Trophoblast giant cells

Trophoblast giant cells (TGCs) are polyploid cells formed via genomic amplification without cell division (endoreduplication) of diploid precursors (reviewed by (231, 232)). The polyploidy genomes of TGCs are also polytene, having distinct chromosome bands of both highly active and highly repressed chromatin. TGCs have extensive rough endoplasmic reticulum indicative of their elevated protein secretion pathways. In these respects, TGCs are similar to hepatocytes. TGCs are the default fate of TE development. In the absence of FGF4 and conditioned media TSCs differentiate within 4-5 days into TGCs (196). The expression of Ascl2 within the EPC (and in TSCs) suppresses the TGC differentiation programing. ASCL2 antagonizes the activity of HAND1, a basic helix-loop-helix transcription factor that promotes TGC differentiation,

37

either through transcriptional repression or competition for common promoters (233, 234).Tthe interacting HAND1 and STRA13 transcription factors negate FGF4 suppression of trophoblast terminal differentiation and promote differentiation into TGC (233-235).

There are four subtypes of TGCs found in the mouse placenta (reviewed by (231, 232)). They can be distinguished by their expression of different members of the prolactin/placental lactogen and prolactin-like gene family. Each of the 23 members of this family is a 22-33 kDa peptide related to growth hormone (225, 227). The earliest TGCs are formed from the mural TE and EPC just prior to implantation. These cells are parietal TGCs (P-TGCs) because they form the outermost surface of the placenta. P-TGCs express proliferin (Prl; Plf1; Prl2c2) and prolactin-1 (Pl1; Prl3d1) at E9.5, and then transition to primarily prolactin-2 (Pl2; Prl3b1) at E12.5 and later gestational time points (232). These cells are vital to implantation and

decidualization. P-TGCs express a multitude of integrins that interact with maternal uterine ECM components fibronectin, laminin, vitronectin and collagen (231). Spiral artery TGCs (SpA-

TGCs) integrate into the maternal spiral arteries to remodel the vascular blood flow. These cells phenocopy many of the functions of normal endothelial cells and are analogous to human endovascular cytotrophoblast (201). The trophoblast decidual invasion is shallower in mice than in human pregnancy, and some of the vascular remodeling is thought to be instigated by uterine natural killer cells in mice (177).

SpA-TGCs are present by E10.5 and express Prl2c2. The chorion gives rise to the C- TGCs and S-TGCs starting at E10.5, these are marked by expression of Prl3b1 but can be distinguished by expression of Prl2c2 in C-TGCs and Ctsq in S-TGCs (231, 232). The

expression of the prolactin gene family, which is found in large cluster of duplicated genes on mouse chromosome band 13qA1, is both a defining and functional feature of TGCs (227, 236).

38

Prolactins are secreted proteins with both paracrine functions that modulate decidualization and have endocrine functions that alter maternal physiology and behavior (225, 227). In the

polypolid P-TGCs there is overamplification of genomic regions harboring gene clusters of the prolactin (including Prl2c2, Prl3b1, and Prl2d1), Cathepsin (including Ctsq1, and Tpbpa), Serpin (intracellular serine proteases) and NK/Clec (Natural-killer/C-type lectins) families (237).

Although the human placenta does not have TGCs the extravillous cytotrophoblast provide many of the same functions during implantation and the more invasive cell types are polypoloid, although not to the same extent as mouse TGCs (238).

Figure 1. Mouse placental development from preimplantation to E12.5+. Abbreviations: Trophectoderm (TE), Epiblast (Epi), Visceral Endoderm (VE), Decidua (Dec), Spiral Artery (SpA), Ecto Placental Cone (EPC), Chorion (Ch), Allantois (Al), Trophoblast Giant Cells (TGCs), Yolk Sac (YS), Fetus (Fe), Spiral Artery TGCs (SpA-TGCs), Parietal TGCs (P-TGCs), Sinusoidal TGCs (S-TGCs), Spongiotrophoblast (SpT), Glycogen Cells (GCs),

Syncytiotrophoblast (SynT), Fetal Vessels (FV). In each stage cell populations are color coded based on lineage origin. At the preimplantation stage the Polar TE is light green, the mural TE is dark green, the Epi is blue and the VE is yellow. At the implantation stage Dec is orange, TGCs are dark green, the EPC is light green, the Ch is purple, the YS is yellow and the Fe and Al are blue. Postimplantation all TGCs are dark green (irrespective of origin), SpT is light green, GC are gray-green, SynT is purple and magenta, Fv are light blue and the chorion is Purple. Maternal and fetal blood (unlabeled are red and dark blue respectively.

Preimplantation E4.5 Implantation E6.5-E7.5 Postimplantation E12.5+` Polar TE Epi VE Mural TE Dec EPC Ch Al TGCs P-TGC SpT Al Ch SynT FV SpA YS Fe GC C-TGC SpA-TGC S-TGC Dec JZ LZ

39

I chose to study 15 out of the 24 known genomic imprints based on their known involvement in prenatal fetal and placental phenotypes. These genomic imprints and the genes within the

clusters they regulate are discussed in detail below. Many of these imprinted gene clusters harbor genes that exhibit parent-of-origin specific monoallelic expression exclusively in extraembryonic tissues. Different types of mouse models have been instrumental in providing insight the role of each genomic imprint and the imprinted genes they regulate. The study of mouse embryos with UPDs and robertsonian translocations revelaed parent-of-origin specific prenatal developmental effects attributable to specific imprinted genomic regions. Targeted mouse genetic approaches including gene deletions and transgenic duplications identified the role of single imprinted genes. Similarly, targeted deletion of DMDs in mice has been used to investigate imprinting

mechansism and their functional significance in development. Although the research reviewed herein has brought to light unique placental functions of imprinted loci, the integrated role of individual imprinted clusters and imprinted DNA methylation per se is largely unknown.

1.5.1 Nnat

The neuronatin (Nnat) imprinted gene cluster is a microimprinted domain found on mouse chromosome band 2qH4 with a syntenic region on human chromosome 20q11.2-12 (239-242). The Nnat gene resides within the single intron of the much larger Blcap (239). The Nnat DMD is located at the Nnat promoter, which transcribes in reverse orientation relative to Blcap (239).