To reveal differentiation potency of yolk-sac endoderm, this tissue from quail embryos was cultured alone or in association with digestive-tract mesenchymes of chick embryos.

When yolk-sac endoderm was cultured alone in vitro, the endoderm of the area vitellina differentiated into the yolk-sac parenchyma, but the endoderm of the extraembryonic area pellucida (EEAP) failed to differentiate into yolk-sac parenchyma, and the endoderm of the area vasculosa became necrotic.

When endoderm of the area vitellina was cultured in association with digestive-tract mesenchymes, all the endodermal cells developed into yolk-sac parenchymal cells after two days. Later, basophilic cells appeared among them, and differentiated into both mesenchymespecific epithelia and intestinal-type epithelium with a striated border, and villi were also formed. Goblet cells appeared in all types of recombinations. The endoderm of the EEAP cultured with digestive-tract mesenchymes gave similar results to that of the area vitellina. In contrast, endoderm of the area vasculosa, when cultured with digestive-tract mesenchymes, became necrotic.

The present investigation demonstrated that the endoderms of the area vitellina and of the EEAP differ in self-differentiation potency, and that their developmental fates can be modified by the influence of digestive-tract mesenchymes. These endoderms can differentiate into the mesenchyme-specific epithelia, though they often differentiate also into the intestinal-type epithelium.

Epithelia of the extraembryonic membranes (such as amnion, chorion, and allantois) of the avian embryo have been used to analyse the inductive ability of various mesenchymes, and a vast number of dissociation and recombination experiments have been carried out (Bonetti, 1959; Kato & Hayashi, 1963; Kato, 1969; Mizuno, 1970, 1972; Sawyer, Abbott & Trelford, 1972; Mizuno & Yasugi, 1973; Yasugi & Mizuno, 1974; Sawyer, 1975, 1978; Yasugi, 1976a, b, 1979; Dhouailly, 1978; Gumpel-Pinot, Yasugi & Mizuno, 1978; Fisher & Sawyer, 1979). However, the reactivity of epithelium of the yolk sac, in spite of its importance in embryonic life, has received little attention.

It has been generally accepted that the yolk-sac endoderm differentiates from the hypoblast and area opaca endoderm (Jacobson, 1938; Vakaet, 1970; Rosenquist, 1972; Wolk & Eyal-Giladi, 1977). In a previous investigation, we studied the normal development of yolk-sac endoderm in the quail embryo and demonstrated that it develops from two different areas: the area opaca and the EEAP (Masui, 1978).

In the present study, dissociation and recombination experiments were performed to reveal the differentiation potency of the endoderms of the area opaca and of the EEAP, when they were cultured in vivo and in vitro in association with or without various mesenchymes of the digestive tract.

Embryos

Chick (Gallus gallus domesticus) and Japanese quail (Coturnix coturnix japónica) embryos were used. Eggs were incubated at 37°C. Yolk sac of quail embryos and digestive organs of chick embryos were used throughout.

Isolation of tissue fragments

Endodermal fragments of the 3- and 5-day area vasculosa and of the 5-day EEAP and mesenchymal fragments of the 5-day digestive tract were, isolated completely with collagenase (Worthington Biochemical Co., Code CLS, 0·03% in Tyrode’s solution for 1 h at 37°C). After isolation, the tissue fragments were washed thoroughly in serum-supplemented Tyrode’s solution, then in Tyrode’s solution. There was no difference between the differentiation of homotypic recombinants obtained by the use of collagenase and that of intact fragments of 5-day digestive organs. The endoderm of the 3- and 5-day area vitellina was isolated without collagenase.

Organ culture of tissue fragments in vitro

The endoderm of the yolk sac was cultured for 2 to 21 days in combination with or without mesenchymes of the digestive tract on an agar clot contained in embryological watch glasses at 37°C by the methods of Wolff & Haffen (1952). The culture medium consisted of seven parts of 1% bactoagar (Difco) in Gey’s solution, three parts of horse serum (Flow Laboratories), three parts of 12-day digestive-tract-free chick embryo extract (50% in Tyrode’s solution). Penicillin G was added. Preliminary experiments showed that the composition of this medium was best for the differentiation of the yolk-sac endoderm. Some endodermal pieces were cultured in fragments of the vitelline membrane according to Wolff (1961) and Mizuno & Sumiya (1974).

The explants were transferred on to new culture medium every seventh day. Intact fragments or homotypic recombinants of 5-day chick digestive organs were also cultured under the same conditions and served as control.

In vivo cultivation of recombinants

After cultivation for 1 to 2 days on the Wolff & Halfen (1952) medium, recombinants were grafted onto the chorio-allantoic membrane (CAM) of 9-day chick embryos for a further 7 to 10 days.

