Digoxygenine-labeled antisense RNA probes complementary to FGF8-like1 and FGF8-like2 transcripts were synthesized and in situ hybridization experiments were performed. No transcripts could be detected in fertilized eggs and syncytial blastoderm stage embryos. Both genes were first expressed at mid-blastula transition in two broad lateral stripes in the prospective neuroectoderm, excluding the ventral domain of the blastoderm. At the beginning of gastrulation, expression of both genes extends to the cephalic furrow in the anterior region and to the posterior midgut invagination in the posterior part of the embryo. During germ band elongation, the expression patterns of the two genes become distinct from one another. Although FGF8-like1 is expressed in the entire germ band, except for a narrow ventral stripe of mesectoderm cells, FGF8-like2 transcripts accumulate in the dorsal-most cells of the germ band. During gastrulation, expression of both genes is confined to the ectodermal cell layer. After gastrulation, expression of FGF8-like1 and FGF8-like2 disappears from the neuroectoderm, and in later embryonic stages the two genes become differentially expressed. These data are consistent with the Northern blot results, which show that in early embryos FGF8-like1 is expressed at high levels in early and mid embryogenesis, whereas FGF8-like2 transcript levels are maintained and even rise during late embryogenesis. These results show that FGF8-like1 and FGF8-like2 are expressed in the cells that serve as substrate for mesoderm cells during migration. Furthermore, the differential expression of FGF8-like1 and FGF8-like2 during mesoderm migration suggests that the gene products might initially work in a redundant fashion and later serve distinct functions in mesoderm morphogenesis (Gryzik, 2004).
In situ hybridization assays were done as a first step toward determining pyramus and thisbe. Initially, the two genes exhibit a very similar expression profile. During cellularization, each gene is expressed in broad lateral stripes within the neurogenic ectoderm. Staining is excluded from the presumptive mesoderm in ventral regions and from the anterior third of the embryo. This ths pattern is maintained during mesoderm invagination and the rapid phase of germ-band elongation. In contrast, at the onset of germ-band elongation, the pyr expression pattern is rapidly refined and expression is detected only in dorsal and ventral regions of the neurogenic ectoderm. At the completion of elongation, both genes are expressed in discrete regions of the epidermis, including different subsets of the ventral neuroblasts (Stathopoulos, 2004).
After the completion of germ-band elongation, ths and pyr exhibit dynamic and, in part, distinct patterns of expression in different tissues. For example, pyr is expressed in ectodermal stripes that refine to smaller spots, which are adjacent to pericardial cells in the dorsal mesoderm. ths exhibits similar, but weaker expression, with additional expression in founders of the visceral muscles. After germ-band retraction, both genes are expressed in the ectodermal derivatives of the gut, the proctodeum and stomodeum, as well as the central nervous system, and in the muscle attachment sites at the segment borders. A recurring theme in the complex expression patterns is the appearance of Ths and Pyr in different epithelial tissues at the time when derivatives of the mesoderm that express Htl become associated with these tissues (Stathopoulos, 2004).
Mesoderm migration is a pivotal event in the early embryonic development of animals. One of the best-studied examples occurs during Drosophila gastrulation. Here, mesodermal cells invaginate, undergo an epithelial-to-mesenchymal transition (EMT), and spread out dorsally over the inner surface of the ectoderm. Although several genes required for spreading have been identified, the inability to visualise mesodermal cells in living embryos has hampered gathering of information about the cell rearrangements involved. Several mechanisms, such as chemotaxis towards a dorsally expressed attractant, differential affinity between mesodermal cells and the ectoderm, and convergent extension, have been proposed. This study resolved the behaviour of Drosophila mesodermal cells in live embryos using photoactivatable-GFP fused to alpha-Tubulin (PAGFP-Tub). By photoactivating presumptive mesodermal cells before gastrulation, it was possible to observe their migration over non-fluorescent ectodermal cells. The outermost (outer) cells, which are in contact with the ectoderm, migrate dorsolaterally as a group but can be overtaken by more internal (inner) cells. Using laser-photoactivation of individual cells, it was then shown that inner cells adjacent to the center of the furrow migrate dorsolaterally away from the midline to reach dorsal positions, while cells at the center of the furrow disperse randomly across the mesoderm, before intercalating with outer cells. These movements are dependent on the FGF receptor Heartless. The results indicate that chemotactic movement and differential affinity are the primary drivers of mesodermal cell spreading. These characterisations pave the way for a more detailed analysis of gene function during early mesoderm development (Murray, 2007).
Using a combination of whole mesoderm and single-cell photoactivation this study has observed the combination of cell behaviours employed by Drosophila mesodermal cells to form a monolayer, providing insights into the mechanisms responsible for this important part of gastrulation. The first observation was that outer cells moved dorsolaterally over the ectoderm. Although this is not unexpected, it nevertheless confirms a central prediction of the chemoattraction model: that cells migrate in a dorsolateral direction. Remarkably, it was then observed that inner cells are able to overtake outer cells to achieve a more dorsal position. Single-cell labelling then showed that these inner cells were likely to have originated from a position adjacent to the centre of the ventral furrow. Significantly, inner lateral (IL) cell progeny invariably move away from the midline, suggesting that they receive a directional guidance cue from the dorsal region of the ectoderm, again consistent with a chemoattraction model (Murray, 2007).
A complication in the simple chemoattraction model is that the two likely chemoattractants, Pyr and Ths, are initially expressed in quite broad lateral domains. During mesoderm migration, however, pyr expression does become restricted to the more dorsal parts of the ectoderm, whereas ths is expressed in a complementary fashion in the ventral regions of the neurogenic ectoderm. It has been suggested that the two ligands may have different binding affinities, and that the refinement of Pyr expression to more dorsal positions could guide mesodermal cells dorsally. An alternative is that those regions of the ectoderm that are not yet covered with mesodermal cells, such as the dorsal ectoderm, are highly attractive to mesodermal cells simply because the FGF ligands that they are producing are not being bound and internalised by outer cells already in contact with the ectoderm (Murray, 2007).
An alternative to chemoattraction that has been suggested is that FGFR activation is permissive rather than instructive and simply imparts a degree of motility to cells, allowing them to disperse until they are able to contact the ectoderm. This motility, combined with a steric hindrance effect, in which cells tended to move into unoccupied territory, could theoretically achieve a monolayer in the absence of directional cues. It would be expected, however, that if IL cell progeny were simply made motile and moved randomly, that cells adjacent to the midline would sometimes cross the midline to contact the ectoderm on the opposing side. This was never observed (Murray, 2007).
The movement of inner cells past the lateralmost outer cells is also consistent with the differential affinity model, according to which mesodermal cells form strong adhesions with the ectoderm. Cells not already in contact with the ectoderm would either intercalate between existing outer cells, or, as seen here, move past them. The fact that intercalation was not seen suggests either that outer cells adhere strongly to the ectoderm and do not easily move apart, or, again, that outer cells are masking FGF produced in the ectoderm. If a differential affinity model is active, the most likely candidate adhesion molecules would be integrins, which are expressed at the interface of the mesoderm and ectoderm, although there is, as yet, no published evidence for a functional role for integrins in this process (Murray, 2007).
