RESEARCH ARTICLE
417
Development 134, 417-426 (2007) doi:10.1242/dev.02726
Differential regulation of imprinting in the murine embryo
and placenta by the Dlk1-Dio3 imprinting control region
Shau-Ping Lin1,2, Phil Coan1, Simao Teixeira da Rocha1, Herve Seitz3,*, Jerome Cavaille3, Pi-Wen Teng1,
Shuji Takada1,† and Anne C. Ferguson-Smith1,‡
Genomic imprinting is an epigenetic mechanism controlling parental-origin-specific gene expression. Perturbing the parental origin
of the distal portion of mouse chromosome 12 causes alterations in the dosage of imprinted genes resulting in embryonic lethality
and developmental abnormalities of both embryo and placenta. A 1 Mb imprinted domain identified on distal chromosome 12
contains three paternally expressed protein-coding genes and multiple non-coding RNA genes, including snoRNAs and microRNAs,
expressed from the maternally inherited chromosome. An intergenic, parental-origin-specific differentially methylated region, the
IG-DMR, which is unmethylated on the maternally inherited chromosome, is necessary for the repression of the paternally
expressed protein-coding genes and for activation of the maternally expressed non-coding RNAs: its absence causes the maternal
chromosome to behave like the paternally inherited one. Here, we characterise the developmental consequences of this
epigenotype switch and compare these with phenotypes associated with paternal uniparental disomy of mouse chromosome 12.
The results show that the embryonic defects described for uniparental disomy embryos can be attributed to this one cluster of
imprinted genes on distal chromosome 12 and that these defects alone, and not the mutant placenta, can cause prenatal lethality.
In the placenta, the absence of the IG-DMR has no phenotypic consequence. Loss of repression of the protein-coding genes occurs
but the non-coding RNAs are not repressed on the maternally inherited chromosome. This indicates that the mechanism of action
of the IG-DMR is different in the embryo and the placenta and suggests that the epigenetic control of imprinting differs in these
two lineages.
INTRODUCTION:
Genomic imprinting in mammals is an epigenetic marking process
that causes a subset of genes to be regulated depending on
their parental origin (Reik and Walter, 2001; Da Rocha and
Ferguson-Smith, 2004). This results in the functional nonequivalence of at least thirteen subchromosomal homologues in the
mouse and the requirement for both a maternal and paternally
inherited copy of these domains for normal development to occur
(http://www.mgu.har.mrc.ac.uk/research/imprinting/largemap.html).
Embryological studies involving the generation of parthenogenetic
and androgenetic conceptuses and chimaeras containing such cells in
the presence of normal biparental cells, showed that imprinted genes
are required for the formation of the placenta and for the development
of particular lineages including mesodermal and ectodermal
derivatives (Barton et al., 1984; McGrath and Solter, 1984; Barton et
al., 1991; Fundele et al., 1991). The function and expression of the
subsequently identified imprinted genes is consistent with this and in
particular emphasises a role for imprinted genes in the regulation of
pre and postnatal resources (Constancia et al., 2004). For example,
all imprinted genes tested to date are expressed in the developing
1
Department of Physiology, Development and Neuroscience, University of
Cambridge, Anatomy Building, Downing Street, Cambridge CB2 3DY, UK. 2Institute
of Biotechnology, College of Bioresources and Agriculture, National Taiwan
University, Taipei, 106, Taiwan. 3LBME-CNRS, UMR 5099, IFR, Toulouse, France.
*Present address: Department of Biochemistry and Molecular Pharmacology,
University of Massachusetts Medical School, Lazare Research Building, Room 870P,
364 Plantation Street, Worcester, MA 01605-2324, USA
†
Present address: Division of Functional Genomics. Jichi Medical School, 3311-1
Yakushiji, Minamikawachimachi, Kawachigun, Tochigi 329-0498, Japan
‡
Author for correspondence (e-mail: [email protected])
Accepted 2 November 2006
extraembryonic lineages where most function to control lineage
specification, morphogenesis and physiological processes within the
placenta (Coan et al., 2005). Indeed, for many imprinted genes, the
imprinting is placenta-specific with other tissues exhibiting biallelic
expression. In addition to a placental function, imprinted genes also
function to control tissue development and growth within the embryo
with alterations in imprinted gene dosage causing defects in a range
of tissues including muscle, skeleton, adipose tissue and liver
(Georgiades et al., 2000; Barton et al., 1991; McLaughlin et al., 1997;
Moon et al., 2002; Charalambous et al., 2003). Finally, defects
involving energy homeostasis and behaviour indicate a role for
imprinted genes in postnatal processes (Plagge et al., 2004; Plagge et
al., 2005; Curley et al., 2005; Charalambous et al., 2007) (S.T.R., S.P.L. and A.F.S., unpublished).
The distal portion of mouse chromosome 12 is subject to genomic
imprinting and harbours a 1 Mb imprinted domain containing
developmentally regulated protein-coding genes and non-coding
RNA genes (Takada et al., 2000; Schmidt et al., 2000; Cavaille et al.,
2002; Tsai et al., 2002; Yevtodiyenko et al., 2002; Seitz et al., 2003;
Seitz et al., 2004; Youngson et al., 2005). The 5⬘ end of the domain
harbours the Dlk1 gene and the 3⬘ end, the Dio3 gene. The need for
correct dosage of imprinted genes at this domain is demonstrated by
the lethality and developmental abnormalities observed in
conceptuses with maternal and paternal uniparental disomy for
chromosome 12 [MatDi(12) and PatDi(12), respectively]. Embryos
with PatDi(12) have two copies of chromosome 12 inherited from
their father with none from their mother. They die late in gestation
and have prenatal muscle hypertrophy and defects in muscle
maturation, costal cartilage defects and hypo-ossification of
mesoderm-derived bones (Georgiades et al., 2000; Sutton et al.,
2003). Placentomegaly is also evident, accompanied by cellular
defects in the junctional and labyrinthine trophoblast zones and in
DEVELOPMENT
KEY WORDS: Genomic imprinting, Dlk1-Dio3 domain, Mouse development
RESEARCH ARTICLE
the decidua basalis (Georgiades et al., 2001). Conceptuses with the
reciprocal MatDi(12) defect, with two copies of maternally inherited
chromosome 12, die perinatally and have proportional growthretardation of both the embryo and the placenta. They have a
reciprocal skeletal muscle phenotype as compared with PatDi(12),
skin defects and defective neural crest-derived middle ear ossicles
(Georgiades et al., 2000; Tevendale et al., 2006). Thus, during
prenatal stages, imprinted genes on chromosome 12 are essential for
fetal viability, the regulation of prenatal growth, the normal
development of extraembryonic tissues and of some mesodermal
and neural crest-derived lineages.
