Protein tyrosine phosphatase regulates the ... - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2011 | 119 | 532–543

doi: 10.1111/j.1471-4159.2011.07411.x

*Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Tokyo, Japan

Abstract The formation and refinement of synaptic connections are key steps of neural development to establish elaborate brain networks. To investigate the functional role of protein tyrosine phosphatase (PTP) r, we employed an olfactory sensory neuron (OSN)-specific gene manipulation system in combination with in vivo imaging of transparent zebrafish embryos. Knockdown of PTPr enhanced the accumulation of synaptic vesicles in the axon terminals of OSNs. The exaggerated accumulation of synaptic vesicles was restored to the normal level by the OSN-specific expression of PTPr, indicating that presynaptic PTPr is responsible for the regulation of synaptic vesicle accumulation. Consistently, transient expression of a dominant-negative form of PTPr in OSNs enhanced the

accumulation of synaptic vesicles. The exaggerated accumulation of synaptic vesicles was reproduced in transgenic zebrafish lines carrying an OSN-specific expression vector of the dominant-negative PTPr. By electron microscopic analysis of the transgenic line, we found the significant increase of the number of OSN-mitral cell synapses in the central zone of the olfactory bulb. The density of docked vesicles at the active zone was also increased significantly. Our results suggest that presynaptic PTPr controls the number of OSN-mitral cell synapses by suppressing their excessive increase. Keywords: docked vesicle, olfactory sensory neuron, presynaptic differentiation, protein tyrosine phosphatase r, synapse formation, synaptic vesicle. J. Neurochem. (2011) 119, 532–543.

The formation and refinement of synaptic connections are key steps of neural development to establish elaborate brain networks providing the basis of perception, learning and cognition. Thus, elucidation of molecular mechanisms that regulate the formation and modulation of central synapses is essential for understanding of neural wiring, brain functions and mental disorders. Trans-synaptic cell adhesion molecules are thought to mediate target recognition and induction of pre- and postsynaptic specializations (Su¨dhof 2008; Sanes and Yamagata 2009; Shen and Scheiffele 2010; Siddiqui and Craig 2011). Furthermore, subsequent remodeling of neural connections often includes activity-dependent addition and elimination of synapses (Goda and Davis 2003). Deregulation of synapse number is implicated in the pathogenesis of mental disorders (Bourgeron 2009). Leukocyte common antigen-related protein (LAR), PTP d and PTPr belong to 2A type receptor-like protein tyrosine phosphatase (RPTP) family (Alonso et al. 2004). Genetic

screening in combination with in vivo imaging in Caenorhabditis elegans and Drosophila revealed that an invertebrate homolog of LAR and its scaffolding protein, liprin, played a role in the morphology of the presynaptic active zone at the

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Received April 22, 2011; revised manuscript received July 11, 2011; accepted July 24, 2011. Address correspondence and reprint requests to Masayoshi Mishina, Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: [email protected] Abbreviations used: dpf, days post-fertilization; ECFP, enhanced cyan fluorescent protein; EGFP, enhanced green fluorescent protein; hpf, hours post-fertilization; IL1RAPL1, interleukin-1 receptor accessory protein-like 1; LAR, leukocyte common antigen-related protein; MO, morpholino oligonucleotide; omp, olfactory marker protein gene; OSN, olfactory sensory neuron; PSD, postsynaptic density; PTP, protein tyrosine phosphatase; RFP, red fluorescent protein; RPTP, receptor-like protein tyrosine phosphatase; VAMP2, vesicle-associated membrane protein 2.

2011 The Authors Journal of Neurochemistry 2011 International Society for Neurochemistry, J. Neurochem. (2011) 119, 532–543

PTPr controls synapse number | 533

neuromuscular junction (Zhen and Jin 1999; Kaufmann et al. 2002). In cultured rat hippocampal neurons, knockdown of LAR reduced the number of dendritic spines (Dunah et al. 2005). However, the role of 2A type RPTPs in formation and refinement of synaptic connections in vivo in vertebrates remains unknown. To investigate the functional role of 2A type RPTPs, we employed the OSN-specific gene manipulation system in combination with in vivo imaging of transparent zebrafish embryos (Yoshida and Mishina 2003). In zebrafish, axons of OSNs reach the target sites, and stop the extension at 50-h post-fertilization (hpf) (Dynes and Ngai 1998; Yoshida et al. 2002). During presynaptic differentiation of zebrafish OSNs, synaptic vesicles visualized with vesicle-associated membrane protein 2 (VAMP2)-enhanced green fluorescent protein (EGFP) fusion protein markedly accumulate in axon terminals between 36 and 84 hpf, whereas the morphological remodeling of axon terminals from complex shapes with filopodia to simple shapes without filopodia proceed between 60 and 84 hpf (Yoshida and Mishina 2005). Here, we showed the morpholino oligonucleotide (MO)-mediated knockdown of PTPr increased the area and number of VAMP2-EGFP puncta in the axon terminal of OSNs. The OSN-specific olfactory marker protein gene (omp) promoter-driven expression of phosphatase-dead form of PTPr (PTPrC1556S) also enhanced the accumulation of synaptic vesicles. Moreover, electron microscopic analyses of transgenic zebrafish carrying an omp promoter-driven expression vector for PTPrC1556S revealed the increase in the density of OSN-mitral cell synapses. These results suggest that PTPr regulates the number of OSN synapses during development.

Materials and methods Animals Zebrafish AB stain was used. Zebrafish embryos were raised at 28.5C in embryo medium containing 17 mM NaCl, 0.27 mM CaCl2, 0.66 mM MgSO4 and 0.4 mM KCl. Cloning of zebrafish PTPr cDNA Detailed procedures for cDNA cloning and in situ hybridization are described in Appendix S1. Construction of OSN-specific expression vectors Procedures for construction of OSN-specific expression vectors are detailed in Appendix S1. Microinjection of expression vectors and MO into zebrafish embryos Expression vectors were linearized by SpeI and dissolved in 100 mM KCl containing 0.05% phenol red. Approximately 0.2– 0.5 nL of the DNA solution at the concentrations of 50–100 ng/lL was injected into the cytoplasm of the 1- to 4-cell embryos. The lissamine-tagged PTPr-MO (5’-CATGACGCAGATGACC TTTGACCTG-3’) complementary to the nucleotide residues –22 to

