Animals

Embryos of White Leghorn chicken (Gallus gallus domesticus) at various developmental stages were used during the experiments.

Epithelial-mesenchymal recombination

Epithelial-mesenchymal recombination techniques are the same as those described in a previous paper (Takiguchi et al., 1986). In brief, epithelia of 6-day esophagus, proventriculus, gizzard, small intestine and 3.5-day allantois and 6-day proventricular mesenchyme were isolated with the aid of collagenase (Cooper Bio-medical, Malvern, USA; Code CLS, 0.03 % in Tyrode's solution, 40 min at 38°C). Epithelial and mesenchymal fragments were recombined after inactivation of collagenase by washing in serum-containing Tyrode's solution and the recombinants were cultured according to the method of Wolff and Haffen (1952) for half a day to ensure the adhesion of two tissue fragments. They were then grafted onto the chorio-allantoic membrane (CAM) of 9-day chicken embryo and cultured for 9 days. Reaggregated proventricular or gizzard mesenchymal cells were prepared as described (Takiguchi-Hayashi et al., 1988; Urase and Yasugi, 1993) and recombined with 6-day proventricular or gizzard epithelium and cultured in vitro for 6 days.

Antibodies

The monoclonal antibodies PKK1 (Labsystems Oy, Helsinki, Finland) and AE3 (ICN Immunobiologicals, Lisle, Illinois, USA) were used to examine cytokeratin expression. PKK1 was raised against cytoskeletal protein isolated from a pig kidney epithelial cell line (Holthofer et al., 1983) and recognizes cytokeratin 8, 18 and 19 in the catalogue of human cytokeratins (Moll et al., 1982). AE3 was prepared against human epidermal cytokeratin (Woodcock-Mitchell et al., 1982) and recognizes most of the basic (type II) cytokeratins. The monoclonal antibody Y37 was raised against embryonic chicken pepsinogen, a secretory protein of the proventricular epithelial cells (Yasugi et al., 1987). As a secondary antibody, fluorescein isothiocyanateconjugated goat anti-mouse IgG (Cooper Biomedical) was used.

Immunohistochemistry

Tissues were removed just after the killing of animals or after culturing and fixed in ice-cold 95% ethanol for 4 hr, embedded in paraffin, and 5 μm sections were subjected to the indirect immunof luorescence according to the method of Sainte-Marie (1962). In brief, deparaffinized sections were first treated with primary antibodies diluted with phosphate-buffered saline for 1 hr at 38°C, and then treated with secondary antibody for 1 hr. Antibody-stained sections were mounted in a glycerin buffer and examined with an Olympus photo-microscope equipped with an epi-illumination fluorescence system.

Distribution of cytokeratins during proventricular epithelial differentiation of chicken embryo

When the proventriculus of 16-day embryo was stained with AE3, both pepsinogen-positive and -negative epithelial cells showed positive reactivity, but the staining pattern of pepsinogen-positive cells was distinctly different from that of pepsinogen-negative cells; in pepsinogen-positive gland cells AE3-positive substances localized only in the apical portion of the cells, while in luminal epithelial cells these substances distributed in entire cytoplasm (Fig. 1a, b). PKK1 stained pepsinogen-negative luminal cells but not pepsino-gen-positive gland cells (Fig. 1a, c).

Fig. 1.

Distribution of pepsinogen and cytokeratin antigens in the proventriculus of 16-day chicken embryo. Immunofluorescence of the pepsinogen antigen (Y37, a), AE3 antigen (b) and PKK1 antigen (c). (d) Histology (hematoxylin-eosin staining) of 16-day proventriculus showing lumen (L), luminal surface epithelium (LE), duct epithelium (DE), gland epithelium (GE) and mesenchyme (M). ×48.

