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ESTABLISHMENT OF VERTEBRATE LEFTRIGHT ASYMMETRY


Hiroshi Hamada, Chikara Meno, Daisuke Watanabe and Yukio Saijoh
The generation of morphological, such as leftright, asymmetry during development is an integral part of the establishment of a body plan. Until recently, the molecular basis of leftright asymmetry was a mystery, but studies indicate that Nodal and the Lefty proteins, transforming growth factor--related molecules, have a central role in generating asymmetric signals. Although the initial mechanism of symmetry breaking remains unknown, developmental biologists are beginning to analyse the pathway that leads to leftright asymmetry establishment and maintenance.
The establishment of the three body axes anteroposterior (A/P), dorsoventral (D/V) and leftright (L/R) is central to the organization of the vertebrate body plan. Although all three axes are associated with morphological asymmetry, L/R asymmetry provides a unique opportunity to study the cellular and molecular mechanisms of asymmetry generation. This is because it is possible to study this process in its entirety, from the initial symmetry-breaking step to the final stages, which involve asymmetric organogenesis. Out of the three body axes, the L/R axis is determined last, and therefore is more accessible and more amenable to experimental manipulation. Finally, defects that arise from abnormal L/R patterning are less severe than abnormal A/P or D/V patterning, which makes experimental approaches easier. Although L/R asymmetry has fascinated biologists for many years, it is only recently that progress has been made at the genetic and molecular levels. Identification of several genes that are expressed asymmetrically with respect to the L/R axis in different vertebrates14 has opened up this field and made molecular-genetic approaches possible for the first time. Expression studies, as well as reverse genetics in mouse and surgical manipulations in chick and frog embryos, have defined the specific roles of these newly identified genes in the L/R pathway. Analysis of mouse mutants and human genetic disorders with L/R defects has also revealed new genes that are involved in this process (for reviews, see REFS 59). The process by which L/R asymmetry is established can be divided into four steps (FIG. 1): initial breaking of L/R symmetry in or near the node, which takes place at the late neural-fold stage10; transfer of L/Rbiased signals from the node to the lateral plate mesoderm (LPM); L/R asymmetric expression of signalling molecules, such as the transforming growth factor- (TGF-)-related molecules Nodal and the Lefty proteins on the left side in the lateral plate; and L/R asymmetric morphogenesis of the visceral organs that are induced by these signalling molecules. This view of the establishment of L/R asymmetry is based on studies of different vertebrates, such as zebrafish, frog, chick and mouse. Xenopus laevis and chick misexpression studies have provided important insights and ideas, whereas genetic studies in the zebrafish and mouse have helped to develop elegant models. This work has led to the identification of the central components in the L/R pathway Nodal, Lefty1 and Lefty2, and the homeobox gene Pitx2 (FIG. 2). In this review, we discuss current views of the molecular mechanisms that are involved in L/R patterning. We focus on genetic data that have been obtained from the mouse, because of the importance of this work to current models of how L/R asymmetry is generated.
Breaking symmetry

Division of Molecular Biology, Institute for Molecular and Cellular Biology, Osaka University, and CREST, Japan Science and Technology Corporation (JST), 13 Yamada-oka, Suita, Osaka 565-0871, Japan. Correspondence to H.H. e-mail: hamada@imcb.osakau.ac.jp
DOI: 10.1038/nrg732

In the early 1990s, Brown, Wolpert and colleagues11,12 proposed a conceptual model for the generation of L/R

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asymmetry in mammalian embryos. Their model had three components: conversion, which biases the generation of asymmetry; random generation of asymmetry; and interpretation, in which individual organs and/or cells recognize asymmetric information. Conversion, which can be regarded as the initial break in symmetry, involves a molecule that is handed, such as the axes of a three-dimensional graph x, y and z. When two of the axes are aligned along the A/P and D/V axes, the remaining axis would be automatically aligned along the future L/R axis. Observations that a loss of conversion results not in symmetry but in random asymmetry (as seen in iv/iv mutant mice and as discussed in more detail below) led Brown and Wolpert11 to propose a separate mechanism for the random generation of asymmetry. Recent observations indicate that, at least in the mouse, the first step conversion might involve rotational movement of the cilia in the node (FIG. 3) (also see a movie of the cilia in the supplementary information online). A link between cilia and L/R determination had been suspected since the discovery of Kartagener syndrome a relatively rare human genetic disorder that includes SITUS INVERSION accompanied by loss of motility of respiratory cilia and sperm flagella13. In Kartagener patients, DYNEIN arms are absent from the respiratory cilia and sperm flagella. Additionally, a mutation in one of the dynein genes has also been identified as the cause of the mouse iv mutant phenotype14. Despite these findings, the connection between cilia and L/R determination remained enigmatic. Cells on the ventral side of the node (node pit cells) each have a MONOCILIUM that projects into the extra-embryonic space15 (FIG. 3). These monocilia belong to the 9+0 class of cilia (BOX 1), which were initially regarded as non-motile, but recent observations16 show that they can rotate rapidly, at a speed of ~600 r.p.m., in a clockwise direction (when viewed from the ventral side). This rotational movement generates a leftward flow of the extra-embryonic fluid around the node that might transport an unknown molecule (also see a movie of the nodal flow in the supplementary information online), which could then act as a left-side determinant. The nodal flow is impaired in several mouse mutants that show SITUS DEFECTS, which support the involvement of the nodal flow in L/R determination. For example, mice that are deficient in the KINESIN superfamily proteins, Kif3a or Kif3b, have randomized situs. They lack the primary cilia in the node and consequently the nodal flow is absent1618. Polaris (TG737) mutant mice also lack the cilia in the node and show L/R randomization, and although the nodal flow has not been carefully examined, it is most probably absent19. iv mutant mouse embryos have morphologically normal, but immotile, cilia in the node20,21, hence the complete absence of the nodal flow in these embryos. Other mutant mice that lack 9+2 cilia, such as those that are deficient in hepatocyte nuclear factor/forkhead homologue 4 (Hfh4, also known as Foxj1)22,23, also have randomized L/R asymmetry. It is not yet known whether these mice also lack the 9+0 cilia in the node.
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Step 1

