slouch

Promoter Structure

To determine the cis-acting regulatory elements controlling the cell-specific expression of NK-1, transiently expressed chloramphencol acetyl transferase (CAT) reporter gene activities from transfected C2C12 myoblasts and NG108-15 neuroblastoma cells were measured using various CAT constructs containing different 5' upstream regions of NK-1. From the initial analysis of 3.9 kb of the 5' upstream region, it has been found that the regions from -1865 to -476 and from -476 to +100 contained strong negative and positive regulatory elements, respectively. Within the positive cis-acting region an 86-bp DNA fragment (from -435 to -350) is sufficient to activate the reporter gene in C2C12 cells, whereas additional regions (from -157 to -28 and from -510 to -425) are required for optimal activity in NG108-15 cells. Gel shift and DNaseI footprinting assays have defined a plausible binding site for C/EBP, 5'-TTTCGCAAG-3' (-424 to -416), and a novel binding site for unknown factors, 5'-AATTACTCACATCC-3' (-370 to -357). Further mutation analysis has revealed that the novel binding sequence for unknown factors is necessary and sufficient for transcriptional activity for reporter gene expression in C2C12 myoblast cells in an orientation-independent manner (Kim, 1999).

Transcriptional Regulation

Ectopic expression of muscle segment homeobox gene in the mesoderm results in altered expression of the slouch/NK1/ S59 and nau/Dmyd genes, leading to a loss of some muscles and defects in the patterning of others, suggesting that the muscle defects are at the level of recruitment and/or patterning of muscle precursor cells (Lord, 1995).

The mechanisms that underlie the segregation of muscle founder cells in the Drosophila embryo are undefined. The proneural gene lethal of scute (l'sc) is expressed in clusters of cells in the somatic mesoderm, from which individual muscle progenitors are singled out by progressive restriction of l'sc expression. Coexpression of l'sc and slouch in a subset of muscle progenitors shows that muscle founders are produced by division of muscle progenitors. In neurogenic mutant embryos the restriction of l'sc expression fails and all cells in a cluster coexpress l'sc and S59. Loss-of-function and overexpression phenotypes indicate a role for l'sc in the segregation of muscle progenitors and the formation of the muscle pattern (Carmena, 1995).

Subsets of differentiating muscles in the Drosophila embryo express putative transcription factors, such as slouch and vestigial. These genes are thought to control the development of specific muscle properties. Myogenesis in embryos mutant for wingless is grossly deranged. Mesodermal expression of slouch is lost, whereas some vestigial-expressing muscles develop. wingless dependence and independence of specific muscle subsets correlates with an early derangement of twist expression in wingless mutants. Ectoderm appears to have a possible role in the patterning of Drosophila mesoderm (Bate, 1993). slouch expression in the midgut corresponds to a region where labial is expressed. Ubx, dpp and wg are required for labial expression and Slouch may be activated in parallel or downstream of labial (Dohrmann, 1990).

During Drosophila embryogenesis, mesodermal cells are recruited to form a complex pattern of larval muscles. The formation of the pattern is initiated by the segregation of a special class of founder myoblasts. Single founders fuse with neighbouring nonfounder myoblasts to form the precursors of individual muscles. Founders and the muscles that they give rise to have specific patterns of gene expression, and it has been suggested that it is the expression of these founder cell genes that determines individual muscle attributes such as size, shape, insertion sites and innervation. The segmentation gene Kruppel is expressed in a subset of founders and muscles. Kruppel protein is expressed in a variety of muscles, including two dorsal muscles (the dorsal acute muscle 1 and the dorsal oblique muscle 1), three lateral muscles (including the lateral longitudinal muscle 1 and the lateral transverse muslces 2 and 4), and four ventral muscles (ventral longitudinal muscle 3, ventral acute muscle2 and ventral oblique muscles 2 and 5). Kruppel regulates specific patterns of gene expression in these cells, specifically the homeodomain gene slouch, also known as S59. Kruppel is required for the acquisition of proper muscle identity. Gain and loss of Kruppel expression in sibling founder cells is sufficient to switch these cells (and the muscles to which they give rise) between alternative cell fates. Thus Kr is not responsible for myogenic differentiation but for the specific characteristics of individual muscles. Ubiquitous expression of Kr does not alter the pattern of slouch expression in muscle progenitors and the onset of slouch expression is normal in the absence of Kr (Ruiz-Gomez, 1997).

