Regulation of Germ Cell Fate During Embryogenesis

胚胎发生过程中生殖细胞命运的调节

基本信息

批准号：
9999114
负责人：
GERALDINE Catherine Joelle SEYDOUX
金额：
$ 40.12万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-01 至 2025-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9999114
关键词：
Affect Animals Biochemical CRISPR/Cas technology Caenorhabditis elegans Cell Differentiation process Cell Proliferation Cell Survival Cells Characteristics Chromatin Complex Cytoplasmic Granules Development Developmental Biology Disease Embryonic Development Family Fertilization Funding Gene Deletion Gene Expression Gene Expression Profile Gene Proteins Gene Silencing Genes Genetic Genetic Transcription Genome Germ Germ Cells Goals Lobular Neoplasia Location Methods Molecular Mutate Oocytes Open Reading Frames Organelles Organism Pattern Phenotype Play Polycomb Process Property Proteins RNA RNA-Binding Proteins Regulation Reproduction Role Scaffolding Protein Shapes Somatic Cell Specific qualifier value Structure of primordial sex cell Surface Time To specify Work arm base chromatin modification genetic analysis genome editing genome-wide innovation interest member mutant nano next generation premature preservation prevent programs protein expression protein function public health relevance rapid technique repaired scaffold tool transcription factor transcriptome transcriptome sequencing whole genome

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of this project is to characterize the mechanisms that specify the fate of the germline, a fundamental problem in developmental biology. In all animals, the founder cells of the germline (primordial germ cells) display two conserved characteristics: they possess germ granules, RNA granules specific to the germline, and they express members of the Nanos family of RNA-binding proteins. The goal of this proposal is to understand how germ granules and Nanos function together to specify the fate of primordial germ cells. In preliminary work, we have found that PGCs that lack Nanos activity prematurely activates the expression of ~1000 genes normally expressed in oocytes and somatic cells, a phenotype also seen in mutants that lack the Polycomb Repressive Complex. Specific Aim I will identify the mechanisms used by Nanos to prevent this massive gene mis-regulation. We have already identified one promising candidate Nanos target: LIN-15B, a likely component of the DRM transcription factor complex that activates gene expression in oocytes. Specific Aim II will examine the role of a new group of germ granule proteins we recently characterized. The MEG proteins form stabilizing scaffolds around the central cores of each germ granule. Our genetic analyses indicate that the MEGs, but not the proteins in the core, are essential for PGC fate. We will investigate how the MEGs, predicted to be intrinsically-disordered with no recognizable domain, function with Nanos to specify PGCs. Two technical breakthroughs support this application. First we have worked out methods to isolate PGCs in large enough numbers to determine their transcriptome by RNAseq. We will use this method to define how Nanos and germ granule proteins affect the PGC transcriptome at a genome-wide level. Second we have developed a highly efficient genome editing method, using CRISPR/Cas9, that allows us to mutate, tag, delete and replace any gene of interest in just 4 days. This method gives us unprecedented genetic and biochemical access to the genes and proteins that are the focus of this application.

描述（由适用提供）：该项目的长期目标是表征指定种系的命运的机制，这是发育生物学中的基本问题。在所有动物中，种系的创始细胞（原始生殖细胞）都显示出两个保守的特征：它们具有胚芽颗粒，特定于种系的RNA颗粒，并表达RNA结合蛋白的纳米家族的成员。该提案的目的是了解生殖颗粒和纳米的功能如何共同指定原始生殖细胞的命运。在初步工作中，我们发现缺乏纳米活性的PGC过早激活了通常在卵母细胞和体细胞中表达的〜1000个基因的表达，这是在缺乏多孔抑制复合物的突变体中看到的表型。具体目的，我将确定纳米用于防止这种大规模基因失调的机制。我们已经确定了一个承诺候选纳米靶标：LIN-15B，这是激活卵母细胞基因表达的DRM转录因子复合物的可能成分。特定的目标II将研究我们最近表征的新系列系列系列系列系列新系列颗粒蛋白的作用。 MEG蛋白在每个细菌颗粒的中心核周围形成稳定的支架。我们的遗传分析表明，MEGS（而不是核心中的蛋白质）对于PGC命运至关重要。我们将研究如何在没有可识别域的纳米功能的情况下进行本质上排序的MEGS来指定PGC。两个技术突破支持此应用程序。首先，我们已经制定了将PGC隔离足够大的方法，以确定RNASEQ的转录组。我们将使用这种方法来定义纳米和细菌颗粒蛋白如何在全基因组水平上影响PGC转录组。其次，我们使用CRISPR/CAS9开发了一种高效的基因组编辑方法，该方法使我们能够在短短4天内突变，标记，删除和替换任何感兴趣的基因。这种方法为我们提供了对本应用重点的基因和蛋白质的前所未有的遗传和生化获取。