+3 of the zebrafish ptprs mRNA (nucleotide residues are numbered from the putative translational initiation codon) and control-MO (5’GTCCAGTTTCCAGTAGACGCAGTAC-3’) were obtained from GeneTools (Philomath, OR, USA). Firstly, zebrafish embryos were injected with either Pomp-CFP-ptprs5’UTR-YFP, Pomp-GG, Pomp-VG or Pomp-VG-PTPr (see Appendix S1) at the 1- to 2cell stages, and then injected with PTPr-MO or control-MO at a concentration of 1–3 ng/nL in 1 · Danieau buffer (Nasevicius and Ekker 2000) at the 4- to 8-cell stages. Distribution of MOs in embryos was monitored with a fluorescent microscope. The PTPrMO suppressed the expression of enhanced yellow fluorescent protein in the OSNs injected with Pomp-CFP-ptprs5’UTR-YFP by 87% and 57% at 60 and 84 hpf, respectively (Figure S1). Fluorescent microscopy and image processing Procedures for fluorescent microscopy, image acquisition and image processing are detailed in Appendix S1. Generation of transgenic zebrafish lines The 14.5-kb NotI-SalI fragment from Pomp-VG-PTPrC1556S (see Appendix S1) was inserted between NotI-SalI sites of pTol2000 (a gift from Dr. Kawakami) to yield pT2-Pomp-VG-PTPrC1556S. Approximately 0.2–0.5 nL of solution containing 50–100 ng/lL pT2-Pomp-VG-PTPrC1556S, 25 ng/lL Tol2 mRNA transcribed in vitro (Kawakami and Shima 1999), 100 mM KCl and 0.05% phenol red was injected into zebrafish fertilized eggs. The injected embryos were raised to adulthood and crossed with wild-type fish to examine the signals of the VAMP2-EGFP in olfactory bulb of embryos by fluorescent microscopy. The number of transgene integrated into the genome of each transgenic zebrafish line was estimated by Southern blot hybridization analysis of genomic DNA from adult fish using exon 11 sequence of ptprs as a probe. Electron microscopy Brains of zebrafish embryos at 60 hpf were fixed and embedded in Epon 812 resin. The blocks were cut coronally at 0.7-lm thickness from posterior to anterior with Reichert Ultracut S ultramicrotome (Leica, Vienna, Austria). The dorsal–ventral and right–left angles of the blocks were adjusted under a stereomicroscope so that the coronal section passed through the posterior edges of the olfactory placodes and the laterally widest edges of the telencephalic ventricle. The posteroanterior level corresponding to the posterior edge of the olfactory pit (see Fig. 6a) is set as zero point for the posteroanterior axis. For the measurement of the area of central zone of jt0041 and jt0051 embryos, the ultra-thin sections with 70 nm width were collected at 8.6, 11.4, 14.2, 15.6, 17, 18.4, 19.8, 21.2 and 22.6 lm from the zero point. For the measurement of synapse density per volume of the OSN axon terminals, the 70 nm serial sections were collected at 11.4, 15.6 and 19.8 lm from the zero point (see Fig. 6b). All quantitative measurements were made on the computer screen using ImageJ 1.37 software in the blind manner with respect to the transgene. The outline of an axon terminal was traced and the enclosed region was defined as area of the axon terminals. The sum of areas of same axon terminal in eight serial sections was multiplied by the thickness (70 nm) of sections to give the volume of an axon terminal. Three-dimensional reconstruction was carried out using the Delta viewer (http://vivaldi.ics. nara-wu.ac.jp/wada/DeltaViewer/index-j.html). The number of


534 | X. Chen et al.

synapses in same axon terminal in eight serial sections was counted, and divided by axon terminal volume to give the synapse density. The number of synaptic vesicles within the axon terminal was counted and divided by the axon terminal area to give the synaptic vesicle density. The electron-dense region on the postsynaptic membrane was traced to measure the length of postsynaptic density (PSD). According to the criterion by Dickinson-Nelson and Reese (1983), synaptic vesicles docked with presynaptic membrane and located up to one-vesicle-diameter (50 nm) distance from the active zone were defined as docked vesicles. The density of docked vesicles was given by dividing the number of docked vesicles by the length of PSD. Procedures for fixation, embedding and preparation of serial sections are detailed in Appendix S1. Statistical analysis Statistical significance was evaluated by one-way or two-way ANOVA. When the interaction was significant, unpaired t-test, Dunnett’s test or Tukey’s post hoc test was employed.

Results Knockdown of PTPr enhances the accumulation of synaptic vesicles in the axon terminals of zebrafish OSNs The 1918-amino-acid sequence of zebrafish PTPr deduced from cloned cDNA shared 70%, 66% and 62% identities with mouse PTPr, PTPd and LAR, respectively. The ptprs mRNA was widely distributed in the brain of zebrafish embryos at 60 hpf (Fig. 1). Coronal sections of the head revealed strong hybridization signals in the olfactory placode

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Fig. 1 Expression of the ptprs mRNA in zebrafish embryos at 60 hpf. (a–d) Dorsal views of whole mount embryos (a, b) and ventral views of the head of embryos (c, d) stained by in situ hybridization with DIGlabeled sense (a, c) and antisense (b, d) probes. (e, f) Sections stained by in situ hybridization with DIG-labeled sense (e) and antisense (f) probes. OP, olfactory placode; RGC, retinal ganglion cell layer. Bars = 1 mm in (a, b) and 100 lm in (c–f).

