Transcriptional Regulation

Nerfin-1 is a nuclear regulator of axon guidance required for a subset of early pathfinding events in the developing Drosophila CNS. Nerfin-1 belongs to a highly conserved subfamily of Zn-finger proteins with cognates identified in nematodes and man. The neural precursor gene prospero is essential for nerfin-1 expression. Unlike nerfin-1 mRNA, which is expressed in many neural precursor cells, the encoded Nerfin-1 protein is only detected in the nuclei of neuronal precursors that will divide just once and then transiently in their nascent neurons. Although nerfin-1 null embryos have no discernible alterations in neural lineage development or in neuronal or glial identities, CNS pioneering neurons require nerfin-1 function for early axon guidance decisions. Furthermore, nerfin-1 is required for the proper development of commissural and connective axon fascicles. Nerfin-1 is essential for the proper expression of robo2, wnt5, derailed, G-oα47A, Lar, and futsch<, genes whose encoded proteins participate in these early navigational events (Kuzin, 2005).

Given the axon guidance defects in nerfin-1null embryos and the fact that Nerfin-1 is a Zn-finger nuclear protein, it was hypothesized that Nerfin-1 may be required for the correct expression of genes involved in axon guidance. Accordingly, the embryonic expression profiles of over 35 genes that have been shown to play important roles in axon guidance were examined. Included in the candidate screen were genes encoding transcription factors, RNA-binding proteins, cell surface receptor proteins, their ligands, signal transduction proteins, and components of the cytoskeleton. Homozygous nerfin-1null embryos were identified by the absence of Nerfin-1 immunoreactivity. Whole-mount in situ hybridization and/or protein immunostaining for altered spatial or temporal expression in nerfin-1null embryos identified six genes that require nerfin-1 function to achieve full wild-type expression levels (Kuzin, 2005).

Two genes involved in anterior vs. posterior commissure choice, those encoding the receptor tyrosine kinase Derailed, and its ligand Wnt5, both required nerfin-1 for full expression. In the absence of nerfin-1, ventral cord expression levels of Robo and Robo3 were unaffected; however, Robo2 expression levels were significantly reduced. Expression of Slit, the ligand for Robo receptors, and Commissureless, a factor responsible for clearing Robo receptors from commissural axons, was unaffected in nerfin-1null embryos (Kuzin, 2005).

Loss of nerfin-1 function also significantly delayed and/or reduced the early expression of the neuron-specific microtubule-associated MAP1B-like gene futsch. futsch expression is normally activated in newborn neurons starting at stage 11; however, in nerfin-1null embryos expression is first detected only at the stage 13. Not until embryonic stage 15 did the level of futsch expression in mutant embryos approach that of wild type. Reduced mRNA steady state levels for the genes encoding Leukocyte-antigen-related-like (Lar), another receptor tyrosine kinase, and G-oα47A gene, which encodes an alpha subunit of heterotrimeric G proteins, were also detected in nerfin-1null embryos. The reduced level of gene expression in mutant embryos was nervous system specific. For example, G-oα47A gene expression in mesodermal derived tissues was not altered in nerfin-1null embryos (Kuzin, 2005).

Mutations in sec15 cause defects in synaptic specificity, axon targeting and localization of axon guidance components

The exocyst is a complex of proteins originally identified in yeast that has been implicated in polarized exocytosis/secretion. Components of the exocyst have been implicated in neurite outgrowth, cell polarity, and cell viability. An exocyst component, sec15, has been isolated in a screen for genes required for synaptic specificity. Loss of sec15 causes a targeting defect of photoreceptors that coincides with mislocalization of specific cell adhesion and signaling molecules. Additionally, sec15 mutant neurons fail to localize other exocyst members like Sec5 and Sec8, but not Sec6, to neuronal terminals. However, loss of sec15 does not cause cell lethality in contrast to loss of sec5 or sec6. The data suggest a role for Sec15 in an exocyst-like subcomplex for the targeting and subcellular distribution of specific proteins. The data also show that functions of other exocyst components persist in the absence of sec15, suggesting that different exocyst components have separable functions (Mehta, 2005).

Elevated levels of chaoptin in photoreceptor terminals have been described for another vesicle-trafficking mutant, the vesicle-SNARE neuronal-synaptobrevin (n-syb). This mutant also exhibits neuronal targeting defects. This observation raises the possibility that vesicle-dependent trafficking of transmembrane or other signaling molecules might be responsible for the neuronal targeting defects of sec15 mutant photoreceptors. Recently, Zhang (2004) identified Rab11 as an interacting partner of Sec15 in mammalian cell culture and proposed that Sec15 is an effector for some but not all Rabs. Indeed, an accumulation or upregulation of Rab11 immunoreactivity was seen in sec15 mutant photoreceptors, consistent with Rab11-positive vesicles failing to fuse with their target sites. To further test this hypothesis, the localization of cell adhesion and signaling molecules was examined in mutant photoreceptor cell bodies as well as terminals during photoreceptor development, precisely when target selection and cartridge formation occur (between P + 5% to P + 40% referring to time after pupation). Proteins were examined that have either been shown to be required for photoreceptor target selection, such as Dlar, N-cadherin, flamingo, and IrreC-rst, or that are likely to be required, based on work in other systems, such as Armadillo, Chaoptin, and Fasciclin II (Mehta, 2005).

Fasciclin II (Fas2) localization was examined in sec15 mutant photoreceptors, since chaoptin upregulation coincides with elevated levels of Fas2 in n-syb mutant photoreceptors. Fas2 appears to be present in aggregates in sec15 mutant photoreceptor cell bodies at P + 20%, in contrast to wild-type photoreceptors. In addition, the neuronal connections of the cell bodies exhibit Fas2 aggregated along the length of the mutant axons. Similarly, overexpression of Fas2 in photoreceptors causes neuronal targeting defects between P + 20% and P + 40%. In contrast to n-syb, however, no elevated levels of Fas2 are observed later in development. Hence, the data suggest that an aberrant localization of Fas2 in a specific developmental time window may at least partially underlie the observed phenotypes (Mehta, 2005).

Similar mislocalization phenotypes in photoreceptor cell bodies were also observed for other cell adhesion molecules such as Dlar and IrreC-rst during the developmental time window of photoreceptor target selection. Dlar is normally restricted apically in developing wild-type photoreceptors, at the center of the ommatidial array. In sec15 mutant photoreceptors it appears much more randomly distributed, such that a basal optical section through the eye shows Dlar at higher levels in mutant ommatidia. Although these results show mislocalization of cell adhesion molecules in the correct cell at the time when they are known to be required for proper target selection, no obvious defects were detected in the localization of Dlar or IrreC-rst in the developing lamina. This leaves open the question of whether mislocalization of Dlar and IrreC-rst beyond the resolution limit of confocal microscopy additionally contributes to the observed targeting defects (Mehta, 2005).

In vertebrates, Lar is known to localize to adherens junctions. Hence, a possible explanation for the mislocalization of Fas2, IrreC-rst, and Dlar in mutant photoreceptor cell bodies is a defect of adherens junctions. The subcellular localization of the adherens junction markers N-cadherin and armadillo was examined in the cell bodies as well as the terminals of mutant photoreceptors, but no mislocalization of N-cadherin was detected in either compartment. However, armadillo displayed localization defects selectively in the developing lamina, but not the photoreceptor cell bodies. Several other cell adhesion and signaling molecules, including flamingo, Crumbs, and Bazooka, were examined, all of which did not display aberrant localization at the level of light microscopy. It is concluded conclude that a specific subset of proteins is mislocalized in sec15 mutants (Mehta, 2005).

Protein Interactions

The synaptic function of the receptor protein tyrosine phosphatase (RPTP), Dlar, and an associated intracellular protein, Liprin-alpha, has been examined at the Drosophila larval neuromuscular junction. Liprin-alpha and Dlar are required for normal synaptic morphology. Synapse complexity is proportional to the amount of Dlar gene product, suggesting that Dlar activity determines synapse size. Ultrastructural analysis reveals that Dliprin-alpha and Dlar are required to define the size and shape of the presynaptic active zone. Accordingly, there is a concomitant decrease in synaptic transmission in both mutants. Finally, epistasis analysis indicates that Dliprin-alpha is required for Dlar's action at the synapse. These data suggest a model where Dliprin-alpha and Dlar cooperate to regulate the formation and/or maintenance of a network of presynaptic proteins (Kaufmann, 2002).

The yeast two hybrid system was used to searched for Drosophila proteins that would bind directly to Dlar in the yeast interaction trap assay. Using the entire cytoplasmic domain of Dlar as bait, approximately ten million Drosophila cDNA clones were examined. Sequence of the first strong interacting clone revealed it to be the Drosophila homolog of Liprin-alpha. Mammalian Liprin-alpha belongs to a family of related mammalian proteins; however, scans of the Drosophila genome identify only one Liprin-alpha gene (Kaufmann, 2002).

The structural motifs in Liprin-alpha family members consist of an N-terminal coiled-coil domain, shown to multimerize the protein and a C-terminal LAR binding region. While Drosophila Liprin-alpha is 47% identical to mammalian Liprin-alpha overall, much higher levels of conservation are seen in individual domains. For example, a region of 70 amino acids near the N terminus of Drosophila Liprin-alpha is over 90% identical to mLiprin-alpha. In addition, the LAR binding domain of Drosophila Liprin-alpha recovered in the protein interaction screen is 75% identical to mLiprin-alpha and 68% identical to its C. elegans homolog (Kaufmann, 2002).

As a first step toward a functional analysis, the expression of Liprin-alpha was examined. Using in situ hybridization, Liprin-alpha mRNA was observed to be abundantly expressed in the embryo, from cellular blastoderm through hatching. A strong signal at early stages (stages 4 and 5) indicates that there is a substantial maternal contribution of Liprin-alpha. Liprin-alpha mRNA accumulates at highest levels in the developing central and peripheral nervous systems (CNS and PNS, respectively) and is not detected outside of the CNS and PNS late in embryonic development. In situs at the third instar stage also reveal persistent expression of liprin-alpha in the larval CNS and bodywall muscle (Kaufmann, 2002).

To determine Liprin-alpha protein localization, polyclonal antibodies were raised and affinity purified. In late stage embryos (stages 16 and 17), the highest levels of Liprin-alpha are restricted to the neuropil of the ventral nerve cord. This precisely matches the pattern of Dlar protein localization at these stages, consistent with direct biochemical association between Liprin and LAR family members. While Liprin-alpha protein localizes to both longitudinal and commissural neuropil in stage 16, it was noticed that the protein becomes restricted to longitudinal neuropil at stage 17 and first instar. Since developing synapses are found primarily in the longitudinal regions, this raised the question of whether Liprin-alpha is primarily synaptic in its localization. To address this, peripheral motor pathways were examined at larval stages when the NMJ is well formed and distinct from the axon. At this stage, Liprin-alpha accumulates specifically at the synapse, showing little or no staining along the axon. Higher resolution confocal images show that Liprin-alpha is both presynaptic and postsynaptic (compared to a neuronal membrane marker), often forming puncta at the membrane reminiscent of active zones. This pattern is nearly identical to the localization of the synaptic protein Discs large (Dlg) and strongly suggests a function for Liprin-alpha at the NMJ. Interestingly, Liprin-alpha localization appears grossly normal at Dlar null mutant NMJs. Although in situ hybridizations confirm that Dlar is expressed throughout the larval CNS, it is not detected in muscle during the same larval stages (Kaufmann, 2002).

The liprin-alpha gene maps to the cytological location 27B1. The closest P element was about 40 kb away from liprin-alpha at that time, though there is now an EP insertion (EP2141) approximately 600 base pairs upstream of liprin-alpha. To generate mutations in liprin-alpha, P elements from two separate lines, l(2)k00605 and l(2)k13315, were remobilized. Two independent strains were recovered from the l(2)k13315 remobilization with insertions in the liprin-alpha transcription unit. Both insertions fall in an intron in the 5'UTR of liprin-alpha within a few nucleotides of each other but are oriented in opposite directions. Staining for Liprin-alpha clearly shows lower levels of the protein in homozygous embryos from one of the insertion lines (J1), while protein levels seemed approximately normal in the other line (F3). Though dramatically lighter than wild-type controls, traces of residual Liprin-alpha are still seen in the CNS of the J1 line. This suggests that J1 is hypomorphic; however, the pattern is also consistent with the perdurance of maternally expressed Liprin-alpha; abundant staining in early embryos (stage 4) supports this theory (Kaufmann, 2002).

To generate stronger mutant alleles, the P element in the F3 line was remobilized. A number of liprin-alpha deficiency lines were generated by this method. One line, liprin-alphaF3ex15, was selected for detailed study. DNA sequence analysis shows that liprin-alphaF3ex15 retains some of the P element and leaves a small piece of the 5'UTR of liprin-alpha while the rest of the gene is deleted. Genomic mapping of this excision shows that the liprin-alpha coding region is completely removed in this line; however, in embryos, traces of protein staining are still observed in the embryonic neuropil, similar to that seen in the liprin-alphaJ1 insertion line; the localization of this antigen appeared normal. It is believed this is due to maternal contribution to the embryo. To generate additional alleles, a P element from the l(2)k00605 mobilization screen, which had inserted 600 base pairs upstream of liprin-alpha, was excised and excisions were analyzed. This latter screen generated deficiencies extending in both directions, some of which removed liprin-alpha, including liprin-alpha77ex17. While generating these alleles, two lines derived from excision of EP2141, liprin-alphaR60 and liprin-alphaR117 became available. Mapping of these excisions shows they are both small deficiencies, removing most of the upstream region between liprin-alpha and an adjacent transcription unit. Liprin-alpha protein levels are reduced in both lines. Dlar expression and localization is normal in all of these liprin-alpha mutant backgrounds (Kaufmann, 2002).

To determine the function of Liprin-alpha, a series of allelic combinations that disrupt liprin-alpha was created with removal of only one copy of flanking genes. Since a percentage of motor axons (~20%) from the intersegmental nerves b and d (ISNb and ISNd, respectively) require Dlar for guidance to their target muscles in embryonic stages, embryonic motor pathways were examined in liprin-alpha mutants. No defects in axon guidance were seen. Thus, no evidence is found that zygotic liprin-alpha is necessary for motor axons to reach their targets, although maternal expression may mask an early function. However, the absence of axon guidance defects in zygotic liprin-alpha mutants afforded the opportunity to examine the role of liprin-alpha in synapse development at the larval NMJ (Kaufmann, 2002).

