5
EPIGENETIC CONTROL OF
EARLY DEVELOPMENT
Unlike unicellulars, metazoans do not produce copies of
themselves. They simply provide gametes with information for
building the Bauplan and the CNS at the phylotypic stage
when the embryo becomes competent of generating the
information for individual development up to adulthood.
All the
early development in metazoans, from egg fertilization
through cleavage, directed cell migration, formation of germ
layers to the formation of the operational central nervous
system at the phylotypic stage, is epigenetically regulated
by parental cytoplasmic factors, deposited in gametes of
both sexes. It is parental cytoplasmic factors, not nuclear
zygotic genes, that in the process of their translation in
the zygote, start and regulate the process of the early
development. The early development depends on epigenetic
factors alone for different periods of time in different
species. This period of exclusive epigenetic control of
regulation of the early development varies from the
two-cell stage in some mammal species to ~5,000-cell stage
in Xenopus. Despite the fact that after these stages zygotic
genes start to be expressed, epigenetic factors, still
continue to direct the early development, until the
phylotypic stage, by regulating expression of zygotic genes.
Early
embryonic development in metazoans is regulated by
epigenetic information that is parentally provided to
gametes (egg- and sperm cells) in the form of parental
cytoplasmic factors. Initially by their own activities, and
later on by determining the temporal order and the spatial
patterning of expression of zygotic genes, these factors
determine cell divisions, establishment of body axes,
formation of the embryonic layers, and gastrulation,
culminating at the phylotypic stage with formation of the
operational CNS (central nervous system) and the resulting
ICS (integrated control system). Thus, the epigenetic
program put in the zygote is an interim developmental
program for early embryonic development, until the
phylotypic stage when the embryonic CNS is operational and
takes over the individual development. It bridges the
physical gap between the parental and embryonic CNSs.
The
recognition of the role of the parental cytoplasmic factors
revealed the fact that the genetic information contained in
the zygotic diploid genome cannot start, drive or control
the early embryonic development. Only epigenetic information
in the form of parental cytoplasmic factors is capable of
starting and accomplishing the early embryonic development.
The absence of that epigenetic information is the reason why
somatic cells, cannot develop into an organism of their kind
in the way an egg (in parthenogenetic organisms) or a zygote
does, even though all of them are in possession of the
complete species-specific genetic information.
Earlier I
have argued and presented supporting evidence that the
synthesis of maternal cytoplasmic factors and their
placement in the egg cell by the follicle and/or nurse
cells, as well as via the active uptake by the oocyte from
the intercellular environment, takes place under the control
of the integrated control system (ICS). Now I will attempt
to prove that the epigenetic information (parental
cytoplasmic factors) determines the early individual
development up to the phylotypic stage, when the CNS, and
the resulting ICS (integrated control system), are
operational.
The entry of
the sperm into animal hemisphere detaches the cortex from
the egg cytoplasm, thus enabling the cortex to rotate by
about 300 in a process that contributes to the
elongation and organization of microtubules into parallel
bundles with their plus (+) ends toward the opposite side of
the sperm entry. This determines the movement of maternal
cytoplasmic factors and formation of the dorsal organizer
there (Weaver and Kimmelman, 2004; figure 5.1).
Figure
5.1. Formation of microtubules and dorsalization after
fertilization of the egg cell. Microtubules (MT) of the
vegetal array arise from several sources. (A) The sperm
centriole introduces polarity by acting as a minus-end
MT-organizing center (-). The resulting radial array of MTs is
called the sperm aster. (B) MTs from the sperm aster grow
toward the periphery of the egg, as do additional MTs from
unknown sources in the core cytoplasm. In addition, short
disorganized MT polymers arise in the vegetal shear zone. (C)
During rotation, MTs from deep in the cytoplasm bend into the
vegetal shear zone and align with peripheral MTs to form the
paral
lel array, with t e plus-e
nds (+) of the rowing MTs
pointing towards the future dorsal (D) side of the embryo.
Abbreviation: V, ventral (From Weaver and Kimelman, 2004).
Parentally,
both maternally and paternally, deposited cytoplasmic factors
in gametes are responsible for egg activation and, after
formation of the zygote, they start translating and induce the
orderly expression of zygotic genes, thus controlling and
regulating the early development until the phylotypic stage.
