Quiescent Dclk1+ stem cells in the mouse intestine

描述（由申请人提供）：真正的静止和长寿的干细胞已经被假定，但迄今为止尚未确定在肠道。双皮质素样激酶1（Dclk1）被认为是肠中的干细胞标志物，但也发现在肠簇细胞中表达。 最近，我们的小组已经产生了Dclk1-Cre-ERT BAC转基因小鼠，我们已经明确表明，Dclk1+簇细胞是长寿命的，静止的干细胞，起源于神经嵴，Dclk1也标记肠神经系统的神经节外祖细胞。我们的假设是，Dclk 1+干细胞发挥作用，既作为神经干细胞，也作为储备肠干细胞，它们共同形成一个生态位的其他干细胞群体在肠道中，主要是通过生成的ENS。我们将探讨这一假设通过四个具体的目标。(1)Dclk1+干细胞和Bmi1+肠道干细胞之间的关系是什么？我们将利用Bmi 1/GFP/+敲入小鼠和Lgr 5/DTR敲入小鼠与Dclk 1转基因系的组合来确定Dclk 1和Bmi 1之间是否存在重叠。(2)去除Dclk1+祖细胞是否会抑制正常肠上皮细胞的稳态和对辐射损伤的反应？与DTR F/F小鼠杂交的Dclk1-Cre-ERT小鼠将用白喉毒素处理，并评估对辐射的再生反应。（三）、放射损伤后Dclk1+祖细胞的激活是否导致肠增殖和再生增加？Dclk1祖细胞将通过条件性缺失PTEN或激活 的IC-Notch1，然后分析对辐射损伤的反应。（四）、Dclk1消融是否 Dclk 1+祖细胞抑制体外ISC生长，Dclk 1+祖细胞的活化促进体外ISC生长。我们将使用来自上述Dclk 1杂交的全肠道培养物来探索Dclk 1消融或激活对体外生长的影响。总的来说，这些研究应该为Dclk1祖细胞作为肠道中的小生境和干细胞的作用及其对肠道再生的贡献提供新的见解。

DESCRIPTION (provided by applicant): Truly quiescent and long-lived stem cells have been postulated but to date not identified in the gut. Doublecortin-like kinase 1 (Dclk1) was proposed as a stem cell marker in the intestine but was also found to be expressed in intestinal tuft cells. Recently, our group has generated Dclk1- Cre-ERT BAC transgenic mice, and we have shown unequivocally that Dclk1+ tuft cells are long-lived, quiescent stem cells that originate from the neural crest, and that Dclk1 also labels extraganglionic progenitors of the enteric nervous system. Our hypothesis is that Dclk1+ stem cells play a role both as neural stem cells and also as reserve intestinal stem cells, which together form a niche for other stem cell populations in the intestine, primarily through generation of the ENS. We will explore this hypothesis through four specific aims. (1) What is the relationship between Dclk1+ stem cells and Bmi1+ intestinal stem cells? We will utilize Bmi1/GFP/+ knockin mice and Lgr5/DTR knockin mice in combination with Dclk1 transgenic lines to determine whether there is overlap between Dclk1 and Bmi1. (2) Does ablation of Dclk1+ progenitors inhibit normal intestinal epithelial homeostasis and the response to radiation injury? Dclk1-Cre-ERT mice crossed to DTR F/F mice will be treated with diptheria toxin and the regenerative response to radiation assessed. (3). Does activation of Dclk1+ progenitors result in intestinal proliferation and increased regeneration following radiation injury? Dclk1 progenitors will be activated by conditional deletion of PTEN or activation of IC-Notch1, followed by analysis of the response to radiation injury. (4). Does ablation of Dclk1 progenitors inhibit in vitro ISC growth, and does activation of Dclk1+ progenitors accelerate in vitro ISC growth. We will use whole intestinal cultures derived from the Dclk1 crosses above to explore the effect of Dclk1 ablation or activation on in vitro growth. Overall, these studies should provide new insights into the role of Dclk1 progenitors as both niche and stem cells in the intestine, and their contribution to intestinal regeneration.