Histological methods

After cultivation, the explants were fixed in Bouin’s fluid, embedded in paraffin, and sectioned at 5 μm. The sections were stained with PAS-haema-oxylin. Morphological characters of the cell nuclei of chick and quail make the distinction between the derivation of tissues (Le Douarin, 1969). Some explants were fixed in Zenker’s solution and the sections were stained by the Feulgcn-Rossenbeck technique (1924).

Analysis of experimental data

The explants were examined according to the following criteria. When various types of differentiation were formed in one explant, they were all scored.

For the oesophageal epithelium, stratified cuboidal epithelium; for the proventricular epithelium, simple or pseudostratified columnar epithelium and proventricular glands; for the gizzard epithelium, high pseudostratified columnar epithelium; for the small intestinal epithelium, simple columnar epithelium with PAS-positive striated border and the formation of villi in typical cases; for the yolk-sac parenchymal cells, large round cells with PAS-positive cytoplasm and large cytoplasmic vacuoles, and often PAS-positive granules.

1 Self-differentiation potency of the yolk-sac endoderm in vitro

The endoderm of the 5-day EEAP in situ is a simple squamous epithelium (Fig. 3). When the isolated endoderm was cultured in vitro enveloped in a fragment of the vitelline membrane, it differentiated mainly into round baso-philic cells arranged irregularly (Fig. 4). Long-term cultivation caused necrosis in the explants, and neither goblet cells nor the striated border differentiated.

Fig. 1

Diagram showing the mode of combination of yolk-sac endoderm with various mesenchymes of the digestive tract. Abbreviations: OE, oesophagus; PRO, proventriculus; GIZ, gizzard; SI, small intestine; EEAP, extraembryonic area pellucida; AVas, area vasculosa; A Vit, area vitellina.

Fig. 1

Diagram showing the mode of combination of yolk-sac endoderm with various mesenchymes of the digestive tract. Abbreviations: OE, oesophagus; PRO, proventriculus; GIZ, gizzard; SI, small intestine; EEAP, extraembryonic area pellucida; AVas, area vasculosa; A Vit, area vitellina.

Fig. 2

Time course of differentiation of 3- and 5-day area vitellina endoderm cultured for 1 to 3 weeks in vitro in association with various mesenchymes of 5-day digestive tract. The percentage of grafts showing mesenchyme-specific induction and self-differentiation potency is compared to the total number of grafts recovered.

Fig. 2

Time course of differentiation of 3- and 5-day area vitellina endoderm cultured for 1 to 3 weeks in vitro in association with various mesenchymes of 5-day digestive tract. The percentage of grafts showing mesenchyme-specific induction and self-differentiation potency is compared to the total number of grafts recovered.

Fig. 3

Intact EEAP of 5-day quail embryo. The endodermal epithelium (EN) is very thin, × 670. In the following recombinations, mesenchyme was obtained from 5-day chick embryos, and yolk-sac endoderm was obtained from quail embryos.

Fig. 3

Intact EEAP of 5-day quail embryo. The endodermal epithelium (EN) is very thin, × 670. In the following recombinations, mesenchyme was obtained from 5-day chick embryos, and yolk-sac endoderm was obtained from quail embryos.

Fig. 4

The endoderm of 5-day EEAP alone cultured for 7 days in vitro enveloped in the vitelline membrane. Undefined cells are seen, × 670.

Fig. 4

The endoderm of 5-day EEAP alone cultured for 7 days in vitro enveloped in the vitelline membrane. Undefined cells are seen, × 670.

The endoderm of the 3- and 5-day area vitellina in situ consists of large cells swollen with PAS-positive yolk drops and irregularly packed in several layers (Fig. 10). When cultured directly on the medium, the area vitellina endoderm differentiated into typical yolk-sac parenchyma (Fig. 11).

Fig. 5

The association of 5-day EEAP endoderm and small intestinal mesenchyme cultured in vitro for 2 weeks. The epithelium is simple columnar, and villi, striated border, and goblet cells are seen (Small-intestinal-type differentiation), × 670.

Fig. 5

The association of 5-day EEAP endoderm and small intestinal mesenchyme cultured in vitro for 2 weeks. The epithelium is simple columnar, and villi, striated border, and goblet cells are seen (Small-intestinal-type differentiation), × 670.

Fig. 6

The association of 5-day EEAP endoderm and proventricular mesenchyme cultured on the CAM for 7 days. Glands are well formed. Mitotic figures are seen (Proventricular-type differentiation), × 340.

