In this article we will discuss about the gene activity during gastrulation period of embryos.
Fragments of sea urchin eggs not containing the nucleus may be treated with agents who cause parthenogenetic activation of the egg (viz., hypertonic seawater). It has been found that the non-nucleated fragments begin to cleave and may reach the morula stage. The animals (embryos), however, do not form a regular blastula and cannot gastrulate, which shows that the presence of nuclei is necessary for development to go beyond cleavage.
During cleavage the main activity of the chromosomes is to duplicate themselves at each cell division. At the completion of cleavage the genes contained in the chromosomes enter a period of new activity, which is the production of large quantities of RNA, particularly messenger RNA.
This production can be recorded and measured in the animal (embryo) with radioactively labeled uridine which is taken up into ribonucleic acid and not into the deoxyribonucleic acid of the genes themselves. After labeling the cells, the ribonucleic acids can be separated chemically.
The product initially obtained is a mixture of different RNA’s; in addition to the messenger RNA, transfer RNA and ribosomal RNA are also present. The different kinds of RNA have different molecular weights, and as a result they are sedimented at different rates when subjected to ultracentrifugation.
Certain fractions isolated in this way represent messenger RNA. The actual amounts can then be estimated by measuring the radioactivity of the sample in an apparatus called a “scintillation counter” (which records the number of particles emitted by the radioactive sample).
Using this kind of method, it has been found that although certain amounts of RNA are produced in the eggs after fertilization and during cleavage; at the approach to gastrulation the production of mRNA increases sharply. The increase is quite sudden, more than tenfold within one hour in the frog Xenopus laevis.
It would be interesting to determine whether or not the mRNA synthesized just before the onset of gastrulation is the same as the mRNA present in the egg cytoplasm or the mRNA produced immediately after fertilization and during earlier cleavage stages. The method of DNA-RNA hybridization has been used in attempts to answer this question.
If a sample of mRNA is mixed with a DNA sample, the RNA molecules will hybridize with corresponding sections of DNA. Once such a hybridization takes place, other RNA molecules, identical to the first ones, cannot find a suitable place on the DNA molecule and thus cannot attach themselves to it.
If a sufficient amount of mRNA is used, and adequate time is allowed for the hybridization to take place, the sites on the purified single-stranded DNA may be saturated with corresponding mRNA’s. If a second sample of mRNA is then added, only those mRNA molecules that are different from the mRNA molecules in the first sample will become attached to the DNA.
For these molecules, corresponding sites on the DNA strand will still be free, whereas the mRNA molecules identical to ones from the first sample will find their places already occupied. Consequently, the proportion of molecules which fails to hybridize is an indication of the degree of similarity between the first and the second samples of mRNA.
An alternative method, which is actually more often used, is to add both samples of mRNA to the same preparation of single-stranded DNA at the same time. If there are similar mRNA molecules in the two samples, they will compete, and the proportion of such molecules hybridizing with the DNA will be reduced, whereas the hybridization of the mRNA molecules which in each sample are different from those in the other sample would proceed unimpeded.
Thus again, a lowering of the proportion of hybridizing molecules is an indication of the presence of the same kinds of mRNA molecules in the samples being compared. Both variations of the method yield essentially similar results.
Using the first variation on sea urchin embryos (animals), it was found that previous hybridization with blastula mRNA reduces subsequent binding of mRNA from late gastrula (to be exact, the “prism” stage) less than previous hybridization with late gastrula mRNA.
The difference, which amounted to about 40 per cent, was due to the fact that some mRNA molecules present in late gastrula (prism stage) mRNA were not identical to blastula mRNA molecules and thus were able to find sites on the DNA molecules not occupied by bound blastula mRNA molecules. These results prove that a proportion of genes which were inactive up to the blastula stage had become active in the gastrula stage.
This experiment is not entirely satisfactory inasmuch as it does not differentiate between repetitive sequences and single copy sequences of DNA (and RNA). It is highly likely, however, that the latter are also among the sequences that are being transcribed in the gastrula and not in the cleaving embryo.
