Retinoic acid signaling controls urothelial development and regeneration

描述（由申请人提供）：尿路上皮是一种复层上皮，由表达Krt 5的基底细胞、中间细胞和伞细胞的管腔层组成， 是泌尿道和血液之间的重要屏障。尿道上皮含有干细胞，可以在急性损伤后再生伞细胞层，但慢性损伤会损害尿道上皮屏障，导致膀胱功能障碍和持续疼痛。鉴定尿路上皮干细胞和控制它们的信号通路对于发展膀胱扩大和修复的策略将是重要的。命运定位研究揭示了在成人尿路上皮中存在表达干细胞群的Sonic hedgehog（Shh），该干细胞群被认为是基底细胞。我们的研究表明Shh+细胞也是胚胎尿路上皮中的干细胞，然而在命运作图实验中使用Krt 5CreERT 2; Rosa 26小鼠对基底细胞的干细胞潜能的直接分析揭示了它们在成人和胚胎尿路上皮中是单能的，这表明不同的尿路上皮细胞类型是干细胞。与此一致，我们发现Shh+细胞群中存在另外两种细胞类型，中间细胞和U-0细胞，这是一种以前没有描述过的细胞群。我们将使用CPP和尿路致病性大肠杆菌在胚胎和成人再生尿路上皮中测试目的1和2中的干细胞潜能中间体和U-0细胞。大肠杆菌作为损伤模型，与Indira Mysorekar实验室合作。 维甲酸（RA）是一种有效的信号分子，可以诱导胚胎干细胞在培养中分化为尿路上皮细胞，这表明类维生素A可以正常调节尿路上皮祖细胞。为了解决这个问题，我们使用ShhCre系来表达显性抑制性RA受体（RaraDN），其已插入到Shh+群体中的Rosa 26基因座中。我们发现在ShhCre/+;RaraDN突变体中既不形成中间细胞也不形成伞细胞，这表明类维生素A通常在尿路上皮特化的祖细胞群体中是重要的。我们将使用细胞类型特异性Cre系直接解决这个问题， 中间和U-0细胞中的RaraDN，以确定哪些尿路上皮群体是发育期间和成人尿路上皮中RA信号传导的介质。这些研究将确定一种新的尿路上皮干细胞群，并将确定RA信号在尿路上皮形成和再生中的作用。RA用于诱导尿路上皮细胞亚型的潜力提高了一种容易获得且廉价的化合物可能具有新的治疗应用的可能性。

DESCRIPTION (provided by applicant): The urothelium is a stratified epithelium composed of Krt5-expressing basal cells, intermediate cells, and a luminal layer of umbrella cells that provide a crucial barrier between the urinary tract and blood. The urothelium contains stem cells that can regenerate the umbrella cell layer after acute damage, but chronic damage can compromise the urothelial barrier, leading to bladder dysfunction and persistent pain. Identification of urothelial stem cells and the signaling pathways that control them will be important for developing strategies for bladder augmentation and repair. Fate mapping studies reveal the existence of a Sonic hedgehog (Shh) expressing stem cell population in the adult urothelium that is proposed to be a basal cell. Our studies indicate that an Shh+ cell is also a stem cell in the embryonic urothelium, however direct analysis of the stem cell potential of basal cells using Krt5CreERT2;Rosa26 mice in fate mapping experiments reveals that they are unipotent in the adult and embryonic urothelium, suggesting that a different urothelial cell type i the stem cell. Consistent with this, we find that two other cell types are present in the Shh+ population, intermediate cells, and U-0 cells, a population that has not been previously described. We will test the stem cell potential intermediate and U-0 cells in Aims 1 and 2 in in the embryonic and adult regenerating urothelium, using CPP and uropathogenic E. coli as injury models, in collaboration with Indira Mysorekar's lab. Retinoic acid (RA) is a potent signaling molecule that can induce embryonic stem cells to differentiate into urothelial cells in culture, suggesting that retinoids may normally regulate urothelial progenitors. To address this, we used the ShhCre line to express a dominant inhibitory RA-receptor (RaraDN) which has been inserted into the Rosa26 locus, in the Shh+ population. We find that neither intermediate cells nor umbrella cells form in ShhCre/+;RaraDN mutants suggesting that retinoids are normally important in progenitor population for urothelial specification. We will directly address this question using cell type specific Cre lines to express RaraDN in intermediate and U-0 cells to determine which urothelial populations are mediators of RA-signaling during development and in the adult urothelium. These studies will identify a novel urothelial stem cell population, and will define the role of RA-signaling in formation and regeneration of the urothelium. The potential for RA to by used for induction of epithelial subtypes in the urothelium raises the possibility that a readily available and inexpensive compound could have novel therapeutic applications.