ON THE ENERGETICS OF DIFFERENTIATION

V. COMPARISON O F THE RATES OF DEVELOPMENT AXD OF OXYGEN

CONSUMPTION OF TIGHT MEMBRANE AND NORMAL

ECHINODERM EGGS

ALBERT TYLER William G. Kerckhoff Laboratories of the Biological Sciences,

California Institute of Teehnologg, Pasadena

POUR FIQURES

It is shown in these experiments that thick-walled embryos arising from tight membrane eggs develop more slowly than do normal embryos. The rate of oxygen consumption is, however, the same in both.

THEORETICAL PART

Embryos from isolated half-blastomeres of the sea urchin develop more slowly than normal. Those from isolated quarter-blastomeres develop still more slowly. On the other hand, giant embryos produced by fusion of two fertilized eggs develop more rapidly than do the normal embryos. These differences in rates of development are interpreted (Tyler, '33, '35) as due to differences in the energy required fo r the differentiation processes in these various embryos. The rate a t which energy is supplied, as measured by the rate of oxygen consumption, was shown to be the same per unit mass for the half-embryos as for the normals. The half-embryos requiring more energy to reach the same stage of development therefore develop more slowly. For the quarter-embryos and giant embryos no oxygen consumption measurements were made, but it is assumed that they too would show the same rate per. unit mass as the normals. It is from dimensional reasoning

395

396 ALBERT TYLEE

that the dwarf embryos are expected to require more energy and the giants less energy for development. An embryo de- veloping from an isolated blastomere of the two-cell stage has half of the surface area of the normal. The wall thickness, as measured in the blastula and gastrula stages is, however, the same in the half-embryo or in the quarter-embryo as in the normal. It is therefore proportionately much too thick. I n the giant embryo, the wall thickness is again the same as in the normal embryo, hence relatively too thin.

For further investigation of this factor of relatively in- creased wall thickness, it was desirable to produce thick- walled embryos of normal size. By inhibiting the elevation of the fertilization membrane in echinoderm eggs, embryos develop that have, in the blastula and gastrula stages, thicker than normal walls. Such embryos, however, are of smaller than normal size. They also differ from the normal in other respects.

EXPERIMENTAL PART

The material used in these experiments was principally the eggs of the sand dollar, Dendraster excentricus. For the respiration measurements, eggs of the sea urchin, Paracen- trotus lividus, were used.

Tight membrane eggs sometimes occur spontaneously in echinoderms. They can be produced artificially by treating the unfertilized eggs with isosmotic non-electrolyte solutions to which a small amount of certain electrolytes have been added (Moore, '32). They can also be obtained by various other agents, notably treatment shortly after insemination with acid sea water (Tyler and Scheer, '37). I n the tight membrane egg the fertilization membrane is not elevated from the surface. Such eggs are readily distinguished in the two- cell stage from what may be called no-membrane-eggs. The blastomeres of the latter are spherical in shape except for the small area of contact, whereas in the former they are hemi- spherical in shape. That the fertilization membrane is actu- ally present is seen clearly at the time of hatching when the blastula from a tight membrane egg ruptures the membrane and swims off leaving it behind.

TIGHT MEMBRANE EGGS 397

The development of the tight membrane eggs has been studied by Tyler and Scheer ('37) and it is shown that in spite of the striking modifications of cleavage, blastula and gastrula stages, normal plutei are formed. Of particular interest here is the increased thickness of wall of the blastulae and gastrulae from tight membrane eggs. The presence of the tight (and presumably rather inelastic) membrane around the egg opposes the increase in diameter that normally takes place as the blastocoel forms. Thus at the time of hatching the blastocoel is considerably smaller and the wall corre- spondingly thicker than normal. After hatching the blastulae from tight membrane eggs increase in diameter, but the rate of increase appears to be no greater than the normal so that at the time of gastrulation, they are still smaller and have a thicker wall than normal. Before gastrulation the normal Dendraster embryo becomes ellipsoidal in shape, the long axis coinciding with the antero-posterior axis of the gastrula. The tight membrane embryo, however, remains spherical.

