Function and Regulation of Sema3 Genes in Palate Development and Innervation

摘要 腭裂是人类常见的先天缺陷，会在进食、言语和营养方面造成严重问题。 死亡率上升。虽然大多数腭裂患者都会接受手术修复，以恢复解剖结构的完整 大约30%的修复后的儿童仍然经历着严重的功能性腭裂和/或 腭咽闭合功能障碍(VPD)，它损害语音并显著影响生活质量。口腔面 畸形，包括腭裂或高弓，鼻桥扁平，以及鼻构音障碍是主要的 先天性面瘫的特征，如Moebius综合征(OMIM 157900)，表明 面/腭神经支配与腭部形态发生和腭咽闭合功能密切相关。然而， 目前尚不清楚调节上颌神经及其整合的分子机制。 具有调节腭部形态发生的整体机制。我们发现缺乏锌的小鼠 手指转录因子OSR2表现为完全性腭裂和显著的腭神经支配中断。我们 以前证明OSR2是腭架生长的一个关键的内在调节因子，并整合了 几个主要分子通路的功能，包括SHH信号和转录因子Pax9- 对腭部形态发生的中介调节。通过RNA-seq转录组图谱，我们发现 Sema3基因家族的三个物理连锁成员Sema3a、Sema3d和Sema3e的表达， 在OSR2突变胚胎中，OSR2基因在发育中的腭间充质中显著上调。《三生三世》 蛋白质最初被认为是中枢神经系统中轴突引导的负性中介。 自那以后，已被证明在许多其他发育过程中发挥着关键作用，包括神经 牙冠迁移、内皮细胞迁移和血管生成、成骨细胞分化。我们的中央 假设在腭部发育过程中，对Sema3基因表达的适当调控是至关重要的。 不仅用于腭部的神经支配，而且还用于腭部的形态发生。最近PLXND1的突变，它编码 SEMA3蛋白的受体，以及破坏SEMA3A和SEMA3A的复杂染色体重排 Sema3d基因与Moebius综合征相关，提示SEMA3-PLXND1 通路在人类脑神经发育和腭部形态发生中也起着至关重要的作用。至 为了更好地了解Sema3基因在腭部发育和神经支配中的功能和调节，我们 将使用多种组学方法、CRISPR/Cas9介导的基因组编辑和复合 突变小鼠研究解开涉及OSR2和Sema3基因调控的分子网络 上颚神经支配和形态发生的关系。这些研究的数据将填补长期存在的一个主要空白 了解腭裂和VPD的发病机制，有助于显著改善 颅面创伤患者的诊断、咨询、治疗和护理策略 条件。

Abstract Cleft palate is a common birth defect in humans and causes significant problems in feeding, speech, and increased mortality. While most cleft palate patients receive surgical repair to restore an anatomically intact palate, about 30% of children with a repaired palate still experience significant functional palate deficit and/or velopharyngeal dysfunction (VPD), which impairs speech and significantly affects quality of life. Orofacial malformations, including cleft or high-arched palate and flattened nasal bridge, and nasal dysarthria are major features of congenital facial palsy conditions such as the Moebius syndrome (OMIM 157900), suggesting that facial/palatal innervation is intimately linked to palate morphogenesis and velopharyngeal function. However, nothing is currently known about the molecular mechanisms regulating palate innervation and their integration with the overall mechanisms regulating palate morphogenesis. We have discovered that mice lacking the zinc finger transcription factor Osr2 exhibit complete cleft palate and dramatic disruption of palatal innervation. We previously demonstrated that Osr2 is a key intrinsic regulator of palatal shelf growth and integrates the functions of several major molecular pathways, including SHH signaling and the transcription factor Pax9- mediated regulation of palate morphogenesis. Through RNA-seq transcriptome profiling we found that expression of three physically linked members of the Sema3 gene family, Sema3a, Sema3d, and Sema3e, was significantly upregulated in the developing palatal mesenchyme in Osr2 mutant embryos. The Sema3 proteins were originally identified as negative mediators of axonal guidance in the central nervous system and have since been shown to play crucial roles in many other developmental processes, including neural crest migration, endothelial migration and vasculogenesis, and osteoblast differentiation. Our central hypothesis is that proper regulation of expression of the Sema3 genes during palate development is crucial for not only palate innervation but also palate morphogenesis. Recently mutations in PLXND1, which encodes a receptor for SEMA3 proteins, and a complex chromosomal rearrangement disrupting both the SEMA3A and SEMA3D genes, have been associated with Moebius syndrome, suggesting that the SEMA3-PLXND1 pathway plays crucial roles in cranial nerve development and palate morphogenesis in humans as well. To better understand the function and regulation of the Sema3 genes in palate development and innervation, we will use a combination of multi-omics approaches, CRISPR/Cas9-mediated genome editing, and compound mutant mouse studies to unravel the molecular network involving Osr2 and the Sema3 genes in the regulation of palate innervation and morphogenesis. Data from these studies will fill a long standing major gap in the understanding of pathogenic mechanisms of cleft palate and VPD, which will help to significantly improve strategies for diagnosis, counseling, treatment and care of patients with these devastating craniofacial conditions.