Some human individuals with uniparental disomy for
chromosome 14 (mUPD14 and pUPD14) have been reported. Part
of chromosome 14 shares syntenic homology with distal mouse
chromosome 12. Maternal and paternal disomy patients have distinct
growth, developmental and neural defects. Clinical features of
mUPD14 include intrauterine growth retardation (IUGR), followed
by postnatal hypotonia. Patients have short stature, precocious
puberty, small hands and feet, obesity, joint laxity, scoliosis,
recurrent otitis media, hydrocephalus and mild mental retardation
(Temple et al., 1991; Mitter et al., 2006; Georgiades et al., 1998;
Sanlaville et al., 2000; Sutton and Shaffer, 2000; Martin et al., 1999).
By contrast, patients with pUPD14 have polyhydramnios, a small
thorax with rib deformities and associated respiratory insufficiency,
facial anomalies, short limbs, ventral wall hernia and moderate to
severe mental retardation (Georgiades et al., 1998; Kurosawa et al.,
2002; Sanlaville et al., 2000; Sutton and Shaffer, 2000). The similar
phenotypes observed in human UPD14 patients and mouse
uniparental disomy 12 mutants suggests some conservation of
growth and neuronal functions related to imprinted genes that reside
in this chromosomal region.
The extent to which the Dlk1-Dio3 domain might be responsible
for most of the defects observed in the MatDi(12) and PatDi(12)
mutant mice and UPD14 patients is not known. The region contains
three protein-coding genes expressed from the paternally inherited
chromosome: delta-like 1 (Dlk1), retrotransposon-like 1 (Rtl1) and
Dio3 (Takada et al., 2000; Schmidt et al., 2000; Tsai et al., 2002;
Seitz et al., 2003; Youngson et al., 2005). On the maternally
inherited chromosome these genes are repressed and several noncoding RNA transcripts are expressed. These include gene trap locus
2 (Gtl2), microRNAs overlapping with and expressed in an antisense
orientation to Rtl1, C/D small nucleolar RNAs (snoRNAs) and the
microRNA-containing gene (Mirg) located in the vicinity of a cluster
containing approximately 40 microRNAs (Seitz et al., 2004). The
parental-allele-specific expression of these imprinted genes is
regulated by an intergenic imprinting control element, the IG-DMR
located 13 kb upstream of Gtl2. When an IG-DMR deletion is
transmitted from the mother, the entire 1 Mb Dlk1-Dio3 imprinted
cluster changes from a maternal epigenotype into a paternal
epigenotype. This results in activation of the normally maternally
repressed imprinted genes (Dlk1, Rtl1 and Dio3), whereas the
transcripts normally expressed from the maternally inherited
chromosome (Gtl2, microRNAs and C/D snoRNAs) become
repressed. Furthermore, the Gtl2 promoter, normally only
methylated after fertilisation on the paternally inherited
chromosome, becomes hypermethylated at the maternal promoter
(Lin et al., 2003). As further evidence of the epigenotype switch, a
region further downstream that is usually methylated on the
maternally inherited chromosome, loses its methylation (N.
Youngson, M. Ito, S.P.L. and A.F.S., unpublished). By contrast, no
significant imprinting defect was observed when the IG-DMR
deletion was transmitted from the father (Lin et al., 2003).
Development 134 (2)
Because of the maternal to paternal epigenotype switch at the
Dlk1-Dio3 domain in this IG-DMR knockout model (⌬IG-DMR/+),
these mutant mice demonstrate the consequence of imprinted gene
dosage alteration in this domain. The main difference between the
⌬IG-DMR/+ mutants and the PatDi(12) is the fact that, in PatDi(12),
both copies of chromosome 12 are paternally-derived hence both
complete chromosomes harbour the paternal epigenotype, whereas
in ⌬IG-DMR/+ embryos only the 1 Mb Dlk1-Dio3 imprinted
domain has switched into a paternal epigenotype on the mutant
maternal chromosome 12. The remainder of the maternal
chromosome 12 in the ⌬IG-DMR/+ mutant maintains its maternal
epigenotype. Therefore, comparing the phenotypes of ⌬IG-DMR/+
conceptuses with those of PatDi(12), enables us to clarify the extent
to which imprinted genes in the Dlk1-Dio3 locus contribute to the
phenotypes observed in the murine PatDi(12) mutants, and provides
insight into whether other imprinted genes might be located
elsewhere on chromosome 12. Here, we have correlated phenotype
to epigenotype in the fetus. In addition, our results have revealed a
difference in the mechanisms regulating imprinting at this domain
between the embryo and the placenta.
MATERIALS AND METHODS
Mice
Animals harbouring deletion of the IG-DMR were maintained on a
C57BL6/J background by crossing males heterozygous for the deletion with
wild-type females. Females carrying the deletion were mated to wild-type
C57BL6/J or DBA/2 males to generate conceptuses with the maternally
inherited deletion. In all experiments, controls were wild-type littermates.
Genotyping was as described previously (Lin et al., 2003). For the
methylation analysis, DNA was isolated from conceptuses derived from
heterozygous intercrosses between animals with the balanced translocation
T(4;12)47H on C3H and 129Sv/101 genetic backgrounds. Conceptuses with
uniparental duplication/deficiency of the distal portion of mouse
chromosome 12 were distinguished from wild-type littermates as described
previously (Tevendale et al., 2006).
