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Human Molecular Genetics Advance Access originally published online on August 21, 2006
Human Molecular Genetics 2006 15(19):2869-2879; doi:10.1093/hmg/ddl228
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Complementary roles of genes regulated by two paternally methylated imprinted regions on chromosomes 7 and 12 in mouse placentation

Manabu Kawahara1, Qiong Wu1, Yukio Yaguchi2, Anne C. Ferguson-Smith3 and Tomohiro Kono1,*

1 Department of BioScience and 2 Electron Microscope Centre, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502, Japan and 3 Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK

* To whom correspondence should be addressed. Tel/Fax: +81 354772543; Email: tomohiro{at}nodai.ac.jp

Received June 22, 2006; Accepted August 8, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Imprinted genes have prominent effects on placentation; however, there is limited knowledge about the manner in which the genes controlled by two paternally methylated regions on chromosomes 7 and 12 contribute to placentation. In order to clarify the functions of these genes in mouse placentation, we examined transcription levels of the paternally methylated genes, tissue differentiation and development and the circulatory system in placentae derived from three types of bi-maternal conceptuses that contained genomes of non-growing (ng) and fully grown (fg) oocytes. The genetic backgrounds of the ng oocytes were as follows: one was derived from the wild-type (ngWT) and another from mutant mice carrying a 13 kb deletion in the H19 transcription unit including the germline-derived differentially methylated region (H19-DMR) on chromosome 7 (ng{Delta}ch7). Another set of oocytes was derived from mutant mice carrying a 4.15 kb deletion in the intergenic germline-derived DMR (IG-DMR) on chromosome 12 (ng{Delta}ch12). Although placental mass was lower in the ngWT/fg placentae compared with that in the WT placentae, it was recovered in the ng{Delta}ch7/fg placentae, but not in the ng{Delta}ch12/fg placentae. The ng{Delta}ch7/fg placental growth improvement was associated with severe dysplasia such as an expanded spongiotrophoblast layer and a malformed labyrinthine zone. In contrast, the ng{Delta}ch12/fg placentae retained the layer structures with expanded giant cells, but their total masses were smaller with a normal circulatory system in order. Our findings demonstrate that the genes controlled by the two paternally methylated regions, H19-DMR and IG-DMR, complementarily organize placentation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
In mammals, imprinted genes, wherein only one of the two parental chromosome copies is expressed, are regulated by epigenetic modifications, including DNA methylation. Acquisition of methylation at key regional controlling elements occurs in the parental germlines, during male or female gametogenesis. Most imprinted genes are regulated by maternally derived methylation, whereas only three have been identified as genes regulated by paternally derived methylation (1). The latter include H19-Igf2 and Gtl2-Dlk1, which are regulated by a differentially methylated region (DMR) on chromosomes 7 (H19-DMR) and 12 (IG-DMR), respectively, and Rasgrf1 on chromosome 9 (2–4). H19-Igf2 and Gtl2-Dlk1 are regulated by paternally derived methylation; however, Igf2 and H19 have opposite expression patterns as do Dlk1 and Gtl2. Imprinted genes also have prominent effects on placentation (5). A series of experiments have demonstrated that in the mouse placenta, Igf2 and Peg1/Mest appear to be involved in growth enhancement, whereas Grb10 and Phlda2 (Ipl, Tssc3) have restraining effects on growth processes. Imprinted genes on chromosome 12 have also been shown to play a role in regulating placental size and organization (6,7). Additionally, Mash2 and many genes linked to the X chromosome play critical roles in placentation (8–14).

Normal parthenotes containing two maternal genomes from matured oocytes showed poorly developed extraembryonic tissues, contributing to their death by E9.5 (embryonic day 9.5) (15,16). The failure of the placenta in parthenotes is due to the inappropriate expression of imprinted genes. This mainly occurs because the maternal epigenotype of the imprinted domain has been imposed on both the two haploid sets resulting in the absence of expression of paternally expressed imprinted genes or the overexpression of maternally expressed imprinted genes (17–19). However, when non-growing (ng) oocytes of WT mice (ngWT), which have erased their maternal imprints and thus are considered to be naïve with respect to most of the maternal imprinting process, were combined with fully grown (fg) oocytes, we observed that ngWT/fg bi-maternal conceptuses could develop to E13.5. Such ngWT/fg bi-maternal conceptuses formed placentae that consisted of three layers, namely the trophoblastic giant cells, spongiotrophoblast and labyrinthine layers. This suggests that failure to acquire maternal imprints in the ng oocyte rescues the placenta to some extent. Further, we have shown that ng/fg bi-maternal conceptuses are infrequently able to develop to term, when the embryos are reconstructed with ng oocytes (ng{Delta}ch7) of mutant mice carrying a 13 kb deletion in the H19 transcription unit with its DMR (20–23). This more successful outcome associated with the provision of Igf2 indicates that its absence in normal parthenotes is a contributory factor to their developmental failure. In addition, in these bi-maternal conceptuses, the IG-DMR on chromosome 12 of the ng{Delta}ch7/fg bi-maternal conceptuses that developed to term became unexpectedly methylated. Expression studies confirmed that the switch from the maternal to the paternal epigenotype in the imprinted domain at the distal regions of chromosomes 7 and 12 resulted in the appropriate transcription of Igf2-H19 and Dlk1-Gtl2 from the ng allele, which in turn enables further development of bi-maternal conceptuses. However, placentation in ng/fg bi-maternal conceptuses has not been investigated to date.

