1 Center for Animal Resources and Development (CARD), Graduate School of Medical
and Pharmaceutical Sciences, Kumamoto University, Honjo 2-2-1, Kumamoto
860-0811, Japan
2 Molecular Genetics Laboratory, Division of Head and Neck Surgery, UCLA School
of Medicine, Los Angeles, CA 90095-7795, USA
3 Department of Cell and Molecular Biology, Tulane University, New Orleans, LA
70118, USA
4 Department of Genome Sciences, Graduate School of Medicine, Kobe University,
Kobe, Japan
5 Molecular Developmental Biology Group, Laboratory of Reproductive and
Developmental Toxicology, National Institute of Environmental Health Sciences,
NC 27709, USA
6 Mammalian Neurogenetics Group, Center for Childhood Communication, The
Children's Hospital of Philadelphia, PA 19104, USA
* Author for correspondence (e-mail: gen{at}kaiju.medic.kumamoto-u.ac.jp)
Accepted 29 August 2003
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SUMMARY |
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Bone morphogenetic protein genes (Bmp genes) and their antagonists were spatiotemporally expressed during GT development. Exogenously applied BMP increased apoptosis of GT and inhibited its outgrowth. It has been shown that the distal urethral epithelium (DUE), distal epithelia marked by the Fgf8 expression, may control the initial GT outgrowth. Exogenously applied BMP4 downregulated the expression of Fgf8 and Wnt5a, concomitant with increased apoptosis and decreased cell proliferation of the GT mesenchyme. Furthermore, noggin mutants and Bmpr1a conditional mutant mice displayed hypoplasia and hyperplasia of the external genitalia respectively. noggin mutant mice exhibited downregulation of Wnt5a and Fgf8 expression with decreased cell proliferation. Consistent with such findings, Wnt5a mutant mice displayed GT agenesis with decreased cell proliferation. By contrast, Bmpr1a mutant mice displayed decreased apoptosis and augmented Fgf8 expression in the DUE associated with GT hyperplasia. These results suggest that some of the Bmp genes could negatively affect proximodistally oriented outgrowth of GT with regulatory functions on cell proliferation and apoptosis.
The DUE region can be marked only until 14.0 dpc (days post coitum) in mouse development, while GT outgrowth continues thereafter. Possible signaling crosstalk among the whole distal GT regions were also investigated.
Key words: External genitalia, Genital tubercle, BMP, FGF8, Noggin, WNT5A, Apoptosis, Cell proliferation, Distal urethral epithelium
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Introduction |
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The GT differentiates into the penis in males and the clitoris in females
(Murakami and Mizuno, 1986).
Epithelialmesenchymal interactions play an essential role in regulating a wide
variety of developmental processes (Capel,
2000
; Hogan, 1999
;
Johnson and Tabin, 1997
;
Kurzrock et al., 1999b
;
Thesleff et al., 1991
;
Thesleff et al., 1995
;
Tickle and Eichele, 1994
).
Such signaling governs many aspects of organogenesis, from the initiation of
organ development to differentiation
(Dassule and McMahon, 1998
;
Tucker et al., 1999
). The
developing limb has long served as a model system for studying such mechanisms
(Duboule, 1993
;
Duboule, 1994
;
Tabin, 1991
;
Tabin, 1995
). The distal
signaling epithelia of the developing limb (apical ectodermal ridge; AER) is
essential for sustained outgrowth and the patterning of a limb through, e.g.
fibroblast growth factors (FGFs) (Laufer
et al., 1994
; Niswander et
al., 1993
; Niswander et al.,
1994
; Pizette and Niswander,
1999
; Sun et al.,
2002
; Zuniga et al.,
1999
).
It has been reported that bone morphogenetic proteins (BMPs) may regulate
the regression and function of the limb AER
(Pizette and Niswander, 1999).
In many cases, these interactions have been associated with the mesenchyme
expressing various Bmp genes. A number of epithelialmesenchymal interactions
have been shown to involve FGF and BMP signals, including development of the
limbs, teeth, feather buds and lung buds
(Martin, 1998
;
Thesleff et al., 1995
;
Tickle, 1999
;
Weaver et al., 2000
).
BMPs are released by a variety of source cells and influence neighboring
target cells (Hogan, 1996).