Left

Right

Node

Step 2

Step 3

NODE

The anterior tip of the primitive streak a structure that gives bilateral symmetry and a midline axis to the embryo. The node gives rise to anterior mesendoderm and has a function that is equivalent to the Spemanns organizer of Xenopus.
LATERAL PLATE MESODERM

Step 4 Pattern L R L R Heart, spleen I Stomach, intestine

(LPM). The mesoderm located in the lateral region of the early somite-stage embryo.
SITUS INVERSION

II

Liver, lung

A congenital malformation in which leftright asymmetry of viscera is completely reversed.


DYNEIN

A microtubule-associated motor protein that is responsible for transporting vesicles and particles towards the proximal (minus) end of microtubules.
MONOCILIUM

III

Blood vessels

A cilium that exists once per cell; each contains the nine outer microtubule doublets but lacks a central doublet. They are therefore referred to as 9+0 cilia.
SITUS DEFECT

A congenital defect in leftright asymmetry.


KINESIN

A microtubule-associated motor protein that is responsible for transporting vesicles and particles towards the distal (plus) end of microtubules.

Figure 1 | Four steps of leftright asymmetric morphogenesis. In the first step, initial symmetry is broken in a process that might involve nodal flow (the NODE is represented in grey; the direction of the flow is indicated with red arrowheads). In the second step, leftright (L/R) asymmetric signals are transferred (arrows) from the node to the LATERAL PLATE MESODERM (LPM). Asymmetric expression of Nodal and Lefty2 in the LPM is established in the third step. The last step involves asymmetric morphogenesis. Three distinct patterns can result from asymmetric morphology: directional looping of a tube (pattern I), differential lobation (pattern II) and one-sided regression of a structure (pattern III). Red and green represent L/R asymmetric signals; grey represents parts of the embryo that are L/R symmetric.

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be the case in practice (also see TABLE 1), although the variation of the phenotype among these mutants might be due to different genetic backgrounds or to a limited number of embryos being examined. One can also argue that the requirement for ciliary components in symmetry breaking might simply reflect a functional requirement for cytoplasmic vesicle and/or organelle transport. Perhaps the most direct approach to test the role of the nodal flow might involve mechanical manipulation of the flow and subsequent examination of its effects on L/R patterning. The role of the nodal flow in other animals is uncertain because no equivalents of monocilia, or the flow, have been found in other vertebrates. If the nodal flow is not a universal mechanism for symmetry breaking, it remains to be seen what mechanisms are used by other vertebrates. It has been suggested that in Xenopus and chick, unidirectional transport of an unknown molecule through intercellular GAP JUNCTIONS might generate its asymmetric distribution in these embryos, therefore breaking the initial symmetry25,26. Although the molecule that might be carried by the nodal flow is unknown, growth differentiation factor 1 (Gdf1) might be a good candidate. The phenotype of Gdf1 / mice 27 a lack of left-sided expression of Nodal, Lefty2 and Pitx2 is consistent with this idea. But Gdf1 is expressed in the node and the LPM, which makes it uncertain whether Gdf1 is required in the node or in the LPM, or both. Another candidate for the L/R determinant is Nodal itself. Nodal is expressed symmetrically on both sides of the node at the late neural-fold stage, before left-sided expression of Nodal and Lefty2 occurs in the lateral plate. Slightly later, at the early somite stage, this expression becomes broader and stronger on the left, but the significance of this asymmetry is not known2. However, because of the many roles of Nodal in earlier events during embryogenesis, such as A/P patterning28 and mesoderm formation29, the full significance of asymmetric Nodal expression in the node remains unclear. The phenotype of most mouse mutants with impaired initial L/R determination can be explained by the lack of nodal flow. One mutation, called Invs (also known as inv) might, however, be an exception; Invs is unique in that most of the homozygotes show L/R inversion instead of randomization30. One would have expected that L/R inversion in these embryos would result from a reversal of the direction of nodal flow. Surprisingly, the direction of the flow is normal but the flow is slow and turbulent20. It is not clear how the slow nodal flow leads to the L/R inversion, although plausible models have been proposed. For example, the delayed activation model proposes that the morphogen is inactive when secreted and is only activated several seconds after secretion20. Invs encodes a protein with 31,32 ANKYRIN repeats , which can interact with calmod33 ulin , but the function of Invs remains elusive. Functional information about Invs might provide important clues for understanding the mechanism behind symmetry breaking.

