Ebf2 is a selective marker of brown and beige adipogenic precursor cells Wenshan Wanga,b, Megan Kissiga,b, Sona Rajakumaria,b, Li Huanga,b, Hee-woong Lima,c, Kyoung-Jae Wona,c, and Patrick Sealea,b,1 aInstitute for Diabetes, Obesity, Metabolism, bDepartment of Cell and Developmental Biology, and cDepartment of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 Edited by C. Ronald Kahn, Joslin Diabetes Center, Harvard Medical School, Boston, MA, and approved August 8, 2014 (received for review July 8, 2014) Brown adipocytes and muscle and dorsal dermis descend from transcriptomic analyses identified a brown-preadipose–specific precursor cells in the dermomyotome, but the factors that regulate gene signature, which included early B-cell factor 2 (Ebf2), a crit- commitment to the brown adipose lineage are unknown. Here, we ical transcriptional regulator in mature brown adipocytes (18). prospectively isolated and determined the molecular profile of We found that brown/beige preadipose activity in embryos and embryonic brown preadipose cells. Brown adipogenic precursor adult fat depots was restricted to Ebf2-expressing cells. Further- activity in embryos was confined to platelet-derived growth factor more, Ebf2 deficiency in brown preadipose cells reduced the ex- + Cre α , myogenic factor 5 -lineage–marked cells. RNA-sequence anal- pression of nearly all brown preadipose signature genes, whereas ysis identified early B-cell factor 2 (Ebf2) as one of the most selec- ectopic expression of Ebf2 in myoblasts activated brown pre- tively expressed genes in this cell fraction. Importantly, Ebf2- GFP adipose-selective genes. Altogether, our study reveals that expressing cells purified from Ebf2 embryos or brown fat tissue Ebf2 is a specific marker of brown/beige preadipose cells and did not express myoblast or dermal cell markers and uniformly dif- that Ebf2 functions at this stage to control precursor identity. ferentiated into brown adipocytes. Interestingly, Ebf2-expressing cells from white fat tissue in adult animals differentiated into Results brown-like (or beige) adipocytes. Loss of Ebf2 in brown preadipose Prospective Isolation of Myf5Cre-Lineage–Marked Brown Preadipose cells reduced the expression levels of brown preadipose-signature Cells. Specific marker genes for brown preadipose cells were pre- genes, whereas ectopic Ebf2 expression in myoblasts activated viously unknown. To identify such factors, we isolated enriched brown preadipose-specific genes. Altogether, these results indicate populations of brown preadipose cells from mouse embryos. that Ebf2 specifically marks and regulates the molecular profile of Brown adipocytes descend from a Myf5Cre-expressing cell lineage brown preadipose cells. that also gives rise to skeletal muscle cells (17). To enable the Cre – beige adipocyte | brown adipose tissue efficient purification of Myf5 -lineage marked cells by flow cytometry, we generated Myf5Cre; R26R-mTmG mice, in which Myf5Cre-expressing cells are genetically and heritably labeled by rown adipose tissue (BAT) expends energy in the form of a membrane-targeted form of GFP [Myf5Cre(GFP)] (19). We heat in response to various stimuli, including cold exposure B noted that GFP was readily detected in somites starting from and certain diets (1). The thermogenic activity of brown adipo- embryonic day (E) ∼E9.5 (Fig. S1A). In agreement with previous cytes is mediated by uncoupling protein 1 (Ucp1), a mitochondrial findings, the majority of peroxisome proliferator-activated receptor membrane protein. Ucp1, when activated, dissipates the electro- chemical gradient that powers ATP synthesis, which allows for low rates of ATP production despite high levels of substrate oxidation Significance (1). Thermogenically competent Ucp1-expressing brown-like adi- pocytes, called beige or brite (brown-in-white) cells, also develop High levels of brown/beige fat activity protects animals against in certain white adipose tissue (WAT) depots in response to cold metabolic disease, but there has been little known about the exposure (reviewed in ref. 2). In mice, BAT and/or beige fat- precursor cells that mediate the expansion of brown or beige mediated thermogenesis suppresses high-fat-diet–associated obesity fat. We discovered that early B-cell factor 2 (Ebf2), a transcrip- – tion factor, is selectively expressed in brown and beige fat cell and metabolic disease (2 8). High levels of activated BAT also + correlate with reduced adiposity in people (9). There is thus great precursors. Through purification of Ebf2 cells, we identified hope that brown/beige fat activity can be increased to reduce obesity a gene profile of brown fat precursors that can be used to and metabolic disease. distinguish these cells from other developmentally related cell BAT depots mature during the embryonic and fetal stages of types. Importantly, Ebf2 was also found to regulate the gene mouse development. In humans, the major BAT depot is lo- expression profile of brown fat precursor cells. Taken together, cated in the interscapular region of newborns, but this tissue this study identifies Ebf2 as a highly specific marker of brown regresses and is absent in adults (10). However, brown and and beige preadipose cells and reveals that Ebf2 functions to beige adipocytes are found in other depots of adult humans, control brown preadipose cell identity. particularly in the supraclavicular area, neck, and along the – Author contributions: W.W. and P.S. designed research; W.W., M.K., S.R., L.H., and H.