Cell D, ifferentiation, 7 (1978) 185--192 © Elsevier/North-Holland Scientific Publishers Ltd.

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L A B I L I Z A T I O N OF T H E S U P E R F I C I A L L A Y E R AND R E D U C T I O N IN SIZE OF Y O L K P L A T E L E T S D U R I N G E A R L Y D E V E L O P M E N T O F XENOPUS L A E V I S

Nancy ROBERTSON*

Department of Molecular Biology and Virus Laboratory, University of California, Berkeley, California 94720, U.S.A.

Accepted 21 February, 1978

Yolk platelets from blastulae and gastrulae of Xenopus laevis were isolated by a standardized procedure and examined by electron microscopy for evidence of stage- dependent changes of structure. Compared to those from blastulae, gastrula platelets frequently lost the superficial layer during isolation, indicating its increased lability. In addition, platelet dimensions were compared for different stages in situ by light micro- scopy, and gastrula platelets near the animal pole were found to be 10--20% smaller along both elliptical axes relative to blastula platelets. It is suggested these alterations represent early steps in the preparation of yolk platelets for breakdown.

Karasaki (1963) investigated the u l t ras t ruc ture o f yo lk platelets (YP) in situ in the ventral e c tode rm region o f Rana pipiens and Triturus pyrrhogaster at ear ly deve lopmen ta l stages. The earliest change o f p la te le t s t ruc tu re involved the d isappearance o f the superficial layer (SL), w i t h o u t removal o f the uni t m e m b r a n e enveloping the platelet , and occur red at the late blastu','~ stage in Rana and at the mid-gastrula stage in Triturus. The removal o f tl~ SL is cons idered the first step of p la te le t b r eakdown , eventua l ly leading to des t ruc t ion o f the crystal l ine main body . Selman and Pawsey (1965) m ad e a comprehens ive survey by light m ic ro scopy and d e t ec t ed SL removal at post-gastrula stages of Xenopus laevis at t imes depending on the e m b r y o n i c tissue. Again, the loss o f SL was fo l lowed by large scale des t ruc t ion o f the main body .

In the presen t s tudy, we have endeavored to de t ec t changes in Xenopus yo lk platelets before SL removal is evident . First, we r e p o r t the d i f ferent ia l s tabil i ty of blastula and gastrula platelets t oward a s tandard ized ex t r ac t ion and pur i f ica t ion p rocedure , and second, we de t ec t a small (10--20%) size r educ t ion in situ in platelets f rom e m b r y o s enter ing the gastrula stage. These changes are suggested to occu r p r epa ra to ry to SL removal .

* Current address: Jules Stein Eye Institute, The Center for Health Sciences, University of California at Los Angeles, Los Angeles, California 90024. Abbreviations: AH, animal hemisphere; bp, blastopore; eq, equator; PVP, polyvinyl- pyrrolidone-10 (tool. wt., 10,000); SL, superficial layer; YP, yolk platelet.

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MATERIALS AND METHODS

1. Embryos

Mature X. laevis were obtained from the South African Snake Farm, Fish Hoek, South Africa, and maintained in fresh tap water at 21°C with biweekly feedings of beef heart. To obtain embryos, a commercial preparation of human chorionic gonadotropin, Antuitrin S (Parke-Davis) was injected into the dorsal lymph sac at a priming dose of 75 and 50 units for females and males, respectively, followed 48 h later by a booster dose of 300 and 100 units respectively. Embryos were dejellied in 1.2% cysteine--HC1, adjusted to pH 7.8 with NaOH.

Embryos of stages 7, 8 and 9 (5--7 h) were chosen as blastulae, and those of stages 10, 11 and 12 (8, 10.5 and 13 h) as gastrulae (Nieuwkoop et al., 1967).

2. YP isolation for SL studies

The original method of Wallace and Karasaki (1963) was modified only in so far as sucrose was replaced by glycerol in all steps of the procedure.

