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).

Transcriptional Regulation

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).

The Drosophila F-box protein Archipelago controls levels of the Trachealess transcription factor in the embryonic tracheal system

The archipelago gene (ago) encodes the F-box specificity subunit of an SCF(skp-cullin-f box) ubiquitin ligase that inhibits cell proliferation in Drosophila and suppresses tumorigenesis in mammals. ago limits mitotic activity by targeting cell cycle and cell growth proteins for ubiquitin-dependent degradation, but the diverse developmental roles of other F-box proteins suggests that it is likely to have additional protein targets. This study shows that ago is required for the post-mitotic shaping of the Drosophila embryonic tracheal system, and that it acts in this tissue by targeting the Trachealess (Trh) protein, a conserved bHLH-PAS transcription factor. ago restricts Trh levels in vivo and antagonizes transcription of the breathless FGF receptor, a known target of Trh in the tracheal system. At a molecular level, the Ago protein binds Trh and is required for proteasome-dependent elimination of Trh in response to expression of the Dysfusion (Dys) protein. ago mutations that elevate Trh levels in vivo are defective in binding forms of Trh found in Dysfusion-positive cells. These data identify a novel function for the ago ubiquitin-ligase in tracheal morphogenesis via Trh and its target breathless, and suggest that ago has distinct functions in mitotic and post-mitotic cells that influence its role in development and disease (Mortimer, 2007).

The biological properties of individual F-box proteins are to a large degree determined by their repertoire of target proteins. In the case of the Drosophila Ago F-box protein, failure to degrade these targets promotes excess proliferation of imaginal disc cells. This observation has led to the identification of Cyclin E and Myc proteins as ago targets. However the broad pattern of Ago expression in the embryo suggests that it might regulate distinct processes and targets in other cell types. In view of the rapidly growing body of work showing that inactivation of human ago/Fbw7 is a common event in a variety of cancers (e.g., Malyukova, 2007; O'Neil, 2007; Thompson, 2007), identification of these targets may provide important insight into the biology of cancers lacking ago function (Mortimer, 2007).

This study shows that Drosophila Ago is required for the post-mitotic morphogenesis of the embryonic tracheal system and that this requirement is due, at least in part, to the ability of Ago to bind directly to a previously unrecognized target, the Trh transcription factor, and stimulate its proteasomal degradation. This ago degradation mechanism appears to fulfill different regulatory roles in different populations of tracheal cells. In non-fusion tracheal cells, ago is required to limit overall levels of Trh, which is normally expressed at moderate levels throughout the tracheal system. In tracheal fusion cells the ago degradation mechanism appears to be strongly potentiated by an unidentified signal generated by Dys, such that Trh is completely eliminated from Dys-expressing cells. At a genetic level, the dependence of ago tracheal phenotypes on trh gene dosage argues that that elevated Trh levels are primarily responsible for branching defects that occur in ago zygotic mutant embryos. In support of this, persistent Trh expression is also observed in ago mutant fusion cells in other tracheal branches. This novel role for Ago in tracheal development is supported by the independent finding that homozygosity for a genomic deletion containing the ago locus is associated with cell migration defects in embryonic tracheal metameres (Mortimer, 2007).

Many important developmental events are controlled by multiple mechanisms that collaborate to regulate a key step in the process. This somewhat redundant control insulates the process from defects in any single pathway, such that major defects only occur when multiple control mechanisms are blocked. The observation that the effect of ago mutations on Trh and btl levels is completely penetrant, but the resulting morphological defects are not, suggests that another pathway acts redundantly to ago to control tracheal development. The strong, dominant enhancement of the ago phenotype by a mutation in the abnormal wing disc (awd) gene (Dammai, 2003) fits very well into a model in which multiple pathways are responsible for the precisely timed down-regulation of the Breathless/FGF pathway: ago attenuates btl transcription by degrading Trh, awd lowers levels of Btl protein on the cell surface by promoting its endocytic internalization (Dammai, 2003), and other pathways act independently to control expression of the FGF ligand branchless in non-tracheal cells. Thus, the incomplete penetrance of the ago phenotype is not indicative of an insignificant role for the gene in tracheal development, but rather may indicate that the tracheal system uses multiple mechanisms to redundantly control a key step in its development (Mortimer, 2007).

The Ago WD repeat region binds Cyclin E and dMyc, and the current work demonstrates that it also binds Trh. Broadly, the Ago-Trh interaction is quite similar to interactions with Cyclin E and dMyc: it is required for the down-regulation of substrate levels in vivo, and its disruption elevates levels of substrate that then drive downstream phenotypes. For substrates like Myc, site-specific phosphorylation generates a motif that binds to the Ago WD-region and stimulates rapid, SCF-mediated protein turnover of the target protein (reviewed in Minella, 2005). In contrast, the data in this study suggest that Trh can physically interact with Ago in two distinct configurations: one that does not require an intact WD-domain and a second WD-dependent mode of binding. The observation that the Ago1 allele can participate in the first complex but not the second and is defective in Trh regulation in vivo, suggests that like other Ago targets, WD-dependent binding is associated with rapid Trh turnover. Expression of Dys appears to shift the balance in favor of this second mode of binding. Combined with the genetic and phenotypic data implicating ago as an in vivo regulator of Trh activity, these molecular data support a model in which Ago can bind to Trh in the absence of Dys and inefficiently stimulate Trh turnover by a WD-dependent mechanism. This inefficient mechanism may be responsible for the fairly mild increase in Trh levels observed in all ago mutant dorsal trunk cells. However, in the presence of Dys, the efficiency of Trh turnover in dorsal trunk fusion cells is enhanced to the degree that the entire pool of Trh is rapidly eliminated. Interestingly, the correlate of this hypothesis - that ectopic expression of dys in non-fusion cells should be sufficient to trigger down-regulation of endogenous Trh - was confirmed in a recent study (Jiang, 2006; Mortimer, 2007).

The nature of the Dys-generated signal responsible for this effect is not currently known. Precedent with other Ago targets suggests that it may involve Trh phosphorylation. Recent work on the mammalian ago ortholog Fbw7 has shown that interactions with substrates can also be modulated by interaction with accessory factors (Punga, 2006), or by conformational changes in the substrate driven by the isomerization of proline residues within the Ago/Fbw7 binding motif (van Drogen, 2006). Proline isomerization has been implicated in the degradation of mammalian c-Myc, but such mechanisms are not currently known to play a role in the degradation of either Myc or bHLH-PAS proteins in Drosophila . An important goal of future studies will be to determine if any of these types of mechanisms are involved in Dys-induced Trh degradation in tracheal cells (Mortimer, 2007).

