Rescue of placental phenotype in a mechanistic model of Beckwith-Wiedemann syndrome
© Oh-McGinnis et al; licensee BioMed Central Ltd. 2010
Received: 4 January 2010
Accepted: 11 May 2010
Published: 11 May 2010
Several imprinted genes have been implicated in the process of placentation. The distal region of mouse chromosome 7 (Chr 7) contains at least ten imprinted genes, several of which are expressed from the maternal homologue in the placenta. The corresponding paternal alleles of these genes are silenced in cis by an incompletely understood mechanism involving the formation of a repressive nuclear compartment mediated by the long non-coding RNA Kcnq1ot1 initiated from imprinting centre 2 (IC2). However, it is unknown whether some maternally expressed genes are silenced on the paternal homologue via a Kcnq1ot1-independent mechanism. We have previously reported that maternal inheritance of a large truncation of Chr7 encompassing the entire IC2-regulated domain (DelTel7 allele) leads to embryonic lethality at mid-gestation accompanied by severe placental abnormalities. Kcnq1ot1 expression can be abolished on the paternal chromosome by deleting IC2 (IC2KO allele). When the IC2KO mutation is paternally inherited, epigenetic silencing is lost in the region and the DelTel7 lethality is rescued in compound heterozygotes, leading to viable DelTel7/IC2KO mice.
Considering the important functions of several IC2-regulated genes in placentation, we set out to determine whether these DelTel7/IC2KO rescued conceptuses develop normal placentae. We report no abnormalities with respect to the architecture and vasculature of the DelTel7/IC2KO rescued placentae. Imprinted expression of several of the IC2-regulated genes critical to placentation is also faithfully recapitulated in DelTel7/IC2KO placentae.
Taken together, our results demonstrate that all the distal chromosome 7 imprinted genes implicated in placental function are silenced by IC2 and Kcnq1ot1 on the paternal allele. Furthermore, our results demonstrate that the methylated maternal IC2 is not required for the regulation of nearby genes. The results show the potential for fully rescuing trans placental abnormalities that are caused by imprinting defects.
Genomic imprinting is the mechanism by which haploid maternal and paternal genomes carry different epigenetic marks, resulting in monoallelic transcription of a subset of genes which are expressed exclusively from either the maternal or paternal allele . To date, over eighty imprinted genes have been identified in humans and mice . Many of these genes are known to have critical roles in embryonic development and placentation  and have also been implicated in the regulation of postnatal behaviour [4–6]. The imprinted region on distal mouse chromosome 7 (Chr 7) shares syntenic homology with human chromosome 11p15.5, a region associated with Beckwith-Wiedemann syndrome (BWS) and Wilms tumor. BWS is an imprinted disorder that is commonly characterized by macroglossia, organomegaly, abdominal wall defects, unusual facial features, hemihypertrophy, hypoglycemia, exomphalos, ear and renal anomalies . Roughly 5-10% of BWS patients are predisposed to a variety of childhood tumours, including hepatoblastoma, rhabdomyosarcoma, neuroblastoma, adrenal carcinoma, with Wilms tumor occurring most frequently [7, 8].
Distal mouse Chr7 contains several key imprinted genes required for fetal development, two of which are also implicated in the etiology of BWS, the paternally expressed insulin-like growth factor-2 (Igf2) gene regulated by imprinting centre 1 (IC1, also known as H19 DMR) located upstream of the maternally expressed H19 gene and the maternally expressed cyclin-dependent kinase inhibitor 1C (Cdkn1c) which is regulated by imprinting centre 2 (IC2, also known as KvDMR1) located within intron 10 of the Kcnq1 gene [9, 10]. In addition to Cdkn1c, the IC2 cluster contains at least eight other maternally expressed genes (Ascl2, Cd81, Tssc4, Kcnq1, Slc22a18, Phlda2, Osbpl5, and Dhcr7) [11–19]. Of these, Ascl2, Cd81, and Osbpl5 have been shown to be expressed and exclusively imprinted in the placenta [20, 21]. The precise functions, if any, of Cd81, Osbpl5, Tssc4, Kcnq1 and Slc22a18 in the placenta remain to be elucidated. However, the remaining genes in the cluster (Ascl2, Phlda2, and Cdkn1c) have well-documented roles in placentation since knockouts of each of these genes result in drastic placental phenotypes. Ascl2 null mice fail to form a spongiotrophoblast layer, a defect leading to embryonic lethality at E10 [15, 22]; Phlda2 null mice are viable but show an expanded spongiotrophoblast and placentomegaly ; Cdkn1c mutants possess varying phenotypes including a reduced labyrinth layer, expanded spongiotrophoblast, placentomegaly and perinatal lethality [24–26].
