Deletion of smn-1, the Caenorhabditis elegans ortholog of the spinal muscular atrophy gene, results in locomotor dysfunction and reduced lifespan.

Abstract

Spinal muscular atrophy is the most common genetic cause of infant mortality and is characterized by degeneration of lower motor neurons leading to muscle wasting. The causative gene has been identified as survival motor neuron (SMN). The invertebrate model organism Caenorhabditis elegans contains smn-1, the ortholog of human SMN. Caenorhabditis elegans smn-1 is expressed in various tissues including the nervous system and body wall muscle, and knockdown of smn-1 by RNA interference is embryonic lethal. Here we show that the smn-1(ok355) deletion, which removes most of smn-1 including the translation start site, produces a pleiotropic phenotype including late larval arrest, reduced lifespan, sterility as well as impaired locomotion and pharyngeal activity. Mutant nematodes develop to late larval stages due to maternal contribution of the smn-1 gene product that allows to study SMN-1 functions beyond embryogenesis. Neuronal, but not muscle-directed, expression of smn-1 partially rescues the smn-1(ok355) phenotype. Thus, the deletion mutant smn-1(ok355) provides a useful platform for functional analysis of an invertebrate ortholog of the human SMN protein.

Full text

Page 1

Deletion of smn-1, the Caenorhabditis elegans
ortholog of the spinal muscular atrophy
gene, results in locomotor dysfunction
and reduced lifespan
Michael Briese1,{, Behrooz Esmaeili1,{, Sandrine Fraboulet1,2, Emma C. Burt1,
Stefanos Christodoulou1, Paula R. Towers1, Kay E. Davies1and David B. Sattelle1,?
1MRC Functional Genomics Unit, Department of Physiology Anatomy and Genetics, University of Oxford,
South Parks Road, Oxford OX1 3QX, UK and2Institut des Neurosciences Grenoble, Centre de recherche Inserm
U836-UJF-CEA-CHU, Universite ´ Joseph Fourier, Ba ˆtiment Edmond J. Safra, Domaine de la Merci, 38706 La Tronche
Cedex, France
Received August 15, 2008; Revised and Accepted September 29, 2008
Spinal muscular atrophy is the most common genetic cause of infant mortality and is characterized by degener-
ation of lower motor neurons leading to muscle wasting. The causative gene has been identified as survival
motor neuron (SMN). The invertebrate model organism Caenorhabditis elegans contains smn-1, the ortholog
of human SMN. Caenorhabditis elegans smn-1 is expressed in various tissues including the nervous system
and body wall muscle, and knockdown of smn-1 by RNA interference is embryonic lethal. Here we show that
the smn-1(ok355) deletion, which removes most of smn-1 including the translation start site, produces a pleio-
tropic phenotype including late larval arrest, reduced lifespan, sterility as well as impaired locomotion and phar-
yngeal activity. Mutant nematodes develop to late larval stages due to maternal contribution of the smn-1 gene
product that allows to study SMN-1 functions beyond embryogenesis. Neuronal, but not muscle-directed,
expression of smn-1 partially rescues the smn-1(ok355) phenotype. Thus, the deletion mutant smn-1(ok355)
provides a useful platform for functional analysis of an invertebrate ortholog of the human SMN protein.
INTRODUCTION
Spinal muscular atrophy (SMA) is an autosomal recessive neu-
romuscular disorder, mostly of childhood-onset, characterized
by degeneration of lower motor neurons in the anterior horn
of the spinal cord leading to proximal muscle wasting. It is
the most common genetic cause of infant mortality with an inci-
dence of around 1 in 10 000 live births (1,2). Childhood-onset
SMA cases show deletions in the survival motor neuron (SMN)
gene located as a telomeric copy (SMN1) and a centromeric
copy (SMN2) on chromosome 5 in an inverted duplicated
region (3). Owing to a single C-to-T transition in exon 7, the
majority of transcripts originating from SMN2 are alternatively
spliced lacking exon 7, and give rise to a truncated, unstable
protein product (4,5). Thus, SMN1, the SMA-determining
gene, produces full-length SMN protein almost exclusively,
whereas SMN2 acts as a disease modifier gene due to the
small amount of functional SMN it produces (6,7).
Even though the genetic defect underlying SMA has been
shown to reside in the SMN gene, important questions con-
cerning the disease etiology remain unresolved. SMN is
expressed widely both within and outside the nervous
system (3). Thus, the specific motor neuron defect seen in
SMA cannot be explained purely in terms of the expression
pattern of SMN but instead may relate to its function (8).
Animal models of SMA have been indispensable for the
study of physiologic roles of SMN in muscle cells and
neurons as well as for the dissection of pathological events
†M.B. and B.E. contributed equally to the work.
?To whom correspondence should be addressed. Tel: þ44 1865272145; Fax: þ44 1865282651; Email: david.sattelle@dpag.ox.ac.uk
# 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Human Molecular Genetics, 2009, Vol. 18, No. 1
doi:10.1093/hmg/ddn320
Advance Access published on October 1, 2008
97–104

