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

Differentiation

Differentiation (1985) 30:47-52

c) Springer-Verlag 1985

Gland formation induced in the allantoic and small-intestinal endoderm by the proventricular mesenchyme is not coupled with pepsinogen expression Sadao Yasugi Susumu Matsusbita ’,and Takeo Mizuno ‘Zoological Institute, Faculty of Science, University of Tokyo, Tokyo, 1 1 3, Japan *Department of Biology, Tokyo Women’s Medical College, Tokyo, 162, Japan

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 to pepsinogens. 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 cytochemical differentiation of the endoderm.

Introduction That the differentiation of avian digestive-tract endoderm is often influenced by the presence of the surrounding mesenchyme has been demonstrated by many experiments involving the in vitro and in vivo culture of recombinates composed of various endoderms and mesenchymes [2-8, 11, 13, 15, 23, 241. Previous investigations dealing mainly with the morphological differentiation of the endoderm induced by the associated mesenchyme have suggested that the mesenchyme plays a decisive role in the morphogenesis of the endoderm. However, recent studies have focused attention on the functional, biochemical differentiation of endodermal cells. In such investigations, antibodies against purified embryonic chick pepsinogen (EPg (251) and chick sucrase [12-141- specific marker proteins of the proventriculus and intestine, respectively - have proved to be useful tools. Yasugi [21] has demonstrated that allantoic endoderm cultured in association with proventricular mesenchyme forms complex glands and exhibits acid-protease activity. However, it has also been shown that allantoic endoderm implanted into the presumptive digestive area of young chick embryos is incorporated into the digestive tract of the host and differentiates into various digestive epithelia

that never produce pepsinogen, even when the implants are located in the proventriculus [22]. In the present study, we further investigated the reactivity of allantoic endoderm to the inductive influence of proventricular mesenchyme, particularly with regard to the appearance of acid proteases, pepsinogens and sucrase. The reactivity of small-intestinal endoderm, which has been found to be rather resistant to the inductive influence of heterologous mesenchymes [24], was also examined. Methods

Materials. Embryos of White-Leghorn chick and Japanese quail were used. Tissue recombination and grafting. The allantoic endoderm of 3.5-day chick embryos and 3-day quail embryos was isolated from the mesenchyme by collagenase treatment (0.03% in Tyrode’s solution, type CLS; Worthington) for 30 min at 37” C. The endoderms and mesenchymes of the proventriculus and small intestine were isolated from 6-day chick embryos and 5-day quail embryos by the same treatment. The isolated tissues were washed thoroughly in serum-supplemented (50%) Tyrode’s solution and then in Tyrode’s solution. The endoderms and mesenchymes were recombined and cultivated for 16 h on a semi-solid agar medium [20] to ascertain the adhesion of both tissues. The recombinates were then grafted onto the chorioallantoic membrane of 9day chick embryos and cultivated for 9-10 days. Mesenchyme was always obtained from chick embryos, while endoderm was obtained from either chick or quail embryos. The use of quail endoderm made it possible to confirm that there was no contamination of the mesenchymal cells in the endoderm preparation.

Immunohistochemistry. The grafts were fixed in ice-cold 95% ethanol for 4 h and then embedded in paraffin. Sections (5 pm) were serially mounted on several slides and then stained with PAS-hematoxylin (PAS-HX) or subjected to indirect immunofluorescence [17]. The antirabbit IgG antiserum conjugated with fluorescein isothiocyanate was purchased from Miles Laboratories (Ind). Immunofluorescence was observed using an Olympus BHF fluorescence microscope. Sections were stained with PAS-HX after immunofluorescence. Antiembryonic pepsinogen antiserum against a purified chick embryonic pepsinogen was raised

48

in rabbits [25] and absorbed as previously described [22]. This antiserum also reacts with a pepsinogen of embryonic quail. The purification of chick sucrase and the specificity of the antiserum to it have been reported elsewhere [12-141. This antiserum also reacts with quail intestinal epithelial cells. In indirect-immunofluorescence and immunoelectrophoretic assays, the antisera to EPg and sucrase specifically react with proventricular and intestinal endodermal cells, respectively.

