Nodal cilia dynamics and the specification of the left/right axis in early vertebrate embryo development

Nodal cilia dynamics is a key factor for left/right axis determination in mouse embryos through the induction of a leftward fluid flow. So far it has not been clearly established how such dynamics is able to induce the asymmetric leftward flow within the node. Herein we propose that an asymmetric two-phase nonplanar beating cilia dynamics that involves the bending of the ciliar axoneme is responsible for the leftward fluid flow. We support our proposal with a host of hydrodynamic arguments, in silico experiments and in vivo video microscopy data in wild-type embryos and inv mutants. Our phenomenological modeling approach underscores how the asymmetry and speed of the flow depends on different relevant parameters. In addition, we discuss how the combination of internal and external mechanisms might cause the two-phase beating cilia dynamics.     

Planar Cell Polarity Enables Posterior Localization of Nodal Cilia and Left-Right Axis Determination during Mouse and Xenopus Embryogenesis

Left-right asymmetry in vertebrates is initiated in an early embryonic structure called the ventral node in human and mouse, and the gastrocoel roof plate (GRP) in the frog. Within these structures, each epithelial cell bears a single motile cilium, and the concerted beating of these cilia produces a leftward fluid flow that is required to initiate left-right asymmetric gene expression. The leftward fluid flow is thought to result from the posterior tilt of the cilia, which protrude from near the posterior portion of each cell''s apical surface. The cells, therefore, display a morphological planar polarization. Planar cell polarity (PCP) is manifested as the coordinated, polarized orientation of cells within epithelial sheets, or as directional cell migration and intercalation during convergent extension. A set of evolutionarily conserved proteins regulates PCP. Here, we provide evidence that vertebrate PCP proteins regulate planar polarity in the mouse ventral node and in the Xenopus gastrocoel roof plate. Asymmetric anterior localization of VANGL1 and PRICKLE2 (PK2) in mouse ventral node cells indicates that these cells are planar polarized by a conserved molecular mechanism. A weakly penetrant Vangl1 mutant phenotype suggests that compromised Vangl1 function may be associated with left-right laterality defects. Stronger functional evidence comes from the Xenopus GRP, where we show that perturbation of VANGL2 protein function disrupts the posterior localization of motile cilia that is required for leftward fluid flow, and causes aberrant expression of the left side-specific gene Nodal. The observation of anterior-posterior PCP in the mouse and in Xenopus embryonic organizers reflects a strong evolutionary conservation of this mechanism that is important for body plan determination.     

Defective Nodal and Cerl2 expression in the Arl13b(hnn) mutant node underlie its heterotaxia

Specification of the left-right axis during embryonic development is critical for the morphogenesis of asymmetric organs such as the heart, lungs, and stomach. The first known left-right asymmetry to occur in the mouse embryo is a leftward fluid flow in the node that is created by rotating cilia on the node surface. This flow is followed by asymmetric expression of Nodal and its inhibitor Cerl2 in the node. Defects in cilia and/or fluid flow in the node lead to defective Nodal and Cerl2 expression and therefore incorrect visceral organ situs. Here we show the cilia protein Arl13b is required for left right axis specification as its absence results in heterotaxia. We find the defect originates in the node where Cerl2 is not downregulated and asymmetric expression of Nodal is not maintained resulting in symmetric expression of both genes. Subsequently, Nodal expression is delayed in the lateral plate mesoderm (LPM). Symmetric Nodal and Cerl2 in the node could result from defects in either the generation and/ or the detection of Nodal flow, which would account for the subsequent defects in the LPM and organ positioning.     

Establishment of visceral left-right asymmetry in mammals: The role of ciliary action and leftward fluid flow in the region of Hensen’s node

