MOLECULAR LINKS FOR CILIOGENESIS AND LEFT RIGHT AXIS

气道上皮细胞高度分化为特定的细胞群，为宿主防御提供多种关键功能，包括粘膜纤毛清除。组织学上，这些气道细胞被分类为属于纤毛细胞，或几种不同类型的非纤毛细胞群之一。最近，在肺发育过程中，已经发现了少数可能调节上皮细胞分化的因素，但尚未发现调节纤毛细胞表型的分子。因此，我们克隆并鉴定了一种名为肝细胞核因子-3/叉头同源物4 （hhh -4）的转录因子，该转录因子在肺发育过程中早期在近端气道上皮细胞中表达。我们发现hhh -4的表达仅限于从鼻到细支气管的气道纤毛上皮细胞。通过在胚胎干细胞中靶向删除产生hhh -4缺陷小鼠，进一步表征了该因子。小鼠没有纤毛，并有倒位。这种表型反映了人类Kartagener综合征，并且首次证实了临床观察，即纤毛发生过程和左右（LR）轴形成是遗传相关的。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，但最近的数据已经导致了LR不对称的分子途径的构建，该途径被认为与纤毛功能有关。hhh -4突变小鼠的独特表型现在为进一步剖析LR途径和深入了解纤毛发生与LR测定的关系提供了有价值的工具。我们假设hhh -4在LR通路的启动中起核心作用，并提出了三个具体目标来评估这一作用：(1)确定hhh -4在发育过程中的早期空间和时间表达及其与胚胎结纤毛的关系。(2)通过对已知LR调控分子的表达特征和遗传分析，评估hhh -4在LR通路中的等级地位。(3)通过分析hhh -4在纤毛发生过程中的基因诱导，在体内通过确定hhh -4调控的基因，表征hhh -4在纤毛发生中的体外调控作用。来自这些研究的信息将有助于更好地理解LR不对称的机制，并确定hhh -4功能与纤毛发生之间的分子伙伴关系。最终，这将有助于理解遗传性肺部疾病和其他纤毛细胞改变的气道疾病中LR不对称障碍和纤毛发生缺陷。

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.