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

描述（由申请人提供）：我们研究工作的总体目标是产生一个定量描述，并最终产生一个数学理论，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Bicoid（Bcd）形态梯度在果蝇胚胎，前-后轴的主要母系决定因素。我们的项目将实验和理论生物物理学的现代方法与分子生物学和遗传学的方法结合在一起，以提供对（1）如何建立和维持床梯度的综合攻击，（2）如何在不同大小的胚胎中按比例缩放，以及（3）如何读出以产生下游基因表达的精确模式。将在表达eGFP-Bcd的活胚胎中测量床梯度的形成和稳定的动力学。从延时双光子显微镜的图像序列将被使用，连同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，以确定蛋白质的半衰期床，并确定绝对浓度的床在细胞核和细胞质在不同的发展阶段。将使用经典染色方法并通过更复杂的图像处理方法扩展来分析具有不同大小但几乎相同比例的密切相关双翅目物种中Bed和gap基因表达模式的缩放。此外，将表达来自不同大小的果蝇物种的eGFP标记的bicoid基因的转化体在黑腹果蝇中表达，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的浓度阈值沿着胚胎的长度，噪音在转录调控的地方限制的准确性与这些阈值可以标记。理论工作将在每个监管步骤的逐步更现实的模型中定义这些限制的性质。为了测试这些模型，将在野生型胚胎以及突变体和遗传嵌合体中测量靶基因（hunchback，orthodenticle）的平均值和方差作为Bed局部浓度的函数，其中水平和活性可以人为操纵。还将测量方差的空间相关性，检验核之间的通信在抑制噪声和提高发育边界的精度中起作用的假设。我们的项目解决了一个基本问题，即信号分子浓度的微小变化如何产生对细胞命运的强大控制。这些信号通路的精确读出是正常发育所必需的。信号的扰动与出生缺陷和成人癌症有关。

DESCRIPTION (provided by applicant): 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 (Bcd) 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.