ph-dependence of the inducing activity of lithium ion

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1 J. Embryo!, exp. Morph., Vol. 15, 3, pp , June With 1 plate Printed in Great Britain ph-dependence of the inducing activity of lithium ion ByYOSHIO MASUI From the Department of Biology, Konan University INTRODUCTION Previous investigations of primary induction in amphibian embryos have shown that there are two kinds of heterogenic inductors, one of which induces the presumptive ectoderm towards neural differentiation alone, the other towards mesodermal or endodermal differentiation, and that they are characteristically distinct from one another in chemical nature (Yamada, 1961; Saxen & Toivonen, 1962). Various substances which are chemically dissimilar to each other have been found to be effective in neural induction, though their inducing activity was always thermostable. On the contrary, mesodermal and endodermal inductions were found to be caused only by certain proteins from vertebrate tissues, and their inducing activity was always thermolabile. The neural induction of gastrula ectoderm can also be brought about by exposing the tissue to saline solution under appropriate conditions but in the absence of specific inductor, whereas this is not the case in the mesodermal and endodermal inductions (Barth, 1941; Holtfreter, 1944, 1945, 1947). Hence, it appears that the mechanism of neural induction is not the same as that of mesodermal and endodermal inductions. However, recent investigations of the action of lithium ion on the differentiation of the gastrula ectoderm have disclosed that the ion induces a broad spectrum of differentiation, not only of neural but also of mesodermal and endodermal character (Barth & Barth, 1959, 1962,1963,1964; Gebhardt & Nieuwkoop, 1964; Masui, 1958,1959, I96,1961; Ogi, 1961). This raises a question of whether the specificity of induction of the differentiation of the ectoderm of amphibian gastrula resides in the inducing substance. A single kind of molecule such as LiCl, which presumably does not carry specific information because of the simplicity of its molecular structure, is capable of inducing a variety of differentiations. Some authors have claimed that the differentiations of gastrula ectoderm are conducted by two different inductors, but that a mutual control system operates between them so that the differentiations are qualified by the co-operative action of these inductors (Yamada, 195, 1958; Nieuwkoop et al 1952; Toivonen & Saxen, 1955; Takaya, 1957, 1959; 1 Author's address: Department of Biology, Konan University, Okamoto, Kobe, Japan.

2 372 YOSHIO MASUI Saxen & Toivonen, 1961). For the purpose of examining this hypothesis I have attempted to test the effect of LiCl on the differentiation of gastrula ectoderm under the influence of varying ph and some neuralizing agents. MATERIALS AND METHODS From gastrulae of Trituruspyrrhogaster at St. 11 or 12 a of Okada & Ichikawa's (1947) developmental table pieces of presumptive ectoderm were dissected with tungsten needles by referring to the map of Nakamura (1942). Care was taken to avoid contamination with the presumptive mesoderm and endoderm, but the purity of material was checked in each experiment by explanting some of the pieces, which were sampled randomly, without. Such control explants examined in fifty-five cases were all found to differentiate into atypical epidermis alone, proving them to be free of contamination. For the twelve pieces of the ectoderm at a time were transferred to a 6 cm wax-coated Petri dish containing 25 ml of the test solution, and each was placed in a separate depression in the wax of 3 mm diameter and 1-5 mm depth, and allowed to stand for a specified period of time and at a specified temperature. As test solutions, LiCl, KC1, NaCl, CaCl 2, MgCl 2, ZnCl 2 and BeCl 2 solutions and LiCl solutions containing one of NaCl, KC1, CaCl 2, MgCl 2, ZnCl 2, NH 4 C1 or urea were used. These solutions were prepared by dissolving each substance in distilled water unless otherwise specified. The ph of the solutions containing LiCl was adjusted with -1 M-LiOH or -12 M-HCI, while the ph of those not containing LiCl was adjusted with -1 M-NaOH or -12 M-HCI. Such ph adjustment had negligible effect on the molarity of the solution. All the solutions were autoclaved for 3 min at 1 C before use. In order to minimize variation of the ph of the test solution during the, the dishes containing the solution and the tissues to be examined were enclosed in an air-tight glass chamber together with a beaker containing 5 ml of 2% NaOH. ph determinations, which were made with a glass electrode immediately before or after the and with test paper during the, showed that the ph usually remained at the specified value ± -2 ph units throughout the. At the end of the the test solution was replaced with Holtfreter's solution by repeated pipettings to rinse the treated tissues. The tissues treated were cultured each in a vessel containing 1 ml of Holtfreter's solution at 18 C for 2 weeks. During culture daily observation of the explants was made with a low-power microscope. A final histological examination was made by usual methods.

