breathless
Spatially and temporally regulated activity of Branchless/Breathless signaling is essential for trachea development in Drosophila. Early ubiquitous
breathless (btl) expression is controlled by binding of Trachealess/Tango heterodimers to the btl minimum enhancer. Branchless/Breathless signaling includes a Sprouty-dependent negative feedback loop. Late btl expression is a target of Branchless/Breathless signaling and hence,
Branchless/Breathless signaling contains a positive feedback loop, which may guarantee a continuous supply of fresh receptors to membranes of growing tracheal branch cells. Branchless/Breathless signaling activates MAP-kinase, which in turn, activates late btl expression and destabilizes Anterior-open (A.K.A. Yan), a repressor for late btl expression. Biochemical and genetic analysis has indicated that the minimum btl enhancer includes binding sites of Anterior-open (Ohshiro, 2002).
The minimum btl enhancer consists of B2 and B3 regions, the latter, a late enhancer. lacZ expression driven by B3 enhancer mimics btl late expression. The B3 enhancer may thus contain binding sites for Aop. Ets-domain containing proteins binds to DNA via conserved Ets domains and the canonical sequence of their targets is 5'GGA. The B3 enhancer contains an inverted repeat of GGA quite near 3' of CME2, a Trh/Tgo binding element. A study was made to determine Aop-capability for binding to the GGA pair. The Ets domain of Aop (ETSAOP) was expressed in Escherichia coli cells and partially purified from cell extracts followed by electrophoresis mobility shift assay (EMSA). Fragment C contains only one GGA, while W is a portion of B3, containing the GGA pair. M1¯M4 are mutants for GGA sites. ETSAOP forms complexes with C and W. The W/ETSAOP complex shows mobility apparently less than that of the C/ETSAOP complex. Each W/ETSAOP complex may contain two molecules of ETSAOP, since W contains two GGAs. Consistent with this, W mutants with a single copy of GGA (M1 or M3) forms complexes with ETSAOP whose mobility was similar to that of the C/ETSAOP complex. No stable ETSAOP/DNA complex is formed subsequent to disruption of two Ets sites, strongly suggesting that targets for Aop in B3 are the GGA pair, ETS1 and ETS2. ETS1 and ETS2 are concluded to be Aop targets required for in vivo transcription of btl (Ohshiro, 2002).
Unlike ETSAOP, the Ets domain of Pnt (ETSPNT) can bind to both W (wild-type B3 enhancer) and M1-M4 (mutant B3 enhancers), suggesting that Pnt is capable of binding to B3 through sequences other than ETS1 and ETS2 or mutated EST sequences. Consistent with this, significant btl-lacZ signals are detected in B3[M2]-lacZ embryos, expressing AopACT (Ohshiro, 2002).
Bnl/Btl signaling in developing trachea not only facilitates cell migration during primary branch formation but also induces the expression of genes required for secondary and terminal branch formation in a subset of tracheal cells. The present study indicates that the btl gene itself is a target of Bnl/Btl signaling and accordingly, Bnl/Btl signaling is regulated by a positive feedback mechanism. The results also indicated that the positive feedback of Bnl/Btl signaling includes MAPK activation. This is the first clear demonstration of the presence of a positive feedback loop in FGF signaling (Ohshiro, 2002).
The positive feedback mechanism may be an important finding, since a negative feedback mechanism has already been shown to be involved in Bnl/Btl signaling. Spry is expressed in many tissues including trachea and functions as an intracellular inhibitor of Ras pathway signal transduction through its binding to Drk and Gapl. In embryos mutant for spry, downstream target genes of Bnl/Btl signaling are misexpressed and extra secondary branch cells are generated. At the onset of secondary branch formation in wild type trachea, only one or a few cells exposed to the highest levels of Bnl overcome the inhibitory action of Spry to acquire secondary-branch-forming cell fate. Receptor molecules activated by ligands are generally considered eliminated from the cell membrane through endocytosis and the mechanism of Bnl/Btl-signaling-activity-dependent btl transcription should thus be indispensable to a constant supply of fresh Btl receptors to membranes of cells receiving Bnl. A proper balance between positive effects from Btl supply and negative effects of Spry on Bnl/Btl signaling may accordingly be essential for properly selecting secondary budding cells in the vicinity of the FGF signaling center and inducing properly terminal branch formation (Ohshiro, 2002).
aop is expressed in most developing tracheal cells and its protein product is localized mainly in the nucleus, except for tip cells that receive the highest levels of Bnl signals. Misexpression of the activated form of Aop in tracheal cells virtually completely abolishes Btl signals and the absence of aop activity leads to btl misexpression in TC, from which btl expression is normally absent. Conversely, the overexpression of bnl, which stimulates Bnl/Btl signal transduction, brings about almost the complete elimination of Aop nuclear signals from most tracheal cells. The destabilization of Aop phosphorylated by the activated form of MAPK would be the most likely reason for this. It thus quite logically follows that the area of normal late btl expression is determined partly by an activity balance between ubiquitous Aop and Bnl/Btl signaling, whose activity should diminish gradually with distance from the Bnl source. Note that in contrast to Spry, whose expression is under the control of Bnl/Btl signaling, aop is transcribed constitutively in a Bnl/Btl-signaling-independent manner. btl is expressed even in growing trachea branch cells associated with nuclear Aop signals. High concentration of Pnt may overcome Aop-dependent repression and activate btl in these cells. Pnt is thought to compete with Aop to activate target gene expression. In pnt mutant embryos, btl expression is lost, again indicating that Pnt activates btl expression. Pnt transcription has been shown to be dependent on Bnl/Btl signaling and, accordingly, Bnl/Btl signaling is expected to activate Pnt expression in Bnl receiving cells. Thus, that the threshold value of Bnl/Btl signaling for btl expression is much lower than that for elimination of nuclear Aop is most likely due to the presence of this Pnt-mediated positive feedback regulation (Ohshiro, 2002).
