1 Department of Cell and Developmental Biology, Vanderbilt University,
Nashville, TN 37232, USA
2 Department of Urologic Surgery, Vanderbilt University, Nashville, TN 37232,
USA
3 Department of Cancer Biology and Vanderbilt-Ingram Cancer Center, Vanderbilt
University, Nashville, TN 37232, USA
4 Laboratory of Metabolic Diseases, The Rockefeller University, New York, NY
10021, USA
5 Mass Spectrometry Research Center, Vanderbilt University, Nashville, TN 37232,
USA
6 Department of Pathology, Vanderbilt University, Nashville, TN 37232, USA
Author for correspondence (e-mail:
robert.matusik{at}vanderbilt.edu)
Accepted 23 May 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Foxa1, Knockout, Prostate, Foxa2, Shh, Androgen, Mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Normal growth and differentiation of prostate epithelium is controlled
through androgen-regulated paracrine signaling from the mesenchyme
(Cunha et al., 1987), as the
stromally located androgen receptor (AR) is essential for prostate epithelial
differentiation (Donjacour and Cunha,
1993
). In the absence of either androgens or stromal AR, the
prostate does not develop (Bardin et al.,
1973
; Cunha et al.,
1987
). Conversely, reciprocal paracrine signals from the
epithelium also patterns stromal cell differentiation
(Cunha et al., 1996
). Emerging
evidence suggests that epithelial-mesenchymal interactions during prostate
organogenesis involves several conserved families of molecules, including
sonic hedgehog (Shh) (Podlasek et al.,
1999
; Lamm et al.,
2002
; Wang et al.,
2003
; Freestone et al.,
2003
; Berman et al.,
2004
), bone morphogenetic protein (Bmp)
(Lamm et al., 2001
),
Fibroblast growth factors (Fgfs) (Thomson
and Cunha, 1999
; Donjacour et
al., 2003
), the Notch-Delta membrane molecule
(Wang et al., 2004
;
Shou et al., 2001
), and the
Nkx3.1 homeobox protein (Bhatia-Gaur et
al., 1999
; Schneider et al.,
2000
; Tanaka et al.,
2000
).
Early prostate ductal budding and morphogenesis is accompanied by a
transient elevation of multiple inductive pathway components (e.g. Shh, Bmp4,
Fgf7, Fgf10 and Notch1), whose activities are dramatically downregulated at
the conclusion of ductal morphogenesis
(Podlasek et al., 1999;
Lamm et al., 2001
;
Thomson et al., 1997
;
Thomson and Cunha, 1999
;
Wang et al., 2004
). Among
these factors, Shh is an epithelium-secreted ligand that plays a crucial role
in prostate morphogenesis. Conflicting reports on Shh-elicited biologic
effects on the growth of the prostate ducts highlights the complex nature of
this pathway. The differences between these studies could reflect a
stage-dependent cellular response
(Podlasek et al., 1999
;
Lamm et al., 2002
;
Freestone et al., 2003
;
Wang et al., 2003
). In
contrast to transient inductive signals, normal ductal growth also requires
the sustained presence of negative modulators. For example, homeobox protein
Nkx3.1-null mice show impaired prostate ductal growth and progressive
epithelial hyperplasia (Bhatia-Gaur et al.,
1999
).
Previous biochemical analysis identified forkhead box a1 (Foxa1) as a key
transcriptional component that interacts with AR on multiple prostatic
enhancers and controls androgen-induced activation of both human and rodent
prostate-specific genes (Gao et al.,
2003). As forkhead transcription factors widely participate in the
development of various organs (Carlsson and
Mahlapuu, 2002
), it was reasonable to expect that Foxa proteins
may play an important role in prostate development, e.g. the
specification/maturation of the prostate epithelial cell. Regulation of gut
differentiation by Foxa homologues is a feature conserved in metazoa
(Weigel and Jackle, 1990
;
Gaudet and Mango, 2002
;
Kalb et al., 1998
;
Horner et al., 1998
) and in
vertebrates, all three Foxa proteins (Foxa1, Foxa2 and Foxa3) are involved in
the epithelial gut tube formation (Zaret,
1999
; Zaret,
2002
). The mouse prostate epithelium is derived from the hindgut
endoderm and expresses Foxa1 throughout prostate development and maturation
(Peterson et al., 1997
;
Kopachik et al., 1998
;
Gao et al., 2003
), whereas it
expresses Foxa2 only during prostate budding
(Mirosevich et al., 2005
).
This suggests a potential role for these forkhead proteins in prostate
development. Furthermore, the induction of multiple mammalian forkhead genes
is dependent on the hedgehog signal in various tissues during embryogenesis
(Chiang et al., 1996
;
Yamagishi et al., 2003
;
Furumoto et al., 1999
;
Mahlapuu et al., 2001
). Thus,
a better view of the precise distribution and regulation between these
molecules in the embryonic urogenital sinus (UGS) is relevant to understanding
the normal developmental process of the prostate. Here, we report the
phenotypic and biochemical characterization of Foxa1-deficient mouse prostate
using organ rescue and tissue recombination experiments. Our data demonstrate
that epithelial Foxa1 plays an early role in prostate ductal morphogenesis,
supporting a regulatory function that is essential for modulating inductive
signals to control prostate cell growth and differentiation.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
E18 rat UGM was prepared for tissue recombination
(Cunha and Donjacour, 1987).
Bladder epithelial tissue fragments were separated from 89 P1 mice (24
Foxa1+/+, 53 Foxa1+/ and 12
Foxa1/), pooled by genotype and individually
recombined with rUGM. Recombinants were grafted into adult male nude mice and
harvested after 4 and 12 weeks. Grafting was performed in triplicate for each
genotype at each time point.
In situ hybridization and RT-PCR
In situ hybridization for Foxa1 and Foxa2 was performed using
digoxigenin-labeled gene-specific probes
(Braissant and Wahli, 1998).
For RT-PCR, total RNA was extracted with QiagenTM RNeasy extraction kit
(Qiagen, Valencia). cDNAs were synthesized with 1.5 µg of total RNA by
Superscript-IITM reverse transcriptase (Invitrogen). PCR was performed
using primer sets (see Table S1 in the supplementary material) to produce
gene-specific fragments with optimal numbers of reaction cycle.
Semi-quantitative analysis was performed by comparing the intensity of PCR
product with normalization to an internal standard gene Gapdh.
ß-Galactosidase staining
Fresh tissues were prefixed for 6 hours in 2% paraformaldehyde, 0.2%
glutaraldehyde in PBS at 4°C, embedded in OCT. Frozen sections were
immersed in the same solution for 10 minutes at 4°C, rinsed in water, and
stained for ß-galactosidase at 37°C for 1-6 hours.
Immunohistochemistry and immunofluorescence
Tissue preparation and staining procedures were as described
(Mirosevich et al., 2005).
