3D imaging and deep sequencing of gene expression in the genital tubercle

描述（由申请人提供）：泌尿生殖系统最常见的异常是尿道下裂，这是一种外生殖器畸形，其特征是尿道下裂无法愈合。 尿道管闭合和包皮（包皮）和腹侧阴茎的不完全形成。尿道下裂的发病率在过去30年中增加了一倍多，没有解释，它现在是人类第二常见的出生缺陷，估计每250个活产中就有1个受到影响。无论尿道下裂的原因是遗传的、环境的还是两者的结合，尿道下裂的预防策略和泌尿系统组织修复的细胞治疗的发展都需要对指导尿道下裂的遗传程序的了解。 细胞命运决定和组织形态发生。这些机制的识别首先需要建立一个基础的描述性数据，在基因表达模式的形式，为下泌尿生殖道的不同组织隔室。此外，转录谱将需要与发育解剖学联系起来，以制定关于形态发生过程中基因功能的假设，并考虑多个基因如何在组织或细胞群体中相互作用。在使用微阵列的初步研究中，我们鉴定了86个El 2.5生殖结节尿道上皮的新标记物。在这里，我们建议使用OPT建立生殖器结节的3D参考模型，并将这些基因中的每一个的表达域映射到参考结节上，以创建发育中的外生殖器中的基因表达的3D多基因图谱。我们将以两种方式巩固这些成果。首先，我们将OPT图谱扩展到外生殖器发育的另外两个阶段。其次，我们将通过使用NexGen测序技术（RNA-seq）深入研究表达谱，以表征基因表达的定量模式和注定形成尿道上皮的内胚层细胞中剪接变体的分布。这种方法的组合将提供丰富的新的细胞类型特异性标记物，揭示尿道祖细胞的定量分子特征，并将这些数据置于3D发育解剖学背景中，研究界可以利用这些数据进行未来的基于假设的研究。

DESCRIPTION (provided by applicant): The most common anomaly of the genitourinary system is hypospadias, a malformation of the external genitalia that is characterized by failure of urethral tube closure and incomplete formation of the prepuce (foreskin) and ventral penis. The incidence of hypospadias has more than doubled over the past 30 years, without explanation, and it is now the second most common birth defect in humans, affecting an estimated 1 of every 250 live births. Whether the causes of hypospadias are genetic, environmental, or a combination of both, development of preventative strategies for hypospadias and of cell-based therapies for repair of urologic tissues will require knowledge of the genetic programs that direct cell fate decisions and tissue morphogenesis. Identification of these mechanisms first requires the establishment of a foundation of descriptive data, in the form of gene expression patterns, for the different tissue compartments of the lower genitourinary tract. Moreover, transcriptional profiling will need to be linked to developmental anatomy in order to formulate hypotheses regarding gene function during morphogenesis, and to consider how multiple genes may interact within a tissue or cell population. In preliminary studies using microarrays, we identifie 86 novel markers of the urethral epithelium of the El 2.5 genital tubercle. Here we propose to use OPT to build a 3D reference model of the genital tubercle, and to map the expression domains of each of these genes onto the reference tubercle to create a 3D, multi-gene atlas of the gene expression in the developing external genitalia. We will build upon these results in two ways. Firstly, we will extend the OPT atlas to two additional stages of external genital development. Secondly, we will drill deeper into the expression profiling by using NexGen sequencing technology (RNA-seq) to characterize the quantitative patterns of gene expression and the distribution of splice variants in the endodermal cells fated to form the urethral epithelium. This combination of approaches will provide a wealth of new cell type-specific markers, uncover the quantitative molecular profiles of urethral progenitors, and place these data in a 3D developmental anatomical context that can be utilized by the research community for future hypothesis-based studies.