Two types of sensory organs, large bristles (macrochaetes) and small bristles (microchaetes), develop in fixed numbers at constant positions on the dorsal part of the mesothorax (also called the notum) of Drosophila. The accurate positioning of the macrochaetes is established within the epithelial sheets of the notum region of the wing imaginal discs during the third larval to early pupal stage. For convenience, this region of the wing disc will be referred to as the 'thoracic disc' to distinguish it from the wing pouch region. Initially, in the thoracic disc, group of cells (termed proneural clusters) are formed and characterized by the expression of the proneural genes achaete (ac) and scute (sc). These proneural clusters form around the positions where macrochaetes will form. Next, one or a few sensory mother cells (SMCs) are singled out from the proneural cluster, and each SMC subsequently undergoes two rounds of cell division to form four progeny cells that differentiate into the components of a sensory bristle. Thus, precise positioning of the macrochaete on the notum depends on the complex expression pattern of the ac and sc genes in the thoracic disc. ac and sc expression patterns are controlled through the action of enhancer-like cis-regulatory elements. These elements are presumed to respond to a prepattern established by local specific combinations of factors. The identity of these pattern producing factors is largely unknown (Tomoyasu, 1998).

Two large bristles, an anterior-dorsocentral bristle (aDC) and a posterior-dorsocentral bristle (pDC) are formed along the anterior/posterior (A/P) axis on the notum. It has been shown that wg activity is necessary for the formation of both aDC and pDC. Wingless is expressed in an anterior-dorsal (medial) to posterior-ventral (lateral) stripe in the thoracic discs. However, the SMCs are not induced all along the wg expression domain, but induced only adjacent to the dorsal posterior side, behind the wg expression domain in the anterior compartment of the thoracic disc. This suggests that Wg signaling alone is insufficient to induce SMCs in aDCs and pDCs, and that another factor(s), which resides on the dorsal posterior side of the thoracic disc, is also required for inducing these SMCs. One candidate factor is Dpp. In the thoracic disc, Dpp is induced in a stripe of cells located posterior to the dorsocentral SMCs. This expression pattern and the property of Dpp as a morphogen suggests that Dpp signaling may also participate in prepattern formation of the macrochaetes on the notum (Tomoyasu, 1998).

The role of Dpp signaling in dorsocentral bristle formation has been examined by either ectopically activating or conditionally reducing Dpp signaling. Ubiquitous activation of Dpp signaling in the notum region of the wing imaginal disc induces additional dorsocentral proneural clusters all along the dorsal side of the wg expression domain, and alters wg expression. Conditional loss-of-function of Dpp signaling during disc development results in the inhibition of dorsocentral proneural cluster formation and expansion of the wg expression domain. These results suggest that Dpp signaling has two indispensable roles in dorsocentral bristle formation: induction of the dorsocentral proneural cluster in cooperation with Wg signaling and restriction of the wg expression domain in the notum region of the wing imaginal disc (Tomoyasu, 1998).

There is a substantial distance between dorsocentral SMCs and the dpp expression domain in wild-type discs. One explanation for the existence of this gap is that the highest level of Dpp signaling inhibits the formation of proneural clusters. A down shift of the Dpp activity slope would release the area in which proneural induction is inhibited by the highest levels of Dpp signaling. The mechanism by which the highest levels of Dpp signaling inhibits proneural induction is unclear and should be studied at the molecular level. It is worth noting that the effective range of wg from its source for proneural cluster induction seem to be different from that of dpp. The dorsocentral proneural cluster is formed within approximately five cell diameters from the wg expression domain, whereas it can be formed more than ten cell diameters from the dpp source. This difference must contribute to the oval shape of the proneural cluster, which is longest along the A/P axis. wg expression is not uniform in the notal stripe: it is lower at the A/P compartment border. It is possible that the difference in wg expression levels along the A/P axis also affects the precise positioning of the dorsocentral proneural cluster (Tomoyasu, 1998).

The secreted signaling protein Dpp acts as a morphogen to pattern the anterior-posterior axis of the Drosophila wing. Dpp activity is required in all cells of the developing wing imaginal disc, but the ligand gradient that supports this activity has not been characterized. A biologically active form of Dpp tagged with GFP was used to examine the ligand gradient. Dpp-GFP forms an unstable extracellular gradient that spreads rapidly in the wing disc. The activity gradient visualized by MAD phosphorylation differs in shape from the ligand gradient. The pMAD gradient adjusts to compartment size when this is experimentally altered. These observations suggest that the Dpp activity gradient may be shaped at the level of receptor activation (Teleman, 2000).

Two modes of active transport have been considered. A possible function for cytonemes might be to transport Dpp from the center of the disc to distant cells. Cytonemes are located in the lumen of the disc, which corresponds to the apical side of the epithelium. Dpp-GFP is not concentrated on the apical side of the wing disc epithelium, as might be expected if cytonemes were involved in Dpp transport. Observations suggest that most of the Dpp-GFP is basolateral. It has also been proposed that receptor-mediated endocytosis plays a role in Dpp gradient formation. According to this view, cells would transport Dpp by repeated cycles of endocytosis and resecretion. Colocalization of Tkv with an endocytic marker suggests that these spots of Tkv represent endocytic vesicles. Thus, the spots of Dpp-GFP could reflect intermediates in the transport process. Alternatively, these vesicles might reflect Dpp targeted for intracellular degradation. With the caveat that both Dpp-GFP and Tkv spots are difficult to visualize, the fact that some Dpp-GFP accumulations colocalize with a Tkv spot, and some do not, might reflect segregation of a recycling receptor and a ligand destined for degradation. The data do not allow for distinguishing between endocytosis-mediated transport and movement through the extracellular space by diffusion (Teleman, 2000).

The Dpp activity gradient gauged by MAD phosphorylation forms a plateau in both A and P compartments and then drops off abruptly. This abrupt transition in pMAD levels does not coincide with abrupt changes in either Dpp ligand or Tkv receptor levels. This observation suggests that additional factors contribute to shaping the activity gradient. Possible modulators include the inhibitory SMAD (Dad) which is induced by Dpp signaling, other Dpp receptors (for example Saxophone), or the levels of the adaptor protein SARA, which has been shown to enhance Smad2/TGF-beta receptor interactions. Alternatively, abrupt transitions are seen when reactions are cooperative. For instance, Dpp receptor binding may be cooperative, or Tkv phosphorylation of MAD may require a cooperative step such as receptor clustering (Teleman, 2000).

The Dpp activity gradient has a remarkable ability to compensate for altered compartment size. Use was made of transgenes expressing constitutively active or dominant negative forms of PI3-kinase. Overexpression of an activated form of PI3K causes tissue overgrowth whereas overexpression of a dominant-negative form causes tissue undergrowth. Compartments can differ by almost a factor of two in size and yet contain all normal pattern elements. Over a broad range of compartment sizes Dpp activity levels are maximal in the center of the disc and minimal at the edge of the compartment. Since adult wings of different sizes are normally patterned, it was expected that the Dpp patterning system would adjust for different tissue sizes at some point in development. Size compensation occurs remarkably early in the Dpp signal transduction cascade, at the level of receptor activation. Presumably, this may result in a continuous coordination between tissue size and patterning. This also suggests that regulators of Dpp target gene expression, such as Brinker, are not responsible for size accommodation, but rather that levels of the Dpp 'upstream signaling components' -- Dpp, Tkv, SARA, Dad, and MAD -- sense and adjust their levels to compartment size (Teleman, 2000).

To examine this problem in a theoretical way, the state of the Dpp activity gradient is considered in two cells that are equidistant from the Dpp source in compartments that are growing at different rates. It is possible for cell A in the larger, faster growing compartment, to have high levels of MAD phosphorylation while cell B does not. There are two differences between cells A and B that can be used to suggest models for how this might be accomplished. (1) Cell A belongs to a faster growing cell population than cell B. If the rate of cell growth and division could influence how quickly cells change their responsiveness to Dpp signaling, the rate of growth might be able to affect the shape of the activity gradient. However, it is found that the presence of a large clone of quickly growing cells does not locally perturb the Dpp activity gradient, suggesting that growth rate per se is not responsible for size accommodation. (2) Cell B is closer to the lateral edge of the disc than cell A. If it is assumed that the edge of the disc provides a sink with a high capacity to degrade or inactivate Dpp, cell B will experience a lower level of Dpp than cell A, despite being equidistant from the source. If Thickveins is involved in Dpp downregulation, the high level in lateral cells might facilitate removal of Dpp by receptor-mediated endocytosis. thickveins has already been shown to limit the effective range of Dpp in the disc, and a correlation between higher Tkv levels in the posterior and a steeper Dpp-GFP gradient is seen. For this mechanism to work it must be assumed that Dpp is not able to downregulate Thickveins laterally. The lateral sink need not degrade Dpp. Other mechanisms for removing Dpp activity laterally could involve secreted antagonists (Teleman, 2000).

It has previously been shown that expression of Dpp target genes spreads slowly around Dpp-expressing clones in the lateral regions of the wing disc. This has been proposed to reflect slow movement of Dpp ligand due to high levels of receptor expression. The current observations indicate that Dpp can, in principle, move rapidly. According to one model, the slow spread of target gene induction in the lateral region might reflect the time required for Dpp-dependent downregulation of the repressor Brinker in tissue surrounding the clones. Alternatively, it might reflect the time needed to accumulate sufficient Dpp in a region where Dpp is rapidly inactivated or cleared from the disc (Teleman, 2000).

Secreted morphogens such as Decapentaplegic are thought to spread through target tissues and form long-range concentration gradients providing positional information. Using a GFP-Dpp fusion, Dpp trafficking was monitored in situ throughout the target tissue during the formation of a long-range concentration gradient. Evidence is presented that long-range Dpp movement involves Dpp receptor and Dynamin functions. The rates of endocytic trafficking and degradation determine Dpp signaling range. A model is suggested wherein the gradient is formed via intracellular trafficking initiated by receptor-mediated endocytosis of the ligand in receiving cells with the gradient slope controlled by endocytic sorting of Dpp toward recycling versus degradation (Entchev, 2000).

Based on the slow expansion of the spalt expression domain during the last three days of larval development, formation of the Dpp gradient has been suggested to be a long-term process. The GFP-Dpp pattern was monitored during different larval stages. During second instar, GFP-Dpp is found only 5 cell diameters away from its source. During early third instar, the gradient is expanded to 10 cells and in late third instar larva, to 25 cells. Thus, as the wing grows, the range of the Dpp gradient expands slowly. The rate is less than 2 cells per 5 hr (around 25 cells in 3 days), consistent with the rate of expansion of Dpp signaling range during development (Entchev, 2000).