Investigating placentation in ng/fg bi-maternal conceptuses may help in elucidating the roles that paternally methylated imprinted genes play in mouse placentation in three ways. First, our system provides the only way of manipulating the expression of multiple imprinted genes, that can be compared with the extremes of full parthenogenesis and androgenesis. Secondly, we can monitor the development of placentae that exclusively possessed the maternal genomes during gestation; that is, we can investigate the phenotypes of the placentae in which all paternally methylated imprinted genes are absent by default. In the case of the ngWT/fg placenta, maternal methylation imprinting was globally modified, differing from the imprinting that is observed in the case of normal parthenotes because one set of maternal imprints are erased and neither paternal methylation nor a second set of maternal methylation imprints are imposed. Thirdly, the evaluation of the ng{Delta}ch7/fg placentae enables an understanding of the relative contributions of paternally methylated imprinted genes on chromosome 7 to the development of the placenta. Furthermore, in this study, we examined the ng{Delta}ch12/fg placentae by using ng oocytes of mice heterozygously carrying a 4.15 kb deletion of the IG-DMR on chromosome 12 (ng{Delta}ch12) (4). It is known that the deletion of the IG-DMR from the maternally inherited chromosome causes loss of imprinting of all genes in the 1 Mb cluster that carries the maternally repressed genes, Dlk1, Dio3 and Rtl1, and the maternally expressed non-coding RNAs, including Gtl2, and microRNAs (4). In the absence of the IG-DMR on the maternal chromosome, Dlk1, Dio3 and Rtl1 are activated and the non-coding RNAs repressed. As expected, the ng{Delta}ch12/fg placentae did not show appropriate expression of imprinted genes on chromosome 7 and exhibited normal expression of imprinted genes on chromosome 12. On the basis of this, it is be possible to address and compare the contributions of paternally methylated imprinted genes on chromosomes 7 or 12 to mouse placentation in a ng/fg bi-maternal epigenetic background.

Morphometric and histological analyses revealed that correction of the expression of paternally methylated imprinted genes on chromosome 7 affected not only the increase in placental mass but also the normal differentiation of giant cells. However, ng{Delta}ch7/fg bi-maternal conceptuses formed placentae with severe dysplasia such as an anomalously expanded spongiotrophoblastic layer and a malformed labyrinthine layer with an anomalous circulatory system. This indicates that these phenotypes are caused by the absence and/or overexpression of other imprinted genes in these placentae. Through the correction of the expression of paternally methylated imprinted genes on chromosome 12, we demonstrated that ng{Delta}ch12/fg bi-maternal conceptuses could develop into at least 18.5-day-old bi-maternal conceptuses, with the placentae comprising the three layers in order. We further observed that the circulatory system of the ng{Delta}ch12/fg placenta was comparable to that of the WT placenta. However, the placental weight barely increased, and the giant cells were abnormally expanded. This suggests that the absence and/or overexpression of chromosome 12 imprinted genes make a major contribution to the dysplasia in ngWT/fg placentae, but cannot rescue the size defect or trophoblast giant cell phenotype that are no longer evident when normal H19 and Igf2 levels are restored. Thus, we provide the first demonstration that the genes regulated by the two paternally imprinted methylated regions on chromosomes 7 and 12 contribute distinct and complementary functions to mouse placentation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The developmental ability of the ngWT/fg and ng{Delta}ch7/fg bi-maternal conceptuses had previously been determined; however, the developmental ability of the ng{Delta}ch12/fg bi-maternal conceptuses remained to be elucidated (17,20). We first examined the ng{Delta}ch12/fg bi-maternal conceptuses carrying the ng-oocyte genome that contained a deletion of the IG-DMR. The results clearly showed that the ng{Delta}ch12/fg bi-maternal conceptuses could develop into at least E18.5 (Kawahara et al., manuscript in preparation). Next, we examined the masses of all the four placental types. The ngWT/fg placentae showed severe growth retardation at E12.5 (Fig. 1A and B). Until E18.5, the ng{Delta}ch7/fg placentae consistently showed the highest masses among the placental types derived from bi-maternal conceptuses, but nonetheless, they were significantly lower than those of the WT placentae (Fig. 1B). Among the ng{Delta}ch7/fg, ng{Delta}ch12/fg and WT placentae, the ng{Delta}ch12/fg placentae maintained the lowest weight until E18.5 (Fig. 1B). To understand the feature of each placenta in detail, we carried out the following analyses: quantitative analysis of gene expression, in situ hybridization, histological analysis and scanning electron microscopic (SEM) studies to observe the placental circulatory system.


