Download Hedgehog Signaling in Development and Homeostasis of the

Physiol Rev 87: 1343–1375, 2007;
doi:10.1152/physrev.00054.2006.
Hedgehog Signaling in Development and Homeostasis
of the Gastrointestinal Tract
GIJS R. VAN DEN BRINK
Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam; Department of
Gastroenterology and Hepatology, Leiden University Medical Center, Leiden, The Netherlands
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
I. Introduction
II. The Hedgehog Pathway
A. Hedgehog secretion
B. Hedgehog reception
C. Hedgehog intracellular signaling
D. Identifying Hedgehog target cells
E. Hedgehog pathway antibodies, a cautionary note
III. Hedgehog Signaling in the Developing Gut
A. Patterning of the developing gut
B. Early development of the gut tube and left-right axis formation
C. Anterior-posterior axis patterning of the gut tube
D. Endodermal cytodifferentiation and radial patterning of the gut tube
IV. Hedgehog Signaling and Homeostasis of the Adult Gut
A. Morphogenesis versus morphostasis
B. Stomach
C. Small intestine
D. Colon
V. Hedgehog Signaling and Gastrointestinal Carcinogenesis
A. Evidence for a role for Hedgehog signaling in initiation of gastrointestinal carcinogenesis?
B. Hedgehog signaling in cancers of the proximal gastrointestinal tract
C. Hedgehog signaling in colorectal cancer
D. Therapeutic targeting of the Hedgehog pathway
VI. Conclusion
1343
1345
1345
1345
1346
1347
1348
1348
1348
1348
1350
1355
1362
1362
1363
1364
1364
1367
1367
1367
1368
1369
1369
van den Brink GR. Hedgehog Signaling in Development and Homeostasis of the Gastrointestinal Tract. Physiol Rev
87: 1343–1375, 2007; doi:10.1152/physrev.00054.2006.—The Hedgehog family of secreted morphogenetic proteins
acts through a complex evolutionary conserved signaling pathway to regulate patterning events during development
and in the adult organism. In this review I discuss the role of Hedgehog signaling in the development, postnatal
maintenance, and carcinogenesis of the gastrointestinal tract. Three mammalian hedgehog genes, sonic hedgehog
(Shh), indian hedgehog (Ihh), and desert hedgehog (Dhh) have been identified. Shh and Ihh are important
endodermal signals in the endodermal-mesodermal cross-talk that patterns the developing gut tube along different
axes. Mutations in Shh, Ihh, and downstream signaling molecules lead to a variety of gross malformations of the
murine gastrointestinal tract including esophageal atresia, tracheoesophageal fistula, annular pancreas, midgut
malrotation, and duodenal and anal atresia. These congenital malformations are also found in varying constellations
in humans, suggesting a possible role for defective Hedgehog signaling in these patients. In the adult, Hedgehog
signaling regulates homeostasis in several endoderm-derived epithelia, for example, the stomach, intestine, and
pancreas. Finally, growth of carcinomas of the proximal gastrointestinal tract such as esophageal, gastric, biliary
duct, and pancreatic cancers may depend on Hedgehog signaling offering a potential avenue for novel therapy for
these aggressive cancers.
I. INTRODUCTION
One of the central problems of biology is the emergence of complexity. How did complex multicellular orwww.prv.org
ganisms evolve, and how is genetic information translated
to a spatially organized body plan? The hallmark of a
developing multicellular organism is the fact that decisions regarding cell fate are not taken at the level of the
0031-9333/07 $18.00 Copyright © 2007 the American Physiological Society
1343
1344
GIJS R. VAN DEN BRINK
FIG. 1. The Drosophila Hedgehog mutant. A: a schematic representation of the Drosophila larva. The larva is patterned into different
segments. The posterior half of each segment or naked cuticle is
smooth, whereas the anterior half is covered with bristles, the so-called
denticles. B: wild-type fruitfly. C: the Hedgehog mutant. Note the absence of most of the naked cuticle in each segment. The resulting
prominence of denticles gave the larve the aspect of a hedgehog. [B and
C from Nusslein-Volhard and Wieschaus (143), with permission from
Nature Publishing Group.]
Physiol Rev • VOL
FIG. 2. The French flag model of the mechanism of action of a
morphogen. In this model, undifferentiated cells (left) can choose three
different cell fates (blue, white, or red). This cell fate is specified in a cell
position-dependent manner by localized production of a morphogen
(middle). Cells respond in a distinct phenotypic manner to different
thresholds of the concentration of the morphogen that depend on their
distance from the source. It is important to realize that in reality many
morphogens and their antagonists generate much more complex patterns. Also, morphogens such as those of the Hedgehog and Wnt pathway are not soluble molecules that spread to a tissue through simple
diffusion but are lipid modified and very poorly soluble. Therefore,
gradients probably form by active only partially resolved mechanisms.
[Modeled after Wolpert (208).]
ingly, of the 15 loci identified in the Nüsslein-Volhard and
Wieschaus screen, 4 play a role in Hedgehog signaling.
The screen identified the Drosophila hedgehog gene,
Hedgehog receptor patched (Ptc), downstream transcription factor ci, and fused, a kinase involved in Hedgehog
signaling (see below). The experiments by NüssleinVolhard and Wieschaus laid the foundation of our current
understanding of the molecular mechanisms of tissue
patterning. Many of the genes that were identified play a
role in morphogenetic pathways.
The existence of morphogens had been inferred at
the beginning of the 20th century from experiments in
developmental biology. The concept was most clearly
voiced by Wolpert in the 1960s (208), who developed the
so-called French flag model (Fig. 2). Morphogens are
substances that originate from a localized source and
form a concentration or activity gradient through a tissue.
A morphogen receiving cell has one or more concentration thresholds that result in the expression of a distinct
set of target genes. Thus morphogen concentration gradients confer positional information by specifying distinct
cellular phenotypes in a field of receiving cells depending
on their distance from the source of the signal (Fig. 2). In
accordance with the limited number of loci identified by
Nüsslein-Volhard and Wieschaus, a very limited number
of highly conserved morphogenetic signaling pathways
have now been identified. Hogan (66) coined the term
morphogenetic code for these pathways that are used
repeatedly in varying constellations to shape different
organs. The four most important pathways that are conserved from fruitfly to human are the Hedgehog, Wnt,
transforming growth factor (TGF)-␤, and receptor tyrosine kinase pathways. The ligands for receptor tyrosine
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
individual cell. To allow the building of complex organs
with intricate patterns of cellular specialization, such decisions are taken at the population level in a cell nonautonomous manner. A breakthrough in the elucidation of
the genetic control of such intercellular communication
in developmental patterning events was made by Nüsslein-Volhard and Wieschaus (143). They performed a
saturating mutagenesis screen in Drosophila (the fruitfly)
and studied the effect of mutations on the segmented
Drosophila embryo. Surprisingly, their mutagenesis
screen in which they were estimated to have mutated
about half the genes in the Drosophila genome only resulted in a small number of mutations (15 different loci)
that affected Drosophila segmentation. In embryos of
wild-type Drosophila, a band of bristles called denticles is
present across the anterior half of each segment, whereas
the posterior half is smooth (the so-called naked cuticle).
In their screen, Nüsslein-Volhard and Wieschaus (143)
identified a group of mutants that affected the patterning
within the segments but at the same time left the number
of segments unaltered. In one of these so-called segment
polarity mutants, the posterior half of each segment failed
to develop, resulting in a larva that is entirely covered by
denticles (Fig. 1). This phenotype gave the larva the aspect of a hedgehog, which inspired the name. Interest-
HEDGEHOG SIGNALING
1345
kinase receptors include, for example, the fibroblast
growth factors (FGF), epidermal growth factor (EGF),
and platelet-derived growth factor (PDGF).
Below I discuss some of the most important aspects
of Hedgehog secretion, reception, and signal transduction. As this review aims primarily to provide an overview
of the role of Hedgehog signaling in the gastrointestinal
tract, many important aspects of Hedgehog biology are
not discussed. Several excellent reviews have been written for those interested in a more in-depth discussion of
various aspects of the Hedgehog pathway (71, 77, 119,
147, 201).
A. Hedgehog Secretion
After the identification of the Hedgehog mutant in
1980, the Drosophila hedgehog gene was published in
1992 by three independent groups (112, 137, 183). Three
murine hedgehog homologs were described a year later
(40). They were named sonic hedgehog (Shh), after a
popular Sega computer game character, desert hedgehog
(Dhh), after an Egyptian species of hedgehog (Hemiechinus auritus), and indian hedgehog (Ihh), a hedgehog
species endemic in Pakistan (Hemiechinus micropus).
These three hedgehog genes are highly conserved between mouse and humans (130). Unfortunately, very little
is known about the factors that control the transcriptional
regulation of the three different hedgehog genes, particularly in the gut.
Hedgehog proteins are extensively processed posttranslationally. Hedgehogs are produced as a ⬃45-kDa
precursor protein that is cleaved autocatalytically, yielding a 19-kDa NH2-terminal fragment that contains all the
signaling functions and a 26-kDa COOH-terminal fragment that catalyzes the cleavage and acts as a cholesterol
transferase (Fig. 3) (16, 111, 156, 157). Although morphogens act in a graded manner through tissues, this is mostly
not through simple diffusion of a soluble protein (127). In
fact, an important feature of Hedgehog proteins is that the
mature NH2-terminal fragments are dually lipid modified
and therefore poorly soluble. The signaling protein is
covalently linked to a palmitate and a cholesterol group
(127). These hydrophobic modifications integrate Hedgehog protein in the cell membrane and play an important
role in the regulation of the range of Hedgehog signaling
in a tissue. Conflicting reports have been published on the
role of the cholesterol moiety in Hedgehog signaling (113,
115). The palmitoylation is essential to both the activity of
the Hedgehog protein and the signaling range (25). Work
from several laboratories has shown that long-range signaling occurs by multimers of NH2-terminal Hedgehog
protein that require both cholesterol and palmitate modPhysiol Rev • VOL
FIG. 3. Posttranslational Hedgehog processing. The 45-kDa hedgehog precursor protein is processed to a 19-kDa NH2-terminal signaling
protein by the COOH-terminal half, which catalyzes this reaction and
acts as a cholesterol transferase. The cholesterol-modified NH2-terminal
protein is further lipid modified at its NH2 terminus by palmitoylation, a
reaction that is catalyzed by Skinny Hedgehog.
ification for their formation (25, 212). Release of lipidmodified Hedgehog protein from the Hedgehog-producing
cell requires Dispatched (Disp), a 12-pass transmembrane
protein with a sterol-sensing domain. Disp1⫺/⫺ mutant
mice have a phenotype similar to mice that lack the
Hedgehog signaling receptor Smoothened (Smo), suggesting that Disp1 is required for all Hedgehog signaling activity (20, 94, 121). A study by Panáková et al. (149) in
Drosophila suggested that Hedgehog multimers may form
by association of the lipid moieties with the outer phospholipid layer of lipoprotein particles and that this association is required for Hedgehog signaling activity. This is
a very interesting finding that may have important implications for vertebrate Hedgehog signaling (see, for example, a recent comment on the possible implications for
atherosclerotic plaques, Ref. 11). Thus Hedgehog proteins
are not soluble signals but dually lipid modified, and their
spread through tissues is only partially understood.
B. Hedgehog Reception
The Hedgehog signal is transmitted by a seven-span
transmembrane receptor Smoothened (Smo). Interestingly, Hedgehogs do not bind to Smo but control the
activity of Smo indirectly by binding to a second receptor,
Patched (Ptc). Two Ptc genes exist in vertebrates, Ptc1
(51) and Ptc2 (139). The Ptc genes encode for 12-span
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
II. THE HEDGEHOG PATHWAY
1346
GIJS R. VAN DEN BRINK
In Drosophila, Smo is localized mainly in the cytoplasm in its inactive form and accumulates at the cell
surface upon binding of Hedgehog to Ptc (33, 216). In
vertebrate systems, inactive Smo is present in the cell
membrane and intracellular vesicles (when overexpressed in cell lines) (23, 30, 76). The most notable change
in Smo cellular distribution upon pathway activation in
vertebrates is rapid accumulation on the primary cilium
(Fig. 5). Corbit et al. (30) have shown that Smo is enriched
in cilia in Hedgehog receiving cells in nonoverexpressing
cells in vivo, that constitutively active Smo is enriched in
the cilium in vitro, and that point mutations in the motif
necessary for translocation to the cilium interfere with
Hedgehog pathway activation in vitro. Interestingly, mutations in intraflagellar transport proteins, necessary for
cilia formation, interfere with Hedgehog signaling (59, 72,
118, 132; for review, see Ref. 173). Together these findings
suggested a functional role of the ciliary enrichment of
activated Smo.
In addition to Ptc, several other proteins have been
identified that also bind Hedgehog and modulate signaling. Hedgehog-interacting protein (Hhip) is a transcriptional target that acts as a negative regulator of Hedgehog
signaling by binding Hedgehog on receiving cells (26).
Recently, a novel class of Hedgehog binding proteins has
been identified in Drosophila that have two vertebrate
homologs, Cdo and Boc. These Hedgehog binding proteins facilitate binding and positively regulate signaling
(189, 210, 214). Boc acts as a Hedgehog receptor on
commissural axons. Given the established role for Cdo
and Boc in myogenic differentiation (84, 85), it is likely
that they act as receptors in the regulation of myogenesis
by Hedgehog signaling (see Ref. 41 for review).
C. Hedgehog Intracellular Signaling
FIG. 4. The current model of the mode of action of the Hedgehog
receptor system. A: in the absence of ligand, Ptc pumps a Smo inhibitory
molecule from one cellular compartment to another where it inhibits
Smo. Upon binding of Hedgehog to Ptc, this pump function is abrogated
and the inhibitory action on Smo released.
Physiol Rev • VOL
The analysis of Hedgehog signaling downstream
from Smo (Fig. 5) is considerably complicated by the
relative lack of conservation between Drosophila and
vertebrates (71, 147). Although the intracellular transduction of the Hedgehog signal is relatively well understood
in Drosophila, understanding of vertebrate signal transduction is far from complete; extensive discussion of the
mechanism of Hedgehog signal transduction is therefore
beyond the scope of this review. In Drosophila, the intracellular transduction of the Hedgehog signal depends on
the processing of downstream transcription factor Ci. Ci
exists in a full-length activating form and truncated repressor form. In the absence of Hedgehog, Ci is processed
to its repressor form by a complex of four kinases [protein kinase A (PKA), casein kinase 1 (CK1), glycogen
synthase kinase 3␤ (GSK3␤), and Fused kinase (Fu)] held
together by Cos2. In the presence of Hedgehog, Cos2 is
recruited by Smo and Ci processing terminated resulting
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
transmembrane receptors with two large hydrophilic extracellular loops that mediate Hedgehog binding. In the
absence of Hedgehog, Ptc inhibits signaling by Smo
(Fig. 4). Ptc1 is a transcriptional target of Hedgehog signaling and acts in a negative-feedback loop to restrict the
range of Hedgehog signaling in a tissue. The mechanism
of the inhibitory action of Ptc on Smo is only partially
understood. Ptc acts catalytically (184) and seems to
control the localization of a secondary Smo-inhibiting
molecule. The nature of this inhibitory molecule is a
matter of active investigation (12, 39). Even though Ptc
may act as a pump that controls the localization of a
secondary Smo antagonist, it has been shown that Ptc
most likely acts in a cell autonomous fashion (on the cell
expressing Ptc and not on surrounding cells) at least in
Drosophila and the chick neural tube (14). This suggests
that Ptc may pump this molecule from the outside to the
inside of the cell or from one cellular compartment to
another. The inhibitory action of Ptc is relieved upon
binding of Hedgehog to Ptc.
HEDGEHOG SIGNALING
1347
in the stabilization of full-length Ci and transcriptional
activation of Hedgehog target genes (for review, see for
example Refs. 67, 71, 83, 147). This function of Cos2 as an
“on/off” switch does not seem to be conserved in vertebrates. Convincing evidence for a vertebrate Cos2 homolog is currently not available (200). Important evidence
has been presented that shows that Suppressor of fused
Su(Fu) may function as a central intracellular on/off
switch in vertebrates (200). Indeed, the Hedgehog pathway is completely on in Su(Fu)⫺/⫺ mice (28, 182), which
strongly resembled the Ptc1 null phenotype (182). Although Drosophila does have a Su(Fu) gene, it does not
seem to play a similar role as Su(Fu) mutant Drosophila
are healthy and fertile (159).
The Hedgehog transcription factors are conserved
end points in intracellular Hedgehog signal transduction.
All aspects of Hedgehog signaling are mediated via the
vertebrate homologs of Drosophila Ci, the Glioblastoma
(Gli) transcription factors Gli1, Gli2, and Gli3 (74). As in
Drosophila, the activity of these transcription factors is
regulated in a ligand-dependent manner. Gli2 and Gli3 are
the major Glis to transduce the Hedgehog signal in the gut
(see below). Both Gli2 and Gli3 can also act as a repressor
of Hedgehog target expression in vivo (116, 148, 203). This
is related to the fact that Gli2 and Gli3 (and not Gli1) can
be processed to a repressor form similar to the Drosophila homolog Ci. However, processing of Gli3 to its
repressor form is much more efficient than that of Gli2
(148). As with Ci, the Gli3 repressor form is formed by a
partial proteolytic processing reaction that requires priming phosphorylation by PKA and subsequent phosphorylation by CK1 and GSK3␤ (188, 203). Tempe et al. (188)
have shown that this phosphorylation can result in ubiquitination and partial proteosomal degradation that generates a truncated version of Gli3 that lacks the transcripPhysiol Rev • VOL
tional activation domain and acts as a dominant negative
Gli (188). This negative regulatory action of PKA, CK1,
and GSK3␤ (Drosophila homolog Shaggy) on Hedgehog
signaling through the generation of a suppressor form of
Ci/Gli protein is conserved from Drosophila to vertebrates. Gli2 processing and degradation and Gli1 degradation are regulated in a similar manner as that of Gli3 (9,
129, 148, 188).
D. Identifying Hedgehog Target Cells
In the analysis of the role of Hedgehog signaling in any
organ, it is essential to identify Hedgehog receiving cells.
Although a number of different target genes have been identified, their regulation often differs in time or per organ. Ptc1
and Gli1 are two target genes that seem to have been
conserved particularly well throughout vertebrate evolution,
and their expression pattern is the best reflection of Hedgehog signaling activity in most if not all situations in vertebrates. A difference may exist for the sensitivity of the
expression of Ptc1 and Gli1 for the range of the Hedgehog
signal in the developing vertebrate gut. In the developing
stomach and colon, both Shh and Ihh are expressed in the
epithelium at E18.5. Ptc1 is expressed at high levels in a
small zone very close to the Hedgehog expressing cells,
whereas expression of Gli1 is also very intense in the
smooth muscle layer at much greater distance (164). It
is, therefore, probably safest to use both Ptc1 and Gli1
as readouts for Hedgehog pathway activity and realize
that these targets may have some level of independent
regulation. As Ptc1 is the receptor for Hedgehog and
required to initiate Hedgehog signaling, it is to be expected that low levels of Ptc1 may be expressed in
tissues in a Hedgehog-independent manner.
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
FIG. 5. A model of Hedgehog signaling in the vertebrate. A: in the absence of Hedgehog signaling, Smo localizes mainly to the cell membrane
and intracellular vesicles. GLI2 and GLI3 are processed by the proteasome upon phosphorylation by protein kinase A (PKA), glycogen synthase
kinase 3␤ (GSK3␤), and casein kinase 1 (CK1). For GLI3, degradation is partial, resulting in a GLI3 protein that lacks the transcriptional activation
domain and represses expression of hedgehog target genes. Degradation of GLI2 is complete. The vertebrate COS2 homolog (possibly KIF7) and
SUFU further repress unprocessed cytoplasmic GLI via an unresolved mechanism. B: in the presence of Hedgehog, repression of SMO by PTC is
relieved, and SMO translocates to the primary cilium where it activates GLI proteins.
1348
GIJS R. VAN DEN BRINK
E. Hedgehog Pathway Antibodies,
a Cautionary Note
immunohistochemistry with those obtained by in situ hybridization until better antibodies become available.
The analysis of the role of Hedgehog signaling in
development and the adult critically depends on the reliable detection of components of the Hedgehog pathway
such as Shh, Ihh, Dhh, Smo, Ptc, and transcription factors
Gli1–3. A large variety of commercial antibodies is currently available (mainly from Santa Cruz). With many if
not most of these antibodies, we have not been able to
obtain credible results with western blot or immunohistochemistry, especially when compared with in situ hybridization. In our hands the specificity of the available
commercial antibodies may even vary between batches.
Some of the antibodies that gave staining patterns that
compared well with in situ hybridization in our hands still
give variable results in the literature. For example, using
the I-19 antibody against Ihh from Santa Cruz, Varnat et al.
(202) observed staining at the base of the crypts, whereas
Jones et al. (80) describe Ihh expression mainly at the tips
of the villi using the same antibody. Since Ihh mRNA is
mainly expressed at the crypt villus junction with a diminishing gradient towards the top of the villus, the staining pattern observed by Jones et al. (80) probably best
reflects the expression of Ihh protein. It is furthermore
important to realize that many anti-Hedgehog antibodies
are produced against the extremely conserved NH2-terminal 19-kDa protein. This results in easy cross-reactivity of
antibodies for different Hedgehog proteins. This may explain, for example, why Jones et al. (80) detect abundant
staining on villi of the small intestine with an anti-Shh
antibody, whereas Shh expression is very low and restricted to a few cells in the small intestinal crypts as
detected by in situ hybridization (6, 198), making it very
unlikely that abundant Shh protein can be detected in the
enterocytes on the villi. Most likely this is explained by
cross-reactivity of the anti-Shh antibody for Ihh protein.
