The receptor protein tyrosine phosphatase CLR-1 is required for synaptic partner recognition

During neural circuit formation, most axons are guided to complex environments, coming into contact with multiple potential synaptic partners. However, it is critical that they recognize specific neurons with which to form synapses. Here, we utilize the split GFP-based marker Neuroligin-1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP) to visualize specific synapses in live animals, and a circuit-specific behavioral assay to probe circuit function. We demonstrate that the receptor protein tyrosine phosphatase (RPTP) clr-1 is necessary for synaptic partner recognition (SPR) between the PHB sensory neurons and the AVA interneurons in C. elegans. Mutations in clr-1/RPTP result in reduced NLG-1 GRASP fluorescence and impaired behavioral output of the PHB circuit. Temperature-shift experiments demonstrate that clr-1/RPTP acts early in development, consistent with a role in SPR. Expression and cell-specific rescue experiments indicate that clr-1/RPTP functions in postsynaptic AVA neurons, and overexpression of clr-1/RPTP in AVA neurons is sufficient to direct additional PHB-AVA synaptogenesis. Genetic analysis reveals that clr-1/RPTP acts in the same pathway as the unc-6/Netrin ligand and the unc-40/DCC receptor, which act in AVA and PHB neurons, respectively. This study defines a new mechanism by which SPR is governed, and demonstrates that these three conserved families of molecules, with roles in neurological disorders and cancer, can act together to regulate communication between cells

CLR-1/RPTP is required during late embryogenesis and the first larval stage.

<p>(A) <i>clr-1/RPTP(e1745)</i> animals shifted from the permissive temperature (16°C) to the restrictive temperature (20°C) or vice versa after hatching display defective behavior (n≥40). <i>clr-1/RPTP(e1745)</i> animals at 16°C during the 3-fold embryo and larval stage 1 (L1) respond normally to SDS. Animals at 20°C during these stages have defective responses (n>65). NS, not significant, ***<i>P</i><0.001, t-test, comparison to wild-type. <i>P</i>-values were adjusted for multiple comparisons using the Hochberg method. Error bars are SEM. (B) Schematics and micrographs of wild-type and <i>clr-1/RPTP</i> animals kept at 20°C or 16°C during the 3-fold embryo and L1 stages. (C) Quantification of reduced NLG-1 GRASP fluorescence intensity in <i>clr-1/RPTP</i> animals kept at 20°C during these stages, in comparison with <i>clr-1/RPTP</i> animals kept at 16°C during these stages. ***<i>P</i><0.001, U-test, comparison to wild-type if directly over bar, or as indicated. <i>P</i>-values were adjusted for multiple comparisons using the Hochberg method. 95% confidence intervals for the medians are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.s005" target="_blank">S1 Table</a>.</p

CLR-1/RPTP acts in postsynaptic neurons, and is localized to the synaptic region.

<p>(A) Schematics and micrographs of normal PHB-AVA NLG-1 GRASP fluorescence in wild-type and <i>clr-1/RPTP(e1745)</i> mutant animals expressing a transgene that drives expression of the <i>clr-1</i> cDNA in AVA neurons (_<i>p</i><i>AVA</i>::<i>clr-1/RPTP</i>), and reduced PHB-AVA NLG-1 GRASP fluorescence in <i>clr-1/RPTP(e1745)</i> mutant animals expressing either a construct that drives expression of the <i>clr-1/RPTP</i> cDNA in PHB neurons (_<i>p</i><i>PHB</i>::<i>clr-1/RPTP</i>), a transgene that drives expression of the <i>clr-1/RPTP</i> cDNA with the extracellular domain deleted in AVA neurons (_<i>p</i><i>AVA</i>::<i>clr-1/RPTPΔxcd)</i>, or a transgene that drives expression of the <i>clr-1/RPTP</i> cDNA with a mutation that inactivates the phosphatase domain (_<i>p</i><i>AVA</i>::<i>clr-1/RPTPpd</i>). (B) Quantification of NLG-1 GRASP fluorescence. Expression of <i>clr-1/RPTP</i> in AVAs, but not PHBs restores NLG-1 GRASP fluorescence in <i>clr-1/RPTP(e1745)</i> mutants (n>75). Expression of the <i>clr-1/RPTP</i> cDNA with the extracellular domain deleted or with a mutation in the active site of the phosphatase domain does not fully restore NLG-1 GRASP fluorescence in <i>clr-1/RPTP(e1745)</i> mutants (n>100). Two or more lines were examined with each transgene, and combined in the graph above. Values for each individual transgenic line are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.s006" target="_blank">S2 Table</a>. NS, not significant, ***<i>P</i><0.001, *<i>P</i><0.05, U-test. Comparison to <i>clr-1/RPTP</i> indicated over individual bars. <i>P</i>-values were adjusted for multiple comparisons using the Hochberg method. 95% confidence intervals for the medians are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.s005" target="_blank">S1 Table</a>. (C) Expression of <i>clr-1/RPTP</i> in AVAs, but not PHBs, rescues the behavioral defect in <i>clr-1/RPTP(e1745)</i> mutants (n>75). Expression of <i>clr-1/RPTP</i> cDNA with the extracellular domain deleted or with a mutation in the active site of the phosphatase domain does not fully rescue the behavioral defect in <i>clr-1/RPTP(e1745)</i> mutants (n≥60). NS, not significant, ***<i>P</i><0.001, t-test. Comparison to <i>clr-1/RPTP</i> indicated over individual bars. <i>P</i>-values were adjusted for multiple comparisons using the Hochberg method. Error bars are SEM. (D) Schematic and micrograph of an animal expressing the <i>clr-1/RPTP</i> cDNA linked to <i>YFP</i> in AVA (_<i>p</i><i>AVA</i>::<i>clr-1/RPTP</i>::<i>YFP</i>). (E) Schematic and micrograph of an animal expressing the <i>clr-1/RPTP</i> cDNA linked to <i>mCherry</i> in AVA (_<i>p</i><i>AVA</i>::<i>clr-1/RPTP</i>::<i>mCherry</i>) and PHB-AVA NLG-1 GRASP, and overlay in a <i>clr-1/RPTP</i> mutant animal. (D-E), CLR-1 localization is brightest in the preanal ganglion (yellow box), and the majority of animals show localization in the anterior half of this region, where PHB-AVA synapses usually form (green fluorescence).</p