The most thoroughly characterized NMJ in the Drosophila larva is the glutamatergic synapse at muscles 6 and 7. This synapse (6/7NMJ) is innervated by two neurons: RP3, which elaborates a terminal decorated with large boutons (type Ib), and MN6/7b, which generates smaller boutons (type Is). The number of boutons and the branch complexity in the 6/7NMJ at abdominal segments A2 and A3 is highly stereotyped during the wandering third larval instar stage. When liprin-alpha mutants are compared to the genetically matched parental control strains using immunohistochemistry, a 30%-50% reduction is found in the size of the 6/7NMJ, as assessed by the number of boutons per NMJ. Both type Ib and type Is boutons are reduced in number. Although more variable than bouton number, branch complexity of the terminal arbor is also decreased compared to wild-type. In addition, the same phenotype is seen in three liprin-alpha loss-of-function combinations derived from two independent parental strains, ruling out genetic background effects. Bouton number is normal in liprin-alpha/+ heterozygotes, demonstrating that liprin-alpha is not haploinsufficient (Kaufmann, 2002).

In order to prove that the reduction in synapse size was due solely to loss of liprin-alpha function, the J1 P element insertion was mobilized and revertants were sought that completely restored the liprin-alpha gene. Two precise excision lines were isolated, and anatomical analysis shows that they have normal bouton numbers and branching complexity at the 6/7NMJ. liprin-alpha is thus required for normal NMJ development (Kaufmann, 2002).

In order to better understand the defects in synaptogenesis, NMJ morphology was examined in liprin-alpha mutants using confocal microscopy. At the tips of wild-type synaptic arbors, a large end bouton is frequently surrounded by smaller boutons, presumably formed by budding of the parent bouton in the process of synapse growth. In contrast, liprin-alpha mutant terminals frequently lack these nascent bouton structures. Quantification of these structures in wild-type and heterozygous controls, compared to two different strong liprin-alpha mutant combinations, reveals a substantial reduction in the mutants. This suggests that Dliprin-alpha is important for the growth of the synapse through formation of new boutons at the ends of terminal branches. The reduction in branch complexity in liprin-alpha mutants is consistent with this hypothesis. Despite the reduction of nascent boutons, the expression and localization of several synaptic markers are unaffected by the liprin-alpha mutations; the markers include the scaffolding protein Discs large (Dlg), the cytoskeletal protein Futsch, and the glutamate receptor GluRII (Kaufmann, 2002).

The function of liprin-alpha at the synapse and the late larval expression of Dlar raised the question of whether Dlar might also be required for NMJ morphogenesis. In this regard, the low penetrance (~20%) of motor axon guidance defects in Dlar null alleles (e.g., Dlar13.2 and Dlar5.5) provided an opportunity to examine NMJ morphology at synapses innervated by the correct number of motor neurons. To identify the ~80% of larval segments where axon guidance was unaffected in Dlar mutants, the analysis was limited to 6/7NMJs that contained both type Ib and type Is boutons from axons that entered the target domain along the correct trajectory (indicating that both RP3 and MN6/7b were present). Antibodies that recognize the synaptic protein Dlg were used in order to distinguish between the two classes of boutons. Examination of bouton number and terminal branch complexity revealed that complete loss of Dlar results in a reduction in synapse size very similar to that observed in liprin-alpha loss-of-function mutants. Both type Ib and type Is boutons were reduced, suggesting that both neurons at the 6/7NMJ require Dlar activity, as observed in liprin-alpha mutants (Kaufmann, 2002).

The synapse is very sensitive to the amount of Dlar expressed. Although a null allele of Dlar (Dlar5.5) would be expected to show more penetrant phenotypes than a weaker hypomorph (Dlarbyp), reduction of Dlar gene dose by 50% (Dlar5.5/+) decreases NMJ size relative to wild-type. This is in contrast to all other known functions of Dlar and suggests that synapse growth is directly proportional to the amount of Dlar activity. If this model is correct, it is predicted that raising Dlar activity above wild-type levels should increase synapse size. Indeed, expression of a full-length Dlar transgene under control of two different postmitotic neural-specific GAL4 drivers increases bouton number in comparison to a wild-type strain. These experiments also suggest that Dlar acts presynaptically, as initially suggested by the neural-specific expression of endogenous Dlar (Kaufmann, 2002).

To determine if the effect of increased Dlar expression was manifest specifically during larval stages when the NMJ undergoes massive growth and to ask if the activity could be supplied by the catalytic domains alone, the cytoplasmic region of Dlar was expressed under a neural-specific GAL4 fused to a hormone receptor (GS-GAL4). This expression system provides both tissue and temporal specificity. Induction of Dlar expression during midlarval development with 50 µg/ml mifepristone increases bouton number (Kaufmann, 2002).

Previous studies have suggested a model where Liprin-alpha acts upstream of Dlar to localize the protein on the cell surface (Serra-Pagès, 1998). The Dlar gain-of-function NMJ phenotype presented an opportunity to perform an epistasis test by simultaneously removing liprin-alpha function while overexpressing Dlar in postmitotic neurons. While overexpression of Dlar does not rescue the liprin-alpha phenotype, the absence of Liprin-alpha protein prevents the increase in NMJ bouton number observed when Dlar is overexpressed in a wild-type background. This puts Liprin-alpha genetically downstream of Dlar, providing evidence that Liprin-alpha is required for the normal output of the LAR pathway (Kaufmann, 2002).

Genetic analysis in C. elegans shows that the Liprin-alpha homolog syd-2 is required to constrain the size of cholinergic active zones (AZs) (Zhen, 1999). To ask whether this Liprin-alpha function is conserved in Drosophila and whether it generalizes to a glutamatergic synapse, an analysis of liprin-alpha mutant NMJ ultrastructure was undertaken. liprin-alpha active zones (AZs) fall across a range of sizes from smaller than normal to far greater in size. Mutant AZs were always abnormal in either total size (area) or shape; the mean maximum dimension of mutant AZs (1319 nm) is nearly double that of wild-type (684 nm). Moreover, the mean total area of mutant AZs is 2.4-fold higher than wild-type (Kaufmann, 2002).

Since Liprin-alpha and Dlar associate directly and display nearly identical gross NMJ phenotypes, it was asked if Dlar is also required to define the dimensions of the presynaptic active zone. Like the liprin-alpha mutants, overall ultrastructure is normal in the Dlar mutants on both presynaptic and postsynaptic sides of the NMJ. However, 61% of individual AZs are larger than the largest AZ found in wild-type NMJs (Kaufmann, 2002).

To elucidate a potential synaptic function of Liprin-alpha and Dlar, the synaptic physiology of mutant larval NMJs were examined by whole-cell recordings of evoked excitatory junctional potentials (EJPs) and spontaneous excitatory junctional potentials (mEJPs). EJPs were reduced 36% in liprin-alpha and 34% in Dlar mutants, compared to precise excision controls; this was highly significant. The resting potential of the mutant muscle was normal (Kaufmann, 2002).

The decrease of synaptic transmission in liprin-alpha and Dlar mutants was not caused by a postsynaptic defect since quantal events (mEJPs) were normal. The mean amplitude and the frequency of unitary mEJPs in liprin-alpha and Dlar larvae also showed no significant differences from control. This indicates that the postsynaptic sensitivity to neurotransmitter and the neurotransmitter content of synaptic vesicles are not altered in mutants. However, the normal postsynaptic neurotransmitter reception pointed to a defect intrinsic to the evoked release process (Kaufmann, 2002).

To better quantify mutant synaptic transmission, the number of quanta released per stimulus was determined. Quantal content is defined by the ratio of EJP/mEJP amplitudes after correcting for nonlinear summation using a reversal potential of 0 mV. The quantal content of evoked release for both liprin-alpha and Dlar was reduced by over 50%. This highly significant reduction in presynaptic vesicle release is consistent with a fundamental defect in active zone structure and function (Kaufmann, 2002).

Analysis of the C. elegans Liprin-alpha gene syd-2 provided the first support for the model that Liprins are integral to the presynaptic protein complex that organizes neurotransmitter release (Zhen, 1999). This function is conserved. Data from C. elegans, Drosophila, and mammals indicate that the protein localization of Liprin-alpha family members to the synapse is also conserved (Zhen, 1999; Wyszynski, 2002). Interestingly, in all organisms, Liprin-alpha is found on both sides of the synapse. Analyses in C. elegans and mammalian neurons support both presynaptic and postsynaptic roles. In C. elegans, loss of syd-2 increases the dimensions of the AZ by approximately 2-fold, with a concomitant decrease in the efficiency of synaptic transmission (Zhen and Jin, 1999). Analysis in Drosophila not only confirms an increase in AZ size, but also reveals that liprin-alpha mutations have a dramatic effect on AZ shape. This latter phenotype is fully penetrant, suggesting that liprin-alpha is a requisite structural component of the AZ (Kaufmann, 2002).

Exactly what Liprin-alpha is doing on the postsynaptic side is unknown. Since Dlar expression is not detected in muscle, this also raises the question of what proteins act in partnership with Liprin-alpha in the subsynaptic reticulum. Studies in mammalian synapses suggest that Liprins interact with several pre- and post-synaptic proteins (Kaufmann, 2002).

But how do Liprins act to support LAR-family receptor function? Several models are possible. Liprin-alpha may act in the signaling mechanism downstream of the receptor. Although Liprins have not been shown to be phosphorylated PTP substrates, they could serve to recruit other necessary signaling proteins. The fact that mLiprin-alpha associates with synaptic scaffolding proteins is consistent with this hypothesis (e.g., GRIP; Wyszynski, 2002). Alternatively, Liprin-alpha may function to deliver LAR-family receptors to their sites of action in the cell, as suggested by experiments in nonneuronal cells. Without a functional delivery system, Dlar overexpression may fail to increase RPTP activity in the appropriate location (Kaufmann, 2002).

Liprin-α has LAR-independent functions in R7 photoreceptor axon targeting

In the Drosophila visual system, the color-sensing photoreceptors R7 and R8 project their axons to two distinct layers in the medulla. Loss of the receptor tyrosine phosphatase LAR from R7 photoreceptors causes their axons to terminate prematurely in the R8 layer. This study has identified a null mutation in the Liprin-α (for LAR-interacting protein) gene based on a similar R7 projection defect. Liprin-α physically interacts with the inactive D2 phosphatase domain of LAR, and this domain is also essential for R7 targeting. However, another LAR-dependent function, egg elongation, requires neither Liprin-α nor the LAR D2 domain. Although human and Caenorhabditis elegans Liprin-α proteins have been reported to control the localization of LAR, LAR localizes to focal adhesions in Drosophila S2R+ cells and to photoreceptor growth cones in vivo independently of Liprin-α. In addition, Liprin-α overexpression or loss of function can affect R7 targeting in the complete absence of LAR. Despite its reported role in axonal transport of synaptic vesicle components, Liprin-α is not required for the transport of endogenous LAR protein to the growth cones of larval R1–R6 photoreceptors. It is concluded that Liprin-α does not simply act by regulating LAR localization but also has LAR-independent functions (Hofmeyer, 2006).

Liprin-α is required for photoreceptor target selection in Drosophila

Classical cadherin-mediated interactions between axons and dendrites are critical to target selection and synapse assembly. However, the molecular mechanisms by which these interactions are controlled are incompletely understood. In the Drosophila visual system, N-cadherin is required in both photoreceptor (R cell) axons and their targets to mediate stabilizing interactions required for R cell target selection. This study identifies the scaffolding protein Liprin-α as a critical component in this process. Mutations were isolated in Liprin-α in a genetic screen for mutations affecting the pattern of synaptic connections made by R1–R6 photoreceptors. Using eye-specific mosaics, a previously undescribed, axonal function for Liprin-α in target selection was demonstated: Liprin-α is required to be cell-autonomous in all subtypes of R1–R6 cells for their axons to reach their targets. Because Liprin-α, the receptor tyrosine phosphatase LAR, and N-cadherin share qualitatively similar mutant phenotypes in R1–R6 cells and are coexpressed in R cells and their synaptic targets, it is inferred that these three genes act at the same step in the targeting process. However, unlike N-cadherin, neither Liprin-α nor LAR is required postsynaptically for R cells to project to their correct targets. Thus, these two proteins, unlike N-cadherin, are functionally asymmetric between axons and dendrites. It is proposed that the adhesive mechanisms that link pre- and post-synaptic cells before synapse formation may be differentially regulated in these two compartments (Choe, 2006).

The heparan sulfate proteoglycan syndecan is an in vivo ligand for the Drosophila LAR receptor tyrosine phosphatase

Receptor tyrosine phosphatases (RPTPs) are essential for axon guidance and synaptogenesis in Drosophila. Each guidance decision made by embryonic motor axons during outgrowth to their muscle targets requires a specific subset of the five neural RPTPs. The logic underlying these requirements, however, is still unclear, partially because the ligands recognized by RPTPs at growth cone choice points have not been identified. RPTPs in general are still 'orphan receptors' because, while they have been found to interact in vitro with many different proteins, their in vivo ligands are unknown. This study uses a new type of deficiency screen to identify the transmembrane heparan sulfate proteoglycan Syndecan (Sdc) as a ligand for the neuronal RPTP LAR. LAR interacts with the glycosaminoglycan chains of Syndecan in vitro with nanomolar affinity. Genetic interaction studies using Sdc and Lar LOF mutations demonstrate that Sdc contributes to LAR's function in motor axon guidance. Overexpression of Sdc on muscles is shown to generate the same phenotype as overexpression of LAR in neurons, and genetic removal of LAR suppresses the phenotype produced by ectopic muscle Sdc. Finally, it is shown that there is at least one additional, nonproteoglycan, ligand for LAR encoded in the genome. Taken together, these results demonstrate that Sdc on muscles can interact with neuronal LAR in vivo and that binding to Sdc increases LAR's signaling efficacy. Thus, Sdc is a ligand that can act in trans to positively regulate signal transduction through LAR within neuronal growth cones (Fox, 2005).

Genetic removal of Sdc from embryos bearing Lar mutations increases the penetrance of the characteristic Lar ISNb bypass phenotype. This effect on penetrance is as large as those usually observed when a second RPTP is genetically removed from a single Rptp mutant (e.g., removal of DPTP69D from a Lar mutant). Removal of Sdc increases penetrance for both hypomorphic and (zygotic) null Lar mutations. The effect on the null penetrance is likely due to reduction of maternally contributed LAR function. However, it was found that Sdc mutations alone do not produce ISNb bypass at a significant frequency, even when both maternal and zygotic Sdc are removed. One explanation for this finding might be that Sdc is partially redundant with Dally and/or Dlp, since these are cell-surface HSPGs expressed in a similar pattern to Sdc (Fox, 2005).

