| Popis: |
Multicolour 3D quantitative imaging of large tissue volumes is pivotal to understand the development and organization of tissues as well as interactions of cells and the distribution of secreted molecules in situ. However, in situ imaging is technically challenging. In particular, the preparation and imaging of large bone and marrow sections, as well as faithful imaging and quantification of individual secreted molecules is not straightforward or possible with current techniques. The localization of most molecules in situ therefore remains unknown. In the work presented in this thesis, I developed together with my colleagues an integrated pipeline to generate reproducible high-dimensional quantitative data from bone marrow and any other tissue. Further, I present my efforts in adapting the Proximity Ligation Assay for large volume in situ imaging of individual molecules, including new 3D quantification strategies and software. Using these approaches, we measured the spatial relationship between hematopoietic cells, bone surfaces and Schwann cells within bone marrow. We further created an atlas of nonhematopoietic cells in the bone marrow and I investigated the process of metastatic colonialization of the bone marrow. I then looked into the distribution of individual C-X-C motif ligand 12 (CXCL12) molecules and found broad CXCL12 distribution with local enrichment at bone surfaces, but no long-range intra-marrow CXCL12 gradients, correcting current assumptions about migration control of hematopoietic stem cells in bone marrow. Overall the here presented technique developments and adaptations will enable new discoveries down to the molecular level in situ. While the biological findings answer some of the current questions in bone marrow biology, the questions raised by my findings represent the basis for more research in this new branch of quantitative molecular in situ research. |
