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.     

Random determination of a developmental process: reversal of normal visceral asymmetry in the mouse.

Situs inversus viscerum in the mouse has been shown to be inherited as an autosomal recessive trait (gene symbol iv) with reduced penetrance. It is hypothesized that the normal allele at the iv locus exhibits complete dominance and controls normal visceral asymmetry. Absence of this control allows the situs of visceral asymmetry to be determined in a random fashion. This hypothesis also appears to apply to the inheritance of situs inversus in man and to the experimental production of situs inversus.     

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.     

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.     

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.     

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.     

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.     

Critical period of rat development when sidedness of asymmetric body structures is determined

We recently reported that rat embryos cultured from the presomite stage in a medium containing the alpha-1 adrenergic agonist, phenylephrine, have a high incidence of situs inversus. In the present study, we have determined more precisely the critical period of development when situs inversus is induced. Rat embryos were harvested at 8 AM on Day 9 of gestation (plug day = Day 0), and divided into different stages of development, namely, early, mid, and late primitive streak stages and early, mid, and late neural plate stages. They then were cultured in rotating bottles to which phenylephrine, 0.5 mM, was added for various durations. After 49 hr of culture, embryos were examined for general morphology including sidedness of the bulboventricular loop, tail, and chorioallantoic placenta. Phenylephrine increased the incidence of situs inversus above control when administered throughout culture from either the early neural plate stage or before, and when administered for 4 hr or more from the early neural plate stage. This increase was significant even at the mid and late primitive streak stages when the control incidence was high. Our results suggest that sidedness of asymmetric body structures is determined during the early neural plate stage. This period is well before the 6-8-somite stage when morphological signs of body asymmetry first appear.     

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).     

Handedness of a Motor Program in C. elegans Is Independent of Left-Right Body Asymmetry

Complex animals display bilaterally asymmetric motor behavior, or “motor handedness,” often revealed by preferential use of limbs on one side. For example, use of right limbs is dominant in a strong majority of humans. While the mechanisms that establish bilateral asymmetry in motor function are unknown in humans, they appear to be distinct from those for other handedness asymmetries, including bilateral visceral organ asymmetry, brain laterality, and ocular dominance. We report here that a simple, genetically homogeneous animal comprised of only ∼1000 somatic cells, the nematode C. elegans, also shows a distinct motor handedness preference: on a population basis, males show a pronounced right-hand turning bias during mating. The handedness bias persists through much of adult lifespan, suggesting that, as in more complex animals, it is an intrinsic trait of each individual, which can differ from the population mean. Our observations imply that the laterality of motor handedness preference in C. elegans is driven by epigenetic factors rather than by genetic variation. The preference for right-hand turns is also seen in animals with mirror-reversed anatomical handedness and is not attributable to stochastic asymmetric loss of male sensory rays that occurs by programmed cell death. As with C. elegans, we also observed a substantial handedness bias, though not necessarily the same preference in direction, in several gonochoristic Caenorhabditis species. These findings indicate that the independence of bilaterally asymmetric motor dominance from overall anatomical asymmetry, and a population-level tendency away from ambidexterity, occur even in simple invertebrates, suggesting that these may be common features of bilaterian metazoans.     

HANDEDNESS IN THE BROWN-THROATED PARAKEET ARATINGA PERTINAX EST RELATION WITH SKELETAL ASYMMETRY

Observations on 56 specimens of Aratinga pertinax when bringing food to the beak prove that 28 birds were right-handed while the other 28 were left-handed. A biometric analysis reveals a slight departure from bilateral symmetry in hindlimb bones in close relationship with handedness. In the right-handed birds, there is a significant predominance in the length of the right hindlimb as a whole and all right limb segments. The opposite holds true in left-handed parrots (except for the femur). Right-handed birds are in general more highly asymmetrical than left-handed ones. There is a positive correlation between asymmetry of homologous segments of a pair of limbs and the asymmetry of the other segments of the same pair of limbs. The absence of significant negative correlations between bilateral differences of the different segments of a limb indicates that departures from bilateral symmetry tend to affect the limb as a whole, and thus is not in agreement with the rule of compensating variations.     

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)     

