DESCRIPTION (provided by applicant): Wilms tumor (WT) is a pediatric renal malignancy thought to arise from aberrant differentiation and persistence of embryonic kidney stem cells. Precisely how nephron progenitors escape pathways of epithelial commitment and conversion, mechanisms that potentially represent 'first hits' in Wilms tumorigenesis, have not been clarified. WT blastema, the putative malignant analogue of these nephron progenitors, retains expression of the transcriptional regulator, Six2, which in mouse development promotes self-renewal of the cap mesenchyme (CM) and prevents its premature epithelial differentiation. Our previous work has shown that SIX2, normally absent in the adult kidney, is persistently expressed across a broad spectrum of human WT. Putting together these observations of SIX2 activity in development and disease, the fundamental question arises whether this gene provides a mechanism for the CM and its progeny to self-perpetuate in the WT sequence. The purpose of developing this mouse model is to test the hypothesis that persistent Six2 expression in the CM impairs epithelial differentiation and promotes retention of this progenitor population as a set-up to develop nephrogenic rests, the putative precursor lesion of WT. We have two aims: 1) to generate and validate a tissue-specific, Cre-activated mouse line that allows temporal control of Six2 expression in nephron progenitors, and 2) to characterize the phenotype of persistent Six2 expression in the embryonic and adult kidney. Briefly, mice will be engineered to express Six2 persistently in nephron progenitors (and daughter cells) under the temporal control of our established tamoxifen-inducible and CM-specific Cited1-Cre-ERT2 transgenic mouse line. To yield temporal control of Six2 expression, a mouse-specific ROSA26-loxP-STOP-loxP-Gfp-2a- Six2 (R26-LSL-Six2, for short) allele will be designed. Once validated to transmit this R26-LSL-Six2 allele in the germline, founders will be inbred to homozygosity for subsequent breeding with heterozygous Cited1-Cre- ERT2 mice. This breeding scheme will produce offspring that all carry the R26-LSL-Six2 allele and that half carry Cited1-Cre-ERT2, which affords control for tamoxifen effects on renal development in Cre-negative mice. Administered to all dams in this scheme, tamoxifen will induce CM-specific production of Cre and thereby excision of the STOP codon in half of the pups, and subsequent Six2 production will occur specifically in the nephron progenitors of the CM and daughter cells. Coinciding with peak Cited1 activity in the CM, tamoxifen will be administered to dams at e15.5, and the resulting phenotype on nephron progenitor differentiation will be evaluated at e19.5 and one month postnatal. In this model, we predict disruption of CM differentiation and retention of these progenitors in the form of nephrogenic rests. The impact of these studies therefore will be to develop a model for exploring persistent Six2 expression in the CM as a candidate mechanism in the initiation of the WT sequence and for evaluating in future work the interactions of Six2 with Wnt/β-catenin signaling as a targetable mechanism of cancer stem cell survival that will reveal new and more efficacious drugs.

PUBLIC HEALTH RELEVANCE: Two transgenic mouse models of Wilms tumor (WT) have been described recently, yet neither was designed specifically to address the 'first hit' mechanisms of nephron progenitor persistence or cancer stem cell self- renewal that fuels WT perpetuity. Using a tamoxifen-inducible Cre-activated system to overexpress Six2 continuously and specifically in nephron progenitors, this novel mouse model will explore the effects of Six2- dependent self-renewal, which normally shuts off on completion of kidney development but remains persistently active across a broad spectrum of WT, on the differentiation potential and retention of this multipotent stem cell population. Because Six2 appears to repress Wnt/β-catenin-dependent differentiation of nephron progenitors, this model will provide a new tool to identify and study targets that perpetuate rogue embryonic and cancer stem cells across the WT sequence.