ReportFormin Is Associated with Left-Right Asymmetry in
the Pond Snail and the FrogHighlightsd Animals tend to be outwardly symmetric but internally are
asymmetric
d Unlike other animals, snails show inherited variation in
asymmetry
d We found that both snails and frogs use a common gene to
define left and right
d Asymmetry is probably an ancient and conserved property of
cells and animalsDavison et al., 2016, Current Biology 26, 654–660
March 7, 2016 ª2016 The Authors
http://dx.doi.org/10.1016/j.cub.2015.12.071Authors
Angus Davison, Gary S. McDowell,
Jennifer M. Holden, ..., Daniel J.
Jackson, Michael Levin, Mark L.
Blaxter
Correspondence
angus.davison@nottingham.ac.uk
In Brief
Davison et al. have discovered a cell
scaffolding protein in snails and frogs that
controls body asymmetry, either the
direction a snail shell coils, or whether a
frog heart is placed to the left or right.
Body asymmetry in animals, including
humans, likely arises from a highly
conserved, intrinsic asymmetry of the
cell.
Accession NumbersKU341240–KU341321
PRJEB11470
ERS1022296
ERS935814
Current Biology
ReportFormin Is Associated with Left-Right Asymmetry
in the Pond Snail and the Frog
Angus Davison,1,* Gary S. McDowell,2 Jennifer M. Holden,1,9 Harriet F. Johnson,1 Georgios D. Koutsovoulos,3
M. Maureen Liu,1 Paco Hulpiau,4 Frans Van Roy,4 Christopher M. Wade,1 Ruby Banerjee,5 Fengtang Yang,5
Satoshi Chiba,6 John W. Davey,4,10 Daniel J. Jackson,7 Michael Levin,2 and Mark L. Blaxter3,8
1School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, UK
2Center for Regenerative and Developmental Biology, and Department of Biology, Tufts University, Medford, MA 02155, USA
3Institute of Evolutionary Biology, University of Edinburgh, Edinburgh EH9 3JT, UK
4Department for Biomedical Molecular Biology, Ghent University, and Inflammation Research Center (IRC), VIB, 9052 Ghent, Belgium
5Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
6Community and Ecosystem Ecology, Division of Ecology and Evolutionary Biology, Graduate School of Life Sciences, Tohoku University,
Aobayama, Sendai 980-8578, Japan
7Department of Geobiology, University of Go¨ttingen, Go¨ttingen 37077, Germany
8Edinburgh Genomics, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JT, UK
9Present address: Warwick Medical School, The University of Warwick, Coventry CV4 7AL, UK
10Present address: Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK
*Correspondence: angus.davison@nottingham.ac.uk
http://dx.doi.org/10.1016/j.cub.2015.12.071
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).SUMMARY
While components of the pathway that establishes
left-right asymmetry have been identified in diverse
animals, from vertebrates to flies, it is striking that
the genes involved in the first symmetry-breaking
step remain wholly unknown in the most obviously
chiral animals, the gastropod snails. Previously,
research on snails was used to show that left-right
signaling of Nodal, downstream of symmetry
breaking, may be an ancestral feature of the Bilateria
[1, 2]. Here, we report that a disabling mutation in
one copy of a tandemly duplicated, diaphanous-
related formin is perfectly associated with symmetry
breaking in the pond snail. This is supported by the
observation that an anti-formin drug treatment con-
verts dextral snail embryos to a sinistral phenocopy,
and in frogs, drug inhibition or overexpression by
microinjection of formin has a chirality-randomizing
effect in early (pre-cilia) embryos. Contrary to expec-
tationsbasedonexistingmodels [3–5],wediscovered
asymmetric gene expression in 2- and 4-cell snail em-
bryos, preceding morphological asymmetry. As the
formin-actin filament has been shown to be part of
an asymmetry-breaking switch in vitro [6, 7], together
these results are consistentwith the view that animals
with diverse body plansmay derive their asymmetries
from the same intracellular chiral elements [8].
