LIM homeodomain transcription factor Isl1 directs normal pyloric development by targeting Gata3.

BACKGROUND: Abnormalities in pyloric development or in contractile function of the pylorus cause reflux of duodenal contents into the stomach and increase the risk of gastric metaplasia and cancer. Abnormalities of the pyloric region are also linked to congenital defects such as the relatively common neonatal hypertrophic pyloric stenosis, and primary duodenogastric reflux. Therefore, understanding pyloric development is of great clinical relevance. Here, we investigated the role of the LIM homeodomain transcription factor Isl1 in pyloric development. RESULTS: Examination of Isl1 expression in developing mouse stomach by immunohistochemistry, whole mount in situ hybridization and real-time quantitative PCR demonstrated that Isl1 is highly expressed in developing mouse stomach, principally in the smooth muscle layer of the pylorus. Isl1 expression was also examined by immunofluorescence in human hypertrophic pyloric stenosis where the majority of smooth muscle cells were found to express Isl1. Isl1 function in embryonic stomach development was investigated utilizing a tamoxifen-inducible Isl1 knockout mouse model. Isl1 deficiency led to nearly complete absence of the pyloric outer longitudinal muscle layer at embryonic day 18.5, which is consistent with Gata3 null mouse phenotype. Chromatin immunoprecipitation, luciferase assays, and electrophoretic mobility shift assays revealed that Isl1 ensures normal pyloric development by directly targeting Gata3. CONCLUSIONS: This study demonstrates that the Isl1-Gata3 transcription regulatory axis is essential for normal pyloric development. These findings are highly clinically relevant and may help to better understand pathways leading to pyloric disease.

Proper development of the outer longitudinal smooth muscle of the mouse pylorus requires Nkx2-5 and Gata3.

BACKGROUND & AIMS: Infantile hypertrophic pyloric stenosis is a common birth anomaly characterized by obstruction of the pyloric lumen. A genome-wide association study implicated NKX2-5, which encodes a transcription factor that is expressed in embryonic heart and pylorus, in the pathogenesis of infantile hypertrophic pyloric stenosis. However, the function of the NKX2-5 in pyloric smooth muscle development has not been examined directly. We investigated the pattern of Nkx2-5 during the course of murine pyloric sphincter development and examined coexpression of Nkx2-5 with Gata3 and Sox9-other transcription factors with pyloric-specific mesenchymal expression. We also assessed pyloric sphincter development in mice with disruption of Nkx2-5 or Gata3. METHODS: We used immunofluorescence analysis to compare levels of NKX2-5, GATA3, and SOX9 in different regions of smooth muscle cells. Pyloric development was assessed in mice with conditional or germline deletion of Nkx2-5 or Gata3, respectively. RESULTS: Gata3, Nkx2-5, and Sox9 are coexpressed in differentiating smooth muscle cells of a distinct fascicle of the pyloric outer longitudinal muscle. Expansion of this fascicle coincides with development of the pyloric sphincter. Disruption of Nkx2-5 or Gata3 causes severe hypoplasia of this fascicle and alters pyloric muscle shape. Although expression of Sox9 requires Nkx2-5 and Gata3, there is no apparent hierarchical relationship between Nkx2-5 and Gata3 during pyloric outer longitudinal muscle development. CONCLUSIONS: Nkx2-5 and Gata3 are independently required for the development of a pyloric outer longitudinal muscle fascicle, which is required for pyloric sphincter morphogenesis in mice. These data indicate that regulatory changes that alter Nkx2-5 or Gata3 expression could contribute to pathogenesis of infantile hypertrophic pyloric stenosis.

Pyloric atresia with epidermolysis bullosa: fetal MRI diagnosis with postnatal correlation.

Pyloric atresia is an uncommon congenital gastric outlet obstruction, accounting for only 1% of gastrointestinal atresias. Up to 55% of cases have associated anomalies, the most common of which is epidermolysis bullosa. Fetal MRI findings of the epidermolysis bullosa-pyloric atresia association have not been previously reported. We present a case of this association diagnosed by prenatal MRI with corroborative postnatal imaging and surgical findings.

Expression pattern of the homeotic gene Bapx1 during early chick gastrointestinal tract development.

