Crebl2, Stat5b, and Klf15—were up-regulated,
whereas transcriptional regulators of chondro-genic and osteogenic lineages—including Sox9 and
-11, Runx1 and -2, Fhl2, and Pitx1—were down-regulated (fig. S14 and supplementary text S3). The
reporter for the ZFP423 transcription factor, which
drives commitment of mesenchymal progenitors
to the adipocyte lineage during embryogenesis
(9), was expressed by dermal cells juxtaposed to
regenerated hair follicles starting on day 21 after
wounding (figs. S17 and S18A). Then, the number
of ZFP423-positive dermal cells increased, before
diminishing by day 28, coincident with the increase
in mature adipocytes (figs. S17 and S18, B and C).
The temporal changes in ZFP423 expression
suggest that wounding activates this embryonic
pathway to facilitate adipocyte regeneration. Supporting this hypothesis, adult Zfp423 mutant
mice (12) failed to regenerate fat completely,
despite forming many new hair follicles after
wounding [n = 9; new adipocyte/follicle ratio,
0.07 ± 0.06, versus 29.6 ± 5.4 in control mice (n =
9)] (Fig. 3D). The critical role of ZFP423 for reprogramming myofibroblasts during wound healing
in the adult contrasts with its nonessential role for
adipocyte development in the embryo because
Zfp423 mutant mice possess skin adipocytes (fig.
S16), likely owing to compensation by redundant
pathways apparently available during development but not regeneration.
To determine the molecular regulation of re-
programming, we considered that bone morpho-
genetic protein (BMP) signaling induces adipogenic
commitment of cells in vitro (9, 13) and that ac-
tively growing hair follicles, which are critical
for myofibroblast-to-adipocyte reprogramming,
strongly express BMP2 and BMP4 (14). Our tran-
scriptomic data also show that endogenous BMP
ligands encoded by Bmp4 and Bmp7 are up-
regulated, whereas the soluble BMP antagonists
encoded by Bambi and Grem1 are down-regulated
in myofibroblasts by day 21 (fig. S14 and supple-
mentary text S4). We also noted marked up-
regulation of expression of pSMAD1/5/8—indicators
of active BMP signaling—in dermal cells next to
regenerated hair follicles at the time of ZFP423
activation (day 21) (fig. S19A).
To test whether BMP signaling modulates
adipocyte regeneration, we studied K14-Noggin
mice, which overexpress Noggin, a soluble BMP
antagonist, in the epithelial cells of the hair follicle. After wounding, these mice failed to regenerate
fat, despite forming normal-appearing hair follicles
[n = 10; new adipocyte/follicle ratio, 0.2 ± 0.1, versus
30.6 ± 6.3 in control mice (n = 10)] (Fig. 3E).
Similarly, treatment of mice during wound healing
with a small-molecule inhibitor of SMAD1, -5,
and -8 phosphorylation largely prevented new
adipocyte regeneration in hair-bearing wounds (n =
7; new adipocyte/follicle ratio, 0.58 ± 0.35) (Fig. 3G).
ZFP423 reporter activity was down-regulated in the
Zfp423-lacZ;K14-Noggin (fig. S20) and inhibitor-treated Zfp423-lacZ mice (fig. S21), indicating that
BMP was activating ZFP423 in myofibroblasts.
To specifically test whether BMP signaling in myofibroblasts is necessary for adipocyte regeneration,
750 17 FEBRUAR Y 2017 • VOL 355 ISSUE 6326
Fig. 3. Molecular profiling and functional studies of adipocyte regenera-
tion reveal that ZFP423 and BMP signaling are necessary for adipocyte
regeneration. (A) Principal component analysis of the myofibroblast transcriptome
reveals distinct changes across four postwounding time points. (B) Differentially
expressed genes (4120 total) from myofibroblasts at days 12 to 26 group into
five distinct clusters (table S5). (C) Differentially expressed genes in several
gene ontologies (GOs) undergo distinct temporal changes in myofibroblasts.
(D) Deletion of Zfp423, (E) overexpression of the soluble BMP antagonist Noggin
in K14-Noggin mice, (F) SMA-CreERT2–driven deletion of BMPR1A, and (G) treat-
ment with the BMP antagonist LDN-193189 (2 mg/kg) during wound healing all
resulted in near-complete loss of regenerated adipocytes in wounds, despite
normal regeneration of hair follicles. WT, wild type. Scale bars in (D) to (G), 100 mm.