breathless


DEVELOPMENTAL BIOLOGY

Embryonic

Early expression of btl is first detected just before onset of gastrulation. Two groups of cells located ventrally at the anterior tip of the embryo express btl as well as some cells at the posterior pole. As gastrulation proceeds [Images], two single-cell-wide stripes on each side of the presumptive mesoderm also express breathless. Later on transcription at the ventral midline is moderated in a segmental manner.

breathless is predominantly expressed in a restricted set of tissues: the developing tracheal system and the delaminating midline glial and neural cells (Glazer, 1991, Klämbt, 1992 and Shishido, 1993).

Distinct roles for two receptor tyrosine kinases in epithelial branching morphogenesis in Drosophila

Branching morphogenesis is a widespread mechanism used to increase the surface area of epithelial organs. Many signaling systems steer development of branched organs, but it is still unclear which cellular processes are regulated by the different pathways. The development of the air sacs of the dorsal thorax of Drosophila was used to study cellular events and their regulation via cell-cell signaling. Two receptor tyrosine kinases play important but distinct roles in air sac outgrowth. Fgf signaling directs cell migration at the tip of the structure, while Egf signaling is instrumental for cell division and cell survival in the growing epithelial structure. Interestingly, Fgf signaling requires Ras, the Mapk pathway, and Pointed to direct migration, suggesting that both cytoskeletal and nuclear events are downstream of receptor activation. Ras and the Mapk pathway are also needed for Egf-regulated cell division/survival, but Pointed is dispensable (Cabernard, 2005).

The air sac of the dorsal thorax grows from a bud that arises during the third larval instar from a wing disc-associated tracheal branch. To illustrate the development of the air sac, a GFP trap line was used that rather ubiquitously expressed membrane bound GFP; tracheal cells were counterstained with an mRFP1-moesin construct under the direct control of the trachea-specific breathless (btl) enhancer. From the early to late third instar stage, a bud-like structure grows out of the transverse connective and spreads on the wing imaginal disc epithelium; this outgrowth corresponds to the primordium of the air sac of the dorsal thorax (Cabernard, 2005).

It has been proposed that the air sac of the dorsal thorax forms de novo from a small group of wing imaginal disc cells, and that the resulting sac subsequently generates a tracheal lumen by an unknown process (Sato, 2002). Since, in the early Drosophila embryo, the lumen arises from an epithelial invagination via cell migration, it was asked whether the cells in the growing air sac are epithelial in nature with a clear apical/basal polarity. For this purpose, a Dα-Catenin-GFP (Dα-Cat-GFP) fusion construct was expressed in the developing air sac and the distribution of GFP from early to late third instar larvae was analyzed. Dα-Cat-GFP labels the adherens junctions (AJs) of epithelial cells. Clearly, the growth of the air sac was accompanied from the early stages onward by an out-bulging of an AJ network, suggesting that most or all of the cells in the growing bud were epithelial in nature, and that a luminal space was generated at the apical side of the epithelial tracheal cells during outgrowth. To confirm this interpretation, use was made of the recent identification of a protein, Piopio (Pio), which is apically secreted into the tracheal lumen in the embryo (Jazwinska, 2003). Indeed, the prospective luminal space in the outgrowing air sac is filled with Pio protein, demonstrating that the air sacs consist of a sac-like epithelial sheet, generating a luminal space as they grow (Cabernard, 2005).

To test whether all cells maintained an apical-basal polarity during air sac budding, single tracheal cells were labelled by using a recently developed assay system that allows for the visualization (and manipulation) of individual tracheal cells in vivo (Ribeiro, 2004). When this scenario was used in the presence of a UAS-Dα-Catenin-GFP chromosome, it was found that, in virtually all cases, such individually labeled air sac cells contacted the lumen and formed AJs with neighboring cells, even when these cells were located at the tip of the outgrowing air sac. The same conclusion was reached when the expression of GFP-moesin was analyzed in single air sac cells; cells at the tip made clear contact with the lumen. Therefore, it is concluded that the air sac is sculpted from an epithelial cell layer, which expands and at the same time generates an apical luminal space filled with secreted proteins (Cabernard, 2005).

It was of interest to better understand how Ras can be used in the same tissue at the same time for different cellular processes. egfr mutant cells can contribute to the tip of the growing air sac, although the clones are relatively small. In the stalk of the air sac, cells lacking Egfr often appeared fragmented, a sign of cell death. Indeed, when egfr mutant cells were sustained with anti-Drice, a marker for apoptotic cells, a strong accumulation of this protein was found. When p35, a viral antiapoptotic protein, was expressed in egfr mutant cell clones, these clones grew to larger sizes and were able to populate the air sac tip at a significantly higher frequency than in the absence of p35. These experiments establish that Egfr is dispensable for migration, and that migration is exclusively triggered by one of the two RTKs, Btl/Fgfr. The experiments also demonstrate that, during the growth phase, Btl/Fgfr signaling is dispensable for cell division; clones can grow to large sizes, although they fail to populate the tip. This same result was obtained with two other components, which are exclusively used by the Fgfr signaling pathway in the air sac (and not the Egfr pathway), namely, Dof and Pointed. Thus, migration and cell division are controlled by two different RTKs, but both RTKs signal via the activation of Ras and the Map kinase pathway to regulate these different cellular outcomes (Cabernard, 2005).

How does Ras control cell migration in the tip and cell division in the remaining air sac? To start to address these questions, whether high levels of constitutive active Ras were compatible with directional cell migration was tested and RasV12 was expressed in wild-type tissue in small cell clones. Interestingly, such clones expanded considerably and grew to large sizes in the center of the air sac or in the stalk, resulting in bulgy outgrowths; however, clones expressing RasV12 never contributed to the tip of the air sac. This finding suggests that unrestricted levels of Ras in a cell perturb its capacity to read out the migratory cues (presumably the Bnl/Fgf ligand); wild-type cells were apparently much better in taking up the leading position. In line with this interpretation, expression of an activated version of Btl (Torso-Btl/Fgfr) also resulted in bulky outgrowths. In addition, cells expressing the chimeric Btl receptor never populated the tip. Quite in contrast, activated Egfr (Egfr fused to a lambda dimerization site) was not able to perturb air sac guidance, but it also triggered higher division rates in clones, generating bulgy outgrowths (Cabernard, 2005).

To test whether single cells expressing activated receptor constructs changed their behavior with regard to cytoskeletal dynamics, the expression of either the activated version of Fgfr or Egfr was induced in early third instar stages and the behavior of such cells was analyzed with live imaging of cultured discs. Cells in the stalk of the air sac expressing activated Fgfr showed extremely dynamic cytoskeletal activity and formed large lamelipodia extending away from the air sac, similar to cells at the tip. Quite in contrast, cells expressing activated Egfr did not show increased lamelipodia formation, and their basal side remained relatively inactive (Cabernard, 2005).

Since the expression of constitutive active versions of the two different RTKs during air sac growth had different effects, whether the endogenous receptors activated the Ras/Mapk pathway to different levels in wild-type air sacs was investigated. In order to monitor the strength of Mapk signaling, an antibody recognizing the double-phosphorylated form of Erk, dpErk were used. Indeed, high levels of dpErk was detected in the nucleus of tip cells; lower dpErk levels were found in the cells in the center of the air sac, and dpErk was mostly cytoplasmic (Cabernard, 2005).

From all of the above-mentioned data, it is concluded that air sac development makes use of two distinct RTKs to control directed organ extension via cell migration (Fgfr) and organ growth via cell division (Egfr). This study carefully analyzed air sac outgrowth from early to late stages, by using a number of different markers labeling either membranes or AJs of individual air sac cells, or the apical luminal compartment. It was found that the thoracic air sac is modeled out of the existing tracheal epithelium, and that a luminal space is generated by the migration of a few cells away from the cuticle of the existing tracheal branch; the luminal space is then expanded by increasing the cell number in the sac-like epithelial structure via cell division. During this process, all cells remain within the epithelium and only round up when they divide. Even those cells that send out filopodia and lamelipodia and migrate in the direction of Bnl/Fgf remain embedded within the epithelium, contact the lumen, and form AJs with their neighbors. Thus, the directed outgrowth of the thoracic air sac during larval development is very similar to the budding of tracheal branches in the early embryo, in that epithelial cells form extensions from the basal side, ultimately resulting in cell movement toward the Fgf ligand. In contrast, during tubule formation of MDCK cells in culture, cells initially depolarize and migrate to form chain-like structures before they repolarize and form the luminal cavity; tubulogenesis is thus accompanied with partial epithelial-to-mesenchymal as well as mesenchymal-to-epithelial transitions. The tube-forming process has been subdivided into different stages such as cyst, extension, chain, cord, and tubule. In the case of the MDCK model system, growth factors have been proposed to trigger branching by inducing a dedifferentiation that allows the monolayer to be remodeled via cell extension and chain formation. Similar to the MDCK system, it was found in Drosophila that growth factor signaling induces the formation of cellular extensions, the first sign of outgrowth. Also, in both systems, cell division is an integral part of the process, but it occurs randomly throughout the structure and not locally at the point of outgrowth. However, two different RTKs are used in the air sac to control extension (migration) and cell division, and chain and chord stages are not observed. It thus appears that both similarities and differences exist between these different cellular systems (Cabernard, 2005).

It has already been reported that cells divide during air sac formation. The cell division rates have been semiquantified and it was found that the elongating structure does not grow preferentially at the tip. The genetic analysis demonstrates that the Egfr is essential for cells to divide and survive efficiently in the air sac. Egfr signals via Ras and the Mapk pathway, but it does not require the Pnt transcription factor to regulate cell division. It is not yet known which ligand activates Egfr, and whether expression of this ligand is induced at early stages of development by Fgf signaling. As shown before (Sato, 2002), the complete lack of Fgfr signaling results in the absence of air sacs; Fgf signaling might thus be used at the onset of the budding process to initiate or trigger cell division, but it is clearly dispensable in later stages. Since cells in the tracheal branch, which gives rise to the air sac primordium, also divide in the absence of Fgf signaling, it is possible that the role of Fgf signaling consists in generating an outgrowth via directed cell movement, triggering cell division indirectly (Cabernard, 2005).

Interestingly, a recent study addressing the role of GDNF/Ret signaling in kidney branching morphogenesis in vivo has shown that ret mutant cells (which are unable to respond to GDNF) can contribute to the primary outgrowth of the ureteric bud, but are excluded from the ampulla that forms at its tip. Apparently, a Ret-dependent proliferation of tip cells under the influence of GDNF controls branch outgrowth. This study found that in Drosophila, in the developing air sac, cells lacking Fgfr are also excluded from the tip. However, evidence is provided that Fgf signaling is translated into directed migration in the leading structure and not into a local increase in cell proliferation. The isolation and cultivation of wing imaginal discs allows for using 4D imaging to document cell behavior during air sac growth. It was found that numerous tip cells extend long filopodia and lamelipodia, similar to the findings reported earlier (Sato, 2002). Tip cells not only produce extensions, but they indeed change their respective position with time, and move forward over the substrate in the direction of the filopodia/lamelipodia. Thus, tip cells are clearly motile and migrate in the direction of Bnl/Fgf. Cell clones incapable of responding to different families of ligands were produced and marked and were examined with regard to their capacity to populate the air sac tip. Among the receptors analyzed, only Btl/Fgfr was strictly required for cells to populate the leading tip of the air sac. Considering the observation that cells in the tip actively migrate, that Btl/Fgfr signaling is required for tracheal cell migration in the embryo, that tracheal cells migrate to ectopic sources of Bnl/Fgf in the embryo and the larva (Sato, 2002), and that cells form numerous filopodia and lamelipodia upon constitutive activation of the Fgf signaling pathway, it is concluded that Fgf steers cell migration in the tip of the air sac and leads to its directional outgrowth on the surface of the wing imaginal disc. The demonstration that the MARCM system can be used to analyze gene function with regard to cell migration in the developing air sac prompted an investigation of the role of Ras and the Mapk pathway in Fgf-directed cell movement (Cabernard, 2005).

Using the MARCM system, it was found that Ras activation is essential for cells to migrate at the tip of the air sac. The requirement for Cnk and Ksr strongly suggests that one important branch downstream of Ras in the control of cell migration is the Mapk pathway. This interpretation is supported by the somewhat surprising finding that the transcription factor Pnt is also strictly required for cell migration. In the Drosophila embryo, genes regulated by Fgf signaling at the transcriptional level and essential for migration have not been identified so far; although both pnt itself and blistered/DSrf are targets of Fgf signaling with important functions in tracheal morphogenesis, they are not required for migration. One possible target of Fgf signaling in the dorsal air sac cells might be the btl/fgfr gene itself. Attempts were made to rescue the pointed defects in air sac development by supplementing a btl transgene under the control of UAS sequences. It was found that even when Btl/Fgfr is provided by the transgenes, pnt mutant clones do not reach the tip. A second gene that might have been a transcriptional target of Pointed is dof; however, it was found that Dof protein is still present in pnt mutant clones (Cabernard, 2005).

The results demonstrate that the outgrowth of the dorsal air sac along the underlying wing imaginal disc is controlled by Btl/Fgfr and Egfr. Fgf signaling is required for directional outgrowth via cell migration, and Egf signaling is required for organ size increase sustaining cell division/cell survival. Both signals use the Ras/Mapk pathway to elicit their cellular responses. To what extent these two pathways regulate different downstream targets is not known at present. However, this study shows that Pointed is only required downstream of Fgf signaling in the control of directed cell migration, and not downstream of Egf signaling in the control of cell division/survival. Since the activation of the Map kinase pathway is much stronger in the cells at the tip as compared to the cells in the central portion or in the stalk of the air sac (according to the levels of dpErk), it is thought that the local availability of the Bnl/Fgf ligand results in a local signaling peak. Egf signaling in more central and proximal cells does not result in a strong activation of the Map kinase pathway, yet this activation is apparently sufficient to control cell division and survival. The independent regulation of cell migration and cell division by two different RTKs might be even more important in later stages of dorsal air sac development, when the growing tip is yet farther away from the main body of the air sac. It will be interesting to find whether other growing branched tissues use similar mechanisms to uncouple directional expansion and size increase (Cabernard, 2005).

Slik and the receptor tyrosine kinase Breathless mediate localized activation of Moesin in terminal tracheal cells

A key element in the regulation of subcellular branching and tube morphogenesis of the Drosophila tracheal system is the organization of the actin cytoskeleton by the ERM protein Moesin. Activation of Moesin within specific subdomains of cells, critical for its interaction with actin, is a tightly controlled process and involves regulatory inputs from membrane proteins, kinases and phosphatases. The kinases that activate Moesin in tracheal cells are not known. This study shows that the Sterile-20 like kinase Slik, enriched at the luminal membrane, is necessary for the activation of Moesin at the luminal membrane and regulates branching and subcellular tube morphogenesis of terminal cells. The results reveal the FGF-receptor Breathless as an additional necessary cue for the activation of Moesin in terminal cells. Breathless-mediated activation of Moesin is independent of the canonical MAP kinase pathway (Ukken, 2014).

