22Sodium and 45Calcium uptake during embryonic induction in Rana pipiens

DEVELOPMENTAL.

BIOLOGY

22Sodium

28, 18-34 (1972)

and 45Calcium

Uptake

during

Embryonic

Induction

in Rana pipiens LESTER Marine

Biological

G. BARTH

AND LUCENA

Laboratory, Accepted

J. BARTH

Woods Hole, Massachusetts Janunry

02543

19, 1972

The uptakes of lZNa and ‘%a by early embryos of Rano pipiens and by hybrid embryos of R. pipiens females x R. syluatica males have been measured. Intracellular Na uptake increases sharply at midgastrula, whereas Ca uptake rises abruptly at neural plate stage. Arrested hybrid gastrulae show lower uptake of **Na and ‘%a, indicating that normal intracellular increases in uptake of isotopes are correlated with development, not with duration of exposure to isotope or changes in permeability with time. Uptake of **Na by hybrid embryos is remarkably increased by prostaglandin E, (a compound whose physiological role involves the transport of Na across cell membranes depending upon Ca mobilization and transport). Curves for uptake of 22Na and %a by the yolk region of the embryo closely resemble those for whole embryos at the same stages. At neural plate, anterior regions of neural plate plus archenteron roof exhibit higher rates of uptake than more posterior explants. The experiments show that, as the embryo develops, the uptake of **Na and ‘“Ca increases. We suggest that Na and Ca are released from previously bound state, possibly from yolk platelets, and thus can exchange with the isotopes. If so, the experiments support the view that normal embryonic induction is initiated by changes in intracellular cations, as suggested by our earlier studies using a cell culture system.

comparison of the blocked hybrid gastrula with the normal gastrula in order to distinguish between changes in permeability to Ca and Na and changes in the intracellular concentrations of exchangeable Ca and Na. Finally we wish to report some relevant data on changes in uptake of 22Na caused by prostaglandin E, (PGE ,), a compound whose physiological role is considered to involve the transport of Na across cell membranes in a manner involving Ca mobilization and transport (Horton, 1969; Ramwell and Shaw, 1970). The experiments reported here show that, as the embryo develops, the uptake of Na and Ca isotopes increases, possibly as a result of the mobilization of compartmentalized intracellular materials from yolk platelets. While the possible involvement of other cations such as potassium and magnesium remains to be investigated, it is noteworthy that workers using other systems are finding that cations are causally involved in cell transformation and growth.

INTRODUCTION

A sustained study (1959-1969) of induction of various cell types from the presumptive epidermis of the frog gastrula has led us to propose that the process of induction is Ca-activated and Na-dependent. The background for this hypothesis is stated in a previous paper (Barth and Barth, 1969). In the present paper we report the results of experiments designed to determine the changes in the concentrations of exchangeable Ca and Na during development, the exact time during gastrulation and neurulation when these changes occur, and the regions of the embryo where the changes take place. During this study we have been led to a Abscissa: Hours of development at 18°C and stage of development. Ordinate: Counts per minute per milligram dry weight. Uptake of 2ZNa by normal embryos at S11 was 5, and by hybrid embryos was 0. Open circles, **Na uptake by normal embryos; filled triangles, ‘%a uptake by normal embryos; open triangles, 22Na uptake by hybrid embryos; filled circles, ‘%a uptake by hybrid embryos. 18 Copyright

0 1972 by Academic

Press, Inc.

BARTH AND BARTH

Cations in Embryonic

As examples, we may list Kroeger (1963), Kroeger and Lezzi (1966), and Lezzi (1970) on salivary gland cell chromosomes; Burnett (1968) on cell differentiation in Hydra; Kostellow and Morrill (1968) on the early amphibian embryo. In still other systems it has been found that activation of sodium and potassium transport is an essential event during transformation of lymphocytes by an “inductor” (Quastel and Kaplan, 1970). In baby hamster kidney cells, external potassium concentration has been shown to control growth rate and DNA synthesis, probably by changing the cell membrane potential (McDonald et al., 1971). METHODS

AND

MATERIALS

1. Solutions The eggs of Rana pipiens are, in usual practice, fertilized in 0.1 Ringer’s solution and raised in this solution for the period from fertilization to hatching. This solution contains a relatively high concentration of Na (0.01 M), and we wished to treat the eggs with the isotope (“‘Na) with high specific activity. We found that doubly distilled water from a Pyrex still was a suitable solution for raising eggs from fertilization to stage 25 (Shumway, 1940). An analysis of this water from the storage bottles by Dr. H. Burr Steinbach’ gave a concentration of Na of 0.005 mM. With the isotope 22Na added to attain an activity of 0.94 PCilml, the concentration of Na was 0.04 mM. Analysis of the total Na content of the egg (0.08 x 10e3 mmole per embryo) showed no changes from blastula to stage 20. Our studies on uptake extended from blastula (stage 9) to tailbud (stage 17). Since the rate of uptake of isotope inside the egg will depend in part on the specific activity, carrier Na as NaCl was added to assure a standard total Na concentration of ’ Dr. H. Burr Steinbach, of the Marine Biological Laboratory, Woods Hole, Massachusetts, performed Na, K, Cl, and osmolarity measurements.

Induction

19

0.15 mM. When 45Ca was used, we added %a to make the solution 0.01 mM. 2. Use of the Isotopes 22Na or 45Ca, obtained from New England Nuclear, was added to our solution and neutralized with KHCO, to give a concentration of 1.0 PCilml unless otherwise stated. One milliliter of eggs (removed from the jelly and washed) was added to 15 ml of isotope solution, mixed, and kept at a constant temperature required by the particular experiment. The resulting concentration of the isotopes is 0.94 pCi/ml (‘X6 x 1.0 PCi). Many preliminary trials showed that the uptake of both 45Ca and 22Na by eggs exposed continuously to the isotopes increased very sharply during gastrulation and early neurulation. For example, in a typical experiment the counts per minute per embryo (cpm/embryo) of 22Na rose from a value of about 50 at stage 10 to about 20,000 at stage 14. The curves for both 22Na and 45Ca were concave upward with rapidly increasing rates of uptake with time. During these preliminary observations it was also noted that the jelly and vitelline membrane enclosing the egg showed high activity. The jelly and vitelline membrane were therefore removed before measuring the cpm of the embryo. In addition, dissection showed that the blastocoel contained isotopes in high concentration, and so the embryos were cut in half in order to wash out the isotopes from the fluid spaces. Thus the measurements which are to be recorded in this paper are of the 22Na and %!a in the cells, do not include the fluids of the blastocoel and archenteron, and do not include isotopes present in the jelly, vitelline membrane, or perivitelline space. Since continuous exposure to the isorate of topes showed an increasing uptake with time, no true equilibrium could be attained. As the egg developed, more isotope was taken up, so that as

