Protein/nucleic Acid Interactions In Vertebrate Embryogenesis

神经嵴在脊椎动物头、面部和颌骨的发育中起着关键作用，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Dlx3，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，我们已经确定了几个TFAP 2a调控的“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。 我们还鉴定了与靶基因编码蛋白相互作用的二级蛋白，开始建立NC的调控网络。 Dlx3的作用更为复杂。根据我们早期对非洲爪蟾的研究，Dlx3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Dlx3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Dlx3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Dlx3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，就像我们对TFAP 2a所做的那样，扩大我们的NC控制网络，这将有助于诊断和治疗基于NC的出生缺陷和疾病。

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 called TFAP2a and Dlx3, the regulation of which 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. We have also gone on to identify secondary proteins that interact with the target gene-encoded proteins, beginning to establish a regulatory network for NC. The role of Dlx3 is more complicated. According to our earlier work with Xenopus, 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, expanding our NC control network, which will help in the diagnosis and treatment of NC-based birth defects and disease.