Notch1 controls development of the extravillous trophoblast lineage in the human placenta

Significance

Progenitor trophoblast cells of the human placenta either fuse to form a syncytium or develop into invasive trophoblasts invading the maternal uterus. However, regulatory pathways controlling their development and distinct differentiation programs are poorly understood. In the present study, we demonstrate that Notch1 is a critical regulator of early pregnancy, promoting development of the invasive, extravillous trophoblast lineage and survival of its progenitors. In vivo, Notch1 is detected in extravillous trophoblast progenitors and clusters of villous trophoblast initiating the invasive differentiation program. In vitro, Notch1 repressed genes involved in self-renewal of fusogenic precursors, but induced genes specifically expressed by extravillous trophoblast progenitors. Our data delineate Notch1 as a key regulator promoting development of the human extravillous trophoblast lineage.

Abstract

Development of the human placenta and its different epithelial trophoblasts is crucial for a successful pregnancy. Besides fusing into a multinuclear syncytium, the exchange surface between mother and fetus, progenitors develop into extravillous trophoblasts invading the maternal uterus and its spiral arteries. Migration into these vessels promotes remodelling and, as a consequence, adaption of blood flow to the fetal–placental unit. Defects in remodelling and trophoblast differentiation are associated with severe gestational diseases, such as preeclampsia. However, mechanisms controlling human trophoblast development are largely unknown. Herein, we show that Notch1 is one such critical regulator, programming primary trophoblasts into progenitors of the invasive differentiation pathway. At the 12th wk of gestation, Notch1 is exclusively detected in precursors of the extravillous trophoblast lineage, forming cell columns anchored to the uterine stroma. At the 6th wk, Notch1 is additionally expressed in clusters of villous trophoblasts underlying the syncytium, suggesting that the receptor initiates the invasive differentiation program in distal regions of the developing placental epithelium. Manipulation of Notch1 in primary trophoblast models demonstrated that the receptor promotes proliferation and survival of extravillous trophoblast progenitors. Notch1 intracellular domain induced genes associated with stemness of cell columns, myc and VE-cadherin, in Notch1⁻ fusogenic precursors, and bound to the myc promoter and enhancer region at RBPJκ cognate sequences. In contrast, Notch1 repressed syncytialization and expression of TEAD4 and p63, two regulators controlling self-renewal of villous cytotrophoblasts. Our results revealed Notch1 as a key factor promoting development of progenitors of the extravillous trophoblast lineage in the human placenta.

Differentiation processes of the human placenta are a prerequisite for fetal development and successful pregnancy outcome. Shortly after implantation, stem cells of the trophectoderm surrounding the blastocyst give rise to the primitive syncytium by cell fusion as well as to proliferative cytotrophoblasts (CTBs) forming primary placental villi (1). Breaking through the multinuclear structures, these villi contact the maternal decidua, the endometrium of pregnancy, and expand laterally to form the so-called trophoblastic shell. The latter encircles the embryo and protects it from oxidative damage during early gestation (2). As pregnancy proceeds, placental villi undergo extensive remodelling involving branching morphogenesis and transformation into secondary and finally tertiary villi by migration of mesenchymal cells and vascularization, respectively. At this stage, two types of villi can be discerned, floating and anchoring villi. Floating villi, which are bathed in maternal blood after establishment of the fetal–maternal circulation, are necessary for hormone production and nutrient and oxygen transport to the developing fetus (3). The outermost epithelial surface of these villi, the multinuclear syncytium, also termed syncytiotrophoblast (STB), is generated by cell fusion of underlying CTB progenitors (4). On the other hand, anchoring villi attached to the decidua form proliferative cell columns giving rise to differentiated, extravillous trophoblasts (EVTs). The latter deeply migrate into uterine tissue and the maternal spiral arteries, provoking vessel remodelling and adaptation of adequate blood flow to the placenta (5, 6). Failures in placentation and artery remodelling have been associated with a variety of pregnancy diseases, such as miscarriage, preeclampsia, fetal growth restriction, and preterm labor (7–10). Besides unfavorable immunological interactions of EVTs with uterine natural killer (uNK) cells (11), abnormal placental development and trophoblast differentiation are thought to contribute to the pathogenesis of gestational disorders. Indeed, CTBs isolated from preeclamptic placentae failed to appropriately differentiate into the invasive lineage in vitro and expressed an antimigratory gene signature (12, 13).

However, our knowledge about human placentation and trophoblast development is only scarce. Bipotential trophoblast progenitor cells have been derived from the chorionic mesenchyme differentiating into EVTs and STBs (14, 15), whereas others identified a specific precursor of the EVT lineage in villous explant cultures (16). Placental structures, trophoblast cell types, and expression patterns of key regulatory transcription factors diverge between mouse and man, thereby hindering comparison of putative regulatory mechanisms (17). Although different transcriptional activators promoting or inhibiting EVT motility have been described (18), it is unknown which factors govern EVT differentiation. Likewise, how regions of column formation are specified and maintained within developing villi remains elusive.

Recent evidence suggested that the developmental Notch pathway could be critically involved in human trophoblast function and differentiation (19–21). Canonical Notch signaling is activated upon direct cell–cell contact involving binding of membrane-anchored ligands, the Serrate-like ligands (Jagged1 and 2), and the Delta-like ligands (DLL1, 3, and 4) to the different Notch receptors (Notch1–4) (22). After two proteolytic cleavage steps, performed by members of a disintegrin and metalloproteinase (ADAM) family and γ-secretase, the Notch intracellular domain (NICD) is released into the cytoplasm. Subsequently, NICD translocates to the nucleus and functions as a coactivator of the transcription factor recombination signal binding protein for Ig kappa J region (RBPJκ) controlling numerous biological processes such as stem cell maintenance, cell lineage determination, and differentiation (23). Human placentae express Notch receptors and their ligands in a cell-specific manner (19, 20). Notch2 is predominantly detected in different EVT subtypes, and inhibition of Notch2 affected trophoblast cell migration (24). In analogy, conditional deletion of Notch2 in murine trophoblast progenitors impaired endovascular invasion and placental perfusion (19). In contrast to that, Notch1, 3, and 4 were shown to be expressed by proliferative CTBs of first trimester placentae (20). Interestingly, Notch1 is absent from second trimester placental tissues (19), suggesting a role of the receptor in early trophoblast development and function.

Therefore, we herein analyzed the specific role of Notch1 in 6th- to 12th-wk human placentae using different primary trophoblast cell models. Our data show that Notch1 is specifically expressed by progenitors of the extravillous trophoblast lineage, located in villi anchoring to the maternal decidua and promotes their proliferation and survival. Moreover, Notch1 repressed syncytialization and genes controlling self-renewal of fusogenic precursors and induced an extravillous trophoblast progenitor-specific gene signature in these cells. The present study delineates Notch1 as a functionally analyzed regulator promoting development of the extravillous trophoblast lineage in the human placenta.

Results

Notch1 Is Specifically Expressed in Progenitors of the Extravillous Trophoblast Lineage.