20

DEVELOPMENTAL BIOLOGY

usual in measuring any parameter during development, it is impossible to isolate totally the time factor from progressive qualitative changes. A series of experiments whereby the developing egg was exposed to the isotopes for intervals instead of continuously showed that some kind of equilibrium was attained during an exposure of 24 hr before any given stage. Exposures to the isotope from 0 to 24 hr; 24 to 48 hr, 48 to 72 hr, 72 to 96 hr resulted in values for cpm/embryo which were the same as continuous exposure of the embryo from 0 to 24 hr, 0 to 48 hr, 0 to 72 hr, and 0 to 96 hr. Shorter exposures, such as 4, 8, 12, and 16 hr before a given stage resulted in lower cpm/embryo as compared with those obtained after continuous exposure to the isotopes. Thus in the body of the experiments reported in this paper, the isotopes were present either continuously or for 24-hr periods. Since the cpm/embryo increase with development and since increase in temperature governs the rate of development, comparisons of cpmfembryo at a given stage at different temperatures were made. Briefly, a comparison of 514 embryos, which had been exposed to 22Na at 10°C and 14°C gave 26,500 cpm/ embryo and 20,300 cpmlembryo, respectively. A comparison of S12 embryos which had been exposed to 22Na at 14.5”C and 22°C resulted in cpmlembryo of 3224 and 774, respectively. Thus the lower the temperature, the higher the cpm/embryo at any given stage. 3. Measurements

After the desired exposure to the isotopes, 3 to 5 embryos were removed from the dish, washed in glass-distilled water and transferred to an operating dish where the vitelline membrane and its adhering jelly was removed by means of watchmakers’ forceps. The embryos were transferred by pipette to another dish, where they were cut in half to wash out the contents of the blastocoel and/or archenteron.

VOLUME 28, 1972

The halves were then pipetted into another dish for a wash and then to a ten Broeck homogenizer, where the volume was made up to 3 or 10 ml, depending on the expected cpm/ml as predicted from preliminary experiments. Aliquots (l-ml and 0.5-ml) of homogenates were pipetted into planchets and dried at 100°C; counts were made with a Model 186 NuclearChicago low background counter. In preliminary experiments the solution used for operating was our standard solution, which contains 88 mM NaCl. Experience showed that when 0.1 Ringer’s solution (10 mM NaCl) was used for operations, the cpm/embryo were higher than in standard solution. After fairly extensive trials we finally chose tris(hydroxymethyl)aminomethane (Tris. HCl) as a substitute for NaCl in our standard solution. Thus the operations were carried out in only a trace of 23Na. Specific activities. For most of the experiments we are not able to calculate specific activities, since we used embryos minus the blastocoel and archenteron fluids and did not have the equipment necessary for determining Na or Ca in parts of an embryo. However, in a few experiments, we used the whole embryo and Dr. Steinbach measured the Na, K, Cl, and osmolarity of these embryos. The amount of Na was constant in the different stages of embryos used. The specific activity of the external solution was 163 x lo* cpm/mmole Na. At stage 9, the specific activity of the whole embryo with fluids was 0.011 x 10”; at Sll-, 0.018 x 108; at S12+, 2.0 x 108. At the latest stage, S14, specific activity rose to 12.4 x 10’ cpm/mmole Na. These results show that only a fraction of the total Na in the embryo as a whole is free to exchange with 22Na, and we would expect a similar situation in our experiments in which we used the cells of the embryo and discarded the fluids. Separate outflux (efflux) experiments performed in glass-distilled water showed

BARTH

BARTH

AND

Cations in Embryonic

very high cpmlembryo even after a day. Even when 0.1 mM Na was added to glassdistilled water, stage 14 embryos, after 24 hr at 145°C still retained 60% of their initial 22Na. Evidently with only traces of 23Na in the external medium exchange with 22Na in the embryo is very slow, and losses during the few minutes of the operation are minimal. 4. Use of the Hybrid

Embryo

The frequently studied hybrid embryo obtained from Rana pipiens egg and R. syluatica sperm develops to beginning gastrula, stage 10, and then is arrested in the sense that in most hybrid embryos no further gastrulation occurs and neural plate is not induced. Thus a variable in development of the normal egg can be examined in the arrested egg to see whether the variable is correlated with development or is simply one that occurs with time. In our case we have a very rapid increase in the rate of uptake of isotopes during gastrulation and neurulation and we can use the arrested gastrula to see whether this increase in rate of uptake is correlated with development or simply with time. Half of the eggs of a gravid R. Treating

solution (+PGE,)

0.1 ml Stock PGE, solution 2.0 ml 22Na stock solution (50 pCi/ml N HCI) 2.0 ml KHCO, (0.018 IV) to neutralize **Na-HCl 96.0 ml Glass-distilled water, pH 7.0

in 0.018 the

21

Induction

Hazen and Company, Alburg, Vermont; males were collected near Plymouth, Massachusetts. R. sylvatica

5. Use of Prostaglandin

E,

Samples of prostaglandin E, (PGE,) were kindly supplied by Dr. John E. Pike of the Upjohn Company, Kalamazoo, Michigan 49001. Stock solutions were prepared by washing the 20-mg sample from its vial, using a total of 2.0 ml of 95% ethanol, then rapidly adding 18.0 ml of a sodium carbonate solution to convert prostaglandin to the sodium salt. The sodium carbonate solution was prepared by dissolving 50 mg of anhydrous Na,CO, in 250 ml of glass-distilled water to give a 20% solution. This stock solution was 2.9 x lo- 3 M PGE, (1 mg/ml of the sodium salt of PGE,). The stock prostaglandin solution was diluted to give final concentrations of 1.5 x lo-* M to 2.9 x 10e6 M of PGE, in solutions used for continuous treatment of eggs and embryos. The actual treating solutions contained also **Na at 0.94 pCi/ml. The complete compositions of solutions used to test the effect of PGE, on **Na uptake are tabulated. Treating

solution

(-PGE,)

0.1 ml Control solution” 2.0 ml ZZNa stock solution 0.018 N HCl 2.0 ml KHCO, (0.018 iV) 96.0 ml Glass-distilled

50 &i/ml

in

water, pH 7.0

0 The control solution listed for 2ZNa treating solution without PGE, consists of 18 ml of 20% Na,CG,, plus 2 ml ethanol. These were the amounts used to prepare the alcoholic solution of the sodium salt of the PGE, samples.

pipiens female are fertilized with R. pipiens sperm and the other half with R. syluatica sperm. Thus we have the same

eggs but in one case they gastrulate and neurulate but in the other they do not. Exposing both sets of eggs to the isotopes we can measure uptake during the period when the normal eggs gastrulate and the arrested eggs do not. Rana pipiens were obtained from J. M.