In wild type, little or no Btl expression occurs in DT and TC but btl misexpression is detected mainly in TC in aop mutants, so that btl expression may perhaps be repressed in DT by some factor other than Aop. It is presumed that Spalt (Sal) is responsible for btl repression in DT. sal encodes a Zn-finger transcription factor and is expressed specifically in DT. sal is essential for DT fate determination and its overexpression throughout the entire trachea almost completely abolishes btl expression. In sal mutants, btl expression is derepressed in presumptive DT cells that fail to migrate, with consequent accumulation at trachea center (Ohshiro, 2002).
In wild type embryo, DTa (anterior DT branch) and DTp (posterior DT branch) cease branch growth and start to fuse with adjacent metamers by stage14, whereas other primary branches continue to grow in a Bnl/Btl-signaling dependent manner. DTa and DTp extend no terminal branches, the formation of which requires Btl activity. The early down-regulation of btl RNA in DT may thus possibly be essential for preventing terminal branch formation in DT (Ohshiro, 2002).
EMSA along with an enhancer assay indicates that btl B3 enhancer, about 120 bp long and responsible for late btl expression contains an inverted repeat consisting of two Aop binding sites (GGAs; ETS1 and ETS2), suggesting that dimerized Aop binds to the B3 enhancer. Pnt is considered to compete with Aop for a common target sequence to activate target gene expression. pnt is essential for late btl expression. As with Aop, the Ets domain of Pnt (ETSPNT) is capable of forming a complex not only with the authentic wild-type B3 enhancer, but with mutant forms of B3 enhancer as well. Pnt may thus be capable of binding to mutated ETSs or B3 sequences other than ETS1/ETS2. The B3 enhancer also contains a sequence similar to the Salr target in chorion s15. This putative Sal/Salr binding site is situated in the B3 enhancer just in the 3' vicinity of ETS1; ETS2 and the putative Sal/Salr binding site may partly overlap. Preliminary experiments have shown that Sal is capable of binding to the B3 enhancer (Ohshiro, 2002).
The B3 enhancer possesses two of three CMEs sites for binding of Trh/Tgo complexes. The disruption of three CMEs in the btl enhancer brings about the complete loss of btl expression in tracheal cells at later stages. Thus, Trh/Tgo may also be required for late btl expression. A POU-Homeobox containing protein, Ventral veinless (Vvl)/Drifter is required for maintenance of btl expression in developing trachea. Pnt, Trh/Tgo, and Ventral veinless/Drifter thus quite likely synergistically activate btl expression in DB, VB, and LTa/p whereas Aop and/or Sal activity represses btl to prevent its expression in TC and/or DT (Ohshiro, 2002).
Spitz, and spitz group genes are at the top of the regulatory hierarchy in the development of salivary ducts. The salivary primordium consists of two regions, a more dorsal pregland anlage and a ventral preduct anlage. Spitz signaling to ventral cells, through the EGF-receptor acts to block forkhead expression in preduct cells, thereby restricting gland identity to more dorsal cells. Forkhead acts in dorsal pregland cells to block duct fate, specifically acting to repress Serrate, a duct specific gene as well as breathless and trachealess, also required for duct formation. The spitz group genes rhomboid and pointed are required for duct fate (Kuo, 1996).
The development of Drosophila trachea is under the control of spatially and/or
quantitatively regulated activity of the FGF receptor known as Breathless, which is also
essential for midline glial migration. This study has identified the minimum enhancer region of breathless. btl is expressed in trachea, midline precursors (MLPs), midgut precursors and salivary duct glands. Three central midline elements (CME) consisting of binding sites for Sim/ARNT (Ah receptor nuclear translocator) heterodimers are identified within a 150 base pair region, from -606 to -447 bases, relative to the P2 transcriptional initiation site. These three sites account for breathless expression in MLPs (Ohshiro, 1997).
breathless expression in developing trachea is regulated by
direct interactions between Trachealess/dARNT heterodimers and the same three central
midline elements (TACGTGs) situated in the minimum enhancer region. As with human and mouse ARNT, the polpeptide contains a basic region, an HLH structure and two PAS domains. 63% of the amino acids in the amino-terminal half (which includes bHLH and the two PAS domains) are invariant for human and Drosophila proteins. dARNT mRNA is distributed homogeneously in early embryos, suggesting maternal deposition. At stage 11, relatively strong signals are transiently detected in tracheal pits, and later in the CNS. These results
also show that Single-minded/dARNT heterodimers, which are essential for
breathless expression in midline precursor cells, share DNA targets in common with
Trachealess/dARNT, indicating that two different basic helix-loop-helix-PAS
protein complexes act through the same target sites in vivo. It is also thought that additional nucleotide sequences flanking CMEs may serve additional cis-regulatory elements for tracheal expression. Late breathless expression might be considered to be under the control of the ligand Branchless which activates genes expressed at late stages, including pointed and blistered/pruned/DSRF (Ohshiro, 1997).
