Roles for genomic imprinting and the zygotic genome in placental development

Abstract

The placenta contains several types of feto-maternal interfaces where zygote-derived cells interact with maternal cells or maternal blood for the promotion of fetal growth and viability. The genetic factors regulating the interactions between different cell types within feto-maternal interfaces and the relative contributions of the maternal and zygotic genomes are poorly understood. Genomic imprinting, the epigenetic process responsible for parental origin-dependent functional differences between homologous chromosomes, has been proposed to contribute to these events. Previous studies showed that mouse conceptuses with an absence of imprinted differences between the two copies of chromosome 12 (upon paternal inheritance of both copies) die late in gestation and have a variety of defects, including placentomegaly. Here we examined the role of chromosome 12 imprinting in these placentae in more detail. We show that the spatial interactions between different cell types within feto-maternal interfaces are defective and identify abnormal behaviors in both zygote-derived and maternal cells that are attributed to the genome of the zygote but not the mother. These include compromised invasion of the maternal decidualized endometrium and the central maternal artery situated within it by zygote-derived trophoblast, abnormalities in the wall of the central maternal artery, and defects within the zygote-derived cellular layer of the labyrinth, which is in direct contact with maternal blood. These findings demonstrate multiple roles for chromosome 12 imprinting in the placenta that have not previously been associated with imprinting effects. They provide insights into the function of imprinting in placental development and have evolutionary and clinical implications.

Figure 1

The major regions and cell types of the mouse placenta at E15.5. (a) Schematic representation of a sagittal section of the mouse placenta. The placenta is oriented with its maternal side at the top and the fetal (flat) side at the bottom. The plane of sectioning is through the center of the placenta and perpendicular to its flat surface. All sections shown in this study were sectioned in this way. The major placental zones (db, decidua basalis; jz, junctional zone; lz, labyrinth zone) are shown; their constituent cell types are depicted by different colors. (b) Magnification of the boxed area in a, showing in more detail the labyrinthine feto-maternal interface (the zygote-derived tissue between fetal and maternal blood). The trilaminar nature of the labyrinthine trophoblast (orange) is only distinguishable with an electron microscope (26) (Fig. 2e). bm, basement membrane; cma, central maternal artery; fbs, fetal blood space; fce, fetal capillary endothelium; fv, fetal vasculature; gc, “glycogen” cell clusters; lt, labyrinthine trophoblast; mbs, maternal blood space; st, spongiotrophoblast cells; tg, trophoblast giant cells; vd, maternal venous drainage. (c and d) H&E staining of normal (c) and pUPD12 (d) E18.5 placentae at low power. (Scale bar = 1 mm.)

Figure 2

Abnormal cell behavior in pUPD12 labyrinthine feto-maternal interfaces. Images are sagittal sections through the labyrinth of E18.5 normal (a, c, e, and g) and pUPD12 placentae (b, d, f, and h) placentae, and g and h are magnifications of the boxed areas in e and f, respectively. (a and b) Immunostaining for laminin α1 (counterstained with hematoxylin), a marker of the fetal endothelium and its associated basement membrane. Note the abnormal shape and density of the fetal vasculature in pUPD12 (b). (c and d) Methylene blue-stained semithin sections showing feto-maternal interfaces situated between the blood of fetal capillaries (f) and maternal blood spaces (m). Note the extensive acellular spaces (asterisks) within the pUPD12 interfaces (d). (e and g) Transmission electron micrographs distinguishing all three trophoblast layers (t1–t3), the fetal capillary endothelium (fc), and its associated basement membrane (bm) in the normal feto-maternal interface (e) as shown previously (25, 26). The outermost trophoblast layer (t3), which is cytotrophoblastic (25), is loosely attached to the middle layer (t2), which is syncytial (25). Note that the t2 layer has many vacuole-like spaces. These were shown to be due to the irregular invaginations of its maternal surface and are in direct contact with maternal blood plasma via the pores present in the t3 layer (25). (f and h) Note that in the pUPD12 labyrinthine interfaces, the acellular discontinuity (asterisks) is between the innermost trophoblast layer (t1) and the basement membrane (bm). Also note the increased thickness of the middle trophoblast layer (t2) in pUPD12. [Scale bars = 50 μm (a and b), 10 μm (c and d), 2 μm (e and f), 1 μm (g and h).]

Figure 3

Morphometric analysis showing the difference in the volume fraction (expressed as a percentage of total volume counted) of four labyrinthine parameters between normal and pUPD12 placentae at E18.5. Values are means; error bars representing the SEM. Statistical significance with P ≤ 0.01 is indicated by one asterisk and that with P ≤ 0.001 by two asterisks.

Figure 4

Junctional zone “glycogen” cells in normal and pUPD12 placentae. (a and b) Adjacent sections of a region of the junctional zone in E15.5 normal placentae. H&E staining (a) shows the morphological distinctiveness of “glycogen” cells (gc) (clear cytoplasm with small, strongly stained nuclei) from spongiotrophoblast cells (st). Together with spongiotrophoblasts, they line venous drainage sinuses (vd) of the junctional zone. Note that strong immunostaining against p57KIP2 protein (b) (counterstaining with hematoxylin) or RNA in situ hybridization for Igf2 gene transcripts (data not shown) marks “glycogen” cells as previously described (2, 27). (c and d) RNA in situ hybridization with an Igf2 transcript-specific probe of E18.5 normal (c) and pUPD12 (d) junctional zone (jz) showing the abnormally high abundance of “glycogen” cells in pUPD12. The white lines in c and d depict the boundaries of the junctional zone from the decidua basalis/trophoblast giant cell layer junction and labyrinth zone (l) as judged from directly adjacent H&E sections. Note that Igf2 is also expressed in the labyrinth as shown previously (27). [Scale bars = 50 μm (a and b) and 100 μm (c and d).]

Figure 5

Defects in the feto-maternal interfaces of the pUPD12 decidua basalis. (a and b) Sagittal sections immunostained with p57KIP2 antibody (without counterstaining) to show shallow invasion of the central decidua basalis by the p57KIP2-positive “glycogen” cells in pUPD12 placentae at E15.5 (b) when compared with normal E15.5 (a) placentae. The maternal surface of the decidua basalis is at the top of each picture. Note that in normal placentae, glycogen cells cluster around the wall of the central maternal artery (a). A similar defective spatial pattern of “glycogen” cells was also seen in pUPD12 placentae upon RNA in situ hybridization with the use of probes specific for Igf2 or 4311 transcripts (data not shown), previously shown to mark “glycogen” cells situated within the decidua basalis (27, 30). (c–f) Sagittal sections of E18.5 normal (c and e) and pUPD12 (d and f) placentae through the central maternal artery situated within the decidua basalis, stained with H&E (c and d) or with an antibody against α smooth muscle actin (e and f) to detect its smooth muscle wall, as previously described (19). For both e and f, control arteries elsewhere in the body are stained positive (data not shown). Sections c, e and d, f are adjacent. Note that in contrast to normal material, the pUPD12 arterial wall lacks considerable acellular eosinophilic material (d) and failed to loose all of its positivity (f). [Scale bars = 100 μm (a and b) and 50 μm (c–f).]