| Description |
Caenorhabditis elegans is constituted of ~1000 somatic cells and a large germline, which start proliferating at the middle of the development cycle (Larvae 3 stage). C. elegans provided fundamental insights in molecular mechanisms used to control gene expression throughout embryonic and post-embryonic development. However, the notorious difficulty to easily extract tissues has impeded the study of tissue-specific aspects of gene regulation. Yet, based on results from other non-"genome-wide" approaches, it has been suggested that the control of gene expression might rely on different molecular mechanisms.
I developed an approach to isolate bulk tissues from whole organisms. A GFP protein is fused to a C. elegans protein anchored to the nuclear envelope. The fusion protein is expressed under the control of a known tissue-specific promoter and the construct is inserted in a single known location in C. elegans genome by mos1-driven transposition. This way, nuclei from a single tissue are fluorescently labelled. Such reporter strain can be collected in large amounts. Subsequently, a nuclei preparation is performed (standard procedure), followed by a sorting procedure. Such procedure requires a bright fluorescent signal from the reporter, so an immunostaining of the reporter is usually performed to enhance its fluorescence. Then, nuclei are resuspended in a specific buffer and their characteristics are recorded in a cell sorter. The DNA content can be labelled by a DNA staining agent, helping the experimenter to identify the nuclei in the suspension being recorded. Specific settings of the cell sorted have to be carefully optimized for C. elegans nuclei, and the gating strategy to select for positive nuclei is empirically chosen to reach a purity of > 95% of positive nuclei. The nuclei after sorting can be used in an Assay for Transposase Accessible Chromatin (ATAC-seq) or for RNA-seq, after specific clean-up steps. |
| Impact |
This technique allowed us to map chromatin accessibility landscape in the five major tissues of C. elegans, as well as its tissue-specific expression, in the young adult developmental stage, when the germline (one of the five major tissues) is functional and produces oocytes. During this project, a report presented the tissue-specific expression landscape of the worm in a much younger developmental stage (Larvae 2, an immature larvae stage) was published. Our data correlates well with this report, and brings additional insights in the gene expression patterns in a functional germline.
Besides, this project uncovered a germline-specific chromatin accessibility landscape, that was not observed at regulatory elements accessible in somatic tissues. This result is under investigation and will soon be ready to be submitted for peer-reviewed publication. |
