Insights into the establishment of left-right asymmetries in vertebrates

The body-plan of vertebrates, while exteriorly essentially symmetric along its medio-lateral plane, displays numerous left-right differences in the disposition and placement of internal organs. Such left-right asymmetries, established during embryogenesis, are controlled by complex epigenetic and genetic cascades that impart laterality information to the different embryo structures and organ primordia. A key and evolutionarily conserved feature of these information cascades among vertebrate embryos is the left-sided transfer of information from the node to the lateral plate mesoderm during early somitogenesis stages. We review here recent evidence concerning the mechanisms that regulate the laterality of such transfer. Furthermore, we propose a model of left-right axis specification that underscores the role of the node as an integrator of laterality information and the evolutionary conservation of the mechanisms that convey such information to and from the node.     

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.     

Left-right asymmetry in the vertebrate embryo: from early information to higher-level integration

Although vertebrates seem to be essentially bilaterally symmetrical on the exterior, there are numerous interior left-right asymmetries in the disposition and placement of internal organs. These asymmetries are established during embryogenesis by complex epigenetic and genetic cascades. Recent studies in a range of model organisms have made important progress in understanding how this laterality information is generated and conveyed to large regions of the embryo. Both commonalities and divergences are emerging in the mechanisms that different vertebrates use in left-right axis specification. Recent evidence also provides intriguing links between the establishment of left-right asymmetries and the symmetrical elongation of the anterior-posterior axis.     

Specification of left-right asymmetry in the embryonic gut of Drosophila

Most animals exhibit stable left-right asymmetries in their body. Although significant progress has been made in elucidating the mechanisms that set up these asymmetries in vertebrates, nothing is known about them in Drosophila. This is usually attributed to the fact that no reversals of stable left-right asymmetries have been observed in Drosophila, although relevant surveys have been carried out. We have focused on the asymmetry of the proventriculus in the embryonic gut of Drosophila, an aspect of left-right asymmetry that is extremely stable in wild-type flies. We show that this asymmetry can be reversed by mutations in the dicephalic and wunen genes, which also cause reversals in the antero-posterior axis of the embryo relative to its mother. This is the first observation to suggest that left-right asymmetries in Drosophila can be reversed by genetic/developmental manipulations. It also suggests that maternal signals may initiate the specification of some left-right asymmetries in the embryo.     

Left-right axis development: examples of similar and divergent strategies to generate asymmetric morphogenesis in chick and mouse embryos

Left-right asymmetry of internal organs is widely distributed in the animal kingdom. The chick and mouse embryos have served as important model organisms to analyze the mechanisms underlying the establishment of the left-right axis. In the chick embryo many genes have been found to be asymmetrically expressed in and around the node, while the same genes in the mouse show symmetric expression patterns. In the mouse there is strong evidence for an establishment of left-right asymmetry through nodal cilia. In contrast, in the chick and in many other organisms left-right asymmetry is probably generated by an early-acting event involving membrane depolarization. In both birds and mammals a conserved Nodal-Lefty-Pitx2 module exists that controls many aspects of asymmetric morphogenesis. This review also gives examples of divergent mechanisms of establishing asymmetric organ formation. Thus there is ample evidence for conserved and non-conserved strategies to generate asymmetry in birds and mammals.     

Knowing left from right: the molecular basis of laterality defects

The apparent symmetry of the vertebrate body conceals profound asymmetries in the development and placement of internal organs. Asymmetric organ development is controlled in part by genes expressed asymmetrically in the early embryo, and alterations in the activities of these genes can result in severe defects during organogenesis. Recently, data from different vertebrates have allowed researchers to put forward a model of genetic interactions that explains how asymmetric patterns of gene expression in the early embryo are translated into spatial patterns of asymmetric organ development. This model helps us to understand the molecular basis of a number of congenital malformations in humans.     

