Larval and pupal stages (continued)

Dpp and the Wing Disc

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).

During vein differentiation dpp is expressed in the pupal veins under the control of genes that establish vein territories in the imaginal disc. Both dpp and thick veins are differentially expressed in vein territories during pupal development. dpp and tkv regulate one another by a feedback mechanism in which Tkv activity represses dpp expression. Dpp, acting through its receptor Thick veins, activates vein differentiation and restricts expression of both veinlet and the Notch-ligand Delta to the developing veins. Ectopic dpp expression or Tkv activation in the wing disc result in the differentiation of ectopic veins. Outside of vein territories, the repression of dpp by the widely expressed Tkv could participate in restricting dpp expression to the veins. It is possible that the observed down-regulation of tkv expression in vein cells participates in generating the levels of Tkv activation necessary to activate vein differentiation, but insufficient to repress dpp expression. The expression of dpp and tkv in vein territories depends (either directly or indirectly) on EGF-receptor activity, because the transcription of these genes is not activated when Egf-R is reduced (as in veinlet and vein mutant wings). Once Dpp is established in the veins, local activation of Tkv in these cells is required both for the maintenance of veinlet and Delta expression and for the veins to differentiate. In dpp mutants, the vein thickening observed in Notch mutants is elimated. Conversely, Notch gain-of-function alleles that lead to the truncation of veins results in very pronounced vein loss in combination with both dpp and tkv mutants. In dpp mutants, Delta and E(spl)mß, which normally takes place in vein territories, is lost. In summary, genetic combinations between mutations that increase or reduce Notch, veinlet and dpp activities suggest that the maintenance of the vein differentiation state during pupal development involves cross-regulatory interactions between these pathways (de Celis, 1997).

Patterning of the developing limbs by the secreted signaling proteins Wingless, Hedgehog and Dpp takes place while the imaginal discs are growing rapidly. Cells born in regions of high ligand concentration may be displaced through growth to regions of lower ligand concentration. A novel lineage-tagging method was used to address the reversibility of cell fate specification by morphogen gradients. Responses to Hedgehog and Dpp in the wing disc are readily reversible. In the leg, cells readily adopt more distal fates, but do not normally shift from distal to proximal fate. However, they can do so if given a growth advantage. These results indicate that cell fate specification by morphogen gradients remains largely reversible so long as the imaginal discs are growing. In other systems, where growth and patterning are uncoupled, nonreversible specification events or 'ratchet' effects may be of functional significance (Weigmann, 1999).

Hh induces dpp expression in anterior cells adjacent to the anteroposterior (AP) boundary of the wing disc. In mature third instar discs, a dpp-lacZ reporter gene is expressed in a narrow stripe of cells in the center of the disc, whereas in young third instar discs, the dpp-lacZ stripe occupies the central third of the disc. This comparison illustrates that the proportion of the disc occupied by Hh-responsive cells is relatively larger in small discs and decreases as the disc grows. Further, it suggests that Hh-responsive cells must be able to lose expression of Hh target genes as the cells are displaced out of range of the Hh signal by growth of the disc. To verify that this is indeed the case, cells born in the Hh-responsive region were lineage-tagged using dppGal4 to direct expression of FLP recombinase. In larvae carrying dppGal4, UAS-Flp and act5c>stop>lacZ, FLP recombinase is expressed in cells expressing dppGal4 and mediates excision of the flip-out 'stop' cassette from the inactive reporter construct to generate an active act5c>lacZ transgene. After excision of the cassette, reporter gene expression is regulated by the actin promoter and is clonally inherited in all the progeny of dppGal4-expressing cells in which the recombination event took place. Cells expressing lacZ fill most of the anterior compartment of the wing pouch, hinge and the notum. By comparison, the dppGal4 domain is much narrower. This indicates that cells born in the dppGal4 domain contribute to most of the A compartment of the wing and that they change their pattern of gene expression as they are displaced out of range of Hh (Weigmann, 1999).

Dpp signaling induces Spalt expression in the wing pouch. Clones of cells unable to transduce the Dpp signal lose Spalt expression, suggesting that expression of Spalt depends on continuous input of the Dpp signal. Spalt is first induced in early third instar discs. To ask whether cells that initiate Spalt expression at this stage revert to a more lateral identity as the disc grows in the course of normal development, cells born in the Spalt domain were lineage-tagged in larvae carrying spaltGal4, UAS-Flp and act5c>stop>lacZ. betaGal-expressing cells are found lateral to the endogenous Spalt domain in both the A and P compartments, indicating that cells can alter their pattern of target gene expression when displaced out of range of the Dpp signal. Taken together, these results suggest that, in general, cells are not committed to maintain a particular threshold response to the Hh or Dpp morphogens. Rather, cells in the wing disc appear to be able to revert to lower threshold responses when morphogen levels decrease (Weigmann, 1999).

The Drosophila EGF receptor (Egfr) is required for the specification of diverse cell fates throughout development. How the activation of Egfr controls the development of vein and intervein cells in the Drosophila wing has been examined. Two distinct events are involved in the determination and differentiation of wing vein cells: (1) the establishment of a positive feedback amplification loop, which drives Egfr signaling in larval stages (at this time, rhomboid, in combination with vein, initiates and amplifies the activity of Egfr in vein cells); (2) the late downregulation of Egfr activity [at this point, the inactivation of MAPK in vein cells is necessary for the maintenance of the expression of decapentaplegic (dpp) and becomes essential for vein differentiation. Subsequently, Egfr becomes activated in intervein territories. During the time that dpp is expressed in vein territories, MAPK activity builds up in intervein territories, probably due to the presence of Vn, a weak Egfr activator. As a consequence, aos expression relocates to intervein territories. Together, these temporal and spatial changes in the activity of Egfr constitute an autoregulatory network that controls the definition of vein and intervein cell types (Martin-Blanco, 1999).

The reiterated use of Egfr is a common effector of differentiation. In the Drosophila eye, Egfr is required for the determination of all cell types. In this system, cell fate depends on the developmental stage at which the receptor is activated. By interfering with Egfr signaling activity, the specification of veins respond to the activation of receptor tyrosine kinase (RTK) signaling during larval stages, but continued activation of RTK signaling results in a failure of vein cells to differentiate. One explanation for these opposite effects could be that early activation of RTK signaling would specify vein cells, while late RTK signaling would implement intervein cell fates. Several observations provide support for this model. In pupae, MAPK is repressed in veins and activated in intervein cells. This activation of MAPK (and the expression of downstream genes, such as argos) responds to Ras signaling activity, and appears to be involved in the suppression of vein cell fates. Indeed, after ectopical activation of D-Raf during the pupal period, promoting intervein cell fates, the MAPK activity remains stimulated all over the wing blade (Martin-Blanco, 1999).

It seems that Egfr is the only receptor tyrosine kinase at work in the wing, able to activate Ras and Raf. While Egfr is ubiquitously expressed during larval imaginal disc development, EGFR mRNA levels are downregulated in the pupal period in presumptive vein cells. This downregulation of Egfr could be involved in the supression of MAPK activity in vein territories. Furthermore, when a dominant negative-Egfr (DN-Egfr) molecule is overexpressed, titrating the endogenous Egfr, in pupae, extra vein tissue is induced. MAPK dephosphorylation in veins could also be induced by other mechanisms; for instance, the early expression of the inhibitor ligand Argos in veins up to 24 hours APF could cooperate in the inactivation of MAPK in these territories (Martin-Blanco, 1999).

What is the function of this change of expression? The first effect of this developmental switch is a modification in the expression of downstream targets. As a consequence of the reduction in MAPK activity from vein cells, aos is eliminated from veins between 24 and 30 hours APF. Conversely, it is upregulated in intervein territories. This scenario is reminiscent of the induction of Egfr ligands in the ventral ectoderm. Here, the primary signal, Spitz induces a relay mechanism by triggering the expression of Vn (and Aos) in adjacent cells. Aos reduces the overall level of Egfr signaling, whereas Vn provides a lower level of activation, capable of inducing only the lateral cell fates. In the larval wing, high levels of Egfr signaling are achieved in veins through a positive feedback loop. Here, Egfr activity promotes the expression of Aos. It is suggested that Aos diffusion from veins could prevent adjacent cells from responding to the vein inductive signals and producing high levels of Egfr activity ('remote inhibition'). Consistently, aos mutant flies display small deltas and extra veins clustered around vein territories. On the contrary, Aos overexpression in larval stages induces the suppression of veins. It is also proposed that, in pupae, while Egfr activity (and Aos) in veins are lost, Vn and Aos expression in intervein cells will reach a competitive balance leading to the activation of Egfr and MAPK, and intervein cell specification (Martin-Blanco, 1999 and references therein).

Several types of cell-cell communication have been proposed to be required during the latter stages of pupal wing development. The dpp gene encodes a member of the TGFbeta superfamily and is expressed during early pupal development in vein primordia. A class of loss-of-function dpp alleles and certain combinations of Dpp receptor mutants lead to vein-loss phenotypes. Mosaic analysis of dpps allele show that mitotic clones affect the differentiation of veins. Meanwhile, the effects of overexpression of dpp or an active form of its receptor thick veins (tkv) indicate that Dpp directs vein differentiation through activation of Tkv in pupal stages. The initiation of dpp expression in pupal stages depends on the activity of early acting genes, and in particular Egfr activity. However, although Egfr signaling is downregulated in vein territories during pupariation, dpp expression is maintained through an autoregulatory loop and remains high in vein cells until their final differentiation. Interestingly, in intervein cells, dpp expression is not activated in response to the Egfr activity described above. On the contrary, these cells express short gastrulation (sog), a gene that exerts an opposing effect to dpp. sog plays a role restricting vein formation to the center of the provein regions. dpp and sog interact antagonistically during vein differentiation. Ectopic activation of Egfr signaling in pupal stages abolishes dpp expression from veins. This suppression of dpp correlates with the loss of veins observed in this condition; it is reminiscent of the effect of Sog overexpression in pupal wings. Moreover, vein plexates induced by compromising Egfr activity in pupal wings, associate with a broadening of dpp-expressing areas (Martin-Blanco, 1999).

It is suggested that Egfr signaling downregulation from vein territories allows dpp to autoregulate dpp expression. It remains to be determined whether sog expression depends on Egfr in intervein territories, or is a consequence of the activity of intervein-specific genes such as blistered. The model presented here on how a single receptor (Egfr), triggering a conserved signal transduction pathway, is used reiteratively to implement two different cell fates in the development of the fly wing serves to reconcile many observations that have been made regarding cell fate specification in the wing. This may well provide a paradigm for the regulation of Egfr signal transduction in other developmental events (Martin-Blanco, 1999).

The dachsous (ds) gene encodes a member of the cadherin family involved in the non-canonical Wnt signaling pathway that controls the establishment of planar cell polarity (PCP) in Drosophila. ds is the only known cadherin gene in Drosophila with a restricted spatial pattern of expression in imaginal discs from early stages of larval development. In the wing disc, ds is first expressed distally, and later is restricted to the hinge and lateral regions of the notum. Flies homozygous for strong ds hypomorphic alleles display previously uncharacterized phenotypes consisting of a reduction of the hinge territory and an ectopic notum. These phenotypes resemble those caused by reduction of Wingless during early wing disc development. An increase in Wg activity can rescue these phenotypes, indicating that Ds is required for efficient Wg signaling. This is further supported by genetic interactions between ds and several components of the Wg pathway in another developmental context. Ds and Wg show a complementary pattern of expression in early wing discs, suggesting that Ds acts in Wg-receiving cells. These results thus provide the first evidence for a more general role of Ds in Wnt signaling during imaginal development, not only affecting cell polarization but also modulating the response to Wg during the subdivision of the wing disc along its proximodistal (PD) axis (Rodríguez, 2004).

Ectopic expression of Dpp in wing cells of DNW discs restores both the formation of the AP border and cell proliferation within the wing pouch, indicating that both Wg and Dpp orchestrate these events. Only cells previously committed to the wing fate by Wg are able to proliferate in response to Dpp, as the UAS-dpp/dpp-Gal4 and UAS-dpp/omb-Gal4 experiments suggest. In the ds mutant background, omb is expressed in anterior wing cells, albeit in the absence of the AP border/Dpp source within the wing pouch, suggesting that this initial omb expression might not be Dpp dependent. Similar results were observed for spalt (sal), another known target gene of dpp. It is proposed that Ds primarily regulates Wg signaling in the initial recruitment of P cells into putative wing territory. Once this initial recruitment has occurred, Dpp expression is established and Dpp signaling can contribute to the further recruitment of P cells. Expression of UAS-dpp in anterior wing pouch cells of ds mutant discs using omb-Gal4 can bypass the initial requirements for Wg in P cell recruitment, leading to the observed wing pouch rescue (Rodríguez, 2004).

In DNW discs, even though the Dpp source is distantly and asymmetrically located with respect to the wing pouch, anterior wing cells differentiate into distinct cell types in a mirror image disposition. This result suggests that specific positional information might be provided independently of dpp. Ap in combination with Wg might contribute to this initial AP positional information. Once P cells are recruited into the wing fate, Dpp takes over and promotes pattern formation along the AP axis, as well as proliferation within the wing pouch (Rodríguez, 2004).

Dpp and the Wing Disc: Dpp gradient formation

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 shits1 clones. Consistent with this, shadows can be seen only under dynamic conditions, when confronting a wave of GFP-Dpp with the shits1 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).

In posterior receiving cells, ectopic expression of DRab7Q67L causes an anterior/posterior compression of the venation pattern and shape of the posterior compartment, suggesting a reduction of the functional range of Dpp signaling. As with Rab5, the distribution of Gal4-driven GFP-Dpp could not be monitored directly under these conditions of enhanced degradation in the receiving cells. Dpp signaling range was therefore monitored by looking at Spalt. Expression of DRab7Q67L in receiving cells causes a reduction of the Sal expression domain, indicating that sorting of endocytic cargo toward degradation limits the range of Dpp signaling. This suggests that Dpp degradation restricts the signaling range (Entchev, 2000).

Growth regulation by Dpp: an essential role for Brinker and a non-essential role for graded signaling levels

Morphogens can control organ development by regulating patterning as well as growth. This study used the model system of the Drosophila wing imaginal disc to address how the patterning signal Decapentaplegic (Dpp) regulates cell proliferation. Contrary to previous models, which implicated the slope of the Dpp gradient as an essential driver of cell proliferation, it was found that the juxtaposition of cells with differential pathway activity is not required for proliferation. Additionally, the results demonstrate that, as is the case for patterning, Dpp controls wing growth entirely via repression of the target gene brinker (brk). The Dpp-Brk system converts an inherently uneven growth program, with excessive cell proliferation in lateral regions and low proliferation in medial regions, into a spatially homogeneous profile of cell divisions throughout the disc (Schwank, 2008).

Morphogen gradients play essential roles in pattern formation during animal development. They direct the transcriptional on and off states of genes in a concentration-dependent manner in various embryonic organ systems. The tight link between organ patterning and organ growth raised the notion that morphogens also determine cell proliferation rates and final tissue size. This latter aspect of the morphogen concept, however, is not well understood. Indeed, it is not clear whether the nuclear response to morphogen signals that directs the transcription of patterning genes also regulates growth. And what property of a morphogen signaling system explains how uniform growth rates can ensue in response to a graded input? This study addresses these questions in the experimental system of the Dpp gradient, a key determinant in pattern formation and growth of the Drosophila wing (Schwank, 2008).

Studies from the past decade have shown that the Dpp gradient in the wing disc does not define the expression boundaries of subordinate patterning genes directly via its nuclear mediators, but does so indirectly by setting up an inverse gradient of the transcriptional repressor Brk. This study investigated the potential role of this indirect mechanism in growth regulation; it was found to be equally important, and essential, for the ability of Dpp to promote growth. Clones of cells with a constitutively active Dpp signaling pathway exhibited qualitatively and quantitatively the same growth behavior as brk- clones, overgrowing when located in the lateral area. Moreover, the phenotype of discs in which Brk levels can no longer be regulated by Dpp (because brk is either lacking genetically, or controlled by a heterologous promoter) are insensitive to experimentally varying Dpp signaling levels. Thus, these experiments demonstrate that the growth output of the Dpp pathway is entirely funneled through the regulation of the brk gene (Schwank, 2008).

The paradigm of Dpp directing pattern formation via brk repression thus also explains how Dpp controls growth. This observation serves to validate the connection between morphogen-mediated patterning and the control of organ size. The results indicate that for the Dpp system, any mechanistic bifurcation of the two outputs occurs downstream of the first tier of transcriptional regulation (Schwank, 2008).

Discs lacking both dpp and brk functions grow to a larger size than wild-type discs. Importantly, in this state, in contrast to the normally uniform profile, cell proliferation also occurs unevenly across the disc, with higher rates in the lateral areas and lower rates in the medial area. Based on this difference, it is concluded that the Dpp-Brk system is not a growth promoter but is rather a growth-modulatory system, ironing out inherent regional differences in proliferation rates (Schwank, 2008).

The origin of the regional proliferative differences in discs devoid of the Dpp-Brk system is unknown. Since such discs lack Dpp, as the only agent known to impose mirror-symmetric differences along the AP axis, no pre-patterning mechanism that depends on it can be postulated. The smooth transitions to higher proliferation rates between medial and lateral areas would be consistent with a diffusible factor that acts in a concentration-dependent manner. This hypothetical factor could originate, for example, at the border between the disc proper and the adjacent peripodial membrane and promote growth laterally. Alternatively, the factor could be a growth inhibitor with high activity in the center of the disc and low activity peripherally. Expression of the factor could be controlled by Hedgehog in a Dpp-independent manner. But this is pure speculation because to date there is no evidence for the existence of such a factor(s) in the developing wing discs (Schwank, 2008).

