Left-right Organizer Research Articles

Bilateral cellular flows display asymmetry prior to left-right organizer formation in amniote gastrulation.

A bilateral body plan is predominant throughout the animal kingdom. Bilaterality of amniote embryos becomes recognizable as midline morphogenesis begins at gastrulation, bisecting an embryonic field into the left and right sides, and left-right (LR) asymmetry patterning follows. While a series of laterality genes expressed after the LR compartmentalization has been extensively studied, the laterality patterning prior to and at the initiation of midline morphogenesis has remained unclear. Here, through a biophysical quantification in a high spatial and temporal resolution, applied to a chick model system, we show that a large-scale bilateral counterrotating cellular flow, termed "polonaise movements", display LR asymmetries in early gastrulation. This cell movement starts prior to the formation of the primitive streak (PS) (the earliest midline structure) and the subsequent appearance of Hensen's node (the LR organizer). The cellular flow speed and vorticity unravel the location and timing of the LR asymmetries. The bilateral flows displayed a Right dominance after 6 h since the start of cell movements. Mitotic arrest that diminishes PS formation resulted in changes in the bilateral flow pattern, but the Right dominance persisted. Our data indicate that the LR asymmetry in amniote gastrula becomes detectable earlier than suggested by current models, which assume that the asymmetric regulation of the laterality signals at the node leads to the LR patterning. More broadly, our results suggest that physical processes can play an unexpected but significant role in influencing LR laterality during embryonic development.

Breaking Left-Right Symmetry by the Interplay of Planar Cell Polarity, Calcium Signaling and Cilia.

The formation of the embryonic left-right axis is a fundamental process in animals, which subsequently conditions both the shape and the correct positioning of internal organs. During vertebrate early development, a transient structure, known as the left-right organizer, breaks the bilateral symmetry in a manner that is critically dependent on the activity of motile and immotile cilia or asymmetric cell migration. Extensive studies have partially elucidated the molecular pathways that initiate left-right asymmetric patterning and morphogenesis. Wnt/planar cell polarity signaling plays an important role in the biased orientation and rotational motion of motile cilia. The leftward fluid flow generated in the cavity of the left-right organizer is sensed by immotile cilia through complex mechanisms to trigger left-sided calcium signaling and lateralized gene expression pattern. Disrupted asymmetric positioning or impaired structure and function of cilia leads to randomized left-right axis determination, which is closely linked to laterality defects, particularly congenital heart disease. Despite of the formidable progress made in deciphering the critical contribution of cilia to establishing the left-right asymmetry, a strong challenge remains to understand how cilia generate and sense fluid flow to differentially activate gene expression across the left-right axis. This review analyzes mechanisms underlying the asymmetric morphogenesis and function of the left-right organizer in left-right axis formation. It also aims to identify important questions that are open for future investigations.

Bilateral cellular flows display asymmetry prior to left-right organizer formation in amniote gastrulation.

Bilaterians are defined by a bilaterally symmetrical body plan. Vertebrates exhibit external bilateral symmetry but display left-right (LR) asymmetry in their internal organs. Amniote embryos switch the patterning of internal organs from bilateral symmetry to LR-asymmetry. Using chick embryos as a model system, here we examined the initiation of LR symmetry breaking. Our biophysical approaches to quantify cellular flows inferred that LR symmetry breaking occurs before the formation of Hensen's node, a LR organizer, which serves as a signaling center for LR patterning-gene programs. Our work demonstrates that quantitative biophysical parameters can help unravel the initiation of LR symmetry breaking, suggesting an involvement of physical mechanisms in this critical biological patterning process.

The Heterotaxy Gene CCDC11 Is Important for Cytokinesis via RhoA Regulation.

