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).

Org-1, the Drosophila ortholog of Tbx1, is a direct activator of known identity genes during muscle specification

Members of the T-Box gene family of transcription factors are important players in regulatory circuits that generate myogenic and cardiogenic lineage diversities in vertebrates. This study shows that during somatic myogenesis in Drosophila, the single ortholog of vertebrate Tbx1, optomotor-blind-related-gene-1 (org-1), is expressed in a small subset of muscle progenitors, founder cells and adult muscle precursors, where it overlaps with the products of the muscle identity genes ladybird (lb) and slouch (slou). In addition, org-1 is expressed in the lineage of the heart-associated alary muscles. org-1 null mutant embryos lack Lb and Slou expression within the muscle lineages that normally co-express org-1. As a consequence, the respective muscle fibers and adult muscle precursors are either severely malformed or missing, as are the alary muscles. To address the mechanisms that mediate these regulatory interactions between Org-1, Lb and Slou, distinct enhancers associated with somatic muscle expression of lb and slou. These lineage- and stage-specific cis-regulatory modules (CRMs) bind Org-1 in vivo, respond to org-1 genetically and require T-box domain binding sites for their activation. In summary, it is proposed that org-1 is a common and direct upstream regulator of slou and lb in the developmental pathway of these two neighboring muscle lineages. Cross-repression between slou and lb and combinatorial activation of lineage-specific targets by Org-1-Slou and Org-1-Lb, respectively, then leads to the distinction between the two lineages. These findings provide new insights into the regulatory circuits that control the proper pattering of the larval somatic musculature in Drosophila (Schaub, 2012).

The musculature and the central nervous system are two examples of tissues featuring a high diversity of cell types. In each hemisegment of the Drosophila embryo, about 30 distinct body wall muscles and six adult muscle precursors are formed and in the CNS the number of neuronal and glial cell types generated is an order of magnitude higher. Currently, only a limited picture is available of the regulatory processes that generate these great cellular diversities, but several steps of muscular and neuronal development in Drosophila known to be accomplished by closely related processes. For example, the earliest progenitor cells, termed muscle progenitors and neuroblasts, respectively, seem to be defined by intersecting anterior-posterior and dorsoventral upstream regulators. In both tissues, these progenitors are preceded by larger clusters of promuscular or proneural cells, respectively, that are characterized by their expression of proneural genes from the Achaete-Scute complex. Lateral inhibition mediated by Delta/Notch signals singles out individual progenitor cells from these clusters. In the case of the muscle progenitors, receptor tyrosine kinase (RTK) signaling, particularly via epidermal growth factor (EGF)- and fibroblast growth factor (FGF)-receptors (Egfr and Htl, respectively), is required as a positive input to antagonize Notch. An additional similarity is the occurrence of asymmetric cell divisions that involve the unequal segregation of the Notch-inhibitor Numb. In the case of muscle progenitors, these asymmetric divisions typically generate two distinct muscle founder cells that go on to form two different larval muscle fibers. In some instances, one sibling of a muscle founder cell gives rise to certain heart precursors or to a stem cell-like adult muscle progenitor that will contribute to the adult body wall musculature. Subsequently, muscle founder myoblasts fuse with surrounding fusion-competent myoblasts to form distinct body wall muscle fibers. During both muscle and neuronal development, the increasing diversity of the newly generated cells is reflected in the distinct combinations of transcription factors expressed in them. Several of these factors are known to have key roles in defining the particular cell identities and have been called muscle identity factors (Schaub, 2012).

Although the broad picture of these diversification events is known, there are major gaps in knowledge with regard to the specific design of the regulatory networks involved. In the case of Drosophila muscle development, a few lineages of muscle progenitors have been characterized in some detail. The first are those marked by the expression of the identity factor Even-skipped (Eve, a homeodomain protein) and have been best characterized with regard to the essential early-acting inputs. These particular muscle progenitors arise in the dorsal portion of the mesoderm and give rise to somatic muscle fibers as well as to specific cells of the dorsal vessel. One of the two adjacent muscle progenitors marked by Eve generates the founder of muscle 1 (DA1), which requires eve function, whereas the other generates the founder of muscle 10 (DO2). The activation of eve in these muscle progenitors requires combinatorial signals from ectodermal Decapentaplegic (Dpp) and Wingless (Wg), the mesodermal competence factor Tinman, which itself is downstream of Dpp, as well as RTK signals via Egfr and Htl. All these combinatorial signaling and transcription factor inputs are directly integrated at the level of a mesoderm-specific enhancer element at the eve locus (Schaub, 2012).