Establishing a left-right axis in the embryo

Vertebrates exhibit evolutionarily conserved asymmetries in the pattern of internal organ placement that are essential for their normal physiological function. Left-right asymmetries in organ situs are dependent upon the formation of an intact left-right axis during embryogenesis. Recently many of the molecular components involved in the initiation and maintenance of the left-right axis have been described. These molecules and their function in promoting left-right asymmetries are reviewed.     

The LIM protein Ajuba is required for ciliogenesis and left-right axis determination in medaka

Cilia are microtubule-based organelles that are present on the surfaces of almost all vertebrate cells. Most cilia function as sensory or molecular transport structures. Malfunctions of cilia have been implicated in several diseases of human development. The assembly of cilia is initiated by the centriole (or basal body), and several centrosomal proteins are involved in this process. The mammalian LIM protein Ajuba is a well-studied centrosomal protein that regulates cell division but its role in ciliogenesis is unknown. In this study, we isolated the medaka homolog of Ajuba and showed that Ajuba localizes to basal bodies of cilia in growth-arrested cells. Knockdown of Ajuba resulted in randomized left-right organ asymmetries and altered expression of early genes responsible for left-right body axis determination. At the cellular level, we found that Ajuba function was essential for ciliogenesis in the cells lining Kupffer’s vesicle; it is these cells that induce the asymmetric fluid flow required for left-right axis determination. Taken together, our findings identify a novel role for Ajuba in the regulation of vertebrate ciliogenesis and left-right axis determination.     

Left-right development from embryos to brains

Bilateran animals have external bilateral symmetry along the dorsoventral (DV) and anteroposterior (AP) axes. Internal left-right asymmetries appear to be consistently aligned along the left-right (LR) axis with respect to the other axes. Left-right development is most apparent in the directional looping of the cardiac tube, the coiling and placement of the intestines, the positioning of internal organs such as liver, gallbladder, pancreas, and stomach. In addition, there are obvious morphological asymmetries in the brains of some vertebrates and functional left-right asymmetries in the activities of the brain, as assessed by psychological testing, MRI, and the analysis of lesions. There are several fundamental questions: What are the origins of the left-right axis, and are they highly conserved across metazoans? Once the left-right axis is established by the initial breaking of bilateral symmetry, what is the genetic pathway that perpetrates left-right development? What are the cellular and tissue mechanics that lead to morphogenesis during, for example, the looping of the cardiac tube, the coiling of the gut, or asymmetric brain development? Finally, do the asymmetric developmental pathways of each organ system take register from the same initial event that establishes the left-right axis, or are there separate mechanisms that orient heart, gut, and brain left-right asymmetry with respect to the DV and AP axes? These questions are beginning to be experimentally addressed, and papers in this issue of Developmental Genetics make contributions to several aspects in the burgeoning field of left-right development. Recent reviews have summarized the emerging genes and pathways in vertebrate left-right development [Wood, 1997; Harvey, 1998; Ramsdell and Yost, 1998]. Here, I give an overview of the contributions in this issue to the fundamental questions in left-right development. Dev. Genet. 23:159–163, 1998. © 1998 Wiley-Liss, Inc.     

Left-right asymmetry in Drosophila

Seminal studies of left-right (L/R) patterning in vertebrate models have led to the discovery of roles for the nodal pathway, ion flows and cilia in this process. Although the molecular mechanisms underlying L/R asymmetries seen in protostomes are less well understood, recent work using Drosophila melanogaster as a novel genetic model system to study this process has identified a number of mutations affecting directional organ looping. The genetic analysis of this, the most evolutionary conserved feature of L/R patterning, revealed the existence of a L/R pathway that involves the actin cytoskeleton and an associated type I myosin. In this review, we describe this work in the context of Drosophila development, and discuss the implications of these results for our understanding of L/R patterning in general.     