Figure 2281
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  		Figure 1. (A) Four types of placentae, namely the WT, ngWT/fg, ng{Delta}ch7/fg and ng{Delta}ch12/fg at E12.5, E15.5 and E18.5. (B) Graphical representation of placental masses (n=5). Values are represented as means±s.d. (indicated by error bars). (a and b) Values with different superscripts are significantly different within the same gestational age (P<0.05).

 
Gene expression of the paternally methylated imprinted genes H19, Igf2, Gtl2 and Dlk1 in the placenta
We performed quantitative expression analysis of the H19, Igf2, Gtl2 and Dlk1 genes in individual WT, ngWT/fg, ng{Delta}ch7/fg and ng{Delta}ch12/fg placentae by using real-time PCR (Fig. 2A). As expected, the expression of the H19 and Igf2 genes at E12.5 was corrected in the ng{Delta}ch7/fg placenta, but not in the ngWT/fg and ng{Delta}ch12/fg placentae. As expected, in both ngWT/fg and ng{Delta}ch7/fg placentae, the Gtl2 RNA expression level was approximately twice that of the mean value in the WT placentae, whereas the Dlk1 RNA expression level remained reduced until E18.5. The ng{Delta}ch12/fg placentae showed appropriate expression patterns of the Gtl2 and Dlk1 genes, except significantly higher expression of the Gtl2 gene at E12.5. Interestingly, the H19 RNA expression level in the ng{Delta}ch12/fg placentae was corrected to that in the WT placentae at E18.5, but not at earlier stages. The Igf2 RNA expression level was repressed in the ng{Delta}ch12/fg placentae. Next, we analysed the localization signal of RNA expression by using in situ hybridization (Fig. 2B). The intensities of the expression signals reflected the results of the quantitative expression analysis. Although, in previous published studies, the signals of Gtl2 RNA expression had not been detected in giant cells (24), other studies have observed Gtl2 transcription in some though not all giant cells (da Rocha et al., manuscript in preparation); signals were detected in the giant cells of the ngWT/fg and ng{Delta}ch7/fg placentae. Dlk1 RNA expression signals were distinctly recognized in the endothelial cells of the fetal blood vessels within the labyrinth of the WT and ng{Delta}ch12/fg placentae; however, no expression signals could be detected in the ngWT/fg and ng{Delta}ch7/fg placentae. Thus, we confirmed that in the ng{Delta}ch7/fg and ng{Delta}ch12/fg placentae, the expression patterns of H19-Igf2 and Gtl2-Dlk1 RNAs were respectively corrected to approximately normal levels seen in the WT placentae.


Figure 2282
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  		Figure 2. Gene expression of the paternally methylated imprinted genes H19, Igf2, Gtl2 and Dlk1 in placentae. (A) Graphical representation of gene expression in the WT, ngWT/fg, ng{Delta}ch7/fg and ng{Delta}ch12/fg placentae at E12.5, E15.5 and E18.5 (n=3). Values are represented as means±s.d. (indicated by error bars). (a–c) Values with different superscripts are significantly different within the same gestational age (P<0.05). (B) In situ hybridization analysis of the paternally imprinted genes H19, Igf2, Gtl2 and Dlk1 to E12.5 placentae (Spo, spongiotrophoblastic layer; Lab, labyrinthine layer). Anti-sense probes were hybridized to the cryosections of the WT, ngWT/fg, ng{Delta}ch7/fg and ng{Delta}ch12/fg placentae. Note that the signals of Gtl2 mRNA expression are evident in the giant cells of the ngWT/fg and ng{Delta}ch7/fg placenta, but not of the WT and ng{Delta}ch12/fg placenta.

