Nodal Cilia Research Articles

Cilia and congenital heart disease

Cilia are membrane-bound organelles that play an important role in motility and sensation.The abnormal structure and function of cilia can cause a broad range of disorders and diseases called ciliopathies.The incidence of congenital heart disease(CHD)was significantly higher in patients with ciliopathies than that in general population.The process of cardiac development involves multiple regulation mechanisms, and the abnormal cardiac development can cause congenital heart disease.Recent studies have indicated that different types of cilia, including nodal cilia and primary cilia, play different roles in the process of cardiac development.And structure defects or abnormal function in cilia can cause congenital heart disease.Some pathogenic genes of congenital heart disease are related to cilia and cilia-transduced cell signaling pathways.Abnormalities of these genes not only lead to the pathogenesis of congenital heart disease, but also influence cilia development or signaling pathways, which can cause other ciliopathies and affect the prognosis of CHD patients.In this review, we present the relationship between cilia and congenital heart disease. Key words: Nodal cilia; Cardiac primary cilia; Congenital heart disease

Nodal cilia-driven flow: Development of a computational model of the nodal cilia axoneme

Cilia-driven nodal flow is important in the determination of left-right asymmetry in the body. Several theoretical and computational models have been proposed to explain the mechanics of ciliary motion, although the full mechanism remains unknown. Here, we developed a three-dimensional nodal cilia axoneme model using a finite element-boundary element coupling method, and investigated the mechanics of nodal ciliary motion. We found that the rotational orbit was strongly dependent on the dynein activation frequency. We also investigated flow field generated by the ciliary rotation, and the flow strength decayed as r-3 at the far field from the cilium. Our numerical results also suggest that experimentally observed tilt angle θ=2π/9 is sufficiently large to make a leftward flow. These findings are helpful in better understanding ciliary motion and nodal flow.

Numerical Computation of Doubly-Periodic Stokes Flow Bounded by a Plane with Applications to Nodal Cilia

AbstractA numerical method is presented for the computation of externally forced Stokes flows bounded by the planez=0 and satisfying periodic boundary conditions in thexandydirections. The motivation for this work is the simulation of flows generated by cilia, which are hair-like structures attached to the surface of cells that generate flows through coordinated beating. Large collections of cilia on a surface can be modeled using a doubly-periodic domain. The approach presented here is to derive a regularized version of the fundamental solution of the incompressible Stokes equations in Fourier space for the periodic directions and physical space for thezdirection. This analytical expression forû(k,m;z) can then be used to compute the fluid velocityu(x,y,z) via a two-dimensional inverse fast Fourier transform for any fixed value ofz. Repeating the computation for multiple values ofzleads to the fluid velocity on a uniform grid in physical space. The zero-flow condition at the planez=0 is enforced through the use of images. The performance of the method is illustrated by numerical examples of particle transport by nodal cilia, which verify optimal particle transport for parameters consistent with previous studies. The results also show that for two cilia in the periodic box, out-of-phase beating produces considerablemore particle transport than in-phase beating.

TTC25 Deficiency Results in Defects of the Outer Dynein Arm Docking Machinery and Primary Ciliary Dyskinesia with Left-Right Body Asymmetry Randomization

Multiprotein complexes referred to as outer dynein arms (ODAs) develop the main mechanical force to generate the ciliary and flagellar beat. ODA defects are the most common cause of primary ciliary dyskinesia (PCD), a congenital disorder of ciliary beating, characterized by recurrent infections of the upper and lower airways, as well as by progressive lung failure and randomization of left-right body asymmetry. Using a whole-exome sequencing approach, we identified recessive loss-of-function mutations within TTC25 in three individuals from two unrelated families affected by PCD. Mice generated by CRISPR/Cas9 technology and carrying a deletion of exons 2 and 3 in Ttc25 presented with laterality defects. Consistently, we observed immotile nodal cilia and missing leftward flow via particle image velocimetry. Furthermore, transmission electron microscopy (TEM) analysis in TTC25-deficient mice revealed an absence of ODAs. Consistent with our findings in mice, we were able to show loss of the ciliary ODAs in humans via TEM and immunofluorescence (IF) analyses. Additionally, IF analyses revealed an absence of the ODA docking complex (ODA-DC), along with its known components CCDC114, CCDC151, and ARMC4. Co-immunoprecipitation revealed interaction between the ODA-DC component CCDC114 and TTC25. Thus, here we report TTC25 as a new member of the ODA-DC machinery in humans and mice.

PIERCE1 is critical for specification of left-right asymmetry in mice.