Some measurements of normal and tight membrane em- bryos are given in table 1, along with the uncleaved eggs for comparison. The diameter of the membrane of the uncleaved tight membrane egg is, of course, practically the same as that of the egg itself. The volumes of the two types of uncleaved eggs are the same. In the blastula stage the outside diameter of the normal embryo is much greater than that of the tight membrane embryo. The volume of the blastula wall is, how- ever, practically the same in both. It is also about the same as that of the uncleaved egg, showing that no significant change in volume of egg material occurs during cleavage. The increased wall thickness of the blastula of the tight mem- brane egg thus corresponds to its smaller diameter.

In the beginning gastrula stage, the normal embryo (fig. 2a) is ellipsoidal in shape, and the wall thickness is different in different parts. It is greatest (25 p ) at the posterior end, slightly less (20 p ) at the anterior end and considerably less (148 p ) at the sides. It would appear from these figures that the ellipsoidal form is attained principally by the stretching

398

173 X 19 1 8 X l 8 18 x 1 9 174 X 18

18

29 X293 2 9 4 x 3 1 291 x 291 30 X 2 8

2 9 t

ALBERT TYLER

7.8 X

8.1 x 106

8.0 X 10'

TABLE 1 Dimensions o f nornlal and tight membrane eggs and blastulae of Deiidraster

Uncleaved normal egg

Average

membrane egg Uncleaved tight

A ver a ge

(just hatching) Normal blastula

Average

blastula ( jus t hatching)

Tight membrane

Average

Normal beginning gastrula

A veratge

beginning gastrula

Tight membrane

Average

OUTSlDE DIAMETEBS

P 113 X 115 1 1 5 x 1 1 4 1 1 4 x 1 1 4 113 X 114

114

115 X 112 114 X 113 115 X 113 114 X 115

114

1 3 6 X 138 139 X 137 134 X 139 1 3 9 x 1 3 5

137

120 x 119 119 X 122 120 x 121 1 1 9 x 1 2 1

120

152 X 123 153 X 124 154 X 122 152 X 123

153 X 123

124 X 123 124 X 125 126 X 123 123 X 125

124 X 124

INSIDE DIAMETEBS

P

1 0 1 x 100 103 X 101 98 x 101

104X 99

101

62 X 60 6 0 x 6 0 6 1 x 6 0 59 X 65

604

109 X 97 108 X 94 1 0 6 x 9 3 1 0 8 x 9 3 108 X 94

72 X 78 70 X 80 71 X 77 70 X 78

7 1 X 78

DIAMETEROF RBTILIZATIOI MEMBBANE

P 148 X 149 147 X 150 148 X 148 149 X 150

149

149 X 148 150 X 148 151 X 147 149 X 147

149

7.8 X l o 1 + 'osterior Anterior

25 25 26 25

25

28 29 30 3 1

2 9 t

18 20 22 19

20 24 25 25 24

243

Lateral

13 15 144 15 144

=?l

223 23 234 23

TIGHT M E M B R A N E EGGS 399

Blastulae (just hatching) Beginning gastrula

of the lateral parts of the wall. At the same time, the figures show an increase in the anterior and posterior portions of the wall of the beginning gastrula as compared with the newly hatched blastula. The tight membrane embryo, in the begin- ning gastrula stage (fig. 2b) is practically spherical in shape. There is much less difference in wall thickness of different regions than in the normal embryo, the corresponding figures being 29+ p, 244 p and 23 p.

Ratios of the dimensions of tight membrane and normal embryos are given in table 2. At the time of hatching, when both are spherical, the outside diameter of the tight membrane blastula is nine-tenths of the normal, while its wall thickness is about 65% greater. At the time of gastrulation, when tlie normal embryo is ellipsoidal, the tight membrane embryo is

WALL THICKNESS LENGTH WIDTH

Posterior Anterior Lateral

0.88 0.88 1.64 1.64 1.64 0.81 1.01 1.18 1.23 1.59

about eight-tenths the length of the normal, while the widths are about the same. The wall thickness ratios differ for dif- ferent regions. In the anterior and posterior regions the wall is only about 20% greater, whereas in the lateral portions it is 60% greater than the normal.

