Cells of the superficial layer which had been explanted from the presumptive ectoderm of Rana japonica early gastrulae at stage 10 differentiated into cement-gland cells (CGCs) when cultured in Barth’s solution containing 90–130 mM-NaCl., and into common epidermal cells and cilia cells when cultured in a solution containing 20–40 mM-NaCl. They failed to differentiate, however, when cultured in a solution in which NaCl is 15 mM or lower.

The optimum condition for inducing the differentiation of CGC was stimulating them with a solution containing 130 mM-NaCl for 6–10 h at 18 °C, followed by culturing in a solution containing 15–40 miu-NaCl for 7 days. The greatest ability to react to the CGC-inducing stimuli resided in the superficial layer of the presumptive ectoderm of the embryo at stages 10–11. Under the optimum condition, the total volume of CGCs induced amounted to about 85% of the explanted tissue. High percentage comparable to this was obtained with stimulation by KC1, RbCl, sucrose or mannitol.

That cell differentiation is induced and regulated by ions is an idea proposed by Barth & Barth. After extensively investigating the differentiation of cell types from amphibian presumptive ectoderm using treatments with various reagents, they have arrived at the hypothesis that the inducing compounds have as a common factor an alteration of membrane properties, resulting in the internal release of inorganic ions to the level necessary for induction (Barth & Barth, 1969, 1972, 1974). At present, we have no direct evidence for the ionic induction of cellular differentiation during normal development, but if one specific type of cell can be induced by a simple, well-known ion, this technique can be made useful in investigating and inferring the normal process of differentiation after the induction.

Given this situation, Picard (1975) and Yoshizaki (1979) aimed to produce an experimental model in which large amounts of cell masses homogeneous with respect to cell population can be obtained for use in the biochemical study of embryonic differentiation. Under the optimum condition of each model, 80–90% of the total explanted tissue differentiated into cement-gland cells (CGCs) in Xenopus (Picard, 1975) and 70% of it differentiated into hatching-gland cells in Rana (Yoshizaki, 1979) after stimulation by 10 mM-NH4Cl and 70mM-LiCl, respectively. The CGC of the amphibian sucker is one of the useful experimental subjects because its differentiation can be monitored under the dissecting microscope by the appearance of adhesive substances on its surface. The present study provides a better method for inducing CGCs from the presumptive ectoderm by NaCl or related alkali metals or sugars.

Matured grass frogs, Rana japónica, were purchased from a dealer in Tokyo during the hibernation period and maintained at 4 °C until use. Fertilized eggs and embryos were obtained as previously described (Yoshizaki, 1976). The stages of development were determined according to Tahara (1959). The jelly coat was digested by immersion in a papain-cysteine mixture (Dawid, 1965) and the vitelline coat was removed manually in sterile Barth’s standard salt solution (Barth & Barth, 1959; 88mM-NaCl, 1 mM-KCl, 0·8 mM-MgSO4, 0·3 mM-Ca(NO3)2, 0·4 mM-CaCl2, 2·3 mM-NaHCO3, 0·2 mM-Na2HPO4,0·3 mM-KH2PO4) in which neither serum nor antibiotics were included. All solutions and instruments used thereafter were sterile.

The technical details were shown in the previous paper (Yoshizaki, 1979). The explanted portion of the presumptive ectoderm (ca. 0·3 × 0·3 mm) was an area about an animal pole of a blastula and an area exactly opposite to the blastopore of a gastrula. Except when otherwise specified, the presumptive ectoderm from a stage-10 embryo (early gastrula) was used. Explants were transferred to Ca-free De Boer’s solution (110 mM-NaCl, 2-2 mM-KCl, pH 8·0 with NaHCO3) and after 15 min of immersion, the pigmented superficial layer was separated from the basal layer by pipetting out the solution surrounding the explants. The explants composed of superficial-layer tissue were then transferred into test solutions containing various concentrations of sodium chloride or related metal ions or sugars which were substituted for sodium chloride in Barth’s standard solution. All the stimulating and culturing solutions were based on Barth’s solution. After incubation in the test solutions for appropriate periods, the explants were washed and cultured in a modified standard solution containing 20 mM-NaCl for 7 days at 18 °C.

At the end of the culturing, the explants were fixed in Bouin’s solution, and serial paraffin sections, 8 pm thick, were stained with PAS (periodic acid-Schiff) after salivary treatment and Mayer’s hematoxylin. The cement-gland cell could be distinguished from the other cell types by the accumulated secretory granules stained with PAS. The volumes of individual tissues were determined by multiplying a thickness of the histological sections by an area of them measured by the point counting planimetry of Hennig & Meyer-Arendt (1963). The results were given as means ± standard errors.

