Regional difference in the responsiveness to the inductive influence of the gizzard mesenchyme among the endoderms of various small intestinal segments of the chick embryo

The differentiation of the endoderms of duodenal, jejunal and ileal segments of the small intestine of 6 day old chick embryos cultured in recombination with the gizzard mesenchyme of 6 day chick embryos was examined. Only the duodenal endoderm differentiated in a mesenchyme-dependent fashion into gizzard-like mucous epithelium forming tubular glands that expressed no sucrase-antigen, while jejunal and ileal endoderms tended to become the sucrase-antigen-positive epithelium most likely according to their developmental fates. The analysis on the differentiation of the duodenal and gizzard endoderms in the presence of various digestive-tract mesenchymes confirmed that the duodenal endoderm had the tendency to differentiate into intestine-type and was different from the gizzard endoderm, which showed the differentiation tendency into gizzard-type. Thus, among the segments of small intestine, only the endoderm of duodenum that was situated next to the gizzard was found to have an ability to respond to the inductive influence of the gizzard mesenchyme and to change its developmental fate.     

The effect of pancreatic mesenchyme on the differentiation of endocrine cells from gastric endoderm

To determine whether mesenchyme plays a part in the differentiation of gut endocrine cells, proventricular endoderm from 4- to 5-day chick or quail embryos was associated with mesenchyme from the dorsal pancreatic bud of chick embryos of the same age. The combinations were grown on the chorioallantoic membranes of host chick embryos until they reached a total incubation age of 21 days. Proventricular or pancreatic endoderm of the appropriate age and species reassociated with its own mesenchyme provided the controls. Morphogenesis in the experimental grafts corresponded closely to that in proventricular controls, i.e. the pancreatic mesenchyme supported the development of proventricular glands from proventricular endoderm. Insulin, glucagon and somatostatin cells and cells with pancreatic polypeptide-like immunoreactivity differentiated in the pancreatic controls. The latter three endocrine cell types, together with neurotensin and bombesin/gastrin-releasing polypeptide (GRP) cells, developed in proventricular controls and experimental grafts. The proportions of the major types common to proventriculus and pancreas (somatostatin and glucagon cells) were in general similar when experimental grafts were compared with proventricular controls but different when experimental and pancreatic control grafts were compared. Hence pancreatic mesenchyme did not materially affect the proportions of these three cell types in experimental grafts, induced no specific pancreatic (insulin) cell type and allowed the differentiation of the characteristic proventricular endocrine cell types, neurotensin and bombesin/GRP cells. However, an important finding was a significant reduction in the proportion of bombesin/GRP cells, attributable in part to a decrease in their number and in part to an increase in the numbers of endocrine cells of the other types. This indicates that mesenchyme may well play a part in determining the regional specificity of populations of gut endocrine cells.     

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.     

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.     

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.     

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.     

Developmental changes in the ability to express embryonic pepsinogen in the stomach epithelia of chick embryos

Summary The avian stomach is subdivided into two parts, the proventriculus and the gizzard. It has been shown that the gizzard epithelium can express embryonic chick pepsinogen (ECPg) antigen, a marker protein of the proventricular epithelium, as well as normal proventricular epithelium, under the appropriate experimental conditions. To study the possible mechanisms involved in the suppression of ECPg synthesis in the gizzard epithelium during normal development, we carried out heterotypic and heterochronic recombination experiments of the epithelium and mesenchyme of these two organ rudiments. When recombined and cultured with 6-day proventricular mesenchyme, gizzard epithelium of 3.5- to 12-day embryos expressed pepsinogen at all stages tested. However, the ratio of ECPg-positive cells to total epithelial cells in the gizzard epithelium decreased rapidly when epithelium older than 7 days was cultured with proventricular mesenchyme. In contrast to proventricular mesenchyme, 6-day gizzard mesenchyme did not allow ECPg expression in associated proventricular epithelium of 3.5- to 7-day embryos. These results indicate that gizzard epithelium does not express pepsinogen in normal development because of both a decrease in ability to express the enzyme in itself in the course of development and a repressive influence of gizzard mesenchyme.     

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.     

