BIOLOGICAL OVERVIEW

The development of muscles (myogenesis) involves cell determination activity as specific as that found in the development of nerves (neurogenesis). The specificity involved in the process of neuroblast origination has to do with the structuring of the neuroectoderm by gap, pair-rule and segment polarity genes. These genes can uniquely define each row of cells in the parasegment, and in this way determine specific fates.

Physical contact between developing tissues is as consequentially relevant as chemical activity. (The former is discussed in additional detail in the biological overviews of muscle segment homeobox and stripe genes). In mesoderm, pair-rule genes define segmentation indirectly, through the physical contact of mesoderm with overlying ectoderm, and the receipt of chemical signals transmitted by the proteins Wingless and DPP.

Some of the same systems operate as in neuroblast and muscle development, including the action of neurogenic genes Delta and Notch and the pro-neural gene lethal of scute (Carmena, 1995).

Three waves of slouch/NK1/S59 expression are found in the development of mesoderm. Initially, after two cycles of mitotic cell division in the mesoderm, slouch is expressed in a single twist expressing cell (I), located ventrally in each hemisegment. After a third round of cell division, the two progeny of cell I as well as a second cluster of cells (II) start to express slouch. More cells express slouch in thoracic and labial segments than in abdominal segments. These cells are at the tips of parasegmental bulges in the mesoderm, corresponding to a posterior position at the parasegmental borders. Type I cells are close to the anterior borders. Soon after the above activity, an additional pair of mesodermal cells (III) express slouch. These type III cells are located in a lateral position at the parasegmental border, and found only in abdominal segments (Carmena, 1995).

The segmental diversity is regulated by the bithorax complex, in one of its most significant roles. Myogenesis continues after this by cell migration and fusion, and attachment to ectodermal cells of the parasegmental border (Dohrmann, 1990).

slouch expression is found in muscle founder cells responsible for the origin of the specific musculature of each segment. Therefore, segmentally repeated musculature is established in a pattern that has its origin in the pattern of the overlying ectoderm. This patterning is communicated to the mesoderm by cell contact and chemical communication. Thereafter, the segmental diversity is governed by the bithorax complex and its constituent homeotic genes.

There is mounting evidence that founder cell identities are determined by the expression of defined combinations of regulatory factors. Notably, loss of function and ectopic expression of ap, Kr, msh and lb can cause transformations in muscle founder identities and aberrant muscle patterns, while loss of nau or coe activity results in arrested differentiation of specific founder cells and absence of the particular muscles that they would normally form. However, for a more complete understanding of muscle diversification, it is necessary to define the functions of additional identity genes, study the hierarchies of their genetic interactions and identify their regulatory targets. A null mutation in the gene encoding S59, named slouch (slou), disrupts the development of all muscles that are derived from S59-expressing founder cells. The observed phenotypes upon mutation and ectopic expression of slouch include transformations of founder cell fates, thus confirming that slouch (S59) functions as an identity gene in muscle development. These fate transformations occur between sibling founder cells as well as between neighboring founders that are not lineage-related. In the latter case, slouch (S59) activity is required cell-autonomously to repress the expression of ladybird (lb) homeobox genes, thereby preventing specification along the lb pathway. Together, these findings provide new insights into the regulatory interactions that establish the somatic muscle pattern (Knirr, 1999).

To assess the role of slou (S59) in muscle development, the muscle pattern in slou mutant embryos was visualized with antibodies against muscle myosin heavy chain (MHC). The pattern of ventral muscles is severely disrupted in these embryos and, notably, there is a complete absence of muscles 25 and 29. In addition, the syncytial fibers at the normal positions of muscles 26 and 27 have aberrant and variable morphologies although their insertion sites and sizes are still reminiscent of those two muscles. Whether they form two separate or one large syncytium in each segment could not be determined. Disruptions are also observed in lateral and dorsolateral areas of the body wall musculature. Specifically, muscle 5 is absent, whereas muscle 8 appears duplicated in each of the segments. At the normal positions of muscles 11 and 18, there is one syncytium (or perhaps two closely associated ones) with a morphology that does not resemble either of these two dorsolateral muscles, although it shares their ventral attachment site. In contrast to the altered ventral and dorsolateral muscle patterns, the pattern of the dorsal somatic muscles is normal in the absence of slou (S59) activity. This is consistent with the observed absence of S59 expression in these areas in wild-type embryos. Together, these observations show that loss of slou activity affects the development of all muscles derived from S59-expressing muscle founders. slou function appears to be strictly required in these muscles, because loss of muscles 5, 25, 29 and abnormalities associated with muscles 11/18 and 26/27 are observed in nearly 100% of all examined abdominal segments from mutant embryos (Knirr, 1999).

