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，被认为是由于胚胎肾干细胞的持续和异常分化引起的。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。建立这一小鼠模型的目的是为了验证SIX2在CM中持续表达会损害上皮细胞分化，并促进这一祖细胞群体的保留和肾源性REST的发展的假说，肾源性REST是WT的前驱病变。我们的目标有两个：1)建立和验证四环素调节的转基因小鼠系，使其能够在胚胎肾脏中暂时控制SIX2的表达；2)鉴定SIX2在胚胎和成人肾脏中持续表达的表型。在四环素反应元件(tre-RFP-2A-SIX2)的控制下，方正转基因小鼠株系将持续表达SIX2。一旦被验证，该小鼠将与提供RTTA(Tet-on)的全局和结构性表达的rosa26-M2-RTTA系杂交。强的环素将在e15.5被用来激活SIX2转基因表达，并将评估其对CM分化和持久性的影响。出生后肾脏内CM细胞的持续存在类似于肾源性休息是预期的表型。这些初步研究将为未来更明确的研究奠定基础，只针对CM的持续SIX2表达。这些未来的研究将使用我们实验室以前设计的CM特异性CITED1-CRE-ERT2小鼠。

DESCRIPTION (provided by applicant): Wilms tumor (WT) is a pediatric renal malignancy thought to arise from persistence and aberrant differentiation 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 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 expression of SIX2 in the CM impairs epithelial differentiation, and promotes retention of this progenitor population and development of nephrogenic rests, the putative precursor lesion of WT. We have two aims: 1) To generate and validate a tetracycline-regulated transgenic mouse line that allows temporal control of SIX2 expression in the embryonic kidney, and 2) To characterize the phenotype of persistent SIX2 expression in the embryonic and adult kidney. A founder transgenic mouse line will be engineered to express SIX2 persistently under the control of a tetracycline response element (TRE-RFP-2A-SIX2). Once validated, this mouse will be crossed with the ROSA26-M2-rtTA line, which provides global and constitutive expression of rtTA (Tet-On). Doxycycline will be administered to dams at e15.5 to activate SIX2 transgene expression, and its effects on CM differentiation and persistence will be evaluated. Persistence of CM cells within the post-natal kidney resembling nephrogenic rests is the anticipated phenotype. These pilot studies will lay the foundation for future, more definitive investigation targeting persisten SIX2 expression to the CM only. These future studies will use the CM-specific CITED1-Cre-ERT2 mouse engineered previously in our laboratory.