Changes in the nuclear lamina composition during early development of Xenopus laevis

Cell, Vol. 41, 191-200,

May 1985, Copyright

0092-86741851050191-10

0 1985 by MIT

Changes in the Nuclear Lamina Composition Early Development of Xenopus laevis Reimer

Stick and Peter Hausen

Max-Planck-lnstitut ftir Entwicklungsbiologie Abteilung ftir Zellbiologie SpemannstraOe 35 D-7400 Tiibingen Federal Republic of Germany

Summary Changes in protein composition of the nuclear lamina were monitored during early development in Xenopus. Lamin LIII, the only lamin present in oocyte nuclei, serves as a lamin pool for the formation of pronuclei and early cleavage nuclei. It is present in embryos up to the tail bud stages. Lamins L, and L,,, the lamins originally found in adult cell nuclei, appear at characteristic times in development. L, first appears at the midblastula transition (MBT), and L,, at the gastrula. Tryptic peptide analysis revealed that all three lamin forms found in the embryo are identical with the adult lamins. De novo synthesis of Llll and L,, observed at MBT, is independent of transcription and must therefore be due to activation of maternal mRNAs. These results are discussed in relation to other nuclear changes occurring during early development. Introduction The nuclear lamina has been demonstrated in interphase nuclei of somatic cells from a variety of organisms and tissues and may be a universal feature of eukaryotic cells (Fawcett, 1981). The rather rigid skeletal structure is composed of only a few major polypeptides, which form a family of related proteins (Gerace et al., 1978; Stick and Hausen, 1980; Krohne et al., 1981). It is localized at the periphery of the nucleus between the inner nuclear membrane and the peripheral chromatin. In interphase nuclei, the DNA is tightly bound to the nuclear lamina, and there is evidence that the lamina is involved in the organization of the DNA in loop domains (Lebkowsky and Laemmli, 1982; Hancock and Hughes, 1982) and in DNA replication (Vogelstein et al., 1980). Other possible functions include spatial organization of the pore complexes and maintenance of nuclear shape. Since the significance of the nuclear lamina for the functional organization of the nucleus is not yet known, we decided to investigate the behavior of the lamina during cellular events that are accompanied by extensive alterations of nuclear morphology and metabolism. Gametogenesis and early development in amphibians are uniquely suited for such studies. The nuclear changes during these processes are prominent and are well characterized. The lamina disappears in meiotic prophase concomitant with the condensation of the chromosomes and the detachment of the chromatin from the nuclear membrane.

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In contrast to maturing sperm, growing oocytes regain the lamina at diplotene (Stick and Schwarz, 1982, 1983), but with its polypeptide composition altered. While the two major lamin components L, and LII (the amphibian homologs to lamin A and B in other vertebrates) exist in nuclei of somatic cells, the lamina of the oocyte nucleus is formed by a single polypeptide (Krohne et al., 1981), which will be called Llll throughout this paper. This difference in lamina composition may be due to the extraordinary features of the germinal vesicle. Three hundred thousand times larger than a normal somatic nucleus, it harbors only the tetraploid chromosome complement. The chromatin is organized in lampbrush chromosomes and is very active in transcription, but unlike the chromatin in somatic nuclei, it is not in contact with the nuclear envelope. The present study extends our previous analysis of gametogenesis (Stick and Schwarz, 1982, 1983) into early embryogenesis. This phase also displays some extensive changes in the functional organization of the nuclei. After fertilization, the compact sperm head undergoes the transition into the male pronucleus. The germinal vesicle has disappeared, and its chromatin becomes concentrated in the female pronucleus. Transcription has stopped and does not appear to be resumed during the cleavage phase that follows. During cleavage, the cell cycle is reduced to an M- and a very short S-period, lacking a Gl and a G2 phase. Nuclear components, which furnish the cleavage nuclei, are provided by the egg or are translated from maternal mRNA. When 2000-4000 nuclei have been formed in the embryo, the cell cycle gradually increases in length, transcription is resumed, and the cells become motile. These changes mark the onset of the midblastula transition (MBT) (Newport and Kirschner, 1982; Broterenbrood et al., 1983), after which the nuclei gradually develop the characteristics of normal somatic nuclei. This study aims to assess the behavior and structure of the nuclear lamina during these phases to find out whether changes in nuclear activity and organization are mirrored in structural alterations of this major skeletal element of the nuclei. Some clues concerning the function of the lamina may then be revealed.