3 ph-dependence of inducing activity 373 RESULTS The effect of lithium and some other metallic ions Solutions of LiCl, KC1, CaCl 2, MgCl 2, ZnCl 2 and BeCl 2 were prepared by dissolving each salt at a concentration of 6 M in a bicarbonate-free Holtfreter's solution of two-thirds the normal strength. The ectodermal explants were immersed in the solution for 3 h at 25 C, and at the ph indicated in Table 1. No dissociation of the ectoderm was brought about in the period of except in the case of with ZnCl 2 solution in which the tissues were always dissociated completely. The zinc-treated ectoderms, however, recovered from dissociation within 24 h to form round bodies with a brown surface. The lithium-treated ectoderms frequently formed humps with a whitish surface Table 1. Differentiation of ectodermal explants treated with salt solution Percentages for dissociation and survivors are of the total number of experimental explants, but for those for differentiation are of the number of survivors. PH LiCl 7 KC1 5-4 MgCl CaCl ZnCl 2 41 BeCl a 4-5 Total number of explants Dissociation during Survivors Epidermis Mesenchyme Neural Mesodermal (overall) Notochord Muscle Pronephros Yolky tissue (endodermal) 29 (%) 28(97%) 25 8 (%) 3(11%) 3 1(35%) 31 (%) 29(94%) 29 (%) (%) (%) 34 (%) 32(94%) 32 1(3%) (%) (%) 31 (%) 29(94%) 27 1(3%) (%) ((%) 34 34(1%) 33(97%) (82%) (%) (%) 24 (%) 14(58%) 14 (%) (%) (%) which were often segregated from the ectodermal vesicles and attached to the bottom of the culture vessel, giving rise to the migration of spindle-shaped cells over the glass surface. All the ectoderms treated with ions other than lithium and zinc were found to form wrinkled bodies covered with atypical epidermis. Generally the explants were found to develop well, except that berylliumtreated explants were liable to disintegrate, expelling necrotic cells during culture. Histological examination showed that in some of the explants treated with lithium chloride solutions muscles and massive yolk-laden tissues were produced (Table 1). On the other hand, in the zinc-treated explants neural tissues were produced frequently and formed archencephalon (Plate 1, fig. A). Neuroid or weakly differentiated neural tissues were found in some of the explants 24 JEEM 15

4 Table 2. Differentiation of ectodermal explants treated with -6 M-LiCl or NaCl at different ph''s Percentages as in Table 1. 4 LiCl ph Total explants Dissociation during. Survivors Epidermis Mesenchyme Neural Mesodermal (overall) Notochord Muscle Pronephros Yolky tissue (endodermal) (1%) 3(88%) 3(45%) 26(76%) (83%) 1(39%) 2(67%) 16(62%) (%) 1(39%) (71%) 14(3%) 1(19%) 11(24%) 3(71%) 3(65%) 28(54%) 3(67%) (37%) 4(13%) 4(14%) 5(17%) 17(57%) 8(17%) 8(29%) 8(27%) (47%) 2(67%) 22(79%) 17(57%) (68%) (68%) 16 8 (18%) (53%) 4 18 (65%) (67%) (92%) 1 3 (3%) (64%) (42%) (1%) (39 %) (68 %) (82%) (5%) o o NaCl PH Total explants Dissociation during Survivors Epidermis Mesenchyme Neural Mesodermal (overall) Notochord Muscle Pronephros Yolky tissue (endodermal) 3-7^ (1%) 28(1%) 16 24(86%) (%) (%) (65%) (71%) 23 (15%) (%) (%) (95 %) 24 (8 %) 54 (1 %) 31 (1 %) 4 (1 %) 33 (85 %) 3 (1 %) 36 (67 %) 26 (84 %) 29 (72 %) (36%) 8(27%) 15(42%) 14(54%) 21(72%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%)