Preparations were done at 4°C. Approximately 100 dejellied embryos were homogenized manually in 0.25 M glycerol with 5% polyvinylpyrrolidone-10 (PVP, mol. wt. = 10,000, Sigma Chemical Co.). The embryo suspension was made to 20 ml with the glycerol-PVP, then layered onto 50 ml of 50% glycerol, also with 5% PVP. The gradient was centrifuged 15 min in an International Clinical tabletop centrifuge at 500 g. After resuspension of the soft pellet in the dilute glycerol, the centrifugation was repeated in some cases to be noted later, using 20% glycerol with 5% PVP as the bo t tom layer. YP pellets were washed free of glycerol-PVP by suspending them in 0.5 ml H20, and repelleting them at 5000 g. This was repeated 4 t imes .

One mm 3 samples of YP pellets were fixed overnight in 2.5% glutaralde- hyde in 0.1 M sodium cacodylate buffer, pH 7.2, postfixed in 1% OsO4 in the same buffer, dehydrated in graded alcohols and propylene oxide, and embedded in Epon. Sections of 900 nm thickness representing 4 non- overlapping samples of each YP batch were collected onto carbon-coated formvar grids and were poststained in uranyl acetate followed by Reynold 's lead citrate, for examination in a Siemens IA electron microscope.

Some blastula-gastrula SL comparisons are of embryos of the same egg clutch, and others are of embryos from unrelated clutches as noted later. In all instances, specific blastula-gastrula comparisons are made on YP isolated with identical solutions and protocols. For each YP batch, a minimum of 12 negatives of non-overlapping fields was taken at a magnification of 2000 or 4000 times. SL was identified as the crescent-shaped cap adhering to part of the YP surface (see Fig. 1). The proport ion of YP with SL was determined for each batch of YP.

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Fig. 1. Isolated yolk platelets used for study of superficial layer lability. A: Blastula, stage 8. Arrow points to superficial layer. B: Gastrula, stage 10. Most platelets lack superficial layer, as shown here. Both pictures, × 5500.

3. Fixation and examination of embryos for in situ YP size measurements

a. Electron microscopy. Healthy-looking embryos from stages 8 and 10 were manually dejellied for fixation and embedment in the same manner as for isolated YP. The embryos were sectioned (500 or 900 nm thickness) near the animal and vegetal poles of both stages. Mid-saggital sections were taken from the equator of blastulae and the blastopore of the gastrulae. Electron microscope negatives were taken at × 4000 and printed at × 12000. Since YP are almost entirely elliptical in cross-section, measurements were taken of both axes.

b. Light microscopy. Embryos of stages 8 and 10 were fixed in 2.5% glutaraldehyde buffered with 0.1 N sodium cacodylate, pH 7.2, and dehyd- rated in graded ~acetones (30, 50, 70 and 95%) for 1 h each and in 2 changes of absolute acetone for 1.5 h each. Two subsequent 1 h immersions in xylene were then followed by embedment in Paraplast. Seven pm serial sections

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were cut from the embryos and were stained in 0.033% toluidine blue in 30% ethanol in 0.1 M sodium citrate buffer, pH 5.0 (Ohno et al., 1964). The sections were then photographed under a Zeiss compound microscope onto Kodak Ektachrome color 35 mm film. The slides were projected, and the major axes of in-focus YP were measured. YP from areas comparable to those in the electron microscope study were selected along the midsaggital plane.

R E S U L T S

1. The superficial layer

YP were extracted and purified by the standardized procedure and examined in the electron microscope. A total of 1039 YP from blastulae and 794 YP from gastrulae were surveyed. Fig. 1 shows typical examples of YP from both stages. YP from gastrulae more frequently lacked adhering and intact SL crescents or even fragments thereof.