6. Localization

Studies

At gastrula or neurula stage, as required by the particular experiment, the eggs were washed in glass-distilled water and 3-5 eggs were transferred to a stender dish of 0.1 Ringer’s solution for removal of the vitelline membrane. Operations by means of glass needle and hairloop were carried out in another dish of 0.1 Ringer’s

22

DEVELOPMENTAL.BIOLIXY

solution, and the samples of neural folds, yolk mass, archenteron roof, or whatever region was being examined, were transferred by means of Spemann pipette through a wash of glass-distilled water and thence onto squares of preweighed aluminum foil measuring about 5 mm2; the foil containers had been placed on aluminum planchets for careful handling during subsequent drying, counting, and weighing procedures. The samples were dried for 30 min at lOO”C, counted with a Model 186 Nuclear Chicago counter, and finally weighed. Dry weights of neural fold explant samples averaged in the range of 0.2 to 0.6 mg; yolk mass explants weighed from 1 to 4 mg dry weight, depending upon size of sample. The neural fold samples included the underlying archenteron roof. Each fold was cut once horizontally to give anterior

VOLUME 28, 1972

and posterior samples. The level of the cross cuts was varied so as to give the four different anteroposterior samples diagrammed in Fig. 3. RESULTS

1. Time Course of Uptake during Normal Development

An overall picture of the changes in uptake of 22Na and 45Caby the cells of the embryo at four stages of development is presented in Fig. 1. These data are from a single batch of eggs and the cpm are of the cells only. Membranes and jelly are excluded, as well as the fluids of the blastocoel and archenteron. The eggs were exposed from the 4-cell stage to the isotopes for 24 hr at 14.5”C, which brought them to stage 9- (blastula). At this time another group of eggs from the same batch was exposed to the isotopes for another 24 hr, by which time they were at stage lO+

15.000-

O-24 Hr* !a-

24-4sHI* so+

48-72”r, 512+

R-96Hr* s14+

FIG. 1. Comparison of the uptake of lZNa and ‘%a from stage 9- to stage 14. Eggs are exposed for 24-hr periods at 145°C to isotopes at a concentration of 0.94 &i/ml in 0.1 mM Na and 0.01 mM Ca. Stages are those of the eggs at the end of the 24-hr period. Uptake of *lNa during 0 to 24 hr was 25; from 24 to 48 hr, uptake was 14 cpm/embryo. Abscissa: Hours of exposure to isotopes and stages of development at end of the 24-hr intervals. Ordinate: Counts per minute per embryo. Filled circles, **Na; open circles, Wa.

BARTH

AND BARTH

Cations in Embryonic

(beginning of gastrulation). Similarly a 24-hr period from stage lO+ to 12-t (end of gastrulation) was treated with isotopes and finally a 24-hr period from 12+ to stage 14 (neural folds) completed this study. In all, four such experiments were carried out and Fig. 1 records the data from one of them. The data from the other three give the same type of curve, but the absolute values differ because different batches of eggs were used. In all four experiments the cpm/embryo at the end of the first 24-hr period are much lower for 22Na as compared with 45Ca (25 and 962 cpm, respectively). The uptake of %a decreases during the next 24-hr interval and 22Na may show a slight decrease, although the cpm are very low for 22Na. During the 24-hr period from SlO+ to 12+, a period characterized by gastrulation, the uptake of 22Na increases from 14 to 3224 cpm/embryo. On the other hand, the uptake of 45Ca continues to decrease to a value of 133 cpm/embryo. Thus during a period when 22Na uptake increases, 45Ca uptake decreases. Finally, during the last 24-hr period from the end of gastrulation (Sl2+) to neural folds (S14) the uptake of both 22Na and 45Ca increases and the curves are concave upward. Other data, not recorded here, show that during the next 24-hr period (96 to 120 hr) both 22Na and 45Ca uptakes continued to increase. Since this period is beyond the periods of induction and neurulation we are not here concerned with it. What does concern us is the precise timing of the sharp increases in uptake of 22Na some time after SIO+ and of 45Ca some time after S12+. For this information we have used a somewhat different approach. Eggs in the 4-cell stage were placed in the isotopes and when the eggs reached stage 11- we measured the uptake of 22Na and then began to take samples at intervals of l-2 hr. When the eggs in 45Ca reached stage 12, we measured

23

Induction

the uptake and began to take samples every l-2 hr. The eggswere at 22°C during the period when samples were taken for measurement and thus the absolute values of uptake for 22Na are lower than the uptake at 145°C (see Methods and Materials). Figure 2 records two curves; one, a plot of the cpm/embryo of 22Na from Sll- to S12 and the other, a curve showing uptake of 45Cafrom Sl2 to S13+. Inspection of the curves reveals that the uptake of 22Na increases with time from SlL to S12 with the curve being concave upward. Figure 1 shows that the uptake continues to increase after S12. The uptake of 45Cadoes not change much until S13 and shows a very sharp rise between S13 and S13+. The uptake continues to rise rapidly and at S14 (8.5 hr) cpm are 3680 (not shown in Fig. 2).

1

3I L

3

4

5

6

70 512

I

2

3

4 5 513

6

7 HOURS s13+

FIG. 2. Comparison of uptake of 22Na and ‘6Ca during gastrulation and neurulation. Eggs are exposed to isotopes continuously from 4-cell stage. Measurements begin at stage llfor 22Na and at stage 12 for ‘%a. Temperature is 22°C; isotopes have activity of 0.94 pCi/ml; Na concentration: 0.1 mM; Ca concentration: 0.01 mM. Abscissa: Times in hours after first measurement; stage of development. Ordinate: counts per minute per embryo. Filled circles, **Na; open circles, ‘%a.

24

DEVELOPMENTAL

BIOLOGY

The chief point of interest at the moment is that the rapid rise in uptake of 22Na occurs during gastrulation, while the rise in uptake of ‘%a occurs during neurulation. Another method of obtaining closely spaced intervals of development is to reduce the temperature. Table 1 compares uptake at 10°C by eggs exposed for 24-hr periods to the isotopes 22Na and ‘%a. The same batch of eggs is used throughout the experiment and the table shows clearly that between SlO- and S13- the uptake of 22Na increases, while the uptake of 45Ca decreases. This fact is of importance in the interpretation of the mechanism of the rapid rise in uptake of the isotopes. If the rapid rise in uptake of 22Na was due to an increase in permeability, then why should we find a decrease in the uptake of 45Ca at the same time? In this same context we have compared the uptake of the isotopes after continuous treatment from the 4-cell stage to stage 14 with uptake when the isotope is applied during the last 24 hr (S12+ to S14). The values (Table 2) are about the same for 96-hr exposures as for 24-hr exposures. If the increase in uptake during development were simply a matter of increase in permeability, the uptake over the 96-hr exposure should be greater than that of a 24-hr exposure. In the discussion we shall use the data of both Table 1 and Table 2 as argu-

COMPARISON

Duration 04 24 to 48 48 to 72 72 to 96 96 to 120 120 to 144

TABLE 1 OF 21Na UPTAKE WITH ‘“Ca UPTAKE BY EMBRYOS AT 10°C” Stage (Shumway) 7to99- to lolo- to 11 11 to 12 12 to 13-

Uptake cpm/ embryo *2Na

‘6Ca

11 8 103 1534 7277

223 184 94 77 47

“Embryos were exposed for 24-hr periods to the isotopes at 0.94 &i/ml in solution with 0.15 mM Na and 0.01 mM Ca.