RESULTS AND DISCUSSION
Bilaterian animals aremore or less symmetrical about themidline
that divides left and right, but internally most organs are asym-
metric in locationor shape.How issymmetrybrokenduringdevel-654 Current Biology 26, 654–660, March 7, 2016 ª2016 The Authorsopment if the ‘‘right’’ and ‘‘left’’ sides are essentially arbitrary? A
longstandingmodel posits that a chiral ‘‘Fmolecule’’ is orientated
relative to the anteroposterior and dorsoventral axes [9]. This
asymmetric molecular reference then determines left-right (LR)
differentiation at the cellular and organismal level.
Given the importance of chiral patterning in the three bilaterian
superphyla, Deuterostomia, Ecdysozoa, and Lophotrochozoa, a
continuing problem is a lack of knowledge of the first symmetry-
breaking steps in the Lophotrochozoa, even though the first
described locus that reverses the whole body structure of an
animal was from the pond snail Lymnaea [10, 11]. Recently, com-
monalities between different species have been discovered
[12–15]. For example, in both vertebrates (Deuterostomia) and
snails (Lophotrochozoa), nodal and pitx encode key signaling
molecules required for the establishment of LR asymmetry, sug-
gesting that these genesmay have been used in the last common
ancestor of Bilateria, but lost in Ecdysozoa [1, 2, 16]. However,
neither nodal nor pitx is the earliest symmetry-breaking determi-
nant in snails, ultimately limiting a knowledge of whether this rep-
resents deep conservation or convergent use of the same genes.
Within Lophotrochozoa, snails are unique in that they exhibit
genetically tractable, natural variation in chirality [17] and so
may aid in understanding of the establishment and conservation
of LRasymmetry.Here,weusegenetics, genomics, andpharma-
cological inhibition to show that the Lymnaea stagnalis chirality
gene is a scaffolding component of the cytoskeleton.We present
strong evidence that this same molecule is one component of
an early chiral cytoskeletal structure that is involved in the
earliest symmetry-breaking steps across the Bilateria.A Gene that Is Associated with Chirality in Pond Snails
ThegastropodmolluscL. stagnalis is naturally variable in left-right
asymmetry, outwardly visible in the chirality of the spiral shell, and
under the control of a single maternally expressed locus. In
L. stagnalis (Figure 1A), maternal D alleles dominantly determine
a clockwise (‘‘dextral’’) twist in offspring [18, 19]. Specifically,
Figure 1. Mapping the Formin Gene, Maternal Expression, and Evolution of Chirality
(A) The snail genera used in this study (image credits: Lymnaea [E. de Roij], Biomphalaria and Physa [creative commons], Partula and Euhadra [A.D.]).
(B) 3,403 offspring were used to infer the recombination breakpoints that bound the D locus. Numbers of mapped recombinants for 1,507 sinistral (dd) snails
are shown on the right and for 1,896 dextral (DD or Dd) on the left. The sinistral mutation must be between loci b6 and b12 (shaded), a region that spans 267
kb (not to scale).
(C) Boxplots show normalized relative quantities (NRQs), on log scale, of quantitative real-time PCR assays of transcripts of three candidate genes and one
control (Larp2/3 1a) in single-cell egg samples from dextral homozygote (DD), dextral heterozygote (Dd), and sinistral recessive homozygote (dd) individuals.
Significant differences in expression were detected for Ldia2 only (DD:dd, p = 0.002; DD:Dd and Dd:dd, p = 0.004).
(D) WMISH of maternal Ldia transcripts in early, dextral L. stagnalis embryos.
(E) Schematic showing two hypotheses for the evolution of chirality in three snail families (dextral = blue; sinistral = red). Either sinistrality evolved once from a
dextral ancestor, with the ancestral Lymnaeid reverting to dextral (bottom), or sinistrality evolved twice (top).
