Mutations in a Guanylate Cyclase GCY-35/GCY-36 Modify
Bardet-Biedl Syndrome–Associated Phenotypes in
Calvin A. Mok1,2,3, Michael P. Healey4, Tanvi Shekhar1, Michel R. Leroux4, Elise He ´on1,3*, Mei Zhen2,3,5*
1The Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Canada, 2Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto,
Canada, 3Institute of Medical Science, University of Toronto, Toronto, Canada, 4Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby,
Canada, 5Department of Molecular Genetics, University of Toronto, Toronto, Canada
Ciliopathies are pleiotropic and genetically heterogeneous disorders caused by defective development and function of the
primary cilium. Bardet-Biedl syndrome (BBS) proteins localize to the base of cilia and undergo intraflagellar transport, and
the loss of their functions leads to a multisystemic ciliopathy. Here we report the identification of mutations in guanylate
cyclases (GCYs) as modifiers of Caenorhabditis elegans bbs endophenotypes. The loss of GCY-35 or GCY-36 results in
suppression of the small body size, developmental delay, and exploration defects exhibited by multiple bbs mutants.
Moreover, an effector of cGMP signalling, a cGMP-dependent protein kinase, EGL-4, also modifies bbs mutant defects. We
propose that a misregulation of cGMP signalling, which underlies developmental and some behavioural defects of C.
elegans bbs mutants, may also contribute to some BBS features in other organisms.
Citation: Mok CA, Healey MP, Shekhar T, Leroux MR, He ´on E, et al. (2011) Mutations in a Guanylate Cyclase GCY-35/GCY-36 Modify Bardet-Biedl Syndrome–
Associated Phenotypes in Caenorhabditis elegans. PLoS Genet 7(10): e1002335. doi:10.1371/journal.pgen.1002335
Editor: Nicholas Katsanis, Duke University, United States of America
Received May 17, 2011; Accepted August 25, 2011; Published October 13, 2011
Copyright: ? 2011 Mok 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: This work was supported by the Canadian Institutes of Health Research grants MOP-93619 (to MZ and EH) and MOP-97956 (to MRL). MZ is a Canada
Research Chair in Neuroscience, EH holds a Mira Godard Chair in Vision Research, MRL holds a Michael Smith Foundation for Health Research award, and CAM
holds a CIHR doctoral research award. 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: email@example.com (MZ); firstname.lastname@example.org (HZ)
The cilium plays diverse cellular functions in metazoans which
include imparting motility, enabling sensory processes and
regulating the activity of cell signalling pathways during develop-
ment . The biogenesis and maintenance of this evolutionarily
conserved organelle relies on intraflagellar transport (IFT) – the
bidirectional transportation of diverse cargo proteins along the
microtubule-based axoneme. Defective IFT or ciliary dysfunction
result in ciliopathies, a growing class of pleiotropic human diseases
with overlapping clinical features, some being of significant
morbidity . Bardet-Biedl syndrome (BBS, OMIM 209900) is
an autosomal recessive and genetically heterogeneous ciliopathy
with hallmark clinical features that include photoreceptor
degeneration, renal abnormalities, obesity, cognitive impairment,
and digit and genital anomalies . To date, sixteen genes are
associated with BBS [3–5]; of these, eight function mostly as a
conserved protein complex (BBSome)  to regulate vesicular
sorting and packaging , IFT [8–9], as well as cilium
maintenance and function (reviewed in ).
Animal models have been instrumental in deciphering the
physiological functions of BBS proteins [2,10]. Initial characteriza-
tion of Caenorhabditis elegans BBS orthologues led to the discovery of
BBS proteins as ciliary components, associating ciliary defects with
the loss of BBS protein function [11–13]. The loss of BBS-7 and
BBS-8 led to shortened cilia, reduced uptake of a lipophilic dye (DiI)
by the cilium, and defective chemo- and thermotaxis [13–14].
Murine Bbs mutants recapitulate several human BBS features
including photoreceptor degeneration, renal anomalies and obesity
[15–16]. Additionally, these models led to the identification of new
features such as neural tube closure defects , anosmia , and
behavioural, mechano- and thermosensory deficits  that
expanded the diagnostic features of human ciliopathies. Morpho-
lino-mediated knockdown of bbs in zebrafish led to developmental
phenotypes such as dorsal thinning, poor somitic definition and
Kupffer’sVesicle malformation[19–21],while defectscharacteristic
of ciliopathy such as delayed retrograde melanosome transport 
and vision impairment  also manifested.
In addition to a role in sensory transduction, the primary cilium
functions as a signalling ‘apparatus’ to regulate development . For
example, IFT-dependent localization of Sonic Hedgehog (Shh)
receptors to primary cilia is required for Shh signalling [23–24].
Disrupting IFT components IFT172, TG737/Polaris and the motor
KIF3A in the mouse resulted in phenotypes typical of Shh mutants
. Similarly, defective planar cell polarity (PCP) signalling [17,26]
and/or aberrant Wnt signalling [27–28] were associated with the
inactivation of BBS, Polaris or KIF3A components. These, and others
studies [29–30] suggest that the cilium may modulate multiple
signalling pathways in a tissue-specific manner. Aberrant PCP, Shh
and Wnt signalling have been implicated in underlying a number of
ciliopathyfeatures,suchasneural tubeclosure,polydactylyand obesity
[17,22,31–32]. The pathology of other features such as photoreceptor
degeneration, remains largely unexplained, indicating the presence of
unidentified cellular processes that are regulated by the cilium.
