© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 3752-3760 doi:10.1242/dev.109231
RESEARCH ARTICLE
Involvement of Wnt, Eda and Shh at defined stages of sweat gland
development
Chang-Yi Cui*, Mingzhu Yin, Jian Sima, Victoria Childress, Marc Michel, Yulan Piao and David Schlessinger
To maintain body temperature, sweat glands develop from embryonic
ectoderm by a poorly defined mechanism. We demonstrate a temporal
cascade of regulation during mouse sweat gland formation. Sweat
gland induction failed completely when canonical Wnt signaling
was blocked in skin epithelium, and was accompanied by sharp
downregulation of downstream Wnt, Eda and Shh pathway genes. The
Wnt antagonist Dkk4 appeared to inhibit this induction: Dkk4 was
sharply downregulated in β-catenin-ablated mice, indicating that it is
induced by Wnt/β-catenin; however, its overexpression repressed Wnt
target genes and significantly reduced gland numbers. Eda signaling
succeeded Wnt. Wnt signaling was still active and nascent sweat gland
pre-germs were still seen in Eda-null mice, but the pre-germs failed to
develop further and the downstream Shh pathway was not activated.
When Wnt and Eda were intact but Shh was ablated, germ induction
and subsequent duct formation occurred normally, but the final stage of
secretory coil formation failed. Thus, sweat gland development shows
a relay of regulatory steps initiated by Wnt/β-catenin – itself modulated
by Dkk4 – with subsequent participation of Eda and Shh pathways.
KEY WORDS: Exocrine gland, Skin appendage, Hair follicle,
Ectodermal dysplasia, Heatstroke, Hyperhidrosis, Mouse
INTRODUCTION
The functions of the skin, the largest human organ, are expanded by
various appendages. Skin exocrine glands are secretory appendages
that include sweat, mammary, salivary, sebaceous, meibomian,
lacrimal and preputial glands – all of which are involved in crucial
human and animal physiology. Sweat glands in particular comprise
a dynamic thermoregulatory organ. Evaporation of sweat from skin
dissipates heat generated by high ambient temperature, physical
activity or fever. Absence or malformation of sweat glands create
the risk of hyperthermia, as frequently encountered in individuals
with skin grafts, due to lack of sweat gland restoration, and in the
hereditary genetic disorder anhidrotic/hypohidrotic ectodermal
dysplasia (EDA/HED) (Cui and Schlessinger, 2006).
Analyzing the process of sweat gland formation is thus essential
for understanding skin biology and also for potential regeneration of
fully functional skin. Sweat gland development starts from an
ectodermal placode consisting of K14+ progenitor cells (Lu et al.,
2012), as seen for hair follicles (Fuchs, 2007; Millar, 2002). Nascent
gland germs grow down progressively into the dermis to form a
sweat duct, ending in a secretory coil. However, developing sweat
glands lack detectable dermal condensations/papillae that regulate
Laboratory of Genetics, National Institute on Aging, National Institutes of Health,
Baltimore, MD 21224, USA.
*Author for correspondence ([email protected])
Received 19 February 2014; Accepted 4 August 2014
3752
the early induction of hair follicles (Headon, 2009). In addition,
distinct from other exocrine glands, which form branched structures
(Mikkola and Millar, 2006; Tucker, 2007), each sweat gland forms
only a single tubular duct (Sato et al., 1989). The molecular
mechanism of sweat gland development remains to be elucidated.
Mice with mutations in the Eda signaling pathway provide models
with which to investigate the molecular basis of the features of sweat
gland development (Cui et al., 2009; Kunisada et al., 2009). Eda
signaling activates NF-κB-mediated transcription through its ligand
ectodysplasin, the receptor Edar and the receptor/adaptor Edaradd
(Botchkarev and Fessing, 2005; Mikkola, 2008). Sweat glands fail to
develop in Eda mutant Tabby mice, but are restored by an Eda-A1
transgene (Cui et al., 2003), and an adaptive variant of EDAR was
recently shown to foster a moderate increase in sweat gland numbers
in an East Asian population and in a related mouse model (Kamberov
et al., 2013). However, neither upstream regulators nor downstream
effectors of Eda in sweat glands have been defined. Here, we examine
a series of mutant mice in conjunction with expression profiling to
demonstrate a regulatory mechanism that operates through a Wnt(Dkk4)-Eda-Shh cascade that is similar, but not identical to, other
skin appendages.
RESULTS
Sweat gland induction fails in Ctnnb1 mutant (β-catenin cKO)
mice
In hair follicle formation, the canonical Wnt pathway acts upstream
of indispensable Eda action (Durmowicz et al., 2002; Laurikkala
et al., 2001; Zhang et al., 2009). We therefore tested the Wnt
pathway for a role in regulation of initiation of sweat gland
development. As an initial approach, we analyzed Wnt activity in
developing mouse sweat glands using TOPGAL Wnt reporter mice
(DasGupta and Fuchs, 1999). During normal sweat gland
development, pre-germ formation began by E16.5, but only in
proximal footpads, and then spread to the distal footpads at E17.5
and E18.5 (supplementary material Fig. S1A). Thus, stages for
pre-induction, pre-germ, germ and advanced germ can all be
simultaneously observed at E17.5. We collected footpads from
TOPGAL reporter mice at E17.5 and carried out X-gal staining. In
pre-induction stages, weak but uniform Wnt activity was detected in
upper dermis, immediately under the basal layer of epidermis
(supplementary material Fig. S1B). When pre-germs and germs
started to form, scattered Wnt-active cells appeared in epidermis,
but Wnt activity declined sharply in dermis (supplementary material
Fig. S1B). Wnt-active epidermal cells were then focalized in sweat
gland germs, with strong activity seen in advanced germs
(supplementary material Fig. S1B). These data implied Wnt
involvement in sweat gland development.
