Development of periderm-specific Cre mouse lines to study palatogenesis

开发周皮特异性 Cre 小鼠品系以研究腭发育

• 批准号: 10112279
• 负责人: VESA M KAARTINEN
• 金额: $ 7.8万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-20 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10112279
• 关键词: Adherence; Adhesions; Biological Assay; Biology; CRISPR/Cas technology; Cells; Cleft Palate; Communities; Confocal Microscopy; Congenital Abnormality; Cre driver; Cytokeratin; Data; Development; Epithelial; Event; Failure; Future; Goals; Histologic; Human; Knock-in; Knock-in Mouse; Knowledge; Ligands; LoxP-flanked allele; Medial; Molecular; Mus; Palate; Periderm; Phase; Play; Process; Reporter; Role; Secondary Palate; Signal Transduction; Syndrome; Tamoxifen; Therapeutic; Time; Transforming Growth Factor beta; arm; base; cell type; experimental study; improved; malformation; mutant; novel; palatal shelves; palatogenesis; premature; prevent; receptor; tool; transforming growth factor beta3

Abstract Our long-term goal is to understand the detailed molecular mechanisms and cellular events that control palate development (palatogenesis). Failure in palatogenesis results in cleft palate, which is one of the most common birth defects in humans. During the final phases of palatogenesis, the palatal periderm (PD, a protective outer cell layer that prevents premature adhesion) must be removed in order for the medial edge epithelia (MEE) to properly adhere, which is prerequisite for appropriate palatogenesis. The previous studies have shown that transforming growth factor-β3 (TGFβ3), plays an irreplaceable role in palate development, both in mice and humans, by regulating the fate of the MEE and adjacent periderm in fusing palatal shelves. Our recent data were the first to suggest that Tgfb3 is necessary for periderm degeneration. However, deeper understanding of the role of periderm-specific signaling events during palate adherence and fusion has been hampered by the fact that until now, there have been no satisfactory periderm-specific Cre-driver lines available for the scientific community. In addition, the previous studies employing a commonly used K14Cre driver line to examine the epithelium-specific signaling in palatogenesis have ignored the fact that K14Cre does not recombine in the periderm, and thus these studies have failed to reveal relevant cell type-specific details of this important signaling mechanism. Due to these impediments, several pertinent questions have remained unanswered. Our present preliminary data demonstrate that the cytokeratin-6a (K6a) locus can effectively be used to generate specific and robust periderm-specific Cre driver lines. We hypothesize that the periderm-specific K6a- based Cre drivers allow us to define the PD-fate and establish the PD-specific cell-autonomous role for TGF-β signaling in induction of adherence between apposing palatal shelves during palatogenesis. The details of these mechanisms will be determined through execution of two specific aims. In Aim 1, we propose to determine the fate of periderm cells during palatogenesis using a novel inducible periderm-specific Cre line; and in Aim 2, we propose to determine the cell-autonomous role of TGF-β signaling in the palatal periderm during palatal epithelial fusion. Successful completion of the proposed experiments will provide improved PD-specific tools for the scientific community and define the cell-autonomous role of TGF-β signaling in the PD; this knowledge may contribute in development of more advanced therapeutic strategies in the future.

摘要 我们的长期目标是了解详细的分子机制和控制 腭部发育(腭部发育)。腭裂是导致腭裂的主要原因之一。 人类常见的先天缺陷。在腭裂形成的最后阶段，腭周皮(Pd，a 防止过早粘连的保护性外层细胞)必须去除，以便内侧边缘 上皮细胞(MEE)正确地附着，这是适当的腭裂发生的先决条件。前人的研究 已有研究表明，转化生长因子-β-3(转化生长因子-β-3)在腭部发育中起着不可替代的作用。 在小鼠和人类中，通过调节MEE和邻近的周皮在融合的腭架中的命运。 我们最近的数据首次表明TGFB3是周皮退化所必需的。然而， 更深入地了解周皮特异性信号事件在腭部粘连和融合过程中的作用 到目前为止，还没有令人满意的周皮特异性CRE驱动器系 可供科学界使用。此外，之前的研究使用了常用的K14Cre 在腭裂发生中研究上皮特异性信号的驱动线忽略了K14Cre确实存在的事实 没有在周皮中重组，因此这些研究未能揭示相关的细胞类型特定的细节 这一重要的信号机制。由于这些障碍，仍然存在几个相关的问题。 无人接听。 我们目前的初步数据表明，细胞角蛋白-6a(K6a)基因座可以有效地用于 生成特定和健壮的Perderm特定的Cre驱动程序行。我们假设周皮特异的K6a- 基于Cre的驱动程序允许我们定义PD-命运，并为转化生长因子-β建立PD特定的细胞自治角色 在腭裂形成过程中，在相对的腭架之间诱导粘连的信号。详细内容如下： 这些机制将通过执行两个具体目标来确定。在目标1中，我们建议 用一种新的可诱导的周皮特异性Cre系确定腭裂发生过程中周皮细胞的命运； 在目标2中，我们建议确定转化生长因子-β信号在腭周胚层中的细胞自主作用。 在腭部上皮融合过程中。 拟议实验的成功完成将为以下方面提供改进的PD专用工具 科学界并确定转化生长因子-β信号在PD中的细胞自主作用；这一知识可能 为未来更先进的治疗策略的发展做出贡献。