Protein/nucleic Acid Interactions In Vertebrate Embryoge

脊椎动物和其他动物一样，外胚层有两个主要的功能：表皮和中枢神经系统（CNS）。该项目的目标是了解决定外胚层细胞如何在这两种途径之间选择的机制，以及随后的组织模式和分化是如何被调节的。在两栖动物胚胎中，骨形态发生蛋白（BMPs）的信号传导是启动表皮发育的主要事件。本实验室采用的实验方法是使用依赖于这些信号作为鉴定可能调节表皮/神经发育程序的基因的标准。人们的注意力集中在远端无同源物Dlx3、5和6以及转录激活子AP-2上。正在使用减法杂交/微阵列技术寻找其他基因。我们发现，三个Dlx基因受到BMP信号的分级反应的差异调节，这至少可以解释原肠中Dlx基因表达空间格局的一些主要特征。Dlx3似乎参与建立颅神经嵴诱导的外侧边界，其方式类似于我们之前发现的该基因的抗神经特性。有趣的是，这种抑制功能似乎不涉及Dlx3对转录的直接调节，而更可能依赖于蛋白质之间的相互作用。Dlx5和Dlx6的作用仍然不清楚，但是这个同源盒基因亚家族解释胚胎形态发生梯度的可能性非常有趣。AP2的表达依赖于bmp，并且该因子的过表达可以挽救bmp失活的外胚层的表皮基因活性，这一发现也非常有趣，因为该基因在几年前被我们的实验室鉴定为潜在的表皮调节因子，使用启动子分析方法。我们也发现AP2在神经嵴细胞的发育中起着重要的作用，目前正在蛋白-蛋白水平上研究AP2、Dlx3等因子之间的相互作用，并利用微阵列分析寻找受这些因子调控的靶基因。

In vertebrates, as in other animals, the ectoderm has two primary fates, epidermis and central nervous system (CNS). The goal of this project is to understand the mechanisms that determine how ectodermal cells choose between these two pathways, and how the ensuing tissue patterning and differentiation are regulated. It has been well established that signaling by bone morphogenetic proteins (BMPs) is the primary event that initiates epidermal development in the amphibian embryo. The experimental approach taken by this laboratory has been to use dependence upon such signaling as a criterion for identifying genes that may regulate the epidermal/neural developmental program. Attention has been focused on the Distal-less homologs Dlx3, 5 and 6 and on the transcriptional activator AP-2. Additional genes are being sought using subtractive hybridization/microarray techniques. We have found that the three Dlx genes are differentially regulated by a graded response to BMP signaling, and that this can account for at least some of the major features of the spatial pattern of Dlx gene expression in the gastrula. Dlx3 appears to be involved in establishing the lateral boundary of cranial neural crest induction, in a manner similar to the anti-neural properties we previously discovered for this gene. Interestingly, this inhibitory function does not appear to involve direct regulation of transcription by Dlx3, but is more likely dependent upon protein-protein interactions. The roles of Dlx5 and Dlx6 remain obscure, but the possibility that this subfamily of homeobox genes function to interpret a morphogenetic gradient in the embryo is very intriguing. The findings that AP2 expression is BMP-dependent, and that over-expression of this factor can rescue epidermal gene activity in BMP-disabled ectoderm are also highly interesting, as this gene was identified by our laboratory several years ago as a potential epidermal regulatory factor, using a promoter analysis approach. We have also found that AP2 plays an important role in the development of neural crest cells, and are now examining the interactions between AP2, Dlx3 and other factors at the protein-protein level, and using microarry analyis to search for target genes regulated by these factors.