Larval (part 2/2)

Expression in wing and leg discs

Although Wingless, Hh and Dpp have been shown to act directly on distant cells in the developing limbs of the Drosophila, little is known about how ligand gradients form in vivo. Wg protein is found in vesicles in Wg-responsive cells in the embryo and imaginal discs. It has been proposed that Wg may be transported by a vesicle-mediated mechanism. A novel method to visualize extracellular Wg protein was used to show that Wg forms an unstable gradient on the basolateral surface of the wing imaginal disc epithelium. Wg movement does not require internalization by dynamin-mediated endocytosis. Dynamin activity is, however, required for Wg secretion. By reversibly blocking Wg secretion, it was found that Wg moves rapidly to form a long-range extracellular gradient. It is concluded that the Wg morphogen gradient forms by rapid movement of ligand through the extracellular space, and depends on continuous secretion and rapid turnover. Endocytosis is not required for Wg movement, but contributes to shaping the gradient by removing extracellular Wg. It is proposed that the extracellular Wg gradient forms by diffusion (Strigini, 2000).

Using conventional antibody labeling methods, Wg protein has been detected in Wg-expressing cells at the dorsoventral (DV) boundary of the wing disc and in an irregular pattern of spots in nearby cells. The intensity and number of spots decreases with distance from the source of Wg, providing indirect evidence that Wg protein forms a gradient across the disc. The conventional antibody labeling protocol involves incubating anti-Wg antibody with fixed and permeabilized wing discs. When discs were incubated with anti-Wg antibody before fixation, a gradient of Wg protein was observed that appeared broader, shallower and less punctate than that observed with the conventional protocol. Control experiments showed that tubulin, an abundant intracellular protein, is readily visualized using the conventional protocol, but is not detected using the extracellular staining protocol. Thus, Wg visualized in this way reflects the distribution of the secreted extracellular protein (Strigini, 2000).

The distribution of extracellular and conventionally labeled Wg protein was compared by sequentially labeling discs using anti-Wg antibodies produced in different species. To determine what proportion of Wg protein is extracellular, intact wing discs were treated with proteinase K. Digestion of Wg was compared with digestion of Fasciclin II, a glycosyl phosphatidyl inositol (GPI)-linked membrane protein, and with cytoplasmic tropomyosin in immunoblots of total disc extracts. In the absence of detergent, Fasciclin II was completely digested, and cytoplasmic tropomyosin was not digested. Wg levels were reduced considerably. Wg and cytoplasmic tropomyosin were completely digested when disc cells were permeabilized with detergent during the protease treatment. These observations indicate that much of the Wg protein in the discs is extracellular and accessible to protease digestion or to antibody binding. The remaining Wg protein is presumably intracellular (Strigini, 2000).

Imaginal discs consist of a single-layered sac of polarized epithelial cells, with the apical surface of the cells oriented towards the lumen of the disc. The polarity of the epithelial cells can be visualized using antibody to Coracle, which labels the junctional complex that separates apical from basolateral surfaces. Using the conventional labeling protocol, most of the Wg appears to be concentrated above the nuclei, near the junctional complex, in Wg-expressing cells. In contrast, extracellular Wg is mainly associated with the basolateral surface of cells. The spots of Wg visualized by conventional labeling in cells away from the source appear to reflect vesicles of internalized Wg protein (Strigini, 2000).

The observation that extracellular Wg appears to be concentrated on the basolateral surface of Wg-expressing and nearby cells prompted an investigation to see whether Wg moves across the apical (that is, lumenal) or basolateral surface of the epithelium. To distinguish between these possibilities, use was made of the observation that overexpression of the Drosophila Wg receptor Frizzled 2 (Dfz2) causes accumulation of Wg on cells at a distance from the DV boundary. Wg accumulation was compared in cells expressing full-length Dfz2 and cells expressing the Wg-binding domain of Dfz2 as a GPI-anchored protein (GPI-Dfz2). Full-length Dfz2 is expressed uniformly on the apical and basolateral surfaces of the epithelium, whereas GPI-anchored proteins localize to the basolateral surface of the imaginal disc cells and so can only accumulate Wg on the basolateral surface. In both cases, Wg accumulates to high levels on the basolateral surface of the epithelial cells but is absent from the region above the nuclei, where most Wg protein was found in Wg-expressing cells. These observations suggest that the extracellular Wg gradient forms on the basolateral surface of the wing disc (Strigini, 2000).

The extracellular Wg gradient correlates with a graded distribution of intracellular Wg vesicles. Does Wg internalization play a role in gradient formation? Previous studies have suggested a role for Shibire-dependent endocytosis in Wg transport in the embryo; shibire encodes the Drosophila homolog of the GTPase dynamin. Dynamins have been implicated in the internalization of clathrin-coated endocytic vesicles and in the internalization of caveolae. To determine whether shibire-dependent vesicle traffic is required for Wg gradient formation, Wg distribution was examined in wing discs carrying clones of shibire temperature sensitive mutant cells. Clones were allowed to grow under conditions in which the temperature-sensitive Shibire protein is active (18°C). The larvae were then shifted to 32oC for 3 hours to inactivate Shibire. Under these conditions, punctate Wg wis not observed in the mutant cells using the conventional labeling protocol, indicating that dynamin activity is required for Wg internalization. Although spots of internalized Wg were not seen in shibire mutant cells, Wg is internalized by wild-type cells adjacent to the clone. The presence of Wg in these cells may reflect movement of Wg across the mutant tissue to reach wild-type cells. In support of this view, extracellular Wg was observed on the surface of shibire mutant cells. The level of extracellular Wg was higher than on nearby wild-type cells. The interpretation that Wg can move across the shibire mutant tissue depends on the assumption that extracellular Wg is not simply stabilized in the shibire mutant tissue. To determine whether removing dynamin function stabilizes extracellular Wg, Wg distribution was examined in shibire mutant discs. At 18°C, extracellular Wg distribution in shibire mutant discs is comparable to that in the wild type. When Shibire is inactivated at 32°C for 3 hours, little or no extracellular Wg is detected. This indicates that extracellular Wg turns over rapidly when the entire disc is shibire mutant. It is concluded that the Wg on the shibire mutant clones reflects Wg secreted by nearby wild-type tissue that has moved across the clone and that the local accumulation reflects impaired endocytosis. Thus, dynamin-mediated internalization does not appear to play a role in Wg transport in the wing disc. In contrast, dynamin-mediated endocytosis appears to play a role in removing secreted Wg from the extracellular space and, therefore, may help to maintain a steep gradient (Strigini, 2000).

The loss of extracellular Wg from homozygous mutant shibire discs suggested that Wg secretion might be impaired by removing dynamin activity. Conventional staining showed an intense band of intracellular Wg accumulation at the DV boundary under these conditions, which resembles the accumulation of Wg in clones of porcupine mutant cells. The porcupine gene encodes a protein that resides in the endoplasmic reticulum and is required for post-translational processing and secretion of Wg in the embryo. Comparable Wg accumulation is observed in shibire mutant clones that include Wg-expressing cells. Wg accumulates only in Wg-expressing mutant cells, even when the clone is small and most of the surrounding cells are wild-type. No intracellular Wg accumulation is seen in shibire mutant clones that abut but do not include Wg-expressing cells (Strigini, 2000).

As shibire encodes the only Drosophila Dynamin identified to date, these results suggest that Shibire protein may have a role in the formation of transport vesicles at the trans-Golgi network, comparable to that reported for dynamin-2, one of Shibire's vertebrate homologs. If so, the traffic of vesicles from the trans-Golgi to the plasma membrane might be blocked in shibire mutant cells. Alternatively, blocking Shibire-dependent endocytosis might impair membrane traffic, and indirectly reduce Wg secretion (Strigini, 2000).

The temperature-sensitive defect in dynamin function caused by the shibire temperature sensative mutation is rapidly reversed by shifting flies back to the permissive temperature. This provides a means to reversibly block Wg secretion. The extracellular Wg gradient was depleted within 3 hours in shibirets mutant discs at 32°C, indicating that extracellular Wg is rapidly lost or degraded even in the absence of endocytosis. Furthermore, internalized Wg is almost entirely cleared from the disc by 3 hours at 32°C. Shifting shibire mutant larvae back to the permissive temperature provides the opportunity to monitor the time course of gradient formation when Wg secretion is reinitiated. With 15 minutes of recovery at 18°C, spots of internalized Wg protein can again be detected across the disc. By 30-60 minutes, the Wg distribution resembles that in control discs kept at 18°C, indicating that Wg protein has traveled across the disc and been internalized by distant cells. These observations indicate that Wg moves rapidly through the tissue to form a gradient covering at least 50 µm in approximately 30 minutes. For comparison, in Xenopus explants, the signaling protein Activin can form a gradient over more than 250 µm by diffusion in a few hours. Thus, the rate of Wg movement is compatible with diffusion through the extracellular space (Strigini, 2000).

Wg binds avidly to glycosaminoglycans. Mutations that affect glycosaminoglycan biosynthesis phenocopy weak wg mutations and genetically interact with wg mutant alleles. Recent reports have implicated the GPI-anchored proteoglycan Dally as a cofactor in Wg signaling. Dally might facilitate Wg signaling by improving retention and movement of Wg along the basolateral surface of the epithelium. No significant alteration could be detected in the distribution of extracellular Wg in dally mutant discs or in dally mutant clones. Overexpression of Dally using the patched promoter to drive expression of the GAL4-encoded transcriptional activator (ptc-GAL4) causes little or no additional accumulation of Wg in cells near the DV boundary. This contrast with the effects of overexpressing Dfz2 using dpp-GAL4 (a weaker GAL4 driver). These observations suggest that Dally may have a relatively low capacity to bind Wg in vivo or that it is present in excess, and that Dfz2 is the limiting factor in Wg binding. These observations strengthen the proposal that Dally might serve as a coreceptor with Dfz2, and suggest that Dally does not play a significant role in shaping the Wg gradient. Clones of cells mutant for sugarless were also examined and no effect on Wg distribution was seen. These observations leave open the question of whether other proteoglycans might contribute to Wg gradient formation (Strigini, 2000).

To identify genes involved in the patterning of adult structures, Gal4-UAS (upstream activating site) technology was used to visualize patterns of gene expression directly in living flies. The gene yellow (y) was made sensitive to Gal4 control and a Gal4-containing P element was inserted randomly into the fly genome. A large number of Gal4 insertion lines were generated and their expression patterns studied. In addition to identifying several characterized developmental genes, the approach revealed previously unsuspected genetic subdivisions of the thorax, which may control the disposition of pattern elements. For example, the pannier expression domain marks a dorsal band along the length of the body, from the occipital head region to the end of the abdomen but excluding the terminalia. In the notum (derived from the wing imaginal disc), the gene labels a territory extending from the dorsal midline laterally to a longitudinal (anterior to posterior) straight line defined by the dorsocentral bristles. Another insertion em462 shows y+ rescue in a territory adjacent to the pannier domain; it is also demarcated medially by the position of the dorsocentral bristles. The em462 domain extends laterally but does not reach the more lateral region of the notum. Possibly, the gene iroquois defines a distinct, more lateral domain. The subdivision of the notum demarcated by the dorsocentral bristles may be significant because this same line also appears to demarcate the most medial expression border of the wingless gene. In the notum, wingless is expressed in a narrow stripe and the wingless domain is included within the em462 domain, extending laterally from the dorsocentral bristles. Apparently there is no overlap between pnr and wg in the scutum (the anterior part of the notum), but they do overlap in the more posterior scutellum. Genetic interaction experiments show that pannier acts as a negative regulator of wingless in the notum and suggest that some of the effects of pannier mutants are produced through an alteration of wingless function. Interestingly, the dorsocentral line is known not to function as a cell lineage border. In addition to bristles, another frequent pattern element in insects is pigmentation, often disposed in longitudial bands and used as a diagnostic criterion for the taxonomy of dipteran species. The medial boundary of a diagnostic pigment band is exactly delimited by the same longitudinal line straddling the dorsocentral bristles that in the Drosophila demarcates pannier, wingless and em462 (Calleja, 1996).

In larvae wingless is expressed in imaginal discs. Unlike the profile found in embryos, wingless does not mark the posterior compartment of parasegments. Instead, it marks the edge of the presumptive wing and the anterior ventral aspect of the leg disc (Wilder, 1995).

wingless expression in the wing is a dynamic process, marked by early and late patterns. The spatial coordinates of wg in the second instar is radically different from the later pattern: wingless is localized ventrally throughout the second instar. Expression is first detected in a cap comprising less than 10 cells in early-mid second instar discs. This is approximately the time vestigial expression is elevated and apterous expression in the dorsal domain is activated. The late wg pattern includes a secondary expression at the Dorsoventral boundary (Williams, 1993).

Wingless has multiple functions in different regions of the wing. A regulatory mutation of wingless similar to the nubbin mutant results in the loss of wg expression in the wing hinge, leaving intact expression at the margin. Wingless is required for local cell proliferation in the hinge, and its absence severly interfers with wing development. Ectopic activation of the wingless pathway in hinge cells leads to overproliferation of cells, indicating that Wingless acts as a mitogen in this part of the wing disc. At the wing margin, the function of Wingless is directed more to patterning and here Wingless has only a secondary affect on proliferation (Neumann, 1996a).

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 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 developmental mechanisms that regulate the relative size and shape of organs have remained obscure despite almost a century of interest in the problem and the fact that changes in relative size represent the dominant mode of evolutionary change. This study investigates how the Hox gene Ultrabithorax instructs the legs on the third thoracic segment of Drosophila to develop with a different size and shape from the legs on the second thoracic segment. Through loss-of-function and gain-of-function experiments, it has been demonstrated that different segments of the leg, the femur and the first tarsal segment, and even different regions of the femur, regulate their size in response to Ubx expression through qualitatively different mechanisms. In some regions, Ubx acts autonomously to specify shape and size, whereas in other regions, Ubx influences size through nonautonomous mechanisms. Loss of Ubx autonomously reduces cell size in the T3 femur, but this reduction seems to be partially compensated by an increase in cell numbers, so that it is unclear what effect cell size and number directly have on femur size. Loss of Ubx has both autonomous and nonautonomous effects on cell number in different regions of the basitarsus, but again there is not a strong correlation between cell size or number and organ size. Total organ size appears to be regulated through mechanisms that operate at the level of the entire leg segment (femur or basitarsus) relatively independently of the behavior of individual subpopulations of cells within the segment (Stern, 2003).

Ubx appears to regulate the final size and shape of the third pair of legs via different mechanisms in different regions of the leg. The most obvious difference is between regulation of femur and basitarsal length, which appears mainly to involve nonautonomous regulation between all the cells of the segment, and the growth of the most proximal femur, which appears to involve autonomous influence of Ubx. In addition, the timing of these controls appears to be different. Ubx influences proximal femur shape after pupation, since the proximal femur shape could be mimicked by overexpressing Ubx in the second leg during pupal development. In contrast, Ubx is required between 24 h AEL and pupation to influence leg length, since clones were induced between 24 and 72 h AEL and overexpression of Ubx in the pupal period does not influence the length of the second leg (Stern, 2003).

The loss of Ubx in cells of the ventral basitarsus causes the most surprising effect: the production of ectopic bristles, a dramatic increase in the width of the basitarsus, and a nonautonomous decrease in bristle number in the adjoining bristle row of the posterior compartment. This is the only case in which the resulting phenotypes cannot be construed as a homeotic transformation from a T3 to a T2 leg. The location of these clones, in the most ventral cells of the anterior compartment, suggests that Ubx may influence wingless (wg) signaling during the development of the T3 basitarsus, because wg is expressed in this domain and is required for driving leg growth and patterning. This view is supported, but not proven, by the observation that loss of wg function during larval development causes a similar widening of the basitarsal segments of all three pairs of legs. Tarsal widening is observed in flies carrying a temperature-sensitive allele of wg; flies were shifted to the restrictive temperature between 88 and 110 h after egg laying. This phenomenon can be seen in a first leg basitarsus. However, no differences were detected in expression of wg protein between the T2 and T3 legs and no obvious change in wg expression was detected in Ubx null clones in developing T3 leg discs (Stern, 2003).

One model consistent with the current observations is that Ubx is required early to upregulate a signaling pathway located in the ventral row of tarsal cells, but that it is required later to repress the same pathway. This two-step model is favored because uniform removal of Ubx from the entire basitarsus from the earliest stages of development (for example, in flies carrying the allelic combination abx1bx3pbx1/Df Ubx) does not cause basitarsal widening or the production of ectopic bristles, but instead transforms the T3 basitarsus to a T2 basitarsus (Stern, 2003).

An alternative interpretation is that Ubx is required to upregulate wg throughout T3 tarsal development. Late removal of Ubx may then cause a drop in wg leading to tarsal widening and the development of ectopic bristles. wg function in the basitarsus is known to be concentration dependent, with more ventral bristle fates requiring higher levels of wg activity. It is therefore worth noting that the ectopic bristles have a thick shape similar to row 8 bristles. This ectopic bristle row may therefore be interpreted as a lateral transformation caused by a slight reduction in wg level in the most ventral cells (Stern, 2003).

Whatever the true model of this function of wg may be, the nonautonomous effects observed are similar to those reported for Ubx control of wg expression in the haltere and doublesex control of wg and decapentaplegic (dpp) functions in the genital imaginal disc. However, whereas control of wg and dpp in the haltere and genital discs causes a dramatic alteration in growth patterns, the effect on the basitarsus is of much smaller magnitude. Thus, Ubx may have only a small effect on this signaling process in the basitarsus. This suggests the intriguing possibility that the major determinants of organ growth, wg and dpp, are regulated by a panoply of patterning genes that subtly control the function of these signaling molecules leading to slight alterations in organ shape in Drosophila. wg has been shown to be required to establish distal elements of the leg before 84 h AEL, but that after this time, wg is apparently required only for patterning ventral elements and also to determine the correct shape of the leg segments. It is therefore possible that other genes, such as Ubx, influence wg action in different ways if they act at different times during leg development. The wg and dpp signaling pathways might therefore commonly play a dual role of controlling proliferation and patterning of the major proximal-distal elements early during development and then contribute to more subtle effects on organ size and shape later during development (Stern, 2003).

Gunage, R. D., Reichert, H. and VijayRaghavan, K. (2014). Identification of a new stem cell population which generates Drosophila flight muscles. Elife: e03126. PubMed ID: 25135939

Identification of a new stem cell population which generates Drosophila flight muscles

How myoblast populations are regulated for the formation of muscles of different sizes is an essentially unanswered question. The large flight muscles of Drosophila develop from adult muscle progenitor (AMP) cells set-aside embryonically. The thoracic segments are all allotted the same small AMP number, while those associated with the wing-disc proliferate extensively to give rise to over 2500 myoblasts. An initial amplification occurs through symmetric divisions and is followed by a switch to asymmetric divisions in which the AMPs self-renew and generate post-mitotic myoblasts. Notch signaling controls the initial amplification of AMPs, while the switch to asymmetric division additionally requires Wingless, which regulates Numb expression in the AMP lineage. In both cases, the epidermal tissue of the wing imaginal disc acts as a niche expressing the ligands Serrate and Wingless. The disc-associated AMPs are a novel muscle stem cell population that orchestrates the early phases of adult flight muscle development (Gunage, 2014. PubMed).

Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes: Transdetermination is induced by Wg ovexpression

Drosophila imaginal disc cells can switch fates by transdetermining from one determined state to another. The expression profiles of cells induced by ectopic Wingless expression to transdetermine from leg to wing were examined by dissecting transdetermined cells and hybridizing probes generated by linear RNA amplification to DNA microarrays. Changes in expression levels implicated a number of genes: lamina ancestor, CG12534 (a gene orthologous to mouse augmenter of liver regeneration), Notch pathway members, and the Polycomb and trithorax groups of chromatin regulators. Functional tests revealed that transdetermination was significantly affected in mutants for lama and seven different PcG and trxG genes. These results validate the described methods for expression profiling as a way to analyze developmental programs, and they show that modifications to chromatin structure are key to changes in cell fate. These findings are likely to be relevant to the mechanisms that lead to disease when homologs of Wingless are expressed at abnormal levels and to the manifestation of pluripotency of stem cells (Klebes, 2005).

When prothoracic (1st) leg discs are fragmented and cultivated in vivo, cells in a proximodorsal region known as the 'weak point' can switch fate and transdetermine. These 'weak point' cells give rise to cuticular wing structures. The leg-to-wing switch is regulated, in part, by the expression of the vestigial (vg) gene, which encodes a transcriptional activator that is a key regulator of wing development. vg is not expressed during normal leg development, but it is expressed during normal wing development and in 'weak point' cells that transdetermine from leg to wing. Activation of vg gene expression marks leg-to-wing transdetermination (Klebes, 2005).

Sustained proliferation appears to be a prerequisite for fate change, and conditions that stimulate growth increase the frequency and enlarge the area of transdetermined tissue. Transdetermination was discovered when fragments of discs were allowed to grow for an extensive period of in vivo culture. More recently, ways to express Wg ectopically have been used to stimulate cell division and cell cycle changes in 'weak point' cells (Sustar, 2005), and have been shown to induce transdetermination very efficiently. Experiments were performed to characterize the genes involved in or responsible for transdetermination that is induced by ectopic Wg. Focus was placed on leg-to-wing transdetermination because it is well characterized, it can be efficiently induced and it can be monitored by the expression of a real-time GFP reporter. These attributes make it possible to isolate transdetermining cells as a group distinct from dorsal leg cells, which regenerate, and ventral leg cells in the same disc, which do not regenerate; and, in this work, to directly define their expression profiles. This analysis identified unique expression properties for each of these cell populations. It also identified a number of genes whose change in expression levels may be significant to understanding transdetermination and the factors that influence developmental plasticity. One is lamina ancestor (lama), whose expression correlates with undifferentiated cells and is shown to control the area of transdetermination. Another has sequence similarity to the mammalian augmenter of liver regeneration (Alr; Gfer -- Mouse Genome Informatics), which controls regenerative capacity in the liver and is upregulated in mammalian stem cells. Fifteen regulators of chromatin structure [e.g. members of the Polycomb group (PcG) and trithorax group (trxG)] are differentially regulated in transdetermining cells, and mutants in seven of these genes have significant effects on transdetermination. These studies identify two types of functions that transdetermination requires -- functions that promote an undifferentiated cell state and functions that re-set chromatin structure (Klebes, 2005).

The importance of chromatin structure to the transcriptional state of determined cells makes it reasonable to assume that re-programming cells to different fates entails reorganization of the Polycomb group (PcG) and trithorax group (trxG) protein complexes that bind to regulatory elements. Although altering the distribution of proteins that mediate chromatin states for transcriptional repression and activation need not involve changes in the levels of expression of the PcG and trxG proteins, the array hybridization data was examined to determine if they do. The PcG Suppressor of zeste 2 [Su(z)2] gene had a median fold repression of 2.1 in eight TD to DWg/VWg comparisons, but the cut-off settings did not detect significant enrichment or repression of most of the other PcG or trxG protein genes with either clustering analysis or the method of ranking median ratios. Since criteria for assigning biological significance to levels of change are purely subjective, the transdetermination expression data was re-analyzed to identify genes whose median ratio changes within a 95% confidence level. Fourteen percent of the genes satisfied these conditions. Among these genes, 15/32 PcG and trxG genes (47%) had such statistically significant changes. Identification of these 15 genes with differential expression suggests that transdetermination may be correlated with large-scale remodeling of chromatin structure (Klebes, 2005).

To test if the small but statistically significant changes in the expression of PcG and trxG genes are indicative of a functional role in determination, discs from wild-type, Polycomb (Pc), Enhancer of Polycomb [E(Pc)], Sex comb on midleg (Scm), Enhancer of zeste [E(z)], Su(z)2, brahma (brm) and osa (osa) larvae were examined. The level of Wg induction was adjested to reduce the frequency of transdetermination and both frequency of transdetermination and area of transdetermined cells was determined. The frequency of leg discs expressing vg increased significantly in E(z), Pc, E(Pc), brm and osa mutants, and the frequency of leg to wing transdetermination in adult cuticle increased in Scm, E(z), Pc, E(Pc) and osa mutants. Remarkably, Su(z)2 heterozygous discs had no vg expression, suggesting that the loss of Su(z)2 function limits vg expression (Klebes, 2005).

Members of the PcG and trxG are known to act as heteromeric complexes by binding to cellular memory modules (CMMs). The functional tests demonstrate that mutant alleles for members of both groups have the same functional consequence (they increase transdetermination frequency). The findings are consistent with recent observations that the traditional view of PcG members as repressors and trxG factors as activators might be an oversimplification, and that a more complex interplay of a varying composition of PcG and trxG proteins takes place at individual CMMs. Furthermore the opposing effects of Pc and Su(z)2 functions are consistent with the proposal that Su(z)2 is one of a subset of PcG genes that is required to activate as well as to suppress gene expression. In addition to measuring the frequency of transdetermination, the relative area of vg expression was examined in the various PcG and trxG heterozyogous mutant discs. The relative area decreased in E(Pc), brm and osa mutant discs, despite the increased frequency of transdetermination in these mutants. There is no evidence to explain these contrasting effects, but the roles in transdetermination of seven PcG and trxG genes that were identified by these results support the proposition that the transcriptional state of determined cells is implemented through the controls imposed by the regulators of chromatin structure (Klebes, 2005).

The determined states that direct cells to particular fates or lineages can be remarkably stable and can persist after many cell divisions in alien environments, but they are not immune to change. In Drosophila, three experimental systems have provided opportunities to investigate the mechanisms that lead to switches of determined states. These are: (1) the classic homeotic mutants; (2) the PcG and trxG mutants that affect the capacity of cells to maintain homeotic gene expression, and (3) transdetermination. During normal development, the homeotic genes are expressed in spatially restricted regions, and cells that lose (or gain) homeotic gene function presumably change the transcriptional profiles characteristic of the particular body part. In the work reported here, techniques of micro-dissection, RNA amplification and array hybridization were used to monitor the transcription profiles of cells in normal leg and wing imaginal discs, in leg disc cells that regenerate and in cells that transdetermine from leg to wing. The results validate the idea that changing determined states involves global changes in gene expression. They also identify genes whose function may be unrelated to the specific fates of the cells characterized, but instead may correlate with developmental plasticity (Klebes, 2005).

Overlap between the transcriptional profiles in the wing and transdetermination lists (15 genes) and with genes in subcluster IV (high expression in wing discs) is extensive. The overlap is sufficient to indicate that the TD leg disc cells have changed to a wing-like program of development, but interestingly, not all wing-specific genes are activated in the TD cells. The reasons could be related to the incomplete inventory of wing structures produced (only ventral wing) or to the altered state of the TD cells. During normal development, vg expression is activated in the embryo and continues through the 3rd instar. Although the regulatory sequences responsible for activation in the embryo have not been identified, in 2nd instar wing discs, vg expression is dependent upon the vgBE enhancer, and in 3rd instar wing discs expression is dependent upon the vgQE enhancer. Expression of vg in TD cells depends on activation by the vgBE enhancer, indicating that cells that respond to Wg-induction do not revert to an embryonic state. Recent studies of the cell cycle characteristics of TD cells support this conclusion (Sustar, 2005), but the role of the vgBE enhancer in TD cells and the incomplete inventory of 'wing-specific genes' in their expression profile probably indicates as well the stage at which the TD cells were analyzed: they were not equivalent to the cells of late 3rd instar wing discs (Klebes, 2005).

Investigations into the molecular basis of transdetermination have led to a model in which inputs from the Wg, Dpp and Hh signaling pathways alter the chromatin state of key selector genes to activate the transdetermination pathway. The analyses were limited to a period 2-3 days after the cells switched fate, because several cell doublings were necessary to produce sufficient numbers of marked TD cells. As a consequence, these studies did not analyze the initial stages. Despite this technical limitation, this study identified several genes that are interesting novel markers of transdetermination (e.g., ap, CG12534, CG14059 and CG4914), as well as several genes that function in the transdetermination process (e.g., lama and the PcG genes). The results from transcriptional profiling add significant detail to a general model proposed for transdetermination (Klebes, 2005).