To gain insights into Notch1 distribution, expression of the receptor was analyzed in placental villi and purified trophoblast subtypes of early pregnancy (Fig. 1). Immunofluorescence in tissue sections, obtained from 6th wk to 7th wk of gestation, revealed that Notch1 specifically localized to a subset of proliferating cell nuclear antigen (PCNA)⁺ CTBs forming multilayered cell columns (Fig. 1A). Coimmunofluorescence of Notch1 with cyclin A or phospho-Histone (p-Histone) H3, labeling trophoblasts in S and M phase, respectively, also indicated that Notch1 specifies cycling progenitors in the proximal cell column coexpressing epidermal growth factor receptor (EGFR) (SI Appendix, Fig. S1 A and B). In contrast, distal human leukocyte antigen G (HLA-G)⁺ cell column trophoblasts (CCTs), differentiating toward EVTs, and the placental syncytium lacked Notch1 expression. Notably, Notch1 was additionally expressed in clusters of single-row villous CTBs (vCTBs) of distal placental villi facing the maternal decidua (Fig. 1A). Closer inspection of adjacent serial sections of a single villus revealed that the Notch1⁺ CTB clusters were parts of developing cell columns (SI Appendix, Fig. S1C). Notch1 expression progressively increased as the columns became multilayered. At the 12th wk of pregnancy, trophoblast-specific Notch1 expression was exclusively detected in proximal CCTs of anchoring villi (Fig. 1B and SI Appendix, Fig. S1A). However, its expression increased in the underlying stroma, compared with earlier weeks (Fig. 1 A and B and SI Appendix, Fig. S1A). Furthermore, localization of Notch1 was analyzed in different regions of a villous tree at the 11th wk of gestation (Fig. 1C). In the proximal and intermediate portion, Notch1 was restricted to cells of the villous core. In contrast, the distal region anchored to the decidua additionally expressed Notch1 in progenitors of the invasive trophoblast lineage. Interestingly, Notch1⁺ CCTs of distal villi lacked expression of the trophoblast stem cell marker caudal-related homeobox transcription factor 2 (Cdx2) (SI Appendix, Fig. S1D). However, the latter was detected in vCTB nuclei of villi residing in the intermediate region of the placenta. Besides its localization at the cell membrane, Notch1 was detected in nuclei of EVT progenitors, suggesting receptor cleavage and activation of canonical Notch signaling in these cells (Fig. 1D). Between the 6th and 12th wk of pregnancy, Notch1 mRNA and protein expression decreased in primary CTB preparations, harboring both proliferative CTBs and differentiated EVTs (SI Appendix, Fig. S1 E and F). Gestation- and differentiation-dependent down-regulation of Notch1 was also observed in isolated vCTBs/CCTs and EVTs, which were immunopurified with EGFR and HLA-G antibodies, respectively (Fig. 1E and SI Appendix, Fig. S1B). The decline of Notch1 toward the end of the first trimester could be associated with the rise in oxygen levels at the time when the placental–maternal circulation and hemotrophic nutrition of the embryo is established (2). In agreement with that assumption, Notch1 expression and activity of a canonical Notch reporter increased in CTBs under hypoxic conditions (SI Appendix, Fig. S1 G and H).

Fig. 1.

Notch1 marks a subset of cycling CCTs and decreases during the first trimester of pregnancy. (A–D) Notch1 IF in first trimester placenta. Stars mark proliferative, Notch1⁻ CCTs or vCTBs underlying the syncytium (S). Nuclei were stained with DAPI. Representative images of cell columns and placental villi from 5th–7th wk of gestation (n = 7) and 10th–12th wk of gestation (n = 5) are shown. VS, villous stroma. (Scale bars, 50 µm.) Tissue sections of 6th (A)- or 12th (B)-wk placentae were immunostained with antibodies against Notch1 and PCNA. In negative controls (Inset pictures) Notch1 primary antibody was replaced by rabbit monoclonal isotype IgG (mAB IgG). Arrowheads indicate clusters of Notch1⁺ vCTBs. (C) IF of different regions of an 11th-wk villous tree using Notch1 and vimentin (VIM) antibodies. Images are representative for (1) the proximal region close to the chorionic plate, (2) the intermediate portion, and (3) the distal part anchoring to the maternal decidua (n = 4). Picture at Left shows the Alcian blue-stained placental villus embedded in paraffin of which different regions (1–3) have been analyzed. (D) IF detecting Notch1 at the cell membrane and in nuclei (arrow) of CCTs. Stippled line denotes boundary between VS and the cell column. (E) Western blot analyses detecting Notch1 in MAC-sorted EGFR⁺ and HLA-G⁺ CTBs at the time of isolation from 7th- and 12th-wk placentae, respectively. Antibodies against EGFR and HLA-G were used to determine purity of CTB cell pools. GAPDH served as loading control. Bar graph denotes mean values ± SD of Notch1 protein levels measured by densitometry in three (12th wk) and four (7th wk) different CTB pools. *P < 0.05.

Notch1 Promotes Cell Column Stability in Anchoring Villi and Survival of Its Progenitors.

For manipulation of Notch1 activity in first trimester villous explant cultures, a specific Notch1-blocking antibody (Notch1-IgG1), inhibiting ADAM-mediated cleavage (25), was used (Fig. 2). Features of the particular antibody were extensively pretested in different trophoblast cell models (SI Appendix, Fig. S2). As previously mentioned (26), treatment with EDTA provoked time-dependent accumulation of Notch1 intracellular domain (N1ICD) (SI Appendix, Fig. S2A), its nuclear recruitment (SI Appendix, Fig. S2B), and induction of the canonical Notch1 target HES1 (SI Appendix, Fig. S2C). Incubation with the Notch1-blocking antibody diminished EDTA-stimulated generation of N1ICD (SI Appendix, Fig. S2 D–F), as well as expression of the full-length Notch1 receptor upon long-term treatment (SI Appendix, Fig. S2G). Moreover, Notch1-IgG₁ dose-dependently decreased luciferase activity of the canonical Notch reporter and impaired EDTA-stimulated HES1 expression (SI Appendix, Fig. S2 H and I). Treatment of villous explants with the blocking antibody reduced Notch1 signals in CCTs and diminished total Notch1 protein expression (Fig. 2 A and B). Upon seeding onto collagen I, Notch1-IgG₁ decreased numbers of anchoring villi per explant, suggesting that either de novo formation and/or stability of preexisting cell columns are affected (Fig. 2 C and D). Indeed, Western blotting and immunofluorescence showed that Notch1 inhibition increased expression of the apoptotic markers cleaved caspase-3, cytokeratin 18 neoepitope, and p53 in protein lysates and proximal CCTs of villous explants (Fig. 2 E–G). Induction of active caspase-3 was also detected upon siRNA-mediated gene silencing of Notch1 in these cultures (SI Appendix, Fig. S3A). Moreover, Notch1-IgG₁ treatment increased apoptosis in Notch1⁺ clusters of vCTBs of 6th wk placentae and in cultivated primary CTBs (SI Appendix, Fig. S3 B–D). In addition, treatment of CTBs with camptothecin (CPT) induced apoptosis and expression of Notch1 (SI Appendix, Fig. S3E). Interestingly, cleaved caspase-3 decreased when Notch1 levels were highest, suggesting that the receptor could, at least partly, counteract CPT-induced apoptosis. Notch1 was shown to promote survival by inhibiting degradation of X-linked inhibitor of apoptosis (XIAP) (27). Stability of the latter, however, was not affected upon silencing of Notch1 in CTBs (SI Appendix, Fig. S3F).