During the initial migration of outer cells over the ectoderm it was found that cells maintained their position relative to their immediate neighbours. This result supports the argument against the convergent extension model. If convergent extension was a primary driving force behind lateral spreading, one would expect to see widespread intercalation throughout the mesoderm as inner cells pushed in between existing outer cells. This was not observed, although the possibility cannot be ruled out that some degree of intercalation does occur during this migration phase. Intercalation does, however, appear to play a part during the later stages of the formation of the monolayer, where inner medial (IM) cell progeny are seen appearing at the ectoderm. The timing of this event, at around the time of the second mitosis, suggests that the sudden lateral spreading that accompanies the second mitotic wave (50 minutes of development) may be due to the intercalation of a pool of inner cells. One possibility is that the adhesion between the mesodermal cells and the surrounding cells, both mesodermal and ectodermal, is decreased as they go through mitosis, permitting the inner cells access to their preferred position in association with the ectoderm. Thus, although a general convergent extension is not in evidence, intercalation does appear to contribute to mesoderm spreading (Murray, 2007).
On the basis of these observations, the following model of mesoderm cell behaviour following ventral furrow formation is presented. Following the breakdown of the epithelium, the first division results in a rapid spreading down onto the ectoderm, presumably due to decreased adhesion between mesodermal cells. Cells that are thereby placed in contact with the ectoderm start to polarise and proceed to migrate dorsolaterally as a group. Outer cells form a strong adhesive contact with the ectoderm, which prevents inner cells from intercalating between them and instead forces inner cells either to take up positions that outer cells vacate near the midline or move past them to more dorsal positions. Inner lateral cells receive a directional cue from the dorsal ectoderm guiding them laterally, over the outer cells. In this manner, by the time of the second mitosis the ectoderm is largely covered by mesodermal cells. Inner medial cell progeny that have failed to contact the ectoderm during the initial spreading are prevented from doing so by cells already strongly adhered to the ectoderm until the time of the second division. The second division then allows the remaining inner cells to contact the ectoderm. This intercalation produces a rapid lateral extension followed by a general retraction as the cells exit mitosis and re-establish adhesive contacts, with the ectoderm finally forming the monolayer (Murray, 2007).
The combination of behaviours observed may represent the most efficient way to rapidly spread one tissue over another. The tendency for cells to migrate dorsolaterally helps to constantly make space for those cells placed nearer the midline. If cells that contacted the ectoderm never moved away, it would mean that internal cells would have to travel further and further dorsally to find space on the ectoderm. In a similar manner, if chemotaxis towards a dorsally placed attractant was the only mechanism operating, one might expect that cells would continue moving dorsally, even if this resulted in an excess of cells in dorsal positions and a deficit closer to the midline. The tendency of mesodermal cells to develop and maintain a strong adhesive contact with the ectoderm would help ensure that all parts of the ectoderm remain covered. Finally, having a period of intercalation serves to give any remaining inner cells a chance to finally contact the ectoderm (Murray, 2007).
The resolution of mesodermal cell behaviour described in this study will make it possible analysis in greater detail of the migration defects in mutants such as htl and pebble. It will also make it possible to test whether cell rearrangements are normal in those situations in which directional information is lost, but in which spreading still occurs (e.g. rescue with activated Htl, or widespread, non-localised expression of FGF ligands). Finally, it will be of interest to determine whether the behaviors observed are typical of mesoderm migration in other systems. In mouse embryos, mesodermal cells emanating from the primitive streak migrate out over the basal surface of the primitive ectoderm to eventually form the mesodermal layer of cells. The cell rearrangements that occur during this process are not known. Photoactivatable GFP, which has provided such a versatile analysis tool here, could be applied to cultured mouse embryos to resolve these events (Murray, 2007).
Mesoderm migration in the Drosophila gastrula depends on the fibroblast growth factor (FGF) receptor Heartless (Htl). During gastrulation Htl is required for adhesive interactions of the mesoderm with the ectoderm and for the generation of protrusive activity of the mesoderm cells during migration. After gastrulation Htl is essential for the differentiation of dorsal mesodermal derivatives. It is not known how Htl is activated, because its ligand has not yet been identified. A genome-wide genetic screen was performed for early zygotic genes and seven genomic regions were identified that are required for normal migration of the mesoderm cells during gastrulation. One of these genomic intervals produces upon its deletion a phenocopy of the htl cell migration phenotype. The genetic and molecular mapping of this genomic region is presented in this study. Two genes, FGF8-like1 and FGF8-like2, were identified that encode novel FGF homologs and were only partially annotated in the Drosophila genome. FGF8-like1 and FGF8-like2 are expressed in the neuroectoderm during gastrulation and present evidence that both act in concert to direct cell shape changes during mesodermal cell migration and are required for the activation of the Htl signaling cascade during gastrulation. It is concluded that FGF8-like1 and FGF8-like2 encode two novel Drosophila FGF homologs, which are required for mesodermal cell migration during gastrulation. These results suggest that FGF8-like1 and FGF8-like2 represent ligands of the Htl FGF receptor (Gryzik, 2004).
Maternal gene products govern cleavage divisions in early embryos until mid-blastula transition, when embryonic development becomes dependent on zygotic transcription. In Drosophila, exploiting this property of early embryos can help to identify early zygotic gene functions in genetic screens. The rationale of these screens is to generate embryos bearing large chromosomal deletions by using chromosomal translocations. An overlapping set of such synthetic deletions that together uncover the entire genome and allow the identification of gene functions required for early morphogenesis, including redundant gene functions, has been generated. This approach was used to identify genes required for mesoderm migration in the gastrula embryo. The correct migration of the mesoderm was visualized by immunolabeling of Twi to mark the presumptive mesoderm cells. After invagination, the mesoderm cells undergo mitosis and subsequently migrate as an aggregate in a dorsolateral direction until a monolayer of mesoderm cells covers the basal surface of the ectoderm. In this screen, approximately 94.5% of the genome was analyzed, and seven genomic regions that contain essential genes for mesoderm migration were identified (Gryzik, 2004).
Two loci mapped to the right arm of chromosome II. A focus was placed on the chromosomal interval uncovered by Tp(2;3)I.707 because embryos deficient for this region exhibited the strongest phenotype, and this phenotype was reminiscent of mutations in htl or dof (downstream of fgf, encoding a Heartless adaptor protein). At the beginning of gastrulation, the mesoderm cells invaginated normally. However, after invagination the mesoderm cells failed to attach to the ectoderm and did not spread out but remained as a tight aggregate, which extended into the interior of the embryo. At later stages some cells were attached to the ectoderm, but many cells remained aggregated and never formed a monolayer. Thus, the chromosomal region 46E to 49E contains a locus that is required for mesoderm migration and exhibits similar phenotypic features as mutations in htl or dof (Gryzik, 2004).