Sex-specific deployment of FGF signaling in Drosophila recruits mesodermal cells into the male genital imaginal disc

The temporal requirement for Breathless was dissected using a dominant-negative form of the receptor. Induction of the dominate negative receptor prior to the onset of tracheal or glial cell migration produced phenotypes that were similar to those observed in the corresponding tissues of breathless null mutant embryos. However, this effect is not detected if the dominant-negative receptor is induced after the initiation of tracheal cell migration, indicating that Breathless is required primarily at the onset of the migration process. Induction of the construct after the tracheal branches are completed, blocked the formation of tracheoles, i.e. extension of cellular processes by the terminal tracheal cells, demonstrating that Breathless plays an essential role in this later process as well (Reichman-Fried, 1995).

A central issue in developmental biology is how the deployment of generic signaling proteins produces diverse specific outcomes. Drosophila FGF is used, only in males, to recruit mesodermal cells expressing the FGF receptor to become part of the genital imaginal disc. Male-specific deployment of FGF signaling is controlled by the sex determination regulatory gene doublesex. The recruited mesodermal cells become epithelial and differentiate into parts of the internal genitalia. These results provide exceptions to two basic tenets of imaginal disc biology -- that imaginal disc cells are derived from the embryonic ectoderm and that they belong to either an anterior or posterior compartment. The recruited mesodermal cells migrate into the disc late in development and are neither anterior nor posterior (Ahmad, 2002).

The extensive sexual dimorphisms of the genitalia and analia suggest that the genital disc is relatively enriched in genes expressed downstream of dsx. To identify such genes, a random collection of enhancer traps was screened for sex-specific expression patterns in late third instar genital discs. Enhancer trap insertions in the bnl and btl genes were both isolated as enhancer traps expressed in male but not female genital discs. The sex specificity and the spatial patterns of expression of these enhancer traps accurately reflect the expression of the bnl and btl genes in the genital disc. Of the three primordia that comprise the genital disc, bnl and btl are both expressed in only one: the A9-derived developing 'male' primordium. bnl and btl are also expressed in adjacent domains: bnl is expressed at the base of two bilateral bowl-like infoldings of the disc epithelium, while btl is expressed in a group of loosely packed cells that fills these bowls and extends over the anterior and ventral surfaces of the disc (Ahmad, 2002).

The juxtaposition of btl- and bnl-expressing cells suggested that their proximity to one another might be the result of FGF-mediated cell-cell signaling. The locations of btl-expressing cells in male genital discs were determined at different stages of larval development. At early third instar (70-75 hr after egg laying), while a few btl-expressing cells are associated with the external surface of the disc, none are detected inside the disc. In mid-third instar (89-99 hr AEL), the btl-expressing cells are lying on the external surface of the disc, as well as adjacent to, and filling shallow invaginations in the disc epithelium. And by late third instar (110-120 hr AEL), these invaginations have become much deeper and are completely filled by btl-expressing cells. Thus, these btl-expressing cells are not originally a part of the disc epithelium but are recruited into invaginations in the epithelium during the third instar. Unlike the disc epithelium, the btl-expressing cells in the third instar disc do not express escargot (esg), a classical marker for ectoderm-derived imaginal cells, indicating that the btl-expressing cells have a different origin than do the other cells of the disc. The btl-expressing cells are, in fact, mesodermal in origin and derived from the adepithelial cells associated with the genital disc (Ahmad, 2002).

A priori, there are two possible explanations for the male-specific expression of FGF. One possibility is that bnl is an A9-specific gene, being expressed only in males where the A9-derived primordium grows significantly. The other possibility is that bnl is a target of the sex determination hierarchy, being either repressed by the female-specific Dsx protein (DsxF) in females and/or activated by the male-specific Dsx protein (DsxM) in males. To distinguish between these possibilities, feminized (Tra protein-expressing) clones of cells were generated in the A9-derived primordium of wild-type male genital discs and the effects of these clones on bnl expression were examined. Whenever feminized clones overlapped domains of bnl expression, the expression of bnl was repressed, indicating that it is cell-autonomous regulation by the sex determination hierarchy that is responsible for the male-specific expression of bnl in the genital disc (Ahmad, 2002).

When a feminized clone completely eliminated bnl expression from one side of a male disc, the lobe lacking bnl expression looked flattened. This was a consequence of btl-expressing cells not migrating into this lobe in the absence of Bnl protein, showing that bnl expression is not simply sufficient, but also necessary for the recruitment of btl-expressing cells. This observation suggests that btl, unlike bnl, is not a target of the sex determination hierarchy, and that the male-specific presence of btl-expressing cells in the genital disc is solely a consequence of Bnl recruiting the btl-expressing cells (Ahmad, 2002).

To examine how dsx regulates bnl expression, bnl expression was examined in wild-type genital discs and discs lacking dsx function. bnl is expressed in the A9-derived primordium of a wild-type male disc, where DsxM is present, but is not expressed in the A8-derived primordium of a wild-type female disc, where DsxF is expressed. However, in a disc in which neither Dsx protein is expressed, both the A8 and A9 primordia proliferate and bnl expression is seen in both primordia. That the A8 primordium grows in both wild-type and dsx mutant females but bnl is expressed in the A8 primordium only when the DsxF protein is absent, implies that bnl expression is repressed in the female genital disc by the presence of DsxF protein (Ahmad, 2002).

The ectopic expression of bnl in the A8-derived 'female' primordia of discs lacking dsx function offers an explanation for a puzzling observation: while wild-type males have only two paragonia (mesodermally derived components of the male disc), dsx mutant flies often have as many as four paragonia-like structures. The finding that the ectopic expression of bnl in flies mutant for dsx results in btl-expressing cells from the ventral surface of the disc being recruited into two ectopic invaginating pockets in the A8-derived female primordium of the disc, in addition to the original bowls in the A9-derived primordium, suggests that these ectopic pockets of btl-expressing cells give rise to the supernumerary paragonia when taken together with the observation that the extra paragonia in dsx mutants arise from the female primordium (Ahmad, 2002).

It is concluded that the sex-specific deployment bnl in the genital disc depends on the sex of the individual bnl-expressing cells. Given that bnl is regulated cell autonomously by DsxF, an obvious question is whether the DsxF protein directly represses bnl. In this regard, it is noted that 0.7 kb and 1.6 kb upstream of the putative bnl transcriptional start site, there are clusters of 5 and 4 sites respectively with at most a 1 bp mismatch to the 13 bp consensus Dsx binding site sequence. This is reminiscent of the 3 Dsx binding sites in a 76 bp stretch of an enhancer for the Yolk protein (Yp) genes, the only known direct targets of dsx (Ahmad, 2002).

The Drosophila sex determination hierarchy acts at multiple levels to control sexual differentiation. Some terminal differentiation genes like the Yp genes are direct transcriptional targets of the Dsx proteins and are continuously subject to their regulation. In other cases, the direct targets of dsx appear to be genes involved in initiating the differentiation of sex-specific tissues; genes expressed subsequently in these sex-specific tissues are governed by a tissue differentiation program, rather than being directly controlled by the sex hierarchy. It seems likely that the targets through which dsx initiates formation of such sex-specific tissues will be the genes where information from several developmental hierarchies are integrated to direct the differentiation of tissues (Ahmad, 2002).

These results suggest that bnl is one of the genes used by the sex determination hierarchy to direct the construction of sex-specific tissues. Bnl recruits btl-expressing cells into the male genital disc, and the recruited cells eventually form the paragonia and vas deferens (another mesodermally derived gonadal organ), tissues that are present only in males. Moreover, three genes expressed in the paragonia, the male-specific transcripts (msts) 316, 355a, and 355b, have been shown to be regulated in a tissue-specific rather than sex-specific manner: while transcription of these three male-specific RNAs begins in the late pupal period, their expression is governed by the sex hierarchy acting earlier, during the third larval instar -- the period when the expression of bnl recruits the paragonia-forming btl-expressing cells into the male genital disc. Thus, the sex-specific expression of the msts is achieved by dsx acting through bnl to generate the sex-specific tissue, the paragonia, in which the msts are subsequently expressed.

bnl also appears to be a gene where information from other regulatory hierarchies and the sex determination hierarchy are integrated in the male genital disc. The genetic hierarchies that control pattern formation and confer positional identity in the thoracic imaginal discs have previously been shown to function analogously in the genital disc. The fact that the bnl expression domain is limited to two specific subsets of the ectoderm-derived disc epithelia in males implies that bnl is also regulated by these pattern formation hierarchies. One area of future exploration will be examining how this coordinated regulation of bnl by dsx and the genes involved in pattern formation is brought about (Ahmad, 2002).

An intriguing aspect of these findings is the gradual transition of the btl-expressing cells, upon recruitment into the male genital disc, from twi-expressing mesodermal cells to epithelial cells with septate junctions. It is not clear if this transformation is also a consequence of FGF signaling, or if it is brought about by a different process. However, three separate observations suggest a role for bnl and btl in this mesoderm-epithelial transition: (1) FGF signaling mediates this process in mice -- during kidney development, FGF2 and leukemia inhibiting factor (LIF) secreted from the epithelial ureteric bud induce the conversion of the undifferentiated mesoderm-derived metanephric mesenchyme to the epithelial tubular structures of the nephron; (2) the converse process can also be mediated by FGF signaling -- FGFR1 regulates the morphogenetic movement and cell fate specification events during gastrulation in mice; it orchestrates the epithelial to mesenchymal transition during morphogenesis at the primitive streak and specifies the mesodermal cell fate of these mesenchymal cells, and (3) stumps, a gene acting downstream of the FGFR-encoding btl, has its expression elevated in the btl-expressing cells undergoing the transition into epithelial cells in the genital disc (Ahmad, 2002 and references therein).

Finally, it is noted that there are striking parallels between the roles of the FGF in sexual differentiation in the fly and FGF9 in sexual differentiation in mice. FGF9 is required for testicular embryogenesis in mice, and in its absence, XY mice undergo male-to-female sex reversal. FGF9 is expressed in the early embryonic gonads of male mice, not in the gonads of female mice, and not in the mesonephros of either sex, while bnl is expressed in the male genital disc, not in the female genital disc, and not in the btl-expressing mesodermal cells that are recruited into the male disc. The mesonephric cells migrate into only the male gonads, and the btl-expressing cells are recruited only into the male genital disc. Exogenous FGF9 induces mesonephric cell migration into female gonads, while ectopic expression of bnl is sufficient to recruit the btl-expressing cells into the female primordium of a dsx disc. The btl-expressing cells are mesodermal in origin, eventually undergo a transition into epithelial cells, and give rise to the vascular paragonia and vas deferens. The mesonephros, too, is derived from the mesoderm, and mesonephric cell migration into the testis contributes to the vascular endothelial, myoepithelial, and peritubular myoid cell populations. Given that there is considerable variation in the earlier aspects of sex determination across species, these findings suggest a possible conserved role for FGF signaling in later aspects of sexual differentiation (Ahmad, 2002 and references therein).

FGF is an essential mitogen and chemoattractant for the air sacs of the Drosophila tracheal system

The Drosophila adult has a complex tracheal system that forms during the pupal period. The derivation of part of this sytem, the air sacs of the dorsal thorax, has been studied. During the third larval instar, air sac precursor cells bud from a tracheal branch in response to FGF, and then they proliferate and migrate to the adepithelial layer of the wing imaginal disc. In addition, FGF induces these air sac precursors to extend cytoneme-like filopodia to FGF-expressing cells. These findings provide evidence that FGF is a mitogen in Drosophila; they correlate growth factor signaling with filopodial contact between signaling and responding cells, and suggest that FGF can act on differentiated tracheal cells to induce a novel behavior and role (Sato, 2002).

Metamorphosis presents special challenges to the tracheal system. The process that transforms the Drosophila larva into an adult fly consumes larval tissues and creates new organs using imaginal cells that were prepared during larval development. A new tracheal system that will satisfy the aerobic requirements of the specialized tissues of the adult must be built. But, in addition, the organs of the pupa must be kept oxygenated while the adult develops. Transformation of the tracheal system begins during the third larval instar, when imaginal tracheoblasts start to divide. These proliferating tracheoblasts spread over the larval tracheal system, using it as a scaffold to form an extensive branching network before the larval cells histolyze. Some tracheoblasts elaborate coiled structures that are unique to the pupa. Others grow to form the air sacs of the adult. Air sacs are large reservoirs that are juxtaposed with major muscle systems and with the brain. These structures have been thought to form as dilations of the main tracheal trunks, which are the direct descendants of the main tracheal trunks of the embryo and larva. The work described in this study shows that in the dorsal thorax these air sacs originate independently from a distinct population of cells (Sato, 2002).

The majority of the adult thorax, including most of the dorsal thoracic epidermis, the wing, and flight muscle, is produced by the wing imaginal disc. This organ arises as a tubular invagination of the epidermis, and when it grows and flattens during the larval periods, it develops four distinct cell types. It has squamous peripodial cells on one surface, columnar epithelial cells on the other surface, a distinct group of adepithelial cells that nestle against the most proximal columnar epithelial cells, and stalk cells that connect the disc to the epidermis. A large tracheal branch attaches in some manner to the columnar epithelial surface. It orients along the dorsal/ventral axis of the disc, but it does not ramify to generate multiple contacts with the disc cells. Therefore, no framework exists to serve as a template for the tracheolar network that provides oxygen to the thoracic cells of the adult. The mechanisms responsible for the branch formation and path finding that produce the extensive and complex tracheolar network in the adult thorax remain to be identified (Sato, 2002).

The work described in this study represents an effort to understand the role of FGF in wing disc development. These investigations led to the identification of a new cell type in the wing disc that migrates and proliferates in response to FGF. These cells contact FGF-expressing cells across at least one cell layer by extending long cytoneme-like filopodia. These cells are destined to form the air sacs that associate with the flight muscles in the adult thorax, but they are distinct from the cells that form the larval trachea or from the group of imaginal precursors that are programmed to generate tracheae in the pupa and adult (Sato, 2002).

To investigate the role of FGF in Drosophila wing disc development, patterns of expression of bnl and the two FGF receptor genes, breathless (btl) and heartless (htl), were examined in third instar discs. bnl/FGF expression is restricted to a small group of cells in the columnar epithelium. An exact count of their number was problematic: since the apparent level of expression in many cells is very low, their number is roughly estimated to be between 15 and 60 in early third instar discs and between 80 and 150 in late third instar discs. These cells straddle the anterior/posterior compartment border and are dorsal to the region of the prospective wing blade. The cells that express bnl most strongly are ventral to the progenitor of the aPA machrochaete, as indicated by double staining for Achaete protein. They are in a region that contributes to the notal wing processes: the cuticle located between the adult scutum and wing hinge (Sato, 2002).

Expression of btl and Htl is restricted to cells in the adepithelial layer of the wing disc and is absent from the cells of the columnar epithelium. The adepithelial cells give rise to the adult musculature; they also express twist, which controls htl and is a signature of all mesodermal cells and muscle precursors. It was confirmed that the wing disc adepithelial cells express both Htl and Twist. In addition, a small group of adepithelial cells was identified that expresses a btl enhancer trap line but expressed neither Htl nor Twist. Stumps, a putative adapter protein required for both Btl and Htl signaling, is expressed in both btl- and Htl-expressing adepithelial cells (Sato, 2002).

