Craniofacial Developmental Dynamics

在这个项目中，我们主要集中在确定唾液腺和其他器官的形态发生机制。我们正在解决以下主要问题： 1.胚胎唾液腺等分支器官是如何在分支形态发生过程中形成其特有的分支构筑的？具体地说，裂隙、花蕾和导管的形成是如何在分子和生物物理水平上调节和协调的？我们如何通过了解分支形态发生和促进特定步骤来促进器官替换的生物工程，特别是唾液腺的生物工程？ 2.细胞外基质、整合素、信号转导、特定基因表达和细胞迁移的选择性调节在分支形态发生以及其他主要组织重排(如脑神经隆起发育)中有何贡献？ 发育中器官的分支形态发生需要协调但仍相对较少了解的上皮细胞-细胞黏附和细胞运动的变化。我们以前的研究已经确定了一个涉及纤维连接蛋白、新的调节因子Btbd7和转录因子Snail2(SLUG)的步进式调节级联，影响涉及E-钙粘附素的细胞黏附和细胞迁移。我们已经将这些初步发现扩展到体外和体内对这一复杂过程的更深入的分析。我们开发了一个Btbd7基因敲除的小鼠模型来验证我们的假设，即Btbd7是体内分支形态发生的关键调节因子。在正常小鼠中，Btbd7水平在分支上皮端芽的外周细胞中升高，在这个位置上它可以增强细胞运动，减少细胞间的粘连，并促进裂隙的形成。Btbd7基因在小鼠体内的遗传消融严重扰乱了胚胎唾液腺、肺和肾脏的分支形态发生。Btbd7的缺失导致外芽细胞更加紧密地堆积和拉长，表现出更强的E-钙粘素定位，降低了细胞的运动性，并减少了与裂隙形成相关的动态的、瞬时的细胞-细胞分离。与之形成鲜明对比的是，内芽细胞没有受到影响。使用培养的MDCK细胞模拟外芽和内芽细胞行为的机制分析证实，Btbd7通过增强迁移和瞬时细胞分离促进细胞-细胞连接中E-钙粘附素的丢失。在机制上，Btbd7通过在MDCK细胞和完整的外周上皮芽细胞中的蛋白酶体活性来促进E-钙粘素的泛素化、内化和降解，以调节细胞动力学。因此，这些研究表明，新的调节分子Btbd7如何在发育中的器官的外围发挥作用，调节上皮分支形态发生过程中细胞黏附和运动的局部动态。 我们之前已经证实，在唾液腺形态发生过程中，影响乙酰化的微管翻译后单个变化可以扰乱分支形态发生。 唾液间充质细胞的这种分子变化改变了间充质微环境，促进了上皮祖细胞亚群的维持和分化，这可能是分支形态发生障碍的原因。具体地说，唾液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+)的祖细胞。这种对K14+祖细胞及其分化的后代肌上皮细胞的影响发生在发育中的唾液腺远端芽区上皮细胞的基底层和基底层上。从机制上讲，这一过程涉及通过转化生长因子β-1和Notch信号通路改变信号。因此，间充质细胞细胞骨架系统的一次翻译后改变可以决定相邻上皮祖细胞的维持和分化，从而改变上皮分支的形态发生。 我们以前已经证明，细胞外基质蛋白anosmin可以通过改变细胞信号来调节神经脊的形成。尽管Anosmin被报道与硫酸乙酰肝素、成纤维细胞生长因子受体和uPA结合，但我们研究了整合素的作用，整合素在细胞黏附和迁移中发挥主要作用。我们鉴定了三种β-1整合素为Anosmin黏附受体。这些研究将整合素-基质相互作用与神经脊发育中的生长因子调控系统联系起来。 这些研究开始阐明复杂的调控系统，对于参与头面部器官发育的细胞和组织动力学非常重要。了解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修复受损的器官功能。

In this project, we are focusing primarily 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 biophysical 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? Branching morphogenesis of developing organs requires coordinated but still relatively poorly understood changes in epithelial cell-cell adhesion and cell motility. Our previous studies had identified a step-wise regulatory cascade involving fibronectin, the novel regulator Btbd7, and the transcription factor Snail2 (Slug) affecting cell adhesion involving E-cadherin and cell migration. We have extended these initial findings to more in-depth analyses of this complex process in vitro and in vivo. We developed a Btbd7 knockout mouse model to test our hypothesis that Btbd7 is a crucial regulator of branching morphogenesis in vivo. In normal mice, Btbd7 levels are elevated in the peripheral cells of branching epithelial end buds, a location at which it could enhance cell motility, reduce cell-cell adhesion, and promote dynamics of cleft formation. Genetic ablation of Btbd7 in mice severely disrupts branching morphogenesis of embryonic salivary gland, lung, and kidney. Loss of Btbd7 results in more tightly packed and elongated outer bud cells, which display stronger E-cadherin localization, reduced cell motility, and decreased dynamic, transient cell-cell separations associated with cleft formation. In striking contrast, inner bud cells remain unaffected. Mechanistic analyses using cultured MDCK cells to mimic outer vs. inner bud cell behavior established that Btbd7 promotes the loss of E-cadherin from cell-cell junctions with enhanced migration and transient cell separation. Mechanistically, Btbd7 serves to enhance E-cadherin ubiquitination, internalization, and degradation via proteasomal activity in MDCK cells and in intact peripheral epithelial bud cells for regulating cell dynamics. Consequently, these studies show how the new regulatory molecule, Btbd7, can function at the periphery of developing organs to regulate the local dynamics of cell adhesion and motility during epithelial branching morphogenesis. We previously established that during salivary gland morphogenesis, a single post-translational alteration in microtubules affecting acetylation can disrupt branching morphogenesis. This molecular alteration in salivary mesenchyme cells alters the mesenchymal microenvironment and promotes the maintenance and differentiation of a subset of epithelial progenitor cells, which can account for the impairment of branching morphogenesis. Specifically, hyperacetylation of microtubules in salivary mesenchymal cells increases cytokeratin 14-positive (K14+) progenitors. This effect on K14+ progenitors and their differentiated progeny, myoepithelial cells, occurs in epithelial basal and suprabasal layers of epithelial cells in the distal endbud region of developing salivary glands. Mechanistically, this process involves alterations in signaling via transforming growth factor beta-1 plus Notch signaling pathways. Thus, a single post-translational alteration in the cytoskeletal system of mesenchyme cells can dictate the maintenance and differentiation of adjacent epithelial progenitor cells to alter epithelial branching morphogenesis. We demonstrated previously that neural crest formation can be regulated by the extracellular matrix protein anosmin by altering cellular signaling. Although anosmin was reported to bind heparan sulfate, FGF receptor, and UPA, we examined for roles of integrins, which play major roles in cell adhesion and migration. We identified three beta-1 integrins as anosmin adhesion receptors. These studies link integrin-matrix interactions to the growth factor regulatory system in neural crest development. These studies are beginning to elucidate the complex regulatory systems important for the cell and tissue dynamics involved in craniofacial organ development. Understanding these underlying morphogenetic mechanisms should promote more effective tissue engineering for restoration of damaged organ function.