Both breathless and ventral veins lacking (also known as drifter) are expressed in all tracheal cells and are
essential for directed cell migrations. Ubiquitously expressed
Breathless protein under control of a heterologous heat-shock promoter is able to rescue
the severely disrupted tracheal phenotype associated with drifter loss-of-function mutations.
In the absence of Drifter function, breathless expression is initiated normally but transcript
levels fall drastically to undetectable levels as tracheal differentiation proceeds. In addition,
breathless regulatory DNA contains seven high affinity Drifter binding sites similar to
previously identified Drifter recognition elements. These results suggest that the Drifter
protein, which maintains its own expression through a tracheal-specific autoregulatory
enhancer, is not necessary for initiation of breathless expression but functions as a direct
transcriptional regulator necessary for maintenance of breathless transcripts at high levels
during tracheal cell migration. This example of a mechanism for maintenance of a committed
cell fate offers a model for understanding how essential gene activities can be maintained
throughout organogenesis (Anderson, 1996).
Salivary eyegone expression is regulated positively by Sex combs reduced and trachealess (trh) but is regulated negatively by forkhead. Scr, the homeotic gene responsible for patterning parasegment 2, is responsible for the activation of every salivary gene that has been tested. As expected, eyg is not expressed in the salivary primordium of Scr-mutant embryos. The trh gene product is necessary for invagination of all salivary duct cells and it is required for expression of downstream duct markers. Because eyg is also expressed in part of the salivary duct primordium, the relationship between trh and eyg was tested in the pathway for duct determination. In wild-type embryos, both trh and eyg expression in the salivary primordium begin early during stage 10. At this stage, eyg expression in trh-mutant embryos is indistinguishable from expression in wild-type embryos. Therefore, initiation of eyg expression in the salivary primordium is independent of trh. In early stage 12, however, eyg expression becomes dependent on trh. Although eyg is expressed strongly in the posterior preduct cells of wild-type embryos, this expression is completely absent in trh-mutant embryos. Therefore it is eyg maintenance, and not its initiation, that depends on trh. Whether trh expression depends on eyg was also tested and it was found that trh expression is unaffected in eyg null-mutant embryos (Jones, 1998).
forkhead plays an important role in establishing the pregland/preduct border by dorsally limiting duct-specific gene expression. trh, like eyg, is also initially expressed throughout the gland primordium. In fkh-mutant embryos, trh transcript never disappears from the pregland cells (Isaac, 1996). Does fkh play a similar negative regulatory role in eyg transcription? When the wild-type eyg expression pattern is compared to that of fkh-mutant embryos, it becomes clear that fkh indeed negatively regulates eyg. eyg expression persists in gland precursors in fkh-mutant embryos. Thus, fkh represses expression of trh and eyg, both of whose expression disappears from the pregland cells at approximately the same time. eyg plays no role in the regulation of fkh expression (Jones, 1998).
Armed with the knowledge that (1) fkh is responsible for the exclusion of both trh and eyg from the pregland cells and (2) trh is necessary for maintenance of eyg expression in the duct cells, it is possible to ask whether fkh represses eyg in the pregland cells simply by repressing trh or if fkh downregulates trh and eyg independently. To address this question, embryos were generated that were doubly mutant for trh and fkh. If the reason for eyg disappearance from the pregland cells in wild-type embryos is disappearance of trh, then it would be predicted that eyg expression would not persist in trh;fkh-mutant embryos. eyg expression, however, does persist in pregland cells in trh;fkh-mutant embryos, suggesting that trh plays no role in eyg repression by fkh. Thus, after
the initial establishment of the salivary primordium by Sex combs reduced, forkhead excludes eyegone expression from the pregland cells so that eyegone's salivary
expression is restricted to the posterior preduct cells. trachealess, in contrast,
activates eyegone expression in the posterior preduct cells (Jones, 1998).
In salivary ducts, breathless (btl) but not Serrate is activated by eyegone. btl codes for a Drosophila FGF receptor homolog that is critical to tracheal and midline glial cell development, while Ser encodes a Notch ligand containing a single EGF repeat. Although neither is required for duct morphogenesis, they are expressed during most of duct development and therefore serve as useful duct markers. Ser and btl expression have been shown to depend on trh (Kuo, 1996). Since trh also regulates eyg, whether eyg might act as an intermediate in the regulation of btl and Ser by trh was also tested. btl expression in duct cells is strongly reduced in embryos lacking eyg. In contrast, expression of Ser is not downregulated in the duct cells of eyg-mutant embryos even though the duct primordia are not fused as in wild-type embryos (Jones, 1998).
Border cells are a small group of follicle cells in the Drosophila ovary. Timely initiation of border cell migration requires the
product of the slow border cells (slbo) locus, which encodes the Drosophila homolog of the
transcription factor C/EBP. One target of SLBO in the control of
border cell migration is breathless. btl
expression in the ovary is border cell-specific, beginning just prior to the migration, and this
expression is reduced in slbo mutants. btl mutations dominantly enhanced the border cell migration
defects found in weak slbo alleles. Furthermore, SLBO-independent btl expression is able to
rescue the migration defects of hypomorphic slbo alleles. Purified SLBO binds eight
sites in the btl 5' flanking region by DNAse I footprinting. Taken together these results suggest that btl
is a key, direct target for SLBO in the regulation of border cell migration (Murphy, 1995).