The btl-expressing cells in the adepithelial layer had not been identified previously, and their presence was unexpected, since one of the principal domains of btl expression is the trachea. Tracheal cells are almost invariably associated with tubules with cuticle-lined lumen, and the adepithelial cells have no such distinct structures. Nevertheless, the adepithelial btl-positive cells appear to maintain continuity with the cells of the main tracheal branch. Evidence is presented that the btl-expressing adepithelial cells are the precursors of the adult tracheal air sacs (Sato, 2002).

To better understand the origin and fate of the btl-expressing adepithelial cells, they were tracked during larval and pupal development. In early third instar wing discs, no btl expression was detected in adepithelial cells; only tracheal branch cells are btl positive. However, as third instar discs mature, btl-positive cells are detected budding from the tracheal branch that adheres to the wing disc. This bud forms at a stereotypical position just dorsal to the wing hinge progenitors and adjacent to the group of 15–60 bnl-expressing cells in the columnar epithelium. During development of the third instar, the number of btl-expressing cells increase, and the bud expands posteriorly toward the region of greatest bnl expression. In late third instar discs, the btl-expressing cells form a coherent group surrounded by Htl-expressing cells. These btl-expressing cells do not express Htl. Possible explanations for the complementarity of the patterns of btl and Htl expression are that btl cells displace Htl-expressing cells or that the expression of these genes is mutually exclusive. However, in the early third instar discs, Htl expression is already absent from the region that will be occupied by btl-expressing cells in older discs; therefore, these are not likely to be sufficient explanations (Sato, 2002).

To characterize these cells further, their relationship to the larval tracheal system was examined. The larval tracheal branch that adheres to the wing disc is called the first transverse connective. It has a small offshoot called the spiracular branch, where imaginal tracheoblasts, precursors of the adult tracheae, are located. These imaginal tracheoblasts do not express btl but do express escargot (esg) and trachealess (trh). Since esg inhibits endoreplication of imaginal cells and the imaginal tracheoblasts are assumed to be diploid, expression of esg in these cells was not unexpected. The presence of Trh, a transcription factor that directly activates btl transcription, may presage btl expression at a subsequent stage. The btl-expressing adepthelial cells also express Trh. They do not express esg early in the third instar; therefore, it is unlikely that they derive from the imaginal tracheoblasts. However, in mid third instar and thereafter, esg expression is evident in the most posterior cells of the group (Sato, 2002).

The capacity of tracheal branch cells to proliferate was unexpected because the cells lining the main tracheal branches had been considered to be both terminally differentiated and polyploid. Neither state is expected to be compatible with a mitotic cell cycle program. The morphology and fluorescence of DAPI-stained discs was examined to better understand the nature of the disc-associated tracheal cells. Most of the cells that line the lumen of the main tracheal branch, the first transverse connective, as well as the imaginal tracheoblasts, have nuclei that are similar in diameter (5–6 µm), are relatively small, and have a similar level of fluorescence. Some cells with large nuclei and >3× the fluorescence intensity of the smaller nuclei were observed populating other branches that connect to the first transverse connective. Assuming that the imaginal tracheoblasts are diploid, these observations suggest that the cells that respond to Bnl-FGF have a similar ploidy and that the tracheal branches that associate with the wing disc include both diploid and polyploid cells (Sato, 2002).

To determine the fate of the btl-positive adepithelial cells, their behavior and movements were examined throughout pupal development. They are located next to the posterior part of the prospective wing hinge in the late third instar disc. Analysis of fixed wing discs prepared at various times during the first 12 hr after puparium formation (APF) revealed that they remain in this location as a tight, rounded cluster of cells next to the prospective wing hinge (Sato, 2002).

To continue following the fate of these cells, five individual pupae that expressed btl-Gal4 UAS-GFP were observed during the pupal period, and photographs were taken at regular intervals. The pupal case is transparent to the GFP fluorescence; therefore, no surgical manipulations were necessary. The btl-positive adepithelial cells were identified by their proximity to the wing hinge at 12 hr APF, consistent with the observations of dissected, fixed discs. These btl-positive cells migrate dorsally between 12 and 23 hr APF then anteriorly and posteriorly to form three branches. At 32 hr APF, they cease their migrations and began to elaborate into air sacs. It is concluded that the btl-positive adepithelial cells are the precursors of the adult air sacs and that the air sacs of the adult thorax are derived from cells that are distinct from the imaginal tracheoblasts (Sato, 2002).

Although the air sac tracheoblasts do not form a tubular structure at 12 hr APF, these pupae do have numerous tracheae that project to the developing muscles. These tracheae are clearly distinct from the air sac tracheoblasts. They derive from the second dorsal branch and are present only during the pupal period. Air sacs are prominent and extensive in older pupae and in adults and are associated with numerous bundles of tracheae that extend from the air sacs and extensively interdigitate with flight muscle cells. At present, the structure of the air sacs is not understood enough to know how these tracheae either connect with or contribute to the function of the air sacs (Sato, 2002).

Thus, bnl/FGF expression is detected in a small group of columnar epithelial cells during the third instar and pupal periods. Although their fate in the adult has not been established, these cells are small in number and therefore cannot produce more than a small part of the adult cuticle. Nevertheless, their effect on the adult is profound. Through the action of Bnl-FGF, they induce a group of tracheal cells to initiate a program of proliferation and migration and to join with the cells in the disc adepithelial layer. Despite this intimate association with these mesodermal progenitors, the FGF-responsive cells retain their tracheal identity and go on to form the prominent adult tracheal air sacs that extend throughout much of the dorsal thorax (Sato, 2002).

The tracheal system of the Drosophila embryo has ten interconnected metameric units on either side of the animal; one unit derives from the second, or mesothoracic, segment. This mesothoracic component consists of portions of the dorsal and lateral trunks, a transverse connective that links these trunks to each other, a dorsal branch that connects the left and right sides, and numerous branches that radiate out to various tissues. During larval development, this general structure is retained, and, although the tracheal cells do not divide, many new branches form, and the diameter of the more proximal tracheae increase. The wing imaginal disc attaches to the transverse connective. Imaginal tracheoblast precursors of the adult tracheae populate a small spiracular branch at a location just dorsal to the disc attachment (Sato, 2002).

In constructing the adult tracheal system, the imaginal tracheoblasts use parts of the larval framework as templates and, in effect, remodel the dorsal and lateral trunks, the transverse connective, the dorsal branch, and the main pupal branches to the wing and leg. In contrast, the large and extensive air sacs do not correspond to earlier branches in any obvious way and have no apparent antecedent. The airs sacs of the dorsal thorax form de novo from a small group of wing imaginal disc cells. This study chronicles the transformation of these air sac tracheoblasts from a tight cluster of adepithelial disc cells to sculpted air sacs. These observations were made by expressing GFP under btl control and by following the GFP-containing cells through the pupal period. It was possible to directly account for three branches of the dorsal thoracic air sacs (e.g. the medioscutal, lateroscutal, and scutellar sacs) as products of the wing disc air sac tracheoblasts. All of the air sacs in the notum appeared to contain GFP in these animals, but the resolution of the analysis was not sufficient to make this a definitive conclusion nor did it allow for a conclusion that all of the air sacs derive from disc tracheoblasts. Nevertheless, this study did establish that the disc tracheoblasts generate air sacs and, by some process that is as yet unknown, form a tracheal lumen and tracheal network. It will be interesting to identify the intrinsic and extrinsic systems that direct the genesis of the air sacs, since they apparently develop in the absence of a preexisting framework (Sato, 2002).

Evidence is presented for air sac tracheoblasts in the wing imaginal disc. Data suggest that there may be similar strategies to make air sacs in other regions of the fly. This statement is based on the presence of nontracheal btl-positive cells and bnl-expressing cells in other imaginal discs. In leg discs, bnl expression is found in the stalk region, and the pattern of expression becomes more extensive and complex in the disc epithelium of pupal discs. In third instar discs, btl-positive cells are localized to an offshoot of a tracheal branch that attaches near the stalk and adjacent to the bnl-expressing cells. After puparium formation, the btl-positive cells migrate along the basal surface of the disc columnar epithelium to a position that roughly correlates with the region where air sacs will later form. In eye-antenna discs, bnl is expressed in cells surrounding the ocelli progenitors. Although btl- expressing cells were not found in larval eye-antenna discs, it was observed that in early pupae btl-positive cells assume a position underlying the presumptive ocelli cells. Air sacs in the adult head underlie most of the medial head cuticle and encircle the region where the ocelli form. These observations led to the suggestion that the process that induces the air sac tracheoblasts in the wing disc may be common to other discs as well (Sato, 2002).

A signaling network for patterning of neuronal connectivity in the Drosophila brain

The precise number and pattern of axonal connections generated during brain development regulates animal behavior. Therefore, understanding how developmental signals interact to regulate axonal extension and retraction to achieve precise neuronal connectivity is a fundamental goal of neurobiology. This question was investigated in the developing adult brain of Drosophila. Extension and retraction is regulated by crosstalk between Wnt, fibroblast growth factor (FGF) receptor, and Jun N-terminal kinase (JNK) signaling, but independent of neuronal activity. The Rac1 GTPase integrates a Wnt-Frizzled-Disheveled axon-stabilizing signal and a Branchless (FGF)-Breathless (FGF receptor) axon-retracting signal to modulate JNK activity. JNK activity is necessary and sufficient for axon extension, whereas the antagonistic Wnt and FGF signals act to balance the extension and retraction required for the generation of the precise wiring pattern (Srahna, 2006).

Based on the observation that blocking Fz2 results in decreased numbers of dorsal cluster neuron (DCN) axons in the medulla, it was reasoned that Fz2 could be a receptor for a putative stabilization signal. Since Fz2 and Fz are partially redundant receptors for the canonical Wnt signaling pathway, expression of the canonical Wnt ligand Wingless (Wg) was investigated in the brain during pupation. However, no Wg expression was detected in the pupal optic lobes, suggesting that Wg is unlikely to be involved in regulating DCN axon extension. Therefore, the expression of Wnt5, which has been shown to be involved in axon repulsion and fasciculation in the embryonic CNS, was investigated. Anti-Wnt5 staining revealed widely distributed Wnt5 expression domains beginning at PF and lasting throughout pupal development and into adult life. Wnt5 is strongly expressed in the distal medulla and is also present on axonal bundles crossing the second optic chiasm.The number of DCN axons crossing to the medulla was examined in wnt5 mutant flies. The number of DCN axons crossing the optic chiasm is reduced from 11.7 to 7.9 in the absence of wnt5, suggesting that it may play a role in stabilizing DCN axons (Srahna, 2006).

Next, the requirement of the Wnt signaling adaptor protein Dsh was tested. In animals heterozygous for dsh6, a null allele of dsh, the average number of DCN axons crossing between the lobula and the medulla is reduced from 11.7 to 7.6 with 78.5% showing less than eight axons crossing. Signaling through Dsh is mediated by one of two domains. Signaling via the DIX (Disheveled and Axin) domain is thought to result in the activation of Armadillo/β-Catenin. DEP (Disheveled, Egl-10, Pleckstrin) domain-dependent signaling results in activation of the JNK signaling pathway by regulation of Rho family GTPase proteins during, for example, convergent extension movements in vertebrates. To uncover which of these two pathways is required for DCN axon extension the dsh1 mutant, deficient only in the activity of the DEP domain, was tested. Indeed, in brains from dsh1 heterozygous animals the number of extending axons was reduced from 11.7 to 7.4. In flies homozygous for the dsh1 allele the average number of axons crossing was further reduced to 4.7, with all the samples having less than six axons crossing. In contrast, the DCN-specific expression of Axin, a physiological inhibitor of the Wnt canonical pathway, did not affect the extension of DCN axons. Similarly, expression of a constitutively active form of the fly β-Catenin Armadillo also had no apparent effect on DCN extension. Finally, whether Wnt5 and Dsh interact synergistically was tested. To this end, wnt5, dsh1 trans-heterozygous animals were generated. These flies show the same phenotype as flies homozygous for dsh1, suggesting that Wnt5 signals through the Dsh DEP domain (Srahna, 2006).

To determine if dsh is expressed at times and places suggested by its genetic requirement in DCN axon outgrowth, the distribution of Dsh protein during brain development was examined. Dsh protein is ubiquitously expressed during brain development. High expression of Dsh is detected in the distal ends of DCN axons at about 15% PF shortly before they extend across the optic chiasm toward the medulla. In general, higher levels of Dsh were observed in the neuropil than in cell bodies (Srahna, 2006).

In summary, these data indicate that the stabilization of DCN axons is dependent on the Dsh protein acting non-canonically via its DEP domain. Importantly, the axons that do cross in dsh mutant brains do so along the correct paths. This suggests that, like JNK signaling, Wnt signaling regulates extension, but not guidance, of the DCN axons (Srahna, 2006).

Wnt signaling to Dsh requires the Fz receptors. To examine if the effect of Wnt5 on DCN axon extension is also mediated by Fz receptors, the number of DCN axons crossing the optic chiasm in was counted fz, fz2, and fz3 mutants. There was no significant change in the number of axons crossing in the brain of fz3 homozygous animals. In contrast, in brains heterozygous for fz and fz2, the number of the axons crossing was reduced from 11.7 to 6.6 (fz) and 6.9 (fz2), with 71% and 85.7%, respectively, showing less than eight axons crossing. These data suggest that DCN axons respond to Wnt5 using the Fz and Fz2 receptors, but not Fz3. To determine whether the Fz receptors act cell-autonomously in individual DCNs, single-cell clones doubly mutant for fz and fz2 were generated and the number of DCN axons crossing the optic chiasm was counted. In contrast to wild-type cells, where 37% of all DCN axons cross, none of the fz, fz2 mutant axons reach the medulla. To test whether wnt5, fz, and fz2 genetically interact in DCNs, flies trans-heterozygous for wnt5 and both receptors were examined. Flies heterozygous for both wnt5 and fz mutations show a strong synergistic loss of DCN axons (11.7 to 3.7) and in fact have a phenotype very similar to that of flies homozygous for dsh1. Flies doubly heterozygous for wnt5 and fz2 also show a significant decrease in DCN axons (5.7), compared with either wnt5 (~8) or fz2 (8.5) mutants. These data indicate that the genetic interaction between wnt5 and fz is stronger than the interaction between wnt5 and fz2 (Srahna, 2006).

Examination of the expression domains of Fz and Fz2 in the developing brain supports the possibility that they play roles in stabilizing DCN axons. Both Fz and Fz2 are widely expressed in the developing adult brain neuropil. In addition, Fz is expressed at higher levels in DCN cell bodies (Srahna, 2006).

The observation that the wnt5 null phenotype can be enhanced by reduction of Fz, Fz2, or Dsh suggests that another Wnt may be partially compensating for the loss of Wnt5. To test this possibility, flies heterozygous for either wnt2 or wnt4 were examined. wnt2 heterozygotes display a reduction of DCN axon crossing from 11.7 to 7.3, whereas no phenotype was observed for wnt4. Thus, wnt2 and wnt5 may act together to stabilize the subset of DCN axons that do not retract during development. In summary, these results support the model that Wnt signaling via the Fz receptors transmits a non-canonical signal through Dsh resulting in the stabilization of a subset of DCN axons (Srahna, 2006).