An entirely different explanation for the experimental observations could be a growth-regulatory mechanism that depends on mechanical forces. It has been proposed that during growth, mechanical compression of cells increases in the center, while cells in the peripheral regions become stretched. Assuming a growth-stimulatory role for stretching and a growth-inhibitory role for compression, growth would be facilitated in the peripheral regions during normal development, and Brk would counter this advantage and thus ensure uniform growth. In the absence of the Dpp-Brk system, the amount and distribution of mechanical stresses are likely to differ significantly, which in turn could feed back on growth and lead to the observed differences between the lateral and medial regions of the disc (Schwank, 2008).

This study has confirmed and extended previous findings that in wing discs with uniform Dpp signaling, lateral cells proliferate faster, and medial cells slower, than cells of wild-type discs. The inhibition of cell proliferation in the medial region is an important pillar for the model which proposes that it is the slope of the Dpp morphogen gradient that serves as the driving force behind medial wing cell proliferation during normal development. Contradicting the proposed requirement for disparate Dpp signaling activities among adjacent cells, it was found that when uniform pathway activity is established in, and limited to, the medial area, no deficit in cell proliferation rates occurs. Indeed, the medial domain of discs with such even Dpp signaling levels expands, and proliferation is uniform. This finding is consistent with results from the twin-spot analysis, which showed that the growth rates of medial tkvQ235D and brkM68 clones are identical to those of wild-type clones. Thus, the transient effect of additional proliferation at clonal boundaries observed by Rogulja (2005) seems to be more important for situations such as wound healing, in which cells of different Dpp signaling levels become juxtaposed, than for the normal growth of a wild-type wing disc. A reduction in proliferation rates in the medial area was found to occur only when Dpp activity is driven in the lateral area, independent of the presence or absence of a Dpp signaling gradient. Ectopic Dpp pathway activation in lateral cells is not only necessary, but also sufficient, to impede proliferation of medial cells. Thus, overproliferating lateral cells appear to exert a proliferation-retardant effect on other cells. Whether this effect underlies a mechanism also used to control proliferation rates during wild-type development, or whether it is 'only' a back-up mechanism used if something goes wrong during development (e.g. wound healing and regeneration), is not known. Moreover, as noted earlier, the mechanistic nature of the communication between lateral and medial cell populations remains speculative. It is possible that high Dpp signaling in lateral cells not only provides them with a growth advantage, but also causes the expression of a factor that spreads within the entire disc to reduce proliferation of cells without an additional growth advantage. Other possible explanations include the competition among wing cells for a limiting proliferation factor (whereby ectopic Dpp-transducing cells prevail), or the negative impact that overproliferating cells might exert on remaining cells via metabolic side-products or increased mechanical compression. These models would also be consistent with the observation that proliferation is reduced in all cells of the wing disc except those with an additional growth advantage (Schwank, 2008).

Dpp-mediated growth control in the wing disc can be summarized as follows. The disc consists of at least two different cell populations, medial and lateral, which have distinct abilities to proliferate. The Dpp signal is required to even out these growth differences and establish a uniform pattern of cell proliferation within the wing primordium. Medial cells must sense high levels of Dpp to shut down brk expression, which consequently promotes medial proliferation. Lateral cells have a growth advantage and must receive little or no Dpp input to allow brk expression. The action of Brk curbs lateral proliferation. It is not knowm how intermediate Brk levels affect the proliferative behavior of cells situated between lateral and medial cells. However, it can be concluded from the present results that differential pathway activity between neighboring cells is not necessary to direct proliferation, since constitutively high Dpp levels in the medial area and nil or low levels in the lateral areas are sufficient for uniform and normal cell proliferation rates throughout the disc (Schwank, 2008).

Dpp gradient formation by dynamin-dependent endocytosis: receptor trafficking and the diffusion model

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).

Three points lead to this conclusion. (1) A 'diffusion, binding and trafficking' DBT model of the 'shibire shadow assay' generates permanent shadows (depletion of Dpp behind the clone), whereas the experimental shadows are transient. (2) The DBT model with saturating cell surface receptor concentration (DBTS) model can generate transient shadows, but only if the surface receptor levels in the clone increase dramatically. This leads to a strong increase in the levels of extracellular ligand in the clone. Using receptor antibodies in the 'shibire shadow assay', these higher levels of surface receptors in the clone were not observed. Similarly, the levels of extracellular ligand were not increased in the clone. (3) In the DBTS model for the 'shibire rescue assay', the levels of both the extracellular Dpp and the surface receptors are dramatically increased in the endocytosis-defective target cells as compared with the WT source. Such an increase is not seen experimentally. Instead, extracellular Dpp enters the receiving tissue over a distance of only 4-5 cells in steady-state. This is in contrast to both DBT and DBTS models of the 'shibire rescue assay' in which ligand can enter the tissue over large distances. Therefore, in addition to downregulating surface receptors, endocytosis is likely to play additional roles in the transport of ligands during gradient formation (Kruse, 2004).

These three caveats of the DBT/DBTS models are actually not caused by the choice of a particular set of parameters. The parameter values used in the calculations were chosen in such a way, that the typical distance over which the ligand gradient extends as well as the characteristic time to reach steady state are consistent with the experimentally observed profiles. Furthermore, if possible, parameters were chosen similar to values measured for the EGF receptor in a cell culture system. The results showing that a high surface receptor concentration inside the clone is required for shadows to appear is independent of any choice of parameters. Furthermore, convincing shadows appear in the DBT and DBTS models only for values of koff, which are small compared with those typically measured in related systems. It will be necessary to estimate the actual parameter values for Dpp during wing morphogenesis in order to ultimately understand its mechanism of spreading (Kruse, 2004).

Therefore, neither the DBT nor the DBTS model can explain the observed ligand and receptor profiles during Dpp spreading in the wing disc. Why should these models fail even though they incorporate many essential phenomena such as ligand diffusion, internalization and resurfacing via receptor recycling? The essential point of both the DBT and the DBTS model is that ligand transport is solely because of diffusion. In other words, this means that ligand bound to the surface receptors when internalized can only resurface at the same position on the cell surface where it was internalized. Only in this case is equation justified and the intracellular transport of the ligand would not contribute to the current of the ligand in the tissue. This implies that simple reaction diffusion models ignore that, in principle, ligand could also be transported by traveling through cells and resurface at other positions on the cell surface when receptors are recycled (Kruse, 2004).

The fact that the DBT and DBTS models (which ignore these effects), cannot account for observed Dpp spreading suggests that contributions of receptor trafficking to transport and ligand current may indeed play an important role. The DBT/DBTS models are currently being generalized to incorporate all relevant transport phenomena (diffusion and transcytosis) in the ligand current as well as the possibility of extracellular degradation of the ligand (Kruse, 2004).

The working hypothesis is that two phenomena contribute to the Dpp current in the developing wing epithelium: extracellular diffusion and intracellular trafficking (i.e. endocytosis plus resecretion). What is the relative importance of these two phenomena to the spreading of the morphogen? Both might be important. Limited by binding to the extracellular matrix and/or degradation, extracellular transport of the morphogen may only account for the spreading of the ligand across a few cell diameters. Intracellular trafficking in turn accounts for the movement of the morphogen across one cell diameter. Both phenomena together then lead to the long-range spreading of the morphogen (Kruse, 2004).

Although it is expected that extracellular diffusion plays a role during morphogen spreading, it has been argued that extracellular diffusion alone is insufficient to understand the reliability and precision of the formed gradient. The important role of intracellular trafficking has been uncovered in experiments in which endocytosis is blocked during morphogenetic signaling. When endocytosis is blocked in the receiving tissue, Dpp spreading does occur, but generates a short-range gradient and thereby signaling responses only within 3 to 5 cells. In particular, in a thermosensitive alpha-adaptin mutant, Dpp activates transcription of its target gene spalt only within 4-5 cells from the source, instead of within 15 cells in WT. Similar results Were obtained by expressing a dominant-negative Rab5 mutant, which impairs endocytosis and endosomal dynamics. These results do not exclude a role for endocytosis in the transduction, rather than on the spreading of Dpp. However, in the 'shibire rescue assay', the reduced range of the extracellular Dpp gradient indicates that impaired endocytosis restricts the spreading of Dpp (Kruse, 2004).

This report is a theoretical and experimental study to address whether diffusion as the sole transport mechanism can explain the spreading of Dpp. The role of different transport mechanisms for Dpp spreading is currently being studying. It has been argued that the rates of endocytosis and recycling known for the EGF receptor in cultured cells are too small to allow for a sufficiently rapid transport by transcytosis. Indeed, the first results based on generalized models (including diffusion and transport by planar transcytosis) show that the parameter values used in this work do not produce consistent gradients during reasonable times. In particular, these models require a faster rate of endocytosis and recycling than those known for the EGF receptor in cultured cells. Therefore, it is essential to measure directly the different dynamic parameters, including the extracellular diffusion coefficient as well as the rates of endocytosis, degradation and recycling of Dpp in the developing wing. To estimate these parameters in situ, photoactivatable fusion proteins are currently being monitored in different cellular locations (extracellular versus endosomal) in the very context of the developing wing epithelium (Kruse, 2004).

Cytoneme-mediated contact-dependent transport of the Drosophila Decapentaplegic signaling protein

Decapentaplegic (Dpp), a Drosophila morphogen signaling protein, transfers directly at synapses made at sites of contact between cells that produce Dpp and cytonemes that extend from recipient cells. The Dpp that cytonemes receive moves together with activated receptors toward the recipient cell body in motile puncta. Genetic loss-of-function conditions for diaphanous, shibire, neuroglian and capricious perturbed cytonemes by reducing their number or only the synapses they make with cells they target; and reduced cytoneme-mediated transport of Dpp and Dpp signaling. These experiments provide direct evidence that cells use cytonemes to exchange signaling proteins, that cytoneme-based exchange is essential for signaling and normal development, and that morphogen distribution and signaling can be contact-dependent, requiring cytoneme synapses (Roy, 2014).

This study revealed an essential role for cytoneme-based transport of signaling proteins in long distance paracrine signaling. This mechanism involves contact-dependent transfer of signaling proteins from producing to responding cells, and although signaling between two epithelial tissues was studied in a Drosophila larva, evidence from other systems supports a general role for cytonemes in paracrine signaling (Roy, 2014).

Studies of cells in culture indicate that filopodia receive and transport signaling proteins that are taken up from culture media. In experiments with human adenocarcinoma cells, uptake of EGF protein from the culture medium led to retrograde transport by filopodia along with activated EGFR, and was sensitive to cytochalasin D, a disruptor of F-actin. Actin-based cytonemes that carry FGFR-rich puncta and that are dependent on the small GTPase RhoD are present in cultured mouse mesenchymal cells (Roy, 2014).

The wing disc has associated trachea whose development depends in part on signaling from the disc. Larval trachea form an interconnected network of oxygen carrying tubes; one, the transverse connective (TC) of Tr2, is bound to the wing disc. During the third larval instar (L3), Branchless, [the fly Fibroblast Growth Factor (FGF)] produced by a group of disc cells induces a new branch, the Air Sac Primordium (ASP), to grow from the TC. The ASP is juxtaposed to the basal surface of the wing disc columnar epithelium; it is a monolayered epithelial tube. At the late L3 stage, the ASP has many cytonemes that extend toward the disc. Cells at the ASP tip extend long cytonemes that contain the FGF receptor Breathless (FGFR) and appear to touch FGF-producing disc cells. The presence and orientation of these cytonemes are dependent on FGF. The late L3 ASP also has shorter cytonemes that contain Tkv and that extend from its lateral flank toward Dpp-expressing disc cells (Roy, 2014).

Some characteristics of Dpp signaling in the ASP are consistent with these cell culture experiments. Dpp that was taken up by an ASP cell was present in motile puncta that translocated along the ASP cell's cytoneme, and some puncta in the ASP cytonemes contained both Dpp and its receptor. However, in contrast to cultured cells, signaling in the ASP did not appear to involve uptake of signaling proteins from the extracellular milieu, but was dependent on synaptic contact between the tip of a cytoneme that extended from a responding ASP cell and the cell body of a Dpp-expressing disc cell. This signaling mechanism appears to involve specific dynamic interactions between signaling and responding cells (Roy, 2014).

ASP cells express both the Tkv Dpp receptor and FGFR, and segregate these receptors to puncta in distinct cytonemes. At the early L3 stage, the ASP is small and does not extend across the disc, and both the Dpp- and FGF-expressing disc cells are distal to its tip. Both Tkv- and FGFR-containing cytoneme extended distally from the tip. The FGFR-conaining cytonemes extended beyond the Dpp-expressing cells and did not take up Dpp. At later L3 stages, the ASP has grown across the disc, and although the FGF-expressing disc cells are distal to it, the Dpp-expressing disc cells lie under its medial region. In these ASPs, the Tkv-containing cytonemes emanated from the medial region and reached as much as 40 microm to pick up Dpp from disc cells. Thus in the ASP, spatially restricted Dpp signal transduction and uptake were associated with cytonemes whose orientation and composition appeared to be specific for Dpp (Roy, 2014).

The dynamism of this signaling system may be inferred from steady-state images. The distribution and orientation of cytonemes changes if expression of signaling protein is compromised and if signaling protein is overexpressed in ectopic locations. These properties suggest that cytonemes are changeable and that their distributions reflect the relative positions of signal-producing and -receiving cells. The different distributions of Tkv-containing cytonemes in the temporal sequence described above are consistent with this idea and with a model of cytoneme impermanence. The observation that some ASP cytonemes contain Tkv, make contact with Dpp-producing cells and take up Dpp whereas other cytonemes contain Tkv but do not make contact with Dpp-producing cells or take up Dpp may also suggest that cytoneme contacts may be transient (Roy, 2014).

Plasticity may be an important attribute of cytonemes that function in a developmental system such as the ASP in which relations between producing and receiving cells change as the larva develops. Cytonemes may have the capacity to regulate release and uptake of signals and to direct signals to a pre-selected target. Regulated release may be implied by the absence of Dpp uptake and Dpp signal transduction in ASP mutant conditions that abolish synaptic contacts by ASP cytonemes. In these experiments, the signal producing cells were not mutant, and the wing discs, which depend on Dpp signaling, developed normally, indicating that the signaling defect was specific to the ASP cells that made defective cytonemes. Because filopodia of cultured cells take up signaling proteins from culture medium and activate signal transduction, it may be assumed that ASP cytonemes are similarly capable of taking up signaling protein that their receptors encounter and that the inability of cytoneme-defective cells to take up Dpp or activate signal transduction may indicate that Dpp was not released in the absence of cytoneme contact (Roy, 2014).

There may be a functional analogy to neuronal signaling. Neurons make asymmetric extensions that send and receive signals, make specific contacts where signal release and uptake are regulated, and require the diaphanous, neuroglian, shibire and capricious genes for contact-mediated signal exchange and signaling. In the developing Drosophila retina, Hh moves to the termini of retinal axons where it signals to post-synaptic laminal neurons in the brain. Perhaps the strongest precedent is Wingless delivery at developing neuromuscular junctions in the Drosophila larva; in this case, Wingless moves to the post-synaptic cell after release in a vesicular form from the pre-synaptic neuron. The current studies have been limited to the cytonemes that are made by receiving cells, but in other contexts, cytonemes extend from cells that deliver signaling proteins, such as the Hh-containing cytonemes of the wing disc, the cytonemes that extend from cap cells in the female germline stem cell niche and that are associated with Notch and EGF signaling. Cytonemes that transport Hh across the chick limb bud from Hh producing cells have also been described. The widespread presence of cytonemes in many cell types and in many contexts suggests that they may provide a general mechanism to move signaling proteins between non-neuronal cells at sites of direct contact (Roy, 2014).

The role of Dpp signaling in maintaining the Drosophila wing anteroposterior compartment boundary

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 tkvbsk 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 tkvbsk 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 madbsk and tkvbsk clones are indistinguishable. Like tkvbsk clones, A madbsk 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) madbrk 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, tkvbsk, and madbsk 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 tkvbsk, madbsk, 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 tkvbsk, madbsk, 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).

How might Omb regulate the segregation behavior of cells at the A/P boundary? Recent work has shown that Omb has at least two roles during the patterning of the Drosophila wing. First, Omb is required for the expression of two Dpp target genes sal and vestigial (vg) (del Alamo Rodriguez, 2004). Since sal mutant clones do respect the A/P boundary, the role of Omb in maintaining the A/P boundary cannot depend on sal induction. Since Vg is required for wing cell proliferation, its role in maintaining the A/P boundary cannot be tested. Second, Omb is involved in shaping the expression pattern of tkv along the A/P axis of the wing disc (del Alamo Rodriguez, 2004). The expression of tkv is reduced in Dpp-producing A cells along the A/P boundary. This reduction of tkv expression is mediated by the transcription factor Master of thickveins (Mtv, also known as Brakeless and Scribbler, which is expressed in these cells in response to the Hh signal. Since both tkv and mtv are upregulated in omb mutant clones, it has been proposed that Omb is required for Mtv to repress tkv (del Alamo Rodriguez, 2004). However, reduction of tkv transcription in A cells does not seem to be important for the segregation of cells at the A/P boundary, because A clones either mutant for mtv, in which tkv levels are increased, or overexpressing tkv, respect the A/P boundary. Thus, neither the role of Omb in repressing tkv nor in activating sal transcription appears to be important for Omb's function in maintaining the A/P boundary. Therefore, other target genes of Omb must exist that mediate Omb's function in maintaining the A/P boundary (Shen, 2005).

Anterior cells at the A/P boundary have been shown to require Hh signal transduction to segregate from P cells. Evidence is provided that A cells in addition need to transduce the Dpp signal for normal segregation. What is the epistatic relationship between Hh and Dpp signaling? The activity of the Hh transduction pathway is not affected in either tkvbsk or madbsk clones as monitored by the expression of the Hh target gene ptc, indicating that Hh signal transduction does not require Dpp signal transduction components for its activity. However, the Dpp target gene omb appears to be important for A cells to interpret the Hh signal because the ability of Ci to specify A-type segregation properties depends, in part, on the activity of Omb. Thus, Dpp signaling acts either downstream of or in parallel with Hh signaling in maintaining the A/P boundary (Shen, 2005).

