Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria
Author for correspondence (e-mail:
annette.neubueser{at}biologie.uni-freiburg.de)
Accepted 5 April 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: FGF signaling, Feather development, FGFR1-Fc, FGFR2-Fc, Su5402, Fgf10, Placode induction
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Members of the BMP, Delta/Notch, SHH, WNT and FGF families of signaling
molecules are expressed during cutaneous appendage development. Several of
these have been shown to play important roles in the communication between
epithelial and mesenchymal cells (reviewed by
Millar, 2002). However, the
identity of the first dermal signal still remains unknown. As a consequence
the molecular mechanisms controlling the earliest stages of appendage
development also remain poorly defined.
Fibroblast growth factors (FGFs) play essential roles in many aspects of
embryogenesis and have previously been implicated as positive regulators of
hair and feather development (reviewed by
Ornitz and Itoh, 2001). Beads
soaked in recombinant FGF protein were sufficient to induce feather buds in
skin explants isolated from scaleless mutant chick embryos that lacked most
feathers because of an ectodermal defect
(Song et al., 1996
).
FGF-soaked beads also induced ectopic buds in explants from apteric regions of
wild-type embryos and bud fusions in regions of ongoing bud development
(Widelitz et al., 1996
).
Expression during epithelial appendage formation has so far only been analyzed
for a subset of the 22 members of the FGF family. Previous studies have shown
that Fgf2 and Fgf4 are expressed in the feather placodes
(Jung et al., 1998
;
Song et al., 1996
). Whether
they are already expressed prior to placode formation is a matter of debate.
Expression of Fgf10 has recently been described in the mesenchyme of
feather and whisker hair primordia, but no expression was detected prior to
placode formation (Ohuchi et al.,
2003
; Tao et al.,
2002
). Fgf5 and Fgf7 are expressed during hair
follicle morphogenesis but are not required for the initiation of hair
development (Guo et al., 1996
;
Hebert et al., 1994
). Based on
these data it has been suggested that FGFs may function as secondary inducers
that promote hair and feather development but are unlikely to be involved in
the initial induction of the placodes.
We have examined the function of fibroblast growth factor (FGF) signaling in the initiation of feather development in the chick. For this purpose we have used replication-competent avian retroviruses to over-express secreted dominant negative versions of FGFR1 and FGFR2 in ovo. This caused a complete block of feather development prior to the initiation of feather placode formation. We further show that Fgf10 is expressed in the dermis of nascent feather buds and is required for placode induction in skin explants. In addition, we demonstrate that FGF10 can stimulate expression of positive and negative regulators of feather development in the overlying ectoderm and can induce its own expression under conditions of low BMP activity. Together these results demonstrate that FGF signaling is required for the initiation of feather placode development and point towards FGF10 as an early dermal signal involved in this process.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Production of retroviruses carrying secreted forms of mFGFR1 (IIIc) and mFGFR2 (IIIb)
The RCAS-FGFR1-Fc retrovirus (referred to as RCAS-R1) was generated by
fusing the extracellular domains of mouse FGFR1 [splice isoform IIIc; GenBank
accession number NM_010206, amino acids 1-30 and 120-367 as described by
Werner et al., (Werner et al.,
1993)] to the Fc-fragment of mouse-IgG (GenBank accession number
AB097849; aa228-463) including a Ser-Ser linker. Similarly, a secreted,
Fc-tagged form of mouse FGF-receptor 2 (splice isoform IIIb; referred to as
RCAS-R2) was constructed by PCR amplifying the extracellular domains of FGFR2
(GenBank accession number M63503; aa1-258) and the Fc fragment as described by
Celli et al., (Celli et al.,
1998
). Both constructs were cloned into the Slax shuttle vector
and then transferred into the RCASBP(A) retroviral vector
(Hughes et al., 1987
). Viral
stocks of 1x109 infectious units/ml were purified according
to the method of Morgan and Fekete (Morgan
and Fekete, 1996
). Viral infection and titers were monitored by
detection of viral gag protein using the anti-gag antibody AMV3C2 developed by
D. Boettiger and obtained from the Developmental Studies Hybridoma Bank
maintained by The University of Iowa, Department of Biological Science, Iowa
City IA52242.
RCAS-R1 virus was initially injected into migrating neural crest cells
adjacent to the midbrain at HH8-9, resulting in widespread infection
throughout the embryos. These embryos were harvested after 14 days (HH38-40,
n=35). In addition, both RCAS-FGFR types were injected into
sub-ectodermal mesenchyme of the back at HH18-20. Some of these embryos were
incubated until HH38 and assayed for morphological changes in feather
development (n=10 each for RCAS-R1 and RCAS-R2). The majority were
incubated until HH27-32 and analyzed by whole-mount in situ RNA detection for
marker gene expression. For each marker and stage, at least three, but more
typically 5-10 infected specimens were examined. Altogether, 15 RCAS-R1- and
17 RCAS-R2-infected embryos were analyzed prior to placode formation
(HH27-28), and 116 RCAS-R1- and 52 RCAS-R2-injected embryos were analyzed at
HH29-33. Except for 13 embryos in which the virus infection apparently had
failed, all specimens analyzed after HH29 showed a large patch devoid of any
sign of feather bud development in the spinal tract. No alterations of feather
development were detected at any stage if control viruses encoding alkaline
phosphatase, the Fc fragment alone, or GFP were injected (n=41)
(Fekete and Cepko, 1993). A
limited number of embryos was also injected subectodermally with RCAS-R2 or
RCAS-AP at HH23-24 and HH26, respectively. These embryos were harvested at
HH30 and analyzed by double whole-mount in situ RNA detection for
Bmp2 and Fgfr2-Fc expression (using an Fc-specific
probe).
