Schröter, 2004).

It is possible that Duf/Kirre and Rst, as well as Sns, are also active in the second series of fusion events leading from the precursor cells to the mature myotubes. In the precursor cells, the Rols/Ants protein concentrates at the membrane (Chen, 2003; Menon, 2001) and it is proposed that Rols/Ants is needed to start the second series of fusion (Rau, 2001). Chen (2001) has shown that in vitro Rols/Ants binds to the intracellular domain of Duf/Kirre, and it is suggested that this might be the signal in the precursor cell that recruits further FCMs for fusion. In the precursor cell, this interaction might initiate the formation of the prefusion complex and subsequently the formation of the electron-dense plaques and finally to membrane breakdown (Schröter, 2004).

It is assumed that Blow and Kette mediate the Duf/Kirre-Rols/Ants interaction signal in the precursor cell. Blow and Kette are also present in the fusion-competent cells, where it is proposed that Rols/Ants function is taken over by an, as yet, unidentified protein that interacts with Sns. In the precursor cell, Rols/Ants is proposed to mediate rearrangement of the cytoskeleton via Mbc/Dock180. The electron-dense plaques and their connection to microfilaments are symmetrical structures at opposing plasma membranes between precursor and fusion-competent myoblasts. It is proposed that the rearrangement of the actin filaments and their connection to electron-dense plaques is dependent on Kette and its interaction with Blow (Schröter, 2004).

The Blow protein is characterised by a pleckstrin homology (PH) domain that is often involved in mediation of membrane binding and in regulation of the cytoskeleton. Because membrane association is important for Kette function, the observed genetic interaction may reflect a contribution of Blow in activating Kette. Interestingly, it has been recently reported that Blow binds to Crk, which in turn is able to associate with the Dock180 homolog Mbc. This adapter protein is proposed to link to the Duf/Kirre protein via Rols/Ants in precursor cells. It is proposed that a similar link between Sns and Mbc in the fusion-competent cells is mediated by a yet unidentified protein. This scenario would link the activation of membrane-bound receptors to the regulation of F-actin dynamics (Schröter, 2004).

The SH2-SH3 adaptor protein Crk has not yet been studied at the functional level in Drosophila but it is known from vertebrates that its orthologue CrkII and Dock180 form a complex after external stimulation and the complex is able to promote Rac1 activation. Rac1, in turn, is acting on the activation of Wave (Schröter, 2004).

The interaction between Blow and Crk is supported by the finding of several potential binding motives in Blow that are described as potential recognition sites by both Crk-SH3-domains. Therefore, it is postulated that the function of Blow in myoblast fusion is dependent on its binding to Crk for which no mutants exist. It is proposed that this interaction leads to the activation of kette (Schröter, 2004).

Combinatorial binding leads to diverse regulatory responses: Lmd is a tissue-specific modulator of Mef2 activity

Understanding how complex patterns of temporal and spatial expression are regulated is central to deciphering genetic programs that drive development. Gene expression is initiated through the action of transcription factors and their cofactors converging on enhancer elements leading to a defined activity. Specific constellations of combinatorial occupancy are therefore often conceptualized as rigid binding codes that give rise to a common output of spatio-temporal expression. This study assessed this assumption using the regulatory input of two essential transcription factors within the Drosophila myogenic network. Mutations in either Myocyte enhancing factor 2 (Mef2) or the zinc-finger transcription factor Lame duck (Lmd) lead to very similar defects in myoblast fusion, yet the underlying molecular mechanism for this shared phenotype is not understood. Using a combination of ChIP-on-chip analysis and expression profiling of loss-of-function mutants, a global view was obtained of the regulatory input of both factors during development. The majority of Lmd-bound enhancers are co-bound by Mef2, representing a subset of Mef2's transcriptional input during these stages of development. Systematic analyses of the regulatory contribution of both factors demonstrate diverse regulatory roles, despite their co-occupancy of shared enhancer elements. These results indicate that Lmd is a tissue-specific modulator of Mef2 activity, acting as both a transcriptional activator and repressor, which has important implications for myogenesis. More generally, this study demonstrates considerable flexibility in the regulatory output of two factors, leading to additive, cooperative, and repressive modes of co-regulation (Cunha, 2010).

Genes that are co-regulated by the same two (or more) transcription factors are generally expected to have very similar spatio-temporal expression profiles. In fact, this assumption has been used by many studies to computationally predict the location of enhancer elements by searching for common TF binding motifs in the vicinity of clusters of co-expressed genes (or synexpression groups). It was therefore surprising when a comparison of experimentally-identified enhancer regions bound by the same two transcription factors uncovered a diverse range of regulatory responses. The 59 genes with enhancer elements co-bound by Lmd and Mef2 at the same stages of development are regulated either in a cooperative, additive or repressive manner depending on the individual enhancers. These data suggest that enhancer regions integrate regulatory inputs more flexibly than previously anticipated. By focusing on individual enhancer elements, how Lmd and Mef2 influence regulatory activity in different contexts was evaluated both in vivo and in vitro. Combining a number of complementary approaches allowed identification of three different modes of TF integration at developmental enhancers leading to additive, cooperative or repressive regulation (Cunha, 2010).

