Notch1 controls development of the extravillous lineage in the human

Sandra 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, 1150 Vienna, 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 epithelial adequate blood flow to the placenta (5, 6). Failures in is crucial for a successful pregnancy. Besides fusing into and artery remodelling have been associated with a variety of a multinuclear syncytium, the exchange surface between mother and pregnancy diseases, such as miscarriage, preeclampsia, fetal growth fetus, progenitors develop into extravillous trophoblasts invading the restriction, and preterm labor (7–10). Besides unfavorable immu- maternal uterus and its spiral arteries. Migration into these vessels nological interactions of EVTs with uterine natural killer (uNK) cells promotes remodelling and, as a consequence, adaption of blood flow (11), abnormal placental development and trophoblast differentia- to the fetal–placental unit. Defects in remodelling and trophoblast tion are thought to contribute to the pathogenesis of gestational differentiation are associated with severe gestational diseases, such disorders. Indeed, CTBs isolated from preeclamptic placentae failed as preeclampsia. However, mechanisms controlling human tropho- to appropriately differentiate into the invasive lineage in vitro and blast development are largely unknown. Herein, we show that Notch1 expressed an antimigratory gene signature (12, 13). is one such critical regulator, programming primary trophoblasts into However, our knowledge about human placentation and tropho- progenitors of the invasive differentiation pathway. At the 12th wk of blast development is only scarce. Bipotential trophoblast progenitor gestation, Notch1 is exclusively detected in precursors of the extra- cells have been derived from the chorionic differentiating villous trophoblast lineage, forming cell columns anchored to the into EVTs and STBs (14, 15), whereas others identified a specific uterine stroma. At the 6th wk, Notch1 is additionally expressed in precursor of the EVT lineage in villous explant cultures (16). Placental clusters of villous trophoblasts underlying the syncytium, suggesting structures, trophoblast cell types, and expression patterns of key reg- that the receptor initiates the invasive differentiation program in dis- ulatory transcription factors diverge between mouse and man, thereby tal regions of the developing placental epithelium. Manipulation of hindering comparison of putative regulatory mechanisms (17). Al- Notch1 in primary trophoblast models demonstrated that the receptor though different transcriptional activators promoting or inhibiting EVT promotes proliferation and survival of extravillous trophoblast pro- motility have been described (18), it is unknown which factors govern genitors. Notch1 intracellular domain induced genes associated with − EVT differentiation. Likewise, how regions of column formation are stemness of cell columns, myc and VE-cadherin, in Notch1 fusogenic precursors, and bound to the myc promoter and enhancer region at specified and maintained within developing villi remains elusive. RBPJκ cognate sequences. In contrast, Notch1 repressed syncytializa- Recent evidence suggested that the developmental Notch path- tion and expression of TEAD4 and p63, two regulators controlling self- way could be critically involved in human trophoblast function and differentiation (19–21). Canonical Notch signaling is activated upon renewal of villous . Our results revealed Notch1 as a – key factor promoting development of progenitors of the extravillous direct cell cell contact involving binding of membrane-anchored trophoblast lineage in the human placenta. ligands, the Serrate-like ligands (Jagged1 and 2), and the Delta- like ligands (DLL1, 3, and 4) to the different Notch receptors – human placenta | trophoblast progenitors | Notch1 | extravillous (Notch1 4) (22). After two proteolytic cleavage steps, performed by trophoblast | cell fusion Significance ifferentiation processes of the human placenta are a pre- Drequisite for fetal development and successful pregnancy out- Progenitor trophoblast cells of the human placenta either fuse to come. Shortly after implantation, stem cells of the trophectoderm form a syncytium or develop into invasive trophoblasts invading surrounding the blastocyst give rise to the primitive syncytium by cell the maternal uterus. However, regulatory pathways controlling fusion as well as to proliferative cytotrophoblasts (CTBs) forming their development and distinct differentiation programs are poorly primary placental villi (1). Breaking through the multinuclear understood. In the present study, we demonstrate that Notch1 is a structures, these villi contact the maternal , the endometrium critical regulator of early pregnancy, promoting development of of pregnancy, and expand laterally to form the so-called tropho- the invasive, extravillous trophoblast lineage and survival of its blastic shell. The latter encircles the embryo and protects it from progenitors. In vivo, Notch1 is detected in extravillous trophoblast oxidative damage during early gestation (2). As pregnancy proceeds, progenitors and clusters of villous trophoblast initiating the in- placental villi undergo extensive remodelling involving branching vasive differentiation program. In vitro, Notch1 repressed genes morphogenesis and transformation into secondary and finally involved in self-renewal of fusogenic precursors, but induced genes specifically expressed by extravillous trophoblast progeni- tertiary villi by migration of mesenchymal cells and vascularization, tors. Our data delineate Notch1 as a key regulator promoting respectively. At this stage, two types of villi can be discerned, floating development of the human extravillous trophoblast lineage. and anchoring villi. Floating villi, which are bathed in maternal blood – after establishment of the fetal maternal circulation, are necessary Author contributions: S.H. and M.K. designed research; S.H. and G.M. performed research; C.F. for hormone production and nutrient and oxygen transport to the contributed new reagents/analytic tools; L.S. and J.P. analyzed data; and M.K. wrote the paper. developing fetus (3). The outermost epithelial surface of these villi, The authors declare no conflict of interest. the multinuclear syncytium, also termed (STB), This article is a PNAS Direct Submission. is generated by cell fusion of underlying CTB progenitors (4). On the Freely available online through the PNAS open access option. other hand, anchoring villi attached to the decidua form proliferative 1 To whom correspondence should be addressed. Email: [email protected]. cell columns giving rise to differentiated, extravillous trophoblasts at. (EVTs). The latter deeply migrate into uterine tissue and the ma- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ternal spiral arteries, provoking vessel remodelling and adaptation of 1073/pnas.1612335113/-/DCSupplemental.

