Alex B Wang et al
The EMBO Journal
Gata6 promotes hair progenitor cell renewal
Expanded View Figures
A
M: morphogenesis C: catagen T: telogen A: anagen
M
C
T
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Hoechst K14 Gata6
A
WT +TM 2d
T-A transition
Hai Germ Differentiation
Early A
Bulge Self-renewal
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Gata6: fl/wt wt/wt
fl/fl
250 bp fl
159 bp wt
Figure EV1. Gata6 is up-regulated in differentiating hair germ cells.
A Experimental scheme for doxycycline chased K5tTA × pTRE-H2B-GFP mice used to obtain divided and un-divided cell populations during the differentiating phase—
telogen (T)–anagen (A) transition—and self-renewing phase of HFSCs during early anagen, as previously described in Zhang et al (2009). Bulge cells were sorted as
divided or un-divided based on H2B-GFP levels and high CD34 and a6-integrin expression; hair germ cells at telogen–anagen transition were the divided cells with
low CD34 levels and high expression of a6-integrin.
B qRT–PCR confirmation of Gata6 expression in differentiating (hair germ, divided), self-renewing (bulge, divided), or non-dividing bulge stem cells (average SD,
n = 3).
C Immunofluorescence image of the skin shows epidermis and infundibulum stained with Hoechst for DNA (blue), basal layer/ORS marker K14 (green), and Gata6 (red).
Scale bar: 30 lm.
D Gel image of DNA genotyped for floxed Gata6 allele. Shown are genotypes from three mice: heterozygote (Gata6fl/wt), homozygous WT (Gata6wt/wt), and homozygous
floxed (Gata6fl/fl).
ª 2016 The Authors
The EMBO Journal
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The EMBO Journal
A 2d TM
Gata6 promotes hair progenitor cell renewal
10d TM
2d TM
4d TM
10d TM
B 2d TM
4d TM
10d TM
4d TM
10d TM
anagen WT
telogen iKO
telogen WT
4d TM
Alex B Wang et al
anagen iKO
2d TM
iKO anagen 10d TM
D 140
# Matrix cells
WT anagen 10d TM
Hoechst CD34
C
120
100
80
60
40
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0
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iKO
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*
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Days post TM (anagen)
Figure EV2. Gata6 is necessary for anagen initiation and maintenance.
A, B Low magnification images of skin with hematoxylin staining, for telogen-induced (A) and anagen-induced (B) mice. Scale bars: 30 lm.
C
Immunofluorescence images of hair follicles 10 days post-anagen induction stained with bulge marker CD34. Scale bars: 30 lm.
D
Quantification of the number of cells in anagen matrix hair follicles post-Gata6 iKO induction (average SD, n = 20 follicles from three mice each). Unpaired
t-test *P-values: 1 day = 0.86, 2 days = 7 × 10 5, 4 days = 4 × 10 10.
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ª 2016 The Authors
Alex B Wang et al
iKO 2d TM
B WT 2d TM
iKO 2d TM
D
Hoechst Gata6 AE15
Hoechst K14 K6
Hoechst Gata6 Gata3
C WT 2d TM
%BrdU+ cells in HS/IRS
iKO 2d TM
Hoechst Gata6 AE13
A WT 2d TM
E
The EMBO Journal
Gata6 promotes hair progenitor cell renewal
60
50
WT
WT 2d TM
iKO 2d TM
iKO
40
30
20
10
0
BrdU 12h
BrdU 2d
Figure EV3. Gata6 is not necessary for terminal differentiation of the hair follicle.
A–D Immunofluorescence staining of skin sections 2 days post-tamoxifen induction for terminally differentiated lineages reveal labeling in the hair bulb, as expected.
Specific stains are indicted in corresponding colors. Scale bars: 30 lm.
E
Quantification of images like those shown in Fig 3 shows percentage of BrdU+ cells located in the differentiated IRS and hair shaft lineages after the initial 12-h
pulse labeling and after the 2 days BrdU chase (average SD, n = 3, P-value = 0.7 for 12 h WT versus iKO and 0.4 for 2 days WT versus iKO).
ª 2016 The Authors
The EMBO Journal
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The EMBO Journal
Gata6 promotes hair progenitor cell renewal
Hoechst Caspase3 γH2A.X
Alex B Wang et al
Figure EV4. Gata6 loss does not cause DNA
damage or apoptosis in the inter-follicular
epidermis or bulge.
Skin sections immunostained for caspase-3 and
cH2A.X show no detectable signal in the interfollicular epidermis or bulge HFSCs. Scale bars: 30 lm.
WT 2d TM
IFE
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lum
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ª 2016 The Authors
Alex B Wang et al
The EMBO Journal
Gata6 promotes hair progenitor cell renewal
200
A
WT
iKO
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140
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Fold change qRT-PCR
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Exo1
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WT pGata6
WT pEdaradd
iKO pGata6
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pMock
pGata6 pEdaradd
Gata6
Edaradd
mock
pGata6
pEdaradd
iKO
WT
B
pGata6 pEdaradd pMock
Figure EV5. Overexpression of Edaradd rescues Gata6 iKO keratinocytes.
A qRT–PCR analysis of Gata6 and Edaradd in Gata6 WT and iKO stably transfected with pMock, pGata6, or pEdaradd (average SD, n = 4).
B Phase contrast images of keratinocytes from cells stably transfected with empty plasmid, pGata6, or pEdaradd. Scale bars: 20 lm.
C qRT–PCR of selected genes found to be overlapping between Gata6 iKO and Eda transgenic analyzed in Gata6 WT and iKO keratinocytes stably expressing pMock,
pGata6, or pEdaradd (average SD, n = 4). Unpaired t-test *P-values: 8 × 10 4 (iKO pGata6 versus pMock), 0.03 (iKO pEdaradd versus pMock). Mann–Whitney U-test
P-values: 0.03 (iKO pGata6 versus pMock), 0.03 (iKO pEdaradd versus pMock).
ª 2016 The Authors
The EMBO Journal
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