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.     

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.     

Heterotypic induction of stomach-like glands in the urogenital sinus endoderm under the influence of stomach mesenchyme of foetal rats

Summary Urogenital sinus endoderm of 16.5-day rat foetuses was combined with stomach mesenchyme and the recombinants were either treated with testosterone and grown in vitro or cultured beneath the kidney capsule of adult male rats of the same strain. It was found that testosterone stimulated mitosis in the urogenital endoderm. In recombinants grown under the kidney capsule a stratified squamous epithelium and stomach-like glands were induced under the influence of the forestomach and glandular stomach mesenchymes. However, the induced glands expressed neither rat pepsinogen nor rat ventral prostatic antigen. They did not produce mRNA for the prostatic steroid-binding protein C1. Thus, stomach mesenchyme of rat foetuses induces organ-specific morphogenesis but not functional differentiation in the heterologous endoderm, indicating that cytodifferentiation does not always accompany morphogenesis.     

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.     

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.     

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.     

A nude mouse xenograft model of fetal intestine development and differentiation.

This report describes a novel in vivo model of intestinal differentiation. Fourteen day, undifferentiated fetal rat small intestine, stripped of the major part of its mesenchyme, suspended in a type I collagen gel and then xenografted into a nude mouse, undergoes small intestinal morphogenesis and cytodifferentiation. All four major epithelial lineages, namely Paneth, goblet, columnar and endocrine are present. Double-label nonisotopic in situ hybridization, employing biotinylated and digoxigenin-labelled whole rat DNA and whole mouse DNA probes, was performed to distinguish donor cells from host cell types. The outer longitudinal smooth muscle layer, and the major part of the lamina propria, including pericryptal fibroblasts, are of host mouse origin; the inner circular smooth muscle layer is of donor rat origin. Cells of the muscularis propria and lamina propria acquired smooth muscle alpha-actin, presumably under the influence of the donor endoderm. Furthermore, this xenograft develops a host vascular network, and cells with the morphological appearance of lymphocytes are present within the intestinal epithelium. The production of chemotactic factors by the endoderm is postulated because grafting of collagen gel alone results in a minimal invasion by stromal cells which do not express smooth muscle alpha-actin.     

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.     

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.     

Differentiation of proventricular epithelium in xenoplastic associations with mesenchymal or fibroblastic cells

Summary Proventricular epithelium (PV epithelium) from 6-day chicken embryos was associated with cultured cells, derived from fetal rat small intestine, or with fetal rat or human skin fibroblasts. The cytodifferentiation of PV epithelium was investigated using antibodies to chicken pepsinogen, a marker protein of PV epithelium, and to chicken sucrase, a marker enzyme of the small-intestinal brush-border membrane. PV epithelium formed complex glands and produced pepsinogen in association with cultured gut mesenchymal cells and skin fibroblasts. Its development was comparable to that achieved under the influence of PV mesenchyme. PV epithelial development was severely inhibited, however, under the influence of intact chicken or rat intestinal mesenchyme. The data are consistent with the idea that during the first step of epithelial-mesenchymal interactions, the epithelium and not the mesenchyme may be responsible for the determination of the developmental fate.     

Keratin expression in rat intestinal crypt and villus cells. Analysis with a panel of monoclonal antibodies.

Seven monoclonal antibodies were prepared against cytoskeletal components of rat intestinal brush borders. In the following paper (Chandler, J. S., Calnek, D., and Quaroni, A., J. Biol. Chem. 266, 11932-11938), three of them were shown to be specific for, respectively, keratin 8 (RK4), keratin 19 (RK7), and a newly identified type I keratin (keratin 21) (RK5). With these antibodies we have investigated the changes in keratin gene expression accompanying intestinal cell differentiation. Keratin 21 was detected exclusively in differentiated villus cells and in goblet, enteroendocrine, and Paneth cells in the crypts; in the proliferative crypt cells keratin 19 was predominant. Analysis of keratins expressed by cultured rat crypt cells (IEC cells) confirmed the absence of keratin 21 in undifferentiated intestinal cells. Changes in keratin's expression similar to those observed with cell differentiation in the adult intestinal mucosa were also demonstrated during early fetal intestinal development: the stratified epithelium present at 15-16 days of gestation contained predominantly keratin 19 with only a small amount of keratin 8; keratin 21 was first detected at 18-19 days of gestation, concomitant with the appearance of a well formed brush border and an apical cytoplasmic terminal web. These results suggest that keratin tonofilaments may play a role in the morphological and structural alterations accompanying intestinal cell differentiation in vivo.     