Results Two different antibody preparations were used to detect Xenopus lamins, an anti-chicken nuclear lamina serum produced in rabbits (anti-l serum, Stick and Hausen, 1980), which cross-reacts with the Xenopus lamins, and a monoclonal antibody (L6-8A7) directed against Xenopus lamins. The two antibodies show the same specificity. In immunoblotting experiments, both recognize all three major Xenopus lamins (L,, 65 kd; L,,, 60 kd; and L,,,, 60 kd). (It should be noted here that the determination of the molecular weight of the lamins may vary greatly, depending on the gel system used.) These results are supported by immunocytochemical findings, which show that both

Cell 192

Figure

1. A Nuclear

Lamina

Is Present

in Female

and Male Pronuclei

Polyester wax sections (6 pm) of Xenopus eggs fixed 30 min after fertilization were stained with anti-lamina serum or preimmune serum and were counterstained with DAPI. (a) animal part of the egg showing the female (Q) and the male (a) pronucleus at a lower magnification; this photograph was taken using a combination of DAPI fluorescence and bright field illumination to demonstrate the two pronuclei as well as the sperm track (SPT) pointing towards the male pronucleus. (c and d) anti-lamina antibody staining of the male and female pronucleus. (b) a similar specimen treated with preimmune serum.

antibodies decorate somatic cell nuclei and the germinal vesicles of oocytes in a way characteristic for nuclear lamina staining (Stick and Krohne, 1982; results for the monoclonal antibody L6-8A7 not shown). Formation of a Nuclear Lamina during Pronuclear Development After fertilization, the female pronucleus is formed by completion of meiosis, whereas the male pronucleus originates from the sperm head. Since the highly compacted sperm nuclei lack a nuclear lamina (Stick and Schwarz, 1982), the question arises whether a lamina is reformed from egg material around the decondensing sperm chromatin in the course of pronucleus formation. This was investigated in serial sections of embryos fixed at different times following fertilization. Male pronuclei can be identified by the sperm track, an array of pigment granules drawn into the egg cytoplasm by the sperm nucleus during its centripetal movement (Figure la). A peripheral lamina can be visualized with lamin-specific antisera in male (Figure lc) and in female (Figure time

Id) formation

pronuclei

30

of a nuclear

min

after lamina

fertilization, structure

at which in pronuclei

seems to be completed. The negative reaction of the preimmunoserum (Figure lb) demonstrates the specificity of the immunostaining.

Large amounts of male pronuclei can be produced in vitro by incubating demembranated sperm nuclei in an extract of activated egg cytoplasm. The in vitro pronucleus formation resembles the in vivo process in several criteria, such as decondensation of the sperm chromatin, formation of the nuclear membranes, and DNA replication (Lohka and Masui, 1983). To test whether this also holds true for the lamina formation, pronuclei formed in vitro were isolated from the incubation mixture by centrifugation, and their lamina proteins were separated on SDS polyacrylamide gels. lmmunoblotting analysis reveals that there is only one lamin present in the preparation (Figure 2, lanes c, e, and g), which comigrates in one-dimensional gel electrophoresis with the L,,, polypeptide of the oocyte nucleus (Figure 2, lane f). The lamina of the pronuclei is clearly different from L, and is also slightly, but reproducibly, distinguished from LII in this assay (Figure 2, lanes d and h). Our experience with 2D gel analyses and tryptic peptide patterns, to be described below, led us to the conclusion that this slight difference is real, and that Llll is in fact the only lamin found in the pronuclei. In vitro formation of a sedimentable lamina structure resistant to detergent and high salt only occurs when sperm nuclei are present in the egg extracts (results not shown). The sperm nuclei prior to incubation show no reaction with lamin-specific antisera in immunoblotting experiments (Figure 2, lane b).

Nuclear 193

Lamina

in Early

Development

a M

b SP

c PN

experiments were probed with monoclonal antibody L68A7 in combination with 1251-labeled secondary antibody. A quantitative comparison of the lamins at different developmental stages is complicated by the rapid change of nuclear numbers and nuclear sizes in the embryos. Sample sizes were therefore arranged in such a way as to contain equal amounts of lamin polypeptides, thus ensuring an accurate estimation of the ratios of the different lamins. As in pronuclei, only one lamina polypeptide is found in early cleavage nuclei up to the 10th or 11th division (Figure 3, lanes b-d, and Figure 4a). By comparing the positions of its isoelectric variants (Figures 5c and 5d) in two-dimensional gel separations, relative to an internal standard, we identified this component as lamin LI,, (see below). Polypeptide L, is first detected in nuclei after the 12th division (Figure 3, lanes e and f; Figure 4b), which corresponds to the onset of midblastula transition (Newport and Kirschner, 1982; Broterenbrood et al., 1983). A more quantitative estimate of the increase of LI relative to Llll was obtained by excising individual spots from the nitrocellulose blot filters and counting Y (Figure 3). The time course of the L, increase resembles that of the other cellular changes observed at the midblastula stages.

d ERY

946860-

0

e PN

f GV

9 PN

--IDFigure 2. Lamin Formed In Vitro

Ilr -L, aD-L,,

-

Llll Is the

Only

Lamin

h ERY -

-L,

-

-b

Constituent

in Pronuclei

Two separate experiments are shown. In the first experiment (lanes a-d), lamina from in vitro formed pronuclei was compared with erythrocyte lamina and sperm. Polyacrylamide gels were loaded with (b) approximately 10’ sperm nuclei predigested with DNAase I, (c) lamina from approximately 10’ pronuclei. (d) erythrocyte lamina preparation. (a) molecular size marker. In the second experiment (lanes e-h), lamina from pronuclei was compared with erythrocyte and germinal vesicle lamina. (e) lamina from lo6 and (g) from 10’ pronuclei. (f) lamina from 20 germinal vesicles, and (h) erythrocyte lamma. After electrophoresis, lamin polypeptides were detected by immunoblotting using anti-lamin serum in combination with the PAP technique (see Experimental Procedures).

