Cilia, calcium and the basis of left-right asymmetry

The clockwise rotation of cilia in the developing mammalian embryo drives a leftward flow of liquid; this genetically regulated biophysical force specifies left-right asymmetry of the mammalian body. How leftward flow is interpreted and information propagated to other tissues is the subject of debate. Four recent papers have shed fresh light on the possible mechanisms.


The role of the node in establishing L-R asymmetry in early mammalian development
In the mouse embryo, the LR axis is established at approxi mately 8.25 days of development. The 8.25day embryo is relatively simple; an obvious head and heart lie at the anterior end and a midline containing the noto chord runs down the middle, with the pit shaped struc ture known as the node at its posterior end ( Figure 1). Immediately to either side of the midline sits the paraxial mesoderm, containing the somites (from which trunk muscle and skeletal tissue develop). This is flanked by the lateral plate mesoderm (LPM), a lineage that will in later development contribute to asymmetric organ structure. Overlying the mesodermal tissue is a thin layer of endoderm that gives rise to the gut. As revealed in the papers to be discussed here, the endoderm also plays a previously unrecognized part in LR patterning.
Over the past 15 years, a general model of the establishment of LR asymmetry has emerged ( Figure 1). The first indication that bilateral symmetry of the embryo has been broken is the LR asymmetric expression of certain genes in regions flanking the node as well as more laterally, in the LPM. Upstream of asymmetric gene expres sion, the rotation of motile cilia within the node (or the equivalent structure in other vertebrates) causes a leftward flow of fluid, called 'nodal flow' [811]. In the mouse, cilia project from the ventral surface of the node; these cilia are polarized with respect to the anterior posterior axis and by rotating in a clockwise direction, drive nodal flow leftwards [12]. Nodal flow has been shown to be both necessary and sufficient to define the left side of the mouse embryo [13,14]. The high incidence of LR patterning defects in humans with immotile cilia suggests that the same is true in humans [15]; approxi mately 50% of patients with immotile or abnormally motile cilia exhibit situs inversus [16]. Downstream of nodal fl ow, asymmetric Ca 2+ signaling is seen at the edges of the node, with stronger signaling on the left side than on the right [17]. In the interests of simplicity, this article will focus on our understanding of the determination of LR asymmetry in the mouse, and will address two ques tions: how is LR asymmetry established at the mouse node; and how is that asymmetry subsequently trans ferred over several cell diameters to the LPM.

Establishing and maintaining asymmetry: the nodal signaling cascade
Downstream of the initial breaking of symmetry at the node, the Nodal signaling cascade is activated in the left, but not the right, LPM ( Figure 1). Nodal, a member of the transforming growth factorbeta (TGFbeta) signal ing family of intercellular signaling proteins, functions as a dimer. Importantly, only those cells in the left and right LPM are competent to respond to Nodal signaling. In the left LPM, Nodal signaling induces expression of the No dal gene itself, the Lefty2 gene, which encodes an antagonist of Nodal signaling, and the Pitx2 gene, which encodes a transcription factor that acts downstream of Nodal. Lefty2 is also a TGFbeta family member, but unlike Nodal it functions as a monomer, and diff uses faster and further than Nodal [1820]. Once Nodal is expressed in the left LPM, the resultant production of Lefty2 sup presses the Nodal cascade in the right LPM, helping to lock in asymmetry [21]. Nodal and Lefty2 are expressed for only 6 to 8 hours. In contrast, once activated, Pitx2 remains asymmetrically expressed in the left LPM for the next two days, so that Pitx2 protein is present in the left LPM during organogenesis [2224]. Th is has led to the proposition that Pitx2 is the ultimate eff ector of leftness [25]. While this is not absolutely the case, asymmetry of Pitx2 expression does underlie asymmetry of many organs [26,27].

