Leftward Fluid Flow Research Articles

R-Spondin 2 governs Xenopus left-right body axis formation by establishing an FGF signaling gradient

Establishment of the left-right (LR, sinistral, dextral) body axis in many vertebrate embryos relies on cilia-driven leftward fluid flow within an LR organizer (LRO). A cardinal question is how leftward flow triggers symmetry breakage. The chemosensation model posits that ciliary flow enriches a signaling molecule on the left side of the LRO that promotes sinistral cell fate. However, the nature of this sinistralizing signal has remained elusive. In the Xenopus LRO, we identified the stem cell growth factor R-Spondin 2 (Rspo2) as a symmetrically expressed, sinistralizing signal. As predicted for a flow-mediated signal, Rspo2 operates downstream of leftward flow but upstream of the asymmetrically expressed gene dand5. Unexpectedly, in LR patterning, Rspo2 acts as an FGF receptor antagonist: Rspo2 via its TSP1 domain binds Fgfr4 and promotes its membrane clearance by Znrf3-mediated endocytosis. Concordantly, we find that at flow-stage, FGF signaling is dextralizing and forms a gradient across the LRO, high on the dextral- and low on the sinistral side. Rspo2 gain- and loss-of function equalize this FGF signaling gradient and sinistralize and dextralize development, respectively. We propose that leftward flow of Rspo2 produces an FGF signaling gradient that governs LR-symmetry breakage.

Nodal flow transfers polycystin to determine mouse left-right asymmetry

Left-dominant [Ca2+]i elevation on the left margin of the ventral node furnishes the initial laterality of mouse embryos. It depends on extracellular leftward fluid flow (nodal flow), fibroblast growth factor receptor (FGFR)/sonic hedgehog (Shh) signaling, and the PKD1L1 polycystin subunit, of which interrelationship is still elusive. Here, we show that leftward nodal flow directs PKD1L1-containing fibrous strands and facilitates Nodal-mediated [Ca2+]i elevation on the left margin. We generate KikGR-PKD1L1 knockin mice in order to monitor protein dynamics with a photoconvertible fluorescence protein tag. By imaging those embryos, we have identified fragile meshwork being gradually transferred leftward involving pleiomorphic extracellular events. A portion of the meshwork finally bridges over the left nodal crown cells in an FGFR/Shh-dependent manner. As PKD1L1 N-term is predominantly associated with Nodal on the left margin and that PKD1L1/PKD2 overexpression significantly augments cellular Nodal sensitivity, we propose that leftward transfer of polycystin-containing fibrous strands determines left-right asymmetry in developing embryos.

CFAP45, a heterotaxy and congenital heart disease gene, affects cilia stability

Congenital heart disease (CHD) is the most common and lethal birth defect, affecting 1.3 million individuals worldwide. During early embryogenesis, errors in Left-Right (LR) patterning called Heterotaxy (Htx) can lead to severe CHD. Many of the genetic underpinnings of Htx/CHD remain unknown. In analyzing a family with Htx/CHD using whole-exome sequencing, we identified a homozygous recessive missense mutation in CFAP45 in two affected siblings. CFAP45 belongs to the coiled-coil domain-containing protein family, and its role in development is emerging. When we depleted Cfap45 in frog embryos, we detected abnormalities in cardiac looping and global markers of LR patterning, recapitulating the patient's heterotaxy phenotype. In vertebrates, laterality is broken at the Left-Right Organizer (LRO) by motile monocilia that generate leftward fluid flow. When we analyzed the LRO in embryos depleted of Cfap45, we discovered “bulges” within the cilia of these monociliated cells. In addition, epidermal multiciliated cells lost cilia with Cfap45 depletion. Via live confocal imaging, we found that Cfap45 localizes in a punctate but static position within the ciliary axoneme, and depletion leads to loss of cilia stability and eventual detachment from the cell's apical surface. This work demonstrates that in Xenopus, Cfap45 is required to sustain cilia stability in multiciliated and monociliated cells, providing a plausible mechanism for its role in heterotaxy and congenital heart disease.

Immotile cilia mechanically sense the direction of fluid flow for left-right determination.

Immotile cilia at the ventral node of mouse embryos are required for sensing leftward fluid flow that breaks left-right symmetry of the body. However, the flow-sensing mechanism has long remained elusive. In this work, we show that immotile cilia at the node undergo asymmetric deformation along the dorsoventral axis in response to the flow. Application of mechanical stimuli to immotile cilia by optical tweezers induced calcium ion transients and degradation of Dand5 messenger RNA (mRNA) in the targeted cells. The Pkd2 channel protein was preferentially localized to the dorsal side of immotile cilia, and calcium ion transients were preferentially induced by mechanical stimuli directed toward the ventral side. Our results uncover the biophysical mechanism by which immotile cilia at the node sense the direction of fluid flow.

