The MicroRNA mir-71 Inhibits Calcium Signaling by
Targeting the TIR-1/Sarm1 Adaptor Protein to Control
Stochastic L/R Neuronal Asymmetry in C. elegans
Yi-Wen Hsieh, Chieh Chang.*, Chiou-Fen Chuang.*
Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center Research Foundation, Cincinnati, Ohio, United States of America
The Caenorhabditis elegans left and right AWC olfactory neurons communicate to establish stochastic asymmetric identities,
AWCONand AWCOFF, by inhibiting a calcium-mediated signaling pathway in the future AWCONcell. NSY-4/claudin-like
protein and NSY-5/innexin gap junction protein are the two parallel signals that antagonize the calcium signaling pathway
to induce the AWCONfate. However, it is not known how the calcium signaling pathway is downregulated by nsy-4 and nsy-
5 in the AWCONcell. Here we identify a microRNA, mir-71, that represses the TIR-1/Sarm1 adaptor protein in the calcium
signaling pathway to promote the AWCONidentity. Similar to tir-1 loss-of-function mutants, overexpression of mir-71
generates two AWCONneurons. tir-1 expression is downregulated through its 39 UTR in AWCON, in which mir-71 is expressed
at a higher level than in AWCOFF. In addition, mir-71 is sufficient to inhibit tir-1 expression in AWC through the mir-71
complementary site in the tir-1 39 UTR. Our genetic studies suggest that mir-71 acts downstream of nsy-4 and nsy-5 to
promote the AWCONidentity in a cell autonomous manner. Furthermore, the stability of mature mir-71 is dependent on nsy-
4 and nsy-5. Together, these results provide insight into the mechanism by which nsy-4 and nsy-5 inhibit calcium signaling
to establish stochastic asymmetric AWC differentiation.
Citation: Hsieh Y-W, Chang C, Chuang C-F (2012) The MicroRNA mir-71 Inhibits Calcium Signaling by Targeting the TIR-1/Sarm1 Adaptor Protein to Control
Stochastic L/R Neuronal Asymmetry in C. elegans. PLoS Genet 8(8): e1002864. doi:10.1371/journal.pgen.1002864
Editor: Andrew D. Chisholm, University of California San Diego, United States of America
Received December 7, 2011; Accepted June 12, 2012; Published August 2, 2012
Copyright: ? 2012 Hsieh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: C-FC was funded by a Whitehall Foundation Research Award, an Alfred P. Sloan Research Fellowship, and NIH grant R01 GM098026. CC was funded by
a Whitehall Foundation Research Award and March of Dimes Foundation. Y-WH was supported by an NIH Organogenesis Training Grant. The funders had no role
in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (C-FC); email@example.com (CC)
. These authors were joint senior authors on this work.
Cell fate determination during development requires both the
induction of cell type specific genes and the suppression of genes that
promote an alternative cell fate [1–4]. For example, both inductive
signaling, mediated by an EGFR-Ras-MAPK pathway, and lateral
inhibition, mediated by LIN-12/Notch activity and microRNA
(miRNA), are required for six multipotential vulval precursor cells to
adopt an invariant pattern of fates in C. elegans . Notch signaling-
mediated lateral inhibition also plays a crucial role in the neuronal/
glial lineage decisions of neural stem cells; as well as the B/T,
alphabeta/gammadelta, and CD4/CD8 lineage choices during
lymphocyte development [6,7]. In the Drosophila eye, the kinase
Warts and PH-domain containing Melted repress each other’s
transcription in a bistable feedback loop to regulate the two
alternativeR8 photoreceptorsubtypes expressingRhodopsinRh5 or
Rh6 . In the C. elegans sensory system, two sets of transcription
factors and miRNAs reciprocally repress each other to achieve and
stabilize one of the two mutually exclusive ASEL and ASER taste
neuronal fates [8–10]. Notch signaling acts upstream of the miRNA-
controlled bistable feedback loop to regulate ASE asymmetry
through a lineage-based mechanism in early embryos .
The C. elegans left and right sides of Amphid Wing Cell C (AWC)
olfactory neurons specify asymmetric subtypes through a novel
mechanism independent of the Notch pathway in late embryo-
genesis . Like ASE neurons, the two AWC neurons are
morphologically symmetrical but take on asymmetric fates, such
that the AWCONneuron expresses the chemoreceptor gene str-2
and the contralateral AWCOFF
Asymmetric differentiation of AWC neurons allows the worm to
discriminate between different odors . In contrast to repro-
ducible ASE asymmetry, AWC asymmetry is stochastic: 50% of
animals express str-2 on the left and the other 50% express it on
the right. Ablation of either AWC neuron causes the remaining
AWC neuron to become AWCOFF, suggesting that AWCOFFis the
default state and the induction of AWCONrequires an interaction
or competition between the AWC neurons . The axons of the
two AWC neurons form chemical synapses with each other; AWC
asymmetry is established near the time of AWC synapse formation
[16,17]. In addition, axon guidance mutants are defective in
inducing the AWCONstate. These results suggest that the synapses
could mediate the AWC interaction for asymmetry .
nsy-4, encoding a claudin-like tight junction protein, and nsy-5,
encoding an innexin gap junction protein, act in parallel to
downregulate the calcium-mediated UNC-43 (CaMKII)/TIR-1
(Sarm1)/NSY-1 (MAPKKK) signaling pathway in the future
AWCONcell [18,19]. Both AWCs and non-AWC neurons in the
NSY-5 gap junction dependent cell network communicate to
neuron does not [12–14].