Detecting fl ow in the node: the three hypotheses
Th e mechanisms by which nodal fl ow is 'perceived' by the embryo remain the subject of debate, with three main hypotheses currently in contention ( Figure 2). Th e 'morphogen hypothesis' argues that a shortlived mole cule becomes enriched on the left side of the node in response to nodal fl ow and that this higher concentration on the left is detected, leading to a LR asymmetric signal [8]. Both computational and experimental investigation argue that such asymmetric enrichment is plausible for molecules between 15 and 50 kDa in size, although the nature of the morphogen and the receptors are unknown [28,29]. A second hypothesis, the 'nodal vesicular parcel (NVP) hypothesis' posits the presence of membrane bounded vesicles that are carried leftwards, breaking in a ciliadependent fashion on the left side of the node where they deliver a cargo of morphogens [30]. Although very appealing, elements of this hypothesis (such as the mecha nism of NVP breaking) clearly need to be modifi ed [31]. Finally, the 'twocilia hypothesis' argues that immotile sensory cilia detect fl ow directly on the left but not the righthand side of the node [17]. Th is model was pre dicated on the known function of the polycystic kidney disease 1 (Pkd1) and polycystic kidney disease 2 (Pkd2) genes in the kidney the proteins encoded by these genes form a complex that detects urine fl ow and gives rise to a Ca 2+ signal in response [32]. Although Pkd1 is not required for LR determination [33], both Pkd2 and the Pkd1 homologue Pkd1like 1 (Pkd1l1) are involved in LR patterning, being needed for the embryo to respond to nodal fl ow [34,35]. However, whether nodal fl ow pushing outwards on the lefthand side of the node can truly be diff erentiated from the pull exerted by nodal fl ow on the righthand side of the node, a requirement of the two cilia model, has been questioned [36]. For all these models, the outcome is an asymmetric (L greater than R) Ca 2+ signal at the node. Currently, only the twocilia hypo thesis (and the known function of Pkd1l1/Pkd2) provides a mechanism to explain how this signal might be generated [17].

investigating ciliary function
A recent study from Hamada and colleagues (Shinohara et al. [37]) utilizes a mixture of genetics, biophysics and imaging to examine the establishment of LR asymmetry at the node. Th e authors make the striking fi nding that just two rotating cilia are suffi cient to break LR sym metry. In previous studies, nodal fl ow has been examined by following the movement of small numbers of particles across the node, allowing overall direction a lity and speed of fl ow to be assessed. For this study Shinohara et al. used an approach called particle image velocimetry (PIV), which they have customized for nodalfl ow analysis [38]. Utilizing a high density of fl uorescent beads within a living node and highspeed confocal imaging of a single optical plane, they followed small variations in particle position over many frames, building up a vector map of fl ow and forces across the entire node. At a local level this provides far more information than the particletracking approaches used up to now, and it seems likely that PIV  will become a new standard for the field. With PIV analysis Shinohara et al. confirm that the early node (8.0 day embryo) has a weak leftward flow, which they demon strate to be present at the time when the first asymmetric gene expression becomes apparent at the node: asymmetry of Cerberuslike 2 (Cerl2; also known as Dand5), an antagonist of Nodal signaling expressed more strongly on the right than the left side of the node. However, asymmetry of the Nodal cascade in the LPM occurs slightly later, once a stronger, more robust, leftwards nodal flow is acting at around the two to three somite stage (8.25day embryo). Using a nontoxic viscous solution (methylcellulose), Shinohara et al. slowed, and even stopped, nodal flow. This allowed them to demon strate that only a weak flow and/or a small temporal window of flow is required to break LR symmetry and drive asymmetric gene expression at the node (Cerl2) and in the left LPM (the Nodal cascade).
In order to investigate the role of altered nodal flow in greater detail, Shinohara et al. sought genetic approaches to perturb it. The Rfx3 locus encodes a transcription factor required for normal ciliogenesis in the node; muta tion of this gene leads to a massive reduction in nodal cilia number and to embryos demonstrating overt LR patterning defects [39]. Loss of the Dpcd (deleted in primary ciliary dyskinesia) locus similarly results in LR patterning defects [40]. Both loci show incomplete penetrance, which led Shinohara et al. to examine Rfx3mutant embryos in greater detail [37]. Analysis of nodal cilia motility by light microscopy revealed that a few rotating cilia were present in these nodes. By maintaining the embryos in culture, the authors were able to image nodal cilia motility and correlate it with subsequent asym metric gene expression. They found mildly affected embryos to have four or five rotating cilia, accompanied by normal LR asymmetric gene expression. In contrast, severely affected embryos had at most a single rotating cilium and showed symmetrical Cerl2 expression at the node and complete absence of LPM Nodal expression. Further analysis revealed that only two rotating cilia were required to establish normal sidedness. In embryos with three or more rotating cilia, the addition of methyl cellulose to reduce flow resulted in loss of sidedness, underlining the role of the remaining level of flow in situs determination. Finally, the position of the rotating cilia within the node (whether they were near the left or right side) was addressed, and strikingly, it emerged that their position within the node bore no relationship to their ability to determine situs: something that might have implications for all three models. Intriguingly, there also seems to be a reduction in the number of immotile cilia at the periphery of the node in these embryos, although this clearly does not affect their ability to detect flow.
