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Development 110, 949-954 (1990)
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The development of handed asymmetry in aggregation chimeras of situs
inversus mutant and wild-type mouse embryo
NIGEL A. BROWN1 *, AFSHAN McCARTHY1 and LEWIS WOLPERT2
l
MRC Experimental Embryology and Teratology Unit, Saint George's Hospital Medical School, Cranmer Terrace, London, SW17 ORE, UK
Department of Anatomy and Developmental Biology, University College and Middlesex Hospital School of Medicine, Windeyer Building,
Cleveland Street, London, W1P 6DB, UK
2
* Author for correspondence
Summary
Mutant iv/iv mice develop as if they have no sense of left
and right, so the development of asymmetry is random:
half normal, half as a mirror-image of normal, situs
inversus. We have made aggregation chimeras of 8-cell
stage iv/iv and + / + embryos, transferred them into
pseudopregnant mice, and examined their phenotype on
day 10 of gestation. The contribution of mutant and
wild-type cells to tissues of the embryo was estimated by
strain-specific isozyme (GPI-1) analysis. We have also
performed reciprocal embryo transfers, iv/iv blastocysts
into + / + mice, and vice versa. These transfers show that
the development of handed asymmetry is determined by
embryonic genotype, and is unaffected by the maternal
environment (at least after day 3), or by the procedures
of embryo collection, culture and transfer. Our observations on the development of 21 viable chimeric
embryos show that neither iv/iv nor + / + cells are
dominant. All embryos (12) with less than 50%
contribution of iv/iv cells to the heart developed with
normal situs. Of 9 embryos with greater than 50 % iv/iv
cells, only 2 developed with inverted situs. These
findings suggests that there was partial 'rescue' of
embryos by some influence of normal over mutant cells.
However, we cannot, statistically, exclude an alternative
interpretation that cells are behaving autonomously.
Interestingly, the embryos that developed with inverted
situs were unique in having greater than two thirds
contribution of iv/iv cells to both the heart and the
visceral yolk-sac.
Introduction
of left and right, so their asymmetry is not handed, but
random (Layton, 1976). In our terms, the conversion or
biasing process is faulty and does not interact with the
random generation of asymmetry; thus, half the
embryos develop with normal visceral situs, half with a
mirror-image pattern: situs inversus (Hummel and
Chapman, 1959). There are also other abnormalities in
iv/iv mice, including isomerisms, heterotaxias, and
heart malformations, and we have discussed these in
relation to our model (Brown and Wolpert, 1990).
There is good evidence that each organ has its
asymmetry specified independently.
In this study, we have attempted to answer two
questions: does the maternal uterine environment play
a role in the development of handed asymmetry in the
mouse? And, primarily, would the presence of some
normal cells in an iv/iv embryo 'rescue' a chimeric
embryo from the effects of the mutation? To examine
the first question, we performed a series of reciprocal
embryo transfer experiments, moving iv/iv blastocysts
into + / + recipients, and vice versa. These studies also
allowed us to evaluate whether embryo collection,
Handed asymmetry is the consistent difference between
left and right sides of the body. For example, the
position of the stomach on the left side; differing
numbers of lung lobes on the two sides; aortic loop to
the left; and so on. Clearly, the embryo must have a
method to distinguish its left from right, in order to
create this body pattern. There is virtually nothing
known about the mechanisms involved, but we have
recently proposed a model, and a conceptual framework (Brown and Wolpert, 1990). The model proposes
that there are three processes: conversion of a
molecular asymmetry to the multicellular level, which
biases a system for the random generation of asymmetry, and finally an interpretation step for individual
organs.
The effects of the mouse mutant gene, iv (situs
inversus viscerum), were important to the genesis of our
model, as they have been to the thinking of others (see
Brown and Wolpert, 1990). The embryos of homozygous iv/iv mice develop as if they have lost their sense
Key words: asymmetry, handedness, chimeras, situs
inversus.
950
N. A. Brown, A. McCarthy and L. Wolpert
handling, culture or transfer had any effect on the
development of asymmetry.
