Skin vasculature and hair follicle cross-talking associated with stem cell activation and tissue homeostasis
- Cornell University, United States
Figures
Figure 1 with 1 supplement
Transient horizontal plexus under hair germ (HPuHG) precedes hair follicle stem cell activation in hair cycle.
(A–E). CD31 images using widefield fluorescence microscopy, with optical Z-stacks and deconvolution from 70 µm thick skin sections, shown as maximal projection images. Yellow-dotted line indicates …
Figure 1—figure supplement 1
Imaging of thin and thick skin sections reveals remodeling of vasculature at different hair cycle stages.
(A–D) Wide field microscopy images of 10 µm skin sections from wild type C57/Bl6 mice at ages and hair cycle stages indicated, stained for endothelial marker CD31 and DAPI for nuclei. Ep, epidermis; …
Figure 2
Quantification of CD31+ skin vasculature organization during hair cycle.
(A) Quantification of total CD31+ area per skin field at different hair cycle stages (as shown in Figure 1) shows significant increase of vasculature at anagen. The total CD31+ area is defined by …
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Figure 2—source data 1
Spreadsheet of original quantification of CD31+ vasculature in total skin (for Figure 2A).
- https://doi.org/10.7554/eLife.45977.005
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Figure 2—source data 2
Spreadsheet of original quantification of CD31+ vasculature in HPuHG region (for Figure 2B).
- https://doi.org/10.7554/eLife.45977.006
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Figure 2—source data 3
Spreadsheet of original quantification of angles of CD31+ vasculature (for Figure 2C).
- https://doi.org/10.7554/eLife.45977.007
Figure 3
CD31+ vasculature undergoes proliferation at anagen and apoptosis at catagen.
(A–F) Skin sections from mice in Figure 1 at late catagen (PD19), telogen (PD20), early anagen (PD21), and anagen (PD28) stained for CD31 and Ki67 or activated Caspase 3, imaged using confocal …
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Figure 3—source data 1
Number of Ki67+ CD31+ double positive cells during hair cycle (for Figure 3F).
- https://doi.org/10.7554/eLife.45977.009
Figure 4 with 1 supplement
Endothelial-specific Alk1 knockout delays HFSC activation and hair cycle.
(A) Schematic of tamoxifen (TM) inductions using the endothelial-specific Cdh5-CreERT2, with hair cycle stages indicated at postnatal days. M, morphogenesis; C, catagen, T, telogen, A, anagen. (B) …
Figure 4—figure supplement 1
Phenotype of Alk1 endothelial knockout skin.
(A) Images of skin sections from Alk1 EndKO and CT mice induced with TM at PD21 show anagen induction by PD31 (see also Table 1). (B) Quantification of Pcadhigh cells of stage-matched Alk1 EndKO and …
Figure 5 with 1 supplement
Increased vasculature density in Alk1 endothelial knockout skin in horizontal plexus under the hair germ (HPuHG).
(A) Skin sections from Alk1flox/flox mice with Cdh5-CreERT2 (EndKO) or without (CT) were injected with tamoxifen (TM) at PD17 and sacrificed at different ages between ~PD 22-PD28, were stage matched …
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Figure 5—source data 1
Spreadsheet of original quantification of HPuHG CD31+ vasculature in Alk1 EndKO and CT (for Figure 5B).
- https://doi.org/10.7554/eLife.45977.015
Figure 5—figure supplement 1
Increase in CD31+ vasculature density in horizontal plexus under the hair germ (HPuHG) in Alk1 endothelial knockout.
(A) Example of Cdh5-CreERT2 x Alk1flox/flox mouse skin (Alk1 EndKO) with regions of interest analyzed in Figure 5. Blue line shows how the CD31+ vasculature in the HPuHG was delineated, dotted …
Figure 6 with 1 supplement
Increased BMP4 protein in the endothelial Alk1 KO skin underneath the hair germ.
(A) Representative images of skin stained with BMP4 antibodies show prominent expression in the HPuHG region in vessel-like structure. Note increased BMP4 in the HPuHG region in the Alk1 EndKO skin. …
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Figure 6—source data 1
Spreadsheet of original quantification of BMP4 in Alk1 EndKO skin (for Figures 6B, C and D).
- https://doi.org/10.7554/eLife.45977.018
Figure 6—figure supplement 1
BMP4 levels in Alk1 EndKO skin.