Page 2

leading to SMA. However, since all model organisms
deployed so far possess only one ortholog of the SMN1
gene, progress has been limited by the lethality resulting
from complete loss of SMN. To circumvent this difficulty,
various strategies have been developed. Expression of
human SMN2 in Smn2/2null mice rescues their embryonic
lethality and generates animals with a movement defect and
motor neuron loss resembling severe SMA (9). In zebrafish,
the reduction of Smn levels using antisense morpholinos pro-
duces motor axon defects such as truncations and aberrant
branching (10). A Drosophila mutant containing a mis-sense
mutation in the fly smn gene survives until late larval stages
due to maternally contributed smn mRNA (11). This mutant
exhibits reduced excitatory postsynaptic currents and struc-
tural defects at neuromuscular junctions in the absence of
motor neuron loss.
A C. elegans ortholog of the human SMN1 gene has been
identified and termed smn-1 (12). A green fluorescent
protein (GFP) reporter construct contains full-length smn-1,
and its upstream sequence produces fluorescence in various
tissues including the nervous system and body wall muscles,
demonstrating widespread expression of smn-1. Additionally,
the SMN-1 protein was detected by immunofluorescence from
the zygotic stage onwards, indicating a maternal contribution
of the protein or its mRNA. So far, the functions of C.
elegans smn-1 have been studied in vivo by RNA interference
(RNAi) induced by injection of double-stranded RNA that pro-
duces knockdown effects in all tissues, including maternal tran-
scripts, although silencing of neuronal genes is less efficient
(13). Targeting smn-1 with RNAi gives rise to severe develop-
mental defects and embryonic lethality, thus limiting further
investigations on postembryonic roles of SMN-1 (12). Here
we have characterized the smn-1(ok355) deletion allele,
which removes most of smn-1 including the translation start
codon, resulting in late larval arrest, a dramatic decrease in life-
span as well as impaired locomotion and pharyngeal pumping.
Early larval development of smn-1(ok355) mutant animals
appears normal due to maternally contributed SMN-1. As it
models aspects of SMA, this smn-1 mutant lends itself to the
functional analysis of an SMN ortholog and has potential
future utilities in screens searching for phenotypic modifiers
as a first step toward potential SMA therapies.
RESULTS
The smn-1(ok355) deletion
Like all model organisms studied to date, C. elegans has only
a single copy of the gene encoding the survival motor neuron
protein. The smn-1(ok355) allele described here removes
975 bp comprising 246 bp of the upstream intergenic region
and most of the smn-1 gene leaving only 87 bp (including
the stop codon) at the 30end (part of exon 5; Fig. 1). The
codingsequencesofthe
F30A10.10 are unaffected by the smn-1(ok355) deletion.
Absence of the smn-1 ATG start codon in the remaining
sequence suggests that smn-1(ok355) is a null allele and
hence the only SMN-1 present is maternal, originating from
balanced smn-1(ok355)/hT2[qIs48] heterozygous mothers
(strain LM99).
adjacentgenes
klp-16
and
Caenorhabditis elegans wild-type animals progress through
four larval stages (L1 to L4) before reaching adulthood. The
most striking phenotype of the smn-1(ok355) mutant is its
late larval arrest (Fig. 2), including defects in gonadogenesis
and germline differentiation (Fig. 3). Arrested smn-1(ok355)
homozygotes become pale due to loss of intestinal pigmenta-
tion giving them a starved appearance. Their mean lifespan is
6.0 days post-L1 compared with 19.9 days for balanced
smn-1(ok355)/hT2[qIs48] and 17.7 days for N2 wild-types
(Fig. 4).
smn-1(ok355) mutants show defects in motility
and pharyngeal pumping
Following larval arrest, smn-1(ok355) nematodes display a pro-
gressive decline in motility. To measure the locomotory activity
we quantified the thrashing rate of animals in M9 buffer.
During early development, i.e. before arrest, the thrashing rate
of smn-1(ok355) homozygotes was similar to balanced
smn-1(ok355)/hT2[qIs48] heterozygotes and N2 wild-types
(Fig. 5). Thrashing of smn-1(ok355) animals then progressively
declined and almost completely ceased after 5 days post-L1. In
contrast, locomotion of smn-1(ok355)/hT2[qIs48] heterozygous
animals was similar to N2 wild-types at all time-points through-
out the assay.
Likewise,smn-1(ok355)mutantsareunabletofeedduetoloss
of pharyngeal function. The rate of pharyngeal pumping was
measured in smn-1(ok355) homozygous mutants over a period
of 4 days and compared with that of N2 wild-type animals and
smn-1(ok355)/hT2[qIs48] heterozygotes (Fig. 6). On the first
day of the assay (1 day after starved L1 larvae were placed on
seeded NGM plates), the pharyngeal pumping rate of the
mutant was indistinguishable from that of wild-type nematodes.
Similar to body wall muscle activity, pharyngeal pumping rates
showed a rapid and progressive decline. Taken together, these
data indicate a general defect in behaviors requiring rhythmic
contractions of muscle tissue in smn-1(ok355) animals.
Neuronal expression of smn-1 partially rescues
the smn-1(ok355) phenotype
It has recently been shown that the phenotype of severe SMA
mice can be corrected by expressing full-length SMN solely in
the nervous system (14). To investigate the sensitivity of
different aspects of the C. elegans neuromuscular system to
smn-1 loss, we sought to provide wild-type smn-1 to either
muscles or neurons of smn-1(ok355) mutants. To this end, res-
cuing constructs were used expressing full-length smn-1 in
body wall and vulval muscles through a myo-3 promoter
(Pmyo-3::smn-1) and in all neurons through an unc-119 pro-
moter (Punc-119::smn-1). Promoters of unc-119 and myo-3
have been used previously for neuronal- and muscle-directed
Figure 1. The smn-1(ok355) deletion allele, showing that only part of smn-1
exon 5 remains. Arrows indicate direction of transcription, the open-reading
frames are depicted as boxes. Scale bar: 500 bp.
98Human Molecular Genetics, 2009, Vol. 18, No. 1

