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

Development of the mouse dermal adipose layer occurs independently of subcutaneous adipose tissue and is marked by restricted early expression of FABP4.

The laboratory mouse is a key animal model for studies of adipose biology, metabolism and disease, yet the developmental changes that occur in tissues and cells that become the adipose layer in mouse skin have received little attention. Moreover, the terminology around this adipose body is often confusing, as frequently no distinction is made between adipose tissue within the skin, and so called subcutaneous fat. Here adipocyte development in mouse dorsal skin was investigated from before birth to the end of the first hair follicle growth cycle. Using Oil Red O staining, immunohistochemistry, quantitative RT-PCR and TUNEL staining we confirmed previous observations of a close spatio-temporal link between hair follicle development and the process of adipogenesis. However, unlike previous studies, we observed that the skin adipose layer was created from cells within the lower dermis. By day 16 of embryonic development (e16) the lower dermis was demarcated from the upper dermal layer, and commitment to adipogenesis in the lower dermis was signalled by expression of FABP4, a marker of adipocyte differentiation. In mature mice the skin adipose layer is separated from underlying subcutaneous adipose tissue by the panniculus carnosus. We observed that the skin adipose tissue did not combine or intermix with subcutaneous adipose tissue at any developmental time point. By transplanting skin isolated from e14.5 mice (prior to the start of adipogenesis), under the kidney capsule of adult mice, we showed that skin adipose tissue develops independently and without influence from subcutaneous depots. This study has reinforced the developmental link between hair follicles and skin adipocyte biology. We argue that because skin adipocytes develop from cells within the dermis and independently from subcutaneous adipose tissue, that it is accurately termed dermal adipose tissue and that, in laboratory mice at least, it represents a separate adipose depot

Trichorhinophalangeal syndrome I (TRPS1) expression is restricted to upper dermal cells at embryonic day 17 (e17), e18 and e19 in murine dermis.

<p>a), DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar = 30 µm. b) The relative mRNA expression for <i>TRPS1</i> was analysed from samples at the e17 (17), e18 (18) and e19 (19) time-points from upper (Area “1”, blue) and lower (Area “2”, red) dermis. The baseline (1-fold change) was established for the e19 upper dermis sample (underlined on the figure) and the mRNA levels of the other samples are shown relative to this. <i>TRPS1</i> mRNA is up-regulated in upper dermis (Area “1”) compared with lower dermis (Area “2”) at all analysed time-points. Data were obtained from triplicate biological replicates. P value refers to the comparison with “19 1” sample (*P≤0.05, **P≤0.01, ***P≤0.001).</p

Relationship between developing skin adipose tissue, the <i>panniculus carnosus</i> and underlying subcutaneous adipose tissue in the dorsal skin of newborn mice.

<p>Mouse dorsal skin sections from the region overlying the anterior (interscapular) subcutaneous adipose depot. Skin sections with underlying subcutaneous adipose tissue were stained with Oil Red O to detect lipids from birth to 19 days postnatally (a–i). The thickness (marked by yellow lines) of the <i>panniculus carnosus</i> layer changes in the specimens over time. An additional thin layer of lipid droplets can be seen under the dermal adipose tissue, for example at day 5 (d - see area close to smaller yellow line). a) 0.5 day old newborn mouse. b) 1 day old newborn mouse. c) 2 day old newborn mouse. d) 5 day old newborn mouse. e) 8 day old newborn mouse. f) 12 day old newborn mouse g) 15 day old newborn mouse. h) 17 day old newborn mouse. i) 19 day old newborn mouse. (a - i) Scale bar = 65 µm.</p

Early accumulation of lipid in cells of the lower dermis follows a spatio-temporal pattern.

<p>Samples of dorsal skin from foetal and early newborn dorsal skin sectioned and stained with Oil Red O to detect lipids (a-h). At e16 no lipid accumulation is detected anywhere in skin (a) note the line of cells representing the precursor of the <i>panniculus carnosus</i> (white arrows). By e18.5–19 the first lipid containing cells are detected, at which point the lipid is often multilocular (b). Sections from the head (c and e) regions of e18.5 dorsal skin show larger numbers of cells with accumulation of lipid in the deep dermis compared with tail regions (d and f) where far fewer lipid producing cells were apparent. In regions between the head and tail, some areas had consistent groups of cells containing lipid (g). Lipid forming cells are occasionally observed in regions where follicle development is apparently less advanced (h). sf = subcutaneous adipose tissue. Scale bars: a, c, d, e, f, g, h = 100 µm, b = 50 µm.</p

Fatty acid binding protein-4 (FABP4) is expressed in the lower dermis of developing mouse back skin from embryonic day 16 (e16). a), e15 (embryonic day 15), b) e16 (embryonic day 16), c) e18 (embryonic day 18), d) P5 (post-natal day 5), e) NC (negative control - no primary antibody).

<p>White dashed line delineates boundary between epidermis and upper dermis. Red dashed line delineates boundary between upper dermis and lower dermis. DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar = 50µ m. f) The relative mRNA expression for <i>Fabp4</i> was analysed from samples at the e17 (17), e18 (18) and e19 (19) time-points from upper (Area “1”, blue) and lower (Area “2”, red) dermis. The baseline (1-fold change) was established for the e19 lower dermis sample (underlined on the figure) and the mRNA levels of the other samples are shown relative to this. <i>Fabp4</i> mRNA is up-regulated in lower dermis (Area “2”) compared with upper dermis (Area “1”) at all analysed time-points. Data were obtained from triplicate biological replicates. P value refers to the comparison with “19 2” sample (*P≤0.05, **P≤0.01, ***P≤0.001).</p

Lipid accumulation in back skin specimens with adipose depots beneath the skin.

<p>Composite image of longitudinally sectioned skin section stained with Oil Red O to show lipid accumulation along the anterior-posterior axis. Samples were prepared from newborn mice at different postnatal time points 0.5 day,1 day, 2 day, 4 day, 5 day, 8 day, 12 day, 15 day, 17 day, 19 day. S = subcutaneous adipose tissue below the anterior and some posterior regions of the skin specimens. (a – j) Scale bar = 300 µm.</p

No significant apoptosis is observed in the adipocyte layer, during its formation, or at any point in the first hair growth cycle as measured by the TUNEL assay.

<p>TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling), a) PC (positive control - nuclease treated 1 day old mouse back skin), b) NC (negative control - 1 day old mouse back skin). In one day specimens labelled cells were occasionally observed in hair follicles (magnified and arrowed in c, d) and in the developing adipose layer (c) but more often they were completely absent from the latter (d). Apart from very occasional follicle-associated labelling virtually no cell death was observed at 5 and 12 days after birth, through the middle stages of the first hair cycle (e, f). Subsequently an increasing amount of cell death was observed associated with regression of the follicles as their growth shut down at 17 and 19 days (magnified and arrowed in g and h) however virtually no cell death labelling was observed in the surrounding adipocyte cells. DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar = 30 µm.</p