Notch1 controls development of the extravilloustrophoblast lineage in the human placentaSandra Haidera, Gudrun Meinhardta, Leila Saleha, Christian Fialab, Jürgen Pollheimera, and Martin Knöflera,1

aDepartment of Obstetrics and Gynaecology, Reproductive Biology Unit, Medical University of Vienna, 1090 Vienna, Austria; and bGynmed Clinic, 1150Vienna, Austria

Edited by R. Michael Roberts, University of Missouri-Columbia, Columbia, MO, and approved October 21, 2016 (received for review July 26, 2016)

Development of the human placenta and its different epithelialtrophoblasts is crucial for a successful pregnancy. Besides fusing intoa multinuclear syncytium, the exchange surface between mother andfetus, progenitors develop into extravillous trophoblasts invading thematernal uterus and its spiral arteries. Migration into these vesselspromotes remodelling and, as a consequence, adaption of blood flowto the fetal–placental unit. Defects in remodelling and trophoblastdifferentiation are associated with severe gestational diseases, suchas preeclampsia. However, mechanisms controlling human tropho-blast development are largely unknown. Herein, we show that Notch1is one such critical regulator, programming primary trophoblasts intoprogenitors of the invasive differentiation pathway. At the 12th wk ofgestation, Notch1 is exclusively detected in precursors of the extra-villous trophoblast lineage, forming cell columns anchored to theuterine stroma. At the 6th wk, Notch1 is additionally expressed inclusters of villous trophoblasts underlying the syncytium, suggestingthat the receptor initiates the invasive differentiation program in dis-tal regions of the developing placental epithelium. Manipulation ofNotch1 in primary trophoblast models demonstrated that the receptorpromotes proliferation and survival of extravillous trophoblast pro-genitors. Notch1 intracellular domain induced genes associated withstemness of cell columns, myc and VE-cadherin, in Notch1− fusogenicprecursors, and bound to the myc promoter and enhancer region atRBPJκ cognate sequences. In contrast, Notch1 repressed syncytializa-tion and expression of TEAD4 and p63, two regulators controlling self-renewal of villous cytotrophoblasts. Our results revealed Notch1 as akey factor promoting development of progenitors of the extravilloustrophoblast lineage in the human placenta.

human placenta | trophoblast progenitors | Notch1 | extravilloustrophoblast | cell fusion

Differentiation processes of the human placenta are a pre-requisite for fetal development and successful pregnancy out-

come. Shortly after implantation, stem cells of the trophectodermsurrounding the blastocyst give rise to the primitive syncytium by cellfusion as well as to proliferative cytotrophoblasts (CTBs) formingprimary placental villi (1). Breaking through the multinuclearstructures, these villi contact the maternal decidua, the endometriumof pregnancy, and expand laterally to form the so-called tropho-blastic shell. The latter encircles the embryo and protects it fromoxidative damage during early gestation (2). As pregnancy proceeds,placental villi undergo extensive remodelling involving branchingmorphogenesis and transformation into secondary and finallytertiary villi by migration of mesenchymal cells and vascularization,respectively. At this stage, two types of villi can be discerned, floatingand anchoring villi. Floating villi, which are bathed in maternal bloodafter establishment of the fetal–maternal circulation, are necessaryfor hormone production and nutrient and oxygen transport to thedeveloping 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 theother hand, anchoring villi attached to the decidua form proliferativecell columns giving rise to differentiated, extravillous trophoblasts(EVTs). The latter deeply migrate into uterine tissue and the ma-ternal spiral arteries, provoking vessel remodelling and adaptation of

adequate blood flow to the placenta (5, 6). Failures in placentationand artery remodelling have been associated with a variety ofpregnancy diseases, such as miscarriage, preeclampsia, fetal growthrestriction, and preterm labor (7–10). Besides unfavorable immu-nological interactions of EVTs with uterine natural killer (uNK) cells(11), abnormal placental development and trophoblast differentia-tion are thought to contribute to the pathogenesis of gestationaldisorders. Indeed, CTBs isolated from preeclamptic placentae failedto appropriately differentiate into the invasive lineage in vitro andexpressed an antimigratory gene signature (12, 13).However, our knowledge about human placentation and tropho-

blast development is only scarce. Bipotential trophoblast progenitorcells have been derived from the chorionic mesenchyme differentiatinginto EVTs and STBs (14, 15), whereas others identified a specificprecursor of the EVT lineage in villous explant cultures (16). Placentalstructures, trophoblast cell types, and expression patterns of key reg-ulatory transcription factors diverge between mouse and man, therebyhindering comparison of putative regulatory mechanisms (17). Al-though different transcriptional activators promoting or inhibiting EVTmotility have been described (18), it is unknown which factors governEVT differentiation. Likewise, how regions of column formation arespecified and maintained within developing villi remains elusive.Recent evidence suggested that the developmental Notch path-

way could be critically involved in human trophoblast function anddifferentiation (19–21). Canonical Notch signaling is activated upondirect cell–cell contact involving binding of membrane-anchoredligands, 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

Significance

Progenitor trophoblast cells of the human placenta either fuse toform a syncytium or develop into invasive trophoblasts invadingthe maternal uterus. However, regulatory pathways controllingtheir development and distinct differentiation programs are poorlyunderstood. In the present study, we demonstrate that Notch1 is acritical regulator of early pregnancy, promoting development ofthe invasive, extravillous trophoblast lineage and survival of itsprogenitors. In vivo, Notch1 is detected in extravillous trophoblastprogenitors and clusters of villous trophoblast initiating the in-vasive differentiation program. In vitro, Notch1 repressed genesinvolved in self-renewal of fusogenic precursors, but inducedgenes specifically expressed by extravillous trophoblast progeni-tors. Our data delineate Notch1 as a key regulator promotingdevelopment of the human extravillous trophoblast lineage.

Author contributions: S.H. and M.K. designed research; S.H. and G.M. performed research; C.F.contributed new reagents/analytic tools; L.S. and J.P. analyzed data; andM.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. Email: martin.knoefler@meduniwien.ac.at.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1612335113/-/DCSupplemental.