and the retinal ganglion cell layer. We examined the possible role of PTPr in presynaptic differentiation of zebrafish OSNs by antisense MO-mediated knockdown of endogenous PTPr in combination with in vivo imaging of synaptic vesicles in the axon terminals of OSNs. Synaptic vesicle accumulation in the axon terminals of OSNs was monitored by the OSNspecific expression of synaptic vesicle marker VAMP2EGFP (Yoshida and Mishina 2005). An antisense MO directed to the 5’-untranslated sequence of the ptprs mRNA (PTPr-MO) suppressed the expression of the omp promoterdriven reporter gene by 87% at 60 hpf (Figure S1). Injection of PTPr-MO or inverted control MO (control-MO) caused no apparent abnormalities in the gross morphology of embryos as well as in the axon projection of OSNs (Fig. 2a). Treatment with PTPr-MO strongly enhanced the accumulation of VAMP2-EGFP signals in the axon terminals of OSNs at 60 hpf compared with control-MO-treated embryos. The VAMP2-EGFP punctate area in the axon terminals of PTPrMO-treated embryos was significantly larger than that of control MO-treated embryos (Tukey’s test, p < 0.01) (Fig. 2c and d). Furthermore, the omp promoter-driven expression of PTPr in the OSNs using Pomp-VG-PTPr vector (Fig. 2b) restored the increase of VAMP2-EGFP punctate areas in embryos injected with PTPr-MO (p < 0.01) (Fig. 2c and d). As each OSN axon terminates within a single specific glomerulus in the olfactory bulb and forms multiple synapses with the target neuron (Oka 1983; Dynes and Ngai 1998), the increase of VAMP2-EGFP signals in the axon terminals could be ascribed to either enhanced accumulation of synaptic vesicles per synapse or increased number of synapses per terminal. To count the number of VAMP2EGFP puncta within the axon terminal, we employed threedimensional surface plots of VAMP2-EGFP signal intensities (Fig. 2c). The number of VAMP2-EGFP puncta in axon terminals was larger in PTPr-MO-treated embryos than in control-MO-treated embryos (p < 0.05) (Fig. 2e). Furthermore, the omp promoter-driven expression of PTPr in the OSNs using Pomp-VG-PTPr vector (Fig. 2b) restored the increased number of VAMP2-EGFP puncta in embryos injected with PTPr-MO to the normal level (p < 0.01) (Fig. 2c and e). Thus, PTPr-MO-mediated knockdown of endogenous PTPr resulted in the increase of the number and area of VAMP2-EGFP puncta. These results suggest that the endogenous PTPr in the presynaptic OSN plays a role in the control of synaptic vesicle accumulation. During the period of synapse formation between OSNs and postsynaptic cells in the olfactory bulb, the morphology of axon terminals of the OSNs is remodeled from large and complex shapes into small and simple ones between 60 hpf and 84 hpf (Yoshida and Mishina 2005). The morphological remodeling can be monitored by the omp promoter-driven expression of EGFP fused with GAP43, which is enriched in the plasma membrane of axonal growth cone (Nakata et al.



1998) (Fig. 3a and Figure S2). PTPr-MO treatment hardly affected the morphological remodeling of the axon terminal from complex into simple ones between 60 and 84 hpf (Fig. 3b and c). Expression of PTPrC1556S in OSNs enhances the accumulation of synaptic vesicles The observation that the knockdown of PTPr resulted in the enhancement of the accumulation of synaptic vesicles in the (a)

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axon terminals of OSNs favors the idea that PTPr may regulate synapse formation at the presynaptic sites. To examine the issue, we tested the effect of OSN-specific expression of a dominant-negative form of PTPr on the synaptic vesicle accumulation by the use of an omp promoter-driven reporter and effector double-cassette vector (Yoshida and Mishina 2003). It was reported that a substitution mutation of the cysteine residue in the active site of the D1 domain of mouse LAR inactivated the phosphatase activity (Streuli et al. 1990) and the corresponding phosphatase-dead form of human LAR was suggested to act as a dominant-negative suppressor of endogenous molecule in cultured neurons (Dunah et al. 2005). We thus constructed the corresponding mutant of zebrafish PTPr (PTPrC1556S), in which serine was substituted for cysteine 1556 in the active site of the D1 domain. The mutant PTPr was expressed together with VAMP2EGFP in the OSNs by the injection of an omp promoterdriven double-cassette vector (Pomp-VG-PTPrC1556S) into zebrafish embryos (Fig. 4a). We also examined the effect of wild-type PTPr by injection of Pomp-VG-PTPr. Embryos injected with Pomp-VG served as controls. In Pomp-VGinjected embryos, VAMP2-EGFP signals were distributed widely along the axon and in the growth cone at 36 hpf and then became gradually restricted and punctate within the axon terminal at 60 hpf (Fig. 4b). At 84 hpf, the VAMP2-

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Fig. 2 Effect of PTPr knockdown on VAMP2-EGFP puncta in the axon terminals of OSNs. (a) The lateral views of whole zebrafish bodies (top row), their lissamine fluorescent signals to monitor the distribution of MOs (second row) and frontal views of the olfactory placode and bulb (bottom row) of control-MO (left)-, PTPr-MO (middle)- and mock (right)-injected embryos carrying the omp promoterdriven tau-EGFP transgene at 60 hpf. Dorsal is to the top and medial is to the right for bottom row panels. Bars = 1 mm in top row panels and 10 lm in bottom row panels. OP, olfactory placode; OB, olfactory bulb. (b) Omp promoter-driven expression vectors for VAMP2-EGFP (top, Pomp-VG) and for PTPr and VAMP2-EGFP (bottom, Pomp-VGPTPr). Black boxes, the 1.4-kb or 2.7-kb 5’ upstream sequence of the omp gene; left-bound hatched box, the 3.0-kb 3’ downstream sequence of the omp gene; hatched boxes, SV40 polyadenylation signal sequence; lines, pBluescript II SK+. (c) Representative VAMP2-EGFP signals in OSN axon terminals of embryos injected with Pomp-VG and control-MO (left), Pomp-VG and PTPr-MO (middle), and Pomp-VGPTPr and PTPr-MO (right) at 60 hpf. The threshold images of VAMP2-EGFP signals are on the middle row and the 3D surface plot vertical images to visualize each VAMP2-EGFP punctum are on the bottom row. Arrowheads point axonal shafts and arrows point the peaks of VAMP2-EGFP signals. Bar = 5 lm. (d, e) The area (d) and number (e) of VAMP2-EGFP puncta in axon terminals of OSNs of embryos injected with Pomp-VG and control-MO (open bars), PompVG and PTPr-MO (filled bars) and Pomp-VG-PTPr and PTPr-MO (gray bars) at 60 hpf (n = 60–79 from 41 to 53 embryos, respectively). All values represent mean ± SEM. *p < 0.05, **p < 0.01, respectively; Tukey’s test.



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Fig. 3 Effect of PTPr knockdown on the axon terminal morphology of OSNs. (a) Omp promoter-driven expression vector for GAP43-EGFP (Pomp-GG). Black box, the 1.4-kb upstream sequence of the omp gene; hatched box, SV40 polyadenylation signal sequence, line, pBluescript II SK+. (b) Representative GAP43-EGFP signals in axon terminals of OSNs in zebrafish embryos injected with Pomp-GG and control-MO (left) and Pomp-GG and PTPr-MO (right) at 84 hpf. Arrowheads point axonal shafts. Bar = 5 lm. (c) The area (left), perimeter (middle) and complexity (right) values of axon terminals of OSNs in zebrafish embryos injected with Pomp-GG and control-MO (open bars) and Pomp-GG and PTPr-MO (filled bars) at 84 hpf (n = 20 each from 14 to 16 embryos, respectively).