The mechanism of silencing of several genes both centromeric and distal to the position of the paternal IC2 over a large domain is not completely understood, though production and elongation of the ncRNA Kcnq1ot1 is thought to play a critical role in this process [27, 28]. Kcnq1ot1 production is accompanied by recruitment of Polycomb group complexes  and acquisition of repressive histone modifications [20, 21]. These observations suggest similarities with the action of other ncRNAs such as the regulation of Igf2r by the ncRNA Airn  and X-inactivation by Xist . The deletion of the paternal IC2 leads to activation of IC2-regulated genes on the paternal chromosome and +/IC2KO mice possess a growth restriction phenotype at least in part because of biallelic expression of Cdkn1c [32, 33]. The placentae of these animals were also reported to be smaller than their wild type litter mates . Recent studies have also shown that IC2 contains CTCF-binding sites which are occupied only on the unmethylated paternal allele however it is still unclear what role these CTCF binding sites might play in silencing the paternal alleles of IC2-regulated genes . It is also not known whether the methylated maternal IC2 allele is important for the activation of nearby genes.
We previously described an engineered truncation of distal Chr 7 with a breakpoint upstream of the Ins2 gene . The resultant 2.7-Mb deletion, called DelTel7, removes the entire IC2-regulated domain along with the telomeric end of the Chr 7 domain, replacing it with an artificial telomere. When the DelTel7 allele is paternally inherited (+/DelTel7 hemizygotes), there is no obvious phenotype, showing absence of paternally expressed imprinted genes required for development or of a haploinsufficiency effect . Maternal transmission of DelTel7 (DelTel7/+ hemizygotes) however, leads to a mid-gestational lethality, consistent with the deletion of all maternally expressed genes in the IC2-regulated sub-domain, and is also accompanied by abnormal placentation including the lack of spongiotrophoblast and an expanded trophoblast giant cell layer . Despite a difference in phenotypic outcome, possibly attributable to the absence of imprinting of essential genes such as ASCL2 in humans [37, 38], maternal inheritance of DelTel7 provides a mechanistic model for BWS, which is often associated with loss of expression of maternally expressed genes . Remarkably, we also demonstrated that the lethality observed upon maternal inheritance of this truncation can be rescued in trans by a paternally inherited IC2KO allele, which was previously shown to abolish Kcnq1ot1 transcription and its associated epigenetic silencing . The compound heterozygous DelTel7/IC2KO pups survive to term but it is unclear whether any placental abnormalities or defects in placental imprinted gene expression remain in the double mutants. Because of the importance of several IC2-regulated genes in placental function and development, we present here a characterization of the rescued DelTel7/IC2KO placentae to determine whether they are comparable to their wild type litter mates and whether deletion of IC2 leads to a full rescue of the placental phenotype caused by the loss of maternal genes from the DelTel7 allele. Our assessment would have direct implications for BWS patients, since placental abnormalities usually accompany BWS fetuses [39–41]. By examining the architecture, vasculature, and imprinted gene expression of the rescued DelTel7/IC2KO placentae we sought to determine whether it is possible to correct imprinting defects by restoring gene expression for a large number of genes in trans and thereby to further elucidate mechanisms of IC2-mediated epigenetic silencing.