See also Table S1 for the mapping data; Figure S3 for further WMISH and quantitative real-time PCR data; and Figure S2C for the full snail phylogeny.during the third cleavage in dextral embryos, four micromeres
simultaneously emerge from four macromeres and twist clock-
wise (‘‘spiral deformation’’; [19]). In homozygous recessive (dd)
sinistral embryos the four micromeres initially emerge neutrally,
without rotation, with a later counterclockwise twist taking place
during furrow ingression [19]. These differences are presaged by
the orientation of metaphase-anaphase spindles. In dextral em-
bryos, micromere spindle orientation is chiral (‘‘spindle inclina-
tion’’; [19]), while in sinistral embryos, the spindles are positioned
radially, such that they do not exhibit chirality.
Previously we defined a 
0.4 Mb region of the 1 Gb
L. stagnalis genome that must contain the chirality locus [18].CuTo identify the chirality-determining gene, we generated two
new resources: a sequenced bacterial artificial chromosome
(BAC) clone walk across the chirality locus interval and draft
genome sequences of DD homozygote and Dd heterozygote in-
dividuals. The BAC clone walk was oriented using three-color fi-
ber fluorescent in situ hybridization (FISH) (Figure S1). We used
the offspring of a large cross to recombination breakpoint map
the position, orientation, and haplotype origin (D or d) of each
BAC clone relative to the original restriction associated DNA
sequencing (RAD-seq) markers (rad4 and rad5) and the chirality
locus D (Table S1). The chirality locus was located between
markers b6 and b12, a region of 267 kb (Figure 1B).We predictedrrent Biology 26, 654–660, March 7, 2016 ª2016 The Authors 655
Figure 2. Impact of Drug Treatment upon 4-Cell Snail Embryos
When applied shortly after the second cleavage had completed, both SMIFH2 and CK-666 reduced the proportion of embryos that survived to the 8-cell stage
(left-hand graphs). Following SMIFH2 treatment (top left), a high proportion of the viable embryos emerged neutrally, without a chiral twist. In contrast, the
proportion of neutral embryos following CK-666 treatment (bottom left) was low. Both drugs reduce the angle of rotation as the micromeres emerge (right-hand
graphs). Mean values for each experiment and SE are shown. See also Table S2 and Movie S1.genes across the scaffolded BAC walk assembly and mapped
DD and Dd whole-genome sequencing data to the assembly to
identify haplotype-specific variation.
Only six genes were in perfect linkage with D in our mapping
cross, all contained on a single BAC scaffold: lysosomal pro-x
carboxypeptidase (Lprcp), furry (Lfry), a tudor domain-contain-
ing protein (Ltud), a major facilitator superfamily domain-
containing protein (Lmfsd), and a pair of tandemly duplicated
diaphanous-related formin genes (Ldia1 and Ldia2; Figure S2A).
In sinistral (dd) snails, Ldia2 was found to contain a homozy-
gous, single base deletion in the 50 of the coding region that
causes a frameshift. The mutation was confirmed by cloning
and sequencing Ldia transcripts from both sinistral and dextral
snails (KU341302–KU341305). No disabling mutations were
discovered in the coding sequences of the other genes,
including the adjacent paralog Ldia1.
Maternal expression of genes was assessed by quantitative
real-time PCR in single-cell embryos from DD, Dd, and dd
mothers. Of the six candidate genes, only Ldia2 showed signifi-
cant differences in expression associated with genotype (Fig-656 Current Biology 26, 654–660, March 7, 2016 ª2016 The Authorsures 1C and S3A). Ldia2 transcripts were readily detected in
dextral embryos fromDDmothers. However, in sinistral embryos
from ddmothers, Ldia2 transcript levels were 
0.6% that of DD
embryos, whereas in embryos from heterozygous Dd mothers,
they were
50%. These differences in expression are consistent
with frameshifted Ldia2sin transcripts being degraded by
nonsense mediated decay.
The tandem duplication perhaps explains why a Ldia2sin mu-
tation is not simply lethal [20–22], in that Ldia1 and Ldia2 may
have overlapping roles in embryonic development, albeit with
some specialized function: while we found that mRNAs for
both Ldia1 and Ldia2 were present in equal quantities in the
dextral 1-cell embryo, Ldia2 was 
3-fold enriched in 1-cell
dextral embryos relative to somatic tissues, whereas Ldia1
was 
10-fold depleted (
30-fold difference overall).
