Skin vasculature and hair follicle cross-talking associated with stem cell activation and tissue homeostasis

9 figures, 3 tables and 1 additional file

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 …

https://doi.org/10.7554/eLife.45977.002
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; …

https://doi.org/10.7554/eLife.45977.003
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 …

https://doi.org/10.7554/eLife.45977.004
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
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
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
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 …

https://doi.org/10.7554/eLife.45977.008
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) …

https://doi.org/10.7554/eLife.45977.010
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 …

https://doi.org/10.7554/eLife.45977.011
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 …

https://doi.org/10.7554/eLife.45977.013
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 …

https://doi.org/10.7554/eLife.45977.014
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. …

https://doi.org/10.7554/eLife.45977.016
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

https://doi.org/10.7554/eLife.45977.017
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 …

https://doi.org/10.7554/eLife.45977.020
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 …

https://doi.org/10.7554/eLife.45977.021
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
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+ …

https://doi.org/10.7554/eLife.45977.022
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 …

https://doi.org/10.7554/eLife.45977.025

Tables

Table 1
Cdh5CreERT2 x Alk1flox/flox KO (EndKO) mice and hair cycle
https://doi.org/10.7554/eLife.45977.012
Mouse IDGenotypeSexHair cycle stage
PD17-20
 F520.01CTMTel
 F520.04CTFTel
 F520.05CTMTel
 F520.06CTFTel
PD17-22
 F167.02CTMAna I
 F64.02CTFTel - AnaI
 F64.01KOFTel
PD17-25
 F81.06CTFTel- Ana I
 F146.05CTMAna I - IIa
 F167.05CTMAna I - IIa
 F433.03CTFTel- Ana I
 F433.04CTFTel- Ana I
 F81.07KOFTel
 F86.02KOFAna I
 F86.04KOMTel
 F99.01KOMTel
 F433.06KOFTel
PD17-28
 E963.02CTMAna IIIc
 F80.04CTFTel - Ana I
 F90.01CTMAna I - Ana IIa
 F146.02CTFAna IIa-c
 F333.01CTMAna I - IIa
 F333.03CTMTel - Ana I
 F333.04CTMTel - Ana I
 F11.04KOFTel - Ana IIa
 F89.01KOMAna I-IIa
 F80.03KOMTel - Ana I
 F90.02KOFTel - Ana I
 F333.02KOFTel - Ana I
PD17-31
 F3.01CTMAna II - Ana III
 F3.03CTMAna II
 F90.05CTMAna II-III
 F146.06CTMAna III
 E906.01KOMTel-Ana I
 F90.07KOFTel-Ana I
PD17-34/35
 E928.03CTFAna III
 E928.05CTFAna IV
 E960.01KOFAna I - Ana II
 E928.01KOFAna I-Ana II
PD21-26
 F218.01CTMAna I - Ana III
 F218.05KOMAna III
PD21-31
 F218.02CTFAna I - Ana II
 F218.04KOFTel - Ana I
PD23-26
 F273.02CTFAna III
 F273.01KOMAna 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 abbreviationFull gene nameKnown functionFold change bu Runx1
TG/WT
Fold change
in WT HG vs
bu
Figfc-fos induced growth factorAgiogenesis, VEGF signaling2x16x
Col5a1Colagen five type a1Ehlers‐Danlos syndrome with lethal arterial events−11x−2.7x
Adamts1A disintegrin-like and metallopeptidase thrombospondin type 1Negative regulation of angiogenesis2x2x
Cxcl1chemokine (C-X-C motif) ligand1Inflammatory response2x3x
Cxcl10chemokine (C-X-C motif) ligand10Negative regulation of angiogenesis6x16x
Edn1endothelin 1Patterning of blood vessels2x2x
Cxcl12chemokine (C-X-C motif) ligand 12Patterning of blood vessels2x2x
Cx3cl1chemokine (C-X3-C motif) ligand1Angiogenesis2x4x
Cxcl9chemokine (C-X-C motif) ligand9Inflammatory response7x10x
Pla2g7phospholipase A2, group VII (platelet-activating factor acetylhydrolase, plasma)Inflammatory response10x6x
Sema3asemaphorin 3ABlood vessel patterning9x3x
Key resources table
Reagent type
(species) or resource
DesignationSource
or reference
IdentifiersAdditional
information
Genetic reagent (M. musculus)Krt14-CreERT2PMID: 11034212RRID: MGI:2177426
Genetic reagent (M. musculus)Cdh5-CreERT2PMID: 20445537RRID: MGI:3848982Dr. Anne Eichmann (Yale University)
Genetic reagent (M. musculus)Alk1floxPMID: 17911384RRID: MGI:4398901Dr. Anne Eichmann (Yale University)
Genetic reagent (M. musculus)Runx1floxPMID:
15784726
RRID: MGI:3043614
Genetic reagent (M. musculus)Rosa26-tdTomatoJackson LaboratoryRRID: MGI:3809523
Genetic reagent
(M. musculus)
C57BL/6Jackson LaboratoryRRID:MGI:3715241
AntibodyAnti-mouse-CD31 (Purified rat monoclonal)BD BiosciencesBD Cat#: 550274,
RRID: AB_393571
1:100
AntibodyAnti-Ki67 (Rabbit polyclonal)AbcamAbcam Cat# ab15580, RRID:AB_4432091:1000
AntibodyAnti-Caspase-3 (Rabbit polyclonal)R and D SystemsR and D Cat# AF835, RRID:AB_22439521:300
AntibodyAnti-neurofilament-1 (Chicken polyclonal)MilliporeMillipore Cat# AB5539, RRID:AB_112121611:1000
AntibodyAnti-BMP4 (Rabbit polyclonal)AbcamAbcam Cat# ab39973, RRID:AB_20635231:1000
AntibodyAnti-EphrinB2 (Rabbit monoclonal)AbcamAbcam Cat #: ab201512,
RRID:AB_2810831
1:500
AntibodyAnti-P-Cadherin
(Rat monoclonal)
Thermo Fisher ScientificThermo Fisher Scientific
Cat# 13-2000Z, RRID:AB_2533006
1:200
Chemical compound, drugTamoxifenMilliporeMillipore
Cat#: T5648
Software, algorithmImageJImageJRRID:SCR_003070
Software, algorithmJMP ProSASRRID: SCR_014242

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