EGFP punctate area in the axon terminal became larger in the axon terminals (Fig. 4b) as previously reported (Yoshida and Mishina 2005). There were significant differences in VAMP2-EGFP punctate area per OSN axon among Pomp-VG-, Pomp-VG-PTPr- and Pomp-VG-PTPrC1556Sinjected embryos (two-way ANOVA: expression vector effect, F = 12.9, p = 3.9 · 10)6; age · expression vector interaction, F = 2.6, p = 0.03) (Fig. 4b–e). The VAMP2-EGFP punctate areas in the axon terminals were comparable among Pomp-VG-, Pomp-VG-PTPr- and Pomp-VG- PTPrC1556Sinjected embryos at 36 hpf during axon path-finding phase (one-way ANOVA: expression vector effect, F = 2.1, p = 0.12) (Fig. 4b–e). However, VAMP2-EGFP punctate areas in the axon terminals of Pomp-VG-PTPrC1556Sinjected embryos at 60 and 84 hpf during synaptogenesis phase were significantly larger than those of Pomp-VG- or Pomp-VG-PTPr-injected embryos (Tukey’s test, p < 0.01 and p < 0.01, at 60 hpf; p < 0.05 and p < 0.01, at 84 hpf, respectively) (Fig. 4e). Furthermore, the number of VAMP2EGFP puncta in the axon terminals of Pomp-VGPTPrC1556S-injected embryos was significantly larger than

that of Pomp-VG- or Pomp-VG-PTPr-injected embryos at 60 hpf (p < 0.01 and p < 0.05, respectively) (Fig. 4f and g). These results suggest that the expression of PTPrC1556S in OSNs stimulated the accumulation of synaptic vesicles in the axon terminals during the period of synaptogenesis. On the other hand, the expression of wild-type PTPr exerted little effect on the accumulation of synaptic vesicles. We also examined the effects of presynaptic PTPr and PTPrC1556S on the morphological remodeling of axon terminals from complex shapes with filopodia to simple shapes without filopodia (Figure S2). There were no significant differences in axon terminal area, perimeter and complexity values among Pomp-GG-, Pomp-GG-PTPr- and Pomp-GG-PTPrC1556S-injected embryos (two-way ANOVA, axon terminal area: expression vector effect, F = 1.30, p = 0.27; age · expression vector interaction, F = 0.39, p = 0.68; axon terminal perimeter: expression vector effect, F = 2.04, p = 0.13; age · expression vector interaction, F = 0.76, p = 0.47; axon terminal complexity: expression vector effect, F = 2.36, p = 0.10; age · expression vector interaction, F = 1.04, p = 0.35). These results suggest that the expressions of PTPr and PTPrC1556S had little effects on the axon terminal remodeling during synaptogenesis. Generation of transgenic PTPrC1556S lines The suppression of PTPr by MO-mediated knockdown or by the expression of a dominant-negative PTPr in OSNs appeared to enhance the synaptic vesicle accumulation and to increase the synapse number in the axon terminal of OSNs. To examine the effect in detail by an electron microscopy, we generated transgenic zebrafish stably carrying Pomp-VG-PTPrC1556S using Tol2 transposon system (Kawakami et al. 2004). Screening by VAMP2-EGFP signals established three independent transgenic lines, Tg(omp: VAMP2-EGFP, omp:ptprsC1556S)jt0051–0053. VAMP2EGFP signals in these transgenic lines were localized mainly in the olfactory bulb and weakly in the olfactory placode at 60 hpf (Fig. 5a). The majority of OSNs were labeled by VAMP2EGFP. Southern blot analysis of genomic DNA revealed that jt0051 and jt0052 lines carried one copy of the transgene, whereas jt0053 line carried multiple copies (Fig. 5b). The effect of PTPrC1556S in the transgenic lines on the accumulation of synaptic vesicles was examined by the injection of an omp promoter-driven VAMP2-red fluorescent protein (RFP) reporter vector (Pomp-VR). An omp promoter-driven VAMP2-enhanced cyan fluorescent protein (ECFP) transgenic line Tg(omp:VAMP2-ECFP)jt0041 (Yoshida and Mishina 2005) and wild-type AB strain served as controls. There were significant differences in the punctate area of VAMP2-RFP signals among control and transgenic lines (one-way ANOVA: F = 9.9, p = 3.6 · 10)6). Punctate areas of VAMP2-RFP signals in the axon terminals of jt0051, jt0052 and jt0053 transgenic embryos were larger than those of the control jt0041 and non-



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Fig. 4 Effects of OSN-specific expressions of PTPr and PTPrC1556S on VAMP2-EGFP puncta in the axon terminals. (a) Omp promoter-driven expression vector for PTPrC1556S and VAMP2EGFP (Pomp-VG-PTPrC1556S). Black boxes, the 1.4-kb or 2.7-kb 5’ upstream sequence of the omp gene; left-bound hatched box, the 3.0kb 3’ downstream sequence of the omp gene; hatched box, SV40 polyadenylation signal sequence; line, pBluescript II SK+. (b–d) Representative VAMP2-EGFP expression signals in the OSN axon terminals of Pomp-VG (b)-, Pomp-VG-PTPr (c)-, and Pomp-VGPTPrC1556S (d)-injected embryos at 36 (left), 60 (middle) and 84 hpf (right). The threshold images of VAMP2-EGFP signals are on the

bottom. (e) The area of VAMP2-EGFP puncta in axon terminals of OSNs in zebrafish embryos injected with Pomp-VG (open bars), Pomp-VG-PTPr (gray bars) and Pomp-VG-PTPrC1556S (filled bars) expression vectors at 36, 60 and 84 hpf (n = 32–71 from 22 to 48 embryos). (f) The representative vertical images obtained by 3D surface plots of VAMP2-EGFP signals of OSN axon terminals at 60 hpf. (g) The number of VAMP2-EGFP puncta in axon terminals of OSNs of embryos injected with Pomp-VG (open bar), Pomp-VG-PTPr (gray bar) and Pomp-VG-PTPrC1556S (filled bar) at 60 hpf (n = 29–46 from 20 to 33 embryos). All values represent mean ± SEM. *p < 0.05 and **p < 0.01, respectively; Tukey’s test. Bars = 5 lm.

transgenic AB embryos at 60 hpf (Dunnett’s test, p < 0.05 or p < 0.01) (Fig. 5c). Furthermore, the numbers of VAMP2-RFP puncta in these PTPrC1556S transgenic embryos were significantly larger than those in control embryos (p < 0.05 or p < 0.01) (Fig. 5d). Thus, the effects of transient expression of PTPrC1556S on the area and number of VAMP2-EGFP puncta were reproduced in these stable transgenic lines. In the subsequent electron micro-

scopic experiments, we utilized jt0051 PTPrC1556S transgenic line as a mutant and jt0041 VAMP2-ECFP transgenic line as a control. Increase in the density of OSN-mitral cell synapses in jt0051 transgenic line For the preparation of appropriate sections for electron microscopic analysis of OSN synapses, we first located the



