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First published online 13 December 2006
doi: 10.1242/dev.02726
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1 Department of Physiology, Development and Neuroscience, University of
Cambridge, Anatomy Building, Downing Street, Cambridge CB2 3DY, UK.
2 Institute of Biotechnology, College of Bioresources and Agriculture, National
Taiwan University, Taipei, 106, Taiwan.
3 LBME-CNRS, UMR 5099, IFR, Toulouse, France.
Author for correspondence (e-mail:
afsmith{at}mole.bio.cam.ac.uk)
Accepted 2 November 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTION:MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Genomic imprinting, Dlk1-Dio3 domain, Mouse development
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INTRODUCTION: |
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TOPSUMMARYINTRODUCTION:MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
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
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 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
growth retardation 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
non-coding 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).
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.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTION:MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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 µm 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 µm 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% 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).
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 µm) 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 µg) 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
(Dlk1-DMR), 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
IG-DMR, 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.
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RESULTS |
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TOPSUMMARYINTRODUCTION:MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IG-DMR/+ mice die pre-/peri-natally and demonstrate skeletal and muscle defects). The overall growth performance of the IG-DMR/+
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).
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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 IG-DMR/+,
compared with 12.7±0.59 in wild-type littermates). Abnormal
ossification was also observed in the sternum of IG-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 IG-DMR/+ 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.
|
).
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 µm2 compared
with 60.17 µm2 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
IG-DMR/+ 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.
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IG-DMR/+ conceptusesIG-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). 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.
|
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.
|
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 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.
|
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 transinteraction 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 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 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).
|
|
DISCUSSION |
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TOPSUMMARYINTRODUCTION:MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IG-DMR/+ placentas is the presence of significant amounts
of non-coding RNAs in IG-DMR/+ 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
Dlk1-Dio3 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 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 protein-coding 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 non-coding 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
RNAi-mediated mechanism (Davis et al.,
2005). It is not known whether 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
placenta-specific 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 IG-DMR 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-germ-line 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.
|
ACKNOWLEDGMENTS |
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|
Footnotes |
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Present address: Division of Functional Genomics. Jichi Medical School,
3311-1 Yakushiji, Minamikawachimachi, Kawachigun, Tochigi 329-0498, Japan
|
REFERENCES |
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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