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Wnt signaling is one of a handful of powerful signaling pathways that play crucial roles in the animal life by controlling the genetic programs of embryonic development and adult homeostasis. When disrupted, these signaling pathways cause developmental defects, or diseases, among them cancer. The gateway of the canonical Wnt pathway, which contains >100 genes, is an essential molecule called beta-catenin (Armadillo in Drosophila). Conditional loss- and gain-of-function mutations of beta-catenin in mice provided powerful tools for the functional analysis of canonical Wnt signaling in many tissues and organs. Such studies revealed roles of Wnt signaling that were previously not accessible to genetic analysis due to the early embryonic lethality of conventional beta-catenin knockout mice, as well as the redundancy of Wnt ligands, receptors, and transcription factors. Analysis of conditional beta-catenin loss- and gain-of-function mutant mice demonstrated that canonical Wnt signals control progenitor cell expansion and lineage decisions both in the early embryo and in many organs. Canonical Wnt signaling also plays important roles in the maintenance of various embryonic or adult stem cells, and as recent findings demonstrated, in cancer stem cell types. This has opened new opportunities to model numerous human diseases, which have been associated with deregulated Wnt signaling. Our review summarizes what has been learned from genetic studies of the Wnt pathway by the analysis of conditional beta-catenin loss- and gain-of-function mice.

The TMEM16 protein family has recently been identified through several different experimental strategies including bioinformatic and microarray-based approaches. In mice and humans, there exist 10 paralogs with each containing eight putative transmembrane domains and a conserved C-terminal domain of unknown function. Mutation of at least one member of this family is associated with a human disorder, and several members of this gene family are overexpressed in different types of cancer. Despite their apparent relevance to normal development and disease, little is known about the expression of TMEM16 paralogs during embryonic development. Here, we provide a phylogenetic analysis of mouse and human TMEM16 paralogs and report the expression of Tmem16a, Tmem16b, Tmem16c, Tmem16f, Tmem16h, Tmem16j, and Tmem16k during murine embryogenesis with an emphasis on the respiratory, digestive, skeletal, and integumentary systems. These data should encourage investigations into the functions of TMEM16 paralogs in vertebrate development.

The ontogeny of the digestive tract was studied histologically and histochemically in sharpsnout sea bream Diplodus puntazzo from hatching (0 DAH, Days After Hatching) until day 57 (57 DAH). At hatching, the digestive tract appeared as a histologically undifferentiated straight tube lying dorsally to the yolk sac. When the mouth opened at 3 DAH, the digestive tract was differentiated into buccopharynx, oesophagus, incipient stomach and intestine. The pancreas, liver and gall bladder were also differentiated at this stage and both the bile and pancreatic duct had opened into the anterior intestine. Active feeding began in 50% of larvae at 4 DAH, although permanence of yolk reserves until 7 DAH suggests a period of both endogenous and exogenous feeding. Nutrient absorption was first visible from 5 DAH, as colourless supra- and infranuclear vacuoles in the anterior intestinal mucosa, suggesting a lipid content, as well as supranuclear, eosinophilic vacuoles, containing protein, in the posterior intestinal mucosa. Early caecal development could be detected from 10 DAH, whereas gastric glands appeared at 30 DAH, indicating the transition from larval to juvenile stage and the acquisition of an adult mode of digestion. Goblet cells appeared in the digestive tract of sharpsnout sea bream larvae shortly after first feeding. The mucus content of goblet cells varied with the digestive region and, in the buccal cavity and oesophagus, also with the developmental phase. This study provides knowledge for better husbandry practices in the aquaculture industry, as well as for the implementation of future nutritional studies.

Microscopic anatomy of the digestive system in embryos and larvae of the terrestrial isopod crustacean Porcellio scaber was investigated by light bright field, fluorescence and electron microscopy. During marsupial ontogenetic development the event-dependent staging was used to discriminate the various embryonic stages. At the late embryo stage the differentiation of the ectodermal part of the gut into the complex filtering foregut and the hindgut with absorptive and transporting functions is accomplished. The gut of the marsupial manca larva is fully developed and similar to that of the adult. In early embryos the endodermal midgut gland primordia are filled with yolk and lipid globules. In late embryos the epithelium of paired midgut gland tubes is composed of two cell types; one of them exhibits orange autofluorescence. The endodermal cells located between the foregut and the midgut glands of late embryos form the prospective midgut. The cells have electron dense cytoplasm, abundant glycogen fields, endoplasmic reticulum, dictyosomes and numerous vesicles. In the adults the endodermal cells of the midgut remain only in the midgut gland ducts which connect the midgut glands and the foregut. Details of the cellular ultrastructure and morphogenesis of the ectodermal and endodermal parts of the digestive system during embryonic development of Porcellio scaber provide data for further phylogenetic and comparative studies in peracaridan crustaceans and other arthropods.

This fate-mapping study reveals that the progenitors of all major parts of the embryonic gut are already present in endoderm of the early-head-fold to early-somite stage (1-9 somites) mouse embryo. The anterior endoderm contributes primarily to the anterior intestinal portal of the early-organogenesis stage (16-19 somites) embryo. Endoderm cells around and lateral to the node are allocated to the open "midgut" region of the embryonic gut. The posterior (post-nodal) endoderm contributes not only to the posterior intestinal portal but also the open "midgut". Descendants of the posterior endoderm span a length of the gut from the level of the 3rd-5th somites to the posterior end of the embryonic gut. The formation of the anterior and posterior intestinal portals is accompanied by similar repertoires of morphogenetic tissue movement. We also discovered that cells on contralateral sides of the anterior endoderm are distributed asymmetrically to the dorsal and ventral sides of the anterior intestinal portal, heralding the acquisition of laterality by the embryonic foregut.

We have investigated the structural basis by which the counterclockwise direction of the amniote gut is established. The chirality of midgut looping is determined by left-right asymmetries in the cellular architecture of the dorsal mesentery, the structure that connects the primitive gut tube to the body wall. The mesenchymal cells of the dorsal mesentery are more condensed on the left side than on the right and, additionally, the overlying epithelium on the left side exhibits a columnar morphology, in contrast to a cuboidal morphology on the right. These properties are instructed by a set of transcription factors: Pitx2 and Isl1 specifically expressed on the left side, and Tbx18 expressed on the right, regulated downstream of the secreted protein Nodal which is present exclusively on the left side. The resultant differences in cellular organization cause the mesentery to assume a trapezoidal shape, tilting the primitive gut tube leftward.

