Lef1 expression in fibroblasts maintains developmental potential in adult skin to regenerate wounds
- School of Molecular Biosciences, Washington State University, United States
- Center for Reproductive Biology, Washington State University, United States
Figures
Figure 1 with 2 supplements
Fibroblasts in developing skin are distinct from regenerating, scarring, and homeostatic conditions.
(a–d) Immunostaining analysis and histological map of the dermis in developing Post-natal day 2 (P2) skin (a), homeostatic skin at P21 (b), skin undergoing regeneration P2 7 days post wound (7dwp) (c…
Figure 1—figure supplement 1
Classifying fibroblast clusters and conditions in development, regeneration, scarring, and homeostasis.
(a) Over-laying gene expression for cell markers on UMAP plots. (b) Dotplot of genes based on expression in cluster.
Figure 1—figure supplement 2
Batch analysis of biological replicates for developing, homeostatic, regenerating, and scarring conditions.
(a–d) Batch analysis of all conditions revealed minimal batch effect.
Figure 2 with 2 supplements
Papillary fibroblast lineage trajectory is defined by Lef1 in RNA Velocity analysis.
(a–c) Immunostaining development (P2) skin for Papillary (a) (Dpp4), Reticular/Hypodermal (b) (Dlk1/Pref1), and Hypodermal makers (c) (Ly6a). (d) Overlaying fibroblast lineage markers on UMAP …
Figure 2—figure supplement 1
Classifying fibroblast clusters in developing fibroblasts.
Over-laying gene expression for fibroblast lineage markers.
Figure 2—figure supplement 2
Clustering and Velocity analysis of regenerating, scarring, and developing fibroblasts.
(a) Comparative clustering analysis of regenerating and scarring fibroblasts projected as a UMAP. (b) Velocity analysis of regenerating and scarring fibroblasts. (c) Expression analysis of …
Figure 3
Dermal Lef1 is required to support hair follicle regeneration in neonatal wounds.
(a) Schematic representation of transgenic strategy to ablate Lef1 expression in dermal fibroblasts. Scale bars are 50 μm (b–e) Verifying dermal Lef1 ablation by immunostaining. White arrows …
Figure 4 with 1 supplement
Lef1 expression in the dermis enhances skin regeneration in large wounds.
(a) Schematic representation of transgenic strategy to ectopically express Lef1 in fibroblasts during development, homeostasis, and wound healing. (b–c) Immunostaining for Lef1 and GFP in P0 Lef1KI …
Figure 4—figure supplement 1
Twist-Lef1KI mice does not show overt phenotypic variations.
(a) Images of P4 litter from Twist-Lef1KI mice revealing viability of line. (b–c) P28 of wild type (b) and Twist-Lef1KI (c) mice. (d) The growth curve of male WT (Lef1KI) mice (24 male mice total) …
Figure 5 with 2 supplements
Lef1 expression in the dermis enhances skin regeneration in permissive macro environments.
(a) Schematic of the differential wound healing analysis from different time points of the hair follicle cycle in murine skin. (b–g,j) Immunostaining analysis of wounds 24dpw from P24 (b–d), P40 (d–e…
Figure 5—figure supplement 1
Analysis of the regenerative potential of Twist-Lef1KI wounds at P40.
(a) Schematic describing the wounding and tissue collection strategy. (b–j) Immunostaining analysis of wild type (Lef1KI) and Twist-Lef1KI mice wounded at P40 and collected at 14 dpw (b–d), 24 dpw (e…
Figure 5—figure supplement 2
Immunostaining analysis of GFP expression in scarring and regenerating wounds of Lef1KI and Twist-Lef1KI mice.
