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

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REVIEWS IN BASIC AND CLINICAL GASTROENTEROLOGY John P. Lynch and David C. Metz, Section Editors

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 endoderm (which gives rise to the epithelium) and the mesoderm (which develops into the smooth muscle 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 diseases. Although the necessity of epithelial-mesenchymal cross talk for GI development is uncontested, the role of the mesoderm remains comparatively less well understood. The transformation of the visceral mesoderm during development is remarkable; it differentiates from a very thin layer of cells into a complex tissue comprising smooth muscle cells, myofibroblasts, neurons, immune cells, endothelial cells, lymphatics, and extracellular matrix molecules, all contributing to the form and function of the digestive system. Understanding the molecular processes that govern the development of these cell types and elucidating 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 maintenance 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.

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nherited or acquired abnormalities of the gastrointestinal (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 transplantation, but their progress requires a better understanding of the processes that govern cell fate decisions, cell proliferation, and tissue differentiation during GI development. Several aspects of GI development are well con-

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: gastrulation, 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 (Figure 1). The outer leaflet becomes the somatic musculature.1 During GI morphogenesis, endoderm and mesoderm undergo extensive regionalization, elongation, and coiling. Concurrently, neural crest cells populate the intestine 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: anterior-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 developAbbreviations 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, gastrointestinal; Hh, Hedgehog; IGF, insulin-like growth factor; ISEMF, intestinal 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

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DORSAL

SM VM

S

N

e

S

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 plasticity diminishing as development proceeds.2 Although the mesoderm has an important role in early GI tract morphogenesis, 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 foregut, 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 proximal 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 contribution of mesoderm components to endoderm patterning 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 metamorphosis 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 myofibroblasts (ISEMFs), both in the stalk and adjacent to the intervillus space, which are important regulators of mesenchymal 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 development of these different, mesodermally derived components is warranted before examining the molecular network governing VM development.

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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 As their name suggests, ISEMFs are cells that are located just below the epithelium of both villi and crypts throughout the length of the intestine (Figure 2). ISEMFs are characterized by ␣–smooth muscle actin expression and are organized in a syncytium between the epithelium and the muscularis mucosa, where they contribute to extracellular matrix (ECM) and basal membrane composition.17 Although it is uncertain whether ISEMFs are derived from the neural crest, mesodermally derived fibroblasts, or smooth muscle cells, they clearly

reside within the mesenchymal layer of the intestine at the base of the crypts, next to the muscularis, starting around week 21 of human gestation.18 In mouse embryos, 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

the adult intestine is to confer shape and motility to this hollow organ and allow for the advancement of its luminal 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 epithelial movement21 (Figure 2). In humans, disorders of enteric myocytes have been described in children with motility syndromes22; a few reports have associated hypoplasia of the muscularis propria with necrotizing enterocolitis 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 actin, as opposed to the more differentiated smooth muscle myocytes, which express ␣– and ␥–smooth muscle actins.25,26 Formation of the smooth muscle layer progresses in a craniocaudal fashion, starting in the esophagus 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 muscularis 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 until 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 manipulation of the developing gut often leads to a Hirschsprung’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 representing 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, zebrafish, 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 Xenopus and zebrafish, the Vent homeobox-containing genes act downstream 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 molecules and transcription factors in both germ layers. For example, during mouse gastrulation, the early mesoderm is patterned by the adjacent endoderm via secreted 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 adjacent endoderm along the anterior-posterior axis, inhibiting 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 endodermal FGF signals, bone morphogenetic proteins (BMPs) pattern the mesoderm of Xenopus embryos into its different components, including the VM. This example of a dorsoventral morphogen in the mesoderm is illustrated in Figure 3. Interactions between Shh, BMP-4, and Foxf1 participate in VM morphogenesis and appear to be conserved

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Table 1. Gene Nomenclature Drosophila

