We are studying the development of handedness, in particular the relationships between handed structures with bilateral symmetry, for example the limbs, and those with lateral asymmetry, such as the heart, lungs and gut. Asymmetric (unilateral) developmental limb abnormalities can be induced by chemical treatment of mouse embryos, either in utero by acetazolamide, or in culture by misonidazole. We have examined these effects in mice homozygous for the iv gene. The development of bilateral symmetry in iv/iv mice is normal, but the control of asymmetry appears to be random, that is 50 % develop normally (situs solitus), 50 % with laterally inverted viscera (situs inversus). We find that the handedness of induced asymmetric limb defects is highly correlated with embryonic visceral situs. Right limb defects are induced in situs solitus embryos, left-sided defects in situs inversus. This suggests that the mechanism of induction of asymmetric defects is not related to any intrinsic difference between the development of left and right limbs, but is connected to visceral asymmetry. In addition, the high correlation of limb defects with situs was observed in culture as well as in utero suggesting that the maternal environment plays no role in the development of asymmetry.

There is a sharp distinction between the handedness that arises in vertebrates from bilateral symmetry, and that in which structures develop consistently on one side, but not the other. The former handedness is related to mirror symmetry, and the latter to asymmetry. Also, the former does not involve any intrinsic difference in morphology between the two sides, whereas the latter does (Brown and Wolpert, 1989).

Thus, it is possible that the development of a left and right limb does not depend on any difference in morphogenesis between the two sides, but is simply due to their developing in mirror symmetry, the plane of symmetry being the midline of the embryo. By contrast, the development of asymmetrical visceral organs like the heart, lung, liver and gut, do not arise from mirror symmetry but must be due to some intrinsic handedness of the embryo. The consistent development, for example, of the heart on the left side thus requires there to be a mechanism by which the embryo can tell its left from its right side, and interpret this positional information in morphogenesis. Nothing is known of this mechanism.

The effect of the carbonic anhydrase inhibitor, acetazolamide, on limb development in mice provides an intriguing possibility for investigating the relationship between symmetrical and asymmetrical handedness. Under appropriate conditions (Layton and Hallesy, 1965; Wilson et al. 1968), acetazolamide causes unilateral forelimb abnormalities on the right side of treated embryos: an asymmetrical defect imposed on supposedly symmetrical structures. A similar unilateral effect can be induced in rat embryos in culture by nitroheterocyclic chemicals (Greenaway et al. 1986; Coakley and Brown, 1986). This raises the possibility that there may actually be intrinsic differences in the development of left and right limbs.

We have been able to investigate the relationship between these handed abnormalities and asymmetrical development by using mice that carry the iv gene. Homozygous iv/iv mice develop as if the embryonic mechanism for distinguishing left from right has been lost. Handedness is thus randomly determined, and 50% of the mice have normal visceral situs (situs solitus), 50% show situs inversus (Hummel and Chapman, 1959; Layton, 1976). Limb development in these mutant mice is normal.

We have studied the relationship between the handedness of visceral organs and the induction of asymmetric limb abnormalities. We were particularly interested to find out if the drug-induced effects were due to an intrinsic difference between left and right limbs, or were related to handedness of the viscera.

Mice

SI/Col mice were obtained from the Jackson Laboratory, Bar Harbour, USA and the colony is maintained by the Animal Services Division, National Institute of Medical Research, Mill Hill. SI/Col is inbred for more than 32 generations and is homozygous for the situs inversus gene (iv/iv). Genetic screening carried out at the MRC Laboratories, Carshalton show that this strain is homozygous at 8 marker loci and is a unique strain.

Outbred MFI (+/+) mice were from the laboratory colony. Relatively few MFI animals were used in the acetazolamide portion of this study because previous work (N. A. Brown, unpublished) had shown that the effect of acetazolamide on limb development in this strain was very similar to the large body of published data.

Acetazolamide treatment in vivo

Mice were housed together overnight and examined for copulatory plugs the following morning, which is termed pregnancy day 1. On day 10, animals were randomly assigned to control and treatment groups (without knowledge of situs, in the case of the SI mice). Mice received two doses of acetazolamide, at 09.00 and 17.00, by intraperitoneal injection. Acetazolamide was dissolved in 0.2% carboxymethyl cellulose at 100 mg ml-1, controls receiving vehicle alone.

