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Published in final edited form as:
Neuron. 2011 July 14; 71(1): 103–116. doi:10.1016/j.neuron.2011.05.034.
The Immunoglobulin super family protein RIG-3 prevents
synaptic potentiation and regulates Wnt signaling
Kavita Babu1,2, Zhitao Hu1,2, Shih-Chieh Chien3, Gian Garriga3,4, and Joshua M. Kaplan1,2,5
1 Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114
2
Department of Neurobiology, Harvard Medical School, Boston, MA 02115
3
Department of Molecular Cell Biology, University of California, Berkeley, CA 94720
4
Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720
Abstract
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Cell surface Ig superfamily proteins (IgSF) have been implicated in several aspects of neuron
development and function. Here, we describe a novel function for a C. elegans IgSF protein,
RIG-3. Mutants lacking RIG-3 have an exaggerated paralytic response to a cholinesterase
inhibitor, aldicarb. Although RIG-3 is expressed in motor neurons, heightened drug
responsiveness was caused by an aldicarb-induced increase in muscle ACR-16 acetylcholine
receptor (AChR) abundance, and a corresponding potentiation of post-synaptic responses at
neuromuscular junctions. Mutants lacking RIG-3 also had defects in the anteroposterior polarity of
the ALM mechanosensory neurons. RIG -3’s effects on synaptic transmission and ALM polarity
were both mediated by changes in Wnt signaling, and in particular by inhibiting CAM-1, a Rortype receptor tyrosine kinase that binds Wnt ligands. These results identify RIG-3 as a novel
regulator of Wnt signaling, and suggest that RIG-3 has an anti-plasticity function that prevents
activity-induced changes in post-synaptic receptor fields.
Keywords
C. elegans; RIG-3; IgSF; neuromuscular junction; synaptic plasticity; Wnt; CAM-1; Ror receptor
tyrosine kinase; nicotinic receptors; ACR-16; neuron polarity
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Introduction
Cell surface IgSF proteins are implicated in diverse aspects of neuronal development,
including: cell and axon migration, target recognition, axon fasciculation, axon
ensheathment by glia, synapse formation and synapse function (Rougon and Hobert, 2003;
Takeda et al., 2001; Walsh and Doherty, 1997). Many IgSF proteins act as either homo- or
heterophilic cell adhesion molecules (CAMs), e.g. NCAM (Yamada and Nelson, 2007).
Other IgSF proteins act as receptors for secreted ligands, or as auxiliary subunits of such
© 2011 Elsevier Inc. All rights reserved.
5
Corresponding author: Joshua M. Kaplan, Ph.D., Department of Molecular Biology, Massachusetts General Hospital, Richard B
Simches Research Building, 185 Cambridge Street CPZN 7250, Boston, MA 02114-2790, Tel: 617-726-5900, Fax: 617-726-5949
kaplan@molbio.mgh.harvard.edu.
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receptors (Barrow and Trowsdale, 2008; Wang and Springer, 1998). IgSF proteins comprise
a large family of proteins (765 in humans, 142 in flies, 80 in worms) (Lander et al., 2001;
Vogel et al., 2003) and mutations in IgSF genes are associated with several human
neurological disorders (Fransen et al., 1997; Sun et al., 2003; Uyemura et al., 1996).
Several CAMs induce synapse formation (Biederer et al., 2002; Kurusu et al., 2008; Linhoff
et al., 2009). For example, Neurexin and Neuroligin induce differentiation of post- and presynaptic specializations, respectively (Nam and Chen, 2005; Scheiffele et al., 2000). Some
CAMs confer specificity for specific types of synapses. Neuroligin-2 induces formation of
GABA synapses, whereas neuroligin-1 promotes formation of glutamatergic synapses (Chih
et al., 2005; Graf et al., 2004). Synaptic CAMs also play an important role in regulating
synaptic transmission. Neurexin-Neuroligin complexes recruit post-synaptic glutamate
receptors, while also altering synaptic vesicle recycling presynaptically (Chubykin et al.,
2007; Futai et al., 2007; Varoqueaux et al., 2006). N-cadherin is required for homeostatic
plasticity (Goda, 2002; Okuda et al., 2007) and integrins promote LTP (Chan et al., 2003).
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Many aspects of neuron and synapse development are regulated by both positive and
negative factors. Axon and cell migrations are shaped by gradients of secreted attractants
and repellents (Tessier-Lavigne, 1994). Similarly, synapse formation is governed by both
positive (e.g. neurexin-neuroligin) and negative factors (e.g. Wnt) (Klassen and Shen, 2007;
Poon et al., 2008; Scheiffele, 2003). Here we show that RIG-3, a cell surface IgSF molecule,
acts as an anti-plasticity molecule, preventing a form of synaptic potentiation, and that it
does so by altering Wnt signaling.
Results
Mutants lacking RIG-3 are hypersensitive to aldicarb
To identify new molecules involved in neuromuscular signaling, we used RNAi to screen
for cell adhesion molecules whose absence alters the responsiveness of C. elegans to the
acetylcholinesterase inhibitor aldicarb. Aldicarb treatment causes acute paralysis due to the
accumulation of acetylcholine (ACh) in the synaptic cleft at the neuromuscular junction
(NMJ). Gene inactivations that alter synaptic function can cause either resistance or
hypersensitivity to aldicarb (Miller et al., 1996; Sieburth et al., 2005; Vashlishan et al.,
2008). For this screen, we selected a collection of 216 putative cell adhesion molecules,
based on the presence of protein domains found in CAMs (data not shown).
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A gene identified in this screen was rig-3, which encodes a GPI-anchored protein containing
two Ig domains and a divergent fibronectin type III (FNIII) domain (Fig. 1A). RIG-3 has a
pattern of protein domains that is similar to the Drosophila proteins Klingon and Wrapper,
and to mammalian NCAMs (Cox et al., 2004; Yamagata et al., 2003). RIG-3 was previously
implicated in axon guidance in C. elegans; however rig-3 single mutants do not show
guidance defects (Schwarz et al., 2009).
Inactivation of rig-3 by RNAi caused significant hypersensitivity to aldicarb (Fig. 1B) and a
similar defect was observed in homozygous rig-3(ok2156) mutants (Fig 1C). The ok2156
mutation deletes 1.5kb of the rig-3 gene, spanning exons 2–5 (including most of the Ig
domains and part of the FNIII domain); consequently, ok2156 is likely to cause a severe loss
of gene function (www.wormbase.org) (Fig. 1A) (Schwarz et al., 2009).
The rig-3 aldicarb hypersensitivity defect was rescued by transgenes driving RIG-3
expression in all neurons (utilizing the snb-1 Synaptobrevin promoter, data not shown) and
in cholinergic neurons (utilizing the unc-17 VAChT promoter) (Fig. 1C). By contrast, rig-3
transgenes expressed in GABA neurons, or in the intestine lacked rescuing activity (Fig.
