DORSOVENTRAL PATTERNING OF THE MAMMALIAN CLOACA

描述（由申请人提供）：大约每2500个肠道畸形中就有一个发生在后肠区域。该区域最常见的出生缺陷是持续性泄殖腔和肛门闭锁，这两种情况都是由于泌尿生殖窦和肛门直肠窦之间关闭不当造成的。在后肠发育过程中，泌尿生殖和肛肠窦最初是一个器官，即泄殖腔。随后的发育导致泄殖腔被尿直肠隔隔开，进一步的形态发生产生膀胱、生殖器官和直肠。众所周知，成熟膀胱、生殖器官和直肠的上皮在形态和功能上是不同的。然而，目前尚不清楚肛门直肠（背侧泄殖腔）和泌尿生殖（腹侧泄殖腔）细胞的早期分化是如何在胚胎泄殖腔中发生的。我们提出的问题是上皮分化是否需要适当的分隔发生，以及上皮分化是否存在信号中心。为了确定分裂是否是这两种细胞系分化的先决条件，我将首先研究泄殖腔分化的时间和启动这些早期细胞命运的分子机制。为了确定胚胎泄殖腔是否在分离前是背侧极化的，我将在小鼠中使用未分裂的胚胎泄殖腔和最近分裂的泄殖腔的腹侧和背侧上皮细胞的深度测序来比较基因表达谱。将从胚胎小鼠中分离出泄殖腔，分为背侧和腹侧两部分。然后对这些细胞进行测序，以确定它们的基因表达谱。发现背腹表达的基因将通过原位杂交绘制其在泄殖腔中的结构域和基因表达边界。为了确定是否需要进行泄殖腔分隔来实现背腹侧极性，我将检查具有持续性泄殖腔的小鼠的背侧和腹侧身份的分子标记。在测序过程中鉴定的基因将在Bmp7突变胚胎中使用原位杂交进行检测。为了确定周围组织是否会影响泄殖腔上皮分化，我将胚胎泄殖腔与脊索和尿囊共培养。将检测培养物的背侧和腹侧形态和分子标记。从共同的肛肠祖细胞池中诱导肛肠和泌尿生殖组织的初始分子遗传学步骤的识别，将通过揭示泌尿生殖和肛肠畸形儿童中哪些机制是开放的扰动，具有重要的临床意义。此外，了解指导祖细胞分化为尿路上皮或肛肠问题的遗传信号将为组织工程提供必要的基础。

DESCRIPTION (provided by applicant): Approximately one out of 2500 gut malformations occur in the hindgut region. The most common birth defects of this region are persistent cloaca and imperforate anus, both of which result from improper closure between the urogenital sinus and anorectal sinus. During hindgut development the urogenital and anorectal sinuses are initially one organ, the cloaca. Subsequent development leads to septation of the cloaca by the urorectal septum and further morphogenesis produces the bladder, genital organs and rectum. It is known that the epithelium lining the mature bladder, genital organs and rectum are morphologically and functionally different. However, it is not known how the early specification of anorectal (dorsal cloaca) versus urogenital (ventral cloaca) cell fate takes place in the embryonic cloaca. We ask the question does epithelial differentiation require proper septation to occur and are there signaling centers for epithelial differentiation. In order to determine if septation is a prerequisite for differentiation of these two cell lineages, I will first investigate the timing of cloaca differentiation and the molecular mechanisms that initiate these early cell fates. To determine whether the embryonic cloaca is dorsoventrally polarized before septation, I will compare gene expression profiles using deep sequencing of the ventral and dorsal epithelial cells of the undivided embryonic cloaca and the recently divided cloaca in mice. Cloacae will be dissected from embryonic mice and divided into dorsal and ventral sections. The cells will then be sequenced to determine their genetic expression profile. Genes found to be dorsoventrally expressed will be mapped for their domains and boundaries of gene expression in the cloaca using in situ hybridization. To then determine whether cloacal septation is required for dorsoventral polarity, I will examine molecular markers of dorsal and ventral identity in mice with persistent cloaca. The genes identified during sequencing will be examined in Bmp7 mutant embryos using in situ hybridization. To determine if surrounding tissues influence cloacal epithelial differentiation, I will co-culture the embryonic cloaca with and without the notochord and allantois. Cultures will be assayed for morphological and molecular markers of dorsal and ventral fate. Identification of the initial molecular genetic steps in the induction of anorectal and urogenital tissues from a common pool of cloacal progenitor cells, will have significant clinical implications by uncovering which mechanisms are open to perturbation in children with urogenital and anorectal malformations. Additionally, knowing the genetic signals that direct progenitor cells to differentiate into either urothelial or anorectal issues will provide an essential foundation for tissue engineering.