INVESTIGATING IN VIVO COMPRESSIVE FORCES: CELL DIVISION, NUCLEAR INTEGRITY, CANCER INITIATION

Cells move through our bodies squeezing into tiny spaces. This stresses them, damaging their DNA or causing errors during cell division, which can promote cancer. However, it is difficult to visualise moving cells inside the body. I will use the transparent embryo of the Zebrafish to ask what happens when cells experience compression. I will study neural crest cells, which move through narrow spaces in fish embryos. These cells can cause a cancer, neuroblastoma. We still don't understand why neuroblastomas happen. One possibility is that they are a consequence of cells squeezing. Can neural crest divide correctly when compressed? Does compression damage chromosomes? To test this, I will generate more space for the cells by using a laser or by using genetic mutants that lack muscle tissue. Conversely, I will compress neural crest cells by filling the embryonic brain with gel. To ask how human neural crest cells react to being physically stressed, I will culture them in very small channels, or transplant them in living fish embryos to observe their behaviour live. These strategies will show how cells cope with physical stress in vivo and shed light on the origin of neuroblastomas.

In vitro studies show that physical confinement causes cellular stress, leading to DNA damage, nuclear envelope ruptures and mitotic errors. However, the consequences of mechanical stress during physiological in vivo migration have not been yet investigated. Using the transparent Zebrafish embryo, I will study migrating neural crest cells (NCCs, progenitors of melanocytes and several neural lineages) in an intact animal amenable to mechanical manipulations. My observations show that these cells are highly confined during their developmental migration, and oil microdroplet measurements of tissue-scale mechanical stresses show that they experience compressive forces of ~200 Pa in magnitude. Furthermore, I found that migrating trunk NCCs experience nuclear deformation and they occasionally undergo mitotic errors. Importantly, NCCs can originate a human childhood cancer, neuroblastoma, which has a high frequency of chromosomal amplifications and rearrangements. Using Zebrafish and human NCCs as a model, I will ask whether compressive mechanical stresses NCCs experience in vivo cause them to suffer DNA damage, mitotic errors, and chromosomal instability. To address these questions, I developed novel in vivo strategies to perturb microenvironmental confinement of Zebrafish NCCs. I will use genetic and laser perturbations to relieve compression from surrounding tissues. Conversely, I will apply ectopic compression to these cells using atomic force microscopy or by filling the embryonic neural tube with stiff gel. I will collaborate with Anestis Tsakiridis (Sheffield, co-I) to ask if human NCCs are prone to chromosomal instability under mechanical stress. I will culture these in microchannels or xenotransplant them into the Zebrafish trunk and perform live imaging to assess chromosomal rearrangements under physical confinement. This work will elucidate how cells cope with physiological mechanical stresses in vivo and shed light on the origin of neuroblastoma.