Left

Midline barrier

Right

Node Initial determination iv, Inv, Kif3, Polaris

Asymmetric expression of signalling molecules

Lefty2

Nodal

Situs-specific morphogenesis

Pitx2

"Leftness"

Figure 2 | Genetic pathway for the determination of leftright asymmetry. The initial leftright (L/R) symmetry breaking takes place in or near the node, at the neural-fold stage of embryogenesis. Analysis of various mouse mutants indicates that proteins of the kinesin superfamily that are encoded by Kif3a and Kif3b, as well as Polaris, Iv and Inv are involved at this early step. Mice with mutations at these loci, except for Inv, lack nodal flow the earliest event in L/R asymmetry determination. The initial symmetry breaking results in left-sided expression of Nodal, a transforming growth factor (TGF)--like signalling molecule. The action of Nodal is restricted by another TGF--like signalling molecule, Lefty2, which acts as a feedback inhibitor of Nodal. The Nodal signal is mediated by a transcription factor, Pitx2; but downstream targets of Pitx2 (blue circles) that could further mediate the Nodal signal are not known. Nodal also induces expression of Lefty1, a third TGF--like signalling molecule that functions as a midline barrier to signals that determine leftness. Inv, inversion of embryonic turning (inversin); iv, inversus viscerum; Kif3a,b, kinesin family member 3a,b; Pitx2, paired-like homeodomain transcription factor 2.

GAP JUNCTION

A junction between two cells through which ions and small molecules can pass.
ANKYRIN

A globular protein that links spectrin, a major cytoskeletal protein, with an integral membrane protein in the erythrocyte plasma membrane. A repetitive motif of this protein ankyrin repeat is found in many other proteins.

So, the nodal flow seems to be important in symmetry breaking, at least in the mouse. But many questions about this first step in the L/R pathway remain. Is the nodal flow a genuine symmetry-breaking event or simply a manifestation of such an event? What is the molecule that might be transported by the flow? How is the leftward flow generated from seemingly synchronized clockwise rotation of the cilia? Is it a conserved mechanism among vertebrates? If the nodal flow is directly responsible for symmetry breaking, all mutations that cause an absence of the nodal flow are expected to have the same downstream effects. As Wagner and Yost24 point out, this might not

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Lefty1

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node to the lateral plate remains unknown. One can predict two possibilities. Asymmetric signals from the node could be relayed by cascades of signalling events; some evidence to support this possibility has been found in the chick 35. Sonic hedgehog (Shh), which is expressed on the left side of the node1, could travel to the paraxial mesoderm on the left side, where it would induce expression of Caronte, which encodes a member of the Cerberus-DAN family3638. Caronte would then travel to the left side of the LPM where it would induce Nodal expression. However, this mechanism is unlikely to be conserved because Caronte homologues have not been identified in other vertebrates. Nonetheless, several lines of indirect evidence indicate the involvement of bone morphogenetic protein (Bmp) in the signalling cascade between the node and LPM. For example, the lack of Smad5 (also known as Madh5, MAD homologue 5), a transducer of Bmp signalling, results in bilateral Nodal expression in the LPM39, which indicates that Bmp is required to repress Nodal expression on the right side of the embryo. Because Bmp expression is bilateral, both sides probably receive Bmp-mediated repression but the left side is released from the repression by an unknown anti-Bmp activity. Alternatively, asymmetric signals that are generated in the node might reach the LPM by diffusion, either passively or by regulated diffusion that involves endocytosis. Importantly, this formal possibility is strengthened by the fact that Nodal can diffuse over a long distance40,41.
Nodal and Lefty

b R A

P L c

node
Figure 3 | The node and monocilia. Scanning electron micrographs of a, b | the node and c | monocilia (arrowhead) in the 8.0 days post coitum (dpc) mouse embryo. The anteroposterior (A/P) and leftright (L/R) axes are indicated. Magnifications: a, 170; b, 600; c, 6,000.
CERBERUS-DAN FAMILY

A family of secreted glycoproteins with a cysteine knot that is characterized by nine conserved cysteines and a cysteine knot region. Some of these family members have antiBMP and/or anti-Nodal activity.
PRECHORDAL PLATE

Several other genes are asymmetrically expressed in the vicinity of the node; they include Dante, which encodes a member of the CERBERUS-DAN FAMILY34, but the significance of their asymmetric expression remains to be examined. It also remains to be seen whether the asymmetric gene expression in or near the node always correlates with that in the LPM.
Transfer of asymmetric signals

The ventral layer of the midline in a region that is rostral to the notochordal plate. It contacts the overlying neural plate at the future forebrain.

For the initial asymmetry to be propagated, asymmetric signals that arise in the node must travel to the lateral plate, but how they are transferred from the

Box 1 | Two types of cilium Two distinct types of cilium are known: 9+2 cilia contain a ring of nine peripheral doublets of microtubles plus a pair of central microtubles, whereas 9+0 cilia, also known as primary cilia or monocilia, lack the central pair of microtubules. 9+2 cilia are typically found in the respiratory epithelium, reproductive system (for example, the oviduct) and central nervous system (for example, ependyma). 9+0 cilia are found in various organs, including skin (keratinocytes), kidney (renal tubular epithelial cells) and blood vessels (in endothelial cells), but their functions remain largely unknown. 9+0 cilia are also found in early mouse embryos, in particular, on the 9+2 9+0 cells of the midline structures, such as PRECHORDAL PLATE, node and notochordal plate15. Central Sulik et al.15 were the pair first to recognize that these cilia are motile, but unidirectional rotation of the cilia was first observed by Microtubule Inner Outer doublet dynein arm dynein arm Nonaka et al.16.