Mef2 and Lmd provide an additive positive input to the regulation of the Act57B locus. Ectopic Mef2 expression in the ectoderm is sufficient to induce Act57B expression, while providing Lmd alone is not. Conversely, enhancer-reporter gene expression is completely lost in lmd mutant embryos and only slightly reduced in Mef2 loss-of-function mutant embryos. Together, these data reveal a role for both transcription factors at this enhancer. Previous studies demonstrated that the initiation of Act57B expression at stage 11 requires Mef2 for its activation. Yet, artificially increasing Mef2 levels at this stage does not lead to premature activation of this locus. The current findings offer an explanation for this result: at this stage of development, combined input from Lmd and Mef2 is required to drive gene expression, while the presence of Mef2 alone is not sufficient to activate transcription. At later stages, when lmd expression is lost, Mef2 concentration has increased sufficiently to maintain Act57B expression. Conversely, the CG14687 locus can be activated by ectopic Lmd in the ectoderm, but not by Mef2 alone and requires lmd, but not Mef2, for its expression in the somatic muscle. Combined ectopic expression of the two TFs, in contrast, leads to a marked increase of reporter signal, again indicating combinatorial positive regulation by both TFs. These findings are supported by the ability of both Lmd and Mef2 to separately activate reporter gene expression in vitro and to yield additive reporter activity in combination (Cunha, 2010).

The blow enhancer shows a different mode of regulation and is synergistically activated by both factors. While neither Mef2 nor Lmd alone are sufficient to activate ectopic gene expression in vivo, supplying both factors simultaneously leads to robust target gene expression. Assaying for reporter gene activation in the two mutant backgrounds yields a complementary result; Mef2 and Lmd activity is required to activate transcription in the somatic mesoderm via the blow enhancer. Moreover, the in vitro reporter assay reveals a positive interaction between the two proteins, indicating that the blow enhancer functions as a cooperative switch (Cunha, 2010).

Analysis of the CG9416 enhancer revealed an antagonistic interaction between Lmd and Mef2. While ectopic expression of Mef2 leads to enhancer activation, simultaneous expression of Lmd markedly attenuates the transcriptional output from this locus. This effect can be reproduced in vitro: while providing Mef2 alone leads to robust activation of the CG9416 enhancer, Lmd is not able to activate gene expression. Instead, Lmd antagonizes the activating input of Mef2 in a concentration-dependent manner. This is the first example of direct negative regulation by Lmd. To identify additional examples of a repressive role for Lmd, the expression profiles of lmd and Mef2 mutant embryos was re-examined. CG9416 is markedly upregulated in lmd mutants, but shows reduced expression in embryos lacking Mef2. Another direct target gene with similar expression changes was selected in these genetic backgrounds, CG30035, and after determining the limits of the ChIP-enriched region its ability to drive reporter gene expression in vitro was assayed. Similar to the CG9416 enhancer, the CG30035 enhancer is robustly activated by Mef2, and this activation is inhibited by Lmd in a dose-dependent manner. This provides a second, independent example for Lmd-mediated repression of gene expression (Cunha, 2010).

In summary, starting from a genomic perspective, a large cohort of genes co-regulated by a pair of tissue-specific transcription factors was identified. Lmd modulates the activity of Mef2 at different enhancers in a context-dependent fashion, allowing for additive, cooperative or antagonistic interactions in the same cells. In this way, the timing and expression levels of Mef2 target genes can be further refined, as exemplified by the Act57B locus, which may owe its early activation during embryonic development to the combined activity of both proteins. Lmd shows homology with the Gli superfamily of transcription factors, which can act both as transcriptional activators and repressors, depending on proteolytic cleavage regulated by the hedgehog signaling pathway. To date, there is no evidence for proteolytic cleavage of Lmd and an irreversible conversion of Lmd from a transcriptional activator to an inhibitor is difficult to reconcile with the observation that Lmd can perform both roles at different loci at the same time, in the same tissue. For the same reason, it is also considered unlikely that Lmd interferes with transcriptional activation simply by binding to Mef2 and sequestering the protein in the cytoplasm. Instead, it is proposed that Lmd exerts a dominant inhibitory influence over a transcriptional activator, either by locally quenching Mef2's activity or through direct repression of the locus, similar to transcriptional repressors described in other developmental networks. These results provide a molecular understanding for the genetic observation that restoring Mef2 activity in lmd mutant embryos is not sufficient to rescue muscle differentiation. Both transcription factors are required to provide different regulatory inputs to a large number of co-regulated target genes during myogenesis. Their associated enhancers have revealed considerable flexibility in integrating regulatory inputs from these two TFs at individual cis-regulatory regions (Cunha, 2010).


REFERENCES

Search PubMed for articles about Drosophila Blown fuse

Cunha, P. M., et al. (2010). Combinatorial binding leads to diverse regulatory responses: Lmd is a tissue-specific modulator of Mef2 activity. PLoS Genet. 6(7): e1001014. PubMed ID: 20617173

Doberstein, S. K., Fetter, R. D., Mehta, A. Y. and Goodman, C. S. (1997). Genetic analysis of myoblast fusion: blown fuse is required for progression beyond the prefusion complex. J. Cell Biol. 136(6): 1249-61. PubMed ID: 9087441

Schröter, R. H., et al. (2004). kette and blown fuse interact genetically during the second fusion step of myogenesis in Drosophila. Development 131: 4501-4509. PubMed ID: 15342475

Schroter, R. H., Buttgereit, D., Beck, L., Holz, A. and Renkawitz-Pohl, R. (2006). Blown fuse regulates stretching and outgrowth but not myoblast fusion of the circular visceral muscles in Drosophila. Differentiation 74(9-10): 608-21. PubMed ID: 17177857


Biological Overview

date revised: 15 April 2011

Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.