To analyze Wnt control further, we generated skin-specific
β-catenin mutant (β-Cat cKO) mice by crossing Ctnnb1loxP/loxP
mice with K14-Cre mice. The resultant β-Cat cKO progeny were
born with open eyes and without whiskers. As expected, β-catenin,
DEVELOPMENT
ABSTRACT
Fig. 1. Ablation of β-catenin from skin epidermis results in blocking of
sweat gland induction. (A) β-catenin and Lef1 are highly expressed in sweat
gland germs in wild type (arrows), but undetectable in β-Cat cKO embryos.
Scale bar: 20 µm. (B) Developmental histology. Pre-germs/early germs can be
occasionally observed at E16.5, germs/advanced germs at E17.5 and early
coiling at around P0 (arrows in wild type). No pre-germ/germ formation in β-Cat
cKO embryos. Right panels show the absence of hair follicle development in
the mutant mice. Scale bars: 25 µm. (C) Cell proliferation and cell death status
in β-Cat cKO footpads. Ki67-positive cells are scattered in the basal layers of
wild-type and β-Cat cKO embryos (upper panels). Caspase 3 is not present in
either wild-type or β-Cat cKO embryos (lower panels). Arrows indicate pregerms. Caspase 3 is occasionally found in cells close to the epidermal ducts in
wild-type adult mice (an arrow in right panel). Scale bars: 25 µm.
which is broadly expressed in skin epidermis and locally
upregulated in sweat gland germs in wild-type embryos, was
absent in mutant skin (Fig. 1A, upper panels). In addition, Lef1,
a partner of β-catenin in transcriptional regulation, was selectively
highly expressed in localized sweat gland germ cells in wild-type
controls but undetectable in mutant skin (Fig. 1A, lower panels).
The β-Cat cKO mice died within a few hours, but the early stage
of sweat gland development could be studied before birth.
Strikingly, β-Cat cKO mice showed no indication of sweat gland
germ formation throughout the period E15.5 to birth (Fig. 1B, β-Cat
cKO). We confirmed that hair follicle induction was also abrogated
in the mutant mice [Fig. 1B, right panel (Huelsken et al., 2001)].
Development (2014) 141, 3752-3760 doi:10.1242/dev.109231
The Wnt pathway is thus required for the initiation of inductive
regulation of both sweat glands and hair follicles.
By Ki67 staining, we found scattered proliferating cells in the
basal layer of footpad skin both in wild-type and β-Cat cKO
embryos (Fig. 1C). Overall, more positive cells were seen in wildtype than in β-Cat cKO mice. Scattered Ki67-positive cells were
also found in nascent sweat gland pre-germs in wild-type embryos
(Fig. 1C, arrow in upper left panel). In more advanced sweat
gland germs, clusters of Ki67-positive cells had formed (Fig. 4C,
upper left panel), consistent with recent findings that major
proliferation occurs in hair follicles at later stages rather than the
early pre-germ formation stage (Ahtiainen et al., 2014). A cell
death marker, caspase 3, was not found in wild-type or β-Cat cKO
footpads at embryonic stages (although a few cells close to the
epidermal ducts were positive in adult footpads; Fig. 1C, lower
and right panels).
We also confirmed the absence of dermal condensation/dermal
papilla formation during normal sweat gland development
(supplementary material Fig. S2; Introduction). Instead, we observed
a connective tissue sheath surrounding sweat gland germs at early
developmental stages. It became thinner but was still discernable at
the subsequent stages of duct and secretory region formation
(supplementary material Fig. S2, arrows; see Discussion).
Wnt/β-catenin action is prerequisite for Eda, Shh and Wnt
pathway activation in sweat gland formation
To determine possible downstream targets of Wnt/β-catenin, we
carried out expression profiling with footpad skin from wild-type and
β-Cat cKO embryos at E15.5, E16.5 and E17.5, representing stages
of pre-induction, early-induction and simultaneous ongoing earlyinduction, late-induction, and early-duct formation, respectively
(Fig. 2A). Among 279 genes significantly altered in expression at
one or more time points in the mutant embryos (Fig. 2B;
supplementary material Fig. S3; full list in supplementary material
Table S1), gene ontology analysis revealed a set of genes – especially
those involved in tissue development, skin exocrine gland/hair/tooth/
limb development or cell-cell signaling – that was significantly
affected in cKO footpads (Table 1). Notably, most had not previously
been analyzed as being expressed in sweat glands. Among them, 40,
including seven morphogenetic genes – Atoh1, Lef1, Edar, Shh,
Dkk4, Wnt10b and Fzd10 – were affected at all three developmental
Fig. 2. Genes regulated by Wnt/β-catenin
during sweat gland development.
(A) Expression profiling was carried out with
whole footpad skin from wild-type and β-Cat
cKO embryos at E15.5, E16.5 and E17.5.
(B) The number of genes significantly
affected in β-Cat cKO embryos. Forty genes
were affected at all three developmental time
points. (C) qRT-PCR assays confirmed
significant downregulation of Edar, Shh,
Dkk4, Wnt10b and Fzd10 in β-Cat cKO
footpads. ***P≤0.05 for Fzd10 and
***P≤0.001 for others. Data are mean±s.e.m.
(D) Edar protein (arrow) was localized to
the membrane of sweat gland germ cells in
wild-type, but not in the mutant, embryos.
