P.R. is supported by the NIH grants R01 DA023999 and R01 NS014841 and the Kavli Institute for Neuroscience at Yale. P.R. also thanks members

of his lab for helpful discussions. D.H.G. is supported by NIH/NIMH grants R01 MH100027 (ACE Network Award), R37 MH060233 (MERiT Award), P50 HD055784 (ACE Center Award), and R01 MH094714, and Simons SFARI Award 206744. D.H.G. thanks T. Grant Belgard, PhD, for very helpful discussions and construction of Table 2 and Lauren Kawaguchi for editorial assistance. “
“It is now clear that individual GSK1120212 order neurons are highly compartmentalized with specific functions and/or signaling that occur in restricted subcellular domains. Extrinsic signals are often spatially localized such that they are “seen” by restricted parts of a neuron, such as synaptic input to a specific dendritic spine or a guidance cue encountered by a growth cone. Twenty-five years ago, when the first issue of Neuron was published, it was well appreciated that the neurons were capable of local information processing, but the potential cellular mechanisms that established and regulated local compartments were not well understood. Dendritic spines had been proposed as biochemical and/or

electrical compartments ( Harris and Kater, 1994 and Koch and Zador, 1993), and polyribosomes had been identified at the base of spines ( Steward and Levy, 1982). However, the view that dominated until nearly

the end of the twentieth century was that the central dogma (DNA-RNA-protein) was carried PI3K inhibitor out centrally—in the nuclei and somata of neurons. In that context, the localization of mRNA observed in some cells was thought to represent a specialized mechanism that operated in unique biological systems, such as egg cells, where storage of mRNAs is needed for subsequent patterning of the early embryo (see Martin and Ephrussi, 2009 for review). Evidence from a number of studies in the last decade, particularly in neurons, has led to a revolution in our thinking. Although the field is still young, it is becoming clear that RNA-based mechanisms provide a highly adaptable link between extrinsic signals old in the environment and the functional responses of a neuron or parts of a neuron. This is accomplished by the localization of both protein-coding and noncoding RNA in neuronal processes and the subsequent regulated local translation of mRNA into protein. Here we discuss some of the key findings that lead us to the view that mRNA localization and RNA-regulated and localized translation underlie many fundamental cellular processes that are regulated by extrinsic signals in neurons, such as memory, dendrite and arbor branching, synapse formation, axon steering, survival, and likely proteostasis. The dynamic regulation of protein synthesis is essential for all cells, including neurons.

To estimate the endogenous mRFP-gephyrin numbers at synapses in vivo, we conducted

decay recordings on fixed spinal cords from 3-month-old KI animals. The tissue was frozen and sliced in sucrose to preserve the mRFP fluorescence (Figure 5A). Unexpectedly, the numbers of clustered gephyrin molecules in spinal cord slices were much higher than in cultured neurons (mean 477 ± 16 molecules, n = 666 clusters from six spinal cord slices; Figure 5B). This disparity could selleck products be attributed either to the size of the gephyrin clusters or to the density of clustered molecules. In order to distinguish between these possibilities, we reconstructed PALM-like images from the detections of blinking mRFP fluorophores at the end of the photobleaching recordings (referred to as nonactivated PALM, or naPALM). The molecule numbers could then be related to the cluster

sizes in the rendered pointillist images (Figure 5C). This analysis showed that gephyrin clusters were, on average, somewhat bigger in spinal cord slices (0.061 ± 0.005 μm2, n = 44 from three slices) than in cultured neurons (0.048 ± 0.002 μm2, n = 115, 11 cells, three experiments). However, this difference was not very pronounced and was partly due to the fact that gephyrin clusters in slices were more often composed of subdomains that may be considered as separate entities. This fits with previous observations that the Bumetanide size of spinal cord synapses Selleckchem MLN0128 varies over a wide range and that larger PSDs have more complex shapes