To test this model, Dally and Dlp expression were reduced in an Sdc mutant background. It is difficult to assess the appropriate extent of reduction for this experiment. Glypicans cannot be completely removed, since this would produce embryos with severe early phenotypes due to loss of Hedgehog and Wingless signaling. Zygotic triple mutants (Sdc dally dlp) were generated, and, also, dally and dlp dsRNAs were injected into Sdc maternal/zygotic mutant embryos (dally/dlp RNAi would affect both maternal and zygotically contributed mRNAs). The genetic triple mutants had CNS phenotypes that are stronger than the Sdc phenotype but displayed few motor axon guidance errors. Dally/Dlp-injected Sdc mutant embryos had more severe phenotypes, but ISNb guidance was not selectively affected. Overall, the data suggest that Sdc is not redundant with Dally or Dlp and that its absence is likely to be compensated for by non-HSPG proteins. Perhaps the second LAR ligand detected by embryo staining is redundant with Sdc. Like Sdc, this ligand is expressed both on CNS axons and in lines in the periphery (Fox, 2005).

A genetic epistasis experiment demonstrates that Sdc acts in trans (as a ligand) to regulate LAR function. However, the data allow this conclusion to be reached only for SNa bifurcation, which is affected by LAR overexpression but not by loss of LAR. In its regulation of the decision of ISNb growth cones to enter the muscle field, Sdc could also act as a ligand, since it is expressed at the appropriate time on patches of cells near the muscle field entry site that could be contacted by LAR-expressing ISNb growth cones at this choice point. Alternatively, Sdc could act as a coreceptor at this choice point since it is expressed on the motor nerves together with LAR. Finally, it is not known if the Sdc that interacts with LAR during ISNb axon guidance is attached to the cell surface or has been shed by proteolytic cleavage. If released then Sdc is the essential ligand; Sdc could be expressed by either muscles or neurons and transported to the choice point (Fox, 2005).

These results show that the cell-surface HSPG Sdc is an in vivo ligand for LAR and indicate that it positively regulates LAR signaling during motor axon guidance. Sdc’s GAG chains bind directly to LAR with high affinity, and this binding requires basic sequences in the first Ig domain of LAR. Further work will be required to determine whether binding to Sdc directly stimulates LAR’s phosphatase activity, relocalizes LAR within the growth cone,or facilitates LAR signaling by another mechanism (Fox, 2005).

The HSPGs Syndecan and Dallylike bind the receptor phosphatase LAR and exert distinct effects on synaptic development

The formation and plasticity of synaptic connections rely on regulatory interactions between pre- and postsynaptic cells. The Drosophila heparan sulfate proteoglycans (HSPGs) Syndecan (Sdc) and Dallylike (Dlp) are synaptic proteins necessary to control distinct aspects of synaptic biology. Sdc promotes the growth of presynaptic terminals, whereas Dlp regulates active zone form and function. Both Sdc and Dlp bind at high affinity to the protein tyrosine phosphatase LAR, a conserved receptor that controls both NMJ growth and active zone morphogenesis. These data and double mutant assays showing a requirement of LAR for actions of both HSPGs lead to a model in which presynaptic LAR is under complex control, with Sdc promoting and Dlp inhibiting LAR in order to control synapse morphogenesis and function (Johnson, 2006).

Converging lines of evidence suggest that membrane-associated HSPGs serve an important purpose in the assembly, function and plasticity of excitatory synapses. The ancient HSPG families of syndecans and glypicans are necessary for Drosophila to regulate distinct aspects of synaptic morphogenesis. Genetic and biochemical data indicate that Sdc and Dlp interact with LAR to control presynaptic properties. Because LAR-family RPTPs have been shown to control the formation of excitatory synapses in Drosophila, C. elegans, and mammals, these findings may represent a more general mechanism for regulating synaptic morphogenesis and function. Despite the importance of LAR-family RPTPs during cellular morphogenesis inside and outside of the nervous system, the lack of physiologically relevant extracellular binding partners has made it challenging to study this well-conserved group of receptors (Johnson, 2006).

The data show that Sdc promotes the formation of presynaptic boutons. This Sdc function appears to be mediated by LAR, as supported by parallel phenotypes, direct binding, in vivo colocalization, and three types of double mutant analysis between Sdc and LAR. Despite the fact that Sdc exhibits gain-of-function activity and endogenous expression on both sides of the synapse, neuronal and muscle-specific rescue experiments show that Sdc function is mainly presynaptic. Because LAR is required only in neurons to promote synapse growth, these findings support a model in which Sdc acts as a neuronal cell-autonomous agonist of LAR. This is somewhat surprising, given the fact that soluble forms of Sdc bind to LAR and that endogenous Sdc appears to be released from the presynaptic membrane to fill the subsynaptic reticulum. One way for Sdc to act presynaptically would be to bind to LAR even before the two proteins are presented on the neuronal surface. Because Dlp has a competitive advantage over Sdc for binding to LAR, a prebound complex of Sdc and LAR would have the ability to stimulate synapse growth before the phosphatase could be inhibited by Dlp. Such a mechanism could provide a time- and/or HSPG concentration-dependent switch from bouton addition to active zone assembly (Johnson, 2006).

Sdc could promote LAR activity in collaboration with an additional cell-type-specific membrane protein. Data from a parallel study has also identified Sdc-LAR interactions during embryonic motor axon guidance. However, in contrast to CNS pathfinding, complete loss of Sdc alone has no significant effect on motor pathfinding, suggesting that additional LAR ligands exist in the early embryo. Recent experiments with the vertebrate LAR ortholog PTP-s suggest that non-HSPG ligands may regulate its ability to promote retinal axon outgrowth. Although it remains a formal possibility, an additional ligand may not be needed to account for the NMJ growth-promoting activity of LAR because the larval synaptic phenotype in Sdc mutants is nearly as strong as the growth defect in LAR mutants (Johnson, 2006).

Active zone assembly is vital for neurotransmission at the synapse and has been proposed as a means to modulate synaptic function over time. Analysis of Dlp reveals that synaptic glypicans are required to regulate active zone morphology and function. Moreover, Dlp is limiting for active zone morphogenesis, consistent with an instructive role. Because the activities of Dlp appear opposite to those of LAR, it is proposed that the high affinity binding of Dlp to LAR induces an inhibition of receptor function. This hypothesis is supported by the double RNAi experiments showing that the LAR effect on Ena phosphorylation is epistatic to the effect of Dlp, indicating that Dlp acts upstream of LAR. Because loss of Dlp at the NMJ did not induce a significant change in the number of presynaptic boutons, the results lead to a model in which Dlp is specialized for control of active zone properties. Such a function might provide a means to independently regulate and spatially distinguish LAR inhibition from LAR activation. In any case, the presence of active zone phenotypes in dlp but not in Sdc reveals specialization among synaptic HSPGs (Johnson, 2006).

LAR regulates both NMJ growth and active zone morphogenesis. Thus, LAR appears to provide a link between two important synaptic properties that are regulated by different extracellular factors. A mechanism to couple bouton growth and active zone formation would make sense because active zones appear early in the nascent bouton. Because LAR catalytic activity is necessary for bouton addition, and yet LAR inhibition by Dlp appears necessary for proper active zone formation, LAR's role at the active zone may be primarily structural. For example, LAR may simply provide an anchorage point for synaptic components like the scaffolding protein Liprin-alpha that regulates active zone formation. Alternatively, LAR may exist in distinct yet active signaling states, one of which is dependent on PTP activity (promoting synapse growth), and one of which is dependent on recruitment of signaling molecules (controlling active zone assembly). Because loss of Dlp or LAR has opposite effects on quantal content at the NMJ, it is attractive to speculate that the Dlp-LAR pathway normally provides a means to modulate the strength of neurotransmission, either during NMJ growth or during synaptic plasticity. LAR PTPs are required for normal physiology and plasticity at mammalian hippocampal synapses (Johnson, 2006).

Sdc and Dlp are both HSPGs that bind to LAR and thus might be expected to act similarly, but the results show that their functions are different. One way to account for the specificity might be a difference in the effect of soluble versus cell-surface HSPGs on LAR. Some ligand molecules such as Ephrins function when clustered at high density (e.g., on a membrane surface) but fail to activate their receptors when presented in a soluble, monomeric form. Another possibility could be that LAR binding or signaling is differentially influenced by direct protein-protein interactions with the Sdc versus Dlp core proteins. The two HSPGs have very different core structures; Sdc is a transmembrane molecule with HS modification sites near the N terminus, whereas Dlp is a GPI-anchored protein with HS sites proximal to the membrane and a large disulphide bonded globular domain located more distally. It may also be relevant that Dlp consistently binds more effectively to LAR than Sdc, with KD measurements in solution showing an affinity approximately 2-fold higher. These results suggest a competition model in which Dlp displaces Sdc, possibly to favor the stabilization of active zones after new growth at the synapse. In this model, presynaptic growth would be initially promoted by Sdc and would then be limited or halted by Dlp binding after formation of close membrane contact between the nerve and muscle. Such a mechanism could insure a transition from growth to synapse stabilization and could participate in subsequent maintenance or plasticity of the synapse (Johnson, 2006).

Of course other molecules influence synapse size, and these might include coligands or coreceptors that may bind to Sdc, Dlp, and/or LAR. Potential candidates might include bone morphogenic protein (BMP), the type II BMP receptor Wishful thinking (Wit), or the Wnt ortholog Wingless (Wg), which have all been shown to regulate NMJ morphology in Drosophila. However, in addition to significant phenotypic differences compared to the HSPGs, overexpression studies indicate that neither BMP nor Wg are limiting for NMJ morphogenesis. In contrast, Sdc and Dlp are both limiting for different aspects of synapse development, consistent with an instructive role in this context. Consistent with this notion, Syndecan-2 is sufficient to promote dendritic spine maturation during hippocampal synaptogenesis in culture. Although vertebrate Syndecan-2 has yet to be tested at the synapse by loss of function, the colocalization and parallel biology of Synecan-2 and vertebrate LAR-family receptors strongly suggest conservation in the regulation of synaptic LAR (Johnson, 2006).

The genetic and biochemical studies described in this study have identified a partnership between HSPGs and LAR in Drosophila NMJ development that sets precedents for (1) the in vivo requirement for members of the syndecan and glypican families in synapse growth and electrophysiological function, (2) the specificity of HSPG function at the synapse, with distinct actions of Sdc and Dlp, and (3) biochemical identification of Sdc and Dlp as LAR binding partners, plus genetic evidence to place them in a pathway regulating biological function at the synapse (Johnson, 2006).

Complex interactions amongst N-cadherin, DLAR, and Liprin-alpha regulate Drosophila photoreceptor axon targeting

The formation of stable adhesive contacts between pre- and post-synaptic neurons represents the initial step in synapse assembly. The cell adhesion molecule N-cadherin, the receptor tyrosine phosphatase DLAR, and the scaffolding molecule Liprin-alpha play critical, evolutionarily conserved roles in this process. However, how these proteins signal to the growth cone and are themselves regulated remains poorly understood. Using Drosophila photoreceptors (R cells) as a model, this study evaluated genetic and physical interactions among these three proteins. DLAR function in this context was shown to be independent of phosphatase activity but requires interactions mediated by its intracellular domain. Genetic studies reveal both positive and, surprisingly, inhibitory interactions amongst all three genes. These observations are corroborated by biochemical studies demonstrating that DLAR physically associates via its phosphatase domain with N-cadherin in Drosophila embryos. Together, these data demonstrate that N-cadherin, DLAR, and Liprin-alpha function in a complex to regulate adhesive interactions between pre- and post-synaptic cells and provide a novel mechanism for controlling the activity of Liprin-alpha in the developing growth cone (Prakash, 2009).

The cytoplasmic adaptor protein Caskin mediates Lar signal transduction during Drosophila motor axon guidance

The multiprotein complexes that receive and transmit axon pathfinding cues during development are essential to circuit generation. Identified and characterized the Drosophila sterile α-motif (SAM) domain-containing protein Caskin, which shares homology with vertebrate Caskin, a CASK [calcium/calmodulin-(CaM)-activated serine-threonine kinase]-interacting protein. Drosophila caskin (ckn) is necessary for embryonic motor axon pathfinding and interacts genetically and physically with the leukocyte common antigen-related (Lar) receptor protein tyrosine phosphatase. In vivo and in vitro analyses of a panel of ckn loss-of-function alleles indicate that the N-terminal SAM domain of Ckn mediates its interaction with Lar. Like Caskin, Liprin-α is a neuronal adaptor protein that interacts with Lar via a SAM domain-mediated interaction. Evidence is presented that Lar does not bind Caskin and Liprin-α concurrently, suggesting they may assemble functionally distinct signaling complexes on Lar. Furthermore, a vertebrate Caskin homolog interacts with LAR family members, arguing that the role of ckn in Lar signal transduction is evolutionarily conserved. Last, several ckn mutants were characterized that retain Lar binding yet display guidance defects, implying the existence of additional Ckn binding partners. Indeed, the SH2/SH3 adaptor protein Dock was identified as a second Caskin-binding protein and it was found that Caskin binds Lar and Dock through distinct domains. Furthermore, whereas ckn has a nonredundant function in Lar-dependent signaling during motor axon targeting, ckn and dock have overlapping roles in axon outgrowth in the CNS. Together, these studies identify caskin as a neuronal adaptor protein required for axon growth and guidance (Weng, 2011).

The Drosophila neuromuscular system is an excellent paradigm to decipher the molecular signals orchestrating the precise matching between individual motorneurons and their muscle partners. A number of guidance cues and receptors coordinately regulate motor axon pathfinding assuring the high fidelity of this process. The axon must integrate these disparate signals as it navigates through its environment. Multidomain adaptor proteins promote such integration since they serve as platforms to facilitate communication between signal transduction cascades (Weng, 2011).

The leukocyte common antigen-related (LAR)-related subfamily of receptor protein tyrosine phosphatases (RPTPs type IIA) are conserved regulators of axon pathfinding and synaptogenesis. This subfamily includes the Drosophila receptors Lar and PTP69D, and the vertebrate receptors LAR, protein tyrosine phosphatase ς (PTPς), and PTPδ. Family members contain a variable number of Ig and fibronectin (FN) III domains extracellularly, and two intracellular phosphatase domains. The membrane-proximal D1 phosphatase domain (D1) confers most if not all of the catalytic activity of the receptor, whereas the membrane-distal D2 domain is catalytically inactive and may contribute to LAR family function via interaction with downstream signaling components. These receptors exhibit neuronal expression patterns, and loss-of-function (LOF) mutants display defects in axon targeting and synapse formation. Heparan sulfate proteoglycans (HSPGs) are binding partners of LAR family members in axon pathfinding and synaptogenesis. In vertebrates, PTPδ is a neuronal receptor for chondroitin sulfate proteoglycan (CSPG) and inhibits axon regeneration after CNS injury. On the intracellular side, Lar activity in some contexts requires phosphatase activity, whereas in other contexts its function is independent of catalytic activity, suggesting a diversity of downstream signaling pathways. Indeed, a number of Lar-interacting proteins have been identified. Lar function in synaptic maturation requires Liprin-α, a sterile α-motif (SAM) domain-containing adaptor protein that interacts with Lar in vertebrates and invertebrates (Weng, 2011).

