Role of the Bradykinin Pathway in Craniofacial Development

描述（由申请人提供）：该提案的广泛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。具体的重点是在颌骨，来自神经嵴，并在嘴。口部从外胚层和内胚层直接并列的“极前区”（EAD）发育而来。缓激肽通路，以前只在成人中描述过，在EAD中是局部必需的，用于口的形成。然而，这一途径对面部形成也有更多的全局影响。我假设EAD是一个颅面组织者，通过分级缓激肽信号传导调节口腔和颅神经嵴发育，并最终产生NO。有两个目标。目的1将描述CPN功能丧失对神经嵴形成的影响。CPN是缓激肽通路介质，在EAD中表达，并且功能丧失导致异常的口和神经嵴组织。我推测，CPN信号调节颅神经嵴的决定，并作为一个迁移停止信号在面部中线。这些假设将在CPN功能局部丧失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的谱系标记。目的将确定缓激肽信号传导在嘴和神经嵴形成中的作用。CPN将配体缓激肽加工成去精氨酸缓激肽。两者都是活性配体并导致一氧化氮NO的产生。我假设缓激肽以浓度依赖性方式通过缓激肽受体起作用，以确定口腔和颅神经嵴。将通过将加载有缓激肽肽的珠植入功能丧失的胚胎中，并分析在距珠不同距离处缺陷的校正来测试这些假设。可响应于缓激肽信号传导的组织谱将通过缓激肽受体功能的分析来确定。这项研究使用了青蛙非洲爪蟾，一个理想的模型，分析颅面缺陷，胚胎发育外的母亲，并作为发展中的脸是很容易接近。可以获得数百个胚胎，这些胚胎可以进行快速检测，因为口和颌的前体在受精后36小时形成，并在3天内发挥作用。大胚胎允许显微操作，并且可以进行功能测定的获得和丧失。面部结构和调控基因在非洲爪蟾和哺乳动物之间是保守的，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以前没有被描述过，也没有EAD作为面部组织者的作用。这些研究与NIDCR的使命相结合，有可能为颅面缺损提供病因学，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.