Mapping of the respective chromosomal interval revealed that none of the available deletions produced a phenotype that resembled that of the translocation. Because the set of deletions used for this analysis did not encompass the entire region uncovered by the translocation, the Drosophila isogenic deficiency kit was employed to construct novel deletions in the region. Of two partially overlapping deletions, Df(2R)ED2230 and Df(2R)ED2238, one produced defects in mesoderm morphogenesis. Although mesoderm migration in Df(2R)ED2230 homozygotes occurred as in the wild-type, Df(2R)ED2238 homozygotes produced a phenotype similar to that of the synthetic deletion embryos identified in the original screen. In the migration phase, mesoderm cells in embryos homozygous for Df(2R)ED2238 did not spread out and remained associated with each other. These defects in mesoderm migration of Df(2R)ED2238 homozygotes presumably also contribute to the failure to produce dorsal mesodermal derivatives, such as pericardial cells, which express even skipped (eve). It is concluded that Df(2R)ED2238 uncovers genes zygotically required for mesoderm migration (Gryzik, 2004).
In order to identify the gene that is uncovered by Df(2R)ED2238 and accounts for its defects in mesoderm morphogenesis, a molecular analysis was performed based upon the molecularly mapped chromosomal breakpoints of Df(2R)ED2238 and Df(2R)ED2230. Because Df(2R)ED2230 did not affect mesoderm migration, it was concluded that the gene must be localized between the distal breakpoints of Df(2R)ED2230 and Df(2R)ED2238. A 179,926 bp interval was identified that is missing in Df(2R)ED2238 but not in Df(2R)ED2230 (Gryzik, 2004).
The gene annotation release 3 of the Drosophila Genome Project predicted 14 genes within this interval. Because the phenotypes of the initial screen were strictly based upon zygotic gene activity, it was reasoned that prime candidates should exhibit a zygotic expression profile. The gene expression data of these 14 predicted genes was reviewed and it was found that only two genes, CG12443 and CG13194, exhibited an early zygotic expression and lacked significant maternal transcripts. The expression profile of CG12443 and CG13194 was confirmed by Northern blotting and in situ hybridization; both genes were found to be expressed zygotically (Gryzik, 2004).
The expression pattern of FGF8-like1 and FGF8-like2 suggested that the two gene products might be required for the activation of Htl. Embryos deficient for both FGF8-like1 and FGF8-like2 exhibit defects in mesoderm migration similar to those seen in htl or dof mutants. In order to prove that FGF8-like1 and FGF8-like2 are indeed required for mesoderm migration, RNA interference experiments were performed. Injection of dsRNA targeting both genes results in a lack of Eve-positive dorsal mesodermal derivatives. However, injection of dsRNA targeting FGF8-like2 alone affected the differentiation of Eve-positive mesoderm derivatives, suggesting that FGF8-like2 might have some nonredundant function for which FGF8-like1 cannot compensate (Gryzik, 2004).
The lack of Eve-positive dorsal mesoderm cells might be due to a function of FGF8-like1 and FGF8-like2 in mesodermal patterning or to defects during the migration of the mesoderm cells. To discriminate between these two possibilities, RNAi of FGF8-like1 and FGF8-like2 was performed in embryos expressing the mesoderm-specific cell surface marker twi::CD2. In the wild-type, twi::CD2 marks cell shape changes occurring during mesoderm migration. The cells extend in the direction of migration and form long cellular protrusions. In embryos mutant for htl, these cell shape changes do not occur. Similarly, in embryos injected with dsRNA targeting FGF8-like1 and FGF8-like2, these cell shape changes are blocked. It is therefore concluded that FGF8-like1 and FGF8-like2 are required for cell shape changes of the mesoderm cells during migration. Because RNAi with FGF8-like1 did not affect differentiation of dorsal mesoderm derivatives, some functions of FGF8-like1 and FGF8-like2 might be redundant (Gryzik, 2004).
The fact that FGF8-like1 and FGF8-like2 are expressed in the ectoderm and are required for cell shape changes of mesoderm cells indicates a non-cell-autonomous function of FGF8-like1 and FGF8-like2. However, the FGF-receptor Htl is specifically expressed in the mesoderm cells. In order to test whether FGF8-like1 and FGF8-like2 are required for the activity of Htl in the mesoderm, the activation of the downstream signaling component MAP kinase was measured by using an antibody that recognizes the activated double-phosphorylated form of MAP kinase. In the wild-type, activated MAP kinase can be detected in the leading-edge cells of the migrating mesoderm. This early activation of MAP kinase in the mesoderm depends on the presence of Htl and its downstream signaling factor Dof. To test whether FGF8-like1 and FGF8-like2 are required for activation of MAP kinase in the mesoderm cells during migration, embryos homozygously mutant for Df(2R)ED2238 or Df(2R)ED2230 were stained with the dpERK antibody. Strikingly, only embryos mutant for Df(2R)ED2238 failed to exhibit dpERK staining in the mesoderm, whereas Df(2R)ED2230 mutant embryos looked like the wild-type. The defect in MAP kinase activation in Df(2R)ED2238 mutant embryos is specific for Htl FGF receptor activation because the staining of other cells that activate the MAP kinase pathway via the EGF receptor remains unimpaired (Gryzik, 2004).
In summary, mesoderm cells in embryos that lack FGF8-like1 and FGF8-like2 fail to exhibit Htl-dependent activation of MAP kinase. These results are consistent with a model in which FGF8-like1 and FGF8-like2 represent Htl receptor ligands, which are required for the early activation of the Htl signaling cascade during gastrulation (Gryzik, 2004).
The similarity of the early expression patterns of FGF8-like1 and FGF8-like2 suggests that their role during early gastrulation might be partially redundant. This idea is consistent with the result that RNAi knockdown of FGF8-like1 alone is not sufficient to produce defects in dorsal mesodermal derivatives. In contrast, during late gastrula stages the expression patterns of FGF8-like1 and FGF8-like2 differ, suggesting that the two genes might have distinct functions during later morphogenesis. This idea is supported by the observation that knockdown of FGF8-like2 alone does produce defects in mesoderm differentiation. It has been shown that the Htl receptor has dual functions in mesoderm morphogenesis. During gastrulation, Htl is required early for adhesive interactions of the mesoderm to the ectoderm and for cell shape changes associated with migration of the mesoderm cells. After gastrulation, Htl is required for specification of dorsal mesodermal derivatives that later will give rise to pericardial cells. The differential expression of FGF8-like1 and FGF8-like2 in later development suggests that the two ligands might act in a nonredundant fashion during mesoderm differentiation (Gryzik, 2004).
Several pieces of evidence suggest that FGF signaling via the Htl receptor is required for setting the correct timing for the interaction of mesoderm to ectoderm in early stages of gastrulation. The most robust migration phenotype of htl loss-of-function mutants occurs during early stages of mesoderm migration, at a time when the cells contact the ectoderm and migrate in dorsolateral direction. In late gastrula embryos, mesoderm cells exhibit directional protrusive activity in htl mutant embryos, indicating that htl is not essential for the migratory properties of the cells in a more general way. Ligand-independent activation of Htl in a htl mutant background is able to rescue the early defects in cell shape changes but fails to completely rescue the late defects in mesoderm migration and differentiation. At the beginning of gastrulation, FGF8-like1 and FGF8-like2 are uniformly expressed in the neuroectoderm, consistent with a permissive function for FGF signaling during early stages of gastrulation. This early expression pattern is likely to depend upon the Dorsal transcription factor because CG12443 has been described as a target of Dorsal (Gryzik, 2004).
