InteractiveFly: GeneBrief

ladybird early and ladybird late: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene names - ladybird early and ladybird late

Synonyms -

Cytological map position - 93D9--93E2

Function - transcription factor

Keyword(s) - segment polarity, CNS, PNS, brain, antenna

Symbols - lbe and lbl

FlyBase ID:FBgn0011278 and FBgn0008651

Genetic map position - 3-[72]

Classification - homeobox

Cellular location - nuclear

NCBI links: Ladybird early Precomputed BLAST | Entrez Gene

NCBI links: Ladybird late Precomputed BLAST | Entrez Gene

The ladybird early and ladybird late genes are homeobox genes arranged in tandem, located in the Drosophila 93E homeobox gene cluster. The pattern of lbe and lbl transcription is complex; in a critical expression domain it overlaps that of wingless in the epidermis. This overlap suggests that lbe and lbl contribute to the segmentation process and that they may more properly belong to the family of segment polarity genes (Jagla, 1994).

While early wingless transcription is dependent on Hedgehog, Hedgehog receptors Patched and Smoothened, and a signal transduction apparatus including Fused and Cubitus interruptus, subsequent expression is maintained by a wingless autoregulation pathway involving Armadillo, Shaggy/Zeste white and cAMP-dependent Protein kinase A. Regulation of wingless expression is even more complex, since wingless expression in the ventral epidermis is maintained by Gooseberry, and in the dorsal epidermis by Lbe and Lbl. winglessseems to have become the prototype for genes subject to complex multistage temporal regulation.

The belief that lbe and lbl are targets of wingless signaling and that they also regulate wingless transcription is based on genetic and transcriptional evidence. lbe and lbl are transcribed in a pattern that matches the expression of wingless. lbe and lbl transcription requires wingless as well as Armadillo, a component of the wingless pathway.

In order to test the influence of lbe and lbl on wingless expression, embryos lacking lbe and lbl have been tested for wingless expression. These embryos lack Wg protein in the labrum and anal plate and have reduced levels of Wg in the dorsal epidermis. Their cuticular denticles are reduced number and have abnormal pigmentation. The abnormal denticles are restricted to those regions posterior to the segmental border row of cells (the primary row) and include only the more posterior tertiary and quaternary cell types (Bokor, 1996 and Jagla, 1997a). It therefore appears that lack of lbe and lbl produces a partial segment polarity phenotype, in which some elements of segment polarity are normal and others are not (Jagla, 1997a).

What is the relationship between lbe and lbl defects and those caused by gooseberry mutation? gooseberry is required for the maintenance of wingless transcription after this transcription is initially established. Jagla (1997a) argue that gsb is required for the maintenance of wg only in the ventral epidermis, and that lbe and lbl are required for late wg expression in the dorsal and in the terminal epidermis. Embryos carrying a null mutation in lbe and lbl do not show a wg-like cuticle phenotype in the ventral epidermis, probably because wg expression in this region is maintained by gsb (Li, 1993).

The embryonic heart precursors of Drosophila are arranged in a repeated pattern of segmental units. There is growing evidence that the development of individual elements of this pattern depends on both mesoderm intrinsic patterning information and inductive signals from the ectoderm. ladybird early and ladybird late, are involved in the cardiogenic pathway in Drosophila. At early stage 12, lb genes are expressed in clusters of about four cells per hemisegment in the developing heart region. These cells represent a segmental subset of tinman-expressing heart progenitors, which form a continuous row at the dorsal crest of the mesoderm at this stage. even-skipped expression begins at a slightly earlier time than lb in similar clusters of cells. It appears that two cells from each segmental eve cluster develop into a particular type of pericardial cells, termed e-PCs. Double stainings for lb and eve expression demonstrate that the e-PC progenitors are distinct from the lb-expressing heart progenitors and located posteriorly adjacent to them in each segment. Similar stainings of embryos at later stages show that the lb-expressing cells give rise to a subpopulation of cardioblasts (CBs) and a second type of pericardial cells, termed l-PCs. Cell rearrangements during stage 12, which involve a 90° clockwise rotation of the heart progenitor clusters within each segment, place the lb-expressing cells at the dorsal side and move the eve-expressing cells ventrally to them. This morphogenetic process results in a dorsal row of cardioblasts and ventrolaterally adjacent rows of pericardial cells on either side of the embryo. At stage 14, generally four out of six cardioblasts per hemisegment express both tin and mef-2. Double stainings with Lb antibodies show that the two anterior tin- and mef-2-expressing cardioblasts in each hemisegment co-express lb. In addition, tin and lb are co-expressed in the l-PCs, which are located ventrally below the cardioblasts. However, lb is not expressed in the e-PCs, which are found in more lateral positions at this stage. These results indicate a diversification among cardioblasts of each segment, as well as among the pericardial cells, that is already apparent during stage 11 (Jagla, 1997b).

LBE and LBL are required for anal plate formation. The anal plate does not develop in lbe and lbl mutants. The activator of lbe and lbl gene activity in the anal plate is most likely the homeotic gene forkhead, which governs terminal development. Alternatively, the homeobox gene caudal, required for the anal pads, tuft and anal sense organ formation, could be part of the genetic circuitry that switches on the wg-ladybird autoregulatory loop in the terminal region (Jagla, 1997a).

The analysis of ladybird regulation of epidermal development suggests that a temporal hierarchy of cuticle and denticle development exists in the epidermis and that lbe and lbl are involved in regulating the later stages of this process.

Genome-wide view of cell fate specification: ladybird acts at multiple levels during diversification of muscle and heart precursors

Correct diversification of cell types during development ensures the formation of functional organs. The evolutionarily conserved homeobox genes from ladybird/Lbx family were found to act as cell identity genes in a number of embryonic tissues. A prior genetic analysis showed that during Drosophila muscle and heart development ladybird is required for the specification of a subset of muscular and cardiac precursors. To learn how ladybird genes exert their cell identity functions, muscle and heart-targeted genome-wide transcriptional profiling and a chromatin immunoprecipitation (ChIP)-on-chip search were performed for direct Ladybird targets. The data reveal that ladybird not only contributes to the combinatorial code of transcription factors specifying the identity of muscle and cardiac precursors, but also regulates a large number of genes involved in setting cell shape, adhesion, and motility. Among direct ladybird targets, bric-a-brac 2 gene was identified as a new component of identity code and inflated encoding αPS2-integrin playing a pivotal role in cell-cell interactions. Unexpectedly, ladybird also contributes to the regulation of terminal differentiation genes encoding structural muscle proteins or contributing to muscle contractility. Thus, the identity gene-governed diversification of cell types is a multistep process involving the transcriptional control of genes determining both morphological and functional properties of cells (Junion, 2007).

Uncovering how the cell fate-specifying genes exert their functions and determine unique properties of cells in a tissue is central to understanding the basic rules governing normal and pathological development. To approach the cell fate determination process at a whole genome level a search was performed for transcriptional targets of the homeobox transcription factor Lb known to be evolutionarily conserved and required for specification of a subset of cardiac and muscular precursors. To this end the targeted expression profiling and the novel ChIP-on-chip method ChEST were combined. The data revealed an unexpectedly complex gene network operating downstream from lb, which appears to act not only by regulating components of the cell identity code but also as a modulator of pan-muscular gene expression at fiber-type level. Of note, the role of Drosophila lb in regulating segment border muscle (SBM) founder motility appears reminiscent of the role of its vertebrate ortholog Lbx1, known to control the migration of leg myoblasts (Vasyutina, 2005) in mouse embryos (Junion, 2007).

Earlier genetic studies revealed that within the same competence domain the cell fate specifying factors acted as repressors to down-regulate genes determining the identity of neighboring cells. Consistent with this finding, lb was found to repress msh and kruppel (kr) during diversification of lateral muscle precursors and even skipped (eve) within the heart primordium. This study found that additional identity code components are regulated negatively by lb. In the lateral muscle domain lb acts as a repressor of the MyoD ortholog nau and the NK homeobox gene slou, both known to be required for the specification of a subset of somatic muscles. This suggests that a particularly complex network of transcription factors (Ap, Msh, Kr, Nau, Slou) controls the specification of individual muscle fates in the lateral domain. Interestingly, none of these factors is coexpressed with lb in the SBM, which appears to be a functionally distinct muscle requiring a specific developmental program. Besides factors with well-documented roles in diversification of muscle fibers, the global approach identified a few novel potential players in the muscle identity network. Among them are expressed in somatic muscle precursors the Pdp1 gene encoding Par domain factor and the CG32611 gene containing a zinc finger motif (Junion, 2007).

Interestingly, in the cardiac domain the data demonstrate that lb is able to positively regulate the expression of tin and the effector of RTK pathway pointed (pnt), both involved in cardiac cell fate specification. These findings are consistent with earlier observations that the forced lb expression leads to the ectopic tin-positive cells within the dorsal vessel. Also, during early cardiogenesis lb directly represses bric a brac 2 (bab2), which emerges as a novel component of the genetic cascade controlling the diversification of cardiac cells. Thus, the ability of Lb to act either as repressor or as activator suggests a context-dependent interaction with cofactors. Of note, several miroarray identified Lb targets have also been found in the RNAi-based screen for genes involved in heart morphogenesis (Junion, 2007).

The data indicate that lb exerts its muscle identity functions via regulation of pan-muscular genes that control cell movements, cell shapes and cell-cell interactions including myoblast fusion, myotube growth, and attachment events (Junion, 2007).

Regulation of if provides an example of how the cell-type-specific fine-tuning of expression can be achieved for genes expressed in all muscle precursors. It was reported previously (Junion, 2005) that if is directly regulated by dMef2 via two intronic CRMs, which most probably ensure a generic muscle-specific if expression. Here, a distinct, Lb-dependent if CRM was identified, able to drive expression in a restricted subset of muscles including the SBM, thus indicating that the if transcription is regulated by a coordinated action of generic and muscle-type-specific CRMs. Identification of Lb-dependent CRM close to if also indicates that lb may contribute to the dMef2-regulated feed-forward loops (Sandmann, 2006). Such a modular regulation of transcription levels would provide an efficient way for precise, fiber-type-specific setting of muscle genes activity. As shown for if in the SBM context, this regulation is expected to be direct via the identified here Lb-binding module. The data also suggest that a similar muscle-type-specific transcriptional regulation can be achieved in an indirect mode. For example, the pan-muscular kettin/sls gene encoding a giant protein required for the formation and maintenance of normal sarcomere structure is regulated by three distinct dMef2-binding CRMs able to drive expression in a muscle-type-specific manner (Junion, 2005). Among them, CRM 1 located upstream the gene was found to drive expression in a subset of ventral and lateral muscles including SBM (Junion, 2005). This study identifies sls as an Lb target in the microarray screen, and demonstrates that in embryos overexpressing Lbe the Sls expression is up-regulated. These findings suggest that Lb can act via an as-yet-unknown factor to modulate sls expression in the SBM (Junion, 2007).

The reported here Lb targets fit into three main categories: (1) genes encoding transcription factors contributing to cell-type-specific identity gene code; (2) genes controlling cell shapes, adhesion, and cell motility; and (3) late-acting genes required for functional properties of cardiac and muscle cells. Importantly, Lb is able to bind to enhancers of genes from all these categories. Also, the functional properties of identified Lb targets strongly suggest that Lb-dependent multistep acquisition of cell identity is executed in a similar way in cardiac and in muscle cells. With respect to the Lb targets encoding transcription factors bab2 was identified as a new component of cardiac cell identity code. Considering the regulation of cell shape, cell adhesion, and motility, a large number of Lb targets code for proteins involved in remodeling of the actin cytoskeleton, for the ECM components and for integrins. Down-regulation of the selected microarray candidates from this category leads to dramatic muscle phenotypes affecting founder migration, myoblast fusion or muscle fiber attachment. Among the late-acting genes, expression and function of a direct, ChEST-identified Lb target gene, CG8698, and demonstrated that it was found to be specifically expressed in differentiated muscles and required for muscle contraction. Thus, it is concluded that the Lb-governed acquisition of cell identity is a long-term process (1) initiated by the spatially restricted expression of a set of transcription factors and (2) executed by precise regulation of genes determining the morphological, dynamic and functional properties specific to a given muscle or heart cell type (Junion, 2007).