The requirement for ago in tracheal cells suggests that the consequences of ago loss vary considerably depending on the proliferative state of the cells, their location within the organism, and their developmental stage. ago mutant clones in the mitotically active larval eye disc show no evidence of excessive Trh levels or deregulated Btl/FGF signaling and conversely, ago zygotic mutant trachea do not display 'extra cell' defects similar to those observed in the eye. The origins of this tissue specificity are currently not clear, although it might simply reflect the differential expression patterns of Ago targets in various mitotic and post-mitotic cell populations. There is currently no evidence that the mammalian Trh homologs NPAS-1 and NPAS-3 are degraded by an Ago/Fbw7 dependent mechanism in mammalian cells. However the finding that Fbw7 knock-out mouse embryos display defects in vascular development (Tetzlaff, 2004; Tsunematsu, 2004) seems to indicate that the Fbw7 ligase may also target proteins involved in tubular morphogenesis, and intriguingly both NPAS1 and NPAS3 have been linked either to this process (Levesque, 2007) or to the transcriptional control of FGFR genes (Pieper, 2005). It has been suggested that the Fbw7 vascular defects arise due to Notch misregulation. However since FGF signaling is known to control branching morphogenesis of the mammalian vasculature and lung, the data presented in this study raise the possibility that vascular phenotypes in Fbw7 knock-out mouse embryos may also reflect the deregulation of developmental pathways that control branching morphogenesis via mammalian homologs of trh and btl (Mortimer, 2007).

Cell autonomy of HIF effects in Drosophila: tracheal cells sense hypoxia and induce terminal branch sprouting

Drosophila tracheal terminal branches are plastic and have the capacity to sprout out projections toward oxygen-starved areas, in a process analogous to mammalian angiogenesis. This response involves the upregulation of FGF/Branchless in hypoxic tissues, which binds its receptor Breathless on tracheal cells. This study show that extra sprouting depends on the Hypoxia-Inducible Factor (HIF)-α homolog Sima and on the HIF-prolyl hydroxylase Fatiga that operates as an oxygen sensor. In mild hypoxia, Sima accumulates in tracheal cells, where it induces breathless, and this induction is sufficient to provoke tracheal extra sprouting. In nontracheal cells, Sima contributes to branchless induction, whereas overexpression of Sima fails to attract terminal branch outgrowth, suggesting that HIF-independent components are also required for full induction of the ligand. It is proposed that the autonomous response to hypoxia that occurs in tracheal cells enhances tracheal sensitivity to increasing Branchless levels, and that this mechanism is a cardinal step in hypoxia-dependent tracheal sprouting (Centanin, 2008).

This study has analyzed the role of the Drosophila HIF-α homolog Sima and the oxygen-sensing prolyl-4-hydroxylase Fga in tracheal terminal branching. It is assumed that during embryonic stages, tracheal development depends on hard-wired developmental cues, and, later, in larval stages, tracheal terminal branching is driven by local hypoxia in the target tissues. The observations carried out in this study indicate that the tracheal system of sima mutant third-instar larvae is indistinguishable from that of wild-type individuals, including the pattern of terminal branches. Thus, the results imply that if terminal branching during normal development was mediated by tissue hypoxia, the mechanism involved in such a local response should be Sima independent. This is a remarkable difference between Drosophila tracheogenesis and the development of the mammalian vascular system, in which HIF proteins are critically required for both vasculogenesis and developmental angiogenesis (Centanin, 2008).

It was also shown that Sima does play a cardinal role in hypoxia-dependent tracheal terminal branch sprouting, as well as in the formation of terminal branches that compensate for poor oxygenation in exceptional situations in which a neighboring branch is missing. Sima-dependent extra sprouting is negatively regulated by the oxygen-sensing prolyl-4-hydroxylase Fga, since fga mutants displayed an extra sprouting phenotype that was even stronger than that observed in wild-type individuals exposed to hypoxia. This extra sprouting phenotype is the first demonstration that loss of function of a HIF-prolyl hydroxylase can provoke an angiogenic-like phenotype. Thus, it seems reasonable to expect that conditional knockdown of mammalian PHDs in an appropriate cell type will promote angiogenesis (Centanin, 2008).

The long-standing paradigm for mammalian angiogenesis is that low oxygen levels trigger HIF accumulation in target tissues, which, in turn, mediates VEGF induction that, upon binding to VEGF receptors on endothelial cells, attracts the outgrowth of newly formed blood capillaries. Nevertheless, this apparently passive role of endothelial cells has recently been challenged. It has been demonstrated that in endothelial cell-specific HIF-α knockout mice the angiogenic response is impaired, highlighting a central role of the oxygen-sensing machinery in endothelial cells (Centanin, 2008).

This study has shown that the specialized Drosophila tracheal cells that respond to hypoxia by projecting angiogenic-like subcellular processes -- i.e., the terminal branches -- are apparently more sensitive to hypoxia than any other cell type in the larva. The sensory threshold to induce Sima-driven gene activation in these cells is shifted to near-normoxic oxygen tension. An alternative interpretation of the data is that tracheal terminal cells are similarly sensitive but more hypoxic than other cells, thereby inducing hypoxia-dependent transcription with higher sensitivity. In either case, the results suggest that Sima-dependent transcription within the tracheal terminal cells is part of the mechanism of oxygen sensing and tracheal extra sprouting (Centanin, 2008).

To test this hypothesis directly, EGFP-labeled sima homozygous mutant terminal cells were generated, and it was found that the ability of these cells to ramify upon a hypoxic stimulus is largely impaired. Furthermore, whether overexpression of Sima in the tracheae can provoke the angiogenic-like response was examined, and it was found that, indeed, expression of Sima restricted to the tracheal system is sufficient to induce extra sprouting. In contrast, overexpression of Sima -- or of a nondegradable variant of Sima -- in flip-out random clones outside the tracheae failed to provoke a similar phenotype, suggesting that accumulation of Sima in these cells is not sufficient for extra sprouting. Interestingly, in these Sima flip-out clones, a cell-autonomous response was observed, in which long subcellular processes projected from the cells that overexpressed Sima. Thus, although it is clear that bnl is induced in hypoxia and attracts the extension of terminal branches, the data support the notion that Sima is necessary, but not sufficient, for bnl induction in hypoxia (Centanin, 2008).

This study investigated which Sima target genes might be responsible for tracheal extra sprouting in fga mutants or upon exposure of wild-type larvae to hypoxia. Northern blot analyses indicated that bnl and btl are both upregulated in mildly hypoxic larvae or fga mutants. However, bnl homozygous EGFP-labeled terminal cells of larvae exposed to hypoxia retained their branching capacity, suggesting that extra sprouting in hypoxia is not mediated by an autocrine effect of Bnl, upon Sima-dependent induction in tracheal cells. In contrast, btl is directly induced by Sima in tracheal cells, and, consistent with this, overexpression of Btl in tracheal cells is sufficient to mimic the phenotypes of larvae exposed to hypoxia. Thus the data suggest that Sima-dependent transcriptional induction of btl in tracheal terminal cells is a critical step of the angiogenic-like response of the tracheal system in hypoxic larvae (Centanin, 2008).

In summary, it is proposed that tracheal cells respond to hypoxia in an autonomous manner, by promoting the accumulation of Sima, which induces expression of the receptor Btl, thereby increasing sensitivity of these cells to the ligand Bnl. Concomitantly, Bnl is induced in hypoxic target tissues through a mechanism that also involves the participation of Sima, and serves to cue the outgrowth of terminal branches toward O2-starved areas (Centanin, 2008).

