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量化的描述，并最终形成一个 数学理论，关于如何在发育中的胚胎中建立基因表达的空间模式。 我们的实验系统是果蝇胚胎中的双类(床)形态原梯度，主要是 前后轴的母体决定因素。我们的项目将现代方法结合在一起 实验和理论生物物理学与分子生物学和遗传学相结合，提供一种 综合攻击(1)如何建立和保持床面坡度，(2)如何按比例调整 不同大小的胚胎之间的比例，以及(3)如何读出它来产生精确的模式 下游基因表达。 床梯度的形成和稳定的动力学将在活胚胎中进行测量。 表达绿色荧光蛋白-BCD。来自时间推移双光子显微镜的图像序列将与 光漂白方法和计算分析，以评估对梯度的被动和主动贡献 动力学，以确定Bed的蛋白质半衰期，并确定Bed在细胞核中的绝对浓度 以及不同发育阶段的细胞质。 Bed和GAP基因表达模式在亲缘关系密切的双翅目物种中的比例 不同大小但比例几乎相同的身体将使用经典的染色方法进行分析， 扩展了更复杂的图像处理方法。此外，表达绿色荧光蛋白的转化子 不同大小果蝇的标记双基因将在果蝇中表达以进行探测 这种伸缩背后的生物物理机制。 尽管基因似乎被Bed激活，在特定的浓度阈值沿着长度 胚胎，转录调控中的噪音限制了这种阈值的准确性 有记号的。理论工作将在每个模型中逐步更接近实际地定义这些限制的性质 监管步骤。为了检验这些模型，目标基因(驼背、正齿)的均值和方差将 在野生型胚胎和突变体中，作为床的局部浓度的函数来测量 基因马赛克，其中的水平和活动可以被人为操纵。地球表面的空间相关性 还将测量方差，以检验原子核之间的通信在 抑制噪声，提高开发边界的精确度。 我们的项目解决了信令集中度的微小变化这一根本问题 分子对细胞命运有强大的控制力。这种信号通路的精确读出需要 正常发育。信号的干扰与成人的出生缺陷和癌症有关。

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.