The Eph receptor tyrosine kinases and their ephrin ligands are cell surface molecules with a wide range of biological functions. Specifically, the Eph/ephrin receptor-ligand family influences cell behaviour during both embryogenesis and adult life, principally through modification of cytoskeletal organisation and cell adhesion. EphA1 (previously referred to as eph) was isolated during a search for novel tyrosine kinases with oncogenic potential. The murine homologue of hEphA1, formerly Esk and now mEphA1, was cloned by reverse transcriptase PCR using degenerate oligonucleotide primers and RNA prepared from embryonic stem cells in culture. Northern blot analysis revealed expression in day 12 mouse embryo, and adult mouse thymus, liver, kidney, lung and placenta. The work in this thesis investigates the expression and function of EphA1 in mutant mouse and other animal models. This has been achieved by a number of different techniques including:- (1) the classical techniques of molecular biology; (2) a search for the zebrafish homologue of mEphA1; (3) generation and phenotypic analysis of the hPLAP EphA1 reporter knockout mouse and (4) generation of the EphA1 conditional knockout mouse. The chromosomal localisation and Southern blotting of genomic digests confirmed that Esk (mEphA1) is the murine homologue of eph (hEphA1). The binding of soluble mEphA1 to a panel of ephrin ligands analysed by surface plasmon resonance (BIACore), and the binding of various ephrin-Fc molecules to cell surface expressed EphA1, confirmed that EphA1 is the cognate receptor for the ephrin-A1 ligand. The mEphA1 genomic sequence was isolated, sequenced and the exon-intron boundaries mapped. Interestingly, Exon 3, which includes the ligand binding domain, is split into two smaller exons (Exon 3a and Exon 3b). This pattern was also found in hEphA1; however, it is a novel finding compared with the other Ephs, and the reason underlying this difference remains speculative. In situ hybridisation analysis confirmed epithelial expression of mEphA1 in the basal layer of the epidermis, developing hair follicles, thymic epithelial cells and adult kidney. At the commencement of the zebrafish (ZF) library screening project in 1997, it seemed likely that there was an ZF orthologue of EphA1. However, over 50 clones were isolated by degenerate PCR of zebrafish cDNA and genomic libraries, and although some of the sequences had homology to known Ephs, none matched EphA1. The ZF genome has now been sequenced completely [http://wwwmap.tuebingen.mpg.de/ ; http://zfin.org/] and has confirmed that there is indeed no zebrafish orthologue of EphA1. The hPLAP EphA1 reporter knockout mouse was generated with the technical assistance of Dr Graham Kay (Queensland Transgenic Laboratory). The homozygous null mice have a kinky tail in two separate embryonic stem cell lines with a high degree of penetrance. A proportion of female null mice display the imperforate vagina phenotype. The null mice are otherwise grossly normal, with equal sex ratios and normal growth, health and life expectancy. The microscopic examination of haematoxylin and eosin stained sections of all the major organs revealed no histological abnormalities. The expression of hPLAP, (hence mEphA1), analysed in frozen sections confirmed the previous work which defined the epithelial expression of mEphA1 to the basal epidermis and hair follicle. There was also previously undescribed hPLAP (mEphA1) expression in the uterus, vagina and small intestine. The EphA1 conditional knockout mouse was also generated with the assistance of the Queensland Transgenic Facility. The homozygous null mice were grossly normal with equal sex ratio and normal health and life expectancy. The kinky tail phenotype was observed infrequently and has not yet been fully characterised in these mice. Similarly the imperforate vagina phenotype has not been observed in this strain of mice. This strain of genetically modified EphA1 knockout mice can be mated with various strains of Cre-deleter mice to achieve tissue specific silencing of EphA1 and consequently allow more precise analysis of EphA1 function. In summary, the studies described in this thesis have confirmed the importance of the Eph/ephrin receptor-ligands in both embryonic development and the maintenance of adult tissues, and have generated several new findings which add to our knowledge of the biology of EphA1. The generation of the hPLAP EphA1 reporter mice and EphA1 conditional knockout mice has provided us with very useful tools. These knockout mice will allow further analysis of the role of EphA1 in mouse models of human diseases, including skin and colon cancer, severe sepsis and post-traumatic injury.
| Identifer | oai:union.ndltd.org:ADTP/256427 |
| Creators | Coulthard, Mark G. |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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