CONTENTS

Zygotic Control of Early Development

by Dr. Derrick Rancourt
Department of Medical Biochemistry
University of Calgary

As we have previously discussed, although the oocyte translational machinery is complete, in general, translation does not occur until fertilization (or oocyte maturation), because of numerous regulatory events. We touched upon some examples of specific translational regulation. We shall now continue with other examples of specific regulation. However, the bulk of this topic will be devoted to the zygotic control of early development.

Key point:

As development continues beyond fertilization, there is a switch from control by the maternal genome (maternal mRNA) to the zygotic genome.

The key features of this topic will be that:

In many organisms transcription of the zygotic genome occurs during the mid blastula transition. This transition is hallmarked by increases in cycle times and nuclear-cytoplasmic ratio which appear to dilute negative transcriptional regulators. Zygotic translation is largely coupled to transcription and is accompanied by the destruction of maternal mRNAs.

Specific Translational Regulation in Early Development

As you will recall, the translation of maternal mRNAs is controlled generally by intracellular pH and calcium. Key molecules within the translational machinery (i.e. initiation factors) could be modified to dampen translation. As well, the structure of maternal mRNAs can be altered (i.e. polyA tail, cap methylation, secondary structure) or mRNAs can be sequestered within the cell or be masked by specific RNP proteins. While alterations in mRNA represent general mechanisms of translational regulation, they can also be specific. As we discussed last time, for instance the cytoplasmic polyadenylation element (CPE) allows certain mRNAs to be preferentially polyadenylated after fertilization. A number of examples of specific translational regulation are shown in your textbook.

An excellent example is that of cyclin A in sea urchin. This protein, as you will recall, is a component of M-phase promoting factor (MPF) and is important in both mitosis and meiosis. In sea urchin embryos, the amount of this protein occurs in waves during cleavage, such that its concentration is highest during interphase. Since the maternal mRNA is stable, this result suggests that not only is translation of cyclin coordinated with the cell cycle but so is its degradation.

(See Browder et al., 1991, Fig. 13.18; Kalthoff, 1996, Figs. 17.11 and 17.12)

In addition to proteins being degraded, maternal mRNAs may also be degraded according to a specific program. Examples of this are the messsages for the ribosomal proteins of Xenopus, the majority of which are degraded at oocyte maturation. Interestingly, three of these RNAs continue to be translated through cleavage and escape degradation.

So in the early developmental program a number of different things can be happening. Maternal mRNAs can be translated during oocyte maturation, after which there is sufficient protein to carry the embryo through early development. Thus this mRNA can be degraded. In other cases, mRNA may need to be stable and translated a number of times throughout cleavage. Finally some maternal mRNAs are not needed until the blastula stage or even in some cases as late as gastrulation.

The most interesting aspect of this is that all of this regulation appears to be able to occur without input from the nucleus (the command centre). We saw this earlier when we were discussing physical and chemical enucleation. Another example is seen in Ilyanassa. You will recall that during cleavage in Ilyanassa an anucleate polar lobe is formed. The polar lobe can be removed from the cleaving cells and yet appropriate proteins will be translated in their appropriate order.

(See Browder et al., 1991, Fig. 5.30, Fig. 13.21; Kalthoff, 1996, Fig. 8.3; Wolpert et al., 1998, Fig. 6.13)

Zygotic Control of Development

If you recall the experiments on interspecies hybridization, you'll remember that early embryonic development mimicked that of the maternal species, while later after the blastula stage signs of paternal contribution became evident. As well, when isozymes are analysed, the expression of the paternal enzyme occurred around the blastula-gastrula stage.

Hybrids made between the sea urchins Strongylocentrotus and Sphaerechinus illustrate the onset of zygotic control. In this experiment, paternal contribution is manifested by the appearance of spicules that do not appear in the maternal Strongylocentrotus embryo. Here we see two examples of hybrids, both of which display spicules suggesting paternal contribution, later in development. The interesting aspect is that the contribution can vary from embryo to embryo, suggesting that there is a lot of plasticity in development and that it is not completely hard-wired. It may be hard-wired early on when the maternal program directs development, but later on, it occurs as a consequence of compromise between different genetic elements. Each time the compromise is different.

