Nodal Cascade Research Articles

Morphological changes and two Nodal paralogs drive left-right asymmetry in the squamate veiled chameleon (C. calyptratus).

The ancestral mode of left-right (L-R) patterning involves cilia in the L-R organizer. However, the mechanisms regulating L-R patterning in non-avian reptiles remains an enigma, since most squamate embryos are undergoing organogenesis at oviposition. In contrast, veiled chameleon (Chamaeleo calyptratus) embryos are pre-gastrula at oviposition, making them an excellent organism for studying L-R patterning evolution. Here we show that veiled chameleon embryos lack motile cilia at the time of L-R asymmetry establishment. Thus, the loss of motile cilia in the L-R organizers is a synapomorphy of all reptiles. Furthermore, in contrast to avians, geckos and turtles, which have one Nodal gene, veiled chameleon exhibits expression of two paralogs of Nodal in the left lateral plate mesoderm, albeit in non-identical patterns. Using live imaging, we observed asymmetric morphological changes that precede, and likely trigger, asymmetric expression of the Nodal cascade. Thus, veiled chameleons are a new and unique model for studying the evolution of L-R patterning.

Bicc1 and Dicer regulate left-right patterning through post-transcriptional control of the Nodal inhibitor Dand5

Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells at the left LRO margin. Ciliary activity of the calcium channel Pkd2 is crucial for flow sensing. How this flow signal is further processed and relayed to the laterality-determining Nodal cascade in the left lateral plate mesoderm (LPM) is largely unknown. We previously showed that flow down-regulates mRNA expression of the Nodal inhibitor Dand5 in left sensory cells. De-repression of the co-expressed Nodal, complexed with the TGFß growth factor Gdf3, drives LPM Nodal cascade induction. Here, we show that post-transcriptional repression of dand5 is a central process in symmetry breaking of Xenopus, zebrafish and mouse. The RNA binding protein Bicc1 was identified as a post-transcriptional regulator of dand5 and gdf3 via their 3′-UTRs. Two distinct Bicc1 functions on dand5 mRNA were observed at pre- and post-flow stages, affecting mRNA stability or flow induced translational inhibition, respectively. To repress dand5, Bicc1 co-operates with Dicer1, placing both proteins in the process of flow sensing. Intriguingly, Bicc1 mediated translational repression of a dand5 3′-UTR mRNA reporter was responsive to pkd2, suggesting that a flow induced Pkd2 signal triggers Bicc1 mediated dand5 inhibition during symmetry breakage.

Brain Lateralization: A Comparative Perspective.

Comparative studies on brain asymmetry date back to the 19th century but then largely disappeared due to the assumption that lateralization is uniquely human. Since the reemergence of this field in the 1970s, we learned that left-right differences of brain and behavior exist throughout the animal kingdom and pay off in terms of sensory, cognitive, and motor efficiency. Ontogenetically, lateralization starts in many species with asymmetrical expression patterns of genes within the Nodal cascade that set up the scene for later complex interactions of genetic, environmental, and epigenetic factors. These take effect during different time points of ontogeny and create asymmetries of neural networks in diverse species. As a result, depending on task demands, left- or right-hemispheric loops of feedforward or feedback projections are then activated and can temporarily dominate a neural process. In addition, asymmetries of commissural transfer can shape lateralized processes in each hemisphere. It is still unclear if interhemispheric interactions depend on an inhibition/excitation dichotomy or instead adjust the contralateral temporal neural structure to delay the other hemisphere or synchronize with it during joint action. As outlined in our review, novel animal models and approaches could be established in the last decades, and they already produced a substantial increase of knowledge. Since there is practically no realm of human perception, cognition, emotion, or action that is not affected by our lateralized neural organization, insights from these comparative studies are crucial to understand the functions and pathologies of our asymmetric brain.

