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Developmental Biology Tutorial
Translational Control of Early Development
Dr. Derrick Rancourt
Department of Medical Biochemistry
University of Calgary
As we discussed previously, the early
developmental program is directed through the translational regulation of
maternal mRNAs. At oocyte maturation and/or fertilization, a significant
surge in protein synthesis is evident. Using fertilization in sea urchin
as our primary model ,we shall discuss how oogenic mRNA translation is regulated
in early development.
The key features of this topic are:
- Translational upregulation at fertilization is accompanied by:
a) increased intracellular pH and Ca++
b) increased polyribosome content and translational efficiency
- Translational suppression before activation may be regulated by:
a) maternal mRNA modification, masking and/or sequestration
b) alterations of key translational regulatory protein
The Oocyte Translational Machinery
The steps of translation (initiation, elongation
and termination) are outlined in your textbook. Be aware of the different
steps in general terms (i.e., what has to come together for initiation to
occur; what is required for elongation?)
(See Browder et al., 1991, Figures 13.4 A and B; Gilbert, 1997,
Figs. 12.10 and 12.11; Kalthoff, 1996, Figs. 17.2 and 17.3; Shostak,
1991, Fig. 4.9)
Since transcription is completely dispensable in early development, this
suggests that most or all of the translational machinery is present in the
oocyte at the time of maturation and/or fertilization.
There are both indirect and direct methods that demonstrate that the
oocyte is full of mRNA. While mRNAs are often found as ribonucleoprotein
particles in cells, during preparations of total embryonic RNA, the RNA
is striped of its proteinaceous components during chemical purification.
mRNA is then purified from total mRNA by virtue of its 3' polyadenlylate
tail, using affinity chromatography. In general, mRNA represents only 1%
of total RNA. Depending on the oocyte, as many as 6 to 11 thousand different
mRNA species will be present.
Based on the number of copies of an individual mRNA in the oocyte, there
are various mRNA classes ranging from abundant to rare. The complexity of
the oocyte mRNA is a function of the total amount of mRNA, the total number
of different transcripts, and the abundance of each class.
(See Browder et al., 1991, Fig. 13.12)
- Which class would encode cytoskeletal proteins, regulatory proteins?
Individual transcripts can be analyzed using Northern hybridization.
Here, mRNA (or total RNA) is separated on a gel, and transferred to a nylon
matrix. Bands representing specific mRNA species, are detected by hybridization
with a radioactive complimentary DNA or RNA probe. The radioactive band
may be exposed on X-ray film.
(For examples, see Browder et al., 1991, Fig. 13.11; Gilbert,
1997, Fig. 12.15; Kalthoff, 1996, Methods 14.3, p. 347)
In another approach, mRNA species can be detected in situ, within
cells, tissues, embryos, etc. Here, the radioactive probe is exposed to
cells and localization of the specific transcript is detected as spots which
appear using a clear photo-emulsion . More recently, in situ hybridization
uses probes that are non-radioactive and have moieties that are recognized
by enzyme-linked antibodies so that mRNA localization can be stained enzymatically.
(For examples, see Browder et al., 1991, Fig. 13.16; Gilbert,
1997, Figs. 12.15, and 12.22; Kalthoff, 1996, Methods 15.1, p. 355 and Fig.
16.19; Wolpert et al., 1998, Box 3B, p. 65)
mRNA can also be observed indirectly using in vitro translation in
the presence of radioactive amino acids. When the products of translation
are separated on a two dimensional gel, hundreds of spots will appear representing
different proteins (and mRNA).
(See Browder et al., 1991, Fig. 13.10).
The fact that in vitro translation can occur with oocyte extracts
suggests that all of the components of translation are at least present.
- Why is this technique only useful for detecting abundant mRNAs?
