twist is initially expressed at stage 5 at the beginning of cellularization in the broad mesodermal anlagen prior to gastrulation. Expression extends around both anterior and posterior poles to label some dorsal cells, even in cells described as ectodermal and endodermal. No label is found in pole cells. As gastrulation [Images] proceeds, cells that have invaginated appear to be less intensely stained than those of the anterior pole that have yet to invaginate. Later, only two mesectodermal cells at the ventral midline appear labelled at the surface of the embryo. As the cell plate sinks to form the amnioproctodeal invagination (late stage 7), no more Twist protein is detected in the posterior midgut rudiment, but the anterior midgut rudiment is labelled with anti-Twist antibodies (Thisse, 1988).

Differential activation of the Toll receptor leads to the formation of a broad Dorsal nuclear gradient that specifies at least three patterning thresholds of gene activity along the dorsoventral axis of precellular embryos. The activities of the Pelle kinase and Twist basic helix-loop-helix (bHLH) transcription factor in transducing Toll signaling have been investigated. Pelle functions downstream of Toll to release Dorsal from the Cactus inhibitor. Twist is an immediate-early gene that is activated upon entry of Dorsal into nuclei. Transgenes misexpressing Pelle and Twist were introduced into different mutant backgrounds and the patterning activities were visualized using various target genes that respond to different thresholds of Toll-Dorsal signaling. These studies suggest that an anteroposterior gradient of Pelle kinase activity is sufficient to generate all known Toll-Dorsal patterning thresholds and that Twist can function as a gradient morphogen to establish at least two distinct dorsoventral patterning thresholds. How the Dorsal gradient system can be modified during metazoan evolution is discussed and it is concluded that Dorsal-Twist interactions are distinct from the interplay between Bicoid and Hunchback, both of which pattern the anteroposterior axis (Stathopoulos, 2002).

The snail, sim, vnd and sog expression patterns represent four different Toll-Dorsal signaling thresholds. snail is activated only by peak levels of the Dorsal gradient; sim and vnd are activated by intermediate levels, and sog is activated by the lowest levels of the gradient. These expression patterns were visualized in mutant and transgenic embryos via in situ hybridization using digoxigenin-labeled antisense RNA probes (Stathopoulos, 2002).

Dorsal target genes are essentially silent in mutant embryos that lack an endogenous dorsoventral Dorsal nuclear gradient. Mutant embryos were collected from females that are homozygous for a null mutation in the gastrulation defective (gd) gene, which blocks the processing of the Spätzle ligand and the activation of the Toll receptor. These mutants permit the analysis of ectopic, anteroposterior Dorsal and Twist gradients in 'apolar' embryos that lack dorsoventral polarity. snail, vnd, and sog are sequentially expressed along the anteroposterior axis of mutant embryos that contain a constitutively activated form of the Toll receptor (Toll10b) misexpressed at the anterior pole using the bicoid (bcd) promoter and 3' UTR. These expression patterns depend on an ectopic anteroposterior Dorsal nuclear gradient. The repression of the vnd and sog patterns at the anterior pole is probably mediated by Snail, which normally excludes expression of these genes in the ventral mesoderm of wild-type embryos (Stathopoulos, 2002).

The activated Pelle-Tor4021 kinase also directs sequential anteroposterior patterns of snail, vnd, and sog expression in gd/gd mutant embryos. As in the case of Toll10b, the activated Pelle kinase was misexpressed at the pole using the bcd 3' UTR. The snail, vnd and sog expression patterns are similar to those obtained with the Toll10b transgene. The vnd and sog expression patterns are probably repressed at the anterior pole by Snail. These results suggest that the levels of Pelle kinase activity are sufficient to determine different Dorsal transcription thresholds (Stathopoulos, 2002).

Similar experiments were carried out with a Pelle-Tor fusion gene that contains the Tor signal peptide, extracellular domain and transmembrane peptide, but lacks the amino acid substitution (Y327C) in the Tor4021 protein that induces dimerization. The Pelle-Tor fusion protein fails to induce snail expression, but succeeds in activating vnd and sog (Stathopoulos, 2002).

There are several similarities between Dorsal and Bicoid (the major determinant of anteroposterior patterning), even though the two morphogens are unrelated. Both gradients activate regulatory genes that are essential for patterning the early embryo. Bicoid activates the zinc finger gene Hunchback, while Dorsal activates the bHLH gene Twist. Loss of the normal Bicoid gradient can be largely compensated by an anteroposterior Hunchback gradient. Hunchback restores posterior head segments and the thoracic segments lost in bicoid/bicoid mutant embryos. The possibility that Twist plays a similar role in dorsoventral patterning was investigated (Stathopoulos, 2002).

Dorsoventral patterning genes exhibit abnormal patterns of expression in twist/twist mutant embryos. For example, snail expression is reduced, and the residual snail pattern exhibits periodicity along the anteroposterior axis. The activities of the constitutively activated Toll10b receptor are significantly impaired in twist/twist mutant embryos. This experiment involved the use of an hsp83-Toll10b-3' bcd UTR transgene, which induces a broad anteroposterior Dorsal nuclear gradient. In an otherwise normal genetic background, the Toll10b transgene leads to the ectopic activation of snail expression in the presumptive head and anterior thorax. However, in twist/twist embryos there is reduced expression of both the endogenous snail expression pattern, and the ectopic pattern in anterior regions. Thus, even abnormally high levels of Toll signaling cannot compensate for the loss of Twist. The gap in snail expression seen at the boundary between the ectopic and endogenous patterns may be due to an unknown repressor that is regulated by low levels of the Dorsal gradient. This gap is more pronounced in twist/twist mutants, suggesting that Twist is not required for the activation of the putative snail repressor (Stathopoulos, 2002).

The preceding results suggest that Twist is necessary for specifying Dorsal gradient thresholds. To determine whether Twist might be sufficient, a twist-bcd transgene was introduced into a number of genetic backgrounds. In wild-type embryos, the twist-bcd transgene induces ectopic activation of the sim gene in anterior regions. This activation is more easily visualized in snail/snail mutant embryos because Snail represses sim expression in the ventral mesoderm. There is a delay in the onset of sim expression in snail/snail embryos, and staining is sporadic in ventral regions where the gene is normally repressed. However, the twist-bcd transgene, when introduced into snail/snail mutant embryos, induces strong expression of sim in anterior regions, although staining is largely restricted to ventral regions where there are high levels of the Dorsal gradient (Stathopoulos, 2002).

In addition, females containing the twist-bcd transgene exhibit a daughterless phenotype, since only male progeny are observed. Sex-lethal expression was examined in embryos obtained from these females. Sex-lethal is required for sex determination in females, and this gene is expressed in female but not male embryos. Sex-lethal expression is repressed in the anterior of embryos obtained from females containing the twist-bcd transgene. This result suggests that Twist may form heterodimeric complexes with Daughterless/Scute, two bHLH proteins required for Sex-lethal expression, thereby titrating the levels of these proteins and making them unable to activate Sex-lethal. The ability of Twist to form homodimeric as well as heterodimeric complexes with other bHLH proteins present in the early embryo may regulate its own dorsoventral patterning activities as well (Stathopoulos, 2002).

In order to determine whether Twist patterning activities require a Dorsal gradient, the twist-bcd transgene was introduced into mutant embryos derived from Tollrm9/Tollrm10 transheterozygous females. This mutant Toll receptor is defective and partially active in a Spätzle-independent fashion. Consequently, mutant embryos contain uniform, low levels of the Dorsal protein in both dorsal and ventral nuclei. The twist-bcd transgene causes a substantial reorganization in the patterning of mutant embryos (Stathopoulos, 2002).

The low, uniform levels of Dorsal present in Tollrm9/Tollrm10 mutant embryos are insufficient to activate snail in precellular or newly cellularized embryos. The twist-bcd transgene activates snail throughout anterior regions of precellular embryos; however, this staining pattern is refined into a broad anterior stripe after cellularization. This ectopic snail expression pattern exhibits a sharp posterior border, suggesting that the Twist gradient is sufficient for the on/off regulation of snail in the absence of a Dorsal gradient (Stathopoulos, 2002).

sog is normally activated throughout the neurogenic ectoderm by the lowest levels of the Dorsal gradient. The low levels of Dorsal present in Tollrm9/Tollrm10 mutant embryos are sufficient to activate sog everywhere except the extreme termini. The twist-bcd transgene leads to the loss of sog expression in anterior regions, probably because of repression by Snail. Snail also appears to repress vnd and sog expression in anterior regions of transgenic embryos that contain the Toll10b or Pelle-Tor4021 transgenes (Stathopoulos, 2002).

The low levels of Dorsal present in Tollrm9/Tollrm10 mutant embryos are insufficient to activate sim, although there is occasional staining in the posterior pole. The twist-bcd transgene leads to the efficient activation of sim in anterior regions. Staining appears to be restricted to those regions where snail expression is lost. These results suggest that a Twist gradient is sufficient to generate multiple dorsoventral patterning thresholds (sim and snail) in the presence of low, uniform levels of Dorsal (Stathopoulos, 2002).

The twist-bcd transgene was introduced into mutant embryos that completely lack Dorsal. Without the transgene these mutants do not express twist, snail, sim, vnd or sog. Introduction of the twist-bcd transgene causes intense expression of twist in the anterior 40% of the embryo. This broad Twist gradient fails to activate snail, but succeeds in inducing weak expression of sim and somewhat stronger staining of vnd at the anterior pole. The activation of vnd in mutant embryos is comparable with the expression seen in wild-type and Tollrm9/Tollrm10 embryos. However, in both wild-type and mutant embryos the vnd pattern is transient, and lost after the completion of cellularization. These results indicate that Twist can activate dorsoventral patterning genes in the absence of Dorsal (Stathopoulos, 2002).

An anteroposterior Twist gradient generates at least two thresholds of gene activity in mutant embryos that contain decreased levels of Dorsal. High levels of Twist activate sim at the anterior pole, whereas lower levels are sufficient to induce the expression of snail in more posterior regions of embryos containing low, uniform levels of the Dorsal protein. These results demonstrate that twist gene activity is not dedicated to mesoderm formation. Instead, Twist supports expression of two regulatory genes, sim and vnd, which pattern ventral regions of the neurogenic ectoderm. The twist-bcd transgene was shown to induce weak expression of both genes even in mutant embryos that completely lack Dorsal (Stathopoulos, 2002).

The Twist gradient exhibits some unexpected activities in Tollrm9/Tollrm10 mutant embryos. In particular, both high and intermediate levels of the gradient initially activate snail in a broad domain of precellular embryos. However, during cellularization snail is repressed at the anterior pole where there are high levels of Twist. Thus, it would appear that high levels of Dorsal + high levels of Twist activate snail expression (e.g. ventral region of wild-type embryos), while low levels of Dorsal + high levels of Twist repress expression (e.g. anterior region of Tollrm9/Tollrm10 embryos containing the twist-bcd transgene). The ratio of Dorsal to Twist might keep Twist patterning activity under control, such that high levels of Twist specify mesodermal targets (i.e., snail); lower levels specify neurogenic targets (i.e., sim and vnd) and the expression of genes such as Sxl remain unaffected (Stathopoulos, 2002).

Alternatively, Twist may differentially interact with a number of bHLH proteins that are present in the early embryo to affect its patterning activity. For example, Twist is thought to form a heteromeric activation complex with other bHLH proteins including the ubiquitous, maternal bHLH protein Daughterless. The loss of Sex-lethal expression at the anterior pole of twist-bcd embryos may result from a failure of Twist-Daughterless heterodimers to activate this gene. It has been demonstrated that Twist-Daughterless heterodimers possess a different patterning activity from Twist-Twist homodimers. It is possible that Twist-Daughterless heterodimers formed in twist-bcd embryos actively repress Sex-lethal expression. Alternatively, Twist might titrate Daughterless levels by forming a sterile heterodimeric complex that is not able to activate Sex-lethal. However, Sex-lethal is normally expressed in ventral regions of wild-type embryos that contain both Twist and the ubiquitous Daughterless, therefore regulation of bHLH patterning activities must be more complex. In relation to dorsovental patterning, Twist-Twist complexes might be favored in anterior regions of embryos that express the twist-bcd transgene, while Twist-bHLH complexes are formed away from the anterior pole where expression of the transgene is decreased. These complexes might fail to activate snail, or even actively repress transcription, since it has been demonstrated that several bHLH proteins can function as repressors of transcription. Regardless of mechanism, the Twist gradient inverts the order of the sequentially expressed snail and sim genes, when compared with the patterns obtained with the normal Dorsal (and Twist) gradient. In the presence of low, uniform levels of Dorsal, high concentrations of Twist specify mesectoderm (sim expression), while lower levels specify mesoderm (snail expression). These observations raise the possibility of evolutionary plasticity in the use of Twist in tissue specification (Stathopoulos, 2002).

snail is activated by Dorsal and Twist in cellularizing embryos. The sharp lateral limits of the snail expression pattern establish the boundary between the presumptive mesoderm and neurogenic ectoderm. It has been suggested that the crude Dorsal gradient triggers a somewhat steeper Twist gradient, and the two activators function synergistically within the snail 5' cis-regulatory DNA to establish the sharp, on/off expression pattern. Dorsal-Twist transcription synergy may provide a means for 'multiplying' the Dorsal and Twist gradients to produce the sharp snail pattern. This model suggests that both proteins must be present in a gradient to generate the sharp snail border. However, while both Dorsal and Twist are required for the activation of snail, a Twist gradient is sufficient to generate a reasonably sharp pattern of snail expression in embryos containing low, uniform levels of Dorsal. It is proposed that cooperative binding of Twist might act as a switch to regulate snail expression when the snail 5' cis-regulatory region is rendered responsive by the Dorsal activator (whether present at uniform levels or in a gradient). Therefore, the ratio of Dorsal to Twist may be important to produce the sharp lateral limits of snail expression (Stathopoulos, 2002).