Lamin L, Is First Detected at the Time of Midblastula Transition (MBT) Polypeptides LI and LII are lamina constituents of somatic nuclei in adult tissue (Krohne et al., 1981), but they are absent from pronuclei. This raises the question as to when these polypeptides appear during development. The cleavage period is characterized in Xenopus by a series of 11-12 rapid metachronous cell divisions. For the isolation of a sufficient number of interphase nuclei from defined cleavage stages, we found it necessary to block the embryonic cell cycle in interphase by inhibiting protein synthesis (Miake-Lye et al., 1983). This procedure allows the collection of nuclei from embryos differing by one cleavage cycle only. Nuclei were isolated from cell-cyclearrested embryos, and lamina fractions were prepared by standard procedures. Lamina composition was monitored by immunoblotting methods after separation on one- or two-dimensional gels. Because of the high sensitivity and the advantage of quantitative evaluation, the blots in these

L, and LII Appear at Different Developmental Stages From the results shown in Figures 4 and 5, it is evident that lamins LI and L,, do not appear at the same time. LI, is immunologically detected for the first time at midgastrula, stage 13 (Figure 4e), and its concentration increases during neurulation (Figure 4f). L, has already reached a high level relative to Llll (Figure 4e cf. Figure 4b) when LII first appears. Therefore, from midgastrula to at least the tail bud stages (about stage 29-30) all three lamins are present in embryonic nuclei, although there is a significant decrease in Llll relative to L, and L,, from late neurula stages onward (cf. Figures 5c and 5d). In the preceding experiments, increases and decreases of lamins have been detected by immunological procedures only. Stage-specific modifications of lamins, however, may alter their binding to antibodies. Such changes might be misinterpreted as changes in the amount of lamin protein present. To rule out such a possibility, crude nuclear lamina preparations from different developmental stages were separated on two-dimensional gels, which were then stained with Coomassie blue. This staining allows an estimation of the amount of lamins relative to reference spots. Although the polypeptide patterns of such crude preparations are complex and change during development, the identity of lamin polypeptide spots was confirmed unambiguously by peptide map analysis (for details see next paragraph). Analysis of the Coomassie blue staining intensity reveals a clear increase in amount of L, from stage 8 (Figure 5a) to stage 27 (Figure 5b), as compared with reference spots (legend). The same is true for L,,, which is not detectable at all at stage 8 (Figure 5a). We therefore conclude that the increase in antibody reaction of L, and L,, observed in the previous immunoblotting experiments reflects the increase in the amount of lamin protein, rather than an altered reactivity to the antibodies.

Cell 194

a GV

bcdefg 9th 10th

11th

12th

13th

ERY

h GV

ij ST18

Figure Nuclei

ERY

3. Lamin L, Appears after the 12th Cleavage

in Embryonic

Nuclear lamina preparations of embryos after the 9th and up to the 13th cleavage cycle (lanes b-f) were separated, together with lamina preparations of germinal vesicle (lanes a and h). erythrocytes (lanes g and j), and stage 16 embryos (lane i), on SDS polyacrylamide gels. Lamins were detected by immunoblotting, using the monoclonal antibody L6-6A7 and Yrabbit-anti-(mouse Fab)-IgG. Different numbers of embryos were used for the preparations, 150 in (b), 125 in (c), 100 in (d), 75 in (e), 63 in (f), and 30 in (i), in order to load approximately equal amounts of lamin Lrrr. Arrows in (e) and (f) denote the position of lamin Lr. The graph shows the ratio of radioactivity in L, versus L,,, counted after immunoblotting of lamina preparations from different cleavage cycles separated on two-dimensional gels (see Figure 4a-4c).