Asymmetric Expression of Maternal Genes Precedes
Asymmetric Morphology
Expression of candidate loci was examined in early L. stagnalis
embryos using whole-mount in situ hybridization (WMISH; [23]).
Figure 3. Tubulin and Actin Staining of
Control and Drug-Treated Embryos
Embryos were fixed and stained with Cy3-b-
tubulin (red) and 488-phalloidin (green) to highlight
the spindle microtubules and filamentous actin,
respectively. DMSO-treated embryos predomi-
nantly showed spindle inclination (left image, 4-cell
stage), with the micromeres usually emerging with
a dextral twist (right image, 8-cell stage). Aminority
of SMIFH2 treated embryos had mitotic spindles
that showed a radial orientation (left image), an
arrangement that was not observed in DMSO
control dextral embryos. In the SMIFH2 right-hand
image (8-cell stage), the top middle and middle left
micromeres are emerging neutrally (arrows), with
the other two showing a partial rotation. Addition
of SMIFH2 did not influence spindle orientation in
4-cell DMSO control or sinistral embryos, with
spindles typically showing a radial orientation.The first two embryonic cleavages in L. stagnalis are equal, so
that the fourmacromeres that are formed from the first two cleav-
ages are indistinguishable [3–5], until contact between the third
quartet of micromeres induces one of the macromeres to
become the future D blastomere (but see 24]). We expected
that mRNA transcripts would initially be equally distributed be-
tween the four macromeres. Surprisingly, however, we found
that Ldia mRNA was asymmetric at the 2-cell stage and largely
confined to one macromere by the 4-cell stage (Figure 1D), with
Lfry sometimes showing an asymmetric pattern, albeit less strik-
ing, and with more variation between individuals (Figure S3B).
While we do not know whether the Ldia2-positive macromere
is the D blastomere, individual macromeres must have an iden-
tity as early as the two-cell stage, contrary to the expectation
based on accepted models [3–5] of development in equal
cleaving snails. This also further emphasizes the general point
that asymmetry is determined very early and intracellularly—
asymmetry of molecules precedes visible morphological asym-
metry, although not necessarily in a causative manner (see [4,
25] for comparison with unequal cleaving embryos). The results
also suggest an explanation for the reduced viability of sinistral
embryos [20–22]—as chiral rotation of micromeres in sinistrals
is sometimes incomplete or error prone, inviability may be
caused by a conflict in identity between individual cells. Further
investigation is necessary, but the sinistral embryos seem to
show more generalized, less obviously asymmetric staining,
consistent with this explanation (perhaps because of reduced
transport on actin microfilaments, [4]) (Figure S3B).
Pharmacological Inhibition of Formin in Early Snail
Embryos Mimics the Sinistral Phenotype
As transgenic approaches are not yet established in L. stagnalis,
andmicroinjection is usually lethal [22], we tested formin involve-
ment in chirality using SMIFH2, an FH2 domain inhibitor [26].
SMIFH2 prevents formin nucleation and processive elongation
of filamentous actin by decreasing the affinity of formin for the
barbed end. Micromolar concentrations of SMIFH2 disrupt theCuformation of formin-dependent, but not Arp2/3 complex-depen-
dent, actin cytoskeletal structures [26].
When 100 mM SMIFH2 was added to genetically dextral 4-cell
embryos shortly after completion of the second cleavage, rela-
tively few embryos (
30%–60%) reached third cleavage at

80 min (Figure 2; Table S2). However, in 
25%–35% of those
that did, all four micromeres emerged neutrally, with no chiral
twist (SMIFH2 treated at 0 min, n = 6 experiments, 274 embryos,
compared to n = 11 control experiments, 219 embryos; p <
0.001, U = 5.5, Mann-Whitney U), with up to 
45% of individual
micromeres emerging neutrally (Figure 2). SMIFH2 treatment of
dextral embryos thus phenocopies normal sinistral embryos.