This study raises some questions about the role of operator binding affinities in the specification of different transcription thresholds. The Dorsal binding sites present in the snail 5' regulatory region bind with lower affinity than the sites present in the rho lateral stripe enhancer (NEE). The analysis of a number of synthetic enhancers prompted the proposal that the activation of Dorsal target genes in the ventral mesoderm versus lateral neurogenic ectoderm depends on the affinity of Dorsal operator sites. However, the demonstration that the twist-bcd transgene can activate snail expression in Tollrm9/Tollrm10 embryos suggests that occupancy of the distal Dorsal-binding sites may not be crucial for determining whether the gene is on or off. It is conceivable that Dorsal occupies one or more sites in mutant embryos, but is unable to trigger expression in the absence of Twist. In general, 'promoter context' (combinations of regulatory factors) might be more critical for defining Dorsal transcription thresholds than the affinities of Dorsal operator sites (Stathopoulos, 2002).

The relationship between Dorsal and Twist appears distinct from the interplay between Bicoid and Hunchback. It has been suggested that the Bicoid gradient is a relatively recent evolutionary innovation for patterning the anterior-posterior axis of long germband insects. By contrast, Hunchback is ancient and is used in the patterning of short germband insects such as grasshoppers. Most of the patterning activity controlled by the Bicoid gradient appears to be mediated by Hunchback, which is a direct target of the Bicoid activator. Only the patterning of the anteriormost head structures requires Bicoid and cannot be compensated by high levels of Hunchback. Thus, the patterning activity of the Bicoid gradient can be explained by the regulation of several target genes, which is consistent with its recent evolution. By contrast, either Dorsal or Twist protein alone produces only a small subset of the five or six dorsoventral patterning thresholds generated by the concerted action of both proteins. It is concluded that Dorsal and Twist work in a highly interdependent and synergistic fashion to regulate a large number of target genes involved in patterning the dorsoventral axis (Stathopoulos, 2002).

Twist acts as a switch in the development of muscle. High levels of Twist are required for somatic myogenesis; this blocks formation of other mesodermal derivatives such as visceral mesoderm and heart. Expression of twist in the ectoderm drives these cells into myogenesis. Thus, after an initial role in gastrulation, twist regulates mesodermal differentiation and propels a specific subset of mesodermal cells into somatic myogenesis (Baylies, 1996).

twist expression in the embryonic mesoderm declines during germ band retraction to leave a residual population of twist-expressing cells in the late embryo (Persistent Twist cells). In the abdomen, the pattern of twist expression is a simple one: a single cell ventrally, pairs of cells laterally and three cells dorsally in each hemisegment. In the thorax, there are patches of cells associated with the imaginal discs and there are additional clusters in A8 and A9 (Bate, 1991).

Establishment of dorsoventral polarity within the Drosophila embryo requires extraembryonic positional information generated during oogenesis. The spatial restriction of Toll activation requires earlier signaling events that occur during oogeneisis to determine the dorsoventral pattern of the follicular epithelium. Toll acts through Dorsal to activate twist. The genes windbeutel, pipe, and nudel are required within the somatic follicle cells of the ovary for production of this spatial cue. Using a novel follicle cell marker system, the effect of mutant follicle cell clones has been evaluated on the embryonic dorsoventral pattern. No spatially localized requirement for nudel activity is found. These results imply that wild-type ndl activity in subpopulations of follicle cells, regardless of their position, must be sufficient to mediate its dorsoventral patterning function. In contrast, windbeutel and pipe are required only within a restricted ventral region of the follicular epithelium. This ventral region can determine lateral embryonic cell fates nonautonomously, indicating that spatial information originating ventrally is subsequently refined, perhaps via diffusion, to yield the gradient of positional information that determines the embryonic dorsoventral pattern (Nilson, 1998).

A determination was made of the minimum clone width along the dorsoventral axis that leads to local elimination of the entire twist stripe. This allowed the definition of a region along the ventral side of the follicular epithelium where wind activity in the follicle cells is required in order to induce embryonic twist expression. Loss of wind acivity in a 1-2 cell wide region does not eliminate the entire twist stripe in the corresponding region of the embryo. However, when wind activity is lost from a region approximately 4-6 cells wide overlying the ventral midline region, local loss of the entire twist expression stripe results. This analysis leads to an initial estimate that wind activity is required in a ventral region approxiately 4-6 follicle cells wide along the dorsoventral axis to induce a normal pattern of twist expression in the embryo. Although these results demonstrate a localized requirement for wind and pipe, they do not imply that either the expression of these genes or their activity is spatially restricted. Rather, these results identify a functionally distinct region within the ventral follicular epithelium that is required to establish the embryonic Dorsal gradient. A likely interpretation is that this region defines a spatially localized process that ultimately generates active Toll ligand. Interestingly, this area corresponds well to the width and postion of the twist expression domain (Nilson, 1998).

The neural receptor-like protein tyrosine phosphatases (RPTPs) DPTP69D, DPTP99A and DLAR are involved in motor axon guidance in the Drosophila embryo. The requirements for these three phosphatases have been analyzed in growth cone guidance decisions along the intersegmental nerve (ISN) and segmental nerve b (SNb) motor pathways. Approximately 40 motoneurons innervate 30 identified muscle fibers in each abdominal hemisegment of the embryo. Axons from these neurons exit the CNS via the segmental nerve (SN) and ISN roots and then extend within five nerve pathways. SNa and SNc emerge from the SN root, while SNb, SNd and the ISN arise from the ISN root.

The growth cone of the aCC neuron pioneers the ISN pathway, exiting the CNS during stage 13 and then growing dorsally past the ventrolateral muscles (VLMs) and lateral muscle 4. During stage 15, ISN growth cones contact one of the three dorsal "persistent Twist" (PT cells). These PT cells do not give rise to embryonic muscles but serve as founder cells for adult muscles (Bate, 1991). During stage 15, ISN growth cones contact one of the PT cells, PT2, and also interact with the peripheral nervous system and muscle fibers. Another PT cell, PT3, is initially located posterior and lateral to PT2, but does not appear to be contacted by the pioneer axons during outgrowth. Later, however, PT3 is contacted by a posteriorly directed side branch of the ISN, and it subsequently migrates toward the main nerve. After passing PT2, the pioneer growth cones extend under the main tracheal trunk and contact a third PT cell, PT1, as well as muscle fibers adjacent to it. By the end of stage 16, the ISN has acquired a highly stereotyped morphology as it innervates the dorsal and ventrolateral muscles, with lateral branches at the proximal edges of muscle 3 (first branch) and 2 (second branch) and a terminal arbor at the proximal edge of muscle 1, just beyond PT1. Pt3 is always at the first branchpoint. ISN axons form synapses on the dorsal muscles during stage 16 and early stage 17, with aCC innervating muscle 1 and RP2 innervating muscle 2 (Desai, 1997).

These results show that the relationships among the tyrosine phosphatases are complex and dependent on cellular context. At growth cone choice points along one nerve, two phosphatases cooperate, while along another nerve these same phosphatases can act in opposition to one another. The results also implicated PT cells in axon guidance decisions (Desai, 1997).

Terminal divisions of myogenic lineages in the Drosophila embryo generate sibling myoblasts that act as founders for larval muscles or form precursors of adult muscles. The formation of individual muscle fibers is seeded by a special class of founder myoblasts that fuse with neighboring mesodermal cells to form the syncytial precursors of particular muscle. Alternative fates adopted by sibling myoblasts are associated with distinct patterns of gene expression. During normal development (embryonic stage 11), two ventrally located progenitor cells divide once to produce three muscle founders and the precursor of an adult muscle (known as a persistent Twist cell because of its continued expression of twist). The more dorsal of the two progenitors divides, first giving rise to the founders of muscles VA1 and VA2, followed by the more ventral progenitors which produce the VA3 founder and the ventral adult persistent Twist precursor (VaP). As the progenitors divide, Numb is included in one of the two dorsal progenitors and in one of the two ventral progenitors. Thus the division of a muscle progenitor produces an unequal distribution of Numb between the founders: one contains Numb, the other does not. In numb mutants, some muscles are lost and others are transformed. For example VA1 and VaP are duplicated and VA2 and VA3 are lost. It is likely that all adult precursors are paired with larval founder cells as alternative fates. Each of the six persistent twist-expressing precursors in an abdominal hemisegment is duplicated in numb mutants. In the case of the lateral adult precursors, this duplication is associated with a loss of the segment border muscle; in the case of the three dorsal precursors, it is associated with the loss of dorsal muscles. Conversely, ectopic expression of numb leads to loss of adult muscle precursor and duplication of larval muscles. Lack of function of inscuteable produces phenotypes in the mesoderm similar to ectopic numb (Gomez, 1997).

Genes expressed in the progenitor cell are maintained in one sibling and repressed in the other. Kruppel, S59 and even skipped expression mark a subset of the developing muscles. In numb mutants the expression of Kruppel, S59 and even skipped is initiated normally but is lost from both founder cells after they are formed. Thus in numb mutants there are no muscles that express Kr, eve or S59. In contrast, when numb is ectopically expressed throughout the mesoderm, Kr, S59 and eve expression are maintained in both founders and in the muscle precursors to which they give rise. In these embryos, Kr, S59 and eve-expressing muscles are duplicated (Gomez, 1997).

Analysis and reconstitution of the genetic cascade controlling early mesoderm morphogenesis in the Drosophila embryo

To understand how transcription factors direct developmental events, it is necessary to know their target or 'effector' genes whose products mediate the downstream cell biological events. Whereas loss of a single target may partially or fully recapitulate the phenotype of loss of the transcription factor, this does not mean that this target is the only direct mediator. For a complete understanding of the pathway it is necessary to identify the full set of targets that together are sufficient to carry out the programme initiated by the transcription factor, which has not yet been attempted for any pathway.In the case of the transcriptional activator Twist, which acts at the top of the mesodermal developmental cascade in Drosophila, two targets, Snail and Fog, are known to be necessary for the first morphogenetic event, the orderly invagination of the mesoderm. A system of reconstituting loss of Twist function by transgenes expressing Snail and Fog independently of Twist was used to analyse the sufficiency of these factors¡Va loss of function assay for additional gene functions to assess what further functions might be needed downstream of Twist. Confirming and extending previous studies, Snail was shown to play an essential role, allowing basic cell shape changes to take place. Fog and at least two other genes are needed to accelerate and coordinate shape changes. Furthermore, this study represents the first step in the systematic reconstruction of the morphogenetic programme downstream of Twist (Seher, 2007).

In addition to Twist, Snail and Fog, there are genes in four regions of the autosomal genome which upon deletion lead to abnormalities during ventral furrow formation. Is it likely that all zygotically active genes that participate in normal mesoderm invagination have been detected? Although the assay proved to be sufficiently sensitive to identify a number of mutants, it is conceivable that further genes with even less pronounced mutant phenotypes were missed. Further, genes with completely redundant functions, for example, because duplicates exist in distinct regions of the genome, might not give a loss-of-function phenotype. Such genes might be identifiable only via sophisticated genetic screens (modifier screens) or appropriate molecular approaches (Seher, 2007).

The loss of the genes uncovered by the deficiencies results only in a delay of furrow formation, and not in the complete failure of invagination. If these genes are Twist targets, there are different possible explanations for the mutants showing such weak phenotypes. The genes might control an essential process parallel to that controlled by Snail, but act in a redundant manner in the pathway, such that disruption of only one of their functions does not lead to the disruption of furrow formation. Alternatively, only the pathway controlled by Snail may be essential, with other genes acting in parallel affecting only the speed and efficiency of furrow formation. The severe phenotype seen in the double mutants of Df(3R)TlP and fog as well as the enhancement of the fog phenotype by the loss of one copy of Snail argue for the latter scenario, i.e. two parallel pathways, both of which are essential (Seher, 2007).

Embryos were created in which the function of the mesodermal transcription factor twist was replaced by two of its downstream targets, snail and fog. The analysis of these embryos concentrated on the first phase of mesoderm morphogenesis, during which cell shape changes internalize the prospective mesoderm. The subsequent epithelial–mesenchymal transition, cell division and cell migration depend on other Twist targets, such as string, htl and dof. Since these are not expressed in the twist,PE::fog;2xPE::sna (twist driven fog and snail) embryos, this aspect of morphogenesis cannot occur in the reconstituted twist embryos (Seher, 2007).

The fog and snail transgenes had distinguishable effects in twist embryos. The PE::fog transgene induces the earliest event of the typical cell shape changes, apical flattening, and enhanced apical constrictions of ventral cells. By contrast, nuclear movement away from the apical cell surface was not significantly improved, nor was cell shortening observed. While the 2xPE::snail transgene also led to some improvement in apical flattening, it had additional effects. A larger number of cells showed distinctive nuclear movement, and a higher frequency of deep invaginations were scored, suggesting a role for Snail in releasing nuclei. With the combination of both transgenes nearly normal cell shape changes occurred which resulted in the formation of proper furrows in a substantial number of embryos. Specifically, many cells assumed the typical wedge-shape of ventral furrow cells, showing that snail and fog are sufficient to induce this shape in the absence of further Twist targets (Seher, 2007).

However, it appears that snail and fog cannot be the only targets downstream of twist to control mesoderm invagination. If they were, they should be able to replace twist function completely and fully restore furrow formation. It is therefore concluded that besides snail and fog other twist target genes must exist which are necessary to orchestrate the formation of the ventral furrow in the accurate, fast and stable fashion in wildtype embryos (Seher, 2007).

The as yet unknown targets must be involved in those events which were not restored in twist,PE::fog;2xPE::sna embryos: the speed of the process, adhesion between apical surfaces, and cell shortening during invagination. The latter process occurs efficiently in snail mutants, confirming that it is not Snail-dependent. Since fog does not contribute to this process it is likely that one or more other twist targets are involved in cell shortening (Seher, 2007).

In summary, the zygotic control of ventral furrow formation branches into separable functions downstream of Twist, the induction of the basic cell shape changes of ventral cells, and the control of the speed, accuracy and coordination of the shape changes. One of the known targets of Twist, the repressor Snail, is necessary to allow the shape changes to occur, whereas Fog and probably other Twist targets are responsible for accuracy and efficiency. Together, they ensure the rapid and regular formation of the ventral furrow. Ventral furrow formation may be an adaptation to the rapid early embryogenesis of long germ insects, serving to position mesodermal cells at a site where they can efficiently begin their FGF-dependent spreading on the inner surface of the ectoderm. The experimental system used in this study may be extended to test the function of the whole set of Twist targets, once they have been identified, for their ability to re-establish mesoderm invagination in the absence of Twist, and thereby reconstruct fully the pathway from a 'selector' gene to the cell biological processes it controls (Seher, 2007).