Fig. 6

The association of 5-day EEAP endoderm and proventricular mesenchyme cultured on the CAM for 7 days. Glands are well formed. Mitotic figures are seen (Proventricular-type differentiation), × 340.

Fig. 7

The association of 5-day EEAP endoderm and proventricular mesenchyme cultured on the CAM for 10 days. The epithelium is simple columnar, and villi are well formed (Small-intestinal-type differentiation), × 340.

Fig. 7

The association of 5-day EEAP endoderm and proventricular mesenchyme cultured on the CAM for 10 days. The epithelium is simple columnar, and villi are well formed (Small-intestinal-type differentiation), × 340.

Fig. 8

The association of 5-day EEAP endoderm and gizzard mesenchyme cultured on the CAM for 7 days. The epithelium is pseudostratified columnar, and PAS-positive granules and secretion are seen (Gizzard-type differentiation), × 670.

Fig. 8

The association of 5-day EEAP endoderm and gizzard mesenchyme cultured on the CAM for 7 days. The epithelium is pseudostratified columnar, and PAS-positive granules and secretion are seen (Gizzard-type differentiation), × 670.

Fig. 9

The association of 5-day EEAP endoderm and small intestinal mesenchyme cultured for ten days on the CAM. The epithelium is simple columnar, and villi are formed (Small-intestinal-type differentiation), × 340.

Fig. 9

The association of 5-day EEAP endoderm and small intestinal mesenchyme cultured for ten days on the CAM. The epithelium is simple columnar, and villi are formed (Small-intestinal-type differentiation), × 340.

Fig. 10

The 5-day quail area vitellina. EC, ectoderm; EN, endoderm. ×400.

Fig. 10

The 5-day quail area vitellina. EC, ectoderm; EN, endoderm. ×400.

Fig. 11

A part of the endoderm of 5-day area vitellina cultured alone in vitro for one week. Yolk-sac parenchymal cells differentiated, × 670.

Fig. 11

A part of the endoderm of 5-day area vitellina cultured alone in vitro for one week. Yolk-sac parenchymal cells differentiated, × 670.

The endoderm of the 3- and 5-day area vasculosa in situ consists of differentiated yolk-sac parenchymal cells. In cultures, the endoderm kept its differentiated state for one week, but longer cultivation resulted in necrosis.

2 The differentiation of yolk-sac endoderm under the influence of digestivetract mesenchymes

2.1 Differentiation of the EEAP endoderm

When endoderm of the 5-day EEAP was cultured in vitro on mesenchyme of 5-day proventriculus or small intestine, it differentiated into the mesenchyme-specific epithelium, and intestinal-type epithelium also developed in all types of recombinations. Goblet cells appeared after two weeks of cultivation. Villi and the striated border differentiated in the recombination with small intestinal mesenchyme after two weeks (Fig. 5). Yolk-sac parenchyma was developed in combination with the gizzard mesenchyme, while it scarcely appeared in the other types of recombinations.

When similar recombinants were cultured on the CAM, the mesenchyme-specific induction and the intestinal differentiation with villi formation took place (Table 1, Figs 69). Striated border was developed best in association with the oesophageal mesenchyme. Villus formation and the differentiation of striated border seemed to occur independently. No yolk-sac parenchyma differentiated.

Table 1

Differentiation of yolk-sac endoderm cultured on CAM in association with 5-day digestive-tract mesenchymes

Differentiation of yolk-sac endoderm cultured on CAM in association with 5-day digestive-tract mesenchymes
Differentiation of yolk-sac endoderm cultured on CAM in association with 5-day digestive-tract mesenchymes

2.2 Differentiation of the area vitellina endoderm

The endoderm of the 3- and 5-day area vitellina was cultured in vitro in association with 5-day digestive-tract mesenchymes. After 2 days of culture, the endodermal cells differentiated into yolk-sac parenchyma (Fig. 12). Cultured longer, they became basophilic and proliferative. These basophilic cells differentiated not only into the mesenchyme-specific epithelia under the influence of the digestive-tract mesenchymes (Table 2, Figs 13,17), but also into epithelium of small intestinal type (Figs 14, 15, 17). When the explants were cultured for more than two weeks, a PAS-positive striated border often developed (Figs 14, 15, 17), and it was as high as that observed in the intact small intestine cultured in vitro. Goblet cells appeared in all types of recombinations. The results are summarized in Fig. 2.

Table 2

Differentiation of 3- and 5-day vitellina endoderm cultured in vitro in association with 5-day digestive-tract mesenchymes

Differentiation of 3- and 5-day vitellina endoderm cultured in vitro in association with 5-day digestive-tract mesenchymes
Differentiation of 3- and 5-day vitellina endoderm cultured in vitro in association with 5-day digestive-tract mesenchymes
Fig. 12

The association of 3-day area vitellina endoderm and oesophageal mesenchyme cultured for 2 days in vitro. Yolk-sac parenchymal cells developed, × 340.

