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.
The expansion of a CGG trinucleotide repeat region on the X-chromosome in the Fragile X mental retardation 1 (FMR1) locus is associated with a number of diseases, ranging from primary ovarian insufficiency (FXPOI) to ataxia (FXTAS) to profound intellectual disability known as Fragile X Syndrome (FXS). One in 151 women is a carrier of a small repeat expansion known as the FMR1 pre-mutation; 15% will develop FXTAS, 25% will be diagnosed with FXPOI, and all carriers risk giving birth to a child with FXS.