1 Shriners Hospital for Children, Portland, OR 97239, USA
2 Oregon Health and Sciences University, Department of Molecular and Medical
Genetics, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA
* Author for correspondence (e-mail: hss{at}shcc.org)
Accepted 8 April 2003
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SUMMARY |
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Key words: Hoxa13, Mouse, Genitourinary development, Hypospadia
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INTRODUCTION |
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Recently, new evidence suggests that androgens are not the sole regulators
of GT patterning. In particular, many genes whose proteins provide the
instructive signals necessary for limb development also play a predominant
role during GT formation, including members of the fibroblast growth factor
(Fgf8 and Fgf10), hedgehog (Shh) and Hox
(Hoxa13 and Hoxd13) gene families
(Haraguchi et al., 2000;
Haraguchi et al., 2001
;
Stadler et al., 2001
;
Zhao and Potter, 2001
;
Warot et al., 1997
;
Mortlock and Innis, 1997
;
Podalesk et al., 1997; Podalesk et al., 1999;
van der Hoeven et al., 1996a
;
van der Hoeven et al., 1996b
).
Finally, human androgen receptor mutations are generally rare in individuals
exhibiting some of the more common genitourinary (GU) malformations such as
hypospadia, a condition characterized by arrested growth, formation and
closure of the external genitalia
(Sutherland et al., 1996
;
Allera et al., 1995
). In males,
hypospadia can affect several different regions of the developing glans penis.
These malformations range from distal defects, impacting the placement and
formation of the urethral opening or meatus (coronal hypospadia), to more
proximal defects, affecting the growth and closure of the ventral urethra
(penile hypospadia), scrotum (scrotal hypospadia) and peritoneum (peritoneal
hypospadia) (Baskin, 2000
;
Silver and Russel, 1999
;
Sutherland et al., 1996
;
Allera et al., 1995
).
To date, little is known about the cellular and molecular mechanisms
underlying this malformation; however, the incidence of hypospadia appears to
be increasing dramatically, affecting as many as 1 in 125 live male births
each year (Baskin et al., 1998;
Baskin et al., 2001
;
Gallentine et al., 2001
;
Paulozzi et al., 1997
).
Recently, mutations in the transcription factor Hoxa13 were shown to
cause Hand-Foot-Genital Syndrome (HFGS), an autosomal dominant disorder that
profoundly affects the development of many genitourinary structures, including
the uterus, bladder, ureters and cervix, as well as causing hypospadia
(Stern et al., 1970
; Pozanski
et al., 1975; Giedion and Prader,
1976
; Mortlock and Innis,
1997
; Goodman et al.,
2000
).
Using gene targeting in mice, similar mutations that phenocopy HFGS
(Stadler et al., 2001;
Fromental-Ramain, 1996; Warot et al.,
1997
) have been generated. From these initial studies, a role for
Hoxa13 in patterning both limb and caudal GU structures has been
established. In particular, mice lacking Hoxa13 exhibited gross
malformations of the rectum, Müllerian ducts, ureters and bladder
(Warot et al., 1997
).
Recognizing both the increasing frequency of hypospadia in the human population, as well as its association with HFGS, we hypothesized that many of the cellular and molecular mechanisms underlying this malformation could be identified in Hoxa13-mutant mice. In this report, we present evidence that mice mutant for Hoxa13 phenocopy the hypospadia in HFGS, causing malformation of the distal glans and meatus, as well as closure defects of the ventral urethra. At the molecular level, we demonstrate that Hoxa13 function is essential for the normal expression of Fgf8 and Bmp7 in the developing urethral epithelium, as well as for the repression of noggin (Nog) expression in the mesenchyme flanking the urethral plate epithelium (UPE). Next, we demonstrate that supplementation with Fgf8 in the urethral epithelium is sufficient to restore proliferation in the developing GT. Furthermore, we show that Bmp7 provides an apoptotic signal to the UPE and flanking mesenchyme, as antibody blocking of Bmp7 is sufficient to reduce most programmed cell death (PCD) in these regions. Interestingly, the blocking of either Bmp4 or Bmp7 signaling during GT outgrowth recapitulated many of the defects exhibited by Hoxa13GFP homozygous mutant mice, confirming that perturbations in Bmp signaling contribute to the coronal and penile hypospadias exhibited by these mice. Finally, a novel role for Hoxa13 in mediating the vascularization of the glans penis was also identified during this study.