Role of Wnt signaling and planar cell polarity in left-right asymmetry.

Wnt signaling plays essential roles in multiple steps of left-right (L-R) determination in development. First, canonical Wnt signaling is required to form the node, where L-R symmetry breaking takes place. Secondly, planar cell polarity (PCP) driven by non-canonical Wnt signaling polarizes node cells along the anterio-posterior (A-P) axis and provides the tilt of rotating cilia at the node, which generate the leftward fluid flow. Thus, reciprocal expression of Wnt5a/5b and their inhibitors Sfrp1, 2, 5 generates a gradient of Wnt5 activity along the embryo's anterior-posterior (A-P) axis. This polarizes cells at the node, by placing PCP core proteins on the anterior or posterior side of each node cell. Polarized PCP proteins subsequently induce asymmetric organization of microtubules along the A-P axis, which is thought to push the centrally localized basal body toward the posterior side of a node cell. Motile cilia that extend from the posteriorly-shifted basal body is tilted toward the posterior side of the embryo. Thirdly, canonical-Wnt signaling regulates the level and expansion of Nodal activity and establishes L-R asymmetric Nodal activity at the node, the first molecular asymmetry in the mouse embryo. Overall, both canonical and non-canonical Wnt signalings are essential for L-R symmetry breaking.

Biophysical Analysis of Mechanical Signals in Immotile Cilia of Mouse Embryonic Nodes Using Advanced Microscopic Techniques.

Immotile cilia of crown cells at the node of mouse embryos are required for sensing leftward fluid flow that gives rise to the breaking of left-right (L-R) symmetry. The flow-sensing mechanism has long remained elusive, mainly because of difficulties inherent in manipulating and precisely analyzing the cilium. Recent progress in optical microscopy and biophysical analysis has allowed us to study the mechanical signals involving primary cilia. In this study, we used high-resolution imaging with mechanical modeling to assess the membrane tension in a single cilium. Optical tweezers, a technique used to trap sub-micron-sized particles with a highly focused laser beam, allowed us to manipulate individual cilia. Super-resolution microscopy allowed us to discern the precise localization of ciliary proteins. Using this protocol, we provide a method for applying these techniques to cilia in mouse embryonic nodes. This method is widely applicable to the determination of mechanical signals in other cilia.

Nodal asymmetry and hedgehog signaling during vertebrate left-right symmetry breaking.

Development of visceral left–right asymmetry in bilateria is based on initial symmetry breaking followed by subsequent asymmetric molecular patterning. An important step is the left-sided expression of transcription factor pitx2 which is mediated by asymmetric expression of the nodal morphogen in the left lateral plate mesoderm of vertebrates. Processes leading to emergence of the asymmetric nodal domain differ depending on the mode of symmetry breaking. In Xenopus laevis and mouse embryos, the leftward fluid flow on the ventral surface of the left–right organizer leads through intermediate steps to enhanced activity of the nodal protein on the left side of the organizer and subsequent asymmetric nodal induction in the lateral plate mesoderm. In the chick embryo, asymmetric morphogenesis of axial organs leads to paraxial nodal asymmetry during the late gastrulation stage. Although it was shown that hedgehog signaling is required for initiation of the nodal expression, the mechanism of its asymmetry remains to be clarified. In this study, we established the activation of hedgehog signaling in early chick embryos to further study its role in the initiation of asymmetric nodal expression. Our data reveal that hedgehog signaling is sufficient to induce the nodal expression in competent domains of the chick embryo, while treatment of Xenopus embryos led to moderate nodal inhibition. We discuss the role of symmetry breaking and competence in the initiation of asymmetric gene expression.

Dmrt2 and myf5 Link Early Somitogenesis to Left-Right Axis Determination in Xenopus laevis.

The vertebrate left-right axis is specified during neurulation by events occurring in a transient ciliated epithelium termed left-right organizer (LRO), which is made up of two distinct cell types. In the axial midline, central LRO (cLRO) cells project motile monocilia and generate a leftward fluid flow, which represents the mechanism of symmetry breakage. This directional fluid flow is perceived by laterally positioned sensory LRO (sLRO) cells, which harbor non-motile cilia. In sLRO cells on the left side, flow-induced signaling triggers post-transcriptional repression of the multi-pathway antagonist dand5. Subsequently, the co-expressed Tgf-β growth factor Nodal1 is released from Dand5-mediated repression to induce left-sided gene expression. Interestingly, Xenopus sLRO cells have somitic fate, suggesting a connection between LR determination and somitogenesis. Here, we show that doublesex and mab3-related transcription factor 2 (Dmrt2), known to be involved in vertebrate somitogenesis, is required for LRO ciliogenesis and sLRO specification. In dmrt2 morphants, misexpression of the myogenic transcription factors tbx6 and myf5 at early gastrula stages preceded the misspecification of sLRO cells at neurula stages. myf5 morphant tadpoles also showed LR defects due to a failure of sLRO development. The gain of myf5 function reintroduced sLRO cells in dmrt2 morphants, demonstrating that paraxial patterning and somitogenesis are functionally linked to LR axis formation in Xenopus.

Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires a Bicc1-Ccr4 RNA degradation complex

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3′ untranslated region (3′-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3′-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3′-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node.

Randomization of Left-right Asymmetry and Congenital Heart Defects: The Role of DNAH5 in Humans and Mice.

Background - Nearly one in 100 live births presents with congenital heart defects (CHD). CHD are frequently associated with laterality defects, such as situs inversus totalis (SIT), a mirrored positioning of internal organs. Body laterality is established by a complex process: monocilia at the embryonic left-right organizer (LRO) facilitate both the generation and sensing of a leftward fluid flow. This induces the conserved left-sided Nodal signaling cascade to initiate asymmetric organogenesis. Primary ciliary dyskinesia (PCD) originates from dysfunction of motile cilia, causing symptoms such as chronic sinusitis, bronchiectasis and frequently SIT. The most frequently mutated gene in PCD, DNAH5 is associated with randomization of body asymmetry resulting in SIT in half of the patients; however, its relation to CHD occurrence in humans has not been investigated in detail so far. Methods - We performed genotype / phenotype correlations in 132 PCD patients carrying disease-causing DNAH5 mutations, focusing on situs defects and CHD. Using high speed video microscopy-, immunofluorescence-, and in situ hybridization analyses, we investigated the initial steps of left-right axis establishment in embryos of a Dnah5 mutant mouse model. Results - 65.9% (87 / 132) of the PCD patients carrying disease-causing DNAH5 mutations had laterality defects: 88.5% (77 / 87) presented with SIT, 11.5% (10 / 87) presented with situs ambiguus; and 6.1% (8 / 132) presented with CHD. In Dnah5mut/mut mice, embryonic LRO monocilia lack outer dynein arms resulting in immotile cilia, impaired flow at the LRO, and randomization of Nodal signaling with normal, reversed or bilateral expression of key molecules. Conclusions - For the first time, we directly demonstrate the disease-mechanism of laterality defects linked to DNAH5 deficiency at the molecular level during embryogenesis. We highlight that mutations in DNAH5 are not only associated with classical randomization of left-right body asymmetry but also with severe laterality defects including CHD.

New insights into ion channel-dependent signalling during left-right patterning.

The left-right organizer (LRO) in the mouse consists of pit cells within the depression, located at the end of the developing notochord, also known as the embryonic node and crown cells lining the outer periphery of the node. Cilia on pit cells are posteriorly tilted, rotate clockwise and generate leftward fluid flow. Primary cilia on crown cells are required to interpret the directionality of fluid movement and initiate flow-dependent gene transcription. Crown cells express PC1-L1 and PC2, which may form a heteromeric polycystin channel complex on primary cilia. It is still only poorly understood how fluid flow activates the ciliary polycystin complex. Besides polycystin channels voltage gated channels like HCN4 and KCNQ1 have been implicated in establishing asymmetry. How this electrical network of ion channels initiates left-sided signalling cascades and differential gene expression is currently only poorly defined.

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

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

Cilia in Left-Right Symmetry Breaking.

Visceral organs of vertebrates show left-right (L-R) asymmetry with regard to their position and morphology. Cilia play essential role in generating L-R asymmetry. A number of genes required for L-R asymmetry have now been identified in vertebrates, including human, many of which contribute to the formation and motility of cilia. In the mouse embryo, breaking of L-R symmetry occurs in the ventral node, where two types of cilia (motile and immotile) are present. Motile cilia are located at the central region of the node, and generate a leftward fluid flow. These motile cilia at the node are unique in that they rotate in the clockwise direction, unlike other immotile cilia such as airway cilia that show planar beating. The second type of cilia essential for L-R asymmetry is immotile cilia that are peripherally located immotile cilia. They sense a flow-dependent signal, which is either chemical or mechanical in nature. Although Ca2+ signaling is implicated in flow sensing, the precise mechanism remains unknown.

Mechanism for generation of left isomerism in Ccdc40 mutant embryos.

