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The vertebrate digestive tract is composed of multiple organs that arise from a common primitive gut tube that extends from the mouth to the anus. In the mouse, gut organogenesis begins shortly after gastrulation, when the definitive endoderm invaginates at its anterior and posterior ends to create the anterior intestinal portal (AIP) and caudal intestinal portal (CIP), respectively. Over time, the open-ended tubes elongate toward one another, and embryo turning enables fusion of the tubes at the yolk stalk, forming a contiguous primitive gut tube (Rosenquist, 1971; Lawson et al., 1986; Fig. 1).
The mammalian primitive gut is an epithelial tube surrounded by splanchnic mesoderm. As the gut tube matures, the foregut, midgut, and hindgut become morphologically distinct. During this time, mesoderm matures into connective tissue and concentric layers of smooth muscle that are innervated by the enteric nervous system and promote contractile movements during digestion. Subsequently, the gut differentiates along its anterior/posterior (A/P) axis into distinct primary organs that have specialized roles in digestion. Accordingly, gut organ epithelia acquire distinct luminal morphologies to help carry out their digestive functions. In general, organs of the foregut (pharynx, esophagus, stomach) ingest food and initiate digestion. Digestion is completed and nutrients are absorbed in the midgut (small intestine), whereas the hindgut (large intestine) resorbs water and ions, and expels undigested waste. Therefore, the basic function of the gut, to digest food, can be accomplished only through the coordinate function of different tissues and organs that comprise the gut.
The discussion below outlines themes based on well-established research in vertebrate gut organogenesis, using pertinent examples from invertebrates as supportive evidence. These themes include molecular mechanisms that drive early development of foregut, midgut, and hindgut from the primitive gut tube, regional patterning of the emerging gut, and coordination of tissue differentiation within the gut. Subsequent development of secondary organs (i.e., thyroid, lungs, liver, pancreas) are not described here but has been the subject of recent reviews (Wells and Melton, 1999; Edlund, 2002; Zaret, 2002; Ober et al., 2003).
DEVELOPMENT OF DIGESTIVE ORGAN PRIMORDIA
Perhaps because vertebrate digestive tract organs arise from a primitive gut tube, subregions of the gut use common molecular mechanisms in their early development. This section will describe three gene classes, FoxA, Gata4, and Sox17, that have well-established roles in early fore-, mid-, and hindgut development (Table 1). The mouse FoxA (fork head box A) transcription factors, FoxA1, 2, 3 (HNF3 α, β, γ), are expressed in primitive gut tube endoderm (with the exception of FoxA3) and in the differentiated gut (Ang et al., 1993; Monaghan et al., 1993; Sasaki and Hogan, 1993). Strikingly, in chimeric mice derived from mutant and wild-type embryonic stem (ES) cells mixed at the blastula stage, FoxA2-/- cells contribute to the hindgut but not the foregut or midgut (Dufort et al., 1998). These findings demonstrate that FoxA2 is required for mouse foregut and midgut development.
Table 1. Expression and Function of Genes That Regulate Early Mouse Gut Development
Gut tube expression
Throughout primitive gut tube endoderm
Early development of foregut and midgut
Anterior intestinal portal
Migration and development of gut primordia
Throughout primitive gut tube endoderm, with decreasing expression in foregut
Growth and morphogenesis of mid- and hindgut endoderm, maintenance of foregut endoderm
Posterior primitive gut tube endoderm
Hindgut development; may regulate expression of Hox genes
Gut endoderm and in nested anterior/posterior patterns in mesoderm (i.e. Abd-B subfamily of HoxA and D clusters)
Gut anterior/posterior patterning
Gut mesoderm differentiation
Gut endoderm differentiation
Of interest, FoxA homologs, for example Drosophila fork head (fkh) and Caenorhabditis elegans pha-4, have conserved roles in gut development. In fkh mutants, foregut and hindgut transform into ectoderm and head structures, whereas the midgut degenerates over time (Jürgens and Weigel, 1988; Weigel et al., 1989). Similarly, C. elegans pha-4 mutants also lack foregut and often hindgut, but the midgut is largely unaffected (Mango et al., 1994). pha-4 functions by directly regulating gene transcription in the foregut primordium, throughout development, and in the adult in all cell types that comprise the foregut (neurons, muscle, epithelia, etc.) (Gaudet and Mango, 2002). In this way, pha-4 functions as a common regulator in most or all gene programs in the foregut.