Additionally, using the Santa Cruz C-20 antibody against
Ptc1, we have previously reported epithelial staining in
the colon of mouse, rat, and human (197). We have now
performed in situ hybridizations for Ptc1 in the mouse
colon and found that although Ptc1 mRNA is expressed in
the epithelium of the anorectum, no Ptc1 mRNA can be
detected in the epithelium of the mouse colon using two
different Ptc1 probes. Instead, Ptc1 localizes to the mesenchyme just underlying the differentiated colonic enterocytes (unpublished observations). Hopefully more reliable anti-Hedgehog pathway antibodies will be available
soon. Of course, it should be realized that Hedgehogs are
proteins that can travel great distances through tissues
and that a perfect correlation between Hedgehog mRNA
and protein may not always be present. However, given
the poor performance of many Hedgehog pathway antibodies, it is important to compare results obtained with
III. HEDGEHOG SIGNALING IN THE
DEVELOPING GUT
The gastrointestinal tract forms from two germ layers, the endoderm and mesoderm, and is innervated by
cells derived from the third germ layer, the ectoderm.
During development the gut evolves from a simple tube
that is morphologically homogeneous to a highly complex
organ that has distinct functional domains along the anterior-posterior and vertical (cross-sectional) axis and develops multiple accessory organs. Three different regions
are normally recognized. The foregut forms the esophagus, stomach, proximal duodenum, thymus, thyroid, airways, pancreas, and liver. The midgut will form most of
the intestine distal from the common bile duct that derives its blood supply from the superior mesenteric artery:
the distal duodenum, the jejunum, ileum, cecum, and the
ascending and proximal transverse colon. The hindgut
forms the distal transverse colon, descending colon, sigmoid, and anorectum that develop around the inferior
mesentery artery. The patterning mechanisms along the
vertical, horizontal, and left-right axes and induction of
growth of accessory organs all involve cross-talk between
cells of the different germ layers. Such interactions between different groups of cells in which a signal is emitted
from one group of cells to change the nature or behavior
of the receiving cells are termed inductive signaling. Two
types of inductive signaling are generally recognized. In
instructive interaction, the signal is necessary to specify
the fate of the receiving cell. In permissive interaction, the
signal simply allows the expression of a phenotype that is
already intrinsic to the receiving cell. Much of this instructive communication between germ layers is through the
action of morphogens among which are members of the
Hedgehog signaling pathway. Below I first review some of
our understanding of the development of the gut and the
role of germ layer interactions, focusing on mouse development. I then discuss the available data that show a role for
Hedgehog signaling in gut development.
B. Early Development of the Gut Tube and
Left-Right Axis Formation
In all animals the primitive gut or archenteron is
formed during gastrulation. The term gastrulation is derived from the Greek word “gaster” and means “to form a
stomach.” At this early point in development, the mouse
embryo is a cup-shaped epithelial layer termed epiblast
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
Physiol Rev • VOL
A. Patterning of the Developing Gut
HEDGEHOG SIGNALING
that is enveloped by the visceral endoderm (Fig. 6). The
epiblast will form three germ layers during gastrulation:
definitive (gut) endoderm, mesoderm, and ectoderm. The
definitive endoderm gives rise to the epithelium of the
gastrointestinal tract, thymus, thyroid, and respiratory
tract; the mesoderm will form the cardiovascular system,
muscles, blood, and bone; the ectoderm forms the epithelium of the skin and the central nervous system. Gastrulation starts at embryonic day (E) 6.25 with the formation
of the primitive streak, a thickening on the posterior
margin of the epiblast (and future posterior end of the
embryo) that forms by epithelial reorganization and is
elongated anteriorly over the epiblast along the midline of
the embryo (176). Epiblast cells undergo a process of
epithelial to mesenchymal transition (EMT) at the primitive streak and ingress through the primitive streak and
node (Fig. 6). The embryonic endodermal cells that have
undergone EMT are inserted in the visceral endoderm,
gradually displacing it. Other cells that have undergone
EMT migrate in between the endoderm and epiblast (now
Physiol Rev • VOL
the primitive ectoderm) forming the mesoderm (176). At
the anterior end of the primitive streak, a small group of
cells forms a notch that is termed the node. Cells of the
node regulate left-right axis formation of the embryo and
are therefore essential for left-right axis formation of the
gut and its derived organs such as looping of the gut tube
and correct positioning of the pancreas and liver (65). The
node has a central depression termed the pit, which is
populated by ciliated cells that generate a leftward flow of
extraembryonic fluid. This leftward flow is the earliest
recognized left-right symmetry breaking event during murine embryogenesis (65) and is essential for the establishment of asymmetric mesodermal expression of genes involved in a program that specifies the left side of the
mouse embryo. Nodal flow may act by causing asymmetric distribution of a soluble signal to the left of the developing embryo (142, 144). A “left-side program” is subsequently activated in the left lateral plate mesoderm, which
contributes to the mesoderm of the gut and has been
shown to play an essential role in gut looping in zebrafish (68).
By E7.5, the mouse embryo has developed “insideout” with an internal layer of ectoderm that is surrounded
by mesoderm and endoderm (206). Formation of the gut
tube is initiated by the formation of the anterior intestinal
portal (AIP; Fig. 7). The AIP is an endodermal invagination at the anterior tip of the embryo at the level of the
cranial neural fold of the ectoderm (91). Formation of the
AIP is closely followed by the formation of a second
endodermal invagination at the caudal end of the embryo,
the caudal intestinal portal (CIP) (91). At E8.5, a complex
turning process of the embryo is initiated that folds the
endoderm to the inside of the embryo and elongates the
AIP and CIP while bringing their openings together
around the yolk sac stalk upon completion of the turning
process at E9.5 (Fig. 7) (91, 206).
1. Expression of hedgehogs during gastrulation
Shh expression is first detected at E7.25 in the midline mesoderm of the head process (40). At E7.75, Shh
expression is initiated in the node, the notochord, and the
definitive endoderm (40, 215). Endodermal expression of
Shh is initiated in the anterior endoderm, and its expression expands to the posterior endoderm (40). Ihh is expressed in the posterior node and the visceral endoderm
at E7.75 (46, 215). Ihh expression in the definitive
endoderm is initiated at later stages of development (13).
2. Hedgehog signaling and left-right axis formation
Hedgehog signaling is dispensable for early gastrulation in the mouse as all three germ layers are normally
formed in mice that lack Smo and therefore lack all
Hedgehog signaling (215). At a later stage of gastrulation,
Hedgehog signaling is involved in gut tube formation as
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
FIG. 6. Germ layer formation. A: at E5.5, the mouse embryo consists
of a single cell layer, the epiblast, enveloped in visceral endoderm.
B: during gastrulation, the posterior end of the epiblast forms a thickened region, the primitive streak. Epiblast cells undergo epithelial to
mesenchymal transition as they pass through the primitive streak and
form the mesenchyme and endoderm. The remaining epiblast cells form
the ectoderm. The endodermal cells migrate anteriorly (arrow) while
displacing the visceral endoderm. The embryo is now still inside-out and
will get its correct orientation during a complex turning movement.
1349
1350
GIJS R. VAN DEN BRINK
FIG. 7. Turning of the embryo. A: at E8, the
ventral side of the cupped embryo is oriented towards the outside. B: after a complex turning
movement, the ventral side ends up at the inside of
the cupped embryo bringing the anterior intestinal
portal (AIP) and caudal intestinal portal (CIP) in
close apposition.
Physiol Rev • VOL
(0.3–5 ␮m) membrane particles (that they termed “nodal
vesicular parcels” or NVPs) in an FGF signaling-dependent manner. The NVPs were transported to the left by
nodal flow and seemed to contain Hedgehog protein.
Recombinant Shh could reverse inhibition of asymmetric
Ca2⫹ signaling by an FGF inhibitor (185). The symmetrical expression pattern of Hedgehog receptor and transcriptional target Ptc1 in the node seems to contradict the
possibility that Hedgehog containing NVPs induces asymmetric Hedgehog signaling in the node. However, the
authors of the node work suggest that Ptc1 may be a
short-range Hedgehog signaling target (at least in the
fruitfly imaginal disks) and that short-range and longrange Hedgehog signaling may have alternative mechanisms of action (65). The authors suggest that the NVPs
may be important in the long-range signaling of Hedgehogs in the node and may be important for the asymmetric induction of Ca2⫹ signaling. Although this remains a
possibility, this suggestion is highly speculative and
awaits further experimental evidence.
In conclusion, Hedgehog signaling plays an important
role in left-right axis specification of the gut and other
organs in the body. To date, the most important evidence
is for a role for Hedgehog signaling in midline specification, which functions as a barrier to restrict diffusion of
asymmetric signals to one side of the developing embryo.
An alternative more direct role for Hedgehog signaling
remains a possibility but awaits further evidence.
C. Anterior-posterior Axis Patterning of the
Gut Tube
1. Instructive signals from the node and mesoderm
establish the anterior-posterior axis
early in development
After gastrulation, the endodermal layer is still a
histologically uniform pseudostratified layer of cuboidal
epithelial cells that is surrounded by a thin layer of me-
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
both Smo and Shh/Ihh compound mutant mice (215) fail
to close the midgut. This defect may be related to that fact
that they do not undergo the embryonic turning process,
something that will impair the normal endodermal folding
movement that results in gut tube closure. Several different mice with mutations in the Hedgehog pathway display
defective left-right axis formation. Smo mutant and Shh/
Ihh compound mutant mice do not initiate cardiac looping (lung or gut phenotype cannot be examined in Smo
mutant or Shh/Ihh compound mutant mice as they do not
survive beyond E9.5) (215). Shh mutant mice have a less
severe cardiac phenotype with delayed and incomplete
looping and fail to develop asymmetrically lobed lungs
(193, 215). Both Ihh and Shh mutant mice display gut
malrotation (164). These data indicate that Shh and Ihh
play redundant roles in left-right axis formation, consistent with the expression of both Shh and Ihh in the node.
Smo and Shh/Ihh mutant mice fail to activate the genetic
program necessary for the specification of the left side of
the body. Both mutants lack expression of nodal in the
left lateral plate mesoderm, a critical component of the
left-side specific gene program (215). As the Hedgehog
target gene Ptc1 is symmetrically expressed in the node, it
is most likely that the effect of Hedgehog signaling on
left-right axis formation is indirect. For example, Hedgehog signaling is important in the specification of the midline, which has been proposed to play an important role in
the stabilization of left-right specific gene expression by
functioning as a barrier that prevents the bilateral diffusion of long-range asymmetric signals (for review, see
Capdevila et al., Ref. 18).
A possible more direct role for Hedgehog signaling in
left-right axis formation has been proposed by Tanaka
et al. (185). It has been shown that nodal flow initiates
asymmetric Ca2⫹ signaling at the left border of the node
(133). Tanaka and colleagues found that this Ca2⫹ signal
depends on FGF signaling. When the authors labeled
membrane lipids with a fluorescent dye, they made the
interesting observation that nodal cells released small
HEDGEHOG SIGNALING
Physiol Rev • VOL
been examined if Hedgehog signaling is involved in these
early AP patterning events.
2. Instructive signals from the endoderm pattern the
gut along the AP axis during later
stages of development
The endodermal layer holds the information necessary for its differentiation along the AP axis at later stages
in development in both mice (177) and rats (38). This
indicates that the mesenchyme mainly has a permissive
role in endodermal development from this point onward.
For example, when undifferentiated E14 rat intestinal
endoderm is recombined with mesenchyme from different regions of the fetal small intestine, the endoderm
seems to determine the region-specific expression of
brush-border enzymes, and when E14 rat gastric or lung
endoderm was recombined with small intestinal mesenchyme, the endoderm developed according to its place of
origin (38). The only exception to the endodermal autonomic region-specific development was in the prospective
colonic endoderm. The colonic endoderm expressed
small intestinal enzymes when combined with small intestinal mesenchyme, indicating that cell fate of the colonic endoderm remains somewhat plastic at E14 (38). It
was even shown that normal intestinal endodermal development is possible when E14 rat endoderm is recombined
with fetal skin fibroblasts, with the fibroblasts developing
into a smooth muscle layer (instead of skeletal muscle)
that was ␣-smooth muscle positive (38, 95). This indicates
that the mesenchyme not only plays mainly a permissive
role at later stages of endoderm development but that
signals from the endoderm play an instructive role in the
development of the mesoderm at this stage. Because
Hedgehogs are expressed in the endoderm and signal to
the mesenchyme, this may implicate the pathway in the
instructive signaling to the mesenchyme at this stage.
3. Endodermal appendage formation
One of the most notable aspects of AP axis formation
of the gut is the formation of endodermal appendages
(Fig. 8). During and right after the turning of the embryo
between E8.5 and E9.5, a series of appendages originate
from the gut tube by a process of budding and elongation.
Buds are formed that generate the thymus (E9.5–E10.5)
(126), thyroid (E8.5) (31), airways (E9.0 –E9.5) (117), liver
(E8.5) (82), and pancreas (E9.5) (100). The specification
of the endodermal zone destined for appendage formation
and the subsequent morphogenetic process of budding
and elongation seems dependent on instructive mesodermal signals. For example, at E8.25–E8.5 signals derived
from the cardiac mesoderm play an essential role in the
development of the liver and airways from a region of the
ventral foregut endoderm that lies in close apposition to
the cardiac mesoderm (56, 81, 174). The notochord, a
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
sodermal cells. Despite its morphological uniformity, the
endodermal layer is already patterned along the anteriorposterior (AP) axis at this point in development (53, 206).
Lawson and Pedersen (108) microinjected single endodermal cells of E6.7 early primitive streak stage mouse embryos with horseradish peroxidase and traced them for up
to 48 h in culture. Their experiment showed that the first
endodermal cells to exit the primitive streak formed the
anterior-most endoderm, whereas cells that were still in
transit through the primitive streak formed more posterior endoderm (108). The earliest AP patterning of
endodermal cells may be related to this timing of the exit
of the primitive streak. For example, the endodermal cells
that were first to exit the primitive streak and have moved
most anteriorly express the homeobox gene Hhex (or
Hex) (192), whereas later endodermal cells express
FoxA2 (or Hnf3␤), and the last endodermal cells to exit
the primitive streak and thus form the posterior
endoderm express Cdx2 (7). Hereafter further AP patterning occurs through instructive and permissive interactions between the endoderm and mesoderm. The major
instructive interactions in early intestinal development
seem to be from the mesoderm to the endoderm. Unfortunately, few studies exist that examined the role of
endodermal mesodermal interactions in the mouse during
early gut development. In a recent study that examined
the role of germ layer interactions in patterning of the
early (E7.5–E7.75) mouse endoderm, the endoderm survived for at least 2 days when it was cultured in vitro in
the absence of mesoderm and maintained the regionspecific expression of two early posterior endodermal
markers the anterior marker ␤-cardiac actin (bCa) and
the posterior marker intestinal fatty acid binding protein (iFabp) gene (205). In this elegant study, the expression of several regulators and markers of endodermal
differentiation was only induced when the endoderm was
cultured with the other two germ layers. The effect was
mediated by soluble factors as it also occurred in the
presence of a membrane that separated the endoderm
from the mesoderm/ectoderm. These results might still
simply indicate that endoderm arrests in development
when cultured without trophic factors derived from the
other germ layers and that the effect would therefore be
an example of permissive induction. However, evidence
for an instructive role for the mesoderm/ectoderm was
provided in further experiments. When anterior
endoderm was cultured with posterior mesoderm/ectoderm, the endoderm expressed the posterior marker
iFabp. When posterior endoderm was cultured with anterior mesoderm/ectoderm it expressed anterior marker
bCa. These data indicate that AP patterning of the early
mouse endoderm occurs through both timing of the exit
of the primitive streak and instructive action of soluble
factors derived from the other two germ layers. It has not
1351
1352
GIJS R. VAN DEN BRINK
mesodermal structure that is formed during gastrulation
and ensures structural support along the axis of the growing embryo, is in close contact with the dorsal endoderm
until E9.0 and specifies the dorsal pancreatic domain in
the chick embryo (61, 102). Inductive signals from endothelial cells are involved in the specification of the thyroid
and both pancreatic buds (see below).
4. Hedgehog signaling and endodermal
appendage formation
A) AIRWAYS. After the formation of a central ventral
tracheal bud and two adjacent ventrolateral lung buds at
E9.5 (19), the trachea and lungs separate from the esophagus by a process of septation and elongation. Shh is
expressed at high levels in the tracheal bud (117). Signaling is from the endoderm to the mesoderm as the Gli
transcription factors are exclusively expressed in the foregut mesoderm (74). One of the roles of Hedgehog signaling to the mesoderm is in the correct specification of
the mesodermal septum that separates the esophagus and
trachea. In Shh⫺/⫺ mice, tracheal budding is delayed and
the elongating trachea is hypoplastic and fails to separate
correctly from the gut tube. The proximal esophagus and
trachea are therefore poorly separated (117, 153). The
lungs of Shh⫺/⫺ mice are severely hypoplastic and do not
lobulate, and the airways fail to branch and recruit proliferating mesenchymal cells, suggesting a role for Hedgehog signaling in branching morphogenesis (117, 153). Gli
transcription factors play a redundant role in airway budding, elongation, and branching. Gli1 mutant mice are
healthy and do not seem to have a phenotype (150).
Gli2⫺/⫺ mice recapitulate many of the aspects of the
Physiol Rev • VOL
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
FIG. 8. A budding phase between E8.5 and E9.5 generates the
endodermal appendages. Schematic depiction of the E9.5 embryo. Red
font indicates exclusion of Shh expression, and green font indicates high
Shh expression. Thb, thyroid bud; tb, tracheal bud; lb, lung bud; hb,
hepatic bud; pb, pancreatic bud.
phenotype of the Shh⫺/⫺ mice with hypoplastic trachea
and lungs and failure to induce mesenchymal proliferation (138). The Gli3⫺/⫺ mouse shows normal airway development but loss of one copy of the Gli3 gene in
Gli2⫺/⫺ Gli3⫹/⫺ mice aggravates the airway phenotype
and is indiscernible from the Shh⫺/⫺ mouse. The phenotype of mice completely deficient for both Gli2 and Gli3
is more severe than that of Shh⫺/⫺ mice as in these mice
there is no development of the trachea and lungs at all
(138). This may suggest a role for another Hedgehog in
airway budding and outgrowth. Expression of Ihh in the
foregut has not been reported, but low expression levels
may have been missed.
A major target and effector of Hedgehog signaling in
airway development is transcription factor Foxf1, which
is expressed in the mesenchyme (124). Expression of
Foxf1can be induced by ectopic expression of Shh, and
Shh⫺/⫺ mice lack Foxf1 expression in the foregut mesenchyme (124). Foxf1⫺/⫺ mice degenerate before endodermal budding begins (125), but Foxf1⫹/⫺ mice do have an
airway phenotype with a failure of the elongating trachea
to separate completely from the esophagus and reduced
branching morphogenesis similar to the Hedgehog pathway mutant mice (124). Thus Hedgehog signaling from
the endoderm to the mesoderm is essential for the formation of the tracheal bud and subsequent branching morphogenesis of the airways and plays a critical role in the
stimulation of growth of the recruited mesenchyme.
B) THYROID. The adult thyroid gland is located in the
cervical region anterior to the trachea in humans and
mice. The bulk of the gland is made up of follicles consisting of thyroid follicular cells that store and release
thyroid hormone. C cells produce calcitonin and are
found in the interfollicular space. Both cell types have a
different embryonic origin as the follicular cells are
endoderm derived whereas C cells are derived from the
neural crest (31). Thyroid morphogenesis is initiated at
E8.5 when the thyroid primordium buds of the ventral
wall of the pharyngeal foregut endoderm that lies in close
contact with the aortic sac endothelium (2, 43). At E9.5,
the thyroid bud separates from the gut tube while it
remains connected by a thin thyroglossal duct and remains in contact with the aortic arch. The thyroglossal
duct disappears and the thyroid becomes completely separated from the gut by E11.5. At E13.5, the thyroid is at its
final position in the midline at the ventral side of the
trachea. It now bifurcates to form two lobes that develop
in close contact with the carotid arteries (2). At E15.5, the
thyroid organizes into a follicular structure, and genes
involved in thyroid hormone production are induced (31).
Similar to the specification of the pancreatic buds (see
below) and in contrast to specification of the airways,
expression of Shh is excluded from the thyroid primordium (44). In the absence of Hedgehog signaling, the
thyroid bud is therefore correctly specified. Although no
HEDGEHOG SIGNALING
Physiol Rev • VOL
FIG. 9. A pancreatic and hepatic endodermal domain are specified
in the ventral endoderm. A population of endodermal cells at the lip of
the ventral endoderm are specified as Pdx1-positive, albumin-negative
ventral pancreatic precursor cells, whereas cells of the more cranial
endoderm that lies in close apposition to the cardiac mesenchyme are
specified as Pdx1-negative albumin-positive hepatic precursor cells.
102). The ventral pancreatic domain is specified at the lip
of the mouse ventral foregut endoderm (Fig. 9). This
endodermal region adopts a pancreatic cell fate around
the six- to eight-somite stage (E8) just before the turning
of the embryo (34). The endoderm that lies slightly more
cranial is closely apposed to the cardiac mesoderm which
secretes FGFs to suppress a pancreatic and induce a
hepatic cell fate in this endodermal region (34, 81). The
ventral and dorsal pancreatic buds develop between E8.5
and E9.5 exactly where the endoderm interacts with the
aorta dorsally and the two vitelline veins ventrally (106,
107). It was shown by a variety of different experimental
approaches that this endodermal contact with the endothelium is both necessary and sufficient to determine the
expression domains of Pdx1 and insulin (106).
