Molecular analysis of the determination of developmental fate in the small intestinal epithelium in the chicken embryo

Determination of the developmental fate in the small intestinal epithelium of the chicken embryo has not been fully analyzed up to the present. This study was carried out to analyze the determination time of the developmental fate of the small intestinal epithelium under the influence of other mesenchymes. The small intestinal epithelium reassociated and cultivated with the proventricular or gizzard mesenchyme or the dermis expressed chicken intestinal fatty acid binding protein, sucrase and CdxA as occurs during the normal development of the small intestinal epithelium. The presumptive intestinal endoderm taken from an earlier stage embryo and associated and cultivated with the proventricular or gizzard mesenchyme, showed gene expression patterns which were the same as those found in normal development. However, when the dermis was associated, the epithelium expressed sonic hedgehog, but never expressed intestinal epithelial- or stomach epithelial-markers. These results indicate that the determination of the developmental fate in the small intestinal epithelium and acquisition of autodifferentiation potency occur at the early stage of the gut development. Moreover the presumptive intestinal endoderm needs the supportive influence of the gut mesenchyme in order to differentiate fully into the intestinal epithelium.     

Regional gene expression in the epithelia of the Xenopus tadpole gut

In recent years much progress has been made in the understanding of the genes and mechanisms involved in specification of the cells of the endoderm, which give rise to the epithelium of the gut and respiratory system. However, little is known about the way in which the gut becomes patterned along its anterior-posterior axis, that is, how boundaries are established between the different epithelia of the gut tube. Here we show that the expression patterns of five genes divide the Xenopus tadpole gut epithelium into at least four regions along this axis in the undifferentiated, 3-day-old gut (stage 41), and that these divisions are maintained until at least 7 days, when cell differentiation is well under way. In addition, the restricted expression patterns of these genes clearly mark the anterior and posterior boundaries of the intestine. Xsox2 is expressed in the anterior gut, spanning the oesophagus and stomach but terminating at the stomach/intestine boundary. Xcad1 and Xcad2, two caudal-type homeobox genes, are expressed in a region with an anterior limit at this boundary and a posterior limit between the colon and proctodeum, therefore covering the whole of the small and large intestines. Intestinal fatty acid binding protein (IFABP) is expressed only in the anterior small intestine, and the even-skipped homeobox gene Xhox3 is expressed in the most posterior part of the gut, the proctodeum.     

Effects of retinoic acid on the endoderm in Xenopus embryos

 When Xenopus embryos from mid-tailbud to early tadpole stages were exposed to retinoic acid (RA), the gut developed with an uncoiled, straight intestinal tube, morphogenesis of the liver and stomach was affected and intestinal epithelial cells developed without a brush border and alkaline phosphatase activity. However, the temporal and spatial expression pattern of XlHbox 8, the only homeobox gene expressed in the endoderm was unaffected. In lateral plate mesodermal cells the expression of α-smooth muscle (SM) actin was delayed. A similar syndrome has been reported in a study of embryos lacking functional FGF receptors in which it was proposed that the uncoiled intestinal tube and the delayed differentiation of the intestinal muscle cells are causally related. Our results support this proposition and further suggest that mesenchymal-epithelial interactions concerned with regional specification of the endoderm may be impaired resulting in other defects in the gut. Received: 3 October 1997 / Accepted: 3 February 1998     

Clonally related cells are restricted to organ boundaries early in the development of the chicken gut to form compartment boundaries

The gut organs are all derived from a simple, undifferentiated, linear gut tube. We analyzed the lineage relationships of cells derived from this gut tube in chicken embryos, determining where the progeny of a single cell are located within the gut. We find that daughter cells derived from a single progenitor can populate both the gizzard (chicken stomach) and the small intestine early in development, but that clonally related cells are restricted to a single organ by stage 12. We also find that clonally related cells can populate different mesodermal layers within the radial axis of the gut throughout all of the stages tested in these experiments. Many genes that have organ-specific expression patterns within the gut have been isolated. The onset of these restricted expression patterns correlates with the time that clonal boundaries appear to form, suggesting that these genes might be involved in the establishment of compartment boundaries, which prevent cells on one side of the boundary from intermingling with cells on the other side of the boundary.     

Regional specification of the endoderm in the early chick embryo

In the avian embryo, the endoderm, which forms a simple flat-sheet structure after gastrulation, is regionally specified in a gradual manner along the antero-posterior and dorso-ventral axes, and eventually differentiates into specific organs with defined morphologies and gene expression profiles. In our study, we carried out transplantation experiments using early chick embryos to elucidate the timing of fate establishment in the endoderm. We showed that at stage 5, posteriorly grafted presumptive foregut endoderm expressed CdxA , a posterior endoderm marker, but not cSox2 , an anterior endoderm marker. Conversely, anteriorly grafted presumptive mid-hindgut endoderm expressed cSox2 but not CdxA . At stage 8, posteriorly grafted presumptive foregut endoderm also expressed CdxA and not cSox2 , but anteriorly grafted presumptive mid-hindgut endoderm showed no changes in its posterior-specific gene expression pattern. At stage 10, both posteriorly grafted foregut endoderm and anteriorly grafted mid-hindgut endoderm maintain their original gene expression patterns. These results suggest that the regional specification of the endoderm occurs between stages 8 and 10 in the foregut, and between stages 5 and 8 in the mid-hindgut.     

