the role of water-regulating mechanisms in the developmen ot f … · embryo/, exp. morph. vol. 28,...

/ . Embryo/, exp. Morph. Vol. 28, 2, pp. 449-462, 1972 4 4 9

Printed in Great Britain

The role of water-regulatingmechanisms in the development of the haploid

syndrome in Xenopus laevis

By LOUIE HAMILTON1 AND P. H. TUFT2

From the Department of Zoology, Edinburgh University

SUMMARYThe uptake of water by haploid and diploid sibling embryos of Xenopus laevis has been

investigated by measuring the density changes which occur during the development of intactembryos from the blastula to the late tail-bud stage, and of explants from which most of thepresumptive endoderm has been removed.

The results show that up to the mid-gastrula stage there is no difference between the haploidand diploid embryos; but from then on, whereas the diploid volume increases steadily, thehaploid gastrulae undergo a series of cyclical volume changes due to loss of fluid through theblastopore. It is concluded that this is the result of an excessive inflow of water through thehaploid ectoderm, because it was found that the volume of haploid ectodermal explantsincreased much more rapidly than the volume of similar diploid explants. Excess flow throughthe haploid ectoderm also accounts for other characteristics of the haploid syndrome-microcephaly and lordosis.

It is suggested that it is the doubling of the cell number in haploid embryos with theconsequent 25 % increase in aggregate cell membrane area which accounts for the differencebetween the uptake of water by the two types of embryos. It is also suggested that changes inthe rate of water flow through the ectoderm and endoderm which are thought to account forthe accumulation of water in the blastocoel and archenteron in the normal diploid embryoarise in a similar way.

INTRODUCTION

The majority of haploid embryos of Xenopus laevis develop into larvae whichshow a characteristic syndrome - large accumulations of water occur under theskin, the nerve cord is short, the brain is small, and there is considerable lordosis.A study of the very few haploid embryos which do not show this syndrome ledFox and Hamilton to conclude that the oedema is a result of an excessive inflowof water through the ectoderm rather than renal failure, because they found thatin these embryos the kidneys were hypertrophied (Fox & Hamilton, 1964).

Accumulation of water in intercellular spaces, however, is a normal feature ofearly development in the diploid embryo; water accumulates at an increasingrate first in the blastocoel, a development of the cleavage cavity, and then in the

1 Author's address: Department of Biology as applied to Medicine, The Middlesex HospitalMedical School, London, W.I, U.K.

2 Author's address: Department of Zoology, West Mains Road, Edinburgh, U.K.

450 L. HAMILTON AND P. H. TUFT

archenteron - a cavity formed by an invagination of the blastula surface andlined by cells derived from the vegetal pole of the blastula. During the formationof the archenteron the blastocoel decreases in volume and disappears.

It is therefore of some interest to know whether the factors which give rise tothe abnormal inflow of water through the haploid larval skin also affect theuptake and distribution of water in the earlier stages of development. We haveaccordingly carried out a series of experiments to determine the rate of wateruptake by decapsulated diploid and haploid siblings from the early blastula tothe late neurula stage, and also the water uptake of vesicles formed by blastulaexplants from which most of the presumptive endoderm has been removed and inwhich gastrulation does not occur. The results of these experiments indicate thatthe rate of transcellular water flow in the presumptive ectoderm and endodermof the haploid embryo is abnormally high from the gastrula stage onwardsbut only gives rise to abnormal volume changes in intact embryos during theneurula stage.

MATERIALS AND METHODS

Embryos

The eggs were obtained from adult Xenopus laevis which were induced tospawn by injection with chorionic gonadotrophin. The eggs were collected at5 min intervals, and 10 min after laying half the eggs were irradiated with u.v.light (2 x 10"4 J mm-2) to produce androgenetic haploids (Gurdon, 1960). Thetreated and control embryos were kept in crystallizing dishes in sterile tap-water at 25 °C in a thermostatic water bath. In what follows we shall assumethat irradiated embryos are haploid and we shall refer to them as such.