A key role of Pox meso in somatic myogenesis of Drosophila: twi and da function as promuscular genes by regulating the activities of competence domain genes, which in turn regulate the combinatorial activities of muscle identity genes and thereby specify the fates of muscle progenitors and founders

The Pax gene Pox meso (Poxm) was the first and so far only gene whose initial expression was shown to occur specifically in the anlage of the somatic mesoderm, yet its role in somatic myogenesis remained unknown. This study shows that it is one of the crucial genes regulating the development of the larval body wall muscles in Drosophila. It has two distinct functions expressed during different phases of myogenesis. The early function, partially redundant with the function of lethal of scute [l(1)sc], demarcates the 'Poxm competence domain', a domain of competence for ventral and lateral muscle development and for the determination of at least some adult muscle precursor cells. The late function is a muscle identity function, required for the specification of muscles DT1, VA1, VA2 and VA3. These results led to a reinterpretation of the roles of l(1)sc and twist in myogenesis and to the proposal of a solution of the 'l(1)sc conundrum' (Duan, 2007).

The development of the complex pattern of the larval body wall muscles of Drosophila provides an excellent paradigm of how a final pattern is established through precise genetic control. Each of the abdominal hemisegments A2-A7 has 30 identifiable individual muscles that develop from the somatic mesoderm. This process is initiated when the invaginated mesoderm migrates dorsolaterally under the ectoderm and is prepatterned by the segmentation genes: the product of sloppy paired (slp), whose activity is maintained by the ectodermal Wingless (Wg) signal, restricts high levels of the bHLH transcription factor Twist (Twi) to the mesodermal regions below the posterior portions of the ectodermal parasegments. These high levels of Twi function as a myogenic switch, separating the posterior somatic and cardiac mesoderm from the anterior visceral mesoderm and fat body. When the dorsal migration of the mesoderm is complete, these metamerically repeated Slp or high Twi domains are further subdivided along the dorsoventral axis by the ectodermal signal Dpp. This signal restricts transcription of tinman (tin) to the dorsal mesoderm, where its homeodomain protein specifies heart and dorsal somatic mesoderm. However, the determinant of the non-dorsal somatic mesoderm remains largely unknown. It appears that Pox meso (Poxm) expression is restricted to the ventral part of the high Twi domain by Dpp (Staehling-Hampton, 1994) to define the lateral and ventral somatic mesoderm anlage. The characterization of the role of Poxm in somatic myogenesis is therefore expected to fill an important gap in understanding of the gene network regulating this process (Duan, 2007).

Soon after this subdivision of the mesoderm, the proneural gene lethal of scute [l(1)sc] begins to be expressed in at least 19 promuscular clusters of cells within the high Twi domain. From these clusters, muscle progenitors are singled out by lateral inhibition through Notch (N) and Ras signaling and are specified by the expression of muscle-identity genes. Cells not singled out begin to express the zinc finger protein Lame duck (Lmd; also known as Minc), which specifies them as fusion-competent myoblasts (FCMs). The progenitors divide to generate different muscle founders, a muscle founder and an adult muscle precursor, or a founder and a cell producing either two adult muscle precursors or two pericardial cells. Each founder forms an individual syncytial muscle precursor by fusing with neighboring FCMs. One of the key steps in muscle pattern formation is the specification of a muscle founder by the expression of a specific set of muscle identity genes. Although an increasing number of these genes have been identified in recent years, the mechanisms that activate their transcription are still poorly understood. Hence, it is important to identify the genes whose products directly regulate the muscle identity genes (Duan, 2007).

This study describes the functional characterization of the Poxm gene. Poxm belongs to the Pax gene family whose members encode transcription factors, including a paired domain. The temporal and spatial expression patterns of Poxm and its loss- and gain-of-function phenotypes reported in this study demonstrate that it is required for most ventral and lateral abdominal muscles to develop properly in all segments and for the activation of muscle identity genes. In addition, Poxm acts itself as muscle identity gene in a few muscles and thus plays a dual role in somatic myogenesis (Duan, 2007).

Since Poxm is expressed during early myogenesis in cells that later give rise to progenitors of most of the ventral and lateral muscles, it may play an important role in the initiation of muscle patterning. To investigate which part of the PoxmR361 muscle phenotype results from the loss of this early Poxm function, a transgene expressing Poxm only during the early myogenic stages, um1-2-Poxm, was introduced into PoxmR361 embryos. In these embryos, the phenotypes of muscles VO4-6, VL2-VL4, VO2, VO1, LO1, LT4 and VT1 are efficiently rescued. The only muscles affected in Poxm mutants that are only slightly rescued by early Poxm are DT1, DO3 and VA1-3, in which Poxm is also expressed during later stages in their founders and/or muscle precursors. These results strongly suggest that Poxm exerts an early function, demarcating a mesodermal domain of competence for ventral, lateral and dorsolateral somatic muscle development (Duan, 2007).

The partial penetrance of the Poxm muscle phenotype suggests that the early Poxm function is largely redundant with that of other genes, an argument also raised to explain the weak muscle phenotype of l(1)sc mutants. The l(1)sc gene encodes a bHLH transcription factor the function of which is thought to be required for the selection of muscle progenitors. Therefore, the effect was examined of Poxm and l(1)sc mutations on larval muscle development in single and double mutant embryos (Duan, 2007).

In agreement with earlier studies, l(1)sc mutants exhibit a weak muscle phenotype, which deviates only slightly from that of wild-type embryos. Although PoxmR361 embryos show a considerably stronger muscle phenotype, most lateral and dorsal muscles are normal. Assuming that Poxm and l(1)sc act independently in muscle development, it was expected that the probability of a muscle being wild-type in Df(1) l(1)sc19/Y; PoxmR361 embryos is the product of the probabilities of the muscle being wild-type in the single mutants. Conversely, if significantly enhanced probabilities are found for muscle defects in double mutants, it may be concluded that Poxm and l(1)sc exhibit partially redundant functions during muscle development. Applying this test, it was found that most muscles are affected independently or nearly independently, with some notable exceptions. These concern muscles VL1-3, SBM, VO1, VO2, DT1, LT3, LT4 and VA3 that are more often absent. Some muscles are strongly affected in Poxm null mutants, such as muscles VO4-6 or muscles VA1-3. Among the other muscles, the more ventral and the more posterior a muscle is located within a segment, the more probable it is that it will show an enhanced phenotype in double mutants. Clearly, there is some redundancy between Poxm and l(1)sc functions in the somatic mesoderm, which is restricted largely to ventral and posterior muscles (Duan, 2007).

In Poxm mutants, only muscle DO3 is frequently duplicated. This duplication results from the transformation of muscle DT1 to DO3, as previously observed for muscles derived from the same progenitor in the absence of a muscle identity gene that is asymmetrically expressed in the two founders and muscle precursors. Thus, late expression of Poxm in the precursor of muscle DT1, but not of DO3, is crucial for their distinction and hence serves a muscle identity function. However, a more detailed analysis shows that muscle DT1 is missing in only two thirds (23/34) of all cases in which muscle DO3 is duplicated. In the remaining 11 cases, muscle DT1 is normal (4), abnormal (6) or duplicated (1). This finding suggests that the late Poxm function is necessary in about 10% (11/108) of all cases to prevent an additional division that generates a second founder of muscle DO3. Absence of Poxm in their founders results in abnormal muscles VA1-3 that cannot be rescued by the early Poxm function, which suggests that their proper specification also depends on the late function of Poxm (Duan, 2007).

These results have demonstrated that the development of larval body wall muscles depends on distinct Poxm functions during two phases. The early function of Poxm specifies, within the high Twi or Slp domain, a subdomain of competence for lateral and ventral muscle development, the 'Poxm competence domain'. This function appears to be analogous to that of tin, which specifies competence for heart and dorsal muscle development in the complementary part of the Slp domain. Poxm and tin thus subdivide the posterior Slp domain into ventral and dorsal subdomains in a manner similar to the partitioning by serpent and bap of the anterior Eve domain into the ventral fat body and the dorsal visceral mesoderm anlagen. After selection of muscle progenitors, proper development of a few muscles still depends on Poxm, which is expressed in muscles DT1 and VA1-3. This late function of Poxm participates in founder specification and muscle differentiation, as is characteristic for muscle identity genes. Finally, the findings suggest a solution to a conceptual problem of the current model of somatic myogenesis, the l(1)sc conundrum (Duan, 2007).

The muscle phenotype of Poxm mutant embryos and its rescue by early Poxm expression shows that the early Poxm function is crucial for the proper development of many ventral and lateral muscles. In addition, the generation of ectopic dorsal and dorsolateral muscles by ectopic Poxm suggests that Poxm has the ability to change cell fates and render cells competent for myogenesis. Therefore, it is proposed that early Poxm demarcates a ventral and lateral domain of competence for somatic myogenesis (Duan, 2007).

The partial penetrance of the Poxm mutant phenotype implies the existence of other competence domain genes performing partially redundant functions. Poxm and L(1)sc partially co-localize in the promuscular clusters and muscle progenitors. In addition, a detailed analysis of l(1)sc and Poxm single and double mutants demonstrates that their functions are partially redundant. Since the muscle phenotype of l(1)sc; Poxm double mutants still shows partial penetrance, additional competence domain genes should be expressed in the Slp domain. One of them is probably tin, which is initially expressed in the entire early mesoderm, because tin mutants affect muscle development in the dorsal as well as lateral and ventral Slp domain. Another candidate is D-six4, which is required for the development of specific muscles that arise from the dorsolateral and ventral regions (Duan, 2007).

Thus, after the initial subdivision of the mesoderm, the high Twi domain is further subdivided by competence domain genes, which specify domains that become competent to select progenitors of distinct subsets of somatic muscles and/or of myocardial and pericardial cells by responding to spatially restricted extracellular signals. These competence domain genes act in a cooperative manner to determine the identities of specific muscles by regulating the expression of the muscle identity genes. When one of them is inactivated, in some cells active competence domain genes can partially compensate for the inactive gene by activating its target genes such that these sometimes, but not always, exceed the threshold levels required for normal development. Hence, muscles derived from these cells exhibit a mutant phenotype with partial penetrance. For other cells, active competence domain genes can compensate completely for the missing gene activity such that these cells will adopt the proper fate and the muscles develop normally. This illustrates that competence is not a matter of 'all' or 'nothing' for muscle development. The deeper reason for this is thought to be that the genetic program regulating myogenesis is not organized in a hierarchical fashion but rather as a complex gene networkthat has an integrated function which is much more stable against mutations within the network than a hierarchical regulation would be (Duan, 2007).

Muscle identity genes usually encode transcription factors, such as Slou, Nau, Ap, Vg, Kr, Eve, Msh, Lb, Run and Kn, that are expressed in subsets of muscle progenitors and founders and determine in a combinatorial fashion the identity of each muscle founder and its subsequent differentiation into a specific muscle of defined size, shape, attachment sites, and innervation. It is envisioned the activation of these genes in promuscular clusters or, after lateral inhibition, in muscle progenitors by Twi and/or the products of competence domain genes and through combinations of localized extracellular signals from the ectoderm and mesoderm. During asymmetric division of progenitors, expression of a muscle identity gene may be maintained in one or both of the two sibling founders, or it may persist in the founder when division generates a founder and an adult muscle precursor. Late expression of Poxm illustrates all three cases. It is expressed in progenitors P26/27 and P29/VaP, which are derived from promuscular cluster 10 and give rise to the founders of muscles VA1 (F26) and VA2 (F27), and to the founder of muscle VA3 (F29) and the ventral adult precursor VaP. Poxm is also expressed in the progenitor derived from cluster 13, P11/18, which generates the founders of muscles DO3 (F11) and DT1 (F18). Although Poxm expression persists in F29 and F18 but not in their siblings, it is maintained in both sibling founders F26 and F27 (Duan, 2007).

The late function of Poxm is identified as a muscle identity function by the high correlation between absence of muscle DT1 and corresponding duplication of muscle DO3 in Poxm mutants. If Poxm was the sole determinant discriminating between F11 and F18, mesodermal ubiquitous expression of Poxm would be expected to transform muscle DO3 into DT1. The results confirm the presence of additional muscles in the region of muscle DT1. It is possible that one of these originates from a transformed F11, but it is impossible to tell whether muscle DO3 is missing because additional muscles have been recruited (Duan, 2007).

It has been shown that in the process of muscle diversification, identity genes may repress or activate other identity genes in progenitors and founders. This study found that the muscle identity gene slou fails to be activated in P11/18 of Poxm mutants. The simplest explanation of this result is that activation and maintenance of slou expression depend on Poxm in P11/18 and its offspring founders. In addition, slou expression is not maintained in F27 of Poxm mutants despite its initial activation in P26/27. It therefore appears that in P26/27 and its offspring F26 and F27, in addition to Kruppel (Kr), Poxm is necessary for the maintenance of slou expression. Although Poxm expression is maintained in both F26 and F27, slou expression is restricted to F27 because Kr is repressed in F26 by N signaling. Apparently, Kr is the crucial determinant that distinguishes F26 from F27, as F27 is altered to F26 in Kr or numb mutants (Duan, 2007).

Since Poxm is expressed in both F26 and F27, whereas its expression is restricted to F18 and not maintained in F11, its late expression in F26 and F27 must be regulated differently from that in F11 and F18 where it appears to be subject to asymmetric N signaling repressing Poxm in F11 (Duan, 2007).

These considerations imply that slou is part of the same gene network as Poxm, a conclusion consistent with the proposed gene network hypothesis since, in the first test of this hypothesis, slou had been isolated as a PRD 9 gene on the basis of its homology to the prd gene (Duan, 2007).