(Triller et al., 1985 and Lushnikova et al., 2011). However, we did observe strong differences regarding the molecule density of gephyrin clusters in adult slices (12,642 ± 749 molecules/μm2) as opposed to cultured neurons (5,054 ± 260 molecules/μm2), suggestive of a greater maturity of inhibitory PSDs in native tissue. We thus looked at the temporal profile of gephyrin clustering during postnatal development. The number of mRFP-gephyrin clusters in 1-μm-thick cortex and spinal cord slices increased with age, reaching about 0.1 clusters/μm2 in adult gray matter (Figure S2A). Surprisingly, the number of mRFP-gephyrin molecules at these clusters differed substantially between mature synapses in spinal cord and cortex (at 6 months), with a mean of 393 ± 19 and 133 ± 10 molecules, respectively (nspc = 427 and ncor = 264 clusters from six or more slices; Figure S2B). Thus, in addition to temporal changes, other factors clearly regulate gephyrin scaffolds. Speculating that the inhibitory receptor types expressed in spinal cord and cortex may have something to do with this, we visualized endogenous GlyRα1 subunits in 6-month-old cortex and spinal cord slices by immunohistochemistry (Figure 5D). Whereas no GlyRs were detected in cortex, many of the PSDs in spinal cord were positive for GlyRα1.

Active boophilin and D1 were efficiently expressed in P. pastoris and purified in a single step by affinity chromatography. Purified recombinant boophilin strongly inhibited HSP inhibitor thrombin, with a dissociation constant in the pM range. Moreover, it also displayed considerable activity against

trypsin (Ki 0.65 nM) and neutrophil elastase (Ki 21 nM). As for purified recombinant D1, it displayed an inhibitory activity against trypsin similar to that of the full-length inhibitor (Ki 2 nM), and also inhibited neutrophil elastase, although with a significantly decreased efficiency (Ki 0.129 μM), suggesting a significant contribution from the C-terminal Kunitz domain to this interaction, compatible with the presence of an alanine residue in the reactive loop P1 position. The three-dimensional structure of the thrombin-boophilin complex revealed a bidentate interaction of boophilin with the active site and the exosite I of α-thrombin. The N-terminal region of the inhibitor binds to and blocks the active site of thrombin while the negatively charged C-terminal Kunitz domain of boophilin docks into the basic exosite I ( Macedo-Ribeiro et al., 2008).

As expected from the thrombin-boophilin complex architecture, isolated D1 does not display inhibitory activity against thrombin, confirming the fundamental contribution of the C-terminal domain-mediated interaction for thrombin inhibition. Further highlighting the importance of the exosite I for thrombin inhibition, Selleckchem RO4929097 boophilin inhibited strongly α-thrombin in vitro but was unable to inhibit the exosite I-disrupted form of the enzyme, γ-thrombin. In contrast to other previously described natural thrombin inhibitors from blood-sucking animals, boophilin may also target additional serine proteases such as trypsin and plasmin (Macedo-Ribeiro et al., 2008). The observed activity of boophilin against neutrophil

elastase corroborated this hypothesis, suggesting a role other than counteracting blood coagulation in the midgut of R. microplus. Blood is a complex mixture of numerous soluble proteins, including plasmin precursor plasminogen, and of different cells, among which the elastase-producing neutrophils. Carnitine palmitoyltransferase II In ticks, blood digestion lasts for several days, during and after the engorgement process, and it is therefore conceivable that boophilin might be used to control any plasmin or elastase activity arising in the midgut during this period, even when complexed with thrombin, avoiding unwanted tissue damage. Boophilin amino acid sequence is 37% identical to that of hemalin (Liao et al., 2009), a thrombin inhibitor described in the tick Haemaphysalis longicornis. However, while hemalin was expressed in all major tissues (including salivary glands, midgut, hemocytes and fat body) of adult female ticks, boophilin was exclusively expressed in the midgut, suggesting an important role in this organ.

