Gradients and the Control of Pattern Formation

最近的实验表明，调节分子的梯度对于建立多细胞动物的身体计划是重要的。然而，关于靶基因如何响应调节分子浓度变化的机制，人们知之甚少。这项申请建议在果蝇身上研究这些机制，这是实现这一目的的理想选择，因为早期胚胎是没有细胞膜的核的合胞体。这使得可以直接研究转录机制，而不会出现细胞间信号传递过程的并发症。这项工作将集中在四个GAP基因上，驼背、巨人、Kruppel和Knirps，它们重叠的蛋白质梯度形成一个整合的系统，建立胚胎中部区域基因表达的条带模式。这项工作是由这样一个假设驱动的，即这些梯度通过充当浓度依赖的抑制因子来建立基因表达边界的位置。对于每个梯度，几个目标基因的边界被设置在相对于源的不同位置。这表明每个靶基因对抑制活性的不同浓度阈值做出反应。为了验证这一假说，我们将使用先前确定的增强子和酵母FLP-FRT重组系统来异位表达每个基因在其正常结构域之外的不同水平。这些实验将测试给定GAP基因的表达是否足以满足特定的反应，并识别潜在的新靶标反应。错误表达系统还将被用来剖析GAP蛋白中表达所需的区域。最初，重点将放在进化上保守的结构域上，这些结构域以前与抑制有关。总之，这些实验将有助于阐明控制早期胚胎中基因表达边界的位置和间距的遗传电路，并提供对控制不同靶基因如何对不同调节蛋白的不同组合和浓度做出反应的一般机制的见解。它们还将为研究在进化过程中改变身体计划的机制提供坚实的基础。

Recent experiments suggest that gradients of regulatory molecules areimportant for establishing the body plans of multicellular animals.However, little is known about the mechanisms whereby target genes respondto changes in the concentration of regulatory molecules. This applicationproposes to study these mechanisms in Drosophila, which is ideal for thispurpose because the early embryo is a syncytium of nuclei that do notpossess cytoplasmic membranes. This permits the study of transcriptionmechanisms directly, without complications from cell-cell signalingprocesses. The work will focus on four gap genes, hunchback, giant,Kruppel, and knirps, whose overlapping protein gradients form an integratedsystem that establishes striped patterns of gene expression in middleregions of the embryo. This work is driven by the hypothesis that thesegradients establish positions of gene expression borders by acting asconcentration-dependent repressors. For each gradient, the borders ofseveral target genes are set at different positions with respect to thesource. This suggests that each target gene responds to a differentconcentration threshold of repressive activity. To test this hypothesis,previously characterized enhancers and the yeast FLP-FRT recombinationsystem will be used to ectopically express different levels of each geneoutside its normal domain. These experiments will test whether expressionof a given gap gene is sufficient for a particular response, and identifypotential new target responses. The misexpression system will also be usedto dissect regions of the gap proteins that are required for repression.Initially, the focus will be on evolutionarily conserved domains that havebeen previously implicated in repression. Together these experiments willhelp clarify the genetic circuitry that controls the positioning andspacing of gene expression boundaries in the early embryo, and provideinsights into general mechanisms that control how different target genesrespond to different combinations and concentrations of regulatoryproteins. They will also provide a firm foundation for studies designed tounravel the mechanisms that change body plans during evolution.