Explants were prepared for electron microscopy as described previously (Yoshizaki & Katagiri, 1975). They were fixed in 5% glutaraldehyde in 0·1 M-cacodylate buffer (pH 7·3) at 4 °C for 3 h, washed in the buffer and postfixed in 1 % osmium tetroxide in cacodylate buffer for 1 h. After dehydration through graded series of ethanol, they were embedded in Epon 812, and ultrathin sections were stained in uranyl acetate and lead citrate and viewed with a Hitachi HS-8 electron microscope.

I. Differentiating cell types depending on the concentration of NaCl

When the explants from stage-10 embryos were cultured in modified solutions for 7 days, they differentiated into three types of cells, viz., cement-gland cell (CGC), cilia cell (CC) and common epidermal cell (CEC), depending on the concentration of NaCl in the solutions (Table 1). In the solutions containing NaCl higher than 90 mM, they differentiated mostly into CGCs. Concentrations of NaCl higher than 130 mM, however, seemed harmful, since the explants in the solution containing 150 mM-NaCl dissociated during the culture period. The percentage of CGC differentiation decreased in the explants in the solution containing 20–40 mM-NaCl and the culture resulted mostly in the differentiation of CCs and CECs. Some explants were composed of only CECs when cultured in the solution containing 20 mM NaCl. When the explants were cultured in the standard solution containing 15 mM or lower concentration of NaCl, no differentiation was observed and the cells were filled with yolk platelets. Ultra-structurally, the differentiated cells had few or no yolk platelets after 7 days culture but did possess each characteristic structure in the form of secretory granules and secreted matter in CGC (Fig. 1), mucous vesicles in CEC and cilia in CC (Fig. 2).

Table 1.

Differentiation of cement gland cell (CGC) in presumptive ectodermal explants cultured in Barth’s standard solution containing various concentrations of NaCl

Differentiation of cement gland cell (CGC) in presumptive ectodermal explants cultured in Barth’s standard solution containing various concentrations of NaCl
Differentiation of cement gland cell (CGC) in presumptive ectodermal explants cultured in Barth’s standard solution containing various concentrations of NaCl
Fig. 1.

Electron micrograph showing cement-gland cells in an explant treated with the standard solution containing 130 mM-NaCl for 8 h and cultured for 7 days in the standard solution containing 20 mM-NaCl. These cells are characterized by the accumulation of secretory granules (SG) and the presence of secreted matter (arrow) near the cell surface. L, lipid droplet; P, pigment granule; Y, yolk platelet. ×2100.

Fig. 1.

Electron micrograph showing cement-gland cells in an explant treated with the standard solution containing 130 mM-NaCl for 8 h and cultured for 7 days in the standard solution containing 20 mM-NaCl. These cells are characterized by the accumulation of secretory granules (SG) and the presence of secreted matter (arrow) near the cell surface. L, lipid droplet; P, pigment granule; Y, yolk platelet. ×2100.

Fig. 2.

Electron micrograph showing common epidermal cells (CEC) and cilia cell (CC) in an explant cultured for 7 days in the standard solution containing 20 mM-NaCl. The CEC is characterized by the presence of a layer of mucous vesicles (MV) beneath the apical plasma membrane. × 2100.

Fig. 2.

Electron micrograph showing common epidermal cells (CEC) and cilia cell (CC) in an explant cultured for 7 days in the standard solution containing 20 mM-NaCl. The CEC is characterized by the presence of a layer of mucous vesicles (MV) beneath the apical plasma membrane. × 2100.

II. Determination of optimum condition to induce CGC by NaCl

(a) NaCl concentration and duration of treatment

Since longer periods of exposure to higher concentrations of NaCl is unfavourable to the viability of the cells, the explants were first treated with the standard solution containing 130 mM-NaCl for 8 h and then cultured in the solutions containing decreasing concentrations of NaCl. As shown in Fig. 3, the percentage of CGC differentiation increased as the concentration of NaCl in the culture medium decreased, and about 85 % of the CGC differentiation occurred when the explants were cultured in the solutions containing 15–40 mM-NaCl.

Fig. 3.

Frequency of induction of the cement-gland cell (CGC) as a function of concentration of NaCl in the culture media. Explants (whose numbers are given) from early gastrulae (stage 10) were treated with standard solution containing 130 mM-NaCl for 8h and cultured in the standard solution containing various concentrations of NaCl for 7 days. The average percent volume of CGC and the standard error are presented.

Fig. 3.

Frequency of induction of the cement-gland cell (CGC) as a function of concentration of NaCl in the culture media. Explants (whose numbers are given) from early gastrulae (stage 10) were treated with standard solution containing 130 mM-NaCl for 8h and cultured in the standard solution containing various concentrations of NaCl for 7 days. The average percent volume of CGC and the standard error are presented.