VOLUME

28, 1972

TABLE

2

COMPARISON OF **Na UPTAKE DURING CONTINUOUS EXPOSURE OF EMBRYOS AND DURING 24-HR INTERVALS OF EXPOSURE TO ISOTOPE’

Expt. No.

1 2 3

Counts per minute per embryo 72 to 96 hr exposure

0 to 96 hr exposure

11,200 18,021 19,607

10,720 16,131 20,357

“Temperature: 0.94 &i/ml.

14.5”C;

z2Na

Final stage of development 1414 14 concentration:

ments that the rapid rise in uptake of the isotopes during development is not the result of an increase in permeability. 2. Uptake of 4sCa and 22Na by Arrested Gastrzhe of a Hybrid Embryo Eggs from three different R. pipiens females were fertilized by three different R. pipiens males and by three different R. syluatica males. At 2-4 cell stage the eggs, both normal and hybrid, were removed from jelly and stored in glass-distilled water overnight at 10°C. At 156-cell stage they were rinsed in glass-distilled water and exposed in the usual way (see Methods and Materials) to either 22Na or 45Ca for continuous treatment with isotopes. At the stages to be measured for uptake, operations and measurements were made in parallel for normal and hybrids, one operator carrying out the procedures for normal eggs and the other operator for hybrids. The measurements apply to cells only; membranes and jelly had been removed, and the embryos were bisected so as to exclude the fluids of blastocoel and archenteron. In Table 3 the data for isotope uptakes by diploid pipiens (N) and by the hybrid R. pipiens 0 x R. syluatica FJare compared. The hybrid embryos, as usual, blocked at early gastrula stage but remained viable for several days after the periods studied here. During late blastula through neural fold (stage 14) normal embryos exceed

BARTHANDBARTH

Cations in Embyonic

Induction

25

neural plate. Sodium uptake had begun to rise sharply at Sll (mid-gastrula) with we11 developed dorsolateral blastoporal Count3 per minute per embryo lips but before any involution at the ventral lip. Thus the uptake in 22Na rises abruptly when the archenteron roof NOrlId Hybrid Hybrid Normal is inducing the overlying presumptive neural plate, and neural plate formation 1456 16 (ll-) 1 coincides with a sudden increase in 45Ca 1653 313 (13%) 2091 2874 uptake. (14) 21 1584 816 159 2 11 These observations indicate that the 1378 1212 302 6423 12+ period of primary embryonic induction is 1891 1649 16899 46190 14 associated with increased 22Na uptake o Concentrations of isotopes in medium: 0.94 followed by an increase in 45Ca uptake. %a. Normal = R. **Na; 2.8 &i/ml &i/ml Thus we were led to examine the localized pipiens p x R. pipiens$; Hybrid = R. pipiem p x R. syluatica 3. Hybrid embryos blocked at early uptakes of these cations within regions of gastrula stage 10. Embryos were exposed continuthe gastrula and neurula, and further to ously to isotopes in glass-distilled water from 4-cell measure Na and Ca isotope uptakes in stage. Stages of blocked hybrid embryos in parenarrested hybrid gastrulae. theses indicate stage of corresponding normal diploid Most significant results of these localiR. pipiens controls. zation studies were (1) the finding that at arrested hybrid embryos in uptake of neurula stages there is an anterior-posterior gradient in 22Na and Wa uptakes both 45Ca and 22Na (Expt. 2). Normal embryos show the usual fall in 45Ca up- in the neural folds; and (2) the fact that take at late gastrula (Sll-12), while 22Na while 45Ca and 22Na uptakes within the yolk mass increase in gastrula and neurula uptake begins its characteristic abrupt stages during normal development, the rise, overtaking and exceeding 45Ca during gastrulation between Sll and S12 hybrid embryo is deficient in uptake of (Expt. 2). The same experiment also both these cations. Hybrid yolk platelets do not take up *“Na and ¶%a at the same shows that in the arrested hybrid embryos rate as do normal embryos, and at neurula the uptake of 45Ca exceeds 22Na for a time. Eventually sodium uptake catches stages values for sodium and calcium uptakes remain very low in the arrested up and exceeds calcium uptake-although at a later stage: at neural fold stage 14 in hybrid embryos. The yolk mass, because it is relatively easy to isolate free of other the hybrid (Expt. 1) rather than during gastrulation, as in normals. These obser- embryonic cell types, provided most of the localization data used in comparing vations of isotope uptakes are consistent hybrid and normal embryos. among the several experiments, although absolute values differ because different The neural folds. Table 4 and Fig. 3 parents were used for eggs and sperm show that the most anterior levels of the from one experiment to the next. neural folds together with underlying mesoderm have the highest 22Naand 45Ca 3. Localization of Uptake of zzNa and 45Ca uptakes. Progressively more posterior within Gastrula and Neuruia Stages samples of neural fold samples decrease and in the Arrested Hybrid Gust&a in values for cpm/mg dry weight for **Na Measurements of the cation uptakes and 45Ca uptakes. Thus the future brain at intervals of l-2 hr (Fig. 2) pinpointed regions take up the isotopes in larger the beginning of the Ca rise precisely at quantities as compared to less anterior the time when neural streak becomes regions, where induction of spinal cord is TABLE

3

UPTAKEOF ‘%a AND 2ZNa BY NORMALAND BY HYBRIDEMBRYOS DURINGDEVELOPMENP

26

DEVELOPMENTAL

TABLE

BIOLOGY

4

UFTAKE OF **Na AND ‘%a BY FOUR REGIONS OF THE NEURAL FOLDS AT EARLY NEIJRU~A STAGESO

Stage

“hW$

Counts/minute/mg azNa

13+

14-

14

1 2 3 4 1 2 3 4 1 2 3 4

6740 4450 5050 2790 8600 5870 6650 3000 12,100 6,570 7,580 3,700

dry weight

%a 3030 2160 1660 1109 3190 1900 1777 1452

4900 3800 4440 2130

DBegion numbers correspond to the diagrams in Fig. 3, where regions of the folds are numbered from anterior (1) through less anterior (3), to posterior (2), through more posterior neural folds, the latter region (4) lying just anterior to the former blastopore. Underlying archenteron roof was included with all operations and each sample included explants from an average of 6 different embryos. The table includes data drawn from operations on embryos from 6 different batches of eggs. Absolute values therefore are not comparable from one experiment to another. Within any one experiment the anterior-posterior gradient is clearly apparent. Embryos were exposed continuously to the isotopes from early cleavage stages until time of operations, as described in Methods and Materials, section 6. Concentrations of isotopes in glassdistilled water were 0.94 &i/ml 22Na and 2.8 pCi/ml “Ca.