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C. elegans BBS orthologues are exclusively expressed by 60
ciliated neurons. Localizing at the base of cilia, they undergo
active IFT, and their absence results in the destabilization of IFT
and sensory defects . C. elegans sensory neurons play key roles in
multiple developmental processes . Some chemosensory
mutants exhibit a reduced body size , indicating that sensory
function may influence this developmental process. Another key
regulator for body size is the cGMP-dependent protein kinase
(PKG) EGL-4 [33–36]; a loss of EGL-4 function leads to increased
body size that is genetically epistatic to that of chemosensory
mutants . The mechanisms for sensory neuron-mediated body
size regulation, however, remain to be fully elucidated. In the
present study, in order to identify additional cilium-regulated
signalling events in C. elegans, we carried out the phenotypic
characterization of bbs mutant animals, and identified genetic
modifiers that associate aberrant cGMP signalling with a subset of
C. elegans bbs mutants share ciliary defects, reduced
body size, delayed developmental timing, and reduced
We performed a thorough phenotypic and behavioural
assessment of severe or complete loss-of-function (lf ) mutants for
the C. elegans bbs-1, -2, -7, -8 and -9 genes (Table S1). Consistent
with previous reports on bbs-7 and -8 , all examined bbs
mutants exhibited a failure in the uptake of a lipophilic dye DiI by
sensory neurons (Figure 1A), confirming a common structural and
functional deficit in the sensory cilia. In addition to sensory defects,
we identified three novel bbs-associated phenotypes: decreased
body size, altered dwelling/exploration behaviour and delayed
Despite grossly normal body morphology, bbs mutants shared a
reduced body length by ,11–28% when compared to wild-type
animals. Defects were visible during early larval stages and
persisted throughout adulthood (Figure 1B and Figure S1). In
these analyses, we defined a 3.5% or greater difference as
biologically relevant in body size change, as this was the upper
range for the coefficient of variance in young adult wild-type
populations. The reduced body length is caused by the loss of BBS
function, as it was fully rescued by introducing a wild-type
genomic or cDNA copy of bbs into the respective mutants
(Figure 1C). A decrease in body width was also characteristic of bbs
mutants (Figure 1D). By DAPI staining of nuclei we did not
observe differences in tissue and cell numbers between wild-type
and bbs-7 animals (data not shown). The overall decrease in body
size is thus best attributed to a smaller, averaged cell size in bbs
bbs double and triple mutants exhibited smaller body sizes that
were no more severe than the smallest bbs single mutant. Although
small differences between the body length of bbs single, double and
triple mutant strains were observed as animals aged, the effects,
however, were not additive (Figure 1B), consistent with BBS
proteins functioning in the same biological processes to regulate
ciliary development and function [6–7,37].
We further examined the body size of a number of IFT mutants,
including che-2, -3, and -11, as well as osm-3, -5, -6 and klp-11, all
of which display defects to cilia structure and abnormal dye filling
(dyf ). Only some exhibited decreases in body size; among them,
che-11 mutants exhibited the most significant decrease (by 11% in
young adulthood), but not as severely as in age-matched bbs
mutants (Figure S2A). Notably, a loss of the IFT motors, KLP-11
and OSM-3 kinesins, and CHE-3 dynein had little or no effect on
body size. Therefore while sensory neurons affect body size, the dyf
phenotype, caused by defective IFT transport, is not indicative of
severe body size defects. The BBSome, exclusively expressed in
ciliated sensory neurons, has a greater influence in the regulation
of body size, indicating a role beyond bridging IFT motors for
BBS proteins in these neurons.
C. elegans exhibits a defined developmental time course .
Multiple bbs mutant strains exhibited slower larval development
(Figure 1E), resulting in a 2.3–6.2 hour delay between the first (L1)
and last (L4) larval stage. During foraging, C. elegans exhibits a
combination of dwelling and exploration/roaming behaviours that
are altered in some chemosensory defective mutants . Multiple
bbs mutants showed a 56% to 76% decrease in overall movement,
or roaming (Figure 1F) when compared to wild-type animals. This
behavioural change does not reflect a general loss of locomotor
activity, as bbs mutant animals exhibit normal locomotion during
roaming. These additional phenotypes support a notion that the C.
elegans cilia regulate cellular processes in addition to taxis
The loss of function of a guanylate cyclase subunit GCY-
35 rescues a subset of endophenotypes in bbs mutants
Unlike all other bbs strains, MT3645 bbs-7(n1606) (received
from the Caenorhabditis Genetics Center) displayed body size, roaming
behaviour and developmental timing characteristics of wild-type
animals. Upon genetic outcrossing, we re-isolated a homozygous
bbs-7(n1606) strain that exhibited phenotypes characteristic of
other bbs mutants. These defects were fully rescued by the
expression of the wild-type genomic copy of bbs-7 (Figure 1D). We
concluded through genetic analyses that a single modifying locus
from the original MT3645 strain rescued a subset of the bbs-7
mutant endophenotypes (Figure 2).
We mapped this modifier allele, hp433, to gcy-35 (Materials and
Methods), a gene encoding the a subunit of a soluble guanylate
cyclase (sGC). sGC proteins are composed of a heme/NO binding
(HNOB), a heme/NO binding associated (HNOBA), and a GC
catalytic domain (reviewed in ). They are heterodimeric
Bardet-Biedl syndrome (BBS) is a genetically heteroge-
neous, multisystemic disorder. Defects to the cilium, an
evolutionarily conserved organelle, cause ciliopathies, a
growing class of diseases that includes BBS. BBS proteins
are involved in the vesicular transport of proteins to the
cilium and in the process of intraflagellar transport. Here
we show that, in addition to sensory defects, Caenorhab-
ditis elegans bbs mutants exhibit reduced body size and
delayed developmental timing. The reduced body size
phenotype is not fully recapitulated by IFT mutants,
suggesting that BBS proteins may have additional
functions beyond bridging IFT motors. We further
identified that the loss of function mutations in the
soluble guanylate cyclase complex, GCY-35/GCY-36, results
in a suppression of these defects. Interestingly, GCY-35/
GCY-36 influences the body size through a cGMP-
dependent protein kinase EGL-4 in a group of body cavity
neurons. BBS proteins, on the other hand, function
through a non-overlapping set of ciliated sensory neurons
to influence cGMP signalling in the body cavity neurons. In
conclusion, this study reveals a non-cell autonomous role
for sensory cilia in regulating cGMP signalling during
development. We propose that aberrant cGMP signalling,
essential for a number of cellular processes, may also
contribute to some ciliopathy features in other systems.