During angiogenesis, vertebrate VEGF receptors are upregulated in endothelial cells of blood vessels that invade hypoxic tissues, and, particularly, Flt-1 induction is HIF dependent. Endothelial-specific overexpression of VEGF receptors might reveal to what extent this induction is a cardinal step in the angiogenic response to hypoxia (Centanin, 2008).

The homeobox transcription factor cut coordinates patterning and growth during Drosophila airway remodeling

A fundamental question in developmental biology is how tissue growth and patterning are coordinately regulated to generate complex organs with characteristic shapes and sizes. This study shows that in the developing primordium that produces the Drosophila adult trachea, the homeobox transcription factor Cut regulates both growth and patterning, and its effects depend on its abundance. Quantification of the abundance of Cut in the developing airway progenitors during late larval stage 3 revealed that the cells of the developing trachea had different amounts of Cut, with the most proliferative region having an intermediate amount of Cut and the region lacking Cut exhibiting differentiation. By manipulating Cut abundance, it was shown that Cut functioned in different regions to regulate proliferation or patterning. Transcriptional profiling of progenitor populations with different amounts of Cut revealed the Wingless (known as Wnt in vertebrates) and Notch signaling pathways as positive and negative regulators of cut expression, respectively. Furthermore, the gene encoding the receptor Breathless (Btl, known as fibroblast growth factor receptor in vertebrates) was identified as a transcriptional target of Cut. Cut inhibited btl expression and tracheal differentiation to maintain the developing airway cells in a progenitor state. Thus, Cut functions in the integration of patterning and growth in a developing epithelial tissue (Pitsouli, 2013).

Targets of Activity

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).

Protein Interactions

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).

Drosophila awd, the homolog of human nm23, regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

Human nm23 has been implicated in suppression of metastasis in various cancers, but the underlying mechanism of such activity has not been fully understood. Using Drosophila tracheal system as a genetic model, this study examined the function of the Drosophila homolog of nm23, the abnormal wing disc (awd) gene, in cell migration. Loss of Drosophila awd results in dysregulated tracheal cell motility. This phenotype can be suppressed by reducing the dosage of the chemotactic FGF receptor (FGFR) homolog, breathless (btl), indicating that btl and awd are functionally antagonists. In addition, mutants of shi/dynamin show similar tracheal phenotypes as in awd and exacerbate those in awd mutant, suggesting defects in vesicle-mediated turnover of FGFR in the awd mutant. Consistent with this, Btl-GFP chimera expressed from a cognate btl promoter-driven system accumulate at high levels on tracheal cell membrane of awd mutants as well as in awd RNA duplex-treated cultured cells. Thus, it is proposed that awd regulates tracheal cell motility by modulating the FGFR levels, through a dynamin-mediated pathway (Dammai, 2003. Full text of article).

p120 Ras GTPase-activating protein associates with fibroblast growth factor receptors in Drosophila

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).

Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis via regulation of Breathless turnover

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).

Drosophila glypican Dally-like acts in FGF-receiving cells to modulate FGF signaling during tracheal morphogenesis

Studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are involved in both breathless (btl)- and heartless (htl)-mediated FGF signaling during embryogenesis. However, the mechanism(s) by which HSPGs control Btl and Htl signaling is unknown. This study shows that dally-like (dlp, a Drosophila glypican) mutant embryos exhibit severe defects in tracheal morphogenesis and show a reduction in btl-mediated FGF signaling activity. However, htl-dependent mesodermal cell migration is not affected in dlp mutant embryos. Furthermore, expression of Dlp, but not other Drosophila HSPGs, can restore effectively the tracheal morphogenesis in dlp embryos. Rescue experiments in dlp embryos demonstrate that Dlp functions only in Bnl/FGF receiving cells in a cell-autonomous manner, but is not essential for Bnl/FGF expression cells. To further dissect the mechanism(s) of Dlp in Btl signaling, the role of Dlp was analyzed in Btl-mediated air sac tracheoblast formation in wing discs. Mosaic analysis experiments show that removal of HSPG activity in FGF-producing or other surrounding cells does not affect tracheoblasts migration, while HSPG mutant tracheoblast cells fail to receive FGF signaling. Together, these results argue strongly that HSPGs regulate Btl signaling exclusively in FGF-receiving cells as co-receptors, but are not essential for the secretion and distribution of the FGF ligand. This mechanism is distinct from HSPG functions in morphogen distribution, and is likely a general paradigm for HSPG functions in FGF signaling in Drosophila (Yan, 2007).

There are three main important findings in this work. First, Dlp was identified as an essential molecule required for tracheal development. Dlp is required for Btl-mediated tracheal branching during embryogenesis while both Dlp and Dally are involved in the formation of air sac tracheoblasts in the wing disc. Second, the data show that other HSPGs cannot replace Dlp for Btl signaling during embryogenesis and that both Dlp and Dally are not essential for Htl-mediated mesodermal cell migration. These data demonstrate that different FGFs may require different HSPGs to execute their effective signaling activities during development. Third and most importantly, strong evidence is provided that Dlp controls Btl signaling only in FGF-receiving cells in both embryonic and larval tracheal systems. This mechanism of HSPG activity in FGF signaling is very different from its roles in regulating the signaling activities of morphogens including Wnt, Hh and Dpp. Together, these new findings further define novel mechanisms and the specificities of HSPGs in FGF signaling during development (Yan, 2007).

Extensive biochemical and cell culture studies suggest that HSPGs are the part of the FGF/FGFR signaling complex. However, the mechanisms of HSPGs in FGF signaling during development are less known. Embryos mutant for two HSPG biosynthesis enzymes, sgl and sfl, exhibit defects in both Btl- and Htl-mediated FGF signaling. An important issue remaining to be solved is which HSPG core proteins are involved in these signaling events. The data in this work provide strong evidence that Dlp is the key molecule required for Btl signaling during embryonic tracheal development, while both Dlp and Dally are involved in the Btl mediated air sac tracheoblasts formation in the wing disc. The results provide several novel insights into the specificity of individual HSPG in FGF signaling. First, Dlp is involved in Btl signaling, but not in Htl signaling. These findings indicate that different FGF/FGFR complexes may require different HSPGs for their signaling activities. Second, Dlp is highly active and specific for Btl signaling; overexpression of the other three Drosophila HSPGs fail to rescue tracheal defects in dlp embryos. The specific activity of Dlp in Btl signaling could be due to the Dlp protein core or the HS GAG chains attached to the Dlp core protein. In this regard, it is especially surprising that Dally, which has 22% identity with Dlp and also bears a GPI anchor, cannot rescue tracheal phenotypes associated with dlp embryos. As Dlp is involved in several other signaling pathways such as Hh, it is unlikely that Dlp core protein interacts with the ligands directly. In this regard, it is worthwhile to note that ectopic expression of Dally also fails to rescue Hh signaling in dlp embryos. It is proposed that Dlp may have unique HS GAG chains that might provide high and specific activity for ligands such as Bnl and Hh (Yan, 2007).