Mutations in CCDC11 (cfap53) have been identified in multiple patients with heterotaxy (Htx), a disorder of left-right (LR) patterning of the internal organs. In Xenopus, depletion of Ccdc11 causes defects in LR patterning, recapitulating the patient phenotype. Upon Ccdc11 depletion, monociliated cells of the Left-Right Organizer (LRO) exhibit multiple cilia per cell. Unexpectedly, we found that Ccdc11 is necessary for successful cytokinesis, explaining the multiciliation phenotype observed in Ccdc11-depleted cells. The small GTPase RhoA is critical for cytokinesis, and our Ccdc11 depletion phenotypes are reminiscent of RhoA loss of function. Here, we demonstrate that during cytokinesis CCDC11 is localized to the cytokinetic contractile ring overlapping with RhoA, and CCDC11 regulates total RhoA protein levels. Our results connect CCDC11 to cytokinesis and LR patterning via RhoA regulation, providing a potential mechanism for heterotaxy disease pathogenesis.

PKD2: An Important Membrane Protein in Organ Development.

PKD2 was first identified as the pathogenic protein for autosomal dominant polycystic kidney disease (ADPKD) and is widely recognized as an ion channel. Subsequent studies have shown that PKD2 is widely expressed in various animal tissues and plays a crucial role in tissue and organ development. Additionally, PKD2 is conserved from single-celled organisms to vertebrates. Here, we provide an overview of recent advances in the function of PKD2 in key model animals, focusing on the establishment of left-right organ asymmetry, renal homeostasis, cardiovascular development, and signal transduction in reproduction and mating. We specifically focus on the roles of PKD2 in development and highlight future prospects for PKD2 research.

Specific Mitotic Events Drive Cytoskeletal Remodeling Required for Left-Right Organizer Development.

Cellular proliferation is vital for tissue development, including the Left-Right Organizer (LRO), a transient organ critical for establishing the vertebrate LR body plan. This study investigates cell redistribution and the role of specific progenitor cells in LRO formation, focusing on cell lineage and behavior. Using zebrafish as a model, we mapped all mitotic events in Kupffer's Vesicle (KV), revealing an FGF-dependent, anteriorly enriched mitotic pattern. With a KV-specific fluorescent microtubule (MT) line, we observed that mitotic spindles align along the KV's longest axis until the rosette stage, spindles that form after spin, and are excluded from KV. Early aligned spindles assemble cytokinetic bridges that point MT bundles toward a tight junction where a rosette will initially form. Post-abscission, repurposed MT bundles remain targeted at the rosette center, facilitating actin recruitment. Additional cells, both cytokinetic and non-cytokinetic, are incorporated into the rosette, repurposing or assembling MT bundles before actin recruitment. These findings show that initial divisions are crucial for rosette assembly, MT patterning, and actin remodeling during KV development.

CC2D1A causes ciliopathy, intellectual disability, heterotaxy, renal dysplasia, and abnormal CSF flow.

Intellectual and developmental disabilities result from abnormal nervous system development. Over a 1,000 genes have been associated with intellectual and developmental disabilities, driving continued efforts toward dissecting variant functionality to enhance our understanding of the disease mechanism. This report identified two novel variants in CC2D1A in a cohort of four patients from two unrelated families. We used multiple model systems for functional analysis, including Xenopus, Drosophila, and patient-derived fibroblasts. Our experiments revealed that cc2d1a is expressed explicitly in a spectrum of ciliated tissues, including the left-right organizer, epidermis, pronephric duct, nephrostomes, and ventricular zone of the brain. In line with this expression pattern, loss of cc2d1a led to cardiac heterotaxy, cystic kidneys, and abnormal CSF circulation via defective ciliogenesis. Interestingly, when we analyzed brain development, mutant tadpoles showed abnormal CSF circulation only in the midbrain region, suggesting abnormal local CSF flow. Furthermore, our analysis of the patient-derived fibroblasts confirmed defective ciliogenesis, further supporting our observations. In summary, we revealed novel insight into the role of CC2D1A by establishing its new critical role in ciliogenesis and CSF circulation.