Epigenetic Control of
Formation of Primordial Germ Cells
Among the first types of embryonic cells that enter the
process of differentiation are the primordial germ cells.
Their fate as gamete precursors is epigenetically determined
by maternal factor(s) (Weidinger et al., 2003). In most
species studied so far, a maternal factor, vasa mRNA,
deposited in the vegetal pole of the egg is the most important
factor for gamete differentiation (primordial germ cells)
during the early development. In Drosophila, it is
involved in formation of the pole plasm at the posterior tip
of the oocyte (Tomancak et al., 1998), where the information
for germ cell differentiation and specification of the abdomen
is stored (Vanzo and
Ephrussi, 2002).
vasa mRNA is found in the germ line cells of zebra fish
(Yoon et al., 1997; Knaut et al., 2000), and Vasa proteins are
detected in chicken (Tsunekawa et al., 2000) and human (Castrillon
et al., 2000) germ cell lineage. As shown earlier, expression
of vasa mRNA in the ovary of the marine fish, Sparus
aurata, is hormonally stimulated by the pituitary hormone
GH (growth hormone) and by E2 (estradiol) (Cardinali et al.,
2003), both synthesized and secreted under neural control.
Epigenetic Control of
Embryonic Directed Cell Migration
Directed cell migration is a common and essential mechanism of
morphogenetic processes taking place during the individual
development. The movement of migrating cells
is determined by
mechanisms that enable them to detect specific cues released
by other cells, or extracellular matrix proteins. The
mechanism of orientation during migration is based on the
presence on the membrane of migrating cells of receptor
molecules that bind their specific ligands. This migration
mechanism is believed to be similar to that which induces
tumor cell metastasis.
Proximate
causes of expression of both the migrating cells’ membrane
receptors and their extracellular ligands are growth factors
(De Felici and Pesce, 1994; Salcedo et al., 1999) and hormones
(Caballero-Campo et al., 2002; Dominguez et al., 2003; Kitaya
et al., 2004), i.e. elements of signal cascades starting with
CNS signals. The process of cell migration during
embryogenesis will be discussed in some details in chapter 6,
subsection.
Cell Migration.
Epigenetic Control of Migration
of Primordial Germ Cells
As pointed out
earlier, the fate of primordial germ cells (PGCs) is
determined by deposition of the maternal vasa mRNA in
the vegetal pole and its asymmetric allocation among the early
blastula cells. In order to fully differentiate into germ
cells, PGCs have to migrate to the genital ridge, the Anlage
of the future gonads, ovaries or testicles.
The directed
migration of PGCs from the vegetal pole to their target sites,
traveling distances exceeding thousand times their diameter,
is made possible by the presence on these cells of specific
membrane receptors, CHCRs (chemokine receptors). PGCs find
their way to the target site by binding
their
receptor, the specific ligand, SDF-1 (chemokine
stromal cell-derived factor-1). This almost universal ligand
of cell migration is a downstream element of signal cascades
originating in the CNS. At the level of hormonal control,
recently it has been demonstrated that E2 (17 beta-estradiol),
via its nuclear receptor, induces expression of the gene
coding for SDF-1 (Coser et al., 2003; Hall and Korach, 2003)
and the latter is a direct target of that hormone (Hall and
Korach, 2003). A down-regulator of the SDF-1 is TGF-beta1
(transforming growth factor-beta1), which inhibits
transcription of the sdf-1 gene in the bone marrow
stromal cells, thus affecting their migration and adhesion
ability (Wright et al., 2003).
Unlike most species studied so far, in Drosophila,
another form of receptor plays the role of the above chemokine
receptor.
This is GPCR (G
protein-coupled receptor), known as Tre1, which is epigenetically
provided to the egg in the form of the maternal
tre1 mRNA.
By specifically binding its ligand, that protein
receptor enables PGCs to migrate through the posterior midgut
epithelium and find their way to the genital ridge (Kunwar et
al., 2003).
Chick and mouse PGCs use a different way of reaching their
target sites; they use the blood flow as a transport vehicle
and leave the blood vessels at specific sites where they sense
the presence of the chemoattractant SDF-1 alpha and, passing
through the blood vessels’ walls, they follow the chemokine to
find their way to the genital ridge (Stebler et al., 2004).
