Science - 06.12.2019

overexpression also did not affect levels of
CCBE1, a protein required for VEGFC-mediated
lymphangiogenesis (fig. S10F).
Because the hair cycle was delayed upon
sustainingAngptl7, we wondered whether
ANGPTL7 dynamics might be important for
other regenerative responses. We focused on
hair plucking (waxing), as it mechanically per-
turbs the SC niche, generating a wound-like
response ( 3 , 34 ). Plucking not only activated
hair regeneration, as expected, it also restricted
lymphatic drainage anddisrupted lymphatic–
SC associations (fig. S11, A and B). More-
over, whenAngptl7was sustained, wound-
induced hair regeneration was abrogated;

conversely, after skin injury in a normal set-

ting,Angptl7gene expression plummeted (fig.

S11, C and D).

Turning to the flip side of the secretome

switch, we engineered doxycycline-inducible

versions ofAngptl4.Wheninducedintelogen,

ANGPTL4 precociously disrupted lymphatic

capillaries surrounding the bulge, reducing

lymphatic drainage and activating HFSCs (Fig.

5, C and D, and fig. S12). Despite a purported

role for ANGPTL4 in blood vasculature ( 35 ),

endothelial vessel density was largely un-

changed, both in AnaII–III of the normal hair

cycle and in telogen of theAngptl4-induced

hair cycle. Similar results were obtained with

NTN4, previously implicated in endothelial

cell biology ( 36 – 38 ) (fig. S12).

Our data suggested that the HFSC-derived

secretome can influence lymphatic dynamics

directly, which we tested by evaluating lymphat-

ic tube formation in a 3D-Matrigel system.

Formation of tubelike structures was enhanced

with ANGPTL7 and impaired with ANGPTL4,

supporting this hypothesis. Consistent with a

nonautonomous role, SC-driven factors in vitro

neither affected HFSC growth nor stimulated

immune cell migration (fig. S13).

Although ANGPTL and NTN receptors are

poorly studied, transcriptional landscaping

of freshly isolated endothelial cells from skin

Gur-Cohenet al.,Science 366 , 1218–1225 (2019) 6 December 2019 5of8

t-SNE1

t-SNE2

Angptl7

Telogen

Bulge Scs

Telogen HG

Anagen HG

High

Low

Relative expression

TelogenAna II-IIIAna IV

0

10

20

30

eGFP

+cells /H2BRFP

+ bulge

(Peak B)

p < 0.0001p < 0.0001

20 μm 20 μm 20 μm

Tel-Ana I Ana II-III Ana IV

Rep-eGFP KRT24 H2BRFP DAPI

VegfbVegfaVegfcAngpt4Angpt2Angpt1Angptl7Angptl6Angptl4Angptl3Angptl2Angptl1

Vash1Flt4Epha2Efnb2Acvr2bAcvrl1EngWnt7bWnt7aAdmNtn1Ntn4Ntn5Edn2Edn3Edn1

Sema3f

Telogen

Ana II

Ana III

0.25

0.5

0.00

-0.5

-0.25

Relative expression

Peak A Peak B

Angptl7

Temporal analysis of Angptl7 reporter expression (Peak B)

ATAC-seq

A SC transcriptome reveals a lymphangiogenic switch

at the onset of SC activation

B

Temporal hierarchical clustering of bulge SCs

Angptl7

t-SNE1

t-SNE2

High

Low

Relative expression

CD E

F

t-SNE1

t-SNE2

Telogen

Bulge SCs

Ana II

Bulge SCs

Ana I

Bulge

SCs

Angptl7

Krt24Igfbp4Ly6AMsx2Krt83Ntn4

Angptl4

200

250

150

50

100

0

TPM

Telo HG

Telo Bu-SC

Ana TAC

EpiSC

Angptl7; Bulge SCs vs HG

ATAC seq of telogen Angptl7

LTR Pgk H2BRFPPeakBSV40eGFP LT R

Telogen

Ana IAna IIIAna V

0.0

0.5

1.0

1.5

Angptl7

; qPCR relative expression

p = 0.0104

p < 0.0001

p = 0.0004

p = 0.0013

Fig. 4. HFSCs promote a lymphovascular switch at hair regeneration onset.
(A) Heatmaps of FACS-purified bulge SCs from telogen, AnaII, and AnaIII HFs.
Boxed transcripts are markedly down (blue) or up (red) upon HFSC activation.
(B) Transcripts per million (TPM) heatmap showing paucity ofAngptl7,Angptl4,
andNtn4in SCA1+epidermal SCs (EpiSCs) or HFSC progeny (MSK2+Krt83+TACs;
IGBP4+KRTnegHGs). (CandD) Single-cell transcriptome clustering of (C) telogen
bulge SCs, telogen HG cells, and AnaI HG cells ( 28 ); and (D) telogen, AnaI, and AnaII
bulge SCs. Each cell is represented as a dot. (E) Quantitative polymerase chain
reaction (qPCR) validation of temporalAngptl7expression in bulge SCs (n=3,

one-way ANOVA; Tukey’s multiple comparisons test). (F) AccessibleAngptl7locus

telogen bulge SC chromatin peaks (left) were used to driveeGFPin mice.

Peak BeGFPdynamics (right) mirror temporalAngptl7transcriptome changes

(n= 5, 5, and 3 mice analyzed for Telogen, AnaII–III, and AnaIV, respectively;

one-way ANOVA; Tukey’s multiple comparisons test). ATAC seq, assay for

transposase-accessible chromatin using sequencing; LTR, long terminal repeats;

Pgk, phosphoglycerate kinase promoter; H2BRFP, histone H2B red fluorescent

protein; SV40, minimal simian virus 40 promoter; Rep-eGFP, reporter-enhanced

green fluorescent protein.

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