Deducing the stage of origin of Wilms' tumours from a developmental series of Wt1-mutant mice

Abstract

Wilms' tumours, paediatric kidney cancers, are the archetypal example of tumours caused through the disruption of normal development. The genetically best-defined subgroup of Wilms' tumours is the group caused by biallelic loss of the WT1 tumour suppressor gene. Here, we describe a developmental series of mouse models with conditional loss of Wt1 in different stages of nephron development before and after the mesenchymal-to-epithelial transition (MET). We demonstrate that Wt1 is essential for normal development at all kidney developmental stages under study. Comparison of genome-wide expression data from the mutant mouse models with human tumour material of mutant or wild-type WT1 datasets identified the stage of origin of human WT1-mutant tumours, and emphasizes fundamental differences between the two human tumour groups due to different developmental stages of origin.

Keywords: Kidney development; Mouse model; WT1; Wilms’ tumour.

Fig. 1.

Lineage tracing of three Cre drivers in cultured kidney rudiments starting from E11.5 kidneys for the indicated time intervals. (A) Nes-Cre Rosa26^eYFP/eYFP. (B) Pax8^+/Cre Rosa26^eYFP/eYFP. (C) Wnt4^+/CreGFP Rosa26^eYFP/eYFP. Scale bars: 200 µm.

Fig. 2.

Renal phenotypes in the three Cre Wt1 conditional models. (A-D) H&E-stained E18.5 embryonic kidneys. (A) Wt1^co/co. (B) Nes-Cre Wt1^co/co. (C) Wnt4^+/CreGFP Wt1^co/co. (D) Pax8^+/Cre Wt1^co/co. CM, cap mesenchyme; CB, comma-shaped body; SB, S-shaped body; UB, ureteric bud; PT, proximal tubule; DT, distal tubule; EM, expanded mesenchyme; IPT, immature proximal tubule. Scale bars: 50 µm. (E) Macroscopic view of Wilms’ tumour in single surviving Nes-Cre Wt1^co/co mouse. Scale bar: 10 mm. (F) Same tumour as in E with left kidney from the same mouse. Scale bar: 10 mm. (G) H&E staining of the tumour in E and F. Scale bar: 500 µm.

Fig. 3.

Genome-wide expression analysis of E18.5 Nes-Cre Wt1^co/co, Wnt4^+/CreGFPWt1^co/co and Pax8^+/CreWt1^co/co kidneys. (A) Comparison at the gene level for genes with increased (left panel) and decreased (right panel) expression. Nes-Cre Wt1^co/co differential genes are shown in blue, Wnt4^+/CreGFP Wt1^co/co differential genes in yellow and Pax8^+/Cre Wt1^co/co differential genes in green. (B) Enrichment for gene sets from cell type/developmental stage-specific GUDMAP datasets of increased genes in the mutant samples. (C) Enrichment for gene sets from cell type/developmental stage-specific GUDMAP datasets of decreased genes in the mutant samples. (D) Enrichment for biological processes (green nodes) and human/mouse phenotypes (brown nodes) coupled to genes increased in the mutant genotypes. (E) Enrichment for biological processes (green nodes) and human/mouse phenotypes (brown nodes) coupled to genes decreased in the mutant genotypes.

Fig. 4.

Time-lapse analysis of control and conditional Wt1 mutants at the indicated time points. (A) Wt1^co/GFP (control). (B) Nes-Cre Wt1^co/GFP. (C) Pax8^+/Cre Wt1^co/GFP. Scale bars: 200 µm.

Fig. 5.

Antibody staining of cultured kidney rudiments (E11.5+6 days in culture) for Wt1, E-cadherin (E-cad), Pax8, pan-Cytokeratin (pan-CK) and Megalin. Genotypes and antibodies are indicated.

Fig. 6.

Antibody staining for nephron progenitor markers (E11.5+6 days in culture). Wt1^GFP signal, antibodies and genotypes as indicated. Arrow indicates loose disorganized cap mesenchyme; asterisk indicates mesenchymal Wt1^GFP-positive cells outside the cap mesenchyme. Scale bars: 100 µm.

Fig. 7.

Branching phenotype in Wt1 mutants. (A) Quantification of branch length, width and angle using time-lapse analysis. Two independent mutant and control kidneys were analyzed and shown individually. T=0 is the moment a branch formed. Error bars indicate the s.e.m. of different branches in the same kidney, n≥6. P-values were calculated using a two-tailed Student's t-distribution. (B) Pan-Cytokeratin antibody staining in indicated genotypes (E11.5+6 day culture). Scale bars: 200 µm. (C) Recombination experiments between wild-type mesenchymes and Pax8^+/Cre mutant ureteric buds stained for calbindin-D-28k antibodies. Scale bars: 100 µm. Panel 1: wild-type kidney (E11.5+2 day culture). Panel 2: Pax8^+/Cre Wt1^co/co kidney (E11.5+2 day culture). Panel 3: recombined wild-type mesenchymes with mechanically dissected Pax8^+/Cre Wt1^co/co ureteric buds (E11.5+2 day culture). Panel 4: recombined wild-type mesenchymes with mechanically dissected Pax8^+/Cre Wt1^co/co ureteric buds (E11.5+2 day culture).

Fig. 8.

Comparison of Nes-Cre Wt1^co/co, Pax8^+/CreWt1^co/co, WT1-mutant and WT1-wild-type Wilms' tumour microarray data. (A) Comparison at the gene level. The 13 genes in the Nes-Cre Wt1^co/co/WT1-mutant Wilms' tumour overlap that give enrichment for muscle functions (see main text) are indicated. (B) Comparison at the GO-term ‘Biological Process'. Red nodes: muscle-related. Light blue nodes: bone/cartilage-related. Yellow nodes: apoptosis-related. Grey nodes: kidney-development-related. Green nodes: histone-modification-related. Orange nodes: cell-cycle-related. (C) Alcian Blue/Alizarin Red staining of E18.5 sections. Scale bars: 50 µm.