Persistent SIX2 expression as a first hit mechanism in Wilms tumorigenesis

 描述(申请人提供)：肾母细胞瘤(WT)是一种儿童肾脏恶性肿瘤，被认为是由胚胎肾干细胞的异常分化和持久性引起的。肾单位祖细胞如何逃脱上皮承诺和转化的途径，这一机制可能代表肾母细胞瘤发生中的“第一次打击”，目前尚不清楚。 WT胚母细胞是这些肾单位前体细胞的恶性类似物，它保留了转录调节因子SIX2的表达，在小鼠发育过程中，SIX2促进了帽间充质(CM)的自我更新，并防止了其过早的上皮分化。我们以前的工作表明，SIX2通常在成人肾脏中缺失，但在人类广泛的WT中持续表达。综合这些对SIX2在发育和疾病中的活性的观察，基本的问题出现了，这个基因是否为CM及其后代在WT序列中自我延续提供了一种机制。建立这个小鼠模型的目的是验证这样的假设，即在CM中持续的SIX2表达损害了上皮分化，并促进了作为设置的这一祖细胞群体的保留 为了发展肾源性REST，可能是WT的前驱病变。我们的目标有两个：1)建立和验证一个组织特异性的、Cre激活的小鼠系，它可以在时间上控制肾单位祖细胞中SIX2的表达，以及2)鉴定SIX2在胚胎和成人肾脏中持续表达的表型。简而言之，在我们建立的三苯氧胺可诱导和CM特异性Cited1-Cre-ERT2转基因小鼠系的时间控制下，小鼠将在肾单位祖细胞(和子细胞)中持续表达SIX2。为了获得SIX2表达的时间控制，将设计一个小鼠特异的rosa26-loxP-Stop-loxP-GFP-2a-SIX2(简称R26-LSL-SIX2)等位基因。一旦被证实可以在生殖系中传递这种R26-LSL-SIX2等位基因，创始人将被近亲交配为纯合，以便随后与杂合的Cited1-Cre-ERT2小鼠进行繁殖。这一育种计划将产生全部携带R26-LSL-SIX2等位基因的后代，其中一半携带Cited1-Cre-ERT2，这为他莫昔芬对Cre阴性小鼠肾脏发育的影响提供了控制。在这个方案中，他莫昔芬应用于所有的母鼠，将诱导CM特异性产生Cre，从而在一半的幼崽中切除终止密码子，随后SIX2的产生将专门发生在CM和子代细胞的肾单位祖细胞中。与CM中Cited1活性高峰期一致的是，他莫昔芬将在e15.5应用于DAMs，并将在e19.5和一个月后评估由此产生的肾单位祖细胞分化的表型。在这个模型中，我们预测了CM分化的中断和这些前体细胞以肾源性休息的形式保留。因此，这些研究的影响将是开发一个模型，以探索持续的SIX2在CM中的表达，作为启动WT序列的候选机制，并在未来的工作中评估SIX2与Wnt/β-catenin信号的相互作用，作为癌症干细胞存活的靶向机制，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.