In this article we will discuss about the manifestation of maternal genes during the early stages of development.
Evidence has been presented that (1) the differences between the various parts of the embryo are not due to differences in their nuclei but to differences in the cytoplasm. From this it may be inferred that (2) the nuclei of the cleavage cells and the genes contained therein do not control the earliest stages of development of the animal egg.
This does not mean, however, that genetic factors have nothing to do with the early stages of development. The genetic factors, the genes, do play a part during this period, but in a very special way. It is not the genes contained in the nuclei of the blastomeres but the genes in the cells of the maternal body that determine the peculiarities of the egg and its early development. The best-known example of this type of gene action is concerned with the inheritance of coiling in gastropod molluscs.
In the snails, as a rule, the shell is coiled spirally, and the internal organization of the animal shows a corresponding dislocation, some of the organs (heart, kidney, and gills) being twisted around through an angle of nearly 180°. The direction of coiling of the shell is clockwise, if viewed from the apex of the shell.
This type of coiling is called dextral. As an exception, the coiling may be counterclockwise, or sinistral. Individuals having a sinistral coiling of the shell have the internal organs dislocated in the opposite direction from that in dextral individuals. In short, dextral and sinistral individuals are in every detail of organization mirror images of each other.
The eggs of gastropod molluscs show a spiral type of cleavage, and it has been noted that the type of cleavage of the egg has a definite relation to the type of coiling of the adult. If the cleavage is dextral—that is, if the cleavage spindles show a clockwise spiraling—the adult snail also has a dextrally coiled shell. If the cleavage is sinistral, the coiling of the shell is likewise sinistral.
The connection between the type of cleavage and the coiling of the shell is established through the position of blastomere D in respect to the other blastomeres. Since it is from blastomere D that the mesoderm develops, its situation is reflected in the position of the internal organs, and the coiling of the shell is one of the secondary expressions of this asymmetry.
There are some species of gastropods in which all the individuals are sinistral, but the main interest attaches to a species in which sinistral individuals occur as a mutation among a population of normal-dextral animals. Such a mutant was discovered in the freshwater snail Limnaea peregra.
Breeding and crossbreeding of dextral and sinistral snails showed that the difference between the two forms is dependent on a pair of allelomorphic genes, the gene for sinistrality being recessive (1), and the gene for the normal dextral coiling being dominant (L). The two genes are inherited according to Mendelian laws, but the action of any genetic combination is manifested only in the next generation after the one in which a given genotype is found.
Thus, if the eggs of a homozygous sinistral individual are fertilized by the sperm of a dextral individual, the eggs cleave sinistrally, and all the snails of this F1 generation show a sinistral coiling of the shell. The genes of the sperm do not manifest themselves, although the genotype of the F1 generation is Ll.
If a second generation (F2) is bred from such sinistral individuals, it is all dextral, instead of showing segregation as would be expected in normal Mendelian inheritance. In fact, segregation does take place in the F2 generation as far as the genes are concerned, but the new genie combinations fail to manifest themselves, since the coiling is determined by the genotype of the mother.
The genotype being Ll, the gene for dextrality dominates and is responsible for the exclusively dextral coiling of the second generation. Only in the third generation (F3) does segregation in the proportion of 3 to 1 become apparent, and then not as segregation among individuals in each brood, but as a segregation of broods. Each brood—that is, the offspring of an individual of the F2 generation—shows a coiling that is determined by the maternal genotype.
Since the individuals of the F2 generation have the genotypes 1 LL, 2 Ll, and 1 ll, three quarters of them, on the average, produce eggs developing into dextral snails, and one quarter produce eggs developing into sinistral individuals. Of the dextral broods, one third breed true, producing only dextral offspring, and two thirds show further segregation among the broods of the next generation.
It is easy to understand that the results of a reciprocal cross (i.e., fertilization of the eggs of a homozygous dextral individual by the sperm of a sinistral individual) will lead to a somewhat different type of pedigree – the F1 generation will be all dextral (with genotype Ll, and the F2 generation again will be all dextral (with genotypes LL, Ll, lL, and ll). The F3 generation will show segregation among broods, just as in the cross examined above.
The whole case becomes clear if it is realized that the type of cleavage depends on the organization of the egg which is established before the maturation divisions of the oocyte nucleus. The type of cleavage is therefore under the influence of the genotype of the parent producing the eggs (the mother).