PLOS Genetics | www.plosgenetics.org1August 2012 | Volume 8 | Issue 8 | e1002864
participate in signaling that coordinates left-right AWC asymme-
try. In addition, non-AWC neurons in the NSY-5 gap junction
network are required for the feedback signal that ensures precise
AWC asymmetry . Once AWC asymmetry is established in
late embryogenesis, both the AWCONand AWCOFFidentities are
maintained by cGMP signaling, dauer pheromone signaling, and
(Sarm1), and nsy-1 (MAPKKK) are also implicated in the
maintenance of AWC asymmetry in the first larval (L1) stage
. Although multiple genes were identified to be involved in the
establishment and the maintenance of AWC asymmetry (for a
review, see ), it is still unknown how the calcium-regulated
signaling pathway is inhibited by nsy-4 and nsy-5 in the AWCON
The TIR-1/Sarm1 adaptor protein assembles a calcium-
signaling complex, UNC-43 (CaMKII)/TIR-1/NSY-1 (ASK1
MAPKKK), at AWC synapses to regulate the default AWCOFF
identity , thus downregulation of tir-1 expression may
represent an efficient mechanism to inhibit calcium signaling in
the cell becoming AWCON. In support of this idea, a prior large
scale examination of potential miRNA targets indicated that tir-1
and unc-43 may be downregulated by this class of RNAs .
Here, we analyze the function of the miRNA mir-71 in stochastic
AWC asymmetry by characterizing its role in downregulation of
the calcium signaling pathway in the AWCONcell. We show that
mir-71 acts downstream of nsy-4/claudin and nsy-5/innexin to
promote AWCONin a cell autonomous manner through inhibiting
tir-1 expression, in parallel with other processes. We also show that
nsy-4 and nsy-5 are required for the stability of mature mir-71. Our
results suggest a mechanism for genetic control of AWC
asymmetry by nsy-4 and nsy-5 through mir-71-mediated downreg-
ulation of calcium signaling.
Identification of miRNAs with predicted target genes in
the AWC calcium signaling pathway
The calcium-regulated UNC-43 (CaMKII)/TIR-1 (Sarm1)/
NSY-1 (ASK1 MAPKKK) signaling pathway suppresses expres-
sion of the AWCONgene str-2 in the default AWCOFFcell
[12,16,25,26]. To establish AWC asymmetry, the calcium-
mediated signaling pathway is suppressed in the future AWCON
cell. miRNAs are small non-coding RNAs that are robust in
mediating post-transcriptional and/or translational downregula-
tion of target genes . In C. elegans, miRNAs are processed from
premature form into mature form by alg-1/alg-2 (encoding the
Argonaute proteins) and dcr-1 (encoding the ribonuclease III
enzyme Dicer) . Gene expression profiling revealed increased
levels of unc-43 and tir-1 in dcr-1 mutants , suggesting that unc-
43 and tir-1 may be downregulated by miRNAs. Thus, we
hypothesized that miRNAs may play a role in downregulation of
the UNC-43/TIR-1/NSY-1 signaling pathway in the cell
To test this hypothesis, we took a computational approach to
identify miRNAs predicted to target the 39 UTRs of known genes,
including unc-2, unc-36, egl-19, unc-43, tir-1, nsy-1, and sek-1, in the
AWC calcium signaling pathway. Only the miRNAs that fit the
following criteria were selected for further analysis: 1) At least 6
nucleotides in the seed region (position 1–7 or 2–8 at the 59 end) of
a miRNA is perfectly matched to the target 39 UTR; 2) The seed
match between a miRNA and its target 39 UTR is conserved
between C. elegans and a closely related nematode species C.
briggsae, since evolutionary conservation between C. elegans and C.
briggsae genomes is useful in identifying functionally relevant DNA
sequences such as regulatory regions [29,30]; and 3) A miRNA is
predicted by both MicroCosm Targets (formerly miRBase
targets/v5/) [31–33] and TargetScan (http://www.targetscan.
