Analysis of a Gene Regulatory Network in Early Animal Development

本研究以海胆为模型系统，探讨了早期动物胚胎形成模式的基本机制。具体而言，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）。这种GRN驱动大微粒-初级间充质细胞（PMC）谱系的规范和分化。大微粒及其后代在发育早期传递关键的诱导信号，随后执行一系列戏剧性的形态发生行为，包括上皮-间质转化、细胞方向迁移、细胞融合和生物矿化骨架的分泌。该提案还解决了长期存在的胚胎调控问题。在未受精卵中携带的胚胎中的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？海胆胚胎以其调控特性而闻名，其中一些最引人注目的例子涉及pmc的异位规范。尽管PMC GRN通常只在大的微粒中被激活，但在适当的实验条件下，早期卵裂期胚胎的任何卵裂球都可以产生PMC。在确定控制早期海胆发育的基因网络和信号通路方面的最新进展为解决现代背景下胚胎调控的历史问题提供了新的机会。具体目标1检查PMC GRN在正常发育期间的部署情况。1) Ettensohn博士将确定通常限制GRN激活的机制，特别是大微粒谱系，重点关注β -连环蛋白水平和不平等细胞分裂的作用。利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的模式，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。这一信息对于阐明基因间潜在的调控相互作用至关重要。3)他将通过识别包括下游“形态调节”基因在内的新成分来扩展PMC GRN。这将通过扩大一个成功的PMC EST试点项目来实现，该项目确定了GRN中的几个关键基因。总体目标是建立一个完整的GRN图谱，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检查了在法规制定期间部署的PMC GRN。利用分子探针和morpholinos，他将分析1)表达，2)功能，以及3)在几种诱导GRN异位激活的不同实验场景下，已知的PMC GRN上游组分的调控相互作用。拟议研究的更广泛影响来自对本科生、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中的胚胎中特定胚胎细胞类型的基因表达程序的最完整画面。这个数据集将是一个有价值的社区资源。

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.