Similar to thyroid development (discussed above),
pancreatic development needs exclusion of Hedgehog expression from both the ventral and dorsal pancreatic primordia (4, 34, 61, 102). At E9.5–E10.5, Shh and Ihh are
expressed uniformly throughout the endoderm of the gut
tube with the exception of Pdx1 positive pancreatic precursor cells (4, 114). Forced expression of Shh in the
pancreatic primordia using a Pdx1 promoter resulted in
abnormal development of the pancreatic endoderm and
mesoderm. The pancreatic mesoderm of these transgenic
animals contained ␣-actin positive smooth muscle cells
and c-kit positive cells of Cajal. Both cell types are normally exclusively found in intestinal mesenchyme. The
pancreatic buds in Pdx1-Shh mice did contain endocrine
and acinar cells, but these failed to cluster into islets and
acinar structures and produced mucins that are typical for
intestinal epithelial cells (4). Thus suppression of Shh
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
Shh is expressed in the thyroid at any point in development, the thyroid does not separate into two distinct
lobes in Shh⫺/⫺ mice but becomes a single unilateral
mass, the same size of a single thyroid lobe in control
mice (44). The single thyroid gland of Shh null mice
formed normal follicles that contained apparently normally differentiating thyrocytes. In an elegant study by Alt
et al. (2), it was shown that the thyroid phenotype is
indirect, in accordance with the lack of Shh expression in
the thyroid. The authors showed that thyroid growth is
directed towards endothelial cells in the zebrafish. Subsequently, the authors examined a mouse that lacks Shh
expression at early stages of development and demonstrated that the aortic arch fails to cross the midline and
both carotid arteries develop on one side of the esophagus. They found that the single thyroid lobe in these mice
is always on the same side as and in close contact with the
mislocated carotid arteries. The fact that Shh expression
is specifically excluded from the thyroid primordium suggests that downregulation of Shh expression may be an
important step in its specification similar to the specification of the pancreatic domain (see below). This idea is
supported by the finding of small foci of ectopic thyroid
tissue in the tracheal tube at 15.5–17.5 days post coitum in
the Shh⫺/⫺ mouse (44), suggesting that Shh acts to suppress thyroid cell fate in the trachea and its loss may be
critical to thyroid primordium formation. Another similarity between the development of the thyroid and pancreas
is the important role of the endothelium in budding (see
below), suggesting that a signal derived from endothelial
cells may be involved in the restriction of Shh expression
from the thyroid and pancreatic buds.
In conclusion, Shh suppresses thyroid cell fate specification in the trachea, and its expression is excluded
from the thyroid primordium, possibly by signals from the
endothelium of the aortic sac. Although the Shh⫺/⫺ mouse
has a thyroid phenotype, this phenotype is indirect and
the result of defective patterning of the cervical vasculature.
C) PANCREAS. The pancreas is a gland with both endocrine and exocrine functions. Most pancreatic tissue
(⬃95–99%) is composed of exocrine acinar cells that produce digestive enzymes and pancreatic ducts that are
lined by duct cells. The rest of the pancreas consists of
small clusters of endocrine cells or islets of Langerhans.
Each cluster of endocrine cells has a core of insulinproducing ␤-cells surrounded by glucagon-producing
␣-cells, somatostatin-producing ␦-cells, and pancreaticpolypeptide cells.
The pancreas develops from two distinct domains of
ventral and dorsal foregut endoderm. The dorsal endodermal domain involved in the formation of the pancreas is
specified by signals from the notochord, which remains in
close contact to the dorsal endoderm until E8.5 and induces the expression of transcription factor Pdx1 (61,
1353
1354
GIJS R. VAN DEN BRINK
TABLE
1.
Endodermal appendage phenotypes in Hedgehog pathway mutant mice
Airways
Shh⫺/⫺
Ihh⫺/⫺
Gli1⫺/⫺
Gli2⫺/⫺
Gli3⫺/⫺
Gli2⫺/⫺ Gli3⫹/⫺
Gli2⫺/⫺ Gli3⫺/⫺
Hhip
(170), but injection of Shh increases the number of pancreatic precursor cells (170) whereas Shh and Smo mutant zebrafish show diminished numbers of pancreatic
precursor cells (35, 170). Experiments by DiIorio et al.
(35) suggest that this role of Hedgehog signaling in early
specification is mediated during gastrulation.
Thus Hedgehog signaling differentially regulates
early pancreatic development in the mouse and zebrafish.
Hedgehog signaling inhibits early pancreatic development
in the mouse, and the exclusion of Shh and Ihh expression from the pancreatic primordia is critical to normal
murine pancreas development.
D) LIVER. Shh is expressed in the liver primordium in
the ventral foregut endoderm (34). It is not clear if there
is a role for Hedgehog signaling in liver budding and
development as liver development seems to progress normally in both Hedgehog and Gli mutant mice (117, 138)
and, for example, in Xenopus injected with a constitutively active form of Smo (213).
In conclusion, instructive mesenchymal signals play
an essential role in the establishment of the AP axis
during early gut development. This is most evident from
the role of mesenchymal factors in the appropriate formation of endodermal appendages. See Table 1 for a
summary of phenotypes in Hedgehog pathway mutant
mice. Much of this instructive signaling seems to act
by modulating the expression of Hedgehogs in the
endoderm, which subsequently acts on the mesenchyme
in a reciprocal manner.
⫺/⫺
Thyroid
Hypoplastic trachea,
severely hypoplastic
lungs
No gross abnormalities
Single-lobed thyroid gland,
thyroid tissue in trachea
Grossly normal
Hypoplastic trachea,
hypoplastic lungs,
lobulation defect right
lung
Hypoplastic lungs
Hypoplastic trachea,
severely hypoplastic
lungs
Complete tracheal atresia,
no lungs
Branching defect
Grossly normal
ND
ND
Pancreas
Grossly normal
Liver
Grossly normal
Annular pancreas, increased numbers
of endocrine cells
Grossly normal
ND
ND
Grossly normal
ND
ND
ND
ND
ND
ND
Normal hepatic bud
ND
ND
Normal hepatic bud
ND
Loss of endocrine cells, exocrine cells
normal
Intestinal type pancreatic
mesenchyme, disorganization of
pancreatic endoderm
Reduced pancreatic mass, intestinal
type pancreatic mesenchyme, loss
of endocrine cells, loss of exocrine
cells
ND
Pdx1-Shh
Pax4-Shh and Ihh
ND, not determined. Note: no gastrointestinal phenotypes are available of the Shh Ihh double-knockout and Ptc and Smo knockout mice as
they are early embryonic lethal.
Physiol Rev • VOL
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
signaling during early pancreatic development is essential
for normal development of both the pancreatic epithelium
and mesoderm. Data from Xenopus further support this
concept (213). Xenopus injected with a constitutive active
form of the Hedgehog receptor Smo completely lack a
pancreas, a phenotype more severe than that of the Pdx1Shh mouse. This may be due to the fact that the transgenic mouse expressed Shh behind a pancreas specific
promoter and that the ectopic Shh transcription therefore
starts after the pancreatic domain has already been specified. Similar to the role of Shh in suppressing thyroid cell
fate in the trachea, expression of Shh in the endoderm
surrounding the pancreatic primordium may be necessary
to suppress pancreatic cell fate in the duodenum and
stomach. Treatment of chick embryos with the Hedgehog
inhibitor cyclopamine in ovo resulted in ectopic pancreatic tissue formation in the duodenum and stomach (103).
Cyclopamine acts at the level of Smo and therefore inhibits the activity of all Hedgehogs (29, 75). No pancreatic
differentiation was observed in the stomach or intestine
of Shh null mice (62), but this may be related to the fact
that Shh and Ihh have overlapping functions in this domain (13, 164). The role of Hedgehog signaling in pancreatic development in zebrafish seems to diverge from that
in the mouse in some aspects. Evidence has been presented that in contrast to the mouse, Hedgehog signaling
is required for the early stages of pancreas development
in the zebrafish (35, 170). As in the mouse, Shh expression
is excluded from the pancreatic anlage in the zebrafish
1355
HEDGEHOG SIGNALING
D. Endodermal Cytodifferentiation and Radial
Patterning of the Gut Tube
Endodermal cytodifferentiation is complete at birth
in humans. It is important to realize that in contrast to
humans, the epithelium is still immature at birth in mice
and rats and that its development continues in the first
four postnatal weeks. Here I discuss the changes that
occur in the first three postnatal weeks as part of the
developing gastrointestinal tract and consider the gastrointestinal tract as “adult” after completion of postnatal
development. The gut tube phenotypes of Hedgehog pathway mutant mice are summarized in Table 2.
Differentiation of the esophageal endodermal layer is
poorly studied in the mouse. A morphological study by
Raymond et al. (165) suggests that differentiation initiates
around E15. At E17, the epithelium is composed of three
to four cell layers and contains both squamous and ciliated cells. Only cells in the basal most epithelial layer
proliferate, and thus a division in a basal compartment of
precursor cells and superficial compartment of differen-
TABLE
2.
Gut tube phenotypes of Hedgehog pathway mutant mice
Esophagus
Shh⫺/⫺
Ihh⫺/⫺
Gli1⫺/⫺
Gli2⫺/⫺
Gli3⫺/⫺
Gli2⫺/⫺ Gli3⫹/⫺
Gli2⫺/⫺ Gli3⫺/⫺
Stomach
Intestine
Esophagus and trachea fail Anterior expansion of Gut malrotation, duodenal
to separate,
glandular stomach
villus overgrowth,
tracheoesophageal
increased gland
abnormal neuronal
fistula
fission
differentiation
No gross abnormalities
No gross
Gut malrotation,
abnormalities
hypoplastic villi,
segmental absence of
neurons,
Hirschsprung’s-like
colonic phenotype
Grossly normal
Grossly normal
Grossly normal
Hypoplastic trachea and
No gross
ND
esophagus, hypoplastic
abnormalities
lungs, lobulation defect
right lung
ND
Anterior expansion of
ND
glandular stomach,
increased glandular
growth
Esophagus and trachea fail
ND
ND
to separate,
tracheoesophageal
fistula
Complete atresia of
ND
ND
esophagus
Hypoplastic branching
villi, precursor cell
accumulation, crypts on
villi
Villln-Hip
Anorectum
Reference Nos.
Imperforate anus, fused 99, 117, 136, 153, 164
anorectum-urinary
tract
No gross abnormalities
164
Grossly normal
150
Imperforate anus, fecto- 99, 136, 138
vesical fistula
Anal stenosis
99, 136, 138
Persistent cloaca,
common outlet guturinary tract
136, 138
Persistent cloaca,
common outlet guturinary tract
136, 138
122
ND, not determined. Note: no phenotypes are available of the Shh⫺/⫺ Ihh⫺/⫺ double-knockout and Ptc and Smo knockout mice as they are
early embryonic lethal.
Physiol Rev • VOL
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
1. Esophageal development and the role of
Hedgehog signaling
tiating cells has been established along the vertical axis at
this point in time. The ciliated cells rapidly disappear
postnatally and are absent 1 wk after birth. The mechanism of this loss of ciliated cells seems to be through
desquamation (134). In contrast to the human esophagus,
which is not keratinized, murine esophageal epithelium
keratinizes ⬃1 mo after birth (37).
Hedgehog signaling plays a critical role in the normal
development of the esophagus (Fig. 10). Shh is initially
expressed throughout the developing esophagus but restricted to the distal esophagus at later stages (117, 164).
The Gli genes are mesodermally expressed, indicating
that Hedgehog signaling is from the endoderm to the
mesoderm (74). At E17.5, the proximal esophagus of
Shh⫺/⫺ mice is hypoplastic and fails to separate completely from the developing trachea; more distally there is
no discernible remaining esophagus in Shh⫺/⫺ mice at
this point in development (117). Similar to the trachea, the
developing lungs [which originate from two lung buds on
each side of the tracheal bud (19)] fail to separate correctly from the gut and are still connected to the gut tube
at E17.5. The importance of Hedgehog signaling to normal
esophageal development was confirmed in Gli mutant
mice. In the Gli2⫺/⫺ mouse, the esophagus has a very
1356
GIJS R. VAN DEN BRINK
small lumen and little mesenchymal cells and fails to
develop a smooth muscle layer. In Gli2⫺/⫺ Gli3⫹/⫺ mutant mice, most of the proximal esophagus is degraded,
and only a very small proximal esophageal remnant can
be observed in the carefully made whole-mount preparations shown by Motoyama et al. (138). A single tube
connects the trachea and lungs to the stomach in Gli2⫺/⫺
Gli3⫹/⫺. Although this has not been carefully examined,
this is most likely the remnant distal esophagus similar to
human with esophageal atresia and tracheoesophageal
fistula (Fig. 10). In Gli2⫺/⫺ Gli3⫺/⫺ mice, the foregut was
hypoplastic and failed to form the appendages for the
trachea and lungs at E9.5. Gli2⫺/⫺ Gli3⫺/⫺ embryos that
survived until E14.5 had a very small remaining proximal
endodermal tube and lacked both trachea and lungs. The
phenotype of these Hedgehog pathway mutant mice indicates that Hedgehog signaling is critical to the maintenance of the developing esophagus and the formation of a
tracheal-esophageal septum. Both phenotypes seem to
result from a block in the mesenchymal growth that is
necessary to support the developing esophagus and separate the gut tube from the airways. This is therefore a
nice example of the critical role of endodermal-derived
Hedgehog in radial patterning of the gut. The role of
Hedgehog signaling in esophageal cytodifferentiation remains to be determined as the severe esophageal phenotype of most Hedgehog mutant mice precludes evaluation
of esophageal cytodifferentiation, and the histology of the
esophageal epithelium of Gli2⫺/⫺ mice has not been reported.
Physiol Rev • VOL
2. Gastric development
Gastric epithelial cytodifferentiation is a highly complex process during which the gastric epithelium is patterned into three distinct zones along the proximal-distal
axis and in precursor cell and differentiated cell compartments along the vertical axis. Whereas the whole stomach
of humans consists of glandular epithelium, mice develop
a squamous epithelium in the first proximal one-third of
the stomach. The distal two-thirds of the stomach consist
of columnar glandular epithelium. A proximal fundic or
zymogenic and distal antral zone can be recognized in the
adult glandular epithelium based on histological features.
Cytodifferentiation of the gastric epithelium is initiated
around E13.5. At E16.5, the epithelium of the forestomach
is a squamous multilayer, whereas the epithelium of the
glandular stomach is a monolayer of columnar cells that
have formed primitive epithelial invaginations (48). Recombination experiments (48) suggest that the endoderm
of the E14.5 stomach does not require mesenchymal instructive signals for appropriate AP patterning, indicating
that the AP pattern has been correctly specified at this
point in time (as is the case in the intestine, see below). It
should be noted, however, that no markers of differentiation were used and that epithelial phenotype was judged
by histology. The authors also performed experiments
with E11.5 and E12.5 endoderm and showed that the
endoderm may still have some plasticity at this stage as 7
out of 10 E11.5/12.5 gastric endoderm explants keratinized when cultured with E14.5 forestomach mesenchyme,
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
FIG. 10. Hedgehog signaling is critical to normal foregut development. A schematic depiction of the phenotype of patients with tracheoesophageal fistula (A), wild-type (B), and Gli2⫺/⫺ Gli3⫹/⫺ (C) mice at E11.5. A: in the most common form of esophageal atresia and tracheoesophageal
fistula, the proximal esophagus is missing and the distal esophagus connects to the lower trachea. C: In Gli2⫺/⫺ Gli3⫹/⫺ mice, the trachea and lungs
fail to separate from the developing gut tube. The proximal esophagus becomes progressively hypoplastic until only a small remnant is present at
E11.5. As a result, the trachea and lungs are connected to the gut tube at E11.5. In humans, a transition from tracheal tissue to esophageal tissue
is found at the point indicated with an arrow in A and C. It is not entirely clear if the tube distal from the lungs, which connects the trachea and
lungs to the stomach, consists of esophageal tissue in Gli2⫺/⫺Gli3⫹/⫺ mice as in humans. [B and C redrawn from Motoyama et al. (138).]
HEDGEHOG SIGNALING
3. Hedgehog signaling in gastric development
From early in stomach development at E11.5 until
after the onset of gastric epithelial cytodifferentiation at
E15.5, Shh is expressed at high levels in the forestomach
and lower levels in the hindstomach, whereas Ihh is expressed in the hindstomach (Fig. 11) (13, 178). Ihh expression in the glandular stomach depends on Fgf signaling as both Fgfr2b⫺/⫺ and Fgf10⫺/⫺ mice lack expression
of Ihh in the stomach (178). Expression of Ptc1 is in the
mesoderm and mirrors that of Shh. There is only very low
expression of Ptc1 in the distal hindstomach at this point
FIG. 11. Hedgehog signaling during early gastric development.
A: Shh is expressed in a gradient with high expression in the forestomach and low to absent expression in the hindstomach. Shh specifies the
zone that will be occupied by squamous forestomach epithelium. Although Ihh is expressed in the hindstomach, it is probably not translated
or otherwise inactive as the expression of Ptc in the mesoderm mirrors
that of Shh. B: expression of Shh is restricted to the forestomach by
FGF10 secreted from the mesoderm, which acts on the FGFR2b in the
endoderm. FGF signaling lies upstream of another negative regulator of
Shh expression, transcription factor Gata4. Activin signaling is another
factor necessary to restrict Shh expression that could hypothetically act
downstream of FGF signaling and upstream of Gata4.
Physiol Rev • VOL
in time, which suggests that Ihh may not be translated or
active (178). Indeed, no gross abnormalities are reported
in the stomachs of Ihh⫺/⫺ mice, although gland formation
may be somewhat reduced [see Fig. 2I in Ramalho-Santos
et al. (164)]. Expression of Shh expands to the hindstomach at later stages of development as both Shh and Ihh are
expressed in the glandular stomach at E18.5 (164). At this
stage of development, Hedgehog signaling may no longer
be exclusive to the mesenchyme as both Ptc1 and Gli1
seem to be expressed also in the epithelial layer (164).
This is similar to the situation in the adult stomach where
Ptc1 is most highly expressed in epithelial gland cells (8,
199). Hedgehog expression in the forestomach has not
been described during later stages of gastric development. The expression of Hedgehogs during epithelial cytodifferentiation and compartmentalization in the first
four postnatal weeks has similarly not been examined.
The analysis of gastric development in Hedgehog
pathway mutant mice is limited by the fact that these
mutants die well before or just after birth, whereas major
events in cytodifferentiation occur postnatally. In Shh⫺/⫺
mice, the area occupied by the squamous epithelium of
forestomach is reduced (99, 164). This forestomach phenotype is in accordance with the Shh expression pattern
and suggests a role for Shh signaling in the specification
of the forestomach domain. Indeed, in mice with mutant
Activin receptors, the domain of Shh expression is expanded from the anterior to the posterior stomach with a
concomitant posterior expansion of the squamous epithelium of the forestomach (101). A similar posterior expansion of Shh expression with posterior expansion of the
squamous epithelium of the forestomach has been observed in Gata4⫺/⫺, Fgfr2b⫺/⫺, and Fgf10⫺/⫺ mice (78,
178). Fgf10 is expressed in the mesenchyme of the glandular stomach, whereas Fgfr2b and Gata4 are expressed
by the endoderm of the glandular stomach (178). As
Gata4 expression is lacking in both Fgfr2b⫺/⫺ and
Fgf10⫺/⫺ mice (178), this places Fgf10 signaling from the
mesoderm to the endoderm upstream of Gata4 in the
restriction of Shh expression to the forestomach. It is not
clear if FGF signaling interacts with Activin signaling in
the suppression of Shh expression, but in the developing
eyelid, Fgf10 is necessary for the expression of Activin
(186) and endodermal Gata4 expression is induced by
TGF-␤ family members (1, 169). It is therefore possible
that Fgf10 signaling induces Activin expression in the
endoderm through Fgfr2b, which would in turn induce the
expression of Gata4 in the glandular stomach.
Shh also plays an important role in the glandular
stomach as glandular growth is increased in Shh⫺/⫺ mice,
resulting in mucosal hyperplasia of the glandular stomach
(99, 164). Kim et al. (99) found that the gastric histology of
Gli3⫺/⫺ mouse phenocopies that of the Shh⫺/⫺ mouse,
indicating that the Shh signal is mainly transduced by Gli3
in the developing stomach. This shows that although Gli3
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
whereas 1 out of 9 were keratinized when cultured with
mesenchyme from the glandular stomach.
At birth, ⬃90% of the cells in the rudimentary gastric
units are precursor cells; this percentage is reduced to
20% in the first 7 days postnatally (P1–P7) as cellular
differentiation occurs while gland size remains stable
(89). The second week after birth (P8 –P14) is marked by
an increase of the number of all cell types and glandular
growth. Between P15 and P28, further cellular differentiation and glandular growth occurs, and the cells in the
glands are compartmentalized (89). Compartmentalization of the gastric glands results in the formation of a
precursor cell compartment (isthmus) somewhere halfway up the unit from where a bidirectional migration of
differentiating cells occurs. Cells that migrate to the luminal surface form the pit or foveolus, whereas cells that
migrate to the bottom of the gland form the gland proper.
1357
1358
GIJS R. VAN DEN BRINK
Physiol Rev • VOL
gland formation completely, resulting in a squamous epithelium that lacks glands. In the glandular stomach much
lower levels of Shh are expressed due to inhibition of Shh
expression by Fgf10 secreted from the mesenchyme. The
resulting low level of Shh in the glandular stomach allows
gland formation but restricts gland branching. The development of gastric epithelial cell lineages seems relatively
undisturbed in the absence of Shh, but there is a defect in
the appropriate restriction of the expression of markers
of the gastric cell lineage. Since Ihh is also expressed in
the glandular stomach, there is redundancy in the Hedgehog pathway, and the phenotype in the absence of both
Shh and Ihh may be more severe. Since the stomachs of
Shh/Ihh double-knockout mice cannot be examined due
to early lethality, further analysis of the role of Hedgehog
signaling awaits conditional inactivation of Hedgehog
pathway components in the stomach endoderm.