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.     

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.     

Induction of functional cytodifferentiation in the epithelium of tissue recombinants. I. Homotypic seminal vesicle recombinants

Functional cytodifferentiation of seminal vesicle epithelium was investigated in tissue recombinants. Neonatal rat and mouse seminal vesicles were separated into epithelium and mesenchyme using trypsin. Epithelium and mesenchyme were then recombined in vitro to form interspecific rat/mouse homotypic recombinants. Growth as renal grafts in adult male athymic mice resulted in seminal vesicle morphogenesis in 70% of the recombinants (the remaining 30% failed to grow). Functional cytodifferentiation was judged by the expression of the major androgen-dependent secretory proteins characteristic of the seminal vesicles of adult rats and mice. Antibodies specific for each of these proteins were used to screen tissue sections by immunocytochemistry and to probe protein extracts by immunoblotting techniques. The heterospecific recombinants synthesized the full range of seminal vesicle secretory proteins that typifies the species providing the epithelium of the recombinant, not the mesenchyme. There was little functional variation between individual recombinants. The time course of development corresponded to that of intact neonatal seminal vesicles grown under the same conditions. Morphogenesis and functional cytodifferentiation were not evident after one week, but were well advanced after two weeks. Seminal vesicle recombinants grown for three weeks were indistinguishable morphologically and functionally from normal adult seminal vesicles. In addition, the ability of adult seminal vesicle epithelium to be induced to proliferate was examined. In association with neonatal seminal vesicle mesenchyme, the epithelium of the adult seminal vesicle proliferated and retained its normal functional activity. Thus, seminal vesicle functional cytodifferentiation can be faithfully reproduced in homotypic tissue recombinants. The methods used in this study will be used to investigate seminal vesicle development in instructive inductions of heterotypic epithelia.     

Effects of retinoic acid on the endoderm in Xenopus embryos

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

(Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: presence of a brush border antigen in the distal large intestine that is immunologically related to beta subunit

The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells (Quaroni, A., and K. J. Isselbacher. 1981. J. Natl. Cancer Inst. 67:1353-1362) was extensively characterized and found to be directed against the beta subunit of (Na+ + K+)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the alpha-beta enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the alpha subunit (Kashgarian, M., D. Biemesderfer, M. Caplan, and B. Forbush. 1985. Kidney Int. 28:899-913), was used to localize (Na+ + K+)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting beta-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na+ + K+)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the beta subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled beta subunit or a beta-subunit-like protein is also transported to the apical cell pole.     

Induction of functional cytodifferentiation in the epithelium of tissue recombinants. II. Instructive induction of Wolffian duct epithelia by neonatal seminal vesicle mesenchyme

When grown as renal grafts in adult male hosts, the upper (cranial), middle and lower (caudal) portions of fetal mouse and rat Wolffian ducts developed into epididymis, epididymis plus ductus deferens, and seminal vesicle, respectively. In heterotypic tissue recombinants, the epithelia from upper and middle Wolffian ducts were instructively induced to undergo seminal vesicle morphogenesis by neonatal seminal vesicle mesenchyme. Functional cytodifferentiation was examined in these recombinants using antibodies against major androgen-dependent, seminal vesicle-specific secretory proteins. The instructively induced Wolffian duct epithelia synthesized normal amounts of all of the secretory proteins characteristic of mature seminal vesicles, as judged by immunocytochemistry on tissue sections and gel electrophoresis plus immunoblotting of secretions extracted from the recombinants. In heterospecific recombinants composed of rat and mouse tissues, the seminal vesicle proteins induced were specific for the species that had provided the epithelium. This showed that the seminal vesicle epithelium in the recombinants was derived from instructively induced Wolffian duct epithelium and not from epithelial contamination of the mesenchymal inductor. Upper Wolffian duct epithelium, instructively induced to undergo seminal vesicle morphogenesis, did not express epididymis-specific secretory proteins, showing that its normal development had been simultaneously repressed.     

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.