The Wilms’ tumour suppressor gene WT1, encodes a structurally diverse and multifunctional protein with tightly controlled expression throughout the development of several organ systems. Although initially defined as a tumor suppressor, WT1 has been found to be overexpressed in some cancers. How WT1 contributes to the shift from normal to aberrant development, or from normal function to oncogenic function, is poorly understood. Recent studies have shown an abundance of bidirectional transcription across metazoan genomes suggesting that non-coding antisense transcripts may have important roles in cell function. WT1-AS transcripts are capable of positively modulating WT1 protein levels in vitro, but relatively little is known about the functions of these antisense transcripts in vivo. The aim of this thesis was to characterize the role of a highly conserved region, Mcr2, located upstream of human and mouse WT1. Our data suggests that Mcr2 is not translated into protein and is transcribed in an antisense orientation. Mcr2 was found partially conserved in fish and well conserved in terrestrial vertebrates. By analysing novel mouse strains with genetically modified Mcr2 we have identified that Mcr2 may have a role in both fertility and embryonic survival, as well as regulating liquid homeostasis in the adult mouse.

The Wilms' tumour suppressor gene Wt1 is a key regulator of embryonic development and tissue homeostasis. In humans, mutation in the gene may lead to childhood kidney cancer, severe glomerular kidney diseases, gonadal dysgenesis and, in rare cases, heart diseases. The importance of WT1 in embryonic development is related to its crucial function in the regulation of two cellular plasticity processes: the epithelial to mesenchymal transition (EMT) and the reverse process, the mesenchymal to epithelial transition (MET). WT1 expression persists during the waves of EMT and MET that generate certain mesodermal tissues. In fact, WT1 is a major regulator of both transitions and it is essential for the survival of mesenchyme progenitors. Furthermore, it has been proposed that WT1 is required for the derivation of progenitors from different mesothelia, possibly through an EMT. Progenitors expressing WT1 are believed to differentiate into different cell types, giving rise to coronary vasculature, adipocytes and hepatic stellate cells. In my PhD I aimed to investigate the instructive role of different WT1 isoforms. To address this, the first goal was to generate a single plasmid that would accommodate all necessary components of an inducible system, in order to derive cellular models for the inducible expression of WT1 single isoforms. Second, I aimed to understand the processes that the single variants were sufficient to drive. Therefore, I started with the establishment of two cellular models for the inducible expression of the main four isoforms of WT1 (with or without the exon 5 and/or the KTS, here referred as +/+, +/-, -/+ and -/-). I cloned different plasmids carrying doxycycline inducible WT1 isoforms and derived single stable clones in two epithelial kidney cell lines that do not express WT1: the MDCK and the IMCD3 cells. I then analysed the expression profiles of the clones, using either microarray or RNA sequencing, and performed cellular assays to characterize the cells after WT1 induction. Overall, WT1 induction did not affect cell growth, cell cycle, cell migration or anchorage independent growth, suggesting that the expression of WT1 in these two cell lines does not change their oncogenic potential. The expression analysis of the MDCK cells suggested that the induction of WT1 isoforms activates an inflammatory response, leading to the overexpression of several cytokines. Moreover, the -/+ isoform speciffically caused the upregulation of fibrotic markers and the rearrangement of the actin cytoskeleton. Interestingly, the expression of the mesothelial marker UPK3B increased following the induction of the -/+ isoform. Because the expression of the -/+ variant led to the most signifficant isoform-specific changes in both cell lines, I focused on this isoform for the validation of the transcriptomic data of the IMCD3 cells. I confirmed that the induction of WT1 -/+ in the IMCD3 cells leads to the upregulation of fibrotic markers, increases cell adhesion and activates the AKT and MAPK pathways. Moreover, there was a significant overexpression of different mesothelial markers and, importantly, of key regulators and markers of developmental processes, such as adipogenesis, skeletal and cartilage development, as well as angiogenesis. I then dissected the timing of expression of some specific markers and regulators, analysing the levels of the genes at different time points after WT1 -/+ induction. The preliminary results intimate that WT1 -/+ might induce epithelial cells in the direction of cartilage-skeletal tissue and fat, possibly through a mesothelial intermediate. The data also suggest that the induction of this isoform initiates an EMT, possibly followed by an MET, as the levels of expression of the differentiation markers and regulators increase. To validate the proposed instructive role of WT1, it will be of crucial importance to determine both RNA and protein levels of markers and regulators at even later time points, both in IMCD3 cells and in a model of inducible embryonic stem cells, which is currently under development. In the future, it will be important to address the relevance of these findings in vivo and to dissect the molecular mechanisms.