In the next experiment, the explants were treated with the standard solutions containing various concentrations of NaCl, ranging from 20 to 150 mM for 8 h, and cultured in the solution containing 20 mM-NaCl. The highest incidence of CGC differentiation was obtained when the explants were treated with 130 mM-NaCl (Fig. 4).

Fig. 4.

Frequency of induction of the cement-gland cell (CGC) as a function of concentration of NaCl in the test solutions. Explants from stage-10 embryos were treated with the standard solution containing various concentrations of NaCl for 8 h and cultured in standard solution containing 20 mM-NaCl.

Fig. 4.

Frequency of induction of the cement-gland cell (CGC) as a function of concentration of NaCl in the test solutions. Explants from stage-10 embryos were treated with the standard solution containing various concentrations of NaCl for 8 h and cultured in standard solution containing 20 mM-NaCl.

The effect of the duration of stimulation by 130 mM-NaCl is shown in Fig. 5. The significantly high percentage of CGC differentiation was obtained when the explants were stimulated for 6–10 h which correspond to an approximate duration of gastrulation.

Fig. 5.

Frequency of induction of the cement-gland cell (CGC) by NaCl as a function of time (h). Explants (whose numbers are given) from early gastrulae (stage 10) were treated with the standard solution containing 130 mM NaCl for various hours, and cultured in standard solution containing 20 mM-NaCl for 7 days. The average percent volume of CGC and standard error are presented.

Fig. 5.

Frequency of induction of the cement-gland cell (CGC) by NaCl as a function of time (h). Explants (whose numbers are given) from early gastrulae (stage 10) were treated with the standard solution containing 130 mM NaCl for various hours, and cultured in standard solution containing 20 mM-NaCl for 7 days. The average percent volume of CGC and standard error are presented.

(b) Temporal and regional differences in the ability to react to NaCl stimulation

Explants of the superficial layer of presumptive ectoderm taken from embryos ranging from blastulae (stage 8) to late gastrulae (stage 12) were stimulated by 130 mM-NaCl for 8 h and cultured in the standard solution containing 20 mM-NaCl for 7 days. As seen in Fig. 6, the percentage of CGC differentiation was low for stages 8 and 9, reached a maximum for stages 10 and 11, and decreased thereafter.

Fig. 6.

Frequency of induction of the cement-gland cell (CGC) by NaCl as a function of developmental stages. Explants from embryos from blastulae (stage 8) to gastrulae (stage 12) were treated with the standard solution containing 130 MM-NaCl for 8 h, and cultured in standard solution containing 20 mM-NaCl.

Fig. 6.

Frequency of induction of the cement-gland cell (CGC) by NaCl as a function of developmental stages. Explants from embryos from blastulae (stage 8) to gastrulae (stage 12) were treated with the standard solution containing 130 MM-NaCl for 8 h, and cultured in standard solution containing 20 mM-NaCl.

An attempt was made to define the regional difference with respect to the ability to react to NaCl stimulation. Several different parts from the superficial layer of presumptive ectoderm of stage-10 embryos were treated in the manner mentioned above, but no significant difference appeared (data not shown).

The next experiment was carried out in order to elucidate the difference in this ability between superficial and basal layers of presumptive ectoderm. For convenience in handling, the explants of each layer were dissected from stage-11 embryos, stimulated by 130 mM-NaCl for 8h and cultured in the standard solution containing 40 mM-NaCl. The results given in Table 2 show that the basal layer is inferior to the superficial one in its ability to react to the stimulation. The major part of the former explants was occupied by the nerve cells. Thus the optimum reaction to the CGC-inducing stimuli occurs in the superficial layer of the presumptive ectoderm of stages-10 and -11 embryos.

Table 2.

Difference between two layers of presumptive ectoderm in the ability to react to stimuli inducing cement-gland cells (CGC)

Difference between two layers of presumptive ectoderm in the ability to react to stimuli inducing cement-gland cells (CGC)
Difference between two layers of presumptive ectoderm in the ability to react to stimuli inducing cement-gland cells (CGC)

III. Induction by various alkali metal ions or sugars replacing Na+

The explants from stage-10 embryos were stimulated by the chlorides of alkali metal ions comparable with Na+(K+, Li+, Rb+ and Cs+) or sugars for 8 h. As a buffer system, 5 mM-Tris-HCl (pH 7·8) was substituted for sodium bicarbonate and phosphates in the test solutions. As shown in Table 3, a high percentage of CGC differentiation comparable to that by NaCl was obtained with stimulation by 130 mM-KCl and RbCl, and 220 mM-mannitol and sucrose. The percentage was low with stimulation by CsCl. No differentiation of CGC was observed with LiCl, but the pigment cells differentiated. The explants dissociated during the culture period when they were stimulated by 130 mM-NH4C1.