The local activation pattern of MAP kinase suggests that during early mesoderm migration Htl is specifically activated in the leading-edge cells of the migrating mesoderm. Because htl is expressed in all mesodermal cells, it has been proposed that the potential ligands might be present in a graded fashion along the dorsoventral axis. Although FGF8-like1 is expressed in a uniform pattern throughout the germ band during gastrulation, FGF8-like2 expression is downregulated in the ventral-lateral ectoderm and only remains expressed in the dorsal-most ectodermal cells. Thus, FGF8-like2 might act as an instructive cue that guides mesoderm cells during the migration to their dorsal targets. The results of the single knockdown of FGF8-like2 by RNAi supports this model (Gryzik, 2004).
The FGF core domains of FGF8-like1 and FGF8-like2 exhibit a high degree of identity with vertebrate FGFs, in particular with mammalian FGF8. During mouse gastrulation, FGF8 is required for progenitor cells to migrate away from the primitive streak. At the primitive streak, epiblast cells undergo an epithelial/mesenchymal transition followed by ingression movement of the endodermal and mesodermal progenitor cells. Interestingly, in FGF8-/- embryos, the epithelial/mesenchymal transition in the streak occurs normally, but the cells fail to migrate and form an aggregate in the streak region. This cellular phenotype is reminiscent of the phenotype of Drosophila embryos mutant for htl. The mesoderm cells of htl mutants invaginate normally and undergo epithelial/mesenchymal transition, but fail to migrate out on the underlying ectoderm. Thus, the cellular functions of FGF8 signaling during gastrulation movements of mesodermal precursor cells in species as different as mouse and Drosophila share similar features (Gryzik, 2004 and references therein).
It is concluded that two FGF receptor homologs, Htl and Btl, are present in the Drosophila genome. Although the ligand of Btl is represented by Bnl, the ligand of the Htl receptor has remained unknown. Two novel FGF family members, FGF8-like1 and FGF8-like2, have been identified that are expressed at the right time and in the right place to serve as ligands for Htl. FGF8-like1 and FGF8-like2 are required for Htl-dependent cell shape changes during mesoderm migration and for signaling events emanating from the Htl receptor but are dispensable for signaling events emanating from other RTKs. It is concluded that FGF8-like1 and FGF8-like2 are required for promotion of mesoderm migration during Drosophila gastrulation and thus represent likely ligands of the FGF receptor Htl (Gryzik, 2004).
The similar early expression profiles of ths and pyr raise the possibility that they function in a redundant fashion to control mesoderm spreading during gastrulation. This would explain why previous genetic screens identified the Htl receptor, but failed to identify the FGF ligands. To date, zygotically lethal mutations have not been identified in either ths or pyr. Perhaps genetic redundancy exists between these two genes such that mutation of both would be required to reveal defects like those seen for htl mutants. To circumvent this potential problem, a small chromosomal deletion Df(2R)BSC252 was identified that removes both genes and was generated by a male-specific recombination event using the P-element P{lacW}walk14026. The exact breakpoints of this deficiency were defined; it removes ~200 kb of genomic DNA, including the entire 110-kb interval that contains ths and pyr. No more than 18 predicted genes are removed, which is not a very big number considering that <1% of all the genes in the Drosophila genome produce specific embryonic patterning defects when disrupted by zygotic mutation. Indeed, none of the predicted genes, several of which encode components of the apoptosis pathway , have been implicated in the control of embryonic development. Embryos were collected from the deficiency strain, and in situ hybridization assays confirm that ths and pyr are not expressed in mutant embryos that are homozygous for the deletion (Stathopoulos, 2004).
In normal embryos, activation of the Htl signaling pathway correlates with the spreading of the mesoderm along the internal surface of the neurogenic ectoderm. This signaling is absent in the mesoderm of htl mutants. To determine whether ths;pyr deficiency mutants have similar defects in mesoderm spreading, sections were analyzed of embryos stained with antibodies recognizing either Twist to observe the mesoderm or dpERK to monitor activation of the Htl pathway. Mesoderm cells begin to migrate at stage 7,8 of embryogenesis, and dpERK staining is detected in those cells that have come into contact with the ectoderm. In contrast, mesoderm migration is defective in ths;pyr deficiency mutants; they also lack dpERK staining (in the mesoderm), as seen in htl mutants. The mesoderm has completed its migration by early stage 10 of embryogenesis. In both wild-type and mutant embryos, the mesoderm comes into contact with Dpp-expressing cells in the dorsal ectoderm but shows various degrees of multilayered and irregular arrangements. The expanded mesoderm forms a monolayer in wild-type embryos, but displays multiple layers in ths;pyr deficiency mutants and htl mutants. dpERK staining is restricted to the dorsal mesoderm of wild-type embryos at stage 9,10 embryos, but is absent in ths;pyr deficiency mutants. Staining is expanded in embryos that ectopically express the ths ligand in the mesoderm. These results are consistent with a requirement of the Ths and Pyr ligands for activation of the Htl receptor in mesoderm migration during gastrulation (Stathopoulos, 2004).
As a result of mesoderm spreading, the dorsal mesoderm comes into contact with the dorsal ectoderm and is induced by a localized Dpp signal to express a variety of regulatory genes required for the differentiation of cardiac tissues and visceral mesoderm. Several marker genes were used to characterize the lethal phenotype caused by the deletion that may result from the inability of dorsal mesoderm lineages to differentiate. These include eve, tin, bagpipe (bap), and mef2. In htl mutants, there is either a loss or reduction in the expression of each of these regulatory genes. Similar disruptions are observed in ths;pyr (BSC25) deficiency homozygotes. There is a complete loss of Eve staining in the pericardial cells and dorsal muscle founder cells of both htl and BSC25 deficiency mutants, but the central nervous system (CNS) pattern is unaffected. After germ-band elongation, tin expression can be seen in two distinct lineages of the dorsal mesoderm, the visceral mesoderm and cardiac mesoderm. There is a severe reduction of tin expression in the cardiac lineage in htl mutants and BSC25 deficiency homozygotes. There is also reduced expression of bap in some segments, although expression is mostly normal (bap is required for visceral mesoderm formation). The minor defects in bap expression may result from incomplete spreading of the mesoderm along the entire anterior-posterior axis in htl and deficiency mutant embryos, whereas the more severe defects in the transcriptional up-regulation of eve and tin may result from a late requirement of Htl activation after mesoderm spreading (Stathopoulos, 2004).