Density gradients

Linear density gradients of colloidal thorium oxide stabilized with dextrinwere made by upward displacement in parallel-sided glass tubes using a simplegradient machine; the gradient tubes were then transferred to a glass-frontedthermostatic bath at 25 °C (±0-001 °C). Details of the gradients are given inTable 1.

The gradients were calibrated by measuring the position of glass densitystandards that had previously been calibrated against droplets of KC1 solutionsin a brom-benzene/kerosene (Solve Esso 15) gradient set-up in a similar wayand saturated with water as described by Linderstrom-Lang & Lanz (1938).Using this technique it is possible to make gradients 20 cm high with a densityrange from top to bottom of 0-045gcm~3givingasensitivityof0-0002gcm~3mm~1

and linearity such that changes in density can be measured to ± 0-0001 g cm"3.In experiments in which the embryos remained in the gradient throughout theirdevelopment, the oxygen tension was maintained by circulating oxygen or airthrough loops of thin-walled polyethylene catheter immersed in the gradients.

The haploid syndrome in Xenopus 451

Table 1. Details of typical density gradient

1. Gradient tube: length 30 cm, diameter 2-5 cm.

2. Density medium:Disperse phase - colloidal thorium oxide (5 nm) stabilized with dextrin ('Troka'

163 Henkel International)Continuous phase - sterile tap-waterConductivity - 5 x 10~5 Cr1 cm"1 (̂ =0-2 mM electrolyte)

3. Method of formation - upward displacement

4. Height of gradient - 25 cm

5. Calibration: glass standards calibrated against KC1 solution in brom-benzenekerosene gradient.

Bottom of gradient ...Density standard

(1)(2)(3)(4)(5)

Top of gradient

6. Height of linear portion

Density1084

10838±00002 gem-3

1-073010606105151043810400

Sensitivity (density change per mm)

7. Temperature of water bath:: 25 ± 0-001 °C

Position (cm)8 0

9-314-119-824-027-833-0

18-7 cm00002 gcm-3

Density measurements

In order to obtain the density data required to calculate the change in volumeof the haploid and diploid embryos three series of experiments were carried out.In the first, successive batches of control and irradiated sibling embryos ofknown age were decapsulated surgically in l/10th Holtfreter solution and theirdensity measured 10 min after they had been introduced into the density gradient.

In the second series, four or five haploid and diploid blastulae were placedin identical gradients set up side by side in the thermostat bath. The gradientswere photographed at intervals of 15 min with an automatic 'Robot' camera.The density of the embryos was determined by projecting the negative image ofthe gradients on to a linear density scale using the calibration beads as referencepoints.

In a third series of experiments the same technique was used to monitor thedensity change of ectodermal explants from irradiated and unirradiated embryos.In these experiments, the bottom third of stage 8 (Nieuwkoop & Faber, 1963)sibling blastulae was removed with fine forceps. The remaining upper two thirdswere allowed to round up inl/lOth Holtfreter solution before being introducedinto the gradients. These explants formed stable vesicles whereas similar explantsfrom a stage 9 blastula containing more ectoderm tended to burst.


The determination of changes in volume and water content

It can be shown that the volume changes which occur during Xenopusdevelopment up to the early tail-bud stage are entirely due to changes in thewater content of the embryo as follows: the weight of the embryo in water, itsreduced weight (RWe) is a function of the mass of the embryo (We), its volume(Ve) and the density of water at the same temperature (/?„.)•

RWe=We-VePw. (1)

The density of the embryo (pe) is in turn a function of its mass and volume:

WPe = jr- (2)

From (1) and (2) the reduced weight can be expressed in terms of its mass anddensity,

A , (3)Pel

or as the sum of the reduced weights of its constituents ( S J R ^ ) and theirweights (Wi) and densities (pt) (Lovtrop, 1953):

RWe = ZRJVi = £ wJl-^\. (4)

Since the reduced weight remains unchanged from cleavage to the early tail-bud stage (Tuft, 1962) it follows from (4) that any change in density must be dueto the uptake or loss of matter with the same density as water.