Previously, three transcription factors, a transcriptional activator form of Ci (hereafter referred to as Ci[act]), En, and Inv, have been shown to be required for the segregation of A and P cells. Evidence exists for the involvement of a fourth transcription factor, the T-box protein Omb. Omb is further shown to act downstream of or in parallel with Ci. How could these four transcription factors regulate the segregation of A and P cells? In a simple model, Ci[act], En, Inv, and Omb could regulate the segregation of A and P cells by controlling the transcription of the same set of target genes that may encode cell affinity molecules or regulate the activity of cell affinity molecules. Omb is activated in both A and P cells in a broad domain centered around the A/P boundary by Dpp signaling where Omb may upregulate the expression of this putative target gene(s). The activity of Ci[act] is restricted to Hh-responding A cells along the A/P boundary. In these A cells, the target gene(s) would be further induced. En and Inv expressions are mainly confined to P cells in which they are known to act as repressors of transcription. Thus, En and Inv would repress the putative target gene(s) in P cells. The abrupt difference in the expression of putative target gene(s) would contribute to the segregation of A and P cells. Anterior clones (but not P clones) of cells lacking Omb would displace the A/P boundary because normally the putative target gene would be highly expressed in A cells, but not in P cells, where it would be repressed by En and Inv. Omb may therefore provide a basal affinity to cells in the center of the wing disc that is modified by Ci[act], En, and Inv to create a sharp difference of this affinity in cells on both sides of the A/P boundary. In an alternative model, Omb, Ci[act], En, and Inv would regulate distinct sets of genes. To distinguish among these models, it will be necessary to identify the Ci[act], En, Inv, and Omb target genes mediating cell segregation (Shen, 2005).

The precise position and shape of the Dpp organizer along the A side of the A/P boundary are important for normal growth and patterning of the wing. It has been proposed that the segregation of cells at the A/P boundary contributes to maintain this precise position and shape of the Dpp organizer in the growing wing disc epithelium. It is intriguing to notice that the Dpp-organizing activity itself plays a role in the segregation of A and P cells, suggesting that the Dpp-organizing activity contributes to maintain its own position. It will be interesting to investigate whether other organizers associated with compartment boundaries have similar functions (Shen, 2005).

Morphogen control of wing growth through the fat signaling pathway

Organ growth is influenced by organ patterning, but the molecular mechanisms that link patterning to growth have remained unclear. The Dpp morphogen gradient in the Drosophila wing influences growth by modulating the activity of the Fat signaling pathway. Dpp signaling regulates the expression and localization of Fat pathway components, and Fat signaling through Dachs is required for the effect of the Dpp gradient on cell proliferation. Juxtaposition of cells that express different levels of the Fat pathway regulators four-jointed and dachsous stimulates expression of Fat/Hippo pathway target genes and cell proliferation, consistent with the hypothesis that the graded expression of these genes contributes to wing growth. Moreover, uniform expression of four-jointed and dachsous in the wing inhibits cell proliferation. These observations identify Fat as a signaling pathway that links the morphogen-mediated establishment of gradients of positional values across developing organs to the regulation of organ growth (Rogulja, 2008).

Studies of regeneration first led to models that proposed that growth could be influenced by gradients of positional values, with steep gradients promoting growth and shallow gradients suppressing growth. Experimental manipulations of Dpp pathway activity in the Drosophila wing supported this concept, but have left unanswered the question of how differences in the levels of Dpp pathway activity perceived by neighboring cells are actually linked to growth. This study has established that the Fat signaling pathway provides this link. Dpp signaling influences the Fat pathway; the expression of upstream Fat pathway regulators, the subcellular localization of Fat pathway components, and downstream transcriptional outputs of Fat signaling are all affected by Dpp signaling. The effects that Tkv and Brk expression have on the expression of Fat target genes parallels their effects on BrdU labeling and depend genetically on Fat signaling (Rogulja, 2008).

Dpp signaling impinges on Fat signaling upstream of Fat, as the expression of both of its known regulators, Fj and Ds, is regulated by Dpp signaling. Although the Fat signaling pathway was only recently discovered, and understanding of Fat signaling and its regulation remains incomplete, the inference that Fat signaling is normally influenced by the Dpp morphogen gradient is supported by the polarized localization of Dachs in wild-type wing discs. Near the D-V compartment boundary, the vector of Dachs polarization parallels the vector of the Dpp morphogen gradient, and the consequences of altered Dpp pathway activity confirm that the correlation between them is reflective of a functional link. The expression of Fj and Ds and the localization of Dachs are also polarized along the D-V axis. The implication that signaling downstream of the D-V compartment boundary thus also impinges on Fat signaling, and indeed may also influence growth through this pathway, is consistent with the observation that normal wing growth requires both A-P and D-V compartment boundary signals, and is further supported here by the observation that Notch activation affects both fj expression and Dachs localization (Rogulja, 2008).

The results argue that Fat signaling is influenced by the graded expression of its regulators: uniform expression of Fj and Ds can activate Fat signaling and thereby inhibit growth, whereas juxtaposition of cells expressing different levels of either Fj or Ds can inhibit Fat signaling and thereby promote growth. Here, a model is proposed to explain how Fat signaling can be modulated by Fj and Ds gradients. Although aspects of the model remain speculative, it provides an explanation for a number of observations that would otherwise appear puzzling, and serves as a useful framework for future studies (Rogulja, 2008).

Central to the model is the inference that the interaction between Ds and Fat activates Fat. This inference is well supported by the observations that mutation or downregulation of ds results in overgrowth and upregulation of Diap1, whereas uniform overexpression of Ds inhibits growth and Diap1 expression. A second key aspect of the model is that once activated by Ds, Fat locally transmits a signal to a complex at the membrane. An important corollary to this is that if Fat and Ds are not engaged around the entire circumference of a cell, then there could be a region where Fat is locally inactive. This is hypothetical, but the Fat-dependent polarization of Dachs implies that there can be regional differences in Fat activity within a cell. Local Fat signaling is then proposed to locally promote Warts stability and activity, and thereby locally antagonize Yki activity. Conversely, a local absence of Fat signaling could result in a local failure to phosphorylate Yki, which could then transit to the nucleus, where it would promote the expression of downstream target genes. Formally, this model treats Fat signaling like a contact inhibition pathway: if Fat is engaged by Ds around the entire circumference of a cell, then Fat is active everywhere and downstream gene expression is off; however, if Fat is not active on even one side of a cell, then Yki-dependent gene expression can be turned on and growth can be promoted (Rogulja, 2008).

In this model, graded expression of Fat regulators, like Fj and Ds, could modulate Fat signaling by polarizing Fat activity within a cell. In theoretical models of PCP, even shallow gradients of polarizing activity can be converted to strong polarity responses through positive-feedback mechanisms. How this might be achieved in Fat signaling is not yet clear, but the polarized localization of Dachs implies that, at some level, Fat activity is normally polarized in wild-type animals, even where the Fj and Ds expression gradients appear relatively shallow. Importantly, this polarization hypothesis provides a solution to the puzzle of how Ds could act as a ligand to activate Fat, yet inhibit Fat along the edges of Ds-expressing clones. In this model, Ds overexpression in clones polarizes Fat activity, possibly through its ability to relocalize Fat. This would allow a strong derepression of Yki on the side of the cell opposite to where Ds and Fat are actually bound, resulting in the induction of Yki:Scalloped target gene expression and promotion of cell proliferation. Propagation of this polarization, e.g., through the influence of Fat-Ds binding on Fat and Ds localization, might explain the spread of effects beyond immediately neighboring cells. Conversely, uniform expression of Ds would generate cells presenting a ligand that activates Fat and dampens the relative difference in expression levels between neighboring cells. Yki would thus remain sequestered around the entire cell circumference, consistent with the reduced growth and Diap1 expression observed. A dampening of gradients could also explain why the induction of Fat/Hippo target gene expression or BrdU labeling associated with clones expressing Ds, Fj, or TkvQ-D is biased toward cells outside of clones (Rogulja, 2008).

The hypothesis of Fat polarization and local signal transduction also suggests a solution to another puzzle. In terms of their effects on tissue polarity and Dachs localization, Fj and Ds always behave as though they have opposite effects on Fat. Conversely, in terms of their effects on cell proliferation and downstream gene expression, Fj and Ds behave as though they have identical effects on Fat. To explain this, it is proposed that Fj acts oppositely to Ds, by, for example, antagonizing Ds-Fat binding. The influence of Ds and Fj on polarity would be a function of the direction in which they polarize Fat activity, which, based on their effects on epitope-tagged protein Dachs:V5, is opposite. In contrast, their influence on downstream gene expression and growth would be a function of the degree to which they polarize Fat activity, which could be the same. In other words, their influence on polarity would be a function of the vector of their expression gradients, and their influence on growth would be a function of the slope. However, since Dachs:V5 generally appears to be strongly polarized, the actual interpretation of Fj and Ds gradients may involve feedback amplification and threshold responses rather than providing a continuous response proportional to the gradient slope (Rogulja, 2008).

The results have provided a molecular understanding of a how a gradient of positional values, established by the morphogen Dpp and reflected, at least in part, in the graded expression of Fj and Ds, can influence growth. However, it is clear that other mechanisms must also contribute to the regulation of wing growth. The relative contribution of Fat gradients to wing growth can be estimated by considering the size of the wing in dachs mutants, or when Fj and Ds are expressed ubiquitously, as, in either case, it would be expected that the derepression of Yki associated with normal Fat signaling gradients was abolished. In both cases, the wing is less than half its normal size. Fat signaling could thus be considered a major, but by no means the sole, mechanism for regulating wing growth. The determination that not all wing growth depends on the regulation of Fat activity fits with the observation that Dpp signaling promotes growth in at least two distinct ways, one dependent upon its gradient, and the other dependent upon its levels. Other models for wing growth, including a Vestigial-dependent recruitment of new cells into the wing, and an inhibition of Dpp-promoted wing growth by mechanical strain, have also been proposed. It is emphasized that these models are not incompatible with the conclusion that a Fat gradient influences growth. Rather, it is plausible, and even likely, that multiple mechanisms contribute to the appropriate regulation of wing growth. Indeed, it is expected that a critical challenge for the future will be to define not only the respective contributions of these or other mechanisms to growth control, but also to understand feedback and crosstalk processes that influence how these different mechanisms interact with each other (Rogulja, 2008).

Dpp of posterior origin patterns the proximal region of the wing

The decapentaplegic (dpp) gene encodes a long-range morphogen that plays a key role in the patterning of the wing imaginal disc of Drosophila. The current view is that dpp is transcriptionally active in a narrow band of anterior compartment cells close to the anterio-posterior (A/P) compartment border. Once the Dpp protein is synthesised, it travels across the A/P border and diffuses forming concentration gradients in the two compartments. A new site of dpp expression has been found in the posterior wing compartment that appears during the third larval period. This source of Dpp signal generates a local gradient of Dpp pathway activity that is independent of that originating in the anterior compartment. This posterior tier of Dpp activity is functionally required for normal wing development: the elimination of dpp expression in the posterior compartment results in defective adult wings in which pattern elements such as the alula and much of the axillary cord are not formed. Moreover, these structures develop normally in the absence of anterior dpp expression. Thus the normal wing pattern requires distinct Dpp organizer activities in the anterior and posterior compartments. It was further shown that, unlike the anterior dpp expression domain, the posterior one is not dependent on Hedgehog activity but is dependant on the activity of the IRO complex gene mirror. Since there is a similar expression in the haltere disc, it is suggested that this late appearing posterior Dpp activity may be an attribute of dorsal thoracic discs (Foronda, 2009).

This study was triggered by a consistent observation of a small region in the P compartment of the wing disc that appeared to be active in dpp transcription. This P compartment expression of dpp has not been properly analysed in previous works about Dpp function in wing disc. dpp expression was carefully examined in third instar wing and haltere discs by in situ hybridization and also with a P-element insertion (P10638) at the disk region of the dpp gene. Transcriptional activity was identified close to and anterior to the A/P border. In addition, dpp transcripts were found in a proximal region of the posterior compartment. A homologous zone of dpp expression was also found in the posterior region of the haltere disc. This dpp posterior transcriptional domain appears during the third larval period; wing discs from early 3rd larval instar do not show it (Foronda, 2009).

According to the fate map, the posterior region containing dpp expression gives rise to proximal adult wing structures, including the alula and the axillary cord. This domain in adult wings was delimited by X-gal staining freshly emerged flies carrying the dpp-lacZ insertion. The area with lacZ activity corresponds mostly to the axillary cord (Foronda, 2009).

To test whether the posterior dpp expression activates the Dpp transduction pathway, use was made of an antibody raised against the phosphorylated (active) form of Mad, an indicator of Dpp pathway activity. As expected from previous work there are high levels of pMad in the centre of the disc, but in addition a domain of pMad activity was observed in the posterior compartment, which includes the dpp expression domain. The zone expressing pMad is bigger than that expressing dpp, consistent with the formation of a diffusion gradient of Dpp activity (Foronda, 2009).

In addition to pMad levels, whether other elements of the Dpp pathway are expressed in the posterior dpp domain was also examined. The gene daughters against dpp (dad) is a target that requires moderate levels of Dpp signalling. dad is expressed in the posterior wing compartment in the same region where pMad is active. Other Dpp-target genes, like optomotor blind and spalt, are also expressed in this region (Foronda, 2009).

The brinker (brk) gene is a special case as it is negatively regulated by Dpp activity. Therefore, it is expressed in the lateral regions of the wing disc where the levels of Dpp activity are lower. The domain of Dpp activity in the proximal posterior compartment should therefore repress brk activity in that region. The comparison of pMad and brk activities in this region clearly shows they occupy mutually exclusive domains, supporting the idea that brk is repressed by the posterior pMad activity. The staining of adult wings of brk-lacZ genotype also argues in the same direction because the region of brk expression does not coincide but abut with that containing Dpp activity. Although the haltere disc was not analyzed with the same detail, there is also a posterior region of pMad activity in the zone where dpp is expressed (Foronda, 2009).

pMad and brk expression were examined in wing discs from second instar larvae. These exhibit the central domain of pMad activity but there is no detectable staining in the posterior compartment. This result is consistent with the late appearance of the posterior dpp transcription domain and indicates that this domain functions only during the second half of the proliferation phase of the disc (Foronda, 2009).

Having shown that the posterior dpp expression domain acts as a source of morphogen the question was addressed whether it has a functional role. Previous experiments analysing dpp mutant clones did not report significant alterations in the posterior wing. However, given the diffusible nature of the Dpp product it is possible that the lack of Dpp in the mutant clones could be rescued by Dpp emanating from neighbour wildtype cells (Foronda, 2009).

An experiment was designed in which all cells in the posterior compartment would be homozygous for the dppd12 mutation, which eliminates dpp activity in the wing disc without affecting embryonic or pupal expression. In discs of genotype dppd12 ck FRT40A/M (2)24F arm-Z FRT40A; hh-gal4/UAS-Flp the high levels of flipase generated by the hh-gal4 driver would induce FRT-mediated mitotic recombination in virtually all the cells in the posterior compartment. The dpp M+ clones produced will have proliferation advantage and are expected to fill the posterior compartment. They can be identified in the disc because they lose the arm-lacZ transgene, and in the adult wing because they are homozygous for the cuticular marker crinkled (ck) (Foronda, 2009).

These clones entirely fill the posterior compartment. Staining for pMad activity shows the normal pattern in the centre of the disc, but the posterior pMad domain is completely absent, in clear contrast with wildtype discs (Foronda, 2009).

Since the genotype used allows good viability of the flies containing dpp M+ clones, a large number of adult wings was examined. In nearly every case the entire posterior compartment is marked with ck, indicating that it lacks dpp expression. The pattern and size of these wings is normal except in the proximal posterior region: the alula does not form and the pattern that appears in its place resembles that of a more distal region, which is specified by Dpp of anterior origin. The axillary cord is much reduced in size and has lost all its characteristic long hairs; some sclerites are also missing. This loss of structures does not appear to be due to death of proximal cells; no indication of caspase activity was found in this region. Moreover, the addition of the apoptosis inhibitor P35 in the posterior compartment does not modify the phenotype of the dpp M+ compartments (Foronda, 2009).

The favored interpretation of the wing phenotype is that in absence of the organising activity of the posterior Dpp, the proximal region is only patterned by Dpp of anterior provenance. It is interesting to note that even though dpp is not expressed in the alula cells, this structure is affected. This result illustrates the role of the posterior Dpp as an organizer, since it affects patterning in a non-autonomous manner (Foronda, 2009).

One intriguing question was if this posterior Dpp could form these proximal structures without Dpp of anterior origin. A transgenic strain carrying UAS-shmiR-dpp2 construct was used which has been shown to degrade the mRNA of Dpp. The smhiR-dpp over-expression in the anterior Dpp domain (by using ptc-gal4 or dppblnk-gal4) is able to silence efficiently Dpp gene activity, as shown by absence of pMad staining and the extended brk expression domain. Neither the posterior dpp expression nor the formation of alula and axillary cord are affected in both size and morphology. It is concluded that Dpp of posterior origin is necessary and sufficient to pattern these structures (Foronda, 2009).

dpp transcription in the P compartment is an intriguing finding and suggests a novel mode of dpp activation. The normal activation of dpp in the A compartment requires Hh signalling, which is blocked in the P compartment. Alternatively, it was possible that a local diminution of en activity in the proximal region of the P compartment would allow Hh and dpp activation by the standard mechanism. This question was addressed by examining in this region the expression of Cubitus interruptus (ci), a transcription factor that is essential for Hh signalling, and of patched (ptc), a Hh target gene. It was found that neither ci nor ptc are expressed in the posterior. However, there was the possibility that these two genes were expressed at low, undetectable levels. To test this possibility in full, Dpp activity was examined in clones of cells mutant for the smoothened (smo) gene, which would be unable to transduce the Hh signal. The result demonstrates that the posterior expression of dpp does not require Hh signalling and must therefore be activated by a different mechanism (Foronda, 2009).