Explant culture and bead implantation
Lumbar skin was isolated prior to placode formation at HH27-28 (referred to
as ppf explants). Alternatively, upper thoracic skin was isolated after the
first feather buds had appeared (HH29-30, referred to as eb explants, compare
Fig. 3A and
Fig. 4A) and cut along the
midline to generate two similar halves. Skin explants were cultured in vitro
for 16-48 hours as previously described
(Neubüser et al., 1997).
In different experiments the following reagents were added to the culture
medium: 25 µM Su5402 (Calbiochem; >95% purity) and 0.5% DMSO, or 0.5%
DMSO alone (16-hour cultures); 10-50 µg/ml FGFR1-Fc (IIIc) or 10-50
µg/ml FGFR2-Fc (IIIb); 5 µg/ml goat anti-FGF10 antibody (SC-7375; Santa
Cruz Biotechnology, Inc) or control goat IgG (SC-2028; Santa Cruz
Biotechnology, Inc). Bead implantations were performed as described previously
(Neubüser et al., 1997
;
Vainio et al., 1993
). Prior to
implantation, heparin acrylic beads (Sigma) were soaked for 2 hours at
37°C in recombinant proteins (0.05, 0.1 or 0.5 mg/ml FGF10; 0.25 mg/ml
Noggin-Fc or in 0.1 mg/ml FGF10 plus 0.25 mg/ml Noggin-Fc; all from R&D)
or PBS. Beads soaked in 0.05-0.5 mg/ml FGF10 had similar effects.
Protein-soaked beads were stored at 4°C for up to 3 weeks. Each experiment
was performed at least twice, but more typically three to five times, and at
least three, but more typically 5-10 specimens were examined for each
condition.
|
|
In situ hybridization was combined with immunohistochemical analysis of
viral infection by embedding the stained embryos into Tissue TekR
(Sakura) after in situ hybridization, followed by cryosectioning. The 10 µm
sections were then processed for antibody staining using the mouse anti-gag
antibody (1:1000 dilution, AMV3C2; Developmental studies Hybridoma Bank,
University of Iowa) and the Vectastain ABC kit (Vector Labs) as described
previously (Matise and Joyner,
1997).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Early feather tract development is normal in embryos over-expressing FGFR1-Fc or FGFR2-Fc
In order to determine at which stage of feather development FGF signaling
is required, we first analyzed whether regions competent to form feathers, the
feather tracts, are specified normally in the presence of FGFR1-Fc or
FGFR2-Fc. Tract formation is characterized by the formation of a dense dermis
(Sengel, 1976). Between HH26
and HH28 several genes are up-regulated in diffuse stripes in the areas where
the first feather buds will appear at HH29. Two such markers are
ß-catenin expression in the ectoderm and Fgfr1
expression in the dermis (Noji et al.,
1993
; Noramly et al.,
1999
; Widelitz et al.,
2000
) (Fig.
2A,D).
In RCAS-R1- and RCAS-R2-infected embryos analyzed at HH27-28, the patterns
of Fgfr1 and ß-catenin expression were
indistinguishable from uninfected or control virus infected embryos
(Fig. 2A-F). Successful
infection was confirmed by subsequent sectioning of the stained embryos,
followed by immunohistochemical detection of the viral gag protein. This,
together with the analysis of Hematoxylin and Eosin (HE)-stained sections of
specimens infected with either RCAS-R1, -R2 or control virus revealed no
obvious difference in the histological appearance and density of the dermis or
the overlying ectoderm in the absence of FGF signaling
(Fig. 2G-J, and data not
shown). Expression of two other markers, the bHLH transcription factor
cDermo1 in the dermis (Scaal et
al., 2001) and Wnt6 in the epidermis
(Chodankar et al., 2003
), was
also unchanged in FGFR1-Fc- and FGFR2-Fc-infected embryos at HH27-28 (data not
shown). Together these results suggest that feather tract formation does not
require FGF signaling.
Forced expression of FGFR1-Fc and FGFR2-Fc prevents feather placode development
We next examined the effects of forced expression of FGFR1-Fc and FGFR2-Fc
on the initiation of feather bud development at HH29-30. During normal
development, ß-catenin, Wnt6 and Fgfr1 are expressed in
diffuse stripes in the feather tracts prior to the initiation of bud
development. Once buds start to form, they are locally up-regulated in the
developing buds and down-regulated in interbud domains
(Fig. 3A)
(Chodankar et al., 2003;
Noji et al., 1993
;
Noramly et al., 1999
;
Noramly and Morgan, 1998
;
Widelitz et al., 2000
).
