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).