Fasciclin 2

DEVELOPMENTAL BIOLOGY

Effects of Mutation and Misexpression

In Fas2 mutants, the CNS displays no gross phenotype, but the MP1 fascicle fails to develop, and growth cones of specific neurons fail to recognize one another or other axons that normally join the MP1 pathway (Grenningloh, 1991). Four classes of abnormal phenotypes are observed: 'bypass' phenotypes, in which axons fail to defasciculate at the choice point where they would normally enter their muscle target region; 'detour' phenotypes, in which these bypass growth cones enter their target regions at different locations; 'stall' phenotypes, in which axons that enter their muscle target region fail to defasciculate; and 'misroute' phenotypes, in which growth cones are diverted into abdormal pathways (Lin, 1994a). Gain of function phenotypes in which Fas2 is expressed in specific axons, show altered fasciculation by abnormally fusing pathways together, and sometimes preventing normal defasciculation (Lin, 1994b).

beaten path codes for a secreted immunoglobulin domain protein that is required for subsets of motor axons to correctly defasciculate from other motor axons at specific choice points. Without this protein, motor axons do not properly enter their muscle target regions. The mutant phenotype resembles a result from previously published experiments where motor axon adhesivity is increased by overexpression of the cell adhesion molecule Fasciclin II. This similarity suggests that Beat may normally work to oppose Fas II-mediated adhesivity and allow defasciculation of otherwise adherent sets of motor axons. Genetic analysis supports this interpretation: when motoneurons have reduced quantities of Fas II, they have less need for beat function, as evidenced by suppression of the beat bypass phenotype double mutant embryos. A similar interaction is observed with the connectin gene, which encodes another cell adhesion protein expressed by a subset of motoneurons (Fambrough, 1996).

The axonal adhesion molecule Fasciclin II and the secreted anti-adhesion molecule Beaten path have critical roles in the development of at least one set of sensory organs, the larval visual organs called Bolwig's organ (BOs). Instead of playing a role in axon defasciculation, in the development of Bolwig's organ, Beat appears to play a role in cell adhesion, a related phenomenon. In normal development, secretion of Beaten path by cells of the optic lobe (a portion of the brain that processes visual information sent from the eyes) allows the Fasciclin II-expressing larval visual organ cells to detach from the optic lobes as a cohesive cell cluster. Thus mechanisms guiding neuronal development may be shared between motoneurons and sensory organs, and adhesion and anti-adhesion is likely to be critical for early steps in development of the larval visual system (Holmes, 1999).

The larval visual system (LVS) is a relatively simple sensory system composed of BOs: two clusters, each composed of 12 photoreceptor cells from which axons extend in a single fascicle to the brain. Development of the LVS differs in several significant ways from that of the motoneurons. Notably, the axon tracks of the Drosophila CNS and PNS develop through axonal outgrowth and migration, characterized by growth cone extension and selective fasciculation. In contrast, the initial connections of the pioneer axons of the LVS are established when the BOs are close to their target cells, the developing larval brain. Thus, it is neuronal cell bodies, the BOs, that move through a complex environment during LVS development, rather than the axons as in development of the CNS and PNS. It is currently unclear whether the anterior relocation of the BOs occurs as a passive consequence of head involution, or whether an active migratory process is involved. Preliminary analysis of two identified mutations, not enough anterior extension and out of place, which result in the failure of the BOs to attain their proper location, reveals that, despite failures in BO development, head involution appears to occur normally (Holmes, 1998a). The apparent genetic separation of BO movement and head involution suggests the processes may also be mechanistically separable. However, the developmental mechanism driving BO migration remains to be determined (Holmes, 1999).

The BOs arise from cells of the optic lobe placodes (OLs), ectodermal tissues that will go on to form the optic lobes of the adult fly. The BOs detach from the OLs and remain at the periphery of the embryo when the OLs invaginate. As the BOs detach from the OLs, axons extend from the BOs and establish initial connections with the brain, which is adjacent to the OLs. Therefore, the axons need only navigate a relatively short distance to reach their targets in the brain at this early stage of development. The BO cells remain clustered for the remainder of development, during which they migrate anteriorly and the Bolwig's nerves (BNs) increase significantly in length. Therefore, proper development of the LVS requires that the BO precursors detach from the OLs, that the pioneer connections between the BOs and the brain be established and maintained, that axons properly fasciculate with the pioneer axons, and that the BOs migrate correctly to their final positions (Holmes, 1999).

By genetic analysis of LVS development, a mutant allele of beat has been identifed that disrupts early stages of BO development (Holmes, 1998). Beat is expressed in a cluster of cells in the OLs before the BOs become distinct. In LVS development, as in motor neuron development, beat interacts genetically with fas II. A mutation in fas II also disrupts LVS development, resulting in a phenotype that is, in some ways, the opposite of that resulting from mutations in beat. Conversely, overexpression of fas II in the BO causes defects in BO development resembling those that result from mutations in beat. By directing expression of beat to either the BOs or the OLs, the LVS phenotype of beat mutations is reversed. Together, these results demonstrate that detachment of the BO precursors from the OL may involve interaction between Beat and Fas II. Thus, at least some of the mechanisms important for regulating intercellular interactions during motor axon development also function to guide development of sensory organs, suggesting that some of the general principles of neuronal development may overlap in these two different neuronal systems (Holmes, 1999).

It has been hypothesized that synaptic strength is directly related to nerve terminal morphology. This has been tested through analysis of synaptic transmission at Drosophila neuromuscular junctions with a genetically reduced number of nerve terminal varicosities. Synaptic transmission would decrease in target cells with fewer varicosities if there is a relationship between the number of varicosities and the strength of synaptic transmission. Animals that have an extreme hypomorphic allele of the gene for the cell adhesion molecule Fasciclin II possess fewer synapse-bearing nerve terminal varicosities; nevertheless, synaptic strength is maintained at a normal level for the muscle cell as a whole. Fewer failures of neurotransmitter release and larger excitatory junction potentials from individual varicosities, as well as more frequent spontaneous release and larger quantal units, provide evidence for enhancement of transmitter release from varicosities in the mutant. Ultrastructural analysis reveals that mutant nerve terminals have bigger synapses with more active zones per synapse, indicating that synaptic enlargement and an accompanying increase in synaptic complexity provide for more transmitter release at mutant varicosities. These results show that morphological parameters of transmitting nerve terminals can be adjusted to functionally compensate for genetic perturbations, thereby maintaining optimal synaptic transmission (Stewart, 1996).

A novel method of P-element mutagenesis is described for the isolation of mutants affecting the development of the Drosophila compound eye. It exploits the interaction between the Bride of Sevenless (Boss) ligand and the Sevenless (Sev) receptor tyrosine kinase; acting in concert, they trigger the formation of the UV-sensitive photoreceptor neuron, R7. In live flies, transposition of a boss cDNA transgene, in an otherwise boss mutant background, was used as a "phenotypic trap" to identify enhancers expressed during a narrow time window in eye development. Using a rapid behavioral screen, more than 400,000 flies were tested for restoration of R7. Because R7 is the primary receptor for UV light, flies containing R7 can be easily separated from flies lacking R7 (either boss or sevenless mutant flies). When given a choice between UV and visible light, 90% of boss mutant flies move toward the visible light; in contrast, 90% of the wild-type flies move toward the UV light. UV-tactic behavior can be conferred upon boss mutant flies by expressing boss in the eye disc during a period in which Sev-expressing precursor cells are competent to respond. Using boss in place of lacZ as an enhancer-trap marker enablesa behavioral screening for genes expressed in the eye imaginal disc within the first 30 hours of ommatidial development. Thus, promoter driven expression of boss in a boss mutant background restores R7 function and UV-tactic behavior. Some 1,800 R7-containing revertant flies were identified. Among these, 21 independent insertions with expression of the boss reporter gene in the R8 cell were identified by a external eye morphology and staining with an antibody against Boss. Among 900 lines with expression of the boss reporter gene in multiple cells assessed for homozygous mutant phenotypes, insertions in the marbles, glass, gap1, and fasciclin II genes were isolated. This phenotypic enhancer-trap facilitates (1) the isolation of enhancer-traps with a specific expression pattern, and (2) the recovery of mutants disrupting development of specific tissues. Because the temporal and tissue specificity of the phenotypic trap is dependent on the choice of the marker used, this approach can be extended to other tissues and developmental stages (Pignoni, 1997).

The molecular mechanisms controlling the ability of motor axons to recognize their appropriate muscle targets were dissected using Drosophila genetics to add or subtract Netrin A, Netrin B, Semaphorin II, and Fasciclin II, either alone or in combination. Discrete target selection by neurons might be specified in a point-to-point fashion such that each motor axon and its appropriate target have unique and complementary molecular labels. Alternatively, specificity might emerge from a dynamic and comparative process in which growth cones respond to qualitative and quantitative molecular differences expressed by neighboring targets and make their decisions based on the relative balance of attractive and repulsive forces. Fas II and Sema II are expressed by all muscles where they promote (Fas II) or inhibit (Sema II) promiscuous synaptogenesis. The level of Sema II expression, while not enough to stop growth cones from exploring their environment, nevertheless provides a threshold that specific attractive signals must overcome in order to permit synapse formation. Decreasing Sema II leads to an increase in innervation. In the absence of Sema II, targeting errors occur, usually in the form of additional ectopic connections to neighboring muscles, although in some cases the absence of the normal connection or inappropriate choice point decisions are observed as well. Increasing Sema II leads to a decrease in innervation. It is concluded that growth cones in this system apparently do not rely solely on single molecular labels on individual targets. Rather, these growth cones assess the relative balance of attractive and repulsive forces and select their targets based on the combinatorial and simultaneous input of multiple cues. Apparently a relative balance model is more valid in this system than a lock-and-key model (Winberg, 1998).

The modest and dynamic level of Fas II helps adjust the threshold for innervation. Prior to synapse formation, Fas II is expressed at a low level across the entire surface of the muscle, making it permissive for growth cone exploration and synapse formation. As the first synapse forms on a muscle, the Fas II level dramatically plummets over the muscle surface while Fas II clusters under the developing synapse. The first successful synapse leads to a rapid reduction in this general attractant, thereby shifting the relative balance in favor of Sema II-mediated repulsion and thus raising the hurdle over which attractive signals must pass in order to promote further synapse formation. In this way, the innervated muscle becomes more refractory to further innervation. Fas II, as a modulator of the balance of attraction and repulsion, becomes a temporal measure of the muscle's synaptic history (Winberg, 1998).

While Sema II generally prevents exuberant synapse formation, it can also play an important role in patterning connections. For example, the two axons that pioneer the transverse nerve (TN) normally meet and fasciculate near muscle 7. In the absence of Sema II, these axons often innervate muscles 7 and 6, and sometimes fail to fasciculate with one another. In this case, Sema II provides a repulsive force (from muscles 7 and 6) at a specific choice point, and in its absence, the TN growth cones make a different decision. Similarly, as the lateral branch of the segmental nerve branch a (SNa) extends posteriorly, one axon branch innervates muscle 5 while another continues posteriorly to innervate muscle 8. In the absence of Sema II, both sometimes stop and innervate muscle 5. In this case, Sema II provides a key repulsive force (from muscle 5) at a specific choice point, and in its absence, the growth cone that usually innervates muscle 8 instead makes a different decision. Both examples show how Sema II can do more than simply sharpen the pattern of innervation; Sema II can also influence specific targeting decisions in a dosage-dependent fashion. The Sema II experiments show that the pattern of expression (i.e., the differential levels expressed by neighboring muscles) can be more important than the absolute level. Simply increasing Sema II on all muscles has little influence on the SNa. But increasing Sema II expression on muscle 5 and not its neighboring muscles does influence the SNa axons, presumably because it presents these axons with a sharp repulsive boundary. This differential expression prevents the lateral branch of the SNa from extending towards muscles 5 and 8 (Winberg, 1998).

