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.

Variable phenotypes and penetrance between and within different zebrafish ciliary transition zone mutants.

Meckel syndrome, nephronophthisis, Joubert syndrome and Bardet-Biedl syndrome are caused by mutations in proteins that localize to the ciliary transition zone (TZ). The phenotypically distinct syndromes suggest that these TZ proteins have differing functions. However, mutations in a single TZ gene can result in multiple syndromes, suggesting that the phenotype is influenced by modifier genes. We performed a comprehensive analysis of ten zebrafish TZ mutants, including mks1, tmem216, tmem67, rpgrip1l, cc2d2a, b9d2, cep290, tctn1, nphp1 and nphp4, as well as mutants in ift88 and ift172. Our data indicate that variations in phenotypes exist between different TZ mutants, supporting different tissue-specific functions of these TZ genes. Further, we observed phenotypic variations within progeny of a single TZ mutant, reminiscent of multiple disease syndromes being associated with mutations in one gene. In some mutants, the dynamics of the phenotype became complex with transitory phenotypes that are corrected over time. We also demonstrated that multiple-guide-derived CRISPR/Cas9 F0 'crispant' embryos recapitulate zygotic null phenotypes, and rapidly identified ciliary phenotypes in 11 cilia-associated gene candidates (ankfn1, ccdc65, cfap57, fhad1, nme7, pacrg, saxo2, c1orf194, ttc26, zmynd12 and cfap52).

Structures and functions of cilia during vertebrate embryo development.

Cilia are hair-like structures that project from the surface of cells. In vertebrates, most cells have an immotile primary cilium that mediates cell signaling, and some specialized cells assemble one or multiple cilia that are motile and beat synchronously to move fluids in one direction. Gene mutations that alter cilia structure or function cause a broad spectrum of disorders termed ciliopathies that impact virtually every system in the body. A wide range of birth defects associated with ciliopathies underscores critical functions for cilia during embryonic development. In many cases, the mechanisms underlying cilia functions during development and disease remain poorly understood. This review describes different types of cilia in vertebrate embryos and discusses recent research results from diverse model systems that provide novel insights into how cilia form and function during embryo development. The work discussed here not only expands our understanding of in vivo cilia biology, but also opens new questions about cilia and their roles in establishing healthy embryos.

Understanding laterality disorders and the left-right organizer: Insights from zebrafish.

Vital internal organs display a left-right (LR) asymmetric arrangement that is established during embryonic development. Disruption of this LR asymmetry-or laterality-can result in congenital organ malformations. Situs inversus totalis (SIT) is a complete concordant reversal of internal organs that results in a low occurrence of clinical consequences. Situs ambiguous, which gives rise to Heterotaxy syndrome (HTX), is characterized by discordant development and arrangement of organs that is associated with a wide range of birth defects. The leading cause of health problems in HTX patients is a congenital heart malformation. Mutations identified in patients with laterality disorders implicate motile cilia in establishing LR asymmetry. However, the cellular and molecular mechanisms underlying SIT and HTX are not fully understood. In several vertebrates, including mouse, frog and zebrafish, motile cilia located in a "left-right organizer" (LRO) trigger conserved signaling pathways that guide asymmetric organ development. Perturbation of LRO formation and/or function in animal models recapitulates organ malformations observed in SIT and HTX patients. This provides an opportunity to use these models to investigate the embryological origins of laterality disorders. The zebrafish embryo has emerged as an important model for investigating the earliest steps of LRO development. Here, we discuss clinical characteristics of human laterality disorders, and highlight experimental results from zebrafish that provide insights into LRO biology and advance our understanding of human laterality disorders.

3D viscoelastic drag forces contribute to cell shape changes during organogenesis in the zebrafish embryo.