At 10.00 on day 19, mice were weighed and killed by cervical dislocation. Uteri were removed and the nature and location of implantation sites were recorded. The visceral situs of mothers was examined. Live and dead fetuses were weighed, examined for external malformations, and for visceral situs and malformation by the method of Staples (1974). All fetuses were fixed, calcified bone was stained with Alizarin Red S, and soft tissue cleared (Staples and Schnell, 1964). Skeletons were examined to confirm the descriptions of limb and digit abnormalities.

Misonidazole treatment in vitro

Embryos were explanted during the morning of day 9, at the 4- to 8-somite stage. They were cultured for 48h in rolling bottles in a medium of 50% immediately-centrifuge heat-inactivated rat serum, 50% Eagle’s Minimal Essential Medium with Earle’s salts, by methods previously described (Freeman et al. 1987). The gas phase of the bottles was 5 % O2 (5% CO2, balance N2) 0-24h; 20% O2 24-32 h; 40% O2 32-48h. Treated embryos were exposed to 0·3m?-misonidazole throughout culture, controls to the vehicle alone, DMSO, at 0.1% v/v.

At the end of culture, yolk-sac diameter; crown-rump length and head length were measured and the morphology of the embryo examined. Situs was determined by: coiling of the caudal trunk (to the right of the head in situs solitus), looping of the heart (bulging to the left in situs solitus) and side of exit of the vitelline vessels (left in situs solitus). There were no heterotaxic day 11 iv/iv embryos in this study, although we have observed several in other investigations (N. A. Brown, unpublished).

Data calculated on a ‘per-litter’ basis were compared using the nonparametric Mann-Whitney ‘U’ test, other values are compared using chi-square analysis, in all cases using the ‘Minitab’ package.

Acetazolamide treatment in vivo

The SI/Col (iv/iv) mice have a poor reproductive performance. In the control SI mice in this study, only 58 % of those that had copulatory plugs were pregnant (i.e. had at least one live fetus) on day 19 (Table 1). There was a mean of 10 implantations per litter, 39 % of which were resorptions on day 19.

Table 1.

Fetal parameters on day 19 of pregnancy in +/+ and iv/iv mice after treatment with acetazolamide on day 10

Fetal parameters on day 19 of pregnancy in +/+ and iv/iv mice after treatment with acetazolamide on day 10
Fetal parameters on day 19 of pregnancy in +/+ and iv/iv mice after treatment with acetazolamide on day 10

The embryonic development of iv/iv mice was more sensitive than that of +/+ animals to the effects of acetazolamide treatment (Table 1). There was no significant increase in embryonic/fetal death following acetazolamide treatment in +/+ mice, nor any effect on the percentage of mothers with live fetuses. At the lower doses of 700 and 800 mg kg-1 the proportion of iv/iv mothers with live fetuses (66% and 64%) was similar to controls, but reduced to 13 % and 23 % at 900 and 1000 mg kg-1. There were increases in the incidence of embryo/fetal death in iv/iv animals at all doses. Embryo/fetal growth was also more markedly affected in iv/iv mice, reflected in reduced mean fetal weights.

Limb development in +/+ animals was affected in all acetazolamide-treated groups (Table 1). There was no obvious dose-response relationship, but there were few litters in each group. In all but one fetus, abnormalities were of the forelimb only and all were ectrodactly (missing digit) malformations. The most common defect (66 % of all limb defects) was missing 5th digit (Fig. 1). The most severe (11% of all defects) was missing 3rd 4th and 5th digits and missing ulna (Fig. 1).

Fig. 1.

Alizarin Red S stained forelimbs of Sl/Col (iv/iv) day 19 fetuses. A and D, control, B, C, E and F exposed to acetazolamide on day 10. A, B and C right limbs from situs solitus fetus, D, E and F left limbs from situs inversus fetuses. Note missing digit 5 in B and E, and missing digits, 3, 4 and 5 and missing ulna in C and F.

Fig. 1.

Alizarin Red S stained forelimbs of Sl/Col (iv/iv) day 19 fetuses. A and D, control, B, C, E and F exposed to acetazolamide on day 10. A, B and C right limbs from situs solitus fetus, D, E and F left limbs from situs inversus fetuses. Note missing digit 5 in B and E, and missing digits, 3, 4 and 5 and missing ulna in C and F.