Mutations in wingless leads to the complete loss of a subset of muscle founder cells characterised by the expression of slouch/S59. Wingless acts directly on the mesoderm to ensure the formation of slouch-expressing founder cells. Wg can signal across germ layers: in the wild-type embryo, Wg from the ectoderm constitutes an inductive signal for the initiation of the development of a subset of somatic muscles (Baylies, 1995).

Muscle founder cells arise from the asymmetric division of muscle progenitor cells, each of which develops from a group of cells in the somatic mesoderm that express lethal of scute. All the cells in a cluster can potentially form muscle progenitors, but owing to lateral inhibition, only one or two develop as such. Muscle progenitors, and the subsequent founder cells, then express transcription factors such as Krüppel, S59 and Even-skipped, all of which confer identity on the muscle. Definition of some muscle progenitors, including three groups that express S59, depends on Wingless signaling. Lateral inhibition requires Delta signaling through Notch and the transcription factor Suppressor of Hairless. Since the Wingless and lateral-inhibition signals are sequential, one might expect that muscle progenitors would fail to develop in the absence of Wingless signaling, regardless of the presence or absence of lateral-inhibition signaling. The development of the S59-expressing muscle progenitor cells has been examined in mutant backgrounds in which both Wingless signaling and lateral inhibition are disrupted. Progenitor cells fail to develop when both these processes are disrupted. This analysis also reveals a repressive function of Notch, required before or concurrent with Wingless signaling that is unrelated to its role in lateral inhibition (Brennan, 2000).

During wild-type development, expression of S59 is first seen during stage 10 in a single muscle progenitor cell either side of the midline in every segment. By stage 11, this pattern has evolved in abdominal segments such that S59 expression is seen both in the nervous system and in two groups of muscle progenitor cells. During stage 12, a third muscle progenitor cell starts to express S59. These muscle progenitor cells give rise to three muscle founder cells that maintain the expression of S59. Fusion of these founder cells with myoblasts results in the S59-expressing muscles seen in late stages of embryogenesis (Brennan, 2000).

Disruption of lateral-inhibition signaling, in either Notch (N) germline-clone, suppressor of Hairless germline-clone or Delta zygotic mutant embryos, increases the number of cells expressing S59 compared with wild type at stage 11. Because of general degeneration of these embryos during germ-band retraction, however, it is difficult to examine the expression of S59 after stage 11, but the mesoderm clusters that can be identified are expanded (Brennan, 2000).

Unlike the disruption of lateral-inhibition signaling, attenuation of Wingless signaling, by removing either wingless (wg) or dishevelled function, blocks the expression of S59 in the mesoderm. In contrast, increasing Wingless signaling, either by overexpressing the Wingless protein in the mesoderm using the GAL4/UAS system (twist-GAL4>UASwg embryos), or by removing shaggy function (sgg^m11 germline-clone embryos), leads to enlarged groups of S59-expressing muscle progenitor cells during stage 11. However, during germ-band retraction, the groups are reduced in size. In the twist-GAL4>UASwg embryos the reduction in cluster size leads to a largely normal set of three muscles, whereas in the sgg^m11 embryos the reduction is more extreme and leads to the loss of S59-expressing muscles (Brennan, 2000).

