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Molecular mechanisms orchestrating EMTs in the cranial neural crest

颅神经嵴中 EMT 的分子机制

基本信息

批准号：
10214990
负责人：
LISA A TANEYHILL
金额：
$ 36.33万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-05 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10214990
关键词：
Address Afferent Neurons Binding Binding Proteins Biochemistry Bioinformatics Biological Biological Assay Biology C cadherin C-terminal Cadherins Cartilage Cell Adhesion Cell-Cell Adhesion Cell-Matrix Junction Cells Cellular Morphology Cephalic Chick Embryo Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Complex Congenital Abnormality Craniofacial Abnormalities DNA Binding Data Dependence Detection Development Developmental Biology Developmental Process Disease Disseminated Malignant Neoplasm Embryo Embryology Epithelial Epithelial Cells Epithelium Extracellular Matrix Extracellular Matrix Degradation Gelatinase A Gene Expression Genes Genetic Transcription Genomics Goals Head Health Heart Human Human Development Human body Image Immunohistochemistry Immunoprecipitation Impairment In Situ Hybridization Intercellular Junctions Knowledge Laser Scanning Confocal Microscopy Lead Ligands Malignant Neoplasms Mass Spectrum Analysis Mediating Mesenchymal Microscopic Microscopy Mission Modeling Molecular N-terminal Nature Neural Crest Neural Crest Cell Oral Pattern Peptide Hydrolases Peptides Peripheral Nervous System Peripheral Nervous System Diseases Pigments Population Prevention Process Proteins Proteolysis Proteomics Public Health Publishing Quality of life Regulation Reporting Research Role Signal Pathway Signal Transduction Site Skeleton Structure System Testing Therapeutic Time Transcriptional Regulation United States National Institutes of Health Work beta catenin bone cell motility cell type chromatin immunoprecipitation craniofacial design developmental disease epithelial to mesenchymal transition extracellular face skin gain of function heuristics human disease improved in vivo in vivo Model innovation insight interdisciplinary approach melanocyte migration nervous system development neuroregulation novel promoter receptor tool transcriptome sequencing transcriptomics treatment strategy vertebrate embryos

项目摘要

PROJECT SUMMARY This proposal aims to address an outstanding issue in developmental biology: What cellular and molecular mechanisms endow immotile cells with the capacity to migrate and ultimately pattern the vertebrate embryo? Neural crest cells (NCCs) provide an exceptional model in which to address this fundamental question, as they differentiate to form various cell types including cranial bones and cartilage, sensory neurons, and melanocytes. Abnormalities in NCC formation are thus responsible for diverse human diseases, such as cancers, craniofacial anomalies, and disorders of the peripheral nervous system. Initially stationary epithelial cells, premigratory NCCs undergo an epithelial-to-mesenchymal transition (EMT), which is typified by changes in cell adhesion and morphology, along with breakdown of extracellular matrix, to become motile. The en masse nature of cranial NCC EMT requires an extraordinary degree of coordination, but how premigratory NCCs initiate and orchestrate this transition remains obscure. Our published and preliminary data have begun to bridge this knowledge gap, revealing the importance of Cadherin-6B (Cad6B) proteolysis during EMT, which reduces cell-cell adhesion to liberate NCCs but also generates peptides (Cad6B N-terminal fragments, NTFs; Cad6B C-terminal fragment 2, CTF2) with novel pro-EMT functions. Cad6B NTFs augment protease activity to facilitate degradation of extracellular matrix substrates, and Cad6B CTF2 modulates expression of critical EMT genes involved in cellular invasion, as identified by both targeted and global transcriptomics approaches. Herein, we propose to elucidate how Cad6B proteolytic peptides function in concert to control NCC EMT, using chick cranial NCCs as a unique in vivo model for EMT that has the advantage of direct translatability to human development. The aims in this proposal will test the hypothesis that EMT is orchestrated by cadherin peptides with distinct activities and functions within the premigratory cranial NCC population. To define the role of Cad6B NTFs during NCC EMT (Aim 1), we will evaluate the interrelationship among NTFs, proteases, and substrates, and determine the expression and function of NTF binding partners previously obtained via a novel mass spectrometry assay in the embryo. To investigate the role of Cad6B CTF2 as a transcriptional modulator during NCC EMT (Aim 2), we will continue characterizing newly identified CTF2 target genes and determine their mechanism of regulation by CTF2, and use ChIP-seq, followed by CTF2-associated sequence mapping and bioinformatics, to reveal new target genes and DNA binding motifs important for CTF2-mediated gene expression. Our proposed research is innovative as it takes a multidisciplinary approach combining embryology, biochemistry, bioinformatics, and novel microscopic analyses to examine the coordination of EMT in vivo. These studies will have great significance to the field by providing a heuristic paradigm to tackle similar questions for other normal developmental processes involving EMTs and for aberrant EMTs in other cell types that lead to birth defects and developmental disorders.

项目摘要该提案旨在解决发育生物学中的一个突出问题：机制赋予不动细胞迁移并最终形成脊椎动物胚胎的能力？神经嵴细胞（NCC）为解决这一基本问题提供了一个特殊的模型，因为它们分化形成各种细胞类型，包括颅骨和软骨、感觉神经元和黑素细胞。因此，NCC形成中的异常导致多种人类疾病，如癌症、颅面神经疾病、脑血管疾病、脑异常和周围神经系统疾病。最初静止的上皮细胞，迁移前NCC 经历上皮向间充质转化（EMT），其典型表现为细胞粘附的变化，形态学，沿着细胞外基质的分解，变得有运动能力。颅骨缺损的完整性 NCC EMT需要高度的协调，但迁移前NCC如何发起和协调这一转变仍不明朗。我们公布的初步数据已经开始弥合这一知识差距，揭示了钙粘蛋白-6B（Cad 6 B）蛋白水解在EMT过程中的重要性，其降低了细胞-细胞粘附，释放NCC，但也产生肽（Cad 6 B N-末端片段，NTF; Cad 6 B C-末端片段2， CTF 2）具有新的pro-EMT功能。Cad 6 B NTF增加蛋白酶活性以促进 Cad 6 B CTF 2调节参与细胞外基质的关键EMT基因的表达，侵袭，如通过靶向和全局转录组学方法鉴定的。在此，我们建议澄清 Cad 6 B蛋白水解肽如何协同作用以控制NCC EMT，使用鸡颅NCC作为独特的 EMT的体内模型，其具有可直接转化为人类发育的优点。这其中的目的该提案将检验EMT是由具有不同活性的钙粘蛋白肽协调的假设，在迁移前颅NCC人群中发挥作用。定义Cad 6 B NTF在NCC EMT期间的作用 (Aim 1），我们将评估NTF、蛋白酶和底物之间的相互关系，并确定先前通过新的质谱分析获得的NTF结合配偶体的表达和功能，胚胎为了研究Cad 6 B CTF 2在NCC EMT（Aim 2）中作为转录调节因子的作用，我们将继续表征新发现的CTF 2靶基因，并通过以下方式确定其调控机制： CTF 2，并使用ChIP-seq，随后是CTF 2相关的序列作图和生物信息学，以揭示新的靶基因和DNA结合基序对CTF 2介导的基因表达很重要。我们的研究计划是创新，因为它采取了多学科的方法，结合胚胎学，生物化学，生物信息学，新的显微镜分析，以检查EMT在体内的协调。这些研究将对通过提供一个启发式的范例来解决其他正常的类似问题，涉及EMT的发育过程和其他细胞类型中导致出生缺陷的异常EMT，发育障碍