Drosophila Dock and vertebrate Nck are neuronal SH2/SH3-containing adaptor proteins that link guidance receptors to cytoskeletal remodeling. Given the widespread expression of Dock in the embryonic CNS and its central position linking guidance receptors to the actin cytoskeleton, it is notable that motor axons in dock LOF mutant embryos display only subtle defects, raising the possibility of compensation or redundancy. Dock/Nck interact directly with a number of receptors, including Robo, DSCAM (Down syndrome cell adhesion molecule), and the insulin receptor and bind directly to cytoskeletal effectors such as p21-activated protein kinase (Pak) and WASP (Wiskott-Aldrich syndrome protein), thereby presumably linking receptor activation to cytoskeletal rearrangement. Of particular relevance, vertebrate Caskin has been identified as a potential Nck interactor. Caskin was first identified as a novel protein binding the CaM kinase domain of CASK. Vertebrate Caskins are predicted scaffolding proteins with multiple ankyrin repeats, an SH3 domain, and two SAM domains, suggesting that Caskin is a component of a multiprotein complex. This study presents genetic, cell-biological, and biochemical evidence arguing that Drosophila Caskin is a Lar-binding partner and is required for Lar signal transduction in motor axon guidance (Weng, 2011).

This study has demonstrate that Caskin mediates a novel Lar RPTP signaling cascade during axonogenesis. Analysis of a panel of ckn LOF alleles indicates that ckn is necessary for motor axon pathfinding, since homozygous mutants display classic bypass defects in the ISNb motor nerve. This phenotype is identical with that displayed by Lar mutants, and genetic and biochemical interaction data demonstrate that Ckn is a Lar-interacting protein. These studies position Caskin to be a core member of a Lar-associated signaling complex that mediates its function during axonogenesis (Weng, 2011).

Vertebrate Caskin was identified as a binding partner of the synaptic adaptor protein CASK and competes for binding to the CaM kinase domain of CASK with the PDZ (postsynaptic density-95/Discs large/zona occludens-1) protein Mint1. The CASK-binding site on Caskin maps to an N-terminal region not conserved in Drosophila, suggesting that fly Caskin does not bind CASK. Consistent with this finding, Drosophila Caskin and CASK do not interact in a yeast interaction assay. However, both mouse and fly Caskin homologs bind LAR family members and Nck/Dock, in support of considerable shared functions. Furthermore, overexpression of the Lar-binding domain of mouse Caskin in Drosophila neurons yields a pathfinding phenotype like that of Lar and ckn LOF, suggesting that mouse Caskin competes with fly Ckn for binding to the Lar receptor to function as a dominant negative. These biochemical studies indicate that, whereas Caskins may have species-specific binding partners, Ckn function in Lar signal transduction is conserved. Drosophila Lar also physically interacts with the Abl tyrosine kinase and its substrate the cytoskeletal regulator Ena (Wills, 1999). This study was unable to detect physical interactions between Ckn and Abl or Ena, suggesting they bind Lar independently. This raises the possibility that Caskin and Abl/Ena constitute parallel pathways downstream of the Lar receptor (Weng, 2011).

The allelic series enabled analysis of the in vitro and in vivo activities of four Caskin mutant proteins: Ckn-A, Ckn-C, Ckn-K, and Ckn-Y. Ckn-A and Ckn-K contain alterations in the first SAM domain and block the interaction of Ckn with Lar, pointing to the importance of this domain for Lar/Ckn complex formation. The in vivo analysis of Ckn-A and Ckn-K is in strong agreement with the in vitro data as motor axon phenotypes are not associated with their overexpression, suggesting they do not interfere with Lar signaling in vivo. The behavior of Ckn-A and Ckn-K contrasts that of Ckn-C, which contains a C-terminal deletion. Ckn-C interacts with Lar, and its neuronal overexpression yields dominant-negative-like effects. In fact, the penetrance of ISNb bypass associated with Ckn-C overexpression is comparable with that observed in embryos lacking both maternal and zygotic Lar, suggesting that it effectively interferes with Lar activity. Although Ckn-C binds Lar, cknC homozygous LOF mutants display a 'Lar-like' ISNb phenotype, indicating that Lar signaling is blocked downstream of receptor binding. Ckn-C does not interact with Dock, but this interaction is insufficient to explain the cknC mutant phenotype since ISNb bypass is not associated with dock LOF. The pathfinding phenotype observed in cknC embryos argues that the allele also disrupts the interaction between Caskin and another downstream protein(s) essential for Lar signaling (Weng, 2011).

Dock/Nck are SH2/SH3-containing adaptor proteins that couple phosphotyrosines on activated receptors to downstream signaling molecules via SH2 and SH3 domain interactions, respectively. Dock also engages in a ligand-regulated SH3 domain interaction with the Robo receptor, demonstrating that it is involved in diverse interactions downstream of guidance receptors. This work has demonstrate that Caskin interacts with the second SH3 domain of Dock (SH3-2). This domain has also been shown to interact with the cytoskeletal effector Pak, raising the issue of the relationship between Caskin and Pak. It will be informative to determine whether Dock forms alternative complexes with Caskin and Pak, or whether Dock binds Caskin and Pak simultaneously (Weng, 2011).

The contrast between the ckn and dock single- and double-mutant phenotypes demonstrates that the adaptors have mostly redundant functions. Single-mutant analyses indicate that ckn plays a nonredundant role in Lar signaling, whereas dock has a unique role in synaptogenesis of the RP3 motorneuron. However, the outgrowth defects observed in dock ckn double mutants argue that these adaptors have overlapping roles in a number of signaling events. These data caution against drawing conclusions of cellular function based solely on single mutant analysis, as this obviously uncovers only the nonredundant functions of a protein. The issue of genetic redundancy may be particularly acute in signaling systems involving multi-subunit complexes with many opportunities for parallel functions. It will be important to identify additional binding partners of dock and ckn to determine whether they have a common set of interactors, or whether they impinge on the cytoskeleton via distinct, yet redundant, paths (Weng, 2011).

The Lar receptor is a member of the type IIA subfamily of RPTPs, comprising Lar and PTP69D in flies. The single-mutant phenotypes of Lar and PTP69D indicate they have nonredundant functions in motor axon guidance, NMJ growth, and photoreceptor axon targeting. Several observations hint that the unique functions implied by the divergent phenotypes of Lar and PTP69D stem in part from distinct ligand-binding activities. Lar and PTP69D alkaline phosphatase fusion proteins have be shown to possess distinct embryonic staining patterns suggesting the presence of unique ligands. Furthermore, overexpression of a chimeric receptor composed of the Lar extracellular domain fused to the PTP69D intracellular domain rescues the LOF photoreceptor defect of Lar, whereas a PTP69D extracellular domain fusion to the Lar intracellular domain does not, arguing that Lar and PTP69D have overlapping intracellular partners and (at least partially) nonoverlapping extracellular ones. However, more recent data open the door for functional differences between the intracellular pathways activated by Lar and PTP69D. R7 photoreceptor axon targeting is independent of Lar phosphatase activity, but dependent on PTP69D phosphatase activity, suggesting that the receptors have distinct binding partners. These findings are consistent with the work presented in this study. Both fly and vertebrate Caskins interact with subsets of LAR family receptors, raising the possibility that the intracellular signaling cascade(s) organized by Ckn contributes to the functional differences between Lar and PTP69D (Weng, 2011).

the physical relationship between Lar, Ckn, and Liprin-α was investigated, and no ternary complex was detected. These binding data support mapping studies indicating that Ckn and Liprin-α both interact with the D2 phosphatase domain of Lar via SAM domain-mediated interactions. They further suggest sequential/competitive binding of Ckn and Liprin-α to the Lar receptor and raise the possibility of distinct neuronal functions. It is conceivable that Ckn and Liprin-α both act downstream of Lar to mediate its activity during axon outgrowth/pathfinding and synaptogenesis, respectively. To determine whether Ckn function is specific for Lar signaling during axonogenesis, it will be informative to test whether ckn LOF mutants exhibit defects in the assembly/localization of presynaptic components similar to that observed in Lar mutants. Alternatively, the function of Liprin-α in Lar signaling may be primarily to localize or maintain Lar at the presynaptic terminal, whereas Ckn functions in downstream signal transduction. This hypothesis is supported by evidence for a conserved function for Liprin-α in synaptic protein targeting or anchoring. A role for Liprin-α in trafficking is further bolstered by conserved physical interactions between Liprin-α and Kinesin, suggesting it is an adaptor protein for anterograde transport of synaptic proteins. In this scenario, it is notable that Liprin-α function is not required for pathfinding, arguing either that another protein serves to localize Lar during guidance or that Lar activity in this process does not require its tight localization to the axon terminal. This model is consistent with the broad axonal localization of Lar during embryogenesis (Weng, 2011).

Extracellularly, LAR family members interact with HSPGs and CSPGs. In Drosophila, mutations in the HSPG syndecan (sdc) interact with Lar in motor axon guidance, but homozygous LOF sdc embryos do not display appreciable bypass phenotypes, arguing that other ligands are involved. Once these ligands are identified, it will be critical to determine whether ligand binding influences the association of intracellular adaptors such as Liprin-α and Caskin with Lar. Recently, vertebrate LAR family members have moved into the spotlight in the field of axon regeneration, as PTPsigma has been shown to be a receptor for CSPGs, which are dramatically upregulated at the lesion site and are strongly inhibitory to axon growth. Strikingly, axons in PTPsigma mutant mice have a greatly enhanced ability for long-distance regeneration relative to wild-type mice. These studies suggest that blocking PTPsigma signaling in injured axons might enhance recovery after spinal cord injury. Hence, the truncated forms of fly and vertebrate Caskins that interfere with Lar signaling are particularly interesting. The identification of such dominant-negative reagents allowing the blockade of Lar signal transduction in vivo may have clinical implications in neuronal regeneration (Weng, 2011).



Localized expression of Dlar is first seen at the onset of germ band retraction. In lateral views of stage 12, expression is observed in the axonal layer at the dorsal (inner) edge of the nerve cord. In stage 14 embryos, expression is restricted to the axonal tracts, anterior and posterior commissures and longitudinal connectives (Tian, 1991).

DLAR mRNA is expressed in the embryo exclusively by neurons; DLAR protein is found exclusively on their developing axons. DLAR is expressed at high levels by both motoneurons and interneurons in the CNS during stages of axon outgrowth and synapse formation. It is also expressed at low levels by a subset of peripheral nervous system neurons. Within the CNS, DLAR is not expressed in all neurons, but rather a large subset. Among these CNS neurons expressing DLAR at high levels are the RP neurons, whose axons comprise much of the SNb, a subset of motor axons that innervate the ventral muscles 7, 6, 13 and 12 as well as 4, 30 and 28 (Krueger, 1996).

LAR negatively regulates Borderless to control synaptic-layer selection

Establishment of synaptic connections in the neuropils of the developing nervous system requires the coordination of specific neurite-neurite interactions (i.e., axon-axon, dendrite-dendrite and axon-dendrite interactions). The molecular mechanisms underlying coordination of neurite-neurite interactions for circuit assembly are incompletely understood. This study identified a novel Ig superfamily transmembrane protein that was named Borderless (Bdl), as a novel regulator of neurite-neurite interactions in Drosophila. Bdl induces homotypic cell-cell adhesion in vitro and mediates neurite-neurite interactions in the developing visual system. Bdl interacts physically and genetically with the Ig transmembrane protein Turtle, a key regulator of axonal tiling. These results also show that the receptor tyrosine phosphatase leukocyte common antigen-related protein (LAR) negatively regulates Bdl to control synaptic-layer selection. It is proposed that precise regulation of Bdl action coordinates neurite-neurite interactions for circuit formation in Drosophila (Cameron, 2013).

The presence of numerous axons and dendrites in the neuropils of the developing CNS makes it a daunting task for establishing specific synaptic connections. Studies over the last two decades have identified a number of cell-surface recognition molecules that mediate specific neurite-neurite interactions for circuit assembly. That many cell-surface recognition molecules are present broadly in developing neuropils throughout embryonic development, however, raises the question how the action of cell-surface recognition molecules is modulated temporally to ensure accuracy in circuit formation (Cameron, 2013).

The assembly of visual circuits in Drosophila is an attractive model for understanding the general mechanisms underlying spatiotemporal control of neurite-neurite interactions. The Drosophila adult visual system is comprised of the compound eye and the optic lobe. The compound eye consists of ∼800 ommatidia, each containing six outer photoreceptor neurons (R1-R6) for processing motion and two inner photoreceptor neurons (R7 and R8) for processing color. R1-R6 axons form synaptic connections in the superficial lamina layer, and R7 and R8 axons project through the lamina into the deeper medulla layer, where they are organized into ∼800 regularly spaced columns. Each R7 and R8 axon from the same ommatidium terminate in a topographic manner in two synaptic layers within the same column. The R8 axon terminates within the M3 layer, and the R7 axon terminates in the deeper M6 layer (Cameron, 2013).

Visual circuit assembly in Drosophila involves complex neurite-neurite interactions. Specific recognition between R-cell axons and their target layers in the optic lobe have been shown to be required for synaptic-layer selection. Visual circuit assembly also requires the interactions among R-cell axons. Selection of postsynaptic targets by R1-R6 axons in the lamina requires specific axon-axon interactions. The assembly of medulla columns requires modulation of both heterotypic and homotypic axon-axon adhesion. For instance, receptor tyrosine phosphatases LAR and protein tyrosine phosphatase 69D (PTP69D) are reported to be involved in negatively regulating the adhesion between R7 and R8 axons for facilitating R7 synaptic-layer selection. And Ig-superfamily transmembrane proteins Dscam2 and Turtle (Tutl) prevent homotypic axon-axon terminal adhesion for tiling L1 and R7 axons, respectively. The exact mechanisms by which those cell-surface recognition molecules negatively regulate axon-axon adhesion, however, remain unknown (Cameron, 2013).

The role of a novel Ig-superfamily transmembrane protein Borderless (Bdl) in Drosophila was investigated in this study. Bdl is expressed in the developing visual system, and functions as a cell-surface recognition molecule to mediate neurite-neurite interactions. The receptor tyrosine phosphatase LAR and the Ig-superfamily transmembrane protein Tutl are key regulators of Bdl-mediated axon-axon interactions in controlling synaptic-layer selection and axonal tiling, respectively. The results shed new light on spatiotemporal control of cell-surface recognition molecules for coordinating circuit assembly (Cameron, 2013).

