RESUMO
Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. Although genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using in situ hybridization, for example, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. Importantly, our technique enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs.
Assuntos
Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica , Análise de Sequência de RNA , Tomografia/métodos , Peixe-Zebra/embriologia , Animais , Imageamento TridimensionalRESUMO
Time-lapse fluorescence microscopy is key to unraveling biological development and function; however, living systems, by their nature, permit only limited interrogation and contain untapped information that can only be captured by more invasive methods. Deep-tissue live imaging presents a particular challenge owing to the spectral range of live-cell imaging probes/fluorescent proteins, which offer only modest optical penetration into scattering tissues. Herein, we employ convolutional neural networks to augment live-imaging data with deep-tissue images taken on fixed samples. We demonstrate that convolutional neural networks may be used to restore deep-tissue contrast in GFP-based time-lapse imaging using paired final-state datasets acquired using near-infrared dyes, an approach termed InfraRed-mediated Image Restoration (IR2). Notably, the networks are remarkably robust over a wide range of developmental times. We employ IR2 to enhance the information content of green fluorescent protein time-lapse images of zebrafish and Drosophila embryo/larval development and demonstrate its quantitative potential in increasing the fidelity of cell tracking/lineaging in developing pescoids. Thus, IR2 is poised to extend live imaging to depths otherwise inaccessible.
Assuntos
Drosophila , Peixe-Zebra , Animais , Imagem com Lapso de Tempo/métodos , Microscopia de Fluorescência , Proteínas de Fluorescência Verde/genéticaRESUMO
Despite the robust healing capacity of the liver, regenerative failure underlies numerous hepatic diseases, including the JAG1 haploinsufficient disorder, Alagille syndrome (ALGS). Cholestasis due to intrahepatic duct (IHD) paucity resolves in certain ALGS cases but fails in most with no clear mechanisms or therapeutic interventions. We find that modulating jag1b and jag2b allele dosage is sufficient to stratify these distinct outcomes, which can be either exacerbated or rescued with genetic manipulation of Notch signaling, demonstrating that perturbations of Jag/Notch signaling may be causal for the spectrum of ALGS liver severities. Although regenerating IHD cells proliferate, they remain clustered in mutants that fail to recover due to a blunted elevation of Notch signaling in the distal-most IHD cells. Increased Notch signaling is required for regenerating IHD cells to branch and segregate into the peripheral region of the growing liver, where biliary paucity is commonly observed in ALGS. Mosaic loss- and-gain-of-function analysis reveals Sox9b to be a key Notch transcriptional effector required cell autonomously to regulate these cellular dynamics during IHD regeneration. Treatment with a small-molecule putative Notch agonist stimulates Sox9 expression in ALGS patient fibroblasts and enhances hepatic sox9b expression, rescues IHD paucity and cholestasis, and increases survival in zebrafish mutants, thereby providing a proof-of-concept therapeutic avenue for this disorder.
Assuntos
Síndrome de Alagille , Ductos Biliares Intra-Hepáticos , Transdução de Sinais , Animais , Humanos , Síndrome de Alagille/genética , Síndrome de Alagille/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Mosaicismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Regeneração , Ductos Biliares Intra-Hepáticos/citologia , Ductos Biliares Intra-Hepáticos/patologia , FibroblastosRESUMO
Lymphatic filariasis (LF) is a chronic debilitating neglected tropical disease (NTD) caused by mosquito-transmitted nematodes that afflicts over 60 million people. Control of LF relies on routine mass drug administration with antiparasitics that clear circulating larval parasites but are ineffective against adults. The development of effective adulticides is hampered by a poor understanding of the processes and tissues driving parasite survival in the host. The adult filariae head region contains essential tissues that control parasite feeding, sensory, secretory, and reproductive behaviors, which express promising molecular substrates for the development of antifilarial drugs, vaccines, and diagnostics. We have adapted spatial transcriptomic approaches to map gene expression patterns across these prioritized but historically intractable head tissues. Spatial and tissue-resolved data reveal distinct biases in the origins of known drug targets and secreted antigens. These data were used to identify potential new drug and vaccine targets, including putative hidden antigens expressed in the alimentary canal, and to spatially associate receptor subunits belonging to druggable families. Spatial transcriptomic approaches provide a powerful resource to aid gene function inference and seed antiparasitic discovery pipelines across helminths of relevance to human and animal health.
