Bicaudal C mutation causes myc and TOR pathway up-regulation and polycystic kidney disease-like phenotypes in Drosophila

Abstract

Progressive cystic kidney degeneration underlies diverse renal diseases, including the most common cause of kidney failure, autosomal dominant Polycystic Kidney Disease (PKD). Genetic analyses of patients and animal models have identified several key drivers of this disease. The precise molecular and cellular changes underlying cystogenesis remain, however, elusive. Drosophila mutants lacking the translational regulator Bicaudal C (BicC, the fly ortholog of vertebrate BICC1 implicated in renal cystogenesis) exhibited progressive cystic degeneration of the renal tubules (so called "Malpighian" tubules) and reduced renal function. The BicC protein was shown to bind to Drosophila (d-) myc mRNA in tubules. Elevation of d-Myc protein levels was a cause of tubular degeneration in BicC mutants. Activation of the Target of Rapamycin (TOR) kinase pathway, another common feature of PKD, was found in BicC mutant flies. Rapamycin administration substantially reduced the cystic phenotype in flies. We present new mechanistic insight on BicC function and propose that Drosophila may serve as a genetically tractable model for dissecting the evolutionarily-conserved molecular mechanisms of renal cystogenesis.

Fig 1. BicC is necessary for proper renal tubule function.

Malpighian (fly renal) tubules were dissected and photographed (light microscopy) at 1 day (A, D, G) or 14 days (B, E, H) or 30 days (C, F, I). Compared to Ore^R wild-type flies (wt, A-C), the BicC^Δ/YC33 (D-F) and the BicC^Δ/IIF34 (G-I) mutant flies both displayed cystic tubules. Both Malpighian tubules degenerated over time, with cysts evident throughout the whole tubule and in both tubules by day 14 (compare A, D, G with B, E, H and C, F, I). Enlargements of the boxed areas in (G, H, I) are shown respectively in (J, K, L), highlighting the tubular deformities. The normal single-cell layer of the renal tubule epithelium appeared disorganized in the mutant flies, which had abnormal tubule budding and branching (arrows). Malpighian tubules dissected ex vivo from BicC mutants contained various crystalline particles apparently floating in the luminal fluid (arrowheads) that were not observed in wild-type flies raised in parallel under identical conditions. Scale bar: 1 mm (A-I) or 312.5 μm (J, K, L).

Fig 2. Quantification of the Malpighian tubule cystic phenotype of BicC flies.

Malpighian tubules from wild type, BicC^Δ/YC33 and BicC^Δ/IIF34 (50 flies each) were dissected at age one day (A), 10 days (B), and 30 days (C) and scored phenotypically ex vivo. Shown are the percentages of tubules affected in the terminal, intermediate, and proximal regions, as well as the observed extra tubular branching. Anterior and posterior tubules were scored separately. Tubular cyst number from the same sample is shown (D). The occurrence of BicC^Δ/YC33 tubules displaying at least four cysts appeared to increase over time. The same class in the BicC^Δ/IIF34 tubules was more numerous at one day of age, was less represented at ten days, and increased again in the older flies (Fig 2D), possibly reflecting mortality of the more severely affected flies (see text).

Fig 3. BicC expression in renal tissue.

Confocal section of Drosophila Malpighian tubules with BicC immunofluorescent staining (red, A, C, D) and DAPI nuclear staining (blue B, C) show that BicC is expressed in the principal cells. Boxed area in (A) is shown enlarged in (D). Epifluorescence microscopy of a 5 μm kidney section from C57BL/6 mice shows Bicc1 accumulation in the cells lining the renal tubule (red, E, G, H). DNA, DAPI (blue, F, G). Panel (H) is an enlargement of the boxed area in (E). Scale bars: 20 μm.

Fig 4. BicC flies exhibit severe renal defects and impaired survival.