Fig. 2.

Notch1 promotes survival of extravillous trophoblast progenitors in anchoring villi. Experiments were performed with placentae between the 6th and 8th wk. (A) IHC analysis of Notch1 in CCTs after treatment of floating explant cultures with Notch1-IgG₁ or ctrl-IgG₁. Sections were stained with Notch1 antibodies (brown color) and hematoxylin (blue) to mark nuclear DNA. Representative cell columns of each 40 explants (derived from four different placentae) analyzed are shown. Stippled line demarcates the cell column from the underlying villous stroma (VS). (Scale bars, 50 μm.) (B) Western blot showing down-regulation of the full-length Notch1 receptor (transmembrane intracellular domain, 120 kDa) in the presence of Notch1-blocking antibody or control. A representative example (10 pooled explants per treatment and placenta, n = 4 placentae analyzed) is shown. (C) Representative pictures of collagen I-attached anchoring villi (arrows) after incubation with Notch1-blocking antibody. Explants (a total of 86 explants per condition, derived from five different placentae) were pretreated with Notch1-IgG₁ or controls for 12 h and seeded onto collagen I. (Scale bars, 500 µm.) (D) After 36 h, numbers of anchoring tips were counted. Bar graph shows mean values ± SD (n = 5). *P < 0.05. (E) Representative images (Scale bar, 50 µm.) showing cytokeratin 18 neoepitope (K18-ne) or p53 IF in Notch1-IgG₁–treated floating explant cultures. CC, cell column; VS, villous stroma. (F) Box plots depict median and interquartile range (IQR) values of K18-ne⁺ or p53⁺ CCTs after Notch1- or ctrl-IgG₁ treatment of each of the 90 explants (n = 5 placentae). (G) Western blot and quantification of cleaved caspase-3 expression after Notch1 inhibition. For each treatment a total of 40 explants (10 pooled explants per placenta, n = 4) were analyzed. Mean values ± SD (n = 4) normalized to GAPDH are shown. *P < 0.05.

Notch1 Maintains Purified Cell Column Progenitors and Inhibits Their Differentiation into Extravillous Trophoblasts.

To analyze the role of Notch1 in purified progenitors, the two different proliferative CTB subtypes, CCT and vCTB, were isolated from first trimester placentae using optimized purification protocols. Upon cultivation on fibronectin, vCTBs underwent cell fusion as indicated by the induction of human chorionic gonadotrophin β (CGβ) and suppression of the CTB marker E-cadherin (SI Appendix, Fig. S4 A, C, and E). The EVT-specific gene HLA-G was not induced upon in vitro differentiation of these cells. In contrast, CCTs seeded on fibronectin up-regulated the EVT markers HLA-G and integrin α1 (ITGA1), but not CGβ (SI Appendix, Fig. S4 B–D). In both progenitor subtypes, Notch1 was down-regulated upon in vitro differentiation (SI Appendix, Fig. S4 D–F). Subsequently, CCTs were isolated from 9th- to 10th-wk placentae, yielding maximal numbers of these cells, and transfected with N1ICD and a mutant variant (N1ICD-∆RAM), lacking the RBPJκ-associated module (RAM) for high-affinity binding to the particular transcription factor (Fig. 3A). After transfection, N1ICD was exclusively detected in nuclei of CCTs, whereas N1ICD-∆RAM localized to both cytoplasm and nuclei of cells (Fig. 3B). Overexpression of N1ICD supressed cleaved caspase-3, but increased cyclin A protein, cyclin D1 mRNA, as well as proliferation of CCTs, measured by EdU-labeling (Fig. 3 C–E). On the contrary, N1ICD-∆RAM did not provoke these effects. Moreover, N1ICD expression prevented differentiation of CCTs into EVTs, because it inhibited up-regulation of HLA-G and maintained expression of hepatocyte growth factor activator inhibitor type 1 (HAI-1), a marker of proliferative vCTBs and CCTs (Fig. 3F and SI Appendix, Fig. S5).

Fig. 3.

N1ICD increases proliferation and survival of purified CCTs and inhibits extravillous trophoblasts differentiation. For each preparation three to five pooled placentae of the 9th–10th wk were used. (A) Schematic representation showing protein domains of full-length Notch1, FLAG-tagged N1ICD, and N1ICD-∆RAM. (B) Notch1 IF detecting localization of N1ICD and its mutant variant after transfection into purified CCTs. (Scale bars, 50 µm.) (C) Representative Western blot and quantification of cyclin A and cleaved caspase-3 after overexpression of Notch1 constructs in CCT preparations (n = 3). Topoisomerase IIβ (TOPOIIβ) was used as loading control. (D) Cyclin D1 mRNA expression (n = 4) and (E) EdU labeling (n = 4) after ectopic expression of wild-type or mutant N1ICD in CCTs. (F) Western blot showing inhibition of HLA-G expression in differentiating cell column progenitors upon N1ICD expression. Mean values ± SD normalized to GAPDH are depicted (n = 3). *P < 0.05; ns, not significant compared with mock control.

Notch1 Expression in Villous CTBs Induces Proliferation and Inhibits Cell Fusion.

To further investigate the role of Notch1 in the placental epithelium, N1ICD was overexpressed in vCTBs isolated from 9th- to 10th-wk placentae (Fig. 4). Compared with 7th to 8th wk of gestation, Notch1 was largely absent from these cells (SI Appendix, Figs. S4F and S6). Similar to its effects in CCTs, N1ICD increased proliferation and cyclin expression in vCTBs, although the effects were more pronounced (Fig. 4 A–C and SI Appendix, Fig. S7 A and B). Again, N1ICD-∆RAM neither changed expression of cell cycle markers nor proliferation. Additionally, N1ICD suppressed in vitro syncytialization and differentiation-dependent induction of CGβ, but maintained HAI-1 and E-cadherin expression in vCTBs (Fig. 4 D–F).

Fig. 4.

N1ICD increases proliferation and inhibits cell fusion of purified vCTBs. FLAG-tagged N1ICD or N1ICD-∆RAM were overexpressed in vCTBs isolated from three to five pooled placentae between the 9th and 10th wk. (A) Representative Western blot showing up-regulation of cyclins and p-Histone H3, a marker of mitosis. As loading control, α-tubulin was used. (B) Percentage of EdU⁺ vCTBs (n = 5) and (C) relative cyclin D1 mRNA levels (n = 3) after expression of N1ICD or its mutant. AU, arbitrary units. (D) Western blot and quantification (n = 5) of markers of undifferentiated (E-cadherin, HAI-1) and differentiated (CGβ) vCTBs after N1ICD transfection. Mean values ± SD normalized to GAPDH are depicted. *P < 0.05; ns, not significant. (E) Representative IF pictures showing E-cadherin⁻ syncytial areas (marked by stippled line) in Notch1-transfected vCTBs at 120 h of in vitro differentiation. (Scale bars, 100 µm.) (F) Box plots depict median and IQR values of the percentage of multinucleated cells (n = 3). *P < 0.05; ns, not significant.

Ectopic Notch1 Induces an Extravillous Trophoblast Progenitor-Specific Gene Signature in Fusogenic Precursors.