 
Histological analyses of the placenta
Histological analysis revealed that the ngWT/fg placentae showed disproportionate expansion of the spongiotrophoblast layer along with a distorted and ambiguous boundary between the spongiotrophoblast layer and the labyrinth; they also showed enlarged giant cells (Fig. 3A and B). However, in the ng{Delta}ch7/fg placentae, enlargement of the trophoblast giant cells was not detected, in contrast to the morphology of the boundary that was not restored. In contrast, the boundary in the ng{Delta}ch12/fg placenta was entirely restored, but enlargement of giant cells seen in the ngWT/fg placenta was not entirely corrected (Fig. 3A and B). Further, morphometric analysis revealed that the labyrinth of the ngWT/fg placentae was reduced in size, and the ratio of the spongiotrophoblast layer to the labyrinth showed an increase of greater than 4-fold (Fig. 3B). This disproportion appeared to be corrected in the ng{Delta}ch7/fg placentae at E12.5. However, the ratio of the spongiotrophoblast layer to the labyrinth in the ng{Delta}ch7/fg placentae was 1.5-fold higher than that in the WT placentae. The ng{Delta}ch7/fg placenta consistently showed disproportionate expansion of the spongiotrophoblast layer and a reduction in the labyrinthine layer. In contrast, the ng{Delta}ch12/fg placentae never showed this type of disproportionate expansion of the spongiotrophoblast layer, and the ratio of the spongiotrophoblast layer to the labyrinth was identical to that in the WT placenta up to E18.5 (Fig. 3B). To gain further insight into the disproportionate expansion of the spongiotrophoblast layer in the ngWT/fg and ng{Delta}ch7/fg placentae, we examined the expression of the Phlda2 gene by real-time PCR and in situ hybridization in the four placental types (Fig. 4A and B). This gene is located on chromosome 7, is exclusively expressed from the maternal allele and encodes a cytoplasmic protein with a pleckstrin-homology domain. It controls placental size via a mechanism that is independent of Igf2 signalling, and its knockout results in global hyperplasia of placental tissues with disproportionate expansion of the spongiotrophoblast layer (11,25). The expression of the Phlda2 gene was repressed in all placental types derived from bi-maternal conceptuses, namely ngWT/fg, ng{Delta}ch7/fg and ng{Delta}ch12/fg placenta (11, 14 and 22%, respectively). This was in accordance with the results of the in situ hybridization (Fig. 4A and B). Hence, these results indicated that the ngWT/fg and ng{Delta}ch7/fg placentae show disproportionate expansion of the spongiotrophoblast layer, independently of the repression of the Phlda2 gene. However, the ng{Delta}ch7/fg placentae showed normal giant cells. Furthermore, the ng{Delta}ch12/fg placentae showed no histological defects except the enlarged giant cells. These results indicate that the spongiotrophoblast expansion and its defective interface with the labyrinthine zone in ngWT/fg is rescued by corrected expression of imprinted genes on chromosome 12 and the trophoblast giant cell expansion by corrected Igf2-H19 expression. More precise gene expression analysis using each placenta tissue might provide further insight into a various placental abnormalities in the bi-maternal conceptuses.


Figure 2283
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  		Figure 3. Histological and morphometric analyses of the WT, ngWT/fg, ng{Delta}ch7/fg and ng{Delta}ch12/fg placentae. (A) Midline placental sections (H&E) at E12.5, E15.5 and E18.5. The blue arrowheads indicate the border between the spongiotrophoblast layer and the labyrinth. High-magnification views of giant cells adjacent to the spongiotrophoblast layer at E12.5 are shown in the bottom row. (B) Spongiotrophoblastic layer (Spo), labyrinthine layer (Lab) and Spo/Lab ratios in the WT, ngWT/fg, ng{Delta}ch7/fg and ng{Delta}ch12/fg placentae (n=4). The average areas of giant cells at E12.5 was also calculated (n=4). (a and b) Values with different superscripts are significantly different within the same gestational age (P<0.05).

 


Figure 2284
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  		Figure 4. Investigation of Phlda2 RNA expression in the WT, ngWT/fg, ng{Delta}ch7/fg and ng{Delta}ch12/fg placentae. Phlda2 RNA transcription was investigated by (A) real-time PCR and (B) in situ hybridization (Spo, spongiotrophoblastic layer; Lab, labyrinthine layer) (n=3) at E12.5. Values are represented as means±s.d. (indicated by error bars). (a and b) Values with different superscripts are significantly different within the same gestational age (P<0.05).