Expression of Bmp4 is observed in the developing limb, lung, kidney,
hair follicle and tooth bud, where inductive interactions occur between the
mesenchyme and adjacent epithelium (Bitgood
and McMahon, 1995
). The essential role of BMP signaling in
organogenesis and the coordinated action of their antagonists have been
demonstrated in many developmental processes. The distribution and activity of
BMPs is regulated by antagonists such as noggin (NOG), chordin (CHD) and
gremlin (CKTSF1B1 Mouse Genome Informatics)
(Khokha et al., 2003
;
Sasai and De Robertis, 1997
).
It has been suggested that the interaction between BMP and its antagonist is
essential for the development and differentiation of the limb appendage
(Brunet et al., 1998
;
Merino et al., 1999
;
Niswander, 2003
).
BMP signaling is mediated by specific receptors composed of
heterodimer/tetramers of two different transmembrane serine/threonine kinase
subunits (Liu et al., 1995),
type I and type II receptors. Upon binding of the ligand, the type II receptor
phosphorylates and activates the type I receptor
(Mishina, 2003
;
Wrana et al., 1992
). In the
mouse, a single type II subunit (BMPR2) has been identified
(Beppu et al., 1997
), while at
least two type 1 subunits [BMPR1A (Alk3) and BMPR1B (Alk6)] have been reported
(ten Dijke et al., 1994
).
Apoptosis is an important process for eliminating regressing tissue regions
and cells during embryonic development. Several Bmp genes have been reported
to be involved in the regulation of apoptosis in various tissues including
limbs (Furuta et al., 1997;
Ganan et al., 1996
;
Jernvall et al., 1998
;
Macias et al., 1997
;
Zou and Niswander, 1996
).
Apoptosis has been further implicated in interdigital cell death
(Merino et al., 1998
;
van der Hoeven et al., 1994
),
and cell death in the AER (Chen and Zhao,
1998
; Pizette and Niswander,
1999
) and tooth enamel knot
(Vaahtokari et al., 1996b
),
which underlie the regression of such transiently formed embryonic regions
(Vaahtokari et al.,
1996a
).
The external genital anlage develops from the posterior embryonic region as
a bud-anlage (Yamada et al.,
2003). The first morphological alteration indicative of GT
outgrowth occurs at 10.5 days post coitum (dpc) in the mouse and continues
throughout the perinatal period (Suzuki et
al., 2002
). Several growth factors, including FGF proteins, sonic
hedgehog (SHH) and WNT have been implicated in the control of external
genitalia development in mice (Haraguchi
et al., 2000
; Haraguchi et
al., 2001
; Perriton et al.,
2002
; Yamaguchi et al.,
1999
). FGF signaling plays a key regulatory component in
orchestrating growth and differentiation of the GT
(Haraguchi et al., 2000
;
Morgan et al., 2003
) and SHH
signaling is required during the initiation of GT outgrowth and subsequent
differentiation, particularly during urethra formation
(Haraguchi et al., 2001
;
Perriton et al., 2002
). The
distal signaling epithelia, the distal urethral epithelium (DUE), has a role
in controlling mesenchymal gene expression and the initial outgrowth of the GT
for early GT development (Haraguchi et
al., 2000
; Ogino et al.,
2001
). In addition to Fgf8 and Shh expression,
we noticed that several BMP signaling molecules were expressed in the DUE and
in the distal-ventral mesenchyme adjacent to the DUE.
The role of BMP signaling in the caudal-end of the developing urogenital
system has been only partially studied so far. Bmp4 expression in the
urogenital sinus has been shown to be important for prostate development
(Lamm et al., 2001). However,
the regulatory role of BMP systems in external genitalia development is
unknown. We report on the roles of BMP signaling in murine external genitalia
development by in vitro organ culture and by analyzing Nog, Wnt5a
mutants and Bmpr1a conditional mutant mice.
The DUE can be observed only until 14.0 dpc in mouse development, whereas GT outgrowth continues thereafter. Detailed study of gene expression and histological analyses revealed that the DUE is located adjacent to the outer-most epithelial layer aligned with the GT surface ectoderm. With the advent of gene expression analysis on Bmpr1a mutant mice, signaling crosstalks among such whole distal GT regions are discussed. These data are consistent with the hypothesis that the actions of `positive' and `negative' signaling involving DUE may be coordinated, depending on the output of BMP signals.