To determine the relative rates of development, the tight membrane and normal embryos were cultured in the same dish, usually in pairs. There is, of course, no difficulty in distingusihing the two types of embryos up to the pluteus stage. The temperature was not controlled in these deter- minations, but since the two types of embryos are cultured in the same dish this was unnecessary. The time to reach the various stages of development is given in table 3. These fig- ures are averages from about twelve determinations, the time

400 ALBERT TYLEE

for five or more different stages being obtained in each. Since the temperature was not the same in the different runs and generally varied throughout one run, the figures are all cor- rected for 20°C. This is done by first determining the factor necessary to correct the time for the normal embryo to reach a given stage to the time in 20", the average time a t this tem- perature having been previously determined (Tyler, '36). Then the time for the tight membrane embryo is multiplied by the same factor. The time for the fastest and the slowest tight membrane embryos as well as the average is given in

TABLE 3

Time of development of normal and tight membrane Dendraster embryos: temperature, ZO'C.

Q gastrulated 4 gastrulated 3 gastrulated 9 gastrulated Early prism Late prism Early pluteus Middle pluteus Late pluteus

NORMAL

hours

21 23t

52

TIGHT MEMBRANE

Range Average

hours

12&14f 14 -16 151-17 17 -21 20 -26 251-273 28 -34 41 -51 60 -72

hours

133

16 19 22 263 31 44 68

153

DELAY

From fertilization

% 8

19 14 27 38 26 32 33 31

From gastrulation

% 200 250 100 13.3 150 61 65 52 40

the table. The variation in rate shown in the table is, to a large extent, due to difficulty in ascertaining the exact stage of development. This is especially true in the gastrula stages in which the crowded mesenchyme cells tend to obscure the extent of invagination. Nevertheless the results are all con- sistent in showing a delay. The difference in stage of de- velopment attained by typical pairs of normal and tight membrane embryos is illustrated in figures 3 and 4.

The tight membrane eggs divide at the same rate as the normal. Also they both apparently begin gastrulation at the same time, or at least within a short time (+ hour) of each

TIGHT MEMBRANE EGGS 401

other. It is therefore reasonable to estimate the delay from the time,of gastrulation (12 hours) as is done in the fourth column of table 3 rather than from fertilization as is done in the third column. According to these figures there is a de- crease in the delay as development proceeds. However, it is evident that the first few values are subject to a large relative error. More significance may be attached to the values for the delay starting a t the three-fourths gastrulated or early

n

2a

n

4a 4b Figs. la, 2a, 3a and 4a Normal embryos. Figs. lb, 2b, 3b and 4b Tight

membrane embryos. 1, newly hatched blastulae; 2, beginning gastrulae; 3 and 4, later gastrulae and prism ; illustrating approximately the difference in dimensions and in rate of development of tight membrane and normal embryos. Drawn from preserved material.

prism stages. The difference between the percentage delay in reaching these stages and that in reaching the later stages appears to be greater than can be accounted for by the range of variation. This would mean then that the retardation occurs chiefly in the gastrulation and in the prism stages.

The respiration experiments were performed on eggs of Paracentrotus at Noscoff using the usual Warburg manom- eters with conical vessels. Some of the runs were started

402 ALBERT TYLER

TIME A* - FERTILIZATION

O F FIRST READING

hours a n 6 8

shortly after fertilization, others in the blastula stage. In the former case the eggs were first divided among the vessels by pipetting the same amounts of a uniform suspension into each. They were’ then inseminated and acid sea water of sufficient strength to produce tight membrane eggs added to certain of the vessels at 15 to 30 seconds later. After 3 or 4 minutes alkaline sea water was added to neutralize the acid. The con- trols were brought to the same volume by the addition of ordinary sea water. The same amount of sperm suspension was also added to the same volume of sea water (usually egg water) in the thermo-barometer vessel and in addition in some cases a thermo-barometer without sperm was included. A comparison of the two thermo-barometers showed that the