Skeletal analysis
The freshly dissected embryos were kept in tap water at room temperature
or 4°C overnight and then incubated at 65°C for 3-5 minutes before skinning
and eviscerating. The carcasses were stained with Alcian Blue and Alizarin
Red using standard procedures (McLeod, 1980; Georgiades et al., 2000).
Haematoxylin and Eosin staining of the sagittal sections was used to assess
thoracic abnormalities in ⌬IG-DMR/+ mutants.
Histology and immunohistochemistry
Immunostaining of muscle was conducted according to Georgiades et al.
(Georgiades et al., 2000) with the following modifications. Whole embryo
or placenta samples were fixed in 4% paraformaldehyde overnight at 4°C,
dehydrated and embedded in paraffin wax using standard protocols. Sections
of 7-10 ␮m were either stained with Haematoxylin and Eosin (Fig. 3), or
with MY32 monoclonal antibody (M4276, Sigma; Fig. 4). The MY-32
antibody is specific for skeletal muscle fibre myosin heavy chain molecules
at the stage analysed, allowing accurate identification and measurement of
all myofibres (Harris et al., 1989; Venuti et al., 1995). The modified protocol
for myosin (MY-32) staining used in this study was as follows. The crosssectionally cut 7 ␮m paraffin-embedded limb sections were first softened on
a hot plate (rotate 360°) for 1 minute then dewaxed in xylene for 10 minutes.
The slides were then rehydrated through ethanol for 10 minutes, methanol
for 5 minutes, 70% methanol for 2 seconds, 50% methanol for 2 seconds,
and 30% methanol for 2 minutes. The slides were finally placed in distilled
water for 5 minutes. Antigen retrieval was conducted by incubating slides in
0.01 M sodium citrate (pH 6.0) for 4 minutes in a pressure cooker. The hot
slides were then washed four times (10 minutes each) in Phosphate Buffer
Saline (PBS). Preblocking took place in 3% H2O2 (in tap water) for 10
minutes, 5% H2O2 in methanol (H1009, Sigma) for 90 minutes, then slides
were washed three times in PBS for 10 minutes each before blocking in 3%
DEVELOPMENT
418
Genomic imprinting differs in embryo and placenta
RESEARCH ARTICLE
Morphometric analysis of skeletal muscle
Comparative morphometrics were carried out on MY32-stained histological
sections through the largest cross-sectional area of the forelimbs.
Comparable sections were carefully selected from the mutants and wild-type
littermates and 3-4 sections (7 ␮m) with 10-section intervals were selected
for each embryo. For example, if section number 300 contained the largest
cross-sectional area for an embryo, sections 285, 295, 305 and 315 would
be chosen. Two skeletal muscle parameters were determined with the
Computer Assisted Stereology Toolbox (CAST) 2.0 system from Olympus
Denmark. These were the mean myofibre cross-sectional area and the mean
proportion of myofibres containing centrally located nuclei. Measurements
were made by randomly selecting the fields across the extensor carpi radialis
longus (ecrl) and values were calculated as mean±s.e.m. Other muscles,
including extensor pollicis longus (epl) and flexor digitorum superficialis
(fds) were also measured and the trend found to be the same. The Bartlett’s
test was performed to evaluate whether raw data obtained were homogenous.
If this was shown to be the case, an unpaired t-test was used to compare
results from mutants with wild-type littermates with P<0.05 for statistical
significance as described for the myofibre area. Conversely, if the raw data
were not homogenous, as for the percentage of centrally located nuclei per
field, the Mann-Whitney U test was introduced to compare results from the
mutants and the wild-type littermates, also with P<0.05 for statistical
significance.
Placenta stereology
Weights were taken for all selected embryos and placentae (n=5 for each
genotype). Placentae were then hemisected using a double-edged razor
blade, each half weighed, and then immediately fixed. The stereology work
was carried out using methods described previously (Coan et al., 2004).
Imprinting and expression levels in placentas isolated from
normal and ⌬IG-DMR conceptuses
Total RNA from the placenta samples without the maternal decidua was
extracted by the acid guanidium thiocyanate-phenol-chloroform (AGPC)
method (Chomczynski and Sacchi, 1987). PolyA+ RNA was extracted from
total RNA using the Dynabeads Oligo (dT)25 Kit (Dynal). Total RNA (120
␮g) was used as the starting material and all procedures and reagents were
as described in the manufacturer’s protocol.
Quantitative northern blot analyses were used to detect Dlk1, Gtl2, Rtl1,
C/D snoRNAs and Dio3 from the mutant and wild-type placenta samples as
described previously (Lin et al., 2003); primer extension was applied to the
microRNAs (Seitz et al., 2004). Quantification of multiple northern blots
and primer extensions, in addition to those shown in Fig. 5A, were used to
generate the histograms shown in Fig. 5B.
The biallelic expression of Dlk1 and Rtl1 from heterozygous placentas
with a maternally inherited deletion [embryonic day (E) 16] was
demonstrated using a single nucleotide polymorphism (SNP) identified
between C57BL/6 and DBA/2 for Dlk1, and between C57BL/6 and Mus
molossinus for Rtl1, as described previously (Lin et al., 2003). Dio3
imprinting was not assessed because allelic differences in the expression of
Dio3 in the placenta have previously been shown to be less pronounced (Tsai
et al., 2002; Yevtodiyenko et al., 2002).
Methylation status of differentially methylated regions in the
placenta
DNA was isolated, digested with restriction enzymes including those
specific for methylated DNA, and assessed by Southern blot analysis with
the following enzymes and probes: for the exon 5 region in Dlk1 (Dlk1DMR), DNA was digested with NheI and HpaII or HhaI and hybridised with
probe D2 as described in Takada et al. (Takada et al., 2000). For the IGDMR, DNA was digested with StuI and MspI or HpaII or HhaI and
hybridised with a NotI-ApaI fragment probe (M4) as described in Takada et
al. (Takada et al., 2002). For the Gtl2-DMR, DNA was digested with HincII
and with either MspI, HpaII or HhaI and hybridised with probe G1 as
described in Takada et al. (Takada et al., 2000). The additional placentalspecific Dlk1-DMR0 was identified using DNA digested with PstI along
with MspI, HpaII or HhaI and hybridised to a 986 bp PstI fragment located
approximately 7.4 kb upstream from the Dlk1 promoter in a CpG-rich region
conserved between mouse and human.