The mechanism of progenitor selection from the somatic mesoderm depends on a process of lateral inhibition very similar to that of neuroblast or sensory organ precursor (SOP) selection in the neuroectoderm from proneural clusters expressing the proneural genes. Because of this similarity, a search among proneural genes for 'promuscular' genes expressed in the somatic mesoderm was performed. This search identified a single proneural gene, l(1)sc, a member of the achaete-scute complex (AS-C), that is expressed in promuscular clusters of the somatic mesoderm. It was, therefore, attractive to consider its function in myogenesis to be analogous to that of proneural genes in neurogenesis. However, whereas proneural genes confer on neuroectodermal cells the ability to become neural precursors rather than epidermal cells, which is their default fate, l(1)sc does not seem to confer on mesodermal cells the ability to undergo somatic myogenesis instead of becoming part of the visceral mesoderm, heart or fat body. When L(1)sc was expressed in the entire mesoderm from stage 8 onward, other mesodermal tissues could not be transformed into somatic mesoderm, whereas a deficiency of l(1)sc resulted in only minor defects of somatic muscle development. In addition, as the l(1)sc muscle mutant phenotype can be rescued by ubiquitous mesodermal L(1)sc expression, its expression in clusters is not decisive for the formation of promuscular clusters and, therefore, l(1)sc cannot play the decisive role in the development of larval body wall muscles that has been proposed. Thus, although l(1)sc serves as an excellent marker for promuscular clusters, it lacks a property expected to be crucial for a promuscular gene. Are there genes that might qualify as promuscular genes and thus extend the close evolutionary relationship of progenitor selection between myogenesis and neurogenesis (Duan, 2007)?

There is indeed a gene that is homologous to proneural genes and expressed in the somatic mesoderm, in the absence of which somatic myogenesis is seriously disturbed. This gene is twi, whose function at stages 10 and 11 more closely corresponds to that of a promuscular gene and which, like l(1)sc, encodes a bHLH transcription factor. Although Twi is also expressed earlier when it is required for mesoderm specification during gastrulation, this early function can be distinguished from its later 'promuscular' function in temperature-sensitive mutants. In these mutants, only high levels of Twi activity, necessary for the formation of the somatic mesoderm, are abolished and no normal somatic muscles develop. Moreover, ubiquitous expression of high levels of Twi in the mesoderm blocks all other mesodermal fates, transforming them to somatic mesoderm. Since the subsequent patterning of somatic muscles depends critically on the relative levels of the products of twi and the proneural gene da, it seems appropriate to consider them both as promuscular genes (Duan, 2007).

In addition to its strict requirement for somatic myogenesis, the proposed promuscular function of twi may be subject to lateral inhibition by N signaling, in further analogy to proneural functions in neurogenesis. This is apparent from experiments demonstrating that the restriction of high Twi levels to the Slp domain during stage 9 depends on N signaling, which downregulates twi in the mesoderm underlying the anterior regions of parasegments where Slp does not override it. Since this process acts directly on an identified twi enhancer during stages 9 and 10, it is conceivable that this enhancer also responds to N signaling during the subsequent lateral inhibition. An alternative, though not mutually exclusive, mechanism for the downregulation of twi implicates the Gli-related zinc finger transcription factor Lmd (Minc), whose expression is maintained by N signaling and in the absence of which twi is not downregulated in fusion-competent myoblasts (Duan, 2007).

During lateral inhibition, loss of Twi precedes that of L(1)sc in the promuscular clusters. It is therefore possible that l(1)sc expression in these cells also depends on high levels of Twi, i.e. on Twi homodimers. Consistent with this interpretation, shifting the equilibrium between Twi homodimers and Twi-Da heterodimers in favor of the latter represses l(1)sc. Since early Poxm expression also depends on Twi, Poxm would be similarly repressed in promuscular clusters through lateral inhibition, either indirectly by repression of twi and/or directly by Twi/Da heterodimers. Such a mechanism might apply generally to both competence domain genes and muscle identity genes during lateral inhibition of promuscular clusters (Duan, 2007).

Thus, twi satisfies two criteria considered to be crucial for a promuscular gene in analogy to those of proneural genes in neurogenesis. However, a third criterion is not fulfilled by twi: its expression, in contrast to that of proneural genes in the neuroectoderm, is ubiquitous rather than restricted to promuscular clusters although this criterion is not a crucial property of proneural genes. Yet promuscular clusters from which the myogenic progenitors are selected exist, as evident from the pattern of l(1)sc expression. These promuscular clusters depend on combinations of the long-range ectodermal signals Wg and Dpp and the localized activities of the EGF signal Spi in the mesoderm and the FGF signals Pyr and Ths in the ectoderm. These signals, together with Twi and/or products of competence domain genes depending on Twi, determine the promuscular clusters by activating specific combinations of muscle identity genes. The identity of the promuscular clusters depends not only on the combination of these signals but, in the case of MAPK signaling elicited by FGF and/or EGF, also on their intensity. In addition, multiple positive and negative feedback loops of the coupled MAPK and N signaling networks ensure a stable selection and specification of muscle progenitors not only within, but also beyond, the limits of a promuscular cluster. Such a conclusion implies that these clusters are not a priori determined, but depend on the range and intensities of the MAPK activating signals, in agreement with the assumption that it is not the expression of l(1)sc that determines the promuscular clusters. In fact, it may be the absence of such a priori determined clusters of equivalent cells in the somatic mesoderm that necessitates such a complex N and Ras signaling circuitry (Duan, 2007).

Therefore, it is proposed that twi and da, instead of l(1)sc, function as promuscular genes by regulating the activities of competence domain genes, which in turn regulate the combinatorial activities of muscle identity genes and thereby specify the fates of muscle progenitors and founders. It is nevertheless surprising that l(1)sc appears to be expressed in all promuscular clusters even though its function is not necessary in most of them. It is possible that this expression pattern is an evolutionary remnant of an atavistic promuscular function of l(1)sc that was later replaced by the promuscular function of twi on whose expression l(1)sc activity depends (Duan, 2007).

Antagonistic function of Lmd and Zfh1 fine tunes cell fate decisions in the Twi and Tin positive mesoderm of Drosophila melanogaster

This study showS that cell fate decisions in the dorsal and lateral mesoderm of Drosophila depend on the antagonistic action of the Gli-like transcription factor Lame duck (Lmd) and the zinc finger homeodomain factor Zfh1. Lmd expression leads to the reduction of Zfh1 positive cell types, thereby restricting the number of Odd-skipped (Odd) positive and Tinman (Tin) positive pericardial cells in the dorsal mesoderm. In more lateral regions, ectopic activation of Zfh1 or loss of Lmd leads to an excess of adult muscle precursor (AMP) like cells. It was also observed that Lmd is co-expressed with Tin in the early dorsal mesoderm and leads to a reduction of Tin expression in cells destined to become dorsal fusion competent myoblasts (FCMs). In the absence of Lmd function, these cells remain Tin positive and develop as Tin positive pericardial cells although they do not express Zfh1. Further, it was shown that Tin repression and pericardial restriction in the dorsal mesoderm facilitated by Lmd is instructed by a late Decapentaplegic (Dpp) signal that is abolished in embryos carrying the disk region mutation dppd6 (Sellin, 2009).

Loss of Lame duck (Lmd) leads to an increase of pericardial cells and adult muscle precursor like cells: In embryos lacking Lmd function, staining for zinc finger homeodomain factor 1 (Zfh1) expression reveals a pericardial hyperplasia phenotype and a general excess of Zfh1 positive mesodermal cells. In wild type embryos, three types of pericardial cells (PCs) have been described: Tin positive (TPCs), Odd positive (OPCs) and Eve positive (EPCs) pericardial cells, all of which express Zfh1 and the handC- GFP reporter. Closer inspection of the pericardial cells in lmd mutant embryos revealed that the number of TPCs and OPCs is dramatically increased, while the number of EPCs is normal. All OPCs co-express the handC- GFP reporter and Zfh1 in wild type and lmd mutant embryos. In contrast, a considerable number of ectopic Tin positive cells, though positive for handC- GFP, do not express Zfh1 in lmd mutant embryos. The absence of β3Tubulin expression in these cells is consistent with earlier reports in which a normal set of cardioblasts was described in lmd mutant embryos. To decide whether the Zfh1 negative/Tin positive cells are atypical pericardial cells or dorsal mesodermal cells that fail to differentiate, a triple staining waa conducted for Tin, Zfh1 and Pericardin (Prc), a collagen that is secreted by differentiated pericardial cells. Prc protein was observed surrounding all Tin positive/Zfh1 negative cells, suggesting that they are ectopic pericardial cells. However, due to the fact that Prc is a secreted protein the possibility cannot be ruled out that there might be occasional Tin positive/Zfh1 negative cells in lmd mutant embryos which do not express Prc themselves, but remain in an uncommitted, dorsal mesodermal state (Sellin, 2009).

For further analysis of the ectopic pericardial cells, the number of OPCs in stage 16-17 embryos was counted. An average of 206.3 OPCs was observed in lmd mutant embryos as compared to 97.8 in wild type embryos, thereby representing a ~2-fold increase. It has been reported that the Odd subgroup of pericardial cells (OPCs) originates from two different lineages: a symmetric lineage (two OPCs from one precursor) and an asymmetric lineage (two OPCs from two precursors), adding up to a total of four OPCs per hemisegment. Of note, the siblings of the asymmetrically derived OPCs, the Seven-up (Svp) positive cardioblasts, are normal in lmd mutants, thus suggesting that the asymmetrically derived OPCs do not contribute to the lmd phenotype. Since the two different types of OPCs can not be distinguished directly because the anti-Svp antibody stains the precursor cells and the cardioblast siblings, but not the final PCs at later stages, their abundance was measured in lmd mutants indirectly. The fact was utilized that in inscutable (insc) mutants, asymmetric cell division fails, and all siblings of the asymmetric OPC lineage become Svp positive cardioblasts. The difference in OPC number between insc; lmd and lmd mutant embryos therefore corresponds to the number of asymmetrically derived OPCs in lmd mutant embryos. A loss of ~45 OPCs was observed in insc; lmd double mutant embryos as compared to lmd mutant embryos. This number is reasonably close to the number of ~38 OPCs that are lost in insc mutant embryos when compared to wild type embryos. In addition, the number of Svp positive precursors, which give rise to the asymmetric Odd lineage, is normal in lmd mutant embryos at early stage 13. Altogether, these data strongly support the initial hypothesis that there is the normal amount of asymmetrically derived OPCs in lmd mutant embryos and the phenotype is not caused by a failure of asymmetric cell division (Sellin, 2009).

An excess of Zfh1 positive cells was also observed in the lateral mesoderm of lmd mutant embryos, where it is normally expressed in the adult muscle precursor cells (AMPs). These imaginal myogenic cells retain Twist (Twi) expression, but do not express any other myogenic genes in the embryo. Instead, they are maintained in a less differentiated state during embryogenesis and are dormant until metamorphosis, when they start to differentiate and give rise to the adult musculature of the fly. In the embryo, they are arranged as groups of cells in the thoracic segments, while six solitary cells (one dorsal, two dorsolateral, two lateral and one ventral) are present in the abdominal hemisegments. It was reported earlier that too many Twi positive cells persist in the lateral mesoderm of lmd mutant embryos. Together with the fact that both Zfh1 and Twi are present in AMPs in the wild type, it appeared likely that both factors are also co-localized in embryos mutant for lmd. Indeed, double staining for Zfh1 and Twi showed a complete overlap in the lateral mesoderm and confirmed that both populations of ectopic cells are identical. They also express the gene holes in muscles (him) which is another marker specific for AMPs. For further characterization, the expression patterns were analyzed of several myogenic markers in lmd mutant embryos. No expression was detected of the muscle specific genes myocyte enhancing factor 2 (Mef2), β3 Tubulin or the reporter rP298 (Duf-lacZ) in Twi/Zfh1/Him positive cells (Sellin, 2009).

Twist expression is normally present during early stages of somatic muscle development in myoblasts that are not yet differentiated. Zfh1, which has been implicated in the repression of mef2, might help in keeping AMPs in the undifferentiated state until metamorphosis. The gene him, which is also expressed in AMPs, was recently reported to be involved in maintaining cells in an undifferentiated state by inhibiting the myogenic signal provided by Mef2 function. Consequently, the ectopic Zfh1/Twist/Him positive cells in the lmd mutant embryos are likely to be cells with myogenic potential, as are the endogenous AMPs, and hence can be considered to be ectopic AMP like cells. To assess if enhanced proliferation is also involved in generating an increased amount of cells in lmd mutant embryos, staining was oerfirned for phosphorylated Histone 3 (pH3), which specifically marks dividing cells. Over-proliferation in the dorsal and lateral mesoderm was not observed in lmd mutant embryos when compared to wild type embryos. Although there is a considerable number of the additional, AMP like cells that persist until the end of embryogenesis, their number is reduced between stage 13 and 16/17. Staining with Nile Blue A revealed a general excess of dying cells during these stages in lmd mutant embryos as compared to wild type, suggesting that not all ectopic cells survive until the end of embryogenesis (Sellin, 2009).

The supernumerary PCs and AMPs originate from the population of fusion competent myoblasts: While no general myogenic genes are expressed in the ectopic AMP like cells, it was however possible to show co-localization of Zfh1 and lmd mRNA in the somatic mesoderm of lmd mutant embryos, which is not observed in wild type embryos. In the wild type, lmd is expressed in fusion competent myoblasts (FCMs), which fail to differentiate in the absence of Lmd function. lmd mRNA is transcribed in a normal pattern in lmd mutant embryos. In situ hybridization with a lmd specific riboprobe therefore allowed visualization of the population of cells destined to become FCMs, although they do not express any other FCM specific genes in lmd mutant embryos. Since the ectopic Zfh1 positive cells co-express lmd mRNA, it is concluded that the ectopic AMP like cells in lmd mutant embryos originate from the FCM population and adopt AMP like characteristics instead. They are therefore generated by cell fate conversions, which is consistent with the observation that there are no additional cell divisions in lmd mutant embryos (Sellin, 2009).

In the dorsal mesoderm of lmd mutant embryos, the additional Zfh1 positive cells express Tin or Odd and Prc, indicating differentiation as pericardial cells. Pericardial cells usually develop from the dorsal cardiac mesoderm specified by Tin expression, while the somatic musculature is situated more laterally and is characterized by prolonged Twi expression. To address the question of whether a conversion of FCMs into PCs could also account for the pericardial hyperplasia phenotype in lmd mutant embryos in an analogous fashion to the ectopic AMP like cells, staining was carried out for Tin and lmd mRNA. It was reasoned that the ectopic Tin cells should also express lmd mRNA if they originate from the pool of mis-specified FCMs. Indeed, there is co-expression of lmd mRNA and Tin in ectopic pericardial cells in stage 13 lmd mutant embryos, indicating that cell fate conversions from FCM to ectopic PC fate are responsible for the observed pericardial hyperplasia phenotype (Sellin, 2009).