Xenopus

Mouse

Chick

Zebrafish

Human

Biniou Dpp Bagpipe Tinman vimar

FoxF1 Smad Xbap; zampogna Nkx2.3,2.5 Not identified

Foxf1/Foxf2 Smad Nkx 3.2/Bapx1 Nkx2.3–2.7 Not identified

Not identified Smad Bapx1 Nkx2.5 Not identified

Not identified Smad bapx1 nkx 2.3–2.7 Not identified

FOXF1 SMAD NKX3.2 NKX 2.3–2.9 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 Xenopus 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 messenger 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 nomenclature). The Drosophila gene biniou and its vertebrate homologues Foxf1 and Foxf2 have emerged as master regulators of VM morphogenesis and differentiation across species.36,37,39 In biniou-null Drosophila embryos, VM development 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 mesenchyme 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 between 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 embryos die at midgestation because of defects in the vascular 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

severe foregut, gallbladder, and lung malformations.38,43 The closely related gene Foxf2 is differentially expressed during mouse GI development, with expression predominating 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 colonic 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 followed by microarray) to identify downstream targets and partners of biniou in Drosophila.39 They showed that although 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 regulatory elements in the absence of biniou.47 Understanding the differences between biniou-dependent and -independent 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 intestine (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 candidates. Further studies to address the differential regional regulation of Foxf1 should elucidate whether the

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development, across species, is that Hh ligands are expressed by the endoderm/epithelium and that Hh receptors and targets are expressed by the mesoderm/mesenchyme, which responds differentially according to developmental 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, homeobox gene products are obvious candidates to integrate these positional cues into regional signals in the

A Nkx2.3-2.9 Nkx3.2 vimar

FoxF1

zampogna

Wnt, FGF, RA

Midgut

Foregut

B

Hindgut

p l

Barx1

Nkx3.2

visceral mesoderm

liver p

Hoxa-5

Hoxa-4

Hoxd-12/13

Foxf1/f2 Nkx2.3 Hlx FoxL

Figure 4. The role of biniou/FoxF1 is conserved across species. Invertebrate example: Drosophila melanogaster. (A) Expression of VM marker fascilin III (Fas III) in stage 12 Drosophila embryos (arrow). Anterior 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

Figure 5. Transcription factors and signaling pathways during anterior-posterior and radial patterning. (A) Early mesoderm formation is under the control of the conserved homeobox genes. Studies in Drosophila have allowed the identification of some early steps in VM formation implicating homeobox genes.180,181 These include tinman (Nkx2.32.9), bagpipe (Nkx3.2), and other genes.181 Tinman is required for mesoderm formation, whereas its downstream target bagpipe promotes VM development.182,183 Vimar, a downstream factor from bagpipe, further specifies VM development.184 In Xenopus, 2 bagpipe homologues 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 mesoderm.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 inhibiting 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 gastrointestinal differentiation, using mouse nomenclature. Not shown is Nkx2.5, which specifies the pylorus in the caudal segment of the foregut. Anterior is to the left, dorsal to the top. Diagram courtesy of Aaron M. Zorn. l, liver; p, pancreas.

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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 Xenopus 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 Although 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 development (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 developing 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. Homeodomain-containing genes belonging to the Nkx2 family (Figure 5A) confer positional information.54 Overexpression 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

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atresias58; Hoxa13 mutations have been described in humans 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 malformation of the intestines of mice, whereas their ectopic expression in chick embryos leads to a homeotic transformation or ectopic expression of region-specific genes. Together with the finding that Hoxa4⫺/⫺ mice have abnormal colonic musculature61 and Hoxa5⫺/⫺ mice have abnormal stomach development,62 these findings indicate that Hox gene products probably participate in regionalization 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 signaling in the VM is necessary for Hox gene induction; subsequent mesenchymal-to-epithelial signaling controls stomach morphogenesis and lung budding,63 and retinoic acid signaling controls endodermal expression of Hox genes such as Pdx1 (pancreatic and duodenal homeobox 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 machinery 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 mesodermal Hox gene products to induce sphincter formation. This is illustrated by Shh⫺/⫺ mice that display homeotic transformation phenotype: intestinal transformation of the stomach, annular pancreas, duodenal stenosis, and an imperforate anus. This homeotic transformation 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 absent 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