Leftward fluid flow in the mouse node is generated by cilia and is critical for initiating asymmetry of the left-right axis. Coiled-coil domain containing-40 (Ccdc40) plays an evolutionarily conserved role in the assembly of motile cilia and establishment of the left-right axis. Approximately one-third of Ccdc40lnks mutant embryos display situs defects and here we investigate the underlying mechanism. Ccdc40lnks mutants show delayed induction of markers of the left-lateral plate mesoderm (L-LPM) including Lefty1, Lefty2 and Nodal. Consistent with defective cilia motility compromising fluid flow across the node, initiation of asymmetric perinodal Cerberus like-2 (Cerl2) expression is delayed and then randomized. This is followed by delayed and then randomized asymmetric Nodal expression around the node. We propose a model to explain how left isomerism arises in a proportion of Ccdc40lnks mutants. We postulate that with defective motile cilia, Cerl2 expression remains symmetric and Nodal is antagonized equally on both sides of the node. This effectively reduces Nodal activation bilaterally, leading to reduced and delayed activation of Nodal and its antagonists in the LPM. This model is further supported by the failure to establish Nodal expression in the left-LPM with reduced Nodal gene dosage in Ccdc40lnks/lnks;NodalLacZ/+ mutants causing a predominance of right not left isomerism. Together these results suggest a model where cilia generated fluid flow in the node functions to ensure robust Nodal activation and a timely left-sided developmental program in the LPM.

Leftward Flow Determines Laterality in Conjoined Twins

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

Cilia are required for asymmetric nodal induction in the sea urchin embryo

BackgroundLeft-right (LR) organ asymmetries are a common feature of metazoan animals. In many cases, laterality is established by a conserved asymmetric Nodal signaling cascade during embryogenesis. In most vertebrates, asymmetric nodal induction results from a cilia-driven leftward fluid flow at the left-right organizer (LRO), a ciliated epithelium present during gastrula/neurula stages. Conservation of LRO and flow beyond the vertebrates has not been reported yet.ResultsHere we study sea urchin embryos, which use nodal to establish larval LR asymmetry as well. Cilia were found in the archenteron of embryos undergoing gastrulation. Expression of foxj1 and dnah9 suggested that archenteron cilia were motile. Cilia were polarized to the posterior pole of cells, a prerequisite of directed flow. High-speed videography revealed rotating cilia in the archenteron slightly before asymmetric nodal induction. Removal of cilia through brief high salt treatments resulted in aberrant patterns of nodal expression. Our data demonstrate that cilia - like in vertebrates - are required for asymmetric nodal induction in sea urchin embryos.ConclusionsBased on these results we argue that the anterior archenteron represents a bona fide LRO and propose that cilia-based symmetry breakage is a synapomorphy of the deuterostomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0128-7) contains supplementary material, which is available to authorized users.

Genetic Analysis Reveals a Hierarchy of Interactions between Polycystin-Encoding Genes and Genes Controlling Cilia Function during Left-Right Determination.

During mammalian development, left-right (L-R) asymmetry is established by a cilia-driven leftward fluid flow within a midline embryonic cavity called the node. This ‘nodal flow’ is detected by peripherally-located crown cells that each assemble a primary cilium which contain the putative Ca2+ channel PKD2. The interaction of flow and crown cell cilia promotes left side-specific expression of Nodal in the lateral plate mesoderm (LPM). Whilst the PKD2-interacting protein PKD1L1 has also been implicated in L-R patterning, the underlying mechanism by which flow is detected and the genetic relationship between Polycystin function and asymmetric gene expression remains unknown. Here, we characterize a Pkd1l1 mutant line in which Nodal is activated bilaterally, suggesting that PKD1L1 is not required for LPM Nodal pathway activation per se, but rather to restrict Nodal to the left side downstream of nodal flow. Epistasis analysis shows that Pkd1l1 acts as an upstream genetic repressor of Pkd2. This study therefore provides a genetic pathway for the early stages of L-R determination. Moreover, using a system in which cultured cells are supplied artificial flow, we demonstrate that PKD1L1 is sufficient to mediate a Ca2+ signaling response after flow stimulation. Finally, we show that an extracellular PKD domain within PKD1L1 is crucial for PKD1L1 function; as such, destabilizing the domain causes L-R defects in the mouse. Our demonstration that PKD1L1 protein can mediate a response to flow coheres with a mechanosensation model of flow sensation in which the force of fluid flow drives asymmetric gene expression in the embryo.

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.

Does stopping left-right nodal flow mirror impaired mechanosensation in the left-right organizer?

Background In most vertebrates organ asymmetries arise in early development through the left-right organizer (LRO). LRO ciliated cells induce a leftward fluid flow responsible for the asymmetric expression of Nodal pathway genes in tissues that will originate internal organs. We have recently published that charon/dand5 transcription is an early flow target in zebrafish by being expressed in the LRO, first symmetrically and later asymmetrically to the right where the fluid flow is weaker. We showed that absence of flow originates symmetric charon expression [1]. Pkd2 has been reported as part of a mechanosensor complex that senses flow and induces a calcium inward flux in kidney cells [2] and mouse LRO [3]. In agreement, mouse and zebrafish Pkd2 mutants have LR defects [4,5].

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

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