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The Role of the Visceral Mesoderm in the Development of the Gastrointestinal

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GASTROENTEROLOGY
BASIC AND CLINICAL
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GASTROENTEROLOGY
The Role of the Visceral Mesoderm in the Development of the
Gastrointestinal Tract
VALÉRIE A. MCLIN,* SUSAN J. HENNING,‡ and MILAN JAMRICH§,储
*Department of Pediatrics, Section of Gastroenterology, Hepatology and Nutrition, §Department of Cellular and Molecular Biology, and 储Department of Molecular and
Human Genetics, Baylor College of Medicine, Houston, Texas; and ‡Departments of Medicine and Cell and Molecular Physiology, University of North Carolina Chapel
Hill, Chapel Hill, North Carolina
The gastrointestinal (GI) tract forms from the en-
doderm (which gives rise to the epithelium) and
the mesoderm (which develops into the smooth mus-
cle layer, the mesenchyme, and numerous other cell
types). Much of what is known of GI development has
been learned from studies of the endoderm and its
derivatives, because of the importance of epithelial
biology in understanding and treating human dis-
eases. Although the necessity of epithelial-mesenchy-
mal cross talk for GI development is uncontested, the
role of the mesoderm remains comparatively less well
understood. The transformation of the visceral me-
soderm during development is remarkable; it differ-
entiates from a very thin layer of cells into a complex
tissue comprising smooth muscle cells, myofibro-
blasts, neurons, immune cells, endothelial cells, lym-
phatics, and extracellular matrix molecules, all con-
tributing to the form and function of the digestive
system. Understanding the molecular processes that
govern the development of these cell types and eluci-
dating their respective contribution to GI patterning
could offer insight into the mechanisms that regulate
cell fate decisions in the intestine, which has the
unique property of rapid cell renewal for the mainte-
nance of epithelial integrity. In reviewing evidence
from both mammalian and nonmammalian models,
we reveal the important role of the visceral mesoderm
in the ontogeny of the GI tract.
I nherited or acquired abnormalities of the gastrointes-
tinal (GI) tract cause many chronic diseases in children
and adults that often require costly, long-term medical
support and organ transplantation. Cell-based therapies
are an attractive alternative to solid organ transplanta-
tion, but their progress requires a better understanding
of the processes that govern cell fate decisions, cell pro-
liferation, and tissue differentiation during GI develop-
ment. Several aspects of GI development are well con-
GASTROENTEROLOGY 2009;136:2074 –2091
John P. Lynch and David C. Metz, Section Editors
served from invertebrates to vertebrates, including the
role of the visceral mesoderm (VM). Understanding the
role of the VM in development may help orient cell-based
therapy research.
Overview of GI Development
Development of the vertebrate GI tract, and of the
VM in particular, is conserved across species. In very
broad terms, it follows this sequence of events: gastrula-
tion, formation of the primitive gut tube from the
endoderm, and apposition of the inner leaflet of the
lateral plate mesoderm against the endoderm.1 This inner
leaflet eventually circles the gut to become the VM (Fig-
ure 1). The outer leaflet becomes the somatic muscula-
ture.1 During GI morphogenesis, endoderm and meso-
derm undergo extensive regionalization, elongation, and
coiling. Concurrently, neural crest cells populate the in-
testine to form the early enteric nervous system (ENS).
This period of rapid growth is characterized by regional
signals that are largely derived from the mesoderm and
pattern the future intestinal domains along 4 axes: ante-
rior-posterior, dorsoventral, left-right, and radial. The
mesoderm sends (or receives) instructive or permissive
signals to the adjacent endoderm; the instructive signals
are sufficient to direct cell fate in the target tissue,
whereas permissive signals enable competent tissue to
activate a previously “primed” genetic program.1 The
most likely scenario in GI patterning is that neither germ
layer holds all the information necessary for GI develop-
Abbreviations used in this paper: BMP, bone morphogenetic protein;
E, embryonic day; ECM, extracellular matrix; ENS, enteric nervous
system; FGF, fibroblast growth factor; GC, glucocorticoid; GI, gastroin-
testinal; Hh, Hedgehog; IGF, insulin-like growth factor; ISEMF, intesti-
nal subepithelial myofibroblast; PDGF, platelet-derived growth factor;
SOCS, suppressor-of-cytokine signaling; VM, visceral mesoderm.
© 2009 by the AGA Institute
0016-5085/09/$36.00
doi:10.1053/j.gastro.2009.03.001
June 2009 VISCERAL MESODERM IN GI DEVELOPMENT 2075

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DORSAL

SM N e
S S
VM

BMP4
Endoderm

Hh
FoxF1

Iro

VENTRAL
Figure 1. Early development of the lateral plate mesoderm. Diagram of a cross section through a Xenopus neurula. Dorsal is to the top. The visceral
mesoderm (VM, orange) and somatic mesoderm (SM, brown) are 2 leaflets that are in continuity with the somitic (S) mesoderm at the lateral plate.
They are situated between the endoderm (yellow) and the ectoderm (e). Endodermal Hh signals produced by the future gut endoderm induce BMP-4
expression in the VM. The ensuing molecular decisions in the VM are shown; BMP-4 activates expression of the transcription factor FoxF1. One of
the early actions of FoxF1 is the inhibition of Iroquois (Iro) in the VM, thereby inhibiting the somatic mesoderm program in cells destined to become
VM. FoxF1 also induces BMP4 expression. N, notochord; S, somitic mesoderm. Modified and reprinted with permission from El-Hodiri et al.167

ment, but rather that the relative contribution of each
tissue varies during ontogeny, with their relative plastic-
ity diminishing as development proceeds.2 Although the
mesoderm has an important role in early GI tract mor-
phogenesis, its formation requires signals from the
endoderm mediated in part by the Hedgehog (Hh) family
of signaling molecules3; this pathway is conserved among
species.4 – 8
Differential regional responses to the Hh signals along
the craniocaudal axis lead to the formation of the fo-
regut, midgut, and hindgut domains.3,5,9 –11 The foregut
gives rise to the esophagus, stomach, duodenum, liver
and bile ducts, pancreas, lung, and thyroid. The midgut is
the main precursor of the intestine that is distal to the
entry of the common bile duct and extends to the prox-
imal transverse colon; this large domain includes the
jejunum, ileum, cecum, and ascending colon. Finally, the
hindgut evolves into the distal transverse, descending,
and rectosigmoid segments of the colon.1,12 The contri-
bution of mesoderm components to endoderm pattern-
ing is well understood for development of the liver and
pancreas. The development of these 2 organs, which also
depends on instructive and permissive signals from the
adjacent mesoderm, is the subject of recent reviews.13–15
Following morphogenesis, the characteristic GI cell
types emerge from the undifferentiated endoderm and
the VM differentiates into its specialized components.
This phase, which begins around embryonic day (E) 15 in
mice and continues postnatally, occurs during metamor-
phosis in the frog and begins toward the end of the first
trimester in humans.16 It coincides with the formation of
the GI tract’s fifth axis, the villus-crypt axis. This axis is
the functional unit of the intestine and is characterized
by finger-like evaginations into the lumen, comprising a
mesenchymal stalk and an epithelial cover composed of
the precursors to the different epithelial lineages. The
mesenchyme contains (intestinal) subepithelial myofi-
broblasts (ISEMFs), both in the stalk and adjacent to the
intervillus space, which are important regulators of mes-
enchymal signaling. In addition, the mesenchyme, also
derived from the VM, forms the lamina propria, together
with blood vessels, lymphatics, resident immune cells,
and the muscularis propria (Figure 2). In the fully mature
GI tract, mesodermal derivatives outnumber endodermal
derivatives (Figure 2). A brief overview of the develop-
ment of these different, mesodermally derived compo-
nents is warranted before examining the molecular net-
work governing VM development.