The approach taken to identify the factor/s behind this posterior Dpp expression was to look for candidate genes or signalling pathways which are expressed in the corresponding place in the posterior compartment. The first one was vein, a ligand of EGFR signalling pathway, which coexpresses with Dpp in late 3rd instar wing disc. vein is the only EGFR ligand required for a proper wing development, so it was a good candidate. The elimination of all posterior vein function has effect neither on posterior Dpp function nor on hinge morphology. Other genes were tested based on expression and/or mutant phenotype in the alula, i.e., homothorax, Zfh2 and empty spiracles, among others. None of them affected Dpp expression (Foronda, 2009).

Another likely candidate was mirror, a member of the Iroquois complex (iro-C), for which a role in alula and axillary cord formation has been described. mirr expression was examined using the mirr-lacZ line and it was found that mirr is expressed in the presumptive alula and axillary cord region (Foronda, 2009).

M+ mirr clones were made to generate posterior compartments that were wholly mirr. They show an adult phenotype more extreme than that of dpp compartments: the alula and the axillary cord are entirely missing. In the discs dpp expression (shown by pMad staining) in the posterior compartment is lost, and consequently brk expression is up-regulated in the presumptive alula region. This result indicates that mirr is necessary for posterior dpp expression. In contrast, Dpp is not required for mirr expression, since the lack of posterior Dpp does not have an effect on mirr-LacZ transcription (Foronda, 2009).

Since the preceding results might suggest a mirr-mediated dpp activation gain of function clones of mirr were generated and whether they gave rise to ectopic Dpp activity was checked. NI significant up-regulation of dpp associated with those clones was detected. These experiments demonstrate that mirr activity is necessary for posterior dpp expression, but it is not sufficient to induce it. Therefore there must be other factors involved in posterior dpp activation (Foronda, 2009).

These results report a novel organizer role of Dpp that occurs during the third instar and is necessary and sufficient to pattern the proximal posterior region of the wing. This is achieved by an hh-independent, mirr-dependent activation of dpp in the posterior compartment (Foronda, 2009).

These findings provide a more complete picture of the development of the wing disc. There is evidence that the three signalling Dpp, Wg and Hh pathways are necessary for normal wing pattern and originate at compartment borders. The results indicate an unforeseen complexity of the function of the Dpp pathway. dpp becomes active in two different body domains, which have independent temporal and spatial regulation. The anterior domain is required for distal wing growth and patterning, whereas the P compartment domain is responsible for the formation of the posterior-proximal structures of the wing. Moreover, the source of Dpp in the posterior domain does not appear to be a compartment border, since no lineage restriction has been reported in the region. A comparable situation has been described for leg development, in which the Dpp and Wg signals originate at the A/P border, but the source of the EFGR signal is not a lineage border (Foronda, 2009).

The fact that this new dpp expression domain is common to wings and halteres may have some evolutionary significance, since this may be an attribute of dorsal thoracic appendages. These results suggest that the posterior tier of dpp expression may have appeared before the mesothoracic and metathoracic appendages diverged. It is therefore, possible that the new model of dorsal appendage development proposed may occur in other species of insects (Foronda, 2009).

Dpp signaling activity requires Pentagone to scale with tissue size in the growing Drosophila wing imaginal disc

The wing of the fruit fly, Drosophila melanogaster, with its simple, two-dimensional structure, is a model organ well suited for a systems biology approach. The wing arises from an epithelial sac referred to as the wing imaginal disc, which undergoes a phase of massive growth and concomitant patterning during larval stages. The Decapentaplegic (Dpp) morphogen plays a central role in wing formation with its ability to co-coordinately regulate patterning and growth. This study asked whether the Dpp signaling activity scales, i.e. expands proportionally, with the growing wing imaginal disc. Using new methods for spatial and temporal quantification of Dpp activity and its scaling properties, it was found that the Dpp response scales with the size of the growing tissue. Notably, scaling is not perfect at all positions in the field and the scaling of target gene domains is ensured specifically where they define vein positions. It was also found that the target gene domains are not defined at constant concentration thresholds of the downstream Dpp activity gradients P-Mad and Brinker. Most interestingly, Pentagone (Pent: see Flybase Magu), an important secreted feedback regulator of the pathway, plays a central role in scaling and acts as an expander of the Dpp gradient during disc growth (Hararatoglu, 2011).

This study measured Dpp pathway activity using an antibody specific to the phosphorylated form of Mad, and compared the P-Mad levels in space and time with the activity levels of direct target genes, such as brk, which plays key roles in both growth and patterning. Transcription of brk is directly repressed via P-Mad binding at defined silencer elements (SEs), resulting in inversely graded brk expression. Brk is the only known regulator affecting the positioning of the expression boundary of omb, while sal and dad translate input from both P-Mad and Brk into their expression boundaries. The dynamics of all of these readouts were analysed using antibodies where possible, to avoid potential misinterpretations due to reporter stability (Hararatoglu, 2011).

P-Mad levels scale very well posterior to 0.4 Lp (Lp stands for the length of the posterior compartment) with the exception of TC5 profiles near the D/V boundary. Previous studies that examined P-Mad scaling reached contradictory conclusions: the P-Mad gradients in late stage discs were reported to correlate with tissue size in a previous study and to have no correlation in another. Similarly, examination of P-Mad gradients across discs of different sizes led to the conclusion that the gradient did not expand, but in another study the P-Mad gradient was shown to scale with tissue size. It is believed that most of the confusion can be attributed to different profile extraction protocols as well as to the use of various definitions of scaling, as discussed below (Hararatoglu, 2011).

Since P-Mad is an early signature of the activation of the Dpp signaling pathway, it was of interest to find out how its scaling properties translate to its immediate key target, the brk gene. In addition to brk being directly repressed by P-Mad, the Brk protein itself is necessary for graded brk transcription. The range of Brk expression was found to strictly follow P-Mad in both wild-type and pent mutant discs. Similar to what was observe for P-Mad, Brk also shows very good scaling for positions posterior to 0.4 Lp. By contrast, levels of Brk increase steadily as the discs grow and cannot be explained by P-Mad dynamics alone. This increase in Brk levels could be due to the build-up of the unknown activator of brk transcription or, alternatively, the SEs in brk could become desensitized to the repressive input of P-Mad. Regardless of the cause of the increase, cells at a given relative position experience increasing levels of the Brk repressor over time (Hararatoglu, 2011).

Traditionally, Dpp and P-Mad gradients have been described by a decaying exponential with characteristic decay length λ. This decay length is different for each profile and corresponds to the position at which the protein levels have decreased by a factor e. The correlation between the decay length and the tissue size has been used as a proxy for scaling, e.g. one study found no significant scaling for the Dpp and P-Mad profiles at the end of 3rd instar stage. Similarly, the width of the P-Mad profile has been used to characterize the spread of the gradient and it was concluded that the width of the P-Mad profile is constant during growth. In contrast, the current study detected that the P-Mad profile expands as the tissue grows. This discrepancy may be due to the fact the previous study lacked time classes TC3 and TC4 in their sample collection, the period where the P-Mad gradient expands before contracting again when measured close to the D/V boundary. Thus, possibly the measurements of the P-Mad profiles were done in the vicinity of the D/V boundary, where at TC5 P-Mad has a sharp profile reminiscent of 30 h younger discs. Hence, the choice of position can significantly alter the final interpretations of the data (Hararatoglu, 2011).

Consistent with these results, another study (Wartlick, 2011) recently showed that the decay lengths of Dpp-GFP, P-Mad, brk-GFP, and dad-RFP do correlate with the length and the area of the posterior compartment during tissue growth. Importantly, the Wartlick study also assessed scaling qualitatively in the whole field by looking at the collapse of the profiles in relative positions and normalized intensities. This method has two advantages: it does not require any fitting of the profiles, and it shows scaling at all positions and not just at the characteristic decay length position (i.e. x = λ) (Hararatoglu, 2011).

A recent mathematical model termed 'expansion-repression integral feedback control' suggests that scaling can emerge as a natural consequence of a feedback loop (BenZvi, 2010). The hypothetical 'expander' molecule facilitates the spread of the morphogen and in turn is repressed by it; scaling is achieved given that the expander is stable and diffusible. The known properties of Pent fit the requirements of this hypothetical agent: Pent is secreted, required for Dpp spreading, and pent transcription is directly inhibited by Dpp signaling. However, it is not known how stable Pent is, and pent transcription is never abolished in the entire field in which the gradient acts during larval stages. To test whether Pent could be a key player involved in scaling of Dpp activity during disc growth, the analyses were repeated at all time points in the absence of Pent. It was found that the P-Mad and Brk gradients indeed fail to scale with the tissue size in this mutant background. Scaling of dad-GFP and Omb are also strongly affected, while Sal still exhibits some degree of Pent-independent scaling in the anterior compartment. Importantly, while the function of Pent is essential for proper scaling of the Dpp activity gradient, it is noted that Pent alone cannot account for the observed selective scaling of Omb and Sal domain boundaries. Scaling of these target genes specifically in those regions in which they have a patterning function points to the involvement of additional players, which will be the subject of future research. Hence, the current findings strongly suggest that Pent is a very good candidate to be the expander in the 'expansion-repression integral feedback control' model and therefore provide the first mechanistic insights into the question of scaling in wing patterning. The exact biochemical functions of Pent have to be determined in order to get a more mechanical view of gradient scaling in the developing wing imaginal disc (Hararatoglu, 2011).

More than 40 years ago, Lewis Wolpert proposed the French flag model to explain pattern formation by morphogens. This study tested whether the activity gradients downstream of Dpp, namely P-Mad and Brk, are read out by their target genes at constant concentration thresholds. Thus, average P-Mad and Brk concentrations were measured at Dad, Omb, and Sal expression boundaries across development. It was found that the amount of P-Mad at these boundaries slightly increased (Dad), slightly decreased (Sal), or was constant throughout development (Omb). Among these three targets, the Omb domain is the widest and it corresponds to a region where the P-Mad gradients scale perfectly; as a result, P-Mad levels fluctuate very little at the Omb domain boundary. Interestingly, the domain boundary of Omb is thought to solely depend on Brk and hence constant P-Mad levels might be a mere coincidence. Remarkably, all the target genes that were considered respond to significantly increasing levels of Brk, suggesting that the target genes desensitize to Brk over time, so that more and more Brk can be tolerated at the domain boundary. Alternatively, if it is considered that the domain boundaries of dad-GFP and Sal do not respond to constant P-Mad levels either, another explanation could be that Brk and P-Mad signals are combined in a non-additive fashion in order to define the boundary position of the target genes. Following this assumption, a simple combination of these signals was sought that is constant at the target gene domain boundary for all TCs. It is proposed that Brk and the unknown activator of Omb could be similarly combined in order to determine the Omb domain boundary (Hararatoglu, 2011).

This paper's data was used to further test a model that was recently proposed to explain the uniform growth in the wing imaginal disc (Wartlick, 2011). The model poses that the temporal changes in Dpp signaling levels drive tissue growth; cells divide when they experience a relative increase of 50% in the levels of Dpp signaling. Since it is the relative differences and not the absolute amount of Dpp signal that regulate cell divisions, the model can account for the uniform growth of the wing disc. Since the relative increase in Dpp activity slows down, the cell cycles lengthen as the disc grows. Growth stops when the cell division time exceeds 30 h. The model of Wartlick is based on the finding that Dpp activity scales with tissue size and that cells at a given relative position experience increasing levels of Dpp signaling over time. In contrast, a general temporal increase was not observed in the level of Dpp signaling at a given relative position in this study. P-Mad is the most upstream and the most dynamic readout available for the activity of the Dpp pathway and it was found that the relative increase in P-Mad levels throughout development is not significantly different from zero at most relative positions. Why is the increase in Dpp-GFP levels not reflected in P-Mad levels? A potential explanation for this might be that the observed accumulation of Dpp-GFP was due to the stability and accumulation of Gal4 since Dpp-GFP was under UAS control. The Wartlick study showed that the half-life of the Dpp-GFP fusion protein is only 20 min, but the Gal4 stability was not considered. Alternatively, the system could get desensitized over time and more and more Dpp would be required to lead to similar P-Mad levels. Finally, increases in Dad levels could counteract the increase in Dpp levels, since Dad is an inhibitory Smad (Hararatoglu, 2011).

Wartlick (2011) monitored Dpp signaling levels using a dad-RFP reporter and found a 5-fold increase in the course of 36 h. In the current analysis, a similar tool, dad-GFP, was used and the Wartlick results could not be fully reproduced. Though it was also found that dad-GFP scales with tissue size, its levels increase merely 2-fold over 40 h, and this increase takes place only in the medial 25% region of the disc, while cells in the lateral part experience a decrease in dad-GFP levels. This disparity in the fold increases is likely due to the higher stability of RFP, since the enhancer used was identical in both studies. Additionally, it was found that the levels of Brk, another direct target of the pathway with a very well-established role in suppressing growth in lateral regions, increase in average 4-fold in the interval studied, an observation not reported by Wartlick. In the lateral areas, increase in Dpp activity (if present) is below detection levels and would be opposed by increasing Brk levels. Importantly, increasing Brk levels, if they were to depend solely on Dpp, would suggest decreasing Dpp activity in lateral areas as Brk expression is directly suppressed by Dpp activity. Hence, the data raise serious questions about the validity of this uniform growth model, especially in the lateral regions of the pouch. An alternative model is favored that does not rely on Dpp activity alone to explain uniform growth in the wing disc (Hararatoglu, 2011).

Crosstalk between epithelial and mesenchymal tissues in tumorigenesis and imaginal disc development

Cancers develop in a complex mutational landscape. Interaction of genetically abnormal cancer cells with normal stromal cells can modify the local microenvironment to promote disease progression for some tumor types. Genetic models of tumorigenesis provide the opportunity to explore how combinations of cancer driver mutations confer distinct properties on tumors. Previous Drosophila models of EGFR-driven cancer have focused on epithelial neoplasia. This study reports a Drosophila genetic model of EGFR-driven tumorigenesis in which the neoplastic transformation depends on interaction between epithelial and mesenchymal cells. Evidence is provided that the secreted proteoglycan Perlecan (Drosophila Trol) can act as a context-dependent oncogene cooperating with EGFR to promote tumorigenesis. Coexpression of Perlecan in the EGFR-expressing epithelial cells potentiates endogenous Wg/Wnt and Dpp/BMP signals from the epithelial cells to support expansion of a mesenchymal compartment. Wg activity is required in the epithelial compartment, whereas Dpp activity is required in the mesenchymal compartment. This genetically normal mesenchymal compartment is required to support growth and neoplastic transformation of the genetically modified epithelial population. This study reports a genetic model of tumor formation that depends on crosstalk between a genetically modified epithelial cell population and normal host mesenchymal cells. Tumorigenesis in this model co-opts a regulatory mechanism that is normally involved in controlling growth of the imaginal disc during development (Herranz, 2014).

Accumulating evidence indicates that tumor progression results from the interaction between tumor cells and the surrounding normal cells that make up the tumor microenvironment. This study has used the Drosophila wing imaginal disc to dissect the crosstalk between tumor cells and surrounding normal cells, in tumors of epithelial origin. In this model, interaction between the two cell populations is required for tumor growth, neoplastic transformation of the epithelium, and metastasis, even though the genetic modifications were introduced into only one of the two cell populations (Herranz, 2014).

Carcinomas express growth factors involved in the communication between cancer cells and tumor-associated normal cells. The role of TGF-β and Wnt signaling pathways in tumor initiation is well known, but their role as mediators of the interaction between tumor cells and stromal cells has been less well studied. This study observed that EGFR overexpression induced expression of the endogenous Wg and Dpp genes in the epithelial compartment of the tumors. Wg, together with EGFR, is needed in the epithelial cells to drive tumorous growth. The role of the Dpp pathway is different. The findings indicate that Dpp, produced by the epithelial cells, acts on the mesenchymal stromal cells. Dpp signaling activity was not required in the epithelial cells themselves for tumorous growth. Instead, downregulation of the Dpp pathway in mesenchymal cells blocked tumorous growth of the epithelial population. This suggests that Dpp signaling elicits a feedback response from the mesenchymal population. As a consequence, the resulting tumors are composed of a mix of mutant epithelial cells and genetically normal mesenchymal cells, resembling organization observed in human tumors (Herranz, 2014).

EGFR is upregulated in many carcinomas. EGFR is able to promote tissue overgrowth, but additional mutations are required for malignant transformation and invasion. The findings of this study have shown that upregulation of Perlecan is sufficient to cooperate with EGFR to produce neoplastic transformation. Perlecan is a secreted HSPG of the ECM that is overexpressed in many human tumor types. TGF-β ligands have been shown to promote changes in the tumor microenvironment in mammals. Perlecan has also been reported to stabilize Wg and promote Wg activity in Drosophila. Thus, Perlecan production could potentiate the effects of Dpp and Wg produced by the epithelial cells. These findings raise the possibility that Perlecans might have a fundamental role in mediating interactions between epithelial tumor cells and mesenchymal stromal cells, in addition to their known roles in tumor angiogenesis (Herranz, 2014).

