FGF receptor 1

DEVELOPMENTAL BIOLOGY

Effects of Mutation or Deletion

Fr1 is required for the specification of even-skipped expressing pericardial and somatic muscle cells. There is a marked reduction in the number of cardial cells as revealed by expression of tinman transcripts as well as myosin heavy chain protein. Most of the dorsal somatic muscles are entirely missing and gaps are seen in the lateral and ventral muscle groups as well. In addition, the visceral musculature fails to undergo its normal morphogenesis in the absence of Fr1 function. Whereas the midgut acquires three constrictions in normal embryos, this fails to occur in Fr1 mutants. Specific muscle precursors expressioning nautilus and Krüppel are also missing in Fr1 mutants. There is a generalized reduction in muscle precursor number as indicated by D-MEF2 expression in the somatic mesoderm, with the most marked effect again evident in the dorsal region. In mutant embryos, the mesodermal cell mass initially remains aggregated and does not undergo the full extent of dorsal migration characteristic of its wild-type counterparts (Gisselbrecht, 1996).

To determine whether mesodermal cells deficient in Fr1 are nevertheless competent to form dorsal mesodermal derivatives, decapentaplegic was ectopically expressed throughout the mesoderm in a Fr1 mutant background. In wild-type embryos, such ectopic DPP expression dorsalizes the mesoderm. When DPP is provided to the entire mesoderm in Fr1 mutants, bagpipe transcription is activated all along the dorsoventral axis where mesodermal cells are found, including in the most ventral postions. These findings demonstrate that the Fr1-deficient mesoderm is competent to respond to DPP in the specification of visceral mesodermal fates (Gisselbrecht, 1996).

Fibroblast growth factor receptor (FGFR) encoded by the heartless (htl) gene is involved in early directional migration of the Drosophila mesoderm. New data is provided that (1) demonstrate a second role for Htl in promoting the specification of the precursors to certain cardiac and somatic muscle cells in the Drosophila embryo, independent of its cell migration function; (2) suggest that Ras and at least one other signal transduction pathway act downstream of Htl, and (3) establish a functional relationship between the Ras pathway and Tinman (Tin), a homeodomain factor that is essential for specifying some of the same dorsal mesodermal cells that are dependent on Htl (Michelson, 1998a).

The involvement of Htl in mesodermal founder cell fate specification was tested by reducing its activity under conditions where earlier cell migration is not compromised. This was accomplished by ectopic expression of a dominant negative of the Htl Fgf receptor. Dominant negative Htl induces numerous defects in mesodermal structures at multiple positions along the dorsoventral axis and at different stages of development. In late stage embryos, somatic muscles are missing from ventral, lateral and dorsal groups, and gaps occur in the rows of cardial and pericardial cells. These defects can be traced to an earlier stage where the corresponding precursor cells are found to be lacking. For example, dominant negative Htl prevents the formation of progenitors of the Eve-expressing pericardial and somatic muscle cells. Small gaps in the normally continuous rows of visceral mesodermal precursors are also observed (Michelson, 1998a).

Ectopic mesodermal expression of a constitutively active form of Ras1 is capable of partially rescuing a strong hypomorphic htl mutant. Partial rescue of a null htl mutation by activated Ras1 also is manifest in the expression of Eve in the dorsal mesoderm. No Eve-positive cells are found in the complete absence of htl function, whereas a hypomorphic mutant contains a markedly reduced number of Eve-expressing segments. Although the Epidermal growth factor, a second receptor tyrosine kinase, is involved in development of Eve muscle founders, all of the Eve-positive cells generated by activated Ras1 in htl mutant embryos are confined to the dorsal mesoderm in their usual segmental pattern, consistent with the involvement of Ras1 in both the migration and cell fate specification functions of Htl (Michelson, 1998a).

Interestingly, loss-of-function mutations in tinman and htl have identical affects on the development of Eve pericardial and somatic muscle cells. Similarities are also seen between the cardial and dorsal somatic muscle phenotypes of these two genes. However, tinman differs significantly from htl in the mechanism of action since mesoderm migration is completely normal in tinman mutants. This implies that tinman is involved in only one of the processes affected by htl, namely the determination of dorsal mesodermal cell fates. Since Ras1 functions in the Htl signaling pathway and activation of this signal transduction has the opposite effect on Eve progenitor development as tin loss-of-function, an epistasis experiment could be performed. Expression of activated Ras1 in a tinman mutant background results in an Eve expression phenotype corresponding to that of tinman. That is, tinman loss-of-function completely blocks the ability of activated Ras1 to promote the formation of Eve pericardial cell and somatic muscle progenitors (Michelson, 1998a).

The following model is proposed for the role of Tinman and Htl in the formation of Dorsal mesoderm. When mesodermal cells reach the dorsal-most region of the ectoderm, they are induced by Dpp to express Tinman, thereby acquiring the competence to differentiate into visceral, cardiac, or dorsal somatic muscle derivatives. Superimposed on this process is the activation of the Ras1 pathway in a small subset of dorsal mesodermal cells. Ras1 activation is mediated by Htl in those cells destined to form the Eve pericardial progenitiors, whereas both Htl and Egf receptor function together to generate a Ras1 signal specific to Eve-positive somatic muscle fate. In this sense, Htl/Egfr/Ras1 signaling serves to distinguish a fate characterized by Eve expression from additional dorsal mesodermal fates that are also dependent on tinman. It should be noted that Tin and Ras1 regulation are not required to function in any particular order; one may precede the other or they may act simultaneously. The essential point is that both are absolutely required for the specification of Eve cardiac and somatic muscle fates in the dorsal mesoderm (Michelson, 1998a).

Embryos with an extensive deletion in the FR1 region show a marked reduction in nautilus expression and a reduction of muscle fiber content. Ventral furrow formation is normal (Shishido, 1993).

Mesodermal progenitors arise in the Drosophila embryo from discrete clusters of lethal of scute (l'sc)-expressing cells. Individual progenitors are specified by the sequential deployment of unique combinations of intercellular signals. Initially, the intersection between the Wingless (Wg) and Decapentaplegic (Dpp) expression domains demarcate an ectodermal prepattern that is imprinted on the adjacent mesoderm in the form of L'sc preclusters. One precluster, preC1, is found in the ventral mesoderm, and the other, preC2, is localized to the dorsal mesoderm. PreC2 encompasses the territory in which dorsal L'sc clusters C2 and C14-C17, the subject of this paper, subsequently develop. All mesodermal cells within preC2 precluster are competent to respond to a subsequent instructive signal mediated by two receptor tyrosine kinases (RTKs), the Drosophila epidermal growth factor receptor (Egfr) and the Heartless (Htl) fibroblast growth factor receptor. By monitoring the expression of the diphosphorylated form of mitogen-associated protein kinase (MAPK), these RTKs are seen to be activated in small clusters of cells within the original competence domain (precluster). Each cluster represents an equivalence group because all members initially resemble progenitors in their expression of both L'sc and mesodermal identity genes. Thus, localized RTK activity induces the formation of mesodermal equivalence groups. The RTKs remain active in the single progenitor that emerges from each cluster under the subsequent inhibitory influence of the neurogenic genes. The singling out of progenitors from mesodermal equivalence groups depends on lateral inhibition mediated by the neurogenic genes. Moreover, Egfr and Htl are differentially involved in the specification of particular progenitors (Carmena, 1998).