Nodal is a typical member of the TGF- superfamily42 that, in the presence of an EGFCFC protein co-factor, transduces signals through its receptors Alk4 and Alk7 (type I receptors), and ActRIIa and ActRIIb (type II activin receptors, also known as Acvr2 and Acvr2b, respectively), to activate Smad2 (MAD homologue 2), Smad4 and Fast (also known as Foxh1, forkhead box H1) transcription factors (FIG. 4; for a review, see REF. 43). Other transcription factors that are known to interact with Smads might also be involved in Nodal signalling (for a review, see REF. 44). Lefty1 and Lefty2 (REFS 4,45) are unusual among the TGF- superfamily members because they lack a cysteine residue that is required for their dimerization, which might be important because TGF- ligands are thought to bind their receptors as dimers. Genetic evidence indicates that Lefty proteins act as antagonists of Nodal, although the mechanism by which this is achieved is unknown.
Expression patterns

Expression of Nodal and the Lefty genes is asymmetric and transient, and Nodal and Lefty2 are both expressed in the LPM exclusively on the left side24,45 (FIG. 5). Their expression on the left side of the LPM begins in a small region adjacent to the node, at the 23-somite stage, it then expands bidirectionally along the A/P axis, and disappears by the 67-somite stage. Lefty1 is mainly expressed in the PROSPECTIVE FLOOR PLATE (PFP) on the left
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side, and its expression is also transient: it begins at the two-somite stage in a region near the node, expands anteriorly and disappears by the 56-somite stage. When the expression patterns of Nodal, Lefty1 and Lefty2 are carefully compared with each other, Nodal expression begins slightly earlier than that of Lefty2 (see below; C. Meno et al., unpublished observations). Regulation. Studies of the transcriptional regulation that underlies asymmetric expression of the Lefty genes and Nodal have revealed interesting regulatory loops (FIG. 6). Both Nodal and Lefty2 have an asymmetric enhancer (ASE), which is necessary and sufficient for their left-sided expression 4648 (FIG. 6). ASEs of Nodal 47,48, Lefty2 (REF. 46) and human LEFTY2 (REF. 49) share several common sequence motifs, the most remarkable of which is the presence of two copies of the sequence AATCCACA, which acts as a Nodalresponsive element. This motif is recognized by Foxh1 (Fast)50, a transcription factor that was originally identified as a transducer of TGF-/Activin signals in Xenopus51. Foxh1 is expressed on both sides of the LPM when Nodal and Lefty2 are expressed on the left side of the LPM, and it can mediate Nodal signals in addition to TGF-/Activin signals50. Experiments on Foxh1/ mice have confirmed that this transcription

PROSPECTIVE FLOOR PLATE

The most ventral part of the neural plate that is located at the midline. It will differentiate into the floor plate and is important in specifying the ventral character of the spinal cord.

Table 1 | Mouse mutants with leftright patterning defects


Gene name ActRIIb Cryptic Fgf8* Foxa2 (Hnf3-) Foxj1 (Hfh4) Furin Gdf1 Inv iv (lrd) Kif3a,b Lefty1 Lefty2 Mgat1 Expected site of function LPM LPM and/or node Node and/or LPM Node Node LPM Node and/or LPM Node Node Node Notochordal plate/floor plate LPM ND Main leftright asymmetry phenotype Right pulmonary isomerism Randomized situs and right isomerism Right isomerism Embryonic lethal (mutants fail to undergo looping and embryonic turning) Situs solitus, situs inversus or heterotaxia Embryonic lethal (mutants fail to undergo looping and embryonic turning) Randomized situs and right pulmonary isomerism Situs inversus or heterotaxia Situs solitus, situs inversus, heterotaxia or isomerism Embryonic lethal (randomized looping; most mutants do not start embryonic turning) Left isomerism Left isomerism Embryonic lethal (mutants often have inverted looping and fail to complete embryonic turning) Randomized looping, right pulmonary isomerism Randomized embryonic turning, defects in the positioning of the abdominal viscera and the heart Right pulmonary isomerism Embryonic lethal (randomized looping, mutants fail to undergo embryonic turning) Embryonic lethal (right isomerism) Embryonic lethal (randomized looping) Looping is partly affected Embryonic lethal (mutants fail to undergo looping and embryonic turning) Embryonic lethal (randomized looping) Embryonic lethal (randomized looping, no initiation of embryonic turning) Embryonic lethal (mutants fail to undergo looping and embryonic turning) Nodal expression in the LPM ND Absent Absent Absent ND Left Absent Right Left, right, bilateral or absent ND (Lefty2 is bilateral or absent) Bilateral Bilateral ND References 59 57 69 2,55 22,23 54 27 30 21,105 1618 56 41 106

Nodal Nodal/Foxa2

LPM and possibly node Possibly node

Absent Bilateral

62 2

Nodal/Smad2 No turning Pitx2 Polaris Shh Shh/Ihh Sil Smad5 Smoothened

LPM and possibly node Notochordal plate/floor plate LPM Node and notochordal plate/floor plate Notochordal plate/floor plate Node and/or LPM Notochordal plate/floor plate LPM Node and/or LPM

ND Left, right or bilateral Left Bilateral Bilateral Absent Bilateral Bilateral Absent

107 67 8183 19 6870 101 68 39 101

*Hypomorphic allele; double heterozygote; double homozygous mutant. ActRIIb, activin receptor IIb; Fgf8, fibroblast growth factor 8; Foxa2 (Hnf3-), forkhead box A2; Foxj1 (Hfh4), forkhead box J1; Gdf1, growth differentiation factor A1; Inv, inversion of embryonic turning (inversin); iv (lrd), inversus viscerum (leftright dynein); Kif3a,b, kinesin family member 3a,b; LPM, lateral plate mesoderm; Mgat1, mannoside acetylglucosaminyltransferase 1; ND, not determined; Pitx2, paired-like homeodomain transcription factor 2; Shh/Ihh, sonic hedgehog homologue/Indian hedgehog homologue; Sil, Tal1 interrupting locus; Smad2,5, MAD homologue 2,5.