RESULTS
⌬IG-DMR/+ mice die pre-/peri-natally and
demonstrate skeletal and muscle defects
Deletion of the unmethylated IG-DMR from the maternal
chromosome results in lethality commencing after E16 (Table 1)
(Lin et al., 2003). The overall growth performance of the ⌬IGDMR/+ mutants was similar to wild-type littermates, except that a
shorter body length and a shorter, broader neck was observed in
many of the mutant mice (Fig. 1). The lethality, short body length
and neck phenotypes have been observed in PatDi(12) and pUPD14
patients (Georgiades et al., 2000; Kamnasaran and Cox, 2002).
By contrast, the reciprocal +/⌬IG-DMR animals inheriting the
deletion paternally did not exhibit lethality or significant growth
abnormalities when measured up to the ninth week postnatally. This
is consistent with a lack of any effect on the expression or imprinting
of genes on the paternally inherited chromosome in the presence of
this deletion (Lin et al., 2003).
At E19, skeletal defects were observed in ⌬IG-DMR/+ embryos.
These included a bell-shaped thorax with abnormally wide
angulation of the ribs relative to the sternum and longer 8th, 9th and
10th ribs (Fig. 2), with the deformed thoracic cage associated with
the protrusion of the abdominal organs (Fig. 3). Hypo-ossification
of the centra and the neural arches of the vertebrae were also
observed in ⌬IG-DMR/+ embryos in relation to their wild-type
littermates (10.3±0.29 ossification centres below the pelvis in ⌬IGDMR/+, compared with 12.7±0.59 in wild-type littermates).
Abnormal ossification was also observed in the sternum of ⌬IG-
Table 1. Maternal transmission of the IG-DMR deletion results in lethality commencing after E16
Age/Genotype
Maternal transmission
Paternal transmission
+/+
KO/+
+/+
+/KO
E14-15
E16-17
E18-19
D1
Post-weaning
7 (1)
11
3
4
39
35 (2)
26
36
58
23 (3)
28
38
9 (1)
1 (11)
5
7
33
0
46
35
Numbers in parentheses represent dead embryos or pups recovered in a cross between C57BL6 or DBA2 males and females heterozygous for the IG-DMR deletion.
DEVELOPMENT
BSA (A7888, Sigma) in PBS for 1 hour. Slides were then incubated in the
first antibody solution (Fast Myosin MY-32; M4276, Sigma; 1:1000
dilution) for 2-3 days at room temperature. After washing three times in
PBS/0.01 M Tween20 (P1379, Sigma) for 10 minutes, slides were incubated
with the secondary antibody (anti-mouse IgG; Sigma, B7264; 1:50 in 1%
BSA in PBS) for 90 minutes at room temperature followed by three 10
minute washes in PBS/Tween. A peroxidase treatment step was then
introduced by incubating the slides in Elite Vectarstain for 30 minutes at
room temperature (the Elite-Vectarstain solution was freshly made according
to manufacturer’s instructions). This was followed by a wash in PBS/Tween
for 2 minutes. Finally, the slides were developed for up to 10 minutes in
DAB (Sigma Fast 3⬘3⬘diaminobenzidine; D4293, Sigma; one tablet was
allowed to dissolve in 5 ml PBS for 30 minutes and urea tablet added 2
minutes before use). The slides were rinsed twice with PBS (15 minutes
each) and counterstained lightly with GillsIII Haematoxylin (352015M,
BDH(Gurr)-VWR) before mounting with DPX mounting medium
(360294H, BDH).
419
420
RESEARCH ARTICLE
Development 134 (2)
Fig. 1. Gross phenotype of E18 ⌬IG-DMR/+ embryos. Comparison
of (A) wild-type littermate (+/+) with (B) an embryo with the maternally
inherited IG-DMR deletion (KO/+). Scale bar: 1 mm.
Fig. 2. Skeletal defects of E19 ⌬IG-DMR/+ embryos. Comparison of
skeletal defects between E19 maternally transmitted IG-DMR knockout
embryos (B,D; KO/+) and wild-type littermates (A,C; +/+). Preparations
are stained with Alcian Blue for cartilage and Alizarin Red for bone. An
upward slanting thoracic cage, with wider angulation of the ribs
relative to the sternum, is observed in B as compared with A. Eight ribs
(instead of 7) were attached to the sternum for IG-DMR knockout
embryos (compare D with C). The green arrow indicates the extraossification observed in KO/+ embryos at the site where the 6th to 8th
ribs attach to the sternum (D). The numbers in C and D refer to the
costal cartilages, where the anterior-most rib is defined as 1. c, costal
cartilage; ste, ossification centre of one of the sternebrae; xp, xiphoid
process. Scale bar: 1 mm.
Absence of placental defects in the ⌬IG-DMR/+
conceptuses
Whereas all of the embryonic phenotypes observed in the PatDi(12)
conceptuses were recapitulated in the ⌬IG-DMR/+ conceptuses in
which the maternal chromosome had acquired a paternal
epigenotype, this was not the case for the placenta. In PatDi(12)
conceptuses, placentomegaly (an increase of 20%), and defects in
all three placental layers – the labyrinthine zone, the junctional zone
and the decidua basalis – were identified. These included
discontinuities and disruption of the trophoblast-endothelial
interface within the labyrinthine zone and shallow/delayed migration
of glycogen cells from the junctional zone into the maternal decidua,
a process normally commencing at around E12 of gestation. As all
of the imprinted genes located in the Dlk1-Dio3 imprinted cluster
are imprinted in the placenta (Takada et al., 2000; Tsai et al., 2002;
Seitz et al., 2003) (Fig. 5), perturbation of their dosage might be
expected to contribute to the abnormal placental phenotypes in the
PatDi(12).