Ectopic expression of slou (S59) in the mesoderm of wild-type embryos also produces severe alterations in the muscle pattern. The patterns of the ventral, lateral and dorsal somatic muscles are severely disrupted in these embryos, thus making it difficult to assign specific identities to individual muscle fibers. Morphological changes have been observed in the dorsal vessel, where some of the cardioblasts appear enlarged or are arranged in clumps instead of single rows and express higher than normal levels of myosin. These observations indicate that expression of S59 in muscle founders and muscles where it is not normally expressed causes abnormal development and perhaps transformations of their identities. The effects in the cardioblasts are reminiscent of those observed upon ectopic expression of nau. These were interpreted as partial transformations from cardial to somatic muscle cell identities (Knirr, 1999).

Initial morphological examination of muscles 26/27 and 11/18 in slou mutants was not sufficient to determine whether these muscles are transformed or retain their identities but undergo abnormal differentiation. For further analysis, S59 was used as a marker for these two muscle types because its expression is maintained in muscles 18 and 27 in late-stage wild-type embryos. S59 mRNA or lacZ driven by an S59/lacZ construct, which mimics the normal pattern of S59 expression, is still expressed in the founders of the morphologically aberrant muscles 11/18 and 26/27 in the slou mutant background. A second marker for muscle 27 is Kr, which has an important role in distinguishing the identity of this muscle from that of muscle 26. In wild-type embryos, Kr is coexpressed with S59 in the progenitor and founders of muscles 26 and 27, and similar to S59, Kr expression is lost in muscle 26 and maintained only in muscle 27. While there is presently no formal proof for these lineage relationships, the data from S59 antibody and S59/lacZ stainings and genetic analysis of lineage gene mutants provide good support for sibling relationships between muscle founders 5 and 25, muscle founders 26 and 27, and muscle founder 29 and the ventral adult precursor cell, respectively. There is also some, albeit less clear, evidence for an origin of muscle founders 18 and 11 from a common progenitor. In slou (S59) mutant embryos, both S59 and Kr expression are initiated normally in the 26/27 progenitors and founders. In contrast to the founder cell stage, during fusion the expression of S59/lacZ, S59 mRNA and S59 nascent transcripts is abolished in most (~85%) of the muscle 26/27 precursors in slou mutant embryos. However, unlike S59, Kr expression is fully maintained in the aberrant 26/27 syncytia until late stages in slou mutants. Expression of nautilus (nau), another marker for muscle 27 , is also not affected in the absence of slou activity. In contrast to Kr and nau, there is no expression of apterous / lacZ (ap / lacZ) in the founders and syncytia of muscle 27 of slou mutant embryos. Together, these observations indicate that, in the absence of slou activity, the progenitors of muscles 26 and 27 are formed normally. Later, the resulting muscle fibers appear to assume features that are intermediate between muscles 26 and 27 because they maintain Kr and nau (as is normally observed in 27) but fail to activate a muscle 27-specific enhancer of ap and lose S59 (as is normally the case for 26) (Knirr, 1999).