Fig. 5 Characterization of PTPrC1556S transgenic lines. (a) Brightfield (top row) and fluorescent (middle row) images of heads of jt0051, jt0052 and jt0053 embryos were merged on the bottom row panels. OB, olfactory bulb; OP, olfactory placode. (b) Southern blot analysis of genomic DNAs from AB, jt0051, jt0052, and jt0053 lines. BglII-, EcoRV- and HindIII-digested genomic DNAs were hybridized with a probe containing ptprs exon 11 sequence. (c, d) The area (c) and number (d) of VAMP2-RFP puncta in axon terminals of OSNs in AB, jt0041, jt0051, jt0052 and jt0053 embryos at 60 hpf (n = 12–29 from 9 to 26 embryos). *p < 0.05; Dunnett’s test.

central zone of the olfactory bulb by following VAMP2ECFP signals of jt0041 control embryos. In the selected coronal level (see Materials and methods), brains of

BODIPY-labeled jt0041 embryos were imaged from posterior to anterior at 60 hpf (Fig. 6a). Three lobes of the glomerular map that correspond to the central zone, dorsal zone, and medial glomerulus can be identified in the BODIPY-labeled olfactory bulb of jt0041 embryos at 60 hpf (Fig. 6a) (Dynes and Ngai 1998). Central zone, which is heavily innervated by VAMP2-ECFP-labeled OSN axons in jt0041 embryos (Fig. 6a), spans approximately 15.2 lm anteroposteriorly in the olfactory bulb at 60 hpf (Fig. 6b). We prepared serial sections of the central zone for electron microscopic analysis. Two types of synapses were discernable in the electron micrographs of the central zone of jt0041 embryos at 60 hpf (Fig. 6c–e). One is the OSN-mitral cell synapse that is characterized by electron-dense presynaptic terminals filled with many small spherical synaptic vesicles and small electron-lucent postsynaptic terminals as described in adult goldfish and zebrafish (Ichikawa 1976; Oka 1983; Byrd and Brunjes 1995). The other is the mitral cell-granule cell synapse that is characterized by small presynaptic and postsynaptic terminals with electron-lucent cytoplasm as described in adult goldfish (Ichikawa 1976). The areas of the central zone estimated from nine coronal sections (see Materials and methods) were comparable between control and PTPrC1556S transgenic embryos (194 ± 17.6 and 158 ± 16.5 lm2 for jt0041 and jt0051, respectively; nine sections from one embryo each; t-test, p = 0.17). The density of OSN-mitral cell synapses in the central zone of jt0051 embryo (3.1 ± 0.2 synapses per 100 lm2) was significantly larger than that of control embryo (1.8 ± 0.4 synapses per 100 lm2) (p < 0.05). On the other hand, the densities of mitral cell-granule cell synapses in the central zone were comparable between jt0051 and control embryos (4.2 ± 0.5 synapses per 100 lm2 and 4.0 ± 0.3 synapses per 100 lm2, respectively; p = 0.76). These results suggest that the number of OSN-mitral cell synapses was selectively increased in the central zone of the PTPrC1556Stransgenic line. We further examined the number of OSN-mitral cell synapses per axon terminal of OSNs in the central zone. Eight serial sections were collected from three regions within the central zone along the posterior-anterior axis (Fig. 6b and see also Materials and methods) to count the number of OSN-mitral cell synapses per volume of the OSN axon terminal in jt0051 and control jt0041 embryos at 60 hpf. The number of OSN-mitral cell synapses per axon terminal in jt0051 embryos (1.9 ± 0.16, n = 99) was significantly larger than that of control embryos (1.2 ± 0.15, n = 95) (p < 0.001) (Fig. 7a and b and Figure S3). There were no significant differences in the volume of OSN axon terminals estimated form eight serial sections between jt0051 and control embryos (0.45 ± 0.04 and 0.43 ± 0.04 lm3, respectively) (p = 0.74). Thus, the density of OSN-mitral cell synapses per volume of axon terminal in jt0051 embryos was significantly larger than that of control embryos (p < 0.001) (Fig. 7c).



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11.4

(c)

Posterior 0

19.8

Central zone 5

10

15

(d)

Fig. 6 Electron micrography of the central zone in the olfactory bulb. (a) Coronal optical sections of the BODIPY-labeled brain of jt0041 embryo at 60 hpf from posterior to anterior. Dorsal is to the top. The posteroanterior level corresponding to the posterior edge of the olfactory pit [asterisks in (v)] is set as zero point for the posteroanterior axis. The posteroanterior level of each optical section is given on the bottom right side of the each panel. A high magnification image of the framed area in (viii) is shown on the right, CZ, central zone; DZ, dorsal zone; MZ, medial zone; OP, olfactory placode. (b) Location of the

20

Anterior 25 μm

(e)

central zone. The central zone spans about 15 lm along posteroanterior axis in the olfactory bulb of jt0041 embryos at 60 hpf (n = 12 embryos). (c) An electron micrograph of the central zone in the olfactory bulb of a jt0041 embryo. A high magnification electron micrograph of the framed area in (c) is shown on the right. (d, e) Electron micrographs of OSN-mitral cell synapses (d) and granule cellmitral cell synapses (e). OSN-mitral cell synapses and granule cellmitral cell synapses are indicated with arrowheads and arrows, respectively. Bars = 100 lm in (a) and 500 nm in (c–e).



(a)

(b)

jt0041 (control)

(c)

jt0051 (PTPσC1556S)

1 jt0041 (control)

2


4

Y

2 X

1

8

3

X

2 4 Y

Y

X 3

1

Z

2

Z

X

**

7 Synapse density (μm–3)

Y

6 5 4 3 2 1 0

Fig. 7 Increase of OSN-mitral cell synapses in PTPrC1556S transgenic embryos. (a, b) Electron micrographs (top) and three-dimensional reconstructions from eight serial electron micrographs (bottom) of axon terminals of OSNs in the central zone of olfactory bulb in control jt0041 embryos (a) and PTPrC1556S transgenic jt0051 embryos (b) at 60 hpf. In the top panels, axon terminals of OSNs are labeled in green and OSN-mitral cell synapses are indicated with arrowheads and numbered. In the bottom panels, OSN-mitral cell

synapses are labeled in red. The positions corresponding to the electron micrographs on the top panels are indicated with broken lines. Bars = 200 nm. (c) Densities of OSN-mitral cell synapses estimated from eight serial sections of OSN axon terminals of control jt0041 line (open bar) and jt0051 line (filled bar) at 60 hpf (n = 95 and 99 axon terminals from three embryos each). The serial sections were prepared at three positions of the central zone indicated by arrowheads in Fig. 6b. All values represent mean ± SEM. **p < 0.01; t-test.