(A) Example of Cdh5-CreERT2 x Alk1flox/flox mouse skin (Alk1 EndKO) from mice injected with TM at PD17 and sacrificed at PD28 stage matched at Tel-Ana I, with regions of interest analyzed in Figure 6…
Figure 7
Varying level of the transcription factor Runx1 in skin epithelium results in rearrangement of CD31+ vasculature around the hair follicle.
(A) Schematics of Runx1 mutant inductions in genetic mouse models to create varying levels of Runx1 in the hair follicle (HF). Runx1 expression in HF is shown in red in wild type follicles (top) and …
Figure 8 with 1 supplement
Epithelial Runx1 knockout results in increased CD31+ vasculature and hence increased BMP4 concentration in the HPuHG region.
(A–B) Runx1flox/flox mice with Krt14-CreERT2 (EpiKO) or without (CT) were injected with tamoxifen (TM) at PD17, sacrificed at ~PD20. Skin sections were DAPI stained, inspected for hair morphology …
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Figure 8—source data 1
Spreadsheet of original quantification on Runx1 EpiKO skin (Figure 8C and D).
- https://doi.org/10.7554/eLife.45977.023
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Figure 8—source data 2
Spreadsheet of original quantification of BMP4 in Runx1 EpiKO skin (for Figure 8E and F).
- https://doi.org/10.7554/eLife.45977.024
Figure 8—figure supplement 1
Runx1 EpiKO skin shows increased vasculature in horizontal plexus under hair germ (HPuHG) at late catagen/telogen and normal vasculature at anagen.
(A) Examples of images from Krt14-CreERT2 x Runx1flox/flox mice (Runx1 EpiKO) and littermate control mice without Cre allele (CT) that were analyzed in Figure 7. Light blue line shows how the CD31+ …
Figure 9
Hypothetical model of HFSCs and vasculature cross-talking in hair cycle.
Left side: During late stages of HFSC quiescence (late catagen/telogen) remodeling of skin vasculature results in assembly of the horizontal plexus under hair germ HPuHG. This results in high …
Tables
Table 1
Cdh5CreERT2 x Alk1flox/flox KO (EndKO) mice and hair cycle
https://doi.org/10.7554/eLife.45977.012| Mouse ID | Genotype | Sex | Hair cycle stage |
|---|---|---|---|
| PD17-20 | |||
| F520.01 | CT | M | Tel |
| F520.04 | CT | F | Tel |
| F520.05 | CT | M | Tel |
| F520.06 | CT | F | Tel |
| PD17-22 | |||
| F167.02 | CT | M | Ana I |
| F64.02 | CT | F | Tel - AnaI |
| F64.01 | KO | F | Tel |
| PD17-25 | |||
| F81.06 | CT | F | Tel- Ana I |
| F146.05 | CT | M | Ana I - IIa |
| F167.05 | CT | M | Ana I - IIa |
| F433.03 | CT | F | Tel- Ana I |
| F433.04 | CT | F | Tel- Ana I |
| F81.07 | KO | F | Tel |
| F86.02 | KO | F | Ana I |
| F86.04 | KO | M | Tel |
| F99.01 | KO | M | Tel |
| F433.06 | KO | F | Tel |
| PD17-28 | |||
| E963.02 | CT | M | Ana IIIc |
| F80.04 | CT | F | Tel - Ana I |
| F90.01 | CT | M | Ana I - Ana IIa |
| F146.02 | CT | F | Ana IIa-c |
| F333.01 | CT | M | Ana I - IIa |
| F333.03 | CT | M | Tel - Ana I |
| F333.04 | CT | M | Tel - Ana I |
| F11.04 | KO | F | Tel - Ana IIa |
| F89.01 | KO | M | Ana I-IIa |
| F80.03 | KO | M | Tel - Ana I |
| F90.02 | KO | F | Tel - Ana I |
| F333.02 | KO | F | Tel - Ana I |
| PD17-31 | |||
| F3.01 | CT | M | Ana II - Ana III |
| F3.03 | CT | M | Ana II |
| F90.05 | CT | M | Ana II-III |
| F146.06 | CT | M | Ana III |
| E906.01 | KO | M | Tel-Ana I |
| F90.07 | KO | F | Tel-Ana I |
| PD17-34/35 | |||
| E928.03 | CT | F | Ana III |
| E928.05 | CT | F | Ana IV |
| E960.01 | KO | F | Ana I - Ana II |
| E928.01 | KO | F | Ana I-Ana II |
| PD21-26 | |||
| F218.01 | CT | M | Ana I - Ana III |
| F218.05 | KO | M | Ana III |
| PD21-31 | |||
| F218.02 | CT | F | Ana I - Ana II |
| F218.04 | KO | F | Tel - Ana I |
| PD23-26 | |||
| F273.02 | CT | F | Ana III |
| F273.01 | KO | M | Ana III |
Table 2
Epithelial-specific Runx1-target genes in hair follicle stem cells with known function in vasculature remodeling.