Ribosomes and tRNA
Greater than 95% of oocyte RNA is rRNA (28S, 18S and 5S). When total
RNA is subjected to sucrose density gradient centrifugation, 28 and 18S
components can be resolved. While we know that
maternal rRNA is in sufficient abundance to take the embryo beyond cleavage,
studies using the anucleolate mutant of Xenopus suggest that maternal
stores of 28 and 18S are sufficient to complete embryogenesis.
(See Browder et al., 1991, Fig. 13.13)
Homozygous recessive mutants fail to make a nucleolus and thus do not generate
28 and 18S rRNA. Heterozygotes make 1 nucleolus and are viable. When heterozygotes
are crossed, 1/4 of the progeny will be mutant. While such mutants do not
generate 28 and 18S rRNA, rRNA originates from the 1-nu mother. Surprisingly,
these embryos will survive to the tadpole stage.
Note that in anucleolate animals 5S rRNA and tRNA (4S) synthesis occurs.
In wild type embryos there is an excess of 5S and tRNA compared to 28 and
18S. For tRNA there is about a 5 fold excess.
- Does that seem like enough tRNA?
Increased Translational Efficiency at Fertilization
In the sea urchin, fertilization
coincides with an increase in protein synthesis.
This is measured by preloading oocytes with radioactive leucine and then
fertilizing them. At different time points post-fertilization, the proteins
are isolated from radioactive amino acids and then counted. As you can see
the synthesis of proteins is considerably enhanced after fertilization.
Fertilization also results in an slight increase in intracellular pH and
Ca^++, both which affect translation positively. The effects of pH on protein
synthesis are illustrated in Browder et al., 1991, Fig. 13.8.
In oocytes protein synthesis can be activated in ammonium chloride-containing
seawater. This increased level of protein synthesis will subside once the
oocytes are returned to normal seawater (panel A). Similarly protein synthesis
can be modulated in fertilized eggs simply by perturbing intracellular pH
(B and C). Here when sodium acetate is used to bring the pH down to pre-fertilization
levels, protein synthesis drops dramatically (down to pre-fertilization
levels), but can be rescued by returning to sea water.
(N.b., the effects of pH on protein synthesis are also illustrated
in Gilbert, 1997, Fig. 12.21)
- Why would subtle pH changes affect translation?
The intracellular release of calcium ions also augments the effect of
pH on protein synthesis. When oocytes are treated with the ionophore A23187
(which stimulates intracellular Ca++ release), in conjunction
with ammonia, protein synthesis rates are comparable to fertilized oocytes.
By itself, Ca++ does not stimulate translation, but
in conjunction with ammonia, rates of protein synthesis are greater than
with ammonia alone.
(See Browder et al., 1991, Fig. 13.9)
Changes in Translational Efficiency
Changes in the efficiency of protein synthesis that occur at fertilization
can be manifested by changes in the appearance of polysomes. Polyribosomes
are resolved from monoribosomes by sucrose density centrifugation and are
measured via optical density at 260nm. Once fractions are collected from
the bottom of the gradient, heavily loaded polysomes will come off first,
followed by less loaded polysomes and then the monosome fraction. Alterations
in the ribosome density of polysomes is measure by changes in the position
of the polysome curve relative to fractions. Alterations in the area under
the polysome curve represent changes to the numbers of polysomes present.
(See Browder et al., 1991, Fig. 13.5)
Theoretically an increase in the density of polysomes (# ribosomes per mRNA)
would reflect an increase in the efficiency of initiation. Similarly a decrease
in the density of polysome would reflect more efficient elongation or a
shorter transit time. Neither of these situations occur during fertilization,
since the relative density of ribosomes remains constant before and after
(See Browder et al., 1991, Fig. 13.7)
Thus, if protein synthesis increases yet polysome density does not change,
this indicates that both initiation and elongation efficiencies are increased
- How could initiation and elongation efficiencies be increased?
Increased translational efficiency can also be manifested by increases
in the numbers of polyribosomes. A dramatic increase in polysome number
is observed after fertilization and accounts for over 50% of the 100-fold
increase in protein synthesis that is observed. Increased initiation and
elongation efficiency accounts for the rest.