(A) Survival curves of fly populations (n = 200, standard deviations are shown) of wild-type (wt), BicC^Δ/YC33 (Δ/YC33) and BicC^Δ/IIF34 (Δ/IIF34). Compared to wild-type, both BicC^Δ/YC33 and BicC^Δ/IIF34 flies have impaired survival, reaching 50% respectively at 48, 28, and 16 days after adult eclosion. (B) Malpighian tubules dissected from a moribund BicC^Δ/IIF34 fly showed severe deformities with large cysts containing apparent impacted materials; scale bar: 100 μm. (C) BicC^Δ/YC33 and BicC^Δ/IIF34 flies exhibited sensitivity to salt stress. Fly populations (wild-type, BicC^Δ/YC33, BicC^Δ/IIF34, n = 150, standard deviations are shown) placed in vials containing cornmeal agar with 0.5M NaCl were at a disadvantage and had shorter life span than the wild-type controls.

Fig 5. BicC regulates d-myc expression in the Malpighian tubule.

(A) RT-PCR of RNA immunoprecipitated with either non-immune control serum (NI, lane 1) or BicC antiserum (αBicC, lane 2). A product specific for d-myc was amplified exclusively from the BicC immunoprecipitate, indicating that BicC regulates d-myc mRNA in the Malpighian tubule. Similar to the situation in the ovary [24], BicC primers used as positive control also yielded a specific product only in the immunoprecipitate. In contrast, tubulin 84B primers did not produce any amplification product, showing that the immunoprecipitation was specific (negative control). Lane 3: PCR negative control (-, no cDNA). Lane 4: PCR positive control (+, cDNA, Malpighian tubule total cDNA). (B) RT-PCR of control RNA immunoprecipitation from wild type and Df(2L)RA5/Df(2L)Osp29 (BicC null, ΔBicC) Malpighian tubules. For both extracts RNP particles (RNPs) were captured with either non-immune control serum (NI, lane 1, 3) or BicC antiserum (αBicC, lane 2, 4). d-myc-specific amplification products were exclusively observed for wild type extracts with the RNP captured by the BicC antiserum, but not with those captured by the non-immune serum. Neither non-immune nor BicC antiserum recovered d-myc containing particles. RNAs extracted from small aliquots of the input extracts (respectively ΔBicC and wild-type) also produced distinct clear PCR products. (C) Representative immunoblots of extracts of Malpighian tubule (four pairs per lane) from wild-type (wt), BicC^Δ/YC33 and BicC^Δ/IIF34 flies probed for d-Myc and tubulin (left). Corresponding graphs of means ± standard deviations of d-Myc levels relative to tubulin (right). Values were calculated from independent biological replicas (n, indicated) and p values (Student’s t test) are shown (right). Myc/tubulin ratios were normalized to the wild-type average. d-Myc protein levels were generally higher in BicC mutants and increased with age.

Fig 6. d-Myc overexpression in the Malpighian tubule causes severe cellular and tubular defects.

Confocal sections of d-Myc immunostaining of Malpighian tubules from wild-type flies (A-C), BicC^Δ/YC33 flies (D-F), and flies with d-Myc Gal4-driven over-expression in both principal and stellate cells (Gal4>d-myc, G-I). d-Myc (red), DNA (DAPI, blue). d-Myc over-expression in the Malpighian tubules caused severe defects. The irregular shape and density of nuclei are suggestive of tissue disorganization. All images in this panel were captured with identical settings and the signal for d-Myc staining in the Gal4>d- myc tubule (G) was saturated, indicating higher levels of d-Myc over-expression relative to those induced by BicC mutation. Scale bar: 50 μm. (J) Representative immunoblot of extracts of Malpighian tubules dissected from wild-type (wt) flies, flies heterozygote for the Gal4-d-myc construct (Gal4/+) and flies over-expressing d-myc c724/+; c42/Gal4>d-myc (Gal4>d-myc) probed for d-Myc and α-tubulin. (k) Graph of means ± standard deviations of d-Myc levels relative to tubulin from five independent biological replicas per each genotype. Values were normalized to the wild-type average; p values (Student’s t test) are shown. d-myc (d-Myc/tubulin) over-expression in Malpighian tubules ranged from three to over 30 times the levels in tubules of control flies.

