The Pancreas Is Altered by In Utero Androgen Exposure: Implications for Clinical Conditions Such as Polycystic Ovary Syndrome (PCOS)

Using an ovine model of polycystic ovary syndrome (PCOS), (pregnant ewes injected with testosterone propionate (TP) (100 mg twice weekly) from day (d)62 to d102 of d147 gestation (maternal injection – MI-TP)), we previously reported female offspring with normal glucose tolerance but hyperinsulinemia. We therefore examined insulin signalling and pancreatic morphology in these offspring using quantitative (Q) RT-PCR and western blotting. In addition the fetal pancreatic responses to MI-TP, and androgenic and estrogenic contributions to such responses (direct fetal injection (FI) of TP (20 mg) or diethylstilbestrol (DES) (20 mg) at d62 and d82 gestation) were assessed at d90 gestation. Fetal plasma was assayed for insulin, testosterone and estradiol, pancreatic tissue was cultured, and expression of key β-cell developmental genes was assessed by QRT-PCR. In female d62MI-TP offspring insulin signalling was unaltered but there was a pancreatic phenotype with increased numbers of β-cells (

Effects of CreERT2, 4-OH Tamoxifen, and Gender on CFU-F Assays

Gene function in stem cell maintenance is often tested by inducing deletion via the Cre-loxP system. However, controls for Cre and other variables are frequently not included. Here we show that when cultured in the presence of 4-OH tamoxifen, bone and marrow cells containing the CreERT2 construct have a reduced colony forming ability. Inactive CreERT2 recombinase, however, has the opposite effect. Young female marrow cells containing the inactive CreERT2 construct grew more colonies than cells lacking the construct altogether. Young female control marrow cells (i.e., negative for CreERT2) also produced significantly greater colony numbers when cultured with 4-OH tamoxifen, compared with the ethanol vehicle control. In conclusion, we report that the use of the Cre-loxP system is inadvisable in combination with CFU-F assays, and that appropriate controls should be in place to extend the future use of Cre-loxP in alternate assays

Visceral and subcutaneous fat have different origins and evidence supports a mesothelial source

International audience: Fuelled by the obesity epidemic, there is considerable interest in the developmental origins of white adipose tissue (WAT) and the stem and progenitor cells from which it arises. Whereas increased visceral fat mass is associated with metabolic dysfunction, increased subcutaneous WAT is protective. There are six visceral fat depots: perirenal, gonadal, epicardial, retroperitoneal, omental and mesenteric, and it is a subject of much debate whether these have a common developmental origin and whether this differs from that for subcutaneous WAT. Here we show that all six visceral WAT depots receive a significant contribution from cells expressing Wt1 late in gestation. Conversely, no subcutaneous WAT or brown adipose tissue arises from Wt1-expressing cells. Postnatally, a subset of visceral WAT continues to arise from Wt1-expressing cells, consistent with the finding that Wt1 marks a proportion of cell populations enriched in WAT progenitors. We show that all visceral fat depots have a mesothelial layer like the visceral organs with which they are associated, and provide several lines of evidence that Wt1-expressing mesothelium can produce adipocytes. These results reveal a major ontogenetic difference between visceral and subcutaneous WAT, and pinpoint the lateral plate mesoderm as a major source of visceral WAT. They also support the notion that visceral WAT progenitors are heterogeneous, and suggest that mesothelium is a source of adipocytes

