The development of asymmetry: the sidedness of drug-induced limb abnormalities is reversed in situs inversus mice

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.     

The development of handed asymmetry in aggregation chimeras of situs inversus mutant and wild-type mouse embryo

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.     

Reversed bodies, reversed brains, and (some) reversed behaviors: of zebrafish and men

Although zebrafish with situs inversus show complete reversal of normal visceral and cerebral asymmetries, they show left-right reversal of only some behaviors, with others continuing to show species-typical lateralization. The implication is that, as in humans, there are at least two independent mechanisms for generating asymmetry.     

Adrenergic neurotransmitters and calcium ionophore-induced situs inversus viscerum in Xenopus laevis embryos

Xenopus laevis embryos at the blastula–early tail bud stage were exposed to norepinephrine or octopamine dissolved in culture saline until they reached the larval stage. The left–right asymmetry of the heart and gut was then examined. We found that these adrenergic neurotransmitters induced situs inversus in the heart and/or gut in up to 35% of tested neurula embryos. Norepinephrine-induced situs inversus was blocked by the α-1 adrenergic antagonist prazosin. Furthermore, A23187, a calcium ionophore, also increased the incidence of situs inversus up to 54% when late-neurula embryos were exposed to the solution. A23187 treatment initiated before neural groove formation was less effective. The incidence of situs inversus induced by these reagents decreased towards the control level (2.2%, 25 untreated embryos out of 1127 embryos in total) in embryos past the stage of neural tube closure. In the present experiments we obtained 22 gut-only situs inversus embryos having an inverted gut and a normal heart. In contrast, such embryos were not observed among the 1127 untreated embryos. An adrenergic signal mediated by an increase in intracellular free calcium may be involved in the asymmetrical visceral morphogenesis of Xenopus embryos.     

Molecular basis of left-right asymmetry

In vertebrates visceral asymmetry is conserved along the left-right axis within the body. Only a small percentage of randomization (situs ambiguus), or complete reversal (situs inversus) of normal internal organ position and structural asymmetry is found in humans. A breakdown in left-right asymmetry is occasionally associated with severe malformations of the organs, clearly indicating that the regulated asymmetric patterning could have an evolutionary advantage over allowing random placement of visceral organs. Genetic, molecular and cell transplantation experiments in humans, mice, zebrafish, chick and Xenopus have advanced our understanding of how initiation and establishment of left-right asymmetry occurs in the vertebrate embryo. In particular, the chick embryo has served as an extraordinary animal model to manipulate genes, cells and tissues. This chick model system has enabled us to reveal the genetic pathways that occur during left-right development. Indeed, genes with asymmetric expression domains have been identified and well characterized using the chick as a model system. The present review summarizes the molecular and experimental studies employed to gain a better understanding of left-right asymmetry pattern formation from the first split of symmetry in embryos, to the exhibition of asymmetric morphologies in organs.     

Duplication/deficiency mapping of situs inversus viscerum (iv), a gene that determines left-right asymmetry in the mouse.

A recessive mutation in the mouse, situs inversus viscerum (iv), results in randomization of organ position along the left-right body axis: approximately 50% of the progeny of homozygous matings exhibit situs solitus and 50% exhibit situs inversus. Recent studies have established genetic linkage between iv and the immunoglobulin heavy chain gene complex (Igh-C), located on distal mouse chromosome 12. In the present study, we have refined the genetic map location of iv relative to the breakpoint of a reciprocal translocation, T(5;12)31H, involving the telomeric region of chromosome 12 distal to Igh-C and the proximal region of chromosome 5. The translocation results in a large 12(5) derivative chromosome and a small 5(12) derivative chromosome. Because mice with either monosomy or tertiary trisomy for the 5(12) chromosomal region are viable, duplication/deficiency mapping is possible. Deficiency mapping was performed by mating iv/iv homozygotes and T31H heterozygotes. Two animals monosomic for distal mouse chromosome 12 were produced. One of the animals with cytogenetically confirmed monosomy for distal chromosome 12 exhibited situs inversus, indicating that the iv mutation is located at or distal to the T31H breakpoint. For duplication analysis, matings were initially carried out between iv/iv homozygotes and unbalanced T31H animals trisomic for distal chromosome 12. Cytogenetically verified tertiary trisomic progeny were identified and backcrossed with iv/iv homozygotes. The resulting trisomic progeny, 50% of which are expected to carry the iv mutation on both cytogenetically normal copies of chromosome 12, were scored for phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Congenital heart disease and the specification of left-right asymmetry

