Download The epigenetic basis of gender in flowering plants and mammals

Review
TRENDS in Genetics Vol.17 No.12 December 2001
705
The epigenetic basis of gender in
flowering plants and mammals
Melissa Spielman, Rinke Vinkenoog, Hugh G. Dickinson and Rod J. Scott
What makes a sperm male or an egg female, and how can we tell? A gamete’s
gender could be defined in many ways, such as the sex of the individual or
organ that produced it, its cellular morphology, or its behaviour at fertilization.
In flowering plants and mammals, however, there is an extra dimension to
the gender of a gamete – due to parental imprinting, some of the genes it
contributes to the next generation will have different expression patterns
depending on whether they were maternally or paternally transmitted. The
non-equivalence of gamete genomes, along with natural and experimental
modification of imprinting, reveal a level of sexual identity that we describe as
‘epigender’. In this paper, we explore epigender in the life history of plants and
animals, and its significance for reproduction and development.
Male and female gametes develop in different cellular
environments, are morphologically distinct and
behave differently at fertilization. However, in
mammals and angiosperms (flowering plants),
parental imprinting1–4 results in a more subtle
distinction between the gametes of the two sexes –
their genomes express different sets of genes after
fertilization, although they inhabit the same cell.
Non-equivalence of gamete genomes has far-reaching
consequences for reproduction; for example,
preventing parthenogenesis in mammals, and at
least partly explaining the difficulties presented by
cloning and other forms of artificial breeding.
The roots of epigender – parental conflict in mammals
and flowering plants
Melissa Spielman
Hugh G. Dickinson
Dept of Plant Sciences,
University of Oxford,
South Parks Road, Oxford,
UK OX1 3RB.
Rinke Vinkenoog
Rod J. Scott*
Dept of Biology and
Biochemistry, University
of Bath, Claverton Down,
Bath, UK BA2 7AY.
*e-mail:
[email protected]
In mammals, many imprinted loci are involved in fetal
growth, and might be particularly important for
placental development2,3,5. Imprinting in angiosperms
disproportionately affects endosperm, a separate
fertilization product that mediates transfer of resources
between seed parent and embryo1,4.(Figure 1 shows a
comparison of reproduction in mammals and flowering
plants.) Increasing the dosage of paternal genomes
(i.e. dosage of active copies of paternally expressed
imprinted genes) tends to promote the growth of
placenta or endosperm, whereas increasing maternal
dosage has the opposite effect1–4,6. The parental conflict
theory explains these observations by presenting
imprinting as a struggle between maternally and
paternally derived genomes over resource allocation
from mother to offspring1,2. In the model, this creates
selection pressure for growth promoters acting during
provisioning of offspring to be expressed when they are
inherited from the father, but silenced when inherited
from the mother. Conversely, growth inhibitors are
selected for expression when maternally derived and
repression when paternally derived.
http://tig.trends.com
Parental imprinting requires epigenetic marking
of alleles in the germline, maintenance of the mark
through cell division, and response to the mark
resulting in uniparental gene expression after
fertilization. In mammals, existing marks are erased
in primordial germ cells (PGCs) and reset during
gametogenesis according to the sex of the animal.
Monoallelic expression of imprinted genes is
maintained by epigenetic regulatory mechanisms such
as DNA methylation and histone acetylation, and
methylation is also a candidate for the primary mark5,7.
We propose the term ‘epigender’to describe the paternal
or maternal character of a genome as defined by the
sex-specific marking of its imprinted genes. Epigender
depends on imprinting but the terms are not
synonymous: imprinting imposes epigender. This
quality has been recognized by others – for example, as
the ‘sex-specific epigenotype’7 – but in this paper, we
seek to extend previous models to incorporate a greater
variety of phenomena. We will discuss epigender as a
spectrum that can be modified either naturally or
experimentally, and consider how epigender changes
during the life-cycle of an organism with imprinting.
Real and virtual changes to genomic balance
Imbalance of entire genomes
Each cell of a mammalian embryo and its extraembryonic membranes contains one genome with
maternal epigender (represented as ‘m’) and one with
paternal epigender (‘p’). Experiments in which mouse
eggs were reconstituted with either two male or two
female pronuclei showed that both maternal and
paternal contributions are essential for embryogenesis.
The resulting androgenetic (0m:2p) or gynogenetic
(2m:0p) embryos aborted with well- or poorly developed
extraembryonic membranes, respectively (reviewed
in Ref. 6). By contrast, viable androgenotes have
been created in zebrafish, which lack a parental
imprinting system (at least one with developmental
consequences)8. In this case, genomes derived from
sperm or egg are interchangeable in the zygote.