Since Wingless signaling is required for the initiation of S59 expression in the mesoderm and lateral-inhibition signaling is required for the subsequent restriction of S59 expression to one or two cells within each cluster, it is expected that in the absence of Wingless signaling S59 will not be expressed, even if lateral-inhibition signaling is also blocked. This appears to be the case in wg^S107.5;Dl^FX3 zygotic and wg^S107.5,Su(H)^SF8 germline-clone embryos. In contrast, mesodermal S59 expression is observed in Df(1)N^81k1,dsh^v26 and Df(1)N^81k1;wg^CX4 germline-clone embryos, in which Wingless signaling is blocked and Notch function is removed. Finally, as with the single-mutant embryos, the double-mutant embryos degenerate during germ-band retraction, making it difficult to examine S59 expression after stage 11 (Brennan, 2000).

These results first confirm that Wingless signaling is required for the initiation of S59 expression and that a Delta-initiated lateral-inhibition signal is required for the restriction of S59 expression to one or two cells of each initial cluster. They also confirm the prediction that, in the absence of a Wingless signal, S59 is not expressed, regardless of whether lateral-inhibition signaling is occurring. Also, even though hyperactivating Wingless signaling leads to initially enlarged groups of S59-expressing muscle progenitor cells, a reasonably normal muscle pattern is obtained (Brennan, 2000).

The observed S59 expression in Df(1)N^81k1, dsh^v26 and Df(1)N^81k1; wg^CX4 embryos can be explained if it is assumed that Notch has a repressive function that precedes Wingless signaling. In this situation, removal of Notch function will lead to the derepression of S59 expression before Wingless signaling. Consequently, it does not matter whether or not Wingless signaling occurs. This repressive function cannot be related to Delta signaling, however, because the removal of Delta or Su(H) function in embryos where Wingless signaling is not occurring does not result in S59 expression. The repressive function of Notch uncovered in these experiments must therefore be distinct from its repressive role during lateral inhibition (Brennan, 2000).

The second observation suggests that in response to increased Wingless signaling there is a linked increase in lateral-inhibition signaling. This would mean that increased Wingless signaling will only lead to a significant increase in the number of muscle progenitors if lateral inhibition cannot occur. The observed difference in the final muscle pattern between twist–GAL4>UASwg and sgg^m11 embryos is probably due to the difference in how Wingless signaling is activated in the different embryos. In the twist–GAL4>UASwg embryos, Wingless signaling is activated only transiently and is restricted to the mesoderm. In contrast, Wingless signaling is activated globally and throughout embryogenesis in sgg^m11 germlineclone embryos. This difference, along with the proposed linkage between Wingless signaling and lateral inhibition would mean that lateral inhibition is much greater in the sgg^m11 embryos. This situation would explain the greater reduction in the size of the groups of S59-expressing muscle progenitor cells observed in the sgg^m11 embryos and the loss of muscles if the restriction is too great (Brennan, 2000).

The link between Wingless signaling and lateral inhibition could occur in a number of ways. For example, Wingless signaling may directly alter a component of the Delta signaling pathway that would then increase the ability of this pathway to transduce the Delta signal. Alternatively, Wingless signaling could affect Delta signaling by altering the transcription of one of the components of the pathway. Either of these mechanisms would allow the organism to generate a lateral-inhibition signal appropriate to the input signal: a strong Wingless signal would lead to a strong lateral-inhibition signal and prevent unnecessary and unwanted development, whereas a weak Wingless signal would lead to a weak lateral-inhibition signal that allows development to proceed even though the input signal is weak. This would allow normal development to occur even if there are fluctuations in the input signal (Brennan, 2000).

It is thought that the muscle progenitor cells develop from a large pool of developmentally equivalent cells that is refined through two steps to produce one muscle progenitor cell. A very large group of cells is initially defined that have the potential to become muscle progenitor cells but are prevented from doing so by the novel function of Notch identified here. Wingless signaling then alleviates this repressive function of Notch within a few cells of the cluster to establish an equivalence group. This triggers the process of lateral inhibition, which subsequently selects a single cell to become a muscle progenitor. In this situation, overexpressing Wingless or constitutively activating Wingless signaling will alleviate the initial repressive function of Notch in all the cells is observed, revealing the larger extent of the initial cluster. The linked increase in lateral-inhibition signaling, however, ensures that the normal number of muscle progenitor cells develop (Brennan, 2000).