RESPIRATION NORUAI, TIGHT TIGHT TIGHT TIME hWRPAL MEMBRANE MEMBRANE h1EXBRANE

how cu.mm. cu.mni. eu.mm. m . m m . cu.mm. c 46.5 48.8 50.4 51.6 52.3

131.5 128.3 130.5 136.6 .... 3 122.3 125.2 126.4 123.8 122.4 5 261.6 253.7 250.2 258.9 * . . .

added sperm accounted for about 1 to 2 mm. pressure change in the first hour and thereafter produced no measurable effect. For the runs started in the blastula stage, equal samples of a uniform suspension were first placed in Petri dishes, the same number of dishes being used as there were vessels available. The insemination, acid treatment and culturing took place in the Petri dishes, then in the blastula stage the eggs were transferred to test tubes from which equal volumes were pipetted into each vessel. In this case, then, the eggs were pipetted twice and the sampling error may be expected to be greater.

I n table 4, the results of four experiments out of seven that were performed are presented. The other three were omitted either because the development was poor, or because the

TIGHT MEMIBRAKE EGGS 403

treatment did not give a sufficiently high percentage of tight membranes, or because the treatment interferred with cleav- age. I t is, of course, difficult to get 100% tight membranes without at the same time interferring with cleavage. Even when a trial test on a sample of the eggs gives perfect results, similar treatment of the eggs to be used in the vessels often fails to work nearly as well, presumably either because the eggs had aged in the interim or because slightly different concentrations of the acid sea water or of the egg suspension mere employed. I n all four sets of experiments presented in table 4, there was between 98 and 100% cleavage and develop- ment. Tight membrane production was generally less, but high enough so that any significant difference in oxygen con- sumption would undoubtedly be noted. In the first experi- ment (started + hour after insemination) there was 95,90 and 8876, respectively, in the three vessels employed. In the second, the percentages of tight membrane eggs were 90 and 85, respectively; in the third, 85, 85, 80; in the fourth, 85 and 90. The figures in table 4 give the oxygen consumption in cubic millimeters. To get the corresponding pressure change in millimeters it is necessary to divide by the vessel constants which are between 1.3 and 1.4 for the five vessels employed. Since the pressure change is read to the nearest 0.5 mm. it is evident that the reading error contributes a negligible frac- tion of the variation shown by the data. This variation is probably principally due to the sampling error. It is, how- ever, relatively small, the biggest difference being less than 5% for the two vessels with normal eggs in the first experi- ment listed. The other duplicates or triplicates differ by less. The average values for the normal and tight membrane eggs are not given in the table, but it may be seen by inspection that the differences between the tight membrane and normal are no greater than the differences in the duplicate normals or the duplicate or triplicate tight membrane.

404 ALBERT TYLER

DISCUSSION

The delay in rate of development of the tight membrane eggs is a result that was to be expected on the basis of the increased thickness of blastula wall and a normal supply of energy. However, it is evident from the data presented here that other factors may be involved. In the beginning gas- trula stage, the shapes of the normal and tight membrane embryo are different. The latter is considerably shorter in its antero-posterior axis while its transverse axes are ap- proximately of normal size. Also the wall thickness at the region of invagination is only slightly greater in the tight membrane embryo. According to the recent vital staining results of Horstadius ('35), it is only this posterior wall that invaginates. With only this region involved, the difference in wall thickness would hardly be sufficient to account for the delay. One might reasonably assume that more of the blas- tula wall than the part actually invaginated is involved in the process. But, in the absence of any satisfactory conception of the mechanism of gastrulation, it is not a t all clear how an increased thickness of the lateral walls would affect the rate of invagination. Another factor that might be involved in the delay is the crowding of the mesenchyme cells in the small blastocoel. It is not likely that this factor would operate in a mechanical way. There is evidently some interaction be- tween the mesenchyme cells and the adjacent wall, as shown by the experiments of Horstadius ('35, '36) of implanting micromeres into blastulae. By such implantation, parts of the wall, that would not ordinarily do so, may be made to invaginate. In the tight membrane egg the mesenchyme cells extend relatively farther anterior than in the normal embryo and can thus interact with the more anterior cells endowing them with the tendency to invaginate. The resuli would be a relatively weakened tendency of the posterior cells to invagi- nate and would conceivably lead to the observed delay in rate of development.