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1C). None of these transgenes altered aldicarb responsiveness of wild type animals (data not
shown). These results suggest that RIG-3 functions in cholinergic neurons to regulate some
aspect of neuromuscular function or development.
Prior work showed that rig-3 is expressed in neurons and in the intestine
(www.wormbase.org) (Schwarz et al., 2009). A construct containing the full rig-3 genomic
region, with mCherry inserted just after the signal sequence (Fig. 1A), was expressed in
ventral cord motor neurons but not in body muscles (Fig. 2A and data not shown). To
identify the rig-3 expressing motor neurons, we performed several double labeling
experiments. The rig-3 construct was co-expressed with acr-2 (A, B, and AS neurons),
unc-4 (VA and DA neurons), unc-129 (DA neurons), but not with ceh-12 (VB neurons)
reporters, which are expressed in the indicated motor neuron classes (Figs. 2 and S2, and
data not shown). These results suggest that RIG-3 is expressed in the VA and DA motor
neurons (and possibly the AS neurons).
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To determine the sub-cellular localization of the RIG-3 protein, we analyzed the expression
of mCherry-tagged RIG-3. The mCherry::RIG-3 genomic construct rescued the rig-3
aldicarb defect (Fig. 1C), demonstrating that this chimeric protein retained RIG-3 function.
mCherry::RIG-3 was distributed in a punctate pattern in dorsal cord axons, and the RIG-3
puncta fluorescence was partially co-localized with the SV protein SNB-1, consistent with
RIG-3 enrichment at pre-synaptic elements (Fig. 2B). RIG-3 fluorescence was also observed
in coelomocytes (Fig. 2C), which are phagocytic cells that internalize proteins secreted into
the body cavity (Fares and Grant, 2002). The coelomocyte fluorescence most likely
corresponds to RIG-3 shed from neuronal membranes (perhaps due to hydrolysis of the GPIanchor). Thus, RIG-3 may function as either a cell surface or a secreted protein. A control
construct expressing cytoplasmic mCherry in cholinergic motor neurons did not produce
coelomocyte fluorescence (Fig. 2D).
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We did several experiments to test the functional importance of membrane-tethered and
secreted RIG-3. A RIG-3 construct lacking the C-terminal GPI-anchoring signal,
RIG-3(ΔGPI), exhibited decreased axonal and increased coelomocyte fluorescence (Fig.
S1), and failed to rescue the aldicarb hypersensitivity defect of rig-3 mutants (Fig. 1C).
Furthermore, RIG-3 expressed in GABA neurons (with the unc-25 promoter) did not rescue
the aldicarb hypersensitivity seen in rig-3 mutants (Fig. 1C), as would be predicted if
secreted RIG-3 lacks rescuing activity. By contrast, a transgene expressing a constitutively
membrane-anchored protein, RIG-3(TMD), in cholinergic neurons produced axonal
fluorescence, lacked coelomocyte fluorescence, and partially rescued the aldicarb
hypersensitivity defect (Fig. S1, Fig. 1C). These results indicate that RIG-3’s synaptic
function is primarily mediated by membrane-associated RIG-3 at presynaptic elements and
not by secreted RIG-3.
Synapse morphology is normal in rig-3 mutants
The rig-3 aldicarb defect could arise from altered development of neurons or synapses. We
did several experiments to address this possibility. The number of ventral cord neurons and
their axon morphologies were unaltered in rig-3 mutants (data not shown), consistent with
prior studies (Schwarz et al., 2009). We also analyzed the morphology of neuromuscular
junctions with several synaptic markers. We found no significant differences in the
morphology, fluorescence intensity, or density of cholinergic and GABAergic NMJs in rig-3
mutants using GFP-tagged SNB-1 Synaptobrevin and SYD-2 α-liprin (an active zone
protein) as markers (Fig. S2C-D and data not shown). Adhesion molecules often anchor the
cortical actin cytoskeleton to the plasma membrane (Leshchyns’ka et al., 2003). To analyze
the actin cytoskeleton at synapses, we expressed two GFP-tagged actin-binding proteins in
cholinergic neurons (GSNL-1 gelsolin and SNN-1 synapsin). GSNL-1 and SNN-1
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fluorescence were unaltered in rig-3 mutants, indicating a relatively normal actin
cytoskeleton (Fig. S2E and data not shown). These results indicate that rig-3 mutants do not
have significant defects in synapse formation or maintenance.
Baseline synaptic transmission is normal in rig-3 mutants
We did several experiments to determine if the rig-3 aldicarb defect is caused by changes in
baseline synaptic transmission. To assay synaptic transmission, we recorded excitatory and
inhibitory post-synaptic currents (EPSC and IPSC) from body muscles. The amplitude and
rate of endogenous EPSCs and IPSCs were not altered in rig-3 mutants, indicating that
baseline cholinergic and GABAergic transmission were both normal (Fig. 3A, and Fig. S3).
The amplitude and total synaptic charge of EPSCs evoked by a depolarizing stimulus were
also unaltered (Fig. 3B). To assess changes in post-synaptic AChRs, we analyzed expression
of ACR-16 receptors. ACR-16::GFP puncta fluorescence was slightly increased in rig-3
mutants compared to wild type controls (15%, p<0.01) (Fig. 4A); however, the amplitude of
currents evoked by bath applied ACh were not altered in rig-3 mutants, suggesting that
muscle sensitivity to ACh was normal (Fig. 3C). Taken together these results indicate that
inactivation of RIG-3 does not significantly alter baseline synaptic transmission.
Aldicarb induces increased muscle sensitivity to ACh in rig-3 mutants
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We recently showed that ACh release at NMJs is enhanced following brief treatments with
aldicarb (Hu et al., 2011). Thus, the rig-3 aldicarb defect could result from an exaggeration
of this aldicarb mediated presynaptic potentiation. To test this idea, we measured the effect
of aldicarb treatment on EPSC rates. A 60 minute aldicarb treatment caused identical
increases in the EPSC rate of both wild type and rig-3 mutants (Fig. 3A). These results
suggest that the rig-3 aldicarb hypersensitivity defect was not caused by increased ACh
release.
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Several results suggest that rig-3 mutant muscles have increased responsiveness to ACh
following aldicarb treatment. We used three assays to measure muscle ACh responses: the
amplitudes of endogenous EPSCs, of stimulus evoked EPSCs, and of currents activated by
exogenously applied ACh. Aldicarb treatment increased the amplitude of endogenous
EPSCs recorded from rig-3 mutant muscles whereas those recorded from wild type animals
were unaltered (Fig. 3A and Fig. S3B, C). Aldicarb had no effect on the decay kinetics of
endogenous EPSCs in rig-3 or in wild type controls (Fig. S3B). The amplitude and total
synaptic charge of evoked responses in aldicarb treated rig-3 mutants were both
significantly greater than that observed in aldicarb treated wild type controls (Fig. 3B).
