Developmental Progression Driving Gastrulation of the Drosophila Early Embryo

 描述（由申请人提供）：拟议的研究计划的主要目标是以果蝇胚胎为模型系统，了解控制原肠胚形成的分子机制。特别是，我们感兴趣的是研究背腹（DV）图案和细胞信号传导过程是如何在早期胚胎的时间调节;在破译控制时空基因表达的顺式调控机制;并研究集体细胞运动是如何编排的。这些都是相互关联的问题，也与所有动物的发展有关，因此这些研究有可能提供深远的见解。为了分析发育事件的进展，我们开发并采用新技术，使用活体成像、包括数学建模在内的计算和分子生物学进行时间相关的观察。我们专注于设计和实施新的成像方法，使我们能够获得精细尺度的时空数据的发展过程中，捕捉转录因子的动态以及细胞运动。在这里，我们提出了三个研究方向，以提供进一步深入了解果蝇原肠胚形成的基因系统。项目1涉及DV模式网络的扩展，重点是时间表达的调节。在早期胚胎中，我们发现沿着DV轴作用的转录因子表现出水平的动态变化。将在野生型与突变型胚胎的单个胚胎分辨率下获得跨越早期发育的多个时间点的推定靶基因的表达谱，以深入了解基因网络如何调节表达的时机。将使用原位杂交进一步研究基因子集的空间表达，并使用实时成像方法实时监测基因表达。项目2将研究协调顺式调节作用的机制和作用，使用方法来分析胚胎每个细胞核内的染色质构象，以及比较协同作用顺式调节元件支持的基因表达水平和时间的测定。项目3将研究FGF信号在迁移细胞中的功能和调节。我们假设FGF信号调节原肠胚形成时中胚层细胞的粘附特性，以支持它们的凝聚力，有组织的运动。硫酸乙酰肝素蛋白聚糖分子和切割状态对FGF活性的作用也将被研究。拟议研究计划的首要目标是通过研究控制模式，信号通路激活以及最终控制早期果蝇胚胎细胞运动的基因网络，了解控制胚胎形态发生的分子机制。所有动物的基因调控机制的保守性保证了这些研究将具有深远的意义。特别是，更好地了解顺式调控机制，一般来说，有许多好处，包括改进，有针对性的基因治疗;而了解细胞迁移是如何控制将提供对细胞转移的调节见解。

 DESCRIPTION (provided by applicant): The overlying goal of the proposed research program is to understand the molecular mechanisms that control gastrulation using the Drosophila embryo as a model system. In particular, we are interested in investigating how dorsal-ventral (DV) patterning and cell signaling processes are temporally regulated in the early embryo; in deciphering the cis-regulatory mechanisms controlling spatiotemporal gene expression; and studying how collective cell movements are orchestrated. These are inter-related questions that also are relevant for the development of all animals, and as such these studies have the potential to provide far-reaching insights. To assay progression of developmental events, we develop and employ novel technologies for making temporally relevant observations using live in vivo imaging, computation including mathematical modeling, and molecular biology. We have focused on the design and implementation of new imaging approaches that allow us to acquire fine-scale spatiotemporal data of developmental processes, to capture transcription factor dynamics as well as cell movements. Here we propose three research directions to provide further insight into the system of genes driving Drosophila gastrulation. Project 1 involves expansion of DV patterning network with a focus on the regulation of temporal expression. In the early embryo, we have found that transcription factors acting along the DV axis exhibit dynamic changes in levels. Expression profiles for putative target genes at multiple time-points spanning the early development will be obtained at single embryo resolution in wildtype versus mutant embryos to provide insight into how timing of expression is regulated by the gene network. Spatial expression of a subset of genes will be further investigated using in situ hybridization, and live imaging methods will be used to monitor gene expression in real-time. Project 2 will investigate the mechanism and role of coordinate cis-regulatory action using methods to analyze chromatin conformation in vivo within each nucleus of the embryo as well as assays to compare levels and timing of gene expression supported by co-acting cis-regulatory elements. Project 3 will investigate the function and regulation of FGF signaling in migrating cells. We hypothesize that FGF signaling modulates the adhesive properties of mesoderm cells at gastrulation to support their cohesive, organized movement. The role of heparan sulfate proteoglycan molecules and cleavage-state on FGF activity will also be investigated. The overlying goal of the proposed research program is to understand the molecular mechanisms controlling morphogenesis of the embryo through study of a network of genes that controls patterning, signaling pathway activation, and, ultimately, cell movements in the early Drosophila embryo. The conservation of gene regulatory mechanisms across all animals promises that these studies will have far reaching implications. In particular, a better understanding of cis-regulatory mechanisms, in general, has many benefits including improved, targeted gene therapy; while understanding how cell migration is controlled will provide insights toward the regulation of cell metastasis.