Shiraki T et al. (2022), Appropriate Amounts and Activity of the Wilms' ...

XB-ART-59483

J Dev Biol 2022 Oct 29;104:. doi: 10.3390/jdb10040046.

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Appropriate Amounts and Activity of the Wilms' Tumor Suppressor Gene, wt1, Are Required for Normal Pronephros Development of Xenopus Embryos.

Shiraki T , Hayashi T , Ozue J , Watanabe M .

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The Wilms' tumor suppressor gene, wt1, encodes a zinc finger-containing transcription factor that binds to a GC-rich motif and regulates the transcription of target genes. wt1 was first identified as a tumor suppressor gene in Wilms' tumor, a pediatric kidney tumor, and has been implicated in normal kidney development. The WT1 protein has transcriptional activation and repression domains and acts as a transcriptional activator or repressor, depending on the target gene and context. In Xenopus, an ortholog of wt1 has been isolated and shown to be expressed in the developing embryonic pronephros. To investigate the role of wt1 in pronephros development in Xenopus embryos, we mutated wt1 by CRISPR/Cas9 and found that the expression of pronephros marker genes was reduced. In reporter assays in which known WT1 binding sequences were placed upstream of the luciferase gene, WT1 activated transcription of the luciferase gene. The injection of wild-type or artificially altered transcriptional activity of wt1 mRNA disrupted the expression of pronephros marker genes in the embryos. These results suggest that the appropriate amounts and activity of WT1 protein are required for normal pronephros development in Xenopus embryos.

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Species referenced: Xenopus laevis
Genes referenced: lhx1 pam pax2 wt1
GO keywords: kidney development
gRNAs referenced: tyr gRNA19 wt1 gRNA2

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	Graphical Abstract
	Figure 1. Schematic diagram of the WT1 protein and the results of CRISPR/Cas9 knockout of wt1. (A) Schematic of the WT1 protein. 17aa and KTS represent splicing variants with or without these insertions (note that 17aa insertion variants are present only in mammals). The target sequence of sgRNA is in the N-terminus. Abbreviations are as follows; RD; transcription repression domain, AD transcription activation domain, ZF; zinc finger DNA binding domain. (B) Details of mutations in wt1 caused by the injection of wt1-sgRNA/Cas9. Mutations in the wt1.L gene are shown on the left and in the wt1.S gene on the right. The sequence of wild-type wt1 is shown above the red line. Blue squares indicate sgRNA target sequences, and green squares indicate PAM sequences. Vertical arrows indicate the positions where the Cas9 protein is thought to cleave DNA. Deleted nucleotides are indicated by dots, replaced nucleotides by red squares, and inserted nucleotides by brown square. Each sequence represents an independent DNA clone. (C) Summary of mutations results from CRISPR/Cas9 method. A total of 22 DNA clones were sequenced, 10 from the wt1.L gene and 12 from the wt1.S gene. The graph summarizes the mutations of both homeologs.
	Figure 2. Expression of the xlim-1 and pax2 genes in embryos injected with wt1-sgRNA/Cas9. (A) Expression of the xlim-1 and pax2 genes in embryos injected with wt1-sgRNA/Cas9 are shown with representative embryos and probe combinations of results. Arrows indicate expression in the pronephric tubules, and arrowheads indicate expression in the pronephric ducts. (B) Expression of the xlim-1 and pax2 genes in embryos injected with tyrosinase-sgRNA/Cas9 are shown with representative embryos and probe combinations of results. Arrows and arrowheads indicate the same as in (A). (C) Edema induced in embryos by injection of wt1-sgRNA/Cas9. These embryos also show the expression of the pax2 gene. The top embryo has edema in the head, the middle embryo in the back, and the bottom embryo in the abdomen (the abdominal edema is collapsed). Arrows indicate the regions edema occurred. Note that the morphology of the embryo is altered due to the development of edema.
	Figure 3. Activation of luciferase reporter DNA by the WT1. (A) Luciferase activity was examined in embryos by injecting reporter DNA (100 pg/embryos) containing canonical WT1-binding sequence (5′ GCGGGGGCG 3′) with or without wt1 mRNA (500 pg/embryos) into the 2-cell stage embryos. The Luciferase activity was examined in the gastrula stage embryos (stage 11). The Luciferase activity of embryos without wt1 mRNA injection was set at 100, and the Luciferase activity of embryos injected with wt1 mRNA was expressed as a relative value. Error bars represent the standard deviations. Experiments were repeated at least three times, and the representative result is shown. (B) Another WT1 binding sequence (5′ GCGTGGGAGT 3′) was injected as in (A), and the representative result is shown.
	Figure 4. The xlim-1 and pax2 gene expression in embryos injected with wild-type, activator, or repressor form of wt1 mRNA. (A) Schematic representation of the right-side view of the 16-cell stage embryo. An injected cell at the vegetal ventral side of the embryo, known to give rise to pronephros at a later stage, was orange-colored. (B) To confirm that the injected cell differentiates into pronephros, mRNA encoding nuclear beta-galactosidase was injected into the cell shown in (A), and the progenies of the injected cell were visualized at stage 34/35. The arrows indicate where the pronephros is formed. (C) Expression of the xlim-1 gene in wt1-mRNA injected embryos. wt1 mRNA of wild-type (500 pg/embryo), activator (100 pg/embryo), or repressor form (200 pg/embryo) was injected into a single cell at the 16-cell stage embryos as indicated in (A). Arrows indicate expression in the pronephric tubules, and arrowheads indicate expression in the pronephric ducts. (D) Expression of the pax2 gene was examined as in (C).

References [+] :

Armstrong, The expression of the Wilms' tumour gene, WT1, in the developing mammalian embryo. 1993, Pubmed