Data showing the accumulation of GFP-Dpp at the side facing the source in the cell clones lacking Tkv and the lack of GFP-Dpp behind the Dynamin-defective clones suggest that receptor-mediated endocytosis of Dpp is essential for the long-range gradient formation. Nondirectional rapid movement of Dpp is seen in the wing epithelium. Therefore, no stable shadow is predicted behind the shi^ts1 clones. Consistent with this, shadows can be seen only under dynamic conditions, when confronting a wave of GFP-Dpp with the shi^ts1 mutant clone. Thus, visualization of GFP-Dpp while traveling through the tissue reveals a role for endocytosis during Dpp transmissionL: this would not have been possible by looking only at expression of the target genes. These data are therefore consistent with a model where Dpp diffusion is limited by extracellular factors and its long-range distribution is mediated by planar transcytosis initiated by Dpp endocytosis (Entchev, 2000).

A mutant of the small GTPase Rab7 (DRab7) was genereated. Rab7 targets endocytic cargo from the early to the late endosome and lysosome for degradation. DRab7 accumulates at a ring-shaped late endosomal structure in the developing wing cells. Overexpression of GFP-DRab7 causes enhanced late endosomal sorting of Texas-red dextran. This phenotype is enhanced by the expression of DRab7Q67L, a dominant gain-of-function mutant blocked in the active GTP-bound state. This indicates that DRab7 controls the sorting of endocytic cargo toward the late endosome. DRab7 mutants can therefore serve as a tool to study late endosomal trafficking of Dpp and to establish the role of this trafficking step in Dpp signaling (Entchev, 2000).

Whether expression of DRab7Q67L causes enhanced degradation of Dpp was addressed. In wild-type secreting cells, endocytosed GFP-Dpp accumulates in vesicular structures which colocalize with internalized Texas-red dextran. GFP-Dpp can also be detected both in the cytoplasm and in vesicular structures that do not colocalize with Texas-red dextran, corresponding to GFP-Dpp which is trafficking through the secretory pathway. GFP-Dpp and DRab7Q67L were coexpressed in the secreting cells. In these cells, cytosolic GFP-Dpp, is found at normal levels, whereas internalized GFP-Dpp is found at much lower levels and cannot be distinguished from cytosolic GFP-Dpp. Furthermore, endocytosed GFP-Dpp is found in the receiving cells, which do not express DRab7Q67L, indicating that secretion of GFP-Dpp from the DRab7Q67L cells is not affected. This is consistent with the proposal that degradation of endocytosed GFP-Dpp is dependent on DRab7 activity (Entchev, 2000).

Developing cells acquire positional information by reading the graded distribution of morphogens. In Drosophila, the Dpp morphogen forms a long-range concentration gradient by spreading from a restricted source in the developing wing. It has been assumed that Dpp spreads by extracellular diffusion. Under this assumption, the main role of endocytosis in gradient formation is to downregulate receptors at the cell surface. These surface receptors bind to the ligand and thereby interfere with its long-range movement. Recent experiments indicate that Dpp spreading is mediated by Dynamin-dependent endocytosis in the target tissue, suggesting that extracellular diffusion alone cannot account for Dpp dispersal. A theoretical study of a model for morphogen spreading was performed based on extracellular diffusion, which takes into account receptor binding and trafficking. Profiles of ligand and surface receptors obtained in this model were compared with experimental data. To this end, the pool of surface receptors and extracellular Dpp was monitored directly with specific antibodies. It is concluded that current models considering pure extracellular diffusion cannot explain the observed role of endocytosis during Dpp long-range movement (Kruse, 2004).

The subdivision of the developing Drosophila wing into anterior (A) and posterior (P) compartments is important for its development. The activities of the selector genes engrailed and invected in posterior cells and the transduction of the Hedgehog signal in anterior cells are required for maintaining the A/P boundary. Based on a previous study, it has been proposed that the signaling molecule Decapentaplegic (Dpp) is also important for this function by signaling from anterior to posterior cells. However, it has not been known whether and in which cells Dpp signal transduction is required for maintaining the A/P boundary. The role of the Dpp signal transduction pathway and the epistatic relationship of Dpp and Hedgehog signaling in maintaining the A/P boundary has been analyzed by clonal analysis. A transcriptional response to Dpp involving the T-box protein Optomotor-blind is required to maintain the A/P boundary. Further, Dpp signal transduction is required in anterior cells, but not in posterior cells, indicating that anterior to posterior signaling by Dpp is not important for maintaining the A/P boundary. Finally, evidence is provided that Dpp signaling acts downstream of or in parallel with Hedgehog signaling to maintain the A/P boundary. It is proposed that Dpp signaling is required for anterior cells to interpret the Hedgehog signal in order to specify segregation properties important for maintaining the A/P boundary (Shen, 2005).

For many years, it was thought that En and Inv regulated the segregation of A and P cells by specifying a P-type cell segregation in a cell-autonomous fashion. Recent work has challenged this view by showing that a unidirectional Hh-mediated signal from P to A cells is required to specify the A-type segregation behavior of A cells and that the role of En and Inv is mainly to control Hh signaling. Based on the findings that A cells signal back to P cells via Dpp and that wings from flies hypomorphic for dpp have a distorted A/P boundary, it has been proposed that A to P signaling by Dpp might also be important to maintain the A/P boundary. However, whether Dpp signal transduction is required for the maintenance of the A/P boundary and in which cells the Dpp signal is required remained unknown. By analyzing clones mutant for tkv, mad, and omb, several independent lines of evidence are provided that Dpp signal transduction is required to maintain the A/P boundary and that it is only required in A cells, but not in P cells. Thus, the results do not support the hypothesis that A to P signaling by Dpp is required to maintain the A/P boundary. Instead, the results suggest that Dpp signaling within Dpp-producing A cells is required to maintain the A/P boundary (Shen, 2005).

Through analysis of mutant clones located at the A/P boundary lacking the activity of the type I Dpp receptor Tkv, evidence is provided that the reception of the Dpp signal in A cells is required to maintain the A/P boundary. When generated in the P compartment, a few tkv⁻bsk⁻ clones displace the A/P boundary to a small extent: this is attributed to the unusual round shape of these clones. However, the majority of P tkv⁻bsk⁻ clones do not displace the A/P boundary, suggesting that the reception of the Dpp signal is not required in P cells to maintain the A/P boundary. In contrast, mutant clones generated in the A compartment at the A/P boundary displace the position of the A/P boundary toward P, indicating that the reception of the Dpp signal is required in A cells to maintain the A/P boundary (Shen, 2005).

How does the reception of the Dpp signal control cell segregation at the A/P boundary? Although the molecular basis is unknown, a cell's segregation behavior presumably depends on its cytoskeletal or surface properties (cell affinity). Members of the TGFβ superfamily have been observed in other systems to be able to activate regulators of the actin cytoskeleton independently of Mad/Smad transcription factors, raising the possibility that Dpp reception could control cell segregation by directly altering structural components of the responding cells. Alternatively, Dpp could control the segregation of cells by regulating the transcription of one or several target genes. To distinguish between these possibilities, the role of downstream components of the Dpp signal transduction pathway were analyzed. Three independent lines of evidence is provided that a transcriptional response to the Dpp signal is required to maintain the A/P boundary. (1) The segregation behaviors of mad⁻bsk⁻ and tkv⁻bsk⁻ clones are indistinguishable. Like tkv⁻bsk⁻ clones, A mad⁻bsk⁻ clones displace the A/P boundary toward P, indicating a role for the transcription factor Mad in A cells to maintain the A/P boundary. (2) mad⁻brk⁻ clones respect the A/P boundary, indicating that repression of brk transcription by Mad is important for normal A/P cell segregation. (3) A omb⁻ clones displace the A/P boundary toward P. The frequency and extent of the boundary displacement of A omb⁻, tkv⁻bsk⁻, and mad⁻bsk⁻ clones is comparable, suggesting that the Dpp target gene omb is the main mediator of this aspect of the Dpp signal. In contrast to omb⁻ clones, most A clones mutant for the Dpp target gene sal do not displace the A/P boundary, indicating that sal does not play an important role in maintaining the A/P boundary. Together, these data suggest that the transduction of the Dpp signal controlling the maintenance of the A/P boundary bifurcates at the level of the Dpp target genes (Shen, 2005).

Cells of tkv⁻bsk⁻, mad⁻bsk⁻, and omb⁻ clones displacing the A/P boundary do not appear to intermingle well with P cells. In fact, within the entire wing disc pouch, these mutant clones have a round shape and smooth borders, suggesting that these mutant cells in general do not intermingle freely with wild-type cells. Similar clone shapes have been reported upon mutation or misexpression of several genes, including mutants in the Dpp target gene sal and misexpression of a constitutively active form of Tkv. The round shapes and smooth borders of clones have been attributed to differences in the affinity of clone cells for their neighbors, suggesting that Tkv, Mad, and the Dpp target genes omb and sal may affect some aspects of wing pouch cell affinity. Therefore, the inability of A tkv⁻bsk⁻, mad⁻bsk⁻, and omb⁻ clones displacing the A/P boundary to intermingle well with P cells is attributed to this particular role (Shen, 2005).

Taken together, this analysis indicates two roles for Dpp signal transduction: (1) it provides some aspects of the cell affinity of both A and P wing pouch cells; (2) it is required in A cells to specify an A cell affinity important for maintaining the A/P boundary. These two roles of Dpp signal transduction could either be related or distinct. The finding that the Dpp target gene sal is required for the first role, but not the second, provides a first indication that these two roles are implemented by partially distinct molecular mechanisms (Shen, 2005).

Programmed cell death or apoptosis plays an important role in the development of multicellular organisms and can also be induced by various stress events. In the Drosophila wing imaginal disc there is little apoptosis in normal development but X-rays can induce high apoptotic levels, which eliminate a large fraction of the disc cells. Nevertheless, irradiated discs form adult patterns of normal size, indicating the existence of compensatory mechanisms. The apoptotic response of the wing disc to X-rays and heat shock has been characterized and also the developmental consequences of compromising apoptosis. The caspase inhibitor P35 was used to prevent the death of apoptotic cells; it causes increased non-autonomous cell proliferation, invasion of compartments and persistent misexpression of the wingless (wg) and decapentaplegic (dpp) signalling genes. It is proposed that a feature of cells undergoing apoptosis is to activate wg and dpp, probably as part of the mechanism to compensate for cell loss. If apoptotic cells are not eliminated, they continuously emit Wg and Dpp signals, which results in developmental aberrations. It is suggested that a similar process of uncoupling apoptosis initiation and cell death may occur during tumour formation in mammalian cells (Pérez-Garijo, 2004).