The dry mass of the embryo would tend to decrease during development asthe result of a loss of solutes and the oxidation of respiratory substrates. Theformer is so small that it cannot be detected (unpublished data) and the latter,which is also small, can be estimated from the oxygen consumption of theembryo. The rate of oxygen consumption rises gradually during development ofthe Xenopus embryo, reaching 4-8 x 10~4ml h"1 per embryo during the lateneurula stage (Tuft, 1953). If we assume that 1 g of respiratory substrate(p = 1-000) involves the uptake of 1-0 x 103 ml O2 then weight will be lost at therate of 4-8 x 10~4 mg h"1; thus for an embryo with a density of 1 -050 g cm"3 andreduced weight of 0-0879 mg, the density will increase at a rate of 1-4 x 10~5gcm"3 h"1. This is below the limit of resolution of the density measuring techniquewe have used.

The relative change in volume of the embryo or its water content (VtIVQ) canthen be calculated from the density of the embryo at t = 0 (p0) and t (pt) and thedensity of water (pw) as follows.


From equations (1) and (2):

vo=——, (5)

Pt — Pw

Y_t _ Po ~Pw /n\v 0 Pt Pw

RESULTS

Morphological differences

Examples of the morphological differences between haploid and diploidembryos from the blastula stage to late gastrula are illustrated in Fig. 1. As willbe seen, there is very little difference between the anatomical appearance of thetwo kinds of embryo except that onset of gastrulation is delayed in the haploidsand, by the time it does begin, the haploid blastocoel is very much larger thanthat in the corresponding diploid embryo. The preparations also show that thedorsal lip of the blastopore is less tightly applied to the yolk plug and that thearchenteron contains less fluid in haploids.

Density changesIntact embryos

The results of the first series of experiments, in which the densities of embryosin a series of different age-groups were measured, showed that up to the lateblastula stage there was no significant difference between the irradiated andcontrol groups, but during the late gastrula and neurula stages the two groupsdiffered considerably. The densities of the irradiated group were more variablethan the control group and had a significantly higher mean density. However,after the control embryos had collapsed and the archenteron had emptied, thedifference between the mean densities was again insignificant.

In the second series of experiments the embryos were allowed to remain in thegradient throughout their development and the density was monitored at 15 minintervals. The results of one such experiment are is shown in Fig. 2, where thedensity changes which took place in each embryo are shown. It will be seen that,as before, the density of both haploid and diploid embryos decreased uniformlyuntil the late gastrula stage. The diploid densities then continued to decreasesteadily until the archenteron collapsed at 22 h, except for a brief transient in-crease at stage 13 when the yolk plug is withdrawn. The density of the haploidembryos on the other hand went through a series of cyclical changes of varyingamplitude which lasted from the mid-gastrula to the late neurula stage.

Results from all experiments in the second series have been pooled and themean density of all embryos of the same age has been calculated. The samples


Diploid

Be

Haploid

Fig. 1. Drawings of half embryos arranged to illustrate the difference between therate of morphological development in haploid and diploid embryos of Xenopuslaevis. Each haploid embryo in the left-hand column was the same age as thecorresponding diploid embryo in the right. Aa = Archenteron, Be = blastocoel.

were tested for homogeneity, and where the F value at the 5 % level was notsignificant the difference between the mean values of treated (androgenetichaploids) and control groups (diploids) was tested using a two tailed t test.When the variances of the samples were not homogeneous, a non-parametrictest was used.