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Materials and methods |
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Mouse genital tubercle (GT) organ culture
Procedures for filter supported organ cultures and a rotating organ culture
system for murine GT were previously described
(Haraguchi et al., 2000).
After 1-3 days in the culture, the GTs were processed for histological
analysis. Recombinant human BMP4, BMP7 (R&D, CA) or NOG (R&D, CA)
proteins were used at a concentration of 1 or 10 µg/ml. Staurosporine
(Sigma) was used at 5-10 nM in the culture. It has been demonstrated that
exposure of mouse embryos to staurosporine at such concentrations (5 nM)
exerted no general toxicity in embryonic development
(Ward et al., 2000
). For
antibody inoculation, anti-SHH (5E1) antibody (DSHB, USA) or control Ig
class-matched antibody (anti-CD90 antibody, Pharmingen) were used at
concentrations of 1-5 µg/ml.
In situ hybridization for gene expression
Whole-mount in situ hybridization was performed by standard procedures with
probes for Bmp7 (kindly provided by M. Yoshida), Bmp4
(Jones et al., 1991),
Bmpr1a (Mishina et al.,
1995
), Bmpr2 (kindly provided by K. Miyazono),
Nog (McMahon et al.,
1998
), cyclin D1 (kindly provided by J. S. Charles),
Bambi (Grotewold et al.,
2001
), Wnt5a (kindly provided by S. Takada),
Fgf8 and Bmp2 (kindly provided by B. L. Hogan).
RT-PCR analysis
Total RNA was isolated by using ISOGEN (Nippon Gene). RT-PCR was performed
with 100 ng of total RNA using One-Step RT-PCR System (Invitrogen). The primer
sequences for EF1: were 5-CTGCTGAGATGGGAAAGGGCTC-3 and
5-ACAGGGACAGTGCCAATGCCTC-3, and the annealing temperature 61°C, as
described for Fgf8 (Ozawa et al.,
1997
) and cyclin D1 (Klein et
al., 2003
). The PCR cycle consisted of a cDNA synthesis step at
50°C for 30 minutes, a pre-denaturation step at 94°C for 2 minutes, a
denatureted step at 94°C for 15 seconds, an annealing step for 30 seconds,
and a extension step at 70°C for 1 minute by using a DNA thermal cycler
(Biometra Co).
Histological and scanning electron microscopy (SEM) analyses
Staining for expression and Phospho-Smad1 immunohistochemical analyses were
performed as described (Ahn et al.,
2001). Tissues were fixed in 4% paraformaldehyde (PFA),
dehydrated, embedded in paraffin wax and sectioned. Hematoxylin and Eosin
staining was performed by standard procedures
(Haraguchi et al., 2001
).
Mouse GTs were fixed in 4% PFA and dehydrated for SEM analysis
(Suzuki et al., 2000
).
lacZ
Preparation of BMP4 protein beads
Recombinant human BMP4 was used at a concentration of 100 µg/ml in
phosphate-buffered saline (PBS). Affigel Blue beads (BioRad) were washed with
PBS and subsequently soaked in each protein for 1 hour at 37°C. Control
beads were treated with PBS containing an equivalent amount of bovine serum
albumin (Sigma).
Analysis of cell proliferation and cell death in vitro
Proliferating cells were detected using rabbit anti-phospho-histone H3 (Ser
10) (Cell Signaling Technology). Cells included in equal areas of control and
treated GT were counted and average numbers were compared. TUNEL analysis for
the detection of apoptotic cells in the GT was performed using the in situ
apoptosis detection kit (TAKARA). Nile Blue (Sigma) was used to localize
apoptotic cells. Dissected GTs were stained with 0.01% Nile Blue in the
medium, then washed with PBS several times and photographed.
