stumps

Transcriptional Regulation

Convergent intercellular signals must be precisely integrated in order to elicit specific biological responses. During specification of muscle and cardiac progenitors from clusters of equivalent cells in the Drosophila embryonic mesoderm, the Ras/MAPK pathway -- activated by both epidermal and fibroblast growth factor receptors -- functions as an inductive cellular determination signal, while lateral inhibition mediated by Notch antagonizes this activity. A critical balance between these signals must be achieved to enable one cell of an equivalence group to segregate as a progenitor while its neighbors assume a nonprogenitor identity. Whether these opposing signals directly interact with each other has been investigated, and how they are integrated by the responding cells to specify their unique fates was been examined. Two distinct modes of lateral inhibition, one Notch based and a second based on the epidermal growth factor receptor antagonist Argos, are described that have complementary and reinforcing functions. Argos/Ras and Notch do not function independently; rather, several modes of cross-talk between these pathways have been uncovered. Ras induces Notch, its ligand Delta, and Argos. Delta and Argos then synergize to nonautonomously block a positive autoregulatory feedback loop that amplifies a fate-inducing Ras signal. This feedback loop is characterized by Ras-mediated upregulation of proximal components of both the epidermal and fibroblast growth factor receptor pathways. In turn, Notch activation in nonprogenitors induces its own expression and simultaneously suppresses both Delta and Argos levels, thereby reinforcing a unidirectional inhibitory response. These reciprocal interactions combine to generate the signal thresholds that are essential for proper specification of progenitors and nonprogenitors from groups of initially equivalent cells (Carmena, 2002).

This study involves the origin of two progenitors from a single cell cluster. The two progenitors are characterized by expression of the segmentation gene eve and are specified in a distinct temporal order in the Drosophila embryonic mesoderm. Progenitor 2 (P2) develops first; it originates from the preC2 cluster which develops into the C2 cluster and subsequently gives rise to a single P2 cell. P2 divides asymmetrically to give rise to two founder cells, one specific for a pair of persistently Eve-positive heart-associated or pericardial cells (EPCs) in every hemisegment and a second of previously undetermined identity. This second founder coexpresses Eve along with the gap gene Runt, with Eve levels rapidly fading but Runt persisting as development proceeds. By the time that Eve is evident in the EPCs, Runt labels a single somatic muscle, dorsal oblique muscle 2 (DO2). Runt is also detected in the muscle DO2 precursor during germband retraction (Carmena, 2002).

The second Eve progenitor, P15, which also has its origin in the preC2 cluster (which gives rise to a C15 cluster) forms later than P2 and divides asymmetrically to yield the founders of dorsal acute muscle 1 (DA1) and another cell whose fate cannot be followed since a specific, stably expressed marker for it is unavailable (Carmena, 2002).

To further substantiate the lineage relationships among these progenitors and founders, observations related to RTK signaling dependence of P2 and P15 specification were used: whereas P15 requires the activities of both Egfr and Htl, only Htl is involved in P2 formation. In this way, targeted mesodermal expression of a dominant negative form of Egfr strongly blocks formation of DA1 but not the EPCs. Also, consistent with DO2 and EPC founders being the progeny of P2, DO2 development, like that of the EPCs, is not affected by dominant negative Egfr. Additional support for the sibling relationship between the DO2 and EPC founders derived from the analysis of targeted expression of a dominant negative form of Htl. Under conditions in which early mesoderm migration is not perturbed, dominant negative Htl generates an incompletely penetrant phenotype in which different hemisegments lose derivatives of P2, P15, or both progenitors. With such partial inhibition of Htl activity, muscle DO2 and the EPCs are consistently either both present or both absent from any given hemisegment; in no cases did one of these cell types develop without the other, as expected for cells derived from a common progenitor. In contrast, muscle DA1 frequently forms in the absence of muscle DO2 and the EPCs, consistent with its derivation from an independent progenitor. Taken together, these data establish that the EPC and DO2 founders are sibling cells of the P2 division, whereas the other Eve-expressing muscle founder arises from a different progenitor (Carmena, 2002).

This model differs from one derived on the basis of clonal analysis in which it was proposed that the two Eve-positive mesodermal cell types originate from the same progenitor. This discrepancy may relate to the fact that muscles form by sequential cell fusions involving both founders and fusion-competent cells of potentially different parental cell origins, thereby confounding the interpretation of clonal analysis in which the cytoplasm of a single myotube is labeled by the lineage tracing marker (Carmena, 2002).

Autoregulation of a signal transduction cascade can cause either enhancement or attenuation of the transduced signal, depending on whether the feedback loop acts positively or negatively. Both types of feedback control occur during the Ras- and N-mediated specification of Eve mesodermal progenitors. Ras activation leads to increased expression of several proximal components of both the Fgfr and Egfr pathways that serve to amplify and/or prolong both fate-inducing RTK/Ras signals in the emerging Eve progenitors. A similar amplification of Egfr signaling occurs via induction of Rho during Drosophila oogenesis and mesothoracic bristle formation, and via upregulation of Egfr expression during C. elegans vulva development. The present analysis also uncovers a positive feedback mechanism for inductive Fgfr signaling, in this case via increased expression of not only the Htl receptor but also its specific signal transducer, Heartbroken (Hbr). Interestingly, the data suggest that the downstream components may respond to different thresholds of Ras activity since Rho exhibits a less robust response than either Htl or Hbr to Ras activation (Carmena, 2002).

Competitive cross-talk between Ras and N is manifest by the ability of the latter to block the expression of proximal components of the two RTK pathwaysónamely Htl/Hbr and Rho -- as well as to prevent the associated activation of MAPK. An antagonistic relationship between the RTK and N pathways is also revealed by the strong genetic interaction between Dl and Egfr, in agreement with previously reported genetic studies. Collectively, these results establish that the RTK and N pathways are not simply acting in parallel to exert opposing influences on progenitor specification; rather, N must be interfering with the generation and/or transmission of the inductive RTK signal. This effect could occur at multiple levels. The ability of activated N to at least partially block MAPK activation induced by constitutive Ras argues that N functions downstream of Ras. An additional direct effect of N on expression of Ras-responsive target genes cannot be excluded, particularly since Enhancer of split repressors are involved in the specification of progenitor cell fates. Such targets could include eve itself, or, given positive autoregulation of RTK signaling, one or more RTK pathway components (Carmena, 2002).