Table 1. Gland formation and the appearance of EPg- and sucrasepositive cells in the recombinates

Electron microscopy. The explants were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.5) for 2 h at 4" C, and then postfixed in 1% osmium tetroxide in the same buffer for 2 h at 4" C. They were dehydrated in ethanol and embedded in Embedding Resin (Taab, UK). Ultrathin sections were stained with uranyl acetate and lead citrate. and observed using a Hitachi H-800 electron microscope.

Allantois" Small intestineb

Biochemical analysis. For the biochemical analysis of acid proteases, recovered grafts were pooled and kept at -20" C. These grafts were homogenized in a glass-Teflon homogenizer in 10 mM sodium phosphate buffer (pH 7.0) containing 50 pg/ml leupeptin and 50 Fg/ml antipain (Peptide Institute, Osaka, Japan). The homogenate was centrifuged at 80,OOOg for 1 h, and the supernatant was used as an enzyme solution. The acid-protedse activity was measured at pH 2.0 using bovine hemoglobin (Sigma, type I) as substrate, according to a modification [25] of Anson's method 111. One unit of enzyme was defined as being the amount which caused an increase of 1.0 absorbance at 280 nm per minute, in the trichloroacetic acid (TCA)-soluble fraction. Whether the acid-protease activity observed in the grafts was due to pepsins was assessed using the following criteria : first, the activity inhibited by the presence of 0.05 pg/ml pepstatin (Peptide Institute) was considered to be 'true acidprotease activity', since pepstatin is a specific inhibitor of acid proteases [19]. Second, the activity inhibited by alkali treatment (PH 9.2) of the enzyme solution (overnight at 4" C) after the activation of pepsinogens in the solution to pepsins by acid treatment (pH 3.2 for 2 h at room temperature) was considered to be due to pepsinogens [25]. The protein level was determined according to the method of Lowry et al. 191 using bovine serum albumin as standard.

Results

There was no significant difference between the differentiation of the chick and quail endoderms. Gland formation and the appearance of the anti-EPg-antiserum- and antisucrase-antiserum-reactive cells in the grafts are summarized in Table 1. Imrnunohistochemical differentiation of allantoic endoderm

The allantoic endoderm that was associated with the proventricular mesenchyme and was cultivated on the chorioallantoic membrane formed complex glands (Fig. 1). As shown in Fig. l a , these glands extended radially from a central lumen, and they were surrounded by well-developed muscular layers. The endoderm differentiated into a pluristratified cuboidal or simple columnar epithelium in which scattered PAS-positive goblet cells were sometimes found

Endoderm

Mesenchyme

Morpho- Number of regenesis combinates with of glands

Proventriculusb Proventriculusb Proventric~lus~ Proventriculusb

41/53

8/8 16/18

Anti-

AntiEPgsucrasereactive reactive glands glands 0120 018 16/18

6/12 818 0118

' 3.5-day chick or 3-day quail &daychick

(Figs. 1 b, 3a). The epithelium was always negative for the anti-EPg antiserum, even when the complex glands had been induced (Fig. 1d), while antisucrase-antiserum-positive cells were detected in 6 out of 12 grafts (Fig. If). The apical surface of some, but not all, simple columnar cells was clearly stained with this antiserum. The percentage of sucrase-positive cells varied from graft to graft, but it never exceeded 20%. This is in agreement with our previously reported results [13]. Immunohistochemical dgferentiation of the small-intestinal endoderm

The small-intestinal endoderm cultivated with proventricular mesenchyme behaved like the allantoic endoderm in that it formed complex glands (Fig. 2 a). Villus-like structures were often seen near the central lumen (Fig. 2b). In the recombinates, no cells reactive with the anti-EPg antiserum were observed (Fig. 2d), but almost all epithelial cells reacted against the antisucrase antiserum (Fig. 20. Electron-microscopic observations