Genetic Modifiers of C. elegans bbs Defects
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Figure 1. Multiple endophenotypes exhibited by bbs mutants. (A) DiI uptake of bbs mutants was reduced compared to wild-type and
transgenic bbs mutants carrying genomic DNA fragments for respective bbs genes. Fisher’s Exact test, *** p,0.001. (B) The body length of young
adult bbs single, double and triple mutants was smaller in comparison to wild-type animals. ANOVA with Tukey, *** p,0.001, n$20. Data represent
mean 6 SD normalized against wild-type body length. (C) The body length defects of bbs mutants were rescued by the expression of wild-type bbs
genes (+) compared to their non-transgenic siblings (2). ANOVA with Tukey, *** p,0.001, n$15. (D) The body width of bbs mutants was also
decreased in comparison to wild-type animals. ANOVA with Tukey, *** p,0.001, n$20. (E) Developmental timing (from L1 to L4 stage) was increased
for bbs mutants in comparison to wild-type animals. ANOVA with Tukey, *** p,0.001, n$50, N$10 replicates. Data represent mean (hours) 6 SD. (F)
bbs mutants roamed less compared to wild-type animals. Kruskal-Wallis with Dunn’s, *** p,0.001, n$25, N$2 replicates. Data represent mean
squares roamed 6 SEM. For bbs-7 dataset, the ok1351 deletion allele was used unless otherwise noted.
Genetic Modifiers of C. elegans bbs Defects
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Genetic Modifiers of C. elegans bbs Defects
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complexes consisting of a and b subunits to catalyze the
conversion of GTP to cGMP. hp433 results in a frame-shift in
the coding sequence and a premature termination codon in the
HNOBA domain, causing a truncation of the GC catalytic domain
(Figure 2A). We also saw that a deletion allele in the GC domain of
gcy-35, ok769, functions as a recessive suppressor of bbs-7 mutants
(Figure 2A and 2B).
While both gcy-35(lf ) alleles exhibit similar body length to that of
wild-type animals, they suppressed the significant body length
defects of multiple bbs mutants (Figure 2B). In contrast, gcy-
35(lf);dyf mutants showed little or no improvement to body length
(Figure S2B). The consistent suppression observed in bbs mutant
animals advocated strongly for the further investigation of gcy-
35(lf) as an epistatic suppressor of bbs-mediated phenotypes. gcy-35
also modified the bbs endophenotypes in developmental timing
and roaming. Both the developmental timing from the first (L1) to
the last (L4) stages, and roaming scores of gcy-35 mutants were
comparable to that of wild-type (Figure 2C and 2D). The
developmental timing of gcy-35;bbs-7 and gcy-35;bbs-2 mutants
was identical to that of wild-type animals, whereas gcy-35;bbs-8
animals showed a partial improvement over that of bbs-8 animals
(Figure 2C). Roaming defects of bbs-2, bbs-7, and bbs-8 mutants
were partially suppressed by gcy-35 (Figure 2D).
Other bbs phenotypes, such as shortened cilia and defective DiI
uptake (Figure 2E and data not shown), were not rescued by gcy-
35(lf) mutants. These results suggest that the ciliary structures
remain impaired in gcy-35;bbs mutants, and that the cellular
pathways regulating body size, developmental timing and roaming
behaviours either function genetically downstream of, or differ
from those involved in sensation.
The GCY-35/GCY-36 sGC complex influences the body
size of bbs mutants through a subset of oxygen sensing
body cavity neurons
GCY-35 and its partner GCY-36 form a heterodimeric sGC that
modulates C. elegans behaviour in response to ambient oxygen
concentrations [40–41]. gcy-36(db66) (lf) mutants  rescued the
body size defect of bbs-7 mutants (Figure 2B). Moreover, gcy-35;
gcy-36;bbs-7 animals exhibited a body size no different from either
gcy-35;bbs-7 or gcy-36;bbs-7 (Figure 2B), consistent with GCY-35 and
We examined in which neurons this sGC influences body size
using bbs mutants. Both GCY-35 and GCY-36 are expressed in the
ciliated body cavity sensory neurons AQR and PQR, and a non-
ciliated body cavity neuron URX; GCY-35 is additionally
expressed in the non-ciliated ALN, PLN, and SDQ neurons
. AQR, PQR and URX expression of wild-type GCY-35 in
gcy-35;bbs-7 animals reverted body size similarly to that of bbs-7
mutants, whereas GCY-35 expression in ALN, PLN, and SDQ
had no effect (Figure 3A). Similarly, the expression of a GFP-
tagged GCY-36 in AQR, PQR and URX neurons in bbs-7;gcy-36
animals was also sufficient to revert body size close to that of bbs-7
mutants (Figure 3A). This suggests a specific requirement of GCY-
35 and GCY-36 in body cavity neurons to regulate body size.
Among them, URX appears to be the most essential neuron, as
restoring GCY-35 in URX (and other neurons not normally
expressing gcy-35/gcy-36) by an exogenous promoter showed a
partial, but significant reversion of the body size in gcy-35;bbs-7
animals (Figure 3A). Similarly, GCY-36 is most essential in URX:
we conducted a mosaic analysis of bbs-7;gcy-36 animals carrying a
functional GFP::GCY-36 transgene, and we observed expression of
GFP::GCY-36 in URX of all rescued animals (Figure 3B). URX
expression of the GCY-35/GCY-36 sGC is therefore essential,
although not fully sufficient, to regulate the body size of bbs mutants.