The Drosophila tracheal system arises from clusters of ectodermal cells that invaginate and migrate to originate a network of epithelial
tubes. Genetic analyses have identified several genes that are specifically expressed in the tracheal cells and are required for tracheal
development. Among them, trachealess (trh) is able to induce ectopic tracheal pits and therefore it has been suggested that it would act
as an inducer of tracheal cell fates; however, this capacity appears to be spatially restricted. The expression of the tracheal
specific genes in the early steps of tracheal development and their crossinteractions have been examined. There is a set of primary genes including trh
and ventral veinless (vvl) whose expression does not depend on any other tracheal gene and a set of downstream genes whose expression
requires different combinations of the primary genes. The combined expression of primary genes is sufficient to induce some
downstream genes but not others. These results indicate that there is not a single master gene responsible for the appropriate expression of the
tracheal genes and support a model where tracheal cell fates are induced by the cooperation of several factors rather than by the activity of a
single tracheal inducer (Boube, 2000).
trh and vvl appear to initiate or to act very early in the genetic hierarchy specifying tracheal development. vvl expression in the tracheal cells is independent of trh function.
It is also found that trh expression in the tracheal cells is
independent of vvl function indicating that the
two genes act in parallel in the control of tracheal cell development. btl, a gene encoding an FGF receptor homolog required for tracheal migration, is a target of trh: btl requires vvl for
the maintenance of its transcription. Transduction of
the FGF signalling also requires the Downstream of FGF (Dof) protein, which is
specifically expressed in the tracheal cells. However, dof is
not a target gene activated as a result of FGF signaling as its
expression is not affected in btl mutant embryos. Conversely, the results show that the specific expression of dof in the tracheal cells is dependent on trh and vvl activity. Thus, trh and vvl enable the
tracheal cells to be competent to FGF signaling by regulating
the expression of at least two elements (btl and dof) acting at
different steps in the Btl pathway (Boube, 2000).
Formation of the trachea occurs by the migration and fusion of clusters of ectodermal cells specified in each side of ten embryonic segments. Morphogenesis of the tracheal tree requires the
activity of many genes, among them breathless (btl) and ventral veinless (vvl), whose mutations abolish tracheal cell migration.
Activation of the btl receptor by branchless (bnl), its putative ligand, exerts an instructive role in the process of guiding tracheal cell
migration. decapentaplegic determines vvl expression along the embryonic dorsoventral axis; expansion of dpp expression results in an increased recruitment of cells to express vvl. These cells are allocated in the expanded tracheal placodes, indicating that expansion of dpp expression causes a concomitant enlargement of the traceal placodes and of vvl expression. vvl is also required for the maintenance of btl expression during tracheal migration (Llimargas, 1997).
vvl is independently required for the specific expression in the tracheal cells of thick veins (tkv) and rhomboid (rho), two
genes whose mutations disrupt only particular branches of the tracheal system. Expression in the tracheal cells
of an activated form of tkv, the Decapentaplegic receptor, induces shifts in the migration of these cells, asserting the role of
the dpp pathway in establishing the branching pattern of the tracheal tree. In addition, by ubiquitous expression of the btl and tkv
genes in vvl mutants it is shown that both genes contribute to vvl function. These results indicate that through activation of its
target genes, vvl makes the tracheal cells competent to further signaling and suggest that the btl transduction pathway could
collaborate with other transduction pathways also regulated by vvl to specify the tracheal branching pattern (Llimargas, 1997).
Breathless interaction with Branchless, the ligand for Breathless
The spatial regularity of Breathless activation is determined by the distribution of Branchless. In the absence of branchless, tracheal cells fail to migrate and branch. bnl is expressed not in the trachea but in ectodermal cells that overly the migrating and branching trachea. The striking feature of bnl expression is its spatial complexity, one of the most complex patterns known in the embryo. For example, at stage 11, just before tracheal branching begins, bnl expression appears in five small clusters of epidermal cells arrayed around the tracheal sac, at positions where the five primary tracheal branches will soon bud. As the primary branches grow by cell migration over the next 2 hours (stages 12 and 13), expression in the clusters decreases. This appears to occur in a specific spatial pattern: the bnl-expressing cells closest to or contacting the growing tracheal branches lose expression first, with the tracheal cells continuing to migrate toward the remaining bnl-expressing cells. Two more cell clusters begin expressing bnl as expression in the other clusters turns off, presaging the subsequent outgrowth of additional specific branches (Sutherland, 1996).
breathless-branchless double mutants exhibit a tracheal phenotype similar to either one's loss-of-function mutation, as expected if the two function in the same signaling pathway. Reduction in the level of breathless can exacerbate the bnl-signaling defects, suggesting that bnl is insufficient in the haploid (single gene copy) state. A constitutively activated form of the BTL receptor can partially ameliorate the effect of the absence of bnl. Under such conditions there is a modest restoration of branching, reflecting the that the normal spatial distribution of activated receptor is not restored. BNL can activate the BTL receptor in vivo. In wild type extracts, a low level of phosphorylated BTL is detected, presumably due to its activation by endogenous BNL. In transgenic embryos that express bnl throughout the body, the level of phosphorylated BTL is increased about 8 fold (Sutherland, 1996).