Data is provided that supports the hypothesis that the regulation of JNK by Rac1 modulates DCN axon extension. As such attempts were made to determine how Wnt signaling might interact with Rac1 and JNK. The opposite phenotypes of dsh and Rac1 loss-of-function suggest that they might act antagonistically. To determine if Rac1 is acting upstream of, downstream of, or in parallel to Dsh in DCN axon extension, dominant-negative Rac1 was expressed in dsh1 mutant flies. If Rac1 acts upstream of Dsh, the dsh1 phenotype (i.e., decreased numbers of axons crossing the optic chiasm) is expected. If Rac1 acts downstream of Dsh, the Rac1 mutant phenotype (i.e., increased number of axons crossing) would be expected If they act in parallel, an intermediate, relatively normal phenotype is expected. Increased numbers of axon crossing were observed, suggesting that Rac1 acts downstream of Dsh during DCN axon extension and that Dsh may repress Rac1 (Srahna, 2006).

Next, whether Dsh control of DCN axon extension is mediated by the JNK signaling pathway acting downstream of Wnt signaling was tested, as the similarity of their phenotypes suggests. If this were the case, activating JNK signaling should suppress the reduction in Dsh levels. Conversely, reducing both should show a synergistic effect. Therefore the JNKK hep was expressed in dsh1 heterozygous flies and it was found that the hep gain-of-function is epistatic to dsh loss-of-function. Furthermore, reducing JNK activity by one copy of BSK-DN in dsh1 mutant animals results in a synergistic reduction of extension to an average of 0.8 axons with 60% showing no axons crossing and no samples with more than three axons. In summary, the results of genetic analyses suggest that Wnt signaling via Dsh enhances JNK activity through the suppression of Rac1 (Srahna, 2006).

Dsh appears to promote JNK signaling and to be expressed in DCN axons prior to their extension toward the medulla early in pupal development. Since JNK signaling is required for this initial extension, it may be that Dsh also plays a role in the early extension of DCN axons. To test this possibility, DCN axon extension was examined at 30% pupal development in dsh1 mutant brains. In wild-type pupae, essentially all (~40) DCN axons extend toward the medulla. In contrast, in dsh1 mutant pupae, a strong reduction in the number of DCN axons crossing the optic chiasm between the lobula and the medulla was observed (Srahna, 2006).

Although the genetic data indicate that Dsh- and Rac-mediated signaling have sensitive and antagonistic effects on the JNK pathway, they do not establish whether the Dsh-Rac interaction modulates JNK's intrinsic activity. To test this, the amount of phosphorylated JNK relative to total JNK levels in fly brains was evaluated by Western blot analysis using phospho-JNK (P-JNK) and pan-JNK specific antibodies. Then it was determined if Dsh is indeed required for increased levels of JNK phosphorylation. Dsh1 mutant brains showed a 25% reduction in P-JNK consistent with a stimulatory role for Dsh on JNK signaling. The reduction caused by loss of Dsh function is reversed, when the amount of Rac is reduced by half, consistent with a negative effect of Rac on JNK signaling downstream of Dsh. These data support the conclusion that Dsh and Rac interact to regulate JNK signaling by modulating the phosphorylated active pool of JNK (Srahna, 2006).

Taken together, these data suggest that during brain development DCN axons extend under the influence of JNK signaling. A non-canonical Wnt signal acting via Fz and Dsh ensures that JNK signaling remains active by attenuating Rac activity. In contrast, activation of the FGFR activates Rac1 and suppresses JNK signaling. These data support a model whereby the balance of the Wnt and FGF signals is responsible for determining the number of DCN axons that stably cross the optic chiasm. To test this model, FGFR levels were reduced, using the dominant-negative btl transgene, in dsh1 heterozygous flies. It was found that simultaneous reduction of FGF and Wnt signaling restored the number of axons crossing the optic chiasm to almost wild-type levels (10.2, with 33% of the samples indistinguishable from wild-type, suggesting that the two signals in parallel, act to control the patterning of DCN axon connectivity (Srahna, 2006).

These data suggest the following model of DCN axon extension and retraction. DCN axons extend due to active JNK signal. These axons encounter Wnt5 and probably Wnt2 as well, resulting in activation of Disheveled. Disheveled, via its DEP domain, has a negative effect on the activity of the Rac GTPase, thus keeping JNK signaling active. After DCN axons cross the second optic chiasm they encounter a spatially regulated FGF/Branchless signal that activates the FGFR/Breathless pathway. Breathless in turn activates Rac, which inhibits JNK signaling in a subset of axons. These axons then retract back toward the lobula. The wide expression of the different components of these pathways and the modulation of JNK phosphorylation by Dsh and Rac in whole-head extracts strongly suggests that this model may apply to many neuronal types (Srahna, 2006).

Effects of Mutation or Deletion

In embryos homozygous for a deletion of the breathless locus, the initial formation of the tracheal pits is not affected. However, the extension of tracheal cell processes leading to the formation of the elaborate tree structure is blocked (Glazer, 1991).

breathless mutants exhibit defects in the two embryonic tissues in which the receptor is expressed: the tracheal system and the midline. The tracheal cells fail to migrate in severe mutants and remain within the tracheal pits. Hypomorphic alleles exhibit partial migration of all tracheal branches. In the midline of the mutant embryos, the posterior pair of midline glial cells begin to migrate anteriorly, but fail to reach the posterior commissure. Abnormalities in cell migration appear to be a common denominator for the btl defects in these two disparate tissues. Irregularities in commissures appear late in development (Klämbt, 1992).

A deregulated Breathless receptor was constructed containing the extracellular and transmembrane regions of the torso dominant allele and the cytoplasmic domain of breathless. Ubiquitous expression of the chimeric receptor at the time of tracheal cell migration did not disrupt migration in wild-type embryos. However, induction of the chimeric receptor corrected the tracheal defects of breathless mutant embryos, allowing the tracheal cells to migrate along their normal tracts. This result indicates that the normal activity of breathless in promoting cell migration does not require spatially restricted cues mediated by the extracellular domain. Deregulated receptors containing the cytoplasmic domains of DFGF-R2 (DFR1), EGF-R, Torso, and Sevenless are all able to partially rescue the migration defects. Constructs containing the activated downstream elements Dras1 and Draf were also able to rescue tracheal migration, demonstrating that these two proteins are key elements in the breathless signaling pathway (Reichman-Fried, 1994).

Although tracheal branching is sequential it is not iterative. The three levels of branching that can be distinguished involve different cellular mechanisms of tube formation. Primary branches are multicellular tubes that arise by cell migration and intercalation; secondary branches are unicellular tubes formed by individual tracheal cells; terminal branches are subcellular tubes formed within long cytoplasmic extensions. Each level of branching is accompanied by expression of a different set of enhancer trap markers. These sets of markers are sequentially activated in progressively restricted domains and ultimately individual tracheal cells that are actively forming new branches. A clonal analysis demonstrates that branching fates are not assigned to tracheal cells until after cell division ceases and branching begins. The Breathless receptor, a tracheal gene required for primary branching, is also required to activate expression of markers involved in secondary branching. Pointed, an ETS-domain transcription factor, is required for secondary branching and also to activate expression of terminal branch markers. The combined morphological, marker expression and genetic data support a model in which successive branching events are mechanistically and genetically distinct but coupled through the action of a tracheal gene regulatory hierarchy (Samakovlis, 1996).

A constitutively active Breathless receptor was prepared by forming a composite BTL receptor containing the dimerization domain of lambda repressor and the transmembrane and cytoplasmic domains of BTL. Since activation of receptor tyrosine kinases depends on ligand-induced dimerization followed by autophosphorylation, it is expected that spontaneously dimerizing receptor would be constitutively active. Constitutively active btl (lambda-BTL) fails to rescue tracheal cell migration defects and fails to restore primary branching patterns when transduced into btl mutants. This contrasts to the ability of transduced normal btl to rescue the defects of btl mutants. It thus seems that regulated BTL activity is essential for the normal pattern of tracheal cell migration. The adverse effects of lambda-BTL could be attributable either to a lack of spatial regulation of activity or to increased levels of BTL activity. These two possibilities were distinguished by expressing lambda-BTL in flies with a normal level of endogeneous BTL, or a reduced level. Lambda-BTL induced tracheal cell migration defects are actually enhanced by reduction of wild-type BTL, suggesting that lack of spatial regulation of BTL leads to migration defects. Lambda-BTL effects secondary and terminal tracheal branching in a different manner than the effects on primary branching. In contrast to the deleterious effects of Lambda-BTL on primary branching, constitutive BTL activity can replace endogenous BTL activity in formation of secondary branches. The normal pattern of secondary branching apparently depends on the restriction of high levels of BTL activity to a small number of cells, because ectopic secondary and terminal branches are observed in embryos in which BTL is overexpressed. Distinct roles for BTL activity in tracheal cell migration versus branching are supported by the obervation that changes in gene expression are required for tracheal cells to produce secondary and/or terminal branches, whereas there is no evidence that tracheal cell migration in response to BTL is mediated throught nuclear signaling (Lee, 1996).

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

Btl activity is required for the migration of tracheal cells to form primary branches, and for the subsequent induction of secondary and terminal tracheal cell fates. Mutations in btl are associated with a marked inhibition of tracheal branching. Given the involvement of hbr in the Htl Fgf receptor signaling pathway, an examination was carried out to see if Hbr might also function with Btl in the tracheal system. Reduction of hbr function is indeed associated with significant defects in tracheal development. In hbrYY202 mutant embryos, numerous primary and secondary tracheal branches are missing and the extension of those that do form is frequently stalled. These results imply that hbr is necessary both for tracheal cell migration and for the acquisition of secondary tracheal cell fates. One hbr allele, hbrems7, exhibits a very similar tracheal phenotype to another, hbrYY202, while a third allele, hbrems6, has a more severe reduction in tracheal branching. Consistent with findings for the mesoderm, both hbrYY202 and hbrems6 are hypomorphic with respect to their effects on tracheal development, since more severe phenotypes occur when either allele is in trans to a deficiency. Interestingly, although hbrems6 has the strongest tracheal phenotype, its mesodermal defects are the least severe of the three hbr alleles. As is the case with heartless and heartbroken, breathless and heartbroken exhibit strong genetic interactions. Thus, hbr is capable of dominantly enhancing a hypomorphic btl allele and btl can dominantly enhance the hbr tracheal phenotype. Together, these genetic interaction experiments suggest that hbr participates in both the Htl and Btl signaling pathways (Michelson, 1998).

The potential requirement for hbr in mediating the effects of Btl hyperactivation was investigated. Ectopic ectodermal expression of Bnl, the Btl ligand, leads to widespread Btl activation, which causes a strong inhibition of primary tracheal branching, accompanied by the induction of disorganized networks of secondary and terminal tracheal branches. A homozygous hbr mutation strongly suppresses this effect of ectopic Bnl - the formation of long primary branches is recovered and the additional fine, higher order branches are markedly reduced in number. Thus, as with activated Htl in the mesoderm, a hypomorphic hbr mutation is capable of at least partially suppressing the effect of Btl hyperactivation (Michelson, 1998).

Expression of activated MapK can be used to follow Rtk involvement in tracheal development. Specific tracheal cell fates are established initially under the influence of Egfr, whose activity is reflected in the expression of activated MapK in the tracheal placodes at stage 10. By stage 11, Btl-dependent expression of diphospho-MapK occurs in the tracheal pits prior to the onset of tracheal branch migration. In either btl or bnl mutant embryos, the Egfr-dependent expression of activated MapK in the tracheal placodes is not affected, while the later expression of activated MapK in the tracheal pits is largely but not completely eliminated. With reduced hbr function, the Egfr-dependent diphospho-MapK pattern at stage 10 is normal, while Btl-dependent expression at stage 11 is very weakly but significantly reduced. The extent to which tracheal pit MapK expression is affected in the bnl and hbr mutants appears to be commensurate with the relative severities of their tracheal migration defects (Michelson, 1998).

Study of the posterior spiracles of Drosophila as a model to understand the genetic and cellular mechanisms controlling morphogenesis

The development of the posterior spiracles of Drosophila may serve as a model to link patterning genes and morphogenesis. A genetic cascade of transcription factors downstream of the Hox gene Abdominal-B subdivides the primordia of the posterior spiracles into two cell populations that develop using two different morphogenetic mechanisms. The inner cells that give rise to the spiracular chamber invaginate by elongating into 'bottle-shaped' cells. The surrounding cells give rise to a protruding stigmatophore by changing their relative positions in a process similar to convergent extension. In the larvae the spiracular chamber forms a very refractile filter, the filzkorper. The opening of the spiracular chamber, the stigma, is surrounded by four sensory organs; the spiracular hairs. Clones labeling the spiracular hairs show that each one is formed by four cells related by lineage, two neural and two support cells, the typical structure of a type I external sensory organ. When the larva is buried in the semi-liquid medium on which it feeds, the stigmatophore periscopes out of the medium allowing the larva to continue breathing. The genetic cascades regulating spiracular chamber, stigmatophore, and trachea morphogenesis are different but coordinated to form a functional tracheal system. In the posterior spiracle, this coordination involves the control of the initiation of cell invagination that starts in the cells closer to the trachea primordium and spreads posteriorly. As a result, the opening of the tracheal system shifts back from the spiracular branch of the trachea into the posterior spiracle cells (Hu, 1999).

The connection of the posterior spiracle to the trachea is a regulated event. In mutants for the Drosophila FGF and FGF-receptor homologs branchless and breathless the tracheal pits do invaginate, but since they do not migrate toward one another, they do not form a continuous network. In contrast, in btl mutants, the posterior spiracle connects normally to the A8 spiracular branch of the trachea. In mutants for Abd-B the stigma of A8 does not slide posteriorly, but stays in the same position as in anterior abdominal segments, where the spiracular branch attaches to the outside epidermis. The contribution of the ems gene to coordination of morphogenetic movements has been examined. The spiracle-trachea connection occurs in cut and sal mutants but not in ems mutants. In ems mutants, invagination of the spiracle cells adjacent to the trachea does not occur, but more posterior cells of the spiracle invaginate normally. The elongation does not occur simultaneously in all cells, but starts in the more anterior ones and, in general, the invaginating cells keep contact with the external surface of the embryo. This results in the cells that have invaginated earlier being deeper in the spiracular chamber and more elongated. The defective invagination in ems mutants results in a spiracle without a lumen and with the tracheal opening located outside it. The results show that cell elongation and formation of a lumen are two independently controlled processes. The spiracles provide a good model for the study of cellular and molecular mechanisms controlling cell shape and cell rearrangements, two mechanisms which are used during the morphogenesis of a variety of organisms (Hu, 1999).