As a prerequisite for the analysis of Notch1 in trophoblast stemness, putative marker proteins of trophoblast self-renewal were studied in placental sections and purified progenitor cell types (Fig. 5). Whereas the transcription factors p63 and TEA domain family member 4 (TEAD4) were predominantly detected in vCTBs, VE-cadherin and myc were mainly expressed in proliferative CCTs (Fig. 5 A–C). Overexpression of N1ICD in vCTB purified from 9th- to 10th-wk placentae, lacking Notch1, induced CCT-specific myc and VE-cadherin expression (Fig. 5 D–F and SI Appendix, Fig. S8). In contrast, ∆Np63 and TEAD4, controlling self-renewal of vCTBs and murine trophoblast stem cells (28, 29), respectively, were suppressed (Fig. 5 D and F and SI Appendix, Fig. S8). In addition, N1ICD increased expression of IFN regulatory factor 6 (IRF6), a negative regulator of p63 (30) (Fig. 5 D and F and SI Appendix, Fig. S8).

Fig. 5.

Expression pattern of markers of cytotrophoblast self-renewal and their regulation by N1ICD and IRF6. Representative pictures of first trimester placentae (A and B) showing coimmunofluorescence of TEAD4, ∆Np63, Notch1, and myc with VIM in serial sections. VE-cadherin expression in trophoblasts of the proximal cell column (CCT) partly overlapped with Notch1. Nuclei were counterstained with DAPI. (Scale bars, 50 µm.) (C) qPCR detecting mRNA expression in purified CCT and vCTB pools (6th–8th wk, three to five placentae per preparation). Mean values ± SD (n = 6) measured in duplicates are shown. AU, arbitrary units. *P < 0.05. Representative Western blots showing (D) ∆Np63, myc, and IRF6 and (E) VE-cadherin protein expression in N1ICD or N1ICD-∆RAM transfected vCTBs. GAPDH was used as loading control. (F) Quantification of Western blots. Mean values ± SD (n = 4 vCTB pools, each consisting of three to five 9th- to 10th-wk placentae) normalized to GAPDH are depicted. *P < 0.05; ns, not significant compared with mock control. Interaction of N1ICD with genomic regions in the (G) myc and (H) IRF6 gene. N1ICD and N1ICD-∆RAM were overexpressed in vCTBs and ChIP was performed using Notch1 antibody. (G) Schematic depiction of the myc gene showing localization of a putative RBPJκ binding site in the promoter (PROM) region at −3.060 bp and the previously identified Notch-dependent myc enhancer (NDME) at +1.4 Mb. (H) Representation of the proximal IRF6 promoter region delineating RBPJκ cognate sequences at −2.4 kb (PROM-1) and −3.6 kb (PROM-2). Bar graphs (G and H) represent PCR signals (mean values ± SD) obtained after ChIP (n = 3) of CTB pools (n = 11 placentae, 6th–8th wk) *P < 0.05; (I) Representative Western blot showing elevation of ∆Np63 expression after silencing of IRF6 in N1ICD-overexpressing vCTBs. Bar graph at Right delineates mean values ± SD (n = 3 vCTB pools, each consisting of three to four 6th- to 8th-wk placentae) normalized to GAPDH. *P < 0.05.

N1ICD binds to the myc and IRF6 gene in CTBs.

Notch1 was recently shown to stimulate myc transcription in T-cell acute lymphoblastic leukemia cells by activating RBPJκ bound to cognate sequences in the proximal promoter (−97 bp) and in the Notch-dependent myc enhancer (NDME) located 1.4 Mb 3′ of the gene (31, 32). Analyses of the myc 5′ flanking region (−5 kb) using Transfac Patch 1.0 identified two putative RBPJκ binding sites, the previously described element at −97 bp (32), and another putative cognate sequence at −3.060 bp. ChIP revealed that N1ICD interacted with the motif at −3.060 bp upon overexpression in vCTBs as well as with the distal NDME used as a control (Fig. 5G and SI Appendix, Fig. S9). As shown (31), N1ICD specifically bound to the c1 element in that region (SI Appendix, Fig. S9). Moreover, ChIP indicated that IRF6 is also a direct target of N1ICD. The latter bound to two different RBPJκ cognate sequences at −3.6 kb and −2.4 kb in the IRF6 promoter (Fig. 5H and SI Appendix, Fig. S9), previously delineated in keratinocytes (33).

Notch1 intracellular domain represses p63 through IRF6 in vCTBs.

To analyze whether Notch-dependent down-regulation of ∆Np63 requires IRF6, vCTBs were transfected with N1ICD and siRNA against IRF6. Silencing of the latter increased ∆Np63 levels, indicating a direct role in N1ICD-induced repression of p63 (Fig. 5I).

Discussion

Failures in trophoblast differentiation and function in a variety of pregnancy disorders strongly warrant a better understanding of the molecular pathways controlling human placental development. However, experimental studies are hampered by ethical constraints to obtain tissues from early human gestation as well as difficulties in establishing self-renewing human trophoblast stem and progenitor cells from trophoblast isolates (34–36). As a consequence, alternative models, such as bone morphogenetic protein (BMP)-treated human ESCs (hESCs), have been developed, allowing in vitro formation of the trophoblast lineage (37, 38). Despite concerns regarding specificity of trophectoderm induction with BMP (37, 39, 40), recent investigations using optimized culture conditions suggested that early stages of human trophoblast development could be mimicked in these cells (41, 42).

Nevertheless, in vivo localization of human trophoblast stem and progenitor cells and their specific features remain poorly characterized. Derivation of a cell line with trophoblast stem cell properties from single blastomers of eight-cell embryos suggested that the trophectoderm cell fate could be initiated at very early stages of pregnancy (43). In addition, trophoblast stem-like cells, expressing Cdx2 and p63, were derived from BMP-induced hESCs generating both EVTs and STBs (42). However, it remains controversial whether bipotential CTBs are maintained during the first trimester of pregnancy (4). Whereas FGF4 was claimed to redirect fusogenic CTBs toward EVT differentiation (44), others showed that EVT precursors isolated from 6th- to 10th-wk placentae were unable to syncytialize, but spontaneously formed 20% HLA-G⁺ cells after 5 d in culture (16). The latter study suggested that distinct trophoblast progenitors, committed to syncytialization and EVT formation, respectively, have developed in first trimester placentae. The present study confirms this assumption. Using sequential trypsin digestions of early placental tissues, removal of STB fragments by gradient centrifugation and immune depletion of differentiated EVTs, CTB progenitor subtypes with high purity were obtained. Upon seeding onto fibronectin, vCTBs fused into STBs, but did not induce the EVT markers HLA-G or ITGA1. In contrast, CCTs differentiated into EVTs lacking STB-specific CGβ expression. Notably, formation of STBs and EVTs occurred under identical culture conditions and on the same matrix, suggesting that differentiation is mainly driven by the intrinsic molecular program of vCTBs and CCTs. Because isolation of the two distinct progenitors is based on the consecutive digestions with different trypsin concentrations, some marginal cross-contamination cannot be avoided (SI Appendix, Fig. S4C). However, the present protocol will allow for further genome-wide gene expression analyses of these CTB subtypes. Thereby, unique surface markers, suitable for additional purification steps, could be identified.