The left-right organizer in zebrafish embryos, Kupffer's Vesicle (KV), is a simple organ that undergoes programmed asymmetric cell shape changes that are necessary to establish the left-right axis of the embryo. We use simulations and experiments to investigate whether 3D mechanical drag forces generated by the posteriorly-directed motion of the KV through the tailbud tissue are sufficient to drive such shape changes. We develop a fully 3D vertex-like (Voronoi) model for the tissue architecture, and demonstrate that the tissue can generate drag forces and drive cell shape changes. Furthermore, we find that tailbud tissue presents a shear-thinning, viscoelastic behavior consistent with those observed in published experiments. We then perform live imaging experiments and particle image velocimetry analysis to quantify the precise tissue velocity gradients around KV as a function of developmental time. We observe robust velocity gradients around the KV, indicating that mechanical drag forces must be exerted on the KV by the tailbud tissue. We demonstrate that experimentally observed velocity fields are consistent with the viscoelastic response seen in simulations. This work also suggests that 3D viscoelastic drag forces could be a generic mechanism for cell shape change in other biological processes.

Loss of vacuolar-type H+-ATPase induces caspase-independent necrosis-like death of hair cells in zebrafish neuromasts.

The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that regulates cellular pH. V-ATPase activity modulates several cellular processes, but cell-type-specific functions remain poorly understood. Patients with mutations in specific V-ATPase subunits can develop sensorineural deafness, but the underlying mechanisms are unclear. Here, we show that V-ATPase mutations disrupt the formation of zebrafish neuromasts, which serve as a model to investigate hearing loss. V-ATPase mutant neuromasts are small and contain pyknotic nuclei that denote dying cells. Molecular markers and live imaging show that loss of V-ATPase induces mechanosensory hair cells in neuromasts, but not neighboring support cells, to undergo caspase-independent necrosis-like cell death. This is the first demonstration that loss of V-ATPase can lead to necrosis-like cell death in a specific cell type in vivo. Mechanistically, loss of V-ATPase reduces mitochondrial membrane potential in hair cells. Modulating the mitochondrial permeability transition pore, which regulates mitochondrial membrane potential, improves hair cell survival. These results have implications for understanding the causes of sensorineural deafness, and more broadly, reveal functions for V-ATPase in promoting survival of a specific cell type in vivo.

Cellular dynamics of EMT: lessons from live in vivo imaging of embryonic development.

Epithelial-mesenchymal transition (EMT) refers to a process in which epithelial cells lose apical-basal polarity and loosen cell-cell junctions to take on mesenchymal cell morphologies and invasive properties that facilitate migration through extracellular matrix. EMT-and the reverse mesenchymal-epithelial transition (MET)-are evolutionarily conserved processes that are used throughout embryonic development to drive tissue morphogenesis. During adult life, EMT is activated to close wounds after injury, but also can be used by cancers to promote metastasis. EMT is controlled by several mechanisms that depend on context. In response to cell-cell signaling and/or interactions with the local environment, cells undergoing EMT make rapid changes in kinase and adaptor proteins, adhesion and extracellular matrix molecules, and gene expression. Many of these changes modulate localization, activity, or expression of cytoskeletal proteins that mediate cell shape changes and cell motility. Since cellular changes during EMT are highly dynamic and context-dependent, it is ideal to analyze this process in situ in living organisms. Embryonic development of model organisms is amenable to live time-lapse microscopy, which provides an opportunity to watch EMT as it happens. Here, with a focus on functions of the actin cytoskeleton, I review recent examples of how live in vivo imaging of embryonic development has led to new insights into mechanisms of EMT. At the same time, I highlight specific developmental processes in model embryos-gastrulation in fly and mouse embryos, and neural crest cell development in zebrafish and frog embryos-that provide in vivo platforms for visualizing cellular dynamics during EMT. In addition, I introduce Kupffer's vesicle in the zebrafish embryo as a new model system to investigate EMT and MET. I discuss how these systems have provided insights into the dynamics of adherens junction remodeling, planar cell polarity signaling, cadherin functions, and cytoskeletal organization during EMT, which are not only important for understanding development, but also cancer progression. These findings shed light on mechanisms of actin cytoskeletal dynamics during EMT, and feature live in vivo imaging strategies that can be exploited in future work to identify new mechanisms of EMT and MET. Video Abstract.

Engineering spatial-organized cardiac organoids for developmental toxicity testing.