org/worm_12/) . Based on these criteria, we identified six
potential miRNAs (mir-71, mir-72, mir-74, mir-228, mir-248, mir-
255) predicted to target unc-2, unc-43, tir-1, nsy-1, and sek-1 (Figure
S1A). A subset of these identified miRNA-target pairs were also
predicted by other miRNA target prediction programs, including
PicTar (http://pictar.mdc-berlin.de/)  and mirWIP (http://
Since most miRNAs are not individually essential and have
functional redundancy [37–40], loss-of-function mutations in a
single miRNA may not show a defect in AWC asymmetry. To
circumvent potential problems that may be posed by functional
redundancy, we took an overexpression approach to determine the
role of these six miRNAs in AWC asymmetry. We generated
transgenic strains overexpressing individual miRNAs in both
AWCs using an odr-3 promoter, expressed strongly in AWC
neuron pair and weakly in AWB neuron pair . Wild-type
animals have str-2p::GFP (AWCONmarker) expression in only one
of the two AWC neurons (Figure 1A and 1E). Since loss-of-
function mutations in the AWC calcium signaling genes (unc-2,
unc-36, unc-43, tir-1, nsy-1, and sek-1) led to str-2p::GFP expression
in both AWC neurons (2AWCONphenotype) (Figure 1B and 1E)
[12,16,25,26], we proposed that overexpression of the miRNA
downregulating one of these calcium signaling genes would also
cause a 2AWCONphenotype. We found that mir-71(OE) animals
overexpressing mir-71, predicted to target tir-1 and nsy-1, had a
strong 2AWCONphenotype (Figure 1C, 1E, and Figure S1B). This
result suggests that mir-71 may downregulate the expression of tir-1
and nsy-1 to control the AWCONfate and that mir-71 is sufficient
to promote AWCONwhen overexpressed. However, overexpres-
sion of the other five miRNAs individually caused a mixed weak
phenotype of 2AWCONand 2AWCOFF(Figure S1B). Since the
activity of the nsy-1 39 UTR in AWC was independent of mir-
71(OE) (Figure S2B), we focused on the investigation of the
potential role of mir-71 in promoting AWCONthrough negatively
regulating tir-1 expression.
Cell identity determination requires a competition be-
tween the induction of cell type–specific genes and the
suppression of genes that promote an alternative cell type.
In the nematode C. elegans, a specific sensory neuron pair
communicates to establish stochastic asymmetric identi-
ties by inhibiting a calcium signaling pathway in the
neuron that becomes an induced identity. However, it is
not understood how cell–cell communication inhibits the
calcium signaling pathway in the induced neuronal
identity. In this study, we identify a microRNA that
represses the expression of a key molecule in the calcium
signaling pathway to promote the induced neuronal
identity. Overexpression of the microRNA causes both
neurons of the pair to become the induced identity, similar
to the mutants that lose function in the calcium signaling
pathway. In addition, the stability of the mature microRNA
is dependent on a claudin-like protein and a gap junction
protein, the two parallel signals that mediate communica-
tion of the neuron pair to promote the induced neuronal
identity. Our results provide insight into the mechanism by
which cell–cell communication inhibits calcium signaling
to establish stochastic asymmetric neuronal differentiation.
miRNA and Stochastic L/R Neuronal Asymmetry
PLOS Genetics | www.plosgenetics.org2August 2012 | Volume 8 | Issue 8 | e1002864
Figure 1. mir-71 promotes the AWCONidentity. (A–D) Expression of a stable transgene str-2p::GFP (AWCONmarker) in wild type (A), tir-1(tm3036)
loss-of-function (lf) mutants (B), mir-71(OE) animals overexpressing the transgene odr-3p::mir-71 in AWCs (C), and tir-1(ky648gf) mutants (D). tir-
1(ky648gf) mutants also carry the transgene odr-1p::DsRed (expressed in both AWCONand AWCOFF) to show that the absence of str-2p::GFP expression
miRNA and Stochastic L/R Neuronal Asymmetry
PLOS Genetics | www.plosgenetics.org3 August 2012 | Volume 8 | Issue 8 | e1002864
odr-3p::GFP transgenic lines are shown. Error bars represent
standard error of the mean. Scale bar, 10 mm.
is not dependent on nsy-4 or nsy-5. The GFP intensity of mir-
71p::GFP was compared between the two AWC cells of the same
animal in wild-type, nsy-4(ky627), and nsy-5(ky634) mutants. The
percentage difference of mir-71p::GFP expression between the two
AWC cells was determined by dividing the higher GFP intensity
with the lower GFP intensity. Error bars represent the standard
error of proportion.
Differential expression of mir-71 in the two AWC cells
71 by stem-loop RT–PCR.
Supplemental Methods: Quantification of mature mir-
We thank members of the Chuang and Chang labs for valuable discussions;
Felicia Ciamacco, Brittany Bayne, and Kalyn Campbell for technical
assistance; Yan Zou for wild-type and alg-1(gk214) RNA samples; and
Jennifer Tucker for comments on the manuscript. We also thank Andy Fire
for C. elegans vectors, Baris Tursun and Oliver Hobert (Columbia
University Medical Center, NY) for the ceh-36p::TagRFP plasmid, the C.
elegans Genetic Center for C. elegans strains, and the WormBase for readily
Conceived and designed the experiments: CC C-FC. Performed the
experiments: Y-WH C-FC. Analyzed the data: Y-WH CC C-FC. Wrote
the paper: Y-WH CC C-FC.
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miRNA and Stochastic L/R Neuronal Asymmetry
PLOS Genetics | www.plosgenetics.org 15August 2012 | Volume 8 | Issue 8 | e1002864