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members of a disintegrin and metalloproteinase (ADAM) familyand γ-secretase, the Notch intracellular domain (NICD) is releasedinto the cytoplasm. Subsequently, NICD translocates to the nucleusand functions as a coactivator of the transcription factor recom-bination signal binding protein for Ig kappa J region (RBPJκ)controlling numerous biological processes such as stem cellmaintenance, cell lineage determination, and differentiation (23).Human placentae express Notch receptors and their ligands in acell-specific manner (19, 20). Notch2 is predominantly detected indifferent EVT subtypes, and inhibition of Notch2 affected tro-phoblast cell migration (24). In analogy, conditional deletion ofNotch2 in murine trophoblast progenitors impaired endovascularinvasion and placental perfusion (19). In contrast to that, Notch1,3, and 4 were shown to be expressed by proliferative CTBs of firsttrimester placentae (20). Interestingly, Notch1 is absent fromsecond trimester placental tissues (19), suggesting a role of thereceptor 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 trophoblastcell models. Our data show that Notch1 is specifically expressed byprogenitors of the extravillous trophoblast lineage, located in villianchoring to the maternal decidua and promotes their pro-liferation and survival. Moreover, Notch1 repressed syncytiali-zation and genes controlling self-renewal of fusogenic precursorsand induced an extravillous trophoblast progenitor-specific genesignature in these cells. The present study delineates Notch1 as afunctionally analyzed regulator promoting development of theextravillous trophoblast lineage in the human placenta.

ResultsNotch1 Is Specifically Expressed in Progenitors of the ExtravillousTrophoblast Lineage. To gain insights into Notch1 distribution,expression of the receptor was analyzed in placental villi andpurified trophoblast subtypes of early pregnancy (Fig. 1).

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 markproliferative, Notch1− CCTs or vCTBs underlying the syncytium (S). Nuclei were stained with DAPI. Representative images of cell columns and placental villifrom 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 replacedby rabbit monoclonal isotype IgG (mAB IgG). Arrowheads indicate clusters of Notch1+ vCTBs. (C) IF of different regions of an 11th-wk villous tree using Notch1and vimentin (VIM) antibodies. Images are representative for (1) the proximal region close to the chorionic plate, (2) the intermediate portion, and (3) thedistal part anchoring to the maternal decidua (n = 4). Picture at Left shows the Alcian blue-stained placental villus embedded in paraffin of which differentregions (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 andthe 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 meanvalues ± SD of Notch1 protein levels measured by densitometry in three (12th wk) and four (7th wk) different CTB pools. *P < 0.05.

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Immunofluorescence in tissue sections, obtained from 6th wk to7th wk of gestation, revealed that Notch1 specifically localized to asubset of proliferating cell nuclear antigen (PCNA)+ CTBs form-ing multilayered cell columns (Fig. 1A). Coimmunofluorescence ofNotch1 with cyclin A or phospho-Histone (p-Histone) H3, labelingtrophoblasts in S and M phase, respectively, also indicated thatNotch1 specifies cycling progenitors in the proximal cell columncoexpressing epidermal growth factor receptor (EGFR) (SIAppendix, Fig. S1 A and B). In contrast, distal human leukocyteantigen G (HLA-G)+ cell column trophoblasts (CCTs), differ-entiating toward EVTs, and the placental syncytium lacked Notch1expression. Notably, Notch1 was additionally expressed in clustersof single-row villous CTBs (vCTBs) of distal placental villi facingthe maternal decidua (Fig. 1A). Closer inspection of adjacent serialsections of a single villus revealed that the Notch1+ CTB clusterswere parts of developing cell columns (SI Appendix, Fig. S1C).Notch1 expression progressively increased as the columns becamemultilayered. At the 12th wk of pregnancy, trophoblast-specificNotch1 expression was exclusively detected in proximal CCTs ofanchoring villi (Fig. 1B and SI Appendix, Fig. S1A). However, itsexpression increased in the underlying stroma, compared withearlier weeks (Fig. 1 A and B and SI Appendix, Fig. S1A). Fur-thermore, localization of Notch1 was analyzed in different regionsof a villous tree at the 11th wk of gestation (Fig. 1C). In theproximal and intermediate portion, Notch1 was restricted to cellsof the villous core. In contrast, the distal region anchored to thedecidua additionally expressed Notch1 in progenitors of the in-vasive trophoblast lineage. Interestingly, Notch1+ CCTs of distalvilli 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 villiresiding in the intermediate region of the placenta. Besides itslocalization at the cell membrane, Notch1 was detected in nuclei ofEVT progenitors, suggesting receptor cleavage and activation ofcanonical Notch signaling in these cells (Fig. 1D). Between the 6thand 12th wk of pregnancy, Notch1 mRNA and protein expressiondecreased in primary CTB preparations, harboring both pro-liferative CTBs and differentiated EVTs (SI Appendix, Fig. S1 Eand F). Gestation- and differentiation-dependent down-regulationof 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 ofNotch1 toward the end of the first trimester could be associatedwith the rise in oxygen levels at the time when the placental–maternal circulation and hemotrophic nutrition of the embryo isestablished (2). In agreement with that assumption, Notch1 ex-pression and activity of a canonical Notch reporter increased inCTBs under hypoxic conditions (SI Appendix, Fig. S1 G and H).

Notch1 Promotes Cell Column Stability in Anchoring Villi and Survival ofIts Progenitors. For manipulation of Notch1 activity in first trimestervillous explant cultures, a specific Notch1-blocking antibody(Notch1-IgG1), inhibiting ADAM-mediated cleavage (25), wasused (Fig. 2). Features of the particular antibody were extensivelypretested in different trophoblast cell models (SI Appendix, Fig. S2).As previously mentioned (26), treatment with EDTA provokedtime-dependent accumulation of Notch1 intracellular domain(N1ICD) (SI Appendix, Fig. S2A), its nuclear recruitment (SI Ap-pendix, Fig. S2B), and induction of the canonical Notch1 targetHES1 (SI Appendix, Fig. S2C). Incubation with the Notch1-blockingantibody diminished EDTA-stimulated generation of N1ICD (SIAppendix, Fig. S2 D–F), as well as expression of the full-lengthNotch1 receptor upon long-term treatment (SI Appendix, Fig. S2G).Moreover, Notch1-IgG1 dose-dependently decreased luciferase ac-tivity of the canonical Notch reporter and impaired EDTA-stimu-lated HES1 expression (SI Appendix, Fig. S2H and I). Treatment ofvillous explants with the blocking antibody reduced Notch1 signalsin CCTs and diminished total Notch1 protein expression (Fig. 2 A

and B). Upon seeding onto collagen I, Notch1-IgG1 decreasednumbers of anchoring villi per explant, suggesting that either denovo formation and/or stability of preexisting cell columns areaffected (Fig. 2 C and D). Indeed, Western blotting and immu-nofluorescence showed that Notch1 inhibition increased expres-sion of the apoptotic markers cleaved caspase-3, cytokeratin 18neoepitope, and p53 in protein lysates and proximal CCTs ofvillous explants (Fig. 2 E–G). Induction of active caspase-3 wasalso detected upon siRNA-mediated gene silencing of Notch1 in