4. Intestinal development
Differentiation of the intestinal endodermal layer occurs in two phases in rodents. The first phase is initiated
at E15 in the mouse and lasts until birth. During this
phase, a wave of differentiation occurs from the duodenum to the colon, transforming the pseudostratified
cuboidal endodermal layer into a polarized columnar epithelial monolayer. The epithelium compartmentalizes
into differentiating and proliferating cell populations. In
the small intestine, this occurs by villus formation along
the vertical axis of the gut. Differentiating cells are located on these fingerlike structures that project into the
lumen of the gut and are extended by upward growth of
the underlying mesenchyme. The formation of villi is
initiated in the proximal duodenum at E14 and occurs in
a proximal to distal wave similar to and just preceding
cytodifferentiation (90). Villus morphogenesis is preceded
by the formation of clusters of condensed mesenchymal
cells that underlie the site of eventual villus formation
(90). The villi are separated from each other by small
patches of intervillus epithelium that contain a population
of proliferating precursor cells.
The colon lacks villi but develops epithelial folds at
E16.5 (151) at the onset of colonic endodermal cytodifferentiation. These folds form in a manner that resembles
small intestinal villus formation with similar mesenchymal condensation and subepithelial expansion (27). The
epithelial folds lie more separated in the proximal colon
(and therefore more villuslike), whereas they are increasingly packed together towards the distal colon.
The endoderm holds all necessary information for
correct patterning of the small intestine along the AP axis
at the onset of cytodifferentiation at E14. Experiments
using E15 whole intestinal explants that were transplanted subcutaneously in young adult syngenic mice (45)
or nude mice (38, 171) have shown that the information
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
may often act as a truncated protein that represses targets
of Hedgehog signaling, it seems to act mainly as a fulllength transcriptional activator in gastric development.
This was further illustrated by the fact that the expression
of both Ptc1 and Gli1, two transcriptional targets of
Hedgehog signaling, was reduced in the stomach of Gli3
mutant mice (101). No change was found in endodermal
proliferation in the gastric mucosa of Shh⫺/⫺ and Gli3⫺/⫺
mice between E14.5 and E18.5 that could account for the
increased mucosal thickness (99, 164). Two different findings can explain this apparent discrepancy. It was found
that apoptosis may be reduced in Shh⫺/⫺ and Gli3⫺/⫺
mice, which could result in net increased glandular
growth. An alternative reason for the mucosal hyperplasia
may be the strongly increased gland branching (99). As
proliferation is judged per gland, an increase in gland
branching will underestimate epithelial proliferation in
Shh⫺/⫺ and Gli3⫺/⫺ mice.
Ramalho-Santos et al. (164) reported histology reminiscent of intestinal transformation of the stomach epithelium in the Shh⫺/⫺ mouse. If true intestinal transformation (or in the adult: intestinal metaplasia) of the stomach would occur in Shh⫺/⫺ mouse, the mucosa should
show the histological hallmark of intestinal transformation, the presence of goblet cells (211). However, there
are no goblet cells in the stomach of Shh⫺/⫺ mice (99),
and one can therefore not compare the phenotype to
intestinal metaplasia of the stomach as it is known in the
adult. The authors do show that there is expression of
markers of intestinal enterocytes (alkaline phosphatase
expression and binding of Wisteria floribunda-agglutinin
lectin) in the gastric epithelium, but it was not examined
if there is loss of gastric epithelial cell markers as is
characteristic for intestinal metaplasia in the adult. In
fact, Kim et al. (99) found that all gastric markers examined are normally expressed in the stomachs of Shh⫺/⫺
and Gli3⫺/⫺ mice. They further show that gastric epithelial cells do not have a brush border (see Ref. 99, Fig. 1i-l)
as would be expected in the case of intestinal transformation. Also, there is no expression of intestinal enterocyte marker villin, intestinal homeobox gene Cdx2 (a
marker of intestinal metaplasia in the adult), or goblet cell
marker Muc2 in the stomachs of Shh⫺/⫺ and Gli3⫺/⫺
mutant mice (99). Together these data suggest that Shh
signaling may be important to appropriately restrict the
expression of markers of the gastric epithelial cell lineage
but that there is no evidence of intestinalization or intestinal metaplasia. This loss of appropriate restriction of the
expression of cell lineage markers has some similarities
with our finding that in rats treated with Hedgehog inhibitor cyclopamine colonic enterocytes aberrantly express a
marker of the goblet cell lineage [see Fig. 1u-x in van den
Brink et al. (197)].
In conclusion, the available evidence suggests that
high levels of Shh expression in the forestomach inhibit
HEDGEHOG SIGNALING
5. Hedgehog signaling in intestinal development
At E11.5 and E12.5, both Shh and Ihh are expressed
throughout the endoderm of developing intestine (13).
Shh expression is downregulated in the small intestine at
E14.5 around the moment of intestinal villus formation
Physiol Rev • VOL
and cytodifferentiation, with the highest remaining expression in the duodenum (13). Ihh remains expressed in
the developing small intestine and colon throughout development (13, 164). At E18.5, the expression of both
genes is restricted to the intervillus epithelium in the
small intestine (122, 164). In the colon Shh is expressed at
the base of the crypts, whereas Ihh is expressed throughout the epithelium (164). Hedgehog signaling in the developing small intestine and colon is from the epithelium to
the mesenchyme as Ptc1 and Gli1 are exclusively expressed in the mesenchyme during development and directly after birth at P0 (13, 122, 164).
Both Shh and Ihh mutant mice show malrotation of
the gut (164). Although there is no reversion of gut situs
in Shh⫺/⫺ or Ihh⫺/⫺ mice, aberrant gut looping may be
related to the role of Hedgehog signaling in left-right axis
formation that is described above. Hedgehog signaling
plays an important role in radial patterning in the developing intestine as it regulates villus formation, smooth
muscle layer formation, and development of the enteric
nervous system. Shh and Ihh seem to play a distinct role
in villus formation. Shh is mainly expressed in the intervillus epithelium of the duodenum. In the duodenum of
the Shh⫺/⫺ mouse, villi appear overgrown and clog the
duodenal lumen, indicating that Shh may act to restrict
villus growth in the duodenum. In contrast, villi in Ihh⫺/⫺
mice are decreased in number and hypoplastic. Madison
et al. (122) used a 12.4-kb fragment of the villin gene to
drive expression of Hhip in the small intestinal epithelium. The 12.4-kb villin promoter drives the expression of
transgenes in the crypts and villi of the small intestine
from E12.5 onwards (123). Unfortunately, the colon cannot be analyzed using the villin promoter as transgene
expression is mostly absent or very low (123).
The phenotype of villin-Hhip transgenic mice was
more severe than that of Ihh⫺/⫺ mice. In mice with high
levels of transgene expression, villus formation was completely abrogated, and regions of epithelium were observed that failed to organize in a single layer of polarized
columnar epithelial cell but seemed instead to have remained pseudocolumnar. As the villin-Hhip transgene
presumably blocks signaling by both Shh and Ihh, this
indicates that the main role for Hedgehog signaling is in
villus growth and development. However, the phenotype
of the Shh null mouse suggests that Shh may play some
role in restricting duodenal villus growth after their establishment. In mice with lower villin-Hhip transgene
levels, the villi were thickened and showed extensive
branching (Fig. 12). Branching was caused by the presence of dispersed cryptlike pockets of proliferating cells
on the villi from which new villi seemed to originate. Thus
Hedgehog signaling not only plays a role in induction of
villus growth, but it also seems to act to restrict precursor
cells to the intervillus epithelium. Interestingly, the phenotype of ectopic crypt formation on the villi was remark-
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
needed to regulate epithelial differentiation is intrinsic to
the gut tube and independent of luminal content or developmental stage-specific hormonal regulation. It was
even shown that when small intestinal segments of E14
rats were transplanted under the skin of nude mice, the
timing of epithelial differentiation of the segments occurs
according to the correct anterior-posterior wave of differentiation (38). Also, as discussed above, endoderm-mesenchymal recombination experiments indicate that the
small intestinal endoderm differentiates according to its
original position along the AP axis and independent from
the type of mesenchyme with which it is recombined.
Only the colonic endoderm inappropriately expresses
small intestinal enzymes when recombined with small
intestinal mesenchyme, indicating an instructive influence
of the colonic mesenchyme at later stages of development. Interestingly, the immature colon epithelium expresses several markers of small intestinal differentiation
and shows absorptive and digestive functions that are
typical for small intestinal enterocytes. Apparently, instructive signals from the colonic mesenchyme are necessarily late in colonic development to suppress small
intestinal cell fate.
The second phase of small intestinal epithelial differentiation occurs after birth, extending from postnatal day
(P) 1 to P28. During the first two postnatal weeks, the
crypts of Lieberkühn are generated in the small intestine.
These crypts replace the intervillus epithelium in a poorly
understood process. The distance between the base of the
intervillus epithelium and the circular smooth muscle
layer remains constant during crypt formation (17). Crypt
formation therefore seems to involve mesenchymal enveloping of the nascent crypt and upwards migration of the
junction between the precursor cell compartment of
the intervillus epithelium and the differentiated cells on
the villi (17). This period coincides with the formation of
morphologically recognizable Paneth cells (15). The number of crypts increases rapidly in the third postnatal week
through crypt fissioning (17). This process of crypt multiplication involves the branching of a crypt to form two
independent crypts and seems to result from symmetric
stem cell division (54). At P28, crypt morphogenesis and
multiplication is complete and the crypt contains a few
Paneth cells at its base (in increasing number from the
duodenum to ileum), one or more stem cells just above
the Paneth cells and/or in between the Paneth cells, and a
number of more or less committed precursor cells above
the stem cell position.
1359
1360
GIJS R. VAN DEN BRINK
FIG. 12. Hedgehog signaling regulates villus formation in the small intestine. A schematic representation of
the P0 small intestine of wild-type (A) and villin-Hhip
transgenic and anti-Hedgehog antibody-treated mice (B)
(122, 204). In the wild-type newborn intestine, proliferating and differentiated cells have compartmentalized. Differentiated enterocytes populate the villi, whereas proliferating precursor cells (characterized by activity of the
Wnt pathway) localize to the epithelium between the
villi. In villin-Hhip mice with low transgene expression,
this compartmentalization is disturbed, and ectopic
patches of precursor cells are found on the villi resulting
in a branching phenotype.
Physiol Rev • VOL
ated via an effect on the precursor cells or the differentiated cells. It could be, for example, that Hedgehog signaling is necessary to repress Wnt signaling and that
precursor cells fail to exit from the cell cycle in the
absence of Hedgehog signaling. Alternatively, Hedgehog
signaling may be necessary for the development and/or
survival of cells that have already exited the cell cycle.
That would result in a similar accumulation of precursor
cells in the epithelium of the villin-Hip mouse secondary
to a lack of differentiated cells. An experiment in Xenopus
may point towards the first possibility. In Xenopus that
were injected with a constitutive active form of Smo, the
endoderm was completely absent from the small intestine, suggesting that Hedgehog signaling may upregulate a
factor in the mesenchyme that suppresses endodermal
precursor cell fate. A major target for Hedgehog signaling
at later stages of intestinal development may be the intestinal subepithelial myofibroblasts (ISEMFs) (158).
ISEMFs are ␣-smooth muscle antigen (␣-SMA)-positive
fibroblast-like cells that lie closely apposed to the basal
membrane of the intestinal epithelium of the colon and
small intestine. Madison et al. (122) have shown that
ISEMFs respond to Hedgehog signaling in vitro. The authors further showed that ISEMFs are increased in number in the mucosa of villin-Hhip mice and abnormally
localized in the villi where they were found in close
proximity to the ectopic cryptlike pockets of precursor
cells (122). These findings have an interesting link with
very elegant work performed by Ormestad et al. (146).
Ormestad and colleagues showed that Foxf1 and Foxf2
transcription factors are mesenchymal Hedgehog target
genes that play a critical role in ISEMFs. At E18.5, Foxf1
and FoxF2 mutant mice show impaired extracellular matrix formation, loss of endodermal differentiation, accumulation of epithelial precursor cells characterized by
increased endodermal Wnt signaling, and accumulation of
ISEMFs (146). In all of these aspects, the phenotype
strongly resembles the phenotype of the villin-Hhip
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
ably similar to the phenotype described in mice that expressed the BMP antagonist noggin behind a villin promoter and mice in which the Bmp receptor 1a was deleted
(58, 60). It has been shown that Bmp4 is a mesenchymal
target of Hedgehog signaling in the small intestine of the
chick (167, 168) and mouse (123, 202), suggesting that
epithelial Hedgehog may be upstream of mesenchymal
Bmp4 in the control of precursor cell compartmentalization. One finding that argues against this possibility is that
the ectopic crypt phenotype in the villin-Noggin mouse
only becomes evident 3 wk after birth, directly after the
completion of intestinal epithelial maturation and crypt
formation (however two different villin promoters were
used in these studies) (58).
The epithelial phenotype of the villin-Hhip mouse
showed a substantial reduction of the number of differentiating cells and accumulation of precursor cells very
similar to phenotype of postnatal mice treated with a
Hedgehog-blocking antibody that had been described before (204) (Fig. 12, discussed below). In the mice that had
lower Hip transgene expression, a normal presence was
observed of the two secretory cell lineages that are
present in the first postnatal week (Goblet cells and endocrine cells), whereas differentiation of the enterocyte
cell lineage was reduced with a poorly developed brush
border and loss of expression of brush-border markers.
This important role of Hedgehog signaling in enterocyte
differentiation correlates with our own experiments (197)
in which we have shown that Hedgehog signaling regulates enterocyte differentiation in the distal colon of the
adult rat (see below). The epithelial phenotype is indirect,
since Hedgehog signaling is exclusively from the epithelium to the mesenchyme in the developing and early
postnatal small intestine and therefore mediated via an as
yet unidentified mesenchymal factor that is controlled by
Hedgehog signaling.
An important question is if the increase in the number of precursor cells in the villin-Hhip mouse is medi-
HEDGEHOG SIGNALING
Physiol Rev • VOL
mal cytodifferentiation. Both Bmp4 and Wnt5a are good
candidates as mediators of this reciprocal signal.
6. Hedgehog signaling in anorectal development
The anorectum develops with a complex interplay of
all three germ layers as it forms at the junction of the
endoderm and ectoderm (32, 168). Hedgehog signaling is
critical to normal anorectal development. Shh⫺/⫺ (and not
Ihh⫺/⫺) mice maintained by Ramalho-Santos et al. (164)
displayed an imperforate anus phenotype. The colon of
Shh⫺/⫺ mice ended in a blind sack and did not form a
connection with the perineum. Shh⫺/⫺ mice maintained
by Mo et al. (136) displayed a failure to separate the
anorectum from the lower urinary tract, that drained in a
common cavity or outlet. Gli2⫺/⫺ mice have an imperforate anus with a rectovesical fistula (between the distal
intestine and the bladder) and a single urethral opening in
the perineum [see also Kim et al. (98)]. The phenotype of
Gli3⫺/⫺ mice is very mild with slight anal stenosis. In
Gli2⫺/⫺ Gli3⫹/⫺ and Gli2⫹/⫺ Gli3⫺/⫺ compound mutant
mice, a persistent cloaca was observed and a common
outlet for the digestive and urogenital tract (136). These
phenotypes indicate that although Gli3 does play a role in
the transduction of the Shh signal to the anorectal mesenchyme, the contribution of Gli2 is much more important similar to the role of Gli signaling in foregut development.
7. Hedgehog signaling in pancreas development
As discussed above, exclusion of Shh and Ihh expression from the prospective pancreatic endoderm plays a
critical role in early pancreatic development. Hedgehog
signaling does play a role at later stages in development
however. Although no Shh is expressed during pancreatic
development, Ihh and Ptc1 are expressed in the pancreas
as detected by RT-PCR from E13.5 onwards, but their
expression has not been localized (and Dhh has not been
examined). Both Ihh and Dhh are expressed in the islets
of the adult pancreas (62, 191). Ptc1 is expressed in the
islets and ducts of the adult pancreas (62), suggesting that
Hedgehog signaling in the islets is autocrine.
Ihh seems to play a role in the morphogenetic movements of the ventral and dorsal pancreatic buds as almost
half of Ihh⫺/⫺ mice develop an annular pancreas in which
an extension of the ventral pancreas encircles the duodenum (62, 164). Histologically, the pancreas of Ihh mutant
mice appears intact and contains normal islets. Since it is
not known if Dhh is expressed during the later stages of
development of the pancreas, it is not clear if presence of
Dhh may rescue a phenotype. More careful analysis of
pancreatic cellular differentiation in Shh⫺/⫺ and Ihh⫺/⫺
mice appeared to show a relative increase in the number
of endocrine cells in the pancreas, especially in the Ihh⫺/⫺
mice. The authors of the study expressed the number of
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
mouse, suggesting that Foxf1 and Foxf2 may be the major
effectors of Hedgehog signaling in the mesenchyme. Interestingly, in Foxf2⫺/⫺ mice, Bmp4 is completely absent
from the mesenchyme, suggesting that loss of mesenchymal Bmp4 expression may indeed explain the endodermal
phenotype in villin-Hhip transgenic mice as discussed
above. Together these data suggest that endodermal
Hedgehog signaling induces Foxf transcription factors in
the mesenchyme, which in turn induce mesenchymal
Bmp4 expression which directly acts on the epithelium to
restrict endodermal precursor cell accumulation and
stimulate endodermal differentiation. An alternative scenario is discussed by Ormestad et al. (135); they show that
mesenchymal Bmp4 acts to suppress mesenchymal
Wnt5a expression and that Wnt5a expression is strongly
induced in Foxf2⫺/⫺ mice. The authors suggest that this
excess Wnt5a may directly be responsible for increased
endodermal Wnt signaling. This is an interesting possibility that remains to be tested as Wnt5a can both activate
and repress canonical Wnt signaling depending on the
receptor context (135).
Both Ihh and Shh mutant mice display a reduced
thickness of the circular smooth muscle layer that surrounds the small intestine and distinct defects in the
development of the enteric nervous system (164). Both
phenotypes are not present in the villin-Hhip mouse. As
a result, the Hirschsprung’s diseaselike phenotype that
has been described in Ihh⫺/⫺ mutant mice (164) is absent
from villin-Hhip mice. The most likely explanation for
this discrepancy is that the effect of Hedgehog signaling
on the size of the developing smooth muscle layer and
development of the enteric nervous system is mainly mediated before expression of the Hhip transgene (i.e., before E12.5). Another possibility is that long-range Hedgehog signaling to the smooth muscle layer and developing
enteric nervous system is not affected by the Hhip transgene.
Wang et al. (204) have studied the role of Hedgehog
signaling in postnatal development of the small intestine
by injecting newborn pups with a Hedgehog blocking or
control antibody. Anti-Hedgehog antibody-treated mice
developed steatorrhea due to lipid malabsorption and a
runting phenotype. The effect on villus morphology was
similar to that later described by Madison et al. (122) in
the prenatal small intestine with shortened villi that show
increased branching, precursor cell accumulation in the
intervillus epithelium, and spreading of precursor cells on
the villus (Fig. 12).
In conclusion, Hedgehog signaling acts on the mesenchyme in the developing small intestine. It regulates the
differentiation of ISEMFs, the smooth muscle layer, and
the developing enteric nervous system. Hedgehog signaling induces the expression of a mesenchymal factor that
acts reciprocally on the developing endoderm and plays a
critical role in precursor cell homeostasis and/or endoder-
1361
1362
GIJS R. VAN DEN BRINK
8. Hedgehog signaling and the VACTERL association
Many of the developmental abnormalities that have
been described in Hedgehog mutant mice above are also
found in human patients. These patients have a variety of
nonrandomly associated developmental abnormalities
that occur sporadically and are known under the acronym
VACTERL. VACTERL stands for vertebral defects (V),
anal atresia (A), cardiac defects (C), tracheoesophageal
fistula (TE), renal anomalies (R), and limb defects (L) (97,
131, 161, 166). Although these abnormalities are often
found together, they are found in varying constellations
and the VACTERL association is not recognized as a
specific syndrome. One study that examined 2,493,999
births only reported a single case with combined recognized deformities in all 6 organs (166). However, all of
these defects have been found in one or more of the
Physiol Rev • VOL
several known human and murine mutants of the Hedgehog pathway (73, 86, 100, 117, 136, 138, 162, 164) [see Kim
et al. for review (98)]. The atresia or stenosis of the
esophagus with associated tracheoesophageal (TE) fistula that was found in Hedgehog mutant mice is observed
in ⬃1 in 2,000 –5,000 live human births (97). Just like in
the Hedgehog mutants this anomaly can be found in association with other gastrointestinal malformations in humans such as anal atresia, midgut malrotation, duodenal
atresia, and annular pancreas (3) and with malformations
of other organs as part of the VACTERL association mentioned above. Even though a strong similarity exists between patients with the VACTERL association and Hedgehog mutant mice, mutations have not yet been described
in patients. The VACTERL association shows considerable overlap with the Feingold syndrome (21). Feingold
syndrome is caused by germline mutations in N-myc
(195), which is an established target of Hedgehog signaling (96). Although no mutations in N-myc have been
found in patients with the VACTERL association, this
suggests that mutations in the Hedgehog pathway or important targets of Hedgehog signaling may indeed be its
cause.