Page 3

rescue (15,16) and are considered strong transcriptional indu-
cers (17). The rescuing constructs were microinjected into the
gonads of smn-1(ok355)/hT2[qIs48] heterozygotes to generate
heritable lines. Transgenic heterozygous mothers carry the
plasmid DNA as multi-copy, extrachromosomal arrays and
transmit the transgene at a certain frequency to their
progeny including smn-1(ok355) homozygotes. Extrachromo-
somal transgenes are frequently lost during cell division result-
ing in mosaic expression of the transgene (18).
Despite having injected a large number of animals, only
one heritable transgenic line each was obtained carrying
extrachromosomal Ex[Pmyo-3::smn-1] or Ex[Punc-119::smn-
1] for as yet unknown reasons. As an indicator for transgene
expression in vivo, the GFP fusions Pmyo-3::GFP and
Punc-119::GFP co-injected with the rescuing constructs
showed expression in the expected tissues (body wall
and vulval muscles for Pmyo-3 and nervous system for
Punc-119, data not shown).
Whereas mutant smn-1(ok355) animals expressing muscle-
directed smn-1 showed only weak phenotypic rescue in a
subset of animals, pan-neuronal expression of smn-1 produced
stronger rescue effects (Fig. 7A). Albeit remaining sterile,
rescued smn-1(ok355); Ex[Punc-119::smn-1] animals had
a larger body size compared with smn-1(ok355) and smn-1
(ok355); Ex[Pmyo-3::smn-1] animals (Fig. 7B) and often
retained their intestinal pigmentation, thus appearing less
starved. However, they did not reach the size of fully-grown
adult
smn-1(ok355)/hT2[qIs48]
partial as opposed to full rescue. We noted that the extent of
the rescuing effects was variable which, most likely, reflects
the mitotic instability of the extrachromosomal transgene. In
agreement with the body length measurements, neuronal-
expressing
smn-1(ok355);Ex[Punc-119::smn-1]
survived longer than smn-1(ok355) and muscle-expressing
smn-1(ok355); Ex[Pmyo-3::smn-1] animals (Fig. 7C) and
showed a partial correction of the pharyngeal pumping
defect (Fig. 6). Taken together, these data suggest that
restoration of neuronal smn-1 activity can, at least partially,
correct aspects of the smn-1(ok355) phenotype.
heterozygotes,indicating
animals
The nervous system of smn-1(ok355) animals
Having shown that neuronal-directed expression of smn-1 is
much more effective than muscle-directed expression in rescu-
ing the larval arrest phenotype of smn-1(ok355), we sought to
test whether smn-1(ok355) exhibits nervous system abnor-
malities in general and loss of cholinergic motor neurons in
particular. To this end we used the pan-neuronal marker
F25B3.3::GFP (19) (strain LM111) and the marker for cholin-
ergic neurons unc-17::GFP (strain LM112) to facilitate neur-
onal identification. On the basis of their connectivity, motor
neurons can be subdivided into classes that innervate dorsal
Figure 2. Developmental comparison of smn-1(ok355) mutants, smn-1(ok355)/hT2[qIs48] heterozygotes and N2 wild-type C. elegans. Age (in days post-L1) is
indicated on the left. The images depict representative phenotypic appearances of synchronized animals at a given age. For all images anterior is on the left and
posterior toward the right.
Human Molecular Genetics, 2009, Vol. 18, No. 1 99