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Results |
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BMP signaling can be antagonized by the activity of several secreted
proteins including NOG (Brunet et al.,
1998; Hirsinger et al.,
1997
; McMahon et al.,
1998
; Merino et al.,
1998
; Zimmerman et al.,
1996
). NOG is known to bind and antagonize BMP2, BMP4 and BMP7. In
developing organs, Bmp gene- and Nog-expressing cells are often
detected in adjacent domains (Brunet et
al., 1998
; Reshef et al.,
1998
). Given the complexity of the expression patterns of several
Bmp genes during GT formation, we also examined the expression of Bmp
antagonist genes. Nog was expressed in the mesenchyme surrounding the
DUE (Fig. 1D, white arrows).
Its expression was also detected in the proximal mesenchyme of the ventral GT
(Fig. 1D, white arrowhead).
Recently, a transmembrane protein that can attenuate BMP signaling, termed
BAMBI (BMP and activin membrane-bound inhibitor) was identified in
Xenopus (Onichtchouk et al.,
1999). It has been shown that the spatiotemporal expression
pattern of Bambi closely matches with that of Bmp4 during
mouse embryonic development (Grotewold et
al., 2001
). Bambi was found to be prominently expressed
in the distal mesenchyme adjacent to the DUE at 12.5 dpc
(Fig. 1E). These dynamic
expression patterns of several Bmp genes and their antagonists has prompted us
to examine their roles during murine GT development.
BMP4 and BMP7 promote apoptosis while NOG inhibits apoptosis in
vitro
Apoptosis was normally observed in the distal epithelium and less
prominently in the distal mesenchyme of the GT at 11.5 dpc and it was confined
mainly to the distal mesenchyme between 12.5 dpc and 13.5 dpc (data not shown)
(Haraguchi et al., 2001). At
14.5 dpc, the number of apoptotic cells decreased in both the epithelium and
mesenchyme (data not shown). Of note is the fact that the spatiotemporal
pattern of apoptosis in both the distal mesenchyme and the DUE is correlated
with the pattern of Bmp4 and Bmp7 expression. This prompted
us to investigate the effect of several BMP(s) on apoptosis. BMP4 beads
implanted into the murine GT increased mesenchymal apoptosis
(Fig. 2C). It was also found
that GT outgrowth was inhibited on the side of the GT implanted with BMP4
beads, compared with the opposite side implanted with control BSA beads
(Fig. 2A; see also
Fig. 4). Inhibition of GT
outgrowth by BMP4 was restored by the addition of NOG and BMP4 beads induced
Nog expression in GT mesenchyme (K.S. and G.Y., unpublished).
|
|
Role of apoptosis in the regulation of GT outgrowth
It has been recently reported that reagents with augmenting activities for
apoptosis have revealed a role, in part, in limb development
(Sanz-Ezquerro and Tickle,
2000). To determine the role of apoptosis in GT development, we
used staurosporine (a protein kinase inhibitor known to induce apoptosis) to
achieve ectopic apoptosis (Jacobson et
al., 1997
). The number of apoptotic cells increased in the distal
epithelium and its adjacent mesenchyme following its treatment
(Fig. 3C-F), which was
accompanied by retarded GT outgrowth (Fig.
3A,B).
|
SHH signaling has previously been shown to be required for the initiation
of GT outgrowth (Haraguchi et al.,
2001). We next examined whether Fgf8 expression in GT
explants was affected by treatment with the anti-SHH antibody, 5E1. GT
explants treated with 5E1 showed a reduction in Fgf8 expression after
24 hours of culture (Fig.
3G,H). These results may suggest that SHH signaling is necessary
for Fgf8 expression, not only for the initiation of GT outgrowth as
shown previously, but also during the outgrowth phase. The treatment of 5E1
antibody retarded GT outgrowth later, at 12.5 dpc with an increase of
apoptosis (Fig. 3I,J). This
raises a possibility that regulation of apoptosis during GT formation through
various signaling outputs may constitute an important mechanism affecting
outgrowing phase of the development.
Exogenous BMP4 down-regulates the expression of Fgf8 and Wnt5a and
suppresses cell proliferation
Fgf8 is expressed in the DUE and has been implicated in the
regulation of initial GT outgrowth
(Haraguchi et al., 2000;
Morgan et al., 2003
;
Perriton et al., 2002
).
Recently, it has been shown that Wnt5a is expressed prominently in
the distal region of the GT mesenchyme
(Oishi et al., 2003
;
Yamaguchi et al., 1999
).