occurring; lowest values are found in invaginated posterior most recently archenteron roof plus overlying neural folds where spinal-caudal structures are being induced. The yolk mass. Figure 4 and Table 5 show that when samples of yolk mass alone are studied for the time course of uptake of 22Na and 45Ca during gastrula and neurula stages, the shapes of the curves closely resemble those plotted from data from whole embryos. Although a number of measurements were made on the cation uptakes in presumptive epidermis and on presumptive neural plate plus underlying

VOLUME

28, 1972

archenteron roof, difficulties in obtaining comparable samples of these regions at successive stages of gastrulation and neurulation have led us to concentrate our attention upon the yolk mass, which is more easily isolated from other types of cells. A number of processes are going on in the whole embryo during the period graphed in Fig. 4 (cell division, duplication of chromosomes, morphogenetic movements such as involution, other cell layer displacements, movements of mesoderm and endoderm with reference to each other and to overlying ectoderm and presumptive neural plate, the specific molecular processes involved in cell differentiation, etc.). When these processes and events do occur, as in normal development, the striking and characteristic changes in isotopic Na and Ca uptakes accompany them. When development is halted at early gastrula stage, the uptakes of 22Na and 45Ca remain low within the embryo as a whole, as well as within yolk regions of the arrested hybrid embryos. 4. Effect of Prostaglandin E, on 22Na Uptake in Normal and Hybrid Embryos

Prostaglandins are long-chain fatty acids endogenous to many tissues whose release can regulate ion permeability of the plasma membrane and as a consequence, that of intracellular organelles

qTJ&

STAGE

$J=Jg

13+

STAGE

14-

FIG. 3. Early neurula stages, showing cuts (dotted lines) made to obtain regions (numbered) which were analyzed for z2Na and ‘%a uptakes. Counts per minute per milligram dry weight are presented in Table 4, where region numbers correspond to numbered areas in Fig. 3.

BARTH AND BARTH

Cations in Embryonic

4.000 1

FIG. 4. Uptake of **Na and ‘%a by yolk regions only at gastrula and neurula stages of normal and hybrid embryos. Data from Table 5. Curves may be compared to Fig. 1 where uptakes by whole embryos were plotted. The similar shape of uptake curves by normal whole embryos and by yolk regions only of normal embryos is evident. The uptake of ‘%a by the yolk region was measured at S13- after Ca uptake had begun to rise; hence the earlier decline during gastrulation noted in whole embryos does not appear in the %a uptake curve for yolk region only. Concentrations of isotopes were 0.94 &i/ml of 22Na and 2.8 &!i/ml of ‘SCa.

(Ramwell and Shaw, 1970). In particular, PGE, (one of the most widely studied of the prostaglandins) displaces membrane Ca, thereby allowing increased Na influx. As we have shown in the foregoing sections, normal gastrulation in the frog embryo is accompanied by a decrease in uptake of %a, with a simultaneous increase in 22Na uptake. Our earlier extensive studies using cell cultures from the gastrula already had shown that induction of new types of cell differentiation in pre-

27

Induction

sumptive epidermis cells by Ca is strongly Na-dependent. Experiments therefore were designed (1) to test the effects of PGE, on 22Na uptake on normal diploid R. pipiens during cleavage through neurula stages, and (2) on arrested gastrulae of the genetic hybrid R. pipiens p x R. syluatica 8; and finally (3) to examine the possibility that PGE 1, by chancing endogenous ion fluxes, might mimic the actions of cations we have been studying by inducing new cell types in cell cultures of the presumptive epidermis of the normal gastrula. Exploratory experiments on normal diploid pipiens embryos were carried out first in order to determine optimal conditions for using the prostaglandin in the present system. (1) It was established that a broad range of concentrations of PGE, are tolerated in the medium for normal development from early cleavage to hatching stages. Prostaglandin is not toxic at concentrations as high as 1.5 x 10m4 n/i and normal development, organogenesis and cellular differentiation TABLE

5

UFTAKE OF 2ZNa AND ‘%a BY YOLK lions ONLY AT GASTRULA AND NEURULA STAGES OF NORMAL AND HYBRID EMBRYOP

Cpm/mg

dry weight

“Data were obtained from eggs of a single R. p&ens p fertilized by R. pipiens sperm to give (N) ormal development or by R. syluatica sperm to give (I-f) ybrid embryos which blocked at early gastrula stage 10. Data from 2 other experimentsusing different batches of eggs gave similar results. Embryos were exposed continuously to the isotopes from early cleavage stages until time of operations, as described in Methods and Materials, section 6. Concentrations of isotopes in glass-distilled water were 0.94 &i/ml zZNa and 2.8 &i/ml Wa.

28

DEVELOPMENTAL BIOLOGY

take place. (2) The compound acts rather rapidly, as evidenced by the fact that an increase in 22Na uptake is noted after only 4 hr exposure at 22°C. (3) PGE, at concentrations of 2.9 x 10e6 M to 5.8 x 1Om6M clearly increases the uptake of 22Na by normal frog embryos exposed from cleavage stages on and measured for uptake at gastrula and early neurula stages. (4) Both precise stage of gastrulation and temperature during treatment affect the extent of PGE, stimulation of **Na uptake. Table 6 summarizes data obtained from embryos of a single female R. pipiens fertilized either by R. pipiens or R. sylvatica sperm on two successive days. Both batches of hybrid embryos blocked at early gastrula stages and survived for four more days without cytolysis; both batches of diploid pipiens embryos developed normally to hatching. Both hybrid (H) and normal (N) embryos were dejellied at 2-4 cell stages and raised in glass-distilled water during early cleavage stages. At approximately 256-300 cell stage (Expt. 1) and 16-32 cell stage (Expt. 2) hybrid and normal embryos were placed in 22Na in glass-distilled water with and without PGE,. Details of composition of treating solutions, volumes of embryos to treating solutions, etc. have been presented above (Methods and Materials, section 5). In hybrid embryos, blocked at early gastrula but equivalent in age to gastrula stages of normal diploids, 22Na uptake is markedly stimulated by the presence of PGE 1 in the culture medium (Table 6). Normal diploid pipiens embryos at these stages are moderately stimulated in 22Na uptake by the prostaglandin (up to 35%). Hybrid embryos, however, show PGE, stimulated uptakes of 22Na amounting to 121-465%, depending upon precise stage of gastrulation in the control diploids. The absolute values in cpm/embryo are consistently lower for hybrids than for normals even in the presence of PGE,.