5 /. Embryo/, exp. Morph., Vol. 15, Part 3 PLATE YOSHIO MASUI facing p. 375

6 ph-dependence of inducing activity 375 treated with calcium or magnesium ions, though true neural differentiation occurred only rarely, to form a small neural vesicle of archencephalon type. All the explants other than those mentioned above were found to form a mass of atypical epidermis alone. The influence ofph upon the effect of lithium ion Explants were exposed to -6 M-LiCl for 3 h at 26 C at a ph ranging from 3- to 1-2. As a control the same was carried out with -6 M-NaCl solution. Results summarized in Table 2 showed that in both NaCl and LiCl s the treated ectoderms were more or less dissociated, the frequency with which complete dissociation was brought about within the period of depending on the ph at which the was carried out. The frequency of dissociation was usually lower in the LiCl than in the NaCl, and it was lowest near ph 6- in the LiCl and near ph 5- in the NaCl. Reaggregation of the dissociated explants was easily brought about by transferring into Holtfreter's solution after with NaCl solution between ph 3-7 and 9-6 or with LiCl solution between ph 3-5 and 1-2. However, the dissociated tissues showed no sign of reaggregation if they had been treated outside the ph ranges indicated above. The NaCl-treated explants showed differentiation of ectodermal tissues alone, mainly resulting in neuralization and sometimes in the formation of mesenchyme and sensory organs (Plate 1, fig. B). On the other hand, the LiCl-treated explants underwent not only neural but also mesodermal differentiations. Most of the neural tissues produced were lacking regional specificity, forming neural masses. Only in a few cases were neural tubes resembling spinal cord formed as a part of axial structures (Plate 1, fig. C). Together with or without neural tissues, PLATE 1 Fig. A. Neural differentiation brought about in an explant treated with ZnCl 2 at ph 41. x9. Fig. B. Differentiation of nasal placode and eye vesicle brought about by with NaCl at ph 9. x9. Fig. C. Neural and medosermal differentiations brought about by with LiCl at ph 4-5. Photograph shows bilateral development of the explant in which neural tube and notochord are located in the median part and somites are in the lateral part, x 9. Fig. D. Mesodermal and endodermal differentiations brought about by with LiCl at ph 1-. A gut-like structure was found beneath the somites, x 9. Fig. E. Completely mesodermal differentiation brought about by with calciumcontaining LiCl solution. The explant shows the differentiation of voluminous notochord and underdeveloped somites, but no neuralization. x 9. Fig. F. Epidermis formation brought about by with calcium-containing LiCl solution. The section photographed shows only the formation of epidermis and a mass of undifferentiated muscle-like tissue which early segregated from the epidermis and attached to the glass surface, but from other sections of the same explant, notochord and somite are shown to be formed in connexion with the undifferentiated tissue in the photograph, x

7 376 YOSHIO MASUI notochord and somites were frequently differentiated, but pronephric tubules were rarely found. No blood cells were ever seen. Besides these tissues masses of yolk-laden cells with a structure resembling primitive gut were found (Plate 1, fig- D). 1% r o.si c <D o iffer c ph Text-fig. 1. The frequency of neuralization as a function of the ph at which ectoderms were treated. Open circles: neuralization of LiCl-treated explants. Filled circles: neuralization of NaCl-treated explants. Vertical bars: confidence interval of the frequencies on a level of confidence coefficient -95; the broken and straight bars corresponding to the open and filled circles respectively. 1% c 6 5 (4-1 o 4 o 3 p 2 1 ion ffere 1 Text-fig. 2. The frequency of mesodermalization and endodermalization of Li-treated explants at varying ph. Filled circles: mesodermalization frequency. Open circles: endodermalization frequency. From Text-figs. 1 and 2 it is apparent that all the modes of differentiation are affected by the ph at which the ectoderms were treated. Neuralization occurred with minimum frequency near ph 5- after both LiCl and NaCl s, but it was brought about with increasing frequency as the ph was raised or

