A major developmental transition in early xenopus embryos: II. control of the onset of transcription

Cell, Vol. 30, 687-696,

October

1982,

Copyright

0 1982

by MIT

A Major Developmental Transition in Early Xenopus Embryos: II. Control of the Onset of Transcription John Newport and Marc Kirschner Department of Biochemistry and Biophysics School of Medicine University of California, San Francisco San Francisco, California 94143

Summary We have shown in the accompanying paper that a developmental transition occurs at the midblastula stage (cleavage 12) in Xenopus embryos, and that this midblastula transition (MBT) is apparently initiated when the ratio of nucleus to cytoplasm reaches a critical value. One manifestation of this transition is the onset of transcription. We show here that a plasmid containing a cloned gene coding for a yeast leucine tRNA comes under developmental control when injected into cleaving eggs. In pre-MBT eggs this plasmid is transiently transcribed and then becomes inactive; however, it becomes transcriptionally active again at the MBT. This pre-MBT suppression of transcription can be reversed by addition of competing DNA. The amount of DNA needed to induce premature transcription is equal to the amount of nuclear DNA present after 12 cleavages (24 ng), suggesting that the MBT is triggered by the DNA through titration of suppressor components present in the egg. Introduction In the accompanying paper we have shown that embryonic development in Xenopus laevis starts with a period of rapid cleavage and undergoes an abrupt and concerted change in the cell cycle and in other properties at cleavage 12. During the period of rapid cleavage all cells within the egg divide nearly synchronously, there is no observable RNA transcription and the cells are not motile. Within 1 hr after the cleavage 12 (-4000 cells per egg), several new cell activities appear, including activation of RNA transcription in all cells of the embryo, onset of lamellipodal formation and general cell motility and the first evidence of Gl and G2 phases in the cell cycle (Newport and Kirschner, 1982). This well defined interval, during which completion of the rapid cleavage period is followed by the immediate activation of a new developmental program, is called the midblastula transition (MBT). We have presented experiments showing that the timing of the MBT does not depend on cell-cell interactions, rounds of cytokinesis, absolute time from fertilization, rounds of DNA replication or initiation of new transcription. Rather, the timing of the MBT depends on reaching a critical ratio of nucleus to cytoplasm (Newport and Kirschner, 1982; also see Kobayakawa and Kubota, 1981). During this early period

of development the total cytoplasmic volume is fixed, whereas the number of nuclei in this fixed volume increases exponentially. We have demonstrated that the onset of transcription and slowdown in DNA synthesis accompanying the MBT also occur in a noncleaving egg, which contains all the embryonic nuclei in a common cytoplasm (coenocytic egg). This suggests that the timing of the MBT could easily be explained by a model in which a substance initially present in excess in the egg cytoplasm is used up as the nuclear content of the egg increases. When this substance is depleted, further nuclear divisions will be blocked (or substantially slowed) and such blockage might in turn allow for expression of previously suppressed cellular processes such as transcription and cell motility. We have attempted to understand the molecular basis of the MBT by microinjection of a plasmid DNA containing a yeast leucine tRNA gene. We demonstrate that transcription of this foreign DNA comes under developmental control when injected into a developing Xenopus egg. After injection into fertilized eggs during the early cleavage period, the plasmid carrying the yeast tRNA gene is transiently active, directing the synthesis of a 120 nucleotide precursor RNA. This transcription completely stops after the plasmid has been in the egg for 1 to 2 hr. However, if the plasmid is injected into a coenocytic egg that has passed the MBT, or if the previously injected egg develops to the MBT stage, transcription remains active or is reactivated. Furthermore, we show that this plasmid can be induced to become transcriptionally active prematurely (prior to the MBT) if the total DNA content of the egg is artificially increased to that normally found in an egg at the MBT, by injection of a second plasmid DNA. These data are consistent with the model described above for triggering of the MBT by a critical level of DNA. We assume that the added DNA depletes some cytoplasmic material that binds stoichiometrically to DNA. Results Although the experiments presented in the accompanying paper demonstrate that RNA transcription which is inactivated in the oocyte during maturation is activated for the first time at the MBT, they reveal nothing about the underlying molecular mechanisms involved in controlling this event. In particular, we do not know whether the lack of transcriptional activity prior to the MBT is due to a lack of transcriptional proteins or cofactors or to a modification of the genomic DNA that inactivates it as a template for transcription. We also do not know whether the inactivation

