ladybird early and ladybird late
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).
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).
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).
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).
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ladybird early and ladybird late:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 20 December 2010
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