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expressed on the right side, but Nodal is not. Therefore, Nodal and Lefty2 might be regulated by additional mechanisms that do not involve the Nodalresponsive Foxh1-dependent ASE. It is also possible that there are functional differences between Nodal and Lefty2 ASEs. Functions. Nodal acts as a determinant for leftness (FIG. 2) because cells that have received Nodal signals will adopt left-side morphology, whereas those that lack Nodal signals will adopt right-side morphology. So, mutant mice, such as Lefty1/ (REF. 56), in which Nodal is bilaterally expressed in the LPM, will eventually develop left isomerism. This means that both sides take the left-side characteristics, such as the presence of monolobed lungs on both sides (the lung has four lobes on the right-hand side). Conversely, those mutants that lack Nodal activity in the LPM, such as Cryptic / (REFS 57,58) and ActRIIb / (REF. 59) mutants, develop right isomerism in the lung (four-lobed lungs on both sides). Perhaps the role of Nodal is to induce expression of genes such as Pitx2 in the cells that lie in the left half of the LPM (mainly SPLANCHNOPLEURE and SOMATOPLEURE) and to instruct them to adopt left-side morphology at later stages. Lefty2 is not a left-side determinant per se, but rather a regulator that restricts the duration and the site of Nodal action. Nodal and Lefty2 set up feedback loops in the left side of the lateral plate. So, in Lefty2ASE/ASE mice that lack the left-sided expression of Lefty2, Nodal expression persists longer than it does in normal mice41: whereas Nodal expression normally terminates at the 67-somite stage, it persists until the 89-somite stage in the Lefty2ASE/ASE mutants. In addition, asymmetric expression of Pitx2 is also upregulated in the mutant embryos. Therefore, the absence of Lefty2, the feedback inhibitor, results in increased expression and functional activity of Nodal. The regulatory loops between Nodal and the Lefty genes are essential not only for L/R patterning, but also for other embryonic patterning processes, such as A/P patterning and formation, and patterning of the mesoderm28,29,6063. For example, during gastrulation, Nodal is required for mesoderm formation and Lefty2 restricts this activity; accordingly, Nodal-null mutant mice fail to form mesoderm, whereas Lefty2-null mutants have an expanded primitive streak and form excess mesoderm.
Reactiondiffusion in leftright determination

Lefty

Nodal

Nodal

ActRII EGFCFC Alk4

ActRII EGFCFC Alk4

Smads FAST

No signals

Nucleus Pitx2

Nucleus

Figure 4 | Actions of Nodal and the Lefty proteins. Genetic evidence indicates that Lefty proteins act as antagonists of Nodal, but the mechanism by which this is achieved remains unknown. Current models indicate that the Lefty proteins and Nodal might compete with each other for ActRII binding. EGFCFC proteins are co-receptors for Nodal and the Lefty proteins that directly interact with Alk4, but not with ActRII103,104. a | The Nodal signal is transduced through the ActRII receptor and Smads and Fast transcription factors that, on their translocation to the nucleus, activate downstream genes, such as Pitx2. b | When Lefty proteins are bound to ActRII, no signals are transduced. ActRII, activin receptor II; Alk4, type I activin receptor; EGFCFC, epidermal growth factorcripto, FRL-1, cryptic; Pitx2, paired-like homeodomain transcription factor 2; Smad5, MAD homologue 5.

SPLANCHNOPLEURE

Dorsal layer of the lateral plate that will develop to form surface structures such as the body wall and limbs.
SOMATOPLEURE

Ventral layer of the lateral plate that will contribute to visceral organs.

factor mediates Nodal signals during embryonic patterning52,53. Nodal induces Lefty2 and its own transcription through ASEs, which indicates that the amplification of Nodal and Lefty2 expression throughout the LPM is brought about by Nodal acting on the ASEs (FIG. 6). On the basis of the above observations, it is possible that the following sequence of events establishes the asymmetric expression of Nodal and Lefty2 in the lateral plate. First, the left-sided expression of Nodal is initiated by an unknown factor. Nodal regulates its own expression in a positive-feedback loop and activates Lefty2 expression on the left side of the LPM. Components of the Nodal-signalling pathway, including an EGFCFC protein (most likely Cryptic), Fast and the Smad2Smad4 regulatory complex, are all required for the maintenance of Nodal expression and for the induction of Lefty2 expression. Lefty2 is an inhibitor of Nodal signalling and, as a result of its antagonism of this signal, the left-sided expression of Nodal and Lefty2 is rapidly terminated. The effect of this feedback mechanism is to restrict the range and duration of Nodal signalling in the developing embryo. However, the relationship between Nodal and Lefty2 described here might be too simplistic. In some cases, for example in Furin-deficient mice54 and in Hnf3-/ (also known as Foxa2, forkhead box A2) aggregation chimaeras55, Lefty2 is ectopically