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.

Cardiovascular and heart diseases are the leading causes of death worldwide. In mammals, when heart damage occurs this organ is unable to regenerate itself. Understanding how to induce a regenerative process has been the focus of a great deal of attention recently. The understanding of heart development and the initial formation of several heart lineages could be used in finding a regenerative approach to heart damage that can mimic developmental processes. The Wilms’ tumour 1 gene (Wt1) is essential in the epicardium, the outer layer of cells around the heart, which during development has a multipotent potential and is the source of progenitors for several heart cell lineages such as: cells of the coronary vasculature, fibroblasts and cardiomyocytes. In my thesis I have focused on using an in-vitro (immortalized epicardial cells where Wt1 can be deleted by adding tamoxifen), and an in-vivo approach (genome wide expression analyses of Wt1 control and Wt1 knock-out epicardial enriched cells), to identify novel targets of Wt1 in the epicardium during development. I found that the chemokines Cxcl10 and Ccl5 are up-regulated in tamoxifen induced immortalized Wt1 knock-out epicardial cells and ex-vivo in heart explants when Wt1 is down-regulated. Ccl5 was found to be able to inhibit cardiomyocyte proliferation and Cxcl10 also inhibited epicardial cell migration, which could further explain ventricular thinning in Wt1 mutant mouse hearts. Wt1 is able to bind directly to the promoter of a chemokine and interferon response regulator gene, Irf7, which is also up-regulated in our in-vivo model. This could provide a mechanism by which Wt1 can inhibit chemokine expression during development, and could link Wt1 with immunological responses, which recently have been shown to play a role in the physiology and development of cells outside immunity, as well as being involved in physiological roles during damage and repair in adult tissues. I have also identified two Wt1-GFP populations (Wt1GFP++ and Wt1GFP+) in the ventricles of Wt1-GFP knock-in mice. The Wt1GFP++ population is enriched for epicardial cells, and a genome wide transcriptome analysis of these cells from E11.5 to E16.5 demonstrates they have a very dynamic regulation of a wide variety of genes, and also it indicates the existence of an early, transient and late Wt1GFP++ gene expression programs. The transcriptome analysis of Wt1GFP++ control and Wt1GFP++ Wt1 knock-out cells, from Gata5-Cre Wt1loxP/gfp mice at E13.5, reveals that Wt1 could regulate a number of previously un-described Wt1 targets related to the early Wt1GFP++ program, and gene ontology analyses indicate that many targets are related to cell to cell signalling and interaction, cell to extracellular matrix interaction, tissue development and morphogenesis. The Wt1GFP+ cell population is positive for a number of cardiomyocyte specific markers and has a low or negative expression of endothelial, epithelial and mesenchymal markers according to my transcriptome analysis. The findings I have described here shed light on the variety of targets of Wt1 and further reveal the function of Wt1 during epicardial development, which could be used in finding a regenerative approach to heart disease.

