Early Determination of Developmental Fate in Presumptive Intestinal Endoderm of the Chicken Embryo

The endodermal epithelia of esophagus, proventriculus and gizzard of 6-day chicken embryos can form glands and express embryonic chicken pepsinogen (ECPg), when they are subjected to the influence of proventricular mesenchyme, while intestinal epithelium of the same age cannot respond to the inductive influence of proventricular mesenchyme. We attempted in this paper to know whether this regional difference of epithelia to respond to mesenchymal influence originates very early in development or it is established gradually in the course of development of digestive tract.
The young presumptive intestinal endoderm taken from embryos having 15–20 somites was associated and cultivated with 6-day proventricular mesenchyme. The presumptive intestinal endoderm never expressed ECPg although it formed gland-like structures. In the control explants composed of presumptive stomach endoderm and proventricular mesenchyme, glands were formed and gland cells expressed ECPg detected by immunocytochemistry and in situ hybridization.
These results indicate that the developmental fate of presumptive intestinal endoderm is determined rather strictly at very early developmental stage, and suggest that the segregation of at least two cell lineages occurs early in the development; one which can express ECPg under the influence of proventricular mesenchyme, and another one which cannot express ECPg and differentiates mainly into 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.     

Demonstration of sucrase immunoreactivity of the brush border induced by duodenal mesenchyme in chick stomach endoderm

Summary When stomach endoderm of chick embryos was recombined and cultured with duodenal mesenchyme, the endoderm developed a brush border structure over a large area and also differentiated into mucous cells in a small area according to its own developmental fate. In the present investigation, we examined whether the induced brush border structure expressed sucrase antigen by immunoelectron microscopy using the antiserum raised against chicken sucrase. Sucrase immunoreactivity could be detected as ferritin particles in the region where the brush border was induced, whereas it was never detected on microvilli of endodermal cells which differentiated into the mucous cells. Thus, almost all of the endodermal cells could be identified as either small intestine-type cells possessing the sucrase antigen or stomach-type cells possessing mucous granules but not the sucrase antigen. The results indicate that stomach endodermal cells of chick embryos can differentiate not only morphologically but also functionally into typical intestinal epithelial cells under the inductive influence of the duodenal mesenchyme.     

Both Humoral Mesenchymal Factors and the Close Association between the Hepatic Endoderm and Mesenchyme can be Involved in Liver Formation of Mouse Embryos

Previous studies with tissue recombination experiments demonstrated that the splanchnic mesenchymes, including hepatic, pulmonary and stomach mesenchymes can support hepatocyte differentiation from the hepatic endoderm in 9.5-day mouse embryos. This phenomenon corresponds to the second hepatic induction. The present study was undertaken to determine whether direct cell-cell contacts between the hepatic endoderm and mesenchyme are required for hepatocyte differentiation, using transfilter experiments in which membrane filters with various pore sizes were inserted between the endoderm and the hepatocyte-inducing mesenchyme (the chick lung mesenchyme). Hepatocyte differentiation occurred even when the direct cell-cell contacts between the hepatic endoderm and the mesenchyme were absent, suggesting that humoral factors may work in this interaction. However, growth of hepatocytes was most prominent in the transfilter experiments with filters having pore sizes of 0.2 and 0.8 mum, which permitted mesenchymal cells or their cell processes to penetrate to the side of the endoderm. These results suggest that two types of tissue interactions, including humoral mesenchymal factors and very local tissue interactions such as direct cell-cell contacts, may be involved in the second step of hepatic induction.     

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.     