Fig. 2. Notch1 promotes survival of extravillous trophoblast progenitors in an-choring villi. Experiments were performed with placentae between the 6th and8th wk. (A) IHC analysis of Notch1 in CCTs after treatment of floating explantcultures with Notch1-IgG1 or ctrl-IgG1. Sections were stained with Notch1 anti-bodies (brown color) and hematoxylin (blue) to mark nuclear DNA. Representativecell columns of each 40 explants (derived from four different placentae) analyzedare shown. Stippled line demarcates the cell column from the underlying villousstroma (VS). (Scale bars, 50 μm.) (B) Western blot showing down-regulation of thefull-length Notch1 receptor (transmembrane intracellular domain, 120 kDa) in thepresence of Notch1-blocking antibody or control. A representative example(10 pooled explants per treatment and placenta, n = 4 placentae analyzed) isshown. (C) Representative pictures of collagen I-attached anchoring villi (arrows)after incubation with Notch1-blocking antibody. Explants (a total of 86 explantsper condition, derived from five different placentae) were pretreatedwith Notch1-IgG1 or controls for 12 h and seeded onto collagen I. (Scale bars, 500 μm.) (D) After36 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 cyto-keratin 18 neoepitope (K18-ne) or p53 IF in Notch1-IgG1–treated floating explantcultures. CC, cell column; VS, villous stroma. (F) Box plots depict median andinterquartile 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 andquantification of cleaved caspase-3 expression after Notch1 inhibition. For eachtreatment a total of 40 explants (10 pooled explants per placenta, n = 4) wereanalyzed. Mean values ± SD (n = 4) normalized to GAPDH are shown. *P < 0.05.

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these cultures (SI Appendix, Fig. S3A). Moreover, Notch1-IgG1treatment increased apoptosis in Notch1+ clusters of vCTBs of6th 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 Notch1levels were highest, suggesting that the receptor could, at leastpartly, counteract CPT-induced apoptosis. Notch1 was shown topromote survival by inhibiting degradation of X-linked inhibitor ofapoptosis (XIAP) (27). Stability of the latter, however, was notaffected upon silencing of Notch1 in CTBs (SI Appendix, Fig. S3F).

Notch1 Maintains Purified Cell Column Progenitors and Inhibits TheirDifferentiation into Extravillous Trophoblasts. To analyze the role ofNotch1 in purified progenitors, the two different proliferative CTBsubtypes, CCT and vCTB, were isolated from first trimester pla-centae using optimized purification protocols. Upon cultivation onfibronectin, vCTBs underwent cell fusion as indicated by the in-duction of human chorionic gonadotrophin β (CGβ) and suppres-sion of the CTB marker E-cadherin (SI Appendix, Fig. S4 A, C, andE). The EVT-specific gene HLA-G was not induced upon in vitrodifferentiation of these cells. In contrast, CCTs seeded on fibro-nectin up-regulated the EVT markers HLA-G and integrin α1(ITGA1), but not CGβ (SI Appendix, Fig. S4 B–D). In both pro-genitor subtypes, Notch1 was down-regulated upon in vitro differ-entiation (SI Appendix, Fig. S4 D–F). Subsequently, CCTs wereisolated from 9th- to 10th-wk placentae, yielding maximal numbersof these cells, and transfected with N1ICD and a mutant variant(N1ICD-ΔRAM), 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 ofCCTs, whereas N1ICD-ΔRAM localized to both cytoplasm andnuclei of cells (Fig. 3B). Overexpression of N1ICD supressedcleaved caspase-3, but increased cyclin A protein, cyclin D1 mRNA,as well as proliferation of CCTs, measured by EdU-labeling (Fig. 3C–E). On the contrary, N1ICD-ΔRAM did not provoke these ef-fects. Moreover, N1ICD expression prevented differentiation ofCCTs into EVTs, because it inhibited up-regulation of HLA-G

and maintained expression of hepatocyte growth factor activatorinhibitor type 1 (HAI-1), a marker of proliferative vCTBs andCCTs (Fig. 3F and SI Appendix, Fig. S5).

Notch1 Expression in Villous CTBs Induces Proliferation and Inhibits CellFusion. To further investigate the role of Notch1 in the placentalepithelium, N1ICD was overexpressed in vCTBs isolated from 9th-to 10th-wk placentae (Fig. 4). Compared with 7th to 8th wk ofgestation, Notch1 was largely absent from these cells (SI Appendix,Figs. S4F and S6). Similar to its effects in CCTs, N1ICD increasedproliferation and cyclin expression in vCTBs, although the effectswere more pronounced (Fig. 4 A–C and SI Appendix, Fig. S7 A andB). Again, N1ICD-ΔRAM neither changed expression of cell cyclemarkers nor proliferation. Additionally, N1ICD suppressed in vitrosyncytialization and differentiation-dependent induction of CGβ, butmaintained HAI-1 and E-cadherin expression in vCTBs (Fig. 4D–F).

