| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam; Department of Gastroenterology and Hepatology, Leiden University Medical Center, Leiden, The Netherlands
ABSTRACT I. INTRODUCTION II. THE HEDGEHOG PATHWAY A. Hedgehog Secretion B. Hedgehog Reception C. Hedgehog Intracellular Signaling D. Identifying Hedgehog Target Cells E. Hedgehog Pathway Antibodies, a Cautionary Note III. HEDGEHOG SIGNALING IN THE DEVELOPING GUT A. Patterning of the Developing Gut B. Early Development of the Gut Tube and Left-Right Axis Formation 1. Expression of hedgehogs during gastrulation 2. Hedgehog signaling and left-right axis formation C. Anterior-posterior Axis Patterning of the Gut Tube 1. Instructive signals from the node and mesoderm establish the anterior-posterior axis early in development 2. Instructive signals from the endoderm pattern the gut along the AP axis during later stages of development 3. Endodermal appendage formation 4. Hedgehog signaling and endodermal appendage formation A) AIRWAYS. B) THYROID. C) PANCREAS. D) LIVER. D. Endodermal Cytodifferentiation and Radial Patterning of the Gut Tube 1. Esophageal development and the role of Hedgehog signaling 2. Gastric development 3. Hedgehog signaling in gastric development 4. Intestinal development 5. Hedgehog signaling in intestinal development 6. Hedgehog signaling in anorectal development 7. Hedgehog signaling in pancreas development 8. Hedgehog signaling and the VACTERL association IV. HEDGEHOG SIGNALING AND HOMEOSTASIS OF THE ADULT GUT A. Morphogenesis Versus Morphostasis B. Stomach C. Small Intestine D. Colon V. HEDGEHOG SIGNALING AND GASTROINTESTINAL CARCINOGENESIS A. Evidence for a Role for Hedgehog Signaling in Initiation of Gastrointestinal Carcinogenesis? B. Hedgehog Signaling in Cancers of the Proximal Gastrointestinal Tract C. Hedgehog Signaling in Colorectal Cancer D. Therapeutic Targeting of the Hedgehog Pathway VI. CONCLUSION GRANTS ACKNOWLEDGMENTS REFERENCES
 |
ABSTRACT |
|---|
 Top NextReferences |
|---|
|
I. INTRODUCTION |
|---|
|
TopPreviousNextReferences |
|---|
|
, and receptor tyrosine kinase pathways. The ligands for receptor tyrosine kinase receptors include, for example, the fibroblast growth factors (FGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF).
|
|
II. THE HEDGEHOG PATHWAY |
|---|
|
TopPreviousNextReferences |
|---|
After the identification of the Hedgehog mutant in 1980, the Drosophila hedgehog gene was published in 1992 by three independent groups (112, 137, 183). Three murine hedgehog homologs were described a year later (40). They were named sonic hedgehog (Shh), after a popular Sega computer game character, desert hedgehog (Dhh), after an Egyptian species of hedgehog (Hemiechinus auritus), and indian hedgehog (Ihh), a hedgehog species endemic in Pakistan (Hemiechinus micropus). These three hedgehog genes are highly conserved between mouse and humans (130). Unfortunately, very little is known about the factors that control the transcriptional regulation of the three different hedgehog genes, particularly in the gut.
Hedgehog proteins are extensively processed posttranslationally. Hedgehogs are produced as a 
45-kDa precursor protein that is cleaved autocatalytically, yielding a 19-kDa NH2-terminal fragment that contains all the signaling functions and a 26-kDa COOH-terminal fragment that catalyzes the cleavage and acts as a cholesterol transferase (Fig. 3) (16, 111, 156, 157). Although morphogens act in a graded manner through tissues, this is mostly not through simple diffusion of a soluble protein (127). In fact, an important feature of Hedgehog proteins is that the mature NH2-terminal fragments are dually lipid modified and therefore poorly soluble. The signaling protein is covalently linked to a palmitate and a cholesterol group (127). These hydrophobic modifications integrate Hedgehog protein in the cell membrane and play an important role in the regulation of the range of Hedgehog signaling in a tissue. Conflicting reports have been published on the role of the cholesterol moiety in Hedgehog signaling (113, 115). The palmitoylation is essential to both the activity of the Hedgehog protein and the signaling range (25). Work from several laboratories has shown that long-range signaling occurs by multimers of NH2-terminal Hedgehog protein that require both cholesterol and palmitate modification for their formation (25, 212). Release of lipid-modified Hedgehog protein from the Hedgehog-producing cell requires Dispatched (Disp), a 12-pass transmembrane protein with a sterol-sensing domain. Disp1–/– mutant mice have a phenotype similar to mice that lack the Hedgehog signaling receptor Smoothened (Smo), suggesting that Disp1 is required for all Hedgehog signaling activity (20, 94, 121). A study by Panáková et al. (149) in Drosophila suggested that Hedgehog multimers may form by association of the lipid moieties with the outer phospholipid layer of lipoprotein particles and that this association is required for Hedgehog signaling activity. This is a very interesting finding that may have important implications for vertebrate Hedgehog signaling (see, for example, a recent comment on the possible implications for atherosclerotic plaques, Ref. 11). Thus Hedgehog proteins are not soluble signals but dually lipid modified, and their spread through tissues is only partially understood.