The step-wise specification of muscle identities has been addressed for muscle founders expressing the identity factor Collier (Col, a COE transcription factor; Knot -- FlyBase). In abdominal segments, Col is expressed in the two adjacent dorsolateral muscle progenitors of muscles 3 (DA3) and 20 (DO5) as well as 18 (DT1) and 19 (DO4) and the corresponding promuscular clusters. The expression is only maintained in the founder and precursor of muscle 3/DA3, which requires Col for its development. This dynamic expression is reflected in the activities of at least two cis-regulatory modules (CRMs) of col. An intronic CRM activates expression within both Col+ promuscular clusters and the progenitors singled out from them, whereas an upstream CRM activates and maintains expression specifically in the muscle founder 3/DA3 and its muscle. The early-acting intronic CRM responds to positional and mesodermal inputs, which might involve binding by Twist and Tinman, whereas activation of the later-acting CRM in the M3/DA3 founder relies on the combinatorial binding of autoregulatory Col, Nautilus (Nau, also known as MyoD), and the Hox factors Antp and Ubx. In this manner, the early patterning and tissue-specific inputs are integrated with the axial inputs of the Hox genes that modulate the muscle pattern and fiber size in different body parts (Schaub, 2012).

The muscle lineages that are the focus of the present study are positioned laterally in the somatic mesoderm and are marked by the expression of the homeodomain factors Slouch (Slou) and Ladybird (Lb), which function as identity genes in these lineages [lb refers herein to the paralogous ladybird early (lbe) and ladybird late (lbl) genes]. Within this area of interest, Slou is expressed in the progenitor and sibling founders of muscles 5 (LO1) and 25 (VT1) and is crucial for the formation of these muscles. Lbe is expressed directly adjacent to Slou in a promuscular cluster and then two muscle progenitors. One of these progenitors forms muscle 8 (SBM) and a lateral adult muscle precursor (lAMP), which continue to express Lb, whereas the other forms a second Lb+ lAMP and a sibling that probably undergoes apoptosis. Genetic and genomic analyses indicate that Lb has important functions in the specification and/or differentiation of muscle 8 (Schaub, 2012).

The expression of slou and lbe appears to be controlled by an overlapping set of upstream regulators, because their mutually exclusive expression in the neighboring progenitors of muscles 5/25 and muscle 8/lAMP requires cross-repression by their respective gene products. However, the nature of these shared regulatory inputs has been unknown. The present study identified org-1, a Drosophila Tbx1 ortholog that had not been characterized extensively prior to this work, as a key activator of both slou and lbe in this pathway. org-1 expression in the mesodermal areas and progenitors that will form muscles 5, 25, 8 and the Lb+ lAMPs precedes that of slou and lbe, and the activation of these identity genes requires org-1 activity. Consequently, the development of these muscles and the lAMPs is disrupted in org-1 mutant embryos. The activation of slou and lbe by org-1 requires T-box binding motifs in their respective founder-specific enhancer elements and show in vivo occupancy of these elements by Org-1. Hence, slou and lbe appear to be direct target genes of org-1 in the developmental pathway of these neighboring muscle lineages. In addition, org-1 is expressed in the progenitor of the alary muscles and its function is needed for the development of these muscles, which form segmental anchors of the dorsal vessel (Schaub, 2012).

Analysis of the expression and function of org-1 in somatic muscle development has established this gene as a new and crucial representative of muscle identity genes in Drosophila. The data have provided new insights into developmental controls in two well-defined muscle lineages and somatic muscle development in general. These lineages include one dependent on the homeobox gene slou, which gives rise to muscles M5 and M25 (also known as 'cluster I'), and another dependent on the lb homeobox genes, which gives rise to the segment border muscle M8 and lateral adult muscle precursors (lAMPs). They arise from promuscular clusters and muscle progenitors abutting each other in the ventrolateral somatic mesoderm. Until now, it was assumed that slou and lb are positioned at the top of the regulatory hierarchy set up within each of these lineages, as their expression and function is already detected at the progenitor cell stage prior to asymmetric divisions into different founder cells. As in the model proposed for the even-skipped-expressing lineage in the dorsal somatic mesoderm, the progenitor-specific expression of slou and lb was assumed to be determined directly by the antagonistic actions of various more broadly active activators and negative influences through lateral inhibition via Notch. Based on genetic assays using the expression of slou and lb, and the formation of the respective muscles as outputs, good candidates for such activators included localized receptor tyrosine kinase (RTK)/Ras/MAPK signals in conjunction with the relatively broadly expressed mesodermal transcription factors Tinman, Six4 (in the case of lb), Twist and Sloppy paired. Ectodermal Wg signals were also found to regulate these Slou+ and Lb+ lineages, although at least in the case of slou these apparently act indirectly by upregulating twi and slp expression (Schaub, 2012).

