The placenta: transcriptional, epigenetic, and physiological integration during development (original) (raw)
Early lineage restriction within the blastocyst. In mice, establishment of the extraembryonic lineages is considered the first differentiation step during early embryonic development. Following compaction (an increase in intercellular adhesion that causes all the cells to adopt a more flattened morphology), two distinct cell populations are created. The outer layer, termed the trophectoderm (TE), exhibits features of polarized epithelia, with apical microvilli and the asymmetric distribution of tight and adherens junctions (Figure 2). The component cells give rise to the placenta (reviewed in ref. 16). An underlying aggregate of irregular nonpolarized cells forms the inner cell mass (ICM). TE cells in direct contact with the ICM, the polar TE, give rise to trophoblast stem (TS) cells in vitro and populate the major structures of the placenta in vivo (Figure 2). The ICM ultimately gives rise to the embryo proper (in vivo) or ES cells (in vitro). Studies in the mouse have revealed the critical importance of a set of lineage-restricted transcription factors for the establishment, maintenance, and pluripotency of ES cell identity (Figure 3): octamer 3/4 (OCT4) (17), SRY-box–containing gene 2 (SOX2) (18), NANOG (19), sal-like protein 4 (SALL4) (20), and Krüppel-like factor 4 (KLF4) (21). This list is not likely to be exhaustive because various sets of transcription factors can induce pluripotency in differentiated cell types (22).
Lineage segregation within the mouse blastocyst and early placenta. (A) At day E3.5, the blastocyst comprises an outer TE destined to populate the placenta and an ICM destined to form the embryo. TE cells not in direct contact with the ICM form the mural TE, whereas those adjacent to the ICM form the polar TE. The polar TE gives rise to the ExE, from which TS cells can be derived in vitro. (B) Following implantation, mural TEs initiate the first wave of TGC differentiation to form 1° TGCs, which contribute directly to the P-TGC population. Cells within the polar TE continue to proliferate and populate the ExE. Some 2° TGCs arise directly from the ExE. Along with 1° TGCs, they compose the P-TGC population that lines the implantation site. Cells within the ExE then differentiate to form the chorionic plate and the EPC. The chorionic plate is responsible for populating the mouse labyrinth with SynTs and a subset of 2° TGCs called S-TGCs. Together, these cells are responsible for the transport functions of the placenta. Cells within the EPC can either differentiate into a population of lineage-committed progenitors known as SpTs, which then differentiate into 2° TGCs, or they can directly differentiate into various 2° TGCs subtypes.
Transcriptional and epigenetic regulation of trophoblast lineage restriction in the mouse. Critical transcriptional regulators are highlighted in green, and epigenetic regulators are highlighted in blue. Undifferentiated ES cells are depicted on the left along with known transcriptional and epigenetic regulators responsible for the maintenance of stemness in mouse ES cells. TS cells are depicted on the right along with the differentiation pathways that give rise to lineage-committed progenitors (CTs and SpTs) as well as the terminally differentiated cells of the placenta (multinucleated SynTs and TGCs). The factors necessary for the derivation or maintenance of TS cells are indicated along with factors that operate in a lineage- and stage-specific manner.
Extraembryonic development also depends on a unique subset of transcriptional regulators (Figure 3). Expression of the Drosophila caudal–related transcription factor Cdx2 is restricted in mice to the TE; its overexpression in mouse ES cells results in the adoption of TE cell fates (23), while its absence results in embryos lacking TE-to-trophoblast differentiation (24). Therefore, while CDX2 is not critical for initially specifying the TE, it is nevertheless required for its maintenance and subsequent differentiation. Specification of the TE in mice also requires the transcription factor TEA-domain family member 4 (TEAD4). Tead4–/– embryos die at the peri-implantation stage and fail to form a blastocoel — the fluid-filled cavity inside the early embryo that is essential for formation of the three embryonic germ layers (25, 26). In these embryos, Cdx2 expression is lost after the morula stage and the entire conceptus consists of ICM derivatives with ubiquitous Oct4 and Nanog expression. Consistent with their critical role in establishing TE cell fates, TS cells cannot be isolated from these embryos, while ES cells can. Unlike Cdx2, Tead4 expression is not restricted to the TE, suggesting that other lineage-specific factors, such as Yes-associated protein 1 (Yap1; also known as Yap) (27–29), may cooperate with TEAD4 to produce the observed effects on TE specification. After TEAD4 and CDX2, the product of the T-box gene eomesodermin homolog (Eomes) is the earliest acting transcription factor known to be required for key postimplantation lineage commitment steps, and mice lacking Eomes gene expression fail to exhibit proper TE-to-trophoblast differentiation (30). Although implantation does occur, the conceptus arrests at a blastocyst-like stage of development (30).