RCAS-R1- and RCAS-R2-infected embryos analyzed at HH29-30 typically showed a large patch devoid of any sign of feather bud development on the back, in the area into which the virus had been injected (Fig. 3). Although the injections were performed unilaterally, these nude patches usually extended across the midline onto the contra-lateral side and thus included the region were the first row of feathers should have formed. In these nude patches Wnt6, ß-catenin and Fgfr1 expression was usually still detectable in the diffuse patterns already observed prior to bud formation (Fig. 3B,C, and data not shown). Occasionally, several weak parallel stripes of expression of these markers were transiently detectable, in particular in RCAS-R2-infected embryos (Fig. 3C, and data not shown). This failure of ß-catenin, Wnt6 and Fgfr1 to undergo a transition from broad, diffuse expression to up-regulated and localized expression in nascent buds indicates that bud formation is not initiated normally in regions infected with RCAS-R1 and RCAS-R2.
Bmp2 is the earliest marker of bud development identified so far
and its localized expression in the ectoderm precedes placode formation
(Jung et al., 1998;
Noramly and Morgan, 1998
).
Slightly later, developing placodes also express Shh
(Fig. 3D)
(Morgan et al., 1998
). In the
nude patches of RCAS-R1- and RCAS-R2-infected embryos expression of the
placode markers Bmp2 and Shh was undetectable
(Fig. 3E-H). Sectioning and
subsequent virus detection of affected embryos after whole-mount in situ
analysis and histological analysis, confirmed widespread infection in the
mesenchyme of the nude patches and absence of any signs of feather placode
formation (Fig. 3I,M',
and data not shown). Similar results were also obtained if embryos were
injected at HH23-HH24, which resulted in smaller areas of infection restricted
to one side of the embryo not including the first row
(Fig. 3K,L). Embryos infected
at HH26, in contrast, showed normal arrangements of Bmp2-expressing
buds in the developing skin, including regions where Fgfr2-Fc
transcripts were detectable (Fig.
3N,O, white circles). Likewise, in embryos infected at HH18-20 or
HH23-24, virus infection was frequently also detectable in bud-containing
regions adjacent to the nude patch (Fig.
3J, and data not shown). Cells in these regions are further away
from the original site of virus injection than the cells inside the nude
patch, and therefore most likely became infected at a later time point.
Together these results show that FGF signaling is necessary for feather
placode formation, the first step of feather bud development, in the first and
subsequent rows. This requirement seems to be transient and at later stages
feather bud development seems to be less sensitive to alterations in the level
of FGF signaling. This conclusions is further supported by the analysis of
additional markers such as delta1, follistatin, Wnt7a and
Wnt3a and Dermo1 (data not shown)
(Crowe et al., 1998
;
Ohyama et al., 2001
;
Patel et al., 1999
;
Scaal et al., 2001
;
Viallet et al., 1998
;
Widelitz et al., 1999
).
FGF signaling is required at the initiation stage of feather placode development
Over-expressing FGFR1-Fc or FGFR2-Fc blocks feather placode development. To
better define the timing when FGF signaling is required, we took advantage of
an in vitro culture system for embryonic skin explants. Pieces of skin were
isolated from the back either shortly before the formation of the first
feather buds (HH28, referred to as prior to placode
formation (ppf) explants), or after the first rows of buds had
appeared (HH29-30, referred to as established buds (eb)
explants). These explants were then cultured in the presence or absence of the
following inhibitors of FGF signaling: the FGF receptor antagonist Su5402,
recombinant FGFR1-Fc or FGFR2-Fc protein
(Mohammadi et al., 1997). To
allow a direct comparison between the number of buds at the onset of culture
with the number present after culture, eb explants were cut in half along the
midline as indicated in Fig. 3A
and Fig. 4A. Of these, only one
half was cultured; the contra-lateral side was immediately fixed and served as
a reference.
Ppf explants cultured for 16 hours in the presence of Su5402 lacked all signs of feather bud development and did not express the placode markers Shh and Bmp2 (Fig. 4B,D). Likewise, ß-catenin and Fgfr1 expression was not locally up-regulated in ppf explants cultured in the presence of the inhibitor (Fig. 4F,H). Instead both genes were expressed in patterns similar to the stage of explantation, a stripe in the case of Fgfr1 and a diffuse low level expression in the case of ß-catenin (compare with Fig. 2A,D). In contrast, explants cultured in control medium developed normal arrays of feather primordia expressing all four markers (Fig. 4A,C,E,G). Similar effects were observed if explants were cultured in the presence of 10-50 µg/ml recombinant FGFR1-Fc or FGFR2-Fc protein (data not shown). Whole-mount TUNEL analysis revealed no increase in the amount of cell death in SU5402-treated explants excluding impaired tissue survival in the presence of the inhibitor as the reason for the failure of feather bud development (data not shown).