Morphogenesis of the facial skeleton depends on inductive interactions between cephalic neural crest cells and cephalic epithelia, including the foregut endoderm. We show that Shh expression in the most rostral zone of the endoderm, endoderm zone I (EZ-I), is necessary to induce the formation of the ventral component of the avian nasal capsule: the mesethmoid cartilage. Surgical removal of EZ-I specifically prevented mesethmoid formation, whereas grafting a supernumerary EZ-I resulted in an ectopic mesethmoid. EZ-I ablation was rescued by Shh-loaded beads, whereas inhibition of Shh signalling suppressed mesethmoid formation. This interaction between the endoderm and cephalic neural crest cells was reproduced in vitro, as evidenced by Gli1 induction. Our work bolsters the hypothesis that early endodermal regionalisation provides the blueprint for facial morphogenesis and that its disruption might cause foetal craniofacial defects, including those of the nasal region.

The enteric nervous system (ENS) is mainly derived from vagal neural crest cells (NCC) that arise at the level of somites 1-7. To understand how the size and composition of the NCC progenitor pool affects ENS development, we reduced the number of NCC by ablating the neural tube adjacent to somites 3-6 to produce aganglionic gut. We then back-transplanted various somite lengths of quail neural tube into the ablated region to determine the 'tipping point', whereby sufficient progenitors were available for complete ENS formation. The addition of one somite length of either vagal, sacral or trunk neural tube into embryos that had the neural tube ablated adjacent to somites 3-6, resulted in ENS formation along the entire gut. Although these additional cells contributed to the progenitor pool, the quail NCC from different axial levels retained their intrinsic identities with respect to their ability to form the ENS; vagal NCC formed most of the ENS, sacral NCC contributed a limited number of ENS cells, and trunk NCC did not contribute to the ENS. As one somite length of vagal NCC was found to comprise almost the entire ENS, we ablated all of the vagal neural crest and back-transplanted one somite length of vagal neural tube from the level of somite 1 or somite 3 into the vagal region at the position of somite 3. NCC from somite 3 formed the ENS along the entire gut, whereas NCC from somite 1 did not. Intrinsic differences, such as an increased capacity for proliferation, as demonstrated in vitro and in vivo, appear to underlie the ability of somite 3 NCC to form the entire ENS.

In the development of mouse gut, longitudinal smooth muscle cells (LMC) and interstitial cells of Cajal (ICC) originate from common precursor cells expressing c-Kit. Recently, some gastrointestinal stromal tumours, which develop from smooth muscle layers of the gut and have gain-of-function mutations of c-kit, have been reported to have gain-of-function mutations of platelet-derived growth factor (PDGF) receptor alpha gene. These data raise the possibility that PDGF signalling might be involved in the development of LMC. Therefore, we examined the expression pattern of the PDGF signal family of embryonic gut by immunohistochemistry and in situ hybridization, and investigated the role of PDGF signals in the development of smooth muscle layers in mouse gut using a new organ culture system. During embryonic development, the circular muscle layer expressed PDGF-A, enteric neurons expressed PDGF-B and common precursor cells of LMC and ICC expressed both PDGF receptor alpha and beta. The selective PDGF receptor inhibitor AG1295 suppressed the differentiation of LMC in gut explants. We conclude that PDGF signals play critical roles in the differentiation of LMC in mouse embryonic gut.

The gut represents a complex organ system with regional differences, which reflect selective digestive and absorptive functions that change constantly in response to bodily requirements and the outside milieu. As a barrier to the external environment, gut epithelium must be renewed rapidly and repeatedly. Growth and renewal of gut epithelial cells is dependent on controlled cell stimulation and proliferation by a number of signaling processes and agents, including gut peptides-both endogenous and exogenous sources. This cascade of events begins during fetal development; with the ingestion of amniotic fluid, this process is enhanced and continued during infancy and early childhood through the ingestion of human milk. Events influenced by amniotic fluid during fetal development and those influenced by human milk that unfold after birth and early childhood to render the gut mature are presented.

Rho GTPases play central roles in the control of cell adhesion and migration, cell cycle progression, growth, and differentiation. However, although most of our knowledge of Rho GTPase function comes from the study of the three classic Rho GTPases RhoA, Rac1, and Cdc42, recent studies have begun to explore the expression, regulation, and function of some of the lesser-known members of the Rho GTPase family. In the present study, we cloned the avian orthologues of RhoV (or Chp for Cdc42 homologous protein) and RhoU (or Wrch-1 for Wnt-regulated Cdc42 homolog-1) and examined their expression patterns by in situ hybridization analysis both during early chick embryogenesis and later on, during gastrointestinal tract development. Our data show that both GTPases are detected in the primitive streak, the somites, the neural crest cells, and the gastrointestinal tract with distinct territories and/or temporal expression windows. Although both proteins are 90% identical, our results indicate that cRhoV and cRhoU are distinctly expressed during chicken embryonic development., ((c) 2008 Wiley-Liss, Inc.)

The European catfish, Silurus glanis L., has become an important aqua cultural fish in Croatia, and it is cultivated extensively in ponds in polyculture with carps. The development of the digestive tract in S. glanis was studied with the aim of improving intensive fish production. Research was carried out on S. glanis larval stadium from 1- to 19-day post-hatching (DPH). The main histological methods used were: haematoxylin and eosin staining, periodic acid Schiff staining (PAS), Alcian blue (AB) and toluidin blue staining (TB). A yolk sac was present during the first 5 days (1-5-DPH). During the initial 3-DPH period, there was no trace PAS and AB activity in the digestive tract. Differentiation of the digestive tract began at 3- to 5-DPH. The oesophagus was positive for AB at 5-DPH, PAS and TB after 7-DPH. Differentiation of enterocytes began at 5-DPG and the intestines were complete at 11-DPH. Development of liver and pancreas was also studied. The analysis of data obtained in this study suggests that after 5-DPH catfish larvae have morphologically completed digestive tracts.

During organogenesis of the C. elegans digestive system, epithelial cells within a cyst-like primordium develop diverse shapes through largely unknown mechanisms. We here analyze two adjacent, dorsal epithelial cells, called pm8 and vpi1, that remodel their shapes and apical junctions to become donut-shaped, or toroidal, single-cell tubes. pm8 and vpi1 delaminate from the dorsal cyst epithelium and migrate ventrally, across the midline of the cyst, on a transient tract of laminin. pm8 appears to encircle the midline by wrapping around finger-like projections from neighboring cells. Finally, pm8 and vpi1 self-fuse to become toroids by expressing AFF-1 and EFF-1, two fusogens that are each sufficient to promote crossfusion between other cell types. Notch signaling in pm8 induces AFF-1 expression, while simultaneously repressing EFF-1 expression; vpi1 expresses EFF-1 independent of Notch. Thus, the adjacent pm8 and vpi1 cells express different fusogens, allowing them to self-fuse into separate, single-cell tubes while avoiding crossfusion.