Myosin1G promotes Nodal signaling to control zebrafish left-right asymmetry

Myosin1D (Myo1D) has recently emerged as a conserved regulator of animal Left-Right (LR) asymmetry that governs the morphogenesis of the vertebrate central LR Organizer (LRO). In addition to Myo1D, the zebrafish genome encodes the closely related Myo1G. Here we show that while Myo1G also controls LR asymmetry, it does so through an entirely different mechanism. Myo1G promotes the Nodal-mediated transfer of laterality information from the LRO to target tissues. At the cellular level, Myo1G is associated with endosomes positive for the TGFβ signaling adapter SARA. myo1g mutants have fewer SARA-positive Activin receptor endosomes and a reduced responsiveness to Nodal ligands that results in a delay of left-sided Nodal propagation and tissue-specific laterality defects in organs that are most distant from the LRO. Additionally, Myo1G promotes signaling by different Nodal ligands in specific biological contexts. Our findings therefore identify Myo1G as a context-dependent regulator of the Nodal signaling pathway.

Natural reversal of cavefish heart asymmetry is controlled by Sonic Hedgehog effects on the left-right organizer.

The direction of left-right visceral asymmetry is conserved in vertebrates. Deviations of the standard asymmetric pattern are rare, and the underlying mechanisms are not understood. Here, we use the teleost Astyanax mexicanus, consisting of surface fish with normal left-oriented heart asymmetry and cavefish with high levels of reversed right-oriented heart asymmetry, to explore natural changes in asymmetry determination. We show that Sonic Hedgehog (Shh) signaling is increased at the posterior midline, Kupffer's vesicle (the teleost left-right organizer) is enlarged and contains longer cilia, and the number of dorsal forerunner cells is increased in cavefish. Furthermore, Shh increase in surface fish embryos induces asymmetric changes resembling the cavefish phenotype. Asymmetric expression of the Nodal antagonist Dand5 is equalized or reversed in cavefish, and Shh increase in surface fish mimics changes in cavefish dand5 asymmetry. Shh decrease reduces the level of right-oriented heart asymmetry in cavefish. Thus, naturally occurring modifications in cavefish heart asymmetry are controlled by the effects of Shh signaling on left-right organizer function.

Molecular anatomy of emerging Xenopus left-right organizer at successive developmental stages.

Vertebrate left-right symmetry breaking is preceded by formation of left-right organizer. In Amphibian, this structure is formed by gastrocoel roof plate, which emerges from superficial suprablastoporal cells. GRP is subdivided into medial area, which generates leftward flow by rotating monocilia and lateral Nodal1 expressing areas, which are involved in sensing of the flow. After successful symmetry breaking, medial cells are incorporated into a deep layer where they contribute to the axial mesoderm, while lateral domains join somitic mesoderm. Here, we performed detailed analysis of spatial and temporal gene expression of important markers and the corresponding morphology of emerging GRP. Endodermal marker Sox17 and markers of superficial mesoderm display complementary patterns at all studied stages. At early stages, GRP forms Tekt2 positive epithelial domain clearly separated from underlying deep layers, while at later stages, this separation disappears. Marker of early somitic mesoderm MyoD1 was absent in emerging GRP and was induced together with Nodal1 during early neurulation. Decreasing morphological separation is accompanied by lateral to medial covering of GRP by endoderm. Our data supports continuous link between superficial mesoderm at the start of gastrulation and mature GRP and suggests late induction of somitic fate in lateral GRP.

Calaxin is a key factor for calcium-dependent waveform control in zebrafish sperm.

Calcium is critical for regulating the waveform of motile cilia and flagella. Calaxin is currently the only known molecule involved in the calcium-dependent regulation in ascidians. We have recently shown that Calaxin stabilizes outer arm dynein (OAD), and the knockout of Calaxin results in primary ciliary dyskinesia phenotypes in vertebrates. However, from the knockout experiments, it was not clear which functions depend on calcium and how Calaxin regulates the waveform. To address this question, here, we generated transgenic zebrafish expressing a mutant E130A-Calaxin deficient in calcium binding. E130A-Calaxin restored the OAD reduction of calaxin -/- sperm and the abnormal movement of calaxin -/- left-right organizer cilia, showing that Calaxin's stabilization of OADs is calcium-independent. In contrast, our quantitative analysis of E130A-Calaxin sperms showed that the calcium-induced asymmetric beating was not restored, linking Calaxin's calcium-binding ability with an asymmetric flagellar beating for the first time. Our data show that Calaxin is a calcium-dependent regulator of the ciliary beating and a calcium-independent OAD stabilizer.