Tiling and self-avoidance, two cellular mechanisms discovered in the early 1980s, are important for patterning neuronal circuitry. Previous studies have identified several cell-surface recognition molecules, such as Dscam, Tutl, Protocadherins, MEGF10, and MEGF11, that mediate homotypic neurite-neurite interactions in tiling and self-avoidance. These cell-surface recognition molecules may act by mediating homotypic repulsion or de-adhesion between adjacent same-type neurites. For instance, molecular and genetic analyses of fly Dscam1 support a role for Dscam1 in mediating homotypic repulsion in dendritic self-avoidance, whereas mammalian Dscams appear to mediate de-adhesion by interfering with some unknown cell-type-specific cell adhesion molecules. The exact mechanisms by which these cell-surface recognition molecules mediate homotypic repulsion or de-adhesion, however, remains elusive (Cameron, 2013).

Several lines of evidence implicate Bdl as a target of Tutl in regulating R7 axonal tiling. First, overexpression of Bdl induced an R7 tiling phenotype similar to that in tutl mutants. Second, Tutl associates with Bdl in cultured cells. And third, loss of bdl rescued the tiling phenotype in tutl mutants. It is proposed that Tutl-mediated surface recognition counteracts the affinity between adjacent R7 axonal terminals by interacting with Bdl. The association of Tutl with Bdl may downregulate the level and/or adhesive activity of Bdl, thus allowing the separation of adjacent R7 axonal terminals. Since co-overexpression of Tutl and Bdl did not affect Bdl-mediated cell-cell aggregation in culture nor the Bdl-overexpression-induced tiling phenotype in flies, it is speculated that the regulation of Bdl by Tutl requires the involvement of additional regulatory molecules. Future studies are needed to determine the exact mechanism by which Tutl downregulates the function of Bdl. It will also be of interest to determine whether other cell-surface recognition molecules implicated in tiling and self-avoidance (e.g., Dscam and Protocadherins), function similarly to modulate certain cell adhesion molecules (Cameron, 2013).

The receptor tyrosine phosphatase LAR and its mammalian homologs have been shown to play important roles in axon guidance, neuronal target selection, and presynaptic development. In the developing Drosophila visual system, LAR is required for target selection of R1-R6 axons in the lamina, and synaptic-layer selection of R7 axons in the medulla. The action of LAR in R7 synaptic-layer selection reportedly involves both stabilization of axon-target interactions and down-regulation of adhesion between R7 and R8 axons. LAR-mediated axon-target interactions may involve the binding between LAR on R7 axons and an unknown ligand in the target layer, which in turn modulates the interaction between LAR and its cytoplasmic domain-binding partner Liprin to stabilize axon-target interactions. It is also reported that LAR negatively regulates an unknown cell adhesion molecule to decrease adhesion between R7 and R8 axons for facilitating synaptic-layer selection of R7 axons (Cameron, 2013).

The current results suggest strongly that LAR downregulates adhesion between R7 and R8 axons by negatively regulating Bdl. That LAR inhibited Bdl-mediated cell-cell adhesion without affecting the level of Bdl suggests that LAR inhibits adhesive activity of Bdl. Although the role of LAR in mediating axon-target interactions requires its binding to Liprin via the cytoplasmic domain, negative regulation of Bdl by LAR appears to involve a Liprin-independent mechanism. This is supported by in vitro analysis showing that a LAR mutant lacking the cytoplasmic domain also inhibited Bdl-mediated adhesion. Consistently, a previous study showed that R8-specific expression of a truncated LAR mutant lacking the cytoplasmic domain in LAR mutants could partially rescue the R7 mistargeting phenotype. LAR may directly modulate Bdl to downregulate R7-R8 adhesion, or act indirectly by interacting with other proteins. Future studies are needed to distinguish between these possibilities (Cameron, 2013).

Although negative regulation of Bdl-mediated axon-axon interactions is necessary for R7 synaptic-layer selection and tiling, it remains unclear how the presence of Bdl contributes to the formation of the R-cell axonal projection pattern in the fly visual system. Cell adhesion molecules, such as NCAM/FasII and L1-CAM/Neuroglian, have been shown to mediate selective fasciculation in axonal pathfinding. Similarly, Bdl-mediated axon-axon interactions may facilitate the projections of R7 and/or R1-R6 axons along the pioneer R8 axon. That the R-cell projection pattern remained normal in bdl mutants may be due to the presence of redundant genes. Functional redundancy among different cell adhesion molecules seems to be common in the developing nervous system, which may account for no or subtle phenotypes in mutants defective in a number of cell adhesion molecules (Cameron, 2013).

In conclusion, this study study identifies Bdl as a novel and important regulator of neurite-neurite interactions in the developing visual system. Tuning of Bdl-mediated axon-axon interactions in axonal tiling and synaptic-layer selection presents an excellent example for modulating the action of cell adhesion molecules in ensuring accuracy in circuit assembly. It is highly likely that similar mechanisms are employed for circuit assembly in mammalian nervous systems (Cameron, 2013).

Effects of Mutation or Deletion

Most Dlar mutants proceed through embryogenesis and early larval stages and die as late instar larvae or when attempting to eclose. A significant number of larvae initiate pupation in the food, a behavior seldom seen in wild type. Mature pupae attempt to eclose and struggle for a considerable period to do so, but are unable to eclose properly (Krueger, 1996)

The neural receptor-like protein tyrosine phosphatases (RPTPs) Protein tyrosine phosphatase 69D, DPTP99A and DLAR are involved in motor axon guidance in the Drosophila embryo. The requirements for these three phosphatases have been analyzed in growth cone guidance decisions along the intersegmental nerve (ISN) and segmental nerve b (SNb) motor pathways. Approximately 40 motoneurons innervate 30 identified muscle fibers in each abdominal hemisegment of the embryo. Axons from these neurons exit the CNS via the segmental nerve (SN) and ISN roots and then extend within five nerve pathways. SNa and SNc emerge from the SN root, while SNb, SNd and the ISN arise from the ISN root.

The growth cone of the aCC neuron pioneers the ISN pathway, exiting the CNS during stage 13 and then growing dorsally past the ventrolateral muscles (VLMs) and lateral muscle 4. During stage 15, ISN growth cones contact one of the three dorsal "persistent Twist" (PT cells). These PT cells do not give rise to embryonic muscles but serve as founder cells for adult muscles (Bate, 1991). During stage 15, ISN growth cones contact one of the PT cells, PT2, and also interact with the peripheral nervous system and muscle fibers. Another PT cell, PT3, is initially located posterior and lateral to PT2, but does not appear to be contacted by the pioneer axons during outgrowth. Later, however, PT3 is contacted by a posteriorly directed side branch of the ISN, and it subsequently migrates toward the main nerve. After passing PT2, the pioneer growth cones extend under the main tracheal trunk and contact a third PT cell, PT1, as well as muscle fibers adjacent to it. By the end of stage 16, the ISN has acquired a highly stereotyped morphology as it innervates the dorsal and ventrolateral muscles, with lateral branches at the proximal edges of muscle 3 (first branch) and 2 (second branch) and a terminal arbor at the proximal edge of muscle 1, just beyond PT1. Pt3 is always at the first branchpoint. ISN axons form synapses on the dorsal muscles during stage 16 and early stage 17, with aCC innervating muscle 1 and RP2 innervating muscle 2 (Desai, 1997).

The Snb motor nerve innervates the ventrolateral muscles and contains the axons of the identified RP1, RP3, RP4 and RP5 motoneurons. RP growth cones leave the common ISN pathway at the exit junction, enter the VLM field and then navigate among the muscle fibers. Synapses form at highly sterotyped postions by late stage 16 (Desai, 1997).

Any one of the three phosphatases suffices for the progression of ISN pioneer growth cones beyond their first intermediate target in the dorsal muscle field. DLAR or Ptp69D can facilitate outgrowth beyond a second intermediate target, and DLAR is uniquely required for formation of a normal terminal arbor. A different pattern of partial redundancy among the three phosphatases is observed for the SNb pathway. Any one of the three suffices to allow SNb axons to leave the common ISN pathway at the exit junction (Desai, 1997).

When DLAR is not expressed, however, SNb axons sometimes bypass their ventrolateral muscle targets after leaving the common pathway, instead growing out as a separate bundle adjacent to the ISN. This abnormal guidance decision can be completely suppressed by also removing DPTP99A, suggesting that DLAR turns off or counteracts a DPTP99A signal that favors the bypass axon trajectory. The complete stall and bypass phenotypes observed in triple mutants might both be caused by the inability of SNb axons to defasciculate. After leaving the ISN at the exit junction, SNb and SNd axons normally follow a short shared pathway that splits at a nearby second junctions. If SNb axons successfully navigate the exit junction but then fail to leave the shared SNb/SNd pathway (defasciculate) and the abnormal combined pathway stops growing, this could produce the club-like SNb morphology characteristic of the complete stall (Desai, 1997).

The results described here indicate that DLAR might conteract or inhibit DPT99A signaling at the muscle entry point. One possible mechanism for inhibition is suggested by the three-dimensional structure of the first catalytic domain of RPTPalpha, a murine RPTPs (Bilwes, 1996). In the crystal, the RPTPalpha domains are arranged as dimers, and the N-terminal region (the so-called "wedge" region of each monomer) appears to block access to the active site of its partner. This arrangement indicates that the dimeric RPTP is likely to be catalytically inactive. Furthermore, earlier work has show that when the cytoplasmic domain of the RPTP CD45 is forced to dimerize, its activity is suppressed. These data suggest that ligand interaction with an RPTP could either suppress its enzymatic activity by forcing dimerization, or activate it by splitting a preformed dimer. The wedge region is conserved in the first PTP domains of these three Drosophila RPTPs (Bilwes, 1996), suggesting that RPTP heterodimers might also be able to form. Thus, the suppression of DPTP99A signaling by DLAR at the VLM entry point could involve the formation of a catalytically inactive DLAR/DPTP99A heterodimer (Desai, 1997).

These results show that the relationships among the tyrosine phosphatases are complex and dependent on cellular context. At growth cone choice points along one nerve, two phosphatases cooperate, while along another nerve these same phosphatases can act in opposition to one another (Desai, 1997).

Previous genetic studies of intersegmental nerve b (ISNb) development have identified several cell-surface proteins required for correct axon guidance to appropriate target muscles. Of all the proteins currently known to control ISNb guidance, Drac1 and Dlar are most likely to mediate target entry as opposed to defasciculation, because both display a parallel bypass phenotype. The small GTPase Drac1 plays a key role in this guidance process. Neuronal expression of the dominant negative mutation Drac1(N17) causes axons to bypass and extend beyond normal synaptic partners. GTPase mutations were placed under the control of the yeast transcriptional activator GAL4. Combination of a neuronal GAL4 'driver' with a GTPase cDNA 'reporter' under the control of the GAL4 upstream activator sequence (UAS) results in specific cDNA expression. Neuronal-specific expression of either Drac1(V12) or Dcdc42(V12) causes ISNb motor growth cones to arrest outgrowth just as axons begin to explore the periphery. This highly penetrant motor phenotype is analogous to the growth cone arrest observed in sensory neurons when they express the same GTPase mutations. SNa axons also require Rac to reach correct targets. The growth cone arrest seen in the V12 backgrounds suggests that hyperactivation of different GTPase pathways disrupts leading edge motility. This phenotype is consistently reproduced by pharmacological blockade of actin assembly, carried out by cytochalasin D treatment. Genetic interactions between Drac1(N17) and the receptor-tyrosine phosphatase Dlar suggest that ISNb guidance requires the integration of multiple, convergent signals. Double mutant Drac1(N17-Dlar compound mutants display a penetrance two- to three-fold higher than expected if the defects were simply additive. Thus, Rac function in ISNb axons is quite sensitive to the dosage of Dlar protein. This synergistic genetic interaction suggests that Rac and Dlar function together, though not in a simple linear pathway. The existence of multiple inputs during target entry may explain why null mutations in Dlar (and other choice point genes) are not completely penetrant on their own (Kaufmann, 1998)

The ISNb and longitudinal pathway defects observed in trio mutants are similar to those of phenotypes observed in embryos mutant for the Abl tyrosine kinase. Previous analysis has shown that a partial reduction in Abl function suppresses the bypass phenotype caused by mutations in the RPTP Dlar, implying an antagonistic relationship between kinase and phosphatase. To address trio function at this ISNb choice point, ISNb pathfinding was examined for dosage-sensitive interactions between Dlar and trio. In strong zygotic Dlar mutants, ISNb bypass was observed at a moderate frequency (18.4%, A2-A7 hemisegments). However, partial reduction of trio activity in this Dlar background enhances the ISNb bypass ~2-fold. Although this potentiation disagrees with a simple model in which trio and Abl function together to oppose phosphatase signaling, it is consistent with the observation that neural expression of Drac1N17 enhances the frequency of bypass in Dlar mutants. Thus, although trio may collaborate with Abl at the CNS midline, it rather appears to cooperate with Dlar and Drac1 during ISNb ventral target entry. The absence of bypass phenotypes in trio single mutants is likely to reflect the existence of additional inputs to Rac family GTPases that would be susceptible to the Drac1N17 dominant-negative effect (Bateman, 2000).

Regulation of actin structures is instrumental in maintaining proper cytoarchitecture in many tissues. In the follicular epithelium of Drosophila ovaries, a system of actin filaments is coordinated across the basal surface of cells encircling the oocyte. These filaments have been postulated to regulate oocyte elongation; however, the molecular components that control this cytoskeletal array are not yet understood. The receptor tyrosine phosphatase (RPTP) Dlar and integrins are involved in organizing basal actin filaments in follicle cells. Mutations in Dlar and the common ß-integrin subunit mys cause a failure in oocyte elongation, which is correlated with a loss of proper actin filament organization. Immunolocalization shows that early in oogenesis Dlar is polarized to membranes where filaments terminate but becomes generally distributed late in development, at which time ß-integrin and Enabled specifically associate with actin filament terminals. Rescue experiments point to the early period of polar Dlar localization as critical for its function. Furthermore, clonal analysis shows that loss of Dlar or mys influences actin filament polarity in wild-type cells that surround mutant tissues, suggesting that communication between neighboring cells regulates cytoskeletal organization. Two integrin alpha subunits encoded by mew and if are required for proper oocyte elongation, implying that multiple components of the ECM are instructive in coordinating actin fiber polarity. It is concluded that Dlar cooperates with integrins to coordinate actin filaments at the basal surface of the follicular epithelium. This is the first direct demonstration of an RPTP's influence on the actin cytoskeleton (Bateman, 2001).