The netrins were initially discovered as long-range chemoattractants that are secreted by midline cells and that attract commissural growth cones toward the midline. Netrins might have another function, and strong evidence is presented supporting this notion. In addition to their CNS midline expression and function in axon guidance, NetA and NetB are also expressed by distinct subsets of muscles where they function as short-range target recognition molecules. Genetic analysis suggests that both types of Netrin-mediated attractive responses (i.e., pathfinding and targeting) require Frazzled, the DCC/UNC-40-like Netrin receptor. In contrast, Fra is not required for NetB-mediated repulsion of the segmental nerve. Even though they are expressed by distinct subsets of muscles and function as target recognition molecules, the two netrins, NetA and NetB, do not act alone in specifying any one of these muscle targets. NetB is expressed by muscles 7 and 6, but NetB is not the sole attractant used by RP3 to innervate these muscles. In the absence of NetB, in 35% of segments RP3 makes the correct pathfinding decisions in the periphery but fails to innervate muscles 7 and 6 properly. However, in the other 65% of segments it does innervate muscles 7 and 6. Clearly, other unknown cues must play a major role in this targeting decision. One potential candidate for an additional targeting cue is the Ig CAM Fasciclin III. However, removal of FasIII does not alter the penetrance of the RP3 phenotype of Netrin or frazzled mutants. NetB functions within the context of the relative balance of general attractants and repellents such as Fas II and Sema II. For example, since the TN axons are attracted by NetB, and muscles 7 and 6 express NetB, why do the TN axons not synapse on muscles 7 and 6? Evidently, they are sufficiently repelled by Sema II to prevent inappropriate synapse formation. Either increasing the level of NetA or NetB or decreasing the level of Sema II leads to ectopic TN synapses. The choice of synaptic partner by TN axons is controlled by the balance of NetB in relation to Sema II and Fas II (Winberg, 1998).

Distinct classes of motor axons respond differentially to NetA and NetB While all motor axons in this system appear to be attracted by Fas II and repelled by Sema II, the different types of motor axons respond differently to NetA and NetB. NetB is expressed by a subset of muscles (7 and 6) where it strongly attracts appropriate (RP3) axons, more weakly attracts certain inappropriate (TN) axons, and repels other inappropriate (SN) axons. RP3 and TN axons can also be strongly attracted by NetA, while SN axons are apparently indifferent to NetA. The TN axons display a stronger responsiveness to NetA than to NetB, as judged by the frequency of ectopic innervation of ventral muscles overexpressing either Netrin. This difference may make biological sense, as TN axons normally extend toward a dorsal stripe of epithelial cells expressing NetA but grow past NetB-expressing ventral muscles without innervating them (Winberg, 1998).

Although all of the molecular signals used for this targeting system are not yet known, four key components have been identified: the pan-muscle expression of Fas II and Sema II and the muscle-specific expression of NetA and NetB. Analysis of these four genes shows that the signals they encode are potent, function as short-range signals in a dosage-dependent fashion, and work in combinations that either amplify or antagonize one another. Fas II and Sema II help control the fidelity and precision of the targeting system, while NetA and NetB provide muscle-specific targeting cues. These results suggest that target selection in this system is not based on absolute attractants or repellents that either ensure or prevent synapse formation, but rather it is based on the balance of attractive and repulsive forces on any given target cell in relationship to its neighboring cells. Targeting molecules such as Netrins, Semaphorins, and IgCAMs sometimes function as antagonists and sometimes as collaborators. This model of target selection is very similar to the current view of axon guidance in terms of a relative balance of attractive and repulsive forces (Winberg, 1998).

A cell-adhesion molecule Fasciclin 2, which is required for synaptic growth, and Still life (Sif), an activator of Rac, were found to localize in the surrounding region of the active zone, defining the periactive zone in Drosophila neuromuscular synapses. betaPS integrin and Discs large, both involved in synaptic development, also decorate the zone. However, Shibire (Shi), the Drosophila dynamin that regulates endocytosis, is found in the distinct region. Mutant analyses show that sif genetically interacts with Fas2 in synaptic growth and that the proper localization of Sif requires Fas2, suggesting that they are components in related signaling pathways that locally function in the periactive zones. It is proposed that neurotransmission and synaptic growth are primarily regulated in segregated subcellular spaces, active zones and periactive zones, respectively (Sone, 2000).

To characterize the Sif localization pattern, particularly in reference to synaptic functional domains, the subcellular distribution of Sif in the boutons of larval neuromuscular junctions was examined by concomitant staining with anti-Pak antibody using laser-scanning confocal microscopy. Anti-Sif antibody labels the synaptic boutons in a network-like pattern, which is strikingly complementary with Pak staining in the boutons. The cross-section profile of the fluorescent intensity also shows that the staining patterns of Sif and Pak are mostly complementary to each other. These staining patterns demonstrate that the areas stained for Sif surround the active zones. Close examinations further reveal that anti-Sif and anti-Pak antibodies produce a number of concentric figures that are occasionally separated from each other. These data suggest that the active zone and the outer ring together form a structural unit that constitutes a synapse. The Sif-positive regions around the active zones are referred to as periactive zones (Sone, 2000).

To characterize the periactive zone, especially in identifying its functional significance, the distribution patterns of other molecules were examined with the aid of Pak staining. Monoclonal antibody, MAb1D4, against Fas2 labels the boutons in a complementary pattern with Pak staining. Fas2 staining surrounds the Pak-positive regions and forms concentric patterns as observed for Sif staining. The cross-section profile also shows similar patterns as Sif and Pak double staining. Sif and Fas2 are indeed co-localized in overlapping network-like patterns. Fas2 is involved in synaptic growth, stabilization and structural plasticity, possibly through its homophilic adhesion. These data suggest that Fas2 controls these synaptic events locally in the periactive zones. Thus, the periactive zone is characterized by the specific localization of two distinct types of molecules: a cell adhesion molecule (Fas2) that controls synaptic development and an intracellular molecule (Sif) that is a GEF to Rac (Sone, 2000).

The polyclonal antibody against Dlg protein stains synaptic boutons in a way similar to MAb6G11. The Dlg staining also appears to be moderately diffused on the muscle surfaces surrounding the bouton. This pattern is complementary with the anti-Pak staining when the bouton is scanned at the surface level. In dlg mutants, the structural properties of synapses, including the formation of subsynaptic reticulum at the postsynapses and the number of active zones at the presynapses, are altered. Furthermore Dlg regulates the synaptic localization of Fas2 by binding directly to the cytoplasmic tail of Fas2. Therefore, one of the roles for Dlg in synaptic development is probably the localization of Fas2 to the periactive zone. These observations indicate that two additional molecules, betaPS integrin and Dlg, are present in synaptic areas including the periactive zones. They are both involved in the structural development of the neuromuscular synapses, and therefore appear to participate in the control of synaptic development in the periactive zones (Sone, 2000).

It has been suggested that a dynamin-rich domain in the presynaptic terminal functions as a site for the vesicular endocytosis. A recent study has also indicated that this domain is distinct from the active zone for exocytosis and instead surrounds the active zone. It is of interest to find out whether the periactive zone is involved in endocytosis and therefore the spatial relationships between the dynamin-rich domain and the periactive zone were examined. Synaptic boutons were co-stained with anti-Fas2 antibody and a polyclonal antibody against the Shi protein, the Drosophila homolog of dynamin. Shi is an essential factor for endocytosis, since endocytosis is completely blocked in the shi mutant. Anti-Shi antibody stains synaptic boutons in donut-like patterns but these patterns are found almost within the holes of the Fas2 rings. This Shi staining may partly overlap with Fas2 staining, but these staining patterns are distinct from each other. Thus, it is concluded that the periactive zone does not coincide with the dynamin-rich zone and therefore does not likely represent a functional domain for endocytosis (Sone, 2000).

Since Sif and Fas2 are co-localized in the periactive zone, a test was performed to see whether there is a genetic interaction between sif and Fas2 loci. The hypomorphic allele of Fas2, Fas2^e76, shows a reduced number of boutons: would changing the dose of sif⁺ affect this bouton number phenotype? Strikingly, the double mutant of Fas2^e76 and sif ^ES11 recovered the bouton number phenotype to the wild-type level, which suggests the presence of a suppressive genetic interaction between the two loci. To assess this genetic interaction further, the effect of Sif overexpression in the Fas2^e76 background was examined. Sif overexpression does not clearly affect the synaptic bouton number in the wild-type background; rather, it causes a significant reduction in Fas2^e76. Moreover, NMJs with extremely few synaptic boutons (less than 20) are observed in the Fas2^e76 larvae overexpressing Sif, when compared with the NMJs in Fas2^e76. Taken together, these data suggest that Sif and Fas2 are the components of related signaling pathways that control synaptic development, and Sif may, in an inhibitory manner, modulate the effect of Fas2 that regulates synaptic growth (Sone, 2000).

The possibility that the molecules in the periactive zone may affect each other in establishing their zonal localization was examined. Because Sif and Fas2 co-localize typically in the periactive zones and interact genetically, focus was placed on these proteins and an investigation was carried out to see if any perturbation occurs in the distribution of several molecular markers in the mutant background of Fas2 or sif. In the sif mutants, the localization of Fas2 is indistinguishable from the wild type. Conversely the Sif localization is normal in a hypomorphic allele of Fas2. However, in the boutons of a more severe hypomorphic allele of Fas2, Fas2^e76, the Sif localization to the periactive zones is perturbed. The Fas2^e76 mutation reduces Fas2 expression to 10% of the wild-type level. In most Fas2^e76 boutons, the network pattern of Sif staining is still observed, but frequently in an irregular or diffused fashion. In more extreme cases, Sif is distributed almost evenly throughout the boutons or is largely concentrated on one side of the boutons so that Sif staining considerably overlaps Pak staining (Sone, 2000).

The mutant larvae could be distinguished from the wild type by the blind test for the Sif and Pak co-staining patterns. Similar results were obtained in the heterozygotes with Fas2^e76 and a Fas2 null allele, suggesting that the alteration of Sif localization is not due to a second-site mutation on the Fas2^e76 chromosome. To investigate the altered distribution of Sif further, the mutant boutons were examined under the electron microscope. A large number of Sif signals are occasionally present in the medial portions of the electron-dense regions in the Fas2^e76 boutons and these signals are still associated with the plasma membrane, as are the signals observed in the wild type. It is therefore concluded that the reduction of Fas2 in the periactive zones results in the improper localization of Sif along the plasma membrane. Previous study has shown that the synaptic localization of Fas2 requires Dlg. Therefore, the localization of Sif was examined in the dlg mutant background, but no apparent alteration was found in the network pattern. A considerable amount of Fas2 is still present in the periactive zones of dlg mutant boutons, while faint or no staining is detected in the Fas2^e76 boutons. This residual Fas2 seems to be sufficient to sustain the proper localization of Sif in the dlg mutants (Sone, 2000).

Previous studies have shown that RAC acts in the neurite outgrowth of neuroblastoma cells that depend on the signal from integrin on the cell surface. The mammalian SIF homolog TIAM1, which functions as a RAC GEF, recruits integrin to specific adhesive contacts at the cell periphery. Moreover, expression of TIAM1 increases cadherin-mediated cell adhesion in epithelial MDCK cells. Therefore, there appear to be signaling links between the RAC and cell-adhesion molecules. Sif activates Rac; sif genetically interacts with Fas2 in synaptic growth and the Sif localization is perturbed in the Fas2 mutants. Taken together, these data suggest that the Sif-Rac pathway is linked to the cell-adhesion molecule Fas2 in close vicinity in the periactive zone (Sone, 2000).