of

maturation,

transcription or

is whether

pressed throughout (1974) has shown

event

specific

to

transcription

an

is

actively

oocyte sup-

the early cleavage period. Roeder that the activities of all three RNA

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polymerases can be assayed from extracts of Xenopus eggs, which are not active in transcription, and that the amount of each of these polymerase activities does not change until after gastrulation (5-6 hr after new transcription has started). Thus the lack of transcription in pre-MBT eggs probably is not due to the absence of active RNA polymerases. Inactivation of Plasmid Templates for Transcription in the Pre-MBT Period To study the regulation of transcription during the first 8 hr of Xenopus development, and to distinguish regulation at the template level from regulation due to changes in the transcriptional apparatus, we have utilized microinjection of a DNA clone containing a yeast leucine tRNA gene (pYLT20). Several studies have demonstrated that when pYLT20 is injected into the germinal vesicle of a Xenopus oocyte it can serve as an active template for the synthesis of a 120 nucleotide immature tRNA transcript (Standring et al., 1981; de Robertis and Olson, 1979). This immature 120 nucleotide transcript contains 9 nucleotides at the 3’ end, 4 nucleotides at the 5’ end and a 33 nucleotide intervening sequence, all of which are normally removed during processing of this transcript to a mature tRNA in yeast (de Robertis and Olson, 1979). Partial processing of this transcript to the mature tRNA occurs in the oocyte system, in the nuclear gel of the germinal vesicle (de Robertis and Nishikura, 1981). We have found that when the plasmid pYLT20 is injected into fertilized Xenopus eggs with radioactively labeled rUTP, a 120 nucleotide transcript is synthesized that is identical with the transcript synthesized in the oocyte system (Figure 1, lane 2). No other transcripts are observable at this time. The fact that pYLT20 is an active template for transcription at a time in Xenopus development when endogenous nuclear transcription is inactive indicates that the suppression of endogenous transcription during this period is not due to lack of transcriptional proteins and cofactors. Rather, it suggests that suppression is an active process involving modification of DNA, chromatin or nuclear structure. To observe the transcriptional competence of pYLT20 injected into eggs at difterent periods of time during early development, we have taken advantage of the fact that the timing of the MBT and the onset of RNA transcription in Xenopus development is independent of celluiarization (Newport and Kirschner, 1982). Thus when one blocks cleavage either by treating the eggs with cytochalasin B or by briefly centrifuging them, they will faithfully carry out their normal developmental program (Newport and Kirschner, 1982). The eggs continue to synthesize DNA at the same rapid rate as cleaving embryos and go through the MBT at the same time as control embryos as judged both by a decrease in their rate of DNA synthesis and by the initiation of RNA transcription

SN

2

58

Figure 1. Transcriptional opment

Regulation

of pYLT20

DNA during

Devel-

Fertilized eggs were blocked from cleaving via centrifugation, as described in the Experimental Procedures. These eggs were injected with 50 nl of a solution containing 20 pg/ml pYLT20 plasmid DNA 60 min after fertilization (1 ng DNA per egg). The w3’P-rUTP was injected at 10 min, 2.5 hr and 7 hr after plasmid DNA injection. The eggs (three per time point) were allowed to incubate for 1 hr after the radioactive label was injected. (Lane 2) Labeling period l-2 hr after fertilization: (lane 5) 4-5 hr after fertilization; (lane 8) 7-8 hr after fertilization. The isolated RNA from these eggs was run on a 5% polyacrylamide-urea gel and autoradiographed for 24 hr. SN: 180 nucleotide snRNA. pYLT20: 120 nucleotide pYLT20-dependent precursor transcript. x: band has not been identified but is labeled at all developmental stages both pre-MBT and post-MST; this band is not sensitive to high concentrations of u-amanitin, suggesting that it is not due to transcription by RNA polymerase. tRNA: endogenous tRNA transcripts.

(Newport and Kirschner, 1982). We have used these cleavage-arrested eggs for pulse-label experiments in which the plasmid pYLT20 is injected at an early time in development and its transcriptional activity is assayed at a later time by injection of radioactively labeled rUTP into these coenocytic eggs. Alternatively, the plasmid itself can be introduced at any time. Using these methods we were able to follow easily the transcriptional competence of pYLT20 before and after the MBT stage of development. When such pulse-label experiments were performed, we found that although pYLT20 was active as a transcriptional template immediately after injection into pre-MBT cytoplasm, it became progressively less

Developmental 689

Regulation

of Transcription

efficient with time and eventually was completely inactivated. Figure 1, lane 2, shows the synthesis of the 120 nucleotide tRNA precursor 10 min after injection. Lane 5 shows the lack of synthesis when the labeled rUTP is injected 2.5 hr after the injection of the plasmid. Figure 2 shows a time course of this inactivation of transcriptional capacity. The plasmid was injected 2 hr after fertilization, and 15 min pulses of CX-~‘PrUTP were given at various times. At 30 min after injection, the transcriptional activity of this template reaches a maximum; it drops to less than one fifth that level 1 hr later, and by 2.5 hr the template is no longer active. We could demonstrate that transcriptional inactivation of the injected plasmid was due to a modification of the plasmid DNA, and not to any change in the transcriptional capacity of the egg 2 hr after plasmid injection, by the fact that plasmid injected at this later time was also initially transcriptionally active, then became inactive over a subsequent 2 hr period (data not shown).

I’ ,

I

I

I

2 Time Figure 2. Developing Plasmid arrested

Kinetics Eggs

-

t

I

4 Since

of Transcriptional

pYLT20 DNA was (via centrifugation)

6

Fertilization Inactivation

injected into 60 min after

I

8 (hrs)

of pYLT20

DNA in

fertilized eggs cleavagefertilization and pulse-la-

beled for 15 min with w3’P-rUTP at later times, as described in the legend to Figure 1. The RNA from the eggs was isolated, and an amount of counts from each sample, normalized for the total CX-~*PrUTP injected, was loaded onto a polyacrylamide-urea gel. Densitometric scanning of the pYLT20-dependent 120 nucleotide band was used to quantitate the amount of pYLT20-dependent RNA synthesized at each time point. All transcriptional rates were graphed relative to the densest band, which was arbitrarily defined as 100% activity.