There are two sets of findings in this report. The first is that cells undergoing apoptosis in the wing disc acquire wg and dpp activity. This can be readily visualised in caspase-inhibited cells that do not die and remain in the disc. The induction of wg and dpp occurred in all the discs examined. During normal apoptosis this expression is transient and is therefore difficult to observe because targeted cells are eliminated rapidly. However, by amplifying wg expression it was possible to show that wg becomes active during normal apoptosis. This result strongly suggests that wg (and by extension dpp) expression is a normal feature of apoptotic cells (Pérez-Garijo, 2004).

The production and emission by the apoptotic cells of the secreted Wg and Dpp signals is probably responsible for the non-autonomous effect on proliferation. These two signals have been shown to control pattern and growth in imaginal discs and therefore may provide a proliferative signal. This mitogenic effect may be responsible for the additional proliferation necessary to compensate for the elimination of apoptotic cells. This provides an explanation for the observation that high levels of induced apoptosis are compatible with final structures of normal size. It might also have a role in generating additional proliferation and signalling during regeneration processes in which the apoptotic programme is likely to be involved. The finding that the Hh pathway is activated during imaginal disc regeneration is also consistent with this possibility (Pérez-Garijo, 2004).

The second set of findings concerns the overall response of compartments to caspase inhibition during apoptosis. These experiments permitted the discrimination of two different aspects of the apoptotic programme: the initiation and execution of apoptosis. By combining pro-apoptotic treatments (X-rays or heat shock) with caspase inhibition the apoptotic programme and cell death can be uncoupled. A particularly interesting consequence of removing death from the apoptotic programme is that it causes a permanent developmental defect. The perdurance of the apoptotic cells generates an abnormal and self-maintained epigenetic programme. It is believed that the reason for this phenomenon lies in the finding that these cells generate the secreted Wg and Dpp signals, which are primary pattern determinants in imaginal discs, although it is conceivable that they may activate other signals as well. The continuous production and emission of these signals by caspase-inhibited cells is expected to produce developmental aberrations and growth defects, especially if apoptotic cells can carry these signals into neighbouring compartments (Pérez-Garijo, 2004).

It is noted that some of the alterations observed after cell death inhibition -- changes of cell size and shape, invasiveness and excess of proliferation -- resembled those of tumorous cells of vertebrates. Since apoptosis inhibition is frequently associated with tumour formation, it could be speculated that some of the cellular transformations leading to tumorogenesis might be provoked not by a series of individual somatic mutations but by the acquisition of an abnormal epigenetic programme triggered by stress events in conditions in which caspase activity is compromised. They could also be caused by the normal developmentally regulated apoptosis when caspase function is defective. It is known that many human cancers are associated with inappropriate activity of the Hh or the Wnt pathway. These two pathways are misexpressed in apoptotic caspase-inhibited cells (Pérez-Garijo, 2004).

In addition, a number of animal viruses are known to promote oncogenic transformations in host mammalian cells. Because some viruses encode caspase inhibitors to prevent death of the host cells (and the baculovirus P35 protein is a typical case), it is possible that some virus infections provoke a process similar to the one reported in this study -- the initiation of the apoptotic pathway in host cells coupled with inhibition of cell death. This may produce abnormal signalling of growth factors, which may result in the acquisition of a permanent and abnormal epigenetic programme by groups of cells (Pérez-Garijo, 2004).

In Drosophila, stresses such as x-irradiation or severe heat shock can cause most epidermal cells to die by apoptosis. Yet, the remaining cells recover from such assaults and form normal adult structures, indicating that they undergo extra growth to replace the lost cells. Recent studies of cells in which the cell death pathway is blocked by expression of the caspase inhibitor P35 have raised the possibility that dying cells normally regulate this compensatory growth by serving as transient sources of mitogenic signals. Caspase-inhibited cells that initiate apoptosis do not die. Instead, they persist in an 'undead' state in which they ectopically express the signaling genes decapentaplegic and wingless and induce abnormal growth and proliferation of surrounding tissue. Using mutations to abolish Dpp and/or Wg signaling by such undead cells, it has been shown that Dpp and Wg constitute opposing stimulatory and inhibitory signals that regulate this excess growth and proliferation. Strikingly, when Wg signaling is blocked, unfettered Dpp signaling by undead cells transforms their neighbors into neoplastic tumors, provided that caspase activity is also blocked in the responding cells. This phenomenon may provide a paradigm for the formation of neoplastic tumors in mammalian tissues that are defective in executing the cell death pathway. Specifically, it is suggested that stress events (exposure to chemical mutagens, viral infection, or irradiation) that initiate apoptosis in such tissues generate undead cells, and that imbalances in growth regulatory signals sent by these cells can induce the oncogenic transformation of neighboring cells (Perez-Garijo, 2005).

Cells that initiate apoptosis in response to x-irradiation normally disappear rapidly, but, as shown in this study, they can persist indefinitely with the help of the caspase inhibitor P35 and maintain characteristics of the apoptotic program, such as Hid, Dronc, and Drice activities and ectopic wg and dpp expression. The same is also true of cells that initiate apoptosis in response to severe heat shock, a stress that is unlikely to change the integrity of the genome, in contrast to x-irradiation. Because undead cells can divide, albeit at a much lower rate than live ones, this epigenetic condition seems to be inherited through cell division. Thus, the developmental aberrations induced by undead cells in surrounding tissue are likely caused by their continuing to send mitogenic signals that might normally be sent only transiently by dying cells (Perez-Garijo, 2005).

The results indicate that the ability of undead cells to induce growth and proliferation depends on their being able to send Dpp signal, a finding that fits with the proposal that Dpp normally regulates cell growth and proliferation in the wing disc. Conversely, the excessive growth induced by undead cells within wg^– P35 clones indicates that Wg acts to inhibit the mitogenic action of Dpp, a role consistent with the finding that Wg functions as a growth repressor during some phases of wing development (Perez-Garijo, 2005).

It is proposed that Dpp and Wg regulate the abnormal growth induced by undead cells by exerting opposite stimulatory and inhibitory effects: Dpp promotes cell division whereas Wg inhibits the response to Dpp, thus constraining the production of new cells and limiting the extent of overgrowth. In normal circumstances in which caspase activity is not blocked by P35 expression, apoptotic cells disappear rapidly, and the transient production of Dpp and Wg may play a role in restoring, but not exceeding, the missing cells. When the apoptotic cells are kept alive with P35, the persistent production of Dpp and Wg and their slightly imbalanced effects cause local overproliferation and outgrowth, which are most clearly observed when an entire compartment is affected. If they cannot send the Dpp signal (e.g., because they are mutant for dpp), there is no proliferative stimulus, but if they can send Dpp but not Wg, the growth promoting function of Dpp acts unimpeded and induces a dramatic over-production of tissue (Perez-Garijo, 2005).

This model accounts at least in part for the remarkable capacity of undead cells within wg^– P35 clones to induce tumors: absence of the proposed inhibitory action of Wg would remove a constraint on the growth-promoting action of Dpp. The lack of wg activity itself may also be a growth-promoting factor, because there is evidence that wg normally acts as a dMyc repressor in cells flanking the prospective wing margin; the increased dMyc levels in the absence of wg activity promote cell cycle progression and growth. However, the tumorous behavior of stressed wg^– P35 clones cannot be explained simply by the uninhibited action of Dpp emitted by undead cells or the consequent elevation of dMyc activity in the surrounding cells, because neither ectopic Dpp signaling nor overexpression of dMyc is sufficient to cause neoplastic transformation in the imaginal discs. It is suggested that undead cells send additional signals that act together with Dpp to induce the neoplastic transformation (Perez-Garijo, 2005).

Another prerequisite for the induction of tumors by undead cells seems to be that the responding cells must also be unable to execute the cell death pathway. It is speculated that unregulated growth within wg^– P35 clones may create new cellular stresses both inside and outside the clones that induce secondary apoptotic events. Apoptotic cells outside the clones would be rapidly eliminated, but those within would be protected by P35 and join a growing population of undead cells that become new sources of Dpp and other tumor-inducing factors. The populations of both undead and live cells within the clone would thus expand at the expense of the surrounding wild-type tissue, eventually eliminating all of the cells that do not express P35. Circumstantial evidence in favor of this view is the large number of undead (Hid-expressing) cells found in discs overgrown by wg^– P35 clones. Given that undead cells proliferate at a low rate, it seems likely that at least some if not most of the undead cells in wg^– P35 tumors will have arisen by secondary apoptotic events, rather than by descent from the initial founder population of stress-induced, undead cells (Perez-Garijo, 2005).

Such a mechanism can account for the dramatic expansion of wg^– P35 clones at the expense of surrounding wild-type tissue, once they are seeded by the initial induction of undead cells. However, it does not explain why only P35-expressing cells, and not neighboring wild-type cells, develop neoplastic properties such as the failure to maintain a normal epithelial morphology. This difference in behavior raises the possibility that P35 expression may have additional consequences, aside from the direct block of the cell death pathway, that predispose cells to neoplastic transformation (Perez-Garijo, 2005).

These results have potential implications for models of tumor transformation in mammals. It is normally argued that oncogenesis is a multistep process that requires a number of successive somatic mutations, but there are also indications that, in some instances, the transformation of cancer cells is associated with epigenetic phenomena, that is, heritable changes in gene function not caused by somatic mutations. This study has provided an example in which cell populations that cannot execute the cell death pathway are predisposed to oncogenic transformation by just such an epigenetic event, namely the induction of undead cells in response to cellular stress. In Drosophila, the ability of such undead cells to induce neighboring cells to become tumorous seems to depend on their sending an abnormal balance of growth regulatory signals that up-regulate activity of the proto-oncogene dMyc in neighboring cells. As a consequence, the responding cells behave as supercompetitors that overproliferate and eventually eliminate surrounding wild-type cells. These findings suggest a mechanism for generating neoplastic tumors in caspase-inhibited cells (Perez-Garijo, 2005).

Imaginal discs of Drosophila provide an excellent system with which to study morphogenesis, pattern formation and cell proliferation in an epithelium. Discs are sac-like in structure and are composed of two epithelial layers: an upper peripodial epithelium and lower disc proper (DP). Although development of the disc proper has been studied extensively in terms of cell proliferation, cell signaling mechanisms and pattern formation, little is known about these same processes in the peripodial epithelium (PE), the cell layer opposing the disc proper. This topic was addressed by focusing on morphogenesis, compartmental organization, proliferation and cell lineage of the PE in wing, second thoracic leg (T2) and eye discs. A subset of peripodial cells in different imaginal discs undergo a cuboidal-to-squamous cell shape change at distinct larval stages. This shape change requires both Hedgehog and Decapentapelagic, but not Wingless, signaling. Additionally, squamous morphogenesis shifts the anteroposterior (AP) compartment boundary in the peripodial epithelium relative to the stationary AP boundary in the disc proper. Finally, by lineage tracing cells in the PE, it was surprisingly found that peripodial cells are displaced into the disc proper during larval development and this movement leads to Ubx repression (McClure, 2005).