Targets of Activity

The similarity of the mutant phenotype of stumps and the FGFRs htl and btl suggests that stumps may be required in the FGFR signaling pathway, but does not indicate whether it acts upstream or downstream of the Htl and Btl receptors. The expression pattern of Stumps mRNA and protein is identical to the combined expression patterns of the two FGFRs. Thus, stumps could be involved in the tissue-specific regulation of htl and btl expression or represent a target gene that is activated as a result of FGF signaling. However, stumps appears to have neither of these roles, since htl and btl expression are not affected in stumps mutant embryos, and conversely, stumps expression is not affected in htl and btl mutants. Signals from FGFRs are transmitted through the Ras/MAPK pathway. The state of activation of the downstream kinase ERK can be monitored in situ by staining with antibodies against the active, dual phosphorylated form of MAP kinase (dp-ERK. dp-ERK is seen in the invaginated mesoderm where it is initially restricted to the cells that contact the ectoderm, and later to cells at the leading edge of the mesoderm as it spreads over the ectoderm. The early dp-ERK staining is seen in those mesodermal cells that express stumps and contact the ectoderm. This mesodermal dp-ERK staining is dependent on Htl, since it is absent in htl mutant embryos. Later, dp-ERK is also seen in tracheal cells as migration is initiated. This staining is absent in btl mutants, showing that, as in the mesoderm, it depends on FGFR function. In stumps mutant embryos, despite the presence of both FGFRs, dp-ERK is detected neither in the mesoderm nor in the tracheae, demonstrating that the MAPK pathway fails to be activated in these organs. In other tissues, where activation of ERK relies on signals transmitted through other receptors (e.g., the EGF receptor), ERK is phosphorylated as in wild-type embryos (Vincent, 1998).

Protein Interactions

Dof is a large molecule essential for signal transduction by the two FGF receptors in Drosophila. It contains two ankyrin repeats and a coiled-coil region, but has no other recognisable structural motif. Dof shares these features with its closest vertebrate relatives, the B-cell signalling molecules BCAP and BANK. In addition, this family of proteins shares a region of homology upstream of the ankyrin repeats, which is called the Dof/BCAP/BANK (DBB) motif. Forty-four proteins have been identified that interact with Dof in a yeast two-hybrid screen. These include the Drosophila FGF-receptor Heartless and Dof itself. The integrity of the DBB motif is required both for Dof and for BCAP to form dimers. Analysis of the interactions between a set of deletion constructs of Dof and the panel of interactors suggests that Dof may adopt different conformations, with a folded conformation stabilized by interactions between the DBB motif and the C-terminal part of the protein (Battersby, 2003).

Fibroblast growth factor (FGF) receptor (FGFR) signaling controls the migration of glial, mesodermal, and tracheal cells in Drosophila melanogaster. Little is known about the molecular events linking receptor activation to cytoskeletal rearrangements during cell migration. A functional characterization has been performed of Downstream-of-FGFR (Dof), a putative adapter protein that acts specifically in FGFR signal transduction in Drosophila. By combining reverse genetic, cell culture, and biochemical approaches, it was demonstrated that Dof is a specific substrate for the two Drosophila FGFRs. After defining a minimal Dof rescue protein, two regions were identified that are important for Dof function in mesodermal and tracheal cell migration. The N-terminal 484 amino acids are strictly required for the interaction of Dof with the FGFRs. Upon receptor activation, tyrosine residue 515 becomes phosphorylated and recruits the phosphatase Corkscrew (Csw). Csw recruitment represents an essential step in FGF-induced cell migration and in the activation of the Ras/MAPK pathway. However, the results also indicate that the activation of Ras is not sufficient to activate the migration machinery in tracheal and mesodermal cells. Additional proteins binding either to the FGFRs, to Dof, or to Csw appear to be crucial for a chemotactic response (Petit, 2004).

Genetic epistasis experiments have shown that Dof functions downstream of the activated FGFRs and upstream or in parallel to Ras. However, the biochemical function of Dof in the interpretation of the chemotactic response to FGFR signaling has not been addressed so far. Using in vivo rescue assays, a minimal Dof protein containing the first 600 amino acids of Dof was identified that allows rescue of both mesodermal and tracheal cell migration. Although the rescue in the tracheal system is not as efficient as the rescue observed with the wild-type construct, all six branches can migrate out, demonstrating that the first 600 amino acids of Dof retain the capacity to read out the local activation state of the FGFRs and to relay the signal to the migration machinery, albeit with somewhat reduced efficiency. Deletion from the C terminus of this dof minigene, as well as internal deletions, results in loss of rescue capacity, thus identifying regions of functional importance (Petit, 2004).

All of the constructs were examined in a Drosophila S2 cell culture assay, in which either the FGFR or the Torso signaling system was activated. Both full-length Dof and Dof600 are phosphorylated on tyrosine residues upon FGF signaling, but Torso cannot use Dof as a substrate. These results are consistent with in vivo data showing that Dof is exclusively needed for FGF-mediated signal transduction and that Torso is able to activate the MAPK cascade in the absence of Dof in dof mutant embryos. Using coimmunoprecipitation experiments, it was shown that Dof forms a complex with both FGFRs and that the first 484 amino acids, although not phosphorylated upon FGF signaling, are required and sufficient for the association with the FGFRs, demonstrating that phosphorylation of Dof is not necessary for complex formation. Cell culture analysis is in line with studies showing that the N-terminal part of Dof directly interacts with the kinase domains of Btl and Htl in yeast two hybrid assays. In addition, it was observed that both the juxtamembrane and the C terminus of Btl can be deleted without affecting considerably the quality of the rescue capacity of the receptor. Thus, it appears that Dof directly docks onto the kinase domain of the FGF receptor, in contrast to the vertebrate FGFR adapter SNT/FRS2, which interacts with a sequence motif in the juxtamembrane region of the receptor (Petit, 2004).

