Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109-0616, USA
* Author for correspondence (e-mail: dgumucio{at}umich.edu)
Accepted 9 November 2004
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SUMMARY |
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Key words: Hedgehog, Wnt, Intestine, Epithelial-mesenchymal crosstalk, Mouse
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Introduction |
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In the small intestine, this inter-tissue communication is first visible at
E14.5. At this time, under the influence of the mesenchyme, the stratified
squamous epithelium is remodeled to form intestinal villi lined with a single
layer of columnar epithelium (Mathan et
al., 1976). Morphological evidence of patterning along the
crypt-villus axis is seen by E16.5 (Calvert
and Pothier, 1990
), when proliferative cells become concentrated
in the flat intervillus regions that will later postnatally give rise to the
crypts, the eventual home of intestinal stem cells. Studies indicate that
crypt development proceeds by anchorage of prospective stem cells to the
intervillus region and remodeling of the villus above these anchored cells to
form flask-shaped crypt structures (Calvert
and Pothier, 1990
), but the signals responsible for the selective
localization of the precrypt structures at the base of the villi have not been
identified. The mechanical process of crypt formation may be assisted by the
action of the contractile subepithelial pericryptal myofibroblasts, or ISEMFs
(Powell et al., 1999
). ISEMFs
are thought to exist as a syncytium of cells, concentrated in the crypt
regions and extending into villus tips where they merge with pericytes
(Powell et al., 1999
). In
addition to their probable mechanical action in shaping the crypts, ISEMFs are
a major source of instructive signals to the epithelium, and are capable of
promoting epithelial proliferation or differentiation via soluble and/or
membrane-tethered signals (Kedinger et
al., 1998
). Alterations in the character and/or distribution of
ISEMFs are associated with several intestinal pathologies, including Crohn's
disease and adenomatous colorectal polyps
(Adegboyega et al., 2002
;
Andoh et al., 2002
).
Both the hedgehog (Hh) and Wnt signaling pathways are likely to play
important roles in intestinal crypt-villus axis formation and stem cell
homeostasis. Several Wnt proteins, as well as their Frizzled receptors, are
expressed in both epithelial and mesenchymal compartments
(Theodosiou and Tabin, 2003).
Tcf4 (Tcf7l2), a transcriptional effector of Wnt signals, is
localized to the intervillus epithelium as early as E16.5
(Korinek et al., 1998
). Mice
lacking Tcf4 exhibit fewer villi, and severely reduced proliferation in the
intervillus regions (Korinek et al.,
1998
), evidence that Tcf4 is required for establishment and/or
maintenance of the crypt stem cell population. Furthermore, proliferation in
the intestinal crypt compartment is Wnt ligand dependent
(Pinto et al., 2003
).
Interrupting Wnt signals by expression of a pan-Wnt inhibitor (Dkk1)
in the intestine profoundly reduces epithelial proliferation. This is
accompanied by decreased Myc expression and increased expression of
the cell cycle inhibitor, p21 (Cdkn1a/Cip1/Waf1). Thus, Wnt
ligands control a master switch between proliferation and differentiation
along the crypt-villus axis (van de
Wetering et al., 2002
).
Sonic (Shh) and Indian (Ihh) hedgehog proteins also play important roles in
small intestinal morphogenesis. Initially expressed throughout the epithelium,
both proteins are redistributed after villus formation, becoming concentrated
in cells of the intervillus region
(Ramalho-Santos et al., 2000).
Mice deficient in Shh or Ihh display extensive
gastrointestinal phenotypes
(Ramalho-Santos et al., 2000
).
Ihh-/- mice die perinatally and exhibit reduced
proliferation in the intervillus region, leading to a depleted progenitor cell
compartment (Ramalho-Santos et al.,
2000
), suggesting that Ihh is critical for the
maintenance of intestinal stem cells. Interestingly,
Shh-/- mice display the opposite phenotype: overgrowth of
duodenal villi (and stomach epithelium). This implies that Shh may
inhibit rather than stimulate proliferation in these regions
(Ramalho-Santos et al., 2000
).