The mef2 gene is expressed in a variety of mesodermal tissues, including the differentiating body wall muscles and the dorsal vessel, or heart. Most aspects of this staining pattern are unaffected in BSC25 deficiency homozygotes, but there is a selective loss of staining in the dorsal mesoderm that forms the heart cells and dorsal somatic muscles. Similar defects have been described for htl mutants, and in both cases, the selective loss of dorsal mesoderm derivatives can be explained by the combined effects of uneven mesoderm spreading and the loss of late Htl signaling after mesoderm spreading (Stathopoulos, 2004).
A late embryonic requirement for Htl signaling has been established for the visceral musculature surrounding the hindgut. Hindgut visceral mesoderm (HVM) migrates over the hindgut ectoderm during dorsal closure, and this migration depends on both Wingless (Wg) and Htl. Unlike the migration of the HVM, subsequent differentiation does not require Wg signaling, but depends solely on Htl. In htl and BSC25 deficiency mutants, Mef2 staining is absent from the hindgut, implying that the hindgut musculature has not differentiated. Although a role for Htl in the differentiation of the pharyngeal muscles has not been previously described, Mef2 staining of pharyngeal muscle is reduced in htl mutants with an even more severe reduction observed in BSC25 deficiency embryos. In addition, the visceral mesoderm associated with both the stomadeum and hindgut in these advanced-stage embryos also expresses bap. This staining is lost in htl and BSC25 deficiency mutant embryos. Both ths and pyr are expressed in the stomadeum and hindgut at the time when these mesodermal derivatives form, and thus it seems likely that the encoded FGFs function as signals to control their movement and/or differentiation at these later stages of embryogenesis (Stathopoulos, 2004).
A dominant-negative form of Htl blocks the formation of the muscle founder cells and the differentiation of the ventral oblique muscles. Most ventral oblique muscles are absent in htl and BSC25 deficiency mutant embryos. This observation is consistent with the model that the localized expression of Pyr and Ths in the ventral ectoderm, possibly within ventral neuroblasts, is required for the specification of muscle founder cells through activation of the Htl receptor (Stathopoulos, 2004).
If Ths and Pyr are the ligands for Htl, then the misexpression of one or both genes in the mesoderm should be sufficient to trigger its activation. A full-length ths cDNA was misexpressed in the mesoderm using a twist-Gal4 transgene. The levels of ectopic ths mRNAs are several-fold higher than the endogenous products, but nonetheless the mesoderm still spreads toward the dorsal ectoderm. There is a substantial increase in the number of Eve-expressing mesodermal cells in embryos ectopically expressing ths in the mesoderm. A similar expansion is obtained with constitutively activated forms of the Htl receptor or Ras kinase or when ths is expressed using an ectodermal driver, 69B-gal4. These results are consistent with Ths acting as a ligand for the Htl receptor (Stathopoulos, 2004).
Mef2 staining was also examined in embryos that mis-express ths throughout the mesoderm. These embryos exhibit no obvious defects in most mesoderm derivatives such as the hindgut musculature with this marker, although the ventral oblique muscles are either absent or unable to extend into ventral regions. One possible explanation is that guidance of the ventral oblique muscles is controlled by pyr and/or ths expression in ventral neuroblasts and that ectopic expression of ths at high levels within the mesoderm masks this guidance mechanism (Stathopoulos, 2004).
The simplest model for the mutant phenotype seen in deficiency homozygotes is that the loss of Ths and Pyr blocks the activation of the Htl receptor, which in turn causes impaired spreading and induction of the mesoderm. To test this model, a genetic complementation experiment was done using a constitutively activated form of the Htl receptor that in theory no longer requires ligand binding for activation. The mutant receptor was expressed in the developing mesoderm of transgenic embryos using a twist-Gal4 transgene. Mutant embryos homozygous for the ths;pyr deficiency never exhibit any eve expression in developing pericardial cells. However, introduction of the activated Htl receptor partially restores Eve expression in the dorsal mesoderm. Staining tends to be stronger in posterior segments, but some of the embryos exhibit Eve staining in anterior regions as well. Similar overexpression of the wild-type htl does not rescue the pericardial Eve expression pattern in mutant embryos. These results indicate that the constitutively activated Htl receptor partially circumvents the need for Ths and Pyr in the differentiation of the dorsal mesoderm (Stathopoulos, 2004).
Tests were performed to see whether ths expression is sufficient to complement the BSC25 deficiency. When ths is expressed in the mesoderm there is strong, but somewhat erratic rescue of the pericardial cells. There is more uniform rescue when ths is expressed in the ectoderm using a 69B-Gal4 driver. These results agree with findings that mesoderm spreading and induction are normally coordinated by FGF signals emanating from the ectoderm (Stathopoulos, 2004).
Fibroblast growth factor (FGF) signaling controls a vast array of biological processes including cell differentiation and migration, wound healing and malignancy. In vertebrates, FGF signaling is complex, with over 100 predicted FGF ligand-receptor combinations. Drosophila presents a simpler model system in which to study FGF signaling, with only three ligands and two FGF receptors (FGFRs) identified. This study analyzed the specificity of FGFR [Heartless (Htl) and Breathless (Btl)] activation by each of the FGF ligands [Pyramus (Pyr), Thisbe (Ths) and Branchless (Bnl)] in Drosophila. It was confirmed that both Pyr and Ths can activate Htl, and that only Bnl can activate Btl. To examine the role of each ligand in supporting activation of the Htl FGFR, genetic approaches were utilized that focus on the earliest stages of embryonic development. When pyr and ths are equivalently expressed using the Gal4 system, these ligands support qualitatively different FGFR signaling responses. Both Pyr and Ths function in a non-autonomous fashion to support mesoderm spreading during gastrulation, but Pyr exhibits a longer functional range. pyr and ths single mutants exhibit defects in mesoderm spreading during gastrulation, yet only pyr mutants exhibit severe defects in dorsal mesoderm specification. This study demonstrated that the Drosophila FGFs have different activities and that cell migration and differentiation have different ligand requirements. Furthermore, these FGF ligands are not regulated solely by differential expression, but the sequences of these linked genes have evolved to serve different functions. It is contended that inherent properties of FGF ligands make them suitable to support specific FGF-dependent processes, and that FGF ligands are not always interchangeable (Kadam, 2009).
These experiments demonstrate that the Drosophila
FGFs Pyr, Ths and Bnl have different functions and that the activation of FGF
receptors by specific ligands affects particular biological processes.
Examination of an allelic series of pyr and ths mutants
suggests that pyr and ths are not redundant in function:
both influence mesoderm spreading, whereas pyr is the dominant player
controlling Eve+ cell specification within the dorsal mesoderm. It has been demonstrated that ectopic expression of ths by twist-Gal4 and
69B-Gal4 in the Df(2R)BSC25 mutant background can support
Htl FGFR activation. However, this study assayed whether the expression
supported in distinct domains would support Htl activation. By a series of
'rescue' experiments, through ectopic expression of one ligand in the
Df(2R)BSC25 mutant background, evidence was obtained that localized expression of the ligands is important for proper mesoderm spreading. It was found, surprisingly, that the ligands exhibit differences in their functional range of action. In addition, using this same approach, it was found that either Pyr or Ths can support Eve+ cell specification within the dorsal mesoderm, but that Bnl cannot. Collectively, these data suggest that the Pyr and Ths FGFs function as ligands for the Htl FGFR and that specificity of FGF-FGFR interactions exists in Drosophila (Kadam, 2009).