Molecular Pathways and Animal Models of Defects in Situs.

Left-right patterning is among the least well understood of the three axes defining the body plan, and yet it is no less important, with left-right patterning defects causing structural birth defects with high morbidity and mortality, such as complex congenital heart disease, biliary atresia, or intestinal malrotation. The cell signaling pathways governing left-right asymmetry are highly conserved and involve multiple components of the TGF-β superfamily of cell signaling molecules. Central to left-right patterning is the differential activation of Nodal on the left, and BMP signaling on the right. In addition, a plethora of other cell signaling pathways including Shh, FGF, and Notch also contribute to the regulation of left-right patterning. In vertebrate embryos such as the mouse, frog, or zebrafish, the specification of left-right identity requires the left-right organizer (LRO) containing cells with motile and primary cilia that mediate the left-sided propagation of Nodal signaling, followed by left-sided activation of Lefty and then Pitx2, a transcription factor that specifies visceral organ asymmetry. While this overall scheme is well conserved, there are striking species differences, including the finding that motile cilia do not play a role in left-right patterning in some vertebrates. Surprisingly, the direction of heart looping, one of the first signs of organ left-right asymmetry, was recently shown to be specified by intrinsic cell chirality, not Nodal signaling, possibly a reflection of the early origin of Nodal signaling in radially symmetric organisms. How this intrinsic chirality interacts with downstream molecular pathways regulating visceral organ asymmetry will need to be further investigated to elucidate how disturbance in left-right patterning may contribute to complex CHD.

Calcium signaling mediates proliferation of the precursor cells that give rise to the ciliated left-right organizer in the zebrafish embryo.

Several of our internal organs, including heart, lungs, stomach, and spleen, develop asymmetrically along the left-right (LR) body axis. Errors in establishing LR asymmetry, or laterality, of internal organs during early embryonic development can result in birth defects. In several vertebrates-including humans, mice, frogs, and fish-cilia play a central role in establishing organ laterality. Motile cilia in a transient embryonic structure called the "left-right organizer" (LRO) generate a directional fluid flow that has been proposed to be detected by mechanosensory cilia to trigger asymmetric signaling pathways that orient the LR axis. However, the mechanisms that control the form and function of the ciliated LRO remain poorly understood. In the zebrafish embryo, precursor cells called dorsal forerunner cells (DFCs) develop into a transient ciliated structure called Kupffer's vesicle (KV) that functions as the LRO. DFCs can be visualized and tracked in the embryo, thereby providing an opportunity to investigate mechanisms that control LRO development. Previous work revealed that proliferation of DFCs via mitosis is a critical step for developing a functional KV. Here, we conducted a targeted pharmacological screen to identify mechanisms that control DFC proliferation. Small molecule inhibitors of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) were found to reduce DFC mitosis. The SERCA pump is involved in regulating intracellular calcium ion (Ca2+) concentration. To visualize Ca2+ in living embryos, we generated transgenic zebrafish using the fluorescent Ca2+ biosensor GCaMP6f. Live imaging identified dynamic cytoplasmic Ca2+ transients ("flux") that occur unambiguously in DFCs. In addition, we report Ca2+ flux events that occur in the nucleus of DFCs. Nuclear Ca2+ flux occurred in DFCs that were about to undergo mitosis. We find that SERCA inhibitor treatments during DFC proliferation stages alters Ca2+ dynamics, reduces the number of ciliated cells in KV, and alters embryo laterality. Mechanistically, SERCA inhibitor treatments eliminated both cytoplasmic and nuclear Ca2+ flux events, and reduced progression of DFCs through the S/G2 phases of the cell cycle. These results identify SERCA-mediated Ca2+ signaling as a mitotic regulator of the precursor cells that give rise to the ciliated LRO.

