Protein/nucleic Acid Interactions In Vertebrate Embryogenesis

神经脊在脊椎动物头部、面部和颌骨的发育中起着至关重要的作用，它提供了颅面骨骼的大部分以及周围神经系统和其他颅骨组织。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。这些步骤中的任何一个步骤的缺陷，无论是由于NC本身的固有缺陷，还是由于NC细胞未能与邻近组织正确地相互作用，都可能导致出生缺陷：多达三分之一的先天性畸形本质上是头面部畸形，主要是由于NC故障。我们使用非洲爪蛙和淡水鱼Danio rerio(斑马鱼)作为实验模式生物来研究NC的发育。这个项目的起点是两个转录因子，TFAP2A和DLX3，我们几年前就证明了它们的调节对于NC发展的早期步骤至关重要。从那时起，我们已经确定了几个TFAP2A调控的靶基因，并对这些基因进行了研究，以帮助了解NC细胞是如何形成、迁移和分化的。我们发现的一个有趣的TFAP2A靶点是新基因INKA。虽然在斑马鱼和小鼠身上印加的丢失显然都是非表型的，但印加蛋白被证明与蛋白激酶PAK4强烈相互作用。这导致了一个关于斑马鱼PAK4功能的有趣项目，将PAK4的母体表达与胚胎发育的几个方面联系起来，暗示了一种由肌动蛋白细胞骨架介导的转录调控形式，其中PAK4是一个重要的调节因子。 DLX3在NC发展中的作用是双段性的。根据我们早期对非洲爪哇的工作，为了发生NC诱导，DLX3必须在早期阶段从NC细胞中排除，而在相邻的侧向细胞中表达。后来，这个因子在NC衍生物中表达，也在终端分化所需的表皮中表达。人类DLX3基因的突变会导致一种影响牙齿、颅面骨和其他结构的显性遗传病。为了更多地了解DLX3在NC中的作用，我们将利用NC特异性启动子元件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，该启动子元件驱动可诱导的激活蛋白。这将使我们能够通过在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活胚胎中的NC发育。最终，我们将使用类似的方法来研究DLX3的目标基因，就像我们对TFAP2A所做的那样，继续阐明NC控制网络 在DLX3项目的同时，我们正在使用相同的可诱导转基因策略来获得对迁移和迁移后NC细胞中细胞-细胞信号的时空控制。我们主要关注骨形态发生蛋白(BMPs)，特别是BMP4，它与几种脊椎动物的头面部模式有关。我们复杂的控制策略将进一步揭示这一信号通路和其他信号通路如何调节脊椎动物的头、脸和颌骨的形状，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, 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.