Drosophila as a model for studying cystic fibrosis pathophysiology of the gastrointestinal system

Abstract

Cystic fibrosis (CF) is a recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The most common symptoms include progressive lung disease and chronic digestive conditions. CF is the first human genetic disease to benefit from having five different species of animal models. Despite the phenotypic differences among the animal models and human CF, these models have provided invaluable insight into understanding disease mechanisms at the organ-system level. Here, we identify a member of the ABCC4 family, CG5789, that has the structural and functional properties expected for encoding the Drosophila equivalent of human CFTR, and thus refer to it as Drosophila CFTR (Dmel\CFTR). We show that knockdown of Dmel\CFTR in the adult intestine disrupts osmotic homeostasis and displays CF-like phenotypes that lead to intestinal stem cell hyperplasia. We also show that expression of wild-type human CFTR, but not mutant variants of CFTR that prevent plasma membrane expression, rescues the mutant phenotypes of Dmel\CFTR Furthermore, we performed RNA sequencing (RNA-Seq)-based transcriptomic analysis using Dmel\CFTR fly intestine and identified a mucin gene, Muc68D, which is required for proper intestinal barrier protection. Altogether, our findings suggest that Drosophila can be a powerful model organism for studying CF pathophysiology.

Keywords: CFTR; Drosophila; cystic fibrosis; gut.

Fig. 1.

Disruption of Cl⁻ and Na⁺ homeostasis in the absence of CG5789. (A) Pairwise sequence identity and similarity of R domain between labeled species. (B) Pairwise sequence identity and similarity of TM8 domain between labeled species. (C) Fluorescent monitoring of intracellular Cl⁻ levels in the midgut epithelium using MQAE. Significant quenching of fluorescence is observed when CG5789 was depleted in the ECs compared to wild-type control. (D) Fluorescent monitoring of intracellular Na⁺ levels in the midgut epithelium using Sodium Green. Enhancement of Sodium Green fluorescence is observed in the absence of CG5789 compared to wild-type control (C and D) (Scale bar, 50 μm.).

Fig. 2.

Intestinal phenotypes in the absence of CG5789. (A) Quantitative measurements of the total EC cell volume. “n” denotes the number of cells of which total cell volume was measured for each genotype. (B) EM cross-sections of posterior midguts. Arrows indicate the PM, mucus (M), and lumen (L). (Scale bar, 800 nm.) (C) Quantitative measurements of the PM thickness. “n” denotes the number of PM thickness measurements for each genotype. (D) qPCR analysis of Crys using total RNA from dissected midguts of indicated genotypes. (E) Survival analysis of wild-type, CG5789 RNAi, and flies coexpressing hCFTRs upon oral infection with P. aeruginosa. Error bars indicate SEM. (F) qPCR analysis of Dpt using total RNA from dissected midguts of indicated genotypes 24 h after Ecc15 oral infection. (G) The posterior midguts of 7- to 10-d-old wild type and RNAi line against CG5789 stained anti–β-gal to mark Dl (green)-expressing ISCs and anti-pH3 to mark mitotic ISCs (red). White arrowheads mark mitotically active ISCs. (Scale bar, 50 μm.) (H) The average number of pH3⁺ cells in the posterior midguts expressing RNAi against CG5789. (I) The average number of pH3⁺ cells in the posterior midguts coexpressing wild-type or mutant hCFTRs with CG5789 RNAi. “n” denotes the number of posterior midguts examined for each genotype. Error bars indicate SEM. ***P < 0.001 (two-tailed t test). n.s., not significant.

Fig. 3.

miR-263a regulates ENaC and osmotic stress downstream of Dmel\CFTR. (A) miR-263a levels are reduced when CG5789 is depleted compared to wild-type controls. (B) Fluorescent monitoring of intracellular Na⁺ levels in the midgut epithelium using Sodium Green. Overexpressing miR-263a in a CG5789-depleted background partially rescues increased fluorescence observed in absence of CG5789 compared to wild-type controls. (C) Quantitative measurements of the total EC cell volume. “n” denotes the number of cells of which total cell volume was measured for each genotype. (D) The number of pH3⁺ cells in the midguts of indicated genotype. Error bars indicate SEM. **P < 0.005 and ***P < 0.0005 two-tailed t test (A) or one-way ANOVA (B–D). n.s., not significant.

Fig. 4.

RNA-Seq analysis of Dmel\CFTR intestine. (A) Pairwise Pearson correlation coefficient (R) of all samples calculated using log2(TPM) (transcript count per million) of 17,490 genes. (B) DEGs called using DESeq2 with P value <0.05 and fold-change ≥1.2 as the cutoff. (C–E) Overlaps between 914 DEGs in a fruit fly model and 151 overlapping DEGs in rat (GSE81114), and 120 (GSE5715) and 47 DEGs (GSE765) in mouse.

Fig. 5.

Intestinal stem cell phenotypes of Muc68D RNAi. (A) The average number of pH3⁺ cells in the posterior midguts expressing RNAi against Muc68D 24 h after Ecc15 oral infection. (B) The average number of pH3⁺ cells in the posterior midguts expressing RNAi against Muc68D, CG5789, and both Muc68D and CG5789. (C) Survival analysis of wild-type and Muc68D RNAi flies upon oral infection with P. aeruginosa. Error bars indicate SEM. (D) Internal bacterial load significantly increased in Muc68D RNAi and CG5789 RNAi flies. (E) The average number of pH3⁺ cells in the posterior midguts expressing RNAi against Muc68D with and without antibiotics treatment. (A, B, and E) “n” denotes the number of posterior midguts examined for each genotype. Error bars indicate SEM. **P < 0.05 and ***P < 0.001 (two-tailed t test). n.s., not significant.

Fig. 6.

Fly intestinal model of CF. Proposed model for the regulation of PM homeostasis, which is required for protection against pathogenic infections. Texts and arrows highlighted in red are consequences of having specific mutation as labeled.