At the molecular level, do FoxA genes function in mouse as they do in C. elegans? In mouse endodermal progenitor cells, FOXA binding sites are among the first to be occupied in the albumin enhancer (Gualdi et al., 1996). Taken together with the finding that FOXA2 can open repressed chromatin, this demonstrates that FoxA genes are “pioneer factors” that render endodermal genes competent to be activated by molecular signals and/or additional transcription factors (Cirillo et al., 2002). This finding supports the idea that FoxA genes are necessary for induction of mouse gut primordia. One possibility is that FoxA 1, 2, 3 each work at different times in development to perform the equivalent functions of pha-4 in establishing gut primordia and driving subsequent development. However, it is not yet known whether, like pha-4, the mouse FoxA genes directly regulate foregut and midgut developmental gene programs.
The zinc-finger transcription factor Gata4 has also been implicated in early gut development. Gata4 is expressed in vertebrate AIP (Arceci et al., 1993; Molkentin et al., 1997; Nemer and Nemer, 2003), and Gata4 null mice lack ventral foregut endoderm. This phenotype is at least partially due to migration defects resulting in malformed AIP and lateral and ventral body folds (Kuo et al., 1997; Molkentin et al., 1997). In mutants of the zebrafish Gata4 homolog faust/gata5 (fau), endodermal migration is also perturbed (Reiter et al., 1999). Furthermore, before migration of endodermal progenitors, there is a vast reduction in the number of cells expressing certain endodermal markers (Reiter et al., 2001). These data suggest that Gata4 functions both in endoderm migration and in early gut endoderm development.
Evidence from Drosophila and C. elegans supports separate roles for Gata4 and FoxA in early gut development. The Gata4 homologs Drosophila serpent (srp) and C. elegans end-1 and end-3 are each expressed in midgut primordia, and accordingly, srp and end-1; end-3 mutant embryos do not develop a midgut (Rehorn et al., 1996; Zhu et al., 1997; Maduro and Rothman, 2002). Furthermore, ectopic expression of end-1 or end-3 in the C. elegans embryo is sufficient to induce midgut fates in nonendodermal cells (Zhu et al., 1998). Thus, in contrast to FoxA homologs in Drosophila and C. elegans, which are important for inducing foregut and hindgut primordia, Gata4 invertebrate orthologs are required for midgut fates. However, it remains unknown whether FoxA and Gata4 function in vertebrates independently to drive development of different gut subregions.
Of interest, in the zebrafish, although the presumptive gut tract contains endodermal progenitors throughout somitogenesis (Ober et al., 2003), subregions of the gut (pharynx and esophagus, intestine, and hindgut) epithelialize and form lumens independently during mid- and late somitogenesis and subsequently connect to form a tube (Wallace and Pack, 2003). One possibility based on these observations is that, as in invertebrates, subregions of the vertebrate gut may arise from separate lineages and/or develop under the control of independent molecular programs.
Sox17 (Sry-related HMG box factor) has recently emerged as an additional regulator of early gut endoderm. Ectopic expression of Xenopus Xsox17 α and -β each cause expansion of posterior gut and can induce ectoderm to differentiate as gut endoderm (Hudson et al., 1997; Clements and Woodland, 2000). Moreover, blocking Xsox17 α and -β function inhibits endodermal development (Clements and Woodland, 2000). Research in mice has revealed a differential requirement for Sox17 in foregut than in mid- and hindgut endoderm. Mouse Sox17 is expressed throughout the primitive gut tube endoderm, although expression in the foregut decreases over time. In Sox17 null mice, foregut endoderm undergoes extensive apoptosis, whereas growth and morphogenesis of mid- and hindgut are severely affected (Kanai-Azuma et al., 2002). These data suggest that Sox17 is critical for development of mid- and hindgut gut endoderm and maintenance of foregut endoderm.
ANTERIOR/POSTERIOR PATTERNING OF THE GUT TUBE
Once cells are specified as gut primordia, they undergo regional patterning, thus setting the stage for differential development of primary organs in the fore-, mid-, and hindgut. This section will describe two gene classes, Cdx2 and Hox paralogues, that have well-established roles in A/P patterning of the gut tube (Table 1). Cdx2, a ParaHox transcription factor, is specifically important for hindgut development. Cdx2 is expressed in the posterior gut tube (Freund et al., 1998), and in mouse chimeras, Cdx2-/- ES cells cannot contribute to the small intestine or colon (Beck et al., 2003). Furthermore, the Drosophila Cdx2 ortholog, caudal, is required for internalization and maintenance of the hindgut primordium (Wu and Lengyel, 1998). Therefore, Cdx2 is critical for hindgut development.
