Creating high-resolution multi-omics molecular atlases for developing urogenital organs

创建用于发育泌尿生殖器官的高分辨率多组学分子图谱

• 批准号: 10673765
• 负责人: FENG CHEN
• 金额: $ 46.01万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10673765
• 关键词: 3-Dimensional; Ablation; Age; Anatomy; Atlases; Cell Nucleus; Cells; Cellular Structures; Childhood; Chromosome Mapping; Communities; Computer Analysis; Data; Databases; Development; Dimensions; Disease; Disease model; Epigenetic Process; Fertilization; Gene Expression; Gene Silencing; Genes; Genitourinary system; Human; Imaging technology; Infrastructure; Institution; Kidney; Light; Lower urinary tract; Lymphatic; Maps; Mesenchyme; Microscopy; Modeling; Molecular; Morbidity - disease rate; Mus; Nerve; Organ; Organ Model; Penetration; Phenotype; Population; Procedures; Process; Prostate; Recurrence; Reproducibility; Research; Research Personnel; Resolution; Signal Transduction; Structure; System; Technology; Time; Ureter; Ureteropelvic junction obstruction; Urinary tract; Work; analysis pipeline; biological systems; body system; congenital anomalies of the kidney; epigenetic profiling; experience; experimental study; male; microscopic imaging; mortality; mouse model; multiple omics; multiplexed imaging; new technology; prenatal; progenitor; reproductive organ; single nucleus RNA-sequencing; transcriptome; transcriptomic profiling; transcriptomics; tumor

The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) has been providing valuable references for the research community studying urogenital development and diseases. It is a recurring theme that rapidly advancing new technologies are instrumental in enhancing and expanding reference databases. Cross fertilization of atlas building efforts spanning various organ systems and disease types will undoubtedly boost the technology penetration across these projects. To build multi-dimensional atlases of developing urogenital organs that incorporate the latest multi-omics and spatial molecular mapping technologies, we have assembled a team with expertise both in urogenital development and multi-dimensional, multi-platform, molecular atlas building. We propose to utilize the infrastructure we developed at our institution for the NCI Human Tumor Atlas Network (HTAN) and other large scale projects as a springboard to help effectively and efficiently propel GUDMAP to the next level with transcriptome-wide coverage, single cell level resolution, and spatial mapping with unprecedented clarity. We will take advantage of our experience in the incorporation of single nucleus (sn) RNA-seq and snATAC-seq to establish a comprehensive epigenetic and transcriptomic landscape in targeted urogenital organs and structures (lower urinary tract (LUT), selected male reproductive organs, kidney vasculature, lymphatics, and nerves) at single cell resolution (Aim 1). We will then add the spatial dimension to this molecular landscape to build 2D and 3D molecular atlases by incorporating spatial transcriptomics (ST), CODEX, and light sheet microscopy (LSM) (Aim 2). In Aim 3, we will extend our study to disease models, focusing on murine models of congenital anomalies of the kidney and the urinary tract (CAKUT). With the proposed experiments, we aim at building multidimensional molecular atlases for developing urogenital organs at unprecedented cellular resolution and gene coverage with the highest efficiency possible. Aim 1: Characterize the epigenetic and transcriptomic landscapes of developing urogenital organs with single cell omics We will perform integrated transcriptomic and epigenetic profiling of the developing/maturing LUT, male reproductive organs, and the kidney at E16.5, NB, and 3 weeks of age. Although the focus for the kidney will be on vasculature, lymphatics, and nerves, since there is a lack of single cell omics data on most of the selected stages, our data will also help to strengthen GUDMAP data for the broadest use by the research community. Aim 2: Construct multi-dimensional molecular atlases for developing urogenital organs using spatial transcriptomics and advanced imaging technologies A major challenge for atlas building in biological systems has been spatially assigning large number of molecular features to the anatomical and cellular structures. We have successfully established experimental procedures and computational analyses pipelines for spatial transcriptomics, CODEX, and light sheet microscopic imaging, to map gene expression data, including transcriptome-wide data to cells and structures. We will use these technologies to analyze the developing/maturing lower urinary tract, male reproductive organs, and the kidney at E16.5, NB, and 3 weeks of age for the construction of truly multi-dimensional, multiplatform molecular atlases. Aim 3: Building molecular atlases for key urogenital structures using murine CAKUT models with cell ablation or gene inactivation CAKUT occurs in many different forms representing a significant cause of morbidity and mortality in the pediatric population. We have generated and analyzed several CAKUT murine models in the past. Building atlases of the target organs for these models will provide high resolution, spatially registered molecular references for key stages of disease initiation and progression. Moreover, such atlases will help researchers better understand normal urogenital development by knowing the tolerance of the systems and processes in dealing with various disturbances. We will use a highly reproducible murine model of CAKUT with inactivation of canonical Smad signaling in ureteral mesenchyme, causing a uniform ureteropelvic junction (UPJ) obstruction phenotype prenatally. We will use the technologies outlined in Aims 1 and 2 to build molecular atlases of the relevant structures (ureter, kidney, UPJ) at key time points and compare the atlases of defective development with those of normal development.