These results indicate that the amplitude increase in KO explants is caused by VIP-dependent enhancement of coupling in SCN cells. Next, to test whether the VPAC2 antagonist can reverse the faster entrainment behavioral phenotype in 4E-BP1 null mice, Eif4ebp1 KO mice were infused with PG99-465 (100 μM, 4 μl) or vehicle (physiological saline, 4 μl) into the lateral ventricle at ZT15, before the light cycle was advanced for 6 hr (light on at ZT18). Both groups of mice re-entrained to the new LD cycle. Notably, however, the mice infused with PG99-465 re-entrained more slowly than those Epigenetic Reader Domain inhibitor infused with saline ( Figure 6G).

From day 2 to day 4 following the LD cycle shift, PG99-465-infused mice exhibited a smaller phase advance than control

(PG99-465 versus vehicle, p < 0.05, ANOVA, Figure 6H). Together, these results demonstrate that VIP overexpression in the SCN underlies the phenotypes of Eif4ebp1 KO mice. mTOR phosphorylates 4E-BP1 and decreases its translational inhibitory activity in the SCN (Cao et al., 2008). To corroborate Baf-A1 molecular weight the regulation of VIP and the clock function by 4E-BP1, we utilized an Mtor+/− mouse strain. In the Mtor+/− SCN, VIP was decreased by ∼50% (Mtor+/− versus Mtor+/+, p < 0.05, Student’s t test; Figure 7A). In the Mtor+/− brain, mTOR activity was decreased, as indicated by decreased phosphorylation of 4E-BP1 (normalized band intensities: Mtor+/− versus Mtor+/+, at Thr70: 0.69 ± 0.04 versus 1 ± 0.07; at Ser65: 0.67 ± 0.07 versus

1 ± 0.19, p < 0.05, Student’s t test), and prepro-VIP was reduced (normalized band intensities: Mtor+/− Oxyphenisatin versus Mtor+/+, 0.43 ± 0.08 versus 1 ± 0.08, p < 0.05, Student’s t test; Figure 7B). To investigate the effects of lower VIP level on the circadian clock function, we monitored circadian behavior of the Mtor+/− mice in LL. Mice were housed in regular cages in LL (200 lx) for 14 days and then transferred to cages equipped with running wheels in LL (55 lx) to record their circadian behavior for 14 days. LL induced three types of behavior (R, AR, and WR) in both Mtor+/− and Mtor+/+animals. A larger percentage of Mtor+/− mice (47.4%, 9/19) exhibited arrhythmic behavior than did Mtor+/+ mice (16.7%, 3/18) (p < 0.05, χ2 test; Figures 7C and 7D), indicating increased susceptibility to LL-induced clock desynchrony in Mtor+/− mice. Taken together, the results demonstrate that mTOR/4E-BP1 signaling bidirectionally regulates VIP level and susceptibility of the SCN clock to desynchronizing effect of LL. In the present study, we found that mTOR signaling promotes Vip mRNA translation by repressing 4E-BP1. Consequently, in Eif4ebp1 KO mice, VIP is increased in the SCN, which is associated with a larger amplitude of PER2 rhythms, accelerated circadian clock entrainment, and enhanced synchrony.

LFP samples neurons over a 300- to 400-μm-wide region (Katzner et al., 2009), so our positive control assesses the synaptic input to the vast majority of neurons in a barrel (∼200–300 μm wide). Amplitudes of sensory-evoked Rapamycin LFPs were proportional to velocity (Figure 4B, middle, black). In individual experiments (Figures S4D and S4E) as in the average (Figure 4B, n = 5), cholinergic blockers consistently decreased LFP responses across velocities (red) with no effect of artificial cerebrospinal fluid (aCSF; green). LFP time course was also impacted by blockers but not vehicle (Figure 4B, right). Blockers ejected 250 μm from the LFP pipette similarly