In the maternally transmitted ⌬IG-DMR/+ conceptuses, however,
there were no significant placental weight differences as compared
with the placentas of wild-type littermates. Indeed, the total placental
volume was also similar in all genotypes (P=0.7) and no genotypespecific morphological defects were observed in the placentas of
either maternally or paternally inherited mutants (data not shown).
DEVELOPMENT
DMR/+ embryos (6 out of 7 embryos having the 5th ossification
centres on the sternum protruding to the 6th sternebra, compared
with 2 out of 11 in the +/+ littermates). The extra ossification centre
observed in the 6th sternebra of ⌬IG-DMR/+ embryos could be
associated with the inappropriate elongation and attachment of the
8th rib to the sternum, suggesting that this abnormal attachment
might signal ectopic ossification (Fig. 2). In addition to the defects
in endochondral ossification, abnormalities in intramembranous
ossification in the skull were seen. The sagittal sutures of the ⌬IGDMR/+ embryos, though variable, were generally wider than in the
wild-type littermates, suggesting delayed ossification of the skull
(data not shown). The above mentioned skeletal defects are similar
to those reported in both human pUPD14 patients and PatDi(12)
mice (Georgiades et al., 2000; Sutton et al., 2003), suggesting that
deregulation of imprinted genes in the Dlk1-Dio3 locus is the cause
of these skeletal abnormalities.
Reciprocal skeletal muscle defects have been demonstrated in
MatDi(12) and PatDi(12) embryos. In PatDi(12) embryos, a larger
myofibre cross-sectional area (muscle hypertrophy) and a
disproportionately high number of myofibres with centrally located
nuclei, have been described (Georgiades et al., 2000). Normally, the
number of myofibres with centrally located nuclei declines after
E16, reflecting the maturation of the fibres as nuclei move to a
peripheral location within the fibre. The abnormally high percentage
of centrally located nuclei found in myofibres of late gestational
PatDi(12) embryos suggests muscle immaturity. Reciprocal defects
are observed in MatDi(12) muscles.
In ⌬IG-DMR/+ mice, we observed that 24.76% of the muscle
cells contained centrally located nuclei as compared with the
significantly lower 3.51% present in wild-type littermates
(P<0.0001; Mann-Whitney U test; Fig. 4G). Muscle hypertrophy
was also observed in ⌬IG-DMR/+ embryos, as indicated by the
significantly larger mean myofibre area of 79.7 ␮m2 compared with
60.17 ␮m2 in +/+ littermates (P<0.0001; unpaired t-test; data from
individual litters are shown in Fig. 4H). The similarities in the
lethality and in the skeletal and muscle phenotypes observed in ⌬IGDMR/+ and PatDi(12) mice indicates that these are caused by the
overexpression of one or more of the three protein-coding genes
and/or by the absence of expression of the non-coding RNAs in both
mouse models.
Genomic imprinting differs in embryo and placenta
RESEARCH ARTICLE
421
Fig. 3. Malformed thoracic cage and abdominal distension of
⌬IG-DMR/+ embryos. Comparable sections of E19 wild-type (A) and
⌬IG-DMR/+ (B) embryos, illustrating the malformed thoracic cage and
abdominal distension of ⌬IG-DMR/+ embryos, which is also associated
with protrusion of the abdominal organs (B). Embryos were stained
with Haematoxylin and Eosin. Scale bar: 1 mm.
Fig. 4. The immature and hypertrophic muscle phenotypes
observed in ⌬IG-DMR/+ embryos. Muscle morphology of normal
(A,C,E) and ⌬IG-DMR/+ (B,D,F) embryos at E19, stained with the
myofibre-specific antibody MY-32. (A,B) Cross-sections of the forelimb.
(C,D) High power views of the extensor carpi radialis longus. (E,F) High
power views of the extensor pollicis longus muscle. (G,H)
Morphometric analyses show that there are statistically significant
differences, both in the proportion of myofibres with centrally-located
nuclei (G; P<0.0001; Mann-Whitney U test), and in the mean myofibre
cross-sectional area (H; P<0.0001, unpaired t-test). epl, extensor
pollucis longus; ecrl, extensor carpi radialis longus; r, radius; u, ulna.
Deletion of the IG-DMR results in activation of
protein-coding genes but only partial repression
of the non-coding RNAs in placenta
Upregulation of paternally expressed genes (Dlk1, Rtl1, Dio3) was
observed in the ⌬IG-DMR/+ placenta samples (Fig. 5A). However,
the effect was different to that seen in embryos (compare to the
inserted panel of Fig. 5B). The expression levels of the paternally
expressed Dlk1, Rtl1 and Dio3 from the ⌬IG-DMR/+ placenta
samples were 148.2%, 181.3% and 150.7%, respectively, relative to
the expression levels from the placentas of wild-type littermates
(normalized as 100%). This is in contrast to the 200%, 450% and
200%, respectively, seen in embryos. The 48.2% further activation
of Dlk1 in placentas with a maternally inherited IG-DMR deletion
was from activation of the normally repressed maternal allele, as
DEVELOPMENT
More detailed stereological measurements were applied. These
included percentage volume fractions of junctional and labyrinthine
zones, the surface areas within the labyrinthine zone (fetal capillaries
and maternal blood spaces), and the interhemal membrane thickness.
These measurements were compared between the ⌬IG-DMR/+
placenta and that of wild-type littermates. The labyrinthine zone
surface areas and the interhemal membrane thickness are of
physiological importance, as these placental components govern the
physical determinants for exchange in gestation (Coan et al., 2004).
No significant differences were observed in any of the criteria tested
between five ⌬IG-DMR/+ placentae and those from five wild-type
littermates (P=0.24-0.85; ANOVA with Fisher’s PLSD test). We
therefore conclude that the ⌬IG-DMR/+ conceptuses do not have
the same placental phenotype as described in the PatDi(12)
conceptuses.
There are at least two possible explanations for this striking
discrepancy between the PatDi(12) and the ⌬IG-DMR/+ placentas:
First, the placenta phenotypes observed in PatDi(12) placentas may
be caused by a currently unidentified imprinted gene or cluster of
imprinted genes on mouse chromosome 12 that is not controlled by
the IG-DMR. Alternatively, the IG-DMR may not confer the same
epigenetic control over the Dlk1-Dio3 imprinted domain in the
placenta as it does in the embryo. These two hypotheses are not
mutually exclusive.