This model contrasts with others in which Wingless signaling is instructive and defines the position at which muscle progenitor cells will develop, but can explain why overexpressing Wingless leads to the development of S59-expressing muscles in their normal position. In this model the Wingless signal is permissive and not instructive: it does not define where S59 will be expressed but merely reveals places defined by earlier mechanisms. Finally, these data suggest that the loss of S59 expression in the absence of a Wingless signal is due to the early repression mediated by Notch (Brennan, 2000).

Specification of individual Slouch muscle progenitors in Drosophila requires sequential Wingless signaling

The patterning of the Drosophila mesoderm requires Wingless. Little is known about how Wg provides patterning information to the mesoderm, which is neither an epithelium nor contains the site of Wg production. By studying specification of muscle founder cells as marked by the lineage-specific transcription factor Slouch, it was asked how mesodermal cells interpret the steady flow of Wg. Through the manipulation of place, time and amount of Wg signaling, it has been observed that Slouch founder cell cluster II is more sensitive to Wg levels than the other Slouch-positive founder cell clusters. To specify Slouch cluster I, Wg signaling is required to maintain high levels of the myogenic transcriptional regulator Twist. However, to specify cluster II, Wg not only maintains high Twist levels, but also provides a second contribution to activate Slouch expression. This dual requirement for Wg provides a paradigm for understanding how one signaling pathway can act over time to create a diverse array of patterning outcomes (Cox, 2005).

In wg mutant embryos, the heart and approximately half the body wall muscles are lost. One subset of these Wg-dependent body wall muscles can be visualized using an antibody to the NK-homeodomain protein Slouch (S59). Slouch expression arises in a precise, stereotypic pattern during embryonic development. It is first expressed in a single progenitor cell during early stage 11 of embryonic development; this cell divides to give rise to two founder cells (Ia and Ib) which together form cluster I (cI). During late stage 11, two additional Slouch-positive progenitors appear at a different ventral location and divide sequentially to form four founder cells that make up cluster II. Still later, at stage 12, a single progenitor arises dorsally and divides to give rise to cluster III. These muscle founder cells contain all the information needed to create a particular subset of muscles and contribute to the stereotypic set of larval muscles in each abdominal segment. After stage 12, Slouch expression is maintained in a subset of these founder cells that give rise, in the final muscle pattern, to muscle VT1 (from cI), VA2 (from cII) and DT1 (from cluster III). Maintenance of Slouch expression in these founder cells is crucial to the development of these muscles; removal of slouch leads to complete (VT1) and partial muscle transformations (VA2; DT1). In this study, focus was placed on the role of Wg in patterning the Slouch muscle founder cells. For simplicity, focus was placed solely on two ventral Slouch clusters (I and II), which develop independently and arise in a similar position along the dorsoventral axis but have different anterior-posterior positions within each abdominal hemisegment (Cox, 2005).

Through the manipulation of the amount and time of exposure to Wg signaling in the Drosophila mesoderm, it is shown that Slouch founder cell cII requires more Wg signaling than its neighbor, cI. Because cII arises in the mesoderm beneath the source of Wg signal, it was initially thought that the sensitivity that was detected would be due to Wg acting as a classic morphogen. Specifically, during stage 11, Wg would directly elicit concentration-dependent responses, leading to Slouch cI specification at low levels and cII at higher levels. Instead, the data suggest an alternative mechanism underlying this sensitivity. For Slouch cI, Wg signaling through Twist is sufficient for fate specification. However, for Slouch cII, a second, Twist-independent Wg signal is also necessary (Cox, 2005).