Regulatory Sequences

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

Expression of lbe and lbl depends on wingless. Previous studies have shown that ubiquitous expression of gooseberry ectopically activates the endogenous gsb gene in cells located anterior to the wild-type stripe. However, this ectopic induction is not observed in a wingless mutant background (Li, 1993). Heat shock gsb is also able to activate the formation of an ectopic strip of lbe. As for gsb, this phenomenon is wg-dependent and cannot be detected in wg mutants. Therefore, it is likely that wg function is required for both activation and maintenance of lbe and lbl expression, and for that matter, gsb as well (Jagla, 1997a).

In the dorsal epidermis, both wg and lbe are gsb-independent. It is concluded that whereas ventral epidermal wg expression may require gsb, in the dorsal epidermis, both wg and lbe are gsb-independent (Jagla, 1997a).

Ladybird is a component of a cardiogenic pathway required for diversification of heart precursors. Expression of lb genes in the subset of cardioblast and pericardial cell precursors is critically dependent on mesodermal tinman function, epidermal Wingless signaling and the coordinate action of neurogenic genes. lb-expressing heart progenitors contribute to the increased number of cardiac precursor cells in Notch, Delta, Enhancer of split, mastermind, big brain and neuralized mutants. Negative regulation by hedgehog is required to restrict ladybird expression to two out of six cardioblasts in each hemisegment. Overexpression of ladybird causes a hyperplasia of heart precursors and alters the identity of even-skipped-positive pericardial cells. Surprisingly, the number of eve-expressing pericardial cells is strongly reduced in overexpressors. These lb expressing cells are transformed into l-paracardial cells. Loss of ladybird function leads to the opposite transformation, suggesting that ladybird participates in the determination of heart lineages and is required to specify the identities of subpopulations of heart cells. Both early Wingless signaling and ladybird-dependent late Wingless signaling are required for proper heart formation. Thus, it is proposed that ladybird plays a dual role in cardiogenesis: (1) during the early phase, it is involved in specification of a segmental subset of heart precursors as a component of the cardiogenic tinman-cascade and (2) during the late phase, it is needed for maintaining wingless activity and thereby sustaining the heart pattern process. These events result in a diversification of heart cell identities within each segment. Since tinman, bagpipe, S59 and ladybird genes are all part of the same homeobox gene cluster, it is likely that their association has to do with the orchestrated diversification of mesoderm (Jagla, 1997b).

Targets of Activity

In the dorsal epidermis and the terminal regions of the body, expression of wingless is independent of gooseberry but requires a wingless-ladybird regulatory feedback loop. Loss of ladybird function reduces the number of wingless-expressing cells in dorsal epidermis and leads to complete inactivation of wingless in the anal plate. Consequently, mutant ladybird embryos fail to develop anal plates and ubiquitous embryonic expression of either one or both ladybird genes leads to severe defects of the dorsal cuticle. Lack of late wingless expression and anal plate formation can be rescued with the use of a heat-shock-ladybird transgene (Jagla, 1997a).

Spatial specificity of mesodermal even-skipped expression relies on multiple repressor sites

Individual cardiac progenitors emerge at defined positions within each segment in the trunk mesoderm. Their specification depends on segmental information from the pre-patterned ectoderm, which provides positional information to the underlying cardiac mesoderm via inductive signals. This pattern is further reinforced by repressive interactions between transcription factors that are expressed in neighboring sets of cardiac progenitors. For example, even-skipped (eve) and ladybird early (lbe) gene products mark adjacent cardiac cell clusters within a segment, and their antagonistic interaction results in mutually exclusive expression domains. Lbe acts directly on the eve mesodermal enhancer (eme) to participate in restricting its expression anteriorly. It is hypothesized that additional repressive activities must regulate the precise pattern of eve expression in the cardiac mesoderm via this enhancer. In this study, two additional repressor motifs: 4 copies of an 'AT'-rich motif (M1a-d) and 2 copies of an 'GC'-rich motif (M2a,b), were identified which when mutated cause expansion of eme-dependent reporter gene expression. Potential negative regulators of eve and were examined and it was found that their overexpression is sufficient to repress eve as well as the eme enhancer via these sites. These data suggest that a combination of factors is likely to interact with multiple essential repressor sites to confer precise spatial specificity of eve expression in the cardiac mesoderm (Liu, 2008).

Although each of the identified repressor sites is necessary, none is individually sufficient for restricting the eme enhancer activity to the eve expression domain. Several additional homeodomain proteins, including Msh, C15 and Lim3, are capable of repressing mesodermal eve expression by interacting with specific sites within the enhancer element. While the repression of mesodermal eve expression by Msh, C15 and Lim3 is likely mediated by the AT-rich M1 sites and the Lb2 site, the repression of eve expression by Lbe requires both the AT-rich M1 and the Lb2 sites as well as the GC-rich M2a site. Therefore, each of the four repressor sites apparently is required on order to confer sensitivity to repression by Lbe. This raises the possibility that repression is the result of a complex in which the cooperation of all four repressor elements is required for successful repression (Liu, 2008).

A prominent feature of the Drosophila is its segmental polarity that includes distinct cardiac cell types that are precisely positioned within each segment. These cardiac progenitors are specified along the anterior-posterior axis during development and are marked by Lbe, Eve or Svp. As the embryo develops, a linear heart tube is formed and this metameric arrangement of cardiac cells types continues to be maintained. Within each hemi-segment, the anterior two pairs form the tinman-expressing 'working myocardium', while the posterior pair that expresses svp and the T-box transcriptional factor Doc form the ostia. Previous studies suggested that repressive interactions between cardiac factors expressed in non-overlapping subtypes of cardiac cells likely contribute to the diversification and maintenance of cellular identities. Svp and Tin have been shown to repress each other's expression during heart tube formation, and the current data suggest that antagonistic interactions between Lbe and Eve are also a part of this mutual repression network. In addition, the data show that eve expression within the cardiac mesoderm is negatively regulated by multiple repressor sites, thus further supporting the idea that transcriptional repression mechanisms play a prominent role in the generation of cellular diversity in the developing heart. Roles were demonstrated for two potential repressors, C15 and Lim3. Although they do not seem to be essential for patterning mesodermal eve expression, they are normally expressed in the cardiac mesoderm in the vicinity of the Eve cells and they do repress the eme enhancer via the identified repressor sites when ectopically expressed. Therefore, it is also possible that they function redundantly with other negative regulators yet to be identified (Liu, 2008).

Default repression is a common mechanism utilized by major signaling pathways, including Wnt, Shh and Notch pathways, to restrict target gene expression. In the absence of signaling, signal-regulated transcription factors function mainly as transcriptional repressors, thus preventing low levels of target gene expression that might be activated by weak, local activators ('default repression'). In response to signals, some transcription factors are then converted into transcriptional activators to promote target gene expression. Thus, transcriptional repression and activation can be mediated by the same binding sites. Default repression mechanisms may also contribute to the restricted mesodermal eve pattern. It has been reported that mesodermal eve expression is under the direct transcriptional control of Wg signaling. Mutating several putative binding sites for dTCF, the transcriptional mediator of Wg signaling, results in an expansion of low-level reporter gene expression within the cardiac mesoderm that is unaffected by reduced wg activity. Thus, dTCF may serve as a default signal to restrict mesodermal eve expression in the absence of wg signaling (Liu, 2008).

It has been shown that Hh signaling not only promotes eve and svp but also inhibits lbe expression in the dorsal mesoderm. One mechanism for Hh signaling may be via inhibition of Cubitus interruptus (Ci)-mediated repression. Interestingly, there is some similarity between the M2a sequence examined in this study (TGGGCCCT) and the consensus sequence for Ci (TGGGTGGTC). This raises the interesting possibility that M2a site may be a putative Ci binding site in eme. Thus, mutations of M2a site, which result in the anterior expansion of eme activity into Lbe expressing cells, may reflect a lack of repression by Ci. Alternatively, the M2a site may mediate transcriptional repression by Lbe or its potential cofactors. The latter hypothesis is more consistent with the observation that reporter gene expression is rendered insensitive to inhibition by Lbe overexpression when the M2a site is mutated in eme. As the M2a site does not resemble the Lbe consensus sequence, the idea is favored that another factor binds to the M2a site, which then cooperates with Lbe in repressing mesodermal eve expression. This interaction may be facilitated by the close proximity of the two sites (Liu, 2008).

In sum, the in vivo functional dissection of eme has revealed that each of two AT-rich sites, M1b or M1c and the previous studied Lb2 site, when mutated, causes reporter gene expansion that encompasses the entire cardiac mesoderm, overlapping with Tinman protein at late stage 12. In addition, the GC-rich site M2a is required for repression anterior to the Eve cluster. The absolute requirement of each repressor site for successful restriction of eve expression within the cardiac mesoderm is in striking contrast to the mechanism of incremental activation of this enhancer in the cardiac mesoderm by activators such as Tinman, dTCF, Mad, E-box and ETS sites. Repression through these repressor sites may require cooperation between the sites, perhaps via a repressor complex. Thus, eliminating the function of any of these sites will disrupt interactions with the complex causing de-repression within the 'activator'-dependent cardiac mesoderm (Liu, 2008).



Activation of lbe precedes that of lbl and appears during germ band elongation in the primordium of the anal plate and subsequently during neuroblast segregation in the epidermis of gnathal segments (mandibular, maxillary and labial), three anterior head segments (labrum, antennal, intercalary) and in some neuroblasts. Later, after completion of germ band elongation, epidermal domains of the terminal regions of the body are found to contain only low levels of LBL protein. Besides the temporal difference, the only difference between LBE and LBL mRNA distribution is the greater abundance of LBE in the trunk epidermis (Jagla, 1997a).

New epidermal and mesodermal domains of lbe and lbl gene expression takes place between 5 and 7 hours after the start of development. Both genes start to be expressed in heart precursors. At the onset of segmental groove formation just posterior to these mesodermal cells in the thoracic and abdominal segments, a one cell wide epidermal stripes appears in which both genes are expressed. These dorsal stripes broaden anteriorly by up to 4-5 cells at 7 hours and then up to 6-7 cells after germ band retraction. The dorsal stripes corresponds to a region of late wingless expression. No expression of either gene is detected in the dorsal epidermal cells of the most posterior abdominal (A8 and A9) segments. At the late extended germ band stage [Images], transcripts of both genes also appear in the ventral, but not the lateral epidermis. At later stages of embryogenesis, expression of both genes disappears progressively from the epidermis and becomes restricted to the segmental border muscles and clusters of cells in the central and peripheral nervous systems (Jagla, 1996).

The Drosophila brain develops from the procephalic neurogenic region of the ectoderm. About 100 neural precursor cells (neuroblasts) delaminate from this region on either side in a reproducible spatiotemporal pattern. Neuroblast maps have been prepared from different stages of the early embryo (stages 9, 10 and 11, when the entire population of neuroblasts has formed), in which about 40 molecular markers representing the expression patterns of 34 different genes are linked to individual neuroblasts. In particular, a detailed description is presented of the spatiotemporal patterns of expression in the procephalic neuroectoderm and in the neuroblast layer of the gap genes empty spiracles, hunchback, huckebein, sloppy paired 1 and tailless; the homeotic gene labial; the early eye genes dachshund, eyeless and twin of eyeless; and several other marker genes (including castor, pdm1, fasciclin 2, klumpfuss, ladybird, runt and unplugged). Based on the combination of genes expressed, each brain neuroblast acquires a unique identity, and it is possible to follow the fate of individual neuroblasts through early neurogenesis. Furthermore, despite the highly derived patterns of expression in the procephalic segments, the co-expression of specific molecular markers discloses the existence of serially homologous neuroblasts in neuromeres of the ventral nerve cord and the brain. Taking into consideration that all brain neuroblasts are now assigned to particular neuromeres and individually identified by their unique gene expression, and that the genes found to be expressed are likely candidates for controlling the development of the respective neuroblasts, these data provide a basic framework for studying the mechanisms leading to pattern and cell diversity in the Drosophila brain, and for addressing those mechanisms that make the brain different from the truncal CNS (Urbach, 2003).

ladybird (lb), a tandem of the homeobox genes ladybird early (lbe) and ladybird late (lbl), both of which encode transcription factors, show a similar expression pattern, with lbe activity slightly preceding that of lbl. At stage 11, both genes are expressed in segmental repetitive patches in the laterodorsal trunk ectoderm and specifically in one NB per hemineuromere, the lateral NB 5-6. Using an antibody against Lbe the protein is first observved by stage 10 in three small procephalic patches in the labral, ocular and antennal ectoderm, and at stage 11 in an additional patch of the intercalary ectoderm. Lbe is selectively expressed in only four brain NBs on either side: one in the tritocerebrum (Td4), one in the deutocerebrum (Dd7) and two in the protocerebrum (Ppv3, Pcv8). Wg/Lbe double labelling demonstrates that Lbe and Wg expression are colocalized in the intercalary, antennal and labral ectoderm, and in Td4 and Dd7; remarkably, the ocular Lbe-positive domain and corresponding NBs (Ppv3 and Pcv8) are Wg negative. Lbe protein is detected in the progeny of the identified brain NBs until the end of embryogenesis (Urbach, 2003).