Assuntos
Anti-Infecciosos , Brugia Malayi , Filariose Linfática , Parasitos , Vacinas , Animais , Anti-Infecciosos/farmacologia , Antiparasitários/farmacologia , Brugia Malayi/genética , Humanos , Parasitos/genética , TranscriptomaRESUMO
BACKGROUND AND AIMS: Alagille Syndrome (ALGS) is a congenital disorder caused by mutations in the Notch ligand gene JAGGED1, leading to neonatal loss of intrahepatic duct (IHD) cells and cholestasis. Cholestasis can resolve in certain patients with ALGS, suggesting regeneration of IHD cells. However, the mechanisms driving IHD cell regeneration following Jagged loss remains unclear. Here, we show that cholestasis due to developmental loss of IHD cells can be consistently phenocopied in zebrafish with compound jagged1b and jagged2b mutations or knockdown. APPROACH AND RESULTS: Leveraging the transience of jagged knockdown in juvenile zebrafish, we find that resumption of Jagged expression leads to robust regeneration of IHD cells through a Notch-dependent mechanism. Combining multiple lineage tracing strategies with whole-liver three-dimensional imaging, we demonstrate that the extrahepatic duct (EHD) is the primary source of multipotent progenitors that contribute to the regeneration, but not to the development, of IHD cells. Hepatocyte-to-IHD cell transdifferentiation is possible but rarely detected. Progenitors in the EHD proliferate and migrate into the liver with Notch signaling loss and differentiate into IHD cells if Notch signaling increases. Tissue-specific mosaic analysis with an inducible dominant-negative Fgf receptor suggests that Fgf signaling from the surrounding mesenchymal cells maintains this extrahepatic niche by directly preventing premature differentiation and allocation of EHD progenitors to the liver. Indeed, transcriptional profiling and functional analysis of adult mouse EHD organoids uncover their distinct differentiation and proliferative potential relative to IHD organoids. CONCLUSIONS: Our data show that IHD cells regenerate upon resumption of Jagged/Notch signaling, from multipotent progenitors originating from an Fgf-dependent extrahepatic stem cell niche. We posit that if Jagged/Notch signaling is augmented, through normal stochastic variation, gene therapy, or a Notch agonist, regeneration of IHD cells in patients with ALGS may be enhanced.
Assuntos
Síndrome de Alagille , Ductos Biliares Extra-Hepáticos , Ductos Biliares Intra-Hepáticos , Proteínas de Ligação ao Cálcio , Proteína Jagged-1 , Regeneração Hepática/fisiologia , Receptores Notch/metabolismo , Proteínas de Peixe-Zebra , Síndrome de Alagille/genética , Síndrome de Alagille/metabolismo , Animais , Ductos Biliares Extra-Hepáticos/crescimento & desenvolvimento , Ductos Biliares Extra-Hepáticos/fisiologia , Ductos Biliares Intra-Hepáticos/crescimento & desenvolvimento , Ductos Biliares Intra-Hepáticos/fisiologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Transdiferenciação Celular , Modelos Animais de Doenças , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
To quantitatively understand biological processes that occur over many hours or days, it is desirable to image multiple samples simultaneously, and automatically process and analyse the resulting datasets. Here, we present a complete multi-sample preparation, imaging, processing and analysis workflow to determine the development of the vascular volume in zebrafish. Up to five live embryos were mounted and imaged simultaneously over several days using selective plane illumination microscopy (SPIM). The resulting large imagery dataset of several terabytes was processed in an automated manner on a high-performance computer cluster and segmented using a novel segmentation approach that uses images of red blood cells as training data. This analysis yielded a precise quantification of growth characteristics of the whole vascular network, head vasculature and tail vasculature over development. Our multi-sample platform demonstrates effective upgrades to conventional single-sample imaging platforms and paves the way for diverse quantitative long-term imaging studies.