8 ph-dependence of inducing activity 311 lowered. Similarly, the mesodermal differentiations of LiCl-treated explants were brought about with minimum frequency near ph 5-, but increased in frequency with shift of ph in either direction. On the other hand, the production of atypical epidermis and yolk-laden tissues was brought about with maximum frequency near ph 6-, and decreased at more acid or alkaline ph. From the result it is evident that while NaCl is effective only in inducing neuralization of ectoderm, LiCl is capable of inducing mesodermal differentiation in addition to neuralization under the same conditions. This seems to imply that mesodermal induction of the ectoderm results from a specific action of the lithium ion which is not shown by the sodium ion. At the same time, it is indicated that the effect of the lithium ion becomes manifest only outside the physiological range of ph, and shows strong ph-dependence. The effect of with alkaline NaCl solution combined with with neutral LiCl solution In order to clarify the significance of ph influence on the inducing effect of lithium ion, of the ectoderm with 6 M-NaCl was tried at ph 9- immediately before or after the with -6 M-LiCl at ph 6-. Both s lasted for 3 h at 26 C. The results indicated in Table 3 showed that Table 3. Differentiation of ectodermal explants passed through alkaline NaCl solution before or after with LiCl solution NaCl and LiCl solutions were used at 6M concentration, and the high and low ph were respectively 9 and 6. Percentages as in Table 1. LiCl alone (ph 61) NaCl alone (ph 8-8) LiCl after NaCl LiCl before NaCl Total explants Dissociation during Survivors Epidermis Mesenchyme Neural Mesodermal (overall) Notochord Muscle Pronephros Yolky tissue (endodermal) 52 1(19%) 28(54%) (14%) 8(29%) 8 22(79%) (1%) 26(84%) (54%) (%) (%) 55 55(1%) 37(67%) (86%) 1(27%) 4 9 1(27%) 45 28(62%) 25(55%) (4%) 1(4%) (56%) all the explants without previous with LiCl underwent complete dissociation after the with alkaline NaCl solution, whereas the explants treated with LiCl beforehand were unaffected by the Na- in one-third

9 378 YOSHIO MASUI of the cases. Moreover, the explants dissociated in NaCl solution after LiCl were found to reaggregate more quickly after being transferred into Holtfreter's solution than those dissociated without the previous LiCl. On the other hand, when the with LiCl was carried out after the with NaCl, it was observed that a mucous substance released from the cells during the NaCl became very viscous and sticky upon transfer to LiCl solution, and the cells were compactly reaggregated more quickly than when they were transferred directly into Holtfreter's solution. This observation may suggest that the action of the lithium ion is to make the surface substance of ectoderm cells sticky and so causes the resistance of the tissue to dissociation following alkaline. It was found that the neuralization following with alkaline NaCl solution occurred less often in the explants which had first been treated with LiCl than in those without previous LiCl (x 2 = *51, -5 > P > -25). On the other hand, the explants treated with LiCl after NaCl were more frequently neuralized than those treated with NaCl solution alone. The increase in the frequency of neuralization was large enough to be significant (X 2 = 6-66,1 >P> 5). Thus, the effect of the lithium ion on neuralization is quite different according to whether the LiCl occurs before or after NaCl. While the lithium ion has the effect of suppressing neuralization if it is applied prior to alkaline NaCl solution, the same ion has an enhancing effect if applied after the NaCl. It was, however, noted that no difference was found in the type of the resulting neural tissues in these cases. In both cases the majority of the neural formation lacked regional character and only in a few cases were hindbrain-like structures found. Mesodermal differentiation was increased in frequency by with alkaline NaCl solution carried out after LiCl. Although the differentiation frequency was not very high in the explants treated with NaCl solution after LiCl as compared with the explants treated with LiCl alone, it is probable that the with alkaline NaCl solution is, if carried out after LiCl, effective in enhancing mesodermal differentiation (X 2 = 3-2,-1 > P > -5). However, no increase in mesodermal differentiation was found when the NaCl was carried out prior to the LiCl. In this case the mesodermal differentiations took place with the same frequency as in the with LiCl alone. Regarding the quality of mesodermal tissues produced, no difference was found between explants treated with alkaline NaCl solution before or after LiCl. In both cases notochord and muscle were the predominant products. In view of the fact that no notochord was produced in the explants treated with neutral LiCl solution alone, the process of mesodermal differentiations seems to be modified by the additional with alkaline NaCl solution so as to result in notochord differentiation.

