Characterization of Cre recombinase models for the study of adipose tissue

Abstract

The study of adipose tissue in vivo has been significantly advanced through the use of genetic mouse models. While the aP2-Cre(BI) and aP2-Cre(Salk) lines have been widely used to target adipose tissue, the specificity of these lines for adipocytes has recently been questioned. Here we characterize Cre recombinase activity in multiple cell populations of the major adipose tissue depots of these and other Cre lines using the membrane-Tomato/membrane-GFP (mT/mG) dual fluorescent reporter. We find that the aP2-Cre(BI) and aP2-Cre(Salk) lines lack specificity for adipocytes within adipose tissue, and that the aP2-Cre(BI) line does not efficiently target adipocytes in white adipose depots. Alternatively, the Adiponectin-CreERT line shows high efficiency and specificity for adipocytes, while the PdgfRα-CreERUCL and PdgfRα-CreERJHU lines do not efficiently target adipocyte precursor cells in the major adipose depots. Instead, we show that the PdgfRα-Cre line is preferable for studies targeting adipocyte precursor cells in vivo.

Keywords: Cre recombinase; adipocyte; adipocyte stem cell; lineage tracing; mouse model.

Figure 1. aP2-Cre^BI primarily labels endothelial cells and brown adipocytes. (A) Confocal microscopy of the indicated adipose tissue depots from aP2-Cre^BI; mT/mG mice. (B) Quantification of cells displaying Cre-mediated expression of eGFP in cell populations of the indicated adipose tissue depots (n = 3). (C) Confocal microscopy of SWAT from aP2-Cre^BI; mT/mG mice stained with isolectin GSIB₄ to label endothelial cells. Scale bars in (A and C) are 100 μm. SWAT, subcutaneous WAT; GWAT, gonadal WAT; RWAT, retroperitoneal WAT; MWAT, mesenteric WAT; BAT, brown adipose tissue; SVF, stromal vascular fraction.

Figure 2. aP2-Cre^Salk labels multiple cell populations within adipose tissue. (A) Confocal microscopy of the indicated adipose tissue depots from aP2-Cre^Salk; mT/mG mice. (B) Quantification of cells displaying Cre-mediated expression of eGFP in cell populations of the indicated adipose tissue depots (n = 3). Scale bars in (A) are 100 μm. SWAT, subcutaneous WAT; GWAT, gonadal WAT; RWAT, retroperitoneal WAT; MWAT, mesenteric WAT; BAT, brown adipose tissue; SVF, stromal vascular fraction.

Figure 3. Adiponectin-CreERT labels mature adipocytes. (A) Confocal microscopy of the indicated adipose tissue depots from Adiponectin-CreERT; mT/mG mice after tamoxifen treatment. (B) Quantification of cells displaying Cre-mediated expression of eGFP in cell populations of the indicated adipose tissue depots (n = 3). Scale bars in (A) are 100 μm. SWAT, subcutaneous WAT; GWAT, gonadal WAT; RWAT, retroperitoneal WAT; MWAT, mesenteric WAT; BAT, brown adipose tissue; SVF, stromal vascular fraction.

Figure 4. PdgfRα-CreER does not efficiently label adipocyte precursor cells. (A) Confocal microscopy of the liver and skeletal muscle (gastrocnemius) from PdgfRα-Cre; mT/mG mice. (B) Quantification of cells displaying Cre-mediated expression of eGFP in liver and skeletal muscle of PdgfRα-Cre; mT/mG mice (n = 3). (C) Immunofluorescence image of the back skin of PdgfRα-CreER^UCL; mT/mG mouse after tamoxifen injection showing recombination in fibroblastic cells of the upper dermis. Perilipin staining of adipocytes indicates the location of the adipocyte layer within the dermal compartment. Representative image from 2 animals. (D) Quantification of cells displaying Cre-mediated expression of eGFP in the adipocyte precursor population of the indicated depots of PdgfRα-CreER^UCL; mT/mG and PdgfRα-CreER^JHU; mT/mG mice after daily treatment of 50mg/kg tamoxifen for 6 d (n = 3–4). Scale bars in (AandC) are 100 μm. SWAT, subcutaneous WAT; GWAT, gonadal WAT; SVF, stromal vascular fraction.