Supraclavicular brown adipocytes originate from Tbx1+ myoprogenitors

Figures

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  Fig 1. scBAT does not arise from Pax3+ progenitor cells. (A) Schematic representation of the location of peri-scapular and neck BAT in mice. (B) Representative photo of major BAT depots examined in this study. (C) Generation of the reporter mice for Pax3+ cells. (D) Fluorescent images of dorsal BAT depots from 5-week-old male Pax3-mTmG mice (green = mG, red = mT, blue = DAPI, scale = 50 μm). (E) Frequency of GFP+ cells within each BAT depot (n = 3). (F) Anatomic location of supraclavicular BAT depot (indicated with red dotted line). (G) Fluorescent images of medial and lateral scBAT from Pax3-mTmG mice. (green = mG, red = mT, blue = DAPI, scale = 50 μm). (H) Quantification of GFP+ cells as a percentage of total adipocytes (n = 3). Data are presented as mean ± SEM. Extended data are listed in S1 Data. BAT, brown adipose tissue; scBAT, supraclavicular brown adipose tissue.

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  Fig 2. scBAT does not arise from Myf5+ precursor cells. (A) Generation of the reporter mice for the Myf5+ lineage cells. (B, C) Fluorescent images of dorsal BAT (B) or scBAT (C) from 2-month-old female Myf5-mTmG mice (green = mG, red = mT, blue = DAPI, scale = 50 μm). (D) Frequency of GFP+ cells within each BAT depot (n = 2–5). Data are presented as mean ± SEM. Extended data are listed in S1 Data. BAT, brown adipose tissue; scBAT, supraclavicular brown adipose tissue.

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  Fig 3. Intact scBAT in mice with PPARγ deficiency in the Myf5+ lineage. (A, B) Indicated dorsal BAT depots from 8-month-old Ppargf/f (mixed sex, n = 10) and PpargΔMyf5 mice (mixed sex, n = 6) were isolated, photographed (A, scale = 1 cm), and weighed (B). (C) Representative immunoblotting of UCP1 and PPARγ with iBAT proteins from Ppargf/f and PpargΔMyf5 mice. Densitometry (n = 9–10) is shown to the right. (D, E) scBAT depots were isolated from 8-month-old Ppargf/f (n = 10) and PpargΔMyf5 mice (n = 6), photographed (D, scale = 1 cm), and weighed (E). (F) Representative UCP1 and PPARγ expression in scBAT from Ppargf/f and PpargΔMyf5 mice. Densitometry (n = 9–10) is shown to the right. Data are presented as mean ± SEM. ***, p < 0.01 by unpaired Student’s t test. Extended data are listed in S1 Data. BAT, brown adipose tissue; iBAT, interscapular brown adipose tissue; PPARγ, peroxisome proliferator-activated receptor gamma; scBAT, supraclavicular brown adipose tissue.

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  Fig 4. scBAT is composed of Tbx1+ lineage adipocytes. (A) Tbx1+ lineage cell labeling with the Tbx1-mTmG mice. (B, C) Fluorescent images of dorsal BAT (B) and scBAT (C) from 6-week-old female Tbx1-mTmG mice (green = mG, red = mT, blue = DAPI, scale = 50 μm). (D) Frequency of GFP+ cells within each BAT depot (n = 3). Data are presented as mean ± SEM. (E) Fluorescent images of scBAT from a 1-year-old Tbx1-mTmG mouse (green = mG, red = mT, blue = DAPI, scale = 500 μm). Salivary gland (epithelial lineage) was labeled by mT. Data are presented as mean ± SEM. Extended data are listed in S1 Data. BAT, brown adipose tissue; scBAT, supraclavicular brown adipose tissue.

