Kupffer's Vesicle Research Articles

Gdf11 regulates left-right asymmetry development through TGF-β signal.

During the embryonic developmental stage in vertebrates, internal organs are arranged along the left-right axis. Disruptions in this process can result in congenital diseases or laterality disorders. The molecular mechanisms of left-right asymmetry in vertebrate development remain largely unclear. Due to its straightforward structure, zebrafish has become a favoured model for studying early laterality events. Here, we demonstrate that growth and development factor 11 (Gdf11) is essential for left-right development via TGF-β signalling. Morphological analysis showed that gdf11 morphants and mutants displayed clear heart and liver laterality disorders in a Nodal signal-dependent manner. Additionally, we found that Kupffer's vesicle formation and ciliogenesis were impaired following gdf11 deletion. We also observed that Gdf11 may form a heterodimer with Spaw, which promotes Smad2/3 phosphorylation and activates TGF-β signalling. Subsequently, Gdf11 promotes left-right laterality by stimulating Foxj1a and its target gene expression. In summary, we reveal a critical role of Gdf11 in left-right patterning, providing fundamental insights into the developmental process of left-right asymmetry.

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.

Ccdc141 is required for left-right axis development by regulating cilia formation in the Kupffer's vesicle of zebrafish

Laterality is a crucial physiological process intricately linked to the cilium-centrosome complex during embryo development. Defects in the process can result in severe organ mispositioning. Coiled-coil domain containing 141 (CCDC141) has been previously known as a centrosome-related gene, but its role in left-right (LR) asymmetry has not been characterized. In this study, we utilize the zebrafish model and human exome analysis to elucidate the function of ccdc141 in laterality defects. The knockdown of ccdc141 in zebrafish disrupts early LR signaling pathways, cilia function, and Kupffer's vesicle formation. Unlike ccdc141-knockdown embryos exhibiting aberrant LR patterns, ccdc141-null mutants show no apparent abnormality, suggesting a genetic compensation response effect. In parallel, we observe a marked reduction in α-tubulin acetylation levels in the ccdc141 crispants. The treatment with histone deacetylase (HDAC) inhibitors, particularly the HDAC6 inhibitor, rescues the ccdc141 crispant phenotypes. Furthermore, exome analysis of 70 patients with laterality defects reveals an increased burden of CCDC141 mutations, with in-vivo studies verifying the pathogenicity of the patient mutation CCDC141-R123G. Our findings highlight the critical role of ccdc141 in ciliogenesis and demonstrate that CCDC141 mutations lead to abnormal LR patterns, identifying it as a candidate gene for laterality defects.

Dynamical forces drive organ morphology changes during embryonic development.

Organs and tissues must change shape in precise ways during embryonic development to execute their functions. Multiple mechanisms including biochemical signaling pathways and biophysical forces help drive these morphology changes, but it has been difficult to tease apart their contributions, especially from tissue-scale dynamic forces that are typically ignored. We use a combination of mathematical models and in vivo experiments to study a simple organ in the zebrafish embryo called Kupffer's vesicle. Modeling indicates that dynamic forces generated by tissue movements in the embryo produce shape changes in Kupffer's vesicle that are observed during development. Laser ablations in the zebrafish embryo that alter these forces result in altered organ shapes matching model predictions. These results demonstrate that dynamic forces sculpt organ shape during embryo development.

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.

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.

Mechanically sensitive HSF1 is a key regulator of left-right symmetry breaking in zebrafish embryos

The left-right symmetry breaking of vertebrate embryos requires nodal flow. However, the molecular mechanisms that mediate the asymmetric gene expression regulation under nodal flow remain elusive. Here, we report that heat shock factor 1 (HSF1) is asymmetrically activated in the Kupffer's vesicle of zebrafish embryos in the presence of nodal flow. Deficiency in HSF1 expression caused a significant situs inversus and disrupted gene expression asymmetry of nodal signaling proteins in zebrafish embryos. Further studies demonstrated that HSF1 is a mechanosensitive protein. The mechanical sensation ability of HSF1 is conserved in a variety of mechanical stimuli in different cell types. Moreover, cilia and Ca2+-Akt signaling axis are essential for the activation of HSF1 under mechanical stress invitro and invivo. Considering the conserved expression of HSF1 in organisms, these findings unveil a fundamental mechanism of gene expression regulation by mechanical clues during embryonic development and other physiological and pathological transformations.

