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).

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).

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).

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).

Adhesion proteins not only control the degree to which cells adhere to each other but are increasingly recognised as regulators of intercellular signalling. Using genetic screening in Drosophila, Fasciclin 2 (Fas2), the Drosophila orthologue of neural cell adhesion molecule (NCAM), has been identified as a physiologically significant and specific inhibitor of epidermal growth factor receptor (EGFR) signalling in development. Loss of fas2 genetically interacts with multiple genetic conditions that perturb EGFR signalling. Fas2 is expressed in dynamic patterns during imaginal disc development, and in the eye it was shown that this depends on EGFR activity, implying participation in a negative-feedback loop. Loss of fas2 causes characteristic EGFR hyperactivity phenotypes in the eye, notum and wing, and also leads to downregulation of Yan, a transcriptional repressor targeted for degradation by EGFR activity. No significant genetic interactions were detected with the Notch, Wingless, Hedgehog or Dpp pathways, nor did Fas2 inhibit the FGF receptor or Torso, indicating specificity in the inhibitory role of Fas2 in EGFR signalling. These results introduce a new regulatory interaction between an adhesion protein and a Drosophila signalling pathway and highlight the extent to which the EGFR pathway must be regulated at multiple levels (Mao, 2009).

These results demonstrate that the NCAM orthologue Fasciclin 2 specifically inhibits EGFR signalling activity during the normal development of the Drosophila eye, notum and wing. Interestingly, like other Drosophila EGFR inhibitors, Fas2 participates in a potential negative-feedback loop to regulate signalling, although the developmental significance of this remains to be established. The evidence for the interaction between Fas2 and EGFR relies on genetic interactions, diagnostic phenotypes of loss of function fas2 mutants, and a direct readout in fas2 clones of reduction of Yan, a transcriptional repressor targeted for degradation by EGFR activity. Furthermore, the results in the eye are supported by similar genetic logic in the developing notum and wing. Despite this, fas2 phenotypes are not identical to those of other known EGFR inhibitors. This is less surprising than it first appears, as the phenotypes of none of the known EGFR inhibitors in Drosophila (which currently include Argos, Kekkon-1, Echinoid, Sprouty, as well as some less specific proteins such as Gap-1) are as strong as constitutive activation of the receptor, and all are distinct. The explanation for the variation in strength and detail of phenotype is that each of the inhibitors has a different molecular mechanism and site of action in the pathway, as well as different sites of expression. For example, Argos is specific to the EGFR and is a diffusible molecule that sequesters ligand. By contrast, Sprouty, a cytoplasmic protein, inhibits a range of receptor tyrosine kinases, whereas Echinoid and Kekkon-1 are cell surface proteins that bind directly to the EGFR. It is evident that EGFR regulation depends on a patchwork of overlapping effects of multiple different types of modulators, each of which has greater or less importance in different developmental contexts. Presumably, this network of regulators underlies the observed precision and robustness of signalling (Mao, 2009)

Loss of Fas2 in the eye triggers at least two distinct types of extra photoreceptor recruitment. The ectopic mini-clusters appear at the same time that the normal outer photoreceptors are recruited and, by analogy with argos mutations, it is believed that they are caused by transformation of the 'mystery cells'. In normal development these form part of the precluster, but are ejected prior to the onset of photoreceptor differentiation. It is also possible that some of the mini-clusters are derived from de novo photoreceptor determination occurring in undifferentiated interommatidial cells, which is known to be triggered by excess EGFR activity. The second recognisable type of extra photoreceptors are the R7-like, Prospero-positive cells. These are presumably the product of abnormal recruitment of cone cell precursors as R7s, a switch of fates within the R7 equivalence group, which is sensitive to altered levels of receptor tyrosine kinase signalling (Mao, 2009).

The genetic data do not reveal a molecular mechanism for the inhibition of EGFR by Fas2 - that will require future biochemical analysis - but its location at the plasma membrane and the non-autonomy that was detected at the border of mutant clones point to three classes of models. (1) Fas2 reduces EGFR ligand production, presumably the TGFα homologues Spitz or Keren, for example by direct sequestration of the mature ligand. (2) Fas2 inhibits EGFR signalling, either by direct interaction with the receptor, or by indirectly downregulating its level or activity; in this case the observed non-autonomy would be indirect and caused by the well established positive feedback loop, whereby EGFR signalling activates expression of Rhomboid 1, which itself generates processed ligands. (3) Perhaps slightly less plausibly, the extracellular domain of Fas2 might be able to span the intercellular gap, thereby interacting with and inhibiting EGFR molecules on adjacent cells (Mao, 2009).

Precedence leads to a favouring of the second model. Two other adhesion proteins, Kekkon-1 and Echinoid, interact directly with the EGFR. Similarly, mammalian E-cadherin can inhibit the EGFR by direct binding. Of particular relevance to this work, it has recently been reported that mammalian EGFR can be inhibited by NCAM, the Fas2 orthologue (Povlsen, 2008). In these experiments using explanted mouse neurons combined with transfected mammalian cell lines, NCAM stimulates neurite outgrowth by blocking EGFR function. Preliminary results lead the authors to favour a mechanism of NCAM-induced downregulation of EGFR levels, although direct parallels with the current work are difficult to draw because the cytoplasmic domains of NCAM and Fas2 are not similar (Mao, 2009)

Beyond the evidence for inhibition of the EGFR described in this study and in the recent paper discussed above, Fas2/NCAM has now been implicated in several other signalling systems. The best characterised of these is an interaction with FGFR signalling, where, both in Drosophila and mammals, FGFR activity is required for Fas2/NCAM induced neurite outgrowth and direct binding of NCAM activates FGFR (Kiselyov, 2003; Christensen, 2006). By contrast, and an illustration of the context dependence of such interactions, it has also recently been reported that NCAM can inhibit FGFR activation by its ligand FGF (Francavilla, 2007). Less well studied links between NCAM and growth factors include the observation that NCAM can act as a signalling receptor for GDNF, and that it participates in the response of oligodendrocyte precursors to PDGF. The work reported in this study is the first genetic evidence to imply a role for Fas2 in the physiological inhibition of EGFR activity. It is important to set this discussion in the context of the well established role of Fas2/NCAM as a neural cell-adhesion molecule, with roles in axonal growth and pathfinding, as well as in synaptic maturation (Mao, 2009)

Overall, it is becoming clear that the EGFR pathway is regulated by multiple partially overlapping mechanisms, presumably because of the importance of regulatory precision and robustness of such a central and pleiotropic pathway. Notably, negative-feedback control is a recurring theme. Much less is known about physiologically significant regulators of EGFR signalling in mammals, and it will be interesting to determine whether feedback control is a conserved strategy. As there are many other signalling pathways and adhesion proteins that contribute to normal development, the total potential number of regulatory interactions between these key cell surface proteins is enormous and, indeed, many have been observed in vivo and in vitro. Of course, some of these might not occur in normal biological contexts, emphasising the value of a genetic approach to revealing which relationships between adhesion proteins and signalling pathways are physiologically relevant (Mao, 2009)