Little is known about when and how disc cells acquire their diverse morphologies. Although Hh and Dpp are well-known for their roles in cell proliferation and patterning it is known that they are also active in epithelial morphogenesis. In eye discs, Hh is both necessary and sufficient to initiate cell shape changes that occur in the morphogenetic furrow. In wing discs, columnar cells require Dpp signaling for normal cytoskeletal organization, shape and pseudostratified organization. This study describes precisely and for the first time when cuboidal, columnar and squamous cell morphologies arise in the epithelia of different imaginal discs. This study examines how the genesis of different morphologies in imaginal discs are affected by loss of a non-autonomous signal (wg, hh and dpp). Hh-dependent Dpp signaling is shown to be required for squamous morphogenesis in the PE of wing and leg discs. Additionally, Dpp signaling is activated as PE cells transition to a squamous morphology. The results indicate that the establishment of columnar morphology in the DP of wing and leg discs is independent of Dpp signaling activity. Clearly, one question still remains: what is the mechanism which causes DP cells to become columnar? The information from these studies provides, at least, an initial framework of how epithelial morphogenesis occurs in imaginal discs (McClure, 2005).

Since both hh and dpp are expressed in wing and leg discs prior to the onset of squamous morphogenesis in the PE, it is clear that their ability to instruct these shape changes must be regulated by additional temporal signals. An obvious candidate for such a temporal signal is ecdysone, which initiates the onset of the larval molts and adult differentiation. The ecdysone signal is mediated by a heterodimer complex consisting of the ecdysone receptor (EcR) and RXR-homolog Ultraspriacle (Usp). To test whether squamous morphogenesis is triggered by ecdysone signaling, usp^–/– clones were induced and it was found that cells of such clones still exhibited normal cuboidal-to-squamous shape changes. Therefore, the temporal cue(s) that initiate disc morphogenesis is independent of ecdysone signaling and remains unknown (McClure, 2005).

Previous studies document that shape change of epithelial cells can activate certain signaling pathways. Thus, squamous morphogenesis of the PE may enhance planar and/or vertical epithelial signaling to promote growth and patterning of the disc. Two observations were made that support this statement: (1) where PE cells fail to undergo squamous morphogenesis, both the disc and adult wing show an obvious reduction in size; (2) in discs that lack PE-specific Dpp signaling, folded clefts in the presumptive wing blade primordia are consistently apposed to a region of squamous PE cells, suggesting communication between the disc epithelia where shape changes do occur. Alternatively, the aberrant apposition of AP compartment boundaries in the PE and DP, owing to a failure in squamous morphogenesis, may result in epithelial abnormalities such as folded clefts in the DP. Resolving the mechanisms by which cell shape can affect disc growth and pattern will integrate both morphogenetic and signaling processes that are crucial for disc development (McClure, 2005).

A lineage analysis of cells has been performed in the wing disc using Ubx-Gal4, UAS flp and act5C>stop>nuclacZ (Pallavi, 2003). Since Ubx-Gal4 is initially expressed in both disc epithelia prior to the second larval instar, cells of both the PE and DP were marked. This analysis concluded that cells of the PE and DP share a common origin in the disc primordium but later become separate lineages, although cells that make up the PE and DP lineages are never specified. The current results, based on lineage-tracing cells born in the PE, are in overall agreement with these conclusions; however, there are some differences. Although Pallavi (2003) identified cell clones spanning both disc epithelia, it could not be determined when or where these clones were born. Furthermore, clones that encompassed cells from both PE and DP were interpreted as either fusions between two independent clones or as clones that originated early in the embryo before separation of the two lineages (PE and DP) (McClure, 2005).

Using four different methods, it has now been found that cells that originate within the PE produce progeny that are a part of the DP. The MARCM and estrogen-inducible systems were used to perform a clonal analysis specific to cells within the PE. These two methods indicate that cells born within the PE produce daughter cells that contribute to the DP. Additionally, a twinspot clonal analysis leads to a similar conclusion and has the advantage of marking cells more directly than either the MARCM- or estrogen-inducible systems. Thus, this analysis indicates a lineage relationship between margin cells in both the PE and DP, and squamous cells in the wing disc, and provides evidence that together these cells comprise the peripodial lineage (McClure, 2005).

As cells are displaced from the PE and into the DP they lose Ubx expression. The loss of Ubx may cause cells to acquire a more distal fate, forging a possible link between displacement and cell fate changes. Similar dynamic cell movements, along with changes in gene expression, have been observed in the chick during somite segmentation. In addition, cell movements and changes in gene expression, similar to what is described here, have been reported by Weigmann (1999), who observed that leg disc cells born in the most proximal regions of the disc contribute to more distal leg segments. Finally, it is proposed that once PE cells are displaced into the DP they may change their cell fate by altered cell signaling. Displaced cuboidal cells at the margins of the disc receive not only planar signals from both epithelial layers, which they are a part of at different stages in larval development, but also vertical signals from overlying PE cells after displacement into the DP. These new planar and/or vertical signals may lead to Ubx repression. It is suggested that the mechanisms that play a role in the development of the imaginal discs may be functionally similar to mechanisms that regulate primary neurogenesis in vertebrates. Neural plate formation and patterning cues arise from two sources: a horizontal source within the plane of an epithelium and a vertical source that arises from the underlying mesodermal cells. The current study suggests that patterning of the imaginal discs is a much more dynamic process with cells exposed to not only signals within the plane of an epithelium but also vertical signals between disc epithelia (McClure, 2005).

Cytonemes are types of filopodia in the Drosophila wing imaginal disc that are proposed to serve as conduits in which morphogen signaling proteins move between producing and target cells. The specificity was investigated of cytonemes that are made by target cells. Cells in wing discs made cytonemes that responded specifically to Decapentaplegic (Dpp) and cells in eye discs made cytonemes that responded specifically to Spitz (the Drosophila epidermal growth factor protein). Tracheal cells had at least two types: one made in response to Branchless (a Drosophila fibroblast growth factor protein, Bnl), to which they segregate the Bnl receptor, and another to which they segregate the Dpp receptor. It is concluded that cells can make several types of cytonemes, each of which responds specifically to a signaling pathway by means of the selective presence of a particular signaling protein receptor that has been localized to that cytoneme (Roy, 2011).

Cells in developing tissues are influenced by multiple signals that they process and integrate to control cell fate, proliferation, and patterning. An example is in the Drosophila wing imaginal disc, where cells depend on several signaling systems that are intrinsic to the disc. Dpp, Wingless (Wg), Hedgehog (Hh), and epidermal growth factor (EGF) are produced and released by different sets of disc cells, and receipt of these signaling proteins programs their neighbors to develop and grow. The mechanisms by which morphogen signaling proteins influence target cells must ensure both specificity and accuracy, and one possibility is that these proteins transfer at points of direct contact. Imaginal discs are flattened sacs that have a monolayer of columnar cells on one side and squamous peripodial cells on the other. Many cells in wing discs make filopodial extensions that lie along the surfaces of the monolayers, oriented toward morphogen-producing cells. These extensions have been termed cytonemes to denote their appearance as cytoplasmic threads and to distinguish them as specialized structures that polarize toward morphogen-producing regions (Roy, 2011).

In wing discs dissected from third instar larvae, cytonemes can be seen as filaments extending from randomly generated somatic clones engineered to express a fluorescent protein such as soluble, cytoplasmic green fluorescent protein (GFP) or a membrane-bound form such as mCD8:GFP (the extracellular and transmembrane domains of the mouse lymphocyte protein CD8 fused to GFP). To image disc cytonemes, unfixed discs were placed peripodial side down on a coverslip, covered with a 1-mm-square glass, and mounted over a depression slide with the disc hanging from the coverslip. Because fluorescence levels in cytonemes were low relative to background, recorded images were processed to increase intensity and were subjected to de-convolution. Expression of CD8:GFP in wing disc clones revealed cytonemes emanating from both the apical and basal surfaces of columnar cells, as well as from peripodial cells (whose apical and basal surfaces could not be distinguished). Most cytonemes were perpendicular to the anterior/posterior (A/P) axis of the disc and oriented toward the cells that produce Dpp at the A/P compartment border; others were oriented toward the cells that produce Wingless at the dorsal/ventral (D/V) compartment border. Disc-associated myoblasts also had filopodia (Roy, 2011).

In the eye disc, cells in the columnar layer organize into ommatidial clusters as a wave of differentiation [the morphogenetic furrow (MF)] passes from posterior to anterior. A second axis, centered at the equator, is orthogonal to the MF and defines a line of mirror-image symmetry where dorsal and ventral ommatidia are juxtaposed. The columnar cells divide during the third instar period but stop or divide only once after the MF passes. CD8:GFP expression was induced in somatic clones and the columnar cells were examined. Whereas clones of six to eight cells were present on both sides of the MF, only cells anterior to the MF had visible cytonemes. Cytonemes emanating from these clones oriented either toward the axis defined by the MF or toward the axis defined by the equator. Single clones with cytonemes oriented both toward the MF and toward the equator were not observed, and there was no apparent correlation between clone position and cytoneme orientation or cytoneme length. Cells in the peripodial layer of the eye disc also had cytonemes (Roy, 2011).

The EGF pathway is a key signaling system for eye development, and cells in the MF express the EGF protein Spitz (Spi). Because one of the two types of anterior cell cytonemes extended toward the MF and to explore the distribution of membrane-bound receptor proteins, clones were induced that expressed an epidermal growth factor receptor:GFP (EGFR:GFP) fusion protein. Anterior cells expressing EGFR:GFP had cytonemes that oriented toward the MF, and most of these cytonemes had fluorescent puncta; no cytonemes that were marked by EGFR:GFP oriented toward the equator. Other than their 'furrow-only' orientation, the cytonemes marked by EGFR:GFP were similar to those marked by CD8:GFP. In contrast, co-expression of CD8:GFP with (nonfluorescent) EGFR marked both furrow-directed and equator-directed cytonemes. Thus, expression of EGFR:GFP does not eliminate the equator-directed cytonemes, suggesting that the specific localization of EGFR:GFP to furrow-directed cytonemes is not a consequence of ectopic (over)expression of this fusion protein (Roy, 2011).