Ectopic Notch1 Induces an Extravillous Trophoblast Progenitor-SpecificGene Signature in Fusogenic Precursors. As a prerequisite for theanalysis of Notch1 in trophoblast stemness, putative marker proteinsof trophoblast self-renewal were studied in placental sections andpurified progenitor cell types (Fig. 5). Whereas the transcriptionfactors p63 and TEA domain family member 4 (TEAD4) werepredominantly detected in vCTBs, VE-cadherin and myc weremainly expressed in proliferative CCTs (Fig. 5 A–C). Over-expression of N1ICD in vCTB purified from 9th- to 10th-wk pla-centae, lacking Notch1, induced CCT-specific myc and VE-cadherinexpression (Fig. 5 D–F and SI Appendix, Fig. S8). In contrast,ΔNp63 and TEAD4, controlling self-renewal of vCTBs and murinetrophoblast stem cells (28, 29), respectively, were suppressed(Fig. 5 D and F and SI Appendix, Fig. S8). In addition, N1ICDincreased expression of IFN regulatory factor 6 (IRF6), a negativeregulator of p63 (30) (Fig. 5 D and F and SI Appendix, Fig. S8).N1ICD binds to the myc and IRF6 gene in CTBs.Notch1 was recently shownto stimulate myc transcription in T-cell acute lymphoblastic leukemiacells by activating RBPJκ bound to cognate sequences in the prox-imal 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

Fig. 3. N1ICD increases proliferation and survival of purified CCTs and inhibits extravillous trophoblasts differentiation. For each preparation three to fivepooled placentae of the 9th–10th wk were used. (A) Schematic representation showing protein domains of full-length Notch1, FLAG-tagged N1ICD, andN1ICD-ΔRAM. (B) Notch1 IF detecting localization of N1ICD and its mutant variant after transfection into purified CCTs. (Scale bars, 50 μm.) (C) RepresentativeWestern 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 mutantN1ICD in CCTs. (F) Western blot showing inhibition of HLA-G expression in differentiating cell column progenitors upon N1ICD expression. Mean values ± SDnormalized to GAPDH are depicted (n = 3). *P < 0.05; ns, not significant compared with mock control.

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5′ flanking region (−5 kb) using Transfac Patch 1.0 identified twoputative 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 bpupon overexpression in vCTBs as well as with the distal NDME usedas a control (Fig. 5G and SI Appendix, Fig. S9). As shown (31),N1ICD specifically bound to the c1 element in that region (SI Ap-pendix, Fig. S9). Moreover, ChIP indicated that IRF6 is also a directtarget of N1ICD. The latter bound to two different RBPJκ cognatesequences at−3.6 kb and −2.4 kb in the IRF6 promoter (Fig. 5H andSI Appendix, Fig. S9), previously delineated in keratinocytes (33).Notch1 intracellular domain represses p63 through IRF6 in vCTBs. To ana-lyze whether Notch-dependent down-regulation of ΔNp63 requiresIRF6, vCTBs were transfected with N1ICD and siRNA againstIRF6. Silencing of the latter increased ΔNp63 levels, indicating adirect role in N1ICD-induced repression of p63 (Fig. 5I).

DiscussionFailures in trophoblast differentiation and function in a variety ofpregnancy disorders strongly warrant a better understanding of themolecular pathways controlling human placental development.However, experimental studies are hampered by ethical constraintsto obtain tissues from early human gestation as well as difficulties inestablishing self-renewing human trophoblast stem and progenitorcells from trophoblast isolates (34–36). As a consequence, alter-native models, such as bone morphogenetic protein (BMP)-treatedhuman ESCs (hESCs), have been developed, allowing in vitroformation of the trophoblast lineage (37, 38). Despite concernsregarding specificity of trophectoderm induction with BMP (37, 39,40), recent investigations using optimized culture conditions sug-gested that early stages of human trophoblast development couldbe 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 cellproperties from single blastomers of eight-cell embryos suggestedthat the trophectoderm cell fate could be initiated at very early stagesof pregnancy (43). In addition, trophoblast stem-like cells, expressingCdx2 and p63, were derived from BMP-induced hESCs generatingboth EVTs and STBs (42). However, it remains controversialwhether bipotential CTBs are maintained during the first trimesterof pregnancy (4). Whereas FGF4 was claimed to redirect fusogenicCTBs toward EVT differentiation (44), others showed that EVTprecursors isolated from 6th- to 10th-wk placentae were unable tosyncytialize, but spontaneously formed 20% HLA-G+ cells after5 d in culture (16). The latter study suggested that distinct tro-phoblast progenitors, committed to syncytialization and EVTformation, respectively, have developed in first trimester placen-tae. The present study confirms this assumption. Using sequentialtrypsin digestions of early placental tissues, removal of STB frag-ments by gradient centrifugation and immune depletion of dif-ferentiated EVTs, CTB progenitor subtypes with high purity wereobtained. Upon seeding onto fibronectin, vCTBs fused into STBs,but did not induce the EVT markers HLA-G or ITGA1. In con-trast, CCTs differentiated into EVTs lacking STB-specific CGβexpression. Notably, formation of STBs and EVTs occurred underidentical culture conditions and on the same matrix, suggestingthat differentiation is mainly driven by the intrinsic molecularprogram of vCTBs and CCTs. Because isolation of the two distinctprogenitors is based on the consecutive digestions with differenttrypsin concentrations, some marginal cross-contamination cannotbe avoided (SI Appendix, Fig. S4C). However, the present protocolwill allow for further genome-wide gene expression analyses ofthese CTB subtypes. Thereby, unique surface markers, suitable foradditional 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

Fig. 4. N1ICD increases proliferation and inhibits cell fusion of purified vCTBs. FLAG-tagged N1ICD or N1ICD-ΔRAM were overexpressed in vCTBs isolated fromthree 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 ofmitosis. 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 itsmutant. AU, arbitrary units. (D) Western blot and quantification (n = 5) of markers of undifferentiated (E-cadherin, HAI-1) and differentiated (CGβ) vCTBs afterN1ICD 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 IQRvalues of the percentage of multinucleated cells (n = 3). *P < 0.05; ns, not significant.