Esrrγa regulates nephron and ciliary development by controlling prostaglandin synthesis.

Cilia are essential for the ontogeny and function of many tissues, including the kidney. Here, we report that transcription factor ERRγ ortholog estrogen related receptor gamma a (Esrrγa) is essential for renal cell fate choice and ciliogenesis in zebrafish. esrrγa deficiency altered proximodistal nephron patterning, decreased the multiciliated cell populace and disrupted ciliogenesis in the nephron, Kupffer's vesicle and otic vesicle. These phenotypes were consistent with interruptions in prostaglandin signaling, and we found that ciliogenesis was rescued by PGE2 or the cyclooxygenase enzyme Ptgs1. Genetic interaction revealed that peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (Ppargc1a), which acts upstream of Ptgs1-mediated prostaglandin synthesis, has a synergistic relationship with Esrrγa in the ciliogenic pathway. These ciliopathic phenotypes were also observed in mice lacking renal epithelial cell (REC) ERRγ, where significantly shorter cilia formed on proximal and distal tubule cells. Decreased cilia length preceded cyst formation in REC-ERRγ knockout mice, suggesting that ciliary changes occur early during pathogenesis. These data position Esrrγa as a novel link between ciliogenesis and nephrogenesis through regulation of prostaglandin signaling and cooperation with Ppargc1a.

Environmental exposure to triazole fungicide causes left-right asymmetry defects and contributes to abnormal heart development in zebrafish embryos by activating PPARγ-coupled Wnt/β-catenin signaling pathway

Triazole fungicides have been widely used all over the world. However, their potential ecological safety and health risks remain unclear, especially their cardiac developmental toxicity. This study systematically investigated whether and how triazole fungicides could activate peroxisome proliferative activity receptor γ (PPARγ) to cause abnormal heart development. Among ten triazole fungicides, difenoconazole (DIF) exhibited the strongest agonistic activity and caused severe pericardial edema in zebrafish embryos, accompanied by a reduction in heart rate, blood flow and cardiac function. In vitro transcriptomic profile implicated that DIF inhibited the Wnt signaling pathway, and in vivo DIF exposure significantly increased the phosphorylation of β-catenin (p = 0.0002) and altered the expression of related genes in zebrafish embryos. Importantly, exposure to DIF could activate PPARγ and inhibit the Wnt/β-catenin signaling pathway, which changed the size of Kupffer's vesicle (KV) (p = 0.02), altered the expression of left-right (LR) asymmetry-related genes, caused cardiac LR asymmetry defect, and eventually led to abnormal heart development. These findings provide evidence for potential developmental toxicity of triazole fungicides and highlight the necessity of assessing their ecological safety and human health risks.

Mild cold stress specifically disturbs clustering movement of DFCs and sequential organ left-right patterning in zebrafish.

In poikilothermic animals, the distinct acclimatization ability of different organs has been previously addressed, while the tissue-specific role of cold stress in early development is largely unknown. In this study, we discovered that despite its role in delaying embryonic development, mild cold stress (22°C) does not disturb multiple-organ progenitor specification, but does give rise to organ left-right (LR) patterning defects. Regarding the mechanism, the data showed that mild cold stress downregulated the expression of cell-adhesion genes cdh1 and cdh2 during gastrulation, especially in dorsal forerunner cells (DFCs), which partially disturbed the clustering movement of DFCs, Kupffer’s vesicle (KV) morphogenesis, and ciliogenesis. As a result, the defects of KV/cilia disrupted asymmetric nodal signaling and subsequent heart and liver LR patterning. In conclusion, our data novelly identified that, in early development, DFCs are more sensitive to mild cold stress, and mild cold stress repressed the expression of cell adhesion-related gene cdh1 and cdh2. This role partially disturbed the clustering movement of DFCs, which resulted in defective KV/cilia development and sequential organ LR patterning defects.