Investigating the role of Wt1 in bone and marrow biology

The bones of the body vary in size and shape, but are fundamentally all composed of the same cell types: osteoblasts, osteoclasts, osteocytes, vascular cells, and sometimes marrow cells. Long bones are formed when mesenchymal stem cells (MSCs) give rise to chondrocytes i.e. cartilage cells, and osteoblasts i.e. bone cells. These develop to form layers of bone encasing a cartilagenous core which eventually becomes the marrow cavity. A recent study showed that deleting the Wilms’ tumour gene, Wt1, in adult mice causes a dramatic loss of bone and fat tissue, fat being another derivative of MSCs. This finding led me to ask whether Wt1 expression is involved in bone biology and whether it plays a functional role in the stem or progenitor populations. Wt1 is a transcription factor that acts as a mesodermal / mesenchymal regulator. It acts as a tumour suppressor gene with mutations leading to the eponymous paediatric kidney tumour. However, in adult cancers it has oncogene characteristics, being highly expressed in the tumours of tissues in which it is not normally present. It also plays a pivotal role in the epithelial to mesenchymal transition (EMT) and vice versa in developing heart and kidney, respectively. There is, however, no evidence of its involvement with EMT / MET in adults. Wt1 is expressed in various developing tissues and is particularly vital for kidney development. Due to its involvement as a regulator of EMT / MET during development and the phenotype observed following its deletion in vivo, we hypothesised that Wt1 is expressed in, and required for the function of mesenchymal stem or progenitor cells populations within the bone marrow. A Wt1-GFP knock in mouse was used to show that Wt1 expressing cells are found in the bone marrow, and also for the first time in the bone. The GFP population overlaps with a non-haematopoietic MSC population defined by 3 cell surface markers in the bone and marrow, as well as an osteoblast (OB) progenitor population. Using a tamoxifen inducible CreERT2 showed that Wt1 loss alters the proportion of GFP cells in the bone and marrow cells that overlap with these MSC and OB progenitor markers, but microarrays were needed to assess the functional effects of Wt1 deletion. Microarrays highlighted various pathways that were altered following the in vitro deletion of Wt1 in total bone and marrow culture, as well as the non-haematopoeitic GFP+ and GFP- populations. In bone cells, deleting Wt1 negatively affects various pathways related to MSCs and their derivatives, including collagen biosynthesis, cartilage development and muscle tissue development. Also negatively affected were Wnt signalling regulation and EMT regulation; this is the first time Wt1 has been shown to be involved in EMT in adult cells. These findings were validated using qRT-PCR to show the down regulation of various genes involved in each pathway, showing that as well as being expressed in these populations it is also playing a functional role. Ossification pathways were negatively altered in the cells not expressing Wt1 following the deletion of the gene suggesting that Wt1 may also be acting in a paracrine manner to play its role in bone homeostasis. As well as in adult tissues, Wt1 was found to be expressed during development in the limb tissue of e11.5 to e16.5 mice. Preliminary results show that Wt1 may also have a functional role during bone development, as loss of expression causes a reduction in the percentage of non-haematopoetic MSC cells in the e18.5 hindlimb. As well as this, preliminary lineage tracing experiments suggest that cells found at the bone surface are of Wt1+ origin. This thesis has also highlighted the importance of experimental conditions and controls, particularly for CFU-F assays. CreERT2, loxP sites, tamoxifen, oxygen tension levels, and gender all exert specific effects on colony formation, independent of Wt1 expression. In conclusion, these data identify Wt1 as a key player in bone development and homeostasis. The microarray results led to the conclusion that Wt1 has a functional role in several mesenchymal pathways and highlights various genes that are potential Wt1 targets and should be further investigated using ChIP-Seq methods

Colony forming abilities are reduced when adult CreER^T2 positive cells are cultured with 4-OH tamoxifen.

<p>(A) Brightfield images of representative colonies stained with Toluidine Blue from male marrow cells cultured under hypoxia. (B) The colony forming assays were performed by culturing male cells from central marrow and enzymatically digested flushed long bone for 10 days following culture in media with 4-OH tamoxifen (1μM) or vehicle control ethanol. Colonies were then stained with Toluidine Blue to assess cartilaginous matrix content. Mean (±SEM) Toluidine Blue positive CFU-F assay colony numbers are reduced following CreER^T2 activation with 4-OH tamoxifen in all Cre⁺ normoxic and hypoxic bone cells and hypoxic marrow cells, compared to culture with ethanol. Mean (±SEM) Toluidine Blue positive CFU-F assay colony numbers are also reduced following CreER^T2 activation with 4-OH tamoxifen in Cre⁺ normoxic bone cells, compared with Cre^- cells cultured with 4-OH tamoxifen. (C) Brightfield images of representative colonies stained with Alkaline Phosphatase from male marrow cells cultured under hypoxia. (D) Colonies were then stained with ALP to assess the degree of bone formation. Mean (±SEM) ALP positive CFU-F assay colony numbers are reduced following CreER^T2 activation with 4-OH tamoxifen in Cre⁺ hypoxic bone cells, compared to culture with ethanol. Mean (±SEM) ALP positive CFU-F assay colony numbers are also reduced following CreER^T2 activation with 4-OH tamoxifen in Cre⁺ normoxic and hypoxic bone cells and hypoxic marrow cells, compared with Cre^- cells cultured with 4-OH tamoxifen. *p<0.05 **p<0.01 ***p<0.001. (Cre^-: n = 3 experiments. Cre⁺: n = 4 experiments. All experiments were performed in technical triplicate). (y axis = mean number of colonies with diameter greater than 1mm).</p

Colony forming abilities are reduced when CreER^T2 positive cells from young mice are cultured with 4-OH tamoxifen. Young female CreER^T2 positive marrow cells form more colonies than CreER^T2 negative cells in the absense of 4-OH tamoxifen, and young female marrow CreER^T2 negative cells cultured with 4-OH tamoxifen give rise to more colonies than when cultured with the ethanol vehicle control.

