There is growing evidence that Notch pathway activation can result in consequences on cell morphogenesis and behaviour, both during embryonic development and cancer progression. In general, Notch is proposed to co-ordinate these processes by regulating expression of key transcription factors. However, many Notch-regulated genes identified in genome-wide studies are involved in fundamental aspects of cell behaviour, suggesting a more direct influence on cellular properties. By testing the functions of 25 such genes it was confirmed that 12 are required in developing adult muscles consistent with roles downstream of Notch. Focusing on three, Reck, rhea/talin and trio, their expression was varified in adult muscle progenitors, and Notch-regulated enhancers in each were identified. Full activity of these enhancers requires functional binding sites for Su(H), the DNA-binding transcription factor in the Notch pathway, validating their direct regulation. Thus, besides its well-known roles in regulating the expression of cell-fate determining transcription factors, Notch signalling also has the potential to directly affect cell morphology/behaviour by modulating expression of genes such as Reck, rhea/talin and trio. This sheds new light on functional outputs of Notch activation in morphogenetic processes (Pezeron, 2014).

Based on the fact that mutations in ena suppress a number of Abl mutant phenotypes, it has been proposed that Abl antagonizes Ena function. In Abl mutant embryos, Ena and actin mislocalize during dorsal closure and cellularization, and apical microvilli are abnormally elongated, indicating that Abl regulates the localization of Ena. In migrating fibroblasts, increasing Ena/VASP levels at the leading edge results in long, unbranched actin filaments, unstable lamellae, and decreased motility due to increased antagonism of capping protein. Interestingly, mutations in the gene encoding Drosophila capping protein ß enhance CNS axon pathfinding defects in Abl mutants, including commissure formation. Therefore, if Fra and/or Abl regulate Ena localization in commissural axons, then in fra, Netrin or Abl mutants, Ena may be mislocalized in the growth cone, leading to inappropriate inhibition of capping protein and excessive F-actin filament elongation. Additionally, reducing regulation of Ena by Fra or Abl may also allow greater Ena regulation by Slit-Robo signaling. In either case, reducing the gene dose of ena in fra, Netrin and trio,Abl mutant embryos would partially relieve these effects, allowing axons to respond more efficiently to other cues and cross the midline, as was observed. Consistent with this idea, it was found that either increasing or decreasing Ena/VASP proteins at the leading edge impairs the elaboration of growth cone filopodia in response to Netrin-DCC signaling, suggesting that Ena/VASP levels must be tightly regulated in order for the growth cone to respond optimally to extracellular signals (Forsthoefel, 2005).

The role of Abl in the growth cone is also likely to be complex. The observations implicate Abl as an effector of attractive Fra signaling. In addition, tyrosine phosphorylation of Robo by Abl is thought to negatively regulate repulsive signaling by Robo. Paradoxically though, loss-of-function mutations in Abl, robo and slit interact genetically, resulting in inappropriate axon crossing at the midline, and indicating that Abl may also promote repulsion in longitudinally migrating growth cones. Obviously, much remains to be understood about the molecular basis for genetic interactions of Abl, particularly how Abl and its various substrates cooperate with different growth cone receptors to yield specific cytoskeletal outputs (Forsthoefel, 2005).

In summary, genetic and biochemical interactions indicate that Abl, Trio and Ena are integrated into a complex signaling network with Fra and the Netrins during commissure formation. These observations identify another receptor that acts through these effectors, and provide a framework for further investigation of signaling by this key, evolutionarily conserved guidance receptor (Forsthoefel, 2005).

Notch is required for many aspects of cell fate specification and morphogenesis during development, including neurogenesis and axon guidance. Genetic and biochemical evidence is provided that Notch directs axon growth and guidance in Drosophila via a 'non-canonical', i.e. non-Su(H)-mediated, signaling pathway, characterized by association with the adaptor protein, Disabled, and Trio, an accessory factor of the Abl tyrosine kinase. Forms of Notch lacking the binding sites for its canonical effector, Su(H), are nearly inactive for the cell fate function of the receptor, but largely or fully active in axon patterning. Conversely, deletion from Notch of the binding site for Disabled impairs its action in axon patterning without disturbing cell fate control. Finally, it was showm by co-immunoprecipitation that Notch protein is physically associated in vivo with both Disabled and Trio. Together, these data provide evidence for an alternate Notch signaling pathway that mediates a postmitotic, morphogenetic function of the receptor (Le Gall, 2008).

Previous studies have led to the speculation that the Abl tyrosine kinase and its associated accessory factors might define an alternate, 'non-canonical', Su(H)-independent signaling pathway for the receptor Notch. The data reported in this study provide strong support for this hypothesis. In extracts of wild type Drosophila, Notch is associated with Disabled and Trio, two proteins that have been associated with the action of Abl tyrosine kinase. The functions of Notch in axon growth and guidance are likely to be executed by these complexes of Notch with Disabled and Trio, and not by its association with Su(H), since deletion of the Disabled binding site from Notch significantly impairs the axon patterning function of the receptor, whereas the Su(H) binding sites are largely dispensable for this process. Moreover, two other Notch derivatives are described that are still capable of executing the axon patterning functions of the receptor despite being completely inactive for specifying Notch-dependent cell fates. Taken together with previous data demonstrating that the genetic interaction of Notch with multiple Abl pathway components is required specifically for Notch-dependent axon patterning, these data provide a molecular picture of a Notch signaling machinery that is distinct from the well-established mechanism by which a proteolytic fragment of Notch enters the nucleus to directly control transcription of Su(H)-dependent target genes (Le Gall, 2008).

The key genetic data in favor of this hypothesis stem from the targeted construction of Notch derivatives that preferentially impair either the canonical, cell fate function of the receptor or its Abl-dependent axon patterning function, respectively. Deletion of the Su(H) binding sites from Notch progressively and dramatically reduces the ability of the receptor to limit neurogenesis, but has only limited effect on growth of CNS longitudinal axons, and no detectable effect on Notch-dependent defasciculation of ISNb motor axons. In contrast, deletion of the Disabled binding site substantially reduces the axon patterning activity of Notch (30%-40%, depending on the assay), while having no effect on cell fate function beyond what can be accounted for by the known Su(H) binding site within the deletion. The properties of these complementary Notch mutants argue for the action of a qualitatively different Notch mechanism in axon patterning. Further supporting this hypothesis is the observation that Notch co-precipitates from wildtype fly extracts with two cytoplasmic signaling proteins, the Abl cofactor, Trio and the adaptor protein, Disabled, potentially providing a molecular machinery to account for the phenotypic data. In principle, a good way to further test the basis of the Notch axonal phenotype would be to examine a disabled mutant, but unfortunately no such mutants are currently available. The phenotype of a trio mutant, in contrast, is consistent with the results documented in this study. The zygotic mutant phenotypes of trio are somewhat subtle, evidently because of persistence of maternally-provided trio RNA and protein, but they include defects in some of the CNS longitudinal axons that are affected in Notch^ts embryos, as well as defects in ISNb motor axon guidance, while trio has not been reported to produce any Notch-like defects in cell fate (Le Gall, 2008).

While deletion of the Disabled binding region of Notch clearly reduces the axonal activity of the protein, substantial residual activity still remains. In the case of Su(H), residual activity of a Notch mutant lacking all in vitro Su(H) binding sites can be traced to an association of Su(H) with the Notch ankyrin repeats that requires the cofactor, Mastermind. By analogy, perhaps Disabled can also associate with Notch via a second site that requires a cofactor present in vivo. Consistent with this idea, preliminary biochemical experiments hint that the RamΔA mutation (Notch deleted for the Disabled binding site) does not wholly ablate recruitment of Disabled and Trio in vivo, though current reagents do not allow this to be assessed rigorously. If so, the ankyrin/cdc10 repeat region would be a plausible candidate for a secondary site of association. Experiments show that the ankyrin repeats, together with the C-terminal portion of the Ram region, contribute substantially to Notch-dependent axon patterning. Since the experiments clearly show that the canonical Notch signaling pathway is dispensable for axonal function, the axonal requirement for this portion of the protein cannot be traced to its function in canonical signaling, and a contribution to formation or activity of Notch/Abl pathway complexes would offer the simplest explanation (Le Gall, 2008).

To date, the role of Disabled in the Abl signaling pathway has been difficult to establish due to the lack of loss-of-function Disabled mutant alleles. The placement of Disabled in the Abl pathway was based initially largely upon the observation that modest overexpression of Disabled suppressed both the embryonic lethality and morphological defects produced by genetic interactions of Abl with its accessory genes Nrt and Fax. The data now show that deletion of the Disabled binding region of Notch specifically impairs a Notch function, axon patterning, that depends on the interaction of Notch with multiple Abl pathway components. Moreover, GAL4-driven overexpression of Disabled modifies the Notch ISNb phenotype in the same way as do other treatment that enhance Abl signaling, such as overexpression of Abl or reduction of enabled. Thus, these data provide further support for association of Disabled with the Abl signaling pathway, though a definitive demonstration awaits the generation and characterization of a disabled mutation (Le Gall, 2008).