泌尿生殖系统发育分子解剖学项目(GUDMAP)一直在为研究泌尿生殖系统发育和疾病的研究界提供有价值的参考。快速发展的新技术有助于加强和扩大参考数据库，这是一个反复出现的主题。跨不同器官系统和疾病类型的地图集建设工作的交叉受精无疑将促进这些项目的技术渗透。为了构建融合了最新的多组学和空间分子图谱技术的泌尿生殖器官发育的多维图谱，我们组建了一支在泌尿生殖器官发育和多维、多平台、分子图谱构建方面具有专业知识的团队。我们建议利用我们为NCI人类肿瘤图谱网络(Htan)和其他大型项目开发的基础设施作为跳板，帮助有效和高效地推动GUDMAP以前所未有的清晰度实现转录组范围的覆盖、单细胞水平的分辨率和空间映射的下一个水平。我们将利用我们在结合单核(Sn)RNA-seq和SnATAC-seq方面的经验，在单细胞分辨率(目标1)下在目标泌尿生殖器官和结构(下尿路、选定的男性生殖器官、肾脏血管、淋巴管和神经)中建立全面的表观遗传和转录图景。然后，我们将添加空间维度到这个分子景观，通过整合空间转录(ST)、法典和光片显微镜(LSM)来建立2D和3D分子图谱(目标2)。在目标3中，我们将把我们的研究扩展到疾病模型，重点是先天性肾脏和尿路异常(CAKUT)的小鼠模型。通过拟议的实验，我们的目标是建立多维分子图谱，以尽可能高的效率以前所未有的细胞分辨率和基因覆盖率发育泌尿生殖器官。 目的1：用单细胞组学方法研究发育中的泌尿生殖器官的表观遗传学和转录图谱 我们将在E16.5、Nb和3周龄对发育/成熟的LUT、雄性生殖器官和肾脏进行综合转录和表观遗传学分析。虽然肾脏的重点将放在血管系统、淋巴管和神经上，但由于缺乏大多数选定阶段的单细胞组学数据，我们的数据也将有助于加强GUDMAP数据，使其最广泛地被研究界使用。 目的2：利用空间转录和先进的成像技术构建多维分子图谱以研究泌尿生殖器官的发育 在生物系统中建立图谱的一个主要挑战是在空间上将大量的分子特征分配给解剖和细胞结构。我们已经成功地建立了空间转录学、CODEX和光片显微成像的实验程序和计算分析管道，将基因表达数据，包括转录组范围的数据映射到细胞和结构。我们将利用这些技术分析发育/成熟的下尿路、雄性生殖器官以及E16.5、Nb和3周龄的肾脏，以构建真正的多维、多平台的分子图谱。 目的3：利用细胞切除或基因失活的小鼠CAKUT模型，建立关键的泌尿生殖系统结构的分子图谱 CAKUT以许多不同的形式发生，是儿科人群发病和死亡的重要原因。在过去，我们已经建立并分析了几个CAKUT小鼠模型。为这些模型建立靶器官图谱将为疾病发生和发展的关键阶段提供高分辨率、空间注册的分子参考。此外，这样的图谱将通过了解系统和过程在处理各种干扰时的耐受性，帮助研究人员更好地了解正常的泌尿生殖系统发育。我们将使用一种具有高度重复性的CAKUT小鼠模型，在输尿管间充质中典型的Smad信号失活，导致产前出现统一的输尿管肾盂连接部(UPJ)梗阻表型。我们将使用AIMS 1和2中概述的技术来构建关键时间点的相关结构(输尿管、肾脏、UPJ)的分子图谱，并将发育缺陷的图谱与正常发育的图谱进行比较。