Dashed lines demarcate the epidermaldermal junction. Scale bar: 20 µm.
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DEVELOPMENT
RESEARCH ARTICLE
Development (2014) 141, 3752-3760 doi:10.1242/dev.109231
Table 1: Functional annotation of genes affected in β-Cat cKO footpads
during development
Category
Gene symbol
Tissue development
Shh, Edar, Wnt7a, Wnt3a, Lef1, Tnf, Tbx3,
Trp73, Emx2, Elf5, Tcfcp2l1, Pthlh,
Col2a1, Otor, Atoh1, Rora, Gjb6, Tmie,
Prlr, Etv4, Celsr3, Ocel1, Chl1, Mmp9, Ivl,
Hrnr, Sprr1b, Sprr2i, Sprr2d, Krt6a, Krt6b
Shh, Edar, Wnt3a, Lef1, Tbx3, Tcfcp2l1
Exocrine gland
development
Hair follicle, tooth and limb
development
Cell-cell signaling
Cell/tissue differentiation
Shh, Edar, Wnt7a, Lef1, Tnf, Mpzl3, Tbx3
Shh, Edar, Wnt10b, Wnt7a, Wnt3a, Dkk4,
Lef1, Fzd10, Kremen2, Epgn, Tnf, Gjb5,
Gjb4, Gjb6, Smpd3, Hnmt, Ereg, Nod2
Ivl, Hrnr, Sprr1b, Sprr2i, Sprr2d, Krt6a,
Krt6b, Ptgs1, Shh, Wnt7a, Wnt3a, Trp73,
Tcfcp2l1, Pthlh, Prlr, Atoh1, Etv4, Celsr3,
Ocel1, Chl1, Emx2
time points studied (Fig. 2B; Table 2). Atoh1 has been reported as
crucial for skin Merkel cell formation, but not for hair follicle
development (Van Keymeulen et al., 2009), and was not further
studied. For the others, qRT-PCR assays confirmed sharp
downregulation in the cKO mice at E15.5, the pre-induction stage
and thereafter (Fig. 2C). This is consistent with their activation
downstream of Wnt/β-catenin. Wnt7a was downregulated in mutant
skin at E16.5 and E17.5, but not yet at E15.5 (Fig. 2C); similarly, Eda
was slightly downregulated at E16.5, the early-induction stage
(Fig. 2C) [this is similar to the observations of Zhang et al. (2009) in
Ctnnb1 mutant back skin]. Immunostaining further confirmed that
Edar was present in the membrane of sweat gland germ cells in wild
type, but absent in the mutant mice (Fig. 2D).
Dkk4 as an antagonist of sweat gland induction by
Wnt/β-catenin
Consistent with previous evidence of Dkk4 as a direct Wnt target
(Bazzi et al., 2007), it was sharply downregulated in β-Cat cKO
embryos. As a Wnt antagonist, however, it might then exert negative
feedback. We therefore examined sweat gland dynamics in mice
bearing a skin-specific Dkk4 transgene (Cui et al., 2010). Dkk4 was
highly expressed in sweat gland germs in wild type, and more
intensely in the Dkk4 transgenic mice (Fig. 3A). Wnt activity was
noticeably reduced in epidermis in the transgenic footpads, as fewer
epithelial cells were positive for X-gal staining compared with wild
type (supplementary material Fig. S1B,C). However, the transgenic
embryos showed prolonged Wnt activity in dermis detectable even
when pre-germs/germs have started to form (supplementary
material Fig. S1B,C); this likely reflects disruption by the Dkk4
transgene of reciprocal signaling interactions between epidermis
Table 2. Morphogenetic genes affected at all three developmental time
points
β-Cat cKO/wild type
Gene
E15.5
E16.5
E17.5
Lef1
Edar
Shh
Dkk4
Wnt10b
Fzd10
Atoh1
0.4
0.5
0.1
0.2
0.3
0.5
0.2
0.5
0.3
0.1
0.1
0.3
0.4
0.1
0.3
0.4
0.1
0.1
0.3
0.5
0.2
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and dermis. We found that sweat gland numbers were significantly
reduced in the transgenic mice. The iodine-starch sweat test revealed
a corresponding reduction of about 40% in sweating spots in adult
transgenics (Fig. 3B,C); and histological analysis of serial sections
further confirmed a decreased number of sweat glands in transgenic
mice, consistent with reduced numbers of X-gal-positive cells
during development. The gland clusters were halved in size
(Fig. 3D, P75). Fewer sweat glands were also evident in
developing transgenics (Fig. 3D, P8). Immunostaining with Lef1
and Edar antibodies revealed fewer sweat gland germs in the
transgenic embryos, but induction at the same time as in wild type
[E16.5 (Fig. 3E, lower panels in Lef1 and Edar) and E17.5 (Fig. 3E,
upper panels)], and showed normal Lef1 and Edar expression
(Fig. 3E). Individual sweat glands formed in the adult mutant mice
appeared normal, with fully open secretory regions and sweat ducts
(Fig. 3D, P75), as well as positive sweat tests. The results thus
indicate that β-catenin activity determines the number of initiation
sites for sweat glands; Dkk4 antagonism would effectively lower
β-catenin activity and therefore the number of sweat glands that are
initiated.
As expected, expression profiling and qRT-PCR assays with
footpad skin from wild-type and transgenic embryos showed that
Dkk4 expression was dramatically upregulated in transgenic embryos
about 160-fold at E15.5 and nearly 80-fold at E16.5. Reflecting an
incomplete suppression of sweat gland induction by the transgene,
changes in expression for downstream genes were milder.