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 experiments of proventriculus (anterior) mesenchyme and gizzard (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 contribution differs according to developmental stage.80,81 The divergent homeobox-containing gene Bapx1 or Nkx3.2 regulates mesodermal BMP-4 and Wnt5a expression in the chick stomach75 and pylorus morphogenesis in the mouse82 (Figure 6). In mice, the regionally restricted homeodomain-containing protein Barx1 is required for mesenchymal expression of Wnt inhibitors and normal development of the stomach epithelium.83 In chick, mesodermal BMP-4 independently induces expression 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 formation of the pylorus. These observations raise 2 important 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 perspective, it seems both economical and practical to modify a preexisting program for the development of a new structure with slightly different functional requirements.

Midgut and Hindgut Development Unlike the stomach, the plasticity of the midgut epithelium, in response to changing mesenchymal signals, appears to be somewhat limited.87,88 Using heterotopic cross-associations of endoderm and mesenchyme from different segments of the developing rat intestine, Ratineau et al showed that segments of intestinal mesenchyme 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 expression when cocultured with proximal or distal intestinal mesenchyme.89 Conversely, colon endoderm did respond to the association with jejunal or ileal mesenchyme by developing into small intestinal epithelium.

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Therefore, the hindgut endoderm, like its anterior counterpart, 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 anteroposterior segments of the intestine secrete varying amounts of growth factors, thereby conferring dosedependent regional characteristics to the adjacent endoderm.19 Similar experiments in chicks showed that recombination of proventricular mesenchyme with midgut 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 program 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 promotes the expression of genes that regulate lateral development, 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 transcription 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. Because 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 Foxf1⫹/⫺ Foxf2⫺/⫺

Xenopus Mouse Mouse

VM VM, foregut VM

Hlx⫺/⫺ Nkx2.3⫺/⫺ Hoxa5⫺/⫺

Mouse Mouse, chick Mouse

VM VM midgut and hindgut VM midgut

organization of the GI tract into concentric layers.80 Differentiation of the crypt-villus axis happens at different times in development in different species and progresses in a craniocaudal fashion. In mice, villus formation 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 cryptvillus axis. Crypt-villus axis formation relies largely on mesenchymal-epithelial interactions that determine villus 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 ubiquitous and Foxf proteins are required for VM development in multiple species. However, because of the pleomorphic 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 interdependence of mesenchymal components among themselves and with the adjacent epithelium. Table 2 summarizes 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.

Phenotype Absent lumen, absent smooth muscle actin expression Esophageal atresia, tracheo-esophageal fistula Abnormal muscularis propria, ECM, absent ENS, dilated colon, increased epithelial proliferation Epithelial proliferation, ENS abnormalities, short gut Thin mesenchyme, abnormal villus formation ENS, smooth muscle, ECM

References 36 38, 41 38, 41 195–197 198, 199 62

Signaling Pathways in Radial Patterning Multiple cell fate and positional decisions contribute to the formation of the organized, tubular gut. To this end, transcription factors regulate and integrate numerous 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 characterized 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 patterning 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 formation in both chick and mouse.35,71 Second, depending on developmental window, endodermal Hh signals are important for induction of reciprocal signaling from the mesenchyme back to the endoderm, because loss of function leads to epithelial proliferation.102 Third, epithelial Hh ligands influence ISEMF localization along the cryptvillus 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 mesenchyme 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.