i -7x--CYCLE

Peptide Map Analysis Reveals Identity of Embryonic and Adult Lamins In the preceding analysis, lamin polypeptides were identified in immunoblotting experiments on the basis of their separation characteristics in one- and two-dimensional gel electrophoresis, taking for granted that these characteristics are unaltered in early development. This assumption, however, does not necessarily need to be valid. Indeed, slight changes in isoelectric points and the number of isoelectric variants in lamins Llll and LII are observed when lamins of different embryonic stages (Figure 4 and Figures 5c and 5d) or lamins from adult tissue and embryonic lamins are compared (not shown, but see Krohne et al., 1981; Figures 6b and se). We therefore used peptide map analysis to compare the tryptic peptide maps of embryonic lamins with those of erythrocyte (L, and Lrr) and germinal vesicle (Lr,,) lamins. Lamin spots indicated with arrows in Figures 5a and 5b were analyzed according to Elder et al., (1977). Representative examples of this analysis are presented in Figure 6. While LIII, L,, and LII can clearly be distinguished by their peptide maps (cf. Figures 6a, 6c and se), the maps of the germinal vesicle lamin Lrrr (Figure 6a), of the Llll of stage 8 (not shown), and of stage 27 embryos (Figure 6b) are identical. This analysis therefore identifies the 60 kd lamin of stage 8 embryos as the Lrrr polypeptide, and it also demonstrates the existence of Lrrr in embryos up to tail bud stages. Identity of the peptide maps is also found for lamin Lr of the erythrocyte (Figure 6c), for L, of stage 27 embryos (Figure 6d), and for the embryonic and adult Lr, lamins (Figures 6e and 6f), respectively. Both Lrr spots marked in Figure 5b, differing slightly in isoelectric point and size, were analyzed; they showed no obvious differences. These results are substantiated by experiments in which mixtures of different lamin peptides were separated. The patterns of the mixtures of homologous lamins were identical with those of each of the corresponding lamins alone. Therefore, it is reasonable to assume that we are dealing with the same lamin proteins in early developmental and in adult tissues.

Appearance of Lamin L, at MBT Is Due to De Novo Synthesis Newly synthesized proteins were isotopically labeled for the duration of two cell cycles by injecting %-methionine into the blastocoel of embryos. Lamins were analyzed by immunoblotting after two-dimensional separation on gels. The binding of anti-lamin antibodies was visualized by the indirect peroxidase-anti-peroxidase (PAP) technique (Sternberger, 1979) instead of using 1251-labeled secondary antibody. This allows the immunological identification of lamins and, after processing the filter for fluorography, the detection of radiolabeled proteins on the same filter. A series of labeling experiments revealed that lamin L, is strongly labeled from its first appearance onward (Figure 7a). The appearance of Lr seems to be exclusively due to de novo synthesis, since in cytoplasmic fractions of eggs or of embryos prior to midblastula stages, we could detect lamin Lrrr, but neither L, nor Lrr, by immunoblotting. From the labeling experiments, it is also evident that lamin L,,, is synthesized at midblastula stages (Figure 7a). As yet, we do not know whether synthesis of Lrrr has already started earlier in development. Synthesis of L, and Llll Is Independent of RNA-Transcription in Early Development The beginning of L, synthesis coincides with the onset of transcription at the MBT The question therefore arises whether the appearance of Lr does in fact depend on transcriptional events. This was tested by examining the biosynthesis of the lamins in the absence of transcription. Hybrid embryos (X. laevis Q x X. borealis o) were injetted at the one-cell stage with a-amanitin sufficient to obtain an internal concentration of approximately 1 pglml. At this concentration of a-amanitin, RNA synthesis directed by polymerase II is inhibited in Xenopus embryos (Newport and Kirschner, 1982). In spite of this inhibition, embryos develop normally beyond the MBT but fail to gastrulate (Newport and Kirschner, 1982). No significant differences in labeling intensity of Lr and L,,, could be detected in a-amanitin-treated embryos (Figure 7b) in com-

Nuclear 195

Lamrna

0

in Early

Development

5,4

605

0

0 10th

ST8

G9

Ll +

12th

ST27

Ll Ll e* 4

13th LI *I,

0

‘I

--I @I b

ST10

Llll

LI -

?!!5 hll

ST27

Go v L, ST13 m

9!2 L, hl

4. Lamin

L, and LII Appear

hl

0 Lll

Frgure 5. The Concentrations of L, and Lrr Increase during velopment as Revealed by Coomassie Blue Staining

0

Figure

-4

Ll e

at Drfferent

Times

in Development

Nuclear lamina preparations from embryos of different developmental stages were separated by two-dimensional gel electrophoresrs, and lamins were detected by rmmunoblotting as described in the legend to Figure 3. (a) Nuclear lamina preparations of 150 embryos after the 10th cleavage cycle; (b) 125 embryos after the 12th: (c) 100 embryos after the 13th; (d) 75 embryos from stage 10; (e) 65 embryos from stage 13; and (f) 50 embryos from stage 20. Only those parts of the filters where spots were visible are shown. The relative intensities of the two most basic isoelectric variants of lamin L,,, varied somewhat in different experiments.

parison with control embryos (Figure 7a) in three independent experiments. This indicates that synthesis of lamin L, and Llll is independent of RNA synthesis at early developmental stages and must therefore be directed by maternal transcripts. The intermediate filament proteins Cl and C2 (Franz et al., 1983) which are present as cytoplasmic contaminants in our nuclear preparation, are also labeled in the presence of a-amanitin, as described for the lamin proteins in these experiments. To control for the efficiency of the inhibition of mRNA synthesis, hybrid embryos were used in these experiments. Biosynthesis of a specific protein, known to be synthesized from maternal mRNA in early development and from newly transcribed mRNA after MBT, was monitored in hybrid embryos (protein lL and 1, respectively in Fig-