Identity, origin, and migration of peripheral glial cells in the Drosophila embryo

Glial cells are crucial for the proper development and function of the nervous system. In the Drosophila embryo, the glial cells of the peripheral nervous system are generated both by central neuroblasts and sensory organ precursors. Most peripheral glial cells need to migrate along axonal projections of motor and sensory neurons to reach their final positions in the periphery. This paper studied the spatial and temporal pattern, the identity, the migration, and the origin of all peripheral glial cells in the truncal segments of wildtype embryos. The establishment of individual identities among these cells is reflected by the expression of a combinatorial code of molecular markers. This allows the identification of individual cells in various genetic backgrounds. Furthermore, mutant analysis of two of these marker genes, spalt major and castor, reveal their implication in peripheral glial development. Using confocal 4D microscopy to monitor and follow peripheral glia migration in living embryos, it was shown that the positioning of most of these cells is predetermined with minor variations, and that the order in which cells migrate into the periphery is almost fixed. By studying their lineages, the origin of each of the peripheral glial cells was uncovered and they were linked to identified central and peripheral neural stem cells (von Hilchen, 2008).

This study has characterized the expression of a collection of cell-specific molecular markers, which allows to identify and distinguish all glial cells in the embryonic peripheral nervous system. The reproducibility with which enhancer-trap lines and marker genes are expressed in the individual peripheral glial cells, indicates that these cells display unique identities. Furthermore, the spatial and temporal pattern of migration and the final arrangement of these cells are relatively stereotypic. This suggests that the specification of the unique identity of each cell does not only define a specific combination of genes to be expressed, but also includes the information about the timing of migration, the nerve tract it is associated with, and to some degree the final position to be occupied along the respective nerve. How the cell receives this information is still unknown. The individual characteristics could be determined (1) by lineage or (2) during migration by cell-cell interactions (between the glial cells or between the glia and other closely associated cells, e.g. neurons, tracheae), or (3) by a combination of both (von Hilchen, 2008).

The master regulatory gene glial cells missing (gcm) is required to induce the glial cell fate. Gcm as a transcription factor switches on downstream target genes, of which the gene encoding for the homeobox transcription factor Reversed polarity (Repo) is well described. As this cascade of gene activation is required for all glial cells in the Drosophila embryo (except the midline glia), it is unlikely to contribute to cell fate diversification among the glia. Whereas central glial cells migrate over rather short distances, in literally any possible direction, to finally occupy stereotypic positions within the CNS, the peripheral glial cells behave differently as they have to migrate over remarkable distances into the periphery. It has been recently shown that the migration of PGs depends on Notch signalling. In Notch mutants or in mutants where Notch signalling is altered in PGs, the migration is impaired or even completely blocked. However, this signalling does not appear to supply the cells with characteristics of their fate apart from the onset and/or maintenance of the migration itself. Sepp (2000) described the developmental dynamics and morphology of a subset of peripheral glial cells and could show that a signalling cascade mediated by the small GTPases RhoA and Rac1 influences the actin cytoskeleton of migrating PGs. Sepp further showed, that, within the analysed population of cells, a 'leading glia’ expresses filopodia-like structures whereas the ‘follower’ cells do not. Similar results were reported by Aigouy (2004). Aigouy established a 4D microscopy technique to record and analyse the developmental dynamics and migratory behaviour of PNS glia during pupal stages in the developing fly wing. In this system, differences between 'leading' and 'follower' glia cells were also observed. The glial cells in the wing PNS migrate along wing veins in a chain with one 'leading' cell in front. If this chain is interrupted by laser ablation of either the leading or intermediate cells, a new 'leading’ cell starts to form filopodia and explores the surrounding. Once this new 'leading' cell catches up with the previous chain or reaches its target area, the filopodia disappear and the cells' morphology changes again. Hence, these differences in glial cell morphology and behaviour in the wing PNS are based on interactions of the glial cells with each other rather than on a predetermined intrinsic cell fate (von Hilchen, 2008).

Findings for the embryonic PNS glia suggest that these cells are predetermined at least to a certain extent. The 4D microscopy approach allowed tracing of the migration of individually identified PGs in living embryos from the moment they leave the CNS until they reach their final position. Apart from the dorsal SOP-derived cells, which never change their position or behaviour, it is always the ePG9 that leaves the CNS first and 'leads' the track. This cell expresses filopodia-like structures, while the following cells do not, although it remains to be experimentally shown whether they can take over the leading function in the absence of ePG9. It is worth mentioning that the SOP-derived ePG12 migrates along trajectories of the ISN prior to ePG9. It is not clear whether ePG12 has any leading function for ePG migration or functions as a guidepost cell for axonal projections. It is the only cell, though, that swaps nerve tracts and finally associates with the TN. Most likely, cell-cell communication between ePG12 and axonal projections and/or neighbouring cells is required for proper pathfinding and positioning. It is always the ePG4 that migrates along and finally enwraps the segmental nerve. As this cell is the only cell associated with the distal part of the segmental nerve, it functions as 'leading' glia for this nerve and expresses filopodia-like structures at least in later stages when it enwraps the SN. This enwrapment occurs in a bidirectional fashion, i.e. the filopodia occur at both ends of the glial cell (von Hilchen, 2008).

Lineage analysis revealed that the PGs mentioned above originate from the CNS neuroblast NB 1-3 and a ventrally located SOP. Interestingly, the two NB 2-5 derived PGs (ePG6 and ePG8) differ from these cells with respect to both identity and behaviour. They express fewer of the analysed PG-specific markers (cas-Gal4 and mirr-lacZ) and it is not possible to distinguish between these two cells so far. Whether the lack of identifying markers is a consequence of or a prerequisite for their different identity and behaviour is not yet clear. The cells migrate along the ISN independently of the NB 1-3- and SOP-derived PGs and frequently overtake them (and occasionally even one another). The correlation of such characteristics with the different origin of these three subpopulations of PGs lends support to the hypothesis that some aspects of cell fate diversification among the PGs may be predetermined by lineage. It is likely, that such predetermined characteristics include the competence to respond to specific external signals that guide the respective cell along the correct nerve to its target position (von Hilchen, 2008).

One incidence for lineage-dependent cell fate determination comes from the analysis of the ladybird homeobox genes. The ladybird genes are expressed in the developing CNS in only few NBs including NB 5-6. The NB 5-6 lineage produces one of the proximal PGs (ePG2) which expresses the Ladybird early (Lbe) protein. It has been shown that a loss of ladybird gene function results in a loss of the ePG2 in a third of all analysed hemisegments, accompanied with a higher number of medially located glial cells in the CNS. An opposite phenotype with excessive cells in the transition zone was observed by ectopic expression of the ladybird genes throughout the CNS. Using an anti-Repo antibody as well as a subset specific reporter transgene (K-lacZ), De Graeve (2004) provided evidence suggesting that the ladybird genes play a role in glial subtype specification in particular NB lineages. Another factor shown to be required for the specification of a lineage-specific set of glial cells (NB1-1-derived subperineurial glia) is Huckebein, which interacts with Gcm to amplify its expression specifically in these cells (von Hilchen, 2008).

Furthermore, in cas mutants, it was shown that the two NB 2-5-derived glia (ePG6 and ePG8) do not migrate into the periphery but most likely stay at their place of birth, although they acquire glial cell fate (as can be deduced from Repo stainings). Thus, similar to Ladybird and Huckebein, Cas seems to be involved in lineage-dependent glial subtype specification rather than determination of glial fate in general. In contrast to ladybird (De Graeve, 2004), though, Cas is not sufficient to ectopically induce glial cell fate or PG subtype specification (von Hilchen, 2008).

This study shows that salm is a likely candidate participating in the control of glial development. Embryos homozygous for salm445 show a pleiotropic and variable phenotype affecting not only glial cells but also PNS neurons, sensory organs, and other tissues. Yet, nearly all ventrally derived PGs stall in the transition zone between CNS and PNS and do not migrate properly into the periphery. In about 50% of the analysed hemisegments, a variable number of one to three PGs are missing, even though these cells could remain in the CNS. salm-lacZ is expressed in the two ventral SOP-derived ePG4 and ePG5, as well as in the dorsal SOP-derived ePG11 along the DLN, and in some of the ligament cells of the lateral chordotonal organ. In salm445 mutants the ePG4 cell can sometimes be detected at its wildtypic position along the SN. If ePG4 is missing along the SN, it could well be a secondary effect, as the SN itself is affected with the SNc shortened or occasionally missing. The ePG5 however, cannot be unambiguously identified in Repo-staining within the group of cells stalling in the transition zone (von Hilchen, 2008).

It needs to be further shown whether the differences between the PGs derived from certain progenitor cells result in functional differences in the larva. The peripheral nerves of the larva are ensheathed by two distinct types of glial cells, the perineurial and the subperineurial glial cells. The subperineurial glia build septate junctions with each other (or themselves) and thereby form the blood-nerve barrier, whereas the perineurial glia form an outer layer and secrete the neural lemma. In order to allow proper electrical conductance, the peripheral nerves must be enwrapped and insulated at the end of embryogenesis when hatching of the larva requires coordinated muscle contractions. It is not known to date which of the embryonic PGs will become perineurial or subperineurial glia, or what other functions they might fulfill (von Hilchen, 2008).

The comprehensive description of the ancestry, identity and dynamics of the developing embryonic peripheral glia, and the molecular markers at hand, provide a crucial basis for further clarification of the mechanisms controlling development, migration, and function of peripheral glia on a single cell level (von Hilchen, 2008).

Drosophila adult muscle precursors form a network of interconnected cells and are specified by the rhomboid-triggered EGF pathway

In Drosophila, a population of muscle-committed stem-like cells called adult muscle precursors (AMPs) keeps an undifferentiated and quiescent state during embryonic life. The embryonic AMPs are at the origin of all adult fly muscles and, as is demonstrated in this study, they express repressors of myogenic differentiation and targets of the Notch pathway known to be involved in muscle cell stemness. By targeting GFP to the AMP cell membranes, it was shown that AMPs are tightly associated with the peripheral nervous system and with a subset of differentiated muscles. They send long cellular processes running along the peripheral nerves and, by the end of embryogenesis, form a network of interconnected cells. Based on evidence from laser ablation experiments, the main role of these cellular extensions is to maintain correct spatial positioning of AMPs. To gain insights into mechanisms that lead to AMP cell specification, a gain-of-function screen was performed with a special focus on lateral AMPs expressing the homeobox gene ladybird. The data show that the rhomboid-triggered EGF signalling pathway controls both the specification and the subsequent maintenance of AMP cells. This finding is supported by the identification of EGF-secreting cells in the lateral domain and the EGF-dependent regulatory modules that drive expression of the ladybird gene in lateral AMPs. Taken together, these results reveal an unsuspected capacity of embryonic AMPs to form a cell network, and shed light on the mechanisms governing their specification and maintenance (Figeac, 2010).

In late Drosophila embryos, each abdominal hemisegment features six AMPs at stereotypical positions associated with differentiating muscle fibres. To better characterize these cells, tests were performed to see whether the Notch pathway, which is known to be required for generation of satellite cells from muscle progenitors and for keeping them ready to engage in muscle regeneration, is also active in AMPs. Analysis of a GFP reporter line, E(spl)M6-GFP, described as a read-out of the Notch pathway in Drosophila, revealed that it is co-expressed with Twist in AMPs. Also, transcripts of another Notch target, Him, specifically accumulated in AMPs. By testing several mesodermal cell markers, it was found that, in addition to Twist, two other transcription factors, Zfh1 and Cut, are expressed in all AMPs. Zfh1 expression in embryonic AMPs has been reported previously, whereas cut has been used to reveal a subset of AMPs associated with larval wing and leg imaginal discs. Despite expressing common markers, the AMPs are heterogenous and differ by the expression of muscle identity genes. For example, slouch (S59) and Pox meso are specifically expressed in ventral (V) AMPs whereas ladybird (lb) and Kruppel (Kr) display lateral (L) AMP-specific expression (Figeac, 2010).

