Plasticity in an embryonic gene regulatory network

 描述（由申请人提供）：拟议研究的主要目标是揭示基因调控网络（GRN）架构确保响应遗传变异的恒定输出的机制，并从机械上剖析将胚胎细胞从多潜能转化为定向分化状态的过程。GRN在C.线虫胚胎发生将被用来研究这些问题。该GRN由母体转录因子SKN-1和三重冗余Wnt、MAPK和src信号系统的联合作用启动。97 C.分析每种具有独特单倍型的秀丽线虫野生分离株（同种型）揭示了内胚层形成中对SKN-1和SKN-2/Wnt的需求的显著变化，允许全面剖析GRN作用中的基因组变化。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，激活了胚胎多潜能性承诺转换（MCT），并防止细胞在发育后期被内胚层GRN的成分重新编程。我们将建立在这些初步的研究结果，揭示内胚层GRN通过三个具体目标的遗传和发育可塑性的机制。在目标1中，我们将描述C.通过全基因组关联研究和QTL分析鉴定相关基因座及其相互作用，研究线虫同种型对SKN-1和Wnt信号传导的需求。我们将量化所选菌株中内胚层GRN组分的表达差异，目的是了解基因型变化如何改变通量，并允许网络中的可塑性。目的2：研究Notch信号系统和两种新的分泌型Notch配体DSL-1和DSL-3对发育可塑性和MCT发生时间的调节作用。我们将评估这样的假设，即该信号系统在主要的外胚层（AB细胞）谱系内自主发挥作用，通过可扩散的分泌分子的作用调节MCT，并且它作用于细胞外基质。 MCT通过调节染色质凝聚。在目标3中，我们将进行基于RNAi的筛选，以确定在胚胎发生过程中调节发育可塑性所需的一系列基因。我们将分析及时执行MCT所需的基因的谱系、区域和时间特异性，评估基因在阻止细胞类型分化的替代方案中的作用范围，并评估基因发挥作用的分子途径。这项研究的发现可能会导致更好地了解再生医学中产生新的替代组织和器官所需的过程。它们还将作为一个范例，用于理解个体的基因型和他们对药物的反应之间的关系，从而有助于个性化医疗的进步。

 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.