Persistent SIX2 expression as a first hit mechanism in Wilms tumorigenesis

 描述（由申请人提供）：肾母细胞瘤（WT）是一种儿科肾脏恶性肿瘤，被认为是由胚胎肾干细胞的异常分化和持续存在引起的。肾单位祖细胞是如何逃脱上皮定型和转化的途径的，这可能是肾母细胞瘤发生中的“首发”机制，尚未阐明。 WT芽基，这些肾单位祖细胞的假定恶性类似物，保留转录调节因子Six 2的表达，Six 2在小鼠发育中促进帽间充质（CM）的自我更新，并防止其过早的上皮分化。我们以前的工作已经表明，SIX 2，通常在成人肾脏中不存在，在广谱的人类WT中持续表达。将发育和疾病中SIX 2活性的这些观察结果放在一起，产生了一个根本问题，即该基因是否为CM及其后代提供了在WT序列中自我延续的机制。开发该小鼠模型的目的是检验以下假设，即CM中持续的Six 2表达损害上皮分化并促进该祖细胞群体作为一种建立的保留 发展为肾源性残余，即WT的假定前驱病变。我们有两个目标：1）产生并验证组织特异性Cre活化的小鼠系，其允许暂时控制肾单位祖细胞中Six 2表达，和2）表征胚胎和成人肾脏中持续Six 2表达的表型。简而言之，在我们建立的他莫昔芬诱导型和CM特异性Cited 1-Cre-ERT 2转基因小鼠系的时间控制下，小鼠将被设计为在肾单位祖细胞（和子细胞）中持续表达Six 2。为了产生Six 2表达的时间控制，将设计小鼠特异性ROSA 26-loxP-STOP-loxP-Gfp-2a-Six 2（简称R26-LSL-Six 2）等位基因。一旦验证在生殖系中传递该R26-LSL-Six 2等位基因，则将建立者近交至纯合性，用于随后与杂合Citedl-Cre-ERT 2小鼠育种。该育种方案将产生全部携带R26-LSL-Six 2等位基因且一半携带Cited 1-Cre-ERT 2的后代，这提供了他莫昔芬对Cre阴性小鼠的肾脏发育的影响的控制。在该方案中，对所有母鼠给药，他莫昔芬将诱导CM特异性产生Cre，从而切除一半幼崽的终止密码子，随后的Six 2产生将特异性发生在CM和子细胞的肾单位祖细胞中。与CM中的Cited 1活性峰值一致，将在e15.5时向母鼠施用他莫昔芬，并在e19.5和出生后1个月评价肾单位祖细胞分化的所得表型。在这个模型中，我们预测中断CM分化和保留这些祖细胞的形式肾休息。因此，这些研究的影响将是开发一种模型，用于探索CM中Six 2的持续表达作为WT序列起始的候选机制，并用于在未来的工作中评估Six 2与Wnt/β-连环蛋白信号传导的相互作用作为癌症干细胞存活的靶向机制，这将揭示新的和更有效的药物。

 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.