Zebrafish Left-right Organizer Research Articles

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.

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.

Tissue Flow Induces Cell Shape Changes During Organogenesis

In embryonic development, cell shape changes are essential for building functional organs, but in many cases, the mechanisms that precisely regulate these changes remain unknown. We propose that fluid-like drag forces generated by the motion of an organ through surrounding tissue could generate changes to its structure that are important for its function. To test this hypothesis, we study the zebrafish left-right organizer, Kupffer’s vesicle (KV), using experiments and mathematical modeling. During development, monociliated cells that comprise KV undergo region-specific shape changes along the anterior-posterior axis that are critical for KV function: anterior cells become long and thin, whereas posterior cells become short and squat. Here, we develop a mathematical vertex-like model for cell shapes that incorporates both tissue rheology and cell motility and constrain the model parameters using previously published rheological data for the zebrafish tailbud as well as our own measurements of the KV speed. We find that drag forces due to dynamics of cells surrounding KV could be sufficient or work in concert with previously identified mechanisms to drive KV cell shape changes during KV development. More broadly, these results suggest that cell shape changes during embryonic development and beyond could be driven by dynamic forces not typically considered in models or experiments.

Chiral Cilia Orientation in the Left-Right Organizer

Chirality is a property of asymmetry between an object and its mirror image. Most biomolecules and many cell types are chiral. In the left-right organizer (LRO), cilia-driven flows transfer such chirality to the body scale. However, the existence of cellular chirality within tissues remains unknown. Here, we investigate this question in Kupffer's vesicle (KV), the zebrafish LRO. Quantitative live imaging reveals that cilia populating the KV display asymmetric orientation between the right and left sides, resulting in a chiral structure, which is different from the chiral cilia rotation. This KV chirality establishment is dynamic and depends on planar cell polarity. While its impact on left-right (LR) symmetry breaking remains unclear, we show that this asymmetry does not depend on the LR signaling pathway or flow. This work identifies a different type of tissue asymmetry and sheds light on chirality genesis in developing tissues.

Vertebrate myosin\xa01d regulates left\u2013right organizer morphogenesis and laterality

Establishing left–right asymmetry is a fundamental process essential for arrangement of visceral organs during development. In vertebrates, motile cilia-driven fluid flow in the left–right organizer (LRO) is essential for initiating symmetry breaking event. Here, we report that myosin 1d (myo1d) is essential for establishing left–right asymmetry in zebrafish. Using super-resolution microscopy, we show that the zebrafish LRO, Kupffer’s vesicle (KV), fails to form a spherical lumen and establish proper unidirectional flow in the absence of myo1d. This process requires directed vacuolar trafficking in KV epithelial cells. Interestingly, the vacuole transporting function of zebrafish Myo1d can be substituted by myosin1C derived from an ancient eukaryote, Acanthamoeba castellanii, where it regulates the transport of contractile vacuoles. Our findings reveal an evolutionary conserved role for an unconventional myosin in vacuole trafficking, lumen formation, and determining laterality.

Mosaic Labeling and 3-Dimensional Morphological Analysis of Single Cells in the Zebrafish Left-right Organizer.

A transient epithelial structure called the left-right organizer (LRO) establishes left-right asymmetry in vertebrate embryos. Developmental defects that alter LRO formation result in left-right patterning errors that often lead to congenital heart malformations. However, little is known about mechanisms that regulate individual cell behaviors during LRO formation. To address this, we developed a Cre-loxP based method to mosaically label precursor cells, called dorsal forerunner cells, that give rise to the zebrafish LRO known as Kupffer's vesicle. This methodology allows lineage tracing, 3-dimensional (3D) reconstruction and morphometric analysis of single LRO cells in living embryos. The ability to visualize and quantify individual LRO cell dynamics provides an opportunity to advance our understanding of LRO development, and in a broader sense, investigate the interplay between intrinsic biochemical mechanisms and extrinsic mechanical forces that drive morphogenesis of epithelial tissues.

Insights into the role of Notch signalling in cilia motility regulation

Background Our previous work has demonstrated that Notch signalling modulates cilia length in the zebrafish left-right organizer (LRO) [1]. However, we also found that the axonemal motor Dnah7, an inner dynein arm motor protein, is upregulated in Notch signalling mutants where motile cilia number is exacerbated related to immotile cilia [1]. Moreover, the knock-down of this motor protein drastically affected cilia motility in the LRO, blocking the nodal flow.

Pkd2 affects the architecture of zebrafish left-right organizer

Background Dorsal anterior clustering (DAC) of motile cilia in the left-right organizer (LRO) is crucial for normal fluid flow dynamics and correct laterality in zebrafish [1]. We directly demonstrated that charon/dand5 transcription is negatively regulated by strong flow in zebrafish LRO [1] which suggests that LRO cells have the ability to sense fluid flow and influence gene expression patterns, but how? Pkd2 ion channel is a good candidate because it participates in a mechanosensory complex that senses fluid flow and induces a calcium inward flux in kidney cells [2] and in nodal cells [3]. In agreement, mouse and zebrafish mutants for Pkd2 have LR defects [4,5]. However, Pkd2 is also involved in cell polarity during migration [6] and in extracellular matrix deposition [7] implying a role for Pkd2 in cell morphogenesis.