The periactive zone has been indicated as a region for the control of synaptic development. The periactive zone surrounds the active zone, which is the site for vesicle exocytosis or neurotransmission. This concentric organization suggests that the two zones specialize for the different cellular functions and constitute an elemental unit for the presynaptic structure. Investigation of how these zones are incorporated into the synaptic bouton during development will be of interest. The segregated distribution of the two zones suggests that the mechanisms controlling synaptic development and neurotransmission may be separable. This view is supported by the mutant analyses for Fas2 and Sif; both mutations affect structural properties of synapses without changing basic electrophysiological functions. In the NMJs of Fas2 mutants, the bouton number is decreased or increased depending on the alleles but the total synaptic strength is maintained at the normal level. Functional strength of the synapse is regulated only through the activity of a transcription factor, cAMP-response-element-binding protein (CREB), which functions independently of Fas2. Also in sif mutants, the basic electrophysiological properties of NMJs are normal. These observations clearly contrast with the mutant phenotypes for the proteins controlling vesicle exocytosis: Synaptotagmin, Cysteine string protein, n-Synaptobrevin and Syntaxin 1A. Mutants in genes coding for all these proteins show impaired EJPs. Taken together, these results indicate that synaptic development and neurotransmission are genetically separable phenomena and are regulated by independent pathways. It is proposed that these genetically separable phenomena are spatially segregated into the two zones on the presynaptic plasma membrane, although the possibility that the two zones interact with each other cannot be excluded (Sone, 2000).

Semaphorins comprise a large family of phylogenetically conserved secreted and transmembrane glycoproteins, many of which have been implicated in repulsive axon guidance events. The transmembrane semaphorin Sema-1a in Drosophila is expressed on motor axons and is required for the generation of neuromuscular connectivity. Sema-1a can function as an axonal repellent and mediates motor axon defasciculation. By manipulating the levels of Sema-1a and the cell adhesion molecules fasciclin II (Fas II) and connectin (Conn) on motor axons, further evidence is provided that Sema-1a mediates axonal defasciculation events by acting as an axonally localized repellent and that correct motor axon guidance results from a balance between attractive and repulsive guidance cues expressed on motor neurons (Yu, 2000).

The failure of axonal defasciculation observed in Sema1a mutant embryos is likely due to the lack of Sema-1a-mediated repulsion among motor axons along efferent trajectories. Reducing adhesion by removal of attractive cues, the CAMs Fas II and Conn, rescues characteristic hyperfasciculation defects (both ISNb and SNa phenotypes) of Sema1a mutants. In contrast, increasing adhesion by overexpression of the CAM Fas II enhances the hyperfasciculation defects in the ISNb and SNa pathways in Sema1a mutant embryos. In addition, reduction in the level of Fas II will also suppress CNS fasciculation defects in Sema1a mutant embryos. These experiments complement those previous studies showing that FasII loss of function can suppress defasciculation defects observed in PlexA mutants. Further, they show that mutations in genes encoding different classes of CAMs, including both Ig superfamily members and LRR-containing proteins, genetically interact with Sema1a mutants, suggesting that CAM-specific signaling events are not involved in this interaction. Taken together, these results demonstrate that Sema-1a regulates axonal fasciculation at specific choice points by countering the attractive functions of at least two CAMs, Fas II and Conn (Yu, 2000).

How might Sema-1a modulate specific defasciculation events at choice points when it appears to be expressed along the entire motor axon? Sema-1a may serve to negatively regulate motor axon adhesion over the entire trajectory and thereby allow extending motor axon growth cones to respond to target recognition cues. A precedent for a reciprocal role for the CAM Fas II comes from a detailed ultrastructural analysis of FasII mutants, where it was observed that although a loss of Fas II does not compromise the extension of axons during early CNS development, it does result in a lack of fasciculation among individual neurons that normally comprise discrete axon bundles. On the basis of analyses of Sema-1a and analysis of Plex A, it seems likely that Sema-1a on CNS axons acts as a generally expressed repellent. The absence of the CAM Fas II, therefore, changes the balance Fas II and Sema-1a and results in defasciculation of CNS bundles. Therefore, a combination of attractants and repellents may serve to allow individual axons within a developing bundle to respond to cues that may reside either at choice points or in adjacent intermediate or final target regions. However, local regulation of the function of these axonally localized cues may also serve to modulate their function, a possibility supported by the observation that Beaten path, which negatively regulates Fas II in motor axons, appears to be localized at certain ventral motor axon choice points (Yu, 2000 and references therein).

Both Conn and Fas II, in addition to being expressed on motor axons, are also expressed on embryonic muscles. However, the interpretation that the observed genetic interactions reveal a balance of repulsive and attractive axonal cues required to regulate motor axon fasciculation is likely not to be compromised by this muscle CAM expression for the following reasons: (1) if loss of CAM expression on motor neurons and target regions were capable of altering axonal fasciculation through alteration of axon/target interactions, one would expect the effect to be a reduction in CAM-mediated motor axon extension on muscle and an enhancement of motor axon fasciculation -- the opposite of what was observe; (2) the levels of Fas II, and to a lesser extent Conn, on embryonic muscles have been shown to be much lower than high axonal levels of these CAMS observed during the embryonic stages analyzed in this study for motor axon pathfinding (Yu, 2000).

Previous studies support the idea that guidance cues act cooperatively to generate the precise pattern of neuromuscular connectivity in Drosophila. Netrins and a secreted semaphorin expressed on muscles and axonal Fas II function in a complementary fashion to regulate motor axon target selection. The dynamic balance of attractive cues (Net A and B, and Fas II) and repulsive cues (Sema-2a and Net B) has been shown to govern the ability of motor axons to undergo normal target recognition and formation of synaptic arborizations. Indeed, alteration of the degree of axonal attraction mediated by Fas II directly affects the ability of motor axons to respond to target-derived attractants and repellents. The results presented here, in combination with published results on PlexA/FasII interactions, extend these observations to the axon bundle itself and illustrate the importance of the interplay between axonal attractants and repellents for complex axon guidance events. It should be noted that although these results support a repulsive role for Sema-1a in motor axon guidance, the related transmembrane semaphorin Sema-1a in the grasshopper has been shown to act as an attractive cue for peripheral afferents in the developing limb (Wong, 1999). In this situation, however, Sema-1a is target derived and not axonal, and it remains to be seen if transmembrane semaphorin-mediated attraction is also plexin dependent. Finally, the simultaneous expression of transmembrane semaphorins and CAMs on axon pathways undergoing complex pathfinding and defasciculation events is not restricted to invertebrates. For example, in the developing vertebrate olfactory system the Ig superfamily members NCAM and olfactory cell adhesion molecules show selective distribution on main and accessory olfactory neurons, suggesting that these CAMs play an instructive role in matching odorant receptor expression zones in the olfactory epithelium and in the olfactory bulb. In addition, both the transmembrane semaphorin Sema6A and the plexin Plexin A1 are expressed in subsets of primary and accessory olfactory neurons. The defasciculation events involved in directing these neurons to their unique glomerular targets in the main and accessory olfactory bulbs are reminiscent of the motor axon guidance events discussed here, suggesting that the maintenance of a balance on axons between repulsive transmembrane semaphorins and attractive CAMs is phylogenetically conserved (Yu, 2000 and references therein).

Drosophila fasciclinII is required for the formation of odor memories and for normal sensitivity to alcohol

Drosophila fasciclinII mutants perform poorly after olfactory conditioning due to a defect in encoding, stabilizing, or retrieving short-term memories. Performance was rescued by inducing the expression of a normal transgene just before training and immediate testing. Induction after training but before testing failed to rescue performance, showing that Fas II does not have an exclusive role in memory retrieval processes. The stability of odor memories in fasII mutants are indistinguishable from control animals when initial performance is normalized. Like several other mutants deficient in odor learning, fasII mutants exhibit a heightened sensitivity to ethanol vapors. A combination of behavioral and genetic strategies have therefore revealed a role for Fas II in the molecular operations of encoding short-term odor memories and conferring alcohol sensitivity. The preferential expression of Fas II in the axons of mushroom body neurons furthermore suggests that short-term odor memories are formed in these neurites (Cheng, 2001).

The results presented here establish a role for Fas II that is significantly different from those known previously. Antibody perturbation experiments and genetic analyses have revealed that the molecule functions in the fasciculation and defasciculation of axon bundles. A second function was discovered through studies of the larval neuromuscular junction, where Fas II is required for the stabilization and growth of the synapse. The molecule is also required for the expression of the proneural genes, atonal and achaete, in the eye-antennal imaginal disc, and for the adhesion or migration of neurons or their precursors in other parts of the nervous system. Most of these biological functions have been ascribed to the molecular function of Fas II as a homophilic cell adhesion molecule. It is surprising given the known developmental roles for Fas II in axon bundling and synapse growth that no morphological defects of the mushroom bodies were observed in the mutants. The possibility that very subtle morphological defects exist in the mushroom bodies of fasII mutants cannot be eliminated. However, a short (1 hr) temperature shift from 18°C to 25°C prior to training is sufficient to rescue the odor learning phenotype and this is reversible with reincubation at 18°C. It is highly unlikely that this temperature shift and subsequent increase in Fas II expression corrects a major morphological defect in the fasII mutants. A more plausible conclusion is that rescue is observed due to a function of Fas II in intercellular and/or intracellular signaling processes that underlie memory formation. How might Fas II be involved in signaling for memory formation? For the role of Fas II in proneural gene expression, there is a dominant interaction of fasII with mutants in the Abelson tyrosine kinase gene, suggestive of a role in signaling through nonreceptor tyrosine kinases. NCAM also signals through nonreceptor tyrosine kinases. Antibody induced clustering of one transmembrane form of NCAM, NCAM-140, induces the association and transient phosphorylation of the nonreceptor kinases, p59fyn and p125fak. Furthermore, NCAM-140 clustering activates the mitogen-activated protein kinases, ERK1 and ERK2, and the transcription factor, CREB. However, NCAM is also linked to other signaling pathways that modulate intracellular inositol phosphates, calcium, and pH. These observations inspire signaling-based models for how Fas II may mediate the formation of memories (Cheng, 2001).

It has been proposed that mushroom body neurons serve as the integrators of the conditioned (CS) and unconditioned stimuli (US) during odor learning. The CS pathway, which conveys odor information, extends from odor receptors on the antennae to the antennal lobe to the calyces of the mushroom bodies. The calyces, however, do not exhibit high levels of Fas II expression, suggesting that the function of Fas II in odor learning is not for the processing of CS information. Fas II is concentrated along the axons of the mushroom bodies (peduncle) and in the neuropil areas that house the mushroom body axon terminals. The mushroom body lobes, in particular, are the targets for projections from modulatory inputs that may convey US information, including dopaminergic inputs and peptidergic inputs from Amnesiac-expressing neuron. These observations are consistent with a role for Fas II in the proper presentation of US information to the mushroom body neurons, or perhaps in events subsequent to the integration of information, such as the strengthening of mushroom body neuron synapses upon follower neurons (Cheng, 2001).