10 ph-dependence of inducing activity 379 The influence of some substances on the effect of lithium ion The influences of NaCl, KC1, CaCl 2, MgCl 2, ZnCl 2, NH 4 C1 and urea on the effect of lithium ion were examined by treating the ectoderm with 6 M-LiCl solution containing one of these substances at a concentration of -4 M. The lasted for 3 h at 26 C at the ph indicated in Table 4. In the solutions Table 4. Differentiation ofectodermal explants treated with LiCl solution containing various substances Percentages as in Table 1 Substance added to PH NaCl KC1 A CaCl ZnCl 2 K Total explants Dissociation during Survivors Epidermis Mesenchyme Neural Mesodermal (overall) Notochord Muscle Pronephros Yolky tissue (endodermal) 46 38(82%) 38(82 / 2 6 1(26 / 2(53 / (74 / 39 3(77%) 33(85%) (27%) 21 (64%) (27%) 41 (%) 33(8%) 9 6 (%) 24(73%) (36%) 35 35(1%) 3(86%) (9%) 1(3%) 1 3(1%) Substance added to O-Ofi M.T in ph Total explants 4 Dissociation during (%) Survivors 32 (;8%) : 24(6 %) 6(14%) 33(94%) Epidermis Mesenchyme Neural 6 5(21 %) 4 (67 %) 12(36%) 9%) Mesodermal (overall) '(%) Notochord Muscle Pronephros Yolky tissue 12 (38%) (endodermal) MgCl 2 A NH 4 C1 7-6-S : (%) 31(7 /o) 35(1%) 19(79 %) 19 5(21 %) (" %) (-%) (%) 6(18%) A Urea A_ (%) 34(77%) (6%) (%) 22(65%) containing only monovalent cation the explants were usually dissociated in the period of, whereas no dissociation was brought about by the with solutions containing a divalent cation with the exception of zinc. Comparing the results shown in Table 4 with those in Table 2, it is seen that the differentiation of the explants treated with LiCl solution containing sodium

11 38 YOSHIO MASUI or potassium ion showed no marked difference from that of the explants treated at the corresponding ph with the solution containing LiCl alone. Treatment with LiCl solution containing calcium caused mesodermal differentiation more frequently than the with the solution containing LiCl alone at the same ph. However, with calcium-containing LiCl solution brought about no neuralization, resulting in the production of completely mesodermalized explants in many cases as shown in Plate 1, fig. E. In the cases in which the ectodermal structure was formed, only epidermis was found to be completely segregated from the mesodermal tissue, as shown in Plate 1, fig. F. Treatment with magnesium-containing LiCl solution was found to give rise to mesodermal differentiation with increased frequency near ph 8- as compared with with the solution containing LiCl alone carried out at the corresponding ph. However, the effect of magnesium ion was peculiar in that the with the LiCl solution containing this ion failed to induce mesoderm, but induced neuralization, near ph 6- or above ph 8-4, and that the solution showed increasing toxicity as the ph was raised so that none of the explants treated at above ph 9- survived. Treatment with LiCl solution containing ZnCl 2 or NH 4 C1 brought about neuralization with considerable frequency, but mesodermal differentiation and the production of yolk-laden tissue were strongly reduced in frequency. The resulting neural tissues were archencephalic, and were sometimes accompanied by the formation of sensory organs. No mesodermal differentiation was found after with the LiCl solution containing urea. In this case, neuralization was brought about in a few cases. These results may indicate that the mesodermalizing effect of lithium ion can be enhanced by calcium or magnesium ion, but it is counteracted by zinc and ammonium ions and urea. DISCUSSION Explants of the gastrula ectoderm of Triturus treated with lithium ion produced various kinds of ectodermal and mesodermal structures. In addition, they formed a mass of yolk-laden cells which sometimes differentiated into a gut-like structure. It has already been demonstrated that definitive endodermal tissues appeared in lithium-treated explants when cultured for a prolonged time (Masui, 196,1961). Hence, the formation of the yolk-laden tissue in the present explants must indicate an occurrence of endodermalization of the ectoderm by lithium. Since among the substances tested in the present experiment only LiCl was effective in inducing mesodermal and endodermal tissues, the meso- and endodermalizations of ectoderm are due to the 'specific' effect of the lithium ion. It is often observed that the ectodermal, mesodermal and endodermal tissues appeared together within a single LiCl-treated explant. Since all the cells uniformly gave rise to epidermis if the explant was untreated, a possible assump-

