Journal: Cell reports
Article Title: Dynamic interplay between IL-1 and WNT pathways in regulating dermal adipocyte lineage cells during skin development and wound regeneration
doi: 10.1016/j.celrep.2023.112647
Figure Lengend Snippet: (A) scRNA-seq analysis of cells isolated from neonatal skin. Pdgfra + dFBs were reclustered into 12 clusters. (B) Neonatal dFBs were grouped into three cell states along differentiation trajectories. (C) Violin plots of indicated genes. (D–G) Neonatal mouse skin sections were immunostained with antibodies against various dFB markers as indicated, and the nuclei were counterstained with DAPI in either blue or white. Scale bars, 50 μm. Abbreviations: p.c., panniculus carnosus muscle layer; p.f., perifollicular; DP, dermal papilla; HF, hair follicle; HB, hair bulb. Brackets show specific anatomical skin layers as indicated. (H) Quantified result of (G) showing the fluorescence intensity (arbitrary unit, A.U.) of PLIN1 (red) and DLK1 (green) across skin section from top to bottom. (I–L) Perifollicular dFBs (CD24 hi LY6A − ), pAds (DPP4 − LY6A + ), and HI-APs (DPP4 + LY6A + ) were sorted and then subjected to qRT-PCR analysis (n = 3/group) of indicated genes (I) or in vitro adipocyte differentiation for adipocytes (stained with oil red O [ORO]) (J). Quantified ORO+ area for each group is shown in (K) (n = 5/group). Sorted cells were also subjected to osteocyte differentiation (stained with alizarin red [AR]; see ), and quantified AR+ area is shown in (L) (n = 5/group). Scale bar, 2 mm. All error bars indicate mean ± SEM; **p < 0.01, ***p < 0.01, ****p < 0.0001. (M) RNA velocity analysis overlaid on tSNE projection of developing dFBs. (N) A model for the proposed spatial location of various dFB subpopulations in the developing skin. Abbreviations: PAP, papillary dFBs; RET, reticular dFBs; RET-AP, reticular adipocyte progenitors; pAd, preadipocytes; Ad, adipocytes; HI-pAd, hypodermal interstitial pAd; HI-AP, hypodermal interstitial AP.
Article Snippet: Cells were then blocked with anti-mouse CD16/32 (eBioscience, 14016185), followed by staining with an antibody cocktail for fibroblasts containing PECy7-CD45 (BioLegend, 147704), PerCP-Cy5.5-CD31 (BioLegend, 102522), FITC-CD26(Biolegend, 137806), AF700-CD24(Biolegend, 101836), PE-THY1 (BioLegend, 105308), APC-PDGFRa (eBioscience, 17140181) and BV605-LY6A (BioLegend, 108133), or an antibody cocktail for immune cells containing FITC-Ly6G (eBioscience, 11593182), PE-F4/80 (eBioscience, 12480182), APC-CD11C (BioLegend, 117310), AF700-MHCII (eBioscience, 56532182), PerCP-Cy5.5-Ly6C (BioLegend, 128012), PECy7-CD11B (Biolegend, 101216), APC-Cy7-CD3 (BioLegend, 100222).
Techniques: Isolation, Fluorescence, Quantitative RT-PCR, In Vitro, Staining
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![(A) scRNA-seq analysis of cells isolated from neonatal skin. <t>Pdgfra</t> + dFBs were reclustered into 12 clusters. (B) Neonatal dFBs were grouped into three cell states along differentiation trajectories. (C) Violin plots of indicated genes. (D–G) Neonatal mouse skin sections were immunostained with antibodies against various dFB markers as indicated, and the nuclei were counterstained with DAPI in either blue or white. Scale bars, 50 μm. Abbreviations: p.c., panniculus carnosus muscle layer; p.f., perifollicular; DP, dermal papilla; HF, hair follicle; HB, hair bulb. Brackets show specific anatomical skin layers as indicated. (H) Quantified result of (G) showing the fluorescence intensity (arbitrary unit, A.U.) of PLIN1 (red) and DLK1 (green) across skin section from top to bottom. (I–L) Perifollicular dFBs (CD24 hi LY6A − ), pAds (DPP4 − LY6A + ), and HI-APs (DPP4 + LY6A + ) were sorted and then subjected to qRT-PCR analysis (n = 3/group) of indicated genes (I) or in vitro adipocyte differentiation for adipocytes (stained with oil red O [ORO]) (J). Quantified ORO+ area for each group is shown in (K) (n = 5/group). Sorted cells were also subjected to osteocyte differentiation (stained with alizarin red [AR]; see ), and quantified AR+ area is shown in (L) (n = 5/group). Scale bar, 2 mm. All error bars indicate mean ± SEM; **p < 0.01, ***p < 0.01, ****p < 0.0001. (M) RNA velocity analysis overlaid on tSNE projection of developing dFBs. (N) A model for the proposed spatial location of various dFB subpopulations in the developing skin. Abbreviations: PAP, papillary dFBs; RET, reticular dFBs; RET-AP, reticular adipocyte progenitors; pAd, preadipocytes; Ad, adipocytes; HI-pAd, hypodermal interstitial pAd; HI-AP, hypodermal interstitial AP.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5379/pmc10765379/pmc10765379__nihms-1953118-f0002.jpg)
![(A–G) Adipoq -C re ERT2; Gsk3 flox/flox mice were administered TAM from w.d. 1 to 6, and w.d. 7 tissues were collected for analysis. (A) Staining of GSK3 (red), PLIN (blue), and DAPI (white), and (B) quantified GSK3 intensity in PLIN1 + cells (n = 4/group). (C) Staining of active β-catenin (red) and ATGL (green) and (D) quantified active β-catenin intensity in ATGL + area (n = 4/group). Scale bars, 100 μm. (E–G) FACS plots and quantified bar graphs (n = 3/group) showing the percentage of ACTA2 + LY6A − myofibroblasts or LY6A + DPP4 − preadipocytes in PDGFRA + dFBs (n = 3/group). (H) FABP4 (red), COLIV (blue), and DAPI (white) staining in healthy control (HC), wound (day 7), and keloid skin sections. Scale bar, 400 μm. (I) Active β-catenin (red), COLIV (blue), and ACTA2 (green) staining in keloid skin sections. Scale bar, 2 mm. (J) Bar graphs showing relative mRNA expression (based on RNA-seq FPKM [fragments per kilobase per million mapped fragments] values) of listed genes in non-lesional and wound tissues from HC or keloid (K) individuals (n = 4–5/group). All error bars indicate mean ± SEM; *p < 0.05, **p < 0.01.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5379/pmc10765379/pmc10765379__nihms-1953118-f0008.jpg)