Molecules related to Fas II such as NCAM in vertebrates and apCAM in Aplysia have been suggested to be involved in learning, memory, and the cellular mechanisms that are thought to underlie these behaviors. Some studies have suggested a role for these cell adhesion receptors in memory consolidation. For example, amnesia of one-trial passive avoidance training can be produced by injecting anti-NCAM antibodies into the chick brain at 6 hr after training but not at earlier times (Rose, 1995). Similar results have been obtained with the rat, with the sensitive period for effects on passive avoidance memory occurring at 6-8 hr post-training. Other studies, however, have suggested a role for NCAM in the early processes of memory formation. For instance, injection of NCAM antisense oligonucleotides before one-trial passive avoidance training blocks memory as early as 3 hr after training. Moreover, application of anti-NCAM antibodies to hippocampal slice preparations reduces the magnitude of long-term potentiation measured in the CA1 region, with no effect upon basal synaptic transmission. Consistent with this observation, a mouse knockout of NCAM exhibits a reduction in the magnitude of hippocampal LTP and spatial learning, although the mutants have marked defects in brain development that prohibit the discrimination of developmental versus physiological roles for NCAM. Thus, the specific role for NCAM and similar molecules in memory processes has remained obscure. The results presented here demonstrate, through the use of powerful gene knockout and replacement strategies, combined with behavioral assays, that the transmembrane form of Fas II is involved early in the encoding of odor memories and not in memory stability or retrieval. Nevertheless, the results do not eliminate the possibility that Fas II and similar molecules participate in later phases of memory. A parsimonious model is that Fas II and related molecules participate in encoding short-term memory through a cell-signaling function and in forming long-term memory through a cell-adhesion function (Cheng, 2001).

Within each temporal phase of memory, the existence of at least three broad operations, including memory formation (encoding), memory stability, and memory retrieval, is expected. In other words, each distinct temporal phase must bracket the molecular operations underlying its formation, stability, and retrieval. The results presented here establish that Fas II is involved in the formation of short-term odor memories. Other events that are probably involved in this operation include the activation of the rutabaga-encoded adenylyl cyclase, a transient elevation of cAMP, and the activation of protein kinase A, the product of the DCO gene. In contrast, the formation of long-term memories likely occurs through alterations in the expression of certain gene products. Stability operations for short- versus long-term memory are also likely to be different. The stability of short-term memory may reside in mechanisms to keep protein kinases active while the stability of long-term memory may reside in feedback systems that maintain gene expression states. Retrieval mechanisms could be distinct for different temporal phases of memory but the simplest idea is that retrieval mechanisms are shared among the temporal phases (Cheng, 2001).

The observation that fasII mutants have heightened ethanol sensitivity extends the overlap between genes involved in odor learning and those involved in alcohol sensitivity. However, several observations distinguish the two behaviors and their underlying genetics and neuroanatomy. Whereas raising fasII mutants carrying the hs-fasII-1 transgene at 25^oC is sufficient to rescue the odor learning phenotype, this treatment fails to rescue the ethanol sensitivity. One possible explanation for this is that the level of expression during development or in the adult produced by hs-fasII-1 in the neural structures mediating ethanol sensitivity is incompatible for normal behavior in the inebriometer. Although the neuroanatomical structures that mediate ethanol sensitivity are not yet defined, they are likely to be distinct from the mushroom bodies, since mushroom body ablation has no effect upon ethanol sensitivity but it abolishes odor learning. Alternatively, it is possible that ethanol sensitivity is caused by a lack of Fas II isoforms other than the transmembrane form expressed from the hs-fasII-1 transgene. Therefore, although the overlap in molecular functions required for normal odor learning and ethanol sensitivity is striking, the two behaviors appear to be mediated by separable neural structures and gene expression requirements (Cheng, 2001).

Regulation of synaptic connectivity: Levels of Fasciclin II influence synaptic growth in the Drosophila CNS

Much of the understanding of synaptogenesis comes from studies that deal with the development of the neuromuscular junction (NMJ). Although well studied, it is not clear how far the NMJ represents an adequate model for the formation of synapses within the CNS. The role of Fasciclin II (Fas II) has been studied in the development of synapses between identified motor neurons and cholinergic interneurons in the CNS of Drosophila. Fas II is a neural cell adhesion molecule homolog that is involved in both target selection and synaptic plasticity at the NMJ in Drosophila. Levels of Fas II are critical determinants of synapse formation and growth in the CNS. The initial establishment of synaptic contacts between identified neurons is seemingly independent of Fas II. The subsequent proliferation of these synaptic connections that occurs postembryonically is, in contrast, significantly retarded by the absence of Fas II. Although the initial formation of synaptic connectivity between these neurons is seemingly independent of Fas II, their formation is, nevertheless, significantly affected by manipulations that alter the relative balance of Fas II in the presynaptic and postsynaptic neurons. Increasing expression of Fas II during embryogenesis, in either the presynaptic or postsynaptic neurons, is sufficient to disrupt the normal level of synaptic connectivity that occurs between these neurons. This effect of Fas II is isoform specific; moreover, it phenocopies the disruption to synaptic connectivity observed after tetanus toxin light chain-dependent blockade of evoked synaptic vesicle release in these neurons (Baines, 2002).

Previous studies of Fas II and synaptogenesis have focused on the accessible synapse formed at the larval NMJ in Drosophila. These studies have been extended to the more complex issue of the formation of central synapses, using a relatively well defined set of synaptic contacts that form during embryogenesis between cholinergic interneurons and identified motor neurons. The analysis at the NMJ shows that Fas II is expressed both presynaptic and postsynaptically but that it is not required for the formation of synaptic connections between motor neurons and their target muscles. However, if Fas II is overexpressed in muscles during a critical period of embryogenesis, it allows additional, ectopic synapses to form and become stabilized on the muscles concerned. These findings together with immunocytochemical studies of Fas II expression have suggested that, during the initial phase of synaptogenesis, Fas II is present in limiting amounts on the postsynaptic cell and that the protein then becomes aggregated under contacts formed by the innervating motor neuron, thereby inhibiting the formation of stable, ectopic contacts by other neurons. These observations imply that, although not essential to synaptogenesis, Fas II can act as a powerful determinant of the distribution and number of contacts on the postsynaptic cell (Baines, 2002).

The first aim of this study was to show whether or not Fas II can act in a similar manner during the embryonic formation of central synapses. Fas II is expressed in motor neurons; it is also expressed in a subset of cholinergic interneurons, although it is not possible to say whether these are immediately presynaptic to the motor neurons being studied. Moreover, the precise distribution of Fas II protein in either the presynaptic or postsynaptic neurons is not known, nor is anything known about the relative expression of Fas II in different neurons. However, the results of both physiological and ultrastructural analyses show that an apparently normal pattern of interneuron to motor neuron synapses develops in the absence of Fas II. In the continued absence of Fas II, however, these synapses clearly fail to proliferate, and, as a consequence, the synaptic drive to motor neurons is reduced. However, increased Fas II expression in either the presynaptic or postsynaptic cells is sufficient to reduce synaptic inputs to the motor neurons as judged physiologically or ultrastructurally. The puzzling aspect of this latter result is that, although it suggests that (as at the NMJ) Fas II can act centrally to influence the pattern of synaptic contacts, it appears to do so in completely the opposite sense: additional Fas II reduces the number of synapses rather than promoting the formation of additional, ectopic contacts. These findings also differ from the observed consequence of disproportionately increasing the mammalian homolog of Fas II, NCAM, in postsynaptic hippocampal neurons maintained in culture; increasing NCAM in culture, as in the NMJ, is sufficient to strengthen synaptic connectivity. The possibility cannot be discounted that increased expression of Fas II in aCC/RP2 results in the additional formation of inappropriate synaptic connections to these neurons, which may be sufficient to weaken, structurally or functionally, the connections that normally form between these neurons and their normal presynaptic partners. However, because a clear reduction is seen in the number of presynaptic input sites in young first instar larvae, under these conditions, it would suggest that any such inappropriate connections are likely to have retracted by this stage. Simply interpreted, the effects reported suggest that, although Fas II is required for postembryonic synapse proliferation, disproportionate increases in levels of Fas II in central neurons has a potentially repressive effect on the formation of synapses between the cell concerned and its putative synaptic partners, regardless of its site of expression. Caution is required in finally adopting this conclusion, because the environment of dendritic arborizations in the embryonic neuropil is likely to be complex, and the contribution of Fas II to dendritic patterning is not known. Thus, although increased levels of Fas II expression, or absence of this CAM, does not alter the gross morphology of aCC based on an analysis of DiI-labeled cells, such manipulations could conceivably alter more subtle aspects of dendritic morphology and disrupt the normal pattern of synaptic connectivity. A detailed analysis of dendritic patterning in these neurons is reliant on, and must wait until, individual presynaptic partner neurons can be visualized (Baines, 2002).

These experiments concentrated on two motor neurons, aCC and RP2, that innervate dorsal muscles. These neurons are identifiable in the embryonic and larval CNS and are relatively accessible to patch-clamp electrodes. In addition, the RRK-Gal4 line allows for the misexpression of proteins such as Fas II selectively in these cells. The results of these experiments were also monitored in a third, control motor neuron, RP3, that innervates ventral longitudinal muscles. The effects of these experiments on RP3 are interesting and revealing. (1) Under control conditions, the frequency of suprathreshold synaptic input to RP3 is approximately one-half that seen in aCC/RP2. This suggests that, under the conditions of these experiments, RP3 (ventral muscles) and aCC/RP2 (dorsal muscles) receive distinct inputs from interneurons involved in generating rhythmic motor outputs. (2) The frequency of input to RP3 remains unchanged when the level of Fas II is increased in aCC/RP2, and the frequency of synaptic input declines in these neurons. This result suggests that the alterations in synaptic communication that were detected are the result of local events in the neurons concerned. (3) Most significant, and reinforcing the interpretation that the effects described depend on the relative levels of Fas II expressed in presynaptic and postsynaptic neurons, is the fact that in experiments in which Fas II is simultaneously expressed in cholinergic interneurons and aCC/RP2, the decline in input frequency to aCC/RP2, seen when Fas II is expressed in either of these sets of neurons alone, fails to occur. This result implies that it is the balance of Fas II in presynaptic and postsynaptic cells that is decisive for the formation of a normal pattern of synaptic inputs. Significantly, in this experiment, the control neuron RP3, with normal levels of Fas II, is innervated by interneurons whose level of Fas II has been increased: it is predicted that synaptic communication should be weakened, and this is indeed the effect that was observed. Thus, alterations in the relative levels of Fas II in presynaptic and postsynaptic cells have local effects that are selective and predictable for individual neurons. This strongly suggests that during synaptogenesis, the balance of Fas II in presynaptic and postsynaptic cells can influence the formation of a normal pattern of synaptic contacts (Baines, 2002).

The strikingly similar results of misexpressing Fas II or tetanus toxin light chain (TeTxLC) in aCC/RP2 suggested that the effects of TeTxLC might be caused at least in part by elevated levels of Fas II in the neurons in which it is expressed. Indeed, the toxin effects are partially rescued by the complete loss of Fas II function. This, together with the observation that an imbalance in presynaptic and postsynaptic levels of Fas II expression is sufficient to interfere with normal synaptogenesis, offers an explanation for the previously puzzling finding that blocking vesicle release from the postsynaptic neuron leads to a reduction in presynaptic input to that cell. If, as in photoreceptor cells, expression of TeTxLC leads to an overall increase in levels of Fas II in the affected cells, then synaptic inputs to those cells would be expected to be disturbed. The finding that the local balance of Fas II influences the formation of central synapses, together with the strong implication that alterations in vesicle trafficking can interfere with this balance, is important for understanding of normal synaptogenesis and its control. Although synaptogenesis can proceed successfully in the absence of Fas II, it is predicted that any (possibly activity-dependent) modulation of Fas II levels in presynaptic or postsynaptic cells has the potential to influence the number and pattern of connections formed in a normal embryo. How activity might regulate levels of Fas II in synaptic terminals remains to be determined. Synaptically targeted membrane proteins, including neurotransmitter receptors, are thought to be constantly moving in to and out of the synaptic membrane, this movement being dependent on successive rounds of vesicular endocytosis and exocytosis. A perturbation at any point in this cycle has the potential to result in an inappropriate surface expression of these proteins and perhaps provide a viable route to influence synaptic plasticity. An example of such a mechanism is long-term sensitization in Aplysia, which involves an activity-dependent downregulation of apCAM (a homolog of Fas II) in the presynaptic sensory neuron. This downregulation appears to be attributable to a cAMP-dependent reduction in gene expression and a simultaneous increase in the rate of endocytotic internalization of preexisting protein from the presynaptic membrane (Baines, 2002).