It had seemed at first reasonable to apply the dimensional analysis to the results with the tight membrane eggs. But in

TIGHT MEMBRANE EGGS 405

view of the complicating factors involved, this becomes im- practical until the contributions of the various factors can be assessed.

From the results of the respiration measurements it is clear that the rise in respiration that occurs after fertilization in sea urchin eggs is unaffected by the failure to elevate the fertilization membrane. It is also evident that the modifica- tion in the shapes of the cells in the cleavage stages, and of the embryo as well in later stages, does not alter the rate of respiration. The respiratory mechanism appears here to be independent of the morphological changes. This independ- ence of the respiratory mechanism appears in many instances (Needham, '33) since the original finding by Warburg ( '10) that cleavage could be suppressed by phenylurethane without affecting the respiratory rate. Evidence for the dependence of the morphological changes on the respiratory changes now appears in the work of Lindahl ('33-'36) in which the effect of lithium and other agents on the respiration is studied. Liithium is shown to inhibit the respiration. The lowered respiration does not simply give a corresponding delay in development, as is the case when temperature is lowered (Tyler, '36), but results in a modification in the type of de- velopment. The difference is evidently that the whole of the respiratory mechanism is sensitive to temperature, whereas, as Lindahl shows, only the increasing fraction of the respira- tion is sensitive to lithium. Potassium, which counteracts more or less the morphological action of lithium (Runnstrom, '28) also overcomes the inhibitory action on the respiration. This cannot be simply interpreted by assuming that it is the increasing fraction of respiration that is associated with the reactions supplying the ener,gy for differentiation. There would be no particular reason for expecting the type of form changes characteristic of the lithium larvae to result from suppression of this fraction of the respiration. Lindahl, him- self, concludes that the morphological effects are only partly due to the inhibition of the respiration.

406 ALBERT TYLER

SUMAIAH Y

1. Embryos developing from eggs in which membrane ele- vation is prevented have, in the blastula and gastrula stages, thicker than normal walls.

2. There is no appreciable change in volume of egg material up to the time of hatching either in the normal or the tight. membrane egg. The thicker wall of the tight membrane em- bryo corresponds to its smaller diameter.

3. The tight membrane egg develops at a slower rate than the normal, the retardation occurring principally in the gas- trula and prism stages.

4. The rate of oxygen consumption of the tight membrane egg in the early cleavage and late blastula stages is the same, within the limits of error, as that of the normal egg.

LITERATURE CITED

HORSTADIUS, S. 1935 Gber die determination im Verlaufe der Eiachse bei Seeigeln. Pubbl. Staz. Zool. Pu’apoli, vol. 14, pp. 251-429.

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1933 Uber ‘ Animalisierte ’ und ‘ vegetativisierte ’ Seeigellarven. Arch. Entw.-mech., Bd. 128, S. 661-664.

Zur Kenntnis der physiologischen Grundlagen der Determina- tion im Seeigelkeim.

The role of unantagonized cations in protecting the mem- brane forming function in the eggs of the sea urchin. Protoplasma, Bd. 15, S. 268-275.

NEEDHAN, J. On the dissociability of the fundamental processes in onto- genesis. Biol. Rev., vol. 8, pp. 180-233.

RUNNSTROM, J. 1928 Zur experimentellen Analgse dcr Wirkung des Lithiurns auf den Seeigelkeim.

TYLER, A. 1933 On the energetics of differentiation. A comparison of the oxygen consumption of ‘half’ and whole embryos of the sea urchin. Pubbl. Staz. Zool. Napoli, vol. 13, pp. 155-161.

11. A comparison of the rates of development of giant and of normal sea-urchin embryos. Biol. Bull., vol. 68, pp. 451460.

1936 On the energetics of differentiation. I11 and IV. Biol. Bull., VOI. 71, pp. 59-100.

Inhibition of fertilization in eggs of marine animals by means of acid.

1910 Uber die Oxydationen in lebenden Zellen nach Versuehen am Seeigelei.

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1936 Acta. Zool., Bd. 17, S. 179-365.

NOORE, A. R. 1932

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Acta. Zool., Bd. 9, S. 365-424.

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W m w , 0.

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