The biosynthesis of HS GAG chains is determined by the HSPG protein core in which the GAG attachment sites and other protein parts such as the N-terminal cystenine-rich domain control both quantity and quality of the attached GAG chains. Detailed structure and functional studies of Dlp will further help to define specific requirements of the core protein or GAG attachment sites in FGF signaling. Furthermore, the unique GAG chains may be modified by specific enzymes. In this regard, it is particularly important to note that 6-O sulfation of HS is critical for Btl signaling, as Drosophila heparan sulfate 6-O-sulfotransferase is specifically expressed in embryonic tracheal system and is required for Btl signaling during embryogenesis. Recent study has shown that the overall sulfation level is more important than strictly defined HS fine structures for FGF signaling in some developmental contexts. In this regard, it is suggested that Dlp may be the optimal substrate for sulfation enzymes during embryogenesis. Therefore, the activity of Dlp in FGF signaling during embryogenesis cannot be replaced by other HSPGs including Dally, Syndecan and Perlecan (Yan, 2007).

Although Dlp is essential for Btl signaling during embryogenesis, both Dally and Dlp are involved in Btl signaling in air sac tracheoblast cells. Similarly, previous studies have shown that both Dally and Dlp are involved in regulating Wg, Hh and Dpp distribution in the wing disc. The different functions of the same HSPG in embryos and discs may reflect temporal and developmental stage dependent regulation of HSPG functions (Yan, 2007).

While it is well established that HSPGs can regulate FGF signaling by facilitating FGF/FGFR interaction, it is unknown whether HSPGs can also control FGF distribution, thereby modulating FGF signaling. This is a particularly important issue as in many developmental contexts FGF ligand is produced in one type of cell and acts on other cells to initiate its biological activity. One important finding of this work is that HSPGs control tracheal morphogenesis by regulating FGF signaling only in FGF-receiving cells, but not by regulating the secretion or distribution of FGF ligand in its producing cells and surrounding cells. Several important results support these conclusion: (1) dlp mutant embryos can suppress the phenotype of overexpressing Bnl in the tracheal cells. (2) Ectopic expression of Dlp in tracheal cells, rather than FGF expression cells, can effectively restore tracheal defects associated with dlp embryos. (3) Embryos rescued by prd-Gal4/UAS-dlp in dlp backbround is very similar to btl mutant embryos rescued by prd-Gal4/UAS-btl-GFP. (4) HSPGs are required for FGF signaling in its receiving cells in the air sac, but are dispensable in the columnar epithelial layer which includes FGF producing cells and other surrounding cells. Detailed analyses thus demonstrate the specific and distinct requirement of HSPGs in FGF signaling during tracheal development. Moreover, embryonic and larval data together suggest this is likely a general mechanism for HSPG function in FGF signaling in Drosophila (Yan, 2007).

Two major models are proposed for the role of HSPGs in FGF signaling. In one model, low affinity HS/GAG chains on the cell surface limit the diffusion of FGF ligand, thereby increasing its local concentration and the probability that it will interact with high-affinity FGFRs. In the second model, HSPGs facilitate the dimerization or oligomerization of FGF ligands thereby inducing receptor clustering and signal transduction. The experimental data cannot exclude either of these mechanisms. However, the results are in favour of the second case, since it is shown that HSPGs are not required in FGF concentration gradient in FGF producing cells, but are essential in FGF-receiving cells. Finally, a recent study showed that dynamin-mediated vesicle internalization is a crucial step to regulate FGF signaling in Drosophila tracheal system. Mutants in awd (abnormal wing disc) or shi (shibire), which encodes for a nucleoside diphosphate kinase and Drosophila dynamin, respectively, have increased levels of Btl in tracheal cell surface, increased FGF signaling activity and ectopic tracheal branching. In this regard, HSPGs may control FGF signaling by stabilizing the FGF/FGFR complex from degradation or internalization in FGF receiving cells. Further experiments using HSPG and awd/shi double mutant are needed to test this possibility (Yan, 2007).

Over the past several years, extensive studies in Drosophila and other model systems have established the essential roles of HSPGs in developmental signaling pathways including Wg, Hh and Dpp. In Drosophila embryo and wing imaginal disc, HSPGs are involved in the transport of morphogens including Wg, Hh and Dpp by a restricted diffusion mechanism. Narrow stripes of clones mutant for HSPGs can impede the movement of morphogens to further cells. However, in all of these cases, the first mutant cells adjacent to the morphogen source can still transduce signals arguing that HSPGs are not essential for morphogen signaling activity, but rather control the distributions or local concentrations of morphogens. The novel results from this work point out a major difference for a role of HSPGs in FGF signaling from their roles in morphogen signaling, as removal of HSPGs (dally-dlp or sfl) from FGF receiving cells can effectively block FGF signaling. Although the graded FGF activity may play an essential role in tracheal morphogenesis, the data from this work argue that the main function of HSPGs in FGF signaling is not to regulate the distribution of FGF ligand. Consistent with the different roles of HSPGs in FGF and morphogen signaling, it was found that Dlp acts cell-autonomously in FGF signaling while it functions non-autonomously in Hh signaling in embryos. These results suggest that Bnl transportation may be different from morphogen movement in the epithelial cells of the wing pouch. Indeed, morphogen molecules diffuse through the same layer of cells, columnar epithelial cells, while FGF is transported between different layers of tissues, from columnar epithelia to tracheoblasts. Moreover, leading air sac cells are always in close proximity with underlying columnar epithelia. They also extend multiple filopodia toward ligand gradient and presumably actively pursue the FGF ligands while wing disc morphogens including Wg, Hh and Dpp need to transport many cell diameters from their sources to reach their receiving cells. Studies in vertebrate also suggest that a graded distribution of FGF8 protein can be generated by the decay of fgf8 mRNA and this RNA gradient is translated into a protein gradient. In this case, no active transport mechanism is required to form a FGF gradient. In mammalian limb and lung development different FGFs are often expressed in different layers of cells, such as epithelium and mesenchyme, and signal through each other. It is interesting to determine whether HSPGs function similarly in these systems as in Drosophila (Yan, 2007).

Regulation of Drosophila embryonic tracheogenesis by dVHL and hypoxia

The tracheal system of Drosophila is an interconnected network of gas-filled epithelial tubes that develops during embryogenesis and functions as the main gas-exchange organ in the larva. Larval tracheal cells respond to hypoxia by activating a program of branching and growth driven by HIF-1α/sima-dependent expression of the breathless (btl) FGF receptor. By contrast, the ability of the developing embryonic tracheal system to respond to hypoxia and integrate hard-wired branching programs with sima-driven tracheal remodeling is not well understood. This study shows that embryonic tracheal cells utilize the conserved ubiquitin ligase (von Hippel-Lindau) (dVHL) to control the HIF-1 α/sima hypoxia response pathway, and two distinct phases of tracheal development with differing hypoxia sensitivities and outcomes were identified: a relatively hypoxia-resistant 'early' phase during which Sima activity conflicts with normal branching and stunts migration, and a relatively hypoxia-sensitive 'late' phase during which the tracheal system uses the dVHL/sima/btl pathway to drive increased branching and growth. Mutations in the archipelago (ago) gene, which antagonizes btl transcription, re-sensitize early embryos to hypoxia, indicating that their relative resistance can be reversed by elevating activity of the btl promoter. These findings reveal a second type of tracheal hypoxic response in which Sima activation conflicts with developmental tracheogenesis, and identify the dVHL and ago ubiquitin ligases as key determinants of hypoxia sensitivity in tracheal cells. The identification of an early stage of tracheal development that is vulnerable to hypoxia is an important addition to models of the invertebrate hypoxic response (Mortimer, 2008).