Table I shows that for each of 3 variations of the glycerol isolation pro- cedure, blastula YP at all tested stages display a higher percentage of YP with SL than do gastrula YP. The reduction between blastula and gastrula stages for each of the 3 procedures is 30--40%. The fact that the overall percentages of YP displaying SL is smallest in the third procedure may be at tr ibutable to the use of a different lot of PVP. Wallace and Karasaki (1963) have noted the importance of PVP for SL stability. Various authors have described the presence of SL on platelets in vivo at these stages, so we conclude the SL of

T A B L E I

S t age -dependen t l ab i l i za t ion of the superficial layer of yo lk platelets .

E x p e r i m e n t Stage I so la t ion N u m b e r of %YP No. a p r o c e d u r e b YP wi th

SL

1 9 Blastula 234 69 .8 2 7--8 Blastula 1 196 69 .3 3 10 Gast rula 311 27.0

2 7--8 Blastula 238 61.3 2 (dup l ica te ) 7--8 Blastula 74 68 .9 2 10- -11 Gas t ru la 2 127 31.5 3 11 - -12 Gas t ru la 120 28.3

4 8 Blastula 143 37.1 4 (dup l ica te ) 8 Blastula 154 32 .5 4 10--11 Gas t ru la 3 93 5.5 4 11 - -12 Gas t ru la 143 1.4

a A d i f f e ren t e x p e r i m e n t n u m b e r indica tes a d i f f e ren t frog used as a source of eggs. b There were 3 i so la t ion p rocedures : 1) 0 .25 M glycerol on 50% glycerol, 1 cen t r i fuga t ion ; 2) 0 .25 M glycerol on 50% glycerol, fo l lowed by 0 .25 M glycerol o n 20% glycerol; 3) Same as 2, b u t a d i f f e ren t b a t c h of PVP. All glycerol so lu t ions c o n t a i n e d 5% PVP.

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T A B L E II

Size of yo lk p la te le t s d e t e r m i n e d f r o m l ight mic rographs (× 2720 or X 2700) f rom each m a j o r level of e m b r y o s of the b las tu la and gastrula stage. All values are given in urn.

Stage o f Level N b 2fi c %S.D. 2b c %S.D. e m b r y o e x a m i n e d a

8 VH 120 9 .92 ± 1.54 16.2 7 .52 ± 1 .30 17.2 Blas tula eq. 90 8.84 ± 1 .80 20.5 6 .78 ± 1.28 18.8

AH 83 4 .18 ± 1.78 42.3 3 .20 -4 1.04 32.8

10 VH 120 9:80 -+ 2 .20 22.5 7 .16 ± 1.78 24.7 Gas t ru la eq. 87 7.54 ± 1 .64 21.9 5 .40 ± 1.24 22.9

AH 58 3 .20 -+ 1.04 32.8 2 .80 ± 0.74 26.4

a VH indica tes vegetal hem i s phe r e ; eq. ind ica tes e q u a t o r ; and AH, an imal hemisphere . b N ind ica tes n u m b e r of p la te le t s measured . c 2~ ind ica tes the m e a n leng th of the longer axis of the ell iptical p la te le t , w i th i ts s t anda rd devia t ion . 2b ind ica tes t he m e a n length o f the s h o r t e r axis, w i th its s t anda rd devia t ion .

gastrula stage platelets is more labile in the conditions of the isolation procedure.

2. YP size changes

a. Light microscope observations Table II gives the mean measurements of YP in 7 pm sections. YP from all 3 levels in gastrula stages were slightly smaller than those from the corresponding level in the blastula stage. The range in major axis length was 4.18 + 1.78 pm in the animal hemisphere to 9.92 + 1.54 in the vegetal hemisphere for blastulae and 3.20 -+ 1.04/~m to 9.80 + 2.20 pm in gastrulae. The percent standard deviation was large, 16--42%. Much of this deviation is attributable to the fact that platelets have

T A B L E III

Signif icance tes t ( t - tes t ) of d i f fe rences b e t w e e n m e a n lengths of the ma jo r and m i n o r axes of ell iptical cross-sect ions o f YP f rom the b las tu la and gastrula stages, as d e t e r m i n e d f rom l ight micrographs . Data are t a k e n f rom Table II. The fo rmu la is given in tex t . I f t >/ 1.96 the d i f fe rence in m e a n axis length i f s igni f icant a t t he 0 .05 level as far as i ts d i f fe rence b e t w e e n the blas tula and gastrula stages, a t equ iva len t pos i t ions in t he embryo . S igni f ican t d i f fe rences are unde r l ined

