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.     

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.     

In vitro analysis of mesenchymal influences on the differentiation of stomach epithelial cells of the chicken embryo

It is well established that epithelial-mesenchymal interactions play important roles in the differentiation of stomach epithelial cells in the chicken embryo. To analyze mesenchymal influences on the differentiation of the epithelial cells, we developed a tissue culture system for stomach (proventriculus and gizzard) epithelia of chicken embryo, and examined their differentiation in the presence or absence of mesenchyme. Stomach epithelium from 6-day chicken embryo did not express embryonic chicken pepsinogen (ECPg), a marker molecule of glandular epithelial cells of proventriculus, while it expressed marker molecules of epithelial cells of the luminal surface of stomach, when cultured alone on the Millipore filter, covered with the gel consisting of extracellular matrix components. When the epithelium was recombined with mesenchyme separated by the filter, differentiation of the epithelium was affected by the recombined mesenchyme. Proventricular and lung mesenchymes induced the expression of ECPg in epithelial cells, and the expression was extensive when the gel contained basement membrane components. Proventricular and gizzard epithelia showed different responses to the mesenchymal action. We tested the effects of some growth factors on the differentiation of epithelial cells using this culture system. Furthermore we devised a "conditioned semi-solid medium experiment" for analysis of the inductive properties of proventricular and lung mesenchymes. The results of this experiment clearly demonstrated for the first time that diffusible factors from mesenchyme induce the differentiation of glandular epithelial cells in the absence of mesenchymal cells.     

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.     

Localization of DNA-synthesizing cells and cell proliferation pattern in developing proventricular (glandular stomach) epithelium of embryonic and hatched chickens

Localization of DNA-synthesizing cells in the developing proventricular (glandular stomach) epithelium of embryonic and hatched chickens was investigated. DNA-synthesizing cells were scattered throughout the proventricular epithelium during all developmental stages studied. The results indicate that there is no clear proliferative zone in the proventricular epithelium of the chicken. The labeling indices (LI) of proventricular epithelial cells were measured. On the 6.5th day of incubation, the LI of glandular epithelium reached 29.5 ± 1.5%. the highest value of all the stages studied. This extremely rapid cell proliferation can be considered to be a driving force for the elongation of the proventricular glands during the following stages. Just after hatching, the LI of both the glandular and luminal (non-glandular) epithelia significantly increased from those on the 18th day of incubation. It is suggested that the rise in LI possibly reflected proventricular growth to fit in the change in the method of nourishment after hatching. In 2 week old chickens, the LI of both the glandular and luminal epithelia were reduced to approximately 1%. The active production of embryonic chicken pepsinogen in all glandular epithelial cells of the embryonic chicken revealed that proliferation and differentiation are not necessarily exclusive during the embryonic stages of proventricular development.     

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.     

Involvement of the signal transduction pathway mediated by epidermal growth factor receptor in the differentiation of chicken glandular stomach

During the development of the chicken proventriculus (glandular stomach), the initially undifferentiated epithelium differentiates into two distinct cell populations: the glandular epithelium, cells of which secrete embryonic chicken pepsinogen (ECPg), and luminal epithelial cells, which express the chicken spasmolytic polypeptide gene (cSP). Based on knowledge of the adult mouse stomach, the ligands of epidermal growth factor (EGF) receptor (EGFR) were expected to affect differentiation of the proventricular epithelium. When EGF was added to the medium in which proventriculi were cultured in vitro, gland formation was suppressed in a dose-dependent manner and the amount of ECPg mRNA decreased, whereas morphological differentiation of luminal epithelium was stimulated. Simultaneous treatment of the proventriculus with EGF and tyrphostin 47 resulted in the attenuation of the effect of EGF, suggesting that EGF, or other ligands of EGFR, may actually be involved in the normal course of development of the proventricular epithelium.     

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.     