Primary antibodies, at indicated dilutions, were AR (rabbit, 1:1000; Santa
Cruz), Ck5 (rabbit, 1:500; Covance Research Products), Foxa2 (goat, 1:1000;
Santa Cruz), Nkx3.1 (rabbit, 1:3000) (Kim
et al., 2002
), SMA (mouse, 1:1500; Sigma),
-actin (mouse,
B4, 1:10,000) (Lessard, 1988
),
Ck14 (mouse, 1:10), Ck8 (mouse, 1:10)
(Wang et al., 2001
),
ß-catenin (mouse, 1:200; BD Transduction Laboratories), p63 (rabbit,
1:200; Santa Cruz), Shh (rabbit, 1:200; Santa Cruz)
(Thayer et al., 2003
;
Niemann et al., 2003
;
Krebs et al., 2003
;
Jaskoll et al., 2004
) and
Ptch1 (goat, 1:200; Santa Cruz) (Canamasas
et al., 2003
; Niemann et al.,
2003
; Di Marcotullio et al.,
2004
; Jaskoll et al.,
2004
; Sheng et al.,
2004
). Fluorescence-conjugated secondary antibodies (A11055,
A21203, A21206, A21207) were purchased from Molecular Probes and diluted
1:200. DAPI staining was performed using Vectashield Mounting Medium (Vector,
H-1200).
Electron microscopy
Tissues were fixed in 2% glutaraldehyde in 0.1M cacodylate buffer at
4°C overnight, dehydrated in alcohol and polymerized. Sections (70 nm)
were cut and ultrastructural analysis was performed on Phillips CM-12
Transmission Electron Microscope equipped with AMT digital camera system.
Matrix assisted laser desorption ionization time-of-flight mass spectrometry
MALDI-TOF MS analysis was performed using a modified protocol
(Chaurand et al., 2004). Using
a cryostat, 10 µm prostate tissue sections were cut and mounted onto a
conductive glass MALDI plate. Three or four sections were obtained per
specimen and were stained with MALDI-compatible Cresyl Violet
(Chaurand et al., 2004
) to
identify regions for profiling, Target areas were then spotted with 150 nl of
saturated sinapinic acid as matrix prepared in acetonitrile/H2O/TFA
(50/50/0.1) by volume and allowed to dry. Each section was then analyzed by
MALDI-TOF MS in the linear mode using an Applied Biosystems Voyager DE-STR
mass spectrometer (Framingham, MA). Acquired spectra were baseline subtracted
and normalized. Spectra from each prostate sample were then averaged before
comparison.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Dual-staining for Shh and patched 1 (Ptch1), the transmembrane receptor for
Shh, showed that Shh was continuously expressed in the E21 UGE with strongest
level observed in the peripheral basally located cells and in the nascent
prostatic epithelial buds (arrowhead in
Fig. 1I), and Ptch1 was
co-expressed in these cells (Fig.
1H,J), consistent with a reported detection of epithelial Ptch1 in
neonatal prostate ductal tips (Pu et al.,
2004). In addition to the UGE compartment, Ptch1 was also detected
in a population of mesenchymal cells close, but not immediately adjacent to,
the Shh-producing UGE (arrow in Fig.
1H). Approximately ten-cell layers separate these stromal
Ptch1-expressing cells and the epithelial cell source of Shh. This distance is
within an effective range of hedgehog signaling
(Ingham and McMahon, 2001
).
Serial sections revealed that these Ptch1-positive stromal cells expressed
smooth muscle
-actin (SMA) (arrow in
Fig. 1K), a mesenchymal target
of Shh (Weaver et al., 2003
).
Detection of Ptch1 in both UGE and UGM suggested that Shh may act in both a
juxtacrine and a paracrine fashion. Interestingly, a scattered Shh staining
was detected in adjacent UGM surrounding the Shh-producing UGE. As this
anti-Shh antibody is raised against the N-terminal epitope (amino acids
41-200) of Shh and is able to detect both the membrane-bound and the secreted
Shh-N peptide; the scattered staining is most probably due to immunoreactivity
with the diffused Shh-N peptides. This has not been reported previously in UGS
(Freestone et al., 2003
;
Berman et al., 2004
).
Expression of AR was detected in all cells of E21 UGS, including the
basally located epithelium (Fig.
1L). However, the staining was stronger in UGM when compared with
UGE. Staining for p63, a basal cell nuclear protein highlighted the peripheral
basal-like cell population (Fig.
1O). Although both p63 and Ck14 are specific for basal cells, p63
has been reported to be expressed in almost all basal cells, whereas Ck14 is
only expressed in a subpopulation
(Signoretti et al., 2000;
Hudson et al., 2001
). In E21
UGS, some UGE co-expressed the basal cell marker p63 and the luminal keratin
Ck8 (Fig. 1M), indicating their
immature/undifferentiated status (Wang et
al., 2001
).
Staining was also performed to localize ß-catenin, a downstream
effector of the Wnt pathway, which can promote endodermal genes including
Foxa1 and Foxa2 (Sinner et al.,
2004). ß-Catenin was specifically localized to the membrane
of both UGE and the surrounding UGM cells
(Fig. 1N), with the strongest
expression seen in p63-expressing basally located cells
(Fig. 1O-P).
In contrast to Foxa1, which continued to be expressed in the postnatal developing prostate (Fig. 1Q) and adult glands (Fig. 1S), Foxa2 mRNA was strongly but transiently expressed only during ductal morphogenesis (Fig. 1R), and its expression decreased to undetectable levels in mature glands (Fig. 1T). Thus, the downregulation of Foxa2 during postnatal prostate development was confirmed by in situ hybridization.
Foxa1 regulates normal prostate ductal morphogenesis
Foxa1 mutant mice were generated using a targeting vector which deletes the
Foxa1 DNA-binding domain and creates an in-frame fusion with the
Escherichia coli lacZ gene (Shih
et al., 1999). Foxal/ mice die
neonatally, thus we used two distinct strategies, renal capsule organ rescue
(Wang et al., 2000
) and tissue
recombination (Cunha and Donjacour,
1987
), to assess the impact of Foxa1-deficiency on prostate
development.
Prostate rudiments (asterisks in Fig. 2A) and adjacent seminal vesicles were dissected from postnatal day 1 (P1) Foxa1 pups. Foxa1/ male mice of P1 contained prostate rudiments (n=13) that were histologically identical to wild type, indicating a normal prostatic induction at this stage. Entire prostate rudiments (n=64) were rescued by renal capsule grafting into intact male athymic nude mice. Grafted prostates were recovered at various intervals between 2 and 15 weeks. Rescued prostates and seminal vesicles were compared after fine dissections. Foxa1/ prostate tissues (asterisk in Fig. 2D) were consistently smaller and solid, whereas controls were enlarged and contained secretions (Fig. 2B,C). As the transgene lacZ was driven by Foxa1 promoter in the mutant allele, ß-galactosidase staining revealed lacZ expression in rescued Foxa1+/ (Fig. 2F) and Foxa1/ prostate epithelium that showed a pronounced epithelial cell disorganization (Fig. 2G).