Page 4

muscles by sending their axons dorsally as commissures to
form the dorsal nerve cord and those classes that project ven-
trally. Motor neurons (classes and individual cells) were identi-
fied by studying their axonal trajectories and cell body position
in the dorsal/ventral nerve cord in LM111 animals (20).
We could not detect any gross abnormalities in nervous
system morphology of smn-1(ok355); F25B3.3::GFP animals
(Fig. 8A). In an attempt to test whether there is loss of
neurons over time, LM112 animals were staged and the
number of cholinergic neurons in the ventral nerve cord was
counted using the marker unc-17::GFP that labels embryonic
and postembryonic motor neurons (21,22). No differences
were detected in the number of GFP-positive neurons in the
ventral cord region between smn-1(ok355) and smn-1(ok355)/
hT2[qIs48] up to 5 days post-L1 (Fig. 8B).
DISCUSSION
In all animal models of SMA studied to date, the ortholog of
human SMN is ubiquitously expressed and exists as a single-
copy gene. We have shown that, in the C. elegans
smn-1(ok355) deletion mutant, the loss of SMN-1 protein
leads to a progressive decline of motor function. Even
though the coding regions of the neighboring genes remain
intact, it remains possible that the upstream regulatory
sequence of klp-16 may be affected by the smn-1(ok355) del-
etion thus disrupting klp-16 expression as well. However, the
klp-16(ok1505) mutant containing a 1.1 kb deletion within the
klp-16 coding sequence has no obvious defects, with normal
movement, body size and brood size (data not shown). Thus,
taken together with the finding that the smn-1(ok355) mutant
phenocopies the less affected progeny originating from
smn-1 RNAi (12), it remains unlikely that the smn-1(ok355)
deletion phenotype described here is caused by the combined
loss of smn-1 and klp-16 function.
The smn-1(ok355) mutation is essentially a null allele, albeit
retaining progressively decreasing levels of maternal SMN-1.
Immunofluorescence analysis of embryos has previously
revealed the presence of SMN-1 at the one-cell stage (P0)
prior to initiation of de novo zygotic transcription (12). Con-
sidering that RNAi-mediated knockdown of smn-1, which
reduces the levels of both maternal and zygotic transcripts,
leads to embryonic lethality, we postulate that the maternal con-
tribution of SMN-1 allows progression of smn-1(ok355)
animals through early development. Consistent with this
notion, the thrashing rate of smn-1(ok355) is unaffected prior
to arrest, indicating normal functioning of the neuromuscular
system. Presumably, the progressive reduction of maternal
SMN-1 results in developmental arrest of homozygous
animals at a late larval stage reflected by a defect in gonadogen-
esis which is followed by paralysis and early lethality.
Closer inspection of the ventral nerve cord of neuronal
GFP-expressing smn-1(ok355) animals failed to reveal any
loss of cholinergic motor neurons. This finding indicates that
embryonic and postembryonic motor neuron development pro-
ceeds normally due to sufficient levels of maternally contri-
buted SMN-1 prior to their larval arrest. The absence of a
motility defect in the early larval stages underlines the
notion that, in smn-1(ok355) animals, motor neurons initially
establish normal functional connections with their target
muscles. When the movement defects first appeared, we
could not detect any loss of cholinergic motor neurons in the
ventral nerve cord. Even when paralysis ensues, motor
neuron numbers remain unaffected. Thus, in smn-1(ok355)
mutants, the maternally contributed SMN-1 can sustain
development to late larval stages when the animals arrest in
response to SMN protein depletion in the absence of any
obvious loss of neuronal cell bodies.
Theabsence ofmotorneuron lossinsmn-1(ok355)animals is
incontrastwithmousemodelsofSMAexhibitingmotordefects
accompanied bymotor neuron degeneration(9),andazebrafish
SMA model showing axon path-finding defects in response to
morpholino-induced
smn
knockdown(10).Rather,the
Figure 4. Survival of smn-1(ok355) mutants (n ¼ 58) in comparison to
smn-1(ok355)/hT2[qIs48]
heterozygotes
C. elegans (n ¼ 54). Each data point represents the fraction of synchronized
animals alive on a given day.
(n ¼ 56) andN2wild-type
Figure 3. Gonad migration defects in C. elegans smn-1(ok355) mutants. (A) In
N2 wild-type hermaphrodites the gonad folds back during the L4 stage and
develops in a U-shaped manner. (B) In arrested smn-1(ok355) the gonad
fails to develop properly.
100Human Molecular Genetics, 2009, Vol. 18, No. 1