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MATERIALS AND METHODS |
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Fluorescent imaging
GFP, CY5 or Texas-Red fluorescence was assessed using a Bio-Rad MRC 1024
confocal imaging system fitted with a Leica DMRB microscope, using filter sets
provided by the manufacturer. Whole-mount images represent 150 to 200 µm
z-series stepped at 2 µm intervals, whereas cryosections were imaged in
single planes using the GFP-tagged nucleus as the primary focal plane. A
Kalman digital averaging filter was used to reduce random noise.
Immunohistochemistry
Primary antibodies used were Cytokeratin 14 (#RDIMCYTOK14abr, Research
Diagnostics), Cytokeratin 5 (#RDIMCYTOK5abr, Research Diagnostics),
Cytokeratin 8/18 (#RDI-Progp11, Research Diagnostics), anti-phosphohistone H3
(#06-570, Upstate Biotechnology), Pecam (CD31; #550274 BD, PharMingen).
Whole-mount analysis
E11.5-15.5 male embryos were fixed for 1 hour in 4%
paraformaldehyde/1xphosphate buffered saline (PBS) at 4°C. The fixed
tissues were rinsed four times (for 5 minutes each time) in 1xPBS, and
then blocked in PBS containing 1% Triton X-100 (PBX), 2% skimmed milk and 5%
donkey serum. Primary antibodies were incubated with the blocked tissues at
4°C overnight using previously determined concentrations. Embryos were
then rinsed in 1xPBX, 2% skimmed milk for 3 hours at room temperature,
followed by overnight incubation with either Texas-Red or CY5-conjugated
secondary antibodies (Jackson Immuno Research) in PBX, 2% skimmed milk, 5%
donkey serum at 4°C.
Cryosection analysis
Embryos were fixed as described above, followed by a 30 minute rinse in
1xPBS. Cryoprotection of the tissues was achieved using a sequential
series of 10-40% sucrose/1xPBS. The embryos were oriented in OCT
(Tissue-Tek) and frozen rapidly on dry ice. The embryos were sectioned to 15
µm using a Leitz Kryostat 1740, and mounted on Superfrost plus slides
(Fisher) for analysis. Sections were rinsed for 10 minutes three times in
1xPBS to remove the OCT. Next the sections were permeabilized and
blocked for 1 hour in PBX, 2% skimmed milk, 5% donkey serum. After blocking,
primary antibodies were incubated on the sections for either 1 hour at room
temperature or overnight at 4°C, using the same blocking solutions at
previously determined concentrations. The sections were then rinsed in
1xPBX, 2% skimmed milk for 2 hours. Species-specific secondary
antibodies conjugated with either Texas-Red or CY5 fluorochromes (Jackson
Immuno Research) were incubated with the sections overnight at 4°C.
RNA in situ hybridization
Antisense riboprobes specific for Bmp4, Bmp7, Msx1, gremlin
(Cktsf1b1Mouse Genome Informatics) and Fgf8 were
generated using plasmids kindly provided by B. Hogan (Bmp4), R.
Beddington (Bmp7), Y. Chen (Msx1), R. Harland (gremlin) and
A. Moon (Fgf8). Hoxa13, Shh, Bmpr1a, Bmpr1b, Bmpr2 and
Msx2 riboprobes were produced using PCR amplifications of
gene-specific exons. The amplifying primers were:
Amplified exons were cloned into a t-tailed vector containing RNA
polymerase T3 and T7 promoters. Embryo preparation, hybridization and analysis
was performed as described by Manley and Capecchi
(Manley and Capecchi,
1995).
TUNEL analysis of apoptosis
TUNEL analysis was performed as described by Stadler et al.
(Stadler et al., 2001).
Genital tubercles from E12.5 heterozygous- and homozygous-mutant embryos were
examined by confocal analysis of 200 µm z-series as described
above. TUNEL analysis on frozen sections followed the same approach as
whole-mount TUNEL assays using 20 µm sections through the genital
tubercle.
