Plasticity in an embryonic gene regulatory network

 描述(申请人提供)：拟议研究的主要目标是揭示基因调控网络(GRN)结构确保对遗传变异的持续输出的机制，并从机械上剖析胚胎细胞从多潜能转化为特定分化状态的过程。描述良好的GRN在线虫胚胎发生过程中指导内胚层的指定和分化，将用于研究这些问题。这种GRN是由母体转录因子SKN-1和三重冗余的Wnt、MAPK和src信号系统共同作用启动的。对97个具有独特单倍型的线虫野生分离株(同型)的分析表明，内胚层形成对SKN-1和MOM-2/WNT的需求有很大的差异，这使得全面剖析基因组在GRN作用中的变化成为可能。此外，我们发现一种新的Notch信号系统在早期胚胎中建立了一种记忆状态，激活了胚胎的多潜能承诺转换(MCT)，并防止细胞在发育后期被内胚层GRN的组成部分重新编程。我们将在这些初步发现的基础上，通过三个特定的目的来揭示内胚层GRN的遗传和发育可塑性的机制。在目标1中，我们将通过全基因组关联研究和QTL分析来确定相关基因座及其相互作用，从而表征线虫在SKN-1和Wnt信号转导需求中广泛变异的分子和遗传基础。我们将量化选定菌株中内胚层GRN成分的表达差异，目的是了解基因变化如何改变网络中的通量，并允许可塑性。在目标2中，我们将研究Notch信号系统的Acton和两个新的分泌型Notch配体DSL-1和DSL-3在调节发育可塑性和MCT发病时间方面的作用。我们将评估这样的假设，即这个信号系统在主要外胚细胞AB细胞的谱系中自主发挥作用，通过可扩散的分泌分子的作用来调节MCT，以及它作用于 MCT通过调节染色质缩合。在目标3中，我们将进行基于RNAi的筛选，以确定在胚胎发育过程中调节发育可塑性所需的一套全面的基因。我们将分析及时执行MCT所需基因的谱系、区域和时间特异性，评估基因在防止细胞类型分化的替代方案中的作用广度，并评估基因发挥作用的分子途径。这项研究的发现可能有助于更好地理解再生医学中产生新的替代组织和器官所需的过程。它们还将作为一种范例，用于了解个体的基因型别与其对药理药剂的反应之间的关系，从而促进个性化医学的进步。

 DESCRIPTION (provided by applicant): The major objectives of the proposed research are to reveal the mechanisms by which gene regulatory network (GRN) architecture ensures a constant output in response to genetic variation and to mechanistically dissect the processes that convert embryonic cells from multipotentiality to a committed state of differentiation. The well-described GRN that directs specification and differentiation of the endoderm during C. elegans embryogenesis will be used to investigate these problems. This GRN is initiated by the combined action of a maternal transcription factor, SKN-1, and a triply redundant Wnt, MAPK, and src signaling system. Analysis of 97 C. elegans wild isolates (isotypes), each with a unique haplotype, revealed dramatic variation in requirements for SKN-1 and MOM-2/Wnt in endoderm formation, allowing comprehensive dissection of genomic changes in GRN action. Further, we found that a novel Notch signaling system establishes a memory state in the early embryo that activates the embryonic multipotentiality ¿ commitment transition (MCT) and prevents cells from being reprogrammed by components of the endoderm GRN later in development. We will build on these preliminary findings to reveal mechanisms of genetic and developmental plasticity in the endoderm GRN through three specific aims. In Aim 1, we will characterize the molecular and genetic basis for broad variation seen among the C. elegans isotypes in the requirement for SKN-1 and Wnt signaling by identifying the relevant loci and their interactions via genome-wide association studies and QTL analysis. We will quantify expression differences in components of the endoderm GRN in selected strains with the goal of understanding how the genotypic changes alter flux, and allow for plasticity, in the network. In Aim 2, we will investigate the acton of the Notch signaling system and two novel secreted Notch ligands, DSL-1 and -3, in regulation of developmental plasticity and the timing of onset of the MCT. We will evaluate the hypotheses that this signaling system functions autonomously within the lineage of the major ectoblast, the AB cell, to regulate the MCT by the action of diffusible secreted molecules and that it acts on the MCT by regulating chromatin condensation. In Aim 3, we will perform RNAi-based screens to identify the comprehensive set of genes required for regulating developmental plasticity during embryogenesis. We will analyze the lineage, regional, and temporal specificity of genes required for timely execution of the MCT, assess the breadth of action of the genes in preventing alternative programs of cell type differentiation, and evaluate the molecular pathways through which the genes function. Findings from this research may lead to a better understanding of the processes required to generate new replacement tissues and organs in regenerative medicine. They will also serve as a paradigm for understanding the relationship between an individual's genotype and their responsiveness to pharmacological agents, thereby contributing to advances in personalized medicine.