These results suggest that the expression of PTPrC1556S in the presynaptic OSNs increases the number of OSN-mitral cell synapses.

in control embryos (p = 0.84 and 0.14, respectively). These results suggest that the expression of PTPrC1556S in presynaptic OSNs increases the densities of synaptic vesicles and docked vesicles in the axon terminals.

Increase of synaptic vesicles and docked vesicles in OSN axon terminals in jt0051 transgenic line We next examined the structural characteristics of synapses in jt0051 and control jt0041 embryos at 60 hpf. There were no significant differences in the terminal area and length of PSD at OSN-mitral cell synapses between jt0051 and control embryos (p = 0.48 and 0.17, respectively) (Fig. 8). On the other hand, the number and density of synaptic vesicles in the OSN axon terminals of jt0051 embryos were significantly larger than those of control embryos (p < 0.05 and p < 0.001, respectively). Furthermore, the number and density of docked vesicles at the active zone of OSN-mitral cell synapses in jt0051 embryos were also significantly larger than those in control embryos (p < 0.001 and p < 0.001, respectively). We also examined the ultrastructures of the mitral cell-granule cell synapse. There were no significant differences in the terminal area and the PSD length at mitral cell-granule cell synapses between jt0051 and control embryos (p = 0.25 and 0.55, respectively) (Fig. 9). The numbers of synaptic vesicles and docked vesicles at the active zone in the presynaptic site of mitral cell-granule cell synapses in jt0051 embryos were also comparable with those

Discussion The formation and refinement of synaptic connections are key steps of neural development to establish elaborate brain networks providing the basis of perception, learning and cognition. Deregulation of synapse number is implicated in the pathogenesis of mental disorders (Bourgeron 2009). However, little is known about the mechanism of the regulation of synapse number. In the present investigation, we revealed a new role of PTPr in regulating the synapse number of OSNs. In zebrafish, the olfactory sensory proto-map is formed as a stereotyped pattern of glomerular arrangement in the olfactory bulb within 3-days post-fertilization (dpf) (Dynes and Ngai 1998). Axo-dendritic contacts of OSNs and mitral cells are first detectable around 50 hpf (Yoshida and Mishina 2005) and glomerular development proceeds thereafter at least beyond 6 dpf (Li et al. 2005). During presynaptic differentiation of OSNs, synaptic vesicles markedly accumulate in axon terminals between 36 and 60 hpf and the morphological remodeling of axon terminals from complex



(a)


jt0041 (control)

(a)

(

(

)

)

(b)


jt0041 (control) PSD length (μm)

0.8 0.6 0.4 0.2

0.2 0.1

60 40 20 0

3 2 1 0

*

60 40 20 0

***

Docked vesicle number –1 /PSD length (μm )

80

***

Docked vesicle number

100

80

0

0 120

Synaptic vesicle number

0.3

1.0

Synaptic vesicle number –2 /terminal area (μm )

Terminal area (μm2)

(b)

12

***

10 8 6 4

Fig. 9 Effect of PTPrC1556S on ultrastructure of mitral cell-granule cell synapses. (a) Electron micrographs of mitral cell-granule cell synapses in the central zone of jt0041 (left) and jt0051 (right) transgenic embryos. Arrows point the synapses. Bar = 200 nm. (b) Axon terminal area, PSD length, synaptic vesicle number, docked vesicle number in mitral cell-granule cell synapse in jt0041 (open bars) and jt0051 (filled bars) transgenic embryos at 60 hpf (61 synapses from 53 axon terminals and 89 synapses from 73 axon terminals, respectively; three embryos each). All values represent mean ± SEM.

2 0

Fig. 8 Ultrastructures of OSN-mitral cell synapses in PTPrC1556S transgenic embryos. (a) Electron micrographs of OSN-mitral cell synapses in the central zone of control jt0041 (left) and jt0051 embryos (right) at 60 hpf. High magnification electron micrographs of the framed areas in the top panels are shown on the bottom. Arrowheads point the synapses. Bar = 200 nm. (b) Quantification of ultrastructures in OSN-mitral cell synapses in the central zone of jt0041 (open bars) and jt0051 embryos (filled bars) at 60 hpf (84 and 163 synapses from 93 to 97 axon terminals, respectively; three embryos each). All values represent mean ± SEM. *p < 0.05 and ***p < 0.001, respectively; t-test.

shapes with filopodia to simple shapes without filopodia proceed between 60 and 84 hpf (Yoshida and Mishina 2005). Correspondingly, odor responses in the olfactory bulb become detectable at 60–72 hpf (Li et al. 2005). In this study, we showed that MO-mediated knockdown of PTPr increased the area and number of VAMP2-EGFP puncta in the axon terminals of OSNs at 60 hpf. These phenotypes of PTPr-MO-treated embryos were rescued by the OSNspecific expression of PTPr, indicating that presynaptic PTPr is responsible for the regulation of synaptic vesicle accumulation. Consistently, the transient expression of dominant-negative form of PTPr in the OSNs stimulated

the development of VAMP2-EGFP puncta during the period of synapse formation between OSNs and mitral cells in the olfactory bulb. On the other hand, both MO-mediated knockdown of PTPr and the expression of dominantnegative form of PTPr had little effect on the morphological remodeling of axon terminals. Thus, PTPr in the OSNs is selectively involved in the regulation of synaptic vesicle accumulation in the axon terminals. We previously showed that protein kinase A, cAMP-response element binding protein and interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) are required for synaptic vesicle accumulation during development (Yoshida and Mishina 2005, 2008; Yoshida et al. 2009). The area of VAMP2-EGFP puncta was decreased by the suppression of protein kinase A, cAMPresponse element binding protein or IL1RAPL1 and was increased by constitutive activation or over-expression of these molecules. Interestingly, over-expression of PTPr in OSNs hardly affected the accumulation of VAMP2-EGFP puncta. Thus, PTPr appears to suppress an exaggerated accumulation of synaptic vesicles, but not their normal accumulation. The increase of synaptic vesicles in the axon terminals could be ascribed to either enhanced accumulation of



