Craniofacial Developmental Dynamics

颅面发育动力学

基本信息

批准号：
10917907
负责人：
Kenneth Yamada
金额：
$ 67.49万
依托单位：
NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10917907
关键词：
3-Dimensional Ablation Acceleration Address Adhesives Architecture Bone Development Brain Cell Adhesion Cell Nucleus Cell-Cell Adhesion Cell-Matrix Junction Cells Characteristics Cloning Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Complex Cytoskeleton Development Embryo Embryonic Development Epithelial Cells Epithelium FGF7 gene Fluorescence Gene Expression Gene Expression Profile Genes Genetic Recombination Gland Goals Guide RNA Integrins Knock-out Laboratories Lentivirus Maintenance Mediating Mesenchymal Methods Microscopy Molecular Morphogenesis Mus Neural Crest Organ Paper Parotid Gland Procedures Process Production Proteoglycan Protocols documentation RNA Recovery Regulation Reporter Research Role Salivary Salivary Glands Serum Signal Transduction Skin Specificity System Tissue Engineering Tissues Ubiquitin biglycan cell motility cellular transduction craniofacial craniofacial development craniofacial tissue design experimental study in vivo matrigel novel organ growth restoration stem cells transcriptomics

项目摘要

This project is focused primarily on determining mechanisms of morphogenesis and maintenance of salivary glands and other organs. We are addressing the following major questions: 1. How do embryonic glands and other organs generate their characteristic architectures during development? 2. What are the contributions of local regulation of organ-specific gene expression, cell adhesion, extracellular matrix, integrins, signal transduction, and local cell migration to organ development? Branching morphogenesis of developing organs requires coordinated but still relatively incompletely understood changes in gene expression, epithelial cell-cell adhesion, cell-matrix adhesion, and cell motility. We had previously performed single-cell and bulk RNA transcriptomic analyses on embryonic submandibular versus parotid salivary glands to characterize their molecular identities at the very early stage of bud initiation. A key finding was the surprising degree of differences in gene expression patterns between these two types of salivary gland quite early in development, indicating gland specificity at even this early single-bud stage. In ongoing studies, recombination experiments in which the mesenchymal tissues and epithelial tissues of these two types of gland are being swapped to characterize alterations in gene expression patterns. Our laboratory has recently developed and refined methods for efficient CRISPR-mediated knockout of specific genes in embryonic mouse salivary gland epithelial ex vivo explants. Such salivary epithelial explants can be cultured successfully under serum-free conditions plus exogenous FGF7 and 5% Matrigel in 96-well plates, which can permit the facile manipulation and recovery of cultured epithelial buds. An efficient workflow for the production of high-titer lentivirus within one week was described, including the use of a fluorescence reporter associated with the guide RNA to highlight the nuclei of transduced cells. Transduction was 80% efficient, and three different genes were readily ablated, including a key integrin gene essential for branching morphogenesis. These procedures should accelerate studies of the roles of specific genes in salivary gland epithelial development. In another protocol paper, highly detailed methods for guide RNA design using CRISPOR, Golden Gate cloning, and production of lentiviruses was described and successfully applied to embryonic salivary epithelial explants. A research collaboration with the laboratories of Achim Werner and Laura Kerosuo applied microscopy methods to contribute to studies identifying roles for a novel tissue-specific ubiquitin switch of functions important for craniofacial, brain, and skin early development. A second study has involved collaboration with the Kerosuo laboratory on neural crest stem cells in development. A third paper contributed to a study implicating the proteoglycan biglycan in bone development. These studies are beginning to elucidate the complex mechanisms that underlie the cell and tissue dynamics involved in craniofacial organ development and maintenance. Understanding these underlying morphogenetic mechanisms during embryonic development should promote more effective tissue engineering for restoration of damaged organ function.

该项目主要集中在确定唾液腺和其他器官的形态发生和维持的机制上。我们正在解决以下主要问题： 1。在开发过程中，胚胎腺体和其他器官如何产生其特征体系结构？ 2。局部调节器官特异性基因表达，细胞粘附，细胞外基质，整合素，信号转导和局部细胞迁移对器官发育的贡献是什么？发育器官的分支形态发生需要协调但仍然相对不完全了解基因表达，上皮细胞 - 细胞粘附，细胞 - 矩阵粘附和细胞运动的变化。我们以前曾在胚胎下颌和腮腺唾液腺上进行了单细胞和大量RNA转录组分析，以表征其在芽启动的早期阶段的分子身份。一个关键的发现是，这两种类型的唾液腺之间的基因表达模式在发育的早期很早就表明了腺体的特异性，即使在这个早期的单个BUD阶段也是如此。在正在进行的研究中，重组实验中，这两种类型的腺体的间充质组织和上皮组织被交换为表征基因表达模式的改变。我们的实验室最近开发了和完善的方法，用于有效的CRISPR介导的胚胎小鼠唾液腺上皮外植体中特定基因的敲除。这种唾液上皮外植体可以在无血清条件下成功培养，再加上96孔板中的外源性FGF7和5％的矩阵，这可以允许培养的上皮芽的便捷操纵和恢复。描述了在一周内生产高细胞慢病毒的有效工作流程，包括使用与导向RNA相关的荧光报告基因突出传递细胞的核。转导效率为80％，并且很容易消融三种不同的基因，包括对分支形态发生必不可少的整合素基因。这些程序应加速研究特定基因在唾液腺上皮发育中的作用的研究。在另一份协议论文中，描述了使用CRIRPOR，金门克隆的高度详细指导RNA设计的方法，以及慢病毒的产生，并成功地应用于胚胎唾液上皮eppep依。与Achim Werner和Laura Kerosuo实验室的研究合作应用了显微镜方法，有助于研究确定新型组织特异性的泛素泛素功能转换对颅面，脑和皮肤早期发育重要的功能的作用。第二项研究涉及与开发神经rest干细胞的Kerosuo实验室合作。第三篇论文介绍了一项研究，暗示了蛋白聚糖在骨发育中。这些研究开始阐明颅面器官发育和维持涉及的细胞和组织动态的复杂机制。了解胚胎发育过程中这些潜在的形态发生机制应促进更有效的组织工程，以恢复受损器官功能。