David R.C. Natale

Early patterning of the chorion leads to the trilaminar trophoblast cell structure in the placental labyrinth

The labyrinth of the rodent placenta contains villi that are the site of nutrient exchange between mother and fetus. They are covered by three trophoblast cell types that separate the maternal blood sinusoids from fetal capillaries - a single mononuclear cell that is a subtype of trophoblast giant cell (sinusoidal or S-TGC) with endocrine function and two multinucleated syncytiotrophoblast layers, each resulting from cell-cell fusion, that function in nutrient transport. The developmental origins of these cell types have not previously been elucidated. We report here the discovery of cell-layer-restricted genes in the mid-gestation labyrinth (E12.5-14.5) including Ctsq in S-TGCs (also Hand1-positive), Syna in syncytiotrophoblast layer I (SynT-I), and Gcm1, Cebpa and Synb in syncytiotrophoblast layer II (SynT-II). These genes were also expressed in distinct layers in the chorion as early as E8.5, prior to villous formation. Specifically, Hand1 was expressed in apical cells lining maternal blood spaces (Ctsq is not expressed until E12.5), Syna in a layer immediately below, and Gcm1, Cebpa and Synb in basal cells in contact with the allantois. Cebpa and Synb were co-expressed with Gcm1 and were reduced in Gcm1 mutants. By contrast, Hand1 and Syna expression was unaltered in Gcm1 mutants, suggesting that Gcm1-positive cells are not required for the induction of the other chorion layers. These data indicate that the three differentiated trophoblast cell types in the labyrinth arise from distinct and autonomous precursors in the chorion that are patterned before morphogenesis begins.

Activin promotes differentiation of cultured mouse trophoblast stem cells towards a labyrinth cell fate.

Prolonged maintenance of trophoblast stem (TS) cells requires fibroblast growth factor (FGF) 4 and embryonic fibroblast feeder cells or feeder cell-conditioned medium. Previous studies have shown that TGF-beta and Activin are sufficient to replace embryonic fibroblast-conditioned medium. Nodal, a member of the TGF-beta superfamily, is also known to be important in vivo for the maintenance of TS cells in the developing placenta. Our current studies indicate that TS cells do not express the Nodal co-receptor, Cripto, and do not respond directly to active Nodal in culture. Conversely, Activin subunits and their receptors are expressed in the placenta and TS cell cultures, with Activin predominantly expressed by trophoblast giant cells (TGCs). Differentiation of TS cells in the presence of TGC-conditioned medium or exogenous Activin results in a reduction in the expression of TGC markers. In line with TGC-produced Activin representing the active component in TGC-conditioned medium, this differentiation-inhibiting effect can be reversed by the addition of follistatin. Additional experiments in which TS cells were differentiated in the presence or absence of exogenous Activin or TGF-beta show that Activin but not TGF-beta results in the maintenance of expression of TS cell markers, prolongs the expression of syncytiotrophoblast markers, and significantly delays the expression of spongiotrophoblast and TGC markers. These results suggest that Activin rather than TGF-beta (or Nodal) acts directly on TS cells influencing both TS cell maintenance and cell fate, depending on whether the cells are also exposed to FGF4.

Phenotypic Analysis of the Mouse Placenta

Placental development is a dynamic and complex process and much of our current understanding of the underlying molecular processes comes from analysis of targeted gene mutations in mice. There are more than 50 strains of mutant mice that have placental defects, and it has become widely appreciated that placental defects should be suspected in cases where embryonic lethality is observed. The degree to which these phenotypes are investigated is highly variable, owing to a general lack of expertise in the field. However, there has been considerable progress in developing techniques and reagents for analyzing placental phenotypes that are relatively simple to apply and that should be accessible to all investigators. This chapter provides a basic outline of the strategies for the general identification and then the subsequent detailed investigation of placental phenotypes.

Establishing Three Blastocyst Lineages—Then What?

Development of the mouse embryo to the blastocyst stage occurs over 3 to 4 days following fertilization of the oocyte. During this time, several molecular and morphological events take place that result in the formation of three distinct cell lineages: the trophectoderm, the epiblast, and the primitive endoderm. Many studies have investigated the processes that control lineage specification in the blastocyst including gene expression, cell signaling, cell–cell contact/positional relationships, and most recently, epigenetics. Here we review, at the molecular level, recent contributions to our understanding of the mechanisms that play a role in formation of these lineages. Additionally, we focus on the next steps in differentiation to highlight processes important in the development of those lineages that contribute to the extraembryonic tissues. In this context, we discuss the establishment of extraembryonic ectoderm and the contributions of parietal and visceral endoderm to yolk sac formation.

Signaling pathways in mouse and human trophoblast differentiation: a comparative review

The mouse is often used as a model for understanding human placentation and offers multiple advantages, including the ability to manipulate gene expression in specific compartments and to derive trophoblast stem cells, which can be maintained or differentiated in vitro. Nevertheless, there are numerous differences between the mouse and human placentas, only the least of which are structural. This review aims to compare mouse and human placentation, with a focus on signaling pathways involved in trophoblast lineage-specific differentiation.

