published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Mdm2 and Mdm4 are critical negative regulators of the p53 tumor suppressor. Mdm4-null mutants are severely anemic and exhibit impaired proliferation of the fetal liver erythroid lineage cells. This phenotype may indicate a cell-intrinsic function of Mdm4 in erythropoiesis. In contrast, red blood cell count was nearly normal in mice engineered to express low levels of Mdm2, suggesting that Mdm2 might be dispensable for red cell production. In the first part of the thesis, we further explore the tissue-specific functions of Mdm2 and Mdm4 in the erythroid lineage by crossing the conditional Mdm4 and Mdm2 alleles to an erythroid-specific-cre (EpoRGFP-Cre ) knock-in allele. Our data show that Mdm2 is required for rescuing erythroid progenitors from p53-mediated apoptosis during primitive erythropoiesis. In contrast, Mdm4 is only required for the high erythropoietic rate during embryonic definitive erythropoiesis. Thus, in this particular cellular context, interestingly, Mdm4 only contributes to p53 regulation at a specific phase of the differientation program. Moreover, a large body of evidence indicates that aberrant expression of either MDM2 or MDM4 impairs p53 tumor suppression function and consequently favors tumor formation. Overexpression of MDM2 was observed in 10% of 8000 human cancers from various sites, including lung or stomach, and MDM4 was found amplified and/or overexpressed in 10-20% of over 800 diverse tumors including lung, colon, stomach and breast cancers. Remarkably, selective MDM4 amplification occurs in about 65% of human retinoblastomas. In contrast, MDM2 amplifications are relatively rare (about 5%) in retinoblastomas, indicating that depending on the tumor context (cell type, initiating oncogene, …), MDM4, rather than MDM2, overexpression might be selected for as a more efficient mean of suppression of p53 function. As part of a large effort to better understand why different cell types require distinct combinations of mutations to form tumours, we will examine the molecular basis for selective up-regulation of Mdm4 in retinoblastomas. In this context, we have successfully generated 2 conditional transgenic mouse lines expressing either mycMdm2 or mycMdm4 driven by the PCAGGs promoters in the ROSA26 locus. Since a cassette containing a floxed transcriptional stop element is inserted upstream of the transgenes, we can achieve tissue-specific expression and spatio-temporal regulation of the transgenes by using different Cre and CreER. By the use of N-terminal myc-tag fused with the transgenes, we are able to compare the expression levels of the transgenes. Finally, due to C-terminal IRES-GFP element, we can easily identify transgene expressing cells. One of our aims is to use this Mdm4 conditional transgenic mouse line as the first, non-chimeric, mouse model of retinoblastoma that can be used as an appropriate preclinical model to improve treatment of this disease.

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Mammalian kidney development proceeds through a series of interactions among metanephric mesenchyme, ureteric bud, extracellular matrix, growth factors and various other signalling molecules. These complex, but well integrated, networks control cell proliferation, differentiation, migration and survival and thus orchestrate kidney development. More and more molecules in these networks have been identified since the past few years. Therefore, it is crucial to explore their functions and the mechanisms by which they work to fill the research gaps. DACTs have recently been reported as pathway-specific regulators in WNT signalling, known to be important in kidney development, but their expressions and functions in mammalian kidneys are yet to be elucidated. The aims of this thesis are to describe the expression patterns of these two new genes, Dact1 and Dact2, in mouse embryonic kidneys and to further investigate their functional roles by applying RNAi to cell culture-based models. The first goal of this thesis is to establish the temporospatial expression patterns of Dact1 and Dact2 in kidneys by conventional end-point RT-PCR, quantitative real time PCR and RNA in situ hybridisation. Based on the expression patterns and preliminary observations in cell cultures, I hypothesize that Dact1 regulates cell proliferation while Dact2 governs cell migration. Experiments including siRNA transfection, BrdU proliferation assay, generation of stable cell lines expressing Dact2 shRNA and wound assay, are designed to test these hypotheses and the results may offer implications of functional roles of both molecules in kidney development. The results obtained are as follows. • Dact1 and Dact2 show different temporal expression patterns in mouse kidneys. In adult kidneys, Dact1 is greatly downregulated while Dact2 is still expressed at a comparable level to that at E14.5. • Dact1 is initially expressed in metanephric mesenchyme and, as development proceeds, shows a characteristic pattern of renal stroma whilst Dact2 is exclusively expressed in ureteric buds throughout embryonic stages. • Dact1 and Dact2 expressions are regulated by known regulators of kidney development including retinoic acid and chlorate. • Silencing of Dact1 facilitates proliferation of embryonic cells. • Silencing of Dact2 hinders migration of renal collecting duct cells. Taken together, I have characterised temporospatial expression patterns of Dact1 and Dact2 in kidneys and provided evidences of functional roles of both novel molecules in cell cultures. Based on this thesis, further studies on Dact1 and Dact2 using either in vitro or in vivo mammalian kidney models will offer more insights into their functions and regulations in renal organogenesis.