We also visualized spindles during the third cleavage of
L. stagnalis by indirect immunofluorescence with anti-b-tubulin
antibody. In line with previous findings [19], control dextral em-
bryos showed the characteristic spindle inclination, especially
in the latter stages of mitosis. In SMIFH2-treated dextral em-
bryos, the spindles were more frequently radially symmetric,
resembling untreated sinistral embryos (Figure 3).
To rule out a non-specific effect of SMIFH2, we compared the
effect of another inhibitor of actin assembly, CK-666, which acts
specifically on Arp2/3 complex-dependent actin patches [27]
and not on formin-dependent actin cables. CK-666 also had a
lethal effect when applied to genetically dextral early four-cell
embryos and also tended to reduce the average angle of emer-
gence of micromeres. However, an achiral phenotype was
observed in rather few CK-666-treated embryos (Figure 2; CK-
666 treated 0 min, n = 3 experiments, 196 embryos, compared
to n = 5 control experiments, 190 embryos; p = 0.107, U = 2.5,
Mann-Whitney U). Therefore, the differential effects of SMIFH2
and CK-666 suggest that formin-mediated actin assembly may
be a critical factor in determining chiral cleavage orientation be-
tween the second and third cleavages.
In SMIFH2-treated, genetically dextral embryos that
continued to develop following neutral emergence of micro-
meres (i.e., like sinistrals), the direction of the subsequent twist
was dextral, rather than sinistral (6/6; Movie S1). The individualrrent Biology 26, 654–660, March 7, 2016 ª2016 The Authors 657
Figure 4. Effect of Drug Treatment and Microinjection of Overexpressed Formin on Chirality in the Frog
(A) Embryos were treated with DMSO, CK-666, or SMIFH2 at the concentrations indicated, allowed to develop, and scored for visceral organ chirality at stage 45.
(B) Embryoswere injected into the animal pole withmRNA encodingmouse dia1 formin and scored for visceral organ situs at stage 45. Images: Examples of organ
situs for experimental microinjection with wild-type mouse dia1 mRNA. The control shows a wild-type (situs solitus) tadpole, ventral view, demonstrating the
normal arrangement of the stomach (yellow arrowhead), heart apex (red arrowhead), and gall bladder (green arrowhead). Heterotaxic tadpoles (ventral view)
resulting from formin overexpression show reversal of all three organs, i.e., situs inversus; the gut position and looping and gall bladder; or the heart.
See also Table S3 and Figure S4.micromeres of genetic sinistrals also sometimes twisted dex-
trally (
0 to 4% in our experiments; the fourth time-lapse in
Movie S1 shows an embryo in which all four micromeres twist
dextrally after neutral emergence; see also [22]). This later twist
is also actin dependent [19], which suggests dextrality may be
the default pathway, independent of FH2 domain function.
A Sinistral Ancestral Pond Snail?
We used genomic and transcriptomic resources, and new se-
quences, to explore links between diaphanous formin and chiral
evolution. First, we found that the chromosomal region that
contains the L. stagnalis chirality locus is deeply conserved,
exhibiting synteny of dia, fry, and tud between L. stagnalis,
Biomphalaria glabrata (planorb snail, sinistral cleaving), andCap-
itella telata (polychaete annelid, dextral cleaving). Second, while
the dia duplication is also evident in Lymnaea trunculata, and so
must pre-date its divergence from L. stagnalis, single-copy dia
genes in B. glabrata and Physa acuta (both sinistral snails: Fig-
ure 1A) are more similar to Ldia1 than Ldia2 (Figure S2B). The
B. glabrata dia gene and Ldia1 also share a non-repetitive UTR
element, absent from Ldia2. Thus, Ldia2 is likely the derived pa-
ralog, which may have evolved specific function in the embryo,
and for which loss leads to a sinistral phenotype.
We mapped chirality onto a new phylogeny of the Hygrophila
(Figure S2C). The predominantly dextral Lymnaeidae and the
sinistral Physidae cluster together, so either sinistrality evolved
once, with a sinistral ancestral Lymnaeid subsequently reverting658 Current Biology 26, 654–660, March 7, 2016 ª2016 The Authorsto dextral, or else, sinistrality evolved on two separate occasions
(Figure 1E). Both explanations are equally parsimonious. How-
ever, only the first is consistent if the duplication was involved
in enabling dextrality in the Lymnaeidae.