The specification of left-right asymmetry of the visceral organs is precisely regulated. The earliest breakage of left-right symmetry occurs as the result of leftward flow generated by asymmetric beating of nodal cilia, which eventually induces asymmetric Nodal/Lefty/Pitx2 expression on the left side of the lateral plate mesoderm. PIERCE1 has been identified as a p53 target gene involved in the DNA damage response. In this study, we found that Pierce1-null mice exhibit severe laterality defects, including situs inversus totalis and heterotaxy with randomized situs and left and right isomerisms. The spectrum of laterality defects was closely correlated with randomized expression of Nodal and its downstream genes, Lefty1/2 and Pitx2. The phenotype of Pierce1-null mice most closely resembled that of mutant mice with impaired ciliogenesis and/or ciliary motility of the node. We also found the loss of asymmetric expression of Cerl2, the earliest flow-responding gene in the node of Pierce1-null embryos. The results suggest that Pierce1-null embryos have defects in generating a symmetry breaking signal including leftward nodal flow. This is the first report implicating a role for PIERCE1 in the symmetry-breaking step of left-right asymmetry specification.

9 + 0 and 9 + 2 cilia are randomly dispersed in the mouse node.

The initial determination of left-right asymmetry is an essential process in embryonic development. In mouse embryo, cilia in the node play an important role generating the nodal flow that subsequently triggers left-right determination in the embryo. Although nodal cilia have historically been thought to have a 9 + 0 axonemal configuration, the existence of 9 + 2 cilia has been reported so far. Because the distribution of those two types of cilia within the node has not yet been reported, we assessed the arrangement of 9 + 0 and 9 + 2 cilia in the node. In this study, we concluded that most of the nodal cilia were 9 + 0 in structure and there were much fewer 9 + 2 cilia than 9 + 0 cilia. Furthermore, the two types of cilia were randomly distributed in the node with no regularity. In addition, we studied the embryonic origin of the crown cells surrounding the node to better understand their identity.

Absence of Radial Spokes in Mouse Node Cilia Is Required for Rotational Movement but Confers Ultrastructural Instability as a Trade-Off

Determination of left-right asymmetry in mouse embryos is established by a leftward fluid flow that is generated by clockwise rotation of node cilia. How node cilia achieve stable unidirectional rotation has remained unknown, however. Here we show that brief exposure to the microtubule-stabilizing drug paclitaxel (Taxol) induces randomly directed rotation and changes the ultrastructure of node cilia. In vivo observations and a computer simulation revealed that a regular 9+0 arrangement of doublet microtubules is essential for stable unidirectional rotation of node cilia. The 9+2 motile cilia of the airway, which manifest planar beating, are resistant to Taxol treatment. However, the airway cilia of mice lacking the radial spoke head protein Rsph4a undergo rotational movement instead of planar beating, are prone to microtubule rearrangement, and are sensitive to Taxol. Our results suggest that the absence of radial spokes allows node cilia to rotate unidirectionally but, as a trade-off, renders them ultrastructurally fragile.

Transportation particles in flows driven by nodal cilia processing D-shaped cones

Transportation particles in flows driven by nodal cilia processing D-shaped cones

A Computational Model of Dynein Activation Patterns that Can Explain Nodal Cilia Rotation

Normal left-right patterning in vertebrates depends on the rotational movement of nodal cilia. In order to produce this ciliary motion, the activity of axonemal dyneins must be tightly regulated in a temporal and spatial manner; the specific activation pattern of the dynein motors in the nodal cilia has not been reported. Contemporary imaging techniques cannot directly assess dynein activity in a living cilium. In this study, we establish a three-dimensional model to mimic the ciliary ultrastructure and assume that the activation of dynein proteins is related to the interdoublet distance. By employing finite-element analysis and grid deformation techniques, we simulate the mechanical function of dyneins by pairs of point loads, investigate the time-variant interdoublet distance, and simulate the dynein-triggered ciliary motion. The computational results indicate that, to produce the rotational movement of nodal cilia, the dynein activity is transferred clockwise (looking from the tip) between the nine doublet microtubules, and along each microtubule, the dynein activation should occur faster at the basal region and slower when it is close to the ciliary tip. Moreover, the time cost by all the dyneins along one microtubule to be activated can be used to deduce the dynein activation pattern; it implies that, as an alternative method, measuring this time can indirectly reveal the dynein activity. The proposed protein-structure model can simulate the ciliary motion triggered by various dynein activation patterns explicitly and may contribute to furthering the studies on axonemal dynein activity.