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development are scarce. Descriptive analyses in first trimestertissues and trophoblast cell models have been performed to gaininsights into the expression patterns and epigenetic profiles of someof these genes (37, 40, 48, 49). However, few of them have beenfunctionally examined by using choriocarcinoma cells as surrogatefor human primary trophoblasts (28, 50). To extend our presentknowledge, we herein analyzed expression of key regulators in thetwo purified CTB progenitor subtypes and performed functionalanalyses in these cells. Immunofluorescence and quantitative PCR(qPCR) indicated that TEAD4, a crucial transcription factor inmurine trophectoderm specification and trophoblast progenitorself-renewal (29, 51), is also present in human vCTBs but largelyabsent from proximal Notch1+ CCTs. Akin to that finding, p63, as-sociated with the CTB progenitor phenotype (28), was predominantly

expressed in vCTBs. However, the stemness markers myc, mark-edly decreasing during the first trimester of pregnancy (52), andVE-cadherin, were mainly detected in CCTs. Besides its classicalrole 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 theintermediate region of the cell column containing proliferativeCCT (Fig. 5B), as also previously shown (54). Its expression partlyoverlaps with Notch1, exclusively found in proximal EVT pro-genitors. Cdx2, the central transcriptional activator factor inmurine trophectoderm development (55), has been detected invCTBs of early placental tissues and rapidly declines toward theend of the first trimester (42, 48, 49). The present study also de-tected Cdx2 in vCTBs of 6th- wk placentae (SI Appendix, Fig.

Fig. 5. Expression pattern of markers of cytotrophoblast self-renewal and their regulation by N1ICD and IRF6. Representative pictures of first trimesterplacentae (A and B) showing coimmunofluorescence of TEAD4, ΔNp63, Notch1, and myc with VIM in serial sections. VE-cadherin expression in trophoblasts ofthe proximal cell column (CCT) partly overlapped with Notch1. Nuclei were counterstained with DAPI. (Scale bars, 50 μm.) (C) qPCR detecting mRNA ex-pression 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) ΔNp63, myc, and IRF6 and (E) VE-cadherin protein expression in N1ICD or N1ICD-ΔRAM transfected vCTBs. GAPDH was used as loading control. (F) Quantification of Western blots. Mean values ± SD (n = 4 vCTB pools, each consisting ofthree to five 9th- to 10th-wk placentae) normalized to GAPDH are depicted. *P < 0.05; ns, not significant compared with mock control. Interaction of N1ICDwith genomic regions in the (G) myc and (H) IRF6 gene. N1ICD and N1ICD-ΔRAMwere 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 previouslyidentified Notch-dependent myc enhancer (NDME) at +1.4 Mb. (H) Representation of the proximal IRF6 promoter region delineating RBPJκ cognate sequencesat −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 = 11placentae, 6th–8th wk) *P < 0.05; (I) Representative Western blot showing elevation of ΔNp63 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.

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S1D). However, the factor was absent from both vCTBs and CCTsof distal villi located at the basal side of the placenta. Instead,CCTs in this region were positive for Notch1, a key regulator ofstem cell niches and cell lineage determination (23). Based onthese expression patterns, we hypothesize that putative Cdx2+

stem cell-like trophoblasts disappear in distally developing regionsof the placenta, at a time when extensive formation of villi, an-choring to the maternal decidua, takes place. Instead, committedprogenitors for cell fusion and EVT differentiation arise, eachexpressing a unique set of self-renewal genes.Development of stable anchoring villi is a prerequisite for a

continuous supply with EVTs invading and remodelling maternaluterine tissues. However, mechanisms controlling establishmentand maintenance of cell columns, replenishing the pool of out-growing trophoblasts, have not been elucidated. Herein, we iden-tified Notch1 as a critical regulator of these processes. Notch1 wasdetected in proliferative/mitotic CCTs of the proximal cell column,thereby defining the EVT progenitor cell niche. Despite its shorthalf-life of ∼1.8 h (56), nuclear N1ICD was occasionally observed inproximal CCTs, suggesting activation of Notch1 cleavage. However,cyclin A and p-Histone H3 were also expressed in neighboringNotch1− CCTs, suggesting that Notch1+ precursors could give riseto committed transient amplifying (TA)-like cells. The latter furtherdevelop into distal HLA-G+ CCTs progressing toward EVTs. TAcells, 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 homeostasisof 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. S1E and F) and is absent from second trimester trophoblasts (19),indicating that the number of progenitor cells diminishes at thetime of oxygen transition. By binding of NICD to HIF-1α, Notchsignaling synergizes with hypoxia to keep cells in an undif-ferentiated state (58, 59). Hence, we speculated that a low-oxygen environment could support mRNA expression and proteinstability of Notch1, as shown in other cell types (59–61), andthereby maintain the proliferative capacity of CCT precursors. In-deed, the present data indicate that hypoxia increases Notch1 aswell as activity of a canonical Notch reporter. After establishmentof the placental–maternal circulation, declining Notch1 levels mightaffect survival and/or proliferation of these cells, thereby slowingdown continuous growth of cell columns and EVT differentiation.On the other hand, Notch1 was detected in EVT progenitorsof 12th-wk placentae, at the time when the receptor was completelyabsent from vCTBs. Maintenance of Notch1+ CCTs in the presence

of increasing oxygen levels could be necessary for on-going EVTformation during the period of spiral artery remodelling.Notch signaling in stem cells is highly complex and exerts dif-

ferent effects in tissues and organs (23, 62). Accordingly, Notchwas shown to promote or inhibit self-renewal, differentiation, andsurvival, depending on the specific cellular context. Previousanalyses of human placental tissues suggested that Notch sig-naling could be predominantly associated with trophoblast pro-genitor cell function (20). Although Notch2 has its main role ininvasive trophoblasts (19, 24), down-regulation of HES1 andRBPJκ reporter activity during EVT formation indicated thatcanonical Notch signaling is associated with undifferentiatedtrophoblasts (20, 21). Herein, functional analyses of Notch1 indifferent primary trophoblast cell models corroborate this evi-dence. Blocking or silencing of Notch1 increased apoptosis invillous explant cultures and isolated CTBs. In contrast, over-expression of N1ICD enhanced proliferation and cyclin expres-sion but suppressed cleaved caspase-3 in purified vCTBs orCCTs. Notch-dependent survival operates through differ-ent mechanisms involving mammalian target of rapamycin(mTOR)–Akt-mediated suppression of p53 or activation of in-hibitors of apoptosis such as XIAP (27, 63). Expression of thelatter was not affected by Notch1 in trophoblasts. However, in-hibition of the receptor induced accumulation of nuclear p53.Notably, silencing of RBPJκ in villous explant cultures did notprovoke apoptosis of CCTs (21), suggesting that N1ICD-mediatedsurvival could be an RBPJκ-independent function, as recentlyshown (64). Different than Notch1, disruption of RBPJκ weaklyincreased proliferation in explant cultures and, as a consequenceEVT formation (21). Several effects could account for this dis-crepancy. Abolishing RBPJκ not only impairs Notch1 but alsoactivities of Notch3 and Notch4, which are abundantly expressedin CTBs (19, 20). Besides binding to RBPJκ, N1ICD interactswith a range of different transcription factors (65), of whichHIF1α, NFκB, GABPA, and ZNF143 are also detected in ourpreviously 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 sys-