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Ischaemia causes irreversible tissue damage in cardiovascular disease. Since regenerative angiogenesis fails to consistently induce sufficient reperfusion to facilitate repair, targeted manipulation of angiogenesis is clinically desirable. The Wilms' tumour suppressor (Wt1) is a transcription factor which regulates numerous genes and cellular processes, including many intrinsic to angiogenesis. We hypothesise that WT1 in the endothelium influences the angiogenic function of endothelial cells. WT1 was identified in endothelial and non-endothelial cells comprising vessel outgrowths generated by cultured aortic rings from WT1-GFP reporter mice. Inducible deletion of WT1 from the endothelium (VE-Wt1 KO) significantly delayed angiogenesis in this assay (p < 0.05 relative to controls). In vivo, WT1 expression was evident in vascular endothelial and perivascular cells of the hindlimb as early as 3 days following femoral artery ligation to induce ischaemia, often in cells expressing epithelial and mesenchymal markers simultaneously. However, VE-Wt1 KO had no effect on hindlimb reperfusion (laser Doppler; days 0-28) or on vessel density (day 28). Similarly, VE-Wt1 KO had no effect on vessel density or expression of angiogenic factors (qRT-PCR) in sponges inserted subcutaneously in mice (20 days). To further understand the role of WT1 in angiogenesis, transcriptomic RNA expression analysis was performed in WT1+ and WT1- cells isolated (FACs) from sponges after implantation in WT1-GFP mice. WT1+ cells exhibited higher expression of genes involved in a number of processes relevant to tissue repair, including angiogenesis (p=3.11x10-8), wound healing (p=3.45x10-7) and epithelial-to-mesenchymal transition (EMT) (p=5.86x10-4). These results shed new light on the role of WT1 in ischaemic angiogenesis. In concurrence with previously published work, we show that deletion of endothelial WT1 can delay angiogenesis however, WT1 is not just instrumental in endothelial cells in this context. WT1 has a broader role in tissue repair in ischaemia, in part through regulation of cell transition (EMT). This work has improved our understanding of the regulatory role of WT1 in angiogenesis and repair, while revealing a number of novel insights into the function of WT1. This highlights WT1 as a potentially beneficial therapeutic target to facilitate regeneration in cardiovascular disease.

No description available.

The Wilms' Tumour Suppressor gene 1, WT1, encodes for a complex protein which is essential in mammals throughout life. Its roles vary with the developmental stages: in the embryo, it regulates the epithelial-mesenchymal balance required for a correct organogenesis and acts as a tumour suppressor; in the adult, it is involved in the maintenance of tissue homeostasis and has been controversially considered as an oncogene. Breast cancer is one of the adult tumours in which WT1 oncogenic function was first hypothesised. This malignancy is the most common in women, with more than one million cases being diagnosed worldwide every year, and represents the leading cause of cancer related deaths. Because of its major health burden, this disease has been extensively studied and special attention has also been paid to normal mammary gland biology: several works have shown that breast cancer can be divided into many molecular subtypes, which may reflect the cell of origin of the tumour; moreover, many genes involved in the normal development of the mammary gland have been proven to also play a role in breast tumorigenesis. WT1 expression has been previously reported in both healthy mammary glands and breast cancer samples, however, its function in this context is not well understood and the evidence gathered so far is extremely contradictory. This thesis aimed to investigate the exact role played by WT1 in both mammary gland and breast cancer biology, using a combination of in vivo and in vitro techniques. Following flow cytometry isolation, Wt1 mRNA expression was detected in the myoepithelial and stem cell subpopulations of the healthy gland. To investigate the effects of WT1 loss, Wt1 conditional mice were crossed with two different mammary specific Cre lines: the knockout animals developed, bred and lactated normally, however, they showed a significant increase of ductular branches during pregnancy, suggesting that WT1 may be involved in the regulation of branching morphogenesis. In order to study WT1 role in mammary tumours, the gene was knocked out in a breast cancer mouse model and knocked down in several breast cancer cell lines, using both constitutive and inducible lentivirus-based systems. WT1 loss did not seem to affect cell proliferation, but resulted in a significant increase in cell migration in vitro and in the upregulation of mesenchymal markers. Furthermore, bioinformatics analysis showed that the WT1-positive tumours mainly belong to the luminal/ER-positive subtypes and express lower levels of mesenchymal markers than the WT1-negative tumours. As a whole, the findings of this thesis characterise WT1 expression in the healthy mammary gland and provide the first evidence of its possible function in this organ; moreover, this work seems to rule out an oncogenic role for WT1 in breast cancer, while suggesting that it could be an upstream regulator of cell migration. Additional experiments are required to confirm this result in vivo and verify whether it could lead to any clinical application.