It has been shown that wg mutants fail to maintain high levels of Twist. Overexpression of Twist leads to expanded somatic mesodermal fates at the expense of other mesodermal fates, such as heart and gut muscle. Conversely, decreasing Twist levels leads to a reduction in somatic mesodermal fate, while heart and gut muscle remain largely unaffected. These findings underscore the importance of high Twist levels for the proper implementation of somatic muscle fate. Because loss of high Twist levels leads to loss of muscle founder cells, including all Slouch-positive clusters of founder cells, it has always appeared that each Slouch cluster requires the same amount of Wg signal (relayed through Twist) to assume its particular fate. In this study, the requirement for Wg in maintaining high Twist levels was uncoupled from the later role of Wg in specifying cII fate. The fact that Twist specifically rescues Slouch cI in a wg mutant background suggests that Slouch cII requires an additional, Twist-independent contribution from Wg for proper patterning. Consistent with these results, wg hypomorphs were found that provided sufficient signaling to maintain high Twist levels during early mesoderm development and therefore pattern cI, but that do not pattern cII. Temperature-shift experiments using wg temperature-sensitive alleles have shown that Slouch cII specification and engrailed expression in the ectoderm require Wg expression at later stages of embryonic development. Thus, the absence of Slouch cII in the different wg alleles, in hh mutant embryos and in a Twist rescued wg mutant embryo, all suggest that proper patterning requires not only an earlier Wg-dependent regulation of Twist, but also an additional Wg contribution to specify its identity (Cox, 2005).

Manipulations of Wg signaling also revealed two additional aspects of Wg signaling to the mesoderm. (1) It was found that the mesoderm, in general, has a different threshold for Wg signaling when compared with the ectoderm. Conditions that completely rescue the ventral ectoderm and epidermis (wg^PE6 at the permissive temperature) fail to completely rescue the mesoderm. (2) It was found that different mesodermal targets respond differently to Wg signaling. For example, expression of the DeltaNTcf (dominant negative form of Pangolin) has mild effects on Twist but significant effects on Slouch cII. Although it is predicted that TCF binds slouch regulatory regions directly, it was found that Wg regulates Twist both directly through TCF and indirectly through the pair-rule gene sloppy-paired. Whether or not the difference in Wg regulation of twist and slouch is due to the structure of the regulatory regions, additional factors that integrate on these promoters in these contexts and the activity of the Arm/dTCF complex remains to be uncovered (Cox, 2005).

This study also underscores the contribution that other factors make to position the Slouch clusters: ectopic Wg expression in the mesoderm does not produce uniform Slouch expression. This aspect of Wg signaling is reflected in other tissues such as the epidermis. The size of Slouch cII could not be further enlarged beyond that seen when Wg signaling was initially increased. This suggests a prepatterning mechanism, perhaps involving the activity of the pair-rule genes that have been shown to be responsible for segmentation of the mesoderm, as well as the integration of other signal transduction pathways, such as EGF/FGF and Notch signaling. The data suggest that Wg signaling then works on this prepattern to regulate the domain of Slouch expression (Cox, 2005).

The effect of Wg on muscle patterning is similar to that described for even-skipped muscle progenitor specification; that is, Wg signaling (in collaboration with such signals as Decapentaplegic) is first required to set up a region of 'competence' through activation of mesoderm-specific factors such as Twist and Tinman. Wg then later cooperates with these intrinsic factors to induce the expression of even-skipped in dorsal muscle progenitors, much as would be suggested for Slouch cII. However, the observations suggest an important variation of Wg signaling in mesodermal patterning. In the case of Slouch patterning, Wg creates temporal as well as spatial diversity, while in patterning eve it only acts temporally. Wg signaling contributes to the expression of Slouch in its two discrete ventral patches by two distinctive mechanisms: through the regulation of an upstream transcription regulator (Twist), which is sufficient for one domain of expression; and through the cooperation of this factor with a second, temporally distinct Wg input for the second domain of expression. The expression of the same gene but at two different times and places, through two Wg-dependent means, gives insight into how an organism may generate diverse tissues in response to the same signal (Cox, 2005).