Chimeric mouse embryos have often been used to
study the interaction of normal and developmentally
mutant cells. Rescue of mutant cells by aggregation of
normal and mutant embryos has been reported for (1)
lethal t12 and jpmsd mutations (Mintz, 1964; Eicher and
Hoppe, 1973), (2) mutations of eye development
(LaVail and Mullen, 1976; Muggleton-Harris et al.
1987) and (3) some murine trisomies (see Cox et al.
1984). It is almost certain that iv is a loss-of-function
mutation, and it is likely that the normal allele codes for
some component of the left/right conversion system. If
the result of the iv mutation is the absence of a
functional molecule, then the presence of non-mutant
cells may be able to restore normality.
We hypothesize that the development of iv/iv plus
wild-type chimeras will follow one of two possibilities: if
the overall determination of left/right positional information is a cell-interactive process then interaction
between cells, mutant and normal, may be able to
rescue iv/iv cells. Alternatively, cells may behave
autonomously with respect to left/right position. Then,
in a chimeric organ, the overall sidedness may be a
'majority decision' - whichever genotype contributes
more than half the cells will dominate. Thus, any organ
primordium with more than 50 % iv/iv cells would be
phenotypically mutant, and half of these would develop
with situs inversus.
We have made chimeras by aggregation of 8-cell iv/iv
and + / + mouse embryos. These were allowed to
develop in utero to day 10 of gestation, the earliest stage
at which visceral situs can be determined. Embryonic
tissues were examined for chimerism using an isozyme
marker. We focused, initially, on the heart because
there is evidence that the development of its asymmetry
is an intrinsic property of the heart, itself (Stalsberg,
1970), and looping of the heart is also the first
observable manifestation of handed asymmetry.
Materials and methods
Mice
The Si/Col inbred strain of mouse is homozygous for the iv
gene, has a pale chinchilla coat colour, and is homozygous for
the Gpi-lsb allele, which codes for the fast-migrating form of
glucose phosphate isomerase (GPI-1B). The MF1 ( + / + )
outbred strain is albino, and homozygous for the Gpi-lj1
allele for slow-migrating GPI-1A. The inbred strain C57BL/
6JLac ( + / + ) is pigmented, and homozygous for Gpi-lsb.
B6CBF1 ( + / + ) mice are Fi hybrids (C57BL/6JxCBA/Ca),
also homozygous for Gpi-lsb). Males of the T145H strain are
genetically sterile and were used to induce pseudopregnancy.
Collection of embryos
Female + / + (MF1 or B6CBF1) and iv/iv (SI/Col) mice (3 to
4 or 6 to 8 weeks old) were superovulated by intraperitoneal
injection of 7.5i.u. or 2.5i.u. of pregnant mares' serum
gonadotrophin, respectively, followed 48h later with 5 i.u. of
human chorionic gonadotrophin. Mice were paired overnight
with their respective males. The presence of a vaginal plug the
following morning was taken as an indication of successful
mating, and this was designated day 1.
On day 3, the oviduct and anterior portion of the uterine
horn were flushed with warm (about 37 °C) Medium 2 (M2,
Fulton and Whittingham, 1978). Fragmenting, lysed, 1-, 2- or
4-cell stage embryos were discarded, and only 8-cell or early
morula stage embryos were collected.
Chimera production
A well-established technique was used to produce chimeras
(Tarkowski, 1961; Mintz, 1962). The zona pellucida was
removed by brief exposure of embryos to warm acid Tyrode's
solution (Nicholson et al. 1975). Zona-free embryos were
washed three times in M2, followed by three washes in warm
Medium 16 (M16, Whittingham, 1971) in small drops on a
60x15 mm plastic Petri dish under paraffin oil, which had
been previously equilibrated with medium.
One iv/iv and one + / + embryo were placed in a drop of
M16 under paraffin oil, the two embryos were brought into
contact with the aid of a glass pipette, and incubated at 37°C
in an atmosphere of 5 % CO2 in air. Embryos were examined
2 to 3h later and, if needed, contact was once again
established, prior to incubation overnight. The following day,
fully aggregated chimeras, either at the late morulae or early
blastocyst stage, were transferred into M2, and 4 to 6 were
transferred into one horn of the uterus of a day 3
pseudopregnant B6CBF1 recipient female. To help establish
pregnancy, the other horn was supplemented with C57BL/
6JLac embryos, which had been collected at the 8-cell stage
and cultured, as above, without removing the zona pellucida.