Though the epithelial phenotypes of Shh vs. Ihh null mice
are disparate, both models exhibit reduced smooth muscle, suggesting that
these two hedgehog signals also have partially redundant functions.
The goal of the work described here was to explore the role of the combined
Shh and Ihh signal in late intestinal development. Since mice doubly deficient
for Shh and Ihh do not develop past early somite stages, we
engineered an intestine-specific blockade of Hh signals by overexpression of
the pan-Hh inhibitor, Hhip. We find that even minor reductions in Hh signals
are sufficient to produce a robust increase in epithelial proliferation. The
epithelial effect of Hh perturbation is indirect since our data indicate that
Hh signals are paracrine and impact ISEMFs and smooth muscle cells (SMCs).
When Hh signals are reduced, ISEMFs, a potential source of Wnt signals
(Brittan and Wright, 2004), are
abnormally located in villus tips in association with proliferating epithelial
cells that express high levels of Tcf4/ß-catenin target genes. Thus, the
dose of the Hh signal to ISEMFs is normally under tight control; through this
signal the epithelium indirectly regulates the size and location of its own
proliferative compartment, effectively controlling the organized patterning of
the crypt-villus axis.
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Materials and methods |
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Villin-noggin transgenic production and immunohistochemistry
The noggin cDNA was amplified by RT-PCR of E12.5 C57BL/6 whole mouse embryo
RNA, TOPO cloned into pCR2.1-TOPO (Invitrogen), and the sequence was verified.
The plasmid was digested with SacII and ApaI and cloned into pBSII-SK+
(Stratagene) digested with SacII and XbaI to create pBSII-noggin. This was
digested with SacII and KpnI to remove the cDNA and cloned into SacII and KpnI
sites 3' of the 12.4Kb villin promoter/enhancer fragment
(Madison et al., 2002) and
5' of an SV40 polyadenylation sequence from the pGL2-basic (Promega)
plasmid to create the p12.4KVil-noggin plasmid with a puc18 backbone for
growth in E. Coli. The transgene fragment was excised by digestion
with PmeI and injected into pronuclei of C57BL6/SJL fertilized oocytes. PCR
genotyping of tail DNA was used to identify transgenic founders. Fourteen
founders were identified, and ten expressed the transgene mRNA.
Protein expression in founders was evaluated by immunohistochemistry for noggin protein using goat anti-noggin antiserum (R&D Systems, 1:250). Immunohistochemistry was performed on 6 µm paraffin sections. Sections were de-paraffinized, rehydrated, and non-specific peroxidase activity was blocked for 30 minutes in 1.5% H2O2 in PBS. Antigen retrieval was performed by boiling for 10 minutes in 0.01 M sodium citrate buffer, pH 6.0, and cooling on ice for 15 minutes. Immunostaining was performed using the TSA-biotin system (Perkin Elmer), and staining developed with SigmaFast DAB (Sigma).
Epithelial-mesenchymal separation of E18.5 embryonic small intestine
Small intestines from E18.5 C57BL/6 embryos were opened longitudinally and
incubated for 8 hours in Cell Recovery Solution (BD Pharmingen) at 4°C.
Gentle shaking separated the epithelium from the mesenchyme. The mesenchyme
was removed with tweezers and homogenized in 1 ml of TRIzol (Invitrogen) to
prepare RNA. The epithelium was pelleted by centrifugation, and resuspended in
1 ml of TRIzol. Using the iScript cDNA synthesis kit (Biorad), cDNA was
synthesized from 1 µg of total epithelial or mesenchymal RNA. To detect
expressed genes, PCR was performed for 30 cycles using primers indicated in
Table S1 in the supplementary material.