The results of this analysis given in Table 2 confirm the results of the earlier


106

105

10410

oQ .o . 0Q/>ax>.goo<)

' Q '0"0 i A * '

12 14 16 18 20Age (h)

Fig. 2. The density of four haploid and four diploid embryos developing in a densitygradient at 25 °C, measured at 15 min intervals, plotted against age.

experiments; they also show that from 24 h to 48 h the mean density of haploidand diploid embryos does not differ significantly. The relative volume changes(Vt/V0) calculated from the mean density values in Table 2 are shown in Fig. 3.

Open embryosDensity measurements on embryos which have been operated on in such a way

that the blastocoel and archenteron are open to the environment ('openedembryos') show that at the early and mid-gastrula stage the mean density of thehaploid cell mass is significantly less than that of the corresponding diploid cellmass. The difference, however, is small and represents an increase in volume ofabout 4 % (Table 3). Measurements made at later stages suggest that this differ-ence does not persist.

Animal pole explants

Attempts to compare the volume changes of vesicles made from the roof ofthe late blastulae failed because, although they formed vesicles, they were un-stable, going through a series of cyclical density changes and finally disintegrating.

However, when stage 8 embryos were used and only the vegetal third of theblastula was removed - that is to say, most of the presumptive endoderm - thevesicles were more stable and behaved very much like normal embryos exceptthat they did not gastrulate.

The relative volume changes VtIVQ of explants of the latter type calculated


Table 2. The difference between the density of diploid and haploid embryos atdifferent ages

Diploid HaploidDifferencebetween

Age

10121416171819202122232427364648

50, 50±53

70, 701

Mean p

106631-06421060310587105421054410506104811048610444105271-05791057710517104151-044810410104131-0329

No.

20263336373632363033283313

51324201421

S.D.

00018000290004000034000590005200053000430005500049000800005000046000330003900030000310001700039

Mean p

106571-0643106081059510602106001058110576105861-058510597105981-05711053410450104611-0449104001 0297

No.

16223534323031303029282912

51217121215

S.D.

00015000330003300068000610005800065000520006700058000520005500070000160003300045000230002500058

NS = not significant. S =*

ivitauj

Odip-Phap)

+ 00006-00001-00008-00008-00059-00056-00075-00095-00100-00141-0-0070-00019+ 0-0060 (d-0-0017-00035-00013-00039+ 0-0013+ 00032

= Significant.Non-parametric test.

t

0-850-370-580-594 0 04094-938006-44

10-253-811-46= 0-25)1022-431163-731-49203

D.F.

34446668676461645860546023

82339302434

P

0-40-80-60-5

< 0-001< 0-001< 0001< 0001< 0001< 0001< 00010-1-0-2

0-300250-30-001

0-1-0-20 1

cance

NSNSNSNSSssssssNSNS*NSNSNSSNSNS

11 -

12 14 16 18 20 22 24 26Age (h)

Fig. 3. The relative volume change (Vt/V10) of haploid and diploid embryos from theage of 10 to 27 h at 25 °C calculated from the mean densities in Table 2.


Table 3

Stage

H i

24-25

Meanof open

<Haploid

1-07231069810683

densityembryos

Diploid

107521071910686

Difference,Pdip ~~ Phap

+ 00029+ 00021+ 00003

D.F.

237

11

t

3-8152-8840-272

P

< 000100250-8

MeanMean

haploiddiploid

10391029100

volumevolume

1-9

1-8

1-7

1-6

' -5

1-4

1-3

1-2

11

10

Haploidvesicles

Diploidvesicles

1 2 3 4 5 6 7 8 9 10 II 12 13 14 15

Hours

Fig. 4. Changes in the relative volume (Vt/V0) of vesicles formed by blastula explantsfrom which most of the vegetal pole material has been removed. Calculated from thedensity of each vesicle measured at 15 min intervals after it was placed in the densitygradient.

from equation (7) are plotted against time in Fig. 4. This shows that the relativevolume of haploid explants increases more rapidly and they reach their maximumsooner than the diploids.