VOLUME 28, 1972 TABLE 6 EFFECT OF PROSTACXANDINE, ON **Na UPTAKE BY NORMAL AND HYBRID EMBRYOSO

Column:

1 1 2 2

1

2

N

11+ W+) 12+

H N H N H N H

Wf) 12 (12) 16

(16)

323

898 253 22,848 7,815 8,409 1,406 19,667

3b

4

669 84 19,360 1,383 6,243 637 22,837

34 201 18 465 35 121 0

a A single R. pipiens Q was fertilized by a R. pipiens or R. sylvatica 6 on 2 successive days. Eggs at 16-32 cell stage (Expt. 2) or at &300 cell stage (Expt. 1) were exposed to 22Na (0.94 pCi/ml) in the presence, or in the absence, of PGE, (2.9 x 10-O d4) as described in Methods and Materials, Section 5. Column 1: Genetic composition: N = diploid pipiens embryos and H = pipiens x sylvatica hybrid embryos. Column 2: Stage at which dejellied embryos were removed from isotope medium, washed, bisected, and analyzed for **Na uptake. Column 3a: Cpm/embryo in eggs exposed to PGE, together with 22Na. Column 3b: Cpm/embryo in eggs raised in 2ZNa medium in absence of PGE,. Column 4: Percent increase due to PGE, = A(+PGE,/-PGEJ x 100.

Thus the normal sharp increase in 22Na uptake during gastrulation (Results, section 1) actually is correlated with development and not simply with age of the cells or duration of exposure to isotope. This observation reinforces the conclusion that changes in uptakes of the isotopes are not due to nonspecific increases in overall permeability with age of embryo, but are truly correlated with reactions significant for development. By the equivalent of stage 16, however, blocked hybrids (morphologically still at early gastrula stage) no longer respond to PGE,. Values for **Na uptake in hybrids equivalent to S16 have apparently reached the same maximal values that were reached earlier at S12+ by normal diploid embryos, and PGE, can cause no additional increases. These data are presented in Table 6. Compare column 3a and 3b for

BARTH AND BARTH

Cati ens in Embryonic

(N)ormal embryos at S12+ in experiment 1 with column 3a and 3b for (H)ybrids (S16) in experiment 2. A final series of preliminary experiments probed the possibility that, using PGE, to cause increased uptake of 22Na in cultures of presumptive epidermis cells, induction of new cell types might result. These experiments were based upon our earlier observations that induction by ions such as Ca and the lithium ion is sodium dependent for completion of the induction during a subsequent time phase. In the two kinds of experiments thus far carried out PGE, has been ineffectual as an inductor. We do not, however, regard these negative results as conclusive with regard to Na transport and normal induction. A variety of combinations of conditions and factors should be tested. DISCUSSION

The stimulus for the research reported in this paper came from the discovery (1) that when small amounts of Ca were added to our standard medium, cultures of presumptive epidermis cells were induced to differentiate into nerve and pigment cells; and (2) that the induction was dependent upon the concentration of Na in the culture medium. The present paper has examined Na and Ca changes within the whole embryo and its parts at gastrula and neurula stages, with the objective of relating the earlier cell culture studies carried out in our laboratory to normal development. Is there any evidence that the concentrations of either of these two cations increase significantly during gastrulation and neurulation? The concentration of either Na or Ca or both could be the controlling factor in the process of normal induction and early differentiation of nerve and pigment cells, as demonstrated in our cell culture studies. The data show that 22Na uptake by the cells of the embryo increases rapidly during gastrulation, while the uptake of %a by the cells of the embryo increases

Induction

29

rapidly during neurulation. Since the total Na, K, and Cl and osmolarity remain constant in the embryo during gastrulation and neurulation, the increased uptakes of 22Na and %a may be a result of (1) an increase in permeability; (2) a release of Na and Ca from a bound or sequestered state within the embryo; or (3) development of a carrier system, because the isotopes enter the embryo against a concentration gradient. Given a membrane-associated carrier system, and the increase in surface membrane area with cell division, the rate of isotope uptake could increase during development. Increases in permeability may possibly account for small increases in uptake. A small increase in permeability to deuterium oxide has been noted by Lovtrup (1960). Our data would call for an increase in permeability to 22Na of 800 x between stages 10 and 14. No such massive permeability increases have been reported. If increases in permeability did occur with time, then a continuous exposure of the embryo from 0 to 96 hr to isotope should result in much higher uptake as compared with a 72 to 96 hr exposure. The uptake from 0 to 24 hr should add to that of 24 to 48 and 48 to 72 and 72 to 96 hr. Actually the uptake from 0 to 96 and 72 to 96 is about the same. This indicates that some kind of equilibrium is reached in the 72 to 96 hr period which is the same for the 0 to 96 hr period. Such a situation would obtain if there were a steady release of Na and Ca into the cytoplasm from yolk platelets during gastrulation and neurulation. An increase in the concentration of these cations would result in a greater uptake of 22Na and 45Ca. Another argument against a massive increase in permeability stems from the facts that, to be consistent with our observed changes in isotope uptakes, a supposed increase in permeability to Na during gastrulation would have to be accompanied by a decrease in permeability to Ca. Finally, a study of uptake in blocked

30

DEVELOPMENTAL. BIOLOGY

hybrid gastrulae showed that even after several days exposure to the isotopes the uptake was still low. These and other considerations led us to explore the literature for evidence concerning the possibility of a massive release of Na and Ca from some sequestered state. Backman and Runnstrijm (1912) found that measurements of the depression of the freezing point of homogenates dropped sharply after fertilization to about one-tenth the value of the ovarian egg, indicating a decrease in free ions. Then with gastrulation and neurulation, the depression of the freezing point increased steadily to regain the value of the ovarian egg in later stages of development. The authors suggest a binding of ions by large molecules at fertilization followed by an intracellular release of these ions during development. Other workers (Abelson and Duryee, 1949) showed that the easily exchangeable Na in the ovarian egg amounted to 12% of the total. Similarly Naora et al. (1962) report that 15% of total Na is easily exchangeable. These researches deal with short exposures to isotope and are not comparable to our study of long-term exchange. They do indicate that only 12-15% of the Na is easily exchangeable in the ovarian egg. A study of the distribution of Na and K between cells and blastocoel fluid was made by Kostellow and Morrill (1968). These workers found the intracellular Na to be only 46 mmoles/kg dry weight while total Na was 80 mmoles at stage 9 ‘/2 (blastula). A separate determination of total Na in the blastocoel gave 31 mmoles. Thus during the formation of the blastocoel fluid the cells lose about 40% of their total Na. At the same time there was no significant loss from cells of K and the content of blastocoel fluid was only 0.6 mmole/kg dry wt of the embryo. From our results showing no loss of Na in the embryo raised in doubly distilled