Although Fas II is not required for the initial formation of an appropriate pattern of synaptic contacts either peripherally (at the NMJ) or centrally, these experiments show that, as at the NMJ, Fas II is essential for the further growth and elaboration of synaptic contacts during postembryonic life. During this larval phase of active feeding and growth, the increasing size of the muscles is matched by an increase in the size and complexity of motor neuron dendritic arbors. Ultrastructural analysis of synaptic inputs to motor neuron arbors during early larval life shows that, at least in these early phases, there is a corresponding increase in the number of presynaptic contacts on the dendrites of aCC/RP2. Strikingly, this increase fails completely in the Fas II null larvae. It is likely that this, together with the previously documented reduced innervation at the NMJ, contributes to the increasing sluggishness and ultimate death of these mutant larvae. The presynaptic contacts formed postembryonically in the series of hypomorphic Fas II alleles has not been analyzed, but, given the relatively normal maturation of the synaptic drive detected in these animals, it seems likely that there is an essential but low level of Fas II that is required for the proper growth and elaboration of presynaptic endings on the motor neuron dendritic arbors. It may well be that, as at the larval NMJ, this critical level of Fas II is a significant determinant of plasticity at central synapses in the fly (Baines, 2002).

Fas2^spin genitalia rotation defects are linked to innervation of the corpora allata

In vertebrate development, the establishment of left-right asymmetry is essential for sidedness and the directional looping of organs like the heart. Both the nodal pathway and retinoic acid play major and conserved regulatory roles in these processes. In order to establish a genetic model of LR asymmetry and organ looping in Drosophila, a screen was carried out for mutations affecting the asymmetric looping of the spermiduct and genitalia in adult flies. A novel mutant, spin, has been isolated in which the looping of the genitalia and spermiduct are incomplete; under-rotation of the genitalia indicates that spin controls looping morphogenesis but not direction, thus uncoupling left-right asymmetry and looping morphogenesis. spin is a novel, rotation-specific allele of the Fasciclin2 gene, which encodes a cell-adhesion protein involved in several aspects of neurogenesis. The focus of Fas2 function in promoting looping was determined to be in synapses of neurosecretory cells. In spin mutants, the synapses connecting specific neurosecretory cells to the corpora allata are affected. The corpus allatum is part of the ring gland and is involved in the control of juvenile hormone titers during development. Genetic and pharmacological results indicate that Fas2^spin rotation defects are linked to an abnormal endocrine function and an elevated level of juvenile hormone. Since juvenile hormone is an insect sesquiterpenoid related to retinoic acid, these results establish a new genetic model for studying organ looping and demonstrate an evolutionarily conserved role for terpenoids in this process (Adam, 2003).

The rotation of genitalia takes place during metamorphosis (in pupae); at this stage, the distal part of the male reproductive apparatus, the genital plate, undergoes a stereotyped 360° clockwise rotation, inducing the spermiduct to loop around the gut in a clockwise direction. The rotation of genitalia can thus be compared with the oriented (dextral or sinistral) looping of internal organs in vertebrates, such as gut and heart. These indeed represent specific LR asymmetry markers, since mutations that affect organ positioning also perturb the direction of organ looping (Adam, 2003).

To initiate a genetic characterization of LR asymmetry and organ looping in flies, a screen was carried out for viable mutations showing defective genitalia rotation. Focus was directed to a novel viable P-element mutation, spin, for which all the adult males show a characteristic mis-rotation of genitalia and are sterile. In spin males, the extent of rotation varies from ~30° to 320°, with a large proportion of males (84%) having their genital plate in a position corresponding to rotation of 135°-225°. Because external mis-rotation does not allow discrimination between under-, hyper- or counter-rotation of the genitalia, males of different phenotypes were dissected and the looping of their spermiduct analyzed. All dissected males showed a clear under-rotation phenotype, indicating that spin is required for the genital plate and spermiduct to undergo complete looping, but has no role in directionality. Dissection of several hundred wild-type males did not reveal any rotation defect, indicating the robustness of this process in normal males (Adam, 2003).

The P-element in spin is inserted in the 5' UTR of the fasciclin 2 (Fas2) gene, suggesting that spin is a novel Fas2 allele (hereafter referred to as Fas2^spin). This conclusion is supported by the following two points. (1) The expression of the Fas2 protein in the original Fas2^spin allele and in a lethal revertant (Fas2^spinRM1) is strongly reduced or absent in embryos, respectively. Significantly, in Fas2^spin third instar larvae, the expression of the Fas2 protein in eye imaginal discs or in whole brain extracts is also strongly reduced. (2) Expression of a UAS-Fas2 transgene under the control of the original spin^P{GAL4} line can fully rescue the rotation and sterility phenotypes (Adam, 2003).

Rotation of genitalia takes place in 2- to 3-day-old pupae over a period of 24 hours. To establish the temporal requirement for Fas2 function in genitalia rotation, a heat-inducible GAL4 line was used to express Fas2 under the UAS promoter. Short egg collections were subjected to a single one hour heat-shock (HS) at 37°C. Adult males were then analyzed and the extent of genitalia rotation rescue determined. A single 1 hour HS at day 7 of development is sufficient to rescue genitalia rotation. Indeed, flies that receive a HS at day 7 of development show a very high degree of rescue (up to 90%). These results indicate that Fas2 is required during a limited period of time during pupal development for rotation to take place normally (Adam, 2003).

In order to identify the tissue(s) and cells that require Fas2 function for genitalia rotation, the GAL4-UAS system was used to drive tissue-specific expression of a UAS-Fas2 transgene in Fas2^spinR5 males. Because Fas2 is required in many aspects of neuronal development, and is expressed mostly in neurons, it was first asked whether Fas2 function was required in the nervous system for rotation. Surprisingly, it was found that the elav-GAL4 line, which drives expression specifically in the CNS during development, is able to rescue Fas2^spinR5 rotation defects fully. This result prompted an examination in detail of the expression pattern of Fas2 protein in the brain and a search for potential nervous system phenotypes in Fas2^spin. This analysis uncovers a previously unknown function of Fas2 in the ring gland (RG). The RG is a composite neuroendocrine organ made of three different specialized regions: the prothoracic gland (PT), the corpora cardiaca (CC) and the corpora allata (CA). The CC probably plays a role in the regulation of blood sugar levels in larvae through adipokinetic hormones. The PT and CA are specialized cells responsible for the secretion of the two primary insect hormones ecdysone and juvenile hormone (JH), respectively. Interestingly, Fas2 expression is restricted to the CC and to specific axonal processes innervating the CA. These neurosecretory cells (nCA) control JH level and can be easily identified using a specific GAL4 line expressed in all CA neurons (Kurs21-GAL4). Since Kurs21-GAL4 driven GFP expression and the anti-Fas2 staining overlap precisely, it is concluded that Fas2 is expressed in all nCA terminals. In Fas2^spin, the overall morphology of the CA synapse is abnormal, showing fused terminal boutons and a reduced number of presynaptic nerve terminals. This result is consistent with the finding that the bouton number is reduced in the neuromuscular junction in strong hypomorph Fas2 flies (Adam, 2003).

In order to demonstrate a direct link between Fas2, the CA, and genitalia rotation, the UAS-GAL4 system was used to express Fas2 in specific subsets of neurons innervating the RG. A few neurons innervate the RG in Drosophila. The PT is innervated by two neurons from each brain hemisphere, whereas the CA is innervated by three neurons. Importantly, neurons innervating the PT and CA are different and map to distinct regions of the brain. A collection of GAL4 lines expressed in different populations of neurons innervating the RG was used to induce neuron-specific expression of Fas2 in Fas2^spinR5 mutants. When GAL4 is expressed strongly in the neurons innervating the CA (nCA) using the Kurs21-GAL4 line, the rotation of genitalia is completely rescued, just as observed with an elav-GAL4 driver. These results show that Fas2 is required in the nCA for normal genitalia rotation. In addition, they indicate that the rotation defects associated with Fas2^spinR5, and probably also with other viable Fas2 alleles, are linked to a defective neuroendocrine function leading to abnormal synthesis of JH during pupal development (Adam, 2003).

The data support a model in which Fas2-expressing nCA neurons control JH titers which in turn remotely control the rotation of the genital plate. This model has two main predictions: (1) if JH is a mediator of Fas2 function during rotation, then Fas2^spin should function cell non-autonomously; (2) JH itself should have the potential to perturb rotation when its level is altered experimentally (Adam, 2003).

The cell non-autonomy of Fas2^spin is manifest in the the rescue experiments using GAL4 lines expressed in subsets of neurons in the brain. If Fas2 is cell non-autonomous for rotation, then it is expected that mosaic animals, in which some cells are mutant while others are wild type, should be rescued. Interestingly, it was found that almost all (99.7%) transposase-induced mosaic males (Fas2^spin/Y; D2-3 transposase/+) have normal, fully rotated genitalia. Consistent with a complete rescue of the external genitalia rotation phenotype, these mosaic males showed normal looping and fertility. Importantly, the comparison of the morphology of the CA synapse in wild type, Fas2^spin and mosaic males (Fas2^spin/Y;D2-3 transposase/+) indicates that all rescued mosaic males had restored a normal CA synapse (Adam, 2003).

The second prediction of this model is that JH, which is proposed to control rotation, should induce looping defects when its level is modified during pupal development. Interestingly, it has been shown that the JH analogs methoprene and pyriproxyfen produce rotation defects at low doses, after topical application to white pre-pupae. Higher doses of these JH analogs induce abdominal defects and lethality. Varying doses of pyriproxyfen were applied to white pre-pupae and the effects on genitalia rotation were monitored. Application of half-lethal doses of pyriproxyfen (0.25 pM/pupae) to wild-type pupae induces rotation defects that are very similar to Fas2^spin phenotypes. Indeed, dissection of the posterior abdomen of unhatched adults (pharate adults) indicates that pyriproxyfen-treated males have an under-rotated phenotype, with some males showing a complete absence of rotation. At higher doses (0.4 pM/pupae), the treatment is lethal; since animals die as pharate adults, their morphology can be analyzed. Dissection reveals a shift toward a no rotation phenotype, with a larger proportion of males having their genital plate in its initial position. Thus, increasing the level of a JH analog produces looping defects ranging from partial to a complete loss of circumrotation (Adam, 2003).

Because pyriproxyfen mimics spin phenotypes in a dose-dependent manner, it is concluded from these experiments that Fas2^spin pupae may have an elevated level of JH. To support this conclusion further, the dosage was modified of the Methoprene-tolerant gene (Met), which encodes a (bHLH)-PAS family of transcriptional regulators (Ashok, 1998). Flies that are mutant for Met are more resistant to JH analogs, indicating that the Met gene participates in JH signal transduction. Conversely, elevated expression of the Met⁺ gene results in flies with higher susceptibility to JH analogs (Ashok, 1998). If JH is elevated in Fas2^spin, then increasing the dose of the Met gene should enhance Fas2^spin genitalia rotation defects, while mutations in Met should suppress them. The effect was tested of Met^K17 (a P element-induced amorphic Met mutation) on Fas2^spin rotation defects. Strikingly, the loss of Met function completely rescues the spin rotation defects, indicating that spin and Met genetically interact and antagonize each other during this process. Conversely, the increase of the dose of the Met+ gene in a Fas2^spinR5 background results in lethality at the pharate adult stage, confirming the strong interaction between the two genes. Dissection of the genital plates reveals exacerbation of rotation defects, with ~25% having a complete lack of rotation of genitalia. This phenotype is very similar to the phenotype of pupae treated with high doses of pyriproxyfen. Altogether, these results indicate that Fas2^spin males have an elevated level of JH causing genitalia rotation defects. This allatotropic (promotion of JH synthesis) phenotype of Fas2^spin is consistent with studies in several insects indicating a role of the CA nerves in negatively controlling levels of JH during metamorphosis (Adam, 2003).