Left-right patterning from the inside out: widespread evidence for intracellular control

The field of left-right (LR) patterning--the study of molecular mechanisms that yield directed morphological asymmetries in otherwise symmetrical organisms--is in disarray. On one hand is the undeniably elegant hypothesis that rotary beating of inclined cilia is the primary symmetry-breaking step: they create an asymmetric extracellular flow across the embryonic midline. On the other hand lurk many early symmetry-breaking steps that, even in some vertebrates, precede the onset of ciliary flow. We highlight an intracellular model of LR patterning where gene expression is initiated by physiological asymmetries that arise from subcellular asymmetries (e.g. motor-protein function along oriented cytoskeletal tracks). A survey of symmetry breaking in eukaryotes ranging from protists to vertebrates suggests that intracellular cytoskeletal elements are ancient and primary LR cues. Evolutionarily, quirky effectors like ciliary motion were likely added later in vertebrates. In some species (like mice), developmentally earlier cues may have been abandoned entirely. Late-developing asymmetries pose a challenge to the intracellular model, but early mid-plane determination in many groups increases its plausibility. Multiple experimental tests are possible.     

Maternal effect of low temperature on handedness determination in C. elegans embryos

C. elegans embryos, larvae, and adults exhibit several left-right asymmetries with an invariant dextral handedness, which first becomes evident in the embryo at the 6-cell stage. Reversed (sinistral) handedness was not observed among > 10,000 N2 adults reared at 16°C or 20°C under standard conditions. However, among the progeny of adults reproducing at 10°C, the frequency of animals with sinistral handedness was increased to ∼0.5%. Cold pulse experiments indicated that the critical period for this increase was in early oogenesis, several hours before the first appearance of left-right asymmetry in the embryo. Hermaphrodites reared at 10°C and mated with males reared at 20°C produced sinistral outcross as well as sinistral self-progeny, indicating that the low temperature effect on oocytes was sufficient to cause reversals. Increased frequency of reversal was also observed among animals developed from embryos lacking the egg shell. Possible mechanisms for the control of embryonic handedness are discussed in the context of these results, including the hypothesis that handedness could be dictated by the chirality of a gametic component. © 1996 Wiley-Liss, Inc.     

H,K-ATPase protein localization and Kir4.1 function reveal concordance of three axes during early determination of left-right asymmetry

Consistent laterality is a fascinating problem, and study of the Xenopus embryo has led to molecular characterization of extremely early steps in left-right patterning: bioelectrical signals produced by ion pumps functioning upstream of asymmetric gene expression. Here, we reveal a number of novel aspects of the H+/K+-ATPase module in chick and frog embryos. Maternal H+/K+-ATPase subunits are asymmetrically localized along the left-right, dorso-ventral, and animal-vegetal axes during the first cleavage stages, in a process dependent on cytoskeletal organization. Using a reporter domain fused to molecular motors, we show that the cytoskeleton of the early frog embryo can provide asymmetric, directional information for subcellular transport along all three axes. Moreover, we show that the Kir4.1 potassium channel, while symmetrically expressed in a dynamic fashion during early cleavages, is required for normal LR asymmetry of frog embryos. Thus, Kir4.1 is an ideal candidate for the K+ ion exit path needed to allow the electroneutral H+/K+-ATPase to generate voltage gradients. In the chick embryo, we show that H+/K+-ATPase and Kir4.1 are expressed in the primitive streak, and that the known requirement for H+/K+-ATPase function in chick asymmetry does not function through effects on the circumferential expression pattern of Connexin43. These data provide details crucial for the mechanistic modeling of the physiological events linking subcellular processes to large-scale patterning and suggest a model where the early cytoskeleton sets up asymmetric ion flux along the left-right axis as a system of planar polarity functioning orthogonal to the apical-basal polarity of the early blastomeres.     