Left-right asymmetric heart jogging increases the robustness of dextral heart looping in zebrafish

Building a left-right (L-R) asymmetric organ requires asymmetric information. This comes from various sources, including asymmetries in embryo-scale genetic cascades (including the left-sided Nodal cascade), organ-intrinsic mechanical forces, and cell-level chirality, but the relative influence of these sources and how they collaborate to drive asymmetric morphogenesis is not understood. During zebrafish heart development, the linear heart tube extends to the left of the midline in a process known as jogging. The jogged heart then undergoes dextral (i.e. rightward) looping to correctly position the heart chambers relative to one another. Left lateralized jogging is governed by the left-sided expression of Nodal in mesoderm tissue, while looping laterality is mainly controlled by heart-intrinsic cell-level asymmetries in the actomyosin cytoskeleton. The purpose of lateralized jogging is not known. Moreover, after jogging, the heart tube returns to an almost midline position and so it is not clear whether or how jogging may impact the dextral loop. Here, we characterize a novel loss-of-function mutant in the zebrafish Nodal homolog southpaw (spaw) that appears to be a true null. We then assess the relationship between jogging and looping laterality in embryos lacking asymmetric Spaw signals. We found that the probability of a dextral loop occurring, does not depend on asymmetric Spaw signals per se, but does depend on the laterality of jogging. Thus, we conclude that the role of leftward jogging is to spatially position the heart tube in a manner that promotes robust dextral looping. When jogging laterality is abnormal, the robustness of dextral looping decreases. This establishes a cooperation between embryo-scale Nodal-dependent L-R asymmetries and organ-intrinsic cellular chirality in the control of asymmetric heart morphogenesis and shows that the transient laterality of the early heart tube has consequences for later heart morphogenetic events.

Dissecting the dynamics of signaling events in the BMP, WNT, and NODAL cascade during self-organized fate patterning in human gastruloids.

During gastrulation, the pluripotent epiblast self-organizes into the 3 germ layers—endoderm, mesoderm and ectoderm, which eventually form the entire embryo. Decades of research in the mouse embryo have revealed that a signaling cascade involving the Bone Morphogenic Protein (BMP), WNT, and NODAL pathways is necessary for gastrulation. In vivo, WNT and NODAL ligands are expressed near the site of gastrulation in the posterior of the embryo, and knockout of these ligands leads to a failure to gastrulate. These data have led to the prevailing view that a signaling gradient in WNT and NODAL underlies patterning during gastrulation; however, the activities of these pathways in space and time have never been directly observed. In this study, we quantify BMP, WNT, and NODAL signaling dynamics in an in vitro model of human gastrulation. Our data suggest that BMP signaling initiates waves of WNT and NODAL signaling activity that move toward the colony center at a constant rate. Using a simple mathematical model, we show that this wave-like behavior is inconsistent with a reaction-diffusion–based Turing system, indicating that there is no stable signaling gradient of WNT/NODAL. Instead, the final signaling state is homogeneous, and spatial differences arise only from boundary effects. We further show that the durations of WNT and NODAL signaling control mesoderm differentiation, while the duration of BMP signaling controls differentiation of CDX2-positive extra-embryonic cells. The identity of these extra-embryonic cells has been controversial, and we use RNA sequencing (RNA-seq) to obtain their transcriptomes and show that they closely resemble human trophoblast cells in vivo. The domain of BMP signaling is identical to the domain of differentiation of these trophoblast-like cells; however, neither WNT nor NODAL forms a spatial pattern that maps directly to the mesodermal region, suggesting that mesoderm differentiation is controlled dynamically by the combinatorial effect of multiple signals. We synthesize our data into a mathematical model that accurately recapitulates signaling dynamics and predicts cell fate patterning upon chemical and physical perturbations. Taken together, our study shows that the dynamics of signaling events in the BMP, WNT, and NODAL cascade in the absence of a stable signaling gradient control fate patterning of human gastruloids.

Structural Asymmetry in the Frontal and Temporal Lobes Is Associated with PCSK6 VNTR Polymorphism.

The nodal cascade influences the development of bodily asymmetries in humans and other vertebrates. The gene PCSK6 has shown a regulatory function during left-right axis formation and is therefore thought to influence bodily left-right asymmetries. However, it is not clear if variation in this gene is also associated with structural asymmetries in the brain. We genotyped an intronic 33bp PCSK6 variable number tandem repeat (VNTR) polymorphism that has been associated with handedness in a cohort of healthy adults. We acquired T1-weighted structural MRI images of 320 participants and defined cortical surface and thickness for each HCP region. The results demonstrate a significant association between PCSK6 VNTR genotypes and gray matter asymmetry in the superior temporal sulcus, which is involved in voice perception. Heterozygous individuals who carry a short (≤ 6 repeats) and a long (≥ 9 repeats) PCSK6 VNTR allele show stronger rightward asymmetry. Further associations were evident in the dorsolateral prefrontal cortex. Here, individuals homozygous for short alleles show a more pronounced asymmetry. This shows that PCSK6, a gene that has been implicated in the ontogenesis of bodily asymmetries by regulating the nodal cascade, is also relevant for structural asymmetries in the human brain.