 2076 MCLIN ET AL GASTROENTEROLOGY Vol. 136, No. 7
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Figure 2. Schematic section through the adult GI tract. Each quadrant represents a different segment of the GI tract, from the stomach to the colon;
mesenchymal structures are shown in detail. The following structures are conserved along the craniocaudal axis: the lamina propria mesenchyme, the
muscularis mucosa, mesenchymal blood vessels, the ISEMFs, the Meissner’s plexus, the longitudinal inner and circular outer layers of the muscularis
propria, the Auerbach plexus, and the serosa. These structures are represented in all quadrants. The stomach (left upper quadrant) is characterized by an
additional oblique inner layer of the muscularis propria. Its epithelium is characterized by deep, bifid glands (g) that fulfill the secretory and endocrine functions
of the stomach. In the duodenum (left lower quadrant), the submucosa is recognizable by its abundance of Brunner’s glands, which are epithelial in origin.
Consistent with the rest of the small intestine, the duodenal epithelium is characterized by the presence of crypts and villi. The other 2 components of the
small intestine are the jejunum and ileum (right lower quadrant). The ileum is characterized by the presence of Peyer’s patches, which are large lymphoid
aggregates in the submucosa. In the jejunum and the colon, the lymphoid aggregates are located in the mucosal tunic rather than in the submucosa.
ISEMFs are adjacent to the epithelium lining crypts and villi. Finally, in the colon (right upper quadrant), the epithelium is characterized by straight glands (g)
but does not include villi or crypts. The characteristic feature of the colon mesenchyme is the thinner, circular outer layer of the muscularis propria. Modified
and reprinted with permission from T. Caceci, DVM (Virginia-Maryland Regional College of Veterinary Medicine).

ISEMFs reside within the mesenchymal layer of the intestine at

As their name suggests, ISEMFs are cells that are
located just below the epithelium of both villi and cry-
pts throughout the length of the intestine (Figure 2).
ISEMFs are characterized by ␣–smooth muscle actin ex-
pression and are organized in a syncytium between the
epithelium and the muscularis mucosa, where they con-
tribute to extracellular matrix (ECM) and basal mem-
brane composition.17 Although it is uncertain whether
ISEMFs are derived from the neural crest, mesodermally
derived fibroblasts, or smooth muscle cells, they clearly
the base of the crypts, next to the muscularis, starting
around week 21 of human gestation.18 In mouse em-
bryos, ISEMFs have been described as early as E18.5.19
Similar to epithelial cells, ISEMFs differentiate as they
migrate up the crypt to villus axis.20 There is increasing
evidence that ISEMFs are major players in GI development.
The Development of the GI Smooth Muscle
The visceral smooth muscle, derived from the VM,
is a very large component of the intestine. Its function in
June 2009
the adult intestine is to confer shape and motility to this
hollow organ and allow for the advancement of its lumi-
nal contents. The muscle layers of the intestine include
the circular inner layer and the longitudinal external
layer. In the stomach, there is an additional oblique layer
(Figure 2). Together, the smooth muscle layers are called
the muscularis propria. Mammals have an additional
muscularis mucosa that separates the epithelium from
the underlying smooth muscle layers and promotes epi-
thelial movement21 (Figure 2). In humans, disorders of
enteric myocytes have been described in children with
motility syndromes22; a few reports have associated hyp-
oplasia of the muscularis propria with necrotizing en-
terocolitis or intestinal perforation in infants.23,24
Little is known about the role of the developing
smooth muscle layer during ontogeny. The early muscle
progenitors differentiate from the loose mesenchyme
that surrounds the primitive gut. The earliest precursor
to both types of enteric smooth muscle cells is a common
progenitor called the smooth muscle myoblast, which is
characterized by the expression of ␣–smooth muscle ac-
tin, as opposed to the more differentiated smooth mus-
cle myocytes, which express ␣– and ␥–smooth muscle
actins.25,26 Formation of the smooth muscle layer pro-
gresses in a craniocaudal fashion, starting in the esoph-
agus at E1125 in mice and 50 hours postfertilization in
zebrafish.27 This first layer of smooth muscle myoblasts
differentiates from undifferentiated mesenchyme to give
rise to the circular inner layer. The second, longitudinal,
external layer appears in the mesenchyme approximately
24 –28 hours later.25,26 Finally, in mammals, the muscu-
laris mucosa forms subjacent to the epithelium. In each
layer, the smooth muscle myoblasts rapidly differentiate
into immature smooth muscle myocytes, but their final
differentiation into mature myocytes does not occur un-
til after birth.25,26
Role of the ENS in GI Development
It is beyond the scope of this review to discuss in
detail the development of the ENS, which is organized
into the submucosal and myenteric plexuses. Each is
derived from neural crest cells, which migrate from the
neural plate between E9 and E13.28 –31 However, for the
purposes of this review, it is relevant to remember that as
the gut develops, it is populated by migrating neural crest
cells that home to the mesenchymal layers. Genetic ma-
nipulation of the developing gut often leads to a Hir-
schsprung’s-like phenotype, suggesting that the integrity
of multiple pathways is required for normal innervation
and muscle development of the gut.
Development of the Lateral Plate
Mesoderm
The molecular network that governs lateral plate
mesoderm development depends on signals from the
endoderm. Cross talk between mesoderm and endoderm
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Figure 3. Early specification of mesodermal tissues. Diagram repre-
senting the action of BMP-4 as a ventralizing morphogen. In most
species, BMP signaling in combination with other pathways patterns
the early mesoderm into different cell types.168 –170 In Xenopus, ze-
brafish, and other species, BMP-4 acts as a mesodermal morphogen
that specifies different mesodermal derivatives along the dorsoventral
axis: blood, muscle, kidney, and notochord.170 –174 According to its
concentration at a given time and place, BMP-4 confers a distinct
dorsoventral identity to different parts of the mesoderm.170,175 In Xeno-
pus and zebrafish, the Vent homeobox-containing genes act down-
stream of BMP-4 to convert the signaling gradient into distinct cellular
responses.176 –178 The essential VM transcription factor FoxF1 is also
downstream from BMP-4.36 Although the exact “dose” of BMP-4 that
regulates the specification and differentiation of lateral plate mesoderm
has not been established, integration of combinatorial regulatory signals
is an important paradigm that is repeated throughout development. The
Vent pathway of ventral mesoderm specification has been confirmed in
zebrafish and amphioxus,170,179 but mammalian studies are still
pending.
is essential for regional changes during morphogenesis
and for cellular differentiation and patterning along the
crypt-villus axis.9,32 This intricate process is the product
of tight spatial and temporal control of signaling mole-
cules and transcription factors in both germ layers.
For example, during mouse gastrulation, the early me-
soderm is patterned by the adjacent endoderm via se-
creted signals. Fibroblast growth factor (FGF)-4 is one of
the earliest characterized signals33; its role persists after
the formation of the gut tube, when it guides the adja-
cent endoderm along the anterior-posterior axis, inhibit-
ing anterior fates and promoting posterior fates in a
concentration-dependent manner.34 The instructive role
of FGF-4 is time sensitive, and as development proceeds
FGF-4 loses its ability to repress anterior development.34
Shortly after gastrulation, possibly in response to en-
dodermal FGF signals, bone morphogenetic proteins
(BMPs) pattern the mesoderm of Xenopus embryos into
its different components, including the VM. This exam-
ple of a dorsoventral morphogen in the mesoderm is
illustrated in Figure 3.
