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 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

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

<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 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