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Title: Investigating the role of Wt1 in bone and marrow biology
Author: McHaffie, Sophie Louise
ISNI:       0000 0004 5991 1853
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2014
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The bones of the body vary in size and shape, but are fundamentally all composed of the same cell types: osteoblasts, osteoclasts, osteocytes, vascular cells, and sometimes marrow cells. Long bones are formed when mesenchymal stem cells (MSCs) give rise to chondrocytes i.e. cartilage cells, and osteoblasts i.e. bone cells. These develop to form layers of bone encasing a cartilagenous core which eventually becomes the marrow cavity. A recent study showed that deleting the Wilms’ tumour gene, Wt1, in adult mice causes a dramatic loss of bone and fat tissue, fat being another derivative of MSCs. This finding led me to ask whether Wt1 expression is involved in bone biology and whether it plays a functional role in the stem or progenitor populations. Wt1 is a transcription factor that acts as a mesodermal / mesenchymal regulator. It acts as a tumour suppressor gene with mutations leading to the eponymous paediatric kidney tumour. However, in adult cancers it has oncogene characteristics, being highly expressed in the tumours of tissues in which it is not normally present. It also plays a pivotal role in the epithelial to mesenchymal transition (EMT) and vice versa in developing heart and kidney, respectively. There is, however, no evidence of its involvement with EMT / MET in adults. Wt1 is expressed in various developing tissues and is particularly vital for kidney development. Due to its involvement as a regulator of EMT / MET during development and the phenotype observed following its deletion in vivo, we hypothesised that Wt1 is expressed in, and required for the function of mesenchymal stem or progenitor cells populations within the bone marrow. A Wt1-GFP knock in mouse was used to show that Wt1 expressing cells are found in the bone marrow, and also for the first time in the bone. The GFP population overlaps with a non-haematopoietic MSC population defined by 3 cell surface markers in the bone and marrow, as well as an osteoblast (OB) progenitor population. Using a tamoxifen inducible CreERT2 showed that Wt1 loss alters the proportion of GFP cells in the bone and marrow cells that overlap with these MSC and OB progenitor markers, but microarrays were needed to assess the functional effects of Wt1 deletion. Microarrays highlighted various pathways that were altered following the in vitro deletion of Wt1 in total bone and marrow culture, as well as the non-haematopoeitic GFP+ and GFP- populations. In bone cells, deleting Wt1 negatively affects various pathways related to MSCs and their derivatives, including collagen biosynthesis, cartilage development and muscle tissue development. Also negatively affected were Wnt signalling regulation and EMT regulation; this is the first time Wt1 has been shown to be involved in EMT in adult cells. These findings were validated using qRT-PCR to show the down regulation of various genes involved in each pathway, showing that as well as being expressed in these populations it is also playing a functional role. Ossification pathways were negatively altered in the cells not expressing Wt1 following the deletion of the gene suggesting that Wt1 may also be acting in a paracrine manner to play its role in bone homeostasis. As well as in adult tissues, Wt1 was found to be expressed during development in the limb tissue of e11.5 to e16.5 mice. Preliminary results show that Wt1 may also have a functional role during bone development, as loss of expression causes a reduction in the percentage of non-haematopoetic MSC cells in the e18.5 hindlimb. As well as this, preliminary lineage tracing experiments suggest that cells found at the bone surface are of Wt1+ origin. This thesis has also highlighted the importance of experimental conditions and controls, particularly for CFU-F assays. CreERT2, loxP sites, tamoxifen, oxygen tension levels, and gender all exert specific effects on colony formation, independent of Wt1 expression. In conclusion, these data identify Wt1 as a key player in bone development and homeostasis. The microarray results led to the conclusion that Wt1 has a functional role in several mesenchymal pathways and highlights various genes that are potential Wt1 targets and should be further investigated using ChIP-Seq methods.
Supervisor: Hastie, Nick Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Wt1 ; bone ; marrow ; mesenchyme