Interactions between Shh, BMP-4, and Foxf1 partici-
pate in VM morphogenesis and appear to be conserved
 2078 MCLIN ET AL
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Table 1. Gene Nomenclature
Drosophila Xenopus Mouse
Biniou FoxF1 Foxf1/Foxf2
Dpp Smad Smad
Bagpipe Xbap; zampogna Nkx 3.2/Bapx1
Tinman Nkx2.3,2.5 Nkx2.3–2.7
vimar Not identified Not identified
NOTE. The names of homologous genes in the different species discussed in the text are summarized. The bagpipe family has 2 related but
different genes in Xenopus. In vertebrates, tinman has multiple homologues belonging to the Nkx2 cluster. Except for the transforming growth
factor ␤ pathway (Dpp/Smad), signaling pathways are not included because molecules referenced in the text have conserved names across
species. In the text, when referring to a gene in a general sense, the murine nomenclature is used. Bapx, bagpipe homeobox-containing gene
1 homologue; Fox, forkhead box.
across species (Figure 1). Studies in chicks have shown
that the endoderm Shh signal activates BMP-4 expression
in the lateral plate mesoderm5 where Smads (intracellular
mediators of BMPs) regulate mesodermal proliferation
and later differentiation into smooth muscle.3,5,35 In Xe-
nopus embryos, BMP-4 activates the expression of FoxF1,
a transcription factor required for lateral plate and VM
formation.36 Furthermore, it is sufficient to induce FoxF1
expression in nonmesodermal tissue, and FoxF1 messen-
ger RNA rescues the phenotype of embryos injected with
a dominant negative BMP-4 receptor.36 In Drosophila,
Dpp (homologous to Smads in vertebrates; see Table 1)
signals are both upstream and downstream of biniou, the
Foxf1 homologue essential for VM development.37 In
mouse, Foxf1 messenger RNA is absent in the foregut
structures of Shh⫺/⫺ embryos.38 Therefore, induction of
the VM via Foxf1 depends on endodermal Hh signals and
mesodermal BMPs (see Table 1 for species-specific no-
menclature).
The Drosophila gene biniou and its vertebrate homo-
logues Foxf1 and Foxf2 have emerged as master regulators
of VM morphogenesis and differentiation across spe-
cies.36,37,39 In biniou-null Drosophila embryos, VM deve-
lopment arrests before GI morphogenesis is complete37
(Figure 4A and B). Likewise, in Xenopus laevis, FoxF1
knockdown leads to severe defects in gut coiling and
elongation, partly because of decreased cell proliferation
in the mesoderm36 (Figure 4C and D). In mouse embryos,
Foxf1 expression localizes to the interfaces between mes-
enchyme and epithelium40; knockout studies have shown
a role in early lateral plate mesoderm formation and later
differentiation. The earliest known function of Foxf1 is in
specifying VM (splanchnic) from somatic mesoderm. In
Foxf1-null embryos, there is incomplete separation be-
tween the visceral and somatic leaflets of the lateral plate;
the homeobox gene Iroquois is ectopically expressed in the
visceral leaflet, suggesting that Foxf1 specifies VM by
inhibiting Iroquois41 (Figure 1). Although Foxf1-null em-
bryos die at midgestation because of defects in the vas-
cular development of the extraembryonic membranes,42
abnormal esophageal development has occurred by this
stage.41 Because both alleles of Foxf1 are required for
normal VM development, Foxf1⫹/⫺ embryos also have
GASTROENTEROLOGY Vol. 136, No. 7
Chick Zebrafish Human
Not identified Not identified FOXF1
Smad Smad SMAD
Bapx1 bapx1 NKX3.2
Nkx2.5 nkx 2.3–2.7 NKX 2.3–2.9
Not identified Not identified Not identified
severe foregut, gallbladder, and lung malformations.38,43
The closely related gene Foxf2 is differentially expressed
during mouse GI development, with expression predom-
inating in the posterior aspect of the developing gut.44,45
This suggests some functional redundancy between Foxf1
and Foxf2 in the distal mesenchyme, which would explain
the predominantly foregut phenotype of the FoxF1⫹/⫺
mice and distal phenotype of Foxf2⫺/⫺ mice. Consistent
with its expression pattern, Foxf2-null embryos have co-
lonic dilatation and anal atresia, whereas the foregut
seems relatively unaffected.46 Although this phenotype is
reminiscent of Hirschsprung’s disease in humans, no
human condition has been associated with absence or
mutations of FoxF proteins. In sum, FoxF proteins are
essential for early VM development and multiple aspects
of intestinal differentiation.
To further characterize the molecular regulation of
early VM development, Jakobsen et al used gene profiling
and ChIP-on-Chip (chromatin immunoprecipitation fol-
lowed by microarray) to identify downstream targets and
partners of biniou in Drosophila.39 They showed that al-
though biniou is expressed throughout VM development,
its binding to specific enhancer elements is under tight
temporal and spatial control. Importantly, they showed
that several biniou targets are conserved in mice; the
expression of Tcf21, Sall4, and Ptk7 were down-regulated
in Foxf1⫺/⫺ or Foxf2⫺/⫺ mouse embryos.39 Bagpipe and
Mef2 are also important regulators of VM development
and differentiation. Each can bind its own set of regula-
tory elements in the absence of biniou.47 Understanding
the differences between biniou-dependent and -indepen-
dent genes will be an important avenue to explore to
advance our understanding of regional and temporal cell
fate decisions during VM development. Of course, it will
be crucial to confirm these findings in vertebrates.
Unlike some of the other transcription factors known
to participate in regional differentiation, biniou/Foxf1 is
expressed throughout the length of the developing intes-
tine (Figure 5A and B). This is of interest because there is
a wealth of genes known to participate in foregut and
hindgut development, whereas there are few midgut can-
didates. Further studies to address the differential re-
gional regulation of Foxf1 should elucidate whether the
June 2009
Figure 4. The role of biniou/FoxF1 is conserved across species. Inver-
tebrate example: Drosophila melanogaster. (A) Expression of VM
marker fascilin III (Fas III) in stage 12 Drosophila embryos (arrow). Ante-
rior is to the left. (B) The VM marker Fas III is absent in the biniou mutant,
which lacks biniou, the Drosophila orthologue of FoxF1.37 Vertebrate
example: effect of FoxF1 loss of function in Xenopus laevis. (C) Ventral
view of a normal gut coiling in a 5-day-old Xenopus embryo. (D) Both
coiling and elongation are severely impaired when FoxF1 is knocked
down using antisense morpholino-oligonucleotides.36 The black cells in
C are melanocytes; they are absent in the knockdown embryos. Cranial
is to the top.
molecular network that regulates midgut development is
a “default” program that needs to be modified for the
development of more recent digestive functions such as
the stomach. What factors participate in regionalization
of the VM?