The relationship between Nodal and the Lefty proteins resembles that of two hypothetical molecules that comprise a reactiondiffusion mechanism as proposed by Alan Turing 64. This theoretical model describes two diffusible molecules, one of which is an activator that stimulates both its own synthesis and the synthesis of its partner, which is an inhibitor. The model requires that the inhibitor diffuses more rapidly than the activator. The outcome of the reactiondiffusion mechanism depends on at least five parameters: initial synthesis rate of the activator, diffusion rates of the activator and of the inhibitor, and degradation rates of the activator and of the inhibitor.
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But how could a reactiondiffusion mechanism be involved in L/R determination? First, the asymmetric expression of Nodal and Lefty2 begins as a small domain on the left side of the LPM, at the level of the node, and gradually expands in both directions along the A/P axis. This rapid expansion of their expression domains could be achieved through a reactiondiffusion mechanism, but a simple version of the model cannot explain why the expression of Nodal and Lefty2 stops at or near the midline and never extends to the right side. To adequately explain the process of L/R determination, additional factors such as Bmp-mediated repression of Nodal on the right of the midline need to be integrated into the model. Second, and perhaps more importantly, the reactiondiffusion system might be the mechanistic basis of generation of random asymmetry the second component of the original Brown and Wolpert model11. A reactiondiffusion system has the potential to amplify an initially small difference. For example, let us imagine a situation in which an activator is induced at two points across the midline (FIG. 7). If the level of the activator on one side of the midline is slightly higher than that on the other side, the former side will take over and the activator level on that side will be amplified, whereas that on the opposite side will disappear. This mechanism is itself random but if there were a bias that would predetermine which side had the initially slightly higher level of the activator, then that side would always win. This bias could be introduced by the nodal flow, which always flows towards the left. So, when the NodalLefty reactiondiffusion system is combined with the nodal flow, it could generate an asymmetry such as the exclusively left-sided Nodal expression. The simplest version of this model, in which Nodal itself would be the determinant that is carried by the flow, can be tested experimentally by the specific removal of Nodal expression in the node. It remains to be seen whether this conceptually attractive model will fit experimental data.
The midline barrier
Nodal

Nodal (dpc) R L

Lefty1,2

Pitx2

8.2

9.5

Figure 5 | Asymmetric expression of Nodal, Lefty1, Lefty2 and Pitx2. Whole-mount in situ hybridization of Nodal, Lefty1, Lefty2 and Pitx2 in mouse embryos at 8.2 and 9.5 days post coitum (dpc) is shown. Lefty1 and Lefty2 expression patterns are shown in the same embryos: blue staining at the midline and that in the left portion of the lateral plate mesoderm represents Lefty1 and Lefty2 expression, respectively. In the 9.5-dpc embryo, only Pitx2 is expressed. Pitx2, paired-like homeodomain transcription factor 2.

Activities of Nodal and the Lefty proteins, and the transcriptional regulatory relationship between Nodal and the Lefty genes described above, indicate that these two genes might comprise a reactiondiffusion system. Continuing studies of Nodal and Lefty2 proteins tagged with green fluorescent protein indicate that they might act over a long range and that Lefty2 might diffuse faster than Nodal (R. Sakuma et al., unpublished observations). If these observations are confirmed, the NodalLefty connection might be the first example of a reactiondiffusion mechanism in a developing embryo.

Smads Fast

Lefty2

Nodal ASE ASE

Lefty2

Figure 6 | Transcriptional relationship between Nodal and the Lefty genes. Nodal regulates its own expression in a positive-feedback loop and activates Lefty2 expression on the left side of the lateral plate mesoderm. This is achieved through its interaction with the Nodal and Lefty2 asymmetric enhancers (ASEs), which are Nodal-responsive Fast/Foxh1-dependent enhancers. Lefty2 then acts as a feedback inhibitor of Nodal. Smads, MAD homologues.

The midline structures, such as the floor plate and the notochord, are important for L/R patterning. Embryological experiments in Xenopus 65,66, and mutant analysis in zebrafish and mouse56, have given rise to an idea that midline structures are required to separate the left and right halves of embryos until L/R patterning is completed. For example, surgical removal of the midline structures, including the notochord, from Xenopus embryos randomizes heart looping and gut coiling, and leads to bilateral expression of Xnr-1, a Nodal-related gene in Xenopus 65,66. Analysis of Lefty1 mutant mice indicates that Lefty1 functions as a specific midline barrier56 (FIG. 2). Midline structures, including the floor plate and the notochord, develop normally even in the absence of Lefty1, but a lack of Lefty1 leads to typical midline barrier defects, such as bilateral expression of the left-specific genes Nodal, Lefty2 and Pitx2, and pulmonary leftisomerism56. This phenotype indicates that Lefty1