Examination of chimeras
The B6CBF1 recipients were killed on day 10, uteri removed
and embryos were dissected out, keeping those from each
horn (potential chimeras and C57BL/6JLac embryos) separate. The gross morphology of all embryos was examined and
recorded, including the two major signs of situs visible at this
time: (a) the side to which the heart tube had looped; and (b)
the direction of embryo turning, which is manifest from two
features: the side of exit/entry of the vitelline vessels; and the
side of the head to which the tail-bud lies. In normal, situs
solitus, embryos the heart tube loops to the right and the
embryo turns to its right, resulting in the vitelline vessels lying
to the left, and the tail bud to the right.
The ectoplacental cone and amnion were removed and
discarded and the embryo and yolk sac washed thoroughly in
isotonic saline. The embryo was dissected into heart (the
pericardium was discarded); head (up to and including the
first branchial arch); body (up to the fore limb bud); and tail
(remaining embryo). The separated tissues were transferred
into 1.5 ml microcentrifuge tubes and stored at minus 20 °C. In
the case of resorptions, any visible conceptual tissue, divided
into the embryo and yolk sac if possible, was thoroughly
washed and stored as above.
Analysis of tissue genotype
Tissue genotype was determined from GPI-1 type by
electrophoretic analysis. First, a sample of heart tissue was
analyzed. If this was found to be chimeric, then the remaining
tissues, as well as the heart for a second time, were analyzed
to assess the degree of chimerism throughout the embryo and
yolk sac.
GPI-1 analysis was carried out using a standard electrophoretic method. Tissues were homogenised by sonication in
30//1 50 mM Tris-HCl buffer pH8.0, using a Branson
ultrasonic generator, duty cycle 30%, output 3, for 6s. 2 to
3 fA of this suspension was applied to cellulose acetate plates
Development of handedness in chimeric embryos
which had been soaked in 25 mM Tris/192mM glycine buffer
pH8.5 for lOmin at room temperature. The running buffer
was also 25 mM Tris/l92mM glycine pH8.5, and the isoenzymes were separated at 120 volts for 90min.
In this system, the isoenzymes move towards the cathode
with the BB form moving much further than the A A form.
The isoenzymes were visualised by applying to the cellulose
acetate plate a mixture containing: 2 ml 1.5% agarose; lml
50mM Tris-HCl buffer pH8.0; lml NADP (lmgmr 1 ); 3
drops MgCU (5.41g/l00ml); 3 drops fructose-6-phosphate
(100mgmr f ); 3 drops 3-(4,5-dimethythiazol-2-yl)-2,5- diphenyltetrazolium bromide (lOmgml"1); 3 drops phenazine
methosulphate (2.5mgml~1) and 9 units glucose-6-phosphate
dehydrogenase. The colour was allowed to develop in the
dark for about 5 min after which the reaction was stopped by
soaking the plates in 5% acetic acid.
The relative amounts of AA and BB isozymes were
estimated from the intensity of staining of the two bands,
measured by an LKB Ultrascan laser densitometer with
integrator. These amounts were taken as a direct measure of
the relative contributions of iv/iv and + / + genotype cells to
the tissue analyzed. Preliminary experiments showed that
equal amounts of iv/iv and + / + embryonic tissue gave the
same intensity of staining on electrophoresis plates. In
addition, electrophoretic analysis of mixed samples of iv/iv
and + / + tissue showed that relative staining intensities were
proportional to amounts of tissue (data not shown).
Reciprocal transfers
Si/Col (iv/iv); MF1 ( + / + ) and B6CBF1 ( + / + ) embryos
were collected on day 3, cultured for 24 h, and transferred into
day 3 pseudopregnant recipients, as described above, without
removal of the zona pellucida. iv/iv embryos were transferred
into B6CBF1 recipients, and + / + embryos into iv/iv
(SI/Col) recipients. Recipient dams were killed on day 10,
and the embryos examined, as described above. In some
cases, recipients were killed on day 18 and fetuses were
examined, as previously described (Brown et al. 1989).