Following implantation in mice, TE cells not in direct contact with the ICM (i.e., the TE cells that form the mural TE) differentiate into primary trophoblast giant cells (1° TGC) that may be analogous to iCTBs in humans (Figures 1 and 2). Unlike secondary TGCs (2° TGCs), which arise from the polar TE and frequently pass through a spongiotrophoblast (SpT) stage of development (Figure 2), 1° TGCs arise directly from the mural TE. 1° and 2° TGCs together compose the parietal TGC (P-TGC) population that anchors the placenta to the uterus and engages in the remodeling of maternal vasculature critical for establishing blood flow to the placenta (Figure 1). TGCs and iCTBs undergo a fascinating process of DNA replication without intervening mitoses (endoreplication), resulting in the formation of highly polyploid cells (reviewed in ref. 31). Some of the molecular mechanisms regulating this process are beginning to be understood. For example, mouse embryos lacking the cell-cycle regulatory protein Geminin contain only polyploid TE and fail to express OCT4 at the blastocyst stage of development (32). Geminin, which is detected as early as the 8-cell stage, had previously been determined to regulate ploidy in cancer cells via its role in replication licensing (33–35). Geminin is downregulated during the S- and G-like phases of endoreplicating TGCs via proteasome-mediated degradation (32). Cyclin E is also required for proper endoreplication in mice, and deficient cyclin E activity results in embryonic lethality due to impaired placentation (36, 37). Therefore, early lineage specification in the mouse blastocyst depends on the proper regulation of cell ploidy. Consistent with this observation, the formation of tetraploid mouse embryos following electrofusion of late 2-cell–stage embryos (38) results in the formation of cells that are largely restricted in their developmental potential to the TE (39), although some early embryonic contribution has also been noted (40). Thus, absolute DNA content can direct cell fate, restricting early differentiation to short-lived extraembryonic structures when deviations from diploidy are detected. In line with these observations, human trophoblast cells exhibit remarkable genetic heterogeneity at the chromosomal level (41).
The interdependence of placental cell fates. Unlike the mural TE, which immediately differentiates into postmitotic 1° TGCs that contribute directly to the P-TGC population, the polar TE continues to proliferate and gives rise to diploid cells that comprise the extraembryonic ectoderm (ExE). Precursors within the ExE proliferate to generate the chorionic plate as well as the ectoplacental cone (EPC) (reviewed in ref. 31). The EPC is itself a stem cell compartment that gives rise to other subpopulations of 2° TGCs either directly or through a lineage-committed progenitor (SpT) population, while cells within the chorionic plate, chorionic trophoblasts (CTs), give rise to the cells that form the transport interface — SynTs and sinusoidal TGCs (S-TGCs) (42). TS cells — stem cells that exclusively contribute to the mouse placenta — can be isolated from the blastocyst (E3.5) or egg cylinder/early streak stage (~E6.5), but this potential is lost soon after (43, 44). In the presence of FGF4, these cells proliferate indefinitely when cocultured with mouse embryonic fibroblasts and, upon growth factor withdrawal, spontaneously differentiate into two of the major cell types of the mature placenta: TGCs and their SpT precursors.