Of the two clusters of dorsal mesodermal cells that express the pair-rule gene even-skipped that arise sequentially from a single precluster, one cluster gives rise to a single Eve-positive progenitor that divides to give rise to a pair of pericardial cells (P2). The second cluster (P15, arising from the same C2 precluster that gives rise to P2) gives rise to at least one dorsal somatic muscle and forms from a second Eve progenitor. The two L'sc clusters from which these progenitors arise are prefigured by a broader domain of L'sc expression (the precluster) that is dependent on the combined activities of Wg and Dpp. The corresponding equivalence groups (clusters) are formed within this prepatterned mesodermal region via localized activation of the Ras1 pathway by two receptor tyrosine kinases (RTKs), Htl, and Egfr. Whereas Htl is required for the Eve cardiac equivalence group, both Egfr and Htl are involved in the Eve muscle cell cluster. These findings demonstrate how positional information initially establishes a mesodermal prepattern, and establish that individual progenitors are progressively determined by unique combinations of intercellular signals. It is concluded that distinct cellular identity codes are generated by the combinatorial activities of Wg, Dpp, Egf, and Fgf signals in the progressive determination of embryonic mesodermal cells (Carmena, 1998).

To examine the question of how Egf and Fgf signals are involved in precluster or cluster development, expression of dominant-negative forms of both receptors were targeted to the embryonic mesoderm. Neither dominant-negative Htl nor dominant-negative Egfr affect preC2 development. However, inhibition of Htl, but not Egfr, signaling leads to loss of C2. This requirement for Htl reflects a direct involvement in cell fate specification because mesoderm migration is completely normal when dominant-negative Htl is expressed under the present conditions. After the singling out of P2 from the C2 precluster, C15 forms at the position occupied previously by C2, a process that requires both Egfr and Htl, in agreement with the demonstration that both RTKs are required for muscle DA1 development. Htl (but not Egfr) also contributes to the formation of other dorsal L'sc clusters. In summary, establishment of the L'sc prepattern does not require the activity of either Egfr or Htl. However, both RTKs are essential for the subsequent organization of L'sc equivalence groups within the mesodermal territory prepatterned by Wg and Dpp (Carmena, 1998).

Transduction of RTK signals occurs, at least in part, via the Ras/MAPK cascade. By use of an antibody that is specific for the diphosphorylated or activated form of mitogen-associated protein kinase (MAPK) (diphospho-MAPK), it is possible to identify localized sites of RTK signaling in the Drosophila embryo. This reagent was used to monitor the spatial and temporal involvement of Egfr and Htl in the formation of mesodermal L'sc clusters and the specification of the corresponding progenitors. The earliest activation of MAPK in the fully migrated mesoderm occurs in C2, coincident with the restriction of L'sc and prior to the appearance of Eve in this cluster. These findings are consistent with the known requirement of Htl for C2 development. Diphospho-MAPK persists in C2 after the onset of Eve expression , but fades from most C2 cells during progenitor selection. Activated MAPK remains transiently in P2 (the paracardial precursor singled out in the C2 cluster) and then disappears. Simultaneously, C15 begins to express both diphospho-MAPK and Eve. The activation of MAPK in C15 is expected from the involvement of Htl and Egfr in C15 formation. P2 then divides to yield sibling founder cells, neither of which initially contains activated MAPK. However, by the time P15 is singled out, MAPK is reactivated in one of the sibling F2s. As is the case with P2, diphospho-MAPK remains at high levels in P15. Moreover, the persistence of MAPK activation correlates with the maintenance of Eve expression in both progenitors. Additional diphospho-MAPK expression is observed in cells derived from C14, C16, and C17, in agreement with the involvement of Htl in the formation of these clusters. Thus, both the temporal and spatial patterns of diphospho-MAPK expression are consistent with a requirement for RTK signaling in the formation of mesodermal L'sc clusters, the induction of mesodermal Eve expression, and the singling out of muscle and cardiac progenitors (Carmena, 1998).

Constitutive activity of DER, Htl, or Ras1 is associated with the development of supernumerary Eve-expressing mesodermal cells. However, it has not been established whether this response is due to activated Ras1-induced proliferation of normal Eve cells or is due to the recruitment of additional cells to an Eve-positive fate. Activated Ras1 does not increase the sizes of L'sc clusters, suggesting that Ras1 is not simply stimulating the division of mesodermal cells. However, to definitively address this question, the effects of constitutive Ras1 activity were examined in string (stg) mutant embryos. Because strong alleles of stg prevent all post-blastoderm cell divisions, a potential cell-proliferation effect of activated Ras1 should be blocked in this genetic background. However, stg should not inhibit the overproduction of Eve-expressing cells if Ras1 promotes their determination. In a stg mutant, only one to two Eve-positive cells are present in each hemisegment. These cells correspond to the Eve progenitors that form in wild-type embryos. The presence of only one Eve progenitor in some segments of stg mutant embryos presumably is due to the smaller number of mesodermal cells that contribute to L'sc clusters when zygotic cell divisions do not occur. Significantly, activated Ras1 generates more Eve progenitors in a stg mutant than are seen in the mutant alone. It is concluded that Ras1 promotes the formation of additional Eve progenitors by inducing more mesodermal cells to assume this fate and not by stimulating the normal progenitors to divide. Next, an assessment was carried out to determine if the Ras1 pathway is sufficient to induce progenitor differentiation by examining the myogenic effects of Ras1 at later developmental stages in both wild-type and myoblast city (mbc) mutant embryos. In the absence of mbc function, muscle fusion does not occur and differentiated muscle founders appear as spindle-shaped myoblasts that express myosin and founder cell markers, such as Eve. In contrast to the mbc mutant in which one Eve-expressing muscle DA1 founder is present in each hemisegment, multiple occurrences of such cells form in mbc embryos under the influence of activated Ras1. All of the Eve plus myosin-positive myoblasts seen in these embryos have the elongated morphology of muscle founders, as opposed to the round shape of neighboring Eve-negative myoblasts. Large syncytia containing many Eve-positive nuclei are present when Ras1 is activated in a wild-type background. Although extra Eve pericardial cells initially appear under the influence of activated Ras1, Eve expression in such cells is lost by later stages. It is concluded that ectopic activation of the Ras1 pathway not only stimulates Eve expression in additional progenitors, but also promotes the formation and differentiation of supernumerary muscle founders (Carmena, 1998).