A novel antigen Apsi is expressed in a half population of endoderm cells in normal and LiCI-treated starfish embryos

A novel antigen, Apsi, revealed a tissue specific expression in the starfish embryo. Apsi was detected in the stomach and intestine of the bipinnaria larva by immunofluorescence microscopy, but was not detected in the esophagus or ectoderm. The expression of Apsi was zygotic and first detected at day 3 after fertilization. Using this antigen as a molecular marker, the effect of LiCI treatment on development was examined by counting the cell number of each germ layer and endoderm tissues on serial paraffin sections. At day 5 larva stage, the ratio of the cell number of ectoderm, esophagus, Apsi-expressing tissue (stomach and intestine) and mesoderm was 75:10:10:5. The corresponding ratio in LiCI-treated embryo was 68:14:14:4. LiCI treatment increased the cell number of endoderm by 40%, at the expense of a 10% decrease in the cell number of ectoderm. In intact embryos, approximately half the endoderm cells expressed Apsi antigen, while the other half did not. LiCI treatment did not change this ratio of Apsi expression in endoderm tissues. These observations indicate that LiCI treatment of early blastulae affects the commitment of ectoderm/endoderm but does not affect the differentiation of the esophagus/stomach and intestine.     

Cdx2 initiates histodifferentiation of the midgut endoderm

Null mutation or haploinsufficiency of Cdx2 results in the development of heterotopic lesions with a gastric phenotype in the midgut endoderm. Conversely transgenic expression of Cdx2 in the stomach causes the endoderm to differentiate into intestinal-type mucosa. We demonstrate that the mesoderm adjacent to intestinal heterotopic areas expresses stomach specific Barx1 while the surrounding mesoderm is Barx1 negative. We conclude that the initiation of gut histodifferentiation lies in the endodermal expression of Cdx2 and that endodermal/mesodermal cross-talk involving Barx1 with appropriate feedback loops results in the development of the postnatal gut phenotype.     

CHRONOLOGICAL CHANGES IN THE INDUCTIVE ABILITY OF THE MESENCHYME OF THE DIGESTIVE ORGANS IN AVIAN EMBRYOS

Dissociation and reassociation experiments were carried out to study the inductive ability of mesenchyme of the oesophagus, gizzard and intestine of the chicken embryo, using 3-day-old quail embryonic allantoic endoderm as an effector tissue. The mesenchyme of the oesophagus and gizzard possesses inductive ability until the Ilth day of incubation. Thereafter, it no longer has inductive influence upon the allantoic endoderm. The intestinal mesenchyme was favourable to differentiation of allantoic endoderm into intestinal epithelium even on the I5th day of incubation. In all types of recombination tested, goblet cells differentiated among allantoic endodermal cells.     

Notch signaling functions as a binary switch for the determination of glandular and luminal fates of endodermal epithelium during chicken stomach development

During development of the chicken proventriculus (glandular stomach), gut endoderm differentiates into glandular and luminal epithelium. We found that Delta1-expressing cells, undifferentiated cells and Notch-activated cells colocalize within the endodermal epithelium during early gland formation. Inhibition of Notch signaling using Numb or dominant-negative form of Su(H) resulted in a luminal differentiation, while forced activation of Notch signaling promoted the specification of immature glandular cells, but prevented the subsequent differentiation and the invagination of the glands. These results suggest that Delta1-mediated Notch signaling among endodermal cells functions as a binary switch for determination of glandular and luminal fates, and regulates patterned differentiation of glands in the chicken proventriculus.     

The murine A33 antigen is expressed at two distinct sites during development, the ICM of the blastocyst and the intestinal epithelium

The expression pattern of the murine A33 antigen has been defined during development using wholemount immunohistochemistry. Two temporally and spatially distinct sites of expression were identified: the inner cell mass of the blastocyst and the endoderm cell layer of the intestinal tract where expression is initiated at E14.5 in the hindgut and subsequently extends throughout the length of the intestine. The onset of mA33 antigen expression in the gut occurs at the beginning of an extensive phase of cell movement involved in the conversion of the endoderm cell layer to a single cell layer of polarized epithelium. Expression of mA33 antigen is then maintained into adulthood, where it is a definitive marker of intestinal epithelium.     

Chronological changes in the sucrase antigen-inducing activity and development of the regional identity in the intestinal mesoderm of the chick embryo

Developmental changes in mesodermal activity to induce intestine-like differentiation expressing sucrase antigen in the endoderm and changes in endodermal reactivity to such an activity in the digestive tract of the chick embryo were analyzed. Digestive-tract endoderms of embryos at 3 days of incubation were highly responsive to the inductive effect of the 5 day duodenal mesenchyme, with the stomach endoderm lying nearest to the intestine having the highest reactivity. Endodermal reactivity decreased with increasing age. It was almost absent in the endoderm of the esophagus or proventriculus of 6 day embryos and in the endoderm of the gizzard of 7 day embryos. The activity of the mesoderm to induce intestine-like differentiation in 5 day gizzard endoderm was high in the 5–10 day duodenal mesenchyme, but was rarely found in 14 day duodenal mesenchyme. This activity was specific to intestinal mesenchymes, among which the duodenal mesenchyme had the highest activity in 5 day embryos. The 3 day intestinal mesenchyme may already have the inductive activity. The presumptive intestinal mesoderm of 1.5 day embryos seemed to have a slight or no activity, but it may have intestinal identity and may manifest a high inductive activity later.