Mechanical Properties of a Primary Cilium As Measured by Resonant Oscillation

Primary cilia are ubiquitous mammalian cellular substructures implicated in an ever-increasing number of regulatory pathways. The well-established ciliary hypothesis states that physical bending of the cilium (for example, due to fluid flow) initiates signaling cascades, yet the mechanical properties of the cilium remain incompletely measured, resulting in confusion regarding the biological significance of flow-induced ciliary mechanotransduction. In this work we measure the mechanical properties of a primary cilium by using an optical trap to induce resonant oscillation of the structure. Our data indicate 1) the primary cilium is not a simple cantilevered beam; 2) the base of the cilium may be modeled as a nonlinear rotatory spring, with the linear spring constant k of the cilium base calculated to be (4.6 ± 0.62) × 10−12 N/rad and nonlinear spring constant α to be (−1 ± 0.34) × 10−10 N/rad2; and 3) the ciliary base may be an essential regulator of mechanotransduction signaling. Our method is also particularly suited to measure mechanical properties of nodal cilia, stereocilia, and motile cilia—anatomically similar structures with very different physiological functions.

Mammalian DRC, Ciliary, Motility and Left‐Right Body Asymmetry

Many advances made in our understanding of the molecular basis of ciliopathies in humans have come through the identification and analysis of candidate ciliary proteins in lower model systems. For example, Growth Arrest Specific 8 (Gas8) has been implicated in cilia motility based on its Chlamydomonas homolog pf2 showing it is a component of the Nexin-dynein regulatory complex (NDRC). However, the study of Gas8 in mammalian systems is very limited, and unexpectedly, recent data indicate that Gas8 localizes to the base of primary non-motile cilia and knockdown studies indicate that Gas8 has a role as a positive regulator of Smoothened activity and its ciliary localization. Here we investigate the in vivo role of Gas8 by generating a mutant mouse line. Gas8 mutant mice did not have any overt phenotypes expected if Hedgehog signaling is affected. Gas8 mutants did present with situs inversus implicating the NDRC in mammalian left-right body axis specification and in the normal function of node cilia. In addition, to the LR defects, Gas8 mutants invariably develop hydrocephalus and were sterile. Our analyses of cilia motility in the ventricles of the brain and trachea show abnormalities in cilia generated fluid movement associated with changes in beat frequency, waveform, and structure of the cilium. We show Gas8 is required for proper assembly of the inner dynein arms (IDA) and the Nexin-Dynein Regulatory Complex (NDRC). Additionally, a screen for GAS8 mutations in human patients with situs defects identified two independent homozygous missense mutations. The two independent mutations at c.595G>A and c.1172C>T interrupt the Gas8 Microtubule Association Domain (GMAD) and the putative Smoothened binding domain, respectively. We hypothesize that both of these mutations will result in disruption of NDRC assembly and cause dyskinetic motile cilia. The pathogenicity of these mutations and their effect on NDRC formation is being analyzed in Chlamydomonas, zebrafish, and mouse lines that express the human disease alleles.

Fluid Dynamics of Flow Driven by Nodal Cilia Precessing Non-circular Cones

Nodal cilia play an important role in the left-right symmetry breaking at the early stage of the mammal embryos and show an apparent rotational motion. This study is about the ow induced by cilia sweeping out circular/elliptical cones above a no-slip plane in the low Reynolds number regime. Using the regularized Stokeslet method, we examine the properties of ows generated by a single cilium and multiple cilia, especially two cilia, which are assumed to be anchored to the embryonic heart wall. For a short-term Lagrangian uid tracer trajectory, epicycles are presented as a nodal cilium precesses in either a circular cone or a non-circular cone shape. For the longterm behavior, the trajectory is periodic around both the circular and elliptical cones. Compared to the circular cone cases, the vertical variances are enhanced in elliptical cone cases. Besides tracer trajectories, uid velocity elds are presented to demonstrate the ow structure. When

Functional diversity of axonemal dyneins as assessed by in vitro and in vivo motility assays of Chlamydomonas mutants.

This review outlines the current knowledge of the functional diversity of axonemal dyneins, as revealed by studies with the model organism Chlamydomonas. Axonemal dyneins, which comprise outer and inner dynein arms, power cilia and flagella beating by producing sliding movements between adjacent outer-doublet microtubules. Outer- and inner-arm dyneins have traditionally been considered similar in structure and function. However, recent evidence suggests that they differ rather strikingly in subunit composition, axonemal arrangement, and molecular motor properties. We posit that these arms make up two largely independent motile systems; whereas outer-arm dynein can generate axonemal beating by itself under certain conditions, inner-arm dynein can generate beating only in cooperation with the central pair/radial spokes. This conclusion is supported by genome analyses of various organisms. Outer-arm dynein appears to be particularly important for nodal cilia of mammalian embryos that function for determination of left-right body asymmetry.