How do these results relate to vertebrate organ looping and LR asymmetry? The fact that JH affects looping morphogenesis in Drosophila suggests an important evolutionary conservation of the role of terpenoids in this process, downstream of LR determination. Like the retinoid hormones, JH is synthesized from the common isoprenoid precursor farnesyl diphosphate via the mevalonate biosynthetic pathway. Furthermore, JH is a sesquiterpenoid that is chemically related to the vertebrate terpene group, represented by retinoic acid. The common terpenoid nature of JH and RA has thus led to the proposal that these molecules may bind a common family of nuclear hormone receptors that might play similar functions in different organisms. In this respect, it is important to note that the JH analog methoprene, the topical application of which leads to genitalia rotation defects, can specifically bind and activate the RXR receptor in mammalian cultured cells. RA signal transduction in vertebrates requires the binding to and the activation of heterodimers composed of RAR and RXR nuclear receptors isoforms. Interestingly, the only insect homolog of vertebrate RXR is encoded by the Drosophila ultraspiracle (usp) gene, which has been shown to bind to JH in vitro (G. Jones, 1997; Jones, 2001). Altogether, these data thus suggest that JH and RA have a related activity (Adam, 2003).

In addition to sharing common chemical features, both RA and JH, when present in excess, have strikingly similar effects on organ looping. In conditions of excess RA, a series of heart defects has been observed, including reversal of symmetry or incomplete looping. In Xenopus, the heart tube fails to loop after continuous exposure to low doses of RA, and incomplete looping of the heart is also observed in mice treated with RA over a long period. It is important to note that the effects of excess RA on heart looping are dose sensitive and stage specific, as are the effects of JH analogs (methoprene and pyriproxyfen) on the looping of genitalia in flies (Adam, 2003).

In addition to blocking organ looping, excess RA can also induce a reversal of LR asymmetry in several vertebrate models. Such a reversal of asymmetry has not been observed after topical application of pyriproxyfen in Drosophila. This apparent discrepancy may be explained by species- and/or stage-specific responsiveness to excess terpenoids, as is found among vertebrates for RA. Another possibility is that JH in flies may have a\ function restricted to organ looping, not sharing the dual role of RA seen in vertebrates (Adam, 2003).

The chemical, and, as shown in this study, the functional and phenotypic similarities associated with JH and RA in flies and vertebrates, respectively, show that terpenoids play an evolutionarily conserved role in handed looping (Adam, 2003).

In Drosophila, genetic control of the establishment of the two major body axes has been well described. LR asymmetry has attracted little interest and thus remained an elusive process for at least two reasons. (1) There are only few and mostly transient (i.e., present during embryonic stages only) LR organs, leading to the view that flies may not represent a good model to study LR axis like vertebrates. In the case of genitalia rotation, this or another LR process have not been clearly validated as candidate LR markers. (2) No mutations had previously been isolated showed a fully penetrant and rotation-specific defect. Though some studies have reported rotation defects in specific allelic combinations, the rotation phenotypes are poorly penetrant and are associated with other developmental defects (Adam, 2003).

Thus, there is a novel parallel between the programs underlying Drosophila and vertebrate asymmetric organ looping. Is this parallel more general? In order to address this question, future work will have to be focused on the identification of new genes involved in genitalia rotation in Drosophila, using genetic screens and reverse genetic approaches. One major goal of future work in Drosophila will be the identification of asymmetrically expressed genes and/or proteins. The fact that no such gene has been identified so far may be due to the lack of appropriate data on the developmental aspects of LR asymmetry in Drosophila. In this respect, this study now allows identification of the male genital disc as a clear candidate tissue for looking at asymmetrically expressed molecular markers. The use of Drosophila and the comparative analysis of the LR asymmetry programs in vertebrates and invertebrates will help provide insights into the molecular mechanisms that underlie the question of symmetry breaking in animals (Adam, 2003).

Genetic analysis of an overlapping functional requirement for L1- and NCAM-type proteins during sensory axon guidance in Drosophila

L1- and NCAM-type cell adhesion molecules represent distinct protein families that function as specific receptors for different axon guidance cues. However, both L1 and NCAM proteins promote axonal growth by inducing neuronal tyrosine kinase activity and are coexpressed in subsets of axon tracts in arthropods and vertebrates. The functional requirements for the Drosophila L1- and NCAM-type proteins, Neuroglian (Nrg) and Fasciclin II (FasII), have been studied during postembryonic sensory axon guidance. The rescue of the Neuroglian loss-of-function (LOF) phenotype by transgenically expressed L1- and NCAM-type proteins demonstrates a functional interchangeability between these proteins in Drosophila photoreceptor pioneer axons, where both proteins are normally coexpressed. In contrast, the ectopic expression of Fasciclin II in mechanosensory neurons causes a strong enhancement of the axonal misguidance phenotype. Moreover, these findings demonstrate that this functionally redundant specificity to mediate axon guidance has been conserved in their vertebrate homologs, L1-CAM and NCAM (Kristiansen, 2005).

This study presents an analysis of the requirements and the functional specificity of Drosophila L1- and NCAM-type proteins during the postembryonic development of the Drosophila peripheral sensory nervous system. The partially penetrant phenotypes, which have been reported for L1- and NCAM-LOF mutants in Drosophila and different vertebrate model systems, suggest that the requirement for these neural CAMs is not absolute and that the lack of either L1- or NCAM-type proteins during nervous system development can be partially compensated for by other gene products. Moreover, considering the unique specificity of L1's and NCAM's homo- and heterophilic adhesive interactions, a molecular redundancy between these protein families may be unexpected. The specificities of the homophilic adhesive interactions within the L1 and the NCAM protein families have undergone considerable evolutionary changes. Drosophila Nrg and FasII exhibit a very low cross-reactivity with their vertebrate homologs, L1-CAM and NCAM. Although only the neuronal isoforms of human (L1-CAM^RSLE+) and of Drosophila Neuroglian (Nrg¹⁸⁰) have been directly tested for their ability to interact with each other, these results indicate that the ability of vertebrate CAMs to rescue the Nrg LOF phenotype most likely relies on homotypic adhesion, rather than on an interaction with endogenous Drosophila CAMs. This conclusion is also supported by the observation that the GOF phenotype in the wing sensory nervous system is only observed when the vertebrate transgene is expressed in both the wing epithelium and the sensory neurons. In addition, endogenous Nrg expression is not required for the production of the GOF axonal misguidance phenotype in the Drosophila wing (Kristiansen, 2005).

Although axonal growth and guidance involve a large array of different neuronal adhesion molecules, there appears to be a limited number of signaling pathways that are shared among structurally different CAM families. The two major signaling pathways, which are triggered by Ig-CAMs, involve nonreceptor tyrosine kinases or receptor tyrosine kinases, such as FGFR and EGFR. Both of these signaling pathways may act synergistically or in a redundant manner. L1-CAM-, NCAM-, as well as N-cadherin-mediated neuronal cell adhesion all activate neuronal FGF receptors and thereby induce neurite outgrowth in vitro. This suggests that structurally different neural CAMs are capable of feeding into the same signaling pathway and that multiple adhesive specificities coordinately influence axonal growth and guidance (Kristiansen, 2005).

Axonal guidance in the Drosophila ocellar sensory system (OSS) and the wing sensory nervous system involves the Nrg-mediated activation of FGF and EGF receptors. Constitutive activation of FGFR or EGFR can rescue the nrg³ LOF phenotype in the OSS, and Nrg GOF axonal misguidance in the developing wing is reversed by a hypomorphic allele of the Drosophila EGF receptor. The two types of neurons in the Drosophila OSS, ocellar pioneer (OP) and bristle mechanosensory (BM) neurons, differ in their expression of Nrg and FasII protein and in their requirement for both proteins during axonal growth and guidance. Whereas the neuron-specific isoforms Nrg¹⁸⁰ and FasII^PEST+ are coexpressed in OP axons, BM axons only express Nrg¹⁸⁰, but not FasII. The surrounding epidermis, which interacts with BM but not with OP axons, expresses the nonneuronal Nrg¹⁶⁷ isoform (Kristiansen, 2005).

The nrg LOF rescue experiments reveal strikingly different requirements for Nrg and FasII protein in the two neuronal cell populations. The requirement for Nrg in OP axons can be sustained by either the neural Nrg¹⁸⁰ or FasII^PEST+, but not by the nonneuronal Nrg¹⁶⁷ isoform. The two Nrg protein isoforms have identical extracellular domains and only differ in the size of their respective cytoplasmic domain. The capacity of FasII to fulfil the Nrg¹⁸⁰ requirement in OP axon guidance suggests that these structurally different proteins share a redundant function in these axons. This conclusion is further supported by the observation that the partially penetrant nrg LOF OP axonal misguidance phenotype is significantly amplified by a reduction of the fasII gene dosage. Remarkably, ectopic FasII^PEST+ expression in BM neurons enhances the deleterious effect of the Nrg loss, a situation that fits within the concept of antiredundancy or opposing functional capacities (Kristiansen, 2005).

The scenario of cell-specific redundant functions of Nrg¹⁸⁰ and FasII^PEST+ is maintained by their vertebrate homologs L1-CAM/Nr-CAM and NCAM¹⁴⁰, respectively. This indicates that the redundant specificities of L1 and NCAM proteins in neuronal subsets and the corresponding molecular interactions have been conserved in both CAM families over a long evolutionary time period. However, in contrast to the nonneuronal Nrg¹⁶⁷ isoform, which exhibits an antiredundant capacity compared with Nrg¹⁸⁰, the nonneuronal (RSLE−) vertebrate L1-CAM isoform is able to rescue the Nrg deficiency in OP axons. In contrast to Drosophila Nrg, the two vertebrate L1-CAM isoforms differ by the inclusion or exclusion of two small exons. The insertion of the five additional amino acid residues, which are encoded by exon2, into the L1-CAM extracellular domain modifies the homo- and heterophilic functions of vertebrate L1-CAMs. The inability of the human L1-CAM^RSLE+ isoform to efficiently interact with Drosophila Neuroglian suggests that the L1-CAM^RSLE+ GOF phenotype is the result of homotypic molecular interactions. Moreover, the rescue of nrg³ OP axonal phenotype by L1-CAM^RSLE− occurs in an Nrg deficient background, suggesting that vertebrate L1-CAM^RSLE− proteins are able to engage in homotypic molecular interactions in Drosophila. Interestingly, the nonneuronal human L1-CAM^RSLE− protein, for which a lower homophilic interaction capacity has been postulated, causes a much weaker GOF phenotype than the neuronal mouse L1-CAM^RSLE+ isoform. Nevertheless, the results indicate that this lower homophilic binding activity of the RSLE− isoform is sufficient to support the functional replacement of Nrg¹⁸⁰ in OP axons in Drosophila (Kristiansen, 2005).

Inclusion of the cytoplasmic miniexon generates a tyrosine-based endocytosis signal (RSLEY) in the neuronal vetebrate L1-CAM isoform. The AP-2-mediated endocytosis of the neuronal L1-CAM^RSLE+ isoform appears to be an important step in the activation of the MAPK signaling cascade by L1-CAM. Since neither Drosophila Nrg isoform contains an equivalent endocytosis signal in their cytoplasmic domain, Drosophila Nrg function either does not involve endocytosis or uses a different type of sorting signal than vertebrate L1 proteins (Kristiansen, 2005).