Limb bone bilateral asymmetry: variability and commonality among modern humans

Humans demonstrate species-wide bilateral asymmetry in long bone dimensions. Previous studies have documented greater right-biases in upper limb bone dimensions--especially in length and diaphyseal breadth--as well as more asymmetry in the upper limb when compared with the lower limb. Some studies have reported left-bias in lower limb bone dimensions, which, combined with the contralateral asymmetry in upper limbs, has been termed "crossed symmetry." The examination of sexual dimorphism and population variation in asymmetry has been limited. This study re-examines these topics in a large, geographically and temporally diverse sample of 780 Holocene adult humans. Fourteen bilateral measures were taken, including maximum lengths, articular and peri-articular breadths, and diaphyseal breadths of the femur, tibia, humerus, and radius. Dimensions were converted into percentage directional (%DA) and absolute (%AA) asymmetries. Results reveal that average diaphyseal breadths in both the upper and lower limbs have the greatest absolute and directional asymmetry among all populations, with lower asymmetry evident in maximum lengths or articular dimensions. Upper limb bones demonstrate a systematic right-bias in all dimensions, while lower limb elements have biases closer to zero %DA, but with slight left-bias in diaphyseal breadths and femoral length. Crossed symmetry exists within individuals between similar dimensions of the upper and lower limbs. Females have more asymmetric and right-biased upper limb maximum lengths, while males have greater humeral diaphyseal and head breadth %DAs. The lower limb demonstrates little sexual dimorphism in asymmetry. Industrial groups exhibit relatively less asymmetry than pre-industrial humans and less dimorphism in asymmetry. A mixture of influences from both genetic and behavioral factors is implicated as the source of these patterns.     

Left-right lineage analysis of the embryonic Xenopus heart reveals a novel framework linking congenital cardiac defects and laterality disease

The significant morbidity and mortality associated with laterality disease almost always are attributed to complex congenital heart defects (CHDs), reflecting the extreme susceptibility of the developing heart to disturbances in the left-right (LR) body plan. To determine how LR positional information becomes ;translated' into anatomical asymmetry, left versus right side cardiomyocyte cell lineages were traced in normal and laterality defective embryos of the frog, Xenopus laevis. In normal embryos, myocytes in some regions of the heart were derived consistently from a unilateral lineage, whereas other regions were derived consistently from both left and right side lineages. However, in heterotaxic embryos experimentally induced by ectopic activation or attenuation of ALK4 signaling, hearts contained variable LR cell composition, not only compared with controls but also compared with hearts from other heterotaxic embryos. In most cases, LR cell lineage defects were associated with abnormal cardiac morphology and were preceded by abnormal Pitx2c expression in the lateral plate mesoderm. In situs inversus embryos there was a mirror image reversal in Pitx2c expression and LR lineage composition. Surprisingly, most of the embryos that failed to develop heterotaxy or situs inversus in response to misregulated ALK4 signaling nevertheless had altered Pitx2c expression, abnormal cardiomyocyte LR lineage composition and abnormal heart structure, demonstrating that cardiac laterality defects can occur even in instances of otherwise normal body situs. These results indicate that: (1) different regions of the heart contain distinct LR myocyte compositions; (2) LR cardiomyocyte lineages and Pitx2c expression are altered in laterality defective embryos; and (3) abnormal LR cardiac lineage composition frequently is associated with cardiac malformations. We propose that proper LR cell composition is necessary for normal morphogenesis, and that misallocated LR cell lineages may be causatively linked with CHDs that are present in heterotaxic individuals, as well as some 'isolated' CHDs that are found in individuals lacking overt features of laterality disease.     

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.     

Enthesis bilateral asymmetry in humans and African apes

Entheses (skeletal muscle and tendon attachment sites) have often been used to infer handedness and activity variability among human populations. However, the specific roles that intensity vs. frequency of muscle contractions play in modifying entheses are not well understood and the assumption that entheses reflect muscle activity levels has been challenged. This study explores the effect of habitual muscular activity on enthesis morphology in humans and African apes by investigating bilateral asymmetry in the forelimbs and hindlimbs of these taxa. Humans have generally more developed entheses in the lower limb while African apes have generally more developed entheses in the forelimbs. All species studied have more asymmetric forelimbs than hindlimbs except humans that show more asymmetrical expression of bony spurs in the lower limbs than in the upper limbs. When comparing species, humans are always more asymmetric in ethesis development than apes for both the forelimbs and hindlimbs, which reflects the relatively greater asymmetry in limb use in humans and the more symmetric use in apes. Enthesis development may reflect cross-symmetry patterns in humans and, more subtly, a moderate handedness in apes during manipulative activities. This study suggests that enthesis morphology provides information on muscle activity levels, with greater development of entheses associated with more habitual or powerful muscle use. The general similarity of ape and human responses to muscle activity suggests that muscle activity influenced enthesis development in Plio-Pleistocene hominins and that interpretation of muscle markings in these fossils can provide data for functional inferences in these extinct species.     

Evidence for an adrenergic mechanism in the control of body asymmetry

The effect of phenylephrine, an alpha-1 adrenergic agonist, on development of body asymmetry was studied using a rat whole embryo culture system. Embryos were explanted at the presomite stage, cultured in 100% rat serum containing various concentrations of phenylephrine, and examined at the 20-25 somite stage for sidedness of asymmetric body structures, namely, bulboventricular loop, allantoic placenta, and tail. Phenylephrine treatment resulted in a dose-dependent increase of situs inversus with a maximum incidence of 52%. Coadministration of prazosin, an alpha-1 adrenergic antagonist, almost completely prevented this effect. Our results suggest that receptor-mediated stimulation of the alpha-1 adrenergic pathway is involved in the control of normal body asymmetry in developing rat embryos.     

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.