Mechanism of ephrin signaling in mammalian palatal fusion

描述(由申请者提供)：次级腭部未能在中线融合会导致腭裂，这是一种可怕的常见出生缺陷，只能通过手术治疗，并造成巨大的健康负担。我们的长期目标是了解腭裂融合的机制，以便更好地治疗腭裂。转化生长因子β3(Tgfss3)被认为是通过引起腭架之间的中线上皮缝(MES)的降解而形成融合间充质的融合腭部所必需的。然而，我们最近发现，在没有Tgfss 3的情况下，激活的ePhin-B信号是导致融合的必要条件和充分条件。肾上腺素反向信号通过涉及磷脂酰肌醇-3激酶(PI3K)的一种机制导致培养的腭部上皮细胞向间充质转化(EMT)。由于已知的肾上腺素也可介导细胞程序性死亡，而细胞凋亡是融合机制的主要部分，因此我们提出假设，即通过诱导内胚层转化和腭部上皮细胞的凋亡而导致腭部融合。我们将通过追求三个目标来检验这一假设。首先，我们将利用我们的发现，即ewitin-B2标记融合腭部的MES细胞，通过共聚焦成像实时跟踪这些细胞的命运。其次，我们将确定已知的腭部肾上腺素如何参与控制MES细胞迁移和凋亡的上皮-间充质相互作用。第三，我们将通过发现：1)与eaffin细胞结构域相互作用的信号蛋白，2)转导eaffin信号的特定PI3K通路，以及3)在细胞质和细胞核中eaffin与已知的腭上皮Tgfss3信号通路的交叉点，从而了解ePhin反向信号在融合中的分子机制。

DESCRIPTION (provided by applicant): Failure of the secondary palate to fuse at midline causes cleft palate, a frighteningly common birth defect that can only be treated with surgery and poses a tremendous health burden. Our long-term goal is to understand the mechanism of palatal fusion so as to better treat cleft palate. Transforming growth factor beta-3 (Tgfss3) is thought to be required to form a fused palate of confluent mesenchyme by causing degradation of the midline epithelial seam (MES) between palatal shelves. However, we recently discovered that activated ephrin-B signaling is necessary and sufficient to cause fusion in the absence of Tgfss3. Ephrin reverse signaling causes epithelial-to-mesenchymal transition (EMT) in cultured palate epithelial cells via a mechanism involving phosphotidyl inositol-3 kinase (PI3K). Because ephrins are also known to mediate programmed cell death, and apoptosis is a major part of the fusion mechanism, we propose the hypothesis that ephrin signaling causes palatal fusion through induction of EMT and apoptosis in palatal epithelia. We will test this hypothesis through pursuit of three aims. First, we will use our discovery that ephrin-B2 marks MES cells in fusing palates to track the fate of these cells in real time with confocal imaging. Second, we will determine how the known ephrins in the palate contribute to the epithelial-mesenchymal interactions that govern MES cell migration and apoptosis. Third, we will learn the molecular mechanism of ephrin reverse signaling in fusion by discovering: 1) the signaling proteins that interact with the ephrin cytodomain, 2) the specific PI3K pathway intermediates that transduce the ephrin signal, and 3) the intersection points in the cytoplasm and the nucleus between the ephrin and the known Tgfss3 signaling pathways in palatal epithelium.