The role of ectodermal primary cilia in murine orofacial development

 描述(申请人提供)：初级纤毛是以微管为基础的细胞器，作为细胞触角，协调多个信号通路的转导。纤毛病是一类以失去初级纤毛为特征的疾病，常伴有口腔错构瘤和牙齿异常等口腔面部缺陷。初级纤毛是Gli蛋白翻译后加工为激活物(Glia)或抑制物(Glir)亚型的重要调节因子。Gli2主要作为转录激活因子(Gli2A)，而Gli3主要作为转录抑制因子(Gli3R)。Gli蛋白有许多转录靶点，包括与增殖、Shh信号和规范-Wnt信号有关的基因。颌面部发育依赖于增殖和Shh/Wnt信号通路的严格空间调控。这项拟议的工作侧重于了解口腔面部发育中外胚层初级纤毛的需求，并利用条件基因Kif3afl/fl；Crect。Kif3a是纤毛发生所必需的顺行鞭毛内蛋白，Crect在发育中的胚胎表面外胚层特异性地驱动Cre重组酶的活性。我的初步数据表明，Kif3afl/fl；Crect在口腔外胚层和间充质中分别有Shh和Lef1的异位表达。我假设外胚层初级纤毛调节下层间充质中的糖加工，以限制Shh和Canonical-Wnt途径的空间域的活性。在目标1中，我将确定初级纤毛的外胚层缺失是否会导致口腔间充质中依赖Gli2A的过度增殖。ShH途径的活性与细胞增殖和肿瘤的发生有关。Kif3afl/fl；Crect突变体在口腔外胚层异位表达Shh。为此，我假设Shh的异位表达是由于外胚层初级纤毛的丧失导致口腔间充质中依赖Gli2A的过度增殖。对于目标2，我将测试口腔外胚层上的初级纤毛是否需要依赖于Gli3R的限制牙齿间充质中的规范-Wnt信号。Kif3afl/fl；Crect突变体发育出异位牙芽，伴随着Lef1的扩展，Lef1是典型的Wnt信号读数，在牙齿间充质中。类似地，典范-Wnt信号功能获得突变体发育出多余的牙齿。已知Gli3R可以限制规范-Wnt信号。我假设外胚层初级纤毛的丧失会导致典型的-Wnt信号的增加，这是由于失去了Gli3R对通路拮抗剂的调节。作为一个整体，这项研究对于促进我们对纤毛病变患者口腔面部缺陷的细胞和分子病因学的理解具有重要意义。目前，这些患者缓解严重疾病的唯一途径 口腔面部缺陷就是外科手术。更深入地了解口腔面部纤毛疾病的发育机制对于开发替代治疗机会是必要的。

 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.