Organ culture with Fgf8b and DHT supplementation
Heparin acrylic beads (#H-5263, Sigma), containing either 0.1 mg/ml BSA
(control) or 0.1mg/ml Fgf8b (R and D Systems), were placed in the UPE of E11.5
male embryos. Tissue explants containing the bead-implanted genital tubercles
were placed on cellulose membranes supported by stainless steel mesh in 60 mm
organ culture dishes containing BGJb media (Sigma-Aldrich), supplemented with
50 U/ml penicillin, 25 µg/ml streptomycin and 0.1 mg/ml ascorbic acid, and
grown for 24 hours in a incubator at 37°C containing air supplemented with
5% CO2. The GT explants were examined for changes in cell
proliferation using the anti-phosphohistone-H3 antibody (Upstate
Biotechnology).
Extended growth response of the GT to Fgf8b was performed using BGJb media, supplemented with 0.1 mg/ml Fgf8b or BSA, in a BTC Engineering rotary culture apparatus. E11.5 GTs were dissected from Hoxa13GFP heterozygous and homozygous mutant embryos and grown for 72 hours. After culture, the developing genitalia were examined for changes in GT outgrowth and morphology, using Hoxa13GFP expression as a marker of the relevant tissues.
Dihydroxytestosterone (DHT; Sigma) was dissolved in 100% ethanol and diluted to 10 nM in BgJb media. GT explants were grown for 6 hours in the presence of 10 nM DHT or the equivalent carrier volume of ethanol. After culture, the explants were fixed in 4% paraformaldehyde and processed for in situ hybridization using the Shh or Hoxa13 riboprobes as described above.
Antibody blocking
E12.5 genital tubercles were dissected from Hoxa13GFP
heterozygous mutant embryos and grown for 72 hours in the BTC rotary culture
apparatus. Culture media consisted of BGJb media (Sigma-Aldrich) buffered with
10 mM HEPES and supplemented with 0.1 mg/ml ascorbic acid. The sealed culture
bottles were continuously supplied with humidified gas containing 40%
O2, 5% CO2 and 55% N2. Polyclonal antibodies
specific for Bmp4 (Bmp4) or Bmp7
(
Bmp7; R and D Systems) were added to the media at a
concentration of 4 µg/ml, whereas control cultures were treated with 2
µg/ml of whole-goat immunoglobulins (IgGs). After 36 hours, 50% of the
culture media was replaced with new aliquots of antibody or control IgGs.
After 72 hours, the genital tubercles were examined for changes in meatus
formation, as well as defects in urethral groove closure using
Hoxa13GFP expression as a marker. To assess changes in PCD
in response to antibody blocking, GT explants from E12.5
Hoxa13GFP-heterozygous embryos were grown, using the same
rotary culture conditions, for 24 hours in media containing 4 µg/ml
Bmp7 or control IgGs. After culture, the explants were fixed
in 4% paraformaldehyde and assessed for changes in programmed cell death by
TUNEL analysis of frozen sections as described.
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RESULTS |
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Hoxa13 mutants exhibit reduced programmed cell death in the
differentiating male GT
During GT development, programmed cell death (PCD) precedes the crucial
fusion events necessary for the growth and closure of the penile urethra
(Haraguchi et al., 2001;
van der Werff et al., 2000
).
To determine if changes in PCD also contribute to the hypospadiac phenotype,
apoptosis in the developing GT was measured using a TUNEL assay. By E11.5, PCD
was readily seen in the distal and proximal UPE of wild-type (not shown) and
heterozygous Hoxa13GFP-mutant embryos
(Fig. 2A). By contrast,
homozygous mutants consistently exhibited reduced PCD in the distal UPE,
whereas the proximal UPE showed no difference in PCD when compared with
controls (Fig. 2A,B).