tems, thereby preserving stemness (62). In agreement with thatfact, N1ICD maintained expression of genes associated with un-differentiated vCTBs and CCTs, E-cadherin and HAI-1, andsuppressed STB formation of fusogenic precursors as well asdifferentiation-dependent induction of CGβ. Moreover, N1ICDalso inhibited differentiation of CCTs into HLA-G+ EVTs. Im-munofluorescence of 5th- to 7th-wk placental tissues revealed thatthe receptor specifically appears in vCTB clusters of distal placentalvilli containing adjacent cell islands with Notch1+ CCTs. Serialsectioning of these villi revealed that single-row Notch1+ vCTBswere parts of developing cell columns. At the 10th–12th wk ofgestation, trophoblast-specific expression of Notch1 was restrictedto proximal CCTs of villi anchoring to the maternal decidua.Therefore, we speculated that Notch1 could initiate the extra-villous trophoblast lineage in distal regions of the placenta, lateron forming the placental basal plate. This view was supported bydata obtained from villous explant cultures, demonstrating thatNotch1 inhibition decreased numbers of outgrowing cell columns.However, Notch1 blocking also provoked CCT apoptosis, asshown above, and small, preformed cell columns cannot be dis-cerned 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 ana-lyzed for markers of vCTB or CCT stemness, defined in the presentstudy. Of note, N1ICD suppressed vCTB-specific p63 and TEAD4expression but induced the CCT markers VE-cadherin and myc.Similar to other cells (31), the latter could be a direct target ofactive Notch1 in CTBs. ChIP revealed that N1ICD binds to the

Fig. 6. Model system depicting the presumptive role of Notch1 in humantrophoblast development. N1ICD programs vCTBs into CCTs, expressing thestemness markers myc and VE-cadherin and prevents EVT differentiation bymaintaining proliferation and survival of these cells. N1ICD also suppressesTEAD4 and p63, the latter by inducing its repressor IRF6, thereby alleviatingself-renewal and cell fusion of vCTBs.

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3′ NDME of the myc gene, previously shown to promote geneexpression (31), as well as to a newly identified RBPJκ motifpresent at −3 kb in the proximal promoter region. In contrast tomyc, N1ICD was recently shown to indirectly suppress p63.N1ICD binds to the IRF6 gene in keratinocytes and activates itsexpression (33). In turn, IRF6 promotes proteasomal degradationof p63 (30). An analogous mechanism could regulate p63 invCTBs, because IRF6 rapidly increased after N1ICD expression.Also, ChIP analyses showed that N1ICD interacts with two RBPJκcognate sequences, recently identified in the proximal IRF6 pro-moter region (33). Moreover, silencing of IRF6 in vCTBs im-paired N1ICD-dependent suppression of ΔNp63. Compared withp63 mRNA, loss of ΔNp63 protein was more pronounced in thepresence 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 ofself-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, indicatingthat it might not be a direct Notch1 target. Notably, immunoflu-orescence revealed that the particular adhesion molecule specifi-cally localizes to proliferative CCTs in the intermediate region ofthe cell column (Fig. 5B) (12). Hence, N1ICD-induced VE-cad-herin expression might suggest that cell column progenitors havefurther developed into TA-like cells. Finally, we speculate thatNotch1-dependent expression of myc and VE-cadherin could besufficient to establish EVT progenitors from Notch1− CTB pre-cursors. On the other hand, suppression of cell fusion and down-regulation of p63/TEAD4 by N1ICD could negatively affect vCTBself-renewal and thereby trigger CCT formation and EVT differ-entiation. Along those lines, silencing of p63 in JEG-3 chorio-carcinoma cells was shown to increase migration, a characteristicfeature 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 itsrestricted expression in the proximal region of developing cell col-umns, functional studies in primary trophoblast models have beenconducted, indicating a crucial role of the receptor in CCT pro-liferation, survival, and differentiation. Notch1 induced markers ofEVT progenitors in Notch1− vCTBs, but suppressed genes controllingvCTB self-renewal (Fig. 6). Further studies are required to delineateNotch ligands involved in this process as well as the mechanism(s)initiating Notch1 expression in the developing placenta.

Materials and MethodsTissue Collection. Placental tissues (6th–12th wk of gestation) were obtainedfrom elective pregnancy terminations. Utilization of tissues and all experi-mental procedures were approved by the Medical University of Viennaethics boards. Written informed consent was obtained from all subjects.

Immunohistochemistry of Paraffin-Embedded Tissue. Serial sections of paraffin-embedded villous explantswere analyzedby immunohistochemistry (IHC) usingDako EnVision+ System-HRP (DAKO, K4011) as instructed by themanufacturer.Sections were incubated with primary antibodies (listed in SI Appendix, TableS1) overnight at 4 °C and nuclei were counterstained with hematoxylin(Merck). Sections were digitally photographed using an Olympus BX50 mi-croscope and CellP software.

Immunofluorescence of Paraffin-Embedded Tissue. Placental tissue was fixed in7.5% (wt/vol) formaldehyde and embedded in paraffin. To analyze Notch1localization in different regions of placental villi (stem villus, intermediate vil-lus, end villus), tissues were stained with Alcian blue solution and carefullyplaced in embedding cassettes. Serial sections (3 μm) of paraffin-embeddedplacental tissue or villous explant cultures were analyzed by immunofluores-cence (IF) as described elsewhere (20). Briefly, sections were deparaffinized inXylol and rehydrated. Antigen retrieval was performed using 1× PT modulebuffer 1 (Thermo Scientific) for 35 min at 93 °C using a KOS microwave his-tostation (Milestone). Sections were incubated with primary antibodies (listedin SI Appendix, Table S1) overnight at 4 °C. Afterward, slides were washedthree 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 wereanalyzed by fluorescence microscopy (Olympus BX50, CellP software) anddigitally 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) andincubated 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/mLof secondary antibodies (listed in SI Appendix, Table S1) and nuclei werestained with DAPI. Slides were analyzed by fluorescence microscopy (OlympusBX50, CellP software) and digitally photographed.