Emerging technologies in stem cell engineering have produced sophisticated organoid platforms by controlling stem cell fate via biomaterial instructive cues. By micropatterning and differentiating human induced pluripotent stem cells (hiPSCs), we have engineered spatially organized cardiac organoids with contracting cardiomyocytes in the center surrounded by stromal cells distributed along the pattern perimeter. We investigated how geometric confinement directed the structural morphology and contractile functions of the cardiac organoids and tailored the pattern geometry to optimize organoid production. Using modern data-mining techniques, we found that pattern sizes significantly affected contraction functions, particularly in the parameters related to contraction duration and diastolic functions. We applied cardiac organoids generated from 600 µm diameter circles as a developmental toxicity screening assay and quantified the embryotoxic potential of nine pharmaceutical compounds. These cardiac organoids have potential use as an in vitro platform for studying organoid structure-function relationships, developmental processes, and drug-induced cardiac developmental toxicity.

PLK1- and PLK4-Mediated Asymmetric Mitotic Centrosome Size and Positioning in the Early Zebrafish Embryo.

Factors that regulate mitotic spindle positioning remain unclear within the confines of extremely large embryonic cells, such as the early divisions of the vertebrate embryo, Danio rerio (zebrafish). We find that the mitotic centrosome, a structure that assembles the mitotic spindle [1], is notably large in the zebrafish embryo (246.44 ± 11.93 µm2 in a 126.86 ± 0.35 µm diameter cell) compared to a C. elegans embryo (5.78 ± 0.18 µm2 in a 55.83 ± 1.04 µm diameter cell). During embryonic cell divisions, cell size changes rapidly in both C. elegans and zebrafish [2, 3], where mitotic centrosome area scales more closely with changes in cell size compared to changes in spindle length. Embryonic zebrafish spindles contain asymmetrically sized mitotic centrosomes (2.14 ± 0.13-fold difference between the two), with the larger mitotic centrosome placed toward the embryo center in a polo-like kinase (PLK) 1- and PLK4-dependent manner. We propose a model in which uniquely large zebrafish embryonic centrosomes direct spindle placement within disproportionately large cells.

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.

Vertebrate myosin 1d regulates left-right 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.

Cell volume changes contribute to epithelial morphogenesis in zebrafish Kupffer's vesicle.

How epithelial cell behaviors are coordinately regulated to sculpt tissue architecture is a fundamental question in biology. Kupffer's vesicle (KV), a transient organ with a fluid-filled lumen, provides a simple system to investigate the interplay between intrinsic cellular mechanisms and external forces during epithelial morphogenesis. Using 3-dimensional (3D) analyses of single cells we identify asymmetric cell volume changes along the anteroposterior axis of KV that coincide with asymmetric cell shape changes. Blocking ion flux prevents these cell volume changes and cell shape changes. Vertex simulations suggest cell shape changes do not depend on lumen expansion. Consistent with this prediction, asymmetric changes in KV cell volume and shape occur normally when KV lumen growth fails due to leaky cell adhesions. These results indicate ion flux mediates cell volume changes that contribute to asymmetric cell shape changes in KV, and that these changes in epithelial morphology are separable from lumen-generated forces.

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.

Paxillin genes and actomyosin contractility regulate myotome morphogenesis in zebrafish.

Paxillin (Pxn) is a key adapter protein and signaling regulator at sites of cell-extracellular matrix (ECM) adhesion. Here, we investigated the role of Pxn during vertebrate development using the zebrafish embryo as a model system. We have characterized two Pxn genes, pxna and pxnb, in zebrafish that are maternally supplied and expressed in multiple tissues. Gene editing and antisense gene knockdown approaches were used to uncover Pxn functions during zebrafish development. While mutation of either pxna or pxnb alone did not cause gross embryonic phenotypes, double mutants lacking maternally supplied pxna or pxnb displayed defects in cardiovascular, axial, and skeletal muscle development. Transient knockdown of Pxn proteins resulted in similar defects. Irregular myotome shape and ECM composition were observed, suggesting an "inside-out" signaling role for Paxillin genes in the development of myotendinous junctions. Inhibiting non-muscle Myosin-II during somitogenesis altered the subcellular localization of Pxn protein and phenocopied pxn gene loss-of-function. This indicates that Paxillin genes are effectors of actomyosin contractility-driven morphogenesis of trunk musculature in zebrafish. Together, these results reveal new functions for Pxn during muscle development and provide novel genetic models to elucidate Pxn functions.