Quantitation of PCD in the distal UPE of homozygous mutants revealed, on
average, a 90% (±8%) loss in TUNEL-positive cells when compared with
controls (Fig. 2A,B).
|
The expression of Bmp7 and Nog are affected in Hoxa13GFP
mutants
To address mechanisms underlying reduced PCD in the mutant GT, we first
determined the localization and timing of pro-apoptotic signals in this
region. In particular, the expression of Bmp4 and Bmp7 was
examined, as these factors regulate many of the apoptotic events necessary for
normal limb (Macias et al.,
1997), craniofacial (Graham et
al., 1996
) and inner ear
(Fekete et. al., 1997
)
development. Although no differences in Bmp4 expression could be
discerned between homozygous-mutant and control embryos (data not shown),
notable changes in Bmp7 expression were seen in the UPE and lateral
shelf mesenchyme (Fig. 3). At
E12.5, wild-type embryos exhibited high levels of Bmp7 expression
along the entire axis of the UPE, as well as in more proximal lateral shelf
mesenchyme (Fig. 3A). By
contrast, mutant embryos express Bmp7 only in distal UPE, with little
expression seen in the lateral shelf mesenchyme
(Fig. 3B). Mutant embryos also
exhibit a thickened layer of epithelium
(Fig. 3B) that physically
separates the growing glans from the more ventral UPE.
|
We next examined whether homozygous-mutant embryos exhibited any changes in the expression of Bmp antagonists or Bmp-target genes that would augment reduced Bmp7 expression in the GT and contribute to the hypospadiac phenotype. In E11.5 wild-type embryos, Nog expression was strongest in the lateral shelf mesenchyme, with no expression in the UPE or mesenchyme adjacent to the UPE (Fig. 3E). By contrast, homozygous mutants exhibited ectopic Nog expression in the mesenchyme immediately flanking the proximal UPE (Fig. 3F). By E12.5, Nog expression was most noticeable in the lateral shelf mesenchyme and mesenchyme flanking the distal UPE (Fig. 3G). In homozygous mutants, ectopic Nog expression was most visible in the mesenchyme adjacent to the medial and proximal UPE (Fig. 3H), a region that overlaps with the normal expression pattern of Bmp4 (Fig. 3H; inset). A second Bmp antagonist, gremlin, exhibited no changes in GT expression between mutant and wild-type embryos (data not shown).
Next we examined whether reduced Bmp7 expression as well as ectopic Nog expression in the GT were sufficient to affect the expression of the Bmp-target genes Msx1 and Msx2. In E11.5 wild-type embryos, Msx1 expression was highest in the mesenchyme immediately adjacent to the proximal UPE (Fig. 3I). In homozygous mutants, Msx1 expression was greatly reduced in the mesenchymal tissues flanking the proximal UPE (Fig. 3J). By E12.5, Msx1 expression is restricted to the distal glans region (Fig. 3K), which is smaller in the mutant male embryo (Fig. 3L).
Interestingly, Msx2 expression is seen throughout the UPE, as well as in the mesenchyme flanking the distal UPE of E11.5 wild-type embryos (Fig. 3M). In homozygous mutants, Msx2 expression is absent in the proximal UPE in the same region as Bmp7 expression is reduced (Fig. 3B) and adjacent to the site of ectopic Nog expression (Fig. 3H). In the rostral GT, Msx2 expression appears normal, which is consistent with the levels of Bmp7 expression in this region (Fig. 3N). By E12.5, Msx2 expression is highest in the developing glans and throughout the UPE (Fig. 3O), whereas in homozygous mutants, no Msx2 expression could be seen in the more proximal UPE (Fig. 3P). The lack of Msx2 expression in the proximal UPE is consistent with the loss of Bmp7 expression in the proximal UPE of Hoxa13GFP-homozygous mutants (Fig. 3B,D).
Bmp-receptor expression is altered in Hoxa13GFP-mutant
embryos
We next examined the expression of Bmp-receptors Bmpr1a, Bmpr1b,
and Bmpr2 in GT of Hoxa13GFP embryos. At E12.5,
all three receptors are uniformly expressed at low levels throughout the
genital shelf mesenchyme (GSM) and UPE of wild-type male embryos
(Fig. 4A,C,E). However, in
Hoxa13GFP-homozygous mutants, elevated levels of
Bmpr1b expression was consistently seen in the condensing rectal
mesenchyme (compare Fig. 4C and
D). At E 13.5, Bmp-receptor expression was confined to the
developing glans, UPE and preputial gland condensations in wild type and
homozygous mutants (Fig. 4G-L). Interestingly, in homozygous mutants, Bmpr1b expression was reduced
in the mid-proximal UPE (Fig.
