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; 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.

泌尿生殖器发育和疾病的研究社区为研究社区提供了有价值的参考，该泌尿生殖器性发育和疾病已为泌尿生殖器解剖项目（GUDMAP）提供了宝贵的参考。这是一个反复出现的主题，快速发展的新技术在增强和扩展参考数据库方面起着重要作用。跨越各种器官系统和疾病类型的地图集的交叉施肥无疑会促进这些项目中的技术渗透。为了构建开发泌尿生殖器官的多维地图，这些图形结合了最新的多词和空间分子映射技术，我们组装了一个在泌尿生殖器开发和多维，多平台的，多层次的分子，分子atlas建筑方面具有专业知识的团队。我们建议利用我们在机构为NCI人类肿瘤网络（HTAN）和其他大规模项目开发的基础设施作为跳板，以有效有效地将GUDMAP推向下一个水平，并通过转录组范围覆盖，单细胞水平分辨率，单个细胞水平分辨率，并与无预防的Clarity进行空间映射。我们将利用我们在纳入单核（SN）RNA-SEQ和SNATAC-SEQ合并方面的经验来建立靶向泌尿生殖器官和结构中的全面表观遗传和转录景观（下尿道（LUT）（LUT），选定的男性生殖器官，肾脏血管，淋巴结式和神经）在单个细胞和单个细胞中。然后，我们将通过合并空间转录组学（ST），法典和轻度显微镜（LSM）（AIM 2），将空间维度添加到该分子景观中，以构建2D和3D分子地图集。在AIM 3中，我们将研究将研究扩展到疾病模型，重点关注肾脏先天异常和尿路（Cakut）的鼠模型。通过提出的实验，我们旨在建立多维分子图谱，以在前所未有的细胞分辨率和基因覆盖范围内开发泌尿生殖器官，并具有最高的效率。 AIM 1：表征具有单细胞Omics的泌尿生殖器官的表观遗传和转录景观 我们将对发育中/成熟的LUT，男性生殖器官以及E16.5，NB和3周龄的肾脏进行整合的转录组和表观遗传分析。尽管肾脏的重点将放在脉管系统，淋巴结和神经上，因为在大多数选定阶段缺乏单细胞磁磁数据，但我们的数据还将有助于加强GUDMAP数据，以便研究社区最广泛使用。 AIM 2：构建使用空间转录组学和高级成像技术开发泌尿生殖器官的多维分子图谱 生物系统中的地图集建筑物的一个主要挑战已在空间上为解剖和细胞结构分配了大量分子特征。我们已经成功地建立了实验程序和计算分析，用于用于空间转录组学，法典和光片显微镜成像的管道，以绘制基因表达数据，包括整个转录组数据到细胞和结构。我们将使用这些技术来分析开发/成熟的较低尿路，男性生殖器官以及E16.5，NB和3周龄的肾脏，以构建真正多维的多维型分子图谱。 AIM 3：使用带有细胞消融或基因失活的鼠Cakut模型来建造关键泌尿生殖器结构的分子图谱 Cakut以许多不同形式出现，代表了小儿人群发病和死亡的重要原因。过去，我们已经生成并分析了几种Cakut Murine模型。这些模型的目标器官的构建图形将提供高分辨率，空间注册的分子参考，以供疾病开始和进展的关键阶段。此外，这样的地图集将通过了解处理各种干扰的系统和过程的耐受性来帮助研究人员更好地了解正常的泌尿生殖器发育。我们将使用高度可重现的Cakut鼠模型，并在输尿管间充质中灭活典型的Smad信号传导，从而导致产前产生均匀的输尿管漏洞（UPJ）阻塞表型。我们将使用目标1和2中概述的技术在关键时间点构建相关结构（输尿管，肾脏，UPJ）的分子地图集，并将有缺陷发展的地图与正常发展的图案进行比较。