Reactivation of Plasmid Templates at the MBT Though the incubation of pYLT20 in embryonic cytoplasm during the rapid cleavage periods leads to the inactivation of this plasmid DNA as a template for transcription, this inactivation is reversible, as shown in Figure 1, lane 8, and in Figure 2, at 7 hr after fertilization, where it can be seen that transcription from pYLT20 resumes again at the MBT. The 120 base precursor RNA is found against a background of cellular RNAs synthesized during the MBT period. As shown in Figure 2, although pYLT20-dependent transcription of tRNA resumed at the MBT, it was not as efficient as that found approximately 30 min after the plasmid was initially injected into the egg at pre-MBT stages. Typically, the amount of pYLT20-dependent transcription observed in post-MBT cytoplasm was only 20%-40% of that found in pre-MBT cytoplasm (before inactivation). This could be due to a number of reasons including plasmid DNA degradation, changes in rUTP pool sizes, competition from endogenous promoters for RNA polymerases and transcription initiation factors or an inability to reverse completely the pre-MBT-acquired transcription block (the last of these possibilities is discussed below). The rUTP pool, as measured by high performance liquid chromatography, at the MBT for both stratified and normal embryos is about the same as it is during the early cleavage stage. We have also shown that DNA degration is not responsible for the apparently decreased synthetic capacity of the post-MBT reactivated plasmid. To study degradation of the injected plasmid, we injected fertilized eggs with a fixed amount of pYLT20 DNA and then allowed them to develop normally. At different times after fertilization, total DNA from these embryos was isolated and run on an agarose gel. The DNA in this gel was then transferred to nitrocellulose paper and probed with radioactively labeled pYLT20 via the method of Southern (1975). The autoradiographs from these blots were scanned with a densitometer to quantitate the amount of pYLT20 DNA remaining in the embryo at different times. Figure 3 shows no significant degradation of injected plasmid DNA for at least 21 hr, by which time neurulation is complete. In fact, about 5% of the plasmid DNA still remains in the embryo 51 hr after fertilization, by which time the now highly developed embryo is swimming vigorously. We conclude from these results that selective degradation of injected plasmid DNA does not occur before 21 hr and, therefore, cannot explain the fact that levels of pYLT20-dependent transcription in the post-MBT period are lower than those observed immediately after injection of the plasmid into pre-MBT egg cytoplasm. These inactivation-reactivation experiments demonstrate three important points. First, suppression of transcription in pre-MBT eggs appears to involve a modification of the template DNA rather than the absence of cofactors. This is shown by the fact that

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when pYLT20 is injected at any time into pre-MBT eggs, transcriptional synthesis occurs for about 2 hr before it terminates. Second, the embryonic cytoplasm is capable of modifying the template at anytime during the rapid cleavage period before the MBT. Such modification therefore is not restricted to the period of oocyte maturation, when transcription is normally first inactivated. This mechanism might also serve to modify newly synthesized endogenous DNA during the rapid cleavage period. Third, the modification of the pYLT20 plasmid template is reversible, and the reversal occurs at the same time during development that transcription from endogenous templates is activated-that is, at the MBT. These observations all seem to indicate that transcription from pYLT20 DNA in fertilized egg cytoplasm is subject to the same regulatory mechanisms which temporally control endogenous transcription during early Xenopus development. As such, the plasmid can be used both to study the regulation of transcription at this time in development and as a probe for better understanding the molecular processes controlling the timing of the MBT in Xenopus. Suppression of Transcription Correlates with Assembly into Chromatin A mechanism that might account for the transcriptional inactivation of pYLT20 incubated in pre-MBT cytoplasm is the assembly of this initially “naked”

Figure 3. Embryos

Stability

of pYLT20

DNA Injected

into Developing

DNA into chromatin. Laskey et al. (1977) have shown that when supercoiled SV40 DNA is injected into the germinal vesicle of the Xenopus oocyte, the DNA is first rapidly converted to a relaxed form and then, by a much slower mechanism, is converted back to a supercoiled form. They showed that the conversion from the relaxed form back to the supercoiled form was due to the assembly of the injected DNA into chromatin (Laskey et al., 1977; Wylie et al., 1978). Several investigators have shown that the mature egg contains a cytoplasmic pool of histones large enough to convert at least 40 ng DNA into chromatin (Adamson and Woodland, 1974; Laskey et al., 1977); that is, much more than would be needed to convert the 2 ng per egg of pYLT20 DNA injected in our experiments into chromatin. We find that 2 ng labeled, supercoiled pYLT20, injected into an egg 80 min after fertilization, goes through the same series of topological transformations that Laskey observed with SV40 DNA injected into the germinal vesicle of oocytes. As shown in Figure 4, the initially supercoiled plasmid runs as the discrete, fastest moving band in lane 0. It is quickly converted to a relaxed circle, and by 5 min about 75% runs as a slow moving band. Over the next 2 hr, this DNA is reconverted to the supercoiled form. Various supercoiled intermediates are visible at 30, 60 and 90 min (Figure 4). Plasmid DNA injected into pre-MBT eggs is assembled into chromatin with roughly the same kinetics with which transcription is extinguished, which might suggest that chromatin assembly is itself sufficient to explain the transcriptional inactivation. However, two experiments argue that although chromatin assembly may be necessary for transcriptional inactivation, it is not sufficient. In the first experiment we show that chromatin assembly occurs in the post-MBT period,