To gain insights into AMP cell shapes and their behaviour, an E(spl) M6-GAL4 line was generated that recapitulates M6-GFP expression, and it was used it to drive a membrane-targeted GFP. It has been previously reported that AMPs are associated with the larval peripheral nervous system (PNS) and that in daughterless mutant embryos lacking all the larval sensory system, the final pattern of AMPs is deranged. This study showed that all embryonic AMPs are closely associated with both the PNS and the differentiated muscles, sitting either at the top of muscle fibres [LAMPs and dorsal (D) AMPs] or on their internal face [dorsolateral (DL) AMPs and VAMPs)]. In late embryos, the AMPs form a network of cells displaying irregular shapes and are interconnected by long cellular processes aligning PNS nerves. Connections between the AMPs initially form within the parasegments, but the AMPs very quickly send filopodia posteriorly and make contact with DLAMPs of the adjacent segment, thus interlinking all AMPs. In addition to the interconnected M6+/twi+ AMPs, a population of morphologically distinct M6+/twi- cells of unknown fate, located more internally in central and posterior regions of the abdomen, was identified (Figeac, 2010).

It has been reported that a subset of muscle progenitors divides asymmetrically and gives rise to numb-positive founder cells that undergo differentiation and to Notch-expressing AMPs. Through this pathway, six AMPs are born in each abdominal hemisegment. In contrast to founders, AMPs express the Notch target Holes in muscle (Him) and Zfh1, the Drosophila homolog of ZEB, both of which are able to counteract Mef2-driven myogenic differentiation. Interestingly, another general AMP marker, E(spl)M6, also corresponds to a Notch target, suggesting that Notch signalling could play an evolutionarily conserved role in muscle cell stemness. It operates not only in vertebrate satellite cells but also, as shown in this study, in Drosophila AMPs. Finally, it is reported that, similar to muscle progenitors, the AMPs are heterogenous and express different muscle identity genes, such as lb or slou. This strongly suggests that AMPs acquire a positional identity that makes them competent to form a given type of muscles during adult myogenesis. For example, the lateral AMPs expressing lb are at the origin of all lateral body wall muscles of the adult fly. In support of the specific positional identities of AMPs comes also the analysis of lame duck (lmd) mutant embryos known to be devoid of fusion-competent myoblasts (FCMs). In this mutant context, the number of Twi-positive and Zfh1-positive AMP-like cells is highly increased, while the number of Lbe- and Twi-positive LAMPs committed to the lateral lineage remains unchanged. Thus in the absence of lmd, some presumptive FCMs can adopt the AMP-like fate but they do not carry positional information transmitted by the identity genes such as lb (Figeac, 2010).

Based on the premise that the AMPs correspond to a novel population of transient stem cells, their shapes and behaviour were analyzed in living embryos carrying M6-GAL4 and UAS-GAP-GFP transgenes. Surprisingly it was found that shortly after their specification, the AMPs start to send cellular processes that align along the nerves of the PNS, with the result that, by the end of embryogenesis, all AMPs become linked together. Interestingly, the intersegmental connections are made via an intermediary M6+ twi- cell of unknown fate. In addition to this particular cell, which ensures the intersegmental link between AMPs, the embryos also contained other M6+ twi- non-neural cells of rounded morphology located more internally that were unconnected to the AMP cell network. The origin and identity of these cells remain unknown (Figeac, 2010).

Exploiting the possibility of following AMPs in vivo, test were performed to see how AMPs would behave if their connections were broken. Since the AMPs separated from the network by laser ablation changed shape and lost their normal positions, it is concluded that one important reason for which AMPs form a cell network is to keep precise spatial positioning. Based on the observation that AMPs send long cellular processes along the peripheral nerves, it is probable that nerves serve as a support for extending AMP cell protrusion. This possibility is supported by the abnormal pattern of AMPs observed in daughterless mutant embryos lacking the PNS and in embryos in which the PNS was affected by the Elav-GAL4 driven expression of the inducer of apoptosis, Reaper. PNS nerves might also represent a source of signals for AMPs such as Delta in order to maintain Notch activity. However, analysis of the lateral domain revealed that Delta expression was associated with the segment border muscle (SBM) precursor but not with the PNS neurons, indicating that Notch activity in lateral AMPs is regulated by Delta produced in the SBM rather than in nerves (Figeac, 2010).

Taking advantage from the restricted number of embryonic AMPs and the genetic tools available in Drosophila, a large-scale gain-of-function screen was performed to identify the genes involved in AMP specification. rho and other components of the EGF signalling pathway were found to be crucially required for both specification and maintenance of AMPs. Importantly, as reported by Krejci (2009), several components of EGF signalling are direct targets of Notch in AMPs, thus creating a link between the two signalling pathways. The high number of AMPs in EGFRCA and RAS gain-of-function contexts provides evidence that RAS signalling not only promotes muscle founder specification, but is also crucial for specifying AMPs when induced by EGF signals. Further support for a key role of the EGFR pathway is the identification of cells sending EGF to lateral AMPs and the demonstration of their role in AMP cell maintenance. It also turns out that the anti-apoptotic role of the EGFR pathway in Drosophila AMPs described in this study is conserved across evolution, since EGF signalling also promotes survival of vertebrate satellite cells (Figeac, 2010).

The evidence for a major role of the EGFR pathway in the specification and maintenance of AMPs raises important questions about EGF targets operating in these muscle-committed stem-like cells in Drosophila. lb genes have been shown to be required for specification of LAMPs, making them candidate targets of EGF signalling in the lateral region. This study shows that lb regulatory modules contain binding sites for ETS factors that act as EGFR effectors and goes on to demonstrate their crucial role in AMP enhancer activity. The proximity of the ETS binding sites and homeodomain binding sites in the AMP element suggests that an adapted spatial conformation of interacting factors is important in allowing simultaneous binding and thus maintenance of the lineage-restricted activity of this enhancer. Interestingly, the main difference between regulatory modules driving expression in differentiated muscle lineages versus regulatory modules that act in non-differentiated AMPs is the responsiveness of the latter category to extrinsic EGF signals. In opposition to this, this study found that intrinsic Mef2 inputs are sufficient to drive expression in differentiated muscle lineage. The ETS and Mef2-driven expression of these two distinct regulatory modules is positively regulated by lb, which is known to play a pivotal role in the specification of muscle lineages in the lateral domain. The specific expression of lb in a subset of AMP cells and of its ortholog Lbx1 in activated satellite cells suggests that similarities in genetic control of Drosophila and vertebrate muscle stem cells might extend beyond those discussed here (Figeac, 2010).


Embryos null for lbe and lbl lack Wg protein in the labrum and anal plate and have reduced levels of Wg protein in the dorsal epidermis subsequent to the 8th hour of development. The most affected region of the dorsal cuticle corresponds to the wg dependent quaternary denticles (reduced in number with abnormal pigmentation) but modifications appear also in tertiary denticles. lbe and lbl deficient mutants also do not develop the anal plate (Jagla, 1997a). It would seem as though primary denticles, present in the segmental border row of cells and cells just posterior to the primary row, which give rise to naked cuticle (Bokor, 1996), are determined early in the establishment of segment polarity.

In the mesoderm of Drosophila embryos, a defined number of cells segregate as progenitors of individual body wall muscles. Progenitors and their progeny founder cells display lineage-specific expression of transcription factors but the mechanisms that regulate their unique identities are poorly understood. The homeobox genes ladybird early and ladybird late are shown to be expressed in only one muscle progenitor and its progeny: the segmental border muscle (SBM) founder cell and two precursors of adult muscles. The only myoblasts with persistent twist expression are the non-differentiated precursors of adult muscles. The SBM progenitor, which co-expresses twi and lb, in comparison to other progenitors, shows some particularities. Unlike S59 and Kr progenitors, it divides giving three progeny: a twi-negative SBM founder cell, which recruits neighbouring myoblasts to built the syncytial SBM fiber, and two adult muscle precursors with persistent twi expression. The position of the latter cells, close to the SBM, indicates that they correspond to lateral adult muscle precursors (LaPs). The distinct fates of lb-positive SBM and LaP myoblasts are already apparent during late stage 12. Neither of the lb-positive myoblasts express Kr, which labels neighbouring lateral and ventral muscle precursors. lb activity is associated with all stages of SBM formation, namely the promuscular cluster, progenitor cell, founder cell, fusing myoblasts and syncytial fiber. The SBM arises from a cluster of 6-7 mesodermal cells, each of which weakly expresses lb. During early extended germ band stage (about 5 hours of development), lb expression becomes restricted to, and upregulated in, only one large cell, the SBM progenitor. This cell, as detected by double staining with a marker of mitosis, undergoes two divisions. The first division gives rise to the SBM founder and is morphologically asymmetric; the second division, most likely symmetric, results in two LaPs. The SBM founder cell starts to migrate dorsally along the segmental border, whereas the LaPs remain at their initial position. The migration of the SBM founder prefigures the final location of SBM syncytial fiber formed by the progressive integration of neighbouring myoblasts. At the onset of dorsal closure, fusion is completed and the SBM contains 6-7 lb-positive nuclei (Jagla, 1998).

The segregation of the ladybird-positive progenitor requires coordinate action of neurogenic genes and an interplay of inductive Hedgehog and Wingless signals from the overlying ectoderm. The SBM progenitor corresponds to the most superficial cell from the promuscular cluster, thus suggesting a role for the overlying ectoderm during its segregation. To investigate this possibility the position of the SBM promuscular cluster with respect to the epidermal anterior and posterior compartments was determined. This cluster is located ventrolaterally below the epidermal posterior compartment. After segregation, the SBM progenitor migrates to a more lateral and posterior position so that, by late stage 11 (7 hours of development), it is detected at the segmental border. Since epidermal Wg and Hedgehog (Hh) signaling has been shown to influence muscle formation, the SBM-associated lb expression was examined in embryos carrying hh and wg thermosensitive mutations. Wg and Hh signalings, mutually dependent at this time, are shown to be required for the promuscular lb activity and/or the segregation of SBM progenitors. The initial influence of these signals is no longer observed later in development. In addition to signals from the epidermis, the activity of the mesodermal gene tinman, initially expressed in the whole trunk mesoderm, is involved in the early events of myogenesis. In tin - embryos, the formation of SBM promuscular clusters and segregation of lb-positive progenitor cells are strongly affected, leading to the absence of the majority of SBM fibers. During promuscular cluster formation, since tin expression becomes restricted to the dorsal mesoderm, its influence on ventrolaterally located SBMs is likely to be indirect and mediated via an unknown factor. The lack of neurogenic gene function, known to be involved in cell-cell interactions during lateral inhibition, generates the opposite phenotype. Mastermind - and Enhancer of split - embryos fail to restrict promuscular lb expression to only one cell; in consequence, they display a hyperplastic lb pattern in later stages. In contrast, the loss of function of a proneural gene, lethal of scute, which is specifically expressed in promuscular clusters and segregating muscle progenitors, has no significant influence on SBM formation (Jagla, 1998).

To investigate the role of lb activity in the specification of SBM and LaP myoblast lineages, the pattern of larval and adult muscle precursors was examined in embryos ectopically expressing lb and in embryos lacking lb activity. The comparison of SBM formation in wild-type, hs-lb and UAS-lb embryos reveals that in about 70% of hemisegments ectopic lb expression leads to the formation of enlarged or duplicated SBMs. Similarly, in 24B-Gal4/UAS-lbe embryos the number of LaPs with persistent twist expression is significantly increased. The overproduction of SBM and LaPs is frequently accompanied by the loss of some neighbouring lateral muscle fibers, suggesting that the ectopic expression of lb may change the identity of a subset of early progenitors (Jagla, 1998).

