Craniofacial Developmental Dynamics

颅面发育动力学

基本信息

批准号：
9339225
负责人：
Kenneth Yamada
金额：
$ 89.45万
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/9339225
关键词：
Ablation Acetylation Address Adhesions Adhesives Affect Antibodies Architecture Basement membrane Binding Biological Assay Biomedical Engineering Cell Adhesion Cell Culture Techniques Cell Nucleus Cell membrane Cell surface Cell-Cell Adhesion Cells Cephalic Characteristics Cleaved cell Complex Cytokeratin-14 Staining Method Cytoskeleton Development Developmental Cell Biology Distal Duct (organ) structure E-Cadherin Embryo Epithelial Epithelial Cells Epithelium Extracellular Matrix Proteins Fibroblast Growth Factor Receptors Fibronectin Receptors Fibronectins Gene Expression Genetic Goals Heparitin Sulfate Human Immunofluorescence Immunologic In Vitro Individual Integrins Kallmann Syndrome Knockout Mice Maintenance Mediating Mesenchymal Mesenchyme Microdissection Microscopy Microtubules Molecular Morphogenesis Mutate Mutation Neural Crest Neurons Notch Signaling Pathway Nuclear Organ Pattern Peptides Phase Process Proteins RNA Interference Regulation Reporting Research Resolution Role Salivary Glands Signal Transduction Signaling Protein Stem cells System Time Tissue Engineering Tissues Transforming Growth Factors adhesion receptor base cell behavior cell motility craniofacial craniofacial development in vivo integrin alpha9 beta1 migration mouse model novel progenitor receptor function reconstitution slug transcription factor

项目摘要

We are focusing on determining the mechanisms of morphogenesis of salivary glands and other organs. We are addressing the following major questions: 1. How do embryonic salivary glands and other branched organs generate their characteristic branched architectures during the process of branching morphogenesis? Specifically, how is the formation of clefts, buds, and ducts mediated and coordinated at molecular and physical levels? How can we facilitate bioengineering for organ replacement, particularly of salivary glands, by understanding branching morphogenesis and by promoting specific steps? 2. What are the contributions of the selective regulation of extracellular matrix, integrins, signal transduction, specific gene expression, and cell migration in branching morphogenesis, as well as in other major tissue rearrangements such as cranial neural crest development? We are applying a variety of approaches to begin answering these complex questions. These approaches include: microdissection, RNA interference, 3D organ explants and cell culture, confocal immunofluorescence and brightfield time-lapse microscopy, genetic ablation, and a variety of other functional inhibition and reconstitution approaches. Our previous research implicated the extracellular matrix protein anosmin in neural crest craniofacial morphogenesis. Genetic defects in anosmin result in human Kallmann syndrome. Its functions include cell adhesion, neuronal migration, and neural crest formation. Anosmin consists of multiple domains, and it has been reported to bind heparan sulfate, FGF receptor, and UPA. We established cell adhesion and spreading assays for anosmin and used them for antibody inhibition analyses to search for anosmin's integrin adhesion receptor(s). Using both anti-functional antibody analyses with a panel of anti-integrin antibodies and specific adhesive peptide competitive inhibition assays, we have identified three integrins that serve to mediate cell adhesion to anosmin. The alpha5-beta1 fibronectin receptor, alpha4-beta1, and alpha9-beta1 integrins are each needed for effective adhesive receptor function in cell adhesion and cell spreading on anosmin; adhesion is also inhibited competitively by both RGD- and CS1-based peptides known to disrupt specific integrin adhesive functions. This identification of anosmin-integrin adhesion receptors should facilitate studies of anosmin function in cell and developmental biology. During salivary gland morphogenesis, we find that a single post-translational change in microtubules affecting acetylation in mesenchymal cells alters the mesenchymal microenvironment and promotes the maintenance and differentiation of a subset of epithelial progenitor cells, which impairs branching morphogenesis. Specifically, hyperacetylation of microtubules in mesenchymal cells increased cytokeratin 14-positive (K14+) progenitors in epithelial cells at the distal endbud region of developing salivary glands. Mechanistically, this process engages the transforming growth factor and Notch signaling pathways. We conclude that a simple post-translational alteration in mesenchyme microtubules dictates the maintenance and differentiation of adjacent epithelial progenitor cells to alter branching morphogenesis of the epithelium. In other descriptive and mechanistic studies of branching morphogenesis, we had recently characterized the overall patterns of both individual cell movement and basement membrane translocation during salivary gland branching morphogenesis. We identified a regulatory process involving fibronectin, the novel regulator Btbd7, and the transcription factor Snail2 (Slug) affecting cell adhesion involving E-cadherin and cell migration. The next phase will be to integrate these findings into an in-depth understanding of this complex process in vitro and in vivo. We will perform more in-depth confocal descriptive analyses at higher resolution combined with further molecular analyses of the roles of regulatory molecules such as Btbd7. For example, we have generated a Btbd7 knockout mouse model that we are characterizing in depth to evaluate its roles in various types of branching morphogenesis. We will also investigate further how matrix proteins signal from the plasma membrane to the nucleus and then to the cytoskeleton to promote dynamic epithelial cell behavior in various epithelial systems in vitro and in vivo.