The development and survival of an organism are dependent on its ability to adapt to changing environmental conditions. Responses to some environmental changes, for example in nutrient availability, temperature, or oxygen concentration, involve alterations in patterns of gene expression that allow the organism to survive periods of environmental stress. In metazoan cells, the cellular response to reduced oxygen is mediated primarily by the HIF (hypoxia inducible factor) family of transcription factors, which are heterodimers composed of α and β subunits belonging to the bHLH Per-ARNT-Sim (bHLH-PAS) protein family. The HIF-1 αβ heterodimer is the primary oxygen-responsive HIF in mammalian cells and binds to a specific DNA sequence termed hypoxia response element (HRE) present in the promoters of target genes involved in energy metabolism, angiogenesis, erythropoiesis, and autophagy. HIF-1 activity is inhibited under normoxic conditions by two hydroxylase enzymes that use dioxygen as a substrate for catalysis to hydroxylate specific proline or aspartate residues in the HIF-1α subunit. These modifications limit HIF-1 activity by either reducing HIF-1α levels or inhibiting its ability to activate HRE-containing target promoters. One of these inhibitory mechanisms involves the 2-oxoglutarate/Fe(II)-dependent HIF-1 prolyl hydroxylase (HPH), which attaches a hydroxyl group onto each of two conserved proline residues in the oxygen-dependent degradation domain (ODD) of mammalian HIF-1α. These modifications create a binding site in the HIF-1α ODD for the Von Hippel-Lindau (VHL) protein, the substrate adaptor component of a ubiquitin ligase that subsequently polyubiquitinates HIF-1α and targets it for degradation by the proteasome. This degradation mechanism operates constitutively in normoxia and is epistatic to otherwise wide spread expression of HIF-1α mRNA. HIF-1α protein is also modified by a second oxygen-dependent hydroxylase termed Factor Inhibiting HIF (FIH) that hydroxylates an asparagine residue in the HIF-1α C-terminal activation domain. This blocks interaction with the CBP/p300 transcriptional co-factor and thus further restricts expression of HIF-1 responsive genes. These parallel O2-dependent hydroxylation mechanisms by HPH and FIH ensure that HIF-1α levels and activity remain low in normoxic conditions. However as oxygen levels become limiting in the cellular environment, rates of hydroxylation decline and HIF-1α is rapidly stabilized in a form that dimerizes with HIF-1β, translocates to the nucleus, and promotes transcription of HRE-containing target genes (Mortimer, 2008).

Evidence suggests that invertebrate homologs of HIF-1 are also regulated in response to changes in oxygen availability. In the fruit fly Drosophila melanogaster, the HPH homolog fatiga (fga) has been shown to genetically antagonize the HIF-1α homolog similar (sima) during development. The Drosophila VHL homolog dVHL has also been shown to be capable of binding to human HIF-1α and stimulating its proteasomal turnover in vitro. In addition, the Drosophila genome encodes a well-characterized HIF-1β homolog tango (tgo), and two potential FIH homologs (CG13902 and CG10133; Berkeley Drosophila Genome Project) that have yet to be analyzed functionally. Spatiotemporal analysis of sima activation using sima-dependent hypoxia-reporter transgenes has shown that exposure to an acute hypoxic stress induces Sima most strongly in cells of the larval and embryonic tracheal system, while induction of reporter activity in other tissues requires more chronic exposure to low oxygen. The larval tracheal system is composed of an interconnected network of polarized, epithelial tubes that duct gases through the organism. As the trachea acts as the primary gas-exchange organ in the larva, it is thus a logical site of hypoxia sensitivity. During larval stages, specific cells within the tracheal system called 'terminal cells' respond to hypoxia by initiating new branching and growth that results in the extension of fine, unicellular, gas-filled tubes toward hypoxic tissues in a manner somewhat analogous to mammalian angiogenesis . Studies have shown that sima and its upstream antagonist fga function within terminal cells to regulate this process. sima is necessary for terminal cell branching in hypoxia and its ectopic activation, by either transgenic overexpression or loss of fga, is sufficient to induce excess branching even in normoxia. These phenotypes have been linked to the ability of sima to promote expression of the breathless (btl) gene, which encodes an FGF receptor that is activated by the branchless (bnl) FGF ligand. This receptor/ligand pair is known to act via a downstream MAP-kinase signaling cascade to promote cell motility and tubular morphogenesis in a variety of systems. Excessive activation of this pathway within tracheal cells by transgenic expression of btl is sufficient to drive excess branching. Reciprocally, misexpression of the bnl ligand in certain peripheral tissues is sufficient to attract excess terminal cell branching. Indeed production of secreted factors such as Bnl may be a significant part of the physiologic mechanism by which hypoxic cells attract new tracheal growth. Sima-driven induction of btl in conditions of hypoxia thus allows larval terminal cells to enter what has been termed an 'active searching' mode in which they are hyper-sensitized to signals emanating from nearby hypoxic non-tracheal cells (Mortimer, 2008 and references therein).

The role of the btl/bnl pathway in tracheal development is not restricted to hypoxia-induced branching of larval terminal cells. It also plays a critical, earlier role in the initial development of the embryonic tracheal system from the tracheal placodes, groups of post-mitotic ectodermal cells distributed along either side of the embryo that undergo a process of invagination, polarization, directed migration, and fusion to create a network of primary and secondary tracheal branches . btl and bnl are each required for this process via a mechanism in which restricted expression of bnl in cells outside the tracheal placode represents a directional cue for the migration of btl-expressing cells within the placode. Accordingly, btl expression is normally highest in pre-migratory and migratory embryonic fusion cells. In contrast to the larval hypoxic response, sima does not appear to be required for morphogenesis of the embryonic tracheal system. Rather, developmentally programmed signals in the embryo dictate a stereotyped pattern of btl and bnl expression that leads to a similarly stereotyped pattern of primary and secondary tracheal branches. The btl/bnl pathway thus responds to developmental signals to drive a fixed pattern of branching in the embryo, while in the subsequent larval stage it responds to hypoxia-dependent sima activity to facilitate the homeostatic growth of larval terminal cells and tracheal remodeling (Mortimer, 2008 and references therein).

