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.     

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.     

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.     

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.     

Gland formation induced in the allantoic and small-intestinal endoderm by the proventricular mesenchyme is not coupled with pepsinogen expression

Abstract. Allantoic and small-intestinal endoderms of chick and quail embryos were associated with the proventricular mesenchyme of chick embryos and then cultivated on chorioallantoic membrane. This resulted in the induction of complex glands, but the recombinates never produced embryo-specific pepsinogens; also, glandular cells developed a brush border, expressed sucrase antigen on their apical surface, and sometimes differentiated into goblet cells, thus indicating that both endoderms have the tendency to differentiate into an intestinal epithelium. In the recombinates composed of allantoic endoderm and proventricular mesenchyme, acid-protease activity was detected, but biochemical analysis revealed that this activity was not due topepsinogens. These results indicate that the gland formation induced in allantoic and small-intestinal endoderms by the proventricular mesenchyme is not accompanied by the expression of pepsinogens, suggesting that independent mechanisms are responsible for the morphogenesis and cyto chemical differentiation of the endoderm.     

DIFFERENTIATION OF THE DIGESTIVE TRACT EPITHELIUM UNDER THE INFLUENCE OF THE HETEROLOGOUS MESENCHYME OF THE DIGESTIVE TRACT IN THE BIRD EMBRYOS

The endoderm of the oesophagus, proventriculus, gizzard or small intestine of the 5-day-old chick or quail embryo was cultivated in combination with homologous or heterologous mesenchyme on a WxxxOLFFyyy and HxxxAFFHNyyy medium for 7 to 21 days or on the chorio-allantoic membrane (CAM) for 8 days. With homologous mesenchyme the epithelium always differentiated homotypically. In association with heterologous mesenchyme, the differentiation of the epithelium was both homotypical and heterotypical depending on the region of the digestive tract. The oesophagus and small intestine differentiate mainly homotypically both in culture and on CAM, but the gizzard and proventriculus show heterotypic differentiation particularly on CAM. Thus, the endoderm of the digestive tract of the 5-day-old chick or quail embryo, though rather "determined", still reacts to the heterologous stimuli of the mesenchyme to some degree.     

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.     

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.     

Localized distribution of a novel mesenchyme-specific antigen in developing chick digestive organs

Summary The mesenchymes of the two avian stomachs, the proventriculus (glandular stomach) and the gizzard (muscular stomach), exert different inductive influences on stomach epithelial morphogenesis and cytodifferentiation. To search for a molecular difference between these two mesenchymes, we have produced monoclonal antibodies directed against chick proventriculi and gizzards and have screened those that differently recognized proventricular and gizzard mesenchymes. Finally, we obtained one monoclonal antibody, T95, and characterized it immunohistochemically. T95 characteristically stains the mesenchymal region just under the gizzard epithelium from 6 days of incubation onward to about 10 days of incubation, while it stains proventricular mesenchyme only weakly during these stages. We also examined immunohistochemically the distribution of well-known extracellular matrix molecules, such as fibronectin, laminin and tenascin, and none of them showed the same localization as T95 antigen in proventricular and gizzard mesenchymes. These results indicate that T95 will be an interesting marker which distinguishes the proventricular and gizzard mesenchymes, at the time when they have different inductive ability.     

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.     

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.     

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.     

Comparison of the early chromatin reaction of the mouse thymocytes incubated with phytohemagglutinin and Concanavalin A

Summary The proventriculus, gizzard and pyloric antrum (region between the gizzard and the duodenum) of 18-day Black Australorp chick embryos and of chicks within 30 h of hatching have been studied by electron microscopy. D and EC cells, and putative G, D₁ and A-like cells were identified (terminology of Solcia et al., 1973) but no ECL cells. No endocrine cells of any kind were revealed in the gizzard.Supported by grants from the Senyte Research Comittee of the University of the Witwaterstrand, Johannesburg     

Analysis of mesenchymal influence on the pepsinogen gene expression in the epithelium of chicken embryonic digestive tract

We performed tissue recombination experiments to discover the mesenchymal influences on differentiation of epithelia in chicken digestive organs. Epithelia and mesenchymes were taken from the lung, esophagus, proventriculus, gizzard, small intestine and large intestine of 6-day chicken embryos and recombined in various associations and cultivated in vitro for 6 days. Rather unexpectedly, embryonic chicken pepsinogen (ECPg) gene, a marker of the proventricular epithelium, was induced in the gizzard epithelium, which does not express ECPg in normal development, by the proventricular and lung mesenchymes. In the second half of this study, we investigated the mode of action of mesenchymal cells on ECPg expression in gizzard epithelial cells more precisely using the cell aggregate culture system, in which gizzard epithelial cells were mixed with proventricular, gizzard or lung mesenchymal cells. We found that supporting action of lung mesenchymal cells on ECPg expression was even stronger than that of proventricular mesenchymal cells, and suggest that the action of lung mesenchyme may be due partly to the enhancement of epithelial cell proliferation. According to the results of this study, together with many facts obtained so far, we will discuss a new model for restricted expression of ECPg in the proventricular epithelium in normal development.     