A number of transcription factors are known to be important for the allocation of cell lineages within the mouse placenta following implantation, and their genetic inactivation can result in large scale disruptions of placental structure. For example, activating enhancer–binding protein 2γ (AP-2γ), which is initially broadly expressed in the mouse blastocyst, becomes restricted to the ExE after E5.5. Disruption of Ap2g gene expression results in a reduction in 1° TGC differentiation, a grossly disorganized ExE, and a smaller than normal EPC (45). Estrogen-related receptor β (ESRRβ) deficiency, on the other hand, causes embryonic lethality at approximately E10.5 (46), when the mouse embryo begins to rely on a well-perfused placenta with a functional circulatory system to fulfill its metabolic needs (47). These embryos fail to vascularize the labyrinth and exhibit large aberrations in the allocation of terminally differentiated cell types, with an overabundance of TGCs and a severe deficit of SpT precursors. This is a recurring theme in many developmental processes, including formation of the placenta, wherein the lack of critical transcriptional regulators results in aberrant proliferation and/or differentiation of lineage-committed precursors. Along these lines, the retinoblastoma tumor suppressor gene (Rb) has surprisingly been found to be critical for extraembryonic development (48). Indeed, placentas developing from embryos lacking Rb gene expression exhibit continued cell proliferation and a failure to develop a normal labyrinth layer. The critical role of Rb in placental development resides solely in early progenitors, and its inactivation exclusively in placental cells derived from the ExE and EPC fails to recapitulate the _Rb_-null phenotype (49). All additional phenotypes observed in _Rb_-null embryos are secondary to the placental defects. Similarly, the paternally imprinted gene mammalian achaete scute homolog 2 (Mash2), which encodes a basic helix-loop-helix (bHLH) transcription factor, is required for the proper formation of all placental cell types (50). In its absence, the chorionic plate — a derivative of the ExE that, along with fetal blood vessels, forms the labyrinth — fails to vascularize, SpTs are lost, and TGC numbers are increased (50). However, chimeric analyses show that Mash2 is required for cell maintenance only in the SpT layer, suggesting that impaired labyrinth formation is a secondary phenomenon (51). Additionally, forkhead box D3–null (_Foxd3_-null) placentas also contain an excess of TGCs due to an inability of progenitor proliferation and premature differentiation (52). These findings highlight the interdependence of the various differentiation pathways that form the mature placenta.
Transcriptional regulation of uterine invasion. Anchoring the conceptus to the uterus is a critical placental function. In mice, TGCs perform this role, which involves iCTBs in humans. The bHLH transcription factor HAND1 is critical for TGC formation, and HAND1-deficient mouse embryos die in utero between E7.5 and E8.5 (53, 54). Both primary and secondary TGCs are affected in these mice, and the invasive capability of Hand1–/– cells in vitro is substantially reduced (55). It has become increasingly apparent that 2° TGCs can be grouped into at least four subtypes with differing origins, morphologies, locations, and functions (42). Despite this fact, HAND1 appears to be required for the formation of all known TGC subtypes, including 1° TGCs, suggesting an early stem cell role. Similarly, the bHLH transcription factor stimulated by retinoic acid 13 (Stra13) also promotes TGC formation (56). Interestingly, retinoic acid treatment promotes TGC formation from TS cells apparently by bypassing the SpT intermediates, consistent with the idea that there are various TGC lineages (42). HAND1 activity is subject to competition from other related factors. The dominant-negative HLH genes inhibitor of DNA binding 1 (Id1) and Id2, for example, are expressed only in the mouse chorion (57) and inhibit TGC formation (58). Id family members appear to play an equally important role in governing human trophoblast differentiation (59). Id2 expression decreases as human cytotrophoblasts differentiate into iCTBs, and this process is diminished in the setting of preeclampsia. Constitutive expression of Id2 in cultured human cytotrophoblasts constrains differentiation and invasion. Similarly, expression of another bHLH antagonist gene, inhibitor of MyoD family a (Imfa), also promotes TGC formation in mice, possibly by inhibiting MASH2 (60).
TGC and iCTB invasion anchor the mouse and human conceptus, respectively, to the uterus, and the endovascular component of this process enables remodeling of maternal spiral arterioles, thereby establishing blood flow to the placenta (reviewed in ref. 61). A subset of iCTBs and TGCs breaches spiral arterioles and differentiates into an endovascular subtype that replaces the resident maternal endothelium and intercalates within the smooth muscle walls of the vessels. In a fascinating transdifferentiation process, human iCTBs with a primarily epithelial phenotype acquire vascular/endothelial characteristics. The component steps include downregulation of integrin α6β4 and E-cadherin and upregulation of integrins αVβ3 and α1β1, VE-cadherin, VCAM-1, and PECAM-1 (reviewed in ref. 62). Additionally, iCTBs produce a number of proteins that are involved in extracellular matrix degradation (reviewed in ref. 63). In humans, the transcriptional basis of this tumor-like gene expression program remains largely unknown. One possibility is that these changes are somehow linked to the aforementioned aberrations in chromosome number that are coincident with iCTB differentiation. Given the large-scale gene expression changes observed (M. Gormley, N. Hunkapiller, and S.J. Fisher, unpublished data), epigenetic mechanisms and environmental factors are also likely to be involved. Importantly, this process transforms maternal spiral arterioles into low-pressure conduits. Understanding the molecular underpinnings of endovascular invasion is critical to maternal-fetal medicine. Aberrations of this “physiological transformation” that result in shallow invasion and underperfusion of the fetal unit are part of the placental component of preeclampsia that somehow triggers maternal hypertension and other manifestations of this serious complication of pregnancy. If untreated, this syndrome can progress to the life-threatening condition eclampsia, which is characterized by maternal seizures (reviewed in ref. 64).