The crosstalk between tumor and microenvironment determines the phenotype of the tumor. Signaling from the tumor microenvironment can suppress the malignant tumor phenotype, yet the tumor microenvironment can also promote malignant transformation. The finding that ablation of the mesenchymal cell population reverted the tumor phenotype in this model suggests that signals from the mesenchymal cells are required for tumor progression. This same cell population was required to support growth of the epithelial population in a nontumorous normal tissue context. It is postulated that signals from the adepithelial mesenchymal cells sustain proliferation of the epithelial cells and likewise that signals from the epithelia drive proliferation of the mesenchymal cells. This normal feedback mechanism can be coopted to drive growth of the two tissues, as, for example, when EGFR and Perlecan were overexpressed. A remarkable, unexpected aspect of these findings is that this feedback loop appears to be sufficient to drive the epithelial tissue beyond hyperplasia, through neoplastic transformation, and into metastasis (Herranz, 2014).

Caspase inhibition during apoptosis causes abnormal Dpp signalling and developmental aberrations in Drosophila

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).

Dpp signaling and the induction of neoplastic tumors by caspase-inhibited apoptotic cells in Drosophila

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).

Evading apoptosis is widely recognized as a hallmark of cancer cells. There is also evidence that caspase activity is inhibited in some aggressive human cancers. These findings may therefore provide a paradigm for the formation of neoplastic tumors in tissues that are unable to die (Perez-Garijo, 2005).

Dependence of Drosophila wing imaginal disc cytonemes on Decapentaplegic

The anterior/posterior (A/P) and dorsal/ventral (D/V) compartment borders that subdivide the wing imaginal discs of Drosophila third instar larvae are each associated with a developmental organizer. Decapentaplegic, a member of the transforming growth factor-ß superfamily, embodies the activity of the A/P organizer. It is produced at the A/P organizer and distributes in a gradient of decreasing concentration to regulate target genes, functioning non-autonomously to regulate growth and patterning of both the anterior and posterior compartments. Wingless is produced at the D/V organizer and embodies its activity. The mechanisms that distribute Dpp and Wg are not known, but proposed mechanisms include extracellular diffusion, successive transfers between neighbouring cells, vesicle-mediated movement, and direct transfer via cytonemes. Cytonemes are actin-based filopodial extensions that have been found to orient towards the A/P organizer from outlying cells. This study shows that in the wing disc, cytonemes orient toward both the A/P and D/V organizers, and that their presence and orientation correlate with Dpp signalling. The Dpp receptor, Thickveins (Tkv), is present in punctae that move along cytonemes. These observations are consistent with a role for cytonemes in signal transduction (Hsiung, 2005).

Cytonemes appear as fluorescent strands emanating from the apical surface of disc cells that express green fluorescent protein (GFP). Using standard epifluorescence microscopy, cytonemes are visible only if neighbouring cells have low background fluorescence, only in unfixed discs, and only if they extend in a single optical plane. The contour of the apical surface of the notum primordium is rather flat, and cytonemes can be imaged in this region in discs that are suspended in liquid. However, the wing pouch primordium is convex, and cytonemes can be imaged only in discs that have been slightly flattened. The fragile nature of disc cells requires that physical and osmotic insults be minimized, and the methods developed to image wing cytonemes avoid rupture, delamination and other responses to injury (Hsiung, 2005).

Small clones (averaging 10-15 cells) expressing CD8-GFP are visible in a speckled pattern. High-magnification views of clones in similar discs reveals cytonemes extending outwards from some, but not all clones. On the basis of the presence or absence of cytonemes and on the orientation of cytonemes, three regions of the disc can be distinguished. In the wing blade primordium, approximately 20% of the clones extended cytonemes oriented toward either the A/P or D/V compartment borders. More than 95% of these clones had cytonemes oriented toward one of the two borders, and <5% had cytonemes oriented toward both. It has not been possible to establish whether all cells extend cytonemes, whether cells can extend more than one cytoneme, or whether a single cell can extend cytonemes toward both axes. A/P cytonemes as long as 80.2 microm have been recorded; the average length in these preparations is 20.8 microm. D/V cytonemes were shorter, averaging 8.8 microm (Hsiung, 2005).

Clones in the notum primordium radiate cytonemes in all directions, without a consistent bias toward either the A/P or D/V axes of the disc. Cytonemes associated with notum clones averaged 7.4 microm in length, almost 65% shorter than A/P cytonemes in the wing primordium. Unlike cells in the wing and notum primordia, cells in the hinge/pleural primordium do not extend cytonemes. These hinge/pleural cells were examined by expressing GFP in clones and by using an enhancer trap expressed in the hinge region, but no cell extensions were observed in either case (Hsiung, 2005).

Although Dpp is essential for cell survival in the notum, there is no indication that the A/P compartment border in the notum has an associated organizing centre, and it is ambiguous whether Dpp functions as a morphogen in the notum region. Dpp is apparently not required for either growth or cell survival in the hinge/pleural region. Thus, directional cytonemes are present in the wing primordium (where Dpp functions as a morphogen); cytonemes are present and 'omni-directional' in the notum (where Dpp may function only to support cell proliferation), and cytonemes are absent in the hinge/pleural region, where Dpp function appears not to be required. These three distinct cell types -- cells with A/P- or D/V-oriented cytonemes, cells with cytonemes lacking a directional bias, or cells without cytonemes -- could each be imaged in a restricted and defined region of the same disc (Hsiung, 2005).

Whether the shape and distribution of cytonemes in wing discs correlates with the presence of Dpp was tested. When Dpp levels were reduced at the A/P organizer using temperature-sensitive mutants of either dpp (dppts) or hedgehog (hhts) (dpp expression depends upon Hh signalling), cytonemes in the wing primordium were affected. At the permissive temperature (18°C) in mutant discs, or at either the permissive or non-permissive (29°C) temperatures in normal discs, cytonemes emanating from cells at the lateral flanks of discs oriented toward the A/P organizer. After incubation at 29°C, however, cytonemes in hhts and dppts mutant discs are more numerous (> twofold), and are not uniformly oriented toward the A/P organizer region. In these mutant discs, curved and bent cytonemes, and cytonemes crossing over each other were observed. Cytonemes with such shapes are never observed under normal conditions, or if mutant larvae are returned to the permissive temperature after a period of incubation at 29°C (Hsiung, 2005).

To test whether Dpp is sufficient for cytoneme induction, cytonemes were imaged in discs in which Dpp was expressed ubiquitously. In control discs, cells project cytonemes toward A/P and D/V axes only, but in discs with heat-shock-induced Dpp (hs-dpp) >50% of the clones projected cytonemes outwards in all directions. These cytonemes were significantly shorter than those in untreated discs, averaging about 10.6 microm in length. Even more striking were the cells in the hinge domain, which normally do not extend cytonemes. Under conditions of ubiquitous Dpp expression, cells in the hinge domain extend cytonemes in apparently random orientations. The ability to image cytonemes is limited to preparations in which discs have been extracted from larvae and the cytonemes are static, but their varied appearance under the conditions tested illustrates that they are dynamic in vivo. Although a model is favored in which they extend from cells in random directions but become stabilized when functional contacts are made with signalling cells, the possibility that their directionality is directly influenced by extracellular cues cannot be excluded (Hsiung, 2005).

The distribution of the Dpp receptor Tkv was monitored, as a Tkv-GFP fusion protein. When expressed in the lateral flanks of wing discs, most of the fluorescence is localized to the plasma membrane of expressing cells. However, bright, motile punctae were also present in more central regions, as far as 30 microm from the edge of the expression domain. These punctae are motile, moving in both anterograde and retrograde directions, and some images clearly revealed their association with cytonemes. Trafficking of these punctae was approximately 5-7 microm s-1, a rate consistent with measured rates of vesicular movement on actin filaments. The resolution of these studies could not establish whether these filaments were inside or on cytonemes (Hsiung, 2005).

The distribution of Tkv-GFP punctae around clones was plotted in discs with normal expression of Dpp or with ubiquitous Dpp expression. In normal discs, Tkv-GFP punctae are polarized in the direction of the A/P border. In contrast, Tkv-GFP punctae in heat-shocked hs-dpp discs are more numerous and project in various directions all around the circumference of the clones. Since these patterns of Tkv-GFP punctae could be imaged in unflattened discs, their distribution was compared in both flattened and unflattened discs. No differences were detected between the two conditions with respect to either the total number of punctae, or to the distance from or position relative to the clones. This confirms that the slight flattening used to image cytonemes does not generate or substantially alter these structures (Hsiung, 2005).

Previous work has demonstrated that cytonemes bind phalloidin (a specific F-actin-binding protein) and can be labelled with an actin-GFP fusion protein, suggesting that cytonemes are actin-based. To test whether cytonemes and the movement of Tkv-GFP punctae is actin-dependent, discs containing clones of Tkv-GFP-expressing cells were treated with cytochalasin D, an actin-binding drug. The number of Tkv-GFP punctae at a distance from the cell bodies was dramatically reduced in treated discs. Bright punctae were observed on the surface of cells expressing Tkv-GFP, and they appeared to move along the surface of the cells even in the presence of drug. In contrast, the bright punctate fluorescence distant from GFP-expressing cells was not motile. These observations suggest that cytonemes can function as vehicles for active, actin-based transport of receptors (Hsiung, 2005).

To better document the structure of cytonemes, optical sections of cytoneme-producing clones were reconstructed to render their three-dimensional structure. In such images, cytonemes labelled with CD8-GFP as well as cytonemes containing Tkv-GFP punctae, were observed that extend from the apical surface of the disc columnar epithelial cells. In contrast, expression of a human guanine nucleotide exchange factor (GEF) protein, Vav-GFP, which has been shown to localize to filopodia in vertebrate cells, labels basal filopodia when expressed in wing disc cells. This preferential placement of proteins into different types of filopodial extensions indicates that apical and basal extensions are structurally distinct: it suggests that these cell extensions may be functionally distinct, and it implies the existence of a mechanism for sorting proteins to specific types of extensions (Hsiung, 2005).

Dpp is synthesized and secreted by a narrow stripe of 5-7 cells adjacent to the A/P compartment border in the wing primordium, and it distributes in a gradient of decreasing concentration that extends across the wing pouch. A concentration gradient does not imply a mechanism for distribution; it is conceivable that cytonemes sense and respond to Dpp but do not ferry it. However, on the basis of the results presented in this study, cytoneme-based transport is considered to be an attractive possibility. As this work shows, the Dpp receptor Tkv is present in cytonemes, and the presence, orientation and shape of cytonemes in wing discs correlates with what is known about the different roles that Dpp has in the wing, notum and hinge primordia. Moreover, cytonemes change in response to conditions of Dpp gain-of-function and loss-of-function. These correlations are consistent with the idea that Dpp moves from its source in an oriented manner imposed by the directionality of these cellular extensions. Several recent studies reported have cellular extensions in Drosophila cells that correlate with signalling by Branchless (a Drosophila FGF), Notch and Scabrous, extensions in spider cells that correlate with signalling by Dpp, and extensions in mammalian cells that correlate with signalling by epidermal growth factor. The widespread occurrence of cytonemes and cytoneme-like filopodia suggests that their role in long-distance signalling might be a general one, one that might permit selective signalling in ways that enable cells to regulate both release and uptake of signals (Hsiung, 2005).

Developmental analysis and squamous morphogenesis of the peripodial epithelium in Drosophila imaginal discs

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).

Modulation of AP and DV signaling pathways by the homeotic gene Ultrabithorax during haltere development

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 dppd6/dppd12 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).

Hox control of organ size by regulation of morphogen production and mobility

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).

It was hypothesized that the more limited pathway activation in the haltere might contribute to its smaller size. If correct, increasing tkv expression in the wing should reduce its size. Adult wings from flies expressing uniform UAS-tkv+ were ~30% smaller than control wings; however, wing cell size remained the same. Similar results were seen in staged imaginal discs and when UAS-tkv+ expression was limited to the wing and the haltere. Conversely, reducing Tkv amounts by uniformly expressing UAS-tkvRNAi in wings and halteres increased haltere size by 30 to 60%. In a complementary experiment, tkv transcription was reduced in the haltere by expressing a known tkv repressor, master of thickveins (mtv). In this experiment, haltere discs were measured instead of the adult appendage; it was consistently found that the appendage-generating region of these discs increased in size by ~40%. Thus, different amounts of Tkv not only affect Dpp pathway activation but also affect organ size. The fact that manipulating only Tkv production does not fully transform the sizes of these appendages suggests that additional mechanisms, such as the reduced amounts of dpp transcription and the modulation of other morphogen pathways by Ubx, also contribute to size regulation. Consistently, when Dpp production is decreased in wing discs that uniformly express UAS-tkv+, wing size was reduced more than it was by either single manipulation (Crickmore, 2006).

Next, how Ubx up-regulates tkv in the haltere was addressed. tkvlacZ expression and amounts of Tkv protein were cell-autonomously reduced in medial Ubx clones, whereas lateral Ubx mutant tissue retained high amounts of Tkv. Because tkv is repressed by Dpp and Hh signaling in the wing, these results suggest that, in the haltere, these signals are not able to repress tkv. Consistently, activation of the Dpp pathway by expressing a constitutively active form of Tkv (TkvQD) resulted in cell-autonomous tkv-lacZ repression in the wing pouch, whereas repression is not observed in the corresponding region of the haltere disc (Crickmore, 2006).

In Ubx mosaic haltere discs, it was also found that medial Ubx+ tissue showed stronger P-Mad staining than Ubx tissue at the same distance from the Dpp source. This observation is interpreted as evidence that Ubx+ tissue is more effective at trapping and transducing Dpp than Ubx tissue because of higher Tkv production in Ubx+ cells (Crickmore, 2006).

To further understand the control of tkv by Ubx, the known tkv repressor, mtv, was examined. In medial wing disc cells, mtv expression is approximately complementary to tkv expression, and mtv clones in this region of the wing disc cell autonomously derepressed tkv. In the haltere, very low mtv-lacZ expression was detected in the cells that stained strongly for P-Mad, suggesting that mtv is repressed by Dpp in this appendage. Accordingly, strong repression of mtv-lacZ was seen in UAS-tkvQD-expressing haltere pouch clones, whereas weak or no repression was seen in analogous wing clones. It was also found that, as expected, Ubx clones in the medial haltere cell autonomously derepressed mtv-lacZ (Crickmore, 2006).

In the wing, Dpp and mtv are mandatory repressors of tkv: In the absence of either, tkv expression is high. In the haltere in the presence of Ubx, Dpp is a repressor of mtv. Consequently, high levels of these obligate tkv repressors (Dpp signaling and mtv) do not coexist in the haltere, resulting in tkv derepression. Consistent with this model, when mtv expression was forced in the medial haltere, where it coexists with Dpp signaling, it repressed tkv-lacZ. It is noted, however, that Ubx is likely to control tkv through additional means, because mtv mutant wing clones did not derepress tkv-lacZ expression to haltere levels, and ectopic mtv in the haltere did not repress tkv-lacZ expression to the extent seen in the medial wing (Crickmore, 2006).

Because of high Tkv production in the wild-type haltere disc, peak Dpp signal transduction occurs in the AP organizer, the same cells that transduce the Hh signal. Thus, in the haltere, the activity profiles for these two signal transduction pathways coincide with each other. In contrast, low tkv expression in the wing AP organizer results in two peaks of Dpp signaling that are on either side of Hh-transducing cells. This difference will have important consequences for the expression of genes that are targets of both pathways. For example, dpp is activated by Hh and repressed by Dpp signaling. In the haltere, these two conflicting inputs occur in the same cells, possibly contributing to reduced dpp expression compared with the wing. Ubx clones cell-autonomously up-regulated dpp-lacZ in the haltere. To test whether Ubx lowers dpp transcription in part by aligning Dpp and Hh signaling, uniform UAS-tkv+ was expressed in the dorsal half of the wing disc. As a result, in this region of the wing disc both signals peaked in the same cells, and dpp-lacZ expression was reduced compared with the ventral half of these wing discs. Conversely, expressing tkvRNAi in dorsal haltere cells increased dpplacZ expression. Thus, Ubx reduces dpp transcription in part by changing where peak Dpp signaling occurs in the disc. Ubx is likely to reduce dpp expression in additional ways, because increasing tkv expression does not lower dpplacZ expression to that observed in wild-type haltere. Nevertheless, varying the relative spatial relationships between signal transduction pathways is a potentially powerful mechanism for modifying the outputs from commonly used pathways. It is suggested that selector genes may work through molecules that control ligand distribution to vary the spatial relationships between these and other signal transduction pathways in diverse contexts during development (Crickmore, 2006).

The finding that increased tkv expression results in decreased dpp transcription reveals an unexpected link between Dpp mobility and Dpp production. Because of this link, the above experiments do not discriminate between growth effects due to differences in Dpp mobility per se as opposed to secondary consequences on Dpp production. To distinguish between these scenarios, use was made of a compartment-specific Ubx regulatory allele, posterior bithorax (pbx), that lacks detectable Ubx in the P compartment when paired with a Ubx null allele but still has normal Ubx expression in the A compartment. Consequently, in pbx/Ubx haltere discs, the P compartment increased in size such that the P:A size ratio was 1.45; the P:A ratio of +/Ubx haltere discs was ~0.35. It is suggested that Dpp more readily diffuses into and through the P compartments of pbx/Ubx discs because of the wing-like expression pattern of tkv and that this wing-like diffusion results in its robust growth (Crickmore, 2006).

To test whether differences in Tkv-regulated Dpp diffusion affect tissue growth independently of an effect on Dpp production, the consequences of expressing UAS-tkv+ uniformly in pbx/Ubx haltere discs were examined. If Tkv's effect on growth is mediated only by lowering Dpp production, both compartments should be reduced in size and thus maintain the same size ratio. However, if reducing Dpp mobility directly affects growth, the P compartment should be reduced in size more than the A compartment, which, in pbx/Ubx discs, already has high tkv expression. It was found that expressing uniform tkv+ in pbx/Ubx discs decreased the size of the P compartment more than the A compartment, resulting in a P:A ratio of 0.83. Because uniform tkv+ returned the P:A ratio back to the wild-type ratio by ~56%, these results suggest that this single variable is sufficient to provide ~50% rescue of the size of an otherwise Ubx mutant P compartment (Crickmore, 2006).