Finally, if the body cavity neurons contribute to the small size
phenotype of bbs mutants, their ablation by the transgene
qaIs2241(Pgcy-36::EGL-1)  in a bbs background should also
have a rescuing effect. While both qaIs2241 and gcy-35;qaIs2241
animals exhibited normal body sizes, bbs-7;qaIs2241 mutants
showed a significantly increased body size when compared to bbs-7
animals (Figure 3C), further supporting that these neurons
modulate the body size of bbs mutants.
The expression and localization of GFP::GCY-35 and
GFP::GCY-36 are grossly normal in bbs mutants
Our genetic analyses indicate that BBS proteins negatively
regulate sGC-mediated signalling activity. We examined if bbs
mutants exhibit an elevated expression and/or expanded locali-
zation of sGC in these neurons. As reported , a functional
GFP::GCY-36 localized largely to the soma and along the
dendrites of AQR, PQR and URX. We did not observe significant
changes in its expression or localization in bbs mutants (Figure S3),
nor did we see a loss of ciliary localization as reported for the loss
of a putative isoprenylation signal at the C-terminus . bbs
mutations did not perturb, at a gross level, the expression and
localization of GFP::GCY-35 either (Figure S3). We did observe a
high degree of morphological variability in the dendritic endings of
URX, AQR, and PQR neurons as previously reported  in
both wild-type and bbs mutants. However, given the morpholog-
ical variability, we cannot exclude the possibility of subtle
alterations in the cilium length of bbs mutants. Therefore, while
AQR, PQR and URX neurons regulate the body size through a
process that involves sGC activity, it does not appear to result
directly from altered sGC protein level or subcellular localization.
The cGMP-dependent protein kinase (PKG) EGL-4 is a sGC
effector in body size regulation
cGMP is a key secondary messenger (reviewed in ). In
C. elegans, cGMP activates a heteromeric cGMP-gated ion channel
TAX-2/TAX-4 for oxygen sensation and other sensory processes
. cGMP also activates a PKG, EGL-4, to regulate olfactory
adaptation, life span, behavioural states and body size [33–34,46–
47]. Specifically, egl-4(lf) mutants exhibit a large body size, and are
epistatic to the reduced body size and roaming behaviour of some
sensory mutants; whereas a constitutively active, gain-of-function
(gf ) egl-4 mutation, causes a small body size . The shared
effects of bbs and egl-4 suggested that EGL-4 could be a
downstream effector of sGCs in body size regulation.
Figure 2. gcy-35(lf) suppresses a subset of bbs endophenotypes. (A) A schematic of the functional domains of GCY-35 and GCY-36 including
allelic information of the respective mutants. (B) gcy-35(lf) and gcy-36(lf) alleles suppressed body length defects in multiple bbs mutant alleles. ANOVA
with Tukey, *** p,0.001, n$20. Data represent mean 6 SD normalized against wild-type body length. (C) gcy-35(lf) fully or partially suppressed bbs
mutant developmental defects. ANOVA with Tukey, *** p,0.001 with respect to wild-type controls; ns – p$0.05, n$50, N$10 replicates. Data
represent mean (hours) 6 SD. (D) Exploration patterns of gcy-35;bbs-7 and gcy-35;bbs-8 animals (light bars) were partially rescued compared to
respective bbs genotypes (dark bars). Kruskal-Wallis with Dunn’s, ** p,0.01; ns – p$0.05, n$25, N$2. Data represent mean squares roamed 6 SEM.
(E) gcy-35 did not rescue the DiI uptake of bbs-7 mutants, while gcy-35(lf) alone did not result in any DiI defects. Fisher’s Exact test, *** p,0.001; ns –
p.0.05. For bbs-7 data depicted, the ok1351 deletion allele was used unless otherwise noted.
Genetic Modifiers of C. elegans bbs Defects
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We examined the egl-4 mutants (Figure 4) for their genetic
interactions with bbs-7 and gcy-35 mutants. egl-4(lf) alleles were
epistatic to bbs-7, gcy-35 and bbs-7;gcy-35 for body size and
developmental timing (Figure 4B and 4C). egl-4(gf) also exhibited
an epistatic effect to bbs-7 and bbs-7;gcy-35 mutants, although egl-
4(gf);bbs-7 and egl-4(gf);bbs-7;gcy-35 mutants may exhibit a slightly
Figure 3. GCY-35 and GCY-36 are required in body cavity neurons to influence body size. (A) The body length of transgenic (+, light bars)
gcy-35;bbs-7 animals expressing GCY-35 or GFP::GCY-36 constructs versus non-transgenic siblings (2, dark bars). Endogenous GCY-35 expressed in
the body cavity neurons (Pgcy-32, Pgcy-36: URX, AQR, PQR), reverted gcy-35(lf)-mediated body size suppression, while expression in other
endogenous GCY-35-expressing neurons (Plad-2: ALN, PLN, SDQ) did not. Expression of GCY-35 in URX and non-overlapping neurons (Pflp-8: URX,
ASE, PVM) partially reverted the suppression in transgenic gcy-35;bbs-7 animals. ANOVA with Tukey, *** p,0.001, ns – p$0.05, n$20. Data represent
mean 6 SD normalized against wild-type body length. (B) Mosaic analysis of bbs-7;gcy-36 transgenic animals expressing GFP::GCY-36 showed a
requirement for URX expression in rescuing suppression effects. Animals were first separated by body size into two groups: small body size (bbs-7-
like, dark bars) or normal body size (bbs-7;gcy-36-like, light bars); they were then examined for the presence or absence of GFP::GCY-36 expression in
the AQR, PQR and URX neurons. Fisher’s Exact test, *** p,0.001; ** p,0.01, n$20. Y-axis indicates the pooled number of animals of respective
neuronal expression patterns. (C) The genetic ablation of AQR, PQR, and URX neurons using the qaIs2241 (pgcy-32::egl-1) transgene rescued the body
size defects of bbs-7 mutants. The body length of wild-type, gcy-35 and bbs-7 animals (dark bars) was compared to those in combination with
qaIs2241 (light bars). ANOVA with Tukey, *** p,0.001; ns – p$0.05, n$20. Data represent mean 6 SD normalized against wild-type body length. For
bbs-7 dataset, the ok1351 deletion allele was used.