Work carried out in the wing imaginal disc suggests that Wg acts as a morphogen. In this tissue, Wg protein was visualized in a graded distribution and it appears to activate multiple target genes directly, in a concentration-dependent manner. Based on these criteria, Wg has been labeled as a classical morphogen. However, careful inspection of the molecular mechanisms underlying Wg activation of both short- and long-range targets in the wing have revealed that the pattern of Wg expression changes during wing imaginal disc development, and that Wg collaborates with other pathways to set up the expression of these genes. These studies have cast doubt on whether Wg is a true morphogen in this tissue (Cox, 2005).

Investigating the molecular mechanisms that govern patterning of the embryonic mesoderm, similarly suggests that Wg does not act on Slouch clusters I and II as a classical morphogen. Wg does not activate cI directly, but instead maintains high levels of Twist, which sets up a somatic mesodermal competency domain that is sufficient to create cI. Additional Wg is then required later to pattern cII. It can be argued that Wg acts as a morphogen to regulate Twist expression (at low levels), and then to control Slouch expression (at high levels) within cells of cII. However, the precise regulation and dependence of Slouch clusters I and II on Wg within both the dorsoventral and anteroposterior axes suggest that there must be additional patterning information available to properly place these two cell types. As more putative morphogens are held up to the lens of molecular biology, it will be interesting to see whether there are unexpected, new twists in the molecular underpinnings of morphogens (Cox, 2005).

Drosophila araucan and caupolican integrate intrinsic and signalling inputs for the acquisition by muscle progenitors of the lateral transverse fate

A central issue of myogenesis is the acquisition of identity by individual muscles. In Drosophila, at the time muscle progenitors are singled out, they already express unique combinations of muscle identity genes. This muscle code results from the integration of positional and temporal signalling inputs. This study identified, by means of loss-of-function and ectopic expression approaches, the Iroquois Complex homeobox genes araucan and caupolican as novel muscle identity genes that confer lateral transverse muscle identity. The acquisition of this fate requires that Araucan/Caupolican repress other muscle identity genes such as slouch and vestigial. In addition, Caupolican-dependent slouch expression depends on the activation state of the Ras/Mitogen Activated Protein Kinase cascade. This provides a comprehensive insight into the way Iroquois genes integrate in muscle progenitors, signalling inputs that modulate gene expression and protein activity (Carrasco-Rando, 2011).

The study of myogenesis in Drosophila has increased the understanding of how the mechanisms that underlie the acquisition of specific properties by individual muscles are integrated within the myogenic terminal differentiation pathway. Thus, the current hypothesis proposes that distinct combinations of regulatory inputs leads to the activation of specific sets of muscle identity genes in progenitors that regulate the expression of a battery of downstream target genes responsible for executing the different developmental programmes. However, the analysis of the specific role of individual muscle identity genes and of their hierarchical relationships is far from complete since the characterisation of direct targets for these transcriptional regulators is very scarce (Carrasco-Rando, 2011).

ara and caup, two members of the Iroquois complex, have been identified as novel type III muscle identity genes. The homeodomain-containing Ara and Caup proteins are necessary for the specification of the lateral transverse (LT) fate. ara/caup appear to be bona fide muscle identity genes. Indeed, similarly to the identity genes Kr and slou, absence of ara/caup does not interfere with the segregation of muscle progenitors or their terminal differentiation, but modifies the specific characteristics of LT1-4 muscles, which are transformed towards VA1, VA2, LL1 and LL1 sib fates. These transformations may be due in part to the up-regulation of slou and vg in the corresponding muscles. Thus, a recent report (Deng, 2010) shows that forced expression of vg in LT muscles induces changes in muscle attachments similar to the ones observed in LT1 in ara/caup mutant embryos. However, it should be stressed that although in ara/caup mutants LT muscles are lost in more than 95% of cases, they are not completely transformed into perfect duplicates of the newly acquired fates. For instance, while the specific LT marker lms is lost in 91% of cases, ectopic slou expression is detected in only 75% of cases. These partial transformations might be due to differences in the signalling inputs acting in the mesodermal region from where these muscles segregate. Unpublished data also showed that forced pan-mesodermal expression of ara/caup alter the fates of many muscles both in dorsal and in ventral regions without converting them into LT muscles (i.e., they do not ectopically express lms). Similarly, Kr and slou ectopic expression is not sufficient to implement a certain muscle fate. The failure to recreate a given muscle identity by adding just one of the relevant muscle identity proteins reveals the importance that cell context, that is, the specific combination of signalling inputs and gene regulators present in each cell, have in determining a specific muscle identity (Carrasco-Rando, 2011).