This study has investigated the mechanism underlying a classic yet poorly understood phenomenon in biology: how size variations are genetically programmed in animal development. Many experiments show that organ size is not governed by counting cell divisions but instead depends on disc-intrinsic yet cell-nonautonomous mechanisms, possibly relying on morphogen signaling. The results support this idea by showing that alterations in a morphogen gradient contribute to size differences between appendages. In the example investigated here, Ubx limits the size of the haltere by reducing both Dpp production and Dpp mobility. Moreover, both of these effects are due, in part, to higher tkv expression in the medial haltere. In many morphogen systems, the receptors themselves have been shown to control the distribution of the ligand and, consequently, pathway activation. This study shows that a selector gene exploits this phenomenon to modify organ size (Crickmore, 2006).

Although the mechanism by which Dpp controls proliferation is not fully understood, recent results argue that, in the medial wing disc, cells may compare the amount of Dpp transduction with their neighbors, whereas lateral cells proliferate in response to absolute Dpp levels. The results suggest several ways in which the altered Dpp gradient in the haltere could limit its growth. First, proliferation of lateral haltere cells may be limited because they perceive less Dpp. Second, the narrower Dpp gradient results in fewer cells exposed to the gradient in the medial haltere. Another notable difference is that, because there are two peaks of Dpp signaling in the wing but only one in the haltere, the wing has four distinct slopes whereas the haltere has only two. The less complex Dpp activity landscape of the haltere may also contribute to its reduced growth (Crickmore, 2006).

On the basis of these results, it is suggested that altering the shape and intensity of morphogen gradients may be a general mechanism by which selector genes affect tissue sizes in animal development. Consistent with this view, wingless (wg), another long-range morphogen in the wing, is partially repressed in the haltere. Intriguingly, some of the size and shape differences in the beaks of Darwin's finches are controlled by alterations in the production of the Dpp ortholog BMP4. The results suggest that differences in the diffusion of this ligand may also contribute to the range of beak morphologies that have evolved in these species (Crickmore, 2006).

Drosophila SnoN modulates growth and patterning by antagonizing TGF-β signalling

Signalling by TGF-β ligands through the Smad family of transcription factors is critical for developmental patterning and growth. Disruption of this pathway has been observed in various cancers. In vertebrates, members of the Ski/Sno protein family can act as negative regulators of TGF-β signalling, interfering with the Smad machinery to inhibit the transcriptional output of this pathway. In some contexts ski/sno genes function as tumour suppressors, but they were originally identified as oncogenes, whose expression is up-regulated in many tumours. These growth regulatory effects and the normal physiological functions of Ski/Sno proteins have been proposed to result from changes in TGF-β signalling. However, this model is controversial and may be over-simplified, because recent findings indicate that Ski/Sno proteins can affect other signalling pathways. To address this issue in an in vivo context, the function of the Drosophila Ski/Sno orthologue, snoN was analyzed. SnoN was found to inhibit growth when overexpressed, indicating a tumour suppressor role in flies. It can act in multiple tissues to selectively and cell autonomously antagonise signalling by TGF-β ligands from both the BMP and Activin sub-families. By contrast, analysis of a snoN mutant indicates that the gene does not play a global role in TGF-β-mediated functions, but specifically inhibits TGF-β-induced wing vein formation. It is proposed that SnoN normally functions redundantly with other TGF-β pathway antagonists to finely adjust signalling levels, but that it can behave as an extremely potent inhibitor of TGF-β signalling when highly expressed, highlighting the significance of its deregulation in cancer cells (Ramel, 2006).

SnoN overexpression produced a range of different patterning and growth phenotypes in the eye, wing and other tissues. The patterning defects observed are consistent with reduced TGF-β signalling, e.g. loss of wing veins with wing GAL4 drivers and failure of thorax closure when expressed in presumptive thoracic body wall epithelium. Dpp and Activin signalling have also been implicated in driving growth in the wing and so the growth inhibitory activity of SnoN in this tissue also fits with its proposed antagonistic function. Growth regulation by TGF-β signalling in the eye has been less extensively studied, but it was possible to show that increasing either Dpp or Activin signalling primarily in differentiating cells promotes overgrowth and this is strongly suppressed by co-overexpression of SnoN. When SnoN is expressed by itself in the differentiating eye, its growth inhibitory effects appear to be entirely due to reduced cell growth and cell size. Even though the disorganized patterning seen in eyes with excess TGF-β signalling may result from changes in cell number as well as cell size, these effects are also potently suppressed by SnoN (Ramel, 2006).

It should be noted that Activin signalling was less efficiently suppressed by SnoN compared to Dpp signalling in both the eye and wing. Although the data support a role for SnoN in inhibiting Activin functions, the possibility that there is cross-talk between the two TGF-β pathways cannot be excluded, particularly in these overexpression assays, and that suppression of Activin’s effects on the Dpp target Smad Mad are being observed. Resolution of this issue will require the identification of a specific Activin target gene in the wing or eye, as has been used for the Dpp pathway (Ramel, 2006).

Overgrowth phenotypes are also observed upon overexpression of components of the InR signalling pathway. Interestingly, SnoN overexpression was unable to suppress the overgrowth generated by Akt1 overexpression, indicating that SnoN specifically acts downstream of TGF-β signalling. In fact, overexpressed Akt1 suppressed the growth inhibitory effects of SnoN. This is consistent with previous observations that the effects of Tkv overexpression on growth are at least partly mediated by components of the InR signalling cascade (Ramel, 2006).

By studying the regulation of an established Dpp target gene, omb, in the wing disc through clonal analysis, it was found that SnoN inhibits Dpp signalling cell autonomously. Not only does this support the hypothesis that SnoN directly modulates TGF-β signalling in flies, but the cell autonomous behaviour of this molecule is consistent with its proposed role as a transcriptional modulator (Ramel, 2006).

In vertebrates, Ski/Sno proteins have been implicated in the regulation of several different signalling cascades, including those involving Hedgehog and Wnt family proteins (Dai, 2002; Chen, 2003), but the relevance of these interactions in vivo has remained unclear. Overexpression data cannot exclude a role for SnoN in these other signalling pathways, but they do suggest a primary function in TGF-β signalling, a conclusion further supported by mutant analysis. Although the SnoN-TGF-β link has been suggested previously in vertebrate systems, primarily through overexpression approaches in cell culture, analysis in Drosophila confirms the importance of this process in vivo. In addition, the results are also consistent with the idea that SnoN’s effects on growth in whole animals may all be mediated through changes in TGF-β signalling, which in Drosophila primarily plays a growth-promoting role. Hence in flies, SnoN acts as a tumour suppressor, but just as in vertebrates, its effects on growth are most clearly observed when overexpressed (Ramel, 2006).

It was surprising to find that flies homozygous for the snoNGS-C517T mutant allele develop with only minor patterning defects (ectopic wing veins). This suggests that SnoN has a highly restricted role in development. Since in situ analysis indicates that snoN is expressed quite broadly in imaginal discs, this restricted role cannot be explained merely by localized gene expression (Ramel, 2006).

It is believed that snoNGS-C517T is likely to represent a strong loss-of-function allele for three reasons. (1) It is predicted to produce a protein that lacks the entire evolutionarily conserved SAND domain, which interacts with Smad4 (co-Smad), and about one third of the Ski/Sno family domain, so it should not be able to antagonize the TGF-β signalling pathway. It also lacks regions required in mammalian Ski for binding to Gli3 (Hedgehog pathway; Dai, 2002) and FHL2 (Wnt pathway; Chen, 2003). Thus, even if fly SnoN could interact with these signalling cascades, it is predicted that the mutant protein would not. (2) Overexpression phenotypes observed with the snoNGS18054 insertion are all completely reverted by the mutation, indicating that the allele has lost its biological activity. (3) Ubiquitous overexpression of the putative dominant negative snoNGS-C517T allele precisely phenocopies the homozygous mutant phenotype. Thus, the results suggest that Drosophila SnoN has no essential role in the majority of TGF-β-dependent events, despite its potent activity as a TGF-β signalling antagonist in flies and vertebrates. The developmental functions of snoN in mammalian development are not yet clear. Indeed, loss of SnoN function has been shown to be embryonic lethal (Shinagawa, 2000), but another group (Pearson-White, 2003) reported that two different snoN mouse mutants are viable and only show T-cell activation defects (Ramel, 2006).

Several lines of evidence confirm that the ectopic wing vein phenotype observed in snoNGS-C517T homozygous flies results from inhibition of a SnoN-dependent function. First and most importantly, expression of wild-type SnoN with bs1348-GAL4 largely suppresses the snoNGS-C517T ectopic wing vein phenotype. In addition, the similarity between the mutant phenotype and the defects observed in situations where TGF-β activity is up-regulated, as well as the genetic interaction data with dpp alleles are fully consistent with the ectopic wing vein phenotype resulting from loss of a TGF-β signalling antagonist, such as SnoN (Ramel, 2006).

The snoNGS-C517T allele also acts in a dominant negative fashion. Indeed, it specifically suppresses the normal function of SnoN (wing vein development inhibition), since strong constitutive overexpression of snoNGS-C517T accurately phenocopies the homozygous mutant phenotype. Moreover, in snoNGS-C517T, tub-GAL4 discs, ectopic P-Mad expression was observed specifically in the region around L5, where the adult phenotype is observed. This last result might appear to contradict models for vertebrate Ski/Sno function, in which these molecules act downstream of Smad activation. However, the pattern of P-Mad expression outside the longitudinal proveins is thought to evolve in a complex process involving long-range signalling and feedback regulation. Thus, increased Dpp transcriptional output in a snoN mutant could subsequently lead to increased P-Mad expression (Ramel, 2006).

Takaesu (2006) suggests that the lethal mutation in the l(2)SH1402 chromosome is caused by loss of the 297{}323 retrotransposon affecting snoN expression, but this transposon was found to be absent even in wild-type flies. Thus, another as yet unidentified mutation must be responsible for the lethality. Surprisingly, it was found that l(2)SH1402 complements the snoN deficiency chromosome used in the current study. The boundaries of this deficiency were confirmed and the absence of the 5′ end of the snoN gene was specifically shown in the deficiency by PCR. l(2)SH1402 also complements the snoNGS-C517T mutant, because l(2)SH1402/snoNGS-C517T animals are viable and display an ectopic wing vein phenotype at a frequency similar to snoNGS-C517T heterozygous flies. These results suggest that the lethality observed in l(2)SH1402 flies is not entirely due to a disruption of SnoN’s function, despite the fact that constitutive overexpression of snoN is reported to rescue the lethal phenotype. In light of these observations, it is therefore believed that the snoNGS-C517T allele has retained little if any normal function and that the homozygous snoNGS-C517T phenotype, therefore, reflects the fact that this gene is not essential for viability in vivo (Ramel, 2006).

One inconsistency in the data is that ectopic wing vein frequency is reduced in snoNGS-C517T /Df(2L)ED12527 flies relative to homozygous snoNGS-C517T animals, suggesting that the effects of snoNGS-C517T are more severe than a complete loss-of-function allele. Evidence is provided that there may be another mutation in the deficiency region that represses TGF-β signalling, since this chromosome also partially suppresses the dominant negative effects of overexpressed SnoNGS-C517T. However, the possibility cannot be eliminated that part of the snoNGS-C517T homozygous mutant phenotype is caused by a dominant effect on other regulators of the TGF-β signalling cascade and this could explain the fact that overexpressed SnoN does not fully rescue this phenotype (Ramel, 2006).

In this regard, it is already known that, in the process of pupal wing vein formation, at least three antagonists of Dpp signalling activity, Short-gastrulation (Sog), Brinker, and Daughter-against-Decapentaplegic, are able to block wing vein development upon overexpression. The intervein expression pattern of SnoN in pupal wings is similar to the expression of Sog, an extracellular molecule that inhibits Dpp ligand binding, and Brinker, which acts as a transcriptional repressor of Dpp target genes. Analysis of sog mutant clones in the wing suggests that Sog is required to limit longitudinal vein formation to the provein regions. The wings of brinker mutant escapers display ectopic wing vein tissue, as do brinker mutant clones. If the dominant negative SnoNGS-C517T protein can still complex to some of the molecules involved in Dpp signalling without inhibiting them, this might partially block the ability of these alternative antagonists to compensate for loss of normal SnoN function and therefore, produce a phenotype more severe than a snoN null allele (Ramel, 2006).

In conclusion, a model is proposed in which Drosophila SnoN normally plays a highly restricted role in TGF-β-dependent events. During development, the sensitivity of TGF-β signalling to SnoN levels may be important in providing a responsive mechanism to fine-tune and balance fluctuations in signalling. One prediction of this model is that snoN mutant phenotypes would be highly sensitive to the genetic background and to levels of TGF-β signalling, both of which were observed. The use of multiple potent, but partially redundant, inhibitors to control a fundamental signalling cascade is a powerful mechanism for maintaining stable levels of signalling activity. However, for snoN, such a mechanism carries the inherent risk that it can cause severe defects if its expression is altered, as is frequently observed in tumours (Ramel, 2006).

A screen for modifiers of Dpp adult phenotypes led to the identification of the Drosophila homolog of the Sno oncogene. The SnoN locus is large, transcriptionally complex and contains a recent retrotransposon insertion that may be essential for SnoN function. This is an intriguing possibility from the perspective of developmental evolution. SnoN is highly transcribed in the embryonic central nervous system and transcripts are most abundant in third instar larvae. SnoN mutant larvae have proliferation defects in the optic lobe of the brain very similar to those seen in baboon (Activin type I receptor) and Smad2 mutants. This suggests that SnoN is a mediator of Baboon signaling. SnoN binds to Medea and Medea/SnoN complexes have enhanced affinity for Smad2. Alternatively, Medea/SnoN complexes have reduced affinity for Mad such that, in the presence of SnoN, Dpp signaling is antagonized. It is proposed that SnoN functions as a switch in optic lobe development, shunting Medea from the Dpp pathway to the Activin pathway to ensure proper proliferation. Pathway switching in target cells is a previously unreported mechanism for regulating TGFß signaling and a novel function for Sno/Ski family proteins (Takaesu, 2006).

Model for the regulation of size in the wing imaginal disc of Drosophila

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 (tkvQ-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).

Temporal regulation of Dpp signaling output in the Drosophila wing

The Decapentaplegic (Dpp) signaling pathway is used in many developmental and homeostatic contexts, each time resulting in cellular responses particular to that biological niche. The flexibility of Dpp signaling is clearly evident in epithelial cells of the Drosophila wing imaginal disc. During larval stages of development Dpp functions as a morphogen, patterning the wing developmental field and stimulating tissue growth. A short time later however, as wing-epithelial cells exit the cell cycle and begin to differentiate, Dpp is a critical determinant of vein-cell fate. It is likely that the Dpp signaling pathway regulates different sets of target genes at these two developmental time points. To identify mechanisms that temporally control the transcriptional output of Dpp signaling in this system, a gene expression profiling approach was undertaken. This study identified genes affected by Dpp signaling at late larval or early pupal developmental time points, thereby identifying patterning- and differentiation-specific downstream targets, respectively. It is concluded that analysis of target genes and transcription factor binding sites associated with these groups of genes revealed potential mechanisms by which target-gene specificity of the Dpp signaling pathway is temporally regulated. In addition, this approach revealed novel mechanisms by which Dpp affects the cellular differentiation of wing-veins (O'Keefe, 2014).

An inwardly rectifying K+ channel is required for patterning.

Mutations that disrupt function of the human inwardly rectifying potassium channel KIR2.1 are associated with the craniofacial and digital defects of Andersen-Tawil Syndrome, but the contribution of Kir channels to development is undefined. Deletion of mouse Kir2.1 also causes cleft palate and digital defects. These defects are strikingly similar to phenotypes that result from disrupted TGFβ/BMP signaling. Drosophila melanogaster was used to show that a Kir2.1 homolog, Irk2, affects development by disrupting BMP signaling. Phenotypes of irk2 deficient lines, a mutant irk2 allele, irk2 siRNA and expression of a dominant-negative Irk2 subunit (Irk2DN) all demonstrate that Irk2 function is necessary for development of the adult wing. Compromised Irk2 function causes wing-patterning defects similar to those found when signaling through a Drosophila BMP homolog, Decapentaplegic (Dpp), is disrupted. To determine whether Irk2 plays a role in the Dpp pathway, flies were generated in which both Irk2 and Dpp functions are reduced. Irk2DN phenotypes are enhanced by decreased Dpp signaling. In wild-type flies, Dpp signaling can be detected in stripes along the anterior/posterior boundary of the larval imaginal wing disc. Reducing function of Irk2 with siRNA, an irk2 deletion, or expression of Irk2DN reduces the Dpp signal in the wing disc. As Irk channels contribute to Dpp signaling in flies, a similar role for Kir2.1 in BMP signaling may explain the morphological defects of Andersen-Tawil Syndrome and the Kir2.1 knockout mouse (Dahal, 2012).

How could Irk2 affect Dpp signaling? It could be that maintenance of membrane potential is important for the production, distribution or propagation of the Dpp signal. Alternatively, there may be communication between Irk channels and the Dpp signaling cascade upstream of Mad activation. Distribution and propagation. Changes in sodium concentrations inhibit sulfation of heparan sulfate and reduce the sensitivity of cells to FGF. It is interesting to speculate that a change in local K+ concentrations could interfere with HSPGs, receptor localization, stabilization of the receptor complex, phosphorylation events or production or distribution of the Dpp ligand. It is also possible that Mad requires K+ for recruitment to the membrane as actin requires Ca2+ for that purpose. This study did not directly established whether Irk2 is required cell-autonomously, but a model if favored that Irk2 function is required for Dpp signaling events outside the cell rather than for intracellular phosphorylation for two reasons. First, en-GAL4 expresses UAS-irk2WT in the posterior compartment of the wing disc, but rescues irk2 deficient phenotypes in the whole wing. Second, when Irk2DN and P35 are expressed together, apoptosis occurs well outside of the region of MS1096 expression, consistent with morphogen causation (Dahal, 2012).