 
Aberrant vasculature of the labyrinth of placenta derived from bi-maternal conceptuses
The labyrinth plays a crucial role in the exchange of nutrients, gases and waste between maternal and fetal blood. The number of blood vessels was counted in the WT and ng{Delta}ch12/fg placentae, and this value was found to increase with gestational age (Fig. 5B). SEM studies of casts additionally revealed that in the ng{Delta}ch7/fg placenta, both the maternal sinusoids and the fetal blood vessels within the labyrinth were defective (Fig. 5A, B and D). The ngWT/fg anfd the ng{Delta}ch7/fg placenta showed irregular dilation of the maternal sinusoids (Fig. 5Bb). At E12.5 and later, on examining the vascular casts of the maternal sinusoids in the WT placentae, we observed that the maternal sinusoids become smaller and more intricate, and the ring-like space at the base of the labyrinth (embryonic side) becomes enlarged as gestation proceeds (Fig. 5Ba, e and h and C). However, in the ng{Delta}ch7/fg placentae, the sinusoidal pattern was disordered and the degree of arborization was reduced, and the development of the ring-like space of the labyrinth was inadequate (Fig. 5Bc, f and i and C). In contrast, the maternal sinusoids of the ng{Delta}ch12/fg placentae showed none of the serious defects that were shown by the ng{Delta}ch7/fg placentae, except that their entire structure was very small (Fig. 5Bd, g and j and C). To visualize the feto-placental circulation, we prepared casts of the fetal circulation by injecting the casting compound into the umbilical cords (Fig. 5D). The umbilical cord of the ng{Delta}ch12/fg placenta was so narrow and thin that the casting compound could not be injected into them and we obtained casts only for the WT and ng{Delta}ch7/fg placentae. We found that in the ng{Delta}ch7/fg placenta, the fetal blood vessels within the labyrinth were strikingly disordered, particularly near the spongiotrophoblast border. Moreover, the blood vessels were remarkably dilated (centre of Fig. 5D). In the middle of the labyrinth, we observed that the fetal blood vessels were abnormally aggregated (right side of Fig. 5D). These results indicate that both the maternal sinusoids and fetal blood vessels in the ng{Delta}ch7/fg placentae are larger and have lower density than those in the WT placentae, whereas the ng{Delta}ch12/fg placentae retain the intact vascular architecture.


Figure 2285
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  		Figure 5. Analyses of placental vasculature. (A) In the morphometric analysis, the number of blood vessels in the WT, ng{Delta}ch7/fg and ng{Delta}ch12/fg placentae was investigated by tracing the surrounding area of each maternal and fetal blood vessel within 114 550 µm2 of the labyrinth (n=4). (a–c) Values with different superscripts are significantly different within the same gestational age (P<0.05). (B) Structure of the maternal sinusoids of the WT (a, e and h), ng{Delta}ch7/fg (c, f and i) and ng{Delta}ch12/fg (d, g and j) placentae at E12.5, E15.5 and E18.5. Structure of the maternal sinusoid of the ngWT/fg placenta at E12.5 is shown in (b). In each panel, the upper parts present an overview of the maternal sinusoids (bar=1 mm) and the lower parts present high-magnification views of the sinusoids in the labyrinth (bar=50 µm). Note that the WT and ng{Delta}ch12/fg placentae show a decrease in the size of the sinusoids with an increase in gestational age. In the ng{Delta}ch7/fg placenta, all these changes were observed to a lesser extent, and the maternal sinusoids were irregularly dilated, as shown by the yellow arrow heads. The ngWT/fg placenta also showed irregular dilation of the maternal sinusoids. (C) The ring-like space at the base of the labyrinth in the WT, ng{Delta}ch7/fg and ng{Delta}ch12/fg placentae at E12.5 and E18.5. The WT and ng{Delta}ch12/fg placentae showed an increase in the size of the ring-like space at the base, but the ng{Delta}ch7/fg placentae did not. (D) Fetal side casts of the labyrinth of the WT (a) and ng{Delta}ch7/fg (b) placentae at E15.5. An overview of the fetal blood vessels is shown on the left (bar=1 mm); the construction of the vasculature up to the spongiotrophoblast border is shown in the centre (bar=100 µm) and the fetal blood vessels in the middle of the labyrinth are shown on the right (bar=100 µm). Note that the organization of blood spaces was disordered; the fetal blood vessels were dilated and aggregated in the ng{Delta}ch7/fg placentae as shown by the blue or yellow arrowheads.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The various analyses reported here of the placental defects caused by the disturbance of paternally methylated imprinted genes located on chromosomes 7 and 12 facilitate the understanding of the manner in which the genes in these regions contribute to mouse placentation. We found that many phenotypes displayed by the ng{Delta}ch7/fg and ng{Delta}ch12/fg placentae were heterogeneous and complementary. This suggests that the two imprinted regions complementarily contribute to mouse placentation and the respective regions have multiple functions in mouse placentation (Fig. 6). Appropriate expression of the two imprinted genes on chromosome 7 results in some corrections to the ngWT/fg placental phenotypes, namely some increase in placental size and normalization of the expanded giant cells arising in the ngWT/fg placentae. Furthermore, when the imprinted genes on chromosome 12 were appropriately expressed in the ng{Delta}ch12/fg placentae, these placentae had the three layers in order and a labyrinthine zone with intricate vasculature.


Figure 2286
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  		Figure 6. Summary of the multiple roles played by the paternally methylated imprinted genes on chromosomes 7 and 12 in mouse placentation. Illustrations present the WT placenta (top), the ngWT/fg placenta (bottom), ng{Delta}ch7/fg placenta (left) and ng{Delta}ch12/fg placenta (right). The roles of the imprinted genes that were elucidated in the present study are listed in the table on the immediate right.

