Cse1l is a negative regulator of CFTR-dependent fluid secretion

Abstract

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.

Figure 1. bao^s866 mutants undergo a dramatic and rapid expansion of the gut lumen between 96 and 120 hpf

A: Brightfield image of 120 hpf WT and bao^s866 mutant larvae. Arrows point to the edges of the gut lumen. B–C: Confocal images of cross-sections of 120 hpf WT (B) and bao^s866 mutant (C). The arrow points to a delaminating cell. f-actin (red), DAPI (blue), sb: swimbladder. Scale bars: 50 μm. D–E: Dramatic shortening of microvilli (arrows) in bao^s866 mutant enterocytes seen by TEM at 120 hpf. Remnants of apoptotic cells (insert) found in the lumen. GC: goblet cell. Scale bars: 10 μm. F–I: Rapid expansion of the gut lumen in bao^s866 mutants expressing H2A:GFP. F: still images (lateral views) from a bao^s866 mutant SPIM recording between (96–120 hpf) showing first and last frames. G–H: Kymographs from bao^s866 mutant (G) and WT (H) gut. Lumen expansion in the mutant occurred in ~200 min, no cell division was observed. I: Still images (lateral views) from the bao^s866 mutant SPIM recording corresponding to the kymograph shown in (G).

Figure 2. Lumen expansion in bao^s866 mutants results from increased CFTR-dependent fluid secretion

A: Inhibition of CFTR blocks lumen expansion in bao^s866 mutants. Ai: Brightfield images of WT and bao^s866 mutants incubated with DMSO (0.1 %) or the CFTR inhibitor T08 (5 μM) from 72 to 120 hpf. Arrows point to the edges of the gut lumen. Aii: Confocal images of cross-sections of 144 hpf control and T08-treated bao^s866 mutants. Arrowheads point to delaminating cells. f-actin (red), DAPI (blue). Scale bars: 50 μm. Aiii: Quantification of the gut phenotype in control and T08-treated bao^s866 mutants. Larvae were placed in three phenotypic categories (no phenotype, mild or severe phenotype) and then genotyped. B: Activation of CFTR in WT phenocopies the gut lumen expansion defect of bao^s866 mutants. Bi: Brighfield image of 144 hpf WT larvae treated with DMSO (0.15%) or CFTR-Act9 (15 μM). Arrows point to the edges of the gut lumen. Bii: Confocal images of cross-sections of 144 hpf control and CFTR-Act9-treated WT larvae. f-actin:red, DAPI:blue. Scale bars: 20 μm. C: CFTR expression and localization is not affected in bao^s866 mutants compared to WT. Ci: Immunoblot of 120 hpf WT and bao^s866 mutants probed against CFTR and β-tubulin. Cii: Confocal images of wholemounts of 120 hpf WT and bao^s866 mutants stained for CFTR. The arrows point to the apical surface of the gut. Anterior to the right.

Figure 3. Isolation of the bao gene

A: Positional cloning of bao. The number of recombinants (rec.) for each marker is shown. B: sequencing of genomic DNA from +/+, −/− and +/− larvae revealed a T to A mutation (arrow) upstream of exon 16’s splice acceptor site. C: RT-PCR on pools of RNA made from WT and bao^s866 mutants demonstrates defective splicing of exon 16 in bao^s866 mutants leading to a premature stop codon in exon 17. Di: Cse1l is depleted in bao^s866 mutants. The asterisk marks the position of a cross-reacting protein band. Dii: The bao^s866 mutation produces a truncated protein that can be detected in immunoblots of transfected HEK293 cells. Ei: Knockdown of Cse1l using an anti-sense morpholino targeting the translation start site phenocopies the bao^s866 mutation (22% penetrance at 5 dpf, n=220). Eii: Immunoblot of 5 dpf un-injected and Cse1l morphants demonstrating knockdown of this protein. F: ISH analysis using an anti-sense probe directed against the 3’ of the cse1l mRNA. G: Immunofluorescence on WT sections showing Cse1l in the sub-apical (arrowhead) and basolateral (arrow) regions of intestinal cells at 120 hpf. Scale bars: 50μm.

Figure 4. Cse1l negatively regulates fluid secretion in mammalian MDCK-C7 cells

Ai: zebrafish GFP-Cse1l but not GFP-Cse1l^s866 co-immunoprecipitates with human CFTR-CTHA. Aii: partial co-localization (arrows) of GFP-Cse1l and CFTR-CTHA in transfected HEK293 cells. Scale bar=10μm; N, nucleus. B: Over-expression of zebrafish GFP-Cse1l abrogates the stimulatory effect of forskolin on CFTR-dependent fluid secretion in 3D cultures of mammalian MDCK-C7 cells. Bi: MDCK-C7 cells were grown on Matrigel^TM for 4 d and then forskolin (1 μM) was added for 16 hrs before the cysts were fixed and imaged. Green:GFP, red:f-actin, blue:β-catenin. Scale bars: 50μm. Bii: quantification of Bi (n=161 for GFP-Cse1l^s866; n=131 for GFP-Cse1l). Error bars: s.e.m. C: Depletion of Cse1l leads to increased fluid secretion in 3D cultures of MDCK-C7 cells. Ci: Immunoblot showing knockdown of dog Cse1l in Sh4 but not in control Sh-scr infected cells. Cii: Knockdown of dog Cse1l leads to lumen expansion in control but not in GFP-Cse1l-expressing MDCK-C7 cells in 3D cultures (5 d in Matrigel^TM) following forskolin treatment. red: f-actin; green:β-catenin (upper panels), GFP (lower panels); blue:Topro. Scale bars: 50 μm. Ciii: quantification of (Cii). n=200 for control; n=149 for GFP-Cse1l. Error bars: s.e.m.