Molecular, Genetic, and Developmental Control of Cleavage

Along with the increase in cell numbers, mammalian cleavage is a period dominated by several critical developmental events. The earliest is the transition from maternally to zygotically produced gene products. Another is the polarization of individual blastomeres, which sets the stage for the developmental decision that results in the subdivision of the cleaving embryo into two distinct types of cells: the trophoblast and the inner cell mass (see Fig. 3.1). Most studies of the molecular biology and genetics of early mammalian development have been done on mice. Until more information on early primate embryogenesis becomes available, results obtained from experimentation on mice must be used as a guide.

Because of the lack of massive storage of maternal ribosomes and RNAs during oogenesis, development of the mammalian embryo must rely on the activation of zygotic gene products at a very early stage. Most maternal transcription products are degraded by the two-cell stage (Fig. 3.4). Some of these, however, stimulate the activation of the embryonic genome, which begins producing RNAs from a significant number of genes (>1500) by the time cleavage has advanced to the four-cell stage. There does not seem to be a sharp transition between the cessation of reliance on purely maternal gene products and the initiation of transcription from the embryonic genome. Some paternal gene products (e.g., isoforms of β-glucuronidase and β₂-microglobulin) appear in the embryo very early, while maternal actin and histone mRNAs are still being used for the production of corresponding proteins. As an indication of the extent to which the early embryo relies on its own gene products, development past the two-cell stage does not occur in the mouse if mRNA transcription is inhibited. In contrast, similar treatment of amphibian embryos does not disrupt development until late cleavage, at which time the embryos begin to synthesize the mRNAs required to control morphogenetic movements and gastrulation.

Mature eggs and sperms are transcriptionally inactive. A major reason for this is that their DNA is highly methylated. Methylation, which occurs on CpG dinucleotides, normally inactivates the associated gene. Such inactivation is often called epigenetic regulation because it does not alter the fundamental DNA sequence. Methylation can inactivate informational genes or their regulators (e.g., enhancers or promoters). Pronounced cycles of global methylation and demethylation occur during the life span of an individual (Fig. 3.5). Within 4 hours after fertilization, the paternally derived genome undergoes rapid, massive demethylation. Demethylation of the maternally derived genome occurs more gradually until the early morula, at which stage all the DNA is maximally demethylated. Remethylation ensues in the inner cell mass, until by the late blastocyst stage it returns to maximal levels. Within the germ cell line, the high methylation levels characteristic of the early embryo fall after the primordial germ cells have entered the genital ridge. During later gametogenesis, remethylation occurs. This remethylation imprints (see p. 43) maternal or paternal characteristics on the gametes and for some genes has profound effects on the embryos produced from these gametes. Epigenetic control is not confined to methylation patterns. Even as early as the zygote, different patterns of histone association with the chromatin account for pronounced differences in gene expression between the male and female pronuclei.

For the first couple of days after fertilization, transcriptional activity in the cleaving embryo is very low. Similarly, fertilized eggs and early mammalian embryos possess a limited capacity for the translation of mRNAs. The factor limiting translational efficiency may be the small number of ribosomes stored in the egg. As cleavage proceeds, products from maternally and paternally derived chromosomes are active in guiding development. Haploid embryos commonly die during cleavage or just after implantation. There is increasing evidence, however, that the control of early development involves more than simply having a diploid set of chromosomes in each cell.

One of the first manifestations of embryonic gene expression is the polarization of the blastomeres of the 8- and 16-cell embryo so that they have clearly recognizable apical and basal surfaces. The polarization of blastomeres leads to one of the most important steps in early mammalian development, namely, the decision that results in the appearance of two separate lines of cells—the trophoblast and the inner cell mass—from the early homogeneous blastomeres. In mice, up to the 8-cell stage all blastomeres are virtually identical. In the 8-cell embryo, the surfaces of the cells are covered with microvilli, and intercellular connections, mediated by E-cadherin, form. Shortly thereafter, differences are noted between polarized cells that have at least one surface situated on the outer border of the embryo and nonpolarized cells that are completely enclosed by other blastomeres. The polarized outer cells are destined to become trophoblast, whereas those cells located in the interior are destined to become the inner cell mass, from which the body of the embryo arises.

The relationship between the position of the blastomeres and their ultimate developmental fate was incorporated into the inside-outside hypothesis. The essence of this hypothesis is that the fate of a blastomere derives from its position within the embryo, rather than from its intrinsic properties. The outer blastomeres ultimately differentiate into the trophoblast, whereas the inner blastomeres form the inner cell mass. If marked blastomeres from disaggregated embryos are placed on the surface of another early embryo, they typically contribute to the formation of the trophoblast. Conversely, if the same marked cells are introduced into the interior of the host embryo, they participate in the formation of the inner cell mass (Fig. 3.6).