BMPs: smooth muscle development and regulation of the proliferative compartment (crypts). BMPs are

members of the transforming growth factor ␤ superfamily; BMPs 2 and 4 have important roles during GI development: 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 phenotypes 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 signals 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 proliferation 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 anteriorposterior patterning, are also necessary for radial patterning and villus formation. In simple terms, BMP-4 in the VM controls smooth muscle proliferation and differentiation 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 differentiation into smooth muscle106 and inhibition of BMP-4 in the stomach is required for the highly developed musculature to meet its functional needs.106 Absence of BMP-4 from the presumptive hindgut mesoderm 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 noggin 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 mesenchyme 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 compartment.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 proliferation.111,112 The obvious next step in elucidating the relative 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 proliferation 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, because regulation of nuclear ␤-catenin accumulation is required for epithelial homeostasis and carcinogenesis.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 developing and adult intestine contains high levels of Wnt pathway components, both canonical and noncanonical.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 components 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 nascent 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 development and homeostasis, probably with different spatial and temporal functions, acting through both the canonical 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 essential 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 positively 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

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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 epithelial effect of adenomatous polyposis coli (APC) germline mutations by increasing the multiplicity of intestinal adenomas.128 The developing intestine expresses Wnt ligands, receptors, and inhibitors, and their roles vary during development. The control of mesodermal Wnt signals is of particular importance because of the contribution of these factors to epithelial tumorigenesis. Tissue-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 muscle 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 mesenchyme compared with epithelium132 and likely mediates autocrine actions via FGFR1 and R2, as well as having a role in crypt morphogenesis via FGFR3133 (Figure 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 signals.134 Mouse studies indicate that epithelial FGF-9 signaling 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 contribution 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 epithelium.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 Furthermore, although explants of intact perinatal colon do not

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 mesenchyme, but not colonic mesenchyme, enables GC induction of maturation in an associated epithelium. Mesenchymal cells alone respond to GCs; these cells express the GC receptor137 and show increased expression of collagen type IV messenger RNA139 following in vivo administration of GCs.139 –141 In addition, in vitro studies have shown that in response to GCs, mesenchymal cells deposit 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 experiments 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 mesenchyme is responsible for the relatively narrow developmental 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 embryos,132 and absence of the thyroid hormone receptor T3R␣ leads to hypoplastic smooth muscle layers in the developing intestine.145 This finding correlates with increased 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 development. The role for thyroid hormone in mammalian epithelial maturation has been difficult to assess because changes in thyroid status cause concomitant changes in circulating GCs.87 However, thyroid hormone can synergize with GCs in eliciting developmental changes in the epithelium,147,148 but it is not known whether the mesenchyme has a part in this synergy. Xenopus species are characterized by a thyroid hormone– driven metamorphosis 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 attractive 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 intestinal epithelial cells are insensitive to thyroid hormone– induced apoptosis when they are cocultured with ECM molecules,149,150 suggesting that the ECM confers survival 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 embryos 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 maturation of smooth muscle precursors via the Wnt pathway.152 PDGF-A and its receptor PDGFR␣ contribute to formation of the crypt-villus axis.153 They are both expressed 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; compared 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 Therefore, PDGF-A must promote cell proliferation and prevent premature differentiation of the mesenchyme. Again, the role of this molecule depends on the developmental 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 epithelial 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 suggesting 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 colonic 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 manner.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 intestinal length.162,163 Stx2-null adult mice have longer intestines 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 epimorphin 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 mesenchymal 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 muscle development, defects in formation of the posteriormost 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 embryos (during metamorphosis). Matrix metalloproteinases 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 during remodeling.165,166 Although the role of matrix metalloproteinase in mammalian GI development is poorly understood, amphibian models offer insight into potential mechanisms.

Conclusions The VM is a complex tissue that gives rise to the smooth muscle, mesenchyme, and other cells that compose 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 permissive signals to the adjacent developing epithelium; this instructional dominance appears to be mediated by soluble factors and signaling pathways that regulate development of the mesoderm and endoderm. Compared with the foregut and hindgut, the development of the midgut is less well understood; it responds to different mesodermal signals. The developmental plasticity of different GI segments is controversial, which could be the result of the different models and conditions used in experiments. Alterations in mesodermal gene expression 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 proliferation. Studying the molecular basis of GI developmental plasticity and the control of epithelial proliferation 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

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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.

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