The current findings have uncovered an additional layer of regulation upstream of slou and lb within the M5/M25 and M8/lAMP lineages that involves org-1 (see Org-1 is a direct regulator of ladybird and slouch and is required for cell fate specification and differentiation in distinct lateral muscle cell lineages). The initial expression of org-1 occurs in segmented areas of the lateral somatic mesoderm, from which the org-1+ M5/M25 and M8/lAMP progenitors emerge. These areas probably delineate the two abutting promuscular clusters from which these progenitors are singled out (as well as a third one dorsolaterally, from which the progenitor and founder of the alary muscle is formed). Although this early expression of org-1 is not required for progenitor formation per se (based upon the normal pattern of org-1:HN39-lacZ in progenitors of org-1 mutants), org-1 is crucial either during this phase or during early phases of progenitor formation for activating slou and lb in the respective progenitors. Hence, it is proposed that the previously identified regulators of slou and lb, including Notch, RTKs, tin, wg, twi and slp, act predominantly in establishing progenitor-specific expression of org-1, which in turn activates slou and lb. However, a plausible alternative to this linear model of regulation would be a feed-forward model, in which some of the above upstream regulators are re-employed during the second step to activate slou and lb together with mandatory Org-1. Whether org-1 activates slou or lb in any given progenitor would depend on the outcome of the previously reported mutual inhibition between slou and lb. It is thought unlikely that the outcome of this process is completely random; instead, a mechanism is favored involving an initial bias towards one or the other. Such biases could, for example, arise through slight differences in the spatial activities of the EGF and FGF receptors (Egfr and Htl), or of transcription factors such as Six4, coupled with differential responses of slou and lb to these regulators. Regardless of the specific mechanism, this principle of joint activation followed by mutual repression allows for the differential specification of directly neighboring cells, in this case of P5/25 and P8/lAMP, that are under the influence of common upstream regulators (Schaub, 2012).

The combination of genetic data and functional enhancer analysis provides convincing evidence that both slou and lb are direct transcriptional targets of Org-1. This conclusion also fits nicely with the observation that a slou enhancer fragment used herein, SK16, is active ectopically in M8 and that Org-1 and the putative Org-1 binding sites are required for its activity not only in M5 and M25, but also in M8. By contrast, an adjacent enhancer fragment, SK10, is not active in M8 and thus reflects the endogenous Org-1-dependent pattern of slou. Presumably unlike SK16, this element still includes lb-dependent repression elements that block its activation by Org-1 in M8 (Schaub, 2012).

The lbl-SBM enhancer (1.36 kb) was identified initially in a bioinformatics approach for sequences near founder cell-expressed genes that are enriched with binding motifs for Twi, Tin, dTCF (Pan - FlyBase; Wg signaling effector), Pointed (RTK signal effector) and Mad (Dpp signaling effector), and also showed increased evolutionary conservation. However, this enhancer had a relatively low score and, furthermore, the Tin and Twi motifs with decent matches to bona fide binding sites for these factors lie outside of a shortened version, lbl-SBMs. Additionally, Smad proteins are not expected to be active in this ventrolateral region. It is possible that this enhancer was picked up with this algorithm largely because of the adjacent high-scoring cardiac enhancer. Nevertheless, some of the remaining binding motifs within lbl-SBMs, e.g. those for dTCF and Pnt, might contribute to the enhancer activity in progenitors and founders together with the essential Org-1 binding sites. The residual activation of the lbl-SBMs enhancer with mutated Org-1 binding motifs in the differentiated M8 shows that the maintenance of enhancer activity at later stages depends on regulators other than Org-1, which agrees with the observation that Org-1 is no longer expressed in M8 during these stages. This maintenance might be regulated by inputs shared with two separate later-acting enhancer elements upstream of lbe (termed LAMPE) and lbl (termed LME). Both of these elements include homeodomain binding motifs that probably mediate autoregulation by Lb, in addition to ETS domain and Mef2-binding motifs, respectively, that also contribute to full activity in M8. In org-1 mutants, the lbl-SBM enhancer is inactive not only early but also late, possibly because Lb protein is not available for autoregulation (Schaub, 2012).