Early

De-

Nuclear lamina preparations of embryos at stage 8 (150 embryos each) (a and c) and stage 27 (75 embryos each) (b and d) were separated by two-dimensional gel electrophoresis and were then either starned with Coomassie brilliant blue (a and b) or were processed for detechon of lamin antigens by immunoblottmg, as described in the legend to Figure 2 (c and d). Lamin spots in (a) and (b), which were used for tryptic pephde map analysis, are indicated by arrows. The arrow heads in (a) and (b) mark three identical reference spots, which can be used to compare intensities of the Coomassie blue staining in (a) and (b). Arrow heads in (c) and (d) mark the position of the basic spot of 1251-BSA, servrng as an internal marker in the immunoblotting experiments. Only those regions of the gels and blotfilters that contain lamin spots are shown.

ure 6i in Woodland and Ballantine, 1980; see also Mohun et al., 1981). The X. borealis (1) and X. laevis (1~) specific homologs of this protein can be distinguished by twodimensional gel electrophoresis. As sperm do not supply mRNA to the egg, in hybrid embryos of X. laevis Q x X. borealis (r crosses, any X. borealis-specific translation products must be derived from newly transcribed mRNA. Therefore biosynthesis of the borealis-specific protein 1 should be suppressed by inhibition of mRNA synthesis, whereas the synthesis of the laevis-specific homolog IL should continue, as translation from a maternal mRNA will not be affected. Thus, labeling of protein 1 and 1 L serves as an internal control of the efficiency of the amanitin inhibition in the hybrid embryos. The result of this analysis is given in Figures 7e and 7f. In marked contrast to the vast majority of proteins, the synthesis of which is not affected by a-amanitin at this stage of development, the synthesis of the borealis-specific protein 1 is completely suppressed in the presence of a-amanitin (Figure 7f), whereas this pro-

Cell 196

LIIIGV @

Lfff ST27

Figure 6. Identification of Embryomc by Tryptic Peptide Analysis

Lamins

The lamin spots L,, Lrr, and Lrrr from the gel in Figures 5a and 5b were cut out, were iodinated within thegel slices, and were trypsinized. lodinated tryptic peptides were also prepared from oocyte and erythrocyte lamins. Samples were applied at the bottom left, and electrophoresis (E) and chromatography(C) were carried out in the indicated directions. (a) L,,, from oocyte, (b) L,,, from stage 27 embryos, (c) L, from erythrocytes, (d) L, from stage 27 embryos, (e) LII from erythrocytes, (f) LII from stage 27 embryos.

Lf ST27

Lff ST 27

E

E L

C

&

C

tein is synthesized in control embryos (Figure 7e). This demonstrates the effective inhibition of mRNA synthesis in our experiments and therefore justifies the conclusion that biosynthesis of lamins is directed by maternal mRNA in early development. Discussion The results presented in this and in the preceding papers (Stick and Schwarz, 1982, 1983) illustrate that the extensive functional and structural changes in the nuclei during gametogenesis and embryogenesis are accompanied by structural changes of the nuclear lamina, suggesting an active participation of the lamina in these nuclear processes. A summary of our findings is given in Figure 8. The lamina structure and lamina proteins disappear in prophase of meiosis while the nuclear membranes persist (Stick and Schwarz, 1983). Disappearance of the lamina correlates with the condensation of the meiotic chromo-

somes. Ll,, appears in oocytes at diplotene. After egg maturation and fertilization, Lrr, is found in pronuclei and in embryonic nuclei up to late stages of development. Lr is first detected at MST, and Lr, at the gastrula. The Lamina of the Germinal Vesicle Serves as a Lamin Pool for the Formation of Embryonic Nuclei To achieve rapid cell divisions in early development, oocytes stockpile large amounts of macromolecules and their precursors (Davidson, 1977). For the lamin polypeptide Lrrr, the only lamin constituent of embryonic nuclei up to MBT, the lamina structure of the germinal vesicle itself may serve as such a store. During germinal vesicle breakdown in the course of egg maturation, the lamina is disassembled, and the lamin polypeptides are dispersed in the ooplasm (Stick and Hausen, unpublished). Indeed, lamin LIII, but not L, and L,,, can be detected by immunoblotting in soluble extracts from mature, unfertilized eggs. How-

Nuclear 197

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in Early

Development

CONTROL

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IMMUNOBLOT

-LI

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)r)-

.

00 -

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i

- .

. . . - -

Figure

7. Synthesis

of Lamin

0

(x-Ah+NITIN

L,,, and L, Is Independent

of Transcription

.

in Early Development

X. laevis Q and X. borealis o hybrid embryos were labeled with %-methionine dimensional gel electrophoresis. (a, c, and e) labeling in the absence of a-amanitin; polypeptides, blot filters were first reacted with anti-lamina serum (see legend to and b) to detect the 35S-labeled polypeptides. Lamin fractions from 75 embryos the cytoplasmic fractions (equivalent to approximately 20 embryos.). Cl and C2 (1983). Arrows in (e) and (f) point toward the X. borealis polypeptide 1 and the X. f) marks the position of actin.

