Role of the Bradykinin Pathway in Craniofacial Development

描述（由申请人提供）：本提案的总体目标是定义指导颅面发育的信号通路，长期目标是诊断和治疗异常。特别的重点是下颌，源自神经嵴，和嘴。口腔从“极前区”（EAD）发育而来，在那里外胚层和内胚层直接并列。缓激肽途径，以前只描述在成人，是局部必需的EAD，口腔形成。然而，这一途径对面部形成也有更全面的影响。我假设EAD是颅面组织者，通过分级缓激肽信号调节口腔和颅神经嵴发育，最终产生NO。有两个目的。目的1将描述CPN功能丧失对神经嵴形成的影响。CPN是缓激肽途径的介质，在EAD中表达，功能丧失导致口腔和神经嵴组织异常。我假设CPN信号调节颅神经嵴的决定，并作为面部中线的迁移停止信号。这些假设将在局部丧失CPN功能后使用反义寡核苷酸和在Sive实验室开发的面部移植试验进行测试。对神经嵴迁移的影响将使用荧光注射示踪剂进行谱系标记。目的是确定缓激素信号在口腔和神经嵴形成中的作用。CPN将配体缓激肽加工成arg缓激肽。两者都是活性配体，导致一氧化氮的产生。我假设缓激肽通过缓激肽受体以浓度依赖性的方式起作用，从而决定口腔和颅神经嵴。这些假设将通过将装载缓激肽的微球植入功能丧失的胚胎，并分析在距离微球不同距离处的缺陷纠正来验证。能对缓激肽信号作出反应的组织谱将通过分析缓激肽受体功能来确定。这项研究使用爪蟾作为分析颅面缺陷的理想模型，因为胚胎在母体外发育，而且发育中的面部很容易接近。可以获得数百个胚胎，这些胚胎可以进行快速检测，因为口腔和颌骨的前体在受精后36小时形成，并在3天内具有功能。大的胚胎允许显微操作，并且可以进行功能的增益和损失分析。在爪蟾和哺乳动物之间，面部结构和调节基因似乎是保守的，这表明从这项研究中获得的信息将与人类生物学直接相关。颅面畸形很普遍，每700个活产婴儿中就有1个出现，但大多数原因不明。这个项目是令人兴奋的，因为缓激肽信号在面部发育中的作用以前没有被描述过，EAD作为面部组织者的作用也没有被描述过。这些研究与NIDCR的任务相结合，有可能提供颅面缺损的病因，并为未来的早期微创治疗提供建议。

DESCRIPTION (provided by applicant): The broad goal of this proposal is to define signaling pathways that direct craniofacial development, with the long-term goal of diagnosing and treating abnormalities. Specific focus is on the jaws, derived from the neural crest, and on the mouth. The mouth develops from the "extreme anterior domain" (EAD), where the ectoderm and endoderm directly juxtapose. The Bradykinin pathway, previously described only in adults, is locally necessary in the EAD, for mouth formation. However, this pathway also has more global effects on face formation. I hypothesize that the EAD is a craniofacial organizer that regulates mouth and cranial neural crest development, through graded Bradykinin signaling, and culminating in NO production. There are two Aims. Aim 1 will characterize the effects of CPN loss of function on neural crest formation. CPN is a Bradykinin pathway mediator, expressed in the EAD, and loss of function results in abnormal mouth and neural crest tissues. I hypothesize that CPN signaling regulates cranial neural crest determination and serves as a migration stop signal at the facial midline. These hypotheses will be tested after local loss of CPN function using antisense oligonucleotides and a face transplant assay developed in the Sive lab. The effect on neural crest migration will use lineage labeling with fluorescent, injected tracers. Aim will determine the role of Bradykinin signaling on mouth and neural crest formation. CPN processes the ligand Bradykinin to desArgBradykinin. Both are active ligands and lead to nitric oxide NO production. I hypothesize that Bradykinins act in a concentration-dependent manner, through the Bradykinin receptor, to determine the mouth and cranial neural crest. These hypotheses will be tested by implanting beads loaded with Bradykinin peptides into loss of function embryos, and analyzing correction of defects at different distances from the bead. The spectrum of tissues that can respond to Bradykinin signaling will be determined by analysis of Bradykinin receptor function. This study uses the frog Xenopus, an ideal model for analysis of craniofacial defects, as embryos develop outside the mother, and as the developing face is readily accessible. Hundreds of embryos can be obtained, and these allow rapid assays, since mouth and jaw precursors form 36 hours after fertilization, and are functional by 3 days. The large embryos allow micromanipulation, and gain and loss of function assays can be performed. Facial structure and regulatory genes appear conserved between Xenopus and mammals, suggesting that the information gained from this study will be directly relevant to human biology. Craniofacial anomalies are prevalent, appearing in 1 out of 700 live births, yet most have unknown cause. This project is exciting since Bradykinin signaling in facial development has not previously been described, neither has the role of the EAD as a facial organizer. These studies interface with the NIDCR mission, and have potential to provide etiologies for craniofacial defects and suggest future early, minimally invasive treatments.