Regulation of embryonic patterning and adult stem cells of oral appendages

描述（由申请人提供）：了解调控牙齿、味觉乳头和丝状乳头等口腔外胚层附属物的发育、形成模式和出生后更新的分子和细胞机制，并确定这些器官中的干细胞和祖细胞群，对于制定再生策略以在先天缺失或疾病导致缺失的情况下替换缺失的牙齿至关重要;用于理解和治疗味觉障碍，包括由放射治疗和小分子抗癌药物引起的味觉障碍;以及用于描绘可能在口腔癌中失调的增殖控制。Wnt/β-连环蛋白信号通路是许多发育过程所必需的，并且在成体干细胞群的增殖和自我更新中起关键作用。小鼠遗传研究揭示了Wnt/β-catenin信号在牙齿和味觉乳头形态发生早期的关键功能。信号传导在牙齿和味觉乳头发育开始之前被广泛激活，并逐渐限制在牙齿和味觉前体发育的部位，确保牙齿和味觉器官的正确定位。基于我们的初步数据，我们假设Wnt信号传导的适当定位需要Wnt配体和分泌的Wnt抑制剂的竞争活性，并且这些通过空间控制口腔上皮细胞的命运、运动和增殖来指导器官形成。 我们将使用小鼠胚胎口腔外植体的实时成像，这些小鼠在Wnt激活的细胞或所有基底上皮细胞中特异性表达染色质定位的GFP，以及膜番茄的普遍表达，以询问Wnt抑制剂的遗传缺失或对牙齿发育重要的Wnt配体的缺失是否会导致细胞运动和/或增殖模式的改变。舌的丝状乳头和味觉乳头在成人生活中不断更新，具有WNT 10A突变的一部分人类患者呈现青春期发作的口腔外胚层缺陷，包括“光滑舌”。我们假设WNT 10A/ -连环蛋白信号传导控制味觉和丝状乳头更新所需的成体干细胞和祖细胞的增殖和/或存活。为了测试这一点，我们将询问小鼠中Wnt 10a的缺失是否影响β-连环蛋白信号传导和舌乳头祖细胞的增殖、存活或分化，并将对成年舌中的Wnt应答细胞进行命运图谱以测试它们是否包括自我更新祖细胞.为了确定Wnt信号传导是否是功能性祖细胞存活所必需的，我们将测试在去除诱导剂后，Wnt抑制剂DKK 1的诱导型转基因表达对丝状和味乳头增殖的抑制是否可逆。这些实验将描绘控制口腔中增殖和器官更新的机制，并将为设计再生策略提供重要信息。

DESCRIPTION (provided by applicant): Understanding the molecular and cellular mechanisms regulating the development, patterning and postnatal renewal of oral ectodermal appendages such as teeth, taste papillae and filiform papillae, and identifying stem and progenitor cell populations in these organs, is critical for developing regenerative strategies to replace missing teeth in cases of congenital absence or loss through disease; for understanding and treating disorders of taste, including those resulting from radiation therapy and small molecule anti-cancer drugs; and for delineating proliferation controls that may be dysregulated in oral cancers. The Wnt/ -catenin signaling pathway is necessary for many developmental processes and plays critical roles in the proliferation and self- renewal of adult stem cell populations. Genetic studies in mice reveal key functions for Wnt/ -catenin signaling at early stages of tooth and taste papilla morphogenesis. Signaling is activated broadly prior to the initiation of tooth and taste papilla development and gradually becomes restricted to sites of tooth and taste precursor development, ensuring correct positioning of tooth and taste organs. Based on our preliminary data, we hypothesize that proper localization of Wnt signaling requires competing activities of Wnt ligands and secreted Wnt inhibitors, and that these direct organ formation by spatially controlling the fates, movements and proliferation of oral epithelial cells. We will use live imaging of embryonic oral explants from mice that express chromatin-localized GFP specifically in Wnt-activated cells or in all basal epithelial cells, together with ubiquitous expression of membrane Tomato, to ask whether genetic deletion of Wnt inhibitors, or loss of a Wnt ligand important for tooth development, cause altered cell movements and/or patterns of proliferation. Filiform and taste papillae of the tongue are continuously renewed in adult life, an a subset of human patients with mutations in WNT10A presents with adolescent onset of oral ectodermal defects including "smooth tongues". We hypothesize that WNT10A/ -catenin signaling controls proliferation and/or survival of adult stem and progenitor cells required for renewal of taste and filiform papillae. To test this we will ask whether loss of Wnt10a in mice affects -catenin signaling and the proliferation, survival, or differentiation of tongue papilla progenitor cells and will fate map Wnt responsive cells in the adult tongue to test whether they include self- renewing progenitors. To determine whether Wnt signaling is necessary for survival of functional progenitors, we will test whether inhibition of filiform and taste papilla proliferaton upon inducible transgenic expression of the Wnt inhibitor DKK1 is reversible after removal of the inducing agent. These experiments will delineate mechanisms controlling proliferation and organ renewal in the oral cavity and will provide important information for designing regenerative strategies.