By
experimentally inhibiting the normal pattern of SDF-1
expression and by supplying SDF-1 to sites where it normally
is absent, changes in the direction of migration are induced
so that PGCs reach ectopic sites of the SDF-1 sources (Doitsidou
et al., 2002; Stebler et al., 2004). Upon arrival in the
genital ridge, the PGC population proliferates under the
influence of FGFs (fibroblast growth factors) (Kawase et al.,
2004) and TGFbeta1 (transforming factor-beta 1), but these
processes are regulated by a balanced proliferation/programmed
cell death or apoptosis (Tres et al., 2004). As it will be
shown later, apoptosis at this stage is also determined by
maternal epigenetic information (read maternal cytoplasmic
factors).
Epigenetic Control of Early
Development in
Insects
In Drosophila, like in insects
in general,
the early embryonic development is epigenetically determined
by parental cytoplasmic factors. Cell division during the
cleavage is regulated by maternal cyclins and the maternal
String protein, whose transcripts are present in the zygote
and are among the earliest mRNAs to be translated. Because of
the uniform distribution of these factors, the early cycles of
nuclear divisions are uniformly executed but no cell divisions
occur. The resulting naked nuclei move to the outer edge of
the embryo, thus leading to formation of a syncytial
blastoderm. This lasts until the 14th cell cycle,
when normal cell divisions begin to occur and the syncytial
blastoderm is transformed into a cellularized blastoderm. This
change is related to the fact that at this point in time the
maternal reserve of the String protein is exhausted and
differential expression of the gene for the String protein in
various parts of the embryo begins. Different regions of the
blastoderm divide at different rates.
After the 17th or 18th cycle, cells in
the epidermis and mesoderm stop dividing, and differentiate.
This cessation of proliferation is caused by the exhaustion of
maternal cyclin E, originally laid down in the egg, which is
required for progression through the cell cycle. (Wolpert et
al. 1998)
In insects, the anterior-posterior axis is established by the
combined action of three systems of maternal cytoplasmic
factors placed in the egg by nurse cells: the bicoid mRNA
in the anterior part of the egg cell, nanos mRNA and caudal mRNA in the posterior, and the
hunchback mRNA
initiate a transcription sequence of genes gap-pair-rule-engrailed-homeotic. In Drosophila
these genes are expressed in phase with the gene for the
neurotransmitter serotonin and serotonin receptor in
characteristic stripes pattern (Colas et al. 1995).
Maternal cytoplasmic factors also induce the synthesis of
neurotransmitters which are among the first products of
zygotic gene activity in the early stages of embryonic
development (Shmukler et al., 1999). A serotonin receptor gene
and the serotonin gene are intensely expressed at 3 hrs (cell
blastoderm stage) of Drosophila embryogenesis. Their
expression is organized in a seven-stripe pattern of the
blastoderm stage and in phase with the pair-rule gene
fushi-tarazu (Colas et al. 1995). Application of serotonin
antagonists causes a transient regression of the first
cleavage furrow in sea urchin embryos, suggesting a
morphogenic role of serotonin in the early embryonic
development (Shmukler et al., 1999).
Maternal signals determine a peak of expression of the
neurotransmitter serotonin and its receptors in Drosophila,
which is coincident with the onset of the germ band extension
(corresponding to the phylotypic stage). How important that
peak of serotonin is for the normal gastrulation is shown by
the fact that mutant Drosophila embryos that fail to reach
that peak do not develop proper germ band extension and die
with a cuticular organization that is characteristic of
embryos that do not express the serotonin receptor (Colas et
al., 1999).
Early during the phylotypic stage, the incipient CNS is
involved in patterning of neighboring embryonic mesoderm and
underlying ectoderm and in determining their cell fates. Among
the inductive effects of the CNS midline cells is the
formation of somatic muscles from mesodermal progenitors (Luer
et al., 1997). Cells of the ventral midline region
of the
Drosophila CNS, and probably other regions of the
CNS (Luer et al., 1997), produce and secrete Spitz, which
induces the synthesis of the EGF receptor in the neighboring
ventral ectoderm, thus determining different fates for cells
of the ventral ectoderm (Golembo et al., 1996; Zhou et al.,
1997).