Since Dof becomes phosphorylated upon FGFR signaling in S2 cells, it was asked whether it was possible to identify functionally important phosphorylation sites, the proteins recognizing these sites in the phosphorylated state, and confirm the results in vivo by making use of the rescue assay and genetic analysis. Two potential phosphorylation target sites were identified by sequence analysis in the essential region comprising amino acids 485 to 600. While mutation of each individual site results in reduced phosphorylation of Dof600 in S2 cells upon FGFR signaling, mutation of only one of these sites, tyrosine 515, abolished the migration rescue capacity in vivo. Since the functionally required tyrosine residue was part of a putative consensus binding site for the SH2 domain of the nonreceptor tyrosine phosphatase Csw/SHP-2, the interaction of Csw with Dof was tested using coimmunoprecipitation experiments; Csw is indeed recruited to the activated signaling complex via Dof. It was found in rescue assays that both the region 485 to 600 as well as the region from 600 to the C terminus (construct dofDelta485-600) are able to confer function to the signaling-deficient N terminus (residues 1 to 484). It is known that the C-terminal sequences also recruit the Csw phosphatase in the absence of tyrosine 515, but it is not known know whether they do so directly or indirectly. Further deletion analyses and biochemical studies will be required to address this question (Petit, 2004).

Genetic evidence supporting an interaction between Dof and Csw was provided some time ago by the finding that mutations in csw produce a phenotype identical to bnl, btl, and dof; i.e., tracheal and mesodermal cells fail to migrate. The sum of these results clearly assign a crucial role for both Dof and Csw downstream of the FGFRs in the migratory response, indicating that the ligand-dependent phosphorylation of Dof leads to the recruitment of Csw to the signaling complex, ultimately triggering cell locomotion. SHP-2, the vertebrate homologue of Csw, has been shown to be required at the initial steps of gastrulation, as mesodermal cells migrate away from the primitive streak in response to chemotactic signals initiated by fibroblast growth factors. In addition, SHP-2 has also been found to be crucial for tubulogenesis and for the sustained stimulation of the ERK/MAPK pathway upon induction of another chemotactic factor, the hepatocyte growth factor/scatter factor, thus placing SHP-2/Csw as a key player in branching morphogenesis induced by diverse chemotactic factors. Therefore, it appears that both in invertebrates and vertebrates, SHP-2/Csw plays a major role in RTK signaling in the control of cell migration. The similarity of the Drosophila FGF signal transduction pathway to the vertebrate FGF pathway make the fly system accessible to address future issues not resolved in vertebrates, such as the targets of SHP-2/Csw involved in Ras activation and/or cell migration (Petit, 2004).

Using the dpERK antibody as a readout for the activation of the Ras/MAPK pathway in vivo, it was found that abolishing the interaction between the Dof600 minimal protein and Csw abolishes the activation of the MAPK cascade upon FGFR signaling. The strong correlation found between migration and MAPK activation when analyzing all mutant dof constructs in this assay might indicate that local activation of the Ras/MAPK pathway in tracheal tip cells is sufficient to trigger the migratory response upon Btl signaling. However, two lines of evidence suggest that this might not be the case (Petit, 2004).

In one case, it has been observed that under conditions in which all tracheal cells sustain high levels of Ras/MAPK activity (upon Ras^V12 overexpression), tracheal cells migrate normally in wild-type embryos. In sharp contrast, ectopic expression of the Bnl ligand leads to a complete disruption of directed migration. Therefore, high levels of Ras/MAPK activity do not appear to produce the same migratory response as ligand-activated FGFR signaling. Indeed, and again in contrast to ectopic Bnl, overexpression of Ras^V12 in wild-type embryos does not produce significant filopodial activity in DT tracheal cells, confirming that the activation of Ras is not sufficient to produce cytoskeletal rearrangements by itself (Petit, 2004).

In the other case, it was also observed that while the Dof600 protein lacking the ankyrin repeats did allow FGFR-dependent activation of the Ras/MAPK pathway and downstream nuclear response genes, this protein failed to induce migration. Thus, even local Ras activation under the control of the endogenous ligand Bnl, Btl, and Dof600DeltaAR is unable to activate the migratory machinery. Interestingly, it has also been reported that Ras activation is insufficient to guide RTK-mediated border cell migration during Drosophila oogenesis (Petit, 2004).

Is Ras activation then required at all for cells to produce a cytoskeletal response and migrate directionally? Unfortunately, genetic analysis cannot be used to directly address this question in the embryo since maternal and zygotic loss of Ras activity results in embryos that do not develop far enough to analyze the tracheal system. However, when activated Ras (Ras^V12) is expressed in the tracheal system or in the mesoderm of dof mutant embryos, a certain rescue of migration can be obtained. This suggests that Ras signaling is essential but not sufficient for efficient FGFR-dependent cell migration; additional proteins binding to the receptor, to Dof or to Csw appear to be crucial for a chemotactic response. To analyze the role of Ras experimentally and in detail, mitotic clones lacking Ras activity should be analyzed with regard to their migration properties. Recent reports concerning the role of FGF signaling in the migration of mesodermal and tracheal cells during late larval development might provide the basis for such analyses (Petit, 2004).

DEVELOPMENTAL BIOLOGY

Embryonic

Stumps mRNA is first detected on the ventral side of the embryo at the late syncytial blastoderm stage, in a region slightly narrower than the mesoderm primordium. It disappears from the mesoderm during germ band extension and is seen in the tracheal placodes by stage 9/10. As the tracheae branch and start to differentiate, the transcript disappears from the primary branches and is seen mainly in the extending secondary branches. These expression patterns resemble those of the Drosophila FGF receptors htl and btl, respectively. The stumps gene is also expressed transiently in the anterior and parts of the posterior midgut primordium (as is btl) and, like htl, in a subset of heart cells and a group of migrating visceral mesodermal cells. Expression is also seen in glia cells and a number of other cells that have not been studied in detail. Later in development, the transcript is detectable in parts of the imaginal discs and the brain (Vincent, 1998).

If stumps acts in the FGFR signaling pathway between the receptor and Ras, then the protein should be found in the cytoplasm. This is indeed seen when embryos are stained with an antibody against Stumps. The antibody stains the same cells that express the mRNA, and these cells are not stained in hbr mutant embryos. Stumps protein is seen throughout the cytoplasm and is often enriched at the periphery of cells. The protein is distributed in a punctate pattern (Vincent, 1998).

Drosophila Heartless acts with Heartbroken/Dof in muscle founder differentiation

The formation of a multi-nucleate myofibre is directed, in Drosophila, by a founder cell. In the embryo, founders are selected by Notch-mediated lateral inhibition, while during adult myogenesis this mechanism of selection does not appear to operate. It is here shown, in the muscles of the adult abdomen, that the Fibroblast growth factor pathway mediates founder cell choice in a novel manner. It is suggested that the developmental patterns of Heartbroken/Dof and Sprouty result in defining the domain and timing of activation of the Fibroblast growth factor receptor Heartless in specific myoblasts, thereby converting them into founder cells. These results point to a way in which muscle differentiation could be initiated and define a critical developmental function for Heartbroken/Dof in myogenesis (Dutta, 2005).