The alternative migratory pathways of the Drosophila tracheal cells are associated with distinct subsets of mesodermal cells

The Drosophila tracheal system is a model for the study of the mechanisms that guide cell migration. The general conclusion from many studies is that migration of tracheal cells relies on directional cues provided by nearby cells. However, very little is known about which paths are followed by the migrating tracheal cells and what kind of interactions they establish to move in the appropriate direction. An analysis has been carried out of how tracheal cells migrate relative to their surroundings and which tissues participate in tracheal cell migration. Cells in different branches are found exploit different strategies for their migration; while some migrate through preexisting grooves, others make their way through homogeneous cell populations. Alternative migratory pathways of tracheal cells are associated with distinct subsets of mesodermal cells and a model is proposed for the allocation of groups of tracheal cells to different branches. These results show how adjacent tissues influence morphogenesis of the tracheal system and offer a model for understanding how organ formation is determined by its genetic program and by the surrounding topological constraints (Franch-Marro, 2000).

Tracheal cells are first specified as clusters of ectodermal cells at the embryonic surface. Since tracheal cells invaginate and form the tracheal pits they occupy the grooves between the muscle precursors of adjacent metameres. The formation of this groove is independent of tracheal invagination because it also forms between metameres that do not have tracheal placodes and it also develops in trh mutant embryos, which do not undergo tracheal invagination. A subset of the tracheal cells moves anteriorly, whereas another subset moves posteriorly until they reach the cells from the adjacent placodes. These cells will form the dorsal trunk, the most prominent tracheal branch that spans the embryo longitudinally. Those cells migrate across the adjacent precursors of somatic muscles and separate the precursors of the most dorsal muscles from the precursors of more ventral muscles. Other cells, those from the dorsal side of the tracheal pit, move dorsally along the longitudinal groove to form the dorsal branches that will end up fusing with the dorsal branches coming from the contralateral hemisegments. In the ventral side, the tracheal cells follow two different paths along the two clusters of lateral muscle precursors at each side of the groove. Anterior ventral cells will form the anterior lateral trunk while the posterior ventral cells will form the posterior lateral trunk. Finally, another group of cells from a midposition in the tracheal pit will migrate inward and will form the visceral branch (Franch-Marro, 2000).

To elucidate how the cells of the dorsal trunk migrate across the precursors of somatic muscles it was first determined whether they recognized some kind of preexisting gap between the most dorsal and the remaining muscle precursors. Thus, an examination was carried out to see whether the gap between these muscle precursors is also established in the absence of migration of the cells of the dorsal trunk or, alternatively, is a consequence of the migration of these tracheal cells. First, the situation of muscle precursors was analyzed in breathless (btl) mutant embryos in which there is no tracheal migration. Second, embryos were studied in which a constitutive form of the Tkv receptor was induced specifically in the tracheal cells; in these embryos, the cells that would normally migrate anteriorly or posteriorly to form the tracheal trunk are instead forced to migrate in the dorsoventral axis. In both cases, no topological segregation between the most dorsal and the remaining precursor cells was observed, indicating that migration of the tracheal cells separates the two subsets of muscle precursors cells at a stage at which the somatic muscle precursors have been specified. Thus, the cells of the dorsal trunk appear to open up their way through a contiguous population of cells (Franch-Marro, 2000).

Specificity of FGF signaling in cell migration in Drosophila

The relationship between receptor tyrosine kinase (RTK) activated signaling pathways and the induction of cell migration was investigated. Using Drosophila tracheal and mesodermal cell migration as model systems, the intracellular domain of the fibroblast growth factor receptors (FGFRs) Breathless (Btl) and Heartless (Htl) can be functionally replaced by the intracellular domains of Torso (Tor) and epidermal growth factor receptor (EGFR). These hybrid receptors can also rescue cell migration in the absence of Downstream of FGFR (Dof), a cytoplasmic protein essential for FGF signaling. These results demonstrate that tracheal and mesodermal cells respond during a specific time window to a receptor tyrosine kinase (RTK) signal with directed migration, independent of the presence or absence of Dof. These findings are discussed in the light of the recent findings that RTKs generate a generic signal that is interpreted in responding cells according to their developmental history (Dossenbach, 2001).

All six major branches of the trachea form in the Btl-Tor hybrid including the dorsal branches and the ganglionic branches, form with high efficiency. In addition, the nuclear target gene DSRF/blistered is activated by the Btl-Tor hybrid, albeit with increased efficiency. Since the Bnl-induced transcriptional activation of pointed (pnt) is a prerequisite for the activation of DSRF/bs, it is presumed that pnt is also activated by Btl-Tor. Therefore, both the cellular events linked to guided migration as well as the nuclear events required for further cell fate specifications can be brought about by the Btl-Tor hybrid protein. It is concluded that the specific interpretation of the RTK signal in tracheal cells, namely its translation into a migratory response and the activation of the appropriate differentiation program, is not due to the specificity of the Bnl/Btl signaling system but rather is a property of tracheal cells. It is possible that tracheal cells (and mesodermal cells) express a particular protein(s) that allows them to trigger migration upon RTK signaling. It has been demonstrated previously that tracheal cells do express distinct proteins that allow them to react to the activated Btl receptors with migration; Dof is activated specifically in tracheal and mesodermal cells to allow FGF signal propagation. However Dof itself is not an essential tracheal factor that allows a link of RTK activation to the cell migration machinery (Dossenbach, 2001)

Besides the essential role of Dof in FGF receptor signaling in Drosophila, little is known about the actual cellular or biochemical function of Dof. Since Btl, Htl and Dof activity are required for guided cell migration, it was asked whether cell motility and guidance induced by the hybrid proteins in the absence of endogenous Btl activity required Dof. None of the FGFRs could induce guided migration in dof mutants, while both the Tor and the EGFR intracellular domain were capable of rescuing migration in the absence of dof; the rescue efficiency of these hybrid receptors is similar to their rescue efficiency in btl mutants, which do have Dof protein. This result clearly demonstrates that tracheal and mesodermal cells respond to an RTK signal with directed migration, independent of the presence or absence of Dof. Dof activity appears to be required for FGF signaling per se, possibly relaying the signal from the activated receptor to downstream components, including Ras. It occurs that within a specific developmental window, tracheal cells respond to RTK activity with directed migration. Later in development, specific genes (pnt, DSRF/bs) are transcriptionally induced as a response to RTK activation. It will be most interesting to identify the cellular factors that impose this specificity and see whether their expression in other cell types results in motility (Dossenbach, 2001)

Recently, it has been shown that border cells rely for their guided migration during oogenesis on EGFR activity. To mediate this guidance function, EGFR signals via a pathway that is independent of Raf-MAP kinase. In addition, the EGFR guidance appears to be receptor-specific, since Htl signaling can not substitute for EGFR signaling. It will be interesting to see whether similar or divergent signaling pathways are required for RTK-mediated migration of border cells, mesodermal and tracheal cells (Dossenbach, 2001)

Differences were found in the rescue efficiencies when the function of the three receptors in the tracheal system and in the mesoderm was compared. The differences might be due (to some extent) to quantitative effects. However, it is also possible that qualitative differences exist and that some of the receptors (for example, EGFR) are less efficient in part because specific, non-essential signal adaptor proteins are absent or reduced in some of the tissues at the stages examined. Without knowing the precise composition of the entire signaling pathways of all three receptors in the tracheal system and in the mesoderm, it is impossible to determine whether the differences in efficiencies observed are due to quantitative and/or qualitative aspects of signaling (Dossenbach, 2001)

Although it has now been shown in many developmental contexts that the intracellular domains of different RTKs are largely interchangeable, it is worth emphasizing that in many cases FGF receptors can not functionally substitute for other Drosophila receptor tyrosine kinases. This is most probably due to the fact that the essential Dof protein is only present in a subset of cells in Drosophila. Thus, the spectrum of cells in which FGF receptors can function is limited to those cells that express Dof and maybe other FGFR-specific signaling components (Dossenbach, 2001)

Grainy head controls apical membrane growth and tube elongation in response to Branchless/FGF signalling

Epithelial organogenesis involves concerted movements and growth of distinct subcellular compartments. Apical membrane enlargement is critical for lumenal elongation of the Drosophila airways, and is independently controlled by the transcription factor Grainy head. Apical membrane overgrowth in grainy head mutants generates branches that are too long and tortuous without affecting epithelial integrity, whereas Grainy head overexpression limits lumenal growth. The chemoattractant Branchless/FGF induces tube outgrowth -- it upregulates Grainy head activity post-translationally, thereby controlling apical membrane expansion to attain its key role in branching. A two-step model for FGF in branching is favored: first, induction of cell movement and apical membrane growth, and second, activation of Grainy head to limit lumen elongation, ensuring that branches reach and attain their characteristic lengths (Hemphälä, 2003).

In order to analyse whether tracheal Grh activity could be targeted by Bnl/Btl signal transduction, GBE-lacZ expression was analyzed in embryos with altered levels of Bnl and Btl activity. When Bnl is ectopically expressed in all tracheal cells, GBE-lacZ expression becomes significantly upregulated, although the levels of Grh protein are not altered. This suggests that Bnl controls Grh activity post-translationally, and surprisingly, upregulates the expression of this artificial Grh target. Nevertheless, the effects of Btl appear specific since with more limited Bnl expression using the Term-Gal4 driver, GBE-lacZ expression becomes enhanced specifically in the cells that respond to Bnl by ectopically expressing the terminal marker DSRF. Similar enhancement of GBE-lacZ expression is evident upon tracheal expression of an activated form of the Btl receptor itself (UASBtl-Tor). In all instances the augmented GBE-lacZ expression is dependent on Grh, since embryos that express ectopic Bnl or the activated form of Btl, but lack Grh activity, do not express GBE-lacZ. Furthermore, ectopic activation of Dpp, another signalling pathway that promotes the growth of dorsal and ganglionic branches during tracheal development, has no effect on GBE-lacZ, indicating that the effects on GBE-lacZ are specific for Bnl/Btl (Hemphälä, 2003).

Whether Bnl signalling is a prerequisite for the transcriptional activity of Grh was tested by analysing the levels of GBE-lacZ expression in mutants for bnl, btl or pointed (pnt). Tracheal GBE-lacZ expression is both reduced and uniform in bnl and btl mutant embryos, but is unchanged in pnt embryos that lack the activity of a downstream transcriptional effector of the ETS family. Since Grh is a substrate for activated MAPK (ERK2) in vitro, its activity could be modulated directly during branching by Bnl-induced phosphorylation. This would account for the fact that GBE-lacZ expression is affected by mutations in bnl and btl, but not by mutations in the nuclear effector pnt (Hemphälä, 2003).

It is concluded that Bnl signalling converts Grh to a more potent activator of its GBE-lacZ target. Since Grh becomes phosphorylated by MAPK in vitro, and MAPK is a downstream effector of Btl signal transduction, the alteration in Grh activity may be brought about by MAPK-mediated phosphorylation of the Grh protein (Hemphälä, 2003).

Drosophila Perlecan modulates FGF and Hedgehog signals to activate neural stem cell division

Mutations in the Drosophila terribly reduced optic lobes (trol) gene cause cell cycle arrest of neuroblasts in the larval brain. trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF (Branchless) and Hedgehog (Hh) signaling. Addition of human FGF-2 to trol mutant brains in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system (Park, 2003).

Trol appears to display functions similar to mammalian Perlecans, which are known to bind FGF-2 and to be required for FGF signaling. Dominant enhancement of the neuroblast proliferation phenotype of two different trol alleles has been observed with mutations in bnl and the Bnl receptor breathless (btl), but not with mutations in the orphan heartless (htl) receptor. The neuroblast proliferation phenotype of trol8 mutant brains was rescued in culture to control levels by addition of human FGF-2. Addition of the MAPK inhibitor PD98059 at 10 hs post-hatching decreased the number of S-phase neuroblasts. Biochemical analysis has shown that FGF-2 can be coimmunoprecipitated with Trol and that the binding of FGF-2 to Trol can be competed by added heparin. This suggests that, like mPerlecan, Trol binds FGF-2 through heparan sulfate residues. These results demonstrate that Trol-mediated FGF signaling is required for initiation of neuroblast proliferation sometime in first larval instar. This similarity to the function of mPerlecans in mammalian FGF signaling and the implications of up-regulation of mPerlecan in tumors strongly imply that trol encodes a functional Drosophila Perlecan homolog (Park, 2003).

Social interactions among epithelial cells during tracheal branching morphogenesis

Many organs are composed of tubular networks that arise by branching morphogenesis in which cells bud from an epithelium and organize into a tube. Fibroblast growth factors (FGFs) and other signalling molecules have been shown to guide branch budding and outgrowth, but it is not known how epithelial cells coordinate their movements and morphogenesis. Genetic mosaic analysis has been used in Drosophila to show that there are two functionally distinct classes of cells in budding tracheal branches: cells at the tip that respond directly to Branchless FGF and lead branch outgrowth, and trailing cells that receive a secondary signal to follow the lead cells and form a tube. These roles are not pre-specified; rather, there is competition between cells such that those with the highest FGF receptor activity take the lead positions, whereas those with less FGF receptor activity assume subsidiary positions and form the branch stalk. Competition appears to involve Notch-mediated lateral inhibition that prevents extra cells from assuming the lead. There may also be cooperation between budding cells, because in a mosaic epithelium, cells that cannot respond to the chemoattractant, or respond only poorly, allow other cells in the epithelium to move ahead of them (Ghabrial, 2006).

The Drosophila tracheal system develops from epithelial sacs of about 80 cells from which primary, secondary and terminal branches sprout without cell division or cell death. Primary branch sprouting is induced by Branchless (Bnl) FGF, a chemoattractant secreted by clusters of cells surrounding each sac, which activates Breathless (Btl) FGF receptor (FGFR), a receptor tyrosine kinase expressed on tracheal cells. Primary branches contain 3-20 cells that organize into a tube as they migrate out from the sac. Bnl also induces the expression of secondary branching genes, such as the transcription factor pointed (pnt), and specifies terminal cells at the ends of outgrowing branches. Terminal cells ramify in the larva in response to Bnl to form fine terminal branches. Other cells at the ends of primary branches become fusion cells that connect with neighbouring branches to form a continuous tracheal network. Terminal and fusion cell fate decisions are also influenced by the Notch, Dpp and Wingless signalling pathways. Dorsal branches, the primary branches that were focused on here in this study, typically consist of five or six cells: two cells near the branch tip, one (DB1) that becomes a terminal cell and another (DB2) that becomes a fusion cell, and three or four cells (DB3-DB6) that form the branch stalk (Ghabrial, 2006).

In a genetic mosaic screen, six mutants (724, 788, 1118, 1187, 1476 and 1684) were identified with a subtle phenotype: mosaic branches (+ / + , +/ - , -/ - cells) were grossly normal, yet homozygous mutant clones (- / - cells) rarely if ever included terminal cells. These were neither general nor terminal-cell-specific lethal mutations because homozygous mutant cells were readily recovered in all other tracheal positions, and there was no decrease in the overall number of cells in mosaic dorsal branches or the number of terminal cells. It was difficult to imagine how mutations could block clone generation in specific cells. It seemed more likely that the mutations caused cells otherwise destined to become terminal cells to switch fates with other tracheal cells (Ghabrial, 2006).

The six mutations compose a single lethal complementation group that mapped to the left arm of chromosome 3 and failed to complement breathlessLG18. DNA sequencing identified a single nucleotide change in each mutant resulting in a nonsense or missense mutation in btl. Five mutations (724, 788, 1118, 1476 and 1684) appear to be null btl mutations, whereas the sixth mutation (1187) causes partial loss of function. Thus, the 'no mutant terminal cells' gene is btl (Ghabrial, 2006).