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more severe phenotype than egl-4(gf) that was only distinguished
by statistical analyses (Figure 4B). By contrast, lf mutants for
another cGMP effector, the cGMP-gated cation channel subunit
TAX-2 and TAX-4, did not exert body size suppression for bbs-7
(Figure S4). Moreover, some TGF-b signalling mutants exhibited
altered body size [48–50]. While EGL-4 was proposed to function
genetically downstream of TGF-b signalling , body size defects
of several TGF-b mutants exhibited additive effects in bbs-7 or bbs-
7;gcy-35 backgrounds (Figure 4D), suggesting that TGF-b
signalling and BBS-mediated body size regulation likely operates
in an additive or parallel manner. Taken together, these results
confirm a specific genetic relationship between bbs-7, gcy-35 and
egl-4 for cilia-mediated body size regulation and developmental
To further test if the body size influence of GCY-35/GCY-36
sGC functions through EGL-4, we expressed egl-4 cDNA
harbouring the ad450(gf) mutation in AQR, PQR and URX
neurons of gcy-35;bbs-7 animals. If GCY-35 regulates body size
through activating EGL-4, a constitutively activated EGL-4(ad450)
in these neurons should abrogate the body size suppression by the
loss of gcy-35. Indeed, this transgene reverted animals to a bbs-7-
like body size (Figure 4E). Wild-type animals expressing the same
transgene did not exhibit changes in body size (Figure 4E). Not
only is this result consistent with elevated cGMP signalling
contributing to the reduced body size in bbs mutants, it further
suggests that GCY-35/GCY-36 regulates body size through EGL-
4 in the body cavity neurons.
BBS proteins are required in multiple sensory neurons to
regulate body size
To investigate whether ciliary functions regulate body size
strictly through body cavity neurons like GCY-35/GCY-36, we
expressed a functional BBS-7 or GFP::BBS-2 in the AQR, PQR
and URX neurons of bbs-7 or bbs-2 mutants, respectively. The
small body size of respective bbs mutants was not rescued
(Figure 5A). We did, however, observe a complete rescue of the
body size with a pan-neuronally expressed GFP::BBS-2 in bbs-2
mutants (Figure 5A). Moreover, we observed a full or partial
rescue of the body size defects when a functional BBS-7 was
expressed in at least two non-overlapping groups of sensory
neurons AWB, AWC, AWA, ADF and ASH, or, ADL, ADF,
ASH, PHA and PHB (Figure 5A). All these neurons have sensory
cilia exposed to outside of the body cavity. Therefore, while the
GCY-35/GCY-36 sGC functions through body cavity neurons to
regulate body size, restoring ciliary function in these neurons alone
does not sufficiently reduce cGMP signalling to restore body size.
Alternatively, other sensory neurons that input onto the body
cavity neurons, may serve to regulate body size. Regardless, the
observation that BBS proteins can influence body size through
different groups of sensory neurons is reminiscent of the previously
reported observation that the body size of egl-4(lf) mutants could
be rescued by restoring EGL-4 expression in non-overlapping sets
of sensory neurons .
GCY-35/GCY-36 acts as a predominant effector in
cilia-mediated body size regulation
That multiple, non-overlapping and non-body cavity ciliated
sensory neurons (CSNs) regulate body size suggests a cumulative
effect on cGMP signalling-mediated body size regulation by
multiple sensory neurons, or, a predominating effect by body
cavity neurons in cilia-mediated body size regulation through
GCY-35/GCY-36. The first model predicts that additional GCs in
other sensory neurons would epistatically suppress body size. The
C. elegans genome encodes 7 sGCs and 27 receptor-like GCs (rGCs)
. Single loss-of-function mutations in other sGCs, GCY-31,
32, 33, 34 and 37 all failed to alter bbs-7 body size defects (Figure
S5A and data not shown). Of 16 rGC mutants tested, four
exhibited modifying effects, two very mildly suppressing and two
significantly exacerbating the smaller body size of bbs-7 animals,
but none suppressed bbs-7 mutant phenotypes comparably to gcy-
35/gcy-36 (Figure 5B and Figure S5A). We examined the effect of
several double and triple rGC mutants on bbs-7 body size to
explore the possibility that multiple rGCs function redundantly
 but we did not observe obvious suppression effect in these
additional mutants (Figure S5A).