Subsequently, it was shown that Wnt5a mutant mice exhibit
proximodistally affected hypoplasia of the external genitalia and limbs
affecting various appendage mesenchymes.
Given the functions of Bmp genes in GT formation shown in this study, it is tempting to speculate that Bmp genes may function in concert with other growth factor systems. Thus, we analyzed the expression of several genes to determine the molecular basis for outgrowth inhibition. The BMP4-treated specimen displayed retarded GT outgrowth (Fig. 4A,B), with a reduction in Fgf8 and Wnt5a expression (Fig. 4C-F). Alteration of Fgf8 expression was observed 6.5 hours after the BMP4 treatment by RT-PCR analysis (Fig. 4J), suggesting that such alteration was elicited by direct or by indirect mechanisms including few cascades in between. By contrast, downregulation of the Wnt5a was not observed at 12 hours but detected after 24 hours of the BMP4 treatment (Fig. 4E,F; data not shown). To examine the role of BMPs in regulating cell proliferation during GT outgrowth, explants were incubated with BMP4 protein. The number of phospho-histone H3-immuno-positive cells decreased to 60% compared with control GT explants (Fig. 4I). To analyze further the effects of BMP4 on cell proliferation of GT, cyclin D1 expression was analyzed. CyclinD1 regulates the G1 phase of the cell cycles through the control of cyclin-dependent kinases. Reduction of cyclin D1 expression was observed 24 hours after the treatment (Fig. 4G,H) and its reduction was initially observed 12 hours after the BMP4 treatment (data not shown). These results imply that inhibition by BMP4 may be mediated either by the action of BMP(s) on cells per se and/or by the modulation of the expression of factors such as Fgf8 and Wnt5a (see Discussion).
Nog mutant mice display GT hypoplasia with decreased Wnt5a, Fgf8
expression and cell proliferation
Although current observations in vitro demonstrate that exogenously
altering BMP signaling could influence GT outgrowth, cell proliferation and
apoptosis, they do not necessarily address their roles in vivo.
Mice lacking Nog have previously been characterized with regard to
defects in somite, forebrain and skeletal formation
(Bachiller et al., 2000;
Brunet et al., 1998
;
McMahon et al., 1998
). To
investigate the possible role of BMP signaling in murine external genitalia
development, we analyzed the phenotype of Nog mutant mice external
genitalia. Nog mutant mice displayed GT hypoplasia and decreased
Wnt5a and Fgf8 expression
(Fig. 5A-F), which is
consistent with the hypothesis that BMP signaling may exert an inhibitory
effect on GT outgrowth. Some Nog mutant mice displayed milder
phenotypes, probably owing to the phenotypic variations by strain backgrounds
(less than 50%; data not shown). To gain insight into the cellular basis of GT
hypoplasia, cell proliferation and apoptosis in Nog mutant mice were
examined. Consistent with the reduced cell proliferation of the organ culture
system (Fig. 4I), Nog
mutant mice also displayed significant reduction in cell proliferation judged
by phospho-histone H3 staining assay (Fig.
5G;
60% compared with wild type).
|
External genital agenesis and reduced cell proliferation of Wnt5a
mutant mice
To further examine the plausible genetic cascade composed by BMP signaling
with Wnt5a, the external genitalia of Wnt5a mutant mice were
examined. Wnt5a mutant mice displayed prominent external genitalia
agenesis, around midembryogenesis (Fig.
6A,B) (Yamaguchi et al.,
1999). It was revealed that Wnt5a mutant mice external
genitalia displayed marked reduction of cell proliferation (
60%),
consistent with the observations for the cell proliferation of Nog
mutant mice (Fig. 6C).
|
|
We next examined whether the apoptosis of GT was affected by the conditional Bmpr1a mutation. A marked reduction of apoptosis in the distal GT mesenchyme in the mutants was observed compared with wild-type mice at 12.5 dpc (Fig. 7H,I). These results are consistent with the hypothesis that Bmp genes may regulate the outgrowth of the GT, at least in part, through the regulation of apoptosis and/or by the regulation of expression of growth-promoting genes.
