Quiescent Dclk1+ stem cells in the mouse intestine

描述(由申请人提供)：真正静止和长期存活的干细胞已经被假设，但到目前为止还没有在肠道中鉴定出来。双重皮质醇样激酶1(Dclk1)被认为是肠道干细胞标记物，但也被发现在肠束细胞中表达。 最近，本课题组成功培育了Dclk1-Cre-ERT BAC转基因小鼠，并明确证明Dclk1+TUFT细胞是一种长期存活的静止干细胞，来源于Lgr5+干细胞，但受神经支配诱导。我们的假设是，Dclk1+簇状细胞既是肠道干细胞巢的一部分，也是肠道干细胞的储备细胞，由外源性神经支配产生。我们将通过三个具体目标来探讨这一假设。(1)神经在Dclk1+簇状细胞的诱导中起什么作用。我们将利用体外培养系统和在体小鼠模型来研究神经支配的作用，重点是NGF-Trk通路在介导神经-绒毛细胞相互作用中的作用。(2)Dclk1+前体细胞的消融是否会抑制正常的肠上皮细胞的动态平衡和生长，以及对辐射损伤的反应？与DTR F/F小鼠杂交的Dclk1-Cre-ERT小鼠将接受白喉毒素治疗，并使用体外和体内系统评估对辐射的再生反应。(3)。辐射损伤后Dclk1+祖细胞的激活是否会导致肠道增殖和再生增加？初步研究表明，Dclk1谱系中APC的缺失和Wnt信号的激活足以将Dclk1簇状细胞转化为活性干细胞。我们将使用以下工具探索Wnt信号的作用 无论是隐蔽培养还是老鼠模型。总体而言，这些研究应该为Dclk1前体作为肠道中的利基细胞和干细胞的作用及其贡献提供新的见解。 到肠道再生。

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 are derived from Lgr5+ stem cells but induced by nervous innervation. Our hypothesis is that Dclk1+ tuft cells play a role both as part of the intestinal stem cell niche and also as reserve intestinal stem cell, and are generated through extrinsic neural innervation. We will explore this hypothesis through three specific aims. (1) What is the role of nerves in the induction of Dclk1+ tuft cells. We will utilize both in vitro culture systems, and in vivo mouse models, to examine the role of nervous innervation, with a focus on NGF-Trk pathway in mediating the nerve-tuft cell interactions. (2) Does ablation of Dclk1+ progenitors inhibit normal intestinal epithelial homeostasis and growth, 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 using both in vitro and in vivo systems. (3). Does activation of Dclk1+ progenitors result in intestinal proliferation and increased regeneration following radiation injury? Preliminary studies suggestion that deletion of Apc in the Dclk1 lineage, and activation of Wnt signaling, is sufficien to convert Dclk1 tuft cells into active stem cells. We will explore the role of Wnt signaling using both crypt cultures and mouse models. 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.