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The RNA-binding protein Bicaudal C is an important regulator of embryonic development in C. elegans, Drosophila and Xenopus. In mouse, bicaudal C (Bicc1) mutants are characterized by the formation of fluid-filled cysts in the kidney and by expansion of epithelial ducts in liver and pancreas. This phenotype is reminiscent of human forms of polycystic kidney disease (PKD). Here, we now provide data that Bicc1 functions by modulating the expression of polycystin 2 (Pkd2), a member of the transient receptor potential (TRP) superfamily. Molecular analyses demonstrate that Bicc1 acts as a post-transcriptional regulator upstream of Pkd2. It regulates the stability of Pkd2 mRNA and its translation efficiency. Bicc1 antagonized the repressive activity of the miR-17 microRNA family on the 3'UTR of Pkd2 mRNA. This was substantiated in Xenopus, in which the pronephric defects of bicc1 knockdowns were rescued by reducing miR-17 activity. At the cellular level, Bicc1 protein is localized to cytoplasmic foci that are positive for the P-body markers GW182 and HEDLs. Based on these data, we propose that the kidney phenotype in Bicc1(-/-) mutant mice is caused by dysregulation of a microRNA-based translational control mechanism.
Fig. 3. Bicc1 regulates Pkd2. (A-C) qPCR analysis for mouse Pkd1, Pkd2 and Pkhd1 mRNA levels in Bicc1+/+ (dark gray) and Bicc1−/− (light gray) littermates. The averages and s.d. from six kidney pairs at E15.5 and four pairs at E18.5 are shown (*, P<0.05, Student's t-test). (D) Western blot analysis comparing Pkd2 protein levels in E15.5 kidneys from two Bicc1 mouse litters (#55 and #65) of the indicated genotypes using the Pkd2-specific antibody from Santa Cruz. Actin served as a loading control. (E) Quantification of multiple Pkd2 western blot analyses comparing several mouse litters at E15.5 and normalized to actin. Average values and s.d. are indicated (*, P<0.05, Student's t-test). (F) Whole-mount in situ hybridization for Pkd2 mRNA on uninjected and xBicC-MO1+2-injected Xenopus embryos at stage 39.
Fig. 4. Bicc1 is epistatic to Pkd2. (A-A′) Analysis of the expression of nbc-1 in the Xenopus late distal tubule at stage 39 by whole-mount in situ hybridization of uninjected control embryos, and embryos radially injected with xBicC-MO1+2 in the presence or absence of a single injection of 2 ng pkd2 mRNA. (B) Quantification of the expression of nbc-1 in the late distal tubule from the experiments shown in A-A′. Black, bilateral expression; white, no expression; gray, unilateral expression rescued by co-injected mRNA. The number of embryos analyzed is indicated. (C-D) Reciprocal experiments to those in A-B using Xenopus embryos injected with either Pkd2-MO alone or together with pkd2-myc or bicc1 mRNA. Co-injection with pkd2-myc rescued nbc-1 expression, whereas co-injection with bicc1 did not. (E) Flow diagram outlining the proposed mechanism of Bicc1 activity.
Fig. 7. Cross-talk between Bicc1 and the miR-17 miRNA family. (A) Alignment of the Xenopus miR-17 family members. Mature forms are highlighted in yellow. The sequence targeted by the miR-17 antisense MO (miR-17-MO) is indicated by the black line. The nucleotides shared between miR-17-MO and the individual members are indicated in red. (B-B′) Analysis of the expression of nbc-1 by whole-mount in situ hybridization of uninjected control embryos, embryos injected with xBicC-MO1+2 alone or with miR-17-MO. Arrowheads indicate the expression of nbc-1 in the Xenopus late distal tubule. Note that this expression domain is rescued upon co-injection of the two antisense MOs. (C) Quantification of the experiments shown in B-B′. Black, bilateral expression; white, reduced or no expression; gray, unilateral, rescued expression in the late distal tubule. (D,D′) Models for the post-transcriptional regulation of Pkd2 mRNA by the miR-17 family in the absence or presence of Bicc1.
Fig. 3. Pkd2 expression at stg. 39
Fig. S5. pkd2 mRNA and protein expression in Xenopus. (A) Whole-mount in situ hybridization detecting expression of pkd2 mRNA in the Xenopus pronephros. Inset is a magnified image of the pronephric tubules and the nephrostomes (arrowheads). (B-C′′) Immunofluorescence using antibodies against Pkd2 (AB9088, Millipore; red) and acetylated α-tubulin (green). Note that Pkd2 is expressed in the cilia of the duct (B-B′′) and those of the multiciliated cells in the nephrostomes (C-C′′) present in the Xenopus pronephros. (D-E′′) Immunofluorescence analysis of the cilia in the pronephric tubules using antibodies against Pkd2 (red) and acetylated α-tubulin (green) comparing uninjected Xenopus embryos with embryos injected with Pkd2-MO (400 fmol) at stage 40. Nuclei were counterstained with DAPI.
Fig. S6. Loss of Pkd2 results in a PKD-like phenotype in Xenopus embryos. (A-B′) Xenopus embryos microinjected with antisense MOs against pkd2 (Pkd2-MO) developed a PKD-like phenotype. Morphological analysis showed severe edema formation (A,A′), while histological analysis using Hematoxylin and Eosin staining detected dilated pronephric tubules (B,B′). en, endoderm; no, notochord; nt, neural tube; pn, pronephros; so, somites, (C) Schematic of the Xenopus pronephros indicating the segments expressing the marker genes nbc-1 and lim1 (lhx1). (D-E′′) Whole-mount in situ hybridization of Xenopus embryos injected with xBicC-MO1+2, Pkd2-MO or uninjected controls at stage 39 with nbc-1 (D-D′′) and Lim1 (E-E′′). Arrowheads indicate the expression of these two genes in the late distal tubule that is lost in the antisense MO-injected embryos.
Fig. S7. Expression of pronephric marker genes in pkd2 morphants. (A-E′) Whole-mount in situ hybridization of uninjected control and Pkd2-MO-injected Xenopus embryos at stage 39 with Nephrin (A,A′), xSGLT-1K (B,B′), NKCC2 (C,C′), NCC (D,D′) and β1-Na/K ATPase (E,E′) mRNA. Note that the expression of NCC, a marker for late distal tubule and pronephric duct, was reduced in Pkd2-MO-injected embryos. (F) Schematic of the Xenopus pronephros indicating the markers analyzed.
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