As no natural variation in chirality has been described in
Biomphalaria or Physa, it is difficult to further test the function
of dia in relation to copy number. Instead, we sampled dia ortho-
logs in two other chirally variable snail genera, Euhadra, a
Japanese land snail, and Partula, an endangered species from
Polynesia (Figure 1A; [28, 29]). In both of these genera, chirality
was not associated with variation in a single-copy dia that was
recovered (Figure S2). Molecular understanding of chiral varia-
tion in these species is therefore likely to reveal additional com-
ponents of the LR asymmetry pathway, including variants in
genes that enable chiral evolution without negative pleiotropic
effects upon fitness.
Formin Also Regulates LR Patterning in the Frog
A popular model of LR symmetry breaking in vertebrates relies
on chiral flow of extracellular fluid during neurulation. However,
this mechanism cannot be universal as many phyla lack the cili-
ated structures required or achieve correct LR patterning prior to
the ciliated structure differentiation (reviewed in [8, 14, 15]).
Inherent asymmetry in the cytoskeleton could provide an
ancient, well-conserved mechanism used by vertebrate em-
bryos at the earliest stages of development to initiate the LR
pattern and instruct the entire body plan [8, 30, 31]. We
investigated whether formin inhibition in early embryos, before
the neurula ciliary flow, could affect chirality in the vertebrate
model Xenopus laevis.
SMIFH2 and CK-666 drug treatments were carried out with
X. laevis embryos at different stages of development and scored
by measuring heterotaxia (independent organ LR inversion) in
tadpoles (Figure 4; Table S3). In early embryos (stages 1–6), dur-
ing which the cytoskeleton instructs LR patterning [30], treat-
ment with 50 mM SMIFH2 had a strong effect on LR patterning
(13% heterotaxia, X2, p < 0.001), with a smaller but significant ef-
fect with CK-666 (7% heterotaxia, X2, p < 0.001), whereas em-
bryos treated at neurula stages (stages 19–21), when ciliary
flow is present, showed a strong effect for both 50 mM SMIFH2
(12%, X2, p < 0.001) and 50 mM CK-666 (16%, X2, p < 0.001).
Treatments spanning late blastula to gastrula (stages 8–14)
had a reduced (3%–4%) but still significant effect (Table S3) on
organ heterotaxia, showing that the efficacy of early treatment
cannot be due to remnant drug persisting to cilia stages.
For a more specific gain-of-function test of formin function in
Xenopus, mouse dia1 mRNA was injected into the animal pole
of frog embryos and organ situs assessed at stage 45. Similar
to the in vitro finding that the spontaneous counterclockwise
alignment of actin bundles can be reversed by overexpression
of alpha-actinin [7], we found that overexpression of dia1 re-
sulted in a high and significant proportion of heterotaxia, whether
injected 30 or 60 min post-fertilization, or in one of two or four
cells (Figures 4 and S4). In addition, targeted injection into the
dorsal left (DL) or ventral right (VR) blastomeres at the 4-cell
stage showed that while significantly different from uninjected
controls (X2, p < 0.001), there is no significant difference between
DL and VR in the effect on heterotaxia (n = 85 DL, 5% hetero-
taxia; n = 135 VR, 9% heterotaxia; p = 0.75 Student’s t test),
which would be expected if the effect were at the point of ciliary
flow, as the left side of the embryo is required for ciliary flow
affecting LR patterning [32].Formin, an F Molecule?