The prevalence of clinical features associated with primary ciliary dyskinesia in a heterotaxy population: results of a web-based survey.

Primary ciliary dyskinesia and heterotaxy are rare but not mutually exclusive disorders, which result from cilia dysfunction. Heterotaxy occurs in at least 12.1% of primary ciliary dyskinesia patients, but the prevalence of primary ciliary dyskinesia within the heterotaxy population is unknown. We designed and distributed a web-based survey to members of an international heterotaxy organisation to determine the prevalence of respiratory features that are common in primary ciliary dyskinesia and that might suggest the possibility of primary ciliary dyskinesia. A total of 49 members (25%) responded, and 37% of the respondents have features suggesting the possibility of primary ciliary dyskinesia, defined as (1) the presence of at least two chronic respiratory symptoms, or (2) bronchiectasis or history of respiratory pathogens suggesting primary ciliary dyskinesia. Of the respondents, four completed comprehensive, in-person evaluations, with definitive primary ciliary dyskinesia confirmed in one individual, and probable primary ciliary dyskinesia identified in two others. The high prevalence of respiratory features compatible with primary ciliary dyskinesia in this heterotaxy population suggests that a subset of heterotaxy patients have dysfunction of respiratory, as well as embryonic nodal cilia. To better assess the possibility of primary ciliary dyskinesia, heterotaxy patients with chronic oto-sino-respiratory symptoms should be referred for a primary ciliary dyskinesia evaluation.

Chemosignalling, mechanotransduction and ciliary behaviour in the embryonic node: Computational evaluation of competing theories.

Precise specification of left-right asymmetry is essential for patterning the internal organs of vertebrates. Within the embryonic node, posteriorly polarised cilia rotate, causing a leftward fluid flow (nodal flow) that establishes left-right asymmetry. The mechanism by which an embryo senses nodal flow remains uncertain. Existing hypotheses argue that either nodal flow carries morphogen(s) or lipid-bounded vesicles towards the left, thereby generating an asymmetric signal, and/or that mechano-sensory cilia sense this unidirectional flow, stimulating left-sided intracellular calcium signalling. To date, direct and definitive evidence supporting these hypotheses has been lacking. In this study, we conduct a multi-scale study to simulate the nodal cilia and the fluidic environment, analysing left-right signal transmission. By employing computational simulation techniques and solving the relevant three-dimensional unsteady transport equations, we study the flow pattern produced by the rotation of active cilia. By importing dilute species and particles into the computational domain, we investigate the transport of morphogens and nodal vesicular parcels, respectively. Furthermore, by extending the analysis to include the solid mechanics of passive deformable cilia and the coupling of their structural behaviour with the emerging fluid mechanics, we study the response of passive cilia to the nodal flow. Our results reproduce the unidirectional nodal flow, allowing us to evaluate the plausibility of both chemo- and mechano-sensing hypotheses. The quantitative measurements of the flow rate, the molecular transport and distribution provide guidance regarding the necessary morphogen molecular weights to break signalling symmetry. The passive sensory ciliary deformation gives indications regarding the plausibility of this mechano-signalling mechanism.

The Zic2 gene directs the formation and function of node cilia to control cardiac situs

The first molecular herald of organ asymmetry during murine embryogenesis is found at the periphery of the node in early-somite stage embryos. Asymmetric gene expression and calcium accumulation at the node occurs in response to a left-ward flow of extracellular fluid across the node, generated by motile cilia within the pit of the node and likely sensed by immotile cilia in the periphery of the node. The ciliation of node cells is controlled by a cascade of node-restricted transcription factor activity during mid-late gastrulation. Mutation of the murine Zic2 transcription factor is associated with random cardiac situs and a loss of asymmetric gene expression at the early-somite node and in the lateral plate. Zic2 is not expressed in these regions but is transiently expressed in the mid-late gastrula node at the time of ciliogenesis. The cilia of the node are overtly abnormal in Zic2 mutant embryos being dysmorphic and short relative to wild-type littermates. The expression of the Noto, Rfx3, and Foxj1 transcription factors known to regulate ciliogenesis is greatly depleted in the mid-gastrula node of mutants, as is the expression of the Pkd1l1 gene required for cilia function. Zic2 appears to be a component of the gene regulatory network that drives ciliation of node cells during gastrulation.