Critical regulators controlling trophoblast stemness and lineage determination have been vastly studied in developing mice (45–48). However, data on key factors controlling human placental development are scarce. Descriptive analyses in first trimester tissues and trophoblast cell models have been performed to gain insights into the expression patterns and epigenetic profiles of some of these genes (37, 40, 48, 49). However, few of them have been functionally examined by using choriocarcinoma cells as surrogate for human primary trophoblasts (28, 50). To extend our present knowledge, we herein analyzed expression of key regulators in the two purified CTB progenitor subtypes and performed functional analyses in these cells. Immunofluorescence and quantitative PCR (qPCR) indicated that TEAD4, a crucial transcription factor in murine trophectoderm specification and trophoblast progenitor self-renewal (29, 51), is also present in human vCTBs but largely absent from proximal Notch1⁺ CCTs. Akin to that finding, p63, associated with the CTB progenitor phenotype (28), was predominantly expressed in vCTBs. However, the stemness markers myc, markedly decreasing during the first trimester of pregnancy (52), and VE-cadherin, were mainly detected in CCTs. Besides its classical role in endothelial cells, VE-cadherin also specifies a transient, hematopoetic stem cell population in the developing liver (53). In anchoring villi VE-cadherin specifically localizes to the intermediate region of the cell column containing proliferative CCT (Fig. 5B), as also previously shown (54). Its expression partly overlaps with Notch1, exclusively found in proximal EVT progenitors. Cdx2, the central transcriptional activator factor in murine trophectoderm development (55), has been detected in vCTBs of early placental tissues and rapidly declines toward the end of the first trimester (42, 48, 49). The present study also detected Cdx2 in vCTBs of 6th- wk placentae (SI Appendix, Fig. S1D). However, the factor was absent from both vCTBs and CCTs of distal villi located at the basal side of the placenta. Instead, CCTs in this region were positive for Notch1, a key regulator of stem cell niches and cell lineage determination (23). Based on these expression patterns, we hypothesize that putative Cdx2⁺ stem cell-like trophoblasts disappear in distally developing regions of the placenta, at a time when extensive formation of villi, anchoring to the maternal decidua, takes place. Instead, committed progenitors for cell fusion and EVT differentiation arise, each expressing a unique set of self-renewal genes.

Development of stable anchoring villi is a prerequisite for a continuous supply with EVTs invading and remodelling maternal uterine tissues. However, mechanisms controlling establishment and maintenance of cell columns, replenishing the pool of outgrowing trophoblasts, have not been elucidated. Herein, we identified Notch1 as a critical regulator of these processes. Notch1 was detected in proliferative/mitotic CCTs of the proximal cell column, thereby defining the EVT progenitor cell niche. Despite its short half-life of ∼1.8 h (56), nuclear N1ICD was occasionally observed in proximal CCTs, suggesting activation of Notch1 cleavage. However, cyclin A and p-Histone H3 were also expressed in neighboring Notch1⁻ CCTs, suggesting that Notch1⁺ precursors could give rise to committed transient amplifying (TA)-like cells. The latter further develop into distal HLA-G⁺ CCTs progressing toward EVTs. TA cells, derived by asymmetric cell division of self-renewing Notch1⁺ stem cells, have been previously described in other epithelial tissues (57). In analogy, we assume that Notch1 could control homeostasis of placental anchoring villi by adapting rates of progenitor self-renewal, TA-like cell formation, and EVT differentiation.

Similar to Cdx2 (49), CTB-specific Notch1 expression markedly decreases during the early weeks of pregnancy (SI Appendix, Fig. S1 E and F) and is absent from second trimester trophoblasts (19), indicating that the number of progenitor cells diminishes at the time of oxygen transition. By binding of NICD to HIF-1α, Notch signaling synergizes with hypoxia to keep cells in an undifferentiated state (58, 59). Hence, we speculated that a low-oxygen environment could support mRNA expression and protein stability of Notch1, as shown in other cell types (59–61), and thereby maintain the proliferative capacity of CCT precursors. Indeed, the present data indicate that hypoxia increases Notch1 as well as activity of a canonical Notch reporter. After establishment of the placental–maternal circulation, declining Notch1 levels might affect survival and/or proliferation of these cells, thereby slowing down continuous growth of cell columns and EVT differentiation. On the other hand, Notch1 was detected in EVT progenitors of 12th-wk placentae, at the time when the receptor was completely absent from vCTBs. Maintenance of Notch1⁺ CCTs in the presence of increasing oxygen levels could be necessary for on-going EVT formation during the period of spiral artery remodelling.

Notch signaling in stem cells is highly complex and exerts different effects in tissues and organs (23, 62). Accordingly, Notch was shown to promote or inhibit self-renewal, differentiation, and survival, depending on the specific cellular context. Previous analyses of human placental tissues suggested that Notch signaling could be predominantly associated with trophoblast progenitor cell function (20). Although Notch2 has its main role in invasive trophoblasts (19, 24), down-regulation of HES1 and RBPJκ reporter activity during EVT formation indicated that canonical Notch signaling is associated with undifferentiated trophoblasts (20, 21). Herein, functional analyses of Notch1 in different primary trophoblast cell models corroborate this evidence. Blocking or silencing of Notch1 increased apoptosis in villous explant cultures and isolated CTBs. In contrast, overexpression of N1ICD enhanced proliferation and cyclin expression but suppressed cleaved caspase-3 in purified vCTBs or CCTs. Notch-dependent survival operates through different mechanisms involving mammalian target of rapamycin (mTOR)–Akt-mediated suppression of p53 or activation of inhibitors of apoptosis such as XIAP (27, 63). Expression of the latter was not affected by Notch1 in trophoblasts. However, inhibition of the receptor induced accumulation of nuclear p53. Notably, silencing of RBPJκ in villous explant cultures did not provoke apoptosis of CCTs (21), suggesting that N1ICD-mediated survival could be an RBPJκ-independent function, as recently shown (64). Different than Notch1, disruption of RBPJκ weakly increased proliferation in explant cultures and, as a consequence EVT formation (21). Several effects could account for this discrepancy. Abolishing RBPJκ not only impairs Notch1 but also activities of Notch3 and Notch4, which are abundantly expressed in CTBs (19, 20). Besides binding to RBPJκ, N1ICD interacts with a range of different transcription factors (65), of which HIF1α, NFκB, GABPA, and ZNF143 are also detected in our previously published microarray data (66) of EGFR⁺ CTBs (accessible at Gene Expression Omnibus, GDS3523). Moreover, RBPJκ has Notch-independent roles in different cell types, mostly as suppressor of gene transcription (67).