Xenopus

Plasmid pYLT20 DNA (50 nl of 20 pg DNA/ml solution) was injected into fertilized eggs before the first cleavage division (60 min after fertilization). These eggs were allowed to develop normally in a dish filled with 25% MMR. DNA was isolated from these embryos 2, 5, 9, 21 and 51 hr after fertilization. This DNA was run on a 1% agarose gel, transferred to nitrocellulose paper and probed with radioktively labeled pBR322 as described in the Experimental Procedures. MBT: embryos at the midblastula transition. N: neurulating embryos. STP: swimming tadpoles. Within experimental error there does not appear to be a significant increase or decrease in the amount of pYLT20 DNA in these embryos until between 21 and 51 hr after fertilization, at which time the amount of DNA remaining decreases to ~5% of the amount originally injected.

Figure

4.

Rate of Assembly

of pYLT20

DNA into Chromatin

Radioactively labeled pYLT20 DNA (2 ng DNA per egg) was injected into eggs 60 min after fertilization and allowed to incubate in egg cytoplasm for 0. 5, 30. 60, 90, 120 or 150 min. The DNA from these eggs was then isolated and run on a 1% agarose gel in TBE buffer for 12 hr at 60 V. The gel was dried and autoradiographed for 24 hr. rc: relaxed circles. SC: supercoiled circles. (Lane A) Relaxed circle standard of pYLT20 obtained by treating the radioactively labeled supercoiled pYLT20 DNA with T4 phage topoisomerase (a gift from B. Alberts). Ladders of bands between relaxed and supercoiled circles result from intermediately supercoiled forms.

Developmental 691

Regulation

of Transcription

but does not lead to extinction of transcriptional activity. The pYLT20 DNA was injected into cleavageblocked coenocytic eggs at the MBT, and its transcriptional activity was monitored over time by injection of radioactively labeled rUTP at different times after the DNA injection (Figure 5). At this stage in development the plasmid was transcribed very efficiently relative to the initial transcription in pre-MBT eggs (about five times better based on densitometric measurements). Furthermore, in marked contrast to the transcriptional inactivation of pYLT20 DNA incubated in pre-MBT eggs, the transcriptional efficiency of the plasmid in these eggs did not decrease significantly over a 3 hr period. To determine whether plasmid injected into post-MBT eggs was assembled into chromatin, we repeated the chromatin assembly experiment described in Figure 4, this time injecting radioactively labeled plasmid into cleavage-blocked eggs that were at the MBT stage. This experiment showed that pYLT20 DNA injected into post-MBT eggs was assembled into chromatin at the same rate as it was when injected into pre-MBT eggs (results not shown). From these two experiments we conclude that in post-MBT

SN

CON7 Figure

7

5. Transcription

8

9

of pYLT20

DNA in Post-MBT

Eggs

Plasmid pYLT20 DNA (50 ng of 20 pg DNA/ml solution) was injected Into fertilized eggs cleavage-arrested (via centrifugation) 7.0 hr after fertilization, which corresponds to the time of the MBT in control eggs (lane CON7, no plasmid injected). Eggs were injected with 2.4 FCi ol-32P-rUTP at 0 hr after the DNA injection (lane 7). 1 hr after injection (lane 8) or 2 hr after injection (lane 9). Labeling was allowed to continue for 1 hr before the RNA was isolated and run on a gel. As seen in lane 7, endogenous transcription is just beginning at 6.5-7.5 hr. SN: 180 nucleotide snRNA. pYLT20: 120 nucleotide pYLT20dependent precursor transcript. pYLT20’: a partially processed form of this transcript. tRNA: endogenous tRNA transcripts.

cytoplasm assembly into chromatin does not inhibit transcriptional activity. On the basis of these observations, it seems unlikely that assembly into chromatin itself causes transcriptional inactivation. However, the similarity of the kinetics of these two processes (chromatin formation and transcriptional inactivation) indicates that although chromatin assembly is not sufficient to bring about transcriptional inactivation, it may be necessary before suppression can occur-that is, chromatin may be the active substrate for either a covalent or a noncovalent modification that would block further transcription in pre-MBT eggs (see below). As mentioned above, the reactivation of pYLT20 as a template for transcription at the MBT was only 20%-40% as efficient as transcription from the template immediately after injection into pre-MBT cytoplasm. We suggested that two explanations for this behavior might be that the plasmid was unable to reactivate transcription completely at the MBT, or that as a yeast template it was a poor competitor for transcriptional proteins against the fairly large amounts (~24 ng per egg) of endogenous nuclear DNA actively engaged in transcription for the first time at the MBT. However, the pYLT20 template injected after the MBT is 20 times more active than the reactivated template. This is very clear when the relative synthesis of the yeast tRNA precursor is compared with the endogenous tRNA synthesis at the MBT (compare Figure 1, lane 8, with Figure 5, lane 8). This comparative inefficiency of the reactivated template suggests that it may have been incompletely reactivated. One noteworthy observation is that if we inject pYLT20 immediately after the MBT, a 120 nucleotide unprocessed tRNA is synthesized during a 1 hr pulse (Figure 5, lane 7). However, in the next 2 hr (Figure 5, lanes 8 and 9) we find another pYLT20-dependent band on the autoradiograph, pYLT20’. This new band corresponds to the 107 nucleotide RNA fragment produced as a result of processing the ends of the original 120 nucleotide immature tRNA transcript. The fact that prqcessing becomes apparent only at this time might be due to the increased number, stability or modification of the now slowly dividing nuclei (where processing apparently occurs; de Robertis and Nishikura, 1981), or to the synthesis at this time, of a new product which is needed to carry out this processing event, such as snRNAs (Newport and Kirschner, 1982; D. Forbes, T. Kornberg and M. Kirschner, manuscript in preparation). Premature Reactivation of pYLT20 Transcription Induced by Injected DNA We have presented evidence that the inactivation of pYLT20 transcription in pre-MBT eggs requires something more than the assembly of the plasmid DNA into chromatin. Furthermore, the model for control of the