The Cdx class may influence hindgut patterning through regulation of Hox (homeobox transcription factor) gene expression, a class of genes that patterns multiple tissues along their A/P axes (see below). Ectopic expression of the Xenopus Cdx gene, Xcad3, activates expression of the posterior Hox genes, HoxA7, HoxC6, and HoxB9, and represses anteriorly expressed HoxB1 and HoxB3 (Isaacs et al., 1998). Similarly, mice heterozygous for Cdx2 display lesions of stomach epithelium in the intestine and colon, suggesting a rostral homeotic shift in the midgut. This phenotype is characteristic of disrupted Hox gene expression (Chawengsaksophak et al., 1997). Taken together with the observation that the Cdx family regulates Hox expression in many other tissues (Subramanian et al., 1995; Charite et al., 1998), these data support a role for Cdx2 patterning the posterior gut by means of Hox gene regulation.
The Hox family's classic role in the A/P patterning of many tissues is likely conserved in the gut. In chick and mouse, subfamilies of Hox genes are expressed in primitive gut tube epithelia, and in mesenchyme are expressed in nested A/P patterns that are maintained through organ differentiation. Moreover, gene expression boundaries of chick Abd-B like Hox genes of the A and D clusters often correlate with positions of morphologic gut organ landmarks, such as sphincters (Roberts et al., 1995; Yokouchi et al., 1995; Warot et al., 1997; Pitera et al., 1999; Kawazoe et al., 2002). However, mouse Hox mutants studied thus far display mild gut phenotypes (Boulet and Capecchi, 1996; Warot et al., 1997), possibly due to redundancy of function within Hox paralogue groups. Although some functional data support a role for Hox genes in gut A/P patterning. Viral expression of chick Hoxd13 in midgut endoderm results in its acquiring a hindgut-like morphology. Moreover, endodermal Hoxa5 loss-of-function results in a possible rostral homeotic shift of specific stomach gastric mucosa cell types (Aubin et al., 2002). Therefore, it seems that, as in other tissues, Hox genes pattern the A/P axis of the gut. One way this might be achieved is by regulating inductive signals that control region-specific differentiation.
CROSSTALK BETWEEN ADJACENT TISSUES COORDINATES REGION-SPECIFIC GUT DIFFERENTIATION
As primary organs emerge, endodermal and mesodermal layers along different A/P positions of the gut tube must coordinate their differentiation. This coordination is achieved through direct signaling between adjacent tissues (Table 1). Several experiments demonstrate that endodermally expressed Shh (Sonic hedgehog) and Ihh (Indian hedgehog) promote differentiation of adjacent mesoderm. Shh is broadly expressed in developing gut tube endoderm, whereas Ihh is expressed in posterior endoderm from the hindgut to the anus. Transducers of hedgehog signaling, Patched (Ptc) 1 and 2 receptors, and the transcriptional effectors Gli 1 and 2 are expressed in adjacent mesoderm, demonstrating that this tissue is a primary target of endodermally derived SHH and IHH (Ramalho-Santos et al., 2000). Consistent with this finding, Shh-/- and Ihh-/- mice each display a reduction in smooth muscle along the small intestine, demonstrating that Hh signaling is required for differentiation of gut mesenchyme (Ramalho-Santos et al., 2000; Sukegawa et al., 2000). Furthermore, when gut endoderm and mesoderm co-cultures are treated with the Hh inhibitor, cyclopamine, mesodermal Bmp4 (Bone morphogenetic protein 4) expression is reduced. This finding suggests that Bmp4 may be a target of endodermal SHH/IHH (Sukegawa et al., 2000). Together these findings show that endodermal Hh signaling is required for differentiation of overlying mesoderm.
Region-specific endodermal gut differentiation is subsequently dependent upon mesoderm signaling back to the endoderm. This phenomenon has been demonstrated most clearly in tissue recombination experiments. For example, chick proventricularis (glandular stomach) mesoderm can instruct gizzard (muscular stomach) endoderm to differentiate as proventricularis epithelia (Koike and Yasugi, 1999). In addition, inhibition of Bmp expression by virally produced Noggin results in the absence of proventricular glandular epithelium (Narita et al., 2000). In chick, Bmp4 expression is excluded from gizzard mesoderm, suggesting that the activity of BMP4 is regionally restricted (Ramalho-Santos et al., 2000; Sukegawa et al., 2000). Therefore, mesodermally expressed molecules, including Bmp4, signal to the endoderm and are a critical means of initiating region specific differentiation.
Despite considerable research on molecular mechanisms regulating development of the fore-, mid-, and hindgut from the primitive gut tube, much remains to be understood. This primer describes well-established research outlining what is known about the roles of several molecules involved in these processes. Research revealing conserved roles of additional regulators, for example Mix-like homeobox genes and FGFs (Fibroblast growth factors), is eagerly anticipated. Topics in gut development that thus far have received little attention include understanding how gene pathways interact with one another, and what is the cascade of molecular events that lead from the primitive gut tube to primary organ differentiation. Recent advances in genetic manipulations, organ culture, and methods of monitoring global changes in gene expression should facilitate our ability to address these questions.