Maternal transfer and biodistribution of citrate and luminogens coated silver nanoparticles in medaka fish

Given the wide applications of silver nanoparticles (AgNPs), it is necessary to evaluate their potentially adverse long-term effects. In this study, we performed a 100-day exposure of medaka fish to citrate and luminogens coated AgNPs and investigated the maternal transfer potentials and biodistribution of AgNPs. Following long-term AgNPs exposure, AgNPs were mainly distributed in the liver, followed by gills, intestine, and brain, but were also detected in the ovary and strongly colocalized with the dissolved Ag+. The quantified transfer efficiency of different Ag species was 1.56–5.07%. Long-term exposure of medaka to small size of AgNPs (20 nm) reduced the hatching rate attributable to the accumulation of AgNPs and their dissolved Ag+. The maternally transferred AgNPs were mainly concentrated in the Kupffer's vesicle of embryos, while their dissolved Ag+ was almost homogeneously distributed in the embryos. In contrast, the newly accumulated AgNPs were mainly absorbed at the chorion of embryos. During initial larval development, the maternally transferred AgNPs and their dissolved Ag+ were consistently concentrated in intestine. Significant dissolution of maternally transferred AgNPs occurred during larval development. Our results showed that long-term exposure to AgNPs caused distinct biodistribution in the next generation of medaka, and may have implications for assessing their potential adverse effects.

Reemployment of Kupffer's vesicle cells into axial and paraxial mesoderm via transdifferentiation.

Kupffer's vesicle (KV) in the teleost embryo is a fluid-filled vesicle surrounded by a layer of epithelial cells with rotating primary cilia. KV transiently acts as the left-right organizer and degenerates after the establishment of left-right asymmetric gene expression. Previous labelling experiments in zebrafish embryos indicated that descendants of KV-epithelial cells are incorporated into mesodermal tissues after the collapse of KV. However, the overall picture of their differentiation potency had been unclear due to the lack of suitable genetic tools and molecular analyses. In the present study, we established a novel zebrafish transgenic line with a promoter of dand5, in which all KV-epithelial cells and their descendants are specifically labelled until the larval stage. We found that KV-epithelial cells undergo epithelial-mesenchymal transition upon KV collapse and infiltrate into adjacent mesodermal progenitors, the presomitic mesoderm and chordoneural hinge. Once incorporated, the descendants of KV-epithelial cells expressed distinct mesodermal differentiation markers and contributed to the mature populations such as the axial muscles and notochordal sheath through normal developmental process. These results indicate that differentiated KV-epithelial cells possess unique plasticity in that they are reemployed into mesodermal lineages through transdifferentiation after they complete their initial role in KV.

Biallelic DNAH9 mutations are identified in Chinese patients with defective left-right patterning and cilia-related complex congenital heart disease.

Defective left-right (LR) pattering results in a spectrum of laterality disorders including situs inversus totalis (SIT) and heterotaxy syndrome (Htx). Approximately, 50% of patients with primary ciliary dyskinesia (PCD) displayed SIT. Recessive variants in DNAH9 have recently been implicated in patients with situs inversus. Here, we describe six unrelated family trios and 2 sporadic patients with laterality defects and complex congenital heart disease (CHD). Through whole exome sequencing (WES), we identified compound heterozygous mutations in DNAH9 in the affected individuals of these family trios. Ex vivo cDNA amplification revealed that DNAH9 mRNA expression was significantly downregulated in these patients carrying biallelic DNAH9 mutations, which cause a premature stop codon or exon skipping. Transmission electron microscopy (TEM) analysis identified ultrastructural defects of the outer dynein arms in these affected individuals. dnah9 knockdown in zebrafish lead to the disturbance of cardiac left-right patterning without affecting ciliogenesis in Kupffer's vesicle (KV). By generating a Dnah9 knockout (KO) C57BL/6n mouse model, we found that Dnah9 loss leads to compromised cardiac function. In this study, we identified recessive DNAH9 mutations in Chinese patients with cardiac abnormalities and defective LR pattering.

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.

CARMIL3 is important for cell migration and morphogenesis during early development in zebrafish