A salient feature of these findings is that equivalence groups form in response to the localized functions of two RTKs acting within the larger prepatterned region. The mesodermal diphospho-MAPK expression pattern not only provides strong support for this hypothesis, but also raises the question of how the upstream receptors are activated in discrete subsets of competent cells. For C15, this may occur through induction by P2, which expresses Rhomboid, a factor that can nonautonomously stimulate Egfr in adjacent cells. The localized activation of Htl could result from restricted expression of its as yet unidentified ligand. Htl itself is enriched in groups of Eve-positive mesodermal cells, a process that might also contribute to the Htl-dependent formation of these clusters. RTK signaling in the Drosophila embryonic mesoderm is important for cell migration and cell fate specification. The latter role can be divided into several distinct functions: (1) Htl and DER promote the formation of L'sc clusters or equivalence groups; (2) Ras1 signaling activates identity gene expression in the entire group of equivalent cells; (3) activated MAPK becomes restricted to progenitors, suggesting a role for RTK signaling in progenitor selection, and (4) MAPK is reactivated in one of the sibling founders derived from a single progenitor, suggesting that RTK activity helps to establish or maintain the founder identity that is initiated by the asymmetric division of its progenitor. Alternatively, RTK function in some founder cells could promote their differentiation. The ability of activated Ras1 to generate supernumerary Eve founders in mbc mutant embryos is consistent with this last possibility. Interestingly, the RTK/Ras1 pathway is similarly utilized in a sequential manner during development of the Drosophila compound eye (Carmena, 1998 and references).

Muscle founder cells are uniquely specified cells that fuse with neighboring myoblasts to generate the complex pattern of body wall muscles in the Drosophila embryo. The positional specification of founder cells for ventral oblique muscles, marked by the restricted expression of Tinman RNA and the activity of a D-mef2 enhancer, has been investigated. The formation of these ventral myoblasts requires the function of the Heartless FGF receptor in the mesoderm and the presence of ventral neuroblasts in the central nervous system. Overproduction of ventral neuroblasts due to the forced expression of the homeodomain protein Vnd leads to increased numbers of founder cells. These results suggest the use of a neuroectoderm-to-mesoderm signaling pathway in the specification of ventral muscle precursors (Schulz, 1999).

A new gene, >heartbroken, has been identified that participates in the signaling pathways of both FGF receptors. heartbroken has been cloned and although it appears to be a novel protein, it possesses several sequences characteristic of a signal transduction protein (Vincent, 1998). Mutations in heartbroken are associated with defects in the migration and later specification of mesodermal and tracheal cells. Genetic interaction and epistasis experiments indicate that heartbroken acts downstream of the two FGF receptors, but either upstream of, or parallel to, Ras1. Furthermore, heartbroken is involved in both the Heartless- and Breathless-dependent activation of Mapk. It has been concluded that heartbroken may contribute to the specificity of developmental responses elicited by FGF receptor signaling (Michelson, 1998b, and Vincent, 1998).

Ras1 is a key signal transducer acting downstream of all (receptor tyryosine kinases) Rtks, including Htl. Since htl and hbr mutants have similar mesodermal phenotypes and genetic interaction studies suggest a functional relationship between the products of these genes, it became interesting to see whether hbr could also be related to Ras1 function. It is known that targeted mesodermal expression of a constitutively activated form of Ras1 can partially rescue the htl mutant phenotype. This conclusion was reached by examining both the activated Ras1-induced migration of Twi-expressing cells and the recovery of dorsally restricted Eve-positive muscle and cardiac progenitors in htl embryos. Using these same assays, it has been found that activated Ras1 is capable of partially rescuing the strong hbr^YY202 mutant. The above results suggest that hbr acts either upstream of Ras1 or on a parallel pathway involved in either initiating or transducing the Htl signal. It was next asked where hbr functions in relation to the receptor by determining if a constitutively activated form of Htl can rescue the hbr phenotype. When expressed in the mesoderm of wild-type embryos, activated Htl induces the formation of additional Eve founder cells but has no effect on mesoderm migration. In a htl mutant background, activated Htl partially corrects the mesoderm migration defect and contributes to the specification of significant numbers of Eve progenitors. Quantitation of the latter effect reveals that activated Htl is significantly more efficient at rescuing loss of htl function than is activated Ras1. In contrast, the influence of activated Htl is completely blocked by a homozygous hbr mutation. These results, as well as the dominant suppression of activated Htl by hbr, argue that hbr acts either downstream of, or parallel to, this mesodermal Fgf receptor (Michelson, 1998b).

The similarity of the mutant phenotype of heartbroken and the FGFRs htl and btl suggests that hbr 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 HBR mRNA and protein is identical to the combined expression patterns of the two FGFRs. Thus, hbr 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, hbr appears to have neither of these roles, since htl and btl expression are not affected in hbr mutant embryos, and conversely, hbr 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 hbr 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 hbr 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).

During Drosophila embryogenesis, the development of the midgut endoderm depends on interactions with the overlying visceral mesoderm. The development of the hindgut also depends on cellular interactions, in this case between the inner ectoderm (hindgut ectoderm) and outer hindgut visceral mesoderm (HVM). In this section of the gut, the ectoderm is essential for the proper specification and differentiation of the mesoderm, whereas the mesoderm is not required for the normal development of the ectoderm. Wingless and the fibroblast growth factor receptor Heartless act over sequential but interdependent phases of hindgut visceral mesoderm development. Wingless is required to establish the primordium and to enhance Heartless expression. Later, Heartless is required to promote the proper differentiation of the hindgut visceral mesoderm itself (Martin, 2001).

The caudal mesoderm gives rise to two populations of visceral mesoderm, the HVM and the LVM (longitudial visceral mesoderm that surrounds the endodermal midgut). The HVM primordium is distinguishable at stage 10 both by the expression of bagpipe (bap) and by virtue of its relatively higher levels of Twist. It includes all mesodermal cells caudal to the 15th Wg stripe, which, unlike mesoderm cells in the trunk, are organised in multiple layers soon after gastrulation. In contrast, the LVM primordium arises from cells adjacent and egg-posterior to the HVM (Martin, 2001).