The results demonstrate that both Pyr and Ths FGF ligands can activate the
Htl FGFR, whereas only the Bnl FGF ligand can activate the Btl FGFR. Specificity of FGFR activation was observed:
pyr or ths, but not bnl, expression is able to
activate Htl to affect expression of Eve, and bnl, but neither
pyr nor ths, is able to support tracheal specification. No
evidence was obtained that other cross-interactions occur (i.e. Pyr-Btl,
Ths-Btl or Bnl-Htl), which demonstrates that Gal4-mediated ectopic expression
does not simply 'swamp the system'. This experimental approach also 'levels
the playing field', since expression of each ligand is driven at the same time
and place and presumably at similar levels. It is concluded that only three
FGF-FGFR combinations function in Drosophila (i.e. Pyr-Htl, Ths-Htl
and Bnl-Btl), which supports the idea that FGFRs exhibit ligand-binding
preferences. Previous studies have investigated FGF signaling specificity by
analyzing the ability of other receptor tyrosine kinases to support cell
migration or by activating particular intracellular signaling pathways to
examine which are required to effect FGFR-dependent cell migration versus cell
differentiation. This work analyzed the specificity of FGF ligand-receptor interactions and how they contribute to particular developmental processes (Kadam, 2009).
When ligand expression is supported by twist-Gal4, Htl FGFRs
presumably become saturated because dpERK is ectopically activated in all
cells and spreading is negatively affected. One explanation for why this might affect mesoderm cell spreading is that these FGF-saturated mesoderm cells may no longer be competent to respond to endogenous ligands that provide directional cues. Recently, it has been shown that movement of the mesoderm cells during gastrulation is in fact
directional (McMahon, 2008). Pyr and Ths ligands are differentially expressed during
gastrulation and this might provide the necessary positional information
required to direct migration of the mesoderm. It is proposed that Pyr and Ths have different activities that fulfil aspects of FGFR activation required to support cell migration. Ectopic expression of Pyr within the ectoderm negatively affects mesoderm spreading, which suggests
that the refined expression domain of pyr within cells of the dorsal
ectoderm is normally required to guide the mesoderm cells toward dorsal
regions. However, even though ectopic expression of ths in the
ectoderm has no effect on mesoderm spreading, ths mutants also
exhibit defects in mesoderm spreading, demonstrating that both genes are
required, perhaps to control different aspects of the migration. The 'rescue'
experiments using the zenVRE.Kr-Gal4 driver support the view that Pyr
has a longer functional range than Ths. These differences
in range of function might correlate with different diffusion capabilities,
but an alternative explanation is that the ligands activate the receptor with
different affinities. Additional experiments will be necessary to distinguish
their exact functions and to uncover the molecular basis for the differential
functions of Pyr and Ths; it is suggested that in vivo imaging and quantitative
analysis (McMahon, 2008) of single-mutant phenotypes will provide insights (Kadam, 2009).
With regard to the FGF-dependent cell differentiation, the 'rescue'
experiments suggest that ectopic expression of either Pyr or Ths is sufficient
to support Eve+ cell specification. The reason why
loss of ths has less of an effect on Eve+ cell
specification is most likely because pyr is prominently expressed in
the vicinity of the future Eve+ cells; normally, Pyr supports this function, but Ths can support this activity if presented at sufficient levels
within the correct domain. Furthermore, it is proposed that FGF signaling might
not play an instructive role in supporting eve expression. Other signaling
pathways already provide positional information required for the specification
of Eve+ cells; FGF signaling pathway activation might simply serve
a permissive role, and in this context either ligand would suffice (Kadam, 2009).
Thisbe (Ths) and Pyramus (Pyr), two closely related Drosophila homologues of the vertebrate fibroblast growth factor (FGF) 8/17/18 subfamily, are ligands for the FGF receptor Heartless (Htl). Both ligands are required for mesoderm development, but their differential expression patterns suggest distinct functions during development. Single mutants were generated and it was found that ths or pyr loss-of-function mutations are semi-lethal and mutants exhibit much weaker phenotypes as compared with loss of both ligands or htl. Thus, pyr and ths display partial redundancy in their requirement in embryogenesis and viability. Nevertheless, it was found that pyr and ths single mutants display defects in gastrulation and mesoderm differentiation. Localised expression of pyr is required for normal cell protrusions and high levels of MAPK activation in migrating mesoderm cells. The results support the model that Pyr acts as an instructive cue for mesoderm migration during gastrulation. Consistent with this function, mutations in pyr affect the normal segmental number of cardioblasts. Furthermore, Pyr is essential for the specification of even-skipped-positive mesodermal precursors and Pyr and Ths are both required for the specification of a subset of somatic muscles. The results demonstrate both independent and overlapping functions of two FGF8 homologues in mesoderm morphogenesis and differentiation. It is proposed that the integration of Pyr and Ths function is required for robustness of Htl-dependent mesoderm spreading and differentiation, but that the functions of Pyr have become more specific, possibly representing an early stage of functional divergence after gene duplication of a common ancestor (Klingseisen, 2009).
The identification of two transposon-associated alleles,
ths02026 and pyr02915, and two
chromosomal deletions, Df(2R)ths238 and Df(2R)pyr36 has been
reported (Kadam, 2009). Genetic complementation analysis with pyr18 and ths759 supports the view that pyr02915 represents a loss-of-function allele, whereas ths02026 is a hypomorphic allele. However, the weaker Eve phenotype of pyr02915 compared with pyr18 indicates that pyr02915 is unlikely to be a null allele. The alleles presented in this study
represent loss-of-function alleles: in pyr18 the entire
pyr gene is deleted, and in ths759 most of the
conserved FGF core domain is deleted. In both cases, neighbouring genes remain
unaffected. Df(2R)ths238 and Df(2R)pyr36 uncover
ths and pyr, respectively, but also delete neighbouring
genes (Kadam, 2009). In summary, whereas a null allele for pyr exists
(pyr18), there is currently no null allele of ths
available that does not simultaneously delete other genes. In
ths759, 84 of the apparent 107 amino acids of the Ths FGF
core domain are deleted. The FGF core domain is conserved in all FGFs, with 28
highly conserved and six identical amino acids. The
core domain contains amino acids important for heparin proteoglycan binding,
glycosylation and FGF receptor activation. Nine conserved amino acids in Ths have been identified that are identical within the FGF8 subfamily, four of which are identical in all FGFs. In the ths759 allele, all of these nine identical amino acids are deleted. Therefore, if the formal
possibility is excluded that the ths759 gene product retains activity independent of the FGF core domain, ths759 represents a functional null allele (Klingseisen, 2009).