Similar to mammals, angiosperms are sensitive to
the balance of parental genomes. Crosses between
plants of different ploidies often result in abnormal
seed development followed by abortion, with
reciprocal endosperm phenotypes depending on the
direction of the cross (reviewed in Ref. 1). Normal
endosperm development in most species depends on
a 2m:1p genomic ratio1,9. In Arabidopsis, crosses
between diploids and tetraploids produce viable
0168-9525/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(01)02519-7
Review
TRENDS in Genetics Vol.17 No.12 December 2001
Flowering plants
Mammals
Pollen
grain
Oocyte
Polar
bodies
Embryo
sac
Fertilization
Fertilization
Sperm
Female
meiosis II
Endosperm
Pronuclei
Embryo
Bi-potential
germ cells
Embryo
Embryo
Placenta
Embryo
Seedling
Germ cell migration into gonads
Flower
Testis
Meiosis I
(prophase)
Ovule
(Female)
Meiosis
Spermatids
Meiosis
Adult
Meiosis I
(complete)
Mature
oocyte
Sperm
Anther
(Male)
Meiosis
Gametogenesis
Gametogenesis
Ovary
Adult
706
Polar
nuclei
Egg
Pollen
grain
Embryo
sac
Sperm
TRENDS in Genetics
triploid embryos, but 2x X 4x crosses (diploid mother
and tetraploid father), generating a 2m:2p endosperm
(and 1m:2p embryo), result in endosperm
overproliferation, whereas 4x X 2x crosses (4m:1p
endosperm, 2m:1p embryo) produce a small
endosperm10. Crosses between diploid and hexaploid
plants produce similar but more extreme reciprocal
phenotypes (Fig. 2a), and seeds almost always abort.
As in mouse, parent-of-origin effects in angiosperms
are usually interpreted as reflecting a change in
balance of active alleles of imprinted genes.
The experiments described above changed the
relative dosage of maternally and paternally derived
http://tig.trends.com
genomes in offspring, but did not alter the epigender
of the individual genomes participating in
fertilization. Below we will discuss experiments in
which offspring were created using genomes that can
be described as neither fully maternal nor paternal.
Hypomethylation
Early experiments designed to disrupt the regulation
of imprinting in mouse involved targeted mutagenesis
of the methyltransferase Dnmt1, which reduces
cytosine methylation in homozygous mutant embryos
by 70% (Refs 11,12). Analysis of three imprinted genes
showed that they lost parent-specific methylation and
Review
TRENDS in Genetics Vol.17 No.12 December 2001
Fig. 1. Reproduction in mammals and flowering plants. Mammals: The oocyte (arrested at metaphase
of meiosis II) is fertilized by a sperm, resumes meiosis, and extrudes the second polar body structure
(polar bodies contain maternal genomes that do not participate in fertilization). Maternal and paternal
genomes remain in separate pronuclei within the fertilized egg (zygote) until the first round of DNA
synthesis is complete. The embryo and extra-embryonic membranes (giving rise to the placenta) both
develop from the zygote; the placenta acquires resources from the mother for transmission to the
developing embryo. The germ-cell lineage is sequestered early in embryogenesis, and later migrates
to the embryonic gonads. In female embryos, oogonia enter meiosis but arrest in prophase I. In adult
females, primary oocytes complete meiosis I, extrude the first polar body, and arrest in metaphase II.
In adult males, spermatocytes undergo meiosis to form spermatids, which differentiate into sperm.
Flowering plants: The pollen grain transmits two sperm to the embryo sac, where one fertilizes the
egg, giving rise to the embryo, and the other fertilizes the fused polar nuclei (central cell nucleus),
producing the endosperm. Endosperm is a terminal tissue that acquires resources from the seed
parent for use in embryo growth and/or germination. The germ-cell lineage does not differentiate
until flower formation in the adult plant. Female meiosis occurs in ovules and male meiosis in anthers.
In most species, there is only one surviving megaspore (black nucleus) from each female meiotic
event. The megaspore divides mitotically to form the embryo sac, which contains two female
gametes: the haploid egg, and the diploid central cell. Male meiosis produces microspores that give
rise to pollen grains, each containing two sperm.
expression patterns in Dnmt1-deficient embryos12.
One of the genes, H19, became biallelically expressed,
but surprisingly – given the association of methylation
with transcriptional repression – the other two were
silent: Igf2 lost expression from the normally active
(a)
707
paternal allele, and Igf2r from the normally active
maternal allele. It has since emerged that Igf2 and
Igf2r are indirectly silenced by hypomethylation, with
methylation of cis-linked sequences having a role in
their activity (reviewed in Ref. 3). Significantly, this
shows that the default state of an imprinted gene is
not necessarily a potential for expression.
Effects of hypomethylation on reproduction have
also been studied in Arabidopsis using an antisense
gene to METHYLTRANSFERASE I (METI a/s),
which reduces cytosine methylation by as much as
85% (although in this case embryos are viable)13,14.
Crosses using one hypomethylated and one wild-type
parent, both of the same ploidy, produced a phenotype
very similar to interploidy crosses with respect to
characteristics diagnostic for parental genomic
imbalance (Fig. 2b, left and middle)14. The
phenotypes suggest that hypomethylation of the
pollen parent ‘maternalises’ sperm genomes –
presumably by removing the methylation mark from
alleles that are normally paternally silenced –
whereas hypomethylating the seed parent
‘paternalises’ central cell genomes.