Analysis of the myogenic requirement of ara/caup has revealed several features about how these genes act to implement LT fates. Thus, although they are expressed in six developing embryonic muscles, only four of them, LT1-4, are miss-specified in the absence of Ara/Caup. The remaining two, DT1 and SBM, seem to develop correctly, according to morphological as well as molecular criteria. It is worth noting that the requirement for ara/caup genes in these six muscles correlates with the onset of their expression. Thus, in the affected LT1-4 muscles Ara/Caup can be first detected at the earliest step of muscle lineages, that is in the promuscular clusters. In contrast, in the unaffected muscles ara/caup start to be expressed later, in the DT1/DO3 progenitor and the SBM founder. This suggests that in muscle lineages ara/caup have to be expressed very early to repress slou and vg to implement the LT fate. Several data support this interpretation. For instance, the observation that ara/caup are co-expressed with slou in DT1, whereas they repress slou in LT3-4, may be related to the fact that slou expression precedes that of ara/caup in the DT1 lineage. Should this be so, one would expect that ectopic expression of ara using the early driver mef2-GAL4, would repress slou in DT1, as it actually does, whereas this repression is not evident using the late driver Con-GAL4. Furthermore, the hypothesis of the relevance of the timing of muscle identity gene expression for muscle fate specification might also apply to the case of slou, where a similar correlation between the strength of the loss-of-function slou phenotypes in specific muscles and the onset of slou expression has also been found (Carrasco-Rando, 2011).

It should be stressed that the generation of the LT code depends not only on the early presence of Ara/Caup on the promuscular clusters but also on the absence (or strong reduction) of DER/Ras activity at that precise developmental stage and location. There is a dynamic regulation of MAPK signalling in the lateral mesoderm. Caup-expressing muscles develop from DER-independent clusters whereas the duplicated muscles observed in ara/caup mutants derive from progenitors that segregate very near the LT progenitors, but originate in DER-dependent promuscular clusters that are specified slightly later in development. Furthermore it was observed both by in vivo and in cell culture that low MAPK activity is required for Caup-dependent slou repression. Therefore, the role of Ara/Caup in the implementation of LT fate is interpreted as follows. At mid stage 11 in the myogenic mesoderm, groups of mesodermal cells acquire myogenic competence as a result of interpreting a combinatorial signalling code that reflects their position along the main body axes, as well as the state of activation of different signalling pathways. Accordingly, these clusters initiate the expression of lethal of scute and a unique code of muscle identity genes, as has been shown in great detail for eve expression in the dorsal mesoderm. In the case of the dorso-lateral mesoderm this code includes ara/caup and Kr and implements the LT fate. Since the level of activation of the Ras/MAPK cascade is low in these clusters, Ara/Caup will behave as transcriptional repressors, preventing the activation of slou or vg in LT1-2 and LT3-4 clusters, which would be otherwise activated in this location. Thus, Ara/Caup implement the LT fate by repressing the execution of the alternative fates (Kr+, Slou+, Con+, Poxm+ and Kr+, Vg+) that would give rise to duplicates of P_VA1/VA2 and P_LL1/LL1sib, respectively, and by allowing a different identity gene code (Kr+, Caup+, Con+, lms+) that generates the LT fate (Carrasco-Rando, 2011).

Slightly later the Ras/MAPK pathway becomes active at the dorsolateral region. This changes the combinatorial signalling code and coincides with a change in the muscle identity genes expressed by the promuscular clusters that segregate from this position, which now accumulate Kr but not Ara/Caup. Progenitors born from them will express either slou or vg and give rise to VA1-2 and LL1/LL1sib fates, all DER-dependent (Carrasco-Rando, 2011).

