Protein/nucleic Acid Interactions In Vertebrate Embryogenesis

在脊椎动物中，外胚层在原肠形成过程中被细分为四个主要命运：分别位于腹面和背面的表皮和中枢神经系统(CNS)，以及位于这两个区域之间的神经脊和感觉安慰剂。这个项目的目标是了解决定外胚层细胞如何在这些途径中选择的机制，以及随后的组织模式和分化是如何调节的，主要集中在神经脊。非洲爪哇神经脊线的诱导需要两个信号，一个是部分衰减的BMP信号，另一个是Wntbeta连环蛋白信号。我们发现转录因子TFAP2Alpha负责将BMP信号传递到基因组，导致广泛的神经突起特异性基因的激活。我们使用激素诱导版本的AP2pha作为工具，结合微阵列分析来识别表皮和神经脊中的靶基因。我们现在的研究重点是确定其中三个基因的功能。一个是我们命名为INCA的基因(AP2在神经峰诱导的意思)。印加在青蛙、老鼠和斑马鱼中的序列和表达模式是保守的。在青蛙和斑马鱼中，我们使用了反义寡核苷酸来表明Inca是头面部软骨以及涉及神经脊的骨骼和其他组织正常发育所必需的。我们还发现，在蛙的发育过程中，印加功能是正常原肠发育所必需的。酵母双杂交分析已经确定p21激活的激酶PAK4是与印加相互作用的伙伴。我们发现INCA的过表达改变了多条信号转导途径，包括Rhoclass GTP酶和丝裂原活化蛋白激酶(MAPK)级联。这些发现支持一种模型，在该模型中，INCA在中胚层诱导和形态发生重塑以及神经脊细胞迁移和分化过程中调节细胞骨架动力学。第二个基因编码一种新的原钙粘附素PCNS，它是一种潜在的黏附分子，在中胚层、神经脊和发育中的体节中瞬时表达。PCNS的缺失会导致中胚层背侧不能收敛延伸，扰乱神经脊细胞的迁移，也会导致体节和轴肌发育异常。第三个基因是一种非肌肉肌球蛋白MyosinX，它在神经脊和感觉安慰剂中表达，在体外也是神经脊细胞在纤维连接蛋白上正常扩散所必需的，在体内也是正常的颅面发育所必需的。由于这些基因在整个脊椎动物系统发育过程中都是保守的，我们预计我们的发现将与生物医学研究以及人类健康和发育广泛相关。

In vertebrates, the ectoderm is subdivided during gastrulation into four primary fates: epidermis and central nervous system (CNS) on the ventral and dorsal surfaces, respectively, and neural crest and sensory placodes located between these two domains. The goal of this project is to understand the mechanisms that determine how ectodermal cells choose among these pathways, and how the ensuing tissue patterning and differentiation are regulated, with a primary focus on the neural crest. Neural crest induction in Xenopus requires two signals, a partially attenuated BMP signal and a Wntbeta catenin signal. We have found that the transcription factor TFAP2alpha is responsible for conveying the BMP signal to the genome, resulting in activation of a broad spectrum of neural crestspecific genes. We used a hormoneinducible version of AP2alpha as a tool in conjunction with microarray analysis to identifiy target genes in both epidermis and neural crest. We are now focusing our research on determining the function of three of these genes. One is a gene we named Inca (for Induced in Neural Crest by AP2). Inca is conserved in sequence and expression pattern in frogs, mice, and zebrafish. In the frog and zebrafish, we have used antisense oligonucleotides to show that Inca is required for proper development of the craniofacial cartilage and bones and other tissues where neural crest is involved. We have also found that in frog development Inca function is required for normal gastrulation movements. Yeast twohybrid analysis has identified a p21activated kinase, PAK4, as an interaction partner with Inca. We find that overexpression of Inca alters multiple signal transduction pathways, including Rhoclass GTPase and mitogenactivated protein kinase (MAPK) cascades. These findings support a model in which Inca functions to regulate cytoskeletal dynamics during both mesoderm induction and morphogenetic remodeling and neural crest cell migration and differentiation. The second gene encodes a novel protocadherin, PCNS, which is a potential adhesion molecule that is transiently expressed in mesoderm, neural crest and developing somites. Loss of PCNS results in failure of convergent extension in dorsal mesoderm, disrupted neural crest cell migration and also in abnormal somite and axial muscle development. The third gene is a nonmuscle myosin, MyosinX, that is expressed in neural crest and sensory placodes, and is also required for proper spreading of neural crest cells on fibronectin in vitro and for normal craniofacial development in vivo. Since these genes are conserved throughout vertebrate phylogeny, we expect our findings to broad relevance to biomedical research and human health and development.