Evolution and Expression of Paxillin Genes in Teleost Fish.

BACKGROUND: Paxillin family proteins regulate intracellular signaling downstream of extracellular matrix adhesion. Tissue expression patterns and cellular functions of Paxillin proteins during embryo development remain poorly understood. Additionally, the evolution of this gene family has not been thoroughly investigated. RESULTS: This report characterizes the evolution and expression of a novel Paxillin gene, called Paxillin-b, in Teleosts. Alignments indicate that Teleost Paxillin-a and Paxillin-b proteins are highly homologous to each other and to human Paxillin. Phylogenetic and synteny analyses suggest that these genes originated from the duplication of an ancestral Paxillin gene that was in a common ancestor of Teleosts and Tetrapods. Analysis of the spatiotemporal expression profiles of Paxillin-a and Paxillin-b using zebrafish revealed both overlapping and distinct domains for Paxillin-a and Paxillin-b during embryo development. Localization of zebrafish Paxillin orthologs expressed in mammalian cells demonstrated that both proteins localize to focal adhesions, similar to mammalian Paxillin. This suggests these proteins regulate adhesion-dependent processes in their endogenous tissues. CONCLUSION: Paxillin-a and Paxillin-b were generated by duplication in Teleosts. These genes likely play similar roles as Paxillin genes in other organisms. This work provides a framework for functional investigation of Paxillin family members during development using the zebrafish as an in vivo model system.

Cilia in vertebrate left-right patterning.

Understanding how left-right (LR) asymmetry is generated in vertebrate embryos is an important problem in developmental biology. In humans, a failure to align the left and right sides of cardiovascular and/or gastrointestinal systems often results in birth defects. Evidence from patients and animal models has implicated cilia in the process of left-right patterning. Here, we review the proposed functions for cilia in establishing LR asymmetry, which include creating transient leftward fluid flows in an embryonic 'left-right organizer'. These flows direct asymmetric activation of a conserved Nodal (TGFß) signalling pathway that guides asymmetric morphogenesis of developing organs. We discuss the leading hypotheses for how cilia-generated asymmetric fluid flows are translated into asymmetric molecular signals. We also discuss emerging mechanisms that control the subcellular positioning of cilia and the cellular architecture of the left-right organizer, both of which are critical for effective cilia function during left-right patterning. Finally, using mosaic cell-labelling and time-lapse imaging in the zebrafish embryo, we provide new evidence that precursor cells maintain their relative positions as they give rise to the ciliated left-right organizer. This suggests the possibility that these cells acquire left-right positional information prior to the appearance of cilia.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.

Eyes shut homolog is required for maintaining the ciliary pocket and survival of photoreceptors in zebrafish.

Mutations in the extracellular matrix protein eyes shut homolog (EYS) cause photoreceptor degeneration in patients with retinitis pigmentosa 25 (RP25). Functions of EYS remain poorly understood, due in part to the lack of an EYS gene in mouse. We investigated the localization of vertebrate EYS proteins and engineered loss-of-function alleles in zebrafish. Immunostaining indicated that EYS localized near the connecting cilium/transition zone in photoreceptors. EYS also strongly localized to the cone outer segments and weakly to the rod outer segments and cone terminals in primate retinas. Analysis of mutant EYS zebrafish revealed disruption of the ciliary pocket in cone photoreceptors, indicating that EYS is required for maintaining the integrity of the ciliary pocket lumen. Mutant zebrafish exhibited progressive loss of cone and rod photoreceptors. Our results indicate that EYS protein localization is species-dependent and that EYS is required for maintaining ciliary pocket morphology and survival of photoreceptors in zebrafish.

Zebrafish Embryo Disinfection with Povidone-Iodine: Evaluating an Alternative to Chlorine Bleach.