An Early Function of Polycystin-2 for Left-Right Organizer Induction in Xenopus

SummaryNodal signaling controls asymmetric organ placement during vertebrate embryogenesis. Nodal is induced by a leftward fluid flow at the ciliated left-right organizer (LRO). The mechanism of flow sensing, however, has remained elusive. pkd2 encodes the calcium channel Polycystin-2, which is required for kidney development and laterality, and may act in flow perception. Here, we have studied the role of Polycystin-2 in Xenopus and show that pkd2 is indispensable for left-right (LR) asymmetry. Knockdown of pkd2 prevented left-asymmetric nodal cascade induction in the lateral plate mesoderm. Defects were due to failure of LRO specification, morphogenesis, and, consequently, absence of leftward flow. Polycystin-2 synergizes with the unconventional nodal-type signaling molecule Xnr3 to induce the LRO precursor tissue before gastrulation, upstream of symmetry breakage. Our data uncover an unknown function of pkd2 in LR axis formation, which we propose represents an ancient role of Polycystin-2 during LRO induction in lower vertebrates.

A Conserved Role of the Unconventional Myosin 1d in Laterality Determination

Anatomical and functional asymmetries are widespread in the animal kingdom [1, 2]. In vertebrates, many visceral organs are asymmetrically placed [3]. In snails, shells and inner organs coil asymmetrically, and in Drosophila, genitalia and hindgut undergo a chiral rotation during development. The evolutionary origin of these asymmetries remains an open question [1]. Nodal signaling is widely used [4], and many, but not all, vertebrates use cilia for symmetry breaking [5]. In Drosophila, which lacks both cilia and Nodal, the unconventional myosin ID (myo1d) gene controls dextralrotation of chiral organs [6, 7]. Here, we studied the role of myo1d in left-right (LR) axis formation in Xenopus. Morpholino oligomer-mediatedmyo1d downregulation affected organ placement in >50% of morphant tadpoles. Induction of the left-asymmetric Nodal cascade was aberrant in >70% of cases. Expression of the flow-target gene dand5 was compromised, as was flow itself, due to shorter, fewer, and non-polarized cilia at the LR organizer. Additional phenotypes pinpointed Wnt/planar cell polarity signaling and suggested that myo1d, like in Drosophila [8], acted in the context of the planar cell polarity pathway. Indeed, convergent extension of gastrula explant cultures was inhibited inmyo1d morphants, and the ATF2 reporter genefor non-canonical Wnt signaling was downregulated. Finally, genetic interference experiments demonstrated a functional interaction between the core planar cell polarity signaling gene vangl2 and myo1d in LR axis formation. Thus, our data identified myo1d as a common denominator of arthropod and chordate asymmetry, in agreement with a monophyletic origin of animal asymmetry.

Vertebrate Left-Right Asymmetry: What Can Nodal Cascade Gene Expression Patterns Tell Us?

Laterality of inner organs is a wide-spread characteristic of vertebrates and beyond. It is ultimately controlled by the left-asymmetric activation of the Nodal signaling cascade in the lateral plate mesoderm of the neurula stage embryo, which results from a cilia-driven leftward flow of extracellular fluids at the left-right organizer. This scenario is widely accepted for laterality determination in wildtype specimens. Deviations from this norm come in different flavors. At the level of organ morphogenesis, laterality may be inverted (situs inversus) or non-concordant with respect to the main body axis (situs ambiguus or heterotaxia). At the level of Nodal cascade gene activation, expression may be inverted, bilaterally induced, or absent. In a given genetic situation, patterns may be randomized or predominantly lacking laterality (absence or bilateral activation). We propose that the distributions of patterns observed may be indicative of the underlying molecular defects, with randomizations being primarily caused by defects in the flow-generating ciliary set-up, and symmetrical patterns being the result of impaired flow sensing, on the left, the right, or both sides. This prediction, the reasoning of which is detailed in this review, pinpoints functions of genes whose role in laterality determination have remained obscure.

