Loss of rad51 in zebrafish (Danio rerio): a novel Fanconi anaemia model

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anaemia (FA) gene, following the discovery of two patients carrying dominant negative mutations. FA is a hereditary DNA repair disorder characterised by various congenital abnormalities, progressive bone marrow failure and cancer predisposition. The cellular and molecular pathology of FA is poorly understood, resulting in a severe lack of effective treatment options. In this thesis, I describe the first viable vertebrate model of RAD51 loss. Phenotypic characterisation of zebrafish rad51 loss-of-function mutants showed that they develop key features of FA, including hypocellular kidney marrow, sensitivity to crosslinking agents and decreased size. Taking advantage of the unique properties of the zebrafish model, I show that some of these symptoms stem from both decreased proliferation, as well as increased apoptosis of embryonic haematopoietic stem and progenitor cells. Co-mutation of p$L was able to rescue the haematopoietic defects seen in the single mutants, but led to tumour development, underscoring the role of rad51 as a tumour suppressor. I further demonstrate that prolonged inflammatory stress can exacerbate the haematological impairment, leading to an additional decrease in kidney marrow cell numbers. In contrast, prolonged aldehyde-derived stress did not induce symptoms in the mutant fish. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signalling during embryogenesis leads to the haematological defects seen later in life in FA. It also strengthens the evidence for the involvement of haematopoietic stress, such as inflammation, in the development of bone marrow failure. Further research on this novel zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.This research was supported by a European Hematology Association PhD Studentshi

Single-cell RNA-sequencing uncovers transcriptional states and fate decisions in haematopoiesis.

The success of marker-based approaches for dissecting haematopoiesis in mouse and human is reliant on the presence of well-defined cell surface markers specific for diverse progenitor populations. An inherent problem with this approach is that the presence of specific cell surface markers does not directly reflect the transcriptional state of a cell. Here, we used a marker-free approach to computationally reconstruct the blood lineage tree in zebrafish and order cells along their differentiation trajectory, based on their global transcriptional differences. Within the population of transcriptionally similar stem and progenitor cells, our analysis reveals considerable cell-to-cell differences in their probability to transition to another committed state. Once fate decision is executed, the suppression of transcription of ribosomal genes and upregulation of lineage-specific factors coordinately controls lineage differentiation. Evolutionary analysis further demonstrates that this haematopoietic programme is highly conserved between zebrafish and higher vertebrates.The study was supported by Cancer Research UK grant number C45041/A14953 (to A.C. and E.A.), European Research Council project 677501 – ZF_Blood (to A.C.) and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute. The authors would like to thank WTSI Cytometry Core Facility for their help with index cell sorting and the Core Sanger Web Team for hosting the cloud web application. The authors would also like to thank the CRUK Cambridge Institute Genomics Core Facility for their contribution in sequencing the data

Overexpression of the oncostatin-M receptor in cervical squamous cell carcinoma is associated with epithelial-mesenchymal transition and poor overall survival.

BACKGROUND: Copy-number gain of the oncostatin-M receptor (OSMR) occurs frequently in cervical squamous cell carcinoma (SCC) and is associated with adverse clinical outcome. We previously showed that OSMR overexpression renders cervical SCC cells more sensitive to the major ligand oncostatin-M (OSM), which increases migration and invasion in vitro. We hypothesised that a major contribution to this phenotype would come from epithelial-mesenchymal transition (EMT). METHODS: We performed a comprehensive integrated study, involving in vitro cell line studies, in vivo animal models and numerous clinical samples from a variety of anatomical sites. RESULTS: In independent sets of cervical, head/neck and lung SCC tissues, OSMR expression levels correlated with multiple EMT-associated phenotypic markers and transcription factors. OSM treatment of OSMR overexpressing cervical SCC cells produced consistent EMT changes and increased tumour sphere formation in suspension culture. In a mouse model, OSMR overexpressing SCC cells treated with OSM showed significant increases in lung colonisation. The biological effects of exogenous OSM were mirrored by highly significant adverse overall survival in cervical SCCs with OSMR overexpression (N=251). CONCLUSIONS: OSM:OSMR interactions are able to induce EMT, increased cancer stem cell-like properties and enhanced lung colonisation in SCC cells. These changes are likely to contribute to the highly significant adverse outcome associated with OSMR overexpression in cervical SCCs.This work was supported by Cancer Research UK (Programme Grant A13080).This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Nature Publishing Group