VOLUME28, 1972

water from gastrula to stage 24, the Na lost from the cells at blastula stages must be retained in the fluids or resorbed by the cells during embryonic development. Our low values for uptake of 22Na in the blastula stage might be in part due to loss of Na to the blastocoel fluid. Possibly the increase in uptake by cells during later development is in part due to a resorption of Na from blastocoel and archenteron fluids. The curves of uptake of 22Na and 45Ca are rapidly rising and are concave upward. It seems worthwhile to consider another variable in development with these same characteristics, The number of cells increases from the fertilized egg to about 1 x lo5 at stage 14 (Sze, 1953). The stage 10 embryo contains 32,000 cells. Thus there is only an increase of 3.1 times over a period (SlO to S14) when uptake of 22Na increases by a factor of 800 and 45Caby a factor of 22. However, the magnitude of increase in number of cells gives an even greater increase in membrane surface area, and this could provide one possible explanation of the increased uptake during gastrulation. The idea that most of the Na is sequestered and is made available for exchange during development receives some support from Riemann et al. (1969); from Century et al. (1970); and from our studies on localization of 22Na uptake in the yolk region of the embryo. Riemann et al. found that the clear cytoplasm of the ovarian egg of Trituru.s contained 63 peq total Na/ml while the yolk contained 54 beq total Nalml. Since in Runu pipiens the clear cytoplasm occupies 16% of the egg by volume and the yolk 78% (McClendon, 1909), a simple calculation shows that the yolk contains 4.3 times the amount of Na in the clear cytoplasm of the ovarian egg. Our measurements show that the Na in the yolk is in part, at least, sequestered and is released with time. The possibility that yolk platelets

BARTH AND BARTH

Cations in Embryonic

sequester a slowly exchangeable Na fraction has been proposed also by Horowitz and Fenichel (1970), although with some reservations, and also by Wallace (19691971). Changes in yolk platelet membranes have been described by Karasaki (1963) for the period of induction during gastrulation, and also during experimental neuralization of Tritums ectoderm. As reported above, in the present experiments the isolated yolk region of normal embryos begins to take up 22Na after stage 11 and ‘%a after stage 13. These times are exactly those when the uptakes of whole embryos show sharp increases. Thus it is suggested that the changes in the yolk platelets observed by Karasaki (1963) during induction result in an increase in exchangeable Na. The fact that yolk regions from pipiens x syluatica hybrid embryos show little increase in uptake of 22Na and %a during gastrulation and neurulation is correlated with the observations of Johnson (1971) that the yolk platelets in the same hybrid do not break down as early as they do in the normal diploid embryos. Our measurements of uptake in hybrid whole embryos at later stages of development show that both 22Na and =‘Y2a uptakes increase rapidly only after stage 14 and later. Thus the failure of yolk platelets to break down during gastrulation and neurulation is correlated with a failure of the yolk region and the whole embryo of hybrids to take up 22Na and 45Ca at the same rate as the normal embryo. When breakdown of yolk platelets does occur later in time in the hybrids, the uptake of the isotopes increases sharply. Since yolk platelets are present in all cells of the normal embryo and contain Na and Ca, probably in sequestered form, any release of Na and Ca into the cytoplasm during gastrulation and neurulation seems most likely to be a result of changes in the yolk platelet.

Induction

31

Localization of Uptake of 2zNa and 45Ca in the Neurulu Our study of localization of isotope uptakes was prompted by the earlier investigations of Mangold and those of Holtfreter on the inductive capacity of the neural plate and also of the archenteron roof of an early neurula. Mangold (1933) by means of cutting up the roof of the archenteron or the neural plate, demonstrated that anterior regions of these structures induced head structures, while posterior regions induced trunk and tail structures. Holtfreter (1933), using a reciprocal method, placed explants of the presumptive epidermis in contact with the mesoderm in various positions along the anteroposterior axis and found that head structures were induced in the anterior positions while trunk structures were induced in posterior positions. Thus it is clear that the anterior neural plate and anterior mesoderm of the neurula contain something which is different from the posterior neural plate and mesoderm. Since our earlier studies showed a difference in the induction of cell types in cultures depending both upon the concentration of the inductor and also the concentration of Na in the medium, it seemed possible that differences in concentration of Ca and Na might be effective in normal induction. If so, we anticipated finding differences in the uptake of 22Na and %a by the anterior regions of neural plate and roof of archenteron as compared with posterior regions. The results presented above supported this prediction in that anterior regions gave greater counts per minute than posterior regions; values for uptake became progressively lower in samples taken from anterior toward posterior ends of the neural plate plus archenteron roof. If the cpm for the isotopes are a measure of the actual exchangeable Na and Ca in the cells, then we can conclude that

32

DEVELOPMENTAL BIOLOGY

the anterior neural plate and archenteron roof have more exchangeable Na and Ca as compared with more posterior regions. We suggest, but do not conclude, that the higher amounts of exchangeable Na and Ca may be responsible for induction of head structures, whereas lower amounts of these cations induce trunk structures. Uptake of zzNa in Normal and Hybrid Embryos Treated with Prostaglandin E,

PGE, increased 22Na uptake markedly in hybrid embryos. However, the block to gastrulation was not lifted; nor did PGE, treatment of cell aggregates serve to induce nerve or pigment cells, even when extra Ca was added in an attempt to reproduce the Ca-induced, sodium-dependent conditions for induction previously reported for cell cultures of presumptive epidermis (Barth and Barth, 1969). These essentially negative results are balanced by the finding that hybrid embryos are remarkably more sensitive than normal diploid embryos to PGE, stimulation of 22Na uptake. Although the reasons for this are not immediately apparent, it might be helpful in designing future experiments to examine PGE, sensitivity in context of earlier studies on the pipiens x sylvatica hybrid arrested gastrula. As recorded in Table 6, although 22Na uptake by hybrid embryos is increased by PGE, as much as 465% at hours of development equivalent to late gastrula, by tailbud stage prostaglandin has no effect and 22Na uptake is the same as in normal diploid late gastrulae. This means that some maximal value for exchangeable sodium ions is reached earlier in normal diploid pipiens embryos, but eventually is also reached at a later “stage” by the arrested hybrid gastrula of pipiens x sylvatica.