 
Roles of the paternally methylated imprinted genes on chromosome 7
The masses of the ng{Delta}ch7/fg placentae were higher than those of the ngWT/fg and ng{Delta}ch12/fg placentae. Previous studies reported that the placental phenotype that was associated with the lack of Igf2 exhibits a reduced mass (8,26,27). On the basis of this, we surmise that these corrections are due to the retrieval of Igf2 expression in the ng{Delta}ch7/fg placentae. However, the masses of the ng{Delta}ch7/fg placentae were never equal to those of the WT placentae. This might be due to multiple histological defects in the ng{Delta}ch7/fg placentae (Fig. 3). Indeed, further correction of the disproportionate growth of both the spongiotrophoblastic layer and labyrinthine zone by the appropriate transcription of the imprinted genes on chromosome 12 may be necessary. This could also ensure that the placental masses of the ng/fg placentae equal those of the WT placentae. Analysis of the placental phenotypes of double mutant ng/fg oocytes will provide further insight into this.

We additionally found that among the three placental types of bi-maternal placentae, only the ng{Delta}ch7/fg placentae had normal giant cells. Middleton et al. (28) also reported the possibility that Igf2 plays an important role in the growth and development of the giant cell tumours of the bone. Our results are consistent with the idea that the level of Igf2 RNA expression is important for the normal formation of trophoblastic giant cells in mouse placentae.

Giant cells contribute to invasiveness and the process of pregnancy by expressing the placental lactogen 1 and proliferin genes (29,30). In addition, oligonucleotide microarray analysis revealed that, at E12.5, the expression of both these genes in the ngWT/fg placentae was greater than twice that in WT placentae (Kawahara et al., unpublished data). In contrast, in the ng{Delta}ch7/fg placentae, the expression of these genes was normal. Taken together, these results and the present findings indicate that the expression of Igf2 and H19 on chromosome 7 contributes to the differentiation of functional giant cells.

Roles of the imprinted genes on chromosome 12
The ng{Delta}ch7/fg placentae showed abnormal expression levels of the paternally methylated imprinted genes on chromosome 12; these placentae evidently had many gross abnormalities. These anomalous phenotypes were entirely restored in the ng{Delta}ch12/fg placenta. The ng{Delta}ch12/fg placenta had a restored boundary between the spongiotrophoblast and labyrinthine zone and the ratio of the spongiotrophoblast layer to the labyrinth totally correlated with that of the WT placentae. The trophoblastic giant cells of the ng{Delta}ch12/fg placentae, however, remained abnormally expanded. Consequently, these findings suggest that the imprinted genes on distal mouse chromosome 12 regulate a balance between spongiotrophoblastic growth and labyrinthine growth.

Dlk1 is a paternally expressed, protein-coding gene on chromosome 12; this gene encodes a transmembrane protein containing epidermal growth factor repeats and is a member of the Notch/Delta/Serrate family of developmental signalling molecules. This gene is involved in several differentiation processes and is expressed in the fetal endothelial cells of the murine placenta; however, its precise function in the placenta is yet to be determined (5,31–35). We examined the subcellular localization of Dlk1 RNA in the mouse placenta. The Dlk1 RNA was specifically transcribed in the endothelial cells of the fetal blood vessels (Fig. 2B). Therefore, the disrupted fetal blood vessels observed in the ng{Delta}ch7/fg placenta might be due to the absence of Dlk1 RNA. In fact, we observed that the ng{Delta}ch12/fg placental vasculature was comparable to the WT placental vasculature. However, we cannot rule out the possibility that other genes located on chromosome 12, such as Rtl1 or Dio3, might be involved in placental vasculogenesis. Although placental defects in Dlk1 knockout animals were not described, conceptuses with paternal uniparental disomy for chromosome 12 have defects in the fetal capillary component of the labyrinthine zone (6,36). It is known that Rtl1 is a retrotransposon-like gene with an open-reading frame of unknown function and that Dio3 is a negative regulator of thyroid hormone metabolism; however, the particular roles of both these genes in placentation has not been reported thus far.

Low level of Phlda2 RNA expression in both the ng{Delta}ch7/fg and ng{Delta}ch12/fg placentae
To understand the reason behind the anomalous expansion of the spongiotrophoblastic layer in the ngWT/fg and ng{Delta}ch7/fg placentae, we investigated the expression pattern of the Phlda2 gene in the four placental types (Fig. 4). It is known that overgrowth along with the expansion of the spongiotrophoblast layer is detected in the Phlda2-null placentae, independent of Igf2 signalling (11). However, all the placental types exhibited low Phlda2 activity, including the ng{Delta}ch12/fg placentae, which did not show the expansion of the spongiotrophoblastic layer.