Distribution of PKK1 antigen was examined from just after the beginning of the proventricular gland formation. At about 6 days of incubation, several cells in the pseudostratified epithelium lessened in height and form an intra-epithelial invaginations, but these ceils in gland rudiments were PKK1-positive and cytokeratins were observed as fibrils running from apical to basal surface (Fig, 2a). During gland elongation, PKK1 antigen decreased at the basal portion and became faint in epithelial cells at the tip of elon-gated glands (Fig. 2b). At 8 days of incubation, epithelial cells of small glands in luminal epithelium, which began to express pepsinogen detected with Y37 antibody, became PKK1-negaive (Fig. 2c). From 9 days of incubation onward, PKK1 antigen was detected only at the apical portion of epithelial cells of glands, while in luminal surface ceils and duct cells of glands, PKK1 antigen persisted from apical to basal surface of the epithelial cells (Fig. 2d, e). From 14 days of incubation onward, PKK1 antigen was not detected in glandular epithelial cells even at the apical portion (Fig. 2f, g). On the other hand, PKK1 antigen was detected in the gizzard epithelium throughout the development (data not shown). All cells other than epithelial cells were PKK1-negative.

Fig. 2.

Distribution of PKK1 antigen during the proventricular gland formation in normal chicken embryos. (a) Day 6. Arrowhead indicates an intra-epithelial invagination. ×413. (b) Day 7. Arrowhead indicates the tip of an elongated gland. ×206. (c) Day 8. Arrowhead indicates a small gland in the luminal epithelium. ×413. (d,e) Day 12. ×103. (f,g) Day 16. ×103. (a-d,f) Immunofluorescence of the PKK1 antigen. (e,g) Immunofluorescence of the pepsinogen antigen in the neighboring sections.

Regulatory influence of the proventricular mesenchyme on the expression of cytokeratins in various digestive tract epithelia

Six-day esophageal, proventricular, gizzard or small-intestinal epithelium or 3.5-day allantoic epithelium was recombined with 6-day proventricular mesenchyme and cultured on the CAM. The esophageal, proventricular and gizzard epithelia showed proventriculus-type complex gland morphology and gland epithelial cells expressed pepsinogen. PKK1 antigen was not detected in gland epithelial cells expressing pepsinogen (Fig. 3). The distribution of AE3 antigen was similar to that in the normal proventriculus (data not shown).

Fig. 3.

Distribution of pepsinogen (a,c,e) and PKK1 (b,d,f) antigens in the recombinants of 6-day esophageal (a,b), proventricular (c,d) or gizzard (e,f) epithelium and 6-day proventricular mesenchyme cultured for 9 days on the CAM. Pepsinogen-producing cells do not express PKK1 antigen. (a,b,d) ×206. (c,e,f) ×103.

Six-day small-intestinal or 3.5-day allantoic epithelium recombined and cultured with the proventricular mesenchyme also formed glands but did not express pepsinogen and PKK1 antigen was detected throughout these epithelia, even in the epithelial cells forming complex glands (Fig. 4).

Fig. 4.

Distribution of pepsinogen (b,d) and PKK1 (a,c) antigens in the recombinants of 6-day small-intestinal (a,b) or 3.5-day allantoic (c,d) epithelium and 6-day proventricular mesenchyme cultured for 9 days on the CAM. Gland epithelial cells are PKK1-positive and do not express pepsinogen. ×103.

When 6-day proventricular of gizzard epithelium was cultured in recombination with 6-day gizzard mesenchyme, these epithelia did not form complex glands nor express pepsinogen, and PKK1 antigen was detected throughout these epithelia (data not shown). These results as to the expression of PKK1 antigen and pepsinogen in various re-combinations are summarized in Fig. 5.

Fig. 5.

Summary of the epithelial-mesenchymal recombination experiments. Four parts of digestive tract epithelium and the allantoic epithelium were isolated and associated with the proventricular mesenchyme. The recombinants were cultured for 9 days on the CAM. Pepsinogen and cytokeratin expression was observed by immunohistochemistry. ES, esophagus; PV, proventriculus; GZ, gizzard; SI, small intestine; AL, allantois.