Under normal circumstances, developing Drosophila tissues do not begin to experience hypoxia until the first larval stage, when organismal growth and movement begin to consume more oxygen than can be provided by passive diffusion alone. As a consequence, the first hypoxic challenge normally occurs after the btl/bnl-dependent elaboration of the primary and secondary embryonic branches is complete. Thus, the ability of the larval tracheal system to drive new branching and remodeling via sima and btl represents the response of a developed 'mature' tracheal system to reduced oxygen availability. By contrast the effect of hypoxia on embryonic tracheal development, which requires tight spatiotemporal control of Btl signaling to pattern the tracheal network, is not as well understood. Given that the trachea does not function as a gas-exchange organ until after fluid is cleared from the tubes at embryonic stage 17, it may be that the transcriptional response of embryonic tracheal cells to hypoxia leads to mainly metabolic changes rather than to a btl-driven program of tubulogenesis and remodeling. However, if the embryonic tracheal system does utilize the sima pathway to induce hypoxia-dependent changes in btl gene transcription, then hypoxic exposure of embryos might be predicted to produce a situation of competing developmental and homeostatic inputs that converge on the btl/bnl pathway. The ability of tracheal cells to integrate such signals may then determine whether or not the embryonic tracheal system is able to adapt to oxygen stress, or whether embryonic tracheal development represents a sensitive period during which the organism's ability to respond to changes in oxygen levels is inherently limited by a pre-programmed pattern of developmental gene expression (Mortimer, 2008).

This study shows that the embryonic tracheal system utilizes the dVHL/sima pathway to respond to hypoxia, but that the type and severity of resulting phenotypes depend on the developmental stage of exposure. Hypoxic challenge while embryonic tracheal cells are responding to developmentally programmed btl/bnl migration signals disrupts tracheal development and results in fragmented and unfused tracheal metameres. In contrast, hypoxic challenge at a somewhat later embryonic stage after fusion is complete results in overgrowth of the primary tracheal branches and the production of extra secondary branches. Interestingly, it was found that the threshold of hypoxia required to induce tracheal phenotypes in the early embryo is higher than that required to induce excess branching phenotypes in later embryonic stages, indicating that tracheal patterning events in the embryo are relatively resistant to hypoxia. Genetic analysis indicates that both types of hypoxic tracheal phenotypes -- stunting and overgrowth -- require sima and can be phenocopied in normoxia by reducing expression of the HIF-1α ubiquitin ligase gene dVHL specifically within tracheal cells. Moreover, it was found that reduced dVHL expression in the larval trachea leads to excess terminal cell branching in a manner quite similar to that observed in fga mutants. Molecular and genetic data indicate that excess btl transcription is a major cause of hypoxia-induced tracheal phenotypes. Consistent with this, mutations in the archipelago (ago) gene, which antagonizes btl transcription in tracheal fusion cells, synergize strongly with dVHL inactivation to disrupt tracheal migration and branching. Interestingly, ago mutations also lower the threshold of hypoxia required to elicit tracheal phenotypes in the 'early' embryo, suggesting that the relative activity of the btl promoter can affect hypoxic sensitivity. These findings show that the dVHL/sima pathway plays an important role in tracheal development, and identify two distinct phases of embryonic development that show different phenotypic outcomes of activating this pathway: an early phase during which sima activity conflicts with developmental control of tracheal branching and migration, and a later phase during which the tracheal system uses the dVHL/sima/btl pathway to adapt to hypoxia by increasing its future capacity to deliver oxygen to target tissues (Mortimer, 2008).

Hypoxia-induced remodeling of tracheal terminal cells represents the response of a developed larval tracheal system to reduced levels of O2 in the environment. By contrast, the response of the developing embryonic tracheal system to systemic hypoxia has not been as well characterized. In light of the observation that embryonic tracheal cells display hypoxia-induced activation of a Sima-reporter) and that sima promotes btl expression in larval tracheal cells, embryonic exposure to hypoxia may thus produce a situation in which hard-wired btl/bnl patterning signals in the embryo come into conflict with the type of sima/btl-driven plasticity of tracheal cell branching seen in the larva. This study examined the effect of hypoxia on embryonic tracheal branching and migration. It was found that hypoxia has dramatic effects on the patterns of morphogenesis of the primary and secondary tracheal branches. Surprisingly, varying the timing and severity of hypoxic challenge is able to shift the outcome from severely stunted tracheal branching to excess branch number and enhanced branch growth. Genetic and molecular data indicate that both classes of phenotypes, stunting and overgrowth, involve regulation of sima activity and btl transcription by dVHL, and that the effects of hypoxia on tracheal development can be mimicked in normoxia by tracheal-specific knockdown of dVHL. This observation confirms a central role for dVHL in restricting the hypoxic response in vivo, and identifies a role for dVHL as a required inhibitor of sima and btl during normal tracheogenesis (Mortimer, 2008).

Since Trh and Sima/HIF-1α share a similar consensus DNA binding site, it is likely that the tracheal phenotypes elicited by either hypoxia or dVHL knockdown are to some degree the product of a combined 'Trh/Sima-like' transcriptional activity in tracheal cells. This conclusion is supported both by the general phenotypic similarity (i.e. migration and overgrowth defects) between hypoxia/dVHL knockdown and trh overexpression, by the modest ability of trh alleles to suppress dVHLi phenotypes, and by the previously demonstrated overlap of transcriptional activity between Trh and human HIF-1α. Indeed, Trh is well-established as a required activator of developmental btl expression. However, because the excess Btl activity that occurs in hypoxia or in the absence of dVHL occurs independently of a change in Trh expression, it thus appears to be mediated largely by increased sima activity (Mortimer, 2008).

This analysis suggests that there are two distinct developmental 'windows' of embryogenesis during which hypoxia has opposite effects on tracheal branching. The first corresponds to a period immediately before and during primary branch migration that is relatively insensitive to hypoxia. Embryos in this stage show a minimal response to 1% O2, but show a nearly complete arrest of migration in 0.5% O2. Interestingly, a prior study found that similarly staged embryos (stage 11) respond to complete anoxia by prolonged developmental arrest, from which they can emerge and resume normal development. These somewhat paradoxical results -- that acute hypoxia is more detrimental to development than chronic anoxia -- might be explained by the observation that chronic exposure to low O2 induces Sima activity throughout the embryo while acute exposure activates Sima only in tracheal cells. The former scenario may result in coordinated developmental and metabolic arrest throughout the organism, while in the latter scenario developmental patterns of gene expression in non-tracheal cells may proceed such that tracheal cells emerging from an 'early' hypoxic response find an embryonic environment in which developmentally hard-wired migratory signals emanating from non-tracheal cells have ceased (Mortimer, 2008).

