miR-263a Regulates ENaC to Maintain Osmotic and Intestinal Stem Cell Homeostasis in Drosophila

Abstract

Proper regulation of osmotic balance and response to tissue damage is crucial in maintaining intestinal stem cell (ISC) homeostasis. We found that Drosophila miR-263a downregulates the expression of epithelial sodium channel (ENaC) subunits in enterocytes (ECs) to maintain osmotic and ISC homeostasis. In the absence of miR-263a, the intraluminal surface of the intestine displays dehydration-like phenotypes, Na⁺ levels are increased in ECs, stress pathways are activated in ECs, and ISCs overproliferate. Furthermore, miR-263a mutants have increased bacterial load and expression of antimicrobial peptides. Strikingly, these phenotypes are reminiscent of the pathophysiology of cystic fibrosis (CF) in which loss-of-function mutations in the chloride channel CF transmembrane conductance regulator can elevate the activity of ENaC, suggesting that Drosophila could be used as a model for CF. Finally, we provide evidence that overexpression of miR-183, the human ortholog of miR-263a, can also directly target the expressions of all three subunits of human ENaC.

Keywords: Drosophila; ENaC; cystic fibrosis; miR-183; miR-263a; microRNA.

Figure 1. Phenotypes of miR-263a mutant

(A–B) The posterior midguts of 14 days old wild-type and miR-263a mutant stained with anti-β-gal to mark Dl (green). White arrowheads mark symmetrically divided ISCs (B). (C–D) The posterior midguts of wild-type and miR-263a mutant stained with anti-pH3 to mark mitotic ISCs (red). (E) The average number of pH3+ cells in the posterior midguts at 7 and 14 days old. “n” denotes the number of posterior midguts examined for each genotype. Error bars indicate SEM. ^***P < 0.001 (two-tailed t-test). (F–I″) MARCM clones are labeled with GFP. White dotted lines outline the miR-263a mutant clones. (F–G″) miR-263a mutant clones can induce ISC proliferation non-cell-autonomously as indicated by the increased number of pH3+ cells (red) outside the mutant clones (orange arrowheads). (H–I″) Non-cell-autonomous increase in the number of ISCs, marked with Dl-LacZ (red), was observed outside the miR-263a mutant clones. See also Figure S1.

Figure 2. Absence of miR-263a activates stress and developmental signaling pathways

(A–B″) Activation of the JNK pathway in miR-263a mutants as visualized by the increased number of puc-LacZ expressing cells. White dotted lines outline miR-263a mutant clones. (C–D) Enhancement of JAK/STAT pathway activity in miR-263a mutants as visualized by the increased expression of 10xSTAT-GFP reporter. (E–F″) Enhancement of EGFR pathway activity in miR-263a mutants as visualized by the increased expression of dpERK. See also Figure S2.

Figure 3. Regulation of ENaC by miR-263a

(A) Relative Nach transcript level in miR-263a mutant midguts. (B) Western blot analysis of Nach in miR-263a mutant midguts. α-Tubulin was used as loading control. (C) Quantitative analysis of the Western blot from (B). (D) Nach 3′ UTR luciferase reporter assay. (E) SCNN1G 3′ UTR luciferase reporter assay. (F) Relative transcript levels of human ENaC subunits after overexpression of miR-183 in CFBE41o- cells. (G) The average number of pH3+ cells in the posterior midguts at 14 days old. Depletion of Nach using two independent RNAi lines and two different Gal4 drivers in the miR-263a mutant background suppressed the increased number of pH3+ cells. The phenotype was also suppressed when other ENaC subunits were depleted in the mutant background. “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). See also Figure S3 and Table S1.

Figure 4. Increased Na⁺ in the miR-263a mutant posterior midgut epithelium

(A-A′) Na⁺ in the midgut epithelium of control midgut. (B-B′) Enhancement of Na⁺ in the miR-263a mutant midgut epithelium. (C–D′) Suppression of increased Na⁺ uptake by overexpressing miR-263a (C-C′) or depleting Nach (D-D′) in the mutant background. (E-E′) Overexpression of Nach partially increased the amount Na+ in the midgut epithelium. See also Figure S4.

Figure 5. Increased ENaC activity disrupts intestinal homeostasis in miR-263a mutants

(A–D) EM cross-sections of posterior midguts. (B) miR-263a mutants show reduced PM thickness compared to wild-type (A). Overexpression of miR-263a (C) and depletion of Nach (D) in the mutant background. Arrows indicate the PM (peritrophic matrix), M (mucus), and L (lumen with digested food). (E) Quantitative measurements of the PM thickness. “n” denotes the number of PM thickness measurements for each genotype. (F–G) Enlarged ECs in the miR-263a mutants visualized by anti-Arm marking the cell membrane. White dotted lines outline the representative ECs. (H) Normalized quantitative measurements of the total EC and their respective nuclei surface area. “n” denotes the number of surface area measurements for each genotype. (I) Quantitative measurements of the total EC volume. “n” denotes the number of cell-volume measurements for each genotype. Error bars indicate SEM. ^**P < 0.05 and ^***P < 0.001 (two-tailed t-test). See also Figure S5.

Figure 6. Increased activation of the Imd pathway and susceptibility to bacterial infection in miR-263a mutants

(A) Significant increase in the relative transcript levels of the AMPs was detected in miR-263a mutant midguts by qPCR. (B–C′) Expression of Drs-GFP in the posterior midgut. The expression of Drs-GFP is significantly lower in wild-type (B-B′) than in miR-263a mutants (C-C′). (D) Internal bacterial load significantly increased in the miR-263a mutants. Error bars indicate SEM. ^***P < 0.001 (two-tailed t-test). Wild-type and miR-263a mutant flies homogenates were spread on the plate. The bacteria colonies were shown on the bottom panel. (E) The average number of pH3+ cells in the posterior midguts after antibiotics treatment for 16 days. Feeding antibiotics had no effect on the hyperproliferation of ISCs observed in the miR-263a mutant midguts. Bottom panel shows the bacteria colonies on medium plate spread with antibiotics fed wild-type and miR-263a mutant homogenate. Internal bacteria were absent in both wild-type and miR-263a mutant flies fed for 16 days with food containing antibiotics. “n” denotes the number of posterior midguts examined for each genotype. Error bars indicate SEM. ^***P < 0.001 (two-tailed t-test). (F) Survival analysis of wild-type and miR-263a mutant flies upon oral infection with P. aeruginosa. The miR-263a mutant flies exhibited significant increase in lethality 3 days after infection. Error bars indicate SEM. See also Figure S6.

Figure 7. Model of regulation of ENaC by miR-263a

A model of miR-263a maintaining ISC and osmotic homeostasis in the midgut epithelium by directly and indirectly regulating the expression of ENaC subunits. See also Figure S7.