During individual development of vertebrates, the anteroposterior, dorsoventral, and left-right axes of the body are established. Although the vertebrates are bilaterally symmetric outside, their internal structure is asymmetric. Of special interest is the insight into establishment of visceral left-right asymmetry in mammals, since it has not only basic but also an applied medical significance. As early as 1976, it was hypothesized that the ciliary action could be associated with the establishment of left-right asymmetry in mammals. Currently, the majority of researchers agree that the ciliary action in the region of Hensen’s node and the resulting leftward laminar fluid flow play a key role in the loss of bilateral symmetry and triggering of expression of the genes constituting the Nodal-Ptx2 signaling cascade, specific of the left side of the embryo. The particular mechanism underlying this phenomenon is still insufficiently clear. There are three competing standpoints on how leftward fluid flow induces expression of several genes in the left side of the embryo. The morphogen gradient hypothesis postulates that the leftward flow creates a high concentration of a signaling biomolecule in the left side of Hensen’s node, which, in turn, stimulates triggering of gene expression of the Nodal-Ptx2 cascade. The biomechanical hypothesis (or two-cilia model) states that the immotile cilia located in the periphery of Hensen’s node act as mechanosensors, activate mechanosensory ion channels, and trigger calcium signaling in the left side of the embryo. Finally, the “shuttle-bus model” holds that left-ward fluid flow carries the lipid vesicles, which are crashed when colliding immotile cilia in the periphery of Hensen’s node to release the contained signaling biomolecules. It is also noteworthy that the association between the ciliary action and establishment of asymmetry has been recently discovered in representatives of the lower invertebrates. In this paper, the author considers evolution of concepts on the mechanisms underlying establishment of visceral left-right asymmetry since 1976 until the present and critically reexamines the current concepts in this field of science. According to the author, serious arguments favoring the biomechanical hypothesis for determination of left-right asymmetry in mammals have been obtained.     

De Novo Formation of Left–Right Asymmetry by Posterior Tilt of Nodal Cilia

In the developing mouse embryo, leftward fluid flow on the ventral side of the node determines left–right (L-R) asymmetry. However, the mechanism by which the rotational movement of node cilia can generate a unidirectional flow remains hypothetical. Here we have addressed this question by motion and morphological analyses of the node cilia and by fluid dynamic model experiments. We found that the cilia stand, not perpendicular to the node surface, but tilted posteriorly. We further confirmed that such posterior tilt can produce leftward flow in model experiments. These results strongly suggest that L-R asymmetry is not the descendant of pre-existing L-R asymmetry within each cell but is generated de novo by combining three sources of spatial information: antero-posterior and dorso-ventral axes, and the chirality of ciliary movement.     

De novo formation of left-right asymmetry by posterior tilt of nodal cilia

In the developing mouse embryo, leftward fluid flow on the ventral side of the node determines left–right (L-R) asymmetry. However, the mechanism by which the rotational movement of node cilia can generate a unidirectional flow remains hypothetical. Here we have addressed this question by motion and morphological analyses of the node cilia and by fluid dynamic model experiments. We found that the cilia stand, not perpendicular to the node surface, but tilted posteriorly. We further confirmed that such posterior tilt can produce leftward flow in model experiments. These results strongly suggest that L-R asymmetry is not the descendant of pre-existing L-R asymmetry within each cell but is generated de novo by combining three sources of spatial information: antero-posterior and dorso-ventral axes, and the chirality of ciliary movement.     

Cell cycle arrest in node cells governs ciliogenesis at the node to break left-right symmetry

Cilia at the node generate a leftward fluid flow that breaks left-right symmetry. However, the molecular mechanisms that regulate ciliogenesis at the node are largely unknown. Here, we show that the epiblast-specific deletion of the gene encoding the BMP type 1 receptor (Acvr1) compromised development of nodal cilia, which results in defects in leftward fluid flow and, thus, abnormalities in left-right patterning. Acvr1 deficiency in mouse embryonic fibroblasts (MEFs) resulted in severe defects in their quiescence-induced primary cilia. Although the induction of quiescence in wild-type MEFs leads to an increase in the level of the cyclin-dependent kinase inhibitor p27(Kip1) and to rapid p27(Kip1) phosphorylation on Ser(10), MEFs deficient in Acvr1 show a reduction in both p27(Kip1) protein levels and in p27(Kip1) Ser(10) phosphorylation. The observed defects in cilium development were rescued by the introduction of p27(Kip1) into Acvr1-deficient MEFs, implying that BMP signaling positively controls p27(Kip1) stability in the G0 phase via p27(Kip1) Ser(10) phosphorylation, which is a prerequisite for induction of primary cilia. Importantly, in control embryos, p27(Kip1) protein is clearly present and strongly phosphorylated on Ser(10) in cells on the quiescent ventral surface of the node. By contrast, the corresponding cells in the node of Acvr1 mutant embryos were proliferative and showed a dramatic attenuation in both p27(Kip1) protein levels and phosphorylation on Ser(10). Our data suggest that cell quiescence controlled by BMP signaling via ACVR1 is required for transient formation of nodal cilia, and provide insight into the fundamental question of how the node represents the mechanistic `node' that regulates the development of left-right symmetry in vertebrates.     

A perspective on inversin

Over the past 5 years, there has been increasing evidence for the role of primary (9+0) cilia in renal physiology and in establishing the left-right axis. The cilia in the renal tract are immotile and thought to have a sensory function. Cilia at the murine embryonic node have a vortical movement that sets up a leftward flow. Inversin, the protein defective in the inv mouse and in patients with type-2 nephronophthisis, localizes to both renal and node primary cilia. However, we present evidence that it is also expressed before the node forms and that its subcellular localization in renal tubular cells is not confined to the cilia. Its role in both the pathway determining left-right axis and renal function remains to be elucidated.     

