The role of ectodermal primary cilia in murine orofacial development

 描述（由申请人提供）：初级纤毛是基于微管的细胞器，用作细胞触角并协调多种信号转导途径。纤毛病是一类以初级纤毛缺失为特征的疾病，常伴有口面部缺陷，如口腔错构瘤和牙齿畸形。初级纤毛是Gli蛋白翻译后加工成其激活子（GliA）或阻遏子（GliR）同种型的重要调节剂。Gli 2主要作为转录激活因子（Gli 2A）发挥作用，而Gli 3主要作为转录抑制因子（Gli 3R）发挥作用。Gli蛋白具有许多转录靶点，包括对增殖、Shh信号传导和经典Wnt信号传导重要的基因。口面部发育依赖于增殖和Shh/Wnt信号通路的紧密空间调节。这项工作的重点是了解外胚层初级纤毛在orofacial发展的要求，并利用条件敲除小鼠Kif 3afl/fl;Crect。Kif 3a是纤毛发生所需的顺行鞭毛内蛋白，Crect特异性地在发育胚胎的表面外胚层中驱动Cre重组酶活性。我的初步数据表明Kif 3afl/fl;Crect分别在口腔外胚层和间充质中异位表达Shh和Lef 1。我假设外胚层初级纤毛调节GLI处理在底层间充质限制空间域的Shh和经典的Wnt途径的活动。在目标1中，我将确定初级纤毛的外胚层损失是否会导致Gli 2A依赖的口腔间充质过度增殖。Shh通路活性与增殖和肿瘤发生增加相关。Kif 3afl/fl;Crect突变体在口腔外胚层异位表达Shh。为了这个目的，我假设，异位Shh表达由于外胚层初级纤毛的损失导致Gli 2A依赖性过度增殖的口腔间充质。对于目标2，我将测试牙齿间充质中经典Wnt信号传导的Gli 3R依赖性限制是否需要口腔外胚层上的初级纤毛。Kif3afl/fl; Crect突变体发育异位牙芽，这是伴随着扩大的Lef 1，读出典型的Wnt信号，在牙齿间充质。类似地，典型的Wnt信号功能获得性突变体会产生多生牙。已知Gli 3R限制经典Wnt信号传导。我推测，外胚层初级纤毛的损失导致增加的经典Wnt信号转导途径拮抗剂的Gli 3R调节的损失。总的来说，这项研究对于促进我们对纤毛疾病患者口面部缺陷的细胞和分子病因学的理解是重要的。目前，这些患者缓解严重疾病的唯一途径 口面缺损是外科手术更深入地了解口面纤毛病变的发展机制是必要的替代治疗机会的发展。

 DESCRIPTION (provided by applicant): Primary cilia are microtubule-based organelles that serve as cellular antennae and coordinate the transduction of multiple signaling pathways. Individuals with ciliopathies, a class of diseases characterized by loss of primary cilia, frequenty present with orofacial defects such as oral hamartomas and dental abnormalities. Primary cilia are important regulators of the post-translational processing of Gli proteins into either their activator (GliA) or repressor (GliR) isoforms. Gli2 primarily functions as a transcriptional activator (Gli2A), while Gli3 is primarily a transcriptional repressor (Gli3R). Gli proteins have many transcriptional targets, including genes important for proliferation, Shh signaling, and canonical-Wnt signaling. Orofacial development depends upon tight spatial regulation of proliferation and Shh/Wnt signaling pathways. This proposed work focuses on understanding the requirement of ectodermal primary cilia in orofacial development and utilizes the conditional mouse knockout Kif3afl/fl;Crect. Kif3a is an anterograde intraflagellar protein that is required fo ciliogenesis, and Crect drives Cre recombinase activity specifically in the surface ectoderm of the developing embryo. My preliminary data indicates that Kif3afl/fl;Crect have ectopic Shh and Lef1 expression in the oral ectoderm and mesenchyme, respectively. I hypothesize ectodermal primary cilia regulate Gli-processing in the underlying mesenchyme to restrict spatial domains of Shh and canonical-Wnt pathway activity. In Aim 1, I will determine if ectodermal loss of primary cilia causes Gli2A-dependent hyperproliferation in the oral mesenchyme. Shh pathway activity is associated with increased proliferation and tumorigenesis. Kif3afl/fl;Crect mutants ectopically express Shh in the oral ectoderm. For this aim, I hypothesize that ectopic Shh expression due to loss of ectodermal primary cilia causes Gli2A-dependent hyperproliferation in the oral mesenchyme. For Aim 2, I will test if primary cilia on the oral ectoderm are required for Gli3R-dependent restriction of canonical-Wnt signaling in dental mesenchyme. Kif3afl/fl;Crect mutants develop ectopic tooth buds, which are accompanied by an expansion of Lef1, a readout for canonical-Wnt signaling, in the dental mesenchyme. Similarly, canonical-Wnt signaling gain-of-function mutants develop supernumerary teeth. Gli3R is known to restrict canonical-Wnt signaling. I hypothesize that loss of ectodermal primary cilia results in increased canonical-Wnt signaling due to loss of Gli3R regulation of pathway antagonists. As a whole, this study is important for advancing our understanding of the cellular and molecular etiology of orofacial defects in ciliopathic patients. Currently, the only avenue these patients have to alleviate severe orofacial defects is surgery. A deeper understanding of the developmental mechanisms of orofacial ciliopathies is necessary for the development of alternate therapeutic opportunities.