The distribution of cells homozygous was quantified for btl null mutations (724 and 788), or homozygous for a wild-type btl allele as a control, in mosaic dorsal branches. Control clones were evenly distributed throughout the branch at the expected frequencies; for example, the ratio of stalk-cell to terminal-cell clones was about 3:1. By contrast, btl-/- cells showed a nearly complete bias against the DB1 position: the ratio of stalk-cell to terminal-cell clones was 51:1. The three exceptional mutant terminal cells may be cases in which the clone was induced after btl began to be expressed, allowing wild-type btl gene products to perdure in mutant cells. Hundreds of mosaic branches with one or more btl-/- cells present in positions DB2-DB6 were recovered without affecting cell or branch morphology. Indeed, branches composed largely or exclusively of btl-/- cells, except for a wild-type terminal cell, were morphologically indistinguishable from wild-type branches. Thus, although all tracheal cells normally express btl, and the receptor is activated by Bnl in most or all cells of budding branches, the receptor appears to be required in just a single leading cell (DB1). All other cells can migrate normally and form tubes in the absence of btl. It is concluded that there are two functionally distinct classes of cells in budding primary branches: lead cells, which require Btl FGFR and directly respond to Bnl FGF, and trailing cells, which do not require Btl but follow the lead cell and form the stalk (Ghabrial, 2006).

What does it take to become the leader? The lead cell (DB1) is specified to become a terminal cell by Bnl-Btl signalling. If terminal cell specification is required, then null mutations in the downstream gene pnt, which abolish this function, should have the same effect as btl mutations. Cell clones homozygous for pntDelta88 or two new pnt alleles isolated in the screen (198 and 1318) failed to develop as terminal cells, as expected. However, unlike btl mosaic branches, pnt mosaic branches often lacked a terminal cell. When a terminal cell was missing, there was usually a pnt-/- cell in the stalk position nearest the tip, presumably the DB1 cell that failed to differentiate into a terminal cell. This suggests that pnt-/- cells are able to assume the lead position but fail to differentiate as terminal cells, and that the bias against btl mutant terminal cells is due to the earlier, pnt-independent, function of Btl in primary branch budding and outgrowth. If cells lacking Btl cannot migrate in response to Bnl during budding, they should not be able to move to the lead position necessary to be selected as a terminal cell. Consistent with this, genetic mosaic analysis of stumps (dof/heartbroken), which encodes a Btl adaptor required for cell migration, showed a dearth of terminal cell clones similar to btl (Ghabrial, 2006).

Two results demonstrate that the ability to sense Bnl and migrate in response to it is not enough to become the leader: cells compete for the lead position. The first involves btlBN (E796K mutation in the kinase domain), a weak btl allele isolated in a separate screen. Unlike btl-/- animals, which die in first larval instar and lack virtually all branches, btlBN homozygotes survived until L3 larval stage or beyond and had a normally patterned tracheal system with a full complement of terminal cells. The only defects detected were a reduced number and altered morphology of terminal branches, presumably due to the dosage-sensitive function of btl in terminal branch outgrowth. The late and subtle phenotype demonstrates that BtlBN protein retains sufficient activity for early migration and terminal cell specification events. However, in genetic mosaic animals, in which btlBN/BN cells must compete with btlBN/+ and btl+/+ tracheal cells, btlBN/BN cells rarely acquired the lead position (DB1) and developed as terminal cells. Indeed, homozygosity for btlBN conferred nearly as complete a bias against becoming a terminal cell as total loss of btl. Thus, Btl activity above the threshold necessary for migration and terminal cell specification is not sufficient to acquire the lead position and become a terminal cell: a cell must have more Btl activity than other cells in the branch (Ghabrial, 2006).

Similar conclusions derive from a second experiment in which marked wild-type (btl+/+) cells were analysed in heterozygous (btl+/-) animals. Whereas btl+/+ clones in control (btl+/+) animals were distributed evenly throughout the branch, btl+/+ clones in btl788/+ heterozygotes preferentially localized to the tip. Cells that did not occupy the lead (DB1) position took positions close to the tip. Similar results were obtained for btl+/+ clones in animals heterozygous for btl1187, a partial-loss-of-function allele. Clones mutant for sprouty, an FGF feedback inhibitor, also preferentially populated the tip. Together, the data show that there is competition for the lead position: cells with highest btl activity assume positions at or near the tip of the branch, whereas those with less or no activity segregate towards its base (Ghabrial, 2006).

Because small differences in btl dosage or activity affect a cell's ability to compete for the lead, whether lateral inhibitory mechanisms that amplify small differences in signalling might be operative was investigated. Data suggest that the Notch pathway, a lateral signalling pathway implicated in cell specification events including cell fate determination at tracheal branch tips, also affects cell arrangement. Nts embryos shifted to the restrictive temperature during budding formed branches in which most DB cells behaved like lead cells, resulting in large clusters of cells congregated at the lead position, whereas expression of constitutively active NACT throughout the tracheal epithelium had the opposite effect, arresting outgrowth and stalling cells near the base of the branch. It is proposed that Notch-mediated lateral inhibition among tracheal cells prevents extra cells from assuming the lead position (Ghabrial, 2006).

These results provide evidence for social stratification and dynamic social interactions between epithelial cells during branching morphogenesis. First, the results show that budding cells are functionally specialized. A cell at the branch tip requires btl and leads outgrowth towards the Bnl signalling center. Trailing cells do not require btl but nevertheless follow the lead cell towards the Bnl source. Because tracheal cells do not migrate or form tubes in btl-/- animals, trailing cells must receive a secondary signal generated by the lead cell that induces them to migrate and also activates their tubulogenesis program. This could be a secreted molecule or physical stimulus such as pulling or stretching the trailing cells (Ghabrial, 2006).

Second, these roles are not pre-specified. Rather, there is competition between cells such that those with high Btl FGFR activity become lead cells whereas those with less or no btl FGFR activity become trailing cells and form the branch stalk. Competition appears to involve Notch-mediated lateral inhibitory signalling between tracheal cells, and it may also be influenced by positive feedback mechanisms such as increased activation and expression of Btl as cells approach the Bnl source. Third, there may be cooperation between cells, because in a genetically mosaic epithelium, tracheal cells with less Btl activity allow those with more activity to move ahead of them (Ghabrial, 2006).

There may be similar social interactions between budding cells in other branching organs. Studies of other branching processes have identified genes selectively expressed in tip cells of budding branches, and in some cases these cells display morphological specializations indicating that they might actively lead outgrowth. However, because most budding branches contain hundreds or thousands of cells, it is difficult to track and manipulate individual cells to investigate social behaviours like those described here. Recent analyses of chimaeric Ret+/Ret- mouse renal ureteric buds in culture and btl mosaic air sacs reveal that cells lacking these receptor tyrosine kinases are excluded from branch tips, indicating that RTK-dependent interactions similar to those described here might be operative in more complex branching events (Ghabrial, 2006).

Dual origin of tissue-specific progenitor cells in Drosophila tracheal remodeling

During Drosophila metamorphosis, most larval cells die. Pupal and adult tissues form from imaginal cells, tissue-specific progenitors allocated in embryogenesis that remain quiescent during embryonic and larval life. Clonal analysis and fate mapping of single, identified cells show that tracheal system remodeling at metamorphosis involves a classical imaginal cell population and a population of differentiated, functional larval tracheal cells that reenter the cell cycle and regain developmental potency. In late larvae, both populations are activated and proliferate, spread over and replace old branches, and diversify into various stalk and coiled tracheolar cells under control of fibroblast growth factor signaling. Thus, Drosophila pupal/adult tissue progenitors can arise both by early allocation of multipotent cells and late return of differentiated cells to a multipotent state, even within a single tissue (Weaver, 2008).

Drosophila larval tissues are composed of differentiated larval cells and imaginal cells. Imaginal cells are pupal and adult tissue progenitors that reside in clusters embedded in or attached to larval tissue. They remain quiescent during embryogenesis and part or all of larval life, then proliferate and differentiate into pupal and adult tissues at metamorphosis. By contrast, larval cells cease dividing and differentiate early in development; however, they typically enlarge and become polyploid during larval life. At metamorphosis, most larval cells die. Although some larval neurons and muscles are retained in adult tissues, no differentiated cells are known to reenter the cell cycle and generate new cells and tissues. This study shows that tracheal (respiratory) system remodeling at metamorphosis is carried out by a classical imaginal cell population and another progenitor population that, like facultative stem cells in mammals, arises from differentiated cells (Weaver, 2008).

During embryogenesis, the tracheal system develops from segmentally repeated groups of ~80 cells that express Trachealess transcription factor and invaginate, forming sacs attached to epidermis by a stalk of spiracular branch (SB) cells. Branches bud from the sacs and cells diversify primarily under control of Branchless FGF (fibroblast growth factor), which activates Breathless FGFR (FGF Receptor) on tracheal cells. At metamorphosis, posterior tracheal segments Tr6 to Tr10 are lost; new branches form in Tr4 and Tr5 to supply posterior tissues and in Tr2 to supply flight muscle. Although most branches in Tr1 to Tr5 are retained, most of their cells are replaced by imaginal cells (Weaver, 2008)..

Previous work indicated that imaginal tracheal cells (tracheoblasts) compose the SB. Unlike other tracheal cells, SB cells express imaginal marker escargot (esg), remain small and quiescent during embryonic and early larval life, and do not form gas transport tubes. Bromodeoxyuridine (BrdU) incorporation studies showed that SB cells enter S phase at the beginning of the third larval period (L3) and divide 12 to 16 hours later. Proliferation continues for 24 hours, generating an expanding cluster of tracheoblasts at the SB-transverse connective (TC) junction. SB cells in the embryo and early larva express trachealess (trh) but, unlike most other tracheal cells, do not express the Trachealess target gene breathless (btl): The tracheal program is apparently arrested at this step. When activated in L3, they turn down esg and turn on btl as they proliferate and leave the SB (Weaver, 2008).

A 'molecular timer' strain was developed that highlights the burst of btl expression in activated tracheoblasts, which allowed distinguishing them from the larval tracheal cells they migrate over and replace. SB tracheoblasts followed stereotyped paths. In Tr4, they migrated along the TC onto the visceral branch; later, some differentiated into coiled tracheolar (CT) cells. Tracheoblasts respected specific boundaries, never spreading into neighboring tracheal segments or populating the dorsal trunk (DT). However, tracheoblasts were observed on the other side of the DT, along the dorsal branch (DB) of Tr4 and other anterior DBs (Weaver, 2008).

If DB tracheoblasts arise from SB tracheoblasts, they would have to move across the DT to reach the DB. However, tracheoblasts were never seen crossing the DT. To exclude this possibility, the fate of SB tracheoblasts was mapped using SB-specific flipase (FLP) recombinase to permanently label SB cells and their descendants. This labeled all tracheoblasts migrating out of the SB, but not DB tracheoblasts. Hence, DB tracheoblasts arise independently (Weaver, 2008).

To identify the source of DB tracheoblasts, early L3 larval DBs, which comprise five to seven cells were scrutinized, but no additional cells or cells were found with the distinctive small size and nuclear morphology of SB tracheoblasts. The positions and number of tracheoblast clones in a clonal analysis of larval DBs suggested that DB tracheoblasts arise from ~4 to 5 progenitors along the DB (Weaver, 2008).

Whether differentiated stalk cells (DB3 to DB7) might be the source of DB tracheoblasts was considered. To test this, a heat-inducible FLP transgene was used to permanently label and trace the fate of individual tracheal cells identified in live L2 larvae. This demonstrated that larval DB stalk cells are the source. Individual stalk cells displayed a range of proliferative capacities, giving rise to 2 to 22 tracheoblasts, although occasionally a labeled stalk cell failed to proliferate or degenerated, the standard fate of DB1 and DB2 cells (Weaver, 2008).

DB stalk cells have a complex morphology unlike typical progenitor or stem cells: They are tubular, with autocellular junctions, some (DB3 cells) forming Y-shaped tubes. Yet these cells become proliferating, migrating DB tracheoblasts while maintaining contacts with neighboring tracheal cells (Weaver, 2008).

Phosphohistone H3 staining showed that DB stalk cells in anterior segments begin dividing 14 to 16 hours after the second molt. Even before they divide, they have smaller nuclei than other larval tracheal cells, including DB stalk cells in posterior segments, which are otherwise indistinguishable but do not give rise to tracheoblasts. BrdU labeling of newly molted third-instar larvae showed that anterior DB stalk cells do not incorporate the label, implying that they do not endoreplicate and presumably remain diploid, unlike posterior DB cells and other differentiated larval cells, most of which endoreplicate and become polyploid (Weaver, 2008).

Although most DB tracheoblasts form multicellular stalks of pupal DBs, in Tr2 they form more elaborate structures. After proliferating and spreading along the DB, they aggregate, form secondary branches, and differentiate into multicellular stalks (MS), unicellular stalks (US), and Blistered (DSRF)-expressing CT cells with coiled intracellular lumens that unfurl on flight muscle. Fate mapping showed that single DB stalk cells in Tr2 routinely formed mixed clones containing MS, US, and CT cells. Thus, DB stalk cells in Tr2 transform into multipotent tracheoblasts that can proliferate and acquire different fates (Weaver, 2008).

Bnl/Btl signaling controls cell fate selection in the embryo. To test for function in pupal tracheoblasts, btl minus clones were generated. These rarely formed CT cells. Likewise, conditional expression of dominant-negative Btl in the L3 tracheal system reduced or eliminated secondary branches and CT cells. Constitutively active receptor induced ectopic secondary branches and CT cells throughout the tracheal system, including all anterior DBs. Thus, DB tracheoblasts in anterior segments can acquire new tracheal fates, and FGF signaling also plays a critical role in reselecting cell fates when DB stalk cells are reactivated (Weaver, 2008).

Anterior DB stalk cells are the first differentiated cells in Drosophila shown to reenter the cell cycle and regain developmental potency. They regain the same abilities to proliferate, spread, and differentiate into various tracheal cell types as SB tracheoblasts, classical imaginal cells that remain quiescent (blocked in tracheal outgrowth and cell diversification) during embryonic and most of larval life. This suggests that both types of tracheal progenitors arrive at a similar state, one by early developmental arrest (SB tracheoblasts), the other by late return to an earlier state (anterior DB stalk cells). The only known features that distinguish these cells from tracheal cells that lack progenitor potential (including posterior DB stalk cells that are otherwise indistinguishable from anterior DB stalk cells) are their small nuclear size and lack of endoreplication. These features may be part of a program that maintains, or allows cells to regain, the proliferative and diversification potential of early tracheal cells. This program is operative in imaginal tracheal cells and can apparently be implemented in other tracheal cells, independent of their differentiation program (Weaver, 2008).

The blurring of the distinction between imaginal and differentiated larval cells in Drosophila parallels a current debate about adult stem cells in mammals. Some mammalian tissues have dedicated stem cells maintained in a primitive state. However, other tissues may rely on facultative stem cells, differentiated cells that reenter the cell cycle to replenish lost cells. The current results show that progenitors with each of these features are present in a single Drosophila tissue and that both play crucial roles. This provides a tractable system for dissection of the arrest of a tissue-specific developmental program and reversal to an earlier, more plastic state, important steps in tissue engineering, repair, and cancer (Weaver, 2008).