Phlda2 is a maternally expressed imprinted gene regulated by the Kvdmr1 located in the 10th intron of the Kcnq1 gene; we have confirmed that this methylation begins postnatally in oocytes that have attained a diameter greater than 40 µm (19). Hence, we had expected the level of Phlda2 transcription in the all placental types to be appropriately modified because the oocytes with a diameter less than 20 µm were selected as ng oocytes for nuclear transfer. We have confirmed the mono-allelic expression of the Phlda2 gene from the fg-oocyte genome in the ngWT/fg placentae at E12.5 (Ogawa et al., submitted for publication). Therefore, the disproportionate expansion of the spongiotrophoblastic layer could not be explained based on the low level of Phlda2 transcription. The reasons for the repression of the Phlda2 gene in ng/fg bi-maternal placentae remain to be elucidated.

From the present study, we could derive the following conclusions (Fig. 6). We confirmed that the deletion of the IG-DMR on chromosome 12 which causes paternalization of the maternal chromosome alone could restore some imbalance imposed by two maternal genomes and facilitate the development of bi-maternal conceptuses to at least E18.5. A combination of this new system and our previous bi-maternal conceptuses production system facilitated a detailed understanding of the contribution of the paternally methylated imprinted genes on chromosomes 7 and 12 towards mouse placentation. The present study provides evidence that imprinted genes transcribed from both the regions, H19-Igf2 and Gtl2-Dlk1, complementarily contribute to mouse placentation. It is probable that the appropriate expression of these imprinted genes would enable ng/fg bi-maternal conceptuses to undergo definitive placentation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Production of ng/fg bi-maternal conceptuses
Fg germinal vesicle (GV) oocytes were collected into M2 medium from the ovarian follicles of B6D2F1 (C57BL/6NxDBA) female mice, 44–48 h after they were injected with equine chorionic gonadotrophin (37). Ovulated MII oocytes were also collected from superovulated B6D2F1 mice, 16 h after they were injected with human chronic gonadotrophin. We collected ng oocytes that were in the diplotene stage of the first meiosis from the ovaries of 1-day-old newborn mice. Serial nuclear transfer was then performed using a previously described method (17,18,22). Ng oocytes derived from WT B6D2F1 (ngWT) females, H19{Delta}13 null mutants (ng{Delta}ch7) or IG-DMR{Delta}4.5 heterozygous mutants were reconstructed with enucleated GV oocytes. After fusion with inactivated Sendai virus, the reconstructed oocytes were cultured for 14 h in {alpha}-MEM medium (GIBCO, Grand Island, NY, USA). A spindle from the reconstructed oocytes was again transferred into ovulated MII oocytes, followed by treatment with 10 mM SrCl2 in Ca2+-free M16 medium for 2 h. These embryos were cultured in M16 medium for 3.5 days in an atmosphere of 5% CO2, 5% O2 and 90% N2 at 37°C (38). The embryos that developed to the blastocyst stage were transferred into the uterine horns of recipient female mice at 2.5 days of pseudopregnancy. The placentae were recovered from the pregnant mice at E12.5, E15.5 and E18.5 and used in the subsequent analyses. In order to distinguish the ng{Delta}ch12/fg bi-maternal conceptuses from the ngWT/fg bi-maternal conceptuses, we confirmed the deletion in IG-DMR by genotyping yolk sacs isolated from all recovered bi-maternal conceptuses.

Quantitative gene expression analysis
Total RNA was extracted using an RNAeasy Mini Kit (QIAGEN K.K., Tokyo, Japan) from the four types of whole placentae: WT placentae derived from fertilized embryos (B6D2F1xC57BL/6N), ngWT/fg, ng{Delta}ch7/fg and ng{Delta}ch12/fg. The cDNAs were then synthesized using the SuperScriptTM. II RnaseH reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA) in a reaction solution (20 µl) containing the total RNA (1 µg) prepared from each placenta. Finally, we performed a quantitative analysis of the gene expression by using real-time PCR (LightCyclerTM System, Roche Molecular Biochemicals, Mannheim, Germany) after preparing a reaction mixture (LightCycler FirstStart DNA Master SYBR Green I, Roche Molecular Biochemicals). The primers used for the analysis were as described in Wu et al. (22). The primers for Phlda2 gene expression analysis were used as follows, sense: 5'-CTT CGA AAA CCG TGA AGA CC-3' and anti-sense primer: 5'-CCT TGT AAT AGT TGG TGA CGA TG-3'.