The observed loss of muscles 5 and 25 in slou (S59) mutant embryos -- muscles that are derived from two daughter cells of a common progenitor (cluster I) -- is strictly correlated with the duplication of the segment border muscle (muscle 8). Because muscle 8 comes from the same lateral area as the progenitor of 5/25, this observation raises the question of whether loss of slou activity causes a transformation of the 5/25 progenitors/founders into muscle 8 progenitors/ founders. Alternatively, slou mutation could have non-autonomous effects on neighboring myoblasts causing a duplication of muscle 8. Recently, it has been shown that the homeobox gene lb is expressed in muscle 8 and its progenitor and functions in specifying this muscle, as well as the lateral adult muscle precursor that is derived from a sibling founder cell. As a first step toward clarifying the developmental relationships between the S59- and lb-expressing lateral muscles, a comparative expression analysis of the two genes in wild-type embryos was performed. During early stage 11, after S59 expression has initiated in the 5/25 progenitor, lb expression is observed in a dorsally abutting cluster of 6-7 cells. Shortly thereafter, lb expression becomes restricted to two progenitors within this promuscular cluster. These progenitors are still closely associated with the S59-expressing progenitor. During late stage 11, the S59 progenitor divides asymmetrically to generate two founders: a larger, dorsally positioned founder 5 and a smaller, more ventral founder 25. Upon completion of this division, each of the two lb progenitors also divides once to generate four founders that are arranged in close proximity. During late stages 12-13, muscle 25 founder migrates ventrally along a diagonal path to form muscle 25 in the posteriorly adjacent segment, whereas the muscle 5 founder migrates slightly dorsally to form muscle 5. At the same time, one of the four lb founders migrates dorsally along the segment border and ultimately fuses with surrounding myoblasts to form muscle 8. Two of the three remaining lb cells stay unfused at their original positions and form two lateral adult precursor cells. The third cell rapidly loses lb expression, which has prevented a definition of its ultimate fate (Knirr, 1999).

To determine whether the duplication of muscle 8 in the absence of slou (S59) activity is due to ectopic expression of lb, a similar analysis was performed in slou mutant embryos. In this analysis, an S59/lacZ line that expresses lacZ in early stages in the progenitor and founder cells for muscle 5 and 25 was used to follow lb cells and S59 founders. slou mutants indeed display lb expression in five founder cells instead of three, and upon division of the second lb progenitor, in six founder cells instead of four. Inspection of S59/lacZ and lb doubly stained embryos clearly demonstrates that the ectopic lb-expressing cells correspond to the cells that are supposed to be muscle 5 and muscle 25 founders. Furthermore, analysis of late-stage slou mutant embryos shows that the supernumerous lb founders now proceed along developmental pathways that are typical for bona fide lb cells. Thus, they form a second lb-expressing muscle 8 and an extra lateral adult muscle precursor in each segment. Because these observations suggest a normal function of slou (S59) in repressing lb in the founders of muscles 5 and 25, a test was performed to see whether ectopic expression of S59 could also repress lb in the authentic founders of muscle 8 and lateral adult muscle precursors. Pan-mesodermal expression of S59 indeed causes a strong reduction in the number of lb-expressing muscle founders, and, in many segments, their complete loss. As a consequence, many segments are lacking muscle 8 and lateral adult precursors in later stages, thus contributing to the strong aberrations in the muscle patterns seen in these embryos (Knirr, 1999).

In addition to muscles 5 and 25, loss of muscle 29 has also been observed in slou mutants. Examination of the fate of the 29 muscle founder in the absence of slou activity reveals the appearance of a second ventral adult muscle precursor cell, a cell type that is normally formed by the sibling of the 29 founder. Thus, in the absence of S59, it appears that the default fate of these two founders is that of ventral adult muscle precursors, and that S59 expression normally promotes a muscle 29 fate in one of them. This is consistent with the transiently higher levels of S59 expression in the muscle 29 founder as compared to the ventral adult muscle precursor. However, the failure to suppress formation of ventral adult precursors upon ectopic S59 expression indicates that slou (S59) is required, but not sufficient to specify one of the two siblings as muscle 29 (Knirr, 1999).