DISCUSSION

The first thing to note about the results obtained in the present experiments isthat the density/time curves for the diploid embryos differ from those previouslypublished (Tuft, 1962, 1964). They do not show a decrease in rate of densitychange during gastrula stages and the collapse of the late neurula occurs 22 hrather than 18 h after fertilization. These differences are the result of the way inwhich the data for the density/time curves were obtained in the two series ofexperiments.


In the earlier experiments embryos were decapsulated surgically immediatelybefore their density was measured and the curves constructed from the meandensities of the different age-groups. In the present experiments, on the otherhand, the curves are based on successive measurements of the density of indivi-dual embryos after decapsulation at the late blastula stage.

Deformation of the embryo during removal of the capsule at the gastrulastage is responsible for the apparent decrease in the rate of density change in theearlier experiments because it tends to cause a loss of fluid from the newlyformed archenteron before the blastopore is tightly closed. This tends to increasethe mean density of embryos at this stage. At later stages when the blastopore istightly closed loss of fluid is less likely to occur.

In encapsulated embryos the elasticity of the capsule opposes the elongationof the notochord and long axis of the embryo at the late neurula stage. Asa result, when the embryo loses its lateral stability it jackknifes, causing a suddenand complete emptying of the archenteron cavity. In decapsulated embryos, onthe other hand, there are no external forces acting on the embryo, and under thesecircumstances the only forces tending to raise the pressure inside the archenteronare elastic forces developed in the body wall itself as the embryo elongates. Theseforces take longer to develop sufficient pressure to collapse the archenteron.

Nevertheless, monitoring the density of naked embryos after decapsulation atthe blastula stage reveal important differences in the uptake of water by haploidand diploid embryos. The density/time curves of the kind illustrated in Fig. 2?

for example, show that from the blastula until the mid-gastrula stage, densitychanges in the two types of embryo do not differ significantly, and this is con-firmed by the combined results in Table 2. The net accumulation of water musttherefore be the same in both types of embryo (see above). This does notnecessarily mean, however, that its distribution within the embryo is the same.From Table 3 it will be seen that the density of the cell mass in haploid embryostends to be slightly less than in the diploid - that is, its volume is greater and thevolume of the large intercellular spaces smaller. The morphological data, on theother hand, suggest that when gastrulation begins in the haploid (Fig. 1) theblastocoel is in fact enlarged. These two observations are not incompatible,because when haploids reach this stage the archenteron in the diploids has al-ready begun to form, and it is not possible to distinguish between density changesdue to water accumulating in the blastocoel and in the archenteron from thedensity of the intact embryo alone.

From the mid-gastrula stage onwards, however, the two types of embryobehave very differently (Table 2). Whereas the mean density of the haploidembryos remains more or less constant, the diploid density decreases continu-ously from 14 to 23 h. The density/time curves of individual embryos (Fig. 2)show that the difference between the two types of embryo is even more striking;the haploid neurulae, unlike the diploid neurulae, undergo a series of cyclicaldensity changes which are the result of successive filling and emptying of their

The haploid syndrome in Xenopus 459archenterons. After 23 h, however, there is again no significant difference be-tween mean density of the haploid and diploid embryos; that is to say, there isno difference between their volumes. This also appears to be true for subsequentstages in spite of the fact that haploid embryos look very abnormal, with largewater-filled cavities under the skin. However, the procedures used at these stagesto immobilize the embryos may alter their water content. For this reason we willonly consider the differences between haploid and diploid embryos in the pre-collapse stages.

For reasons that have been given in earlier papers (Tuft, 1962, 1964) it hasbeen suggested that water-regulating mechanisms in the cell membranes main-tain the relatively constant cell volume observed during the early stages ofdevelopment, and are so arranged that they also give rise to a net transcellularinflow of water through the animal pole and a net outflow through the vegetalpole of the blastula. The difference between the magnitudes of these two flowsresults in an accumulation of water in the blastocoel. But when, subsequently,the derivatives of the vegetal pole cells come to line the archenteron cavity, asa result of invagination, both flows are directed inwards and give rise to a veryrapid increase in the volume of the archenteron cavity (Tuft, 1962). This isillustrated diagrammatically in Fig. 5.