Background: Necrotizing enterocolitis (NEC) is the most common gastrointestinal (GI) emergency seen in neonatal units. Many factors have been considered as potentially important aetiologically, including gut ischaemia, sepsis and feeding. However, evidence remains equivocal., Objective: This study investigated whether preterm babies born to mothers with abnormal antenatal umbilical Dopplers (absent or reversed end diastolic flow--AREDF), that is exposed to antenatal gut ischaemia, are at an identical risk of developing NEC early in life, compared to babies born to mothers with normal Dopplers., Methods: All preterm (

Cyclo-oxygenase (COX), which catalyses the conversion of arachidonic acid to prostaglandin endoperoxide and prostanoids, is widely expressed in mammalian organs. The aim of the study was to evaluate the immunoexpression of the constitutive and inducible cyclo-oxygenase isoforms (COX-1 and COX-2 respectively) in the oesophagus, stomach and the small and large bowels of untreated rat dams and foetuses on gestational day 21. The localisation of the COX isoforms was similar in the maternal and foetal organs, although the intensity of the reaction for COX-2 was stronger in the foetuses. Cytoplasmic COX-1 immunostaining was found in myocytes of the muscularis propria, muscularis mucosae and the blood vessels. It was also positive in the endothelial cells, scattered stromal cells of the lamina propria and the ganglion cells of the nerve plexus in the bowels. Apart from the keratinised layer, a strong reaction was revealed in the stratified squamous epithelium of the oesophagus and forestomach. Negative or weakly positive staining was found in the mucus-secreting cells covering the surface, gastric pits and pyloric glands, as well as in the parietal cells and the chief cells. Weakly positive COX-1 immunostaining was observed in epithelial cells of the small intestine crypts, but in some cases enterocytes and goblet cells covering villi were also positive. In the colonic mucosa weak COX-1 staining was typical of the absorptive, and goblet cells. The COX-2 immunostaining was nuclear and/or cytoplasmic. An inconsistent positive reaction was seen in the muscle of the muscularis mucosae, muscularis propria and the blood vessels. Positive staining was also found in scattered stromal cells of the lamina propria and adventitia and the ganglion cells. Weak nuclear staining was found in the stratified squamous epithelium of the oesophagus and forestomach. Unlike the strong foetal reactivity in the epithelial cells of the glandular stomach, a negative or weakly positive reaction was seen in the maternal parietal and/or mucous-secreting surface stomach cells. Some epithelial cells of the crypts both in the small and large bowel were also COX-2 positive. In conclusion, constitutive and inducible COX isoforms were detected in the digestive tract of pregnant female and in foetuses. COX-1 was the predominant isoform in both the adult and foetal organs.

To program pluripotent cells into blood, a knowledge of the locations of precursors during their journey through the embryo and the signals they experience would be informative. The anterior (a) and posterior (p) ventral blood islands (VBIs) in Xenopus are derived from opposite sides of the pregastrula embryo. The aVBI goes through a "hemangioblast" state, characterized by coexpression of blood and endothelial genes at neurula stages, whereas the pVBI expresses these genes in a nonoverlapping fashion several hours later, after commitment to either a blood or an endothelial fate. We describe a novel role for fibroblast growth factor (FGF) in controlling the timing of Scl, Lmo2, and Runx1 expression in the 2 VBI compartments. Blocking FGF signaling during gastrulation expands expression at neurula stages into posterior regions. We show, by lineage labeling, explant analysis, and targeted blocking of FGF signaling, that this is due to the pVBI prematurely expressing these genes with the timing of the aVBI. In contrast, overexpression of FGF in aVBI precursors eliminates the anterior hemangioblast program. Using this information, we have recapitulated the anterior hemangioblast program in pluripotent cells in vitro by inhibiting FGF signaling in anterior mesoderm induced by activin and exposed to bone morphogenetic protein (BMP) signaling.

Background: Alpha 2 Macroglobulin family members have been studied extensively with respect to their roles in physiology and human disease including innate immunity and Alzheimer's disease, but little is known about a possible role in liver development loss-of-function in model systems., Principal Findings: We report the isolation of the zebrafish alpha2 macroglobulin-like (A2ML) gene and its specific expression in the liver during differentiation. Morpholino-based knock-down of A2ML did not block the initial formation of the liver primordium, but inhibited liver growth and differentiation., Significance: This report on A2ML function in zebrafish development provides the first evidence for a specific role of an A2M family gene in liver formation during early embryogenesis in a vertebrate.

Most bilaterian animals have evolved a through gut that is regionally specialized along the anterior-posterior axis. In the polychaete annelid, Capitella sp. I, the alimentary canal is subdivided into a buccal cavity, pharynx, esophagus, midgut, and hindgut. Members of the Fox and GATA families of transcription factors have conserved functions in patterning ectodermal and endodermal gut components. We have isolated and characterized expression of one FoxA gene (CapI-foxA) and four GATA genes (CapI-gataB1, CapI-gataB2, CapI-gataB3, and CapI-gataA1) from Capitella sp. I. Both gene families are expressed in the developing gut of this polychaete. CapI-foxA, an ortholog of the FoxA subgroup, is expressed in vegetal hemisphere micromeres of cleavage-stage embryos, in multiple blastomeres within and surrounding the blastopore during gastrulation, and throughout morphogenesis of the pharynx, esophagus, and hindgut. The CapI-gataB genes group within the vertebrate GATA4/5/6 subfamily, appear to be products of lineage-specific gene duplication, and are expressed in specific domains of endomesoderm. CapI-gataB1 is expressed in endoderm precursors and throughout developing midgut endoderm, and is particularly prominent at anterior and posterior midgut boundaries. CapI-gataB2 is co-expressed with CapI-gataB1 in midgut endoderm, and is also expressed in visceral mesoderm. CapI-gataB3 is limited to and coexpressed with CapI-gataB2 in visceral mesoderm. CapI-gataA1 groups within the vertebrate GATA1/2/3 subfamily and is expressed primarily in ectodermal tissues of the brain, ventral nerve cord, lateral trunk, and both pharyngeal and esophageal regions of the foregut. Collectively, the CapI-foxA and CapI-gata genes show patterns of expression that span almost the entire length of the developing alimentary canal, consistent with a role in gut development.