2x X 6x
Nuclear transfer from ‘pre-imprinted’ stages
2x X 2x
6x X 2x
Peripheral
endosperm
Chalazal
endosperm
Embryo
(b)
2x X 2xMETIa/s
2xMETIa/s X 2x
2xfie-1 X 2x
Fig. 2. Manipulation of epigender in seeds. Confocal micrographs showing seeds produced by wildtype Arabidopsis plants of different ploidies (top) and hypomethylated or mutant plants (bottom).
Embryos and endosperms are artificially coloured green and red, respectively. (a) Balance of
maternal and paternal genomes is altered by interploidy crosses. A normal seed from a 2x X 2x cross
(middle) is compared with a maternalized seed (6x X 2x, left) and a paternalized seed (2x X 6x, right),
all at eight days after pollination. The maternalized seed is small with a reduced peripheral and
chalazal endosperm (yellow circle). The paternalized seed is large with overgrown peripheral and
chalazal endosperm, and endosperm does not cellularize. Scale bar is 100 µm. (b) Hypomethylation
and a fie-1 mutation both phenocopy effects of interploidy crosses, suggesting that these changes
also alter epigender in seeds. All images are at the same magnification. A 2x seed parent with normal
methylation crossed with a 2x pollen parent hypomethylated by the METI antisense gene (METIa/s)
(left) produces seeds resembling those from 4x X 2x crosses (in mature seed weight, and cell
number, timing of cellularization, and morphology of endosperm) where both parents have normal
methylation. A 2x METIa/s mother crossed with a wild-type 2x father (middle) produces a similar
phenotype to 2x X 4x crosses (see also Ref. 14). A maternal fie-1 mutation (right) produces a
similar phenotype to 2x X 6x crosses (lethal paternal excess) (see also Ref. 31).
http://tig.trends.com
Another series of experiments in mouse had
comparable results to hypomethylation. Oocytes were
reconstituted using nuclei from cells at developmental
stages before establishment of imprints. Obata et al.15
created parthenogenetic embryos using one nucleus
from a fully grown (fg) oocyte and one from an
immature non-growing (ng) oocyte. H19, normally
maternally expressed, was active in the ng genome.
However, the normally maternally repressed
Peg1/Mest, Peg3 and Snrpn were also active,
indicating that full maternal epigender had not yet
been imposed in the ng oocyte. Igf2, Igf2r and the
normally maternal-specific p57KIP2 were silent in the
ng genome. Kato et al.16 transplanted nuclei from
(male) PGCs, taken after the stage of imprinting
erasure, into enucleated oocytes. In the resulting
‘germ-cell embryos’, H19 as well as four genes that are
normally paternal-specific were detected, but Igf2,
Igf2r and p57KIP2 were silent. Both fg/ng and germ-cell
embryos aborted. These experiments show that
immature gametogenetic cells are not in the same
epigender state as mature gametes, and also that the
oocyte cannot impose the correct epigender for normal
development on an introduced nucleus.
Mutations affecting epigender
Disruptions to imprinted mammalian genes in the
form of mutation, loss of imprinting or abnormal
transmission have been extensively studied17. These
can cause major phenotypic effects in offspring. For
example, mice inheriting a paternally transmitted
deletion in Igf2 are small18, consistent with the
phenotype predicted by parental conflict theory for
maternalization. In humans, Prader–Willi and
Angelman syndromes (PWS and AS) are genetic
708
Review
TRENDS in Genetics Vol.17 No.12 December 2001
disorders caused by loss of expression from the same
cluster of imprinted genes on chromosome 15, but
paternal expression is lost in PWS, and maternal
expression in AS (Ref. 19). Therefore, maternalization
and paternalization of this chromosomal region cause
distinct disease phenotypes. Strikingly, abnormal
biallelic expression of imprinted growth promoters,
or deletion of imprinted genes involved in growth
inhibition or apoptosis, occur in many cancers, both
hereditary and sporadic (reviewed in Refs 20,21).
Overexpression of Igf2 occurs in a wide variety of
cancers, suggesting a correlation of paternalization
with overproliferation of malignant cells. It is perhaps
not surprising that imprinted gene expression is
frequently disrupted in cancer: according to parental
conflict (as well as some other theories of imprinting
evolution22), imprinted genes have been selected for
parent-specific expression precisely because of their
importance in growth control. However, we would
not expect paternalization in an adult cell always to
cause overgrowth, or maternalization always to
inhibit growth, as imprinting is mainly a feature
of embryogenesis.