IV. HEDGEHOG SIGNALING AND
HOMEOSTASIS OF THE ADULT GUT
A. Morphogenesis Versus Morphostasis
As discussed above, the developing embryo is patterned along several different axes. This patterning involves inductive signals that are exchanged between different germ layers through morphogenetic signaling pathways that are highly conserved throughout evolution. It is
important to realize that cell fate regulation along different spatial axes is not a feature unique to the developing
embryo. Cell fate is similarly regulated in a positiondependent manner along different axes in rapidly regenerating epithelial tissues such as those of the gastrointestinal tract (63). Equally spaced stem cell niches along the
length of the gut continuously generate a fresh supply of
epithelial cells. Cell fate of stem cells and their descendants is regulated along the vertical axis of the gut. The
second axis of cell fate determination is the longitudinal
axis (esophagus, stomach, small intestine, colon), which
determines the phenotype of epithelial cells that are generated by a particular stem cell. It is increasingly being
recognized that morphogens not only play a role in the
spatial regulation of cell fate during morphogenesis but
also play a critical role in the maintenance of tissue
homeostasis or morphostasis in the adult along these two
different axes. This also explains why mutations in morphogenetic pathways such as the Wnt and BMP pathway
can initiate carcinogenesis (10, 58, 60, 69). Knowledge of
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
endocrine cells relative to body weight (62). This distorts
the calculations as body weight was much reduced in
Shh⫺/⫺ mice, whereas the pancreas was of normal size (as
may be expected given the absence of Shh expression at
any point in pancreatic development). If the number of
endocrine cells is expressed per pancreas weight, their
relative number is only modestly increased in Shh⫺/⫺
mice (22% increase), whereas the number of endocrine
cells/pancreas weight is increased by 75% in the Ihh⫺/⫺
mice (62). This seems more consistent with a role for Ihh
signaling in later pancreatic development and suggests
that Ihh acts as a negative regulator of the endocrine cell
fate in the developing pancreas. This finding was corroborated by the phenotype of mice that lack the Hedgehog
antagonist hedgehog interacting protein (Hhip). In
Hhip⫺/⫺ mice, excess Hedgehog signaling in the pancreas
results in loss of endocrine cells (92). Similarly, overexpression of Shh or Ihh in the pancreas from E13.0 onwards using a Pax4 promoter resulted in excess mesenchymal growth, inhibition of epithelial expansion, and
loss of endocrine cells. At E18.5, this resulted in a pancreas that was ⬃60% reduced in size mainly due to a
dramatic reduction in epithelial cell content (93). In conclusion, all mouse mutants in the Hedgehog pathway that
have been analyzed show that Hedgehog signaling represses endocrine cell fate at later stages of pancreatic
development. Interestingly, Pax6, a gene that is repressed
by Hedgehog signaling in the developing neural tube (42),
pituitary gland (104), and retina (154), plays a critical role
in pancreatic development. Pax6 is a transcription factor
that is expressed at least from E9.0 onwards in the pancreatic endoderm (180), and mice that lack Pax6 have
reduced numbers of endocrine cells and disturbed islet
morphology (172, 180). It would be interesting to see if the
observed negative regulatory effect of Hedgehog signaling
may (in part) be mediated through repression of Pax6
expression.
HEDGEHOG SIGNALING
the role of Hedgehog signaling in the adult gut is still
limited, since it has received little attention to date. Here
I will discuss what is currently known about the role of
Hedgehog signaling in adult gut and how this pathway may
be deregulated during gastrointestinal carcinogenesis.
B. Stomach
Physiol Rev • VOL
Recently, Fukaya et al. (49) performed a careful analysis of Hedgehog pathway component mRNAs in microdissected pit isthmus and gland cells of the human stomach. Unfortunately, the exact provenance of the specimens in this study was not specified; it is therefore not
entirely clear if all specimens shown are of fundic origin,
which makes the interpretation of some of the results
somewhat difficult. Their results showed that all three
Hedgehogs can be detected by PCR in gastric epithelium
in humans. The authors found Ihh expression in gastric
pit cells and Shh and Dhh expression in the gastric gland.
Ihh was expressed by the differentiated pit cells as determined by immunohistochemistry, whereas both Shh and
Dhh localized to parietal cells immunohistochemically.
Given the fact that the Dhh antibody used in this study
recognizes the 19-kDa active fragment of Dhh which is
extremely conserved between all Hedgehogs, it may also
be that the anti-Dhh antibody cross-reacts with Shh in
parietal cells, and this result therefore needs to be confirmed by in situ hybridization. In the hands of Fukaya
et al. (49), a Ptc1 antibody reacted only with gastric pit
cells. This result contradicts previously obtained results
with two different Ptc1 antibodies (C-20 from Santa Cruz
and an antibody obtained from Dr. R. Toftgard) (36, 199),
in situ hybridization (141), and a Ptc1-LacZ reporter
mouse (8) which all localized Ptc1 to the gastric glands of
the gastric fundus. Together these results may suggest
that Ptc1 is mainly expressed in the fundic glands but that
low levels may be present in the fundic pit cells where it
is induced by Ihh that is produced by differentiated pit
cells. This conclusion is supported by the observation that
cyclopamine treatment depletes pit cells from primary
cultures of mouse gastric epithelial cells (49).
Although Hedgehog signaling maintains proliferation
of some gastric cancer cells (see below), we found that
treatment of mice with cyclopamine increased epithelial
proliferation in vivo by 60 –70% (199). Similarly, Fukaya
et al. (49) observed that treatment of primary cultures of
normal mouse gastric epithelial cells with Smo antagonist
cyclopamine doubled their proliferation. This may indicate that there is a difference in the role of Hedgehog
signaling in cell cycle regulation between normal and
cancer cells. More likely, one of the factors that may
explain the difference observed in the response of gastric
carcinoma cells and primary gastric epithelial cells in vivo
and in culture is that the effects on cancer cells have been
observed in pure populations of cultured cancer cells,
whereas both our in vivo experiment and the primary
cultures of gastric epithelial cells of Fukaya et al. included
all gastric cell types and stromal cells. The fact that Ptc1
is not expressed in the isthmus (49) (which contains the
precursor cells) suggests that Hedgehogs may not act
directly on precursor cells and that the observed effects
on proliferation are indirect and mediated via effects of
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
One month after birth a stable regenerating system is
established in which mucus-secreting pit cells migrate
towards the lumen from the precursor cell compartment
in the isthmus (87, 109). The proximal glandular stomach
(fundus) has small pit regions and large glands, whereas
the distal stomach (antrum) has large pit regions and
small glands. Glands are composed of several cell types
that migrate down to the gland base from the stem cell
position. Fundic glands are composed of parietal cells
which secrete acid, endocrine cells, and mucous neck
cells, a cell type with uncertain function and which transdifferentiates when halfway down the gland into zymogenic cells that secrete digestive enzymes (88). Antral
glands are exclusively composed of mucous and endocrine cell types (110). We found that Shh protein and
mRNA were expressed in the parietal cells of the fundic
glands in humans (198, 199). The protein formed a concentration gradient with highest expression in the parietal
cells closest to the isthmus and gradually decreasing expression as the parietal cell matured towards the base of
the gland (199). In contrast to the restricted expression
pattern in humans, we found Shh in a variety of gland
cells in rats and mice (199), a finding that was confirmed
by in situ hybridization for Shh in the Mongolian gerbil
(181). No Shh protein or mRNA is found in the glands of
the antrum (198). Hedgehog receptor and transcriptional
target Ptc1 was similarly expressed in the epithelium of
the gastric fundic glands (199). Treatment of mice with
Smo antagonist cyclopamine resulted in downregulation
of potential Hedgehog targets such as islet-1, Bmp4, and
Foxa2 (Hnf3␤). Bmp4 was expressed in mesenchymal
fibroblast-like cells and Foxa2 mainly by parietal cells
with much weaker staining in other gastric epithelial cell
types. These results indicated that fibroblasts and parietal
cells were the major targets of Hedgehog signaling in the
fundus. The role of Hedgehog signaling in parietal cell
physiology was further explored in isolated canine parietal cells by Stepan et al. (179). It was shown that Shh
expression in parietal cells could be strongly induced by
EGF and that Shh positively regulates the expression of
the H⫹-K⫹-ATPase ␣-subunit through Gli binding elements in the promoter region and potentiates histamineinduced gastric acid secretion. Treatment with cyclopamine completely blocked EGF-induced H⫹-K⫹-ATPase
expression but not induction of the c-Fos promoter, indicating that induction of Shh mediates some but not all
aspects of EGF signaling in parietal cells.
1363
1364
GIJS R. VAN DEN BRINK
Hedgehog signaling on stromal cell types or on the differentiated epithelial cells.
In conclusion, much work remains to be done in
understanding the role of Hedgehog signaling in the adult
stomach. Progress will be made by more careful analysis
of expression patterns of Hedgehog pathway components
by in situ hybridization and the study of inducible mutant
mice of different components of the Hedgehog pathway.
So far the available data have shown a role for Shh
signaling in parietal cell physiology and suggest that Ihh
may be involved in the differentiation and maintenance of
the pit cell lineage.
The adult small intestinal epithelium is established
after the completion of crypt formation in the third postnatal week. A stem cell just above the base of the crypts
generates enterocytes, goblet cells, and endocrine cells
that migrate up towards the villus and Paneth cells that
migrate down and fill the base of the crypts (52).
Very little is known about the role of Hedgehog signaling in the adult small intestine. We found that low
levels of Shh mRNA can be detected just above the Paneth
cell position in the crypt, but expression levels are too
low to be detected by immunohistochemistry when using
gastric specimens as a positive control (198). This low
expression of Shh in the small intestine and colon compared with the stomach was confirmed by others by quantitative PCR (181). In situ hybridization by Batts et al. (6)
showed that Ihh is expressed at the crypt-villus junction
with gradually diminishing expression towards the tip of
the villus, whereas Ptc1 is expressed at low levels in the
mesenchyme (Fig. 13). Ihh protein is mainly expressed by
the enterocytes on the upper half of the villus (80). This
partially overlapping expression pattern of mRNA and
protein is typical for many enterocyte genes and explained by the fact that cells that have moved to the top of
the villus often no longer transcribe mRNA from a gene
but still express the translated protein. Immunohistochemical analysis suggests that rare Ptc1-positive epithelial cells may also exist around the stem cell position just
above the Paneth cell (202). Varnat et al. (202) found Ihh
protein expression in the Paneth cells at the base of the
crypts; it is not entirely clear how this can be reconciled
with the in situ hybridization and immunohistochemistry
done by others. This issue needs to be resolved by further
experimentation that would also take into account the
exact provenance of the small intestinal tissue along its
AP axis as the concentration of Hedgehogs diminishes
towards the distal small intestine (204), and differences in
the expression patterns of different Hedgehog pathway
components may exist along the AP axis of the small
intestine.
Physiol Rev • VOL
FIG. 13. Hedgehog signaling in the adult small intestine. Ihh is the
predominant Hedgehog expressed in the adult small intestine. Ihh
mRNA is expressed at highest levels at the crypt villus junction, with
diminishing levels towards the top of the crypt. Ihh protein is expressed
from halfway up the villus to its tip. Ptc mRNA expression is mainly in
the mesenchyme, but rare Ptc protein-expressing cells may exist in the
crypts. Shh mRNA is expressed by a few epithelial cells in each crypt.
We found that treatment of mice with cyclopamine
results in a modest ⬃10% reduction of proliferation in the
duodenum. The only available data concerning the role of
Hedgehog signaling in the adult small intestine are from a
study by Varnat et al. (202). These authors studied the role
of Ppar␤ (⫽Ppar␦) in small intestinal homeostasis and
showed that Ppar␤ is important for Paneth cell differentiation. The expression of Ihh mRNA and protein was
induced approximately threefold in Ppar␤ null mice, and
Ihh mRNA could be strongly reduced by treatment of
wild-type mice with a Ppar␤ agonist, showing that Ppar␤
negatively regulates Ihh expression. Treatment of wildtype and Ppar␤⫺/⫺ mutant mice with cyclopamine increased the number of Paneth cells in the duodenum,
suggesting that at least part of the effect of Ppar␤ is
mediated through repression of Ihh expression. This effect was recapitulated in vitro as the expression of lysozyme, a Paneth cell marker, was reduced in HT-29 colon
cancer cells by treatment with recombinant Shh, whereas
treatment with cyclopamine increased lysozyme expression (202).
D. Colon
The mucosa of the colon is covered by a flat epithelium that lacks a villus structure but contains numerous
crypts. Three cell types exist in the adult colon: absorptive enterocytes, goblet cells, and endocrine cells. The
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
C. Small Intestine
HEDGEHOG SIGNALING
Physiol Rev • VOL
showed loss of markers of enterocyte differentiation such
as brush-border staining for villin and carbonic anhydrase
IV but inappropriately expressed intestinal trefoil factor
(Itf), a marker of the goblet cell lineage. These data
suggested that Hedgehog signaling is necessary for enterocyte maturation and the appropriate restriction of the
enterocyte transcriptome to markers of the enterocyte
lineage. This phenomenon of the inappropriate induction
of Itf expression in the enterocyte lineage may be similar
to the induction of alkaline phosphatase expression in the
gastric epithelium that has been observed in the developing stomach of Hedgehog mutant mice (discussed above).
Interestingly, since Ptc1 seems to be expressed uniquely
in the mesenchyme underlying the differentiated enterocytes (at least in the mouse), this effect on enterocyte
differentiation is likely indirect similar to the effect on
epithelial differentiation in the developing small intestine
observed in the villin-Hip mouse (discussed above).
We have confirmed the role of Hedgehog signaling in
enterocyte maturation in the HT-29 cell differentiation
model. This model is based on the observation that HT-29
cells grow into a confluent monolayer and can be made to
differentiate upon glucose withdrawal or stimulation with
butyrate, a short-chain fatty acid (5, 218). We found that
butyrate induces Hedgehog expression in HT-29 cells. We
further demonstrated that Hedgehog signaling is necessary and sufficient for induction of differentiation markers p21 (WAF/CIP1) and villin in HT-29 cells, experiments
that have been confirmed by Varnat et al. (202). HT-29
cells do express PTC1 and GLI1 mRNA (160, 217), explaining the observed effects of Hedgehog on HT-29 cells.
It is, however, questionable if experiments examining a
direct effect of Hedgehog signaling on epithelial cells are
of any relevance since we fail to detect Ptc1 mRNA in the
colonic epithelium of mice in vivo (unpublished observations discussed above), making it much more likely that a
so far unidentified factor from the mesenchyme mediates
the observed effects on enterocyte differentiation in vivo.
Clearly additional experiments are required to reassess
PTC1 expression and examine GLI1 expression in normal
human colonic epithelium and colorectal cancers using
in situ hybridization to avoid problems of antibody
aspecifity.
In rats treated with cyclopamine, proliferation increased by ⬃50% similar to the effect observed in the
mouse stomach and in contrast to the effect on the small
intestine where we observed a modest reduction of epithelial proliferation. As in the stomach, this probably
reflects the fact that differentiating epithelial cells and not
the precursor cells are the major target of Hedgehog
signaling in vivo or that the effect on proliferation is
indirectly mediated via the stroma in vivo as our recent in
situ hybridization for Ptc1 seems to suggest. In contrast to
the inhibitory effect of Hedgehog signaling on colonic
precursor cells studied in a whole tissue context in vivo,
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
proximal (ascending) and distal (descending) colon have
a different histology. Histology of the proximal colon of
the mouse has been poorly studied; the mucosa is organized in large folds, and epithelial precursor cells are
localized halfway up the crypts, indicating that some cells
migrate down towards the base of the crypt. In the distal
colon precursor cells localize to the bottom of the crypt,
and the different cell lineages are present in gradients
(22). Enterocytes differentiate in a gradient from the base
to the top of the crypt, and differentiated enterocytes are
found at the top of the crypt and on the intercrypt tables.
Goblet cells are mainly found in the midcrypt region,
whereas most endocrine cells are found at the base of the
crypt (22).
We found very low levels of Shh at the base of the
crypts by in situ hybridization. Similar to the small intestine, we have not been able to detect Shh protein in these
cells by immunohistochemistry, using gastric biopsies as
positive control (198). As mentioned above, this correlates with data from Suzuki et al. (181), who have shown
by quantitative PCR that levels of Shh are very low in the
small intestine and colon compared with the stomach.
Using different antibodies we detected a strong signal for
Hedgehog protein in the absorptive enterocytes of the
distal colon (Fig. 14) (197). These same cells were positive for Ihh by in situ hybridization showing that differentiated enterocytes of the distal colon produce Ihh. We
have previously described Ptc1 protein expression in the
epithelium and mesenchyme of the human, rat, and
mouse colon (197) and Ptc1 mRNA in the epithelium and
mesenchyme of the distal human colon (141), suggesting
that Hedgehog signaling acts both paracrine and autocrine in the adult colon. As some doubts were raised
about the specificity of the anti-Ptc1 antibodies, we have
now performed novel in situ hybridizations for Ptc1 in the
mouse colon and anorectum and find that although Ptc1
mRNA is expressed in the epithelium of the anorectum,
no Ptc1 mRNA is detected in colonic epithelium (unpublished observations). Instead, a clear Ptc1 mRNA signal is
detected in the mesenchyme underlying the Ihh-positive
differentiated colonic enterocytes in the mouse. This
clearly suggests that Hedgehog signaling in the adult colon of the mouse is uniquely from the epithelium to the
mesenchyme similar to the adult and developing small
intestine (discussed above) and not partially autocrine as
we have suggested previously based on immunohistochemical results (197). We will also reexamine the human
colon as it is possible that differences between species
exist and that PTC1 mRNA is expressed in the human
colonic epithelium as we have found previously (141).
Additionally, Gli1 localization will have to be examined
by in situ hybridization in both mice and humans.
In rats treated with Smo antagonist cyclopamine,
enterocyte maturation was disturbed in the distal colon,
whereas goblet cells matured normally. The enterocytes
1365
1366
GIJS R. VAN DEN BRINK
Hedgehog signaling seems to stimulate proliferation if
primary colonic enterocytes are isolated from their context and cultured in pure populations (145). This opposite
effect of Hedgehog signaling on isolated precursor cells
and precursor cells in vivo is similar to the situation in the
stomach discussed above. It indicates that some effects of
Hedgehog signaling on epithelial cells may be indirect and
mediated via the stroma and that care has to be taken in
the interpretation of results when isolated gastrointestinal precursor cells are studied.
One of the effects we observed when looking for
possible targets of Hedgehog signaling in the distal colon
Physiol Rev • VOL
was that the expression of Bmp4 was increased in the
epithelium of cyclopamine-treated rats. In the distal colon
Bmp4 is an epithelial Wnt target that is expressed mainly
at the base of the crypt (196). Signaling by the Wnt pathway is critical in the regulation of intestinal precursor cell
maintenance and proliferation (55, 196). When we examined the Bmp4 expression pattern in cyclopamine-treated
rats, we observed that the normal expression gradient
was lost and high Bmp4 expression extended all the way
up to the abnormally differentiated enterocytes at the top
of the crypt (197). These data suggested that Hedgehog
signaling may act to restrict Wnt signaling to the base of
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
FIG. 14. Hedgehog signaling in the adult colon. Hedgehog protein is expressed by the differentiated colonic enterocytes at the top of the crypt
in the human (A) and rat (B) distal colon. C–J show colon of control rats (C, E, G, I) and rats treated with Smo inhibitor cyclopamine (D, F, H, J).
C: in controls, nuclei of the differentiated enterocytes at the top of the crypt have a dense slender appearance. D: in cyclopamine-treated rats,
differentiation of the enterocytes is disturbed (open arrowheads). A few remaining normal enterocytes can be observed (closed arrowhead). E:
normal colonic enterocytes express the brush-border enzyme carbonic anhydrase IV (CAIV; arrows). F: expression of CAIV is lost in cyclopaminetreated rats (remaining normal cells with brush-border staining indicated with arrows). G and I: in control rats, the expression of intestinal trefoil
factor (ITF) is appropriately restricted to the goblet cell lineage (asterisk in I indicates ITF positive goblet cell). H and J: in cyclopamine-treated
animals, expression of ITF is induced in the enterocyte lineage (arrows indicate ITF positive enterocytes). [From van den Brink et al. (197), with
permission from Nature Publishing Group.]
HEDGEHOG SIGNALING
the crypt. Experiments are currently underway in our
laboratory to confirm this negative effect of Hedgehog
signaling on the Wnt pathway in conditional Hedgehog
pathway mutant mice in vivo.
In conclusion, differentiated colonic enterocytes of
the distal colon produce Ihh which plays a role in their
appropriate maturation. Given our recent finding that
Ptc1 is expressed in the mesenchyme and not the epithelium of the mouse colon (unpublished observations), this
effect of enterocyte differentiation is likely the result of
epithelial-mesenchymal interactions, but additional experiments are required to address this issue.
A. Evidence for a Role for Hedgehog Signaling in
Initiation of Gastrointestinal Carcinogenesis?
The role of Hedgehog signaling in carcinogenesis of
several tissues has become evident from the analysis of
patients with inherited cancer syndromes and genetically
modified mice. Mutations in the Hedgehog receptor PTC1
lead to constitutive activity of the Hedgehog pathway and
cause basal cell carcinomas (BCC) and medulloblastomas
in patients with Gorlin syndrome (57, 79), a phenotype
that is recapitulated in mice with the same mutations. The
relevance of PTC1 mutations for the biology of these
cancers is underscored by the fact that these activating
mutations in the Hedgehog pathway are also found in
sporadically occurring cases of the same cancers (155,
163, 194, 209). Two germline and two sporadic mutations
in the intracellular Hedgehog signaling antagonist Suppressor of Fused have been described in 46 children with
medulloblastoma (187). Subsequent screening for SUFU
mutations in 2 independent sets of 134 (105) and 33
medulloblastomas (140) found 0 and 2 SUFU mutations,
respectively, suggesting that SUFU mutations exist but
are a rare phenomenon.