The presence of Trio in Notch complexes suggests that Rho family GTPases, particularly Rho and Rac, are good candidates for a downstream readout of Notch/Abl signaling. Consistent with this, dominant genetic interactions of Notch were observed with mutations in the three Drosophila Rac genes, but not, for example, with Cdc42. Such a readout would make sense in the context of the effects of Notch on growth cone guidance and would be consistent with previous studies of Drosophila Trio. It is interesting to note that identification of Rac as an effector of Notch/Abl signaling might suggest the possibility of Notch/Abl signaling having a non-Su(H) nuclear component in some developmental contexts in addition to its cytoskeletal targets. Rho family GTPases typically have multiple downstream targets, including nuclear gene regulation in addition to cytoskeletal structure and dynamics (Le Gall, 2008).

The key step in canonical Notch signaling is the proteolytic cleavage of the receptor by γ-secretase to release the active, intracellular moiety of the molecule, NICD. Does γ-secretase also play a role in Notch/Abl signaling. Since Disabled and Trio associated were found with full-length Notch prior to cleavage, and since Notch/Abl signaling in the growth cone presumably targets the cortical actin cytoskeleton, one possibility is that γ-secretase cleavage terminates the Notch/Abl signal by separating the receptor-bound complex from membrane-tethered components of the pathway such as Abl kinase and Rho GTPases. Alternatively, in contexts such as ISNb, perhaps displacement of Disabled and Trio away from the membrane is part of the mechanism by which Notch antagonizes Abl pathway activity. Moreover, while proteolytic activity is the most apparent function of γ-secretase there have been suggestions that the complex may also have a separate function in Notch trafficking, aside from cleavage. If so, this activity could modulate Notch/Abl signaling independent of any role for protease cleavage. Clearly, additional experiments will be necessary to assess the various possible models (Le Gall, 2008).

Is the interaction of Notch with Abl pathway proteins limited to just a few Drosophila growth cones, or is it of more general biological significance. The ability to detect Notch complexes with Disabled and Trio in extract of whole embryos argues for the latter, as does the strong phylogenetic conservation of all the components of the pathway. Good candidates for potential Notch/Abl-dependent processes are provided by those developmental contexts in which non-Su(H) Notch signaling has been proposed previously. In Drosophila, these include organization of actin structure at the D/V boundary of the developing wing; in mammals, they include myogenesis and B-cell development, as well as a ligand-stimulated cytoplasmic signaling process of Notch that is essential for the survival of mouse neural stem cells and human embryonic stem cells (Le Gall, 2008).

Axon guidance and classic lateral inhibition seem to represent limit cases in which the Notch signal is largely transduced selectively through either the Abl pathway or the Su(H) pathway, respectively. It seems likely, however, that in each case both pathways make some contribution to Notch function: deletion of Su(H) binding sites does have some deleterious effect on growth of CNS longitudinal axons, while mutation of Abl and Nrt cause small but reproducible decreases in the efficacy of the classic Notch function that discriminates the identities of sibling cells. Perhaps two parallel Notch signals, one through the canonical Su(H) pathway and the other mediated by the Notch/Abl interaction, can be used in concert to provide a richer nuclear readout, or to coordinate nuclear gene regulation with cortical properties such as cytoskeletal structure and cell adhesion. It will be of great interest to determine whether some classic functions of Notch, such as dendritic patterning or oncogenesis, reflect more balanced contributions both from canonical Notch signaling and from the Notch/Abl pathway (Le Gall, 2008).

The Drosophila neuromuscular junction (NMJ) is capable of rapidly budding new presynaptic varicosities over the course of minutes in response to elevated neuronal activity. Using live imaging of synaptic growth, this dynamic process was characterized, and it was demonstrated that rapid bouton budding requires retrograde bone morphogenic protein (BMP) signaling and local alteration in the presynaptic actin cytoskeleton. BMP acts during development to provide competence for rapid synaptic growth by regulating the levels of the Rho-type guanine nucleotide exchange factor Trio, a transcriptional output of BMP-Smad signaling. In a parallel pathway, it was found that the BMP type II receptor Wit signals through the effector protein LIM domain kinase 1 (Limk) to regulate bouton budding. Limk interfaces with structural plasticity by controlling the activity of the actin depolymerizing protein Cofilin. Expression of constitutively active or inactive Cofilin (Twinstar) in motor neurons demonstrates that increased Cofilin activity promotes rapid bouton formation in response to elevated synaptic activity. Correspondingly, the overexpression of Limk, which inhibits Cofilin, inhibits bouton budding. Live imaging of the presynaptic F-actin cytoskeleton reveals that activity-dependent bouton addition is accompanied by the formation of new F-actin puncta at sites of synaptic growth. Pharmacological disruption of actin turnover inhibits bouton budding, indicating that local changes in the actin cytoskeleton at pre-existing boutons precede new budding events. It is proposed that developmental BMP signaling potentiates NMJs for rapid activity-dependent structural plasticity that is achieved by muscle release of retrograde signals that regulate local presynaptic actin cytoskeletal dynamics (Piccioli, 2014).

Activity-dependent changes in synaptic structure play an important role in developmental wiring of the nervous system. The Drosophila larval neuromuscular junction (NMJ) has emerged as a model glutamatergic synapse that is well suited to study activity-dependent structural plasticity. The NMJ can be imaged in vivo during developmental periods of rapid synaptic growth when the axonal terminal expands ~5- to 10-fold in size over 5 d. Forward genetic screens to identify mutations that alter synaptic growth have revealed essential roles for retrograde bone morphogenic protein (BMP) signaling mediated by the secreted ligand Glass bottom boat (Gbb). Mutations that disrupt BMP signaling lead to synaptic undergrowth and neurotransmitter release defects. Multiple pathways downstream of retrograde BMP signaling through the type II receptor Wishful thinking (Wit) have been linked to synaptic growth, synapse stability, and homeostatic plasticity in Drosophila. BMP signaling via the Smad transcription factor Mothers against Dpp (Mad) regulates the expression of the Rho-type guanine nucleotide exchange factor (GEF) trio to control normal synaptic growth. Wit also interacts with LIM domain kinase 1 (Limk) to enhance synaptic stabilization in a pathway parallel to canonical Smad-dependent signaling. BMP signaling through Wit also potentiates synapses for homeostatic plasticity in a pathway that is independent of limk and synaptic growth regulation (Piccioli, 2014).

The NMJ displays acute structural plasticity in the form of rapid presynaptic bouton budding in response to elevated levels of neuronal activity. These rapidly generated presynaptic varicosities, referred to as ghost boutons, lack presynaptic and postsynaptic transmission machinery when initially formed. The budding of ghost boutons requires retrograde signaling mediated by the postsynaptic Ca2+-sensitive vesicle trafficking regulator synaptotagmin (Syt) 4 (Korkut, 2013). Syt4 also participates in developmental synaptic growth and controls retrograde signaling that mediates enhanced spontaneous release at the NMJ (Yoshihara, 2005; Barber, 2009). Beyond the role of Syt4 in ghost bouton budding, little is known about the signaling pathways that underlie this rapid form of structural synaptic plasticity. In particular, it is unclear whether pathways that regulate synaptic growth over the longer time scales of larval development also trigger acute structural plasticity. To address these issues, this study identified synaptic pathways that are required for rapid structural plasticity at Drosophila NMJs. Ghost bouton budding was found to be locally regulated at the synapse level, occurring in axons that have been severed from the neuronal cell body. In addition, activity-induced ghost bouton formation requires Syt1-mediated neurotransmitter release and postsynaptic glutamate receptor function. Like developmental growth, retrograde BMP signaling is required for ghost bouton budding. BMP signaling functions through a permissive role mediated by developmental Smad and Trio signaling, as well as through a local Wit-dependent modulation of Limk and Cofilin (Twinstar) activity that alters presynaptic actin dynamics (Piccioli, 2014).

Experimental analysis of ghost bouton budding at the Drosophila NMJ indicates that rapid activity-dependent synaptic growth requires retrograde BMP signaling at this synapse. The current data support a model in which BMP signaling through the type II receptor Wit is required developmentally to potentiate synapses for budding in response to elevated synaptic activity. This pathway requires Smad-dependent expression of the Rho-type GEF trio, and parallels a requirement for BMP signaling and Trio in developmental synaptic growth that occurs during the larval stages. In a parallel pathway, Wit interaction with Limk inhibits bouton budding through regulation of Cofilin activity. Both pathways regulate the synaptic actin cytoskeleton and may converge on similar actin regulatory molecules such as Limk and Cofilin via Rac1 or RhoA. Manipulating Cofilin activity levels by the overexpression of Limk or the expression of constitutively active/inactive Cofilin demonstrates that high Cofilin activity favors bouton budding, while low Cofilin activity inhibits budding. Local changes in the actin cytoskeleton that accompany activity-dependent bouton budding were also observed at sites of new synaptic growth. In addition, pharmacological disruption of normal actin turnover inhibits budding, suggesting that increased actin turnover mediated by Cofilin potentiates rapid activity-dependent synaptic plasticity (Piccioli, 2014).