4; compare I and J), whereas Bmpr2 expression appeared
elevated in the distal UPE when compared with controls
(Fig. 4K,L).
|
|
GT proliferation defects result from reduced Fgf8 signaling and can
be rescued in vitro by Fgf8 supplementation
As Fgf8 produced by the UPE directs many of the proliferative events during
GT development (Haraguchi et al.,
2000), we examined whether loss of Hoxa13 function
affects Fgf8 expression and signaling in this region. In the GT,
Fgf8 expression was highest at E11.5 in both wild type and homozygous
mutants (Fig. 6A,B). Section
analysis of Fgf8 expression in the UPE revealed marked differences in the
localization of Fgf8 transcripts between wild-type and homozygous mutant
embryos. Specifically, wild-type embryos express Fgf8 throughout the UPE,
providing a proliferative signal along the entire GT axis
(Fig. 6C). By contrast,
Hoxa13GFP-homozygous mutants exhibit a dramatic reduction
of Fgf8 expression in the proximal UPE, whereas expression in the
distal UPE appears unaffected (Fig.
6D). Interestingly, the genital shelf mesenchyme immediately
adjacent to the proximal UPE is also the site of poor proliferation in E11.5
embryos lacking Hoxa13 (Fig.
6; compare I,J with K,L), which suggests that reduced Fgf8
expression in this region causes this defect in proliferation.
|
Stratification and signaling defects in the UPE are independent of
sonic hedgehog function in the developing GT
To determine if the loss of Fgf8 expression in the proximal UPE
reflects a change in the polarization of the UPE, we examined the expression
of Shh in the GT of E12.5 Hoxa13GFP embryos.
Analysis of Shh expression in the UPE showed no differences in
expression between wild-type and homozygous-mutant embryos
(Fig. 7A,B), which confirms
that polarizing signals are normally produced in the mutant UPE. The presence
of Shh expression in the mutant proximal UPE strongly suggests that
Hoxa13 is required for Fgf8 expression in the proximal UPE,
as a more generalized effect would have also affected Shh expression
in this region.
|
Embryos lacking Hoxa13 exhibit aberrant vascular development in
distal GT
Next, because changes in Tgfß signaling can also affect tissue
vascularization, we examined whether changes in the expression of the
Tgfß family member Bmp7 and its antagonist, Nog, might
also affect GT vascularization. Section analysis of the distal urethra and
glans revealed dramatic differences between heterozygous- and
homozygous-mutant embryos in the vascularization of these tissues. In E13.5
heterozygous and wild-type (not shown) embryos, the mesenchyme immediately
flanking the UPE is vascularized by fine capillary vessels 10-15 µm in
diameter (Fig. 8A). By
contrast, the same region in homozygous mutants was consistently (10/10)
vascularized by vessels typically ranging from 70-150 µm in diameter
(Fig. 8B). Cellular analysis of
these enlarged vessels confirmed them to be functional vascular tissue,
containing an endothelium expressing the angiogenic marker Pecam-1 (CD31;
PecamMouse Genome Informatics) (Fig.
8A,B) as well as red blood cells
(Fig. 7B). Interestingly,
vessel endothelial cells of heterozygous embryos strongly co-express Pecam and
Hoxa13GFP as determined the yellow co-localized signal
(Fig. 8A), whereas in
homozygous mutants the degree of Pecam and Hoxa13GFP
co-localization was greatly reduced in the endothelial cells
(Fig. 8B).
|
Androgen signaling is reduced in Hoxa13-deficient embryos
To characterize the relationship between Hoxa13 and the androgen
signals necessary for GT growth and development, we examined the expression of
the androgen receptor (AR) in the developing glans and urethra of E15.5
littermates. In the distal urethra of heterozygous embryos, AR proteins are
detected in the surface ectoderm as well as in the condensing mesenchyme
proximal and lateral to the urethral epithelium
(Fig. 9A). In homozygous
mutants, AR expression is reduced in the surface ectoderm as well as in the
mesenchyme proximal and lateral to the hypospadiac urethral epithelium
(Fig. 9B). Interestingly the
medial mesenchymal condensation (Fig.