Indirect proof that new kinds of messenger RNA are produced in the gastrula stage is provided by the fact that when protein synthesis becomes enhanced at this stage, the new proteins are qualitatively different from those present in the egg. This has been proved in sea urchins and in amphibians by applying the extremely sensitive methods of immunology. It has been found that the gastrula contains antigens, capable of causing the formation of antibodies, which was not present before, besides containing antigens already present in the egg at the beginning of development.
Another approach to the checking of the changing pattern of protein synthesis in development, and in gastrulation in particular, is by the use of the method of two-dimensional gel electrophoresis. In essence, the method consists of subjecting a protein mixture to electrophoresis in two stages.
In the first stage, known as “iso-electro-focusing,” the protein mixture is passed through a gel column in which there is maintained a pH gradient from 3.5 to 10.5. The proteins migrate along the column depending on their own electric charge. For each protein there is a characteristic pH (isoelectric point) where it does not migrate further in the electric field of the column, since at this pH its oppositely charged sites are balanced.
The gel column is then subjected to a flow at right angles to the first in a medium of acrylamide and sodium dodecyl sulfate. The latter chemical equalizes the electric charges of the protein molecules, and their migration is then solely dependent on their molecular weight.
As a result, the various proteins become distributed within the gel plate in a pattern depending on both the electric charge and the molecular weight of the individual proteins. If the proteins are radioactively labeled, they can be subsequently visualized, by exposing the gel to an x-ray photographic film, as a “fluorogram.”
The spots on the fluorogram, corresponding to the individual kinds of protein, do not of course indicate directly the chemical nature of each. If desired, however, bits of the gel producing individual spots may be cut out and subjected to further chemical analysis.
In one experiment using this method, mRNA was extracted from Xenopus embryos in mid-cleavage stages (Nieuwkoop and Faber’s stages 6½ – 7½) and in mid-gastrula stages (Nieuwkoop and Faber’s stages 11 – 11+). The mRNASs were then allowed to direct protein synthesis in vitro, with the amino acids (including35 S-labeled methionine) supplied in a medium containing reticulocyte lysate.
It may be noted that a number of spots in the two fluorograms are identical. These, incidentally, can serve to establish a reference grid for the differing spots. The more obvious spots, which are present in the second fluorogram and are absent in the first, are marked by arrows.
These spots indicate that the corresponding proteins were not coded for by the mRNA in the cleavage stage, but were synthesized under control of mRNA which only appeared in the gastrula stage, or had only become active at that stage. The spots that are similar in the two fluorograms are due to mRNA which was present both at cleavage and during gastrulation.
It should be noted that the fluorogram of the gastrula stage was obtained after a much shorter exposure than the fluorogram of the cleavage stage, which indicates that the gastrula mRNA was much more active under the conditions of this experiment.
The new activities of genes at the beginning of gastrulation are not restricted to producing new kinds of mRNA (which enables new kinds of proteins to be synthesized). The other kinds of RNA, transfer RNA and ribosomal RNA, must also be supplied in adequate quantities if development is to proceed normally, and we have already seen that these RNA’s, which have a supporting function in protein synthesis, are likewise transcribed from nuclear genes at the start of gastrulation.
What happens if one of these supporting factors is lacking has been shown by studies on a peculiar mutation in the frog Xenopus laevis. In this mutation, the section of DNA which codes for ribosomal RNA is missing. As a result, individuals carrying this character in a homozygous state are unable to synthesize rRNA and are devoid of nucleoli (the “anucleolate” mutation).
In the absence of rRNA there can be no ribosomes and no protein synthesis. The mutation can be perpetuated in the heterozygous state, in which the non-mutant chromosome set provides the locus for rRNA transcription and thus for protein synthesis.
Homozygous individuals, produced by mating two heterozygous parents, start their lives normally, since enough ribosomes are supplied in the egg by the heterozygous genotype of the female to take the animal (embryo) through cleavage and early gastrulation stages. The production of new rRNA and thus of new ribosomes, however, which normally occurs during gastrulation, cannot take place. Protein synthesis as a result slows to a halt, and the embryos die in the early tadpole stage.