The Hedgehog mutant mice that have been analyzed
thus far do not develop gastrointestinal cancers except in
the pancreas, indicating that it is unlikely that overactivation of the Hedgehog pathway is sufficient to initiate
carcinogenesis in most of the gastrointestinal tract. In
fact, a mouse with an inducible constitutive active form of
Smo developed rhabdomyosarcoma, BCC, and medulloblastoma as expected, but no neoplastic lesions were
observed in the esophagus, stomach, biliary epithelium,
small intestine, or colon (128). The only gastrointestinal
organ with a role for Hedgehog signaling in tumor initiation may be the pancreas. Thayer et al. (190) have shown
that Shh is overexpressed in human preneoplastic pancreatic lesions and reported that a mouse that expressed Shh
behind a pancreas specific promoter developed preneoPhysiol Rev • VOL
plastic lesions, so-called PanIN lesions [for review, see
Hezel et al. (64)]. In a recent consensus report on mouse
models of pancreatic cancer it was concluded that although the pancreas of the Pdx1-Shh mouse shows intestinal metaplasia and cellular atypia, no epithelial lesions
were observed that resemble human PanIN lesions (70). A
mouse with inducible activation of a dominant active
GLI2 gene (152) developed undifferentiated pancreatic
tumors that showed invasive growth. Similar to the Pdx1Shh mouse, no lesions formed in these mice that resembled human preneoplastic lesions, arguing for an alternative pathway of tumorigenesis. Activating mutations in
RAS play an important role in pancreatic carcinogenesis
in humans and mice, with such mutations recapitulating
many aspects of the disease, including the formation of
PanIN lesions (64, 70). When the dominant active GLI2
inducible mice were crossed with mice that express an
inducible active Ras mutant (KrasG12D) in a pancreas
specific manner, dominant active GLI2 increased the formation of PanIN lesions, and mice succumbed from invasive cancers within weeks after birth (152). This suggests
that activation of Hedgehog signaling is not an initiator of
pancreatic carcinogenesis but plays an early role in pancreatic tumor progression in humans. Interestingly, Pasca
di Magliano et al. (152) found that mutant Ras strongly
induced pancreatic expression of both Shh and Ihh. This
suggests that Hedgehog ligand overexpression may be
one of the first steps that facilitate Ras-mediated carcinogenesis in the pancreas.
In contrast, in two organs that seem to express high
levels of Hedgehog protein in the adult gut, expression is
lost rather than increased in preneoplastic lesions. In the
proximal stomach, expression of SHH is reduced by Helicobacter pylori infection and lost in intestinal metaplasia
and gastric atrophy, factors that predispose to gastric
carcinogenesis (36, 175, 198). A similar loss of Shh expression was observed in the Mongolian gerbil model of
Helicobacter pylori-associated gastric carcinogenesis
(181). Likewise, in the distal colon, IHH expression is lost
very early in the process of the adenoma to carcinoma
sequence of colorectal cancer (197). There is therefore no
evidence that mutations in or altered regulation of the
Hedgehog pathway can initiate gastrointestinal carcinogenesis. As discussed below, there is important evidence
that Hedgehog signaling may play a role in the maintenance of tumor cell viability at later stages of tumorigenesis.
B. Hedgehog Signaling in Cancers of the Proximal
Gastrointestinal Tract
In an important study by Berman et al. (8), it was
shown that cancer cell lines derived from the esophagus,
stomach, pancreas, and biliary tract showed high auton-
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
V. HEDGEHOG SIGNALING AND
GASTROINTESTINAL CARCINOGENESIS
1367
1368
GIJS R. VAN DEN BRINK
Physiol Rev • VOL
C. Hedgehog Signaling in Colorectal Cancer
As discussed above, we have shown that Ihh signaling regulates differentiation of the absorptive enterocyte
lineage in the colon of the rat. We further found that IHH
expression is lost very early in the process of colorectal
carcinogenesis in humans (197). We have studied neoplastic lesions in patients with familial adenomatous polyposis (FAP) who carry a germline mutation in the adenomatous polyposis coli (APC) gene (50). These patients
develop hundreds to thousands of polyps in adolescence
upon loss of expression of the second APC allele in
intestinal epithelial precursor cells. Loss of APC expression results in uncontrolled activity of the Wnt signaling
pathway, which results in clonal precursor cell accumulation (55). Although FAP may be a rare syndrome, it is
believed that up to 90% of colorectal cancers are similarly
caused by mutations in APC or other mutations that result
in uncontrolled Wnt pathway activity (55). We found that
IHH expression was lost from polyps in patients with FAP
and in flat lesions that precede the polyp stage. These data
indicated that loss of IHH expression may be associated
with one of the first steps in colorectal carcinogenesis and
a direct result from the overactivation of the Wnt pathway. Similar to the results in APC mutant polyps, we were
able to show that IHH expression is very low in DLD-1
colorectal cancer cells which carry a mutation in the APC
gene. Expression of IHH could be restored in DLD-1 cells
by blocking Wnt pathway activation (197). These results
indicated that loss of IHH expression in APC mutant
polyps indeed resulted directly from Wnt pathway activation.
There is little evidence for activation of Hedgehog
signaling at later stages of colorectal carcinogenesis. In
the study by Berman et al. (8), it was found that in
contrast to proximal gastrointestinal cancer cells, colon
cancer cells did not depend on Hedgehog signaling for
their survival. The authors showed that this lack of sensitivity of colorectal cancer cells to cyclopamine correlated with the absence of active Hedgehog signaling in
these cells. This corroborates our findings that Hedgehog
signaling is low to absent in undifferentiated colorectal
cancer cells. We similarly found that inhibition of Hedgehog signaling with cyclopamine in colorectal cancer cells
did not affect their viability or proliferation (unpublished
observations). In contrast, Qualtrough et al. (160) observed increased cell death in a variety of colonic epithelial cell lines treated with cyclopamine; these experiments
included HT-29 cells that have also been used by us. We
currently have no explanation for this difference, but the
sensitivity of the viability of colorectal cancer cells to the
effects of cyclopamine may depend on cell culture conditions, such as the level of confluence. Berman et al. (8)
did not detect any PTC1 mRNA in colorectal cancer cells.
However, GLI1 expression correlated with PTC1 expres-
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
omous Hedgehog pathway activity. This was supported by
the demonstration that PTC1 mRNA expression was
highly induced in gastric and pancreatic carcinomas compared with adjacent normal tissue, indicating increased
pathway activity. It was shown that cell lines and cells
derived from pancreas carcinoma xenografts that express
high PTC1 levels depended on Hedgehog signaling for
their survival in vitro. The overactivity of the Hedgehog
pathway resulted from overexpression of Hedgehog ligand as it could be blocked by both the SMO antagonist
cyclopamine and a Hedgehog blocking monoclonal antibody. The data correlated with a jointly published study
by Thayer et al. (190) in which it was found that expression of SHH and PTC1 protein is low or absent
from the normal pancreas, induced at advanced stage
preneoplastic lesions, and highest in adenocarcinomas.
Six out of 13 pancreatic cell lines were sensitive to
treatment with cyclopamine in this study when grown
in vitro or as shown for some as xenografts in nude
mice. Importantly, some of the cell lines that were
unresponsive to cyclopamine treatment did express
high levels of Hedgehog target genes, suggesting that
the pathway may be activated at a level downstream of
SMO in these cells. It is important in the interpretation
of all of the available data to realize that the role of
Hedgehog signaling on gastrointestinal carcinoma cells
has so far been studied in isolated populations and not
in the context of the tumor stroma (the stroma in
xenografts is unlikely to adequately reflect the stroma
found in real cancers). As discussed above in section
IVB, the effect of Hedgehog signaling on epithelial cells
may depend on the presence of stromal cells. It is
therefore now important to examine the role of Hedgehog signaling in these tumors in their in vivo context
for example in genetically modified mice.
Samples of large patient cohorts should be examined
for Hedgehog pathway activation, to further address the
role of Hedgehog signaling in proximal gastrointestinal
carcinomas. Little data are currently available to accurately assess the frequency of Hedgehog pathway activation in proximal gastrointestinal carcinomas. Ma et al.
(120) examined 99 gastric adenocarcinomas and found
evidence for Hedgehog activity (PTC1 expression detected by in situ hybridization) in ⬃65% of all gastric
carcinomas except signet-ring cell carcinomas. The frequency of Hedgehog activation in tumors correlated with
advanced stage and poor differentiation (120). In conclusion, important evidence indicates that Hedgehog signaling is active at later stages of tumorigenesis of the proximal gastrointestinal tract. Although this may often be
associated with ligand overexpression, the data by Thayer
et al. (190) suggest that activation at a level below Smo
may also play a role.
HEDGEHOG SIGNALING
D. Therapeutic Targeting of the Hedgehog Pathway
The spectacular effect of SMO inhibition on the viability of many gastrointestinal cancer cell lines suggests
that the Hedgehog pathway may be an attractive target for
cancer therapy. It is important to further examine in large
numbers of cancers if Hedgehog pathway activity is
mostly due to ligand overexpression (8) or if pathway
Physiol Rev • VOL
activation at a level downstream of Smo plays a role in
some tumors as the data of Thayer et al. suggest (190). It
seems preferable to try to target Hedgehog signaling as far
downstream as possible. This allows treatment of cancers
in which the pathway is activated at a level downstream
of Smo and may help to minimize the chance of the
development of drug resistance. The Gli transcription
factors may therefore ultimately be the ideal target for
therapy. This may be a difficult target for drug development, however, as this would probably require a drug that
interferes with protein-DNA binding. Several compounds
have now been described that inhibit Hedgehog signaling
at the level of Smo (24, 47, 207). Hopefully one or more of
these compounds may have favorable pharmacokinetic
properties and a low toxicity profile that would allow the
first trials in human cancer patients.
VI. CONCLUSION
The Hedgehog signaling pathway is a highly conserved signaling pathway that plays an important role in
pattern formation in many, if not most, of the organs of
the body. The pathway affects patterning of the gut along
all of its axes. The first effect mediated by Hedgehog
signaling that is of relevance for the developing gut is the
regulation of left-right axis formation. Hedgehog signaling
allows the establishment of this axis by establishing a
midline barrier that is necessary to contain long-range
acting signals to one side of the body to allow the induction of left-right specific gene expression. During the early
formation of the gut tube, instructive signals from the
mesenchyme establish specific domains of Hedgehog expression along the AP axis that are critical to the normal
outgrowth of endodermal appendages, such as the airways and pancreas. At a later stage, Hedgehog signaling
affects, for example, AP axis formation of the stomach by
specifying the location of the forestomach-glandular
stomach boundary. Hedgehog affects the radial axis by
regulating the size of the smooth muscle layer of the gut,
development of the intestinal nervous system, and villus
formation in the small intestine.
Analysis of the role of Hedgehog signaling in the
adult has thus far been hampered by the embryonic lethality of most of the Hedgehog pathway mutant mice.
More knowledge will hopefully soon be available by the
generation of inducible mutant mice. From the analysis of
the expression of Hedgehog pathway components, it
seems that at least two domains exist in the adult gut with
Hedgehog pathway activity: the epithelium of the proximal stomach and the distal colon. In the stomach, Hedgehog signaling seems to play a role in parietal cell physiology and possibly pit cell survival and/or differentiation.
In the distal colon, Hedgehog signaling regulates colonic
enterocyte differentiation.
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
sion in all cell lines examined except in the colorectal
cancer cells. Correlation between PTC1 and GLI1 expression may be expected as both are transcriptional targets
of Hedgehog signaling. Also, since SMO is expressed in
colorectal cancer cells (160, 217), absence of PTC1 expression would be expected to activate the pathway and
would not result in low to absent activity of Hedgehog
signaling as observed by Berman et al. (8). Indeed, several
other labs have shown that PTC1 mRNA is expressed in
colorectal cancer cells. Qualtrough et al. (160) demonstrated PTC1 expression in two out of two colon adenoma
cell lines and three out of three colorectal cancer cell
lines. Zhu et al. (217) detected PTC1 mRNA in six out of
eight colorectal cancer cell lines. Both Zhu et al. and
Qualtrough et al. demonstrated PTC1 expression in HT-29
cells that were used in our experiments. Furthermore,
PTC1 mRNA has been demonstrated by PCR in normal
colonic epithelial cells on microdissected cells (145) and
by in situ hybridization in normal human colon (141). As
discussed above, we have recently performed in situ hybridizations for Ptc1 mRNA in the mouse colon, since
questions arose about the specificity of the anti-Ptc1 antibodies we previously used. Although we found that Ptc1
mRNA is expressed in the epithelium of the anorectum,
we did not detect any Ptc1 mRNA in the epithelium of the
mouse colon where it was exclusively expressed in the
mesenchyme underlying the differentiated enterocytes
(unpublished observations). Expression of PTC1 in colorectal cancer cells may therefore reflect their loss of
differentiation along the anterior-posterior axis of the
gastrointestinal tract (colorectal cancer cells are, for example, known to express several markers of gastric epithelium). We will reassess PTC1 mRNA expression in the
normal colonic epithelium and in colorectal cancers in
humans.
It remains questionable, however, if PTC expression
and Hedgehog responsiveness in colorectal cancer cell
lines is of any relevance if Hedgehog signaling is mainly
towards the mesenchyme in vivo. Clearly, additional experiments are required to resolve this issue, and these are
currently underway in our laboratory.
In conclusion, the relevance of the loss of Hedgehog
signaling in colorectal carcinogenesis is not yet clear. It
may be involved in the loss of epithelial differentiation
that is a hallmark of adenomatous polyps.
1369
1370
GIJS R. VAN DEN BRINK
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence: G. R.
van den Brink, Dept. of Gastroenterology & Hepatology, Leiden
University Medical Center, Building 1, C4-P012, PO Box 9600, 2300
RC Leiden, The Netherlands (e-mail: [email protected]).
GRANTS
This study was supported by a VENI grant from the Netherlands Organization for Scientific Research.
REFERENCES
1. Afouda BA, Ciau-Uitz A, Patient R. GATA4, 5 and 6 mediate
TGFbeta maintenance of endodermal gene expression in Xenopus
embryos. Development 132: 763–774, 2005.
2. Alt B, Elsalini OA, Schrumpf P, Haufs N, Lawson ND,
Schwabe GC, Mundlos S, Gruters A, Krude H, Rohr KB. Arteries define the position of the thyroid gland during its developmental relocalisation. Development 133: 3797–3804, 2006.
3. Andrassy RJ, Mahour GH. Gastrointestinal anomalies associated
with esophageal atresia or tracheoesophageal fistula. Arch Surg
114: 1125–1128, 1979.
4. Apelqvist A, Ahlgren U, Edlund H. Sonic hedgehog directs specialised mesoderm differentiation in the intestine and pancreas.
Curr Biol 7: 801– 804, 1997.
Physiol Rev • VOL
5. Augeron C, Laboisse CL. Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after
treatment with sodium butyrate. Cancer Res 44: 3961–3969, 1984.
6. Batts LE, Polk DB, Dubois RN, Kulessa H. Bmp signaling is
required for intestinal growth and morphogenesis. Dev Dyn 235:
1563–1570, 2006.
7. Beck F, Erler T, Russell A, James R. Expression of Cdx-2 in the
mouse embryo and placenta: possible role in patterning of the
extra-embryonic membranes. Dev Dyn 204: 219 –227, 1995.
8. Berman DM, Karhadkar SS, Maitra A, Montes De Oca R,
Gerstenblith MR, Briggs K, Parker AR, Shimada Y, Eshleman
JR, Watkins DN, Beachy PA. Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours. Nature
425: 846 – 851, 2003.
9. Bhatia N, Thiyagarajan S, Elcheva I, Saleem M, Dlugosz A,
Mukhtar H, Spiegelman VS. Gli2 is targeted for ubiquitination
and degradation by beta-TrCP ubiquitin ligase. J Biol Chem 281:
19320 –19326, 2006.
10. Bienz M, Clevers H. Linking colorectal cancer to Wnt signaling.
Cell 103: 311–320, 2000.
11. Bijlsma MF, Peppelenbosch MP, Spek CA. Hedgehog morphogen in cardiovascular disease. Circulation 114: 1985–1991, 2006.
12. Bijlsma MF, Spek CA, Zivkovic D, van de Water S, Rezaee F,
Peppelenbosch MP. Repression of smoothened by patched-dependent (Pro-) vitamin D3 secretion. PLoS Biol 4, 2006.
13. Bitgood MJ, McMahon AP. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse
embryo. Dev Biol 172: 126 –138, 1995.
14. Briscoe J, Chen Y, Jessell TM, Struhl G. A hedgehog-insensitive
form of patched provides evidence for direct long-range morphogen activity of sonic hedgehog in the neural tube. Mol Cell 7:
1279 –1291, 2001.
15. Bry L, Falk P, Huttner K, Ouellette A, Midtvedt T, Gordon JI.
Paneth cell differentiation in the developing intestine of normal
and transgenic mice. Proc Natl Acad Sci USA 91: 10335–10339,
1994.
16. Bumcrot DA, Takada R, McMahon AP. Proteolytic processing
yields two secreted forms of sonic hedgehog. Mol Cell Biol 15:
2294 –2303, 1995.
17. Calvert R, Pothier P. Migration of fetal intestinal intervillous cells
in neonatal mice. Anat Rec 227: 199 –206, 1990.
18. Capdevila J, Vogan KJ, Tabin CJ, Izpisua Belmonte JC. Mechanisms of left-right determination in vertebrates. Cell 101: 9 –21,
2000.
19. Cardoso WV, Lu J. Regulation of early lung morphogenesis: questions, facts and controversies. Development 133: 1611–1624, 2006.
20. Caspary T, Garcia-Garcia MJ, Huangfu D, Eggenschwiler JT,
Wyler MR, Rakeman AS, Alcorn HL, Anderson KV. Mouse
Dispatched homolog1 is required for long-range, but not juxtacrine,
Hh signaling. Curr Biol 12: 1628 –1632, 2002.
21. Celli J, van Bokhoven H, Brunner HG. Feingold syndrome:
clinical review and genetic mapping. Am J Med Genet A 122:
294 –300, 2003.
22. Chang WW, Leblond CP. Renewal of the epithelium in the descending colon of the mouse. I. Presence of three cell populations:
vacuolated-columnar, mucous and argentaffin. Am J Anat 131:
73–99, 1971.
23. Chen JK, Taipale J, Cooper MK, Beachy PA. Inhibition of
Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev 16: 2743–2748, 2002.
24. Chen JK, Taipale J, Young KE, Maiti T, Beachy PA. Small
molecule modulation of Smoothened activity. Proc Natl Acad Sci
USA 99: 14071–14076, 2002.
25. Chen MH, Li YJ, Kawakami T, Xu SM, Chuang PT. Palmitoylation is required for the production of a soluble multimeric Hedgehog protein complex and long-range signaling in vertebrates. Genes
Dev 18: 641– 659, 2004.
26. Chuang PT, McMahon AP. Vertebrate Hedgehog signalling modulated by induction of a Hedgehog-binding protein. Nature 397:
617– 621, 1999.
27. Colony PC, Conforti JC. Morphogenesis in the fetal rat proximal
colon: effects of cytochalasin D. Anat Rec 235: 241–252, 1993.
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
Hedgehog signaling is initially lost in preneoplastic
lesions of the stomach and colon. It seems, however, that
at later stages of carcinogenesis there is a gain of Hedgehog signaling that maintains viability of carcinoma cells
in vitro and when grown as xenografts in nude mice. This
gain of Hedgehog signaling may be both at the level of
Hedgehog ligand overexpression and activation downstream of Smo, which has important implications for
possible therapeutic strategies.
Many important questions remain, especially for our
understanding of the role of Hedgehog signaling in the
adult gut. What exactly is the role of Hedgehog signaling
in parietal cell physiology in vivo? Hedgehog signaling is
lost early in the process of neoplastic change of the distal
colon. Is this an epiphenomenon or does it play a causal
role in loss of cellular differentiation? Is Hedgehog signaling in the colon exclusively from the epithelium to the
mesenchyme or also partially autocrine? Hedgehog signaling seems to antagonize Wnt signaling in the distal
colon; does this mean that Hedgehog signaling can act as
a tumor suppressor in the colon? Hedgehog signaling
seems essential to maintain the viability of cell lines derived from cancers of the proximal gastrointestinal tract
in vitro and when grown as xenografts. Is this reflective of
effects on these tumor cells when they are in their natural
environment of tumor stroma which is known to play a
critical role in carcinoma behavior? Will it be possible to
treat patients with systemic Hedgehog antagonists without major side effects? In the coming years many of these
questions will be addressed in experiments in animal
models and hopefully also in the first clinical trials in
human cancer patients.
HEDGEHOG SIGNALING
Physiol Rev • VOL
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
moda T, Nimura Y, Yoshida T, Sasaki H. Hedgehog signal activation in gastric pit cell and in diffuse-type gastric cancer. Gastroenterology 131: 14 –29, 2006.
Galiatsatos P, Foulkes WD. Familial adenomatous polyposis.
Am J Gastroenterol 101: 385–398, 2006.
Goodrich LV, Johnson RL, Milenkovic L, McMahon JA, Scott
MP. Conservation of the hedgehog/patched signaling pathway from
flies to mice: induction of a mouse patched gene by Hedgehog.
Genes Dev 10: 301–312, 1996.
Gordon JI, Hermiston ML. Differentiation and self-renewal in the
mouse gastrointestinal epithelium. Curr Opin Cell Biol 6: 795– 803,
1994.
Grapin-Botton A. Antero-posterior patterning of the vertebrate
digestive tract: 40 years after Nicole Le Douarin’s PhD thesis. Int J
Dev Biol 49: 335–347, 2005.