Epigenetic Control of Early Development in Other Invertebrates
As a
representative example of the maternal control of the
development of germ layers in other vertebrates may be
mentioned the GRN (gene regulatory network) of the marine
echinoderm Strongylocentrotus purpuratus (Davidson et al.,
2003; figure 5.2). The ultimate source of information for
activation of the endomesoderm GRN in this species is the
epigenetic information provided to the egg cell in the form of
maternal cytoplasmic factors, which determines expression of
zygotic and early embryonic genes leading to formation of the
germ layers in this echinoderm.
Epigenetic Control of Early Development in Vertebrates
The dorsal
side of the embryo is established opposite of where the sperm
cell enters the egg cell. As a result of the cortical
reaction, beta-catenin, a maternal transcription factor, which
belongs to the Wnt signal transduction pathway, and other
maternal cytoplasmic factors such as mRNAs for Vg1, Xwnt11,
Noggin, and Activin, are accumulated in the dorsal side of the
developing embryo. The first divisions of the zygote are
stimulated by the synthesis of the cyclin protein Cdc6 by the
maternal Cdc6 mRNA that is stored in the egg cell during the
maturation of the oocyte shortly after the GVBD (germinal
vesicle breakdown). The maternal Cdc6 mRNA makes possible the
replication of zygotic chromosomes by activating the MCM (minichromosome
maintenance) helicase complex (Lemaitre et al., 2002).
Upon each cell
division, cyclins are destroyed and new cyclins are
synthesized from the reserve of maternal cyclin mRNAs. They
combine with a cyclin-dependent kinase to form the
MPF (maturation promoting factor), a phosphoprotein that is
responsible for cell division since the early stages of the
embryonic development. When treated with MPF complex, the
cleaved cells arrested in S phase resume their mitotic cycle
(Gilbert, 1997a). In this context, one has to remember that
MPF is an active form of the pre-MPF, which in turn, is
induced by progesterone (Murray and Kirschner, 1989).
Progesterone
causes polyadenylation of the maternal c-mos mRNA, whose
translation’s product is a phosphoprotein (pp 39mos) (Sheets
et al., 1995). This phosphoprotein stimulates resumption of
meiosis. Its complex with the cyclin-dependent kinase 2 (cdk2)
represents the cytostatic factor (CSF), which prevents
degradation of MPF (Watanabe et al., 1991). So, progesterone,
whose secretion is under binary neural control, may be crucial
for cell division during the early stages of embryonic
development.
By the 14th
cycle of cell divisions, the reserve of maternal cyclin mRNA
and the String, a protein phosphatase, are exhausted. The
zygotically coded String protein (cdc 25 phosphatase) is
rhythmically translated to phosphorylate the pre-MPF
(maturation promoting factor) and transform it into active MPF,
just before the mitotic cell division. The zygotic string gene
will be differentially expressed, only in the cells that have
inherited transcription factors of the gap, pair ruled, and
other early patterning genes, which are expressed in phase
with the gene for the neurotransmitter serotonin and serotonin
receptor in characteristic stripes pattern. This, ultimately,
explains the fact that some parts of the embryo (those that
express the string), will grow faster than others.
Maternal
transcripts of three types of retinoic acid receptors (RAR)
are detected during all the stages from the oocyte through the
hatched blastocyst in bovines (Mohan et al., 2001). A sequence
in the transcription of homeobox genes that are essentially
involved in axis formation and morphogenesis during early
bovine embryonic development, from the zygote to the
blastocyst stage (Ponsuksill et al., 2001). In view of the
well-established fact of RA control of expression of various
homeobox genes, and of the fact that RA (retinoic acid) and
its receptors are present as maternal factors in oocytes and
embryonic cells during the early development, it is reasonable
to assume that
initially maternal and later zygotic RA
and its receptors also regulate expression of homeobox genes
in bovines.
Expression of
various types of RA receptors in the inner cell mass and
trophoectoderm of blastocysts suggests that maternal RA is
likely to directly regulate gene expression during
preimplantation development
(Mohan et al., 2001) of bovine embryos.