To date, no imprinted genes located outside the Dlk1-Dio3
domain have been identified on chromosome 12. Multiple candidate
genes, selected through array-based expression profiling of
MatDi(12) and PatDi(12) placentas and through in silico and
functional predictions, have shown bi-allelic expression in attempts
to experimentally validate imprinting (D. Gray and A.F.S.,
unpublished). Nevertheless, we currently cannot rule out the
presence of other imprinted genes outside the Dlk1-Dio3 domain on
mouse chromosome 12 that might cause the placental defects in
uniparental disomy conceptuses. In order to address whether
imprinting in the placenta was governed by the IG-DMR in the same
way as in the embryo, expression levels of all the imprinted
transcripts were measured in the placenta.
422
RESEARCH ARTICLE
Development 134 (2)
Fig. 5. Expression of imprinted genes in
placentas as a consequence of the IG-DMR
deletion. (A) Northern blot analysis for
imprinted genes in the Dlk1-Dio3 domain
(Gtl2, Dlk1, Rtl1, Dio3), and primer extension
assay (for micro-RNAs miR-127 and miR-410).
MBII-48, MBII-49 and MBII-78 are snoRNA
genes. Gapdh and U3 (Rnu3 – Mouse Genome
Informatics) are controls. Gene symbols in blue
and red denote those expressed from the
paternally and maternally inherited
chromosomes, respectively. Each genotype is
represented by duplicate tracks of RNA isolated
from different placentas. (B) Bar chart
comparing expression levels between E16
placentas carrying the maternally-derived IGDMR deletion (red; KO/+), with the paternally
derived deletion (blue; +/KO), and wild-type
littermates (yellow; +/+). Error bars represent
s.e.m. Values were calculated from data
generated for each gene using control (n=5-8)
and mutant (n=2-5) placentas from different
litters (n=2-4). Statistically significant
differences from normal are: *, P<0.05; **,
P<0.005; ***, P<0.0005; ANOVA + Fisher’s
PLSD test. Values were normalised against
Gapdh except the small RNAs which were
normalised against the unlinked, nonimprinted snoRNA U3. For comparison, the
inserted panel shows the equivalent expression
analysis in E16 embryos (see Lin et al., 2003).
(C) Biallelic expression of Dlk1 in placentas of
conceptuses with a maternally transmitted IGDMR deletion. Sequence analysis of RT-PCR
products from control littermate and
heterozygote placenta upon maternal
transmission of the IG-DMR deletion.
Activation of Dlk1 from the normally repressed
maternally inherited allele is only observed
when the IG-DMR deletion is maternally
transmitted. The Dlk1 expression in the normal
placenta is exclusively from the paternal allele.
interaction between Rtl1 transcripts and the microRNAs results in
RNAi-mediated degradation of a proportion of (but not all) Rtl1
transcripts (Davis et al., 2005). Compared with the 450% increase
in Rtl1 transcripts in ⌬IG-DMR/+ embryos in the absence of the
expression of the complementary microRNAs, the expression levels
of miR-127 and Rtl1 in ⌬IG-DMR/+ placentas were 66.21% and
181.25% of those in wild-type littermates, respectively (Fig. 5B).
This less striking upregulation of Rtl1 in ⌬IG-DMR/+ placentas
could, in part, be due to the presence of significant levels of the
microRNAs expressed in the placenta.
Differential methylation in the placenta and
embryos
Previous IG-DMR deletion studies in the embryo indicated that the
unmethylated locus on the maternal chromosome was required for
expression and hypomethylation at the maternally inherited Gtl2
promoter. In its absence, the associated repression of Gtl2 was
correlated with hypermethylation of the promoter. Recently,
maintenance methylation was shown to be dispensable for
imprinting at the Kcnq1 domain on distal chromosome 7 in the
mouse placenta (Lewis et al., 2004; Umlauf et al., 2004). Results
presented here suggest that the IG-DMR is not alone responsible for
expression of the non-coding RNAs in the placenta, unlike in the
DEVELOPMENT
demonstrated by allele-specific RT-PCR sequencing (Fig. 5C). A
similar loss of imprinting was also seen for Rtl1 (data not shown).
Because Dio3 already exhibits significant biallelic activity in the
normal placenta (Tsai et al., 2003; Yevtodiyenko et al., 2004), allelic
changes were not assessed.
In contrast to the ⌬IG-DMR/+ embryo, the maternally expressed
non-coding RNA genes [Gtl2, miR-127 (Mirn127 – Mouse Genome
Informatics) and the snoRNAs MBII-48, MBII-49 and MBII-78]
showed considerable expression in the placenta. Some
downregulation of the maternally expressed genes was observed.
Partly owing to the large variation in expression levels, the
downregulation of Gtl2 is not statistically significant when
compared with wild-type littermates (P=0.1724; ANOVA + Fisher’s
PLSD test). Therefore, although there is loss of imprinting of the
normally paternally expressed protein-coding genes on the maternal
chromosome resulting in their overexpression, the non-coding
RNAs expressed from the maternal chromosome are not fully
repressed but rather display significant activity. Therefore, IG-DMR
deletion from the maternally inherited chromosome does not cause
a complete maternal to paternal epigenotype switch in placentas.
We demonstrated previously that, in embryos, the absence of
microRNAs antisense to Rtl1 is associated with increased Rtl1
transcript levels (Lin et al., 2003). Normally, a direct trans-
embryo. We therefore assessed the placental methylation status of
previously characterised DMRs in the region to determine whether
this differed from the embryo. We used mice with uniparental
duplications of the distal portion of mouse chromosome 12
(Tevendale et al., 2006) and their normal littermates to compare the
two parental chromosomes. Our results show a different methylation
pattern at the Dlk1-Dio3 domain in placentas and embryos (Fig. 6)
(Takada et al., 2000). Importantly, the methylation profile of the
germ-line derived IG-DMR was the same in the placenta as in the
embryo (Fig. 6A). However, the other differentially methylated
regions in the placentas had different methylation profiles than in the
embryo. Overall, this profile indicates a reduction in the methylation
differences between the maternal and paternal chromosomes and
suggests that, with the exception of the germ-line mark, differential
DNA methylation at other DMRs at this locus may not be as relevant
RESEARCH ARTICLE
423
as in the embryo (Fig. 6B-E). Methylation at these regions was not
analysed in the IG-DMR mutant as the differences between the two
parental chromosomes in the placenta is so small. Despite this
reduction in differential methylation in the normal placenta, Dlk1
and Gtl2 are imprinted (Takada et al., 2000).