Aldicarb treatment also significantly increased the amplitude of ACh-activated currents in
rig-3 mutants whereas those recorded from wild type animals were significantly reduced
(Fig. 3C). The rig-3 defects in endogenous EPSC, evoked EPSC, and ACh-activated
currents were all rescued by a rig-3 transgene expressed in cholinergic neurons (Figs. 3 and
S3C). By contrast, aldicarb pretreatment had no effect on the amplitude of endogenous
IPSCs recorded from either wild type or rig-3 mutant muscles, suggesting that body muscle
responses to GABA were unaltered (Fig. S3A). Taken together, these results suggest that
aldicarb enhances body muscle ACh responses in rig-3 mutants (but not in wild type
controls) and that this effect is specific for ACh responses.
Aldicarb increases the synaptic abundance of ACR-16 receptors in rig-3 mutants
Increased ACh responses could be caused by altered expression or activity of nicotinic
AChRs. C. elegans body muscles express two classes of nicotinic AChRs, homomeric
ACR-16 receptors and heteropentameric αβ-type receptors that are sensitive to a synthetic
agonist levamisole. Levamisole (Lev) receptors account for only 20% of the synaptic and
ACh-activated currents in body muscles (Francis et al., 2005; Touroutine et al., 2005).
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Following aldicarb treatment, ACR-16::GFP puncta fluorescence was significantly increased
in rig-3 mutants (35%, p<0.001), while levels in wild type animals were unaltered (Fig. 4A).
By contrast, aldicarb treatment had no effect on UNC-29::GFP Lev receptor fluorescence
nor on UNC-49::GFP GABAA receptor fluorescence (consistent with the unaltered IPSC
amplitudes) in both wild type and rig-3 mutants (Fig. S4), indicating that this effect was
specific for ACR-16 receptors. This increase in ACR-16 fluorescence was fully rescued by a
transgene expressing RIG-3 in cholinergic neurons (Fig. 4A). Collectively, these results
demonstrate that inactivation of rig-3 reveals an aldicarb-induced potentiation of synaptic
transmission, which may result from increased synaptic abundance of ACR-16 receptors.
RIG-3’s effect on ACR-16 is restricted to adjacent post-synaptic targets
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Pre-synaptic RIG-3 could regulate post-synaptic receptors by either of two general
mechanisms. RIG-3 could act in a spatially restricted manner, regulating ACR-16 levels in
adjacent post-synaptic membranes. Alternatively, RIG-3 expressed in one neuron could
regulate ACR-16 abundance at NMJs formed by neighboring neurons. To distinguish
between these possibilities, we examined the effect of RIG-3 expression in the DA motor
neurons. DA neurons have cell bodies in the ventral midline, they extend a dendritic process
in the ventral cord (which receives synaptic input from interneurons), and an axonal process
in the dorsal cord (which forms NMJs with dorsal body muscles) (Fig. 4B). mCherry-tagged
RIG-3 expressed in DA neurons was targeted to puncta in dorsal cord axons whereas little
RIG-3 fluorescence was observed in the DA ventral cord processes (Fig. 4B), consistent
with pre-synaptic targeting of RIG-3 (Fig. 2B). Transgenes expressing RIG-3 in DA neurons
rescued the rig-3 ACR-16 fluorescence defect in the dorsal cord, but did not rescue the
ACR-16 defect in the ventral cord (Fig. 4C and D) nor the rig-3 aldicarb paralysis defect
(Fig. S4C). These results suggest that the active form of RIG-3 is restricted to axons and that
RIG-3 functions in a spatially restricted manner, regulating ACR-16 levels in adjacent postsynaptic membranes.
ACR-16 receptors are required for aldicarb-induced potentiation of rig-3 mutant synapses
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Increased ACR-16 targeting to synapses could provide a mechanism to explain the aldicarbinduced enhancement of synaptic transmission in rig-3 mutants. Consistent with this idea,
the aldicarb hypersensitivity, the increased EPSC amplitudes, and the increased AChactivated current following aldicarb treatment were all eliminated in acr-16; rig-3 double
mutants (Fig. 5). The residual ACh-activated current in acr-16 mutants are a direct measure
of Lev receptor function; consequently, this double mutant analysis demonstrates that
ACR-16 receptors are absolutely required for RIG-3’s synaptic effects, and that changes in
Lev receptor mediated currents are not observed in rig-3 mutants. Over-expression of
ACR-16 in wild type body muscles also produced hypersensitivity to aldicarb (Fig. 5A),
suggesting that increased ACR-16 levels are sufficient to cause this defect. However,
increased expression of the acr-16 gene is unlikely to explain the rig-3 mutant phenotype
because quantitative PCR did not detect significant changes in acr-16 mRNA levels
following aldicarb treatment: acr-16 mRNA levels after aldicarb treatment (normalized to
untreated controls) rig-3 = 0.80 ± 0.09, WT= 0.77 ± 0.13. These results suggest that aldicarb
regulates ACR-16 in a post-transcriptional manner in rig-3 mutants, thereby enhancing
synaptic transmission. These results also indicate that changes in ACR-16 can account for
all of the rig-3 synaptic defects.
ACR-16 mobility in the nerve cord is increased in rig-3 mutants
The receptors present at a synapse are provided by the dynamic exchange between a mobile
pool of receptors, and receptors bound at post-synaptic elements (Opazo and Choquet,
2011). To determine how RIG-3 alters this equilibrium, we analyzed fluorescence recovery
after photobleaching (FRAP) of ACR-16::GFP puncta in the dorsal nerve cord (Fig. 6). The
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ACR-16 FRAP observed in untreated wild type controls and rig-3 mutants were not
significantly different. Following aldicarb treatment, FRAP was significantly increased in
rig-3 mutants, but was unaltered in wild type controls. By contrast FRAP of UNC-49::GFP
(GABAA receptor) was unaltered by aldicarb treatment in both wild type and rig-3 mutants
(Fig. S5). These experiments indicate that aldicarb treatment significantly increased the
population of mobile ACR-16 receptors in rig-3 mutants, but not in wild type controls.
These results support the idea that RIG-3 restricts the exchange between synaptic and
mobile ACR-16 receptors, and that it does so by controlling the number of mobile receptors
available for synaptic recruitment.
CAM-1 is required for RIG-3 regulation of ACR-16
A prior study showed that CAM-1, a Ror-type receptor tyrosine kinase (RTK), promotes
ACR-16 delivery to NMJs, but does not regulate Lev receptor levels (Francis et al., 2005).