Notch1 effectively inhibits differentiation in many cellular systems, thereby preserving stemness (62). In agreement with that fact, N1ICD maintained expression of genes associated with undifferentiated vCTBs and CCTs, E-cadherin and HAI-1, and suppressed STB formation of fusogenic precursors as well as differentiation-dependent induction of CGβ. Moreover, N1ICD also inhibited differentiation of CCTs into HLA-G⁺ EVTs. Immunofluorescence of 5th- to 7th-wk placental tissues revealed that the receptor specifically appears in vCTB clusters of distal placental villi containing adjacent cell islands with Notch1⁺ CCTs. Serial sectioning of these villi revealed that single-row Notch1⁺ vCTBs were parts of developing cell columns. At the 10th–12th wk of gestation, trophoblast-specific expression of Notch1 was restricted to proximal CCTs of villi anchoring to the maternal decidua. Therefore, we speculated that Notch1 could initiate the extravillous trophoblast lineage in distal regions of the placenta, later on forming the placental basal plate. This view was supported by data obtained from villous explant cultures, demonstrating that Notch1 inhibition decreased numbers of outgrowing cell columns. However, Notch1 blocking also provoked CCT apoptosis, as shown above, and small, preformed cell columns cannot be discerned from de novo forming tips in this system.

Hence, N1ICD was specifically overexpressed in vCTBs isolated from 9th- to 10th-wk placentae, largely lacking Notch1, and analyzed for markers of vCTB or CCT stemness, defined in the present study. Of note, N1ICD suppressed vCTB-specific p63 and TEAD4 expression but induced the CCT markers VE-cadherin and myc. Similar to other cells (31), the latter could be a direct target of active Notch1 in CTBs. ChIP revealed that N1ICD binds to the 3′ NDME of the myc gene, previously shown to promote gene expression (31), as well as to a newly identified RBPJκ motif present at −3 kb in the proximal promoter region. In contrast to myc, N1ICD was recently shown to indirectly suppress p63. N1ICD binds to the IRF6 gene in keratinocytes and activates its expression (33). In turn, IRF6 promotes proteasomal degradation of p63 (30). An analogous mechanism could regulate p63 in vCTBs, because IRF6 rapidly increased after N1ICD expression. Also, ChIP analyses showed that N1ICD interacts with two RBPJκ cognate sequences, recently identified in the proximal IRF6 promoter region (33). Moreover, silencing of IRF6 in vCTBs impaired N1ICD-dependent suppression of ∆Np63. Compared with p63 mRNA, loss of ∆Np63 protein was more pronounced in the presence of ectopic N1ICD (Fig. 5 D and F and SI Appendix, Fig. S8), suggesting that its stability could be affected. Additionally, Notch1-mediated inhibition of p63 could diminish numbers of self-renewing TEAD4⁺ vCTBs and, as a consequence, cell fusion. Unlike myc, p63, or IRF6, N1ICD-dependent induction of VE-cadherin was only noticed upon long-term expression, indicating that it might not be a direct Notch1 target. Notably, immunofluorescence revealed that the particular adhesion molecule specifically localizes to proliferative CCTs in the intermediate region of the cell column (Fig. 5B) (12). Hence, N1ICD-induced VE-cadherin expression might suggest that cell column progenitors have further developed into TA-like cells. Finally, we speculate that Notch1-dependent expression of myc and VE-cadherin could be sufficient to establish EVT progenitors from Notch1⁻ CTB precursors. On the other hand, suppression of cell fusion and down-regulation of p63/TEAD4 by N1ICD could negatively affect vCTB self-renewal and thereby trigger CCT formation and EVT differentiation. Along those lines, silencing of p63 in JEG-3 choriocarcinoma cells was shown to increase migration, a characteristic feature of invasive EVTs (28).

In summary, the present study shows that Notch1 is a critical regulator of EVT development in the human placenta. Based on its restricted expression in the proximal region of developing cell columns, functional studies in primary trophoblast models have been conducted, indicating a crucial role of the receptor in CCT proliferation, survival, and differentiation. Notch1 induced markers of EVT progenitors in Notch1⁻ vCTBs, but suppressed genes controlling vCTB self-renewal (Fig. 6). Further studies are required to delineate Notch ligands involved in this process as well as the mechanism(s) initiating Notch1 expression in the developing placenta.

Fig. 6.

Model system depicting the presumptive role of Notch1 in human trophoblast development. N1ICD programs vCTBs into CCTs, expressing the stemness markers myc and VE-cadherin and prevents EVT differentiation by maintaining proliferation and survival of these cells. N1ICD also suppresses TEAD4 and p63, the latter by inducing its repressor IRF6, thereby alleviating self-renewal and cell fusion of vCTBs.

Materials and Methods

Tissue Collection.

Placental tissues (6th–12th wk of gestation) were obtained from elective pregnancy terminations. Utilization of tissues and all experimental procedures were approved by the Medical University of Vienna ethics boards. Written informed consent was obtained from all subjects.

Immunohistochemistry of Paraffin-Embedded Tissue.

Serial sections of paraffin-embedded villous explants were analyzed by immunohistochemistry (IHC) using Dako EnVision+ System-HRP (DAKO, K4011) as instructed by the manufacturer. Sections were incubated with primary antibodies (listed in SI Appendix, Table S1) overnight at 4 °C and nuclei were counterstained with hematoxylin (Merck). Sections were digitally photographed using an Olympus BX50 microscope and CellP software.

Immunofluorescence of Paraffin-Embedded Tissue.

Placental tissue was fixed in 7.5% (wt/vol) formaldehyde and embedded in paraffin. To analyze Notch1 localization in different regions of placental villi (stem villus, intermediate villus, end villus), tissues were stained with Alcian blue solution and carefully placed in embedding cassettes. Serial sections (3 µm) of paraffin-embedded placental tissue or villous explant cultures were analyzed by immunofluorescence (IF) as described elsewhere (20). Briefly, sections were deparaffinized in Xylol and rehydrated. Antigen retrieval was performed using 1× PT module buffer 1 (Thermo Scientific) for 35 min at 93 °C using a KOS microwave histostation (Milestone). Sections were incubated with primary antibodies (listed in SI Appendix, Table S1) overnight at 4 °C. Afterward, slides were washed three times and incubated for 1 h with secondary antibodies (2 µg/mL) listed in SI Appendix, Table S1. Nuclei were stained with 1 µg/mL DAPI. Tissue were analyzed by fluorescence microscopy (Olympus BX50, CellP software) and digitally photographed.

Immunofluorescence of Cultured Cells.

Cells were fixed with 3.7% (wt/vol) paraformaldehyde (10 min), treated with 0.1% Triton X-100 (5 min) and incubated with primary antibodies overnight at 4 °C (listed in SI Appendix, Table S1). Subsequently, cells were washed and incubated for 1 h with 2 µg/mL of secondary antibodies (listed in SI Appendix, Table S1) and nuclei were stained with DAPI. Slides were analyzed by fluorescence microscopy (Olympus BX50, CellP software) and digitally photographed.

Isolation and Cultivation of Placental Primary CTBs.

CTBs were isolated by adapted enzymatic dispersion and Percoll density gradient centrifugation [10–70% (vol/vol); GE Healthcare] of pooled first trimester placentae (n = 3–6 per isolation) as described (68, 69) and plated (45 min) in culture medium (DMEM/Ham’s F-12, 10% (vol/vol) FCS, 0.05 mg/mL gentamicin, 0.5 µg/mL fungizone; Gibco) allowing for adherence of contaminating stromal cells. Nonadherent trophoblasts were collected and CTBs were either seeded in culture medium onto fibronectin-coated (20 µg/mL; Millipore) dishes (2.5 × 10⁵ cells per square centimeter) or further purified using positive selection with EGFR-phycoerythrin (PE) or HLA-G-PE antibodies (listed in SI Appendix, Table S1) and anti-PE MicroBeads (MACS Miltenyi Biotec) according to the manufacturer’s instructions. The contamination with stromal cells was routinely tested by IF with antibodies detecting cytokeratin 7 (trophoblast cells) and vimentin (nontrophoblast cells). Vimentin⁺ cells were fewer than 1%. The purity of isolated EGFR⁺ and HLA-G⁺ CTB populations was verified using qPCR and Western blotting.