Formation of the maternal-fetal transport interface. Glial cells missing-1 (GCM1) is the earliest-acting transcription factor known to function during formation of the mouse placental labyrinth. Its expression in the chorionic plate region marks the first lineage-committed progenitors destined to differentiate into the multinucleated SynTs that form the interface between maternal and fetal vessels (65). Similar to TGC differentiation, formation of the labyrinth in mice involves the generation of multiple labyrinth-specific subtypes that can be characterized based on marker gene expression and localization. The chorionic plate, which is composed of CTs, is derived from the ExE, while SpTs and most secondary TGCs are derived from the EPC (Figure 2). Clusters of cells within the chorionic plate initiate Gcm1 gene expression at day E7.5. In its absence, the chorionic plate remains compact, fetal vessels do not invade into the placenta, and similarly to the effect of HAND1 on TGC differentiation, SynT differentiation does not occur. Interestingly, Gcm1 gene expression as well as terminal differentiation within the chorionic plate is dependent on the expression of the Ets-domain transcriptional repressor Ets2 repressor factor (Erf) (66). _Erf_-null mice fail to induce Gcm1 gene expression in the chorionic plate and maintain Esrrb expression, thereby inhibiting differentiation of the chorionic plate into the mature labyrinth. _Erf_-null TS cells exhibit delayed differentiation, maintaining expression of the TS cell marker genes Cdx2, Esrrb, and Eomes and expressing reduced levels of the SpT-specific gene trophoblast-specific protein α (Tpbpa; also known as 4311). Erf and Gcm1 expression thus define a population of lineage-committed progenitors destined to form the labyrinth, and its absence precludes SynT differentiation. Another Ets-domain transcription factor gene, Ets2, is also required for early trophoblast differentiation. In its absence, there is substantially reduced CT development and decreased expression of ExE markers (67). _Ets2_-null TS cells grow more slowly than their WT counterparts and express less Cdx2 (68). Required slightly later for formation of the labyrinth, CCAAT/enhancer–binding protein α (C/EBPα) and C/EBPβ are coexpressed in the early-gestation chorionic plate and later in trophoblasts of the labyrinthine layer. In placentas lacking both C/EBPα and C/EBPβ, blood vessels invade the chorion but vessel expansion and development of the labyrinthine layer are impaired (69). Interestingly, in mice, fetal endothelial cells are separated from maternal blood by 3 trophoblast layers: a mononucleated TGC subtype (S-TGCs) and two separate SynT layers (70) (Figure 1, D–F). These cells express unique marker genes and, like TGCs, appear to arise from distinct precursors in the chorion that differentiate along their respective paths before morphogenesis begins. Interestingly, S-TGCs may also derive from the chorionic plate in mice, as opposed to the EPC, highlighting the diverse origin of TGC subtypes (42). In humans, as gestational age advances, the precursor villous CTB (vCTB) layer that underlies the SynT (Figure 1, B and C) becomes discontinuous, which may limit the ability of the human placenta to repair itself.
A major difficulty with attempting to study SynT function is the inability of TS cells to readily form these cells in vitro. Surprisingly, we have demonstrated that the oxygen-sensitive transcriptional regulator HIF actively suppresses SynT formation from TS cells in culture (71). This occurs in an oxygen-independent manner and is due to the modulation of cellular histone deacetylase (HDAC) activity by the HIF family of transcription factors. HIF deficiency, as well as generalized HDAC inhibition, prevents TGC and SpT formation from mouse TS cells and promotes the formation of SynTs (71). This unexpected finding has now made it feasible to study SynT function more reliably in vitro, and studies of the mechanisms involved should yield new insights into key lineage commitment steps necessary for the formation of this critical placental cell type. Additionally, the results of these studies highlight the interrelationship of genetic, epigenetic, and environmental factors in TS cell fate determination (72).