Leftward Flow Determines Laterality in Conjoined Twins

Conjoined twins fused at the thorax display an enigmatic left-right defect: although left twins are normal, laterality is disturbed in one-half of right twins [1-3]. Molecularly, this randomization corresponds to a lack of asymmetric Nodal cascade induction in right twins [4]. We studied leftward flow [5, 6] at the left-right organizer (LRO) [7, 8] in thoracopagus twins in Xenopus, which displayed a duplicated, fused, and ciliated LRO. Cilia were motile and produced a leftward flow from the right LRO margin of the right to the left margin of the left twin. Motility was required for correct laterality in left twins, as knockdown of dynein motor dnah9 prevented Nodal cascade induction. Nodal was rescued by parallel knockdown of the inhibitor dand5 [9, 10] on the left side of the left twin. Lack of Nodal induction in the right twin, despite the presence of flow, was due to insufficient suppression of dand5. Knockdown of dand5 at the center of the fused LRO resulted in asymmetric Nodal cascade induction in the right twin as well. Manipulation of leftward flow and dand5 in a targeted and sided manner induced the Nodal cascade in a predictable manner, in the left twin, the right one, both, or neither. Laterality in conjoined twins thus was determined by cilia-driven leftward fluid flow like in single embryos, which solves a century-old riddle, as the phenomenon was already studied by some of the founders of experimental embryology, including Dareste [11], Fol and Warynsky [12], and Spemann and Falkenberg [13] (reviewed in [14]).

A novel form of PCD that impacts nodal, but not tracheal cilia

Motile cilia, in the embryonic node, drive a leftward fluid flow (termed nodal flow) that establishes the left-right axis. We identified lrm5 in a genetic screen for mouse left-right patterning mutants - the embryos exhibited disturbed situs. Mapping and sequencing revealed a novel mutation in the axonemal dynein heavy chain locus Dnah11: loss of function gives rise to immotile cilia in mice; many human primary ciliary dyskinesia (PCD) patients with DNAH11 mutations have hyper-motile cilia. To our surprise, unlike the previously characterised Dnah11iv mutant, lrm5 tracheal ciliary beat frequency (CBF) was normal. However, the number of lrm5 homozygotes at weaning was lower than Mendelian ratios would predict. Age of death analysis identified a 50% reduction in embryos between E14.5 and E15.5, consistent with death from embryonic cardiac failure. Expression analysis of early molecular markers of left-right asymmetry revealed randomised or bilateral activation of the normally left-sided Nodal Cascade. As this suggested an early, primary patterning defect, we analysed nodal flow by particle image velocimetry (PIV); rather than the wild-type leftward flow, or the absent flow in Dnah11iv, we observed a chaotic fluid flow in lrm5 nodes. We therefore assessed nodal CBF and ciliary amplitude by DIC microscopy; lrm5 cilia beat at 1.5x normal frequency, but with an abnormal motion. In summary, lrm5 is a novel form of PCD, impacting nodal but not tracheal ciliary beating. We would predict that equivalent mutations in humans might underlie situs defects and congenital heart disease in the absence of respiratory disease.

Serotonin Signaling Is Required for Wnt-Dependent GRP Specification and Leftward Flow in Xenopus

In vertebrates, most inner organs are asymmetrically arranged with respect to the main body axis [1]. Symmetry breakage in fish, amphibian, and mammalian embryos depends on cilia-driven leftward flow of extracellular fluid during neurulation [2-5]. Flow induces the asymmetric nodal cascade that governs asymmetric organ morphogenesis and placement [1, 6, 7]. In the frog Xenopus, an alternative laterality-generating mechanism involving asymmetric localization of serotonin at the 32-cell stage has been proposed [8]. However, no functional linkage between this early localization and flow at neurula stage has emerged. Here, we report that serotonin signaling is required for specification of the superficial mesoderm (SM), which gives rise to the ciliated gastrocoel roof plate (GRP) where flow occurs [5, 9]. Flow and asymmetry were lost in embryos in which serotonin signaling was downregulated. Serotonin, which we found uniformly distributed along the main body axes in the early embryo, was required for Wnt signaling, which provides the instructive signal to specify the GRP. Importantly, serotonin was required for Wnt-induced double-axis formation as well. Our data confirm flow as primary mechanism of symmetry breakage and suggest a general role of serotonin as competence factor for Wnt signaling during axis formation in Xenopus.

