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Rap2 and TNIK control Plexin-dependent tiled synaptic innervation in C. elegans

  1. Xi Chen
  2. Akihiro CE Shibata
  3. Ardalan Hendi
  4. Mizuki Kurashina
  5. Ethan Fortes
  6. Nicholas L Weilinger
  7. Brian A MacVicar
  8. Hideji Murakoshi
  9. Kota Mizumoto  Is a corresponding author
  1. The University of British Columbia, Canada
  2. National Institute for Physiological Sciences, Japan
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Cite this article as: eLife 2018;7:e38801 doi: 10.7554/eLife.38801

Abstract

During development, neurons form synapses with their fate-determined targets. While we begin to elucidate the mechanisms by which extracellular ligand-receptor interactions enhance synapse specificity by inhibiting synaptogenesis, our knowledge about their intracellular mechanisms remains limited. Here we show that Rap2 GTPase (rap-2) and its effector, TNIK (mig-15), act genetically downstream of Plexin (plx-1) to restrict presynaptic assembly and to form tiled synaptic innervation in C. elegans. Both constitutively GTP- and GDP-forms of rap-2 mutants exhibit synaptic tiling defects as plx-1 mutants, suggesting that cycling of the RAP-2 nucleotide state is critical for synapse inhibition. Consistently, PLX-1 suppresses local RAP-2 activity. Excessive ectopic synapse formation in mig-15 mutants causes a severe synaptic tiling defect. Conversely, overexpression of mig-15 strongly inhibited synapse formation, suggesting that mig-15 is a negative regulator of synapse formation. These results reveal that subcellular regulation of small GTPase activity by Plexin shapes proper synapse patterning in vivo.

https://doi.org/10.7554/eLife.38801.001

eLife digest

Genes do more than just direct the color of our hair or eyes. They produce proteins that are involved in almost every process in the body. In humans, the majority of active genes can be found in the brain, where they help it to develop and work properly – effectively controlling how we move and behave.

The brain’s functional units, the nerve cells or neurons, communicate with each other by releasing messenger molecules in the gap between them, the synapse. These molecules are then picked up from specific receptor proteins of the receiving neuron.

In the nervous system, neurons only form synapses with the cells they need to connect with, even though they are surrounded by many more cells. This implies that they use specific mechanisms to stop neurons from forming synapses with incorrect target cells. This is important, because if too many synapses were present or if synapses formed with incorrect target cells, it would compromise the information flow in the nervous system. This would ultimately lead to various neurological conditions, including Autism Spectrum Disorder.

In 2013, researchers found that in the roundworm Caenorhabditis elegans, a receptor protein called Plexin, is located at the surface of the neurons and can inhibit the formation of nearby synapses. Now, Chen et al. – including one author involved in the previous research – wanted to find out what genes Plexin manipulates when it stops synapses from growing. Knowing what each of those genes does can help us understand how neurons can inhibit synapses.

The results revealed that Plexin appears to regulate two genes, Rap2 and TNIK. Plexin reduced the activity of Rap2 in the neuron that released the messenger, which hindered the formation of synapses. The gene TNIK and its protein on the other hand, have the ability to modify other proteins and could so inhibit the growth of synapses. When TNIK was experimentally removed, the number of synapses increased, but when its activity was increased, the number of synapses was strongly reduced.

These findings could help scientists understand how mutations in Rap2 or TNIK can lead to various neurological conditions. A next step will be to test if these genes also affect the formation of synapses in other species such as mice, which have a more complex nervous system that is structurally and functionally more similar to that of humans.

https://doi.org/10.7554/eLife.38801.002

Introduction

During nervous system development, various instructive and repulsive signaling cues cooperatively direct neurons to form chemical synapses with their appropriate targets. Studies have identified some molecules and elucidated their downstream mechanisms that instruct synaptogenesis such as FGF, Ephrin/Eph, Ig-family of cell adhesion molecules (IgCAMs) and synaptic cell adhesion molecules (SynCAMs) (Shen and Bargmann, 2003; Shen et al., 2004; Dabrowski and Umemori, 2016; Dabrowski et al., 2015; Feng et al., 2000; Kayser et al., 2008; Terauchi et al., 2010; Yamagata et al., 2003). Several axon guidance cues and their receptors also play critical roles to inhibit synapse formation (Inaki et al., 2007; Klassen and Shen, 2007; Poon et al., 2008). Semaphorins (Sema) and their receptors, Plexins, are two conserved families of molecules that have a well-established function to repel axons during development (Kolodkin et al., 1993, 1992; Negishi et al., 2005a; Tran et al., 2007) and play prominent roles contributing to immune system, cardiovascular development and cancer regulation (Epstein et al., 2015; Neufeld et al., 2005; Takamatsu and Kumanogoh, 2012).

In addition to its function as a long-range axon guidance cue during neuronal development, Sema/Plexin signaling plays a critical role as a negative regulator of synapse formation. The role of Sema/Plexin signaling to inhibit synapse formation was first observed in Drosophila, where ectopic expression of Sema2a causes elimination of specific neuromuscular junctions (Matthes et al., 1995). In mammals, Sema3E/PlexinD1 specifies sensory-motor connections (Pecho-Vrieseling et al., 2009). Secreted Sema3F locally inhibits spine development through its receptors PlexinA3 and Neuropilin-2 in hippocampal granule cells (Tran et al., 2009). Sema5A/PlexinA2 signaling inhibits excitatory synapse formation in dentate granule cells (Duan et al., 2014). Sema5B diminishes synaptic connections in cultured hippocampal neurons (O'Connor et al., 2009). However, little is known about the intracellular mechanisms through which Sema/Plexin signaling inhibits synapse formation.

The cytoplasmic domain of Plexin contains a GAP (GTPase-activating protein) domain that inactivates small GTPases (Oinuma et al., 2004; Rohm et al., 2000). Upon activation by Semaphorins, Plexins repel axon outgrowth by inhibiting R-Ras (Negishi et al., 2005b; Hota and Buck, 2012; Tasaka et al., 2012). Recent biochemical and structural analyses demonstrated that the GAP domain of mammalian PlexinA3 is specific for Rap GTPases, which belong to the Ras family of GTPases and regulate the actin cytoskeleton (Wang et al., 2012, 2013). PlexinA3 dimerization by Semaphorin activates its GAP domain, thereby inhibiting Rap1 from inducing neurite retraction. Drosophila PlexA and zebrafish PlexinA1 promote remodeling of epithelial cells by inhibiting Rap1 GTPase during wound healing (Yoo et al., 2016). Another Rap GTPase, Rap2, can inhibit neurite outgrowth (Kawabe et al., 2010). Similar to Sema/Plexin signaling, Rap GTPases regulate synapse formation and function. Rap2 negatively regulate spine number in cultured hippocampal neurons (Fu et al., 2007). Rap1 and Rap2 regulate synaptic activity by removing AMPA receptors from spines during long-term depression and depotentiation, respectively (Zhu et al., 2002, Zhu et al., 2005). While the GAP domain of Plexin is critical to inhibit synapse formation (Duan et al., 2014; Mizumoto and Shen, 2013a), we still do not know whether Plexin regulates synapse patterning via Rap GTPases at presynaptic sites.

In Caenorhabditis elegans, Sema/Plexin signaling functions in vulva formation and male ray development (Dalpé et al., 2005, 2004; Fujii et al., 2002; Ikegami et al., 2004; Liu et al., 2005; Nakao et al., 2007; Nukazuka et al., 2008, 2011). Using this model system, we previously reported that Sema/Plexin signaling in the nervous system mediates a critical inter-axonal interaction for the tiled synaptic innervation of two DA-class cholinergic motor neurons (DA8 and DA9) (Mizumoto and Shen, 2013a). Cell bodies of nine DA neurons in C. elegans reside in the ventral nerve cord, sending dendrites ventrally and axons dorsally to form en passant synapses onto the dorsal body wall muscles. Even though axons of DA neurons show significant overlap, each motor neuron forms synapses onto muscles within specific sub-axonal domains, which do not overlap with those from neighboring DA neurons. This unique synaptic innervation creates tiled synaptic patterns along the nerve cord (White et al., 1986).

Tiled synaptic innervation occurs within most motor neuron classes and may contribute to the sinusoidal locomotion pattern of C. elegans (White et al., 1986). Using a combination of two fluorescent proteins (GFP and mCherry) fused with the presynaptic vesicle protein, RAB-3, and two tissue specific promoters (Figure 1) (Mizumoto and Shen, 2013a), we can visualize this synaptic tiling between DA8 and DA9 neurons. We reported that PLX-1 localizes at the anterior edge of the DA9 synaptic domain in axon-axon interactions in a Semaphorin-dependent manner, where it locally inhibits formation of the presynaptic specialization via its GAP domain. Loss of Semas or plx-1 causes anterior expansion of DA9 synaptic domain and posterior expansion of DA8 synaptic domain. This result indicates loss of inter-axonal interactions between DA8 and DA9 neurons. Consistently, Tran et al., also observed excess dendritic spine formation, specifically within the region close to the cell body, in the plexin knockout mouse (Tran et al., 2009). These findings suggest a conserved mechanism by which Sema/Plexin locally inhibits synapse formation.

Figure 1 with 3 supplements see all
Gain- and loss-of-function rap-2 mutants show synaptic tiling defects.

(A and B) Representative image of synaptic tiling of wild type (A) and plx-1 mutant (B) animals. Images show wyIs446 marker to label synapses in DA8 (GFP::RAB-3) and DA9 (GFP::RAB-3+mCherry::RAB-3). Dotted box represents the magnified images from A and B of the synaptic tiling border. Schematics of DA8 (green) and DA9 (magenta) neurons with parameters for analysis shown on the right. (C–F) Representative images of wyIs446 strains with the following genotypes: rap-2(G12V) overexpression in DA neurons (C), rap-2 G12V (miz18) (D), rap-2 null (gk11) (E) and rap-2 S17A (miz19) (F). Synaptic domains from DA8 and DA9 are highlighted with green and magenta lines, respectively. Asterisks: DA9 cell body. Arrows: dorsal commissure of DA9. Scale bars: 20 μm. (G) Quantification of overlap between DA8 and DA9 synaptic domains. Each dot represents a single animal. Blue bars indicate mean ± SEM. n.s.: not significant; ***p<0.001.

https://doi.org/10.7554/eLife.38801.003

We previously reported that let-60/KRas gain-of-function mutants showed very mild synaptic tiling defects. Since mammalian Plexin acts as a RapGAP, we hypothesized that Rap GTPase is the major downstream effector of PLX-1 to regulate synaptic tiling of DA neurons. Here, we report that rap-2, a C. elegans ortholog of human Rap2A, and its effector kinase mig-15 (TNIK: Traf2- and Nck-interacting kinase) act genetically downstream of plx-1 to regulate synaptic tiling. PLX-1 delineates the border of synaptic tiling by locally inhibiting RAP-2 along the DA9 axon. We also discovered an unexpected role for mig-15 in inhibiting synapse formation. Our results reveal the mechanism underlying Plexin signaling to form fine synaptic map connectivity.

