PDGFRα controls the balance of stromal and adipogenic cells during adipose tissue organogenesis

Abstract

Adipose tissue is distributed in depots throughout the body with specialized roles in energy storage and thermogenesis. PDGFRα is a marker of adipocyte precursors, and increased PDGFRα activity causes adipose tissue fibrosis in adult mice. However, the function of PDGFRα during adipose tissue organogenesis is unknown. Here, by analyzing mice with juxtamembrane or kinase domain point mutations that increase PDGFRα activity (V561D or D842V), we found that PDGFRα activation inhibits embryonic white adipose tissue organogenesis in a tissue-autonomous manner. By lineage tracing analysis, we also found that collagen-expressing precursor fibroblasts differentiate into white adipocytes in the embryo. PDGFRα inhibited the formation of adipocytes from these precursors while favoring the formation of stromal fibroblasts. This imbalance between adipocytes and stromal cells was accompanied by overexpression of the cell fate regulator Zfp521. PDGFRα activation also inhibited the formation of juvenile beige adipocytes in the inguinal fat pad. Our data highlight the importance of balancing stromal versus adipogenic cell expansion during white adipose tissue development, with PDGFRα activity coordinating this crucial process in the embryo.

Keywords: Adipocyte; Cell fate; Lipodystrophy; Mouse; Myf5; Platelet-derived growth factor.

Fig. 1.

PDGFRα activation inhibits WAT but not BAT development in Myf5-D842V mutants. (A-D) Gross morphology of the indicated adipose tissue depots of a Myf5-D842V mouse and littermate control at P18. Orange outlined areas show the region of iBAT. Yellow outlined areas show the region of WAT. (E) Mass of the indicated tissues at P18. n=3 littermate tissue samples per data point. (F) Mass of the indicated tissues at P18 relative to whole body mass. n=3 littermates per data point. (E,F) Mean±s.e.m. ND, non-detected. (G-J) H&E-stained images of adipose depots derived from Myf5-Cre⁺ lineage (iWAT, rWAT) or Myf5-Cre^neg lineage (ingWAT, pWAT) tissues at P21 from a Myf5-D842V mouse and littermate control. Arrows indicate the remnant of stromal tissue that remains in place of WAT. Scale bars: 100 μm.

Fig. 2.

PDGFRα activation causes lipodystrophy in Sox2-V561D mutants. (A-D) Gross morphology of the indicated adipose tissue depots of a Sox2-V561D mouse and littermate control at P18. Orange outlined areas show the region of iBAT. Yellow outlined areas show the region of WAT. (E) Mass of the indicated tissues at P18. n=3 tissue samples per data point. (F) Mass of the indicated tissues at P18 relative to whole body mass. n=3 mice per data point. All data are presented as mean± s.e.m. *P<0.05, **P<0.01, ***P<0.001, Student's t-test.

Fig. 3.

PDGFRα activation inhibits adipose tissue organogenesis and causes fibrosis in Sox2-V561D mutants. (A-D) H&E-stained images of tissue sections from a Sox2-V561D mouse and littermate control at P18. Arrows indicate the tissue margin enriched for stromal fibroblasts. (E) Quantification of iWAT and ingWAT adipocyte size at P18. n=30 adipocytes per data point. (F) Picrosirius Red-stained images of ingWAT at P18, with polarized light microscopy to visualize collagen fibers. (G) Quantification of collagen fiber area as a percentage of total area. n=3 polarized light images per genotype. All data are presented as mean±s.e.m. **P<0.01, ****P<0.0001, Student's t-test. Scale bars: 100 μm.

Fig. 4.

Emergence of stromal fibroblasts and adipocytes during embryogenesis. (A) Analysis of ingWAT organogenesis in Col1a1-EGFP mice at the indicated embryonic time points, with immunofluorescence staining for Plin1. (B) Higher magnification of A. Arrows indicate colocalization of Col1a1-EGFP reporter and Plin1. (C) Quantification of the percentage of Plin1⁺ cells colabeled with EGFP. n=3. (D) Lineage tracing analysis by breeding Col1a2-CreER mice and R26-tdTomato reporter mice, as shown in E,F. (E) Immunofluorescence of Col1a2-CreER;R26-tdTomato lineage tracing in ingWAT, iWAT and iBAT at E18.5, with staining for Plin1. Arrows indicate colocalization of Tomato and Plin1. (F) Quantification of percentage of Tomato⁺ cells colabeled with Plin1. n=4 embryos. All data are presented as mean±s.e.m. **P<0.01, ****P<0.0001, Student's t-test. Scale bars: 100 μm in A; 50 μm in B,E.

Fig. 5.

Disrupted fibroblast-adipocyte balance in newborn Sox2-V561D mice. (A,B) H&E-stained images of iWAT and ingWAT from a Sox2-V561D mouse and littermate control at P0. (C,D) Immunofluorescence analysis of iWAT and ingWAT from a Sox2-V561D mouse and littermate control at P0, stained for Plin1. (E) Quantification of the percentage of Plin1⁺ adipocytes and Plin1^neg fibroblasts from iWAT and ingWAT at P0. n=3-4 sections per data point. (F) Quantification of the total number of Plin1⁺ adipocytes and Plin1^neg fibroblasts from iWAT and ingWAT at P0. n=3-4 sections per data point. All data are presented as mean±s.e.m. *P<0.05, **P<0.01, Student's t-test. Scale bars: 100 μm.

Fig. 6.

PDGFRα activation inhibits preadipocyte commitment. (A,B,G,H) Fold change in adipocyte marker and adipogenic transcription factor mRNA levels in ingWAT at the indicated time points, as determined by quantitative RT-PCR. Results are shown as fold change in mutant over control. n=3 tissue samples per genotype. (C) Representative flow cytometry plots of the stromal-vascular fraction from ingWAT in control and Sox2-V561D mice at E18.5. Pseudocolor plots show Lin^neg (CD45⁻ CD31⁻) cells. The Lin^neg (CD29⁺ CD34⁺) adipocyte progenitor population and Lin^neg (CD29⁺ CD34⁻) non-adipogenic populations are outlined. (D) Representative flow cytometry plots of Lin^neg (CD29⁺ CD34⁺) cells from C. Pseudocolor plots show the Lin^neg (CD29⁺ CD34⁺ Sca1⁺ CD24⁺) Q1 and Lin^neg (CD29⁺ CD34⁺ Sca1⁺ CD24⁻) Q4 adipocyte precursor populations. (E,F) Quantification of data in C and D plus additional biological replicates (not shown). n=3 mice per genotype. All data are presented as mean±s.e.m. Red significance identifier refers to subpopulation shown in red; black significance identifier refers to the total cell population. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student's t-test.

Fig. 7.

PDGFRα inhibits the emergence of beige adipocytes. (A) H&E-stained images of ingWAT from a Sox2-V561D mouse and littermate control at P18. Boxed areas are shown at higher magnification to the right. (B) Immunofluorescence analysis of ingWAT from a Sox2-V561D mouse and littermate control at P18, stained for Plin1 and UCP1. (C) Fold change in beige adipocyte marker mRNA levels in ingWAT at P18, as determined by quantitative RT-PCR. Results are shown as fold change in mutant over control. n=3 tissue samples per genotype. All data are presented as mean±s.e.m. *P<0.05, **P<0.01, Student's t-test. Scale bars: 200 μm in A; 100 μm in B.