The data suggested that Ara/Caup might act as repressors of slou in the Drosophila mesoderm. Therefore whether slou might be a direct target of Ara/Caup was investigated. An 'in silico' search of a previously reported slou cis-regulatory region identified two putative Iro binding sites (BS) at positions +129 (BS1) and -1642 (BS2), relative to the transcription start site, which match the consensus ACAN_2-8TGT. This regulatory region was cloned in a Luciferase reporter vector and Luciferase activity was measured in Drosophila Schneider-2 (S2) cells transiently transfected with this construct and increasing amounts of HA-tagged Caup. Contrary to expectations, it was found that addition of Caup-HA increased the basal Luciferase activity driven by the slou regulatory region in a dose dependent manner, indicating that Caup acts as a transcriptional activator of slou under these conditions. The reported regulation of the chicken Irx2 factor by MAPK (that switches it from repressor to activator) could explain this result. Since Western Blot analysis of S2 lysates using an antibody against diphospho-extracellular-signal related kinase (dpErk) showed the MAPK pathway to be active in S2 cells, and experimental evidence has been obtained showing the presence of phosphorylated Caup in S2 cells with constitutively active MAPK pathway, it was hypothesized that the activation effect of Caup in S2 cells could be due to the Ras/MAPK cascade turning Caup from transcriptional repressor into activator. Indeed, the inhibition of the Ras/MAPK pathway by the PD98059 MAP-erk kinase-1 (MEK1) inhibitor induced a Caup-dose dependent decrease in Luciferase activity driven by the slou regulatory sequences. This result could not be attributed to a direct effect of the inhibitor over the slou promoter, since its addition did not modify the basal Luciferase activity of the construct (Carrasco-Rando, 2011).

Thus S2 cell experiments suggest a molecular mechanism by which the Ras/MAPK pathway modulates the transcriptional activity of Ara/Caup on slou. Low MAPK activity and direct binding of Caup to BS1 site of the slou gene would favour strong repression of slou. BS1 could be embedded in a silencer regulatory element or its binding to Caup may block transcription of the downstream located luciferase gene. On the contrary, Caup-dependent activation of slou would be dependent on MAPK signalling. It is hypothesized that MAPK-dependent Caup phosphorylation could modulate its interaction with different transcriptional co-factors or/and its binding site affinity (Carrasco-Rando, 2011).

Furthermore, in vivo evidence indicates a repressor function of presumably non-phosphorylated Caup on slou since forced activation of the Ras pathway allows co-expression of slou and caup. On the other hand, the ectopic expression of slou induced by caup-over-expression is suggestive of a possible activator function of phosphorylated Caup (Carrasco-Rando, 2011).

The role of IRO proteins in cell fate specification is conserved in both vertebrates and invertebrates. This study has shown that the interplay between MAPK signalling and IRO activity found in vertebrate neuroepithelium is also at work in Drosophila myogenesis. This study has identifed potential direct target of Ara/Caup, slou and has proposed vg as a candidate gene to be regulated by Ara/Caup. In both cases the genes subordinated to ara/caup encode transcription factors that might in turn regulate the expression of other genes, genes that must be repressed in LT muscles in order to acquire the LT fate. These results, therefore, provide insights into the way Ara/Caup control lateral muscle identity and on the role of signalling pathway inputs to modulate the activity of these transcription factors, with consequences in their downstream targets. It also highlights the importance that the specific combination of muscle identity genes, their hierarchical relationships and their temporal activation have in determining the identity of a given muscle cell, very alike to what is at work during the acquisition of neural fates (Carrasco-Rando, 2011).

Protein interactions

The PRD-repeat domains of Slouch and PRD protein are sufficient to mediate protein-protein interaction, suggesting that the PRD-domain functions as a protein-binding interface and thereby may increase the DNA binding specificity of homeodomain transcription factors (Kim, 1995).

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