Taste organs: formation and regulation

描述(申请人提供)：味蕾细胞，将化学刺激转化为神经信号传递到中枢神经系统的感觉末端器官，驻留在哺乳动物舌头的味觉乳头中。因此，味觉乳头承载着将分化为包括味蕾细胞的上皮细胞。然而，味觉生物学领域缺乏对：(1)在乳头发育过程中可能的味觉细胞前体是什么；(2)这些前体如何以及何时在舌上皮中分化以获得味觉乳头和味蕾细胞表型；以及(3)潜在的间质如何以及通过什么因素向舌乳头和味蕾中的舌上皮发出信号。我们对舌上皮中神经脊(NC)衍生细胞的分布以及间充质NC衍生细胞的遗传修饰引起的舌和味觉乳头的表型改变的初步数据表明，神经脊在味觉器官的形成中对上皮和间充质都有贡献。Cre介导的、组织特异性的、遗传标记和修饰为这些细胞谱系分析和功能分析提供了强大的工具。使用两个特征良好的转基因小鼠品系，WNT1-Cre和P0-Cre，用于NC检测。我们发现：(1)在舌上皮细胞中，Wnt1-Cre和P0-Cre标记的NC来源细胞包括味觉乳头和味蕾(P0-Cre丰富，Wnt1-Cre少见)，提示可能存在味觉细胞的NC来源，这导致了该领域的一个新概念。P0-Cre标记细胞是第一个 早期以单一、分散的元素分布，后来在味乳头和味蕾中呈簇状分布。我们认为，单个分散的P0-Cre标记的上皮细胞是NC的前体细胞，这些细胞将在味觉乳头和味蕾中分化为特定类型的细胞。(2)在舌间充质中，WNT1-Cre标记的NC来源细胞与味觉乳头和味蕾密切相关。WNT1-Cre驱动的I型受体Alk2和Alk3对骨形态发生蛋白(BMP)的条件遗传修饰以水平、阶段和受体特定的方式显著改变舌头、味乳头和味蕾的形成和维持。这表明，舌间充质中的BMP信号通过不同的受体与上皮细胞相互作用，在舌头、味乳头和味蕾的发育过程中发挥不同的作用。拟议的研究将利用现代技术(Cre介导的基因修饰)以及体内和体外研究相结合的方式，解决味觉乳头器官形成的基本问题。我们的目标是：(1)证明NC细胞向舌原基上皮迁移的最佳阶段(1a)和初级颅区(中脑或后脑)(1b)；这些细胞在舌上皮中分化的时间和数量，以获得特定的细胞表型(1c)；NC(1d)来源的味蕾细胞的类型和比例；(目的2)研究在舌间充质NC来源的细胞中，BMP信号如何通过不同的I型受体(ALK2和ALK3)在舌头、味乳头和味蕾的发育和维持过程中与上皮细胞相互作用，并进行基因修饰，以(2a)通过Wnt1-Cre驱动的Alk2或Alk3的条件性敲除下调BMP信号的活性；以及(2b)通过Wnt1-Cre驱动的每个受体的条件性成分激活上调BMP信号。总之，关于味觉乳头和味蕾细胞起源、分化和间充质相互作用的研究将有助于理解味觉器官的发育，并将为味觉乳头和味蕾细胞的神经峰贡献领域带来新的信息和新的视角。

DESCRIPTION (provided by applicant): Taste bud cells, the sensory end organs that transduce chemical stimuli into neural signals conveyed to the central nervous system, reside in taste papillae in the mammalian tongue. Therefore, taste papillae host the epithelium that will differentiate to include taste bud cells. However, the field of taste biology lacks a complete understanding of: (1) what constitutes possible taste cell precursors in developing papillae; (2) how and when the precursors differentiate in lingual epithelium to acquire taste papilla and taste bud cell phenotypes; and (3) how and via what factors the underlying mesenchyme signals to tongue epithelium in papilla and taste bud development. Our preliminary data on neural crest (NC) derived cell distributions in lingual epithelium, and of phenotypic alterations of tongue and taste papillae induced by genetic modifications in mesenchymal NC derived cells, suggest neural crest contributions to both epithelium and mesenchyme in the formation of taste organs. Cre- mediated, tissue-specific, genetic labeling and modifications provide powerful tools for these cell lineage assays and functional analyses. Two well-characterized transgenic mouse lines for NC assays, Wnt1-Cre and P0-Cre, are used. We have found that: (1) In tongue epithelium, both Wnt1-Cre and P0-Cre labeled NC derived cells are seen including taste papillae and taste buds (abundant in P0-Cre, infrequent in Wnt1-Cre) suggesting a potential NC derivation of taste cells which leads to a new concept in the field. P0-Cre labeled cells are first distributed in single, scattered elements at early stages to a clustered pattern later in taste papillae and taste buds. We propose that the single, scattered P0-Cre labeled epithelial cells are NC precursors and these will undergo cell differentiation to specific cell types in taste papillae and taste buds. (2) In tongue mesenchyme, Wnt1-Cre labeled NC derived cells are closely associated with taste papillae and taste buds. Wnt1-Cre driven, conditional genetic modifications of type I receptors Alk2 and Alk3 for bone morphogenetic proteins (BMPs) significantly alter the tongue, taste papilla and taste bud formation and maintenance in a level-, stage- and receptor-specific manner. This suggests that BMP signaling in tongue mesenchyme, via distinct receptors, interacts with the overlying epithelium for different roles in the development of tongue, taste papillae and taste buds. The proposed studies will address fundamental issues about formation of the taste papilla organ, using modern techniques (Cre-mediated genetic modifications) and combination of in vivo and in vitro studies. Our goals are to: (Aim 1) demonstrate the optimal stage (1a) and primary cranial region (midbrain or hindbrain) (1b) of NC cell migration into the epithelium of tongue primordium; when and how many these cells differentiate in lingual epithelium to acquire specific cell phenotypes (1c); what types and proportions of taste bud cells are derived from NC (1d); (Aim 2) characterize how the BMP signaling in tongue mesenchymal NC derived cells, via distinct type I receptors (ALK2 and ALK3), interacts with the overlying epithelium in the development and maintenance of tongue, taste papillae and taste buds, with genetic modifications to (2a) down-regulate BMP signaling activity with Wnt1-Cre driven conditional knockout of Alk2 or Alk3; and (2b) up-regulate BMP signaling with Wnt1-Cre driven conditional constitutive activation of each receptor. Overall, the proposed studies for demonstration of cell origin, differentiation and mesenchymal interactions in taste papillae and taste buds will contribute to understanding development of the taste organ and will bring new information and novel perspectives to the field for neural crest contributions to taste papillae and taste bud cells.