(See Browder et al., 1991, Fig 13.22)

Treatment with actinomycin D demonstrates that the formation of these structures is dependent upon zygotic transcription.

Activation of Zygotic Transcription

After fertilization, organisms appear to differ in their regulation of zygotic transcription. Xenopus and Drosophila have a period of transcriptional quiescence that occurs during cleavage. At this time, cells are undergoing rapid cellular divisions. In Xenopus, transcription occurs at the midblastula transition, at which time cleavage has slowed down considerably.

(See Browder et al., 1991, Fig. 13.23; Shostak, 1991, Fig. 15.14, Wolpert et al., 1998, Figs. 3.33 and 3.34)

Similarly in Drosophila, transcription is observed by the cellular blastoderm stage, which is at cleavage cycle 10. In Drosophila and Xenopus, it takes roughly 10 and 20 minutes respectively for early cleavage cells to divide. That's incredibly fast when you bear in mind that a bacterium takes roughly 20 minutes to do the same thing.

Question:

How does Drosophila, which has 100 times more DNA than E. coli, or Xenopus, which has 1000 times more DNA than E. coli, manage to replicate its DNA in equal or less time during cleavage?

Because Drosophila and Xenopus are so busy replicating their genomes, it has been suggested that the general down-regulation of transcription is related to relatively short interphase periods. It is not until the cell cycle slows down that transcription is even possible.

In sea urchin, there is no quiescent period. mRNA transcription increases gradually over the time of cleavage. For instance, northern blotting demonstrates two zygotic forms of histone H2B. The early form, which is both maternal and zygotic, declines in its levels immediately following fertilization and then accumulates during cleavage. Interestingly, sea urchins synthesize an early set of histones that are found predominantly during cleavage. The expression of the later set of histones appears at the blastula stage, and by the gastrula stag the histones that are synthesized are specifically of the late form.

(See Browder et al., 1991, Fig. 13.26)

Question:

Why synthesize a cleavage-specific set of histones?

Whereas mRNA transcription increases through cleavage, tRNA and rRNA transcription do not occur until the blastula stage.

Mouse is similar to sea urchin in that mRNA transcription is detected as early as the 2 cell stage, even in the late zygote. New rRNA is observed, as well, at the 2 cell stage, while tRNA appears at the 4 cell stage. Treatment with transcription inhibitors affect cleavage at the two cell stage.

Is There a Unifying Mechanism Regulating Zygotic Transcription?

One thing that bothers developmental biologists is that there isn't a unifying principle regarding the regulation of zygotic transcription since the sea urchin and mouse do not display a type of mid-blastula transition. It has been noted that the cell cycle may be one regulatory point. In mouse the cell cycle is very long and it can be argued that there is ample time for transcription to occur. Recall, as well, that that sea urchin has a number of blastomeres of varying size, which undergo asynchronous cleavage.

Question:

Can asynchronous cleavage be used to explain why the sea urchin does not appear to have a midblastula transition? How would you test this?

One observation that refutes the notion that zygotic transcription is linked to the cell cycle is that in mouse, transcription can occur even if the cell cycle is arrested in S phase. Interestingly, zygotic transcription occurs regardless of replicative state or what cell cycle stage the embryo is in. As a result it has been suggested that mouse zygotic transcription might be regulated by a time dependent mechanism....the so-called zygotic clock.

Experiments in Xenopus have refuted that idea that there is a zygotic clock. When embryos are ligated such that two separate cleavage embryos result where cleavage is delayed on one side, transcription is observed only on the side where the blastula resides and not the side of the immature blastula. Since transcription occurs only on the side of the later embryo, this suggests that a clock is not working.

(See Browder et al., 1991, Fig. 13.29)

Maternal Cytoplasmic Factors May Regulate Zygotic Transcription

Developmental biologists believe that negative cytoplasmic factors may in fact regulate zygotic transcription and that these are mediated by nuclear/cytoplasmic ratios. This idea first emerged from work on Xenopus where it was observed that transcription from actively transcribing nuclei was repressed when the nucleus was transferred to a quiescent cytoplasm. This was confirmed by the experiment just discussed, because only in the embryo with the right number of cells (therefore correct nuclear-cytoplasmic ratio) does transcription occur.