Regulatory influence of reaggregated proventricular mesenchymal cells on the expression of cytokeratins in digestive tract epithelia

In in vitro organ culture, 6-day proventricular and gizzard rudiments showed their typical morphology, and the distribution of PKK1 antigen was consistent with that during early development of the digestive tract; PKK1 antigen dis-appeared only in the gland cells of the proventriculus (data not shown). The reaggregated 6-day proventricular mesenchymal cells induced glands and pepsinogen in the homologous proventricular epithelium and PKK1 antigen disappeared in the pepsinogen-expressing epithelial cells (Fig. 6a, b), while reaggregated 6-day gizzard mesenchymal cells suppressed the gland formation and pepsinogen expression in the homologous gizzard epithelium and PKK1 antigen was detected throughout the epithelium (Fig. 6c). When 6-day gizzard epithelium was recombined and cultured with the reaggregated proventricular mesenchymal cells, the gizzard epithelium formed glands and expressed pepsinogen, and PKK1 antigen disappeared in the pepsinogen-expressing epithelial cells (Fig. 6d, e).

Fig. 6.

Distribution of the pepsinogen (a,d) and PKK1 (b,c,e) antigens in the recombinants of 6-day proventricular (a,b) or gizzard (d,e) epithelium and reaggregated 6-day proventricular mesenchymal cells, or in the recombinants of 6-day gizzard epithelium and reaggre-gated 6-day gizzard mesenchymal cells (c). Immunofluorescence at the apical surface of luminal epithelial cells (a,d) is due to pepsinogen secreted by the gland epithelial cells; the luminal epithelial cells themselves are pepsinogen-negative. ×206.

Nature of cytokeratins present in the chicken digestive tract epithelial cells

Our present results showed that the majority of epithelial cells of the embryonic chicken digestive tract possess substances recognized by PKK1 and AE3 monoclonal anti-bodies, which were raised against mammalian cytokeratins. Whether these substances are in fact cytokeratins is crucial for evaluating the results of the present study. We tried several times to detect the antigen recognized by PKK1 on immunoblots after SDS-PAGE of the crude extracts or of the keratin fractions (Mathinsen and Miller, 1987) of the digestive organs but could not obtain positive signals. AE3 anti-body gave clear band of 54kDa on the immunoblot (data not shown). For this reason we could not identify the molecular weight of cytokeratins recognized by PKK1. We can conceive, however, that the substances recognized by PKK1 is cytokeratin because its localization is restricted to endodermal epithelial cells and it appeared as fine fibrils on the sections and because this antibody recognizes also fish cytokeratins (manufacturer's information). Moreover the re-ported amino acid sequences of mammalian and chicken cytokeratins show high similarity (Charlebois et al., 1990).

We have recently cloned a cDNA of chicken cytokeratin gene from cDNA library of embryonic digestive organs. It has a sequence similarity with human cytokeratin 19 and its expression pattern was almost identical to that of PKK1-reactive substances (Sato et al., unpublished results). These results suggest that the reactivity to PKK1 antibody found in the chicken digestive tract epithelium is in fact due to the presence of cytokeratin in these cells.

Correlation of pepsinogen expression and cytokeratin disappearance in the proventricular epithelial cells

Proventricular epithelial cells can be subdivided into gland cells, duct cells and luminal epithelial cells (Fig. 1d). Among these cell types, only gland cells are PKK1 -negative, while duct and luminal epithelial cells are PKK1-positive (Figs. 1 and 2). In all vertebrates studied except mammals, pepsinogen-expressing cells in glandular stomach are PKK1-negative, while in mammals pepsinogen-expressing cells are PKK1-positive (Takiguchi-Hayashi and Yasugi, 1988).

It is still unclear why pepsinogen-expressing proventricular epithelial cells are negative to PKK1. We cannot exclude the possibility that the PKK1-negative epithelial cells may contain very small amount of PKK1-reactive cytokeratins. However it is known that the structure of gland epithelial cells, which actively secrete pepsinogen zymogen, is different from that of other epithelial cells (Toner, 1963). Also the distribution of AE3-reactive cytokeratin is different between pepsinogen-expressing and -non-expressing cells (Fig. 1a, b). These facts suggest that disappearance of PKK1 antigen and altered localization of AE3 antigen in proventricular gland epithelial cells may be related to some cellular physiological functions, such as production and transport of zymogen granules.

Recently we have found that expression of some genes coding substances important for development such as cSox 2 (Ishii et al., unpublished result) and sonic hedgehog (Narita et al., unpublished result) decreases dramatically in proventricular gland epithelial cells from just after the onset of gland formation. Also the expression of a mucus-associated antigen decreases abruptly in proventricular gland epithelial cells (Yoshida et al., 1996). These results indicate that proventricular gland epithelial cells constitute special sub-types among digestive tract epithelium.

