Persistent SIX2 expression as a first hit mechanism in Wilms tumorigenesis

描述（申请人提供）：肾母细胞瘤（Wilms tumor， WT）是一种儿童肾脏恶性肿瘤，被认为是由胚胎肾干细胞持续和异常分化引起的。确切地说，肾素祖细胞如何逃避上皮细胞的承诺和转化途径，以及在肾母细胞瘤发生中潜在的“首发”机制尚不清楚。WT囊胚，被认为是这些肾元祖细胞的恶性类似物，保留了转录调节因子SIX2的表达，SIX2在小鼠发育中促进帽间质（CM）的自我更新，并防止过早的上皮分化。我们之前的工作表明，SIX2在成人肾脏中通常不存在，但在人类WT的广泛范围内持续表达。将这些SIX2在发育和疾病中的活性观察结合起来，基本问题出现了，该基因是否为CM及其后代在WT序列中自我延续提供了一种机制。建立该小鼠模型的目的是验证CM中SIX2的持续表达是否会损害上皮分化，并促进该祖细胞群的保留和肾源性休息的发展，肾源性休息是假定的WT的前体病变。我们有两个目的：1)建立并验证四环素调控的转基因小鼠系，使SIX2在胚胎肾脏中的表达得到时间控制；2)表征SIX2在胚胎和成人肾脏中持续表达的表型。我们将在四环素应答元件（trer - rfp - 2a -SIX2）的控制下，构建一种能够持续表达SIX2的创始转基因小鼠系。一旦验证，将该小鼠与ROSA26-M2-rtTA细胞系杂交，该细胞系提供rtTA的全局和组成性表达（Tet-On）。在e15.5时给予多西环素以激活SIX2转基因表达，并评估其对CM分化和持久性的影响。CM细胞在出生后肾脏内的持久性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针对CM持续表达SIX2的研究奠定基础。这些未来的研究将使用我们实验室先前设计的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.