DORSOVENTRAL PATTERNING OF THE MAMMALIAN CLOACA

描述（由申请方提供）：大约2500例肠道畸形中有1例发生在后肠区域。该地区最常见的出生缺陷是持续泄殖腔和肛门失禁，这两种缺陷都是由于尿生殖窦和肛门直肠窦之间的关闭不当造成的。在后肠发育过程中，尿生殖窦和肛门直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分隔，进一步的形态发生产生膀胱、生殖器官和直肠。已知成熟膀胱、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）与泌尿生殖（腹侧泄殖腔）细胞命运的早期规格发生在胚胎泄殖腔。我们提出这样的问题：上皮细胞分化是否需要适当的分隔才能发生？上皮细胞分化是否存在信号中心？为了确定分隔是否是这两种细胞谱系分化的先决条件，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隔前背腹极化，我将使用未分裂的胚胎泄殖腔和最近分裂的小鼠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。将从胚胎小鼠中解剖出Clobium，并分成背侧和腹侧切片。然后将对细胞进行测序以确定其基因表达谱。发现背腹侧表达的基因将被映射为他们的结构域和泄殖腔中的基因表达的边界使用原位杂交。为了确定泄殖腔分隔是否是背腹极性所必需的，我将研究持续泄殖腔小鼠的背腹身份的分子标记。将使用原位杂交在Bmp7突变胚胎中检查测序期间鉴定的基因。为了确定周围组织是否会影响泄殖腔上皮的分化，我将在有或没有脊索和尿囊的情况下共培养胚胎泄殖腔。将对培养物进行背侧和腹侧命运的形态学和分子标记物分析。从泄殖腔祖细胞的共同池中诱导肛门直肠和泌尿生殖器组织的初始分子遗传步骤的鉴定，将通过揭示哪些机制对患有泌尿生殖器和肛门直肠畸形的儿童的扰动开放而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门直肠组织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.