Results

rap-2 functions downstream of plx-1 to regulate synaptic tiling

Three Rap genes exist in the C. elegans genome (rap-1, rap-2 and rap-3). To delineate which Rap GTPase functions downstream of PLX-1 in synaptic tiling between DA8 and DA9 neurons, we first examined the expression patterns of all three rap genes (Figure 1—figure supplement 1). Among them, only rap-2, an ortholog of mammalian Rap2a, was expressed in motor neurons including DA8 and DA9, while rap-1 and rap-3 were not expressed in these cells (Figure 1—figure supplement 1). In wild type animals, synaptic domains of DA8 and DA9 neurons did not show significant overlap, creating tiled synaptic innervation (Figure 1A and G). In the plx-1(nc36) null mutant, synaptic domains of DA8 and DA9 expanded posteriorly and anteriorly, respectively. As a result, synaptic domains of these neurons overlapped significantly (Figure 1B and G).

Since the intracellular domain of Plexin contains a RapGAP domain, we hypothesized that RAP-2 preferentially exists in a GTP-bound form in the plx-1 mutants. The G12V mutant is widely used as a constitutively GTP-form of small GTPases including mammalian Rap2A and C. elegans RAP-1 (Kawabe et al., 2010; Pellis-van Berkel et al., 2005). Expression of a constitutively GTP-bound form of rap-2(G12V) under the A-type neuron specific promoter, Punc-4, elicited a similar synaptic tiling defect as plx-1 mutants (Figure 1C and G). Expression of wild type rap-2 under the unc-4 promoter did not affect the synaptic tiling pattern, suggesting that G12V mutation but not over-expression of rap-2 caused the synaptic tiling defect (Figure 1G). We then generated rap-2(G12V) mutants using CRISPR/Cas9 genome editing. We observed the same synaptic tiling defects in three independent rap-2(G12V) mutant alleles (miz16, miz17 and miz18) as in plx-1 mutants (Figure 1D and G and Figure 1—figure supplement 2). We found a comparable level of gene expression among all three rap-2(G12V) mutants to wild type rap-2 using RT-qPCR (Figure 1—figure supplement 2). These results confirm that the rap-2(G12V) mutation itself, not changes in gene expression, underlie the synaptic tiling defect in rap-2(G12V) mutants.

Surprisingly, the null mutant of rap-2(gk11) also showed the same synaptic tiling defect, as did the constitutively GTP-form of rap-2(G12V) mutants (Figure 1E and G). This result suggests that synaptic tiling requires both the GTP- and GDP-bound forms of RAP-2. Indeed, the constitutively GDP-bound form of rap-2 mutants (S17A: miz19, miz20) showed the identical synaptic tiling defect as rap-2(G12V) and rap-2(gk11) mutants (Figure 1F and G and Figure 1—figure supplement 2). These results suggest that the cycling between GTP- and GDP-forms of RAP-2 is critical to regulate the spatial patterning of synapses.

Similar to plx-1 mutants, the synaptic tiling defect in all rap-2 mutants is caused by both the posterior expansion of DA8 synaptic domain and the anterior expansion of the DA9 synaptic domain (Figure 1—figure supplement 2). However, none of rap-2(G12V), rap-2(S17A) and rap-2(gk11) mutants enhanced or suppressed the synaptic tiling defect in plx-1 mutants (Figure 1G). These results suggest that plx-1 and rap-2 function in the same genetic pathway. Consistent with the expression patterns of rap-1 and rap-3, neither rap-1(pk2082) nor rap-3(gk3975) null mutants showed significant synaptic tiling defects by themselves and did not enhance the synaptic tiling defect in rap-2(gk11) null mutants (Figure 1—figure supplement 1). All mCherry::RAB-3 puncta co-localized with active zone markers, CLA-1 (Xuan et al., 2017) and UNC-10/Rim (Wu et al., 2013) in the DA9 neurons of plx-1(nc36) and rap-2(gk11) mutants, suggesting that RAB-3 puncta represent bona fide synapses (Figure 1—figure supplement 3). Taken together, these data indicate that plx-1 and rap-2 act in the same genetic pathway for synaptic tiling in DA neurons.

RAP-2 functions cell autonomously in DA neurons

We next determined the cellular location for rap-2 function. Since rap-2(gk11) null mutants showed a synaptic tiling defect, we conducted tissue specific rescue experiments using tissue-specific promoters as previously described (Mizumoto and Shen, 2013a). Expression of rap-2 in the post-synaptic body wall muscle cells under the hlh-1 promoter or in another class of cholinergic motor neurons in the dorsal nerve cord (DB neurons) under the truncated unc-129 promoter did not rescue the synaptic tiling defect in rap-2(gk11) animals (Figure 2A, B and G). However, DA neuron-specific expression using the unc-4c promoter strongly rescued the synaptic tiling defect (Figure 2C and G). DA9-specific expression of rap-2 under the mig-13 promoter partially rescued the synaptic tiling defect (Figure 2E and G). DA9-specific expression of rap-2 rescued the phenotype of anterior expansion of the DA9 synaptic domain but not the posterior expansion of DA8 synaptic domain (Figure 2H and I). These results suggest that rap-2 regulates synapse patterning in a cell-autonomous manner. In contrast to the DA9-specific rescue experiment in rap-2 mutants, DA9-specific expression of plx-1 cDNA was sufficient to rescue synaptic defects in both DA9 and DA8 (Mizumoto and Shen, 2013a) (see Discussion).

rap-2 functions in DA neurons.

(A–F) Representative images of rap-2; wyIs446 animals expressing Phlh-1::rap-2 (A), Punc-129::rap-2 (B), Punc-4c::rap-2 (C), Punc-4c::Rap2a (human) (D), Pmig-13::rap-2 (E) and Punc-4c;;rap-1 (F). Synaptic domains of DA8 and DA9 are highlighted with green and magenta lines, respectively. Asterisks: DA9 cell body. Arrows: dorsal commissure of DA9. Scale bars: 20 μm. (G–I) Quantification of DA8/DA9 overlap (G), DA9 synaptic domain (H) and DA8 asynaptic domain (I). Each dot represents a single animal. Blue bars indicate mean ± SEM. n.s.: not significant; ***p<0.001; **p<0.01; *p<0.05.

https://doi.org/10.7554/eLife.38801.007

We also observed that expression of human Rap2a in DA neurons rescued the synaptic tiling defect of rap-2 mutants, suggesting the function of rap-2 in synapse patterning is conserved across species (Figure 2D and G). Previous work suggested a partial functional redundancy between rap-1 and rap-2 in C. elegans (Pellis-van Berkel et al., 2005). However, we found that rap-1 expression in DA neurons did not rescue the synaptic tiling defect of rap-2 mutants, suggesting functional diversity between rap-1 and rap-2 (Figure 2F and G). Taken together, we conclude that rap-2 functions cell autonomously in DA neurons to regulate synaptic tiling.

RAP-2 activity is spatially regulated by PLX-1

Previously, we demonstrated that PLX-1::GFP is localized at the anterior edge of the DA9 synaptic domain, where it negatively regulates synapse formation through its cytoplasmic GAP domain (Figure 3A and E) (Mizumoto and Shen, 2013a). In the rap-2(gk11) mutant background, we observed no change in PLX-1::GFP localization but did observe ectopic synapses in the axonal region anterior to the PLX-1::GFP domain (Figure 3B and F). This result is consistent with our hypothesis that rap-2 acts downstream of plx-1 to regulate synaptic tiling. Together with our finding that synaptic tiling requires both GTP- and GDP-bound forms of RAP-2, we speculate that PLX-1 acting at the anterior edge of the DA9 synaptic domain regulates the spatial activity of RAP-2 along the axon.

PLX-1::GFP localization is not affected in rap-2 and mig-15 mutants.

(A–D) Representative image of PLX-1::GFP alone (top) and PLX-1::GFP with mCherry::RAB-3 (middle) labeled with wyIs320 in DA9 of wildtype (A), rap-2 (gk11) (B) and mig-15(rh148) (C). Bracket indicates the PLX-1::GFP patch localized at the anterior edge of the DA9 synaptic domain. Arrowheads indicate ectopic synapses formed anterior to the PLX-1::GFP patch. Scale bars: 20 μm. (E–G) Quantification of the normalized mCherry::RAB-3 signal (magenta) and PLX-1::GFP signal (green) in the dorsal axon of DA9 in wildtype (E), rap-2 (gk11) (F) and mig-15(rh148) (G). Animal were aligned according to the PLX-1::GFP patch at the anterior edge of the DA9 synaptic domain (orange arrow). Light colors indicate SEM.

https://doi.org/10.7554/eLife.38801.008

We then sought to determine the spatial distribution of GTP-RAP-2 in DA9 axon. We conducted Fluorescence Lifetime Imaging Microscopy (FLIM)-based FRET (Förster Resonance Energy Transfer) measurements using EGFP-Rap2A (human) and mRFP-RalGDS(RBD: Ras Binding Domain)-mRFP (Yasuda et al., 2006). As RalGDS-RBD specifically binds to GTP-Rap2 but not GDP-Rap2 (Ohba et al., 2000), FRET from EGFP-Rap2A to mRFP-RalGDS(RBD)-mRFP can be used as a readout of Rap2 activity. We detected FRET signal as a change of GFP fluorescence lifetime (Figure 4A). In HeLa cells, we observed a shorter lifetime of constitutively bound GTP construct EGFP-Rap2A(G12V) compared to GDP-bound EGFP-Rap2A(S17A), indicating that the FRET sensor can detect the nucleotide state of Rap2A (Figure 4B and C). Due to the low expression of C. elegans RAP-2 constructs in HeLa cells, we were not able to test whether the mammalian FRET sensor can detect C. elegans RAP-2 activity (data not shown).

PLX-1 locally inhibits Rap2 activity in DA9.

(A) Schematic of Rap2 FRET sensor system. Binding of mRFP- RalGDS(RBD)-mRFP to EGFP-Rap2 induces FRET from EGFP to mRFP, leading to decreased EGFP fluorescence lifetime. (B) Representative fluorescence (top) and fluorescence lifetime images (bottom) of HeLa cells expressing Rap2 FRET sensor (mRFP-RalGDS(RBD)-mRFP) with either constitutively GTP(G12V)- or GDP(S17A)- forms of human Rap2A. (C) Quantification of the binding fraction of Rap2 mutants in HeLa cells. Binding fractions measured from fluorescence decay curves of individual cells (G12V, n = 25; S17A, n = 25), as described previously (Murakoshi et al., 2011). Data shown as mean ± SEM. Asterisks denote statistical significance (p<0.05, student’s t-test). (D) Representative images of fluorescence lifetime (top) and fluorescence (bottom) of mizIs19 in wild type (left) and plx-1 mutants (right). White arrowheads indicate the position of the putative synaptic tiling border, as judged by the slight dorsal shift of the DA9 axon, as reported previously (Mizumoto and Shen, 2013a). (E) Representative images of fluorescence lifetime (top) and fluorescence (bottom) of mizIs19 in unc-104 (left) and unc-104; plx-1 double mutants (right). (F) Quantification of the difference in binding fraction of GTP-Rap2a and RalGDS(RBD) between synaptic region (dotted boxes in D) and anterior asynaptic region (solid boxes in D). The binding fraction measured in synaptic region (dotted rectangle) was subtracted by that in non-synaptic region (solid rectangle). Five micrometers along the axon line from the synaptic tiling border were used for quantification. Data presented as mean ± SEM (wild type, n = 22; plx-1, n = 17). (G) Quantification of the difference in binding fraction of GTP-Rap2A and RalGDS(RBD) between synaptic region (dotted boxes in E) and anterior asynaptic region (solid boxes in E). The binding fraction measured in synaptic region (dotted rectangle) subtracted from that in non-synaptic region (solid rectangle). Five micrometers along the axon line from the synaptic tiling border were used for quantification. Data are presented as mean ± SEM (unc-104, n = 21; unc-104; plx-1, n = 23). (H) Quantification of the difference in binding fraction of GTP-Rap2a and RalGDS(RBD) between synaptic region and anterior asynaptic region in plx-1 mutants and plx-1 mutants expressing rescuing constructs. Five micrometers along the axon line from the synaptic tiling border were used for quantification. Data are presented as mean ± SEM (no array, n = 35; Ex[Pmig-13::plx-1(RA)], n = 39; Ex[Pmig-13::plx-1(Δsema)], n = 41; Ex[Pmig-13::plx-1], n = 51).

