MOLECULAR LINKS FOR CILIOGENESIS AND LEFT RIGHT AXIS

气道上皮细胞高度分化成特定的细胞群，为宿主防御提供多种关键功能，包括粘膜纤毛清除。 在组织学上，这些气道细胞已被分类为属于纤毛，或几种不同类型的非纤毛细胞群之一。 近年来，虽然已经发现了一些在肺发育过程中调控上皮细胞分化的因子，但还没有发现调控纤毛细胞表型的分子，因此我们克隆并鉴定了一个转录因子，即肝细胞核因子3/叉头同源物4（hepatocyte nuclear factor-3/forkhead homologue 4，HFH-4），它在肺发育过程中早期表达于近端气道上皮细胞。 我们发现HFH-4表达仅限于从鼻到细支气管的气道中的纤毛上皮细胞。 通过在胚胎干细胞中靶向缺失产生HFH-4缺陷小鼠来进一步表征该因子。 小鼠无纤毛，并有内脏逆位。 这种表型反映了人类Kartagener综合征，并且首次证实了临床观察结果，即纤毛发生和左右（LR）轴形成的过程是遗传相关的。 虽然哺乳动物纤毛发生的分子机制在很大程度上是未知的，最近的数据已经导致LR不对称性的分子途径的构建，这是pueretically连接到纤毛功能。 HFH-4突变小鼠的独特表型现在提供了一个有价值的工具，以进一步剖析LR途径，并深入了解纤毛发生与LR测定的关系。 我们假设HFH-4在LR通路的启动中起着核心作用，并提出了三个具体的目标来评估这种作用：（1）确定HFH-4在发育过程中的早期时空表达以及与胚胎结纤毛的关系。 (2)通过表征已知LR调节分子的表达和遗传分析，评估HFH-4在LR途径中的分级位置。 (3)通过分析纤毛发生过程中的基因诱导来表征HFH-4在体外纤毛发生调节中的作用，并通过测定HFH-4调节的基因来表征HFH-4在体内纤毛发生调节中的作用。 从这些研究中获得的信息将导致LR不对称机制的更好理解，并确定连接HFH-4功能纤毛发生的分子伴侣。 最终，这将有助于了解LR不对称性疾病和遗传性肺病和其他纤毛细胞改变的气道疾病中纤毛发生缺陷。

Airway epithelial cells are highly differentiated into specific cell populations providing a variety of critical functions for host defense including mucociliary clearance. Histologically, these airway cells have been classified as belonging to ciliated, or one of several different types of non-ciliated cell populations. Recently, a small number of factors that may regulate epithelial cell differentiation during lung development have been identified however, molecules that regulate the ciliated cell phenotype have not been found. Accordingly, we have cloned and characterized a transcription factor called hepatocyte nuclear factor-3/forkhead homologue 4 (HFH-4) that is expressed early in proximal airway epithelial cells during lung development. We found HFH-4 expression was restricted to ciliated epithelial cells in the airway, from the nose to the bronchiole(s). This factor was further characterized by generating HFH-4 deficient mice by targeted deletion in embryonic stem cells. Mice had no cilia and had situs inversus. This phenotype mirrors the human Kartagener's syndrome and is the first confirmation of the clinical observation that the processes of ciliogenesis and left-right (LR) axis formation are genetically linked. While molecular mechanisms of mammalian ciliogenesis are largely unknown, recent data has led to the construction of a molecular pathway for LR asymmetry that is putatively linked to cilia function. The unique phenotype of the HFH-4 mutant mouse now provides a valuable tool to further dissect the LR pathway and to gain insight into the relationship of ciliogenesis to LR determination. We hypothesize that HFH-4 plays a central role in the initiation of the LR pathway, and propose three specific aims to evaluate this role: (1) Determine the early spatial and temporal expression of HFH-4 in development and the relationship to cilia at the embryonic node. (2) Assess the hierarchical position of HFH-4 in the LR pathway by characterization of expression of known LR regulatory molecules and by genetic analysis. (3) Characterize the role of HFH-4 in the regulation of ciliogenesis in vitro by analysis of gene induction during ciliogenesis and in vivo by determination of genes regulated by HFH-4. Information from these studies will lead to a greater understanding of mechanisms of LR asymmetry and identify molecular partners that link HFH-4 function to ciliogenesis. Ultimately, this will contribute to an understanding of disorders in LR asymmetry and defects in ciliogenesis in genetic lung diseases and other airway diseases with alterations in ciliated cells.