(a–b) Immunostain of histological sections of 8 mm wounds from P40 harvested at 24 dpw. Immunostains were for GFP and Lef1.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (M. musculus) | Dermo1-Cre or Twist2-Cre, C57Bl6/BalbC | Jackson Laboratory | RRID:MGI:3840442 | |
| Strain, strain background (M. musculus) | Lef1KI, C57Bl6/BalbC | Engelhardt lab - University of Iowa | Other | A gift from the Engelhardt lab |
| Strain, strain background (M. musculus) | Lef1KO, C57Bl6/BalbC | Xue lab - University of Iowa | RRID:MGI:5447957 | A gift from the Xue lab |
| Sequencing kit | Single Cell 3’ Library and Gel Bead Kit v2 | 10X Genomics | Cat#: PN-120267 | |
| Antibody | Anti-Integrin alpha 8 (Itga8) (goat polyclonal) | R and D Systems | Cat#: AF4076 | IF(1:200) |
| Antibody | Anti-Pref1/Dlk1 (rabbit monoclonal) | R and D Systems | Cat#: AF8277 | IF(1:200) |
| Antibody | Anti-Dpp4/CD26 (goat polyclonal) | R and D Systems | Cat#: AF954 | IF(1:200) |
| Antibody | Anti-Pdgfra (goat polyclonal) | R and D Systems | Cat#: AF1062 | IF(1:200) |
| Antibody | Anti-Lef1 (rabbit monoclonal) | Cell Signaling | Cat#: 2230S | IF(1:200) |
| Antibody | Anti-Alpha smooth muscle actin (Acta2) (rabbit polyclonal) | abcam | Cat#: ab5694 | IF(1:200) |
| Antibody | Anti GFP (chicken polyclonal) | abcam | Cat#: ab13970 | IF(1:200) |
| Antibody | Anti-Ly6a/Sca1 (rat monoclonal) | Biolegend | Cat#: 108108 | IF(1:200) |
| Antibody | Anti-CD49f/Integrin alpha 6 (Itga6) (rat monoclonal) | BD Biosciences | Cat#: 555735 | IF(1:200) |
| Antibody | Anti-goat Alexa Fluor 488 (Donkey polyclonal) | Invitrogen | Cat#: A-11055 | IF(1:500) |
| Antibody | Anti-goat Alexa Fluor 555 (Donkey polyclonal) | Invitrogen | Cat#: A-21432 | IF(1:500) |
| Antibody | Anti-goat Alexa fluor 647 (Donkey polyclonal) | Invitrogen | Cat#: A-21447 | IF(1:500) |
| Antibody | Anti-rabbit Alexa fluor Plus 555 (Donkey polyclonal) | Invitrogen | Cat#: A32794 | IF(1:500) |
| Antibody | Anti-rabbit Alexa fluor 647 (Donkey polyclonal) | Invitrogen | Cat#: A-31573 | IF(1:500) |
| Antibody | Anti-chicken Alexa fluor 488 (Goat polyclonal) | Invitrogen | Cat#: A-11039 | IF(1:500) |
| Peptide, recombinant protein | Dispase | Corning | Cat#: 3254235 | |
| Peptide, recombinant protein | Collagenase Type I | ThermoFisher | Cat#: 17100017 | |
| Software, algorithm | Cellranger v3.0.2 | 10X Genomics | RRID:SCR_017344 | |
| Software, algorithm | scanpy | scanpy | RRID:SCR_018139 | |
| Software, algorithm | scvelo | scVelo | RRID:SCR_018168 |
Additional files
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Supplementary file 1
Markers used to identify cell populations in scRNA-seq analysis.
Genes utilized to identify clusters in scRNA analysis.
- https://cdn.elifesciences.org/articles/60066/elife-60066-supp1-v1.xlsx
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Supplementary file 2
Velocity genes for all developing fibroblast populations.
The genes driving Velocity Trajectory in developing fibroblasts for all clusters.
- https://cdn.elifesciences.org/articles/60066/elife-60066-supp2-v1.xlsx
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Supplementary file 3
Inhibitory genes during hair follicle cycling.
List of previously identified genes hypothesized to be inhibitory during hair follicle cycle (Wang et al., 2017). The list also includes the relative expression values of microarray analysis (Lin et al., 2009).
- https://cdn.elifesciences.org/articles/60066/elife-60066-supp3-v1.xlsx
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Supplementary file 4
Differential expression of all regenerating fibroblasts compared to scarring fibroblasts.
This is a list of genes upregulated in regenerating or scarring fibroblasts when compared to each other in our SCANPY analysis.
- https://cdn.elifesciences.org/articles/60066/elife-60066-supp4-v1.csv
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Supplementary file 5
Panther pathway analysis of velocity genes from developing fibroblasts.
List of pathways generated from velocity genes of developing fibroblasts.
- https://cdn.elifesciences.org/articles/60066/elife-60066-supp5-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/60066/elife-60066-transrepform-v1.docx