Histological analysis of rescued prostates (between 2 and 15 weeks) showed
that Foxa1/ prostate developed many solid
epithelial cell cords with cribriform patterns, and no normal-appearing lumen
was observed (Fig. 2J,P,R; see
Fig. S1 A,C in the supplementary material). In 4-week-old renal-rescued
wild-type control prostates (Fig.
2K,M,O; see Fig. S1B,F in the supplementary material), a normal
lumen lined with monolayer of luminal epithelial cells was observed,
reflecting normal prostate development, as previously reported
(Kurita et al., 2004;
Berman et al., 2004
). Although
in the rescue experiments it is difficult to precisely define each lobe, based
upon careful microdissection (with attention on the anatomic location
referring to adjacent seminal vesicles) and determination of gross morphology,
we compared rescued tissues as closely as possible on a lobe basis. Identical
Foxa1-deficient phenotype was consistently observed in distinct prostate lobes
dissected from all Foxa1/ prostates examined
(n=13), suggesting that Foxa1 regulates prostate epithelial ductal
pattern regardless of lobe identity. Tentatively identified lobes are listed
as ventral prostates (VPs) in Fig.
2, and dorsolateral prostates (DLPs) in Fig. S1 in the
supplementary material.
AR staining did not suggest a protein level change in Foxa1-deficient prostate epithelial cells (Fig. 2L-O). The same results were obtained when distinct lobes were dissected (see Fig. S1A-H in the supplementary material). Notably, when extending the rescue period up to 12-15 weeks, Foxa1/ prostates continued to demonstrate the growth of epithelial cords with no obvious ductal canalization or luminal formation (see Fig. S1C,G in the supplementary material). To confirm this alteration in epithelial ductal patterning and to rule out the possibility of histological artifacts during section preparation, 1 µm frozen sections from 12-week-old rescued prostates were stained with Toluidine Blue to define the prostate ductal structure. As shown in Fig. 2P, Foxa1/ prostate contained `balls' of disorganized epithelial cells with impaired luminal structure (arrowhead), while the wild-type prostate showed highly organized luminal cells with a normal lumen (arrowhead in Fig. 2Q). Staining for E-cadherin, an adhesion molecule expressed specifically on epithelial cell membrane, demonstrated a total loss of cell polarity in Foxa1-deficient epithelial cells (Fig. 2R and inset). By contrast, wild-type luminal epithelial cells lined the lumen in a highly organized pattern (arrowhead in Fig. 2S), as indicated by focused E-cadherin staining at epithelial cell conjunctions (inset).
Using normal embryonic inductive UGM, we performed tissue recombination
experiments with Foxa1/ epithelium to
determine if normal ductal patterning would be obtained. Neonatal
Foxa1+/+ or Foxa1/ mouse
bladder epithelium were recombined with wild-type E18 rat (r) UGM.
Fig. 2H shows 12-week-old
grafted recombinants that were derived from wild-type (left) and
Foxa1-deficient epithelium. The size and the wet weight
(Fig. 2I) of null recombinants
(n=15) were significantly smaller than control recombinants
(n=15), even though the same number of wt rUGM cells were used
(P<0.01). Histological analysis indicated that the embryonic rat
UGM cells instructively elicited normal-appearing prostate glandular structure
from wild-type bladder epithelium (Fig.
2U) as previously documented
(Cunha et al., 1987); however,
the same UGM cells failed to induce normal prostate architecture from
Foxa1/ epithelium
(Fig. 2T), indicating a pivotal
role for Foxa1 in determining the epithelial cell responsiveness to inductive
mesenchymal signals.
|
Foxa1-deficient prostate shows an altered epithelial cell population and a modified stromal pattern
To define this phenotype, cell populations were examined from both rescue
and recombinant prostates. In the normal mouse prostate, mature epithelium
contains only a small proportion of basal cells (around 10%), whereas the
luminal cells comprise the remaining epithelial cell type. Basal keratin Ck5
staining demonstrated a clear expansion of basal-like cells in
Foxa1/ prostates (4-week-old rescued
prostate in Fig. 3A).
Remarkably, in some Foxa1/ epithelial ducts, these
basal keratin-expressing cells became the predominant cell type, and their
location was extended into the epithelial cords
(Fig. 3B). Such a distribution
of basal cells was not observed in any rescued wild-type prostates, rather
basal cells are localized as a discontinuous layer between the luminal cells
and the basement membrane (Fig.
3C). Basal keratin staining on tissue recombinants derived from
wild-type and Foxa1/ epithelium showed a
similar expansion of basal-like cells in null recombinants (see Fig. S3A in
the supplementary material).
|
Given that the loss of Foxa1 led to a failure of the prostate epithelium to
mature, it seems likely that stromal patterning, which itself is dependent
upon epithelial differentiation (Cunha et
al., 1996), could also be abnormal. Staining for SMA revealed an
expansion in smooth muscle layer that immediately surrounds the
Foxa1/ epithelial cords
(Fig. 3H,I), suggesting
mesenchymal hypercellularity. In the age-matched 4-week rescued wild-type
prostate, only a thin layer of smooth muscle surrounded the ducts
(Fig. 3J). Surprisingly, SMA
staining on tissue recombinants revealed a more pronounced expansion of smooth
muscle cells in recombinants that were derived from
Foxa1/ epithelium plus wild-type rUGM (see
Fig. S3I in the supplementary material). The expression of smooth muscle
-actin, a late marker for smooth muscle differentiation
(Qian et al., 1996
), was
detected equally in wild-type and null recombinants (see Fig. S3K in the
supplementary material), suggesting that although perturbed paracrine
signaling from Foxa1/ epithelium modified
stromal pattern, smooth muscle cell differentiation was complete.
Foxa1/ prostate is immature and devoid of secretory luminal cell
Semi-quantitative RT-PCR was performed on rescued prostates to examine the
expression of several key inductive signaling molecules that correlate with
early prostate morphogenesis (Podlasek et
al., 1999; Thomson et al.,
1997
; Thomson and Cunha,
1999
; Lamm et al.,
2001
; Wang et al.,
2004
) (Fig. 4).
Gene expression profiles were compared after normalization to an internal
standard gene Gapdh. Elevated mRNA levels of Shh, Gli2, Fgf7, Fgf10,
Bmp4, ß-catenin and Notch1 were detected in 4-week-old rescued
Foxa1/ prostates compared with controls. The
continued presence of these molecules in the null prostate indicated that
Foxa1 may be required for the maturation of the prostate. Interestingly, the
expression of two other Gli proteins was either slightly increased (Gli1) or
equally expressed (Gli3) in control and null tissues. However, the expression
of Ihh, Bmp7 and Nkx3.1 were undetectable or decreased in the null. As the
expression of Shh (Fig. 1F,I)
and Notch1 (Wang et al., 2004
)
are associated with basally located epithelial cells in early developing
prostate, elevation in both proteins supported an increased basal-like cell
population in Foxa1/ prostate.