ever, there is no indication for a cytoplasmic store of lamins in oocytes, as revealed by the same technique using enucleated oocytes (our preliminary results). The absence of lamin LIIr from the nucleoplasm of germinal vesicles has been reported previously (Stick and Krohne, 1982). Since both egg maturation (Drury and SchorderetSlatkine, 1975; Wassermann and Masui, 1975) and formation of nuclei in the eggs (Forbes et al., 1983) can proceed in the absence of translation, lamin polypeptides found in these nuclei are probably derived from the lamina of the germinal vesicle. Forbes et al. (1983) have calculated that

.

for 1 hr at stage 8-9, and polypeptides were separated by two(b, d, and f) in the presence of a-amanitin. To visualize the lamin Figure 2) (c and d) and were then processed for fluorography (a were loaded to each gel in (a) and (b). (e and f) Fluorograms of in (a and b) mark cytokeratins 1 and 2 according to Franz et al. laevis polypeptlde lL (Woodland and Ballantine, 1980). A (e and

the egg’s store of nuclear constituents is large enough to integrate a quantity of DNA equivalent to 1000 nuclei into nuclear-like structures. This number comes close to the number of nuclei present at MBT (2000-4000). At least from the MBT onward, the embryo is supplied with additional L,rr protein by de novo synthesis. A shift of IPs of the Llll lamin is observed after germinal vesicle breakdown in meiosis (unpublished results). In CHO cells, the disassembly of the lamina structure during mitosis is accompanied by phosphorylation of the lamina polypeptides, resulting in a shift of their isoelectric points

Cell 196

oocoHID

L

2

:

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g ; m

: 3 ‘.

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Figure 6. Pattern of the Lamina genesis and Early Development

YIDBLISWLl ST 8

CllTAULl

NEURYLA

TAlLBUD

ST 12

*T 20

5110

Polypeptide of Xenopus

Composition laevis

during

Oo-

Lamin composition of oogonia has not been analyzed in detail. Oocyte stages according to Dumont (1972), embryonic stages according to Nieuwkoop and Faber (1967).

(IPs) (Gerace and Blobel, 1980). Since the factors regulating nuclear envelope breakdown and chromosome condensation, such as the maturation-promoting factor and cytostatic factor, are effective both in mitosis and in meiosis (Sunkara et al., 1979), the same mechanisms may be effective in nuclear envelope breakdown during both of these events. This may also hold for the mechanisms by which disassembly and reaggregation of the lamins is achieved in these processes. The Time Course of the Appearance of Lamin L, in Nuclei of Blastula Embryos Coincides with the MBT Lamin LI is first detected in embryonic nuclei at midblastula stage. The time course of its accumulation in the embryonic nuclei coincides with several other changes in cellular function, which mark the onset of the MBT. Since no cytoplasmic store of LI polypeptide could be detected by immunoblotting samples from cytoplasm of embryos taken prior to midblastula stage, we conclude that the appearance of L, is due exclusively to the de novo synthesis revealed by labeling experiments. This de novo synthesis, like the synthesis of LIII, is independent of RNA synthesis and must therefore be achieved by the activation of a maternal transcript. While synthesis of the majority of proteins translated from maternal messengers starts just after fertilization (Ballantine et al., 1979), activation of maternal messengers at the onset of the MBT has been reported for a restricted number of proteins, for example, histon Hi (Woodland et al., 1979), fibronectin (Lee et al., 1984), and some other proteins of yet unknown function (Woodland and Ballantine, 1980). It would be interesting to know whether the mRNAs of these proteins form a particular class of maternal messengers the activation of which is affected by a common mechanism. From Gastrula Stages Onward, All Three Lamins Are Present in Embryonic Nuclei There is a significant gap between the appearance of lamin L, and lamin L,,. The concentration of the latter increases from gastrula stages onward, when the embryo becomes independent of the store of maternal messengers (Woodland et al., 1979; Woodland and Ballantine, 1980). However, we do not yet know whether the synthesis of LII at gastrula stages is indeed directed by newly transcribed mRNA rather than by maternal messenger. In contrast to the blastula stages, inhibition of transcription in