Fluid extraction from the left-right organizer uncovers mechanical properties needed for symmetry breaking.

Humans and other vertebrates define body axis left-right asymmetry in the early stages of embryo development. The mechanism behind left-right establishment is not fully understood. Symmetry breaking occurs in a dedicated organ called the left-right organizer (LRO) and involves motile cilia generating fluid-flow therein. However, it has been a matter of debate whether the process of symmetry breaking relies on a chemosensory or a mechanosensory mechanism (Shinohara et al., 2012). Novel tailored manipulations for LRO fluid extraction in living zebrafish embryos allowed us to pinpoint a physiological developmental period for breaking left-right symmetry during development. The shortest critical time-window was narrowed to one hour and characterized by a mild counterclockwise flow. The experimental challenge consisted in emptying the LRO of its fluid, abrogating simultaneously flow force and chemical determinants. Our findings revealed an unprecedented recovery capacity of the embryo to re-fil and re-circulate new LRO fluid. The embryos that later developed laterality problems were found to be those that had lower anterior angular velocity and thus less anterior-posterior heterogeneity. Next, aiming to test the presence of any secreted determinant, we replaced the extracted LRO fluid by a physiological buffer. Despite some transitory flow homogenization, laterality defects were absent unless viscosity was altered, demonstrating that symmetry breaking does not depend on the nature of the fluid content but is rather sensitive to fluid mechanics. Altogether, we conclude that the zebrafish LRO is more sensitive to fluid dynamics for symmetry breaking.

Single cell RNA analysis of the left–right organizer transcriptome reveals potential novel heterotaxy genes

The establishment of left–right patterning in mice occurs at a transient structure called the embryonic node or left–right organizer (LRO). Previous analysis of the LRO has proven challenging due to the small cell number and transient nature of this structure. Here, we seek to overcome these difficulties to define the transcriptome of the LRO. Specifically, we used single cell RNA sequencing of 0–1 somite embryos to identify LRO enriched genes which were compared to bulk RNA sequencing of LRO cells isolated by fluorescent activated cell sorting. Gene ontology analysis indicated an enrichment of genes associated with cilia and laterality terms. Furthermore, comparison to previously identified LRO genes identified 127 novel LRO genes, including Ttll3, Syne1 and Sparcl1, for which the expression patterns were validated using whole mount in situ hybridization. This list of novel LRO genes will be a useful resource for further studies on LRO morphogenesis, the establishment of laterality and the genetic causes of heterotaxy.

A spatiotemporal barrier formed by Follistatin is required for left–right patterning

How left-right (LR) asymmetry emerges in a patterning field along the anterior-posterior axis remains an unresolved problem in developmental biology. Left-biased Nodal emanating from the LR organizer propagates from posterior to anterior (PA) and establishes the LR pattern of the whole embryo. However, little is known about the regulatory mechanism of the PA spread of Nodal and its asymmetric activation in the forebrain. Here, we identify bilaterally expressed Follistatin (Fst) as a regulator blocking the propagation of the zebrafish Nodal ortholog Southpaw (Spaw) in the right lateral plate mesoderm (LPM), and restricting Spaw transmission in the left LPM to facilitate the establishment of a robust LR asymmetric Nodal patterning. In addition, Fst inhibits the Activin-Nodal signaling pathway in the forebrain thus preventing Nodal activation prior to the arrival, at a later time, of Spaw emanating from the left LPM. This contributes to the orderly propagation of asymmetric Nodal activation along the PA axis. The LR regulation function of Fst is further confirmed in chick and frog embryos. Overall, our results suggest that a robust LR patterning emerges by counteracting a Fst barrier formed along the PA axis.

Rab8, Rab11, and Rab35 coordinate lumen and cilia formation during zebrafish left-right organizer development.