Gizzard formation and the role of Bapx1

The anterior-posterior gut pattern is formed from three broad domains: fore-, mid-, and hindgut that have distinct functional, morphological, and molecular boundaries. The stomach demarcates the posterior boundary of the foregut. Avian stomachs are composed of two chambers: the anterior chamber (proventriculus) and the thick muscular posterior chamber (gizzard). Expression of candidate pattern formation control factors are restricted in the chick stomach regions such that Bmp4 and Wnt5a are not expressed in the gizzard. We previously implicated Bmp4 as controlling growth and differentiation of the gut musculature. Bmp4 is not expressed in the developing gizzard but is expressed in the rest of the gut including the adjacent proventriculus and midgut. Bapx1 (Nkx3.2) is expressed in the gizzard musculature but not in the proventriculus or midgut. We show ectopic expression of Bapx1 in the proventriculus results in a gizzard-like morphology and inhibits the normal proventricular expression of Bmp4 and Wnt5a. Overexpression of a reverse-function Bapx1 construct can result in a small stomach and ectopic extension of Bmp4 and Wnt5a expression into the gizzard. We suggest the role of Bapx1 is to regulate the expression of Bmp4 and Wnt5a to pattern the avian stomach.     

Tissue interaction regulates expression of a spasmolytic polypeptide gene in chicken stomach epithelium

The primitive epithelium of embryonic chicken proventriculus (glandular stomach) differentiates, after day 6 of incubation, into luminal epithelium, which faces the lumen and abundantly secretes mucus, and glandular epithelium, which invaginates into mesenchyme and later expresses embryonic chicken pepsinogen (ECPg). So far it is not well understood how undifferentiated epithelial cells differentiate into these two distinct cell populations. Spasmolytic polypeptide (SP) is known to be expressed in surface mucous cells of mammalian stomach. In order to obtain the differentiation marker for proventricular luminal epithelial cells, we cloned a cDNA encoding chicken SP ( cSP ). Sequence analysis indicated that cSP has the duplicated cysteine-rich domain characteristic of SP. Examination of the spatial and temporal expression pattern of cSP gene revealed that, during embryogenesis, cSP was expressed in luminal epithelial cells of the proventriculus, gizzard, small intestine, and lung, but not the esophagus. In the proventriculus, cSP mRNA was first detected on day 8 of incubation and was localized to differentiated luminal epithelial cells. By using cSP as a molecular marker, the effects of mesenchyme on the differentiation of epithelium were analyzed in vitro . On the basis of these data, a model is presented concerning the differentiation of proventricular epithelium.     

Epithelial Invagination: Adhesive Properties of Cells Can Govern Position and Directionality of Epithelial Folding

The aim of the present study was to investigate the mesenchymal influence on cultured epithelioid cells originating from an already differentiated intestine. Epithelioid cell cultures of 6-day-old suckling rat intestine were established by sequential trypsinizations of the mucosa. Embryonic intestinal monolayers of quail cells (13 days) were used as control because of their natural cell marker. Six to thirty days after plating, both types of epithelioid cells were associated in heterospecific combination with 5½-day-old chick embryonic small intestinal mesenchyme, after removal of the endoderm by collagenase treatment. In order to test the differentiation capabilities of the associations, they were grafted for 10–12 days into 3-day-old chick embryos. The results show that in such an in vivo culture system, the chimeric associations gave rise to well differentiated intestinal structures indicating that the epithelioid cell cultures derived from late embryonic or neonatal intestine will go through organotypic differentiation when recombined with an appropriate mesenchyme.     

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.     

Expression of Fgf10 and Fgf receptors during development of the embryonic chicken stomach

Fibroblast growth factor 10 (FGF10) is involved in numerous different aspects of embryonic development and especially in active epithelial-mesenchymal interactions during morphogenesis of many organs as a mesenchymal regulator by activating its receptors (FGFR1b and FGFR2b) expressed in the epithelial tissue. FGFR2b is also activated by FGF7 although FGF7 does not bind to FGFR1b. To provide basic data to analyze function of FGFs in the developing gut, here we cloned Fgf7 and studied expression patterns of Fgf7, Fgf10 and Fgfr1-4 during the development of chicken stomach (glandular stomach; proventriculus and muscular stomach; gizzard). Fgf10 is expressed both in the proventricular and gizzard mesenchyme while Fgf7 is expressed only in gizzard mesenchyme. Fgfr1-4 are expressed both in the epithelium and mesenchyme with a different spatial expression patterns. Furthermore, RT-PCR analysis reveals that Fgfr1b and Fgfr2b are expressed only in epithelia of both organs.