In Arabidopsis endosperm, FIS1/MEA is imprinted
and expressed only from the maternal genome; and
the other two FERTILIZATION-INDEPENDENT
SEED loci, FIS2 and FIS3/FIE, might also be
maternal-specific, at least early in seed
development23–29. For any of these loci, mutations in
the maternally derived allele lead to seed abortion,
with endosperm phenotypes that we interpret as
diagnostic of paternal excess: overproliferation, failure
of cellularization, and large chalazal endosperm
(Fig. 2b, right)30–32. FIS1/MEA and FIS3/FIE encode
Polycomb proteins that interact, whereas FIS2
encodes a candidate zinc-finger transcription
factor25,28,29,33–35. In Drosophila and other animals,
Polycomb proteins participate in complexes that
repress transcription of target genes, probably
through epigenetic modification of chromatin36,37.
The phenotype of fis mutants suggests that one role of
FIS proteins in Arabidopsis is to repress transcription
of loci in the maternally derived genome that are
normally expressed only when paternally contributed;
that is, to suppress p epigender in the maternal
genome. Accordingly, when a FIS gene is mutated, the
maternal genome acquires some p quality, explaining
the strong paternal-excess phenotype. This is
supported by the observation that fertilization of a fis
mutant mother by a hypomethylated pollen donor
(which has a maternalizing effect; see above) can
rescue the seed-abortion phenotype, in many cases
even when the pollen did not contribute a relevant
wild-type FIS allele26,28–31. The evidence above
suggests that FIS genes: (1) are imprinted and
expressed from the maternal genome, and (2) in turn
regulate expression of imprinted genes that are
silenced in the maternal genome. Therefore, FIS genes
could have a multi-level role in establishment and/or
maintenance of epigender in flowering plants.
http://tig.trends.com
Epigender and reproduction
Parthenogenesis
Epigender has profound consequences for the breeding
systems available to an organism, both in terms of
potential for asexual reproduction and ability to
hybridize. Many animals, including vertebrates such
as amphibians, fish and birds, are able to reproduce by
parthenogenesis, but this has never been reported in
mammals, and it can not be induced experimentally
(reviewed in Refs 38,39). It has been suggested that
absence of parthenogenesis in mammals is due to the
imprinting system1,6,38. Therefore, the requirement for
genomes with maternal and paternal epigender in
mammalian embryos appears to present an
insurmountable obstacle to natural cloning.
By contrast, many hundreds of angiosperm
species reproduce by apomixis, production of a seed
containing an embryo descended only from the seed
parent40,41. However, most apomicts still require
fertilization of the central cell to produce an
endosperm. Haig and Westoby1 argued that apomixis
has been able to evolve in angiosperms because the
embryo is relatively insensitive to imprinting so can
develop without a paternal genome, provided it
associates with a sexual endosperm. Therefore
epigender is also a barrier to self-cloning in plants,
although more easily circumvented than in mammals.
Interspecific incompatibility
Interspecific crosses among angiosperms illustrate the
impact of epigender on ability to hybridize. Some pairs
of species at the same ploidy level behave in crosses as
though they have different ploidies, suggesting an
effective genomic imbalance in the seed (reviewed in
Ref. 1). Johnston et al.42 accounted for this with the
endosperm balance number (EBN) hypothesis. In this
system, each species has an EBN that reflects effective
ploidy, and it is EBNs rather than chromosome sets
per se that must be in a 2m:1p ratio for successful
endosperm development. Haig and Westoby1 argued
that the EBN system reflects the existence of
imprinted genes that either promote or inhibit resource
acquisition. By extension, a species’ EBN could reflect
the effectiveness with which its paternally transmitted
genome can extract resources for offspring and its
maternally transmitted genome can inhibit resource
transfer. For example, the paternal genome from a
species with a high EBN might silence more inhibitors
of endosperm growth than a species with low EBN,
or silence inhibitors for longer. Johnston and
Hanneman43 found that manipulating EBN ratios in
endosperm by changing the ploidy of polar nuclei or
sperm allowed normally incompatible species to set
seed. We propose that modifying the epigender of one
partner’s gametes should also allow crosses between
species with otherwise incompatible EBNs.
The effect of hybridization on expression of
imprinted genes has been directly tested in deer
mouse. Peromyscus polionotus and Peromyscus
maniculatus are of similar size, but when female
Review
(a)
TRENDS in Genetics Vol.17 No.12 December 2001
Mammals
709
(b)
Flowering plants
pre-m
pre-p
m
p
m
p
pre-m
pre-p
Imprinting
Sporocyte
PGC
p
Interploidy
cross
2x X 6x
Hypomethylation
Imprinting
m
p
Egg
Sperm
m
p
m
2m
Egg
p
2p
Polar
nuclei
Sperm
m
m
Sperm
p
p
Gametes
Gametes
2x METIa/s X 2x
Loss of maternal
imprinting?
m
p
m
p
Zygote
m
p
m
p
m
p
m
p
m
p
p
m
p
Lethal
paternal
excess
p
Paternal
excess
m
(p)
m
(p)
m
p
m
p
p
Lethal
paternal
excess
Endosperm
Zygote
fie-1 X 2x
m
TRENDS in Genetics
Fig. 3. Models for epigender states in mammalian and flowering plant reproduction. (a) Mammals:
genomes of primordial germ cells (PGCs) after imprinting erasure are pre-maternal (pre-m) and prepaternal (pre-p). Imprinting results in potential for expression of maternal-specific genes and repression
of paternal-specific genes (i.e. maternal epigender, m) in the egg genome, and conversely potential for
paternal-specific but not maternal-specific expression (i.e. paternal epigender, p) in the sperm genome.