mRNA in situ hybridization
In order to prepare cryosections, we collected and fixed the four types of placentae at E12.5 with 4% paraformaldehyde, incubated them at 4°C overnight in 20% sucrose in PBS and subsequently embedded them in OCT compound for 10 min at room temperature. This was followed by freezing at –80°C until use. The sections were allowed to dry and were immediately processed for RNA in situ hybridization after slicing. For in situ hybridization, digoxigenin (DIG)-labelled RNA probes were prepared according to manufacturer's instructions by using a DIG RNA labelling kit (Roche Diagnostics GmbH, Mannheim, Germany). To prepare RNA probes for each gene, a 27emsp14;kb mouse H19 cDNA clone (39) was used. For other probes, the following genes were amplified: Igf2 1346 bp (sense primer 5'-GCT GAC CTC ATT TCC CGA TAC-3' and anti-sense primer 5'-AAA ATT TTG GGT CCC CTT CCT-3'), Gtl2 (sense primer 5'-AAC CCA CTA CCA TAC AGA GGA-3' and anti-sense primer 5'-CGA GAG AAT GGT TGA GAC ACA-3') and Dlk1 (sense primer 5'-CCTCTTGC TCCTGCTGGCTTTC-3' and anti-sense primer 5'-GAT GTG TTG CTC GGG CTG CTG A-3'). Following amplification, these genes were inserted into a pGEM®-T Easy Vector (Promega). The Phlda2 probe was prepared as described previously (25). None of these sense control probes produced significant signals.

Histological and morphometric analyses
In order to measure the areas of the labyrinthine and spongiotrophoblast layers, we captured digitized images of the midline paraffin sections stained with haematoxylin/eosin (H&E). Placental entire images were saved as high-quality tif-files and analysed by using the MetaMorph software (Universal Imaging Co., Downingtown, PA, USA). The total number of pixels in each layer was calculated by using the ‘measurement’ tool of the MetaMorph software. Additionally, we analysed the average areas of giant cells at E12.5 and the number of blood vessels within the labyrinth by using PALM Robo Software 2.2-0103 (PALM Microlaser Technologies, AG). We calculated the number of blood vessels by tracing the area surrounding each blood vessel, that is, within 114 550 µm2 of the labyrinth. In the case of giant cells, average areas of five giant cells per H&E section from each placental type were calculated.

Scanning electron microscopy of vascular corrosion casts
Vascular corrosion casts of the placentae were prepared based on methods described previously (29,40). To prepare casts of the maternal vasculature, pregnant mice at E12.5, E15.5 and E18.5 were anaesthetized and the left ventricle of the beating heart was injected with a heparinized saline solution. An incision that served as an exit point for perfusion was made in the right atrium. Subsequently, the placentae were perfused with Mercox solution (Okenshoji, Tokyo, Japan), a casting compound, by infusion via the left ventricle.

For preparing fetal side casts at E15.5, the pregnant mice were sacrificed by cervical dislocation, and the uterus was excised and immersed in PBS. An implantation site was excised from the uterus and placed in a Petri dish under a stereomicroscope. The uterus was cut to expose the yolk sac, and the embryo and placenta were then exposed. The embryo and placenta were transferred onto a dried paper, and a 32G needle was introduced into the umbilical artery; this was followed by injection with heparinized saline solution, followed by perfusion with Mercox solution. An incision that served as an exit point for perfusion was made in the umbilical vein.

For complete hardening, each cast was polymerized in hot water at 60°C for 2 hours, corroded in 20% KOH, washed overnight in water and air-dried. The air-dried samples were frozen, cracked with a cooled razor blade to observe the internal structure of the labyrinth and then sputter-coated with platinum. Using a SEM (Hitachi S-4000, Tokyo) at low voltage, we examined five to 12 casts of the maternal vasculature and three to five casts of the fetal vasculature obtained from several pregnant mice.

Statistical analyses
Statistical analyses of all data for comparison were carried out using analysis of one-way analysis of variance and Fisher's PLSD test by using the statistical analysis software Statview (Abacus Concepts, Inc., Berkeley, CA, USA). A P-value of <0.05 was considered significant. When depicting statistical significance in the figures, the use of a, b and c indicates that within a gestational stage, a is significantly different from b and c and b is significantly different from c. A value marked as ab is not significantly different from the values marked a or b.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Dr Shirley Tilghman, Princeton University, for providing the H19{Delta}13 mutant mice and Dr Tom Moore, University College Cork, for the discussions. We would also like to thank Dr Eimei Sato, Tohoku University, Dr Yosuke Shiraishi of Shinjuku Acupuncture, Moxibustion and Judo Therapy Special School, Dr Miya Kobayashi, Kinjo Gakuin University and Dr Kazuyo Suzuki, Nagoya University, for their advice on corrosion casts and morphometry. This study was supported by a grant from the Bio-oriented Technology Research Advancement Institution (BRAIN), Japan.

Conflict of Interest statement. No conflicts declared.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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M. Kawahara, S. Morita, N. Takahashi, and T. Kono
Defining Contributions of Paternally Methylated Imprinted Genes at the Igf2-H19 and Dlk1-Gtl2 Domains to Mouse Placentation by Transcriptomic Analysis
J. Biol. Chem., June 26, 2009; 284(26): 17751 - 17765.
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