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Ebf2 Is a Selective Marker of Brown and Beige Adipogenic Precursor Cells

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Ebf2 Is a Selective Marker of Brown and Beige Adipogenic Precursor Cells

Wenshan Wang et al. Proc Natl Acad Sci U S A.

Abstract

Brown adipocytes and muscle and dorsal dermis descend from precursor cells in the dermomyotome, but the factors that regulate commitment to the brown adipose lineage are unknown. Here, we prospectively isolated and determined the molecular profile of embryonic brown preadipose cells. Brown adipogenic precursor activity in embryos was confined to platelet-derived growth factor α(+), myogenic factor 5(Cre)-lineage-marked cells. RNA-sequence analysis identified early B-cell factor 2 (Ebf2) as one of the most selectively expressed genes in this cell fraction. Importantly, Ebf2-expressing cells purified from Ebf2(GFP) embryos or brown fat tissue did not express myoblast or dermal cell markers and uniformly differentiated into brown adipocytes. Interestingly, Ebf2-expressing cells from white fat tissue in adult animals differentiated into brown-like (or beige) adipocytes. Loss of Ebf2 in brown preadipose cells reduced the expression levels of brown preadipose-signature genes, whereas ectopic Ebf2 expression in myoblasts activated brown preadipose-specific genes. Altogether, these results indicate that Ebf2 specifically marks and regulates the molecular profile of brown preadipose cells.

Keywords: beige adipocyte; brown adipose tissue.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Myf5Cre-lineage–derived brown adipogenic precursors are Pdgfrα+. (A) Cells from E14.5 Myf5Cre; mTmG embryos were fractionated based on expression of GFP, Pdgfrα, and Itga7. The percentage of each cell fraction (of total cell number) is reported as mean ± SD, n = 3. (B) FACS-purified fractions were induced to differentiate into adipocytes and stained with Oil Red O. (C) mRNA levels of general adipogenic genes, brown-fat–selective markers in indicated cell cultures. Pα, Pdgfrα; Ia7, Itga7; ba, brown adipocyte. Values are mean ± SD, n = 3; *P < 0.05, **P < 0.01. (D) Brown adipogenic conversion of sorted precursor populations was evaluated by immunofluoresent staining of Pparγ and Prdm16.
Fig. 2.
Fig. 2.
Prospective identification of Ebf2+ brown adipogenic precursors. (A) Cells from E14.5 Ebf2GFP embryos were fractionated based on GFP and Pdgfrα expression. The percentages of each population are reported as mean ± SD, n = 3. (B) Flow-sorted cell populations (from A) were induced to undergo adipocyte differentiation and stained with Oil Red O. (Scale bar, 100 µm.) (C) mRNA levels of general adipocyte markers, brown-fat–selective genes, and mitochondrial genes in indicated cultures. Values are mean ± SD, n = 3; *P < 0.05, **P < 0.01. (D) Immunofluorescence staining of Pparγ and Prdm16 in indicated cell cultures. (Scale bar, 50 µm.) (E) mRNA levels of Ebf2, Dermo1, and MyoD in freshly sorted cell fractions. Values are mean ± SD, n = 3; *P < 0.05, **P < 0.01.
Fig. 3.
Fig. 3.
Ebf2 marks beige adipogenic precursors in WAT. (A) FACS plots and percentage of stromal cells expressing Ebf2-GFP in iWAT from mice housed at thermoneutrality (30 °C) or cold (4 °C) for 3 d. FSC, forward scatter. The percentage is mean ± SD, n = 3 (six mice per experiment). (B) Sort-purified cell populations from iWAT were induced to undergo adipocyte differentiation and stained with Oil Red O. (C and D) mRNA levels of Gfp, general adipogenic genes, and (D) brown-fat–selective genes in cell cultures from B. Values are mean ± SD, n = 3; *P < 0.05, **P < 0.01.
Fig. 4.
Fig. 4.
Ebf2 and MyoD are expressed in distinct cells within the somitic mesoderm. (A) Immunofluorescence staining of tdTomato, GFP (Myf5Cre), and Ebf2 (red) on adjacent sagittal sections of E10.5 and E11.5 Myf5Cre; mTmG embryos. (B) Costaining of Ebf2 (red) and MyoD (cyan) on transverse sections of anterior somites of E12.5 Myf5Cre; mTmG embryos. GFP (Myf5Cre)/tdTomato double staining is shown (far Left). White dashed boxes show magnified regions 1 and 2. (C) mRNA levels of Ebf2 and MyoD in freshly sorted cells from E12.5, E13.5, and E14.5 embryos. Values are mean ± SD, n = 3 experiments; *P < 0.05, **P < 0.01.
Fig. 5.
Fig. 5.
Ebf2 regulates the brown-preadipose–specific gene signature. (A) Scatter plot showing global gene expression in freshly sorted Ebf2+ versus Ebf2 (all Pdgfrα+) precursor cells (x axis) and Pdgfrα+ versus Pdgfrα [all Myf5Cre(GFP)+] cells (y axis). (B) mRNA levels of select signature genes in established cell lines. (C) mRNA levels of signature genes in wild-type (WT) and Ebf2-knockout (KO) brown preadipose cells. (D) C2C12 myoblasts were transduced with control (ctl) puromycin (Puro)- or Ebf2-expressing retrovirus. Western blot analysis of Ebf2 protein levels (Left). mRNA levels of muscle-specific genes and brown-preadipose–selective genes (Right). Values are mean ± SD, n = 3; *P < 0.05, **P < 0.01.

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