In the recombinates composed of allantoic endoderm and proventricular mesenchyme, epithelial cells with an immature brush border were occasionally observed, but the microvilli were short (less than 300 nm; Fig. 3a). In the recombinates composed of small-intestinal endoderm and proventricular mesenchyme, most of the epithelial cells had a welldeveloped brush border with longer microvilli (70& 800 nm; Fig. 3 b). Biochemical analysis of acid proteases induced in allantoic endoderm by proventricular mesenchyme

In a previous study [21], we detected acid-protease activity at pH 2.0 in recombinates composed of allantoic endoderm and proventricular mesenchyme, and we referred to this activity as peptic activity. The already mentioned results prompted us to analyze whether this protease activity is due to pepsinogens or not. The recombinates composed of allantoic endoderm and proventricular mesenchyme as well as those composed of allantoic endoderm and small-intestinal mesenchyme exhibited definite but lower levels of acid-protease activity than the recombinates composed of proventricular endoderm and proventricular mesenchyme and of similarly cultivated

49

Fig. 1a-f. Morphology and immunohistochemistry of recombinates of allantoic endoderm and proventricular mesenchyme cultivated on chorioallantoic membrane for 9 days. a Welldeveloped glands are arranged radially (PAS-HX). b Higher magnification of the glands ; the pluristratified epithelia are cuboidal or simple columnar. Scattered, PAS-positive goblet cells are visible (urrows; PAS-HX). c Section stained with PAS-HX after immunofluorescence. d The same section as that shown in c; immunofluorescence for EPg. e Section stained with PAS-HX after immunofluorescence. f The same section as that shown in e; immunofluorescence for sucrase. a x 36; b x 436; c f x 215

intact 6 d a y proventriculi (Table 2), thus confirming our previous results [21]. The results obtained by biochemically analyzing the inhibition of acid proteases are summarized in Table 3. Pepstatin, a specific inhibitor of acid proteases, inhibited the acid-protease activity of all kinds of recombinates by about go%, thus indicating that most of the activity found was due to true acid proteases. Alkali treatment, which is known to inhibit the activity of embryonic chick pepsin [25], also lowered the activity in control recombinates of proventricular endoderm and proventricular mesenchyme by about SO%, while the activi-

ty in intact 15-day proventriculi was reduced by about 80%. However, alkali treatment had hardly any effect on the activities of recombinates of allantoic endoderm and proventricular or small-intestinal mesenchyme (Table 3).

Discussion The results obtained in the present study confirmed previous observations that the proventricular mesenchyme can affect the morphological differentiation of the endoderms both of the digestive tract and of the extraembryonic membranes [lo, 13, 16, 22, 271: the allantoic and small-intestinal

50

Fig. 2a-f. Morphology and immunohistochemistry of recombinates of small-intestinal endoderm and proventricular mesenchyme cultivated on chorioallantoic membrane for 9 days. a Well-developed glands are formed (PAS-HX). b Villus-like structures are visible near the central lumen of the graft (PAS-HX). c Section stained with PAS-HX after immunofluorescence. d The same section as that shown in c; immunofluorescence for EPg. The fluorescence seen in the mesenchyme represents the autofluorescence of erythrocytes. e Section stained with PAS-HX after immunofluorescence. f The same section as that shown in e; immunofluorescence for sucrase. All of the apical surface of the glandular epithelium is positive. a x 138; b x 165; c-f x 138

endoderms underwent heterotypic morphogenesis under the influence of the proventricular mesenchyme, and formed complex glands never found during the normal course of development of the allantois and small intestine. The present study also revealed that allantoic and smallintestinal endoderms have a tendency to differentiate into a small-intestinal epithelium that has sucrase antigen on the apical-cell surface. Concomitantly, some endodermal cells differentiated into goblet cells, one of the cell types of the intestinal epithelium. We have previously shown that allantoic endoderm is apt to differentiate into intestinal epithelium when it is cultured in combination with heterolo-

gous mesenchymes [22, 231, a finding that has been confirmed by Matsushita [I 31. The small-intestinal endoderm also expresses sucrase when it is associated with gizzard or oesophageal mesenchyme (unpublished results). The appearance of the brush border in these recombinates was examined using electron microscopy. The brush border in the combination of allantoic endoderm and proventricular mesenchyme was rather immature, but the sucrase antigen has been found to be associated with such immature, developing microvilli during the normal development of embryonic intestinal cells [14]. Allantoic and small-intestinal endoderm, however,