Androgen stimulation induces the differentiation of immature prostate
epithelium into luminal cells that produce prostate-specific secretory
proteins (Kasper and Matusik,
2000). Transmission electron microscopy (EM) provides an unbiased
way to determine secretory features of prostate epithelial cells at an
ultrastructural level. EM analysis, on 12-week-old rescued wild-type prostate,
showed tall columnar luminal epithelium
(Fig. 5A), with enlarged Golgi
complexes, and numerous dense secretory materials, the `prostasomes'
(Sahlen et al., 2002
), within
apical vesicles or at luminal surfaces
(Fig. 5C). These
ultrastructural features demonstrating secretory activities were completely
absent in rescued Foxa1/ epithelium
(Fig. 5B,D).
|
|
RT-PCR was performed to validate the results obtained from IMS. Pbsn mRNA
was completely undetectable in Foxa1-deficient prostates, while Sbp was
dramatically decreased when compared with controls
(Fig. 5F). Loss of Pbsn in
Foxa1/ prostates strongly supported our
previous study that two forkhead response elements were essential for
androgen-induced Pbsn expression (Gao et
al., 2003). Interestingly, Sbp gene was identified in this study
as a novel Foxa1 target. Examination of Sbp promoter revealed an organization
of forkhead and androgen response elements that are similar to those seen in
other androgen-regulated prostatic enhancers (see Fig. S4 in the supplementary
material) (Gao et al., 2003
).
Whether these Foxa1-binding sites within the Sbp promoter are
transcriptionally functional requires further investigation; however, the
absence of secretory features and prostate differentiation markers in
Foxa1/ prostates do indicate a disruption in
luminal epithelial cell maturation.
Perturbed epithelial-stromal interactions alter the null prostate ductal pattern
As phenotypic and molecular analysis on
Foxa1/ prostates indicated a perturbed
epithelial-mesenchymal interaction, we examined potential pathways involving
in signaling. Given that Shh regulates prostate ductal morphogenesis and
deregulated hedgehog activity has been implicated in prostate diseases
(Fan et al., 2004;
Karhadkar et al., 2004
;
Sanchez et al., 2004
), we
examined Shh expression in rescued tissues. Strong and focused Shh expression
was detected in rescued Foxa1/ epithelial
cell cords (Fig. 6A) and
Shh-positive ductal epithelial buds were evident
(Fig. 6B). As Ihh is absent the
null prostate (Fig. 4), the
pattern of Shh staining seen in Foxa1/
prostate cannot be due to the antibody crossreactivity with Ihh. The same
Shh-producing epithelium was positive for Ptch1
(Fig. 6C). However, no focused
staining for Shh or Ptch1 was detected in rescued wild-type prostates (see
Fig. S5A-C in the supplementary material and data not shown). By comparing the
serial sections stained with SMA (Fig.
3H), we noted that Shh-expressing null epithelium (arrows in
Fig. 6B) were surrounded by
thick smooth muscle layers (see Fig. S5D,E in the supplementary material), in
agreement with the finding that SMA is a mesenchymal target of Shh
(Weaver et al., 2003
). This
suggests that the deregulated focal Shh activation in Foxa1-deficient
epithelium may contribute to the altered epithelial-stromal interaction.
ß-Catenin nuclear activation has been implicated in abnormal prostate
epithelial cell growth (Bierie et al.,
2003; Cheshire and Isaacs,
2003
) and we observed an increase of ß-catenin mRNA level in
Foxa1/ prostates
(Fig. 4). However, we did not
detect an increased nuclear level of ß-catenin (red) in the
p63-expressing basal-like cells (green) in
Foxa1/ epithelium
(Fig. 6D). Instead, the signals
are primarily localized to the cell membrane in both
Foxa1+/+ and Foxa1/
epithelium (Fig. 6D,E).
Nevertheless, the staining demonstrated a disrupted null epithelial cell
polarity (Fig. 6D).
|
Foxa2 is a target of Shh in the neural tube
(Chiang et al., 1996;
Hynes et al., 1997
) and
pharyngeal endoderm (Yamagishi et al.,
2003
); we have shown that Foxa2 expression overlaps with Shh in
embryonic UGE (Fig. 1E-G),
whereas Foxa2 is only transiently detected during prostate budding
(Fig. 1R). Nuclear Foxa2
expression was retained in Foxa1/ epithelium
(Fig. 6J) even in prostates
rescued for 15 weeks (see Fig. S5F in the supplementary material). These
Foxa2-expressing cells tend to form tiny buds at ductal tips, suggesting that
Foxa2 may closely correlate with epithelial cell growth and budding, a role
consistent with its expression pattern during prostate budding
(Fig. 1E). A similar
correlation between elevated Foxa2 in Foxa1-null lung epithelium has been
reported (Wan et al., 2005
).
Upon comparison of serial sections, nuclear Foxa2 staining was seen in
Shh-expressing Foxa1/ cells
(Fig. 6K). Consistent with
previous studies (Kopachik et al.,
1998
; Mirosevich et al.,
2005
), no detectable Foxa2 was seen in rescued
Foxa1+/+ prostates
(Fig. 6L). Furthermore, this
deregulated Foxa2 expression was confirmed in tissue recombinants derived from
Foxa1/ epithelium plus wild-type rUGM (see
Fig. S5G in the supplementary material).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The finding that Foxa1 participates in ductal morphogenesis is consistent
with its early expression in embryonic UGE, during prostate budding, and
ductal initiation. Although both Foxa1 and its closely related family member,
Foxa2, are expressed in the early developing prostate epithelial buds, the
distribution of Foxa2 is restricted to the basally located cell population,
while Foxa1 is broadly expressed in almost all epithelium. Temporally, Foxa2
expression is downregulated to barely detectable levels shortly after birth,
while Foxa1 is continuously expressed from early development to the maturation
of the gland. This pattern is different from that observed in several other
endodermal organs (e.g. lung and liver) where Foxa1 and Foxa2 co-express
during adulthood (Zaret,
1999). In the prostate epithelium, the distinct spatial and
temporal distribution of Foxa1 and Foxa2 suggest that these two transcription
factors play different roles in prostate development. In Foxa1-deficient
prostate epithelium, we observed sustained Foxa2 expression; however,
continued Foxa2 expression does not rescue the Foxa1-deficient phenotype by
compensating the loss of Foxa1, strongly arguing that the functions of these
two proteins are divergent in the prostate. This result in the mouse prostate
is different from the observation made in lung morphogenesis where Foxa1 and
Foxa2 are functionally redundant (Wan et
al., 2005
). It is important to note that Foxa1 and Foxa2 are
co-expressed in lung endoderm throughout adulthood, whereas only Foxa1 is
expressed in the mature prostate epithelium.