reduced responses (Figure S4F), indicating that drugs impacted an area of at least an entire barrel. We conclude that cholinergic receptors in rat barrel cortex modulate sensory responses and are antagonized by our local perfusion method. Together, these results show that ACh is not necessary to

produce awake patterns of Vm in cortical neurons. The locus coeruleus (LC)-norepinephrine (NE) system is also a plausible mechanism of the switch in cortical dynamics. Pharmacologically stimulating LC desynchronizes EEG (Berridge et al., 1993), and the firing rates of noradrenergic LC neurons change with arousal (Aston-Jones and Bloom, 1981). To examine a possible role of NE, we initially locally perfused 1 mM antagonists of α1 (prazosin), α2 (yohimbine), and β (propranolol) click here noradrenergic receptors while recording from L4 neurons with thalamus intact. This high concentration prevented cells from achieving/maintaining prolonged depolarization under both anesthesia and wakefulness (Figure 5A, Figure S5A). Ipsilateral LC lesion also prevented sustained depolarization (Figure 5B), indicating that our pharmacology results were due to NE receptor blockade rather than nonspecific drug

effects. Thus, some minimal amount of NE appears required for prolonged depolarizations normally observed during sleep/anesthesia, consistent with tonic LC firing under these conditions (Aston-Jones and Bloom, 1981). We predicted that clear slow-wave fluctuations selleck kinase inhibitor should emerge in awake animals for low levels of NE. To test this, we locally perfused lower concentrations of antagonists in L4 barrels, again after thalamic lesion to ensure that measurements reflected synaptic input from the local network and not thalamic afferents (Figure 5C, left). A wide range of concentrations of NE blockers (1–100 μM; Figure S5B) were sufficient to induce periodic synaptic quiescence in awake animals (Figure 5C). In stark contrast to ACh antagonists, NE blockers induced clear bimodality of cortical Vm during wakefulness (Figure 5D). Under NE blockade, wakefulness and anesthesia had comparably long quiescent states (Figure 5E, red; n = 7, p = 0.69; Figure S5B, right), whereas perfusion of DMSO vehicle resembled control (green; n = 5).

We next wanted to assess whether target-derived BDNF has a physiological role in regulating the levels of SMAD1/5/8 in axons in developing embryos. During early embryonic development, BDNF expression is principally localized to the maxillary and ophthalmic mesenchyme, with highest expression toward the

epithelium, but is absent from the mandibular mesenchyme (Arumäe et al., 1993 and O’Connor and Tessier-Lavigne, 1999). The absence of BDNF in the mandibular mesenchyme matches with the absence of SMAD1/5/8 from the mandibular branch in E12.5 mouse embryos (Figures 3A and S3A). This correspondence suggests a PF2341066 causal role for BDNF in controlling axonal SMAD1/5/8 levels in vivo. To determine if BDNF physiologically regulates axonal SMAD levels, we examined axonal SMAD levels in maxillary and ophthalmic axons of the trigeminal ganglia in E12.5 BDNF−/− mouse embryos. CDK and cancer BDNF−/− embryos exhibit normal trigeminal ganglion development, as well as normal trigeminal axon growth and pathfinding in early embryonic development ( Ernfors et al., 1994 and O’Connor and Tessier-Lavigne, 1999). While SMAD1/5/8 was readily detectable in

maxillary axons of BDNF+/− littermate control embryos, axonal SMAD1/5/8 levels were markedly reduced in BDNF−/− embryos ( Figures 8A and S8A–S8C). Similarly, SMAD1/5/8 levels were markedly reduced in the ophthalmic bundle in BDNF−/− embryos compared to BDNF+/− littermate controls ( Figure S8D). These data suggest that target-derived BDNF physiologically regulates the expression of SMAD1/5/8 in axons. Our experiments using cultured neurons suggest that BDNF promotes the ability of BMP4 to retrogradely induce the expression of Tbx3, a positional identity marker for maxillary/ophthalmic trigeminal neurons. CYTH4 To further examine this idea, we asked whether mandibular neurons can be induced to express maxillary/ophthalmic positional