Breathless, the Ras pathway and Downstream of FGF (DOF)
To determine whether Corkscrew plays a role in signaling from the Breathless receptor tyrosine kinase in tracheal development, phenotypes of mutant csw embryos were examined using a tracheal-specific marker. In mutant embryos, tracheal cell precursors are produced normally, but their subsequet migration, which generates the tracheal tree, appears defective. An incomplete and disconnected system of tracheal branches is the final result. Thus it appears that CSW operates positively in BTL signaling for the formation of the mature larval tracheal network (Perkins, 1996).
The involvement of Breathless, a Drosophila FGF receptor tyrosine kinase homolog, in border cell migration has prompted an inquiry as to whether RAS, a downstream effector for receptor tyrosine kinases, contributes
to receptor tyrosine kinase-mediated motility. A dominant-negative RAS protein inhibits cell migration when expressed specifically in
border cells during the period when these cells normally migrate. When expressed prior to
migration, dominant-negative RAS promotes premature initiation of migration. Conversely,
expression of constitutively active RAS prior to migration results in a significant delay in the initiation
step. Furthermore, the defect in initiation of border cell migration found in slbo1, a mutation at the
locus that encodes the Drosophila C/EBP homolog, is largely rescued by reducing RAS activity in border cells
prior to migration. Taken together, these observations indicate that RAS activity plays two distinct
roles in the border cells: (1) reduction in RAS activity promotes the initiation of that migration
process and (2) RAS activity is required during border cell migration. The
possible involvement of two downstream effectors of Ras in border cell migration was also examined. Raf activity was
dispensable to border cell migration while reduced Ral activity inhibited initiation. Ra1 is a small GTPase that is activated by RAS. Therefore, RAS plays a critical role in the dynamic regulation of border cell migration via a
Raf-independent pathway. It is believed that reducing RAS activity bypasses the normal requirement for SLBO expression for cell migration. The alternative explanation, that SLBO activates the expression of specific receptor tyrosine kinases is here held as not tenable (Lee, 1996).
The active state of receptor tyrosine kinases (RTKs) and the RTK signaling cascade
pathways were followed in situ. This was achieved by monitoring, with a specific monoclonal antibody, the distribution of
the active, dual phosphorylated form of MAP kinase (ERK). A dynamic pattern is observed during
embryonic and larval phases of Drosophila development, which can be attributed, to a large extent, to
the known RTKs. Torso-dependent, Egfr-dependent, Breathless-dependent, and Heartless dependent activation profiles have all been identified. This specific detection has enabled the determination of the time of receptor activation, the visualization of
gradients and boundaries of activation, and has allowed the postulation of the distribution of active ligands. A novel pattern is observed in the visceral mesoderm at stage 11 that is not Heartless dependent, as patches of cells display activated ERK at normal intensity in heartless mutants. Since
the antibody was raised against the phosphorylated form of a conserved ERK peptide containing the
TEY motif, this approach is applicable to a wide spectrum of multicellular organisms (Gabay, 1997).
Receptor tyrosine kinases (RTKs) transmit signals to the cell nucleus via the MAP kinase (MAPK) cascade, using specific molecules to link the activated
receptors to the MAPK cascade activator, Ras. A component of the FGF receptor (FGFR) signal transduction pathway, Downstream of
FGFR (Dof), also termed heartbroken, has been identified. Dof is an intracellular protein that is essential for signal transmission by the FGFR and acts downstream of the receptor and upstream of Ras.
Unlike other signaling molecules that act downstream of RTKs, Dof is not expressed ubiquitously but is present exclusively in cells that express FGFRs. DOF mRNA is first expressed on the ventral side of the embryo at the late syncytial blastoderm stage, in a region slightly narrower than the mesoderm primordium. It disappears from the mesoderm during germ band extension, and is seen in the tracheal placodes by stage 9/10. As the tracheae branch and start to differentiate, the transcript disappears from the primary branches and is seen mainly in the extended secondary branches. These expression patterns resemble those of the Drosophila FGF receptors htl and btl. The dof gene is also expressed transiently in the anterior and parts of the posterior midgut primordium (as is btl) and, like htl in a subset of heart cells and a group of migrating visceral mesodermal cells. Expression is also seen in glia cells.
Dof is needed in these cells for activation of the MAPK cascade via FGF signaling, but not for activation via other RTK ligands (Vincent, 1998).
The open reading frame of transcript II of DOF encodes a protein of 1009 amino acids. Database searches fail to identify any other proteins with significant overall homology to Dof. However the protein has two ankyrin repeats and another region (predicted to fold into a coiled coil) with some similarity to the myosins. The protein is likely to be phosphorylated and phosphorylated residues may serve as binding sites for the SH2 domain of the Drosophila Grb2 homolog. The context of another tyrosine suggests that it might represent a binding site for the SH2 domain of the regulatory subunit of phosphatidyl 3-kinase, while yet another tyrosine could represent a binding site for RasGAP. There is also a potential binding site for the protein tyrosine phosphatase SH-PTP2 (coded for by the corkscrew gene). Dual phosphorylated MAP kinase is absent in dof mutant embryos. Dof therefore appears to be
committed exclusively to FGFR-mediated signal transduction. Dof is unlikely to act as an adaptor protein sensing the activated (phosphorylated) state of the receptor, because it does not possess SH2 or SH3 domains (Vincent, 1998).