Supplementary material Table S2 lists significantly affected genes
and supplementary material Table S3 shows functional annotation.
Edar expression showed a downward trend of 30% at E15.5, when
Shh was the most strikingly affected morphogen (Fig. 3F). By E16.5,
both Edar and Shh were sharply downregulated, and Eda, Fzd10 and
Wnt10b were also statistically significantly reduced (Fig. 3F). Wnt7a
and Lef1 were also slightly lower, but Wnt7b and Ctnnb1 were
unaffected, and may thus be regulated in a manner unresponsive to
Dkk4 levels.
Disruption of Eda signaling results in abortive sweat gland
pre-germ formation
Turning to the Eda pathway, the genes whose expression was
significantly affected in β-catenin cKO footpads (Table 2) included
Edar – like Dkk4, previously shown to be a direct Wnt target (Zhang
et al., 2009). In addition, mice depleted of Eda pathway genes,
including Tabby, Downless and Crinkled mutants, lack sweat
glands (Grüneberg, 1971); however, early stage developmental
defects have not been characterized. We found that Tabby mice do
form pre-germ like structures at early developmental stages. A few
elevated clusters of basal keratinocytes in footpads (Fig. 4A, arrows
in Ta) were faintly positive for Lef1 at E16.5, and more intensely
and extensively stained at E17.5; by E18.5 the staining was
subsiding (Fig. 4B, Ta) and thereafter disappeared. Thus, full ‘gland
germs’ never formed in Tabby mutants. In wild-type controls,
pre-germs, germs and early stage straight ducts are apparent at these
stages. Lef1 expression was comparably found in the clustered
pre-germ and germ cells, though only in the tip of growing ducts
(it became undetectable in advanced ducts starting to coil; Fig. 4B,
wild type). Similar to wild-type pre-germs (Fig. 1C), some pre-germ
such as cells in Tabby mutants were positive, but weakly so, for
Ki67 (Fig. 4C, left panels). Caspase 3 was not present in either
wild-type or Tabby footpads (Fig. 4C, right panels).
Thus, Eda and Dkk4 are both located downstream of Wnt/
β-catenin (Fig. 2C; Table 2), but have distinctive effects on sweat
gland development (Figs 3 and 4). Nevertheless, because both affect
DEVELOPMENT
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Development (2014) 141, 3752-3760 doi:10.1242/dev.109231
Shh expression profoundly (Fig. 3F and Fig. 4D) (Kunisada et al.,
2009), we asked whether they interact in their effects. We generated
Dkk4 transgenic Tabby mice (TaDk4TG) (see Materials and
Methods). Sweat glands were not formed in TaDk4TG mice, but
immunostaining with Lef1 revealed formation of pre-germ-like
structures that were similar to Tabby pre-germs (Fig. 4E). Again,
this is consistent with the action of Dkk4 feedback at the initiation of
sweat glands, and of Eda at the subsequent pre-germ formation.
Shh is downstream of Eda and regulates final secretory
region formation
Morphogenetic genes, such as Edar, Wnt10b and Fzd10, were
significantly affected in β-Cat cKO mice but not in Tabby mice,
consistent with pure Wnt-mediated regulation independent of Eda
(Fig. 4D). However, as seen in β-Cat cKO, expression profiling in
Tabby mice indicates that Shh is the most strikingly affected gene
from induction through secretory region formation (Kunisada
et al., 2009). qRT-PCR assays confirmed sharp downregulation at
early developmental stages (confirmed in Fig. 4D). These results
suggested that Shh is activated downstream of Eda.
To increase resolution of its action, we analyzed skin-specific
Shh mutant mice (Shh cKO) (Cui et al., 2011). Consistent with a
regulatory location for Shh downstream of the Wnt-Eda cascade,
qRT-PCR assays showed unchanged expression levels for Eda, Edar,
Wnt10b and Fzd10 in the Shh mutant mice at E15.5 and E16.5; Dkk4
was transiently highly expressed only at E15.5 (supplementary
material Fig. S4A). At the histological level, as in conventional
knockout mice (Chiang et al., 1999), hair follicle formation was
arrested at the early germ stage (Fig. 5A; see Discussion). By contrast,
sweat gland germs formed and developed further to form sweat ducts
in the cKO mice (Fig. 5B, arrows in cKO P8 show sweat ducts).
Consistent with normal induction and duct formation, Lef1 and Edar
proteins were normally expressed in the nucleus and membrane of
sweat gland germs in the mutant mice (Fig. 5C).
However, in absence of Shh, formation of the final secretory
region showed a characteristic defect. The coiling process was
initiated, but its progression was curtailed in the cKO mice, yielding
a secretory region much smaller than wild type, clearly discernible
at P5 and decisive at P8 (Fig. 5B). Immunostaining for K14 and K8,
markers for the sweat duct and secretory regions, respectively,
further confirmed that sweat ducts appeared normal in the Shh cKO
mice but secretory regions were rudimentary (Fig. 5D, secretory
regions circled with broken lines). Ki67-positive cells were clearly
sparser than in the wild type at both P5 and P8 (supplementary
material Fig. S4B, left panels), but caspase 3 was negative in both
mutant and wild type (supplementary material Fig. S4B, right
panels). Thus, the developmental defect of secretory region
formation appears to reflect depressed cell proliferation rather
than accelerated loss.