51

Fig. 3a, b. Apical surface of endodermal cells of the allantois (a) and small intestine (b) cultivated with proventricular mescnchyme. A goblet cell is seen in a. x 10,OOO

never produced embryonic pepsinogen that could be detected using the antiserum to a pepsinogen purified from embryonic chick proventriculi. This is consistent with results obtained after the implantation of allantoic endoderm into the presumptive proventricular region [22]. There was a possibility, however, that the endoderm might produce pepsinogens other than the pepsinogen against which the antiserum had been raised, since embryonic proventriculi contain several pepsinogen species [26, 281. This possibility was investigated by assessing the effects of pepstatin and alkali treatment on the acid-protease activity detected in recombinates consisting of allantoic endoderm and proventricular mesenchyme. Biochemical analysis demonstrated Table 2. Acid-protease activities or recombinates and control grafts Endoderm

Mesenchyme

Allantois' Proventriculusd Allantois' Small intestined Proventriculusd ProventricuIusd Intact proventriculi'

Number of determinations.

Acid-protease activity (units/mg protein)b

7

0.047 f0.016 0.035 f0.01 5 0.083f0.014 0.169 0.022

5 5

4

' For each determination,

10-50 grafts were pooled and assayed Mean+SD 3.5-day chick or 3-day quail &day chick 6day intact proventriculi were cultivated for 9-10 days

that the protease activity detected in the recombinates was due to true, nonspecific acid proteases, but not to pepsinogens. This conclusion was further confirmed by the fact that the similar acid-protease activity was detected in recombinates of allantoic endoderm and small-intestinalmesenchyme (Table 2). These results do not necessarily indicate that the proventricular mesenchyme is unable to induce pepsinogens in heterologous endoderms, since we have previously found that the primary hypoblast of young avian embryos - which is destined to differentiate into the yolk-sac endoderm during the normal course of development - expresses pepsinogen-antigen and forms glands when it is recombined with the proventricular mesenchyme [271. Moreover, we have also shown that gizzard and oesophageal endoderms can also respond to the stimulus of the proventricular mesenchyme by producing pepsinogens (unpublished results). Thus, it can be concluded that the failure of pepsinogen to appear in allantoic and small-intestinal endoderms is due to the absence of reactivity in these endoderms to the pepsinogen-inducing signals coming from the proventricular mesenchyme. In this respect, a further possibility is that the mRNAs for the pepsinogens are induced in allantoic or small-intestinal endoderm by the proventricular mesenchyme, but their translation cannot be realized in endodermal cells. This possibility is currently being examined using DNA complementary to the mRNAs of embryonic pepsinogens. In conclusion, when allantoic and small-intestinal endoderms were cultured in combination with the proventricular

Table 3. Inhibition of acid-protease activities of recombinates and control proventriculi Endoderm

~

Mesenchyme

~

Allantois' Allantois" Proventriculus lntact proventriculi

Number of grafts

Acid-protcase activity (units/mg protein)

Inhibition (YO) Pepstatin

Alkali treatment

89

1 13 52 82

~

Proventrialus Small intestine Proventriculus

3.5-day chick or 3-day quail 6-daychick 15-day chick proventriculi (not cultured)

56 35 25

7

0.026 0.036 0.080 0.30

95

87 97

mesenchyme, these recombinates formed complex glands, but although they produced sucrase, they never expressed pepsinogens. These results confirm the view that, in epithelial-mesenchymal interactions that occur during organogenesis, heterotypic morphogenesis of effector tissues is not always coupled with the production of proteins specific to the epithelia of the organs from which the associated mesenchyme is derived [18, 22, 231. Acknowledgements. This study was supported by Grants-in-aid from the Ministry of Health and Welfare of Japan for cancer research (59-7), from the Cancer Research Foundation, from the Ministry of Education, Science and Culture of Japan for Special Project Research (Project No. 59213004), as well as from The Naito Foundation (Grant for 1983).

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Received April 1985 / Accepted in revised form September 1985