ISEMF cell culture
Rat intestinal ISEMF cell lines MIC101, MIC216, and MIC316 were obtained
from Dr Michele Kedinger (INSERM, Strasbourg, France) and cultured in DMEM-F12
nutrient mixture, with 10% fetal bovine serum (FBS) at 37°C, 5%
CO2. The human colonic ISEMF cell line CCD-18Co (ATCC, #CRL-1459)
was cultured according to ATCC specifications. Shh-conditioned medium was
prepared by Fugene (Roche) transfection of Cos7 cells (maintained in DMEM, 10%
FBS) with 4 µg of pcDNA3.1 or pcDNA3.1-Shh expression plasmid per 10 cm
plate. The pcDNA3.1-Shh expression vector was obtained by cloning the
full-length cDNA for Shh (obtained by RT-PCR of E18.5 small intestine
RNA) into an EcoR1 site in pcDNA3.1 by standard techniques. Eighteen hours
after transfection, Cos7 medium was replaced with medium specific to each
ISEMF cell line and incubated for 30 additional hours. Conditioned medium was
filtered through a 0.22 µm filter and stored at -80°C. ISEMF cell lines
grown in six-well plates were treated with conditioned medium for 24 and 72
hours. During the 72-hour incubation, fresh conditioned medium was added after
48 hours. Medium was then aspirated, RNA prepared using TRIzol, and cDNA
synthesized as described above. All tissue reagents are from Invitrogen
Corporation, unless indicated otherwise.
E18.5 intestinal mesenchyme culture and stimulation with Shh-N
Small intestines from E18.5 C57BL/6 embryos were opened longitudinally and
incubated for 8 hours in Cell Recovery Solution (BD Pharmingen) at 4°C.
Gentle shaking separated the epithelium from the mesenchyme. The mesenchyme
was removed with tweezers, finely minced with a scalpel, and cultured on
collagen-coated 12-well plates in DMEM, 10% FBS, 10 mM Hepes. Medium was
changed after 24 hours, replaced with new medium, and cultured for an
additional 24 hours. Tissue was then treated for 12, 36, or 48 hours with 0.1,
0.5, or 2.5 µg/ml recombinant mouse Shh N-terminal polypeptide (Shh-N,
R&D Systems) or equivalent amount of vehicle (5% trehalose, 12.5 mg/ml
BSA, in PBS). All reagents were obtained from Invitrogen Corporation, unless
stated otherwise.
Histology, immunohistochemistry, immunofluorescence, and in situ hybridization
Standard histological procedures were used for H and E staining, Alcian
Blue staining, and Oil Red-O staining. X-gal stain of
Ptch1+/nLacZ neonatal jejunum was performed on cryostat
sections as previously described (Madison
et al., 2002). For subsequent
-smooth muscle actin
(
-SMA) and desmin immunofluorescence, slides were washed three times
with PBS and incubated overnight with mouse Cy3-conjugated anti-
-SMA,
(Sigma, 1:500) and rabbit anti-desmin (AbCam, 1:250), followed by incubation
with goat anti-rabbit Alexafluor 488 (Molecular Probes, 1:1000).
Ptch1+/nLacZ mice are from Jackson Laboratories, strain
B6;129-Ptchtm1Mps/J, #003081.
Immunohistochemistry was performed on 6 µm paraffin sections. Sections were de-paraffinized, rehydrated, and nonspecific peroxidase activity blocked for 30 minutes in 1.5% H2O2 in PBS. Antigen retrieval was performed by boiling for 10 minutes in 0.01 M sodium citrate buffer, pH 6.0, and cooled on ice for 15 minutes. Immunostaining was performed using the TSA-biotin system (Perkin Elmer), and developed with SigmaFast DAB (Sigma). Antibodies used were: mouse anti-Hh (Developmental Studies Hybridoma Bank, clone 5E1, 1:1000), goat anti-Hhip (Santa Cruz, 1:50), rabbit anti-Ki67 (Novocastra, 1:3000), rabbit anti-Cdx1 (courtesy of Dr Debra Silberg, 1:500), goat anti-EphB2 (R&D Systems, 1:50), rat anti-CD44v6 (Bender MedSystems, 1:1000), rabbit anti-iFABP (courtesy of Dr Jeffrey Gordon, 1:400), rabbit anti-Chromogranin A (ImmunoStar, 1:10,000).