https://doi.org/10.7554/eLife.38801.009

We then expressed EGFP-Rap2A and mRFP-RalGDS(RBD)-mRFP FRET sensors in DA9 neurons in C. elegans. As human Rap2a rescued the synaptic tiling defect of rap-2(gk11) mutants (Figure 2G), we reasoned that the activity pattern of human Rap2A should recapitulate that of endogenous RAP-2. We indeed observed lower Rap2A activity at the anterior edge of the DA9 synaptic domain compared to within the synaptic domain (Figure 4D and F). This observation is consistent with the localization of PLX-1::GFP at the anterior edge of DA9 synaptic domain (Figure 3A) (Mizumoto and Shen, 2013a). Local inhibition of Rap2a activity was strongly diminished in the plx-1 mutant background (Figure 4D and F). Higher Rap2 activity in the synaptic region could simply indicate the presence of synapses within the synaptic domain, rather than Rap2 inactivation by Plexin at the anterior edge of the synaptic domain. To exclude this possibility, we examined Rap2 activity in unc-104/Kif1A mutants, which show no synapses are formed in DA9 axon (Ou et al., 2010). We showed previously that PLX-1::GFP localization to the synaptic tiling border was independent of synapses, since it was unaffected in unc-104/Kif1A mutants (Mizumoto and Shen, 2013a). In unc-104 mutants, we observed the same local inhibition of Rap2A activity at the putative synaptic tiling border, but not in unc-104; plx-1 double mutants (Figure 4E and G), indicating that Plexin controls local Rap2 activity independent of synapses.

To understand that this local Rap2 inactivation at the synaptic tiling border depends on the localized RapGAP activity of PLX-1, we examined the rescue activity of two PLX-1 mutant constructs, PLX-1(RA) and PLX-1(ΔSema), neither of which rescued the synaptic tiling defect of plx-1 mutants (Mizumoto and Shen, 2013a). PLX-1(RA) is a GAP-deficient mutant but localizes normally at the anterior edge of the DA9 synaptic domain. PLX-1(ΔSema) contains intact GAP domain but cannot be activated by the endogenous ligand and shows diffused localization due to deletion of the extracellular SEMA domain (Mizumoto and Shen, 2013a). We observed no local Rap2 inactivation in plx-1 mutant animals expressing these mutant PLX-1 constructs in DA9, while expression of wild type PLX-1 cDNA rescued local Rap2 inactivation at the anterior edge of the DA9 synaptic domain (Figure 4H).

While we do not fully exclude the possibility that PLX-1 indirectly regulates local Rap2 activity, these data taken together with the biochemical evidence that mammalian Plexin acts as RapGAP (Wang et al., 2013, 2012) strongly suggests that Plexin localized at the anterior edge of the DA9 synaptic domain locally inactivates Rap2 GTPase to delineate the synaptic tiling border in DA9.

mig-15(TNIK) regulates synaptic tiling

In mammals, TNIK (Traf2 and Nck1-interacting kinase) acts with Rap2 to regulate neurite extension, AMPA receptor trafficking in hippocampal neurons and microvilli formation in intestinal cells (Hussain et al., 2010; Kawabe et al., 2010; Gloerich et al., 2012). In C. elegans, mig-15 is the sole ortholog of mammalian TNIK and its paralog MINK1 (Misshapen-like kinase 1), which also is an effector of Rap GTPase (Nonaka et al., 2008 ). mig-15 can regulate various cellular processes, such as axon guidance and cell migration (Chapman et al., 2008; Poinat et al., 2002; Shakir et al., 2006; Teulière et al., 2011). We found that mig-15(rh148) hypomorphic mutants showed a severe synaptic tiling defect (Figure 5A and D). Similar to plx-1 and rap-2 mutants, the synaptic tiling defect followed the anterior expansion of the DA9 synaptic domain and the posterior expansion of the DA8 synaptic domain (Figure 5E and F). All RAB-3 puncta in mig-15(rh148) mutants co-localized with active zone markers, CLA-1 and UNC-10 (Figure 1—figure supplement 3), suggesting that these RAB-3 puncta represent bona fide synapses. We observed axon guidance defects (23%, n = 100) or ectopic branch formation (56%, n = 100) in DA9 of mig-15 mutant animals (Figure 5—figure supplement 1). These were excluded from our analysis of synaptic tiling phenotypes. While only half of the mig-15 mutant animals showed axon guidance defects or ectopic branch formation, the synaptic tiling defect of mig-15 mutants was almost fully penetrant (Figure 5D). We did not observe significant synaptic tiling defects in cdh-4(rh310) mutants (DA8/DA9 overlap: 4.6 ± 1.02 μm, n = 21), which can exhibit an axon defasciculation phenotype in the dorsal nerve cord neurons (Schmitz et al., 2008). These data suggest that the synaptic tiling defect in the mig-15 mutants is not a secondary effect of axon outgrowth and guidance.

Figure 5 with 2 supplements see all
mig-15(TNIK) mutants show a severe synaptic tiling defect.

(A–C) Representative images of synaptic tiling marker (wyIs446) in mig-15(rh148) (A), rap-2(gk11); mig-15(rh148) (B) and plx-1(nc36); mig-15(rh148) (C) mutants. Synaptic domains of DA8 and DA9 are highlighted with green and magenta lines, respectively. Asterisks: DA9 cell body. Arrows: dorsal commissure of DA9. Scale bars: 20 μm. (D–F) Quantification of overlap between DA8 and DA9 synaptic domains (D), DA9 synaptic domain (E) and DA8 asynaptic domain (F) in respective mutant backgrounds. Each dot represents measurement from a single animal. Blue bars indicate mean ± SEM. n.s.: not significant; ***p<0.001.

https://doi.org/10.7554/eLife.38801.010

The other two nonsense alleles (rh326: Q439Stop, rh80: W898Stop) also showed identical synaptic tiling defects as mig-15(rh148) (Figure 5—figure supplement 2). mig-15(rh80) has a nonsense mutation within the highly conserved CNH (citron/NIK homology) domain, which is required to interact with Rap2 in both mammals and C. elegans (Taira et al., 2004). This suggests a physical interaction between RAP-2 and MIG-15 for synaptic tiling. plx-1 or rap-2 mutants did not enhance the synaptic tiling defect in mig-15 mutants (Figure 5B–F). This result is consistent with our hypothesis that mig-15 acts in the same genetic pathway as plx-1 and rap-2. The PLX-1::GFP patch at the putative synaptic tiling border was unaffected in mig-15 mutants, even though the position of the PLX-1::GFP patch has shifted slightly posteriorly compared with wild type (Figure 3C and G). Taken together, these results suggest that mig-15 acts downstream of plx-1 to regulate synaptic tiling.

Interestingly, the degree of overlap between DA8 and DA9 synaptic domains was even larger in mig-15 mutants than those observed in plx-1 and rap-2 mutants (compare Figures 1G and 5D), suggesting that mig-15 also acts downstream of additional signaling pathways (see discussion).

mig-15 functions in DA neurons

We then determined in which cells mig-15 functions by conducting tissue specific rescue experiments. Since several mig-15 isoforms (wormbase and data not shown) exist, we used the mig-15 genomic sequence for the rescue experiments. Expression of mig-15 under the DA neuron specific promoter (Punc-4c) strongly rescued the synaptic tiling defect of mig-15(rh148) mutants (Figure 6A and F), consistent with our hypothesis that mig-15 acts in the same genetic pathway as plx-1 and rap-2.

mig-15 functions in DA neurons.

(A–D) Representative images of synaptic tiling marker (wyIs446) in the following backgrounds: mig-15(rh148); Ex[Punc-4c::mig-15] (A), mig-15(rh148); Ex[Pmig-13::mig-15] (B), wild type (C) and wild type animals expressing dominant-negative mig-15(K50A) in DA neurons (D). Synaptic domains of DA8 and DA9 are highlighted with green and magenta lines, respectively. Asterisks: DA9 cell body. Arrows: dorsal commissure of DA9. Scale bars: 20 μm. (E) Amino acid alignment of amino-terminal region of MIG-15 and TNIK. A kinase-dead mutation in TNIK (K54A) and corresponding mutation in MIG-15 (K50A) are highlighted in red. (F–I) Quantification of overlap between DA8 and DA9 synaptic domains (F and I), DA9 synaptic domain (G), DA8 asynaptic domain (H) in respective mutant backgrounds. Each dot represents measurements from a single animal. Blue bars indicate mean ± SEM. n.s.: not significant; ***p<0.001; *p<0.05.

https://doi.org/10.7554/eLife.38801.013

Expression of mig-15 in both DA8 and DA9 rescued both posterior expansion of the DA8 synaptic domain and anterior expansion of the DA9 synaptic domain (Figure 6G and H). DA9 specific expression of mig-15 under the mig-13 promoter rescued anterior expansion of the DA9 synaptic domain, suggesting that mig-15 functions cell autonomously in DA9 (Figure 6B and G). We observed that Pmig-13::mig-15 weakly rescued the posterior expansion of the DA8 synaptic domain (Figure 6H). This is likely due to the leaky expression of mig-15 in DA8, as the mig-15 genomic fragment without promoter showed slight rescue of the synaptic tiling defect in mig-15(rh148) mutants (Figure 6F). Kinase dead TNIK mutants act as a dominant-negative (Mahmoudi et al., 2009). In DA neurons, expression of mutant mig-15(kd), which carries the same mutation at the corresponding amino acid of the dominant-negative TNIK (Figure 6E), in DA neurons caused a severe synaptic tiling defect (Figure 6C, D and I). Based on these results, we conclude that mig-15 functions cell autonomously in DA neurons.

mig-15 inhibits synapse formation

We observed that DA9-specific expression of mig-15 under the mig-13 promoter in mig-15 mutants often exhibited a shorter synaptic domain compared to wild type (Figure 6B and G). So, we speculated that an excess amount of mig-15 inhibits synapse formation. We tested the effect of mig-15 overexpression in the wild type background. Strikingly, DA9-specific mig-15 overexpression in wild type (mig-15(OE)) significantly reduced synapse number compared to wild type (Figure 7A, C and D and Figure 7—figure supplement 1). This reduction occurred without affecting the overall morphology of the DA9 neuron (Figure 5—figure supplement 1). Conversely, DA9 synapse number was significantly increased in the mig-15(rh148) mutants (Figure 7B and D and Figure 7—figure supplement 1). mig-15 overexpression also significantly reduced synapse number in DD-type GABAergic motor neurons (Figure 7—figure supplement 2). These results indicate that mig-15 is a negative regulator of synapse formation. Further, pan-neuronal expression of mig-15 under the rab-3 promoter caused severe uncoordinated locomotion in wildtype animals (Figure 7—figure supplement 3). These locomotor defects occurred concomitant with significantly reduced GFP::RAB-3 intensity in the dorsal nerve cord in mig-15 over-expressing animals and without causing significant axon guidance defects (Figure 7—figure supplement 3). Taken together, these data indicate that reduced synapse number by mig-15 overexpression disrupted proper functioning of the motor circuit. Importantly, we observed no significant increase in synapse numbers in plx-1 or rap-2 mutants (Figure 7—figure supplement 1), suggesting that the role of mig-15 in negatively regulating synapse number is independent of its role in PLX-1/RAP-2 -mediated synaptic tiling (Figure 8G).