Fetal magnetic resonance imaging (MRI) is considered a valuable second line imaging tool for confirmation, completion and correction of complex fetal ultrasonography findings. Fetal MRI has proven its value in pathologies of the central nervous system. Few studies have focussed on the value of fetal MRI in abdominal pathologies. With the continuing advances in hardware and software, fetal MRI is progressively valuable in fetal abdominal pathologies. A proper knowledge of the normal fetal abdominal anatomy and signal intensities is essential. The current manuscript reviews normal fetal abdominal anatomy.

Background: The regulation of intestinal growth and development in human neonates is incompletely understood, which hinders the provision of nutrients enterally. The "hindgut" hormones glucagon-like peptides 1 and 2 have been shown to play an important role in the regulation of nutrient assimilation, intestinal growth, and function., Objective: Our goal was to investigate the production of glucagon-like peptides 1 and 2 in premature human infants and examine the effects of prematurity and feeding on hormone release., Patients and Methods: With informed consent, premature infants who were admitted to a tertiary neonatal intensive care nursery (gestational age: 28-32 weeks) were monitored with weekly determinations of postprandial glucagon-like peptide 1 and 2 levels. Comparison studies with groups of normal infants and adults were performed. Hormone levels were obtained by using specific radioimmunoassay for glucagon-like peptide 1 (1-36) and glucagon-like peptide 2 (1-33), modified for small sample volumes; accurate monitoring of enteral intake was performed at all of the sampling time points., Results: Forty-five infants with a mean gestational age of 29.6 +/- 1.9 weeks were studied; fasting levels of both glucagon-like peptides 1 and 2 were elevated. There was no correlation between gestational age and glucagon-like peptide 2 output. However, both glucagon-like peptide 1 and 2 levels were correlated with the caloric value of feeds., Conclusions: The premature human neonate has significantly higher fasting levels of glucagon-like peptides 1 and 2 compared with adults; feeding increases these levels further. These findings suggest that the proglucagon-derived peptides may have a role in normal intestinal development and nutrient handling.

The Hand proteins of the bHLH family of transcriptional factors play critical roles in vertebrate cardiogenesis. In Drosophila, the single orthologous Hand gene is expressed in the developing embryonic dorsal vessel (heart), lymph glands, circular visceral musculature, and a subset of CNS cells. We demonstrate that the absence of Hand activity causes semilethality during the early larval instars. The dorsal vessel and midgut musculature are unaffected in null mutant embryos, but in a large fraction the lymph glands are missing. However, homozygous adult flies lacking Hand possess morphologically abnormal dorsal vessels characterized by a disorganized myofibrillar structure, reduced systolic and diastolic diameter, and abnormal heartbeat contractions, and suffer from premature lethality. In addition, their midguts are highly deformed; in the most severe cases, there is midgut blockage and a massive excess of ectopic peritrophic membrane tubules exiting a rupture in an anterior midgut bulge. Nevertheless, the visceral musculature appears to be relatively normal. Based on these phenotypes, we conclude that the expression of the Drosophila Hand gene in the dorsal vessel and circular visceral muscles is mainly required during pupal stages, when Hand participates in the proper hormone-dependent remodeling of the larval aorta into the adult heart and in the normal morphogenesis of the adult midgut endoderm during metamorphosis.

Although bilateral animals appear to have left-right (LR) symmetry from the outside, their internal organs often show directional and stereotypical LR asymmetry. The mechanisms by which the LR axis is established in vertebrates have been extensively studied. However, how each organ develops its LR asymmetric morphology with respect to the LR axis is still unclear. Here, we showed that Drosophila Jun N-terminal kinase (D-JNK) signaling is involved in the LR asymmetric looping of the anterior-midgut (AMG) in Drosophila. Mutant embryos of puckered (puc), which encodes a D-JNK phosphatase, showed random laterality of the AMG. Directional LR looping of the AMG required D-JNK signaling to be down-regulated by puc in the trunk visceral mesoderm. Not only the down-regulation, but also the activation of D-JNK signaling was required for the LR asymmetric looping. We also found that the LR asymmetric cell rearrangement in the circular visceral muscle (CVM) was regulated by D-JNK signaling and required for the LR asymmetric looping of the AMG. Rac1, a Rho family small GTPase, augmented D-JNK signaling in this process. Our results also suggest that a basic mechanism for eliciting LR asymmetric gut looping may be conserved between vertebrates and invertebrates.

Mouse embryonic stem (ES) cells are pluripotent and retain their potential to form cells, tissues and organs originated from three embryonic germ layers. Recently, we developed in vitro organ--gut-like structures--from mouse ES cells. They had basically similar morphological features to a mouse gastrointestinal tract in vivo composed of three distinct layers (i.e., epithelium, connective tissue and musculature). Gut-like structures showed spontaneous contractions derived from pacemaker cells (interstitial cells of Cajal) in the musculature. We also examined their formation process and expression pattern of transcription factors crucial for gut organogenesis such as Id2, Sox17, HNF3beta/Foxa2 and GATA4. We found that they mimic the development of embryonic gut in vivo and showed a similar expression pattern of common transcription factors. They also maintain their developmental potential after transplantation to a renal capsule. Therefore, gut-like structures are suitable for in vitro models of gastrointestinal tracts and their development. In addition, we pointed out several unique features different from gut in vivo that provide useful and advantageous tools to investigate the developmental mechanism of the gastrointestinal tract.

The Hedgehog family of secreted morphogenetic proteins acts through a complex evolutionary conserved signaling pathway to regulate patterning events during development and in the adult organism. In this review I discuss the role of Hedgehog signaling in the development, postnatal maintenance, and carcinogenesis of the gastrointestinal tract. Three mammalian hedgehog genes, sonic hedgehog (Shh), indian hedgehog (Ihh), and desert hedgehog (Dhh) have been identified. Shh and Ihh are important endodermal signals in the endodermal-mesodermal cross-talk that patterns the developing gut tube along different axes. Mutations in Shh, Ihh, and downstream signaling molecules lead to a variety of gross malformations of the murine gastrointestinal tract including esophageal atresia, tracheoesophageal fistula, annular pancreas, midgut malrotation, and duodenal and anal atresia. These congenital malformations are also found in varying constellations in humans, suggesting a possible role for defective Hedgehog signaling in these patients. In the adult, Hedgehog signaling regulates homeostasis in several endoderm-derived epithelia, for example, the stomach, intestine, and pancreas. Finally, growth of carcinomas of the proximal gastrointestinal tract such as esophageal, gastric, biliary duct, and pancreatic cancers may depend on Hedgehog signaling offering a potential avenue for novel therapy for these aggressive cancers.