The second type of tracheal response occurs during a later 'window' of embryogenesis after btl/bnl-driven primary and secondary branch migration and fusion are largely complete. It involves sinuous overgrowth of the primary and secondary branches, and duplication of secondary branches. As in the 'early' response, 'late' hypoxic phenotypes are controlled by the dVHL/sima pathway, yet unlike the 'early' response, these phenotypes occur at high penetrance even at 1% O2. Thus the 'late' embryonic tracheal system is relatively sensitized to hypoxia and responds with increased branching in a manner similar to larval terminal cells. Indeed, much as larval branching increases with decreasing O2 levels, it was observed that dorsal trunk growth in the late embryo is graded to the degree of hypoxia. The mechanism underlying the differential sensitivity of the 'early' and 'late' tracheal system may be quite complex. However, it was found that tracheogenesis can be sensitized to hypoxia by reducing activity of ago, a ubiquitin ligase component that restricts btl transcription in tracheal cells via its role in degrading the Trh transcription factor. Increasing transcriptional input on the btl promoter thus appears to sensitize 'early' tracheal cells to hypoxia. As Sima also controls btl transcription, one explanation of the difference in sensitivity between different embryonic stages may thus lie in differences in the activation state of the btl promoter. If so then the activity of the endogenous btl regulatory network may be an important determinant of the threshold of hypoxia required to elicit changes in tracheal architecture (Mortimer, 2008).

An organism can have its hypoxic response triggered in two ways, either by systemic exposure of the whole organism to a reduced O2 environment or by localized hypoxia produced by increased O2 consumption in metabolically active tissues. Data from this study and others suggests there may be distinctions between these two triggers. Exposing larvae or embryos to a systemic pulse of hypoxia results in a 'btl-centric' response specifically in tracheal cells. Outside of an 'early' vulnerable period which corresponds to embryonic branch migration and fusion, elevated Btl activity in embryonic tracheal cells promotes branch duplications and overgrowth similar to that seen in larvae. By contrast, tracheal growth induced by localized hypoxia in the larva has been suggested to involve a 'bnl-centric' model in which the hypoxic tissue secretes Bnl and recruits new tracheal branching. Whether this type of mechanism operates in embryos, or whether embryos ever experience localized hypoxia in non-tracheal cells, has not been established (Mortimer, 2008).

tHE data indicate that dVHL is a central player in the hypoxic response pathway in embryonic and larval tracheal cells. A prior study found that injection of dVHL dsRNA into syncytial embryos disrupted normal tracheogenesis, but was technically limited in its ability to conduct a detailed analysis of dVHL function in development and homeostasis. The current study found that dVHL knockdown specifically in tracheal cells mimics the effect of systemic hypoxia on embryonic tracheal architecture and larval terminal cell branching. dVHL knockdown thus phenocopies loss of the HPH gene fga, which normally functions to target Sima to the dVHL ubiquitin ligase in normoxia. Moreover, all phenotypes that result from reduced dVHL expression can be rescued by reducing sima activity, suggesting that Sima is the major target of dVHL in the tracheal system. These data support a model in which dVHL, fga, and sima function as part of a conserved VHL/HPH/HIF-1α pathway to control tracheal morphogenesis in embryos and larvae. The btl receptor appears to be an important target of this pathway in embryonic (this study) and larval tracheal cells. Knockdown of dVHL elevates btl transcription in embryonic placodes and tracheal branches, and removal of a copy of the gene effectively suppresses dVHL tracheal phenotypes. Reciprocally, overexpression of wild type btl in embryonic tracheal cells can produce migration defects and sinuous overgrowth, while expression of a constitutively active btl chimera (btlλ) also leads to primary branch stunting and duplication of secondary branches. Interestingly, pupal lethality associated with tracheal-specific knockdown of dVHL is not sensitive to the dose of btl, but is dependent on sima. Thus the dVHL/sima pathway may have btl independent effects on tracheal cells in later stages of development (Mortimer, 2008).

Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

The respiratory (tracheal) system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. A unique embryonic tracheal tubulogenesis phenotype has been described caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr) tyrosine kinase (TK). Ptp10D physically interacts with Egfr. This study demonstrates that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, it was shown that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. It was also shown that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways (Jeon, 2012).

The Drosophila tracheal system is an intricate branched network of air-filled tubes that delivers oxygen to tissues. Tube formation in the tracheal system involves complex morphogenetic events that differ between tube types. Multicellular tubes have lumens that are surrounded by the apical surfaces of several cells. Unicellular tubes are formed by rolling up of single cells to form junctions with themselves. Seamless tubes are intracellular structures within terminal cells. Many genes have been identified that affect the formation and morphology of tracheal tubes (Jeon, 2012).

The absence of the two Type III RPTPs, Ptp4E and Ptp10D, changes the geometries of the tubes in unicellular and terminal branches, so that they form spherical cysts in place of continuous tubular lumen. The phenotype involves a loss of negative regulation of the Egfr RTK, and Ptp10D physically associates with Egfr (Jeon, 2012).

One of the mammalian Ptp4E/Ptp10D orthologs, PTPRJ (DEP-1), is a direct regulator of multiple growth factor receptor TKs. This led to a test of the hypothesis that Btl and Pvr, the other two Drosophila growth factor receptor TK orthologs that are expressed in embryonic tracheae, are also required for the Ptp4E Ptp10D phenotype (Jeon, 2012).

A quantitative analysis of cyst size at the TC/LT junction showed that tracheally expressed DN mutants of Egfr, Btl, and Pvr all suppress the Ptp4E Ptp10D phenotype almost to wild-type, suggesting that dysregulation of all three RTKs is required for the replacement of linear tubes by spherical cysts. Also, CA mutants of each RTK enhance the phenotype, producing enlarged cysts. These effects cannot be produced by all RTKs, since expression of a CA mutant of InR did not enhance the phenotype (Jeon, 2012).

If the Ptp4E Ptp10D cyst phenotype is the consequence of simultaneous deregulation of all three RTKs, it might be possible to generate the phenotype in a wild-type background by expressing multiple RTK CA mutants. This did not work, even when all three CA mutants were expressed at once. Possible explanations include: 1) because PTP activity normally dominates over RTK activity, the effect on PTyr levels of removing negative regulation by the RPTPs is much greater than that produced by expressing CA RTK mutants in the presence of the RPTPs; 2) other TKs are important for the phenotype, and their activities must also be increased; 3) the RPTPs have PTP-independent activities as adhesion molecules, and generation of the phenotype requires both elevation of RTK activity and the absence of the PTP-independent functions of the RPTPs (Jeon, 2012).

Whether Ptp4E and Ptp10D both regulate all three RTKs was investigated. If they have specificity for particular RTKs, one might be able to generate the cyst phenotype by removing only one RPTP in the presence of a CA RTK mutant. Such combinations were made for Egfr (Ptp4E, Btl>Egfr-CA and Ptp10D, Btl>Egfr-CA), but neither of them had cysts. Since the PTP domains of Ptp4E and Ptp10D are 89% identical, it is likely that they have the same enzymatic targets. The idea that the RPTPs have redundant functions is also consistent with the observation that Ptp4E and Ptp10D single mutants have no detectable phenotypes, while the double mutant is lethal and has both tracheal and nervous system defects (Jeon, 2012).