Figure 7 with 3 supplements see all
mig-15 negatively regulates synapse number.

(A–C) Representative images of DA9 presynaptic marker, GFP::RAB-3 (wyIs85), in wild type (A), mig-15(rh148) (B) and mig-15 overexpressing animals (C). Brackets represent DA9 synaptic domain. Asterisks: DA9 cell body. Arrows: dorsal commissure of DA9. Scale bars: 20 μm. (D) Quantification of DA9 synapse number. Each dot represents measurements from a single animal. Blue bars indicate mean ± SEM. n.s.: not significant; ***p<0.001; *p<0.05. (E–H) Representative images of synaptic branched F-actin labeled with GFP::ut-CH (wyIs329) in wild type (E), rap-2(gk11) (F), mig-15(rh148) (G) and mig-15 overexpressing animals (H). (I) Quantification of the length of GFP::ut-CH. Distance from the dorsal commissure to the most anterior and brightest GFP spot was measured. Blue bars indicate mean ± SEM. n.s.: not significant; ***p<0.001; **p<0.01, *p<0.05.

https://doi.org/10.7554/eLife.38801.014
PLX-1/RAP-2 signaling coordinates synapse number and synaptic tiling border.

(A–C) Representative images of synaptic tiling marker (wyIs446) overexpressing mig-15 in DA9 from wild type (A), plx-1(nc36) (B) and rap-2(gk11) (C). Synaptic domains of DA8 and DA9 highlighted with green and magenta lines, respectively. Asterisks: DA9 cell body. Arrows: dorsal commissure of DA9. Scale bars: 20 μm. (D) Schematic illustration of synapse distribution in wild type (left), animals overexpressing mig-15 in DA9 (middle) and synaptic tiling mutants overexpressing mig-15 in DA9 (right). Arrows indicate the synaptic tiling border. Colored arrows represent expanded or shortened synaptic domain from DA8 (green) and DA9 (magenta). (E) Quantification of the DA8 asynaptic domain. (F) Quantification of the DA9 synaptic domain. Each dot represents measurements from a single animal. Blue bars indicate mean ± SEM. n.s.: not significant; ***p<0.001. (G) A model of synaptic tiling. PLX-1/RAP-2 signaling controls synaptic tiling via MIG-15, while MIG-15 also plays crucial roles in inhibiting the number of synapses. Solid lines indicate putative direct regulations, dotted lines represent indirect or unknown mode of regulations.

https://doi.org/10.7554/eLife.38801.018

Rap GTPase and TNIK are well-known actin cytoskeleton regulators (Lin et al., 2010, 2008; Taira et al., 2004). Previous studies demonstrated that presynaptic development requires ARP2/3-dependent branched F-actin (Chia et al., 2012, 2014). Branched F-actin visualized by GFP::ut-CH (utrophin calponin homology domain) is enriched within the DA9 synaptic domain (Figure 7E) (Chia et al., 2012; Mizumoto and Shen, 2013a). We predicted that mig-15 negatively regulates synapse formation by re-organizing branched F-actin at the anterior edge of the synaptic domain. Consistently, we observed longer synaptic F-actin distribution in rap-2(gk11) and mig-15(rh148) mutants (Figure 7F, G and I). While GFP::ut-CH was observed in the posterior asynaptic axonal region or in the dendrite of DA9, synapse formation is likely inhibited by Wnt and Netrin signaling as previously reported (Klassen and Shen, 2007; Poon et al., 2008). Conversely, overexpression of mig-15 in DA9 significantly decreased the length of synaptic F-actin (Figure 7H and I). Overexpression of mig-15 also appeared to decrease the overall amount of synaptic F-actin (Figure 7H). This result suggests that mig-15 inhibits synapse formation by negatively regulating the formation of synaptic F-actin.

plx-1 and rap-2 coordinate synaptic tiling border positioning and synapse number 

mig-15(OE) reduced the number of synapses in DA9. As a result, the length of the DA9 synaptic domain was significantly reduced in mig-15(OE) animals than in wild type (Figure 8A and E). Yet, synaptic tiling is maintained without a significant gap between DA8 and DA9 synaptic domains in mig-15(OE) animals, suggesting that the position of synaptic tiling border shifted posteriorly in mig-15(OE) animals. Indeed, the length of the posterior asynaptic domain of DA8 was significantly shorter in mig-15(OE) animals, indicating that the DA8 synaptic domain expanded posteriorly (Figure 8D). The PLX-1::GFP patch at the anterior edge of the DA9 synaptic domain also shifted posteriorly in mig-15(OE) animals with reduced synapse number (Figure 3D). These results strongly suggest that the PLX-1/RAP-2 signaling pathway may specify the position of the synaptic tiling border according to the available number of synapses in each DA neuron (Figure 8G). We propose that synaptic tiling is a mechanism to maintain a uniform distribution of synapses from one class of motor neuron in the nerve cord. Consistently, we found that DA8 synaptic domain did not shift posteriorly when mig-15 was overexpressed in DA9 of the synaptic tiling mutants, plx-1 or rap-2 (Figure 8B–E). This result suggests that DA8 no longer senses the reduction of DA9 synapse number in the synaptic tiling mutants. Synapse number was not different between mig-15(OE) and in rap-2(gk11); mig-15(OE) animals (Figure 7—figure supplement 1), suggesting that mig-15 is not dependent on the Plexin/Rap2 signaling pathway to inhibit synapse number (Figure 8G).

In summary, we demonstrate that synaptic tiling maintains a uniform distribution of synapses from one class of motor neurons along the nerve cord. Further, our results indicate plx-1 and rap-2 play critical roles in this process by coordinating the position of the synaptic tiling border.

Discussion

While much is known about the morphogenic triggers for axon guidance and patterned synapse formation, the downstream sequelae of these intracellular effectors have remained unclear. We discovered the role of Rap2 GTPase and TNIK in synapse pattern formation in C. elegans. Since Sema/Plexin signaling processes to inhibit synapse formation are well conserved across species, we propose that Sema/Plexin in mammals also utilize Rap2 and TNIK to regulate synapse patterning.

Cell autonomous and non-autonomous functions of Sema/Plexin signaling components

Previously we showed that both smp-1 and plx-1 are necessary and sufficient in DA9, which suggests that smp-1 and plx-1 act cell-autonomously in DA9 and non-autonomously in DA8 to regulate synaptic tiling. We proposed that Sema/PLX-1 in DA9 send a retrograde signal to DA8 through an unidentified signaling molecule (X) to induce the synaptic tiling pattern in DA8 (Mizumoto and Shen, 2013a). However, we found that both rap-2 and mig-15 act cell autonomously, since our DA9-specific rescue experiment only rescued the DA9 phenotype, but not the DA8 phenotype. This conclusion is further supported since the synaptic tiling defects of these mutants were fully rescued when both neurons express functional rap-2 cDNA or mig-15 genomic DNA. We propose that each neuron utilizes a different set of cell surface proteins but share common intracellular mechanisms to specify synapse patterning. Diverse signaling and cell adhesion molecules, such as atrial natriuretic peptide receptor (NPR) and GPCRs, regulate Rap activity (Gloerich and Bos, 2011; Birukova et al., 2008; Weissman et al., 2004). Screening for these potential Rap regulators should identify novel molecules that interact with Sema/Plexin and act in DA9.

Cycling of Rap GTPase activity in synaptic tiling

We showed that proper synapse patterning requires both GDP- and GTP-forms of RAP-2. Considering that PLX-1 regulates the spatial distribution of RAP-2 activity and mig-15 acts genetically downstream of plx-1 in synaptic tiling, RAP-2 may also locally regulate MIG-15 (TNIK). While we did not observe a specific subcellular localization of GFP-MIG-15 in DA9 (data not shown), PLX-1 and RAP-2 may instead regulate MIG-15 activity rather than its spatial localization. Further biochemical characterization of MIG-15 regulation by GTP-RAP-2 or GDP-RAP-2 will elucidate the exact functions of RAP-2 in synapse patterning.

Small GTPase activity is regulated by GAP and GEF (Guanine nucleotide exchange factor) proteins. Yet, we did not observe significant synaptic tiling defects in mutants of putative RAP-2 GEFs, which include pxf-1(RAPGEF2/6) and epac-1(RAPGEF3/4/5) (data not shown) (Frische et al., 2007; Pellis-van Berkel et al., 2005). We speculate that multiple RapGEFs act redundantly to activate RAP-2 in synaptic tiling.

mig-15(TNIK) may integrate multiple inhibitory cues during synapse formation

mig-15 mutants show a greater degree of overlap between DA8 and DA9 synaptic domains than plx-1 or rap-2 mutants. This effect partially occurs from excess synaptogenesis in the posterior asynaptic domain of both DA8 and DA9 neurons. Previously, we demonstrated that Wnt morphogens and their receptors, Frizzled, instruct synaptic topographic patterning by locally inhibiting synapse formation. Indeed, synaptic tiling defects in mig-15 mutants was somewhat similar to the combined effect of plx-1 and wnt mutants (Mizumoto and Shen, 2013b). TNIK can act as a positive regulator of the canonical Wnt signaling pathway in colorectal cancer cells (Mahmoudi et al., 2009; Shitashige et al., 2010). While we do not know whether the canonical Wnt signaling pathway contributes to local inhibition of synapse formation, we propose that TNIK integrates multiple signaling pathways for precise synapse pattern formation.

In addition to its role in synapse pattern formation, our data indicate that mig-15 also plays a role as a negative regulator of synapse number. Since neither plx-1 nor rap-2 mutants showed a significant increase in synapse number in DA9, mig-15 may inhibit synapse formation in a different signaling pathway (Figure 8G). As we observed a global reduction of synaptic actin staining in animals over-expressing mig-15, we propose that mig-15 controls synapse number by regulating synaptic F-actin.

The exact mechanisms of synaptic actin regulation by TNIK remain undetermined. TNIK could activate the JNK kinase pathway (Taira et al., 2004; Fu et al., 1999). The MIG-15/JNK-1 signaling pathway inhibits axonal branch formation in s C. elegans ensory neurons (Crawley et al., 2017). In contrast to these well-established roles of MIG-15/TNIK as an activator of the JNK pathway, we did not observe any synaptic tiling defect nor change in synapse number in jnk-1 mutant animals (Figure 5—figure supplement 2 and data not shown). Our results suggest mig-15 does not inhibit synapse formation through the JNK pathway.