Fig 7. d-myc knockdown can rescue the Malpighian tubule defects of BicC mutants.

Light microscopy of dissected Malpighian tubules from sibling BicC^YC33/YC333 (A, B, C, control) and BicC^YC33/YC33; myc^RNAi (D, E) flies in which RNAi was induced in both principal and stellate cells with the c42 and c724 drivers respectively. d-myc RNAi rescued greatly the morphology of the Malpighian tubule. Boxed regions in (A) and (D) are shown enlarged respectively in (B, C) and (E). (F) Representative d-Myc and tubulin immunoblots of extracts from five Malpighian tubules dissected from flies of the following genotypes: wild-type (wt); heterozygotes for the Gal4 driver c724 and c42 constructs (Gal4/+); heterozygotes for the d-myc^RNAi construct (myc^RNAi/+); myc RNAi driven by c724 and c42 (Gal4>myc^RNAi); BicC^YC33/YC33 homozygotes (BicC^YC33); myc RNAi driven in BicC^YC33 homozygotes (BicC^YC33; Gal4>myc^RNAi). The latter two genotypes were sibling flies from the same crosses. (G) Corresponding graph summarizing quantitative immunoblots of means ± standard deviations of d-Myc/tubulin ratios from three independent biological replicas per each genotype. Values were normalized to the wild-type average. The p value (Student’s t test) is shown for the BicC mutants. Reducing d-myc expression in BicC^YC33/YC33 mutants restored the d-Myc protein to control levels. (H) Cystic scoring of the BicC^YC33/YC33; Gal4/+ (red, n = 100) and BicC^YC33/YC33; Gal4/myc^RNAi (blue) sibling flies. Results are shown for the lines TRiP.JF01762 (dark blue, n = 100) shown above, and TRiP.JF01761 (light blue, n = 80, see supplemental information). RNAi-induced d-Myc reduction decreased cystic deformities in the terminal and intermediate tubules. Results for the anterior and posterior tubules are shown separately. (I-K) Confocal sections of Malpighian tubules dissected from BicC^YC33/YC33 flies (I, control) and BicC^YC33/YC33; myc^RNAi flies (J, K) stained with DAPI. The distribution of cell nuclei in BicC Malpighian tubules (I) appeared disturbed (compared with wild-type in Figs 3B and 6B) with disrupted cell arrangement and cystic enlargements. Defects were largely rescued by reducing d-myc expression via RNAi specifically in the principal and stellate cells of the Malpighian tubules (J, K). Scale bar: 100 μm.

Fig 8. TOR upregulation and rapamycin rescue of BicC mutant flies.

(A) Control wild-type (wt) and BicC^Δ/IIF34 (Δ/IIF34) flies that were administered equal volumes of solvent (ethanol, vehicle) showed the characteristic impaired survival of BicC mutants compared to wild-type. (B) In contrast, sibling flies of both genotypes that were administered rapamycin exhibited markedly improved survival of the BicC mutants compared to control flies that were administered vehicle. A, B n = 200, standard deviations are shown. Rapamycin administration did not compromise survival of the wild-type flies. Rapamycin appeared to induce almost complete rescue at early time points and substantial rescue over time. Malpighian tubules of vehicle (C) and rapamycin-treated (D) BicC^Δ/IIF34 flies showed marked morphological rescue with fewer cysts and more regular tubule structure. (E) Total and phosphorylated S6K immunoblots of extracts from wild type and BicC^ΔIIF34 Malpighian tubules from flies administered rapamycin or vehicle for 8 days post-eclosion indicated apparent mitigation of the TOR up-regulation in the BicC mutants.

Fig 9. Bicc1 downregulation in Pkd1^-/- mice.

(A) In accordance to our analyses of human microarrays of PKD patients, Bicc1 immunoblots of kidney extracts from newborn littermates Pkd^+/+ and Pkd1^-/- showed that all Pkd1^-/- renal tissues exhibited considerable Bicc1 downregulation. GAPDH: loading control.