In eb explant halves cultured in the presence of Su5402, expression of all four markers was detectable in previously formed buds (Fig. 4J,N, and data not shown), but no additional rows of buds had formed laterally compared to the reference halves fixed immediately after dissection (Fig. 4I,M, and data not shown). In contrast, additional rows of buds not present at the time of dissection had formed in explant halves cultured in the presence of DMSO (Fig. 4K,L). Importantly, even buds at the earliest stage of bud development, evident in the reference half by weak localized BMP2 expression (Fig. 4M, arrows), had continued to develop in the presence of Su5402 as indicated by increased Bmp2 expression (Fig. 4N, arrows). Western blot analysis confirmed that the concentration of Su5402 used in these experiments was sufficient to block ERK phosphorylation in eb explants, arguing against an incomplete block of FGF signaling as the reason for the maintenance of established feather placodes (Fig. S2, http://dev.biologists.org/supplemental/). Together these results show that FGF signaling is required for feather placode development prior to the initiation of Bmp2 expression, the earliest available placode marker, but is not required to maintain developing placodes during the culture period once Bmp2 expression has been established.
Fgf3 and Fgf10 are expressed in the dermis of developing feather buds and Fgf10 expression in the future first row of buds precedes placode formation
We next searched for members of the FGF family that could function as the
placode-inducing FGF signal identified by the previous experiments. Since
tissue recombination experiments have shown that feather placode development
is initiated by signals from the underlying dermis, we focused on members of
the FGF family that function as mesenchyme to ectoderm signals in other
developmental settings (Bei and Maas,
1998; Min et al.,
1998
; Sekine et al.,
1999
). By RT-PCR, we detected Fgf3 and Fgf10
transcripts in HH29 chick back skin (data not shown). Fgf3 is
expressed in the condensed mesenchyme underlying the ectodermal placodes of
already formed buds at HH29 (Fig.
5B,I). No Fgf3 expression, however, was detectable at
HH28, prior to the formation of the first feather buds
(Fig. 5A). In contrast,
Fgf10 was already expressed in the dermis at HH28. Expression was
detected in a continuous stripe underlying the regions where the future first
rows of buds will develop (Fig.
5D,G, arrowheads) and was subsequently up-regulated in the
mesenchyme of nascent feather buds (Fig.
5E,H). In subsequent rows, no continuous stripe of expression was
detected. Instead Fgf10 was expressed in initially very weak and
slightly diffuse spots (Fig. 5F
unaffected area, and data not shown). Comparison of Fgf10 expression
in the dermis with Bmp2 expression in the epidermis on adjacent
sections suggested that the expression of both starts at about the same time
(data not shown).
|
FGF10 is required for feather placode formation
In order to directly test whether FGF10 is required for the initiation of
feather bud development, we analyzed feather bud development in ppf explants
cultured in the presence of FGF10-blocking antibodies. These antibodies have
previously been used to inactivate FGF10 in tooth explants
(Harada et al., 2002). They
recognize recombinant FGF10 and a band of similar size in protein extracts
from chicken skin, but show no cross-reactivity to FGF2, 3, 4, 5, 7, 8 or 18
in western blot experiments (Fig. S3,
http://dev.biologists.org/supplemental/).
In ppf explants cultured for 16 hours in the presence of these antibodies,
Shh and Bmp2 expression was undetectable and no thickened
feather placodes or dermal condensations formed
(Fig. 6B,E). Previously formed
Bmp2-positive buds were maintained in the presence of the anti-FGF10
antibodies, but no additional buds developed
(Fig. 6F). In contrast,
multiple Shh- and Bmp2-expressing feather buds formed in
explants cultured in the presence of control IgG
(Fig. 6A,D). Feather buds also
developed in explants cultured in medium containing both the FGF10-blocking
antibodies and recombinant FGF10 protein
(Fig. 6C), but not in specimens
cultured in the presence of the antibodies and FGF3, FGF4 or FGF7 protein
(data not shown). Thus only FGF10 is able to titrate and neutralize the
antibodies. Under the same conditions FGF3-blocking antibodies
(Harada et al., 2002
) had no
effect on bud development in explants (data not shown). Together these results
provide evidence that FGF10 is required for the initiation of feather bud
development.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FGF2 and FGF4 can promote feather bud formation and can induce feathers in
skin explants (Song et al.,
1996; Widelitz et al.,
1996
). Both are expressed in the epidermal placodes and have been
suggested to serve as locally acting promoters of placode development
(Jung et al., 1998
).
Consistent with this suggested function our results show that FGF signaling is
required for feather placode formation. Retroviral over-expression of soluble
versions of FGFR1 or FGFR2 had no effect on the expression of early feather
tract markers suggesting that FGF signaling is not required for feather tract
specification. However, markers such as Fgfr1, Wnt6 or
ß-catenin subsequently failed to undergo the transition from
broad diffuse expression in the early tract to localized expression in the
forming buds. Moreover, Bmp2, the earliest known marker of placode
development (Jung et al.,
1998
; Noramly and Morgan,
1998
), and Shh, a slightly later marker of feather
placodes (Chuong et al., 2000
;
Morgan et al., 1998
), were not
detectable at all in the presence of FGFR1-Fc or FGFR2-Fc. The same effects
were also observed if skin explants were dissected shortly before the onset of
placode development and cultured in the presence of inhibitors of FGF
signaling. Together these results point towards a requirement for FGF
signaling just prior to or during the initiation of feather placode
development.