It is striking that the findings of Shinohara et al. [37] argue for only a few motile cilia being required to estab lish LR asymmetry, when perhaps 200 such cilia are present within a wildtype node. This raises the question of whether this apparent excess of cilia is truly required, or whether it is an evolutionary aberration or hangover. Unused function tends to be lost when evolutionary selection pressure is removed, as in the case of eye and pigment loss in cavedwelling animals [41]. Three obvious possible explanations present themselves. First, the pre sence of higher numbers of motile cilia and prolonged flow could have subtle effects on LR determination that are not being assessed in these studies, possibly influ encing the precise timing or extent of asymmetric gene expression, or some other unknown event downstream of nodal flow. The final outcome of these events on the anatomy and physiology of the adult mouse is what is being selected. Second, the presence of many motile cilia might add robustness to the symmetrybreaking event such that deleterious outcomes (affecting cardiac pattern ing, for example) become extremely rare. Third, the system may not currently be under selection. In this case we might expect a loss of function to be occurring, and perhaps for variation to be evident between different strains and species of mice. Studies in other types of organism and the production of adult mice that have developed from embryos with small numbers of motile cilia may be able to shed some light on this.
Shinohara et al. do not directly address the question of what mechanism might underlie flow sensing in the node. The weak flow produced by two rotating cilia would primarily change morphogen concentrations only very locally, and the authors surmise that this would slow down, but not necessarily destroy, a morphogenbased mecha nism. The impact of weak flow on mechanosensa tion (in the twocilia model) would also be noticeable, although the authors argue how it might be possible for forces created by these cilia to be directly transduced (almost instantaneously) across the node. On the basis of developmental timing, they argue that the twocilia model is more likely to be true.
In a subsequent study, Hamada and colleagues (Yoshiba et al. [42]) have addressed aspects of the mechanism down stream of nodal flow. They have investigated the role of cilia and Pkd2 in the crown cells that surround the edge of the node (Figure 1), cells that contain primarily immotile cilia. As a result of the speed at which the early embryo grows and develops, conditional gene deletion in the node is technically challenging, and it is difficult to establish whether all protein has been lost from a cell. The authors have elegantly overcome such worries by analyzing nullmutant embryos into which regionalized gene expression has been reintroduced by transgenesis. In this way they reveal that expression of Pkd2 (a gene normally broadly expressed in the early embryo) is required solely in the node crown cells for normal LR patterning to occur; expression driven specifi cally in the remainder of the node did not rescue LR patterning. Th ey then showed that Pkd2 protein must be localized to cilia for it to function in LR patterning. Finally, utilizing the Kif3anull mutant (Kif3a encodes a motor protein required for cilia formation), Yoshiba et al. created embryos in which cilia were present only in the node crown cells. By then applying an artifi cial fl ow across the node, they were able to activate the normal downstream LR pathway in these mice. Th is led them to propose a model in which fl ow is detected through cilialocalized Pkd2 protein in node crown cells, which in turn leads to repression of Cerl2 on the left side of the node. While these data fi t well with the twocilia hypothesis, the mechanism by which fl ow or a morphogen is detected remains unaddressed. Clearly, Pkd1l1 must be a prime candidate for performing this function, although it remains to be established whether Pkd1l1 responds to a morphogen or to fl ow [34,43].

Transferring asymmetry from the node: intra-or extracellular communication?
