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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Sonic hedgehog expression in developing chicken digestive organs is regulated by epithelial–mesenchymal interactions

Sonic hedgehog (Shh) gene encodes a secreted protein that acts as an important mediator of cell–cell interactions. A detailed analysis of Shh expression in the digestive organs of the chicken embryo was carried out. Shh expression in the endoderm begins at stage 7, when the formation of the foregut commences, and is found as narrow bands in the midgut. Shh expression around the anterior intestinal portal at stage 15 is restricted to the columnar endoderm lined by the thick splanchnic mesoderm, suggesting that the existence of thick splanchnic mesoderm might be necessary for Shh expression in the columnar endoderm. After the gut is closed, Shh expression is found universally in digestive epithelia, including the cecal epithelium. However, its expression ceases in the epithelium of the proventricular glands, the ductus choledochus and ductus pancreaticus that protrude from the main digestive duct. When the gizzard epithelium differentiated into glands under the influence of the proventricular mesenchyme, the glandular epithelium lost the ability to express Shh . These findings suggest that Shh expression in the epithelium may be regulated by surrounding mesenchyme throughout organogenesis of the digestive organs and is closely involved in epithelial–mesenchymal interactions in developing digestive organs.     

家兔胚泡ICM在体外培养系统中的行为

一、用显微外科方法从兔早期胚泡分离来的ICM在所用的体外培养系统中的行为,因ICM的生长状态而不同。呈带状生长的ICM从近端向远处延伸,而细胞的分化则从远端逐渐向近端进行,它具有明显的极性。细胞的分化秩序井然,层次分明。因此呈带状生长的ICM适用于进行细胞分化和细胞谱系的研究。二、呈团块状生长的ICM,没有明显的极性,细胞分化开始于团块的外表面,逐渐向中央进行。细胞分化开始稍晚一些,速度也较慢,这种生长状态的ICM适用于分离胚胎干细胞。三、兔子胚外内胚层从ICM分化来,它经历了二次分化的过程。第一次发生于培养后第三天(相当于交配后第七天),形成第一胚外内胚层-胚外体壁内胚层,它向远处迁移,远离原始外胚层。培养后四天(相当于交配后第八天),形成第二种胚外内胚层-胚外脏壁内胚层,它尾随体壁内胚层向远处迁移,其后缘的部分细胞向原始外胚层附近的滋胚层下面侵润。四、胚外体壁内胚层和脏壁内胚层的细胞是由少数决定了的和离开ICM的细胞分化和增殖而来。五、文中讨论了早期胚胎细胞分化的谱系问题。     

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.