synaptic vesicles per synapse or increased number of synapses per terminal, as each OSN axon terminates within a single specific glomerulus in the olfactory bulb and forms multiple synapses with the target neuron (Oka 1983; Dynes and Ngai 1998). Three-dimensional surface plots of VAMP2-EGFP signal intensities showed that the number of VAMP2-EGFP puncta within the axon terminal was increased in OSN axon terminals of embryos treated with PTPr-MO or dominant-negative PTPr. To further examine the issue at the electron microscopic level, we established transgenic zebrafish lines carrying an OSN-specific expression vector of the dominant-negative PTPr. Both the area and number of VAMP2-RFP puncta were increased in OSN axon terminals of all three transgenic lines, thus reproducing the effects of MO-mediated PTPr knockdown and transient dominant-negative PTPr expression. By electron microscopic analyses of serial sections of the central zone, we found that the number of OSN-mitral cell synapses characterized by electron-dense presynaptic terminals filled with many small spherical synaptic vesicles and small electronlucent postsynaptic terminals (Ichikawa 1976; Oka 1983; Byrd and Brunjes 1995) was selectively increased in the transgenic line. Our results suggest that PTPr regulates the number of OSN-mitral cell synapses in zebrafish OSNs. Recently, presynaptic PTPr in cultured rat hippocampal neurons was reported to induce pre- and postsynaptic differentiation through the interaction with postsynaptic netrin-G ligand-3 or TrkC (Kwon et al. 2010; Takahashi et al. 2011). However, MO-mediated knockdown of PTPr or suppression of PTPr by the expression of a dominantnegative form resulted in the enhanced accumulation of synaptic vesicles in OSN axon terminals and the increase of the number of OSN-mitral cell synapses rather than the impairments of presynaptic differentiation in zebrafish embryos. Thus, our results revealed a new role of presynaptic PTPr in keeping the proper number of synapses by suppressing their excessive increase. This finding raises a question of how PTPr controls the number of OSN-mitral cell synapses. The initial axo-dendritic contacts trigger intracellular signals that induce assembly of active zone and accumulation of synaptic vesicles at presynaptic terminals, whereas subsequent refinement of synaptic connections often includes activity-dependent elimination of synapses. As the number of synapses is controlled by synapse formation and elimination during development, PTPr may be involved in synapse elimination rather than synapse formation. Drosophila hypomorphic mutant with decreased expression of synaptic adhesion molecule Fasciclin II showed the increase of synaptic boutons at the neuromuscular junction in third instar larvae suggesting a role of Fasciclin II in activitydependent regulation of the synapse (Schuster et al. 1996a,b). Thus, one possibility is that PTPr might regulate the synapse number by modulating cell adhesion molecules. Interestingly, N-cadherin and b-catenin are substrates for

PTPr (Siu et al. 2007) and it was reported that the disruption of cadherin-b-catenin interaction by tyrosine phosphorylation of b-catenin increases synapse density in cultured rat hippocampal neurons (Bamji et al. 2006). In addition, the trans-synaptic interaction of presynaptic PTPd and postsynaptic IL1RAPL1 organizes synaptogenesis of mouse cortical neurons (Yoshida et al. 2011). Electron microscopic analysis of OSN-mitral cell synapse also revealed the significant increase of the densities of docked synaptic vesicles at the active zone in the transgenic line carrying an OSN-specific expression vector of the dominant-negative PTPr. Zebrafish PTPr interacted with presynaptic scaffold protein liprin-a through its D2 domain (data not shown) as reported for mouse PTPr (Pulido et al. 1995). In mouse olfactory bulb, strong immunoreactivities for liprin-a1 and liprin-a2 were found in the glomerular layer and the olfactory nerve layer but not in the external plexiform layer suggesting that liprin-a1 and liprin-a2 are present in the axon of mouse OSNs (Spangler et al. 2011; Zu¨rner et al. 2011). Through various protein-protein interactions liprin-a can link to various active zone and synaptic vesicle proteins such as RIM, Munc-18 and Rab3 that regulate the number of docked vesicles (Wang et al. 1997; Toonen et al. 2006; Coleman et al. 2007). Thus, it is possible that PTPr may regulate docked vesicle clusters through liprin-a. In conclusion, our results suggest that presynaptic PTPr controls the number of OSN-mitral cell synapses by suppressing their excessive increase.

Acknowledgements This work was supported in part by the Grant-in-Aid for Scientific Research and the Global Center of Excellence program from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Dr K. Kawakami for pTol2000 plasmid, Dr Y. Masuda for helpful advice and Ms. A. Uotsu, Ms. A. Kakihara and Dr T. Shiroshima for help in zebrafish breeding.

Supporting information Additional supporting information may be found in the online version of this article: Appendix S1. Supplementary Materials and methods. Figure S1. Evaluation of MO-mediated protein knockdown. Figure S2. Effects of PTPr and PTPrC1556S on morphological remodeling of OSN axon terminals. Figure S3. Serial electron micrographs of axon terminals of OSNs. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.