Anterior-Posterior Patterning of the
Mesoderm and GI Tract
GI development progresses in a craniocaudal
manner, but the mechanisms by which this occurs are
not fully understood. The conserved paradigm of GI
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development, across species, is that Hh ligands are
expressed by the endoderm/epithelium and that Hh re-
ceptors and targets are expressed by the mesoderm/mes-
enchyme, which responds differentially according to de-
velopmental stage and anterior-posterior position.4 – 8,48
Multiple signaling pathways confer positional cues to the
developing intestine (Figure 6). Because of their known
role in segmental and anterior-posterior patterning, ho-
meobox gene products are obvious candidates to inte-
grate these positional cues into regional signals in the
A
Nkx2.3-2.9
Nkx3.2
vimar FoxF1 zampogna
Wnt, FGF, RA
Foregut Midgut Hindgut
B
p
visceral
mesoderm
l liver p
Barx1
Nkx3.2
Hoxa-5
Hoxa-4 Hoxd-12/13
Foxf1/f2
Nkx2.3
Hlx
FoxL
Figure 5. Transcription factors and signaling pathways during anter-
ior-posterior and radial patterning. (A) Early mesoderm formation is
under the control of the conserved homeobox genes. Studies in Dro-
sophila have allowed the identification of some early steps in VM forma-
tion implicating homeobox genes.180,181 These include tinman (Nkx2.3-
2.9), bagpipe (Nkx3.2), and other genes.181 Tinman is required for
mesoderm formation, whereas its downstream target bagpipe pro-
motes VM development.182,183 Vimar, a downstream factor from bag-
pipe, further specifies VM development.184 In Xenopus, 2 bagpipe ho-
mologues have been identified and their expression has been analyzed,
suggesting that these the role of these homeobox genes is conserved in
development; Xbap (Nkx3.2) is expressed in the foregut musculature of
Xenopus and mouse82,185 and zampogna in the musculature of the
Xenopus midgut, with some overlap in the posterior foregut meso-
derm.185,186 Expression of zampogna in the posterior indicates early
anterior-posterior differentiation in Xenopus. Vimar is indicated in green
in the figure because it has been identified in Drosophila but not in
Xenopus. Foxf1, downstream of Nkx3.2 (bagpipe), is first expressed in
the late gastrula and then throughout the VM of the embryo and the
adult. Although the exact relationship between these genes and major
signaling pathways is incompletely understood, in the early embryo,
FGFs, Wnts, and retinoic acid (RA) promote posterior fates while inhib-
iting anterior fates. Figure of Xenopus embryo at stage 28: © 1994
Pieter D. Nieuwkoop and J. Faber.187 (B) Spatial distribution of selected
mesodermal forkhead and homeobox genes involved in vertebrate gas-
trointestinal differentiation, using mouse nomenclature. Not shown is
Nkx2.5, which specifies the pylorus in the caudal segment of the fo-
regut. Anterior is to the left, dorsal to the top. Diagram courtesy of Aaron
M. Zorn. l, liver; p, pancreas.
 2080 MCLIN ET AL
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Figure 6. Heterotopic recombinations of mesoderm and endoderm.
Detailed dissection and recombination experiments in neurula-stage
embryos (during regional specification of the primitive gut tube) of Xe-
nopus laevis, the African clawed frog (equivalent to mice with 7– 8
somites or a 22-day-old human embryo), have shown that early
endodermal regionalization depends on mesodermal signals.188,189 Al-
though the nature of these signals is incompletely understood, the
major signaling pathways appear to be involved. For example, in the
chick, the endoderm needs instructive BMP/activin family signals from
the lateral plate mesoderm to differentiate64; retinoic acid (RA) in the VM
confers a posteriorizing gradient to the developing GI tract.190 In mouse,
Xenopus, and zebrafish, mesodermal Wnt signals confer temporally
regulated anteroposterior information to the adjacent endoderm.191,192
Wnt antagonists in the anterior VM are necessary for stomach devel-
opment (anterior)83 and Wnts probably posteriorize the VM, but this has
not been shown. Because this paradigm has been studied in detail in
liver and pancreas development, we refer the reader directly to those
studies.191–193 Modified and reprinted with permission from Horb and
Slack.189
presumptive intestine (Figure 5A). Detailed expression
studies have mapped Hox gene products in the develop-
ing gut; the majority are expressed in the mesoderm.49 –53
There are 3 important points about the role of Hox
gene products in GI patterning. The first is that their role
appears to have been selected for during evolution. Ho-
meodomain-containing genes belonging to the Nkx2
family (Figure 5A) confer positional information.54 Over-
expression of vertebrate Nkx2 genes can partially rescue
the phenotype of Drosophila embryos null for the tinman
gene in the same cascade, indicating a highly conserved
function for these genes (tinman is upstream of Nkx2;
Figure 5A).55
Another take-home point concerning Hox genes is that
they confer regional information that contributes to the
formation of boundaries by conveying permissive signals
to the mesenchyme.3 In mice carrying a homozygous
deletion of the Hoxd3-13 cluster,56 which is normally
expressed in the mesoderm, sphincter formation of both
the pylorus and anus is absent.56 Furthermore, ectopic
expression of Hoxd12 and Hoxd13 causes abnormal or
absent ceca.57 Hoxa13⫺/⫺ mice develop GI stenoses and
GASTROENTEROLOGY Vol. 136, No. 7
atresias58; Hoxa13 mutations have been described in hu-
mans with limb, GI, and genitourinary abnormalities.59,60
Misexpression of Hoxd13 in the chick midgut leads to a
hindgut phenotype (homeotic transformation). These
findings suggest that loss of Hox genes leads to malfor-
mation of the intestines of mice, whereas their ectopic
expression in chick embryos leads to a homeotic trans-
formation or ectopic expression of region-specific genes.
Together with the finding that Hoxa4⫺/⫺ mice have ab-
normal colonic musculature61 and Hoxa5⫺/⫺ mice have
abnormal stomach development,62 these findings indi-
cate that Hox gene products probably participate in re-
gionalization in part through the regulation of smooth
muscle precursor cell fate and proliferation.