Endoderm- and mesenchyme-dependent commitment of the differentiated epithelial cell types in the developing intestine of rat

During organogenesis, the intestinal tract progressively acquires a functional regionalization along the antero-posterior axis. Positional information needed for enterocytes has been studied, but the mechanisms that control Paneth and endocrine cell differentiation are poorly understood. We have used a model of endoderm/mesenchyme cross-associations to evaluate the respective roles of endoderm and mesenchyme in the cytodifferentiation of these epithelial cells. Heterotopic cross-associations comprising endoderm and mesenchyme from the presumptive proximal jejunum and colon were developed as xenografts in nude mice. Our results show that endoderm from the presumptive proximal jejunum when associated with colonic mesenchyme generate small intestinal enterocytes. Interestingly, no lysozyme-producing cells were generated. On the other hand, associations comprising colon endoderm and jejunal mesenchyme showed heterodifferentiation with typical small intestinal morphology with sucrase-isomaltase expression and Paneth cell differentiation. Heterotopic associations developed enteroendocrine cell patterns according to the normal fate of the endodermal moiety. As enteroendocrine cell commitment seems to occur before the other intestinal cell types, we cannot exclude a role of instructive signals from the mesenchyme on endocrine cell differentiation earlier in the development. These results identified a complex pattern of cell commitment, dependent of the differentiation type of the epithelial cell, on the regional origin of the endoderm and the associated mesenchyme.     

Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice

During embryogenesis, the pancreas arises from dorsal and ventral pancreatic protrusions from the primitive gut endoderm upon induction by different stimuli from neighboring mesodermal tissues. Recent studies have shown that Retinoic Acid (RA) signaling is essential for the development of the pancreas in non-mammalian vertebrates. To investigate whether RA regulates mouse pancreas development, we have studied the phenotype of mice with a targeted deletion in the retinaldehyde dehydrogenase 2 (Raldh2) gene, encoding the enzyme required to synthesize RA in the embryo. We show that Raldh2 is expressed in the dorsal pancreatic mesenchyme at the early stage of pancreas specification. RA-responding cells have been detected in pancreatic endodermal and mesenchymal cells. Raldh2-deficient mice do not develop a dorsal pancreatic bud. Mutant embryos lack Pdx 1 expression, an essential regulator of early pancreas development, in the dorsal but not the ventral endoderm. In contrast to Pdx 1-deficient mice, the early glucagon-expressing cells do not develop in Raldh2 knockout embryos. Shh expression is, as in the wild-type embryo, excluded from the dorsal endodermal region at the site where the dorsal bud is expected to form, indicating that the dorsal bud defect is not related to a mis-expression of Shh. Mesenchymal expression of the LIM homeodomain protein Isl 1, required for the formation of the dorsal mesenchyme, is altered in Raldh2--/-- embryos. The homeobox gene Hlxb9, which is essential for the initiation of the pancreatic program in the dorsal foregut endoderm, is still expressed in Raldh2--/-- dorsal epithelium but the number of HB9-expressing cells is severely reduced. Maternal supplementation of RA rescues early dorsal pancreas development and restores endodermal Pdx 1 and mesenchymal Isl 1 expression as well as endocrine cell differentiation. These findings suggest that RA signaling is important for the proper differentiation of the dorsal mesenchyme and development of the dorsal endoderm. We conclude that RA synthesized in the mesenchyme is specifically required for the normal development of the dorsal pancreatic endoderm at a stage preceding Pdx 1 function.     

Importance of a fibroblastic support for in vitro differentiation of intestinal endodermal cells and for their response to glucocorticoids

Microexplants of 14- or 15-day-old fetal rat intestinal endoderm, separated from mesenchyme by collagenase, were placed on culture dishes coated with different extracellular matrix components or on confluent monolayers of intestinal mesenchymal cells or of fetal skin fibroblasts. Only small variations in the attachment or spreading of the endodermal cells could be observed when they were cultured on the different acellular substrata, and their survival never exceeded one week. When cocultured with intestinal or skin fibroblasts, endodermal cells proliferated and the survival time was prolonged to 2 or 3 weeks. Furthermore, differentiation, as assessed by the polarization of the cells, occurred and was characterized by the maturation of apical brush borders and by the synthesis of microvillar digestive enzymes visualized immunocytochemically with monoclonal antibodies. Glucocorticoids accelerated structural differentiation and stimulated or induced brush border enzymes only in the coculture conditions. These experiments emphasize the role of a fibroblastic support without tissue specificity on the cytodifferentiation of intestinal endodermal cells. They also suggest a mesenchymal dependence on the hormonal response.     