The implication of a key cytoskeletal protein in LR patterning of
both molluscan and vertebrate embryos is consistent with a
view of asymmetry as a highly conserved, ancient property in
which diverse body plans leverage asymmetry from the same
intracellular chiral elements. Bilaterian LR asymmetry may be
dependent upon thephysical orientationof theactin cytoskeleton,
which, by exerting mechanical stresses on the cell, results in heli-
cal rotation [6, 7].Whilemultiple elements potentially contribute to
the establishment of this asymmetry, formins may have pivotal
roles in coordinating functions that depend upon both the actin
and microtubule cytoskeleton [7, 30, 33, 34]. Pond snails are
now an experimentally tractable, comparative model in which to
integrate understanding of the action of downstream patterning
genes, such as nodal, and the dynamics of cellular interaction
and movement in the embryo to generate handedness.ACCESSION NUMBERS
The accession numbers for sequences in the INSDC are: PRJEB11470 (all li-
braries), ERS1022296 (genomic libraries), and ERS935814 (BAC libraries).
GenBank accession numbers are KU341240–KU341321. Dryad accession is
http://doi.org/10.5061/dryad.r4342.CuSUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
four figures, three tables, and one movie and can be found with this article on-
line at http://dx.doi.org/10.1016/j.cub.2015.12.071.
AUTHOR CONTRIBUTIONS
Conceptualization, A.D., M.L., and M.L.B; Methodology and Investigation,
A.D. and C.M.W. (phylogenetics); Methodology and Investigation, H.F.J.,
A.D. (quantitative real-time PCR), J.M.H., and A.D. (snail microscopy and phar-
macology); Methodology and Investigation, D.J.J. (WMISH), R.B., and F.Y.
(FISH, with help from A.D. and M.M.L.); Methodology and Investigation, A.D.
and M.M.L. (BAC walk, with BioS&T); Methodology and Investigation,
G.S.M. and M.L. (laboratory - frog); Methodology and Investigation, A.D. and
S.C. (Japan fieldwork); Methodology and Investigation, A.D., C.M.W., D.J.J.,
G.D.K., J.W.D., M.L.B., P.H., and F.V.R. (bioinformatics); Writing – Original
draft, A.D. and M.L.B.; Writing – Review & Editing, A.D., C.M.W., D.J.J.,
G.S.M., G.D.K., H.F.J., J.M.H., M.M.L., M.L., and M.L.B. Supervision and
Data Analysis, A.D., M.L., and M.L.B; Discovered the mutation, A.D. The order
in which G.S.M. and J.M.H. appear in the author list was decided by a ‘‘best of
three’’ coin toss.
ACKNOWLEDGMENTS
The work was principally funded by BBSRC grant BB/F018940/1 to A.D.,
M.L.B., and Aziz Aboobaker, with additional funding provided by the Univer-
sities of Edinburgh and Nottingham, the Wellcome Trust Sanger Institute
(WT098051), BBSRC grants G00661X and F021135, MRC grant (G0900740),
NERC grant (R8/H10/56) to M.L.B., Leverhulme Trust grant F/00114U to
C.M.W., grants from the G. Harold and Leila Y. Mathers Charitable Foundation
and the Physical Science Oncology Center supported by Award Number
U54CA143876 from the National Cancer Institute to M.L., and DFG funding
to D.J.J. (JA2108/1-1), with additional funding from the JSPS and the Daiwa
Foundation to A.D. G.D.K. and H.F.J. were supported by BBSRC PhD student-
ships. The authors would like to thank BioS&T/Areti Karadimos for BAC library
construction; Edinburgh Genomics staff for sequencing support; Nigel P.
Carter for support in initiating the FISH assay; Takahiro Asami, EdmundGitten-
berger, and Gerhard Falkner for the initial finding and breeding of sinistral
snails; and Aziz Aboobaker, William Brown, John Armour, and David Lambert
for providing plentiful, useful advice. Nebis Navarro, Paul Richards, Cendrine
Hudelot, and Sheila Keeble assisted with some laboratory work; Yuta Morii
and Kazuki Kimura assisted with fieldwork. Partula mooreanawere kindly sup-
plied by Ross Poulter at the Zoological Society of Scotland. Helpful reviews
were providedbyEdmundGittenberger and two anonymous referees. ForXen-
opus laevis, this studywas carried out in strict accordancewith the recommen-
dations in the Guide for the Care and Use of Laboratory Animals of the NIH.
Received: August 24, 2015
Revised: December 1, 2015
Accepted: December 29, 2015
Published: February 25, 2016
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