<p>(A) Brightfield images of representative colonies from female marrow cells cultured under hypoxia (scale bar = 5mm). (B) The colony forming assays were performed by culturing cells from central marrow and enzymatically digested flushed long bone for 10 days following culture in media with 4-OH tamoxifen (1μM) or vehicle control ethanol. Mean (±SEM) CFU-F assay colony numbers are reduced following CreER^T2 activation with 4-OH tamoxifen in all Cre⁺ marrow and bone cells, irrespective of sex or culture conditions, compared to culture with ethanol. Mean (±SEM) CFU-F assay colony numbers are also reduced following CreER^T2 activation with 4-OH tamoxifen in Cre⁺ male marrow and bone cells, and female bone cells, compared to Cre^- culture with 4-OH tamoxifen. Mean (±SEM) CFU-F assay colony numbers are higher from female Cre⁺ marrow cells cultured under both normoxia and hypoxia with ethanol, compared to Cre^- marrow cells with ethanol. Mean (±SEM) CFU-F assay colony numbers are increased in female Cre^- marrow cells cultured with 4-OH tamoxifen compared to ethanol, when cultured under both normoxia and hypoxia. *p<0.05 **p<0.01 ***p<0.001. (n = 3 experiments. All experiments were performed in technical triplicate). (y axis = mean number of colonies with diameter greater than 1mm).</p

Colony forming abilities are reduced when adult CreER^T2 positive cells are cultured with 4-OH tamoxifen.

<p>(A) Brightfield images of representative colonies from male bone cells cultured under hypoxia. (B) The colony forming assays were performed by culturing cells from central marrow and enzymatically digested flushed long bone for 10 days following culture in media with 4-OH tamoxifen (1μM) or vehicle control ethanol. Mean (±SEM) CFU-F assay colony numbers are reduced following CreER^T2 activation with 4-OH tamoxifen in all Cre⁺ marrow and bone cells, irrespective of sex or culture conditions, compared to culture with ethanol. Mean (±SEM) CFU-F assay colony numbers are also reduced following CreER^T2 activation with 4-OH tamoxifen in Cre⁺ male marrow and bone cells, female bone cells, and female hypoxic marrow, compared to Cre^- culture with 4-OH tamoxifen. *p<0.05 **p<0.01 ***p<0.001. (Cre^-: n = 3 experiments. Cre⁺: n = 4 experiments. All experiments were performed in technical triplicate). (y axis = mean number of colonies with diameter greater than 1mm).</p

Effects of CreER^T2, 4-OH Tamoxifen, and Gender on CFU-F Assays

<div><p>Gene function in stem cell maintenance is often tested by inducing deletion via the Cre-<i>loxP</i> system. However, controls for Cre and other variables are frequently not included. Here we show that when cultured in the presence of 4-OH tamoxifen, bone and marrow cells containing the CreER^T2 construct have a reduced colony forming ability. Inactive CreER^T2 recombinase, however, has the opposite effect. Young female marrow cells containing the inactive CreER^T2 construct grew more colonies than cells lacking the construct altogether. Young female control marrow cells (i.e., negative for CreER^T2) also produced significantly greater colony numbers when cultured with 4-OH tamoxifen, compared with the ethanol vehicle control. In conclusion, we report that the use of the Cre-<i>loxP</i> system is inadvisable in combination with CFU-F assays, and that appropriate controls should be in place to extend the future use of Cre-<i>loxP</i> in alternate assays.</p></div

Fetal pancreatic expression of key developmental genes and <i>in vitro</i> insulin secretion is dependent upon fetal sex and timing of exposure to excess androgens.

<p><b>A: </b><b><i>Female fetuses</i></b>: At d90, (vehicle n = 5, MI-TP n = 8), d62 MI-TP significantly increased expression of <i>IGF1R</i> (<i>P</i><0.05), <i>PDX1</i> (<i>P</i><0.01), <i>INSR</i> (<i>P</i><0.05) and <i>INS</i> (<i>P</i><0.05) mRNA. <b><i>Male fetuses</i></b>: Whilst there were differences in expression attributable to the sex of the fetuses (see results section for full description), there were no differences in expression of any genes studied attributable to <i>in utero</i> treatment in male fetuses studied at d90 (vehicle n = 6, MI-TP n = 6) of gestation. Different superscripts denote significant differences (P<0.01–0.05, see results sections for full details).</p