Regulation of cytokeratin expression by the proventricular mesenchyme

We demonstrated that the proventricular mesenchyme can regulate cytokeratin expression in the digestive tract epithelia of chicken embryos. Disappearance of PKK1 antigen and altered distribution of AE3 antigen in the esophageal, proventricular and gizzard epithelia cultured in recombination with the proventricular mesenchyme were shown by grafting of recombinants on the CAM (Fig. 4). It was also shown that reaggregated proventricular mesenchymal cells can regulate cytokeratin expression in the stomach epithelia just as intact proventricular mesenchyme (Fig. 6). These results indicate that the regulatory influences on cytokeratin expression in the digestive tract epithelia comes from the mesenchymal cells, not from hormone-like factors.

We also revealed that the competence of the epithelium to respond to the cytokeratin-regulating action of the proventricular mesenchyme is closely related to whether pepsinogen can be induced in the epithelium or not. It was also demonstrated that the change of cytokeratin expression in the epithelium is not necessary for gland formation since small-intestinal and allantoic epithelia formed complex glands while they maintain the reactivity to PKK1. Various epithelial-mesenchymal recombination experiments have shown that, in some cases, epithelial morphogenesis can be induced by the heterologous mesenchymes but the epithelial cytodifferentiation cannot be altered by the same mesenchyme (Lawson, 1972; Mizuno and Yasugi, 1990; Sakakura et al., 1976). As for the digestive tract of chicken embryo, the small-intestinal and allantoic epithelia formed proventriculus-like complex glands but did not express pepsinogen when recombined and cultured with the proventricular mesenchyme (Haffen et al., 1982; Yasugi, 1984; Yasugi et al., 1985). These results suggest that the process of epithelial cytodifferentiation requires some mechanisms different from those of epithelial morphogenesis.

It was claimed that appropriate cell shape or polarity is prerequisite for the expression of some genes. For example, mammary gland epithelial cells express casein actively when cultured on floating collagen gels, but not on attached collagen gels, and cell shape and polarity are quite different in these two cases (Emerman and Pitelka, 1977; Haeuptle et al., 1983; Lee et al., 1985). The importance of laminin was also stressed for the synthesis of casein in mammary epithelial cells (Streuli et al., 1995). It was also reported that the cytoskeleton is important for the polarity of intestinal epithelial cells (Fath et al., 1993). The present study may also suggest that the disappearance of PKK1 antigen and altered distribution of AE3 antigen are necessary to maintain the specific physiological characteristics of gland cells which secrete pepsinogen.

An experiment showing the importance of tissue inter-actions in the regulation of cytokeratin gene expression is reported (Kurpakus et al., 1992), in which bovine conjunctival epithelium was cultivated on the corneal substrate with or without basement membrane. The conjunctival epithelium does not express K12 cytokeratin normally whereas the cor-neal epithelium does. Under the presence of the corneal basement membrane, the conjunctival epithelium came to express this cytokeratin, but without basement membrane it remained silent as to the expression of K12 cytokeratin, indicating the involvement of basement membrane in the cytokeratin gene expression.

In conclusion, the present study reveals that the mesenchymal influences regulate at the same time the expressions of pepsinogen and cytokeratin in epithelial ceils. Our recent study showed that the mesenchymal influence on the expression of pepsinogen gene is mediated by the promoter region of this gene (Fukuda et al., 1994). Whether the regulatory pathways are common for the expressions of these two different proteins is very important in considering the nature of the mesenchymal influences on the epithelial differentiation, and the problem deserves further analysis. It has been demonstrated, using cDNA for embryonic chicken pepsinogen as a probe, that the proventricular mesenchyme regulates the expression of pepsinogen gene at transcriptional level (Hayashi et al., 1988). The same kind of study is necessary for cytokeratin genes. In this context, it is note-worthy to mention that the tissue-specific expression of K18 cytokeratin is regulated by DNAase-hypersensitive sites located in promoter regions and exon 6 of this gene (Nenzaniv and Oshima, 1993).