If inwardly rectifying K+ channels are necessary for Dpp signaling to designate wing patterning, how could Irk subunits have been missed in Dpp modifier screens? The partial redundancy of the Irk subunits make severe phenotypes unlikely unless all of the subunits are compromised. Phenotypes are not severe at 25°C, the temperature at which development occurs for most screens. Third, the necessity of Irk2 seems to be tissue specific. Dpp is required for patterning of multiple structures, but in screening multiple GAL4 drivers, expression of Irk2DN causes defects in only a few of these (Dahal, 2012).

Many BMP/TGFβ-dependent processes go awry when Kir2.1 is not functional in humans and in mice. The phenotypes of Kir2.1 knockout mice are reminiscent of the morphological defects of ATS: cleft palate, incomplete dentition and digit abnormalities. All of these phenotypes have also been associated with defects in TGFβ superfamily signaling. For example, TGFβ2 and TGFβ3 knockout animals have cleft palate. Loss of BMP4 impedes proper tooth development. Deletion of Pax9, which activates BMP4 transcription, causes cleft palate, incomplete dentition, and extra digits in mice. A conditional knockout of BMP2 and BMP4 in the forepaw causes extra digits like the Kir2.1 knockout. BMP signaling is responsible for inhibiting growth of extra digits and initiating apoptosis to separate digits in mice. Although the developmental defects associated with Kir2.1 knockout are incompletely characterized in the mouse, these BMP-like phenotypes support the hypothesis that disruption of Kir2.1 interferes with TGFβ/BMP signaling in mammals (Dahal, 2012).

The finding that inwardly rectifying K+ channels are necessary for BMP signaling in the Drosophila wing alters the landscape of current research by demonstrating that K+ channels contribute significantly to development. How broadly can these findings be applied to other K+ channels? A gain-of-function GYG to SYG change in the selectivity filter of the G protein-coupled Irk2 (GIRK2) channel allows it to pass Na+ and Ca2+, which alters cerebellar development in the weaver mouse. However, deletion of GIRK2 allows mice to develop normally. Therefore, developmental defects of weaver mice are presumed to be due to changes in Na+ and Ca2+, rather than changes in K+, levels. By contrast, the mutations that are associated with ATS cause loss of Kir2.1 function and the Kir2.1 knockout mouse has severe developmental defects. The data presents a rationale for inquiry into the putative contribution of other K+ channels to developmental signaling (Dahal, 2012). Altering Irk channels interferes with BMP signaling to contribute to morphological abnormalities in Drosophila. It is likely that the developmental defects associated with ATS and the Kir2.1 knockout mouse are similarly due to defective TGFβ/BMP signaling. If Kir2.1 channel function is necessary for TGFβ/BMP signaling in mammals, the Kir channels could represent new potential therapeutic targets for slowing tumor growth and metastasis (Dahal, 2012).

Dpp and the Leg Disc

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 dacP-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 wgts 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 Egfrts 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 Egfrts 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 Egfrts clones do not survive in distal regions, raising the possibility that rn expression may be lost in Egfrts discs simply because of reduced growth or cell survival in this region. However, expression of other genes, including wg and dpp, appears normal in Egfrts 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).

Free extracellular diffusion creates the Dpp morphogen gradient of the Drosophila wing disc

How morphogen gradients form has long been a subject of controversy. The strongest support for the view that morphogens do not simply spread by free diffusion has come from a variety of studies of the Decapentaplegic (Dpp) gradient of the Drosophila larval wing disc. In the present study, it was initially shown how the failure, in such studies, to consider the coupling of transport to receptor-mediated uptake and degradation has led to estimates of transport rates that are orders of magnitude too low, lending unwarranted support to a variety of hypothetical mechanisms, such as 'planar transcytosis' and 'restricted extracellular diffusion.' Using several independent dynamic methods, this study obtained data that are inconsistent with such models and shows directly that Dpp transport occurs by simple, rapid diffusion in the extracellular space. The implications are discussed of these findings for other morphogen systems in which complex transport mechanisms have been proposed. It is believed that these findings resolve a major, longstanding question about morphogen gradient formation and provide a solid framework for interpreting experimental observations of morphogen gradient dynamics (Zhou, 2012)

The Gyc76C receptor Guanylyl cyclase and the Foraging cGMP-dependent kinase regulate extracellular matrix organization and BMP signaling in the developing wing of Drosophila melanogaster

The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, this study has identified Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. This study identified a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases and shows that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. Parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues are discussed (Schleede, 2015).

The vein cells that develop from the ectodermal epithelia of the Drosophila melanogaster wing are positioned, elaborated and maintained by a series of well-characterized intercellular signaling pathways. The wing is easily visualized, and specific mutant vein phenotypes have been linked to changes in specific signals, making the wing an ideal tissue for examining signaling mechanisms, for identifying intracellular and extracellular crosstalk between different pathways, and for isolating new pathway components (Schleede, 2015).

The venation defect, the loss of the posterior crossvein (PCV), is used to identify and characterize participants in Bone Morphogenetic Protein (BMP) signaling. The PCV is formed during the end of the first day of pupal wing development, well after the formation of the longitudinal veins (LVs, numbered L1-L6), and requires localized BMP signaling in the PCV region between L4 and L5. Many of the homozygous viable crossveinless mutants identified in early genetic screens have now been shown to disrupt direct regulators of BMP signaling, especially those that bind BMPs and regulate BMP movement and activity in the extracellular space. The PCV is especially sensitive to loss of these regulators because of the long range over which signaling must take place, and the role many of these BMP regulators play in the assembly or disassembly of a BMP-carrying 'shuttle' (Schleede, 2015).

The BMP Decapentaplegic (Dpp) is secreted by the pupal LVs, possibly as a heterodimer with the BMP Glass bottom boat (Gbb). This stimulates autocrine and short-range BMP signaling in the LVs that is relatively insensitive to extracellular BMP regulators. However, Dpp and Gbb also signal over a long range by moving into the intervein tissues where the PCV forms. In order for this to occur, the secreted BMPs must bind the D. melanogaster Chordin homolog Short gastrulation (Sog) and the Twisted gastrulation family member Crossveinless (Cv, termed here Cv-Tsg2 to avoid confusion with other 'Cv' gene names). The Sog/Cv-Tsg2 complex facilitates the movement of BMPs from the LVs through the extracellular space, likely by protecting BMPs from binding to cell bound molecules such as their receptors. In order to stimulate signaling in the PCV, BMPs must also be freed from the complex. The Tolloid-related protease (Tlr, also known as Tolkin) cleaves Sog, lowering its affinity for BMPs, and Tsg family proteins help stimulate this cleavage. Signaling is further aided in the PCV region by a positive feedback loop, as BMP signaling increases localized expression of the BMP-binding protein Crossveinless 2 (Cv-2, recently renamed BMPER in vertebrates). Cv-2 also binds Sog, cell surface glypicans and the BMP receptor complex, and likely acts as a co-receptor and a transfer protein that frees BMPs from Sog. The lipoprotein Crossveinless-d (Cv-d) also binds BMPs and glypicans and helps signaling by an unknown mechanism (Schleede, 2015).

PCV development takes place in a complex and changing extracellular environment, but while there is some evidence that PCV-specific BMP signaling can be influenced by changes in tissue morphology or loss of the cell-bound glypican heparan sulfate proteoglycans, other aspects of the environment have not been greatly investigated. During the initial stages of BMP signaling in the PCV, at 15-18 hours after pupariation (AP), the dorsal and ventral wing epithelia form a sack that retains only a few dorsal to ventral connections from earlier stages; the inner, basal side of the sack is filled with extracellular matrix (ECM) proteins, both diffusely and in laminar aggregates. As BMP signaling in the PCV is maintained and refined, from 18-30 hours AP, increasing numbers of dorsal and ventral epithelial cells adhere, basal to basal, flattening the sack. The veins form as ECM-filled channels between the two epithelia, while in intervein regions scattered pockets of ECM are retained basolaterally between the cells within each epithelium; a small amount of ECM is also retained at the sites of basal-to-basal contact. This changing ECM environment could potentially alter BMP movement, assembly of BMP-containing complexes, and signal reception, as has been demonstrated in other developmental contexts in Drosophila (Schleede, 2015).

This study demonstrates the strong influence of the pupal ECM on PCV-specific long-range BMP signaling, through the identification of a previously unknown ECM-regulating pathway in the wing. In a screen conducted for novel crossveinless mutations on the third chromosome, a mutation was found in the guanylyl cyclase at 76C (gyc76C) locus, which encodes one of five transmembrane, receptor class guanylyl cyclases in D. melanogaster. Gyc76C has been previously characterized for its role in Semaphorin-mediated axon guidance; Malpighian tubule physiology, and the development of embryonic muscles and salivary glands. Like the similar mammalian natriuretic peptide receptors NPR1 and NPR2, the guanylyl cyclase activity of Gyc76C is likely regulated by secreted peptides, and can act via a variety of downstream cGMP sensors (Schleede, 2015).

The evidence suggests that Gyc76C influences BMP signaling in the pupal wing by changing the activity of the cGMP-dependent kinase Foraging (For; also known as Dg2 or Pkg24A), also a novel role for this kinase. But rather than controlling BMP signal transduction in a cell-autonomous manner, evidence is provided that Gyc76C and Foraging regulate BMP signaling non-autonomously by dramatically altering the wing ECM during the period of BMP signaling in the PCV. This effect is largely mediated by changing the levels and activity of matrix metalloproteinases (Mmps), especially Drosophila Mmp2. Genetic interactions suggest that the ECM alterations affect the extracellular mobility and activity of the BMP-binding protein Sog (Schleede, 2015).

This provides the first demonstration of Gyc76C and For activity in the developing wing, and the first evidence these proteins can act by affecting Mmp activity. Moreover, the demonstration of in vivo link from a guanylyl cyclase to Mmps and the ECM, and from there to long-range BMP signaling, may have parallels with findings in mammalian cells and tissues. NPR and NO-mediated changes in cGMP activity can on the one hand change matrix metalloproteinase expression secretion and activity, and on the other change in BMP and TGFβ signaling (Schleede, 2015).

Mutation 3L043, uncovered by a genetic screen to identify homozygous lethal mutations required for PCV development, is a novel allele of gyc76C, a transmembrane peptide receptor that, like vertebrate NPRs, acts as a guanylyl cyclase. gyc76C is likely linked by cGMP production to the activity of the cGMP-dependent kinase For, and that Gyc76C and For define a new pathway for the regulation of wing ECM. This pathway appears to act largely through changes in the activity of ECM-remodeling Mmp enzymes. Loss of gyc76C or For alter both the organization of the wing ECM and the levels of the two D. melanogaster Mmps, and the gyc76C knockdown phenotype can be largely reversed by knockdown of Mmp2. This is the first indication of a role for cGMP, Gyc76C and For function in the developing wing, and their effects on the ECM provides a novel molecular output for each (Schleede, 2015).

Gyc76C and For are necessary for the normal refinement and maintenance of long-range BMP signaling in the posterior crossvein region of the pupal wing; in fact, crossvein loss is the most prominent aspect of the adult gyc76C knockdown phenotype. The evidence suggests that this effect is also mediated by changes in Mmp activity, and most likely the Mmp-dependent reorganization of the ECM. In fact, analysis using genetic mosaics finds no evidence for a reliable, cell autonomous effect of cGMP activity on BMP signal transduction in the wing. Thus, this apparent crosstalk between receptor guanylyl cyclase activity and BMP signaling in the wing is mediated by extracellular effects (Schleede, 2015).

It is noteworthy that the cGMP activity mediated by NPR or nitric oxide signaling can change also Mmp gene expression, secretion or activation in many different mammalian cells and tissues. Both positive and negative effects have been noted, depending on the cells, the context, and the specific Mmp. Given the strong role of the ECM in cell-cell signaling, the contribution of cGMP-mediated changes in Mmp activity to extracellular signaling may be significant. There is also precedent for cGMP activity specifically affecting BMP and TGFβ signaling in mammals. cGMP-dependent kinase activity increases BMP signaling in C2C12 cells, and this effect has been suggested to underlie some of the effects of nitric oxide-induced cGMP on BMP-dependent pulmonary arterial hypertension. Conversely, atrial natriuretic peptide stimulates the guanylyl cyclase activities of NPR1 and NPR2 and can inhibit TGFβ activity in myofibroblasts; this inhibition has been suggested to underlie the opposing roles of atrial natriuretic peptide and TGFβ during hypoxia-induced remodeling of the pulmonary vasculature. However, unlike the pathway observed in the fly wing, these mammalian effects are thought to be mediated by the intracellular modulation of signal transduction, with cGMP-dependent kinases altering BMP receptor activity or the phosphorylation and nuclear accumulation of receptor-activated Smads. Nonetheless, it remains possible that there are additional layers of regulation mediated through extracellular effects, underscoring the importance of testing cell autonomy (Schleede, 2015).

Aside from its role in adult Malpighian tubule physiology, Gyc76C was previously shown to have three developmental effects: in the embryo it regulates the repulsive axon guidance mediated by Semaphorin 1A and Plexin A, the proper formation and arrangement of somatic muscles, and lumen formation in the salivary gland. All these may have links to the ECM. Loss of gyc76C from embryonic muscles affects the distribution and vesicular accumulation of the βintegrin Mys, and reduces laminins and the integrin regulator Talin in the salivary gland. The axon defects likely involve a physical interaction between Gyc76C and semaphorin receptors that affects cGMP levels; nonetheless, gyc76C mutant axon defects are very similar to those caused by loss of the perlecan Trol (Schleede, 2015).

The parallels between the different contexts of Gyc76C action are not exact, however. First, only the wing phenotype has been linked to a change in Mmp activity. Second, unlike the muscle phenotype, the wing phenotype is not accompanied by any obvious changes in integrin levels or distribution, beyond those caused by altered venation. Finally, most gyc76C mutant phenotypes are reproduced by loss of the Pkg21D (Dg1) cytoplasmic cGMP-dependent kinase, instead of For (Dg2, Pkg24A) as found in the wing, and thus may be mediated by different kinase targets (Schleede, 2015).

For has been largely analyzed for behavioral mutant phenotypes, and the overlap between Pkg21D and For targets is unknown. While many targets have been identified for the two mammalian cGMP-dependent kinases, PRKG1 (which exists in alpha and beta isoforms) and PRKG2, it is not clear if either of these is functionally equivalent to For. One of the protein isoforms generated by the for locus has a putative protein interaction/dimerization motif with slight similarity to the N-terminal binding/dimerization domains of alpha and beta PRKG1, but all three For isoforms have long N-terminal regions that are lacking from PRKG1 and PRKG2. In fact, a recent study suggested that For is instead functionally equivalent to PRKG2: Like PRKG2, For can stimulate phosphorylation of FOXO, and is localized to cell membranes in vitro. But For apparently lacks the canonical myristoylation site that is thought to account for the membrane localization and thus much of the target specificity of PRKG2. FOXO remains the only identified For target, and foxo null mutants are viable with normal wings (Schleede, 2015).

The loss of long range BMP signaling in the PCV region caused by knockdown of gyc76C can, like the ECM, be largely rescued by knockdown of Mmp2. Two results suggest that it is the alteration to the ECM that affects long-range BMP signaling, rather than some independent effect of Mmp2. First, the BMP signaling defects caused by gyc76C knockdown were rescued by directly manipulating the ECM through the overexpression of the perlecan Trol. Second, when Mmp activity is inhibited by overexpression of the diffusible Mmp inhibitor TIMP, this not only rescued the PCV BMP signaling defects caused by gyc76C knockdown, but also led to ectopic BMP signaling, not throughout the region of TIMP expression, but only in those regions with abnormal accumulation of ECM (Schleede, 2015).

The Mmp2-mediated changes in the ECM likely affect long-range BMP signaling by altering the activity of extracellular BMP-binding proteins, particularly Sog. The BMPs Dpp and Gbb produced in the LVs bind Sog and Cv-Tsg2, shuttle into the PCV region, and are released there by Tlr-mediated cleavage of Sog and transfer to Cv-2 and the receptors. Genetic interaction experiments suggest that knockdown of gyc76C both increases Sog's affinity for BMPs and reduces the movement of the Sog/Cv-Tsg2/BMP complex into the crossvein region (Schleede, 2015).

Collagen IV provides the best-studied example for how the ECM might affect Sog activity. The two D. melanogaster collagen IV chains regulate BMP signaling in other contexts, and they bind both Sog and the BMP Dpp. Results suggest that collagen IV helps assemble and release a Dpp/Sog/Tsg shuttling complex, and also recruits the Tld protease that cleaves Sog cleavage and releases Dpp for signaling. D. melanogaster Mmp1 can cleave vertebrate Collagen IV. Since reduced Gyc76C and For activity increases abnormal Collagen IV aggregates throughout the wing and diffuse Collagen IV in the veins, it ishypothesized that these Collagen IV changes both foster the assembly or stability of Sog/Cv-Tsg2/BMP complexes and tether them to the ECM, favoring the sequestration of BMPs in the complex and reducing thelong-range movement of the complex into the region of the PCV (Schleede, 2015).