Regulation of the Bone Morphogenetic Protein (BMP) signaling pathway is essential for the normal development of vertebrate gastrointestinal (GI) tract, but also for the differentiation of the digestive mesenchymal layer into smooth muscles and submucosal layer. Different studies demonstrated that Bapx1 (for bagpipe homeobox homolog 1) negatively regulates the BMP pathway, but its precise expression pattern during the development and the differentiation of the GI tract mesenchyme actually remains to be examined. Here, we present the spatio-temporal expression profile of Bapx1 in the chick GI tract. We show that Bapx1 is first expressed in the undifferentiated mesenchyme of the gizzard and the colon. After the differentiation of the digestive mesenchyme, we found Bapx1 strongly expressed in the gizzard smooth muscle and in the submucosa layer of the colon. This expression pattern provides new insights into the roles of Bapx1 during the regionalization of the GI tract and the differentiation of the digestive mesenchyme of the colon and the stomach.

Dividing the tubular gut: generation of organ boundaries at the pylorus.

The discrete organs that comprise the gastrointestinal tract (esophagus, stomach, small intestine, and large intestine) arise embryonically by regional differentiation of a single tube that is initially morphologically similar along its length. Regional organ differentiation programs, for example, for stomach or intestine, involve signaling cross-talk between epithelium and mesenchyme and result in the formation of precise boundaries between organs, across which dramatic differences in both morphology and gene expression are seen. The pylorus is a unique area of the gut tube because it not only marks an important organ boundary in the tubular gut (the stomach/intestinal boundary) but is also the hub for the development of multiple accessory organs (liver, pancreas, gall bladder, and spleen). This chapter examines: (a) our current understanding of the molecular and morphogenic processes that underlie the generation of the dramatic epithelial tissue boundary that compartmentalizes stomach and intestine; (b) the tissue interactions that promote development of the accessory organs in this area; and (c) the molecular interactions that specify patterning of the pyloric sphincter. Though the focus here is primarily on the mouse as a model organism, the molecular underpinnings of organ patterning near the pylorus are shared by chick and frog. Thus, further study of these conserved developmental programs could potentially shed light on the mechanisms underlying human pyloric malformations such as infantile hypertrophic pyloric stenosis.

Dynamic patterning at the pylorus: formation of an epithelial intestine-stomach boundary in late fetal life.

In the adult mouse, distinct morphological and transcriptional differences separate stomach from intestinal epithelium. Remarkably, the epithelial boundary between these two organs is literally one cell thick. This discrete junction is established suddenly and precisely at embryonic day (E) 16.5, by sharpening a previously diffuse intermediate zone. In the present study, we define the dynamic transcriptome of stomach, pylorus, and intestinal tissues between E14.5 and E16.5. We show that establishment of this boundary is concomitant with the induction of over a thousand genes in intestinal epithelium, and these gene products provide intestinal character. Hence, we call this process intestinalization. We identify specific transcription factors (Hnf4 gamma, Creb3l3, and Tcfec) and examine signaling pathways (Hedgehog and Wnt) that may play a role in this process. Finally, we define a unique expression domain at the pylorus itself and detect novel pylorus-specific patterns for the transcription factor Gata3 and the secreted protein nephrocan.

Six2 activity is required for the formation of the mammalian pyloric sphincter.

The functional activity of Six2, a member of the so/Six family of homeodomain-containing transcription factors, is required during mammalian kidney organogenesis. We have now determined that Six2 activity is also necessary for the formation of the pyloric sphincter, the functional gate at the stomach-duodenum junction that inhibits duodenogastric reflux. Our data reveal that several genes known to be important for pyloric sphincter formation in the chick (e.g., Bmp4, Bmpr1b, Nkx2.5, Sox9, and Gremlin) also appear to be required for the formation of this structure in mammals. Thus, we propose that Six2 activity regulates this gene network during the genesis of the pyloric sphincter in the mouse.

The development of fetal pylorus during the fetal period.

OBJECTIVE: The objective of this study was to explore the localization of the pylorus, its macroscopic and microscopic development and relationship with neighboring structures. MATERIALS AND METHODS: The study is carried out on 160 human fetuses aged between 9 and 40 weeks of gestation. Abdomen was divided into four quadrants by horizontal and vertical planes passing through the umbilicus. Topographical localization of the pylorus in reference to these quadrants and its distance were determined. Pylorus was divided into pre-pyloric, pyloric, and post-pyloric regions. Starting from the pre-pyloric end, serial sections spanning whole pyloric part were obtained. Wall thickness, the thickness of the muscular coat were measured under light microscope using sections stained with hematoxylin eosin. Sections with the thickest muscular coat were considered as the region where pyloric sphincter was. FINDINGS: Pylorus was located in the right upper quadrant, on the median plane and in the left upper quadrant. There was a significant relation between the thickness of the muscular coat in the stomach, duodenum and the pyloric region and gestational age. In the region of the pyloric sphincter, the rate of increase in the thickness of the muscular coat was higher in the first and the first half of the second trimesters than term fetuses. CONCLUSION: We believe that data obtained in the present study will contribute to the assessment of development of the pyloric region in intra-uterine cases.