(1) It is reported that ectopic wg expression results in statistically significant changes in the expression of 15 PcG and trxG genes. Moreover, although the magnitudes of these changes were very small for most of these genes, functional assays with seven of these genes revealed remarkably large effects on the metrics used to monitor transdetermination -- the fraction of discs with TD cells, the proportion of disc epithelium that TD cells represent, and the fraction of adult legs with wing cuticle. These effects strongly implicate PcG and trxG genes in the process of transdetermination and suggest that the changes in determined states manifested by transdetermination are either driven by or are enabled by changes in chromatin structure. This conclusion is consistent with the demonstrated roles of PcG and trxG genes in the self-renewing capacity of mouse hematopoietic stem cells, in Wg signaling and in the maintenance of determined states. The results now show that the PcG and trxG functions are also crucial to pluripotency in imaginal disc cells, namely that pluripotency by 'weak point' cells is dependent upon precisely regulated levels of PcG and trxG proteins, and is exquisitely sensitive to reductions in gene dose (Klebes, 2005).

The data do not suggest how the PcG and trxG genes affect transdetermination, but several possible mechanisms deserve consideration. A recent study (Sustar, 2005) reported that transdetermination correlates with an extension of the S phase of the cell cycle. Several proteins involved in cell cycle regulation physically associate with PcG and trxG proteins, and Brahma, one of the proteins that affects the metrics of transdetermination, has been shown to dissociate from chromatin in late S-phase and to reassociate in G1. It is possible that changes in the S-phase of TD cells are a consequence of changes in PcG/trxG protein composition (Klebes, 2005).

Another generic explanation is that transdetermination is dependent or sensitive to expression of specific targets of PcG and trxG genes. Among the 167 Pc/Trx response elements (PRE/TREs) predicted to exist in the Drosophila genome, one is in direct proximity to the vg gene. It is possible that upregulation of vg in TD cells is mediated through this element. Another factor may be the contribution of targets of Wg signaling, since targets of Wg signaling have been shown to be upregulated in osa and brm mutants. These are among a number of likely possible targets, and identifying the sites at which the PcG and trxG proteins function will be necessary if an understand is to be gained of how transdetermination is regulated. Importantly, understanding the roles of such targets and establishing whether these roles are direct will be essential to rationalize how expression levels of individual PcG and trxG genes correlate with the effects of PcG and trxG mutants on transdetermination (Klebes, 2005).

(2) The requirement for lama suggests that proliferation of TD cells involves functions that suppress differentiation. lama expression has been correlated with neural and glial progenitors prior to, but not after, differentiation, and it is observed that lama is expressed in imaginal progenitor cells and in early but not late 3rd instar discs. lama expression is re-activated in leg cells that transdetermine. The upregulation of unpaired in TD cells may be relevant in this context, since the JAK/STAT pathway functions to suppress differentiation and to promote self-renewal of stem cells in the Drosophila testis. It is suggested that it has a similar role in TD cells (Klebes, 2005).

(3) A role for Notch is implied by the expression profiles of several Notch pathway genes. Notch may contribute directly to transdetermination through the activation of the vgBE enhancer [which has a binding site for Su(H)] and of similarly configured sequences that were found to be present in the regulatory regions of 45 other TD genes. It will be important to test whether Notch signaling is required to activate these co-expressed genes, and if it is, to learn what cell-cell interactions and 'community effects' regulate activation of the Notch pathway in TD cells (Klebes, 2005).

(4) The upregulation in TD cells of many genes involved in growth and division, and the identification of DNA replication element (DRE) sites in the regulatory region of many of these genes supports the observation that TD cells become re-programmed after passing through a novel proliferative state (Sustar, 2005), and suggests that this change is in part implemented through DRE-dependent regulation (Klebes, 2005).

There was an interesting correlation between transdetermination induced by Wg mis-expression and the role of Wg/Wnt signaling for stem cells. Wg/Wnt signaling functions as a mitogen and maintains both somatic and germline stem cells in the Drosophila ovary, and mammalian hematopoetic stem cells. Although the 'weak point' cells in the Drosophila leg disc might lack the self-renewing capacity that characterizes stem cells, they respond to Wg mis-expression by manifesting a latent potential for growth and transdetermination. It seems likely that many of the genes are conserved that are involved in regulating stem cells and that lead to disease states when relevant regulatory networks lose their effectiveness (Klebes, 2005).

The prevalence of transcription factors among the genes whose relative expression levels differed most in the tissue comparisons was intriguing. It is commonly assumed that transcription factors function catalytically and that they greatly amplify the production of their targets, so the expectation was that the targets of tissue-specific transcription factors would have the highest degree of tissue-specific expression. In these studies, tissue-specific expression of 15 transcription factors among the 40 top-ranking genes in the wing and leg data sets (38%) is consistent with the large number of differentially expressed genes in these tissues, but these rankings suggest that the targets of these transcription factors are expressed at lower relative levels than the transcription factors that regulate their expression. One possible explanation is that the targets are expressed in both wing and leg disc cells, but the transcription factors that regulate them are not. This would imply that the importance of position-specific regulation lies with the regulator, not the level of expression of the target. Another possibility is that these transcription factors do not act catalytically to amplify the levels of their targets, or do so very inefficiently and require a high concentration of transcription factor to regulate the production of a small number of transcripts. Further analysis will be required to distinguish between these or other explanations, but it is noted that the prevalence of transcription factors in such data sets is neither unique to wing-leg comparisons nor universal (Klebes, 2005).

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).

dachsous is required for Wg-dependent pattern formation in the Drosophila wing disc

The dachsous (ds) gene encodes a member of the cadherin family involved in the non-canonical Wnt signaling pathway (see Eisenmann's Wnt Signaling) 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).

In second instar larvae, ds-lacZ expression is essentially confined to the distal part of the wing disc, but is absent in those distal A cells in which Wg strongly accumulates. This Wg expression constitutes the earliest marker for the nascent wing pouch. Soon thereafter, when Wg expression is expanded to the adjacent P cells, ds-lacZ expression fades away and becomes confined to a ring of cells around the prospective wing pouch. At this stage, most of the hinge cells located between the prospective notum and wing pouch express ds-lacZ at high levels, as revealed by the Iro and Nub markers. A weak expression of ds-lacZ overlaps with the periphery of the Nub domain and marks the region that will become the proximal wing. At third instar, ds-lacZ expression is also observed within the lateral regions of the prospective notum. An antibody directed against the cytoplasmic region of Ds protein reveals a Ds protein distribution similar to the ds-lacZ expression pattern and an apical location at the plasma membrane. From these results, it is concluded that ds-lacZ expression is one of the earliest and most specific markers of the prospective hinge during the second and early third larval instar (Rodríguez, 2004).

During patterning and growth of the wing blade, Wg distribution has been proposed to signal to distant cells in a concentration-dependent manner. Several mechanisms, such as the interaction of Wg with heparin sulfate-containing proteoglycans, as well as regulated endo- and exocytosis, are involved in shaping the gradient and delimiting the range of signaling. Wg protein is predominantly located at the apical surface in the producing cells, and in the embryo it has been demonstrated that this sub-cellular location is essential for its signaling activity. When the hinge territory is already specified at early third instar, wg is activated in these cells and acts as a cell proliferation signal necessary for the development of most structures. Wg expression within the hinge can be described as two rings, the 'inner ring’ (IR) and the 'outer ring’ (OR), which overlap with the areas of low (ring I) and high expression of ds-lacZ (ring II), respectively. In this context, since Ds is also apically located, it was of interest to determine whether Ds has a role in Wg-producing cells. To test this, the distribution of Wg was examined in large clones of ds mutant cells. Two results were obtained from these experiments: (1) the level of Wg in the producing cells was slightly increased with respect to neighbouring wild-type cells, and (2) the Wg gradient within mutant tissue appears to be broader. These results should imply a higher signaling capacity of Wg in ds mutant cells; however, that was not the case. In ds clones, Wg accumulation was less marked apically and was relatively more abundant in the baso-lateral region than in the wild-type cells. Interestingly, this phenomenon seems not to be strictly cell autonomous, because adjacent wild-type cells also displayed a similar abnormal sub-cellular localization of Wg protein. This effect could be due to basal Wg protein diffusing more rapidly to adjacent cells than apical protein does, as has been observed in the embryo. Taken together, these observations suggest that Ds protein contributes to the apical localization of Wg protein at the plasma membrane. It is thought unlikely that this function of Ds is responsible for the early PD patterning defects in DNW discs, since ds and wg are expressed in complementary domains during early larval development (Rodríguez, 2004).

Whether Ds is required for Wg-mediated patterning in imaginal discs other than the wing disc was examined by analyzing the genetic interactions between ds and several components of the Wg pathway during leg development. Homo- and hetero-allelic combinations of ds cause a reduction of the segment size and fusion of the tarsal segments, with partial elimination of the tarsal joints. This phenotype resembles some defects associated with the loss of function of pangolin/dTCF, and legless/BCL9. Therefore, the levels of Wg signaling were manipulated in mid-strength heteroallelic combinations of ds. The loss of one wild-type copy of dsh enhanced the fusion of leg tarsi and shortened the leg segments. By contrast, the leg phenotype of ds showed a complete recovery of the tarsal joints and an increase in the length of the segments when one dose of the nkd gene, an antagonist of the Wg pathway, was eliminated. Taken together, these findings support a more general role for ds in Wg-mediated patterning processes (Rodríguez, 2004).

The wing primordium is specified as a few anterior cells that express wg at the distal-most part of the wing imaginal disc at second larval instar. Slightly later, wg is also expressed in P cells and these cells are recruited into the wing fate. In 'double-notum-winglet' (DNW) ds strongly hypomorphic mutant discs, the level of Ds protein is highly reduced and only the initial anterior group of Wg-expressing cells becomes specified into the wing fate. The levels of this initial Wg expression seems not to be affected in DNW discs. However, neither the P cells abutting the initial anterior Wg domain nor the surrounding cells of this early wing primordium are able to respond to Wg, leading to the formation of a wing pouch composed exclusively by A cells. Moreover, the activation of Wg target genes (such as hth, required for the specification of hinge cells) fails in DNW discs, and, consequently, the proximal wing and hinge structures do not develop. The significantly reduced rings of ds-lacZ, hth and zfh2 expression in DNW discs most likely reflect the residual Ds activity retained in the ds38k mutant. Cells close to the Wg source might thus still be able to respond to high Wg levels during early stages of wing development. However, under null conditions for ds (dsD36) the expression of zfh2 is eliminated (Rodríguez, 2004).

Thus, in addition to its function in PCP, ds plays a role in early patterning when the specification of the different territories along the PD axis takes place in response to Wg. Initially, ds facilitates the recruitment of P cells into the wing fate in response to Wg. Subsequently, Ds promotes the activation of Wg target genes in the surrounding cells to specify the hinge. Note that once the hinge cells have been specified in response to Wg signaling, ds seems to be dispensable for global wing disc patterning, as the ‘classical’ ds38k phenotype shows. In this case, only mild defects such as slight tissue overgrowth or polarity defects were observed, suggesting additional functions of ds related to cell adhesion (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 vertebrates, during telencephalon formation, the organization into different structures requires the expression of different cadherins in adjacent regions to maintain a compartment boundary based on differential cell affinity features. It has been suggested that the expression pattern of each of these cadherins is under the control of specific signaling cascades (Rodríguez, 2004).

In Drosophila, during imaginal disc development, indirect evidence has suggested that cell adhesion might be under the control of the same signaling pathways that control cell proliferation and patterning. The smooth borders of clones mutant for thick vein (tkv), the receptor of Dpp, or smoothened (smo), a downstream component of the Hedgehog (Hh) signaling pathway, indicate that mutant cells change their affinity properties and therefore try to minimise the contact with surrounding wild-type cells. Nevertheless, little is known about the molecules involved in these adhesiveness differences. Recent work has proposed that both tartan and capricious (caps), two transmembrane proteins regulated by ap, are putative candidates to maintain the affinity boundary between dorsal and ventral cells. However, whereas clones ectopically expressing tartan and caps in V cells tend to contact D cells, the elimination of tartan and caps in clones from D cells had no effect on DV boundary formation (Rodríguez, 2004).

In the DNW phenotype, the ectopic notum develops from cells of the hinge territory. According to the proposed subdivision into concentric rings (I to III), cells from the outermost ring III expressing Tsh and Ds will give rise to that part of the body wall that is excluded from the notum region. In DNW discs, the absence of Ds produces an expansion of notal-specific iro-C expression to more distal positions to fill up the Tsh domain. These distal cells acquire a notum fate, generating an ectopic notum similar to wg1 mutant flies (Rodríguez, 2004).

Thus, Ds protein contributes to hinge/notum boundary formation by means of an affinity border. This process would occur at early second instar when Iro-C expression is capable of specifying the notum fate. This finding provides the first evidence that a cadherin is able to maintain the cell boundary between two adjacent territories in Drosophila (Rodríguez, 2004).

How does ds participate in Wg signaling? Several findings point out a specific role of Ds in the modulation of Wg signaling: (1) the elimination of zfh2 expression in ds mutant clones; (2) the genetic interactions of ds alleles with several components of the Wg signaling pathway, and (3) the rescue of the DNW phenotype by increasing Wg levels. It has been shown that Ds is associated with adherens junctions at the apical surface of the imaginal cells, to mediate cell-cell adhesion. A major step of the cell adhesion mechanism requires interaction of the cytoplasmic tail with Arm/ß-catenin to connect the cadherin-catenin complex to the actin cytoskeleton. Thus, the phenotype could reflect changes in the balance between cytoplasmic Arm versus Arm anchored to the plasma membrane. If this were the case, then a reduction of ds function would increase Wg signaling; however, the results presented above indicate that loss of ds decreases Wg signaling. Moreover, sequence analysis has shown that the ß-catenin binding motifs in the Ds protein, which have to be in tandem to be functional, are separated by a stretch of amino acids, further discarding the possibility that Ds binds directly to Arm to modulate its cytoplasmic levels (Rodríguez, 2004).

Alternatively, the apical plasma membrane acts as a structural center that contains crucial components that modulate the Wg pathway, such as Dsh, E-APC and Axin. Axin and E-APC, promote the degradation of cytoplasmic Arm, the main effector of the Wg cascade. Previous work has shown that, upon binding of Wg in the receiving cells, the Axin/E-APC complex becomes anchored to the plasma membrane to prevent Arm degradation. In this context, Ds protein, as part of the adherens junctions, could be the cadherin required to anchor this degradation complex to the plasma membrane. In ds mutant cells, the cytoplasmic levels of the Axin/E-APC complex would be higher and, therefore, Wg signaling would decrease. In agreement with this hypothesis, mild ds phenotypes are enhanced when a copy of dsh gene is eliminated. Still, Ds could act at the level of Wg reception, by increasing the Fz/Wg-binding affinity or by recruiting Fz molecules to the apical plasma membrane, as has been demonstrated for the cadherin Fmi in the PCP processes (Rodríguez, 2004).

Early anterior Wg activity initiates specification of the PD axis in the wing disc: To date, the current model explaining the specification of the territories along the PD axis assumes that the initial anterior Wg expression at second instar is required only for cells to acquire the wing fate. It is only later, when wg is expressed in two concentric rings that its function is required to specify the hinge territory (Rodríguez, 2004).

Wg has been shown to be required for the development of the hinge. In contrast, Wg activates downstream genes such as hth or zfh2 to specify the hinge fate. In contrast, Wg controls cell proliferation when it is expressed from early third instar into the IR and OR rings. It has been established that the specification of the hinge takes place later than the wing; however, the data show that an early and timely limited depletion of Wg activity causes a failure in hinge specification. This is mainly based on the observation that only early-induced ds clones abolish zfh2 expression required for hinge formation. In ds mutant clones induced later, hinge development is unaffected, although a perdurance of ds activity in these clones cannot be excluded. Still, the rescue of hinge development in DNW discs that ectopically express Wg under dpp-Gal4 further support an early specification of the hinge. In these discs, ectopic Wg expression stays confined to the AP border. At early stages, the AP border must be located close enough to the nascent wing primordial to allow the spreading of Wg into regions destined to become hinge territory. At late stages, the narrow stripe of ectopic Wg expression can no longer account for the maintenance of the whole hinge territory. It is rather the Wg within the IR and OR that maintains hth expression and, with it, the specification of the hinge fate. At this stage, either Wg works independently of ds or its requirements for ds are lower. Thus, if hinge specification is not initiated early upon ds and wg activities, wg expression cannot be established and the development of the hinge is aborted (Rodríguez, 2004).

The present results provide insights that help in the understanding of how the PD axis is established in the wing disc. The initial event in this process would be the early activity of Wg. When Wg is expressed at the distal part of the wing disc in a small group of anterior cells, it not only promotes the activation of target genes like vg, nub or scalloped (sd) in the wing cells, but also the expression of hth and zfh2 to specify the hinge. At the same time, Wg would repress tsh or vein (vn) at the distal part of the wing disc to separate the proximal wing and hinge regions from the body wall where Egfr signaling activates notum-specific genes like iro-C. Thus, in cooperation with dpp, wg establishes the AP and PD axis in the prospective wing and hinge regions (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).

fat, four-jointed, dachsous and dachs influence the expression of Wingless in the proximal wing

In the Drosophila wing, distal cells signal to proximal cells to induce the expression of Wingless, but the basis for this distal-to-proximal signaling is unknown. Three genes that act together during the establishment of tissue polarity, fat, four-jointed and dachsous, also influence the expression of Wingless in the proximal wing. fat is required cell autonomously by proximal wing cells to repress Wingless expression, and misexpression of Wingless contributes to proximal wing overgrowth in fat mutant discs. Four-jointed and Dachsous can influence Wingless expression and Fat localization non-autonomously, consistent with the suggestion that they influence signaling to Fat-expressing cells. dachs is identified as a gene that is genetically required downstream of fat, both for its effects on imaginal disc growth and for the expression of Wingless in the proximal wing. These observations provide important support for the emerging view that Four-jointed, Dachsous and Fat function in an intercellular signaling pathway, identify a normal role for these proteins in signaling interactions that regulate growth and patterning of the proximal wing, and identify Dachs as a candidate downstream effector of a Fat signaling pathway (Cho, 2004).

There is a progressive elaboration of patterning along the PD axis over the course of wing development. During the second larval instar, interactions among the Epidermal Growth Factor Receptor, Dpp and Wg signaling pathways divide the wing disc into a dorsal region, which will give rise to notum, and a ventral region, from which the wing will arise. An initial PD subdivision of the wing is then effected by signaling from the AP and DV compartment boundaries, which promotes the expression of two genes, scalloped and vestigial, that encode subunits of a heterodimeric transcription factor (Sd-Vg) in the center of the wing. This subdivides the wing into distal cells, which give rise to the wing blade, and surrounding cells, which give rise to proximal wing and wing hinge structures. The proximal wing is further subdivided into a series of molecularly distinct domains. Studies of Sd-Vg function in the wing led to the realization that the elaboration of this finer pattern depends in part upon signaling from the distal, Sd-Vg-expressing cells, to more proximal cells. Thus, mutation of vg leads to elimination, not only of the wing blade, where Vg is expressed, but also of more proximal tissue. Conversely, ectopic expression of Vg in the proximal wing reorganizes the patterning of surrounding cells (Cho, 2004 and references therein).

A key target of the distal signal is Wg, which during early third instar is expressed in a ring of cells that surround the SD-VG-expressing cells, and which later becomes expressed in a second, more proximal ring. Wg expression in the inner, distal ring within the proximal wing is regulated by an enhancer called spade-flag (spd-fg), after an allele of wg in which this enhancer is deleted (Neumann, 1996a). Studies of this allele, together with ectopic expression experiments, have revealed that Wg is necessary and sufficient to promote growth of the proximal wing. Wg also plays a role in proximal wing patterning; it acts in a positive-feedback loop to maintain expression of Homothorax (Hth). The rotund (rn) gene has been identified as an additional target of distal signaling (Cho, 2004 and references therein).

This work identified Four-jointed (Fj), Dachsous (Ds), Fat and Dachs as proteins that influence signaling to proximal wing cells to regulate Wg and rn expression. Fj is a type II transmembrane protein, which is largely restricted to the Golgi. Null mutations in fj do not cause any obvious defects in the proximal wing. However, fj plays a role in the regulation of tissue polarity, yet acts redundantly with some other factor(s) in this process. Mutations in fat or ds can also influence tissue polarity. Although the molecular relationships among these proteins are not well understood, genetic studies suggest that fj and ds act via effects on fat, and both fj and ds can influence Fat localization in genetic mosaics (Cho, 2004 and references therein).

Interestingly, alleles of fj, ds and fat, as well as alleles of another gene, dachs, can result in similar defects in wing blade and leg growth. The similar requirements for these genes during both appendage growth and tissue polarity, together with the expression patterns of fj and ds in the developing wing, led to this investigation of their requirements for proximal wing development. All four genes influence the expression of Wg in the proximal wing, and genetic experiments suggest a pathway in which Fj and Ds act to modulate the activity of Fat, which then regulates transcription via a pathway that includes Dachs. These observations lend strong support to the hypothesis that Fj, Ds and Fat function as components of an intercellular signal transduction pathway, implicate Dachs as a key downstream component of this pathway, and identify a normal role for these genes in proximodistal patterning during Drosophila wing development (Cho, 2004).

The common feature of all of the manipulations of FJ and DS expression carried out in this study is that Wg expression, and by inference, Fat activity, can be altered when cells with different levels of Fj or Ds are juxtaposed. In the case of Fj, its normal expression pattern, and effects of mutant and ectopic expression clones are all consistent with the interpretation that juxtaposition of cells with different levels of Fj is associated with inhibition of Fat in the cells with less Fj and activation of Fat in the cells with more Fj. The influence of Ds, however, is more variable. Studies of tissue polarity in the eye suggest that Ds inhibits Fat activity in Ds-expressing cells, and/or promotes Fat activity in neighboring cells. The predominant effect of Ds during early wing development is consistent with this, but its effects in late discs are not. Studies of tissue polarity in the abdomen suggest that the Ds gradient might be interpreted differently by anterior versus posterior cells, and it is possible that a similar phenomena causes the effects of Ds to vary during wing development (Cho, 2004).

The influence of ds mutation on gene expression and growth in the wing is much weaker than that of fat. It has been suggested that Fj might influence Fat via effects on Ds, and fj mutant clones have been observed to influence Ds protein staining. The observations are consistent with the inference that both Ds and Fj can regulate Fat activity, but they do not directly address the question of whether Fj acts through Ds. They do, however, indicate that even the combined effects of Fj and Ds cannot account for FAT regulation, and, assuming that the strongest available alleles are null, other regulators of Fat activity must exist. It is presumably because of the counteracting influence of these other regulators that alterations in Fj and Ds expression have relatively weak effects. In addition, according to the hypothesis that Fat activity is influenced by relative rather than absolute levels of its regulators, the effects of Fj or Ds could be expected to vary depending upon their temporal and spatial profiles of expression, as well as on the precise shape and location of clones (Cho, 2004).

The observations imply the existence of at least two intracellular branches of the Fat signaling pathway. One branch involves the transcriptional repressor Grunge, influences tissue polarity, certain aspects of cell affinity, and fj expression, but does not influence growth or wg expression. An alternative branch does not require Grunge, but does require Dachs. Dachs is implicated as a downstream component of the Fat pathway, based on its cell autonomous influence on Fat-dependent processes, and by genetic epistasis. The determination that it encodes an unconventional myosin, and hence presumably a cytoplasmic protein, is consistent with this possibility. It also suggests that Dachs does not itself function as a transcription factor, and hence implies the existence of other components of this branch of the Fat pathway. This Grunge-independent branch influences Wg expression in the proximal wing and imaginal disc growth. However, further studies will be required to determine whether Dachs functions solely in Grunge-independent Fat signaling, or whether instead Dachs is required for all Fat signaling (Cho, 2004).

The observations that fj expression is regulated by Sd-Vg, and that fj is both necessary and sufficient to modulate the distal ring of Wg expression in the proximal wing, suggest that Fj influences the activity of a distal signal, which then acts to influence Fat activity. However, the relatively weak effects of fj indicate that other factors must also contribute to distal signaling, just as fj functions redundantly with other factors to influence tissue polarity. Since Ds expression is downregulated in a domain that is broader than the Vg expression domain, a direct influence of Vg on the Ds gradient is unlikely, and the essentially normal appearance of Wg expression in the proximal wing in fj ds double mutants implies that Ds is not a good candidate for the hypothetic factor Signal X. Rather, it is suggested that Ds acts in parallel to signaling from Vg-expressing cells to modulate Fat activity. This Vg-independent effect would account for the remnant of the distal ring that sometimes appears in vg null mutants. Importantly though, the observation that the phenotypes of hypomorphic dachs mutant clones on Wg expression are more severe than fj and ds suggests that the hypothesized additional factors also act via the Fat pathway. It is also noted that the limitation of Wg expression to the proximal wing even in fat mutant clones implies that Wg expression both requires Nubbin, and is actively repressed by distally-expressed genes (Cho, 2004).

The recovery of normal Wg expression by later stages in both fj and dachs mutant clones implies that the maintenance of Wg occurs by a distinct mechanism. Prior studies have identified a positive-feedback loop between Wg and Hth that is required to maintain their expression. It is suggested that once this feedback loop is initiated, Fat signaling is no longer required for Wg expression. Moreover, the recovery of normal levels of Wg at late stages suggests that this positive-feedback loop can amplify reduced levels of wg to near normal levels (Cho, 2004).

The distinct consequences of Vg expression and Fj expression in clones in the proximal wing suggest that another signal or signals, which are qualitatively distinct from the Fj-dependent signal, is also released from VG-expressing cells. When Vg is ectopically expressed, Wg is often induced in a ring of expression that completely encircles it. However, this is not the case for Fj-expressing clones. Both Vg- and Fj-expressing clones can activate rn and wg only within NUB-expressing cells, but Vg expression can result in non-autonomous expansion of the Nub domain, and this expansion presumably facilitates the expression of Wg by surrounding cells. Another striking difference between Vg- and Fj-expressing clones is that in the case of ectopic Fj, enhanced Wg expression is only in adjacent cells. By contrast, in the case of Vg, Wg expression initiates in neighboring cells, but often moves several cells away as the disc grows, resulting in a gap between Vg and Wg expression. This gap suggests that a repressor of Wg expression becomes expressed there, and recent studies have identified Defective proventriculus (Dve) as such a repressor (Cho, 2004).

In strong fat mutants, the wing discs become enlarged and have extra folds and outgrowths in the proximal wing. The disproportionate overgrowth of the proximal wing is due to upregulation of Wg in this region, as demonstrated by its suppression by wgspd-fg. At the same time, clones of cells mutant for fat overgrow in other imaginal cells, and fat wgspd-fg discs are still enlarged compared with wild-type discs. Thus, Fat appears to act both by regulating the expression of other signaling pathways (e.g. Wg), and via its own, novel growth pathway. The identification of additional components of this pathway will offer new approaches for investigating its profound influence on disc growth (Cho, 2004).

Caspase inhibition during apoptosis causes abnormal Wg 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).

Apoptotic cells can induce compensatory cell proliferation through the JNK and the Wingless signaling pathways

In many metazoans, damaged and potentially dangerous cells are rapidly eliminated by apoptosis. In Drosophila, this is often compensated for by extraproliferation of neighboring cells, which allows the organism to tolerate considerable cell death without compromising development and body size. Despite its importance, the mechanistic basis of such compensatory proliferation remains poorly understood. Apoptotic cells are shown to express the secretory factors Wingless and Decapentaplegic. When cells undergoing apoptosis were kept alive with the caspase inhibitor p35, excessive nonautonomous cell proliferation was observed. Significantly, Wg signaling is necessary and, at least in some cells, also sufficient for mitogenesis under these conditions. Finally, evidence is provided that the DIAP1 antagonists reaper and hid can activate the JNK pathway and that this pathway is required for inducing wg and cell proliferation. These findings support a model where apoptotic cells activate signaling cascades for compensatory proliferation (Ryoo, 2004).

To investigate how the inhibition of diap1 may lead to mitogen expression, attention was focused on Dronc and the Jun N-terminal Kinase (JNK) pathway. Dronc has been implicated in compensatory proliferation, and its activity can be inhibited by the expression of droncDN. In addition, the JNK signaling pathway was considered as a candidate, since its activity is known to correlate with many forms of stress-provoked apoptosis, including disruption of morphogens, cell competition, and rpr expression. In Drosophila, the JNK pathway can be effectively blocked by the expression of puckered (puc), which encodes a phosphatase that negatively regulates JNK (Ryoo, 2004).

To induce patches of undead cells, wing imaginal discs were generated with mosaic clones expressing hid and p35. 48 hr after induction, these imaginal discs contained hid-expressing clones that autonomously induced wg. Using this experimental setup, it was asked whether additional expression of either droncDN or puc would block wg induction in undead cells. When droncDN was coexpressed, a subset of the hid-expressing population was still able to induce wg. In contrast, when puc was coexpressed, wg induction by hid was almost completely blocked. These results provide evidence that the JNK pathway is required for wg induction under these conditions but fail to uncover a similar requirement for Dronc (Ryoo, 2004).