Cell migration is important during early animal embryogenesis. Cell migration and cell shape are controlled by actin assembly and dynamics, which depend on capping proteins, including the barbed-end heterodimeric actin capping protein (CP). CP activity can be regulated by capping-protein-interacting (CPI) motif proteins, including CARMIL (capping protein Arp2/3 myosin-I linker) family proteins. Previous studies of CARMIL3, one of the three highly conserved CARMIL genes in vertebrates, have largely been limited to cells in culture. Towards understanding CARMIL function during embryogenesis in vivo, we analyzed zebrafish lines carrying mutations of carmil3. Maternal-zygotic mutants showed impaired endodermal migration during gastrulation, along with defects in dorsal forerunner cell (DFC) cluster formation, which affected the morphogenesis of Kupffer's vesicle (KV). Mutant KVs were smaller, contained fewer cells and displayed decreased numbers of cilia, leading to defects in left/right (L/R) patterning with variable penetrance and expressivity. The penetrance and expressivity of the KV phenotype in carmil3 mutants correlated well with the L/R heart positioning defect at the end of embryogenesis. This in vivo animal study of CARMIL3 reveals its new role during morphogenesis of the vertebrate embryo. This role involves migration of endodermal cells and DFCs, along with subsequent morphogenesis of the KV and L/R asymmetry.

Zebrafish Model as a Screen to Prevent Cyst Inflation in Autosomal Dominant Polycystic Kidney Disease.

In autosomal dominant polycystic kidney disease (ADPKD), kidney cyst growth requires the recruitment of CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel that is defective in cystic fibrosis. We have been studying cyst inflation using the zebrafish Kupffer’s vesicle (KV) as model system because we previously demonstrated that knocking down polycystin 2 (PC2) induced a CFTR-mediated enlargement of the organ. We have now quantified the PC2 knockdown by showing that it causes a 73% reduction in the number of KV cilia expressing PC2. According to the literature, this is an essential event in kidney cystogenesis in ADPKD mice. Additionally, we demonstrated that the PC2 knockdown leads to a significant accumulation of CFTR-GFP at the apical region of the KV cells. Furthermore, we determined that KV enlargement is rescued by the injection of Xenopus pkd2 mRNA and by 100 µM tolvaptan treatment, the unique and approved pharmacologic approach for ADPKD management. We expected vasopressin V2 receptor antagonist to lower the cAMP levels of KV-lining cells and, thus, to inactivate CFTR. These findings further support the use of the KV as an in vivo model for screening compounds that may prevent cyst enlargement in this ciliopathy, through CFTR inhibition.

Genetic compensation for cilia defects in cep290 mutants by upregulation of cilia-associated small GTPases.

ABSTRACTMutations in CEP290 (also known as NPHP6), a large multidomain coiled coil protein, are associated with multiple cilia-associated syndromes. Over 130 CEP290 mutations have been linked to a wide spectrum of human ciliopathies, raising the question of how mutations in a single gene cause different disease syndromes. In zebrafish, the expressivity of cep290 deficiencies were linked to the type of genetic ablation: acute cep290 morpholino knockdown caused severe cilia-related phenotypes, whereas deficiencies in a CRISPR/Cas9 genetic mutant were restricted to photoreceptor defects. Here, we show that milder phenotypes in genetic mutants were associated with the upregulation of genes encoding the cilia-associated small GTPases arl3, arl13b and unc119b. Upregulation of UNC119b was also observed in urine-derived renal epithelial cells from human Joubert syndrome CEP290 patients. Ectopic expression of arl3, arl13b and unc119b in cep290 morphant zebrafish embryos rescued Kupffer's vesicle cilia and partially rescued photoreceptor outer segment defects. The results suggest that genetic compensation by upregulation of genes involved in a common subcellular process, lipidated protein trafficking to cilia, may be a conserved mechanism contributing to genotype-phenotype variations observed in CEP290 deficiencies.This article has an associated First Person interview with the first author of the paper.

Esrrγ Regulates Nephron and Cilia Development by Modulating Prostaglandin Synthesis