The hindgut ectoderm is derived from a ring of cells that lies just anterior to the posterior midgut primordium at the cellular blastoderm stage. At stage 7, these cells invaginate into the embryo and, by stage 10, form a hollow tube that extends by cell division and rearrangement. HVM cells become closely associated with the invaginated hindgut ectoderm at stage 11. Later in this stage, all HVM cells begin to express Connectin and this together with Twist expression, can be used to follow the cells as they move over the hindgut ectoderm tube (Martin, 2001).

As the germband retracts, the hindgut tube undergoes considerable morphological rearrangements. By stage 13, it lies longitudinally from the anus and bends at right angles to join the posterior midgut. Over time, the bend flattens laterally and the hindgut lengthens, revealing morphological subdivisions. HVM cells continue to cover the ectodermal tube during these stages and as they mature, they begin to express Myosin (Martin, 2001).

To determine whether hindgut ectoderm requires interactions with mesoderm to form normally, its development was followed in twist mutant embryos that lack mesoderm. In these embryos, cells of the hindgut ectoderm can form a tube that bends anteriorly, lengthens partially at stage 13 and expresses Wg and Dichaete in restricted domains, similar to wild-type embryos. Thus, characteristic features of ectodermal gut development occur in the complete absence of surrounding visceral mesoderm (Martin, 2001).

To test whether hindgut ectoderm is needed for the proper development of the HVM, hindgut ectoderm cells were selectively killed early in their embryogenesis using the GAL4/UAS system. 455.2GAL4, which is expressed in the primordium of the hindgut ectoderm from stage 9 onward but not in the caudal mesoderm, was used to drive expression of reaper, a gene whose protein product promotes death of those cells in which it is expressed (Martin, 2001).

As in wild-type embryos, Twist is expressed strongly in the prospective HVM at early stage 11 in embryos carrying 455.2GAL4;UASreaper constructs. However, even though the morphology of the hindgut ectoderm appears normal at this stage, the bulk of the HVM is not closely associated with the hindgut ectoderm. By late stage 11, the number of HVM cells expressing Connectin or Twist is clearly reduced. This is concomitant with the severe disruption in the morphology of the hindgut ectoderm. During stage 13, many HVM cells die. The surviving cells are those that attach to any remaining hindgut ectoderm; these cells persist, differentiating to form a variable amount of visceral muscle. Thus it is concluded that the hindgut ectoderm acts as a template to promote the development and differentiation of the HVM (Martin, 2001).

The establishment of the HVM primordium at stage 10 relies on Wg. Since Wg is not required after mid stage 11, the requirement for the hindgut ectoderm could reflect the need for a second signaling pathway in the interactive process that underlies HVM differentiation. Indeed htl, which encodes the FGF receptor tyrosine kinase DFR1, is essential for HVM development. Although the development of the hindgut ectoderm appears relatively normal in htl embryos, hindgut visceral muscle fails to differentiate altogether. The early stages of HVM development appear relatively normal, thus at stage 10, bap expression is established normally in the caudal visceral mesoderm. Defects in HVM development first appear at stage 11, once the HVM has begun to move over the hindgut ectoderm. Initial Connectin expression is reduced both in intensity and in cell number, while Twist expression is rapidly lost from HVM cells late in stage 11. Expression of both markers is lost completely between stages 12 and 13. Thus, htl is necessary to promote the development of the HVM as it migrates over the hindgut ectoderm and to allow its further differentiation (Martin, 2001).

htl is initially expressed throughout the mesoderm, though its expression is particularly strong in the primordium of the HVM where it is maintained throughout embryogenesis. This is consistent with the possibility that the visceral mesoderm responds to an FGF signal from the hindgut ectoderm. If so, activation of signaling cascades downstream of htl in HVM cells would be expected. The double phosphorylated form of ERK (diphospho-ERK) is expressed weakly in HVM cells between stage 10-12 and this expression is reduced in htl embryos. The fact that htl functions through the activation of the MAPK pathway in the development of the HVM is suggested by two additional observations. (1) The loss of Connectin and Myosin expression in the HVM of htl embryos can be partially rescued when an activated form of Ras (a component of the MAPK signaling pathway) is expressed throughout the mesoderm. (2) Targetted mesodermal expression of a dominant negative form of htl (DNhtl) or of a dominant negative form of Raf (also a component of the MAPK signaling cascade), both lead to a reduction in the number of Connectin-expressing HVM cells (Martin, 2001).

This analysis shows that the establishment of the HVM primordium at stage 10 relies on Wg but not on htl. Consequently, there is an early htl-independent role for Wg. However, in this first phase of HVM development, Wg is also required for the normal development of htl expression in the HVM and in wg embryos, expression of htl in the cells that normally give rise to the HVM does not become enhanced at stages 9-10. At stages 11 and 12, Wg and htl partly function independent of one another in controlling Connectin expression in the HVM. Thus, the loss of Connectin expression in the HVM is more severe in embryos mutant for both wg and htl than in either wg or htl mutant embryos. All wg;htl double mutant embryos lose expression of Connectin in the HVM by stage 12, whereas at the same stage, some wg embryos maintain Connectin expression in a few cells and there is weak expression of Connectin in htl embryos. If Wg can act in parallel with htl to promote Connectin expression in the HVM, this explains why misexpression of Wg throughout the hindgut ectoderm of a htl mutant embryo directs strong expression of Connectin in HVM cells at stage 12. Differentiation of the HVM requires htl. Wg is not required past stage 11 for the continued development of the HVM, and the HVM fails to differentiate and express Myosin when Wg is misexpressed in the hindgut ectoderm of a htl mutant embryo (Martin, 2001).

The Drosophila sugarless and sulfateless genes encode enzymes required for the biosynthesis of heparan sulfate glycosaminoglycans. Biochemical studies have shown that heparan sulfate glycosaminoglycans are involved in signaling by fibroblast growth factor receptors, but evidence for such a requirement in an intact organism has not been available. sugarless and sulfateless mutant embryos are shown to have phenotypes similar to those lacking the functions of two Drosophila fibroblast growth factor receptors, Heartless and Breathless. sfl and sgl mutants are shown to phenocopy the mesoderm migration defect associated with loss of heartless function and sfl and sgl are required for Btl-dependent tracheal cell migration. Moreover, both Heartless- and Breathless-dependent MAPK activation are significantly reduced in embryos which fail to synthesize heparan sulfate glycosaminoglycans. Consistent with an involvement of Sulfateless and Sugarless in fibroblast growth factor receptor signaling, a constitutively activated form of Heartless partially rescues sugarless and sulfateless mutants, and dosage-sensitive interactions occur between heartless and the heparan sulfate glycosaminoglycan biosynthetic enzyme genes. Overexpression of Branchless, the Breathless ligand, can partially overcome the requirement of Sugarless and Sulfateless for Breathless activity. These results provide the first genetic evidence that heparan sulfate glycosaminoglycans are essential for fibroblast growth factor receptor signaling in a well defined developmental context, and support a model in which heparan sulfate glycosaminoglycans facilitate fibroblast growth factor ligand and/or ligand-receptor oligomerization (Lin, 1999).

