The C. elegans Germline: A Test Tube for Cell and Developmental Biology

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SUMMARY/ABSTRACT The earliest stages of embryonic development are guided by mRNAs, proteins, and organelles provided by the maternal germline. As the embryo switches from maternal to zygotic control, mechanisms are deployed to clear maternal messenger RNAs (mRNAs) from the embryo after they have completed their functions. The molecular mechanisms of maternal mRNA clearance are incompletely understood but are of intensive interest for the field. We recently discovered that a highly conserved RNA-binding protein is required for maternal mRNA clearance in the nematode Caenorhabditis elegans. We defined the mRNAs associated with this protein, the conserved Rbfox homolog SPN-4, in oocytes using RNA-binding protein immunopurification and RNA sequencing. Remarkably, we found that many SPN-4-associated are rapidly destabilized and cleared after fertilization. We are testing the hypothesis that SPN-4 clears many of its associated mRNAs in somatic blastomeres through its association with the CCR4-NOT deadenylase complex, which functions in mRNA destabilization. Because the CCR4-NOT deadenylase complex is required for viability and germline development, we developed new tools to study its role during early embryogenesis using the auxin-inducible degradation system. A key technology needed to complete these studies is quantitative single-molecule fluorescence in situ hybridization (smFISH). This administrative supplement to the parent award would enable us to upgrade our existing fluorescence microscope for high-throughput quantitative smFISH studies by adding a motorized stage and deconvolution software. Because mRNAs are inherently unstable, our experimental protocols require the microscopic imaging to be completed immediately within a day of detection. The motorized stage will enable rapid and automated image acquisition of the many embryos of different stages required to assess and document the RNA clearance mechanisms. Deconvolution algorithms are needed to achieve optimal resolution to image and quantify single mRNA transcripts. We expect that these upgrades to our existing microscope will enable us to define the molecular mechanisms by which a conserved RNA-binding protein recruits the CCR4-NOT deadenylase complex to clear maternal mRNAs shortly after fertilization to remodel the transcriptome of the early embryo. These experiments in the C. elegans experimental organism will instruct our understanding of early postfertilization development by analogy if not homology.