Complex congenital heart disease (CHD) is often seen in conjunction with heterotaxy, the randomization of left-right visceral organ situs. However, the link between cardiovascular morphogenesis and left-right patterning is not well understood. To elucidate the role of left-right patterning in cardiovascular development, we examined situs anomalies and CHD in mice with a loss of function allele of Dnaic1, a dynein protein required for motile cilia function and left-right patterning. Dnaic1 mutants were found to have nodal cilia required for left-right patterning, but they were immotile. Half the mutants had concordant organ situs comprising situs solitus or mirror symmetric situs inversus. The remaining half had randomized organ situs or heterotaxy. Looping of the heart tube, the first anatomical lateralization, showed abnormal L-loop bias rather than the expected D-loop orientation in heterotaxy and nonheterotaxy mutants. Situs solitus/inversus mutants were viable with mild or no defects consisting of azygos continuation and/or ventricular septal defects, whereas all heterotaxy mutants had complex CHD. In heterotaxy mutants, but not situs solitus/inversus mutants, the morphological left ventricle was thin and often associated with a hypoplastic transverse aortic arch. Thus, in conclusion, Dnaic1 mutants can achieve situs solitus or inversus even with immotile nodal cilia. However, the finding of abnormal L-loop bias in heterotaxy and nonheterotaxy mutants would suggest motile cilia are required for normal heart looping. Based on these findings, we propose motile nodal cilia patterns heart looping but heart and visceral organ lateralization is driven by signaling not requiring nodal cilia motility.     

Morphogenesis in an asymmetric medium

Some key experiments of artificial production ofsitus inversus viscerum are briefly reviewed and a two-step mechanism for the explanation of the systematic asymmetric visceral arrangement in vertebrates is proposed. A two-variable reaction-diffusion system displaying a symmetry-breaking bifurcation is considered, and it is demonstrated that a slight asymmetry of the boundary conditions can give rise to a marked asymmetry in the resulting dissipative structure in both one-and three-dimensional systems. A criterion is formulated allowing classification of reaction-diffusion systems operating in a three-dimensional space with regard to their ability to incorporate slight asymmetries at the boundaries in the form of a chiral dissipative structure.     

Asymmetry Revisited

Interest in the developmental basis of symmetry and asymmetry,as old as experimental embryology itself, has recently becomereactivated. A brief survey is made of recent or current activitiesin the following areas: (i) development of asymmetry in thechick limb bud, as related to the so-called zone of polarizingactivity (Saunders et al.); (ii) situs inversus viscerum inamphibians, as induced by transplantation and defect experiments,radiation, and chemical treatment (von Woellwarth, von Kraft,Wehrmaker); (iii) situs inversus viscerum in mammals as relatedto (a) twinning (Cockayne, Torgerson, Baker-Cohen); (b) genetics(Feldman, Cockayne, Torgerson for man, Tihen et al., Hummeland Chapman for mouse); and (c) teratogens (Layton and associates);(iv) ultrastructure in snails (Morrill and Perkins); (v) asymmetrydeveloping as a result of changes in chemical instability inhomogeneous systems (Ortoleva and Ross); (vi) asymmetrical mentality(Sperry and associates). No general conclusions are attempted.     

Right isomerism of the brain in inversus viscerum mutant mice

Left-right (L-R) asymmetry is a fundamental feature of higher-order neural function. However, the molecular basis of brain asymmetry remains unclear. We recently reported L-R asymmetry of hippocampal circuitry caused by differential allocation of N-methyl-D-aspartate receptor (NMDAR) subunit GluRepsilon2 (NR2B) in hippocampal synapses. Using electrophysiology and immunocytochemistry, here we analyzed the hippocampal circuitry of the inversus viscerum (iv) mouse that has a randomized laterality of internal organs. The iv mouse hippocampus lacks L-R asymmetry, it exhibits right isomerism in the synaptic distribution of the epsilon2 subunit, irrespective of the laterality of visceral organs. This independent right isomerism of the hippocampus is the first evidence that a distinct mechanism downstream of the iv mutation generates brain asymmetry.     