How asymmetric information moves from the node out to the LPM is also the subject of competing hypotheses (Figure 3). When Brueckner and colleagues [17] originally described the generation of asymmetric Ca 2+ at the node, they commented that (at least on occasions) the asym metric signal spread as far as the lateral plate. Th is clearly provides one possible mechanism by which asymmetric information might travel out from the node by Ca 2+ moving intracellularly from cell to cell. In contrast, Hamada and colleagues built on two other facts: that Nodal expression at the node is required for Nodal activation in the LPM [44,45]; and the recognized ability of Nodal to activate its own expression [46,47]. Th ey proposed that asymmetrically distributed extracellular Nodal protein at the node is transported more readily leftwards, through the extracellular matrix [48]. Two recent papers provide signifi cant advances in our under standing of the mechanisms of these processes.
In one paper, Hadjantonakis and colleagues (Viotti et al. [49]) report that Sox17null embryos exhibit defective LR patterning; Sox17 encodes an Srybox containing protein that is required for normal defi nitive endoderm formation [50]. Th ese embryos do not express the Nodal cascade in either the left or the right LPM but, signifi cantly, retain asymmetric gene expression at the node. Th is clearly suggests that communication between the node and the LPM requires the defi nitive endoderm, a result reminiscent of the observations of McGrath et al. [17]. Together, these data suggested that calciuminduced calcium release was signaling between cells in the defi ni tive endoderm; such signals are known to travel via gap junctions. By surveying gapjunction protein expression in normal embryos, Viotti et al. found that the core gap junction protein connexin 43 (also known as Gja1) was expressed in the defi nitive endoderm. However, it proved to be absent from the endoderm of Sox17 mutant embryos. Loss of connexin 43 does not, of course, directly prove a loss of gapjunction function. Viotti et al. therefore assessed this by injecting dye into defi nitive endoderm cells, revealing that in wildtype embryos small, but not large, dye molecules moved between cells through gap junctions. Th e dyes did not cross into other cell lineages and, signifi cantly, never moved into or crossed the midline, demonstrating that the left and right sides of the embryo are distinct and not linked by gap junctions. In the absence of such a barrier, both sides of the embryo  Figure 2) and tissues lateral to the node. The cells of the node are shown in blue, the endoderm in green, the ectoderm in red, paraxial mesoderm in yellow and lateral plate mesoderm in purple. Viotti and colleagues' [49] and Saund and colleagues' work [51] argues that calcium signaling, via gap junctions, carries signals from the node leftwards through the endoderm. Oki and colleagues' analyses [48] argue that Nodal protein itself travels leftwards through an extracellular, but intraembryonic, route and directly activates the Nodal locus in the lateral plate. would be activated by any signal mediated through the endoderm. When Sox17-null embryos were examined, the dyes did not migrate between cells, demonstrating a loss of gapjunction connections. The role of gap junctions was firmly established when pharmacological agents were used to block gapjunction function in wild type embryos, and this reproduced the LR patterning defects seen in the Sox17-null embryos. This work shows that the definitive endoderm and gap junctions are required for the transfer of LR asymmetry signals from the node to the LPM.

Gap junction Nodal Ca 2+
While Viotti et al. have shown that a gapjunction dependent Ca 2+ signal can travel from the node to the left LPM, they stop short of providing a mechanism by which Ca 2+ might activate Nodal expression in the LPM. In contrast, a model proposed by Oki et al. [48] does provide an explanation for Nodal activation in the LPM Nodal moving out from the node. However, the Oki model provides no explanation of the role of asymmetric Ca 2+ signaling in the endoderm. It is of course tempting to combine these two models: a simple combined Viotti Oki model might propose that calcium signaling in the endoderm influences the underlying cell matrix, which in turn affects the ability of Nodal protein to propagate from the node to the left LPM.In a contemporaneous study, Saijoh and colleagues (Saund et al. [51]) have independently identified the role of Sox17 and the definitive endoderm in LR patterning [51]. These authors specifically investigated the link between loss of Sox17 function and the proposed intraembryonic, extra cellular route for Nodal. They examined extracellular matrix proteins previously implicated in the translocation of Nodal protein, revealing a defect in a proportion of Sox17 mutant embryos. However, a smaller proportion of embryos exhibited such defects than exhibited abnormal LR patterning. Saund et al. therefore argue that this change is not the primary cause of the LR defects in the Sox17 mutants. Of course, this does not fully exclude the possibility that a combination of similar defects, includ ing those changes that they detected, are influencing intraembryonic extracellular transport of Nodal to the left LPM.