Involvement of Bmp and FoxA1 in auxiliary regulation
Two other genes are strongly implicated in sweat gland formation. At
an early stage, ablation of one, Bmpr1a, results in failure of sweat
gland formation, and when Bmp was suppressed by its antagonist
noggin (Nog), hair follicles formed in mouse footpads, where they are
usually not seen (Leung et al., 2013; Plikus et al., 2004). As an aid to
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DEVELOPMENT
Fig. 3. Sweat gland numbers were significantly reduced in
Dkk4 transgenic mice. (A) In situ hybridization revealed
selective expression of Dkk4 in sweat gland germs in wild-type
embryos, and high expression in the basal layer of skin
epidermis and sweat gland germs in the transgenic mice.
Arrows indicate sweat gland germs. Scale bar: 50 µm.
(B) Sweat test showed decreased numbers of sweating spots in
the transgenic mice. (C) A reduction of ∼40% of sweating
spots was seen in the transgenic mice. *P≤0.01. Data are
mean±s.e.m. (D) The sizes of sweat gland clusters (outlined) in
the transgenic mice were about half of wild-type controls in adult
(P75) and developing (P8) mice. Secretory regions and ducts
opened normally in the transgenic mice (P75). Scale bar:
100 µm. (E) Formed sweat gland germs at E16.5 (arrows) and
E17.5 (arrowheads) normally expressed Lef1 and Edar in the
transgenic embryos. Scale bar: 20 µm. (F) qRT-PCR assays
show moderate but significant downregulation of several Wnt
target genes in transgenic embryos. * and **P≤0.005 for Edar
and Shh; P≤0.05 for the others. Data are mean±s.e.m.
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determining its time of action, our expression profiling showed no
significant changes in expression of any Bmp pathway genes in β-Cat
cKO footpads. In further targeted qRT-PCR assays, Bmp4 expression
was unchanged in the mutant embryos at all time points analyzed
(supplementary material Fig. S5A). Nog was slightly upregulated in
the mutant embryos at E15.5, but unchanged at E16.5 or E17.5; and
Bmp2 was slightly downregulated only at E17.5. In addition, the
pattern of expression was very similar in Tabby footpads
(supplementary material Fig. S5B), i.e. in the absence of Eda.
Immunostaining for Bmpr1a was undetectable at pre-germ/germ
stages in wild-type, β-Cat cKO or Tabby footpads, but showed weak
positivity only in advanced germs in wild-type mice (supplementary
material Fig. S5C). By contrast, its downstream effector pSmad1/5
was highly expressed in sweat gland germs in wild type, broadly
expressed in the basal layer of β-Cat cKO embryos and positive for
pre-germ like structures in Tabby (supplementary material Fig. S5D).
Thus, Bmp likely acts upstream of the Wnt-Eda cascade, as in hair
follicles (Botchkarev et al., 1999), but its role requires further
characterization.
The other pathway known to be involved in sweat gland
maturation, which requires Foxa1, acts at the late stage of
development of sweating capacity (Cui et al., 2012). In Shh mutant
mice, as in wild type, immunostaining revealed nuclear localization of
Foxa1 in scattered developing secretory cells (supplementary material
Fig. S5E), consistent with its established role in addition to Shh.
DISCUSSION
The Wnt-Eda cascade in early stage sweat gland
development
Sweat secretion requires coordinated action of sympathetic nerve
impulses, regulatory transcription factors and resultant secretory
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structures assembled during development (Cui et al., 2012; Sato et al.,
1989). Sweat gland development includes successive induction, duct
formation and secretory region formation (Fig. 6). Putatively
implicated at an early stage in Wnt/β-catenin activation are the
Bmp/Nog pathway and engrailed 1, a negative regulator of β-catenin
(Bachar-Dahan et al., 2006; Botchkarev et al., 1999). We find here
that Wnt/β-catenin, once activated, then controls the regulatory
hierarchy for sweat gland induction and duct formation involving
Eda/Edar and Dkk4 (Fig. 6). It likely sets the number of gland
initiation sites.
Dkk4 and Dkk1 are members of the Wnt antagonist Dkk family,
which suppresses Wnt signaling by binding to Wnt co-receptors
Lrp5/6 (Niehrs, 2006). In skin, however, Dkk1 is expressed in
mesenchyme surrounding hair germs, whereas Dkk4 is in the
epidermal part of hair germs (Andl et al., 2002; Bazzi et al., 2007).
Dkk1 completely blocked hair follicle induction when overexpressed
in mice (Andl et al., 2002); however, Dkk4 transgenic mice showed
comparable numbers of hairs with wild-type controls, though hair
subtypes were severely distorted (Cui et al., 2010).
Dkk4, but not Dkk1, was significantly downregulated in β-Cat
cKO footpads. Dkk4 is regulated by canonical Wnt (Bazzi et al.,
2007) and has also been shown to be directly regulated by Edar
during hair follicle development (Zhang et al., 2009). As in
hair follicles, Dkk4 is selectively expressed in the epidermal
compartment of sweat gland germs (Fig. 3A). However, our data
suggest that it is primarily regulated by Wnt rather than Eda in sweat
glands (Fig. 2C). We showed that Dkk4 levels are lowered at early
developmental stages in hair follicles (e.g. E13.5, E14.5) (Cui et al.,
2006), but lowering occurs at late stages in sweat glands (E17.5,
E18.5) in Tabby (Fig. 4D; Kunisada et al., 2009). The apparent
functional effect of Dkk4 in sweat glands is also very different from
DEVELOPMENT
Fig. 4. Abortive sweat gland pre-germ
formation in Tabby. (A) A nascent pre-germ
like structure formed in Tabby at E17.5 (arrows
in Ta). Scale bar: 25 µm. (B) Lef1 staining
shows developing sweat glands in wild-type
and nascent pre-germs in Tabby (arrows).