Of note, there is a distinct overlap of lmd mRNA and Tin expression in the dorsal mesoderm of stage 12 embryos, both in wild type and lmd mutant background. This observation is consistent with the observed cell fate switch from FCM to PC fate and indicates that in wild type embryos, dorsal FCMs are specified in the dorsal, Tin positive mesoderm rather than the Twi positive somatic mesoderm. Indeed, dorsal muscle phenotypes can be observed in embryos mutant for tin, consistent with the conclusion that dorsal muscle cell types (i.e., FCMs) develop from the early dorsal mesoderm specified by Tin expression: If this domain is not specified, it can not generate dorsal FCMs (or other dorsal mesodermal derivatives, like heart or visceral mesoderm) (Sellin, 2009).

Co-expression of Tin and lmd mRNA is no longer detectable after germ band retraction (stage 13) in wild type embryos, but persists in lmd mutant embryos until the lmd mRNA signal fades (at about stage 14-15). Thus, it seems that repression of Tin in the dorsal mesoderm depends on the presence of Lmd protein. To substantiate this observation, Lmd was overexpressed in the whole mesoderm with the twi-Gal4 driver to assess its influence on Tin expression. Indeed, a reduction was observed of Tin expression in stage 12 embryos overexpressing Lmd compared to wild type, further confirming a negative influence of Lmd on Tin expression in the dorsal mesoderm. At later stages, the number of TPCs (and OPCs) remains reduced, while the cardioblasts are not affected. A model is therefore proposed in which the initial dorsal mesoderm specified by Tin expression is subdivided by Lmd into cardiac mesoderm and dorsal musculature by repression of Tin in lateral regions and induction of a myogenic differentiation program instead. During this process, Tin expression is maintained only in the cells that are destined to become pericardial cells (or cardioblasts), while Tin is repressed by Lmd in the dorsally localized FCMs. Loss of Lmd function consistently leads to an increased amount of Tin positive cells in the dorsal mesoderm from stage 13 onwards, which then can differentiate as ectopic pericardial cells as indicated by the expression of Prc. Taken altogether, these data suggest that, in the absence of Lmd function, the pool of unspecified FCMs can develop as ectopic PCs in the Tin-positive dorsal mesoderm and as AMP-like cells in the lateral and ventral mesoderm. However, increased cell death, and the possibility that a small number of ectopic Tin positive cells might exist without Prc/Zfh1 expression as mentioned earlier, suggest the possibility that not all cells of the FCM population follow alternative cell fates. Instead, some cells might remain in an uncommitted mesodermal state in lmd mutant embryos (Sellin, 2009).

Normally, instructive Dpp signals from the ectoderm are responsible for the specification of cardiac cell types by maintaining Tin expression solely in the dorsal mesoderm, while Twist activity in the lateral and ventral mesoderm leads to the development of the somatic musculature. To test if reduced Dpp signaling has a similar effect on PC number as overexpression of Lmd, by reducing the size of the Tin domain, embryos carrying the mutation mad1-2 were examined. mad1-2 is a weak hypomorphic allele of the Dpp effector Mad and causes larval lethality, thereby allowing observation of late stages of embryogenesis. Indeed, a decreased number of OPCs and TPCs was observed in mad1-2 mutant embryos, without any effect on cardioblast number, as is the case when overexpressing Lmd. Of note, the number of OPCs is decreased to a similar extent in mad1-2; lmd double, as compared to lmd single mutant embryos. Therefore, it is concluded that in the presence of the hypomorphic mad1-2 mutation, the dorsal mesoderm that is specified by Dpp-dependent Tin expression is reduced, resulting in a reduction of PCs in a Lmd independent manner. However, Lmd further restricts the number of PCs in the mad1-2 mutant background, as revealed by an increased number of PCs and the presence of TPCs without Zfh1 expression in mad1-2; lmd double mutants when compared to mad1-2 single mutants (Sellin, 2009).

Pericardial cells share their developmental origin with the myogenic cardioblasts in a similar fashion as AMPs with founder cells in the somatic musculature. During lateral inhibition, Notch activation promotes myogenic FCM fate as opposed to the progenitors of founder cells in the lateral mesoderm or cardiogenic progenitors in the dorsal mesoderm. Subsequently, during the process of asymmetric cell division, Notch activation renders the daughter cell always non-myogenic (PC or AMP fate). Although the AMPs have the potential to develop into muscle cells during metamorphosis, they are considered non-myogenic in this context because they do not yet express any myogenic genes, such as mef2, lmd or muscle structural genes in the embryo. In the case of pericardial cells, there is surprisingly little data available about their physiological role. While it is known that the OPCs contribute to the population of nephrocytes in postembryonic stages, TPCs and EPCs are not correlated with any function at all, and their developmental fate after embryogenesis is still unknown. A recent study described the development of adult muscular structures, the so called wing hearts, from a specialized subset of EPCs. This is the first hint that some pericardial cells might be considered as imaginal myogenic cells in an analogous fashion to AMPs, and it highlights the necessity to further characterize pericardial cells (Sellin, 2009).

It is currently known that PCs and AMPs have in common a dependency on active Notch signaling although they stem from different cell lineages and mesodermal primordia (Tin vs. Twi domain). FCMs, which adopt AMP or PC like characteristics in lmd mutant embryos, also need active Notch signaling. In fact, Lmd is a downstream target of N signaling and induces the FCM differentiation program. The observed lmd phenotype could be explained if, in the absence of Lmd, Notch activity always promoted AMP or PC (non-myogenic) fate, but not FCM fate, independently of the original pathway that is involved (lateral inhibition or asymmetric cell division). To assess this hypothesis, double mutants for lmd and genes involved in the Notch pathway were established. For this analysis kuzbanian and mastermind alleles were chosen because loss of either gene causes lethality only late in embryogenesis due to a maternal component, thereby allowing the analysis of later events in heart and muscle development. Both genes have also been well studied with respect to their molecular function and developmental implications. Kuzbanian (Kuz) is an ADAM metalloprotease that is known to process the Notch receptor following ligand binding. Zygotic loss of function mutations lead to defects in both lateral inhibition and asymmetric cell division in heart and muscle development, although the phenotype is far weaker than in embryos carrying N loss of function alleles. mastermind (mam) is involved in transducing the Notch signal and displays a stronger heart phenotype than kuz and a mild Notch-like muscle phenotype. Staining was perfomed for expression of Krüppel (Kr) and him mRNA, which are specific for a subset of muscle founders and AMPs/ PCs, respectively, and an increase of Notch negative cell types, corresponding to founders, was observed in the somatic mesoderm of kuz mutant embryos. This is accompanied by a reduction of AMPs, confirming the expected function of Kuz in facilitating N function in muscle cell differentiation. Furthermore, the number of FCMs as marked by Lmd expression is strongly reduced in kuz mutants, although the effect is not as complete as in N loss of function alleles (Sellin, 2009).

In kuz; lmd double mutant embryos, the increase of AMPs is milder than in lmd mutant embryos, which is consistent with a failure in lateral inhibition and a concomitant reduction of FCMs that are available for conversion to AMPs. The number of Kr-positive founder cells is increased to comparable levels in kuz and kuz; lmd mutant embryos, suggesting that Notch inactive cell fates (muscle founders and cardioblasts) are not influenced by the absence of Lmd, and that Notch acts as a permissive signal to allow the cell fate switch in lmd mutant embryos. mam; lmd double mutant embryos display a similar phenotype. Altogether, these findings suggest that in the double mutants, a general reduction of cell types with Notch activity (i.e. FCMs) occurs, followed by the conversion of the remaining potential FCMs to AMP or PC fate under the influence of N signaling in the absence of Lmd. Lame duck is present in stages 12-14, which is later than the period during which Notch activity is involved in facilitating cell fate decisions within the musculature. Hence, it appears that Notch can promote AMP or PC fate at a relatively late time point in the absence of Lmd (Sellin, 2009).

It was of interest to know if the endogenous set of AMPs, which develop through asymmetric cell divisions of muscle progenitors, is specified correctly in lmd mutant embryos. For example, the lateral AMPs are the siblings of the segment border muscle founder (SBM), and share with the latter the expression of the identity factor Ladybird early (Lbe). To discern ectopic cells and endogenous AMPs in lmd mutant embryos, co-staining was performed for Lbe and Twi expression. Indeed, the normal number of lateral AMPs, as marked by Lbe expression, is present in lmd mutant embryos, while far too many Twi-positive cells was observed in general. The latter are the ectopic AMP like cells that are presumed to be recruited from the FCM population. This observation further confirms that individual mesodermal lineages, such as the asymmetrically derived OPCs or individual AMPs, are not influenced by the loss of Lmd function (Sellin, 2009).

The proposed model of cell fate switches from myogenic FCM fate to non-myogenic AMP like or PC fate, but not myogenic fates (cardioblasts or founder cells), is consistent with the observation that Notch signaling is often employed to delay or inhibit the differentiation of stem cells or progenitor cells, especially in myogenesis. In vertebrates, Notch signaling prevents satellite cells (muscle stem cells) from entering a myogenic differentiation program in cell culture as well as in vivo, and impaired upregulation of its ligand Delta-like 1 in satellite cells has been correlated with a decreased capacity of aging muscle tissue to regenerate. While the data are consistent with the general function of Notch in preventing cells to enter the myogenic differentiation program by promoting the AMP or PC fate, they also highlight the special and unusual properties of Lmd - as a target of Notch signaling - in Drosophila muscle development. Although it is activated by Notch, it has the ability to induce myogenic differentiation. The data strongly suggest that the AMP or PC fate is the default consequence of Notch signaling in Drosophila myogenesis and that Lmd function overrules this signal to induce the FCM differentiation program in lateral or dorsal competence domains. It was shown that N has a biphasic function in heart differentiation analogous to the situation in the somatic mesoderm. At an early phase, N activity restricts the number of the sum of CBs and PCs, reflecting a function in the definition of early cardiac progenitors, likely by lateral inhibition. Subsequently, N activity is needed to promote pericardial cell fates in asymmetric cell division of the early progenitors. Although the last division step is in many cases a symmetric division seem to indicate that the majority of cardiac cell types is generated by asymmetric cell divisions segregating cardiac and pericardial fates. This might occur in some cases at one of the earlier division steps of the progenitor(s). Since these data indicate the generation of FCMs from the dorsal mesoderm, as reflected by co-expression of Tin and Lmd in stage 12 embyos, it might be suggested that dorsal FCMs originate from dorsal competence domains which also give rise to the above mentioned cardiac progenitors. These progenitors divide asymmetrically to generate CBs and PCs analogous to FC/AMP sibling pairs from more lateral competence fields, while it ia proposes that all or some of the remaining cells of the competence domains begin to express Lmd and generate FCMs under instructive influence of N signaling. In the absence of Lmd function (either in wild type in the N active daughter cells of the progenitors, or in lmd mutant embryos in all N active cells of the competence domains), the N signal promotes non-myogenic cell fates according to the mesodermal context (i.e., dorsal vs. lateral mesoderm). This would then result in the differentiation of the non-segregating population (normally developing as FCMs) as PCs in the Tin domain and AMPs in the somatic mesoderm (Sellin, 2009).

Lame duck and Zfh1 act antagonistically in mesodermal cell fate decisions: While loss of Lmd function results in an increased number of Zfh1-positive cell types, overexpression of Lmd leads to the opposite phenotype. The pan-mesodermally active twi-Gal4 driver line was used to induce Lmd expression in the whole mesoderm, and a reduction of OPCs, TPCs and AMPs was observed. To assess whether pericardial cell reduction might be a secondary effect of the early Tin repression caused by ectopic Lmd activity, the later and cardiac specific handCA-Gal4 driver, which is active in the heart from stage 12 onwards, was used. At this time point, the OPC precursors are already specified and are no longer expressing Tin. Since hand>Lmd overexpression severely reduces the number of all pericardial cells, it is concluded that their reduction is not only a secondary effect of the narrower Tin domain in embryos overexpressing Lmd. To further confirm this conclusion, the phenotype of zfh1 mutant embryos was compared with that of embryos overexpressing Lmd. The number of OPCs and TPCs is also reduced in zfh1 mutant embryos quite similarly to embryos overexpressing Lmd, although the early Tin expression pattern is normal in the absence of Zfh1 function. It is therefore unlikely that Lmd acts negatively on Zfh1 expression only by reducing Tin expression, but rather also independently of Tin function (Sellin, 2009).

There are however important differences in the phenotypes of twi > Lmd and zfh1 mutant embryos. Zfh1 appears to be involved in maintaining, but not in specification of OPCs, because it has been observed that loss of Zfh1 does not affect the number of OPC precursors at stage 13, but rather leads to a decrease of OPCs at later stages. This is in contrast to a reduced number of OPC precursors in stage 13 embryos overexpressing Lmd. Therefore, Zfh1 repression alone can not account for the loss of PCs in embryos ectopically expressing Lmd. Instead, it might be that the reduction of the dorsal Tin domain by ectopic Lmd expression results in the specification of fewer OPC precursor cells, followed by further reduction of the remaining OPCs by the negative effect of ectopic Lmd on Zfh1 expression. Consistently, a much stronger reduction of OPCs was observed after ectopic expression of Lmd as compared to the loss of OPCs in zfh1 mutant embryos. The observation that loss of Lmd function leads to the appearance of TPCs that do not express Zfh1, but Prc as a marker of pericardial differentiation, is another hint that both effects occur independently of each other and that pericardial differentiation can be accomplished in the absence of Zfh1 in lmd mutant embryos (Sellin, 2009).

Taken altogether, it does not seem likely that Tin and Zfh1 act in an epistatic hierarchy in dorsal mesodermal cell fate decisions. Instead, the data support the conclusion that Lmd regulates OPC and TPC number by two independent mechanisms: (1) Initially, Lmd restricts the cardiac field in general through repression of Tin, which leads to the reduction of early OPC precursors and the elimination of Tin expression in cells that do not express Zfh1 (which can differentiate as TPCs, as indicated by Prc expression, in the absence of Lmd function). (2) Later, it represses Zfh1, thereby reducing further the number of OPCs and TPCs. This is consistent with previous findings which described Zfh1-dependent and Zfh1-independent mechanisms for the regulation of OPC and TPC number (Sellin, 2009).