To gain further insight into RPTP function, attempts were made to identify phenotypes caused by loss of Dlar in nonneuronal Drosophila tissues. During this analysis, an egg-shaped defect was found among late-stage oocytes in dissected mutant ovaries. In wild-type ovaries, oocytes begin as small and relatively spherical bodies and grow more elongated through their later stages. In Dlar mutants, some oocytes fail to elongate properly, such that late-stage oocytes appear rounded. This phenotype is moderate in penetrance (14.1% defective stage 14 oocytes), similar to the mild effect caused by loss of Dlar in the embryonic nervous system. No defects are found in major aspects of oocyte patterning in Dlar mutants; both the micropyle and the dorsal appendages are formed in their correct positions, although the latter are often shortened relative to those of the wild-type. Similarly, the oocyte nucleus is correctly positioned in the dorsal-anterior compartment in rounded oocytes. Thus, mutations in Dlar disrupt normal oocyte shape determination without altering the gross polarity of the oocyte (Bateman, 2001).

Previous studies suggested that insect oocyte elongation is mediated by the follicular epithelium. In particular, mutations in the 'round egg' gene kugel disrupt the polarity of follicular basal F-actin, which is postulated to restrict oocyte growth in the short axis. Follicle cell actin structures were examined in Dlar mutant egg chambers (Bateman, 2001).

Wild-type and Dlar mutant oocytes stained with Texas Red-phalloidin display actin bundles at the basal surface of the follicular epithelium throughout the vitellogenic stages of development (stages 8-13). In wild-type stage 8 oocytes, bundles are strictly perpendicular to the A-P axis. This wild-type pattern is also observed in many Dlar mutant oocytes at this stage, consistent with the partial penetrance of the elongation defect. However, in some Dlar mutant egg chambers, actin bundles are poorly organized; the strict polarization perpendicular to the A-P axis is lost, although the actin filaments that do form appear polarized within a given cell. Additionally, cell shapes are generally less regular than the wild-type array, with F-actin accumulating abnormally at the boundaries of some cells. Thus, loss of Dlar causes a distinct disruption of the actin cytoskeleton in follicle cells (Bateman, 2001).

At early stages, it is difficult to correlate cytoskeletal defects with oocyte elongation phenotypes due to the relatively spherical shape of wild-type oocytes at this time in development. However, polarized basal actin arrays are also seen at late stages (stage 12), when disruptions are observed in Dlar mutants, and oocyte elongation can be assessed. At this stage, a strong correlation is found between defects in actin polarity and a failure to elongate the oocyte in various genetic backgrounds. Mutant oocytes that elongate properly display a wild-type pattern of basal actin filaments, while oocytes that fail to elongate show disruptions in the organization of these fibers. It is concluded that Dlar functions in either establishing or maintaining the polarity of actin filaments in the follicular epithelium and thereby influences oocyte morphology (Bateman, 2001).

It was next asked whether Dlar functions within follicle cells to affect actin fiber polarity. The wild-type pattern of Dlar expression was determined by using an anti-Dlar antibody. Consistent with Dlar's role in actin filament organization, staining of stage 7-8 oocytes shows that Dlar is specifically concentrated at basal follicle cell contacts, with most of the protein localized in a polarized manner at membranes where actin filaments terminate. At medial planes of focus, Dlar is concentrated where the borders of three follicle cells meet. This site is also rich in F-actin and may represent a specialized region for cytoskeletal assembly (Bateman, 2001).

At later stages, follicle cells begin to migrate over the germ tissue, which is associated with a loss of polarized Dlar localization. Instead, oocytes at stage 10 and beyond show an even distribution of Dlar staining around the borders of cells at all planes of focus. No Dlar staining is found in germ tissues, implying a function specific to follicle cells (Bateman, 2001).

To further explore Dlar function in organizing actin, Dlar mutant clones were generated in follicle cells by using the FLP recombinase. As expected from the incomplete penetrance of Dlar nulls, many individual clones show no obvious defects in cytoskeletal organization. However, in cases where disruptions in F-actin polarity are evident, defects are always observed in both mutant cells and in wild-type cells surrounding the clone, indicating that the influence on the actin cytoskeleton is nonautonomous in Dlar mutants. Although the global organization of actin polarity is lost around these clones, actin filaments tend to be similarly polarized in adjacent cells, implying that neighboring cells influence one another's cytoskeletal organization. Similar disruptions were observed in and around mutant clones in late-stage oocytes, where they correlated with a failure to elongate the oocyte, as observed in homozygous females (Bateman, 2001).

In addition to nonautonomous influences on the cytoskeleton, defects were also observed in the normal pattern of Dlar localization in wild-type cells surrounding mutant clones; rather than showing the polarized localization observed at the basal surface of wild-type stage 8 oocytes, Dlar becomes evenly distributed around the borders of many wild-type cells surrounding mutant tissue. A failure to properly localize Dlar was occasionally observed around clones with no obvious effect on actin filaments, implying that Dlar localization is not a strict determinant of actin filament polarity. Rather, it is likely that Dlar plays a regulatory role in ensuring the fidelity of basal F-actin polarity in early follicle cells (Bateman, 2001).

The finding that Dlar influences but does not dictate basal F-actin organization led to a search for other components that may interact with actin structures. Actin filaments in Drosophila follicle cells are highly reminiscent of stress fibers, which are bundles of F-actin observed at the basal surface of many cultured cells. Stress fiber formation is dependent upon the activation of integrins, transmembrane proteins that link the actin cytoskeleton to the outlying ECM through the focal adhesion complex. To assess whether the actin bundles of follicle cells are related to stress fibers, it was asked whether Drosophila integrins play a role in their formation and maintenance (Bateman, 2001).

Wild-type oocytes stained with anti-ß-integrin show intense labeling at the basal surface throughout development, consistent with a strong association with basal actin structures and the underlying laminin-rich ECM. Higher magnification views of stage 7-8 follicles show that ß-integrin staining is somewhat diffuse at the basal surface, with significant staining over cell-cell contacts and where actin filaments terminate. Often, integrin staining is observed along an individual actin filament, reflecting a close association with the actin cytoskeleton. A similar pattern of staining is observed for the ECM component laminin, a ligand for PS1 integrin heterodimers. As with Dlar, integrin staining is intense at cell-cell contacts where 3 cells meet at this early stage. However, unlike the pattern observed for Dlar, ß-integrin remains localized to the terminals of actin bundles until late stages of oogenesis, primarily highlighting cell membranes parallel to the A-P axis. This continued association of ß-integrin with actin bundles throughout development is consistent with a role in actin filament formation and maintenance, as observed between stress fibers and focal adhesions (FAs) in cultured cells (Bateman, 2001).

The coupling of integrins to the cytoskeleton is mediated by a group of intracellular proteins that bind the cytoplasmic tails of receptors and interact with actin filaments. For example, members of the Vasodilator-stimulated phosphoprotein (VASP) family localize to FAs, where they are thought to regulate actin assembly. To explore the relationship between stress fibers and basal actin filaments of follicle cells, oocytes were stained with antibodies to the Drosophila VASP homolog Enabled (Ena). At early stages (stage 7-8), Ena is relatively diffuse throughout the cytoplasm, with little obvious concentration at cell membranes. However, at stages 10-12, significant Ena staining at actin filament terminals is observed, coinciding with the period of specific ß-integrin association with filaments. Staining was also observed along actin filaments that traverse the cell, consistent with the VASP family's proposed role in actin regulation. The association of actin bundles with multiple FA markers supports the hypothesis that the basal cytoskeleton of Drosophila follicle cells is analogous to stress fibers observed in vitro (Bateman, 2001).

Due to the association between actin filaments and ß-integrin in late-stage oocytes, it was asked if integrin staining was affected in cells within and surrounding Dlar mutant clones. Indeed, cells lacking Dlar show improper localization of integrin associated with a loss of F-actin polarity. ß-integrin staining remains highest at the filament terminals of mutant cells regardless of their orientation, resulting in a loss of staining at membranes parallel to the A-P axis. In some cases, ß-integrin staining shows no restriction to opposite sides of a cell as it does in the wild-type but instead appears generally distributed around the cell border (Bateman, 2001).

Because strong defects in ß-integrin localization are only observed in cells with actin defects, it is unclear whether these errors reflect a direct effect of Dlar on integrins or whether integrin localization is dependent upon prior orientation of F-actin by Dlar. To address this issue, it was asked whether integrins determine the polarity of basal actin filaments, which would suggest an upstream instructive role rather than a downstream dependence. Strong integrin mutants do not survive to adulthood, therefore, clones of mutant tissue were created. A screen for genes required in follicular development identified a mutation (968) with a round-egg phenotype indistinguishable from Dlar mutants; mapping data placed the insertion near the myospheroid (mys) locus encoding ß-integrin. The 968 mutant was shown to be allelic to mys by its failure to complement the allele mysnj42 and by loss of ß-integrin staining within 968 clones. As observed with Dlar, Texas Red-phalloidin staining of oocytes carrying mys968 clones shows disruptions in basal actin fiber polarity within mutant cells and in wild-type cells beyond the clonal boundary. Follicle cell clones of the independent allele mys10 also result in round eggs, confirming that integrins are required in follicle cells for elongation of the oocyte (Bateman, 2001).

Drosophila integrins function as heterodimers of a common ß subunit (mys) with a series of alpha subunits, creating functional receptors with different binding properties. For example, the alpha integrin encoded by multiple edematous wings (mew) is a receptor for laminin when combined with ß-integrin, while the alpha-integrin encoded by inflated (if) confers specificity to ECM components carrying the amino acid motif RGD. To determine if these ligands are relevant to integrin function in follicle cells, clones of if and mew were created. Rounded eggs were observed in oocytes with clones of either subunit, implying that both laminin and RGD components of the ECM are involved in mediating follicle cell control of oocyte shape (Bateman, 2001).

The similarity in phenotypes caused by loss of either Dlar or integrins suggests a cooperative relationship in organizing F-actin. To explore this, whether a genetic relationship exists between Dlar and mys was investigated. It was reasoned that the mild penetrance of round eggs in Dlar mutants may be sensitive to the dosage of genes in the same pathway. Indeed, removal of half of the gene dosage of mys in a Dlar null background causes a substantial increase in the penetrance of the round egg phenotype. For example, Dlar5.5/Dlar13.2 females show defective rounding in 14.1% of their stage 14 oocytes, while in mys1/+;Dlar5.5/Dlar13.2 females, the penetrance is increased nearly 4-fold to 48.7%. This enhancement is not due to changes in the overall fitness of mutant flies since Dlar mutants carrying the balancer TM6B, which decreases the viability of Dlar escapers, show little change in mutant oocyte penetrance (22.0% round stage 14 oocytes). Thus, genetic interactions support a model wherein Dlar and integrins cooperate in organizing basal actin filaments (Bateman, 2001).

Functions of LAR in oogenesis

The follicle cell monolayer that encircles each developing Drosophila oocyte contributes actively to egg development and patterning, and also represents a model stem cell-derived epithelium. Mutations in the receptor-like transmembrane tyrosine phosphatase Lar have been identified that disorganize follicle formation, block egg chamber elongation and disrupt Oskar localization, which is an indicator of oocyte anterior-posterior polarity. Alterations in actin filament organization correlate with these defects. Actin filaments in the basal follicle cell domain normally become polarized during stage 6 around the anterior-posterior axis defined by the polar cells (follicle cells lie at the anterior and posterior poles of ovarian egg chambers beginning at stage 3), but mutations in Lar frequently disrupt polar cell differentiation and actin polarization. Lar function is only needed in somatic cells, and (for Oskar localization) its action is autonomous to posterior follicle cells. Polarity signals may be laid down by these cells within the extracellular matrix (ECM), possibly in the distribution of the candidate Lar ligand Laminin A, and read out at the time Oskar is localized in a Lar-dependent manner. Lar is not required autonomously to polarize somatic cell actin during stages 6. Lar acts somatically early in oogenesis, during follicle formation, and it is postulated that Lar functions in germarium intercyst cells that are required for polar cell specification and differentiation. These studies suggest that positional information can be stored transiently in the ECM. A major function of Lar may be to transduce such signals (Frydman, 2001).

The structure and properties of Lar that have been revealed by studies of both the Drosophila and mammalian counterparts suggest how it might act as a polarity transducer. Lar is a receptor-like tyrosine phosphatase that is important for axon pathfinding in Drosophila. A family of closely related Lar-like phosphatases also exists in the mouse. Mouse Lar is required for the development of the mammary epithelium, while the related PTPsigma functions in neuronal and epithelial development. The Lar extracellular domain contains three immunoglobulin and several fibronectin type III domains and is thought to transduce signals via its cytoplasmic tyrosine phosphatase domains after activation by adhesion to the ECM or by small protein ligands. However, physiological Lar ligands have not been documented. In mammalian cells, binding of the ECM laminin-nidogen complex to a specific fibronectin domain in mammalian Lar causes changes in the actin cytoskeleton, suggesting that Lar can transduce information from the ECM (Frydman, 2001 and references therein).

A clue to the mechanism of Lar action comes from studies on its role in Oskar localization. Posterior follicle cells must express Lar to ensure that Oskar is localized properly at the oocyte posterior. When posterior follicle cells lack the ECM component Laminin A (LanA), Oskar localization is usually disrupted. These studies suggest that LanA and ECM mediate the posterior follicle cell-oocyte signal. As Lar has been reported to bind to the laminin-nidogen complex, Lar might act as the LanA receptor in this pathway. However, it remains less clear how a signal initiated by an interaction between LanA in the ECM and Lar on a posterior follicle cell would be transduced into the oocyte. Some LanA-containing ECM resides between the apical surface of the posterior follicle cells and the oocyte, and it has been proposed LanA interacts directly with the oocyte surface. An alternative model is proposed. LanA was observed only on the basal side of the follicle cells, and LanA clones induce round eggs. These observations and the follicle cell autonomous requirement of Lar for Oskar localization argue that the LanA signal is received by Lar on the basal surface of the follicle cells and leads to some change in the receiving cells that is transduced to the oocyte. This could be via a secondary signal, or by changes in the structural or adhesive properties of the cells that can locally affect the oocyte surface with which they come into contact. Lar mutation does not affect the apical basal polarity of follicle cells, because the apical-basal asymmetry of actin staining is maintained and multiple-layered follicle cells are never observed (Frydman, 2001).