Multiple genetic perturbations of BMP signaling were identified that altered the frequency of activity-dependent bouton budding at the NMJ. Although several of these mechanisms are shared with those previously characterized to control BMP-mediated developmental synaptic growth, several manipulations separated rapid activity-dependent BMP-mediated bouton budding from the slower forms of developmental growth. In the case of wit mutants or motor neuron overexpression of dad, a reduction in baseline bouton number was observed that showed varying degrees of severity. Wit mutants displayed strongly undergrown synapses, while dad overexpression animals had only modest synaptic undergrowth. In contrast, both these manipulations strongly suppressed ghost bouton budding. Additionally, synaptic undergrowth with partial knockdown of Gbb using postsynaptic RNAi was not observed, while this manipulation caused a strong reduction in ghost bouton budding. These observations indicate that rapid ghost bouton budding is more sensitive to modest perturbations in BMP signaling compared with developmental synaptic growth. One explanation for this differential sensitivity is that BMP signaling potentiates NMJs for activity-dependent bouton budding via transcriptional regulation of molecular components that are not required for normal synaptic growth. Alternatively, similar molecular pathways are required, but at different levels of output. In particular, trio mutants display a less severe synaptic undergrowth phenotype than wit mutants, but show similarly severe defects in ghost bouton budding. Because trio expression is strongly dependent on BMP signaling (Ball, 2010), a modest reduction in BMP output could reduce Trio levels such that ghost bouton budding is significantly reduced, while normal synaptic growth is less affected. It will be interesting to determine in future studies whether the developmental role for BMP signaling for acute structural plasticity shares a critical period as has recently been found for BMP function during developmental synaptic growth (Piccioli, 2014).

Given the requirement of the postsynaptic Ca2+ sensor Syt4 for normal levels of ghost bouton budding, an attractive model is that BMP is released acutely in response to elevated activity through the fusion of Syt4-positive postsynaptic vesicles. However, the current analysis indicates that retrograde BMP signaling through trio transcriptional upregulation is unlikely to be an instructive cue for bouton budding, as the severing of axons and the inhibition of retrograde trafficking of P-Mad before stimulation does not reduce budding in response to elevated activity. It is possible that synaptic P-Mad may play an instructional role in ghost bouton budding, as a local decrease in budding frequency was observed when Gbb expression was specifically reduced in muscle 6. Neuronal overexpression of dad also reduced synaptic P-Mad. Therefore, dad overexpression could inhibit ghost bouton budding by decreasing synaptic P-Mad signaling, in addition to decreasing nuclear Smad signaling. However, no dosage-dependent genetic interactions were observed between syt4 and wit, suggesting that Syt4 may participate in a separate pathway to regulate ghost bouton budding. Activity-dependent fusion of Syt4 postsynaptic vesicles (Yoshihara, 2005) could release a separate unidentified retrograde signal that provides an instructive cue for budding that would function in parallel to a developmental requirement for retrograde BMP signaling (Piccioli, 2014).

In addition to instructive cues from the postsynaptic compartment that trigger ghost bouton budding, the presynaptic nerve terminal must have molecular machines in place to read out these signals and execute the budding event. The regulation of Rho GTPases via Rho GEFs and GAPs downstream of extracellular cues is an attractive mechanism, as these proteins play critical roles in the regulation of neuronal morphology and axonal guidance. Several studies have shown that retrograde synaptic signaling regulates Rho GTPase activity to alter synaptic function and growth in Drosophila (Tolias, 2011). Ghost bouton budding mediated by developmental BMP signaling also shares some similarities with mechanisms underlying homeostatic plasticity at Drosophila NMJs. The Eph receptor is required for synaptic homeostasis at the NMJ, and it interfaces with developmental BMP signaling via Wit. While Eph receptor-mediated homeostatic plasticity predominantly requires the downstream RhoA-type GEF Ephexin, the Eph receptor may also signal through Rac1. Drosophila VAP-33A may also act as a ligand for synaptic Eph receptors, as it has been shown to regulate NMJ morphology and growth, while preferentially localizing to sites of bouton budding. The current analysis indicates that the levels of Trio, which functions as a Rho-type GEF, are bidirectionally correlated with ghost bouton budding activity and that overexpressed Trio is localized to ghost boutons after budding. As such, acute Trio regulation represents another attractive pathway for rapidly modifying bouton budding activity (Piccioli, 2014).

Rho GTPase signaling can produce distinct effects in differing systems and cell types depending on the presence or absence of downstream effectors, although most of these pathways ultimately impinge on regulation of the actin cytoskeletal. Indeed, this study has found a key role for Limk regulation of Cofilin activity in the control of ghost bouton budding. The current findings indicate that Limk activity normally functions to inhibit the formation of ghost boutons, as neuronal overexpression of Limk strongly suppressed activity-dependent bouton budding. Consistent with an inhibitory role for Limk, Cofilin activity promotes budding, while the overexpression of an inactive Cofilin inhibited budding. The expression of mutant Cofilin transgenes resulted in visible changes to the presynaptic actin cytoskeleton at NMJs, indicating that these manipulations likely alter rapid budding events by changing local actin dynamics at sites of potential growth. Using live imaging of F-actin dynamics before and after bouton budding, the formation of new F-actin puncta was observed at sites of bouton budding. Elevated Cofilin activity is sufficient to increase ghost bouton budding frequency, and is predicted to increase actin turnover and the formation of F-actin structures. Pharmacological disruption of actin polymerization dynamics also disrupts rapid bouton addition in response to elevated activity (Piccioli, 2014).

These findings support a model whereby Wit has opposing signaling roles with respect to bouton budding. Providing a permissive role via Smad signaling and an inhibitory role via Limk activation may provide for a system in which increased potential for rapid synaptic expansion is directly coupled to enhanced synaptic stability. This coupling could set a threshold for ghost bouton budding downstream of synaptic activity. In the background of moderate or low synaptic activity, Limk prevents ghost bouton budding. When synaptic activity is elevated, additional signaling events promote new synaptic growth by either reducing or outcompeting Limk activity, with a concurrent activation of Cofilin. Decreased Limk activity downstream of extracellular cues has been shown to regulate cell morphology in other systems as well, providing an attractive mechanism for rapid activity-dependent regulation of synaptic structure at Drosophila NMJs (Piccioli, 2014).

DEVELOPMENTAL BIOLOGY

Embryonic

Trio mRNA is abundant and ubiquitous early in embryogenesis; this indicates a substantial maternal contribution. At stages 11-12, Trio message is broadly distributed, with highest levels in invaginating gut (anterior and posterior) and in a repeating pattern within the developing central nervous system (CNS). At stages 13-15, CNS expression is maintained at high levels, while a new pattern of epidermal expression at segment boundaries emerges. This peripheral staining corresponds to the epidermal muscle attachment (EMA) cells, a specialization of the body wall epidermis involved in patterning and maintaining the integrity of muscle attachment sites. EMA cell expression is transient and is undetectable by stage 16, while CNS expression is maintained at a reduced level throughout stage 16. The abundant and sustained expression of Trio RNA in the developing CNS is consistent with a role in axonogenesis (Bateman, 2000).

To examine the temporal and spatial expression of trio during development, in situ analysis of Canton-S wild-type embryos was performed with antisense riboprobes. Extensive accumulation of Trio mRNA was observed in early cleavage stage embryos, indicating maternal contribution of the Trio mRNA. By the cellular blastoderm stage, levels of Trio mRNA are greatly reduced compared with the earlier maternal contribution. As germband extension proceeds, mRNA accumulation in the invaginating mesodermal layer is detected. The first accumulation of Trio mRNA in the developing nervous system is evident during stage 10. Expression in the nervous system persists through approximately stage 15. It appeared that most, if not all, neurons of the CNS express Trio mRNA. In addition, low levels of trio expression are observed throughout the epidermis at these stages. Patches of Trio expression in the lateral epidermis become evident in stage 13 embryos. This accumulation corresponds to muscle attachment sites for the somatic musculature. During the process of dorsal closure, increased levels of Trio mRNA accumulation were observed in leading edge cells that enclose the yolk sack along the dorsal surface. By stage 16, the majority of Trio mRNA accumulation becomes restricted to cells surrounding the developing gut. Throughout embryonic development, Trio expression appears most prominently in cells that are migrating or undergoing dynamic cytoskeletal rearrangements (Liebl, 2000).

To assess the function of Trio, its distribution patterns in Drosophila tissues were examined. Trio protein fused to GST was expressed in bacteria and used to generate rabbit polyclonal and mouse monoclonal (mAb 9.4A) antibodies. In the embryonic CNS, Trio staining is initially detected at stage 12, when axons start extending from the neuronal cell bodies. At stage 13, Trio is detected in the growing axon fascicles running on the longitudinal tracts and on those crossing the midline of the ventral cord. As the CNS develops, the axonal expression becomes more robust in pattern and is detected preferentially in the longitudinal fascicles and weakly in commissural fascicles of stage 16 embryos. These expression patterns suggest that Trio may be involved in axonogenesis that includes axonal extension, fasciculation, or pathway selection. In addition to the neural tissue, Trio is found in the epidermis and strongly in the muscle attachment sites. Trio expression is observed throughout development (Awasaki, 2000).

Larval and adult

In the adult brain, while a large number of cells in the cortex and most neuropil regions are weakly labeled with anti-Trio antibody, strong Trio staining is detected in several groups of neurons, including the MB neurons. A pair of MBs are located in the central brain, and each exhibits a characteristic structure that consists of calyx, peduncle, and five (alpha, alpha', beta, beta' and gamma) lobes. These parts are formed by the neurites emanating from clusters of neurons, Kenyon cells, located in the dorsocaudal cortical regions. All of these neurons extend their processes throughout the peduncle but are classified into three types by their further projection patterns. The individual axons extended from the alpha/beta or alpha'/beta' lobe neurons bifurcate into the alpha and beta, or alpha' and beta', lobes, respectively, and the axons of the gamma lobe neurons project to the gamma lobe after passing through the peduncle. Trio is expressed in a subset of MB neurons and distributed in the cell bodies, calyx, central and lateral peduncles, and alpha', beta', and gamma lobes but not in alpha and beta lobes. To confirm this assignment, MBs were doubly stained with mAb 1D4, which strongly labels the alpha and beta lobes and a part of the peduncle, and weakly labels the gamma lobe but not the alpha' and beta' lobes. These staining patterns were complementary to each other in most MB regions, which confirm the subregional distribution of Trio in MB (Awasaki, 2000).