In the prospective first row of buds we have detected FGF10 expression in the dermis prior to the localized expression of any of the known placode markers in the overlying epidermis. Such a stripe, however, was not detectable in subsequent rows. There, we first detected Fgf10 expression in weak and slightly diffuse spots in the dermis at roughly the same time when Bmp2 expression became detectable in the overlying epidermis. We have furthermore shown that blocking antibodies recognizing FGF10 mimic the effects of soluble FGF receptors and block the initiation of placode development in skin explants. Together these observations make FGF10 a candidate for the FGF signal necessary for feather placode development. Whether FGF10 serves to initiate placode development or whether it collaborates with other signals and functions to promote and stabilize placode development after an initial induction by a different signal is still unclear.
Secreted versions of FGFR2-IIIb or FGFR1-IIIc are expected to
preferentially bind to and sequester FGF10, FGF7 and FGF3, or FGF2, FGF4 and
related FGFs, respectively (reviewed by
Ornitz and Itoh, 2001). Both
types of receptors also bind other members of the FGF family, but with lower
affinity (Ornitz et al., 1996
;
Orr-Urtreger et al., 1993
)
(M.M. and A.N., unpublished). In our experiments, retroviral over-expression
of either of the two receptor isoforms blocked feather development. Except for
a more widespread effect of FGFR2-IIIb-Fc, the resulting phenotypes were
highly similar, most likely because both receptor isoforms block feather
development by sequestering FGF10 and/or a related FGF. The more widespread
effect of FGFR2-IIIb-Fc would then be explained by the higher affinity of this
receptor isoform to members of this subfamily of FGFs. Alternatively, FGFR1-Fc
could block feather development by sequestering FGF2 and FGF4 produced in
feather placodes, a block of feather bud development after initiation of
placode development, followed by bud regression. However, this appears less
likely since culture of skin explants in the presence of FGF signaling
inhibitors after the onset of placode development did not result in bud
regression.
In skin explants, FGF10 was sufficient to up-regulate ectodermal expression
of Wnt6, Bmp2 and follistatin, molecules that have been
implicated as positive and negative regulators of feather development. Several
previous studies have shown that canonical WNT signaling functions as an
essential, positively acting signal during early epithelial appendage
development (Andl et al., 2002;
Huelsken et al., 2001
;
Kratochwil et al., 1996
;
Noramly et al., 1999
;
Widelitz et al., 2000
).
However, it is so far not clear whether for placode development WNT signaling
is required in the ectoderm or the dermis. Furthermore, it is unclear which
member of the Wnt family activates the pathway in either of the tissues.
Keratin-14 (K14)-promoter driven over-expression of Dkk1, a soluble Wnt
antagonist, in the epidermis of transgenic mice lead to a complete failure of
placode formation and the subsequent absence of all hairs, a phenotype similar
to the one we observed in the chick after over-expression of FGFR1-Fc or
FGFR2-Fc (Andl et al., 2002
).
Given these similarities, it is tempting to speculate that Wnt/ß-catenin
and FGF signaling could cooperate in a common pathway in initiating feather
placode development. Such a situation has recently been shown for the
initiation of limb development (Barrow et
al., 2003
; Kawakami et al.,
2001
). There, Wnt/ß-catenin in the early limb mesenchyme
appears to be required to initiate Fgf10 expression in the same
tissue. Mesenchymally derived FGF10 then regulates expression of
Wnt3a in the overlying ectodermal cells, which is required to
activate expression of Fgf8 in the same cells. In analogy, Wnt
signaling in the dermis could act upstream of dermal Fgf10 expression
during feather development. FGF10-mediated up-regulation of expression of
Wnt6, and maybe other members of the Wnt family, in the ectoderm
could then activate the pathway in the epidermis and promote placode
formation.
FGF10 also induces expression of Bmp2, a negative regulator of
feather development. According to the current model, BMP2 controls bud spacing
and bud size through lateral inhibition
(Jiang et al., 1999;
Jung et al., 1998
;
Noramly and Morgan, 1998
). The
buds themselves, expressing high levels of Bmp2, are thought to be
protected from the inhibitory effect of BMP signaling, e.g. through the
production of BMP antagonists. We have found that FGF10 also upregulates
expression of the secreted BMP antagonist Follistatin. A uniform stripe of
follistatin expression normally precedes feather placode formation
and has been suggested to label a region competent to form feather buds
(Patel et al., 1999
). Feather
placodes therefore always develop within a region of high follistatin
expression in which BMP activity might be low. Our finding that FGF10 protein
can induce Fgf10 expression, but only in the context of low BMP
activity, achieved through application of beads soaked in a mixture of FGF10
and Noggin-Fc suggests that the level of BMP signaling is critical in
determining the response to FGF10. The high level of Bmp2 expression
induced in response to beads soaked in FGF10 protein alone could result in
increased BMP2 signaling, in spite of the simultaneous induction of
follistatin, and in inhibition of further bud development. A recent
study by Tao et al. (Tao et al.,
2002
) supports this idea. These authors used a retroviral
construct to broadly over-express FGF10 in chicken skin and found a complete
failure of feather development, associated with widespread induction of
Bmp2, and a loss of all periodic gene expression. Thus, in spite of
its requirement for placode development and its dermal expression, from
current knowledge, FGF10 lacks important properties that would be expected of
the primary inductive signal from the dermis that initiates feather
development.