|
The Hedgehog signal is transmitted by a seven-span transmembrane receptor Smoothened (Smo). Interestingly, Hedgehogs do not bind to Smo but control the activity of Smo indirectly by binding to a second receptor, Patched (Ptc). Two Ptc genes exist in vertebrates, Ptc1 (51) and Ptc2 (139). The Ptc genes encode for 12-span transmembrane receptors with two large hydrophilic extracellular loops that mediate Hedgehog binding. In the absence of Hedgehog, Ptc inhibits signaling by Smo (Fig. 4). Ptc1 is a transcriptional target of Hedgehog signaling and acts in a negative-feedback loop to restrict the range of Hedgehog signaling in a tissue. The mechanism of the inhibitory action of Ptc on Smo is only partially understood. Ptc acts catalytically (184) and seems to control the localization of a secondary Smo-inhibiting molecule. The nature of this inhibitory molecule is a matter of active investigation (12, 39). Even though Ptc may act as a pump that controls the localization of a secondary Smo antagonist, it has been shown that Ptc most likely acts in a cell autonomous fashion (on the cell expressing Ptc and not on surrounding cells) at least in Drosophila and the chick neural tube (14). This suggests that Ptc may pump this molecule from the outside to the inside of the cell or from one cellular compartment to another. The inhibitory action of Ptc is relieved upon binding of Hedgehog to Ptc.
|
|
C. Hedgehog Intracellular Signaling
The analysis of Hedgehog signaling downstream from Smo (Fig. 5) is considerably complicated by the relative lack of conservation between Drosophila and vertebrates (71, 147). Although the intracellular transduction of the Hedgehog signal is relatively well understood in Drosophila, understanding of vertebrate signal transduction is far from complete; extensive discussion of the mechanism of Hedgehog signal transduction is therefore beyond the scope of this review. In Drosophila, the intracellular transduction of the Hedgehog signal depends on the processing of downstream transcription factor Ci. Ci exists in a full-length activating form and truncated repressor form. In the absence of Hedgehog, Ci is processed to its repressor form by a complex of four kinases [protein kinase A (PKA), casein kinase 1 (CK1), glycogen synthase kinase 3 (GSK3), and Fused kinase (Fu)] held together by Cos2. In the presence of Hedgehog, Cos2 is recruited by Smo and Ci processing terminated resulting in the stabilization of full-length Ci and transcriptional activation of Hedgehog target genes (for review, see for example Refs. 67, 71, 83, 147). This function of Cos2 as an "on/off" switch does not seem to be conserved in vertebrates. Convincing evidence for a vertebrate Cos2 homolog is currently not available (200). Important evidence has been presented that shows that Suppressor of fused Su(Fu) may function as a central intracellular on/off switch in vertebrates (200). Indeed, the Hedgehog pathway is completely on in Su(Fu)–/– mice (28, 182), which strongly resembled the Ptc1 null phenotype (182). Although Drosophila does have a Su(Fu) gene, it does not seem to play a similar role as Su(Fu) mutant Drosophila are healthy and fertile (159).
The Hedgehog transcription factors are conserved end points in intracellular Hedgehog signal transduction. All aspects of Hedgehog signaling are mediated via the vertebrate homologs of Drosophila Ci, the Glioblastoma (Gli) transcription factors Gli1, Gli2, and Gli3 (74). As in Drosophila, the activity of these transcription factors is regulated in a ligand-dependent manner. Gli2 and Gli3 are the major Glis to transduce the Hedgehog signal in the gut (see below). Both Gli2 and Gli3 can also act as a repressor of Hedgehog target expression in vivo (116, 148, 203). This is related to the fact that Gli2 and Gli3 (and not Gli1) can be processed to a repressor form similar to the Drosophila homolog Ci. However, processing of Gli3 to its repressor form is much more efficient than that of Gli2 (148). As with Ci, the Gli3 repressor form is formed by a partial proteolytic processing reaction that requires priming phosphorylation by PKA and subsequent phosphorylation by CK1 and GSK3 (188, 203). Tempe et al. (188) have shown that this phosphorylation can result in ubiquitination and partial proteosomal degradation that generates a truncated version of Gli3 that lacks the transcriptional activation domain and acts as a dominant negative Gli (188). This negative regulatory action of PKA, CK1, and GSK3 (Drosophila homolog Shaggy) on Hedgehog signaling through the generation of a suppressor form of Ci/Gli protein is conserved from Drosophila to vertebrates. Gli2 processing and degradation and Gli1 degradation are regulated in a similar manner as that of Gli3 (9, 129, 148, 188).
D. Identifying Hedgehog Target Cells
In the analysis of the role of Hedgehog signaling in any organ, it is essential to identify Hedgehog receiving cells. Although a number of different target genes have been identified, their regulation often differs in time or per organ. Ptc1 and Gli1 are two target genes that seem to have been conserved particularly well throughout vertebrate evolution, and their expression pattern is the best reflection of Hedgehog signaling activity in most if not all situations in vertebrates. A difference may exist for the sensitivity of the expression of Ptc1 and Gli1 for the range of the Hedgehog signal in the developing vertebrate gut. In the developing stomach and colon, both Shh and Ihh are expressed in the epithelium at E18.5. Ptc1 is expressed at high levels in a small zone very close to the Hedgehog expressing cells, whereas expression of Gli1 is also very intense in the smooth muscle layer at much greater distance (164). It is, therefore, probably safest to use both Ptc1 and Gli1 as readouts for Hedgehog pathway activity and realize that these targets may have some level of independent regulation. As Ptc1 is the receptor for Hedgehog and required to initiate Hedgehog signaling, it is to be expected that low levels of Ptc1 may be expressed in tissues in a Hedgehog-independent manner.