12 ph-dependence of inducing activity 381 tion is that a single test-piece of the ectoderm consists of a heterogeneous cell population, the cells differing from each other in their competence. Under such conditions lithium ions may induce the mesoderm from the cells withmesodermal competence, while those with endodermal competence develop into the endoderm. The cells which are less competent for induction by lithium ion may undergo the formation of ectodermal structures. An alternative assumption is that the cells of the explant are homogeneous with respect to the competence, but difference of the conditions under which the cells respond to lithium ion may lead to different ways of differentiation. Besides, we should consider possible secondary interactions between the structures developed. For instance, the ectodermal structures in the LiCl-treated explant such as neural tube and ear vesicle may be induced secondarily by the mesoderm. It is known that the anterior neural plate does not develop into an archencephahc structure, as would be normally expected, when it is attached to the trunk mesoderm (Takaya, 1955, 1959). Thus, the mesoderm may exert the influence on the adjoining neural tissues to inhibit archencephalic differentiation. This can be a reason why no archencephahc structure was formed in the LiCl-treated explants. It is interesting that in the with calcium-containing LiCl-solution mesodermalization occurred in the absence of neuralization. The explants thus treated wholly transformed into mesoderm, or formed epidermis in the absence of contact with mesoderm. In the latter case (Plate 1, fig. F), it seems that early complete segregation of mesodermal and ectodermal components takes place, as observed by Gebhardt & Nieuwkoop (1964). Therefore, in this there must be no chance for the inductive interaction between the mesoderm and ectoderm. Secondary interaction may take place between the mesodermal and endodermal components, as it was found that the factors regulating the differentiation of endoderm reside in the mesenchyme and mesodermal tissues (Okada, 196; Takata, 196). The effect of lithium ion is strongly ph-dependent. Apparently the at high or low ph enhanced the mesodermalizing effect of the ion. The result seems to contradict the finding using the ectoderm of Ambystoma mexicanum. In this material Gebhardt & Nieuwkoop (1964) showed that the mesodermalization occurred frequently by the with LiCl near neutral ph. It has been known that Ambystoma ectoderm is more sensitive to inductive stimuli from the surrounding saline solution than Triturus ectoderm: in the former the neuralization occurs at any ph, while in the latter it occurs only outside the physiological range of ph (Barth, 1941; Holtfreter, 1944, 1945, 1947; Yamada, 195; Karasaki, 1957). Recent data (Englander, 1962; Johnen, 1964) indicate that Ambystoma ectoderm responds more quickly to both the neural and mesodermal inductors than Triturus ectoderm. Therefore, it may be assumed that there is some difference in the competence between Ambystoma and Triturus ectoderms for mesodermalization as well as for neuralization by the action of ions. Probably in Ambystoma the ectoderm is already provided with the meso-