The presence of primitive prostate structures in Foxa1-deficient mice
indicates that this protein is not absolutely required for prostatic induction
and the initial budding processes. The same observation has been made in Shh
mutant mice whose prostatic buds can be induced upon renal capsule grafting or
in vitro culture (Berman et al.,
2004). However, different from Shh mutant prostates, whose ductal
pattern is only minimally affected (Berman
et al., 2004
), the buds that are formed in Foxa1-deficient
prostate do not follow the normal pattern of development and differentiation.
Instead, structurally aberrant epithelial ducts demonstrate a
hyperproliferative feature reminiscent of solid primitive epithelial cords
that are surrounded by thick stromal layer. The abnormal ductal phenotype of
Foxa1-deficient prostate is accompanied by a series of molecular aberrations,
including maintained elevation in a number of early signaling molecules, most
of which are involved in epithelial-mesenchymal interactions that induce and
promote ductal morphogenesis. As Foxa1 is exclusively expressed in the
epithelium, the altered expression of several stromal factors is probably due
to a secondary effect caused by a perturbed epithelium-to-mesenchyme signaling
induced by the Foxa1-null epithelium. Thus, persistent detection of early
signaling molecules reflects a developmentally arrested feature of these
Foxa1-deficient prostates.
We confirmed that the activation of Shh correlated with multiple reported
targets (Ptch1, Gli, Foxa2 and Sma) in Foxa1-deficient prostate, where
pathological features indicated cellular hyperproliferation in both epithelial
and stromal cells. Taken together, our data suggest a correlation between Shh
activity and the hyperproliferative features of the null prostate. Deregulated
hedgehog pathway signaling has been identified in proliferative diseases such
as prostate cancer (Fan et al.,
2004; Karhadkar et al.,
2004
; Sanchez et al.,
2004
). It is conceivable that in Foxa1-deficient prostate, the
deregulated hedgehog activity may be one of the causal factors that contribute
to the abnormal ductal pattern. Our results agree with the fact that Shh is
capable of inducing the growth of both epithelial and mesenchymal cells
(Bellusci et al., 1997
), and
exert mitogenic effects on the development of various tissues
(Lamm et al., 2002
;
Yu et al., 2002
;
Jaskoll et al., 2004
;
Thibert et al., 2003
).
Collectively, our data demonstrate that, in addition to a paracrine mechanism,
Shh signaling may also act within the prostatic epithelium in a juxtacrine (or
autocrine) manner. This provides an explanation for the hyperproliferation
seen in Foxa1/ epithelium. A similar model
for Shh has been proposed in the mouse embryonic salivary gland epithelium
(Jaskoll et al., 2004
).
|
We observed an overlapped expression of Shh and Foxa2 in the basally
located epithelial cell population within the embryonic UGS. The Foxa2
promoter contains a Shh-responsive element
(Sasaki et al., 1997), and Shh
secreted from the notochord induced Foxa2 expression in the floorplate of the
neural tube; reciprocally Foxa2 maintained Shh expression in a positive
feedback loop (Chiang et al.,
1996
; Hynes et al.,
1997
; Echelard et al.,
1993
; Sasaki et al.,
1997
). Detection of both Shh and Foxa2 in Foxa1-deficient prostate
epithelium indicates that a reciprocal regulation between two proteins is
actively present in these mutant prostates. This observation also suggests
that Foxa1 could be a negative regulator that modulates the expression of
Foxa2 or Shh in normal situation.
A novelty of Foxa1-deficent prostate phenotype is that the null epithelial
cells have many features of the basally located epithelial cells that occur in
embryonic UGS. These basal cells of the UGS are postulated to act as the
transit/amplifying population in the prostate, and are widely believed to be
capable of acting as luminal cell precursors
(Abate-Shen and Shen, 2000).
However, fully differentiated basal cells cannot be essential precursors to
luminal cells as the p63-null prostates develop despite a lack of basal cells
but they are required for ductal integrity
(Kurita et al., 2004
). In our
study, no mature luminal epithelial cells were observed as defined by
ultrastructural features or the expression of differentiation markers,
suggesting that Foxa1 is essential for epithelial cell maturation. In
addition, the prostate-specific transcription factor Nkx3.1 is also reduced in
the Foxa1-deficient epithelium. Given the presence of forkhead binding sites
in mouse and human Nkx3.1 gene enhancers (N.G., unpublished), one would
speculate a potential regulatory mechanism may exist.
The relationship between various genetic changes and defects in prostatic
development has been explored by a number of groups (summarized in
Fig. 7). The normal prostate
grows as a result of androgenically driven mesenchymal-epithelial cell
interactions. In the normal prostate, this process results in the generation
of solid epithelial cords that undergo ductal branching morphogenesis and
cytodifferentiation giving rise to distinct basal and luminal cell
populations. The luminal cells of the normal prostate express copious
quantities of secretions (Cunha et al.,
1987).
In the absence of epithelial AR (but in the presence of mesenchymal AR),
prostatic development and differentiation can occur
(Fig. 7). Tissue recombination
experiments with AR-null epithelium results in prostatic tissue containing
apparently normal basal and luminal cells. Glands have a well-defined lumen,
but the luminal epithelial cells lack expression of prostatic secretory
proteins (Cunha and Chung,
1981; Donjacour and Cunha,
1993
). This demonstrates that epithelial AR is not required for
prostatic development and cytodifferentiation but is required for the
initiation and maintenance of secretory activity, the principle differentiated
function of the luminal epithelial cells.
In the Nkx3.1-null mouse, the prostate develops a relatively normal ductal
structure, albeit with reduced ductal branching and ductal tip number.
Epithelial differentiation is somewhat disrupted with the formation of
multilayered hyperplasia, notably in the anterior prostate, and in papillary
tufts (Bhatia-Gaur et al.,
1999). Epithelial cytodifferentiation, as reflected by secretory
activity, is reduced (Fig.
7).
The Foxa1-deficient prostate is severely impeded in terms of ductal
branching morphogenesis, even when compared with the Nkx3.1-null prostate.
Ductal canalization and epithelial cytodifferentiation are profoundly
inhibited. Differentiated secretory protein expression (e.g. Pbsn and Sbp) is
absent even though there is a normal level of AR expression in the Foxa1-null
epithelium. Previous work (Donjacour and
Cunha, 1993) and our study have provided clear evidence that both
AR and Foxa1 regulate prostate development, but Foxa1 plays an early role in
promoting glandular morphogenesis and cytodifferentiation.
The most severe prostatic phenotype (a total failure to develop) can be
elicited by loss of either stromal or total AR (for example in Tfm mice or in
AIS humans) (Cunha et al.,
1987). This demonstrates that stromal and epithelial AR functions
are separate and distinct. Notably, Foxa1 probably functions at two levels
acting both to control glandular morphogenesis and cytodifferentiation
(present study) and secretory function
(Gao et al., 2003
).
Taken together, our study demonstrates that Foxa1 plays a pivotal role in prostate ductal morphogenesis and implies that this protein may crucially involve in modulating the balance of inductive and negative regulators to control prostate cell growth, differentiation and patterning.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/15/3431/DC1
* These authors contributed equally to this work
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abate-Shen, C. and Shen, M. M. (2000).