Fertilization produces a diploid zygote nucleus where one genome has maternal epigender and one
paternal epigender. Half tones indicate nuclei where resetting of imprinting status has occurred.
Flowering plants: plant somatic cells appear to be insensitive to imprinting, so are represented as both
maternal and paternal. The mechanism for insensitivity is unknown but could involve failure to maintain
or recognize imprints. It is not known when imprints are set in plants, but hypomethylation experiments
suggest that at least some component of imprinting is imposed before meiosis14. Imprinting produces
an egg and polar nuclei with maternal epigender and sperm with paternal epigender. Each pollination
results in two fertilization products, endosperm and embryo. The zygote is apparently insensitive to
imprinting, while the endosperm requires a balance of two maternal genomes to one paternal genome
for normal development. (b) Examples of altering epigender in Arabidopsis seeds. A cross between a
2x seed parent and 6x pollen parent (top) results in a balance of two maternal to three paternal genomes
in endosperm, causing a lethal paternal-excess phenotype. Hypomethylation of the seed parent
(middle) adds paternal epigender to the seed, possibly by allowing expression of genes in the
maternally derived endosperm genomes that would normally be expressed only if paternally derived. A
fie-1 mutation (bottom) also adds paternal epigender to the seed, in this case to a lethal degree. Half
tones indicate nuclei where resetting of imprinting status has occurred. See text for further explanation.
P. maniculatus is crossed with male P. polionotus,
offspring are smaller than either parent and have
small placentas; the reciprocal cross produces
abnormally large offspring (with large placentas),
which usually abort44. Vrana et al.45 found widespread
disruption to methylation and expression of imprinted
genes in hybrid progeny, suggesting that the
mechanism for maintaining or responding to epigender
has diverged during evolution of these species.
Synthesis
Qualities of epigender
In the model presented here, the test for the
maternal or paternal value of a genome’s epigender
is to introduce it into an egg (or central cell in the
case of angiosperms), then assay imprinted gene
http://tig.trends.com
expression at the time this becomes parent specific
(i.e. post-implantation for most mammalian imprinted
genes46). According to this test, a genome with paternal
epigender has the potential to express all of the genes
that would normally be active when contributed by a
sperm, but not those genes that are expressed
exclusively from the maternal genome. Likewise,
maternal epigender denotes the potential to express all
of the genes that are active when contributed by an egg
(in mammals) or central cell (in flowering plants), and
only those genes. Experimental manipulation and
mutations show that maternal or paternal epigender
are not the only states that can be occupied by a
genome, but represent the endpoints of a spectrum
where intermediate values are also possible. The
range of the spectrum is fixed within a species, but
varies between species: this is reflected in the
different ‘imprinting strengths’ revealed by crossing
angiosperms with different EBNs. Hybridization might
also disrupt the system for maintenance or response
to epigender, as suggested by Peromyscus crosses.
Epigender in life history
Because different genomes in the same organism
have different epigenders, it is possible to construct
a scheme for epigender in the life history of an
individual (Fig. 3). Current evidence supports three
basic epigender states in mammalian development:
• State 1 diploid: primordial germ cell after erasure
of imprinting but before new imprints have been
applied: ‘pre-m’ and ‘pre-p’. This is not a completely
‘naive’ state in which every gene destined for
imprinting has a potential for expression, as some
loci require the imprinting process for activation.
710
Review
TRENDS in Genetics Vol.17 No.12 December 2001
• State 2 haploid: gametogenic cell after imposition
of imprinting: m or p, depending on the sex of the
individual that produced it (the sex chromosome
carried by the gamete is irrelevant, as all sperm are
p, though half contain an X chromosome).
• State 3 diploid: somatic cell after fertilization: one
genome is m, the other is p. The right balance of
m and p is critical for normal development.
An analogous model could be proposed for plants,
though not in detail, as little is known about the
stages at which imprints are imposed and erased. For
angiosperms, the tissue in which state 3 is critical is
endosperm, where two genomes are m and one is p.
in prophase of meiosis I, suggesting that imprinting is
complete by this stage55. Human embryos have also been
produced by fertilizing oocytes with spermatids, which
are post-meiotic but not fully differentiated (reviewed
in Ref. 56). It has been noted by others that gametes
used for in vitro fertilization should have the full set of
imprints56; that is, they must have fully maternal or
paternal epigender to ensure normal offspring. The
epigender model also illustrates the difficulty of
single-sex reproduction; for example, to create a viable
embryo from two egg genomes would require a switch
from maternal to paternal epigender in one of them.
