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命运，即表皮和中枢神经系统(CNS)。这个项目的目标是了解决定外胚层细胞如何在这两条途径之间选择的机制，以及随后的组织图案和分化是如何受到调控的。已经证实，骨形态发生蛋白(BMPs)的信号转导是启动两栖类胚胎表皮发育的主要事件。该实验室采取的实验方法是使用对这种信号的依赖作为识别可能调节表皮/神经发育程序的基因的标准。人们的注意力集中在无末端的同系物DLX3、5和6以及转录激活因子AP-2上。正在使用消减杂交/微阵列技术寻找更多的基因。我们发现，这三个DLX基因是通过对BMP信号的分级反应来进行差异调控的，这至少可以解释DLX基因在原肠胚中表达的空间模式的一些主要特征。DLX3似乎以类似于我们之前发现的该基因的抗神经特性的方式参与了建立颅神经脊诱导的侧边界。有趣的是，这种抑制功能似乎不涉及DLX3对转录的直接调节，但更可能依赖于蛋白质-蛋白质的相互作用。Dlx5和Dlx6的作用仍然不清楚，但这个同源异型盒基因亚家族解释胚胎形态发生梯度的可能性非常耐人寻味。AP2的表达依赖于BMP，并且该因子的过度表达可以挽救BMP缺失的外胚层中的表皮基因活性，这一发现也非常有趣，因为我们的实验室几年前利用启动子分析的方法将该基因鉴定为潜在的表皮调节因子。我们还发现AP2在神经脊细胞的发育中起着重要的作用，目前正在蛋白质-蛋白质水平上研究AP2、DLX3和其他因子之间的相互作用，并利用基因芯片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.