Unlike in slou mutants or embryos with ectopic lb expression, in org-1 mutants the muscles derived from the progenitors of M5/25 and M8/lAMP are not transformed into other muscles with recognizable identities. Instead, they form syncytia with variable shapes that do not appear to be properly attached to tendon cells. This phenotype is explained by the requirement of org-1 for the activation of both slou and lb. Whereas in slou mutants the progenitor of M5/M25 expresses ectopic lb, which transforms it into a second progenitor of M8/lAMP, in org-1 mutants de-repression of lb is not possible as org-1 is also needed for the activation of lb. Hence, neither of the two progenitors is able to express any of these identity genes that would allow them to form identifiable muscle fibers, although they still divide into founder cells that form syncytia. Only the founder of M25 forms a syncytium that resembles M25 to some extent, perhaps because it still expresses an unknown, org-1-independent identity gene that can establish some of the characteristics of M25 in the absence of slou and org-1 function (Schaub, 2012).

Vertebrate Tbx1 is also expressed prominently in mesodermal areas that form muscles, although in this case not in the trunk region but rather in the core mesoderm of the pharyngeal arches that form branchiomeric muscles of the head. Notably, Tbx1 is also expressed in pharyngeal arch core mesoderm derived from anterior portions of the second heart field, which makes prominent contributions to the outflow tract and right ventricle of the heart. All of these muscle and heart tissues are disrupted upon mutation of Tbx1 in the mouse and are also affected in patients suffering from the DiGeorge (or velo-cardio-facial) syndrome, in which mutations and deletions cause haploinsufficiency of TBX1. No obvious connections are seen between Drosophila org-1 function and the cardiogenic roles of vertebrate Tbx1, as org-1-expressing cells do not contribute to the dorsal vessel proper and the org-1+ alary muscles are specialized skeletal muscle fibers rather than genuine cardiac muscles. However, conceivably some of the regulatory interactions upstream or downstream of Tbx1/org-1 during the development of vertebrate head muscles and Drosophila trunk muscles have been conserved. This could, for example, be the case during tongue muscle development, in which the lb ortholog Lbx1 is co-expressed with Tbx1. The identification of larger numbers of upstream regulators and targets of both org-1 and Tbx1 will be required in order to obtain a clearer picture of the evolutionary conservation and divergence of the developmental functions of these genes (Schaub, 2012).

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 (sggm11 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 sggm11 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 wgS107.5;DlFX3 zygotic and wgS107.5,Su(H)SF8 germline-clone embryos. In contrast, mesodermal S59 expression is observed in Df(1)N81k1,dshv26 and Df(1)N81k1;wgCX4 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)N81k1, dshv26 and Df(1)N81k1; wgCX4 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 sggm11 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 sggm11 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 sggm11 embryos. This situation would explain the greater reduction in the size of the groups of S59-expressing muscle progenitor cells observed in the sggm11 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 (wgPE6 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 lateral muscles scarcer (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 PVA1/VA2 and PLL1/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 ACAN2-8TGT. 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).

Targets of Activity

Molecular mechanism underlying the regulatory specificity of a Drosophila homeodomain protein that specifies myoblast identity

A subfamily of Drosophila homeodomain (HD) transcription factors (TFs) controls the identities of individual muscle founder cells (FCs). However, the molecular mechanisms by which these TFs generate unique FC genetic programs remain unknown. To investigate this problem, genome-wide mRNA expression profiling was applied to identify genes that are activated or repressed by the muscle HD TFs Slouch (Slou) and Muscle segment homeobox (Msh). Protein-binding microarrays were used to define the sequences that are bound by Slou, Msh and other HD TFs that have mesodermal expression. These studies revealed that a large class of HDs, including Slou and Msh, predominantly recognize TAAT core sequences but that each HD also binds to unique sites that deviate from this canonical motif. To understand better the regulatory specificity of an individual FC identity HD, the functions of atypical binding sites that are preferentially bound by Slou were evaluated relative to other HDs within muscle enhancers that are either activated or repressed by this TF. These studies showed that Slou regulates the activities of particular myoblast enhancers through Slou-preferred sequences, whereas swapping these sequences for sites that are capable of binding to multiple HD family members does not support the normal regulatory functions of Slou. Moreover, atypical Slou-binding sites are overrepresented in putative enhancers associated with additional Slou-responsive FC genes. Collectively, these studies provide new insights into the roles of individual HD TFs in determining cellular identity, and suggest that the diversity of HD binding preferences can confer regulatory specificity (Busser, 2012).