To independently investigate the role of puc and droncDN in compensatory proliferation, the size of wing discs harboring undead cells was measured and they were compared with those of the sibling controls. Under the experimental conditions, wing discs harboring hid- and p35-expressing clones were on average 53% larger than their sibling controls. Coexpression of puc within these undead clones significantly limited growth, resulting in only a small increase in wing disc size that was not statistically significant. In contrast, coexpression of droncDN did not limit growth. Wing size measurements also correlated with the degree of wg induction. The larger size of discs harboring hid- and p35-expressing cells is not due simply to extra cell survival: (1) these undead cells are derived from the normal lineage; (2) the size of wing discs expressing hid, p35, and puc serves as a control. In this case, although a large number of undead cells were generated, no significant increase in disc size was observed, in stark contrast to the discs expressing hid and p35 only. It is concluded that the JNK pathway is required for the nonautonomous growth promoting activity of the undead cells (Ryoo, 2004).

To confirm a role of puc in imaginal disc growth, rpr and p35 werecoexpressed in wild-type and puc−/+ imaginal discs. Like hid, rpr is a DIAP1 antagonist, but with a weaker cell killing activity when overexpressed in imaginal disc cells. In a puc+/+ background, a small amount of ectopic wg expression was observed, indicative of rpr's weaker DIAP1 inhibiting activity. In contrast, ectopic wg expression was strongly enhanced in puc−/+ discs. Because the puc allele used, pucE69, also acts as a lacZ reporter, JNK pathway induction could be monitored simultaneously. wg induction in undead cells correlates very well with puc-lacZ expression, with a stronger induction at the center of the wing pouch. These results further support the role of JNK in the induction of wg (Ryoo, 2004).

Next to be tested was whether the reduction of puc had an effect on apoptosis-induced cell proliferation. Whereas puc−/+ discs expressing only p35 had BrdU incorporation similar to wild-type discs, coexpression of rpr and p35 in puc−/+ led to a significant increase in BrdU incorporation. Also, the size of these discs were on average 41% larger than those coexpressing rpr and p35 in a puc+/+ background. Taken together, these results show that diap1 inhibition leads to JNK activation and that JNK activity promotes wg induction and cell proliferation (Ryoo, 2004).

To directly test if JNK signaling can activate wg and dpp expression, hepCA, a constitutively active form of hemipterous (hep), the Drosophila JNK kinase was conditionally expressed. Expression of hepCA causes induction of wg-lacZ within 22 hr and to a lesser extent also dpp-lacZ. These ß-gal-expressing cells shifted basally and were apoptotic as assayed by anti-active caspase-3 antibody labeling. Hid protein levels were also elevated in these cells. Significantly, since p35 was not use to block apoptosis in this experiment, this demonstrates that wg and dpp can be induced not only in undead cells, but also in 'real' apoptotic cells (Ryoo, 2004).

This study provides evidence that the central apoptotic regulators can control the activity of mitogenic pathways. In particular, inhibition of DIAP1, either via expression of Reaper and Hid or by mutational inactivation, leads to the induction of the putative mitogens wg and dpp. When apoptosis was initiated through DIAP1 inhibition but cells were kept alive by blocking caspases, the resulting 'undead cells' exhibited strong mitogenic activity and stimulated tissue overgrowth. Inhibiting wg signaling with a conditional TCFDN blocked cell proliferation in imaginal discs, indicating that wg has an essential mitogenic function. Finally, evidence was provided that the JNK pathway mediates mitogen expression and imaginal disc overgrowth in response to rpr and hid. Based on these results, it is proposed that apoptotic cells actively signal to induce compensatory proliferation. DIAP1 inhibits both caspases as well as dTRAF1. According to this model, when DIAP1 is inhibited in response to cellular injury, the JNK pathway is activated and wg/dpp are induced in apoptotic cells. Secretion of these factors stimulates growth of proliferation-competent neighboring cells and leads to compensatory proliferation (Ryoo, 2004).

This study provides clear genetic evidence that diap1 is involved in compensatory proliferation. Overall, similar results were obtained with hypomorphic diap1 alleles (diap122-8s, diap133-1s), a null allele (diap1th5), and inactivation of diap1 by expression of Reaper and Hid. However, whereas expression of p35 effectively blocked apoptosis of diap122-8s/22-8s cells and in response to Reaper/Hid, it only partially suppressed the death of diap1th5/th5 cells. Consequently, the generation of undead cells was less efficient with the diap1th5 mutation. Moreover, these results suggest that the JNK pathway transduces the signal to activate mitogen expression and cell proliferation. Since IAPs have been shown to ubiquitylate TRAFs in both mammals and Drosophila and since no evidence was found for Dronc in growth promotion, it is attractive to speculate that JNK is regulated through direct DIAP1/TRAF1 interaction (Ryoo, 2004).

Many of the results were obtained with undead cells, i.e., cells stimulated to undergo apoptosis but rescued from death by caspase inhibition. Consequently, it may be argued that mitogenic signaling under these conditions is a unique property of undead cells and not physiologically relevant. However, it is shown that JNK activation induces expression of wg and dpp autonomously in 'genuine' apoptotic cells. Therefore, the view is favored that extending the life of doomed cells by p35 expression simply enhances a phenomenon that is otherwise transient and difficult to observe. Rapid clearance of apoptotic cells is presumably important to limit their mitogenic activity to achieve compensatory growth, as opposed to the tissue overgrowths seen with undead cells. It has been reported that wg, dpp, and hh are involved in the regeneration of fragmented imaginal discs, a phenomenon in which cut and cultured imaginal disc fragments undergo massive proliferation. In this paradigm, mitogen expression persists at the wound sites, resulting in cell fate respecification as well as proliferation. This aspect of persistent mitogen expression is similar to the situation in undead cells. However, apoptotic cells seldom interfere with cell fate, likely because they do not persist long enough to secrete levels of mitogens that are sufficient to alter cell fate decisions. In contrast, it is conceivable that cells with impaired apoptosis, such as cancer cells, may have excessive and undesirable mitogenic and morphogenetic activity (Ryoo, 2004).

Another important unresolved question is why compensatory proliferation is seen only in response to cellular injury, but not during normal developmental apoptosis. In particular, inactivation of DIAP1 by Reaper, Hid, and Grim is restricted not only to injury-provoked apoptosis, but also underlies most developmental cell deaths. One possible explanation is that activation of the JNK pathway is key to mitogenic signaling of apoptotic cells. Consistent with this idea, the JNK pathway is activated in response to tissue stress and injury, but not during developmental apoptosis. Furthermore, this study shows that JNK signaling can induce the expression of wg/dpp and nonautonomous cell proliferation. Therefore, it is possible that robust JNK activation and compensatory proliferation require the combined input of stress and apoptotic signals (Ryoo, 2004).

The role of wg signaling in cell proliferation has remained controversial. wg is required for disc growth and is sufficient to promote proliferation in proximal discs. However, mosaic analyses of wg pathway genes in imaginal discs also indicate that wg signaling constrains cell proliferation in distal parts of imaginal discs. These previous studies relied on clonal analyses, which are typically performed several days after manipulation. Such a long time course allows for a multitude of secondary effects. This study attempted to improve upon such pitfalls by using a conditional gene activation system to examine the mitogenic effect of wg signaling over a short time course (12 hr). The results indicate that wg signaling is required for cell proliferation and hence may contribute to the regulation of normal disc growth. In addition, compensatory proliferation cannot bypass the requirement for wg signaling. However, the results do not exclude the possibility that additional mitogens are required (Ryoo, 2004).

Besides its contribution to compensatory proliferation in Drosophila imaginal discs, mitogenic signaling in response to stress-induced apoptosis may be a more general and an evolutionary conserved phenomenon. For example, hallmarks of cancer include apoptotic stress, such as genetic instability, as well as defective apoptosis. This combination may generate undead cells in tumor tissues that promote further tumor growth. Also, mice with disrupted apoptosis genes exhibit brains that resemble hyperplasia (e.g., in caspase-9 and Apaf-1 mutants). Although this has been largely attributed to the failure to eliminate excessive cells through apoptosis, a recent study has reported extraproliferation as a secondary consequence of defective apoptosis. Based on the behavior of undead cells in Drosophila imaginal discs, one might expect mutations that block or delay apoptosis to cause secondary proliferation and hyperplasia. It remains to be tested if such a mechanism contributes to hyperplasia in mouse models and human malignancies (Ryoo, 2004).

The role of Dpp and Wg in compensatory proliferation and in the formation of hyperplastic overgrowths caused by apoptotic cells in the Drosophila wing disc

Non-lethal stress treatments (X-radiation or heat shock) administered to Drosophila imaginal discs induce massive apoptosis, which may eliminate more that 50% of the cells. Yet the discs are able to recover to form final structures of normal size and pattern. Thus, the surviving cells have to undergo additional proliferation to compensate for the cell loss. The finding that apoptotic cells ectopically express dpp and wg suggested that ectopic Dpp/Wg signalling might be responsible for compensatory proliferation. This hypothesis was tested by analysing the response to irradiation-induced apoptosis of disc compartments that are mutant for dpp, for wg, or for both. There is compensatory proliferation in these compartments, indicating that the ectopic Dpp/Wg signalling generated by apoptotic cells is not involved. However, this ectopic Dpp/Wg signalling is responsible for the hyperplastic overgrowths that appear when apoptotic ('undead') cells are kept alive with the caspase inhibitor P35. The ectopic Dpp/Wg signalling and the overgrowths caused by undead cells are due to a non-apoptotic function of the JNK pathway. It is proposed that the compensatory growth is simply a homeostatic response of wing compartments, which resume growth after massive cellular loss until they reach the final correct size. The ectopic Dpp/Wg signalling associated with apoptosis is inconsequential in compartments with normal apoptotic cells, which die soon after the stress event. In compartments containing undead cells, the adventitious Dpp/Wg signalling results in hyperplastic overgrowths (Pérez-Garijo, 2009).

The involvement of Dpp/Wg signalling in compensatory proliferation was suggested by the finding that dpp and wg are expressed in apoptotic cells. This, together with the observation of increased proliferation in the vicinity of the apoptotic cells, led to the model that compensatory proliferation is caused by the mitogenic activity of the ectopic Dpp and Wg signals emitted by apoptotic cells (Pérez-Garijo, 2009).

In irradiated discs, the ectopic Dpp/Wg signalling generated by the apoptotic cells is superimposed on the normal Dpp/Wg signalling. The latter is essential for the normal growth of the wing compartments; in dppd12 homozygous discs the wings are reduced to a rudiment, and is shown in this study the lack of wg activity results in smaller compartments. These experiments have tested the role of the ectopic Dpp/Wg signalling in size restoration of irradiated discs, that is, the contribution of the apoptotic cells to the process (Pérez-Garijo, 2009).

The ability of P compartments to compensate growth in conditions in which apoptotic cells can produce neither the Dpp nor the Wg signal, or are defective in both signals, was analyzed. The results indicate that the model of compensatory proliferation mentioned above is incorrect. The elimination of ectopic dpp and wg functions in wing discs subjected to massive apoptosis does not impede the restoration of normal size and pattern; in other words, there is compensatory growth without contribution of the Dpp and Wg signals emitted by the apoptotic cells (Pérez-Garijo, 2009).

Having studied compensatory growth only in P compartments, it is just conceivable that Dpp and Wg originated by apoptotic cells in the A compartment might diffuse to the P compartment where they could induce the additional growth necessary to compensate size. This is very unlikely for two reasons. (1) The undead apoptotic cells induce additional proliferation only in their own vicinity. Thus, it is hard to imagine that Dpp/Wg of anterior origin could have an affect on proliferation extending to the entire posterior compartment. Moreover, in the current experiments the cells are not protected by P35; they are not undead cells but regular apoptotic cells that die soon after initiating apoptosis. Therefore the proliferation stimulus they provide would be very short lived. (2) If the Dpp and Wg of apoptotic origin were able to travel a long way across compartment borders, it would be expected that the overgrowths produced by undead cells were not restricted to compartments. For example, in irradiated hh>p35 discs or in en>hid + p35 (and other similar genotypes), in which undead cells belong to the P compartment, the A compartment should also overgrow, stimulated by the Dpp and Wg of posterior origin. In all cases reported, the effect is essentially restricted to the posterior compartment (Pérez-Garijo, 2009).

Thus, although the dpp and wg genes are activated in apoptotic cells, their function appears to be inconsequential. So what is the mechanism responsible for the compensatory growth? One possibility is the existence of some other hitherto undetected signal with mitogenic properties. Although this possibility cannot be ruled out, it appears unlikely because Dpp and Wg are the major growth signals identified in the wing disc after many years of studies. The Dpp pathway has been shown to play a major role in inducing growth in the wing disc; in absence of Dpp activity wing growth is much reduced and an excess of Dpp activity causes additional growth. Moreover, in the experiments in which apoptotic cells are protected with P35, it was found that the absence of Dpp and Wg prevents the appearance of overgrowths, strongly suggesting that these signals are responsible for the additional growth associated with apoptotic cells (Pérez-Garijo, 2009).

It is believed that compensatory growth does not require any special mechanism involving the participation of apoptotic cells. It is the normal process that regulates compartment size that is responsible for restoring normal size after massive apoptosis. It has been shown recently that A and P compartments are autonomous units of size control in the wing disc, i.e., A and P compartments grow autonomously until they reach the final correct size. It has also been shown that the size control mechanism is highly homeostatic. It can adjust to changes in cell size and number, and to differential cell division rates -- alterations in any of these parameters do not produce changes in the final compartment size. As stated above, only the overproduction of Dpp results in breakdown of the size control mechanism (Pérez-Garijo, 2009).

It thought that the compensatory growth after the loss of cells because of irradiation (or any other stress event) is another example of the versatility of the size control mechanism. It is proposed that the massive cell death caused by the irradiation would be equivalent to making the compartment smaller. The irradiated compartment would then restore the correct size simply by performing some additional division. It would be, in effect, an overall regeneration process of the entire blastema, which would be achieved by lengthening the proliferation period, an idea that is supported by observations such that damage to growing discs results in a prolonged growth period. Even a loss of 50% of the cells can be restored if all of the surviving cells divided once. In the wing disc, the length of the division period is about 8-12 hours and therefore only a short delay may be sufficient to allow time for recovery. Thus, irradiated discs would, after some delay caused by the stress, resume growth and the normal control mechanism would stop growth once compartments have reached the final size (Pérez-Garijo, 2009).

Although the ectopic Dpp and Wg signals do not have a role in compensatory proliferation, they are required for the appearance of overgrowths caused by undead cells. A key difference between undead cells and normal apoptotic cells is that the former persistently express Dpp and Wg (probably as a result of JNK activity). In irradiated posterior compartments that comprise undead and non-apoptotic cells, such as, for example, in irradiated hh>p35 discs, the undead cells keep producing the Dpp and Wg signals from shortly after the irradiation and until the end of the proliferation period of the disc. Thus, the non-apoptotic cells receive a continuous supply of the Dpp and Wg mitogens from the undead ones. The result is an overgrowth, which is also associated with abnormal cell differentiation. Both additional growth and abnormal differentiation would be expected in these circumstances, as Dpp and Wg are growth inducers as well as morphogens determining cell pattern and differentiation (Pérez-Garijo, 2009).

The overall conclusion from the above is that the ectopic Dpp and Wg signals generated by apoptotic cells are irrelevant for compensatory proliferation, but are prime factors in the generation of hyperplastic overgrowths caused by undead cells. The question then is why are dpp and wg activated in normal apoptotic cells. It is thought that their activity is a collateral effect of the activation of the JNK pathway after an apoptotic stimulus: γ-irradiation induces JNK activity in the wing disc and radiation-induced apoptosis depends on JNK activity. As expected, in these experiments X-irradiation also induced JNK activity (Pérez-Garijo, 2009).

The function of the JNK pathway appears to be required for the ectopic expression of wg and dpp in apoptotic cells. In experiments in which cell death is blocked with P35 after apoptosis induction, the JNK pathway becomes continuously activated in undead cells and appears to be associated with ectopic wg expression. It is therefore possible that the ectopic activation of dpp and wg in the apoptotic cells could be a consequence of JNK function, rather than a consequence of the apoptotic program. The results strongly support this view: direct activation of JNK via the UAS-hepact construct in dronc mutant discs, in which apoptosis is much reduced, induces wg and dpp expression. Furthermore, these mutant discs show hyperplastic overgrowths in the spalt domain, where JNK is active (Pérez-Garijo, 2009).

It has been shown that JNK activity induces several cellular functions: the initiation of the apoptotic program, and also other non-apoptotic functions, such as the capacity for cell migration and the ability to induce dpp. It is probable that normal apoptotic cells acquire these other JNK-dependent properties, but that they die very quickly and so these other functions have minimal effects. This is different in undead cells because the JNK activity becomes persistent and, therefore, they can manifest some or all of the JNK non-apoptotic functions: these cells can move and invade neighbouring compartments, and express dpp and wg continuously. It is thought that it is the persistent manifestation of these two non-apoptotic JNK-mediated properties, dpp/wg activation and the induction of cell migration that causes the hyperplastic overgrowth (Pérez-Garijo, 2009).

The implication of the Dpp and Wg signals in hyperplastic overgrowths in Drosophila might have some general significance as their vertebrate homologues, BMP/TGFβ and Wnt, are known to be involved in the generation of tumours in mammals. Moreover, inappropriate function of the JNK pathway is also connected with tumour formation in vertebrates. It is speculated that situations similar to those described in this study might also occur in mammalian cells in which caspase activity is blocked, by virus infections or other causes. This could result in continuous activation of the JNK pathway and, subsequently, of BMP/TGFβ and Wnt, and could eventually produce a tumour (Pérez-Garijo, 2009).

Fat and Wingless signaling oppositely regulate epithelial cell-cell adhesion and distal wing development

Development of organ-specific size and shape demands tight coordination between tissue growth and cell-cell adhesion. Dynamic regulation of cell adhesion proteins thus plays an important role during organogenesis. In Drosophila, the homophilic cell adhesion protein DE-Cadherin regulates epithelial cell-cell adhesion at adherens junctions (AJs). This study shows that along the proximodistal (PD) axis of the developing wing epithelium, apical cell shapes and expression of DE-Cad are graded in response to Wingless, a morphogen secreted from the dorsoventral (DV) organizer in distal wing, suggesting a PD gradient of cell-cell adhesion. The Fat (Ft) tumor suppressor, by contrast, represses DE-Cad expression. In genetic tests, ft behaves as a suppressor of Wg signaling. Cytoplasmic pool of ß-catenin/Arm, the intracellular transducer of Wg signaling, is negatively correlated with the activity of Ft. Moreover, unlike that of Wg, signaling by Ft negatively regulates the expression of Distalless (Dll) and Vestigial (Vg). Finally, Ft is shown to intersect Wnt/Wg signaling, downstream of the Wg ligand. Fat and Wg signaling thus exert opposing regulation to coordinate cell-cell adhesion and patterning along the PD axis of Drosophila wing (Jaiswal, 2006).

Cells of the dorsoventral (DV) boundary in the wing imaginal disc synthesize Wg. The DV boundary marks the distal end of the growing appendage, while the future hinge region, displaying Wg expression in two concentric rings, marks the proximal wing. The lacZ reporter of the quadrant enhancer of vestigial (vg), Q-vg-lacZ marks the entire distal wing [i.e. the presumptive wing blade (pouch) (Jaiswal, 2006).

In optical sections of the imaginal disc epithelium, AJs are visualized in the XY or XZ planes based on immunolocalization of DE-Cad and ß-catenin/Arm, besides binding with fluorochrome conjugated Phalloidin to F-actin. Both ß-catenin/Arm and DE-Cad display characteristic upregulation across the DV boundary along the PD axis of the wing imaginal disc. Optical sections along the XY plane reveal higher levels of DE-Cad localization and narrower apical circumferences in the AJs of cells flanking the DV boundary when compared with those of the more proximally located cells. Optical sections along the XZ plane further confirmed upregulation of DE-Cad. Thus, along the PD axis of the wing disc, cell shapes and DE-Cad levels are graded (Jaiswal, 2006).

Whether the PD gradient of cell shape and DE-Cad levels are linked to Wg signaling was tested. Somatic clones displaying constitutive Wg signaling (induced by overexpression of Dsh or of a degradation resistant variant of ß-catenin/Arm, ArmS10) induce cell-autonomous upregulation in the levels of DE-Cad and apical cell constrictions. Somatic clones expressing secreted Wg, however, are expected to induce non-cell-autonomous effects. Indeed, these clones induced non-cell autonomous and graded upregulation in the levels of DE-Cad in the AJs and changes in apical cell shapes. In the presumptive hinge region, Wg overexpression produces a more striking pattern of non-cell autonomous changes in cell shapes: cells neighboring the Wg-expressing cells appear to organize as whorls around the former and display epithelial misfolding (Jaiswal, 2006).

Furthermore, expression of GPI-anchored DFz2 receptor GPI-DFz2, which compromises Wg signaling, obliterates the characteristic PD gradient in the levels of DE-Cad and F-actin in the AJs. Finally, loss of Wg expression in the DV boundary of wing imaginal disc of Nts mutants grown at a restricted temperature also abolishes the PD gradient of DE-Cad and apical cell shapes. To further test if apical cell constrictions are linked to elevated levels of DE-Cad in AJs, DE-Cad was expressed in somatic clones. These clones were apically constricted, consistent with the role of DE-Cad/E-Cad in remodeling cell shape and tissue architecture. These results thus link Wg signaling to the PD gradient in the levels of DE-Cad and apical cell shapes in the wing imaginal discs (Jaiswal, 2006).

Somatic clones with altered cell-cell adhesion sort out from their neighbors and display smooth clone borders. Indeed, somatic clones displaying gain of Wg signaling owing to Dsh or ArmS10 misexpression sort out from their neighbors and display smooth clone borders, akin to those misexpressing DE-Cad. Wg signaling may alter cell-cell adhesion by enhancing recruitment of ß-catenin/Arm to the AJs and/or by its transcriptional input. In many cell types, for example, expression of cadherins rather than the levels of catenins appears to be the rate-limiting step of Catenin-Cadherin complex formation at AJs and cell-cell adhesion. Wild type ß-catenin/Arm (ArmS2), when overexpressed, does not transduce Wg signaling. Somatic clones overexpressing ArmS2 display 'wiggly' clone borders, unlike those expressing Dsh or ArmS10. Thus, expression of ß-catenin/Arm alone, without a concomitant enhancement of Wg signaling, fails to alter cell-cell adhesion. Cell-cell adhesion in wing imaginal disc epithelium is therefore likely to be regulated by transcriptional input from Wg signaling (Jaiswal, 2006).

To test if canonical Wg signaling regulates DE-Cad expression, the response of its lacZ reporter, DE-Cad-lacZ, was examined. Cells receiving high threshold of Wg signaling in the wing imaginal discs, as in those flanking the DV boundary, displayed higher levels of DE-Cad-lacZ reporter activity when compared with those further away from the source of Wg expression. Furthermore, somatic clones expressing ArmS10 or Dsh display cell-autonomous activation of the DE-Cad-lacZ. Finally, clones expressing the secreted Wg induce non-cell-autonomous activation of DE-Cad-lacZ: i.e., in cells within and surrounding the clones. Together, these results suggest that regulation of DE-Cad by the long-range activity of the Wg morphogen sets up the PD gradient of cell-cell adhesion and cell shape in the distal wing (Jaiswal, 2006).

Somatic clones lacking Ft (ft-/ft-), marked by loss of GFP, display overgrowth and altered cell-cell adhesion with characteristic circular and smooth clone borders, unlike the 'wiggly' borders of their wild type (ft+/ft+) twins that are marked by brighter GFP. Furthermore, cells lacking Ft displayed upregulation of DE-Cad in their AJs and DE-Cad-lacZ. By contrast, when Ft was overexpressed, levels of both DE-Cad or DE-Cad-lacZ were downregulated. Besides, following overexpression of Ft in the posterior wing compartment, cells flanking the DV boundary displayed wider apical circumferences when compared with those of the anterior wing compartment. These results suggest that Ft regulates DE-Cad expression, cell-cell adhesion and apical cell shapes in the distal wing (Jaiswal, 2006).

The results suggest that by regulating DE-Cad expression, Wg signaling integrates cell-cell adhesion with tissue growth and pattern. Regulation of DE-Cad expression could be a prevalent mechanism for coordination of the emerging pattern in an organ primordium with the spatial control of its cell-cell adhesion. For example, DE-Cad levels are also upregulated in cells flanking the stripe of cells along the AP boundary that express the morphogen Decapentaplegic (Dpp); misregulation of Dpp signaling also affects DE-Cad expression. The Ft tumor suppressor, by contrast, negatively regulates DE-Cad expression in the distal wing. This may also explain the inverse correlation between the levels of DE-Cad in AJs and the activity of Ft. Thus, besides its heterophilic binding with Ds, Ft controls cell-cell adhesions at AJs by regulating DE-Cad expression (Jaiswal, 2006).

Apart from cell-cell adhesion, DE/E-Cad regulation may impact a variety of other cellular processes and developmental mechanisms. E-Cad has been shown to mark the sites of actin assembly on cell surface. Cadherin complexes regulate cytoskeletal networks and cell polarity, while disruption of AJ associated components affects asymmetric cell division. Fat1, a mammalian homolog of Drosophila Ft, modulates actin dynamics. Interestingly, Ft also regulates orientated cell division (OCD) in imaginal epithelium, which is mirrored by orientation of the spindles of the dividing cells; OCD may also regulate organ shape along the PD axis. Misregulation of DE-Cad may thus affect the cytoskeleton and produce OCD phenotype in ft mutant discs (Jaiswal, 2006).

In both loss- and gain-of-function assays, this study shows that Ft downregulates Dll and Vg/Q-vg-lacZ in the distal wing. Although Vg/Q-vg-lacZ and Dll have not been ascertained to be the direct targets of Wg, all available evidence so far suggests that these targets positively respond to Wg signaling. These results also show that Ft and Wg signaling intersect and control distal wing growth and pattern, presumably through their opposing regulation of a common set of targets, namely, DE-Cad, Vg and Dll. Apart from Wg signaling, Dpp signaling also regulates Q-vg-lacZ; however, its long-range target, Omb is not upregulated in ft mutant clones, suggesting that regulation of distal wing targets by Ft is mediated by its intersection with Wg signaling (Jaiswal, 2006).

The results show that Ft negatively regulates Wg signaling. Loss or gain of Ft induces a telltale sign of perturbations in Wg signaling, namely, changes in the cellular pool of ß-catenin/Arm, consistent with its role as a suppressor of Wg signaling in genetic tests. The results further reveal intersection of Ft with Wg signaling downstream of the Wg ligand, while with respect to its receptor, Ft is likely to act either upstream of or parallel to Fz/Fz2. It is interesting to note here that the role of Ft in PCP regulation has also been suggested to be either parallel to or upstream of the Fz receptor. It is also noted that Ft co-localizes with neither Fz nor Fz2 and does not mediate their subcellular localization, thereby suggesting that Ft interacts with Fz indirectly. Unraveling the genetic and molecular basis of this interaction may explain how Ft straddles both the canonical (growth and cell-cell adhesion) and non-canonical (PCP) Wnt signaling pathways (Jaiswal, 2006).

One of the remarkable aspects of development of an organ primordium is that a stereotypic PCP is achieved even while it passes through dynamic changes in its size and shape. The fact that changing organ sizes/shapes does not alter PCP suggests an in-built mechanism to regulate constancy of PCP during animal development. A link between PCP and growth through the activity of Ft has been speculated, since it regulates both. Intersection of Ft and the canonical Wg signaling seen here might provide a mechanism to coordinate PCP and organ growth (Jaiswal, 2006).

Drosophila wing growth is under dynamic spatial and temporal regulation by Wg signaling. Furthermore, different thresholds of Wg signaling impact cell proliferation in their characteristic ways and activate distinct sets of PD markers. Although at a very high threshold, Wg signaling inhibits cell proliferation, at a modest threshold it has been shown to stimulate growth. It is noted that loss of Ft fails to activate Wg targets that demand a high threshold of Wg signaling, e.g., Ac, which is required for wing margin specific bristle development. Conversely, overexpression of Ft also does not lead to loss of margin bristles, suggesting that it is not a strong repressor of Wg signaling either. The short-range Wg target, fz3-lacZ, which responds to a high threshold of Wg signaling, is also not upregulated by loss of Ft. Dll responds to a higher threshold of Wg signaling than that required for Vg/Q-vg. Dll and Vg display modest and strong upregulation respectively, following loss of Ft. These results suggest that loss of Ft upregulates Wg signaling to only modest thresholds, consistent with the growth-promoting effect of the latter (Jaiswal, 2006).

