Osr1 Interacts Synergistically with Wt1 to Regulate Kidney Organogenesis

<div><p>Renal hypoplasia is a common cause of pediatric renal failure and several adult-onset diseases. Recent studies have associated a variant of the <i>OSR1</i> gene with reduction of newborn kidney size and function in heterozygotes and neonatal lethality with kidney defects in homozygotes. How OSR1 regulates kidney development and nephron endowment is not well understood, however. In this study, by using the recently developed CRISPR genome editing technology, we genetically labeled the endogenous Osr1 protein and show that Osr1 interacts with Wt1 in the developing kidney. Whereas mice heterozygous for either an <i>Osr1</i> or <i>Wt1</i> null allele have normal kidneys at birth, most mice heterozygous for both <i>Osr1</i> and <i>Wt1</i> exhibit defects in metanephric kidney development, including unilateral or bilateral kidney agenesis or hypoplasia. The developmental defects in the <i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> mouse embryos were detected as early as E10.5, during specification of the metanephric mesenchyme, with the <i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> mouse embryos exhibiting significantly reduced Pax2-positive and Six2-positive nephron progenitor cells. Moreover, expression of <i>Gdnf</i>, the major nephrogenic signal for inducing ureteric bud outgrowth, was significantly reduced in the metanephric mesenchyme in <i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> embryos in comparison with the <i>Osr1</i>^<i>+/-</i> or <i>Wt1</i>^<i>+/-</i> littermates. By E11.5, as the ureteric buds invade the metanephric mesenchyme and initiate branching morphogenesis, kidney morphogenesis was significantly impaired in the <i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> embryos in comparison with the <i>Osr1</i>^<i>+/-</i> or <i>Wt1</i>^<i>+/-</i> embryos. These results indicate that Osr1 and Wt1 act synergistically to regulate nephron endowment by controlling metanephric mesenchyme specification during early nephrogenesis.</p></div

<i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> embryos exhibit defects in ureteric bud branching.

<p>(A-C) HE staining of P0 kidney sections from <i>Osr1</i>^<i>+/-</i> (A), <i>Wt1</i>^<i>+/-</i> (B) and <i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> (C) mice. (D-F) Immunofluorescent staining of Six2 (red) in P0 sections from <i>Osr1</i>^<i>+/-</i> (D), <i>Wt1</i>^<i>+/-</i> (E) and <i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> (F) mice. Nuclear are co-staining with DAPI (Blue). (G-L) Whole mount immunofluorescence detection of Jag1 (red, G-I) or Wt1 (green, J-L) and pan-cytokeratin (CK, green in G-I, and red in J-L) in cultured <i>Osr1</i>^<i>+/-</i> (G, J), <i>Wt1</i>^<i>+/-</i> (H, K) and <i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> (I, L) kidney explants. (M-O) Whole mount immunofluorescent detection of Pax2 (Green) in <i>Osr1</i>^<i>+/-</i> (M), <i>Wt1</i>^<i>+/-</i> (N) and <i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> (O) kidney at E12.5. Scale bar, 200 μm.</p

Analysis of cell apoptosis and proliferation during early kidney development in <i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> embryos and littermates.

<p>(A-H) Cell apoptosis is analyzed by TUNEL (green) and counterstained with DAPI (blue). TUNEL assay detected no obvious change in cell apoptosis in <i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> metanephric mesenchyme (C, D, G, H) compared with <i>Osr1</i>^<i>+/-</i> (A, E) and <i>Wt1</i>^<i>+/-</i> (B, F) metanephric mesenchyme at E10.5 (A-D) and E11.5 (E-H). (I-P) Analysis of cell proliferation by BrdU incorporation at E10.5 (I-L) (n = 18) and E11.5 (n = 7) (M-P) embryos. BrdU index is calculated by the ratio of BrdU-positive cells (green) versus Six2-positive cells (red). Scale bar, 100 μm.</p

<i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> mice exhibit kidney defects.

