Fluid secretion is essential to organ development and function, yet relatively little is known about the roles of fluid secretion in vivo. Early in development, fluid secretion plays important roles during the process of lumen formation and is necessary for organ homeostasis throughout life. A human disease, cystic fibrosis (CF) is caused by loss of cystic fibrosis transmembrane conductance regulator (CFTR) function, a chloride channel and key regulator of vertebrate fluid secretion. CFTR regulates fluid secretion by governing ion transport and osmotic gradients across epithelia.
To identify the developmental requirements for cftr function, we generated cftr mutant zebrafish using transcription activator like effector nucleases (TALENs). In cftr mutant zebrafish, we observed defects in the specification of left-right (LR) asymmetry. In the zebrafish, LR asymmetry is specified in part by directional fluid flow within a ciliated structure, Kupffer's vesicle (KV). Using live imaging of several transgenic markers in KV, we determined that lumen expansion is impaired in cftr mutants, which prevents directional fluid flow necessary for KV function. To examine cftr expression, we generated bacterial artificial chromosome (BAC) transgenic zebrafish expressing fluorescent Cftr fusion proteins under the control of the cftr promoter. These transgenes express Cftr within the KV epithelium and the protein localizes to the apical membrane. These transgenes rescue the KV function and the specification of LR asymmetry. These studies reveal a new role for cftr during KV morphogenesis and function in the zebrafish.
In the zebrafish pancreas, we found that loss of cftr function leads to defects reminiscent of CF including destruction of exocrine tissue and changes in islet morphology. Additionally, we observed exocrine pancreatic destruction by 3 weeks post fertilization (wpf). Analysis of cftr BAC expression in the adult and larval zebrafish pancreata revealed that cftr is expressed specifically within the ducts, localized to the apical membrane throughout life. Adult cftr mutant pancreata developed substantial degeneration of exocrine tissue and experienced reduced growth rates. In contrast, we found that cftr is not necessary for the specification or initial development of the larval pancreas. Exocrine and endocrine tissues developed similarly in WT and cftr mutant larvae. These results indicate that cftr-dependent fluid secretion is important for maintenance of the zebrafish pancreas. Altogether, these studies of cftr function in KV and the pancreas demonstrate that fluid secretion is an essential component of lumen morphogenesis and organ function.
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