Unlike the progenitors described thus far, but similar to the neuroblasts, the ladybird-positive progenitor undergoes morphologically asymmetric division. Ectopic ladybird expression is sufficient to change the identity of a subset of progenitor/founder cells and to generate an altered pattern of muscle precursors. When ectopically expressed, ladybird transforms the identity of neighbouring, Krüppel-positive progenitors, leading to the formation of giant segmental border muscles and supernumerary precursors of lateral adult muscles. In about 70% of hemisegments, the ectopic lb expression leads to the formation of enlarged or duplicated SBMs. The number of LaPs with persistent twi expression is significantly increased. The overproduction of SBM and LaPs is frequently accompanied by the loss of some neighbouring lateral muscle fibers, suggesting that the ectopic expression of lb may change the identity of a subset of early myoblasts (progenitors/founder cells) and modify the muscle pattern. The number of Kr-expressing muscle precursors just adjacent to the SBM is dramatically reduced, indicating lb-induced transformation of myoblast identities. In embryos lacking ladybird gene function, specification of two ladybird-expressing myoblast lineages is affected. The segmental border muscles do not form or have abnormal shapes and insertion sites, while the number of lateral precursors of adult muscles is dramatically reduced. Altogether, these results provide new insights into the genetic control of diversification of muscle precursors and indicate a further similarity between the myogenic and neurogenic pathways (Jagla, 1998).

In Drosophila embryos, founder cells that give rise to cardiac precursors and dorsal somatic muscles derive from dorsally located progenitors. Individual fates of founder cells are thought to be specified by combinatorial code of transcription factors encoded by identity genes. To date, a large number of identity genes have been identified; however, the mechanisms by which these genes contribute to cell fate specification remain largely unknown. Regulatory interactions of ladybird (lb), msh and even skipped (eve), the three identity genes specifying a subset of heart and/or dorsal muscle precursors, have been analyzed. Deregulation of each of them alters the number of cells that express the other two genes, thus changing the ratio between cardiac and muscular cells, and the ratio between different cell subsets within the heart and within the dorsal muscles. Specifically, mutation of the muscle identity gene msh and misexpression of the heart identity gene lb leads to heart hyperplasia with similar cell fate modifications. In msh mutant embryos, the presumptive msh-muscle cells switch on lb or eve expression and are recruited to form supernumerary heart or dorsal muscle cells, thus indicating that msh functions as a repressor of lb and eve. Similarly, overexpression of lb represses endogenous msh and eve activity, hence leading to the respecification of msh and eve positive progenitors, resulting in the overproduction of a subset of heart cells. As deduced from heart and muscle phenotypes of numb mutant embryos, the cell fate modifications induced by gain-of-function of identity genes are not lineage restricted. Consistent with all these observations, it is proposed that the major role of identity genes is to maintain their restricted expression by repressing other identity genes competent to respond positively to extrinsic signals. The cross-repressive interactions of identity genes are likely to ensure their localized expression over time, thus providing an essential element in establishing cell identity (Jagla, 2002).

Ectopically expressed lb has been shown to inhibit eve in the founder cell of the DA1 muscle. This effect may be due to either a specific inhibition of eve by lb or a more general regulatory mechanism of fate specification. Data presented here favour the latter possibility, showing that the gain of lb function affects expression of several identity genes and consequently influences fates of cells in which these genes are expressed. Specifically, embryos that ectopically express lb have an increased number of tin-positive heart cells with a concomitant reduction of dorsal muscles. To demonstrate that the supernumerary cardiac cells result from cell fate switches, rather than from additional proliferation, mshDelta mutants, displaying heart hyperplasia similar to that observed in embryos overexpressing lb, were used. In this particular msh mutant, the presumptive msh-positive muscle cells monitored by lacZ start to express cardiac markers. This suggests that switches from muscular to cardiac fates contribute to heart hyperplasia induced by deregulation of identity genes. Interestingly, the ectopic expression of lb and msh leads to reciprocal phenotypes, and indicates that the identity genes specifically expressed in the heart promote dorsal mesodermal cells to enter the cardiogenic pathway, while the muscle identity genes promote the myogenic pathway. However, more detailed analysis shows that ectopic lb promotes only specific cardiac fates and ectopic msh only specific muscle identities, thus indicating that the identity genes instruct dorsal mesodermal cells to adopt the specific cardiac or muscular fates, rather than make a choice between cardiac and muscular development. This property is particularly well illustrated by the phenotypes generated by the ectopic eve, which is involved in the specification of a subset of heart and dorsal muscle cells and when ectopically expressed promotes specification of supernumerary cells of both types. Moreover, deregulated heart and dorsal muscle identity genes preferentially affect fates of mesodermal cells located in dorsal but not in ventral regions, thus suggesting that the identity gene action is instructive only in a permissive context (Jagla, 2002).

This observation is in complete agreement with the model of competence domain. According to this concept, the high level of Wg and Dpp signals present in the anterodorsal region (under the intersection of Wg and Dpp epidermal domains) provides a major cue that direct mesodermal cells into cardiac or dorsal muscle development. In relation to this model, these data design a new regulatory mechanism that provides a paradigm of how the intrinsic transcription factors and extrinsic signaling molecules converge to specify cell fates (Jagla, 2002).

The findings suggest cross-repressive interactions that occur between transcription factors that specify adjacent and non-overlapping populations of muscle and heart cells. Most likely, in normal development, these interactions have a functional relevance once the progenitor cells segregate, and then continue to play an important role in the next step of cell fate diversification, namely in founder cells. The gain- and loss-of-function experiments presented indicate that the identity genes may function as repressors starting from the progenitor stage onwards. However, the earliest activation of inappropriate identity gene as a result of the loss of function of repressor (in mshDelta embryos) was documented in founder cells (Jagla, 2002).

It is proposed that cross-repressive interactions allow the refinement of the potentially imprecise pattern of identity gene expression induced by the interplay of Wg and Dpp signaling pathways. Wg and Dpp create a permissive context for the development of cardiac and dorsal muscle precursors. In such a context, the transcription factors that specify these two types of cells (e.g. lb, eve and msh) are expected to be activated in all dorsal mesodermal cells. The local restriction of identity gene expression is, however, provided by a combinatorial signaling code mediated by two receptor tyrosine kinases, the Drosophila epidermal growth factor receptor and the Heartless (Htl) fibroblast growth factor receptor. Transient localized activity of these two mesodermal signaling pathways is thought to subdivide the large competence domain into small clusters of equivalent cells from which individual progenitors segregate. Depending on the combination of RTKs activities, the individual identity genes are activated only in a defined equivalence group and in the resulting progenitor. This study defines an additional step to the aforementioned model. It is proposed that the major role of identity genes is to maintain their restricted expression in progenitors and subsequently in founder cells by repressing other identity genes competent to respond positively to Wg and Dpp signals. These cross-repressive interactions are likely to ensure constant localized identity gene expression over time, thus providing a crucial element in establishing cell identity (Jagla, 2002).

The homeobox genes ladybird in Drosophila and their vertebrate counterparts Lbx1 genes display restricted expression patterns in a subset of muscle precursors, and both of them are implicated in diversification of muscle cell fates. In order to gain new insights into mechanisms controlling conserved aspects of cell fate specification, a gain-of-function (GOF) screen was performed for modifiers of the mesodermal expression of ladybird genes using a collection of EP element carrying Drosophila lines. Among the identified genes, several have been previously implicated in cell fate specification processes, thus validating the strategy of the screen. Observed GOF phenotypes have led to the identification of an important number of candidate genes, whose myogenic and/or cardiogenic functions remain to be investigated. Among them, the EP insertions close to rhomboid, yan and rac2 suggest new roles for these genes in diversification of muscle and/or heart cell lineages. The analysis of loss and GOF of rhomboid and yan reveals their new roles in specification of ladybird-expressing precursors of adult muscles (LaPs) and ladybird/tinman-positive pericardial cells. Observed phenotypes strongly suggest that rhomboid and yan act at the level of progenitor and founder cells and contribute to the diversification of mesodermal fates. Analysis of rac2 phenotypes clearly demonstrate that the altered mesodermal level of Rac2 can influence specification of a number of cardiac and muscular cell types, including those expressing ladybird. The finding that in rac2 mutants ladybird and even skipped-positive muscle founders are overproduced, indicates a new early function for this gene during segregation of muscle progenitors and/or specification of founder cells. Intriguingly, rhomboid, yan and rac2 act as conserved components of Receptor Tyrosine Kinase (RTK) signalling pathways, suggesting that RTK signalling constitutes a part of a conserved regulatory network governing diversification of muscle and heart cell types (Bidet, 2003).

The presented rho, yan and rac2 gain and loss-of-function phenotypes, clearly demonstrate that these genes play critical roles in the specification of lb-expressing mesodermal lineages. When over-expressed, the regulator of EGF-ligand maturation rho is able to induce specification of an increased number of lb-positive lateral adult muscle precursors (LaPs). Consistent with this observation, the GOF of a negative effector of RTKs signalling yan leads to the loss of LaPs. Interestingly, the large number of LaPs in rho GOF embryos suggests that during segregation of the LaPs progenitor, the Notch-mediated lateral inhibition is affected. Antagonistic activities of the EGFR and the Notch signalling pathways have been reported, thus indicating that the excess of EGFR signalling can overrule the lateral inhibition during specification of muscular progenitors. The highly restricted mesodermal expression of rho suggests, however, that in wild type embryos the rho-triggered EGF signals can interfere with lateral inhibition only in a subset of promuscular clusters. This indicates that other RTKs contribute to the negative interactions with Notch. Taking into consideration all the available information, it is speculated that the ectopically expressed rho induces the EGFR pathway that antagonizes Notch dependent lateral inhibition, specifically during segregation of the LaP progenitor. This results in promoting the LaP fate. Since in rho and yan mutants the segmental border muscle (SBM) is duplicated, it is proposed that during specification of SBM founder the repressive action of yan is relieved by a Rho/EGFR-independent RTK pathway (Bidet, 2003).

The loss of both, the SBM and the LaPs, is also observed in embryos over-expressing Rac2. This was surprising as previous reports suggested the involvement of rac2 in myoblast fusion processes (Hakeda-Suzuki, 2002). Since loss of rac2 function confirms its role in cell fate specification decisions and leads to the overproduction of lb positive muscle cells, it is hypothesised that rac2 might exert this new function by interacting with RTK signalling components. One potential way by which rac2 might exert the cell fate specification functions is the control of growth factor receptor trafficking and degradation. This possibility is in agreement with the previously described implication of vertebrate Rho-GTPases, RhoA, RhoB and Rac in cellular trafficking of the EGFR. It has been shown that the ligand-bound EGFR undergoes trafficking events that relocalize the receptor to the clathrin coated pits on the cellular membrane and then promote its internalization. The most important step in intracellular processing of EGFR is the formation of Multivesicular Bodies (MVB), which direct the EGFR either to the recycling or to the degradation pathways. One of the small Rho-GTPases, RhoB, was found to be specifically associated with MVB, and when over-expressed, was able to promote the EGFR degradation. The potential RhoB-like role of Drosophila rac2 in directing the RTKs to degradation is in agreement with the overproduction of lb-expressing muscle cells in rac2 mutants. The phenotype is reminiscent of that observed in mutants for the negative RTK effector Yan (Bidet, 2003).

These data also demonstrate new roles for rho, yan and rac2 in the specification of cardiac lineages. Interestingly, mutations of rho and rac2 affect specification of pericardial cells with no major effects on cardioblast identity. yan loss and GOF leads to even more pronounced phenotypes suggesting that, in addition to EGFR, other RTKs are involved in diversification of cardiac fates. rho and Ras/MAPK pathway have been shown to influence specification of eve-expressing pericardial cells. In addition, this study shows that rho represses and yan promotes specification of lb-positive pericardial cells. Surprisingly, in rho mutants, the supernumerary lb-positive pericardial cells co-express eve, a situation never observed in wild type embryos because of mutual repressive activities of eve and lb. This suggests that cross-repression requires the co-ordinated action of identity gene products and effectors of RTK signalling pathway. The overproduction of tin/eve-positive pericardial cells observed in rho GOF and in rac2 loss of function mutants suggests that the diversification of this particular cell type involves a rac2-dependent trafficking of EGF receptor. A future challenge will be to unravel whether Drosophila rac2 indeed co-operates with cell fate specification machinery by controlling the intracellular processing of EGFR and others RTKs (Bidet, 2003).