FGF10 is required for cell proliferation and gland formation in the stomach epithelium of the chicken embryo

The development of digestive organs in vertebrates involves active epithelial-mesenchymal interactions. In the chicken proventriculus (glandular stomach), the morphogenesis and cytodifferentiation of the epithelium are controlled by the inductive signaling factors that are secreted from the underlying mesenchyme. Previous studies have shown that Fgf10 is expressed in the developing chicken proventricular mesenchyme, whereas its receptors are present in the epithelium. In our present study, we show that FGF10 is an early mesenchymal signal that is critically associated with the developmental processes in the proventricular epithelium. Furthermore, virus-mediated Fgf10 overexpression in ovo results in a hypermorphic epithelial structure and an increase in epithelial cell number. In contrast, the overexpression of a secreted FGFR2b (sFGFR2b), an FGF10 antagonist, blocks cell proliferation and gland formation in the proventricular epithelium in ovo. This downregulation of proliferative activity was subsequently found to retard gland formation and also to delay differentiation of the epithelium. These results demonstrate that FGF10 signaling, mediated by FGFR1b and/or FGFR2b, is required for proliferation and gland formation in the epithelium in the developing chick embryo.     

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.     

Growth and Differentiation of Primary Cultures of Mouse Mammary Epithelium Embedded in Collagen Gel

Mammary glands from BALB/cfC3H midpregnant (9–11 days) mice were dissociated with collagenase and pronase, separated on a Percoll gradient, and the epithelial cells were cultured inside collagen gel. The cell number increased three-to five-fold when cultured for 6–8 days in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), cortisol (1.0 μg/ml), prolactin (10 μg/ml), transferrin (10 μg/ml), and epidermal growth factor (0.01 μg/ml). The casein level, as determined by radioimmunoassay, at the end of this growth phase, was much lower than that present in freshly dissociated cells. In order to stimulate casein production, the gels were released from the sides of the plastic dish and allowed to float for eight days in Waymouth's medium, containing insulin (10 μg/ml), cortisol (5 μg/ml), prolactin (10 μg/ml), and 0.25% bovine serum albumin. The casein level at the end of this differentiation phase was found to be comparable to that seen in the original freshly dissociated cells. Cells grown in DME/F12 (1: 1) medium containing 3% swine serum, insulin (10 μg/ml), and transferrin (10 μg/ml) were still capable of undergoing casein production, indicating that the presence of exogenous lactogenic hormones such as cortisol and prolactin, as well as exogenous growth factors such as epidermal growth factor, is not necessary during the growth phase for subsequent casein production during the differentiation phase. Two factors that seemed more important for subsequent casein stimulation were: (1) releasing collagen gels at the beginning of the differentiation phase, and (2) switching to'differentiation' medium. This present two-step protocol has allowed primary cultures of dissociated midpregnant mouse mammary epithelial cells to undergo several rounds of division inside a collagen gel matrix and to be subsequently stimulated to produce the mammary-specific protein, casein.     

Down-regulation of endodermal Shh is required for gland formation in chicken stomach

During the development of the proventriculus (glandular stomach) of the chicken embryo, the endodermal epithelium invades into the surrounding mesenchyme and forms glands. The glandular epithelial cells produce pepsinogen, while the non-glandular (luminal) epithelial cells secrete mucus. Sonic hedgehog is expressed uniformly in the proventricular epithelium before gland formation, but its expression ceases in gland cells. Here we present evidence that down-regulation of Sonic hedgehog is necessary for gland formation in the epithelium using a specific inhibitor of Sonic hedgehog signaling and virus mediated overexpression of Sonic hedgehog. We also show that gland formation is not induced by down-regulation of Sonic hedgehog alone; a mesenchymal influence is also required.     

Expression and function of Wnt5a in the development of the glandular stomach in the chicken embryo

The epithelium of the chicken embryonic glandular stomach (proventriculus) differentiates into both a glandular and a luminal epithelium, the cells of which express specific marker genes. The subsequent formation and differentiation of the glands then proceed under the influence of the mesenchyme. To search for possible candidates for the mesenchymal factors involved, we have now investigated the expression and function of Wnt5a in this process. Our current results show that Wnt5a is expressed in the mesenchyme during active gland formation and that overexpression of this gene in ovo results in the increased and ectopic expression of some of the marker genes of the luminal and glandular epithelia. In particular, the overexpression of Wnt5a markedly enhances the expression of the embryonic chicken pepsinogen gene, a marker of the glandular epithelium, indicating its role as a mesenchymal factor that regulates the differentiation of the proventricular epithelium.     