Third, Hox genes likely confer positional information
along the mouth-to-anus axis by integrating multiple
regulatory inputs. In the early mouse embryo, BMP sig-
naling in the VM is necessary for Hox gene induction;
subsequent mesenchymal-to-epithelial signaling controls
stomach morphogenesis and lung budding,63 and reti-
noic acid signaling controls endodermal expression of
Hox genes such as Pdx1 (pancreatic and duodenal ho-
meobox 1) or Cdx2 (caudal type homeobox transcription
factor 2).64 Cdx genes are regulated by Wnts, and it is
hypothesized that the Wnt pathway “co-opted” Hox ma-
chinery to execute their anterior-posterior program.65– 69
Further studies are required to determine if this holds
true in VM development. Finally, considering that the
mesoderm appears to respond differentially to Hh signals
along its anterior-posterior axis, Hh might induce me-
sodermal Hox gene products to induce sphincter for-
mation. This is illustrated by Shh⫺/⫺ mice that display
homeotic transformation phenotype: intestinal transfor-
mation of the stomach, annular pancreas, duodenal ste-
nosis, and an imperforate anus. This homeotic transfor-
mation of the Shh⫺/⫺ stomach supports the concept of an
Hh-to-Hox pathway, which has now been demonstrated
in the foregut and hindgut of chick embryos, mice, and
rats.5,62,70
Although Shh becomes restricted to the foregut and
hindgut after E14.5, Ihh is expressed throughout the
length of the intestinal epithelium during development
and adulthood.6 These expression patterns are consistent
with the phenotypes of knockout animals.6,7 In contrast
to the Shh⫺/⫺ phenotype, Ihh⫺/⫺ mice have a phenotype
that resembles human Hirschsprung’s disease, nearly ab-
sent enteric neurons and a thin muscularis propria,71
which is also observed in the Foxf2⫺/⫺ mice.46 Although
an Hh-to-Fox axis has not been explored in detail, the
role of Fox genes in this process has been supported by
lack of Foxf1 expression in the GI tract of mice with Gli2
and Gli3 mutations.72 Importantly, these phenotypes
mimic human syndromes with GI malformations that
have been associated with Hh pathway defects, such as
Pallister–Hall syndrome73 and the VATER association.74
June 2009
Mesenchyme-to-Endoderm Signaling in
Stomach Development
Stomach development is a unique example of the
differential response of the mesenchyme-to-endodermal
signals. The stomach of the developing chick has an
anterior and a posterior chamber. Recombination exper-
iments of proventriculus (anterior) mesenchyme and giz-
zard (posterior) epithelium (and vice versa) have shown
that the epithelium often adopts the fate of the adjacent
mesenchyme.3,75,76 However, this is not the case in other
parts of the GI tract.77–79 Signaling by BMPs 2 and 4
participate in this cross talk, although their relative con-
tribution differs according to developmental stage.80,81
The divergent homeobox-containing gene Bapx1 or
Nkx3.2 regulates mesodermal BMP-4 and Wnt5a expres-
sion in the chick stomach75 and pylorus morphogenesis
in the mouse82 (Figure 6). In mice, the regionally re-
stricted homeodomain-containing protein Barx1 is re-
quired for mesenchymal expression of Wnt inhibitors
and normal development of the stomach epithelium.83 In
chick, mesodermal BMP-4 independently induces expres-
sion of Nkx2.5 and Sox 9 in a cell-autonomous manner,
leading to the formation of the pyloric sphincter.84,85
Consistent with the role of BMP in anterior-posterior
patterning, constitutive activation of BMP signaling in
distal segments of the developing gut mesenchyme leads
to ectopic, cell-autonomous expression of the pyloric
sphincter marker Nkx2.5.86 In summary, regulation of
major growth factors by mesodermal homeobox genes is
necessary for epithelial stomach morphogenesis and for-
mation of the pylorus. These observations raise 2 impor-
tant points. First, regionalization of the developing GI
tract is governed in part by circumscribed transcription
factors in response to local signals.83 Second, precise
regulation of signaling pathways in the stomach suggests
that gastric development requires modification of the
midgut “default” program. From a developmental per-
spective, it seems both economical and practical to
modify a preexisting program for the development of a
new structure with slightly different functional re-
quirements.
Midgut and Hindgut Development
Unlike the stomach, the plasticity of the midgut
epithelium, in response to changing mesenchymal sig-
nals, appears to be somewhat limited.87,88 Using hetero-
topic cross-associations of endoderm and mesenchyme
from different segments of the developing rat intestine,
Ratineau et al showed that segments of intestinal mes-
enchyme from rat fetuses did not confer equal regional
information to adjacent endoderm. In the 14-day-old
fetus, midgut endoderm did not change its enzyme ex-
pression when cocultured with proximal or distal intes-
tinal mesenchyme.89 Conversely, colon endoderm did
respond to the association with jejunal or ileal mesen-
chyme by developing into small intestinal epithelium.
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Therefore, the hindgut endoderm, like its anterior coun-
terpart, shows relative plasticity late in development in
response to instructive signals from the VM. ISEMFs
appear to have a role in this process; ISEMFs from an-
teroposterior segments of the intestine secrete varying
amounts of growth factors, thereby conferring dose-
dependent regional characteristics to the adjacent
endoderm.19 Similar experiments in chicks showed that
recombination of proventricular mesenchyme with mid-
gut endoderm did not alter epithelial gene expression in
the midgut but did in the foregut.90 Taken together,
these findings suggest that the midgut endoderm pro-
gram might be specified earlier than the cranial- and
caudal-most segments of the gut.
The Lateral Plate Mesoderm and
Mesenchyme in Left-Right Asymmetry
of the GI Tract
In all vertebrates, shortly after gastrulation, the
lateral plate mesoderm participates in left-right cell fate
decisions. The transforming growth factor ␤ family
member Growth and differentiation factor-1 (Gdf-1)91 pro-
motes the expression of genes that regulate lateral devel-
opment, such as nodal, Lefty, and Pitx2 in the left lateral
plate mesoderm.92–96 Recently, events downstream of
Pitx2 (paired-like homeodomain transcription factor 2)
and its partner LIM-homeodomain containing transcrip-
tion factor Isl1 (islet-1) were identified in the dorsal
mesentery. The dorsal mesentery, which is continuous
with the visceral mesenchyme, derives from the lateral
plate mesoderm and anchors the gut to the dorsal wall of
the vertebrate embryo. Pitx2 and Isl1 control expression
of glycosaminoglycans and the ECM protein N-cadherin;
expression is asymmetrically distributed in the dorsal
mesentery of chick and mouse, leading to changes in cell
shape and intercellular connections that induce the first
gut tilt to the left.97 This study has shown a role for the
mesoderm-derived mesentery in gut morphogenesis. Be-
cause Shh⫺/⫺ and Ihh⫺/⫺ mice display malrotation, it is
probable that endodermal Hh signals are upstream of
this pathway.71 In Xenopus, retinoic acid–metabolizing
enzymes are expressed in the VM of the gut during the
early stages of gut coiling.98 Addition of retinoic acid or
retinoic acid inhibitors to embryonic culture medium
before the onset of gut coiling leads to abnormal chirality
and organ heterotaxy.98 Thus, the Gdf-1/Pitx2/ECM
pathway likely results from the integration of multiple
signaling gradients in the mesenchyme.
The Role of the VM in Radial
Patterning, Villus Formation, and the
Differentiation of the Epithelial and
Mesenchymal Layers
In addition to its role in anterior-posterior and
left-right patterning, the mesoderm is required for radial
patterning and villus formation. Radial patterning is the
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Table 2. Transcription Factors in Radial Patterning of the Gut
Transcription factor Model Expression
FoxF1 Xenopus VM
Foxf1⫹/⫺ Mouse VM, foregut
Foxf2⫺/⫺ Mouse VM
Hlx⫺/⫺ Mouse VM
Nkx2.3⫺/⫺ Mouse, chick VM midgut and hindgut
Hoxa5⫺/⫺ Mouse VM midgut
organization of the GI tract into concentric layers.80
Differentiation of the crypt-villus axis happens at differ-
ent times in development in different species and
progresses in a craniocaudal fashion. In mice, villus for-
mation begins around E15, but crypt formation does not
occur until the postnatal period. In Xenopus, the trough/
intestinal fold axis is the product of metamorphosis, and
in humans, the newborn infant has a complete crypt-
villus axis. Crypt-villus axis formation relies largely on
mesenchymal-epithelial interactions that determine vil-
lus length, crypt depth, and spacing between villi.77,99 –101
The inaugural event in villus formation is mesenchymal
condensation, which depends on ubiquitous signaling
pathways that vary according to the developmental time
period. Certain transcription factors that participate in
early morphogenetic processes also contribute to radial
patterning and villus formation.
Transcription Factors That Contribute to
Radial Patterning
The transcription factors Foxf1/f2 are expressed
throughout development; apart from their roles in early
patterning of the lateral plate, they also have a role in
later differentiation.36,46 Studies of mouse knockout
models have shown that Foxf proteins are required for
multiple aspects of VM development, including radial
patterning. Foxf1/2 expression in the early VM is ubiq-
uitous and Foxf proteins are required for VM develop-
ment in multiple species. However, because of the pleo-
morphic phenotypes of knockout animals, it is difficult
to determine the precise role of Foxf genes. Several other
transcription factors participate in radial patterning,
with their respective roles still incompletely understood.