13 382 YOSHIO MASUI dermal competence to respond to lithium ion at any ph, while in Triturus it may become as competent as in Ambystoma only when exposed to extreme ph. On the other hand, the endodermalization of LiCl-treated explants of the ectoderm occurred mostly at neutral ph in Triturus. So, the competence of the ectoderm for the induction by lithium ion may change from the endodermal to the mesodermal with the shift from neutral ph. Mesodermalization of the LiCl-treated explants was also enhanced when the ectoderm was exposed to alkaline NaCl solution after pre with LiCl at neutral ph. Similar results were obtained by the application of killed organizer to the LiCl-pretreated ectoderm or by shock- with ammonia of the LiCl-pretreated ectoderm (Masui, 196, 1961). Recent works of Barth & Barth (1963, 1964) also suggest the enhancement of the lithium effect by adding neuralizing agents. In their experiments ectoderm cells only underwent neuralization when lithium ion was applied for a short period or at a low dose, but differentiated frequently into pigment cells and sporadically into mesoderm cells when the ion was applied for a prolonged time or in combination with other neuralizing agents. It seems likely that the neuralizing agents may sensitize the mesodermal competence of the ectoderm so as to respond to the induction by lithium ion more readily. It should be remembered that some neuralizing agents such as ZnCl 2, NH 4 C1 and urea failed to enhance the mesodermalization of the lithium-treated explants, as shown in the present results. Probably the strong neuralizing action of the agent may affect the explant so profoundly that it is biased only towards neuralization without being sensitized to induction by lithium ion. It is important to note that in the cell culture of Barth & Barth (1963, 1964) mesodermal differentiation occurred only sporadically after lithium. This may imply that in order for the ectoderm cells to accomplish the mesodermal differentiation after induction by lithium ion, they require some complex factors from the environment which are lacking in their experimental system of cell culture in dispersed form. A mass effect caused by aggregation of a large number of cells, as found by Muchmore (1957) in the case of somite differentiation, may be critical for the mesodermal differentiation of the ectodermal cells induced by lithium ion as well. From the foregoing discussion it emerges that for the mesodermalization of ectoderm under influence of lithium ion it is necessary not only to sensitize the cells to make them competent, but also to maintain them in an aggregate of a sufficient size until the induction is stabilized. According to Ranzi (1957, 1962) the effect of lithium ion is referable to its action on proteins of embryonic cells. He found that lithium ion increased the viscosity of the protein solutions. This makes, according to his interpretation, the proteins resistant to the action of such agents as thiocyanate and urea. However, it is still open to question how lithium ion interacts with the ions normally present in the embryonic cell, though some investigations have been done (Lallier, 1955; Ranzi, 1962; Vahs & Zenner, 1965). In so far as the present

14 ph-dependence of inducing activity 383 experiment was concerned, sodium and potassium ions showed neither marked synergetism nor antagonism to lithium ion. Calcium and magnesium ions were found to act on lithium ion synergistically near neutral ph. Both Ca and Mg ions did not have mesodermalizing activity when applied singly, although they showed weak neuralizing action (Barth & Barth, 1963, 1964; Flickinger, 1964). These divalent cations, therefore, can only sensitize mesodermal competence so as to render the ectoderm more reactive to lithium ion near neutral ph. On the other hand, zinc and ammonium ions were strong antagonists of lithium ion, suppressing the mesodermalization as well as endodermalization by lithium ion. Thus, we may tentatively assume that ions having weak or moderate neuralizing action, such as hydrogen, hydroxyl, calcium and magnesium, act on the mesodermalizing activity of lithium ion synergistically, whereas the ions having strong neuralizing action, such as zinc and ammonium, appear to act antagonistically. For detailed analysis of the mechanism of interaction between lithium and other ions, however, new approaches are necessary. SUMMARY 1. Using ectodermal explants isolated from early gastrulae of Triturus pyrrhogaster, the inductive effect of lithium ion was examined at different ph's and under the influence of neuralizing substances. 2. Treatment with -6 M-LiCl brought about the differentiation of axial mesoderm together with neuralization and the production of yolk-laden tissues which sometimes formed gut-like structures. The neuralization and mesodermalization were brought about with minimum frequency by carried out near ph 5-, but they increased when was carried out at alkaline or acid ph. On the contrary, the production of yolk-laden tissue was brought about with maximum frequency by the near ph 6- and decreased as the ph at which the was done was raised or lowered. 3. Treatment with alkaline NaCl solution carried out after the with LiCl solution was found to be effective in enhancement of mesodermalization, but the same carried out before the LiCl had no effect on incidence of mesodermalization. 4. Treatment with LiCl solution containing calcium or magnesium ion which alone have a weak neuralizing action brought about considerable increase of the mesodermalization near neutral ph. On the other hand, zinc and ammonium ions and urea, which act as strong neuralizing agents, had an inhibitory effect or mesodermalization by lithium ion. 5. The results are discussed in relation to the significance of the influence of ph and neuralizing agents on the mesodermalizing effect of lithium ion, and it is suggested that these agents sensitize the mesodermal competence of the ectoderm to the inductive action of the ion, resulting in enhancement of the mesodermalization.