我们专注于确定唾液腺和其他器官形态发生的机制。我们正在解决以下主要问题： 1。在分支形态发生过程中，胚胎唾液腺和其他分支器官如何产生其特征性的分支结构？具体而言，在分子和物理水平上介导并协调的裂口，芽和管道的形成如何？我们如何通过理解分支形态发生并促进特定步骤来促进生物工程的替代，特别是唾液腺的替代？ 2。在分支形态发生的细胞外基质，整合素，信号转导，特定基因表达和细胞迁移以及其他主要的组织重排（如颅神经crest发育）中，选择性调节的贡献是什么？我们正在应用各种方法开始回答这些复杂的问题。这些方法包括：显微解剖，RNA干扰，3D器官外植体和细胞培养物，共聚焦免疫荧光和明亮的延时显微镜，遗传消融以及各种其他功能性抑制和重构方法。我们以前的研究暗示了神经颅骨形态发生中细胞外基质蛋白厌氧菌蛋白。阳离子中的遗传缺陷导致人类卡尔曼综合征。它的功能包括细胞粘附，神经元迁移和神经rest形成。 Anosmin由多个结构域组成，据报道它可以结合硫酸肝素，FGF受体和UPA。我们建立了细胞粘附并扩散了厌氧菌素的测定法，并将其用于抗体抑制分析来搜索阳离子的整联蛋白粘附受体（S）。使用抗功能性抗体分析与一组抗整合蛋白抗体和特定的粘附肽竞争性抑制测定法，我们已经确定了三种整合素，可介导细胞粘附于厌氧蛋白。 Alpha5-Beta1纤连蛋白受体，α4-Beta1和alpha9-Beta1整合素都是有效的粘附受体功能在细胞粘附和细胞上散布在Anosmin上的有效粘附受体功能所必需的。 RGD和基于CS1的肽也可以抑制粘附，从而破坏特定整合素粘合剂功能。这种鉴定阳离子蛋白 - 整合素粘附受体应促进细胞和发育生物学中厌食的功能的研究。在唾液腺形态发生过程中，我们发现影响间充质细胞乙酰化的微管的单个翻译后变化改变了间充质微环境的改变，并促进了上皮祖细胞子集的维持和分化，从而损害了分支分支形态发生。具体而言，间充质细胞中微管的高乙酰化增加了细胞角蛋白14阳性（K14+）祖细胞的祖细胞中上皮细胞的祖细胞的远端末端末端区域的乳腺发育腺体发展。从机械上讲，此过程与转化的生长因子和缺口信号通路相关。我们得出的结论是，间充质微管中简单的翻译后改变决定了相邻上皮祖细胞的维持和分化，以改变上皮的分支形态发生。在分支形态发生的其他描述性和机械研究中，我们最近表征了唾液腺分支形态发生过程中单个细胞运动和基底膜易位的总体模式。我们确定了涉及纤连蛋白，新型调节剂BTBD7的调节过程以及影响涉及E-钙粘蛋白和细胞迁移的细胞粘附的转录因子Snail2（SLUG）。下一阶段将是将这些发现在体外和体内深入了解这一复杂过程中。我们将在较高分辨率的情况下进行更深入的共焦描述性分析，并进一步对调节分子（例如BTBD7）的作用进行进一步的分子分析。例如，我们已经生成了一个BTBD7敲除小鼠模型，我们正在深入表征它，以评估其在各种类型的分支形态发生中的作用。我们还将进一步研究基质蛋白如何从质膜发出信号到细胞核，然后是细胞骨架，以在体外和体内促进各种上皮系统中的动态上皮细胞行为。