Loss of imprinted gene expression in DelTel7/+ placentae
Other then the entire IC2 sub-domain, DelTel7 also deletes the very distal end of Chr7. This region contains close to 20 annotated genes. Unlike genes in the IC1 and IC2 domains, we find that most of these distal genes are not expressed in the placenta at significant levels (Additional file 1). The absence of an obvious phenotype in paternal hemizygotes for DelTel7 argues against haploinsufficiency effects in these mutants.
Germline transmission from DelTel7/IC2KO mice
Transmission of the DelTel7 (Δ) and IC2KO alleles from compound heterozygotes
DelTel7/IC2KO placentae exhibit normal architecture
DelTel7/IC2KO placentae exhibit normal vasculature
DelTel7/IC2KO placentae exhibit proper expression of imprinted genes
Placental abnormalities such as placentomegaly  and placental mesenchymal dysplasia  are commonly observed in mothers carrying BWS fetuses. Mouse models of the BWS imprinted region such as the Igf2 transgenic mouse model  and Cdkn1c null mouse models [24–26] which accurately recapitulate several phenotypic characteristics of the human disorder, are also accompanied by severe placental abnormalities, highlighting the importance of determining whether any placental abnormalities exist in the DelTel7/IC2KO mouse model. In these compound heterozygotes, IC2-regulated imprinted genes such as Cdkn1c are deleted from the maternal allele and can only be expressed from the paternal chromosome because of the IC2 deletion. Our studies have shown that rescuing the deleted IC2 imprinted domain results in appropriate placentation with respect to placental architecture, placental vasculature and imprinted gene expression. Our findings lead us to propose that rescuing an imprinting defect can also rescue the associated placental phenotype, which may have implications for BWS patients who have associated placental defects.
It has been postulated that mechanisms other than transcription and elongation of Kcnq1ot1 may influence repression of the paternal distal Chr 7 genes. For example, Cdkn1c imprinting has previously been shown to be controlled by an element some distance away from the gene itself in a bacterial artificial chromosome (BAC) mouse model of Cdkn1c, which failed to reproduce appropriate Cdkn1c imprinting . In another study, a yeast artifical chromosome (YAC) transgene containing the entire IC2 domain was shown to recapitulate faithful imprinting of most of the IC2-regulated genes (Phlda2, Slc22a18, Kcnq1 and Cdkn1c), but loss of imprinting of Ascl2 and Tssc4 was observed when the YAC was paternally inherited . Both of these genes lie centromeric to IC2 and suggests that perhaps these genes require additional control elements beyond Kcnq1ot1 transcription on the paternal allele that are absent on the YAC transgene. The results of our study differ from the above in that we have demonstrated that the absence of Kcnq1ot1 transcription and/or deletion of other sequences in IC2 not only leads to full and normal expression of the paternal distal Chr 7 genes but to a full rescue of our DelTel7 placental phenotype, suggesting that this locus is the single most critical component in maintaining epigenetic silencing in this region. Since both normal placental phenotype and placental expression of IC2 genes are observed in DelTel7/IC2KO conceptuses, our results also suggest that the normally methylated maternal allele of IC2 and its potential association with methyl-CpG-binding factors is not necessary in regulating the expression of the genes analyzed.
The placentomegaly resulting from maternal inheritance of a Phlda2 null allele has been shown to be almost completely rescued by paternal inheritance of the same IC2KO allele used in this study , providing evidence of proper Phlda2 expression levels from the paternal IC2KO allele. To the best of our knowledge, the DelTel7/IC2KO model is the first example of rescue of a large disrupted imprinted region, not just a single gene. We demonstrate that restoration of gene expression for a large number of genes in trans is possible and our work demonstrates the potential ability to rectify placental abnormalities caused by imprinting defects. Future studies include developing RNAi to knock down Kcnq1ot1 during preimplantation stages in DelTel7 pregnant females to determine if this can rescue placental and embryonic phenotypes. Such experiments would suggest that it may be possible to therapeutically target the ncRNA as a step towards aiding BWS patients with maternal defects at IC2 that result in maternal Kcnq1ot1 reactivation.