https://doi.org/10.7554/eLife.45977.019| Gene abbreviation | Full gene name | Known function | Fold change bu Runx1 TG/WT | Fold change in WT HG vs bu |
|---|---|---|---|---|
| Figf | c-fos induced growth factor | Agiogenesis, VEGF signaling | 2x | 16x |
| Col5a1 | Colagen five type a1 | Ehlers‐Danlos syndrome with lethal arterial events | −11x | −2.7x |
| Adamts1 | A disintegrin-like and metallopeptidase thrombospondin type 1 | Negative regulation of angiogenesis | 2x | 2x |
| Cxcl1 | chemokine (C-X-C motif) ligand1 | Inflammatory response | 2x | 3x |
| Cxcl10 | chemokine (C-X-C motif) ligand10 | Negative regulation of angiogenesis | 6x | 16x |
| Edn1 | endothelin 1 | Patterning of blood vessels | 2x | 2x |
| Cxcl12 | chemokine (C-X-C motif) ligand 12 | Patterning of blood vessels | 2x | 2x |
| Cx3cl1 | chemokine (C-X3-C motif) ligand1 | Angiogenesis | 2x | 4x |
| Cxcl9 | chemokine (C-X-C motif) ligand9 | Inflammatory response | 7x | 10x |
| Pla2g7 | phospholipase A2, group VII (platelet-activating factor acetylhydrolase, plasma) | Inflammatory response | 10x | 6x |
| Sema3a | semaphorin 3A | Blood vessel patterning | 9x | 3x |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (M. musculus) | Krt14-CreERT2 | PMID: 11034212 | RRID: MGI:2177426 | |
| Genetic reagent (M. musculus) | Cdh5-CreERT2 | PMID: 20445537 | RRID: MGI:3848982 | Dr. Anne Eichmann (Yale University) |
| Genetic reagent (M. musculus) | Alk1flox | PMID: 17911384 | RRID: MGI:4398901 | Dr. Anne Eichmann (Yale University) |
| Genetic reagent (M. musculus) | Runx1flox | PMID: 15784726 | RRID: MGI:3043614 | |
| Genetic reagent (M. musculus) | Rosa26-tdTomato | Jackson Laboratory | RRID: MGI:3809523 | |
| Genetic reagent (M. musculus) | C57BL/6 | Jackson Laboratory | RRID:MGI:3715241 | |
| Antibody | Anti-mouse-CD31 (Purified rat monoclonal) | BD Biosciences | BD Cat#: 550274, RRID: AB_393571 | 1:100 |
| Antibody | Anti-Ki67 (Rabbit polyclonal) | Abcam | Abcam Cat# ab15580, RRID:AB_443209 | 1:1000 |
| Antibody | Anti-Caspase-3 (Rabbit polyclonal) | R and D Systems | R and D Cat# AF835, RRID:AB_2243952 | 1:300 |
| Antibody | Anti-neurofilament-1 (Chicken polyclonal) | Millipore | Millipore Cat# AB5539, RRID:AB_11212161 | 1:1000 |
| Antibody | Anti-BMP4 (Rabbit polyclonal) | Abcam | Abcam Cat# ab39973, RRID:AB_2063523 | 1:1000 |
| Antibody | Anti-EphrinB2 (Rabbit monoclonal) | Abcam | Abcam Cat #: ab201512, RRID:AB_2810831 | 1:500 |
| Antibody | Anti-P-Cadherin (Rat monoclonal) | Thermo Fisher Scientific | Thermo Fisher Scientific Cat# 13-2000Z, RRID:AB_2533006 | 1:200 |
| Chemical compound, drug | Tamoxifen | Millipore | Millipore Cat#: T5648 | |
| Software, algorithm | ImageJ | ImageJ | RRID:SCR_003070 | |
| Software, algorithm | JMP Pro | SAS | RRID: SCR_014242 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.45977.026