Evidence that the furrow-directed cytonemes depend on Spi/EGF signaling was obtained by expressing a dominant negative form of EGFR. Although EGFR is required for cell proliferation in the disc, small clones expressing EGFR^DN were recovered that co-expressed EGFR^DN and CD8:GFP; in these clones, only cytonemes that appeared to be randomly oriented were present, indicating that the long, furrow-directed cytonemes may require EGFR signal transduction in the cytoneme-producing cells (Roy, 2011).

Wing disc-associated tracheal cells also make cytonemes. The transverse connective (TC) is a tracheal tube that nestles against the basal surface of the wing disc columnar epithelium and that sprouts a new branch [the air sac primordium (ASP)] during the third instar period in response to Branchless (Bnl) expressed by the wing disc. Tracheal tubes are composed of a monolayer of polarized cells whose apical surfaces line a lumen. Expression of CD8:GFP throughout the trachea (btl-Gal4 UAS-CD8:GFP) made it possible to detect GFP fluorescence in several types of cytonemes emanating from the basal surfaces of the TC and ASP. Cytonemes at the tip of the ASP (length range, 12 to 50 μm; average length of 23 μm) contained the Breathless (Btl); the Drosophila fibrobast growth factor receptor (FGFR) and appeared to contact disc cells that express Bnl. Short cytonemes (length range, 2 to 15 μm; average length of 8.5 μm) extended from the TC cells in the vicinity of the ASP (Roy, 2011).

Tests were carried out to se whether Dpp, Spi, Bnl, and Hh affected wing disc, eye disc, and tracheal cytonemes differentially. Ubiquitous expression of Spi, Bnl, or Hh (induced by heat shock) did not alter the A/P-oriented apical cytonemes in the wing disc, and, in the eye disc, the long cytonemes of the columnar layer were unaltered after ubiquitous expression of Dpp, Bnl, or Hh. In contrast, long oriented cytonemes were absent in wing discs after ubiquitous expression of Dpp, and only short cytonemes that appeared to be randomly oriented were observed. Similarly, 0.5 to 3 hours after cSpi, a constitutively active form of EGF, was expressed ectopically by heat shock induction, clones expressing CD8:GFP in the eye disc had many short cytonemes that lacked apparent directional bias; in contrast to controls, no long cytonemes oriented toward the MF were observed. Cytonemes with normal orientation and length (including MF-directed cytonemes) were present in eye discs that were examined later, 8 hours after a pulse of cSpi expression. To monitor EGFR-containing cytonemes for sensitivity and responsiveness to Spi, cSpi was expressed by heat shock induction, and cells in clones expressing EGFR:GFP were examined. After a pulse of cSpi expression, the extensions oriented outward without apparent directional bias, and the EGFR:GFP puncta were present in all cytonemes (Roy, 2011).

To examine responses of the ASP tip cytonemes, Hh, Spi, Dpp, and Bnl were overexpressed by heat shock and GFP-marked cytonemes at the ASP tip were examined. No differences in number of cytonemes were detected until about 3 hours after heat shock. Four to 5 hours after heat shock, expression of Bnl increased the number of tip cytonemes by ~2.6 times, and although most of the cytonemes were <30 μm, the cytonemes >30 μm also increased (~3.2 times). Most of the long cytonemes in these preparations were oriented in directions other than toward the cells that normally express Bnl. The number of long cytonemes >30 μm did not change after overexpression of Hh, Spi, and Dpp (0.6 to 0.8 times); the number of short cytonemes increased after Dpp overexpression (~1.7 times) but not after overexpression of Hh or Spi (Roy, 2011).

Thus, the responses of apical wing disc cytonemes to overexpressed Dpp, of eye disc cytonemes to ubiquitous Spi, and of ASP tip cytonemes to exogenous Bnl (Drosophila FGF) are similar. These results suggest that the cytonemes detected in the wing discs and eye discs may have orientations and lengths that are dependent specifically on the respective sources of Dpp and Spi, whereas the ASP may extend cytonemes in response to more than one signaling protein. These results are, however, complicated by the heat shock mode of induction because both the cells that expressed GFP (and extended marked cytonemes) as well as the surrounding cells expressed the signaling proteins. To overcome this problem, a method was developed to induce two types of somatic clones in the same tissue, one that expressed GFP and another that expressed Dpp (Roy, 2011).

The GAL4 system was used to label cytonemes with CD8:GFP. Clones of GAL4-expressing cells were generated with heat shock-induced flippase (FLP recombinase). The second type of clone expressed a Dpp:Cherry fusion and was generated with a variant Cre-progesterone receptor recombinase that could be activated with a regime of heat shock and RU486. By adjusting the timing and strength of induction, wing discs were produced with small, independent, and relatively infrequent clones. In discs with clones that expressed ectopic Dpp as well as clones that expressed CD8:GFP, apical cytonemes tagged with GFP were detected that oriented toward nearby Dpp:Cherry-expressing cells and not toward either the A/P or D/V signaling centers. Such 'abnormally directed' cytonemes were never observed in control discs. The abnormally oriented cytonemes suggest that apical cytonemes in the wing blade respond directly to sources of Dpp and that their orientation reflects extant sources of signaling protein (Roy, 2011).

To characterize the relationship between tracheal ASP tip cytonemes and FGF signaling from the wing disc, the distribution of Btl (FGFR) was examined in ASP cells and in ASP cytonemes. In preparations from larvae with tracheal expression of both CD8:GFP and Btl:Cherry (btl-GAL4 UAS-CD8:GFP;UAS-Btl:Cherry), cytonemes were marked by CD8:GFP, some of which had fluorescent Btl:Cherry puncta. Each ASP had only a few long (>30 μm) cytonemes, most of which contained Btl:Cherry puncta. Few of the more numerous short cytonemes (<30 μm) contained Btl:Cherry puncta. To characterize Btl:Cherry after overexpression of Bnl, focus was placed on preparations obtained 1 to 2 hours post-induction (genotype btl-GAL4 UAS-CD8:GFP/HS-Bnl;UAS-Btl:Cherry/Gal80^ts), because during this time interval the ASP morphology was close to normal but cytonemes had changed. ASPs were ignored after longer postinduction intervals because of major malformations to ASP morphology after 3 to 4 hours. Long cytonemes with Btl:Cherry puncta were present 1 hour after a pulse of Bnl expression; but 2 hours after the pulse, most ASPs had no long cytonemes, and the number of short puncta-containing cytonemes increased at the tip and along the shaft of the ASPs. After control heat shock or heat shock-induced expression of Dpp, the distribution of Btl:Cherry puncta in the ASP tip cytonemes was similar to normal controls: Long cytonemes had Btl:Cherry puncta, but most short cytonemes did not (Roy, 2011).

Because the number of small cytonemes at the ASP tip may have increased after ectopic Dpp expression, whether the thickveins (tkv) gene, which encodes a subunit of the Dpp receptor, is expressed in the ASP was investigated. Expression of the tkv reporter, tkv-lacZ (P{lacW}tkv¹⁶⁷¹³), was detected in the ASP. When Tkv:GFP and Btl:Cherry were expressed together, Tkv:GFP and Btl:Cherry segregated to separate tip cytonemes at the ASP tip. Whereas Tkv-containing cytonemes were short (<30 μm), most of the Btl-containing cytonemes were longer (three of four of the Btl:Cherry-containing cytonemes were longer than 30 μm), and they lay in focal planes closer to the disc. These properties were consistent in all preparations examined in which both green Tkv and red Btl cytonemes were intact. Imaging these marked ASPs revealed that overexpressed Tkv:GFP and Btl:Cherry were present not only in the plasma membranes (as expected) but also in separate puncta in the cell bodies. This shows that Tkv and Btl receptors also segregated to separate locations in the ASP cell bodies (Roy, 2011).

These findings suggest that the ASP has long cytonemes that are specific to Bnl and specifically harbor Btl-containing puncta and that the ASP also has cytonemes that are specific to Dpp and specifically harbor Tkv. Similarly in the eye disc, the presence of EGFR:GFP in furrow-oriented cytonemes and not in equator-oriented cytonemes suggests that cytonemes in the eye disc also selectively localize receptors. And as was previously shown, apical cytonemes in the wing disc selectively localize Tkv. The apparent ligand specificities and contrasting makeup of these cytonemes suggest a diversity of functionally distinct subtypes: Cells appear to make cytonemes that respond specifically to the Dpp, EGF, or Bnl signaling proteins. The basal filopodia implicated in Delta-Notch signaling in the wing disc may represent yet another type (Roy, 2011).

The mechanism that endows cytonemes with specificity for a particular signaling protein cannot be based solely on tissue-specific expression of a receptor. Spi, Dpp, and Hh are active in eye discs, but only changes in Spi signaling affected the furrow-directed cytonemes. And in the wing disc, both the Hh and EGF signal transduction pathways are active in cells at the A/P compartment border, but the apical cytonemes only responded to overexpressed Dpp. The findings that tracheal cells in the ASP respond to both Dpp and Bnl and that the Tkv and Btl receptors are present in different cytonemes that the ASP cells extend suggest that specificity may be a consequence of the constitution of the cytoneme, not on which receptors the cells make. The mechanism that localizes receptors to different cytonemes is not known, but because the marked receptors that were expressed also segregated to different intracellular puncta, the processes that concentrate these receptors in separate locations may not be exclusive to cytonemes. There is a precedent for segregation of proteins to different cellular extensions, neurons segregate proteins to dendrites or axons, so extending projections with specific and distinct attributes may be a general property of cells (Roy, 2011).

Suppression of wing fate and specification of haltere fate in Drosophila by the homeotic gene Ultrabithorax is a classical example of Hox regulation of serial homology (Lewis, E. B. 1978. Nature 276: 565–570) and has served as a paradigm for understanding homeotic gene function. DNA microarray analyses was used to identify potential targets of Ultrabithorax function during haltere specification. Expression patterns of 18 validated target genes and functional analyses of a subset of these genes suggest that down-regulation of both anterior–posterior and dorso-ventral signaling is critical for haltere fate specification. This is further confirmed by the observation that combined over-expression of Decapentaplegic and Vestigial is sufficient to override the effect of Ubx and cause dramatic haltere-to-wing transformations. These results also demonstrate that analysis of the differential development of wing and haltere is a good assay system to identify novel regulators of key signaling pathways (Mohit, 2005).