The DelTel7 deletion offers an opportunity to assess the phenotypic consequences of loss of function for a cluster of maternally expressed genes on distal Chr7. Here we show that deletion of the Kcnq1ot1 promoter and nearby sequences (IC2KO) on the paternal allele can fully rescue the placental phenotype associated with maternal inheritance of DelTel7. In the viable conceptuses, monoallelic expression of imprinted genes is reversed and is now provided by the non-silencing alleles on the paternal IC2KO chromosome. Our results show that all the genes required for normal placentation, missing in the DelTel7 allele are regulated by IC2 and the ncRNA Kcnq1ot1 on the paternal allele during normal development. The methylated maternal allele of IC2 is not implicated in the regulation of these genes. Our work establishes the possibility of rescuing imprinted disorders such as BWS in trans, by interfering with the expression or the long-range silencing function of the regulatory ncRNA Kcnq1ot1.
Mice and genotyping
For all genotypes, the maternal allele is always given first. We previously presented the generation of the DelTel7 mouse line and genotyping of the DelTel7 allele . Genotyping of the IC2KO allele (KvDMR1) is as previously described . Females heterozygous for the deletion allele (+/DelTel7) maintained on the outbred CD-1 background were mated to +/IC2KO males on the C57BL/6 background and placentae at various developmental stages were dissected. All animal experiments were performed under certificate A07-0160 from the UBC Animal Care Committee and complied with the national CCAC guidelines to the ethical care and use of experimental animals.
RT-PCR and quantitative PCR
E9.5 and E14.5 placentae were dissected in cold PBS and snap frozen on dry ice. Placental RNA was extracted by Trizol (Invitrogen) and cDNA was synthesized using SuperScript II (Invitrogen). For Kcnq1ot1 detection, a gene specific primer for Kcnq1ot1 was designed (Kcnq1ot1 GSP 5'CACATACACACACCCAACTCG 3') and PCR was performed under standard conditions. Primers for the Ppia reaction have been previously described . Quantitative RT-PCR was performed in technical triplicate on five individual placental samples of each genotype at E9.5 and three samples at E14.5 (+/+ and DelTel7/IC2KO) for all genes analyzed. For Cdkn1c, forward primer (5' GCGCAAACGTCTGAGATGAG 3') and reverse primer (5' CAGCCGAAGCCCAGAGTTC 3') were used. For Phlda2, forward primer (5' CCCGCCAAGGAGCTGTTT 3') and reverse primer (5' CCTTGTAATAGTTGGTGACGATGGT 3') were used. For Ascl2, forward primer (5' TCCTGGTGGACCTACCTGCTT 3') and reverse primer (5' AGGTCAGTCAGCACTTGGCATT 3') were used. For Tssc4, forward primer (5' ACGGGTGTCAGGTCGTATGG 3') and reverse primer (5' TGAGGGAGACGGTGTCAGAAG 3') were used. For Kcnq1, forward primer (5' AGAAGCAGAGGCAGAAGCACT 3') and the reverse primer has been previously described . For Cd81, forward primer (5' CTGGCTGGAGGCGTGATC 3') and reverse primer (5' TGGGTGCCGGTTTGTTTC 3') were used. For 4311, forward primer (5' CAGCTTTGGACATCACAGGTACTT 3') and reverse primer (5' TGCGCTTCAGGGACTATAGCA 3') were used. For Peg1, forward primer (5' TGTCCATCCCCATTCATTTT 3') and reverse primer (5' GAGTTCCAGCTGCCTGATTC 3') were used.