Statistical analyses
The analyses of the proportions of normal and inverted
embryos of different genotypes were by chi-square, with
Fisher's exact or Yates correction where appropriate, using
the SPSS/PC programme. The correlations of iv/iv contributions between different tissues was by Pearson product
moment correlation coefficient and analysis of variance, using
the Minitab/PC programme.
Results
Reciprocal embryo transfer
+/+ embryos into iv/iv recipients
A total of 147 + / + embryos (100 B6CBF1, 47 MF1),
were transferred into 14 iv/iv recipients (5 inverted, 9
normal phenotype). Eight recipients (3 inverted, 5
normal), containing 81 embryos, did not become
pregnant. Of the remaining 66 embryos, 40 (61%)
implanted, and of these, 26 (65 %) were viable fetuses
when examined on day 18. All 26 (100%) fetuses had
normal visceral situs.
iv/iv embryos into +/+ recipients
228 iv/iv embryos were transferred into 16 B6CBF1
+ / + recipients. Eight recipients, containing 67 em-
951
bryos, did not become pregnant. Of the remaining 161
embryos, 90 (56%) implanted, and there were 46
(51 %) viable embryos upon examination on day 10. 22
(48 %) had normal situs, 20 (43 %) were situs inversus,
and 4 (9%) were heterotaxic. All the heterotaxic
embryos had inverted heart loops, but normal cephalocaudal turning.
Chimeras
The superovulation and mating of 472 iv/iv mice
produced 201 (43 %) plugged animals, from which 1201
embryos were flushed. Of these, 658 (55 %) were at the
8- cell stage. 502 of these iv/iv embryos were
aggregated with 8-cell or early morulae + / + embryos.
After 24 h of culture, 384 (76%) of the aggregates had
formed morulae, and were transferred into day 3
B6CBF1 pseudopregnant recipients.
When recipients were killed on day 10 and their uteri
examined, 135 implantation sites, containing 137
embryos or resorptions (36% of the number transferred), were collected and examined. Observations on
these 137 conceptuses are summarised in Fig. 1. 55
(40 %) of the conceptuses were classified as resorptions
as they did not contain a viable embryo. In 24 of these,
some resorbing embryonic material was recovered, and
6 of these embryonic samples were chimeric, but in no
case could embryonic situs be determined, so these are
not informative.
Of the 82 viable embryos, only 7 showed the situs
inversus phenotype. Five of these appeared not to
contain any + / + tissue, based on GPI-1 analysis, so are
not chimeric, or contain only a very small proportion of
normal cells. Both other situs inversus embryos
contained mostly iv/iv tissue. One embryo was actually
heterotaxic, with normal turning but inverted heart,
and came from an implantation site containing a
second, resorbing, embryo which was also chimeric
(about 50:50 chimerism).
A majority of the embryos with normal situs (47,
64%) appeared not to contain any iv/iv tissue, while
some (9, 12%) were 100% iv/iv. A total of 19 werechimeric, 10 of which were mostly + / + tissue.
The data in Fig. 1 show the poor developmental
potential of iv/iv embryos. Of the 137 conceptuses, 54
were wholly + / + derived, but only half as many (25)
were wholly of iv/iv origin. Similarly, only 6 of the 27
chimeric conceptuses were mostly iv/iv, whereas 14
were mostly + / + , and 7 equal. This suggests a selective
advantage of + / + embryos, but this may be unrelated
to the iv locus since the strains used differ at many loci.
The genotype of the 31 completely resorbed conceptuses is unknown, but of the partially resorbed
embryos 11 were iv/iv, only 7 + / + . This suggests a
higher rate of embryonic death for iv/iv embryos.
The degree of chimerism in different tissues of the 21
chimeric viable embryos is shown in Table 1. In
general, the degree of chimerism was similar in the
heart (H), head (D), body (B) and tail (T) portions of
the embryos, and the percentage iv/iv contribution was
highly correlated for all these tissues (for the 17
chimeras with complete data, the correlation coefficient
952
N. A. Brown, A. McCarthy and L. Wolpert
Total Conceptuses
137
Resorptions
55 (40%)
Viable Embryos
82 (60%)
Situs Solitus
75 (91%)
Situs Inversus
7 (9%)
100% iv/lv
5 (71%)
Chimeras
2 (29%)
100% iv/iv
9 (12%)
100% • / •
47 (64%)
Some conceptus
24 (44%)
No conceptus
31 (56%)
Chimeras
19 (26%)
100% • / •
7 (29%)
100% iv/iv
11 (46%)
Mostly • / •
10 (53%)
Mostly Iv/lv
' 2 (100%)
Chimeric
6 (25%)
_ Mostly • / •
4 (67%)
Mostly iv/iv
3 (16%)
Mostly iv/iv
' 1 (17)
Equal
6 (32%)
Equal
1 (17%)
Fig. 1. Summary of the fate of iv/iv/'+/+ embryos, aggregated at the 8-cell/early morula stage, cultured overnight,
transferred into day 3 pseudopregnant recipients, and examined on day 10.