Since MBs undergo dynamic morphological changes during development, it needed to be clarified how Trio expression is associated with the changes. Trio is distributed in the larval MB, which includes the larval vertical (LV) and medial (LM) lobes, peduncle, calyx, and cell bodies throughout larval life. During the pupal stages, the Trio expression pattern in MB alters as metamorphosis proceeded. Trio staining is found in the vertical and medial lobes at 12 hr after puparium formation (APF), is then confined in the approximate anterior region of the peduncle, with no signals in any lobes at 24 hr APF, and is only detected in the gamma lobe at 48 hr APF. These successive changes in Trio staining correspond to the remodeling processes of the gamma lobe neurons. The gamma lobe neurons are the first neurons generated that form vertical and medial lobes during the larval stages, then undergo degeneration that results in the loss of both lobes around 18 hr APF, and finally regenerate medially to form adult gamma lobes at 24–36 hr APF. Trio is found continuously distributed in the gamma lobe neurons in varying patterns during the larval, pupal, and adult stages. Trio is not observed in the alpha/beta and alpha'/beta' lobe neurons at 24 hr or 48 hr APF (Awasaki, 2000).

Trio is not only expressed in neurons extending neurites at the developing stages, but is also abundantly found in adult brains. This adult expression suggests that Trio functions in some cellular events other than neurite development. To assess the possible Trio function, immunoelectron microscopy was performed to examine the subcellular localization of Trio in adult brains. Trio signals are distributed in a patched pattern in the axons and cell bodies of neurons in the central brain and optic lobes. The patches are, in many cases, associated with clusters of vesicles in the axoplasm. Occasionally, small areas of the plasma membrane close to the patches are stained. This staining, however, may have resulted from diffusion of the dye from the patches. Furthermore, in the lamina neuropil, which exhibits an array of lamina cartridges consisting of synaptic pairs, photoreceptor cells, and lamina neurons, Trio is found in the dendritic terminals of the lamina neurons that contact or intrude into the photoreceptor cells. Occasionally the terminals with the Trio signals form postsynapses, and the signals are largely associated with the plasma membrane in the terminals. No staining is detected in the presynaptic terminals of the photoreceptor cells in adult brains (Awasaki, 2000).

Effects of Mutation or Deletion

Correct pathfinding by Drosophila photoreceptor axons requires recruitment of p21-activated kinase (Pak) to the membrane by the SH2-SH3 adaptor Dock. The guanine nucleotide exchange factor (GEF) Trio has been identified as another essential component in photoreceptor axon guidance. Regulated exchange activity of one of the two Trio GEF domains is critical for accurate pathfinding. This GEF domain activates Rac, which in turn activates Pak. Mutations in trio result in projection defects similar to those observed in both Pak and dock mutants, and trio interacts genetically with Rac, Pak, and dock. These data define a signaling pathway from Trio to Rac to Pak that links guidance receptors to the growth cone cytoskeleton. It is proposed that distinct signals transduced via Trio and Dock act combinatorially to activate Pak in spatially restricted domains within the growth cone, thereby controlling the direction of axon extension (Newsome, 2000).

The development of different axon pathways was assessed in order to examine the trio loss-of-function phenotype during embryonic nervous system development. Embryos from different allelic combinations were collected and stained with the anti-Fasciclin II (Fas II) antibody mAb 1D4, an excellent marker for motor axon pathways. Analysis of motor axon pathfinding revealed defects in the ability of nerve branches to reach their target muscles in trio mutants. The two branches most sensitive to perturbation of small GTPase function, ISNb and SNa, are also most sensitive to the loss of trio activity. These phenotypes were observed in all allelic combinations tested, with some variation in penetrance depending on genetic background. Occasionally, defects in target muscle attachment to the underlying epidermis were observed, which likely reflect a role for trio in EMS cells. To avoid scoring guidance errors that could be caused by the target rather than the growth cone, all segments with abnormal muscle patterning were excluded from the analysis (Bateman, 2000).

To ensure that the observed defects in ISNb guidance result from the loss of trio, embryos carrying trio^BX4, a precise excision of the P[1372/3] insertion that restores the locus, were examined. These embryos display wild-type levels of ISNb stop short (2.8%), demonstrating that the phenotype is insertion dependent. The defects observed in SNa development were similar to those of the ISNb phenotype. In trio mutants, SNa sometimes fails to extend either its lateral branch, to contact muscles 5 and 8, or its vertical branch, to contact longitudinal muscles 21-24. Occasionally, SNa fails to reach its target domain altogether; instead it stalls beneath the ventral target domain of ISNb. Although not highly penetrant, these SNa defects were seen in all allelic combinations examined but not in wild-type or precise excision controls. Consistent with defects observed in motoneurons, analysis of axon trajectories in the CNS reveal an inability of axons to pathfind correctly. During the early development of the CNS, longitudinal axons are required to cross segment boundaries and extend into neighboring segments, such that by the late stages of embryonic development (stage 17), distinct 1D4-positive fascicles form continuous pathways along the length of the CNS. In mutant embryos lacking trio function, defects are observed in the formation of these pathways. The most dramatic and persistent disruption was seen in the lateralmost Fas II-positive longitudinal pathway, where breaks and/or inappropriate direction of these interneuronal axons are often observed. In trio^1372/3/trio^1372/3 embryos, 20.6% of stage 17 A2–A8 hemisegments fail to connect to the neighboring segment, compared with only 1.2% (n = 320) in wild-type embryos. Similar defects are seen in multiple allelic combinations (Bateman, 2000).

Mutations in trio cause specific defects in the formation of multiple embryonic axon pathways, implying a role for trio activity in developing axons. However, because trio expression is not restricted to neurons, it is possible that its activity is required elsewhere and that the axonal phenotypes observed represent functions outside the nervous system. To exclude this possibility, axon pathway formation was examined in mutant embryos while simultaneously expressing a wild-type trio construct in postmitotic neurons using the GAL4 driver elaV-GAL4. These embryos show a marked reduction in pathfinding errors in both the CNS and the motor nervous system, indicating that the axonal defects in trio mutants result from a lack of trio function in neurons (Bateman, 2000).

Dosage-sensitive genetic interactions between trio and Abl have been documented. A number of observations support the interaction of Abl and Trio in a common regulatory network. First, the dosage-sensitive genetic interactions between trio and Abl are reciprocal, as assayed by either viability or CNS architecture. Heterozygous mutations in trio worsen the Abl mutant phenotype, while heterozygous mutations in Abl worsen trio mutant phenotypes. A background of compromised signaling (Abl¹/Abl⁴, Df(3L)FpaI/trio^M89, or trio^P0368/10/trio^M89) is enhanced by reduced activity of another member of this network (Liebl, 2000).

As further evidence for the involvement of Abl and Trio in a common signaling network, the Abl and trio homozygous mutant phenotypes show a synergistic interaction. Neither the Abl mutant background nor the trio mutant background have dramatic phenotypic consequences on CNS architecture. Phenotypes similar to those reported here have been observed in a variety of trio mutant combinations (Awasaki, 2000; Bateman, 2000). However, combining these two backgrounds to generate trio, Abl homozygous mutant embryos results in dramatic disruption of the CNS scaffolding. Taken alone, this synthetic enhancement may represent common or independent signaling pathways involving Abl and Trio. However, combined with the dosage-sensitive interactions between Abl and trio observed, it is likely that these molecules are involved in overlapping or interdependent networks. Similar synergistic effects between Abl and fax, and Abl and dab, have been reported. In addition to the reciprocal genetic enhancement between Abl and trio, a null allele of fax (fax^M7) greatly worsens the trio hypomorphic mutant's viability, while a dab null allele weakly modifies this background (Liebl, 2000).

The ena gene was identified through its ability to suppress the Abl mutant phenotype. Since reductions in ena compensate for the absence of Abl, it has been hypothesized that a precise balance between Abl and Enabled activity is required for viability. Similar to the genetic interaction between Abl and ena, heterozygous mutations in ena can partially alleviate the trio mutant phenotype. One interpretation of this interaction is that a balance between trio and ena is required, and Trio may possess a biochemical function that is antagonistic to Enabled's. Since neural enriched isoforms of Mena, the murine homolog of Enabled, are believed to be involved in filopodia formation to extend the growth cone, a potential antagonistic role for Trio is the retraction of growth cones. Drosophila Trio's second DH domain stimulates the formation of stress fibers in REF-52 cells (Newsome, 2000). In neurons, the formation of similar actin-myosin contractile filaments leads to neurite retraction. Therefore, a balance between the biochemical activities of Enabled and Drosophila Trio may be required for a proper balance between extension and retraction of the growth cone in response to attractive and repulsive pathfinding cues (Liebl, 2000).

It is not envisioned that the stimulation of stress fiber formation leading to neurite retraction is the only possible biochemical function of Trio. In fibroblasts, human Trio activates Rac1 with its first DH domain, inducing the formation of lamellipodia and membrane ruffles (Bellanger, 1998; Seipel, 1999). Similar biochemical activity reported for the first DH domain of Drosophila Trio (Newsome, 2000) would presumably lead to lamellipodia/filopodia formation analogous to the activity of Enabled in the growth cone. The elucidation of the biochemical interrelationships between Abl, Ena, Fax, and Trio suggested by their genetic interactions awaits detailed analyses (Liebl, 2000).