Lef1 is found in pre-germs, germs and cells in
the tip of growing ducts in wild type, and in pregerms in Tabby (arrows). Lef1 is undetectable
in a sweat duct ready to coil in wild type
(right panel). Scale bar: 25 µm. (C) Ki67 is
found at high levels in advanced germs in wild
type and at lower levels in pre-germ cells in
Tabby (arrows). Caspase 3 is not present in
either wild-type or Ta footpads. Scale bar:
20 µm. (D) qRT-PCR assays show sharp
downregulation of Eda and Shh in Tabby.
Dkk4 was significantly downregulated only at
E17.5. ***P≤0.002. Data are mean±s.e.m.
(E) Dkk4 transgenic Tabby mice lack sweat
glands, but form pre-germ like structures, as
seen in Tabby. Arrows indicate pre-germs and
germs of sweat glands for each genotype.
Scale bar: 25 µm.
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Development (2014) 141, 3752-3760 doi:10.1242/dev.109231
throughout duct formation (Cui et al., 2009). Shh intervenes later
(Fig. 5 and below).
Various appendages display distinctive signaling interaction
patterns
that in hair follicles. Overexpressed Dkk4 blocked induction of
about 40% of mouse sweat glands, but had no apparent effect on
hair follicle number. Differential regulatory networks may thus
delimit Dkk4 action in various skin appendages.
We also found that several Wnt target genes were moderately but
significantly downregulated in the Dkk4 transgenic sweat glands,
consistent with a negative-feedback interaction of Dkk4 with Wnt
(Fig. 6). Because the glands formed in the presence of various levels
of Dkk4 are structurally and functionally very similar, the increased
level of Dkk4 apparently only limits the probability of successful
sweat gland germ formation, leading to an overall reduction in
number.
Wnt/β-catenin also directly activates the downstream Eda
pathway. Ablation of β-catenin thus results in a complete
absence of sweat gland induction, and Eda mutant Tabby mice
show only primitive pre-germ-like structures. Eda is then required
Shh action: an example of variable involvement and timing in
different skin appendages
Shh is key to post-induction development
appendages (Chuong et al., 2000). In hair
required for hair germ invagination and dermal
but not for hair germ induction (Chiang et al.,
of several skin
follicles, Shh is
papilla formation
1999; St-Jacques
Fig. 6. Schematic representation of sweat gland
development. Wnt/β-catenin regulates sweat gland
induction through Eda/Edar and Dkk4, and possibly also
through Wnt10b, Fzd10 and Epgn (see text). Dkk4
suppresses gland induction through a negative-feedback
effect on Wnt/β-catenin. The Bmp pathway and Engrailed
may regulate Wnt/β-catenin upstream, and Eda/Edar may
regulate Dkk4 at late developmental stages. Eda and Edar
are required for sweat duct formation downstream of Wnt/
β-catenin; Wnt10b, Fzd10, Tbx3 and Mmp9 may also be
involved. Shh is required for secretory region formation, and
Foxa1 is likely involved in the completion of sweat gland
formation.
3757
DEVELOPMENT
Fig. 5. Ablation of Shh from skin epidermis results in rudimentary
secretory region formation. (A) Hair follicles were arrested at the germ stage
in the mutant mice (arrows). Scale bar: 100 µm. (B) Secretory regions started
to coil, but further progression was interrupted in Shh cKO mice (P5 and P8).
Arrows indicate sweat ducts with normal spacing in the mutant mice. Scale bar:
100 µm. (C) Gland germs were induced as normal, and Lef1 and Edar
were expressed as normal in the mutant mice. Scale bar: 20 µm. (D) Krt14 and
Krt8 staining shows normal sweat ducts, but rudimentary secretory regions
(circled by broken lines) formed in Shh cKO mice. Scale bar: 20 µm.
Although skin appendages are thought to form by reciprocal
signaling interactions between ectoderm and mesenchyme (Millar,
2002), the use and phase of action of signaling pathways vary. Here,
we discuss steps of sweat gland development compared with other
skin appendages.
In hair follicles, use and phasing are similar to sweat glands,
especially at the early developmental stages. Wnt is required for
early induction and Eda for late induction through hair peg/sweat
duct formation (Chen et al., 2012; Cui et al., 2009; Millar, 2002;
Schmidt-Ullrich et al., 2006; Zhang et al., 2009) (Figs 1 and 4). The
specification of appendage type may then be effected at least in part
by variations in the Shh pathway (see below). In contrast to hair
follicles and sweat glands, salivary gland early stage development
depends instead on Fgf and Pitx2 pathways (Jaskoll et al., 2004b;
Tucker, 2007). Eda and Shh pathways are then required for latestage branching morphogenesis (Häärä et al., 2011; Patel et al.,
2011). In early stage mammary gland development, yet another
pattern is seen. Both Fgf and Wnt are required for induction, and
Eda and PTHrP for ductal and branching morphogenesis (Lindfors
et al., 2013; Voutilainen et al., 2012).
At the structural level, a striking feature seen in hair follicles, the
dermal papilla, is not found in the exocrine appendages that have been
studied (Headon, 2009). Wnt and Shh pathways are required for the
formation of well-described dermal papillae for hair follicles (Chen
et al., 2012; Millar, 2002; Zhang et al., 2009); however, salivary
glands and mammary glands have no apparent dermal papillae.