Assuntos
Sistema Cardiovascular/embriologia , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência/métodos , Animais , Fenômenos Biológicos , Análise por Conglomerados , Embrião não Mamífero , Proteínas de Fluorescência Verde/metabolismo , Software , Peixe-ZebraRESUMO
In biology, we are often confronted with information-rich, large-scale trajectory data, but exploring and communicating patterns in such data can be a cumbersome task. Ideally, the data should be wrapped with an interactive visualisation in one concise packet that makes it straightforward to create and test hypotheses collaboratively. To address these challenges, we have developed a tool, linus, which makes the process of exploring and sharing 3D trajectories as easy as browsing a website. We provide a python script that reads trajectory data, enriches them with additional features such as edge bundling or custom axes, and generates an interactive web-based visualisation that can be shared online. linus facilitates the collaborative discovery of patterns in complex trajectory data.
Assuntos
Biologia Computacional/métodos , Disseminação de Informação/métodos , Internet , Linguagens de Programação , Interface Usuário-ComputadorRESUMO
During gastrulation, embryonic cells become specified into distinct germ layers. In mouse, this continues throughout somitogenesis from a population of bipotent stem cells called neuromesodermal progenitors (NMps). However, the degree of self-renewal associated with NMps in the fast-developing zebrafish embryo is unclear. Using a genetic clone-tracing method, we labelled early embryonic progenitors and found a strong clonal similarity between spinal cord and mesoderm tissues. We followed individual cell lineages using light-sheet imaging, revealing a common neuromesodermal lineage contribution to a subset of spinal cord tissue across the anterior-posterior body axis. An initial population subdivides at mid-gastrula stages and is directly allocated to neural and mesodermal compartments during gastrulation. A second population in the tailbud undergoes delayed allocation to contribute to the neural and mesodermal compartment only at late somitogenesis. Cell tracking and retrospective cell fate assignment at late somitogenesis stages reveal these cells to be a collection of mono-fated progenitors. Our results suggest that NMps are a conserved population of bipotential progenitors, the lineage of which varies in a species-specific manner due to vastly different rates of differentiation and growth.
Assuntos
Mesoderma/citologia , Células-Tronco Neurais/metabolismo , Medula Espinal/crescimento & desenvolvimento , Células-Tronco/citologia , Animais , Padronização Corporal , Divisão Celular , Linhagem da Célula , Rastreamento de Células , Gastrulação , Mesoderma/metabolismo , Modelos Biológicos , Células-Tronco Neurais/citologia , Especificidade de Órgãos , Somitos/citologia , Somitos/metabolismo , Medula Espinal/citologia , Células-Tronco/metabolismo , Cauda , Peixe-ZebraRESUMO
Repetitive aeroallergen exposure is linked to sensitization and airway remodeling through incompletely understood mechanisms. In this study, we examine the dynamic mucosal response to cat dander extract (CDE), a ubiquitous aero-allergen linked to remodeling, sensitization and asthma. We find that daily exposure of CDE in naïve C57BL/6 mice activates innate neutrophilic inflammation followed by transition to a lymphocytic response associated with waves of mucosal transforming growth factor (TGF) isoform expression. In parallel, enhanced bronchiolar Smad3 expression and accumulation of phospho-SMAD3 was observed, indicating paracrine activation of canonical TGFßR signaling. CDE exposure similarly triggered epithelial cell plasticity, associated with expression of mesenchymal regulatory factors (Snai1 and Zeb1), reduction of epithelial markers (Cdh1) and activation of the NFκB/RelA transcriptional activator. To determine whether NFκB functionally mediates CDE-induced growth factor response, mice were stimulated with CDE in the absence or presence of a selective IKK inhibitor. IKK inhibition substantially reduced the level of CDE-induced TGFß1 expression, pSMAD3 accumulation, Snai1 and Zeb1 expression. Activation of epithelial plasticity was demonstrated by flow cytometry in whole lung homogenates, where CDE induces accumulation of SMA+Epcam+ population. Club cells are important sources of cytokine and growth factor production. To determine whether Club cell innate signaling through NFκB/RelA mediated CDE induced TGFß signaling, we depleted RelA in Secretoglobin (Scgb1a1)-expressing bronchiolar cells. Immunofluorescence-optical clearing light sheet microscopy showed a punctate distribution of Scgb1a1 progenitors throughout the small airway. We found that RelA depletion in Secretoglobin+ cells results in inhibition of the mucosal TGFß response, blockade of EMT and reduced subepithelial myofibroblast expansion. We conclude that the Secretoglobin-derived bronchiolar cell is central to coordinating the innate response required for mucosal TGFß1 response, EMT and myofibroblast expansion. These data have important mechanistic implications for how aero-allergens trigger mucosal injury response and remodeling in the small airway.