Molecular genetics of prostate cancer. Genes Dev.
14,2410
-2434.
Bardin, C. W., Bullock, L. P., Sherins, R. J., Mowszowicz, I. and Blackburn, W. R. (1973). Androgen metabolism and mechanism of action in male pseudohermaphroditism: a study of testicular feminization. Recent Prog. Horm. Res. 29, 65-109.[Medline]
Bellusci, S., Furuta, Y., Rush, M. G., Henderson, R., Winnier,
G. and Hogan, B. L. (1997). Involvement of Sonic
hedgehog (Shh) in mouse embryonic lung growth and morphogenesis.
Development 124,53
-63.
Berman, D. M., Desai, N., Wang, X., Karhadkar, S. S., Reynon, M., Abate-Shen, C., Beachy, P. A. and Shen, M. M. (2004). Roles for Hedgehog signaling in androgen production and prostate ductal morphogenesis. Dev. Biol. 267,387 -398.[CrossRef][Medline]
Bhatia-Gaur, R., Donjacour, A. A., Sciavolino, P. J., Kim, M.,
Desai, N., Young, P., Norton, C. R., Gridley, T., Cardiff, R. D.,
Cunha, G. R. et al. (1999). Roles for Nkx3.1 in prostate
development and cancer. Genes Dev.
13,966
-977.
Bierie, B., Nozawa, M., Renou, J. P., Shillingford, J. M., Morgan, F., Oka, T., Taketo, M. M., Cardiff, R. D., Miyoshi, K., Wagner, K. U. et al. (2003). Activation of beta-catenin in prostate epithelium induces hyperplasias and squamous transdifferentiation. Oncogene 22,3875 -3887.[CrossRef][Medline]
Braissant, O. and Wahli, W. (1998).
Differential expression of peroxisome proliferator-activated receptor-alpha,
-beta, and -gamma during rat embryonic development.
Endocrinology 139,2748
-2754.
Canamasas, I., Debes, A., Natali, P. G. and Kurzik-Dumke, U.
(2003). Understanding human cancer using Drosophila: Tid47, a
cytosolic product of the DnaJ-like tumor suppressor gene l2Tid, is a novel
molecular partner of patched related to skin cancer. J. Biol.
Chem. 278,30952
-30960.
Carlsson, P. and Mahlapuu, M. (2002). Forkhead transcription factors: key players in development and metabolism. Dev. Biol. 250,1 -23.[CrossRef][Medline]
Chang, C. S., Saltzman, A. G., Hiipakka, R. A., Huang, I. Y. and
Liao, S. S. (1987). Prostatic spermine-binding
protein. Cloning and nucleotide sequence of cDNA, amino acid sequence, and
androgenic control of mRNA level. J. Biol. Chem.
262,2826
-2831.
Chaurand, P., DaGue, B. B., Ma, S., Kasper, S. and Caprioli, R. M. (2001). Strain-based Squence Variations and Structure Analysis of Murine Prostate Specific Spermine Binding Protein Using Mass Spectrometry. Biochemistry 40,9725 -9733.[CrossRef][Medline]
Chaurand, P., Schwartz, S. A., Billheimer, D., Xu, B. J., Crecelius, A. and Caprioli, R. M. (2004). Integrating histology and imaging mass spectrometry. Anal. Chem. 76,1145 -1155.[CrossRef][Medline]
Cheshire, D. R. and Isaacs, W. B. (2003).
Beta-catenin signaling in prostate cancer: an early perspective.
Endocr. Relat. Cancer
10,537
-560.
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383,407 -413.[CrossRef][Medline]
Cunha, G. R. and Chung, L. W. (1981). Stromal-epithelial interactionsI. Induction of prostatic phenotype in urothelium of testicular feminized (Tfm/y) mice. J. Steroid Biochem. 14,1317 -1324.[CrossRef][Medline]
Cunha, G. R. and Donjacour, A. (1987). Mesenchymal-epithelial interactions: technical considerations. Prog. Clin. Biol. Res. 239,273 -282.[Medline]
Cunha, G. R., Donjacour, A. A., Cooke, P. S., Mee, S., Bigsby, R. M., Higgins, S. J. and Sugimura, Y. (1987). The endocrinology and developmental biology of the prostate. Endocr. Rev. 8,338 -362.[Medline]
Cunha, G. R., Hayward, S. W., Dahiya, R. and Foster, B. A. (1996). Smooth muscle-epithelial interactions in normal and neoplastic prostatic development. Acta Anat. (Basel) 155, 63-72.[Medline]
Di Marcotullio, L., Ferretti, E., De Smaele, E., Argenti, B.,
Mincione, C., Zazzeroni, F., Gallo, R., Masuelli, L., Napolitano, M.,
Maroder, M. et al. (2004). REN(KCTD11) is a suppressor of
Hedgehog signaling and is deleted in human medulloblastoma. Proc.
Natl. Acad. Sci. USA 101,10833
-10838.
Donjacour, A. A. and Cunha, G. R. (1993). Assessment of prostatic protein secretion in tissue recombinants made of urogenital sinus mesenchyme and urothelium from normal or androgen-insensitive mice. Endocrinology 132,2342 -2350.[Abstract]
Donjacour, A. A., Thomson, A. A. and Cunha, G. R. (2003). FGF-10 plays an essential role in the growth of the fetal prostate. Dev. Biol. 261, 39-54.[CrossRef][Medline]
Echelard, Y., Epstein, D. J., St Jacques, B., Shen, L., Mohler, J., McMahon, J. A. and McMahon, A. P. (1993). Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75,1417 -1430.[CrossRef][Medline]
Fan, L., Pepicelli, C. V., Dibble, C. C., Catbagan, W., Zarycki,
J. L., Laciak, R., Gipp, J., Shaw, A., Lamm, M. L., Munoz, A. et
al. (2004). Hedgehog signaling promotes prostate xenograft
tumor growth. Endocrinology
145,3961
-3970.
Freestone, S. H., Marker, P., Grace, O. C., Tomlinson, D. C., Cunha, G. R., Harnden, P. and Thomson, A. A. (2003). Sonic hedgehog regulates prostatic growth and epithelial differentiation. Dev. Biol. 264,352 -362.[CrossRef][Medline]
Furumoto, T. A., Miura, N., Akasaka, T., Mizutani-Koseki, Y., Sudo, H., Fukuda, K., Maekawa, M., Yuasa, S., Fu, Y., Moriya, H. et al. (1999). Notochord-dependent expression of MFH1 and PAX1 cooperates to maintain the proliferation of sclerotome cells during the vertebral column development. Dev. Biol. 210, 15-29.[CrossRef][Medline]
Gao, N., Zhang, J., Rao, M. A., Case, T. C., Mirosevich, J.,
Wang, Y., Jin, R., Gupta, A., Rennie, P. S. and Matusik, R. J.