For -SMA, Ki67, and desmin immunofluorescence, 6 µm paraffin
sections were prepared as described above. Sections were blocked for 30
minutes at room temperature with 10% goat serum, 1% BSA, and 0.3% Triton-X
100, in PBS. Cy3-conjugated mouse anti-
-SMA (Sigma, 1:500), rabbit
anti-Ki67 (Novocastra, 1:1000), and rabbit anti-desmin (Abcam, 1:250) were
incubated overnight at 4°C, followed by incubation with goat anti-rabbit
Alexafluor 488 (Molecular Probes, 1:1000).
Frozen sections were prepared by freezing tissue in OCT (TissueTek), sectioning at 4 µm, and fixing for 5 minutes in ice-cold 4% formaldehyde in PBS. The following antibodies were used: mouse anti-ß-catenin (Transduction Laboratories, 1:1000), rabbit anti-CCK (Chemicon, 1:1000), rabbit anti-peripherin (Chemicon, 1:1000), rabbit anti-cGKII (courtesy of Dr Michael Uhler, 1:500), and Phalloidin-FITC (Molecular Probes, 1:250). Fluorophore-labeled secondary antibodies used were goat anti-rabbit Alexafluor 568 and goat anti-mouse Alexafluor 488 (Molecular Probes, 1:1000).
For in situ hybridization, a digoxigenin-labeled cRNA probe was created
from a 959 bp cDNA fragment of the Ihh mRNA (nucleotides 828-1786, NCBI
Accession Number BC046984) covering the exon 2-3 boundary and part of the
3' UTR, and hybridized at 100-200 ng/µl on 10 µM cryosections from
E18.5 C57BL/6 intestinal tissue. All other procedures were performed as
described (Prado et al.,
2004).
Quantitative RT-PCR analysis (Q-RT-PCR)
Cultured intestinal mesenchyme or whole tissue from wild-type or transgenic
jejunum was homogenized in TRIzol (Invitrogen). Using the iScript cDNA
synthesis kit (Biorad), cDNA was synthesized from 1 µg of total RNA.
Q-RT-PCR was performed using SybrGreen incorporation on a BioRad iCycler using
primers listed in Table S1 (see supplementary material). Threshold cycles were
normalized to threshold cycles for HPRT.
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Results |
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Blockade of Hh signals in villin-Hhip mice
Hhip attenuates Hh activity by binding to all Hh proteins with high
affinity (Chuang and McMahon,
1999). The phenotype of Hhip-/- mice is
consistent with increased Hh signaling
(Chuang et al., 2003
;
Kawahira et al., 2003
), while
overexpression of Hhip mirrors loss of Hh signaling
(Chuang and McMahon, 1999
).
The Hhip cDNA used in the villin-Hhip transgene contains a
deletion of the C-terminal 22 amino acid transmembrane domain; this secreted
form of the Hhip protein is an effective inhibitor of Shh as well as Ihh
signaling, in vitro and in vivo
(Chuang and McMahon, 1999
;
Treier et al., 2001
).
The mouse villin promoter directs high-level transgene expression
exclusively in the intestinal epithelium as early as E12.5, and is largely
resistant to chromosomal position effects
(Madison et al., 2002). Our
study of villin-Hhip transgenic mice encompassed analysis of 18
independent founders, 14 of which demonstrated a phenotype, though the level
of transgene expression varied widely (Fig.
1G,H, top). Villin-Hhip mice exhibited a variable, but
significant reduction of two direct transcriptional targets of Hh signals
(Ptch1 and Gli1) for all eight founders assayed
(Fig. 1H, bottom), confirming
that Hhip
TM overexpression attenuated Hh signaling to varying
degrees.