-w.L. spinal column (10 14). Recent lineage studies showed that performed research; S.R. contributed new reagents/analytic tools; W.W., H.-w.L., K.-J.W., brown adipocytes originate from precursor cells that express and P.S. analyzed data; and W.W. and P.S. wrote the paper. myogenic factor 5 (Myf5), paired box protein 7 (Pax7), Pax3,and The authors declare no conflict of interest. engrailed 1 (En1) in the somitic mesoderm (15–17). However, “ This article is a PNAS Direct Submission. the factors that control brown preadipose cell fate in this tri- Data deposition: The sequences reported in this paper have been deposited in the Gene potent” compartment are unknown. Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. In the absence of molecular markers for committed brown pre- GSE60443). adipocytes, such cells can only be recognized retrospectively after See Commentary on page 14318. + their differentiation into Ucp1 adipocytes. Here, we com- 1To whom correspondence should be addressed. Email: [email protected]. bined lineage and cell surface markers to prospectively isolate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. brown preadipose cells during embryogenesis. Global mRNA 1073/pnas.1412685111/-/DCSupplemental. 14466–14471 | PNAS | October 7, 2014 | vol. 111 | no. 40 www.pnas.org/cgi/doi/10.1073/pnas.1412685111 Downloaded by guest on October 1, 2021 γ (Pparγ)-expressing adipocytes in the interscapular BAT as well as E14.5 Ctrl GFP- GFP+ A Cre 104 B Myf5 ;mTmG SEE COMMENTARY the Desmin-expressing skeletal myofibers in the trunk and limb 103 Pdgfrα- Pdgfrα+ Itga7+ Pdgfrα+ + 102 101 were GFP at E16.5 (Fig. S1B) (17). GFP was also expressed in GFP- GFP+ 101 102 103 adipocytes within the axillary and cervical BAT (Fig. S1C)andin the majority of preadipocytes from postnatal BAT (Fig. S1D). Pdgfrα- Pdgfrα+ + GFP cells also contributed to the dorsal dermis (Fig. S1B). These Itga7- Itga7+ Cre GFP+;Pdgfrα- data indicate that Myf5 cells contribute to at least three different 105 45±2.4 20.1±2.5 104 Oil-red-O staining α mesodermal tissue types in late stage embryos: BAT, skeletal α 104 103 103 102 Pdgfr muscle, and dorsal dermis. Consistent with recent studies, GFP was Pdgfr 102 101 17.8±1.5 17.2±1.6 5.4±0.7 also found in some adipocytes in the retroperitoneal and back s.c. 101 102 103 104 101 102 103 Cre Phase WAT of adult mice (20) (Fig. S1E). Thus, Myf5Cre (GFP) is Myf5 (GFP) Itga7 C General adipocyte genes Brown fat-selective genes a selective but not specific marker of developing brown fat cells. Pparγ AdipoQ Ucp1 Cidea Pparα Prdm16 250 700 14 BAT forms prenatally in mice, but the embryonic stages as- ** 12 ** 200 600 sociated with the morphological and molecular differentiation of 500 10 150 400 ** 8 brown adipocytes were unclear. To assess this differentiation, we 100 300 6 200 4 ** 50 performed hematoxylin and eosin (H&E) staining and immu- 100 2 relative mRNA 0 0 0 - + + + - + + - - + + + - + + - + – + α α α α α α α α α α α α α α α α α nohistochemistry on transverse sections of E13.5 E16.5 embryos. α P P P P Ia7+ P P Ia7+ P Ia7+ P P P Ia7+ P P P P Ia7+ P Ia7+ P P + P ba cell ba cell ba cell ba cell ba cell ba cell Cre 3T3-L1 3T3-L1 3T3-L1 3T3-L1 3T3-L1 At E14, we detected distinctive clusters of Myf5 (GFP) pre- GFP - + - + 3T3-L1 GFP - + - + GFP - + - + sumptive brown adipogenic cells that expressed Pparγ (Fig. S2). D GFP+;Itga7+ GFP-; Pdgfrα+ GFP+;Pdgfrα+ Consistent with previous studies, BAT was easily recognized by GFP Pparγ Merge GFP Pparγ Merge GFP Pparγ Merge H&E staining at E15.5 (21, 22) and contained cells that expressed γ the mature adipocyte marker perilipin (Fig. S2). This suggested that Ppar the dorsal interscapular region of E14 embryos is a rich source of pioneering brown adipose precursors. Prdm16 We next considered whether particular cell surface markers could GFP Prdm16 Merge GFP Prdm16 Merge GFP Prdm16 Merge be combined with Myf5Cre-lineage marking to provide additional + selectivity for brown preadipose cells. Platelet-derived growth factor Fig. 1. Myf5Cre-lineage–derived brown adipogenic precursors are Pdgfrα .(A) α α Cells from E14.5 Myf5Cre;mTmGembryos were fractionated based on ex- (Pdgfr ) is a cell surface marker of adipogenic precursors in adult α WAT and muscle (23–26); this prompted us to examine Pdgfrα pression of GFP, Pdgfr , and Itga7.