Role of the Bradykinin Pathway in Craniofacial Development

缓激肽通路在颅面发育中的作用

• 批准号: 8904316
• 负责人: Laura Anne Jacox
• 金额: $ 4.32万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2016-05-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8904316
• 关键词: Adult; Anterior; Anterior nares; Antisense Oligonucleotides; Biological Assay; Bradykinin; Bradykinin Receptor; Cell Death; Cell Proliferation; Cells; Cephalic; Complex; Congenital Abnormality; Craniofacial Abnormalities; Data; Defect; Development; Diagnosis; Ectoderm; Elements; Embryo; Endoderm; Etiology; Face; Fertilization; Future; Goals; Hour; Human Biology; Implant; In Situ Hybridization; Jaw; Kininogens; Label; Lead; Ligands; Live Birth; Mammals; Mediator of activation protein; Micromanipulation; Migration Assay; Mission; Modeling; Mothers; National Institute of Dental and Craniofacial Research; Neural Crest; Nitric Oxide; Oral cavity; Pathway interactions; Peptides; Phenotype; Process; Production; Rana; Regulator Genes; Role; Signal Pathway; Signal Transduction; Structure; Testing; Tissues; Tracer; Xenopus; blood pressure regulation; craniofacial; craniofacial development; facial transplantation; in vivo; loss of function; migration; minimally invasive; receptor expression; receptor function

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.

描述（由申请人提供）：该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。特别关注的是源自神经嵴的下颌和嘴巴。嘴从“最前部区域”（EAD）发育而来，外胚层和内胚层直接并置。之前仅在成人中描述的缓激肽途径在 EAD 中对于口腔形成是局部必需的。然而，这条通路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。目标 1 将描述 CPN 功能丧失对神经嵴形成的影响。 CPN 是一种缓激肽途径介质，在 EAD 中表达，功能丧失会导致口腔和神经嵴组织异常。我假设 CPN 信号调节颅神经嵴决定并充当面部中线的迁移停止信号。这些假设将在 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检测进行测试。对神经嵴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对口腔和神经嵴形成的作用。 CPN 将配体缓激肽加工成 desArgBradykinin。两者都是活性配体并导致一氧化氮 NO 的产生。我假设缓激肽通过缓激肽受体以浓度依赖性方式发挥作用，以确定口腔和颅神经嵴。这些假设将通过将载有缓激肽肽的珠子植入丧失功能的胚胎中并分析距珠子不同距离处的缺陷校正来进行测试。可对缓激肽信号传导作出反应的组织范围将通过缓激肽受体功能的分析来确定。这项研究使用青蛙非洲爪蟾，这是分析颅面缺陷的理想模型，因为胚胎在母体外发育，并且发育中的面部很容易接近。可以获得数百个胚胎，并且可以进行快速检测，因为口和颌前体在受精后 36 小时内形成，并在 3 天后发挥功能。大胚胎允许显微操作，并且可以进行功能获得和丧失的检测。非洲爪蟾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。颅面畸形很普遍，每 700 名活产儿中就有 1 人出现颅面畸形，但大多数原因不明。这个项目令人兴奋，因为以前从未描述过面部发育中的缓激肽信号传导，也没有描述过 EAD 作为面部组织者的作用。这些研究与 NIDCR 的使命相结合，有可能提供颅面缺陷的病因，并建议未来的早期微创治疗。