Function and Regulation of Sema3 Genes in Palate Development and Innervation

摘要 腭裂是一种常见的人类出生缺陷，并导致喂养，言语和听力方面的严重问题。 增加死亡率。虽然大多数腭裂患者接受手术修复，以恢复解剖完整 腭，约30%的儿童修复腭仍然经历显着的功能性腭缺陷和/或 咽喉功能障碍（VPD），损害言语并显著影响生活质量。口面 畸形，包括腭裂或高弓腭和扁平鼻梁，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/腭神经支配与腭的形态发生和咽喉功能密切相关。然而，在这方面， 目前对调节腭神经支配及其整合的分子机制尚不清楚 与调节腭形态发生的整体机制有关。我们发现缺乏锌的老鼠 指转录因子Osr 2表现出完全腭裂和腭神经支配的显著破坏。我们 以前的研究表明，Osr 2是腭架生长的关键内在调节因子， 几个主要的分子途径的功能，包括SHH信号和转录因子Pax 9- 介导的腭形态发生调节。通过RNA-seq转录组分析，我们发现， Sema 3基因家族的三个物理连锁成员Sema 3a、Sema 3d和Sema 3e的表达， 在Osr 2突变体胚胎发育中的腭间充质中显著上调。关于Sema 3 蛋白质最初被鉴定为中枢神经系统中轴突导向的负介质 并已被证明在许多其他发育过程中起着关键作用，包括神经 嵴迁移、内皮迁移和血管生成以及成骨细胞分化。我们的中央 假设在腭发育过程中，Sema 3基因表达的适当调节是至关重要的 不仅对腭神经支配而且对腭形态发生都有重要作用。最近，PLXND 1基因发生突变， SEMA 3蛋白的受体，以及一种复杂的染色体重排， SEMA 3D基因与Moebius综合征有关，这表明SEMA 3-PLXND 1 通路在人类颅神经发育和腭形态发生中也起着至关重要的作用。到 为了更好地了解Sema 3基因在腭发育和神经支配中的功能和调控，我们 将使用多组学方法，CRISPR/Cas9介导的基因组编辑和化合物 突变小鼠研究，以解开涉及Osr 2和Sema 3基因调控的分子网络 腭神经支配和形态发生的过程。这些研究的数据将填补长期存在的重大空白， 了解腭裂和VPD的发病机制，这将有助于显着提高 诊断，咨询，治疗和护理这些毁灭性的颅面患者的策略 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.