Since some slou (S59) mutant animals escape lethality, the consequences of slou mutation for adult muscle patterning could be studied. Shortly after eclosion, the larval segment border muscles (muscles 8) in wild-type flies still persist, whereas other larval muscles have been histolyzed and replaced by the adult muscle groups derived from corresponding adult muscle precursors. Freshly eclosed slou mutant flies display a duplicated muscle 8 in every abdominal hemisegment. Because slou mutant embryos were found to have one supernumerary ventral and lateral adult muscle precursor, one might expect to find corresponding increases in the number of ventral and lateral adult muscle fibers. However, these muscle groups contain essentially the same numbers of muscle fibers as in wild-type flies (~6 ventral fibers and ~20 lateral fibers per abdominal hemisegment). This result indicates that there is not a strict one-to-one relationship between the number of embryonic muscle precursors and the corresponding adult muscle fibers and suggests that there are mechanisms to readjust the number of muscles. Alternatively, it is possible that the extra adult muscle precursors in slou mutants are not fully functional and unable to form adult muscle fibers (Knirr, 1999).

slou mutant flies display sluggish movements, are unable to fly and the majority have outspread, upheld or drooping wing postures. Because embryonic thoracic segments, which produce the flight muscles, contain a large number of S59-expressing myoblasts, adult slou mutant flies were sectioned to determine whether these phenotypes might be due to abnormalities in flight muscle development. slou mutant flies have a full complement of direct and indirect flight muscles, and their overall morphology closely resembles that of corresponding muscles from wild-type flies. However, it is possible that loss of slou activity causes more subtle defects in thoracic muscle differentiation. Because S59 is also expressed in specific neurons of the CNS, the observed motoric and wing posture phenotypes could also result from defects in the development and function of motoneurons, which were not analyzed in the present study (Knirr, 1999).

The observations with respect to cluster I provide novel clues about the regulatory pathways from promuscular clusters to identified muscles. Muscle progenitors from neighboring clusters segregate at different time points in early mesoderm development. Specifically, at the time when lb expression initiates in the entire promuscular 8/LaP cluster, the muscle 5/25 progenitor of the adjacent cluster is already specified and expresses S59. It is proposed that this is due to sequential regulatory inputs received by these cell clusters. An initial input (A) may serve to activate lb at intermediate levels in all cells of the promuscular cluster, and perhaps at the same time define this cluster whereas another input (B), which is received by the neighboring cluster, triggers S59 expression in the 5/25 progenitor in a process that also involves lateral inhibition through Notch signaling. In a subsequent step, a functionally analogous input (C) would then serve to restrict lb expression to two progenitors in the promuscular 8/LaP cluster. Interestingly, based upon observations in slou mutants, input C appears not to be restricted to this cluster, but may cover both clusters or at least include the areas where they abut. The reason for the exclusive response of the 8/LaP cluster to this input is that slou (S59) and active Notch apparently repress lb activation by input C in the 5/25 cluster. This model would explain why, in the absence of slou activity, the cell normally destined to become a 5/25 progenitor, (in which the Notch pathway is inactive) expresses lb ectopically. The final effects on the muscle pattern are very similar to those obtained with GAL4/UAS-driven ectopic lb expression. The model is also in agreement with data showing that ectopic expression of S59 can repress lb in the 8/LaP cluster, producing muscle phenotypes similar to those of lb loss of function. An additional requirement for a response to these spatially localized inputs, which is shared by the cells in both clusters, is the activity of tinman, which may act as a mesoderm-specific coactivator for slou (S59) and lb activation by external inputs (Knirr, 1999).