Instead, they form rather broad areas of dermal mesenchyme
(Harunaga et al., 2011; Mikkola and Millar, 2006). As shown by
heterotypic tissue recombination experiments, dynamic dermalepidermal signaling interactions nevertheless occur during both
salivary gland and mammary gland development (Kratochwil, 1969;
Kusakabe et al., 1985). We find that sweat glands are also comparably
surrounded by sheaths of connective tissue (supplementary material
Fig. S2), though less abundantly than in salivary or mammary gland
mesenchyme. We infer that sweat gland development also requires
dermal-epidermal interactions, with the sweat gland mesenchyme/
connective tissue sheath likely providing dermal signaling.
et al., 1998). In exocrine appendages, it was dispensable for
mammary gland development (Gallego et al., 2002; Michno et al.,
2003), but required for late stage salivary gland branching
morphogenesis (Jaskoll et al., 2004a). In sweat glands, Shh is not
required for germ formation or for subsequent duct invagination
(Fig. 5), but is indispensable for final stage secretory coil formation,
more similar to its role in salivary glands.
How can Shh vary in its determinative role in different skin
appendage lineages? Among the possible factors to be assessed
are: specific local niche matrices; pre-determinative differences
in effectors in placodes; and the involvement of additional
pathways such as that regulating sweating capacity, which is
initiated by FoxA1 (for example) (Cui et al., 2012). However, the
level and/or timing of Shh expression might be an important
effector. Suggestively, inhibition of Shh signaling resulted in
glandular tissue formation in back skin, which otherwise harbors
only hair follicles (Gritli-Linde et al., 2007). In addition,
consistent with this notion, we found that Shh expression was
very low in early sweat gland induction stages and then increased
sharply during postnatal secretory coil formation (Kunisada
et al., 2009).
The expression profiling of appendages derived from wild type
compared with the mutant mouse models also provided additional
genes as candidates for involvement in sweat gland development
(Table 1). For example, among the known targets downstream of
Wnt/β-catenin, Epgn was upregulated in the β-catenin-ablated
mice from early induction stages (Fig. 6; supplementary material
Table S1); overexpression of this gene has recently been shown to
suppress primary hair formation and promote sebaceous gland
hyperplasia (Dahlhoff et al., 2010). By contrast, Tbx3 and Mmp9
were downregulated at late induction/early duct formation stages.
Tbx3 mutations cause the Ulnar-mammary syndrome that affects
limb, apocrine sweat gland and hair follicle development
(OMIM#181450), and might be involved in late stage eccrine
sweat gland formation. We believe that Mmp9, which is also
downregulated in Tabby mutants (Kunisada et al., 2009), is
also involved in tissue remodeling during sweat duct invagination.
How the candidate genes might affect differential cell lineage
specification and further development now becomes an area for
future study.
MATERIALS AND METHODS
Mouse models
All research was conducted according to the guidelines as defined by the
Office of Animal Care and Use in the NIH Intramural Program, and all
animal study protocols were approved by the NIA Institutional Review
Board. We used six genetically modified mouse strains for this study. The
Tg(TCF/Lef1-lacZ)34Efu/J (TOPGAL reporter, stock#: 004623) mice,
B6.129-Ctnnb1 tm2Kem/KnwJ (Ctnnb1loxP/loxP, stock#: 004152) mice, the
B6.129-Shh tm2Amc/J (ShhloxP/loxP, stock#: 004293) mice, the Tg(KRT14cre)1Amc/J (K14-Cre, stock#: 004782) mice and the C57BL/6J Aw-J-Ta6J
(Tabby, stock#: 000338) mice were purchased from the Jackson Laboratory
(Bar Harbor, MA, USA) and set up in colonies at the NIA animal facility.
Skin-specific β-catenin mutant mice (β-Cat cKO) and skin-specific Shh
mutant mice (Shh cKO) were generated by crossing the Ctnnb1loxP/loxP mice
or the ShhloxP/loxP mice with the K14-Cre mice. Genotyping for TOPGAL,
Ctnnb1, Shh and K14-Cre mutant mice was carried out by PCR with
protocols provided by the Jackson Laboratory. Tabby genotyping was
carried out by PCR and subsequent DdeI or AvaI digestion, as previously
described (Cui et al., 2006).
The skin-specific Dkk4 transgenic mice (K14-Dkk4) were generated in
our laboratory and have been reported previously (Cui et al., 2010). Briefly,
the full-length open reading frame of mouse Dkk4 cDNA with insertion of a
Flag tag in the 3′ end was subcloned into a K14 promoter vector and
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Development (2014) 141, 3752-3760 doi:10.1242/dev.109231
microinjected into pronuclei of one-cell C57BL/6J mouse embryos.
Potential founders were mated to C57BL/6J mice to establish transgenic
lines. Dkk4 transgenic TOPGAL mice were generated by crossing K14Dkk4 transgenic mice with TOPGAL reporter mice, and Dkk4 transgenic
Tabby male mice by crossing K14-Dkk4 transgenic male mice with Tabby
female mice.