9A,C,D) reflects the anlagen from which the penian bone (P)
develops postnatally, under the control of androgen signaling
(Murakami, 1987). In
Hoxa13GFP-homozygous mutants, this condensation is absent
in the more distal sections of the penis
(Fig. 9B), and appears
disorganized in more proximal sections when compared with heterozygous
controls (Fig. 9C,D). This lack
of mesenchymal condensation is remarkably similar to the phenotype exhibited
in the autopod of Hoxa13GFP-mutant mice
(Stadler et al., 2001
), which
suggests a similar role for Hoxa13 in regulating mesenchymal cell
adhesion and provides an explanation for hypoplasia of the os-penis in
Hypodactyly mice (Post and Innis,
1999
).
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DISCUSSION |
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In a model of Hoxa13 function during GT development
(Fig. 10A,B,D), we conclude
that Hoxa13 is required for the normal expression of Fgf8
and Bmp7 in the UPE, as well as the repression of Nog in the
mesenchyme flanking the UPE. In the absence of Hoxa13 function
(Fig. 10C,E), defects in the
external genitalia can be attributed to perturbations in three essential
processes. First, reduced Fgf8 signaling from the proximal UPE causes a
dramatic decrease in the initial proliferation of the GT mesenchyme, a process
previously shown to be essential for the development of the external genitalia
(Haraguchi et al., 2000;
Perriton et al., 2002
).
Second, reduced Bmp7 signaling lowers the amount of Msx1 and Msx2
expression in the mesenchyme flanking the proximal UPE, causing a decrease in
PCD necessary for the removal of overlying ectoderm, urethral tube closure and
meatus formation. Finally, Hoxa13 is required for the repression of
Nog in the mesenchyme flanking the medial-proximal UPE, a site where
Bmp4 signaling has an essential role in mediating urethral tube
closure. Surprisingly, we did not see an elevation in PCD in the distal
urethra and glans, which are noticeably smaller in
Hoxa13GFP homozygous mutants. This finding is consistent
with the normal levels of Shh expression in the UPE of
Hoxa13GFP mutants, which was previously shown to confer
cell survival in tissues derived from the hindgut or foregut endoderm
(Perriton et al., 2002
;
Litingtung et al., 1998
). By
this same mechanism, elevated levels of Shh in the UPE of
Hoxa13GFP-homozygous mutants could contribute to the
reduced PCD seen in these tissues, although we see no evidence for elevated
Shh expression in the UPE of Hoxa13GFP homozygous
mutant embryos.
|
Similarly, because no changes in Fgf10 or Wnt7a
expression could be detected in GT of Hoxa13GFP mutants
(data not shown), it is likely that Bmp7, Bmp4 and Fgf8
function as the predominant factors required for GT outgrowth. This conclusion
is supported by the rescue of GT proliferation by ectopic Fgf8 application
(this work) (Haraguchi et al.,
2000), as well as studies in chick where the overexpression of
truncated Hoxa13 proteins caused reduced Fgf8 expression and
severe malformations of the gut and genitourinary regions
(de Santa Barbara and Roberts,
2002
). Recently, studies of ß-catenin function in the
developing limb (Barrow et al.,
2003
) also revealed a role for this molecule in mediating GT
outgrowth, as mice lacking ß-catenin in the GT exhibit hypoplasia of the
GT and its derivatives (J. Barrow, personal communication).
Failure to stratify the UPE contributes to the hypospadiac
phenotype
By E12.5 the UPE exhibits changes in cell morphology and cytokeratin
expression that are indicative of maturation and stratification of the
epithelial signaling center. In particular, cells lining the UPE shift from a
layer of simple epithelium to several cuboidal cell layers (stratified) that
now express K-14, a cytokeratin that is normally found in differentiating
epithelium (Kuzrock et al.,
1999; Kivela and Uusitalo,
1998
; Coulombe and Omary,
2002
). In homozygous-mutant littermates, stratification and K-14
expression are compromised, resulting in the disorganized arrangement of
rounded cells in the UPE. This phenotype may be attributed to the focal loss
of K-14 expression in the UPE, as mice lacking K-14 exhibit defects in the
stratification of the skin and corneal epithelium
(Lloyd et al., 1995
).