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  Fig 5. PPARγ deficiency in the Tbx1+ lineage impairs scBAT function. (A–D) Weights of supraclavicular (A), interscapular (B), subscapular (C), and supraspinal (D) BAT depots from 4-month-old Ppargf/f (n = 7) and PpargΔTbx1 (n = 6) female mice. (E) Representative HE staining of scBAT from Ppargf/f and PpargΔTbx1 female mice (scale = 100 μm). # indicates areas of adipose whitening and tissue degeneration. (F, G) Thermogenic gene expression in scBAT (F) and iBAT (G) of female mice was determined by RT-qPCR and adjusted by total tissue RNA to calculate the relative total transcript levels. (H) Changes in core body temperature of Ppargf/f (n = 9) and PpargΔTbx1 (n = 7) female mice during cold challenge in 4°C. (I–L) Weights of supraclavicular (I), interscapular (J), subscapular (K), and supraspinal (L) BAT depots from 4-month-old Ppargf/f (n = 14) and PpargΔTbx1 (n = 10) male mice after 3 weeks of cold challenge. (M) Representative HE staining of scBAT from Ppargf/f and PpargΔTbx1 male mice (scale = 100 μm). (N, O) Thermogenic gene expression in scBAT (N) and iBAT (O) of male mice was determined by RT-qPCR and adjusted by total tissue RNA to calculate the relative total transcript levels.…

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  Fig 6. Characterization of HFD-fed PpargΔTbx1 mice. (A, B) Body weight of female (A, n = 6–7) and male (B, n = 10–12) Ppargf/f and PpargΔTbx1 mice after HFD feeding. (C) Glucose tolerant test of HFD-fed Ppargf/f (n = 8) and PpargΔTbx1 (n = 10) mice (mixed sexes). (D) HFD-fed Ppargf/f (n = 10) and PpargΔTbx1 (n = 10) male mice were fasted overnight, orally gavaged with 4 μg LPS (in 200 μl olive oil), and sera were collected at indicated time for LPS measurement. (E–H) HFD-fed Ppargf/f (10 males, 7 females) and PpargΔTbx1 (8 males, 5 females) mice were fasted and gavaged with 50 μl of 0.1 μg/μl LPS in water per 20 g of body weight. scBAT (E–G) and liver (H) were collected 3 h later for RT-qPCR quantification the expression of Ucp1 (E), Adipoq (F), and inflammatory genes (G, H). Data are presented as mean ± SEM. *, p < 0.05; **, p < 0.01 by unpaired Student’s t test (E, F) or two-way ANOVA (D). Extended data are listed in S1 Data. HFD, high-fat diet; LPS, lipopolysaccharide; scBAT, supraclavicular brown adipose tissue.

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  Fig 7. scBAT dysfunction in Prdm16ΔTbx1 mice. (A) Weight of indicated tissues as a percentage of body weight in Prdm16f/f (n = 8) and Prdm16ΔTbx1 (n = 5) female mice. (B) Core body temperature of Prdm16f/f (n = 8) and Prdm16ΔTbx1 (n = 5) female mice during cold challenge in 4°C. (C) Representative HE staining of scBAT from Prdm16f/f and Prdm16ΔTbx1 female mice (scale = 100 μm). (D) RT-qPCR measurements of gene expression in scBAT and iBAT of Prdm16f/f (n = 8) and Prdm16ΔTbx1 (n = 5) female mice. (E) Core body temperature of Prdm16f/f (n = 5) and Prdm16ΔTbx1 (n = 6) male mice during cold challenge in 4°C. (F) Weight of indicated tissues as a percentage of body weight in Prdm16f/f (n = 5) and Prdm16ΔTbx1 (n = 6) male mice after 3 weeks of cold challenge. Data are presented as mean ± SEM. *, p < 0.05; **, p < 0.01 by unpaired Student’s t test (D) or two-way ANOVA (B, E). Extended data are listed in S1 Data. iBAT, interscapular brown adipose tissue; scBAT, supraclavicular brown adipose tissue.

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  Fig 8. TBX1 gene expression in human deep neck BAT. (A) Taqman RT-qPCR of TBX1 gene expression, normalized to GAPDH, in subcutaneous (subQ) neck WAT and deep neck BAT from 3 donors. (B) SubQ neck WAT and deep neck BAT preadipocytes from 4 donors were differentiated to adipocytes in the presence or absence of rosiglitazone for 14 days. TBX1 gene expression normalized to GAPDH was determined using Taqman probes. (C) Differentiated adipocytes from SubQ WAT and deep neck BAT (n = 4 donors) were treated with or without 500 μm dibutyril-cAMP for 10 h. TBX1 gene expression was normalized to GAPDH. Data are presented as mean ± SD. *, p < 0.05; **, p < 0.01 by paired Student’s t test (A) or two-way ANOVA (B, C). Extended data are listed in S1 Data. BAT, brown adipose tissue.