Page 5

smn-1(ok355) phenotype described here resembles that of the
Drosophila SMA model bearing a mis-sense mutation in smn
similar to some of the mutations found in SMA patients (11).
The mutant smn flies also survive past embryogenesis due to
maternally contributed mRNA and die as late larvae following
depletionofthematernalcomponent.However,whereashomo-
zygous smn mutant fly larvae produce both mutant and wild-
type Smn protein due to the presence of both transcripts,
smn-1(ok355) larvae contain only wild-type smn-1 transcript.
Nevertheless, even though the smn-1(ok355) mutant relates to
the vast majority of SMA cases showing deletions in the
SMN1 gene, the situation in this nematode model of SMA
differs from SMA in several facets. Caenorhabditis elegans
produces full-length SMN-1 protein from only one gene
encoding an SMN ortholog, smn-1. In contrast, in addition to
SMN1, humans possess SMN2 which in SMA patients acts
as a phenotypic modifier gene producing low levels of func-
tional SMN protein. As a consequence, SMA is caused by
insufficiency rather than a complete loss of SMN protein. Fur-
thermore, unlike the case for C. elegans and Drosophila, there
is no maternal contribution of SMN mRNA in mammals. For
example, homozygous loss of Smn in mice is embryonic
lethal (23), which has been overcome by expressing human
SMN2 in Smn2/2mice (9,24). Thus, the absence of a C.
elegans SMN2 ortholog combined with maternally contributed
SMN-1 causes smn-1(ok355) mutants to exhibit a gradual
decline in SMN-1 levels ultimately leading to a complete
loss of the protein as opposed to the low remaining SMN
levels seen in SMA patients. Nevertheless, even though
maternal transmission may mask possible functions of
SMN-1 during development, the smn-1(ok355) mutant pro-
vides the opportunity to study postembryonic roles of
SMN-1 in motor neurons and at neuromuscular junctions.
To further understand the pathological events leading to
SMA, it is critical to elucidate whether the SMN reduction
in motor neurons is a primary defect in the disease or
whether there are independent muscle defects as well.
The answer to this question is particularly important for the
development of therapeutic strategies aiming to up-regulate
the SMN levels in selected cell types. Whether the
smn-1(ok355) phenotype originates due to defects in muscles
or neurons has been addressed here by rescue experiments in
which smn-1 was delivered locally to either muscle cells or
neurons. It emerged that neuronal-directed, but not muscle-
directed,expressionof
smn-1
smn-1(ok355) phenotype. When assessing the rescue exper-
iments, it has to be taken into account, however, that even
though the unc-119 promoter used here exhibits pan-neuronal
activity we cannot exclude the possibility of promoter activity
levels below the detection limit in cell types other than
neurons. Furthermore, we cannot rule out the possibility that
the structure and composition of the extrachromosomal
arrays may be different thus producing variability in the
extent of mosaicism and transgene expression levels.
In the light of our findings, it is of interest to note that neur-
onal expression of full-length human SMN by the Prion protein
(PrP) promoter rescues severe SMA mice containing two
copies of human SMN2 and lacking mouse Smn (Smn2/2;
SMN2þ/þ) (14). These SMA mice normally have an average
lifespan of 5 days (9), which could be extended to 210 days
in one line homozygous for the PrP-SMN transgene
(Smn2/2; SMN2þ/þ; PrP-SMN). When SMN was targeted
only to skeletal muscle using the human skeletal actin
(HSA) promoter neither improvement of the SMA phenotype
nor any extension of survival was seen. One HSA–SMN line
showing low expression of SMN in the spinal cord did
affect the SMA phenotype with mice living for 160 days on
average. Thus, a small increase of SMN in neurons was suf-
ficient to extend the survival of SMA mice considerably
whereas high SMN levels in mature skeletal muscle alone
had no effect. In comparison to these data we observed only
partial as opposed to full rescue when expressing smn-1 in
the nervous system of smn-1(ok355) animals. This difference
may be explained by the complete loss of maternally con-
tributed SMN-1 activity in the non-neuronal tissues of
rescued smn-1(ok355); Ex[Punc-119::smn-1] animals in con-
trast with the low remaining levels in rescued Smn2/2;
SMN2þ/þ; PrP-SMN mice.
Inconclusion, the C. elegans mutant smn-1(ok355) provides a
disease model mimicking aspects of SMA such as reduced
SMN-1 protein levels throughout the organism. Mutant
partiallyrescuedthe
Figure 6. Pharyngeal pumping analysis of C. elegans smn-1(ok355) mutants,
balanced smn-1(ok355)/hT2[qIs48] heterozygotes and N2 wild-types as well
as muscle-expressing smn-1(ok355); Ex[Pmyo-3::smn-1] and neuronal-
expressing smn-1(ok355); Ex[Punc-119::smn-1] animals. Asterisks: P ,
0.0001. Numbers at the base of each bar indicate number of animals used.
The graphs depict mean+SEM.
Figure 5. Progressive development of movement defects in C. elegans
smn-1(ok355) animals, balanced smn-1(ok355)/hT2[qIs48] heterozygotes and
N2 wild-type animals. For each group, n ¼ 20. Asterisks: P , 0.001. The
graphs depict mean+SEM.
Human Molecular Genetics, 2009, Vol. 18, No. 1101

Page 6

animals proceed through embryogenesis but arrest as late larvae
indicating that, in C. elegans, SMN-1 has functions later during
development and in adult life. In future studies, smn-1(ok355)
mutants may serve as a starting point for the identification of
modifier genes in genetic or genome-wide RNAi screens that
enhance or suppress the smn-1(ok355) phenotype and for devel-
oping novel therapeutic strategies in drug screens.
MATERIALS AND METHODS
Strains
Strains were grown and maintained as described previously
(25). The following strains were used in this study: N2,
NW1229 [evIs111[F25B3.3::GFP; dpy-20(þ)]], RM1872
[mdEx72[unc-17::GFP; pha-1(þ)]], LM97 [smn-1(ok355)/þ I],
LM99 [smn-1(ok355) I/hT2[bli-4(e937) let-?(q782) qIs48]
(I;III)], LM111 [smn-1(ok355) I/hT2[bli-4(e937) let-?(q782)
qIs48](I;III);evIs111],LM112
[smn-1(ok355) I/hT2
[bli-4(e937) let-?(q782) qIs48] (I;III); mdEx72] and VC1005
[klp-16(ok1505) I].
Isolation of the smn-1(ok355) deletion
The smn-1(ok355) deletion allele of smn-1 located on chromo-
some I was obtained from the C. elegans Gene Knockout Con-
sortium. From the genetically heterogeneous population, we
cloned individual animals heterozygous for the deletion and
out-crossed them six times to wild-type (N2) nematodes.
The unbalanced smn-1(ok355)/þ I strain (LM97) was main-
tained by picking heterozygous animals and confirming
the presence of the deletion by PCR using primers
50-GACTTCAGATTTGATCAACGGTC-30and 50-ACACCA
ATTACCAAACGATCAAC-30. Stable maintenance of the
deletion was achieved by introducing the genetic balancer
hT2 (I;III), which is a reciprocal translocation of chromosomal
segments between chromosomes I and III, generating strain
LM99. Todistinguish homozygous fromheterozygous
Figure 7. Neuronally expressed smn-1 partially rescues the C. elegans smn-1(ok355) phenotype. (A) Examples of mutant animals carrying extrachromosomal
Pmyo-3::smn-1 inducing expression of smn-1 in body wall and vulval muscles or Punc-119::smn-1 for smn-1 expression in all neurons. For comparison, arrested
smn-1(ok355) and adult smn-1(ok355)/hT2[qIs48] animals are shown. (B) Comparison of body lengths of arrested smn-1(ok355), adult smn-1(ok355)/hT2[qIs48]
as well as fully-grown smn-1(ok355); Ex[Pmyo-3::smn-1] and smn-1(ok355); Ex[Punc-119::smn-1] animals. Graphs show the median, n ¼ 100 for each group.
Asterisks: P , 0.001. (C) Survival of smn-1(ok355); Ex[Pmyo-3::smn-1] (n ¼ 26) and smn-1(ok355); Ex[Punc-119::smn-1] (n ¼ 24) animals in comparison
with smn-1(ok355)/hT2[qIs48] heterozygotes (n ¼ 30) and smn-1(ok355) homozygotes (n ¼ 15, data taken from an independent experiment). Scale bar in
(A): 200 mm.
102Human Molecular Genetics, 2009, Vol. 18, No. 1