Reduction in Hh signaling affects villus morphogenesis and epithelial proliferation
Villin-Hhip founders were sacrificed at P0, P2, P3, or P5, and
intestines were examined histologically. In the three founders expressing the
highest levels of the transgene and exhibiting over 75% reduction in
Ptch1 mRNA (Founders #4, 5, 6,
Fig. 1H) the epithelium was
flattened (Fig. 2B-D). Ki67
staining revealed that the flattening was not due to loss of proliferative
cells; rather, the epithelium was hyperproliferative
(Fig. 2F-H) and in some
regions, exhibited a pseudostratified rather than simple columnar form
(Fig. 2F, arrows, insets). In
some areas from high-expressing (but not low-expressing) founders, the
muscularis externa was severely reduced (see
Fig. 4I), a feature also
observed, though to lesser extent, in Ihh and Shh null mice
(Ramalho-Santos et al., 2000).
Hyper-proliferation and stratification of the epithelium concomitant with
blunted villi suggests that strong attenuation of Hh signaling interferes with
epithelial remodeling and villus formation in the fetal small intestine.
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As intestinal cells differentiate along the crypt-villus axis, the apical
brush border matures; F-actin becomes more concentrated at the apical surface
(Stappenbeck and Gordon, 2000)
and microvilli increase in length. In high-expressing founders, electron
microscopy revealed a poorly developed brush border on villus tips
(Fig. 4G). Brush
border-associated F-actin was significantly reduced
(Fig. 4I,J). Together, these
data indicate that the normal program of cellular differentiation along the
crypt-villus axis is compromised in villin-Hhip mice. Defects in
enterocyte function (and thus nutrient absorption) could contribute to
neonatal wasting of villin-Hhip mice.
Though earlier studies documented somewhat reduced numbers of
cholecystokinin (CCK) cells in Ihh-/- mice, these cells
were normally represented in villin-Hhip mice, as were goblet cells
(see Fig. S1A-F in the supplementary material). Similarly, while alterations
in the number or positioning of enteric neurons were observed in both
Shh-/- and Ihh-/- models, no obvious
changes in neuronal patterning were observed in villin-Hhip mice
(Fig. 4I,J). This latter
discrepancy may be explained by temporal differences. Since differentiated
enteric neurons can be found in mouse intestine as early as E10.5
(Young et al., 1999) and the
villin promoter drives expression no earlier than E12.5
(Madison et al., 2002
),
neuronal differentiation probably precedes Hhip
TM expression. Together,
the data indicate that enteric neurons, once established, can be maintained in
the presence of reduced Hh signals.
Villin-Hhip mice exhibit increased Tcf4/ß-catenin activity in the intestinal epithelium
The Wnt pathway, through its control of target genes such as
c-myc, regulates the master switch between proliferation vs.
differentiation in the intestinal epithelium
(Pinto et al., 2003;
van de Wetering et al., 2002
).
Clearly, this switch is perturbed in villin-Hhip mice. Wnt signals
are also critical for maintenance of the crypt compartment
(van de Wetering et al., 2002
)
and villin-Hhip mice exhibit an increase in precrypt-like structures.
Finally, branched villi, in association with increased epithelial
proliferation, were also seen in transgenic mice in which the canonical Wnt
pathway was enhanced by overexpression of a constitutively active form of
ß-catenin in the intestinal epithelium
(Wong et al., 1998
). All of
these features prompted us to examine markers of epithelial Wnt signaling in
the villin-Hhip model. In wild-type mice, known Tcf4/ß-catenin
target genes (Cdx1, Cd44 and Ephb2) are normally restricted
to the crypt compartment (Batlle et al.,
2002
; Lickert et al.,
2000
; Wielenga et al.,
1999
). All three of these markers were highly expressed throughout
the epithelium of transgenic founders (Fig.
5B,D,F). In P2 to P5 transgenic mice, increased expression of
these markers was apparent in the ectopic crypt-like structures
(Fig. 5D,F, insets). The
c-myc proto-oncogene, a known Tcf4/ß-catenin target, was also
significantly upregulated (Fig.
5G). To further investigate this apparent increase in canonical
Wnt signaling, we examined the intracellular localization of ß-catenin.