While few other D. melanogaster Mmp targets have been identified, it is likely that Mmp1 and Mmp2 share the broad specificity of their mammalian counterparts, so other ECM components, known or unknown, might be involved. For instance, vertebrate Perlecan and can be cleaved by Mmps. Trol regulates BMP signaling in other D. melanogaster contexts, and Trol overexpression rescue gyc76C knockdown's effects on BMP signaling. But while null trol alleles are lethal before pupal stages, normal PCVs were formed in viable and even adult lethal alleles like trolG0023, and actin-Gal 4-driven expression of trol-RNAi using any of four different trol-RNAi lines did not alter adult wing venation. Loss of the D. melanogaster laminin B chain shared by all laminin trimers strongly disrupts wing venation, and a zebrafish laminin mutation can reduce BMP signaling (Schleede, 2015).

Finally, it was recently shown that Dlp, one of the two D. melanogaster glypicans, can be removed from the cell surface by Mmp2. While gyc76C knockdown did not detectably alter anti-Dlp staining in the pupal wing, it is noteworthy that Dlp and the second glypican Dally are required non-autonomously for BMP signaling in the PCV and that they bind BMPs and other BMP-binding proteins.

A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system

Brain areas each generate specific neuron subtypes during development. However, underlying regional variations in neurogenesis strategies and regulatory mechanisms remain poorly understood. In Drosophila, neurons in four optic lobe ganglia originate from two neuroepithelia, the outer (OPC) and inner (IPC) proliferation centers. Using genetic manipulations, this study found that one IPC neuroepithelial domain progressively transformed into migratory progenitors that matured into neural stem cells (neuroblasts) in a second domain. Progenitors emerged by an epithelial-mesenchymal transition-like mechanism that required the Snail-family member Escargot and, in subdomains, Decapentaplegic signaling. The proneural factors Lethal of scute and Asense differentially controlled progenitor supply and maturation into neuroblasts. These switched expression from Asense to a third proneural protein, Atonal. Dichaete and Tailless mediated this transition, which was essential for generating two neuron populations at defined positions. It is proposed that this neurogenesis mode is central for setting up a new proliferative zone to facilitate spatio-temporal matching of neurogenesis and connectivity across ganglia. (Apitz, 2014).

Recent studies have distinguished three neurogenesis modes in the Drosophila CNS. First, type I neuroblasts arise from neuroepithelia and generate GMCs, which produce neuronal and glial progeny. Second, Dpn+ type II neuroblasts in the dorsomedial central brain go through a transit-amplifying Dpn+, Ase+ population, called intermediate neural precursors, which generate GMCs and postmitotic offspring. Third, lateral OPC neuroepithelial cells bypass the neuroblast stage and generate lamina precursor cells (LPCs) that divide once to produce lamina neurons. The current results provide evidence for a fourth strategy: p-IPC neuroepithelial cells give rise to progenitors that migrate to a second neurogenic domain, where they mature into type I neuroblasts. These progenitors are distinct, as they originate from the neuroepithelium, do not express markers for neuroblasts, intermediate neural precursors, GMCs or postmitotic neurons, and acquire NSC properties after completing their migration (Apitz, 2014).

Migratory progenitors arise from the p-IPC by a mechanism that shares cellular and molecular characteristics with EMT. On the basis of data on gastrulation and neural crest formation, EMT is commonly associated with cells adopting a mesenchymal state, enabling them to leave their epithelial tissue and migrate through the extracellular matrix to new locations. A recent study also reported an EMT-like process in the mammalian neocortex, whereby newborn neurons and intermediate progenitors delaminate from the ventricular neuroepithelium and radially migrate to the pial surface. This study observed that neuroepithelial cells at the p-IPC margins and migratory progenitors upregulated the Snail homolog Esg, whereas E-cad levels were decreased. Moreover, esg knockdown caused the formation of ectopic E-cad-expressing clusters adjacent to the p-IPC. Although this is a previously uncharacterized role of Drosophila esg, these findings are consistent with the requirement of two Snail transcription factors, Scratch1 and 2, and downregulation of E-cad in cortical EMT migration (Apitz, 2014).

Although TGFβ signaling is well known to induce EMT, it was unclear whether it could have such a role in the brain. Two lines of evidence are consistent with a requirement of the Drosophila family member Dpp. First, it is expressed and downstream signaling is activated in dorsal and ventral p-IPC subdomains and emerging cell streams. Second, tkv mutant cells form small neuroepithelial clusters in p-IPC vicinity. Similar to the neural crest, where distinct molecular cascades control delamination in the head and trunk, region-specific regulators may also be required in p-IPC subdomains. Because neuroblasts derived from Dpp-dependent cell streams map to defined areas in the d-IPC, this pathway could potentially couple EMT and neuron subtype specification (Apitz, 2014).

Cell migration is an essential feature of vertebrate brain development. Commonly, postmitotic immature neurons migrate from their proliferation zones to distant regions, where they further differentiate and integrate into local circuits. Examples include the radial migration of projection neurons and tangential migration of interneurons in the embryonic cortex, as well as migration of interneuron precursors in the rostral migratory stream to the olfactory bulb in adults. In contrast, IPC progenitors develop into NSCs (neuroblasts) after they migrated. A recent study found that NSCs relocating from the embryonic ventral hippocampus to the dentate gyrus act as source for adult NSCs in the subgranular zone. In addition, cerebellar granule cell precursors migrate from the rhombic lip to the external granule layer, where they proliferate during early postnatal development. The migration of neural cell types that become proliferative in a new niche could therefore constitute a more general strategy. IPC progenitors form streams of elongated, closely associated cells. Despite their different developmental state, their organization is notably similar to the neuronal chain network in the lateral walls of the subventricular zone and the rostral migratory stream in mammals, or of migratory trunk neural crest cells in chick. Further studies will need to identify the determinants directing migratory progenitors into the d-IPC (Apitz, 2014).

Several constraints could shape a neurogenesis mode that requires migratory progenitors in the larval optic lobe. The OPC is located superficially and the IPC is positioned centrally. If medulla and lobula neurons arose by neuroepithelial duplications, these new populations would need to be integrated into an ancestral visual circuit consisting of lamina and lobula plate neurons. Cellular migration may therefore be a derived feature and serve as an essential spatial adjustment of the IPC to the newly added medulla. In principle, the migratory population could consist of immature neurons. However, migratory progenitors help to establish a new superficial proliferative niche, and to align OPC and d-IPC neuroblast positions. This in turn enables the OPC and IPC to use spatially matching birth order-driven neurogenesis patterns for establishing functionally coherent connections across ganglia (Apitz, 2014).

IPC progenitors were primed to mature into neuroblasts, but were prevented to do so in cell streams. Consistently, progenitors showed weak cytoplasmic Mira expression and prematurely differentiated into neuroblasts following loss of Pcl. Although Dichaete has been shown to repress ase to maintain embryonic neuroectodermal cells in an undifferentiated state, this study did not identify such a role in the IPC. Future studies are therefore required to distinguish whether this block in neuroblast maturation is released in the d-IPC by cell-intrinsic mechanisms or locally acting signals (Apitz, 2014).

The p-IPC and d-IPC consecutively expressed three proneural factors. esg-positive p-IPC neuroepithelial cells transiently expressed L'sc as they converted into progenitors. Following arrival in the d-IPC, progenitors matured into neuroblasts, which switched bHLH protein expression from Ase to Ato. This correlated with a change in cell division orientations from toward the lamina to the optic lobe surface and the generation of two lineages, distal cells and lobula plate neurons. The progression of neuroblasts through two stages is supported by the observations that progenitors solely entered the lower d-IPC, all neuroblasts were labeled with Ase in this area, and idpp reporter gene expression in a progenitor subset persisted in both lower and upper d-IPC neuroblasts and their progeny (Apitz, 2014).

Late l'sc knockdown reduced the number of d-IPC neuroblasts and both neuron classes, whereas p-IPC formation and EMT of progenitors appeared to be unaffected. This supports the idea that l'sc promotes neuroblast formation by controlling the rate of conversion and the progenitor supply. In contrast, ase loss severely decreased the amount of lower d-IPC neuroblasts and distal cells. This revealed a central role in the maturation of progenitors into neuroblasts, endowing them with the potential to proliferate and generate a specific lineage. Although these functions are the opposite of those observed in the OPC, they align with the role of a murine Ase homolog, Achaete-scute homolog 1 (Ascl1), in the embryonic telencephalon. Ase- neuroblasts with type I proliferation patterns have not previously been described. Further underscoring the context-dependent activities of proneural bHLH factors, ato does not have the equivalent role of ase in conferring neurogenic properties to upper d-IPC neuroblasts, but acts upstream of differentiation programs controlling the projections of lobula plate neurons (Apitz, 2014).

Although Ase and Ato each regulated distinct aspects of d-IPC development, they were not required for either the transition or the extent of their expression domains. These functions were fulfilled by Dichaete and tll, whose cross-regulatory interactions were essential for the transition from Ase+ to Ato+, Dac+ expression. To link birth order and fate, temporal identity transcription factors are sequentially expressed by neuroblasts and inherited by GMCs and their progeny born during a given developmental window. Acting as the final two members of the OPC-specific series of temporal identity factors, Dichaete is required for Tll expression, whereas tll is sufficient, but not required, to inhibit Dichaete Although OPC and d-IPC neuroblasts shared the sequential expression of Dichaete and Tll, key differences include the fact that d-IPC progeny did not maintain Dichaete, that Tll was transiently expressed in newborn progeny of the upper d-IPC and was not maintained in older lineages, that Dichaete in the lower d-IPC was not required in its own expression domain for neurogenesis, and that Dichaete was required to activate tll, and tll to repress Dichaete and ase, as well as to independently upregulate Ato and Dac. Although the mechanisms that trigger the timing of the switch require further analysis, these observations support the notion that, in the d-IPC, Dichaete and tll do not function as temporal identity factors, but as switching factors between two sequential neuroblast stages. The vertebrate homologs of Dichaete and tll, Sox2 and Tlx, are essential for adult NSC maintenance and Sox2 positively regulates Tlx expression, suiggesting that core regulatory interactions between Dichaete and tll family members may be conserved (Apitz, 2014).

These studies uncovered molecular signatures for generating a migratory neural population by EMT and subsequent NSC development that are in part shared between the fly optic lobe and vertebrate cortical neurogenesis. The unexpected parallels suggest that ancestral gene regulatory cassettes imparting specific cellular properties may have been re-employed during vertebrate brain development. Analysis of p-IPC and d-IPC neurogenesis in the Drosophila optic lobe therefore opens new possibilities for systematically identifying genes regulating EMT, cell migration and sequential NSC specification (Apitz, 2014).

Haemocytes control stem cell activity in the Drosophila intestine

Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes were recruited to the intestine and secreted the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switched their response to DPP by inducing expression of Thickveins, a second type I receptor that had previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promoted infection resistance, but also contributed to the development of intestinal dysplasia in ageing flies. The study proposes that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans (Ayyaz, 2015).

Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes are recruited to the intestine and secrete the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switch their response to DPP by inducing expression of Thickveins, a second type I receptor that has previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in ageing flies. It is proposed that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans (Ayyaz, 2015).

The results extend the current model for the control of epithelial regeneration in the wake of acute infections in the Drosophila intestine. It is proposed that the control of ISC proliferation by haemocyte-derived DPP integrates with the previously described regulation of ISC proliferation by local signals from the epithelium and the visceral muscle, allowing precise temporal control of ISC proliferation in response to tissue damage, inflammation and infection (Ayyaz, 2015).

The association of haemocytes with the intestine is extensive, and can be dynamically increased on infection or damage. In this respect, the current observations parallel the invasion of subepithelial layers of the vertebrate intestine by blood cells that induce proliferative responses of crypt stem cells during infection. A role for macrophages and myeloid cells in promoting tissue repair and regeneration has been described in adult salamanders and in mammals, where TGFβ ligands secreted by these immune cells can inhibit ISC proliferation, but can also contribute to tumour progression. The results provide a conceptual framework for immune cell/stem cell interactions in these contexts (Ayyaz, 2015).

The observation that DPP/SAX/SMOX signalling is required for UPD-induced proliferation of ISCs suggests that SAX/SMOX signalling cooperates with JAK/STAT and EGFR signalling in the induction of ISC proliferation. Accordingly, while constitutive activation of EGFR/RAS or JAK/STAT signalling in ISCs is sufficient to promote ISC proliferation cell autonomously, this study found that this partially depends on Smox. Even in these gain-of-function conditions, ISC proliferation can thus be fully induced only in the presence of basal SMOX activity. As short-term overexpression of DPP in haemocytes does not induce ISC proliferation, it is further proposed that DPP/SAX/SMOX signalling can activate ISCs only when JAK/STAT and/or EGFR signalling are activated in parallel. However, long-term overexpression of DPP in haemocytes results in increased ISC proliferation, suggesting that chronic activation of immune cells disrupts normal signalling mechanisms and results in ISC activation even in the absence of tissue damage (Ayyaz, 2015).

BMP TGFβ signalling pathways are critical for metazoan growth and development and have been well characterized in flies. Multiple ligands, receptors and transcription factors with highly context-dependent interactions and function have been described. This complexity is reflected by the sometimes conflicting studies exploring DPP/TKV/SAX signalling in the adult intestine. These studies consistently highlight two important aspects of BMP signalling in the adult Drosophila gut: ISCs can undergo opposite proliferative responses to BMP signals; and there are various sources of DPP that differentially influence ISC function in specific conditions. By characterizing the temporal regulation of BMP signalling activity in ISCs, the results resolve some of these conflicts: it is proposed that early in the regenerative response, haemocyte-derived DPP triggers ISC proliferation by activating SAX/SMOX signalling, and ISC quiescence is re-established by muscle-derived DPP as soon as TKV becomes expressed. Of note, some of the conflicting conclusions described in the literature may have originated from problems with the genetic tools used in some studies. This study have used two independent RNAi lines (BL25782 and BL33618) that effectively decrease dpp mRNA levels in haemocytes when expressed using HmlΔ::Gal4 (Ayyaz, 2015).

The close association of haemocytes with the type IV collagen Viking suggests that the stimulation of ISC proliferation by haemocyte-derived DPP may also be controlled at the level of ligand availability, as suggested previously for DPP from other sources. The regulation of SAX/SMOX signalling by DPP observed in this study is surprising, but consistent with earlier reports showing that SAX can respond to DPP in certain contexts. Biochemical studies have suggested that heterotetrameric complexes between the type II receptor PUNT and the type I receptors SAX and TKV can bind DPP, and complexes with TKV/TKV homodimers preferentially bind DPP, and complexes with SAX/SAX homodimers preferentially bind GBB. In the absence of TKV, SAX has been proposed to sequester GBB, shaping the GBB activity gradient, but to fail to signal effectively. Expression of GBB in the midgut epithelium has recently been described, and ligand heterodimers between GBB and DPP are well established. Consistent with earlier reports, this study found that GBB knockdown in ECs significantly reduces ISC proliferation in response to infection. Complex interactions between haemocyte-derived DPP, epithelial GBB, and ISC-expressed SAX, PUNT and TKV thus probably shape the response of ISCs to damage, and will be an interesting area of further study (Ayyaz, 2015).

Similar complexities exist in the regulation of transcription factors by SAX and TKV. Canonically, SMOX is regulated by Activin ligands (Activin, Dawdle, Myoglianin and maybe more), and the type I receptor Baboon. This study has tested the role of Activin and Dawdle in ISC regulation, and, in contrast to DPP, this study could not detect a requirement for these factors in the induction of ISC proliferation after Ecc15 infection. Furthermore, the data establish a requirement for haemocyte-derived DPP as well as for SAX expression in ISCs in the nuclear translocation of SMOX after a challenge. This study thus indicates that in this context, SAX responds to DPP and regulates SMOX. Regulation of SMOX by SAX has been described before, yet SAX is also known to promote MAD phosphorylation, but only in the presence of TKV. Consistent with such observations, this study has detected MAD phosphorylation in ISCs only in the late recovery phase on bacterial infection, when TKV is simultaneously induced in ISCs. During this recovery phase, ISCs maintain high SAX expression, but SMOX nuclear localization is not detected anymore, suggesting that SAX cannot activate SMOX in the presence of TKV, and might actually divert signals towards MAD instead. The data also suggest that Medea (the Drosophila SMAD4 homologue) is not required for SMOX activity. Although surprising, this observation is consistent with recent reports that SMAD proteins in mammals can translocate into the nucleus and activate target genes in a SMAD4-independent manner. The specific signalling readouts in ISCs when these cells are exposed to various BMP ligands and are expressing different combinations of receptors are thus likely to be complex (Ayyaz, 2015).

The current findings demonstrate that the control of ISC proliferation by haemocyte-derived DPP is critical for tolerance against enteropathogens, but contributes to ageing-associated epithelial dysfunction, highlighting the importance of tightly controlled interactions between blood cells and stem cells in this tissue. Nevertheless, where haemocytes themselves are required for normal lifespan, loss of haemocyte-derived DPP does not impact lifespan. One interpretation of this finding is that beneficial (improved gut homeostasis) and deleterious (for example, reduced immune competence of the gut epithelium) consequences of reduced haemocyte-derived DPP cancel each other out over the lifespan of the animal. It will be interesting to test this hypothesis in future studies. Ageing is associated with systemic inflammation, and a role for immune cells in promoting inflammation in ageing vertebrates has been proposed. In humans, recruitment of immune cells to the gut is required for proper stem cell proliferation in response to luminal microbes, and prolonged inflammatory bowel disease further contributes to cancer development. It is thus anticipated that conserved macrophage/stem cell interactions influence the aetiology and progression of such diseases. The data confirm a role for haemocytes in age-related intestinal dysplasia in the fly intestine, and provide mechanistic insight into the causes for this deregulation. It can be anticipated that similar interactions between macrophages and intestinal stem cells may contribute to the development of IBDs, intestinal cancers, and general loss of homeostasis in the ageing human intestine (Ayyaz, 2015).

Dpp and the Genital Disc

decapentaplegic Developmental Biology part 3/3 | back to part 1/3

decapentaplegic: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Targets of activity | Protein Interactions | Post-transcriptional Regulation | Effect of mutation | References

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