Polar cells are likely to play a key role in polarizing actin in stage 5-6 follicle cells. Several observations support the idea that polar cells organize the actin planar polarity. Actin polarity focuses around both the anterior and posterior polar cell pairs, and spreads from the poles towards the equator of the follicle. Additionally, ectopic polar cells induced by Hedgehog (Hh) expression sometimes have actin polarizing activity. These findings suggest that polar cells send a signal that orients the actin alignment in circumferential direction. In follicles where actin fails to become aligned, the polar cell signal may have been blocked or reduced, despite the presence of morphologically recognizable polar cells. Not all the ectopic polar cells induced by Hh expression affect the actin alignment of nearby follicle cells, supporting the idea that polar cells can express differentiation markers, but still be incompetent as polarizing centers. However, Lar was not required in polar cells, because follicles with normally oriented actin are observed, despite the presence of a Lar mutant polar cell pair (Frydman, 2001).

The autonomy of the effects of Lar on Oskar localization contrasts with its apparently non-autonomous action on follicle cell planar polarity at stages 6-8. There was no relationship between actin alignment and the Lar genotype of particular somatic cells in stage 7 or later follicles. This observation can be rationalized by postulating that Lar acts on a subset of cells that is required for polar cell specification and differentiation. Lar is required in region 2b of the germarium, a time when polar cells are not yet fully specified, and in its absence somatic cell behavior and follicle formation is compromised. It is proposed that the intercyst cells interact with the polar cell precursors in a Lar-dependent manner. Intercyst cells mostly become main body follicle cells and do not remain a recognizable subpopulation; hence, it is not possible to infer their genotype from later stage egg chambers and compare it with the state of actin polarization. A correlation was noted between mutant intercyst cells and pinching defects. Thus, a relationship may exist between the intercyst cell genotype and actin polarization that cannot be followed with existing markers (Frydman, 2001).

One possibility is that Lar acts in a similar manner in intercyst cells and in posterior follicle cells. The germarium contains peripheral somatic cells whose basal actin fibers are aligned perpendicular to the AP axis. Integrin is aligned in a similar manner, suggesting that the basement membrane is correspondingly organized. As budding proceeds, Lar may be required to interpret polarity information from the basement membrane as part of the process that partitions cells into main body and polar cells. Alternatively, Lar may act in a different manner within these intercyst cells to assist in polar cell specification and differentiation (Frydman, 2001).

The Lar requirement for polar cell determination provides an explanation for another interesting fact. The phenotypic effects of Lar mutations observed are very similar to weak mutations in Notch or other Notch-pathway genes. Notch mutants, like those in Lar, cause the production of extra polar cells, interfere with egg chamber budding and disrupt the anterior-posterior axis of the oocyte. Notch signaling is required for polar cell specification. Mutations in either Lar or Notch may cause similar disruptions in polar cell differentiation and hence similar downstream effects on egg chamber development and patterning (Frydman, 2001).

These experiments suggest a novel function for the ECM during follicle cell development -- the storage of patterning information for later use. Somatic cells maintain an ECM that surrounds the germarium; when a follicle buds off, it contains a portion of this ECM in the basement membranes of its component cells. These studies emphasize that this ECM may be a repository of polarity information that is used at critical times when polarization and cell specification are taking place. During follicle budding, the AP axis of the new chamber is correlated with the differentiation of two pairs of polar cells at each terminus. An interaction between posterior follicle cells and the oocyte ensures that the germline AP axis will correspond to the somatic axis. It is suggested that at about the same time, somatic cell interactions ensure that exactly four correctly positioned polar cells differentiate per follicle. This requires Lar-dependent readouts from the same ECM that the interacting cells contributed to polarizing. In stage 8, posterior follicle cells are likewise guided to maintain localized Oskar over an appropriately sized polar region. In both cases, cells that helped synthesize an ordered ECM, later use it in a Lar-dependent manner for additional and possibly more refined patterning. The interactions studied may serve as a model for the roles of the ECM and of Lar signaling in the development of other epidermal and neural cells (Frydman, 2001).

Four receptor-linked protein tyrosine phosphatases are selectively expressed on central nervous system axons in the Drosophila embryo. Three of these (DLAR, DPTP69D, DPTP99A) regulate motor axon guidance decisions during embryonic development. The role of the fourth neural phosphatase, DPTP10D, has been examined by analyzing double-, triple-, and quadruple-mutant embryos lacking all possible combinations of the phosphatases. This analysis shows that all four phosphatases participate in guidance of interneuronal axons within the longitudinal tracts of the central nervous system. In the neuromuscular system, DPTP10D works together with the other three phosphatases to facilitate outgrowth and bifurcation of the SNa nerve, but acts in opposition to the others in regulating extension of ISN motor axons past intermediate targets. These results provide evidence for three kinds of genetic interactions among the neural tyrosine phosphatases: partial redundancy, competition, and collaboration (Sun, 2001).

Receptor-linked protein tyrosine phosphatases (RPTPs) regulate axon guidance and synaptogenesis in Drosophila embryos and larvae. DPTP52F, the sixth RPTP to be discovered in Drosophila, is described. Genomic analysis indicates that there are likely to be no additional RPTPs encoded in the fly genome. Five of the six Drosophila RPTPs have C. elegans counterparts, and three of the six are also orthologous to human RPTP subfamilies. DPTP52F, however, has no clear orthologs in other organisms. The DPTP52F extracellular domain contains five fibronectin type III repeats and it has a single phosphatase domain. DPTP52F is selectively expressed in the CNS of late embryos, as are DPTP10D, DLAR, DPTP69D and DPTP99A. To define developmental roles for DPTP52F, RNA interference (RNAi)-induced phenotypes were examined as a guide to identify Ptp52F alleles among a collection of EMS-induced lethal mutations. Ptp52F single mutant embryos have axon guidance phenotypes that affect CNS longitudinal tracts. This phenotype is suppressed in Dlar Ptp52F double mutants, indicating that DPTP52F and DLAR interact competitively in regulating CNS axon guidance decisions. Ptp52F single mutations also cause motor axon phenotypes that selectively affect the SNa nerve. DPTP52F, DPTP10D and DPTP69D have partially redundant roles in regulation of guidance decisions made by axons within the ISN and ISNb motor nerves (Schindelholz, 2001).

Ptp52F mutants display a variety of SNa guidance defects. The most common defect, as in Ptp52F RNAi embryos, is a failure to bifurcate. In other hemisegments, the SNa has extra branches, or stalls near the bifurcation point. The penetrances of such SNa phenotypes in Ptp52F18.3 homozygotes or Ptp52F18.3/Df(2R)JP4 transheterozygotes are 37% and 41%, respectively. The two other Ptp52F alleles and the transheterozygous combinations of the three Ptp52F alleles with Df(2R)JP8 have a lower penetrance of SNa defects (22-28%) (Schindelholz, 2001).

Single mutants that lack any of the other four neural RPTPs do not display SNa phenotypes. However, combinations of Rptp mutations do affect the SNa. To evaluate how removal of other RPTPs might affect Ptp52F SNa phenotypes, double mutants lacking both DPTP52F and each of the other RPTPs were made. The absence of DPTP10D, DPTP69D or DLAR increases the penetrance of the Ptp52F18.3 defects, particularly those in which the SNa stalls near the bifurcation point. No new phenotypes are observed in double mutants, however. Removal of DPTP99A does not affect the overall penetrance of SNa phenotypes, but does decrease the frequency of ectopic branches (Schindelholz, 2001).

The ISNb motor nerve innervates the VLMs and contains the axons of the identified RP1, RP3, RP4 and RP5 motoneurons. RP growth cones leave the common ISN pathway at the exit junction, enter the VLM field, and then navigate among the muscle fibers. Synapses begin to form at stereotyped positions by late stage 16. Ptp52F mutations produce any detectable ISNb phenotypes only at low frequencies (Schindelholz, 2001).

Ptp10D mutations produce no ISNb phenotypes. Removal of both DPTP10D and DPTP52F, however, generates a strong phenotype in which the ISNb stalls within the VLMs, often at the proximal edge of muscle 13. This stall phenotype is observed in Ptp52F single mutants, but its frequency can be dramatically increased in double mutants for addition of a Ptp10D mutation to the hypomorphic mutation Ptp52F8.10.3;. Removal of DPTP69D also greatly enhances the Ptp52F stall phenotype (Schindelholz, 2001).

Dlar Ptp52F double mutants have parallel bypass phenotypes identical to those of Dlar single mutants. Ptp99A mutations cause no ISNb phenotypes on their own or in combination with Ptp52F (Schindelholz, 2001).

The ISN passes its first (FB) and second (SB) lateral branchpoints before reaching the position of its terminal arbor at the proximal edge of muscle 1. In Ptp52F mutants, most ISNs are normal. Dlar mutations produce SB phenotypes with a similar penetrance (19% for null alleles). When Dlar and Ptp52F mutations are combined, the frequency of the SB termination phenotype is similar to that of the single mutants. Ptp99A mutations have no effects on ISN on their own, and also cause no enhancement of the Ptp52F phenotype (Schindelholz, 2001).

Ptp10D and Ptp69D single and double mutants have no ISN phenotypes. However, removal of either of these RPTPs from a Ptp52F mutant background enhances the penetrances of the Ptp52F ISN phenotypes. Ptp10D Ptp52F double mutants have a reduced terminal arbor (T) phenotype that is less frequently observed in Ptp52F single mutants. Removal of DPTP69D does not affect the T phenotype, but produces an increase in the SB phenotype. In summary, these results indicate that DPTP52F, DPTP10D and DPTP69D have partially redundant functions in regulation of ISN outgrowth. It is interesting that Ptp52F mutations do not produce synergistic phenotypes when combined with Dlar mutations, which are the only other Rptp mutations that generate strong ISN phenotypes on their own. Perhaps there are two separate 'functions' needed for normal ISN outgrowth, one of which involves DLAR and the other DPTP52F (Schindelholz, 2001).

DPTP52F is the only RPTP whose removal produces clear phenotypes in the 1D4-positive longitudinal bundles of the CNS. The 1D4 pathways are usually indistinguishable from wild type in single mutants lacking each of the other four RPTPs. Removal of DPTP10D or DPTP69D from a Ptp52F background strengthens the Ptp52F CNS phenotype. The longitudinal 1D4-positive bundles become more irregular, and frequent breaks and discontinuities in the middle bundle are observed. No new synergistic phenotype like that produced by removal of DPTP10D and DPTP69D together is observed. Removal of DPTP99A does not affect the Ptp52F CNS phenotype (Schindelholz, 2001).

In contrast to these results, when a Dlar mutation is introduced into a Ptp52F mutant background, the morphology of the 1D4-positive bundles reverts to wild type. In a few segments of Dlar Ptp52F double mutants, breaks in the outer 1D4-positive bundle are still seen, but defasciculation and irregularities in the inner two bundles are not observed. The suppression is specific to the CNS phenotypes detected at late stage 16, because the introduction of Dlar mutations into a Ptp52F mutant background does not correct the failure of the pCC growth cone to extend at the appropriate time. DLAR also participates in another competitive interaction: the Dlar ISNb parallel bypass phenotype is absent in Dlar Ptp99A double mutants. Here, however, it is a Dlar phenotype that is suppressed by removal of another RPTP, rather than the reverse. Ptp52F mutations do not affect Dlar parallel bypass phenotypes. Determination of the mechanisms that underlie these genetic interactions will require biochemical analysis of DPTP5F and of the signaling pathways in which it participates (Schindelholz, 2001).

Functions of LAR in R7 photoreceptor axon targeting

During Drosophila visual system development, photoreceptors R7 and R8 project axons to targets in distinct layers of the optic lobe. The LAR receptor tyrosine phosphatase is required in the eye for correct targeting of R7 axons. In LAR mutants, R7 axons initially project to their correct target layer, but then retract to the R8 target layer. This targeting defect can be fully rescued by transgenic expression of LAR in R7, and partially rescued by expression of LAR in R8. The phosphatase domains of LAR are required for its activity in R7, but not in R8. These data suggest that LAR can act both as a receptor in R7, and as a ligand provided by R8. Genetic interactions implicate both Enabled and Trio in LAR signal transduction. As a receptor in the R7 growth cone, LAR might recognize cues that promote attraction or adhesion to the target. As a ligand in R8, LAR might provide a signal that promotes the repulsion or defasciculation of the R7 growth cone from R8. Together, these two signals could help to initiate and stabilize contacts between the R7 axon and its target (Maurel-Zaffran, 2001).

The autonomous function of LAR is firmly established by two key findings: single mutant R7 axons are frequently mistargeted, and, in a homozygous LAR mutant background, transgenic expression of LAR in R7 is sufficient to restore normal axon targeting. That LAR may also have a nonautonomous function is, at this point, more speculative. Evidence in support of this idea comes from the finding that the mistargeting of LAR mutant R7 axons is more frequent when R8 is also mutant. While this is consistent with a nonautonomous requirement for LAR in R8, it might also be explained by the more effective removal of LAR function from R7 in these experiments. The case for a nonautonomous function of LAR gains further support from transgenic rescue experiments. In the LAR null mutant background, expression in R8 of either the full-length LAR protein or a truncated version lacking most of the cytoplasmic domain is sufficient to restore the correct targeting of at least some R7 axons. Again, some caution is needed. Transgenic rescue experiments, by their nature, do not necessarily tell how the endogenous LAR protein operates. For example, the level or timing of transgenic LAR expression in R8 could conceivably allow it to signal to R7, even though the endogenous protein does not. Despite these caveats, the fact that two complementary sets of observations both suggest a nonautonomous role for LAR; it may be inferred that 'reverse' signaling by LAR in R8 does indeed contribute to R7 axon target selection even under normal conditions (Maurel-Zaffran, 2001).

If this idea is correct, then presumably the R7 growth cone must have some means of detecting a LAR signal from R8 above the background 'noise' resulting from strong LAR expression throughout the medulla. This might be achieved, for example, if LAR acts in combination with other factors specific to R8. Alternatively, the LAR signal from R8 may be spatially and temporally separated from signaling events in the medulla. R7 and R8 axons are in intimate contact long before they arrive in the medulla, providing ample opportunity for a LAR signal to pass from R8 to R7 before it is drowned out in the medulla (Maurel-Zaffran, 2001).

Nonautonomous functions for LAR family RPTPs have also been observed in other systems. For example, Drosophila LAR helps to organize the structure of actin filaments in the follicle cell epithelium during oogenesis. Mosaic analysis reveals a nonautonomous requirement for LAR within the epithelium, because actin organization is perturbed not only in patches of LAR minus follicle cells, but also in the LAR plus cells that surround them. What is unclear in this case is whether this involves a nonautonomous function of LAR signaling to polar follicle cells, or merely a 'ripple effect' by which cytoskeletal disorganization spreads within the epithelium in the neighborhood of LAR minus clones. A clearer example of a LAR family RPTP acting as a ligand is provided by vertebrate PTP-delta, in this case probably through homophilic interactions. A soluble form of the PTP-delta extracellular domain can stimulate both the growth and turning of CNS neurites in vitro, evidently via distinct mechanisms (Maurel-Zaffran, 2001 and referneces therein).