Assuntos
Remodelação das Vias Aéreas , Asma/genética , Regulação da Expressão Gênica , Miofibroblastos/metabolismo , NF-kappa B/genética , Secretoglobinas/metabolismo , Fator de Crescimento Transformador beta/genética , Alérgenos/efeitos adversos , Animais , Asma/metabolismo , Asma/patologia , Bronquíolos/metabolismo , Bronquíolos/patologia , Gatos , Transdiferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/patologia , NF-kappa B/biossíntese , Transdução de Sinais , Fator de Crescimento Transformador beta/biossínteseRESUMO
We identify a novel endothelial membrane behaviour in transgenic zebrafish. Cerebral blood vessels extrude large transient spherical structures that persist for an average of 23 min before regressing into the parent vessel. We term these structures "kugeln", after the German for sphere. Kugeln are only observed arising from the cerebral vessels and are present as late as 28 days post fertilization. Kugeln do not communicate with the vessel lumen and can form in the absence of blood flow. They contain little or no cytoplasm, but the majority are highly positive for nitric oxide reactivity. Kugeln do not interact with brain lymphatic endothelial cells (BLECs) and can form in their absence, nor do they perform a scavenging role or interact with macrophages. Inhibition of actin polymerization, Myosin II, or Notch signalling reduces kugel formation, while inhibition of VEGF or Wnt dysregulation (either inhibition or activation) increases kugel formation. Kugeln represent a novel Notch-dependent NO-containing endothelial organelle restricted to the cerebral vessels, of currently unknown function.
Assuntos
Vasos Sanguíneos/citologia , Encéfalo/citologia , Células Endoteliais/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Neovascularização Fisiológica/genética , Peixe-Zebra/embriologia , Actinas/antagonistas & inibidores , Actinas/genética , Actinas/metabolismo , Animais , Animais Geneticamente Modificados , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/ultraestrutura , Encéfalo/irrigação sanguínea , Encéfalo/embriologia , Encéfalo/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Circulação Cerebrovascular/genética , Embrião não Mamífero , Células Endoteliais/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Miosina Tipo II/antagonistas & inibidores , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Óxido Nítrico/metabolismo , Organelas/metabolismo , Organelas/ultraestrutura , Polimerização/efeitos dos fármacos , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Tiazolidinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Antifungal therapy can fail in a remarkable number of patients with invasive fungal disease, resulting in significant morbidity worldwide. A major contributor to this failure is that while these drugs have high potency in vitro, we do not fully understand how they work inside infected hosts. Here, we used a transparent larval zebrafish model of Aspergillus fumigatus infection amenable to real-time imaging of invasive disease as an in vivo intermediate vertebrate model to investigate the efficacy and mechanism of the antifungal drug voriconazole. We found that the ability of voriconazole to protect against A. fumigatus infection depends on host innate immune cells and, specifically, on the presence of macrophages. While voriconazole inhibits fungal spore germination and growth in vitro, it does not do so in larval zebrafish. Instead, live imaging of whole, intact larvae over a multiday course of infection revealed that macrophages slow down initial fungal growth, allowing voriconazole time to target and kill A. fumigatus hyphae postgermination. These findings shed light on how antifungal drugs such as voriconazole may synergize with the immune response in living hosts.
Assuntos
Antifúngicos/uso terapêutico , Aspergilose/tratamento farmacológico , Aspergilose/imunologia , Aspergillus fumigatus , Voriconazol/uso terapêutico , Animais , Aspergilose/microbiologia , Imunidade Celular , Larva , Macrófagos/imunologia , Testes de Sensibilidade Microbiana , Esporos Fúngicos/imunologia , Peixe-ZebraRESUMO
The impact of light-sheet fluorescence microscopy (LSFM) is visible in fields as diverse as developmental and cell biology, anatomical science, biophysics and neuroscience. Although adoption among biologists has been steady, LSFM has not displaced more traditional imaging methods despite its often-superior performance. One reason for this is that the field has largely conformed to a do-it-yourself ethic, although the challenges of big image data cannot be overstated. With the most powerful implementations of LSFM available to only a few groups worldwide, the scope of this technique is unnecessarily limited. Here we elucidate the key developments and define a simple set of underlying principles governing LSFM. In doing so, we aim to clarify the decisions to be made for those who wish to develop and use bespoke light-sheet systems and to assist in identifying the best approaches to apply this powerful technique to myriad biological questions.