There are two well characterized HSPGs in Drosophila: Dally, a Glypican-like cell surface molecule that has been implicated in both Decapentaplegic and Wg signaling, and a transmembrane proteoglycan related to the vertebrate Syndecan family. It has been suggested that syndecans particpate in signaling by vertebrate FGFRs, although other HSPGs may also be involved in this process. It is also possible that different HSPGs could be specific for particular FGF ligand-receptor combinations in individual tissues or at distinct developmental stages. Genetic analysis in Drosophila should provide a useful approach for addressing these important questions (Lin, 1999 and references).

Cell adhesion molecules (CAMs) implement the process of axon guidance by promoting specific selection and attachment to substrates. In Drosophila, loss-of-function conditions of either the Neuroglian CAM, the FGF receptor coded by the gene heartless, or the EGF receptor coded by Egfr display a similar phenotype of abnormal substrate selection and axon guidance by peripheral sensory neurons. Moreover, neuroglian loss-of-function phenotype can be suppressed by the expression of gain-of-function conditions of heartless or Egfr. The results are consistent with a scenario where the activity of these receptor tyrosine kinases is controlled by Neuroglian at choice points where sensory axons select between alternative substrates for extension (Garcia-Alonso, 2000).

The ocellar sensory system (OSS) offers a simple scenario for the study of axon guidance at the cellular level. During axon guidance, growth cones make decisions at choice points. In order to change trajectories at these choice points, it is assumed that signal transduction mechanisms should operate to transform specific extracellular information in the modulation of their actin cytoskeleton. In the OSS, the initial decision to attach or not to attach to the head epithelium appears to be a key choice (at the first choice point) for two types of sensory axons as they navigate to their respective targets in the brain. Due to the process of head eversion, ocellar pioneer (OP) axons must navigate in the extracellular matrix (ECM), free of adhesion to the underlying epidermis. Reciprocally, bristle mechanosensory (BM) axons should follow the epidermis before and after head eversion, since they do not reach the brain until this later stage. Should BM axons initially extend apart from the epithelium, they might possibly be unable to follow a physical substrate toward their brain targets after head eversion. Therefore, the process of head eversion establishes a constraint that prevents substrate redundancy between ECM and epithelium. OSS axons must make a second decision in order to leave the surface of the head and project to the brain (at the second choice point). OP axons leave the ECM surrounding the head capsule toward the brain before head eversion. In contrast, BM axons leave the head's internal epithelial surface after head eversion, when several BM axons have converged together. Each of these decision processes are abnormal in nrg, htl, and Egfr mutant individuals. OP axons can decide to attach to the epithelium, preventing them from reaching the brain. OP axons can fail to leave the internal surface of the head (even when extending free of epithelial attachment) and project to ectopic positions within the head after eversion. BM axons can be found extending, although they are abnormally separated from the epidermis after head eversion, suggesting a failure of attachment to the epithelium before head eversion. BM axons also can stall in the epidermis, suggesting that Nrg may also promote axon extension. Finally, BM axons can fail to leave the epidermis toward the brain, as they should, remaining instead within the epidermal layer, where they project in abnormal directions. In such cases, they can sometimes be observed to perforate the epidermis and project outside of the head, suggesting that BM axons navigate in the epithelial surface, using proteases to facilitate their movements. In contrast, when attached to the epithelium, OP axons seem to have difficulty extending properly. One possibility is that extension in the epithelium requires this perforating activity that BM axons have and that may be missing in OP axons that normally extend in the ECM (Garcia-Alonso, 2000).

Since nrg, htl, and Egfr mutants exhibit similar OSS axon phenotypes, it seems possible that they function in a common mechanism during OSS axon guidance. In order to test genetically if Nrg behaves as an upstream regulator of the Fgfr and the Egfr, different double mutant combinations were constructed between nrg and gain-of-function conditions for htl and Egfr (which should function independently of upstream regulation). Gain-of-function conditions of htl can partially suppress the nrg phenotype. Elp alleles behave as gain-of-function conditions of Egfr and behave as strong suppressors of nrg OSS axon phenotype. Therefore, although it is still possible that Nrg could also perform some role based on pure adhesion, the results are most consistent with the idea that both Htl and Egfr mediate Nrg function in OSS neurons (Garcia-Alonso, 2000).

Htl and Egfr exhibit some specificity in their effects on OSS axon guidance. Htl seems to be preferentially required by OP axons and BM axons to project to the brain, while Egfr seems to be more involved in BM axon attachment and extension in the epithelium. In contrast to in vitro studies in vertebrates, no evidence has been found that Htl promotes axon growth. Rather, in the Drosophila OSS, it seems that the Egfr is preferentially required for outgrowth. This discrepancy with the vertebrate in vitro studies could be explained if the Drosophila in vivo situation were more prone to the deployment of compensatory molecular interactions (which might mask a role of Htl in axon growth) than the in vitro situation. In such a case, a deficit of one RTK could be partially compensated by an increase in the activity of the other RTK. This could happen if, for example, different RTKs were regulated through a common negative feedback loop. This explanation would also help explain the presence of a mild phenotype in consititutively active lambda-Htl individuals and would account for those cases in which some nrg OSS alteration is enhanced by an increase or suppressed by a reduction in RTK activity. This explanation is also consistent with the lack of effect of the gain of function of one RTK over the loss of function of the other (since this condition would itself increment the activity of the former RTK) (Garcia-Alonso, 2000).

It is likely that the RTKs also mediate the function of signals other than Nrg during OSS axon guidance. This is suggested by the fact that the nrg phenotype is weaker than the RTK phenotype and by the fact that it can be enhanced by a reduction of 50% in the amount of Egfr. These signals could represent other CAMs or growth factor ligands diffusing from the brain. One suggestive possibility is that OP and BM axons depart from the head capsule in response to some as yet unidentified diffusible signal from their brain targets (acting in addition to Nrg signaling). Further studies will be necessary to evaluate this possibility (Garcia-Alonso, 2000).