Stomach development is dependent on fibroblast growth factor 10/fibroblast growth factor receptor 2b-mediated signaling.

BACKGROUND & AIMS: Fibroblast growth factors (Fgfs) and their receptors (Fgfrs) are important intercellular signaling molecules that are essential to mammalian embryonic development. The signaling pathways between endoderm-derived gastric epithelium and the surrounding mesenchyme are largely unknown; however, the developmental expression profile of the IIIb isoform of Fgfr2 (Fgfr2b) and its main ligand, Fgf10, suggest that they may be strong candidates. Mice lacking either component (Fgfr2b-/- or Fgf10-/-) were examined to determine the role of Fgfr2b-mediated signaling during gastric organogenesis. METHODS: Stomachs from embryonic day 13.5-18.5 Fgfr2b-/-, Fgf10-/-, and wild-type littermates were collected and analyzed by conventional histology, immunohistochemistry, in situ hybridization, and electron microscopy. RESULTS: Fgfr2b-/- and Fgf10-/- fetuses had stomachs smaller than wild-type, consisting of relatively proportionate forestomach but disproportionately reduced glandular stomach, the mucosa of which has low cytoarchitectural complexity with a spiral arrangement of large mucosal folds. During mid to late fetal stages (embryonic day 15.5-18.5), epithelial differentiation to mucous and chief cell lineages was rudimentary, with no expression of several early cytodifferentiation markers including GATA4, GATA6, and H+/K+-adenosine triphosphatase and abnormal expression of members of the hedgehog family of signaling molecules. CONCLUSIONS: Fgfr2b and Fgf10 are part of a signaling network with Sonic hedgehog and Indian hedgehog that are essential to anterior-posterior and radial patterning in gastric development.

Sox9 and Nkx2.5 determine the pyloric sphincter epithelium under the control of BMP signaling.

The organs of the digestive tract are specified by coordinated signaling between the endoderm and mesoderm during development. These epithelial-mesenchymal interactions lead to the organ-specific morphogenesis and differentiation of regions along the gut tube. In this paper, we show that in the chick, the SRY-related transcription factor Sox9 is a marker for the posterior gizzard. Viral misexpression of Sox9 in the gizzard mesoderm is sufficient to specify epithelium characteristic of the pyloric sphincter. Sox9 expression is normally limited to the region of the posterior gizzard under the regulation of BMP signaling from the adjacent midgut. Misexpression of an activated form of BMPR1b in the gizzard upregulates Sox9 expression, while the BMP antagonist noggin down-regulates Sox9 expression in the gizzard mesoderm. Previously, Nkx2.5 was identified as a marker for the mesoderm of the pyloric sphincter. As with Sox9, BMP signaling appears to regulate Nkx2.5 and its ability to determine the pyloric epithelium. Despite these similarities, our evidence suggests that Sox9 and Nkx2.5 are regulated independently by BMP signaling, and act coordinately to specify the pyloric sphincter.

SOX9 specifies the pyloric sphincter epithelium through mesenchymal-epithelial signals.

Gastrointestinal (GI) development is highly conserved across vertebrates. Although several transcription factors and morphogenic proteins are involved in the molecular controls of GI development, the interplay between these factors is not fully understood. We report herein the expression pattern of Sox9 during GI development, and provide evidence that it functions, in part, to define the pyloric sphincter epithelium. SOX9 is expressed in the endoderm of the GI tract (with the exclusion of the gizzard) and its derivate organs, the lung and pancreas. Moreover, SOX9 is also expressed at the mesoderm of the pyloric sphincter, a structure that demarcates the gizzard from the duodenum. Using retroviral misexpression technique, we show that Sox9 expression in the pyloric sphincter is under the control of the BMP signaling pathway, known to play a key role in the development of this structure. By misexpressing SOX9 in the mesoderm of the gizzard, we show that SOX9 is able to transdifferentiate the adjacent gizzard epithelium into pyloric sphincter-like epithelium through the control of mesodermal-epithelial signals mediated in part by Gremlin (a modulator of the BMP pathway). Our results suggest that SOX9 is necessary and sufficient to specify the pyloric sphincter epithelial properties.

Villin: A marker for development of the epithelial pyloric border.