References Alonso A., Sasin J. and Bottini N. et al. (2004) Protein tyrosine phosphatase in the human genome. Cell 117, 699–711. Bamji S. X., Rico B., Kimes N. and Reichardt L. F. (2006) BDNF mobilizes synaptic vesicles and enhances synapse formation by disrupting cadherin-b-catenin interactions. J. Cell Biol. 174, 289–299. Bourgeron T. (2009) A synaptic trek to autism. Curr. Opin. Neurobiol. 19, 231–234. Byrd C. A. and Brunjes P. C. (1995) Organization of the olfactory system in the adult zebrafish: histological, immunohistochemical, and quantitative analysis. J. Comp. Neurol. 358, 247–259. Coleman W. L., Bill C. A. and Bykhovskaia M. (2007) Rab3a deletion reduces vesicle docking and transmitter release at the mouse diaphragm synapse. Neurosci. 148, 1–6. Dickinson-Nelson A. and Reese T. S. (1983) Structural changes during transmitter release at synapses in the frog sympathetic ganglion. J. Neurosci. 3, 42–52. Dunah A. W., Hueske E., Wyszynski M., Hoogenraad C. C., Jaworski J., Park D. T., Simonetta A., Liu G. and Sheng M. (2005) LAR receptor protein tyrosine phosphatases in the development and maintenance of excitatory synapses. Nat. Neurosci. 4, 458–467. Dynes J. L. and Ngai J. (1998) Pathfinding of olfactory neuron axons to stereotyped glomerular targets revealed by dynamic imaging in living zebrafish embryos. Neuron 20, 1081–1091. Goda Y. and Davis G. W. (2003) Mechanisms of synapse assembly and disassembly. Neuron 40, 243–264. Ichikawa M. (1976) Fine structure of the olfactory bulb in the goldfish, Carassius auratus. Brain Res. 115, 43–46. Kaufmann N., DeProto J., Ranjan R., Wan H. and Vactor D. V. (2002) Drosophila liprin-a and the receptor phosphatase Dlar control synapse morphogenesis. Neuron 34, 27–38. Kawakami K. and Shima A. (1999) Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene 240, 239– 244. Kawakami K., Takeda H., Kawakami N., Kobayashi M., Matsuda N. and Mishina M. (2004) A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev. Cell 7, 133–144. Kwon S. K., Woo J., Kim S. Y., Kim H. and Kim E. (2010) Transsynaptic adhesions between netrin-G ligand-3 (NGL-3) and receptor tyrosine phosphatases LAR, protein-tyrosine phosphatase d (PTPd), and PTPr via specific domains regulate excitatory synapse formation. J. Biol. Chem. 285, 13966–13978. Li J., Mack J. A., Souren M., Yaksi E., Higashijima S., Mione M., Fetcho J. R. and Friedrich R. W. (2005) Early development of functional spatial maps in the zebrafish olfactory bulb. J. Neurosci. 25, 5784–5795. Nakata T., Terada S. and Hirokawa N. (1998) Visualization of the dynamics of synaptic vesicle and plasma membrane proteins in living axons. J. Cell Biol. 140, 659–674. Nasevicius A. and Ekker S. C. (2000) Effective targeted gene ‘knockdown’ in zebrafish. Nat. Genet. 26, 216–220. Oka Y. (1983) Golgi, Electron-microscopic and combined golgi-electron- microscopic studies of the mitral cells in the goldfish olfactory bulb. Neurosci. 8, 723–742. Pulido R., Serra-Page`s C., Tang M. and Streuli M. (1995) The LAR/PTPd/ PTPr subfamily of transmembrane protein-tyrosine-phosphatases: multiple human LAR, PTPd, and PTPr isoforms are expressed in a tissuespecific manner and associate with the LAR-interacting protein LIP.1. Proc. Natl Acad. Sci. USA 92, 11686–11690. Sanes J. R. and Yamagata M. (2009) Many paths to synaptic specificity. Annu. Rev. Cell Dev. Biol. 25, 161–195.

Schuster C. M., Davis G. W., Fetter R. D. and Goodman C. S. (1996a) Genetic dissection of structural and functional components of synaptic plasticity I. Fasciclin II controls synaptic stabilization and growth. Neuron. 17, 641–654. Schuster C. M., Davis G. W., Fetter R. D. and Goodman C. S. (1996b) Genetic dissection of structural and functional components of synaptic plasticity II. Fasciclin II controls presynaptic structural plasticity. Neuron. 17, 655–667. Shen K. and Scheiffele P. (2010) Genetics and cell biology of building specific synaptic connectivity. Annu. Rev. Neurosci. 33, 473–507. Siddiqui T. J. and Craig A. M. (2011) Synaptic organizing complexes. Curr. Opin. Neurobiol. 21, 132–143. Siu R., Fladd C. and Rotin D. (2007) N-cadherin is an in vivo substrate for protein tyrosine phosphatase r (PTPr) and participates in PTPr-mediated inhibition of axon growth. Mol. Cell Biol. 27, 208– 219. Spangler S. A., Jaarsma D., Graaff E., Wulf P. S., Akhmanova A. and Hoogenraad C. C. (2011) Differential expression of liprin-a family proteins in the brain suggests functional diversification. J. Comp. Neurol. 519, 3040–3060. Streuli M., Krueger N. X., Thai T., Tang M. and Saito H. (1990) Distinct functional roles of the two intracellular phosphatase like domains of the receptor-linked protein tyrosine phosphatases LCA and LAR. EMBO J. 9, 2399–2407. Su¨dhof T. C. (2008) Neuroligins and neurexins link synaptic function to cognitive disease. Nature 455, 903–911. Takahashi H., Arstikaitis P., Prasad T., Bartlett T. E., Wang Y. T., Murphy T. H. and Craig A. M. (2011) Postsynaptic TrkC and presynaptic PTPr function as a bidirectional excitatory synaptic organizing complex. Neuron 69, 287–303. Toonen R. F. G., Wierda K., Sons M. S., Wit H., Cornelisse L. N. and Brussaard A. (2006) Munc18–1 expression levels control synapse recovery by regulating readily releasable pool size. Proc. Natl Acad. Sci. USA 103, 18332–18337. Wang Y., Okamoto M., Schmitz F., Hofman K. and Su¨dhof T. C. (1997) RIM: a putative Rab3-effector in regulating synaptic vesicle fusion. Nature 388, 593–598. Yoshida T. and Mishina M. (2003) Neuron-specific gene manipulations in transparent zebrafish embryos. Methods Cell Sci. 25, 15–23. Yoshida T. and Mishina M. (2005) Distinct roles of calcineurin-nuclear factor of activated T-cells and protein kinase A-cAMP response element-binding protein signaling in presynaptic differentiation. J. Neurosci. 25, 3067–3079. Yoshida T. and Mishina M. (2008) Zebrafish orthologue of mental retardation protein IL1RAPL1 regulates presynaptic differentiation. Mol. Cell. Neurosci. 39, 218–228. Yoshida T., Ito A., Matsuda N. and Mishina M. (2002) Regulation by protein kinase A switching of axonal pathfinding of zebrafish olfactory sensory neurons through the olfactory placode-olfactory bulb boundary. J. Neurosci. 22, 4964–4972. Yoshida T., Uchida S. and Mishina M. (2009) Regulation of synaptic vesicle accumulation and axon terminal remodeling during synapse formation by distinct Ca2+signaling. J. Neurochem. 111, 160–170. Yoshida T., Yasumura M., Uemura T., Lee S., Ra M., Taguchi R., Iwakura Y. and Mishina M. (2011) IL1RAPL1 associated with mental retardation and autism mediates synapse formation by transsynaptic interaction with PTPd. J. Neurosci. in press. Zhen M. and Jin Y. (1999) The liprin protein SYD-2 regulates the differentiation of presynaptic termini in C. elegans. Nature 401, 371–375. Zu¨rner M., Mittelstaedt T., Dieck S., Becker A. and Schoch S. (2011) Analyses of the spatiotemporal expression and subcellular localization of liprin-a proteins. J. Comp. Neurol. 519, 3019– 3039.