The cell polarity model offers an alternative explanation for the conversion of generic blastomeres to trophoblast or inner cell mass. According to this hypothesis, if the plane of cell division of a blastomere at the eight-cell stage is parallel to the outer surface of the embryo, the outer daughter cell develops a polarity, with its apical surface facing the zona pellucida (Fig. 3.7). The inner daughter cell remains apolar and goes on to form part of the inner cell mass. Experimental evidence suggests that a key element underlying a daughter cell’s becoming an outer cell is inheritance of a patch of outer cell membrane containing microvilli and the actin microfilament-stabilizing protein, ezrin. The proteins that produce polarity in the outer cells are postulated to direct their differentiation toward the trophoblastic lineage. Common to the inside-outside hypothesis and the cell polarity model is the recognition that a cell that does not contact the surface does not differentiate into trophoblast, but rather becomes part of the inner cell mass.

Even though by the 16-cell stage the embryo consists of clearly recognizable polar outer cells and nonpolar inner cells, cells of either type still can become transformed into cells of the other type. Thus, cells of the inner cell mass, if transplanted to the outer surface of another embryo, can become trophoblast, and at least some of the outer cells can turn into inner cell mass if transplanted into the interior. By the 32-cell stage, this capability for phenotypic transformation has become largely lost. Investigators have shown that cells of the inner cell mass of 16-cell embryos still retain the molecular machinery to turn into trophoblastic cells, because if the cells are exposed to the surface, they undergo the transformation into trophoblastic cells without new mRNA synthesis. Experiments of this type show that the developmental potential, or potency (the types of cells that a precursor cell can form) of many cells is greater than their normal developmental fate (the types of cells that a precursor cell normally forms).

The changes in phenotype of the inner and outer cells are accompanied by important molecular differences. Critical to the formation of cells of the trophoblast is the transcription factor, Cdx-2. Cdx-2 is essential for trophoblastic differentiation, and it also antagonizes the expression of molecules that are associated with the inner cell mass. Increased Cdx-2 levels both enhance the formation of molecules associated with polarization and increase the proportion of cells that undergo symmetrical cell division, thus increasing the number of trophoblastic cells. Cdx2 mutants fail to implant into the endometrial epithelium.

In contrast to cells of the trophoblast, which increasingly take on an epithelial character, cells of the inner cell mass express molecules that are associated with great developmental flexibility. Three such molecules are oct-4, Nanog, and Sox-2.

The oct4 gene codes for a specific transcription factor that binds the octamer ATTTGCAT on DNA. There is a close relationship between the expression of the oct4 gene and the highly undifferentiated state of cells. In mice, maternally derived oct-4 protein is found in developing oocytes and is active in the zygote. After the experimentally induced loss of oct-4 protein, development is arrested at the one-cell stage. This shows that maternally derived oct-4 protein is required to permit development to proceed to the two-cell stage, when transcription of the embryonic genes begins.

Oct-4 is expressed in all blastomeres up to the morula stage. As various differentiated cell types begin to emerge in the embryo, the levels of oct4 gene expression in these cells decrease until it is no longer detectable. Such a decrease is first noted in cells that become committed to forming extraembryonic structures and finally in cells of the specific germ layers as they emerge from the primitive streak (see Chapter 5). Even after virtually all cells of the embryo have ceased to express the oct4 gene, it is still detectable in the primordial germ cells as they migrate from the region of the allantois to the genital ridges. Because of its pattern of distribution, oct-4 protein is suspected to play a regulatory role in maintenance of the undifferentiated state and in establishing and maintaining the pluripotency of the germ cells.

Two other important genes in early development are Nanog and Sox2. Inner cells resulting from the division of cells in the eight-cell embryo begin to produce Sox-2, which binds onto DNA in partnership with oct-4 to regulate the expression of genes that control cellular differentiation. Nanog first appears in the late morula and along with Oct-4 functions to maintain the integrity of the inner cell mass. In the absence of Nanog function, cells of the inner cell mass differentiate into primitive endoderm (hypoblast), whereas lack of function of oct-4 causes inner cell mass cells to differentiate into trophoblast. Overall, but through different mechanisms, cells of both the trophoblast and inner cell mass are normally inhibited from becoming transformed into the other type.