FGF ligands in Drosophila have distinct activities required to support cell migration and differentiation

Fibroblast growth factor (FGF) signaling controls a vast array of biological processes including cell differentiation and migration, wound healing and malignancy. In vertebrates, FGF signaling is complex, with over 100 predicted FGF ligand-receptor combinations. Drosophila presents a simpler model system in which to study FGF signaling, with only three ligands and two FGF receptors (FGFRs) identified. This study analyzed the specificity of FGFR [Heartless (Htl) and Breathless (Btl)] activation by each of the FGF ligands [Pyramus (Pyr), Thisbe (Ths) and Branchless (Bnl)] in Drosophila. It was confirmed that both Pyr and Ths can activate Htl, and that only Bnl can activate Btl. To examine the role of each ligand in supporting activation of the Htl FGFR, genetic approaches were utilized that focus on the earliest stages of embryonic development. When pyr and ths are equivalently expressed using the Gal4 system, these ligands support qualitatively different FGFR signaling responses. Both Pyr and Ths function in a non-autonomous fashion to support mesoderm spreading during gastrulation, but Pyr exhibits a longer functional range. pyr and ths single mutants exhibit defects in mesoderm spreading during gastrulation, yet only pyr mutants exhibit severe defects in dorsal mesoderm specification. This study demonstrated that the Drosophila FGFs have different activities and that cell migration and differentiation have different ligand requirements. Furthermore, these FGF ligands are not regulated solely by differential expression, but the sequences of these linked genes have evolved to serve different functions. It is contended that inherent properties of FGF ligands make them suitable to support specific FGF-dependent processes, and that FGF ligands are not always interchangeable (Kadam, 2009).

These experiments demonstrate that the Drosophila FGFs Pyr, Ths and Bnl have different functions and that the activation of FGF receptors by specific ligands affects particular biological processes. Examination of an allelic series of pyr and ths mutants suggests that pyr and ths are not redundant in function: both influence mesoderm spreading, whereas pyr is the dominant player controlling Eve+ cell specification within the dorsal mesoderm. It has been demonstrated that ectopic expression of ths by twist-Gal4 and 69B-Gal4 in the Df(2R)BSC25 mutant background can support Htl FGFR activation. However, this study assayed whether the expression supported in distinct domains would support Htl activation. By a series of 'rescue' experiments, through ectopic expression of one ligand in the Df(2R)BSC25 mutant background, evidence was obtained that localized expression of the ligands is important for proper mesoderm spreading. It was found, surprisingly, that the ligands exhibit differences in their functional range of action. In addition, using this same approach, it was found that either Pyr or Ths can support Eve+ cell specification within the dorsal mesoderm, but that Bnl cannot. Collectively, these data suggest that the Pyr and Ths FGFs function as ligands for the Htl FGFR and that specificity of FGF-FGFR interactions exists in Drosophila (Kadam, 2009).

The results demonstrate that both Pyr and Ths FGF ligands can activate the Htl FGFR, whereas only the Bnl FGF ligand can activate the Btl FGFR. Specificity of FGFR activation was observed: pyr or ths, but not bnl, expression is able to activate Htl to affect expression of Eve, and bnl, but neither pyr nor ths, is able to support tracheal specification. No evidence was obtained that other cross-interactions occur (i.e. Pyr-Btl, Ths-Btl or Bnl-Htl), which demonstrates that Gal4-mediated ectopic expression does not simply 'swamp the system'. This experimental approach also 'levels the playing field', since expression of each ligand is driven at the same time and place and presumably at similar levels. It is concluded that only three FGF-FGFR combinations function in Drosophila (i.e. Pyr-Htl, Ths-Htl and Bnl-Btl), which supports the idea that FGFRs exhibit ligand-binding preferences. Previous studies have investigated FGF signaling specificity by analyzing the ability of other receptor tyrosine kinases to support cell migration or by activating particular intracellular signaling pathways to examine which are required to effect FGFR-dependent cell migration versus cell differentiation. This work analyzed the specificity of FGF ligand-receptor interactions and how they contribute to particular developmental processes (Kadam, 2009).

When ligand expression is supported by twist-Gal4, Htl FGFRs presumably become saturated because dpERK is ectopically activated in all cells and spreading is negatively affected. One explanation for why this might affect mesoderm cell spreading is that these FGF-saturated mesoderm cells may no longer be competent to respond to endogenous ligands that provide directional cues. Recently, it has been shown that movement of the mesoderm cells during gastrulation is in fact directional (McMahon, 2008). Pyr and Ths ligands are differentially expressed during gastrulation and this might provide the necessary positional information required to direct migration of the mesoderm. It is proposed that Pyr and Ths have different activities that fulfil aspects of FGFR activation required to support cell migration. Ectopic expression of Pyr within the ectoderm negatively affects mesoderm spreading, which suggests that the refined expression domain of pyr within cells of the dorsal ectoderm is normally required to guide the mesoderm cells toward dorsal regions. However, even though ectopic expression of ths in the ectoderm has no effect on mesoderm spreading, ths mutants also exhibit defects in mesoderm spreading, demonstrating that both genes are required, perhaps to control different aspects of the migration. The 'rescue' experiments using the zenVRE.Kr-Gal4 driver support the view that Pyr has a longer functional range than Ths. These differences in range of function might correlate with different diffusion capabilities, but an alternative explanation is that the ligands activate the receptor with different affinities. Additional experiments will be necessary to distinguish their exact functions and to uncover the molecular basis for the differential functions of Pyr and Ths; it is suggested that in vivo imaging and quantitative analysis (McMahon, 2008) of single-mutant phenotypes will provide insights (Kadam, 2009).

With regard to the FGF-dependent cell differentiation, the 'rescue' experiments suggest that ectopic expression of either Pyr or Ths is sufficient to support Eve+ cell specification. The reason why loss of ths has less of an effect on Eve+ cell specification is most likely because pyr is prominently expressed in the vicinity of the future Eve+ cells; normally, Pyr supports this function, but Ths can support this activity if presented at sufficient levels within the correct domain. Furthermore, it is proposed that FGF signaling might not play an instructive role in supporting eve expression. Other signaling pathways already provide positional information required for the specification of Eve+ cells; FGF signaling pathway activation might simply serve a permissive role, and in this context either ligand would suffice (Kadam, 2009).

Sen, A., Yokokura, T., Kankel, M. W., Dimlich, D. N., Manent, J., Sanyal, S. and Artavanis-Tsakonas, S. (2011). Modeling spinal muscular atrophy in Drosophila links Smn to FGF signaling. J Cell Biol 192: 481-495. PubMed ID: 21300852

Modeling spinal muscular atrophy in Drosophila links Smn to FGF signaling

Spinal muscular atrophy (SMA), a devastating neurodegenerative disorder characterized by motor neuron loss and muscle atrophy, has been linked to mutations in the Survival Motor Neuron (SMN) gene. Based on an SMA model developed in Drosophila, which displays features that are analogous to the human pathology and vertebrate SMA models, the fibroblast growth factor (FGF) signaling pathway was functionally linked to the Drosophila homologue of SMN, Smn through interaction of the FGF receptor breathless with Smn. This study functionally characterize this relationship and demonstrates that Smn activity regulates the expression of FGF signaling components and thus FGF signaling. Furthermore, it was shown that alterations in FGF signaling activity are able to modify the neuromuscular junction defects caused by loss of Smn function and that muscle-specific activation of FGF is sufficient to rescue Smn-associated abnormalities (Sen, 2011).

Given the variability of the SMA phenotype and the proven relationship between the severity of the disease and small changes in wild-type SMN activity, there is a significant possibility that any modifiers of SMN activity, either direct or indirect, will have therapeutic value. To systematically explore the genome for genes that are capable of modulating SMN function in vivo, advantage was taken of the existence of an SMA model offered by Drosophila to search for Smn genetic interacters. The model that was developed is based on the lethality and an associated neuromuscular junction phenotype linked to loss of Smn function, a phenotype remarkably similar to the NMJ phenotype reported for human patients. Though the role of SMN in biogenesis of snRNPs has been well documented, its regulators and downstream effectors have not been systematically delineated, nor has the link between mutations in SMN and the specific loss of motor neurons seen in SMA patients been uncovered. It may be the case that the specificity of this phenotype is reflective of either specialized SMN functions at the NMJ or a particular sensitivity of motor neurons to the loss of SMN activity. Among the genes the genetic strategy revealed as Smn loss of function modifiers was breathless, encoding an FGF receptor, thus establishing a link between Smn and the FGF pathway (Sen, 2011).

Importantly, in addition to this link, it was also found that FGF signaling is independently involved in NMJ morphogenesis, a function demonstrated in vertebrates but not previously attributed to this pathway in Drosophila despite extensive characterization of its essential role in branching morphogenesis of the tracheal system, migration of multiple cell types, as well as the proper patterning of the mesoderm. The morphological effects that were observed, caused by the modulation of several pathway elements, plainly reveal an involvement of FGF signaling at the NMJ, a role confirmed by the electrophysiological analyses. The down-regulation of FGF signals in muscle results in a reduction of bouton numbers and is associated with increased mEJP amplitudes. The opposite effect is observed when FGF signaling is increased in muscles, suggesting that FGF signaling inversely regulates quantal size. Thus, FGF perturbation in muscle alters both presynaptic growth and specific aspects of synaptic transmission. These observations imply the existence of functional trans-synaptic homeostatic mechanisms, which have been previously shown to compensate for similar changes by increasing presynaptic bouton numbers and transmitter release. However, in this specific instance, only synaptic growth (bouton number) but not transmitter release (quantal content) is affected, the precise mechanisms for which remain unclear. Moreover, the fact that mEJP amplitudes are affected suggests that postsynaptic receptivity to glutamate release from the presynapse is altered. Similar quantal size phenotypes have been observed in several instances previously. For instance, postsynaptic PKA and NF-kappaB are known to regulate quantal size through changes in DGluRs. Directly altering the expression of various GluR subunits also predictably influences quantal size. The genetic interaction this study has demonstrated between FGF and Smn can be described as an epistatic relationship in which the FGF pathway functions downstream of Smn and is consistent with the observation that neuromuscular defects associated with loss of Smn function in muscle can be rescued by muscle-specific activation of FGF signaling. Intriguingly, the relationship described in this study between Smn and FGF is valid beyond the NMJ, as loss of Smn function genetic mosaics in the wing disc clearly result in the down-regulation of FGF signaling. Although the precise molecular mechanism underlying this relationship is still elusive, Smn activity affects transcript and protein levels of the FGF receptor, as well as the expression of additional elements of the FGF pathway. Whether this defines a cascade of interrelated events or whether each of these changes reflects an independent Smn-related regulatory event remains to be determined. Given the fact that Smn mutants in Drosophila display altered postsynaptic currents and severely compromised postsynaptic receptor clustering in muscles, it is conceivable that FGF signaling represents a link between Smn activity and postsynaptic glutamate receptor levels (Sen, 2011).

It should be noted that a link between SMN and the FGF pathway has been suggested by a series of studies in vertebrates where a molecular interaction between an FGF-2 isoform and the SMN protein has been described.These studies raise the possibility that FGF-2 may negatively interfere with SMN complex function through SMN itself. Such observations would, on first appearance, suggest that the epistatic relationship between SMN and FGF signaling in vertebrate cells may be the reverse of what was observed in Drosophila. In point of fact however, the differences in the experimental parameters and approaches between these studies do not allow meaningful comparisons (Sen, 2011).

An important question raised by the above phenotypic analyses is whether the abnormalities associated with FGF and/or Smn perturbations reflect developmental or maintenance issues. It may be the case that the larval system in Drosophila is not ideally suited to differentiate between these alternatives as larval tissue is destined to undergo programmed cell death (histolysis) during metamorphosis. One advantage that flies do offer, however, is the ability to dissociate the development of the adult neuromuscular system from its maintenance as the entirety of its development occurs during the pupal stage, before emergence of the adult. Thus, the Drosophila pupa/adult may provide a platform to address these issues, as Drosophila displays Smn-dependent adult phenotypes. In light of the relationship that was established between Smn and FGF signaling and the known involvement of FGF signaling in the development of both the larval and adult musculature, it will be particularly interesting to examine the effects of modulating FGF activity on the aforementioned processes. Such studies may be of particular relevance to SMA where it is quite difficult to discern the developmental consequences of SMN loss in humans, as neurodegenerative symptoms displayed by patients may obscure basic problems resulting from altered developmental programs such as neuronal pathfinding, initial NMJ formation, etc (Sen, 2011).

In vertebrates, synaptic development and maintenance use at least three distinct signaling mechanisms: the TGF-β, wingless, and FGF pathways. In Drosophila, it is noteworthy that the first two have been demonstrated to function in a similar fashion at the NMJ. Remarkably, the genetic screens involving Smn have identified elements of all three of these pathways as modifiers of Smn-related phenotypes. These connections are considered particularly significant as they raise the possibility that Smn may serve as a node, integrating signaling events crucial for NMJ function, potentially leaving this structure particularly vulnerable to the loss of Smn. Though further correspondence between the Drosophila model and the human condition remains to be determined, the Smn-FGF relationship observed in Drosophila raises the possibility that pharmacological manipulation of FGF signals might mitigate SMN motor neuron-related abnormalities (Sen, 2011).

Mitotic cell rounding accelerates epithelial invagination

Mitotic cells assume a spherical shape by increasing their surface tension and osmotic pressure by extensively reorganizing their interphase actin cytoskeleton into a cortical meshwork and their microtubules into the mitotic spindle. Mitotic entry is known to interfere with tissue morphogenetic events that require cell-shape changes controlled by the interphase cytoskeleton, such as apical constriction. However, this study shows that mitosis plays an active role in the epithelial invagination of the Drosophila tracheal placode. Invagination begins with a slow phase under the control of epidermal growth factor receptor (EGFR) signalling; in this process, the central apically constricted cells, which are surrounded by intercalating cells, form a shallow pit. This slow phase is followed by a fast phase, in which the pit is rapidly depressed, accompanied by mitotic entry, which leads to the internalization of all the cells in the placode. It was found that mitotic cell rounding, but not cell division, of the central cells in the placode is required to accelerate invagination, in conjunction with EGFR-induced myosin II contractility in the surrounding cells. It is proposed that mitotic cell rounding causes the epithelium to buckle under pressure and acts as a switch for morphogenetic transition at the appropriate time (Kondo, 2013).

The invagination of epithelial placodes converts flat sheets into the three-dimensional structures that form complex organs, and it is a key morphogenetic process in animal development. A major mechanism of invagination is apical constriction, which is driven by actomyosin contraction. However, not all constricted cells invaginate, and some cell internalization occurs without apical constriction, suggesting that additional mechanisms of inward cell movement contribute to invagination (Kondo, 2013).