(2003). The role of hepatocyte nuclear factor-3 alpha (Forkhead
Box A1) and androgen receptor in transcriptional regulation of prostatic
genes. Mol. Endocrinol.
17,1484
-1507.
Gaudet, J. and Mango, S. E. (2002). Regulation
of organogenesis by the Caenorhabditis elegans FoxA protein PHA-4.
Science 295,821
-825.
Hayward, S. W., Baskin, L. S., Haughney, P. C., Cunha, A. R., Foster, B. A., Dahiya, R., Prins, G. S. and Cunha, G. R. (1996). Epithelial development in the rat ventral prostate, anterior prostate and seminal vesicle. Acta Anat. (Basel) 155,81 -93.[Medline]
Horner, M. A., Quintin, S., Domeier, M. E., Kimble, J.,
Labouesse, M. and Mango, S. E. (1998). pha-4, an HNF-3
homolog, specifies pharyngeal organ identity in Caenorhabditis elegans.
Genes Dev. 12,1947
-1952.
Hudson, D. L., Guy, A. T., Fry, P., O'Hare, M. J., Watt, F. M.
and Masters, J. R. (2001). Epithelial cell
differentiation pathways in the human prostate: identification of intermediate
phenotypes by keratin expression. J. Histochem.
Cytochem. 49,271
-278.
Hynes, M., Stone, D. M., Dowd, M., Pitts-Meek, S., Goddard, A., Gurney, A. and Rosenthal, A. (1997). Control of cell pattern in the neural tube by the zinc finger transcription factor and oncogene Gli-1. Neuron 19, 15-26.[CrossRef][Medline]
Ingham, P. W. and McMahon, A. P. (2001).
Hedgehog signaling in animal development: paradigms and principles.
Genes Dev. 15,3059
-3087.
Jaskoll, T., Leo, T., Witcher, D., Ormestad, M., Astorga, J., Bringas, P., Jr, Carlsson, P. and Melnick, M. (2004). Sonic hedgehog signaling plays an essential role during embryonic salivary gland epithelial branching morphogenesis. Dev. Dyn. 229,722 -732.[CrossRef][Medline]
Kalb, J. M., Lau, K. K., Goszczynski, B., Fukushige, T., Moons,
D., Okkema, P. G. and McGhee, J. D. (1998). pha-4 is
Ce-fkh-1, a fork head/HNF-3alpha, beta, gamma homolog that functions in
organogenesis of the C. elegans pharynx. Development
125,2171
-2180.
Karhadkar, S. S., Bova, G. S., Abdallah, N., Dhara, S., Gardner, D., Maitra, A., Isaacs, J. T., Berman, D. M. and Beachy, P. A. (2004). Hedgehog signalling in prostate regeneration, neoplasia and metastasis. Nature 431,707 -712.[CrossRef][Medline]
Kasper, S. and Matusik, R. J. (2000). Rat probasin: structure and function of an outlier lipocalin. Biochim. Biophys. Acta 1482,249 -258.[Medline]
Kim, M. J., Cardiff, R. D., Desai, N., Banach-Petrosky, W. A.,
Parsons, R., Shen, M. M. and Abate-Shen, C. (2002).
Cooperativity of Nkx3.1 and Pten loss of function in a mouse model of prostate
carcinogenesis. Proc. Natl. Acad. Sci. USA
99,2884
-2889.
Kopachik, W., Hayward, S. W. and Cunha, G. R. (1998). Expression of hepatocyte nuclear factor-3alpha in rat prostate, seminal vesicle, and bladder. Dev. Dyn. 211,131 -140.[CrossRef][Medline]
Krebs, O., Schreiner, C. M., Scott, W. J., Jr, Bell, S. M.,
Robbins, D. J., Goetz, J. A., Alt, H., Hawes, N., Wolf, E. and Favor,
J. (2003). Replicated anterior zeugopod (raz): a
polydactylous mouse mutant with lowered Shh signaling in the limb bud.
Development 130,6037
-6047.
Kurita, T., Medina, R. T., Mills, A. A. and Cunha, G. R.
(2004). Role of p63 and basal cells in the prostate.
Development 131,4955
-4964.
Lamm, M. L., Podlasek, C. A., Barnett, D. H., Lee, J., Clemens, J. Q., Hebner, C. M. and Bushman, W. (2001). Mesenchymal factor bone morphogenetic protein 4 restricts ductal budding and branching morphogenesis in the developing prostate. Dev. Biol. 232,301 -314.[CrossRef][Medline]
Lamm, M. L., Catbagan, W. S., Laciak, R. J., Barnett, D. H., Hebner, C. M., Gaffield, W., Walterhouse, D., Iannaccone, P. and Bushman, W. (2002). Sonic hedgehog activates mesenchymal Gli1 expression during prostate ductal bud formation. Dev. Biol. 249,349 -366.[CrossRef][Medline]
Lessard, J. L. (1988). Two monoclonal antibodies to actin: one muscle selective and one generally reactive. Cell Motil. Cytoskeleton 10,349 -362.[CrossRef][Medline]
Mahlapuu, M., Enerback, S. and Carlsson, P. (2001). Haploinsufficiency of the forkhead gene Foxf1, a target for sonic hedgehog signaling, causes lung and foregut malformations. Development 128,2397 -2406.[Medline]
Marker, P. C., Donjacour, A. A., Dahiya, R. and Cunha, G. R. (2003). Hormonal, cellular, and molecular control of prostatic development. Dev. Biol. 253,165 -174.[CrossRef][Medline]
Mirosevich, J., Gao, N. and Matusik, R. J. (2005). Expression of Foxa transcription factors in the developing and adult murine prostate. Prostate 62,339 -352.[CrossRef][Medline]
Niemann, C., Unden, A. B., Lyle, S., Zouboulis, C., Toftgard, R.
and Watt, F. M. (2003). Indian hedgehog and
beta-catenin signaling: role in the sebaceous lineage of normal and neoplastic
mammalian epidermis. Proc. Natl. Acad. Sci. USA
100,11873
-11880.
Peterson, R. S., Clevidence, D. E., Ye, H. and Costa, R. H. (1997). Hepatocyte nuclear factor-3 alpha promoter regulation involves recognition by cell-specific factors, thyroid transcription factor-1, and autoactivation. Cell Growth Differ. 8, 69-82.[Abstract]
Podlasek, C. A., Barnett, D. H., Clemens, J. Q., Bak, P. M. and Bushman, W. (1999). Prostate development requires Sonic hedgehog expressed by the urogenital sinus epithelium. Dev. Biol. 209,28 -39.[CrossRef][Medline]
Pu, Y., Huang, L. and Prins, G. S. (2004). Sonic hedgehog-patched Gli signaling in the developing rat prostate gland: lobe-specific suppression by neonatal estrogens reduces ductal growth and branching. Dev. Biol. 273,257 -275.[CrossRef][Medline]
Qian, J., Kumar, A., Szucsik, J. C. and Lessard, J. L. (1996). Tissue and developmental specific expression of murine smooth muscle gamma-actin fusion genes in transgenic mice. Dev. Dyn. 207,135 -144.[CrossRef][Medline]
Sahlen, G. E., Egevad, L., Ahlander, A., Norlen, B. J., Ronquist, G. and Nilsson, B. O. (2002). Ultrastructure of the secretion of prostasomes from benign and malignant epithelial cells in the prostate. Prostate 53,192 -199.[CrossRef][Medline]
Sanchez, P., Hernandez, A. M., Stecca, B., Kahler, A. J.,
DeGueme, A. M., Barrett, A., Beyna, M., Datta, M. W., Datta, S. and
Altaba, A. (2004). Inhibition of prostate cancer
proliferation by interference with SONIC HEDGEHOG-GLI1 signaling.