E. Hedgehog Pathway Antibodies, a Cautionary Note
The analysis of the role of Hedgehog signaling in development and the adult critically depends on the reliable detection of components of the Hedgehog pathway such as Shh, Ihh, Dhh, Smo, Ptc, and transcription factors Gli1–3. A large variety of commercial antibodies is currently available (mainly from Santa Cruz). With many if not most of these antibodies, we have not been able to obtain credible results with western blot or immunohistochemistry, especially when compared with in situ hybridization. In our hands the specificity of the available commercial antibodies may even vary between batches. Some of the antibodies that gave staining patterns that compared well with in situ hybridization in our hands still give variable results in the literature. For example, using the I-19 antibody against Ihh from Santa Cruz, Varnat et al. (202) observed staining at the base of the crypts, whereas Jones et al. (80) describe Ihh expression mainly at the tips of the villi using the same antibody. Since Ihh mRNA is mainly expressed at the crypt villus junction with a diminishing gradient towards the top of the villus, the staining pattern observed by Jones et al. (80) probably best reflects the expression of Ihh protein. It is furthermore important to realize that many anti-Hedgehog antibodies are produced against the extremely conserved NH2-terminal 19-kDa protein. This results in easy cross-reactivity of antibodies for different Hedgehog proteins. This may explain, for example, why Jones et al. (80) detect abundant staining on villi of the small intestine with an anti-Shh antibody, whereas Shh expression is very low and restricted to a few cells in the small intestinal crypts as detected by in situ hybridization (6, 198), making it very unlikely that abundant Shh protein can be detected in the enterocytes on the villi. Most likely this is explained by cross-reactivity of the anti-Shh antibody for Ihh protein. Additionally, using the Santa Cruz C-20 antibody against Ptc1, we have previously reported epithelial staining in the colon of mouse, rat, and human (197). We have now performed in situ hybridizations for Ptc1 in the mouse colon and found that although Ptc1 mRNA is expressed in the epithelium of the anorectum, no Ptc1 mRNA can be detected in the epithelium of the mouse colon using two different Ptc1 probes. Instead, Ptc1 localizes to the mesenchyme just underlying the differentiated colonic enterocytes (unpublished observations). Hopefully more reliable anti-Hedgehog pathway antibodies will be available soon. Of course, it should be realized that Hedgehogs are proteins that can travel great distances through tissues and that a perfect correlation between Hedgehog mRNA and protein may not always be present. However, given the poor performance of many Hedgehog pathway antibodies, it is important to compare results obtained with immunohistochemistry with those obtained by in situ hybridization until better antibodies become available.
|
III. HEDGEHOG SIGNALING IN THE DEVELOPING GUT |
|---|
|
TopPreviousNextReferences |
|---|
The gastrointestinal tract forms from two germ layers, the endoderm and mesoderm, and is innervated by cells derived from the third germ layer, the ectoderm. During development the gut evolves from a simple tube that is morphologically homogeneous to a highly complex organ that has distinct functional domains along the anterior-posterior and vertical (cross-sectional) axis and develops multiple accessory organs. Three different regions are normally recognized. The foregut forms the esophagus, stomach, proximal duodenum, thymus, thyroid, airways, pancreas, and liver. The midgut will form most of the intestine distal from the common bile duct that derives its blood supply from the superior mesenteric artery: the distal duodenum, the jejunum, ileum, cecum, and the ascending and proximal transverse colon. The hindgut forms the distal transverse colon, descending colon, sigmoid, and anorectum that develop around the inferior mesentery artery. The patterning mechanisms along the vertical, horizontal, and left-right axes and induction of growth of accessory organs all involve cross-talk between cells of the different germ layers. Such interactions between different groups of cells in which a signal is emitted from one group of cells to change the nature or behavior of the receiving cells are termed inductive signaling. Two types of inductive signaling are generally recognized. In instructive interaction, the signal is necessary to specify the fate of the receiving cell. In permissive interaction, the signal simply allows the expression of a phenotype that is already intrinsic to the receiving cell. Much of this instructive communication between germ layers is through the action of morphogens among which are members of the Hedgehog signaling pathway. Below I first review some of our understanding of the development of the gut and the role of germ layer interactions, focusing on mouse development. I then discuss the available data that show a role for Hedgehog signaling in gut development.
B. Early Development of the Gut Tube and Left-Right Axis Formation
In all animals the primitive gut or archenteron is formed during gastrulation. The term gastrulation is derived from the Greek word "gaster" and means "to form a stomach." At this early point in development, the mouse embryo is a cup-shaped epithelial layer termed epiblast that is enveloped by the visceral endoderm (Fig. 6). The epiblast will form three germ layers during gastrulation: definitive (gut) endoderm, mesoderm, and ectoderm. The definitive endoderm gives rise to the epithelium of the gastrointestinal tract, thymus, thyroid, and respiratory tract; the mesoderm will form the cardiovascular system, muscles, blood, and bone; the ectoderm forms the epithelium of the skin and the central nervous system. Gastrulation starts at embryonic day (E) 6.25 with the formation of the primitive streak, a thickening on the posterior margin of the epiblast (and future posterior end of the embryo) that forms by epithelial reorganization and is elongated anteriorly over the epiblast along the midline of the embryo (176). Epiblast cells undergo a process of epithelial to mesenchymal transition (EMT) at the primitive streak and ingress through the primitive streak and node (Fig. 6). The embryonic endodermal cells that have undergone EMT are inserted in the visceral endoderm, gradually displacing it. Other cells that have undergone EMT migrate in between the endoderm and epiblast (now the primitive ectoderm) forming the mesoderm (176). At the anterior end of the primitive streak, a small group of cells forms a notch that is termed the node. Cells of the node regulate left-right axis formation of the embryo and are therefore essential for left-right axis formation of the gut and its derived organs such as looping of the gut tube and correct positioning of the pancreas and liver (65). The node has a central depression termed the pit, which is populated by ciliated cells that generate a leftward flow of extraembryonic fluid. This leftward flow is the earliest recognized left-right symmetry breaking event during murine embryogenesis (65) and is essential for the establishment of asymmetric mesodermal expression of genes involved in a program that specifies the left side of the mouse embryo. Nodal flow may act by causing asymmetric distribution of a soluble signal to the left of the developing embryo (142, 144). A "left-side program" is subsequently activated in the left lateral plate mesoderm, which contributes to the mesoderm of the gut and has been shown to play an essential role in gut looping in zebrafish (68).