prospects and questions
Although significant advances in the understanding of LR determination have been made, gaps still remain. Even following these most recent studies, it is evident that we do not fully understand how nodal flow leads to LR asymmetry. Both the twocilia and morphogen hypo theses remain entirely plausible, and both have their champions within the field. Discriminating between them is not simple, and short of the identification of the putative ligand central to the morphogen hypothesis, may remain so, as if the twocilia hypothesis is correct, no such morphogen exists. These studies are moving into the realm of the biophysicist, and will increasingly require an understanding of equations and modeling. An appreciation of the node as a (low Reynolds number) microfluidic environment, in which inertia effectively disappears, is required. In such an environment, our 'realworld' experiences can lead us to expect outcomes that are in fact incorrect, taking us far from reality; a very accessible discussion of such environments, and life at low Reynolds number, is available in the excellent article by Purcell [52].
Is it possible that both the twocilia and morphogen mechanisms are acting in concert, providing two signals of 'leftness' in the node? Clearly, in such a scenario these might both have an impact on Nodal expression, but it is also possible to envisage that they might have different targets. Indeed, Pitx2, the most downstream gene of the Nodal signaling cascade, does not affect gross cardiac situs [26], implying that there must be additional asym metrically expressed loci controlling this process. The simple explanation is that such loci are directly controlled by asymmetric Nodal expression in the LPM; in other words, that there are additional unidentified Nodal target genes at the end of the Nodal cascade. However, arguments also exist for Nodalindependent asymmetric gene expression in the LPM: analysis of the Ablim1 locus reveals it to be asymmetrically expressed in the left but not right LPM in the absence of Nodal expression [53]. Moreover, both galanin (Gal), a neuropeptide with a role in neuronal inhibition, and Pitx2 retain LR asymmetric expression in early cardiac tissue (at the anterior end of the LPM) in the absence of the Nodal coreceptor Cryptic, which is required for Nodal expression in the LPM [54]. Whether these loci are affected by one rather than another of the putative mechanisms remains uninvestigated. Further uncertainty remains downstream of Pitx2, where the target genes that facilitate asymmetric morphogenesis remain to be identified.
The presence of very early LR asymmetry, established by the initial cleavage of the embryo, has been strongly argued in Xenopus [55]. However, few such suggestions have been made for the mouse. The one exception is a purely embryological study by Gardner [56], which revealed that manipulation of the early blastomeres can affect the direction of embryonic axial rotation, but not other aspects of situs. While at present there is no explanation of how this might function at a molecular level, it implies that another system of LR determination may be acting in addition to that driven by nodal flow.
The intriguing question of how neural LR asymmetry is established in mammals remains largely unanswered. Is it linked with, or independent of, visceral asymmetry? In zebrafish, there is asymmetric expression of the Nodal cascade in the habenular nucleus of the brain (on the left but not right side), but no such association has been detected in the mouse or suggested in humans. The human LMO4 locus shows asymmetric expression in the brain of 12weekold human embryos, with stronger rightsided than leftsided expression [57]. This is, however, at a much later stage of development than the establishment of visceral asymmetry, suggesting that it may be a downstream event and/or that neural asym metry is entirely independent of visceral asymmetry. Intriguingly, expression of the mouse Lmo4 locus, while also asymmetrical, appears to be random, with individual embryos demonstrating a left or a rightsided preference in their expression [57]. Whether this reflects innate differences between mouse and human brains (and neural asymmetry) remains to be determined.
Continued study, in the mouse as well as in other organisms, will be required to unravel the nature of LR determination and its evolution. Understanding the differences between the mechanisms by which various organisms pattern their LR axes should reveal which elements of the process have remained constant and which have varied. Ultimately, this knowledge will provide insight into the evolution of LR asymmetry, how processes such as nodal flow have been gained and lost in different organisms, and perhaps to our understanding the evolutionary driving forces involved.