Fig 2: Schematic model depicting lineage relationships and an increase in molecular and cellular heterogeneity during mammary gland ontogeny. a A profound shift in gene expression occurs between pre-puberty and the onset of puberty. New intermediate clusters identified include a luminal intermediate (Lum Int) population and a mixed-lineage or ‘lineage-primed’ subset. Lum Int expresses luminal genes at levels intermediate between LP and ML, but a small number of genes (four indicated) showed at least 20% higher average expression. CD55+ cells with intermediate properties were readily detectable in puberty and become restricted to the luminal lineage during development. b Proposed position of the newly identified clusters along the epithelial hierarchy. The mixed-lineage subset within the basal compartment may represent a transient population that precedes commitment to the luminal lineage. Molecular evidence places the Lum Int cluster between the luminal progenitor and mature ductal/alveolar cells

Fig 3: Loss of LXRα from KCs Recapitulates Main Features of Zeb2−/− KCs(A) Expression of CD64 and F4/80 by live CD45+Ly6G−Ly6C− liver cells in Clec4f-crexNr1h3fl/fl and Nr1h3fl/fl mice. Liver macs as a percentage of total live CD45+ cells and absolute number per gram of liver.(B) Expression of Clec4F and Tim4 by total liver macs in Clec4f-crexNr1h3fl/fl and Nr1h3fl/fl mice and percentage of total macs expressing Clec4F and Tim4. Data are pooled from two experiments with n = 12 per group. ∗∗∗p < 0.001 Student’s t test.(C) Percentage total chimerism of total Clec4F+ KCs in Nr1h3fl/fl and Clec4f-crexNr1h3fl/fl mice. Data are pooled from two experiments with n = 6–8 per group. ∗∗∗p < 0.001; Student’s t-test.(D) t-SNE plot of SC-RNA-Seq data from KCs from Clec4f-crexNr1h3fl/fl and Nr1h3fl/fl mice, showing clusters, assigned groups and CRE− (Red) and CRE+ (Teal) overlay.(E) Heatmaps showing top DE genes (15 downregulated, 15 upregulated) based on LogFC in KCs with loss of LXRα and expression of the same genes by the indicated groups of KCs from Zeb2fl/fl and Clec4f-crexZeb2fl/fl mice.(F and G) Histogram and MFI of CD55 expression in (F) Zeb2+/+ (Zeb2fl/fl), SiglecF+Zeb2−/− KCs, and SiglecF−Zeb2+/− KCs from Clec4f-crexZeb2fl/fl mice and (G) Nr1h3+/+ (Nr1h3fl/fl) and Nr1h3−/− KCs from Clec4f-crexNr1h3fl/fl mice.(H) Venn diagram showing DE genes specific to Zeb2−/− KCs, Nr1h3−/− KCs, or shared between both mac populations. See also Figure S4.

Fig 4: COMET identifies favorable multi‐gene marker panels for splenic follicular B cells A, BCOMET outputs for two highly ranked 2‐gene marker panels predicted by COMET to isolate the splenic follicular B‐cell population, based on analysis of the Tabula Muris dataset (Tabula Muris Consortium, 2018). Shown are binarized values of gene expression following binarization by the XL‐mHG threshold for each gene separately (left, middle) and when using both genes combined (right).C, DFlow cytometry staining for the marker combinations CD62L+CD44− (C) and CD55+CD62L+ (D) confirms that COMET's candidate multi‐gene marker panels are favorable for flow cytometry staining of splenic follicular B cells. Both marker combinations included a significantly higher frequency of follicular B cells (CD23+) and a lower frequency of other B‐cell subpopulations (DN and CD21+) than the single staining for CD62L+ and CD55+, respectively. (DN = double negative). The marker combinations were selected based on availability of established antibodies. Bars and error bars indicate the mean and standard deviation. *P < 0.05; **P < 0.01; ***P < 0.001; n = 6 biological replicates; unpaired, two‐tailed t‐test.

Fig 5: Muscle PGC-1α overexpression prevents the accumulation of intramuscular adipocytes(A) Representative images of H&E staining (scale bar: 200 μm).(B) Heatmap showing relative expression of Myh3, Plin1, Adipoq, Fapb4, Cd55 and Cd142 genes by RT-qPCR quantification of 3-month-old WT and MCK-PGC-1α mice injected with 50% glycerol (Gly) at 21 dpi. Data are normalized by Actb expression and displayed as relative expression (2–ΔΔCt; mean) vs WT-Sham group. Significance of the differences: ∗p<0.05 vs WT-Sham or MCK-PGC-1α-Sham group (two-way ANOVA+ Holm-Šídák test; n = 7 mice/group).(C) Representative images of ORO staining (scale bar: 200 μm).(D and F) Representative images of perilipin/Hoechst immunostaining of TA muscle (scale bar: 500 μm) of (D) 3-month-old WT and MCK-PGC-1α mice injected with Gly at 21 dpi and (F) 12-month-old WT and MCK-PGC-1α mice injected with BaCl2 at 14 dpi. White dotted lines indicate the boarders of the TA muscle. In D, red-doted rectangles highlight the same area shown in panels A and C.(E and G) Densitometric quantification of Alexa488 signal, proportional to anti-perilipin antibody labeling. Significance of the differences: ∗p<0.05 (Student’s t test; n = 7 mice/group).