During myogenesis in the Drosophila embryo a single precursor cell is chosen by Notch-mediated lateral inhibition. The daughters of the precursor cell form two embryonic muscle founder cells -- each with a characteristic pattern of expression of markers that specify its identity -- or they form an embryonic muscle founder cell and an adult myoblast progenitor. This latter cell type proliferates during larval life and its progeny, the adult myoblasts, are associated with imaginal discs and larval nerves. While embryonic founder cells shut down the expression of Twi, a marker of myoblast identity, the adult myoblasts retain Twi expression during their proliferative phase during larval life. At the onset of metamorphosis, Twi levels decline in a group of cells, the adult founders, that express duf-lacZ at high levels and are located at the sites of myofibre formation. Twi expression is also shut off in other myoblasts as they fuse with the founder to form multi-nucleate cells (Dutta, 2005).

Interestingly, adult myoblasts, like the embryonic founders from whose siblings they are derived, express duf-lacZ (albeit at low levels) throughout larval life. As adult muscle differentiation begins, this low-level expression changes dramatically to a pattern in which one founder cell -- expressing duf-lacZ at high levels -- is chosen to seed each muscle fibre. How is this founder cell chosen? Removal of Notch signalling in adult myoblasts does not result in an increase in the number of founders. This suggests that lateral inhibition mediated by Notch, the process that operates in the embryo, is not the mechanism by which adult founders are chosen. Indeed, the requirements are quite different; adult myoblasts all express duf-lacZ at low levels, suggesting (consistent with their origins as siblings of embryonic founders) that they all already have some properties similar to founder cells. In choosing adult founder cells, therefore, duf-lacZ is to be up-regulated in cells that will become founders and down-regulated in others that will become fusion-competent cells. The results of this study show that the Htl pathway plays a key role in choosing adult founders. It is suggested that Htl does this using an unusual mechanism in which an intracellular positive regulator plays an important role (Dutta, 2005).

Adult myoblasts in the third larval instar express Twi, Hbr/Dof, Htl, and sty-lacZ. At the onset of adult abdominal myogenesis, Twi expression declines. With this, the expression of Hbr and Sty declines in myoblasts. It is suggested that, in the third instar larva, the presence of Sty prevents the activation of the Htl receptor, even if the ligand and Hbr/Dof are available. However, since both Hbr/Dof and sty-lacZ expression decline with Twi, at the onset of myogenesis, the Htl receptor will still be unable to function, because Hbr/Dof is necessary for the function of the Htl receptor. It is suggested that, as Sty and Hbr/Dof expression decline (as Twi expression shuts down at the onset of myogenesis), the Htl receptor is active in some myoblasts. Htl signalling maintains Hbr/Dof expression in these cells by a positive feedback mechanism. Maintenance of Hbr/Dof expression reinforces the Htl signal, which in turn up-regulates the expression of founder-specific genes such as duf in these cells, thereby imparting them with founder properties. Consistent with this hypothesis, activating the Htl receptor results in the maintenance of Hbr/Dof in adult myoblasts. This prolonged activation of Hbr/Dof, and therefore of duf, could be the cause of morphological changes associated with the excess founder cells (Dutta, 2005).

How could this localised activation of the receptor occur? One way is via the localised availability of the Htl ligand. Proximity of some of the cells to the source of the ligand could cause higher levels of Htl signalling in those cells than others, thus biasing their fate towards that of a founder. Examining the expression pattern of the recently identified ligands of Htl should be able to resolve whether this indeed is the case. A second, and more likely, mechanism for localised activation of receptor is via a process that does not involve the localised presence of the ligand. This possibility is suggested because the continued mis-expression of Hbr/Dof in all adult myoblasts results in an increased number of founders and muscle fibres. Since Hbr/Dof function is dependent on ligand activation of the receptor, the ligand must be available to Htl on all myoblasts. Local activation of the receptor could occur by Hbr/Dof being maintained briefly in a founder cell pattern in some myoblasts even as all of the others down-regulate Sty and Hbr/Dof at the onset of myogenesis (with the decline of Twi expression). This continued expression of Hbr/Dof in some myoblasts, and the absence of Sty, could allow local activation of the receptor and the consequent maintenance of Hbr/Dof in a founder pattern (Dutta, 2005).

The problem then shifts to deciphering the mechanism by which the (hypothetical) localised activation of Hbr/Dof takes place. Since abdominal myoblasts are associated with nerves, one possibility is that the signal could come from the nerves. This 'solution' has two problems, however. (1) It is not clear how a precise periodicity of signal, expressed along the nerve and seen by associated myoblasts, would be generated to organise the correct spacing of founder cells. More pertinent perhaps is the observation that (2) surgical removal of the nerve does not affect the number of muscle fibres. Thus, nerves are unlikely to be the source for the signal that organises myoblasts in a founder pattern. Another possible source for a signal that maintains and elevates Hbr/Dof expression in a founder pattern could be the epidermis. The abdominal epidermis develops from ectodermal cells, the histoblasts. As the epidermis differentiates during metamorphosis, muscle tendon precursor cells (specified by and expressing the stripe locus) can be identified. The tendon precursor cells, given that they are in proximity to the differentiating myoblasts, could possibly be a source of organising signal that modulates Hbr/Dof expression to a founder pattern. Thus, the precise segmental and regional patterning of the epidermis could organise the pattern of founder cells in the developing abdominal musculature. In favour of this hypothesis is the finding that reduction of stripe-expressing cells in the dorsal thoracic disc results in the reduction of duf-lacZ expression in the larval templates that give rise to the thoracic dorsal longitudinal muscles, and increasing stripe expression in the ectoderm results in the increase of duf-lacZ expression in the developing dorsal longitudinal muscles. It is not known yet if these results apply to the abdomen (Dutta, 2005).

A third possible mechanism of localised activation of Htl, not exclusive of either of the ones mentioned earlier, is that a dynamic interaction between ligands, other activators, and repressors results in the activation of Htl in a specific pattern. Such a process has been described in the embryo, e.g., in the anterior patterning of follicle cells in the Drosophila egg (Dutta, 2005).