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timing of the MBT presented in the accompanying paper predicts that this change in developmental programming depends on reaching a critical ratio of nucleus to cytoplasm-that is, the nucleus must deplete the cytoplasm of a component necessary to continue rapid division and to suppress other cellular processes such as transcription and cell motility. If the inactivation of pYLT20 transcription in pre-MBT eggs is due to the association of plasmid DNA with a component present in the egg cytoplasm, then transcriptional reactivation of plasmid DNA at the MBT might be caused by the removal of this component from the plasmid by the increasing amount of endogenous nuclear DNA. According to this model, it should be possible to reactive pYLT20 transcription before the MBT if one injects into pre-MBT eggs an amount of nonspecific competing DNA equal to that normally present at the MBT. To do this, we injected pYLT20 into a pre-MBT cleavage-arrested fertilized egg and allowed the plasmid to incubate in this cytoplasm long enough to cause complete suppression of pYLT20dependent transcription (2 hr). The eggs were then injected with a second plasmid DNA (pBR322) at a concentration equal to that present in an embryo at the MBT (24 ng per egg). Following the second injection, the eggs were injected with a-3’P-rUTP at different times to assay the transcriptional activity of the pYLT20 template. We found that the 120 nucleotide pYLT20-dependent transcript was synthesized within 1 hr of the injection of the second competing plasmid DNA (Figure 6). During the first 1 hr labeling period very little pYLT20 transcription was observed. However, in the second 1 hr period, lane 5, appreciable transcription was observed 3 hr before the MBT. This result demonstrates that transcriptional suppression of pYLT20 can be reversed when 24 ng of DNA or an amount of DNA equivalent to 4000 nuclei is present prior to the time at which the MBT would normally occur. As shown in Figure 6, not only is pYLT20 transcription reactivated, but there is also evidence that endogenous nuclear transcripts are prematurely activated. As shown in lanes 6 and 7, a transcript comigrating with snRNA U2 is visible 3 hr before the MBT would normally occur. Unfortunately, the coenocytic egg does not allow for the study of the onset of ceil motility. However, these experiments indicate that the injection of excess plasmid DNA will initiate the MBT early, as measured by transcription. We have studied the level of DNA needed to reactive transcription of both pYLT20 and endogenous Xenopus genes and induce the slowdown of DNA synthesis. To determine how much exogenous DNA was needed to induce this apparent premature MBT, we repeated the experiment described in Figure 6, this time varying the amount of pBR322 injected and assaying the transcriptional reactivation of previously injected pYLT20 1 hr after the injection of pBR322 (Figure 7).

4 5678 Figure 6. Reactivation Excess DNA

of pYLT20

Transcription

in the Presence

of

Plasmid pYLT20 DNA (50 nl of 20 PQ DNA/ml solution) was injected into eggs cleavage-arrested (via centrifugation) 60 min after fertilization and was allowed to incubate in this cytoplasm for 2 hr. At 3 hr after fktilization, these eggs were injected with pBR322 DNA (50 nl of a 0.5 mQ/ml DNA solution, 25 nQ DNA per egg). After this procedure, the eggs were injected with 2.5 pCi or-32P-rUTP and allowed to label for 1 hr. Injections of a-32P-rUTP were at 0 hr (lane 4). 1 hr (lane 5). 2 hr (lane 6), 3 hr (lane 7) or 4 hr (lane 8) after injection of pBR322 DNA. Lane numbers correspond to total time after fertilization. The RNA from these eggs was then isolated and run on a 5% polyacrylamide-urea gal. In control eggs the MBT occurs at 7 hr. The pBR322 DNA induction of pYLT20 transcription required approximately 1 hr (compare lanes 4 and 51, whereas the first signs of endogenous transcription become detectable 2 hr after pBR322 injection-that is, the first signs of small nuclear RNA (SN) and mature tRNA transcripts CtRNA) are present in lane 6. pBR: high molecular weight bands resulting from pBR322-DNA-dependent transcription. pYLT20: 120 nucleotide pYLT20-dependent precursor transcript. x: unidentified, a-amanitin-insensitive component (see legend to Figure 1).