Over-proliferation in ft mutant imaginal discs is induced by perturbation of as yet unidentified disc-intrinsic mechanisms that determine the discs' characteristic final sizes. The imaginal discs of ft mutants continue to grow and the extent of their over-proliferation appears to be constrained only by the developmental time available during the extended periods of their larval life. By contrast, growth in wild-type imaginal discs is determinate, which ceases after they attain their predetermined sizes even under conditions of unlimited developmental time; for example, on transplantation into wild-type adult host abdomen that can sustain development. ft mutant imaginal discs thus acquire unlimited proliferative potential, akin to immortalization, a crucial step during tumorigenesis. It is significant that the Ft tumor suppressor downregulates Wg/Wnt signaling, a pathway implicated in cancers. Several orthologs of Ft have been identified in vertebrates with diverse functions. It will thus be interesting to explore if these orthologs of Ft in higher vertebrates also interact with Wnt signaling and thereby behave as tumor suppressors (Jaiswal, 2006).

Wingless and synapse differentiation

At vertebrate neuromuscular junctions (NMJs), Agrin plays pivotal roles in synapse development, but molecules that activate synapse formation at central synapses are largely unknown. Members of the Wnt family are well established as morphogens, yet recently they have also been implicated in synapse maturation. Wingless is essential for synapse development. Wg and its receptor are expressed at glutamatergic NMJs, and Wg is secreted by synaptic boutons. Loss of Wg leads to dramatic reductions in target-dependent synapse formation, and new boutons either fail to develop active zones and postsynaptic specializations or these are strikingly aberrant. It is suggested that Wg signals the coordinated development of pre- and post-synaptic compartments (Packard, 2002).

Wg immunoreactivity is strongest at glutamatergic type Ib boutons from the earliest stages of larval development, but is absent, or at very low levels at type Is, II, and III boutons. Comparison of Wg immunoreactivity with presynaptic markers [including anti-HRP, which stains the presynaptic membrane, and vesicle markers, including Cysteine string protein (CSP) and Synapsin] revealed that Wg was localized both presynaptically and postsynaptically. Postsynaptic localization of Wg was observed around synaptic boutons; the postsynaptic region being defined as the NMJ region that fails to colocalize with any presynaptic marker. Postsynaptic Wg localization was also confirmed by double labeling anti-DLG and anti-Wg. DLG is both pre- and post-synaptic, but is particularly enriched at the muscle postsynaptic membrane complex (subsynaptic reticulum-SSR), a system of folded membranes that appears continuous with the plasma membrane (Packard, 2002).

Wg is likely to interact with DFz2, which is clustered around synaptic boutons, either postsynaptically or both pre- and postsynaptically. Studies with shi mutants also suggest that secreted Wg is likely to be endocytosed by muscles. Under low Wg levels, synapse formation is severely impaired, with many boutons lacking active zones and post-synaptic structures. In those mutant boutons that develop active zones, release sites are abnormally shaped and the postsynaptic apparatus is markedly abnormal. It is suggested that Wg provides an essential signal for active zone development, and that this signal is critical for proper development of the postsynaptic apparatus. Two models are proposed by which Wg may signal synaptic development. Secretion of Wg and its interaction with pre- and post-synaptic DFz2 may initiate a cascade of events that signals both pre- and postsynaptic differentiation. Alternatively, Wg secretion initiates a postsynaptic response, which elicits a retrograde signal required for proper formation of active zones and new synaptic boutons. It is further proposed that endocytosis of Wg by muscles might regulate Wg concentration at the synapse. The presence of DFz2 in motorneurons suggests that the receptor is present pre- as well as post-synaptically and lends support to the first model (Packard, 2002).

Several lines of evidence support these models. Mutations blocking Wg secretion dramatically reduce postsynaptic accumulation of Wg. In the absence of Shi function, postsynaptic Wg is dramatically increased and this can be mimicked by selectively blocking postsynaptic Shi. The physiological significance of Wg endocytosis might be to terminate the Wg signaling cascade by receptor-mediated endocytosis. Interestingly, shi mutant synapses were shown to contain abnormally large pockets apposed to active zones. The presence of these pockets in wg mutant boutons of intermediate severity, suggests that endocytosis might be required for normal development of the postsynaptic apparatus. Dynamin's role in endocytosis has been extensively documented. A recent report, nonetheless, provides evidence that in disc epithelium Shibire may be required for Wg secretion. However, the current results significantly differ from this observation, in that in the wing disc, mutations in porc had the same effect as mutations in shi, confining Wg immunoreactivity to Wg producing cells. In contrast, mutations were found in porc that prevented postsynaptic Wg accumulation, while mutations in shi enhance postsynaptic accumulation (Packard, 2002).

Additional support for Wg being secreted by the boutons emerged in studies with Wg overexpression. Overexpressing Wg at type I boutons, but not within muscle cells, results in a substantial increase in postsynaptic Wg at the NMJ. Further, driving GFP-tagged Wg, using a strain containing the wg promoter fused to Gal4, results in postsynaptic GFP, even though the wg-Gal4 strain does not show reporter gene expression in muscle cells (Packard, 2002).

These studies show that Wg has a dramatic influence on synaptic bouton formation and on determination of bouton structure. At the light level, mutations in wg result in a drastic reduction in target-dependent synapse formation, and in an increase in the proportion of boutons associated with a dynamic cytoskeleton. Muscle dependent NMJ expansion has been widely documented in Drosophila and precise correlation between muscle growth and synaptic bouton number suggests the presence of a complex signaling mechanism that couples synapse formation to muscle growth. This work provides compelling evidence that Wg is involved in this process. The results also show that the influence of Wg on synapse formation has a degree of specificity for different neurons, since alterations in Wg levels affect only the glutamatergic type I endings (Packard, 2002).

The Wg pathway, through inactivation of GSK-3ß, has been implicated in the regulation of microtubules in axons. In these studies, it was found that the organization of Futsch, a MAP1B-related protein, is altered at growing regions of wg mutant NMJs. While direct phosphorylation of Futsch by GSK-3ß has not yet been demonstrated, GSK-3ß is enriched at presynaptic boutons, and alteration in Wg levels leads to alterations of the microtubular network. Based on previous observations of growth cones and of Drosophila mutants that alter NMJ expansion, these results are consistent with the idea that the defect in NMJ expansion in wg mutants is not the result of a premature stabilization of the arbor. In wg mutants, the synaptic cytoskeleton is undergoing dynamic changes, but factors required to promote formation of the entire bouton are lacking. This view is supported by the finding that a significant proportion of boutons that did develop under decreased Wg levels, lacked essential synaptic elements such as active zones and mitochondria, and had abnormal morphology. Thus, the cytoskeletal changes underlying NMJ expansion are probably necessary, but not sufficient to ensure synaptic growth (Packard, 2002).

These results are consistent with studies that show that activation of a Wnt-7a-dependent pathway results in an increase in growth cone complexity and in changes in microtubule bundling. wnt-7a knockout mice have a delay in the development of multisynaptic glomerular rosettes in the cerebellum and in the accumulation of Synapsin. An apparent discrepancy is that, in the cerebellum, it is the postsynaptic cell that secrets Wnt-7, and which retrogradely affects the development of the presynaptic region. It would be interesting to determine whether different Wnt isoforms may operate in different cells (eg: pre-versus postsynaptic cells) to establish synapse morphology, or whether the difference with this study simply reflects the divergence of the two tissues examined (Packard, 2002).

While many of the molecular components of the active zone have been identified, the mechanisms that trigger their differentiation are unknown. One of the most remarkable phenotypes encountered in wgts mutants was the lack of active zones in about a third of the boutons examined. This result points to Wg as a pivotal factor during presynaptic differentiation. Interestingly, boutons lacking active zones are also devoid of postsynaptic specializations, suggesting that active zones might be required for postsynaptic development. This argument is supported by the finding that in most mutant boutons that developed active zones, these active zones are abnormal, and these boutons develop aberrant postsynaptic specializations. This model is also supported by studies showing that active zone formation precedes SSR development. Mutations in the Drosophila Rho-type guanine nucleotide exchange factor dPix also led to a severe reduction of the SSR. However, active zones are still present in dpix mutants and minimal SSR persists. In contrast, mutations in kakapo also display active sites that are lacking T bars, but in these mutants the structured electron dense apposition of pre- and post-synaptic membranes remains intact. In Drosophila, active zones can form in the absence of target muscles, suggesting that the absence of active zones in wg mutants is not due to defective muscles (Packard, 2002).

Recent studies point to a role for bone morphogenetic proteins (BMP) in synapse development. Drosophila mutants lacking wishful thinking (wit), a type II BMP receptor, like mutations in wg, display reduced bouton number. In addition, although active zones do form in wit mutants, many have an abnormal morphology. At the NMJ, wit is localized and appears to function in the presynaptic cell, raising the intriguing possibility that the ligand is secreted by the postsynaptic cell. Thus, as in the wing disc, Wg and Dpp-like ligands may collaborate in the coordinated development of pre- and post-synaptic structures (Packard, 2002).

A subset of boutons in wg mutants appears to have normal active zones, but the morphology of the postsynaptic membrane directly juxtaposed to active zones is dramatically altered. Strikingly, synaptic proteins, such as Dlg and DGluRIIA, are abnormally localized in wg mutants, and this phenotype can be replicated by mutations in porc or by postsynaptic overexpression of either DFz2 or DFz2DN. The aberrant distribution of DGluRIIA and Dlg observed in wg mutants may be the consequence of grossly abnormal postsynaptic structure. Alternatively, the diffuse appearance of glutamate receptors may point to a direct role of Wg in glutamate receptor clustering (Packard, 2002).

Taken together, these results are consistent with a model by which Wg is required for the formation of active zones, and that active zone formation is essential for proper development of the postsynaptic apparatus. In this context, Wg may serve a function similar to Agrin at the vertebrate NMJ, in which an extracellular matrix associated protein secreted by the presynaptic cell signals postsynaptic differentiation. Remarkably, mutant mice lacking Agrin have widespread abnormalities in the presynaptic terminal, and blocking Agrin function by antibodies blocks presynaptic differentiation, providing compelling evidence that Agrin is also required for presynaptic development. An Agrin homolog does not appear to be present in the Drosophila genome, suggesting that a different set of secreted proteins, such as Wg, may subserve Agrin's function (Packard, 2002).

Erect wing regulates synaptic growth in Drosophila by integration of multiple signaling pathways

Formation of synaptic connections is a dynamic and highly regulated process. Little is known about the gene networks that regulate synaptic growth and how they balance stimulatory and restrictive signals. This study shows that the neuronally expressed transcription factor gene erect wing (ewg) is a major target of the RNA binding protein ELAV and that EWG restricts synaptic growth at neuromuscular junctions. Using a functional genomics approach it was demonstrated that EWG acts primarily through increasing mRNA levels of genes involved in transcriptional and post-transcriptional regulation of gene expression, while genes at the end of the regulatory expression hierarchy (effector genes) represent only a minor portion, indicating an extensive regulatory network. Among EWG-regulated genes are components of Wingless and Notch signaling pathways. In a clonal analysis it was demonstrated that EWG genetically interacts with Wingless and Notch, and also with TGF-β and AP-1 pathways in the regulation of synaptic growth. These results show that EWG restricts synaptic growth by integrating multiple cellular signaling pathways into an extensive regulatory gene expression network (Haussmann, 2008).

Several pathways have been identified that stimulate synaptic growth at NMJs of Drosophila larvae (Wnt/Wingless, TGF-β/BMP and jun kinase). Overexpression of AP-1 and mutants in regulatory genes involved in Wnt/Wingless and TGF-β/BMP pathways (spinster, highwire, shaggy and the proteasome) can increase bouton numbers, suggesting that synaptic growth is regulated through the balance of stimulatory and restrictive signals. This study has identified such a restrictive role for the transcription factor EWG and, through the analysis of EWG-regulated genes, for the N pathway in the regulation of synaptic growth. Using genetic mosaics, it was further demonstrated that EWG's role in synaptic growth regulation is cell-autonomous, suggesting that the transcriptional regulator EWG mediates this restrictive effect through the alteration of transcription pre-synaptically (Haussmann, 2008).

Analysis of genes differentially expressed in ewgl1mutants revealed a rather unexpected set of genes involved in synaptic growth regulation, besides an expected number of metabolic genes due to homology of EWG to human NRF-1. Most genes that could account for the phenotype of ewgl1mutants, and that are thus expressed in the nervous system, are involved in transcriptional and post-transcriptional regulation of gene expression. Although changes of transcript levels in ewgl1mutants were mostly moderate, their significance was validated through mRNA profiling with rescued ewgl1mutants under the same conditions of RNA preparation and microarray hybridization. In addition, differences in gene expression in ewgl1mutants were validated using quantitative RT-PCR and biochemical assays with regard to predicted changes in glycogen levels based on differential regulation of genes involved in gluconeogenesis. Furthermore, genetic interaction experiments in double mutants with increased bouton numbers support that these co-regulated genes are functionally connected in regulating synaptic growth (Haussmann, 2008).

The group of neuronal genes among those differentially regulated in ewgl1mutants that have been demonstrated to have roles in synaptic growth or could account for it, is remarkably small. In particular, from the large number of cell adhesion molecules and cytoskeletal proteins present in the Drosophila genome only a handful is differentially regulated. Similar results have also been obtained in response to JNK and AP-1 signaling. These results are in contrast to changes in gene expression induced by acute or chronically enhanced neuronal activity in Drosophila seizure mutants, which also result in synaptic overgrowth. Here, the vast majority of differentially regulated genes are for cell adhesion molecules and cytoskeletal proteins or their regulators, and genes involved in synaptic transmission and neuronal excitability; transcriptional or post-transcriptional regulators comprise only a minor portion. These differences could be explained by separate pathways regulating growth independent of neuronal activity (Haussmann, 2008).

Particularly striking is the large number of genes involved in RNA processing among genes differentially expressed in ewgl1mutants. Although local regulation of gene expression is required in growth cones of navigating axons, a prominent role for pre-synaptic regulation of gene expression at the RNA level is only just emerging, but is a hallmark of post-synaptic plasticity. Several RNA binding proteins have been implicated in memory storage . osk and CPSF (cleavage and polyadenylation specificity factor) are among the genes differentially regulated in ewgl1mutants. Other genes involved in RNA processing differentially regulated in ewgl1mutants comprise the whole spectrum of regulation at the post-transcriptional level, from nuclear organization (otefin), alternative pre-mRNA processing (Pinin, CPSF, Rox8) and export/import (Segregetion distorter, Nxf2, CG11092, Karyopherin, Transportin) to transport, localization and translation (oskar, swallow, ribosomal protein genes S5 and Rpl24), and likely also include the regulation of mRNA stability (Rox8) (Haussmann, 2008).

An intriguing connection between ewg and signaling pathways involved in regulating synaptic growth is indicated by differentially regulated components of the Wg and N pathways (gro and Hairless) in ewgl1mutants. Consistent with a role of the co-repressor gro in Wg and N mediated transcriptional regulation of synaptic growth, Wg and N signaling pathways do not operate independently of ewg in genetic interaction experiments. The transcriptional regulatory networks of EWG, Wg and N seem to be highly interwoven. Overexpression of pan, the transcriptional mediator of canonical Wg signaling, which is repressed by gro, does not lead to a further expansion of synaptic growth in ewg mutants, suggesting a requirement for ewg-regulated genes. This effect could be mediated by deregulated N signaling, which is also repressed by gro, but antagonistic to Wg in synaptic growth. Thus, removal of gro, as in ewg, will relieve the repressive effect of N and antagonize the stimulatory effect of pan. In the complementary situation, removal of N increased bouton numbers further in the absence of EWG, which is consistent with an increase in Wg signaling as a result of down-regulated gro in ewg mutants. Antagonism between N and Wg pathways has also been found in wing discs, where N inhibits armadillo (arm), the transcriptional co-activator of canonical Wg signaling. Intriguingly, gro has also been found to be a target of receptor tyrosine kinase signaling and, thus, can combine additional pathways with N and Wg signaling. In addition to transcriptional hierarchies, chromatin remodeling has also been implicated in synaptic plasticity. Strikingly, CG6297, a Drosophila homologue of the histone deacetylase RPD3, is differentially expressed in ewgl1mutants and physically interacts with gro (Haussmann, 2008).

How ewg exerts its effect on TGF-β signaling is less clear. A prominent regulatory step in this pathway is the regulated degradation of the SMAD co-factor Medea by Highwire. Several genes involved in regulating protein stability are differentially down-regulated in ewg mutants (CG6759, CG3431, CG4973, CG7288, CG3455, CG9327 and CG9556). Lower expression levels of these genes might interfere with stabilization of Medea and explain why the effect of activated TGF-β signaling is not additive in the absence of EWG (tkvA GOF ewg LOF). Bouton numbers in wit null mutants are marginally increased in the absence of EWG, suggesting further that genes regulated by SMADs are involved in mediating synaptic overgrowth in ewgl1mutants. Potentially, ewg could also regulate TGF-β signaling through the endosomal pathway involving spinster and/or spichthyin (Haussmann, 2008).

Functionally related genes have been shown to be co-regulated, suggesting additional ELAV targets in EWG-regulated gene networks. Indeed, ELAV negatively regulates alternative splicing of the penultimate exon in armadillo (arm). Exclusion of this exon, which truncates the carboxyl terminus of arm, reduces Wg signal transduction, which is in agreement with ewg's antagonistic role relative to Wg signaling. Another known ELAV target gene is neuroglian (nrg), where a role in synapse formation has recently been demonstrated in the giant fiber system. Taken together, the establishment of a gene network regulated by EWG will now serve as valuable tool to identify further ELAV regulated modules that shape the synapse (Haussmann, 2008).

The transcription factor EWG is a major target of the RNA binding protein ELAV, which regulates EWG protein expression via a splicing mechanism. EWG is required pre-synaptically and cell-autonomously at third instar neuromuscular junctions to restrict synaptic growth, demonstrating that restrictive activities at gene expression levels are also required for synaptic growth regulation. EWG mediates regulation of synaptic growth primarily by increasing transcript levels of genes involved in transcriptional and post-transcriptional regulation of gene expression. Genes at the end of the gene expression hierarchy (effector genes) represent only a minor portion of EWG-regulated genes. Since analysis of mutants in genes differentially regulated in ewgl1mutants revealed that these genes are involved in both stimulatory and restrictive pathways of synaptic growth, and since ewg genetically interacts with a number of signaling pathways (Wingless, Notch, TGF-β and AP-1), the results suggest that synaptic growth in Drosophila is regulated by the interplay of multiple signaling pathways rather than through independent pathways (Haussmann, 2008).

Dendritic refinement of an identified neuron in the Drosophila CNS is regulated by neuronal activity and Wnt signaling

The dendrites of neurons undergo dramatic reorganization in response to developmental and other cues, such as stress and hormones. Although their morphogenesis is an active area of research, there are few neuron preparations that allow the mechanistic study of how dendritic fields are established in central neurons. Dendritic refinement is a key final step of neuronal circuit formation and is closely linked to emergence of function. This is a study of a central serotonergic neuron in the Drosophila brain, the dendrites of which undergo a dramatic morphological change during metamorphosis. Using tools to manipulate gene expression in this neuron, the refinement of dendrites during pupal life was examined. This study shows that the final pattern emerges after an initial growth phase, in which the dendrites function as 'detectors', sensing inputs received by the cell. Consistent with this, reducing excitability of the cell through hyperpolarization by expression of K(ir)2.1 results in increased dendritic length. Sensory input, possibly acting through NMDA receptors, is necessary for dendritic refinement. These results indicate that activity triggers Wnt signaling, which plays a 'pro-retraction' role in sculpting the dendritic field: in the absence of sensory input, dendritic arbors do not retract, a phenotype that can be rescued by activating Wnt signaling. These findings integrate sensory activity, NMDA receptors and Wingless/Wnt5 signaling pathways to advance understanding of how dendritic refinement is established. The maturation of sensory function is shown to interact with broadly distributed signaling molecules, resulting in their localized action in the refinement of dendritic arbors (Singh, 2010).

This study focuses on a specific phase during the metamorphosis of the dendrites of a central serotonergic neuron, in which excess growth is removed by a process that has been termed refinement. Genetic analyses using loss-of-function mutants and RNAi-mediated knockdown of specific genes has led to a postulated a link between neuronal activity, synaptic input and Wnt signaling in this process. The sparse dendrites innervating the adult antennal lobe, present on the wide-field serotonergic neurons (CSDn) during the larval stage, are removed early in pupation by pruning, followed by a period of exuberant growth. The arrival of sensory neurons at the antennal lobe correlates well with when growth of the CSDn dendrites ceases and removal of the excess branches occurs. The CSDn must be active for the refinement process to occur, as refinement fails when neuronal activity is inhibited or when the sensory neurons are absent. Phenotypes observed in the latter case can be rescued by ectopic activation of the neuron using the temperature-sensitive dTrp-A1 channel. It is suggested that activity within the CSDn, possibly together with activity in presynaptic neurons, acts to provide the correlated activity required to trigger activation of NMDARs. Knockdown of NMDARs affects the refinement process, although identifying its specific action requires further study. A possible consequence of the activity-dependent process is activation of the Wg pathway, as the phenotype observed in aristalless mutants can also be rescued by ectopic expression of Dishevelled (Dsh) in the CSDn. It seems unlikely that activity within the CSDn leads to the release of Wnt ligands, but rather that dendrites respond locally to Wnt ligands in the region of a dendrite that is receiving input. Although other interpretations of the data are possible, a hypothesis is favored whereby specific synapses are stabilized as a result of correlated neuronal activity, and that excess dendritic branches are removed by Wnt signaling (Singh, 2010).

Perturbations in neuronal activity can be compensated by changes at multiple levels, including alterations in the expression of ion channels and in synaptic strength. Tripodi (2008) provides evidence for structural homeostasis whereby alterations in afferent input during development can be compensated by changes in dendritic geometry. This suggests that dendritic arbors serve as sensors for input levels, thus allowing the self-organization of circuits that is necessary for robust behavioral outputs (Tripodi, 2008). The current studies in the CSDn support these observations: reduced activation of the cell by targeted expression of Kir2.1 results in a greatly enlarged dendritic field in the adult. This phenotype can be explained by a mechanism in which the absence of electrical activity results in a failure of the signaling mechanisms that stop growth of the arbors and that remove additional branches. Reduced excitability could also drive the homeostatic mechanisms towards making more arbors and to suppress the refinement program (Singh, 2010).

Dendritic growth and refinement are closely associated with input activity and synapse formation during development. Activity-dependent development of circuits is thought to utilize mechanisms similar to those involved in Hebbian learning and plasticity. NMDARs are ideal candidates for detecting correlated pre- and postsynaptic activity, which is crucial in the Hebbian model of learning and plasticity. Strengthening of synapses, as in this study, leads to the stabilization and extension of dendrites, whereas weakening of synapses leads to the destabilization and elimination of dendritic branches (Espinosa, 2009; Cline, 2008; Constantine-Paton, 1998). During vertebrate hippocampal development, NMDAR activation has been shown to limit synapse number and reduce dendritic complexity. The stabilization of a particular synapse or arbor possibly attenuates the formation of new branches or synapses, thus limiting further dendritic growth. In such a scenario, knocking down NMDAR levels would be expected to result in increased dendritic complexity, as indeed has been observed in this study. The mechanism by which 'appropriately connected' synapses are strengthened, whereas suboptimal contacts are eliminated, needs to be studied in thus system. In other systems, Ca2+, which is released upon NMDAR activation, impinges on various intracellular effectors that regulate dendritic morphogenesis. In addition, selective stabilization/destabilization of dendritic arbors could be affected by the local release of growth factors in response to activity (Singh, 2010).

This study shows that activity-dependent activation of the Wnt pathway facilitates retraction of dendritic arbors. Arbors that receive appropriate input are somehow protected and stabilized. These experiments suggest that Wnt-dependent refinement functions through a non-nuclear pathway and could act by impinging directly on cytoskeletal dynamics (Schlessinger, 2009; Salinas, 2008). Disruption of the microtubule cytoskeleton is a key feature of dendritic pruning in Drosophila during metamorphosis. GSK3β (Shaggy in Drosophila) an intracellular inhibitor of the Wnt pathway, has been shown to act as a sensor of inputs for neuronal activity (Chiang, 2009) and a potent regulator of microtubule dynamics in axons. In the Drosophila embryonic CNS, the Src family of tyrosine kinases (SFKs) is required for Wnt5/Drl-mediated signaling. Interestingly, SFKs seem to act as a crucial point of convergence for multiple signaling pathways that enhance NMDAR activity and hence are thought to act as molecular hubs for the control of NMDARs. It is tempting to envisage a scenario in which there is cross-talk between Wnt5/Drl signaling-mediated activation of SFKs and NMDAR signaling during refinement (Singh, 2010).

In summary, this study shows that the dendritic refinement of a central modulatory serotonergic neuron is regulated by electrical activity, NMDAR and Wnt signaling. Similar mechanisms have been implicated in dendritic growth and refinement of excitatory neurons in vertebrates. This study provides a model neuron preparation in which the dendritic growth and refinement of a modulatory neuron can be analyzed genetically. It was demonstrated that the dendrites of CSDn receive input from sensory neurons from the arista, supporting previous suggestions that mechanosensory input could alter sensitivity to odorant stimuli. In both Drosophila (Dacks, 2009) and the mammalian olfactory bulb (Petzold, 2009), serotonin gates the odor-evoked sensory response. CSDn sends projections to higher brain centers and multiglomerular projections to the contralateral antennal lobe and hence it is likely to influence the overall properties of the olfactory circuit. This study suggests that the structural and resulting functional properties of this neuron emerge from an interaction between partner neurons, together with input from intrinsic and extrinsic cues (Singh, 2010).

Synaptic homeostasis is consolidated by the cell fate gene gooseberry, a Drosophila pax3/7 homolog

In a large-scale screening effort, the gene gooseberry (gsb) was identified as being necessary for synaptic homeostasis at the Drosophila neuromuscular junction. The gsb gene encodes a pair-rule transcription factor that participates in embryonic neuronal cell fate specification. This study defines a new postembryonic role for gooseberry. gsb becomes widely expressed in the postembryonic CNS, including within mature motoneurons. Loss of gsb does not alter neuromuscular growth, morphology, or the distribution of essential synaptic proteins. However, gsb function is required postembryonically for the sustained expression of synaptic homeostasis. In GluRIIA mutant animals, miniature EPSP (mEPSP) amplitudes are significantly decreased, and there is a compensatory homeostatic increase in presynaptic release that restores normal muscle excitation. Loss of gsb significantly impairs the homeostatic increase in presynaptic release in the GluRIIA mutant. Interestingly, gsb is not required for the rapid induction of synaptic homeostasis. Furthermore, gsb seems to be specifically involved in the mechanisms responsible for a homeostatic increase in presynaptic release, since it is not required for the homeostatic decrease in presynaptic release observed following an increase in mEPSP amplitude. Finally, Gsb has been shown to antagonize Wingless signaling during embryonic fate specification, and initial evidence is presented that this activity is conserved during synaptic homeostasis. Thus, gsb was identified as a gene that distinguishes between rapid induction versus sustained expression of synaptic homeostasis and distinguishes between the mechanisms responsible for homeostatic increase versus decrease in synaptic vesicle release (Marie, 2010).

This study has advanced understanding of synaptic homeostasis in several important ways. First, gsb was identified as required in postmitotic, postembryonic neurons for synaptic homeostasis at the Drosophila NMJ. Drosophila gsb is the homolog of vertebrate pax3/pax7. Thus, these data identify a new function for a conserved gene family that has been traditionally studied in the context of neuronal fate specification. Second, it was demonstrated that loss of gsb selectively disrupts the expression of synaptic homeostasis without impairing the rapid induction of synaptic homeostasis. These data suggest that genetically separable mechanisms exist for the induction versus the expression of synaptic homeostasis at the Drosophila NMJ. Third, it was demonstrated that loss of Gsb selectively disrupts the mechanisms responsible for a homeostatic increase in presynaptic release without impairing the mechanisms responsible for homeostatic decrease in presynaptic release. Therefore, these two forms of homeostatic modulation, both expressed at the Drosophila NMJ, appear to involve genetically separable mechanisms. Fourth, because gsb is a transcription factor, these data highlight the possibility that the persistent expression of synaptic homeostasis in the GluRIIA mutant is consolidated through transcription-/translation-dependent mechanisms, while the rapid induction of homeostatic signaling is independent of new protein synthesis. Thus, there may exist genetically separable phases of homeostatic signaling at the Drosophila NMJ analogous to the induction versus expression of long-term synaptic plasticity in other systems. Finally, evidence is presented to support the hypothesis that Gsb functions similarly during cell fate specification and synaptic homeostasis. According to the emerging model, Gsb may antagonize Wingless signaling in motoneurons to facilitate the consolidation of synaptic homeostasis. While there remains considerable work to prove this model, the data, in combination with prior work during embryonic cell fate specification, provide the basis for a compelling model that can be examined in greater detail in future studies (Marie, 2010).