<i>clr-1/RPTP</i> mutants display defective synaptic partner recognition.

<p>(A) Schematic diagrams of PHB and AVA neurons. (B) Schematic diagrams of the trans-synaptic marker NLG-1 GRASP in pre- and postsynaptic neurites (red circles represent cross-sections of neurites in a region with <i>en passant</i> synapses). Split GFPs are linked to the synaptically localized protein NLG-1, so that specific synapses are labeled with green fluorescence in wild-type animals. If a neurite fails to form synapses with the correct partner, NLG-1 GRASP will not reconstitute. (C) Diagram of the PHB and ASH chemosensory circuits including synapses (triangles) connecting sensory neurons (ovals) and interneurons (rectangles) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.ref004" target="_blank">4</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.ref010" target="_blank">10</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.ref052" target="_blank">52</a>]. (D) Outline of a behavioral assay that tests PHB circuit function. Backward movement is induced with a nose touch. Function of PHB-AVA synapses halts backward movement in response to 0.1% SDS. (E and F) Schematics and micrographs of mCherry-labeled PHB neurons and AVA neurites in wild-type (E) and <i>clr-1/RPTP(e1745)</i> mutant animals (F), displaying normal morphology and axon guidance to the ventral nerve cord, followed by anterior projection. (G) Schematics and micrographs of PHB-AVA NLG-1 GRASP in wild-type and <i>clr-1/RPTP(e1745)</i> mutant animals, showing reduced synapses in <i>clr-1/RPTP(e1745)</i> mutant animals. (H) Quantification of NLG-1 GRASP fluorescence demonstrates a reduction in <i>clr-1/RPTP</i> mutant animals including <i>clr-1/RPTP(e1745)</i>, <i>clr-1/RPTP(e2530)</i>, and <i>clr-1/RPTP(n1992)</i> as compared with wild type (n>80 except for the low-brood size allele <i>e2530</i>, (n = 38)). ***<i>P</i><0.001, **<i>P</i><0.01, Mann-Whitney U-test, comparison to wild-type. <i>P</i>-values were adjusted for multiple comparisons using the Hochberg method. 95% confidence intervals for the medians are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.s005" target="_blank">S1 Table</a>. (I) <i>clr-1/RPTP(e1745)</i> mutants display a defect in the behavioral response to SDS (n>75). ***<i>P</i><0.001, t-test, comparison to wild-type. Error bars are standard error of the mean (SEM).</p

CLR-1/RPTP acts in the UNC-6/Netrin and UNC-40/DCC pathway to direct SPR.

<p>In this model, limiting amounts of UNC-6/Netrin secreted from AVA interneurons bind UNC-40/DCC expressed in PHB neurons. CLR-1/RPTP expressed in AVA neurons acts genetically downstream of UNC-6/Netrin in the SPR pathway, and its extracellular domain is required for full SPR function, suggesting that it may interact with UNC-6/Netrin, UNC-40/DCC, or another unidentified ligand or receptor. The requirement of CLR-1/RPTP’s phosphatase domain for rescue indicates that phosphatase activity is also required for its function in SPR.</p

CLR-1/RPTP is sufficient to drive increased synaptogenesis.

<p>(A) Representative schematics and micrographs of NLG-1 GRASP fluorescence labeling PHB-AVA synapses in wild-type animals and animals overexpressing <i>clr-1/RPTP</i> in AVA neurons (_<i>p</i><i>AVA</i>::<i>clr-1/RPTP OE)</i>. (B) Quantification of NLG-1 GRASP fluorescence. Overexpression of <i>clr-1/RPTP</i> in AVA neurons results in increased NLG-1 GRASP fluorescence (n>100). Two lines were examined with this transgene, and combined in the graph above. Values for each individual transgenic line are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.s006" target="_blank">S2 Table</a>. ***<i>P</i><0.001, U-test, comparison to wild-type. 95% confidence intervals for the medians are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007312#pgen.1007312.s005" target="_blank">S1 Table</a>. (C) Overexpression of <i>clr-1/RPTP</i> in AVAs results in a faster behavioral response (n = 80). *<i>P</i><0.05, t-test, comparison to wild-type. Error bars are SEM.</p