Shells and heart: Are human laterality and chirality of snails controlled by the same maternal genes?

The body of most animals display left-right asymmetry of internal organs. Alteration of such asymmetry results in severe congenital defects particularly affecting the cardiovascular system. The earliest known genes involved in asymmetry, the Nodal signalling cascade, are expressed asymmetrically during embryonic development. Nodal was discovered in the mouse, but orthologs (also involved in left-right specification) were reported in ascidians, sea-urchins, and snails. Mutations in Nodal-pathway genes cause alteration of several aspects of chirality, but not entirely mirror phenotypes of the body. Other factors upstream of nodal must be involved in the generation of left-right asymmetry. In snails, breeding experiments have demonstrated that chirality is controlled by a nuclear gene with maternal effect. Given the available evidence, we propose that an evolutionarily conserved genetic basis of chirality (the same that controls left-right asymmetry in snails) is a major synapomorphy of the Bilateria. This hypothesis fits with the observation that: (a) the proportion of patients with heterotaxy and a detected mutation in a gene of the Nodal cascade is actually low, and (b) horizontal recurrence of laterality defects is remarkably more frequent than vertical recurrence, and includes a notable number of affected sibs and/or repeated abortions from unaffected mothers. Identification of the maternal gene(s) involved will allow for the identification of homozygous females at risk of having affected children and spontaneous abortions, and would provide a general medical framework for understanding the genetics of most alterations of chirality.

The Nodal Inhibitor Coco Is a Critical Target of Leftward Flow in Xenopus

Vertebrate laterality, which is manifested by asymmetrically placed organs [1], depends on asymmetric activation of the Nodal signaling cascade in the left lateral plate mesoderm [2]. In fish, amphibians, and mammals, a cilia-driven leftward flow of extracellular fluid acts upstream of the Nodal cascade [3-6]. The direct target of flow has remained elusive. In Xenopus, flow occurs at the gastrocoel roof plate (GRP) in the dorsal midline of the embryo [4, 7]. The GRP is bordered by a second, bilaterally symmetrical Nodal expression domain [8]. Here we identify the Nodal inhibitor Coco as a critical target of flow. Coco and Xenopus Nodal-related 1 (Xnr1) are coexpressed in the lateralmost ciliated GRP cells. Coco becomes downregulated on the left side of the GRP as a direct readout of flow. Ablation of flow prevented Coco repression, whereas Xnr1 expression was independent of flow. Loss of flow-induced laterality defects were rescued by knockdown of Coco on the left side. Parallel knockdown of Coco and Xnr1 in GRP cells restored laterality defects in flow-impaired embryos, demonstrating that Coco acted through GRP-expressed Xnr1. Coco thus acts as a critical target of flow, suggesting that symmetry is broken by flow-mediated left-asymmetric release of Nodal repression at the midline.