Page 7

animals visually, we used the hT2[bli-4(e937) let-?(q782)
qIs48] (I;III) derivative of the chromosomal balancer, where
qIs48 is an insertion of extrachromosomal ccEx9747 carrying
the promoter::GFP fusions myo-2::GFP (expressed in the
pharynx), pes-10::GFP (expressed in 4–60 cell embryos)
and a gut-specific enhancer fused to GFP (26,27). Hetero-
zygous smn-1(ok355) I/hT2[bli-4(e937) let-?(q782) qIs48]
(I;III) (referred to as ‘smn-1(ok355)/hT2[qIs48]’) segregate
smn-1(ok355)
homozygotes,
smn-1(ok355)/hT2[qIs48] heterozygotes and arrested aneu-
ploid progeny. Since hT2[qIs48] is homozygous lethal, all
animals expressing GFP are heterozygous for smn-1(ok355).
Animals homozygous for the smn-1(ok355) deletion can be
identified due to absence of GFP fluorescence in the pharynx.
hT2[qIs48]
homozygotes,
Physiological assays
Embryos were released by subjecting gravid N2 and LM99
hermaphrodites to bleach and were subsequently left in M9
overnight for obtaining hatched synchronized smn-1(ok355)
homozygotes, balanced smn-1(ok355)/hT2[qIs48] heterozy-
gotes and N2 wild-types at the L1 larval stage. Synchronized
L1 larvae were placed on nematode growth medium (NGM)
plates seeded with E. coli OP50, and left for 1 day at 228C.
Motility was then assessed every day by placing animals in
M9 buffer and measuring their movement as thrashing rate
in a 1 min period. A single thrash was defined as a change
in the direction of bending at the mid-body. Animals were
left in M9 for at least 5 min prior to measurement. For quanti-
fication of pharyngeal pumping synchronized smn-1(ok355)
homozygotes, smn-1(ok355)/hT2[qIs48] heterozygotes, N2
wild-type and tissue-specific rescue animals (see below)
aged 1 day post-L1 were placed on OP50-seeded plates at
228C. Terminal bulb pumps were measured each day under
a dissection microscope for periods of 15 s. Between 2 and
10 periods of 15 s were counted for each animal.
Lifespan and brood size assays
For the lifespan assay synchronized smn-1(ok355) homo-
zygotes, smn-1(ok355)/hT2[qIs48] heterozygotes and N2
wild-types aged 1 day post-L1 were placed on OP50-seeded
plates (5 cm diameter) at 228C. Survival was monitored
daily, and animals were scored as dead when they no longer
responded to head or tail touch using a platinum wire pick.
To avoid crowding due to progeny production and to ensure
the availability of sufficient food, animals were transferred
to new plates when deemed necessary. Animals that crawled
off the NGM agar were excluded from the data. For brood
size assays, klp-16(ok1505) and wild-type N2 animals were
synchronized and their brood size determined over a 5 day
period post-L4 at 208C. PCR analysis was used to confirm
the presence of the klp-16(ok1505) deletion using primers
recommended in WormBase (http://www.wormbase.org/).
Tissue-specific rescue of the smn-1(ok355) mutant
To rescue the smn-1(ok355) phenotype in a tissue-specific
manner,
smn-1(ok355)/hT2[qIs48]
microinjected with the promoter::smn-1 fusion constructs
Pmyo-3::smn-1 or Punc-119::smn-1 expressing smn-1 in
body wall muscle or neuronal tissue, respectively. These res-
cuing constructs contain smn-1 from the ATG start to the
TAA stop codon (amplified from N2 genomic DNA) placed
under transcriptional control of 2 kb upstream of myo-3 for
Pmyo-3::smn-1 (generating muscle-directed expression) or
2.2 kb upstream of unc-119 in Punc-119::smn-1 (for pan-
neuronal expression). To visualize promoter activity, smn-1
wasreplacedby
GFP
Punc-119::GFP and Pmyo-3::GFP. For neuronal rescue,
Punc-119::smn-1 and Punc-119::GFP were co-injected at
10 ng/ml each together with pRF4 at 200 ng/ml encoding the
marker rol-6(su1006) the expression of which induces a
‘rolling’ phenotype (28). Analogously, for muscle-directed
heterozygoteswere
togeneratethereporters
Figure 8. The nervous system of C. elegans smn-1(ok355) animals. (A) Arrangement of nerve cell bodies in the ventral nerve cord of L3 smn-1(ok355)/
hT2[qIs48] heterozygotes and smn-1(ok355) homozygotes. F25B3.3::GFP labels all neuron types whereas unc-17::GFP is restricted to cholinergic neurons.
(B) Cholinergic motor neuron number in the ventral nerve cord of smn-1(ok355) and smn-1(ok355)/hT2[qIs48] animals. GFP-positive neurons expressing
the cholinergic marker unc-17::GFP were counted from and including the VA2 (anterior) to the VA11 neuron (posterior). For each group, n ¼ 10. The
graphs depict mean+SEM.
Human Molecular Genetics, 2009, Vol. 18, No. 1103