Table 1. Genotype of tissues in iv/iv + / + chimeric
day 10 embryos
Percentage contribution of iv/iv to tissue
No.
Heart
Head
Body
Tail
YSac
Situs
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
79
71
68
68
66
55
54
54
53
49
43
39
28
28
28
25
23
21
15
15
2
57
51
53
46
62
42
37
33
54
63
50
58
82
67
47
40
45
39
52
50
54
75
60
50
40
43
44
46
73
71
6
55
61
52
50
54
41
52
34
20
37
43
36
21
60
45
38
31
24
16
16
19
25
20
18
13
24
18
18
7
43
67
30
70
36
49
58
21
38
N
I
H
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
— = Tissue not analyzed.
* Situs, N=Normal; I=Inverted; H=Heterotaxic.
Table 2. Summary of tissue genotype and embryo
phenotype in iv/iv plus + / + aggregated embryos
examined on day 10 of gestation
Tissue genotype
iv/iv contribution
to heart tissue
All
>50%
<50%
None
Embryo phenotype
Number of embryos
Normal
Inverted
9
7
12
47
Embryos aggregated at the 8-cell/early morula stage and
examined on day 10. Genotype determined by GPI analysis.
more than two thirds contribution of iv/iv tissue to both
the heart and the visceral yolk sac.
A summary of the tissue genotype and embryo
phenotype of all the 82 viable embryos produced in the
chimera study is given in Table 2.
Discussion
The maternal environment did not play a role in the
development of handed asymmetry in these experiments. The results of the reciprocal embryo transfer
r=0.91 B v T; 0.90 H v B; 0.86 H v D; 0.83 H v T; 0.83 experiments were clear-cut. Normal + / + embryos
D v B; and 0.82 D v T). In contrast, the visceral yolk-sac transferred into iv/iv recipients developed with normal
visceral situs, in all cases. Similarly, iv/iv embryos
(Y) was often quite different, and was not correlated
transferred into + / + recipients developed as they
with the other tissues (r=0.23 Y v D; 0.17 Y v H; -0.10
would have done had they not been transferred: half
Y v B; -0.03 Y v T). The mean contribution of iv/iv
cells to the yolk sac in these 17 chimeras was 50%, with normal situs, half with situs inversus. This suggests
that the maternal environment plays no role in the
apparently greater than for any tissue of the embryo
determination of the left/right axis, unless the influence
proper (39 to 46%), but these are not statistically
is irreversibly exerted during the first 2 days after
significant differences (f>0.05, ANOVA). The two
fertilization, prior to embryo collection in these
embryos with abnormal situs were unique in having
Development of handedness in chimeric embryos
experiments. It is unlikely that left/right position is
encoded within maternal cytoplasm since mutant (iv/iv)
oocytes fertilized with + / + sperm develop completely
normally (Layton, 1976; Brown, unpublished). Thus,
left/right positional sense seems intrinsic to the
embryo.
These observations also show that the manipulations
involved in collection, culture and transfer of iv/iv
embryos did not have a detectable influence on the
50:50 ratio of normal:inverted visceral development.
Thus, any effects on this ratio in the chimera studies can
not be attributed to such artifacts.
Three categories of viable embryos were produced in
the chimera study, based on heart tissue genotype
analysis: (a) all-+/+; (b) all-/v/iv; and (c) chimeras.
The phenotype of the all—1-/ + embryos was, as
expected, 100% normal visceral situs. Of the 14 alliv/iv embryos, 9 were normal, 5 were situs inversus.
