Genomic characterization of Wilms' tumor suppressor 1 targets in nephron progenitor cells during kidney development

Abstract

The Wilms' tumor suppressor 1 (WT1) gene encodes a DNA- and RNA-binding protein that plays an essential role in nephron progenitor differentiation during renal development. To identify WT1 target genes that might regulate nephron progenitor differentiation in vivo, we performed chromatin immunoprecipitation (ChIP) coupled to mouse promoter microarray (ChIP-chip) using chromatin prepared from embryonic mouse kidney tissue. We identified 1663 genes bound by WT1, 86% of which contain a previously identified, conserved, high-affinity WT1 binding site. To investigate functional interactions between WT1 and candidate target genes in nephron progenitors, we used a novel, modified WT1 morpholino loss-of-function model in embryonic mouse kidney explants to knock down WT1 expression in nephron progenitors ex vivo. Low doses of WT1 morpholino resulted in reduced WT1 target gene expression specifically in nephron progenitors, whereas high doses of WT1 morpholino arrested kidney explant development and were associated with increased nephron progenitor cell apoptosis, reminiscent of the phenotype observed in Wt1(-/-) embryos. Collectively, our results provide a comprehensive description of endogenous WT1 target genes in nephron progenitor cells in vivo, as well as insights into the transcriptional signaling networks controlled by WT1 that might direct nephron progenitor fate during renal development.

Fig. 1.

Distribution of WT1 bound promoter regions and the WT1 matrix site. (A) Weblogo of the WT1 matrix site identified by THEME, comprising multiple permutations of a high-affinity, EGR1-like WT1 consensus site. (B) Histogram showing distribution of the defined peak regions of WT1-bound promoters identified by ChIP-chip in relation to the transcriptional start site (TSS). Most WT1-bound regions (~85%) and the WT1 matrix sites contained within these defined peak regions are localized within 1 kb of the TSS.

Fig. 2.

ChIP-PCR validation of ChIP-chip results. (A-R) Plots of the mean fold-enrichment of WT1 ChIP versus input DNA (n=3) for selected WT1-bound genes expressed in the developing kidney. The numeric peak fold-enrichment value from the microarray is noted above the peak. The chromosomal position of the ChIP-PCR amplicon is noted along the X axis (black box). The position of the transcript is noted below the graph. The corresponding ChIP-PCR result is shown underneath. (A-H) WT1-binding sites with fold-enrichment scores less than 8-fold were generally not validated by ChIP-PCR (see Table S6 in the supplementary material for quantification of ChIP-PCR results). (I-R) By contrast, ChIP-PCR consistently confirmed WT1-specific enrichment in >90% of target genes with enrichment scores 8-fold or higher.

Fig. 3.

Reduced WT1 expression and arrested development in WT1 morphant kidney explants. (A,A′,C,C′) E12.5 kidney explants cultured for 24 hours in media alone (A,A′) or in 10 μM control morpholino (cntl MO; C,C′) exhibit strong WT1 (red) expression in the cap of nephron progenitors (dashed outlines) surrounding the tip of the ureteric bud (green), as well as in epithelialized nephrogenic structures (arrows). Arrowheads denote regions shown in high magnification in adjacent panels. (B,B′) Background auto-fluorescence in negative control explants incubated with rabbit IgG. (D,D′) WT1 expression is markedly reduced in the cap region and moderately reduced in epithelialized nephrogenic structures of explants treated with 10 μM WT1 MO. The number of WT1-expressing nephrogenic tubules is also reduced in these explants. (E,E′) Explants treated with 20 μM cntl MO exhibit moderate reductions in ureteric bud branching compared with explants cultured in media alone but continue to express WT1 in nephron progenitor cells and form WT1-expressing epithelialized nephrogenic structures. (F,F′) By contrast, WT1 expression is not detected in explants treated with 20 μM WT1 MO, and explants do not undergo growth or ureteric bud branching.

Fig. 4.

Expression of kidney development WT1 target genes is reduced in WT1 morphant kidney explants. (A,A′) Control morphant explants exhibit a characteristic expression pattern of Six2, a marker of nephron progenitors. (A′) Higher magnification of region in A denoted by arrowhead. Dashed lines demarcate the ureteric bud.(B,B′) The discrete pattern of cap-specific Six2 expression is lost in WT1 morphants, which instead exhibit an expanded Six2 expression domain. (C-J′) Control morphants express Bmp7 (C,C′), Pax2 (E,E′), Sall1 (G,G′) and HeyL (I,I′) in nephron progenitors and other lineages. In all cases, WT1 vivo-morpholino treatment results in a specific and marked reduction of gene expression in nephron progenitor cells (D,D′,F,F′,H,H′,J,J′). Arrowheads denote regions shown in higher magnification in adjacent panels.

Fig. 5.

Increased apoptosis in WT1 morphant explants, shown by TUNEL labeling. (A,B) Control morphant kidney explants treated with low (10 μM) or higher doses (20 μM) of control vivo-morpholino exhibit TUNEL-positive cells (green) mainly in the mesenchyme peripheral to the Pax2 (red) expression domain of nephron progenitors and ureteric bud. (C,D) Low (C) and high (D) doses of WT1 vivo-morpholino result in an expansion of this outer apoptotic zone, which extends further in towards the centre of the explant. The number of apoptotic cells in the nephron progenitor cap region and in ureteric bud is increased in WT1 morphants (C,D versus A,B). At high doses of WT1 vivo-morpholino, a marked increase in the number of apoptotic cells is observed in the cap region adjacent to the ureteric bud (D). Note that Pax2 expression is reduced in progenitor cells of WT1 morphant explants compared with controls (C,D versus A,B). (E) Negative control explant processed without Tdt enzyme showing absence of TUNEL-positivity.

Fig. 6.

Expression of novel kidney WT1 target genes is reduced in WT1 morphant kidney explants. Expression of Cxxc5 (A,A′), Lsp1 (C,C′), Pbx2 (E,E′), Rps6ka3 (I,I′), Scx (K,K′) and Sox11 (M,M′) in control morphant explants, demonstrating expression in nephron progenitor cells and other lineages. Plxdc2 (G,G′) is expressed predominantly in the outer region of the metanephric mesenchyme, a weak WT1-expression domain. In all cases, WT1 vivo-morpholino treatment results in a specific and marked reduction of target gene expression in nephron progenitor cells (B,B′,D,D′,F,F′,J,J′,L,L′,N,N′) or in mesenchyme peripheral to the nephron progenitors (H,H′). Dashed outlines demarcate the ureteric bud. Arrowheads denote regions shown in higher magnification in adjacent panels.