Consistent with this study, we observed modestly reduced synaptic ACR-16::GFP
fluorescence (78% wild type, p< 0.01) and endogenous EPSC amplitudes (80% wild type,
p< 0.001) in cam-1 mutants (Fig. 7A and B). The cam-1 null mutation did not eliminate
synaptic ACR-16 receptors, as indicated by the residual ACR-16 synaptic fluorescence (Fig.
7B), and by the fact that the endogenous EPSC amplitude observed in acr-16 mutants (48%
wild type, p<0.001; Fig. 7A) were significantly smaller than those observed in cam-1 null
mutants. Thus, synaptic ACR-16 levels are reduced but not eliminated in cam-1 mutants.
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CAM-1 and RIG-3 have opposite effects on synaptic ACR-16 levels and both selectively
regulate ACR-16, having little effect on Lev receptors (Francis et al., 2005). Prompted by
these results, we tested the idea that RIG-3’s effects on ACR-16 are mediated by changes in
CAM-1 activity. Consistent with this idea, the aldicarb hypersensitivity, the increased
endogenous EPSC amplitudes, and the increased ACR-16::GFP levels following aldicarb
treatment were all eliminated in cam-1; rig-3 double mutants (Fig. 7A-C). To determine if
RIG-3 regulates CAM-1 levels, we analyzed GFP-tagged CAM-1 fluorescence in body
muscles. Aldicarb treatment significantly increased CAM-1 puncta fluorescence in the nerve
cord of rig-3 mutants, but had no effect on CAM-1 levels in wild type controls (Fig. 7D).
Taken together, these results suggest that RIG-3 negatively regulates CAM-1 levels at
NMJs, and that increased CAM-1 activity is required for RIG-3’s effects on ACR-16.
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Several prior studies showed that CAM-1 binds secreted Wnt ligands and functions as a Wnt
receptor or as an antagonist inhibiting signaling by other Wnt receptors (Green et al., 2008).
Prompted by these results, we wondered if RIG-3’s effects on synaptic transmission could
result from changes in Wnt signaling at the NMJ. Consistent with this idea, we found that a
mig-14 Wntless mutation, which reduces Wnt secretion (Myers and Greenwald, 2007; Pan et
al., 2008; Yang et al., 2008), confers resistance to aldicarb-induced paralysis and eliminates
the rig-3 aldicarb hypersensitivity defect in mig-14; rig-3 double mutants (Fig. 7E),
implying that Wnt secretion is required for RIG-3’s effects on aldicarb responsiveness.
MIG-14 and CAM-1 regulate Wnt signaling in several developmental pathways, and have
not been implicated in any other (i.e. non-Wnt) signaling pathways; consequently, these
results strongly support the idea RIG-3’s effects on the NMJ are mediated by changes in
Wnt signaling.
RIG-3 alters Wnt regulation of ALM polarity
RIG-3’s effects on CAM-1 at NMJs suggest that RIG-3 might also regulate Wnt signaling in
other tissues. To test this idea, we analyzed the anteroposterior polarity of the ALM
mechanosensory neurons. Several prior studies showed that ALM polarity is regulated by
Wnt signaling (Hilliard and Bargmann, 2006; Prasad and Clark, 2006). The mCherry-tagged
rig-3 genomic construct was expressed in ALM neurons (Fig. 8A), suggesting that RIG-3
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could play a role in Wnt mediated control of ALM polarity. In wild type animals, ALM
neurons have a single anteriorly directed process (Fig. 8B-C). In mutants with decreased
Wnt signaling, ALM neurons exhibit either of two defects, with some neurons having both
an anterior and a posterior process (bipolar ALMs) while others have a single posteriorly
directed process (reversed ALMs) (Fig. 8B-C) (Fleming et al., 2010; Prasad and Clark,
2006). The prevalence of bipolar and reversed ALM neurons differs among Wnt mutants.
These differences in ALM defects likely result from the fact that C. elegans has five Wnt
ligands, which have distinct effects on ALM polarity. For example, two prior studies
showed that the effects of two Wnts (CWN-1 and EGL-20) on ALM polarity are
antagonized by a third Wnt (LIN-44) (Fleming et al., 2010; Prasad and Clark, 2006). Thus,
the precise ALM phenotype observed is determined by how each mutation alters signaling
by the different Wnt ligands.
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To further investigate if RIG-3 plays a role in Wnt signaling, we analyzed the effect RIG-3
inactivation on ALM polarity in several genetic backgrounds. Although ALM polarity was
unaltered in rig-3 single mutants, the rig-3 mutation significantly altered ALM polarity
defects caused by other Wnt pathway mutations in double and triple mutants. Inactivating
RIG-3 in cwn-1; egl-20 double mutants decreased the severity of ALM polarity defects:
ALM reversals were significantly reduced in cwn-1; egl-20; rig-3 triple mutants (p <0.01,
Fishers exact test), while the number of bipolar ALMs was unaffected (p = 0.21, Fishers
exact test). Inactivating RIG-3 in mig-14 Wntless mutants decreased ALM reversals and
increased bipolar ALMs (Fig. 8B-C). The different outcome in mig-14 mutants likely results
from the fact that MIG-14 is required for secretion of all Wnt ligands. By contrast, the rig-3
mutation had no effect on ALM polarity in two strains lacking CAM-1, i.e. cam-1; rig-3
double mutants and cam-1 mig-14; rig-3 triple mutants (Fig. 8C). These results lead to three
conclusions. First, RIG-3 plays an important role in Wnt regulation of ALM polarity.
Second, CAM-1 is absolutely required for RIG-3’s effects on ALM polarity. Third, RIG-3’s
effects on ALM polarity and on ACR-16 levels at the NMJ can both be explained by
changes in Wnt signaling.
Discussion
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Here we define a novel function for an IgSF protein, RIG-3. Our results lead to six primary
conclusions. First, RIG-3 acts in motor neurons to prevent a novel form of post-synaptic
plasticity that is induced by aldicarb treatment. Second, inactivating RIG-3 has no effect on
baseline synaptic transmission, suggesting that RIG-3’s function is required only during
aldicarb-induced plasticity. Third, the synaptic potentiation observed in rig-3 mutants is
mediated by aldicarb-induced accumulation of post-synaptic ACR-16 nAChR receptors.
Fourth, RIG-3 decreases the number of mobile ACR-16 receptors available for recruitment
into post-synaptic receptor fields. Fifth, inactivating RIG-3 also alters the polarity of ALM
neurons. And sixth, RIG-3’s effects on cholinergic transmission and on ALM polarity are
both mediated by changes in Wnt signaling, and in particular by inhibiting the activity of a
Wnt-binding protein (CAM-1). Below, we discuss the significance of these findings.