What would be the signal that triggers Nrg-dependent RTK activation? Nrg, like its vertebrate homolog L1, can behave as a homophilic CAM. It has been proposed that the homophilic interaction of L1 would activate the function of the Fgfr. Therefore, it is proposed that the homophilic interaction between Nrg¹⁸⁰ (the neural-specific form) molecules would give a positive input on RTK activity in both OP axons (from the beginning of axon extension) and BM axons (after several mechanosensory axons have converged), which would signal the axons to lift off from the epidermis and project to brain targets. However, BM axons initially extend as single processes and interact with the Nrg¹⁶⁷ form in the epithelium, where this interaction would result in the activation of the RTKs, and RTK signaling would promote extension in the epidermis. This shift in the RTK's activity outcome might be caused by the involvement of some other molecule specifically interacting with Nrg¹⁶⁷. In agreement with this idea, rescue experiments of nrg using Nrg¹⁸⁰ reveal that BM axon extension on the epidermis cannot be implemented by this molecular form. Since homophilic binding between the different Nrg forms is possible, this high degree of specificity suggests the existence of additional molecules (specifically interacting with Nrg¹⁶⁷) that mediate BM axon association with the epithelium. Some observations are consistent with this model. (1) OP axons fasciculate with one another (50 or so per ocellus) from the very beginning of axon extension. These axons fasciculate together due to the presence of Neurotactin and other CAMs in the membrane. Defasciculation of OP axons (caused by a lack of Neurotactin) increases the chances that OP axons extend abnormally in the epidermis. These results suggest that fasciculation helps generate a robust process of axon guidance. In this model, defasciculation would reduce the probability of Nrg¹⁸⁰–Nrg¹⁸⁰ interactions between OP axons, producing a deficit of RTK activity, therefore, making them more likely to extend attached to the epidermis. (2) BM axons initially follow the epidermis in isolation from other axons but begin to converge as they approach the dorsal antennal field. After several BM axons have converged together, the BM fascicle lifts off from the epidermis. Thus, the signal for BM nerves to leave the epidermis might be a given threshold of Nrg¹⁸⁰–Nrg¹⁸⁰ interactions between the different BM axons. If this model is correct, the specificity of the OP and BM growth cone interaction with the epidermis would reside in the way Nrg¹⁶⁷ and Nrg¹⁸⁰ differ in their interactions with other molecule(s). Nrg¹⁶⁷ differs from Nrg¹⁸⁰ in the cytoplasmic domain. It has been previously shown that the cytoplasmic domain can regulate the adhesive properties of the extracellular part of the protein. Therefore, it is possible that some CAM molecule might specifically interact with Nrg¹⁶⁷ to help promote initial RTK activation and attachment to the epithelium in BM axons. This molecule could represent the Drosophila homolog of some of the known vertebrate heterophilic partners of L1 (Garcia-Alonso, 2000).

In summary, these results strongly support the idea that Neuroglian functions in axon guidance by regulating the activity of both the Fgfr and the Egfr and suggest a scenario where other CAMs and growth factor activities would act in concert with Neuroglian for RTK regulation. In addition, the possible existence of cross-regulatory circuits between RTKs would add another level of control that might help to understand the high degree of canalization displayed by the axon guidance process from Drosophila to mammals (Garcia-Alonso, 2000).

A mutation in heartless reveals that posterior migration of the salivary gland requires an intact visceral mesoderm and integrin function

The final overall shape of the salivary gland and its position within the developing embryo arise as a consequence of both its intrinsic properties and its interactions with surrounding tissues. This study focuses on the role of directed cell migration in shaping and positioning the Drosophila salivary gland. The salivary gland turns and migrates along the visceral mesoderm to become properly oriented with respect to the overall embryo. Salivary gland posterior migration requires the activities of genes that position the visceral mesoderm precursors, such as heartless, thickveins, and tinman, but does not require a differentiated visceral mesoderm. A role for integrin function in salivary gland migration is demonstrated. Although the mutations affecting salivary gland motility and directional migration cause defects in the final positioning of the salivary gland, most do not affect the length or diameter of the salivary gland tube. These findings suggest that salivary tube dimensions may be an intrinsic property of salivary gland cells (Bradley, 2003).

Embryos homozygous for EMS2, a mutation isolated in a screen to identify genes required for normal salivary gland formation, have a wide range of defects in salivary gland shape and position that appear to result from abnormal migration. The EMS2 defects included secretory tubes oriented on the DV axis, relatively straight tubes with only the distal tips misoriented, and tubes with multiple kinks along their lengths. EMS2 defects were first apparent during embryonic stage 12, when wild-type distal salivary cells normally reach the turning point and initiate posterior migration. In some EMS2 stage 12 mutants, salivary cells were observed in positions dorsal to the wild-type turning point, suggesting that the cells failed to turn posteriorly and, instead, had continued to migrate dorsally. Earlier events, including secretory cell internalization and early dorsal movement, appear normal in EMS2 mutants. Unlike in rib mutants, where the salivary glands fail to migrate past the turning point, EMS2 salivary glands migrate in abnormal directions. Thus, the ability of EMS2 mutant salivary cells to migrate per se is unaffected, and instead, either a cue that guides posterior salivary cell migration or the ability of the salivary cells to respond to such a cue is disrupted in EMS2 embryos (Bradley, 2003).

In a fortuitous cross to balance EMS2 over a different balancer chromosome, it was found that EMS2 fails to complement a null heartless allele (htl^AB42). Subsequent complementation tests with an independent htl allele and a deficiency removing htl revealed complete noncomplementation, indicating that EMS2 has a lesion in the htl gene. Embryos homozygous for htl^AB42 or transheterozygous for htl^AB42 and EMS2 have salivary gland defects similar to those of EMS2 homozygotes, suggesting that the mutation in htl, and not a second site mutation, causes the salivary gland migration defects. Sequence analysis of the htl ORF from EMS2 genomic DNA reveals a nucleotide change (C2638T) in codon 491, resulting in a nonsense mutation; the HTL protein encoded by EMS2 lacks the carboxy-terminal 225 residues. HTL is a fibroblast growth factor receptor (FGFR1) with an extracellular ligand-binding domain and a cytoplasmic tyrosine kinase domain. The HTL protein encoded by the EMS2 mutant allele disrupts the essential kinase domain. These data demonstrate that EMS2 is a new allele of htl (htl^EMS2) and that htl is required for normal salivary gland formation (Bradley, 2003).