That the hybrid block represents a delay rather than complete absence of processes at various levels of organization

VOLUME 28, 1972

is an idea proposed some years ago by Gregg. It will be recalled that careful studies by Gregg (1960) on the pipiens x sylvatica hybrid led to the conclusion that intracellular changes facilitating respiratory enzyme-substrate union take place in the hybrid but at a slower rate than in normal embryos. Gregg proved that there is nothing qualitatively missing from the hybrid respiratory machinery, because homogenates prepared from hybrids respire at rates quantitatively similar to R. pipiens homogenates. That the hybrid is slow or deficient in membrane differentiation is implied by Gregg’s work and by our own present results. Although the uptake of isotope by the hybrid eventually attains that of the normal gastrula, the increase in **Na uptake may occur too late in development to permit the necessary sodium-dependent enzyme reactions to take place at the right time. It is interesting that the order of magnitude noted by Gregg for DNP stimulation of hybrid respiration (300-400%) and that of PGE, stimulation of 22Na uptake (200-475%) are similar, in view of the fact that activation of ATPase and oxygen consumption can be triggered by changes in cytoplasmic sodium (Ramwell and Shaw, 1970). The marked stimulating effect that PGE, has on 22Na uptake by the hybrid gastrula suggests that there may be more than the normal number of PGE, binding sites in hybrid gastrulae. PGE, is thought to displace membrane-bound Ca*+ and thereby allow increased 22Na influx (Horton, 1969; Ramwell and Shaw, 1970). It is possible that one aspect of cellular differentiation includes a reduction in Ca-binding sites and that this membrane differentiation is delayed in the genetic hybrid. It has been shown that a single genetic locus changes the cation transport characteristics (of red blood cell membranes in sheep) in a manner that reflects differentiation of Na: K

BARTH

AND BARTH

Cations in Embryonic

transport sites in the membrane (Dunham and Hoffman, 1971). The experiments with hybrid embryos prove that when development is blocked, 22Na uptake is reduced. Thus the normal sharp increase in 22Na uptake during gastrulation is correlated with development and not simply with age of embryo or duration of exposure to isotope. The argument that levels of exchangeable Na and Ca within the embryo are directly involved in cell differentiation, and the evidence suggesting yolk as a source of these ions during primary induction have been strengthened and supplemented by the above report. This work was supported by a grant from the National Science Foundation (GB 23026) to the Marine Biological Laboratory, Woods Hole, Massachusetts 02543. Dr. John E. Pike, Upjohn Company, Kalamazoo, Michigan 49001, kindly supplied samples of prostaglandin E,. We are indebted to Dr. H. Burr Steinbach and to Dr. Robin A. Wallace for constructive criticisms of the manuscript. To Dr. Steinbach we owe also the analyses recorded in footnote 1. REFERENCES P. H., and DURYEE, W. R. (1949). Radioactive sodium permeability and exchange in frog eggs. Biol. Bull. 96, 205-217. BACKMAN, E. L., and RUNNSTR~M, J. (1912). Der osmotische Druck wahrend der Embryonalentwicklung von Rana temporaria. Pfluegers Arch. Gesamte Physiol. Menschen Tiere 144, 287-345. BARTH, L. G., and BARTH, L. J. (1969). The sodium dependence of embryonic induction. Deuelop. Biol. 20, 236-262. BURNETT, A. L. (1968). The acquisition, maintenance, and lability of the differentiated state in Hydra. In “The Stability of the Differentiated State” (H. Ursprung, ed.), Vol. 1, 109-127. Springer-Verlag New York Inc. CENTURY, T. J., FENICHEL, I. R., and HOROWITZ, S. B. (1970). The concentrations of water, sodium and potassium in the nucleus and cytoplasm of amphibian oocytes. J. Cell Sci. 7, 5-13. DUNHAM, P. B., and HOFFMAN, J. F. (1971). Active cation transport and ouabain binding in high potassium and low potassium red blood cells of sheep. J. Gen. Physiol. 58, 94-116. ABELSON,

Induction

33

GREGG, J. R. (1960). Respiratory regulation in amphibian development. Biol. Bull. 119,428-439. HOLTFRETER, J. (1933). Der Einfluss von Wirtsalter und verschiedenen Organbezirken auf die Differenzierung von angelagerten Gastrulaektoderm. Wilhelm Roux Arch. Entwicklungsmech. Organismen 127, 619-775. HOROWITZ, S. B., and FENICHEL, I. R. (1970). Analysis of Na transport in the amphibian oocyte by extractive and radioautographic techniques. J. Cell Biol. 47, 120-131. HORTON, E. W. (1969). Hypotheses on physiological roles of prostaglandins. Physiol. Reu. 49, 122-161. JOHNSON, K. E. (1971). A biochemical and cytological investigation of differentiation in the interspecific hybrid amphibian embryo Rana pipiens p x Rana syloatica 6. J. Exp. Zool. 177, 191-206. KARASAKI, S. (1963). Studies on amphibian yolk. 5. Electron microscopic observations on the utilization of yolk platelets during embryogenesis. J. Ultrastract. Res. 9, 225-247. KOSTELLOW, A. B., and MORRILL, G. A. (1968). Intracellular sodium ion concentration changes in the early amphibian embryo and the influence on nuclear metabolism. Exp. Cell Res. 50, 639-644. KROEGER, H. (1963). Chemical nature of the system controlling gene activities in insect cells. Nature (London) 200, 1234-1235. of KROEGER, H., and LEZZI, M. (1966). Regulation gene action in insect development. Anna Reu. Entomol. 11, l-22. LEZZI, M. (1970). Differential gene activation in isolated chromosomes. ht. Reu. Cytol. 29, 127-168. L~VTRUP, S. (1960). Water permeation in the amphibian embryo. J. Exp. Zool. 145, 139-149. MCCLENDON, J. F. (1909). Cytological and chemical studies of centrifuged frogs eggs. Wilhelm Roux Arch. Entwicklungsmech. Organismen 27,247-257. MCDONALD, T. F., SACHS, H. G., ORR, C. W., and EBERT, J. D. (1971). The effect of potassium on BHK cells: intracellular ions, ATP, growth, DNA synthesis and membrane potential. Anna. Rep. Director, Dept. Embryol., Carnegie Inst. Washington Yearb. 70. MANGOLD, 0. (1933). Uber die Induktionsfahigkeit der verschiedenen Bezirke der Neurula von Urodelen. Naturwissenschaften 43, 761-766. NAORA, H., NAORA, H., IZAWA, M., ALLFREY, V. G., and MIRSKY, A. E. (1962). Some observations on differences in composition between the nucleus and cytoplasm of the frog oocyte. Proc. Nat. Acad. Sci. U.S.A. 48, 853-859. QUASTEL, M. R., and KAPLAN, J. G. (1970). Lymphocyte stimulation: The effect of ouabain on nucleic acid and protein synthesis. Exp. Cell Res. 62, 407-420. RAMWELL, P. W., and SHAW, J. E. (1970). Biological

34 significance

DEVELOPMENTAL BIOLOGY of the prostaglandins.

Recent Progr.

Horm. Res. 26, 139-187. RIEMANN, W., MUIR, C., and MACGREGOR, H. C. (1969). Sodium and potassium in oocytes of Tritunes cristatus. J. Cell. Sci. 4, 299-304. SHUMWAY, W. (1940). Stages in the normal development of Rana pipiens. And. Rec. 78, 139-147.

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SZE, L. C. (1953). Changes in the amount of desoxyribonucleic acid in the development of Rana pipiens. J. Exp. Zool. 122, 577-601. WALLACE, R. A. (1969-1971). Personal communications.