Rhomboid belongs to a large family of intermembrane serine proteases regulating the EGF-like ligand maturation in different species from prokaryotes to Human. One of the mouse rho homologs, ventrhoid, exhibits a very dynamic expression in central nervous system and forming somites, suggesting it may regulate early cell fate specification genes in a manner similar to that in which rho regulates lb in Drosophila. Several yan-like genes have also been identified in vertebrates. Two human yan homologs, named tel1 and tel2 share similar mesodermal embryonic expression pattern restricted to hematopoietic lineages. In addition, in adult mouse, tel1 is expressed in the heart and in skeletal muscles. As in Drosophila, yan functions with its closely related partner pointed. It is important to note that the vertebrate pnt genes ets-1 and ets-2 are involved in early embryonic heart and muscle development. The numerous vertebrate homologs of the third candidate gene of this study, rac2, control a variety of cellular processes including actin polymerization, integrin complex formation, cell adhesion, membrane trafficking, cell cycle progression, and cell proliferation. The majority Rho-GTPases are ubiquitously expressed, including the developing muscular and cardiac tissues, but their myogenic functions have not yet been investigated. The vertebrate Rac2 gene is specifically required for hematopoiesis. Its mutation in mice leads to the defective neutrophil cellular functions reminiscent of human phagocyte immunodeficiency. The only described link between Rho-GTPases and muscle concerns the binding and activation of a Serine/Threonine protein kinase homologous to myotonic dystrophy kinase by a small GTP binding protein Rho. It is speculated, however, that given the involvement of RhoB in EGFR trafficking, the vertebrate Rho GTPase can contribute to RTK-controlled myogenic pathways (Bidet, 2003).

Altogether, these data suggest that the RTK signalling involving rho, yan and rac2 might play an important and at least partially conserved role in diversification of cardiac and muscular lineages (Bidet, 2003).

Downstream of identity genes: muscle-type-specific regulation of the fusion process

In all metazoan organisms, the diversification of cell types involves determination of cell fates and subsequent execution of specific differentiation programs. During Drosophila myogenesis, identity genes specify the fates of founder myoblasts, from which derive all individual larval muscles. To understand how cell fate information residing within founders is translated during differentiation, this study focused on three identity genes, eve, lb, and slou, and how they control the size of individual muscles by regulating the number of fusion events. They achieve this by setting expression levels of Muscle protein 20 (Mp20), Paxillin (Pax), and M-spondin (mspo), three genes that regulate actin dynamics and cell adhesion and, as is shown in this study, modulate the fusion process in a muscle-specific manner. Thus, these data show how the identity information implemented by transcription factors is translated via target genes into cell-type-specific programs of differentiation (Bataillé, 2010).

The myoblast fusion is asymmetric and takes place between founder cells (FCs) and fusion competent myoblasts (FCMs). Previous reports originated the idea that FCMs are not 'naive' myoblasts and contribute to the modulation of fusion process. In contrast, the current results support a view that FCs rather than FCMs carry the instructive information and lead to the conclusion that FCMs do not play an active role in setting the number of fusion events. However, because the spatial distribution of FCMs seems to be nonuniform, it is conceivable that the local distribution of FCMs is coordinated with the requirements of FCs to facilitate fusion process (Bataillé, 2010).

The identity genes lb, slou, and eve are required to specify FCs at the origin of five muscles the DA1, DT1, SBM, VA2, and VT1. This study provides evidence that these identity genes are also required for setting the muscle-specific number of fusions and demonstrates how this identity information is executed. After specification step, FCs fuse, between the embryonic stage 12 and 15, with a determined number of FCMs to generate muscles with a specific number of nuclei. During this time period eve, lb, and slou continue to be expressed in subsets of developing muscles and the data show that they are sufficient to establish the muscle-specific fusion programs in DA1, SBM, and VT1 (11, 7, and 4 nuclei, respectively). Furthermore, slou in combination with other factors contributes to two other programs that end up with seven to eight fusion events in muscles DT1 and VA2. To regulate number of fusion events eve, lb, and slou act by modulating expression of genes involved in dynamics of actin cytoskeleton or cell adhesion. Starting from stage 13, they establish a muscle-specific combinatorial code of expression levels of three targets: Mp20, Pax, and mspo. The combination of expression of the targets leads to the muscle-specific control of the number of fusion events. This notion is supported by the fact that each of identity genes is able to impose at ectopic locations the combinatorial realisator code of Mp20, Pax, and mspo expression, and thus, ectopically execute its fusion program. Given that the code of Mp20, Pax, and mspo is not sufficient to explain fusion programs in all muscles, it is hypothesized that other identity gene targets exist that modulate fusion counting (Bataillé, 2010).

The data support a two-step model of myoblast fusion according to which a muscle precursor is formed between stage 12 and 13 by an initial fusion, and then, between stage 13 and 15, fuses with additional myoblasts until the muscle reaches its final size. The fact that Mp20, Pax, and mspo are expressed from stage 13 suggests that the transition point between the two steps depends not only on the timing of FCM migration but also on the activation of limiting factors such as the identity gene targets which modulate the number of additional fusions. Since no nuclear divisions were observed in FCs or in growing myotubes in any of the genetic contexts analyzed, it can confidently be said that the number of nuclei present in each muscle is determined only by the number of fusion events (Bataillé, 2010).

Specification of FCs requires combinatorial code of activities of identity genes. This study shows that the same identity genes play instructive roles in subsequent muscle-type-specific differentiation process. Importantly, the data enlighten the fact that the identity genes are not equivalent and have distinct, context-dependent mode of action. eve, lb, and slou are sufficient to set the fusion programs in DA1, SBM, and VT1 muscles; however, in VA2 and DT1 slou functions in a different way and seems not to have a decisive role in this process. Because the specification of the VA2 and DT1 FCs also involves functions of Poxm, Kr, and ap, it is hypothesized that they act together with slou in setting fusion programs of VA2 and DT1. This raises an important question about hierarchy of identity genes during execution of muscle identity programs and their roles in acquisition of specific properties of muscles such as number of nuclei, attachment points, and innervation (Bataillé, 2010).

The data presented in this study demonstrate that the number of fusion events in developing muscles is regulated by a muscle-specific combinatorial realisator code of identity gene targets. In contrast to the previously identified fusion genes acting in all muscles, the identified identity targets, Mp20, Pax, and mspo, display muscle-type specific expression and modulate fusion in a muscle-type-specific manner proportionally to the level of their expression. The loss and gain of function of each of them lead to subtle fusion phenotypes indicating that the range of fusion events controlled by these three candidates is limited. Indeed, the loss of function of Mp20 results in loss of two nuclei in a subset of muscles, whereas its overexpression induces the recruitment of maximum two FCMs. A similar range of defects in number of fusion events is observed in Pax and mspo mutant embryos indicating that they influence fusion process at the same level (Bataillé, 2010).

Mp20 encodes a cytoskeletal protein displaying restricted expression in adult muscles and sharing sequence homology with the lineage-restricted mouse proteins SM22alpha, SM22beta, and NP25. These proteins contain calponin-like repeats, and, in mammals, interact with F-actin and participate in the organization of the actin cytoskeleton. In Drosophila S2R cells, the RNAi knockdown of Mp20 induces a phenotype of round and nonadherent cells supporting its role in regulation of fusion process (Bataillé, 2010 and references therein).

The second candidate, Pax (DPxn37), is a scaffold protein that recruits structural and signaling molecules to the sites of focal adhesion. Pax has been shown to be involved in the actin cytoskeleton organization, cell adhesion, cell migration, and cell survival. In the developing Drosophila muscles, Pax protein localizes at muscle-tendon junctions suggesting that it may play a role in muscle attachment. The current analyses of Pax mutant embryos do not reveal muscle-tendon adhesion defects but show discrete myoblast fusion phenotypes, which correlate with differential muscle-specific expression of Pax. The role of Pax in modulating fusion is consistent with previously described implications of Pax interacting proteins, including ARF6 in myoblast fusion in both Drosophila and vertebrates, and FAK in vertebrates (Bataillé, 2010 and references therein).

Finally, mspo belongs to the F-Spondins, a conserved family of ECM proteins, which maintain cell-matrix adhesion in multiple tissues. In vertebrates, F-Spondins have context-dependent effects on axon outgrowth and cell migration. As Mp20, Pax, and Mspo are expressed in FC cells and growing myotubes, one possibility is that they modify the spreading and/or motility of FC protrusions required to attract FCMs. Alternatively, by modulating actin cytoskeleton, Mp20, Pax, and Mspo may also influence the stability of adhesion between the growing muscle and the FCM creating permissive conditions or blocking the progression of fusion process (Bataillé, 2010).

The muscle-type-specific regulation of fusion programs by the identity genes and their targets raises an intriguing question of how this regulation is executed from the mechanistic point of view. Because different levels of expression of Mp20, Pax, and mspo correlate with different fusion programs in both wild-type and genetically manipulated embryos, it was thought that by following kinetics of fusion in small and big muscles insights would be gained into how the fusion programs are modulated. It turns out that the rate of fusion is proportional to the size of muscle, meaning the number of fusion events, thus revealing that the identity genes acting via their targets set up the frequency of fusion events. Accordingly, loss and gain of function of identity genes and their targets identified here results in modulations of fusion programs by accelerating or slowing down the fusion rate. This finding provides insights into mechanistic understanding of muscle-type-specific regulation of fusion process and raises an important question about whether this mechanism is broadly conserved (Bataillé, 2010).


Two novel homeobox genes are the mouse (Lbx1) and human (LBX1) homologs of the Drosophila ladybird genes. They are highly related not only within the coding region but also in 5' and 3' untranslated regions. Several amino acid residues inside and around the homeodomain, have been conserved between the mammalian Lbx genes and their Drosophila counterparts. The mouse Lbx1 gene is located on chromosome 19 (region D) and the human LBX1 gene maps to the related q24 region of chromosome 10, known as a breakpoint region in translocations t(7;10) and t(10;14) involved in T-cell leukemias. Thus, LBX1 and the protooncogene HOX11 map to a common chromosomal region, as do their Drosophila counterparts, the ladybird and 93Bal genes. The mouse Lbx1 gene is specifically expressed during embryogenesis. From 10.5 days of gestation, Lbx1 expression is detected in the central nervous system and some developing muscles. In the CNS, Lbx1 transcripts are expressed in the dorsal part of the mantle layer of the spinal cord and hindbrain, up to a sharp boundary within the developing metencephalon. Thus, Lbx1 may be involved in spinal cord and hindbrain differentiation and/or patterning; its restricted expression pattern could depend upon evolutionarily conserved inductive signals involving some mammalian Wnt and Pax genes, as is the case for Drosophila ladybird genes and wingless or gooseberry (Jagla, 1995).

In vertebrates all skeletal muscles of trunk and limbs are derived from condensations of the paraxial mesoderm, that is from the somites. Limb muscle precursor cells migrate during embryogenesis from somites to limb buds, where migration stops and differentiation occurs. lbx1 homeobox genes (related to the Drosophila ladybird genes) have been characterized in chicken and mice. These genes are expressed in migrating limb muscle precursor cells in both species. Analysis of splotch mutant mice shows that lbx1 and c-met are differently affected by the lack of Pax-3 (Drosophila homolog: Paired). Limb buds of splotch (Pax-3 mutant) mice are devoid of lbx1 transcripts, while expression of c-met is still detectable at a low level. The presence of c-met-positive cells in splotch mice entering the limbs indicates that migration of cells from somites to limbs is not entirely dependent on Pax-3. Induction of epithelial to mesenchymal transition of Pax-3-positive cells by SF/HGF is not sufficient to induce ectopic lbx-1 expression at the inter-limb level, while ectopic limb formation is able to activate lbx1 expression. It is postulated that Pax-3 is necessary for lbx1 expression in the lateral tips of somites but additional, yet unknown signals derived from limb buds are needed to initiate lbx1 expression. The role of limb bud-derived signals involved in targeted muscle precursor cell migration, and lbx1 activation was further confirmed by analysis of explanted somite/limb bud co-cultures in collagen gels (Mennerich, 1998).

Myogenic differentiation can be initiated by a limited number of molecules. Overexpression of Lbx1 in vivo and in vitro leads to a strong activation of various muscle markers. Cell proliferation, which is strongly stimulated by Lbx1 and Pax3, is required for Lbx1- or Pax3-dependent myogenic activation. Inhibition of cell proliferation prevents expression of muscle differentiation markers, while the activation of other putative downstream targets of Pax3 and Lbx1 is not affected. These findings imply that a critical function of Pax3 and Lbx1 during muscle cell formation is the enlargement of muscle cell populations. The growth of the muscle precursor cell population may increase the bias for myogenic differentiation and thus enable myogenic cells to respond to environmental cues. These results might provide an explanation of how Pax3 and Lbx1 induce myogenesis by amplification of the myogenic precursor cell pool with an increasing potential for myogenic differentiation, and emphasize the critical role of the size of cell populations biased for differentiation (Mennerich, 2001).