We further examined the combinational effect of gcy-35 and
other rGC mutants on bbs-7, including a mildly suppressing allele
(gcy-4(tm1653)), two exacerbating alleles (gcy-7(tm901) and gcy-
16(ok2538)) and three ‘‘neutral’’ alleles (gcy-23(ok797), gcy-
28(tm2411) and gcy-25(tm4300)). We did not observe a significant
body size improvement between gcy-35;bbs-7 and these triple
mutants (Figure S5B). Therefore with the caveat that we have not
exhausted the examination of all single or combinational GC
mutants, GCY-35/GCY-36, through the body cavity neurons, act
uniquely as a predominating effector for BBS-mediated body size
In the present study, we show that C. elegans bbs mutants exhibit
reduced body length, delayed development and altered roaming
pattern, in addition to known sensory defects. These endopheno-
types depend, fully or in part, on the GCY-35/GCY-36 sGC
complex, through its effector EGL-4 PKG, in the AQR, PQR and
URX body cavity neurons. On the other hand, body size can also
be regulated via multiple, non-overlapping sets of non-body cavity
sensory neurons. We propose that the loss of C. elegans BBS
function in ciliated sensory neurons leads to non-cell autonomous,
aberrant cGMP-PKG signalling in body cavity neurons, which
contributes to abnormal body size and delayed development.
C. elegans bbs mutants exhibit non-cell autonomous
Ciliated sensory neurons transduce environmental cues into
behavioural responses. In C. elegans bbs mutants, defective IFT and
ciliary functions are reflected by chemosensory and thermosensory
deficits [13–14]. Given the restricted expression of C. elegans BBS
Figure 4. EGL-4 is an effector of GCY-35/GCY-36 in influencing body length and developmental timing. (A) A schematic of EGL-4
showing allelic information on the ad450sd (gain-of-function) and n478 (loss-of-function). egl-4(gf) and egl-4(lf) are epistatic to bbs-7(ok1351) (gray
bars), gcy-35(hp443) (diagonal striped bars) and gcy-35;bbs-7 (dark bars) for both body size (B) and developmental timing (C). ANOVA with Tukey,
*** p,0.001; ns – p$0.05 or body length difference ,3.5%. For body length, data represent mean 6 SD normalized against wild-type body length,
n$20. For developmental timing, data represent mean (hours) 6 SD, n$50, N$10 replicates. (D) Loss of function mutations in the TGF-b pathway
(BMP-5/dbl-1 and lon-2, white bars) had an additive effect on regulating body size in conjunction with bbs mutants (light bars), while being unaffected
by the loss of gcy-35(ok769) (dark bars). ANOVA with Tukey, *** p,0.001; ns – p$0.05, n$20. Data represent mean 6 SD normalized against wild-
type body length. (E) The transgenic expression (+, light bars) of egl-4(gf) in body cavity neurons reverted the body size suppression in gcy-35;bbs-7 in
comparison to non-transgenic siblings (2, dark bars). EGL-4(gf) expression within body cavity neurons did not affect the body size of wild-type
transgenic controls. ANOVA with Tukey, *** p,0.001; ns – p$0.05, n$20. Data represent mean 6 SD normalized against wild-type body length.
Genetic Modifiers of C. elegans bbs Defects
PLoS Genetics | www.plosgenetics.org8 October 2011 | Volume 7 | Issue 10 | e1002335
Genetic Modifiers of C. elegans bbs Defects
PLoS Genetics | www.plosgenetics.org9 October 2011 | Volume 7 | Issue 10 | e1002335
proteins in sensory neurons, the additional bbs endophenotypes
such as developmental timing, body and inferred cell size, and
roaming indicate that in addition to sensory perception, sensory
neurons also participate in developmental regulation in a non-cell
autonomous manner. These bbs endophenoptypes are not
recapitulated by several dyf/IFT motor mutants, further implying
that BBS proteins affect sensory neuron function in addition to
their role in IFT.
While all bbs mutants share these endophenotypes, they exhibit
small differences in the severity of phenotypic expression that
could be attributed to specific allelic effects. Alternatively, BBS
proteins could possess certain degrees of unknown functional
specificity. This may not be so surprising given the difference in
phenotypic expression among BBS patient populations [53–55], as
well as the observation that tissue-specific BBS isoforms are
responsible for some syndromic features [22,56].
The involvement of primary cilia in signalling during develop-
ment  also positions them to affect development in a non-
autonomous fashion. For example, mouse BBS proteins are
required in the hypothalamus to regulate leptin receptor
trafficking and to prevent the onset of obesity . Ciliary
dysfunction therefore contributes to increased adiposity partly in a
non-cell autonomous manner. The additional phenotypes of C.
elegans bbs mutants, highlights the global and non-cell autonomous
consequence of sensory ciliary dysfunction, which may also
account for some phenotypic features in other ciliopathy models.
The GCY-35/GCY-36 sGC regulates body size through a
mechanism divergent from oxygen sensing
Previous studies established that the GCY-35/GCY-36 sGC
can regulate oxygen sensation through either the body cavity
neurons, or another group of neurons [40–42]. Activated by
oxygen, this complex catalyzes the conversion of GTP to cGMP,
which subsequently activates the cGMP-gated cation channel
TAX-2/TAX-4 to initiate hyperoxic avoidance responses .
Additional sGCs can act in body cavity neurons or other neurons
under specific hypoxic conditions [45,58].
GCY-35/GCY-36 modifies body size through a mechanism
partly divergent from that of hyperoxic avoidance. GCY-35 is only
necessary and sufficient in body cavity neurons that either have
ciliated dendrites  or express some ciliated neuron-specific
genes . Furthermore, the loss of EGL-4, but not TAX-2 or
TAX-4, suppresses the body size defects of bbs and dyf mutants.
The loss of TAX-2 and TAX-4, in fact, slightly exacerbated bbs
phenotypes (Figure S4), which may reflect an increased cGMP
pool for EGL-4 activation or the loss of a potential EGL-4
phosphorylation target . As well, despite some sGCs having
overlapping expression profiles with GCY-35/GCY-36, other
oxygen-responsive sGC mutants failed to suppress bbs-7 body size
defects under standard culture conditions - possibly due to low
activity under normoxia. Therefore, body cavity neurons, through
GCY-35/GCY-36 activity, participate in developmental regula-
tion through an alternate cGMP effector.