Tissue recombination between mouse and chick tissues have shown that the
inductive signals exchanged between dermis and epidermis that initiate
cutaneous appendage formation are conserved between birds and mammals. We
would therefore predict that FGF signaling also functions in the initiation of
hair follicle development. Indeed, Celli et al.
(Celli et al., 1998) have
described a complete absence of hair follicles in transgenic mice
over-expressing FGFR2-IIIb-Fc under the control of a metallothionein promoter
in 50% of the founder animals but did not characterize these defects further.
Nevertheless, this study supports the idea that ligands capable of binding to
FGFR2-IIIb are also involved in the initiation of hair follicle formation in
the mouse. Knockouts of individual members of the FGF family and individual
FGFR isoforms have so far failed to clearly reveal this requirement, most
probably because of redundancy (Arman et
al., 1999
; De Moerlooze et
al., 2000
; Guo et al.,
1996
; Hebert et al.,
1994
; Min et al.,
1998
; Petiot et al.,
2003
; Sekine et al.,
1999
; Suzuki et al.,
2000
). Ultimately, the generation of mice deficient in several
members of the FGF or FGF receptor family will therefore be necessary to
determine which members of the FGF family collaborate in the initiation of
hair follicle development.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
* Present address: Developmental Biology Unit, Institute of Biology I,
University of Freiburg, Hauptstrasse 1, D-79104 Freiburg, Germany
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andl, T., Reddy, S. T., Gaddapara, T. and Millar, S. E. (2002). WNT signals are required for the initiation of hair follicle development. Dev. Cell 2, 643-653.[Medline]
Arman, E., Haffner-Krausz, R., Gorivodsky, M. and Lonai, P.
(1999). Fgfr2 is required for limb outgrowth and lung-branching
morphogenesis. Proc. Natl. Acad. Sci. USA
96,11895
-11899.
Barrow, J. R., Thomas, K. R., Boussadia-Zahui, O., Moore, R.,
Kemler, R., Capecchi, M. R. and McMahon, A. P. (2003).
Ectodermal Wnt3/beta-catenin signaling is required for the establishment and
maintenance of the apical ectodermal ridge. Genes Dev.
17,394
-409.
Bei, M. and Maas, R. (1998). FGFs and BMP4
induce both Msx1-independent and Msx1-dependent signaling pathways in early
tooth development. Development
125,4325
-4333.
Celli, G., LaRochelle, W. J., Mackem, S., Sharp, R. and Merlino,
G. (1998). Soluble dominant-negative receptor uncovers
essential roles for fibroblast growth factors in multi-organ induction and
patterning. EMBO J. 17,1642
-1655.
Chodankar, R., Chang, C. H., Yue, Z., Jiang, T. X., Suksaweang,
S., Burrus, L., Chuong, C. M. and Widelitz, R. (2003).
Shift of localized growth zones contributes to skin appendage morphogenesis:
role of the Wnt/beta-catenin pathway. J. Invest.
Dermatol. 120,20
-26.
Chuong, C. M., Patel, N., Lin, J., Jung, H. S. and Widelitz, R. B. (2000). Sonic hedgehog signaling pathway in vertebrate epithelial appendage morphogenesis: perspectives in development and evolution. Cell. Mol. Life Sci. 57,1672 -1681.[Medline]
Compagni, A., Wilgenbus, P., Impagnatiello, M. A., Cotten, M.
and Christofori, G. (2000). Fibroblast growth factors
are required for efficient tumor angiogenesis. Cancer
Res. 60,7163
-7169.
Crowe, R., Henrique, D., Ish-Horowicz, D. and Niswander, L.
(1998). A new role for Notch and Delta in cell fate decisions:
patterning the feather array. Development
125,767
-775.
De Moerlooze, L., Spencer-Dene, B., Revest, J., Hajihosseini,
M., Rosewell, I. and Dickson, C. (2000). An important
role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in
mesenchymal-epithelial signalling during mouse organogenesis.
Development 127,483
-492.
Fekete, D. M. and Cepko, C. L. (1993). Replication-competent retroviral vectors encoding alkaline phosphatase reveal spatial restriction of viral gene expression/transduction in the chick embryo. Mol. Cell. Biol. 13,2604 -2613.[Abstract]
Francis, P. H., Richardson, M. K., Brickell, P. M. and Tickle,
C. (1994). Bone morphogenetic proteins and a signalling
pathway that controls patterning in the developing chick limb.
Development 120,209
-218.
Guo, L., Degenstein, L. and Fuchs, E. (1996). Keratinocyte growth factor is required for hair development but not for wound healing. Genes Dev. 10,165 -175.[Abstract]
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88,49 -92.
Harada, H., Toyono, T., Toyoshima, K. and Ohuchi, H. (2002). FGF10 maintains stem cell population during mouse incisor development. Connect Tissue Res 43,201 -204.[Medline]
Hartmann, C. and Tabin, C. J. (2000). Dual
roles of Wnt signaling during chondrogenesis in the chicken limb.
Development 127,3141
-3159.
