Mechanism of ephrin signaling in mammalian palatal fusion

描述（由申请人提供）：腭裂是一种非常常见的出生缺陷，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融合的机制，以便更好地治疗腭裂。转化生长因子β -3 （Tgfss3）被认为是通过引起腭架之间中线上皮缝（MES）的降解来形成融合间质融合腭所必需的。然而，我们最近发现激活的ephrin-B信号是在缺乏Tgfss3的情况下引起融合的必要和充分条件。Ephrin反向信号通路通过磷脂酰肌醇-3激酶（PI3K）介导腭上皮细胞上皮向间质转化（EMT）。由于已知ephrin介导细胞程序性死亡，而细胞凋亡是融合机制的主要部分，我们提出了ephrin信号通过诱导腭上皮细胞EMT和细胞凋亡导致腭融合的假设。我们将通过追求三个目标来检验这一假设。首先，我们将利用我们发现的ephrin-B2标记融合腭中的MES细胞，通过共聚焦成像实时跟踪这些细胞的命运。其次，我们将确定上颚中已知的ephrins如何参与控制MES细胞迁移和凋亡的上皮-间质相互作用。第三，我们将通过发现：1)与ephrin细胞结构域相互作用的信号蛋白，2)转导ephrin信号的特异性PI3K通路中间体，以及3)腭上皮中ephrin与已知Tgfss3信号通路在细胞质和细胞核中的交点来了解融合中ephrin反向信号的分子机制。

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.