Of note, it was previously shown that Zfh1 overexpression leads to an increase in pericardial cell number (both OPCs and TPCs) and a concomitant loss of dorsal somatic muscle cells, indicating that overexpression of Zfh1 phenocopies the pericardial hyperplasia in lmd mutant embryos. It was shown further that overexpression of Zfh1 with the twist-Gal4 or 24B-Gal4 driver leads to an increased number of AMP like cells in the dorsal mesoderm although the effect is rather weak when compared to lmd mutant embryos. Zfh1 overexpression does not however alter the pattern of Lmd expression, indicating that Zfh1 does not antagonize Lmd function at the transcriptional level. To verify whether Zfh1 has an influence on Lmd at the posttranscriptional level, the intracellular distribution of Lmd was analyzed in embryos overexpressing Zfh1, because Lmd function has been shown to be modulated by its subcellular localization in wild type embryos. In embryos overexpressing Zfh1, the subcellular localization of Lmd does not appear to be altered, suggesting that Zfh1 does not influence the subcellular distribution of the Lmd protein (Sellin, 2009).

Taken together, these data indicate that Lmd and Zfh1 have generally opposite effects on dorsal mesoderm differentiation: Lmd loss-of-function or Zfh1 gain-of-function leads to increased AMPs or PCs, whereas Lmd gain-of-function and Zfh1 loss-of-function reduce these cell types. Consequently, Lmd and Zfh1 can be considered to be functional antagonists, although their repression is not mutual. One possible explanation for the antagonistic effect of Zfh1 overexpression might be due to its direct negative influence on mef2 expression, thereby counteracting the mef2 activating function of Lmd. The vertebrate functional orthologue of Zfh1, ZEB2 (or Sip1), also inhibits myotube development in culture and represses a number of myogenic genes, and is able to rescue Zfh1 function in Drosophila (Sellin, 2009).

Lmd is instructed to restrict Tin expression by a late, pro-myogenic Dpp signal: While in wild type embryos Tin expression is repressed in cells destined to become dorsal FCMs between stages 12 and 13, there is a prolonged co-localization of Tin and lmd mRNA in cells of the dorsal mesoderm in lmd mutant embryos. As a consequence, dorsal FCMs adopt pericardial cell fates in the absence of Lmd function. Of note, this effect can also be observed in embryos carrying the dppd6 disk region mutation. These embryos lack a late Dpp signal (beginning at about stage 12) that is involved in pericardial restriction. Early Dpp signaling does not seem to be affected since the dorsal mesoderm (characterized by Dpp-dependent Tin expression) is normal in dppd6 mutant embryos. Quite contrary to embryos with otherwise decreased Dpp signaling and a reduced pericardial field, such as mad1-2 embryos, the dppd6 mutant embryos display a pericardial hyperplasia phenotype that resembles in many aspects the phenotype observed in lmd mutant embryos. Too many OPCs, TPCs and atypical TPCs without Zfh1 expression are also detected, although the dppd6 mutant phenotype is milder than the lmd mutant phenotype. This resemblance in phenotypes suggested an epistatic relationship of Lmd and the late Dpp signal. In addition, the accumulation of phosphorylated Mad (pMad) has been traced in PCs and cells within the dorsal musculature that are not positive for founder specific Kr or Eve expression, and hence are likely to be FCMs. Altogether, these findings lead to the hypothesis that Lmd might be a target of the late Dpp signal in FCMs. However, Lmd is expressed in a normal pattern (both at the mRNA and protein levels) in dppd6 mutant embryos, indicating that Lmd expression is independent of Dpp signaling. Nevertheless, co-staining with anti-Tin antibody revealed a prolonged co-localization of Tin and lmd mRNA in dppd6 mutant embryos until stage 14/15, as observed in lmd mutant embryos, suggesting a requirement for late Dpp signaling in the process of pericardial restriction by Lmd. To assess if the restrictive influence of late Dpp signaling on Tin expression is indeed relayed by Lmd in the dorsal mesoderm, or if both negative effects are independent of each other, the late Dpp signal was enhanced in the lmd mutant background. For this purpose, the leading edge driver LE-Gal4 was used to overexpress Dpp, which was shown to reduce the number of OPCs and TPCs in the wild type background. It was reasoned that this effect would be lost in lmd mutant embryos if Lmd is responsible for the restricting effect on PC number. The number of OPCs was counted in LE > Dpp; lmd embryos in comparison to lmd mutant embryos. While overexpression of Dpp with the LE-Gal4 driver in the wild type background led to a reduction of OPCs by ~1.2-fold, no reduction of OPCs was observed in the lmd mutant background, indicating that Lmd is indeed necessary to interpret the late Dpp signal as pro-myogenic. Altogether, these data suggest that the pro-myogenic effect of the late Dpp signal is Lmd dependent, although not by inducing Lmd expression. Instead, the presence of Dpp activity seems to be a prerequisite for the negative influence of Lmd on Tin expression and might act as an instructive signal to modify Lmd activity to allow repression of Tin. If the late Dpp signal is lost -- as is the case in embryos carrying the hypomorphic allele dppd6 - repression of Tin fails even in the presence of Lmd protein, indicating that repressive activity of Lmd is dependent on Dpp signaling (Sellin, 2009).

A model is proposed in which the subdivision of the early Tin positive primordium into pericardial and dorsal muscle tissues is mediated via the antagonistic action of Lmd and Zfh1 under the instructive influence of late Dpp signals. While the early function of Dpp restricts Tin expression to the dorsal mesoderm, subsequent Dpp signaling provides pro-myogenic input to modulate the pericardial field in favor of the dorsal musculature. The present data show that the function of this late Dpp signal requires Lmd activity, strongly suggesting that Lmd is a target of Dpp for establishing the boundary between the dorsal musculature and pericardial field. Repression of Tin also appears to be dependent on Dpp signaling. The previous observation that pMad accumulation occurs in PCs and dorsal muscle cells, which are likely to be FCMs, is consistent with the finding that Lmd is needed to relay the pro-myogenic function of late Dpp signaling. These cells originate from the Tin-expressing dorsal mesoderm, and co-expression of Tin and lmd mRNA in wild type embryos at stage 12 can be observed. In the presence of Lmd protein, this co-expression is not maintained after stage 12 due to a repressive function of Lmd on Tin. Of note, it was previously shown that Lmd function depends on posttranscriptional mechanisms that modulate its specific subcellular localization and activity, and it might be speculated that Dpp signaling is involved in changing Lmd function into a repressive form. However, there is no evidence that the negative influence of Lmd on Tin expression is of a direct nature, or if there are other factors that are involved in the process. In this context, the following explanation for the antagonistic effect of Zfh1 overexpression without repression of Lmd could also be considered. Since the vertebrate homologue ZEB2 was shown to inhibit activation of target genes by Smads, an excess of Zfh1 might antagonize the late Dpp signal by repressing pMad-dependent interaction partners of Lmd, thereby preventing the repression of Tin (and/or other targets) in the dorsal mesoderm. Lmd expression and function would not be affected elsewhere which would be consistent with the observation that Zfh1 is not a general repressor of Lmd (Sellin, 2009).

Pulsed contractions of an actin-myosin network drive apical constriction

Apical constriction facilitates epithelial sheet bending and invagination during morphogenesis. Apical constriction is conventionally thought to be driven by the continuous purse-string-like contraction of a circumferential actin and non-muscle myosin-II (myosin) belt underlying adherens junctions. However, it is unclear whether other force-generating mechanisms can drive this process. This study shows, with the use of real-time imaging and quantitative image analysis of Drosophila gastrulation, that the apical constriction of ventral furrow cells is pulsed. Repeated constrictions, which are asynchronous between neighbouring cells, are interrupted by pauses in which the constricted state of the cell apex is maintained. In contrast to the purse-string model, constriction pulses are powered by actin-myosin network contractions that occur at the medial apical cortex and pull discrete adherens junction sites inwards. The transcription factors Twist and Snail differentially regulate pulsed constriction. Expression of snail initiates actin-myosin network contractions, whereas expression of twist stabilizes the constricted state of the cell apex. These results suggest a new model for apical constriction in which a cortical actin-myosin cytoskeleton functions as a developmentally controlled subcellular ratchet to reduce apical area incrementally (Martin, 2009).

During Drosophila gastrulation, apical constriction of ventral cells facilitates the formation of a ventral furrow and the subsequent internalization of the presumptive mesoderm. Although myosin is known to localize to the apical cortex of constricting ventral furrow cells, it is not known how myosin produces force to drive constriction. Understanding this mechanism requires a quantitative analysis of cell and cytoskeletal dynamics. Methods were developed to reveal and quantify apical cell shape with Spider-GFP, a green fluorescent protein (GFP)-tagged membrane-associated protein that outlines individual cells. Ventral cells were constricted to about 50% of their initial apical area before the onset of invagination and continued to constrict during invagination. Although the average apical area steadily decreased at a rate of about 5 microm2 min-1, individual cells showed transient pulses of rapid constriction that exceeded 10-15 microm2 min-1. During the initial 2 min of constriction, weak constriction pulses were often interrupted by periods of cell stretching. However, at 2 min, constriction pulses increased in magnitude and cell shape seemed to be stabilized between pulses, leading to net constriction. These two phases probably correspond to the 'slow/apical flattening' and 'fast/stochastic' phases that have been described previously. Overall, cells underwent an average of 3.2 ± 1.2 constriction pulses over 6 min, with an average interval of 82.8 ± 48 s between pulses (mean ± s.d., n = 40 cells, 126 pulses). Constriction pulses were mostly asynchronous between adjacent cells. As a consequence, cell apices between constrictions seemed to be pulled by their constricting neighbours. Thus, apical constriction occurs by means of pulses of rapid constriction interrupted by pauses during which cells must stabilize their constricted state before reinitiating constriction (Martin, 2009).

To determine how myosin might generate force during pulsed constrictions, myosin and cell dynamics were simultaneously imaged by using myosin regulatory light chain (spaghetti squash, or squ) fused to mCherry (Myosin-mCherry) and Spider-GFP. Discrete myosin spots and fibres present on the apical cortex formed a network that extended across the tissue. These myosin structures were dynamic, with apical myosin spots repeatedly increasing in intensity and moving together (at about 40 nm s-1) to form larger and more intense myosin structures at the medial apical cortex. This process, which is referred to as myosin coalescence, resulted in bursts of myosin accumulation that were correlated with constriction pulses. The peak rate of myosin coalescence preceded the peak constriction rate by 5-10 s, suggesting that myosin coalescence causes apical constriction. Between myosin coalescence events, myosin structures, including fibres, remained present on the cortex, possibly maintaining cortical tension between constriction pulses. Contrary to the purse-string model, no significant myosin accumulation was seen at cell-cell junctions. To confirm that constriction involved medial myosin coalescence and not contraction of a circumferential purse-string, constriction rate was correlated with myosin intensity at either the medial or junctional regions of the cell. Apical constriction was correlated more significantly with medial myosin, suggesting that, in contrast to the purse-string model, constriction is driven by contractions at the medial apical cortex (Martin, 2009).

Myosin coalescence resembled contraction of a cortical actin-myosin network. Therefore, to determine whether apical constriction is driven by pulsed contractions of the actin-myosin network, the organization of the cortical actin cytoskeleton was examined. In fibroblasts and keratocytes, actin network contraction bundles actin filaments into fibre-like structures. Consistent with this expectation was the identification of an actin filament meshwork underlying the apical cortex in which prominent actin-myosin fibres spanning the apical cortex appeared specifically in constricting cells. An actin-myosin network contraction model would predict that myosin coalescence results from myosin spots exerting traction on each other through the cortical actin network. To test whether myosin coalescence requires an intact actin network, the actin network was disrupted with cytochalasin D (CytoD). Disruption of the actin network with CytoD resulted in apical myosin spots that localized together with actin structures and appeared specifically in ventral cells. Myosin spots in CytoD-injected embryos showed more rapid movement than those in control-injected embryos, suggesting that apical myosin spots in untreated embryos are constrained by the cortical actin network. Although myosin movement was uninhibited in CytoD-treated embryos, myosin spots failed to coalesce and cells failed to constrict. Because myosin coalescence requires an intact actin network, it is proposed that pulses of myosin coalescence represent contractions of the actin-myosin network (Martin, 2009).

Because actin-myosin contractions occurred at the medial apical cortex, it was unclear how the actin-myosin network was coupled to adherens junctions. Therefore E-Cadherin-GFP and Myosin-mCherry were imaged to examine the relationship between myosin and adherens junctions. Before apical constriction, adherens junctions are present about 4 microm below the apical cortex. As apical constriction initiated, these subapical adherens junctions gradually disappeared and adherens junctions simultaneously appeared apically at the same level as myosin. This apical redistribution of adherens junctions occurred at specific sites along cell edges (midway between vertices). As apical constriction initiated, these sites bent inwards. This bending depended on the presence of an intact actin network, which is consistent with contraction of the actin-myosin network generating force to pull junctions. Indeed, myosin spots undergoing coalescence were observed to lead adherens junctions as they transiently bent inwards. Thus, pulsed contraction of the actin-myosin network at the medial cortex seems to pull the cell surface inwards at discrete adherens junction sites, resulting in apical constriction (Martin, 2009).