Lbx1 staining resides within the expression domain of the lateral marker Sim1, a basic helix-loop-helix transcription factor, indicating that Lbx1 belongs to the lateral program of the somite. However, Sim1 staining extends throughout the lateral half of the dermomyotome and, at lower levels, encompasses the lateral sclerotome. Similar to Sim1, the putative VEGF receptor Quek1 and the recently cloned divergent homeobox gene Meis2 also occupy a wide lateral territory. Thus, Lbx1 marks a sub-population of lateral somite cells only. Within the lateral somite, expression of Lbx1 co-localizes with elevated signals for the paired and homeobox containing transcription factor Pax3, and with signals for the receptor tyrosine kinase c-Met, both thought to be indicative of prospective hypaxial myoblasts. However, Pax3 expression commences throughout the dorsoventral perimeter of the anterior segmental plate, and continues in the medial and lateral dermomyotome before the up-regulation in the lateral dermomyotomal lip takes place. Likewise, c-Met displays an additional expression domain in the medial dermomyotomal lip. Therefore, while the mechanisms leading to the dorsally restricted expression pattern of Sim1, the lateral up-regulation of Pax3 and the lateral activation of c-Met may be related to those stimulating Lbx1, Lbx1 is the only known marker that is exclusive for the lateral dermomyotomal lip. Unlike Sim1, Pax3 and c-Met, Lbx1 expression is excluded from flank regions, where the lateral dermomyotomal lips remain epithelial, and hypaxial muscle precursors are added to the lateral myotome during demomyotomal elongation. In such flank regions myoblasts begin to express muscle-determining genes as soon as they enter the lateral myotome. In contrast, where Lbx1 is expressed, the lateral dermomyotomal lips de-epithelialise, and activation of muscle determining genes appears delayed. Thus, the expression pattern of Lbx1 along the anteroposterior axis prefigures the different fates of lateral dermomyotomal lips, before they become morphologically evident: the gene labels lateral dermomyotomal lips bound to de-epithelialise and to generate late differentiating hypaxial muscle precursors. Occipitally and at the level of the limbs, the dispersing lateral dermomyotomal lips release migratory muscle precursors that head toward branchial arches and limbs to provide the intrinsic tongue and limb musculature, respectively. During the course of their migration, these cells continue to express Lbx1. In mammals, cervical cells populating the septum transversum to form the muscular diaphragm are also Lbx1 positive. In chick and mouse, Lbx1 is down-regulated when the migrating cells settle at their target sites and initiate expression of the muscle determining genes. Thus, Lbx1 serves as a bona fide marker for muscle precursors capable of long range migration (Dietrich, 1998 and references).

During development of the amniote embryo, the dorsolateral territory of the somite is destined to give rise to the hypaxial skeletal musculature. The lateral mesoderm has been suggested to emit signals required for somite lateralization. In line with these studies, separation of paraxial mesoderm from intermediate and lateral mesoderm prevents expression of Lbx1 and upregulation of Pax3. Similarly, when an additional strip of paraxial mesoderm is inserted parallel to the endogenous one, only the strip in contact with the lateral structures expresses the lateral markers. Lastly, when strips of paraxial mesoderm are grafted on top of the notochord after neural tube removal, the lateral markers are lacking in the graft, while the medial markers are found in their normal dorsoventrally restricted domains. This emphasizes that the formation of the hypaxial musculature belongs to the lateral developmental pathway, since it depends on appositional signaling by the lateral structures (Dietrich, 1998 and references).

To study the mechanisms that lead to the formation of this musculature, Lbx1 was used as a marker for the anatomical structures that produce the signals necessary for the specification of the hypaxial musculature. These signals have been characterized by ablating muscles or transplanting them to ectopic locations in the chick embryo. In addition, BMP4 soaked beads were inserted medial to the somite. The data suggest that lateralizing signals from intermediate and lateral mesoderm have to synergize with dorsalizing signals from the surface ectoderm to induce the formation of the hypaxial musculature. However, the lateralizing function of the lateral mesoderm can only in part be mimicked by BMP4. The following model is proposed for the induction of hypaxial musculature: in the paraxial mesoderm, lateral identities are induced and medial identities are repressed by intermediate and lateral mesoderm, involving BMP4 as a general lateralizing signal, and possibly additional factors involved in the specification of the hypaxial musculature. Within the lateral somite half, dorsal identities are induced by the surface ectoderm, possibly via WNT signalling. The dorsalizing signals may be antagonized by signals released by the underlying aorta or endoderm. However, where the lateralizing and dorsalizing signals synergize, formation of the hypaxial musculature is induced, as monitored by the upregulation of Pax3, and at occipital, cervical and limb levels, by the expression of the novel marker Lbx1 (Dietrich, 1998).

During vertebrate embryogenesis, myogenic precursor cells of limb muscles delaminate from the ventro-lateral edge of the somitic dermomyotome and migrate to the limb buds, where they congregate into dorsal and ventral muscle masses. It has been proposed that the surrounding connective tissue controls muscle pattern formation in limbs. Regulatory molecules such as receptor tyrosine kinases like c-Met and those encoded by homeobox-containing genes, including c-Met, Tbx1, Mox2, Six1 and Six2, Pitx2, Pax3 and Lbx1h (a homolog of Drosophila Ladybird genes), are expressed in migrating limb precursor cells. The role of these genes in the patterning of limb muscles is unknown, although mutation of Pax3 or Met causes disruption of limb muscle development at an initial step, disturbing the epithelial-to-mesenchymal transition of the somitic epithelium. No limb muscle cells form in these mutants, and the early loss of myogenic precursor cells prevents an analysis of later functions of these genes during limb muscle development. Based on quail-chick chimaera studies, it was assumed that a cell-autonomous contribution of myogenic cells to the formation of individual limb muscles is negligible, and that an instructive role of limb mesenchyme is critical in this process. Lbx1h determines migratory routes of muscle precursor cells in a cell-autonomous manner, thereby leading to the formation of distinct limb muscle patterns. Inactivation of Lbx1h, which is specifically expressed in migrating muscle precursor cells, leads to a lack of extensor muscles in forelimbs and an absence of muscles in hindlimbs. The defect is caused by the failure of all muscle precursor cells of hindlimbs and of precursor cells of extensor muscles of forelimbs to migrate to their corresponding muscle anlagen. These results demonstrate that Lbx1h is a key regulator of muscle precursor cell migration and is required for the acquisition of dorsal identities of forelimb muscles (Schafer, 1999).

Described here is the cloning, expression pattern, and genomic organization of Lbx2, a murine homolog of the Drosophila and mammalian ladybird genes. Lbx2 includes a homeodomain motif most closely related to those of Lbx1 and the Drosophila Ladybird proteins. Lbx2 transcripts are first detected at E10.5 when they are located in the gonadal component of the urogenital ridge. Expression of Lbx2 dramatically increases by E11.5 in the urogenital ridges, and in the cranial surface ectoderm. At later stages, Lbx2 transcripts are expressed in the brain and organs derived from the urogenital ridge, including the gonadal tubercle, kidneys, and adrenal glands. From E14.5 to birth, Lbx2 expression is evident in the developing retinal neuroepithelium and the vibrissa (Chen, 1999).

The homeobox gene Lbx1 is expressed in migrating hypaxial muscle precursor cells during development. These precursors delaminate from the lateral edge of the dermomyotome and form distinct streams that migrate over large distances, using characteristic paths. The targets of migration are limbs, septum transversum and the floor of the first branchial arch where the cells form skeletal muscle of limbs and shoulders, diaphragm and hypoglossal cord, respectively. Gene targeting was used to analyse the function of Lbx1 in the mouse. Myogenic precursor cells delaminate from the dermomyotome in Lbx1 mutants, but migrate in an aberrant manner. Most critically affected are migrating cells that move to the limbs. Precursor cells that reach the dorsal limb field are absent. In the ventral limb, precursors are present but distributed in an abnormal manner. As a consequence, at birth some muscles in the forelimbs are completely lacking (extensor muscles) or reduced in size (flexor muscles). Hindlimb muscles are affected strongly, and distal limb muscles are more affected than proximal ones. Other migrating precursor cells heading towards the floor of the first branchial arch move along the appropriate path in Lbx1 mutants. However, these cells migrate less efficiently and reduced numbers of precursors reach their distal target. At birth, the internal lingual muscle is therefore reduced in size. It is suggested that Lbx1 controls the expression of genes that are essential for the recognition or interpretation of cues that guide migrating muscle precursors and maintain their migratory potential (Brohmann, 2000).

The somitic mesoderm gives rise to multiple tissues in the developing embryo including bone, connective tissue and muscle. Somites form as an epithelial ball of cells that later segregate into sclerotome and dermomyotome in response to patterning signals that arise from adjacent tissues. Cells in the ventral half of the nascent somite undergo a transition from epithelium to mesenchyme, forming the sclerotome. Subsequently, sclerotome differentiates further to give rise to the axial skeleton. Cells in the dorsolateral half of the somite retain their epithelial morphology, forming the dermomyotome, which contains precursors for both the dermis and for skeletal muscles. Dermomyotome derived muscle precursors not only generate the epaxial muscles that attach to the vertebral column, but also the hypaxial musculature of the limb, tongue, diaphragm and ventral body wall. Whereas the epaxial muscles arise from cells in the medial dermomyotome, hypaxial muscles are derived from precursors in the lateral half of the dermomyotome. The precursors for hypaxial muscles exhibit markedly different morphogenetic behaviours at different axial levels of the embryo. Cells in the ventral dermomyotome at limb and cervical levels delaminate and undergo long range migration to form diaphragm, tongue muscles and appendicular muscles. In contrast, hypaxial muscle precursors at interlimb levels do not migrate. Instead, they retain their epithelial morphology, forming a bud that extends ventrally toward the midline to give rise to ventral body wall muscles (Gross, 2000).

Previous studies have shown that Pax3, together with the c-Met receptor tyrosine kinase and its ligand Scatter Factor (SF) are necessary for the migration of hypaxial muscle precursors in mice. Lbx1 and Pax3 are co-expressed in all migrating hypaxial muscle precursors, raising the possibility that Lbx1 regulates their migration. To examine the function of Lbx1 in muscle development, the Lbx1 gene was inactivated by homologous recombination. Mice lacking Lbx1 exhibit an extensive loss of limb muscles, although some forelimb and hindlimb muscles are still present. The pattern of muscle loss suggests that Lbx1 is not required for the specification of particular limb muscles, and the muscle defects that occur in Lbx1 mutant mice can be solely attributed to changes in muscle precursor migration. c-Met is expressed in Lbx1 mutant mice and limb muscle precursors delaminate from the ventral dermomyotome but fail to migrate laterally into the limb. Muscle precursors still migrate ventrally and give rise to tongue, diaphragm and some limb muscles, demonstrating that Lbx1 is necessary for the lateral, but not ventral, migration of hypaxial muscle precursors. These results suggest that Lbx1 regulates responsiveness to a lateral migration signal that emanates from the developing limb (Gross, 2000).

Association and relay neurons that are generated in the dorsal spinal cord play essential roles in transducing somatosensory information. During development, these two major neuronal classes are delineated by the expression of the homeodomain transcription factor Lbx1. Lbx1 is expressed in and required for the correct specification of three early dorsal interneuron populations and late-born neurons that form the substantia gelatinosa. In mice lacking Lbx1, cells types that arise in the ventral alar plate acquire more dorsal identities. This results in the loss of dorsal horn association interneurons, excess production of commissural neurons, and disrupted sensory afferent innervation of the dorsal horn. Therefore, Lbx1 plays a critical role in the development of sensory pathways in the spinal cord that relay pain and touch (Gross, 2002).

It is generally held that vertebrate muscle precursors depend totally on environmental cues for their development. Instead, it is shown that somites are predisposed toward a particular myogenic program. This predisposition depends on the somite's axial identity: when flank somites are transformed into limb-level somites, either by shifting somitic boundaries with FGF8 or by overexpressing posterior Hox genes, they readily activate the program typical for migratory limb muscle precursors. The intrinsic control over myogenic programs can only be overridden by FGF4 signals provided by the apical ectodermal ridge of a developing limb (Alvares, 2003).

Since the competence to start a particular program of hypaxial myogenesis is linked to the position of the somites along the anteroposterior body axis, it was reasoned that this competence may be a consequence of the somite's axial identity. Indeed, when the ability was exploited of FGF8 to move the somitic boundaries, thereby changing the axial identity of somites in the flank into the identity of hindlimb-level somites, these somites gained the ability to produce Lbx1-expressing migratory muscle precursors (MMPs). This suggests that the positional values intrinsic to the somites determine their competence to generate either migratory or nonmigratory hypaxial muscle precursors (Alvares, 2003).