Although the analysis of the two Nrg isoforms indicates a specific requirement for Nrg¹⁸⁰ in OSS neurons, analysis of GOF conditions in the wing peripheral nervous system reveals an underlying common ability to activate RTK signaling. Since the Nrg-mediated activation of EGFR kinase only requires the extracellular Nrg domain for its interaction with the EGFR, both Nrg isoforms are able to exhibit an identical RTK-dependent axonal misguidance GOF phenotype. The ability of homologous vertebrate L1- and NCAM proteins to elicit the same response in Drosophila sensory neurons indicates a common, conserved specificity to influence RTK activity and thereby to regulate axonal growth and guidance. However, the different ability of the neuronal versus the nonneuronal Nrg isoform to rescue the nrg LOF phenotype in the OSS indicates that Nrg-mediated axonal guidance is also regulated by cytoplasmic interactions (Kristiansen, 2005).

Since the separation of arthropods and chordates, there has been an enormous diversification in the size and organization of metazoan nervous systems. At the same time, there has also been an increase in the number of L1- and NCAM-type paralogous genes in vertebrates (but not in Drosophila), as well as structural divergence and acquisition of new specific functions within each protein family. Both types of proteins have conserved an average of 25%–30% amino acid identity between their vertebrate and Drosophila homologues. The two groups of genes are of roughly similar size, and both have undergone independent events that resulted in the generation of different tissue-specific isoforms in Drosophila and vertebrates. Although both the vertebrate and invertebrate proteins are normally coexpressed in specific axonal tracts, their respective realms of expression have shifted in insect versus vertebrate nervous systems. As a result, NCAM expression is more widespread than L1-CAM or Nr-CAM in vertebrates, while FasII is more restricted than Nrg in insects. Therefore, all these genes are evidently highly accessible to mutation and genetic drift, and the current situation most probably reflects a selective pressure to maintain NCAM- and L1-type protein coexpression in specific axonal tracts of the nervous system. Nevertheless, although both L1 and NCAM proteins have acquired many new functions in both arthropod and chordate species, it appears that they initially had at least partially overlapping roles in growth cone signaling during axon guidance. Both CAM families have apparently maintained some of these shared functions and a common specificity, including a basic function as activators of RTK signaling, over a long time period (Kristiansen, 2005).

Therefore, it seems that the functional redundancy between L1- and NCAM-type proteins could constitute an important evolutionary constraint. It prevents the drift of these molecules into completely different functional entities, while at the same time, it allows their structures to further diverge and acquire separate and additional specificities. It has been proposed that functional redundancy is one mechanism for the canalization (stability after developmental perturbation and during evolution) of developmental processes. The requirement for a shared specificity between L1- and NCAM-type proteins in the control of RTK signaling during axon guidance might therefore reflect a requisite for redundancy that is found in any complex communication process. Redundancy is an essential component in any communication process for ensuring reliability by compensating the naturally occurring perturbations. Neuronal wiring is a cell communication-driven process where a highly complex set of signaling systems operates in parallel. As the number of different signals involved in axon guidance enlarged concomitant with an increase in complexity during evolution, the system noise affecting growth cone signal integration during development also increased. Unspecific adhesive interactions may also constitute a major source of noise for navigating growth cones. Therefore, cooperative redundancy might contribute to establishing a “buffered” physiological context required for ensuring process fidelity. It is postulated that this is the reason why the ancestral functional redundancy between L1- and NCAM-type molecules has been conserved over the last 600 million years of evolution (Kristiansen, 2005).

Basolateral junctions utilize warts signaling to control epithelial-mesenchymal transition and proliferation crucial for migration and invasion of Drosophila ovarian epithelial cells

Fasciclin2 (Fas2) and Discs large (Dlg) localize to the basolateral junction (BLJ) of Drosophila follicle epithelial cells and inhibit their proliferation and invasion. To identify a BLJ signaling pathway a genome-wide screen was performed for mutants that enhance dlg tumorigenesis. Two genes were identified that encode known BLJ scaffolding proteins, lethal giant larvae (lgl) and scribble (scrib), and several not previously associated with BLJ function, including warts (wts) and roughened eye (roe/rotund), which encode a serine-threonine kinase and a transcription factor, respectively. Like scrib, wts and roe also enhance Fas2 and lgl tumorigenesis. Further, scrib, wts, and roe block border cell migration, and cause noninvasive tumors that resemble dlg partial loss of function, suggesting that the BLJ utilizes Wts signaling to repress EMT and proliferation, but not motility. Apicolateral junction proteins Fat (Ft), Expanded (Ex), and Merlin (Mer) either are not involved in these processes, or have highly spatio-temporally restricted roles, diminishing their significance as upstream inputs to Wts in follicle cells. This is further indicated in that Wts targets, CyclinE and DIAP1, are elevated in Fas2, dlg, lgl, wts, and roe cells, but not Fat, ex, or mer cells. Thus, the BLJ appears to regulate epithelial polarity and dynamics not only as a localized scaffold, but also by communicating signals to the nucleus. Wts may be regulated by distinct junction inputs depending on developmental context (Zhao, 2008).

The purpose of this work was to gain greater insight into how the BLJ suppresses epithelial tumorigenesis and invasion by identifying and understanding the function of new genes important for BLJ function. To do so, a genomewide screen was completed for enhancers of dlg, which encodes a scaffolding protein that is a crucial organizer of the BLJ and is a potent repressor of follicle epithelial cell tumorigenesis and invasion. Deficiencies that cumulatively span ∼80% of the autosomes, or 64% of the Drosophila genome were systematically screened. A relatively small number of enhancers, ∼1 per 1000 genes screened, were detected indicating that the screen selected for loci specifically required for dlg function. Thus, the novel dlg enhancer genes that were identified, wts, roe, ebi, as well as at least two genes yet to be identified, are likely to be key collaborators with dlg in suppressing epithelial invasion. The specificity of the interactions between dlg and these enhancers is further indicated in that more than one allele of each gene showed an interaction, in several dlg backgrounds, and the strengths of enhancement were similar to deficiencies defining each locus. wts, roe, and ebi also enhanced Fas2 and lgl, indicating that they are not just important for dlg function, but for the function of the BLJ as a whole. In addition, overexpression of all enhancers except ebi suppressed dlg and Fas2 tumorigenesis, further confirming that the identified genes function in a BLJ network (Zhao, 2008).

BLJ pathway components in the nucleus and their putative relationship to Notch: ebi encodes an F-box protein with WD repeats that promotes protein degradation of specific targets. The failure of ebi overexpression to suppress Fas2 or dlg, and the relatively mild ebi phenotypes (midoogenesis small-nucleus and epithelial-organization defects, but no defects in germinal vesicle localization), suggest that ebi may function in only one of the three branches of BLJ signaling or in a parallel pathway to the BLJ. In the eye, ebi is important for promoting differentiation and inhibiting proliferation, which appear to be separable functions. Thus ebi could enhance Fas2 and dlg tumorigenesis by functioning within the proliferation-repressing branch of the BLJ, or the importance of ebi for differentiation suggests that it could function in the EMT branch of the BLJ or both. In contrast, ebi promotes protein degradation in response to Notch (N) and Drosophila EGF receptor (EgfR) signals, suggesting that it may act in a parallel pathway. Both Ebi and its mammalian homolog, TBL1, function in a corepressor complex through association with nuclear hormone transcriptional corepressor SMRTER/SMRT (Zhao, 2008).

Interestingly, although most N appears to be localized on the apical surface of follicle cells, some N is also localized in BLJs. Thus, it is possible that N localized to the BLJ may signal directly to Ebi. Consistent with this possibility, it was found that all of the genes in the BLJ network share some midoogenesis defects with N, including the small nucleus phenotype, epithelial stratification defects, and mislocalization of the germinal vesicle. The epithelial defects are also reminiscent of N-pathway mutants brainiac and egghead, which are required in the germ line for regulating N that is localized on the apical surface of the follicle cells abutting the germ line. Thus one possibility is that N signaling activity is regulated by its localization to apical vs. basolateral junctions in response to several signaling pathways acting during midoogenesis (Zhao, 2008).

The other modest dlg enhancer that was identified, roe, encodes a Krüppel-family zinc-finger protein that appears to be a transcription factor. Roe is also implicated in Notch signaling and thus may function with Ebi in N-dependent processes as proposed above. However, in contrast to ebi, roe loss caused follicle cell tumors, suggesting that roe may function more directly in a BLJ pathway than ebi. Consistent with a direct role for Roe in BLJ signaling, it was found that roe overexpression suppressed Fas2 and dlg tumorigenesis. Further, as for Fas2, dlg, and wts, roe represses CycE and DIAP1 expression (Zhao, 2008).

Warts was of special interested because of the many similarities observed in the quality and strength of wts and scrib phenotypes, suggesting that they are components in a BLJ signaling pathway, rather than a parallel pathway that cross talks with BLJ signaling. wts encodes a serine/threonine kinase that is an ortholog of human tumor suppressors Lats1 and Lats2, both of which have been linked to highly aggressive breast cancers. The prevailing model for Wts signaling in Drosophila is based on signaling in eye and wing tissue. Wts appears to relay signals from apicolateral junction proteins Ft, Ex, and Mer in wing and eye tissues. However, the results from almost every assay, including early tumor formation, border cell migration, BrdU, PH3, CycE, and DIAP1 expression, indicated little functional overlap between Ft, ex, mer, or mer; ex and wts, thus diminishing the importance of apicolateral Ft-Ex-Mer for Wts activation in follicle cells. The exceptions were that during midoogenesis, Mer is required for border cell migration and Ex is required for the endocycle switch, while both are required for maintenance of epithelial integrity and positioning of the germinal vesicle. However, the involvement of Ex and Mer in these processes are fundamentally distinct from how they act in Wts-dependent processes in other tissues. (1) Ft is not involved; (2) no indication was observed of Ex-Mer synergism; (3) ex, mer, and mer; ex phenotypes are relatively mild when compared to wts. It is concluded that the model for Wts activation in which apicolateral junction proteins Ft, Ex, and Mer play the predominant role cannot be universally applicable in all cell types. Rather, the relative importance of Ex and Mer for Wts regulation appears to depend on developmental context (Zhao, 2008).

Consistent with this proposal, strong functional interdependence and phenotypic similarities were found between Fas2, dlg, lgl, scrib, and wts, thus indicating that the BLJ, not the apicolateral junction, plays the predominant role in Wts regulation during oogenesis. Although genetic evidence alone cannot completely rule out that Wts may act in a parallel pathway to the BLJ and impinge on a set of downstream targets that overlap with those targeted by the BLJ, the following observations favor a model in which the BLJ is more directly involved in Wts regulation (it is noted that these are not mutually exclusive alternatives): (1) over 50 tumor suppressor genes have been identified in Drosophila, but lgl, scrib, and wts were the only strong dlg enhancers identified in this genomewide screen; (2) wts showed strong genetic interactions with Fas2, dlg, and lgl, similar to or stronger than scrib, which encodes a known BLJ protein; (3) wts has early tumor phenotypes similar to dlg partial loss of function and to scrib; (4) wts has the same border cell migration phenotype as scrib; (5) wts has similar small nucleus, epithelial stratification, and germinal vesicle defects as Fas2, dlg, lgl, and scrib; (6) like lgl and scrib, wts overexpression suppressed Fas2 and dlg tumorigenesis; (7) Fas2, dlg, and wts have similar proliferation defects, and (8) Fas2, dlg, and wts similarly repress CycE and DIAP1 expression, which is especially crucial, because CycE and DIAP1 are downstream targets of Wts signaling, and ex and mer had no impact on their expression, contrary to results in other tissues. Thus, the data strongly indicate that the BLJ signals through Wts, and may impinge on Roe in the nucleus, thus suggesting the first BLJ signaling pathway in animal cells. This implies that the BLJ not only acts as a localized scaffold, but also signals to the nucleus to control gene expression, both of which cooperate to regulate epithelial polarity and dynamics (Zhao, 2008).