Questions remain, however, as to how such ablation of BMP signaling affects Fgf8 expression in the DUE where Cre activity was not detected. Roles of possible signaling crosstalks within the whole distal GT epithelia are discussed in relation to the structure and expression status (see below; Fig. 8).
|
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Discussion |
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External genital anlage, genital tubercle (GT), is one of the organs
outgrowing from the posterior body trunk. GT initiates from the cloaca region
forming as a bud-anlage from 10.5 dpc and continues to differentiate
throughout the perinatal period (Suzuki et
al., 2002; Yamada et al.,
2003
). A coordinated mode of development of the caudal embryonic
region including the cloaca, tail gut and VER (ventral ectodermal ridge) has
been reported previously (Goldman et al.,
2000
; Miller and Briglin,
1996
). Regulated apoptosis has been described as underlying such
posterior-embryonic development, e.g. tail gut regression. Of note, the
emergence and presence of apoptosis during GT development was observed in
parallel with the onset and maintenance of the expression of Bmp genes. In
this study, series of gain-of-function studies by organ culture and
loss-of-function studies with several knockout mice indicated an importance of
the regulation of apoptosis and cell proliferation during GT formation.
Apoptosis in the distal region of GT was markedly reduced in Bmpr1a
conditional mutants or by NOG treatment in vitro. By contrast, apoptosis was
increased mainly in the distal GT mesenchyme by BMP, staurosporine and
anti-SHH antibody treatment suggesting an importance of apoptotic regulation
depending on several signaling outputs.
The activity of BMP has also been associated with the developmentally
regulated onset of apoptosis (Coucouvanis
and Martin, 1999; Furuta et
al., 1997
). Bmp genes are responsible for the regulated outgrowth
of the limb (Macias et al.,
1997
; Merino et al.,
1999
). In the case of urogenital development, Bmp4 is
normally expressed in the mesenchyme adjacent to the cloaca and induced
ectopically in the affected cloaca-like region with augmented apoptosis in
Shh mutant embryos, which show GT agenesis
(Haraguchi et al., 2001
).
Taken together, the output of BMP signaling may affect GT outgrowth, in part,
through a mechanism including apoptosis.
In addition to the regulation of apoptosis, the clear reduction of GT cell
proliferation, shown by organ culture studies or by Nog and
Wnt5a mutant mice analyses, suggested a plausible regulation of cell
proliferation by the BMP system. The role of Wnt5a in regulating cell
proliferation as a possible downstream gene of Bmp4 is suggested. In
limb morphogenesis, Wnt5a is expressed in the distal limb mesenchyme
close to AER. Wnt5a mutant mice showed decreased cell proliferation
in such regions (Yamaguchi et al.,
1999). But they showed increased cell proliferation of lungs
suggesting tissue-dependent variability in the role of WNT5A signaling. We
found a significant decrease of cell proliferation in Wnt5a mutant
GTs associated with GT agenesis.
The current data might imply plausible differences in between cell survival
factors and growth stimulatory factors (mitotic factors) because increased
apoptotic signals were not detected in the hypoplasic Wnt5a mutant
GTs without alteration of Fgf8 expression (data not shown).
Thus,Wnt5a might work as a cell mitotic (growth stimulatory) factor
rather than as a cell survival factor. It may not be primarily responsible as
a `close' downstream effector of BMP signaling because its expression was not
altered after 12 hours of BMP4 treatment. Bmpr1a mutant mice
hyperplasic GTs did not show alteration of cell proliferation nor alteration
of Wnt5a expression (data not shown). However they showed clear
upregulation of Fgf8 expression and loss of apoptosis. It has been
also suggested that FGF8 might be one of the survival factors because its
downregulation induced augmented apoptosis for limb formation
(Sun et al., 2002). BMP4
treatment in vitro altered both Fgf8 and Wnt5a expression
leading to augmented apoptosis and decreased cell proliferation (Figs
2,
4). Reduction of cell
proliferation in the Nog mutant mice with loss of Fgf8 and
Wnt5a expression may also suggest a possibility of alteration of both
factor systems. The two activities described above, however, can not be
strictly distinguished as mutually distinct pathways and further analysis on
the relationships for both processes, will be required.
