Craniofacial Developmental Dynamics

这个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。我们正在解决以下主要问题： 1.胚胎腺和其他器官是如何在发育过程中产生其特有的结构的？ 2.局部调节器官特异性基因表达、细胞黏附、细胞外基质、整合素、信号转导和局部细胞迁移对器官发育有何贡献？ 发育中器官的分支形态发生需要基因表达、上皮细胞-细胞黏附、细胞-基质黏附和细胞运动性的协调但仍不完全了解的变化。我们之前对胚胎颌下腺和腮腺进行了单细胞和批量RNA转录分析，以确定它们在芽起始的非常早期的分子特性。一个关键的发现是，这两种类型的唾液腺在发育相当早的时候，基因表达模式的差异程度令人惊讶，这表明即使在这个早期的单芽阶段，腺体也具有特异性。在正在进行的研究中，重组实验将这两种类型的腺体的间充质组织和上皮组织互换，以表征基因表达模式的变化。 我们实验室最近开发并改进了CRISPR介导的有效敲除胚胎小鼠唾液腺上皮体外培养组织中特定基因的方法。这样的唾液上皮外植体可以在96孔板中，在无血清条件下，添加外源Fgf7和5%Matrigel成功培养，这使得培养的上皮芽可以方便地操作和恢复。描述了一种在一周内生产高滴度慢病毒的有效工作流程，包括使用与引导RNA相关联的荧光报告程序来突出转导细胞的细胞核。转导效率为80%，三个不同的基因很容易被去除，其中包括一个对分枝形态发生至关重要的整合素基因。这些程序应该会加速特定基因在唾液腺上皮发育中的作用的研究。在另一篇协议文件中，描述了使用CRISPOR、金门克隆和慢病毒生产来指导RNA设计的非常详细的方法，并成功地应用于胚胎唾液上皮外植体。 与Achim Werner和Laura Kerosuo的实验室合作的研究应用显微镜方法，帮助研究确定一种新的组织特异性泛素开关的角色，该开关对颅面、大脑和皮肤的早期发育至关重要。第二项研究涉及与Kerosuo实验室合作开发神经脊干细胞。第三篇论文参与了一项研究，该研究表明蛋白多糖二聚糖与骨骼发育有关。 这些研究开始阐明涉及头面部器官发育和维持的细胞和组织动力学的复杂机制。了解胚胎发育过程中的这些潜在的形态发生机制应该会促进更有效的组织工程来恢复受损的器官功能。

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.