In this study, motoneurons with decreased levels of Gsb are unable to express synaptic homeostasis in the background of a GluRIIA mutation. By contrast, the rapid, protein synthesis-independent induction of synaptic homeostasis following application of the glutamate receptor antagonist PhTx is normal. One possible explanation for this difference is that PhTx and the GluRIIA mutant cause different postsynaptic perturbations and initiate separate homeostatic signaling systems, only one of which is affected by loss of gsb. This seems unlikely, however, because previously published data indicate that PhTx primarily acts upon postsynaptic glutamate receptors including those that contain the GluRIIA receptor subunit. Furthermore, several mutations have been shown to block synaptic homeostasis both in the GluRIIA mutant and following PhTx application, demonstrating that these two perturbations share, at some level, a common molecular mechanism of homeostatic signaling. It is hypothesized, therefore, that loss of Gsb impairs a molecular process that is selectively involved in the sustained expression of synaptic homeostasis. This would be consistent with the sustained expression of synaptic homeostasis requiring new protein synthesis (Marie, 2010).

The possibility that Gsb participates specifically in the sustained expression or consolidation of synaptic homeostasis has interesting implications. In one model of homeostatic signaling, the GluRIIA mutation represents a persistent stress that induces a continuous, rapidly induced form of homeostatic compensation. In this model, the homeostatic modulation of presynaptic release is continually updated and never consolidated. An alternative model is that, once induced, the homeostatic modulation of presynaptic release is consolidated and maintained for prolonged periods of time. If this is the case, it should be possible to selectively disrupt the consolidation of synaptic homeostasis independently of the mechanisms of induction. Loss of Gsb appears to do just this. It is not possible to persistently inhibit protein synthesis during larval development. However, the demonstration that decreased Gsb disrupts synaptic homeostasis in the GluRIIA mutant suggests that transcription and translation may be involved in the mechanisms that consolidate a homeostatic change in presynaptic release (Marie, 2010).

In Drosophila, like in vertebrates, combinations of transcriptional regulators determine the fate of neurons. Indeed, transcription factors control all stages of early neuronal development and neuronal circuit formation, from the direction in which the axon initially extends from the neuronal cell body, the location of the terminal zone of the axonal arborization, and the specificity of synaptic targeting to the choice of neurotransmitter. More recently, some evidence suggests that expression of the transcription factor evenskipped during early embryogenesis could affect a motoneuron's complement of ion channels and neuron excitability. However very little is known regarding the role of transcriptional regulators in mature neurons. Recently, it was demonstrated that mild perturbation of the engrailed gene lead to mice with an adult phenotype that resembles key pathological features of Parkinson's disease. In this study, the expression of Gsb-RNAi using a postmitotic neuronal GAL4 driver leads to the conclusion that Gsb has a postmitotic activity that is essential to the maintenance of synaptic homeostasis. These data provide evidence that the transcription factors involved in embryonic development may have potent postembryonic functions that are necessary for the maintenance of stable neural function (Marie, 2010).

How do embryonic transcriptional regulators influence the expression of synaptic homeostasis? This study presents data to support a model in which Gsb function is conserved during embryonic patterning and synaptic homeostasis. Specifically, decreased wg levels rescue synaptic homeostasis in GluRIIA; gsb/+ double mutant animals. According to this model, Wg antagonizes the expression or consolidation of synaptic homeostasis, providing new insight into the activity of this potent intercellular, synaptic signaling molecule. It could be important, for example, to suppress homeostatic signaling during anatomical synaptic plasticity, a process in which Wg has been implicated. These data are strengthened by two observations. First, these data are supported by the well established embryonic activity of Gsb. Second, since partial loss of wg rescues the homeostatic defect in GluRIIA; gsb/+ double mutant animals, it is unlikely that this represents a nonspecific genetic interaction. Many additional experiments will be necessary to prove the function of wg as an antagonist of synaptic homeostasis. The data, however, take this model beyond the stage of pure speculation and suggest that this will be an important avenue of future experimental investigation (Marie, 2010).

Activity-dependent retrograde laminin A signaling regulates synapse growth at Drosophila neuromuscular junctions

Retrograde signals induced by synaptic activities are derived from postsynaptic cells to potentiate presynaptic properties, such as cytoskeletal dynamics, gene expression, and synaptic growth. However, it is not known whether activity-dependent retrograde signals can also depotentiate synaptic properties. This study shows that laminin A (LanA) functions as a retrograde signal to suppress synapse growth at Drosophila neuromuscular junctions (NMJs). The presynaptic integrin pathway consists of the integrin subunit βν and focal adhesion kinase 56 (Fak56), both of which are required to suppress crawling activity-dependent NMJ growth. LanA protein is localized in the synaptic cleft and only muscle-derived LanA is functional in modulating NMJ growth. The LanA level at NMJs is inversely correlated with NMJ size and regulated by larval crawling activity, synapse excitability, postsynaptic response, and anterograde Wnt/Wingless signaling, all of which modulate NMJ growth through LanA and βν. These data indicate that synaptic activities down-regulate levels of the retrograde signal LanA to promote NMJ growth (Tsai, 2012).

This study proposes a plasticity mechanism by which the synapse growth (or size) can be modulated by larva crawling and synaptic activities. These activities modulate LanA-integrin signaling that functions to constrain NMJ growth. This trans-synaptic signaling functions in a retrograde manner, which requires postsynaptic muscle-derived LanA and presynaptic integrin. The model suggests various activities modulate NMJ growth by regulating the LanA level and integrin signaling (Tsai, 2012).

Regulation of LanA levels at NMJs is the major mechanism underlying this synaptic structural plasticity. The LanA levels at NMJs are tightly coupled to several synaptic activities that are involved in synaptic structural plasticity at NMJs. Wg signaling in both pre- and postsynaptic compartments are shown to modify synaptic structure at Drosophila NMJs. The channel mutations para and eag Sh alter both synaptic potential and NMJ size. Finally, manipulation of postsynaptic responses by altering the GluRIIA and GluRIIB compositions also fine tunes synapse size and pFAK levels. Activities that promote NMJ growth also down-regulated LanA levels at NMJs. In contrast, NMJ growth suppression was accompanied with LanA accumulation, establishing an inverse correlation between the LanA level and the NMJ size. Importantly, manipulation of the gene dosage of LanA (or βν) could override these synaptic activities in NMJ growth regulation. This study also showed that LanA down-regulation at NMJs preceded synaptic structural remodeling induced by larval crawling, further supporting that LanA is a major mediator of these activities to modulate NMJ growth (Tsai, 2012).

Integrin signaling activities play important roles in synapse development and plasticity. In mammalian central synapses, various integrin subunits are important to transmit postsynaptic signaling in various plasticity models may function redundantly with βν to mediate integrin signaling. This study indicates a distinct presynaptic integrin pathway that is likely composed of βν and αPS3 (encoded by Volado), as suggested by their strong genetic interaction in NMJ growth. In response to postsynapse-secreted LanA signals, activation of the presynaptic integrin is transmitted through Fak56 activation. Interestingly, the signaling activity is rather local, limited by the range of LanA distribution, and shown by muscle 6-specific rescue, although this does not exclude the involvement of signaling to the nuclei of motor neurons. The presynaptic integrin/Fak56 signaling is in turn mediated by two downstream signaling activities. The activation of NF1/cAMP signaling, which suppressed NMJ overgrowth induced by crawling activity or βν mutation. The integrin/Fak56 pathway also suppresses Ras/MAPK signaling Tsai, 2008), as shown by diphospho-ERK (dpERK) accumulation and Fas2 reduction at NMJs in high crawling condition. These pathways have been shown to regulate cell adhesion and cytoskeletal organization, leading to the stabilization of synapses. The activity-dependent depletion of the LanA laminins in the synaptic cleft would allow the remodeling of synapses and further growth of NMJs (Tsai, 2012).

The activity-dependent structural plasticity is specific to the presynaptic integrin pathway. hiw mutants that show large NMJ size still retained the structural plasticity and constant pFAK levels at NMJs. Interestingly, LanA levels were increased in hiw mutants, in contrast to other NMJ overgrown mutants. Two nonmutually exclusive mechanisms can regulate activity-dependent LanA expressions at NMJs. First, within hours of activity induction, the LanA levels can be regulated at NMJs by putative ECM regulators such as matrix metalloproteinases. Second, transcription regulation of LanA can provide long-term changes of LanA levels at NMJs. Activity-triggered presynaptic Wg secretion promotes Wg receptor DFz2 activation on both post- and presynaptic compartments. The LanA level is regulated by the anterograde Wg signaling that is transduced through nuclear entry of the DFz2 intracellular domain and its transcription activity. However, LanA is unlikely to mediate all aspects of Wg signaling activity as overexpression of LanA in postsynapses suppressed ghost bouton formation, a hallmark in disrupting Wg signaling. Postsynaptic BMP/Gbb functions as a retrograde signal to activate presynaptic BMP type II receptor Wit in response to synaptic activity. With the lack of genetic interaction between BMP/Gbb and integrin signaling components, and constant levels of phosphorylated Mothers against dpp (pMad) in different crawling activities, it is proposed that both BMP/Gbb and LanA pathways can function in parallel by retrograde mechanisms to regulate NMJ growth (Tsai, 2012).

Expression in genital disc

Unlike the thoracic discs, the anterior and posterior compartmental organization of the genital imaginal disc is compound, consisting of three primordia ­ the female genital, male genital, and anal primordia. Each primordium is divided into anterior and posterior compartments. Genes that are known to be expressed in a compartment-specific manner in other discs (engrailed, hedgehog, patched, decapentaplegic, wingless and cubitus interruptus) are expressed in analogous patterns in each primordium of the genital disc. Specifically, engrailed and cubitus interruptus are expressed in complementary domains, while patched, decapentaplegic and wingless are expressed along the border between the two domains. en and inv are required in the posterior comparment of the genital disc to repress dpp and activate hh. Mitotic clones induced at the beginning of the second larval instar do not cross the boundary between the engrailed-expressing and cubitus interruptus-expressing domains, indicating that these domains are true genetic compartments (Chen, 1997).

Comparison of the wg expression pattern with the fate maps of the genital discs suggests that the wg expression domains in both the female and male genital discs correspond to the internal, but not the external, genitalia. wg mutation results in deletion of all internal structures in both males and females. In addition, the male external genitalia show deletions in certain structures, such as clasper teeth, the lateral plate bristles and the penis apparatus. The female external genitalia are generally unaffected (Chen, 1997).

The adult clonal phenotypes of protein kinase A and engrailed-invected mutants provide a more detailed map of the adult genitalia and analia with respect to the anterior/posterior compartmental subdivision. A new model has been proposed to describe the anterior and posterior compartmental organization of the genital disc. Each of the three primordia (female, male and anal) is composed of its own anterior and posterior compartments. Each primordium has a larger anterior compartment and a smaller posterior compartment. Each genital disc is divided into anterior and posterior compartment (Chen, 1997).

cAMP-dependent protein kinase A and engrailed-invected are genes known to play compartment-specific functions in other discs. The anterior/posterior patterning functions of these genes are conserved in the genital disc. en-inv mutant clones cause posterior to anterior transformations in adult terminalia. Pka is required to repress ptc, dpp and wg expression in the anterior compartment of the genital disc. Pka mutant clones result in pattern duplications in adult terminalia (Chen, 1997).

The genital disc consists of three primordia: moving from anterior to posterior they are the female genital primordium, the male genital primordium and the anal primordia. Only one of the two genital primordia develops, depending on the individual's sex, whereas the anal primordium develops in both sexes. It is proposed here that the genital disc, which is of ventral origin, is organized in a manner similar to the antennal and leg discs: the expression domains of decapentaplegic and wingless are mostly complementary and abut engrailed expression. An analysis was made of the roles of the genes hedgehog, patched, dpp and wg in the development of the three primordia that form the genital disc. The morphogenetic alterations produced by ectopic expression of hh mimic a lack of ptc function. Both genetic conditions cause derepression of dpp and wg. Ectopic expression of either of these genes causes non-autonomous duplications and/or reductions of genital and anal structures. Some of these alterations are explained by the mutual repression of wg and dpp. In the development of the genital disc, the functional relationships between these genes seem to be analogous to those described for leg and antennal discs: dpp and wg are induced in the anterior compartment by Hh protein blocking the repressive effect of Ptc, and the mutual repression of dpp and wg restrict one another to their respective domains. It may be concluded that dpp and wg act as general organizers for development of the genital disc (Sánchez, 1997).

The development of the genital primordia is based on two processes: cell proliferation and sexual differentiation. Cell proliferation refers to the capacity of each genital primordium to grow or to be kept in the repressed state. Sexual differentiation refers to the type of adult structure formed by each genital primordium. It is proposed that the control of cell proliferation in the male and female genitalia requires the concerted action of Abd-B and doublesex, either directly or indirectly, through the expression of the genes dpp and wingless. Thus, in female genital discs, the repressed male primordium does not express dpp whereas the repressed female primordium of the male genital discs expresses a reduced level of dpp. This reduced level seems to be insufficient to stimulate cell growth. In contrast, when strong dpp levels are obtained in the repressed female primordium of male discs, repressed female primordia overproliferate in mutants for patched or costal-2, as well as in the discs where uniform ectopic expreession of hedgehog is produced. The genes dpp and wg, however, do not participate in the sexual differentiation process, which depends on sexual cytodifferentiation genes. Thus the growth of repressed female primordia of the patched mutant male discs would give rise to no adult female genital structures since the genetic sex is male (Sánchez, 1997 and references).

Wingless and the structuring of the abdomen

The adult abdominal epidermis develops during the pupal stage from groups of cells called histoblast nests, which differ from imaginal discs in two important respects: (1) abdominal histoblasts do not invaginate during embryogenesis, but remain part of the larval epidermis and secrete larval cuticle, and (2) they do not proliferate during the larval stages. After pupariation, histoblasts multiply rapidly and migrate to replace the polyploid larval epidermal cells (LEC). As the individual nests grow and merge, LEC are destroyed only upon contact with histoblasts, so that the continuity of the pupal epidermis is maintained at all times. The replacement of LEC by histoblasts is completed by 40-42 hours after puparium formation (APF). The epidermis of each abdominal segment is produced by three bilateral pairs of histoblast nests: the anterior dorsal nests produce the tergite; the posterior dorsal nests form the flexible intertergal cuticle, and the ventral nests produce the sternite and pleura. In addition, a spiracular nest produces a small patch of epidermis around each spiracle. Dorsally, each segment is composed of a sclerotized, pigmented tergite and flexible, unpigmented intertergal cuticle that is normally folded underneath the tergite. All cells in the sternites, pleura and tergites secrete 3-4 trichomes per cell. The wide-based, curved trichomes secreted by pleural cells are distinct from the thinner, straighter sternal and tergal trichomes. In addition, sternites and tergites, but not the pleura, contain arrays of bristles. Dorsoventral patterning is also present within tergites, since the dark pigment band at the posterior edge of each tergite is wider medially than laterally. Some segments deviate from the typical pattern. For example, the first abdominal segment (A1) lacks a sternite. Also, in the male, A7 lacks both a sternite and a tergite, A6 lacks bristles on its sternite, and A5 and A6 have uniformly darkly pigmented tergites (Kopp, 1999 and references).

The adult abdominal epidermis is formed during the first 40-42 hours of pupal development. At pupariation, the abdominal epidermis is composed predominantly of the polyploid LEC, which are easily distinguishable from the much smaller, diploid histoblasts. At this stage, the anterior dorsal histoblast nest (aDHN) contains 13-19 cells; the posterior dorsal nest (pDHN) contains 5-8 cells, and the ventral nest (VHN) contains 9-13 cells. Histoblasts begin to proliferate and migrate to supplant the LEC soon after pupariation. At 18-20 hours APF, the aDHN and pDHN merge to form a single dorsal histoblast nest (DHN). The DHN merges with the VHN and the spiracular anlage between 20 and 28 hours APF. The spiracle, located at the lateral midline, marks the boundary between ventral and dorsal histoblasts, and eventually the boundary between the pleura and the tergite. The fusion of histoblast nests of consecutive segments begins at 28 hours APF and proceeds until 40-42 hours APF, when the formation of a continuous adult epidermis is completed by the fusion of contralateral nests at the dorsal and ventral midlines. Morphological differentiation of the epidermis into sternite, tergite and pleural territories becomes evident shortly thereafter. These regions can be distinguished at 45 hours APF by differences in the shape and arrangement of cells and by the pattern of developing adult muscles (Kopp, 1999).

The origin of the adult wg and dpp patterns can be traced to the early pupal stage. At the time of fusion of the aDHN and pDHN (18 hours APF), wg and dpp expression domains encompass both adult and larval cells, and are limited to the posterior region of the anterior compartment. Within this zone, the patterns of wg and dpp are largely complementary along the DV axis. wg is expressed in a dorsal-posterior sector of the aDHN and the adjacent dorso-lateral LEC, as well as in a ventral sector of the VHN and the adjacent ventro-lateral LEC. wg is not expressed in the dorsal part of the VHN, in the ventral part of the aDHN or in the lateral LEC between the two nests. wg expression is also weak or absent in the LEC near the dorsal and ventral midlines. dpp is expressed in a dorsal sector of the VHN and in a few cells at the dorsal margin of the aDHN. dpp expression is also seen in the lateral LEC between the VHN and aDHN, and in the dorsal LEC between contralateral dorsal nests. The pupal expression of wg evolves from the pattern present in the larva, where wg is expressed in a circumferential stripe along the AP compartment boundary. The early pupal pattern develops by gradual elimination of expression at the ventral, dorsal and lateral midlines. dpp is not expressed in the epidermis of third instar larvae. However, the expression of dpp in the pupa is reminiscent of the embryo, where it is expressed in mid-dorsal and ventro-lateral stripes. Thus, the pupal expression of dpp may reflect some memory of this embryonic pattern (Kopp, 1999 and references).

The patterns of wg and dpp expression established by 18 hours APF are maintained during the subsequent growth of the histoblast nests. At the time of fusion of the VHN and DHN (24-28 hours APF), wg expression is seen in sectors in the ventral third of the VHN and in the dorsal half of the DHN. dpp is expressed in a stripe in the dorsal two-thirds of the VHN and in a group of 30-40 cells at the dorsal DHN margin. dpp expression also extends transiently into the ventral DHN margin; this expression lasts for only a few hours, and encompasses about 15 cells at its peak (Kopp, 1999).

The complementary expression patterns of wg and dpp are retained in the newly formed adult epidermis at 40-42 hours APF. dpp is expressed in a transverse stripe in the presumptive pleura and in a wedge-shaped stripe along the dorsal midline of the tergite. The limits of pleural expression of dpp coincide precisely with the sternite-pleura and tergite-pleura boundaries. wg is expressed in the sternite and in the medial tergite, but is excluded from the dorsal midline. Neither gene is expressed in a large lateral region of the tergite. The expression of wg and dpp remains restricted to the posterior region of the anterior compartment, with sharply defined posterior and graded anterior boundaries. Double labelling with Engrailed shows that the posterior limit of dpp expression coincides with the compartment boundary. Based on morphological landmarks and on the pattern of lacZ expression in wg-lacZ/dpp-lacZ pupae, the same appears to be true for wg (Kopp, 1999).

The division into dorsal tergite, ventral sternite and ventro-lateral pleural cuticle is largely specified during the pupal stage by Wingless, Decapentaplegic and Egf receptor signaling. Expression of wg and dpp is activated at the posterior edge of the anterior compartment by Hedgehog signaling. Within this region, wg and dpp are expressed in domains that are mutually exclusive along the dorso-ventral axis: wg is expressed in the sternite and medio-lateral tergite, whereas dpp expression is confined to the pleura and the dorsal midline. Neither gene is expressed in the lateral tergite. Tergite and sternite cell fates are specified by Wg signaling. Egfr acts synergistically with Wg to promote tergite and sternite identities, and Wg and Egfr activities are opposed by Dpp signaling, which promotes pleural identity. Wg and Dpp interact antagonistically at two levels:(1) their expression is confined to complementary domains by mutual transcriptional repression and (2) Wg and Dpp compete directly with one another by exerting opposite effects on cell fate. Egfr signaling does not affect the expression of wg or dpp, indicating that it interacts with Wg and Dpp at the level of cell fate determination. Within the tergite, the requirements for Wg and Egfr function are roughly complementary: Wg is required mainly in the medial region, whereas Egfr is most important laterally. Dpp signaling at the dorsal midline controls dorso-ventral patterning within the tergite by promoting pigmentation in the medial region (Kopp, 1999).

The major conclusion of this report is that much of the dorso-ventral (DV) patterning of the adult abdomen is determined by antagonistic interactions between Dpp, which specifies pleural cell fate, and Wg and Egfr signaling, which together specify tergite and sternite fates. Expression of wg and dpp is activated at the posterior edge of the anterior compartment by Hh signaling. Within this zone, wg and dpp are expressed in complementary patterns along the DV axis: wg is expressed in the presumptive sternite and in the medio-lateral region of the tergite, whereas dpp is expressed in the presumptive pleura and at the dorsal midline of the tergite. Although the pattern of Egfr activation in the abdomen has not been determined, Egfr signaling is most important in the lateral tergite, a region where neither wg nor dpp are expressed (Kopp, 1999).

The kinase Sgg modulates temporal development of macrochaetes in Drosophila by phosphorylation of Scute and Pannier

Evolution of novel structures is often made possible by changes in the timing or spatial expression of genes regulating development. Macrochaetes, large sensory bristles arranged into species-specific stereotypical patterns, are an evolutionary novelty of cyclorraphous flies (see The development and evolution of bristle patterns in Diptera) and are associated with changes in both the temporal and spatial expression of the proneural genes achaete (ac) and scute (sc). Changes in spatial expression are associated with the evolution of cis-regulatory sequences, but it is not known how temporal regulation is achieved. One factor required for ac-sc expression, the expression of which coincides temporally with that of ac-sc in the notum, is Wingless (Wg). Wingless downregulates the activity of the serine/threonine kinase Shaggy (Sgg; also known as GSK-3). This study demonstrates that Scute is phosphorylated by Sgg on a serine residue and that mutation of this residue results in a form of Sc with heightened proneural activity that can rescue the loss of bristles characteristic of wg mutants. It is suggested that the phosphorylated form of Sc has reduced transcriptional activity such that sc is unable to autoregulate, an essential function for the segregation of bristle precursors. Sgg also phosphorylates Pannier, a transcriptional activator of ac-sc, the activity of which is similarly dampened when in the phosphorylated state. Furthermore, it was shown that Wg signalling does not act directly via a cis-regulatory element of the ac-sc genes. It is suggested that temporal control of ac-sc activity in cyclorraphous flies is likely to be regulated by permissive factors and might therefore not be encoded at the level of ac-sc gene sequences (Yang, 2012).

achaete-scute products become detectable in wing discs only at mid third larval instar. The known upstream regulators, Pnr and the Iro-C genes, are selector genes that pattern the medial and lateral halves of the notum, respectively. Therefore their activity is not restricted to ac-sc activation and bristle patterning and they are expressed for a considerable period before ac-sc gene products are detected. Furthermore, although activation of ac-sc in proneural clusters by Pnr and Iro-C dramatically increases transcription at these sites, the ac-sc genes are also expressed at low levels over the entire disc epithelium, presumably through activity of the basal promoters. Indeed maintenance of proneural genes in an active state of basal transcription is a general feature of neuroepithelia. So what prevents accumulation of Ac-Sc at earlier stages in disc development (Yang, 2012)?

This study has shown that Sc is phosphorylated by Sgg, an enzyme that is expressed constitutively. Furthermore a mutated form of Sc that is resistant to phosphorylation has significantly greater bristle-forming activity than the wild-type protein. This suggests reduced transcriptional activity of phospho-Sc. One possibility is that the turnover of phospho-Sc is rapid, owing to phosphorylation-dependent ubiquitination and degradation. It has been reported that mutations in the GSK-3β consensus motif in β-catenin abolishes ubiquitination and leads to protein stability. GSK-3β also induces ubiquitination and degradation of Drosophila myc protein through the proteasome pathway and mutation of residues in the phosphorylation domain affects stability of this protein. Indeed it has been shown that mutation of the phosphorylation site SPTS to APAA stabilizes the Sc protein. This suggests that before expression of wg at the mid third larval instar, the stability and transcriptional activity of any Sc present, whether derived from transcription mediated by the basal promoter or enhanced by Pnr and the Iro-C proteins, would be reduced through phosphorylation by Sgg (Yang, 2012).

Development of neural precursors requires high levels of Sc, which are needed for the process of lateral inhibition and singling out of precursors as well as for autoregulation. During this process in Drosophila, Sc binds its own promoter, through a specific regulatory sequence, the sensory organ precursor enhancer (SOPE), to further activate transcription in presumptive precursors (Culi, 1998). Therefore, any factors that diminish the activity of Sc itself have the potential to prevent sufficient accumulation to allow selection of precursors and maintenance of precursor cell fate. Expression of wg at mid third larval instar would lead to inactivation of Sgg. The consequent accumulation of a more active nonphosphorylated form of Sc might allow levels of Sc to accumulate sufficiently for precursor cell development. Achaete does not appear to be a target for Sgg. However, this protein has been shown to be dispensable for bristle development (Yang, 2012).

Pnr is also a target for phosphorylation by Sgg and, like Sc, a mutated phosphorylation-resistant form of Pnr is hyperactive. So phosphorylation of Pnr might also result in ubiquitination and increased degradation, a situation that would be modified by Wg signalling at mid third larval instar. The effects of phosphorylation on Pnr and Sc appear to be quantitative, rather than all or nothing. Pannier has other targets before Wg signalling and activation of ac-sc (the iro genes and wg itself) and if the sole function of Wg were to be the inactivation of Sgg then one would expect loss of sgg function to have no bristle phenotype. So de-phosphorylation might just give an extra little boost to the system. Interestingly it has been shown that the Drosophila transcription factor Mad is also a target of Sgg and that phosphorylation-resistant Mad proteins are hyperactive (Eivers, 2009). Mad is activated by Dpp/TGFβ signalling, which in turn regulates expression of both pnr and the Iro-C genes in the thorax. Thus, it appears that inactivation of Sgg by the Wg signal can stimulate the levels of Sc via multiple routes: by increasing the levels of expression of pnr and the Iro-C genes as well as the activity of Pnr and Sc themselves. Thus, expression of wg at mid third larval instar might result in levels of Sc sufficient for macrochaete development. It is not known how the second phase of ac-sc expression for microchaetes is regulated (Yang, 2012).

Wingless is unlikely to be the only factor regulating temporal ac-sc expression. Indeed, although loss of sgg function can affect bristles over the entire notum, the effects of wg appear to be restricted to the medial notum. Other factors must be involved on the lateral notum. One possibility is NFkappa-B/Rel, a factor that is required for functioning of the the sensory organ precursor enhancer (SOPE) and singling out of precursors, and that also indirectly affects the stability of sc transcripts (Culi, 1998; Ayyar, 2007). Another event that coincides with the accumulation of ac-sc products at mid third larval instar is a small peak of 20H-ecdysteroid (not associated with a moult). Indeed ecdysone has been implicated in temporal regulation of expression of the proneural gene atonal and the development of atonal-dependent sense organs (Yang, 2012).