Page 8

rescue
Pmyo-3::GFP and pRF4.
For body length measurements, photomicrographs were
taken of mobile but growth-arrested smn-1(ok355) animals,
which were identified by their starved, pale appearance, and
adult smn-1(ok355)/hT2[qIs48] animals from mixed-stage
populations. For body length analysis of transgenic rescue
nematodes, individual animals were monitored daily when
deemed necessary due to variability in the extent of rescue
observed. Photomicrographs of transgenic rescue animals
were taken either when they were considered fully grown or
when they appeared starved, i.e. growth-arrested, or immobile.
Images of animals were obtained using a Nikon SMZ1000
stereomicroscope and analyzed using ImageJ software from
NIH (http://rsb.info.nih.gov/ij/) to measure the body length
along the midline from the tip of the head to the base of the
tail. Lifespan of rescue animals was assessed by picking syn-
chronized animals aged 3 days post-L1 and monitoring their
survival as described above.
attempts,
Pmyo-3::smn-1
wasco-injected with
FUNDING
This work was funded by the Medical Research Council of
the UK. M.B. was supported by a Wellcome Trust 4-Year
Doctoral Programme in Neuroscience.
ACKNOWLEDGEMENTS
The authors would like to thank the C. elegans Gene Knockout
Consortium and the Caenorhabditis Genetics Center for pro-
viding strains.
Conflict of Interest statement. None declared.
REFERENCES
1. Roberts, D.F., Chavez, J. and Court, S.D. (1970) The genetic component
in child mortality. Arch. Dis. Child., 45, 33–38.
2. Ogino, S. and Wilson, R.B. (2002) Genetic testing and risk assessment for
spinal muscular atrophy (SMA). Hum. Genet., 111, 477–500.
3. Lefebvre, S., Burglen, L., Reboullet, S., Clermont, O., Burlet, P., Viollet,
L., Benichou, B., Cruaud, C., Millasseau, P., Zeviani, M. et al. (1995)
Identification and characterization of a spinal muscular
atrophy-determining gene. Cell, 80, 155–165.
4. Lorson, C.L. and Androphy, E.J. (2000) An exonic enhancer is required
for inclusion of an essential exon in the SMA-determining gene, SMN.
Hum. Mol. Genet., 9, 259–265.
5. Vitte, J., Fassier, C., Tiziano, F.D., Dalard, C., Soave, S., Roblot, N.,
Brahe, C., Saugier-Veber, P., Bonnefont, J.P. and Melki, J. (2007) Refined
characterization of the expression and stability of the SMN gene products.
Am. J. Pathol., 171, 1269–1280.
6. Lorson, C.L., Hahnen, E., Androphy, E.J. and Wirth, B. (1999) A single
nucleotide in the SMN gene regulates splicing and is responsible for
spinal muscular atrophy. Proc. Natl Acad. Sci. USA, 96, 6307–6311.
7. Monani, U.R., Lorson, C.L., Parsons, D.W., Prior, T.W., Androphy, E.J.,
Burghes, A.H. and McPherson, J.D. (1999) A single nucleotide difference
that alters splicing patterns distinguishes the SMA gene SMN1 from the
copy gene SMN2. Hum. Mol. Genet., 8, 1177–1183.
8. Briese, M., Esmaeili, B. and Sattelle, D.B. (2005) Is spinal muscular
atrophy the result of defects in motor neuron processes? Bioessays, 27,
946–957.
9. Monani, U.R., Sendtner, M., Coovert, D.D., Parsons, D.W., Andreassi, C.,
Le, T.T., Jablonka, S., Schrank, B., Rossol, W., Prior, T.W. et al. (2000)
The human centromeric survival motor neuron gene (SMN2) rescues
embryonic lethality in Smn(2/2) mice and results in a mouse with spinal
muscular atrophy. Hum. Mol. Genet., 9, 333–339.
10. McWhorter, M.L., Monani, U.R., Burghes, A.H. and Beattie, C.E. (2003)
Knockdown of the survival motor neuron (Smn) protein in zebrafish
causes defects in motor axon outgrowth and pathfinding. J. Cell Biol., 162,
919–931.
11. Chan, Y.B., Miguel-Aliaga, I., Franks, C., Thomas, N., Trulzsch, B.,
Sattelle, D.B., Davies, K.E. and van den Heuvel, M. (2003)
