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

泌尿生殖系统发育分子解剖项目（GUDMAP）为研究泌尿生殖系统发育和疾病提供了有价值的参考。迅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图谱构建工作的交叉受精无疑将促进这些项目的技术渗透。为了结合最新的多组学和空间分子定位技术构建发育中的泌尿生殖器官的多维地图集，我们组建了一支在泌尿生殖器官发育和多维、多平台、分子地图集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，帮助有效和高效地将GUDMAP推进到转录组范围覆盖，单细胞水平分辨率和前所未有的清晰度空间制图的下一个水平。我们将利用我们在结合单核RNA-seq和snATAC-seq方面的经验，在单细胞分辨率下建立目标泌尿生殖器官和结构（下尿路（LUT），选定的男性生殖器官，肾脏血管系统，淋巴管和神经）的综合表观遗传学和转录组学景观（目的1）。然后，我们将通过结合空间转录组学（ST）、CODEX和薄层显微镜（LSM），将空间维度添加到这个分子景观中，构建2D和3D分子图谱（Aim 2）。在Aim 3中，我们将把我们的研究扩展到疾病模型，重点是小鼠先天性肾和尿路异常（CAKUT）模型。通过本实验，我们旨在以前所未有的细胞分辨率和最高的效率建立多维分子图谱，用于发育泌尿生殖器官。

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.