The experiment shows that at pBR322 DNA concentrations of 8 ng or less per egg, pYLT20 transcription remains inactivated, whereas at DNA concentrations of 16 ng or more per egg, this transcription is again activated, with maximum reactivation occurring when 32 ng of DNA is present in the egg. Thus, as shown in Figure 7, the DNA-dependent transcriptional reactivation of pYLT20 occurs at a DNA concentration equal to the amount of DNA that would be present in a normal egg after 11 to 13 synchronous cell divisions. The fact that the amount of DNA needed to reactivate pYLT20 transcription in pre-MBT cytoplasm is approximately equal to the amount of endogenous Xen-

Developmental 693

Regulation

of Transcription

Figure 7. DNA-Concentration-Dependent Reactivation of pYLT20 Transcription

l

Plasmid pYLT20 DNA (2 ng per egg) was injected into cleavage-blocked eggs 50 min after fertilization. These eggs were injected with pBR322 DNA and w3’P-rUTP 2 hr later, incubated for 1 hr, then lysed. The isolated RNA was run on a 5% polyacrylamide-urea gel and quantitated by densitometric measurement of the 120 nucleotide pYLTPO-dependent band. Brackets: total amount of DNA contained in an egg after I 1, 12, 13 and synchronous cleavages.

0.5

I .o

1.5

2.0

2.5

log (ng DNA/egg) opus DNA present at the MBT strongly suggests that the timing of the MBT is controlled by a mechanism that measures the amount of DNA in the embryo. Discussion The earliest developmental stage of Xenopus embryogenesis consists of a period of rapid cleavage divisions. These divisions are nearly synchronous for ail cells within the embryo and occur approximately every 35 min. This rapid cleavage program terminates abruptly after 12 divisions (-4000 ceils per embryo) and, as we have shown in the accompanying paper, is followed by the activation of several new cell activities. These include desynchronization of the embryonic cell cycles, elongation of these cell cycles as a result of the inclusion of Gl and G2 phases and elongation in the lengths of both S and M phases; the onset of cell motility; and activation of RNA transcription in all ceils of the egg (Newport and Kirschner, 1982). The transition from the completion of the rapid cleavage period to the onset of these new activities occurs within 1 hr of cleavage 12 (-7 hr after fertilization) and is known as the midblastula transition (Signoret and Lefresne, 1971; Gerhart, 1980). We have investigated the mechanism regulating transcription during early embryogenesis in Xenopus and used this transcriptional regulation as a probe for determining which parameters are involved in timing the onset of the MBT. We have shown that after injection into a Xenopus egg the transcriptional activity of a DNA plasmid (pYLT20) containing a yeast leucine tRNA gene changes in a developmentally regulated fashion. When this plasmid is injected into fertilized eggs prior to the time of the MBT, it is initially active as a template for transcription; however, it loses all template activity within 2 hr. We have shown that

transcriptional inactivation of pYLT20 in the pre-MBT cytoplasm is not due to DNA degradation but rather to a modification of this template. However, the injected template is active again for RNA transcription at the MBT, showing that this modification is reversible and that this reversal occurs at the same time that endogenous transcription becomes activated for the first time. These results strongly suggest that the injected plasmid is modified in such a way that its transcriptional activity becomes regulated by the same molecular factors controlling the transcription of endogenous DNA both before and after the MBT. These experiments were all carried out with a plasmid carrying a tRNA gene; therefore, the conclusions might be restricted to endogenous tRNA synthesis (that is, transcription dependent on RNA polymerase Ill). However, we feel that the suppression of pYLT20 transcription in pre-MBT embryos is not due to the binding of a specific tRNA gene repressor, since we injected approximately 6 x 10’ copies of pYLT20 per egg, or about 500 times the number of tRNA genes present in MBT embryos, if one assumes that the Xenopus genome contains 1000 tRNA gene copies. If a specific repressor were present that was responsible for suppressing tRNA synthesis in stoichiometric amounts, it should have inactivated less than 1% of the injected plasmid. On the basis of this argument and the experiments described below we think it unlikely that the inactivation of transcription of pYLT20 in pre-MBT cytoplasm is due to the binding of a specific repressor-like protein to the promotor of the leucine tRNA gene carried by this plasmid. Rather, it appears much more likely that inactivation is due to a less specific event involving modification of all of the DNA carried by the plasmid, which would prevent further transcription from occurring. In an attempt to understand the molecular nature of

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the transcriptional suppression of pYLT20 DNA in preMBT eggs, we have shown that the plasmid is assembled into chromatin when injected into fertilized eggs. These results are similar to those obtained by Laskey with SV40 DNA injected into Xenopus oocytes (Laskey et al., 1977; Wylie et al., 1978). By using a method that prevented cleavage in fertilized eggs but allowed DNA synthesis to continue normally (Newport and Kirschner, 1982), we could inject pYLT20 DNA at any time during the early developmental period. This DNA was converted into chromatin in approximately 2 hr in either pre-MBT or post-MBT eggs. However, when pYLT20 plasmid DNA was injected into post-MBT cleavage-arrested eggs, it remained transcriptionally active for at least 3 hr, demonstrating that the inactivation of pYLT20 DNA transcription injected into pre-MBT eggs is not due exclusively to the assembly of the DNA into chromatin. Nevertheless, the fact that it takes 2 hr for the plasmid to become transcriptionally inactive in pre-MBT eggs and to assemble into chromatin in this cytoplasm suggests that assembly into chromatin is a necessary step along the pathway to transcriptional inactivation. As we have shown, the timing of the MBT depends on reaching a critical ratio of nucleus to cytoplasm (Newport and Kirschner, 1982; see also Kobayakawa and Kubota, 1981). This was achieved by varying independently either the initial nuclear content or the initial cytoplasmic content. We propose that when this ratio is reached, a component initially piesent in the cytoplasm of an unfertilized egg is depleted from the cytoplasm via titration with nuclear material. Thus in this model we would predict that the pool of this component in an unfertilized egg is sufficient to allow for 12 cleavage divisions, or the accumulation of approximately 4000 nuclei, before the pool is depleted. The nucleus is an extremely complex structure consisting of numerous molecular components including DNA, chromatin and nuclear matrix materials, as well as associated structures such as centrioles. The titratable element whose cytoplasmic depletion results in the MBT could be a factor necessary to form any one of these components, or it could bind to one of these structures once they have formed. We have CLEAVAGES