The transcription factors Twist and Snail regulate the apical constriction of ventral furrow cells. Snail is a transcriptional repressor whose target or targets are currently unknown, whereas Twist enhances snail expression and activates the expression of fog and t48, which are thought to activate the Rho1 GTPase and promote myosin contractility. To examine the mechanism of pulsed apical constriction further, how Twist and Snail regulate myosin dynamics was tested. In contrast to wild-type ventral cells, in which myosin was concentrated on the apical cortex, twist and snail mutants accumulated myosin predominantly at cell junctions, similarly to lateral cells. These ventral cells failed to constrict productively, which supported the cortical actin-myosin network contraction model, rather than the purse-string model, for apical constriction. twist and snail mutants differentially affected the coalescence of the minimal myosin that did localize to the apical cortex. Although myosin coalescence was inhibited in snail mutants, it still occurred in twist mutants, as did pulsed constrictions. This difference was also observed when Snail or Twist activity was knocked down by RNA-mediated interference. However, the magnitude of constriction pulses in twistRNAi embryos was greater than that of twist mutant embryos, suggesting that the low level of Twist activity present in twistRNAi embryos enhances contraction efficiency by activating the expression of snail or other transcriptional targets. Myosin coalescence was inhibited in snail twist double mutants, demonstrating that the pulsed constrictions in twist mutants required snail expression. Thus, the expression of snail, not twist, initiates the actin-myosin network contractions that power constriction pulses (Martin, 2009).

Net apical constriction was inhibited in both snailRNAi and twistRNAi embryos. It was therefore asked why the pulsed contractions that were observed in twistRNAi embryos failed to constrict cells. Using Spider-GFP to visualize cell outlines, it was found that although constriction pulses were inhibited in snailRNAi embryos, constriction pulses still occurred in twistRNAi embryos. However, the constricted state of cells in twistRNAi embryos was not stabilized between pulses, resulting in fluctuations in apical area with little net constriction. This stabilization defect was not due to lower snail activity, because these fluctuations continued when snail expression was driven independently of twist by using the P[sna] transgene. Although the frequency and magnitude of constriction pulses in such embryos were similar to those in control embryos, stretching events were significantly higher in twistRNAi; P[sna] embryos, suggesting a defect in maintaining cortical tension. This defect might result from a failure to establish a dense actin meshwork, because both twist mutants and twistRNAi embryos had a more loosely arranged apical meshwork of actin spots and fibres than constricting wild-type cells did. twist expression therefore stabilizes the constricted state of cells between pulsed contractions (Martin, 2009).

Thus, a 'ratchet' model is proposed for apical constriction, in which phases of actin-myosin network contraction and stabilization are repeated to constrict the cell apex incrementally. In contrast to the purse-string model, it was found that apical constriction is correlated with pulses of actin-myosin network contraction that occur on the apical cortex. Pulsed cortical contractions could allow dynamic rearrangements of the actin network to optimize force generation as cells change shape. Because contractions are asynchronous, cells must resist pulling forces from adjacent cells between contractions. A cortical actin-myosin meshwork seems to provide the cortical tension necessary to stabilize apical cell shape and promote net constriction. The transcription factors Snail and Twist are critical for the contraction and stabilization phases of constriction, respectively. Thus, Snail and Twist activities are temporally coordinated to drive productive apical constriction. Despite the dynamic nature of the contractions in individual cells, the behaviour of the system at the tissue level is continuous, in a similar manner to convergent extension in Xenopus. Pulsed contraction may therefore represent a conserved cellular mechanism that drives precise tissue-level behaviour (Martin, 2009).

Discrete Levels of Twist activity are required to direct distinct cell functions during gastrulation and somatic myogenesis

Twist (Twi), a conserved basic helix-loop-helix transcriptional regulator, directs the epithelial-to-mesenchymal transition (EMT), and regulates changes in cell fate, cell polarity, cell division and cell migration in organisms from flies to humans. Analogous to its role in EMT, Twist has been implicated in metastasis in numerous cancer types, including breast, pancreatic and prostate. In the Drosophila embryo, Twist is essential for discrete events in gastrulation and mesodermal patterning. In this study, a twi allelic series was derived by examining the various cellular events required for gastrulation in Drosophila. By genetically manipulating the levels of Twi activity during gastrulation, it was found that coordination of cell division is the most sensitive cellular event, whereas changes in cell shape are the least sensitive. Strikingly, it was shown that by increasing levels of Snail expression in a severe twi hypomorphic allelic background, but not a twi null background, gastrulation can be reconstituted and viable adult flies can be produced. The results demonstrate that the level of Twi activity determines whether the cellular events of ventral furrow formation, EMT, cell division and mesodermal migration occur (Wong, 2014).

Hypomorphic twi alleles were some of the earliest identified Drosophila embryonic mutants. Our thorough genetic characterization of these alleles and the establishment of a twi allelic series has helped to fine-tune understanding of twi function during gastrulation and mesodermal development. By genetically titrating Twi, insight has been gained into the mechanisms by which activation of twi target genes translates to cellular process, such as mesoderm invagination, EMT, proliferation and migration. Finally, it was shown that expression of twi target genes in the twiRY50/twi1 background can rescue certain aspects of mesoderm and somatic muscle development. In the case of Sna, this rescue was nearly complete and included adult viability. These findings have deepened understanding of how twi controls multiple target genes during mesoderm and muscle development, and can be more broadly applied to vertebrate development and human cancer progression (Wong, 2014).

Based on embryonic phenotypes, the allelic series from least to most severe is: twiV50/twiV50, twiV50/twi1, twiRY50/twiRY50, twiRY50/twi1and twi1/twi1. Analysis of mutant embryos has shown that certain cellular processes have a greater sensitivity to the twi genetic background than others. Previously studies have shown that somatic myogenesis is exceptionally sensitive to twi levels. Similarly, the number of invaginated cells during gastrulation has a direct correspondence to the twi allelic series, with each step down yielding 2 fewer invaginated cells. In contrast, formation of the ventral furrow is a robust process, with no disruption except for the strong allelic combinations of twiRY50/twi1 and twi1/twi1. One way to explain the current data is to assign different levels of twi activity to these allelic combinations, with twiV50/twiV50 having the greatest twi activity level and twiRY50/twi1 the least (and twi1/twi1 having none). Following this logic, ventral furrow formation would require the least amount of twi activity and somatic myogenesis would require a large amount of activity. This explanation is complicated by the pleiotropic nature of twi function, and the possibility that twi acts in different ways at its gene targets to regulate their function in different cell types. Factors contributing to the differential response of particular twi target genes could include: the number of twi binding sites, the chromatin landscape, the twi binding partner and the requirement for other cofactors. Further experiments will be required to assign specific cellular functions to particular twi alleles, and help us to elucidate the particular roles twi plays in each discrete process (Wong, 2014).

An extension of this analysis provides a hypothesis for the developmental delays that occur in even the weakest twi allelic combinations: inefficient activation of twi target genes due to reduced twi activity requires the embryo to put cell processes on hold until these gene products have built up sufficient expression to advance the process in question. This hypothesis is best illustrated in twiV50/twi1 embryos, where levels of Htl and Dmef2 appear low, fewer cells make up the VF to become mesodermal cells, and the development of these embryos is delayed. Nevertheless, the final muscle pattern in twiV50/twi1 embryos was relatively normal, with only some missing and mispatterned muscles. This recovery suggests that, ultimately, mesoderm and muscle development is robust and can proceed in twi hypomorphs with a reduced number of founding mesodermal cells (Wong, 2014).

Though processes such as VF formation and muscle development were only affected in twi hypomorphs, synchronized mesoderm mitosis is one process that is disrupted in both twi hypermorphs and hypomorphs. Mesodermal mitosis occurs asynchronously in twiRY50/twiRY50 embryos, as well as twi overexpression embryos. Additionally, Sna rescue of twiRY50/twi1 embryos causes mesodermal mitosis to occur asynchronously. This sensitivity may be due to the Twi's role in both negatively and positively regulating the activity and expression of the cell cycle regulator String/cdc25. Previous work has shown that String, a Ser/Thr phosphatase, is precisely regulated by twi to achieve synchronized cell divisions in the mesoderm. This finding has important implications for disease, as twi has been shown to regulate cell proliferation both in cancer cells and mesenchymal stem cells (Wong, 2014).

The Sna rescue data also illustrates how twi can function with other transcriptional regulators. twi and Daughterless are examples of regulators that switch between transcriptional activation and repression depending on binding partner and tissue context. Moreover, twi and Sna have been shown to concomitantly bind enhancers associated with htl and tinman. Independently, Sna has been shown to repress number of genes, such as single-minded, rhomboid, wntD and short gastrulation, in the ventral-most mesodermal cells, restricting their expression to lateral regions. The repression of these target genes, however, functions to promote gastrulation, as Sna is essential for the initiation of cell shape changes, even in the absence of twi. For example, twi null mutant embryos, which briefly express Sna in a Dl-dependent/Twi-independent manner, exhibit cell shape changes that are required for mesodermal invagination. These cell shape changes, however, are entirely missing in twi/sna double mutants (Wong, 2014).

Full Sna rescue was specific to the twiRY50/twi1 hypomorphic background, while Sna overexpression in twi1 homozygous null embryos had a limited rescue function. What mechanism could explain the ability of Sna overexpression to rescue hypomorphic mutant embryos, but not homozygous null embryos? While Sna was initially characterized as a transcriptional repressor, recent work has uncovered a role for Sna in the activation of mesodermal target genes. A subset of Sna target genes, including Dmef2, htl and tin, are positively regulated by both twi and Sna. It is known, however, from the inability of overexpressed Sna to rescue gastrulation in twi1 homozygous mutants that this Sna activation of target genes is not sufficient Additionally, Sna binds to the twi enhancer and positively regulates its transcription. Consistent with this finding, this study observed that twiRY50/twi1 embryos rescued by Sna overexpression showed wild-type levels of twi expression. These results suggested one possible model where Sna rescue increases twiRY50 expression levels, thereby providing sufficient twi activity to drive gastrulation and mesoderm formation in combination with Sna. Given the data from recent whole genome studies, this rescue is likely direct (Wong, 2014).

Gastrulation relies on the level of twi activity to regulate the EMT and cell migration. Strikingly, in twiRY50/twi1 embryos, invaginated mesodermal cells appeared to undergo EMT initially, but, at later stages, these mesodermal cells were no longer observed. Because apoptosis was not detected in these embryos, one possible explanation is that the twiRY50/twi1 mesodermal cells underwent a mesenchymal-to-epithelial transition (MET) to revert back to their epithelial morphology. This effect suggests one role of twi is to prevent the EMT from reverting. This finding has important implications for human cancers, suggesting that therapeutic knockdown of twi could be crucial for halting the initiation of metastasis. The MET observed in twiRY50/twi1 embryos is also relevant to the next step of metastasis, as recent studies have found that metastatic cancer cells must revert back to an epithelial state in order to proliferate and form secondary tumors. Another possibility is that twiRY50/twi1 mesodermal cells are unable to maintain mesodermal cell fate and no longer express mesodermal markers. This possibility suggests that the cells have dedifferentiated, a process that is relevant for tumor formation and the development of cancer stem cells. Overall, these results highlight the parallels between mesodermal development and tumor formation, which suggests that nuanced regulation of twi is also critical for different stages of tumor development and metastasis. In fact, twi and its target genes, particularly Sna, have been implicated in various metastatic tumors including breast, esophageal and uterine cancers. Additionally, expression of twi in human tumors correlates to resistance to a number of chemotherapeutics as well as poor outcomes. Similar to their role in Drosophila gastrulation, both the Twist and Snail families of proteins control genes that direct cell shape changes and EMT in humans (Wong, 2014).

Finally, the allelic series developed in this study has provided a tractable genetic system for the study of other factors affecting cell shape changes, the EMT, cell proliferation and cell cycle regulation. The results are quantitative and provide benchmarks such as the number of invaginated cells in each genetic background. Therefore, this system provides an extremely sensitive in vivo read-out for testing the role of other genes, and potentially drugs, that affect processes directly relevant to human cancers (Wong, 2014).


During larval life, the twist-expressing cells proliferate and, in the abdomen, they form ventral, lateral and dorsal clusters that are the precursors of the adult abdominal muscles, while in the thorax, they form populations of cells in the imaginal discs that correspond to the adepithelial cells (Bate, 1991).

During metamorphosis, the adult muscles of the abdomen develop from pools of myoblasts that are present in the larva. The adult myoblasts express twist in the third larval instar and the early pupa and are closely associated with nerves. Growing adult nerves and the twist-expressing cells migrate out across the developing abdominal epidermis, and as twist expression declines, the myoblasts begin to synthesize Beta 3 tubulin. There follows a process involving cell fusion and segregation into cell groups to form multinucleate muscle precursors. These bipolar precursors migrate at both ends to find their correct attachment points (Currie, 1991).

In Drosophila the precursors of the adult musculature arise during embryogenesis. These precursor cells have been termed Persistent Twist Cells (PTCs), since they continue to express the transcription factor Twist after that gene ceases expression elsewhere in the mesoderm. In the larval abdomen, the PTCs are associated with peripheral nerves in stereotypic ventral, dorsal, and lateral clusters, which give rise, respectively, to the ventral, dorsal, and lateral muscle fiber groups of the adult. The developmental potential of the PTCs was tested by using a microbeam laser to ablate specific clusters in larvae. The ablation of a single segmental PTC cluster does not usually result in the deletion of the corresponding adult fibers of that segment. Instead, normal or near normal numbers of adult fibers can form after the ablation. Examination of pupae following ablation shows that migrating PTCs from adjacent segments are able to invade the affected segment, replenishing the ablated cells. However, the ablation of homologous PTCs in multiple segments does result in the deletion of the corresponding adult muscle fibers. These data indicate that the PTCs in an abdominal segment can contribute to the formation of muscle fibers in adjacent abdominal segments, and thus are not inherently restricted to the formation of muscle fibers within their segment of origin (Farrell, 1999).

In insects, specialized mesodermal cells serve as templates to organize myoblasts into distinct muscle fibers during embryogenesis. In the grasshopper embryo, large mesodermal cells called muscle pioneers extend between the epidermal attachment points of future muscle fibers and serve as foci for myoblast fusion. In the Drosophila embryo, muscle founder cells serve a similar function, organizing large numbers of myoblasts into larval muscles. During the metamorphosis of Drosophila, nearly all larval muscles degenerate and are replaced by a set of de novo adult muscles. The extent to which specialized mesodermal cells homologous to the founders and pioneers of the insect embryo are involved in the development of adult-specific muscles has yet to be established. In the larval thorax, the majority of imaginal myoblasts are associated with the imaginal discs. Reported in this study is the identification of a morphologically distinct class of disc-associated myoblasts called imaginal pioneers that prefigure the formation of at least three adult-specific muscles (the three largest adult muscles in the thorax): the mesothoracic (segment T2) tergal depressor of the trochanter (TDT) and dorsoventral muscles I and II (DVM-I and -II). Like the muscle pioneers of the grasshopper, the imaginal pioneers attach to the epidermis at sites where the future muscle insertions will arise and erect a scaffold for developing adult muscles. These findings suggest that a prior segregation of imaginal myoblasts into at least two populations, one of which may act as pioneers or founders, must occur during development (Rivlin, 2000).