Btl (breathless) and Htl (heartless), the two FGFRs (fibroblast growth factor receptors) in Drosophila melanogaster, control cell migration and differentiation in the developing embryo. These receptors signal through the conserved Ras/mitogen-activated protein kinase pathway, but how they regulate Ras activity is not known. The present study shows that there is a direct interaction between p120 RasGAP (Ras GTPase-activating protein), a negative regulator of Ras, and activated FGFRs in Drosophila. The interaction is dependent on the SH2 (Src homology 2) domains of RasGAP, which have been shown to interact with a phosphotyrosine residue within the consensus sequence (phospho)YXXPXD. A potential binding site that matches this consensus is found in both Btl and Htl, located between the transmembrane and kinase domains of each receptor. A peptide corresponding to this region is capable of binding
RasGAP only when the tyrosine residue is phosphorylated. This tyrosine residue
appears to be conserved in human FGFR-1 and mediates the association with the
adapter protein CrkII, but no association between dCrk (Drosophila homologue of
CrkII) and the activated FGFRs was detected. RasGAP is a substrate of the
activated FGFR kinase domain, and mutation of the tyrosine residue within the
potential binding site on the receptor prevents tyrosine phosphorylation of
RasGAP. RasGAP attenuates FGFR signalling in vivo and this ability is
dependent on both its SH2 domains and its GAP activity. On the basis of these
results, it is proposed that RasGAP is directly recruited into activated FGFRs in
Drosophila and plays a role in regulating the strength of signalling through
Ras and the mitogen-activated protein kinase pathway (Woodcock, 2004).
Alignments of the mammalian FGFR-1 to the Drosophila FGFRs revealed a potential
binding site for the RasGAP SH2 domains, conserved between species, in the form
of a tyrosine residue within the consensus sequence, (phospho)YXXPXD, lying
between the transmembrane domain and the cytoplasmic tyrosine kinase domain of
the receptors. The fact that the corresponding tyrosine residue was shown to be
both an autophosphorylation site and a docking site for SH2 domains in mammalian
FGFR-1 suggested that this juxtamembrane tyrosine is a good candidate
for a RasGAP-binding site on Btl and Htl. This was shown to be the case in three
separate experiments. (1) A peptide corresponding to this region of Btl and
Htl can precipitate RasGAP expressed in Drosophila only when the peptide is
phosphorylated on the tyrosine. (2) This phosphorylated peptide
readily competes with Tor-Htl for binding to immobilized RasGAP SH2 domains,
whereas the non-phosphorylated peptide can not compete with this binding.
(3) If the juxtamembrane Tyr402 of Htl is mutated, RasGAP is no longer
tyrosine-phosphorylated when expressed in S. pombe. This suggests that RasGAP
can not be recruited to the mutated receptor and therefore is not in a
position to be phosphorylated by the active receptor kinase domain. The evidence
provided indicates that the juxtamembrane tyrosine residue of Btl and Htl is
both an autophosphorylation site and a binding site for the SH2 domains of
RasGAP (Woodcock, 2004).
Why does RasGAP have tandem SH2 domains? Two possible reasons for the existence
of tandem SH2 domains in the adapter region of RasGAP are (1) they may be
required to bind two separate docking sites simultaneously and (2) they may
stabilize the interaction with one docking site by increasing the avidity. The
individual requirement of the SH2 domains in the interaction with Btl and Htl
was tested in two experiments. One of them showed that the immobilized pTyr
peptide of Btl/Htl was unable to precipitate RasGAP with single N- and
C-terminal mutant SH2 domains to the same extent as wild-type RasGAP. This shows
that the two SH2 domains in RasGAP work together to increase the strength of the
interaction with the juxtamembrane phosphotyrosine of FGFRs. The second
experiment showed that both SH2 domains of RasGAP were required for its maximal
tyrosine phosphorylation in response to FGFR signalling in Drosophila. This
suggests that when RasGAP has only one intact SH2 domain, it cannot form a
stable enough complex with an activated tyrosine kinase, probably the FGFR in
this case, to be phosphorylated efficiently. These results indicate that the
tandem SH2 domains of the RasGAP adapter region are essential for both the
initial interaction with FGFRs and its subsequent phosphorylation on tyrosine.
The fact that the activated FGFRs will be dimerized means that there will be two
juxtamembrane phosphotyrosine-binding sites in the receptor complex, both of
which may be bound to the tandem SH2 domains of RasGAP, adding to the avidity of
the association and stabilizing the interaction. The requirement for both SH2
domains in the binding of RasGAP to FGFRs is consistent with the requirement of
both SH2 domains for the attenuation of ectopic FGFR signalling by RasGAP in the
wing (Woodcock, 2004).
