Protein/nucleic Acid Interactions In Vertebrate Embryogenesis

脊椎动物胚胎发生中的蛋白质/核酸相互作用

• 批准号: 8736832
• 负责人: THOMAS D sargent
• 金额: $ 74.81万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8736832
• 关键词: Actins; Adult; Adverse effects; Affect; Animal Model; Apoptotic; Automobile Driving; BMP4; Blood Cells; Bone Morphogenetic Proteins; Cartilage; Cell Differentiation process; Cell-Cell Adhesion; Cells; Cephalic; Confocal Microscopy; Congenital Abnormality; Cytoskeleton; Defect; Development; Diagnosis; Disease; Dominant-Negative Mutation; Elements; Embryo; Embryonic Development; Epidermis; Experimental Models; Face; Failure; Fishes; Fresh Water; Gene Deletion; Gene Expression; Gene Targeting; Genes; Genetic; Genotype; Goals; Growth Factor; Head; Health; Hereditary Disease; Human; Human Genetics; Jaw; Lateral; Lead; Learning; Life; Ligand Binding Domain; Link; Mediating; Monitor; Morphogenesis; Mus; Muscle; Mutate; Mutation; Myeloid Cells; Myelopoiesis; Nature; Neural Crest; Neural Crest Cell; Nucleic Acids; Oligonucleotides; Pattern; Peripheral Nervous System; Phenotype; Play; Production; Protein Kinase; Proteins; Rana; Regulation; Regulator Genes; Role; Shapes; Signal Pathway; Skeleton; Staging; Structure; System; Testing; Tissues; Tooth structure; Transcriptional Regulation; Transgenes; Transgenic Organisms; Water; Work; Xenopus; Zebrafish; base; blastomere structure; bone; cell motility; craniofacial; ecdysone receptor; egg; gain of function; gene discovery; intercellular communication; interest; migration; novel; polypeptide; promoter; research study; transcription factor; tricho-dento-osseous syndrome; zebrafish development

The neural crest (NC) plays a critical role in the developmental of the vertebrate head, face and jaws, providing the bulk of the craniofacial skeleton as well as peripheral nervous system and other cranial tissues. Normal craniofacial development depends on proper induction, migration and differentiation of NC cells and derivatives. Deficiencies at any of these steps, whether due to intrinsic defects in NC itself, or in failure of NC cells to interact properly with adjacent tissues, can lead to birth defects: up to a third of all congenital malformations are craniofacial in nature and mostly due to such NC failures. We have used the frog Xenopus and the freshwater fish Danio rerio(zebrafish)as experimental model organisms to study NC development. The starting point for this project was two transcription factors, TFAP2a and Dlx3, the regulation of which we showed several years ago to be critical for the early steps in NC development. Since then we have identified several target genes for TFAP2a regulation, and have studied these genes to help understand how NC cells form, migrate and differentiate. One interesting TFAP2a target we discovered was the novel gene Inka. While loss of Inka has turned out to be apparently non-phenotypic in both zebrafish and mouse, the Inka protein was shown to interact strongly with the protein kinase PAK4. Thiis has led to an interesting project on PAK4 function in zebrafish, linking maternal expression of PAK4 to several aspects of embryonic development, implicating a form of transcriptional control mediated by the actin cytoskeleton, of which PAK4 is an important regulator. The role of Dlx3 in NC development is biphasic. According to our earlier work with Xenopus, in order for NC induction to occur, Dlx3 must be excluded from NC cells at early stages, being expressed in adjacent, lateral cells. Later, this factor is expressed in NC derivatives, as well as in epidermis, where it is required for terminal differentiation. Mutation of the human Dlx3 gene results in a dominant genetic disease affecting tooth, craniofacial bone and other structures. To learn more about Dlx3 in NC we will express the mutated and also wild-type versions of this gene in zebrafish NC using transgenes controlled by a NC-specific promoter element driving an inducible activator protein. This will allow us to control the level of Dlx3 proteins in NC cells by adding inducer to the fish water. We will then monitor NC development in live embryos by confocal microscopy. Eventually we will use similar approaches to investigate target genes for Dlx3, as we have for TFAP2a, continuing to elucidate the NC control network In parallel with the Dlx3 project, we are using the same inducible transgenic strategy to gain temporospatial control of cell-cell signaling in migrating and postmigratory NC cells. Our primary focus is on bone morphogenetic proteins (BMPs), especially BMP4 which has been implicated in craniofacial patterning in several vertebrate species. Our sophisticated control strategy should reveal further aspects of how this and other signaling pathways regulate the shape of the vertebrate head, face and jaws, which will help in the diagnosis and treatment of NC-based birth defects and disease.

神经嵴（NC）在脊椎动物头、面部和颌骨的发育中起着至关重要的作用，提供了颅面骨骼以及周围神经系统和其他颅组织的主体。 正常的颅面发育依赖于NC细胞及其衍生物的适当诱导、迁移和分化。 在任何这些步骤中的缺陷，无论是由于NC本身的内在缺陷，还是由于NC细胞与邻近组织的相互作用失败，都可能导致出生缺陷：高达三分之一的先天性畸形是颅面畸形，大多数是由于这种NC失败。我们使用蛙爪蟾和淡水鱼Danio rerio（斑马鱼）作为实验模式生物来研究NC的发展。该项目的起点是两个转录因子，TFAP 2a和Dlx 3，几年前我们发现它们的调节对于NC发育的早期步骤至关重要。从那时起，我们已经确定了TFAP 2a调控的几个靶基因，并研究了这些基因，以帮助了解NC细胞如何形成，迁移和分化。我们发现的一个有趣的TFAP 2a靶点是新基因Inka。 虽然在斑马鱼和小鼠中Inka的丢失已经被证明是明显的非表型，但Inka蛋白显示出与蛋白激酶PAK 4强烈相互作用。这导致了一个有趣的项目PAK 4在斑马鱼的功能，连接PAK 4的母体表达胚胎发育的几个方面，涉及一种形式的转录控制介导的肌动蛋白细胞骨架，其中PAK 4是一个重要的调节器。 Dlx 3在NC发育中的作用是双相的。根据我们早期对非洲爪蟾的研究，为了使NC诱导发生，Dlx 3必须在早期阶段从NC细胞中排除，在相邻的侧细胞中表达。后来，该因子在NC衍生物中以及在表皮中表达，在表皮中其是终末分化所需的。人类Dlx 3基因的突变导致影响牙齿、颅面骨和其他结构的显性遗传疾病。 为了更多地了解Dlx 3在NC中的作用，我们将使用由NC特异性启动子元件驱动诱导型激活蛋白控制的转基因在斑马鱼NC中表达该基因的突变和野生型版本。这将允许我们通过向鱼水中添加诱导剂来控制NC细胞中Dlx 3蛋白的水平。然后，我们将通过共聚焦显微镜监测NC在活胚胎中的发育。最终，我们将使用类似的方法来研究Dlx 3的靶基因，就像我们研究TFAP 2a一样，继续阐明NC控制网络 与Dlx 3项目平行，我们正在使用相同的诱导型转基因策略来获得迁移和迁移后NC细胞中细胞-细胞信号传导的时空控制。 我们的主要重点是骨形态发生蛋白（BMP），特别是BMP 4已牵连在几个脊椎动物物种的颅面图案。我们复杂的控制策略应该进一步揭示这个和其他信号通路如何调节脊椎动物头部，面部和颌骨的形状，这将有助于诊断和治疗NC为基础的出生缺陷和疾病。