To obtain three-dimensional information about cell behaviour during invagination, live imaging was performed of the Drosophila tracheal placode. Ten pairs of tracheal placodes, each of which is composed of about 40 cells, are formed in the ectoderm at mid-embryogenesis, and each placode initiates invagination simultaneously. Using an adherens junction marker, DE-cadherin-green fluorescent protein (E-cad-GFP), it was found that the adherens junctions of the central placode cells slowly created a depression by apical constriction, which became the tracheal pit. After 30 to 60 min of slow movement (slow phase), the tracheal pit was suddenly enlarged, and the tracheal cells were rapidly internalized (fast phase) and eventually formed L-shaped tube structures (Kondo, 2013).

After the fast transition, all the tracheal cells and surrounding epidermal cells entered mitosis 16, the final round of embryonic mitosis. It was noticed that the fast invagination was always associated with the mitotic entry of central cells that were frequently the first to enter mitosis 16. Intriguingly, mitotic rounding of the central constricted cells occurred simultaneously with the rapid depression of their apices, followed by chromosome condensation 10 min later. In this study, this atypical mitotic rounding associated with apical depression in an internalized cell is called 'rounding', to distinguish it from canonical surface mitosis (surface cell rounding) (Kondo, 2013).

To determine whether cell rounding is required for invagination, zygotic mutants were examined of the cell-cycle gene Cyclin A (CycA), which fail to enter mitosis 16, and double parkeda3 (dupa3), which show a prolonged S phase 16 and delayed entry into mitosis 16. Tracheal invagination was initiated normally in the CycA and dupa3 mutants, but proceeded more slowly than in controls, indicating that entry into mitosis 16 is required for proper timing of the fast phase (Kondo, 2013).

Although delayed, the accelerated invagination in the CycA or dupa3 mutants eventually occurred, allowing the formation of tube structures and suggesting that additional mechanisms are involved. After invagination, fibroblast growth factor (FGF) signalling is activated in the tracheal cells to induce branching morphogenesis through chemotaxis. To examine the contribution of FGF signalling to invagination, mutants of the FGF ligand branchless (bnl) or the FGF receptor breathless (btl) were analyzed. These mutants invaginated normally, indicating that chemoattraction to FGF is dispensable for invagination (Kondo, 2013).

Next, to assess FGF's role in the mitosis-defective condition, double mutants were analyzed for CycA and bnl or CycA and btl, and it was found that they showed slower invagination than CycA single mutants. Furthermore, the invagination in these double mutants was incomplete, in that the cells failed to form L-shaped tubular structures. Therefore, FGF signalling is critical for invagination when mitosis is blocked, serving a back-up role. Tracheal-specific CycA expression rescued the defects in invagination speed and tube structure in the CycA btl mutants. In addition, mitosis of cells outside the pit was occasionally observed that occurred before the mitosis of the central apically constricted cells and was not correlated with the fast invagination phase. Thus, mitosis of the surrounding epidermal cells is dispensable for tracheal invagination. Taken together, it is concluded that mitotic entry of central cells is a major mechanism for accelerating tracheal invagination (Kondo, 2013).

To distinguish the role of cell rounding from that of cell division in the fast phase, the microtubule inhibitor colchicine was used to arrest the cell cycle after cell rounding. Colchicine treatment after mitosis 15 induced M-phase arrest at mitosis 16, but the fast invagination movement accompanied by cell rounding was not affected. This result indicates that cell rounding, but not cell division, is responsible for the acceleration phase of the tracheal invagination (Kondo, 2013).

Mitosis of cells in the columnar epithelium normally occurs at the apical surface after surface rounding. It was next asked how the apical surface of the central cells becomes depressed during internalized cell rounding. One possible model explains internalized cell rounding as cell-autonomously controlled by the association of the cells with the basement membrane or underlying mesodermal cells. However, genetic removal of basement-membrane adhesion by the maternal and zygotic mutation of βPS-integrin (also known as mys) did not compromise the speed of invagination, and snail-twist double-mutant embryos, which lack mesodermal cells, still showed tracheal invagination with internalized cell rounding. These results suggest that anchoring to the basal side is probably not required (Kondo, 2013).

A second model proposes that the apical depression of the rounding cells is driven by local planar interactions among the tracheal cells. Before and during tracheal invagination, myosin II is enriched at the cell boundaries tangential to the centre of the placode and regulates cell intercalation. It was noted that the myosin II level in the central cells was lower than in the surrounding, intercalating cells. Nevertheless, the apices of the central cells were constricted during the slow phase, strongly suggesting that the surrounding cells exerted centripetal pressure on the central cells through myosin II cables. Myosin II cables fail to form in EGFR signalling mutants (such as rho, the rhomboid endopeptidase required for EGF ligand maturation, and Egfr), and apical constriction is impaired in these mutants. The first few cells undergoing mitosis 16 in the tracheal placode of rho or Egfr mutants showed surface cell rounding with expanded apices, indicating that EGFR signalling is required to couple the mitotic cell rounding with fast apical depression. It is speculated that the columnar shape of the central cells resists centripetal movements, resulting in the accumulation of inward pressure during the slow phase. The existence of such resistance was supported by the results of a physical perturbation experiment using a pulsed ultraviolet lase. The cell rounding associated with mitotic entry would release the stored inward pressure by means of cytoskeletal remodelling that causes rapid depression of apical surface together with the active shortening of cell height, leading to rapid buckling of the apical surface and the fast phase of invagination (Kondo, 2013).

Even with the loss of both EGFR and FGF signalling, the tracheal placodes form moderately invaginated structures, compared to the flat tracheal placode observed in the rho-bnl-CycA triple mutant at the same stage, indicating that cells needed to undergo mitosis 16 to induce invagination, independent of EGFR and FGF signalling. In rho bnl double mutants, although the cells undergoing the earliest mitoses showed surface cell rounding, some of the subsequent mitotic events were coupled to apical depression and internalized cell rounding. Unlike the earlier mitotic events on the surface, the internalized rounding cells in the rho bnl embryos showed constricted apices and were surrounded by apically rounded cells before mitosis. Internalized rounding with a constricted apical surface were shared properties of cells in mitoses leading to invagination, in both control and rho bnl embryos. It is suggested that the first few cells undergoing surface cell rounding compress the adjacent interphase cells and restrict their apical area, so that they are forced to move internally after rounding, causing the epithelial layer to buckle and invaginate (Kondo, 2013).

Although invagination was largely blocked in the rho-bnl-CycA triple mutants, any double mutant combination permitted invagination to some degree, indicating that three qualitatively distinct mechanisms, mitotic cell rounding, myosin II contractility (EGFR) and active cell motility (FGFR), can independently trigger invagination. In the normal context of wild-type development the combination of cell rounding and EGFR signalling may optimize the timing and speed of invagination, and then invaginated tracheal sacs subsequently respond to FGF emanating from several target tissues guiding branching morphogenesis (Kondo, 2013).

These observations demonstrates a new role for mitosis in tissue morphogenesis to generate mechanical force through cell rounding, independent of cell division. This is distinct from previously described invagination mechanisms involving cell-autonomous constriction by the apical activation of actomyosin contractility, which is incompatible with mitosis. Mitosis 16 outside the tracheal placode occurs in clusters on the ectoderm surface, but does not lead to invagination, suggesting that the tracheal placode is sensitized to invaginate upon mitosis, independent of EGFR and FGFR signalling. Future research to uncover the properties of the tracheal placode that enables it to respond to clustered mitosis will explain not only this new mode of morphogenesis, but also the homeostasis mechanisms of epithelial architecture (Kondo, 2013).

Progenitor outgrowth from the niche in Drosophila trachea is guided by FGF from decaying branches

Although there has been progress identifying adult stem and progenitor cells and the signals that control their proliferation and differentiation, little is known about the substrates and signals that guide them out of their niche. By examining Drosophila tracheal outgrowth during metamorphosis, this study showed that progenitors follow a stereotyped path out of the niche, tracking along a subset of tracheal branches destined for destruction. The embryonic tracheal inducer branchless FGF (fibroblast growth factor) is expressed dynamically just ahead of progenitor outgrowth in decaying branches. Knockdown of branchless abrogates progenitor outgrowth, whereas misexpression redirects it. Thus, reactivation of an embryonic tracheal inducer in decaying branches directs outgrowth of progenitors that replace them. This explains how the structure of a newly generated tissue is coordinated with that of the old (Chen, 2014).

Many adult stem cells reside in specific anatomical locations, or niches, and are activated during tissue homeostasis and after injury. Although considerable effort has been made to identify factors that control stem cell proliferation and differentiation, how stem or progenitor cells move out of the niche and how they form new tissue are not well understood. Tissue formation in mature animals faces challenges not present in the embryo. The new cells migrate longer distances and navigate around and integrate into a complex milieu of differentiated tissues. This work investigated the substratum and signals that guide Drosophila tracheal imaginal progenitor cells into the posterior during metamorphosis to form the pupal abdominal tracheae (PAT) that replace the posterior half of the larval tracheal system (tracheal metameres Tr6 to Tr10), which decays at this time (Chen, 2014).

The PAT extend from the transverse connective (TC) branches in Tr4 and Tr5. Each PAT consists of a multicellular stalk with many secondary branches, each of which has dozens of terminal cells that form numerous fine terminal branches (tracheoles). There are two known tracheal progenitor populations at metamorphosis: dedifferentiated larval tracheal cells and spiracular branch (SB) imaginal tracheal cells set aside during embryonic tracheal development. Lineage tracing showed that PAT derive from imaginal progenitors (Chen, 2014).

To determine how progenitors in Tr4 and Tr5 reach the posterior, a btl-RFP-moe transgene (RFP, red fluorescent protein) was used to label activated progenitor cells, and ppk4>GFP (GFP, green fluorescent protein) was used to label larval tracheal branches. Before metamorphosis, there are 7 to 10 quiescent progenitor cells in each SB niche. In early third larval instar (L3), progenitors proliferate but remain in the niche. Later in L3, progenitors leave the niche, moving onto the larval TC branches toward the dorsal trunk (DT), while progenitors within the niche continue to proliferate. Progenitors in other metameres also proliferate but do not move out of the niche. Migrating progenitors in Tr4 and Tr5 crawl along the basal surface of larval tracheal cells, with cytoplasmic projections emanating from cells at the leading edges of the progenitor cluster. Progenitors maintain epithelial polarity and a lumen continuous with the SB and TC branches, forming a saclike structure. By wandering L3, progenitors reach the DT, where they pause (~12 hours) until the onset of puparium formation (Chen, 2014).

Around 1 hour after puparium formation (APF), progenitors move onto the DT and turn posteriorly. Posterior migration continues for 9 hours, extending half the animal's length (~0.8 mm) past Tr9. Live imaging showed that progenitors move at ~1.7 μm/min, crawling along and wrapping around the DT as they migrate (Chen, 2014).

Differentiation begins as progenitors migrate. At the beginning of puparium formation (0 hours APF), a subset of progenitors that have exited the niche begins to express the terminal cell master regulator Pruned (Blistered) SRF (serum response factor), initiating cell specialization). As progenitors migrate along the DT, budlike structures composed of Pruned-expressing cells are detected at the tips of progenitor clusters, whereas Pruned-negative cells form the stalks of new trachea. By 6 hours APF, Pruned-expressing progenitors in the tips adopt an elongated and differentiated morphology, flattening along the DT as they extend further posteriorly. Around 13 hours APF, the PAT mature and fill with gas as posterior tracheal branches collapse (Chen, 2014).

What guides tracheal progenitors on their stereotyped path along specific branches of the larval tracheal system? Expression of breathless (btl) FGFR (fibroblast growth factor receptor) is induced in PAT progenitors, as shown by the btl-RFP-moe reporter. Whether the Btl pathway, which directs tracheal branch outgrowth in embryos and larvae and induces adult air-sac primordium formation, is involved was tested. Expression of dominant-negative Btl FGFR in the progenitors and their descendants blocked migration and diminished or eliminated PAT formation. To determine the source of the only known Btl ligand, Branchless (Bnl) FGF, a bnl reporter, bnl-Gal4 enhancer trap line NP2211 driving UAS-GFP was used. Unlike previously described examples of tracheal outgrowth, bnl was not expressed in surrounding tissue. Instead, it was expressed within the tracheal system, specifically by larval tracheal cells along which progenitors migrate. The expression pattern is dynamic and precise, almost perfectly matching the positions and timing of progenitor migration. In L3 animals, when progenitors are observed along the TC branches, bnl>GFP was expressed in TC larval cells in Tr4 and Tr5, but not in other metameres. Shortly after puparium formation, when PAT progenitors turn to migrate toward the posterior, DT larval cells in the segment just posterior to PAT progenitors express bnl>GFP. As progenitors continue along the DT, DT larval cells activate bnl>GFP expression one segment at a time from anterior to posterior, matching progenitor movement (Chen, 2014).

This dynamic bnl expression along the migration path is required for progenitor outgrowth. Knockdown of bnl expression by RNA interference (RNAi) in larval tracheal cells blocked migration and resulted in diminished or absent PAT. Mosaic expression of bnl RNAi in small patches along the path also arrested migration, so long as the patch encompassed the full DT circumference. Thus, Bnl is required all along the migration path, and the signal does not cross even short gaps (Chen, 2014).

Ectopic bnl expression in GFP-labeled clones of larval tracheal cells induced by dfr-FLP redirected progenitor migration. Depending on the location of the clones, ectopic bnl caused incorrect exit from the niche, premature entry onto the DT, or wrong turns on the DT. Dual clones induced bifurcation with groups of progenitors moving toward each ectopic bnl source. Clones in Tr3 and posterior metameres caused progenitors in these regions to leave the niche, even though they do not normally do so. When there was a large clone, progenitors migrated throughout the clone, implying that progenitors do not require a gradient and will spread to cover an entire region of cells expressing bnl at equivalent levels. When bnl-expressing clones failed to induce migration, the clones appeared to be too far from the progenitors or there was competition from another clone close by. Ectopic bnl expression within the progenitor cluster arrested migration (Chen, 2014).

The results show that the embryonic tracheal inducer Bnl FGF guides tracheal progenitors out of the niche and into the posterior during tracheal metamorphosis. The source of Bnl is the larval tracheal branches destined for destruction, which serve both as the source of the chemoattractant and as the substratum for progenitor migration. Several days earlier in embryos, these larval tracheal branches were themselves induced by Bnl provided by neighboring tissues. But after embryonic development, most tracheal cells, including those in the decaying larval branches, down-regulate btl FGFR expression and thus do not respond to (or sequester) the Bnl signal they later express. One of the most notable aspects of this larval Bnl is its exquisitely specific pattern in decaying larval branches, which presages progenitor outgrowth. It is unclear how Bnl expression is controlled, though it does not appear to require signals from migrating progenitors because the bnl reporter expression front progressed normally when progenitor outgrowth was stalled by a tracheal break. Perhaps expression of Bnl involves gradients in the tracheal system or spatial patterning cues established during embryonic development in conjunction with temporal signals mediated by molting hormones (Chen, 2014).

Because the signal guiding progenitor migration is provided by tracheae destined for destruction, progenitors become positioned along the larval branches they replace. Perhaps during tissue repair and homeostasis, recruitment of adult stem or progenitor cells from the niche is similarly guided by signals from decaying tissue, thereby ensuring that new tissue is directed to the appropriate sites (Chen, 2014).


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

date revised: 15 October 2013

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