Suppression of wing fate and specification of haltere fate by Ubx is a classical example of Hox regulation, which has served as a paradigm for understanding the nature of homeotic gene function. Using microarray analyses and subsequent downstream validation by methods other than microarray, 18 potential targets have been identified of Ubx function during haltere specification. In addition, differential expression of Dpp at the transcriptional level has been observed between wing and haltere imaginal discs. Including previously known 13 targets, there are now as many as 32 well-established direct or indirect targets of Ubx function during haltere specification. Although Ubx may regulate additional downstream targets, the expression patterns of the genes identified suggest that negative regulation of D/V and A/P signaling is one of the important mechanisms by which Ubx specifies haltere development (Mohit, 2005).

The functional significance of down-regulation of these signaling pathways is confirmed by the dramatic homeotic transformations caused by ectopic activation of Dpp and/or Vg in developing haltere discs. These transformed halteres still lacked veins and wing margin bristles, indicating that Ubx specifies haltere development by additional mechanisms. Indeed, the EGFR pathway, which plays a significant role in specifying wing veins, is directly repressed by Ubx in haltere discs (S. K. Pallavi, unpublished observations reported in Mohit, 2005). Furthermore, over-expression of Dad in wing discs does not cause any obvious wing-to-haltere transformation nor do dpp^d6/dpp^d12 wings show such phenotypes. Thus, while over-expression of Dpp causes partial haltere-to-wing transformations, down-regulation of Dpp in wing discs has no such effect. Further investigation is needed to identify all the critical steps downstream of Ubx required to completely transform haltere to a wing or vice versa. Nevertheless, the dramatic homeotic transformations induced by the co-expression of just two genes (Dpp and Vg) suggest that down-regulation of these two steps by Ubx is critical to specify haltere fate (Mohit, 2005).

Although both Vg and Dpp are known to induce growth, it is believed that the observed homeotic transformation is due to re-patterning and trans-differentiation and not due to simple over-growth. Induction of over-growth in haltere leads to larger appendages, but not homeotic transformations. Furthermore, a recent report suggests that changes in cell division patterns alone do not lead to cell fate changes. Thus, Dpp/Vg-induced homeosis is a specific mechanism that overrides the effect of Ubx and suggests an important mechanism for Ubx function during haltere specification. Interestingly, in the mouse, signaling molecules such as Bmp2, Bmp7 and Fgf8 are downstream targets of Hoxa13 during the development of limbs and genitalia. Thus, down-regulation of Dpp and Wnt/Wg signaling pathways in Drosophila and Bmp and Fgf in mouse suggest a common theme underlying Hox gene function during appendage specification and development (Mohit, 2005).

The results presented in this report are significant in two ways. First, they suggest a mechanism by which halteres may have evolved from hind wings of lepidopteran insects. Ubx protein itself has not evolved among the diverse insect groups, although there are significant differences in Ubx sequences between Drosophila and crustacean Arthropods. Nevertheless, over-expression of Ubx derived from either a non-winged arthropod such as Onychophora or a four-winged insect such as Tribolium is sufficient to induce wing-to-haltere transformations in Drosophila. This suggests that, in the dipteran lineage, certain wing patterning genes have come under the regulation of Ubx. In such a scenario, it is likely that only a small number of genes will have their cis-regulatory sequences modified (converging mutations) to enable their regulation by Ubx. Considering the gross morphological differences between lepidopteran hind wings and halteres, any new target of Ubx will have greater influence on the entire hind wing morphology. Indeed, over-expression of Dpp and/or Vg caused dramatic haltere-to-wing homeotic transformations. Since such transformations were not observed by over-expressing their upstream regulators such as Hh, Ci, N or Wg, it is likely that direct targets of Ubx would be closer to Dpp and Vg in the hierarchy of gene regulation. Currently, chromatin immunoprecipitation experiments using haltere extracts are underway to identify those target genes (Mohit, 2005).

The second significant conclusion from the results described here is on the utility of differential development of wing and haltere as a good model system to identify additional components of both A/P and D/V signaling. Nine such genes have been identified, 8 of which show modulation of their expression patterns along the D/V axis. Based on restricted expression patterns and biochemical features of the encoded proteins, their possible involvement in maintaining the integrity of the D/V boundary as well as differences between dorsal and ventral compartments is predicted. Indeed, preliminary characterization of two genes suggests their probable roles to restrict Wg expression to the D/V boundary (Mohit, 2005).

A recent report has identified 16 potential genes downstream of mouse Hoxd cluster during the development of the most distal parts such as digits and genitals. Most of them have not been previously implicated in the early stages of either limb or genital bud development or as components of the known signal transduction pathways. Considering tissue- and developmental stage-specific expression of those genes, it is possible that those targets too could be novel modulators of known signal transduction pathways. Taken together, these results provide a framework for understanding the mechanisms by which Hox genes specify segment-specific developmental pathways (Mohit, 2005).

Selector genes modify developmental pathways to sculpt animal body parts. Although body parts differ in size, the ways in which selector genes create size differences are unknown. This study investigated how the Drosophila Hox gene Ultrabithorax (Ubx) limits the size of the haltere, which, by the end of larval development, has ~fivefold fewer cells than the wing. It was found that Ubx controls haltere size by restricting both the transcription and the mobility of the morphogen Decapentaplegic (Dpp). Ubx restricts Dpp's distribution in the haltere by increasing the amounts of the Dpp receptor, thickveins. Because morphogens control tissue growth in many contexts, these findings provide a potentially general mechanism for how selector genes modify organ sizes (Crickmore, 2006).

Changes in body part sizes have been critical for diversification and specialization of animal species during evolution. The beaks of Darwin's finches provide a famous example for how adaptation can produce variations in size and shape that allowed these birds to take advantage of specialized ecological niches and food supplies. Sizes also vary between homologous structures within an individual. For example, vertebrate digits and ribs vary in size, likely due to the activities of selector genes such as the Hox genes. Although the control of organ growth by selector genes is likely to be common in animal development, little is known about the mechanisms underlying this control (Crickmore, 2006).

The two flight appendages of Drosophila, the wing and the haltere, provide a classic example of serially homologous structures of different sizes. Halteres, appendages used for balance during flight, are thought to have been modified from full-sized hindwings during the evolution of two-winged flies from their four-winged ancestors. All aspects of haltere development that distinguish it from a wing, including its reduced size, are under the control of the Hox gene Ultra-bithorax (Ubx), which is expressed in all haltere imaginal disc cells but not in wing imaginal disc cells. At all stages of development, haltere and wing primordia (imaginal discs) are different sizes. In the embryo, the wing primordium has about twice as many cells as the haltere primordium. By the end of larval development, the wing disc has ~five times more cells (~50,000) than the haltere disc (~10,000). The wing and haltere appendages will form from the pouch region of these mature discs. The final step that contributes to wing and haltere size differences occurs during metamorphosis, when wing, but not haltere, cells flatten, thus increasing the surface area of the final appendage (Crickmore, 2006).

To confirm that Ubx has a postembryonic role in limiting the size of the haltere disc, Ubx^– clones were generated midway through larval development. Haltere discs–bearing large Ubx^– clones generated at this time become much larger than wild-type discs. Ubx could limit haltere size cell-autonomously by, for example, slowing the cell cycle of haltere cells relative to wing cells. This was tested by comparing the sizes of isolated Ubx^– clones in the haltere with those of their simultaneously generated wild-type twin clones. Contrary to the prediction of a cell-autonomous function for Ubx in size control, Ubx mutant clones did not grow larger than their twins, a result that is consistent with earlier experiments suggesting that wing and haltere cells have similar mitotic rates during development. Hence, Ubx limits the size of the haltere during larval development by modifying pathways that control organ growth cell-nonautonomously (Crickmore, 2006).

In the fly wing, Decapentaplegic (Dpp) [a long-range morphogen of the bone morphogenetic protein (BMP) family] has been shown to promote growth. In both the wing and the haltere, Dpp is produced and secreted from a specialized stripe of cells called the AP organizer, which is induced by the juxtaposition of anterior (A) and posterior (P) compartments, two groups of cells that have separate cell lineages. The AP organizer is a stripe of A cells that are instructed to synthesize Dpp by the short-range morphogen Hedgehog (Hh) secreted from adjacent P compartment cells. Dpp has a positive role in appendage growth. When more Dpp is supplied to the wing disc, either ectopically or within the AP organizer, more cells are incorporated into the developing wing field. Conversely, mutations that reduce the amount of Dpp lead to smaller wings (Crickmore, 2006).

A comparison of the expression patterns of Dpp pathway components in the wing and the haltere demonstrates that Ubx is modifying this pathway. Compared with the wing, the stripe of dpp expression in the haltere was reduced in both its width and intensity, as reported by a lacZ insertion into the dpp locus (dpp-lacZ). There was also a difference in the profile of Dpp pathway activation, as visualized by an antibody that detects P-Mad, the activated form of the Dpp pathway transcription factor Mothers against Dpp (Mad). In the wing, P-Mad staining was low in the cells that transcribe dpp. Immediately anterior and posterior to this activity trough, P-Mad labeling peaked in intensity and then gradually decayed further from the Dpp source, revealing a bimodal activity gradient. In contrast, in the haltere intense P-Mad staining was detected only in a single stripe of cells that overlaps with Dpp-producing cells of the AP organizer (Crickmore, 2006).

Because of the coincidence between dpp transcription and peak P-Mad staining in the haltere, it was hypothesized that Dpp might be less able to move from haltere cells that secrete this ligand. This idea was tested by generating clones of cells in both wing and haltere discs in which the actin5c promoter drove the expression of a green fluorescent protein (GFP)–tagged version of Dpp (Dpp:GFP). By using an extracellular staining protocol to analyze simultaneously generated clones, Dpp:GFP and P-Mad were observed much further from producing cells in the wing than in the haltere. These observations strongly suggest that, compared with the wing, Dpp's mobility—and consequently the range of Dpp pathway activation—is reduced in the haltere (Crickmore, 2006).

Whether the decreased production of Dpp in the haltere contributes to the different pattern of pathway activation observed in this tissue compared with the wing was tested. This is unlikely because, even in haltere discs that overexpress Dpp in its normal expression domain, peak P-Mad staining was still observed close to Dpp-expressing cells. Despite increased dpp expression, no P-Mad activity trough was observed in these haltere discs. Further, although they become larger, these discs remained smaller than wild-type wing discs. It is concluded that the decreased Dpp production in the haltere contributes to its reduced growth, but there must be mechanisms that also limit the extent of Dpp pathway activation, even in the presence of increased Dpp production (Crickmore, 2006).

