Role of the Bradykinin Pathway in Craniofacial Development

描述(由申请人提供)：这项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。具体的焦点是源自神经脊部的颌骨和嘴巴。口腔由外胚层和内胚层直接并列的“极前区”(EAD)发育而来。以前只在成人中描述的缓激肽途径，在EAD中是局部必需的，用于口腔形成。然而，这种途径对面孔的形成也有更多的全局性影响。我推测，EAD是一个颅面组织者，通过分级的缓激肽信号调节嘴巴和脑神经脊线的发育，最终不产生任何东西。有两个目标。目的1描述CPN功能丧失对神经脊形成的影响。CPN是一种缓激肽途径的介导物，在EAD中表达，功能丧失会导致口腔和神经脊组织的异常。我推测，CPN信号调节着脑神经脊线的确定，并在面部中线起着迁移停止信号的作用。这些假说将在Cpn功能局部丧失后使用反义寡核苷酸和Sive实验室开发的面部移植实验进行验证。对神经脊迁移的影响将使用带有荧光注射示踪剂的谱系标记。目的将确定缓激肽信号在口腔和神经脊形成中的作用。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，并导致一氧化氮的产生。我推测，缓激肽通过缓激肽受体以一种浓度依赖的方式起作用，决定口腔和脑神经的波峰。这些假说将通过将装载缓激肽的珠子植入丧失功能的胚胎中，并分析在距离珠子不同距离处缺陷的纠正来检验。能够对缓激肽信号做出反应的组织的光谱将通过对缓激肽受体功能的分析来确定。这项研究使用了非洲爪蛙，这是分析头面部缺陷的理想模型，因为胚胎在母亲之外发育，而且发育中的脸很容易接触到。可以获得数百个胚胎，这些胚胎可以进行快速分析，因为口腔和下巴前体在受精后36小时形成，并在3天后起作用。大胚胎可以进行显微操作，并可以进行功能获得和丧失的分析。非洲爪哇和哺乳动物之间的面部结构和调控基因似乎是保守的，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。颅面畸形很常见，每700名活产儿中就有1名出现，但大多数原因不明。这个项目令人兴奋，因为缓激肽信号在面部发育中的作用以前从未被描述过，EAD作为面部组织者的作用也是如此。这些研究与NIDCR的任务相结合，有可能提供头面部缺陷的病因，并建议未来的早期、微创治疗。

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.