Ecdysteroid concentration inducing cell proliferation brings about the imaginal differentiation in the wing disc of Bombyx mori in vitro

This study was undertaken to evaluate the range of 20-hydroxyecdysone (20HE) concentrations which induce cell proliferation and imaginal differentiation in lepidopteran wing discs in vitro . Wing discs were cultured in medium containing various doses of 20HE. During imaginal differentiation in vitro , wing discs were observed histologically and the number of mitosis was counted every day. Wing discs differentiated adult features in medium containing 0.02–0.2 μg/mL 20HE, and these doses also increased the number of mitosis in disc cells. Wing discs developed the same in vitro as they do in vivo . The concentration of 20HE over 0.2 μg/mL inhibited both mitosis and imaginal differentiation. Cell proliferation, cuticle deposition and tissue elongation were successively observed in vitro the same as observed in vivo . These results suggest that a moderate concentration of ecdysteroid can induce cell proliferation followed by imaginal differentiation.     

The characteristics of endothelial progenitor cells derived from mononuclear cells of rat bone marrow in different culture conditions

Endothelial progenitor cells (EPCs) derived from bone marrow are known to be heterogeneous. In this study, we tried to find favorable conditions that induce the differentiation of mononuclear cells (MNCs) from bone marrow into EPCs. The differentiation capacity of MNCs from rat bone marrow was investigated in different conditions, such as different media, different induction times and different culture surfaces. The cell morphology and endothelial biomarkers associated with differentiated MNCs were studied. Our results indicated that MNCs cultured in EGM-2MV (Endothelial cell basal medium-2, plus SingleQuots of growth supplements) developed a bursiform shape, a late EPC-like morphology, while MNCs cultured in complete medium (CM, M199 with 10% FBS, 20 ng/mL VEGF and 10 ng/mL bFGF) showed a spindle shape, an early EPC-like morphology. Cells of both morphologies were able to incorporate DiI-ac-LDL and bind lectin in vitro. MNCs cultured in EGM-2MV exhibited a higher proliferation rate and higher eNOS expression than MNCs cultured in CM. MNCs cultured in EGM-2MV had the ability to form tubes on Matrigel. Flow cytometry results indicated that CD133 expression was highest at day 12 and that the greatest number of cells positive for both FLK-1 and CD133 appeared at day 20 from cells cultured in dishes without fibronectin coating. In addition, the expression levels of CD133, CD31 and FLK-1/CD133 were not significantly different between cells of different shapes. Our experiments suggest that MNCs from bone marrow can be differentiated into late EP-like cells in EGM-2MV, which have the ability to rapidly proliferate. These MNCs can also be differentiated into early EP-like cells in CM. Additionally, fibronectin may not be necessary for the differentiation of EPCs to mature ECs after three generations. Differentiated MNCs from bone marrow in EGM-2MV have the characteristics of EPCs, although the expression levels of EPC markers were lower than previously reported.     

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.     

Epidermal growth factor inhibits morphogenesis of the embryonic quail uropygial gland cultured in vitro

Formation of the uropygial papilla and glandular lumena was inhibited when the uropygial rudiment of a day 8 1/3 quail embryo was cultured for 2 days in a chemically defined medium in the presence of 50 ng/mL of epidermal growth factor (EGF). The epithelium of EGF-treated explants remained at the placode stage, or underwent minor invagination into the mesenchyme and became stratified like that of a 12- or 13-day-old embryo. EGF promoted cellular proliferation in the uropygial epithelium and the epidermis adjacent to the gland and it shortened the lag phase of proliferation and markedly stimulated epithelial DNA synthesis, detected immunocytochemically by labeling explants with 5-bromodeoxyuridine (BrdU). The maximal labeling index in EGF-treated uropygial epithelium was 55% higher than in the control. Electron microscopic observation revealed that the basal lamina had become irregular in the EGF-treated explants and that epithelial cytoplasmic processes penetrated through the basal lamina toward the mesenchyme. These same phenomena are observed in vivo when the glandular buds are formed during day 12–13. Some precocious changes occurred in the uropygial epithelium when the rudiment was cultured in the presence of EGF.