Greaves LC, Preston SL, Tadrous PJ, Taylor RW, Barron MJ,
Oukrif D, Leedham SJ, Deheragoda M, Sasieni P, Novelli MR,
Jankowski JA, Turnbull DM, Wright NA, McDonald SA. Mitochondrial DNA mutations are established in human colonic stem
cells, and mutated clones expand by crypt fission. Proc Natl Acad
Sci USA 103: 714 –719, 2006.
Gregorieff A, Clevers H. Wnt signaling in the intestinal epithelium: from endoderm to cancer. Genes Dev 19: 877– 890, 2005.
Gualdi R, Bossard P, Zheng M, Hamada Y, Coleman JR, Zaret
KS. Hepatic specification of the gut endoderm in vitro: cell signaling and transcriptional control. Genes Dev 10: 1670 –1682, 1996.
Hahn H, Wicking C, Zaphiropoulous PG, Gailani MR, Shanley
S, Chidambaram A, Vorechovsky I, Holmberg E, Unden AB,
Gillies S, Negus K, Smyth I, Pressman C, Leffell DJ, Gerrard
B, Goldstein AM, Dean M, Toftgard R, Chenevix-Trench G,
Wainwright B, Bale AE. Mutations of the human homolog of
Drosophila patched in the nevoid basal cell carcinoma syndrome.
Cell 85: 841– 851, 1996.
Haramis AP, Begthel H, van den Born M, van Es J, Jonkheer
S, Offerhaus GJ, Clevers H. De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science 303:
1684 –1686, 2004.
Haycraft CJ, Banizs B, Aydin-Son Y, Zhang Q, Michaud EJ,
Yoder BK. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function. PLoS
Genet 1: e53, 2005.
He XC, Zhang J, Tong WG, Tawfik O, Ross J, Scoville DH, Tian
Q, Zeng X, He X, Wiedemann LM, Mishina Y, Li L. BMP signaling inhibits intestinal stem cell self-renewal through suppression of
Wnt-beta-catenin signaling. Nat Genet 36: 1117–1121, 2004.
Hebrok M, Kim SK, Melton DA. Notochord repression of
endodermal Sonic hedgehog permits pancreas development. Genes
Dev 12: 1705–1713, 1998.
Hebrok M, Kim SK, St Jacques B, McMahon AP, Melton DA.
Regulation of pancreas development by hedgehog signaling. Development 127: 4905– 4913, 2000.
Hermiston ML, Wong MH, Gordon JI. Forced expression of
E-cadherin in the mouse intestinal epithelium slows cell migration
and provides evidence for nonautonomous regulation of cell fate in
a self-renewing system. Genes Dev 10: 985–996, 1996.
Hezel AF, Kimmelman AC, Stanger BZ, Bardeesy N, Depinho
RA. Genetics and biology of pancreatic ductal adenocarcinoma.
Genes Dev 20: 1218 –1249, 2006.
Hirokawa N, Tanaka Y, Okada Y, Takeda S. Nodal flow and the
generation of left-right asymmetry. Cell 125: 33– 45, 2006.
Hogan BL. Morphogenesis. Cell 96: 225–233, 1999.
Hooper JE, Scott MP. Communicating with Hedgehogs. Nat Rev
Mol Cell Biol 6: 306 –317, 2005.
Horne-Badovinac S, Rebagliati M, Stainier DY. A cellular
framework for gut-looping morphogenesis in zebrafish. Science
302: 662– 665, 2003.
Howe JR, Bair JL, Sayed MG, Anderson ME, Mitros FA, Petersen GM, Velculescu VE, Traverso G, Vogelstein B. Germline
mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis. Nat Genet 28: 184 –187, 2001.
Hruban RH, Adsay NV, Albores-Saavedra J, Anver MR,
Biankin AV, Boivin GP, Furth EE, Furukawa T, Klein A,
Klimstra DS, Kloppel G, Lauwers GY, Longnecker DS, Luttges
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
28. Cooper AF, Yu KP, Brueckner M, Brailey LL, Johnson L,
McGrath JM, Bale AE. Cardiac and CNS defects in a mouse with
targeted disruption of suppressor of fused. Development 132: 4407–
4417, 2005.
29. Cooper MK, Porter JA, Young KE, Beachy PA. Teratogenmediated inhibition of target tissue response to Shh signaling.
Science 280: 1603–1607, 1998.
30. Corbit KC, Aanstad P, Singla V, Norman AR, Stainier DY,
Reiter JF. Vertebrate Smoothened functions at the primary cilium.
Nature 437: 1018 –1021, 2005.
31. De Felice M, Di Lauro R. Thyroid development and its disorders:
genetics and molecular mechanisms. Endocr Rev 25: 722–746, 2004.
32. De Santa Barbara P, Roberts DJ. Tail gut endoderm and gut/
genitourinary/tail development: a new tissue-specific role for
Hoxa13. Development 129: 551–561, 2002.
33. Denef N, Neubuser D, Perez L, Cohen SM. Hedgehog induces
opposite changes in turnover and subcellular localization of
patched and smoothened. Cell 102: 521–531, 2000.
34. Deutsch G, Jung J, Zheng M, Lora J, Zaret KS. A bipotential
precursor population for pancreas and liver within the embryonic
endoderm. Development 128: 871– 881, 2001.
35. diIorio PJ, Moss JB, Sbrogna JL, Karlstrom RO, Moss LG.
Sonic hedgehog is required early in pancreatic islet development.
Dev Biol 244: 75– 84, 2002.
36. Dimmler A, Brabletz T, Hlubek F, Hafner M, Rau T, Kirchner
T, Faller G. Transcription of sonic hedgehog, a potential factor for
gastric morphogenesis and gastric mucosa maintenance, is upregulated in acidic conditions. Lab Invest 83: 1829 –1837, 2003.
37. Duan H, Gao F, Li S, Nagata T. Postnatal development and aging
of esophageal epithelium in mouse: a light and electron microscopic radioautographic study. Cell Mol Biol 39: 309 –316, 1993.
38. Duluc I, Freund JN, Leberquier C, Kedinger M. Fetal
endoderm primarily holds the temporal and positional information
required for mammalian intestinal development. J Cell Biol 126:
211–221, 1994.
39. Dwyer JR, Sever N, Carlson M, Nelson SF, Beachy PA, Parhami F. Oxysterols are novel activators of the hedgehog signaling
pathway in pluripotent mesenchymal cells. J Biol Chem. In press.
40. Echelard Y, Epstein DJ, St-Jacques B, Shen L, Mohler J,
McMahon JA, McMahon AP. Sonic hedgehog, a member of a
family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75: 1417–1430, 1993.
41. Ehlen HW, Buelens LA, Vortkamp A. Hedgehog signaling in
skeletal development. Birth Defects Res C Embryo Today 78: 267–
279, 2006.
42. Ericson J, Rashbass P, Schedl A, Brenner-Morton S,
Kawakami A, van Heyningen V, Jessell TM, Briscoe J. Pax6
controls progenitor cell identity and neuronal fate in response to
graded Shh signaling. Cell 90: 169 –180, 1997.
43. Fagman H, Andersson L, Nilsson M. The developing mouse
thyroid: embryonic vessel contacts and parenchymal growth pattern during specification, budding, migration, and lobulation. Dev
Dyn 235: 444 – 455, 2006.
44. Fagman H, Grande M, Gritli-Linde A, Nilsson M. Genetic deletion of sonic hedgehog causes hemiagenesis and ectopic development of the thyroid in mouse. Am J Pathol 164: 1865–1872, 2004.
45. Falk P, Roth KA, Gordon JI. Lectins are sensitive tools for
defining the differentiation programs of mouse gut epithelial cell
lineages. Am J Physiol Gastrointest Liver Physiol 266: G987–
G1003, 1994.
46. Farrington SM, Belaoussoff M, Baron MH. Winged-helix,
Hedgehog and Bmp genes are differentially expressed in distinct
cell layers of the murine yolk sac. Mech Dev 62: 197–211, 1997.
47. Frank-Kamenetsky M, Zhang XM, Bottega S, Guicherit O,
Wichterle H, Dudek H, Bumcrot D, Wang FY, Jones S, Shulok
J, Rubin LL, Porter JA. Small-molecule modulators of Hedgehog
signaling: identification and characterization of Smoothened agonists and antagonists. J Biol 1: 10, 2002.
48. Fukamachi H, Mizuno T, Takayama S. Epithelial-mesenchymal
interactions in differentiation of stomach epithelium in fetal mice.
Anat Embryol 157: 151–160, 1979.
49. Fukaya M, Isohata N, Ohta H, Aoyagi K, Ochiya T, Saeki N,
Yanagihara K, Nakanishi Y, Taniguchi H, Sakamoto H, Shi-
1371
1372
71.
72.
73.
74.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
J, Maitra A, Offerhaus GJ, Perez-Gallego L, Redston M, Tuveson DA. Pathology of genetically engineered mouse models of
pancreatic exocrine cancer: consensus report and recommendations. Cancer Res 66: 95–106, 2006.
Huangfu D, Anderson KV. Signaling from Smo to Ci/Gli: conservation and divergence of Hedgehog pathways from Drosophila to
vertebrates. Development 133: 3–14, 2006.
Huangfu D, Liu A, Rakeman AS, Murcia NS, Niswander L,
Anderson KV. Hedgehog signalling in the mouse requires intraflagellar transport proteins. Nature 426: 83– 87, 2003.
Hui CC, Joyner AL. A mouse model of greig cephalopolysyndactyly syndrome: the extra-toesJ mutation contains an intragenic
deletion of the Gli3 gene. Nat Genet 3: 241–246, 1993.
Hui CC, Slusarski D, Platt KA, Holmgren R, Joyner AL. Expression of three mouse homologs of the Drosophila segment
polarity gene cubitus interruptus, Gli, Gli-2, Gli-3, in ectoderm- and
mesoderm-derived tissues suggests multiple roles during postimplantation development. Dev Biol 162: 402– 413, 1994.
Incardona JP, Gaffield W, Kapur RP, Roelink H. The teratogenic Veratrum alkaloid cyclopamine inhibits sonic hedgehog signal transduction. Development 125: 3553–3562, 1998.
Incardona JP, Gruenberg J, Roelink H. Sonic hedgehog induces
the segregation of patched and smoothened in endosomes. Curr
Biol 12: 983–995, 2002.
Ingham PW, Placzek M. Orchestrating ontogenesis: variations on
a theme by sonic hedgehog. Nat Rev Genet 7: 841– 850, 2006.
Jacobsen CM, Narita N, Bielinska M, Syder AJ, Gordon JI,
Wilson DB. Genetic mosaic analysis reveals that GATA-4 is required for proper differentiation of mouse gastric epithelium. Dev
Biol 241: 34 – 46, 2002.
Johnson RL, Rothman AL, Xie J, Goodrich LV, Bare JW,
Bonifas JM, Quinn AG, Myers RM, Cox DR, Epstein EH Jr,
Scott MP. Human homolog of patched, a candidate gene for the
basal cell nevus syndrome. Science 272: 1668 –1671, 1996.
Jones RG, Li X, Gray PD, Kuang J, Clayton F, Samowitz WS,
Madison BB, Gumucio DL, Kuwada SK. Conditional deletion of
␤1 integrins in the intestinal epithelium causes a loss of Hedgehog
expression, intestinal hyperplasia, early postnatal lethality. J Cell
Biol 175: 505–514, 2006.
Jung J, Zheng M, Goldfarb M, Zaret KS. Initiation of mammalian liver development from endoderm by fibroblast growth factors.
Science 284: 1998 –2003, 1999.
Kaestner KH. The making of the liver: developmental competence
in foregut endoderm and induction of the hepatogenic program.
Cell Cycle 4: 1146 –1148, 2005.
Kalderon D. The mechanism of hedgehog signal transduction.
Biochem Soc Trans 33: 1509 –1512, 2005.
Kang JS, Mulieri PJ, Hu Y, Taliana L, Krauss RS. BOC, an Ig
superfamily member, associates with CDO to positively regulate
myogenic differentiation. EMBO J 21: 114 –124, 2002.
Kang JS, Mulieri PJ, Miller C, Sassoon DA, Krauss RS. CDO,
a robo-related cell surface protein that mediates myogenic differentiation. J Cell Biol 143: 403– 413, 1998.
Kang S, Graham JM Jr, Olney AH, Biesecker LG. GLI3 frameshift mutations cause autosomal dominant Pallister-Hall syndrome.
Nat Genet 15: 266 –268, 1997.
Karam SM, Leblond CP. Dynamics of epithelial cells in the corpus of the mouse stomach. II. Outward migration of pit cells. Anat
Rec 236: 280 –296, 1993.
Karam SM, Leblond CP. Identifying and counting epithelial cell
types in the “corpus” of the mouse stomach. Anat Rec 232: 231–246,
1992.
Karam SM, Li Q, Gordon JI. Gastric epithelial morphogenesis in
normal and transgenic mice. Am J Physiol Gastrointest Liver
Physiol 272: G1209 –G1220, 1997.
Karlsson L, Lindahl P, Heath JK, Betsholtz C. Abnormal gastrointestinal development in PDGF-A and PDGFR-␣ deficient mice
implicates a novel mesenchymal structure with putative instructive
properties in villus morphogenesis. Development 127: 3457–3466,
2000.
Kaufman M. The Atlas of Mouse Development. San Diego, CA:
Academic, 1992.
Physiol Rev • VOL
92. Kawahira H, Ma NH, Tzanakakis ES, McMahon AP, Chuang
PT, Hebrok M. Combined activities of hedgehog signaling inhibitors regulate pancreas development. Development 130: 4871– 4879,
2003.
93. Kawahira H, Scheel DW, Smith SB, German MS, Hebrok M.
Hedgehog signaling regulates expansion of pancreatic epithelial
cells. Dev Biol 280: 111–121, 2005.
94. Kawakami T, Kawcak T, Li YJ, Zhang W, Hu Y, Chuang PT.
Mouse dispatched mutants fail to distribute hedgehog proteins and
are defective in hedgehog signaling. Development 129: 5753–5765,
2002.
95. Kedinger M, Simon-Assmann P, Bouziges F, Arnold C,
Alexandre E, Haffen K. Smooth muscle actin expression during
rat gut development and induction in fetal skin fibroblastic cells
associated with intestinal embryonic epithelium. Differentiation
43: 87–97, 1990.
96. Kenney AM, Widlund HR, Rowitch DH. Hedgehog and PI-3
kinase signaling converge on Nmyc1 to promote cell cycle progression in cerebellar neuronal precursors. Development 131: 217–228,
2004.
97. Khoury MJ, Cordero JF, Greenberg F, James LM, Erickson
JD. A population study of the VACTERL association: evidence for
its etiologic heterogeneity. Pediatrics 71: 815– 820, 1983.
98. Kim J, Kim P, Hui CC. The VACTERL association: lessons from
the Sonic hedgehog pathway. Clin Genet 59: 306 –315, 2001.
99. Kim JH, Huang Z, Mo R. Gli3 null mice display glandular overgrowth of the developing stomach. Dev Dyn 234: 984 –991, 2005.
100. Kim SK, Hebrok M. Intercellular signals regulating pancreas development and function. Genes Dev 15: 111–127, 2001.
101. Kim SK, Hebrok M, Li E, Oh SP, Schrewe H, Harmon EB, Lee
JS, Melton DA. Activin receptor patterning of foregut organogenesis. Genes Dev 14: 1866 –1871, 2000.
102. Kim SK, Hebrok M, Melton DA. Notochord to endoderm signaling is required for pancreas development. Development 124: 4243–
4252, 1997.
103. Kim SK, Melton DA. Pancreas development is promoted by cyclopamine, a hedgehog signaling inhibitor. Proc Natl Acad Sci USA
95: 13036 –13041, 1998.
104. Kioussi C, O’Connell S, St-Onge L, Treier M, Gleiberman AS,
Gruss P, Rosenfeld MG. Pax6 is essential for establishing ventraldorsal cell boundaries in pituitary gland development. Proc Natl
Acad Sci USA 96: 14378 –14382, 1999.
105. Koch A, Waha A, Hartmann W, Milde U, Goodyer CG, Sorensen N, Berthold F, Digon-Sontgerath B, Kratzschmar J,
Wiestler OD, Pietsch T. No evidence for mutations or altered
expression of the Suppressor of Fused gene (SUFU) in primitive
neuroectodermal tumours. Neuropathol Appl Neurobiol 30: 532–
539, 2004.
106. Lammert E, Cleaver O, Melton D. Induction of pancreatic differentiation by signals from blood vessels. Science 294: 564 –567,
2001.
107. Lammert E, Cleaver O, Melton D. Role of endothelial cells in
early pancreas and liver development. Mech Dev 120: 59 – 64, 2003.
108. Lawson KA, Pedersen RA. Cell fate, morphogenetic movement
and population kinetics of embryonic endoderm at the time of germ
layer formation in the mouse. Development 101: 627– 652, 1987.
109. Lee ER. Dynamic histology of the antral epithelium in the mouse
stomach. III. Ultrastructure and renewal of pit cells. Am J Anat 172:
225–240, 1985.
110. Lee ER, Leblond CP. Dynamic histology of the antral epithelium
in the mouse stomach. IV. Ultrastructure and renewal of gland
cells. Am J Anat 172: 241–259, 1985.
111. Lee JJ, Ekker SC, von Kessler DP, Porter JA, Sun BI, Beachy
PA. Autoproteolysis in hedgehog protein biogenesis. Science 266:
1528 –1537, 1994.
112. Lee JJ, von Kessler DP, Parks S, Beachy PA. Secretion and
localized transcription suggest a role in positional signaling for
products of the segmentation gene hedgehog. Cell 71: 33–50, 1992.
113. Lewis PM, Dunn MP, McMahon JA, Logan M, Martin JF,
St-Jacques B, McMahon AP. Cholesterol modification of sonic
hedgehog is required for long-range signaling activity and effective
modulation of signaling by Ptc1. Cell 105: 599 – 612, 2001.
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
75.
GIJS R. VAN DEN BRINK
HEDGEHOG SIGNALING
Physiol Rev • VOL
135. Mikels AJ, Nusse R. Purified Wnt5a protein activates or inhibits
beta-catenin-TCF signaling depending on receptor context. PLoS
Biol 4: e115, 2006.
136. Mo R, Kim JH, Zhang J, Chiang C, Hui CC, Kim PC. Anorectal
malformations caused by defects in sonic hedgehog signaling. Am J
Pathol 159: 765–774, 2001.
137. Mohler J, Vani K. Molecular organization and embryonic expression of the hedgehog gene involved in cell-cell communication in
segmental patterning of Drosophila. Development 115: 957–971,
1992.
138. Motoyama J, Liu J, Mo R, Ding Q, Post M, Hui CC. Essential
function of Gli2 and Gli3 in the formation of lung, trachea and
oesophagus. Nat Genet 20: 54 –57, 1998.
139. Motoyama J, Takabatake T, Takeshima K, Hui C. Ptch2, a
second mouse Patched gene is co-expressed with Sonic hedgehog.
Nat Genet 18: 104 –106, 1998.
140. Ng D, Stavrou T, Liu L, Taylor MD, Gold B, Dean M, Kelley
MJ, Dubovsky EC, Vezina G, Nicholson HS, Byrne J, Rutka
JT, Hogg D, Reaman GH, Goldstein AM. Retrospective family
study of childhood medulloblastoma. Am J Med Genet A 134:
399 – 403, 2005.
141. Nielsen CM, Williams J, van den Brink GR, Lauwers GY,
Roberts DJ. Hh pathway expression in human gut tissues and in
inflammatory gut diseases. Lab Invest 84: 1631–1642, 2004.
142. Nonaka S, Tanaka Y, Okada Y, Takeda S, Harada A, Kanai Y,
Kido M, Hirokawa N. Randomization of left-right asymmetry due
to loss of nodal cilia generating leftward flow of extraembryonic
fluid in mice lacking KIF3B motor protein. Cell 95: 829 – 837, 1998.
143. Nusslein-Volhard C, Wieschaus E. Mutations affecting segment
number and polarity in Drosophila. Nature 287: 795– 801, 1980.
144. Okada Y, Takeda S, Tanaka Y, Belmonte JC, Hirokawa N.
Mechanism of nodal flow: a conserved symmetry breaking event in
left-right axis determination. Cell 121: 633– 644, 2005.
145. Oniscu A, James RM, Morris RG, Bader S, Malcomson RD,
Harrison DJ. Expression of Sonic hedgehog pathway genes is
altered in colonic neoplasia. J Pathol 203: 909 –917, 2004.
146. Ormestad M, Astorga J, Landgren H, Wang T, Johansson BR,
Miura N, Carlsson P. Foxf1 and Foxf2 control murine gut development by limiting mesenchymal Wnt signaling and promoting
extracellular matrix production. Development 133: 833– 843, 2006.
147. Osterlund T, Kogerman P. Hedgehog signalling: how to get from
Smo to Ci and Gli. Trends Cell Biol 16: 176 –180, 2006.
148. Pan Y, Bai CB, Joyner AL, Wang B. Sonic hedgehog signaling
regulates Gli2 transcriptional activity by suppressing its processing
and degradation. Mol Cell Biol 26: 3365–3377, 2006.
149. Panakova D, Sprong H, Marois E, Thiele C, Eaton S. Lipoprotein particles are required for Hedgehog and Wingless signalling.
Nature 435: 58 – 65, 2005.
150. Park HL, Bai C, Platt KA, Matise MP, Beeghly A, Hui CC,
Nakashima M, Joyner AL. Mouse Gli1 mutants are viable but
have defects in SHH signaling in combination with a Gli2 mutation.
Development 127: 1593–1605, 2000.
151. Park YK, Franklin JL, Settle SH, Levy SE, Chung E, Jeyakumar LH, Shyr Y, Washington MK, Whitehead RH, Aronow BJ,
Coffey RJ. Gene expression profile analysis of mouse colon embryonic development. Genesis 41: 1–12, 2005.