|
|
Shh expression is first detected at E7.25 in the midline mesoderm of the head process (40). At E7.75, Shh expression is initiated in the node, the notochord, and the definitive endoderm (40, 215). Endodermal expression of Shh is initiated in the anterior endoderm, and its expression expands to the posterior endoderm (40). Ihh is expressed in the posterior node and the visceral endoderm at E7.75 (46, 215). Ihh expression in the definitive endoderm is initiated at later stages of development (13).
2. Hedgehog signaling and left-right axis formation
Hedgehog signaling is dispensable for early gastrulation in the mouse as all three germ layers are normally formed in mice that lack Smo and therefore lack all Hedgehog signaling (215). At a later stage of gastrulation, Hedgehog signaling is involved in gut tube formation as both Smo and Shh/Ihh compound mutant mice (215) fail to close the midgut. This defect may be related to that fact that they do not undergo the embryonic turning process, something that will impair the normal endodermal folding movement that results in gut tube closure. Several different mice with mutations in the Hedgehog pathway display defective left-right axis formation. Smo mutant and Shh/Ihh compound mutant mice do not initiate cardiac looping (lung or gut phenotype cannot be examined in Smo mutant or Shh/Ihh compound mutant mice as they do not survive beyond E9.5) (215). Shh mutant mice have a less severe cardiac phenotype with delayed and incomplete looping and fail to develop asymmetrically lobed lungs (193, 215). Both Ihh and Shh mutant mice display gut malrotation (164). These data indicate that Shh and Ihh play redundant roles in left-right axis formation, consistent with the expression of both Shh and Ihh in the node. Smo and Shh/Ihh mutant mice fail to activate the genetic program necessary for the specification of the left side of the body. Both mutants lack expression of nodal in the left lateral plate mesoderm, a critical component of the left-side specific gene program (215). As the Hedgehog target gene Ptc1 is symmetrically expressed in the node, it is most likely that the effect of Hedgehog signaling on left-right axis formation is indirect. For example, Hedgehog signaling is important in the specification of the midline, which has been proposed to play an important role in the stabilization of left-right specific gene expression by functioning as a barrier that prevents the bilateral diffusion of long-range asymmetric signals (for review, see Capdevila et al., Ref. 18).
A possible more direct role for Hedgehog signaling in left-right axis formation has been proposed by Tanaka et al. (185). It has been shown that nodal flow initiates asymmetric Ca2+ signaling at the left border of the node (133). Tanaka and colleagues found that this Ca2+ signal depends on FGF signaling. When the authors labeled membrane lipids with a fluorescent dye, they made the interesting observation that nodal cells released small (0.3–5 µm) membrane particles (that they termed "nodal vesicular parcels" or NVPs) in an FGF signaling-dependent manner. The NVPs were transported to the left by nodal flow and seemed to contain Hedgehog protein. Recombinant Shh could reverse inhibition of asymmetric Ca2+ signaling by an FGF inhibitor (185). The symmetrical expression pattern of Hedgehog receptor and transcriptional target Ptc1 in the node seems to contradict the possibility that Hedgehog containing NVPs induces asymmetric Hedgehog signaling in the node. However, the authors of the node work suggest that Ptc1 may be a short-range Hedgehog signaling target (at least in the fruitfly imaginal disks) and that short-range and long-range Hedgehog signaling may have alternative mechanisms of action (65). The authors suggest that the NVPs may be important in the long-range signaling of Hedgehogs in the node and may be important for the asymmetric induction of Ca2+ signaling. Although this remains a possibility, this suggestion is highly speculative and awaits further experimental evidence.
In conclusion, Hedgehog signaling plays an important role in left-right axis specification of the gut and other organs in the body. To date, the most important evidence is for a role for Hedgehog signaling in midline specification, which functions as a barrier to restrict diffusion of asymmetric signals to one side of the developing embryo. An alternative more direct role for Hedgehog signaling remains a possibility but awaits further evidence.
