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

Abstract

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.

Figure 1. Wt1+ cells reside in the SVF in visceral WAT but not subcutaneous WAT or BAT depots

Using the Wt1-GFP mouse model, representative FACS plots showing Wt1+ cells (GFP) are not detected in the mature adipocytes in the (a) epididymal, (b) mesenteric, (c) subcutaneous, or (d) BAT fat pad. In the SVF, Wt1+ cells are found in the (e) epididymal, (f) mesenteric, (g) retroperitoneal, (h) omental, (i) perirenal, and (j) epicardial depots; however, Wt1+ cells are not detected in the SVF from (k) BAT or (l) subcutaneous fat pads. GFP signal is indicated in the x-axis. Gates are chosen using cells from Wt1-GFP negative mice (n=3). (m) The level of WT1 mRNA in human visceral and subcutaneous fat is measured by Q-PCR (y-axis is expressed in arbitrary unit). V indicates ‘visceral’ (omental fat in this case). S indicates subcutaneous fat. (n), The level of WT1 mRNA from human ‘BAT’ and adjacent white adipose tissue (WAT) is measured by Q-PCR. Sample ‘4V’ is visceral fat which acts as a positive control (y-axis is expressed in arbitrary units).

Figure 2. Extensive long term contribution of the Wt1+ cells, induced at E14.5 to mature adipocytes in epididymal WAT but not in subcutaneous WAT or BAT. Some Wt1+ cells in the adult adipose tissues can contribute to mature adipocytes in visceral but not subcutaneous WAT or BAT

One dose of tamoxifen was given to pregnant animals at E14.5 and mice were harvested at 1.2-years old (n=3). Sections from various fat pads are stained with GFP antibody (red), Wt1-antibody (green), and DAPI (blue). a,b, epididymal fat pad; c, absence of Wt1-lineaged cells or endogenous Wt1-expressing cells in the subcutaneous and (d) BAT. In (e), immunostaining of sections of fat dissected from the constitutively active Wt1-Cre;R26RYFP indicates presence of the GFP positive adipocytes (indicate in green) in the visceral fat and absence of GFP positive cells in the subcutaneous WAT (f). (g), Wt1+ cells in the epididymal fat pad from the Wt1−^CreERT2.mTmG model induced at 3-weeks old gave rise to mature adipocytes (GFP= red, endogenous Wt1= green, cell nuclei= DAPI, blue) when mice were harvested at 3 months-old. h, mature adipocytes are indicated by perilipin antibody staining (GFP= red, perilipin= green, cell nuclei= blue). i, Absence of GFP+ cells in the subcutaneous or BAT fat pad (j). k, The mesothelium structure in adipose tissue arises from Wt1+ cells (red). Mature adipocytes are stained with perilipin antibody (green). (l), Mesothelium in adipose tissues (epididymal) express endogenous Wt1 (indicated in green) and is Wt1-lineage positive (indicated in red).

Figure 3. Wt1+ cells in adult adipose tissues express adipose progenitor surface markers

a, A schematic representation of the FACS strategy taken from Rodeheffer et al is shown. b, Dot plots showing representative FACS staining profiles and gating of adipose SVF from adult Wt1-GFP mice. Lin−and CD31− populations (‘P8’) from live singlets are selected. The Lin−CD31− population is further separated on the basis of expression of CD34 and CD29. The CD34 and CD29 double positive cells are further analysed based on the expression of Sca1 and CD24. The majority of the Wt1-GFP positive cells (indicated in green) are in the (Lin−CD31−CD34+CD29+Sca1+CD24−) population. c, The percentage of each populations in (Lin−CD31−GFP+) from different fat pads is shown (n=4. Data represent the mean ±s.e.m). d, The percentage of the cells in each population that are Wt1-GFP positive. Epi= epididymal, Mes= mesenteric, RP= retroperitoneal, and OM= omentum. (n=4. Data represent the mean ±s.e.m.). (e), Effect of deleting Wt1 on the percentage of different populations of adipose progenitors. FACS analysis of percentage of adipose progenitors in fat pad from 3-weeks old female WGER mice (CAGG^CreERT2.Wt1^loxp/GFP) that were injected with tamoxifen and harvested at day 7 post-injection (n=4 for each group. Data represent the mean ±s.e.m. and one-tailed Student’s t-tests were used to assess statistical significance. *P< 0.0.5). Cre-negative (CAGG^+/+.Wt1^loxp/GFP) littermates injected with tamoxifen are included as the control.

Figure 4. Characterising an ex vivo model of mesothelium/epididymal appendage differentiation into adipocytes using multiphoton microscopy

a, Multiphoton microscope images showing epididymal appendage explants (Wt1^CreERT2;mTmG/+) cultured in adipogenesis differentiation medium at Day 1, Day 7, and Day 14. Membranous GFP signal is indicated in green. CARS is used to detect lipid (indicated in red). An enlarged image of Day 14 is also included. Lipid droplet analysis of the Wt1-derived adipocytes (green) and the neighbouring adipocytes (red) from the explants culture system (b) or from epididymal fat pad induced by tamoxifen in vivo (c). (green = GFP, red = tdTomato, cyan = CARS). Analysis includes quantification of number of lipid droplets / cell, quantification of lipid droplet diameter, quantification of cell size, and quantification of % lipid content / cell. Statistics source data for (c) can be found in Supplementary Table 5.

Figure 5. FACS profiling of mesothelium and mesothelium-derived cells by adipose progenitor markers

(a) Mesothelium and the mesothelium-derived cells collected from the Wt1-GFP line at different developmental stages and adult were stained with adipose progenitor surface markers. Representative FACS plots showing GFP+ cells stained with the markers. (b) Immunofluorescence staining showing the mesothelium staining at the surface of visceral organs (E15.5), with and without collagenase treatment. GFP is indicated in green and cell nuclei are stained with DAPI (blue). (c) Summary of Wt1-expressing WATs and their possible origins during development. In E8.5, Wt1 is first expressed in the intermediate mesoderm and lateral plate mesoderm (highlighted in green). Wt1+ mesenchymal cells from intermediate mesoderm later give rise to pro/meso/metanephros, genital ridge, ovary, testis, and adrenal glands. Wt1-expressing coelomic epithelium (derived from lateral plate mesoderm) contributes to mesothelium, mesenchyme, and also genital ridge, ovary, testis, and adrenal glands. At E9.5, Wt1 expression is also found in septum transversum, which contributes to gut mesenteries, hepatic sinusoids, peri/epicardium, ventricular myocardium see note above, and diaphragm. At E14.5, Wt1 is expressed in the developing kidney, gonads, adrenal glands, spleen, omentum, mesenteries, the mesothelial layer of all visceral organs (including epicardium and pericardium), and the peritoneum, as well as mesenchyme located near these tissues; the mesothelium and mesenchyme are represented in green. In the adult, we found Wt1 expression in all the visceral fat pads including omentum (1), retroperitoneal (2), perirenal (3), mesenteric (4), epididymal (5) in the abdominal cavity, and epicardial (6) in the thoracic cavity. Its expression is not detected in the subcutaneous (7) and BAT (8) which are both outside the body cavity. It has been shown previously that the myf5+ population of cells in the paraxial mesoderm contributes to BAT as well as retroperitoneal WAT. The origin of subcutaneous WAT (7) is currently not known.