Due to the pleiotropic phenotype of the mig-15 mutants, our genetic and phenotypic analyses of mig-15 did not exquisitely reveal the mechanistic relationship between PLX-1 and MIG-15 in synaptic tiling regulation. Further biochemical studies of MIG-15 regulation by Plexin/Rap2 in synaptic tiling will elucidate the molecular mechanisms that underlie the role of MIG-15/TNIK in synapse pattern formation.

Plexin signaling and diseases

Aberrant neuronal wiring underlies many neurological disorders. Not surprisingly, Semaphorin and Plexin genes are associated with various neurodevelopmental disorders and intellectual disabilities, including autism spectrum disorders (ASD) and schizophrenia (Mah et al., 2006; Gene Discovery Project of Johns Hopkins & the Autism Consortium et al., 2009). For example, PLXNB1, SEMA3A, SEMA4D and SEMA6C are significantly upregulated in the prefrontal cortices of schizophrenic patients (Eastwood et al., 2003; Gilabert-Juan et al., 2015). On the other hand, non-synonymous variations in the Sema3D gene had a significant protective effect against developing schizophrenia (Fujii et al., 2011). More recent work showed that loss of Sema5A/PlexA2 signaling induces excess excitatory synapse formation in granule cells, which caused ASD-like behavioural defects in mice (Duan et al., 2014).

Similar to Sema/Plexin signaling, TNIK is also associated with various neurological disorders, including schizophrenia and intellectual disabilities (Anazi et al., 2016; Potkin et al., 2010). TNIK can also physically bind and act with DISC1 (Disrupted in Schizophrenia 1) to regulate synaptic composition (Wang et al., 2011). So, we propose that the Sema/Plexin/Rap2/TNIK signaling pathway plays a critical role to precisely define synaptic connections and its disruption may induce serious neurological disorders.

Interestingly, SNPs in Plexin genes are also associated with extremely high IQ (Spain et al., 2016). Recent work suggests that loss of PlexinA1 confers better motor control in rodents due to increased synaptic connectivity in the corticospinal cord (Gu et al., 2017). Further studies on the Plexin/Rap2/TNIK signaling pathway in synapse map formation, as presented here, will likely reveal the genetic basis of these disorders and conditions.

Materials and methods

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Gene
(C. elegans)
rap-2NAC25D7.7
Gene
(C. elegans)
plx-1NAY55F3AL.1
Gene
(C. elegans)
mig-15NAZC504.4
Strain, strain background
(C. elegans)
rap-2(gk11)C. elegans stock center (CGC)VC14
Strain, strain background
(C. elegans)
mig-15(rh148)C. elegans stock center (CGC)NJ490
Strain, strain background
(C. elegans)
plx-1(nc36)C. elegans stock center (CGC)ST36
Strain, strain background
(C. elegans)
rap-2(miz18)This studyUJ401G12V mutant
Strain, strain background
(C. elegans)
rap-2(miz19)This studyUJ402S17A mutant
Strain, strain background
(C. elegans)
wyIs446This studyTV14517synaptic tiling marker
Strain, strain background
(C. elegans)
mizIs19This studyUJ397Rap2 FRET sensor strain

Strains

All C. elegans strains were derived from Bristol N2 and raised on OP50 Escherichia coli-seeded nematode growth medium (NGM) plates at 20C and maintained as described previously (Brenner, 1974). The following mutants were used in this study: unc-104(e1265)II, plx-1(nc36)IV, rap-1(pk2082)IV, rap-3(gk3975)IV, jnk-1(gk7)IV rap-2(gk11)V, rap-2(miz16)V, rap-2(miz17)V, rap-2(miz18)V, rap-2(miz19)V. rap-2(miz20)V, mig-15(rh148)X, mig-15(rh80)X, mig-15(rh326)X.

CRISPR/Cas9 genome editing 

rap-2(miz16)V, rap-2(miz17)V, rap-2(miz18)V, rap-2(miz19)V. rap-2(miz20)V were generated using Co-CRISPR method (Kim et al., 2014). unc-22 or dpy-10 co-CRISPR markers were used for selecting candidate animals (Kim et al., 2014; Arribere et al., 2014). Vectors for sgRNA and Cas9 were obtained from Addgene (Plasmid ID: 46169 and 46168, respectively) (Friedland et al., 2013). The rap-2 guide RNA sequence (5’ – gTAGTGGAGGTGTCGGAAAAT-3’) was designed using MIT CRISPR design tool (crispr.mit.edu:8079) and inserted into sgRNA vector using Q5 Site-Directed Mutagenesis kit (NEB). Repair templates with either G12V (miz17 and miz18) and S17A (miz19 and miz20) mutation were generated by PCR with primer sets carrying corresponding mutations (see supplemental Experimental Procesures). Synonymous mutations were also introduced in the sgRNA recognition sequence to avoid Cas9 recruitment to the edited genome. PCR products were cloned into EcoRI site of the pBluescript SK(+) vector. Synthesized double-stranded DNA (GeneArt, Thermo Fisher) was used as a repair template for generating rap-2(miz16) mutant.

Plasmid constructions

C. elegans expression clones were made in a derivative of pPD49.26 (A. Fire), the pSM vector (kind gift from S. McCarroll and C. I. Bargmann). Primer sets used in this study are listed in the Supplemental Experimental Procedures. The following constructs were used and transgenes were generated using standard microinjection method (Mello et al., 1991): wyIs446 (Punc-4::2xGFP-rab-3; Pmig-13::mCherry-rab-3; Podr-1::RFP), wyIs85 (Pitr-1::GFP-rab-3; Podr-1::RFP), wyIs442 (Pflp-13::2xGFP-rab-3; Pplx-2::mCherry-rab-3; Podr-1::RFP), wyIs320 (Pitr-1::plx-1::GFP; Pmig-13::mCherry;;rab-3; Podr-1::GFP), wyIs329 (Pmig-13::GFP-ut-CH; Pmig-13::mCherry::rab-3; Podr-1::GFP), wyIs524 (Punc-4::2xGFP-rab-3; Pmig-13::mCherry-rab-3; Podr-1::RFP), wyIs685 (Pmig-13::mCherry::rab-3; Pmig-13::GFPnovo2::cla-1; Podr-1::GFP) mizIs1 (Pitr-1::GFPnovo2-CAAX; Pvha-6::zif-1; Pitr-1::mCherry::rab-3; Podr-1::GFP), mizIs19 (Pmig-13::eGFP::hRap2a; Pmig-13::mRFP-RalGDS(RBD)-mRFP; Podr-1::GFP), mizIs33 (Prab-3::mig-15; Podr-1::GFP), jsIs682 (Prab-3::GFP::rab-3; lin-15(+)), wyEx5445 (Prap-1::GFP; Punc-4::myr-mCherry; Podr-1::RFP), wyEx5464 (Prap-2::GFP; Punc-4::myr-mCherry; Podr-1::RFP), mizEx194 (Prap-3::GFP; Pmig-13::myr-mCherry; Podr-1::RFP), mizEx165 (Phlh-1::rap-2; Podr-1::GFP), mizEx164 (Punc-129::rap-2; Podr-1::GFP), mizEx174 (Punc-4c::rap-2; Podr-1::GFP), mizEx157 (Pmig-13::rap-2; Podr-1::GFP), mizEx156 (Punc-4c::hRap2a; Podr-1::GFP), mizEx177 (Punc-4c::rap-1; Podr-1::GFP), mizEx151 (Pmig-13::mig-15; Podr-1::GFP), mizEx147 (Punc-4c::mig-15; Podr-1::GFP), mizEx153 (ΔpSMmig-15; Podr-1::GFP), mizEx178 (Punc-4c::mig-15(K50A); Podr-1::GFP), mizEx173 (Punc-4::rap-2(G12V); Podr-1::GFP), mizEx179 (Pflp-13::mig-15; Podr-1::GFP), mizEx170 (Pmig-13::mig-15; Podr-1::GFP), mizEx197 (Pmig-13::mig-15; Podr-1::GFP), mizEx210 (Pmig-13::mig-15; Podr-1::RFP), mizEx257 (Prab-3::GFP; Prab-3::mCherry::rab-3, Podr-1::mScarlet::CAAX), mizEx272 (Podr-1::GFP; Pmig-13::unc-10::TdTomato); mizEx309 (Pmig-13::plx-1(RA); Podr-1::RFP), mizEx312 (Pmig-13::plx-1(Δsema); Podr-1::RFP); mizEx314 (Pmig-13::plx-1; Podr-1::RFP).

Cloning of rap-1, rap-2 and mig-15

cDNAs of rap-1 and rap-2 were obtained from cDNA library prepared from N2 RNA. Trizol (Invitrogen) was used to purify total RNA from N2, and the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) was used for the reverse-transcription. mig-15 genomic DNA was amplified from the N2 genomic DNA purified using GeneJET Genomic DNA Purification Kit (Thermo Scientific). Phusion (NEB) or Q5 (NEB) DNA polymerases were used for all PCR reactions for amplifying cDNA and genomic DNA fragments. Amplified fragments were cloned into the AscI and KpnI sites of pSM vector using SLiCE method (Motohashi, 2015), Gibson assembly (Gibson et al., 2009) or T4 ligase (NEB). List of primers used in this study is available in the Supplemental Experimental Procedures.

Confocal microscopy

Images of fluorescently tagged fusion proteins were captured in live C. elegans using a Zeiss LSM800 confocal microscope (Carl Zeiss, Germany). Worms were immobilized on 2% agarose pad using a mixture of 7.5 mM levamisole (Sigma-Aldrich) and 0.225M BDM (2,3-butanedione monoxime) (Sigma-Aldrich). Images were analyzed with Zen software (Carl Zeiss) or Image J (NIH, USA). Definition of each parameter is as follows (Mizumoto and Shen, 2013a): DA8/9 overlap: a distance between the most anterior DA9 synapse and the most posterior DA8 synapse, DA8 asynaptic domain: a distance from commissure to the most posterior DA8 synapse, DA9 synaptic domain: a distance between the most anterior and posterior DA9 synapses. Middle L4 (judged by the stereotyped shape of developing vulva) animals were used for quantification. Averages were taken from at least 20 samples. For GFP::Utrophin-CH, we measured the length from the posterior end of dorsal axon to the anterior end of GFP::Utrophin-CH domain. For each marker strain, the same imaging setting (laser power, gain pinhole) and image processing were used for comparing different genotypes.

Two-photon FLIM-FRET experiment

Expression vector for cultured cells (pCI-eGFP-hRap2a, pCI-eGFP-RAP-1, pCI-eGFP-RAP-2, pCI-eGFP-RAP-2(G12V), pCI-eGFP-RAP-2(S17A)) were generated by replacing Ras in pCI-eGFP-Ras (Yasuda et al., 2006) with hRap2a and rap-2 cDNAs with XhoI and BamHI. pCI-mRFP-RalGDS-mRFP plasmid is a kind gift from Dr. Yasuda. Rap2 and FRET sensor plasmids were mixed in 1:2 ratio and transfected into HeLa cells using Lipofectamine 3000 (ThermoFisher). FLIM was conducted 24 hr after transfection. For expression of FRET sensor in the DA9 neuron, each fusion protein constructs were cloned into AscI and KpnI sites of the pSM vector containing mig-13 promoter using SLiCE method.