(See Browder et al., 1991, Fig. 13.6)
Mechanisms of Translational Suppression in Oocytes
Because oocyte extracts are sufficient to translate proteins in vitro,
this suggests that all of the translational components are present in the
oocyte, perhaps in short supply. Since there are many components in translation,
the general suppression that occurs in oocytes may be regulated by a number
Alterations in Maternal mRNA
Recall that mRNAs having a short (or absent) poly(A) tail are not translated
efficiently. During early development, maternal mRNAs that become translationally
activated also have increased poly(A) tail length. Cytoplasmic polyadenylation
occurs on maternal mRNAs that have a CPE element. Interestingly, when this
CPE element is added to heterologous mRNA (i.e., globin), then the translation
of this new mRNA is developmentally-activated.
- While poly(A) control of translation is a general translation control
point, CPE's are found only in specific maternal mRNAs. How could other
maternal mRNAs act through this same control mechanism?
Another mRNA structural change that is correlated with translational
activation is cap methylation. Oogenic mRNAs from unfertilized eggs are
unmethylated and often become capped after fertilization. Whether this is
really a true translational control mechanism remains to be seen.
Finally, RNA secondary structure can also affect mRNA translatability. Often
because of thermodymic stability, regions of mRNA will fold on itself to
form double stranded regions (hairpin structures). These structures make
certain mRNAs more difficult to translate than others. Oocyte factors that
unwind mRNA secondary structure are more active post fertilization.
Within the cell, untranslated mRNAs are complexed with proteins forming
ribonucleoprotein (RNP) particles. As such, RNP proteins have the potential
to mask maternal mRNA. While the composition of mRNPs has been found to
differ between fertilized and unfertilized oocytes, only recently has evidence
supporting the masked mRNA hypothesis been found. In Xenopus, it
has been observed that histone H1 mRNA synthesized in vivo, fails
to be translated in oocytes, while transcripts synthesized in vitro
do not. A key RNP protein FRGY2 has been directly implicated in masking
mRNA following transcription.
- How could maternal mRNAs become unmasked?
mRNA Sequestration (localization)
Another way to keep mRNA away from the translational machinery is to
sequester it within the cell. For instance in sea urchin ooctyes, histone
mRNA is found in the female pronucleus, and it is not until nuclear envelope
breakdown, that the mRNA is released. While compartmentalization may prevent
specific mRNAs from being translated, localization might also allow certain
mRNAs to be preferentially translated. In oocytes, the translational machinery
has been found to be associated with the cytoskeleton. Therefore the cytoskeletal
localization of specific mRNAs may enhance their chances of being translated.
In Drosophila, for instance specific maternal mRNAs associated with
polarity of the egg, are localized to cytoskeletal elements and are translated
The Translational Machinery
Experiments have demonstrated that mRNA is not the factor that limits
translation in the oocyte. For instance, when globin mRNA is microinjected
into freshly-fertilized sea urchin oocytes, its translation affects the
synthesis of other oocyte proteins. This result suggests that the translational
machinery is limiting.
As you know, fertilization acts as a trigger to initiate a program of
events starting with cleavage and continuing with gastrulation and neurulation,
etc. However, although fertilization results in union of maternal and paternal
genomes, zygotic gene activity is not required until the blastula.
In fact, after fertilization and through cleavage, the maternal, paternal
and zygotic genomes are completely irrelevant in many species. This is because
in the egg, there exists a stockpile of maternally derived mRNAs which govern
embryogenesis through cleavage to the blastula stage. After the formation
of the blastula, zygotic gene transcription is activated, which carries
the embryo through the rest of embryogenesis.
Take-Home Message: Early embryonic events are orchestrated through the
postranscriptional control of maternal mRNA.
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.
Kalthoff, K. 1996. Analysis of Biological Development. McGraw-Hill.
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.