The dorsal aortic compartment is a developmental source of brown adipose tissue in mice

Figures

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  Fig. 1. Osr1 is expressed in adipogenic progenitors and preadipocyes. a–c Immunolabeling of thoracic level sections of E13.5 (representative image of n = 2) and E14.5 (representative images of n = 4) Osr1GCE/+ embryos, respectively, for eGFP (Osr1 reporter; green), PPARγ (preadipocytes; red) and MyHC (muscle; white); iBAT: interscapular BAT; sBAT: subscapular BAT; latBAT: lateral BAT; axilWAT: axillary WAT; cBAT: cervical BAT. Boxed regions are shown as magnifications right. Orientation is indicated: d: dorsal; l: lateral; m: medial; v: ventral. d Labeling of E18.5 Osr1GCE/+ thoracic sections (sBAT area shown; representative image of n = 3) for eGFP (Osr1 reporter, green), Oil red O (lipids, red) and DAPI (nuclei; blue). sBAT depot indicated by dashed lines. e Genetic lineage tracing of E11.5 Osr1+ cells to E18.5 (schematic depiction top) using Osr1GCE/ +;RmTmG/ + embryos; representative images of n = 3. Indicated BAT depots are shown, tissue sections were labeled for mGFP (Osr1 lineage; green), FABP4 (adipocytes; red) and DAPI (nuclei; blue). Arrowheads indicate cells shown in magnification inserts. Box plot quantification shown right (Averages of n = 3 biological replicates shown as…

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  Fig. 2. E11.5 Osr1+ cells comprise dermomyotomal adipogenic progenitors. a Cross-sections of E11.5 Osr1GCE/+ embryos at thoracic level labeled for eGFP (Osr1 reporter; green), PAX7 (red) and PPARγ (preadipocytes; white). Cells coexpressing eGFP (Osr1) and PPARγ indicated by arrowheads, see magnification inserts. Abdominal wall (aw) indicated by magenta line; nt: neural tube. Orientation is indicated: d: dorsal; v: ventral; representative image of n = 4. b Parasagittal sections of E11.5 Osr1GCE/+ embryos at thoracic level labeled for OSR1 (green) and EBF2 (magenta). Area of OSR1 and EBF2 coexpression is indicated by an arrowhead, see magnification inserts. Areas of the dermomyotomes are indicated by dashed lines, the abdominal wall (aw) indicated by a purple line; representative image of n = 3. c Genetic lineage tracing of Pax7+ cells (top and middle rows; representative images of n = 2 for each condition), and Myf5+ cells (bottom row; representative image of n = 4). Pax7CreERt2 was induced by tamoxifen administration at E9.5, Myf5Cre is constitutive. Analysis at E11.5 (top row) or E12.5 (middle and bottom rows); see schematic depictions to the left. Tissue sections were labeled for mGFP…

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  Fig. 3. At E9.5, Osr1 labels distinct, non-dermomyotomal BAT progenitors. a Section of E9.5 wild type mouse embryo at the level as indicated, labeled for OSR1 (green), PAX7 (red), PECAM1 (white) and DAPI (nuclei; blue). Boxed area shown as magnification right. Abbreviations: da dorsal aorta; dm dermomyotome; fg: foregut; lu lung bud; nt neural tube; representative image of n = 3. b Mapping of Osr1 expression on an E9.5 embryo section of the MOSTA atlas; cell type annotation shown left, Osr1 spatial expression and heatmap depiction of expression shown right. The aorta-gonad-mesonephros region (AGM) is pointed out by an arrowhead. c Genetic lineage tracing of E9.5 Osr1+ cells to E18.5 (schematic depiction left) using Osr1GCE/+;RmTmG/+ embryos; representative images of n = 3. Indicated BAT depots are shown, tissue sections were labeled for mGFP (Osr1 lineage; green), FABP4 (adipocytes; red) and DAPI (nuclei; blue). Arrowheads indicate cells shown in magnification inserts. Box plot quantification shown right (Averages of n = 3 biological replicates shown as dots, error bar depicts standard error of means; each embryo was investigated on multiple sections). The sBAT box plot shows a range of…