In addition to a role for trio during development of the CNS, observations of trio expression in leading edge cells during dorsal closure and an uninflated, blistered wing phenotype in the trio hypomorphic background suggest additional roles for trio during development. Dominant-negative Rho subfamily constructs can disrupt the actin cytoskeleton in leading edge cells, with subsequent effects on dorsal closure. Intense trio expression in leading edge cells may indicate that Trio plays a role in this process. Expressing dominant-negative Cdc42 proteins in wing discs can produce wing blisters similar to those observed in the trio hypomorphic background. Mutations in inflated, a Drosophila integrin, produce similar wing blisters, as well. Since the mammalian c-Abl kinase is activated in response to integrin-mediated cell adhesion, and Abl is expressed in wing imaginal disc epithelial cells, the blistered wing phenotype seen in trio hypomorphic animals potentially presents a modifiable phenotype with which to explore additional aspects of Trio signaling networks (Liebl, 2000).

The trio^E4.1/Df(3L)FpaI embryos appear normal as a whole structure, and the gross morphology of the CNS also look similar to wild type. When the embryos are stained with mAb 1D4, however, defects in the axon patterning in the CNS are found. mAb 1D4 stains three longitudinal fascicles at each lateral side of the ventral nervous system in wild-type embryos at stage 17. In trio^E4.1/Df(3L)FpaI embryos, however, the stained fascicles are arranged in an abnormal pattern. The outermost fascicles are very discontinuous and fused to the adjacent inner fascicles. This phenotype is more pronounced in the mutant first instar larvae, in which the outermost fascicles are thin and frequently disrupted, producing gaps. Portions of the axons turn vertically to the inner fascicles, showing orthogonal patterns. Axons in other fascicles also exhibit irregular arrangements. These abnormal mAb 1D4 staining patterns are similarly observed in trio^E4.1 homozygous embryos and larvae, indicating that trio^E4.1 is a functionally strong or null allele for these axonal phenotypes. To trace the axon pathways more accurately in the mutants, embryos expressing Tau-Myc protein under the control of the lim3 promoter in a small number of neurons were further examined. lim3 is expressed in a subset of interneurons and motor neurons, including the RP motor neurons in embryos and first instar larvae. The Tau-positive axons extending medially from a lateral cluster of neurons turn vertically to navigate on the longitudinal tract and form a fascicle with the axons extended from other segments in the wild-type larva. In the trio^E4.1/Df(3L)FpaI larvae, these axons do not faithfully extend on the longitudinal tracts and exhibit a wavy pattern or frequently turn along three-dimensional axes to follow the wrong tracts. These misrouting phenotypes demonstrate that Trio has an essential role for axon patterning in the embryonic and larval CNS (Awasaki, 2000).

In contrast to the clear defects in the mutant CNS, mild aberrations have been observed in the motor axons projecting to the body wall muscles. ISNb, a motor axon fascicle, innervates the ventrolateral muscles 6, 7, 12, and 13 in a stereotypical fashion. In mutant embryos, while most ISNb fascicles correctly extend toward the target muscles, 10% of ISNb exhibit a stall or fusion phenotype; 7% of the fascicles stall on muscle 7 or prior to entering the muscle territory after exiting the CNS, and 3% of the fascicles fuse to another fascicle ISNd and stall shortly (Awasaki, 2000).

Since Trio is strongly expressed in MB throughout development, the effects of trio mutations on the MB structure were examined. mAb 1D4 labels MB of the wandering third instar larvae, which include the peduncles and the LV and LM lobes but not the cell bodies of the MB neurons and calyx. The core regions of the peduncle, LV, and LM are clearly unstained. These staining patterns indicate that mAb 1D4 is a useful marker for visualizing the whole neurite structure of the larval MB. In both trio^P0368/10/Df(3L)FpaI and trio^P0368/10/trio^E4.1 larvae, the LV and LM lobes are found to be abnormally developed: thin or small lobes in the position of the LV lobe (15 of 18 MBs in trio^P0368/10/trio^E4.1); short lobes in the position of the LM lobe (15 of 18 in trio^P0368/10/trio^E4.1). In most cases, the peduncles appeared normal, with the core region remaining unstained, whereas the core regions of LV and LM are ambiguous when compared with wild type. The degree of defects and altered morphology vary among the individual mutant MBs. These observations suggest that the MB neurons in the trio mutant extend their axons normally in the peduncle but often fail to project further along the lobe-forming tracts (Awasaki, 2000).

In the adult MB, mAb 1D4 labels the alpha and beta lobes strongly, and the gamma lobe weakly. In trio^P0368/10/Df(3L)FpaI and trio^P0368/10/trio^E4.1 flies, shortened or deformed lobes exhibiting weak mAb 1D4 staining are found in the position of the medial lobes, while the peduncles are formed in an apparently normal shape. Based on the position and staining intensity, the abnormal lobes possibly arise from the gamma lobe neurons, in which Trio is continuously expressed during their differentiation in wild type. Moreover, in place of the alpha and beta lobes, strangely shaped lobes with strong staining are found at the anteriormost region of the peduncle, and sometimes unexpectedly close to the calyx. Since no expression of Trio is observed in the alpha/beta lobe neurons at any stages in wild type, the aberrant formation of the alpha and beta lobes is likely caused by an indirect consequence of the defects in the preexisting larval lobes that the alpha/beta lobe axons later follow (Awasaki, 2000).

Since Trio expression is not confined to the MB neurons, it is uncertain whether the defects in MB are caused by the loss of Trio function in the MB neurons or are a secondary effect resulting from structural alterations in the adjacent brain regions involved in MB development. To discriminate between these possibilities, clonal analyses were performed using the MARCM system, with which only mutant clones can be labeled. Clones of the MB neuroblasts were induced in first instar larvae and analyzed in wandering third instar larvae. The wild-type MB neurons extend their axons through the peduncle and bifurcate into the LV and LM lobes. Mutant MB clones exhibit an abnormal axonal pattern. The axons emanating from the trio^E4.1 clones appear to navigate normally through the peduncle to the approximate region of bifurcation, but the axons found in the two lobes are sparsely distributed, with an apparent reduction in the overall fluorescent intensity of the lobes. The fluorescent intensity of larva-specific spur-shaped lateral (LSL) projection remains high. These observations indicate that the axons in the mutant clones are either stalled on the tracts or misrouted to LSL. In addition, the trio neuroblast clones interestingly exhibits a bundle of neurites overextended from the calyx, a major dendritic cluster of MBs. It remains to be revealed, however, whether the neurites have the properties of axons or dendrites. Taken together, these phenotypes are primarily caused by an alteration in the intrinsic nature of the mutant clones. Thus, it is concluded that Trio plays an essential role in the development of MBs through controlling the directional extension of the axons and/or dendrites (Awasaki, 2000).

Rac GTPases regulate the actin cytoskeleton to control changes in cell shape. To date, the analysis of Rac function during development has relied heavily on the use of dominant mutant isoforms. Here, loss-of-function mutations have been used to show that the three Drosophila Rac genes, Rac1, Rac2 and Mtl, have overlapping functions in the control of epithelial morphogenesis, myoblast fusion, and axon growth and guidance. They are not required for the establishment of planar cell polarity, as had been suggested on the basis of studies using dominant mutant isoforms. The guanine nucleotide exchange factor, Trio, is essential for Rac function in axon growth and guidance, but not for epithelial morphogenesis or myoblast fusion. Different Rac activators thus act in different developmental processes. The specific cellular response to Rac activation may be determined more by the upstream activator than the specific Rac protein involved (Hakeda-Suzuki, 2002).

Endogenous Rac GTPases thus function in morphogenesis of the epidermis, mesoderm, and nervous system. Are they regulated by the same or different upstream activators in each of these tissues? The guanine nucleotide exchange factor Trio activates Rac1, Rac2 and Mtl in vitro, and loss of trio function in the visual system results in projection errors of photoreceptor axons similar to those observed in Rac triple mutants. Axon guidance errors and occasional stalling defects also occur in embryos lacking zygotic trio function. Axon stalling becomes severe in both the CNS and PNS if the maternal trio function is also eliminated. As with the Rac proteins, low levels of Trio activity are sufficient but essential for axon growth. This critical requirement for Trio in axon growth is particularly striking, given that the Drosophila genome encodes at least 22 other Rho family GTPase exchange factors, several of which are also expressed in the developing nervous system (Hakeda-Suzuki, 2002).

In the embryonic nervous system and adult visual system, loss of trio function thus results in defects remarkably similar to those observed upon loss of Rac function, consistent with the idea that Trio and Rac proteins act in a common pathway in vivo. An epistasis experiment was performed to test this. Overexpression of the Trio GEF1 domain using the eye-specific GMR promoter results in a severely disrupted eye morphology and highly aberrant photoreceptor axon projections. If Trio signals through Rac proteins in vivo, then these defects should be dependent on Rac function. This is indeed the case. Both the eye morphology and axon projection defects are almost completely suppressed in animals homozygous for loss-of-function mutations in either Rac1 or Rac2. Mtl alone does not suppress this trio gain-of-function phenotype. The Rac1;Rac2;Mtl triple mutant phenotype is completely epistatic to the trio gain-of-function phenotype. These data demonstrate that Trio GEF1 does indeed act through Rac proteins in vivo, and further suggest that Rac1 and Rac2 are its preferred substrates. The trio loss-of-function phenotype is however much more severe than the Rac1;Rac2 double mutant phenotype, suggesting that endogenous Trio may also activate Mtl, at least when Rac1 and Rac2 are lacking (Hakeda-Suzuki, 2002).