DE-Cadherin regulates unconventional Myosin ID and Myosin IC in Drosophila left-right asymmetry establishment

In bilateria, positioning and looping of visceral organs requires proper left-right (L/R) asymmetry establishment. Recent work in Drosophila has identified a novel situs inversus gene encoding the unconventional type ID myosin (MyoID). In myoID mutant flies, the L/R axis is inverted, causing reversed looping of organs, such as the gut, spermiduct and genitalia. We have previously shown that MyoID interacts physically with β-Catenin, suggesting a role of the adherens junction in Drosophila L/R asymmetry. Here, we show that DE-Cadherin co-immunoprecipitates with MyoID and is required for MyoID L/R activity. We further demonstrate that MyoIC, a closely related unconventional type I myosin, can antagonize MyoID L/R activity by preventing its binding to adherens junction components, both in vitro and in vivo. Interestingly, DE-Cadherin inhibits MyoIC, providing a protective mechanism to MyoID function. Conditional genetic experiments indicate that DE-Cadherin, MyoIC and MyoID show temporal synchronicity for their function in L/R asymmetry. These data suggest that following MyoID recruitment by β-Catenin at the adherens junction, DE-Cadherin has a twofold effect on Drosophila L/R asymmetry by promoting MyoID activity and repressing that of MyoIC. Interestingly, the product of the vertebrate situs inversus gene inversin also physically interacts with β-Catenin, suggesting that the adherens junction might serve as a conserved platform for determinants to establish L/R asymmetry both in vertebrates and invertebrates.     

Dextrocardia with situs inversus totalis: Cardiovascular surgery in three patients with concomitant coronary artery disease

Three patients with situs inversus totalis (mirror-image dextrocardia) and concomitant coronary artery disease were admitted to our institution for evaluation. In all cases, aortocoronary bypass grafting was successful. Patients with situs inversus and mirror-image dextrocardia are believed to have normal longevity, and, as these studies suggest, they have the same long-term prognosis after coronary bypass grafting as patients with situs solitus.     

Axonemal dynein intermediate-chain gene (DNAI1) mutations result in situs inversus and primary ciliary dyskinesia (Kartagener syndrome)

Kartagener syndrome (KS) is a trilogy of symptoms (nasal polyps, bronchiectasis, and situs inversus totalis) that is associated with ultrastructural anomalies of cilia of epithelial cells covering the upper and lower respiratory tracts and spermatozoa flagellae. The axonemal dynein intermediate-chain gene 1 (DNAI1), which has been demonstrated to be responsible for a case of primary ciliary dyskinesia (PCD) without situs inversus, was screened for mutation in a series of 34 patients with KS. We identified compound heterozygous DNAI1 gene defects in three independent patients and in two of their siblings who presented with PCD and situs solitus (i.e., normal position of inner organs). Strikingly, these five patients share one mutant allele (splice defect), which is identical to one of the mutant DNAI1 alleles found in the patient with PCD, reported elsewhere. Finally, this study demonstrates a link between ciliary function and situs determination, since compound mutation heterozygosity in DNAI1 results in PCD with situs solitus or situs inversus (KS).     

Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis

Cilia, as motile and sensory organelles, have been implicated in normal development, as well as diseases including cystic kidney disease, hydrocephalus and situs inversus. In kidney epithelia, cilia are proposed to be non-motile sensory organelles, while in the mouse node, two cilia populations, motile and non-motile have been proposed to regulate situs. We show that cilia in the zebrafish larval kidney, the spinal cord and Kupffer's vesicle are motile, suggesting that fluid flow is a common feature of each of these organs. Disruption of cilia structure or motility resulted in pronephric cyst formation, hydrocephalus and left-right asymmetry defects. The data show that loss of fluid flow leads to fluid accumulation, which can account for organ distension pathologies in the kidney and brain. In Kupffer's vesicle, loss of flow is associated with loss of left-right patterning, indicating that the 'nodal flow' mechanism of generating situs is conserved in non-mammalian vertebrates.     

