MOLECULAR LINKS FOR CILIOGENESIS AND LEFT RIGHT AXIS

呼吸道上皮细胞高度分化为特定的细胞群，为宿主防御提供了包括粘液纤毛清除在内的各种关键功能。组织学上，这些呼吸道细胞被归类为纤毛细胞，或几种不同类型的无纤毛细胞群之一。近年来，在肺发育过程中发现了一些可能调节上皮细胞分化的因子，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4(HfH-4)的转录因子，该转录因子在肺发育过程中早期在近端呼吸道上皮细胞中表达。我们发现hfh-4的表达仅限于从鼻子到细支气管处的呼吸道纤毛上皮细胞(S)。该因子的进一步特征是通过在胚胎干细胞中定向缺失来产生HfH-4缺陷小鼠。小鼠无纤毛，有内翻位。这一表型反映了人类Kartag‘s综合征，并首次证实了临床观察到纤毛发生和左右(LR)轴的形成过程存在遗传联系。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，该分子途径被认为与纤毛功能有关。HfH-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测定的关系提供了一个有价值的工具。我们假设HfH-4在LR通路的启动中起着中心作用，并提出了三个具体目标来评价这一作用：(1)确定HfH-4在发育中的早期时空表达及其与胚胎结节纤毛的关系。(2)通过已知的LR调节分子的表达特征和遗传分析，评估HfH-4在LR途径中的层级位置。(3)通过分析纤毛发生过程中的基因诱导和体内HfH-4调控基因的表达，研究HfH-4在体外纤毛发生调控中的作用。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，并确定将HfH-4功能与纤毛发生联系起来的分子伙伴。最终，这将有助于理解遗传性肺部疾病和其他具有纤毛细胞改变的呼吸道疾病中LR不对称的障碍和纤毛发生的缺陷。

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.