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How could Lefty1 function as the midline barrier? The barrier would have to restrict the passage of a signal (factor X) across the midline. In the simplest model, Lefty1 protein itself could physically interact with factor X to prevent its diffusion. Caronte, an antagonist of Bmp that has been identified in chick36,37, is thought to be a good candidate for factor X, because Caronte is expressed in the paraxial mesoderm on the left side and it can induce Nodal expression. However, so far, there is no evidence for a direct interaction between the Caronte and Lefty proteins, and Caronte homologues have not been found in other vertebrates. Alternatively, Nodal itself could be factor X. In support of this possibility, recent findings41 indicate that Nodal is a long-range-acting molecule (see above). Lefty1 from the floor plate might bind to Nodal receptors in the adjacent area and prevent Nodal activity from being propagated across the midline. The midline barrier might be necessary only when the left-side signals, such as Nodal and Lefty2, are present. In fact, Lefty1 expression in the floor plate begins slightly earlier than that of Nodal expression on the left side of the LPM. Continued Lefty1 expression seems to require Nodal signals because Lefty1 expression is lost in Cryptic mutants57, which lack the ability to encode a co-receptor for Nodal. Therefore, Lefty1 might be induced by Nodal that has diffused from the left side of the LPM to the floor plate. Understanding the exact mechanism that underlies Lefty1 expression in the midline will require analysis of its transcriptional regulatory elements. There is indirect evidence that the anterior and posterior portions of the midline barrier are formed and/or maintained by distinct mechanisms. When Lefty1 is absent, ectopic expression of the left-side-specific genes is mainly detected in the anterior region of the right side of the LPM56. So, Lefty1 might be a component of the anterior midline barrier, and the posterior portion of the midline barrier might involve a different mechanism. Consistent with this idea, the posterior border of Lefty1 expression ends at the node. The nature of the posterior midline barrier that lies posterior to the node remains unclear, although it is known that excess retinoic acid specifically perturbs the posterior midline function71,72.
Situs-specific morphogenesis

Left

Right

Step 1 node LPM LPM

Step 2

Step 3

RD

Step 4

RD

Step 5

Figure 7 | Model for the reactiondiffusion system in leftright determination. Nodal expression is induced in the peri-node regions on both sides (red arrows, step 1). Nodal protein travels bidirectionally to the lateral plate mesoderm (LPM) (black horizontal arrows, step 1) and induces Nodal and Lefty2 (step 2). The next step involves a tug-of-war between the left and right sides of the LPM. In the simplest model, the signalling molecule transported by the nodal flow (green arrow, step 3) is Nodal. The left side of the LPM therefore receives a higher level of Nodal and expresses Nodal and Lefty2 at a higher level than the right side (step 4). Consequently, Nodal expression on the right disappears, whereas that on the left is amplified (step 5). RD, reaction diffusion; red and blue indicate gene expression on the left and the right, respectively.

induces or functions as the midline barrier that prevents an unknown left-side-specific signalling molecule from crossing the midline. This idea is supported by observations of other mutants with midline defects. For example, Shh/, Sil/ (Tal1 interrupting locus) and nt (no turning) mutant embryos, which show bilateral expression of the left-side-specific genes, all lack Lefty1 expression in the prospective floor plate (PFP)6770. Therefore, the phenotype of these mutants can be explained by the lack of Lefty1. In fact, there are no mouse mutants at present that have typical midline-barrier defects, but retain normal Lefty1 expression in the PFP.

Shortly after asymmetric Nodal expression disappears from the left side of the LPM, situs-specific morphogenesis begins. The cardiac tube loops towards the right side and the embryo turns along the A/P axis in a clockwise direction, followed by asymmetric lobe formation in the lungs, rotation and coiling of the digestive tract, and regression of parts of the vascular system on one side (FIG. 1, STEP 4). This asymmetric morphogenesis occurs in response to Nodal signalling organ primordia will adopt left-side morphology in the presence of Nodal signals, but in the absence of Nodal activity they will adopt right-side morphology.
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Pitx2, a bicoid-type homeobox transcription factor, is important in asymmetric morphogenesis in response to Nodal7378. Pitx2 is asymmetrically expressed in the left side of the LPM, as are Nodal and Lefty2. Asymmetric Pitx2 expression begins in the left LPM concomitantly with Nodal and Lefty2, but persists for much longer (FIG. 5). So, Pitx2 expression is still apparent in the primordia of most asymmetric organs at the late somite stage: main blood vessels, such as the sinus venosus, vitelline vein and cardinal vein, the common atrial chamber of the heart, the septum transversus (which will contribute to the liver) and a foregut region that corresponds to the future lung bud all of these organs will lie on the left side of the body. Recent analysis of transcriptional regulation of Pitx2 (REF. 79) showed that its asymmetric expression is conferred by an enhancer that contains three Fast-binding sites and a binding site for the homeobox transcription factor Nkx2. The Fast-binding sites are essential for initiating asymmetric expression, whereas the Nkx2-binding site is dispensable for the initiation but necessary for the maintenance of late-stage expression. Therefore, the left-sided expression of Pitx2 is initiated by Nodal signalling and is maintained by Nkx2. This L/R asymmetric expression pattern of Pitx2 is conserved among all the vertebrates that have been examined so far. Pitx2 is responsible for generating left-side morphology of at least some of the visceral organs. For example, Pitx2-null mutant mice show typical right isomerism in the lung (four lobes on both sides)8084. But, because of the additional severe defects in the Pitx2-null mutants, L/R defects of other visceral organs, such as the stomach, gut, liver, spleen and vascular system, have not been analysed. So, it is not clear whether Pitx2 is required for normal development of all the asymmetric organs or only a subset. To understand the exact role of Pitx2 in asymmetric morphogenesis, it will be necessary to examine Pitx2-null mutants that specifically lack asymmetric expression of Pitx2, and to compare their visceral organ phenotype with that of Nodal conditional mutants. Data available at present indicate that Nodal induces left-side-specific morphology both by Pitx2dependent and -independent mechanisms. Pitx2 is asymmetrically expressed in many primordial organs, but if it induces distinct sets of target genes in different organs, the readout of Nodal signalling could be different in each location: it could involve accelerated or decelerated growth rate, apoptosis or cell migration, depending on the type of gene that was induced. Unfortunately, no Pitx2 targets have yet been identified that are relevant to L/R asymmetry. So far, few vertebrate genes have been found that are only expressed on the right, although some genes show a subtle right-sided bias. cSna, a Snail-related gene in the chick, is expressed exclusively on the right side of chick embryos85 and has been proposed to negatively regulate Pitx2. The mouse homologue of cSna (Sna) is also expressed in the LPM on the right side86, but it is not as obviously asymmetric as in the chick. Sna might participate in right-side-specific morphogenesis, but so far its role has not been tested genetically. Nkx3.2 (also known
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as Bapx1, bagpipe homeobox gene 1 homologue) is also expressed asymmetrically36,87. In mouse, this is expressed symmetrically in the paraxial mesoderm, but only on the right in the LPM. Mutant mice that lack Nkx3.2 do not have obvious situs defects8890, but they lack a spleen and have morphological abnormalities of the gastroduodenal portion of the gut, which might be regarded as L/R positional defects. So, the role of Nkx3.2 in L/R asymmetric morphogenesis needs to be examined further before any firm conclusions can be drawn.
Conservation of the leftright pathway