Gastrointestinal and pancreatic neuroendocrine tumors originate from the cells of the diffuse endocrine system. Their molecular genetic mechanism of development and progression is complex and remains largely unknown, and they are different in genetic composition from the gastrointestinal epithelial tumors. The current literature suggests that multiple genes are involved in their tumorigenesis with significant differences for tumors of different embryological derivatives: foregut, midgut and hindgut. The MEN1 gene is involved in initiation of 33% of foregut gastrointestinal neuroendocrine tumors. 18q defects are present almost exclusively in mid/hindgut neuroendocrine tumors. X-chromosome markers are associated with malignant behavior in foregut tumors only. Analysis of poorly differentiated neuroendocrine carcinomas of any site demonstrates high chromosomal instability and frequent p53 alterations similar to other poorly differentiated carcinomas. Several factors played a limiting role in the molecular studies published to date: the tumors are rare and heterogeneous, it is difficult to predict their behavior and prognosis, and several different tumor classifications are used by the investigators in the studies. Future studies need to evaluate molecular genetic composition of large series of gastrointestinal and pancreatic neuroendocrine tumors of each specific tumor type. Understanding of specific genetic alterations characteristic for gastrointestinal and pancreatic neuroendocrine tumors might lead to their improved diagnosis, morphologic and molecular characterization and treatment.

The digestive tract of vertebrates is a complex organ system required for the digestion of food and the absorption of nutrients. The colon evolved as a water absorption organ essential for vertebrates to survive on land. In contrast to land vertebrates, the Chondrichthyes (sharks, skates and rays) are nearly iso-osmotic with their ocean environment and do not reabsorb water from food waste. To understand the origin of the vertebrate colon, we examined the distribution of sulfated and sialyated mucus-producing cells in the little skate, Raja erinacea, as an indication of water absorption function in the chondrichthian digestive tract. The percentage of acid mucin producing goblet cells was analyzed in the spiral valve and hindgut of little skate and the small intestine and colon of mouse embryos. Levels of acid mucins in the hindgut of the little skate was comparable to that of the small intestines of terrestrial vertebrates, whereas the distal region of the spiral valve contained high levels of acid mucin producing cells similar to the colon of mouse and chick. The low numbers of acid mucins in the little skate hindgut confirms that a functional colon for water absorption is absent in the Chondrichthyes. Interestingly, the presence of high levels of acid mucins in the posterior spiral valve provides evidence for a possible primordial water-absorbing organ in the elasmobranchs. Hoxd13 patterns acid mucins in the colons of terrestrial vertebrates. Expression of Hoxd13 and Hoxa13 in R. erinacea suggests conserved roles for Hox genes in patterning the early hindgut.

Little is known about how the endoderm germ layer is patterned along the anterior-posterior (A-P) axis before the formation of a gut tube (embryonic day [e] 7.5-8.5 in mouse), largely due to a paucity of molecular markers of endoderm. In particular, there are few genes that mark posterior domains of endoderm that give rise to the midgut and hindgut. We have identified 8 molecular markers that are expressed in discrete domains of the gastrula stage endoderm (e7.5), suggesting that a significant level of pattern exists in the endoderm before the formation of a gut tube. Three genes Tmprss2, NM_029639, and Dsp are expressed in a presumptive midgut domain overlying the node, a domain for which molecular markers have not previously been identified. Two genes, Klf5 and Epha2 are expressed in posterior endoderm associated with the primitive streak. Expression of these five genes persists in the midgut and/or hindgut at e8.5, 9.5 and 10.5, suggesting that these genes are markers of these domains throughout these stages of development. We have identified three genes Slc39a8, Amot, and Dp1l1, which are expressed in the visceral endoderm at e7.5. Starting at e9.5, Dp1l1 is expressed de novo in the liver, midgut, and hindgut. Our findings suggest that presumptive midgut and hindgut domains are being established at the molecular level by the end of gastrulation, earlier than previously thought, and emphasize the importance of endoderm patterning before the formation of the fetal gut., (Copyright 2007 Wiley-Liss, Inc.)

Mowat-Wilson syndrome is a recently delineated autosomal dominant developmental anomaly, whereby heterozygous mutations in the ZFHX1B gene cause mental retardation, delayed motor development, epilepsy and a wide spectrum of clinically heterogeneous features, suggestive of neurocristopathies at the cephalic, cardiac and vagal levels. However, our understanding of the etiology of this condition at the cellular level remains vague. This study presents the Zfhx1b protein expression domain in mouse embryos and correlates this with a novel mouse model involving a conditional mutation in the Zfhx1b gene in neural crest precursor cells. These mutant mice display craniofacial and gastrointestinal malformations that show resemblance to those found in human patients with Mowat-Wilson syndrome. In addition to these clinically recognized alterations, we document developmental defects in the heart, melanoblasts and sympathetic and parasympathetic anlagen. The latter observations in our mouse model for Mowat-Wilson suggest a hitherto unknown role for Zfhx1b in the development of these particular neural crest derivatives, which is a set of observations that should be acknowledged in the clinical management of this genetic disorder.

The vertebrate enteric nervous system is formed by a rostro-caudally directed invasion of the embryonic gastrointestinal mesenchyme by neural crest cells. Failure to complete this invasion results in the distal intestine lacking intrinsic neurons. This potentially fatal condition is called Hirschsprung's Disease. A mathematical model of cell invasion incorporating cell motility and proliferation of neural crest cells to a carrying capacity predicted invasion outcomes to imagined manipulations, and these manipulations were tested experimentally. Mathematical and experimental results agreed. The results show that the directional invasion is chiefly driven by neural crest cell proliferation. Moreover, this proliferation occurs in a small region at the wavefront of the invading population. These results provide an understanding of why many genes implicated in Hirschsprung's Disease influence neural crest population size. In addition, during in vivo development the underlying gut tissues are growing simultaneously as the neural crest cell invasion proceeds. The interactions between proliferation, motility and gut growth dictate whether or not complete colonization is successful. Mathematical modeling provides insights into the conditions required for complete colonization or a Hirschsprung's-like deficiency. Experimental evidence supports the hypotheses suggested by the modeling.

