Fmr1 Function and Repeat Expansion in the Developing Germline

项目摘要 这项提案的目标是将胎儿生殖细胞发育的事件与生殖细胞的起源和遗传联系起来， 在Fmr 1基因座突变，这是与脆性X疾病。有证据表明，FMR 1 三核苷酸重复扩增发生在生殖细胞的发育过程中，然而， 重复扩增，代际传递机制，和功能的Fmr 1蛋白（FMRP）， 这一谱系尚不清楚，因为很少有研究对整个配子库或其前体进行过研究。 Fmr 1缺陷的小鼠模型以及Fmr 1-PM提供了阐明这些机制的机会 参与发展.我们的初步数据支持Fmr 1在保护胎儿生殖基因组完整性中的作用 细胞，并表明胎儿生殖细胞的预定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测试整个假设，即胚胎生殖的发育 细胞提供了一个窗口的机会，Fmr 1-PM扩增的基础上的功能，Fmr 1在基因组 完整性，但另一方面，发展选择反对生殖细胞的代际传递 与雌性生殖系相比，在雄性生殖系中具有扩增。脆性X综合征，沿着 其他三核苷酸重复疾病，被称为“双刃剑”，因为病理重复 扩增发生在DNA修复所需的基因中。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鉴定其RNA靶点。鉴于有证据表明 病理性CGG扩增发生在Fmr 1转录过程中，这些研究将提供关键的洞察力， 生殖细胞发育过程中易受Fmr 1重复扩增影响的时期。虽然规模 遗传性Fmr 1前突变随着母亲年龄的增加而增加，这一信息来自成功使用 卵母细胞，而对整个配子池一无所知。在目标2中，我们将检验以下假设： FMRP 1增加胎儿卵原细胞减数分裂I的保真度，而Fmr 1 CGG重复扩增干扰减数分裂I。 减数分裂和妥协的质量卵母细胞在成年人。这些研究将阐明 Fmr 1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， 将同时使用基于Crispr的成像和下一代测序来揭示大规模海啸的影响。 细胞凋亡发生在正常男性和女性生殖细胞发育的多样性重复 在Fmr 1突变前小鼠中的扩增。这些研究将有助于深入了解 病理性Fmr 1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.