The difficulty in understanding the precise role of a given
transcription factor stems from the developmental inter-
dependence of mesenchymal components among them-
selves and with the adjacent epithelium. Table 2 summa-
rizes the phenotypes associated with loss of function of
forkhead or homeobox-containing transcription factors
in the VM. Smooth muscle, enteric neurons, and ECM
appear to be the most vulnerable components of the
developing VM in response to genetic manipulation. In
addition to changes in the VM, genetic manipulation of
the VM usually leads to aberrant epithelial development
and proliferation.
GASTROENTEROLOGY Vol. 136, No. 7
Phenotype References
Absent lumen, absent smooth muscle actin expression 36
Esophageal atresia, tracheo-esophageal fistula 38, 41
Abnormal muscularis propria, ECM, absent ENS, 38, 41
dilated colon, increased epithelial proliferation
Epithelial proliferation, ENS abnormalities, short gut 195–197
Thin mesenchyme, abnormal villus formation 198, 199
ENS, smooth muscle, ECM 62
Signaling Pathways in Radial Patterning
Multiple cell fate and positional decisions contrib-
ute to the formation of the organized, tubular gut. To
this end, transcription factors regulate and integrate nu-
merous secreted signals, leading to the differentiation of
each mesodermal component and its orientation along
the crypt-villus or epithelium-serosa axes. Although these
different signaling pathways overlap and their relative
contribution is still incompletely understood, we will
now discuss their individual roles as they are character-
ized to date.
Hh: smooth muscle proliferation and positioning
of the crypt. Loss-of-function studies of different com-
ponents of the Hh pathway have led to the comparatively
well-understood role of this pathway in the radial pat-
terning of the GI tract. In very broad terms, its functions
are 4-fold, keeping in mind, however, that these vary
according to developmental window. First, Hh ligands
act as radial morphogens for the development of the
smooth muscle layer; in other words, too much or too
little ligand inhibits smooth muscle development, while
the right concentration, regulated by the distance from
the endodermal source, induces smooth muscle forma-
tion in both chick and mouse.35,71 Second, depending on
developmental window, endodermal Hh signals are im-
portant for induction of reciprocal signaling from the
mesenchyme back to the endoderm, because loss of func-
tion leads to epithelial proliferation.102 Third, epithelial
Hh ligands influence ISEMF localization along the crypt-
villus axis, probably because these cells serve to integrate
the epithelial signal, thereby regulating the proliferative
effect of the Hh signal.103 Fourth, Shh and Ihh have
complementary roles in ENS development; Shh inhibits
ENS development, while Ihh is essential.35,71 In summary,
the Hh pathway serves as a radial morphogen affecting
both cell fate and proliferative decisions in the layers of
the mesenchyme and epithelium. In the future, targeted,
tissue-specific modification of Hh signaling to the mes-
enchyme should reveal the relative contributions of Ihh or
Shh to the development of the different concentric layers.
Further, it may help elucidate the concentration and
temporal needs of each cell type, thereby facilitating the
direction of cell fate in vitro.
June 2009
BMPs: smooth muscle development and regula-
tion of the proliferative compartment (crypts). BMPs are
members of the transforming growth factor ␤ superfam-
ily; BMPs 2 and 4 have important roles during GI devel-
opment: signaling via the BMP receptor (BMPR)-1a.80,104
BMP transcription in the VM is initiated in response to
endodermal Hh signals,5 so many of the BMP-related
phenotypes are reminiscent of Hh loss-of-function phe-
notypes discussed in the previous section. Although the
role of BMPs in GI development also varies according to
developmental stage and tissue layer, studies in the early
developing gut of avian embryos suggest that BMP sig-
nals are required by all gut layers that are distal to the
stomach105 and contribute to the morphogenesis and
differentiation of all components of the GI tract.106
Figure 7. Molecular cross talk between the VM and the endoderm
during crypt-villus formation and in the adult. (A) Signaling pathways
during crypt-villus formation. Hh ligands signal to the mesenchyme. In
response, probably via ISEMFs, mesodermal Wnt ligands promote pro-
liferation in the intervillus space and in the villus (starting at E16.25) and
BMP signals regulate proliferation of the epithelium. Arrows indicate
direction of signal. (B) In the adult with a fully developed crypt-villus axis.
On the left, pathways expressed in the mesenchyme; on the right,
pathways expressed in the epithelium. The signals in the adult likely
occur simultaneously. Modified and reprinted with permission from
Crosnier et al.194
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BMPs, previously discussed for their role in anterior-
posterior patterning, are also necessary for radial pattern-
ing and villus formation. In simple terms, BMP-4 in the
VM controls smooth muscle proliferation and differen-
tiation throughout most of the developing vertebrate
GI tract107; this is probably conserved across species.
Whereas the small intestine needs sufficient expression of
BMP-4 in the mesenchyme for the development of an
adequate muscularis propria, overexpression of BMP-4 in
the mesenchyme of the embryonic chick gut delays dif-
ferentiation into smooth muscle106 and inhibition of
BMP-4 in the stomach is required for the highly devel-
oped musculature to meet its functional needs.106 Ab-
sence of BMP-4 from the presumptive hindgut meso-
derm promotes aberrant endoderm proliferation and
ENS development, indicating the importance of BMP-4
not only in smooth muscle development but also in the
regulation of epithelial proliferation.81
BMP signaling is important for villus formation and
endodermal/epithelial patterning (Figure 7A and B). Its
role in epithelial morphogenesis and differentiation is
complex and involves cross talk between epithelium and
mesenchyme; several studies have attempted to address
this issue.106 –109 When the secreted BMP inhibitor nog-
gin is constitutively expressed in the developing mouse
epithelium, an excess number of crypts develop.107,110
Depending on the model and the developmental stage
analyzed, epithelial and mesenchymal proliferation and
differentiation are affected, leading to aberrant crypt and
villus formation, similar to that associated with the
pathogenesis of human juvenile polyposis.110 However,
when BMP signals to only the epithelium are specifically
blocked using a villin-dnBMPR1a (dominant negative BMP
receptor 1a) transgene, only epithelial proliferation and
secretory lineage differentiation are affected; villus or
crypt number are not.109 Taken together, these studies
support a model in which BMP signaling in the mesen-
chyme sends antiproliferative signals to the crypts and
epithelial stem cells, probably through a signaling relay
in the pericrypt mesenchyme (possibly ISEMFs), that
regulates the size and location of the proliferative com-
partment.108 It is unclear whether the mesodermal BMP
brake is conserved across species and whether it is in
effect at all developmental time points. In contrast to the
evidence presented from mice, during gut remodeling
associated with Xenopus metamorphosis, mesenchymal
BMPs promote rather than inhibit epithelial prolifera-
tion.111,112 The obvious next step in elucidating the re-
lative roles of BMP signaling in the epithelium and
mesenchyme is the conditional modification of BMP
signaling in the mesodermal layer, something impeded
by lack of appropriate tools.