Highlights in DD

‘Highlights’ calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Calculating what's left (Fluid dynamics of nodal flow and left-right patterning in development by Julyan H.E. Cartwright, Nicolas Piro, Oreste Piro, Idan Tuval, Dev Dyn 237:3477–3490) Like it or not, most biological phenomena cannot be adequately explained without the help of physics. Luckily for the physics-phobe, there are gifted teachers such as Cartwright and colleagues who are exceptionally skilled at explaining biology's numerical roots. This review covers their and others' work modeling the fluid dynamics behind nodal flow–fluid flow induced by monocilia within the node, one of the earliest steps in establishing vertebrate left–right asymmetry. In the interest of examining models with predictive power, they discuss what happens when as of yet experimentally untested variables are included in fluid dynamical models. Biological concepts are nimbly intertwined with the derivation of models that can be used to understand them, giving even the most mathematically challenged something onto which they can grasp. Change of heart (Cellular nonmuscle myosins NMHC-IIA and NMHC-IIB and vertebrate heart looping by Wenge Lu, Steven H. Seeholzer, Mingda Han, Anne-Sophie Arnold, Maria Serrano, Barbara Garita, Nancy J. Philp, Cassandra Farthing, Peter Steele, Jizhen Chen, Kersti K. Linask, Dev Dyn 237:3557–3564) Galanin is a neuropeptide, well known for regulating various nervous system functions, such as metabolism and regulation of food intake. Therefore, it is surprising that Schweickert and colleagues found that Gal, the gene that encodes Galanin, is asymmetrically expressed in the linear heart tube, and is later localized to the second pacemaker, the atrioventricular node and ring. Furthermore, like asymmetric expression of left–right (LR) patterning gene Nodal, and downstream genes Lefty2 and Pitx2c in the lateral plate mesoderm (LPM) in the early embryo, left-sided heart Gal expression at later stages is dependent on leftward flow of cilia in the posterior notochord (commonly termed as node). This finding demonstrates that Gal is regulated by the LR pathway. Therefore, it is surprising that Gal heart expression remains unchanged in mutants for the Nodal co-receptor cryptic, even though expression of Nodal cascade genes disappears in the LPM. Also surprising is that asymmetric heart expression of Pitx2c also remains unchanged in half of cryptic homozygotes. This discovery led the authors to hypothesize that asymmetric gene expression in the heart is cryptic independent. Neither Galanin nor cryptic are behaving as expected—perhaps they had a change of heart. MANning the barrier (Man1, an inner nuclear membrane protein, regulates left-right axis formation by controlling nodal signaling in a node-independent manner by Akihiko Ishimura, Shinsuke Chida, Shin-lchi Osada, Dev Dyn 237:3565–3576) During specification of the left–right axis, the midline acts as a barrier that keeps asymmetrically expressed signals from becoming bilateral. Here, Ishimura et al., provide evidence for the first time that Man1, an inner nuclear membrane protein, regulates activity of a midline barrier. Transgenic mice bearing truncated MAN1 that lacks an R-SMAD interacting domain (R-SMADs are downstream effectors of TGFβ family signaling), show bilateral expression of the left–right determination gene Nodal, and downstream genes Lefty2 and Pitx2. Of interest, ectopic expression of the three genes occurs despite that Lefty1, a marker for the midline barrier, is still present. How could this be? One possible answer lies in the discovery that expression of coreceptors for Nodal, Cryptic and Cripto, are up-regulated. The event may enhance competence for Nodal signaling. Another possibility stems from their finding that bilateral Nodal expression is maintained in Man1 mutants crossed with Nodal hypomorphs, despite that these double mutants lack a node, and thus early node-derived Nodal. This means that, instead of under the control of node-derived Nodal, ectopic Nodal may be controlled by an unknown molecule that is derepressed in a Man1 mutant background. One candidate is Bmp2, whose expression is also augmented in Man1 mutants. The observation that Man1 is transiently expressed in the posterior midline bolsters the idea that the gene also directly or indirectly regulates activity of a midline barrier.

Xenopus, an ideal model system to study vertebrate left‐right asymmetry

Vertebrate organ laterality is manifested by the asymmetric morphogenesis and placement of inner organs. Asymmetric induction of the Nodal signaling cascade in the left lateral plate mesoderm (LPM) precedes and is essential for asymmetric organ morphogenesis. While the Nodal cascade is highly conserved, symmetry breakage is considered to vary between the different classes of the vertebrates. In Xenopus, early determinants at cleavage stages were thought to break symmetry, opposed to cilia-driven leftward flow in mammals and fish. The main objectives of this review are to emphasize the conserved nature of symmetry breakage, and to demonstrate the power of Xenopus embryology to analyze and manipulate flow. In addition, mutant phenotypes described in other model organisms can easily be mimicked in frog by single or multiple knockdowns in combination with experimental manipulations and flow analysis. Xenopus, therefore, is ideally suited to address the major open questions in the field. Developmental Dynamics 238:1215-1225, 2009. (c) 2009 Wiley-Liss, Inc.