Electron microscopic study of in vitro differentiation of the small intestinal endoderm in young bird embryo in the presence or absence of mesenchyme

Electron microscopical studies demonstrated that the small intestinal endoderm of young avian embryos cultures in vitro in the presence or absence of mesenchyme can differentiate into an absorptive epithelium with the brush border, and that, in the absence of mesenchyme the brush border develops much earlier than in the presence of mesenchyme, but goblet cells do not appear and morphogenesis of villi does not occur. These results show that the intestinal mesenchyme controls the endodermal differentiation, though the undifferentiated endoderm possesses self-differentiation potency.     

Effects of human fetal gastroenteric mesenchymal cells on some developmental aspects of animal gut endoderm

Human intestinal and gastric mesenchymal cells were associated with chick and rat intestinal endoderm in order to test their species-specific capacity on epithelial differentiation. Primary cell cultures were established from human intestinal and gastric mesenchyme. Animal intestinal endoderms were associated with both cell types, grafted in ovo and allowed to develop for 12 days. The morphologic and enzymatic differentiation of the recombinants demonstrated two types of inductive properties exerted by human fetal intestinal and gastric mesenchymal cells, respectively. Firstly, human intestinal mesenchymal cells triggered intrinsic developmental capacities in chick and rat endoderm, i.e. enhanced structural brush-border maturation in both species and precocious sucrase induction in rat endoderm. Secondly, human gastric mesenchymal cells provoked the partial conversion of chick intestinal endoderm into gastric structures. Such properties were not found in homologous animal mesenchymes.     

Intestinal mesenchyme provokes differentiation of intestinal endocrine cells in gizzard endoderm

The gizzard (muscular stomach) of chicks is deficient in endocrine cells at hatching. It has previously been shown that proventricular types and proportions of endocrine cells can be induced in gizzard endoderm under the influence of proventricular (glandular stomach) mesenchyme. In order to test its capacity to form nongastric endocrine cell types, gizzard endoderm of 3.75- to 5-day chick embryos was combined with mesenchyme from the small intestine of 3.5- to 4-day quail embryos. The combinations were grown as chorio-allantoic grafts until they attained an incubation age comparable to that of hatching chicks. Controls comprised reassociated endoderm and mesenchyme of chick gizzard and of quail intestine. In the experimental grafts, morphogenesis was predominantly intestinal but some grafts showed gizzard-like features, particularly if the endoderm had been provided by older donors. All intestinal endocrine cell types, including those also found in the normal proventriculus (serotonin-, glucagon-, pancreatic polypeptide-, neurotensin- and somatostatin-immunoreactive cells) differentiated in experimental grafts, some even where morphogenesis was gizzard-like. Hence progenitors of not only gastric, but also intestinal, endocrine cells are indeed present in gizzard endoderm. The possibility that gizzard mesenchyme is inhibitory to endocrine cell differentiation is mooted. Motilin- and secretin-immunoreactive cells, which are characteristic of the intestine but not of the proventriculus of chicks at hatching, were respectively sparse or absent when the endoderm was derived from older donors. Thus the ability of gizzard endoderm to differentiate into nongastric endocrine cell types declines before its capacity to form gastric types. The unexpected appearance of gastrin-releasing peptide (GRP)-immunoreactive cells, a proventricular type not found in normal chick intestine, suggests that the intestinal mesenchyme, at least in this instance, was exercising a permissive role.     

Fetal endoderm primarily holds the temporal and positional information required for mammalian intestinal development