One way in which Dpp's activation profile can be modified is by varying the production of the type I Dpp receptor, Thick veins (Tkv). In the wing, tkv expression is low within and around the source of Dpp, resulting in low Dpp signal transduction in these cells and robust Dpp diffusion. Low tkv expression in the medial wing is due to repression by both Hh and Dpp. Accordingly, tkv expression is highest in lateral regions of the wing disc, where Hh and Dpp signaling are low. In contrast to the wing, tkv transcription and protein levels were high in all cells of the haltere. Thus, the more restricted Dpp mobility and P-Mad pattern in the haltere may result from a failure to repress tkv medially. To test this idea, all cells of the wing disc were supplied with uniform UAS-tkv⁺ expression, to mimic the haltere pattern. The resulting P-Mad pattern in these wing discs was very similar to that found in the wild-type haltere: The P-Mad trough was gone, and the activity gradient was compacted into a single stripe that coincides with Dpp-producing cells. Conversely, lowering the amount of Tkv in the haltere by expressing an RNA interference (RNAi) hairpin construct directed against tkv (UAS-tkvRNAi) in Dpp-producing cells induced a bimodal pattern of P-Mad staining similar to that of the wild-type wing disc. Thus, different amounts of Tkv result in qualitative differences in the P-Mad profiles of the wing and the haltere (Crickmore, 2006).

For animal development it is necessary that organs stop growing after they reach a certain size. However, it is still largely unknown how this termination of growth is regulated. The wing imaginal disc of Drosophila serves as a commonly used model system to study the regulation of growth. Paradoxically, it has been observed that growth occurs uniformly throughout the disc, even though Decapentaplegic (Dpp), a key inducer of growth, forms a gradient. This paper presents a model for the control of growth in the wing imaginal disc, which can account for the uniform occurrence and termination of growth. A central feature of the model is that net growth is not only regulated by growth factors, but by mechanical forces as well. According to the model, growth factors like Dpp induce growth in the center of the disc, which subsequently causes a tangential stretching of surrounding peripheral regions. Above a certain threshold, this stretching stimulates growth in these peripheral regions. Since the stretching is not completely compensated for by the induced growth, the peripheral regions will compress the center of the disc, leading to an inhibition of growth in the center. The larger the disc, the stronger this compression becomes and hence the stronger the inhibiting effect. Growth ceases when the growth factors can no longer overcome this inhibition. With numerical simulations it is shown that the model indeed yields uniform growth. Furthermore, the model can also account for other experimental data on growth in the wing disc (Aegerter-Wilmsen, 2007).

Since the wing imaginal disc serves as a model system to study the regulation of growth, a large amount of experimental data is already available. The model has been evaluated with experimental results from the literature. When clones with increased Dpp signaling are generated, they grow larger in the lateral regions than in the medial part. Furthermore, clones with decreased Dpp signaling survive better laterally than medially. A common explanation for these findings is that the medial cells are more competitive than the lateral cells because they receive higher levels of Dpp. Therefore, a clone with a fixed level of Dpp signaling is hindered more when growing in the medial part than when growing more laterally. The model may offer an additional, alternative explanation. A clone is stretched more and compressed less when growing laterally than when growing medially. Therefore, it grows faster laterally as long as its level of Dpp signaling is fixed. It is expected that both competition and differences in compression contribute to the difference of size among different clones (Aegerter-Wilmsen, 2007 and references therein).

Discs with homogeneous Dpp signaling are expanded along the dorsoventral boundary. According to the model, the total growth factor activity in these discs is highest along the dorsoventral boundary, thus accounting for the expansion along this boundary. Furthermore, it has been found that discs with homogeneous Dpp signaling do not show uniform growth. Instead the growth rate of cells in the lateral regions, close to the dorsoventral boundary, is higher than the growth rate of cells in the medial part of the disc. According to the model, the high growth factor activity along the dorsoventral boundary will promote additional growth along the whole boundary. This stretches the regions further away from the dorsoventral boundary. This stretching pulls the cells along the dorsoventral boundary toward the center of the disc. The cells in the center are thus being compressed. The closer the cells are located to the center, the more they are compressed and the more growth is inhibited, thus leading to the observed differences in growth rate (Aegerter-Wilmsen, 2007).

The Dpp pathway can be activated locally by expressing a constitutively active form of one of its receptors (tkv^Q-D). Recently, it has been shown that activating the Dpp pathway in clones in this way can stimulate transient non-autonomous cell proliferation. When inhibiting the pathway, similar effects were seen. Clones with increased Dpp activity were modeled as a region with increased Dpp activity compared to its surrounding tissue with lower homogeneous Dpp activity. In that case, the cells with high Dpp signaling initially grow faster than the surrounding cells, thus stretching them. As in the wild-type situation at the start of growth, the stretching is highest in the cells closest to the region with high Dpp signaling and therefore growth is induced in these cells. This non-autonomous growth increases the stretching in the cells further away from the clone, which will increase their growth. Therefore, after some time, growth in the cells surrounding the clone will be homogeneous again, comparable with the situation in the wild type disc. Thus, the model accounts for the non-autonomous effect as well as for the observation that it only occurs transiently (Aegerter-Wilmsen, 2007).

Clones with decreased Dpp activity were modeled in a similar way. The cells surrounding the clone get stretched between the slow growing cells in the clone and the faster growing cells further away from the clone. Therefore growth is also induced non-autonomously in cells surrounding clones with decreased Dpp signaling, which is again in agreement with the data (Aegerter-Wilmsen, 2007).

Non-autonomous effects on cell proliferation were also assessed for clones in which growth is increased by overexpressing CyclinD and Cdk4 instead of by increased Dpp signaling. The non-autonomous proliferation was not observed in that case, even though this would in principle be expected based on the model. However, cell divisions are only slightly increased in these clones and apoptosis is increased, which is generally accompanied by basal extrusion. Therefore, it seems as if co-expression of CyclinD and Cdk4 causes only very little net overgrowth at the stage measured. For such clones the non-autonomous stimulation of proliferation is expected to be less pronounced and to occur at a relatively late point in time, which may explain why it has not been observed (Aegerter-Wilmsen, 2007).

Experimentally induced alterations in cell proliferation are often compensated for by changes in cell size, such that the final wing disc size is not changed. This suggests that wing disc size is not a function of cell numbers. In the model, the wing disc is considered as an elastic sheet with certain mechanical properties. As long as the mechanical properties of the tissue as a whole are not influenced by cell size, the final disc size is indeed not a function of cell numbers according to the model. Furthermore, according to the model, it would be expected that a reduction of growth in the center of the disc automatically leads to a reduction of growth in the peripheral regions. Indeed, when the size of the wing blade was decreased by down-regulating vestigial (vg) expression, non-autonomous reductions in surrounding WT territories were observed along all axes of growth. Lastly, the model predicts that stretching occurs in the peripheral regions. Therefore, it also predicts that, upon cutting the disc from the end toward the middle, tissue at both sides of the cut moves apart. In wound healing experiments, this was indeed observed. In contrast, the model predicts that the central region of the disc becomes compressed. The increased thickness of the (columnar layer of the) wing disc could be seen as an indication that compression indeed occurs (Aegerter-Wilmsen, 2007).

This paper has presented a model for the determination of final size in the wing imaginal disc. In the model, growth is negatively regulated by mechanical stresses, which are automatically generated as a result of growth rate differences in an elastic tissue. With the use of numerical simulations, it was demonstrated that the model naturally leads to uniform growth as was shown experimentally and that it leads to the observed final size of the wing disc. Furthermore, it was argued that the model can also account for other experimental data in literature (Aegerter-Wilmsen, 2007).

Homothorax and Extradenticle are expressed in the proximal domain of the leg (the Hth domain) : all the other factors studied (Wingless, Decapentaplegic and Distalless) are expressed in more distal regions (the Dll domain). Dachsund (Dac) is expressed in an intermediate domain, dorsal and lateral to the more distal Dll domain (the Dac domain). What follows is a more complete description of these domains. The expression of several targets of the signaling molecules Wg and Dpp were examined in relation to the hth expression domain. dpp expression in the leg disc at the early third larval instar stage consists of a sector that originates at the center of the disc, extends dorsally to the periphery and shows extensive overlap with Hth. omb, a target of the Dpp-signaling pathway, is expressed in a dorsal sector that, in contrast to dpp, extends dorsally only to abut, but not overlap with, the hth domain. wg expression consists of a ventral sector of cells that extends from the center to the periphery of the disc, whereas H15, an enhancer trap line that requires wg signaling for its activation, is largely not transcribed in the hth domain. The restriction of these Wg and Dpp target genes to non-hth-expressing cells suggests that hth restricts signaling by these two molecules. By the late third larval instar stage, there is a small degree of overlap between hth and omb expression as well as between hth and H15. This expression corresponds to the trochanter domain where gene activation can occur independent of the Wg- and Dpp-signaling pathways. Unlike omb and H15, the Dll and dac genes require input from both the Dpp and Wg signal transduction pathways to be activated in leg discs. Dll encodes a homeodomain protein present in the central portion of leg discs, and its activation requires the highest concentrations of Wg and Dpp. dac encodes a nuclear protein and a putative transcription factor whose expression is repressed by high concentrations, and activated by intermediate concentrations, of Wg and Dpp. By performing triple stains for the dac^P-lacZ reporter gene, and Dll and Hth proteins at the early third larval instar stage, it was found that the leg disc is defined by three non-overlapping domains of gene expression. The distal-most domain of the leg disc contains Dll protein (the Dll domain). Dorsal and dorsolateral, but not ventral, to the Dll domain are cells that express dac (the Dac domain). The proximal-most cells of the disc, which surround the dac and Dll domains, express hth (the Hth domain). At the mid 3rd larval instar stage (~96 hours after egg lay, or AEL), the distal-most cells express only Dll and are surrounded by a ring of cells that express both Dll and dac. At this stage, there is also a dorsal patch of cells that express dac but not Dll. hth expression remains limited to the proximal-most cells of the disc and shows no overlap with dac or Dll. By the late 3rd larval instar stage (~120 hours AEL), hth is still not co-expressed with dac or Dll, with the exception of a thin band of cells corresponding to the trochanter domain, where all three genes are co-expressed. Gene expression in the trochanter domain is likely to represent secondary patterning events, because it is not dependent on Wg- or Dpp-signaling. At this stage dac expression also surrounds and partially overlaps the Dll expression domain. It is proposed that the Dll and Dac domains, where hth transcription is off and Exd is cytoplasmic, are Dpp- and/or Wg-responsive domains, as demonstrated by the ability of these cells to respond to these signals by activating the target genes Dll, dac, omb and H15. In contrast, the hth domain, where hth is active and Exd is nuclear, is a Wg- and/or Dpp-non- responsive domain, where these signals are present but cannot activate these targets (Abu-Shaar, 1998).

