3-Dimensional modeling of basal cell function in pseudostratified epithelia

描述（由申请人提供）：生物学中的当前范例是存在于假复层上皮中的所谓基底细胞从不与器官的腔侧接触。与这一教条相反，我们最近发现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，Cell 135; 1108-1117，2008年12月）。这项研究提案是由这一范式转变的发现驱动的。我们发现，基底细胞扫描选定的上皮细胞的腔侧，并通过与其他上皮细胞的串扰来调节它们的功能。我们观察到“顶端到达”基底细胞在几个组织中的男性生殖道和上呼吸道，表明腔采样属性的基底细胞是一个普遍的现象。在这项研究计划中，我们将研究体内基底细胞的空间，时间和运动行为，我们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，我们将产生新的小鼠表达红色荧光蛋白，mRaspberry，只在基底细胞。我们将这些小鼠与我们实验室现有的小鼠杂交，包括在树突细胞中表达黄色荧光蛋白的CD 11 c-YFP小鼠，以及在附睾透明细胞、肺的非纤毛细胞和肾闰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，其中几种细胞类型将同时成像，在活的动物，使用活体多光子显微镜配备了小型化的目标，并在其中离子和一氧化氮流量将被测量在真实的时间使用选择性微电极。我们将集中在两个上皮，附睾，这是我们的研究计划的核心，和气管。附睾连接睾丸和输精管，参与精子的成熟和储存，因此在男性生育力中起着至关重要的作用。本申请的另一个目标组织，气管，不断地被外来过敏性和致病性物质侵入，并在气道和身体之间提供结构屏障。我们认为，基底细胞是探测附睾中调节男性生育能力的管腔因子和气管中吸入分子的前线传感器。更好地了解基底细胞的新型顶端感觉作用以及它们如何将其发现传递到相邻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治疗这些疾病的创新诊断和治疗干预措施。此外，这项研究计划将对我们对上皮细胞的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疾病中的干扰方式提供前所未有的见解。 公共卫生相关性：身体中的许多器官，包括生殖道和肺的器官，都由一个由形成上皮的细胞排列的小管系统组成，上皮是一种在器官的血液侧和由管形成的腔之间形成屏障的结构。普遍的观点是，这些上皮中所谓的“基底细胞”从不与充满液体或空气的腔（称为管腔）接触，但我们最近发现，这些细胞实际上延伸出细长的突起，扫描管腔，并通过将其发现传达给相邻细胞来调节器官功能。我们建议创建新的模型系统，其中不同上皮细胞类型的三维关系和功能（在活体动物中通过不同颜色的荧光标记物的存在来识别）可以被真实的实时监测，因为基底细胞检测并响应于出现在器官腔中的各种药物、激素、化学物质和病原体;我们获得的数据将为包括男性不育症、慢性阻塞性气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.