Having identified Trio as the primary activator of Rac proteins during axon growth, whether Trio is required for any of the other Rac functions was investigated. Dorsal closure occurs normally in embryos lacking both maternal and zygotic trio function. Myoblast fusion also appears complete in these embryos, but myotubes often fail to attach themselves correctly to the epidermis. Thus, although it is expressed in both the epidermis and mesoderm, Trio is not required for either dorsal closure or myoblast fusion (Hakeda-Suzuki, 2002).

Thus endogenous Rac proteins control cell-sheet spreading, cell fusion, and axon growth and guidance, and they also regulate axon branching. Each of these processes involves its own characteristic restructuring of the cytoskeleton, and hence is likely to be mediated by a different set of Rac effectors. What determines which of these effector pathways will be stimulated when Rac proteins are activated? One possibility would be that distinct Rac proteins have distinct effectors. This may well be the case for myoblast fusion, which can be mediated by Rac1 or Rac2, but not Mtl. However, in most cases Rac1, Rac2 and Mtl have largely overlapping functions, indicating that they also share a common set of effectors. A similar pattern of overlapping functions in diverse processes has also recently been reported for the three C. elegans Rac genes. In general, the cellular response is therefore unlikely to be dictated by the specific Rac protein involved. These results suggest an alternative possibility. Trio, despite its widespread expression, is required for only a limited subset of Rac functions. This suggests that the set of effectors a Rac protein engages, and hence the cellular response it induces, might also depend on how or where it has been activated. Trio, for example, might activate Rac proteins to a level, for a duration, or in a subcellular location, that allows it to stimulate only those effector pathways that control motility and guidance. Exploring the basis for specificity in Rac function is an important task for the future (Hakeda-Suzuki, 2002).

The Drosophila L1CAM homolog Neuroglian signals through distinct pathways to control different aspects of mushroom body axon development

The spatiotemporal integration of adhesion and signaling during neuritogenesis is an important prerequisite for the establishment of neuronal networks in the developing brain. This study describes the role of the L1-type CAM Neuroglian protein (NRG) in different steps of Drosophila mushroom body (MB) neuron axonogenesis. Selective axon bundling in the peduncle requires both the extracellular and the intracellular domain of NRG. A novel role was uncovered for the ZO-1 homolog Polychaetoid (PYD) in axon branching and in sister branch outgrowth and guidance downstream of the neuron-specific isoform NRG-180. Furthermore, genetic analyses show that the role of NRG in different aspects of MB axonal development not only involves PYD, but also TRIO, SEMA-1A and RAC1 (Goossens, 2011).

This study demonstrates a requirement for Neuroglian signaling in different steps of mushroom body (MB) axonogenesis, namely (1) axonal projection into the peduncle, and (2) branching, outgrowth and guidance of axonal sister branches. The two steps in mushroom body axonogenesis are genetically separable and seem to involve distinct NRG signaling complexes (Goossens, 2011).

In peduncle formation, NRG signaling does not rely on the NRG-180-specific intracellular domain, but on the extracellular domain and the part of the cytoplasmic domain common to both NRG isoforms. The extracellular domain contributes intercellular adhesive properties, necessary for axon fasciculation into a peduncle. This conclusion is supported by the defective adhesive properties of the NRG⁸⁴⁹ mutant protein in cell aggregation assays, and by the fact that Nrg⁸⁴⁹ hemizygotes frequently lack the peduncle. Interaxonal fasciculation in the peduncle probably involves binding to and stabilization by the actin cytoskeleton network via the ankyrin-binding domain shared by the two NRG isoforms. This conclusion is supported by previous aggregation experiments in Drosophila S2 cells in which it was shown that homophilic binding of NRG leads to recruitment of ankyrin to the contact sites and by the observation that RNAi-mediated knockdown of neuron-specific ank2-RNA results in MB phenotypes similar to those seen in Nrg mutants (Goossens, 2011).

MB lobe development, on the other hand, requires the NRG-180-specific intracellular fragment. This study showed that PYD acts downstream of NRG-180 during the formation of α and γ lobes. Consistent with this, axon stalling defects (i.e. lack of peduncle formation) were never observed in pyd mutants, whereas defects were observed in lobe outgrowth, branching and guidance. Furthermore, the neuron-specific NRG-180 isoform can bind directly to the first PDZ-domain of this MAGUK protein. The observation that NRG and PYD interact to mediate proper sister neurite projections defines a novel role for the ZO-1 homolog PYD in axonogenesis. Thus far, the best-known role of MAGUKs in the nervous system has been in synapse development and function, as is the case for one of the prototypic MAGUKs, Drosophila Discs Large 1 (Dlg1), whereas PYD is known as a component of adherens junctions (Goossens, 2011).

Sema-1a, trio and Rac1 were also found to be a part of the genetic network that interacts with Nrg. The observation that heterozygosity for mutations in Sema-1a and trio both suppress NRG-180 overexpression induced MB phenotypes indicates that Sema-1a and trio are genetically downstream of Nrg and possibly in the same pathway. By contrast, the introduction of a Rac1 mutation in a Nrg gain- or loss-of-function background results in both cases in enhancement of MB phenotypes. This argues against a one-to-one signaling model between NRG and RAC1, in which RAC1 acts only downstream of NRG-180. Consistent with the genetic data, no direct physical interaction could be detected between NRG-180 and RAC1, but preliminary co-immunoprecipitation data suggest that NRG-180 can bind to TRIO. Further experiments will be necessary to assess whether this binding also occurs in vivo, and whether it is instrumental for NRG-180-dependent modulation of RAC1 signaling (Goossens, 2011).

Contrary to the observed genetic interaction between Nrg and Sema-1a, this study found no evidence for interaction between Nrg and two genes that code for well-characterized Semaphorin receptors, plexin A and plexin B. This is an unexpected observation in light of the fact that Sema-1a and plexin A and plexin B interact during mushroom body development. Therefore, this suggests that during mushroom body development Sema-1a acts both in a plexin-dependent and a plexin-independent way. A plexin-independent role in axon outgrowth has previously been described for vertebrate Sema7a (Goossens, 2011).

The distinct requirement for NRG in peduncle and lobe formation is reminiscent of what has been shown for DSCAM. This protein has an early and essential role for selective fasciculation of young axons in the peduncle and is subsequently required for bifurcation and branch segregation. In light of this, it is interesting to note that the different cell-adhesion molecules that have been implicated in MB development have a different MB expression pattern or temporal requirement for MB development. NRG is expressed in the MBs throughout its entire development, but no essential function was found for Neuroglian in larval MB development. By contrast, DSCAM expression disappears with fiber maturation and mutants have larval MB phenotypes. Likewise, Fas2 mutants display larval lobe defects, whereas no lobe defects were found in adult mutants. Taken together, these observations suggest that different steps in MB axonogenesis depend on combinations not only of isoforms of the same cell-adhesion molecule (e.g. DSCAM) but also of different cell surface molecules (e.g. NRG, FAS2 and SEMA-1A). Jointly, the cell-surface molecule complement of any given axon combined with guidance signals will then control the signaling required for proper neural circuit formation in the MBs (Goossens, 2011).

Loss of syd-1 from R7 neurons disrupts two distinct phases of presynaptic development

Genetic analyses in both worm and fly have identified the RhoGAP-like protein Syd-1 (RhoGAP100F) as a key positive regulator of presynaptic assembly. In worm, loss of syd-1 can be fully rescued by overexpressing wild-type Liprin-α, suggesting that the primary function of Syd-1 in this process is to recruit Liprin-α. This study shows that loss of syd-1 from Drosophila R7 photoreceptors causes two morphological defects that occur at distinct developmental time points. First, syd-1 mutant R7 axons often fail to form terminal boutons in their normal M6 target layer. Later, those mutant axons that do contact M6 often project thin extensions beyond it. The earlier defect coincides with a failure to localize synaptic vesicles (SVs), suggesting that it reflects a failure in presynaptic assembly. The relationship between syd-1 and Liprin-α in R7s was analyzed. It was found that loss of Liprin-α causes a stronger early R7 defect and provide a possible explanation for this disparity: Liprin-α was shown to promote Kinesin-3/Unc-104/Imac-mediated axon transport independently of Syd-1 and that Kinesin-3/Unc-104/Imac is required for normal R7 bouton formation. Unlike loss of syd-1, loss of Liprin-α does not cause late R7 extensions. It was shown that overexpressing Liprin-α partly rescues the early but not the late syd-1 mutant R7 defect. It is therefore concluded that the two defects are caused by distinct molecular mechanisms. Trio overexpression was found to rescues both syd-1 defects and that trio and syd-1 have similar loss- and gain-of-function phenotypes, suggesting that the primary function of Syd-1 in R7s may be to promote Trio activity (Holbrook, 2012).

