Functional analysis of the Argonaute family proteins during early embryonic and germ cell development in the mouse

  • Stefano Comazzetto

    Student thesis: Doctoral ThesisDoctor of Philosophy


    Argonaute proteins are known regulators of gene expression through their association with small non-coding RNAs. Specifically in mammals, microRNAs (miRNA) and short interfering RNAs (siRNAs) bind to Ago-like subclade of the Argonaute family to regulate gene expression, while Piwi-interacting RNAs (piRNAs) associate with PIWI proteins to silence transposons in the germ line.
    In mammals, genetic experiments revealed that Ago2 is the central effector for both siRNA and miRNA silencing pathways in several developmental processes. Phosphorylation of serine 388 of murine protein is dependent on the MK2 and Akt/PKB pathways and regulates Ago2 localization. I created a nonphoshorylatable Ago2S388A allele to study this phosphorylation event during mouse development. Lack of Ago2 phosphorylation is dispensable for adult hematopoiesis, oogenesis, and for the MK2-dependent endotoxic stress response. Furthermore, I discovered a partially penetrant lethal phenotype of Ago2S388A/S388A embryos at peri-implantation in mixed 129P2xC57Bl6N and 129S2 genetic backgrounds, but not in C57Bl/6N background. In conclusion, I demonstrated for the first time that mutation of a post-translational modification site has a differential phenotypic outcome depending on the genetic background.
    Genetic ablation of PIWI-proteins in the mouse leads to a block in male meiosis. Strikingly, loss of Miwi2 causes an additional progressive loss of germ cells, reminiscent of a stem cell phenotype. However, Miwi2 levels are high in fetal testis and become undetectable soon after birth. I generated a Miwi2tdTomato transcriptional reporter allele and proved that Miwi2 expression marks a subpopulation of spermatogonial cells that possesses surface markers, cell cycle and gene expression profile reminiscent of stem cells in juvenile testis. Comparison of juvenile and adult Miwi2-expressing cells revealed a significant difference in Thy-1 surface expression, which could be indicative of a higher self-renewal capacity in young cells. In summary, these data posit that Miwi2 expression defines a population of bona fide stem cells in the mouse testis.
    Date of Award2013
    Original languageEnglish
    SupervisorStephen Keyse (Supervisor) & Dónal O'Carroll (Supervisor)

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