Analysis of a Gene Regulatory Network in Early Animal Development

早期动物发育中的基因调控网络分析

• 批准号: 0517214
• 负责人: Charles Ettensohn
• 金额: $ 26万
• 依托单位: Carnegie-Mellon University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0517214&HistoricalAwards=false
• 关键词: Analysis; Gene; Regulatory; Network; Early

This proposal examines fundamental mechanisms that underlie the patterning of early animal embryos, using the sea urchin as a model system. Specifically, the proposal examines a key transcriptional gene regulatory network (GRN) in early sea urchin development. This GRN drives the specification and differentiation of the large micromere-primary mesenchyme cell (PMC) lineage. The large micromeres and their descendants transmit critical inductive signals early in development and later execute a dramatic sequence of morphogenetic behaviors, including epithelial-mesenchymal transition, direction cell migration, cell fusion, and secretion of a biomineralized skeleton.The proposal also addresses the long-standing problem of embryonic regulation. How can early differential gene expression in embryos, entrained in the unfertilized egg, be reconciled with labile blastomere fates during early development? Sea urchin embryos are famous for their regulative properties and some of the most spectacular examples involve ectopic specification of PMCs. Although the PMC GRN is normally activated only in the large micromeres, any blastomere of the early cleavage stage embryo can give rise to PMCs underappropriate experimental conditions. Recent progress in identifying gene networks and signaling pathways that control early sea urchin development provides a new opportunity to address the historic problem of embryonic regulation in a modern context.Specific Aim 1 examines the PMC GRN as it is deployed during normal development.1) Dr. Ettensohn will identify mechanisms that normally restrict activation of the GRN specifically to the large micromere lineage, focussing on the roles of beta-catenin levels and unequal cell division. Using molecular biological and pharmacological methods, we will manipulate levels of nuclear beta-catenin and patterns of cell division in early blastomeres and determine whether this causes ectopic activation of the PMC GRN.2) He will use fluorescent, multiplex in situ hybridization and intron probes to determine the temporal order of activation of genes in the network. This information will be critical in elucidating potential regulatory interactions between genes. 3) He will expand the PMC GRN by identifying new components, including downstream "morphoregulatory" genes. This will be accomplished by scaling-up a successful pilot PMC EST project which identified several critical genes in the GRN. The overarching goal is to develop a complete picture of this GRN that links the earliest molecular patterning events in the embryo to specific morphogenetic cell behaviors during gastrulation.Specific Aim 2 examines the PMC GRN as it is deployed during regulative development. Using molecular probes and morpholinos, he will analyze the 1) expression, 2) function, and 3) regulatory interactions of the known upstream components of the PMC GRN under several different experimental scenarios that induce ectopic activation of the GRN. The broader impact of the proposed research is derived from the training of undergraduate students, graduate students, and postdoctoral fellows. In addition, the work will contribute to the development and analysis of a large-scale PMC "gene catalogue" that will represent the most complete picture of the program of gene expression in a specific embryonic cell type in any developing embryo. This data set will be a valuable community resource.

该提案使用海胆作为模型系统，研究了早期动物胚胎模式的基本机制。具体来说，该提案研究了早期海胆发育中的关键转录基因调控网络（GRN）。该 GRN 驱动大微米原代间充质细胞 (PMC) 谱系的规范和分化。大微米及其后代在发育早期传递关键的诱导信号，随后执行一系列引人注目的形态发生行为，包括上皮-间质转化、定向细胞迁移、细胞融合和生物矿化骨架的分泌。该提案还解决了胚胎调控的长期问题。未受精卵中夹带的胚胎早期差异基因表达如何与早期发育过程中不稳定的卵裂球命运相协调？海胆胚胎以其调节特性而闻名，其中一些最引人注目的例子涉及 PMC 的异位规范。虽然 PMC GRN 通常仅在大微米中被激活，但早期卵裂阶段胚胎的任何卵裂球都可以在适当的实验条件下产生 PMC。最近在识别控制早期海胆发育的基因网络和信号通路方面取得的进展，为解决现代背景下胚胎调控的历史性问题提供了新的机会。具体目标 1 检查正常发育过程中部署的 PMC GRN。1) Ettensohn 博士将确定通常将 GRN 的激活限制在大微米谱系上的机制，重点关注 β-连环蛋白水平和 细胞分裂不均等。使用分子生物学和药理学方法，我们将操纵早期卵裂球中核β-连环蛋白的水平和细胞分裂模式，并确定这是否会导致 PMC GRN 的异位激活。2) 他将使用荧光、多重原位杂交和内含子探针来确定网络中基因激活的时间顺序。这些信息对于阐明基因之间潜在的调控相互作用至关重要。 3) 他将通过识别新的组件（包括下游“形态调节”基因）来扩展 PMC GRN。这将通过扩大成功的 PMC EST 试点项目来实现，该项目确定了 GRN 中的几个关键基因。总体目标是开发该 GRN 的完整图像，将胚胎中最早的分子模式事件与原肠胚形成过程中特定的形态发生细胞行为联系起来。具体目标 2 检查 PMC GRN 在调节发育过程中的部署情况。他将使用分子探针和吗啉，在几种不同的诱导 GRN 异位激活的实验场景下，分析 PMC GRN 已知上游组件的 1) 表达、2) 功能和 3) 调节相互作用。拟议研究的更广泛影响来自于对本科生、研究生和博士后的培训。此外，这项工作将有助于大规模 PMC“基因目录”的开发和分析，该目录将代表任何发育胚胎中特定胚胎细胞类型的基因表达程序的最完整图片。该数据集将成为宝贵的社区资源。