C. Anterior-posterior Axis Patterning of the Gut Tube
1. Instructive signals from the node and mesoderm establish the anterior-posterior axis early in development
After gastrulation, the endodermal layer is still a histologically uniform pseudostratified layer of cuboidal epithelial cells that is surrounded by a thin layer of mesodermal cells. Despite its morphological uniformity, the endodermal layer is already patterned along the anterior-posterior (AP) axis at this point in development (53, 206). Lawson and Pedersen (108) microinjected single endodermal cells of E6.7 early primitive streak stage mouse embryos with horseradish peroxidase and traced them for up to 48 h in culture. Their experiment showed that the first endodermal cells to exit the primitive streak formed the anterior-most endoderm, whereas cells that were still in transit through the primitive streak formed more posterior endoderm (108). The earliest AP patterning of endodermal cells may be related to this timing of the exit of the primitive streak. For example, the endodermal cells that were first to exit the primitive streak and have moved most anteriorly express the homeobox gene Hhex (or Hex) (192), whereas later endodermal cells express FoxA2 (or Hnf3), and the last endodermal cells to exit the primitive streak and thus form the posterior endoderm express Cdx2 (7). Hereafter further AP patterning occurs through instructive and permissive interactions between the endoderm and mesoderm. The major instructive interactions in early intestinal development seem to be from the mesoderm to the endoderm. Unfortunately, few studies exist that examined the role of endodermal mesodermal interactions in the mouse during early gut development. In a recent study that examined the role of germ layer interactions in patterning of the early (E7.5–E7.75) mouse endoderm, the endoderm survived for at least 2 days when it was cultured in vitro in the absence of mesoderm and maintained the region-specific expression of two early posterior endodermal markers the anterior marker -cardiac actin (bCa) and the posterior marker intestinal fatty acid binding protein (iFabp) gene (205). In this elegant study, the expression of several regulators and markers of endodermal differentiation was only induced when the endoderm was cultured with the other two germ layers. The effect was mediated by soluble factors as it also occurred in the presence of a membrane that separated the endoderm from the mesoderm/ectoderm. These results might still simply indicate that endoderm arrests in development when cultured without trophic factors derived from the other germ layers and that the effect would therefore be an example of permissive induction. However, evidence for an instructive role for the mesoderm/ectoderm was provided in further experiments. When anterior endoderm was cultured with posterior mesoderm/ectoderm, the endoderm expressed the posterior marker iFabp. When posterior endoderm was cultured with anterior mesoderm/ectoderm it expressed anterior marker bCa. These data indicate that AP patterning of the early mouse endoderm occurs through both timing of the exit of the primitive streak and instructive action of soluble factors derived from the other two germ layers. It has not been examined if Hedgehog signaling is involved in these early AP patterning events.
2. Instructive signals from the endoderm pattern the gut along the AP axis during later stages of development
The endodermal layer holds the information necessary for its differentiation along the AP axis at later stages in development in both mice (177) and rats (38). This indicates that the mesenchyme mainly has a permissive role in endodermal development from this point onward. For example, when undifferentiated E14 rat intestinal endoderm is recombined with mesenchyme from different regions of the fetal small intestine, the endoderm seems to determine the region-specific expression of brush-border enzymes, and when E14 rat gastric or lung endoderm was recombined with small intestinal mesenchyme, the endoderm developed according to its place of origin (38). The only exception to the endodermal autonomic region-specific development was in the prospective colonic endoderm. The colonic endoderm expressed small intestinal enzymes when combined with small intestinal mesenchyme, indicating that cell fate of the colonic endoderm remains somewhat plastic at E14 (38). It was even shown that normal intestinal endodermal development is possible when E14 rat endoderm is recombined with fetal skin fibroblasts, with the fibroblasts developing into a smooth muscle layer (instead of skeletal muscle) that was 
-smooth muscle positive (38, 95). This indicates that the mesenchyme not only plays mainly a permissive role at later stages of endoderm development but that signals from the endoderm play an instructive role in the development of the mesoderm at this stage. Because Hedgehogs are expressed in the endoderm and signal to the mesenchyme, this may implicate the pathway in the instructive signaling to the mesenchyme at this stage.
3. Endodermal appendage formation
One of the most notable aspects of AP axis formation of the gut is the formation of endodermal appendages (Fig. 8). During and right after the turning of the embryo between E8.5 and E9.5, a series of appendages originate from the gut tube by a process of budding and elongation. Buds are formed that generate the thymus (E9.5–E10.5) (126), thyroid (E8.5) (31), airways (E9.0–E9.5) (117), liver (E8.5) (82), and pancreas (E9.5) (100). The specification of the endodermal zone destined for appendage formation and the subsequent morphogenetic process of budding and elongation seems dependent on instructive mesodermal signals. For example, at E8.25–E8.5 signals derived from the cardiac mesoderm play an essential role in the development of the liver and airways from a region of the ventral foregut endoderm that lies in close apposition to the cardiac mesoderm (56, 81, 174). The notochord, a mesodermal structure that is formed during gastrulation and ensures structural support along the axis of the growing embryo, is in close contact with the dorsal endoderm until E9.0 and specifies the dorsal pancreatic domain in the chick embryo (61, 102). Inductive signals from endothelial cells are involved in the specification of the thyroid and both pancreatic buds (see below).