II

shown that both pYLT20 DNA and endogenous Xenopus DNA are reactivated for transcription in pre-MBT eggs when excess pBR322 DNA is injected into these eggs. This suggests that the element normally suppressing transcription of both pYLT20 and endogenous DNA in pre-MBT cytoplasm can be removed from these templates by competing DNA. The fact that this competition does not become effective in reactivating transcription until the competing DNA reaches a value equivalent to that which would be present at the MBT (-24 ng DNA per egg) argues that in unfertilized eggs the suppressing factor is present in quantities sufficient to control the timing of the MBT-that is, it is not synthesized in significant quantities during the rapid cleavage period. On the basis of this evidence, we suggest that the timing of at least the activation of transcription seen at the MBT is due to the depletion of a factor that binds to chromatin. This model predicts, as illustrated in Figure 8, that the unfertilized egg contains a large cytoplasmic pool of a factor which, when bound to chromatin, is capable of suppressing RNA transcription. At the end of each synchronous round of DNA synthesis the total amount of DNA in the whole egg is doubled and the new chromatin titrates a portion of the factor from the cytoplasmic pool. The depletion process continues until the completion of round 12 of DNA synthesis, at which time the cytoplasmic pool is depleted. After cleavage 12 the factor bound to DNA becomes diluted with each subsequent round of DNA synthesis, and this dilution process allows for the activation of developmentally programmed transcription. Because the amount of chromatin present in an egg during the rapid cleavage period is increasing exponentially, a titration scheme such as that described above could account for the highly coordinated fashion in which all cells of the embryo go through the MBT within a very short time of each other; for example, two cells with as much as a twofold difference in the amount of this regulating protein would be separated by only 0.5 hr in terms of the time at which they would undergo the MBT. The experiments presented in this report allow us to present a model for only the regulation of the activation of transcription at the MBT. However, this

12

Figure 8. Titration Transcription

Model

for

the

Onset

of

At cleavage 11, excess suppressor molecules (stippled circles) are present in the cytoplasm and saturate the DNA template. At cleavage 12, the DNA level has doubled and stoichiometric titration is achieved. Subsequent cleavage leads to lack of saturation of the DNA template and activation of transcription. RNA synthesis

-

GI. G2

-

motiliry

-

-

+ + +

Developmental 695

Regulation

of Transcription

not the only new cellular activity activated at this time (Newport and Kirschner, 1982). At the MBT the cell cycles of the cells within the embryo elongate as a result of the inclusion of Gi and G2 phases and the elongation of S and M phases and become asynchronous relative to each other, and most of the cells becomes motile (see Newport and Kirschner, 1982). We do not know whether the factors that suppress RNA transcription during rapid cleavage also regulate other processes during this period. One way in which all of the events associated with the MBT could be controlled by a common regulatory mechanism would be if the rapid DNA synthesis and cleavage in this period prior to the MBT itself precluded transcription and cell motility from occurring. For example, all of these events, including transcription, may require a Gl or G2 period for initiation in the Xenopus embryo. When nuclei from different tissues and species are transplanted into Xenopus eggs they stop RNA transcription, swell in size (as a result of the uptake of Xenopus proteins) and begin DNA synthesis, all within 90 min of transplantation (Gurdon and Woodland, 1968). This demonstrates that the egg cytoplasm contains all the proteins necessary to convert a nucleus which normally replicates its DNA, a small segment at a time during a 6-10 hr S phase, into a nucleus which will replicate all of its DNA rapidly. Such rapid DNA synthesis probably requires substantial rearrangement and modification of both the nucleus and the chromatin. If chromatin modification is a prerequisite for rapid DNA synthesis, one effect of this modification might be the inactivation of RNA transcription on this DNA. Thus the inactivation of pYLT20 DNA transcription in pre-MBT cytoplasm could be due to the binding of a factor or factors that would normally play a role in activating chromatin for rapid replication. Whether all the cellular functions that seem to be coordinatedly controlled at the MBT are regulated by a single factor has not been determined. If they are, an understanding of the MBT would allow us to integrate the phenomena of initiation of DNA replication, cell cycie control, transcription and cell motility, all of which are important in early development.