Wnts: proliferation versus differentiation. Wnts
are accepted as the master regulators of epithelial prolif-
eration and differentiation in the developing and adult
GI tract.113–115 Most studies have focused on the role of
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Wnts in endoderm specification and differentiation, be-
cause regulation of nuclear ␤-catenin accumulation is
required for epithelial homeostasis and carcinogene-
sis.83,116,117 The origin of these Wnt signals is mostly
mesodermal118,119 (Figure 7A and B). The exact cellular
origin of the Wnt signal in vivo is still unclear, but
ISEMFs are likely to contribute because they express Wnt
ligands and because they are adjacent to the proliferative
compartment of the epithelium, the intestinal crypts.118
In situ hybridization analyses of mouse and chick
embryos have shown that the mesenchyme of the devel-
oping and adult intestine contains high levels of Wnt
pathway components, both canonical and noncanoni-
cal.119,120 In the adult, Wnt signals are necessary for
maintenance of the proliferative compartment of the
epithelium.121 During development, it has been accepted
that the intervillus mesenchyme expresses Wnt compo-
nents because signs of Wnt activity were found in the
epithelium.122,123 However, novel findings suggest the
adult pattern of crypt-predominant Wnt activity only
begins around postnatal day 3 in mice; before this time
point, canonical Wnt activity predominates in the nas-
cent villi coincident with the mesenchymal expression of
several Wnt ligands, including Wnt5a119,124 (Figure 7A).
Importantly, in this study, ␤-catenin activity does not
correlate with epithelial proliferation, suggesting that
Wnt signaling may be more than just an on/off switch for
proliferation versus differentiation.124
Recent studies have highlighted multiple roles for
Wnt5a in the mesenchyme.119,125 Wnt5a is expressed both
in the embryonic and adult mesenchyme, suggesting that
it is an important signaling molecule in intestinal devel-
opment and homeostasis, probably with different spatial
and temporal functions, acting through both the canon-
ical and noncanonical pathways.119 Wnt5a⫺/⫺ mice have
multiple defects: thinner muscularis propria, improper
midgut closure, and a dramatically shortened midgut.
This latter effect is in part mediated by defective post–
mitotic cell intercalation in the epithelium in a non—␤-
catenin– dependent manner.125 This finding joins that of
others in highlighting a role for the mesenchyme in gut
elongation and morphogenesis125,126 and highlights the
importance of Wnt signaling in more than epithelial
proliferation and differentiation.
Together with other Wnt ligands, Wnt5a is also essen-
tial during radial patterning when it cooperates with Hh,
BMPs, and Forkhead genes. As outlined previously, Foxf
mutants have altered collagen production and decreased
mesenchymal BMP4 signaling, which leads to Wnt5a
overexpression and increased epithelial proliferation.46
Similarly, Foxl1 participates in the regulation of Wnt
signals to the adjacent epithelium. Although Foxl1 posi-
tively regulates BMP-4 and BMP-2 expression, its main
mechanism of action involves proteoglycan synthesis in
the mesenchyme.127,128 Ectopic syndecan-1 synthesis in
the villus mesenchyme of Foxl1-null mice is associated
GASTROENTEROLOGY Vol. 136, No. 7
with aberrant epithelial expression of nuclear ␤-catenin
and increased epithelial proliferation. Histologically,
Foxl1-null mice have delayed formation of intestinal villi
compared with wild-type mice, but this is not observed in
Foxf mutant mice, in which the mesodermal structures
appear to be normal.127,129 Adult Foxf mutant mice have
multiple epithelial abnormalities, including increased cell
proliferation in the villi, cystic inclusions, and abnormal
stomach histology.127,129 Loss of Foxl1 augments the ep-
ithelial effect of adenomatous polyposis coli (APC) germline
mutations by increasing the multiplicity of intestinal
adenomas.128 The developing intestine expresses Wnt li-
gands, receptors, and inhibitors, and their roles vary
during development. The control of mesodermal Wnt
signals is of particular importance because of the contri-
bution of these factors to epithelial tumorigenesis. Tis-
sue-specific modulation of Wnts in the mesenchyme will
improve our understanding of the role of Wnts both in
the mesenchyme proper and in the adjacent epithelium.
FGFs. The role of the FGF pathway in radial
differentiation is less well understood that that of the
Hh, BMP, or WNT signaling pathways. In Xenopus, FGF
signaling is necessary for expression of the smooth mus-
cle cell marker XSM22␣130 and smooth muscle actin and
patterning of the early intestine.131 In the E18.5 mouse
embryo, FGF13 is highly enriched (47.6-fold) in the mes-
enchyme compared with epithelium132 and likely medi-
ates autocrine actions via FGFR1 and R2, as well as
having a role in crypt morphogenesis via FGFR3133 (Fig-
ure 7B). Normal cecum development is characterized by
an outward growth of both mesenchymal and epithelial
proliferation134 and requires mesenchymal FGF-10 and
BMP-4 signaling in response to epithelial FGF-9 sig-
nals.134 Mouse studies indicate that epithelial FGF-9 sig-
naling controls gut length by regulating proliferation
and differentiation of subepithelial fibroblasts, possibly
through putative mesenchymal stem cells.126 FGFs are
required for normal development of the gut because they
regulate mesenchymal signaling, but we need to learn
more about the time periods and regions in which these
potent signaling molecules are active or inactive. Like
other pathways, the understanding of their relative con-
tribution to mesodermal and epithelial patterning will be
vastly aided by the development of mesoderm-specific
tools.
Glucocorticoid and Thyroid Hormones
in GI Development
Glucocorticoids (GCs) promote maturation and
differentiation of the human and rodent intestinal epi-
thelium.87,135 However, the mesenchyme appears to be
essential for this effect. In vitro studies have shown that
fetal rat intestinal endoderm cells do not respond to GCs,
but when they are cultured with mesenchymal cells and
GCs, ␣-glucosidase expression is induced.136,137 Further-
more, although explants of intact perinatal colon do not
June 2009
respond to GCs, sucrase expression is induced when
explants derived from colonic endoderm are associated
with small intestinal mesenchyme and exposed to GCs.138
These findings indicate that small intestinal mesen-
chyme, but not colonic mesenchyme, enables GC induc-
tion of maturation in an associated epithelium. Mesen-
chymal cells alone respond to GCs; these cells express the
GC receptor137 and show increased expression of collagen
type IV messenger RNA139 following in vivo administra-
tion of GCs.139 –141 In addition, in vitro studies have
shown that in response to GCs, mesenchymal cells de-
posit laminin at the mesenchymal-epithelial interface,
which appears to be essential for the effects of GCs on
the epithelium because the effects can be blocked in vitro
by the addition of anti-laminin antibodies.142 These ex-
periments suggest that the mesenchyme acts as either a
direct GC target that signals to the epithelium or that the
mesenchyme induces the adjacent epithelium to respond
to the hormone.142 Regardless of the specific mechanism,
questions of clinical significance are whether the mesen-
chyme is responsible for the relatively narrow develop-
mental stage during which the intestinal epithelium can
respond to GCs143,144 and whether these mesenchymal
effects are permissive or instructive.