Gap junctions relay FGF8‐mediated right‐sided repression of Nodal in rabbit

In vertebrate gastrula/neurula embryos, a cilia-driven leftward flow asymmetrically activates the Nodal cascade in the left lateral plate mesoderm (LPM). In frog embryos left-right axis formation was postulated to depend on gap junctions (GJs) during cleavage. Here, we show that GJs cooperate with fibroblast growth factor-8 (FGF8) to specify asymmetric Nodal in the rabbit embryo at gastrula/neurula. GJs and FGF signaling were manipulated in whole embryo and explant cultures of rabbit blastodiscs. These experiments demonstrate that right-sided inhibition of Nodal by FGF8 depended on intercellular communication by means of GJs, and that left-sided induction of Nodal required attenuation of gap junctional communication (GJC). Before flow, the left and right side were equally competent but actively prevented from Nodal induction through FGF8/GJ. Our data suggest that flow unilaterally attenuates FGF8/GJ-mediated repression of Nodal on the left side, integrating GJC and FGF8 into the flow-based mechanism of symmetry breakage in the vertebrate embryo.

Cilia-Driven Leftward Flow Determines Laterality in Xenopus

Determination of the vertebrate left-right body axis during embryogenesis results in asymmetric development and placement of most inner organs. Although the asymmetric Nodal cascade is conserved in all vertebrates, the mechanism of symmetry breakage has remained controversial. In mammalian and fish embryos, a cilia-driven leftward flow of extracellular fluid is required for initiation of the Nodal cascade. This flow is localized at the posterior notochord ("node") and Kupffer's vesicle, respectively. In frog and chick embryos, however, molecular asymmetries are required earlier, from cleavage stages through gastrulation. The validity of a cilia-based mechanism for all vertebrates therefore has been questioned. Here we show that a cilia-driven leftward flow precedes asymmetric nodal expression in the frog Xenopus. Motile monocilia emerged on the gastrocoel roof plate during neurulation and lengthened and polarized from an initially central position to the posterior pole of cells. Concomitantly, a robust leftward fluid flow developed from stage 15 onward, significantly before asymmetric nodal transcription started in the left-lateral-plate mesoderm at stage 19. Injection of 1.5% methylcellulose into the archenteron prevented leftward flow and resulted in laterality defects, demonstrating that the flow itself was required for asymmetric gene expression and organ placement.

Symmetry Breaking and the Evolution of Development

Because of its simplicity, the binary-switch nature of left-right asymmetry permits meaningful comparisons among many different organisms. Phylogenetic analyses of asymmetry variation, inheritance, and molecular mechanisms reveal unexpected insights into how development evolves. First, directional asymmetry, an evolutionary novelty, arose from nonheritable origins almost as often as from mutations, implying that genetic assimilation ("phenotype precedes genotype") is a common mode of evolution. Second, the molecular pathway directing hearts leftward-the nodal cascade-varies considerably among vertebrates (homology of form does not require homology of development) and was possibly co-opted from a preexisting asymmetrical chordate organ system. Finally, declining frequencies of spontaneous asymmetry reversal throughout vertebrate evolution suggest that heart development has become more canalized.

The Ion Channel Polycystin-2 Is Required for Left-Right Axis Determination in Mice

Generation of laterality depends on a pathway which involves the asymmetrically expressed genes nodal, Ebaf, Leftb, and Pitx2[1–3]. In mouse, node monocilia are required upstream of the nodal cascade [4]. In chick and frog, gap junctions are essential prior to node/organizer formation [5, 6]. It was hypothesized that differential activity of ion channels gives rise to unidirectional transfer through gap junctions, resulting in asymmetric gene expression [3, 6]. PKD2, which if mutated causes autosomal dominant polycystic kidney disease (ADPKD) in humans, encodes the calcium release channel polycystin-2 [7–11]. We have generated a knockout allele of Pkd2 in mouse. In addition to malformations described previously [12], homozygous mutant embryos showed right pulmonary isomerism, randomization of embryonic turning, heart looping, and abdominal situs. Leftb and nodal were not expressed in the left lateral plate mesoderm (LPM), and Ebaf was absent from floorplate. Pitx2 was bilaterally expressed in posterior LPM but absent anteriorly. Pkd2 was ubiquitously expressed at headfold and early somite stages, with higher levels in floorplate and notochord. The embryonic midline, however, was present, and normal levels of Foxa2 and shh were expressed, suggesting that polycystin-2 acts downstream or in parallel to shh and upstream of the nodal cascade.