|
A) AIRWAYS. After the formation of a central ventral tracheal bud and two adjacent ventrolateral lung buds at E9.5 (19), the trachea and lungs separate from the esophagus by a process of septation and elongation. Shh is expressed at high levels in the tracheal bud (117). Signaling is from the endoderm to the mesoderm as the Gli transcription factors are exclusively expressed in the foregut mesoderm (74). One of the roles of Hedgehog signaling to the mesoderm is in the correct specification of the mesodermal septum that separates the esophagus and trachea. In Shh–/– mice, tracheal budding is delayed and the elongating trachea is hypoplastic and fails to separate correctly from the gut tube. The proximal esophagus and trachea are therefore poorly separated (117, 153). The lungs of Shh–/– mice are severely hypoplastic and do not lobulate, and the airways fail to branch and recruit proliferating mesenchymal cells, suggesting a role for Hedgehog signaling in branching morphogenesis (117, 153). Gli transcription factors play a redundant role in airway budding, elongation, and branching. Gli1 mutant mice are healthy and do not seem to have a phenotype (150). Gli2–/– mice recapitulate many of the aspects of the phenotype of the Shh–/– mice with hypoplastic trachea and lungs and failure to induce mesenchymal proliferation (138). The Gli3–/– mouse shows normal airway development but loss of one copy of the Gli3 gene in Gli2–/– Gli3+/– mice aggravates the airway phenotype and is indiscernible from the Shh–/– mouse. The phenotype of mice completely deficient for both Gli2 and Gli3 is more severe than that of Shh–/– mice as in these mice there is no development of the trachea and lungs at all (138). This may suggest a role for another Hedgehog in airway budding and outgrowth. Expression of Ihh in the foregut has not been reported, but low expression levels may have been missed.
A major target and effector of Hedgehog signaling in airway development is transcription factor Foxf1, which is expressed in the mesenchyme (124). Expression of Foxf1can be induced by ectopic expression of Shh, and Shh–/– mice lack Foxf1 expression in the foregut mesenchyme (124). Foxf1–/– mice degenerate before endodermal budding begins (125), but Foxf1+/– mice do have an airway phenotype with a failure of the elongating trachea to separate completely from the esophagus and reduced branching morphogenesis similar to the Hedgehog pathway mutant mice (124). Thus Hedgehog signaling from the endoderm to the mesoderm is essential for the formation of the tracheal bud and subsequent branching morphogenesis of the airways and plays a critical role in the stimulation of growth of the recruited mesenchyme.
B) THYROID. The adult thyroid gland is located in the cervical region anterior to the trachea in humans and mice. The bulk of the gland is made up of follicles consisting of thyroid follicular cells that store and release thyroid hormone. C cells produce calcitonin and are found in the interfollicular space. Both cell types have a different embryonic origin as the follicular cells are endoderm derived whereas C cells are derived from the neural crest (31). Thyroid morphogenesis is initiated at E8.5 when the thyroid primordium buds of the ventral wall of the pharyngeal foregut endoderm that lies in close contact with the aortic sac endothelium (2, 43). At E9.5, the thyroid bud separates from the gut tube while it remains connected by a thin thyroglossal duct and remains in contact with the aortic arch. The thyroglossal duct disappears and the thyroid becomes completely separated from the gut by E11.5. At E13.5, the thyroid is at its final position in the midline at the ventral side of the trachea. It now bifurcates to form two lobes that develop in close contact with the carotid arteries (2). At E15.5, the thyroid organizes into a follicular structure, and genes involved in thyroid hormone production are induced (31). Similar to the specification of the pancreatic buds (see below) and in contrast to specification of the airways, expression of Shh is excluded from the thyroid primordium (44). In the absence of Hedgehog signaling, the thyroid bud is therefore correctly specified. Although no Shh is expressed in the thyroid at any point in development, the thyroid does not separate into two distinct lobes in Shh–/– mice but becomes a single unilateral mass, the same size of a single thyroid lobe in control mice (44). The single thyroid gland of Shh null mice formed normal follicles that contained apparently normally differentiating thyrocytes. In an elegant study by Alt et al. (2), it was shown that the thyroid phenotype is indirect, in accordance with the lack of Shh expression in the thyroid. The authors showed that thyroid growth is directed towards endothelial cells in the zebrafish. Subsequently, the authors examined a mouse that lacks Shh expression at early stages of development and demonstrated that the aortic arch fails to cross the midline and both carotid arteries develop on one side of the esophagus. They found that the single thyroid lobe in these mice is always on the same side as and in close contact with the mislocated carotid arteries. The fact that Shh expression is specifically excluded from the thyroid primordium suggests that downregulation of Shh expression may be an important step in its specification similar to the specification of the pancreatic domain (see below). This idea is supported by the finding of small foci of ectopic thyroid tissue in the tracheal tube at 15.5–17.5 days post coitum in the Shh–/– mouse (44), suggesting that Shh acts to suppress thyroid cell fate in the trachea and its loss may be critical to thyroid primordium formation. Another similarity between the development of the thyroid and pancreas is the important role of the endothelium in budding (see below), suggesting that a signal derived from endothelial cells may be involved in the restriction of Shh expression from the thyroid and pancreatic buds.
In conclusion, Shh suppresses thyroid cell fate specification in the trachea, and its expression is excluded from the thyroid primordium, possibly by signals from the endothelium of the aortic sac. Although the Shh–/– mouse has a thyroid phenotype, this phenotype is indirect and the result of defective patterning of the cervical vasculature.
C) PANCREAS.
The pancreas is a gland with both endocrine and exocrine functions. Most pancreatic tissue (95–99%) is composed of exocrine acinar cells that produce digestive enzymes and pancreatic ducts that are lined by duct cells. The rest of the pancreas consists of small clusters of endocrine cells or islets of Langerhans. Each cluster of endocrine cells has a core of insulin-producing -cells surrounded by glucagon-producing -cells, somatostatin-producing 
-cells, and pancreatic-polypeptide cells.
