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In vivo and in vitro studies of adipogenesis with particular reference to adipocyte development in rodent skin

Skin comprises an epidermis, dermis, skin appendages including hair follicles, and a fat layer. There is a growing interest in the biology of specific fat depots, and skin fat is relatively poorly studied. Importantly, most knowledge about the molecular control of adipocyte differentiation comes from in vitro studies on cell lines. This thesis aimed to provide new insights into adipogenesis in vivo by directly studying development of the skin fat layer and its relationship to the surrounding skin and hair follicles. Work, presented in Chapter 2, investigated the timing and localisation of developing fat cells in back skin of rodent embryos. Analysis of the adipogenic transcription factor C/EBPalpha and lipid accumulation revealed preadipocytes in the lower dermis of embryonic mouse skin at e17 and the start of lipid accumulation by e19. The dermal fat cells then created an adipose layer between and beneath hair follicles apparently independently of subcutaneous fat tissue. In Chapter 3, a combined laser capture microdissection and microarray approach generated gene expression profiles of cells from upper and lower dermis over three time points. Verification of the microarray data by qPCR and immunohistochemistry, and bioinformatics analysis confirmed a subdivision of the lower dermis with enriched fat-related pathways. Comparison of this microarray data with published information on adipogenesis of 3T3-cells in vitro showed important early differences with regard to transcription factor, cell cycle, cytoskeletal and extracellular matrix gene expression. Later time points revealed greater similarities between in vivo and in vitro data involving genes characteristic of mature adipocytes. In Chapter 4, the involvement of the Egfr gene (selected from generated microarray lists) in dermal fat development was investigated functionally using a skin organ culture model. In Chapter 5, a marker gene selected from the arrays (Cd36) was successfully used to develop a method of isolating dermal preadipocytes by fluorescence activated cell sorting. Specialised organ culture techniques, presented in Chapters 4 and 5, allowed the adipogenic capabilities of cells from different mouse embryonic skin compartments to be investigated. This revealed a high plasticity of dermal cells at earlier embryonic time points (e15 - 15.5) and their specialisation into either non-fatty cells (upper dermis) or adipocytes (lower dermis) later (e18.5 - 19). As summarised in Chapter 6, this thesis confirmed that the fat layer that develops from cells of the lower dermis should have a distinct nomenclature (dermal adipose tissue) from the subcutaneous fat depot and could be under different regulatory mechanisms. The work has established a new in situ model of in vivo adipogenesis and the microarray data obtained has provided novel information on molecular control of adipogenesis in general, as well as pointers as to why the lower, but not the upper compartment of the late embryonic dermis turns into fat.
Date January 2012
CreatorsWojciechowicz, Kamila
PublisherDurham University
Source SetsEthos UK
Detected LanguageEnglish
TypeElectronic Thesis or Dissertation

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