In 1899, the German surgeon Max Wilms hypothesised that different cell types in a variety of childhood kidney cancers were all derived from the mesodermal layer during embryonic development. Nearly a century later, the WT1 gene was identified on the short arm of chromosome 11, and was thought to be inactive in ~20% of nephroblastomas (Wilms’ tumours). The expression of WT1 after birth appears to be restricted to a finite number of tissues, namely, the glomerular podocytes, mesothelium and ~1% of bone marrow cells. Emerging evidence suggests WT1 is required not only for development, but also for tissue homeostasis, regeneration, repair and angiogenesis. Interestingly, WT1 has been implicated in the response to myocardial infarction and tumour angiogenesis, yet its precise role remains unclear. This thesis aims to address the hypothesis that activation of the WT1 gene in the vascular endothelium is essential for physiological and pathophysiological angiogenesis in the adult. In order to assess whether Wt1 was expressed in quiescent endothelial cells (ECs) immunofluorescence was used to analyse a variety of tissues in the adult mouse. Whilst Wt1 was detected in renal podocytes, no endothelial Wt1 expression was discovered in the lung, heart, kidney, spleen and gastrocnemius muscle. In contrast, tissues known to undergo physiological angiogenesis (endometrium and breast) did exhibit Wt1 expression in the vascular endothelium. Moreover, tubular EC outgrowths generated by aortic rings embedded in collagen ex vivo were positive for Wt1. The role of Wt1 in ischaemic angiogenesis was assessed using models of hind-limb and coronary ischaemia in the mouse. Wt1 was detected in ECs and non-vascular cells following ischaemic injury by a combination of immunofluorescence and qualitative real-time polymerase chain reaction (qRT-PCR). Using a time course analysis of these experimental models the chronology of this relationship was demonstrated, alongside the association with key angiogenic factors, such as Vegf. Given the findings in ischaemic tissue the C3(1)/Tag transgenic mammary cancer model was used to test the hypothesis that Wt1 would be upregulated in the tumour vasculature. Endothelial Wt1 was up regulated in these tumours compared to healthy control tissue. This finding was mirrored in a sub-set of aggressive breast cancers, confirming that the results obtained in mice can be translated to humans. Quantitative PCR revealed no association between histopathological grade of the tumours, oestrogen receptor status, and WT1 expression. In order to delineate the cell types involved in vessel formation, Wt1+ cells were sorted using fluorescent activated cell sorting (FACS) from transgenic mice with a green fluorescent protein knocked into the Wt1 locus following sponge implantation. Distinct sub-populations of Wt1+ cells were identified, some of which expressed EC and pericyte markers. Moreover, these Wt1+ sub-populations changed in composition and number over time. These findings were confirmed by genetic fate mapping of Wt1+ cells in this model. Finally, a conditional knockout mouse was generated to allow the selective deletion of Wt1 from vascular ECs in the sponge model of angiogenesis. The results demonstrated that deletion of Wt1 from this cellular compartment led to a dramatic reduction in vessel formation supporting a potential role in regulating angiogenesis. These results support the hypothesis that expression of WT1 in the vascular endothelium contributes to the regulation of angiogenesis in tumours and ischaemic tissue, and provides evidence that selective deletion of the gene inhibits new vessel formation. This suggests that targeting WT1 may have a therapeutic benefit in cancer and could aid regeneration of ischaemic tissues following injury in conditions such as myocardial infarction and critical limb ischaemia.

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