In conclusion, while many mechanistic details still remain elusive, the implication of the FGF pathway as a key player in adult founder cell choice provides the molecular tools to identify missing elements in the pathway. Integrated within the broad question of founder cell specification are more specific questions pertinent to the different muscle groups. Activation of Htl signalling produces a less prominent effect on the dorsal muscles than on the lateral muscles. Also, the extra founders of the dorsal muscles are located in a characteristic fashion (altered in orientation) that is different from that observed for the excess lateral founders. These observations raise questions about whether the dorsal and lateral groups of founders have different levels of sensitivity to the FGF pathway and whether they employ the pathway in different ways (Dutta, 2005).

The results allow the testing of whether this pathway operates in a similar manner during myogenesis in other contexts in Drosophila and in other animals, in particular the higher vertebrates. Vertebrate muscles are composed of multiple fibres, which make them similar to Drosophila adult muscles. Vertebrate myogenesis shares several features with Drosophila myogenesis, at the level of genetic and molecular regulatory mechanisms. The FGF pathway in vertebrates, mediated by multiple isoforms of the receptor and the ligand, has been found to play an instructive role in induction and commitment of myogenic cells. In Xenopus, for instance, an FGF-mediated pathway controls specification and differentiation of myotomal progenitors. Also, signalling via FGFR4 positively regulates myogenic differentiation during avian limb muscle development. The present study, showing the role of Htl in muscle differentiation, highlights yet another similarity. This study also provides directions for probing how the number and location of fibres are regulated in vertebrates, questions that remain to be resolved in the field of vertebrate myogenesis (Dutta, 2005).

Effects of Mutation or Deletion

In a search for mutations affecting morphogenetic movements in the Drosophila embryo, a mutation was found that in homozygous mutant embryos causes defects in the mesoderm and the tracheal system. Although determination of tracheal cell fate and cell division are unaffected in this mutant, the cells do not migrate and, consequently, the tracheal network fails to form. This phenotype is highly reminiscent of that seen in embryos mutant for the genes encoding the FGFR Breathless and its ligand Branchless. The earliest abnormal phenotype detected is in the mesoderm, during late stages of gastrulation, when the invaginated mesoderm would normally spread out on the underlying ectoderm. In homozygous mutant embryos, this spreading does not occur properly, and the mesoderm does not form as an evenly spread cell layer. Later, no heart precursors can be detected; the musculature is disrupted, and an insufficient amount of visceral mesoderm is produced. These phenotypes closely resemble those of the mesodermally expressed FGFR htl. The defects observed in the embryos of P1740 mutants are thus the combined defects seen in mutants of the two known Drosophila FGFRs. For reasons described below, the corresponding gene was named downstream of FGFR (dof), and the allele described above, dof1 (Vincent, 1998). The Interactive Fly follows the convention set by FlyBase and uses the term stumps when referring to this gene.

stumps is required for the activation of the MAPK cascade in the FGFR-expressing cells. Constitutively activated signaling components can partially rescue the defects observed in htl and btl mutants. This approach was used to determine at what level in the signal transduction pathway stumps mutations interfere with the activation of the MAPK cascade. Analysis of embryos expressing activated components in the developing tracheae enabled an examination of both morphogenetic effects (rescue of migration and branching) and effects on gene regulation. The transcriptional target of FGF signaling that was monitored was the Drosophila Serum Response Factor, DSRF, which is expressed in terminal tracheal cells from stage 14 and is strictly dependent on the activity of btl. Expression of a constitutively active Btl protein (Torso-Btl) in the tracheal system of wild-type embryos leads to the ectopic expression of DSRF in dorsal and ganglionic branches; in addition, excessive terminal branching can occur. In btl mutants, expression of Torso-Btl protein also leads to the expression of DSRF and tracheal cell migration is partially rescued; as in wild-type embryos, additional branching is seen. Similar effects are seen in wild-type and btl mutant embryos following expression of activated forms of Ras or Raf. A test was performed to see if these activated signaling components were also able to rescue the defects seen in stumps mutants. In addition to the defects in tracheal cell migration, it was found that, as in btl mutants, DSRF is not expressed in stumps mutants. Activated Ras and activated Raf partially rescue the tracheal cell migration defects and lead to the expression of DSRF expression in stumps mutant embryos. These results demonstrate that the requirement for stumps can be overcome by constitutive activation of the MAPK cascade and suggest that stumps is required upstream of Ras and Raf. In contrast to activated Ras and Raf, expression of Torso-Btl fails to rescue tracheal migration defects and DSRF expression in stumps mutants, strongly suggesting that Dof is essential to transmit the activated state of the Btl receptor to the MAPK cascade (Vincent, 1998).

Since many signal transduction components have been shown to be shared by different RTKs, it seemed possible that Stumps might also be required for signaling by other RTKs. Therefore, the effect of ectopic expression of activated Torso on the tracheae was examined. The tracheal branching pattern shows essentially the mutant phenotype, because activated Torso was expressed under heat-shock control after the initial migration of tracheal cells. However, in contrast to activated Btl, activated Torso is able to induce DSRF expression in stumps mutants. Stumps thus does not appear to be required for signaling through other RTKs. This result is in agreement with the finding that EGF receptor-dependent dp-ERK phosphorylation is normal in stumps mutants (Vincent, 1998).

Since FGF signaling is thought to be associated with cell migration, an analysis was carried out to see whether other migrating cells that express stumps are affected in their migratory capacity in stumps mutants. A population of highly motile cells that express both stumps and htl arises at the posterior end of the germ band and can be visualized with antibodies directed against beta-galactosidase in a strain that contains lacZ under control of the croc promoter. These cells migrate along the visceral mesoderm , covering a distance of more than half the length of the embryo. In stumps mutants, these cells migrated at the same rate as in the wild type, but in a slightly less well-organized manner. In addition, no major defects in cell migration are seen in the anterior midgut primordium, which expresses stumps and btl and migrates over a long distance. Thus, these cells either do not use Stumps and the FGF receptors at all or they use them for processes other than cell migration (Vincent, 1998).

One allele of stumps, hbr ^YY202, was obtained in the original screen and two additional alleles, hbr^ems6 and hbr^ems7, were obtained in a subsequent mutagenesis. Of the three, hbr^YY202 has the strongest mesodermal phenotype, although comparison to a deficiency that covers stumps indicates that this allele is not null but rather is a strong hypomorph (Michelson, 1998).

