Roles of Cftr-dependent Fluid Secretion During Organ Morphogenesis and Function

Abstract

<p>Fluid secretion is essential to organ development and function, yet relatively little is known about the roles of fluid secretion <italic>in vivo</italic>. 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. </p><p>To identify the developmental requirements for <italic>cftr</italic> function, we generated <italic>cftr</italic> mutant zebrafish using transcription activator like effector nucleases (TALENs). In <italic>cftr</italic> 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 <italic>cftr</italic> mutants, which prevents directional fluid flow necessary for KV function. To examine <italic>cftr</italic> expression, we generated bacterial artificial chromosome (BAC) transgenic zebrafish expressing fluorescent Cftr fusion proteins under the control of the <italic>cftr</italic> 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 <italic>cftr</italic> during KV morphogenesis and function in the zebrafish. </p><p>In the zebrafish pancreas, we found that loss of <italic>cftr</italic> 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 <italic>cftr</italic> BAC expression in the adult and larval zebrafish pancreata revealed that <italic>cftr</italic> is expressed specifically within the ducts, localized to the apical membrane throughout life. Adult <italic>cftr</italic> mutant pancreata developed substantial degeneration of exocrine tissue and experienced reduced growth rates. In contrast, we found that <italic>cftr</italic> is not necessary for the specification or initial development of the larval pancreas. Exocrine and endocrine tissues developed similarly in WT and <italic>cftr</italic> mutant larvae. These results indicate that <italic>cftr</italic>-dependent fluid secretion is important for maintenance of the zebrafish pancreas. Altogether, these studies of <italic>cftr</italic> function in KV and the pancreas demonstrate that fluid secretion is an essential component of lumen morphogenesis and organ function.</p>