Kartagener's syndrome and the syndrome of immotile cilia

Summary Kartagener's syndrome (KS) is a hereditary disease with typical symptoms of situs inversus, bronchiectasis, and chronic infections of the nasal mucosa. Autosomal recessive inheritance cannot be doubted on account of repeated observations of affected sibs and parental cansanguinity. The bronchopulmonary symptoms in sibs, however, cannot be explained by this mode of inheritance.Recent clinical findings and electron microscope investigations suggest that KS is a special form of manifestation within the immotile cilia syndrome. This disease combines the typical bronchial and nasal symptoms of KS with sterility in the male due to immotile sperm tails and, as a facultative symptom, situs inversus. Thus, sibs with bronchiectasis but without situs inversus are also classified under this syndrome. The symptoms mentioned are caused by an abnormal morphology of bronchial cilia and sperm tails, which can be demonstrated by electron microscopy. The dynein arms normally attached to the nine microtubular doublets and providing a normal ciliary movement are lacking.It is assumed that during early embryonic life ciliary beats in the growing embryo determine the type of laterality. When ciliary movements are absent laterality may develop fortuitously, thus effecting a situs inversus in about half the affected cases. The numerical evaluation of pedigrees from the literature supports this assumption.     

Abnormal nodal flow precedes situs inversus in iv and inv mice.

We examined the nodal flow of well-characterized mouse mutants, inversus viscerum (iv) and inversion of embryonic turning (inv), and found that their laterality defects are always accompanied by an abnormality in nodal flow. In a randomized laterality mutant, iv, the nodal cilia were immotile and the nodal flow was absent. In a situs inversus mutant, inv, the nodal cilia was motile but could only produce very weak leftward nodal flow. These results consistently support our hypothesis that the nodal flow produces the gradient of putative morphogen and triggers the first L-R determination event.     

Handedness and situs inversus in primary ciliary dyskinesia

...The limbs on the right side are stronger. [The] cause may be ... [that] ... motion, and abilities of moving, are somewhat holpen from the liver, which lieth on the right side. (Sir Francis Bacon, Sylva sylvarum (1627).)Fifty per cent of people with primary ciliary dyskinesia (PCD) (also known as immotile cilia syndrome or Siewert-Kartagener syndrome) have situs inversus, which is thought to result from absent nodal ciliary rotation and failure of normal symmetry breaking. In a study of 88 people with PCD, only 15.2% of 46 individuals with situs inversus, and 14.3% of 42 individuals with situs solitus, were left handed. Because cerebral lateralization is therefore still present, the nodal cilia cannot be the primary mechanism responsible for symmetry breaking in the vertebrate body. Intriguingly, one behavioural lateralization, wearing a wrist-watch on the right wrist, did correlate with situs inversus.     

Left-right asymmetry defect in the hippocampal circuitry impairs spatial learning and working memory in iv mice

Although left-right (L-R) asymmetry is a fundamental feature of higher-order brain function, little is known about how asymmetry defects of the brain affect animal behavior. Previously, we identified structural and functional asymmetries in the circuitry of the mouse hippocampus resulting from the asymmetrical distribution of NMDA receptor GluR ε2 (NR2B) subunits. We further examined the ε2 asymmetry in the inversus viscerum (iv) mouse, which has randomized laterality of internal organs, and found that the iv mouse hippocampus exhibits right isomerism (bilateral right-sidedness) in the synaptic distribution of the ε2 subunit, irrespective of the laterality of visceral organs. To investigate the effects of hippocampal laterality defects on higher-order brain functions, we examined the capacity of reference and working memories of iv mice using a dry maze and a delayed nonmatching-to-position (DNMTP) task, respectively. The iv mice improved dry maze performance more slowly than control mice during acquisition, whereas the asymptotic level of performance was similar between the two groups. In the DNMTP task, the iv mice showed poorer accuracy than control mice as the retention interval became longer. These results suggest that the L-R asymmetry of hippocampal circuitry is critical for the acquisition of reference memory and the retention of working memory.