In normal villus tip cells, where Wnt signals are low or absent,
ß-catenin is membrane associated. In contrast, intervillus epithelial
cells display cytoplasmic and nuclear ß-catenin, reflecting Wnt activity
(Fig. 5I, arrows). In
villin-Hhip mice this pattern is perturbed: increased levels of
cytoplasmic and nuclear ß-catenin are observed, even in villus tip
epithelial cells (Fig. 5J,K,
arrows).
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Villin-noggin mice were also generated by Haramis et al.
(Haramis et al., 2004).
Consistent with our own findings, neonatal villin-noggin mice showed
no obvious phenotype. However, after P28, these mice exhibited an expanded
stromal compartment and developed ectopic crypt structures on villi - features
very similar to our villin-Hhip mice. This ultimately leads to the
formation of intraepithelial neoplastic lesions similar to those seen in
patients with juvenile polyposis syndrome (JPS). It is possible that the
villin-Hhip model, in which two signaling pathways (Bmps and Hh) are
perturbed in the neonate, unveils an early cooperation between Bmp and Hh
signals to spatially restrict formation of the crypt compartment to the
intervillus base, a function that may be mainly served by Bmps alone later in
life.
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Discussion |
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Hh signals impact subepithelial myofibroblasts
ISEMFs actively participate in epithelial-mesenchymal crosstalk, regulating
both epithelial proliferation and differentiation (reviewed by
Kedinger et al., 1998;
Powell et al., 1999
). Though
normally concentrated in pericryptal regions, ISEMFs can be mobilized by
chemotactic signals such as TGFß, endothelin 1, and PDGF-BB, and this is
important during injury repair (De Wever
and Mareel, 2003
; Marra et
al., 1999
; Powell et al.,
1999
). In adenocarcinomas, ISEMFs are amplified
(Adegboyega et al., 2002
;
Sappino et al., 1989
), and
ISEMF-derived factors are thought to be important in the proliferation,
adhesion, and migration of tumor cells
(Bhowmick et al., 2004
;
Dignass et al., 1994
). The
work presented here shows that ISEMFs represent a major target for Hh
signaling in the intestine. In villin-Hhip mice, these cells are
abnormally distributed in villus tips, in close association with ectopic
proliferating epithelial cells and mislocalized precrypt structures. Thus, we
conclude that Hh signals sent to epithelially associated ISEMFs localize the
precrypt structure and maintain the organization of the crypt-villus axis.
Mitogenic and inhibitory effects of Hh on intestinal smooth muscle (SM) patterning
Besides ISEMFs, two additional cell types in the intestinal mesenchyme
respond to Hh as measured by Ptch1+/nLacZ staining: SMCs of the
innermost layer of the ME; and -SMA(-)/desmin(+) cells within the
lamina propria. The SM phenotypes of villin-Hhip mice may reflect
alterations in both these populations; transgene expression level also clearly
plays a role in these differential responses. In mice with high levels of
Hhip
TM, the ME is severely reduced. This reduction is more severe than
is seen in either Shh-/- or Ihh-/-
mice, suggesting that Shh and Ihh play cooperative mitogenic roles in the
development of this structure. Previous studies in the kidney
(Yu et al., 2002
), lung
(Miller et al., 2004
;
Weaver et al., 2003
) and gut
(Roberts et al., 1998
;
Smith et al., 2000
) are
consistent with a mitogenic effect of Hh signals on SM progenitors. In
villin-Hhip mice with mild reductions in Hh signaling,
desmin(+)/
-SMA(-) or
-SMA(lo) cells (probably immature SMC) are
expanded in villus core lamina propria. This suggests that Hh acts to inhibit
the proliferation or differentiation of SM in this compartment. In fact,
epithelial Hh has been shown to direct the circumferential patterning of chick
gut by inhibiting SM differentiation in the proximal lamina propria, thereby
ensuring the development of the ME only in the outermost layers of the gut
tube (Sukegawa et al., 2000
).