RIG-3 is a novel regulator of Wnt signaling
Several results suggest that RIG-3 inhibits the function of CAM-1. First, aldicarb treatment
increased CAM-1 levels at NMJs in rig-3 mutants, but not in wild type controls. Second, a
cam-1 null mutation eliminated RIG-3’s effects on aldicarb responses, EPSCs, ACR-16
levels, and ALM polarity. This double mutant analysis is particularly informative. The
cam-1 mutation completely occludes the effects of RIG-3 on ACR-16 trafficking, despite the
fact that ACR-16 levels are only modestly reduced in cam-1 mutants (~80% wild type
levels). These results provide strong genetic evidence that CAM-1 acts downstream of
RIG-3. Thus, both double mutant analysis and imaging data support the idea that RIG-3
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regulates ACR-16 and ALM polarity through changes in CAM-1 activity. RIG-3’s effects on
CAM-1 levels could occur through a variety of mechanisms. RIG-3 and CAM-1 both
contain Ig domains, which could mediate direct binding interactions between these proteins.
Alternatively, RIG-3 could inhibit Wnt secretion or Wnt binding to CAM-1 or other Wnt
receptors.
CAM-1 can function as a receptor mediating the effects of Wnt ligands or as an antagonist
inhibiting Wnt binding to other Wnt receptors (Green et al., 2008). Despite this ambiguity,
all of CAM-1’s known effects on development are mediated by changes in Wnt signaling
(Green et al., 2008). Thus, RIG-3’s absolute requirement for CAM-1 suggests that RIG-3’s
effects on synaptic transmission and on ALM polarity are both mediated by changes in Wnt
signaling.
RIG-3 inhibition of CAM-1 could potentially promote or inhibit Wnt signaling, depending
on whether CAM-1 functions as a receptor or an antagonist. Consequently, to assess how
RIG-3 alters Wnt signaling, we compared the effect of rig-3 mutations to those caused by
mutations inactivating Wnt ligands or decreasing Wnt secretion. At the NMJ, a mig-14
Wntless mutation and a rig-3 mutation had opposite effects on aldicarb-induced paralysis
and the effect of RIG-3 on aldicarb-responsiveness was eliminated in mig-14; rig-3 double
mutants. These results suggest that RIG-3 regulates aldicarb responses by inhibiting Wnt
signaling at the NMJ.
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For ALM polarity, the results are more complicated. Prior studies showed that four Wnt
ligands play a role in dictating ALM polarity but that distinct ALM defects (i.e. bipolar
versus reversed ALM neurons) are observed when different combinations of Wnt’s are
inactivated (Fleming et al., 2010; Prasad and Clark, 2006). Two results suggest that a global
reduction in Wnt signaling primarily leads to reversed ALM neurons: quintuple mutants
containing mutations in all five Wnt ligands (55% reversed, 5% bipolar) (Fleming et al.,
2010) and mig-14 mutants (which reduce secretion of all Wnt ligands) (69% reversed, 12%
bipolar) (Fig. 8). These data indicate that the different ALM phenotypes observed in Wnt
mutants are analogous to an allelic series whereby more extreme Wnt defects cause
primarily ALM reversals while less severe defects cause fewer reversals and increased
bipolar ALMs. Inactivating RIG-3 in cwn-1; egl-20 double mutants significantly decreased
reversed ALMs and had no effect on bipolar ALMs, indicating that RIG-3 and these two
Wnt ligands have opposite effects on ALM polarity. Inactivating RIG-3 in mig-14 mutants
also resulted in a less severe phenotype (with decreased ALM reversals and increased
bipolar ALMs). In both experiments, rig-3 mutations and mutations inactivating Wnt
signaling had opposite effects on ALM polarity. Thus, analysis of RIG-3’s effects on the
NMJ and on ALM polarity both support the idea that RIG-3 normally inhibits Wnt
signaling. These results do not exclude the possibility that RIG-3 promotes Wnt signaling in
other contexts. In particular, in cases where CAM-1 functions as a Wnt antagonist, RIG-3
inhibition of CAM-1 could enhance Wnt signaling.
Wnt’s have been implicated in many aspects of neuronal development, including axon
guidance, cell migrations, and synapse formation (Budnik and Salinas, 2011). Although
Wnt’s are often involved in regulating development, several results suggest that RIG-3 and
CAM-1’s effects on ACR-16 trafficking are not mediated by changes in synapse
development. Inactivation of RIG-3 had no effect on synapse morphology nor on baseline
synaptic transmission at adult cholinergic and GABAergic NMJs, suggesting that
development of these synapses had not been altered. Instead, a rig-3 synaptic defect was
apparent only after treating adult animals with aldicarb, implying the RIG-3 is required for
aldicarb-induced plasticity. Post-synaptic responses at these cholinergic NMJs are mediated
two classes of nicotinic receptors (i.e. ACR-16 and Lev receptors). In rig-3 mutants, aldicarb
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treatment increased ACR-16 levels and ACR-16-mediated currents, but had no effect on
UNC-29 Lev receptor levels nor on Lev receptor-mediated currents. These results argue
strongly against a developmental basis for the rig-3 synaptic defect because disruptions of
synapse or muscle development would alter both post-synaptic receptors equally, and would
not be contingent upon aldicarb treatment. For these reasons, we propose that RIG-3
regulates Wnt signals involved in both neural development (ALM polarity) and synaptic
plasticity (ACR-16 trafficking).
Wnt’s are implicated in several other examples of synaptic plasticity. For example, activity
evokes Wnt secretion in both Drosophila and in rodent hippocampal neurons, mediating
activity dependent plasticity in both cases (Ataman et al., 2008; Chen et al., 2006).
Implications for Wnt signaling
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Several other Wnt antagonists have been described (Kawano and Kypta, 2003). For
example, secreted frizzled-related proteins (SFRPs) bind Wnt ligands, inhibiting their
signaling. Secreted Dickkopf proteins bind to low density lipoprotein receptor related
proteins (LRPs), which are accessory subunits for Frizzled receptors, thereby inhibiting Wnt
signaling. We show that RIG-3 inhibits CAM-1, a receptor mediating non-canonical Wnt
signaling. These results suggest that different inhibitors are utilized to inhibit canonical and
non-canonical Wnt pathways. Prior studies focused primarily on the developmental effects
of Wnt antagonists. Our results suggest that RIG-3 (and potentially other Wnt antagonists)
could also regulate activity-induced synaptic plasticity in mature animals. Like Wnts,
several other secreted morphogens have also been implicated in regulating synaptic
function, including IGFs, BMPs, and EGF related ligands (Chiu and Cline, 2010; Keshishian
and Kim, 2004; Mei and Xiong, 2008). Antagonists have been identified for each of these
morphogens (Fernandez-Gamba et al., 2009; Ghiglione et al., 1999; Schweitzer et al., 1995;
Smith, 1999). It will be interesting to see if other morphogen antagonists also act as antiplasticity molecules.