Hebert, J. M., Rosenquist, T., Gotz, J. and Martin, G. R. (1994). FGF5 as a regulator of the hair growth cycle: evidence from targeted and spontaneous mutations. Cell 78,1017 -1025.[Medline]
Huelsken, J., Vogel, R., Erdmann, B., Cotsarelis, G. and Birchmeier, W. (2001). beta-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell 105,533 -545.[CrossRef][Medline]
Hughes, S. H., Greenhouse, J. J., Petropoulos, C. J. and Sutrave, P. (1987). Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors. J. Virol. 61,3004 -3012.[Medline]
Jiang, T. X., Jung, H. S., Widelitz, R. B. and Chuong, C. M.
(1999). Self-organization of periodic patterns by dissociated
feather mesenchymal cells and the regulation of size, number and spacing of
primordia. Development
126,4997
-5009.
Jung, H. S., Francis-West, P. H., Widelitz, R. B., Jiang, T. X., Ting-Berreth, S., Tickle, C., Wolpert, L. and Chuong, C. M. (1998). Local inhibitory action of BMPs and their relationships with activators in feather formation: implications for periodic patterning. Dev. Biol. 196,11 -23.[CrossRef][Medline]
Kawakami, Y., Capdevila, J., Buscher, D., Itoh, T., Rodriguez Esteban, C. and Izpisua Belmonte, J. C. (2001). WNT signals control FGF-dependent limb initiation and AER induction in the chick embryo. Cell 104,891 -900.[CrossRef][Medline]
Kratochwil, K., Dull, M., Farinas, I., Galceran, J. and Grosschedl, R. (1996). Lef1 expression is activated by BMP-4 and regulates inductive tissue interactions in tooth and hair development. Genes Dev. 10,1382 -1394.[Abstract]
Matise, M. P. and Joyner, A. L. (1997).
Expression patterns of developmental control genes in normal and Engrailed-1
mutant mouse spinal cord reveal early diversity in developing interneurons.
J. Neurosci. 17,7805
-7816.
Millar, S. E. (2002). Molecular mechanisms
regulating hair follicle development. J. Invest.
Dermatol. 118,216
-225.
Min, H., Danilenko, D. M., Scully, S. A., Bolon, B., Ring, B.
D., Tarpley, J. E., DeRose, M. and Simonet, W. S.
(1998). Fgf-10 is required for both limb and lung development and
exhibits striking functional similarity to Drosophila branchless.
Genes Dev. 12,3156
-3161.
Mohammadi, M., McMahon, G., Sun, L., Tang, C., Hirth, P., Yeh,
B. K., Hubbard, S. R. and Schlessinger, J. (1997).
Structures of the tyrosine kinase domain of fibroblast growth factor receptor
in complex with inhibitors. Science
276,955
-960.
Morgan, B. A. and Fekete, D. M. (1996). Manipulating gene expression with replication-competent retroviruses. Methods Cell Biol. 51,185 -218.[Medline]
Morgan, B. A., Orkin, R. W., Noramly, S. and Perez, A. (1998). Stage-specific effects of sonic hedgehog expression in the epidermis. Dev. Biol. 201, 1-12.[CrossRef][Medline]
Neubuser, A., Peters, H., Balling, R. and Martin, G. R. (1997). Antagonistic interactions between FGF and BMP signaling pathways: a mechanism for positioning the sites of tooth formation. Cell 90,247 -255.[Medline]
Noji, S., Koyama, E., Myokai, F., Nohno, T., Ohuchi, H., Nishikawa, K. and Taniguchi, S. (1993). Differential expression of three chick FGF receptor genes, FGFR1, FGFR2 and FGFR3, in limb and feather development. Prog. Clin. Biol. Res. 338B,645 -654.
Noramly, S., Freeman, A. and Morgan, B. A.
(1999). beta-catenin signaling can initiate feather bud
development. Development
126,3509
-3521.
Noramly, S. and Morgan, B. A. (1998). BMPs
mediate lateral inhibition at successive stages in feather tract development.
Development 125,3775
-3787.
Ohuchi, H., Nakagawa, T., Yamamoto, A., Araga, A., Ohata, T.,
Ishimaru, Y., Yoshioka, H., Kuwana, T., Nohno, T., Yamasaki, M. et
al. (1997). The mesenchymal factor, FGF10, initiates and
maintains the outgrowth of the chick limb bud through interaction with FGF8,
an apical ectodermal factor. Development
124,2235
-2244.
Ohuchi, H., Tao, H., Ohata, K., Itoh, N., Kato, S., Noji, S. and Ono, K. (2003). Fibroblast growth factor 10 is required for proper development of the mouse whiskers. Biochem. Biophys. Res. Commun. 302,562 -567.[CrossRef][Medline]
Ohyama, A., Saito, F., Ohuchi, H. and Noji, S. (2001). Differential expression of two BMP antagonists, gremlin and Follistatin, during development of the chick feather bud. Mech. Dev. 100,331 -333.[CrossRef][Medline]
Ornitz, D. M. and Itoh, N. (2001). Fibroblast growth factors. Genome Biol. 2, REVIEWS3005.