In rodents, the intestinal tract progressively acquires a functional regionalization during postnatal development. Using lactase-phlorizin hydrolase as a marker, we have analyzed in a xenograft model the ontogenic potencies of fetal rat intestinal segments taken prior to endoderm cytodifferentiation. Segments from the presumptive proximal jejunum and distal ileum grafted in nude mice developed correct spatial and temporal patterns of lactase protein and mRNA expression, which reproduced the normal pre- and post-weaning conditions. Segments from the fetal colon showed a faint lactase immunostaining 8-10 d after transplantation in chick embryos but not in mice; it is consistent with the transient expression of this enzyme in the colon of rat neonates. Heterotopic cross-associations comprising endoderm and mesenchyme from the presumptive proximal jejunum and distal ileum developed as xenografts in nude mice, and they exhibited lactase mRNA and protein expression patterns that were typical of the origin of the endodermal moiety. Endoderm from the distal ileum also expressed a normal lactase pattern when it was associated to fetal skin fibroblasts, while the fibroblasts differentiated into muscle layers containing alpha-smooth- muscle actin. Noteworthy, associations comprising colon endoderm and small intestinal mesenchyme showed a typical small intestinal morphology and expressed the digestive enzyme sucrase-isomaltase normally absent in the colon. However, in heterologous associations comprising lung or stomach endoderm and small intestinal mesenchyme, the epithelial compartment expressed markers in accordance to their tissue of origin but neither intestinal lactase nor sucrase-isomaltase. A thick intestinal muscle coat in which cells expressed alpha-smooth- muscle actin surrounded the grafts. The results demonstrate that: (a) the temporal and positional information needed for intestinal ontogeny up to the post-weaning stage results from an intrinsic program that is fixed in mammalian fetuses prior to endoderm cytodifferentiation; (b) this temporal and positional information is primarily carried by the endodermal moiety which is also able to change the fate of heterologous mesodermal cells to form intestinal mesenchyme; and (c) the small intestinal mesenchyme in turn may deliver instructive information as shown in association with colonic endoderm; yet this effect is not obvious with nonintestinal endoderms.     

Pepsinogen Induction in Chick Stomach Epithelia by Reaggregated Proventricular Mesenchymal Cells In Vitro

In vitro organ culture system which permits embryonic chick proventriculus (glandular stomach) to synthesize pepsinogen de novo was developed. Explants of the proventricular rudiment were cultured on Millipore filters in Medium 199 with Earle's salts supplemented with 50% 12-day embryo extract at 38°C in 95% air and 5% CO₂.
In these culture conditions, pepsinogen, a functional marker protein of proventriculus, was first detected after 3 days of cultivation of 6-day chick proventricular rudiment. When recombined and cultured with 6-day proventricular mesenchyme, 6-day oesophageal, proventricular or gizzard (muscular stomach) epithelium expressed pepsinogen while small intestinal epithelium did not. These results were consistent with the previous results obtained by chorioallantoic membrane (CAM) grafting, and showed that the culture conditions are permissive for pepsinogen expression.
When recombined and cultured with reaggregated mesenchymal cells isolated from 6-day proventricular mesenchymal fragments, both 6-day proventricular and gizzard epithelia formed glandular structure and expressed pepsinogen. This indicates that the proventricular mesenchymal cells retain the ability to induce morphogenesis and cytodifferentiation of the proventricular epithelium even if the normal organization of proventricular mesenchyme is once destroyed.     

Inductive properties of fibroblastic cell cultures derived from rat intestinal mucosa on epithelial differentiation

The present study represents a first attempt to elucidate the regulatory properties displayed by the non-epithelial portion of the intestinal mucosa, growing as fibroblasts in monolayer cultures. Thus, we compared the inductive action of 6-day suckling rat duodenal fibroblasts with that displayed by chick embryonic intestinal mesenchyme on the heterotypic cytodifferentiation of 5 1/2-day chick embryonic gizzard endoderm. The latter, isolated by 0.03% collagenase, was surrounded by intestinal intramucosal fibroblastic cell sheets. As control experiments, fibroblastic cells derived from the intestinal muscle or from 20-day fetal rat skin and lung were used. Every type of association was grafted into the coelomic cavity of 3-day chick embryos for 11 to 12 days, a system providing their vascularization and growth. The results clearly demonstrate that the mucosal fibroblastic cells of rat intestine were as potent as embryonic intestinal mesenchyme in inducing brush-border enzymes like sucrase and maltase, in conformity with an induced intestinal morphology. In contrast, the control fibroblastic cells were completely ineffective.     

