Notch Signaling and the Somitogenesis Oscillator

描述(申请人提供)：在分子、细胞和多细胞水平上，生物组织的时空生成是生物网络中许多独立成分相互作用的产物。这些网络构成了复杂的系统，其性质大于各组成部分之和。在体细胞发生过程中，一种时间模式机制，即振荡器，产生了一种新出现的空间模式，预示着形态节段的形成。这种振荡器产生了基因表达的条纹/波，在即将进行分割的细胞场中反复传播。我们的第一个目标是利用活体成像、仔细分期的胚胎和荧光原位杂交，建立更详细的斑马鱼体细胞发育过程中基因表达和形态的周期性变化的年表。最近的工作，包括我们自己的工作，发现Notch信号通路在振荡器机制和更直接调节形态分割的后续过程中都具有连续的功能。在斑马鱼中，我们已经证明了Notch配体AEI/deltaD、Bea/deltaC和Notch靶基因her1都在振荡器机制中发挥作用，但每个都有不同的突变表型。我们的第二个目标是通过两个Delta突变体的比较表型分析、双突变分析、异位表达实验和遗传嵌合体来区分这两个Delta同源基因的功能。我们假设AEI/DeltaD和beaJdeltaC在振荡器电路中具有不同的信号作用，并且Delta信号的相对功能和强度在分割组织中的不同位置有所不同。第三个目的是研究her1在振荡器中的功能。我们将确定Her1蛋白水平是否振荡，以及蛋白质的稳定性是否决定振荡器的频率。最终目标是开发振荡器的实时读数，以便可视化活胚胎中的振荡。总而言之，这些实验解决了细胞与其近邻之间如何通过Notch途径进行局部信号相互作用，从而在旁轴中胚层内产生新的、更高水平的组织。这种对支配体细胞发生的遗传网络的分析是我们理解脊椎动物发育的核心，并对生物回路的复杂属性具有广泛的影响。

DESCRIPTION (provided by applicant): At the molecular, cellular and multicellular level, the spatial and temporal generation of biological organization is the product of the interaction of many independent components within biological networks. These networks constitute complex systems with properties greater than the sum of the constituent parts. During somitogenesis, a temporal patterning mechanism, the oscillator, gives rise to a emergent spatial pattern that presages the formation of morphological segments. This oscillator creates stripes/waves of gene expression that repeatedly travel through the field of cells about to undergo segmentation. Our first objective is to establish a more detailed chronology of the cyclical changes in gene expression and morphology that occur during zebrafish somitogenesis using vivo imaging, carefully staged embryos and fluorescent in situ hybridization. Recent work, including our own, has found that the Notch signaling pathway has successive functions in both the oscillator mechanism and in a later process that more directly regulates morphological segmentation. In the zebrafish, we have shown that the Notch ligands aei/deltaD, bea/deltaC and Notch target gene her1 each function within the oscillator mechanism, but each has a distinct mutant phenotype. Our second aim is to distinguish the functions of the two delta homologs by comparative phenotypic analysis of the two delta mutants, double mutant analysis, ectopic expression experiments and genetic mosaics. We hypothesize that aei/deltaD and beaJdeltaC have distinct signaling roles within the oscillator circuit and that the relative function and intensities of the delta signals vary at different positions within the segmenting tissue. The third aim examines the function of her1 within the oscillator. We will determine if Her1 protein levels oscillate and if the stability of the protein determines the frequency of the oscillator. The final aim is to develop a real-time readout of the oscillator in order to visualize the oscillations in live embryos. Cumulatively, these experiments address how local signaling interactions between a cell and its immediate neighbors via the Notch pathway give rise to an emergent, higher-level of organization within the paraxial mesoderm. This analysis of the genetic network that governs somitogenesis is central to our understanding of vertebrate development and has broad implications concerning the complex properties of biological circuits.