There is a corresponding juxtamembrane tyrosine residue
equivalent to that of Btl and Htl in mammalian FGFR-1 that is autophosphorylated
and binds the SH2 domain of mammalian CrkII. However, when dCrk, the
Drosophila homologue of CrkII, was tested for its ability to bind the FGFR
homologues in Drosophila, no association could be detected. This suggests that
the interaction between Crk and FGFRs is not conserved between mammals and
Drosophila. Therefore it is unlikely that in Drosophila, RasGAP and dCrk compete
in vivo for binding to the juxtamembrane tyrosine residues of Btl and Htl. In
mammals, it is possible that CrkII and p120 RasGAP may both compete with each
other for binding to the juxtamembrane tyrosine residue of FGFR-1 (Woodcock, 2004).
Drk, the Drosophila homologue of mammalian Grb2, has been shown to act upstream
of Ras in RTK signalling pathways and acts by recruiting the Ras activator, Sos,
to the site of receptor activation. Drk binds
directly to activated RTKs or to substrates of the activated RTKs.
Since activation of Ras has been shown to partially rescue both btl and htl
mutant phenotypes, the ability of Drk to interact directly with these FGFRs
was tested. However, a GST fusion protein of wild-type Drk was not capable of
associating with activated Btl or Htl expressed in adult Drosophila. This result
suggests that the recruitment of Drk to the site of FGFR activation in
Drosophila is not directly to the receptor, but requires a receptor substrate,
as in the case of mammals that utilize the adapter-like protein FRS2. The lack
of an FRS2 homologue in Drosophila suggests the involvement of a novel adapter
in FGFR signalling; one candidate is the cytoplasmic protein Dof, which is
essential for FGFR signalling in Drosophila. It acts between FGFRs and Ras, and
it contains a number of tyrosine residues that lie within a consensus binding
sequence for the Drk SH2 domain (Woodcock, 2004).
In conclusion, a model is proposed in which, after ligand binding, the FGFRs Btl
and Htl undergo autophosphorylation on the juxtamembrane tyrosine residue,
thereby providing a docking site for RasGAP. This association is stabilized by
the fact that RasGAP possesses two SH2 domains, both of which are required for
maximal binding to the FGFRs. Once recruited, RasGAP becomes a substrate for the
active kinase domain of the receptor, potentially providing further docking
sites at the location of receptor activation. Recruitment of RasGAP to the
activated FGFRs would allow it to act on its substrate, namely RasGTP, thus
negatively regulating the downstream signal through Ras effector pathways, such
as the MAPK pathway. This model is consistent with the observations that the
ability of RasGAP to attenuate FGFR signalling in vivo requires its GAP activity
and both its SH2 domains, but not its SH3 domain, which is dispensable for FGFR
binding. However, the physiological relevance of the association of RasGAP with
FGFRs remains to be established. The recent identification of mutants defective
in the gene encoding RasGAP, vacuolar peduncle (vap), will make it possible to examine the effects of loss of RasGAP activity on FGFR signalling pathways regulating morphogenesis and differentiation in the Drosophila embryo (Woodcock, 2004).
While studying the developmental functions of the Drosophila dopamine synthesis pathway genes, interesting and unexpected mutant phenotypes were noticed in the developing trachea, a tubule network that has been studied as a model for branching morphogenesis. Specifically, Punch (Pu) and pale (ple) mutants with reduced dopamine synthesis show ectopic/aberrant migration, while Catecholamines up (Catsup) mutants that over-express dopamine show a characteristic loss of migration phenotype. Expression of Punch, Ple, Catsup and dopamine was seen in tracheal cells. The dopamine pathway mutant phenotypes can be reproduced by pharmacological treatments of dopamine and a pathway inhibitor 3-iodotyrosine (3-IT), implicating dopamine as a direct mediator of the regulatory function. Furthermore, these mutants genetically interact with components of the endocytic pathway, namely shibire/dynamin and awd/nm23, that promote endocytosis of the chemotactic signaling receptor Btl/FGFR. Consistent with the genetic results, the surface and total cellular levels of a Btl-GFP fusion protein in the tracheal cells and in cultured S2 cells are reduced upon dopamine treatment, and increased in the presence of 3-IT. Moreover, the transducer of Btl signaling, MAP kinase, is hyper-activated throughout the tracheal tube in the Pu mutant. Finally it was shown that dopamine regulates endocytosis via controlling the dynamin protein level (Hsouna, 2007).
This report demonstrates that genes involved in DA biosynthesis also regulate tracheal cell migration, and that this function is mediated by DA. This is unexpected since DA is normally associated with neuronal function. However, this novel developmental function is not fortuitous because of the strong and highly specific expression of Punch in tracheal cells during the migratory phase of tracheal development. In addition, the ectopic migration tracheal phenotypes in Pu/GTPCH mutants can be rescued by expressing a Pu/GTPCH transgene in developing trachea using a btl promoter, demonstrating the trachea-specific function of the DA pathway. Moreover, the expression of this transgene shows a dosage-dependent progression of phenotypic outcomes. That is, mild expression rescues ectopic migration, while over-expression tips the balance to blocked migration (Hsouna, 2007).