Vertebrate kidney development is a conserved and nuanced process that requires a complex concert of signals to facilitate patterning and differentiation of nephron functional units. Each nephron is comprised of over a dozen specialized cell types, characterized by the expression of solute transporters and other distinct cellular features such as the formation of cilia, which are hair-like projections that facilitate mechano- and chemo-sensing. Since deficiency of the transcription factor Esrrγ has been linked to kidney dysfunction, typified by cyst formation and kidney failure, we hypothesize that Esrrγ also plays a role during renal ontogeny. Zebrafish offer a distinct model to study organogenesis, as highly conserved nephrons are fully formed by 24 hours post fertilization (hpf), and function beginning at 48 hpf. Zebrafish nephrons are comprised of both mono- and multi- ciliated cells, further aiding in the study of cilia formation. Using this conserved zebrafish model, we found that esrrγa regulates both nephron cell specification and ciliogenesis. esrrγa deficient embryos exhibited altered nephron composition with increased proximal segments, decreased distal segment populations and decreased number of multiciliated cells. Further, the formation of cilia was disrupted, where both cilia length and number of ciliated basal bodies were significantly reduced in mono- and multi- ciliated cell types. Cilia formation was disrupted in a non-tissue specific manner, as cilia length was also decreased in the Kupffer's vesicle at 14 hpf, and the ear structure at 96 hpf. These phenotypes were consistent with a disruption of prostaglandin signaling, and we found that expression of the prostaglandin cyclooxygenase enzyme (ptgs1) was reduced in esrrya deficient embryos. Treatment with dmPGE2 or ptgs1 overexpression was sufficient to rescue renal and cilia defects. These data position esrrγa as a key regulator of prostaglandin signaling and nephron cell development, and Esrrγ may be a potential target for future ciliopathic treatments. This study has far reaching implications, as cilia are key features of various tissues, including the kidney, reproductive system, central nervous system, and respiratory system.

Derivation and Characterization of a New Embryonic Cell Line from the Olive Flounder Paralichthys olivaceus

A new embryonic cell line that consisted predominantly of fibroblast cells has been established from embryos at Kupffer’s vesicle (KV) stage of the olive flounder Paralichthys olivaceus, designated as the PoEKC. PoEKC cells have been subcultured for 61 passages over a period of 18 months. The cells were cultured in DMEM/F-12 medium supplemented with antibiotics, FBS, and growth factors at temperature of 25°C. The growth curve of the PoEKC cells comprised an exponential growth phase and a long plateau phase. Chromosome analysis revealed that the cells possessed the normal flounder diploid karyotype of 2n = 48t. The origin of the cell line was confirmed by testing the partial sequences of cytochrome oxidase c subunit I (COI) gene of mtDNA. The pluripotency markers, genes OCT4-1, SOX2, KLF4, and NANOG showed positive signals in the PoEKC cells, and the cells also presented high ALP activity. According to the above results, the PoEKC cells might be pluripotency, though the pluripotency needs further confirmation. The cells were also successfully transfected with GFP reporter gene suggesting that it could be utilized for gene function study in the flounder.

Biliary-Atresia-Associated Mannosidase-1-Alpha-2 Gene Regulates Biliary and Ciliary Morphogenesis and Laterality.

Background/AimsInfectious and genetic factors are invoked, respectively in isolated biliary atresia (BA), or syndromic BA, with major extrahepatic anomalies. However, isolated BA is also associated with minor extrahepatic gut and cardiovascular anomalies and multiple susceptibility genes, suggesting common origins.MethodsWe investigated novel susceptibility genes with genome-wide association, targeted sequencing and tissue staining in BA requiring liver transplantation, independent of BA subtype. Candidate gene effects on morphogenesis, developmental pathways, and ciliogenesis, which regulates left-right patterning were investigated with zebrafish knockdown and mouse knockout models, mouse airway cell cultures, and liver transcriptome analysis.ResultsSingle nucleotide polymorphisms in Mannosidase-1-α-2 (MAN1A2) were significantly associated with BA and with other polymorphisms known to affect MAN1A2 expression but were not differentially enriched in either BA subtype. In zebrafish embryos, man1a2 knockdown caused poor biliary network formation, ciliary dysgenesis in Kupffer’s vesicle, cardiac and liver heterotaxy, and dysregulated egfra and other developmental genes. Suboptimal man1a2 knockdown synergized with suboptimal EGFR signaling or suboptimal knockdown of the EGFR pathway gene, adenosine-ribosylation-factor-6, which had minimal effects individually, to reproduce biliary defects but not heterotaxy. In cultured mouse airway epithelium, Man1a2 knockdown arrested ciliary development and motility. Man1a2–/– mice, which experience respiratory failure, also demonstrated portal and bile ductular inflammation. Human BA liver and Man1a2–/– liver exhibited reduced Man1a2 expression and dysregulated ciliary genes, known to cause multisystem human laterality defects.ConclusionBA requiring transplantation associates with sequence variants in MAN1A2. man1a2 regulates laterality, in addition to hepatobiliary morphogenesis, by regulating ciliogenesis in zebrafish and mice, providing a novel developmental basis for multisystem defects in BA.