When each of the DN RTK mutants was expressed together with a CA mutant of one of the other RTKs in the Ptp4E Ptp10D background, the DN mutant was now unable to suppress the phenotype back to wild-type. Instead the phenotype returned to the strength of unmodified Ptp4E Ptp10D mutants. This shows that if the activity of one RTK is sufficiently elevated, it can replace the requirement for another RTK. Thus, none of the three RTKs is uniquely required to generate the phenotype. Rather, the formation of tubes vs. cysts is controlled by the total amount of activity of certain RTKs in tracheal cells. This implies that the downstream pathways whose increased activity causes cyst formation use SH2-domain effectors that can bind to PTyr sites on any of the three RTKs (Jeon, 2012).

It is interesting that the three RTKs can substitute for each other in regulating the tube vs. cyst decision when they are deregulated by loss of the RPTPs, since the RTKs do not seem to have interchangeable activities in wild-type tracheal cells (or in other tissues). Loss or gain of Btl function produces defects in primary, secondary, and terminal tracheal branching. Loss of Egfr function produces a much more subtle tracheal phenotype affecting tissue integrity. Maintenance of tissue integrity requires signaling through the MAP kinase pathway downstream of Egfr, but is unaffected by reduction of MAP kinase signaling downstream of Btl (Jeon, 2012).

These findings can be explained by the fact that growth factor receptor TKs are usually in an inactive state, due to insufficient levels of ligands and to negative regulation by PTPs. They become active only when they come into contact with elevated levels of their ligands at specific times and places. The activities of Type III RPTPs that dephosphorylate the RTKs might also be transiently reduced at some of these times and places, possibly through interactions of their XC domains with as yet unidentified ligands. As a consequence of the tight control of RTK activity, only those downstream signaling pathways that are most responsive to a particular RTK are likely to be activated by that RTK at any time in wild-type embryos, and the outcomes of signaling through these pathways may also be controlled by the subcellular distributions of ligands, RTKs, RPTPs, and downstream effectors. By contrast, in the absence of Ptp4E and Ptp10D, basal levels of RTK ligands may be able to drive all of the growth factor RTKs to a high level of activity, resulting in strong signaling through all of the downstream pathways they can control. Loss of negative regulation might also cause delocalization of signaling, so that effectors whose activity is normally restricted to particular parts of the cell become activated in a cell-wide manner. Under these conditions, a reduction in the activity of any one of the RTKs by a DN mutant will decrease signaling through multiple downstream pathways. Adding a CA mutant of another RTK can then turn signaling through all of these pathways back up, compensating for the effects of the DN mutant (Jeon, 2012).

The ability of RTKs to substitute for each other in control of cyst formation is conceptually similar to cell transformation through elevation of RTK signaling. RTK activity in cultured cells is tightly controlled, and only a few endogenous RTKs are normally involved in cellular responses to the mitogenic growth factors in their culture medium. Many RTKs can signal through the Ras/MAP kinase pathway, however, and elevated Ras/MAP kinase transduction is sufficient to cause transformation of established cell lines. Thus, oncogenic (CA) mutants of a variety of RTKs are able to transform fibroblastic cell lines when expressed at high levels, regardless of whether the endogenous versions of the RTKs are normally used to regulate proliferation in those cell lines (or are even expressed there) (Jeon, 2012).

The observations on the interchangeability of the RTKs suggests that the decision to form cysts rather than tubes in Ptp4E Ptp10D mutants is very sensitive to the levels of PTyr on the effector binding sites on autophosphorylated RTKs, and that cysts appear when total PTyr rises above a critical threshold. This conclusion is based on the complete suppression of the Ptp4E Ptp10D cyst phenotype that is produced by expression of any of the three DN mutants, even though each DN would eliminate only about 1/3 of total PTyr on the RTKs, if they have roughly equal activities. In wild-type embryos, negative regulation by the RPTPs keeps RTK signaling well below this threshold, so the system is insulated against random fluctuations in phosphorylation or downstream signaling (Jeon, 2012).

The Ptp4E Ptp10D GB cyst number phenotype is completely suppressed by Btl-DN, but not suppressed at all by Egfr-DN and only slightly by Pvr-DN. This might be taken as evidence that elevation of Btl activity is uniquely required to replace GB tubes with cysts. However, when Btl-DN is competed against Pvr-CA, it is only able to suppress the Ptp4E Ptp10D, Btl>Pvr-CA phenotype back to that of unmodified Ptp4E Ptp10D, indicating that Btl can be replaced by Pvr if its activity is sufficiently elevated. These findings can be explained if Btl activity is much higher than Egfr or Pvr activity in GB cells, so that a DN mutant that knocks down endogenous Btl activity by a DN mutant has a greater effect on phosphorylation of effector binding sites than mutants that reduce Egfr or Pvr activities. The activity of CA RTK mutants is independent of the endogenous levels of RTKs, so the Pvr CA mutants could still reverse the effect of Btl-DN even if Pvr-DN has no effect on its own (Jeon, 2012).

To evaluate whether the MAP kinase signaling pathway is involved in the determination of tube geometry, a CA mutant of Phl, the Drosophila Raf kinase, was expressed in the Ptp4E Ptp10D background. Phl-CA enhances the TC/LT phenotype almost as strongly as Egfr-CA. When Phl-CA is combined with Egfr or Btl DN mutants, the phenotype is suppressed back to that of unmodified Ptp4E Ptp10D. Since these RTKs are upstream of Raf, the fact that suppression occurs suggests that pathways other than the MAP kinase pathway are required to generate the phenotype. However, these other pathways may be stimulated by elevation of MAP kinase signaling, since one would expect suppression back to a near-wild-type phenotype if they were completely independent of the MAP kinase pathway (Jeon, 2012).

The involvement of Rho GTPases was tested by expressing DN mutants of Rho1, Rac1, and Cdc42 in the Ptp4E Ptp10D background. Rho1-DN and Rac1-DN completely suppress the TC/LT phenotype, and Cdc42-DN and a tracheally expressed Rho1 RNAi construct produces partial suppression. The DN mutant data do not necessarily show that Rho and Rac are both required for generation of the phenotype. DN mutants may occlude binding of wild-type Rho family GTPases to their GEFs, some of which can act on both Rho and Rac. Therefore high-level expression of Rac-DN might inhibit Rho activation, and vice versa. However, the suppression of the TC/LT phenotype by Rho1 RNAi, which is a specific inhibitor, implicates Rho1 itself in the phenotype. Rho1-CA enhances the Ptp4E Ptp10D phenotype, producing cysts on most DBs (Jeon, 2012).

The differences in the abilities of the three RTK DN mutants to suppress the effects of Phl-CA and Rho-CA may provide clues to the pathways that act downstream of these kinases. The MAP kinase and Rho pathways are not necessarily independent, since Ras/MAP kinase pathway activation can increase Rho-GTP in some cell lines. Also, each of the three RTKs is likely to activate both pathways to some extent, as well as many other downstream pathways. Defining the specific outputs of the MAP kinase and Rho pathways that control tracheal tube geometry and identifying other RTK pathways that regulate tube formation is likely to require genome-wide screens for suppressors and enhancers of the Ptp4E Ptp10D phenotype (Jeon, 2012).

breathless: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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