Ornitz, D. M., Xu, J., Colvin, J. S., McEwen, D. G., MacArthur,
C. A., Coulier, F., Gao, G. and Goldfarb, M. (1996).
Receptor specificity of the fibroblast growth factor family. J.
Biol. Chem. 271,15292
-15297.
Orr-Urtreger, A., Bedford, M., Burakova, T., Arman, E., Zimmer, Y., Yayon, A., Givol, D. and Lonai, P. (1993). Developmental localization of the splicing alternatives of fibroblast growth factor receptor-2 (FGFR2). Dev. Biol. 158,475 -486.[CrossRef][Medline]
Patel, K., Makarenkova, H. and Jung, H. S. (1999). The role of long range, local and direct signalling molecules during chick feather bud development involving the BMPs, follistatin and the Eph receptor tyrosine kinase Eph-A4. Mech. Dev. 86,51 -62.[CrossRef][Medline]
Patstone, G., Pasquale, E. B. and Maher, P. A. (1993). Different members of the fibroblast growth factor receptor family are specific to distinct cell types in the developing chicken embryo. Dev. Biol. 155,107 -123.[CrossRef][Medline]
Peters, K., Werner, S., Liao, X., Wert, S., Whitsett, J. and Williams, L. (1994). Targeted expression of a dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung. EMBO J. 13,3296 -3301.[Abstract]
Petiot, A., Conti, F., Grose, R., Revest, J., Hodivala-Dilke, K.
and Dickson, C. (2003). A crucial role for Fgfr2-IIIb
signalling in epidermal development and hair follicle patterning.
Development 130,5493
-5501.
Riddle, R. D., Johnson, R. L., Laufer, E. and Tabin, C. (1993). Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75,1401 -1416.[Medline]
Scaal, M., Fuchtbauer, E. M. and Brand-Saberi, B. (2001). cDermo-1 expression indicates a role in avian skin development. Anat. Embryol. (Berl) 203, 1-7.[CrossRef][Medline]
Schubert, F., Mootoosamy, R., Walters, E., Graham, A., Tumiotto, L., Munsterberg, A., Lumsden, A. and Dietrich, S. (2002). Wnt6 marks sites of epithelial transformations in the chick embryo. Mech. Dev. 114, 143.[CrossRef][Medline]
Sekine, K., Ohuchi, H., Fujiwara, M., Yamasaki, M., Yoshizawa, T., Sato, T., Yagishita, N., Matsui, D., Koga, Y., Itoh, N. et al. (1999). Fgf10 is essential for limb and lung formation. Nat. Genet. 21,138 -141.[CrossRef][Medline]
Sengel, P. (1976). Morphogenesis of the Skin. Cambridge, UK: Cambridge University Press.
Sengel, P. (1990). Pattern formation in skin development. Int. J. Dev. Biol. 34, 33-50.[Medline]
Song, H., Wang, Y. and Goetinck, P. F. (1996).
Fibroblast growth factor 2 can replace ectodermal signaling for feather
development. Proc. Natl. Acad. Sci. USA
93,10246
-10249.
Suzuki, K., Yamanishi, K., Mori, O., Kamikawa, M., Andersen, B., Kato, S., Toyoda, T. and Yamada, G. (2000). Defective terminal differentiation and hypoplasia of the epidermis in mice lacking the Fgf10 gene. FEBS Lett. 481, 53-56.[CrossRef][Medline]
Tao, H., Yoshimoto, Y., Yoshioka, H., Nohno, T., Noji, S. and Ohuchi, H. (2002). FGF10 is a mesenchymally derived stimulator for epidermal development in the chick embryonic skin. Mech. Dev. 116,39 -49.[CrossRef][Medline]
Vainio, S., Karavanova, I., Jowett, A. and Thesleff, I. (1993). Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 75,45 -58.[Medline]
Viallet, J. P., Prin, F., Olivera-Martinez, I., Hirsinger, E., Pourquie, O. and Dhouailly, D. (1998). Chick Delta-1 gene expression and the formation of the feather primordia. Mech. Dev. 72,159 -168.[CrossRef][Medline]
Werner, S., Weinberg, W., Liao, X., Peters, K. G., Blessing, M., Yuspa, S. H., Weiner, R. L. and Williams, L. T. (1993). Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation. EMBO J. 12,2635 -2643.[Abstract]
Widelitz, R. B., Jiang, T. X., Noveen, A., Chen, C. W. and Chuong, C. M. (1996). FGF induces new feather buds from developing avian skin. J. Invest. Dermatol. 107,797 -803.[Abstract]
Widelitz, R. B., Jiang, T. X., Chen, C. W., Stott, N. S. and
Chuong, C. M. (1999). Wnt-7a in feather morphogenesis:
involvement of anterior-posterior asymmetry and proximal-distal elongation
demonstrated with an in vitro reconstitution model.
Development 126,2577
-2587.
Widelitz, R. B., Jiang, T. X., Lu, J. and Chuong, C. M. (2000). beta-catenin in epithelial morphogenesis: conversion of part of avian foot scales into feather buds with a mutated beta-catenin. Dev. Biol. 219,98 -114.[CrossRef][Medline]
Wilkinson, D. G. and Nieto, M. A. (1993). Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Methods Enzymol. 225,361 -373.[Medline]