How can these results in follicle cells, which suggest that Wts acts predominantly downstream of the BLJ, be reconciled with findings in eye tissue, which indicate that Wts acts downstream of the apicolateral junction? Interestingly, the genetic data in the eye suggest that Ft, Ex, and Mer cannot account for all of the signals that activate Wts, because wts overgrowth and tissue disorganization phenotypes are more severe than ft or mer; ex. On the basis of these findings in follicle cells, it is possible that Wts activation in the eye requires additional input from the BLJ. This possibility may have been overlooked thus far because dlg does not appear to have an overgrowth phenotype in the eye. dlg may be essential for additional functions in the eye that are epistatic to its tumor suppressor function, thus preventing loss of cells from the epithelium that could mask an overgrowth phenotype. Consistent with this, when activated Ras^v12 is combined with dlg loss, dramatic tumors develop that are larger and more invasive than those produced by Ras^v12 alone (Zhao, 2008).

In contrast, Dlg may have a diminished role in Wts signaling in the eye, much as the evidence indicates a diminished role for Ex and Mer in Wts signaling in the ovary. According to this model, Wts receives predominant input from distinct lateral junctions depending on tissue context. One distinction is that ovarian follicle cells are derived from a mesodermal lineage, while the eye and wing tissues are from ectodermal lineages. Further, many genes that disrupt apical-basal polarity and epithelial morphology have only subtle phenotypes in the eye by comparison to the ovary or embryo. Finally, the follicular epithelium requires input from junctions on all three follicle cell surfaces, lateral, apical, and basal, whereas most epithelia require only two, lateral and apical or basal. Thus, ovarian and imaginal tissues are likely to organize signaling pathways acting downstream of epithelial junctions in similar, yet fundamentally different ways to meet the unique organizational requirements of their cell-tissue morphologies. Some or all of these differences may contribute to the suggested specificity observed in Wts signaling downstream of BLJs in follicle cells. In general, these findings raise the possibility for future investigation that depending on the cell-tissue morphologies of a given organ, one lateral junction may play a predominant organizational role, and Wts signaling may act as a universal signaling adapter for mediating contact inhibition from that junction (Zhao, 2008).

An especially interesting aspect of Mer and Ex function that was uncovered in follicle cells is that it appears to be restricted to predominantly postmitotic, differentiated cells, in contrast to the role of Mer and Ex in other tissues. Further, given the absence of an involvement of Ft and lack of Mer-Ex synergism it is concluded that if Mer and Ex would be involved in Wts activation in follicle cells, they would have to function via a fundamentally distinct mechanism than in other tissues. It is proposed that during early oogenesis, the BLJ alone may provide the predominant input to Wts. Then, during midoogenesis, Ex and Mer may become involved in novel interactions with Dlg or other components of the BLJ to activate Wts in spatiotemporally distinct populations of differentiating cells to help achieve their unique developmental functions (Zhao, 2008).

How do wts, scrib, and roe promote motility? It is proposed that Scrib, Wts, and Roe are all crucially involved in EMT. In EMT, cells (1) loose apical-basal polarity and become mesenchymal-like, and (2) adopt a polarity conducive to movement. scrib, wts, and roe cells clearly lose epithelial polarity and become mesenchymal-like as indicated by their rounded morphology and lateralized phenotype. However, scrib, wts, and roe tumors do not invade, and scrib, wts, and roe border cells do not move, suggesting that the second aspect of EMT, adoption of a polarity conducive to movement, is defective. Consistent with this, mammalian Scrib is required for migration and epithelial wound healing of cultured human breast epithelial cells, and is also required in vivo for wound healing in mice. Human Scrib directs migration by organizing several polarities crucial for migration, including the orientation of the microtubule and Golgi networks and the localization of Cdc42 and Rac1 to the cell's leading edge. Thus Scrib has a conserved function in directed cell migration by organizing a polarity conducive to movement. In mammalian PC12 cells Scrib is in complex with Rac1. Fly Rac1 is essential for border cell migration and invasion of Fas2 and dlg tumors, suggesting that an essential role of Scrib in Rac1 function may be of crucial importance for movement. The apparent conserved role of BLJ proteins in organizing EMT, and both promoting and repressing movement, reemphasizes the suggestion that BLJ proteins do more than merely maintain apical-basal polarity, but rather repress a cellular transformation from epithelial polarity to a mesenchymal, lateralized signature conducive to movement (Zhao, 2008).

How is the function of scrib, wts, and roe in promoting border cell movement consistent with the requirement of Fas2, dlg, and lgl in repressing border cell movement? Further, how do scrib and wts act as enhancers of dlg tumor invasion even though scrib and wts tumors are noninvasive? For border cell movement, Fas2 and dlg mutations not only accelerate movement, but also delay border cell delamination. The delay in border cell delamination suggests that the BLJ normally promotes motility, but this promoting function can be bypassed when the repression of motility branch of the BLJ pathway is simultaneously lost. Cumulative data indicate that scrib, wts, and roe act predominantly within the EMT and proliferation branches of the BLJ pathway, and not the repression of motility branch. It is suggested that without simultaneous loss of the repression of motility branch of the BLJ pathway, scrib and wts border cells cannot bypass the essential requirement for the second step of EMT, thus border cell motility is blocked (Zhao, 2008).

This interpretation is also consistent with the seemingly paradoxical function of scrib and wts as enhancers of dlg tumor invasion, even though Scrib and Wts promote rather than repress border cell movement. The noninvasive scrib and wts tumor phenotypes indicate that they are crucial for repressing the first step of EMT, loss of epithelial polarity and adoption of a lateralized, mesenchymal-like phenotype. It has been suggested that scrib and wts enhance dlg invasive tumorigenesis by increasing the rate at which dlg mutant follicle cells undergo EMT and further facilitate invasion by depressing proliferation control and increasing the number of follicle cells available for movement. Thus, even though scrib and wts are required to promote movement, it is suggested that in dlg; scrib/+ or dlg; wts/+ tumors this requirement can be bypassed because the branch of the BLJ pathway that represses motility is simultaneously disrupted (Zhao, 2008).

The noninvasive tumor phenotypes of scrib and wts are very similar to the phenotypes of dlg mutants that specifically disrupt Dlg SH3 and GuK domains. Thus Scrib and Wts may act specifically downstream of the Dlg SH3 and GuK domains. Consistent with this, Scrib appears to associate with the Dlg GuK domain in neuronal synapses via the linker protein GuK-holder. Further, whereas Fas2, dlg, and lgl cause faster border cell migration, border cell migration is very similar to wild type in the dlg SH3/GuK-specific mutants, suggesting that Dlg SH3/GuK predominantly represses the first step of EMT and proliferation but not motility. On the basis of this specificity, it is suggest that one reason that lgl may be a stronger dlg enhancer than scrib and wts is that lgl represses motility in addition to EMT and proliferation. For example, the de novo tumor formation observed when one copy of lgl, scrib, or wts is removed in dlg^hf/dlg^sw ovaries suggests that a threshold level of BLJ activity essential for maintenance of polarity has been lost. However, the lgl interaction may be much stronger than scrib and wts because lgl additionally represses motility (Zhao, 2008).

Increased expression of CycE and DIAP1, known Wts targets, was observed in Fas2, dlg, lgl, scrib, wts, and roe cells. Thus the importance of CycE for proliferation control, and DIAP1 for control of EMT and motility, suggests that part of the mechanism by which Fas2-Dlg represses tumorigenesis is through activating Wts signaling. DIAP1 is in a complex with Rac1 and Profilin and enables border cell motility apparently by promoting actin turnover. Further, in the embryo, DIAP1 loss leads to Dlg cleavage and cellular rounding and dispersal. Too much DIAP1 also appears to be deleterious to movement, because targeted overexpression of DIAP1 specifically in border cells slows their migration (data not shown). Thus maintaining the proper balance of DIAP1 is critical for directed movement, and it may be part of the mechanism by which Scrib and Wts influence border cell movement, suggesting that interaction with Dlg and Rac1 may be another level at which Scrib regulates EMT and movement, consistent with the possibility that it functions downstream of Scrib and Wts in follicle cells to repress both EMT and proliferation (Zhao, 2008).

In contrast to the strong enhancement of dlg by scrib, Fas2 was only weakly enhanced by scrib. Given the complexity of coordinating EMT, proliferation, and motility within an epithelial field, perhaps the simplest model is that multiple Dlg complexes reside within the BLJ, each with a distinct set of ligands that control one or more morphogenetic activities (Zhao, 2008).

Another interesting difference in the enhancement of dlg and Fas2 by lgl, scrib, wts, and roe was that they all enhanced both dlg tumorigenesis and invasion, but only enhanced Fas2 tumorigenesis, without invasion. An important difference between these experiments may be that in Fas2^null follicle cells, Dlg is missing Fas2 as a ligand, whereas in dlg^hf/dlg^sw, dlg^hf/dlg^ip20, and dlg^hf/dlg^lv55 follicle cells, Fas2 is localized at sites of contact between follicle cells in both the native epithelium and in streams of invading cells, suggesting that Fas2 continues to act as a Dlg ligand in these cells. This is probably an important difference because Fas2-Dlg binding is expected to control the conformation of Dlg. Dlg conformations in turn may specify Dlg intra- and intermolecular interactions that determine the relative balance of EMT, proliferation, and invasion factors that associate with the BLJ scaffold. For example, in neuronal cells intramolecular interactions between Dlg SH3 and GuK domains regulate the strength of intermolecular binding of GuK-holder, which binds Scrib. The SH3-GuK intramolecular interaction is further modulated by intramolecular interactions with PDZ3, which are regulated by intermolecular interactions with neurolignin, a transmembrane ligand for PDZ3 (Zhao, 2008).

On the basis of this molecular model, it is proposed that in the absence of Fas2, Dlg has a distinct conformation that tilts the balance toward EMT and proliferation over invasion, when Lgl, Scrib, Wts, or Roe are reduced. This study has shown that lgl, scrib, wts, and roe are expected to act predominantly downstream of Dlg SH3 and GuK domains to repress EMT and proliferation. Thus, removal of one copy of lgl, scrib, wts, or roe in Fas2 cells may tip the ratio of factors controlling EMT, motility, and proliferation toward derepression of EMT and proliferation, masking the Fas2 requirement for invasion. One possibility is that lgl, scrib, wts, or roe are especially important for expression of a protein in the apicolateral junction, such as Par-3/Bazooka, which is essential for dlg invasion. Consistent with this, Ex upregulation is seen in both dlg and wts clones. Further, lgl enhancement at the lgl^ts permissive temperature showed essentially the opposite trend from Fas2. Rather than enhance tumorigenesis over invasion, removal of one copy of Fas2, dlg, scrib, wts, or roe in lgl egg chambers favored invasion. Thus, it is suggested that tumor invasiveness associated with particular combinations of mutated BLJ proteins may be masked or unmasked on the basis of the balance of activities that are disrupted, rather than disruption of particular activities per se (Zhao, 2008).

In summary, this study has identified the first signaling pathway that acts downstream of the BLJ that specifically controls EMT and proliferation, and important clues have been gained as to how this signaling may be organized. Like the Drosophila follicular epithelium, the human ovarian surface epithelium, which is thought to be the site of origin of most ovarian cancers, is derived from a mesodermal lineage. The data suggest that the BLJ plays an especially crucial role in the follicle cells compared to ectodermal lineages in repressing epithelial invasion and that the follicular epithelium appears to organize signaling from epithelial junctions in distinct ways compared to other epithelia. Given the conservation in the lineage of the fly and human epithelia, and the sensitivity of this screen for detecting molecules important for invasive carcinogenesis, it is proposed that the fly egg chamber may serve as a prototype for identifying early molecular events that are crucial for invasion of human ovarian cancer and possibly other malignancies that remain undetected before they start to invade (Zhao, 2008).

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