Fmr1 Function and Repeat Expansion in the Developing Germline

项目摘要 这项建议的目标是将胎儿生殖细胞发育事件与胎儿生殖细胞的起源和遗传联系起来。 与脆性X疾病相关的Fmr1基因突变。有证据表明，Fmr1 三核苷酸重复扩增发生在生殖细胞的发育过程中，然而Fmr1的准确时间 Fmr1蛋白(FMRP)的重复扩增、代际传递机制和功能 这一血统尚不清楚，因为很少有研究检查整个配子池或其前体。 Fmr1缺乏的小鼠模型以及Fmr1-PM为阐明这些机制提供了机会 正在开发中。我们的初步数据支持Fmr1在保护胎儿胚胎基因组完整性方面的作用 细胞，提示胎儿生殖细胞的程序性凋亡期在睾丸中以不同的方式起作用 而不是卵巢。这里提出的研究将检验胎儿胚胎发育的总体假设 基于基因组中Fmr1的功能，细胞为Fmr1-PM扩增提供了机会之窗 完整性，但另一方面，发育不利于生殖细胞的代际传递 与雌性生殖系相比，雄性生殖系中的扩增。脆性X综合征，以及 其他三核苷酸重复疾病，因其病理性重复而被称为一把‘双刃剑’ DNA修复所需的基因发生了扩张。在目标1中，我们将研究FMRP在 保护发育中的生殖细胞的完整性并识别其RNA靶标。鉴于有证据表明 病理性CGG扩增发生在Fmr1转录过程中，这些研究将为 生殖细胞发育过程中对Fmr1重复扩增的脆弱时期。尽管它的尺寸 遗传性Fmr1前突变随着母亲年龄的增加而增加，这一信息源于成功使用 卵母细胞，而对整个配子池一无所知。在目标2中，我们将检验假设 FMRP1增加胎儿卵原细胞减数分裂I的保真度，而Fmr1 CGG重复扩增干扰 减数分裂并影响成年卵母细胞的质量。这些研究将阐明两者之间的关系 Fmr1前突变、卵母细胞质量和胎儿卵巢减数分裂进入动力学之间的关系。在《目标3》中我们 将使用基于Crispr的成像和下一代测序来揭示 发生在正常男性和女性生殖细胞发育过程中的凋亡重复序列的多样性 Fmr1前突变小鼠的扩增。这些研究将提供对儿童发育起源的洞察。 病理性Fmr1重复扩增和潜在的其他突变。

PROJECT ABSTRACT The goal of this proposal is to connect the events of fetal germ cell development to the origin and inheritance of mutations in the Fmr1 locus, which are associated with Fragile X diseases. Evidence suggests that Fmr1 trinucleotide repeat expansions occur during development of germ cells, however the precise timing of Fmr1 repeat expansions, mechanism for intergenerational transmission, and function of the Fmr1 protein (FMRP) in this lineage remains unclear, as few studies have examined the entire pool of gametes or their precursors. Mouse models of Fmr1 deficiency as well as Fmr1-PM provide an opportunity to elucidate these mechanisms in development. Our preliminary data support a role for Fmr1 in protecting the genomic integrity of fetal germ cells and suggest that the period of scheduled apoptosis of fetal germ cells acts in divergent ways in the testis versus the ovary. The studies proposed here will test the overall hypothesis that the development of fetal germ cells provides a window of opportunity for Fmr1-PM amplifications based on the function of Fmr1 in genomic integrity, but on the other hand development selects against the intergenerational transmission of germ cells with amplifications in the male germline as compared to the female germline. Fragile X syndrome, along with other trinucleotide repeat diseases, has been called a `double-edged sword' because the pathological repeat expansion occurs in a gene required for DNA repair. In Aim 1, we will investigate the function of FMRP in protecting the integrity of developing germ cells and identify its RNA targets. Given the evidence that pathological CGG amplification occurs during Fmr1 transcription, these studies will provide critical insight into the periods of vulnerability to Fmr1 repeat amplification during germ cell development. Although the size of inherited Fmr1 pre-mutations increases with maternal age, this information is derived from successfully used oocytes, whereas nothing is known about the entire gamete pool. In Aim 2, we will test the hypothesis that FMRP1 increases fidelity of meiosis I in fetal oogonia whereas Fmr1 CGG repeat expansions interfere with meiosis and compromise the quality of oocytes in the adult. These studies will elucidate the relationship between Fmr1 pre-mutation, oocyte quality, and the dynamics of meiotic entry in the fetal ovary. In Aim 3 we will use both Crispr-based imaging and next generation sequencing to reveal the impact of massive waves of apoptosis that occur during normal male and female germ cell development on the diversity of repeat expansions in Fmr1 pre-mutation mice. These studies will provide insight into the developmental origin of pathological Fmr1 repeat expansions and potentially other mutations.