Fig 2: Classification of different cell populations within the adipose tissue.a, Flow cytometry dot plots show the new gating strategy used to sort eP1, eP2 and eP3. b, Quantification of adipogenesis (left) and cell number (right) of Lin-Sca1+ cells, eP1 cells, eP2 cells, VAP1+CD142+ APCs, VAP1-CD55- cells, DN:CD142- cells and eP3 induced by A Cocktail (1 µM dexamethasone, 0.5 mM isobutylmethylxanthine and 1 µM insulin). Data are shown as mean ± s.e.m., n = 8 independent wells. Data were analyzed with one-way analysis of variance (ANOVA); F(6,49) = 69.7002, P < 0.0001. c, Microscopy images of different cell populations shown in b on differentiation day 6. Experiment was repeated twice. d, Relative mRNA levels of P1 marker (Cd55, Pcsk6, Efhd1, Pi16 and Smpd3), P2 markers (Vap1, Col4a1, Sparcl1 and Sdc1) and Areg cell-specific marker (Cd142, Gdf10, Igfbp3, Fmo2 and Clec11a) genes in different cell populations; n = 3 biological replicates. Data show mean ± s.e.m. e, Quantification of adipogenesis (left) and cell number (right) of Lin-Sca1+ cells, eP1 cells, eP2 cells and eP3 induced by A Cocktail. Data show mean ± s.e.m.; n = 6 independent wells. Data were analyzed with one-way ANOVA; F(3,20) = 280.8, P < 0.0001 (left); multicomparison with Lin-Sca1+ group was performed by two-stage step-up method with false discovery rate (FDR) = 0.05. F(3,20) = 2.838, P = 0.064 (right). f, Quantification of adipogenesis (left) and cell numbers (right) of Lin-Sca1+ cells, eP1 cells, eP2 cells and eP3 induced by C Cocktail (1 µM insulin). Data are shown as mean ± s.e.m., n = 6 independent wells. Data were analyzed with one-way ANOVA. F(3,20) = 48.27, P < 0.0001 (left), multicomparison with Lin-Sca1+ group was performed by two-stage step-up method with FDR = 0.05. F(3,20) = 0.189, P = 0.903 (right). g, Microscopy images of different cell populations shown in e and f on differentiation day 6. In all panels, nuclei were stained with Hoechst 33342 (blue) and lipids were stained with LD540 (yellow). Scale bars, 100 µm.Source data

Fig 3: other markers expression in subpopulations of adipogenitors.a) Scheme for HFD induced obesity. b) eP3 percentage in ingWAT quantified by FACS. Data show as mean ± SD, n = 6 biological replicates. Data was analyzed using two-tailed Student’s t-test. c) Circulating RSPO2 levels in chow and HFD fed mice. Data shown as mean ± SD, n = 6 biological replicates. Data was analyzed using two-tailed Student’s t-test. d) RSPO2 expression quantified by western blot in ingWAT, visWAT and liver. HSP90 served as loading control. Data show the mean ± SD, n = 6 biological replicates. Data was analyzed by two-tailed Student’s t-test. e) Rspo1-4 mRNA levels in Lin-Sca1+ cells and eP3 cells of ingWAT. Data are presented as mean + /- SEM, n = 4 biological replicates. Data was analyzed by one-way ANOVA, in Lin-Sca1+ group, F(3,12)=602, P < 0.0001; in eP3 group, F(3,12)=326, P < 0.0001. Source data

Fig 4: Laminin a5 is needed for HR+ luminal epithelial cell identity. (A) Representative CD24/CD29 FACS plot showing relative amounts of lineage marker negative luminal and basal MECs from 10-week-old Lama5fl/fl;K8-CreERT2 and control animals. Gating strategy for the same sample is shown in Fig. S3A. (B,C) Quantification of the percentages of basal and luminal MECs (+/+, n=3; fl/fl, n=3 individuals analyzed) and (C) luminal HR+ and HR- subpopulations from 10-week-old Lama5fl/fl;K8-CreERT2 and control mice (+/+, n=6; fl/fl, n=6 individuals analyzed). Data points indicate samples analyzed from individual mice. (D) Mean intensity of Sca1+ luminal population from Lama5fl/fl;K8-CreERT2 and control mice. Data show mean from n=5 separate experiments, in which 3-4 individual animals were analyzed per group. Data points indicate mean values from five separate experiments. (E) Representative Sca1-BV711 histograms of individual Lama5+/+;K8-CreERT2 and Lama5fl/fl;K8-CreERT2 mice. (F) qRT-PCR analysis of Esr1, PR and RANKL expression compared with GAPDH in luminal MECs from 10-week-old Lama5fl/fl;K8-CreERT2 and control mice (Esr1 +/+, n=5; fl/fl, n=4; PR +/+, n=9; fl/fl, n=8; RANKL +/+, n=4; fl/fl, n=3). Data points indicate samples analyzed from individual mice. Data are mean±s.d. *P<0.05, **P<0.01 [unpaired two-tailed Student's t-test (B,C,F); paired t-test (D)].

Fig 5: Gli1-labeled meniscus cells possess mesenchymal progenitor properties.(A) Representative immunofluorescence images of Sca1, Cd90, Cd200, PDGFRa, Cd248, and Prg4 staining in 3-month-old Gli1ER/Td meniscus. Scale bars, 200 µm. Boxed areas are shown at high magnification in corresponding panels at the bottom. Dashed lines indicate meniscus surface. Yellow cells are double positive for progenitor marker and Td. F: femur; T: tibia; M: meniscus. Blue: DAPI. (B) Quantification of the expression level of mesenchymal progenitor markers in Gli1+ and Gli1- cells from meniscus. Digested meniscus cells from 3-month-old Gli1ER/Td mice were subjected to flow cytometry analysis. n = 3 independent experiments. (C) CFU-F assay of digested meniscus cells. Td+colonies and Td- colonies were counted from 1 × 104 seeded cells. n = 3 independent experiments. (D) Representative bright-field images of the scratch-wound closure in Gli1+ or Gli1- meniscus cells at 0 and 48 hr. Scale bars, 200 µm. Solid lines indicate the remaining area not covered by meniscus cells. (E) The relative migration rate was measured by the percentage of scratched area being covered by migrated cells at 48 hr. n = 3 independent experiments. (F) Representative adipogenic (AD), osteogenic (OB), and chondrogenic (CH) differentiation images of Gli1+ and Gli1- cells. Cells were stained by Oil Red, Alizarin red, and Alcian blue, respectively. (G) qRT-PCR analysis of lineage markers in Gli1- or Gli1+meniscal cells after being cultured in adipogenic, osteogenic and chondrogenic differentiation media for 1, 2, and 3 weeks, respectively. n = 4 independent experiments. Statistical analysis was performed using unpaired two-tailed t-test. Data presented as mean ± s.e.m. *p<0.05, **p<0.01, ***p<0.001.Figure 2—source data 1.Raw data for Figure 2B,C,E,G.