Wingless signalling has important functions in the thorax, likely to be ancient, that are linked to the development and patterning of flight muscles. So wg was probably already expressed on the notum of the ancestor of the Cyclorrapha, before the evolution of macrochaetes. The rapid development of the notum and short pupal period in many Nematocera leaves little requirement for any temporal control of expression. By contrast, the prolonged period of growth and patterning during the larval and pupal life of Drosophila allows time for two discrete phases of proneural gene expression. Wingless might then have been co-opted for the regulation of ac-sc and the evolution of macrochaetes in the lineage leading to the Cyclorrapha. The current results suggest that the Wg signal does not involve transcriptional regulation of target genes but instead is mediated simply through inactivation of Sgg. The phosphorylation sites are strongly conserved in the sc genes of C. vicina and C. capitata, two other species of Schizophora, suggesting a conserved mechanism of regulation by Wg and Sgg. By contrast, the same sites are not conserved in the other genes of the Drosophila AS-C, or in the ac-sc homologues of A. gambiae, although other potential Sgg phosphorylation sites can be detected in these proteins. Phosphorylation of Sc by Sgg could have been recently acquired in the Cyclorrapha. The ac and sc genes themselves have arisen from duplication events thought to have taken place during evolution of the Cyclorrapha (see Negre, 2009). Phosphorylation of Pnr by Sgg might also have been acquired in the lineage leading to the Schizophora, as one of the sites is conserved in the pnr protein of C. vicina, but not that of Megaselia abdita or A. gambiae (Yang, 2012).

Uniform proneural gene expression, together with Notch-mediated lateral inhibition, is sufficient to generate a pattern of evenly spaced, but randomly positioned, bristles such as that seen in Nematocera and for the microchaetes of the Cyclorrapha. For this process, the SOPE, a very ancient regulatory element that predates the Diptera (Ayyar, 2010), is the only cis-regulatory element of ac-sc that would be required. Factors that act through the SOPE could be co-opted to modulate the temporal activity of ac-sc. This includes factors regulating activity of Sc, which itself binds the SOPE (Culi, 1998). Control at this level could be superimposed on the ancestral state without the need to acquire new regulatory sequences for the binding of novel transcriptional repressors and activators. By contrast, the spatially restricted expression underlying the macrochaete pattern is linked to changes at the AS-C complex and the acquisition of novel cis-regulatory elements that possibly arose in association with gene duplication events. This illustrates the power of evolution to make use of factors acting both in cis and in trans to effect morphological change (Yang, 2012).

Optic lobe glia express Wingless: Glial migration to the optic lobe is directed by retinal axons

In the developing Drosophila visual system, glia migrate into stereotyped positions within the photoreceptor axon target fields and provide positional information for photoreceptor axon guidance. Conversely, glial migration depends on photoreceptor axons, as glia precursors stall in their progenitor zones when retinal innervation is eliminated. These results support the view that this requirement for retinal innervation reflects a role of photoreceptor axons in the establishment of an axonal scaffold that guides glial cell migration. Optic lobe cortical axons extend from dorsal and ventral positions toward incoming photoreceptor axons and establish at least four separate pathways that direct glia to proper destinations in the optic lobe neuropiles. Photoreceptor axons induce the outgrowth of these scaffold axons. Most glia do not migrate when the scaffold axons are missing. Moreover, glia follow the aberrant pathways of scaffold axons that project aberrantly, as occurs in the mutant dachsous. The local absence of glia is accompanied by extensive apoptosis of optic lobe cortical neurons. These observations reveal a mechanism for coordinating photoreceptor axon arrival in the brain with the distribution of glia to multiple target destinations, where they are required for axon guidance and neuronal survival (Dearborn, 2004).

The Drosophila optic ganglia are derived from a pair of neuroectodermal proliferative centers known as the inner and outer anlage, located on the ventrolateral surface of each larval brain hemisphere. The onset of Wingless (Wg) expression has been described in a few cells located at the presumptive dorsal and ventral margins of the disc-shaped outer anlagen in young larvae. These two `Wg domains' are positioned as adjacent wedges within the disc-shaped outer anlage. In mid-metamorphosis, the disc opens at a fissure located between the two Wg domains; the disc unfurls linearly, such that the Wg domains move to the future dorsal and ventral poles of the ganglion. Interestingly, the Wg domains are roughly coincident with sites revealed by clonal analyses to be the origins of glia that migrate into the lamina neuropile. This relationship was investigated in greater detail (Dearborn, 2004).

A marker of wingless expression, the lacZ reporter construct 17en40 (or wg-lacZ) was used to examine wg expression with respect to glial cell development and movement. In cells expressing wg-lacZ, cytoplasmic ß-galactosidase fills cellular processes and permits the visualization of their detailed morphology. The paths of glial cell migration are observed to coincide with a stereotyped pattern of cytoplasmic extensions emanating from wg-lacZ expressing cells. The extensions follow stereotyped routes and terminate at specific destinations in the lamina, medulla and lobula. Glia form chains along these extensions and accumulate in neuropile destinations beyond the extension's termini. Marker analysis indicates that the glia undergo differentiation as they progress along these paths. Lamina glia express the early glial differentiation factor glial cells missing (gcm) at their points of entry onto the extensions. Further along the path toward the neuropile, glia commence expression of the homeodomain protein Repo, a marker downstream of Gcm expression. Thus, in the case of lamina marginal glia, differentiation can be assessed relative to glial progression along the migratory pathway (Dearborn, 2004).

The wg-lacZ positive extensions are evidently axons. Their cell membranes label by anti-horseradish peroxidase antibody, a neuronal marker. Their cell bodies label positive for Elav, also a neuronal marker. The axons extending toward the same neuropile destination bundle together in a fascicle, as indicated by labeling of individual axons in mosaic animals. Four wg-lacZ labeled fascicles were resolved emerging from each of the dorsal and ventral Wg domains. One fascicle from each domain extends to the border of the lamina field, terminating at a position adjacent to layers of glia known as the lamina epithelial (Ep) and marginal (Ma) glia (established nomenclature is found at Flybrain). These layers of glia lie, respectively, above and below the layer of the axon termini of the R1-R6 photoreceptors. Glia can be observed migrating in a chain along the fascicles. Owing to the absence of specific markers, epithelial and marginal glia could not be distinguished prior to their separation into distinct layers. It seems likely, however, that both glial types migrate on the same pathway. One fascicle from each Wg domain extends to the cortex, neuropile boundary of the medulla, and is associated with the chain-like migration of medulla neuropile (MNG) glia. One fascicle from each domain extends to the boundary between the medulla and lobula, and corresponds to a pathway for migration of inner chiasm (Xi) glia, which demarcate the border between medulla and lobula neuropiles. The final pair of fascicles extends into the lobula neuropile and forms a pathway associated with lobula neuropile glia (LoG). These four sets of putative migratory guides are referred to as 'scaffold axons'. Notably, the migration of these four glia types depends on retinal innervation of the optic lobe, a requirement that is explored in this study (Dearborn, 2004).

Earlier stage specimens were examined in order to determine the temporal relationship between glial migration and the outgrowth of scaffold axons. During the first two larval stages, most neuroectodermal cells of the outer anlagen express the cell adhesion protein Fasciclin 2 (Fas2), with the exception of those in the two Wingless domains; Wingless-expressing cells are Fas2 negative. As development proceeds to the third instar stage, the neuroectodermal populations mostly convert to blast cells that produce the neurons and glia of the lamina and medulla. The differentiation of wg-lacZ positive scaffold neurons and the extension of their axons follow this general temporal scheme, a small population of glia precedes the arrival of the first photoreceptor axons in the target field of the retinal axon. A small population of centrally located glia have been described that precede the arrival of photoreceptor axons in the lamina. At these early time points, when the Wg domains consist of fewer than a hundred cells; scaffold axon fascicles are not detected. Photoreceptor axons subsequently arrive in the temporal order that follows the posterior to anterior pattern of eye development. The elaboration of wg-lacZ positive scaffold axons is first detected as the first photoreceptor axons arrive in the target field, when only the first one or two columns of ommatidia have initiated differentiation in the eye disc. As retinal innervation continues, the number of glia in the neuropile regions increases steadily. Thus, Wingless-positive cells, though present long before the arrival of photoreceptor axons in the brain, appear to first extend axons when retinal axons begin to arrive in the brain (Dearborn, 2004).

The wg-lacZ labeled-axons also were examined in pupal stage animals, where mature axon projections into the optic lobe neuropiles can be resolved. wg-lacZ positive axons can still be detected extending from cell bodies located at dorsal and ventral cortical positions. The axons project, respectively, to dorsal and ventral targets in the medulla and lobula neuropiles. It is evident that the wg-lacZ-positive neurons include intrinsic neurons of the proximal medulla (Pm neurons), which extend arbors tangentially within small regions of the proximal medulla. Projections into a specific tangential layer of the lobula were also observed. Projections into the lamina neuropile were not observed; at the third instar stage the extensions terminate at the border of the developing lamina neuropile. These axons thus may not have become part of lamina circuitry, or alternatively have ceased wg-lacZ expression at the pupal stages examined (Dearborn, 2004).

Attempts were made to determine the location of progenitors that give rise to the distinct types of migratory glia and the neurons that form their migratory pathways. The Wingless expressing cells of the dorsal and ventral domains are located in areas of complex gene expression controlled by Wingless (Wg) signaling activity. Adjacent to the Wg domains are non-overlapping cell populations that express the TGF-ß family member Decapentaplegic (Dpp). Both the Wg- and Dpp-positive cell populations express the transcription factor Optomotor Blind (Omb). Dachsous (Ds), a Cadherin family member, is expressed in a graded fashion with respect to the Wg domains. These three genes, though expressed in different patterns, are under the control of Wg activity (Dearborn, 2004 and references therein).

By performing clonal analysis with tissue labeled to provide positional landmarks, it was possible to localize glial progenitors and scaffold neurons to distinct sites of origin. The FLP/FRT system was used to generate somatic clones that were positively marked by membrane-bound GFP (UAS-CD8::GFP). Rare recombination events were induced such that most specimens harbored only one or a few labeled cell clones in the developing optic ganglia, which were examined in late third instar larvae. Clones including lamina marginal and epithelial glia were found to label progenitors located at the dorsal and ventral margins of the outer anlagen. Clones that included lamina epithelial glia, lamina marginal glia, medulla neuropile glia, inner chiasm glia or lobula neuropile glia were all found within the domain of cells that express Wg, Omb and Ds (Domain I). A majority of these clones contained both labeled scaffold neurons and glia (16/26); clones with only neurons or glia were less frequent. In the majority of specimens in which glia were labeled, the clone extended into multiple domains and included Domain I. With rare exception, these larger clones also contained neurons. Thus, at the time that somatic recombination was induced (mid-second instar, starting 75 hours after egg laying), most progenitor cells retained the potential to produce both neurons and glia. When the specimens were analyzed with respect to the glial types that were labeled, an interesting pattern emerged with regard to the position of labeled progenitor cells in the Domain I region. Labeled progenitors for each of the glial cell types appeared in distinct domains on the proximal-distal axis. For example, the lamina epithelial and marginal glial progenitors were found in a more lateral position than medulla neuropile glial progenitors. Inner chiasm glial progenitors were observed in an even more proximal location. Hence, the progenitor domains for distinct glial types appear to be organized into a proximal distal stack within Domain I (Dearborn, 2004).

Labeled scaffold neurons were most often included in the glial clones, and were found in close proximity to glial progenitors that used the axons as migratory guides. In a minority of cases, small clones were recovered which included only scaffold neurons. These clones were contained within Domain I (Wg, Omb, Ds expression) in all but two of nineteen cases. These data do not resolve when the glial and neuronal lineages diverge. However, they do permit the conclusion that glia and the neurons that appear to establish their migratory pathways are generated in close proximity and with a lineage relationship (Dearborn, 2004).

The migration of glia from the prospective dorsal and ventral margins of the developing optic lobe depends on the arrival of photoreceptor axons in the target field. When photoreceptor axons are absent, as occurs in mutants that eliminate ommatidial development (sine oculis, eyes absent and eyeless) most glia remain stalled in their progenitor domains. When photoreceptor axons innervate only part of the lamina field, glia migrate to the region that receives retinal innervation. It has thus been supposed that photoreceptor axons attract glia into the lamina target field. It was thus thought it might be informative to examine the glial migratory scaffold under conditions where glial migration did not occur (Dearborn, 2004).

To this end, the axon scaffold was examined in sine oculis1 (so) and EyelessD (ey) animals. These mutants display variable penetrance, such that photoreceptor neurons can be completely absent, or develop in variably sized clusters in a particular region of the developing retina. A lack of retinal innervation has compound effects on optic lobe development. Lamina neurons fail to develop because of the absence of axon-borne signals. The medulla is greatly reduced in cell number by extensive apoptosis. Employing mosaic analysis, Fischbach and Technau (1984) showed that so1 acts in the eye to bring about these effects on the brain (Dearborn, 2004).

In the current analysis, the scaffold axons were labeled by the expression of wg-lacZ. When photoreceptor axons were absent, the scaffold axons were likewise missing. However, wg-lacZ positive cells seemed to be present in normal number in the Wg domains, and expressed the neuronal HRP antigens. In prior work, a number of markers expressed in the vicinity of the Wg domains were expressed normally in the absence of retinal innervation (e.g., Dpp and Omb). Therefore, it is thought unlikely that retinal innervation is required for the differentiation of these optic lobe neurons. When the scaffold axons were absent in so1 and eyD animals, glia accumulated at the edges of the Wg domains near the point where they would have joined axon fascicles on paths toward neuropile destinations. Most so1 and eyD animals develop part of an eye, such that the corresponding optic lobe receives partial innervation. In these cases, photoreceptor axons project to appropriate retinotopic locations despite the absence of the usual array of neighboring axons. In such specimens, scaffold axons were found only in parts of the brain that received retinal innervation and not in regions that completely lacked innervation. A correlation between retinal innervation, scaffold axon extension and glial migration was found in each of 32 animals with partial eye development restricted to either dorsal or ventral regions. These observations thus argue strongly that scaffold axon outgrowth and glial migration depend on retinal innervation (Dearborn, 2004).

Retinal innervation might elicit scaffold axon outgrowth, thereby establishing a necessary pathway for glial migration. Conversely, glial migration, elicited by retinal innervation directly, might establish a necessary pathway for scaffold axon outgrowth. Two approaches were undertaken in an attempt to resolve this issue. In the first, scaffold axons were eliminated by neuronal expression of activated Ras1 (Ras1N17) in order to determine whether glial migration would occur in their absence. In the second, mutant animals with misdirected scaffold axons were examined in order to determine whether migratory glia follow the aberrant axon projections. Both approaches indicated that scaffold axons are necessary as glial migratory guides (Dearborn, 2004).

Prior work has revealed extensive apoptosis in the optic lobes of 'eyeless' mutants of Drosophila. In the mutant sine oculis (so), mosaic analysis has revealed that extensive cell death in the optic lobe is due to the lack of so function in the retina and not the brain. These and additional observations have led to the conclusion that the optic lobe phenotype of sine oculis is due to lack of retinal innervation. The 'trophic' function of photoreceptor axons could be direct, via provision of a survival factor, or indirect, e.g., by eliciting the migration of glia that provide a survival factor (Dearborn, 2004).

To address the issue, wild-type and so1 animals were examined for the onset of apoptosis by their expression of activated caspase, which can be monitored in Drosophila with an antibody against the activated human caspase 3 protein. In third instar stage wild-type animals, few cortical cells are labeled by the anti-Caspase labeling. By contrast, so1 third instar larval optic lobes display an increased number of caspase 3-positive cells throughout the medulla cortex. Putative apoptotic cells were concentrated in regions where glia were particularly few or absent. Caspase 3-positive cells were also particularly prevalent in cortical areas immediately adjacent to the neuropile. Areas particularly deficient in glia also displayed an irregular neuropile structure. These observations were subjected to a quantitative analysis. Areas of 50 µm2 in 1.3 µm confocal optical sections of the medulla cortex were counted for the number of caspase 3-positive cells. In wild-type animals, an average of 0.6 activated caspase-positive cells were found per 50 µm2 area. No regional differences in the density of apoptotic cells were observed in wild-type animals. so1 specimens displayed an average of 5.5 apoptotic cells in 50 µm2 areas adjacent to medulla neuropile regions that lacked medulla neuropile glia. Elevated apoptosis, an average of 6.5 activated caspase-positive cells, was also observed in distal cortical cell populations whose axons would normally innervate glia-starved regions of neuropile. By contrast, cell populations adjacent to glia-rich regions of medulla in the same so1 animals showed only 2.4 apoptotic cells per 50 µm2, while in the corresponding distal cortical cell populations that innervate these glia-rich regions, only 2.5 apoptotic cells per 50 µm2 were found. Thus, although so1 animals displayed an overall increase in caspase-positive cells, the frequency was significantly greater in cell populations that innervated glia-starved regions of neuropile. The results of this analysis are statistically significant: for medulla neuropile proximal regions within so1 animals, a paired t-test analysis yielded a P-value of 1.3e-06 (comparing glia-poor and glia-rich regions), while the same statistical analysis comparing cortical regions yielded a P value of 4.8e-07. These observations suggest that both local and long-range trophic cues are provided by glia to cortical neurons. Additional studies show that the absence of glia, rather than photoreceptor axons, appears the more likely cause of extensive apoptosis and neural cell loss observed in eyeless strains of Drosophila (Dearborn, 2004).

It is concluded that Drosophila optic lobe glia use axon fascicles as migratory guides and that the extension of these axon fascicles is induced by the ingrowth of photoreceptor axons from the developing retina. The migratory scaffold axons emerge from optic lobe regions that are in close proximity to sites where glial cells originate; both arise in the dorsal and ventral domains where cells express the morphogen Wingless. When the scaffold axons were eliminated by the autonomous expression of an activated Ras transgene, glia failed to migrate and stalled at the borders of their progenitor sites. Extensive cortical cell apoptosis ensued. When the scaffold axons projected aberrantly (in animals mutant for the cadherin Dachsous), glia followed the aberrant routes to incorrect destinations. The longstanding observation that glial migration does not occur in eyeless mutant Drosophila might thus be explained by an indirect mechanism in which innervation controls the establishment of an axon scaffold necessary to direct glial migration (Dearborn, 2004).

The migratory scaffold axons were identified by their cytoplasmic expression of ß-galactosidase from lacZ under the control of a wingless promoter. The neurons are thus residents of the Wg domains, a point additionally supported by labeling small numbers of neurons that projected their axons toward glial destinations. In total, four different wg-lacZ delineated pathways were identified. These appear to account for all the pathways taken by optic lobe glia that have been identified as migratory by clonal studies. Separate pathways were identified for medulla neuropile glia, lobula neuropile and inner chiasm glia. A single scaffold axon pathway was observed leading to the marginal and epithelial glial layers of the lamina, suggesting that both of these glial types follow the same pathway. Perhaps these glia become separated only on the interposition of photoreceptor R1-R6 growth cones as they arrive in the lamina. Whether the epithelial and marginal glia arise from distinct precursors that migrate on the same pathway is unclear. In all cases, glia were observed to form migratory 'chains' along axonal extensions, resembling a similar organization of migratory glia on retinal axons en route from the optic stalk to the eye field and from midline progenitor sites to destinations in the PNS. In pupal stage animals, the wg-lacZ labeled neurons were observed in dorsal and ventral cortical locations, sending projections into neuropile targets consistent with the patterns of glial migration. However, no axons from wg-lacZ positive neurons were observed extending into the lamina neuropile at this stage (Dearborn, 2004).

The optic lobe regions surrounding the Wg domains display complex patterns of gene expression, mainly because of the signaling activity of Wingless. Clonal analysis indicates that all five migratory glial cell types examined arise from these domains. Interestingly, the sites from which particular glia arise are stacked on the proximal distal axis in a manner that correlates with target destinations in the developing ganglia. Thus, for example, somatic clones that label the medulla neuropile glia are located at a position that corresponds to the medial/distal position of MNG glia relative to the lamina and lobula glia. Furthermore, on the basis of their expression of wg-lacZ, as well as clonal analysis, the neurons that extend scaffold axons arise in close proximity to the sites of glial origin. Indeed, as somatic clones induced in mid-second instar larval animals often labeled both scaffold neurons and migratory glia, the glia and neurons must share common progenitors. It is curious that all of these distinct cell types express wingless. These is no evidence that axonally transported Wg functions in optic lobe development, as it does in the development of the neuromuscular junction. Partial elimination of wg+ activity (by the use of a conditional wgts allele) did not result in a specific defect in glial migration in the optic lobe but other possible functions of Wg were not addressed by this analysis (Dearborn, 2004).

These observations suggest a developmental mechanism for the control of glial cell migration that depends on the establishment of an axon scaffold for guidance of migrating glia. In normal development, a small number of glia migrate into the target field of the photoreceptor axon prior to the arrival of the first photoreceptor axons. These glia, which migrate independently of retinal innervation, may serve a necessary early role in photoreceptor axon guidance. They may be targets for the first retinal axons to arrive in the optic lobe, and provide the first signals that differentiate the outgrowth termination points of the R1-R6 and R7/8 axons. It is proposed that the first photoreceptor axons to arrive in the optic lobe elicit outgrowth of the scaffold axons from neurons at the dorsal and ventral margins. Subsequent migration of glia from the dorsal and ventral margin progenitors is then both permitted and directed along the specific pathways of the scaffold. After the erection of the migratory scaffold, glial migration may be independent of continued photoreceptor axon ingrowth. On this point, it is noted that glia migrate into the lamina in approximately normal numbers in hh1 animals, in which ommatidial development ceases after 11-13 columns form at the posterior of the developing retina. Therefore, glial migration may not depend on continued arrival of new retinal axons in the lamina primordium. Nonetheless, this observation cannot rule out the alternative interpretation that retinal axons emit a continuous attractive signal for glial migration that functions in adjunct with the migratory axon scaffold (Dearborn, 2004).

How do photoreceptor axons control the outgrowth of axons from the Wg domains? This does not appear to be a consequence of an affect of retinal innervation on neuronal development in the Wg domains, which appear normal in size and organization in eyeless mutant Drosophila strains. Hedgehog, which is brought into the brain by retinal axons, is not required for the expression of either Wg or Dpp at the dorsal and ventral margins of the optic lobe. It seems that the induction of scaffold axon outgrowth by the photoreceptor axons may be direct, since the outgrowth occurs in the hedgehog mutant, hh1, in which the first steps of lamina neuronal development fail to occur. Thus, one might suppose that retinal axons emit a chemoattractant for scaffold axon outgrowth, either synthesized in the retina or acquired from environmental sources and redistributed by retinal axons (Dearborn, 2004).

The system for glial migration guidance permits diversified cell types to originate from a common site, and yet target specific locations in complex neuropiles. One could imagine that as more complex and diversified neuropiles evolved, relatively simple changes in the developmental pathway of glial progenitors and the projections of scaffold axons would deliver glial support to new structures. This system also has the feature of functioning as a developmental timing 'checkpoint' that fine-tunes the general hormonal coordination of imaginal development. Thus, upon their initial arrival in the brain, retinal axons provide a fine level of local cellular control over the movement of glia, preparing the target field for the next steps of optic lobe development (Dearborn, 2004).

Homeotic proboscipedia function modulates hedgehog-mediated organizer activity to pattern adult Drosophila mouthparts: negative regulation of wingless and decapentaplegic downstream of hh signaling

Drosophila proboscipedia (HoxA2/B2 homolog) mutants develop distal legs in place of their adult labial mouthparts. How pb homeotic function distinguishes the developmental programs of labium and leg has been examined. The labial-to-leg transformation in pb mutants occurs progressively over a 2-day period in mid-development, as viewed with identity markers such as dachshund (dac). This transformation requires hedgehog activity, and involves a morphogenetic reorganization of the labial imaginal disc. These results implicate pb function in modulating global axial organization. Pb protein acts in at least two ways. (1) Pb cell autonomously regulates the expression of target genes such as dac; (2) Pb acts in opposition to the organizing action of hedgehog. This latter action is cell-autonomous, but has a nonautonomous effect on labial structure, via the negative regulation of wingless and decapentaplegic. This opposition of Pb to hedgehog target expression appears to occur at the level of the conserved transcription factor cubitus interruptus/Gli that mediates hedgehog signaling activity. These results extend selector function to primary steps of tissue patterning, and leads to the notion of a homeotic organizer (Joulia, 2005).

The labial palps, the drinking and taste apparatus of the adult fly head, are highly refined ventral appendages homologous to legs and antennae. As for most adult structures, these mouthparts are derived from larval imaginal discs, the labial discs. Wild-type pb selector function acts together with a second Hox locus, Scr, to direct the development of the labial discs giving rise to the adult proboscis. In the absence of pb activity, the adult labium is transformed to distal prothoracic (T1) legs, reflecting the ongoing expression and function of Scr in the same disc. Though the pb locus shows prominent segmental embryonic expression, as for the other Drosophila homeotic genes of the Bithorax and Antennapedia complexes, it is unique in that it has no detected embryonic function and null pb mutants eclose as adults that are unable to feed. Thus, normal pb selector function is required relatively late, in the labial imaginal discs that proliferate and differentiate during larval/pupal development to yield the adult labial palps. Though the genetic pathway guiding development of the ventral labial imaginal discs to adult mouthparts remains relatively unexplored both in flies and elsewhere, study of P-D patterning has identified several genes subject to pb regulation in the labial discs (notably Dll, dac, and hth) and a distinct organization of normal labial discs has been indicated compared to other imaginal discs (Joulia, 2005).

This study pursued an investigation of how pb homeotic function distinguishes between labial and leg developmental programs. The results implicate pb function at the level of global axial organization. Employing identity markers such as dachshund (dac), a 2-day period late in larval development has been identified when normal pb function is required for labial development. The labial-to-leg transformation occurs during the third larval instar stage, involves a progressive morphogenetic reorganization of the labial imaginal disc, and is hedgehog-dependent. This analysis of the transformation indicates that normal pb action is required at least at two distinct levels. One is in the cell-autonomous regulation of target genes such as dac likely to be implicated in cell identity. A second level involves an autonomous action with a nonautonomous effect on labial structure, through the negative regulation of wingless and decapentaplegic downstream of hh signaling. This opposition to hh targets is likely to occur at the level of the transcription factor cubitus interruptus/Gli, a crucial and conserved mediator of hh signaling activity. These results led to a proposal that homeotic function may exist in intimate functional contact with the hedgehog organizer signaling system: the 'homeotic organizer' (Joulia, 2005).

Segmental organization in the imaginal discs involves the reiterated deployment of segment polarity genes that organize the fundamental segmental form. This involves a cascade proceeding from posteriorly expressed Engrailed protein through a short-range Hh morphogen gradient in anterior cells favoring the activator form of Ci transcription factor, which in turn activates wg and dpp to establish two concurrent, instructive concentration gradients that structure gene expression along the proximo-distal axis. In contrast with this elaborate choreography of the segment polarity genes, the homeodomain proteins encoded by Hox genes are expressed in a segmental register, which obscures how they can direct the differentiation of distinct cell types within the segment. The present investigation of homeotic proboscipedia function during labial palp formation indicates a multipronged action for pb in the labial disc. Pb acts cell-autonomously in the negative regulation of target genes including dac, which is normally extinguished in Pb-expressing cells of labial or leg imaginal discs but is activated in labial discs in the absence of pb activity. This activation of dac in mutant labial cells is hh-dependent and is likely a response to wg and dpp morphogen signals as for leg discs. The data further indicate that pb acts cell autonomously to regulate the level of both wg and dpp expression in response to hh. Thus, pb appears to negatively regulate dac expression directly, but also by withholding positive instructions from Wg and Dpp morphogens. The interweaving of homeotic selector proteins with strategic target genes including morphogens (wg, dpp) and targets of signaling activity (dac, Dll) may influence segment patterning from global size and shape to specific local pattern and cell identity. This positioning offers a powerful yet economical mode of selector function that helps to better understand how a single selector gene can integrate global patterning with cellular identity (Joulia, 2005).