To verify specificity of the Notch1 blocking, CTBs were allowed to attach for 2 h, washed with prewarmed PBS, and preincubated with culture medium containing either 0.5 µg/mL ctrl-IgG₁ (ctrl-IgG₁ ABIN376845; Antibodies-Online) or 0.5 µg/mL Notch1-IgG₁ (Notch1-IgG₁, Genentech) antibodies for 1 h. Subsequently, 5 mM EDTA was added for another 30 min, inducing receptor cleavage/generation of N1ICD. For long-term Notch1 inhibition, CTBs were seeded for 2 h and antibodies were added for an additional 48 h. To analyze prosurvival effects of Notch1, CTBs were seeded for 2 h, washed, and incubated with culture medium containing either DMSO (ctrl) or 1 µM CPT for 1, 2, 4, 6, and 24 h. To verify whether the Notch1 could inhibit apoptosis via XIAP stabilization, CTBs were preincubated with a mixture of four different siRNAs against Notch1 (si-Notch1; l-007771–00-0005, ON-TARGETplus SMARTpools, Dharmacon-Thermo Fisher Scientific) or nontargeting siRNA (si-ctrl, d-001810-10-20) overnight and subsequently treated with DMSO (−) or 1 µM CPT (+) for 24 h.

Isolation and Cultivation of Purified Primary vCTBs and CCTs.

CCT and vCTB cell populations were isolated by two consecutive digestion steps followed by Percoll density gradient centrifugation. Precisely, placental tissue (6th–8th and 9th–10th wk of gestation, n = 3–5 per isolation) was minced into small pieces (1–3 mm). For isolation of CCTs, the first digestion was performed with 0.125% trypsin (Gibco) and 12.5 mg/mL DNase I (Sigma-Aldrich) in Mg²⁺/Ca²⁺-free HBSS (1× HBSS, Gibco) for 30 min at 37 °C. Subsequently, digestion was stopped using 10% (vol/vol) FBS (PAA Laboratories) and cells were filtered through a 100-µm cell strainer (BD Biosciences). To isolate vCTB cells, a second digestion step of the remaining tissue was performed with 0.25% trypsin and 12.5 mg/mL DNase I for 30 min at 37 °C and processed as described above. Digestion solutions containing either CCTs or vCTBs were each layered on top of a Percoll gradient [10–70% (vol/vol)] and cells between 35 and 50% of the Percoll layer were collected. Contaminating RBCs were removed by incubating cells with erythrocyte lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA, pH 7.3) for 5 min at room temperature and subsequently washed with 1× HBSS. Contaminating stromal cells were depleted from CCT preparations as mentioned above. Finally, differentiated CCTs/EVTs were removed from proliferative CCTs or vCTBs, using HLA-G-PE antibodies (Exbio) and anti-PE MicroBeads (MACS Miltenyi Biotec) as instructed by the manufacturer. CCT and vCTB cells were seeded onto fibronectin-coated dishes at a density of 2.5 × 10⁵ cells per square centimeter and 3.25 × 10⁵ cells per square centimeter, respectively. CCT and vCTB cells were harvested after 24 h (undifferentiated cells) and between 72 and 120 h (differentiated cells). Cells were either fixed for IF analyses or snap frozen for qPCR and Western blotting.

Notch1 Plasmids and CTB Transfection.

Full-length human N1ICD (residues 1762–2556) and a N1ICD-ΔRAM domain (residues 1877–2556) were a generous gift of C. O. Joe, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea. Notch1 variants are cloned into pFLAG-CMV-2 (Sigma) as described elsewhere (70). Empty pFLAG-CMV-2 served as negative control (mock-CTRL). Isolated CCTs and vCTBs were transfected with the 4D-Nucleofector (program EO-100, Lonza) using the AMAXA SG Cell Line Kit according to the manufacturer’s instructions. Subsequently, cells were seeded at a density of 3.5 × 10⁵ cells per square centimeter. Transfection with a pmaxGFP (Lonza) revealed an average transfection efficiency of 44 ± 18%. Transfected trophoblasts were incubated up to 96 h at 37 °C. To evaluate localization of transfected N1ICD and N1ICD-ΔRAM, IF was performed as described above. For down-regulation of IRF6 in N1ICD-overexpressing CTBs, siRNA-mediated IRF6 gene silencing was performed using a mixture of four different siRNAs targeting IRF6 (si-IRF6) or a nontargeting (si-ctrl) control pool (l-012227–005 and d-001810–10-20 ON-TARGETplus SMARTpools, Dharmacon-Thermo Fisher Scientific). Notch1-transfected cells were seeded in the presence of si-ctrl or si-IRF6 and incubated for 24 h.

Proliferation Assays.

Purified CCTs and vCTBs were transfected with mock-CTRL, N1ICD, or N1ICD-ΔRAM, seeded onto fibronectin-coated 48-well dishes, and incubated for 24 h. Afterward, 10 µM 5-ethynyl-2′-deoxyuridine (EdU) (EdU-Click 488, BaseClick) was added for 4 h. Subsequently, cells were fixed and EdU was detected according to the manufacturer’s instructions. Nuclei were stained with DAPI (Roche). Cells were digitally photographed (seven pictures per condition) using the EVOS FL Color Imaging System and EdU⁺ nuclei were counted using Adobe Photoshop CS5.

Notch1 Inhibition in First Trimester Villous Explant Cultures.

Villous explant cultures were performed as described elsewhere (71). In detail, pieces of villous tissues (5–6 mm) were dissected from the placental basal plate (7th–8th wk of gestation), and further divided into two equal parts. To evaluate efficiency to Notch1-blocking antibodies, villous explants were treated with either 0.5 µg/mL ctrl-IgG1 or 0.5 µg/mL Notch1-IgG1 for 1 h. The Notch1-blocking antibody, a generous gift of C. W. Siebel, Genentech, San Francisco, binds and stabilizes the negative regulatory region of the receptor, thereby inhibiting conformational changes, ADAM-mediated cleavage, and N1ICD-dependent activation of canonical signaling (25). Subsequently, 5 mM EDTA was added for up to 60 min and explants were homogenized to isolate protein lysates. For outgrowth analyses, floating explant pairs were supplemented with 0.5 µg/mL ctrl-IgG₁ or 0.5 µg/mL Notch1-IgG₁ and kept overnight in explant culture medium (DMEM/Ham’s F-12, 0.05 mg/mL gentamicin), respectively. Subsequently, explants were seeded onto rat tail collagen I (attachment for 4 h) and subsequently covered with medium containing either 0.5 µg/mL ctrl-IgG₁ or 0.5 µg/mL Notch1-IgG₁. After another 24 h, explants were digitally photographed and differences in anchorage and outgrowth were evaluated using the EVOS FL Color Imaging System. For long-term antibody- or siRNA-mediated Notch1 inhibition, explants (7th–8th wk of gestation) were incubated for 48 h in culture medium supplemented with 0.5 µg/mL Notch1-IgG₁ or Notch1 siRNA. Next, explants were either fixed with 7.5% (wt/vol) formaldehyde and embedded in paraffin or homogenized for Western blotting as described below.

