Hochgeschwindigkeitszellsortierer
Final Report Abstract
Project Elke Roovers „Sorting of zebrafish primordial stem cells (PGCs)“ (AG Prof. Dr. Ketting, Institut für Molekulare Biologie gGmbH, Mainz): For an in vivo approach of single cell RNA sequencing (scRNA-seq), GFP-positive cells of zebrafish embryos were sorted at different stages during early development in a control and a mutant background. The GFP-transgene marks the primordial germ cells (PGCs) that will form oocytes or sperm cells in the adult gonad. The mutant shows defects in PGC formation early during development, which prompted us to look at this defect at a transcript level of single cells. The cells were collected at 3 different stages of development: 1) The moment PGCs are first being specified (3.5 hours post fertilization, hpf), 2) the moment they migrate to the future gonad area (8hpf) and 3) when they have arrived at the gonadal ridge (24hpf). After overcoming various problems, sample preparation due to mechanical stress, gating out unwanted cell types, mostly from the york being the tissue at the most apparent at that embryonic state, gating on the GFP-positive cells (<0.1%, see figure 2) or diminishing the mechanical forces during the sorting process, the single target cells were sorted into 96 well microtiter plates in order to sequence for RNA. In order to deal with the high autofluorescence of the sample cells, wildtype samples were always measured to define the cut-off. Project Natalia Soshnikova (AG Dr. Soshnikova, Institute of Molecular Biology gGmbH, Mainz): During the past years, our research was focused on in-depth molecular characterization of the embryonic and adult intestinal epithelium on both transcriptional and chromatin levels. To gain insight into the molecular properties of mouse embryonic intestinal epithelium, we performed whole transcriptome RNA- sequencing of EpCAM positive epithelial cells from small intestines at E12.5 as well as of Lgr5-EGFPhigh adult ISCs isolated by flow cytometry. Our work supports a model where the embryonic intestinal epithelium consists of transcriptionally heterogeneous cell populations, which are in turn different from the adult ISCs. We defined the molecular signatures of these intestinal epithelial progenitor pools by sequencing the transcriptomes of sorted cells from the embryonic small intestines at E13.5. To assess phenotypic differences between the progenitor cells we analysed their potential to form intestinal organoids in vitro. The progenitor and corresponding control cells from the embryonic small intestines were sorted at E13.5 and plated in Matrigel for 3D organoid cultures. As a result, we identified and characterized at the molecular and phenotypic levels distinct populations of embryonic intestinal epithelial progenitors that contribute to the adult ISCs pool. To define the role of chromatin during intestinal stem cell formation, maintenance and differentiation, we have generated genome-wide chromatin state maps for two embryonic stages (E12.5 and E14.5), adult ISCs, enterocytes and Paneth cells using ChIP and MBD-sequencing for low amount of sorted cells (2x105). Specifically, we assessed the distribution of 5meC, H3K4me3, H3K27me3, H3K27Ac and H2Az marks. Clustering and meta-analysis of all transcriptome and chromatin data defined a large number of potential regulatory elements, including distal, proximal as well as intergenic enhancers.
Publications
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Oligodendrocyte Precursor Cells Synthesize Neuromodulatory Factors. PLoS ONE, vol. 10, no. 5:e0127222
Dominik Sakry, Yigit H, Dimou L and Trotter J
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NeuroD1 reprograms chromatin and transcription factor landscapes to induce the neuronal program. The EMBO Journal, vol. 35, p. 24-45, 2016
Abhijeet Pataskar, Johannes Jung, Pawel Smialowski, Florian Noack, Federico Calegari,Tobias Straub & VijayTiwari