The haploid state of the egg nucleus continues for only a very short time and cannot materially affect the organization of the egg. The sperm enters the egg after this organization is already established. Similarly, we should expect that the elaboration of cytoplasmic organ-forming substances is under the control of maternal genes, even though this cannot be proved because no mutants are known which produce a difference in these substances.
It is conceivable that maternal genes might produce conditions in the egg leading to morphogenetic processes, which take place at a later stage of development, without visibly modifying the cleavage pattern. Several cases of such an influence are actually known.
In the axolotl (Ambystoma mexicanum) a lethal gene “o” has been discovered which has a maternal effect. The gene is recessive, so that heterozygote individuals (the + o individuals) are completely normal. Homozygote animals (oo), produced by a cross between two heterozygote parents, develop normally in the early stages but in later life show a slight retardation of growth.
Their regeneration capacity is severely reduced, so that amputated legs are not restored as would occur in normal axolotls. The homozygote males are sterile; their testes are underdeveloped and spermatogenesis does not go beyond the spermatogonial stages.
The homozygote females produce eggs capable of fertilization and normal cleavage, but at the onset of gastrulation the development is retarded. The embryos start gastrulation but usually do not go beyond the crescentic blastopore stage and then die.
Occasionally gastrulation is completed but the affected embryos never enter the stage of organogenesis. This abnormal course of development is in no way affected by the genotype of the spermatozoon; the spermatozoon may carry the normal + gene or a mutant o gene (if the male is a heterozygote).
The arrest in development is in both cases exactly the same, showing that the particular type of abnormal early development does not depend on the genes present in the cells of the developing embryo, but exclusively on the genes of the mother (maternal effect).
The effect of the maternal genotype oo may be neutralized by injecting into freshly fertilized eggs or into eggs at the beginning of cleavage (two blastomeres) the cytoplasm from a normal, ripe egg. As little as 1 to 5 per cent normal cytoplasm improves development and causes most of the embryos to complete gastrulation, enter organogenesis, and sometimes produce swimming larvae.
This experiment proves that the lethal embryos lack something which is present in the cytoplasm of the normal embryo. This “something,” which has been termed the “corrective factor,” must be the result of the activity in the mother of the un-mutated + gene. In the homozygous female the o gene is unable to produce the corrective factor; the maternal lethal effect is thus due to a specific deficiency.
In the oocyte a high concentration of the corrective factor is contained in the nuclear sap (0.2 to 0.5 per cent is sufficient to improve development) but that the cytoplasm of the oocyte when injected into a lethal egg has hardly any effect.
It is only when the nuclear membrane of the oocyte ruptures in the process of maturation and the nuclear sap mixed with the cytoplasm that the cytoplasm acquires the ability to correct development of the lethal embryos. Centrifugation of the cytoplasm of normal eggs showed that the corrective agent remains in the supernatant after sedimentation of all cellular inclusions (yolk, pigment mitochondria, and ribosomes) but that it sediments after further prolonged high speed centrifugation.
It is thus a macromolecular substance which is not bound to any cellular organoids. The substance is inactivated by heating between 50 and 55° C. or by treatment with trypsin, and it may be sedimented by ammonium sulfate. Thus, it shows characteristics of a protein.
From this work it emerges that the normal allele of the gene o is responsible for the accumulation in the nuclear sap of immature oocytes of a protein-like substance, which, upon the nuclear membrane’s breaking up during maturation, enters the cytoplasm and is indispensable if the embryo is to go through gastrulation and enter the phase of organogenesis.
The corrective substance is, of course, not the only substance produced under the influence of the maternal genes while the egg is developing in the ovary. Many, if not all, cytoplasmic substances in the egg must have a similar origin. The number of different chemical substances in an oocyte is very great. Using very refined methods of study it has been estimated that perhaps as many as 10,000 different genes are producing mRNA’s in the oocyte of the frog Xenopus laevis.
It would be expected that most of the mRNA’s, or at least a large proportion of them, would be directing the synthesis of proteins in the oocytes or during cleavage. The cytoplasmic substances determining the properties of blastomeres during cleavage may thus be an aftereffect of the action of genes at a previous stage of the reproductive cycle.