This view invoking multiple and overlapping modes of regulation by a homeotic selector protein supports and extends the vision from analyses seeking to explain how Ultrabithorax (Ubx) selector function differentiates between the serially homologous wing and haltere appendages. This analysis supports a role for Ubx in fruit flies transforming a dorsal default state (wing) to haltere, by repressing the accumulation of Wg in the posterior part of the haltere, and by regulating a subset of Dpp or Wg activated targets such as vestigial and spalt related. Additionally, clear evidence has been presented for a nonautonomous action of Ubx via its activity in cells of the D-V organizer where wg is expressed. Ubx thus acts to down-regulate wg in the haltere, but also intervenes to modulate the expression of targets of both dpp and wingless signaling pathways. An analysis of mutants for maxillopedia (mxp), the Tribolium pb homolog, revealed augmented transcription of flour beetle wg within the transformed labial segment. This observation, in full accord with the above results for Ubx, and the current results for Drosophila pb, supports a conserved role for homeotic regulation of nonautonomous signaling input in appendage development. At the same time, mxp mutants show a precocious maxilla-to-leg transformation in larvae, demonstrating a prior, embryonic requirement for mxp. This result is of particular interest since it highlights a temporal aspect of pb action in the fly labial disc: the absence of pb function early has no apparent effect on the labial discs in early L3 larvae, which appear normal. It is only subsequently that these diverge toward leg structure. Thus, the globally conserved activity of mxp/pb in equivalent beetle or fly organs is nonetheless employed in temporally different ways among species. Though it is not clear whether this reflects the existence of species-specific co-factors or rather of the effects of expression dosage and timing, such modifications might offer important possibilities for changing form. Variations on all these themes can probably contribute to the diversification of organism form, within and among species (Joulia, 2005)

The roles of diffusible Wg and Dpp morphogens induced by Hh at the A-P boundary, and the transcriptional programs they induce according to their concentrations within a gradient, are considered central to organizing the group of cells constituting a segment. The present work indicates that pb normally acts downstream of Hh within the organizer, where it maintains Wg and Dpp at low levels in labial imaginal tissue. Overexpressing Wg or Dpp in the labial discs results in malformed, overgrown or transformed 'labial' tissue. These observations support the viewpoint that limiting morphogen accumulation is essential to ensuring that the labial program is correctly applied. This study underlines the potential importance of the absolute levels of wg and dpp-encoded signaling molecules deployed for tissue organization. While a gradient may in principle be formed from any source, part of the spectrum of threshold levels necessary for stimulating specific gene responses is likely removed from the repertoire in the labial environment. The absolute level of activation or inhibition of diverse signaling pathways thus may be in itself a tissue-specific property, allowing gradients of related form but with different instructive capacities that can be a distinctive element in guiding tissue formation and specifying ultimate identity. This integration of diverse sorts of information -- the hh organizer linked to the Hox selector -- may confer order to tissue organization and identity (Joulia, 2005).

The fine-tuning of morphogen signals by Hox selectors coupled with the concomitant regulation of downstream targets thus appears to offer a strategic control point for achieving reliable developmental control coupled with evolutionary flexibility. The modulation of different cell signaling pathways by pb activity implies it can regulate both the tissue “context” generated by the signaling pathways activated in a tissue, and the cellular response to this context. This capacity to meld large-scale patterning with cellular identities merits emphasis (Joulia, 2005).

While the logic described above appears to be conserved, its application leads to widely different results according to the species and the tissue. Quite recently, an analysis of vertebrate Hox function has led to the identification of an intimate developmental link between Hox selector function and hedgehog signaling. This analysis reveals a direct physical interaction between the mouse Ci homolog Gli and Hox homeodomain transcription factors. It thus provides a compelling complement to the present work, since the molecular framework of a direct link between Gli and Hox proteins goes far to rationalise the dose-sensitive interplay between Ci and Pb that was observed in Drosophila. If Hox proteins indeed compete for available nuclear Gli/Ci, this molecular mechanism may also help to understand other phenomena including phenotypic suppression in flies or posterior prevalence in mice. Correspondingly, the current data place Pb in antagonism to Ci within the hedgehog organizer, where it modulates output from the wg and dpp genes and the instructive morphogens they encode. These complementary observations from insect and vertebrate models suggest the existence of an evolutionarily conserved machinery with enormous potential for generating morphological diversity. It will be exciting to know more about how the homeotic selector function is integrated in known cascades that make use of conserved molecules both to ensure the fidelity of normal form, as well as to generate new form (Joulia, 2005).

Robustness and stability of the gene regulatory network involved in DV boundary formation in the Drosophila wing

Gene regulatory networks have been conserved during evolution. The Drosophila wing and the vertebrate hindbrain share the gene network involved in the establishment of the boundary between dorsal and ventral compartments in the wing and adjacent rhombomeres in the hindbrain. A positive feedback-loop between boundary and non-boundary cells and mediated by the activities of Notch and Wingless/Wnt-1 leads to the establishment of a Notch dependent organizer at the boundary. By means of a Systems Biology approach that combines mathematical modeling and both in silico and in vivo experiments in the Drosophila wing primordium, this regulatory network was modeled and tested; evidence is presented that a novel property, namely refractoriness to the Wingless signaling molecule, is required in boundary cells for the formation of a stable dorsal-ventral boundary. This new property has been validated in vivo, promotes mutually exclusive domains of Notch and Wingless activities and confers stability to the dorsal-ventral boundary. A robustness analysis of the regulatory network complements the results and ensures its biological plausibility (Buceta, 2007).

In silico evidence is presented that refractoriness to the Wg signal in boundary cells provides stability to the gene regulatory network. Boundary cells are characterized by high levels of Notch activity, thus suggesting Notch is responsible for making boundary cells refractory to the Wg signal. The role of Notch in this process was analyzed in the developing wing primordium. Ectopic activation of Notch in non-boundary cells represses Wg target gene expression. Note that Notch, in this case, causes ectopic Wg expression in non-boundary cells, which induces target gene expression only in Wg non-expressing cells. By contrast, ectopic expression of Wg alone induces the expression of target genes in both Wg-expressing and non-expressing cells. When boundary cells lack Notch activity, either by mutation or by expression of a dominant negative form of Delta known to titrate out the Notch receptor, these cells start to express target genes of Wg. It can then be concluded that either Notch activity itself, or one or several of its target genes inhibits the expression of Wg target genes in boundary cells (Buceta, 2007).

High levels of Notch activity induce expression of the homeobox gene cut in boundary cells and Cut has been previously shown to be required to repress Delta and Serrate expression in these cells. Then, whether Cut mediates the activity of Notch in inhibiting the expression of other Wg target genes was examined. In the absence of Cut activity, either in a homozygous mutant background or in clones of mutant cells, boundary cells start expressing genes regulated by the Wg signal, and ectopic Notch activation in non-boundary cells is now unable to repress Wg target gene expression. Note that Notch, in this case, causes ectopic expression of Wg, which induces target gene expression in both Wg-expressing and non-expressing cells. Finally, forced expression of Cut in non-boundary cells represses the expression of Wg target genes. Taken together, these results indicate that Cut is not only required but also sufficient to inhibit Wg target gene expression in boundary cells downstream of Notch (Buceta, 2007).

Cut might exert its function either by blocking the Wg signaling pathway or, alternatively, by inhibiting the expression of every Wg target gene. The Wg signaling pathway is activated by controlling the levels and subcellular localization of the transcriptional co-activator Armadillo (Arm, known as β-catenin in vertebrates). In the absence of Wg signal, Arm levels are kept low through degradation. This degradation depends on the phosphorylation of Arm by the kinase Shaggy/Zeste white-3/Glycogen synthase kinase-3β (GSK-3β). Phosphorylated Arm is recognized rapidly by the proteasome and destroyed. Following Wg ligand binding, this degradation is inhibited, which enables Arm to accumulate, enter the nucleus and activate a transcriptional response. In the Drosophila wing, Arm protein levels are severely reduced in boundary cells, when compared with adjacent cells, even though extracellular Wg protein is available in both types of cells. This observation indicates that the activity of the Wg signaling pathway is repressed in these cells at the level or upstream of Arm. Consistent with this observation, a dominantly activated form of Arm (ArmS10), which lacks the GSK-3ß phosphorylation sites and escapes degradation, induces expression of Wg targets in boundary cells. Overexpression of any other limiting factor of the Wg pathway that acts upstream of Arm is unable to induce Wg target gene expression in these cells (Buceta, 2007).

Cut appears to mediate this type of repression of the Wg signaling pathway. In the absence of Cut activity, Arm protein levels are not reduced in boundary cells, and ectopic expression of Cut in non-boundary cells reduces Arm protein levels and represses the expression of Wg target genes. Moreover, ArmS10 can bypass the effects of ectopic Cut expression and restores Wg target gene expression in non-boundary cells. Co-expression of limiting factors of the Wg pathway acting upstream of Arm does not cause this effect. Taken together, these results indicate that Cut blocks the Wg signaling pathway at the level or upstream of Arm. Cut might exert its function through transcriptional regulation of a gene product involved in regulating the degradation of Arm (Buceta, 2007).

So far in vivo evidence has been provided that Cut is required in boundary cells to repress the Wg signaling pathway and also, by means of in silico experiments, it has been shown that such repression leads to a stable DV boundary formation. In silico implementation of the refractoriness to the Wg signal via Cut leads to stable DV boundary formation. The stationary pattern of gene expression and activity observed in this case is in agreement with in vivo results (Buceta, 2007).

The conclusions can be extended further with regard to the role played by Cut in DV boundary formation. In the absence of refractoriness to the Wg signal (provided by the activity of Cut in boundary cells) an initial increase in Notch activity and Wg expression takes place. This result suggests that Cut is dispensable for the onset of the DV boundary. This and the evolution predicted by modeling are in agreement with the in vivo results. In cut mutant discs, the early activation of Notch at the DV boundary, as shown by the expression of Wg, is comparable to wild-type discs. However, in mature third instar discs Notch activity and Wg expression are not maintained in the mutant background. Taken together, these results indicate that refractoriness of boundary cells to the Wg signal provided by the activity of Cut is required to shape a stationary and stable DV boundary in the developing wing primordium (Buceta, 2007).

This study analyzed the properties of the regulatory network for the establishment and maintenance of the DV organizer in the Drosophila wing imaginal disc. Evidence is provided that that a mathematical model can convert the initial DV asymmetric expression pattern of Notch ligands into the DV symmetric and mutually exclusive domains of active receptor and Notch ligands in boundary and non-boundary cells, respectively. To model the network 'circuitry', and test and verify the proposal, advantage was taken of a combination between in vivo and in silico experiments that has allowed checking of the analytical and predictive capacity of the modeling (Buceta, 2007).

The most striking finding of this research is that a novel property is required in the regulatory network for a robust and stable maintenance of the DV organizer: namely boundary cells must be refractory to the Wg signal. This property is conferred by the activity of Notch through its target gene cut. The role of Cut in repressing the Wg signaling pathway in boundary cells, and Wg in repressing Notch in non-boundary cells, generates two mutually exclusive domains of Notch and Wg activities, corresponding to boundary and non-boundary cells, respectively. Consequently, Notch ligands and receptors are expressed in two distinct non-overlapping cell populations. This helps to restrict the width of the boundary population to few (two-three) cells and contributes to polarizing ligand-receptor signaling towards the boundary and not against it, i.e., flanking ligands signal Notch towards the boundary but not against it since down-regulation of the Notch pathway in non-boundary cells inhibits the receptors' activity in those cells. In addition, light has been shed on several dynamical properties of the network, such as the refinement of Notch activity (Buceta, 2007).

At the time the role of Cut in the repression of Delta and Serrate expression was described, Cut and the concomitant restriction of ligand expression to non-boundary cells were postulated to be essential for the stability of the DV boundary. However, the other negative input of Wg into the Notch pathway through the activity of Dishevelled was not taken into account. In silico results have predicted that a general repression of the Wg pathway is required for stable activity of Notch at the DV boundary. In vivo results indicate that this repression takes place at the level or upstream of Armadillo. In order to be refractory to the inhibitory effect of Dishevelled on Notch, this repression should be taking place close to Dishevelled if not further upstream in the Wg signaling cascade (Buceta, 2007).

Finally, the conclusions are placed into a broader context. Boundary formation between adjacent rhombomeres in vertebrates relies on the same Wnt/Notch-dependent regulatory network. Therefore, it is speculated that boundary cells also need to be refractory to the Wnt signal to generate stable boundaries. To close, it is concluded that the robustness and stability of this network, in which the interconnectivity of the elements is crucial and even more important than the value of the parameters used, might explain its use in boundary formation in other multicellular organisms (Buceta, 2007).

A screen for genes that function in wg-induced leg-to-wing transdetermination

Many diverse animal species regenerate parts of an organ or tissue after injury. However, the molecules responsible for the regenerative growth remain largely unknown. The screen reported in this study aimed to identify genes that function in regeneration and the transdetermination events closely associated with imaginal disc regeneration using Drosophila melanogaster. A collection of 97 recessive lethal P-lacZ enhancer trap lines were screened for two primary criteria: first, the ability to dominantly modify wg-induced leg-to-wing transdetermination and second, for the activation or repression of the lacZ reporter gene in the blastema during disc regeneration. Of the 97 P-lacZ lines, six genes (Krüppelhomolog- 1, rpd3, jing, combgap, Aly and S6 kinase) were identified that met both criteria. Five of these genes suppress, while one enhances, leg-to-wing transdetermination and therefore affects disc regeneration. Two of the genes, jing and rpd3, function in concert with chromatin remodeling proteins of the Polycomb Group (PcG) and trithorax Group (trxG) genes during Drosophila development, thus linking chromatin remodeling with the process of regeneration (McClure, 2008).

There are three different mechanisms that organisms use to re-grow and replace lost or damaged body parts, and often, more than one mechanism can function within different tissues of the same organism. Muscle and bone, for example, repair themselves by activating a resident stem cell population, while the liver regenerates by compensatory proliferation of normally quiescent differentiated cells. Appendage/fin regeneration in lower vertebrates occurs by a process termed epimorphic regeneration, which proceeds in three distinct stages: (1) wound healing and migration of the surrounding epithelial cells to form the wound epidermis, (2) formation of the regeneration blastema -- a mass of undifferentiated and proliferating cells of mesenchymal origin and (3) regenerative outgrowth and pattern re-formation. Whether these diverse modes of regeneration share a common molecular and genetic basis is not known (McClure, 2008).

Regeneration in the Drosophila imaginal discs, the primordia of the adult fly appendages, closely parallels epimorphic limb/fin regeneration in lower vertebrates. Cells in the imaginal discs are rigidly determined to form specific adult structures (e.g., legs and wings) by the third larval instar. If the discs are fragmented at this time and cultured in vivo, they will regenerate. Disc regeneration begins 12 h after wounding, when transient heterotypic contacts are made between peripodial (squamous epithelium) and columnar cells (disc proper) near the cut edges of the wound. These initial contacts involve microvilli-like extensions and provide temporary wound closure. Then, approximately 24 h after wounding, homotypic cell contacts (between columnar or between squamous cells) are made involving the close apposition of cell membranes and cellular bridges, which eventually (48 h after wounding) restore the physical continuity of the disc. Before and during wound healing, cell division is randomly distributed throughout the disc. However, once completed (36-48 h after wounding), division is observed only in cells near the wound site. These cells are known as the regeneration blastema. Thus, like appendage regeneration in lower vertebrates, disc regeneration involves wound healing followed by blastema formation (McClure, 2008).

Blastema cells are responsible for the regeneration and repatterning of the entire missing disc fragment. Thus, these cells exhibit remarkable developmental plasticity. For example, in anterior- only leg disc fragments, some blastema cells will switch to posterior identity and establish a novel posterior compartment in the regenerate. This anterior/posterior conversion occurs during heterotypic wound healing, when hedgehog (hh)- expressing peripodial cells induce ectopic engrailed (en) expression in the apposing anterior columnar cells. In addition, the disc blastema, like its vertebrate counterpart, is able to form a normal regenerate (complete leg disc and adult leg) when isolated from the remaining disc fragment. Regenerative plasticity is also observed when a few blastema cells switch fate to that of another disc type (e.g., leg-to-wing), in a phenomenon known as transdetermination. Transdetermination events are closely associated with regenerative disc growth. Clonal analysis, for example, has shown that blastema cells first regenerate the missing disc structures, and only then, are they competent to transdetermine (McClure, 2008).

Little is known about how the regeneration blastema forms in the fragmented leg disc, although ectopic Wingless (Wg/Wnt1) expression is detected along the cut site, both prior to and during blastema formation. Wg is a developmental signal in many different tissues and animals; in flies Wg patterns all of the imaginal discs, functioning as both a morphogen and mitogen to regulate disc cell fate and growth. In lower vertebrates, Wnt ligands are key regulators of blastema formation during epimorphic regeneration. Thus, activation of Wg within the disc blastema is potentially important for regeneration. This idea is consistent with the observation that ubiquitous expression of wg during the second or third larval instars, in unfragmented leg discs, is sufficient to induce a regeneration blastema in the proximodorsal region of the disc, known as the weak point. Moreover, ubiquitous expression of wg mimics the pattern deviations associated with leg disc fragmentation and subsequent regeneration, including the duplication of ventral with concomitant loss of dorsal pattern elements and leg-to-wing transdetermination events. Thus, leg disc regeneration can be examined using two experimental protocols: fragmentation or ubiquitous wg expression. However, it is important to point out that only fragmentation-induced regeneration involves wound healing (McClure, 2008).

Precisely which molecules and signaling pathways are required for the process of regeneration remain poorly understood, partly because the organisms historically used to study regeneration (e.g., newts and salamanders) have been refractory to genetics and molecular manipulations. Recently, however, the use of new genetic techniques together with 'regeneration' model systems -- such as planarians, hydra and zebrafish have given researchers the opportunity to examine the mechanisms of regeneration and to identify the genes, proteins and signaling pathways that regulate different regenerative processes. For example, a large scale RNAi-based screen was performed to survey gene function in planarian tissue homeostasis and regeneration. Out of ~1000 genes examined, RNAi knock-down of 240 displayed regeneration-related phenotypes, including defects in wound healing, blastema formation and blastema cell differentiation. Despite these studies, however, it remains unclear whether regeneration requires only the modulation of genes expressed at the time of injury, the reactivation of earlier developmental genes and/or signaling pathways, or the activation of novel genes specific to the process of regeneration. Thus, a major interest in the field of regenerative biology is the identification of gene products that regulate blastema formation, blastema growth and regenerative cellular plasticity. A genetic screen, using wg-induced leg disc regeneration, aimed at identifying genes that regulate cellular plasticity and regeneration using Drosophila was carried out prothoracic leg discs. A collection of 97 recessive lethal P-element lacZ (PZ) insertion lines were screened for ectopic lacZ expression during wg-induced leg disc regeneration, and six genes were identified that function in wg-induced leg disc regeneration, including genes with functional ties to Wg signaling as well as chromatin remodeling proteins (McClure, 2008).

This study consisted of an enhancer trap screen designed to identify genes with changed gene expression during leg disc regeneration as well as required for regenerative proliferation and growth. The screen identified 19 genes that when heterozygous mutant (PZ/+), dominantly modify wg-induced leg-to-wing transdetermination, which serves as a functional assay for disc regeneration. Of the 19 genes, 37% are transcription factors or involved in transcriptional regulation (tai, Krh1, ken, jing, combgap (cg), rpd3 and Aly), 21% function in cell cycle regulation and growth (oho23B, S6k, polo and cycA), 10.5% play a role in protein secretion (Secβ61 and Syx13), and 31% are of other or unknown function [l(3)01629, CG30947, l(2)00248, l(3)05203, l(3)01344, Nup154]. The identification of transcription factors as the most frequent class of genes that modify wg-induced leg disc regeneration was similarly observed in a DNA microarray screen designed to identify genes enriched in leg disc cells that transdetermine to wing (Klebes, 2005). Together, these findings strongly suggest that transcription factors and their downstream targets play a prominent role in disc cell plasticity (McClure, 2008).

Using lacZ expression analyses, together with whole mount in situ hybridization experiments, the expression patterns of the 19 genes that modified wg-induced leg-to-wing transdetermination were verified. This analysis identified several different expression patterns upon wg-induced regeneration, including a loss of gene expression, ubiquitous expression and genes with expression limited to the regeneration blastema. Such observations indicate that a complex change of gene expression, both negative and positive, mediates the process of epimorphic regeneration. Six (jing, Alyi cg, rpd3, Kr-h1 and S6k) of the 19 modifiers displayed expression limited to the regeneration blastema, indicating that novel markers of regeneration and transdetermination have been identified. The blastema-specific expression patterns of jing, Aly, cg, Kr-h1, rpd3 and S6k raised the intriguing possibility that these genes may be functionally involved in the formation, cell proliferation or maintenance of the blastema during disc regeneration. Indeed, upon ubiquitous wg expression jing/+ animals rarely formed a regeneration blastema, indicating that two wild-type copies of jing are required for the initiation of the regenerative process. In contrast, Aly/+ and cg/+ animals formed a normal blastema, but only after a one-day delay. Therefore, two wild-type copies of the Aly and cg genes are required for the proper timing of regeneration. In addition, it was found that the frequency of blastema formation was reduced in rpd3/+ animals, implicating this gene in the process of regeneration. Interestingly, heterozygous mutations in all four of these genes (jing, Aly, cg and rpd3) strongly suppress wg-induced leg-to-wing transdetermination. It is speculated that the transdetermination frequency declines in these mutant animals because the initiation and/or timing of blastema formation is delayed. This idea is consistent with all previous work which has shown that blastema cells are only competent to transdetermine after they have regenerated the missing disc structures. Heterozygous mutations in Kr-h1 and S6k did not significantly alter the formation of the wg-induced regeneration blastema, however, these genes did affect regeneration-induced transdetermination. Such results suggest that Kr-h1 and S6k specifically function to modulate the cell fate changes that occur as a consequence of regeneration (McClure, 2008).

Investigations into the molecular basis of transdetermination have shown that inputs from the Wg, Decapentapelagic (Dpp) and Hedgehog (Hh) signaling pathways activate key selector genes out of their normal developmental context, such as ectopic Vg activation in the leg disc, which then drives cell-fate switches. Several of the genes identified in this screen have functional ties to Wg, Dpp and Hh signaling pathways. For example, Cg is a zinc-finger transcription factor that is required for proper transcriptional regulation of the Hh signaling effector gene Cubitus interruptus (Ci). In cg mutant wing and leg discs, Ci expression is lowered in the anterior compartment, resulting in the ectopic activation of wg and dpp and significant disc overgrowth. Another gene identified in this screen -- ken, functions in concert with Dpp to direct the development of the Drosophila terminalia. Further characterizations of whether these genes and other modifiers of transdetermination and regeneration affect Wg, Dpp and Hh expression and/or signaling may shed light on the regulation of regeneration and regeneration-induced proliferation and cell fate plasticity (McClure, 2008).

Notch signalling coordinates tissue growth and wing fate specification in Drosophila; Tissue size modulates the activity range of the signalling molecules Wingless and Vein

During the development of a given organ, tissue growth and fate specification are simultaneously controlled by the activity of a discrete number of signalling molecules. These two processes are extraordinarily coordinated in the Drosophila wing primordium, which extensively proliferates during larval development to give rise to the dorsal thoracic body wall and the adult wing. The developmental decision between wing and body wall is defined by the opposing activities of two secreted signalling molecules, Wingless and the EGF receptor ligand Vein. Notch signalling is involved in the determination of a variety of cell fates, including growth and cell survival. Evidence is presented that growth of the wing primordium mediated by the activity of Notch is required for wing fate specification. The data indicate that tissue size modulates the activity range of the signalling molecules Wingless and Vein. These results highlight a crucial role of Notch in linking proliferation and fate specification in the developing wing primordium (Rafel, 2008).

The expression of Wg in the most ventral part of the wing disc specifies the wing field at the same time as restricting Vn expression to the most dorsal part. Vn is required to block the responsiveness of body wall cells to Wg. Thus, the relative concentration of the diffusible proteins Wg and Vn experienced by disc cells directs their wing versus body wall fate. It is interesting to note that the expression of these two molecules is established long before the wing field is induced in the presumptive wing primordium. Wg expression starts long before wing field specification takes place, as revealed by the later induction of Nub expression and the reduction in the expression of the body wall cell marker Tsh. It is therefore proposed that tissue growth modulates the cellular response to these signalling molecules and controls, in time, wing fate specification. In the early wing primordium, Vn might reach every wing cell, thereby blocking responsiveness to Wg and repressing wing fate specification. Growth induced by Notch activity might pull the sources of Wg and Vn apart and, thus, most ventral cells might not sense sufficient Vn levels, so Wg would be able to induce wing fate. Interestingly, the overexpression of Wg or overactivation of its signalling pathway is able to bypass the requirement of growth in this process, indicating that the cells sense the relative levels of Wg and Vn. Once the wing field has been specified, Wg starts to be expressed along the presumptive wing margin, where it exerts a fundamental function in the maintenance of the Notch-dependent organizing center along the DV boundary. Note that the organizing activity of Notch at the DV boundary takes place long after the early function of Notch revealed in this work, which is involved in promoting growth and facilitating wing fate specification. As revealed by the expression of the Notch target E(spl)m-β, it is not until late in the second instar that the expression of Notch is restricted to the DV boundary. During the process of wing fate specification that takes place during second instar, it is uniformly expressed in the whole wing disc. These results imply that growth also facilitates the reiterative use of signalling molecules, such as Wg and Notch, to exert different functions during the development of a multicellular organ like the wing primordium (Rafel, 2008).

At the same time that wing and body wall fate specification takes place in the wing primordium, Vn is involved in the induction of apterous expression in the dorsal region. Consistent with the model proposed above, the activity of Vn, as monitored by the expression of apterous, was modulated by tissue growth. In the absence of Notch activity, even though Vn expression is not affected, Vn appears to reach every wing cell, as apterous expression was expanded ventrally. Increased levels of Wg expression or growth promoted by CycE appear to re-establish the dorsally restricted range of activity of Vn, as apterous expansion was blocked under these circumstances (Rafel, 2008).

Growth promoted by Notch has also been shown to be directly involved in the specification of the eye within the Drosophila eye-antenna primordium, a process that also depends upon the opposing activities of two secreted signalling molecules, in this case Dpp and Wg. Thus, Notch coordinates in a very elegant manner both eye and wing primordia tissue growth and eye/wing specification, by modulating the response of the cells to the activities of signalling molecules. These results indicate that the same mechanism might be commonly used in animal development to coordinate tissue growth and fate specification (Rafel, 2008).

The evolution of wings was crucial in the process of adaptation, allowing insects to escape predators or colonize new niches. The loss and recovery of wings has occurred during the course of evolution. This would suggest that wing developmental pathways are conserved in wingless insects and are being re-used. According to the current results, it is speculated that adaptive changes in animal size could modulate the cellular response to signalling molecules such as Wg, thereby helping to drive some of these extraordinary reversible transitions (Rafel, 2008).

Wg and Wnt4 provide long-range directional input to planar cell polarity orientation in Drosophila

Planar cell polarity (PCP) is cellular polarity within the plane of an epithelial tissue or organ. PCP is established through interactions of the core Frizzled (Fz)/PCP factors and, although their molecular interactions are beginning to be understood, the upstream input providing the directional bias and polarity axis remains unknown. Among core PCP genes, Fz is unique as it regulates PCP both cell-autonomously and non-autonomously, with its extracellular domain acting as a ligand for Van Gogh (Vang). This study demonstrates in Drosophila wings that Wg (Wingless) and dWnt4 (Drosophila Wnt homologue) provide instructive regulatory input for PCP axis determination, establishing polarity axes along their graded distribution and perpendicular to their expression domain borders. Loss-of-function studies reveal that Wg and dWnt4 act redundantly in PCP determination. They affect PCP by modulating the intercellular interaction between Fz and Vang, which is thought to be a key step in setting up initial polarity, thus providing directionality to the PCP process (Wu, 2013).

The data indicate that Wg/dWnt4 regulate the establishment of Fz–PCP axes by modulating the Fz–Vang intercellular interactions in a graded, dosage dependent manner. Consequently they might generate different levels of Fz–Vang interactions across a Wg/dWnt4 gradient experienced by cells. This process is reiterated across the tissue, and the directionality of Fz–Vang binding is subsequently reinforced by intracellular core PCP factor interactions. The data are consistent with a model in which Wg/dWnt4 generate a Fz ‘activity'), suggesting that both of these light sensors are necessary for light avoidance behavior.' gradient models. Accordingly, PCP axes are orientated towards the Wg/dWnt4 source, which is evident in (at least) the wing and eye. The early wing PCP axis (late larval to early pupal stages) correlates well with Wg/dWnt4 margin expression and, similarly, in the eye polarity is oriented in the dorsoventral axis towards the poles where Wg/Wnt4 are expressed. This model, relying on a Fz–Vang interaction, is also compatible with the addition of Fmi to this scenario, with intercellular (homophilic) Fmi–Fmi interactions also being required for PCP specification. As Fmi forms complexes with both Fz and Vang, the full complement of intercellular interactions includes Fz/Fmi–Fmi/Vang complexes, and these interactions would also be modulated by Wnt binding to Fz, either directly as proposed in this model or possibly by modulating the Fmi–Fmi interactions by Fmi being associated with Fz that is bound to different levels of Wg/Wnt4. In vivo, Fmi helps to enrich both Fz and Vang to the subapical junctional region, and Fmi–Fmi interactions bring Fz and Vang to close molecular proximity (Wu, 2013).