15 384 YOSHIO MASUI RESUME Dependance de Vactivite inductrice de Vion lithium a regard de ph 1. L'action inductrice de l'ion Li+ a ete examinee a differents ph et sous l'influence de substances neuralisantes, sur des explants ectodermiques isoles de jeunes gastrulas de Triturus pyrrhogaster. 2. Un traitement au LiCl,6 M a provoque la differentiation du mesoderme axial en meme temps qu'une neuralisation et la production de tissus charges de vitellus qui ont quelquefois forme des structures de type intestinal. La frequence la plus basse de neuralisation et de mesodermalisation a suivi un traitement mene aux environs de ph 5, mais elle s'est accrue apres un traitement realise en ph alcalin ou acide. Au contraire, la formation de tissu charge de vitellus a ete la plus frequente apres un traitement aux environs de ph 6 et a diminue quand le ph auquel s'etait fait le traitement etait plus eleve ou plus bas. 3. Un traitement par une solution alcaline de NaCl, realise apres le traitement par une solution de LiCl, a accru la mesodermalisation, mais si le meme traitement etait realise avant le traitement au lithium, il n'accroissait pas le mesodermalisation. 4. Un traitement par une solution de LiCl contenant des ions Ca ++ ou Mg ++ qui, agissant isolement, ont une faible action neuralisante, a provoque un accroissement considerable de la mesodermalisation aux environs de ph 7 (neutralite). D'autre part, les ions Zn ++, NH + 4 et l'uree, qui sont des agents fortement neuralisants, ont eu un effet inhibiteur sur la mesodermalisation par l'ion Li+. 5. La discussion des resultats est faite sous le rapport de la signification de l'influence du ph et des agents neuralisants sur l'effet mesodermalisant de l'ion Li +, et on suppose que ces agents sensibilisent la competence mesodermique de l'ectoderme a l'action inductrice de l'ion, provoquant un accroissement de la mesodermalisation. The author thanks Professor D. R. Newth for his kind criticisms, and is indebted to Dr T. S. Okada for reading the manuscript and for his criticisms. REFERENCES BARTH, L. G. (1941). Neural differentiation without organizer. /. exp. Zool. 87, BARTH, L. G. & BARTH, L. J. (1959). Differentiation of cells of the Rana pipiens gastrula in unconditioned medium. /. Embryol. exp. Morph. 7, BARTH, L. G. & BARTH, L. J. (1962). Further investigations of the differentiation in vitro of presumptive epidermis cells of the Rana pipiens gastrula. /. Morph. 11, BARTH, L. G. & BARTH, L. J. (1963). The relation between intensity of inductor and type of cellular differentiation of Rana pipiens presumptive epidermis. Biol. Bull. 124, BARTH, L. G. & BARTH, L. J. (1964). Sequential induction of the presumptive epidermis of the Rana pipiens gastrula. Biol. Bull. 125, ENGLANDER, H. (1962). Die Differenzierungsleistungen des Triturus- und Ambystoma- Ektoderms unter der Einwirkung von Knochenmark. Wilhelm Roux Arch. EntwMech. Org. 154,

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