Fig. 12

The association of 3-day area vitellina endoderm and oesophageal mesenchyme cultured for 2 days in vitro. Yolk-sac parenchymal cells developed, × 340.

Fig. 13

The association of 3-day area vitellina endoderm and proventricular mesenchyme cultured for 3 weeks in vitro. The epithelium is simple columnar, and typical glands and goblet cells are seen (Proventricular-type differentiation), × 340.

Fig. 13

The association of 3-day area vitellina endoderm and proventricular mesenchyme cultured for 3 weeks in vitro. The epithelium is simple columnar, and typical glands and goblet cells are seen (Proventricular-type differentiation), × 340.

Fig. 14

The association of 5-day area vitellina endoderm and proventricular mesenchyme cultured for 2 weeks in vitro. All the epithelial cells are simple columnar with striated border. Glands are also well formed. Goblet cells appear, × 340.

Fig. 14

The association of 5-day area vitellina endoderm and proventricular mesenchyme cultured for 2 weeks in vitro. All the epithelial cells are simple columnar with striated border. Glands are also well formed. Goblet cells appear, × 340.

Fig. 15

The association of 5-day area vitellina endoderm and proventricular mesenchyme cultured for 2 weeksin vitro. Undulated, simple columnar epithelium, well developed striated border, and goblet cells are seen (Small-intestinal-type differentiation), × 340.

Fig. 15

The association of 5-day area vitellina endoderm and proventricular mesenchyme cultured for 2 weeksin vitro. Undulated, simple columnar epithelium, well developed striated border, and goblet cells are seen (Small-intestinal-type differentiation), × 340.

Fig. 16

The association of 5-day area vitellina endoderm and gizzard mesenchyme cultured for 2 weeks in vitro. Simple columnar epithelium composed of well developed yolk-sac parenchymal cells, × 670.

Fig. 16

The association of 5-day area vitellina endoderm and gizzard mesenchyme cultured for 2 weeks in vitro. Simple columnar epithelium composed of well developed yolk-sac parenchymal cells, × 670.

Fig. 17

The association of 5-day area vitellina endoderm and small intestinal mesenchyme cultured for 3 weeks in vitro. High simple columnar epithelium, well developed striated border, and goblet cells are seen (Small-intestinal-type differentiation). × 670.

Fig. 17

The association of 5-day area vitellina endoderm and small intestinal mesenchyme cultured for 3 weeks in vitro. High simple columnar epithelium, well developed striated border, and goblet cells are seen (Small-intestinal-type differentiation). × 670.

When the endoderm of the area vitellina of 3- or 5-day embryos was cultured on the CAM in combination with the digestive-tract mesenchymes, the results were similar to those obtained in vitro (Table 1). The endoderm of the area vitellina differentiated into mesenchyme-specific epithelia and also into intestinal type epithelium with goblet cells and well-developed villi in all types of combinations. In association with the oesophageal mesenchyme, striated border developed on a stratified cuboidal epithelium (Fig. 18).

Fig. 18

The association of 5-day area vitellina endoderm and oesophageal mesenchyme cultured for 10 days on the CAM. The epithelium is pluristratified. There also differentiated striated border and goblet cells (Oesophageal-type differentiation). ×670.

Fig. 18

The association of 5-day area vitellina endoderm and oesophageal mesenchyme cultured for 10 days on the CAM. The epithelium is pluristratified. There also differentiated striated border and goblet cells (Oesophageal-type differentiation). ×670.

2.3 Differentiation of the area vasculosa endoderm in vitro

Endoderm of the 3- and 5-day area vasculosa was cultured in vitro in association with 5-day digestive-tract mesenchymes. The endoderm became necrotic and scarcely differentiated into mesenchyme-specific epithelia for intestinal-type epithelium.

Self-differentiation of yolk-sac endoderm

In normal development, the area vitellina endoderm differentiates into yolksac parenchyma according to the expansion of the mesoderm (Bellairs, 1963; Bennett, Dubois & Chapeville, 1972; Bennett, 1973; Masui, 1978). The present investigation clearly demonstrated that isolated endoderm of the area vitellina can self-differentiate into yolk-sac parenchyma in the absence of mesenchyme, when the endoderm was cultured alone in vitro. Moreover, even when cultured with inductively active mesenchymes of the digestive-tract, the area vitellina endoderm differentiated into yolk-sac parenchyma in the first place. Therefore, it is conceivable that the area vitellina endoderm has an intense potency to differentiate into the yolk-sac parenchyma by itself.