The pancreas develops from two distinct domains of ventral and dorsal foregut endoderm. The dorsal endodermal domain involved in the formation of the pancreas is specified by signals from the notochord, which remains in close contact to the dorsal endoderm until E8.5 and induces the expression of transcription factor Pdx1 (61, 102). The ventral pancreatic domain is specified at the lip of the mouse ventral foregut endoderm (Fig. 9). This endodermal region adopts a pancreatic cell fate around the six- to eight-somite stage (E8) just before the turning of the embryo (34). The endoderm that lies slightly more cranial is closely apposed to the cardiac mesoderm which secretes FGFs to suppress a pancreatic and induce a hepatic cell fate in this endodermal region (34, 81). The ventral and dorsal pancreatic buds develop between E8.5 and E9.5 exactly where the endoderm interacts with the aorta dorsally and the two vitelline veins ventrally (106, 107). It was shown by a variety of different experimental approaches that this endodermal contact with the endothelium is both necessary and sufficient to determine the expression domains of Pdx1 and insulin (106).
|
-actin positive smooth muscle cells and c-kit positive cells of Cajal. Both cell types are normally exclusively found in intestinal mesenchyme. The pancreatic buds in Pdx1-Shh mice did contain endocrine and acinar cells, but these failed to cluster into islets and acinar structures and produced mucins that are typical for intestinal epithelial cells (4). Thus suppression of Shh signaling during early pancreatic development is essential for normal development of both the pancreatic epithelium and mesoderm. Data from Xenopus further support this concept (213). Xenopus injected with a constitutive active form of the Hedgehog receptor Smo completely lack a pancreas, a phenotype more severe than that of the Pdx1-Shh mouse. This may be due to the fact that the transgenic mouse expressed Shh behind a pancreas specific promoter and that the ectopic Shh transcription therefore starts after the pancreatic domain has already been specified. Similar to the role of Shh in suppressing thyroid cell fate in the trachea, expression of Shh in the endoderm surrounding the pancreatic primordium may be necessary to suppress pancreatic cell fate in the duodenum and stomach. Treatment of chick embryos with the Hedgehog inhibitor cyclopamine in ovo resulted in ectopic pancreatic tissue formation in the duodenum and stomach (103). Cyclopamine acts at the level of Smo and therefore inhibits the activity of all Hedgehogs (29, 75). No pancreatic differentiation was observed in the stomach or intestine of Shh null mice (62), but this may be related to the fact that Shh and Ihh have overlapping functions in this domain (13, 164). The role of Hedgehog signaling in pancreatic development in zebrafish seems to diverge from that in the mouse in some aspects. Evidence has been presented that in contrast to the mouse, Hedgehog signaling is required for the early stages of pancreas development in the zebrafish (35, 170). As in the mouse, Shh expression is excluded from the pancreatic anlage in the zebrafish (170), but injection of Shh increases the number of pancreatic precursor cells (170) whereas Shh and Smo mutant zebrafish show diminished numbers of pancreatic precursor cells (35, 170). Experiments by DiIorio et al. (35) suggest that this role of Hedgehog signaling in early specification is mediated during gastrulation. Thus Hedgehog signaling differentially regulates early pancreatic development in the mouse and zebrafish. Hedgehog signaling inhibits early pancreatic development in the mouse, and the exclusion of Shh and Ihh expression from the pancreatic primordia is critical to normal murine pancreas development.
D) LIVER. Shh is expressed in the liver primordium in the ventral foregut endoderm (34). It is not clear if there is a role for Hedgehog signaling in liver budding and development as liver development seems to progress normally in both Hedgehog and Gli mutant mice (117, 138) and, for example, in Xenopus injected with a constitutively active form of Smo (213).
In conclusion, instructive mesenchymal signals play an essential role in the establishment of the AP axis during early gut development. This is most evident from the role of mesenchymal factors in the appropriate formation of endodermal appendages. See Table 1 for a summary of phenotypes in Hedgehog pathway mutant mice. Much of this instructive signaling seems to act by modulating the expression of Hedgehogs in the endoderm, which subsequently acts on the mesenchyme in a reciprocal manner.
|
Endodermal cytodifferentiation is complete at birth in humans. It is important to realize that in contrast to humans, the epithelium is still immature at birth in mice and rats and that its development continues in the first four postnatal weeks. Here I discuss the changes that occur in the first three postnatal weeks as part of the developing gastrointestinal tract and consider the gastrointestinal tract as "adult" after completion of postnatal development. The gut tube phenotypes of Hedgehog pathway mutant mice are summarized in Table 2.
|
Differentiation of the esophageal endodermal layer is poorly studied in the mouse. A morphological study by Raymond et al. (165) suggests that differentiation initiates around E15. At E17, the epithelium is composed of three to four cell layers and contains both squamous and ciliated cells. Only cells in the basal most epithelial layer proliferate, and thus a division in a basal compartment of precursor cells and superficial compartment of differentiating cells has been established along the vertical axis at this point in time. The ciliated cells rapidly disappear postnatally and are absent 1 wk after birth. The mechanism of this loss of ciliated cells seems to be through desquamation (134). In contrast to the human esophagus, which is not keratinized, murine esophageal epithelium keratinizes 1 mo after birth (37).