RIG-3 constrains post-synaptic plasticity
NIH-PA Author Manuscript
C. elegans has been extensively utilized as a model to study synapse development and
function. One limitation of this model has been the absence of a paradigm for studying
synaptic plasticity. Our analysis of rig-3 mutants identified a novel form of post-synaptic
potentiation whereby a brief treatment with aldicarb induces an increased abundance of
post-synaptic ACR-16 receptors and a corresponding increase in post-synaptic currents. By
contrast, none of these effects were observed following aldicarb treatment of wild type
controls. Collectively, these results demonstrate that inactivation of RIG-3 reveals a novel
form of plasticity whereby the activity-dependent delivery of ACR-16 receptors to synapses
is enhanced.
Analysis of vertebrate synapses has shown that receptors mediating post-synaptic responses
are supplied by a mobile pool of receptors in the plasma membrane that are retained at
synapses by diffusional trapping (Opazo and Choquet, 2011). We propose that RIG-3
regulates synaptic delivery of ACR-16 by an analogous mechanism. C. elegans NMJs are
formed in the dorsal and ventral nerve cords by en passant contacts between motor neuron
axons and processes extending from body muscles (which are termed muscle arms). In rig-3
mutants, aldicarb treatment increases the mobile fraction of GFP-tagged ACR-16 receptors
in the nerve cord. These mobile ACR-16 receptors are likely in the plasma membrane of
muscle arms, as receptors residing in intracellular organelles are typically immobile (Tardin
et al., 2003). An increased number of mobile ACR-16 receptors available for diffusional
trapping would be expected to cause a corresponding increase in synaptic ACR-16 receptors
(Opazo and Choquet, 2011).
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Although RIG-3 regulates post-synaptic receptor trafficking, RIG-3 functions in the presynaptic membrane. RIG-3 is expressed in cholinergic motor neurons, is enriched near presynaptic elements, and the active form of RIG-3 is tethered to the pre-synaptic plasma
membrane. Thus, RIG-3’s effects on ACR-16 levels are triggered from a pre-synaptic
location. Trans-synaptic regulation of ACR-16 levels by RIG-3 could occur by a variety of
mechanisms. Pre-synaptic RIG-3 could antagonize signaling by secreted Wnt molecules. In
this scenario, one might expect that RIG-3 expressed in one motor neuron would regulate
ACR-16 levels at synapses formed by neighboring neurons. Contrary to this idea, we found
that RIG-3’s effects on ACR-16 are spatially restricted to nearby post-synaptic elements,
and possibly to direct post-synaptic targets. Other potential mechanisms for trans-synaptic
regulation of ACR-16 levels include direct binding of RIG-3 to post-synaptic CAM-1
receptors, or local regulation of Wnt binding to CAM-1 expressed in post-synaptic partners.
Further experiments will be required to determine the precise mechanisms by which RIG-3
and CAM-1 regulate ACR-16 trafficking.
RIG-3 regulated plasticity is similar in some respects to LTP at hippocampal synapses in
rodents. In both synapses, post-synaptic currents are a composite of receptors with fast
(ACR-16 and AMPA) and slow (Lev receptors and NMDA) kinetics, and potentiation is
mediated by increased delivery of fast receptors. In this context, it is intriguing that some
forms of LTP are disrupted by interfering with Wnt signaling (Chen et al., 2006).
NIH-PA Author Manuscript
Aldicarb treatment also induces a form of pre-synaptic potentiation (Hu et al., 2011). This
pre-synaptic effect is mediated by aldicarb-induced secretion of an endogenous neuropeptide
(NLP-12), which enhances ACh release at NMJs. Thus, the C. elegans body wall cholinergic
NMJ exhibits pre- and post-synaptic forms of plasticity, both of which are induced by
aldicarb treatment, but which are mediated by distinct signaling pathways. It will be
interesting to determine if these two forms of aldicarb induced plasticity are coordinately
regulated.
Implications for circuit function
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Several adhesion molecules are known to promote recruitment of post-synaptic receptors. In
particular, Neuroligin-1 promotes recruitment of glutamate receptors to synapses, whereas
Neuroligin-2 promotes recruitment of GABA receptors (Chih et al., 2005; Graf et al., 2004).
Several other families of cell surface molecules also promote synaptic targeting of receptors,
including auxiliary subunits (e.g. TARPs) and CUB domain containing proteins (e.g. SOL-1
and LEV-10) (Chen et al., 2000; Gally et al., 2004; Zheng et al., 2004). Our results suggest
that cell surface IgSF proteins (like RIG-3) can also stabilize synaptic signaling, by
preventing plastic changes in post-synaptic receptor fields. Thus, we propose that the
dynamic behavior of post-synaptic receptors is regulated by both positive and negative
factors.
Anti-plasticity molecules like RIG-3 could play important roles in circuit development or
function. In particular, we envisage two potential functions for anti-plasticity molecules.
During development, morphogens typically induce bistable signals, producing switch like
changes in cellular fates. This property of morphogen signaling could be particularly useful
in the context of developing circuits. During development, many circuits undergo activitydependent synaptic refinement, where plasticity in each circuit is restricted to specific times
during development, often referred to as critical periods (Hensch, 2004). Thus, these forms
of critical period plasticity occur in a switch like manner, opening and closing during
specific developmental time windows. We speculate that morphogens and their antagonists
may provide a biochemical mechanism for spatial and temporal patterning of synaptic
plasticity during development.
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Anti-plasticity molecules could also stabilize circuit function. It has long been proposed that
mechanisms must exist that oppose correlation based rules for activity-dependent plasticity
(e.g. LTP and LTD) (Miller, 1996). These correlation based plasticity rules are thought to
confer instability on circuits because repeated potentiation or depression would
systematically shift all synapses to higher or lower activities. Homeostatic plasticity (or
metaplasticity) has been proposed as a potential solution to this problem (Pratt et al., 2003).
We propose that anti-plasticity molecules may also perform this stabilizing function.
Inappropriate changes in circuit activity could be prevented by expression of molecules such
as RIG-3, whose function is to prevent expression of plasticity. Conversely, mutations in
anti-plasticity molecules would perturb circuit activity, and may contribute to cognitive and
behavioral disorders.
Methods
Strains
Strains were maintained as described previously at 20°C (Brenner, 1974). OP50 Escherichia
coli were used for feeding. The wild-type reference strain was N2 Bristol. Descriptions of
allele lesions can be found at http://www.wormbase.org. The mutant strains used were:
eri-1(mg366), lin-15B(n744), rig-3(ok2156), acr-16(ok789), cam-1(ak37), mig-14(ga62),
cwn-1(ok546) and egl-20(n585).