Level in the e m b r y o t

2a 2b

AH .4:.08 2 .92 E q u a t o r 5 .00 7.26 VH 0.50 1 .80

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the shape of ell ipsoids o f r evo lu t ion and are r a n d o m l y o r i en ted in the sect ions, so t h a t a 2 d imens iona l mic rog raph ic p ro j ec t ion o f the axes rare ly reveals the t rue ma jo r and m i n o r axes. In addi t ion , the 7 g m sect ions will cu t m a n y pla te le ts in to par t s and genera te cross-sect ions wi th axes smaller than the t rue m a j o r and m i n o r axes of the in tac t p la te le t .

Table I I I gives the resul ts o f a s ignif icance test , the t - test , used to evalua te the observed reduc t ions in p la te le t size. The a p p r o p r i a t e equa t ion (Arkin e t al., 1970) is:

Zl - X2 t =

+ - - N1 N2

where X1 = m e a n length of el l ipt ical YP axis, 2a or 2b, b las tu la X2 = same, b u t fo r gastrula, N~ = n u m b e r of b las tu la YP measured , N2 = n u m b e r of gastrula YP measu red , o ~ = s t andard dev ia t ion of YP length, blastula , a2 = same, b u t for gastrula.

Since N is a lways greater than 30, the value o f t fo r the 95% conf idence level m u s t be > 1.96.

b. Electron microscope observations Table IV gives the m e a n measure - m e n t s o f YP in 900 p m sect ions. The d i a m e t e r o f the m a j o r axis, 2a, o f YP ranges f r o m 2.78 -+ 1.04 p m in the an imal hemisphe re to 8 .88 -+ 3 .38 u m in the vegetal hemisphe re fo r stage 8 and 4 .42 -+ 2 .52 p m - - 7 . 9 6 -+ 2 .98 p m for s tage 10. The pe rcen tage s t andard devia t ion fo r all values is large, 30- -60%.

In the th in sec t ions fo r e l ec t ron m i c r o s c o p y , we rare ly ob ta in a sec t ion t h rough the t rue m a j o r and m i n o r axis o f the p la te le t . Ins tead , a great var ie ty of p la te le t cross-sect ions are genera ted . R o u g h ca lcula t ion shows t h a t

TABLE IV

Size of yolk platelets in electron micrographs, × 12,000, from each major level of embryos of the blastula and gastrula stages. Sections were 500 nm in thickness. All values are given in urn. Abbreviations are defined in the footnotes of Table II.

Stage of Level N 2a %S.D. 2b %S.D. embryo examined

8 VH 49 8.88 +- 3.38 38.2 6.84 -+ 2.76 40.4 Blastula eq. 59 7.84 +- 2.76 30.1 5.92 +- 1.92 32.3

AH 128 3.05 ± 1.12 36.6 2.54 ± 0.92 36.1

10 VH 58 7.96 ± 2.98 37.6 5.56 +- 2.20 39.5 Gastrula bp. 53 7.54 ± 2.48 32.9 5.20 ± 1.86 35.7

AH 42 4.44 ± 2.52 56.6 3.40 +- 1.96 54.8

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a thin-sectioned ellipsoid will give rise to a set of cross sections, the mean dimensions of which are 78% the true major and minor axes of the ellipsoid, and a standard deviation of 30% on that mean. Thus, we expect a large variation inherent in the thin-sectioned material. The variation is so great that all the figures but one fail the t-test. Therefore, we present Table IV only as illustrative of the problem. It is interesting that the mean axis lengths for the thin-sectioned platelets are generally smaller by 10--20% compared to the mean lengths determined from light micrographs. This difference is consistent with the problem mentioned above, namely, thin-sectioning generates a greater variety of small cross-sections.