gastrula greatly damages the embryos, making interpretation of inhibitor experiments difficult. Lamins L, and L,, are present in roughly equal amounts from late neurula stages onward, as in erythrocytes and other somatic nuclei. Although the concentration of L,,, decreases relative to L, and LII from neurula stages to tail bud stages, it is unambiguously identified at these late stages of development. A more detailed analysis would have to reveal whether the decrease of Llll is due to a decrease of this lamin component from all nuclei or whether it is due to the disappearance of Llll from particular tissues or cell types, as has been reported for other nuclear proteins originally located in the germinal vesicle (Dreyer et al., 1981). It is quite possible that the lamina of embryonic nuclei is transiently composed of all three lamin polypeptides. Considerations Concerning the Function of the Nuclear Lamina In discussing different membrane- and chromatin-binding properties of mammalian lamin A and B, Gerace and Blobel(l981) proposed a functional specialization of these two polypeptides, though definite proof is still lacking. In this context, it is noteworthy that lamin L,,, alone can build up a lamina structure in early cleavage nuclei. Therefore in early development, the different functions of the lamins, such as membrane- and chromatin-binding, must be carried out by only this one lamin component. Lamin Llll builds up the lamina structure of nuclei as diverse as the germinal vesicle, the pronuclei, and the cleavage nuclei. The great differences in the structural and functional organization of these nuclei, described before, can obviously be maintained in the absence of a difference in the lamina composition. The spatial organization of the chromatin in these nuclei deserves special attention. In the embryonic nuclei, the DNA is attached to the nuclear lamina, while the germinal vesicle chromosomes are localized in the center of the nucleus, with no contact with the nuclear envelope. Factors other than lamina composition must be responsible for the different chromosomal arrangements in these nuclei. The appearance of L, correlates with the midblastula transition. The question of a causal relationship between the integration of L, into the lamina and the impending changes in nuclear activity remains to be clarified. As the nuclear lamina seems to be involved in loop-size organization (Lebkowsky and Laemmli, 1982), a change in the lamina may be linked up with changes in loop domain (Buongiorno-Nardelli et al., 1982) and replicon size (Blumenthal et al., 1973; Callan, 1973). Hancock and Boulikas (1982) discussed DNA loop domains as regulatory units of transcription, which links the above-mentioned processes with the onset of RNA synthesis at the MBT However, as long as such considerations remain speculative, the possibility that the different lamins are functionally equivalent cannot be ruled out. The observed pattern of lamina polypeptide composition may then be explained by other than functional principles, such as gene regulatory constraints in embryogenesis. Recently, Smith and Fisher (1984) reported a change in

Nuclear 199

Lamina

in Early

Development

lamina composition during early development in Drosophila. Their data is consistent with the hypothesis that in Drosophila, too, lamina composition changes at a time analogous to the MBT in amphibians, although more analysis is necessary to verify this. Experimental

Procedures

Animals Xenopus laevis were obtained from the South African Snake Farm, Fish-Hoek, South Africa. Xenopus boreahswere bred from pairs kindly donated by Dr. M. Fischberg, Geneva. Embryos of X. laevls or hybrids (X. laevis x X. borealis) were produced by artificial insemination. Jelly coats were removed IO min after fertilization with 2% cysteln in H,O, pH 8.0. Embryos were kept in 5% Ficoll in 1110 strength modified Barth’s solution (MESH) at 23°C r 0.5%. At the time of gastrulation, they were transferred to 1110 MBS-H. Embryos were staged according to Nieuwkoop and Faber (1967), or cleavage cycles were counted directly under the dissecting microscope, and cell cycle length was measured. The number of cleavage cycles from the 8th-9th cycle onward was calculated, using these cell cycie times. For the experiments shown in Figure 3 and m Figure 4, batches of highly synchronouslycleaving embryos were used. Antisera The rabbit anti-chicken lamina serum has been described previously (Stick and Hausen, 1980). To establish hybridoma cell lines (Kijhler and Milstein, 1975) producing lamin specific monoclonal antibodies, we used spleen cells from mice that had been injected 4-5 times each with 25 pg of a total Xenopus laevis germinal vesicle lamina preparation (Stick and Krohne, 1982). Cell culture supernatants were tested for lamin-specific antibodies, using indirect immunofluorescence microscopy on TCA-fixed and wax-embedded Xenopus ovaries (see histological procedures) and using immunoblotting techniques of lamin preparations from Xenopus erythrocyte nuclei or germinal vesicles. Histological Procedures Embryos were fixed at various times after fertilization in 2% TCA (30 min), were dehydrated, and were embedded in polyethyleneglycol distearate (Steedman, 1957). Sections 6 pm thick were cut. Indirect immunofluorescence staining was carried out as described (Stick and Schwarz, 1983). In addition, sections were counterstained with Eriochrome-schwarzT(Chroma, Stuttgart, FRG) to shift autofluorescence of the yolk to a red color that is easily distlnguished from the green fluorescein signal (Schenk and Churukian, 1974). Photographs were taken with a Zeiss ICM 405 photomicroscope equipped with BP 485120, FT 510, BP 515-565 (FITC), and BP 365, FT 395, LP 397 (DAPI) fluorescence filter sets (Zeiss, Oberkochen, FRG) on Kodak Ektachrome EL 135 film. In Vitro Pronuclear Formation In vitro pronuclear formation was performed according to Lohka and Masui (1983) with one modification. Sperm were isolated by homogenizing whole testis that had been carefully cleaned from erythrocytes. The homogenate was layered on a cushion of 70% percoll (Pharmacia, Uppsala, Sweden) in MBS-H, and sperm were sedimented by centrifugation for 15 min at 1500 x g. Isolation of Embryonic Nuclei and Preparation of a Crude Lamina Fraction To isolate embryonic nuclei, embryos up to stage 12 were blocked in protein synthesis by incubating them in 0.4 mglml cycloheximide in l/IO MESH for 1 hr to arrest cells in interphase. Embryos were gently homogenized in nuclear isolation buffer (0.005 M Tris-HCI, pH 8.0; 0.002 M MgCI,; 0.0005 M spermine; 0.25 M sucrose; 0.5% Triton X-100; 0.0001 M phenylmethylsulphonylfluoride; and 20 U/ml Kallikrein inhibltar, Trasylol) in the cold. The homogenate was layered on 60% percoll in the same buffer and was centrifuged for 10 min at 3000 2( g. The supernatant was separated from the nuclear fraction that formed an interband on top of the percoll cushion. The nuclei were washed once in nuclear isolation buffer, were resuspended in the same buffer without Triton X-100, and were treated with DNAase I (Worthington, USA)