What might be the nature of these sequential inputs? The observation of the occurrence of sequential events during promuscular cluster/muscle progenitor formation is reminiscent of previous observations made in the dorsal somatic mesoderm. In these studies, it was shown that an initial, FGF-receptor (Htl)-dependent signal is involved in delineating a promuscular cluster, C2, and in specifying a progenitor within it, which in turn gives rise to a pair of eve-expressing pericardial cells. A subsequent signal, which is mediated by the EGF-receptor (DER/Flb, perhaps together with Htl), functions analogously to define an adjacent cluster, C15, and an eve-expressing progenitor within it that gives rise to muscle 1. Whereas the Htl-activating signal has not been identified, it has been suggested that localized Rhomboid expression in the initially formed progenitor P2, in conjunction with the more broadly expressed ligands Spitz and Vein, may trigger Egfr-activation in the adjacent C15 cluster and P15 progenitor. It is possible that promuscular cluster formation and progenitor specification in the lateral somatic mesoderm follows a similar sequence of events. Since embryos with reduced Htl activity appear to lack muscles 5, 8 and 25 and embryos with reduced Egfr activity lack muscle 8, but not muscles 5 and 25, it is conceivable that input B activates S59 through Htl, whereas input C activates lb through Egfr (in the absence of S59). It is not known whether the progenitor of muscles 5/25 expresses Rhomboid or another Egfr-activating signal but, if so, slou is not required for activating the expression of such signals. Rather, slou functions in a strictly cell-autonomous manner, since its mutation affects only muscles that are derived from slou (S59)-expressing progenitor and founder cells. Mechanistically, one of these functions is likely to include direct repression of lb transcription in the progenitor of muscles 5/25 (Knirr, 1999).

Besides its involvement in early cell fate decisions, it is plausible that slou continues to function in the developing muscles that maintain its expression. Of note, in situ hybridization experiments with slou (S59) intron probes show that upon division of the progenitor 5/25, the muscle 5 founder maintains transcription until fusion, whereas the muscle 25 founder initially stops transcribing slou (S59) but resumes expression upon fusion with myoblasts at its final position. In addition, the nuclei of newly fused cells also initiate slou (S59) transcription, thus providing high levels of Slou (S59) protein in muscles 18, 25 and 27 during their later stages of development. Although no direct evidence is available, this intricate regulation suggests that the differential maintenance of slou (S59) could be directly involved in the control of certain late aspects of muscle development, such as muscle differentiation and morphogenesis, and possibly also innervation (Knirr, 1999).

GENE STRUCTURE

slouch/NK1/S59 is a homeodomain protein in a class by itself. It is expressed regionally in very restricted cell groups, including endodermal, mesodermal (muscle) and neural. It is found closely linked to other homeobox genes, including ladybird-early, ladybird-late, tinman and bagpipe (Kim, 1989, Jagla, 1994).

Bases in 5' UTR - 325

Exons - four

Bases in 3' UTR - 372

PROTEIN STRUCTURE

Amino Acids - 661

Structural Domains

Slouch/NK1 has a paired class homeodomain, meaning it has a homedomain DNA binding region of the paired class, and a paired box which can mediate protein-protein interaction. The paired repeat, rich in amino acids His and Pro, is similar to that of Drosophila genes paired and bicoid. Other domains include a polyalanine stretch and an acidic domain followed by a polyglycine stretch amino terminal to the homeodomain (Dohrmann, 1990).

The Engrailed homeoprotein is a dominantly acting, so-called 'active' transcriptional repressor, both in cultured cells and in vivo. When retargeted via a homeodomain swap to the endogenous fushi tarazu gene (ftz), Engrailed actively represses ftz, resulting in a ftz mutant phenocopy. Functional regions of Engrailed have been mapped using this in vivo repression assay. In addition to a region containing an active repression domain identified in cell culture assays, there are two evolutionarily conserved regions that contribute to activity. The one that does not flank the HD is particularly crucial to repression activity in vivo. This domain is present not only in all engrailed-class homeoproteins but also in all known members of several other classes, including goosecoid, Slouch, Nk2 (vnd) and muscle segment homeobox. The repressive domain is located in the eh1 region, known as 'region three', found several hundred amino acids N-terminal to the homeodomain. The consensus sequence, arrived at by comparing Engrailed, Msh, Gsc, Slouch and NK2 proteins from a variety of species, consists of a 23 amino acid homologous motif found in all these proteins. Thus Engrailed's active repression function in vivo is dependent on a highly conserved interaction that was established early in the evolution of the homeobox gene superfamily. Using rescue transgenes it has been shown that the widely conserved in vivo repression domain is required for the normal function of Engrailed in the embryo (Smith, 1996).

date revised: 26 October 99

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