These defects were predominantly of the right forelimb (Table 2). Pooling data across all treatment groups, 87 % of affected fetuses had limb defects either on the right side only, or clearly more severe on the right side. In 8% of fetuses, defects were similar on both sides, and in 6% the left side was more affected. Thus, out of 52 affected fetuses 37 had exclusively right forelimb defects, only one had exclusively left forelimb defects. All +/+ mothers and fetuses examined had normal visceral situs.

Table 2.

Sidedness of limb malformations in day 19 +/+ fetuses following acetazolamide treatment on day 10

Sidedness of limb malformations in day 19 +/+ fetuses following acetazolamide treatment on day 10
Sidedness of limb malformations in day 19 +/+ fetuses following acetazolamide treatment on day 10

Limb development of iv/iv embryos was also affected by acetazolamide treatment, with 80 % of fetuses affected at the higher doses, although few survived to term (Table 1). The types and incidences of defects were very similar to those of the +/+ fetuses, predominantly missing digit 5, but more severe ectrodactyly was also observed (Fig. 1).

The sidedness of the limb defects was highly correlated with the visceral situs of the fetus (Table 3). Of 28 situs solitus fetuses with limb defects, 23 had exclusively right forelimb defects, none had left-sided effects. Thus, the right side was more affected in 93 % of situs solitus fetuses, with equal effects on both sides in 7 % of fetuses (Table 3). This distribution (fetuses with right/ even/left defects=26/2/0) is not significantly different to that in +/+ fetuses (45/4/3, P>0.05, chi-squared= 1.7).

Table 3.

Sidedness of limb malformations in day 19 iv/iv fetuses following acetazolamide treatment on day 10

Sidedness of limb malformations in day 19 iv/iv fetuses following acetazolamide treatment on day 10
Sidedness of limb malformations in day 19 iv/iv fetuses following acetazolamide treatment on day 10

In contrast, the left side was predominantly affected in situs inversus iv/iv fetuses. Of 25 affected fetuses 13 had exclusively left forelimb defects, 2 had only right-sided effects. Overall, the left side was more affected in 76% of fetuses, 8% equal and 16% right sided. This distribution (4/2/19) is highly significantly different from that of situs solitus iv/iv fetuses (P<0.01, chi-squared =35.1) and +/+ fetuses (P<0.01, chi-squared =42.4). If the distribution is inverted (19/2/4), to express the sidedness of defects concordant with fetal situs, this is not statistically different from either of the other groups (P>0.05, chi-squared 4.9 and 2.2).

Some aspects of the visceral situs of iv/iv mothers and fetuses are shown in Table 4. Of the 91 mothers in the study, 8 were not examined fully (3 situs solitus, 5 situs inversus) and these are not included in the table. All 230 live iv/iv fetuses examined in the study are included, regardless of treatment group. The overall incidence of abnormality includes any aberration of visceral situs in relation to the general situs of the fetus or mother.

Table 4.

Visceral situs of 83 iv/iv mothers and 230 iv/iv fetusesa

Visceral situs of 83 iv/iv mothers and 230 iv/iv fetusesa
Visceral situs of 83 iv/iv mothers and 230 iv/iv fetusesa

The ratio of situs solitus to situs inversus was close to 50:50 for both mothers and fetuses (49:51 and 50:48 [with 3% heterotaxic, ie mixed, for example: situs solitus thoracic, situs inversus abdominal], respectively). The overall incidence of abnormality was higher in fetuses (46%) than mothers (28%). In both cases, the incidence of abnormality was not significantly different in situs solitus and situs inversus animals (14/41 vs 9/42, P>0.05, chi-squared=1.68, and 44/114 vs 56/110, P>0.05, chi-squared=3.4). The most common abnormality was a ‘missing’ liver lobe, ie a single lobe on the right side in situs solitus, or on the left in situs inversus. This was associated with an abnormal path of the inferior vena cava in all cases. The incidence of this defect was similar in mothers and fetuses, and in normal and inverted situs.

