Roles of Ascl1 for generation and differentiation of taste cells

总结 味道有助于生活质量，以及健康的饮食习惯。在人类和其他脊椎动物中， 味觉传导发生在味蕾，当味觉刺激与成熟的（终末分化的） 专门的细胞通常被分类为I型、II型和III型细胞。味觉细胞的正常连续更新 是味觉内稳态的基础;当周转被破坏时，味觉也会被破坏。味细胞 来源于干/祖细胞，在动物的一生中不断提供新的味觉细胞。 然而，味觉细胞周转的机制知之甚少。我们最近的研究表明， Pou 2f 3转录因子对于II型细胞的产生是必需的，并且II型细胞的产生， 介导甜味、鲜味和苦味的第三型细胞与第三型细胞密切相关， 介导酸味和咸味，可能参与II型和III型之间的细胞间横向通讯 细胞我们的初步研究结果表明，Ascl 1，一个在基底前体和基底膜中表达的转录因子， 味蕾内的分化的III型细胞，参与III型细胞和可能II型细胞的产生。 在小鼠胚胎发育过程中部分细胞。因此，我们建议阐明Ascl 1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新过程中。我们还将研究Ascl 1在II型细胞生成中的作用。 由于传统的Ascl 1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Ascl 1条件性 敲除（cKO）小鼠。这些小鼠具有“floxed”Ascl 1等位基因，其在Ascl 1等位基因的5'和3'处具有loxP位点。 编码序列，以及在特定细胞类型中表达并驱动Cre-loxP介导的Ascl 1的Cre重组酶 在这些细胞中删除。由于Ascl 1在基础前体和分化的III型中可能发挥不同的作用， 细胞，我们将进行目标1和2的实验，以区分这些作用。在目标1中，我们将诱导 分化的III型细胞中的Ascl 1 cKO。本实验将检验Ascl 1在细胞中表达的假设， III型细胞对于这些细胞的功能分化/维持是必需的。在目标2中，我们将诱导 Ascl 1 cKO在味觉祖细胞/干细胞中产生基底前体细胞。这个实验将测试 假设Ascl 1对于III型和II型细胞的产生是必需的。在这两个目标中，我们将分析 Ascl 1缺乏是否影响特定类型味觉细胞的分子和功能特征， 将在这两种类型的Ascl 1 cKO小鼠中评估对味觉系统的影响， 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 从这项研究中获得的信息将有助于加深对味觉细胞更新和味觉丧失机制的理解 伴随着放疗和化疗，感染性疾病和衰老。

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.