Notch is an evolutionarily conserved local cell signaling mechanism that participates in a variety of cellular processes: cell fate specification, differentiation, proliferation, apoptosis, adhesion, epithelial-mesenchymal transition, migration, and angiogenesis. These processes can be subverted in Notch-mediated pathological situations. In the first part of this review, we will discuss the role of Notch in vertebrate central nervous system development, somitogenesis, cardiovascular and endocrine development, with attention to the mechanisms by which Notch regulates cell fate specification and patterning in these tissues. In the second part, we will review the molecular aspects of Notch-mediated neoplasias, where Notch can act as an oncogene or as a tumor suppressor. From all these studies, it becomes evident that the outcome of Notch signaling is strictly context-dependent and differences in the strength, timing, cell type, and context of the signal may affect the final outcome. It is essential to understand how Notch integrates inputs from other signaling pathways and how specificity is achieved, because this knowledge may be relevant for future therapeutic applications.

Prokineticins (Prok-1 and Prok-2) belong to a newly identified AVIT protein family. They are involved in variety of activities in various tissues, including smooth muscle contraction of the gastrointestinal tract and promoting proliferation of endothelial cells derived from adrenal gland. Importantly, they also act as the survival factors to modulate growth and survival of neurons and hematopoietic stem cells. In this study we demonstrated that Prok-1 (but not Prok-2) protein is expressed in the mucosa and mesenchyme of the mouse embryonic gut during enteric nervous system development. Its receptor, PK-R1 is expressed in the enteric neural crest cells (NCCs). To elucidate the physiological role(s) of Prok-1 in NCCs, we isolated the NCCs from the mouse embryonic gut (E11.5) and cultured them in the form of neurospheres. In an in vitro NCC culture, Prok-1 was able to activate both Akt and MAPK pathways and induce the proliferation and differentiation (but not migration) of NCCs via PK-R1. Knock-down of PK-R1 using siRNA resulted in a complete abolishment of Prok-1 induced proliferation. Taken together, it is the first report demonstrating that Prok-1 acts as a gut mucosa/mesenchyme-derived factor and maintains proliferation and differentiation of enteric NCCs.

At the end of embryogenesis of Lepisma saccharina L. (Insecta, Zygentoma), when the stomodaeum and proctodaeum are completely formed, the midgut epithelium is replaced by the primary midgut, a yolk mass is surrounded by a cell membrane. Midgut epithelium formation begins in the 1st larval stage. Energids migrate toward the yolk periphery and aggregate just beneath the cell membrane. They are gradually enclosed by cell membrane folds of the primary midgut. Single cells are formed. Succeeding energids join just formed cells. Thus, groups of cells, regenerative cell groups, are formed. Their number gradually increases. The external cells of the regenerative cell groups transform into epithelial cells and their basal regions spread toward the next regenerative cell groups. Epithelial cells of neighboring regenerative cell groups join each other to form the epithelium. At the end of the 2nd larval stage, just before molting, degeneration of newly the formed epithelium begins. Remains of organelles and basal membrane occur between the regenerative cell groups. The new epithelium is formed from the regenerative cell groups, which are now termed stem cells of the midgut epithelium., (2007 Wiley-Liss, Inc.)

Many animals have genetically determined left-right (LR) asymmetry of their internal organs. The midline structure of vertebrate embryos has important roles in LR asymmetric development both as the signaling center for LR asymmetry and as a barrier to inappropriate LR signaling across the midline. However, in invertebrates, the functions of the midline in LR asymmetric development are unknown. To elucidate these roles, we studied the involvement of single-minded (sim) in the LR asymmetry of the Drosophila embryonic gut, which develops in a stereotypic, asymmetric manner. sim encodes a bHLH/PAS transcription factor that is required for the development of the ventral midline structure. Here we report that sim was expressed in the midline of the foregut and hindgut primordia. The handedness of the embryonic gut was affected in sim mutant embryos and in embryos overexpressing sim. However, midline-derived events, which involve Slit/Robo and EGFr signaling and direct the development of the tissues adjacent to the midline, did not affect the laterality of this organ, suggesting a crucial role for the midline itself in LR asymmetry. In the sim mutants, the midline structures of the embryonic anal pad were deformed. The mis-expression of sim in the anal-pad primordium induced LR defects. We also found that different portions of the embryonic gut require sim functions at different times for normal LR asymmetry. Our results suggest that the midline structures are involved in the LR asymmetric development of the Drosophila embryonic gut.

Gastrointestinal (GI) motility results from the coordinated actions of enteric neurons, interstitial cells of Cajal (ICC), and smooth muscle cells. The GI tract of the zebrafish has a cellular anatomy that is essentially similar to humans. Although enteric nerves and smooth muscle cells have been described, it is unknown if ICC are present in the zebrafish. Immunohistochemistry and PCR were used determine expression for the zebrafish Kit orthologue in the zebrafish gastrointestinal tract. Cells displaying Kit-like immunoreactivity were identified in the muscular layers of the adult zebrafish gastrointestinal tract. Two layers of Kit-positive cells were identified, one with multipolar cells located between the longitudinal and circular smooth muscle layers and one with simple bipolar cells located deep in the circular muscle layer. Primers specifically designed to amplify mRNA coding for two zebrafish kit genes, kita and kitb, and two kit ligands, kitla and kitlb, amplified the expected transcript from total RNA isolated from zebrafish GI tissues. The Sparse mutant, a kita null mutant, showed reduced contraction frequency and increased size of the GI tract indicating a functional role for kita. These data establish the presence of a cellular network with Kit-like immunoreactivity in the myenteric plexus region of the zebrafish GI tract, adjacent to enteric neurons. Expression of kita and kitb, and the ligands kitla and kitlb, were verified in the adult GI tract. The anatomical arrangement of the Kit-positive cells strongly suggests that they are ICC.

Our understanding of the relationship between microbes and their animal hosts have changed dramatically in the last decade. The development of powerful new molecular methods as well as different animal models, particularly germ free rodents, has enabled precise characterisation of the ways in which commensal bacteria in the gastrointestinal tract and other areas interact with their hosts. It is now clear that animal development, far from being genetically determined is intimately bound up with the microbial flora of that particular animal. In germ free mice, the addition of Bacteroides thetaiotaomicron mediates the maturation and function of the gastrointestinal tract in several different ways. Similarly Bacteroides fragilis directs the development of the immune system both in and outside the gastrointestinal tract. The relevance of these findings and others to our understanding of human development and disease is discussed.

