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，并最终建立一个数学理论，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 （Bcd）形态梯度，这是母体决定前后轴的主要因素。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起来，提供了一个综合的攻击(1)床梯度是如何建立和维持的，(2)它是如何在不同大小的胚胎中按比例缩放的，以及(3)它是如何被读出以产生下游基因表达的精确模式的。床梯度的形成和稳定的动力学将在表达eGFP-Bcd的活胚胎中进行测量。延时双光子显微镜的图像序列将与光漂白方法和计算分析一起使用，以评估对梯度动力学的被动和主动贡献，确定Bed的蛋白质半衰期，并确定不同发育阶段细胞核和细胞质中Bed的绝对浓度。在具有不同大小但几乎相同比例的密切相关的双翅目物种中，床和间隙基因表达模式的缩放将使用经典的染色方法进行分析，并通过更复杂的图像处理方法进行扩展。此外，来自不同大小蝇种的表达eGFP标记的bicoid基因的转化子将在黑腹果蝇中表达，以探索这种缩放背后的生物物理机制。尽管基因似乎在胚胎长度的特定浓度阈值上被Bed激活，但转录调节中的噪音限制了标记这些阈值的准确性。理论工作将逐步在每个监管步骤的更现实的模型中定义这些限制的性质。为了测试这些模型，靶基因（驼背、正齿）的均值和方差将作为局部Bed浓度的函数进行测量，无论是在野生型胚胎中，还是在水平和活动可以人为操纵的突变体和遗传嵌合体中。变异的空间相关性也将被测量，以检验原子核之间的交流在抑制噪声和提高发育边界精度方面发挥作用的假设。我们的项目解决了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的基本问题。这种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.