The cascade in which Pitx2 is positively regulated by Nodal and the Lefty genes (FIG. 2) forms the central part of the L/R pathway and is conserved among vertebrates from the zebrafish to mouse77,91100. So, Pitx2, Nodal and the Lefty genes not only have similar roles, but also are regulated in a similar manner77,91100. It is possible, however, that the mechanisms that lead to left-sided Nodal expression have not been conserved. As described earlier, a mechanism that involves the nodal flow and rotation of the nodal cilia might not operate in vertebrates other than mammals. Also, Shh has different roles in chick and in mouse. In the chick, Shh is expressed on the left side of the node and can induce a left-side programme1, but in the mouse, Shh is required for the formation of the midline, including the floor plate. Shh/ mutant mouse embryos lose Lefty1 expression and have midline defects and bilateral Nodal expression, which is associated with left isomerism of the lung66,67. Mutant mice that lack both Shh and Indian hedgehog homologue (Ihh), lose Gdf1 expression in the node and the LPM, as well as Cryptic expression in the LPM101. Therefore, the Hedgehog signal, made up of a combination of Shh and Ihh signals, also functions as a basic component that is required for left-side specification. In addition, fibroblast growth factor 8 (Fgf8) is also involved; it seems to function upstream of Nodal both in the chick and the mouse, but in the chick it acts as one of the right-side determinants102, whereas in the mouse it is required for left-side signalling 69. Therefore, vertebrates seem to have adopted diverse strategies for setting up asymmetric Nodal expression.
Conclusion

The past few years have seen rapid progress in our understanding of L/R patterning mechanisms in vertebrates. But much remains to be learnt. Obviously, one of the most challenging questions concerns the breaking of the initial symmetry. In the mouse, nodal flow is an attractive mechanism, but do cilia have a conserved function among vertebrates? If not, is there a universal mechanism for symmetry breaking? Although genetics has shown that Nodal and the Lefty proteins have crucial functions, we need to know how these proteins act in vivo. In particular, it will be important to know what happens to these signalling molecules in the embryo once they have been secreted. How far and how rapidly do they diffuse from the site of their synthesis? What mechanism is responsible for their transport is passive diffusion or active transport involved?

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There are also questions concerning the regulation of Nodal. How is its asymmetric expression initiated? How is Nodal expression restricted to the left side? Although Pitx2 is a mediator of Nodal signalling, we have no idea how Nodal induces asymmetric morphologies in various organs. Many of the key elements have been revealed and need to be analysed more completely. However, the new era of functional genomics and systematic mutagenesis in zebrafish and mouse should also bring many more genes into the L/R pathway. Finally, L/R patterning and A/P patterning seem to share a common mechanism that involves the NodalLefty regulatory loops. It will be interesting to see how much is common between these two pathways. Studies of L/R asymmetry might yet reveal a fundamental mechanism that underlies symmetry breaking in general.

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Acknowledgements
We thank other members of H. Hamadas lab for their stimulating discussion, S. Nonaka for recording the nodal flow shown in the supplementary information online, and S. Kondo and H. Meinhardt for their advice on the reactiondiffusion system. The work done in H. Hamadas lab was supported by CREST.

Online links
DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ ActRIIa | ActRIIb | Cryptic | Dante | Egf | Fast2 | Fgf8 | Gdf1 | Hfh4 | Hnf3- | Ihh | Invs | Kif3a | Kif3b | Lefty1 | Lefty2 | Nkx2 | Nkx3.2 | Nodal | nt | Pitx2 | Polaris | Shh | Sil | Smad2 | Smad4 | Smad5 | Sna | Snail OMIM: http://www.ncbi.nlm.nih.gov/Omim/ Kartagener syndrome FURTHER INFORMATION Encyclopedia of Life Sciences: http://www.els.net/ Chordate and vertebrate body plans | Vertebrate embryo: establishment of leftright asymmetry Access to this interactive links box is free online.

NATURE REVIEWS | GENETICS

VOLUME 3 | FEBRUARY 2002 | 1 1 3

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