Follistatin-like 1 (Fstl1) is a distantly related homolog of the Activin and Bone Morphogenetic Protein antagonist Follistatin. Interestingly, this molecule also has homology with the extracellular matrix modifying protein BM-40/SPARC/osteonectin. Previous studies in chick have identified Fstl1 as a regulator of early mesoderm patterning, somitogenesis, myogenesis and neural development. In this study, we determine the developmental expression pattern of Fstl1 in the mouse. We find that Fstl1 is ubiquitously expressed in the early embryo, and that expression becomes regionalized later during development. In the majority of tissues, Fstl1 is strongly expressed in the mesenchymal component and excluded from the epithelium. Notable exceptions include the central nervous system, in which Fstl1 expression is entirely absent with the exception of the choroid plexi and floor plate, the lung, in which Fstl1 expression can be seen in airway epithelia and the kidney, in which collecting ducts and nascent nephron epithelia express the highest levels of Fstl1.

Owing to their high turnover, the intestinal mucosal cells have a particularly high requirement for polyamines. Therefore, they are an excellent charcol for the study of polyamine function in rapid physiological growth and differentiation. After a cursory introduction to the major aspects of polyamine metabolism, regulation, and mode of action, we discuss the contribution of the polyamines to the maintenance of normal gut function, the maturation of the intestinal mucosa, and its repair after injuries. Repletion of cellular polyamine pools with (D,L)-2-(difluoromethyl)ornithine has considerably improved our understanding of how the polyamines are involved in the regulation of normal and neoplastic growth. Unfortunately, the attempts to exploit polyamine metabolism as a cancer therapeutic target have not yet been successful. However, the selective inactivation of ornithine decarboxylase appears to be a promising chemopreventive method in familial adenomatous polyposis. Presumably, it relies on the fact that ornithine decarboxylase is a critical regulator of the proliferative response of the protooncogene c-myc, but not of its apoptotic response.

The enteric nervous system arises predominantly from vagal level neural crest cells that migrate into the foregut and then colonize the entire length of the gastrointestinal tract. Previous studies have demonstrated that glial cell line-derived neurotrophic factor (GDNF) promotes the migration of enteric neural crest-derived cells (ENCs) in vitro, but a role for GDNF in the migration of ENCs in vivo has yet to be demonstrated. In this study, the effects of Gdnf haploinsufficiency on ENC rate of migration and number during mid embryonic development were examined. Although the entire gut of embryonic Gdnf(+/-) mice was colonized, a significant delay in the migration of ENCs along the embryonic hindgut was found. However, significant effects of Gdnf haploinsufficiency on ENC number were detected before the stage at which migration defects were first evident. As previous studies have shown a relationship between ENC number and migration, the effects of Gdnf haploinsufficiency on migration may be due to an indirect effect on cell number and/or a direct effect of GDNF on ENC migration. Gdnf haploinsufficiency did not cause any detectable change in the rate of neuronal differentiation of ENCs.

The basic helix-loop-helix DNA binding protein Hand2 is expressed in neural crest-derived precursors of enteric neurons and has been shown to affect both neurogenesis and neurotransmitter specification of noradrenergic sympathetic ganglion neurons. In the current study, our goal was to determine whether Hand2 affects neurogenesis and/or expression of vasoactive intestinal polypeptide and choline acetyltransferase in developing enteric neurons. Gain-of-function of Hand2 in HNK-1(+) immmunoselected precursor cells resulted in increased neurogenesis. The number of neurons expressing vasoactive intestinal polypeptide increased in response to Hand2 overexpression although choline acetyltransferase was not affected. Targeted deletion of Hand2 in neural crest cells resulted in loss of all neurons expressing vasoactive intestinal polypeptide along the length of the gastrointestinal tract, patterning defects in the myenteric plexus of the stomach, and altered number and morphology of neurons expressing TH. Our data demonstrate that expression of Hand2 is sufficient and necessary for neurogenesis and expression of a subset of cell type-specific markers in the developing enteric nervous system.

An intact enteric nervous system is required for normal gastrointestinal tract function. Several human conditions result from decreased innervation by enteric neurons; however, the genetic basis of enteric nervous system development and function is incompletely understood. In an effort to increase our understanding of the mechanisms underlying enteric nervous system development, we screened mutagenized zebrafish for changes in the number or distribution of enteric neurons. We also established a motility assay and rescreened mutants to learn whether enteric neuron number is correlated with gastrointestinal motility in zebrafish. We describe mutations isolated in our screen that affect enteric neurons specifically, as well as mutations that affect other neural crest derivatives or have pleiotropic effects. We show a correlation between the severity of enteric neuron loss and gastrointestinal motility defects. This screen provides biological tools that serve as the basis for future mechanistic studies.

Interstitial cells of Cajal (ICC) are specialized cells in smooth muscle organs that generate and propagate pacemaker activity, receive inputs from motor neurons, and serve as mechanosensors. In the gastrointestinal tract, development and maintenance of the ICC phenotype have been linked to intracellular signaling via Kit, but its role in development of ICC during embryogenesis is controversial. Here we have studied the development of functional ICC-MY during the late gestational period in mice. Blocking Kit with a neutralizing antibody before and after development of spontaneous electrical activity (E17 to P0) caused loss of ICC-MY networks and pacemaker activity. ICC-MY and pacemaker activity developed normally in W/+ and W(V)/+ heterozygotes, but failed to develop between E17 to P0 in W/W(V) embryos with compromised Kit function. Muscles treated with Kit neutralizing antibody or the tyrosine kinase inhibitor, imatinib mesylate (STI571), from E17-P0 for 3 days caused loss of functionally developed ICC-MY networks, but ICC-MY and pacemaker activity recovered within 9 days after discontinuing treatment with neutralizing antibody or imatinib mesylate. These data suggest that Kit signaling is an important factor in lineage decision and in the development of functional ICC in late gestation. ICC-MY demonstrate significant plasticity in gastrointestinal tissues. Manipulation of the ICC phenotype might provide useful therapies in gastrointestinal disease where the Kit-positive cell population is either lost or amplified.