Htl signaling is essential for normal mesoderm development. Since stumps and htl have very similar phenotypes, it was interesting to see whether stumps interacts genetically with htl, implying a possible role for Stumps in Htl signaling. This possibility was investigated in two ways. (1) stumps was examined to determine its capacity to dominantly enhance a partial loss-of-function htl allele. In contrast to a null allele of htl, the hypomorphic hbr^YY202 allele does not completely eliminate mesodermal Eve expression. On average, each hbr^YY202 mutant embryo has 1.5 hemisegments with at least one Eve-positive dorsal mesodermal cell. This is reduced to 0.8 Eve-positive hemisegments in the presence of one mutant copy of stumps, a highly significant difference. Thus, stumps not only has a heartless-like mesodermal phenotype, but it also exhibits a dosage-sensitive genetic interaction with htl. (2) It was asked if stumps could dominantly suppress a hyperactivated form of Htl. The latter was generated by replacing the extracellular domain of the Htl receptor with the dimerization domain of the bacteriophage lambda cI repressor. This strategy constitutively activates receptor tyrosine kinases by causing ligand-independent receptor dimerization. Such a construct was placed under GAL4 UAS control and ectopically expressed in transgenic embryos using a twi-GAL4 driver. Under the influence of activated Htl, increased numbers of Eve-expressing muscle and cardiac progenitors are formed in an otherwise wild-type genetic background. This response is equivalent to that obtained with activated forms of either Ras1 or Egfr and reflects the involvement of both Rtks in mesodermal cell fate specification. Heterozygous stumps alone does not affect mesodermal Eve expression or mesoderm migration. However, one mutant copy of stumps markedly suppresses the influence of activated Htl on the formation of Eve-positive mesodermal cells. This suggests that stumps is required for the cell fate specification function of Htl, in addition to its involvement in Htl-dependent cell migration. These findings establish a strong genetic interaction between stumps and htl, suggesting that stumps is involved in mesodermal Fgf receptor signaling (Michelson, 1998).

The inability of stumps mutant cells to migrate into the Dpp expression domain in the dorsal region of the embryo can account for the associated absence of dorsal mesodermal structures. However, stumps might also be required in order for mesodermal cells to respond to this growth factor. This possibility was investigated by assessing the effects of ectopic Dpp on the expression of the Dpp target gene, bagpipe (bap). bap normally is expressed in a set of dorsally restricted, segmentally repeated patches of cells that give rise to the visceral mesoderm. This expression is markedly reduced in a stumps mutant, consistent with the previously documented defect in visceral mesoderm development. In an otherwise wild-type genetic background, ectopic Dpp induces bap transcription in ventral and lateral mesodermal cells. The same response to ectopic Dpp is seen in stumps mutant embryos. Thus, competence to be induced by Dpp does not require stumps. This mesodermal response to Dpp also was found to be independent of htl (Michelson, 1998).

stumps, a Drosophila gene required for fibroblast growth factor (FGF)-directed migrations of tracheal and mesodermal cells

Fibroblast growth factors (FGFs) bind to FGF receptors, transmembrane tyrosine kinases that activate mitogenic, motogenic, and differentiative responses in different tissues. While there has been substantial progress in elucidating the Ras-MAP kinase pathway that mediates the differentiative responses, the signal transduction pathways that lead to directed cell migrations are not well defined. A Drosophila gene called stumps, termed heartbroken in other studies, is described that is required for FGF-dependent migrations of tracheal and mesodermal cells. These migrations are controlled by different FGF ligands and receptors, and they occur by different cellular mechanisms: the tracheal migrations occur as part of an epithelium whereas the mesodermal migrations are fibroblast-like. In the stumps mutant, tracheal cells fail to move out from the epithelial sacs, and only rudimentary tracheal branches form. Mesodermal cells fail in their dorsal migrations after gastrulation. The stumps mutation does not block all FGF signaling effects in these tissues: both random cell migrations and Ras-MAP kinase-mediated induction of FGF-specific effector genes occurs upon ectopic expression of the ligand or upon expression of a constitutively activated Ras protein in the migrating cells. The results suggest that stumps function promotes FGF-directed cell migrations, either by potentiating the FGF signaling process or by coupling the signal to the cellular machinery required for directed cell movement (Imam, 1999).

An important consideration with regard to the role of stumps in FGF-mediated tracheal migrations is that the gene does not appear to be required for all aspects of the tracheal FGF signaling pathway. All downstream effects of the Branchless FGF pathway examined in addition to cell migration fail in the stumps mutant, including the activation of MAP kinase, the induction of secondary and terminal branching genes, and the formation of secondary and terminal branches. However, all of the gene inductive and differentiative effects could be restored by ectopic expression of the Branchless FGF or expression of the activated Ras. These treatments also induce some tracheal cell migration, although the migrations are limited in extent and do not follow the normal outgrowth pathways. Thus, the stumps1 mutant has the ability to generate an active FGF signal, receive the signal, and transduce it through MAP kinase and on to downstream target genes. The residual signaling capability is unlikely to result from leakiness of the stumps1 allele because stumps1 behaves as a null allele in genetic tests, and the same residual signaling capability is found in deficiency homozygotes that presumably lack the stumps locus (Imam, 1999).

Given that aspects of the FGF pathway are intact in the stumps mutant, including expression of the Branchless FGF and Breathless FGF receptor genes, two general models are suggested for how stumps may function in the tracheal FGF pathway. The first is that stumps acts as a potentiator in the pathway. In this model, stumps is not absolutely required for any step in the process, but it is necessary to amplify or focus one or more steps. For example, it might facilitate transport or reception of the ligand, as heparan sulfate proteoglycans are believed to function in presenting the FGF ligand to FGF receptors in cultured mammalian cells. Or, it might function like the LIN-2, LIN-7, and LIN-10 proteins in C. elegans to concentrate the receptor tyrosine kinase at the basal (signal receiving) side of the tracheal cells. It could also function downstream as a scaffold that concentrates components of the signal transduction machinery, thereby amplifying the signal. If the potentiator model is correct, then the data do not allow for the placing of stumps more precisely in the FGF pathway, because overexpression of the ligand or introduction of the activated Ras could either force signaling through or bypass the signaling bottleneck caused by the absence of stumps (Imam, 1999 and references).