Table 3.

Induction of cement-gland cell (CGC) by 8 h treatment with various kinds of chlorides or sugars

Induction of cement-gland cell (CGC) by 8 h treatment with various kinds of chlorides or sugars
Induction of cement-gland cell (CGC) by 8 h treatment with various kinds of chlorides or sugars

Sequential induction of the cement-gland cell (CGC), cilia cell (CC) and common epidermal cell (CEC) from the superficial layer of presumptive ectoderm could be made only by changing the concentration of Na+ in the culture medium. A similar phenomenon was reported by Barth (1965, 1966) and Barth & Barth (1963) in regard to the induction of the nerve cell, pigment cell, cilia cell and epithelial cell from the basal layer of presumptive ectoderm by sucrose, K+, Li+, Mg++, Ca++ or Na+. Attempts to obtain cell masses homogeneous with respect to cell population have already been made with CGC in Xenopus(Picard, 1975) and with the hatching-gland cell in Rana(Yoshizaki, 1979), in which the presumptive ectoderm was stimulated by NH4+ and Li+, respectively. Under the optimum condition defined here of 6–10 h treatment with the standard solution containing 130 mM-NaCl, about 85% of the total explanted tissue was induced to differentiate into CGC. The exact mechanism of the action of these inducing compounds on the receptive cells remains an open question, but that they act to keep the intracellular concentration of the ions at the level necessary for induction of each cell type (Barth & Barth, 1974) might be one of the possible explanations for the results. Barth & Barth (1972) have suggested the essential role of intracellular Na+ for normal development based on the finding of a specific increase of 22Na uptake after the beginning of gastrulation.

The effectiveness of sucrose or mannitol in the induction of CGC might be explained by the tonicity that it withdraws the water from the cell to elevate the concentration of intracellular ions to an appropriate level. The tonicity alone, however, seems insufficient to explain the effects by alkali metal ions because of the greatness of the difference of inducing ability among used ions and the occurrence of occasional higher induction by ions than that by sugars.

There is as yet no direct evidence of ionic induction of differentiation in the normal development of amphibian embryos. Kostellow & Morrill (1968) have reported that in Rana the intracellular sodium passes from the cells into the blastocoel fluid during the blastocoel formation, and the blastocoel fluid comes to contain a high concentration of sodium, about 71 mM which was calculated by Barth & Barth (1974). The concentration is about 100 mM in Xenopus(Slack, Warner & Warren, 1973). Thus the concentration of sodium in the blastocoel fluid of the late blastulae is less than the optimum concentration for CGC induction (130 mM), but within the range in which some induction can occur. The sodium concentration of the fluids both in the blastocoel and the intercellular spaces to which the presumptive cells are exposed will change with the advance of gastrulation.

Except for Li+, the alkali metal ions used were more or less effective in inducing CGC differentiation in Rana, whereas they were ineffective in Xenopus(Picard, 1975). This difference may be due to different potencies of presumptive ectoderm in two genera. A difference in potency was observed between different layers of Rana ectoderm, too, the basal layer having a lower potency to react to CGC-inducing stimuli than the superficial layer.

The yolk platelets of the amphibian embryonic cells are important organelles as a reservoir of both nutrients and inorganic ions (Morrill, Kostellow & Murphy, 1971). There is an evidence that yolk-platelet breakdown is correlated with embryonic induction and cellular differentiation. As Karasaki (1963) observed, the disappearance of the superficial layer of platelets is one of the first changes in induced cells. The same situation was true in the present study in that the differentiated cells had few or no platelets after 7 days culture, whereas the undifferentiated cells in the standard solution containing 15 mM or lower NaCl were filled with platelets still possessing the superficial layer. Remarkable in the latter cells was an abnormal accumulation of ribosome-like particles in a regular arrangement around the nucleus (Yoshizaki, unpublished). Once stimulated, however, presumptive ectodermal cells can embark on a programme leading to differentiation even in the solution containing 15 mM-NaCl, as shown in Fig. 3. On the other hand, such cells from embryos later than the late gastrula stage could continue their development in this solution without artificial stimulation (Yoshizaki, unpublished). One cause of the arrest of development and the resultant absence of differentiation must be the lack of available precursors for the synthesis of protein due to an inadequate intracellular environment for yolk lysis. Analysis of the arrested cells is now under way to reveal the basic metabolism in amphibian embryonic cells.

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