This does not differ, statistically (P>0.05, chi-square),
from the expected 7:7 ratio, but the sample is small, in
statistical terms. However, if the apparent shift towards
a larger proportion of normal embryos were real, it
could be due to rescue by the presence of a small
amount of + / + tissue, either in the heart below the
limit of detection of our analysis (about 1%), or in
other tissues, which were not analyzed in those embryos
that did not show apparent chimerism of the heart.
The proposition of a rescue of iv/iv cells by normal
cells in a chimera appears to be supported by our
observations, although rescue is clearly not complete.
There were 21 viable chimeric embryos in this study,
but only 2 had abnormal situs. No embryo with less than
two thirds iv/iv tissue (17 in total) showed abnormal
situs, including 7 embryos with approximately equal (43
to 66 % iv/iv) proportions of normal and mutant tissue.
This suggests that + / + cells exert some correcting
influence over mutant tissue. Of course, these studies
cannot suggest any mechanism for such an influence,
although it is compatible with the possibility that + / +
cells provide a molecule that the mutant cells lack.
We cannot, however, reject the alternative possibility
of cell-autonomy; indeed, our observations can be
interpreted as generally consistent with this hypothesis.
If cells were autonomous, we might expect that an
embryonic organ with more than 50 % + / + cells would
have the + / + (normal) phenotype, whereas one with
more than 50% iv/iv cells would develop as a mutant,
and thus half would be situs inversus. Of 12 chimeras
with more than 50% + / + contribution to the heart, all
had normal situs. Of the 9 chimeras with more than
50% iv/iv contribution to the heart, 2 had inverted
heart loops. Although this 7:2 ratio appears to favour
normal development, it is not statistically different
(P>0.05, Fisher's exact) to the ratio of 4:5, or 5:4, that
would be expected if the hypothesis were correct.
Again, the numbers are small, and would have to be
increased four-fold for the difference in the observed
ratios to achieve statistical significance. It is possible
that the asymmetry of an organ is determined by some
critical sub-population of cells, which would be masked
by our whole-organ analyses. Use of a cell-based
953
marker for chimerism would address this possibility
(see below).
While we cannot conclusively distinguish between the
two hypotheses, it is clear that neither iv/iv nor + / +
cells in an embryo are dominant. Only 2 of 21 chimeras
developed with situs inversus, which is highly statistically different (P<0.01, chi-square) to the expected
10:11 ratio if iv/iv cells were dominant over +/+•
Similarly, + / + cells are not dominant. The two
embryos that had inverted situs contained as much as
50% + / + cells in some tissues, and about 30% in the
heart. Thus, to answer our original question: the
presence of a small proportion of non-mutant tissue is
not sufficient to ensure normal development.
The number of informative chimeras produced in this
study was small, despite aggregating more than 500
iv/iv with + / + embryos. This is partially because of the
poor developmental potential of Si/Col iv/iv embryos,
as we have previously described (Brown et al. 1989). In
addition, these iv/iv embryos seem to be at a
disadvantage in aggregation chimeras with MF1 + / +
embryos. It does not seem profitable to attempt to
increase the numbers with further experiments of the
same design. Rather, we intend to examine further
chimeras using a cell-based marker, on histological
preparations, rather than the whole-tissue based
marker used here. In this way, we will gain information
of the spatial distribution of chimerism within tissues, in
relation to the development of asymmetry.
One interesting observation can be made from our
current data; the embryos with abnormal development
of situs, in particular heart looping, were those with a
high proportion of iv/iv cells in the heart, itself, rather
than in the surrounding tissue. This supports the
suggestion (Stalsberg, 1970) that the control of the
direction of heart looping is intrinsic to the heart. In
addition, it was only those embryos that had high iv/iv
contribution to both heart and visceral yolk-sac that
developed abnormally. It may not be coincidence that
the early paired cardiac primordia develop in very close
proximity to the visceral yolk-sac endoderm. Again,
cell-based information on chimerism may allow some
conclusions on the relative importance of endoderm
and mesoderm components of the heart and visceral
yolk sac.
We thank the Biological Services Division of the National
Institute for Medical Research for breeding the SI/Col mice.
This work was supported, in part, by the Wellcome Trust, to
whom we are grateful.
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{Accepted 21 August 1990)