Hedgehog signaling plays a critical role in the normal development of the esophagus (Fig. 10). Shh is initially expressed throughout the developing esophagus but restricted to the distal esophagus at later stages (117, 164). The Gli genes are mesodermally expressed, indicating that Hedgehog signaling is from the endoderm to the mesoderm (74). At E17.5, the proximal esophagus of Shh–/– mice is hypoplastic and fails to separate completely from the developing trachea; more distally there is no discernible remaining esophagus in Shh–/– mice at this point in development (117). Similar to the trachea, the developing lungs [which originate from two lung buds on each side of the tracheal bud (19)] fail to separate correctly from the gut and are still connected to the gut tube at E17.5. The importance of Hedgehog signaling to normal esophageal development was confirmed in Gli mutant mice. In the Gli2–/– mouse, the esophagus has a very small lumen and little mesenchymal cells and fails to develop a smooth muscle layer. In Gli2–/– Gli3+/– mutant mice, most of the proximal esophagus is degraded, and only a very small proximal esophageal remnant can be observed in the carefully made whole-mount preparations shown by Motoyama et al. (138). A single tube connects the trachea and lungs to the stomach in Gli2–/– Gli3+/–. Although this has not been carefully examined, this is most likely the remnant distal esophagus similar to human with esophageal atresia and tracheoesophageal fistula (Fig. 10). In Gli2–/– Gli3–/– mice, the foregut was hypoplastic and failed to form the appendages for the trachea and lungs at E9.5. Gli2–/– Gli3–/– embryos that survived until E14.5 had a very small remaining proximal endodermal tube and lacked both trachea and lungs. The phenotype of these Hedgehog pathway mutant mice indicates that Hedgehog signaling is critical to the maintenance of the developing esophagus and the formation of a tracheal-esophageal septum. Both phenotypes seem to result from a block in the mesenchymal growth that is necessary to support the developing esophagus and separate the gut tube from the airways. This is therefore a nice example of the critical role of endodermal-derived Hedgehog in radial patterning of the gut. The role of Hedgehog signaling in esophageal cytodifferentiation remains to be determined as the severe esophageal phenotype of most Hedgehog mutant mice precludes evaluation of esophageal cytodifferentiation, and the histology of the esophageal epithelium of Gli2–/– mice has not been reported.
|
Gastric epithelial cytodifferentiation is a highly complex process during which the gastric epithelium is patterned into three distinct zones along the proximal-distal axis and in precursor cell and differentiated cell compartments along the vertical axis. Whereas the whole stomach of humans consists of glandular epithelium, mice develop a squamous epithelium in the first proximal one-third of the stomach. The distal two-thirds of the stomach consist of columnar glandular epithelium. A proximal fundic or zymogenic and distal antral zone can be recognized in the adult glandular epithelium based on histological features. Cytodifferentiation of the gastric epithelium is initiated around E13.5. At E16.5, the epithelium of the forestomach is a squamous multilayer, whereas the epithelium of the glandular stomach is a monolayer of columnar cells that have formed primitive epithelial invaginations (48). Recombination experiments (48) suggest that the endoderm of the E14.5 stomach does not require mesenchymal instructive signals for appropriate AP patterning, indicating that the AP pattern has been correctly specified at this point in time (as is the case in the intestine, see below). It should be noted, however, that no markers of differentiation were used and that epithelial phenotype was judged by histology. The authors also performed experiments with E11.5 and E12.5 endoderm and showed that the endoderm may still have some plasticity at this stage as 7 out of 10 E11.5/12.5 gastric endoderm explants keratinized when cultured with E14.5 forestomach mesenchyme, whereas 1 out of 9 were keratinized when cultured with mesenchyme from the glandular stomach.
At birth, 90% of the cells in the rudimentary gastric units are precursor cells; this percentage is reduced to 20% in the first 7 days postnatally (P1–P7) as cellular differentiation occurs while gland size remains stable (89). The second week after birth (P8–P14) is marked by an increase of the number of all cell types and glandular growth. Between P15 and P28, further cellular differentiation and glandular growth occurs, and the cells in the glands are compartmentalized (89). Compartmentalization of the gastric glands results in the formation of a precursor cell compartment (isthmus) somewhere halfway up the unit from where a bidirectional migration of differentiating cells occurs. Cells that migrate to the luminal surface form the pit or foveolus, whereas cells that migrate to the bottom of the gland form the gland proper.
3. Hedgehog signaling in gastric development
From early in stomach development at E11.5 until after the onset of gastric epithelial cytodifferentiation at E15.5, Shh is expressed at high levels in the forestomach and lower levels in the hindstomach, whereas Ihh is expressed in the hindstomach (Fig. 11) (13, 178). Ihh expression in the glandular stomach depends on Fgf signaling as both Fgfr2b–/– and Fgf10–/– mice lack expression of Ihh in the stomach (178). Expression of Ptc1 is in the mesoderm and mirrors that of Shh. There is only very low expression of Ptc1 in the distal hindstomach at this point in time, which suggests that Ihh may not be translated or active (178). Indeed, no gross abnormalities are reported in the stomachs of Ihh–/– mice, although gland formation may be somewhat reduced [see Fig. 2I in Ramalho-Santos et al. (164)]. Expression of Shh expands to the hindstomach at later stages of development as both Shh and Ihh are expressed in the glandular stomach at E18.5 (164). At this stage of development, Hedgehog signaling may no longer be exclusive to the mesenchyme as both Ptc1 and Gli1 seem to be expressed also in the epithelial layer (164). This is similar to the situation in the adult stomach where Ptc1 is most highly expressed in epithelial gland cells (8, 199). Hedgehog expression in the forestomach has not been described during later stages of gastric development. The expression of Hedgehogs during epithelial cytodifferentiation and compartmentalization in the first four postnatal weeks has similarly not been examined.
|