Interestingly, the loss of epithelial keratins is often associated with
elevated PCD (McGowan et al.,
2002
; Oshima,
2002
). However, in proximal UPE of
Hoxa13GFP-homozygous mutants no significant changes in PCD
were observed. This result is explained by the maintenance of K-8/18
expression in mutant UPE, which protects epithelium from PCD by directly
binding pro-apoptotic proteins, including the Tradd domain of Tnf receptor 1,
as well as providing structural support to minimize PCD initiated by
mechanical stress (Inada et al.,
2001
; Marceau et al.,
2001
; Baribault et al.,
1993
; Baribault et al.,
1994
). Similarly, changes in the stratification and keratin
expression patterns in the UPE could also affect cell-cell interactions
between the progressing mesenchyme and the underlying epithelium, preventing
the efficient movement of mesenchymal cells necessary for urethral
closure.
PCD as a mechanism to remove the distal UPE and its secreted
factors
An examination of PCD during GT development suggests a highly dynamic
interplay between mesenchymal proliferation and apoptosis. Using a TUNEL
assay, PCD was first detected in the cloacal membrane (CM) covering the GT
(data not shown). Within 12 hours, a rapid shift in PCD had occurred, moving
from CM ectoderm to the endodermal tissues of the UPE. What is intriguing
about this shift in PCD is its coincidence with peak Fgf8 expression in the
UPE. Developmentally, the localization of PCD in the UPE could serve two
important functions. First, the removal of the UPE by PCD could modulate
levels of Fgf8 expression, providing a signal for differentiation to
occur; a similar use of PCD is seen in the developing limb, where removal of
the AER reduces Fgf8 signaling to stimulate differentiation of the
underlying mesenchyme (Zwilling,
1955; Saunders et al.,
1957
; Macias et al.,
1997
; Niswander et al.,
1994
). Second, to achieve closure of the urethra, the progressing
mesenchyme must cover the medial epithelial layer. In
Hoxa13GFP-homozygous mutants, the epithelial layer is
elevated and thickened, which would inhibit closure of the medial urethral by
the proliferating mesenchyme. After UPE removal, PCD shifts again to the
mesenchymal tissues immediately flanking the UPE, occurring as early as E12.5
and persisting in the mesenchyme until closure of the penile urethra
(Baskin et al., 2001
;
van der Werff et al., 2000
).
Mechanistically, the reduction of PCD in the urethral mesenchyme of
Hoxa13GFP-mutant mice helps to explain the pathology of
the hypospadiac phenotype, where the persistence of thickened urethral
epithelium places a physical barrier between the progressing mesenchymal
shelves.
Bmp signaling and GT development
Our analysis of GT development in the presence of Bmp4 and Bmp7 blocking
antibodies strongly suggests a role for these factors in mediating growth and
closure of the meatus and urethra. Here, an analysis of GT development in
embryos lacking either Bmp7 or Bmp4, or combinations of
both, would provide the most corroborative data to define the complete
function of these molecules during the formation of the external genitalia. To
date, no characterizations of Bmp7 function in the external genitalia
have been reported, although Bmp7 has been shown to play an essential
role in mediating kidney tubule formation, as well as in the production of
meiotic germ cells (Luo et al.,
1995; Dudley et al.,
1995
; Dudley and Robertson,
1997
; Zhao et al.,
2001
). Similarly, the function of Bmp4 in the developing
GT is unknown as embryos lacking Bmp4 die between E6.5 and E9.5
(Winnier et al., 1995
).
Recognizing the similarity in phenotypes between blocked Bmp signaling and
those exhibited by Hoxa13GFP-homozygous mutants, it is
probable that the defects in meatus formation and urethral tube closure seen
in Hoxa13GFP-mutant mice reflect the combined effects of
reduced Bmp7 expression and those elicited by Nog
antagonism.
Hoxa13 is essential for maintaining capillary vessel diameter and
morphology
Normally, vascularization of the developing glans is provided by two
well-defined rings of capillary beds supplying both the prepuce and the
mesenchyme immediately adjacent the UPE. In Hoxa13GFP
mutants, capillary vessel morphology, placement and diameter are dramatically
altered in the distal glans. Interestingly, this phenotype is also present in
the glans clitoris of homozygous mutant female mice (data not shown),
reflecting the common origin of these two structures and conservation of the
mechanisms underlying this gross change in vessel diameter. Aberrant
vascularization of the glans is also associated with human hypospadias, and
was interpreted as a default state stemming from the aborted differentiation
of the glans, meatus and urethral spongiosum
(Baskin et al., 1998;
Baskin, 2000
). Clearly the
enlargement of the capillary vessels in the glans of
Hoxa13GFP homozygous mutant mice reflects more than a
default vascular state as only the distal vessels supplying the glans are
affected, whereas the entire glans and urethra are hypoplastic in these
mice.