FGF receptor activation initiates a signal transduction cascade that elicits the cellular responses required for many processes, including cell differentiation, cell proliferation, and cell migration. The ligand for HTL has not been identified, but a downstream effector molecule known as Heartbroken (Hbr; also called Downstream of FGFR or Stumps) is required for HTL signal transduction. hbr mutant embryos have the same range of salivary defects as observed in htl mutants, indicating that the FGFR1 signaling pathway is required for normal salivary gland shape and position (Bradley, 2003).

To determine how FGFR1 signaling is required for salivary gland migration, the embryonic expression patterns of htl and hbr were examined No salivary gland expression of htl RNA, HTL protein, or hbr RNA was detected at any stage. Moreover, a dominant-negative htl transgene (UAS-htl^DN, expressed specifically in salivary cells using the fkh-Gal4 driver, has no effect on salivary gland shape or position. These results suggest that HTL signaling is not required in the salivary cells for their migration, and thus must act indirectly (Bradley, 2003).

To learn how FGFR1 signaling functions in salivary gland migration, known roles for htl were examined. htl is expressed to high levels in mesodermal primordia and is required for the migration of mesodermal cells to specific sites in the embryo where they will differentiate into distinct mesodermal structures. The mesodermal structures most severely affected by htl mutations are those whose formation requires the farthest migration of precursor cells, specifically the dorsal vessel (or heart) and the visceral mesoderm (VM). Of these structures, the VM is most relevant to salivary gland migration since it is closest to the turning point. To correlate the VM with all stages of salivary gland migration, VM structure and salivary gland positioning were simultaneously examined by immunostaining for the VM protein, Fasciclin3 (FAS3), and various salivary gland proteins. In wild-type embryos, internalized salivary cells moved dorsally until they reached the VM. The observation that distal salivary cells that have already migrated posteriorly are in close proximity to the VM suggested that the VM is the 'turning point' at which cells begin to migrate posteriorly. Examination of histological sections revealed that dorsal salivary cells directly contact the overlying VM. In subsequent stages of salivary gland migration, as the tube becomes oriented along the A/P axis, the dorsal side of the salivary tube is observed in close proximity to the VM, suggesting that dorsal salivary cells may interact with the VM throughout posterior migration (Bradley, 2003).

htl mutants have defective VM; unlike the intact, cohesive structure observed in wild-type embryos, the VM in htl mutants is disrupted, with variably sized fragments of VM at the normal position of the VM. Salivary gland migration was examined in relation to the VM fragments in htl mutants and a correlation was found at early stage 12. In embryos where no mesoderm is present at the turning point, the distal tip of the salivary gland is observed dorsal to the level of the remaining VM fragments, indicating that salivary cells continued to migrate dorsally instead of reorienting posteriorly. In embryos where a small VM fragment is present at the turning point, posterior migration of the salivary gland varies. In embryos where VM is present at the turning point, salivary gland cells initiate posterior migration as in wild-type. Thus, the VM is required at the turning point for the posterior redirection of salivary distal cells. Due to the dynamic nature of the VM, whose position changes relative to the salivary gland in subsequent stages, it was difficult to definitively correlate disruptions in VM structure with abnormal salivary gland shape at later stages. Occasionally salivary glands were seen that initially had turned posteriorly, but were kinked in a position that correlated with a break in VM, indicating that intact VM is required for the duration of the migratory process. Thus, the variability of the VM structure explains the range of salivary gland phenotypes in the htl mutants (Bradley, 2003).

These studies suggest that the VM is required for salivary gland migration and predicts that other mutations affecting VM formation would cause similar salivary gland defects. During development, mesodermal precursor cells that migrate dorsally are exposed to the dorsally localized Decapentaplegic (DPP) signaling molecule, which is required to specify dorsal mesoderm derivatives, including cardiac and VM precursors. In embryos lacking thickveins (tkv), which encodes a DPP receptor, breaks of variable width and position in the VM are observed, similar to the breaks observed in the htl VM. Correspondingly, salivary gland migration defects were observed in tkv mutants similar to those seen in htl mutants. Such migration defects were not observed, however, in embryos expressing a negative regulator of the DPP pathway, Daughters against dpp. Together, these results suggest that DPP signaling, like FGFR1, is not required in the salivary gland cells for their normal migration, but rather that DPP signaling is required for VM formation, which in turn is required for the directed migration of the salivary gland (Bradley, 2003).

In htl, tkv, and tinman, the residual fragments of VM express Fas3, have a VM-like structure, and are able to direct salivary gland migration if present along its migratory path. Thus, the residual structures appeared to be differentiated VM with wild-type properties. To determine whether salivary gland migration requires a differentiated VM, embryos with mutations in the VM-specific gene biniou (bin) were examined. In bin mutant embryos, VM precursors segregate from dorsal mesoderm and move internally where they coalesce into the typical VM band; however, all tested VM-specific genes, including Fas3, fail to be expressed in bin mutants. Thus, an intact structure formed from VM precursors is present in bin mutants, but the VM precursor cells fail to express markers indicative of differentiation from a general mesodermal cell into a VM-specific cell. The salivary glands in bin mutants had no defects in turning or posterior migration, suggesting that guidance of salivary gland posterior migration by the VM requires neither the terminal differentiation of the precursors nor the function of any VM gene whose expression is bin-dependent (Bradley, 2003).

The VM forms a contiguous structure that may physically block salivary cells from further dorsal movement, thereby causing the cells to move posteriorly, in the path of least resistance. Alternatively or additionally, there may be a bin-independent factor (or factors) that guides salivary gland migration in a more instructive way, perhaps via a secreted signal or a transmembrane guidance molecule. If the mesodermal cue were informational, a signaling pathway functioning within salivary gland cells would have to be involved. A screen of several candidate pathways revealed that mutations disrupting the FGFR1-, FGFR2-, EGF-, DPP-, JNK-, or Wg-signaling pathway did not have phenotypes consistent with a role in the salivary cells for their migration. Thus, focus was placed on molecules known to have a more direct role in migration, specifically the integrin family of cell adhesion molecules, which are heterodimers of two transmembrane proteins, an alpha and a ß subunit. In Drosophila, each of the five identified alpha subunits (alpha_PS1-5) is thought to dimerize with the ß_PS subunit encoded by the myospheroid (mys) gene. The alpha subunit of alpha_PS2ß_PS (PS2) integrin is expressed in all mesodermal cells beginning at a very early stage, suggesting that PS2 integrin is likely to be present in the VM precursor cells prior to bin-dependent differentiation. Indeed, alpha_PS2 RNA expression was observed in the mesoderm of bin^R22 homozygotes. In embryos mutant for inflated (if), the gene encoding the alpha_PS2 subunit, migration of two tissues along the VM is affected, the endoderm and the tracheal visceral branch. Thus, the PS2 integrin is required to make the VM a suitable substrate for the migration of at least two distinct cell populations (Bradley, 2003).