Genetic steps to organ laterality in zebrafish

All internal organs are asymmetric along the left-right axis. Here we report a genetic screen to discover mutations which perturb organ laterality. Our particular focus is upon whether, and how, organs are linked to each other as they achieve their laterally asymmetric positions. We generated mutations by ENU mutagenesis and examined F3 progeny using a cocktail of probes that reveal early primordia of heart, gut, liver and pancreas. From the 750 genomes examined, we isolated seven recessive mutations which affect the earliest left-right positioning of one or all of the organs. None of these mutations caused discernable defects elsewhere in the embryo at the stages examined. This is in contrast to those mutations we reported previously (Chen et al., 1997) which, along with left-right abnormalities, cause marked perturbation in gastrulation, body form or midline structures. We find that the mutations can be classified on the basis of whether they perturb relationships among organ laterality. In Class 1 mutations, none of the organs manifest any left-right asymmetry. The heart does not jog to the left and normally leftpredominant BMP4 in the early heart tube remains symmetric. The gut tends to remain midline. There frequently is a remarkable bilateral duplication of liver and pancreas. Embryos with Class 2 mutations have organotypic asymmetry but, in any given embryo, organ positions can be normal, reversed or randomized. Class 3 reveals a hitherto unsuspected gene that selectively affects laterality of heart. We find that visceral organ positions are predicted by the direction of the preceding cardiac jog. We interpret this as suggesting that normally there is linkage between cardiac and visceral organ laterality. Class 1 mutations, we suggest, effectively remove the global laterality signals, with the consequence that organ positions are effectively symmetrical. Embryos with Class 2 mutations do manifest linkage among organs, but it may be reversed, suggesting that the global signals may be present but incorrectly orientated in some of the embryos. That laterality decisions of organs may be independently perturbed, as in the Class 3 mutation, indicates that there are distinctive pathways for reception and organotypic interpretation of the global signals.     

Asymmetries in H+/K+-ATPase and cell membrane potentials comprise a very early step in left-right patterning

A pharmacological screen identified the H+ and K+ ATPase transporter as obligatory for normal orientation of the left-right body axis in Xenopus. Maternal H+/K+-ATPase mRNA is symmetrically expressed in the 1-cell Xenopus embryo but becomes localized during the first two cell divisions, demonstrating that asymmetry is generated within two hours postfertilization. Although H+/K+-ATPase subunit mRNAs are symmetrically localized in chick embryos, an endogenous H+/K+-ATPase-dependent difference in membrane voltage potential exists between the left and right sides of the primitive streak. In both species, pharmacologic or genetic perturbation of endogenous H+/K+-ATPase randomized the sided pattern of asymmetrically expressed genes and induced organ heterotaxia. Thus, LR asymmetry determination depends on a very early differential ion flux created by H+/K+-ATPase activity.     

A conserved role for the nodal signaling pathway in the establishment of dorso-ventral and left-right axes in deuterostomes

Nodal factors play crucial roles during embryogenesis of chordates. They have been implicated in a number of developmental processes, including mesoderm and endoderm formation and patterning of the embryo along the anterior-posterior and left-right axes. We have analyzed the function of the Nodal signaling pathway during the embryogenesis of the sea urchin, a non-chordate organism. We found that Nodal signaling plays a central role in axis specification in the sea urchin, but surprisingly, its first main role appears to be in ectoderm patterning and not in specification of the endoderm and mesoderm germ layers as in vertebrates. Starting at the early blastula stage, sea urchin nodal is expressed in the presumptive oral ectoderm where it controls the formation of the oral-aboral axis. A second conserved role for nodal signaling during vertebrate evolution is its involvement in the establishment of left-right asymmetries. Sea urchin larvae exhibit profound left-right asymmetry with the formation of the adult rudiment occurring only on the left side. We found that a nodal/lefty/pitx2 gene cassette regulates left-right asymmetry in the sea urchin but that intriguingly, the expression of these genes is reversed compared to vertebrates. We have shown that Nodal signals emitted from the right ectoderm of the larva regulate the asymmetrical morphogenesis of the coelomic pouches by inhibiting rudiment formation on the right side of the larva. This result shows that the mechanisms responsible for patterning the left-right axis are conserved in echinoderms and that this role for nodal is conserved among the deuterostomes. We will discuss the implications regarding the reference axes of the sea urchin and the ancestral function of the nodal gene in the last section of this review.