E9.5 and E14.5 placentae were dissected in cold PBS and fixed in fresh 4% paraformaldehyde overnight at 4°C. Paraffin-embedded sections were baked, cleared of paraffin and rehydrated by a series of xylene and ethanol washes. Antigen retrieval was done by microwaving slides in 1 mM EDTA, pH 7.5. Endogenous peroxidase blocking was done in 0.3% hydrogen peroxidase for 30 minutes. Blocking was done with 5% goat normal serum (Vector Labs) in 1× TBS-T with 0.5% BSA for 30 minutes. To detect Phlda2 protein in the labyrinth layer, and to identify fetal capillaries of the labyrinth layer, a 1:1000 dilution of the Phlda2 antibody and a 1:25 dilution of rabbit polyclonal anti-laminin antibody (Sigma) were used, respectively. Incubation was done overnight at room temperature for Phlda2 antibody and 1 hour for laminin antibody. A 1:500 dilution of the biotinylated anti-rabbit IgG secondary antibody (Vector Labs) was used for 1 hour and after a series of TBS-T washes, the staining was performed with the Vectastain Elite ABC kit and DAB substrate (Vector Labs). Counterstain was done with hematoxylin (Sigma) for about 1 minute and the slides were dehydrated in a series of ethanol and xylene washes before mounting with Entellan mounting medium (EM Science) and coverslipped with glass.
In situ hybridization
Antisense and sense strand probes were DIG-labelled using 10× DIG labeling mix (Roche) and T7, SP6, or T3 RNA polymerases (Roche). Probes were digested with DNaseI and purified by LiCl precipitation. Placentae at various stages were dissected in PBS and fixed in fresh 4% paraformaldehyde/1× PBS (RNase free) overnight at 4°C. The next day, they were washed in PBS and left overnight in 30% sucrose in PBS. They were allowed to equilibrate in OCT (Tissue-Tek) for half an hour at room temperature before embedding in OCT on dry ice. The blocks were stored at -80°C until ready to be used. Cryostat sections were cut at 10 microns and stored at -20°C until next day when hybridized. Sections were first thawed at 50°C, fixed in 4% PFA, treated with Proteinase K (final concentration of 40 ug/ml) and acetylated in acetic anhydride to reduce background. Sections were then prehybridized in hybridization buffer (50% formamide, 5× SSC, 5× Denhardt's, 0.25 mg/ml Yeast tRNA) at 60°C for 3 hours before hybridization at 55°C overnight. The next day, non-specific probe was removed by a series of SSC washes and treated with RNase A before blocking for 1 hour in 1% blocking buffer (Roche) at 37°C. Sheep anti-DIG-alkaline phosphatase conjugated antibody (1:2000) (Roche). The BCIP (Roche) and NBT (Roche) reaction was used to detect the signal. After three 1× PBS washes, sections were fixed in a solution containing 3.7% formaldehyde, counterstained with nuclear fast red (Sigma) and dehydrated before mounting with Entellan mounting medium and coverslipping with glass. Multiple sections (between 16 and 36) were analyzed per genotype per marker.
Placental fetal vasculature casts
Fetal vascular corrosion casts of E14.5 placentae were generated by following the procedure previously described by Whiteley et al. . Vascular casts were analyzed by SEM and morphometric analysis of capillary diameter was carried out using Openlab software (Improvision).
List of abbreviations used
Canadian Council on Animal Care
- Chr 7:
mouse chromosome 7
trophoblast glycogen cells
imprinting center 2, KvDMR1
in situ hybridization
periodic acid Schiff
reverse transcription polymerase chain reaction
scanning electron microscope
yeast artificial chromosome.
The authors thank Jay Cross for the Pcdh12 cDNA clone, Rosalind John for the Cdkn1c cDNA clone, Ben Tycko for the PHLDA2 antibody, Janet Rossant for the TpbpA (4311) and Prl3b1 (Pl-II) cDNA clones, and Xiaoli Wu for her guidance with in situ hybridization. LL holds a Canada Research Chair. This work was funded by a grant from the Canadian Institutes of Health Research to LL (MOP-82863) and NCI/NIH Grant 2RO1 CA089426 to MJH. ROM has a Four Year Doctoral Scholarship from UBC.
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