GFP-fused SV proteins, such as Syt-GFP, are classic tools for studying presynaptic development but have not been used previously to analyze R7s. This study found that, as expected, Syt-GFP within R7s is enriched at sites known by electron microscopy to contain active zones. Loss of LAR, Liprin-α, or syd-1 causes R7 terminals to fail to contact their normal, M6, target layer. This study demonstrated that this morphological defect correlates temporally with a failure to localize SVs to presynaptic sites and is therefore likely to reflect a defect in R7 presynaptic development rather than simply in target layer selection (Holbrook, 2012).

Liprin-α is not only a scaffold for the assembly and retention of presynaptic components, including SVs, at presynaptic sites but also a positive regulator of Kinesin-3/Unc-104/Imac-dependent axon transport of those components. This study shows that, unlike Liprin-α, Syd-1 is not required for normal Kinesin-3/Unc-104/Imac-mediated transport. However, SVs are similarly mislocalized in Liprin-α and syd-1 mutant R7 axons that contact M6. A simple interpretation is that this mislocalization reflects a requirement for Liprin-α and syd-1 in retaining SVs within R7 terminals; in support of this, it was found that SVs are localized normally to syd-1 mutant R7 axon terminals at 24 h APF, before synaptogenesis. It was hypothesized that the additional disruption of axon transport in Liprin-α mutant R7s is reflected in their greater inability to maintain contact with M6; in support of this, it was found that imac mutant R7 axons also lose contact with M6 (Holbrook, 2012).

Although both Liprin-α and syd-1 are required for the clustering of SVs at en passant synapses in worm, syd-1 is not required for the localization of SVs to NMJ terminals in fly. The molecular mechanisms underlying presynaptic development at NMJ and in R7s have been shown previously to differ in several respects. The current finding further highlights the importance of analyzing synapse development using multiple neuron types (Holbrook, 2012).

Although mitochondria are often enriched at synapses, it remains unclear what proportion of them might be stably associated with presynaptic sites rather than transported there in response to acute energy needs. Within at least some axons, most clusters of stationary mitochondria reside at nonsynaptic sites. In R7s, Mito-GFP was found to be enriched at presynaptic sites. Because arthropod photoreceptor neurons continuously release neurotransmitter in response to light, this enrichment might simply be caused by continuous energy needs. However, this study found that mitochondria remained enriched at R7 terminals even in the absence of light-evoked activity, indicating that either spontaneous release is sufficient for their recruitment or an activity-independent mechanism is responsible. It is speculated that the permanently high energy demands at photoreceptor synapses may have selected for the activity-independent association of mitochondria with R7 synapses and that this localization requires syd-1 and Liprin-α. Mito-GFP is mislocalized in imac mutant R7s, despite previous work indicating that Kinesin-3/Unc-104/Imac is not required for transport of mitochondria. It is therefore thought that mitochondria are normally tethered at R7 presynaptic sites and that loss of imac indirectly causes their mislocalization by disrupting transport of the components required for tethering to occur (Holbrook, 2012).

Previous work identified two different phenotypes associated with loss of the LAR/Liprin/trio pathway: loss of LAR or Liprin-α caused R7 axons to terminate before their M6 target layer, whereas loss of Liprin-β or trio caused R7 axons to project extensions beyond M6. One possibility is that these two defects are simply different manifestations of the same cellular defect: a decrease in the stability of the synaptic contact between R7s and their targets. However, this study has shown that loss of a single gene, syd-1, causes both defects and that the defects occur at distinct developmental time points, suggesting that they occur by distinct mechanisms. In support of this, Liprin-α overexpression can rescue the early but not the late syd-1 defect (Holbrook, 2012).

The earlier defect, failure to contact M6, correlates with the failure to localize SVs, suggesting, as mentioned above, that this represents a failure to assemble synapses. However, the cause of the later morphological defect and the precise nature of the extensions remain unclear. It is noted that the extensions often terminate in small varicosities that can contain Syt-GFP, and Mito-GFP, indicating that they are not simply filopodia but may instead represent sites of ectopic presynaptic assembly. One possibility is that, as at NMJ, loss of syd-1 causes ectopic accumulations of Liprin-α, Brp, Nrx-1, or other presynaptic proteins and that these might then promote ectopic, abnormal presynaptic assembly. A second possibility is that the extensions may instead be an indirect consequence of the role of syd-1 in postsynaptic development: perhaps the extensions are the response of the syd-1 mutant R7 terminal to defects in its postsynaptic target. Loss of Liprin-α causes no such postsynaptic effect, providing an explanation for why Liprin-α mutant R7s do not form extensions. A third possibility is that R7s form distinct types of synapses at different time points. Failure to assemble one type of synapse, which R7s assemble first, causes decreased contact with M6, whereas failure to assemble a second type, which occur later, results in extensions. Consistent with this model, R7s form synapses with more than one neuron type (Holbrook, 2012).

Loss of syd-1 has a significantly weaker effect on fly NMJ development than does loss of Liprin-&alpha. Likewise, this study shows that the early phase of R7 terminal development, during which presynaptic components are localized, is less affected by loss of syd-1 than by loss of Liprin-α. A possible explanation for this difference is identified: loss of Liprin-α, but not of syd-1, significantly decreases Kinesin-3/Unc-104/Imac-mediated axon transport, and Kinesin-3/Unc-104/Imac is required for R7s to form boutons in M6 (Holbrook, 2012).

In both worm and fly, Syd-1 is required for the normal localization of Liprin-α and Brp/ELKS to presynaptic sites. In worm, loss of syd-1 can be rescued either by overexpressing full-length wild-type Liprin-α, or by overexpressing a domain of Liprin-α that promotes oligomerization of Liprin-α proteins, or by a mutation that enhances the ability of Liprin-α to bind Brp/ELKS. These results suggest that the primary function of Syd-1 is to potentiate Liprin-α activities. However, this sutyd found that Liprin-α overexpression only partially rescues the early defect that syd-1 mutant R7s have in assembling synapses. This suggests that, as in worm, Liprin-α can act partly independently of Syd-1 during presynaptic assembly but that, unlike in worm, Syd-1 also has some Liprin-α-independent function. In contrast, Liprin-α overexpression does not at all rescue the late extensions caused by loss of syd-1. As it speculated above, one possibility is that these extensions might be caused by mislocalized Liprin-α, Brp, or Nrx-1 (Holbrook, 2012).

Unlike Liprin-α, Trio overexpression fully rescues the early and partly rescues the late defect caused by loss of syd-1, suggesting that Syd-1 promotes R7 synaptic terminal development primarily by potentiating Trio activity. Consistent with this model, loss of trio phenocopies loss of syd-1 from R7s, and overexpressing Syd-1 or Trio bypasses the need for LAR to similar degrees. At fly NMJ, Trio promotes presynaptic development by acting as a GEF for Rac1. Syd-1 has a RhoGAP domain, albeit one that has not been shown to interact with GTPases. Syd-1 may act distantly upstream of Trio. However, it is also possible that Syd-1 might instead regulate one or more small GTPases in parallel with Trio. GAPs and GEFs have opposite effects on GTPases, but loss of trio or syd-1 causes similar defects at both NMJ and in R7s. One possibility, therefore, is that Syd-1 acts as a GAP not for Rac1 but for Rho, which often functions in opposition to Rac. Alternatively, Syd-1 might act as an atypical GAP for Rac1 -- perhaps lacking GAP activity but able to bind and protect Rac1-GTP from conventional GAPs -- or Syd-1 might yet act as a conventional GAP for Rac1 if it is the rate of cycling between GDP- and GTP-bound states of Rac1 (rather than simply the amount of the GTPase that is in the 'active,' GTP-bound, state) that promotes presynaptic development (Holbrook, 2012).

An allele of sequoia dominantly enhances a trio mutant phenotype to influence Drosophila larval behavior

The transition of Drosophila third instar larvae from feeding, photo-phobic foragers to non-feeding, photo-neutral wanderers is a classic behavioral switch that precedes pupariation. The neuronal network responsible for this behavior has recently begun to be defined. Previous genetic analyses have identified signaling components for food and light sensory inputs and neuropeptide hormonal outputs as being critical for the forager to wanderer transition. Trio is a Rho-Guanine Nucleotide Exchange Factor integrated into a variety of signaling networks including those governing axon pathfinding in early development. Sequoia is a pan-neuronally expressed zinc-finger transcription factor that governs dendrite and axon outgrowth. Using pre-pupal lethality as an endpoint, a screened was performed for dominant second-site enhancers of a weakly lethal trio mutant background. In these screens, an allele of sequoia has been identified. While these mutants have no obvious disruption of embryonic central nervous system architecture and survive to third instar larvae similar to controls, they retain forager behavior and thus fail to pupariate at high frequency (Dean, 2013).

Serial synapse formation through filopodial competition for synaptic seeding factors

Following axon pathfinding, growth cones transition from stochastic filopodial exploration to the formation of a limited number of synapses. How the interplay of filopodia and synapse assembly ensures robust connectivity in the brain has remained a challenging problem. This study developed a new 4D analysis method for filopodial dynamics and a data-driven computational model of synapse formation for R7 photoreceptor axons in developing Drosophila brains. Live data support a 'serial synapse formation' model, where at any time point only 1-2 'synaptogenic' filopodia suppress the synaptic competence of other filopodia through competition for synaptic seeding factors. Loss of the synaptic seeding factors Syd-1 and Liprin-alpha leads to a loss of this suppression, filopodial destabilization, and reduced synapse formation. The failure to form synapses can cause the destabilization and secondary retraction of axon terminals. This model provides a filopodial 'winner-takes-all' mechanism that ensures the formation of an appropriate number of synapses (Ozel, 2019).