Timed-mating, RNA isolation, expression profiling and qRT-PCR
Timed-mating was set up for TOPGAL, β-Cat cKO, Tabby, Shh cKO and
Dkk4 transgenic mice to obtain embryos. Except for footpads, mouse skin
is covered entirely by hair; by contrast, sweat glands are located
exclusively in the footpads, facilitating selective analyses. We collected
non-hairy footpad skin from embryos at E15.5, E16.5 and E17.5 under a
dissection microscope, and fixed in 10% formaldehyde (Ricca Chemical)
for histological analysis or snap-froze them in dry ice and stored them in a
−80°C freezer for RNA isolation. Frozen whole footpad skin samples from
β-Cat cKO (E15.5, E16.5 and E17.5) or Dkk4 transgenic (E15.5 and E16.5)
embryos were then separated into two groups for each genotype at each
time point for biological duplicates, a minimum replication, but sufficient
to reveal a number of crucial genes in sweat glands, including Foxa1 and
Shh (Kunisada et al., 2009). Total RNAs were isolated with Trizol reagent
(Invitrogen), and cyanine-3-labeled cRNAs were hybridized to the NIA
Mouse 44K Microarray v3.0 (Agilent Technologies). Duplicated data were
analyzed by ANOVA (Kunisada et al., 2009). Genes with FDR<0.05, fold
difference>2.0 and mean log intensity>2.0 were considered to be
significant for β-Cat cKO versus wild type comparison. Given that the
expression changes were less abundant, FDR<0.05, fold difference>1.5
and mean log intensity>2.0 were considered to be significant for Dkk4
transgenic mice. Hybridization data have been deposited in the Gene
Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) with
accession number GSE50859 for β-Cat cKO and GSE50862 for Dkk4
transgenic embryos. One-step Taqman qRT-PCR was carried out with
ready-to-use probe/primer sets for Eda, Edar, Shh, Dkk4, Wnt10b, Fzd10,
Wnt7a, Ctnnb1, Lef1, Bmp2, Bmp4 and Nog (Applied Biosystems). Each
of the two sets of RNAs for each genotype (wild-type, β-Cat cKO, Tabby,
Dkk4 transgenic or Shh cKO) was assayed in triplicate by qRT-PCR.
Reactions were normalized to GAPDH and data were analyzed using
Student’s t-test.
X-gal staining, general histology, immunofluorescent staining,
in situ hybridization and sweat test
X-gal staining was carried out with TOPGAL and Dkk4 transgenic
TOPGAL embryos using a lacZ tissue staining kit (rep-lz-t, InvivoGen).
Briefly, 12 µm frozen sections from E17.5 mouse footpads were fixed in
0.5% glutaraldehyde for 10 min at room temperature and then incubated
with X-gal staining solution overnight at room temperature. Positive signals
were visualized after washing sections in PBS.
For general histology, footpad skin fixed in 10% formaldehyde was
dehydrated and embedded in paraffin (P3558, Sigma). Sections (5 μm) were
cut, deparaffinized and ‘unmasked’ by heating at 121°C for 2 min in an
Antigen Unmasking Solution (H-3300, Vector Laboratories). Hematoxylin
and Eosin (H&E) staining was carried out according to the manufacturer’s
standard protocol (Sigma). Indirect immunofluorescence staining was
carried out with primary and secondary antibodies listed below. Sections
were blocked with a serum-free protein blocking solution (X0909, Dako) for
30 min before antibody application, and antibodies were diluted in an
Antibody Dilution solution (S0809, Dako).
For immunofluorescent staining, rabbit antibodies against β-catenin
(#9587, Cell Signaling, 1:1000 dilution), Lef1 (#2230, Cell Signaling,
1:300), Edar (18032-1-AP, Proteintech, 1:150), Ki67 (ab15580, Abcam,
1:100), caspase 3 (#9661, Cell Signaling, 1:200), Bmpr1a ( pa5-27280,
Thermo Scientific, 1:200), pSmad1/5 (#9516, Cell Signaling, 1:50) and K14
(PRB155P, Covance, 1:1500), and guinea pig antibodies for K8 (GPK8,
Progen, 1:200) and K14 (GP-CK14, Progen, 1:1500) were incubated with
deparaffinized, antigen-unmasked sweat gland sections. AlexaFluorconjugated secondary antibodies were used to detect primary antibodies
(Invitrogen), and images were taken using a Deltavision Microscope System
(Applied Precision).
DEVELOPMENT
RESEARCH ARTICLE
For in situ hybridization, a 1 kb fragment spanning exons I-IV of mouse
Dkk4 cDNA was amplified by PCR and subcloned into a pGEM-T vector
(Promega). Primers used were: forward, 5′-TGGAGCTCCGAGAGACCAGAGTGA-3′; reverse, 5′-TGAGTGTAACATGCTCGCCTGTAA-3′.
Mouse footpads were fixed in 4% paraformaldehyde, dehydrated with 30%
sucrose and embedded in OCT compound. Cryosections (20 μm) were
hybridized with 1 μg/ml digoxin-labeled antisense probes at 65°C in
hybridization solution (50% formamide, 5× SSC, 300 µg/ml yeast tRNA,
100 ng/ml heparin, 1 mM EDTA, 1× Denhardt’s solution, 0.1% Tween 20,
0.1% CHAPS, 5 mM EDTA), followed by washing with 2× SSC and 0.2×
SSC, and incubated with anti-digoxin antibody (Roche). Positive signal was
detected with NBT/BCIP solution (Roche).
For the sweat test, a hind paw of a wild-type or mutant mouse was painted
with iodine/alcohol solution (2%). Once dry, the surface was painted with
starch-oil (10 g starch/10 ml castor oil). Purple sweating spots start to form
1-2 min later and peak at around 10 min.
Acknowledgements
The authors thank Ramaiah Nagaraja for critical reading, and Yong Qian and Alexei
Sharov for technical help.
Competing interests
The authors declare no competing financial interests.
Author contributions
C.-Y.C. and D.S. developed the concepts and designed the experiments; C.-Y.C.,
M.Y., J.S., V.C., M.M. and Y.P. performed the experiments; C.-Y.C. and D.S.
analyzed the data; C.-Y.C. and D.S. wrote the paper.
Funding
This work was supported by the Intramural Research Program of the National
Institute on Aging, National Institutes of Health. Deposited in PMC for release after
12 months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.109231/-/DC1
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