Both mitogenic and inhibitory effects of Hh have also been demonstrated in the
kidney (Yu et al., 2002
). In
the villin-Hhip model, these effects are dose dependent and could
reflect the morphogenic effects of Hh
(Tabata and Takei, 2004
), or
may be secondary to the timing of transgene expression (the transgene is
activated earlier in high expressors). However, since two SM cell types of the
ME and the lamina propria are Hh targets, the apparently opposite responses
may reflect cell-intrinsic differences, perhaps modified by the distinct
cellular neighborhoods inhabited by these cells.
Hh signals and epithelial patterning
The effects of Hh attenuation on the epithelium are dramatic. These effects
are indirect - the result of altered secondary signals from the mesenchyme.
Mice expressing the highest levels of HhipTM exhibit an immature
epithelium with poorly formed villi and impaired conversion from a
pseudostratified epithelium to a simple columnar morphology. Previous studies
in the mouse have demonstrated that villus morphogenesis is partially
coordinated through epithelial secretion of platelet-derived growth factor A
(PDGFA), which stimulates mesenchymal condensation, proliferation, and
evagination of overlying epithelium to form villi
(Karlsson et al., 2000
).
However, Pdgfa null mice do not demonstrate complete loss of villi.
It is possible that Hh and PDGF, both epithelial signals, cooperate to mediate
this mesenchymally driven remodeling process.
Our studies further demonstrate that subsequent to villus formation, Hh
signals are required to organize the crypt-villus axis such that the
proliferative compartment is properly localized to the intervillus base.
Interestingly, though the epithelial cells expressing Hh are the same cells
that normally proliferate in response to Wnt signals
(Korinek et al., 1998;
Pinto et al., 2003
), our
results reveal that Hh signals are not required for epithelial proliferation,
maintenance of epithelial Wnt target gene activity, or for formation of
precrypt structures, since all of these activities are stimulated in
villin-Hhip mice. In light of the concurrent finding of mislocalized
ISEMFs in villin-Hhip mice, we speculate that reduced Hh levels may
interfere with an anchoring activity responsible for restricting the precrypt
cells to the intervillus region. Though details of this anchoring mechanism
are not complete, these studies clearly demonstrate that Hh-mediated paracrine
signaling is responsible for organizing the proliferative compartment of the
intestinal epithelium. It is intriguing that while both the adult colon and
neonatal small intestine both employ Hh signals to restrict epithelial
proliferation and Wnt target gene activity, this action is mediated by very
different routes (van den Brink et al.,
2004
). In the colon, Ihh and Tcf4 mRNAs are
found in two different cellular compartments (differentiated and proliferative
cells, respectively) and the two signaling pathways mutually repress each
other in a manner that requires autocrine signaling by Ihh.
Hh signaling and tumorigenesis
We demonstrate here that epithelial proliferation in the neonatal small
intestine is highly sensitive to the level of Hh signaling. This predicts that
alterations in components of the Hh pathway that lead to reduced Hh signaling
could potentially predispose to tumors. That is, in a slight twist to the
original Kinzler and Vogelstein description
(Kinzler and Vogelstein,
1998), the epithelial Hh signal has an `auto-landscaper' effect
via its control of mesenchymal to epithelial crosstalk. The pro-proliferative
effect of reduced Hh signaling appears contrary to recent findings that in
several other endodermally derived organs (lung, stomach, biliary tree, and
pancreas), overexpression of Hh ligands in the epithelium is associated with
cancer (Berman et al., 2003
;
Thayer et al., 2003
;
Watkins et al., 2003
).
Importantly, these tumors rely on Hh ligands for autocrine rather than
paracrine stimulation of tumor growth. In fact, inhibition of Hh signals has
been proposed as a potential therapy for these aggressive tumors
(Berman et al., 2003
;
Thayer et al., 2003
;
Watkins et al., 2003
). It
seems clear that reduced Hh signaling activity promotes epithelial
proliferation in both neonatal small intestine (this report) and adult colon
(van den Brink et al., 2004
),
though this has yet to be tested in adult small intestine. Thus, therapies
aimed at shrinking gastric, pulmonary, or pancreatic cancers by untargeted
pharmacological inhibition of Hh could potentially augur disastrous intestinal
pathology.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/2/279/DC1
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