Recently, we reported the formation of gut-like structures from mouse ESCs in vitro. To determine whether ESCs provide an in vitro model of gastrointestinal (GI) tracts and their organogenesis, we investigated the morphological features, formation process, cellular development, and regional location within the GI tract by immunohistochemistry, electron microscopy, and reverse transcription-polymerase chain reaction. We also examined the developmental potential by transplantation into kidney capsules. The results demonstrated that Id2-expressing epithelium developed first, alpha-smooth muscle actin appeared around the periphery, and finally, the gut-like structures were formed into a three-layer organ with well-differentiated epithelium. A connective tissue layer and musculature with interstitial cells of Cajal developed, similar to organogenesis of the embryonic gut. Enteric neurons appeared underdeveloped, and blood vessels were absent. Many structures expressed intestinal markers Cdx2 and 5-hydroxytryptamine but not the stomach marker H(+)/K(+) ATPase. Transplants obtained blood vessels and extrinsic nerve growth from the host to prolong life, and even grafts of premature structures did not form teratoma. In conclusion, gut-like structures were provided with prototypical tissue components of the GI tract and are inherent in the intestine rather than the stomach. The formation process was basically same as in gut organogenesis. They maintain their developmental potential after transplantation. Therefore, gut-like structures provide a unique and useful in vitro system for development and stem cell studies of the GI tract, including transplantation experiments.

Preterm birth is associated with immature digestive function that may require the use of total parenteral nutrition and special oral feeding regimens. Little is known about the responses to oral food in the preterm neonate and how enteral nutrients affect the immature gastrointestinal tract (GIT). In vivo studies are difficult to perform in laboratory rodents because of their small body size and that of immature organs at birth, and this makes the large farm animals (e.g., pigs, cattle, sheep) more attractive models in this field. In these species, preterm delivery at 88%-95% gestation is associated clinical complications and degrees of GIT immaturity similar to those in infants born at 70%-90% gestation. Studies in both animals and infants indicate that the immature GIT responds to the first enteral food with rapid increases in gut mass and surface area, blood flow, motility, digestive capacity, and nutrient absorption. To a large extent, the enteral food responses are birth independent, and can be elicited also in utero, at least during late gestation. Nevertheless, preterm neonates show compromised GIT structure, function, and immunology, particularly when delivered by caesarean section and fed diets other than mother's milk. Formula-fed preterm infants are thus at increased risk of developing diseases such as necrotizing enterocolitis, unless special care is taken to avoid excessive nutrient fermentation and bacterial overgrowth. The extent to which results obtained in preterm animals (most notably the pig) can be used to reflect similar conditions in preterm infants is discussed.

This review considers the hypothesis that nutrition during infancy affects developmental epigenetics in the gut, causing metabolic imprinting of gastrointestinal (GI) structure and function. Fundamentals of epigenetic gene regulation are reviewed, with an emphasis on the epigenetic mechanism of DNA methylation. Recent data indicate that transient nutritional and other stimuli during critical periods of prenatal and early postnatal development can affect the establishment or developmental maturation of gene-specific DNA methylation, thereby inducing permanent changes in gene expression. Although epigenetic processes are clearly involved in postnatal mammalian GI development, we currently know very little about these mechanisms. Data indicating that epigenetic gene regulation plays an important role in GI development and pathology are reviewed, and suggestions for future research in this area are offered.

Canonical and noncanonical Wnt signaling regulate crucial events in the development of vertebrates and invertebrates. In this work we show that vertebrate Diversin, a potential orthologue of Drosophila Diego, controls fusion of heart precursors and gastrulation movements in zebrafish embryogenesis. These events are regulated by noncanonical Wnt signaling, which is independent of beta-catenin. We found that Diversin directly interacts with Dishevelled and that this interaction is necessary and sufficient to mediate signals of the noncanonical Wnt pathway to downstream effectors like Rho family GTPases and Jun N-terminal kinase. The ankyrin repeats of Diversin are required for the interaction with Dishevelled, for the activation of noncanonical Wnt signaling, and for the biological responses. The mutation K446M in the DEP domain of vertebrate Dishevelled, which mimics a classical Drosophila loss of function mutation, prevents functional interaction with Diversin's ankyrin repeats. Diversin also affects planar cell polarity in Drosophila, which is controlled by the noncanonical Wnt signaling pathway. Our data thus demonstrate that Diversin and Dishevelled function together in a mutually dependent fashion in zebrafish gastrulation and organ formation.

Vagal sensory axons and migrating neural crest-derived precursor cells follow similar pathways to reach the gut. The crest-derived cells express the netrin receptor deleted in colorectal cancer (DCC) and migrate toward netrins expressed by the intestinal mucosa and pancreas; this attraction is required for the formation of submucosal and pancreatic ganglia. We tested the hypothesis that enteric netrins also attract vagal sensory fibers. These axons were located as a function of age in fetal mice by applying the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) bilaterally to nodose ganglia. DiI-labeled axons were found in the esophagus and proximal stomach by E12 and, more distally, in the small bowel at E14-E16. Transcripts encoding DCC were expressed in the nodose ganglia of mice from E12 to adulthood but were developmentally regulated. Paraesophageal anterior and posterior vagal trunks were DCC immunoreactive from E12 to E16. Transcripts encoding netrin-1 were expressed in the developing foregut and midgut; netrin-1 immunoreactivity was detected in the outer gut mesenchyme and mucosal epithelium. Neurites from explanted E14 nodose ganglia grew selectively toward cocultured E14 distal foregut explants (P < 0.01). Antibodies to DCC specifically abolished this preferential outgrowth (P < 0.05). Nodose axons also grew selectively toward cocultured netrin-secreting 293-EBNA cells (P < 0.005); antibodies to DCC again blocked this preferential outgrowth (P < 0.05). These data suggest that netrins, which are expressed in the bowel, attract DCC-expressing vagal sensory axons.

The Drosophila transcription factor Grainyhead (grh) is expressed in ectoderm-derived tissues where it regulates several key developmental events including cuticle formation, tracheal elongation and dorsal closure. Our laboratory has recently identified three novel mammalian homologues of the grh gene, Grainyhead-like 1, -2 and -3 (Grhl1-3) that rewrite the phylogeny of this family. Using gene targeting in mice, we have shown that Grhl3 is essential for neural tube closure, skin barrier formation and wound healing. Despite their extensive sequence homology, Grhl1 and Grhl2 are unable to compensate for loss of Grhl3 in these developmental processes. To explore this lack of redundancy, and to gain further insights into the functions of this gene family in mammalian development we have performed an extensive in situ hybridisation analysis. We demonstrate that, although all three Grhl genes are highly expressed in the developing epidermis, they display subtle differences in the timing and level of expression. Surprisingly, we also demonstrate differential expression patterns in non-ectoderm-derived tissues, including the heart, the lung, and the metanephric kidney. These findings expand our understanding of the unique role of Grhl3 in neurulation and epidermal morphogenesis, and provide a focus for further functional analysis of the Grhl genes during mouse embryogenesis.