Science - 06.12.2019

Focusing first onAngptl7,wefindthatprior
RNA sequencing (RNA-seq) data ( 28 )showed
thatAngptl7was not even expressed in the
primed HG cells of telogen or early anagen
HFs (Fig. 4C and fig. S8D). To delineate the
detailed temporal changes ofAngptl7within
bulgeSCs,weperformedstage-specific single-
cell RNA-seq. Three distinct temporal patterns
emerged from t-distributed stochastic neigh-
bor embedding (t-SNE) and unsupervised hier-
archical clustering (Fig. 4D and fig. S8E). Using
machine learning–based cell-cycle allocation
analysis ( 29 ), it was concluded that prolifer-
ative cells resided in AnaII SCs, in agreement

with our 5-ethynyl-2′-deoxyuridine (EdU) label-

ing studies. Additionally, in contrast to SC

markerSox9, which was maintained in bulge

SCs at all stages,Angptl7transcription in bulge

SCs diminished shortly after anagen onset,

even sooner than that of established quiescence

regulatorNfatc1( 28 ). Moreover, theAngptl7

expression dip was transient, occurring only

between AnaII and AnaIII (Fig. 4E).

Angptl7dynamics were recapitulated at the

chromatin level. As judged by chromatin immu-

noprecipitation sequencing (ChIP-seq) analyses

of fluorescence-activated cell sorting (FACS)–

purified HF cells ( 30 ), theAngptl7chroma-

tin state was active in telogen (H3K27ac+),

poised in AnaIII (H3K27acnegH3K4me1+),

and silent in AnaVI differentiated cells

(H3K27acnegH3K4me1neg) (fig. S9A). More-

over, when either of the twoAngptl7locus–

accessible chromatin elements ( 30 )wereused

as enhancers to drive enhanced green fluores-

cent protein (eGFP) expression in vivo, report-

ers faithfully recapitulatedAngptl7’s temporal

dynamics in bulge expression during the hair

cycle (Fig. 4F and fig. S9B). These results

intimated that changes in SC-derivedAngptl7

transcription are involved in reshaping the

regenerative microenvironment of the niche.

Gur-Cohenet al.,Science 366 , 1218–1225 (2019) 6 December 2019 3of8

Prox1-CreER+ ; iDTRfl

TAM

TAMTAMTAMDT Analysis

2 nd

Telogen

P60 (Day 0)

Full Ana

P15

Day 1-10

x1x1 Crx1 Cr-Cr

AM

CrreEERRR++;iiDTRRRfl

TTTA

TTTA

rreErER;iiDTR

A

TTTA

reEERR; iDTRi

AMAM

AMDTT Analysis

LYVE1 pattern

Prox1-CreER+;iDTRfl

Ctrl depleted (D10)

Ctrl

Targeted lymphatic vessel depletion

LYVE1 LYVE1

Prox1-CreER+;iDTR-depleted (D10)

HFs precociously enter anagen upon lymphatic vessel depletion

50 μm

DAPI P-CAD SOX9 LYVE1

30 μm

SCs exit quiescence upon depletion

Ctrl

Prox1-

depleted

0

20

40

60

80

100

120

%

Hair follicles

Tel-Ana I

Ana II-III

Ana IV-V

P < 0.0001

P = 0.0347

P = 0.0013

DyeCycle Violet

Day 1 post depletion

S/M/G2

G0/G1

A

0.0

0.5

1.0

1.5

2.0

2.5

Lymphatic capillar

ies volume

(per 1

μm

3 area)

p = 0.0183

Ctrl

Prox1-

depleted

0

500

1000

1500

2000

LYVE1 intensity(per μm

3 ; a.u.)

p = 0.0232

Ctrl

Prox1-

depleted

IgG Ctrl

VEGFR3-Fc

0

10

20

30

40

50

EdU

+ cell

s per folli

cle

IgG Ctrl p < 0.0001

VEGFR3-Fc

KRT24 P-CAD EdU DAPI30 μm 30 μm 30 μm

IgG/VR3-Fc Analysis

2 nd

Telogen

(P60)

IgG/VR3-FcIgG/VR3-FcIgG/VR3-Fc

P65

IgG/VR3-Fc

Lymphatic regression upon disruption of VEGFR3 signaling induces anagen entry

IgG Ctrl

VEGFR3-Fc

0

20

40

60

80

100

120

% Hair follicles

Telogen-Ana I

P < 0.0001

P < 0.0001

P = 0.9036

Ana II-III

A Ana IV-V

-Fc

IgIgG/VRIg Ig IgG/VR3-FIgGG/VR/

3-F

c

IgG

/VR3-F

c

IgG

/VR3-Fc

IgG

/VR3-

IgG/

IgG/VEGFR3-Fc

ID injection

B C

D

Ctrl

Depleted D1

0

20

40

60

80

100

120

Cell cycle profile in HFSCs

G0/G1

S/M/G2

p = 0.0045

p = 0.0051

+DT

ID injection

Ctrl

Depleted

0

20

40

60

80

100

-10^30103104105

Fig. 2. Disrupting lymphatic capillaries triggers hair regeneration.
(A) Timeline of experimental model and diphtheria toxin (DT)–induced
depletion of lymphatic vessels during the long quiescence of second telogen.
Quantifications: lymphatic intensity (n= 3); volume (n=5and4forCtrl
andProx1-depleted, respectively; two-tailed unpairedttest). ID, intradermal;
TAM, tamoxifen; P, postnatal day; Ctrl, control; D, day; a.u., arbitrary units.
(B) Precocious anagen induction after lymphatic depletion, as demonstrated

by HF growth and association with LYVE1+capillaries (n= 5, two-way ANOVA

with Sidak’s multiple comparisons test). P-CAD, P-cadherin. (C) Flow cytometry

of HFSC cell cycle after lymphatic depletion (n= 3, two-way ANOVA with Sidak’s

multiple comparisons test). (D) Experimental strategy using decoy VEGF3-Fc to

interrupt VEGFR3 signaling in dermal lymphatic vessels. EdU incorporation reveals

anagen entry after treatment (n= 4, multiple EdU measurements, two-tailed

unpairedttest). IgG, immunoglobulin G.

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