If this hypothesis is correct then the difference between the behaviour of thehaploid and diploid embryos could be explained in one of two ways: either theblastopore lips in the haploid neurulae are weakened in some way and are unableto withstand the normal pressures developed within the archenteron by a normalinflow of water, or alternatively the net inflow across the haploid ectoderm andendoderm is abnormally high and the blastopore lips cannot withstand theexcess pressure developed.

The behaviour of animal pole explants enables us to distinguish between thesetwo alternative explanations. It will be recalled that in these experiments mostof the vegetal (that is, endodermal) surface of the blastula was removed and theremaining portion, comprising mainly presumptive ectoderm, was allowed toround up. It will be seen from the results illustrated in Fig. 4 that the volume ofvesicles formed by haploid explants increased very much more rapidly than thevolume of similar diploid vesicles, and after reaching a maximum also decreasemore rapidly, suggesting that both the inflow and outflow are increased.

The decrease in volume is not due to bursting of the vesicle, because the rateof change is too small, but probably results from an increase in the net outflowof water due to the growth of vegetal pole material left in the explants when theywere made. This is consistent with the observation that diploid vesicles takeabout 6 h to empty whereas in intact diploid embryos, which have more vegetalpole surface, the blastocoel empties in 3 h.

These experiments demonstrate that the flow through the haploid ectoderm isgreater than that through the diploid, and it follows from what has been saidearlier that the flow through the haploid endoderm is also greater. We can con-

30 EMB 28


>'• -V

Blastula

* -V

Mid-blastula Early gastulaNeuruki

Fig. 5. Diagram to illustrate the net water flows through presumptive ectoderm andendoderm in the Xenopus embryo at different stages in its development after Tuft(Tuft, 1962). Aa = Archenteron; Be = blastocoel.

elude therefore that the successive filling and emptying of the intact haploidarchenteron is at least in part the result of an increased inflow of water.

It is interesting to note that an increased inflow through the ectoderm couldalso account for dorsal flexure and microcephaly - two other characteristics ofthe haploid syndrome. The neural tube is formed by invagination of the neur-ectoderm, and in the normal diploid embryo it is flexed ventrally at first, but aswater is removed from its lumen it straightens out. An increase in this outflowwould therefore be expected to maintain a very much reduced neural volume.

The haploid neurulae differ from the diploid in one other important respect- they have twice as many cells, and the cells are half the size of those in thediploid. Although the aggregate cell volume is the same in both haploid anddiploid embryos, doubling the cell number gives rise to an increase of 25 % inthe aggregate cell surface area in the haploids. (See Appendix.)

If the hypothesis outlined earlier is correct, an increase in cell number willtherefore increase the number of water-regulating sites responsible for the nettranscellular water flows across the ectodermal and endodermal cells.

The explant experiments lend support to this view. It will be recalled that theexplants used in these experiments, which consisted of all the presumptiveectoderm and a little of the endoderm, were made at the mid-blastula stage andwere allowed to heal before being introduced into the density gradient. That isto say the measurements began when the intact embryo would normally be atstage 9. At this stage the rate of cell multiplication in diploid embryos decreasesrapidly until it reaches the relatively low rate characteristic of later stages indevelopment. In haploid embryos on the other hand the high rate of divisioncharacteristic of the earlier stages is maintained for about an hour until the cellnumber has been doubled, after which the rate becomes the same as the diploid.

The aggregate cell surface in the haploid explants will therefore tend toincrease very much more rapidly than it does in the diploid, and, if our hypo-thesis is correct, the explants should reach their maximum rate of volume changesooner than the diploids, which, as we have seen, is exactly what the experimentalresults show (Fig. 4).