NIH-PA Author Manuscript
RNAi Feeding assay
RNAi assays were performed in the eri-1; lin-15b background (Wang et al., 2005). RNAi
clones utilized were previously described (Kamath and Ahringer, 2003; Kamath et al.,
2003). Acute aldicarb assays were performed in triplicate on young adult worms by an
experimenter unaware of the identity of the RNAi clone utilized, all as described (Lackner et
al., 1999). Aldicarb (Sigma and Roche) concentration was 1 mM.
Fluorescence Microscopy and Quantitative Analysis
All quantitative imaging was done using a Olympus PlanAPO 100x 1.4 NA objective and an
ORCA100 CCD camera (Hamamatsu). Worms were immobilized with 30 mg/ml BDM
(Sigma). Imaging was done in either untreated animals or after a 60 minute exposure to
1mM aldicarb. Line scans of dorsal cord fluorescence were analyzed in Igor Pro
(WaveMetrics) using custom-written software (Burbea et al., 2002; Dittman and Kaplan,
2006). For coelomocyte imaging, the posterior coelomocyte was imaged in larval stage 4
(L4) and early adult worms (Sieburth et al., 2007). All p-values indicated were based on
student t-tests.
NIH-PA Author Manuscript
ALM polarity was visualized with the integrated transgene zdIs5 [Pmec-4::gfp], in animals
immobilized with 1% sodium azide, using a Zeiss Axioskop2 microscope. The zdIs5
transgene is expressed in six mechanosensory neurons, ALMs, PLMs, AVM and PVM (Pan
et al., 2008). For ALM, the bipolar phenotype was defined as a normal anterior process and
a posterior process that is longer than five ALM cell diameters in length. P-values were
determined by the Fisher exact test.
FRAP experiments were performed using the Olympus FV1000 confocal microscope. Image
stacks were captured, and maximum intensity projections were obtained using Metamorph
7.1 software (Universal Imaging). For FRAP experiments, the worms were immobilized in
10% agarose containing 0.5 μl of 0.1 μm polystyrene microspheres (polysciences). Animals
were imaged at 5 and 10 minute intervals until 55 or 65 minutes after photobleaching. To
control for motion artifacts, we measured fluorescence of neighbouring unbleached ACR-16
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Babu et al.
Page 12
puncta. We excluded any experiments where the fluorescence of neighboring puncta
changed by >10% over the course of the experiment.
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Electrophysiology
Electrophysiology was done on dissected adults as previously described (Richmond and
Jorgensen, 1999). All recording conditions were as described previously (Sieburth et al.,
2007). Aldicarb treatment refers to 60 minutes in 1mM aldicarb. For comparing average
electrophysiological values, statistical significance was determined using the Mann-Whitney
test or student’s t test. For cumulative probability distributions, the Kolmogorov-Smirnov
test was used to determine statistical significance.
RIG-3 constructs and transgenes
Transgenic strains were generated by microinjection using several co-injection markers:
KP#1338 (pttx-3::GFP), KP#1480 (pmyo-2::NLS-mCherry) or KP#1106 (pmyo-2::NLSGFP) (Mello et al., 1991). Integrated transgenes containing pmyo-3::ACR-16::GFP
(nuIs299) and the pmyo-3::CAM-1::GFP (nuIs465) were generated by UV mutagenesis. All
of these constructs were derivatives of pPD49.26 or pPD95.75 (Fire, 1997).
NIH-PA Author Manuscript
Five RIG-3 constructs rescued rig-3(ok2156) mutants: KP#5918 (psnb-1::RIG-3), KP#5914
(punc-17::RIG-3), KP#6005 (punc-17::mCherry::RIG-3), KP#6292
(punc-17::mCherry::RIG-3TMD) and KP#6298 (prig-3::mCherry::RIG-3). Three RIG-3
constructs did not rescue rig-3(ok2156) mutants: KP #5915 (punc-25::RIG-3), KP#6012
(pvha-6::RIG-3) and KP#6305 (punc-17::mCherry::RIG-3ΔGPI). The
punc-129::mCherry::RIG-3 expresses RIG-3 in DA neurons, and was used to assess rescue
of ACR-16 defects in the dorsal versus ventral cords (KP#6008). All rescue constructs
contained the RIG-3 cDNA, with mCherry inserted between the amino acids 42 and 43. The
genomic mCherry::RIG-3 line was made using a 11kb RIG-3 genomic fragment with
mCherry inserted between amino acids 42 and 43. RIG-3(ΔGPI) contains a deletion of the
carboxy-terminal 23 amino acids. In RIG-3(TMD), the carboxy-terminal 23 residues or
RIG-3 were replaced by transmembrane and cytoplasmic domains derived from NLG-1.
Several transgenes containing GFP-tagged synaptic proteins were previously described:
nuIs321 (punc-17::mCherry), nuEx379 (pacr-2::GFP), nuIs152 (punc-129::GFP::SNB-1),
nuIs159 (punc-129::SYD-2::YFP), nuIs169 (punc-129::GSNL-1::YFP) (Sieburth et al.,
2005; Sieburth et al., 2007), zdIs5 (pmec-4::GFP) (Pan et al., 2008), akIs38 (UNC-29::GFP)
(Francis et al., 2005) and nuIs283 (pmyo-3::UNC-49::GFP) (J. Bai and J.K., unpublished).
Quantitative PCR experiments
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For real-time PCR experiments, late L4 and early adult worms were transferred to mock
treatment plates or plates containing 1 mM aldicarb for 1 hour after which the RNA from
these animals was harvested and quantitative PCR performed as detailed in (Simon et al.,
2008).
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
This work was supported by grants from the NIH, NS32196 (J.M.K.) and NS32057 (G.G.). We thank the following
for strains, reagents, and advice: C. elegans Genetic Stock Center, Villu Maricq, Jihong Bai, Ed Pym, and Eyleen
O’Rourke. We also thank members of the Kaplan laboratory for suggestions and comments on this manuscript.
Neuron. Author manuscript; available in PMC 2012 July 14.
Babu et al.
Page 13
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Figure 1. Inactivation of rig-3 causes hypersensitivity to aldicarb
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(A) A schematic of the RIG-3 protein is shown, indicating the signal sequence (ss), Ig,
FNIII, and GPI- anchoring domains, and the site utilized for mCherry tagging. The domains
deleted in rig-3(ok2156) mutants are indicated by the bar. (B) Aldicarb-induced paralysis is
compared following RNAi treatments with rig-3 and two negative controls, empty vector
(vector) and eri-1. The number of replicate experiments for each RNAi treatment (>20
animals/replicate) is indicated. (C) Aldicarb-induced paralysis is shown for the indicated
genotypes. RIG-3 transgenes are as follows: ACh neurons (unc-17 promoter), gut (vha-6
promoter), GABA (unc-25 promoter), RIG-3 (mCherry-tagged rig-3 genomic construct),
TMD (membrane-anchored RIG-3 expressed in ACh neurons), ΔGPI (constitutively
secreted RIG-3 expressed in ACh neurons). The number of trials (~20 animals/trial) is
indicated for each genotype. Values that differ significantly from wild type (***, p < 0.001)
and from rig-3 mutants (###, p < 0.001) are indicated. Error bars indicate SEM.