The product of GTPCH enzymatic activity, BH4, is also a stabilizing cofactor of nitric oxide synthase (NOS), which is needed for hypoxia-induced outgrowth of terminal tracheal branches. However, the Pu mutant phenotypes reported in this study are most likely unrelated to this developmental process because of the following reasons: (1) Terminal branching occurs near the end of embryogenesis and during larval development whereas the defects in primary and secondary branch migration occur during mid-embryogenesis. (2) Mutations in ple, which has no role in NOS function, also results in ectopic migration phenotypes. (3) DA and the DA pathway inhibitor can phenocopy the genetic mutants. (4) DA treatment can rescue Pu/GTPCH mutant phenotypes, implicating a direct role of DA in primary and secondary branch migration. Interestingly, DA is enriched in the trachea (Hsouna, 2007).
An inhibitory role for DA in the regulation of cell migration has been reported for a number of cell types over recent years. DA is capable of blocking the migration of vascular smooth muscle cells, a factor in the formation of atherosclerotic lesions, in a process mediated through the D1 class of DA receptors (Yasunari, 2003). DA also was reported to interfere with activated neutrophil transendothelial migration. Similarly, DA, acting through D1 receptors, is reported to reduce the migration of regulatory T cells in damaged neural regions, thereby providing protection against T cell-mediated neurodegeneration (Kipnis, 2004). Finally, DA acting through the D2 class of DA receptors, can inhibit tumor angiogenesis in highly vascularized gastric and ovarian tumors. It would be interesting to determine whether the mammalian GTPCH-TH-DA enzymatic pathways also have a non-neurological function in modulating tubulogenesis during development (Hsouna, 2007).
DA pathway mutants show tracheal abnormalities that are strikingly similar to the perturbations of tracheal cell migration in embryos with abnormalities in FGF signaling. Diminution of DA expression in Drosophila embryos, either by mutations (Pu and ple) or by pharmacological depletion (3-IT treatment), has dramatic effects on the stereotyped migratory behavior of tracheal cells and patterning of the resulting branched structure. Entire branches are misdirected and individual cells move away from the tracheal branches, a phenotype that has been termed 'run-away cells'. Under conditions of excess DA, via mutations (Catsup) or pharmacological modification (DA treatment), the converse phenotype, one of blocked migration, is evident. This phenotype is often associated with clustering of tracheal cells near the stunted ends of tracheal tubes. These opposing phenotypes are correlated with the increased or decreased levels of MAPK activation, the mediator of FGFR signaling (Hsouna, 2007).
The data further demonstrate that DA regulates FGF receptor turnover. Btl;;GFP fusion protein is down-regulated in the presence of DA and up-regulated in the presence of 3-IT, either in trachea or in cultured S2 cells. These results are consistent with the model that DA promotes internalization of Btl/FGFR, leading to its degradation through the endocytic pathway. The role of DA in internalization of FGFR is further suggested by the genetic interaction between the DA synthesis and endocytic pathway genes. Mutations in the human tumor suppressor nucleoside diphosphate kinase (NDK) gene (nm23) are strongly associated with tumor metastatic activity. Its functional homolog in Drosophila, abnormal wing discs (awd), has been shown to genetically interact with a temperature-sensitive allele of the shibire gene (shits), which encodes dynamin, a large GTPase required for the formation of clathrin-coated endocytic vesicles. A function for awd/nm23 in the migratory phase of tracheal development has been demonstrated and functional interactions occur with shi/dynamin that are required for the modulation of FGFR/Btl levels in tracheal cells. This report shows that Pu and ple phenotypes are exacerbated by awd and rescued by btl, while the Catsup phenotypes are rescued by awd but exacerbated by btl. These results indicate that Pu/GTPCH and Ple/TH, and by extension, DA, are positive regulators of endocytosis (Hsouna, 2007).
This analysis was extended by implicating direct involvement of DA in regulating the Shi/dynamin protein itself. Mutations in Pu/GTPCH, which regulates DA pools, result in reduction of the Shi/dynamin levels and, in consequence, the subsequent accumulation of FGFR/Btl in the plasma membranes of tracheal cells, thus accounting for ectopic migratory behavior of these mutant cells. Importantly, down-regulation of Shi/dynamin level in the Pu mutants can be rescued by treatment with DA. A direct correlation between DA and dynamin levels is also demonstrated in cultured S2 cells. Thus, the genetic and pharmacological evidence in this report supports the hypothesis that the diminished DA pools that accompany loss-of-function mutations in the Pu/GTPCH and ple/TH genes result in deficits of DA-mediated signaling necessary for Shi/dynamin accumulation (Hsouna, 2007).
Evidence of a role of DA in receptor endocytosis has emerged recently. For instance, DA can promote VEGFR endocytosis in cultured human endothelial cells. DA D3 receptor-mediated modulation of GABAA receptor and DA-regulated endocytosis of the renal cell Na+, K+-ATPase are similarly dynamin-mediated events. Why is a neurohormone also a mediator of endocytosis in other cell types? It is interesting to consider the possibility that the endocytic activity of DA may in fact be its ancestral function, which was adopted by the neurons and tubular cells later. Indeed, primitive, nerve-less multicellular organisms such as sponge can produce dopamine. The precise mechanism(s) by which DA regulates dynamin assembly is not yet clear. However, DA signaling promotes dynamin stabilization and assembly at the plasma membrane in cultured human kidney cells, and thus endocytosis, by activating protein phosphatase 2A which dephosphorylates dynamin-2. It is possible that a similar mechanism of action occurs in the tracheal cells and future experiments will help to address this possibility (Hsouna, 2007).
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