In addition to the previously identified Dlk1-DMR, IG-DMR and
Gtl2-DMR, an additional differentially methylated region, DlkDMR0, was found upstream of Dlk1. This DMR was differentially
methylated in the placenta exhibiting hypermethylation on the
paternal chromosome and less methylation on the maternal allele,
but was completely unmethylated on both chromosomes in the
embryo (Fig. 6D,E). Any function for this element remains to be
determined. These findings suggest that either there is a different set
of imprint marks in the placenta and/or that the placenta possesses a
different mechanism for interpreting imprinted marks than in the
Fig. 6. Methylation studies of the
Dlk1-Gtl2 imprinted domain in
placenta. (A) The IG-DMR region is fully
methylated on the paternal chromosome
and unmethylated in the maternal
chromosome in placenta. The Dlk1-DMR
(B) and Gtl2-DMR (C) are partially
methylated on both parental alleles in the
placentas, with methylation level being
slightly higher on the paternal allele. (D) A
placenta-specific differentially methylated
region, Dlk1-DMR0, is identified ~7.4 kb
upstream of the Dlk1 transcriptional start
site. (E) Summary of the methylation
status of the Dlk1-Gtl2 domain in
embryos, sperm and placentas. White and
black circles represent unmethylated and
fully methylated regions, respectively.
One, two and three quarters-filled circles
represent alleles methylated by
approximately 25%, 50% and 75%,
respectively. M, maternal allele; P, paternal
allele
DEVELOPMENT
Genomic imprinting differs in embryo and placenta
RESEARCH ARTICLE
embryo. Regardless, as in the embryo, the IG-DMR is necessary for
repression of the paternally expressed imprinted genes on the
maternal chromosome in the placenta.
DISCUSSION
Our results indicate that the Dlk1-Dio3 domain is responsible for all
the fetal anomalies described for PatDi(12). This was not the case
for the placental defects. According to the expression levels of
imprinted genes in the Dlk1-Dio3 imprinted domain in the IG-DMR
knockout placentas, we found that the IG-DMR deletion from the
maternal chromosome does not cause a complete maternal to
paternal epigenotype switch in placenta as it does in the embryos.
One explanation for this may be that imprinting is regulated
differently in the placenta and that the IG-DMR itself is not
sufficient to confer full imprinting in that organ. The major
difference observed between PatDi(12) and ⌬IG-DMR/+ placentas
is the presence of significant amounts of non-coding RNAs in ⌬IGDMR/+ placentas. This finding might explain why placental defects
are absent in ⌬IG-DMR/+ conceptuses, as compared with the
PatDi(12) conceptuses, and is indicative of these non-coding RNAs
having an important biological function. Although it cannot be ruled
out that imprinted genes located elsewhere on chromosome 12
contribute to the previously described placental abnormalities, our
results indicate that the IG-DMR functions differently in the
placenta than in the embryo.
Deletion of the unmethylated IG-DMR on the maternal
chromosome causes the 1 Mb Dlk1-Dio3 imprinted locus on that
chromosome to undergo an epigenotype switch and behave like the
paternal one in the mutant embryos. This involves inappropriate
activation of three normally repressed protein-coding genes and the
repression of a series of non-coding RNAs on the mutant maternal
chromosome that are likely to play important roles. The
consequences of this epigenotype switch are that mutant embryos
are no longer viable, and that they share most of the abnormal
developmental phenotypes previously described for animals with
paternal uniparental duplication of the entire chromosome 12. This
finding suggests that the 1 Mb Dlk1-Dio3 imprinted domain is the
key imprinted domain on chromosome 12 that is responsible for the
developmental defects and lethality of the PatDi(12) conceptuses.
One exception to this is the placenta. Mutant phenotypes described
for PatDi(12) placentas were not recapitulated in the IG-DMR
knockout in placenta. This finding indicates that the developmental
abnormalities in the fetus, and not the placenta, are responsible for
the prenatal lethality in both genetic models.
It is not yet clear which, if any, of the imprinted genes in the Dlk1Dio3 imprinted locus is the primary cause of the embryonic lethality
described here. Overexpressing Dlk1 to 200% alone does not cause
lethality in hemizygous Dlk1 transgenic mice. However,
homozygous Dlk1 transgenic conceptuses die late in gestation and
exhibit a similar skeletal phenotype to that described for PatDi(12)
and ⌬IG-DMR/+ embryos (S.T.R., S.P.L. and A.F.S., unpublished).
This is consistent with the expression of Dlk1 during skeletogenesis
(Abdallah et al., 2003; Yevtodiyenko and Schmidt, 2006). Targeted
deletion of Dlk1 in mice results in skeletal malformations that
include rib fusions and asymmetrical fusion of the ribs to the
sternum (Moon et al., 2002), indicating that Dlk1 is indeed required
for normal skeletal development. Transgenic mice overexpressing
the soluble Pref-1/hFc fusion protein (the soluble form of Dlk1 fused
to the human immunoglobulin-gamma constant region) under the
control of the adipocyte fatty acid-binding protein (aP2) promoter,
also have skeletal abnormalities, primarily in the distal vertebrae.
The severity of these abnormalities correlates with the levels of
Development 134 (2)
circulating Pref-1/hFc, and includes a smaller thoracic cavity with
short ribs, and fused and disorganised vertebrae resulting in scoliosis
(Lee et al., 2003). In addition to its role in skeletal development,
Dlk1 is also implicated in myogenesis. Dlk1 is strongly expressed
during myogenesis (Yevtodiyenko and Schmidt, 2006) and ectopic
expression of an ovine Dlk1 cDNA in muscle results in post-natal
muscular hypertrophy (Davis et al., 2004).