Apomixis in angiosperms
Switching epigender: prospects for artificial breeding
Somatic cloning and in vitro fertilization in mammals
The epigender scheme is relevant not just for natural
systems, but for artificial breeding systems such as
cloning. For example, the non-equivalence of states 1
and 3 is consistent with the observation that mammals
can be cloned by reconstituting an enucleated egg with
an adult somatic cell nucleus47 (state 3), but not with
a PGC nucleus16 (state 1). Even where somatic cells
are used in cloning, imprinting has been proposed as
one reason for the low success rate46. A possible
explanation for failure is the complex stage- and tissuespecific nature of imprinting48, which means that a
differentiated cell is not necessarily in the full m and p
state of the developing embryo. Embryonic stem (ES)
cells are derived from undifferentiated cells, so their
use as nuclear donors might be expected to circumvent
this problem. However, ES cell lines and mice cloned
from them show widespread misregulation of
imprinted gene methylation and expression49,50. For
both differentiated and ES cells, decay or instability
of state 3 epigender seems to present an obstacle to
cloning. It should also be noted that many other factors
are important to the success of cloning in mammals,
such as the type of recipient cell (e.g. only oocytes
appear to have the ‘reprogramming’ activity required
to restore totipotency to a nucleus from a differentiated
cell without also erasing its imprints51); and the extent
of reprogramming of the donor nucleus46.
Imprinting aberrations have also been suggested as
an explanation for growth defects following not only
nuclear transfer, but also in vitro embryo culture and
other manipulations of early embryos52,53. Fetal
overgrowth of sheep has recently been correlated with
maternal hypomethylation and reduced expression
of Igf2r (Ref. 54). In this experiment embryos were
produced by in vivo fertilization so epigender of the
early zygote was presumably normal; however, during
the subsequent in vitro culture, epigenetic modification
of imprinted genes was proposed to occur. In the model
presented here, manipulation of embryos in state 3
added p epigender to the maternally derived genome.
In contrast to embryos produced by transfer of ng
oocyte or PGC nuclei (above), mouse embryos generated
by fertilizing an oocyte with an immature spermatogenic
cell are viable, even when the male genome donors are
http://tig.trends.com
A goal of plant breeders is to develop crops that
reproduce through apomixis40. As endosperm is
required to support embryogenesis and/or
germination the sensitivity of endosperm to epigender
presents a major obstacle to breeding apomicts.
In Arabidopsis, some mutant allelles of the three
FIS genes (see above) allow the central cell to proliferate
without fertilization, but region-specific differentiation
(e.g. chalazal endosperm development) does not
occur23,24,31. However, if fie-1 mutants are
hypomethylated by the METI antisense gene,
autonomous endosperm development progresses
further31. An obvious component of normal seed
development missing from unfertilized fie-1 mutant
ovules is a genome contributed by a sperm: perhaps it
is lack of a paternal genome that prevents autonomous
endosperms in fie-1 mutants (or any other fis mutants)
from developing fully. In sexually produced seeds,
hypomethylation of the mother appears to add p
epigender14 (see above). Similarly, hypomethylation
could overcome the block to endosperm development
in fie-1 mutants by, in effect, supplying the missing
paternal genome. However, evidence presented above
suggests that in sexual seeds, a fis mutation itself has
a paternalizing effect. One explanation for the additive
effects of a fie-1 mutation and hypomethylation is that
the FIS complex and DNA methylation could cooperate
to prevent p-specific gene expression in the maternal
genome. Such an interaction between Polycomb
proteins and other epigenetic regulators is supported
by the finding that Drosophila homologues of
FIS1/MEA and FIS3/FIE associate physically with
histone deacetylase57. Although the mechanism
remains to be investigated, the phenotype of
hypomethylated fie-1 mutants shows that modification
of epigender could promote apomixis.
Conclusion
All genomes in an organism with imprinting possess
epigender. The maternal or paternal quality of a
genome is determined by the epigenetic marks on its
imprinted alleles, but the extent to which the genome’s
epigender is reflected in a gene expression pattern or
a developmental phenotype depends on the cellular
environment. Therefore, both the epigender of a donor
nucleus and the status of the recipient cell affect the
Review
Acknowledgements
We are grateful to
Andrew Ward and
Jonathan Slack for helpful
comments. M.S. is funded
by BBSRC grant P12018
and R.V. by BBSRC grant
P08575.
TRENDS in Genetics Vol.17 No.12 December 2001
outcome of cloning; and in interspecific crosses, the
epigender of gamete genomes and the mechanisms
for maintaining and responding to epigender are
all important to the success of hybridization. The
epigender model provides a simple conceptual
framework encompassing a wide variety of natural
and experimental phenomena, and also suggests
further investigations, such as screening for mutants
References
1 Haig, D. and Westoby, M. (1991) Genomic
imprinting in endosperm: its effect on seed
development in crosses between species, and
between different ploidies of the same species,
and its implications for the evolution of apomixis.