A custom-made two-photon fluorescence lifetime imaging microscope was used as described elsewhere (Murakoshi et al., 2011). Briefly, EGFP-Rap2a was excited with a Ti-sapphire laser (Mai Tai; Spectra-Physics) tuned to 920 nm. The X and Y scanning galvano mirrors (6210 hr; Cambridge Technology) were controlled with ScanImage software (Pologruto et al., 2003). EGFP photon signals were collected an objective lens (60×, 1.0 NA; Olympus) and a photomultiplier tube (H7422-40p; Hamamatsu) placed after a dichroic mirror (FF553-SDi01; Semrock) and emission filter (FF01-510/84; Semrock). A fluorescence lifetime curve was recorded by a time-correlated single-photon-counting board (SPC-150; Becker and Hickl) controlled with the software described previously (Yasuda et al., 2006). For construction of a fluorescence lifetime image, the mean fluorescence lifetime values (τm) in each pixel were translated into a color-coded image. We quantified free EGFP-Rap2a and EGFP-Rap2a undergoing FRET (binding fraction) as described elsewhere (Yasuda et al., 2006). Briefly, we calculated the proportion of EGFP undergoing FRET in individual ROIs using the following formula:

(1) PFRET=τfree(τfreeτm)(τfreeτFRET)(τfree+τFRETτm)PFRET=τfree(τfreeτm)(τfreeτFRET)(τfree+τFRETτm)

where τfree and τFRET are the fluorescence lifetime of free EGFP and EGFP undergoing FRET, respectively.

Statistics

Prism (GraphPad) software was used for statistical analysis. One-way ANOVA was done and corrected for multiple comparisons with posthoc Tukey's multiple comparisons tests done between all genotypes. Student’s t-test was used for pairwise comparison. Sample numbers were pre-determined before conducting statistical analyses.

Supplemental experimental procedures

The method for qRT-PCR and sequences of primers and repair templates used in this study are available in Supplementary file 1.

References

  1. 1
  2. 2
  3. 3
  4. 4
    The genetics of Caenorhabditis elegans.
    1. S Brenner
    (1974)
    Genetics 77:71–94.
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
  19. 19
  20. 20
  21. 21
    Caenorhabditis elegans PlexinA, PLX-1, interacts with transmembrane semaphorins and regulates epidermal morphogenesis
    1. T Fujii
    2. F Nakao
    3. Y Shibata
    4. G Shioi
    5. E Kodama
    6. H Fujisawa
    7. S Takagi
    (2002)
    Development 129:2053–2063.
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
  30. 30
  31. 31
  32. 32
  33. 33
  34. 34
  35. 35
  36. 36
  37. 37
  38. 38
  39. 39
  40. 40
  41. 41
  42. 42
  43. 43
  44. 44
  45. 45
    Efficient gene transfer in c.elegans: extrachromosomal maintenance and integration of transforming sequences.
    1. CC Mello
    2. JM Kramer
    3. D Stinchcomb
    4. V Ambros
    (1991)
    The EMBO Journal 10:3959–4029.
  46. 46
  47. 47
  48. 48
  49. 49
  50. 50
  51. 51
  52. 52
  53. 53
  54. 54
  55. 55
  56. 56
  57. 57
  58. 58
  59. 59
  60. 60
  61. 61
  62. 62
  63. 63
  64. 64
  65. 65
  66. 66
  67. 67
  68. 68
  69. 69
  70. 70
  71. 71
  72. 72
  73. 73
  74. 74
  75. 75
  76. 76
  77. 77
  78. 78
  79. 79
  80. 80
  81. 81
  82. 82
  83. 83
  84. 84
  85. 85
  86. 86
  87. 87
  88. 88
  89. 89
  90. 90
  91. 91
  92. 92

Decision letter

  1. Piali Sengupta
    Reviewing Editor; Brandeis University, United States
  2. K VijayRaghavan
    Senior Editor; National Centre for Biological Sciences, Tata Institute of Fundamental Research, India

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "Rap2 and TNIK control Plexin-dependent synaptic tiling in C. elegans" for consideration by eLife. Your article has been reviewed by a Senior Editor, a Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation among the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

All reviewers appreciate the focus on identifying new genes and pathways regulating synaptic tiling. However, to some extent, many of the molecular players implicated in this manuscript have previously been identified in the Sema/Plexin pathway in other contexts (Plexin implicated by this lab in synaptic tiling, Rap is a known target of Plexin signaling, MIG-15/TNIK is a known regulator of Rap). A strength of your manuscript is the description of the roles of all these components in a pathway that regulates synaptic tiling. However, given this, as the reviewers note, there are several gaps/weaknesses in the current analyses tying these components together. The reviewers suggest several experiments to strengthen the work.

Reviewer #1:

This is an interesting study that is the next step in defining the signaling pathway(s) downstream of PLX-1 signaling in regulating tiling of DA8 and DA9 motor neuron synapses. The data supporting a cell-autonomous role for rap-2 downstream of plx-1 are convincing, however they are also expected (that at least one C. elegans Rap homolog would be downstream) based on other studies of Plexin signaling.

While mig-15 is clearly doing something interesting in terms of inhibiting synapse formation throughout the worm, the data implicating MIG-15 downstream of PLX-1 RAP-2 signaling is less convincing. The authors' results with respect to MIG-15 feel like the beginning of a new story, and not just a new molecule downstream of Plexins.

Reviewer #2:

In this manuscript, the authors use C. elegans DA9 and DA8 neurons as the model to investigate synaptic tilling. The authors describe a potential important role of Rap2 and TNIK in regulating the synaptic tilling of DA neurons. The authors present genetic evidence to support the function of Rap2 and TNIK in this process, however the evidence to link Semaphorin receptors with Rap2 or TNIK was relatively weak.

1) The authors show that both gain-of function and loss-of-function of rap-2 have similar phenotypes. They use rap-2 (G12V) as a gain-of-function mutation for RAP-2, is this mutation really cause gain-of-function in C. elegans neurons? The authors need either cite published results or test it by themselves.

2) The authors use the co-localzation of mCherry::RAB-3 with CLA-1 as evidence to show those synapses are "functional synapses". To really reach the conclusion that those synapses are functional synapses, one needs to show either those synapses have similar structures as "normal" synapses, or to show those synapses have similar ability to release neuronal transmitters. I would suggest the authors either use other term (rather than functional synapse) to describe the phenotype or carry out EM or electrophysiology studies to prove that those synapses are functional synapses. It will also be helpful if the authors can test other synaptic markers such as SNB-1, SID-1, SYD-2, to confirm the results.

3) The link between PLX-1 with Rap2 activation was wake. The authors show the activation of RAP-2 has correlation with the localization of PLX-1, but this doesn't mean the activation of RAP-2 is directly regulated by the PLX-1 or depend the activation of PLX-1. Since this is one of the major conclusions of the paper, some direct evidence is needed to prove the activation of RAP2 is indeed locally and directly regulated by PLX-1 and the PLX-1 ligand. Otherwise, the authors need to revise their conclusion.

4) As shown in Figure 7, mig-15(OE) seems to affect the tilling border DA8/DA9/. Does mig-15(OE) also affect the localization of PLX-1 and the local activation of Rap2?

Reviewer #3:

Chen et al., identify the small GTPase rap-2 and its effector mig-15/TNIK as novel regulators of synaptic tiling in DA8/DA9 motor neurons in C. elegans, and further show that MIG-15 inhibits synapse formation in multiple neuron types. Mizumoto et al. (2013) previously showed that Semaphorin/Plexin signaling regulates branched actin via the Plexin GAP domain to control synaptic tiling between DA8 and DA9. Semaphorin/Plexin signaling is known to inhibit synapse formation (Matthes, 1995, Tran, 2009, Duan, 2014, O'Connor, 2009), however the intracellular signaling mechanisms that connect Plexin to synapse development are not known. Based on published data, cited by the authors, Plexins are known to inhibit the Rap family of small GTPases to regulate actin remodeling (Wang, 2012, Wang, 2013), and TNIK family kinases are known downstream effectors of Rap-GTP (Hussain, 2010, Kawabe, 2010, Nonaka, 2008). Thus, the authors test if C. elegans Rap family members and mig-15/TNIK regulate synaptic tiling downstream of PLX-1. Chen et al. found that rap-2 and mig-15 mutants have strong defects in synaptic tiling. The authors use FLIM-based FRET to show that RAP2A is inhibited locally at the synaptic tiling border between DA8 and DA9, and this local effect is abrogated in plx-1 mutants. They show that rap-2 functions in DA9 neurons to regulate synaptic tiling and use genetic double mutants to show that plx-1 and rap-2 act in the same pathway. Chen et al., identify the Rap-GTP effector kinase, mig-15/TNIK as a novel regulator of synaptic tiling and, interestingly, as a general inhibitor of synapse formation throughout the nervous system.

1) The images showing PLX-1::GFP localization to the tiling border are not clear. Mizumoto et al., previously showed that PLX-1::GFP is highly expressed and diffuse in the ventral dendrite and asynaptic regions of the axon including in the axon commissure and also anterior to the synaptic tiling border, with some dimmer puncta within the synaptic region (Neuron 2013). In this manuscript (Figure 3A-B), PLX-1::GFP is much dimmer and punctate at the tiling border. Better images and quantification of PLX-1::GFP distribution, as previously performed, would improve this data.

2) In Figure 3F-G, the authors show that local inhibition of Rap2A at the tiling border does not happen in the absence of plx-1 and conclude that the data "strongly suggest that Plexin localized at the anterior edge of the DA9 synaptic domain locally inactivates Rap2A GTPase to delineate the synaptic tiling border in DA9." Since plx-1 mutants are missing PLX-1 in the whole animal including throughout DA9, a better experiment might be to rescue the mutant with PLX-1(ΔSema)::GFP, which the authors previously showed was mislocalized throughout DA9, and show that mislocalized, inactive PLX-1 does not rescue the local inactivation of Rap2A. And importantly, does GAP-deficient PLX-1(RA)::GFP, which the authors previously showed was properly localized to the tiling border, show no change in local Rap2A activity?

3) Can the authors show that there are no subtle defects in DA8/DA9 axon contact in mig-15 mutants that could explain the stronger synaptic tiling defects? The authors show that mig-15 mutants have strong defects in axon outgrowth and branching in about 50% of the animals (Figure 5—figure supplement 1). In addition, mig-15 mutants have a more dramatic DA8/DA9 overlap phenotype (50-55um overlap in Figure 4) compared to plx-1 or rap-2 mutants (which have ~30um overlap in Figure 1). The authors previously showed (Mizumoto et al., 2013) that in axon guidance mutants unc-34 and unc-129, DA9 is misguided and does not make axon contacts in the dorsal cord leading to strong DA8/DA9 synaptic tiling overlap defects (up to 50um). Thus, it is not clear if the DA8/DA9 overlap defects observed in mig-15 mutants are primarily due to axon guidance defects (i.e. more subtle defects where DA8 and DA9 do not contact each other) or to a more direct role for mig-15 in synaptic tiling.