Mechanistically, changes in Tgfß or Smad signaling also affect blood
vessel development, morphology and diameter (reviewed by
Dennler et al., 2002;
ten Dijke et al., 2002
;
Weinstein et al., 2000
;
Oh et al., 2000
;
Vargesson and Laufer, 2001
).
In particular, mice lacking Smad5 (Madh5Mouse Genome Informatics)
exhibit many of the same defects shown by Bmp4- and Tgf-beta
1- (Tgfb1Mouse Genome Informatics) null mice, including
enlarged blood vessels (Yang et al.,
1999
; Chang et al.,
1999
). Our examination of Bmp4 and Bmp7 function
in the developing GT indicates that although Bmp7 is uniquely
expressed in the UPE, both molecules function similarly in the glans to
regulate the formation of the meatus. Here the combinatorial effects of
perturbed Bmp4 and Bmp7 signaling may contribute the
enlargement of the glans capillary vessels. We cannot exclude the possibility
that changes in Bmp-receptor expression in the developing glans, could also
contribute to the enlarged capillary vessel phenotype, although the expression
of receptors Bmpr1a, Bmpr1b and Bmpr2 in the glans was not significantly
different between E13.5 homozygous-mutant and control embryos.
Hoxa13 is required for normal androgen receptor expression in the
developing GT
Our examination of AR localization in the developing murine GT revealed a
high degree of co-localization between Hoxa13 and the AR in the
proximal urethral mesenchyme. By contrast, only minor levels of AR protein
could be detected in the same mesenchymal regions of
Hoxa13GFP-homozygous mutants. This reduction in AR
expression appears specific to the mesenchyme immediately adjacent to the
urethral epithelium, and suggests a role for Hoxa13 in mediating the
responsiveness of these tissues to androgens by modulating the levels of AR in
the urethral epithelium. This modulation in AR expression would provide a
developmental link between hypospadias associated with environmental
antagonists of the AR and hypospadias associated with loss of Hoxa13
function, placing Hoxa13 as an important modulator of AR signaling
during the development of the external genitalia
(Baatrup and Junge, 2001;
Curtis, 2001
;
Baskin et al., 2001
).
Alternatively, environmental compounds could affect Hoxa13 expression
directly, in a manner similar to the ectopic expression of Hox genes in
response to exogenous retinoids (Simeone
et al., 1991
; Whiting,
1997
).
In mice, the prenatal response of the GT to AR signaling is the growth and
closure of urethra and glans penis, as mice lacking AR signaling also exhibit
hypospadia (Murakami, 1987).
Postnatally, AR signaling is required for the formation of the os-penis
(Murakami, 1987
). Here, the
function of Hoxa13 in mediating the postnatal development of the
external genitalia is unknown as Hoxa13 mutations derived from
homologous recombination are not compatible with life beyond E15.5
(Stadler et al., 2001
;
Fromental-Ramain et al.,
1996
). An examination of os-penis formation in hypodactyly mice
(Post and Innis, 1999
)
suggests that Hoxa13 mediates similar mesenchymal condensation events
as described in the limb (Stadler et al.,
2001
). However, it is important to note that the hypodactyly
mutation in Hoxa13 also affects the expression of Hoxd13
causing phenotypes more similar to those of
Hoxa13/Hoxd13-compound mutants (Robertson et al., 1996;
Robertson et al., 1997
;
Fromental-Ramian, 1996).
Using conditional mutagenesis, Hoxa13 function could be completely assessed throughout genitourinary development. This analysis would provide important clues regarding the developmental pathology underlying hypospadias beyond E15.5 while also providing an important resource to examine both neonatal development and adult maintenance of genitourinary tissues affected by loss of Hoxa13 function.
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ACKNOWLEDGMENTS |
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