Philos. Trans. R. Soc. London Ser. B 333, 1–13
2 Moore, T. and Haig, D. (1991) Genomic imprinting
in mammalian development: a parental tug-ofwar. Trends Genet. 7, 45–49
3 Tilghman, S.M. (1999) The sins of the fathers and
mothers: genomic imprinting in mammalian
development. Cell 96, 185–193
4 Alleman, M. and Doctor, J. (2000) Genomic
imprinting in plants: observations and evolutionary
implications. Plant Mol. Biol. 43, 147–161
5 Arney, K.L. et al. (2001) Epigenetic reprogramming
of the genome – from the germ line to the embryo
and back again. Int. J. Dev. Biol. 45, 533–540
6 Surani, M.A.H. (1987) Evidences and
consequences of differences between maternal
and paternal genomes during embryogenesis in
the mouse. In Experimental Approaches to
Mammalian Embryonic Development (Rossant, J.
and Pedersen, R. A., eds), pp. 401–435, Cambridge
University Press
7 Surani, M.A. (1998) Imprinting and the initiation
of gene silencing in the germ line. Cell 93, 309–312
8 Corley-Smith, G.E. et al. (1996) Production of
androgenetic zebrafish (Danio rerio). Genetics
142, 1265–1267
9 Lin, B-Y. (1984) Ploidy barrier to endosperm
development in maize. Genetics 107, 103–115
10 Scott, R.J. et al. (1998) Parent-of-origin effects on
seed development in Arabidopsis thaliana.
Development 125, 3329–3341
11 Li, E. et al. (1992) Targeted mutation of the DNA
methyltransferase gene results in embryonic
lethality. Cell 69, 915–926
12 Li, E. et al. (1993) Role for DNA methylation in
genomic imprinting. Nature 366, 362–365
13 Finnegan, E.J. et al. (1996) Reduced DNA methylation
in Arabidopsis results in abnormal plant development.
Proc. Natl. Acad. Sci. U. S. A. 93, 8449–8454
14 Adams, S. et al. (2000) Parent-of-origin effects on
seed development in Arabidopsis thaliana require
DNA methylation. Development 127, 2493–2502
15 Obata, Y. et al. (1998) Disruption of primary
imprinting during oocyte growth leads to the
modified expression of imprinted genes during
embryogenesis. Development 125, 1553–1560
16 Kato, Y. et al. (1999) Developmental potential of
mouse primordial germ cells. Development
126, 1823–1832
17 Pfeifer, K. (2000) Mechanisms of genomic
imprinting. Am. J. Hum. Genet. 67, 777–787
18 De Chiara, T.M. et al. (1991) Parental imprinting
of the mouse insulin–like growth factor II gene.
Cell 64, 849–859
19 Nicholls, R.D. et al. (1998) Imprinting in
Prader–Willi and Angelman syndromes. Trends
Genet. 14, 194–200
http://tig.trends.com
711
that change epigender. Consideration of epigender
is vital to understanding the results of in vitro
fertilization or cloning in mammals, and controlled
modification of epigender could perhaps solve some of
the problems associated with these techniques. The
same approach could also help achieve goals of plant
breeders, such as crossing species with different
endosperm balance number, or triggering apomixis.
20 Feinberg, A.P. (1997) Genomic imprinting as a
developmental process disturbed in cancer. In
Genomic Imprinting (Reik, W. and Surani, A.,
eds), pp. 165–176, IRL/Oxford University Press
21 Malik, K. and Brown, K.W. (2000) Epigenetic gene
deregulation in cancer. Br. J. Cancer 83, 1583–1588
22 Hurst, L.D. (1997) Evolutionary theories of
genomic imprinting. In Genomic Imprinting
(Reik, W. and Surani, A., eds), pp. 211–237,
IRL/Oxford University Press
23 Ohad, N. et al. (1996) A mutation that allows
endosperm development without fertilisation.
Proc. Natl. Acad. Sci. U. S. A. 93, 5319–5324
24 Chaudhury, A.M et al. (1997) Fertilizationindependent seed development in Arabidopsis
thaliana. Proc. Natl. Acad. Sci. U. S. A.
94, 4223–4228
25 Grossniklaus, U. et al. (1998) Maternal control of
embryogenesis by MEDEA, a Polycomb group
gene in Arabidopsis. Science 280, 446–450
26 Vielle-Calzada, J.P. et al. (1999) Maintenance of
genomic imprinting at the Arabidopsis medea
locus requires zygotic DDM1 activity. Genes Dev.
13, 2971–2982
27 Kinoshita, T. et al. (1999) Imprinting of the
MEDEA Polycomb gene in the Arabidopsis
endosperm. Plant Cell 11, 1945–1952
28 Luo, M. et al. (2000) Expression and parent-of-origin
effects for FIS2, MEA, and FIE in the endosperm
and embryo of developing Arabidopsis seeds. Proc.