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Figure 2. RIG-3 is expressed in ACh neurons
(A) The mCherry-tagged rig-3 genomic construct is expressed in cholinergic motor neurons,
which are identified by expression of the acr-2::gfp reporter. Arrows indicate the cholinergic
neurons that express RIG-3. (B) Distribution of mCherry::RIG-3 (expressed with the rig-3
promoter) and GFP::SNB-1 (expressed in DA neurons with the unc-129 promoter) are
compared in dorsal cord axons. (C) mCherry::RIG-3 fluorescence in coelomocytes is shown.
(D) Soluble mCherry (expressed in cholinergic neurons) did not label coelomocytes.
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Figure 3. Aldicarb treated rig-3 mutants show an increased post-synaptic responses
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Endogenous EPSCs (A), stimulus-evoked EPSCs (B), and ACh-evoked currents (C) were
recorded from body wall muscle of adult worms of the indicated genotypes, with (grey) and
without (black) a 60 minute aldicarb treatment. Representative traces of endogenous EPSCs,
averaged traces of stimulus-evoked responses and ACh-evoked responses, and summary
data for all three are shown. Rescue (resc) refers to transgenic animals expressing RIG-3 in
cholinergic neurons (with the unc-17 promoter). The number of animals analyzed is
indicated for each genotype. Values that differ significantly from untreated wild type (***, p
<0.001) and from untreated rig-3 mutants (###, p <0.001) are indicated. Error bars indicate
SEM.
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Figure 4. Aldicarb increases ACR-16 synaptic abundance in rig-3 mutants
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(A) Representative images of dorsal cord ACR-16::GFP fluorescence in aldicarb treated
animals is shown for the indicated genotypes. Summary data (Right) for dorsal ACR-16
puncta fluorescence is shown for control and aldicarb treated animals of the indicated
genotypes. ACh rescue refers to rig-3 mutants containing a transgene expressing RIG-3 in
all cholinergic neurons (using the unc-17 promoter). (B) A schematic illustrating the
morphology of a cholinergic DA motor neuron (right) and representative images of RIG-3
fluorescence in the dorsal and ventral cord processes of DA neurons are shown (left).
mCherry-tagged RIG-3 was expressed in DA neurons (using the unc-129 promoter). (C-D)
Representative images and summary data are shown for dorsal (C) and ventral (D) ACR-16
puncta fluorescence in the indicated genotypes. DA rescue refers to rig-3 mutants containing
a transgene expressing RIG-3 in DA neurons (using the unc-129 promoter). The number of
animals analyzed is indicated for each genotype. A GFP-tagged ACR-16 construct was
expressed in body muscles (with the myo-3 promoter). ACR-16 puncta in the dorsal and
ventral cords correspond to post-synaptic receptors at dorsal and ventral NMJs, respectively.
Values that differ significantly from aldicarb treated wild type controls are indicated (**, p
< 0.01; ***, p < 0.001). Error bars indicate SEM.
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Figure 5. ACR-16 is required for aldicarb-induced potentiation of post-synaptic responses in
rig-3 mutants
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(A) Summary data is shown for aldicarb-induced paralysis in the indicated genotypes.
Transgenic animals over-expressing ACR-16 (ACR-16 OE) are indicated. The number of
trials (~20 animals/trial) is indicated for each genotype. (B-C) Traces and summary data for
endogenous EPSCs (B), evoked EPSCs (C), and ACh-activated currents (D) are shown for
control and aldicarb treated animals of the indicated genotypes. For endogenous EPSCs,
representative traces are shown. For evoked EPSCs and ACh-activated EPSCs, averaged
traces are shown. The number of animals analyzed is indicated for each genotype. Values
that differ significantly from wild type controls are indicated (***, p < 0.001; *, p <0.05).
Error bars indicate SEM.
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Figure 6. RIG-3 regulates ACR-16 delivery to post-synaptic elements
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Representative images (A) and summary data (B) are shown for FRAP of ACR-16::GFP at
dorsal cord NMJs of control and aldicarb treated animals. At time 0, ACR-16 fluorescence
in a 2 μm box encompassing a single punctum was photobleached. ACR-16 fluorescence
was subsequently measured at the photobleached and a neighboring control punctum. The
fractional recovery of fluorescence 45 minutes after photobleaching is shown (B). The
fluorescence of control unbleached puncta did not change significantly after 45 minutes of
imaging (data not shown). The number of animals analyzed is indicated for each genotype.
Values that differ significantly from wild type controls are indicated (**, p <0.01). Error
bars indicate SEM.
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Figure 7. CAM-1 Wnt receptors are required for RIG-3’s effects on ACR-16
(A) Averaged traces and summary data are shown for endogenous EPSCs of control and
aldicarb treated animals. The number of animals analyzed is indicated for each genotype.
(B-C) Representative images and summary data are shown for dorsal cord ACR-16::GFP
(B) and ventral cord CAM-1::GFP fluorescence (C) in control and aldicarb treated animals.
The number of animals analyzed is indicated for each genotype. GFP-tagged ACR-16 and
CAM-1 were expressed in body muscles (using the myo-3 promoter). (D-E) Summary data
is shown for aldicarb-induced paralysis in the indicated genotypes. The number of trials
(~20 animals/trial) is indicated for each genotype. In panel E, all strains (including the WT
control) contain the zdIs5 transgene, which expresses GFP in the touch neurons. Values that
differ significantly from wild type controls are indicated (***, p < 0.001; *, p <0.05). Error
bars indicate SEM.
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Figure 8. RIG-3 antagonizes the effects of Wnt on ALM polarity
(A) Expression of the mCherry-tagged rig-3 genomic construct is shown in ALM neurons.
The ALM neurons were visualized with the zdIs5 transgene, which expresses GFP in the
touch neurons (with the mec-4 promoter). (B) Representative images and schematic
drawings are shown illustrating wild type, bipolar (less severe), and reversed (more severe)
ALM defects. (C) Summary data for ALM polarity defects are shown for the indicated
genotypes. The number of animals analyzed is indicated for each genotype. All strains
contain the zdIs5 transgene, to allow visualization of ALM neurons. Values that differ
significantly from wild type controls are indicated (***, p< 0.001; **, p< 0.01).
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