Isolation and Cultivation of Placental Primary CTBs. CTBs were isolated by adaptedenzymatic dispersion andPercoll density gradient centrifugation [10–70% (vol/vol);GE Healthcare] of pooled first trimester placentae (n = 3–6 per isolation) asdescribed (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) allowingfor adherence of contaminating stromal cells. Nonadherent trophoblasts werecollected and CTBs were either seeded in culture medium onto fibronectin-coated (20 μg/mL; Millipore) dishes (2.5 × 105 cells per square centimeter) orfurther purified using positive selection with EGFR-phycoerythrin (PE) or HLA-G-PE antibodies (listed in SI Appendix, Table S1) and anti-PE MicroBeads (MACSMiltenyi Biotec) according to the manufacturer’s instructions. The contamina-tion with stromal cells was routinely tested by IF with antibodies detectingcytokeratin 7 (trophoblast cells) and vimentin (nontrophoblast cells). Vimentin+

cells were fewer than 1%. The purity of isolated EGFR+ and HLA-G+ CTB pop-ulations was verified using qPCR and Western blotting.

To verify specificityof theNotch1blocking, CTBswereallowed toattach for2h,washedwith prewarmed PBS, and preincubatedwith culturemedium containingeither 0.5 μg/mL ctrl-IgG1 (ctrl-IgG1 ABIN376845; Antibodies-Online) or 0.5 μg/mLNotch1-IgG1 (Notch1-IgG1, Genentech) antibodies for 1 h. Subsequently, 5 mMEDTA was added for another 30 min, inducing receptor cleavage/generation ofN1ICD. For long-term Notch1 inhibition, CTBs were seeded for 2 h and antibodieswere added for an additional 48 h. To analyze prosurvival effects of Notch1,CTBs were seeded for 2 h, washed, and incubated with culture medium con-taining either DMSO (ctrl) or 1 μM CPT for 1, 2, 4, 6, and 24 h. To verify whetherthe Notch1 could inhibit apoptosis via XIAP stabilization, CTBs were pre-incubated with a mixture of four different siRNAs against Notch1 (si-Notch1;L-007771–00-0005, ON-TARGETplus SMARTpools, Dharmacon-Thermo Fisher Sci-entific) or nontargeting siRNA (si-ctrl, D-001810-10-20) overnight and sub-sequently treated with DMSO (−) or 1 μM CPT (+) for 24 h.

Isolation and Cultivation of Purified Primary vCTBs and CCTs. CCT and vCTB cellpopulations were isolated by two consecutive digestion steps followed by Percolldensity gradient centrifugation. Precisely, placental tissue (6th–8thand9th–10thwkof gestation, n = 3–5 per isolation) was minced into small pieces (1–3 mm). Forisolation 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 (BDBiosciences). To isolate vCTB cells, a second digestion step of the remaining tissuewas performed with 0.25% trypsin and 12.5 mg/mL DNase I for 30 min at 37 °Cand processed as described above. Digestion solutions containing either CCTs orvCTBs were each layered on top of a Percoll gradient [10–70% (vol/vol)] and cellsbetween 35 and 50% of the Percoll layer were collected. ContaminatingRBCs 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 roomtemperature and subsequently washed with 1× HBSS. Contaminatingstromal cells were depleted from CCT preparations as mentioned above.Finally, differentiated CCTs/EVTs were removed from proliferative CCTs orvCTBs, using HLA-G-PE antibodies (Exbio) and anti-PE MicroBeads (MACSMiltenyi Biotec) as instructed by the manufacturer. CCT and vCTB cellswere seeded onto fibronectin-coated dishes at a density of 2.5 × 105 cellsper square centimeter and 3.25 × 105 cells per square centimeter, respectively.CCT and vCTB cells were harvested after 24 h (undifferentiated cells) and be-tween 72 and 120 h (differentiated cells). Cells were either fixed for IF analyses orsnap frozen for qPCR and Western blotting.

Notch1 Plasmids and CTB Transfection. Full-length human N1ICD (residues 1762–2556) and a N1ICD-ΔRAMdomain (residues 1877–2556) were a generous gift ofC. O. Joe, Department of Biological Sciences, Korea Advanced Institute of Sci-ence and Technology, Daejeon, South Korea. Notch1 variants are cloned intopFLAG-CMV-2 (Sigma) as described elsewhere (70). Empty pFLAG-CMV-2 servedas negative control (mock-CTRL). Isolated CCTs and vCTBs were transfectedwith the 4D-Nucleofector (program EO-100, Lonza) using the AMAXA SG CellLine Kit according to the manufacturer’s instructions. Subsequently, cells were

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seeded at a density of 3.5 × 105 cells per square centimeter. Transfection with apmaxGFP (Lonza) revealed an average transfection efficiency of 44 ± 18%.Transfected trophoblasts were incubated up to 96 h at 37 °C. To evaluate lo-calization of transfected N1ICD and N1ICD-ΔRAM, IF was performed as de-scribed above. For down-regulation of IRF6 in N1ICD-overexpressing CTBs,siRNA-mediated IRF6 gene silencing was performed using a mixture of fourdifferent 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 presenceof si-ctrl or si-IRF6 and incubated for 24 h.