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Matthew-Wood syndrome is a rare human birth defect condition defined by the phenotypic constellation of clinical anophthalmia, diaphragmatic hernia, pulmonary hypoplasia and cardiac defects. Matthew-Wood syndrome has a high mortality rate, with most patients dying due to respiratory insufficiency as a consequence of pulmonary hypoplasia, within the first year of life. Mutations within STRA6 are causative for Matthew-Wood syndrome. STRA6 acts as a retinol transporter for retinol bound to its physiological carrier RBP4 allowing regulated entry of retinol into the cell. A mammalian model for Matthew-Wood syndrome was not found within the literature; however a morpholino knockdown of stra6 in the zebrafish did show phenotypic features consistent with those observed in human patients. The desire to create a mammalian model of Matthew-Wood syndrome drove the work contained within this thesis. Stra6-/- mice do not represent a model for Matthew-Wood syndrome with homozygous animals being viable, found in the expected ratio and demonstrating none of the developmental abnormalities observed in human patients. Retinal defects, cataracts and persistent hyperplastic primary vitereous affect the microphthalmic eye of Stra6-/- offspring of Stra6-/- mothers fed a retinoid-free diet from plug to birth indicating that Stra6 is required for normal eye development under low-retinoid stress. The disparity in phenotype between human Matthew-Wood patients and Stra6-/- mice may be the result of functional redundancy in the mouse between Stra6 and its paralogue, Stra6.2. Stra6.2 is well conserved through evolution and is found in diverse species, including the basal eumetazoan Trichoplax. STRA6.2 has become split across its resident chromosome with an associated break in gene synteny, in humans and great apes, causing most of the gene to no longer be transcribed. However a small portion of the gene, representing the final transmembrane domain and the C-terminal intracellular tail of the protein, remains expressed in human. stra6.2 is required for normal development in the zebrafish with stra6.2 morphants being phenotypically distinguishable from control injected embryos from the 10-somite stage by a larger head-tail distance indicating an axial extension defect. stra6.2 morphants also display microphthalmia, jaw malformation, shortened and curved body axis and retinal lamination defects. stra6.2 was found to be required to prevent an excess of retinoic acid resulting in an upregulation of retinoic acid-dependent gene expression through an increase in RA synthesis by Raldh enzymes in morphants. Stra6.2-/- mice are viable and fertile and phenotypically normal, even under retinoid-stress, supporting the notion of functional redundancy. In compound knockouts, normal development and postnatal survival can be maintained by a single copy of Stra6 in Stra6+/-;Stra6.2-/- animals. Stra6.2 is less able to support normal development and survival with ~50% of Stra6-/-;Stra6.2+/- animals dying before weaning or showing reduced growth although the remaining animals are indistinguishable from their littermates. Stra6 and Stra6.2 are functionally redundant for development under normal dietary conditions in the mouse and a single copy of either is able to support development in at least 50% of animals. Stra6-/-;Stra6.2-/- mice were therefore hypothesised to be the logical mouse model of Matthew-Wood syndrome, however these mice die early in gestation between E7.5-E9.5. The early embryonic lethality in Stra6-/-;Stra6.2-/- mouse embryos compared to postnatal survival in human Matthew-Wood patients, to which they are the comparable genetic model, could be attributed to the shortened STRA6.2 remaining within the human genome. The equivalent portion of Stra6 has validated signalling motifs, which may still be active in STRA6.2, allowing development to proceed in human ‘STRA6-/-’ embryos.