Adipocytes are derived from PdgfRα+ precursor cells in subcutaneous WAT. (a) Confocal images of whole-mounted SWAT from indicated 4-week old Cre:mT/mG male mice (red: membrane-targeted dTomato; green: membrane-targeted eGFP, indicating Cre excision of dTomato). (b) Confocal images of membrane targeted eGFP and Isolectin GS-IB4 Alexa Fluor 647 staining endothelial cells of Cdh5-Cre:mT/mG SWAT. (c) Quantification of flow cytometry analysis of SVF populations from indicated 4-week old Cre:mT/mG mice (n=3). (d) Quantification of qPCR analysis of PdgfRα in mature adipocytes and FACS sorted SVF, Lin−:CD29+:CD34+:Sca-1+:CD24+ (CD24+) and Lin−:CD29+:CD34+:Sca-1+:CD24− (CD24−) cell populations (n=5 RNA extractions from independently isolated cell samples, *** p<0.001). (e) Quantification of flow cytometry analysis of anti-PdgfRα-PE antibody staining in indicated cell populations from 6-week old male C57BL/6 SWAT (n=3 SWAT SVF preparations). (f) A histogram of the distribution of CD24 staining in PdgfRα+:Lin−:CD29+:CD34+:Sca-1+ cells from (e). All error bars represent S.E.M. All scale bars represent 100 μm.

CD24+ adipocyte progenitors give rise to CD24− cells in vivo. (a) Flow cytometry plots of SVF from a time course SWAT development in C57BL/6 mice at the indicated embryonic (e) and post-natal (P) days. Dot plots show Lin−:CD29+:CD34+ cells. (b) CD24 staining in Lin−:CD29+:CD34+:Sca1+ cells from e17.5 and P42 SWAT. (c) Oil Red O staining of differentiated FACS sorted cell populations from e17.5 SWAT. Scale bar represents 100 μm. (d) Representative images of flow cytometry plots of SWAT Lin−:CD29+:CD34+ cells from 6-week old Rag2-/-;IL2Rγ-/-;A-Zip mice at indicated days post transplantation of 100,000 FACS-isolated dTomato+, Lin−:CD29+:CD34+:Sca-1+:CD24+ (CD24+) cells from 6-week old mT/mG mice. Plots show overlay of dTomato+ transplanted cells (white) on Rag2-/-;IL2Rγ-/-;A-Zip recipient SVF cells (blue). (e) Quantification of flow cytometry analysis of dTomato+ CD24+ transplantations into Rag2-/-;IL2Rγ-/-;A-Zip mice (n=4 transplantations, **** p<0.0001, error bar represent S.E.M.). (f). Representative flow cytometry plot of SWAT Lin−:CD29+:CD34+ cells from 6-week old Rag2-/-;IL2Rγ-/-;A-Zip mice at 1 day post transplantation of 100,000 FACS isolated dTomato+, CD24− cells from 6-week old mT/mG mice. The dot plot shows overlay of dTomato+ transplanted cells (white) on Rag2-/-;IL2Rγ-/-;A-Zip recipient SVF cells (blue). (g) Quantification of flow cytometry analysis of dTomato+ CD24− transplantations into Rag2-/- ;IL2Rγ-/-;A-Zip mice (n=3 transplants, error bars represent S.E.M.).

CD24− cells express late adipogenic genes. (a) A representative dot plot is shown with boxes representing the sort parameters for the CD24+ (blue) and CD24− (red) populations. (b,c) qPCR of CD24 expression, late adipogenic and mature adipocyte specific genes in purified mature adipocytes and FACS isolated cell populations (n=5 RNA extractions from independently isolated cell samples; *** p<0.001 vs. all other populations; ^a p<0.001 vs. CD24− and adipocytes (Adi); ^b p<0.001 vs. CD24+ and SVF; ^c p<0.001 vs. Adi and p<0.05 vs. CD24+; error bars represent S.E.M.).

CD24− cells are further committed to an adipogenic fate. (a) Breakdown of PdgfRα+ (α+) SVF into subpopulations. Flow cytometry plots and percentages are shown in . α+:CD24−, α+:CD24+, and α+:Sca1− are Lin−:CD29+:CD34+. α+:CD34− are Lin−:CD29+. (b) Luminescence in 12-week old FVBN/J or A-Zip mice at 6 weeks post subcutaneous sternum transplantation of indicated FACS isolated populations from 6-week old leptin-luciferase BAC transgenic mice. 50,000 cells were transplanted per experiment. (c) Quantification of luminescence from (b) (n=5 for α+:CD24+ and α+:Lin+ transplants into FVB; n=10 for α+:CD24− and SVF transplants into FVB; n=4 for α+:CD24− transplants into AZIP. *p<.05, error bars represent S.E.M.). (d) Brightfield and fluorescent images of tissue formed 6 weeks post subcutaneous sternum transplantation of 2×10⁵ Adiponectin-Cre:mT/mG α+:CD24− cells. Blue arrow indicates tissue formed from transplanted cells. Yellow dashes outline a small piece of recipient SWAT placed on top of sternum as a negative imaging control. Scale bars represent 1 mm. (e) Representative confocal images of Lipidtox stained tissues from (d). Scale bars represent 100 μm. (f) Blood glucose measurement of A-Zip mice before and 6 weeks post subcutaneous sternum transplantation of 50,000 α+CD24− as shown in (b) and (c). (g) A model of in vivo adipogenesis.