After pathfinding, axon growth cones transition to become terminal structures with presynaptic active zones. How axon terminals form a defined number of synaptic contacts with a specific subset of partners is a daunting problem in dense brain regions. Stochastically extending and retracting filopodial extensions occur during both pathfinding and synapse formation and are thought to facilitate interactions between synaptic partners. However, little is known about the role of stochastic filopodial dynamics for robust synapse formation (Ozel, 2019).

Presynaptic active zone assembly is a key step in synapse formation and regulated by a conserved set of proteins. An early active zone 'seeding' step has been defined through the functions of the multidomain scaffold proteins Syd-1 and Liprin-α in C. elegans and Drosophila neuromuscular junction (NMJ). Syd-1 is a RhoGAP-domain-containing protein that recruits Liprin-α to the active zone. Liprin-α is an adaptor protein named after its direct interaction with the receptor tyrosine phosphatase Leukocyte common antigen-related (LAR). The Liprin-α and LAR interaction has been directly implicated in active zone assembly across species. Downstream, Liprin-α and Syd-1 recruit core active zone components and ELKS/CAST family protein Brp. Finally, the RhoGEF Trio has been proposed to function downstream of the Lar/Liprin-α/Syd-1 and has recently been suggested to regulate active zone size (Ozel, 2019).

Remarkably, the proposed Lar-Liprin-α-Syd-1-Trio pathway has been characterized in parallel for its role in axon guidance, independent of active zone assembly. In the Drosophila visual system, mutants in all four genes have been implicated in the layer-specific targeting of photoreceptor R7 axons in the medulla neuropil. It is unclear whether any of the four mutants affect active zone assembly in R7 neurons. Dual roles in axon pathfinding and synapse formation have been shown or proposed for all four genes. Independent implications in active zone assembly and axon pathfinding raise the question what functions are primary or secondary (Ozel, 2019).

This study investigated the relationship between filopodial dynamics and synapse assembly in the presynaptic R7 terminal. The early synaptic seeding factors Liprin-α and Syd-1 accumulate in only a single filopodium per terminal at any given point in time. Consequently, only 1-2 filopodia per terminal are stabilized, suggesting that only 1-2 filopodia are synaptogenic at any time. A data-driven computational model shows that this 'serial synapse formation model' is supported by the measured dynamics and could be tested in mutants for liprin-α, syd-1, lar, and trio. Specific defects in filopodial dynamics precede all other defects, including axon terminal retractions. A quantitative 'winner-takes-all' model, from stochastic filopodial dynamics to the formation of a limited number of synapses, is presented as well as a model for axon terminal stabilization based on filopodia and synapses (Ozel, 2019).

The data link bulbous filopodia to synapse formation based on three findings: (1) in the wild type, these are the only filopodia that specifically occur during the time window of synapse formation and do not exhibit stochastic dynamics, and wild-type R7 photoreceptor axons stabilize 1-2 bulbous filopodia at a time; (2) the synaptic seeding factors Liprin-α and Syd-1 non-randomly localize to 1-2 bulbous filopodia at a time; and (3) loss of liprin-α or syd-1 selectively affects the stabilization of bulbous filopodia. Loss of the upstream receptor lar similarly selectively affects bulbous filopodia but, in addition to bulb destabilization, also strongly affects bulb initiation. Together, these findings support a model whereby stochastic filopodial exploration leads to bulb stabilization and synapse formation one at a time. In this model, restrictive synaptogenic filopodia formation 'paces' the formation of ~25 synapses over 50 h, effectively controlling synapse numbers within the available developmental time window (Ozel, 2019).

The key mechanism of this model is the inhibitory feedback of synaptogenic filopodium formation. In contrast to all other filopodia, the dynamics of bulbous filopodia are not independent events. How are synaptic seeding factors competitively distributed between these filopodia? Live-imaging data suggest that Liprin-α or Syd-1 can traffic in and out of filopodia but overexpressed proteins accumulate in the axon terminal trunk and do not enter to more than 1-2 filopodia, indicating that trafficking into filopodia is restricted. Morphologically, filopodia are very thin structures that may not provide much space for freely diffusing proteins or organelles. On the other hand, the bulbous tip provides a much larger volume that may be required for sufficient amounts of synaptic seeding factors and other building material to initiate synapse formation. Furthermore, computational tests show that 'winner-takes-all' dynamics can arise from the dynamic distribution of a limited resource that confers a competitive advantage (longer lifetime, which leads to further accumulation) without the need for active filopodial communication (Ozel, 2019).

Since Syd-1 and Liprin-α are not required for bulb initiation, it is speculated that filopodial contact with a synaptic partner may initiate the bulb and precede active zone formation. The data suggest that Lar is a good candidate for a presynaptic receptor with such a role, but it is unlikely to be the sole upstream receptor. Neurexin and PTP69D, for example, are other known candidates. In the absence of an upstream receptor or the seeding factors themselves, synapse assembly fails and bulbs destabilize. New bulb generation following loss of bulbs in the absence of seeding factors can also be explained with seeding factors as a limiting resource with a competitive advantage. This is reminiscent of other competitive processes that shape neuronal morphology, e.g., the restricting role of building material in the competitive development of dendritic branches in a motorneuron (Ryglewski, 2017). Mutant analyses suggest that stable bulbs are linked to negative feedback on other bulbs via the function of the RhoGEF Trio. While the exact mechanism is unclear, it is tempting to speculate about a role of actin-dependent signaling downstream of synaptic seeding (Ozel, 2019).

The current model only considers the presynaptic axon terminal. The main postsynaptic partner of R7 are amacrine-like Dm8 cells, whose elaborate dendritic processes are present in direct vicinity to the R7 filopodia throughout the developmental period of synapse formation. Currently the dynamics of the postsynaptic processes and whether they restrict availability or are 'easily found' as postsynaptic partners are unknown. The presynaptic model presented in this study could explain the observed slow, serial synapse formation even in the presence of abundant postsynaptic partner processes (Ozel, 2019).

Mutations in the proposed pathway components Lar, Liprin-α, Syd1, and Trio have been independently characterized for their roles in active zone assembly (mostly at the larval neuromuscular junction) and axon targeting, in large part in the visual system. It is likely that all four genes exert more than one function in different contexts. Defects in synapse formation and retraction are captured by the measured parameters and the current model. However, some differences in overall morphology, including overextensions in the syd-1 mutant, may be described by parameters not considered in the model, e.g., filopodial length, and due to some differences in their molecular function. Similarly, for Lar, independent context-dependent functions have been characterized based on different downstream adaptors (Ozel, 2019).

It was asked to what extent a primary role for lar, trio, syd-1, and liprin-α in synapse formation could explain previously observed phenotypes. All filopodial defects in the four mutants occur independently and prior to possible retraction events. Combined live imaging and computational modeling suggests that defects in the syd-1 and liprin-α mutants are consistent with a primary defect in bulb stabilization and synapse formation. These defects may in turn lead to axon destabilization or represent independent functions; lar may have an additional earlier adhesion function and trio does not play a critical role in the formation of the correct number of synapses, while its effect on general filopodial dynamics may sensitize mutant axons to other changes (Ozel, 2019).

The conclusion that Lar, Liprin-α, and Syd-1 have a primary function in synapse formation is based on three pieces of evidence: (1) all mutants initially target correctly and exhibit normal filopodial dynamics prior to synapse formation; (2) the mutants start retracting only when synaptic contacts initiate, in the order and severity from the receptor to the downstream elements; and (3) all three mutants exhibit the loss of competitive bulb stabilization. Taken together, these observations support a direct role in synapse formation following bulb stabilization, but other molecular functions cannot be excluded. For example, in both C. elegans and Drosophila, Lar has been shown to function independently in axon guidance and synapse formation. This study found that syd-1ΔRhoGAP mutants have normal terminal morphology and only a mild decrease in the number of BrpD3-puncta. This is consistent with recent findings that a RhoGAP-deficient Syd-1 fragment is sufficient to rescue early active zone seeding events at the Drosophila neuromuscular junction but not the recruitment of Brp as the active zones mature (Spinner, 2018). However, since homozygous syd-1ΔRhoGAP flies have no obvious connectivity defects, synapse numbers are apparently sufficient for axon terminal stabilization (Ozel, 2019).

Finally, these observations suggest that loss of the primary functions of these proteins in filopodial dynamics and synapse formation are sufficient to cause axon retractions. The phenotypes observed in this study for lar, liprin-α, and syd-1 are somewhat similar but in contrast to cadN only occur at or after the time of synaptic partner identification. While filopodia continuously decrease, synapses continuously increase, thereby allowing a takeover of the axon terminal stabilization function. The modeling fits the wild type, liprin-α, syd-1, and trio remarkably well. In contrast, while retractions in the lar mutant are qualitatively predicted, the model fails to explain retractions quantitatively. A partial explanation may be that the model was parameterized only based on the lar mutant axon terminals that are still unretracted at P60. These are only 30% of terminals by that time, and this study has effectively selected for terminals with dynamics that prevented retractions thus far. It is likely that earlier retractions are caused by defects in filopodial adhesion or synaptic contacts. In sum, the data and modeling support a role for synapses in the stabilization of R7 axon terminals, which can lead to probabilistic axon retractions in mutants affecting synapse formation (Ozel, 2019).

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date revised: 10 December 2019

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