Roles of Ascl1 for generation and differentiation of taste cells

摘要 品味有助于提高生活质量，也有助于养成健康的饮食习惯。在人类和其他脊椎动物身上， 味觉传导发生在味蕾中，当味觉刺激与成熟(终末分化)相互作用时。 特化细胞通常被归类为I、II和III型细胞。味觉细胞的正常持续周转 这是味觉动态平衡的基础；当周转被打乱时，味觉也同样被打乱。味觉细胞是 来源于干细胞/祖细胞，在动物一生中不断提供新的味觉细胞。 然而，味觉细胞更替的机制却知之甚少。我们最近的研究表明， Pou2f3转录因子是II型细胞生成所必需的，而II型细胞的生成， 它调节甜味、鲜味(可口的)和苦味，与III型细胞密切相关，后者 调节酸味和咸味，可能参与III型和III型之间的细胞间横向通讯 细胞。我们的初步发现表明，转录因子Ascl1在基础前体和 味蕾内分化的III型细胞参与了III型细胞和II型细胞的产生 小鼠胚胎发育过程中的部分细胞。因此，我们建议阐明Ascl1在 III型细胞生成和功能分化/维持的分子和细胞机制 在成年小鼠的味觉细胞更替过程中。我们还将研究Ascl1的S在II型细胞生成中的作用。 由于传统的Ascl1基因敲除小鼠在出生后不久就会死亡，我们将使用两种类型的Ascl1条件性 基因敲除(CKO)小鼠。这些小鼠具有Ascl1等位基因，该等位基因在Ascl1的5‘和3’端有loxP位点 编码序列和Cre重组酶在特定细胞类型中的表达及驱动Cre-loxP介导的Ascl1 这些细胞中的缺失。因为Ascl1可能在基础前体和分化的III型中发挥不同的作用 对于细胞，我们将进行目标1和目标2的实验，以区分这些角色。在目标1中，我们将归纳 ASCL1 CKO在分化的III型细胞中的表达。本实验将验证Ascl1的S在细胞中表达的假设 III型细胞是这些细胞功能分化/维持所必需的。在目标2中，我们将归纳 ASCL1 CKO在味觉祖细胞/干细胞中，产生基本的前体细胞。这项实验将测试 假设Ascl1是产生III型和II型细胞所必需的。在这两个目标中，我们将分析 Ascl1缺乏是否会影响特定类型的味觉细胞的分子和功能特征 将在这两种类型的Ascl1 CKO小鼠身上进行味觉系统的评估，这些小鼠具有(A)组织化学基因和 蛋白质表达分析，以检查组织化学分析和味觉神经记录。知识 这项研究将有助于加深对味觉细胞周转和味觉丧失机制的理解 伴随着放射和化疗、传染病和衰老。

Summary Taste contributes to quality of life, as well as to healthy eating habits. In humans and other vertebrate animals, taste transduction occurs in taste buds, when taste stimuli interact with mature (terminally differentiated) specialized cells that are often classified as type I, II, and III cells. Normal continuous turnover of taste cells underlies the homeostasis of taste; when turnover is disrupted, taste is likewise disrupted. Taste cells are derived from stem/progenitor cells, which continuously supply new taste cells throughout an animal's life. However, the mechanisms of taste cell turnover are poorly understood. Our recent studies have revealed that the Pou2f3 transcription factor is necessary for type II cell generation, and that the generation of type II cells, which mediate sweet, umami (savory), and bitter tastes, is intimately related with that of type III cells, which mediate sour and salty tastes and may be involved in intercellular lateral communication between type II and III cells. Our preliminary findings indicate that Ascl1, a transcription factor expressed in basal precursor and differentiated type III cells within taste buds, is involved in the generation of type III cells and probably type II cells in part during embryonic development in mice. We therefore propose to elucidate the roles of Ascl1 in molecular and cellular mechanisms in the generation and functional differentiation/maintenance of type III cells during taste cell turnover in adult mice. We will also examine Ascl1's role in the generation of type II cells. Because conventional Ascl1 knockout mice die shortly after birth, we will use two types of Ascl1 conditional knockout (cKO) mice. These mice have a “floxed” Ascl1 allele that has loxP sites at 5' and 3' of an Ascl1 coding sequence, and Cre recombinase expressed in specific cell types and driving Cre-loxP mediated Ascl1 deletion in these cells. Because Ascl1 may play different roles in basal precursor and differentiated type III cells, we will conduct experiments of Aims 1 and 2 to distinguish between these roles. In Aim 1, we will induce Ascl1 cKO in differentiated type III cells. This experiment will test the hypothesis that Ascl1's expression in type III cells is necessary for functional differentiation/maintenance of these cells. In Aim 2, we will induce Ascl1 cKO in taste progenitor/stem cells that give rise to basal precursor cells. This experiment will test the hypothesis that Ascl1 is necessary for the generation of type III and II cells. In both Aims, we will analyze whether Ascl1 deficiency affects molecular and functional characteristics of specific types of taste cells, using on the taste system will be assessed in these two types of Ascl1 cKO mice with (a) histochemical gene and protein expression analyses to examine histochemical analyses and gustatory nerve recordings. Knowledge obtained from this research will improve understanding of taste cell turnover and the mechanisms of taste loss accompanying radio- and chemotherapy, infectious conditions, and aging.