It will be interesting to explore further the potential for bidirectional signaling by RPTPs and their ligands. An urgent priority in this regard is the identification of the relevant RPTP ligands. The highly penetrant and specific axon targeting defect in LAR mutants makes the Drosophila visual system an ideal setting in which to search for functionally relevant LAR ligands. Another important task for the future will be elucidating the molecular mechanisms by which LAR regulates R7 axon target selection. There are already several hints. The strong dosage-sensitive genetic interactions observed between LAR and both ena and trio point to a possible intracellular signaling pathway leading from LAR to the actin cytoskeleton. LAR may also act by modulating the activities of cell adhesion molecules, such as N-cadherin. Evidence in support of this idea comes from the similarity of the LAR and N-cadherin visual system phenotypes, and the finding that vertebrate relatives of LAR associate with the N-cadherin/catenin complex and modulate ß-catenin phosphorylation (Maurel-Zaffran, 2001).

Finding out exactly how LAR regulates photoreceptor axon targeting in Drosophila may also shed light on RPTP functions in the vertebrate visual system. Mammalian PTP-delta and chick CRYPalpha, both members of the LAR subclass of RPTPs, are also expressed on retinal axons. CRYPalpha antibodies and ectodomain fusions have been used to interfere with its function in vitro, resulting in defects in retinal axon growth cone morphology and motility that bear at least superficial similarity to the abnormalities observed for R7 growth cones in Drosophila LAR mutants. Ectodomain fusions have also been used as probes to assess the distribution of CRYPalpha ligands in the developing chick visual system. Binding partners for CRYPalpha appear to be widely distributed in the chick visual system, including the optic stalk and the retinorecipient layers of the tectum. Signaling by RPTPs may thus underlie the layer-specific targeting of retinal axons in vertebrates just as it does in Drosophila (Maurel-Zaffran, 2001).

Receptor tyrosine phosphatases regulate birth order-dependent axonal fasciculation and midline repulsion during development of the Drosophila mushroom body

Receptor tyrosine phosphatases (RPTPs) are required for axon guidance during embryonic development in Drosophila. This study examined the roles of four RPTPs during development of the larval mushroom body (MB). MB neurons extend axons into parallel tracts known as the peduncle and lobes. The temporal order of neuronal birth is reflected in the organization of axons within these tracts. Axons of the youngest neurons, known as core fibers, extend within a single bundle at the center, while those of older neurons fill the outer layers. RPTPs are selectively expressed on the core fibers of the MB. Ptp10D and Ptp69D regulate segregation of the young axons into a single core bundle. Ptp69D signaling is required for axonal extension beyond the peduncle. Lar and Ptp69D are necessary for the axonal branching decisions that create the lobes. Avoidance of the brain midline by extending medial lobe axons involves signaling through Lar (Kurusu, 2008).

The mushroom bodies (MBs) are highly conserved paired structures in the insect brain that are essential for olfactory learning and other higher-order functions. MBs vary greatly in size between insect species, but their overall organization is very similar in all insects. In Drosophila, an adult MB contains about 2500 principal neurons, known as Kenyon cells. All Kenyon cells are generated from four neuroblasts (NBs) in each brain hemisphere. Each NB produces three types of Kenyon cells (γ, α′/β′, and α/β) in a strict temporal order, and the four lineages are indistinguishable. An MB is thus a fourfold-symmetric structure. γ neurons are generated by the mid-third instar stage, α′/β′ neurons between mid-third instar and puparium formation, and α/β neurons after puparium formation (Kurusu, 2008).

Kenyon cell dendrites extend into a glomerular structure called the calyx, which receives olfactory input from the projection neurons of the antennal lobe (AL). The axons of Kenyon cells from each lineage group fasciculate into a bundle, and the four bundles merge to form the peduncle, a massive parallel tract that extends ventrally and then splits into two branches, each composed of intertwined lobes (Kurusu, 2008).

The three types of Kenyon cells differ with respect to their dendritic and axonal projection patterns in the calyx and lobes. These anatomical subdivisions are likely to be important for the analysis of complex sensory inputs, because γ, α′/β′, and α/β neurons receive projections from different sets of AL glomeruli (Kurusu, 2008).

In larvae, the axonal structures formed by the splitting of the peduncle are called the dorsal and medial lobes. Every Kenyon cell axon bifurcates during outgrowth and sends a branch into each lobe. The γ axons extend first and may serve as pathways to guide the axons of later-born α′/β′ neurons in the larva. α/β neurons follow similar trajectories during the pupal phase. The distal portions of the γ axons degenerate during pupal development, and their medial branches later regrow to form the adult γ lobe. The adult MB has a dorsal branch composed of the intertwined α and α′ lobes, and a medial branch containing the β, β′, and γ lobes. The lobes within the medial branch extend toward the corresponding lobes of the MB in the other hemisphere, but stop at the edge of a midline region that is devoid of MB axons (Kurusu, 2008).

The temporal order of Kenyon cell birth is reflected in the organization of axons in the peduncle and lobes, with the axons of the youngest neurons (the core fibers) in the center and those of older neurons arranged as a series of concentric rings around the core. The core fibers and the layers formed by older axons can be distinguished using antibody markers, GAL4 drivers, and staining for polymerized actin using phalloidin. The actin-rich core fibers are a transient population, because the ingrowth of new axons from sequentially generated later-born neurons occurs successively at the center and displaces the old core fibers outward into the innermost ring. Axons lose bright phalloidin staining as they move outward within the peduncle. This organization is consistent with the idea that the core fibers form a pioneer pathway used to guide extension of the axons of later-born neurons, which then in turn become the new core and are used as pathways by still later axons. To understand axon guidance and lobe morphogenesis during MB development, it is thus valuable to define and genetically characterize receptors and adhesion molecules expressed on these core fibers (Kurusu, 2008).

This study shows that four receptor tyrosine phosphatases (RPTPs) are selectively expressed on core fibers within the peduncle and lobes, and that RPTP signaling regulates segregation of core fibers into a single bundle. RPTPs have cellular adhesion molecule (CAM)-like extracellular regions containing immunoglobulin (Ig) domains and/or fibronectin type III (FN3) repeats. The ligands that interact with these extracellular domains are largely unknown. However, vertebrate and Drosophila Lar RPTPs bind to heparan sulfate proteoglycans (HSPGs), and the cell-surface HSPG Syndecan (Sdc) contributes to Lar's functions during axon guidance and synaptogenesis (Kurusu, 2008).

The four RPTPs localized to core fibers are Lar, Ptp10D, Ptp69D, and Ptp99A. They are all selectively expressed on central nervous system (CNS) axons in the embryo. There are a total of six RPTPs encoded in the Drosophila genome. The other two RPTPs, Ptp52F and Ptp4E, were not studied because Ptp52F mutants die as embryos and Ptp4E mutants have not been characterized. Also, good antibodies against these two proteins are not available (Kurusu, 2008).

RPTPs have been extensively analyzed as regulators of motor axon guidance in the embryonic neuromuscular system. Each guidance decision made by motor axons can be defined by a requirement for a specific subset of the five RPTPs that have been examined. There is substantial redundancy among RPTPs, so that high-penetrance alterations in a guidance decision are usually observed only when two or more RPTPs are removed. For example, axons of the ISNb nerve fail to leave the common ISN pathway and thus remain fasciculated to ISN axons ('fusion bypass' phenotype) when Lar, Ptp69D, and Ptp99A are all absent. When only Lar is missing, ISNb axons leave the ISN but then sometimes fail to enter their target muscle field (Kurusu, 2008).

RPTPs also regulate CNS axon guidance in a redundant manner. A subset of longitudinal axons abnormally cross the midline in Ptp10D Ptp69D double mutants, while all longitudinal axons are rerouted into midline-crossing commissural pathways in Lar Ptp10D Ptp69D Ptp99A quadruple mutants. All four RPTPs thus participate in midline crossing decisions in the embryo. This paper shows that Lar regulates avoidance of the brain midline by MB axons in the larval brain, and that high-penetrance ectopic crossing phenotypes are observed when only Lar is absent (Kurusu, 2008).

RPTP function has also been characterized during later development and in adulthood. Lar mutants have reduced numbers of boutons at neuromuscular junctions (NMJs) in larvae, and Lar and Ptp69D mutants display alterations in photoreceptor axon guidance and synaptic maintenance in the optic lobes. Finally, hypomorphic Ptp10D mutants are defective in long-term memory formation, and this phenotype can be rescued by restoration of Ptp10D expression in the MB or by acute induction of Ptp10D in adults. This result indicates that RPTPs are likely to regulate synaptic function in mature animals. Consistent with this, this study shows that all four RPTPs are expressed in specific patterns within the neuropil of the adult brain (Kurusu, 2008).

This paper demonstrates that RPTPs are selectively expressed on the core fibers of the MB. At any one time during larval development, the core fiber bundle is composed of the youngest axons within the peduncle and lobes. Core fibers are likely to serve as pathways for guidance of axons of later-born neurons. These later axons then become the new core fiber bundle and displace the old core fibers outward into concentric rings (Kurusu, 2008).

Two of the RPTPs, Ptp69D and Ptp10D, are expressed only on the innermost core fibers, while Lar and Ptp99A are expressed within an expanded core region including somewhat older axons. The data indicate that Ptp10D and Ptp69D are involved in segregation of core fibers. Split-core phenotypes, in which two or more phalloidin-rich, OK107::mCD8-GFP-low bundles are observed within the peduncle, are observed in MBs lacking Ptp69D function. They were also observed in larvae hemizygous for a complete deletion of Ptp10D and the adjacent bif gene, Δ59. The split-core phenotype occurs only when both Ptp10D and Bif are absent. These two genes have been shown to genetically interact in other contexts (Kurusu, 2008).

Ptp69D is also required for outgrowth of axons from the peduncle into the lobes, because regions near the junctions of the peduncle and the lobes are expanded at the expense of the lobes in MBs lacking Ptp69D function. Another distinctive phenotype seen in these experiments is abnormal extension of medial lobe axons across the brain midline. This is seen in Lar mutant larvae and in larval and adult Lar mutant NB clones (Kurusu, 2008).

Prior to this work, only two genes had been identified as having mutant phenotypes affecting core fiber segregation. Both of these encode adhesion molecules. mRNA from the Dscam gene, which encodes homophilic Ig domain CAMs, is alternatively spliced to generate up to 38,000 different protein isoforms. Like the RPTPs, Dscam is selectively expressed on young axons that travel within the core fiber bundle. Dscam mutant MBs and NB clones exhibited multiple core fiber bundles within the peduncle. The similarities between Dscam, Ptp69D, and Ptp10D phenotypes and expression patterns suggest that these RPTPs could be involved in Dscam signaling during outgrowth of young axons within the peduncle. Alternatively, the RPTPs could regulate adhesion via other pathways that also affect core fiber segregation and are partially redundant with Dscam (Kurusu, 2008).

Core fiber segregation defects were also observed in ~25% of NB clones bearing null mutations eliminating expression of the homophilic Ig-domain CAM FasII. FasII, unlike Dscam and the RPTPs, is expressed only on older axons outside the core region, and engagement between FasII molecules on different cell surfaces is thought to trigger adhesion rather than repulsion. This suggests that core fiber segregation defects in FasII mutants arise by a different mechanism. Perhaps older axons lacking FasII fail to adhere sufficiently to each other and thus open passageways that allow young axons to pioneer multiple pathways within the peduncle (Kurusu, 2008).

Mutants lacking expression of Lar exhibit phenotypes in which one or both of the medial lobes of the bilaterally symmetric MBs extend across the midline, so that they appear fused to one another. It was also found that Lar mutant NB clones extended axons across the midline into the territory occupied by the other MB. These phenotypes suggest that a repulsive signal emanates from the brain midline region, and that Lar participates in reception of this signal by growing MB axons within the medial lobe (Kurusu, 2008).

This is formally similar to repulsion from the CNS midline in the embryo, where binding of midline Slit to Roundabout (Robo) receptors on neuronal growth cones causes them to navigate away from the midline. Axons abnormally cross the midline in Ptp10D Ptp69D double mutant embryos, and genetic interaction studies showed that these RPTPs are positive regulators of Robo signaling in the embryo. Lar is also implicated in the decisions of axons to cross the embryonic midline (Kurusu, 2008).

The MB midline crossing phenotypes seen in Lar mutants are probably not mediated through alterations in Slit-Robo signaling. Robo1, 2, and 3 mutant phenotypes in the larval MB have been described, and they do not include fusion of medial lobes across the midline. The Lar ligand Sdc is not selectively expressed at the brain midline, so binding of Lar to Sdc probably does not trigger repulsion of MB axons. Perhaps other, as yet unidentified, Lar ligands are expressed in the midline region, and interactions between these ligands and the RPTP facilitates repulsion. Studies showed that Lar binds to at least one non-HSPG ligand in the embryo (Kurusu, 2008).

A number of other genes that regulate repulsion of MB axons from the midline have been identified in other studies. Mutations in derailed (linotte), which encodes a protein related to Ryk receptor tyrosine kinases, cause overgrowth of medial lobes across the midline. The Derailed receptor responds to a Wnt5 signal, and mediates exclusion of axons from posterior commissural pathways that cross the embryonic midline. Medial lobe fusion across the midline is also observed in fmr1 mutants and in FMR1 overexpression animals. FMR1 is an RNA-binding protein that is orthologous to the human gene affected in Fragile X mental retardation syndrome (Kurusu, 2008).

Other genes potentially involved in repulsion from the brain midline were identified in a microarray screen for MB-expressed genes. ML fusion was observed in some larvae when expression of these genes was inhibited using RNAi methods. The gene with a known function for which RNAi produced ML fusion with the highest penetrance is CG6083, encoding an aldehyde reductase. RNAi for a gene encoding a cGMP phosphodiesterase also produced ML fusion. cGMP signaling has been implicated in growth cone repulsion in vertebrate neuronal cultures (Kurusu, 2008).

The work described in this paper shows that four RPTPs are localized to growing MB axons and are important for the creation of the distinctive architecture of the MB's axonal network. Examination of Rptp mutant phenotypes shows that these CAM-like signaling molecules control several different axon guidance decisions that occur during outgrowth. Ptp10D and Ptp69D regulate segregation of the growing axons into a single core bundle within the peduncle. Ptp69D and Lar are necessary for the later axonal extension and branching events that create the dorsal and medial lobes. Medial lobe axons cease outgrowth before they reach the brain midline, and their decision to stop involves Lar (Kurusu, 2008).

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).

Leukocyte-antigen-related-like/Dlar: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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