Pubertal mouse mammary gland development - transcriptome analysis and the investigation of Fbln2 expression and function.
PhD thesis, University of Glasgow.
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Mouse mammary gland morphogenesis at puberty is a complex developmental process, regulated by systemic hormones, local growth factors and dependent on the epithelial/epithelial and epithelial/stromal interactions. TEBs which invade the fat pad are important in laying down the epithelial framework of the gland at this time point. The objective of this thesis was to use a combination of ‘pathway-‘ and ‘candidate gene analysis’ of the transcriptome of isolated TEBs and ducts and associated stroma, combined with detailed analyses of selected proteins, to further define the key proteins and processes involved at puberty. Using GeneChip® Mouse Exon 1.0 ST Arrays we identified the epithelial-, epithelial-stromal- and stromal transcriptomes of TEBs and ducts and defined the major functional pathways/biological processes in each compartment. By ranking the transcripts according to their expression levels and known functions in other systems, we identified genes of potential importance for pubertal mammary morphogenesis. We focused our study on Upk3a and Fbln2 and their protein products. Upk3a could only be detected at mRNA level and thus further analysis was based on Fbln2. We demonstrated that Fbln2 V1 and Fbln2 protein are predominantly expressed in the epithelium and stroma of TEBs. Using hormone primed mice we demonstrated that Fbln2 expression and localisation in the mouse mammary gland is positively regulated by E2 and P. Furthermore, by a combination of further in silico analysis, in vitro functional assays, IHC and IF we identified Vcan, Lama1, Fbn1, ColVIαIII, ColIVαI, ColXVIIIαI, Eln, Per, Acan, Nid, Itgb3 and Itga5 as potential binding partners of Fbln2 in mammary gland. Finally, we reported lack of an obvious mammary phenotype in Fbln2 KO-/- mice at puberty but demonstrated that this may be attributed to the over-compensation by Fbln1.
This thesis demonstrates the benefit of DNA microarray analysis in studying pubertal development of mouse mammary gland. It identifies Fbln2 as a potential pubertal mammary regulator which by interacting with various ECM proteins at different sites of mammary milieu may contribute to an array of structural and migratory functions during mammary morphogenesis. These data substantially add to the understanding of the development of mammary gland at puberty and reveal many potential avenues for further investigations.
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