20 rg/ml for 15 min at 37%. To isolate a crude lamina fraction, treated nuclei were extracted two times with 1 M KCI, 1% Triton X-100 in 0.01 M Tris-HCI, pH 7.5, and were washed once in distilled water. Radiolabeling of Embryos Seventy-five embryos were each injected into the blastocoel with 40 nl of L-%-Methionine (33 Cilml) (Amersham Braunschweig, F.F.G.). Embryos were labeled for 1 hr and nuclei were isolated as described above. In experiments where a-amanitin was used, embryos were injected at the one-cell stage with 25 nl of o-amanitin (40 pglml) in 0.088 M NaCI, 0.015 M Tris-HCI, pH 7.5. Gel Electrophoresis and lmmunoblotting Isoelectric focusing and SDS gel electrophoresls were carried out according to O’Farrell(l975) with the following modifications: Ampholines (Servalyte, Serva, Heidelberg, F.R.G.), 20% ampholines pH 2-11, and 40% ampholines each pH 4-6 and pH 5-7. 12% acrylamide gels were used for the separation in the second dimension. Proteins were electrophoretically transferred to nitrocellulose sheets (Schleicher und Schtill, F.R.G.) for 3 hr at 0.4 A in 0.025 M Tris base, 0.2 M Glycine, 20% methanol (v/v). Filters were processed for immunostaining as described (Stick and Krohne, 1982). Binding of mouse monoclonal antibodies was detected using ‘%labeled rabbit anti-mouse Fab fragments (Stick and Krohne, 1982). In experiments where rabbit anti-chicken lamin antisera were used, detection was by the indirect peroxidase-anti-peroxidase (PAP) technique according to Sternberger (1979). Goat anti-rabbit serum was a gift from Dr. H. Schwarz (Max Planck-lnstitut ftir Virusforschung, Ttibingen), PAP was from Miles-Yeda Ltd. (Israel). I-Chloro-1-naphtol (0.5 mglml in 0.083 M Tris-HCI, pH 7.6, 16% ethanol (v/v), 0.15% H,O,) was used for the color reaction. In some experiments ‘%BSA was used as an internal marker. Radiolodination was performed as described by Greenwood et al. (1963). %-label was detected by fluorography. The dry filters were dipped for 30 set in toluene contaming 27% (w/v) 2,5-diphenyl-oxazol (PPO). Peptide Peptide (1977).

Mapping mapping

was done

essentially

as described

by Elder et al.

Acknowledgments We would like to thank Karin Herrmann for excellent help in these experiments, and Gerd Klein, who established the hybridoma cell lines used. We are grateful to Dr. ChrIstine Dreyer for helpful suggestions, and to Dr. Hartmut Beug for critical reading of the manuscript. Dr Rudolf Winklbauer we wish to thank for stimulating discussions during the whole period of this work. We also thank Roswitha GrZjmke-Lutz and Susanne Haase for their help in the photographic work, and Brigitte Hieberfor typing the manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

November

16, 1984; revised

February

19, 1985

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Note Added

Krohne, G., Dabauvalle, M.-C., and Franke, W. W. (1981). Cell typespecific differences in protein composition of nuclear pore complex-lamina structures in oocytes and erythrocytes of Xenopus laevis. J. Mol. Biol. 757, 121-141.

Recent results show that lamin Llli is translated at only a very low ratio before MBT. Its rate of synthesis increases after the onset of MBT, parallel to the activation of the maternal transcripts of lamin L,.

Lebkowsky, long-range 325-344.

J. S., and Laemmli, U. K. (1982). Non-histone proteins and organization of HeLa interphase DNA. J. Mol. Biol. 756,

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M. (1984). Temporal and spatial Xenopus development. Cell 36,

Lohka, M. J., and Masui, Y. (1983). Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220, 719-721. Miake-Lye, R., Newport, J., and Kirschner, M. (1983). Maturationpromoting factor induces nuclear envelope breakdown in cycloheximide-arrested embryos in Xenopus laevis. J. Cell Biol. 97, 81-91. Mohun, T. J., Brownson, S., and Wylie, C. C. (1981). Protein in interspecies hybrid embryos of the amphibian Xenopus. Res. 732, 281-288.

synthesis Exp. Cell

Nieuwkoop, P. D., and Faber, J. (1967). Normal Table of Xenopus (Daudin). (Amsterdam: North-Holland Publishing Co.).

laevis

in Proof