identity markers. Explants derived from either the maxillary/ophthalmic or the mandibular portion of E13.5 rat trigeminal ganglia were cultured in microfluidic chambers. Application of BDNF/BMP4 to the axonal compartment led to Tbx3 expression in both maxillary/ophthalmic and mandibular explants ( Figure S8E). These results suggest that the mandibular neurons have the capacity to express maxillary/ophthalmic positional identity markers, but most likely do not because they are not physiologically exposed to BDNF and BMP4. To address the physiological role of BDNF in regulating the patterning of the trigeminal ganglia, we examined positional identity markers in BDNF−/− embryos. In E12.5 control (BDNF+/−) embryos, pSMAD1/5/8 and Tbx3 are highly expressed in the nuclei of maxillary- and ophthalmic-innervating neurons of the trigeminal ganglia ( Figure 8B).

Figure 2B shows corresponding results for feature-based coding. These cells encoded the conjunction of relative magnitude with color and/or shape, although for convenience we refer to them by color. The scatter plot shows each cell’s preference for higher-magnitude red stimuli (positive values) or higher-magnitude blue stimuli (negative values). As with order-based

magnitude coding, only a minority of cells (31%) encoded relative magnitude in both tasks, but of those 76 cells, 73 (96%) had the same preference in both tasks (inset of Figure 2B, dark blue bar). Figure 2B2 shows that among cells with significant coding in both tasks, there was a strong correlation in preferences (r = 0.81, p < 0.001). Figure 2B3 shows an analogous comparison for the BMS-754807 cell line duration and matching tasks. Of the 76 cells with significant feature-based magnitude coding in both tasks, 51 were also tested in the matching task. Of these 51 cells, 47 (92%) shared the same feature preference in the matching task as in both discrimination tasks. Figure S4B2 shows the same data as a normalized index. Because the matching task did not require any decisions about magnitude, we conclude that these cells encoded the nonspatial goal chosen by the monkey on each trial: red or blue. Cells with check details significant relative-magnitude

coding in both main tasks showed a strong correlation between the duration and matching tasks (r = 0.95, p < 0.001), as well as between the distance and matching tasks (r = 0.81, p < 0.001). For the 37 neurons with significant effects in all three tasks, these correlations were r = 0.85, r = 0.97, and r = 0.86, respectively,

for duration versus distance, duration versus matching, and distance versus matching (p < 0.001). Because the monkeys could not know which response to make until the two stimuli reappeared at the end of the D2 delay period (target on, “go”), the goal representation during the decision period specified the object that served as the target of a response and not the motor response per se or the spatial goal. Thus, of the cells showing feature-based coding ( Figure 2B), we found three separate populations of neurons: cells that encoded conjunctions of features with relative distance (e.g., red-farther), click here cells that encoded conjunctions of features with relative duration (e.g., red-longer), and cells that encoded the chosen goal (e.g., a red target stimulus in all three tasks). Figure S3B shows a neuron with magnitude coding specific to the duration task, Figure S3C shows one for the distance task, and Figure S3D shows a cell that encoded its preferred goal in all three tasks. Figure S4 confirms these results for normalized indices. Figure 3 examines whether the properties just described for the decision period persisted through the S2 and D2 periods.