<p>(A-F) At P0, compared with wildtype (A), <i>Osr1</i>^<i>+/-</i> (B), and <i>Wt1</i>^{<b><i>+/-</i></b>} (C) mice, <i>Osr1</i>^<i>+/-</i><i>Wt1</i>^{<b><i>+/-</i></b>} mice exhibit kidney malformations, including bilateral renal agenesis (5/44) (D), unilateral renal agenesis (15/44) (E) and renal hypoplasia (22/44) (F). (G) Quantification of the number of nephrons per kidney in the wildtype (<i>+/+</i>), <i>Osr1</i>^<i>+/-</i>, <i>Wt1</i>^<i>+/-</i>, and <i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> mice at P21. Error bar represents SEM. ***, p < 0.005.</p

Molecular marker expression in the metanephric mesenchyme in <i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> embryos and littermates.

<p>Whole mount <i>in situ</i> hybridization detection of <i>Gdnf</i> (A-C), <i>Eya1</i> (D-F), and <i>Sall1</i> (G-I) mRNA expression in E10.5 embryos. Scale bar, 200 μm. (J) Real-time RT-PCR analysis of the levels of expression of <i>Gdnf</i>, <i>Eya1</i>, and <i>Sall1</i> mRNAs in Wt1-GFP+ cells from E9.5 <i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> embryos and <i>Wt1</i>^<i>+/-</i> littermate. *, p < 0.05.</p

<i>Osr1</i>^<i>+/-</i><i>Wt1</i>^<i>+/-</i> embryos exhibit defects in metanephric mesenchyme.

<p>(A-H) Whole mount immunofluorescent staining for Pax2 (red) at E10.5 (A-D) and E11.5 (E-H). The white dot line outlines the ureteric bud epithelium. The green dashed line outlines the MM domain. Asterisk in D indicates that no condensed Pax2+ MM domain was detected. (I) Quantification of MM volume at E10.5 (n = 10). The MM volume does not include UB or duct epithelium. Error bar represent SEM. *, p < 0.05; ***, p < 0.005. (J-M) Immunofluorescent staining for Pax2 (green) in E11.5 sections. Scale bar, 100 μm.</p

Osr1 and Wt1 form protein interaction complex when co-expressed.

<p>(A) HEK293T cells were co-transfected with plasmids expressing Myc-tagged Osr1 and Flag-tagged Wt1, Lhx1, Six2, or a Flag-tag vector, respectively. Immunoprecipitation was carried out with an anti-Myc antibody, and the resulting protein complexes resolved by western blotting and detected with an anti-Flag antibody. Note that Flag-Wt1 and Flag-Six2 were co-immunoprecipitated with Myc-Osr1. (B) HEK293T cells were co-transfected with plasmids expressing Flag-tagged Osr1 and Myc-tagged Pax2. Immunoprecipitation was carried out with an anti-Flag antibody, and the resulting protein complexes resolved by western blotting and detected with an anti-Myc antibody.</p

Osr1 and Wt1 are co-expressed in the early metanephric mesenchyme during kidney development and physically interact <i>in vivo</i>.

<p>(A) Schematic diagram of the <i>Osr1</i> gene structure and strategy for CRISPR/Cas9-mediated insertion of the 2xTY1 epitope tag at the N-terminus of the endogenous Osr1 protein. The gRNA target sequence is shown in red font. The sequence of the mid-portion of the oligonucleotide donor template for homology directed repair contains the 2xTY1 tag-coding sequence (shown in green font). The endogenous <i>Osr1</i> ATG codon is shown in blue font. The tag sequence contains its own ATG initiation codon at the 5’ end (indicated by M* underneath the sequence). (B) <i>Osr1</i>^<i>TY/+</i>and <i>Osr1</i>^{<i>TY1/TY1</i>} mice were identified by PCR genotyping. (C-E) Immunofluorescent staining for the TY1 epitope (red) and the Wt1 protein (green) on sections of E10.5 (C), E11.5 (D), and E13.5 (E) <i>Osr1</i>^<i>TY1/+</i> embryos. (F-H) Immunofluorescent staining for eGFP (red), Wt1 (green) and Jag1 (blue) on sections of E10.5 (F), E11.5 (G), and E13.5 (H) <i>Osr1</i>^<i>GCE/+</i> embryos. The ureteric bud is outlined with white dashed circle in (C-H). (I) E16.5 kidneys were collected from <i>Osr1</i>^{<i>TY1/TY1</i>}and wildtype embryos. Immunoprecipitation was carried out with an anti-TY1 antibody, and the resulting protein complexes resolved by western blotting and detected with anti-Six2 and anti-Wt1 antibodies. Scale bar, 50 μm.</p