This study has used an integrated genomics approach to interrogate the molecular mechanisms of action of a subset of identity HD TFs that have been proposed to control the unique gene expression programs of muscle FCs. It was first shown that founder cell (FC) genes are differentially responsive to Slou and Msh, which suggests functional specificity in the regulation of FC genes by these FCI-HD TFs, and is consistent with the known effects of these TFs on muscle cell fates. Protein-binding microarray (PBM) assays defined the specific sequences that are bound by these HDs, revealing that the majority of binding sites contain TAAT core sequences that are shared by all founder cell identity homeodomain (FCI-HD) TFs, but that each HD also binds to a small number of unique, atypical sequences. In each of two Slou-responsive FC enhancers, it was found that the transcriptional specificity of Slou is mediated by its binding to a single motif that is preferred by Slou and that is not bound by other mesodermally expressed HDs that were examined. Genome-wide computational studies provide further evidence for the potential importance of HD-preferred binding sites within the myogenic network of FC genes. Nevertheless, mesodermal HD proteins do not exclusively act through these atypical motifs as Hox TFs have been documented to regulate other muscle enhancers through HD-common binding sites (Busser, 2012).

The data show that the diversity of HD-binding preferences may confer the cell-specific effects of HDs by controlling which member of a related TF family is able to bind to and function at a particular site in a given CRM. This feature of enhancers may be especially important in developmental contexts where multiple family members that have different activities are co-expressed, resulting in potential competition for TF binding to shared sites. Such would be the case for FCI-HD and Hox TFs, both of which participate in the myogenic program but with distinct regulatory functions. Given the high level of conservation of these individual binding sites, there appears to be strong evolutionary selection for a particular HD-preferred sequence, a process that may be driven by the requirement for maintaining essential interactions with other TFs in a given regulatory context. For example, the DNA specificity of Hox HDs is known to be modified by interactions with co-factors such as the PBC and MEIS subclasses of TALE HD proteins. Although there is currently no evidence that these co-factors interact with Drosophila FCI-HD TFs, PBC proteins are thought to interact with similar classes of vertebrate TFs. Other forms of collaboration with FCI-HD TFs may also occur, including TF heterodimerization, cooperative interactions with other co-factors, or formation of multi-protein complexes of signal-activated and tissue-restricted TFs that have convergent effects on mesodermal gene expression (Busser, 2012).

The existence of functional HD-preferred binding sites raises the issue of how such sequences mediate their regulatory effects, especially as site specificity swap experiments revealed that the particular nucleotide sequence of a Slou-preferred site appears to be crucial for its function. It is possible that the specific sequences of HD-preferred DNA-binding sites form unique structures that are recognized by some HDs and not by others in certain contexts. Alternatively, binding to such sequences may induce a distinct protein conformation that is essential for enabling the HD to activate or repress the corresponding CRM, for example, by facilitating interactions with co-factors or other regulatory proteins (Busser, 2012).

Although the results support a central role for sequences preferred by one particular HD TF, the complexity of FC gene expression makes it likely that additional HD input occurs through sequences preferred by other co-expressed HDs. As many FCI-HD TFs have mutually exclusive expression patterns, a DNA binding site specific to, for example, Slou, Msh and Lb will be used by each TF in the cells in which they are differentially expressed. Thus, the HD-binding profile of enhancers should be re-examined as a collection of sequences with the ability to bind one or many HDs and where the functions of those sites in individual cells are dependent on the expression of the corresponding TF. The cumulative effects of these cell-specific binding events will then direct the discrete regulatory responses of the target genes (Busser, 2012).

In conclusion, presents a previously uncharacterized mechanism by which different members of the FCI-HD class of TFs determines the unique genetic programs of single myoblasts in a developing embryo. This regulatory process involves the selective recognition of particular DNA sequences by individual HDs. The ability of distinct DNA-binding sequences to generate an additional level of regulatory complexity may be of general importance in the architecture of transcriptional networks and in the evolution of TF families and CRMs. Finally, the approach used here provides a general strategy for investigating similar issues about the specialized roles played by individual members of other TF families, and how those functions may be precisely encoded in the cis-regulatory language of the genome (Busser, 2012).

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).

slouch: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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