An essential process during Danio rerio's left-right organizer (Kupffer's Vesicle, KV) formation is the formation of a motile cilium by developing KV cells which extends into the KV lumen. Beating of motile cilia within the KV lumen directs fluid flow to establish the embryo's left-right axis. However, the timepoint at which KV cells start to form cilia and how cilia formation is coordinated with KV lumen formation have not been examined. We identified that nascent KV cells form cilia at their centrosomes at random intracellular positions that then move towards a forming apical membrane containing cystic fibrosis transmembrane conductance regulator (CFTR). Using optogenetic clustering approaches, we found that Rab35 positive membranes recruit Rab11 to modulate CFTR delivery to the apical membrane, which is required for lumen opening, and subsequent cilia extension into the lumen. Once the intracellular cilia reach the CFTR positive apical membrane, Arl13b-positive cilia extend and elongate in a Rab8 dependent manner into the forming lumen once the lumen reaches an area of 300 μm2. These studies demonstrate the need to acutely coordinate Rab8, Rab11, and Rab35-mediated membrane trafficking events to ensure appropriate timing in lumen and cilia formation during KV development.

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.

Congenital heart defects caused by FOXJ1.

FOXJ1 is expressed in ciliated cells of the airways, testis, oviduct, central nervous system and the embryonic left-right organizer. Ablation or targeted mutation of Foxj1 in mice, zebrafish and frogs results in loss of ciliary motility and/or reduced length and number of motile cilia, affecting the establishment of the left-right axis. In humans, heterozygous pathogenic variants in FOXJ1 cause ciliopathy leading to situs inversus, obstructive hydrocephalus and chronic airway disease. Here, we report a novel truncating FOXJ1 variant (c.784_799dup; p.Glu267Glyfs*12) identified by clinical exome sequencing from a patient with isolated congenital heart defects (CHD) which included atrial and ventricular septal defects, double outlet right ventricle (DORV) and transposition of the great arteries. Functional experiments show that FOXJ1 c.784_799dup; p.Glu267Glyfs*12, unlike FOXJ1, fails to induce ectopic cilia in frog epidermis in vivo or to activate the ADGB promoter, a downstream target of FOXJ1 in cilia, in transactivation assays in vitro. Variant analysis of patients with heterotaxy or heterotaxy-related CHD indicates that pathogenic variants in FOXJ1 are an infrequent cause of heterotaxy. Finally, we characterize embryonic-stage CHD in Foxj1 loss-of-function mice, demonstrating randomized heart looping. Abnormal heart looping includes reversed looping (dextrocardia), ventral looping and no looping/single ventricle hearts. Complex CHDs revealed by histological analysis include atrioventricular septal defects, DORV, single ventricle defects as well as abnormal position of the great arteries. These results indicate that pathogenic variants in FOXJ1 can cause isolated CHD.

Pathogenic variants in CLXN encoding the outer dynein arm docking–associated calcium-binding protein calaxin cause primary ciliary dyskinesia

PurposePrimary ciliary dyskinesia (PCD) is a heterogeneous disorder that includes respiratory symptoms, laterality defects, and infertility caused by dysfunction of motile cilia. Most PCD-causing variants result in abnormal outer dynein arms (ODAs), which provide the generative force for respiratory ciliary beating and proper mucociliary clearance. MethodsIn addition to studies in mouse and planaria, clinical exome sequencing and functional analyses in human were performed. ResultsIn this study, we identified homozygous pathogenic variants in CLXN (EFCAB1/ODAD5) in 3 individuals with laterality defects and respiratory symptoms. Consistently, we found that Clxn is expressed in mice left-right organizer. Transmission electron microscopy depicted ODA defects in distal ciliary axonemes. Immunofluorescence microscopy revealed absence of CLXN from the ciliary axonemes, absence of the ODA components DNAH5, DNAI1, and DNAI2 from the distal axonemes, and mislocalization or absence of DNAH9. In addition, CLXN was undetectable in ciliary axonemes of individuals with defects in the ODA-docking machinery: ODAD1, ODAD2, ODAD3, and ODAD4. Furthermore, SMED-EFCAB1-deficient planaria displayed ciliary dysmotility. ConclusionOur results revealed that pathogenic variants in CLXN cause PCD with defects in the assembly of distal ODAs in the respiratory cilia. CLXN should be referred to as ODA-docking complex–associated protein ODAD5.