Thyroid hormone also has a role in the development of
the vertebrate GI mesenchyme. Thyroid-responsive genes
are expressed in the mesenchyme of E18.5 mouse embr-
yos,132 and absence of the thyroid hormone receptor T3R␣
leads to hypoplastic smooth muscle layers in the devel-
oping intestine.145 This finding correlates with increa-
sed expression of T3R␣ in the intestinal muscle layers
of wild-type animals,145,146 which might suggest that
smooth muscle cells require a thyroid signal for develop-
ment. The role for thyroid hormone in mammalian epi-
thelial maturation has been difficult to assess because
changes in thyroid status cause concomitant changes in
circulating GCs.87 However, thyroid hormone can syner-
gize with GCs in eliciting developmental changes in the
epithelium,147,148 but it is not known whether the mes-
enchyme has a part in this synergy. Xenopus species are
characterized by a thyroid hormone– driven metamor-
phosis during which the GI tract undergoes remodeling
characterized by extensive changes of the mesenchyme
and development of the muscular layers. Although this
developmental step is unique to anurans, it is an attrac-
tive model to examine intestinal mesoderm/mesenchyme
development; in 7 days, the 2-cell-thick VM proliferates
into longitudinal and circular muscle layers as well as
numerous other cell types.146 Cultures of Xenopus intes-
tinal epithelial cells are insensitive to thyroid hormone–
induced apoptosis when they are cocultured with ECM
molecules,149,150 suggesting that the ECM confers sur-
vival signals to the epithelium. Therefore, the study of GI
remodeling during Xenopus metamorphosis should offer
important insight into the development of the VM and
its components.151
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Cytokines and Other Signaling
Molecules in GI Development
In addition to modulators of the major signaling
pathways and hormones, the VM of E18.5 mouse em-
bryos is enriched in multiple peptide growth factors and
cytokines.132 These include endothelins, insulin-like
growth factor (IGF), and platelet-derived growth factor
(PDGF). For example, PDGF is believed to regulate mat-
uration of smooth muscle precursors via the Wnt path-
way.152 PDGF-A and its receptor PDGFR␣ contribute to
formation of the crypt-villus axis.153 They are both ex-
pressed in the developing mouse small intestine starting
around E15.5; PDGF-A is expressed in the epithelium,
and PDGFR is expressed in the mesenchyme. PDGF-A⫺/⫺
and PDGFR␣⫺/⫺ mice have similar phenotypes; com-
pared with wild-type mice, they have fewer and broader
villi, a thinner mesenchyme, and premature expression of
smooth muscle actin in the villus mesenchyme.153 There-
fore, PDGF-A must promote cell proliferation and pre-
vent premature differentiation of the mesenchyme.
Again, the role of this molecule depends on the develop-
mental stage, because mutations in the PDGFR␣ are
associated with GI stromal tumors in humans.154 –158
IGF-1 is expressed in the mesenchyme of the E18.5
intestines of mice,132 and IGF-1 receptors are expressed in
the submucosal region of the neonatal small intestine;
expression is down-regulated after birth.132,159 IGF-1 and
IGF-2 binding to the IGF receptor promotes epithe-
lial differentiation.159 Suppressor-of-cytokine signaling
(SOCS) 2 is normally required for regulating the effect of
IGFs. Consistent with this role, SOCS2-null mice have
increased size and weight, intestine length, and thickness
of the lamina propria.160 Members of the SOCS family
are highly expressed in the E18.5 mesenchyme,132 sug-
gesting that, like other growth factors, IGF and cytokine
signaling in the mesenchyme is tightly regulated.
The peptide endothelin-1 and its receptors are also
expressed in the E18.5 mesenchyme of mice.132 The co-
lonic mesenchyme of rat embryos expresses high levels of
endothelin 3 at E16.5, which promotes differentiation of
adjacent epithelia in a dose- and region-dependent man-
ner.161
Epimorphin (encoded by Stx2) is also up-regulated in
the mesenchyme,132,162,163 where it is secreted by ISEMFs
to mediate epithelial morphogenesis and control intesti-
nal length.162,163 Stx2-null adult mice have longer intes-
tines and colons than their wild-type counterparts.162
Three-week-old Stx2-null pups have increased crypt cell
number and increased villus length, suggesting that epi-
morphin controls cell proliferation during the final
stages of crypt-villus axis formation. This effect has been
proposed to result from decreased BMP-4 signaling and
increased expression of ␤-catenin target genes.162 It is
unclear why loss of the protein confers a regenerative
advantage, but therapeutic manipulation of this pathway
may be attractive to pursue.
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Smooth muscle genes are up-regulated in the mesen-
chymal compartment,132 and studies in zebrafish have
shown that visceral smooth muscle cells are involved in
GI ontogeny.27,164 Zebrafish expressing a mutant smooth
muscle myosin heavy chain have abnormal smooth mus-
cle development, defects in formation of the posterior-
most segment of the intestine, uncontrolled epithelial
proliferation, and aberrant epithelial expression of ECM
proteins.164 This phenotype is similar to that of Ihh⫺/⫺
and Foxf2⫺/⫺ mice. It is possible that smooth muscle
myosin heavy chain is a downstream target of a putative
Ihh-Foxf pathway that controls GI radial differentiation.
Matrix metalloproteinase molecules are expressed in
the mesenchyme of E18.5 mouse embryos,132 and high
levels of matrix metalloproteinases are transcribed in the
mesenchyme during intestinal remodeling in Xenopus em-
bryos (during metamorphosis). Matrix metalloprotein-
ases cleave various ECM components and are target genes
for thyroid hormone; therefore, they are likely to regulate
ECM composition, basement membrane fenestrations,
and subsequent signaling to the adjacent epithelium dur-
ing remodeling.165,166 Although the role of matrix metal-
loproteinase in mammalian GI development is poorly
understood, amphibian models offer insight into poten-
tial mechanisms.
Conclusions
The VM is a complex tissue that gives rise to the
smooth muscle, mesenchyme, and other cells that com-
pose the outer layers of the mature intestine and has
multiple essential functions during the development of
the GI tract. The development of each VM cell type is
intricately linked to that of its neighbors, and although
many of the molecular interactions are incompletely
characterized, abnormal mesenchymal signaling almost
invariably leads to aberrant epithelial proliferation. The
VM and its components confer instructive and permis-
sive signals to the adjacent developing epithelium; this
instructional dominance appears to be mediated by sol-
uble factors and signaling pathways that regulate devel-
opment of the mesoderm and endoderm.
Compared with the foregut and hindgut, the develop-
ment of the midgut is less well understood; it responds to
different mesodermal signals. The developmental plastic-
ity of different GI segments is controversial, which could
be the result of the different models and conditions used
in experiments. Alterations in mesodermal gene expres-
sion patterns lead to abnormal mesoderm differentiation
but also to epithelial proliferation, so one of the major
roles of the VM appears to be control of epithelial pro-
liferation. Studying the molecular basis of GI develop-
mental plasticity and the control of epithelial prolifera-
tion by the mesenchyme could identify new therapeutic
targets for cancer and other diseases of the GI tract.
Improving our knowledge of the spatial and temporal
control of GI cell fates might ultimately contribute to
GASTROENTEROLOGY Vol. 136, No. 7
developing cell-based therapies for treatment of these
disorders.
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sophila homeobox gene tinman: differential expression of cNKx-
2.3 and cNKx-2.5 during heart and gut development. Mech Dev
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Received September 19, 2008. Accepted March 4, 2009.
Reprint requests
Address requests for reprints to: Valérie A. McLin, MD, Unite de
Gastroenterologie, Hepatologie et Nutrition Pediatrique, Hopital des
Enfants, CH-1211 Geneva, Switzerland. e-mail: valerie.mclin@
hcuge.ch.
Acknowledgments
The authors thank all the reviewers for their encouragement
and in particular reviewers 1 and 3 for extremely constructive
suggestions. V.A.M. thanks Peter M. Carson for enduring
support.
Conflicts of interest
The authors disclose no conflicts.
Funding
V.A.M. is supported by the National Institutes of Health
(K08DK078656) and a Young Investigator Award from the
Children’s Digestive Health and Nutrition Foundation, S.J.H. is
supported by the National Institutes of Health (R01DK069585), and
M.J. is supported by the Retinal Research Foundation.