152. Pasca di Magliano M, Sekine S, Ermilov A, Ferris J, Dlugosz
AA, Hebrok M. Hedgehog/Ras interactions regulate early stages of
pancreatic cancer. Genes Dev 20: 3161–3173, 2006.
153. Pepicelli CV, Lewis PM, McMahon AP. Sonic hedgehog regulates
branching morphogenesis in the mammalian lung. Curr Biol 8:
1083–1086, 1998.
154. Perron M, Boy S, Amato MA, Viczian A, Koebernick K, Pieler
T, Harris WA. A novel function for Hedgehog signalling in retinal
pigment epithelium differentiation. Development 130: 1565–1577,
2003.
155. Pietsch T, Waha A, Koch A, Kraus J, Albrecht S, Tonn J,
Sorensen N, Berthold F, Henk B, Schmandt N, Wolf HK, von
Deimling A, Wainwright B, Chenevix-Trench G, Wiestler OD,
Wicking C. Medulloblastomas of the desmoplastic variant carry
mutations of the human homologue of Drosophila patched. Cancer
Res 57: 2085–2088, 1997.
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
114. Li H, Arber S, Jessell TM, Edlund H. Selective agenesis of the
dorsal pancreas in mice lacking homeobox gene Hlxb9. Nat Genet
23: 67–70, 1999.
115. Li Y, Zhang H, Litingtung Y, Chiang C. Cholesterol modification
restricts the spread of Shh gradient in the limb bud. Proc Natl Acad
Sci USA 103: 6548 – 6553, 2006.
116. Litingtung Y, Chiang C. Specification of ventral neuron types is
mediated by an antagonistic interaction between Shh and Gli3. Nat
Neurosci 3: 979 –985, 2000.
117. Litingtung Y, Lei L, Westphal H, Chiang C. Sonic hedgehog is
essential to foregut development. Nat Genet 20: 58 – 61, 1998.
118. Liu A, Wang B, Niswander LA. Mouse intraflagellar transport
proteins regulate both the activator and repressor functions of Gli
transcription factors. Development 132: 3103–3111, 2005.
119. Lum L, Beachy PA. The Hedgehog response network: sensors,
switches, routers. Science 304: 1755–1759, 2004.
120. Ma X, Chen K, Huang S, Zhang X, Adegboyega PA, Evers BM,
Zhang H, Xie J. Frequent activation of the hedgehog pathway in
advanced gastric adenocarcinomas. Carcinogenesis 26: 1698 –1705,
2005.
121. Ma Y, Erkner A, Gong R, Yao S, Taipale J, Basler K, Beachy
PA. Hedgehog-mediated patterning of the mammalian embryo requires transporter-like function of dispatched. Cell 111: 63–75,
2002.
122. Madison BB, Braunstein K, Kuizon E, Portman K, Qiao XT,
Gumucio DL. Epithelial hedgehog signals pattern the intestinal
crypt-villus axis. Development 132: 279 –289, 2005.
123. Madison BB, Dunbar L, Qiao XT, Braunstein K, Braunstein E,
Gumucio DL. Cis elements of the villin gene control expression in
restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine. J Biol Chem 277: 33275–33283,
2002.
124. Mahlapuu M, Enerback S, Carlsson P. Haploinsufficiency of the
forkhead gene Foxf1, a target for sonic hedgehog signaling, causes
lung and foregut malformations. Development 128: 2397–2406,
2001.
125. Mahlapuu M, Ormestad M, Enerback S, Carlsson P. The forkhead transcription factor Foxf1 is required for differentiation of
extra-embryonic and lateral plate mesoderm. Development 128:
155–166, 2001.
126. Manley NR. Thymus organogenesis and molecular mechanisms of
thymic epithelial cell differentiation. Semin Immunol 12: 421– 428,
2000.
127. Mann RK, Beachy PA. Novel lipid modifications of secreted protein signals. Annu Rev Biochem 73: 891–923, 2004.
128. Mao J, Ligon KL, Rakhlin EY, Thayer SP, Bronson RT, Rowitch D, McMahon AP. A novel somatic mouse model to survey
tumorigenic potential applied to the Hedgehog pathway. Cancer
Res 66: 10171–10178, 2006.
129. Marcotullio LD, Ferretti E, Greco A, De Smaele E, Po A, Sico
MA, Alimandi M, Giannini G, Maroder M, Screpanti I, Gulino
A. Numb is a suppressor of Hedgehog signalling and targets Gli1
for Itch-dependent ubiquitination. Nat Cell Biol 8: 1415–1423, 2006.
130. Marigo V, Roberts DJ, Lee SM, Tsukurov O, Levi T, Gastier
JM, Epstein DJ, Gilbert DJ, Copeland NG, Seidman CE, Jenkins NA, Seidman JG, McMahon AP, Tabin C. Cloning, expression, chromosomal location of SHH and IHH: two human homologues of the Drosophila segment polarity gene hedgehog. Genomics 28: 44 –51, 1995.
131. Martinez-Frias ML, Bermejo E, Rodriguez-Pinilla E. Anal atresia, vertebral, genital, urinary tract anomalies: a primary polytopic
developmental field defect identified through an epidemiological
analysis of associations. Am J Med Genet 95: 169 –173, 2000.
132. May SR, Ashique AM, Karlen M, Wang B, Shen Y, Zarbalis K,
Reiter J, Ericson J, Peterson AS. Loss of the retrograde motor
for IFT disrupts localization of Smo to cilia and prevents the
expression of both activator and repressor functions of Gli. Dev
Biol 287: 378 –389, 2005.
133. McGrath J, Somlo S, Makova S, Tian X, Brueckner M. Two
populations of node monocilia initiate left-right asymmetry in the
mouse. Cell 114: 61–73, 2003.
134. Menard D, Arsenault P. Maturation of human fetal esophagus
maintained in organ culture. Anat Rec 217: 348 –354, 1987.
1373
1374
GIJS R. VAN DEN BRINK
Physiol Rev • VOL
176.
177.
178.
179.
180.
181.
182.
183.
184.
185.
186.
187.
188.
189.
190.
191.
192.
193.
194.
progressing to gastric cancer. Am J Gastroenterol 100: 581–587,
2005.
Shook D, Keller R. Mechanisms, mechanics and function of epithelial-mesenchymal transitions in early development. Mech Dev
120: 1351–1383, 2003.
Soriano L. Differentiation of epithelium of the digestive tube
in vitro. J Embryol Exp Morphol 14: 119 –128, 1965.
Spencer-Dene B, Sala FG, Bellusci S, Gschmeissner S, Stamp
G, Dickson C. Stomach development is dependent on fibroblast
growth factor 10/fibroblast growth factor receptor 2b-mediated
signaling. Gastroenterology 130: 1233–1244, 2006.
Stepan V, Ramamoorthy S, Nitsche H, Zavros Y, Merchant JL,
Todisco A. Regulation and function of the sonic hedgehog signal
transduction pathway in isolated gastric parietal cells. J Biol Chem
280: 15700 –15708, 2005.
St-Onge L, Sosa-Pineda B, Chowdhury K, Mansouri A, Gruss
P. Pax6 is required for differentiation of glucagon-producing alphacells in mouse pancreas. Nature 387: 406 – 409, 1997.
Suzuki H, Minegishi Y, Nomoto Y, Ota T, Masaoka T, van den
Brink GR, Hibi T. Down-regulation of a morphogen (sonic hedgehog) gradient in the gastric epithelium of Helicobacter pyloriinfected Mongolian gerbils. J Pathol 206: 186 –197, 2005.
Svard J, Heby-Henricson K, Persson-Lek M, Rozell B, Lauth
M, Bergstrom A, Ericson J, Toftgard R, Teglund S. Genetic
elimination of Suppressor of fused reveals an essential repressor
function in the mammalian Hedgehog signaling pathway. Dev Cell
10: 187–197, 2006.
Tabata T, Eaton S, Kornberg TB. The Drosophila hedgehog
gene is expressed specifically in posterior compartment cells and is
a target of engrailed regulation. Genes Dev 6: 2635–2645, 1992.
Taipale J, Cooper MK, Maiti T, Beachy PA. Patched acts catalytically to suppress the activity of Smoothened. Nature 418: 892–
897, 2002.
Tanaka Y, Okada Y, Hirokawa N. FGF-induced vesicular release
of Sonic hedgehog and retinoic acid in leftward nodal flow is
critical for left-right determination. Nature 435: 172–177, 2005.
Tao H, Shimizu M, Kusumoto R, Ono K, Noji S, Ohuchi H. A
dual role of FGF10 in proliferation and coordinated migration of
epithelial leading edge cells during mouse eyelid development.
Development 132: 3217–3230, 2005.
Taylor MD, Liu L, Raffel C, Hui CC, Mainprize TG, Zhang X,
Agatep R, Chiappa S, Gao L, Lowrance A, Hao A, Goldstein
AM, Stavrou T, Scherer SW, Dura WT, Wainwright B, Squire
JA, Rutka JT, Hogg D. Mutations in SUFU predispose to medulloblastoma. Nat Genet 31: 306 –310, 2002.
Tempe D, Casas M, Karaz S, Blanchet-Tournier MF, Concordet
JP. Multisite protein kinase A and glycogen synthase kinase 3beta
phosphorylation leads to Gli3 ubiquitination by SCFbetaTrCP. Mol
Cell Biol 26: 4316 – 4326, 2006.
Tenzen T, Allen BL, Cole F, Kang JS, Krauss RS, McMahon
AP. The cell surface membrane proteins Cdo and Boc are components and targets of the Hedgehog signaling pathway and feedback
network in mice. Dev Cell 10: 647– 656, 2006.
Thayer SP, di Magliano MP, Heiser PW, Nielsen CM, Roberts
DJ, Lauwers GY, Qi YP, Gysin S, Fernandez-del Castillo C,
Yajnik V, Antoniu B, McMahon M, Warshaw AL, Hebrok M.
Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature 425: 851– 856, 2003.
Thomas MK, Rastalsky N, Lee JH, Habener JF. Hedgehog
signaling regulation of insulin production by pancreatic beta-cells.
Diabetes 49: 2039 –2047, 2000.
Thomas PQ, Brown A, Beddington RS. Hex: a homeobox gene
revealing peri-implantation asymmetry in the mouse embryo and
an early transient marker of endothelial cell precursors. Development 125: 85–94, 1998.
Tsukui T, Capdevila J, Tamura K, Ruiz-Lozano P, RodriguezEsteban C, Yonei-Tamura S, Magallon J, Chandraratna RA,
Chien K, Blumberg B, Evans RM, Belmonte JC. Multiple leftright asymmetry defects in Shh(⫺/⫺) mutant mice unveil a convergence of the shh and retinoic acid pathways in the control of
Lefty-1. Proc Natl Acad Sci USA 96: 11376 –11381, 1999.
Unden AB, Holmberg E, Lundh-Rozell B, Stahle-Backdahl M,
Zaphiropoulos PG, Toftgard R, Vorechovsky I. Mutations in the
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
156. Porter JA, Ekker SC, Park WJ, von Kessler DP, Young KE,
Chen CH, Ma Y, Woods AS, Cotter RJ, Koonin EV, Beachy PA.
Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell 86:
21–34, 1996.
157. Porter JA, von Kessler DP, Ekker SC, Young KE, Lee JJ,
Moses K, Beachy PA. The product of hedgehog autoproteolytic
cleavage active in local and long-range signalling. Nature 374:
363–366, 1995.
158. Powell DW, Adegboyega PA, Di Mari JF, Mifflin RC. Epithelial
cells and their neighbors. I. Role of intestinal myofibroblasts in
development, repair, cancer. Am J Physiol Gastrointest Liver
Physiol 289: G2–G7, 2005.
159. Preat T. Characterization of Suppressor of fused, a complete
suppressor of the fused segment polarity gene of Drosophila melanogaster. Genetics 132: 725–736, 1992.
160. Qualtrough D, Buda A, Gaffield W, Williams AC, Paraskeva C.
Hedgehog signalling in colorectal tumour cells: induction of apoptosis with cyclopamine treatment. Int J Cancer 110: 831– 837,
2004.
161. Quan L, Smith DW. The VATER association. Vertebral defects,
Anal atresia, T-E fistula with esophageal atresia, Radial and Renal
dysplasia: a spectrum of associated defects. J Pediatr 82: 104 –107,
1973.
162. Radhakrishna U, Bornholdt D, Scott HS, Patel UC, Rossier C,
Engel H, Bottani A, Chandal D, Blouin JL, Solanki JV,
Grzeschik KH, Antonarakis SE. The phenotypic spectrum of
GLI3 morphopathies includes autosomal dominant preaxial polydactyly type-IV and postaxial polydactyly type-A/B; no phenotype
prediction from the position of GLI3 mutations. Am J Hum Genet
65: 645– 655, 1999.
163. Raffel C, Jenkins RB, Frederick L, Hebrink D, Alderete B,
Fults DW, James CD. Sporadic medulloblastomas contain PTCH
mutations. Cancer Res 57: 842– 845, 1997.
164. Ramalho-Santos M, Melton DA, McMahon AP. Hedgehog signals regulate multiple aspects of gastrointestinal development. Development 127: 2763–2772, 2000.
165. Raymond C, Anne V, Millane G. Development of esophageal
epithelium in the fetal and neonatal mouse. Anat Rec 230: 225–234,
1991.
166. Rittler M, Paz JE, Castilla EE. VACTERL association, epidemiologic definition and delineation. Am J Med Genet 63: 529 –536,
1996.
167. Roberts DJ, Johnson RL, Burke AC, Nelson CE, Morgan BA,
Tabin C. Sonic hedgehog is an endodermal signal inducing Bmp-4
and Hox genes during induction and regionalization of the chick
hindgut. Development 121: 3163–3174, 1995.
168. Roberts DJ, Smith DM, Goff DJ, Tabin CJ. Epithelial-mesenchymal signaling during the regionalization of the chick gut. Development 125: 2791–2801, 1998.
169. Rossi JM, Dunn NR, Hogan BL, Zaret KS. Distinct mesodermal
signals, including BMPs from the septum transversum mesenchyme, are required in combination for hepatogenesis from the
endoderm. Genes Dev 15: 1998 –2009, 2001.
170. Roy S, Qiao T, Wolff C, Ingham PW. Hedgehog signaling pathway is essential for pancreas specification in the zebrafish embryo.
Curr Biol 11: 1358 –1363, 2001.
171. Rubin DC, Swietlicki E, Roth KA, Gordon JI. Use of fetal
intestinal isografts from normal and transgenic mice to study the
programming of positional information along the duodenal-to-colonic axis. J Biol Chem 267: 15122–15133, 1992.
172. Sander M, Neubuser A, Kalamaras J, Ee HC, Martin GR,
German MS. Genetic analysis reveals that PAX6 is required for
normal transcription of pancreatic hormone genes and islet development. Genes Dev 11: 1662–1673, 1997.
173. Scholey JM, Anderson KV. Intraflagellar transport and ciliumbased signaling. Cell 125: 439 – 442, 2006.
174. Serls AE, Doherty S, Parvatiyar P, Wells JM, Deutsch GH.
Different thresholds of fibroblast growth factors pattern the ventral
foregut into liver and lung. Development 132: 35– 47, 2005.
175. Shiotani A, Iishi H, Uedo N, Ishiguro S, Tatsuta M, Nakae Y,
Kumamoto M, Merchant JL. Evidence that loss of sonic hedgehog is an indicator of Helicobater pylori-induced atrophic gastritis
HEDGEHOG SIGNALING
195.
196.
197.
199.
200.
201.
202.
203.
204.
Physiol Rev • VOL
205. Wells JM, Melton DA. Early mouse endoderm is patterned by
soluble factors from adjacent germ layers. Development 127: 1563–
1572, 2000.
206. Wells JM, Melton DA. Vertebrate endoderm development. Annu
Rev Cell Dev Biol 15: 393– 410, 1999.
207. Williams JA, Guicherit OM, Zaharian BI, Xu Y, Chai L, Wichterle H, Kon C, Gatchalian C, Porter JA, Rubin LL, Wang FY.
Identification of a small molecule inhibitor of the hedgehog signaling pathway: effects on basal cell carcinoma-like lesions. Proc Natl
Acad Sci USA 100: 4616 – 4621, 2003.
208. Wolpert L. One hundred years of positional information. Trends
Genet 12: 359 –364, 1996.
209. Xie J, Murone M, Luoh SM, Ryan A, Gu Q, Zhang C, Bonifas
JM, Lam CW, Hynes M, Goddard A, Rosenthal A, Epstein EH
Jr, de Sauvage FJ. Activating Smoothened mutations in sporadic
basal-cell carcinoma. Nature 391: 90 –92, 1998.
210. Yao S, Lum L, Beachy P. The ihog cell-surface proteins bind
Hedgehog and mediate pathway activation. Cell 125: 343–357, 2006.
211. Yuasa Y. Control of gut differentiation and intestinal-type gastric
carcinogenesis. Nat Rev Cancer 3: 592– 600, 2003.
212. Zeng X, Goetz JA, Suber LM, Scott WJ Jr, Schreiner CM,
Robbins DJ. A freely diffusible form of Sonic hedgehog mediates
long-range signalling. Nature 411: 716 –720, 2001.
213. Zhang J, Rosenthal A, de Sauvage FJ, Shivdasani RA. Downregulation of Hedgehog signaling is required for organogenesis of
the small intestine in Xenopus. Dev Biol 229: 188 –202, 2001.
214. Zhang W, Kang JS, Cole F, Yi MJ, Krauss RS. Cdo functions at
multiple points in the Sonic Hedgehog pathway, Cdo-deficient mice
accurately model human holoprosencephaly. Dev Cell 10: 657– 665,
2006.
215. Zhang XM, Ramalho-Santos M, McMahon AP. Smoothened mutants reveal redundant roles for Shh and Ihh signaling including
regulation of L/R symmetry by the mouse node. Cell 106: 781–792,
2001.
216. Zhu AJ, Zheng L, Suyama K, Scott MP. Altered localization of
Drosophila Smoothened protein activates Hedgehog signal transduction. Genes Dev 17: 1240 –1252, 2003.
217. Zhu Y, James RM, Peter A, Lomas C, Cheung F, Harrison DJ,
Bader SA. Functional Smoothened is required for expression of
GLI3 in colorectal carcinoma cells. Cancer Lett 207: 205–214, 2004.
218. Zweibaum A, Pinto M, Chevalier G, Dussaulx E, Triadou N,
Lacroix B, Haffen K, Brun JL, Rousset M. Enterocytic differentiation of a subpopulation of the human colon tumor cell line HT-29
selected for growth in sugar-free medium and its inhibition by
glucose. J Cell Physiol 122: 21–29, 1985.
87 • OCTOBER 2007 •
www.prv.org
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on June 17, 2017
198.
human homologue of Drosophila patched (PTCH) in basal cell
carcinomas and the Gorlin syndrome: different in vivo mechanisms
of PTCH inactivation. Cancer Res 56: 4562– 4565, 1996.
Van Bokhoven H, Celli J, van Reeuwijk J, Rinne T, Glaudemans B, van Beusekom E, Rieu P, Newbury-Ecob RA, Chiang
C, Brunner HG. MYCN haploinsufficiency is associated with reduced brain size and intestinal atresias in Feingold syndrome. Nat
Genet 37: 465– 467, 2005.
Van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I,
Hurlstone A, van der Horn K, Batlle E, Coudreuse D, Haramis
AP, Tjon-Pon-Fong M, Moerer P, van den Born M, Soete G,
Pals S, Eilers M, Medema R, Clevers H. The beta-catenin/TCF-4
complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111: 241–250, 2002.
Van den Brink GR, Bleuming SA, Hardwick JC, Schepman BL,
Offerhaus GJ, Keller JJ, Nielsen C, Gaffield W, van Deventer
SJ, Roberts DJ, Peppelenbosch MP. Indian Hedgehog is an
antagonist of Wnt signaling in colonic epithelial cell differentiation.
Nat Genet 36: 277–282, 2004.
Van den Brink GR, Hardwick JC, Nielsen C, Xu C, ten Kate
FJ, Glickman J, van Deventer SJ, Roberts DJ, Peppelenbosch
MP. Sonic hedgehog expression correlates with fundic gland differentiation in the adult gastrointestinal tract. Gut 51: 628 – 633,
2002.
Van den Brink GR, Hardwick JC, Tytgat GN, Brink MA, Ten
Kate FJ, Van Deventer SJ, Peppelenbosch MP. Sonic hedgehog
regulates gastric gland morphogenesis in man and mouse. Gastroenterology 121: 317–328, 2001.
Varjosalo M, Li SP, Taipale J. Divergence of hedgehog signal
transduction mechanism between Drosophila and mammals. Dev
Cell 10: 177–186, 2006.
Varjosalo M, Taipale J. Hedgehog signaling. J Cell Sci 120: 3– 6,
2007.
Varnat F, Heggeler BB, Grisel P, Boucard N, Corthesy-Theulaz I, Wahli W, Desvergne B. PPARbeta/delta regulates paneth
cell differentiation via controlling the hedgehog signaling pathway.
Gastroenterology 131: 538 –553, 2006.
Wang B, Fallon JF, Beachy PA. Hedgehog-regulated processing
of Gli3 produces an anterior/posterior repressor gradient in the
developing vertebrate limb. Cell 100: 423– 434, 2000.
Wang LC, Nassir F, Liu ZY, Ling L, Kuo F, Crowell T, Olson D,
Davidson NO, Burkly LC. Disruption of hedgehog signaling reveals a novel role in intestinal morphogenesis and intestinal-specific lipid metabolism in mice. Gastroenterology 122: 469 – 482,
2002.
1375