EGL-4 is present fairly ubiquitously, but the activation of EGL-
4 in sensory neurons exerts a dominant influence on body size
. The genetic epistasis of both egl-4(lf) and egl-4(gf) alleles over
that of bbs and bbs;gcy-35 argues in favour of BBS proteins and
EGL-4 functioning through a shared cellular pathway to regulate
body size and developmental timing. Expression of EGL-4(gf ) in
the body cavity neurons of gcy-35;bbs-7 mutants specifically
alleviated the rescuing effect on body size, suggesting that
increased EGL-4 activity, driven by increased availability of
cGMP in body cavity neurons, contributes to the body size defects
of some ciliary mutants (Figure 5C).
BBS proteins affect body size indirectly through body
The body size defects of ciliary mutants are rescued by non-
overlapping sets of sensory neurons. However, restoring BBS
function in body cavity neurons is insufficient to rescue the
observed body size defects, giving rise to a possibility that the effect
of cGMP signalling by body cavity neurons is indirectly moderated
by a non-cell autonomous function of BBS proteins in ciliated
sensory neurons. Furthermore, that URX, a pair of non-ciliated
neurons, play a necessary role in this suppression indicates that
BBS proteins are not directly influencing body size in these
neurons. Our genetic analyses of the modifying effect of other GC
mutants also support this scenario, as we have not found additional
GC mutants that potently restore the body size of bbs mutants.
These results do not exclude the possibility that other GCs
function redundantly in non-body cavity sensory neurons to
influence body size through EGL-4/PKG (Figure 5D). The
overexpression of egl-4(gf) in body cavity neurons was incapable
of further reducing the body size of gcy-35;bbs-7 animals beyond
that of bbs-7 mutants. This is in concordance with the ablation of
body cavity neurons, which did not phenocopy the large body size
of EGL-4 loss of function mutants, suggesting additional neuronal
groups influence body size through EGL-4/PKG signalling. This
study, however, establishes body cavity neurons as a predominat-
ing cGMP/PKG effector in body size regulation, and the ciliated
sensory neurons as playing a key role in moderating cGMP
signalling of these effector neurons.
Mechanisms on how dysfunctional ciliary sensory neurons lead
to elevated cGMP/PKG signalling in these neurons are unknown.
The body cavity neurons, AQR, PQR and URX do not receive
extensive or direct synaptic inputs from sensory neurons where
BBS proteins are sufficient to rescue body size. The non-cell
autonomous effect of ciliated sensory neurons therefore suggests a
potential involvement of indirect synaptic inputs, or other forms of
neuronal communications, such as peptidergic and/or hormonal
signalling between these neuronal groups. For example, body
cavity neurons express the C. elegans homologue of the neuropep-
tide NPY receptor , making their activity susceptible to
modulation by neuropeptides, some of which could be secreted by
sensory neurons . Sensory neurons also secrete insulin/IGF-
like ligands, some of which may systematically affect neuronal
Figure 5. Multiple sensory neurons influence the body size of bbs mutants. (A) The expression of wild-type BBS protein in body cavity
neurons was insufficient to rescue body length defects of bbs-7 and bbs-2 mutants, while expression in other ciliated sensory neurons (light bars)
restored body length. ANOVA with Tukey, *** p,0.001; ns – p$0.05, n$11. Data represent mean 6 SD normalized against wild-type body length. (B)
GCY-35, but not other GCY mutants, exhibited a strong suppression effect on bbs-7 mutant body length. Box and whisker plot of additional gcy
mutants showing partially improved body length (light bars), decreased body length (black bars), and no change (dark bars) in comparison to bbs-7
mutants. Boxes represent 25th–75thpercentile of populations with maximum and minimum values as whiskers. ANOVA with Tukey, *** p,0.001; ns –
p$0.05 compared to bbs-7 mutants. Data represent mean 6 SD normalized against wild-type body length. (C–D) A model for ciliary and cGMP-
mediated body size regulation. (C) GCY-35/GCY-36 activity produces cGMP in the body cavity neurons and activates EGL-4 to inhibit body size. This
process is influenced by BBS proteins present in other ciliated sensory neurons. (D) Some ciliated sensory neurons producing other GCs undergo a
BBS-independent EGL-4 activation to influence body size and developmental timing.
Genetic Modifiers of C. elegans bbs Defects
PLoS Genetics | www.plosgenetics.org10 October 2011 | Volume 7 | Issue 10 | e1002335
states [63–64]. Indeed, insulin and leptin have been shown to
regulate the activity of specific hypothalamic neurons [65–66].
Speculatively, C. elegans BBS proteins could affect the secretion of
multiple signals by ciliated sensory neurons to regulate cGMP/
EGL-4 signalling in the body cavity neurons.
A potential involvement of aberrant cGMP signalling in
While aberrant PCP, Shh and Wnt signalling underlie a
number of ciliopathy features, the biology behind other ciliopathy
features such as photoreceptor degeneration, and reduced body
size in Bbs mice  remains unexplained. cGMP signalling plays
key roles in biological processes such as phototransduction, axonal
guidance, and synaptic plasticity (reviewed in [67–68]). PKGs
have also been implicated in photoreceptor degeneration and
dwarfism [69–70]. It is worth exploring the involvement of cGMP
signalling in the underlying pathology of BBS and other ciliopathy
Materials and Methods
All strains were maintained on NGM plates at 20uC. C. elegans
bbs, gcy and egl-4 strains were obtained from the CGC. CX7102 was
obtained from the Bargmann lab. Genotypes for all strains are
listed in Text S1.
