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The development and function of many internal organs requires precisely regulated fluid secretion. A key regulator of vertebrate fluid secretion is an anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Loss of CFTR function leads to defects in fluid transport and cystic fibrosis (CF), a complex disease characterized by a loss of fluid secretion and mucus buildup in many organs including the lungs, liver, and pancreas. Several animal models including mouse, ferret and pig have been generated to investigate the pathophysiology of CF. However, these models have limited accessibility to early processes in the development of CF and are not amenable for forward genetic or chemical screens. Here, we show that Cftr is expressed and localized to the apical membrane of the zebrafish pancreatic duct and that loss of cftr function leads to destruction of the exocrine pancreas and a cystic fibrosis phenotype that mirrors human disease. Our analyses reveal that the cftr mutant pancreas initially develops normally, then rapidly loses pancreatic tissue during larval life, reflecting pancreatic disease in CF. Altogether, we demonstrate that the cftr mutant zebrafish is a powerful new model for pancreatitis and pancreatic destruction in CF. This accessible model will allow more detailed investigation into the mechanisms that drive CF of the pancreas and facilitate development of new therapies to treat the disease.

By contrast with mammals, adult zebrafish have a high capacity to regenerate damaged or lost myocardium through proliferation of spared cardiomyocytes. The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received recent attention as a target in cardiac repair strategies. While it is recognized that epicardium is required for muscle regeneration and itself has high regenerative potential, the extent of cellular heterogeneity within epicardial tissue is largely unexplored. In this study, we performed transcriptome analysis on dozens of epicardial lineage cells purified from zebrafish harboring a transgenic reporter for the pan-epicardial gene tcf21. Hierarchical clustering analysis suggested the presence of at least three epicardial cell subsets defined by expression signatures. We validated many new pan-epicardial and epicardial markers by alternative expression assays. Additionally, we explored the function of the scaffolding protein and main component of caveolae, caveolin-1 (cav1), which was present in each epicardial subset. In BAC transgenic zebrafish, cav1 regulatory sequences drove strong expression in ostensibly all epicardial cells and in coronary vascular endothelial cells. Moreover, cav1 mutant zebrafish generated by genome editing showed grossly normal heart development and adult cardiac anatomy, but displayed profound defects in injury-induced cardiomyocyte proliferation and heart regeneration. Our study defines a new platform for the discovery of epicardial lineage markers, genetic tools, and mechanisms of heart regeneration.

Summary Transport of chloride through the cystic fibrosis transmembrane conductance regulator (CFTR) channel is a key step in regulating fluid secretion in vertebrates [1, 2]. Loss of CFTR function leads to cystic fibrosis [1, 3, 4], a disease that affects the lungs, pancreas, liver, intestine, and vas deferens. Conversely, uncontrolled activation of the channel leads to increased fluid secretion and plays a major role in several diseases and conditions including cholera [5, 6] and other secretory diarrheas [7] as well as polycystic kidney disease [8–10]. Understanding how CFTR activity is regulated in vivo has been limited by the lack of a genetic model. Here, we used a forward genetic approach in zebrafish to uncover CFTR regulators. We report the identification, isolation, and characterization of a mutation in the zebrafish cse1l gene that leads to the sudden and dramatic expansion of the gut tube. We show that this phenotype results from a rapid accumulation of fluid due to the uncontrolled activation of the CFTR channel. Analyses in zebrafish larvae and mammalian cells indicate that Cse1l is a negative regulator of CFTR-dependent fluid secretion. This work demonstrates the importance of fluid homeostasis in development and establishes the zebrafish as a much-needed model system to study CFTR regulation in vivo.

Mechanical interactions are essential for bending and shaping tissues during morphogenesis. A central feature of nearly all internal organs is the formation of a tubular network consisting of an epithelium that surrounds a central lumen. Lumen formation during organogenesis requires precisely coordinated mechanical and biochemical interactions. Whereas many genetic regulators of lumen formation have been identified, relatively little is known about the mechanical cues that drive lumen morphogenesis. Lumens can be shaped by a variety of physical behaviors including wrapping a sheet of cells around a hollow core, rearranging cells to expose a lumenal cavity, or elongating a tube via cell migration, though many of the details underlying these movements remain poorly understood. It is essential to define how forces generated by individual cells cooperate to produce the tissue-level forces that drive organogenesis. The transduction of mechanical forces relies on several conserved processes including the contraction of cytoskeletal networks or expansion of lumens through increased fluid pressure. The morphogenetic events that drive lumen formation serve as a model for similar mechanical processes occurring throughout development. To understand how lumenal networks arise, it will be essential to investigate how biochemical and mechanical processes integrate to generate complex structures from comparatively simple interactions.

Networks of interconnected tubes form the basic structural element of many organs. Tubes are composed of polarized epithelia that enclose a lumen. During organogenesis, lumens form by several distinct mechanisms, ranging from wrapping of an epithelial sheet to generation of a lumen de novo within a rod of cells. Nevertheless, all tube formation processes share some basic common principles that result in the generation of a single, continuous lumen. Interactions with the surrounding environment direct epithelial cell polarization that governs physiological regulators of lumen formation including the actin cytoskeleton, adhesion, vesicular transport, and fluid secretion. Polarized physiological processes mediate the mechanical interactions between epithelial cells and their environment. Polarity within actin cytoskeleton generates contractile forces generating morphogenetic movements. Secretion of fluid or matrix into the lumen generates outward forces driving lumen opening and expansion. Thus, cell polarity is crucial for vectorial transport processes and structural asymmetries during lumen formation. Here we focus on recent discoveries illuminating the relationship between lumen formation and cell polarity in vivo.

Over several decades genetic studies have unraveled many molecular mechanisms that underlie the signaling networks guiding morphogenesis, but the mechanical forces at work remain much less well understood. Accumulation of fluid within a luminal space can generate outward hydrostatic pressure capable of shaping morphogenesis at several scales, ranging from individual organs to the entire vertebrate body-plan. Here, we focus on recent work that uncovered mechanical roles for fluid secretion during morphogenesis. Identifying the roles and regulation of fluid secretion will be instrumental for understanding the mechanics of morphogenesis as well as many human diseases of complex genetic and environmental origin including secretory diarrheas and scoliosis.

Regulated fluid secretion is crucial for the function of most organs. In vertebrates, the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) is a master regulator of fluid secretion. Although the biophysical properties of CFTR have been well characterized in vitro, little is known about its in vivo role during development. Here, we investigated the function of Cftr during zebrafish development by generating several cftr mutant alleles using TAL effector nucleases. We found that loss of cftr function leads to organ laterality defects. In zebrafish, left-right (LR) asymmetry requires cilia-driven fluid flow within the lumen of Kupffer’s vesicle (KV). Using live imaging we found that KV morphogenesis is disrupted in cftr mutants. Loss of Cftr-mediated fluid secretion impairs KV lumen expansion leading to defects in organ laterality. Using bacterial artificial chromosome recombineering, we generated transgenic fish expressing functional Cftr fusion proteins with fluorescent tags under the control of the cftr promoter. The transgenes completely rescued the cftr mutant phenotype. Live imaging of these transgenic lines showed that Cftr is localized to the apical membrane of the epithelial cells in KV during lumen formation. Pharmacological stimulation of Cftr-dependent fluid secretion led to an expansion of the KV lumen. Conversely, inhibition of ion gradient formation impaired KV lumen inflation. Interestingly, cilia formation and motility in KV were not affected, suggesting that fluid secretion and flow are independently controlled in KV. These findings uncover a new role for cftr in KV morphogenesis and function during zebrafish development.