Natl. Acad. Sci. U. S. A. 97, 10637–10642
29 Yadegari, R. et al. (2000) Mutations in the FIE and
MEA genes that encode interacting Polycomb proteins
cause parent-of-origin effects on seed development by
distinct mechanisms. Plant Cell 12, 2367–2381
30 Kiyosue, T. et al. (1999) Control of fertilizationindependent endosperm development by the
MEDEA Polycomb gene in Arabidopsis. Proc.
Natl. Acad. Sci. U. S. A. 96, 4186–4191
31 Vinkenoog, R. et al. (2000) Hypomethylation
promotes autonomous endosperm development
and rescues postfertilization lethality in fie
mutants. Plant Cell 12, 2271–2282
32 Sørensen, M.B. et al. (2001) Polycomb group genes
control pattern formation in plant seed. Curr.
Biol. 11, 277–281
33 Ohad, N. et al. (1999) Mutations in FIE, a WD
Polycomb group gene, allow endosperm
development without fertilization. Plant Cell
11, 407–415
34 Luo, M. et al. (1999) Genes controlling fertilizationindependent seed development in Arabidopsis
thaliana. Proc. Natl. Acad. Sci. U. S. A. 96, 296–301
35 Spillane, C. et al. (2000) Interaction of the Arabidopsis
Polycomb group proteins FIE and MEA mediates
their common phenotypes. Curr. Biol. 10, 1535–1538
36 Pirotta, V. (1997) PcG complexes and chromatin
silencing. Curr. Opin. Genet. Dev. 7, 249–258
37 Seydoux, G. and Strome, S. (1999) Launching the
germline in Caenorhabditis elegans: regulation of
gene expression in early germ cells. Development
126, 3275–3283
38 Solter, D. (1988) Differential imprinting and
expression of maternal and paternal genomes.
Annu. Rev. Genet. 22, 127–146
39 Vinkenoog, R. et al. (2001) Genomic imprinting in
plants. In Methods in Molecular Biology: Genomic
Imprinting (Ward, A., ed.), pp. 327–370,
Humana Press
40 Koltunow, A. (1993) Apomixis: embryo sacs and
embryos formed without meiosis or fertilization in
ovules. Plant Cell 5, 1425–1437
41 Richards, A.J. (1997) Plant Breeding Systems 2nd
edn, Chapman and Hall
42 Johnston, S.A. et al. (1980) The significance of
genic balance to endosperm development in
interspecific crosses. Theor. Appl. Genet. 57, 5–9
43 Johnston, S.A. and Hanneman, R.E., Jr (1982)
Manipulations of endosperm balance number
overcome crossing barriers between diploid
Solanum species. Science 217, 446–448
44 Dawson, W.D. (1965) Fertility and size inheritance
in a Peromyscus species cross. Evolution 19, 44–55
45 Vrana, P.B. et al. (1998) Genomic imprinting is
disrupted in interspecific Peromyscus hybrids.
Nat. Genet. 20, 362–365
46 Solter, D. (2000) Mammalian cloning: advances
and limitations. Nat. Rev. Genet. 1, 199–207
47 Wilmut, I. et al. (1997) Viable offspring derived
from fetal and adult mammalian cells. Nature
385, 810–813
48 Ohlsson, R. et al. (1995) Plasticity of imprinting.
In Genomic Imprinting: Causes and Consequences
(Ohlsson, R. et al. eds), pp. 182–194, Cambridge
University Press
49 Dean, W. et al. (1998) Altered imprinted gene
methylation and expression in completely ES cellderived mouse fetuses: association with aberrant
phenotypes. Development 125, 2273–2282
50 Humpherys, D. et al. (2001) Epigenetic instability
in ES cells and cloned mice. Science 293, 95–97
51 Surani, M.A. (1999) Reprogramming a somatic
nucleus by trans-modification activity in germ
cells. Semin. Cell Dev. Biol. 10, 273–277
52 Moore, T. and Reik, W. (1996) Genetic conflict in
early development: parental imprinting in normal
and abnormal growth. Rev. Reprod. 1, 73–77
53 Young, L.E. et al. (1998) Large offspring syndrome
in cattle and sheep. Rev. Reprod. 3, 155–163
54 Young, L.E. et al. (2001) Epigenetic change in
IGF2R is associated with fetal overgrowth after
sheep embryo culture. Nat. Genet. 27, 153–154
55 Ogura, A. et al. (1998) Development of normal
mice from metaphase I oocytes fertilized with
primary spermatocytes. Proc. Natl. Acad. Sci.
U. S. A. 95, 5611–5615
56 Tesarik, J. et al. (1998) Spermatids as gametes:
indications and limitations. Hum. Reprod.
13 (Suppl. 3), 89–107
57 Tie, F. et al. (2001) The Drosophila Polycomb
Group proteins ESC and E(Z) are present in a
complex containing the histone-binding protein
p55 and the histone deacetylase RPD3.
Development 128, 275–286