Other observations that support this theory in Xenopus include:

polyspermy or removal of cytoplasm advances the MBT
haploid embryos have a retarded MBT
suppressed gene transcription can be alleviated by injection of extraneous DNA

(See Browder et al., 1991, Fig. 13.28)

(Interestingly, when male and female pronuclei of newly-fertilized mouse oocytes are microinjected with a DNA construct, transcription is observed only from male pronuclear injections, suggesting that these negative elements have not yet begun to occupy the male pronucleus.)

Question:

Since blastular cells differ in size within the Xenopus embryo, how can nuclear/cytoplasmic ratios regulate transcription?

Cytoplasmic Inhibitor Theory Confirmed using Drosophila Genetics

Because Drosophila genetics has uncovered a number of zygotic lethal mutations, a number of markers of early zygotic transcription have been discovered. These represent the gap and pair rule genes which will be discussed in greater detail in the next section of lectures. By definition, zygotic lethal genes represent anything that is lethal when homozygous. The earliest zygotic lethals such as the gap gene Krupple and the pair rule gene fushi tarazu (ftz) are expressed as early as cell cycle 10, when the cellular blastoderm is forming.

Expression is of these genes is detected by in situ hybridization; by cycle 10 both genes appear to be expressed according to their normal pattern. Interestingly, expression can be detected as early as cell cycle 8; however at this time expression is limited to a few scattered nuclei in the periphery of the embryo. This result suggests that while the bulk of early transcription occurs at cycle 10, the regulation of zygotic transcription is leaky, and transcription at cycle 8 is stochastic. This observation is consistent with the nuclear/cytoplasm theory, since negative regulators within embryo may be locally depleted in a stochastic manner. (Recall that at cycle 8, the Drosophila embryo is still a syncytium.)

A second experiment, which confirms the first, relates to the use of a mutant gs(1)N26. In this mutant, nuclei within the syncitium migrate non-uniformly to the periphery, first in the anterior and later in the posterior. In this mutant, ftz expression is first observed in the anterior of the embryo and later followed by correct posterior expression. Because the nuclear/cytoplasm ratio is increased anteriorly first, depletion of cytoplasmic inhibitors occur there first.

Tramtrack is a Maternal Inhibitor of Zygotic Gene Expression

Tramtrack (ttk) is a maternal effect lethal gene that encodes a zinc finger protein which in part acts as a repressor of ftz transcription. Interestingly, when the dosage of ttk loaded into the oocyte is altered, a significant effect on zygotic ftz expression is observed. When maternal ttk levels are lowered using heterozygous mothers, ftz transcription is observed one cell cycle earlier (In this experiment, homozygous mothers cannot be used since very abnormal embryos result.). Alternately, if extra ttk protein is loaded into the oocyte using exogenously added ttk genes, ftz expression is delayed.

The Switch from Oogenic to Zygotic Translation

Compared with maternal mRNA in oocytes, the translation of zygotic mRNA is largely linked to transcription. The efficiency with which zygotic translation occurs relates to the degradation of maternal mRNA after fertilization. In mouse, this degradation is very rapid, occurring the two cell stage, after which translation of these products occur directly from zygotic sources. This translation correlates with the appearance of new proteins derived from zygotic mRNA. This example serves to indicate that many proteins have both a maternal and a zygotic phase.

(See Browder et al., 1991, Fig. 13.25)

Question:

How could you distinguish between maternal and zygotic phases? What type of proteins do you think would have both maternal and zygotic phases?

Interestingly, maternal effect genes isolated in Drosophila have indicated that the programmed degradation of maternal mRNAs may also be related to the nuclear/cytoplasmic ratio. How this occurs is unknown.

References

Browder, L.W., Erickson, C.A. and Jeffery, W.R. 1991. Developmental Biology. Third edition. Saunders College Pub. Philadelphia.

Gilbert, S.F. 1997. Developmental Biology. Fifth Edition. Sinauer. Sunderland, MA.

Kalthoff, K. 1996. Analysis of Biological Development. McGraw-Hill. New York.

Shostak, S. 1991. Embryology. HarperCollins. New York.

Wolpert, L., Beddington, R., Brockes, J., Jessell, T., Lawrence, P. and Meyerowitz, E. 1998. Principles of Development. Current Biology. London.

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Last revised Thursday, July 16, 1998