4) It is not clear whether mig-15 acts upstream, downstream or in parallel to plx-1 and rap-2 to regulate synaptic patterning. If MIG-15 acts downstream of PLX-1, and Rap-2-GTP binds and activates TNIK/MIG-15 to inhibit synapse formation, then MIG-15(OE) should inhibit synapse formation independent of plx-1 and rap-2. Instead, Figure 7E appears to show that the effects of MIG-15(OE) on DA9 synapses are suppressed by plx-1 or rap-2. The interpretation of these results should be clarified. Also, it would be helpful if plx-1 and rap-2 single mutants are shown for comparison in Figure 7E to determine whether MIG-15 functions upstream of plx-1 and rap-2 or in parallel.

5) Have the authors tested if there are any functional/ behavioral consequences to defects in synaptic tiling between DA8 and DA9?

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for submitting your article "Rap2 and TNIK control Plexin-dependent synaptic tiling in C. elegans" for consideration by eLife. Your article has been reviewed by K VijayRaghavan as the Senior Editor, a Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

While all reviewers appreciate the additional data and analyses presented in this manuscript, and the extent to which previous comments have been addressed, the major remaining concern is about the placement of MIG-15 in the PLX-1 pathway. The data supporting the notion that MIG-15 is downstream of PLX-1 are weak. While this issue is addressed in part in Figure 8, the manuscript should be revised to address this concern. Specifically, the manuscript should overtly state the limitations of the genetic and phenotypic analyses and only speculate about the mechanistic relationship between MIG-15 and PLX-1.

https://doi.org/10.7554/eLife.38801.023

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

Reviewer #1:

This is an interesting study that is the next step in defining the signaling pathway(s) downstream of PLX-1 signaling in regulating tiling of DA8 and DA9 motor neuron synapses. The data supporting a cell-autonomous role for rap-2 downstream of plx-1 are convincing, however they are also expected (that at least one C. elegans Rap homolog would be downstream) based on other studies of Plexin signaling.

We appreciate the reviewer’s enthusiasm in our work. While it is indeed already shown that Plexin functions as a RapGAP, our work is the first to show the role of Plexin/Rap signaling in synapse pattern formation in vivo. More importantly, using FLIM, we show for the first time that Plexin controls the spatial activity of Rap2 small GTPase within the specific axonal segment. With our additional data to support this observation as described below, we believe that this work provides a significant advance in our knowledge of the Plexin/Rap2 signaling pathway in synapse pattern formation.

While mig-15 is clearly doing something interesting in terms of inhibiting synapse formation throughout the worm, the data implicating MIG-15 downstream of PLX-1 RAP-2 signaling is less convincing. The authors' results with respect to MIG-15 feel like the beginning of a new story, and not just a new molecule downstream of Plexins.

We absolutely agree with the reviewer’s comment. This work is a beginning of our next exciting challenge in understanding the roles of TNIK/MIG-15 in inhibiting presynaptic development. While the larger synaptic tiling defects we observed in mig-15 mutants clearly suggests that mig-15 functions in multiple pathways as we discussed in the Discussion section, we still believe that mig-15 functions downstream of plx-1 in synaptic tiling. The critical observation we added this time was PLX-1::GFP localization was not affected in the mig-15(rh148) mutant background, while ectopic synapses are formed beyond the PLX-1::GFP patch at the putative synaptic tiling border. This data strongly suggests that mig-15 controls synapse patterning downstream of PLX-1. We therefore believe that it is appropriate to include mig-15 as a Plexin/Rap2 signaling component in this manuscript.

Reviewer #2:

In this manuscript, the authors use C. elegans DA9 and DA8 neurons as the model to investigate synaptic tilling. The authors describe a potential important role of Rap2 and TNIK in regulating the synaptic tilling of DA neurons. The authors present genetic evidence to support the function of Rap2 and TNIK in this process, however the evidence to link Semaphorin receptors with Rap2 or TNIK was relatively weak.

1) The authors show that both gain-of function and loss-of-function of rap-2 have similar phenotypes. They use rap-2(G12V) as a gain-of-function mutation for RAP-2, is this mutation really cause gain-of-function in C. elegans neurons? The authors need either cite published results or test it by themselves.

As we showed in Figure 1, expression of rap-2(G12V) in wild-type background caused synaptic tiling defects, suggesting that rap-2(G12V) is a gain-of-function mutant. We observed no synaptic tiling defects when we injected wild-type rap-2 under the same unc-4 promoter in wild-type. This confirms that the synaptic tiling defect observed in animals expressing rap-2(G12V) is not due to the over-expression but rather the G12V mutation itself. This data is included in the revised Figure 1. We also rephrased ‘over-expression’ to ‘expression’ in the following sentence:

“Expression of a constitutively GTP-bound form of rap-2 (G12V) under the A-type neuron specific promoter, Punc-4, elicited a similar synaptic tiling defect as plx-1 mutants (Figures 1C and 1G). Expression of wild type rap-2 under the unc-4 promoter did not affect the synaptic tiling pattern, suggesting that G12V mutation but not over-expression of rap-2 caused the synaptic tiling defect (Figure 1G).

hRap2A 1 MREYKVVVLGSGGVGKSALTVQFVTGTFIEKYDPTIEDFYRKEIEVDSSPSVLEILDTAG 60

MRE+KVVVLGSGGVGKSALTVQFV+ TFIEKYDPTIEDFYRKEIEVD PSVLEILDTAG

RAP-2 1 MREFKVVVLGSGGVGKSALTVQFVSSTFIEKYDPTIEDFYRKEIEVDGQPSVLEILDTAG 60

In addition, considering the high sequence identity between C. elegans RAP-2 and human Rap2A (please see above sequence comparison), and the fact that G12V mutation is widely used as a constitutively active mutant for many small GTPases including Ras, Rac, Cdc42, Rap (including RAP-1 in C. elegans: MBC 2005 16(1): 106–116), we believe that C. elegans RAP-2 (G12V) is also a gain-of-function mutant.

We added the following sentence to explain G12V mutation generally used in Rap GTPase field.

The G12V mutant is widely used as a constitutively GTP-form of small GTPases including mammalian Rap2A and C. elegans RAP-1 (Kawabe et al., 2010; Pellis van Berkal et al., 2005).

We also conducted GST-RalGDS(RBD) pull-down assay in 293T cell line, which is a well-established assay to detect GTP-form of Rap small GTPase (McLeod et al., JBC 1998; Lin et al., Oncogene 2010) and found that C. elegans RAP-2(G12V) was coimmunoprecipitated with RalGDS (See Author response image 1). We were a little bit hesitant to put this figure in the manuscript since we did not detect C. elegans RAP-2 in the lysate due to low expression in 3 replicates (right gel), which hindered us from conducting quantitative analysis. Nevertheless, this data is also consistent with the idea that C. elegans RAP-2(G12V) is a constitutively active form.

The FLIM-FRET experiment we showed in response to reviewer1’s comment 2 also suggests that RAP-2(G12V) is a constitutively GTP-bound form.

2) The authors use the co-localzation of mCherry::RAB-3 with CLA-1 as evidence to show those synapses are "functional synapses". To really reach the conclusion that those synapses are functional synapses, one needs to show either those synapses have similar structures as "normal" synapses, or to show those synapses have similar ability to release neuronal transmitters. I would suggest the authors either use other term (rather than functional synapse) to describe the phenotype or carry out EM or electrophysiology studies to prove that those synapses are functional synapses. It will also be helpful if the authors can test other synaptic markers such as SNB-1, SID-1, SYD-2, to confirm the results.

We now examined another active zone markers, UNC-10::TdTomato, which colocalized with RAB-3 synaptic vesicle markers in the plx-1, rap-2 and mig-15 mutants. The representative images are in Figure S3. We also re-phrased ‘functional synapses’ to ‘bona fide synapses’.

3) The link between PLX-1 with Rap2 activation was wake. The authors show the activation of RAP-2 has correlation with the localization of PLX-1, but this doesn't mean the activation of RAP-2 is directly regulated by the PLX-1 or depend the activation of PLX-1. Since this is one of the major conclusions of the paper, some direct evidence is needed to prove the activation of RAP2 is indeed locally and directly regulated by PLX-1 and the PLX-1 ligand. Otherwise, the authors need to revise their conclusion.

We appreciate the reviewer’s comment. This is also raised by the reviewer 3. As reviewer 3 suggested, we conducted additional FLIM experiments using two mutant PLX-1 rescuing constructs; one cannot be activated by Semaphorin; the other has no GAP activity but can localize normally (please see our response to reviewer 3, comment 2 for details). Neither of them rescued the local Rap2 inactivation pattern. These data strongly support our model that PLX-1 controls local RAP-2 activity.

Regarding whether PLX-1 directly regulates Rap2, we do not have clear evidence for this and therefore did not mention that it was direct. However, we think it is highly likely since structural and biochemical experiments using mammalian Plexin nicely showed that Plexin is a RapGAP. To clarify our conclusion, we added the following sentence.

“While we do not fully exclude the possibility that PLX-1 indirectly regulated local Rap2 activity, together with the biochemical evidence that mammalian Plexin acts as RapGAP (Wang et al., 2012; 2013), these data strongly suggests that Plexin localized at the anterior edge of the DA9 synaptic domain locally inactivates Rap2 GTPase to delineate the synaptic tiling border in DA9.”

4) As shown in Figure 7, mig-15(OE) seems to affect the tilling border DA8/DA9/. Does mig-15(OE) also affect the localization of PLX-1 and the local activation of Rap2?

We indeed observed the posterior shift of PLX-1::GFP in animals expressing mig-15. A representative image is now included in Figure 3.Due to the variability of the phenotype caused by mig-15 overexpression from an extrachromosomal array, we were not able to quantify the shift of PLX-1::GFP. For the same variability issue, we were not able to conduct FLIM experiment.

Reviewer #3:

[…] 1) The images showing PLX-1::GFP localization to the tiling border are not clear. Mizumoto et al., previously showed that PLX-1::GFP is highly expressed and diffuse in the ventral dendrite and asynaptic regions of the axon including in the axon commissure and also anterior to the synaptic tiling border, with some dimmer puncta within the synaptic region (Neuron 2013). In this manuscript (Figure 3A-B), PLX-1::GFP is much dimmer and punctate at the tiling border. Better images and quantification of PLX-1::GFP distribution, as previously performed, would improve this data.

We appreciate the reviewer’s suggestion. We re-set the imaging condition for PLX-1::GFP so that the PLX-1::GFP patch is now more obvious than the original ones. We also added the quantification of the signals in Figure 3.

2) In Figure 3F-G, the authors show that local inhibition of Rap2A at the tiling border does not happen in the absence of plx-1 and conclude that the data "strongly suggest that Plexin localized at the anterior edge of the DA9 synaptic domain locally inactivates Rap2A GTPase to delineate the synaptic tiling border in DA9." Since plx-1 mutants are missing PLX-1 in the whole animal including throughout DA9, a better experiment might be to rescue the mutant with PLX-1(ΔSema)::GFP, which the authors previously showed was mislocalized throughout DA9, and show that mislocalized, inactive PLX-1 does not rescue the local inactivation of Rap2A. And importantly, does GAP-deficient PLX-1(RA)::GFP, which the authors previously showed was properly localized to the tiling border, show no change in local Rap2A activity?

We again appreciate the reviewer’s insightful suggestion. We conducted the suggested experiments and indeed observed no rescue in Rap2 activity pattern when PLX-1(ΔSema) or PLX-1(RA) was expressed in DA9. On the other hand, expression of wildtype PLX-1 nicely recreated the region with low Rap2 activity in the plx-1 mutant background. This observation strengthens our conclusion that PLX-1 locally inhibits Rap2 activity at the synaptic tiling border.