Intercellular Fmi–Fmi interactions are strong, as Fmi-expressing S2 cells form cell aggregates through homophilic Fmi interactions. The interaction between Fz and Vang is weaker, and cell–cell contacts between the two cell groups are infrequent. It was suggested that PCP signal sensing complexes include both Fmi and Fz on one cell interacting with Fmi/Vang at the surface of a neighbouring cell. Within these complexes, Fz is required for sending a polarity signal, whereas Fmi and Vang are involved in its reception, consistent with the data and model. Although it has been suggested that Fmi is capable of sensing Fz/Fmi signals in the absence of Vang, the 'Fz-sensing' capability of cells with Fmi alone (lacking Vang) is much weaker than that of cells with Vang. It will be interesting to determine if there are other PCP regulators directly involved in modifying Fmi–Fmi interactions (Wu, 2013).

How do these data relate to previous models and why was the Wg/Wnt4 requirement not observed before? Previous work attempted to address the role for the wing margin in PCP by examining either mutants affecting wing margin cells without eliminating wg/Wnt expression or in clones. Although cellular hairs near the site of wing margin loss point towards remaining wing margin areas, the effect Is considered weak. Potential effects were examined of Wnt LOF clones of Df(2L)NL, lacking wnt4, wg, wnt6 and wnt10. In contrast to the global reduction of Wg/Wnt4 through the temperature sensitive wg allele, such clones cause only mild PCP perturbations. There are several reasons why clonal loss of Wnt expression in the margin only mildly affects PCP orientation: cells can respond to Wnts from several sources/cells from remaining Wnt-expressing wing margin regions; polarization strengths (measured by nematic order) in the first few rows of cells near the margin are much weaker than those in cells further away (at 14-17 h APF) and weak PCP reorientation in cells neighbouring wing margin clones could thus reflect the initial weak polarization in these cells; and PCP orientation changes from its initial radial polarity towards the proximodistal polarity during hinge contraction morphogenesis and associated cell flow, probably leading to significant corrections of subtle defects near the margin. Similarly, PCP orientation in cells near the margin is only very weak early (at 14-16 h APF), probably because cells close to the Wnt-producing cells are exposed to saturated Wnt levels (and not a Wnt gradient), or because the presence of other organizers (directing polarity parallel to the margin) weakens the effect of Wnts. PCP in these cells is established/corrected through more local interactions during the feedback loops among neighbouring cells (Wu, 2013).

To determine the direct role for Wg/Wnts on Fz–PCP signalling, it was examined at pupal stages, as the patterning role for canonical Wg signalling is much reduced then and PCP still correlates well with Wg/Wnt4 expression. Importantly, Wnt4 does not affect expression of patterning genes through canonical signalling at larval or pupal stages, yet Wnt4 alters PCP orientation, consistent with the model that Wnt4/Wg act directly on Fz-PCP interactions. The observation that Wnt4 requires Fz to affect neighbouring cells further supports this model. It is likely that, as well as the Wg/Wnt4 input and mechanism identified in this study, both early and late PCP axes depend on further cues, provided for instance by the parallel Ft/Ds-PCP system or other morphogenetic organizers. Strikingly, such a scenario would suggest that Wg regulates PCP directionality through both PCP systems, affecting Fz-PCP interactions directly and through canonical Wg signalling transcriptionally regulating graded fj and ds expression in eyes and wings. In summary, these data provide insight into Wnt-mediated mechanisms to directly regulate long-range Fz–PCP orientation by modulating Fz–Vang/PCP interactions during tissue morphogenesis (Wu, 2013).

Dual role of Wingless signaling in stem-like hematopoietic precursor maintenance in Drosophila

In Drosophila, blood development occurs in a specialized larval hematopoietic organ, the lymph gland (LG), within which stem-like hemocyte precursors or prohemocytes differentiate to multiple blood cell types. This study shows that components of the Wingless (Wg) signaling pathway are expressed in prohemocytes. Loss- and gain-of-function analysis indicates that canonical Wg signaling is required for maintenance of prohemocytes and negatively regulates their differentiation. Wg signals locally in a short-range fashion within different compartments of the LG. In addition, Wg signaling positively regulates the proliferation and maintenance of cells that function as a hematopoietic niche in Drosophila, the posterior signaling center (PSC), and in the proliferation of crystal cells. These studies reveal a conserved function of Wg signaling in the maintenance of stem-like blood progenitors and reveal an involvement of this pathway in the regulation of hemocyte differentiation through its action in the hematopoietic niche (Sinenko, 2009).

The results demonstrate a requirement for Wg signaling in hematopoiesis, adding to the role of the Notch, Hedgehog, Jak/STAT, and Ras/Raf/MAPK pathways in various aspects of blood development in Drosophila. It is known that the Wg/Wnt signaling pathway is required for hematopoietic development in mammals and its deregulation is involved in leukemogenesis in humans. Although a large number of studies have explored its function, the role of Wnt signaling in mammalian hematopoiesis remains unresolved due to the often conflicting results obtained from in vitro, in vivo, and misexpression experiments. Independent studies of β-catenin knockout in mice have shown reduced self-renewal capacity of HSCs, while other studies have suggested that Wnt signaling may still occur in HSCs lacking both β- and γ-catenins. Loss of Wnt signaling derived from the endosteal HSC niche inhibits the ability of HSCs to reconstitute an immune system due to loss of quiescence (Fleming, 2008). It is not clear from these studies if the defects in reconstitution are due to engraftment problems or stem cell exhaustion. In the current studies advantage was taken of Drosophila genetics to help clarify the role of Wg signaling in hematopoiesis. The lymph gland allows an in vivo model system in which to visualize the dynamic interactions of Wg with other pathways and dissect the genetic networks involved in the maintenance of stem-like blood progenitors (Sinenko, 2009).

As in vertebrates, hematopoietic development in Drosophila is controlled at the level of multipotential stem-like cells that are maintained and eventually differentiate into various mature hemocyte lineages. Time course studies show that Wg and other components of the pathway are withdrawn from cells upon their differentiation but maintained in stem-like hemocyte precursors. Loss- and gain-of -function analyses indicate that Wg signaling operates through a short-range mechanism in hemocyte precursors, where local concentrations of Wg negatively control their differentiation. It is important to note that this function of Wg signaling is later than its requirement for promoting development of cardiogenic mesoderm during early embryogenesis, as dome-Gal4 and Wg expressions in LG overlap only during larval instar. The function of Wg signaling in stem-like hemocyte precursors is conserved, as the loss of Wnt3a or β-catenin in mammalian HSCs significantly impairs their self-renewal, while activation of the pathway exhausts HSCs through a block in differentiation that increases their self-renewal. Drosophila E-cadherin has been identified as a target of Wg function in prohemocytes, where it likely contributes to proper zonation of the LG. Control of E-cadherin expression by the Wnt/Wg pathway has been shown in other invertebrate and vertebrate tissues, indicating a conservation of this function. The observed deficiency in DE-cadherin expression in stem-like progenitors lacking Wg signaling may provide an additional similarity to the lack of engraftment of mammalian HSCs exposed to a Wnt-deficient niche (Sinenko, 2009).

During hematopoietic development, Wg signaling operates locally in different compartments of the LG. Importantly, Wg is required for maintaining proper development of the PSC, the hematopoietic niche. This indicates that Wg signaling controls maintenance of hematopoietic progenitors by a dual mechanism, a direct cell autonomous function in prohemocytes, and an indirect regulation that depends on its role in the development of the niche. An analogous mechanism has been shown in the mouse system, where a microenvironment lacking Wnt signaling fails to maintain HSCs in a quiescent state, reducing their long-term reconstituting activity (Sinenko, 2009).

The maintenance of stem-like hemocyte precursors is mediated by integration of a number of signaling pathways. In addition to the intrinsic function of the Wg pathway in prohemocytes, other niche generated extrinsic signals, such as Hh and the JAK/STAT pathways, participate in this process. Wg/Wnt signaling function is important in intestinal stem cells of Drosophila and mammals, as well as in mammalian HSCs, T cells, and B cells. It is clear that the functions of Wg signaling in hematopoietic processes of Drosophila are conserved during mammalian hematopoiesis (Sinenko, 2009).

Previous studies have suggested that the function of Fz and DFz2 receptors can be either redundant or distinct depending on the signaling context. In this regard, it was found that function of Fz receptors is redundant in prohemocyte maintenance while only DFz2 is utilized for Wg signaling in cells of the hematopoietic niche and the crystal cells (Sinenko, 2009).

The ability to observe the dynamic pattern of wingless expression across developmental stages established its in vivo role in the maintenance of stem-like progenitors. The advantage of being able to manipulate individual cell populations within each compartment of the lymph gland permitted dissection of the direct versus indirect effects of Wg on blood progenitors. This versatility in analysis emphasizes the advantages of Drosophila blood development as a model of hematopoiesis in which to further investigate the complex events of Wg signaling and its intricate interaction with other signaling networks during hematopoietic development (Sinenko, 2009).


The spatial and temporal pattern of expression of enhancer trap lines reporting on the wingless and engrailed genes was characterized in the adult antenna of Drosophila. wg is expressed in a subset of cells in the third and fifth segments of the antenna. In the third segment expression is restricted to a crescent-shaped area in the ventral-most region of the segment. In the fifth segment, part of the base of the arista, a subset of cells express wg, while engrailed expression is restricted to subsets of cells in the second, third, and fifth segments. The time courses of expression seen for wg and en, although different from one another, reveal a complex well-controlled pattern of temporal expression, providing evidence that regulatory mechanisms are preserved throughout the life span of the adult fly. Upon eclosion (hatching) the level of wg expression is at its maximum and the levels decline so that between days 10 and 15, expression is barely detectable. Re-expression is seen and peaks between 20 and 50 days of adult life. The temporal pattern of en expression is different from that seen with wingless. Expression is at its maximum upon eclosion and declines slowly over the entire life span of the fly. Altering the life span demonstrates that the temporal patterns of expression of both wg and en are linked to life span. One correlation that has been noted is an inverse relationship between transcription from the wg gene and fertility. These studies suggest that the expression of wg and en in the adult antenna is controlled by age-dependent mechanisms (Rogina, 1997).

Non-autonomous crosstalk between the Jak/Stat and Egfr pathways mediates Apc1-driven intestinal stem cell hyperplasia in the Drosophila adult midgut

Inactivating mutations within adenomatous polyposis coli (APC), a negative regulator of Wnt signaling, are responsible for most sporadic and hereditary forms of colorectal cancer (CRC). This study used the adult Drosophila midgut as a model system to investigate the molecular events that mediate intestinal hyperplasia following loss of Apc in the intestine. The results indicate that the conserved Wnt target Myc and its binding partner Max are required for the initiation and maintenance of intestinal stem cell (ISC) hyperproliferation following Apc1 loss. Importantly, it was found that loss of Apc1 leads to the production of the interleukin-like ligands Upd2/3 and the EGF-like Spitz in a Myc-dependent manner. Loss of Apc1 or high Wg in ISCs results in non-cell-autonomous upregulation of upd3 in enterocytes and subsequent activation of Jak/Stat signaling in ISCs. Crucially, knocking down Jak/Stat or Spitz/Egfr signaling suppresses Apc1-dependent ISC hyperproliferation. In summary, these results uncover a novel non-cell-autonomous interplay between Wnt/Myc, Egfr and Jak/Stat signaling in the regulation of intestinal hyperproliferation. Furthermore, evidence is presented suggesting potential conservation in mouse models and human CRC. Therefore, the Drosophila adult midgut proves to be a powerful genetic system to identify novel mediators of APC phenotypes in the intestine (Cordero, 2012).

Using the Drosophila adult midgut as a model system this study has uncovered a key set of molecular events that mediate Apc-dependent intestinal hyperproliferation. The results suggest that paracrine crosstalk between Egfr and Jak/Stat signaling is essential for Apc1-dependent ISC hyperproliferation in the Drosophila midgut (Cordero, 2012).

Previous studies have demonstrated that Myc depletion prevents Apc-driven intestinal hyperplasia in the mammalian intestine. This study provides evidence that such a dependency on Myc is conserved between mammals and Drosophila. It was further demonstrated that endogenous Myc or Max depletion causes regression of an established Apc1 phenotype in the intestine. Taken together, these data highlight the importance of developing Myc-targeted therapies to inhibit Apc1-deficient cells. Since not all roles of Myc are Max dependent, present efforts are focused on developing inhibitors that interfere with Myc binding to Max and would therefore be less toxic. These data provide the first in vivo evidence in support of the Myc/Max interface as a valid therapeutic target for CRC (Cordero, 2012).

Recent work showed that loss of the tuberous sclerosis complex (TSC) in the Drosophila midgut leads to an increase in cell size and inhibition of ISC proliferation. Reduction of endogenous Myc in TSC-deficient midguts restored normal ISC growth and division. These results might appear contradictory to the current work, where Myc is a positive regulator of ISC proliferation. However, in both scenarios, modulation of Myc levels restores the normal proliferative rate of ISCs (Cordero, 2012).

Previous work in mouse showed that Myc upregulation is essential for Wnt-driven ISC hyperproliferation in the intestine. However, Myc overexpression alone only recapitulates some of the phenotypes of hyperactivated Wnt signaling. This study shows that overexpression of Myc is capable of mimicking some aspects of high Wnt signaling in the Drosophila midgut, such as the activation of Jak/Stat, but is not sufficient to drive ISC hyperproliferation. Multiple lines of evidence have shown that forced overexpression of Myc in Drosophila and vertebrate models results in apoptosis partly through activation of p53. Therefore, driving ectopic myc alone is unlikely to parallel Apc deletion in the intestine, where the activation of multiple pathways downstream of Wnt signaling is likely to contribute cooperatively to hyperproliferation (Cordero, 2012).

Understanding the contribution of Jak/Stat signaling to the Apc phenotype in the mammalian intestine has been complicated by genetic redundancy between Stat transcription factors. Constitutive deletion of Stat3 within the intestinal epithelium slowed tumor formation in the ApcMin/+ mouse, but the tumors that arose were more aggressive and ectopically expressed Stat1. Using the Drosophila midgut, direct in vivo evidence is provided that activation of Jak/Stat signaling downstream of Apc1/Myc mediates Apc1-dependent hyperproliferation (Cordero, 2012).

The data on the Drosophila midgut and in mouse and human tissue samples suggest that blocking Jak/Stat activation could represent an efficacious therapeutic strategy to treat CRC. Currently, there are a number of Jak2 inhibitors under development and it would be of great interest to examine whether any of these could modify the phenotypes associated with Apc loss (Cordero, 2012).

Previous studies have demonstrated that enterocytes (ECs) are the main source of Upds/interleukins in the midgut epithelium. The results show that activation of Wnt/Myc signaling in ISCs leads to non-autonomous upregulation of upd3 within ECs. Furthermore, Spitz/Egfr signaling appears to mediate the paracrine crosstalk between Wnt/Myc and Jak/Stat in the midgut. Overexpression of a dominant-negative Egfr in ECs blocks upd3 upregulation and ISC hyperproliferation in response to high Wnt signaling. A previous EC-specific role for Egfr has been demonstrated during midgut remodeling upon bacterial damage. Nevertheless, the downstream signaling that mediates such a role of Egfr remains unclear given that the activation of downstream MAPK/ERK occurs exclusively within ISCs. Therefore, the current evidence would suggest that Egfr activity in ECs does not involve cell-autonomous ERK activation. Consistent with these observations, p-ERK (Rolled -- FlyBase) localization was not detected outside ISCs in response to either Apc loss or overexpression of wg in the Drosophila midgut. Reports on the Apc murine intestine have also failed to detect robust ERK activation. Since MAPK/ERK is only one of the pathways activated downstream of Egfr, it is possible that ERK-independent mechanisms are involved. It is important to explore this further because ERK-independent roles of Egfr signaling have not yet been reported in Drosophila. Thus, what mediates Upd3 upregulation in ECs in response to Egfr signaling activation and whether Spitz-dependent upregulation of Upd3 involves a direct role of Egfr in ECs remain unclear. A potential alternative explanation is that intermediate factors induced in response to Spitz/Egfr activation in ISCs might drive Upd3 expression (Cordero, 2012).

In summary, this study has elucidated a novel molecular signaling network leading to Wnt-dependent intestinal hyperproliferation. Given the preponderance of APC mutations in CRC, the integration of Egfr and Jak/Stat activation might be a conserved initiating event in the disease (Cordero, 2012).

wingless signaling regulates the maintenance of ovarian somatic stem cells

Identifying the signals involved in maintaining stem cells is critical to understanding stem cell biology and to using stem cells in future regenerative medicine. In the Drosophila ovary, Hedgehog is the only known signal for maintaining somatic stem cells (SSCs). Wingless (Wg) signaling is also essential for SSC maintenance in the Drosophila ovary. Wg is expressed in terminal filament and cap cells, a few cells away from SSCs. Downregulation of Wg signaling in SSCs through removal of positive regulators of Wg signaling, dishevelled and armadillo, results in rapid SSC loss. Constitutive Wg signaling in SSCs through the removal of its negative regulators, Axin and shaggy, also causes SSC loss. Also, constitutive wg signaling causes over-proliferation and abnormal differentiation of somatic follicle cells. This work demonstrates that wg signaling regulates SSC maintenance and that its constitutive signaling influences follicle cell proliferation and differentiation. In mammals, constitutive ß-catenin causes over-proliferation and abnormal differentiation of skin cells, resulting in skin cancer formation. Possibly, mechanisms regulating proliferation and differentiation of epithelial cells, including epithelial stem cells, are conserved from Drosophila to man (Song, 2003).

Two or three GSCs, surrounded by three groups of somatic cells at the germarial tip [terminal filament cells (TFs), cap cells (CPCs) and inner germarial sheath (IGS) cells] produce all germline cells in the ovariole. These stem cells directly contact cap cells and are posterior to terminal filament cells. After a GSC divides, the daughter still in contact with cap cells remains a stem cell, whereas the daughter that is more distant from cap cells differentiates into a cystoblast. However, if both daughters remain in contact with cap cells, they both become stem cells. Consistent with the existence of niches, terminal filament, cap cells and IGS cells express several genes that are important for GSC function. In addition, the failure of GSCs to stay in their niches, because of defects in DE-cadherin-mediated cell adhesion, results in stem cell loss (Song, 2003).

Two or three SSCs, located in the middle of the germarium, generate several different types of somatic follicle cells in egg chambers. These stem cells directly interact with a posterior group of IGS cells that may function as the niche for SSCs. Their immediate progeny, also known as follicle cell progenitors, can divide and generate differentiated follicle cells that encapsulate germ cysts to form individual egg chambers. hh is expressed in terminal filament and cap cells, and directly regulates SSC maintenance and proliferation. fs(1)Yb regulate S follicle cell proliferation through the regulation of hh expression in the Drosophila ovary (Song, 2003).

Wg produced from terminal filament and cap cells may reach SSCs at a distance of a few cells by either diffusion or active transport, and then Wg directly controls SSC maintenance. Furthermore, correct intermediate levels of wg signaling seem to be important for maintaining SSCs in the Drosophila ovary. Reduction of wg signaling in SSCs by removal of positive regulators such as arm and dsh causes rapid SSC loss, as does constitutive wg signaling in SSCs by removal of negative regulators such as Axn and sgg. wg signaling maintains SSCs through several possible mechanisms: (1) wg signaling could be required for SSC self-renewal and/or survival; (2) it could maintain the association of SSCs with IGS cells, and/or (3) both mechanisms could work simultaneously. DE-cadherin-mediated cell adhesion has been shown to be important for keeping SSCs in their niche; it also shares arm as a common component with wg signaling. wg signaling is known to regulate levels of arm, which are also important for DE-cadherin-mediated cell adhesion. Thus, it is possible that wg signaling regulates cell adhesion between SSCs and their niches. In addition, arm mutant clonal analysis strongly argues that wg signaling must also directly regulate SSC self-renewal and/or survival. arm2 mutant SSC clones are lost very quickly over time in comparison with wild-type SSC clones, and the arm2 mutation primarily affects wg signaling but does not disrupt DE-cadherin-mediated cell adhesion. Therefore, wg signaling controls SSC maintenance through regulating SSC self-renewal/survival and/or cell adhesion between SSCs and their niche cells. The temperature-sensitive allele of wg gives very mild phenotypes in follicle cell production, however, removal of wg downstream components has a dramatic impact on SSC maintenance. In Drosophila, there are six other wg-related genes. This raises an interesting possibility that other wg-like molecules could also be involved in regulating SSC maintenance (Song, 2003).

In addition to wg signaling, hh signaling is also essential for SSC maintenance and proliferation. Hyperactive hh signaling causes follicle cell over-proliferation and abnormal differentiation of follicle cells. Disrupting hh signaling in SSCs by removing the function of hh downstream components such as Smoothened and Cubitus interruptus results in rapid SSC loss. Similarly, reduction or elimination of wg signaling also causes rapid SSC loss. Removal of patched, a negative regulator of the hh pathway, stabilizes SSCs. However, SSCs mutant for negative regulators for the wg pathway, sgg and Axn, are destabilized. All the evidence indicates that wg and hh may use different mechanisms to regulate SSCs in the Drosophila ovary (Song, 2003).

Constitutive wg signaling increases the division rates of early follicle cell progenitors in the germarium. When Fz2, dsh and activated arm are over-expressed, extra follicle cells accumulate in the ovarioles, suggesting that hyper-activation of wg signaling causes over-proliferation of follicle cells. Furthermore, sgg or Axn mutations cause over-proliferation of follicle cells, resulting in the formation of extra follicle cells that accumulate outside egg chambers. These cells are not mitotically active and usually assume some stalk cell characteristics. These results suggest that production of extra follicle cells by excessive wg signaling is because of higher mitotic activities of progenitors and/or SSCs in the germarium. It is important to note that sgg mutations are more potent than Axn in stimulating the proliferation of follicle cell progenitors. The different potencies may be because of differences in how these mutations affect wg signaling. Alternatively, because sgg negatively regulates hh signaling, sgg could be involved in negatively regulating both hh and wg signaling in the ovary. It has been demonstrated that excessive hh signaling causes extra follicle cells to accumulate outside egg chambers. Therefore, it might be probable that sgg is involved in regulating both hh and wg signaling pathways in follicle cells of the Drosophila ovary (Song, 2003).

This study also demonstrates that constitutive wg signaling disrupts the normal differentiation of somatic follicle cells. Mutant Axn or sgg follicle cells in and outside the germarium express higher levels of Hts in their membranes and tend to accumulate between egg chambers. In ovarioles that contain a majority of mutant follicle cells, germline cysts fail to undergo normal morphological changes necessary for proper encapsulation by follicle cells, although they are wild type, suggesting that the mutant follicle cells are defective in their interactions with germ cells. Although some of them are recruited to egg chambers, these mutant follicle cells have abnormal morphologies (e.g. smaller and irregular sizes). Huli tai shao is present not only on spectrosomes in GSCs, cystoblasts and fusomes in early germline cysts, but also on the membranes of somatic follicle cells. The abnormal follicle cell phenotype may be because of abnormal levels of Hts, which may prevent follicle cells from shape changes and growth. The extra mutant follicle cells accumulating outside egg chambers express Lamin C and do not divide similar to stalk cells. However, unlike stalk cells, they express high levels of Fas3. Similar to the mutant follicle cells in the germarium, the mutant follicle cells that are recruited to egg chambers also express high levels of Hts. Unlike the follicle cells in the germarium, the cells fail to express high levels of Fas3. These results indicate that constitutive wg signaling in follicle cells disrupts proper follicle cell differentiation (Song, 2003).

A dynamic population of stromal cells contributes to the follicle stem cell niche in the Drosophila ovary

Epithelial stem cells are maintained within niches that promote self-renewal by providing signals that specify the stem cell fate. In the Drosophila ovary, epithelial follicle stem cells (FSCs) reside in niches at the anterior tip of the tissue and support continuous growth of the ovarian follicle epithelium. This study demonstrates that a neighboring dynamic population of stromal cells, called escort cells, are FSC niche cells. Escort cells produce both Wingless and Hedgehog ligands for the FSC lineage, and Wingless signaling is specific for the FSC niche whereas Hedgehog signaling is active in both FSCs and daughter cells. In addition, this study shows that multiple escort cells simultaneously encapsulate germ cell cysts and contact FSCs. Thus, FSCs are maintained in a dynamic niche by a non-dedicated population of niche cells (Sahai-Hernandez, 2013).

Taken together, the results of this study challenge the notion that the FSC niche is maintained by gradients of ligands produced solely at distant sites. Instead, the data indicate that the FSC niche has a more canonical architecture in which at least some key niche signals are produced locally, although the FSC niche might also differ from other well-characterized niches in some ways, such as the extent to which it remodels during adulthood. Notably, the results do not contradict the observation that Hh protein relocalizes from apical cells to the FSC niche during changes from a poor to a rich diet, as the flies were consistently maintained on nutrient-rich media. It will be interesting to investigate how such distantly produced ligands interact with locally produced niche signals to control FSC behavior during normal homeostasis and in response to stresses (Sahai-Hernandez, 2013).

In addition, the results confirm and extend the conclusion that Wg acts specifically on FSCs and ISCs, thus highlighting the role of Wg as a specific epithelial stem cell niche factor. As in other types of stem cell niches, this specificity could be achieved through multiple mechanisms, including local delivery of the Wg ligand to the niche and crosstalk with other pathways such as Notch and Hh, which are known to interact with the Wg pathway. Although the precise function(s) of Wg signaling in FSCs is unclear, the observation that a reduction in Wg ligand results in a backup of cysts near the FSC niche at the region 2a/2b border and fused cysts downstream from the FSC niche suggests that one role is to promote FSC proliferation. In addition, the finding that FSC daughter cells with ectopic Wg signaling fail to form into a polarized follicle epithelium suggests that Wg signaling might also promote self-renewal in FSCs by suppressing the follicle cell differentiation program. By contrast, observations and published studies indicate that Hh signaling is not specific for the FSC niche but instead constitutes a more general signal that derives from multiple sources and regulates proliferation and differentiation in both FSCs and prefollicle cells. Consistent with this conclusion, Hh signaling is active throughout the germarium and is required both in FSCs to promote self-renewal and in prefollicle cells to promote development toward the stalk and polar lineages (Sahai-Hernandez, 2013).

Finally, multicolor labeling of somatic cells in the germarium indicated that multiple densely packed escort cell membranes surround region 2a cysts and contact the FSC niche. Although the possibility cannot be ruled out that one or more cells in this region are dedicated FSC niche cells, observations strongly suggest that at least some escort cells contribute to both germ cell development and the FSC niche. Since these escort cells are dynamic, constantly changing their shape and position to facilitate the passage of germ cell cysts, it is perhaps somewhat surprising that the FSCs are so stable in the tissue. Indeed, the rate of FSC turnover is comparable to that of female GSCs, which are maintained by a dedicated and more static niche cell population. It will be interesting to investigate how this dynamic population of escort cells is able to maintain such a stable microenvironment for the FSCs. One possibility is that redundant sources of niche signals may allow niches of this type to partially break down and reform as needed to rapidly accommodate the changing demands of the tissue (Sahai-Hernandez, 2013).

The current observations reinforce several themes that are emerging from recent studies of stem cell niches in different epithelial tissues. First, as in the FSC niche, the Wnt/Wg signaling pathway is a key stem cell niche signal in many Drosophila and mammalian epithelial tissues. Second, in several epithelial tissues, the stem cell self-renewal signals are also known to be produced by differentiated cells rather than a dedicated niche cell population. For example, Drosophila ISCs of the gut receive self-renewal signals from both nearby enterocytes and the surrounding visceral muscle. Likewise, mammalian ISCs at the base of the crypt receive self-renewal signals from Paneth cells, which are adjacent secretory cells with antimicrobial functions. Lastly, several epithelial niches have recently been shown to have a transitory capacity that may resemble the dynamic nature of the FSC niche. For example, stem cell niches can form de novo in the Drosophila intestine to accommodate increased food availability, and in the mammalian skin in response to hyperactive Wnt signaling. In addition, mammalian ISCs produce niche cells in vivo and can spontaneously reform a niche in culture. In all of these examples, it seems likely that the relationship between the epithelial stem cell and its niche is not static, but instead flexible and dynamic. Further studies of the Drosophila FSC niche and these other experimental models will continue to provide insights into the mechanism by which a dynamic epithelial stem cell niche functions (Sahai-Hernandez, 2013).

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).

Return: Wingless Developmental biology part 1/2

wingless continued: Biological Overview | Evolutionary Homologs | Transcriptional regulation |Targets of Activity | Protein Interactions | mRNA Transport | Effects of mutation | References

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