In contrast, the EEAP endoderm hardly differentiated into yolk-sac parenchyma in normal development (Masui, 1978) and when cultured alone in vitro (in the present study). It has been reported, however, that area pellucida endoderm of early stages can self-differentiate into yolk-sac parenchyma when it is cultured in vitro enveloped in the vitelline membrane (Sumiya, 1976). In the present investigation, it was demonstrated that the EEAP endoderm differentiated into yolk-sac parenchyma when cultured with gizzard mesenchyme. It might be that the EEAP endoderm can barely self-differentiate alone into yolk-sac parenchyma, but still has that potency, and, in adequate conditions can differentiate into yolk-sac parenchyma.

Inductive ability of digestive-tract mesenchymes and the differentiation potency of yolk-sac endoderm

Specific induction has been studied between the digestive-tract mesenchymes and heterologous epithelia: gizzard or proventricular mesenchyme and epi-dermis (McLoughlin, 1961), gizzard mesenchyme and proventricular epithelium (Sigot, 1963; Sigot & Marin, 1970), proventricular or intestinal mesenchyme and ureter (Bishop-Calame, 1966), proventricular, gizzard, or intestinal mesenchyme and bronchial epithelium (Dameron, 1968), digestive-tract mesenchymes and allantoic endoderm (Mizuno & Yasugi, 1973; Yasugi & Mizuno, 1974; Yasugi, 1976a, b, 1979; Gumpel-Pinot et al. 1978), and mesenchymes and epithelia of digestive tract (Yasugi & Mizuno, 1978; Gumpel-Pinot et al. 1978).

In the present investigation we demonstrated that, when the area vitellina endoderm was cultured with digestive-tract mesenchymes, the endodermal cells differentiated into yolk-sac parenchymal cells at first, but later they became basophilic and proliferative, and lastly they differentiated not only into the mesenchyme-specific epithelia of the digestive tract but also into small intestinal type epithelium. The EEAP endoderm, which is originally basophilic, also can differentiate into mesenchyme-specific epithelia and the small intestinal type epithelium under the influence of digestive-tract mesenchymes.

The results of the present investigation confirm the instructive induction of the digestive-tract mesenchymes, and demonstrate clearly the competence of the area vitellina and EEAP endoderm to the induction by the digestive-tract mesenchymes.

The endoderm of the area vitellina and EEAP does not differentiate into intestinal type epithelium with goblet cells or striated border, during normal development (Bellairs, 1963; Bennett et al. 1972; Bennett, 1973; Masui, 1978; Mobbs & McMillan, 1979) nor in in vitro culture (the present investigation). However, when cultured with digestive-tract mesenchymes, the yolk-sac endoderm sometimes differentiated into small intestinal type epithelium with goblet cells, striated border and villi, in all types of recombinations. These results indicate that the endoderm of the area vitellina and EEAP can manifest its differentiation potency to intestinal epithelium under non-organ-specific stimuli of digestive-tract mesenchymes.

In the recombination experiments, it is not conceivable that the mere increase in cell number by the association with mesenchymes could affect the differentiation pattern of the yolk-sac endoderm, because the yolk-sac endoderm differentiated into mesenchyme-specific epithelia according to the types of combined digestive-tract mesenchymes. Further, when cultured alone without any mesen-chymal cells, the area vitellina endoderm differentiated only into yolk-sac parenchyma, even if the volume of endoderm in an explant was increased.

The influence of yolk-sac endoderm on digestive-tract mesenchymes

Little is known about the inductive influence of the epithelium to the mesenchyme: the small intestinal epithelium induced villus formation in intrasplanchnopleural grafting (Gumpel-Pinot, et al. 1978) and in the CAM grafting after recombination with heterologous mesenchymes (Yasugi & Mizuno, 1978). The present investigation revealed that villi were formed when the area vitellina or EEAP endoderm was cultured on the CAM with digestive-tract mesenchymes. The villus formation did not take place unless the epithelium differentiated into intestinal type epithelium. Therefore the villus formation seems to occur only when the epithelium manifests intestinal differentiation.

Mechanisms of differentiation of the area vitellina endoderm into mesenchyme-specific epithelia and those of the intestinalization under the influence of the digestive-tract mesenchymes are problems worth further studies. We are now following the course of the differentiation of the yolk-sac endoderm and giving attention to the expression of tissue-specific enzymes.

The author wishes to express his deep gratitude to Professor Takeo Mizuno of the University of Tokyo for his valuable advice and encouragement during the course of this work.

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