Proliferation Assays. Purified CCTs and vCTBs were transfected withmock-CTRL,N1ICD, or N1ICD-ΔRAM, seeded onto fibronectin-coated 48-well dishes, andincubated for 24 h. Afterward, 10 μM 5-ethynyl-2′-deoxyuridine (EdU)(EdU-Click 488, BaseClick) was added for 4 h. Subsequently, cells were fixedand EdU was detected according to the manufacturer’s instructions. Nucleiwere stained with DAPI (Roche). Cells were digitally photographed (sevenpictures 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 explantcultures were performed as described elsewhere (71). In detail, pieces ofvillous tissues (5–6 mm) were dissected from the placental basal plate (7th–8th wk of gestation), and further divided into two equal parts. To evaluateefficiency to Notch1-blocking antibodies, villous explants were treated witheither 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, therebyinhibiting conformational changes, ADAM-mediated cleavage, and N1ICD-dependent activation of canonical signaling (25). Subsequently, 5 mM EDTAwas added for up to 60 min and explants were homogenized to isolateprotein lysates. For outgrowth analyses, floating explant pairs were sup-plemented with 0.5 μg/mL ctrl-IgG1 or 0.5 μg/mL Notch1-IgG1 and keptovernight in explant culture medium (DMEM/Ham’s F-12, 0.05 mg/mL gen-tamicin), respectively. Subsequently, explants were seeded onto rat tail col-lagen I (attachment for 4 h) and subsequently covered with mediumcontaining either 0.5 μg/mL ctrl-IgG1 or 0.5 μg/mL Notch1-IgG1. After another24 h, explants were digitally photographed and differences in anchorageand outgrowth were evaluated using the EVOS FL Color Imaging System. Forlong-term antibody- or siRNA-mediated Notch1 inhibition, explants (7th–8thwk of gestation) were incubated for 48 h in culture medium supplementedwith 0.5 μg/mL Notch1-IgG1 or Notch1 siRNA. Next, explants were eitherfixed with 7.5% (wt/vol) formaldehyde and embedded in paraffin or ho-mogenized for Western blotting as described below.

Cultivation and Luciferase Reporter Assay in Trophoblastic SGHPL-5 Cells and PrimaryCTBs. 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. Inhibitionof Notch1 cleavage was performed with ctrl-IgG1 or Notch1-IgG1 as mentionedabove. For detection of canonical Notch activity subconfluent SGHPL-5 cells werecotransfected with 2 μg/mL of a luciferase reporter containing four RBPJκ bindingsites (mutant or wild-type plasmids) and 0.5 μg/mL pCMV–β-galactosidase (CMV-βGal; normalization control) using Lipofectamine 2000 (Invitrogen) as recentlydescribed (20). Primary CTBs isolated from pooled 6th-to 8th-wk placentae weretransfected with reporter plasmids using the AMAXA system asmentioned 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 incubatedunder 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. Luciferaseactivity and β-galactosidase activity were determined as previously published (72).

Western Blotting. Whole-cell lysates were prepared using standard protocols asrecently described (72). Villous explants were additionally homogenized for 20 sat 3,640 × g with a Precellys 24 (PeqLab). Nuclear extracts were isolated usingthe 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 primaryantibodies (SI Appendix, Table S1). Subsequently, filters were washed andincubated for 1 h with HRP-conjugated secondary antibodies (SI Appendix,Table S1). Signals were developed using ECL Prime Detection Kit (GE Health-care) 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 per-formed as indicated by the manufacturers. Villous explants were homoge-nized with the Precellys 24 (PeqLab) before RNA isolation. qPCR analyses (induplicates) were performed using the 7500 Fast Real-time PCR System (Ap-plied Biosystems, ABI) as described (73). The following TaqMan Gene Ex-pression 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 innuMIXqPCR MasterMix Probe (Biometra), and 0.2 μL ROX Reference Dye (Invi-trogen) were used per sample. Signals (ΔCt) were normalized to TATA-boxbinding protein (TBP) (ABI, 4333769F). Relative expression levels were de-termined 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 themanufacturer. 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 minwith 2 mM disuccinimidyl glutarate (DSG) and 1% formaldehyde (15 min).After nuclei preparation, chromatin digestion, and sonication, purified chro-matin lysates were incubated either with Notch1 antibody (1 μg, Cell Signaling,3608), normal rabbit IgG (negative control) (1 μg; Cell Signaling, 2729) or His-tone H3 XP rabbit mAb (positive control) (1 μg; Cell Signaling, 4620) overnightat 4 °C. Immunoprecipitated chromatin was captured with ChIP-Grade ProteinG magnetic beads and eluted. Purified DNA was assessed by semi-qPCR usingTaq Polymerase (Fermentas, EP0402). PCR conditions were as follows: 5 min at96 °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 PROMprimers were designed to spanthe region harboring a putative RBPJκ DNA binding motif (CTGCGGGAA)identified by Transfac Patch 1.0 at −3.060 bp, which differs by one nucleotidefrom 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): forward5′-ATGAGGTCAAGCTGGACCTAC-3′ and reverse 5′-TGACGGTGTCTGATCACTTA-3′;MYC NDME-c1 (200 bp): forward 5′-GCTGCCACATGCTGATGAAC-3′ and reverse5′-CCAGGTAGGGGCATTACGTC-3′; MYC NDME-c2 (174 bp): forward 5′-GAG-GCCCCCATTCATTACCC-3′ and reverse 5′-GCAGTTCTTCCTACGCTGGT-3′; IRF6PROM-1: forward 5′-ACCCTCCCAGCTTGAGTTTT-3′ and reverse 5′-AAACCCCA-GTGGCATACAAG-3′ (142 bp); IRF6 PROM-2: forward 5′-TCAATGGAGGGCAA-AATGAT-3′ and reverse 5′-ACGCCTCATCTGCTTGATCT-3′ (162 bp); humanRPL30 (control) (Cell Signaling, 7014). PCR products were analyzed on1.5% (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 exam-ined with Kolmogorov–Smirnov and Levene tests, respectively. Statistical analysisof data between two means was performed with Student’s t test or Mann–Whitney u test using SPSS 14. Comparisons of multiple groups were evaluatedwith one-way ANOVA and appropriate post hoc tests or Kruskal–Wallis testsfollowed by pairwise Mann–Whitney u tests and Shaffer’s correction. A P value of<0.05 was considered statistically significant. Unless otherwise noted, all experi-ments were conducted in duplicates and replicated at least three times.

ACKNOWLEDGMENTS. We thank G. S. Whitley (St. George’s University ofLondon) for providing SGHPL-5 cells and C. Siebel (Genentech) for providingthe Notch1 blocking antibody. This study was supported by the AustrianScience Fund (Grant P-28417-B30).

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Haider et al. PNAS | Published online November 14, 2016 | E7719

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