Nodal flow and the generation of left-right asymmetry

The establishment of left-right asymmetry in mammals is a good example of how multiple cell biological processes coordinate in the formation of a basic body plan. The leftward movement of fluid at the ventral node, called nodal flow, is the central process in symmetry breaking on the left-right axis. Nodal flow is autonomously generated by the rotation of cilia that are tilted toward the posterior on cells of the ventral node. These cilia are built by transport via the KIF3 motor complex. How nodal flow is interpreted to create left-right asymmetry has been a matter of debate. Recent evidence suggests that the leftward movement of membrane-sheathed particles, called nodal vesicular parcels (NVPs), may result in the activation of the non-canonical Hedgehog signaling pathway, an asymmetric elevation in intracellular Ca(2+) and changes in gene expression.     

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

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

The left-right determinant Inversin is a component of node monocilia and other 9+0 cilia

Inversin (Inv), a protein that contains ankyrin repeats, plays a key role in left-right determination during mammalian embryonic development, but its precise function remains unknown. Transgenic mice expressing an Inv and green fluorescent protein (GFP) fusion construct (Inv::GFP) were established to facilitate characterization of the subcellular localization of Inv. The Inv::GFP transgene rescued the laterality defects and polycystic kidney disease of Inv/Inv mice, indicating that the fusion protein is functional. In transgenic embryos, Inv::GFP protein was detected in the node monocilia. The fusion protein was also present in other 9+0 monocilia, including those of kidney epithelial cells and the pituitary gland, but it was not localized to 9+2 cilia. The N-terminal region of Inv (InvDeltaC) including the ankyrin repeats also localized to the node cilia and rescued the left-right defects of Inv/Inv mutants. Although no obvious abnormalities were detected in the node monocilia of Inv/Inv embryos, the laterality defects of such embryos were corrected by an artificial leftward flow of fluid in the node, suggesting that nodal flow is impaired by the Inv mutation. These results suggest that the Inv protein contributes to left-right determination as a component of monocilia in the node and is essential for the generation of normal nodal flow.     

Two populations of node monocilia initiate left-right asymmetry in the mouse

The vertebrate body plan has conserved handed left-right (LR) asymmetry that is manifested in the heart, lungs, and gut. Leftward flow of extracellular fluid at the node (nodal flow) is critical for normal LR axis determination in the mouse. Nodal flow is generated by motile node cell monocilia and requires the axonemal dynein, left-right dynein (lrd). In the absence of lrd, LR determination becomes random. The cation channel polycystin-2 is also required to establish LR asymmetry. We show that lrd localizes to a centrally located subset of node monocilia, while polycystin-2 is found in all node monocilia. Asymmetric calcium signaling appears at the left margin of the node coincident with nodal flow. These observations suggest that LR asymmetry is established by an entirely ciliary mechanism: motile, lrd-containing monocilia generate nodal flow, and nonmotile polycystin-2 containing cilia sense nodal flow initiating an asymmetric calcium signal at the left border of the node.     

The active and passive ciliary motion in the embryo node: A computational fluid dynamics model

The breaking of left–right symmetry in the mammalian embryo is believed to occur in a transient embryonic structure, the node, when cilia create a leftward flow of liquid. The two-cilia hypothesis proposes that the node contains two kinds of primary cilia: motile cilia that rotate autonomously to generate the leftward fluid flow and passive cilia that act as mechano-sensors, responding to flow. While studies support this hypothesis, the mechanism by which the sensory cilia respond to the fluid flow is still unclear. In this paper, we present a computational model of two cilia, one active and one passive. By employing computational fluid dynamics, deformable mesh computational techniques and fluid–structure interaction analysis, and solving the three-dimensional unsteady transport equations, we study the flow pattern produced by the movement of the active cilium and the response of the passive cilium to this flow. Our results reveal that clockwise rotation of the active cilium can generate a counter-clockwise elliptical rotation and overall lateral displacement for its neighboring passive one, of measurable magnitude and consistent pattern. This supports the plausibility of the two-cilia hypothesis and helps quantify the motion pattern for the passive cilium induced by this regional flow.     