Acid protease expression and regulation in the embryonic proventriculus of birds

Normal embryonic proventriculi and the heterospecific recombinants of proventricular endoderm and mesenchyme were transplanted onto the chorio-allantoic membrane, and electrophoretic patterns of acid proteases in the explants were analyzed. The results demonstrated that the 6-day chick and 5-day quail proventricular endoderm produces acid proteases according to its own genetic information even under the influence of heterospecific mesenchyme, and that the production of acid proteases is regulated by some humoral factors of the hosts.     

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.     

Gizzard epithelium of chick embryos can express embryonic pepsinogen antigen,a marker protein of proventriculus

Summary The avian stomach is composed of two distinct organs, the proventriculus and the gizzard. Pepsinogen expression in the proventricular and gizzard epithelia of chick embryos was investigated immunohistochemically with anti-embryonic chick pepsinogen (anti-ECPg) antiserum. In normal development, the ECPg antigen was expressed only in the glandular epithelial cells of the embryonic proventriculus from the 8th day of incubation onwards. However, both proventricular and gizzard epithelia of 6-day embryos expressed the ECPg antigen when recombined and cultured with the proventricular mesenchyme. Chronological studies revealed that the ECPg antigen was first detected in a few epithelial cells at 3 days of cultivation. The percentage of ECPg-positive cells among the total epithelial cells in each recombinant increased with the length of the culture period and all the glandular epithelial cells were positive at 9 days. During this process, the percentage of ECPg-positive cells in each cultured recombinant was similar in proventricular and gizzard epithelia. Moreover, both epithelia could express the ECPg antigen when recombined and cultured with the oesophageal or small-intestine mesenchyme for 9 days, though the percentage of ECPg-positive cells in each cultured recombinant was much lower than that in the cultured recombinant with the proventricular mesenchyme. These results indicate that the gizzard epithelium of 6-day chick embryos possesses a similar potential for pepsinogen expression as the proventricular epithelium of the same age.     

Thinning of the basement Membrane and Localized Cell Proliferation during Gland Formation of the Chick Proventriculus

The early processes of proventricular gland formation in the chick embryo were investigated. The glands appeared as intra-epithelial invaginations of the proventricular endoderm on day 6 of incubation. By day 6.5 they began to protrude into the mesenchyme and elongated without branching until day 9. Before elongation of the glands, the immunofluorescence of laminin and the ultrastructure of the basal lamina were consistently observed in the intra-epithelial invaginations as well as in other regions, and the mitotic activity in the gland rudiments was not different from that in other regions. However, at the tips of the elongating glands, little laminin was detected and the basal lamina were thin and discontinuous. The mitotic activity at the tip of the glands was higher than that in non-glandular epithelium or in the stalk of the glands. These results suggest causal relationships between thinning of the basement membrane and localized epithelial cell proliferation at the tip of the elongating glands.     

Chronological analysis of the intestinalization of chick stomach endoderm induced in vitro by duodenal mesenchyme

Summary Inductive action of duodenal mesenchyme on stomach endoderm in the chick embryo was chronologically analysed in vitro by the use of electron microscopy and immunofluorescence techniques. The behaviour of the endoderm-mesenchyme interfaces was particularly studied during the induction. In recombinates of 4-day stomach endoderm and 6-day duodenal mesenchyme, all the endodermal cells were undifferentiated at the start of cultivation. Small-intestinal sucrase antigen could first be detected on the 5th day of cultivation in one-third of the stomach endoderm, and a striated border on the 7th day. With a longer cultivation period, intestine-type cells increased in number in the stomach endoderm and the density of microvilli on the apical surface became higher. At the endoderm-mesenchyme interfaces a number of direct contacts between endodermal and mesenchymal cells were observed from the beginning to the end of cultivation. These were especially abundant in the early period before the appearance of signs of intestinal cytodifferentiation. These results suggest that the mesenchymal cells adjacent to the endodermal tissue play an important role in the intestinal induction which occurs during the early period of cultivation, probably via direct cell-to-cell contracts.     