The second general class of models for stumps function requires a bifurcation in the FGF signaling pathway. In these models, stumps is required only for one fork in the pathway: the fork needed for directed cell migration (guidance). The other fork would remain intact in the stumps mutant. Because MAP kinase activation and downstream effector genes are able to be induced in the stumps mutant, FGF signal production, reception, and this fork of the signal transduction pathway would be independent of stumps. General cell motility would also not require stumps, since tracheal cells in the stumps mutant could be induced to move, albeit erratically, upon ectopic expression of the Branchless FGF or expression of an activated Ras. Ectopic expression of activated Ras in the wild type does not perturb directed cell migration, demonstrating that the cells continue to receive the normal FGF guidance cues under these conditions. According to the bifurcating pathway models, all of the other FGF signaling failures observed in the stumps mutant would be secondary consequences of the tracheal cells' inability to follow the FGF signal toward its source and to receive continuous high levels of signal. Thus, if the signal were artificially brought close to the tracheal cells, as in the ectopic expression experiments, the mutant cells could respond normally in many respects, except for proper directional migration, just as was observed (Imam, 1999).

In the bifurcating pathway models, stumps functions downstream of the receptor. It might serve, for example, to mark the highest point of signaling activity on the membrane of the receiving cell and couple the activated receptor to the cytoskeletal machinery required for directed cell movement. The data, however, do not allow the split to be placed in the pathway with respect to Ras (or MAP kinase). Although the analysis of the mesodermal migration defects in the stumps mutant is more limited because the ligand for the mesodermal FGF pathway is not known, the available data also fit well with a bifurcating signal transduction pathway. Mesodermal cells in the stumps mutant fail in their directed migrations, although they were still able to undergo limited, apparently random migrations. Expression of activated Ras stimulates motility and restores some dorsal mesodermal development. If the bifurcation model is correct, it would suggest that the Heartless and Breathless FGF receptors use related signal transduction machineries to mediate directed cell migrations. Indeed, the intracellular portion of the Heartless FGF receptor can partially rescue defective tracheal outgrowth in a Breathless FGF receptor mutant, implying common downstream effectors in migration (Imam, 1999 and references).

Many FGF receptors and other receptor tyrosine kinase pathways stimulate a variety of cellular effects including mitogenic, motogenic, and differentiative responses. There is growing evidence that directed cell migrations are mediated by a special branch in RTK signal transduction pathways that is not shared by the mitogenic and differentiative responses. Various intracellular signaling molecules including Ras, Rac, phospholipase C-, phosphoinositide-3' kinase, and Src have been implicated in signal transduction processes associated with migration. Molecular characterization of the stumps gene and biochemical analysis of its products should help elucidate the FGF signal transduction pathways that control tracheal and mesodermal cell migrations and may provide insight into how these and other RTK pathways guide cell movements (Imam, 1999 and references).

A functional domain of Dof that is required for fibroblast growth factor signaling

Signal transduction by fibroblast growth factor (FGF) receptors in Drosophila depends upon the intracellular protein Dof, which has been proposed to act downstream of the receptors and upstream of Ras. Dof is the product of a fast-evolving gene whose vertebrate homologs, BCAP and BANK, are involved in signaling downstream of the B-cell receptor. Mapping functional domains within Dof revealed that neither of its potential interaction motifs, the ankyrin repeats and the coiled coil, is essential for the function of Dof. However, a region has been identified within the N terminus of the protein with similarity to BCAP and BANK, that is referred to as the Dof, BCAP, and BANK (DBB) motif; the DBB motif is required for FGF-dependent signal transduction and is necessary for efficient interaction of Dof with the FGF receptor Heartless. In addition, Dof is phosphorylated in the presence of an activated FGF receptor and tyrosine residues can contribute to the function of the molecule (Wilson, 2004).

Three different ways are envisioned by which Dof could function, all of which are consistent with the physical interaction of Dof with the FGF receptors. (1) Dof could be required for the transport of the FGF receptors to the cell surface. However, the detection of Heartless at the peripheries of cells in the absence of Dof argues against such a function. (2) Dof may have a role in the activation of the receptors. It could, for example, facilitate or stabilize conformational changes or autophosphorylation of the receptors. No data is available that would specifically support this model, but it is not ruled out by any of the results. (3) Dof is phosphorylated in the presence of an activated FGF receptor and could be involved in transmission of the signal from the receptors (Wilson, 2004).

Although Dof is a large protein, the only motifs that can be identified in the primary sequence are two ankyrin repeats and a coiled coil. Comparison of the Drosophila protein with its Anopheles homolog and the most closely related vertebrate proteins, BCAP and BANK, suggests that dof may be an example of a fast-evolving gene used for FGF signaling in Drosophila but which has acquired a novel function with the development of the immune system in higher vertebrates. Dof shares a number of distinct parts with these proteins, namely, the ankyrin repeats, the coiled coil, and the region adjacent to the ankyrin repeats, which has been termed the DBB motif. Surprisingly, despite the conservation of these domains, only the DBB domain appears to be indispensable for FGF signaling in Drosophila. In this respect, it is interesting that the association of BANK with the IP-3 receptor, which stimulates the release of calcium from intracellular stores upon activation of the B-cell receptor, also depends upon the N-terminal part of the protein but not upon the ankyrin repeats that are present within this region. However, two caveats apply to the in vivo assays that were used to test the function of Dof: (1) there are aspects of FGF signaling besides those examined in the assays, such as feedback regulation and the response to oxygen deprivation, which could be affected by the Dof mutations; (2) the consequence of a particular deletion was determined based on the overexpression of the mutant protein, and this may have masked certain physiological requirements for particular domains of the protein. Thus, all of the functional domains of Dof might not yet been determined; nevertheless, this approach has revealed a critical part of the protein (Wilson, 2004).

The most important region for the function of Dof corresponded to the DBB motif, which is critical for FGF signaling and for the efficient interaction of Dof with the receptor. Dof mutants with deletions that disrupted the DBB motif have only miminal biological activity and are no longer capable of interacting efficiently with the FGF receptor. These observations suggest that the DBB domain interacts directly with the FGF receptors. This is unlikely to be the only function of the DBB motif, since this domain is conserved in BCAP and BANK, which are expressed in B cells and macrophages and are required for the function of the B-cell receptor and thus are unlikely to interact with FGF receptors. Indeed, it was found that the DBB domain in both Dof and BCAP is required to mediate self-association in yeast cells, indicating that this domain may have a more general role in mediating protein-protein interactions (Wilson, 2004).