In the thorax, the majority of myoblasts that will contribute to the developing adult muscles are associated with the imaginal discs. In the embryo, the imaginal discs arise as invaginations of the epidermis. By the end of the third larval instar, the mature discs appear as sac-like structures that are connected to the epidermis via an attachment stalk. Each disc and its stalk are surrounded by a common basal lamina. The disc-associated myoblasts, also called 'adepithelial cells' to distinguish them from the epithelial cells of the imaginal discs, reside in a cavity between the disc epithelium and the basal lamina (Rivlin, 2000).

An examination was carried out to see whether a diverse population of precursors contributes to the developing adult muscles. Two techniques were used in an effort to identify those cells that contribute to the developing adult muscles: an antibody against twist and twist-lacZ. Both of these techniques rely on the persistent expression of twist within the population of adult muscle precursors. An antibody against Twist was used to examine diversification within the population of adult muscle precursors in embryos and larval Drosophila. To track the fate of these precursors in the pupa, and to identify the first appearance of anlagen for the DVMs and TDT, the transgene twist-lacZ was used. Although twist expression ceases after myoblasts fuse, the syncytial precursor or anlage can be identified due to the perdurance of the lacZ product. By the end of embryogenesis, 13-15 Twist-expressing adult muscle precursors in T1 and 17-18 precursors each in T2 and T3 are associated with the primordia for the imaginal discs. During the first two larval instars, while there is a steady increase in the number of adult muscle precursors in each thoracic segment, all myoblasts appear to be uniform in size. However, by the start of the third and final larval instar, the population of adult muscle precursors is diversified. At this stage, up to five Twist-expressing cells with large nuclei are seen on the stalk connecting the T2 leg disc to the epidermis. Measuring 9-10 mm in diameter, the large nuclei were more than twice the size of the other disc-associated twist-expressing nuclei (Rivlin, 2000).

These large twist-expressing cells prefigure a subset of the adult thoracic muscles and are here referred to as imaginal pioneers (IPs). Between 4 and 6 h APF, the imaginal discs evert and elongate to form the adult appendages and epidermis of the thorax. By the end of disc eversion, the unfolding leg and wing disc epithelia meet and fuse to form the lateral part of the thorax. At this time, a continuous pool of myoblasts extends between the lumen of the leg and the lumen of the wing. In whole mounts, IPs could be identified by their large nuclei just anterior and posterior to the lumen of the leg, in the region where DVM-I and -II, respectively, will develop. Unlike earlier stages, by 6 h APF, the IP nuclei could be reliably identified in a lacZ background. 2.82 +/- 0.12 (mean +/- SEM), and 1.88 +/- 0.17 IP nuclei were identified in the regions where DVM-I and -II will develop, thus suggesting that each DVM fiber is prefigured by a single IP (Rivlin, 2000).

From 8 to 12 h after puparium formation (APF), the TDT anlage retains its ovoid shape but increases in size and density. In whole mounts, from 13 to 16 IP nuclei can be resolved in the developing TDT at 12 h APF. Because sections through the TDT anlage at 4 h APF reveal that the anlage is composed of 12 to 13 IPs, it is concluded that IP number does not vary significantly from 4 to 12 h APF. From 14 to 18 h APF, the developing TDT extends dorsally and nearly doubles in length. Due to the high density of nuclei during this period, individual IP nuclei could not be resolved in whole mounts. The fate of the IP nuclei in the TDT after 12 h APF could therefore not be determined. From 20 to 24 h APF, the developing TDT increases in width. A close examination of sections through the TDT at this time reveals that the TDT is comprised of an array of thin fibers that surround a large central core containing a mass of loosely packed myoblasts. Myoblasts are also clustered at the dorsal end of the muscle, suggesting that elongation may occur, in part, by the fusion of myoblasts with the dorsal end of the muscle. Myoblasts that are mitotic are observed in the core and at the dorsal end of the TDT. A survey of other tubular muscles (i.e., pleural sternal muscle) suggests that they are also comprised of an array of fibers surrounding a core of available myoblasts and may therefore follow a similar developmental strategy (Rivlin, 2000).

In contrast, the development of the DVMs does not involve a central core of myoblasts like that observed in the development of the TDT. At 24 h APF, each DVM fiber appears as a syncytium of nuclei surrounded by individual myoblasts. Unlike the TDT, IP nuclei are identifiable in the developing DVMs after 12 h APF. Using twist-lacZ and the indirect flight muscle (IFM) marker, act88F-lacZ, the fate of the IP nuclei was followed in the developing DVMs from 6 to 24 h APF. No variation in the number and size of the IP-derived nuclei was observed during this period. By 16 h APF the three and two fibers of DVM-I and -II, respectively, are clearly visible. At this stage, each fiber contains a single IP nucleus, typically located in the middle of the fiber. This nucleus is still present at 24 h APF when the fiber becomes contractile. Individual IP nuclei could not be resolved beyond 24 h APF due to the highly contracted state of the DVM fibers (Rivlin, 2000).

While these findings strongly suggest that the IPs preconfigure a subset of adult thoracic muscles, a second issue that needs to be addressed is whether the IPs also serve as muscle founders. During embryogenesis, the formation of larval muscles is seeded by a set of specialized mesodermal cells called muscle founders. Each founder cell subsequently fuses with a set of undifferentiated, fusion-competent mesodermal cells, also called feeder myoblasts, to produce a syncytial precursor. According to the founder cell model, the founder cell nucleus entrains feeder nuclei within the syncytium to follow a pattern of gene expression and, hence, a particular developmental pathway. The key feature of this hypothesis is that the founder cell acts as a conduit for the transfer of information to feeder myoblasts. At this point, there no direct evidence that IPs function as muscle founders. However, several pieces of indirect evidence suggest that muscle founders participate in the development of adult muscles. Transplantation experiments have demonstrated that disc-associated myoblasts are competent to fuse with and develop into a diverse group of thoracic and abdominal muscles. Furthermore, it has been demonstrated that the pattern of gene expression in thoracic donor nuclei is “entrained” by those of the host abdominal muscle to which they fuse. This has led many to argue that the disc-associated myoblasts are imaginal feeder myoblasts and, like the fusion-competent myoblasts in the embryo, represent an undifferentiated set of equivalent cells that require a founder cell for their proper differentiation. The IPs identified in this study may represent the founder cells with which these feeders fuse (Rivlin, 2000).

Effects of Mutation or Deletion

In the mutants twist and snail, which fail to differentiate the ventrally derived mesoderm, mitoses specific to the mesoderm are absent. The lateral mesectodermal domain shows a partial ventral shift in twist mutants but a proportion of ventral cells do not behave characteristically, suggesting that twist has a positive role in the establishment of the mesoderm. In contrast, snail is required to repress mesectodermal fates in cells of the presumptive mesoderm. In the absence of both genes, the mesodermal and the mesectodermal anlage are deleted (Arora, 1992).

In Drosophila, ventral furrow formation and mesoderm differentiation are initiated by two regulatory genes, twist and snail. Sna is sufficient to initiate the invagination of the ventral-most embryonic cells in the absence of twi+ gene activity, but the invagination process is subsequently arrested (Ip, 1994).

twist and snail have a critical role in development of the endoderm. Aspects of midgut development, migration of anterior midgut (AMG) primordium and the posterior midgut (PMG) primordium, and transition to an endodermal epithelium -- all depend on the mesoderm. The extension of the midgut primordia is achieved by cell migration along the visceral mesoderm which forms a continuous layer of cells within the germ band. In mutant embryos lacking the entire mesoderm or failing to differentiate the visceral mesoderm, AMG and PMG are formed but do not migrate properly. In addition, they fail to form an epithelium and instead either remain as compact cell masses anterior and posterior to the yolk (in twist and snail mutant embryos) or only occasionally wrap around the yolk before embryogenesis is completed (in tinman-deficient embryos). Thus the visceral mesoderm serves as a substratum for the migrating endodermal cells. Contact between visceral mesoderm and endoderm is required for the latter to become an epithelium (Reuter, 1993).

Both the extracellular neural lamella, which ensheaths the CNS, and its source, the underlying perineurial sheath cell layer, fail to develop in Drosophila embryos that are homozygous for a loss of function mutation in the twist gene, and which thus lack mesodermal derivatives. The cell layer immediately below the perineurial sheath cells composed of barrier glial cells, constitutes the ion permeability barrier in wild-type embryos. This barrier, present in twist mutant embryos, appears to be normal at the ultrastructural level, and functions as a blood-brain ion barrier (Edwards, 1993).

Developing sensory axons have been studied in Drosophila embryos that carry a mutation in the trachealess and/or the twist gene. In these embryos, the tracheae and/or somatic muscles that represent part of the substrate on which sensory axons normally grow, are absent. The results demonstrate that in each of these three mutant backgrounds, the majority of sensory nerves form normally. This indicates that neither the tracheae nor the somatic musculature is absolutely required for pathfinding of the embryonic sensory axons. However, the incidence of misrouted axons is significantly increased, most strongly in the trh, twi double mutant. Furthermore, axonal elongation is considerably slowed down, and sensory neurons frequently fail to send out an axon (Younossi-Hartenstein, 1993).

Hemocytes derive exclusively from the mesoderm of the head and disperse along several invariant migratory paths throughout the embryo. The origin of hemocytes from the head mesoderm is further supported by the finding that in Bicaudal D, a mutation that lacks all head structures, and in twist snail double mutants, where no mesoderm develops, hemocytes do not form. All embryonic hemocytes behave like a homogeneous population with respect to their potential for phagocytosis. Thus, in the wild type, about 80-90% of hemocytes become macrophages during late development. In the Drosophila embryo, apoptosis can occur independently of macrophages, since mutations lacking macrophages show abundant cell death (Tepass, 1994).

During Drosophila embryogenesis the Malpighian tubules evaginate from the hindgut anlage and in a series of morphogenetic events form two pairs of long narrow tubes, each pair emptying into the hindgut through a single ureter. Some of the genes that are involved in specifying the cell type of the tubules have been described. Mutations of previously described genes were surveyed and ten were identified that are required for morphogenesis of the Malpighian tubules. Of those ten, four block tubule development at early stages; four block later stages of development, and two, rib and raw, alter the shape of the tubules without arresting specific morphogenetic events. Three of the genes, sna, twi, and trh, are known to encode transcription factors and are therefore likely to be part of the network of genes that dictate the Malpighian tubule pattern of gene expression (Jack, 1999).

twi and sna, were found to be necessary for development of the tubules beyond evagination. These genes could function downstream of Kruppel and parallel to cut to implement the Malpighian tubule specific pattern of gene expression. Both twi and sna function in determining mesodermal fate. The effect of sna mutations on the morphogenesis of the tubules, which are of ectodermal origin, can be explained by the fact that sna is also expressed in the Malpighian tubules. twi expression has not been reported in the Malpighian tubules although the distribution of the protein throughout embryogenesis has been described. One possibility is that twi is expressed in the Malpighian tubules at a level that was undetected or at a time before the budding of the tubules. twi is active in the development of the embryonic termini. Lack of twi activity could lead to the failure of the tubules to develop by virtue of a defect in terminal specification that occurs prior to budding of the tubules. Alternatively, the effect of twi may be indirect, possibly occurring through an inductive interaction of another tissue on the Malpighian tubules (Jack, 1999).

The Drosophila tracheal system is a model for the study of the mechanisms that guide cell migration. The general conclusion from many studies is that migration of tracheal cells relies on directional cues provided by nearby cells. However, very little is known about which paths are followed by the migrating tracheal cells and what kind of interactions they establish to move in the appropriate direction. An analysis has been carried out of how tracheal cells migrate relative to their surroundings and which tissues participate in tracheal cell migration. Cells in different branches are found exploit different strategies for their migration; while some migrate through preexisting grooves, others make their way through homogeneous cell populations. Alternative migratory pathways of tracheal cells are associated with distinct subsets of mesodermal cells and a model is proposed for the allocation of groups of tracheal cells to different branches. These results show how adjacent tissues influence morphogenesis of the tracheal system and offer a model for understanding how organ formation is determined by its genetic program and by the surrounding topological constraints (Franch-Marro, 2000).

Tracheal cells are first specified as clusters of ectodermal cells at the embryonic surface. Since tracheal cells invaginate and form the tracheal pits they occupy the grooves between the muscle precursors of adjacent metameres. The formation of this groove is independent of tracheal invagination because it also forms between metameres that do not have tracheal placodes and it also develops in trh mutant embryos, which do not undergo tracheal invagination. A subset of the tracheal cells moves anteriorly, whereas another subset moves posteriorly until they reach the cells from the adjacent placodes. These cells will form the dorsal trunk, the most prominent tracheal branch that spans the embryo longitudinally. Those cells migrate across the adjacent precursors of somatic muscles and separate the precursors of the most dorsal muscles from the precursors of more ventral muscles. Other cells, those from the dorsal side of the tracheal pit, move dorsally along the longitudinal groove to form the dorsal branches that will end up fusing with the dorsal branches coming from the contralateral hemisegments. In the ventral side, the tracheal cells follow two different paths along the two clusters of lateral muscle precursors at each side of the groove. Anterior ventral cells will form the anterior lateral trunk while the posterior ventral cells will form the posterior lateral trunk. Finally, another group of cells from a midposition in the tracheal pit will migrate inward and will form the visceral branch. To ascertain the role of the mesoderm in tracheal cell migration, an examination was carried out of what tracheal structures develop in embryos that lack mesoderm derivatives. In twi mutant embryos, formation of the tracheal tree is severely perturbed: there is no dorsal trunk and there is only some development of dorsal and ventral branches. Although twi mutant embryos are severely affected, their tracheal phenotype suggests an important role for the mesoderm in tracheal cell migration (Franch-Marro, 2000).

twist: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity and Protein Interactions | Developmental Biology | References

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