ApoE Receptor 2 Mediation of Trophoblast Dysfunction and Pregnancy Complications Induced by Antiphospholipid Antibodies in Mice

Pregnancies in women with the antiphospholipid syndrome (APS) are frequently complicated by fetal loss and intrauterine growth restriction (IUGR). How circulating antiphospholipid antibodies (aPL) cause pregnancy complications in APS is poorly understood. We sought to determine whether the low‐density lipoprotein receptor family member apolipoprotein E receptor 2 (ApoER2) mediates trophoblast dysfunction and pregnancy complications induced by aPL.

IFPA Meeting 2008 Workshops Report

Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topics were discussed in 12 themed workshops. Topics covered included: immunology of placentation; galectins and trophoblast invasion; signaling in implantation and invasion; markers to identify trophoblast subpopulations; placental pathology; placental toxicology; stereology; placental transport of fatty acids; placental mesenchymal stem cells; comparative placentation; trophoblast and neoplasia; trophoblast differentiation. This report is a summary of the various topics covered.

Ly6e expression is restricted to syncytiotrophoblast cells of the mouse placenta

In the present study, we characterized the expression of lymphocyte antigen 6, locus E (Ly6e) in mouse placental trophoblast. We identified Ly6e mRNA expression in trophoblast stem (TS) cells by a gene expression screen. In vivo, Ly6e was first detectable by mRNA in situ hybridization in the chorion beginning at E8.5 with spatial expression similar to Syncytin a (Syna). At later stages of gestation, Ly6e was restricted to syncytiotrophoblast in the labyrinth. Northern blot confirmed that Ly6e was expressed in both undifferentiated and differentiated TS cell cultures but that its expression increased with differentiation. FACS analysis confirmed these results and allowed us to isolate LY6E⁺ cells, which we found to express Syna at a much higher level than did LY6E⁻ cells. Our findings suggest that LY6E is expressed in differentiated syncytiotrophoblast and may also be useful as an early marker, expressed in progenitors of this cell-type.

Cell–cell adhesion defects in Mrj mutant trophoblast cells are associated with failure to pattern the chorion during early placental development

Early placental development in mice involves patterning of the chorion into distinct layers, though little is understood regarding the interactions that regulate its organization. Here we demonstrate that keratin aggregates found in Mrj−/− chorionic trophoblast cells are associated with abnormal cell morphology, collapse of the actin cytoskeleton, E‐cadherin and β‐catenin misexpression and extracellular matrix (ECM) disorganization. Accordingly, Mrj−/− trophoblast cells in vitro are nonadherent and display erratic migratory behavior. These cells also fail to differentiate into syncytiotrophoblast cells since Rhox4b expression, a marker of syncytiotrophoblast progenitors, was maintained and Gcm1, Synb, and Syna expression failed to increase. This differentiation defect was not solely attributable to E‐cadherin misexpression or ECM disorganization. However, plating Mrj‐deficient cells on exogenous laminin‐511 normalized their cell behavior. Lastly, we show that Mrj−/− chorions at embryonic day 8.5 have expanded Rhox4b expression domains and do not form normal layers of gene expression suggesting that chorion patterning requires Mrj. Developmental Dynamics 240:2505–2519, 2011.

Contribution of a Non-β-Cell Source to β-Cell Mass during Pregnancy

β-cell mass in the pancreas increases significantly during pregnancy as an adaptation to maternal insulin resistance. Lineage tracing studies in rodents have presented conflicting evidence on the role of cell duplication in the formation of new β-cells during gestation, while recent human data suggest that new islets are a major contributor to increased β-cell mass in pregnancy. Here, we aim to: 1) determine whether a non-β-cell source contributes to the appearance of new β-cells during pregnancy and 2) investigate whether recapitulation of the embryonic developmental pathway involving high expression of neurogenin 3 (Ngn3) plays a role in the up-regulation of β-cell mass during pregnancy. Using a mouse β-cell lineage-tracing model, which labels insulin-producing β-cells with red fluorescent protein (RFP), we found that the percentage of labeled β-cells dropped from 97% prior to pregnancy to 87% at mid-pregnancy. This suggests contribution of a non-β-cell source to the increase in total β-cell numbers during pregnancy. In addition, we observed a population of hormone-negative, Ngn3-positive cells in islets of both non-pregnant and pregnant mice, and this population dropped from 12% of all islets cells in the non-pregnant mice to 5% by day 8 of pregnancy. Concomitantly, a decrease in expression of Ngn3 and changes in its upstream regulatory network (Sox9 and Hes-1) as well as downstream targets (NeuroD, Nkx2.2, Rfx6 and IA1) were also observed during pregnancy. Our results show that duplication of pre-existing β-cells is not the sole source of new β-cells during pregnancy and that Ngn3 may be involved in this process.