Arf4 Is Required for Mammalian Development but Dispensable for Ciliary Assembly

The primary cilium is a sensory organelle, defects in which cause a wide range of human diseases including retinal degeneration, polycystic kidney disease and birth defects. The sensory functions of cilia require specific receptors to be targeted to the ciliary subdomain of the plasma membrane. Arf4 has been proposed to sort cargo destined for the cilium at the Golgi complex and deemed a key regulator of ciliary protein trafficking. In this work, we show that Arf4 binds to the ciliary targeting sequence (CTS) of fibrocystin. Knockdown of Arf4 indicates that it is not absolutely required for trafficking of the fibrocystin CTS to cilia as steady-state CTS levels are unaffected. However, we did observe a delay in delivery of newly synthesized CTS from the Golgi complex to the cilium when Arf4 was reduced. Arf4 mutant mice are embryonic lethal and die at mid-gestation shortly after node formation. Nodal cilia appeared normal and functioned properly to break left-right symmetry in Arf4 mutant embryos. At this stage of development Arf4 expression is highest in the visceral endoderm but we did not detect cilia on these cells. In the visceral endoderm, the lack of Arf4 caused defects in cell structure and apical protein localization. This work suggests that while Arf4 is not required for ciliary assembly, it is important for the efficient transport of fibrocystin to cilia, and also plays critical roles in non-ciliary processes.

Targeted NGS gene panel identifies mutations in RSPH1 causing primary ciliary dyskinesia and a common mechanism for ciliary central pair agenesis due to radial spoke defects

Primary ciliary dyskinesia (PCD) is an inherited chronic respiratory obstructive disease with randomized body laterality and infertility, resulting from cilia and sperm dysmotility. PCD is characterized by clinical variability and extensive genetic heterogeneity, associated with different cilia ultrastructural defects and mutations identified in >20 genes. Next generation sequencing (NGS) technologies therefore present a promising approach for genetic diagnosis which is not yet in routine use. We developed a targeted panel-based NGS pipeline to identify mutations by sequencing of selected candidate genes in 70 genetically undefined PCD patients. This detected loss-of-function RSPH1 mutations in four individuals with isolated central pair (CP) agenesis and normal body laterality, from two unrelated families. Ultrastructural analysis in RSPH1-mutated cilia revealed transposition of peripheral outer microtubules into the ‘empty’ CP space, accompanied by a distinctive intermittent loss of the central pair microtubules. We find that mutations in RSPH1, RSPH4A and RSPH9, which all encode homologs of components of the ‘head’ structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. By high-resolution immunofluorescence we identified a loss of RSPH4A and RSPH9 along with RSPH1 from RSPH1-mutated cilia, suggesting RSPH1 mutations may result in loss of the entire spoke head structure. CP loss is seen in up to 28% of PCD cases, in whom laterality determination specified by CP-less embryonic node cilia remains undisturbed. We propose this defect could arise from instability or agenesis of the ciliary central microtubules due to loss of their normal radial spoke head tethering.

B113 体の左右を決めるマウス胚ノード繊毛の回転運動と構造に関する研究(B1-3 細胞バイオメカニクス3,軟骨)

B113 体の左右を決めるマウス胚ノード繊毛の回転運動と構造に関する研究(B1-3 細胞バイオメカニクス3,軟骨)

Mutation of HES7 in a large extended family with spondylocostal dysostosis and dextrocardia with situs inversus

Spondylocostal dysotosis (SCD) is a rare developmental congenital abnormality of the axial skeleton. Mutation of genes in the Notch signaling pathway cause SCD types 1-5. Dextrocardia with situs inversus is a rare congenital malformation in which the thoracic and abdominal organs are mirror images of normal. Such laterality defects are associated with gene mutations in the Nodal signaling pathway or cilia assembly or function. We investigated two distantly related individuals with a rare combination of severe segmental defects of the vertebrae (SDV) and dextrocardia with situs inversus. We found that both individuals were homozygous for the same mutation in HES7, and that this mutation caused a significant reduction of HES7 protein function; HES7 mutation causes SCD4. Two other individuals with SDV from two unrelated families were found to be homozygous for the same mutation. Interestingly, although the penetrance of the vertebral defects was complete, only 3/7 had dextrocardia with situs inversus, suggesting randomization of left-right patterning. Two of the affected individuals presented with neural tube malformations including myelomeningocele, spina bifida occulta and/or Chiari II malformation. Such neural tube phenotypes are shared with the originally identified SCD4 patient, but have not been reported in the other forms of SCD. In conclusion, it appears that mutation of HES7 is uniquely associated with defects in vertebral, heart and neural tube formation, and this observation will help provide a discriminatory diagnostic guide in patients with SCD, as well as inform molecular genetic testing.