, 1998, Lu et al., 2001, Patterson Tenofovir price et al., 2010 and Yang et al., 2008). Here we focus on the role of postsynaptic complexin, which

unlike core SNARE proteins is not generally involved in membrane fusion events but is specifically required for calcium-dependent synaptic vesicle exocytosis. Although mice lacking complexin-2 have been reported to exhibit impaired LTP (Huang et al., 2000 and Takahashi et al., 1999), the interpretation of this result is ambiguous since effects on transmitter release during LTP induction cannot be ruled out. Using viral-mediated expression of shRNAs to complexin-1 and -2 in vivo, we find that knockdown of complexin-1 and -2 in hippocampal CA1 pyramidal cells impairs LTP without detectably altering basal synaptic transmission. Rescue experiments reveal that the postsynaptic function of complexin in LTP requires binding to SNARE complexes and its N-terminal activation domain. Identical results were obtained in a culture model of LTP in which NMDAR-triggered trafficking of AMPARs to synapses was assayed. In forebrain neurons, complexins function in presynaptic Dolutegravir research buy vesicle exocytosis with synaptotagmin-1, but we find that postsynaptic synaptotagmin-1 is not essential for LTP. Together, these results suggest that the mechanisms underlying regulated

postsynaptic exocytosis of AMPARs during LTP are unexpectedly similar to those regulating presynaptic vesicle exocytosis in that both require complexins. However, the requirement for synaptotagmin-1 in calcium-triggered presynaptic vesicle exocytosis but not for AMPAR delivery during LTP indicates that complexins act in conjunction with distinct regulators on the pre- versus postsynaptic sides Sarcosine oxidase of excitatory synapses. To test the postsynaptic

role of complexin in modulating excitatory synaptic transmission, we used a lentiviral molecular replacement strategy. Consistent with previous work (Maximov et al., 2009), simultaneous expression of two shRNAs targeted to complexin-1 and -2 (shCpx1/2) and complexin-2 alone (shCpx2) in a multipromoter lentivirus (Figure 1A) efficiently knocked down endogenous complexin-1 and -2 (Cpx KD) in dissociated cultured neurons (Figure 1B; Figure S1 available online), resulting in a dramatic decrease in evoked EPSCs (Figure 1C). This presynaptic effect on evoked synaptic transmission in cultured neurons was rescued by simultaneous expression of an shRNA-resistant complexin-1 fused to the GFP variant, Venus, at its N terminus (Figures 1A–1C). In contrast, a mutant form of complexin-1 (Cpx14M) that is unable to bind SNARE complexes due to four amino acid substitutions in its central α-helix domain (R48A/R59A/K69A/Y70A) (Maximov et al., 2009) did not rescue evoked EPSCs (Figures 1A–1C). These results demonstrate the effectiveness and specificity of the lenitiviral Cpx KD and confirm that the interaction of complexin with the SNARE complex is required for controlling presynaptic vesicle fusion.

Subsequently, stimulation was discontinued and responses OSI-906 nmr at the active port had no effect (“extinction”). After a further 30 min had elapsed, brief “priming” stimulation trains were delivered to indicate to the rat that stimulation was once again available (“reacquisition”). We found that Th::Cre+ rats rapidly extinguished and then reacquired responding for DA ICSS, performing significantly fewer active nosepokes during extinction as compared to both maintenance and reacquisition (two-tailed Wilcoxon signed-rank test;

p < 0.01 for maintenance versus extinction, p < 0.05 for extinction versus reacquisition, Figures 6F and 6G). The extinction of active responding was rapid; within 5 min after extinction onset, rats had decreased their average rate of responding at the active nosepoke to less than 10% of the rate sustained during maintenance. Importantly, by the last 5 min of the extinction phase Th::Cre+ rats no longer responded preferentially at the active nosepoke ( Figure 6H), instead responding

at equivalently this website low levels at both active and inactive nosepoke ports. Next, we asked whether the contingency between behavioral responses and optical stimulation was required to sustain responding. Rats were allowed to respond for stimulation over 30 min (“maintenance”), followed by a period of contingency degradation (“CD”) during which stimulation trains were delivered pseudorandomly at intervals matched to the average rate at which they were earned by each rat during FR1 responding in a previous session. Rats could continue to respond at the active port during this phase, but the delivery of stimulation trains occurred independently of these responses. After 30 min had passed, noncontingent stimulation ceased and reinforcement was once again made contingent on responses Vorinostat (SAHA, MK0683) in the active port (“reacquisition”). We found that Th::Cre+ rats were sensitive to degradation of the contingency between response and reinforcement, as they performed significantly fewer active nosepokes during CD than they had during maintenance (two-tailed Wilcoxon signed-rank test,