The upregulated Dio3 in ⌬IG-DMR/+ embryos may also
contribute to the lethality phenotype by downregulating the active
form of thyroid hormone (T3), thyroid hormone being important in
controlling many cellular, metabolic and physiological functions
(Forhead et al., 2003; Fowden, 1995; Fowden and Silver, 1995;
Harakawa et al., 1989; Oppenheimer et al., 1987). Upregulation of
Dio3 in the ⌬IG-DMR/+ and PatDi(12) embryos may contribute to
the skeletal phenotypes also through downregulating the active form
of thyroid hormone. Indeed, disrupted utilization of thyroid
hormones has previously been shown to retard growth and bone
maturation (Gothe et al., 1999). A contribution from Dio3 in the
muscle phenotypes observed in PatDi(12) and ⌬IG-DMR/+ mice
also cannot be ruled out. Thyroid hormone has been demonstrated
to be important in inducing myoblasts to exit the cell cycle, to
differentiate and to express a muscle-specific phenotype, prenatally
(Muscat et al., 1995). As a negative regulator of thyroid hormone,
the excess level of Dio3 in PatDi(12) and ⌬IG-DMR/+ embryos
could reduce the contribution of thyroid hormone to the promotion
of myoblast differentiation, consistent with the defects observed.
The developmental effects of overexpressing Rtl1 and silencing the
maternally expressed non-coding RNAs from the Dlk1-Dio3 locus
have not as yet been demonstrated.
In addition to mouse, the contribution of the DLK1-GTL2
imprinted locus towards a mutant muscle phenotype has also been
demonstrated in sheep. A single nucleotide substution occurring
between DLK1 and GTL2 (around 12 kb upstream of the IG-DMR)
causes overexpression of several imprinted genes in the cluster
without affecting their parental-allele-specific gene expression
pattern. Overexpression of the genes expressed on the paternal
chromosome, leads to a post-natal muscle hypertrophy phenotype
termed Callipyge (Georges et al., 2003). This muscular hypertrophy
is characterised by a rostro-caudal gradient (i.e. it is more
pronounced in the hind quarters), and manifests itself only after three
to four weeks of age (Georges et al., 2003). This phenotype only
occurs when there is net upregulation of DLK1and PEG11/RTL1
expression in +/Callipyge animals. No muscle hypertrophy
phenotype is observed when the non-coding RNA genes (GTL2,
anti-PEG11 and MEG8) are upregulated on the maternal
chromosome (Callipyge/+) (Bidwell et al., 2004; Charlier et al.,
2001). In homozygous Callipyge animals, transcripts from all of the
above mentioned genes are upregulated on both the maternal and
paternally inherited chromosomes. Interestingly, the muscle defect
manifest in paternal heterozygotes is abrogated in the homozyous
animals, suggesting that maternally expressed non-coding RNAs
might be negatively regulating the paternally expressed proteincoding genes post-transcriptionally, at least in muscle.
Such a mechanism may explain the absence of the defective
placenta in the IG-DMR knockout conceptuses. Interestingly,
although biallelic, the measured transcript levels of the paternally
expressed protein-coding genes are not as high as in the embryo,
correlating with the expression of the maternally expressed noncoding RNAs. It is known that the microRNAs expressed antisense
to Rtl1 from the maternally inherited chromosome are able to target
Rtl1 post-transcriptionally and guide its degradation by an RNAimediated mechanism (Davis et al., 2005). It is not known whether
DEVELOPMENT
424
the other paternally expressed imprinted genes might also be
negatively regulated by the non-coding RNAs on the maternally
inherited chromosome at this locus. Nonetheless, these results
indicate that there is an inverse relationship between the levels of
protein-coding genes and non-coding RNAs in more than one
animal model and organ system, and the placental model here
provides another experimental system in which to consider the
relationship between protein-coding genes and non-coding RNAs at
this locus.
Although all the imprinted genes in the distal 12 cluster are
expressed in the placenta, the IG-DMR deletion experiment
demonstrated that this imprinting control element is not sufficient
for full control of imprinting at this domain in the placenta. This may
be the reason that we did not observe placental abnormalities in
⌬IG-DMR/+ conceptuses, in contrast to the PatDi(12) conceptuses.
Consistent with this idea is the finding that a slightly different pattern
of methylation is observed in the placenta than in the embryo. Our
data suggest that the IG-DMR is necessary and sufficient for
imprinting in the embryo. In the placenta, however, the IG-DMR is
necessary for the repression of the protein-coding genes, but not for
the activity of the non-coding RNAs. It is possible that chromatin is
regulated differently in the placenta than in the embryo.
Several findings indicate that epigenetic control in
extraembryonic tissues might be different from that in embryos
(Chapman et al., 1984; Wagschal and Feil, 2006), and several
imprinted genes show tissue-specific imprinting only in the placenta
(Coan et al., 2005). Although maintaining DNA methylation at
imprinted domains is required for imprinting maintenance in the
embryo, it has been shown for one imprinted domain that placentaspecific imprinting does not require maintenance methylation
(Lewis et al., 2004). These findings are consistent with results
presented here. In placenta, although the germ-line-derived IGDMR shows clear and comparable differential methylation to the
embryo, the methylation state of those secondary DMRs is different,
with the two parental chromosomes appearing more similar in their
methylation state. This suggests that methylation at the non-germline DMRs may not be important in the placenta. The identification
of an additional placenta-specific DMR 5⬘ of Dlk1 in a region that is
hypomethylated in embryos again emphasises the difference in the
epigenetic profiles of embryonic versus extraembryonic tissues.
More extensive epigenetic profiling of this domain in the placenta is
underway.
S.-P.L., P.C. and S.T.R. were supported by PhD studentships from the Taiwanese
National Government, The Anatomical Society, and FCT Portugal respectively.
The research was funded by grants from the MRC and CR-UK.
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