Generalized disruption of inherited genomic imprints leads to wide-ranging placental defects and dysregulated fetal growth

Abstract

Monoallelic expression of imprinted genes, including ones solely expressed in the placenta, is essential for normal placental development and fetal growth. To better understand the role of placental imprinting in placental development and fetal growth, we examined conceptuses developing in the absence of maternally derived DNA (cytosine-5-)-methyltransferase 1o (DNMT1o). Absence of DNMT1o results in the partial loss of methylation at imprinted differentially methylated domain (DMD) sequences in the embryo and the placenta. Mid-gestation E9.5 DNMT1o-deficient placentas exhibited structural abnormalities of all tissue layers. At E17.5, all examined placentas had aberrant placental morphology, most notably in the spongiotrophoblast and labyrinth layers. Abnormalities included an expanded volume fraction of spongiotrophoblast tissue with extension of the spongiotrophoblast layer into the labyrinth. Many mutant placentas also demonstrated migration abnormalities of glycogen cells. Additionally, the volume fraction of the labyrinth was reduced, as was the surface area for maternal fetal gas exchange. Despite these placental morphologic abnormalities, approximately one-half of DNMT1o-deficient fetuses survived to late gestation (E17.5). Furthermore, DNMT1o-deficient placentas supported a broad range of fetal growth. The ability of some DNMT1o-deficient and morphologically abnormal placentas to support fetal growth in excess of wild type demonstrates the importance of differential methylation of DMDs and proper imprinting of discrete gene clusters to placental morphogenesis and fetal growth.

Fig. 1

At E9.5, DNMT1o-deficient placentas have variable loss of DMD methylation at H19, Kcnq1, Snrpn and Mest. Methylation patterns of a representative wild type and three DNMT1o-deficient placentas are shown. M= maternal alleles. P= paternal alleles. Position of methylated CpGs dinucleotides are indicated as filled circles. The median methylation at the normally methylated allele is shown with the interquartile range in parentheses. * Denotes significantly different methylation of CpGs compared to wild type by Kruskal Wallis with p<0.05.

Fig. 2

At E17.5, DNMT1o-deficient placentas have variable loss of DMD methylation at Snrpn and Mest, but DMD methylation at H19 and Kcnq1 did not differ from wild type in placentas examined. Methylation patterns of a representative wild type and three DNMT1o-deficient placentas are shown. M= maternal alleles. P= paternal alleles. Position of methylated CpGs dinucleotides are indicated as filled circles. The median methylation at the normally methylated allele is shown with the interquartile range in parentheses. * Denotes significantly different methylation of CpGs compared to wild type by Kruskal Wallis with p<0.05.

Fig. 3

Expression of imprinted genes from DNMT1o-deficient placentas at E9.5, E12.5, E15.5 and E17.5 evolves across gestation. The histograms summarize mean expression (+SEM) of imprinted genes from seven imprinted gene clusters in DNMT1odeficient placentas compared to wild type (n=5). Each qPCR measurement was performed in triplicate, normalized to the L32 gene, and analyzed with the ΔΔCt method. (n=number of placentas studied). * Denotes significantly different expression compared to wild type by Kruskal Wallis with p < 0.05.

Fig. 4

A broad range of morphologic and gene expression abnormalities is present in E9.5 DNMT1o-deficient placentas. Hematoxylin/Eosin (H&E) staining and in situ hybridization (ISH) of frozen sections of wt and DNMT1o-deficient E9.5 conceptuses from a single litter. Multiple sections from placentas of each genotype were assessed and representative sections are shown. Each column of sections was obtained from a single conceptus. ISH probes are listed on the left include placenta lineage markers Prl2c2, Tpbpa and Tfeb as well as the imprinted genes Ascl2, Phlda2 and Igf2. wt= Wild type. M= DNMT1o-deficient mutant. Scale bar 500uM.

Fig. 5

At E17.5, DNMT1o-deficient placentas demonstrate characteristic morphologic abnormalities and have a broader distribution of embryonic/placental (E/P) weight ratios compared to wild type. (A) H&E staining of paraffin-embedded placental sections at E17.5 with the labyrinth, spongiotrophoblast, and maternal decidua delineated. (B) Scatter plot of embryonic to placental weight of E17.5 wild type and DNMT1o-deficient conceptuses. (C, D) Box plots showing median values, upper and lower quartiles and range of embryonic and placental weights among wild type and DNMT1o-deficient conceptuses. (E,F) Stereologic analysis of volume fraction of spongiotrophoblast and labyrinth depicted as box plots. Scale bars, 400µm (A). wt= Wild type. M= DNMT1odeficient mutant. de= Decidua. sp= Spongiotrophoblast. lb= Labyrinth. *p <0.05 by Kruskal Wallis.

Fig. 6

At E17.5, DNMT1o-deficient placentas had abnormal spongtotrophoblast layer and glycogen cell distribution. (A–D) ISH with Tpbpa in representative wild type and DNMT1o-deficient placentas. (E–H) PAS staining localizes glycogen staining largely to maternal decidua in wild type placentas. DNMT1o-deficient placentas have abnormal accumulation of glycogen in spongiotrophoblasts. This is prominent in most placentas examined (F,G) but less notable in other placentas (H). de= Decidua. sp= Spongiotrophoblast. lb= Labyrinth. Scale bar (A–D) 400µm, (E–H) 400µm (left panel) and 100µm (right panel).

Fig. 7

The labyrinth layer in DNMT1o-deficient placentas at E17.5 has both abnormal fetal vasculature and maternal blood pools. (A–E) ISH with Leptin receptor in representative wild type and DNMT1o-deficient placentas. Leptin receptor is present in the trophoblasts that line the maternal blood pools. (F–J) Immuno-staining with CD31 in wild type and DNMT1o-deficient placentas. * Maternal blood pools that are dilated in DNMT1o- deficient placentas. Scale bar (A–E) 400µm (F–J) 50um.

Fig. 8

Shared and distinct gene expression patterns are present in E17.5 DNMT1odeficient placentas with distinct E/P ratios. (A) Dendrograms describing genome-wide expression differences in E17.5 placentas. The X-axis is 1 minus the correlation coefficient. The E17.5 DNMT1o-deficient placentas were pooled into two groups based on the ratio of embryo/placental weight ratio (E/P) ratio with 4 placentas in each group. (B) Venn diagrams show the proportion of over-lapping and distinct genes that are down or up regulated in placentas with either a high E/P ratio or a low E/P ratio. Genes were included for consideration at a q < 0.05. Data are stratified by fold changes, 2.0 (top), 1.5 (middle), and 1.3 (lower). (C) Gene lists show results of functional analysis performed using Ingenuity Pathway Analysis. P value was less 0.01 for all genes in the functional analysis.