Lhx2 is expressed in the septum transversum mesenchyme that becomes an integral part of the liver and the formation of these cells is independent of functional Lhx2

Liver development is based on reciprocal interactions between ventral foregut endoderm and adjacent mesenchymal tissues. Targeted disruption of the LIM-homeobox gene Lhx2 has revealed that it is important for the expansion of the liver during embryonic development, whereas it appears not to be involved in the induction of hepatic fate. It is not known whether Lhx2 is expressed in the endodermal or mesenchymal portion of the liver, or if the cells normally expressing Lhx2 are absent or present in the liver of Lhx2(-/-) embryos. To address this we have analyzed gene expression from the Lhx2 locus during hepatic development in wild type and Lhx2(-/-) mice. Lhx2 is expressed in cells of the septum transversum mesenchyme adjacent to the liver bud from embryonic day 9. The hepatic cords subsequently migrate into and intermingle with the Lhx2+ cells of the septum transversum mesenchyme. Lhx2 expression is thereafter maintained in a subpopulation of mesenchymal cells in the liver until adult life. In adult liver the Lhx2+ mesenchymal cells co-express desmin, a marker associated with stellate cells. At embryonic day 10.5, cells expressing the mutant Lhx2 allel are present in Lhx2(-/-) livers, and expression of Hlx, hepatocyte growth factor, Hex and Prox1, genes known to be important in liver development, is independent of functional Lhx2 expression. Thus, Lhx2 is specifically expressed in the liver-associated septum transversum mesenchyme that subsequently becomes an integral part of the liver and the formation of these mesenchymal cells does not require functional Lhx2.     

Origin of pancreatic precursors in the chick embryo and the mechanism of endoderm regionalization

To study the developmental origin of the pancreas we used DiI crystals to mark regions of the early chick endoderm: this allowed correlations to be established between specific endoderm sites and the positions of their descendants. Endodermal precursor cells for the stomach, pancreas and intestine were found to segregate immediately after completion of gastrulation. Transplantation experiments showed that region-specific endodermal fates are determined sequentially in the order stomach, intestine, and then pancreas. Non-pancreatic endoderm transplanted to the stomach region generated ectopic pancreas expressing both insulin and glucagon. These results imply that a pancreas-inducing signal is emitted from somitic mesoderm underlying the pre-pancreatic region, and this extends rostrally beyond the stomach endoderm region at the early somite stage. Transplantation experiments revealed that the endoderm responding to these pancreatic-inducing signals lies within the pre-pancreatic region and extends caudally beyond the region of the intestinal endoderm. The results indicate that pancreatic fate is determined in the area of overlap between these two regions.     

Foregut endoderm is specified early in avian development through signal(s) emanating from Hensen's node or its derivatives

In this study, the initial specification of foregut endoderm in the chick embryo was analyzed. A fate map constructed for the area pellucida endoderm at definitive streak-stage showed centrally-located presumptive cells of foregut-derived organs around Hensen’s node. Intracoelomic cultivation of the area pellucida endoderm at this stage combined with somatic mesoderm resulted in the differentiation predominantly into intestinal epithelium, suggesting that this endoderm may not yet be regionally specified. In vitro cultivation of this endoderm for 1–1.5 day combined with Hensen’s node or its derivatives but not with other embryonic structures/tissues elicited endodermal expression of cSox2 but not of cHoxb9, which is characteristic of specified foregut endoderm. When the anteriormost or posteriormost part of the area pellucida endoderm at this stage, whose fate is extraembryonic, was combined with Hensen’s node or its derivatives for 1 day, then enwrapped with somatic mesoderm and cultivated for a long period intracoelomically, differentiation of various foregut organ epithelia was observed. Such epithelia never appeared in the endoderm associated with other embryonic structures/tissues and cultured similarly. Thus, Hensen’s node and its derivatives that lie centrally in the presumptive endodermal area of the foregut are likely to play an important role in the initial specification of the foregut. Chordin-expressing COS cells or noggin-producing CHO cells transplanted into the anteriormost area pellucida of the definitve streak-stage embryo could induce endodermal expression of cSox2 but not of cHoxb9, suggesting that chordin and noggin that emanate from Hensen’s node and its derivatives, may be involved in this process.