Possible crosstalk among the genes for growth factors
In the mouse, Bmp4 is frequently expressed adjacent to
Fgf-expressing regions and is involved in the regulation of cell proliferation
and differentiation (Hogan,
1996). Several Bmp genes are expressed in the mouse dorsal
forebrain and facial primordia (Barlow and
Francis-West, 1997
; Furuta et
al., 1997
). Ectopic application of BMP4 reduces the expression of
both Shh and Fgf8
(Ohkubo et al., 2002
) and
represses anterior neural gene expression promoting apoptosis in mouse
forebrain explants (Graham et al.,
1994
). Bmp4 and Fgf10 are often expressed in
adjacent domains during organogenesis
(Weaver et al., 2000
). During
limb morphogenesis, BMP-FGF crosstalk has been suggested as functioning
during, for example, the apical ectodermal ridge (AER) formation or
differentiation of the inter-digit necrotic zone. Inactivation of BMP
signaling results in the loss of Fgf8 expression in the AER of the
limb (Ahn et al., 2001
;
Pizette et al., 2001
). In
addition, it has been reported that syndactyly of heterozygous Fused toes
(Ft) mice (van der Hoeven et al.,
1994
), correlates with an imbalance in Bmp4 and
Fgf8 expression (Heymer and
Ruther, 1999
). Apart from limbs, developmental budding processes
are often influenced by the interaction of positive growth regulators (e.g.,
the FGF family or SHH) and negative growth regulators (such as BMP2, BMP4 and
BMP7) (Hogan, 1999
;
Jung et al., 1998
). The BMP
and FGF signaling pathways are known to interact antagonistically in many
developmental contexts, including branchial arch development
(Neubuser et al., 1997
;
St Amand et al., 2000
).
BMP-FGF crosstalk may also function during GT development. Downregulation
of the Fgf8 expression after 6.5 hours of the BMP4 treatment
suggested a close, but not necessarily direct, relationship between
Bmp4 and Fgf8 by this study. Later, Bmp4 and
Fgf10 are both expressed during bilateral mesenchymal differentiation
adjacent to the midline urethral plate epithelium during urethra formation
(Haraguchi et al., 2001).
During GT development, the expression pattern of Bmp7 in the DUE
overlapped, at least in part, with that of Fgf8
(Morgan et al., 2003
).
Combinatorial BMP-FGF crosstalk in the whole distal GT region, such as
BMP7-FGF8, BMP4-FGF8 or with BMP antagonists remains to be tested.
In Drosophila, Decapentaplegic (DPP) is a downstream target gene
of HH signaling. There is also evidence that BMP4 may be a downstream target
of SHH (Ingham and McMahon,
2001; McMahon et al.,
2003
) or located upstream of SHH by regulating its expression in
the mouse dental epithelium and limb bud
(Ahn et al., 2001
;
Zhang et al., 2000
). It has
been shown that SHH possess some outgrowth-promoting activities by GT organ
cultures, because blocking SHH signaling induced the GT apoptosis and
downregulated the Fgf8 expression of DUE (this study)
(Haraguchi et al., 2000
;
Haraguchi et al., 2001
).
Further studies are necessary for BMP(s)-SHH interaction during GT
formation.
It has been reported that BMP-WNT signaling pathways may be interactive in
limb and lung morphogenesis (Barrow et al.,
2003; Li et al.,
2002
; Soshnikova et al.,
2003
) and have antagonistic functions in the specification of the
trunk neural crest (Jin et al.,
2001
). BMP4 treatment reduced Wnt5a expression in vitro
and Nog mutant mice showed decreased Wnt5a expression with
GT hypoplasia. The current study based on gain-or-loss-of-function assays
showed marked alterations of gene expression profiles of the DUE region,
suggesting its orchestrating functions in early GT development. Detailed
analysis of genes related to DUE regions of Bmpr1a mutant mice
indicated additional aspects of modulation of BMP signaling through whole
distal GT regions (see below).
DUE formation and BMP signaling during GT development: the DUE as
part of the signaling cascades
The AER, a transient specialized distal epithelium, is essential for
vertebrate embryonic limb outgrowth along the proximodistal (PD) axis. The
SHH/FGF feedback signaling loop that operates between the polarizing region
and the AER, may coordinate growth and patterning for the limb
(Haramis et al., 1995;
Niswander et al., 1994
;
Zuniga et al., 2002
). Members
of the Bmp and Fgf gene families have been suggested as regulating various
epithelial-mesenchymal interactions during limb development
(Martin, 1998
) including
opposite roles during limb outgrowth depending on the context of the PD
development (Ganan et al.,
1996
; Niswander and Martin,
1993
).