Arthropods and higher vertebrates both possess appendages, but these are morphologically distinct and the molecular mechanisms regulating patterning along their proximodistal axis (base to tip) are thought to be quite different. In Drosophila, gene expression along this axis is thought to be controlled primarily by a combination of transforming growth factor-ß and Wnt signalling from sources of ligands, Decapentaplegic (Dpp) and Wingless (Wg), in dorsal and ventral stripes, respectively. In vertebrates, however, proximodistal patterning is regulated by receptor tyrosine kinase (RTK) activity from a source of ligands, fibroblast growth factors (FGFs), at the tip of the limb bud. This study revises the understanding of limb development in flies and shows that the distal region is actually patterned by a distal-to-proximal gradient of RTK activity, established by a source of epidermal growth factor (EGF)-related ligands at the presumptive tip. This similarity between proximodistal patterning in vertebrates and flies supports previous suggestions of an evolutionary relationship between appendages/body-wall outgrowths in animals (Campbell, 2002).

Initially, tests were performed to see whether Wg and Dpp directly pattern the proximodistal axis of the tarsus by determining their role in activation of the aristaless (al) gene in the center of the disc. al encodes for a homoeodomain protein required for development of structures found at the tip of the leg, including the claws. Previous studies indicated that al expression is activated by Wg and Dpp and this was confirmed with loss of function studies: al expression is absent from the center of wg and dpp mutant discs. However, this does not rule out al being activated by a secondary signal, which in turn is activated by Wg and Dpp. To test this, al expression was monitored in discs containing clones of cells mutant for genes required for transduction of Wg or Dpp signals, including arrow (arr), which encodes for a Wg co-receptor, and thickveins (tkv), which encodes a Dpp receptor (clone founder cells were generated before the onset of al expression). Central al expression is absent in discs consisting largely of arr mutant clones, but, as in wg^ts discs, such large clones would remove any putative secondary signal, and, in fact, further analysis revealed that al is still expressed in arr mutant cells located outside of the very center. Similarly, al can still be detected in tkv mutant cells. Thus, Wg and Dpp signalling are required, but not directly, to induce al, suggesting that it is activated by a secondary signal, which in turn is activated by Wg and Dpp (Campbell, 2002).

Similar results were obtained analyzing marker gene expression in Egfr^ts discs, including al, Bar (B, expressed in segments IV and V) and rotund (rn, expressed in segments II–IV). Loss of EGFR activity results in loss of al, B and rn expression, but al is lost at a lower temperature than B, which in turn is lost at a lower temperature than rn, indicating that the more distal the marker, the higher the EGFR activity level required for expression. Clonal analysis with Egfr^ts showed that this response to EGFR is cell autonomous, and that again, al requires higher EGFR activity than B. It was not possible to do similar tests for rn because at temperatures above 31°C Egfr^ts clones do not survive in distal regions, raising the possibility that rn expression may be lost in Egfr^ts discs simply because of reduced growth or cell survival in this region. However, expression of other genes, including wg and dpp, appears normal in Egfr^ts discs. This is also true for the Wg and Dpp target Dll, clearly demonstrating that Wg, Dpp and Dll are not sufficient to distalize the leg (Campbell, 2002).

Ectopic activation of EGFR signalling results in autonomous, ectopic expression of al and B in mid-third-instar discs. Curiously, not all regions of the disc respond identically, with ventral regions being the most responsive and lateral regions the least. The reason for this is unclear but it indicates that factors in addition to EGFR may be regulating expression of tarsal genes such as al, at least outside of their normal domains. Only the presumptive tarsus is responsive to ectopic EGFR activity; this is most evident in adult appendages where no defects in patterning can be observed outside of here. Other regions may be refractive to ectopic EGFR activity because expression of tarsal genes requires Dll, which is expressed only in distal regions under Wg and Dpp control (Campbell, 2002).

The role of buttonhead and Sp1 in the development of the ventral imaginal discs: activation of wg and dpp

The related genes buttonhead (btd) and Drosophila Sp1 (the Drosophila homolog of the human SP1 gene) encode zinc-finger transcription factors known to play a developmental role in the formation of the Drosophila head segments and the mechanosensory larval organs. A novel function of btd and Sp1 is reported: they induce the formation and are required for the growth of the ventral imaginal discs. They act as activators of the headcase (hdc) and Distal-less (Dll) genes, which allocate the cells of the disc primordia. The requirement for btd and Sp1 persists during the development of ventral discs: inactivation by RNA interference results in a strong reduction of the size of legs and antennae. Ectopic expression of btd in the dorsal imaginal discs (eyes, wings and halteres) results in the formation of the corresponding ventral structures (antennae and legs). However, these structures are not patterned by the morphogenetic signals present in the dorsal discs; the cells expressing btd generate their own signalling system, including the establishment of a sharp boundary of engrailed expression, and the local activation of the wingless and decapentaplegic genes. Thus, the Btd product has the capacity to induce the activity of the entire genetic network necessary for ventral imaginal discs development. It is proposed that this property is a reflection of the initial function of the btd/Sp1 genes that consists of establishing the fate of the ventral disc primordia and determining their pattern and growth (Estella, 2003).

In a search for genes with restricted expression in the adult cuticle, the MD808 Gal4 line was found to direct expression in the ventral derivatives of the adult body; proboscis, antennae, legs and genitalia. In the abdomen and analia no clear expression was discerned. It was also noticed that the insertion was located in the first chromosome and associated with a lethal mutation. The mutant larvae showed a head phenotype resembling that described for mutants at the btd gene: loss of antennal organ and the ventral arms of the cephalopharyngeal skeleton, and complementation analysis indicated that the chromosome carrying the insert contained a mutation at btd. The expression pattern found in MD808/UAS-lacZ embryos was also similar to that reported for btd, suggesting that the Gal4 insertion was located at this gene. In addition, the imaginal expression of MD808 and of btd was largely coincident (Estella, 2003).

Further to the genetic analysis and the expression data, DNA fragments at the insertion site were cloned to map the position of the P-element on the genome. It is located 753 bp 5' of the btd gene. The related gene Sp1 is immediately adjacent. It is likely that btd and Sp1 have originated by a tandem duplication of a primordial btd-like gene (Estella, 2003).

One particularly significant result about the mode of action of btd comes from the analysis of the ectopic leg patterns observed with ectopic btb expression in the wing and halteres. The clones of cells ectopically expressing btd tend to recapitulate the complete development of leg and antennal discs. For example, the whole genetic network necessary to make a leg appears to be activated. btd induces the activity of hth, dac and Dll, the domains of which account for the entire disc. Furthermore, hth, dac and Dll are activated in a spatially discriminated manner. The formation of the dac and Dll domains is dependent on signalling from Wg and Dpp, although they require different signal thresholds. In one clone, for example hth is expressed only in the peripheral region, resembling the normal expression in the leg disc; in another clone the discriminate expressions of dac and Dll define three distinct regions. The formation of the dac and Dll domains is dependent on signalling from Wg and Dpp, although they require different signal thresholds, but the hth domain is independent from Wg and Dpp (Estella, 2003).

The generation of distinct hth, dac and Dll domains within the clones suggested that btd-expressing cells in the wing and haltere generate their own signalling process. Indeed, within these clones there is local activation of en, the transcription factor that initiates Hh/Wg/Dpp signalling in imaginal discs. btd-expressing clones also acquire wg and dpp activity in subsets of cells. It is probably in the boundary of en-expressing with non expressing cells where the Wg and Dpp signals are generated de novo; subsequently, their diffusion initiates the same patterning mechanism which operates during normal leg development. The result of this process is that the hth, dac and Dll genes are expressed in different domains contributing to form leg patterns containing DV and PD axes. One question for which there is no clear answer is how the initial asymmetry is generated, so that a few cells within the group gain (or lose) en activity. The cells expressing en within the clones are those closer to the posterior compartment cells. It is conceivable that there might be an external signal, perhaps mediated by Hh, which triggers the initial asymmetry (Estella, 2003).

The ability of cells expressing btd to build their own patterning mechanism is also indicated by the observation that inducing btd activity in different parts of the wing disc results in the production of similar sets of leg pattern elements. For example, in MD743/UAS-btd and omb-Gal4/UAS-btd flies, btd is induced in different, non-overlapping wing regions, and yet all leg pattern elements are produced in both genotypes. Thus, the effect of btd is by and large independent of the position where it is induced, e.g., it does not depend on local positional signals (Estella, 2003).

A relevant issue is whether the ability of the Btd product to induce the formation of the full array of ventral structures has a functional significance in normal development. This property may be a faithful reflection of the original btd/Sp1 function: the activation of the developmental program to build the ventral adult patterns. btd/Sp1 function can be envisaged as follows. During the embryonic period, the conjunction of several regulatory factors (Wg, Dpp, EGF, Hox genes) allows activation of btd/Sp1 in a group of cells in each thoracic segment (it is assumed that a similar process takes place in the head). These cells become the precursors of the ventral imaginal discs and will eventually form the ventral thorax of the adult -- these include the trunk (the hth domain) and appendage (the Dll domain) regions. The activity of btd/Sp1 is instrumental in segregating these ventral discs precursors from those of the larval epidermis and determining their imaginal fate. It is involved in specifying their segment identity (in collaboration with the Hox genes) and in establishing their pattern and growth. To achieve the latter role btd/Sp1 induces the production of the growth signals Wg and Dpp, probably in response to localized activation of en and subsequent signalling by hedgehog (hh) (Estella, 2003).

A problem with this model is that in normal development all the genes involved, hth, en, hh, wg and dpp, are expressed in embryos prior to btd/Sp1. Why should a new round of activation be necessary? Although a totally satisfactory answer can not be provided, it is noted that clones of btd-expressing cells in wing or haltere lose their memory of en expression. Those that originated in the A compartment had no previous en expression, but gained it in some cells. Conversely, all cells in P compartment clones contained en activity but some lose it. The best explanation for this unexpected behavior is that btd provokes a 'naïve' cell state in which the previous commitment for en activity is lost. Later, en activity is re-established. This phenomenon may reflect the process that occurs in normal development. The initial btd/Sp1 domain may not inherit the previous developmental commitments and has to build a new developmental program. It is worth considering that the btd/Sp1function appears to determine ventral imaginal fate as different from larval fate. This is a major developmental decision, which may require de novo establishment of the genetic system responsible for pattern and growth. A key aspect of this would be the localized activation of en in part of the btd/Sp1 domain. It is speculated that there might be a short-range signal, perhaps Hh, emanating from nearby en-expressing embryonic cells, that acts as an inducer in the btd/Sp1 primordium. There is evidence that Hh can induce en activity (Estella, 2003).

Dpp and the Genital Disc