Constructs lacking this domain still provide above-background biological activity, suggesting that perhaps other parts of the molecule participate in receptor binding, allowing residual signal transmission in the absence of the DBB. Conversely, the DBB region in itself is clearly not sufficient for transmission of the signal, showing that other essential functions reside elsewhere in the molecule. The smallest N-terminal fragment of Dof with biological activity was Dof[1-522]. A mutant with a more extensive C-terminal truncation, Dof[1-446], had no biological activity but was still able to interact with the cytoplasmic domain of the FGF receptor Htl. Together, these findings imply that in addition to the DBB domain there are essential properties of Dof that are located within the 76 amino acids between residues 446 and 522. Intriguingly, there are two tyrosine residues in this region that are potential binding sites for PI 3-kinase and Corkscrew. The mutation of the potential Corkscrew binding site has a clear effect upon the activation of Even-skipped within the mesoderm, suggesting that this binding site contributes to the function of the molecule (Wilson, 2004).

In summary, two parts of Dof are important for its function. The DBB motif is necessary for the efficient interaction of Dof with the receptor, and tyrosines between the ankyrin repeat and the coiled-coil region also contribute to the function of Dof, possibly by recruiting Csw. Thus, similar to FRS2 in vertebrate FGF signal transmission, Dof uses a protein-protein interaction domain to interact with the receptor and acts as a substrate for phosphorylation, and it can therefore recruit other signaling molecules. It is intriguing that although the FGF signaling pathway must have existed in the common precursor of insects and vertebrates, different molecules have taken on this role in the two lineages, while their respective homologs in the other lineage have diverged in sequence and function (Wilson, 2004).

FGF signalling and the mechanism of mesoderm spreading in Drosophila embryos

FGF signalling is needed for the proper establishment of the mesodermal cell layer in Drosophila embryos. The activation of the FGF receptor Heartless triggers the di-phosphorylation of MAPK in the mesoderm, which accumulates in a graded fashion with the highest levels seen at the dorsal edge of the mesoderm. This study examines the specific requirement for FGF signalling in the spreading process. Only the initial step of spreading, specifically the establishment of contact between the ectoderm and the mesoderm, depends upon FGF signalling, and unlike the role of FGF signalling in the differentiation of heart precursors this function cannot be replaced by other receptor tyrosine kinases. The initiation of mesoderm spreading requires the FGF receptor to possess a functional kinase domain, but does not depend upon the activation of MAPK. Thus, the dispersal of the mesoderm at early stages is regulated by pathways downstream of the FGF receptor that are independent of the MAPK cascade. Furthermore, the activation of MAPK by Heartless needs additional cues from the ectoderm. It is proposed that FGF signalling is required during the initial stages of mesoderm spreading to promote the efficient interaction of the mesoderm with the ectoderm rather than having a long-range chemotactic function, and this is discussed in relation to the cellular mechanism of mesoderm spreading (Wilson, 2005).

As the mesoderm spreads out over the surface of the ectoderm, the mesodermal cells that are in contact with the ectoderm accumulate high levels of the active form of MAPK. The fact that this accumulation of active MAPK is seen only in embryos with a functional FGF-signalling system in the mesoderm, but not in htl or dof (stumps or heartbroken) mutant embryos, indicates that it is triggered by the FGF receptor. Htl and Dof are expressed throughout the mesoderm, which suggests that the local activation of MAPK is induced by the local availability of a ligand, consistent with the expression pattern of the recently discovered ligands for Htl in the ectoderm. However, even a constitutively active form of Heartless expressed throughout the mesoderm, which is able to rescue spreading in htl mutants, only mediates MAPK activation at early stages in the cells directly apposed to the ectoderm. It is concluded that the presence of an activated form of the FGF receptor is not sufficient to trigger MAPK activation in mesodermal cells (Wilson, 2005).

This result may appear to contradict earlier studies showing the ability of activated FGF-receptors to trigger MAPK activation throughout the mesoderm, but the embryos in these studies were not analysed during the phase of the earliest contact of the mesoderm with the ectoderm, but rather at later stages, just before the time when MAPK activation normally occurs in the heart precursors in the dorsal region of the mesoderm. This phase of FGF-dependent MAPK activation in the mesoderm clearly has different requirements from the early phase, as is also shown by the results using other RTKs or downstream effectors of the RTK signalling pathway. These experiments demonstrate that signals from activated Raf cannot be transduced to MAPK in the cells during the early phase, except in the presence of an activated FGF receptor. It is concluded that, in addition to the signal from an activated RTK via Raf, a second event is necessary for MAPK to become phosphorylated. This event could either generate a second positive signal, or it could lead to the release of a negative, inhibitory signal (Wilson, 2005).

Two points suggest that the event depends on contact of the mesodermal cells with the ectoderm: (1) Lambda-htl (receptor that dimerizes spontaneously and becomes autophosphorylated in a ligand-independent fashion) induces MAPK phosphorylation only in mesodermal cells contacting the ectoderm, although it is expressed at uniform levels in all mesodermal cells; (2) the phenotype of pbl mutants supports this view. As in htl and dof mutants, the early contact of the mesoderm with the ectoderm fails to be made in pbl mutants, and mesoderm spreading is impaired. At later stages, Htl is able to trigger MAPK phosphorylation in the dorsal part of the mesoderm of pbl mutants, showing that FGF signalling in the mesoderm as such does not depend on pbl. By contrast, the early activation of MAPK is abolished. It is therefore argued that contact is a prerequisite for early FGF-receptor induced MAPK activation (Wilson, 2005).

Spreading of the mesoderm on the ectoderm leads to a redistribution of mesodermal cells away from the site of invagination towards the dorsal edge of the ectoderm. This is often considered to be a process of directed cell migration. In this view, the graded distribution of activated MAPK levels in the nuclei of the mesodermal cells is suggestive of a response to a chemotactic signal originating from the target region. Both the expression pattern of the Htl ligands and the phenotypes of mutants in which the fate of the target region has been changed are inconsistent with this view. The activation of Heartless appears to be permissive for mesoderm spreading and it is suggested that FGF signalling functions primarily to promote the efficient interaction of the entire mesodermal primordium with the surface of the ectoderm and that this could act to impose order during the transition from an epithelial to a mesenchymal state. Simple spatial constraints could lead to an apparently directed migration. With the mass of mesodermal cells initially concentrated near the site of invagination, the only direction available for migration is away from this site. Hence, a signal-inducing motility would automatically promote directional movement. The dispersal of the mesoderm mass in dof mutants is noticeably improved by blocking cell division, and it is believed that this might be due to the smaller number of cells in the mesodermal primordium having greater access to the surface of the ectoderm (Wilson, 2005).

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date revised: 10 May 2006

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