Whether PS2 integrin is required for salivary gland migration was examined by staining if mutant embryos for several salivary gland proteins. In if homozygotes, salivary cells appear to invaginate normally. The first group of salivary cells to be internalized reaches the approximate level of the wild-type turning point but fails to migrate. During subsequent stages, the remaining if salivary cells continue to internalize, but the distal tip remains at the approximate VM turning point, and the tube is often slightly bent. By late stages, if salivary tubes are frequently folded in half with the distal tips oriented anteriorly. The apparent lack of salivary gland migration in if mutants is distinct from the mismigration phenotypes in htl, hbr, tkv, and tin mutants (Bradley, 2003).

The migration defect is identical whether integrin function is missing in the salivary gland or in the mesoderm. This equivalent integrin function is not seen for endoderm migration; loss of integrin function within migrating endodermal cells results in a stronger defect than loss of integrin function in the mesoderm, which also provides the substratum for endodermal migration. Perhaps the substrate tissue is less crucial for migrating endodermal cells because these cells undergo an epithelial-to-mesenchymal transition and migrate as individual cells, which may have a propensity to migrate, even on suboptimal substrata. The mode of salivary gland migration may be different because cells remain part of an epithelium in which cellular movements must be coordinated for migration. Perhaps the concerted migration of an organized tissue is more dependent on its substratum (Bradley, 2003).

The PS1 integrin is likely to function at the surface of salivary cells to promote their migration, since alpha_PS1 is expressed in the salivary gland and not in the surrounding mesoderm. alpha_PS2, which is not expressed in salivary cells, is expressed in all mesoderm, including the mesodermal cell population through which the salivary tube traverses on its way to the VM. In the absence of early VM-specific (or other mesoderm-specific) Gal4 drivers, additional roles for PS2 function in this population of mesodermal cells cannot be ruled out (Bradley, 2003).

Several possible roles for the integrins in their respective tissues can be envisioned. The PS1 integrin may function in the salivary gland to attach the migrating cells to the mesoderm and/or to recognize the mesoderm as a suitable substratum. Alternatively, PS1 may promote motility of salivary gland cells by activating intracellular signaling events. The PS2 integrin is likely to organize the mesodermal ECM, creating a suitable substratum for migration. A PS2-organized matrix may assemble or concentrate ligands for PS1, potentially explaining the identical phenotypes. Given that integrins have been shown to regulate gene expression through intracellular signaling, a less direct role for PS1 and PS2 in modulating expression of a gene or genes required for migration cannot be ruled out (Bradley, 2003).

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).

Morphogenesis of the mesodermal cell layer has been considered to depend entirely on FGF signalling, but in fact, FGF signalling is essential only for the initial establishment of contact between mesoderm and ectoderm, and for the late heart-differentiation signal, and these two processes are independent and experimentally separable. Dominant-negative FGF-receptor constructs disrupt differentiation, but do not affect spreading when expressed after the initial contact has been made. Conversely, constitutively active tyrosine kinases other than FGF receptors expressed in htl mutants rescue late differentiation, but not early spreading. Similarly, in the mutants of the RhoGEF pbl, no early contact is made, and spreading is therefore inefficient, but cells that reach the dorsal region of the mesoderm are able to respond to FGF, activate MAPK, and differentiate into heart precursors (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).

Both the establishment of mesoderm-ectodermal cell contact and the activation of MAPK require the kinase domain of the FGF receptor to be intact, which suggests that these events depend upon a substrate of the FGF receptors not recognised by other activated receptor tyrosine kinases. One possibility is that this substrate is Dof, which is specifically phosphorylated by an activated FGF receptor. In this situation, Dof would provide a unique function that cannot be substituted by other activated receptor tyrosine kinases. Alternatively, this substrate could be a second receptor that is activated upon contact of the mesoderm with the ectoderm, or a component that acts in, or on, a pathway triggered by the engagement of the mesoderm with the ectoderm (Wilson, 2005).

It seemed likely that the early morphogenetic activity might require changes in subcellular architecture involving cytoskeletal regulators. Indeed, the establishment of contact between the mesoderm and the ectoderm is affected by mutations in the gene encoding the RhoGEF Pebble, and, as shown in this study, a reduction in the level of Rho and Rac proteins within the embryo. It is not known whether the Rho-family GTPases act downstream of or in parallel with FGF signalling. The defects of htl mutants cannot be rescued by the expression of an activated form of Rac or Cdc42. Thus, if Rac acts downstream of the FGF receptor, it is not in a simple epistatic pathway but requires the activation of other pathways as well. Alternatively, FGF signalling may act in conjunction with a separate pathway that directs the activity of the Rac proteins to promote contact between the mesoderm and the ectoderm (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).

These observations raise the questions of how mesodermal cells spread over the surface of the ectoderm, and how activated MAPK accumulates in a graded fashion. A number of possibilities can be envisioned to account for the migration of the mesoderm. For example, the first cell that makes contact with the ectoderm could crawl over the ectoderm and function as the 'leading' cell of the mesodermal sheet. The other cells of the mesoderm tube would make contact with the ectoderm sequentially to follow the leading cell as it migrates dorsally. In this case, the MAPK gradient would be explained by the accumulation of the highest levels of activated MAPK in the cells that had been in contact with the ectoderm for the longest period of time. Alternatively, the cell that makes the initial contact with the ectoderm could remain largely stationary, and other mesodermal cells would reach the ectoderm by crawling over that cell. Once a mesodermal cell is in contact with the ectoderm, motility of the cell would cease, as in the process of 'boundary capture' described for mesodermal cells in Xenopus reaching the notochord during convergence movements. In this model, contact between the ectoderm and mesoderm would have an important role in establishing the single cell layer of mesoderm that covers the surface of the ectoderm at later stages. The MAPK gradient can be explained in this case by a transient activation of MAPK, which is downregulated once motility ceases, a model that is more consistent with known feedback mechanisms that operate during signal transduction. This model implies that the cells with the highest level of MAPK at the edge of the mesoderm would have only just come into contact with the ectoderm. In order to distinguish between the two mechanisms, cell labelling experiments will be required (Wilson, 2005).

FGF signalling is only one of many mechanisms that contribute to the establishment of the mesodermal cell layer. It is not essential for migration as such, but is clearly important for the orderly dispersal of mesodermal cells away from their site of invagination. These results suggest that FGF-signalling facilitates cell spreading by promoting the apposition of the invaginated mesodermal epithelium against the ectoderm (Wilson, 2005).

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