3-Dimensional modeling of basal cell function in pseudostratified epithelia

描述(申请人提供)：目前生物学中的一个范例是，所谓的基底细胞，存在于假复层上皮细胞中，永远不会与器官的管腔一侧接触。与这一教条相反，我们最近表明，这些细胞延伸细长的身体突起，穿过紧密连接屏障到达管腔(Shum等人。牢房135；1108-1117,2008年12月)。这项研究建议是由这一范式转换的发现推动的。我们发现，基底细胞扫描选定的上皮细胞的管腔一侧，并通过与其他上皮细胞的串扰来调节它们的功能。我们在男性生殖道和上呼吸道的几个组织中观察到“顶端”的基底细胞，表明基底细胞的腔采样特性是一种普遍的现象。在这项研究中，我们将研究体内基底细胞的空间、时间和运动行为，并将表征基底细胞和相邻细胞之间的细胞间通讯网络。为了做到这一点，我们将培育出在基底细胞中专门表达红色荧光蛋白mRaspberry的新型小鼠。我们将把这些小鼠与我们实验室现有的小鼠进行杂交，包括在树突状细胞中表达黄色荧光蛋白的CD11c-YFP小鼠，以及在附睾透明细胞、肺非纤毛细胞和肾脏嵌入细胞中表达绿色荧光蛋白EGFP的B1-EGFP小鼠。这将产生一种新的动物模型，在活体动物中，将使用配备微型物镜的活体多光子显微镜同时对几种细胞类型进行成像，并将使用选择性微电极实时测量离子和一氧化氮的通量。我们将专注于两种上皮细胞，附睾，这是我们研究计划的核心，以及气管。附睾连接睾丸和输精管，参与精子的成熟和储存，因此对男性生育能力起着至关重要的作用。这种应用的另一个目标组织是气管，它不断受到外来致敏和致病物质的侵袭，并在呼吸道和身体之间提供了结构性屏障。我们认为，基底细胞是前线的传感器，探测调节附睾中男性生育能力的管腔因子，并吸入气管中的分子。更好地理解基底细胞新的尖端感觉作用以及它们如何将研究结果传递给邻近细胞，将有助于确定男性不育和肺部疾病(包括哮喘、慢性阻塞性肺疾病(COPD)和囊性纤维化(CF))的病理生理机制。因此，监测和解码附睾和气管中的细胞间对话将促进对这些疾病的创新诊断和治疗干预。此外，这项研究计划将对我们对上皮细胞的总体理解产生更广泛的影响，因为这里产生的数据将为复杂组织建立的通信网络以及它如何在疾病中受到干扰提供前所未有的见解。 与公共卫生相关：人体内的许多器官，包括生殖道和肺部的器官，都由一个由形成上皮的细胞构成的小管系统组成，这种结构在器官的血液侧和由管子形成的空腔之间形成屏障。流行的观点是，这些上皮细胞中的所谓“基底细胞”永远不会与充满液体或空气的腔(称为管腔)接触，但我们最近表明，这些细胞实际上延伸了细长的投射，扫描管腔，并通过与相邻细胞交流他们的发现来调节器官功能。我们建议创建新的模型系统，在该系统中，随着基底细胞检测并对器官腔中出现的各种药物、激素、化学物质和病原体做出反应，可以实时监测不同上皮细胞类型的三维关系和功能(通过不同颜色的荧光标记在活体动物中识别)；我们产生的数据将为包括男性不育症、慢性阻塞性呼吸道疾病和囊性纤维化在内的疾病提出新的诊断和治疗策略。

DESCRIPTION (provided by applicant): A current paradigm in biology is that so-called basal cells, present in pseudostratified epithelia, are never in contact with the luminal side of an organ. In contrast to this dogma, we recently showed that these cells extend slender body projections that cross the tight-junction barrier to reach the lumen (Shum et al. Cell 135; 1108-1117, Dec 2008). This research proposal is driven by this paradigm-shifting discovery. We found that basal cells scan the luminal side of selected epithelia and modulate their function via crosstalk with other epithelial cells. We observed "apical-reaching" basal cells in several tissues of the male reproductive and upper respiratory tracts indicating that the luminal sampling property of basal cells is a generalized phenomenon. In this research program, we will examine the spatial, temporal and motional behavior of basal cells in vivo and we will characterize the intercellular communication networks between basal cells and adjacent cells. To do so, we will generate novel mice expressing the red fluorescent protein, mRaspberry, in basal cells exclusively. We will cross-breed these mice with current mice available in our laboratory, including CD11c-YFP mice that express the yellow fluorescent protein in dendritic cells, and B1-EGFP mice that express the green fluorescent protein EGFP in epididymal clear cells, non- ciliated cells of the lung, and kidney intercalated cells. This will generate a novel animal model in which several cell types will be imaged simultaneously, in live animals, using intravital multiphoton microscopes equipped with miniaturized objectives, and in which ionic and nitric oxide fluxes will be measured in real time using selective microelectrodes. We will focus on two epithelia, the epididymis, which is at the core of our research program, and the trachea. The epididymis, which connects the testis to the vas deferens, is involved in the maturation and storage of spermatozoa and, therefore, plays a crucial role in male fertility. The other target tissue of this application, the trachea, is constantly invaded by foreign allergenic and pathogenic substances, and provides a structural barrier between the airway and the body. We propose that basal cells are front-line sensors that probe luminal factors that regulate male fertility in the epididymis, and inhaled molecules in the trachea. A better understanding of the novel apical sensory role of basal cells and how they transmit their findings to adjacent cells will help define the pathophysiological mechanisms underlying male infertility, and diseases of the lung, including asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). Monitoring and decoding intercellular conversations in the epididymis and trachea will, thus, promote innovative diagnostic and therapeutic interventions for the treatment of these diseases. In addition, this research program will have broader implications for our understanding of epithelia in general, as data generated here will provide unprecedented insights into the communication network established by complex tissues and on how it is perturbed in disease. PUBLIC HEALTH RELEVANCE: Many organs in the body, including those of the reproductive tract and the lungs, are comprised of a system of tubules lined by cells that form an epithelium, a structure that creates a barrier between the blood side of the organ and the cavity formed by the tube. The prevailing view is that so-called "basal cells" in these epithelia are never in contact with the fluid or air-filled cavity (known as the lumen), but we showed recently that these cells in fact extend long, slender projections that scan the lumen and modulate organ function by communicating their findings to adjacent cells. We propose to create new model systems in which the three-dimensional relationship and functions of different epithelial cell types (identified in live animals by the presence of different colored fluorescent markers) can be monitored in real time as the basal cells detect and respond to various drugs, hormones, chemicals and pathogens that appear in the cavity of the organ; the data we generate will suggest new diagnostic and therapeutic strategies for diseases including male infertility, chronic obstructive airway disease and cystic fibrosis.