The haploid syndrome in Xenopus 461We may therefore conclude from these experiments that the abnormal develop-

ment of the haploid embryo, like that of the tadpole described by Fox &Hamilton (1964), is the result of an excessive inflow of water. Furthermore if, ashas been suggested, the uptake of water by the embryo involves water-regulatingsites in the cell membranes, the excess flow into the haploid archenteron can beattributed to the increase in total cell membrane area which is a consequence ofdoubling the number and halving the size of the cells at the late blastula stage.

Changes in the relative rate of water flow through the presumptive ectodermand endoderm which are thought to account for the accumulation of water inthe blastocoel and archenteron of the normal diploid embryo may arise ina similar way. Thus during the formation of the blastocoel the rate of celldivision in the presumptive ectoderm is very much greater than it is in theendoderm, whereas the reverse is true during gastrulation and formation of thearchenteron. If this is so the simple model put forward to account for the uptakeand distribution of water by the Xenopus embryo in earlier papers (Tuft, 1962,1964) will have to be modified and detailed information obtained about theultrastructure and dimensions of the cells in the two layers.

APPENDIX I

Effect of cell number on total area of cell membrane

The aggregate cell surface area A in an embryo consisting of nx cells andhaving a total cell volume V can be calculated as follows: let vl5 rx, ax be thevolume, radius, and area respectively of the individual cells. Then assuming thecells are spherical,

vi = I = W , (1)

/ 1V \ -2-1 - 4^)*. (3)

Then the aggregate surface areaAx = nxax (4)

and from (3) and (4)Ax = /ii(47r)4(3K)*. (5)

Similarly, for an embryo with the same volume V but with «2 cells,

A% = 4(4ar)*(3F)f • (6)

The ratio of the aggregate cell surface area in the two embryos from (5) and (6)is then

A2 \n2)(7)


For diploid and haploid embryos where Fhap = Fdip and «hap = 2«dlp,

A'dip

= 2 * . (8)•A,

This work was carried out in the Department of Zoology, University of Edinburgh. Wewould like to thank Professor Mitchison for the facilities provided, Mr Holmes who did theillustrations, and Mrs Ann Muir for technical assistance. One of us, P. H. Tuft, also wishesto thank the Distillers Company for the grant in aid.

REFERENCESFox, H. & HAMILTON, L. (1964). Pronephric system in haploid and diploid larvae of Xenopus

laevis. Experientia 20, 289.GURDON, J. B. (1960). The effects of ultraviolet irradiation on uncleaved eggs of Xenopus

laevis. Q. Jl Microsc. Sci. 101, 299-311.LINDERSTROM-LANG, K. & LANZ, H. (1938). Studies on enzymatic histochemistry. XXIX.

Dilatometric micro-estimation of peptidase activity. C. r. Trav. Lab. Carlsberg, (Chim.) 21,315-338.

L0VTROP, S. (1953). Energy sources of amphibian embryogenesis. C. r. Trav. Lab. Carlsberg28, no. 14, 372-396.

NIEUWKOOP, P. D. & FABER, J. (ed.) (1956). Normal Table of Xenopus laevis (Daudiri).Amsterdam: North Holland Publishing Co.

TUFT, P. H. (1953). Energy changes in development. Archs. neerl. Zool. 10, (Suppl 1), 59-75.TUFT, P. H. (1962). The uptake and distribution of water in the embryo of Xenopus laevis.

J. exp. Biol. 39, 1-19.TUFT, P. H. (1964). The uptake and distribution of water in the developing amphibian embryo.

Symp. Soc. exp. Biol. no. xix, pp. 385-402. Cambridge University Press.

{Manuscript received 20 March 1972)

the role of water-regulating mechanisms in the developmen ot f … · embryo/, exp. morph. vol. 28,...

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