Proc. Natl. Acad. Sci. USA
101,12561
-12566.
Sasaki, H., Hui, C., Nakafuku, M. and Kondoh, H.
(1997). A binding site for Gli proteins is essential for
HNF-3beta floor plate enhancer activity in transgenics and can respond to Shh
in vitro. Development
124,1313
-1322.
Schneider, A., Brand, T., Zweigerdt, R. and Arnold, H. (2000). Targeted disruption of the Nkx3.1 gene in mice results in morphogenetic defects of minor salivary glands: parallels to glandular duct morphogenesis in prostate. Mech. Dev. 95,163 -174.[CrossRef][Medline]
Shih, D. Q., Navas, M. A., Kuwajima, S., Duncan, S. A. and
Stoffel, M. (1999). Impaired glucose homeostasis and neonatal
mortality in hepatocyte nuclear factor 3alpha-deficient mice. Proc.
Natl. Acad. Sci. USA 96,10152
-10157.
Sheng, T., Li, C., Zhang, X., Chi, S., He, N., Chen, K., McCormick, F., Gatalica, Z. and Xie, J. (2004). Activation of the hedgehog pathway in advanced prostate cancer. Mol. Cancer 3,29 .[CrossRef][Medline]
Shou, J., Ross, S., Koeppen, H., De Sauvage, F. J. and Gao, W.
Q. (2001). Dynamics of notch expression during murine
prostate development and tumorigenesis. Cancer Res.
61,7291
-7297.
Signoretti, S., Waltregny, D., Dilks, J., Isaac, B., Lin, D.,
Garraway, L., Yang, A., Montironi, R., McKeon, F. and Loda, M.
(2000). p63 is a prostate basal cell marker and is required for
prostate development. Am. J. Pathol.
157,1769
-1775.
Sinner, D., Rankin, S., Lee, M. and Zorn, A. M.
(2004). Sox17 and beta-catenin cooperate to regulate the
transcription of endodermal genes. Development
131,3069
-3080.
Tanaka, M., Komuro, I., Inagaki, H., Jenkins, N. A., Copeland, N. G. and Izumo, S. (2000). Nkx3.1, a murine homolog of Ddrosophila bagpipe, regulates epithelial ductal branching and proliferation of the prostate and palatine glands. Dev. Dyn. 219,248 -260.[CrossRef][Medline]
Thayer, S. P., di Magliano, M. P., Heiser, P. W., Nielsen, C. M., Roberts, D. J., Lauwers, G. Y., Qi, Y. P., Gysin, S., Fernandez-del Castillo, C., Yajnik, V. et al. (2003). Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature 425,851 -856.[CrossRef][Medline]
Thibert, C., Teillet, M. A., Lapointe, F., Mazelin, L., Le
Douarin, N. M. and Mehlen, P. (2003). Inhibition of
neuroepithelial patched-induced apoptosis by sonic hedgehog.
Science 301,843
-846.
Thomson, A. A. and Cunha, G. R. (1999).
Prostatic growth and development are regulated by FGF10.
Development 126,3693
-3701.
Thomson, A. A., Foster, B. A. and Cunha, G. R.
(1997). Analysis of growth factor and receptor mRNA levels during
development of the rat seminal vesicle and prostate.
Development 124,2431
-2439.
Wan, H., Dingle, S., Xu, Y., Besnard, V., Kaestner, K. H., Ang, S. L., Wert, S., Stahlman, M. T. and Whitsett, J. A. (2005). Compensatory roles of Foxa1 and Foxa2 during lung morphogenesis. J. Biol. Chem. 14,13809 -13816.[CrossRef]
Wang, B. E., Shou, J., Ross, S., Koeppen, H., De Sauvage, F. J.
and Gao, W. Q. (2003). Inhibition of epithelial ductal
branching in the prostate by sonic hedgehog is indirectly mediated by stromal
cells. J. Biol. Chem.
278,18506
-18513.
Wang, X. D., Shou, J., Wong, P., French, D. M. and Gao, W.
Q. (2004). Notch1-expressing cells are indispensable for
prostatic branching morphogenesis during development and re-growth following
castration and androgen replacement. J. Biol. Chem.
279,24733
-24744.
Wang, Y., Hayward, S. W., Donjacour, A. A., Young, P., Jacks,
T., Sage, J., Dahiya, R., Cardiff, R. D., Day, M. L. and Cunha, G.
R. (2000). Sex hormone-induced carcinogenesis in Rb-deficient
prostate tissue. Cancer Res.
60,6008
-6017.
Wang, Y., Hayward, S., Cao, M., Thayer, K. and Cunha, G. (2001). Cell differentiation lineage in the prostate. Differentiation 68,270 -279.[CrossRef][Medline]
Weaver, M., Batts, L. and Hogan, B. L. (2003). Tissue interactions pattern the mesenchyme of the embryonic mouse lung. Dev. Biol. 258,169 -184.[CrossRef][Medline]
Weigel, D. and Jackle, H. (1990). The fork head domain: a novel DNA binding motif of eukaryotic transcription factors? Cell 63,455 -456.[CrossRef][Medline]
Wells, J. M. and Melton, D. A. (1999). Vertebrate endoderm development. Annu. Rev. Cell Dev. Biol. 15,393 -410.[CrossRef][Medline]
Yamagishi, H., Maeda, J., Hu, T., McAnally, J., Conway, S. J.,
Kume, T., Meyers, E. N., Yamagishi, C. and Srivastava, D.
(2003). Tbx1 is regulated by tissue-specific forkhead proteins
through a common Sonic hedgehog-responsive enhancer. Genes
Dev. 17,269
-281.
Yu, J., Carroll, T. J. and McMahon, A. P. (2002). Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney. Development 129,5301 -5312.[Medline]
Zaret, K. (1999). Developmental competence of the gut endoderm: genetic potentiation by GATA and HNF3/fork head proteins. Dev. Biol. 209,1 -10.[CrossRef][Medline]
Zaret, K. S. (2002). Regulatory phases of early liver development: paradigms of organogenesis. Nat. Rev. Genet. 3,499 -512.[CrossRef][Medline]