The complex expression patterns of htl and its two ligands,
pyr and ths, in post-gastrulation stages suggested that Htl
signalling functions directly in cell fate decisions during mesoderm
differentiation. pyr is required for Eve expression in dorsal
mesoderm derivates, whereas ths is dispensable
(Kadam, 2009). In addition, overexpression of Pyr leads to an expansion of mesodermal Eve-positive clusters in a similar fashion to experimental overactivation of the Ras1 (Ras85D) pathway. Ths exhibits similar gain-of-function effects to Pyr with respect to expansion of Eve-positive clusters in dorsal mesoderm, suggesting that Ths and Pyr have similar signalling properties
(Kadam, 2009). However, as Eve expression is unaffected in ths single mutants, it is unlikely that Ths contributes to the
expression of Eve in these cells (Kadam, 2009; Klingseisen, 2009).
Expression of Eve in the precursors of the pericardial cells and DA1 muscle
founders depends on the activation of several signalling pathways in a group
of mesodermal pre-clusters expressing lethal of scute.
Wingless (Wg) and Dpp signalling define a dorsal domain of mesoderm cells that
are competent to activate transcription of eve in response to
localised activation of Ras1. This localised Ras1 activation is largely
dependent on Htl signalling. During this specification process, Pyr is expressed in
segmental dorsal ectodermal patches in close proximity to the sites in the
mesoderm where the dorsal Eve-positive clusters form. Whereas the effect on
Eve expression is fully penetrant, the generation of other dorsal mesodermal
precursors, e.g. those expressing Lb, is only mildly affected in pyr
mutant embryos. Interestingly, it was observed that overexpression of Pyr results in strong activation of MAPK and ectopic Eve expression in the absence of normal dorsolateral migration. These results indicate that Pyr expression
causes cells to become more sensitive to Dpp and Wg signalling and thus
represents a limiting factor of the signalling network that triggers
specification of Eve-positive dorsal mesoderm (Klingseisen, 2009).
With the exception of the lack of Eve-expressing mesodermal precursors,
none of the other mesoderm differentiation defects in pyr single
mutants occurred with similar expressivity; for instance, the defects in
formation of specific somatic muscles (SBM, VO4, VO5 and VO6) were penetrant
at a low expressivity as they did not occur in each segment. In addition, the
defects in SBM and VO muscles were also evident in ths homozygotes
and became even more severe when one copy of the ths gene was removed
in a pyr homozygous background. These observations suggest
overlapping functions of pyr and ths in the specification of
these muscles. In summary, it is concluded that both ligands are involved in the differentiation of specific subsets of muscles (Klingseisen, 2009).
Whereas htl mutants exhibit severe defects, ths and
pyr single mutants exhibit weak defects in mesoderm spreading
(Kadam, 2009). Nevertheless, this study found that both ligands are required for equal attachment of the mesoderm cells on to the ectoderm after invagination. As this phenotype occurs in both single mutants, either the overall level of FGF
ligand at this stage is crucial, or both of the ligands need to bind to
Htl-expressing cells, or each of the FGFs exerts independent functions in this
process. When the gene dosage of both of the ligands is reduced by half, early
mesoderm morphogenesis was normal, excluding the possibility that the overall
level of FGF plays a major role.
Furthermore, it was recently shown that each ligand is able to signal in the
absence of the other, suggesting that Ths and Pyr do not directly cooperate in
Htl activation (Kadam, 2009). It will be interesting to
determine how each of the ligands might independently support particular
aspects of early mesoderm movements (Klingseisen, 2009).
Although both ligands are required for the early stages, only pyr
mutants exhibited defects in dorsolateral migration and mesoderm monolayer
formation (Kadam, 2009). The defects in monolayer formation observed in in the curren study are only subtle, in contrast to the defects reported by Kadam
(2009). These
discrepancies might reflect differences in the alleles used in the two
studies. No monolayer defects were observed in ths759
mutant alleles, whereas a deletion uncovering ths exhibits defects in
monolayer formation (Kadam, 2009). This raises the possibility that domains other than the FGF core domain present in the protein encoded by the ths759
allele might exert some function in monolayer formation. This is thought
unlikely since the non-conserved C-terminal tail is dispensable for activation of Htl. The deletion that was used to eliminate
ths function, Df(2R)ths238, eliminates ths and ten
proximal genes raising the alternative possibility that deletion of a gene (or
genes) within Df(2R)ths238 contributes to the rather severe mesoderm
spreading defect presented by Kadam (2009). Rescue
experiments using full-length genomic constructs will be informative to
further characterise these ths deletion alleles (Klingseisen, 2009).
The presently available data are consistent with a role of the localised
expression of Pyr at the dorsal edge of the ectoderm in providing an
instructive cue for the cells to migrate in a dorsal direction. For example, Pyr expression might produce an instructive cue that promotes dorsolateral
movement of the mesoderm. It has been shown previously that FGFs can exhibit
characteristics of chemoattractants in other systems. Although
loss- and gain-of-function analyses demonstrate that pyr is required
for normal protrusive activity during dorsolateral migration, monolayer
formation is much less affected than in htl mutants or ligand double
mutants (Kadam, 2009). Therefore, although Pyr might provide a directional cue,
non-polarised expression of Ths alone can compensate to some extent for the
absence of this putative directional cue. In this sense, the two ligands
differ slightly in their requirements for mesoderm spreading, but it is the
directional movement through localised expression of pyr that causes
this to be a robust morphogenetic process (Klingseisen, 2009).
The FGF8-like ligands exhibit overlapping functions except for the
induction of mesodermal Eve expression, the formation of the SBM and
dorsolateral migration. They cooperate to provide robustness of Htl-dependent
mesoderm morphogenesis and differentiation. These imperfect redundancies
become obvious in the single mutant phenotypes and might reflect the fact that
pyr and ths are likely to be derived from a gene duplication
event in the Drosophilids. In more basic insects,
such as Anopheles gambia and Tribolium castaneum, only one
FGF8-like gene exists, and this is more similar to ths and might
represent a common ancestor. It has therefore been suggested that
ths might have retained some of the ancestral functions of the
Fgf8 homologue in Drosophila melanogaster. This would imply
that the establishment of a localised dorsal expression domain and the
hypothesised instructive role of Pyr are derived qualities. The data presented
in this study indicate that localised expression of pyr renders mesoderm
spreading more robust than in the absence of pyr expression. It would
be of interest to analyse the expression and function of FGF8-like signalling
in more basic insects that exhibit long germ band development and contain only
one FGF8 homologue. Gene duplication has been proposed as a general mechanism
in vertebrates to explain the expansion of FGF genes. Studies in dipteran
species might provide insights into the evolution of the requirements for
localised expression of a growth factor in directional cell movement during
gastrulation (Klingseisen, 2009).