Mucus-associated antigen in epithelial cells of the chicken digestive tract: Developmental change in expression and implications for morphogenesis-function relationships

The embryonic chicken digestive tract consists of endodermal epithelium and mesenchyme derived from splanchnic mesoderm. Interactions between these two tissues are important for the establishment of regionality and the subsequent differentiation of digestive organs. In the present study we obtained a monoclonal antibody that reacted with mucus-associated antigen and named it the MA antibody. From 6 days of incubation, this antibody reacted with the esophageal, proventricular and gizzard epithelia. In the proventriculus, the MA antigen was expressed in luminal epithelial cells, while pepsinogen-producing gland cells became MA antigen-negative. The intestinal goblet cells, which secrete mucus, became positive to the antibody from day 13 of incubation. When the esophageal, proventricular or gizzard epithelium of a 6 day embryo was associated and cultivated with the proventricular mesenchyme, the luminal epithelial cells remained reactive to the MA antibody while gland cells were negative or only weakly positive. If the small-intestinal epithelium was cultivated with the proventricular or gizzard mesenchyme, the antigen was detected on the apical surface of the epithelium, suggesting that the expression of the MA antigen was induced by mesenchymal influences in the small-intestinal epithelium. These results suggest that spatio-temporally regulated expression of the MA antigen is controlled by the epithelial-mesenchymal interactions.     

Role of epithelial-mesenchymal interactions in the ontogenesis of intestinal brush-border enzymes

The respective roles of embryonic intestinal mesenchyme and endoderm in the biochemical differentiation of brushborder enzymes have been investigated. As a first step of this study, the prenatal developmental pattern of several enzymes (maltase, sucrase, lactase, alkaline phosphatase), measured in brush-border membranes purified from chick and rat intestine, has been established. Xenoplastic recombinations between the intestinal tissue components of $\text{5-}\text{1}\text{2}\text{-}\text{day-old}$ chick embryos and 14-day-old fetal rats have been performed. After 11 days of intracoelomic graft in 3-day-old chick embryos, the combinations composed of chick mesenchyme and rat endoderm (Cm/Re) showed enzyme activities characteristic of the fetal rat intestine: high lactase activity and traces of sucrase activity. The inverse combinations composed of rat mesenchyme and chick endoderm (Rm/Ce) exhibited a chicken-like pattern: high sucrase activity and traces of lactase activity. In the latter combinations, the specific enzyme activities were similar to those present in the intestine of 15- to 16-day-old chick embryos (theoretical level reached after the grafting period). Conversely, the levels of enzyme activities of the Cm/Re combinations remained lower than those present in the normally developed rat intestine. These results show that the endodermal tissue carries the specific characteristics of its future biochemical differentiation. They also suggest that the important maturation events, which occur shortly before birth in the rat, are dependent upon other factors, presumably hormones.     

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.     

Differentiation of intestinal and ectopic endocrine cells from avian gastric and pancreatic endoderm

The chorio-allantoic grafts analysed were prepared from avian proventricular endoderm combined with its own or pancreatic mesenchyme and from re-associated pancreatic layers. Intestine developed ectopically in some grafts: in these, endocrine cells typical of intestine differentiated irrespective of the source of the endoderm or mesenchyme. In addition, endocrine cells inappropriate for the surrounding histology were detected in small numbers in grafts of all categories. Clearly it is not the mesenchyme that is responsible but perhaps some aspect of the procedure, which may relate to stressful stimuli thought to provoke intestinal metaplasia. The differentiation of inappropriate cells aids in understanding the occurrence of ectopic endocrine tumours.     

Enzymatic response to glucocorticoids of the chick intestinal endoderm associated with various mesenchymal cell types

The aim of the present study was to test the morphological and functional maturation of recombinants composed of chick intestinal endoderms associated to different mesenchymal supports and their enzymatic response to glucocorticoids. For this purpose 5.5-day chick embryonic intestinal endoderm has been associated to 14-day fetal rat gut mesenchyme, to rat intestinal fibroblasts (6-day neonatal rat intramucosal fibroblasts) or to rat control fibroblasts, originating from 20-day fetal rat skin and lung and from 6-day neonatal rat intestinal muscle. The recombinants were grown as intracoelomic grafts either for 12 days or for 10 days plus 2 days in organ culture in the presence of dexamethasone. The data show that heterospecific recombinants achieve subnormal morphogenesis and enzymatic maturation. The organ culture experiments further reveal that sucrase activity is insensitive to dexamethasone in all types of recombinants whereas, alkaline phosphatase is highly stimulated over the levels present in the intestine developed in situ whatever the stromal support, except when this support is provided by rat gut mesenchyme. These results support the view that in the intestine the hormonal response is mediated by epithelial-mesenchymal interactions.     

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.