Novel transgenic reporter/deleter allele for Type I taste cells

描述（申请人提供）：味蕾是味觉感觉的传导元件。因此，它们代表了检测营养物质（如钠盐、糖和氨基酸）和有害物质（如酸和有毒苦味化合物）的过程的第一步。了解这一过程中的步骤可能会导致治疗干预，可用于调节食物摄入，这是控制肥胖的关键因素。每个味蕾包括三种细长的味觉细胞和一群基底细胞。了解每种细胞类型的功能作用是理解每种刺激如何被检测以及这些信息如何传递到神经系统的基础。在三种梭状细胞中，表达苦、甜、鲜味转导的受体和信号效应器的II型梭状细胞是了解得最好的。它们释放ATP来激活传入神经纤维上的嘌呤能受体。III型，或“突触前”细胞，检测酸性刺激并释放血清素和去甲肾上腺素。I型，或“胶质样”细胞是味蕾中最丰富的细胞，但也是最不为人所知的细胞。与神经系统中的神经胶质细胞类似，它们的膜紧密地包裹着其他味觉细胞，并表达ntpdes2，一种降解II型细胞释放的ATP的外ATP酶。最近的数据表明，I型细胞可能具有其他功能，包括Na+盐的转导和II型细胞的调节。不像II型和III型细胞

DESCRIPTION (provided by applicant): Taste buds are the transducing elements of gustatory sensation. As such, they represent the first step in the process by which nutritious substances, such as Na+ salts, sugars, and amino acids can be detected and distinguished from harmful substances, such as acids and toxic bitter compounds. Understanding the steps in this process may lead to therapeutic interventions that can be used to modulate food intake, a critical factor in controlling obesity. Each taste bud comprises three types of elongate taste cells and a population of basal cells. Understanding the functional role of each cell type is fundamental to understanding how each type of stimulus is detected and how this information is transmitted to the nervous system. Of the three types of fusiform cells, the Type II cells, which express the receptors and signaling effectors for bitter, sweet, and umami transduction, are the best understood. They release ATP to activate purinergic receptors on afferent nerve fibers. Type III, or "presynaptic" cells, detect sour stimuli and release serotonin and noradrenalin. Type I, or "glial-like" cells are the most abundant cells in the taste bud but also the least understood. Similar to glial cells in the nervous system, their membranes closely envelope other taste cells and they express NTPDase2, an ectoATPase that degrades ATP that is released from Type II cells. Recent data suggest that Type I cells may have additional functions, including transduction of Na+ salts and modulation of Type II cells. Unlike Type II and Type III cells, there are no fluorescent reporters for identification of Type I taste cells, making identification and functional characterization difficult. The proposed studies will utilize existing reagents and technology to develop a gene-targeted mouse that will express a nuclear-targeted fluorescent reporter (GFP) and Cre recombinase from the NTPDase2 promoter. Nuclear localization of GFP will insure that Type I cells can be distinguished from the other cell types, both within the bud and after isolation. Moreover, Cre recombinase will allow selective deletion of sequence from Type I taste cells, when these mice are crossed with mice carrying an appropriate "floxed" allele. In the first aim, we will validate the expression of the targeted allele, by crossing the mce to a commercially available Cre reporter line, Rosa26-tdTomato, which will express a red fluorescent reporter upon Cre-mediated excision of sequence flanked by loxp sites upstream of the reporter. In the second aim, we will utilize the mice to test the hypothesis that Type I cells are necessary for amiloride-sensitive salt taste. Whole cell recording will test whether Type I cells express functional amiloride- sensitive Na+ currents. Further, we will develop the methodology to specifically ablate type I taste cells, using the diphtheria toxin receptor targeted to Type I taste cells. In summary, these experiments will allow us define the Type I cell population and provide a new tool to dissect the functions of this most common taste cell type.