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  Fig. 4. Single-cell analysis of Osr1+ cells at E9.5 and E11.5. a UMAP visualization of E9.5 (top) and E11.5 (bottom) Osr1-eGFP single-cell sequencing analysis. Density plots shown right. b UMAP visualization and cluster identities of combined E9.5 and E11.5 Osr1-eGFP cells. c Dot plot depiction of pre-BAT signature (left) and Ebf2 expression (right) mapped to E9.5 or E11.5 Osr1-eGFP single-cell data. d Dot plot depiction of P3 peri-aortic adipogenic progenitor signatures from Angueira et al. 2021, mapped to E9.5 or E11.5 Osr1-eGFP single-cell data. e Deconvolution analysis of Osr1/preBAT cell signatures on the MOSTA atlas at E11.5 (annotation shown left; arrowheads point out the anterior dermomyotome region). Expression of Ebf2 and Osr1 are shown for comparison; right side: heatmap depiction of Osr1 expression according to MOSTA annotation.

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  Fig. 5. Multilineage potential of Osr1+ cells at E9.5. a Dot plot expression analysis of indicated genes in the E9.5 versus E11.5 Osr1-eGFP clusters 1 (left) and 0 (right). b Enrichr based gene ontology analysis of Osr1-eGFP clusters 1 (left) and 0 (right). c Velocity analysis of E9.5 (top) and E11.5 (bottom) Osr1-eGFP single-cell sequencing datasets without the small clusters 3, 4, 9, 10, 11; boxed region shown as magnification to the right. d STREAM inferred trajectories in high-dimensional space visualized for clusters 0, 1 and 2 of the Osr1-GFP E9.5 or E11.5 datasets, respectively. e Expression pattern of genes that label specific stages among the trajectory of cl1 into cl0 and cl2 within the Osr1-GFP E9.5 dataset. f Genetic lineage tracing analysis of E9.5 Osr1+ cells analyzed at E18.5. Tissue section shows the shoulder region; orientation is indicated: d: dorsal; l: lateral; m: medial; v: ventral. Labeling: mGFP (Osr1 lineage; green), Perilipin (adipocytes; red), MyHC (muscle, white) and DAPI (nuclei; blue). Magnifications of boxed regions shown right. Orange Arrowheads indicate E9.5 Osr1 lineage contribution to skeletal muscle, cartilage and blood vessel walls. AxiWAT axillary…

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  Fig. 6. A migratory mesoangioblast-like Osr1+ cell population as alternative BAT progenitor at E9.5. a Tissue sections (section plane indicated) of E9.5 wild-type mouse embryos labeled for OSR1 (green), PAX7 (red), PECAM1 (endothelial cells, white) and DAPI (nuclei; blue). Boxed areas are shown as magnifications right, arrowheads indicate PECAM1+ /OSR1+ cells; representative image of n = 3. b Feature plot (left) and dot plot (right) depiction of mesoangioblast (MAB) signature mapped to the Osr1 datasets. c–f Tissue sections of Osr1GCE/+;RmTmG/+ embryos induced with tamoxifen at E9.5 (C-E) or E11.5 (F) and analyzed at indicated time points (see schematic depictions to the left). c, d are labeled for mGFP (Osr1 lineage; green), PECAM1 (magenta) and DAPI (nuclei; blue), (e, f) are labeled for mGFP (Osr1 lineage; green), MyHC (skeletal muscle; magenta) and DAPI (nuclei; blue). BAT: presumptive area of brown adipose tissue; da: dorsal aorta; es: esophagus; li: limb bud; nt: neural tube; tr: trachea. Arrowheads indicate Osr1 lineage cells associated with blood vessels in e1 and f1, Osr1 lineage cells within the presumptive BAT depot in e2, and Osr1 lineage contributing to myofibers in e3.…