Dynamics, scaling, and precision of morphogen gradients in the Drosophila embryo

我们研究工作的总体目标是产生一个定量描述，并最终 数学理论，基因表达的空间模式是如何在发育中的胚胎中建立的。 我们的实验系统是果蝇胚胎中的Bicoid（Bed）形态基梯度， 前后轴的母体决定因素。我们的项目汇集了现代的方法， 实验和理论生物物理学与分子生物学和遗传学，以提供一个 综合攻击（1）如何建立和维持床梯度，（2）如何缩放 不同大小的胚胎之间的比例，以及（3）它是如何读出以产生精确的模式， 下游基因表达。 将在活胚胎中测量床梯度的形成和稳定的动力学 表达eGFP-Bcd。将使用延时双光子显微镜的图像序列，以及 光漂白方法和计算分析，以评估被动和主动的贡献梯度 动力学，以确定Bed的蛋白质半衰期，并确定细胞核中Bed的绝对浓度 和细胞质的不同发育阶段。 床和间隙基因表达模式在密切相关的双翅目物种， 不同大小但几乎相同比例的体将使用经典染色方法进行分析， 通过更复杂的图像处理方法进行扩展。此外，表达eGFP的转化体 来自不同大小的果蝇物种的标记的bicoid基因将在黑腹果蝇中表达，以探测 这种缩放背后的生物物理机制。 虽然基因似乎被激活床在特定浓度阈值沿沿着的长度 在胚胎中，转录调控中的噪音限制了这种阈值可以被精确地识别的准确性。 标记。理论工作将界定这些限制的性质，在逐步更现实的模型， 监管步骤。为了检验这些模型，将靶基因（hunchback，orthodenticle）的均值和方差 作为野生型胚胎和突变体中Bed局部浓度的函数进行测量， 基因镶嵌，水平和活动可以人为操纵。空间相关性 方差也将被测量，测试假设，即核之间的通信发挥了作用， 抑制噪音，提高发展边界的精确性。 我们的项目解决了一个基本问题，即信号浓度的微小变化 分子产生对细胞命运的稳健控制。需要精确读出这些信号通路， 正常发展。信号的扰动与出生缺陷和成人癌症有关。

The overall goal of our research effort is to produce a quantitative description, and ultimately a mathematical theory, of how spatial patterns of gene expression are established in a developing embryo. Our experimental system is the Bicoid (Bed)morphogen gradient in the Drosophila embryo, the primary maternal determinant of the anterior-posterior axis. Our project brings together modern methods of experimental and theoretical biophysics with those of molecular biology and genetics to provide an integrated attack on (1) how the Bed gradient is established and maintained, (2) how is it scaled proportionately across embryos of different size, and (3) how is it read out to produce precise patterns of downstream gene expression. The dynamics of the formation and stabilization of the Bed gradient will be measured in living embryos expressing eGFP-Bcd. Image sequences from time-lapse two photon microscopy will be used, together with photobleaching methods and computational analysis, to assess passive and active contributions to gradient dynamics, to determine the protein half life of Bed, and to determine absolute concentrations of Bed in nuclei and cytoplasm at various stages of development. The scaling of Bed and gap gene expression patterns across closely related dipteran species that have bodies of different size but almost identical proportions will be analyzed using classical staining methods, extended by more sophisticated image processing methods. In addition, transformants expressing eGFP labeled-bicoid genes from different sized fly species will be expressed in Drosophla melanogaster to probe the biophysical mechanisms behind this scaling. Although genes appear to be activated by Bed at specific concentration thresholds along the length of the embryo, noise in transcriptional regulation places limits on the accuracy with which such thresholds can be marked. Theoretical work will define the nature of these limits in progressively more realistic models of each regulatory step. To test these models, the mean and variance of target genes (hunchback, orthodenticle) will be measured as functions of local concentration of Bed,both in wild type embryos, and in mutants and genetic mosaics where levels and activities can be artificially manipulated. Spatial correlations in the variance will also be measured, testing the hypothesis that communication among nuclei plays a role in suppressing noise and enhancing the precision of developmental boundaries. Our project addresses the fundamental question of how small changes in the concentration of signaling molecules produce robust control of cell fate. Precise read-out of such signaling pathways is required for normal development. Perturbations in signaling are associated with birth defects and cancer in adults.