Mycobacteriosis is a common bacterial infection in laboratory zebrafish caused by several different species and strains of Mycobacterium, including both rapid and slow growers. One control measure used to prevent mycobacterial spread within and between facilities is surface disinfection of eggs. Recent studies have highlighted the effectiveness of povidone-iodine (PVPI) on preventing propagation of Mycobacterium spp. found in zebrafish colonies. We evaluated the effect of disinfection using 12.5-50 ppm PVPI (unbuffered and buffered) on zebrafish exposed at 6 or 24 h postfertilization (hpf) to determine if this treatment is suitable for use in research zebrafish. Our results show that 6 hpf embryos are less sensitive to treatment as fewer effects on mortality, developmental delay, and deformity were observed. We also found that buffered PVPI treatment results in a greater knockdown of Mycobacterium chelonae and Mycobacterium marinum, as well as results in decreased harmful effects on embryos. Treatments of shorter (2 min vs. 5 min) duration were also more effective at killing mycobacteria in addition to resulting in fewer effects on embryo health. In addition, we compared the efficacy of a rinsing regimen to rinsing and disinfecting. Based on the findings of this study, we recommend disinfecting embryos for 2 min with buffered PVPI at 12.5-25 ppm.

Mutations in zebrafish pitx2 model congenital malformations in Axenfeld-Rieger syndrome but do not disrupt left-right placement of visceral organs.

Pitx2 is a conserved homeodomain transcription factor that has multiple functions during embryonic development. Mutations in human PITX2 cause autosomal dominant Axenfeld-Rieger syndrome (ARS), characterized by congenital eye and tooth malformations. Pitx2(-/-) knockout mouse models recapitulate aspects of ARS, but are embryonic lethal. To date, ARS treatments remain limited to managing individual symptoms due to an incomplete understanding of PITX2 function. In addition to regulating eye and tooth development, Pitx2 is a target of a conserved Nodal (TGFß) signaling pathway that mediates left-right (LR) asymmetry of visceral organs. Based on its highly conserved asymmetric expression domain, the Nodal-Pitx2 axis has long been considered a common denominator of LR development in vertebrate embryos. However, functions of Pitx2 during asymmetric organ morphogenesis are not well understood. To gain new insight into Pitx2 function we used genome editing to create mutations in the zebrafish pitx2 gene. Mutations in the pitx2 homeodomain caused phenotypes reminiscent of ARS, including aberrant development of the cornea and anterior chamber of the eye and reduced or absent teeth. Intriguingly, LR asymmetric looping of the heart and gut was normal in pitx2 mutants. These results suggest conserved roles for Pitx2 in eye and tooth development and indicate Pitx2 is not required for asymmetric looping of zebrafish visceral organs. This work establishes zebrafish pitx2 mutants as a new animal model for investigating mechanisms underlying congenital malformations in ARS and high-throughput drug screening for ARS therapeutics. Additionally, pitx2 mutants present a unique opportunity to identify new genes involved in vertebrate LR patterning. We show Nodal signaling-independent of Pitx2-controls asymmetric expression of the fatty acid elongase elovl6 in zebrafish, pointing to a potential novel pathway during LR organogenesis.

Kupffer's vesicle size threshold for robust left-right patterning of the zebrafish embryo.

BACKGROUND: Motile cilia in the "organ of asymmetry" create directional fluid flows that are vital for left-right (LR) asymmetric patterning of vertebrate embryos. Organ function often depends on tightly regulated organ size control, but the role of organ of asymmetry size in LR patterning has remained unknown. Observations of the organ of asymmetry in the zebrafish, called Kupffer's vesicle (KV), have suggested significant variations in KV size in wild-type embryos, raising questions about the impact of KV organ size on LR patterning. RESULTS: To understand the relationship between organ of asymmetry size and its function, we characterized variations in KV at several developmental stages and in several different zebrafish strains. We found that the number of KV cilia and the size of the KV lumen were highly variable, whereas the length of KV cilia showed less variation. These variabilities were similar among different genetic backgrounds. By specifically modulating KV size and analyzing individual embryos, we identified a size threshold that is necessary for KV function. CONCLUSIONS: Together these results indicate the KV organ of asymmetry size is not tightly controlled during development, but rather must only exceed a threshold to direct robust LR patterning of the zebrafish embryo.