p < 0.01; Figures 6I and 6J) despite the fact that the number of stimulation trains delivered did not differ across the two epochs (two-tailed Wilcoxon signed-rank test, p > 0.05, Figure 6J). Interestingly, by the last 5 min of the CD phase Th::Cre+ rats still showed a small but significant preference for responding at the active nosepoke ( Figure 6K). Additionally, on average rats increased responding at the active port during reacquisition, although when summed across the 30 min epoch this change was not statistically significant (two-tailed Wilcoxon signed-rank test, p > 0.05; Figure 6J). Together, the extinction and contingency degradation manipulations demonstrate that the robust maintenance of Th::Cre+ rat responding at the active port arises from response-contingent optical stimulation of DA neurons.

Interestingly, one is Ptp10D itself, indicating that the Ptp10D XC domain may have homophilic

binding activity. This was also observed in a cell aggregation assay (see below). sas encodes two cell-surface proteins of 1,693 and 1,348 amino acids (aa). The mRNA for the larger isoform includes an alternatively spliced exon encoding a 345 aa sequence that is inserted at aa 929. Sas proteins have two XC domains with defined structures: a von Willebrand factor type C (VWC) domain (aa ∼750–825) and two FN3 repeats (aa ∼1,400–1,600). There is a single predicted transmembrane domain, and a short cytoplasmic domain of 37 aa ( Schonbaum et al., 1992). Western blotting of Sas from embryos reveals multiple bands between 100 kD and 220 kD, this website suggesting that there are extracellular Sas proteins that lack transmembrane and cytoplasmic sequences. Sas does MAPK Inhibitor Library clinical trial not have an obvious vertebrate ortholog, but there are numerous vertebrate proteins with VWC domains and FN3 repeats homologous to those in Sas. To confirm that Sas was responsible for ectopic binding of 10D-AP to embryos from GE24911 crosses, we made transgenic lines containing a UAS-linked cDNA construct encoding the large Sas isoform. When these were crossed to a variety

of GAL4 driver lines, we observed ectopic 10D-AP staining in patterns that corresponded to the expression patterns of the drivers. Ventrolateral and ventral muscles were brightly labeled by 10D-AP in embryos expressing Sas from the 24B-GAL4 driver ( Figures 1F and 1G). Embryos expressing Sas in glia from Repo-GAL4 showed increased 10D-AP staining along peripheral nerves ( Figures why 1H and 1I). Embryos expressing Sas from 5053A-GAL4, which is selectively expressed in muscle 12, displayed ectopic 10D-AP staining only on that muscle ( Figures 1J and 1K). We compared 10D-AP and anti-Sas staining in ectopic expression embryos, and found that the two patterns were superimposable ( Figure S2). Although ectopic 10D-AP staining is observed wherever Sas is overexpressed, Sas is not required for axonal

staining by 10D-AP. In embryos homozygous for a sas point mutation or a Df that removes sas, a normal axonal pattern of 10D-AP staining was observed (data not shown). This indicates that Ptp10D has other binding partners (see Figure S1). The fact that ectopic Sas expression confers ectopic staining with an exogenous Ptp10D fusion protein does not prove that Sas itself binds to Ptp10D. To determine whether exogenous Sas can interact with endogenous Ptp10D, we performed reverse staining experiments, incubating embryos with purified Sas-Fc, a dimeric human Fc fusion protein containing the XC domain of the large Sas isoform. In live-dissected wild-type embryos, we observed only weak staining with Sas-Fc, and we could not see changes in the staining pattern when we ectopically expressed Ptp10D using the Ptp10DEP1172 insertion line.