Neuromuscular defects in a Drosophila survival motor neuron gene
mutant. Hum. Mol. Genet., 12, 1367–1376.
12. Miguel-Aliaga, I., Culetto, E., Walker, D.S., Baylis, H.A., Sattelle, D.B.
and Davies, K.E. (1999) The Caenorhabditis elegans orthologue of the
human gene responsible for spinal muscular atrophy is a maternal product
critical for germline maturation and embryonic viability. Hum. Mol.
Genet., 8, 2133–2143.
13. Timmons, L., Court, D.L. and Fire, A. (2001) Ingestion of bacterially
expressed dsRNAs can produce specific and potent genetic interference in
Caenorhabditis elegans. Gene, 263, 103–112.
14. Gavrilina, T.O., McGovern, V.L., Workman, E., Crawford, T.O.,
Gogliotti, R.G., DiDonato, C.J., Monani, U.R., Morris, G.E. and Burghes,
A.H. (2008) Neuronal SMN expression corrects spinal muscular atrophy
in severe SMA mice while muscle-specific SMN expression has no
phenotypic effect. Hum. Mol. Genet., 17, 1063–1075.
15. Feng, Z., Li, W., Ward, A., Piggott, B.J., Larkspur, E.R., Sternberg, P.W.
and Xu, X.Z. (2006) A C. elegans model of nicotine-dependent behavior:
regulation by TRP-family channels. Cell, 127, 621–633.
16. Wolkow, C.A., Kimura, K.D., Lee, M.S. and Ruvkun, G. (2000)
Regulation of C. elegans life-span by insulinlike signaling in the nervous
system. Science, 290, 147–150.
17. Timmons, L., Tabara, H., Mello, C.C. and Fire, A.Z. (2003) Inducible
systemic RNA silencing in Caenorhabditis elegans. Mol. Biol. Cell, 14,
2972–2983.
18. Stinchcomb, D.T., Shaw, J.E., Carr, S.H. and Hirsh, D. (1985)
Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol.
Cell Biol., 5, 3484–3496.
19. Altun-Gultekin, Z., Andachi, Y., Tsalik, E.L., Pilgrim, D., Kohara, Y. and
Hobert, O. (2001) A regulatory cascade of three homeobox genes, ceh-10,
ttx-3 and ceh-23, controls cell fate specification of a defined interneuron
class in C. elegans. Development, 128, 1951–1969.
20. White, J.G., Southgate, E., Thomson, J.N. and Brenner, S. (1986) The
structure of the nervous system of the nematode Caenorhabditis elegans.
Philos. Trans. R. Soc. Lond. B Biol. Sci., 314, 1–340.
21. Lickteig, K.M., Duerr, J.S., Frisby, D.L., Hall, D.H., Rand, J.B. and
Miller, D.M. III (2001) Regulation of neurotransmitter vesicles by the
homeodomain protein UNC-4 and its transcriptional corepressor UNC-37/
groucho in Caenorhabditis elegans cholinergic motor neurons.
J. Neurosci, 21, 2001–2014.
22. Rand, J.B., Duerr, J.S. and Frisby, D.L. (2000) Neurogenetics of vesicular
transporters in C. elegans. FASEB J, 14, 2414–2422.
23. Schrank, B., Gotz, R., Gunnersen, J.M., Ure, J.M., Toyka, K.V., Smith,
A.G. and Sendtner, M. (1997) Inactivation of the survival motor neuron
gene, a candidate gene for human spinal muscular atrophy, leads to
massive cell death in early mouse embryos. Proc. Natl Acad. Sci. USA, 94,
9920–9925.
24. Hsieh-Li, H.M., Chang, J.G., Jong, Y.J., Wu, M.H., Wang, N.M., Tsai,
C.H. and Li, H. (2000) A mouse model for spinal muscular atrophy. Nat.
Genet., 24, 66–70.
25. Brenner, S. (1974) The genetics of Caenorhabditis elegans. Genetics, 77,
71–94.
26. Mathies, L.D., Henderson, S.T. and Kimble, J. (2003) The C. elegans
Hand gene controls embryogenesis and early gonadogenesis.
Development, 130, 2881–2892.
27. Edgley, M.L. and Riddle, D.L. (2001) LG II balancer chromosomes in
Caenorhabditis elegans: mT1(II;III) and the mIn1 set of dominantly and
recessively marked inversions. Mol. Genet. Genomics, 266, 385–395.
28. Mello, C.C., Kramer, J.M., Stinchcomb, D. and Ambros, V. (1991)
Efficient gene transfer in C.elegans: extrachromosomal maintenance and
integration of transforming sequences. EMBO J, 10, 3959–3970.
104 Human Molecular Genetics, 2009, Vol. 18, No. 1