DISCUSSION

1. SL breakdown

From these studies, the first sign of YP alteration during early development appears to be labilization of the SL in a way such that it detaches during isolation in glycerol gradients. This change occurs in the blastula to gastrula transition. Labilization may portend actual SL breakdown of the sort observed in Rana and Triturus. Selman and Pawsey (1965) equated the SL with the fastgreen staining borders of Xenopus platelets and observed that these borders are lost by stage 25 in myotome, notochord and ectodermal tissues, and by stage 45 in endoderm derivatives. These consti tute the earliest observable changes in YP at the light microscope level, a result which implies that SL removal in Xenopus may commence at a later stage than in Rana or Triturus. Clearly, more data on YP demolition in situ from stages 8 through 20 are required in order to determine when SL breakdown can first be detected at the electron microscope level. It remains, however, that SL labilization beings at gastrula, perhaps preparatory to SL removal.

Yolk platelets are complex organelles containing not only the proteins of the SL and crystalline main body, but also neutral and amino sugars (Robertson, in preparation) and lipids. These different components may be withdrawn from the platelets at different periods in development, with SL and main body disintegration being rather late and drastic steps in platelet catabolism.

2. YP size changes

In these studies there were no detectable size changes occurring in vegetal YP during the blastula to gastrula transition. On the other hand, animal hemisphere YP do exhibit a size decrement of roughly 10--20% during this period. YP from the polar region of the animal hemisphere were chosen because these cells exhibit virtually no migration after stage 8.25 (Nakatsuji, 1975) and therefore redistribution of platelets will be minimized for the comparison.

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Blastula equatorial YP and blastoporal YP also show statistically signifi- cant size differences. However, cell migrations at the embryo surface in the blastoporal region during these stages have been well-documented (Baker, 1965; Keller, 1977). T h e extent to which these YP size differences reflect migration only, instead of being indicative of real YP size reduction, is a moo t question at this time. If size reduction does occur in the equatorial region, then certain studies by others on internal cell movements at gastrula- tion will have to be reassessed. For example, Nakatsuji (1975) classifies cells of Xenopus embryos on the basis of the size the largest YP present within 1 pm sections of a cell, and reports that the equator is occupied by 'A', 'B' and 'C' cells at different times from stages 8 through 10. There are several difficulties with using the largest YP as the basis for cell classification. First, from the animal pole data, there is a possibility that equatorial YP might be undergoing size reduction. Second, upon division, one daughter cell may retain the single largest YP which conferred the original class assignment, while the other cell would be reassigned to the next lower size class. Even without cell movement, the assignment of cell class would be expected to change merely due to cell division and concomitant YP apportionment. Studies of cell movement would be more secure in the use of some other indicator besides YP size.

ACKNOWLEDGEMENTS

This work was done in partial fulfillment of the requirements of the Ph.D. degree. I want to thank Drs. Robley Williams and John Gerhart for use of their facilities and for very helpful discussion of the work. Supported by NIH grants AI-10845 (RCW) and GM 19363 (JCG) and NSG grant PCM-76- 01549 (RCW).

REFERENCES

Arkin, H. and R. Colton: In: Statistical Methods, Chapter XIV 5th edn. (Barnes and Noble Books, New York) pp. 141--162 (1970).

Baker, P.C.: J. Cell Biol., 24, 95--116 (1965). Karasaki, S. : J . Ultrastruct. R es. 9, 225--247 (1963). Keller, R. and G.C. Schoenwolf: Wilhelm Roux'Arch. , 182 , 165--186 (1977). Nakatsuji, N.: Wilhelm Roux ' Arch. 178, 1--14 (1975). Ohno, S., S. Karasaki and K. Takata: Exp. Cell Res. 33 ,310--318 (1964). Selman, G. and G. Pawsey : J. Embryol. Exp. Morphol. 14, 191--212 (1965). Wallace, R. and S. Karasaki: J. Cell Biol. 18, 153--166 (1963).