The formation and positioning of cilia in Ciona intestinalis embryos in relation to the generation and evolution of chordate left-right asymmetry

In the early mouse embryo monocilia on the ventral node rotate to generate a leftward flow of fluid. This nodal flow is essential for the left-sided expression of nodal and pitx2, and for subsequent asymmetric organ patterning. Equivalent left fluid flow has been identified in other vertebrates, including Xenopus and zebrafish, indicating it is an ancient vertebrate mechanism. Asymmetric nodal and Pitx expression have also been identified in several invertebrates, including the vertebrates' nearest relatives, the urochordates. However whether cilia regulate this asymmetric gene expression remains unknown, and previous studies in urochordates have not identified any cilia prior to the larval stage, when asymmetry is already long established. Here we use Scanning and Transmission Electron Microscopy and immunofluorescence to investigate cilia in the urochordate Ciona intestinalis. We show that single cilia are transiently present on each ectoderm cell of the late neurula/early tailbud stage embryo, a time point just before onset of asymmetric nodal expression. Mapping the position of each cilium on these cells shows they are posteriorly positioned, something also described for mouse node cilia. The C. intestinalis cilia have a 9+0 ring ultrastructure, however we find no evidence of structures associated with motility such as dynein arms, radial spokes or nexin. Furthermore the 9+0 ring structure becomes disorganised immediately after the cilia have exited the cell, indicative of cilia which are not capable of motility. Our results indicate that although cilia are present prior to molecular asymmetries, they are not motile and hence cannot be operating in the same way as the flow-generating cilia of the vertebrate node. We conclude that the cilia may have a role in the development of C. intestinalis left-right asymmetry but that this would have to be in a sensory capacity, perhaps as mechanosensors as hypothesised in two-cilia physical models of vertebrate cilia-driven asymmetry.     

Regional cell shape changes control form and function of Kupffer's vesicle in the zebrafish embryo

Cilia-generated fluid flow in an 'organ of asymmetry' is critical for establishing the left-right body axis in several vertebrate embryos. However, the cell biology underlying how motile cilia produce coordinated flow and asymmetric signals is not well defined. In the zebrafish organ of asymmetry-called Kupffer's vesicle (KV)-ciliated cells are asymmetrically positioned along the anterior-posterior axis such that more cilia are placed in the anterior region. We previously demonstrated that Rho kinase 2b (Rock2b) is required for anteroposterior asymmetry and fluid flow in KV, but it remained unclear how the distribution of ciliated cells becomes asymmetric during KV development. Here, we identify a morphogenetic process we refer to as 'KV remodeling' that transforms initial symmetry in KV architecture into anteroposterior asymmetry. Live imaging of KV cells revealed region-specific cell shape changes that mediate tight packing of ciliated cells into the anterior pole. Mathematical modeling indicated that different interfacial tensions in anterior and posterior KV cells are involved in KV remodeling. Interfering with non-muscle myosin II (referred to as Myosin II) activity, which modulates cellular interfacial tensions and is regulated by Rock proteins, disrupted KV cell shape changes and the anteroposterior distribution of KV cilia. Similar defects were observed in Rock2b depleted embryos. Furthermore, inhibiting Myosin II at specific stages of KV development perturbed asymmetric flow and left-right asymmetry. These results indicate that regional cell shape changes control the development of anteroposterior asymmetry in KV, which is necessary to generate coordinated asymmetric fluid flow and left-right patterning of the embryo.     

Abnormal nodal flow precedes situs inversus in iv and inv mice.

We examined the nodal flow of well-characterized mouse mutants, inversus viscerum (iv) and inversion of embryonic turning (inv), and found that their laterality defects are always accompanied by an abnormality in nodal flow. In a randomized laterality mutant, iv, the nodal cilia were immotile and the nodal flow was absent. In a situs inversus mutant, inv, the nodal cilia was motile but could only produce very weak leftward nodal flow. These results consistently support our hypothesis that the nodal flow produces the gradient of putative morphogen and triggers the first L-R determination event.     

Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis

Cilia, as motile and sensory organelles, have been implicated in normal development, as well as diseases including cystic kidney disease, hydrocephalus and situs inversus. In kidney epithelia, cilia are proposed to be non-motile sensory organelles, while in the mouse node, two cilia populations, motile and non-motile have been proposed to regulate situs. We show that cilia in the zebrafish larval kidney, the spinal cord and Kupffer's vesicle are motile, suggesting that fluid flow is a common feature of each of these organs. Disruption of cilia structure or motility resulted in pronephric cyst formation, hydrocephalus and left-right asymmetry defects. The data show that loss of fluid flow leads to fluid accumulation, which can account for organ distension pathologies in the kidney and brain. In Kupffer's vesicle, loss of flow is associated with loss of left-right patterning, indicating that the 'nodal flow' mechanism of generating situs is conserved in non-mammalian vertebrates.