Migration is a complex, dynamic process that has largely been studied using qualitative or static approaches. As technology has improved, it is now possible to take quantitative approaches towards understanding cell migration using in vivo imaging and tracking analyses. In this manner, a four-step model of mesoderm migration during Drosophila gastrulation was establised: (I) mesodermal tube formation, (II) collapse of the mesoderm, (III) dorsal migration and spreading and (IV) monolayer formation. The data provide evidence that these steps are temporally distinct and that each might require different chemical inputs. To support this, the role was analyzed of fibroblast growth factor (FGF) signaling, in particular the function of two Drosophila FGF ligands, Pyramus and Thisbe, during mesoderm migration. It was determined that FGF signaling through both ligands controls movements in the radial direction. Thisbe is required for the initial collapse of the mesoderm onto the ectoderm, whereas both Pyramus and Thisbe are required for monolayer formation. In addition, it was uncovered that the GTPase Rap1 regulates radial movement of cells and localization of the beta-integrin subunit, Myospheroid, which is also required for monolayer formation. These analyses suggest that distinct signals influence particular movements, since it was found that FGF signaling is involved in controlling collapse and monolayer formation but not dorsal movement, whereas integrins are required to support monolayer formation only and not earlier movements. This work demonstrates that complex cell migration is not necessarily a fluid process, but suggests instead that different types of movements are directed by distinct inputs in a stepwise manner (McMahon, 2010).
Mesoderm migration was found to be a combination of complex three-dimensional movements involving many molecular components. live imaging, coupled with quantitative analyses, is important for studying complex cell movements, as it allowed migration to be decomposed into different movement types and thus has allowed description of subtle phenotypes. First, analysis of the directional movements of mesoderm cells within wild-type embryos was extended, focusing on the temporal sequences of events. Cells were found follow a sequential and distinct set of trajectories: movement in the radial direction (tube collapse: -5 to 15 minutes, 0=onset of germband elongation), followed by movement in the angular direction (dorsal migration: 15 to 75 minutes) and ending with small intercalation movements in the radial direction (monolayer formation: 75 to 110 minutes). These movements appear temporally distinct (i.e. stepwise), and thus molecular signals controlling each process were sought (McMahon, 2010).
Which mesoderm movements were FGF-dependent were investigated and, in particular, either Ths- or Pyr-dependent. The interaction between Htl and its two ligands provides a simpler system relative to vertebrates (which exhibit over 120 receptor-ligand interactions) in which to study how and why multiple FGF ligands interact with the same receptor. Previously, it was found that FGF signaling via the Htl FGFR controls collapse of the mesodermal tube but not dorsal-directed spreading (McMahon, 2008). This study demonstrated that FGF signaling is also required for monolayer formation. In addition, distinct, non-redundant roles were defined for the FGF ligands: Ths (but not Pyr) is required for collapse of the mesodermal tube, whereas both Pyr and Ths are required for proper intercalation of mesoderm cells after dorsal spreading (McMahon, 2010).
This analysis raises questions about ligand choice during collapse and monolayer formation. Within the mesodermal tube, cells at the top require a long-range signal in order to orient towards the ectoderm during tube collapse, whereas the signals controlling intercalation during monolayer formation can be of shorter range. It is suggested that the ligands have different activities that are appropriately tuned for these processes. In fact, recent studies of the functional domains of these proteins suggest that Ths has a longer range of action than Pyr, in agreement with the analysis that Pyr does not support tube collapse, but does have a hand in monolayer formation (McMahon, 2010).
This study has demonstrated that Rap1 mutants have a similar mesoderm phenotype to the FGFR htl mutant, with defects in collapse and monolayer formation. It was not possible to establish whether Rap1 acts downstream of FGF signaling, as the complete loss of Mys in Rap1 mutants is more severe than the patchy expression of Mys seen in htl mutants. Therefore, Rap1 could be working in parallel to or downstream of FGF signaling during mesoderm migration. Rap1 has been implicated in several morphogenetic events during Drosophila gastrulation and probably interacts with many different signaling pathways. Further study of Rap1, along with other GTPases, will shed light onto their role during mesoderm migration, how they interact with one another and what signaling pathways control them (McMahon, 2010).
Focus was placed on the more specific phenotype of mys mutants, as its localization is affected in htl mutants and it exhibits a monolayer defect that is similar to pyr and ths mutants. Integrins are important for cell adhesion, so it is not surprising that cells fail to make stable contact with the ectoderm through intercalation in mys mutants. However, some cells do contribute to monolayer formation in the absence of Mys, implying that other adhesion molecules are involved in maintaining contact between the mesoderm and ectoderm. These other adhesion molecules might be activated downstream of FGF signaling as the htl mutant monolayer phenotype is more severe than the mys mutant. Discovering the downstream targets of Htl, which might regulate cell adhesion properties, will help to shed light on the mechanisms supporting collapse of the mesodermal tube (which is not dependent on Mys) and monolayer formation (which is Mys-dependent) (McMahon, 2010).
Cell protrusions, such as filopodia, are important for sensing chemoattractants and polarizing movement during migration. Previous studies have focused on protrusive activity at the leading edge during mesoderm migration in Drosophila and shown that these protrusions are FGF-dependent. In this study, it was found that protrusions exist in all mesoderm cells, not just the leading edge, and that these protrusions also extend into the ectoderm (McMahon, 2010).
The study demonstrates that FGF signaling, as well as integrin activity, is required to support protrusive activity into the ectoderm; this is a potential mechanism by which FGF signaling and Mys could control movement toward the ectoderm during monolayer formation. The function of protrusions at the leading edge remains unclear, as they appear to be reduced in pyr and mys mutants, but migration in the dorsal direction still occurs in both mutant backgrounds. One interpretation is that FGF and Mys are important for generalized protrusive activity and that extensive protrusions are required for intercalation but not dorsal migration (McMahon, 2010).
Based on this study, it is proposed that mesoderm migration is a stepwise process, with each event requiring different molecular cues to achieve collective migration. Invagination of the mesoderm is the first step in this process and is dependent on Snail, Twist, Concertina, Fog and several other genes. Next, collapse of the mesoderm tube onto the ectoderm requires Htl activation via Ths. Rap1 might be involved in this process as well but the phenotype of Rap1 mutants is complex and it is unclear which phenotypes are primary defects (McMahon, 2010).
Following collapse, mesoderm cells spread dorsally by an unknown mechanism. Dorsal migration is unaffected in pyr and ths mutants and occurs in all cells that contact the ectoderm in htl mutants, implying that FGF signaling is, at most, indirectly involved in this step owing to the earlier tube collapse defect (McMahon, 2008). Whether dorsal migration requires chemoattractive signals or whether the cells simply move in this direction because it is the area of least resistance remains unclear (McMahon, 2010).
Finally, after dorsal spreading is complete, any remaining cells not contacting the ectoderm intercalate to form a monolayer. This process is controlled by a combination of both Pyr and Ths interacting through Htl and also by Rap1 and Mys. In other systems, intercalation can lead to changes in the properties of the cell collective, for instance, lengthening of a body plan. However, this study has shown that dorsal migration and spreading are not a result of intercalation, as intercalation occurs after spreading has finished (McMahon, 2010).
Coordination of these signals to control collective migration enables the mesoderm to form a symmetrical structure, which is essential for embryo survival. This model begins to address the question of how hundreds of cells move in concerted fashion and is relevant for a generalized understanding of embryogenesis and organogenesis. It was found that mesoderm migration is accomplished through sequential movements in different directions, implying that collective migration might be best achieved by distinct phases of movement (McMahon, 2010).
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date revised: 5 August 2011
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