Cultivation and Luciferase Reporter Assay in Trophoblastic SGHPL-5 Cells and Primary CTBs.

SGHPL-5 cells were cultivated in DMEM/Ham’s F-12 supplemented with 10% (vol/vol) FCS and 0.05 mg/mL gentamicin at standard cell culture conditions. Notch1 cleavage was induced by adding 5 mM EDTA for up to 30 min. Inhibition of Notch1 cleavage was performed with ctrl-IgG₁ or Notch1-IgG₁ as mentioned above. For detection of canonical Notch activity subconfluent SGHPL-5 cells were cotransfected with 2 μg/mL of a luciferase reporter containing four RBPJκ binding sites (mutant or wild-type plasmids) and 0.5 μg/mL pCMV–β-galactosidase (CMV-βGal; normalization control) using Lipofectamine 2000 (Invitrogen) as recently described (20). Primary CTBs isolated from pooled 6th-to 8th-wk placentae were transfected with reporter plasmids using the AMAXA system as mentioned above. After 6 h, medium was changed. SGHPL-5 cells were incubated either with DAPT, ctrl-IgG₁, or Notch1-IgG₁ (80 ng/mL and 400 ng/mL). Primary CTBs were incubated under 20% (vol/vol) oxygen (ctrl), 5% (vol/vol) oxygen, or 20% (vol/vol) oxygen/50 µM CoCl₂. Protein lysates were prepared after an additional 24 h. Luciferase activity and β-galactosidase activity were determined as previously published (72).

Western Blotting.

Whole-cell lysates were prepared using standard protocols as recently described (72). Villous explants were additionally homogenized for 20 s at 3,640 × g with a Precellys 24 (PeqLab). Nuclear extracts were isolated using the NE-PER nuclear and cytoplasmic protein extraction kit (Thermo Scientific). Protein extracts were separated on SDS/PAA gels, transferred onto Hybond-P PVDF (Amersham) membranes and incubated overnight at 4 °C with primary antibodies (SI Appendix, Table S1). Subsequently, filters were washed and incubated for 1 h with HRP-conjugated secondary antibodies (SI Appendix, Table S1). Signals were developed using ECL Prime Detection Kit (GE Healthcare) and visualized with FluorChemQ Imaging System (Alpha Innotech). Quantification was performed using ImageJ software.

qPCR.

RNA isolation (PeqGold Trifast, PeqLab) and reverse transcription (RevertAid H Minus Reverse Transcriptase, Fermentas, EP0451) were performed as indicated by the manufacturers. Villous explants were homogenized with the Precellys 24 (PeqLab) before RNA isolation. qPCR analyses (in duplicates) were performed using the 7500 Fast Real-time PCR System (Applied Biosystems, ABI) as described (73). The following TaqMan Gene Expression Assays were used: NOTCH1 (ABI, Hs01062014_m1), CCND1 (ABI, Hs00277039_m1), TEAD4 (ABI, Hs01125032_m1), p63 (ABI, Hs00978340_m1), MYC (ABI, Hs00153408_m1), VE-cadherin (ABI, Hs00901465_m1), and HES1 (ABI, Hs00172878_m1). A total of 1 µL cDNA, 0.5 µL primers, 5 µL innuMIX qPCR MasterMix Probe (Biometra), and 0.2 µL ROX Reference Dye (Invitrogen) were used per sample. Signals (ΔCt) were normalized to TATA-box binding protein (TBP) (ABI, 4333769F). Relative expression levels were determined by using values of controls as a calibrator (ΔΔCt).

ChIP.

For ChIP analyses, SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling, 9003) was used as mentioned by the manufacturer. Briefly, vCTBs were isolated, transfected with N1ICD or N1ICD-ΔRAM, seeded onto fibronectin-coated six-well dishes, and incubated for 20 h. Subsequently, cells were fixed for 45 min with 2 mM disuccinimidyl glutarate (DSG) and 1% formaldehyde (15 min). After nuclei preparation, chromatin digestion, and sonication, purified chromatin lysates were incubated either with Notch1 antibody (1 µg, Cell Signaling, 3608), normal rabbit IgG (negative control) (1 µg; Cell Signaling, 2729) or Histone H3 XP rabbit mAb (positive control) (1 µg; Cell Signaling, 4620) overnight at 4 °C. Immunoprecipitated chromatin was captured with ChIP-Grade Protein G magnetic beads and eluted. Purified DNA was assessed by semi-qPCR using Taq Polymerase (Fermentas, EP0402). PCR conditions were as follows: 5 min at 96 °C (initial denaturation); 45 s at 95 °C, 45 s at 58 °C, 45 s at 72 °C (35 cycles), and 5 min at 72 °C (final extension). MYC PROM primers were designed to span the region harboring a putative RBPJκ DNA binding motif (CTGCGGGAA) identified by Transfac Patch 1.0 at −3.060 bp, which differs by one nucleotide from the consensus sequence CTGTGGGAA. MYC NDME-c1, MYC NDME-c2, IRF6 PROM-1, and IRF6 PROM-2 primers were published elsewhere (31, 33). The following primer sequences were used: MYC PROM (229 bp): forward 5′-ATGAGGTCAAGCTGGACCTAC-3′ and reverse 5′-TGACGGTGTCTGATCACTTA-3′; MYC NDME-c1 (200 bp): forward 5′-GCTGCCACATGCTGATGAAC-3′ and reverse 5′-CCAGGTAGGGGCATTACGTC-3′; MYC NDME-c2 (174 bp): forward 5′-GAGGCCCCCATTCATTACCC-3′ and reverse 5′-GCAGTTCTTCCTACGCTGGT-3′; IRF6 PROM-1: forward 5′-ACCCTCCCAGCTTGAGTTTT-3′ and reverse 5′-AAACCCCAGTGGCATACAAG-3′ (142 bp); IRF6 PROM-2: forward 5′-TCAATGGAGGGCAAAATGAT-3′ and reverse 5′-ACGCCTCATCTGCTTGATCT-3′ (162 bp); human RPL30 (control) (Cell Signaling, 7014). PCR products were analyzed on 1.5% (wt/vol) agarose gels containing Midori green (Nippon Genetics, MG04) and digitally photographed under UV. Relative occupancy of N1ICD or N1ICD-ΔRAM was normalized to normal rabbit IgG.

Statistical Analyses.

Gaussian distribution and equality of variances were examined with Kolmogorov–Smirnov and Levene tests, respectively. Statistical analysis of data between two means was performed with Student’s t test or Mann–Whitney u test using SPSS 14. Comparisons of multiple groups were evaluated with one-way ANOVA and appropriate post hoc tests or Kruskal–Wallis tests followed by pairwise Mann–Whitney u tests and Shaffer’s correction. A P value of <0.05 was considered statistically significant. Unless otherwise noted, all experiments were conducted in duplicates and replicated at least three times.