It is established that the axial identity of somites is controlled by the overlapping expression of Hox/HOM genes. In particular, a code of Hox gene expression is conserved for crucial anatomical landmarks such as the neck-thorax transition. Significantly, conserved Hox gene expression boundaries also demarcate the transition of limb-flank somites. Moreover, Hox gene expression patterns are maintained when somites are heterotopically grafted, as is the ability to express the MMP marker Lbx1. It was therefore reasoned that Hox genes may control the somitic competence to initiate a particular myogenic program. To test this possibility, expression constructs were engineered for HoxD9 and HoxA10, both normally present in hindlimb-level somites but not in the flank. Upon electroporation of either of the constructs into flank somites, these somites readily expressed Lbx1, while control constructs were unable to evoke expression of the Lbx1 gene. Thus, Lbx1 expression-directly or indirectly-is under the control of Hox genes (Alvares, 2003).

Evolving enhancer-promoter interactions within the tinman complex of the flour beetle, Tribolium castaneum

Modifications of cis-regulatory DNAs, particularly enhancers, underlie changes in gene expression during animal evolution. This study presents evidence for a distinct mechanism of regulatory evolution, whereby a novel pattern of gene expression arises from altered gene targeting of a conserved enhancer. The tinman gene complex (Tin-C) controls the patterning of dorsal mesodermal tissues, including the dorsal vessel or heart in Drosophila. In Drosophila, all members of the Tin-C are involved in muscle cell differentiation, and many of the mesodermal patterning functions of Tin-C genes are conserved between flies, annelids and vertebrates. For example, the founding member, tinman (also known as NK4), is expressed in the cardiac mesoderm of all three major animal groups. Moreover, bagpipe (NK3) is involved in patterning both fly and vertebrate visceral mesoderm, whereas ladybird/Lbx, slouch/Nk1, C15/Txl and Msh (Dr)/Msx regulate the patterning of somatic muscle precursors in both flies and annelids. Despite broad conservation of Tin-C gene expression patterns in the flour beetle (Tribolium castaneum), the honeybee (Apis mellifera) and the fruit fly (Drosophila melanogaster), the expression of a key pericardial determinant, ladybird, is absent from the dorsal mesoderm of Tribolium embryos. Evidence is presented that this loss in expression is replaced by expression of C15, the neighboring gene in the complex. This switch in expression from ladybird to C15 appears to arise from an inversion within the tinman complex, which redirects a conserved ladybird 3' enhancer to regulate C15. In Drosophila, this enhancer fails to activate C15 expression owing to the activity of an insulator at the intervening ladybird early promoter. By contrast, a chromosomal inversion allows the cardiac enhancer to bypass the ladybird insulator in Tribolium. Given the high frequency of genome rearrangements in insects, it is possible that such enhancer switching might be widely used in the diversification of the arthropods (Cande, 2009).

The homeodomain protein Ladybird late regulates synthesis of milk proteins during pregnancy in the Tsetse fly (Glossina morsitans)

Regulation of tissue and development specific gene expression patterns underlies the functional specialization of organs in multi-cellular organisms. In the viviparous tsetse fly (Glossina), the female accessory gland is specialized to generate nutrients in the form of a milk-like secretion to support growth of intrauterine larva. Multiple milk protein genes are expressed specifically in the female accessory gland and are tightly linked with larval development. Disruption of milk protein synthesis deprives developing larvae of nutrients and results in extended larval development and/or in abortion. The ability to cause such a disruption could be utilized as a tsetse control strategy. This study identified and delineated the regulatory sequence of a major milk protein gene (milk gland protein 1:mgp1) by utilizing a combination of molecular techniques in tsetse, Drosophila transgenics, transcriptomics and in silico sequence analyses. The function of this promoter is conserved between tsetse and Drosophila. In transgenic Drosophila the mgp1 promoter directs reporter gene expression in a tissue and stage specific manner orthologous to that of Glossina. Analysis of the minimal required regulatory region of mgp1, and the regulatory regions of other Glossina milk proteins identified putative homeodomain protein binding sites as the sole common feature. Annotation and expression analysis of Glossina homeodomain proteins identified ladybird late (lbl) as being accessory gland/fat body specific and differentially expressed between lactating/non-lactating flies. Knockdown of lbl in tsetse resulted in a significant reduction in transcript abundance of multiple milk protein genes and in a significant loss of fecundity. The role of Lbl in adult reproductive physiology is previously unknown. These results suggest that Lbl is part of a conserved reproductive regulatory system that could have implications beyond tsetse to other vector insects such as mosquitoes. This system is critical for tsetse fecundity and provides a potential target for development of a reproductive inhibitor (Attardo, 2014).


Search PubMed for articles about Drosophila ladybird early and ladybird late

Aigouy, B., et al. (2004). Time-lapse and cell ablation reveal the role of cell interactions in fly glia migration and proliferation. Development 131: 5127-5138. PubMed Citation: 15459105

Alvares, L. E., et al. (2003). Intrinsic, Hox-dependent cues determine the fate of skeletal muscle precursors. Dev. Cell 5: 379-390. 12967558

Attardo, G. M., Benoit, J. B., Michalkova, V., Patrick, K. R., Krause, T. B. and Aksoy, S. (2014). The homeodomain protein Ladybird late regulates synthesis of milk proteins during pregnancy in the Tsetse fly (Glossina morsitans). PLoS Negl Trop Dis 8: e2645. PubMed ID: 24763082

Bataillé, L., et al. (2010). Downstream of identity genes: muscle-type-specific regulation of the fusion process. Dev. Cell 19(2): 317-28. PubMed Citation: 20708593

Bidet, Y., et al. (2003). Modifiers of muscle and heart cell fate specification identified by gain-of-function screen in Drosophila. Mech. Dev. 120: 991-1007. 14550529

Bokor, P. and DiNardo, S. (1996). The roles of hedgehog, wingless and lines in patterning the dorsal epidermis in Drosophila. Development 122: 1083-1092. PubMed Citation: 8620835

Brohmann, H., Jagla, K. and Birchmeier, C. (2000). The role of Lbx1 in migration of muscle precursor cells. Development 127: 437-445. PubMed Citation: 10603359

Cande, J. D., Chopra, V. S. and Levine, M. (2009). Evolving enhancer-promoter interactions within the tinman complex of the flour beetle, Tribolium castaneum. Development. 136(18): 3153-60. PubMed Citation: 19700619

Chen, F., Liu, K. C. and Epstein, J. A. (1999). Lbx2, a novel murine homeobox gene related to the Drosophila ladybird genes is expressed in the developing urogenital system, eye and brain. Mec. Dev. 84 (1-2):181-184. PubMed Citation: 10473138

De Graeve, F., et al. (2004). The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells. Dev. Biol. 270: 122-134. PubMed Citation: 15136145

Dietrich, S., et al. (1998). Specification of the hypaxial musculature. Development 125(12): 2235-2249. PubMed Citation: 9584123

Figeac, N., Jagla, T., Aradhya, R., Da Ponte, J. P. and Jagla, K. (2010). Drosophila adult muscle precursors form a network of interconnected cells and are specified by the rhomboid-triggered EGF pathway. Development 137(12): 1965-73. PubMed Citation: 20463031

Gross, M. K., et al. (2000). Lbx1 is required for muscle precursor migration along a lateral pathway into the limb. Development 127: 413-424. PubMed Citation: 10603357

Gross, M. K., Dottori, M. and Goulding, M. (2002). Lbx1 specifies somatosensory association interneurons in the dorsal spinal cord. Neuron 34: 535-549. 12062038

Hakeda-Suzuki, S., et al. (2002). Rac function and regulation during Drosophila development. Nature 416: 438-442. 1191963

Jagla, K., et al. (1993). A novel homeobox nkch4 gene from the Drosophila 93D/E region. Gene 127: 165-171. PubMed Citation: 8099053

Jagla, K., et al. (1994). A distinct class of homeodomain proteins is encoded by two sequentially expressed Drosophila genes from the 93D/E cluster. Nucleic Acids Res 22: 1202-7. PubMed Citation: 7909370

Jagla, K., et al. (1995). Mouse Lbx1 and human LBX1 define a novel mammalian homeobox gene family related to the Drosophila lady bird genes. Mech. Dev. 53: 345-356. PubMed Citation: 8645601

Jagla, K., et al. (1997a). ladybird, a tandem of homeobox genes that maintain late wingless expression in terminal and dorsal epidermis of the Drosophila embryo. Development 124: 91-100. PubMed Citation: 9006070

Jagla, K., et al. (1997b). ladybird, a new component of the cardiogenic pathway in Drosophila required for diversification of heart precursors. Development 124: 3471-3479. PubMed Citation: 9342040

Jagla, T., et al. (1998). ladybird determines cell fate decisions during diversification of Drosophila somatic muscles. Development 125(18): 3699-3708. PubMed Citation: 9716535

Jagla, T., et al. (2002). Cross-repressive interactions of identity genes are essential for proper specification of cardiac and muscular fates in Drosophila. Development 129: 1037-1047. 11861486

Junion, G., Jagla, T., Duplant, S., Tapin, R., Da Ponte, J.P. and Jagla, K. (2005). Mapping Dmef2-binding regulatory modules by using a ChIP-enriched in silico targets approach. Proc. Natl. Acad. Sci. 102: 18479-18484. PubMed citation: 16339902

Junion, G., Bataille, L., Jagla, T., Da Ponte, J. P., Tapin, R. and Jagla, K. (2007). Genome-wide view of cell fate specification: ladybird acts at multiple levels during diversification of muscle and heart precursors. Genes Dev. 21(23): 3163-80. PubMed citation: 18056427

Krejci, A., et al. (2009). Direct response to Notch activation: signaling crosstalk and incoherent logic. Sci. Signal. 2: ra1. PubMed Citation: 19176515

Li, X. and Noll, M. (1993). Role of the gooseberry gene in Drosophila embryos: maintenance of wingless expression by a wingless--gooseberry autoregulatory loop. EMBO J. 12: 4499-509. PubMed Citation: 8223460

Liu, J., Qian, L., Han, Z., Wu, X. and Bodmer, R. (2008). Spatial specificity of mesodermal even-skipped expression relies on multiple repressor sites. Dev. Biol. 313(2): 876-86. PubMed Citation: 18067885

Mennerich, D., Schafer, K. and Braun, T. (1998). Pax-3 is necessary but not sufficient for lbx1 expression in myogenic precursor cells of the limb. Mech. Dev. 73(2): 147-158. PubMed Citation: 9622616

Mennerich, D. and Braun, T. (2001). Activation of myogenesis by the homeobox gene Lbx1 requires cell proliferation. EMBO J. 20: 7174-7183. 11742994

Sandmann, T., et al. (2006). A temporal map of transcription factor activity: Mef2 directly regulates target genes at all stages of muscle development. Dev. Cell 10(6): 797-807. PubMed citation: 16740481

Schafer, K. and Braun, T. (1999). Early specification of limb muscle precursor cells by the homeobox gene Lbx1h. Nat. Genet. 23(2): 213-6. PubMed Citation: 9622616

Schaub, C., Nagaso, H., Jin, H. and Frasch, M. (2012). Org-1, the Drosophila ortholog of Tbx1, is a direct activator of known identity genes during muscle specification. Development 139: 1001-1012. PubMed ID: 22318630

Sepp, K. J., Schulte, J. and Auld, V. J. (2000). Developmental dynamics of peripheral glia in Drosophila melanogaster. Glia 30: 122-133. PubMed Citation: 10719354

Urbach, R. and Technau, G. M. (2003). Molecular markers for identified neuroblasts in the developing brain of Drosophila. Development 130: 3621-3637. 12835380

Vasyutina, E., Stebler, J., Brand-Saberi, B., Schulz, S., Raz, E. and Birchmeier, C. (2005). CXCR4 and Gab1 cooperate to control the development of migrating muscle progenitor cells. Genes Dev. 19: 2187-2198. PubMed citation: 16166380

von Hilchen, C. M., et al. (2008). Identity, origin, and migration of peripheral glial cells in the Drosophila embryo. Mech. Dev. 125: 337-352. PubMed Citation: 18077143

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date revised: 25 March 2013

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