ladybird early and ladybird late

DEVELOPMENTAL BIOLOGY

Embryonic

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 gliaí expresses filopodia-like structures whereas the ëfollowerí 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 'leadingí 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).

Effects of Mutation or Deletion

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, msh^Delta 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 msh^Delta 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).

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

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

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.

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

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

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.

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.

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

date revised: 15 September 2008 

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

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