In the adult gastrointestinal tract, the morphologic borders between esophagus and stomach and between stomach and small intestine are literally one cell thick. The patterning mechanisms that underlie the development of these sharp regional divisions from a once continuous endodermal tube are still obscure. In the embryonic endoderm of the developing gut, region-specific expression of certain genes (e.g., intestine-specific expression of the actin bundling protein villin) can be detected as early as 9.0 days post coitum, although the morphologic differentiation of the gut epithelium proper does not begin until 4 to 5 days later. By using a mouse model in which a beta-galactosidase marker has been inserted into the endogenous villin locus, we examined the development of the stomach/intestinal (pyloric) border during gut organogenesis. The data indicate that the border is not sharp from the outset. Rather, the initial border region is characterized by a decreasing gradient of villin/beta-galactosidase expression that extends into the distal stomach. A sharp epithelial border of villin/beta-galactosidase expression appears abruptly at day 16 and is further refined over the next 3 weeks to form the distinct one-cell-thick border characteristic of the adult. These results indicate that an important previously unrecognized patterning event occurs in the gut epithelium at 16 days; this event may define an epithelial compartment boundary between the stomach and the intestine. The villin/beta-galactosidase mouse model characterized here provides an excellent substrate with which to further dissect the mechanisms involved in this patterning process.

Hunterian Lecture. The ontogeny of the peptide innervation of the human pylorus with special reference to understanding the aetiology and pathogenesis of infantile hypertrophic pyloric stenosis.

Infantile hypertrophic pyloric stenosis is the most common cause for urgent abdominal surgery in infancy. The aetiology of the condition is unknown. The ontogeny of the innervation and structure of the normal infant pylorus is unknown. A variety of differing histological features have been attributed to this condition and a number of animal models have been described. The histological changes in the human condition and those in the animal models have not been quantified and statistically verified. Thus, precise comparisons cannot be made. Immunohistochemistry was the principal technique employed in this study. Using this technique, the ontogeny and structure of the normal infant pylorus have been documented. The morphological and immunohistochemical changes underlying infantile hypertrophic pyloric stenosis have been quantified for the first time and compared with the quantified changes in natural and experimental animal models of this condition.

Roles of BMP signaling and Nkx2.5 in patterning at the chick midgut-foregut boundary.

Patterning of the gut into morphologically distinct regions results from the appropriate factors being expressed in strict spatial and temporal patterns to assign cells their fates in development. Often, the boundaries of gene expression early in development correspond to delineations between different regions of the adult gut. For example, Bmp4 is expressed throughout the hindgut and midgut, but is not expressed in the early gizzard. Ectopic BMP4 in the gizzard caused a thinning of the muscularis. To understand this phenotype we examined the expression of the receptors transducing BMP signaling during gut development. We find that the BMP receptors are differentially expressed in distinct regions of the chicken embryonic gut. By using constitutively activated versions of the BMP type I receptors, we find that the BMP receptors act similarly to BMP4 in the gizzard when ectopically expressed. We show that the mesodermal thinning seen upon ectopic BMP signaling is due to an increase in apoptosis and a decrease in proliferation within the gizzard mesoderm. The mesodermal thinning is characterized by a disorganization and lack of differentiation of smooth muscle in the gizzard mesoderm. Further, ectopic BMP receptors cause an upregulation of Nkx2.5, the pyloric sphincter marker, similar to that seen with ectopic BMP4. This upregulation of Nkx2.5 is a cell-autonomous event within the mesoderm of the gizzard. We also find that Nkx2.5 is necessary and sufficient for establishing aspects of pyloric sphincter differentiation.

Control systems of gastrointestinal motility are immature at birth in dogs.

Networks of interstitial cells of Cajal (ICC) in the myenteric plexus (Myp) or circular muscle (CM) function as pacemakers for gastrointestinal slow waves. ICC in contact with muscle and closely associated with nerves in the CM may mediate inhibitory neurotransmission. We wondered if ICC in Myp and CM and their connections are immature at birth and mature first in the proximal gut in association with nerves. Tissues from lower esophageal sphincter (LES), pylorus (PYL), small intestine (SI) and colon (CO) of 18 term fetal dogs taken from six females were fixed and prepared for ultrastructural examination and studied. Ganglia were present where expected in the Myp and submucous plexus (SMP). ICC cells were present in the Myp of PYL, SI and CO and appeared to have normal relationships to the outer border of CM as in adults. ICC in CM were found associated with nerves in the LES and in PYL, but not in SI or CO. However, axons in CM were everywhere usually free of glial covering, indicating ongoing migration or development. No organized deep muscular plexus (DMP) in SI or submuscular plexus (SP) in colon was present. Visible gap junctions were absent everywhere except for very rare ones between circular muscle cells. We conclude that at birth the neural and ICC networks of CM are more immature in intestine and colon than in oesophagus and stomach. Development of nerve and ICC of CM in oesophagus and stomach apparently precedes that in the remaining gut. However networks in these regions have not achieved adult organization and ICC and smooth muscle cells are anatomically poorly coupled. These findings suggest the reasons that gut motility at birth will not be adult in pattern are because ICC, nerve and muscle control systems are not fully differentiated. Further developmental delays in ICC and nerve maturation could have serious consequences for feeding of infant animals.