Parental Imprinting

Experimentation, coupled with observations on some unusual developmental disturbances in mice and humans, has shown that the expression of certain genes derived from the egg differs from the expression of the same genes derived from the spermatozoon. Called parental imprinting, the effects are manifest in different ways. It is possible to remove a pronucleus from a newly inseminated mouse egg and replace it with a pronucleus taken from another inseminated egg at a similar stage of development (Fig. 3.8). If a male or female pronucleus is removed and replaced with a corresponding male or female pronucleus, development is normal. If a male pronucleus is removed and replaced with a female pronucleus (resulting in a zygote with two female pronuclei), however, the embryo itself develops fairly normally, but the placenta and yolk sac are poorly developed. Conversely, a zygote with two male pronuclei produces a severely stunted embryo, whereas the placenta and yolk sac are nearly normal.

Parental imprinting occurs during gametogenesis. Methylation of DNA, effected through specific imprinting centers, is one of the major means of imprinting and results in the differential expression of paternal and maternal alleles of the imprinted genes. Imprinted genes are transcriptionally silenced. The imprinted genes are maintained during development and possibly into adulthood, but a given imprint is not passed onto that individual’s progeny. Instead, the parental imprints on the genes are erased, and new imprints, corresponding to the sex of that individual, are established in the oocytes and sperm during gametogenesis.

Not all genes are parentally imprinted. Present estimates suggest that up to 2100 human genes are imprinted. Clinical Correlation 3.1 discusses some conditions and syndromes associated with disturbances in parental imprinting.

Clinical Correlation 3.1   Conditions and Syndromes Associated with Parental Imprinting

A striking example of paternal imprinting in humans is a hydatidiform mole (see Fig. 7.16), which is characterized by the overdevelopment of trophoblastic tissues and the extreme underdevelopment of the embryo. This condition can result from the fertilization of an egg by two spermatozoa and the consequent failure of the maternal genome of the egg to participate in development or from the duplication of a sperm pronucleus in an “empty” egg. This form of highly abnormal development is consistent with the hypothesis that paternal imprinting favors the development of the trophoblast at the expense of the embryo.

Several other syndromes are also based on parental imprinting. Beckwith-Wiedemann syndrome, characterized by fetal overgrowth and an increased incidence of childhood cancers, has been mapped to the imprinted region on chromosome 11, which contains the genes for insulinlike growth factor-II (IGF-II, which promotes cell proliferation) and H19 (a growth suppressor). It occurs when both alleles of the IGF2 gene express a paternal imprinting pattern. Another instructive example involves deletion of regions in the long arm of chromosome 15, specifically involving the gene UBE3A. Children of either sex who inherit the maternal deletion develop Angelman’s syndrome, which includes severe mental retardation, seizures, and ataxia. A child who inherits a paternal deletion of the same region develops Prader-Willi syndrome, characterized by obesity, short stature, hypogonadism, a bowed upper lip, and mild mental retardation.

X-Chromosome Inactivation

Another example of the inequality of genetic expression during early development is the pattern of X-chromosome inactivation in female embryos. It is well known from cytogenetic studies that one of the two X chromosomes in the cells of females is inactivated by extreme condensation. This is the basis for the sex chromatin, or Barr body, which can be shown in cells of females, but not in the cells of normal males. The purpose of X-chromosome inactivation is dosage compensation, or preservation of the cells from an excess of X-chromosomal gene products.

X-chromosome inactivation is initiated at the X-inactivation center, a unique locus on the X chromosome. XIST (X–inactive specific transcript), one of the genes in the X-inactivation center, produces a large RNA with no protein coding potential. XIST RNA remains in the nucleus and coats the entire inactive X chromosome, thus not allowing any further transcription from that chromosome. In the inactivated X chromosome, the XIST gene is unmethylated and expressed, whereas in the active X chromosome, this gene is methylated and silent.

Genetic studies show a complex ontogenetic history of X-chromosome inactivation (Fig. 3.9). In the female zygote, both X chromosomes are transcriptionally inactive, although not through the actions of XIST, because of the global inactivation of transcription in the early cleaving embryo. By the four-cell stage and into the morula stage, the paternally derived X chromosome becomes inactivated as the result of parental imprinting. Then, as the embryo forms the blastocyst, the paternally derived X chromosomes in the trophoblast and the hypoblast (see Fig. 5.1) remain inactivated, but within the cells of the inner cell mass both X chromosomes become active. As the cells of the inner cell mass begin to differentiate, the somatic cells undergo random permanent XIST-based X-chromosome inactivation of either the maternal or the paternal X chromosome. Within the germ cell line, activation of both X chromosomes occurs during the first meiotic division.

Developmental Properties of Cleaving Embryos

Early mammalian embryogenesis is considered to be a highly regulative process. Regulation is the ability of an embryo or organ primordium to produce a normal structure if parts have been removed or added.* At the cellular level, this means that the fates of cells in a regulative system are not irretrievably fixed, and the cells can still respond to environmental cues. Because the assignment of blastomeres to different cell lineages is one of the principal features of mammalian development, identifying the environmental factors that are involved is important.