There were abnormalities of lung lobation in about 10% of iv/iv fetuses. Normal +/+ mice have three lung lobes on the right, a caudate lobe which originates from the right, and single lobe on the left. Some iv/iv animals have ‘two right sides’ with either 3/2/3 or 3/1/3 lobes, others have ‘two left sides’: either 1/0/1 or 1/1/1. The incidences of these two phenotypes in iv/iv fetuses in this study were approximately equal, as were the incidences in situs solitus and situs inversus (Table 4). In mothers the incidence was about half that in fetuses, with one of each phenotype in situs solitus and situs inversus animals. There was a wide variety of other abnormalities of situs in iv/iv fetuses (data not shown).

Misonidazole treatment in vitro

The results of culturing day 9 +/+ and iv/iv embryos for 48 h in the presence and absence of misonidazole are summarized in Table 5. The first 24 h of these cultures were performed in a gas phase of 5% O2, which we found in our previous studies with rat embryos (Coakley and Brown, 1986) to be necessary to induce high incidences of unilateral limb-bud defects. These conditions were not ideal for the mouse embryos used in this study (Table 5), but sufficient embryos grew normally to allow a comparison of situs and limb-bud abnormality.

Table 5.

Sidedness of limb-bud defects in +/ + and iv/iv embryos after 48 hours culture from day 9, with and without exposure to 0.3 m? misonidazole

Sidedness of limb-bud defects in +/ + and iv/iv embryos after 48 hours culture from day 9, with and without exposure to 0.3 m? misonidazole
Sidedness of limb-bud defects in +/ + and iv/iv embryos after 48 hours culture from day 9, with and without exposure to 0.3 m? misonidazole

In all embryos with limb-bud abnormality (73 in total) except one, the side of the defect was concordant with the situs of the embryo: right in solitus; left in inversus. This was true for both misonidazole-treated and control embryos. The exception was a single untreated iv/iv embryo of normal situs which had a very slight reduction of the left limb bud. The defects observed in untreated embryos are most likely a reflection of the low oxygen concentration in the culture environment, as we have observed in rat embryos (Coakley and Brown, 1986).

There are some other potentially interesting features of the data in Table 5, but the small numbers of embryos in individual groups, and the potential complication of the suboptimal culture conditions prevent firm conclusions. For example, it appears that situs inversus iv/iv embryos have a higher incidence of limb defects, but also grow better in culture, than situs solitus embryos of the same genotype. Overall, however, the Sl/Col embryos were less sensitive than MFI to the effects of both culture and treatment.

The most important feature to emerge from these results is the very high correlation between the handedness of the induced limb abnormalities and the handedness of the viscera. This clearly suggests that whatever the mechanism for the asymmetry of acetazolamide and misonidazole actions, it is not related to any intrinsic difference between left and right limb development, but rather to the disposition of the visceral organs in the animal. Thus, it is not just the position of the limb on the left or right side.

The same conclusions are reached from effects induced in utero or in culture. This clearly shows that the maternal environment plays no role in determining the effect.

Our results do not illuminate the mechanism of induction of unilateral limb defects, but the most likely explanation lies in the vascular supply to the limbs. It is thought that the asymmetric development of the cardiovascular system leads to subtle differences in the vessels serving the left and right limbs. We have shown, indirectly, that oxygen tension is lower in the right limb than the left (Coakley and Brown, 1986), and that this is reversed in situs inversus embryos (Brown, unpublished).

The data on visceral handedness in iv/iv mutant mice confirm and extend the previous observations of Chapman and Hummel (1959) and Layton (1976). It is noteworthy that the phenotypic effects of the mutation have remained very stable over 30 years, and independent of genetic background. Of particular note is the almost exact 50:50 proportion of situs solitus and inversus. The high incidence of visceral abnormalities, which is similar regardless of situs, suggests that the mutation results in more than simple loss of left/right identity. For lung lobation, the presence ‘double rightsided’ and ‘double left-sided’ fetuses, each present at a similar frequency, regardless of situs, is also intriguing. The significance of these features, in relation to a model for handedness, is discussed elsewhere (Brown and Wolpert, 1989).

The authors thank Dr Colin Hetherington and Brian Edmonds for maintaining the SI/Col colony, John Larsson and Ian Yendall for the figure, Lynn Dean for preparing the manuscript, and the Wellcome Trust for support.

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