The ontogeny of innervation of the human pylorus.

PURPOSE: The aim of this study was to document the vagal innervation and expression of neuropeptides, neuronal nitric oxide synthase (nNOS), and neural cell adhesion molecule (NCAM) in the neuromuscular system of the developing human pylorus. METHODS: Specimens of human pylorus (n = 54; age range, 8 weeks' gestation to 6 months postnatal) were studied. Vagal innervation was determined by Dil autofluorescence. A wide range of neuropeptides, NCAM, and the neural isoform of NOS were examined by immunohistochemistry. RESULTS: Vagal innervation was first recognized in the myenteric plexus in the 12-week-old fetus as was vasoactive intestinal polypeptide (VIP) expression. Neuropeptides were present from 8 weeks' gestation and appeared to be expressed progressively from the adventitia toward the mucosa and showed an adultlike profile by 23 weeks' gestation. A craniocaudal pattern of expression was noted for VIP and nNOS. Alpha smooth muscle actin was expressed by muscle fibers of the muscularis propria from 8 weeks and the muscularis mucosae by 14 weeks. All the isoforms of NCAM examined were expressed from 8 weeks in the muscularis propria and by 12 weeks in the submucosa. CONCLUSION: The expression of the antigens studied correlated with the gestational age and development of the pylorus.

Developmental changes in mucosubstances revealed by immunostaining with antimucus monoclonal antibodies and lectin staining in the epithelium lining the segment from gizzard to duodenum of the chick embryo.

The mucosubstances in the epithelium lining the segment from gizzard to duodenum during development of the chick embryo was studied histochemically using monoclonal antibodies against gizzard mucus and lectins, with attention to the regional differentiation of the epithelium in this segment. The anterior limit of epithelial CdxA mRNA expression detected by in situ hybridisation, which served as the position of the gizzard-duodenal boundary, was clearly found from d 3. Granules positive for some antibodies or lectins were found in the region ranging from the posterior part of the gizzard to the duodenum at d 3, which was followed by an increase in the number of granules and a gradual enlargement of the granule-positive area to the anterior part of the gizzard over 4-6 d. From d 4, the epithelia of the gizzard body and of the pyloric or duodenal region came to be differently stained with some antibodies or lectins. From d 10, each region showed a specific pattern of staining. The epithelia of the gizzard body and pyloric region contained abundant mucus granules with a different staining pattern. In the duodenum the number of stained granules was low except in occasional goblet cells. Thus the epithelia of the gizzard body, pyloric region and duodenum may produce different mucosubstances and the regional differentiation in these epithelia may start at rather early stages soon after the formation of digestive tube.

The ontogeny and distribution of glucagon- and pancreatic polypeptide-immunoreactive cells in the gastrointestinal tract of the chicken.

The times of first appearance and the distribution of APP- and glucagon-immunoreactive cells have been established in the embryonic chick gut between 11 days of incubation and hatching. These immunoreactive cell types appeared for the first time at 13 days of incubation, APP-immunoreactive cells in the duodenum and upper ileum and glucagon-immunoreactive cells in the proventriculus and duodenum. At 14 days, APP-immunoreactive cells were detected in the proventriculus and lower ileum and glucagon-immunoreactive cells in the pyloric region, upper and lower ileum. Thereafter both APP- and glucagon-immunoreactive cells increased in frequency until the numbers at hatching were approximated, APP-immunoreactive cells at 19 days and glucagon immunoreactive cells at 17 1/2 days of incubation. No APP- or glucagon-immunoreactive cells were found in the gizzard, caecum or rectum at any of the selected stages examined. When these types of endocrine cells first appeared, the surface epithelium of the gastrointestinal tract was relatively undifferentiated. A few glands were present in the proventriculus only, at this stage. Thereafter immunoreactive cells of both types were found in the glandular epithelium of the proventriculus, pyloric region and small intestine soon after morphogenesis had begun.