(pH 7.0), mM EDTA and SDS by being passed through a Pipetman tip several times. This solution was immediately extracted twice with an equal volume of phenol, equilibrated with 10 mM Tris-HCI (pH 7.01, ether-extracted once with 5 volumes of ether (to remove phenol); following this procedure, the solution was brought to a concentration of 300 mM in sodium acetate, and RNA was precipitated at -70°C for 1 hr after addition of 2.5 volumes of ethanol. The precipitated RNA was centrifuged for 5 min in an Eppendorf centrifuge, washed once with 100% ethanol, repelleted, dried briefly, then resuspended in 5-20 ~1 TEE, 5 M urea and bromophenol blue. Typically, 5 ~1 of this solution was loaded onto a 40 cm long, 0.3 mm thick, 5% polyacrylamide gel made up in TBE plus 7 M urea and run at 1800 V for 2 hr in TBE buffer. The gels were autoradiographed for 1-7 days at -70°C with an intensifying screen. Plasmid pYLT20 DNA was labeled in viva in E. coli. The bacteria were grown in L broth plus tetracycline and ampicillin until they had reached a density of 5 x lO’/cc. They were then pelleted and transferred to M9 plus 5% glucose and 200 pg/ml choramphenicol plus 5 mCi/liter ‘*P-PO4 and grown for another 12 hr, and plasmid DNA was isolated. Radioactively labeled supercoiled pYLT20 DNA was isolated from relaxed circles via acid-phenol extraction. The labeled DNA had a specific activity of 1 O5 cpm/$g. TBE is 100 mM Tris base, 100 mM boric acid and 2 mM EDTA. MMR is 5.0 mM HEPES, 100 mM NaCI. 2 mM KCI, 1 .O mM MgSO+ 2.0 mM CaCI, and 0.1 mM EDTA at pH 7.8.

Experimental

Gerhart, J. G. (1980). Mechanisms regulating pattern formation in the amphibian egg and early embryo. In Biological Regulation and Development, 2, R. F. Goldberger, ed. (New York: Plenum Press).

is

Procedures

Materials Radioactively labeled nucleotides were obtained from Amersham. Plasmid pYLT20 DNA was a gift from D. Standring. Xenopus eggs were obtained and fertilized as previously described (Newport and Kirschner, 1982). Cleavage-Blocked Eggs To block cleavage, the eggs (in MMR plus 5% Ficoll) were centrifuged on a cushion of 50% Ficoll at 500 g for 10 min, 40-60 min after fertilization. These eggs will occasionally form small but incomplete cleavage furrows. They remain healthy for at least 12 hr, as judged by their ability to continue to synthesize both DNA and RNA. Eggs were injected in MMR plus 5% Ficoll. Normally, three to five eggs were lysed in 100 (11of 10 mM Tris-HCI

Acknowledgments We thank John Gerhart (U. C. Berkeley) for helpful discussions and David Standring (U. C. San Francisco) for advice on the plasmid transcription experiments and for providing the pYLT20 plasmid. We thank Sumire Kobayashi for the HPLC analysis of the rUTP pools. We thank Marilyn Hersh for help in preparing this manuscript. This work was supported by grants from the American Cancer Society and the National Institutes of Health. J. N. is a fellow of the National Institutes of Health. 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

June 11, 1982;

revised

July 23, 1982

Adamson, E. D. and Woodland, H. R. (1974). early development: histone and DNA synthesis J. Mol. Biol. 88, 263-285.

Histone synthesis in are not coordinated.

de Robertis, E. M. and Nishikura, K. (1981). RNA processing oocytes microinjected with cloned Qenes. In International Cell H. G. Schweiger, ed. (Berlin: Springer-Verlag), pp 60-65. de Robertis, E. M. and Olson, M. W. (1979). processing of cloned yeast tyrosine tRNA genes frog oocytes. Nature 278, 137-143.

in frog BiOlOQy,

Transcription microinjected

and into

Gurdon, J. B. and Woodland, H. R. (1968). The cytoplasmic control of nuclear activity in animal development. Biol. Rev. 43, 233-267. Kobayakawa, Y. and Kubota, H. (1981). Temporal pattern of cleavages and the onset of gastrulation in amphibian embryos developed from eggs with reduced cytoplasm. J. Embryol. Exp. Morphol. 62, 83-94. Laskey. R. A., Mills, A. D. and Morris, N. R. (1977). Assembly of SV40 chromatin in a cell-free system from Xenopus eggs. Cell 70, 237-243. Newport, J. N. and Kirschner, M. W. (1982). A major developmental transition in early Xenopus embryos: I. Characterization and timing of cellular changes at the midblastula stage. Ceil 30, 675-686.

Cell 696

Roeder, R. G. (1974). Multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in Xenopus laevis. J. Biol. Chem. 249, 249-256. Signoret, J. and Lefresne, J. (1971). Contribution of I’etude segmentation de I’oef d’axolotl. I. Definition de la transition uleenne. Ann. Embryol. Morphogen. 4, 113-l 23. Southern, fragments 503-517.

de la blast-

E. M. (1975). Detection of specific sequences among DNA separated by gel electrophoresis. J. Mol. Biol. 98,

Standring, D. M.. Venegas, A. and Rutter, W. J. (1981). Yeast tRNAJ leu gene transcribed and spliced in a HeLa cell extract. Proc. Nat. Acad. Sci. 78, 5963-5867. Wylie, A. H., Laskey, R. A., Finch, J. and Gurdon, J. 6. (1978). Selective DNA conservation and chromatin assembly after injection of SV40 into Xenopus oocytes. Dev. Biol. 64, 178-l 88.