As for external genitalia formation, the DUE has roles in controlling
mesenchymal gene expression and initial GT outgrowth
(Haraguchi et al., 2000;
Haraguchi et al., 2001
). Our
current analysis of the DUE revealed dynamic and complex gene expression
including Fgf8 and Bmp4. Several BMP signaling molecules
were found to be dynamically expressed in and adjacent to the DUE. In addition
to the Fgf8 expressing DUE region, the outer-most epithelial layer
and aligned GT surface ectoderm regions were identified
(Fig. 8B; the yellow region and
black broken surface epithelial region, respectively). It was found that
Fgf8, Bmp4, Bmp7 and Shh were not detected in the above two
regions (Fig. 8B; data not
shown). This may represent an intriguing structural contrast with other
signaling epithelia, e.g. the AER, which is composed of distinct apical
ectodermal epithelia. Although the current analysis agrees with the hypothesis
of an essential function of the distal GT region orchestrating initial GT
outgrowth as a whole, this study also raised a question of the composition of
whole distal GT regions and the functions of each included region
(Fig. 8C,D). Down- or
upregulation of the Fgf8 expression observed in
gain-or-loss-of-function studies are consistent with the idea of the
importance of the DUE for early GT development. Alteration of the pSMAD1
expression, however, suggested that the primary effect of Bmpr1a
conditional mutation resides in the outer-most epithelial layer
(Fig. 8D, yellow region) and
aligned GT surface ectoderm regions (Fig.
8D; such influences shown by black doubleheaded arrows may be
mediated also by dorsal epithelial regions). The enlargement of the DUE
associated with upregulation of Fgf8 appears to reflect an indirect
consequence of altered BMP signaling and cellular alterations being relayed
from whole distal GT epithelia, thereby eliciting GT dysmorphogenesis
(Fig. 7, Fig. 8D). To what extent could
the GT hyperplasia phenotype of Bmpr1a mutants be derived from DUE
and/or from distal GT epithelia? Although current analyses showed a clear
alteration of marker gene expression in the DUE by both
gain-or-loss-of-function assays, this study does not yet answer this question.
Normal Fgf8 expression in the DUE remains until
14 dpc
(Haraguchi et al., 2000
),
while GT outgrowth continues further later. Bmpr1a mutant GT
hyperplasia was observed in newborn samples. This might reflect
DUE-less-dependent GT outgrowth for late-staged GTs. In this aspect previous
findings suggesting the importance of normal GT ectoderm for proper GT
development would be intriguing, albeit based on broad ectoderm-mesenchymal
recombination assays available at that time
(Murakami and Mizuno, 1986
).
Another recent related observation is that ectodermal overexpression of
Nog by a K14-transgene, results in the prominent GT
hyperplasia observed for similarly late-staged GT specimens (C.-M. Chuong,
personal communication). During other appendage development, it has been shown
that Bmp genes are expressed in the early ventral limb ectoderm and that BMP
signaling is required for dorsoventral (DV) patterning in limb buds
(Ahn et al., 2001
;
Pizette et al., 2001
). Both DV
patterning and growth of the limb require signals from the limb ectoderm to
the underlying mesenchyme (Chen and
Johnson, 1999
). Further studies on whole distal GT epithelial
regions related to the PD, DV aspect of molecular cascades, will be
required.
It remains to be investigated as to what extent outer-most epithelial layer
and DUE could be derived from surface ectoderm and/or endoderm origin,
respectively, because of the lack of detailed molecular and cellular analyses
available (Kurzrock et al.,
1999a). The DUE region has received some attention as a unique
epithelia related as the cloacal plate epithelium for early stages, and the
solid urethral plate for later stages, although its molecular nature has
remained completely elusive (Kurzrock et
al., 2000
; Penington and
Hutson, 2002
). This study also indicates, for the first time, the
possibility of complex signaling in the whole distal GT region.
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ACKNOWLEDGMENTS |
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