Cftr controls lumen expansion and function of Kupffer’s vesicle in zebrafish

Abstract

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.

Mitotic cell rounding and epithelial thinning regulate lumen growth and shape

“…We demonstrate that this process requires the atp1a1 gene, while putative channels and transporters involved can be speculated from reported gene expression data (Supplementary Note 1). We also show that epithelial thinning occurs during brain ventricle expansion and, while not reported, we also observe this in published images of other lumen forming systems (gut and Kuppfer vesicle 5,23 ). This suggests that epithelial thinning is a widespread driver of lumen growth and shape beyond the inner ear cavity.…”

“…We explored the cellular mechanisms responsible for this anisotropic growth. While the relevance of fluid movement in increasing the size of the cavity has been demonstrated in several experimental models 5,10,11 , with the involvement of paracellular transportation and epithelial chloride secretion 9,23 , its role in shaping the lumen has still not been evaluated. It is widely assumed that the increased intraluminal concentration of chloride (and other ions) promotes the movement of water from outside the organ into the lumen (by paracellular or transcellular transport), similar to what occurs in secretory epithelial organs 12,13 .…”

Epigenetic control of intestinal barrier function and inflammation in zebrafish

“…A BAC containing the upstream sequence and first exon of tnfa (CH211-176D22) was modified as described (45). Recombination was performed to replace the tnfa start site with GFP by using the following homology primers: tnfa 5ʹ homology, 5ʹ-CCTTATCAA-AAGCATTTACACTTGTAGAATCTTTAAGACATACAGCAATTATGGTGAGCAAG-GGCGAGGAG-3ʹ; tnfa 3ʹ homology, 5ʹ-TAAAGGCAACTCTCCTTCTTCAACAT-CCAAAAACGCCCGACTCTCAAGCTTATTGGAGCTCCACCGCGGTG-3ʹ.…”

Single continuous lumen formation in the zebrafish gut is mediated by smoothened-dependent tissue remodeling

“…A BAC containing cldn15la was modified as previously described (Navis et al, 2013). For the C-terminal GFP fusion, a 20-aa spacer (DLPAEQKAASEEDLDPPVAT) was used.…”