Assuntos
Células/citologia , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Animais , Disciplinas das Ciências Biológicas/instrumentação , Disciplinas das Ciências Biológicas/métodos , Imagem Molecular/métodosRESUMO
Fluorescently labeled structures can be spectrally isolated and imaged at high resolution in living embryos by light sheet microscopy. Multimodal imaging techniques are now needed to put these distinct structures back into the context of the surrounding tissue. We found that the bright-field contrast of unstained specimens in a selective plane illumination microscopy (SPIM) setup can be exploited for in vivo tomographic reconstructions of the three-dimensional anatomy of zebrafish, without causing phototoxicity. We report multimodal imaging of entire zebrafish embryos over several hours of development, as well as segmentation, tracking and automatic registration of individual organs.
Assuntos
Microscopia/métodos , Tomografia Óptica/métodos , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologiaRESUMO
During embryonic development, vascular networks remodel to meet the increasing demand of growing tissues for oxygen and nutrients. This is achieved by the pruning of redundant blood vessel segments, which then allows more efficient blood flow patterns. Because of the lack of an in vivo system suitable for high-resolution live imaging, the dynamics of the pruning process have not been described in detail. Here, we present the subintestinal vein (SIV) plexus of the zebrafish embryo as a novel model to study pruning at the cellular level. We show that blood vessel regression is a coordinated process of cell rearrangements involving lumen collapse and cell-cell contact resolution. Interestingly, the cellular rearrangements during pruning resemble endothelial cell behavior during vessel fusion in a reversed order. In pruning segments, endothelial cells first migrate toward opposing sides where they join the parental vascular branches, thus remodeling the multicellular segment into a unicellular connection. Often, the lumen is maintained throughout this process, and transient unicellular tubes form through cell self-fusion. In a second step, the unicellular connection is resolved unilaterally, and the pruning cell rejoins the opposing branch. Thus, we show for the first time that various cellular activities are coordinated to achieve blood vessel pruning and define two different morphogenetic pathways, which are selected by the flow environment.
Assuntos
Fusão Celular , Endotélio Vascular/embriologia , Animais , Animais Geneticamente Modificados , Neovascularização Fisiológica , Peixe-Zebra/embriologiaRESUMO
How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using Drosophila germband extension (GBE) as a model. We found previously that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole Drosophila embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis.
Assuntos
Drosophila/embriologia , Embrião não Mamífero/anatomia & histologia , Endoderma/embriologia , Gastrulação , Modelos Anatômicos , Morfogênese , Animais , Biomarcadores/metabolismo , Forma Celular , Tamanho Celular , Drosophila/genética , Drosophila/metabolismo , Drosophila/ultraestrutura , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Endoderma/metabolismo , Endoderma/ultraestrutura , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fenômenos Mecânicos , Proteínas de Fusão de Membrana/genética , Proteínas de Fusão de Membrana/metabolismo , Microscopia Eletrônica de Varredura/veterinária , Microscopia de Vídeo/veterinária , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reologia , Imagem com Lapso de Tempo/veterinária , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismoRESUMO
The formation of a single lumen during tubulogenesis is crucial for the development and function of many organs. Although 3D cell culture models have identified molecular mechanisms controlling lumen formation in vitro, their function during vertebrate organogenesis is poorly understood. Using light sheet microscopy and genetic approaches we have investigated single lumen formation in the zebrafish gut. Here we show that during gut development multiple lumens open and enlarge to generate a distinct intermediate, which consists of two adjacent unfused lumens separated by basolateral contacts. We observed that these lumens arise independently from each other along the length of the gut and do not share a continuous apical surface. Resolution of this intermediate into a single, continuous lumen requires the remodeling of contacts between adjacent lumens and subsequent lumen fusion. We show that lumen resolution, but not lumen opening, is impaired in smoothened (smo) mutants, indicating that fluid-driven lumen enlargement and resolution are two distinct processes. Furthermore, we show that smo mutants exhibit perturbations in the Rab11 trafficking pathway and demonstrate that Rab11-mediated trafficking is necessary for single lumen formation. Thus, lumen resolution is a distinct genetically controlled process crucial for single, continuous lumen formation in the zebrafish gut.