We believe this experiment also answered the question 3 from reviewer 2. The new data is now added to Figure 4.

3) Can the authors show that there are no subtle defects in DA8/DA9 axon contact in mig-15 mutants that could explain the stronger synaptic tiling defects? The authors show that mig-15 mutants have strong defects in axon outgrowth and branching in about 50% of the animals (Supplemental Figure 4). In addition, mig-15 mutants have a more dramatic DA8/DA9 overlap phenotype (50-55um overlap in Figure 4) compared to plx-1 or rap-2 mutants (which have ~30um overlap in Figure 1). The authors previously showed (Mizumoto et al., 2013) that in axon guidance mutants unc-34 and unc-129, DA9 is misguided and does not make axon contacts in the dorsal cord leading to strong DA8/DA9 synaptic tiling overlap defects (up to 50um). Thus, it is not clear if the DA8/DA9 overlap defects observed in mig-15 mutants are primarily due to axon guidance defects (i.e. more subtle defects where DA8 and DA9 do not contact each other) or to a more direct role for mig-15 in synaptic tiling.

In our previous paper, we showed that the PLX-1::GFP localization is dependent on the axon-axon interaction. In the axon guidance mutants in which axon-axon interaction between DA8 and DA9 is disrupted, PLX-1::GFP was distributed evenly throughout the axon. In mig-15 mutants, PLX-1::GFP localization was largely unaffected, suggesting that the synaptic tiling defect in mig-15 is not due to the loss of axon-axon interaction between DA8 and DA9. Now this data is included in Figure 3. We added the following sentence in the revised manuscript.

“The PLX-1::GFP patch at the putative synaptic tiling border was unaffected in mig-15 mutants, even though the position of the PLX-1::GFP patch has shifted slightly posteriorly compared with wild-type animals (Figure 3C), suggesting that mig-15 acts downstream of PLX-1 in regulating synaptic tiling.”

In addition, we did not observe severe synaptic tiling defect in cdh-4(rh310), which showed axon defasciculation phenotype in the dorsal nerve cord neurons (Schmitz et al., 2008). The overlap between DA8 and DA9 synaptic domains was 4.6 µm (n=21, SEM ± 1.02). This observation also supports the idea that the large overlap between DA8 and DA9 synaptic domains in mig-15 mutants is not due to the loss of axon-axon interaction. We included the following sentence:

“In addition, we did not observe significant synaptic tiling defects in cdh-4(rh310) mutants (4.6 ± 1.02 µm, n=21), which exhibits axon defasciculation phenotype in the dorsal nerve cord neurons (Schmitz et al., 2008). These data suggest that the synaptic tiling defect in the mig-15 mutants is not a secondary effect of axon outgrowth and guidance.”

4) It is not clear whether mig-15 acts upstream, downstream or in parallel to plx-1 and rap-2 to regulate synaptic patterning. If MIG-15 acts downstream of PLX-1, and Rap-2-GTP binds and activates TNIK/MIG-15 to inhibit synapse formation, then MIG-15(OE) should inhibit synapse formation independent of plx-1 and rap-2. Instead, Figure 7E appears to show that the effects of MIG-15(OE) on DA9 synapses are suppressed by plx-1 or rap-2. The interpretation of these results should be clarified. Also, it would be helpful if plx-1 and rap-2 single mutants are shown for comparison in Figure 7E to determine whether MIG-15 functions upstream of plx-1 and rap-2 or in parallel.

We believe that mig-15 has two roles. First, mig-15 acts downstream of plx-1 and rap-2 in synapse patterning (or synaptic tiling). We propose this based on the synaptic tiling defect (but normal PLX-1 localization) in mig-15 mutants. Second, mig-15 functions as a key negative regulator of synapse number. Since we did not observe a significant increase in synapse numbers in plx-1 or rap-2 mutants, the role of mig-15 in controlling synapse number is independent of Plexin signaling pathway. Indeed, the reduction of synapse number in mig-15-overexpressing animals is not suppressed in plx-1 or rap-2 mutants. We added the following sentence in the revised manuscript to clarify the relationship between Plex/Rap pathway and mig-15.

“Synapse number was not different in mig-15(OE) and rap-2(gk11); mig-15(OE) animals, suggesting that the role of mig-15 in inhibiting synapse number is not dependent on Plexin/Rap2 signaling pathway (Figure S6).”

5) Have the authors tested if there are any functional/ behavioral consequences to defects in synaptic tiling between DA8 and DA9?

All synaptic tiling mutants we have isolated so far, except for mig-15 mutantswhich are sick and hence do not move well, exhibit superficially normal locomotion. This is probably due to the functional redundancy in the motor circuit, in which subtle synaptic pattern defect could be compensated by other mechanisms. Most of the C. elegans mutants with synapse patterning defects indeed show minimal locomotion defects, and hence have been missed from the traditional locomotion-based genetic screenings.

[Editors' note: the author responses to the re-review follow.]

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

While all reviewers appreciate the additional data and analyses presented in this manuscript, and the extent to which previous comments have been addressed, the major remaining concern is about the placement of MIG-15 in the PLX-1 pathway. The data supporting the notion that MIG-15 is downstream of PLX-1 are weak. While this issue is addressed in part in Figure 8, the manuscript should be revised to address this concern. Specifically, the manuscript should overtly state the limitations of the genetic and phenotypic analyses and only speculate about the mechanistic relationship between MIG-15 and PLX-1.

We agree with the reviewers’ comments.

In the revised manuscript, we emphasized that our works revealed the genetic relationship between mig-15 and plx-1 by adding ‘genetically’ in the following sentences.

Abstract:

“Here, we show that Rap2 GTPase (rap-2) and its effector, TNIK (mig-15), act genetically downstream of Plexin (plx-1) to restrict presynaptic assembly and form tiled synaptic innervation in C. elegans.”

Introduction:

“Here, we report that rap-2, a C. elegans ortholog of human Rap2A, and its effector kinase mig-15 (TNIK: Traf2- and Nck-interacting kinase) act genetically downstream of PLX-1 to regulate synaptic tiling.”

In the following sentence, rap-2 was replaced to plx-1 as we did not show that mig-15 acts downstream of rap-2 in synaptic tiling.

Discussion section:

“We showed that proper synapse patterning requires both GDP- and GTP-forms of RAP-2. Considering that PLX-1 regulates the spatial distribution of RAP-2 activity and mig-15 acts genetically downstream of plx-1 in synaptic tiling, RAP-2 may also locally regulate MIG-15 (TNIK).”

The following sentences were added to describe the limitation of our analyses and the needs of future biochemical analysis to fully understand the mechanisms of PLX-1/Rap2/TNIK signaling in synapse pattern formation.

Discussion section:

“Due to the pleiotropic phenotype of the mig-15 mutants, our genetic and phenotypic analyses of mig-15 did not exquisitely reveal the mechanistic relationship between PLX-1 and MIG-15 in synaptic tiling regulation. Further biochemical studies of MIG-15 regulation by Plexin/Rap2 in synaptic tiling will elucidate the molecular mechanisms that underlie the role of MIG-15/TNIK in synapse pattern formation.”

https://doi.org/10.7554/eLife.38801.024

Article and author information

Author details

  1. Xi Chen

    Department of Zoology, The University of British Columbia, Vancouver, Canada
    Contribution
    Formal analysis, Investigation, Visualization
    Competing interests
    No competing interests declared
  2. Akihiro CE Shibata

    Supportive Center for Brain Research, National Institute for Physiological Sciences, Okazaki, Japan
    Contribution
    Formal analysis, Validation, Investigation, Visualization, Writing—review and editing
    Contributed equally with
    Ardalan Hendi
    Competing interests
    No competing interests declared
  3. Ardalan Hendi

    Department of Zoology, The University of British Columbia, Vancouver, Canada
    Contribution
    Formal analysis, Validation, Investigation, Visualization, Writing—review and editing
    Contributed equally with
    Akihiro CE Shibata
    Competing interests
    No competing interests declared
  4. Mizuki Kurashina

    Department of Zoology, The University of British Columbia, Vancouver, Canada
    Contribution
    Formal analysis, Validation, Investigation, Visualization, Writing—review and editing
    Competing interests
    No competing interests declared
  5. Ethan Fortes

    Department of Zoology, The University of British Columbia, Vancouver, Canada
    Contribution
    Formal analysis, Validation, Investigation, Visualization, Writing—review and editing
    Competing interests
    No competing interests declared
  6. Nicholas L Weilinger

    Department of Psychiatry, The University of British Columbia, Vancouver, Canada
    Contribution
    Investigation, Writing—review and editing
    Competing interests
    No competing interests declared
  7. Brian A MacVicar

    Department of Psychiatry, The University of British Columbia, Vancouver, Canada
    Contribution
    Resources, Supervision
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4596-4623
  8. Hideji Murakoshi

    Supportive Center for Brain Research, National Institute for Physiological Sciences, Okazaki, Japan
    Contribution
    Resources, Supervision, Validation, Investigation, Writing—review and editing
    Competing interests
    No competing interests declared
  9. Kota Mizumoto

    Department of Zoology, The University of British Columbia, Vancouver, Canada
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Writing—original draft, Writing—review and editing
    For correspondence
    mizumoto@zoology.ubc.ca
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8091-4483

Funding

Human Frontier Science Program (CDA-00004/2014)

  • Kota Mizumoto

Canadian Institutes of Health Research (PJT-148667)

  • Kota Mizumoto

Natural Sciences and Engineering Research Council of Canada (RGPIN-2015-04022)

  • Kota Mizumoto

Canada Research Chairs

  • Kota Mizumoto

Michael Smith Foundation for Health Research (16869)

  • Kota Mizumoto

Tomizawa Jun-ichi and Keiko Fund of Molecular Biology Society of Japan for Young Scientists

  • Kota Mizumoto

Canada Foundation for Innovation (34722)

  • Kota Mizumoto

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are grateful to Donald Moerman and his lab members for generating rap-3 mutant strain, providing other mutant strains, sharing reagents and for general discussions. We also thank Ryohei Yasuda for FRET sensor plasmids, Hiroshi Kawabe for human Rap2a cDNA, Richard Ikegami for plx-2 promoter construct, Harald Hutter and Peri Kurshan for sharing strains, May Dang-Lawson and Michael Gold for unpublished biochemical experiments, Lisa Fernando for the technical support, all Mizumoto lab members and general discussions, Kang Shen, Shaul Yogev and Maulik Patel for comments on the manuscript. Some strains used in this study were obtained from the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440) and C. elegans gene knock out consortium. Mizumoto Lab is funded by HFSP (CDA-00004/2014), CIHR (PJT-148667), NSERC (RGPIN-2015–04022), CFI-JELF (34722) and Tomizawa Jun-ichi and Keiko Fund for Young Scientist. KM is a recipient of Canada Research Chair and Michael Smith Foundation for Health Research Scholar.

Senior Editor

  1. K VijayRaghavan, National Centre for Biological Sciences, Tata Institute of Fundamental Research, India

Reviewing Editor

  1. Piali Sengupta, Brandeis University, United States

Publication history

  1. Received: May 31, 2018
  2. Accepted: July 11, 2018
  3. Version of Record published: July 31, 2018 (version 1)

Copyright

© 2018, Chen et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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