Mapping and cloning of hp433
bbs-7(n1606);hp433 mutants were outcrossed twice against N2
by selecting animals that were genotyped for n1606 mutation, but
exhibited normal body size. The hp433 mutation was crossed into
bbs-7(ok1351) mutants and mapped based on the suppression of
small body size and roaming defects using the SNP markers in the
CB4856 strain, which placed it at a 93.5 kb interval between the
SNPs pkp1133 and uCE1-1426. We conclude that hp433 encodes
gcy-35 by: 1) Injection of three overlapping fosmids covering gcy-
35, T04D3.5, and T04D3.t2, reverted the body size suppression in
hp433; bbs-7 animals. A fragment of WRM641cB09 that
encompassed a truncated gcy-35, but complete T04D3.5 and
T04D3.t2 failed to revert the hp433 suppression; A genomic
fragment containing only gcy-35 fully reverted the suppression. 2)
gcy-35(ok769) animals shared the same synthetic phenotypes and
genetic interactions with bbs-7 as hp433, while hp433;bbs-7(ok1351)
animals also failed to complement gcy-35(ok769);bbs-7(ok1351). 3)
Sequencing of gcy-35 identified a 2 bp deletion in exon 8.
Molecular biology, C. elegans phenotype examination
See Text S1.
and adulthood stages. The body length measurements of bbs
mutants at L4 (A) and 66-hours post-L4(B) showed consistent size
defects when compared to similarly staged wild-type animals.
ANOVA with Tukey, *** p,0.001 in comparison to wild-type
animals, n$20. Data represent mean 6 SD normalized against
wild-type body length.
bbs mutants exhibit a smaller body size in late larvae
genetic interaction with gcy-35(lf). (A) The relative length of dyf
mutants in comparison to wild-type animals. Some dyf mutants
dyf (dye-filling) mutants show variable body size and
had variable degrees of body length defects, while others showed
little to no change in body length. ANOVA with Tukey, ***
p,0.001; ns – p$0.05 or length difference ,3.5% relative to wild-
type animals, n$30. Data represent mean 6 SD normalized
against wild-type body length. (B) The body size of only a subset of
dyf mutants (dark boxes) was mildly altered by the loss of gcy-35
(light boxes). Boxes represent 25th–75thpercentile of populations
with maximum and minimum values as whiskers. ANOVA with
Tukey, *** p,0.001; * p,0.05; ns – p$0.05 or length difference
,3.5%, n$30. Data represent mean 6 SD normalized against
wild-type body length.
GCY-36 localisation. GFP signals by Pgcy-36::GFP::GCY-35 or
Pgcy-32::GFP::GCY-36 expressed in gcy-35(hp433) (A–C) or gcy-
36(db66) (G–I) mutants. Strong signals were observed in the soma
(orange arrowheads) and tips of the dendrites (white arrowheads)
in AQR, PQR, and in the soma and dendrites of URX neurons.
Expression of the same constructs in a gcy-35;bbs-7 (D–F) or bbs-
7;gcy-36 (J–L) backgrounds exhibited no gross changes to
localization in comparison to wild-type animals. Shown here are
representative images of young adult animals.
bbs mutants show no visible defects in GCY-35 or
suppress the body size defects of bbs mutants (A) tax-2 and tax-4
failed to rescue bbs-7 mutant body size defects. ANOVA with
Tukey, *** p,0.001; ns – p$0.05 or length difference ,3.5%,
n$20. Data represent mean 6 SD normalized against wild-type
body length. (B) Developmental timing in tax-2;bbs-7 mutants was
further delayed in comparison to bbs-7 mutant populations.
ANOVA, *** p,0.001, n$50, N$10 replicates. Data represent
mean (hours) 6 SD. (C) Roaming defects of tax-2;bbs-7 and tax-4
bbs-7 animals were no different compared to bbs-7 single animals.
Kruskal-Wallis with Dunn’s, ns – p$0.05, n$25, N$2 replicates.
Data represent mean squares roamed 6 SEM.
The cGMP-gated ion channel TAX-2/4 does not
bbs-7 body size defects. (A) Mutant alleles of multiple sGC genes
(gray bars) and rGC genes (dark bars) did not exhibit a significant
modifying effect on bbs-7 mutants. The loss of functionally
redundant rGCs (gcy-8, -18, -23)  does not significantly modify
bbs-7 body size defects either (black bars). ANOVA with Tukey, ns
– p,0.05, n$30. (B) Suppression of bbs-7 body size by the loss of
gcy-35 was not significantly influenced by the loss of additional
rGCs. ANOVA with Tukey, *** p,0.001; ** p,0.01; ns –
p$0.05, n$20. All data represent mean 6 SD normalized against
wild-type body length.
Mutations in other guanylate cyclases do not suppress
outlining the type of change or deletion characterized in these
strains and the resulting changes to protein translation or domains.
A list of the bbs mutant alleles used in this study,
this study, and methods for molecular biology, phenotype analyses,
statistical analyses and fluorescent microscopy.
Text S1 includes a list of strains generated and used in
We thank Y. Wang and H. Li for technical support, C. Bargmann and M.
de Bono for strains and constructs, and the Caenorhabditis Genetics Center and
C. elegans Knockout Consortium for mutants.
Genetic Modifiers of C. elegans bbs Defects
PLoS Genetics | www.plosgenetics.org11October 2011 | Volume 7 | Issue 10 | e1002335
Conceived and designed the experiments: CAM EH MZ. Performed the
experiments: CAM MPH TS MZ. Analyzed the data: CAM MPH TS MZ.
Contributed reagents/materials/analysis tools: CAM MPH TS MRL EH
MZ. Wrote the paper: CAM EH MZ.
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Genetic Modifiers of C. elegans bbs Defects
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Genetic Modifiers of C. elegans bbs Defects
PLoS Genetics | www.plosgenetics.org13 October 2011 | Volume 7 | Issue 10 | e1002335