E7710–E7719 | PNAS | Published online November 14, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1612335113 Downloaded by guest on September 29, 2021 members of a disintegrin and metalloproteinase (ADAM) family Therefore, we herein analyzed the specific role of Notch1 in 6th- PNAS PLUS and γ-secretase, the Notch intracellular domain (NICD) is released to 12th-wk human placentae using different primary trophoblast into the cytoplasm. Subsequently, NICD translocates to the nucleus cell models. Our data show that Notch1 is specifically expressed by and functions as a coactivator of the transcription factor recom- progenitors of the extravillous trophoblast lineage, located in villi bination signal binding protein for Ig kappa J region (RBPJκ) anchoring to the maternal decidua and promotes their pro- controlling numerous biological processes such as stem cell liferation and survival. Moreover, Notch1 repressed syncytiali- maintenance, cell lineage determination, and differentiation (23). zation and genes controlling self-renewal of fusogenic precursors Human placentae express Notch receptors and their ligands in a and induced an extravillous trophoblast progenitor-specific gene cell-specific manner (19, 20). Notch2 is predominantly detected in signature in these cells. The present study delineates Notch1 as a different EVT subtypes, and inhibition of Notch2 affected tro- functionally analyzed regulator promoting development of the phoblast cell migration (24). In analogy, conditional deletion of extravillous trophoblast lineage in the human placenta. Notch2 in murine trophoblast progenitors impaired endovascular invasion and placental perfusion (19). In contrast to that, Notch1, Results 3, and 4 were shown to be expressed by proliferative CTBs of first Notch1 Is Specifically Expressed in Progenitors of the Extravillous trimester placentae (20). Interestingly, Notch1 is absent from Trophoblast Lineage. To gain insights into Notch1 distribution, second trimester placental tissues (19), suggesting a role of the expression of the receptor was analyzed in placental villi and receptor in early trophoblast development and function. purified trophoblast subtypes of early pregnancy (Fig. 1). BIOLOGY DEVELOPMENTAL

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.

Haider et al. PNAS | Published online November 14, 2016 | E7711 Downloaded by guest on September 29, 2021 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 form- ing 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.S1A and B). In contrast, distal human leukocyte + antigen G (HLA-G) cell column trophoblasts (CCTs), differ- entiating 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). Fur- thermore, 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 in- + vasive 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 Fig. 2. Notch1 promotes survival of extravillous trophoblast progenitors in an- decreased in primary CTB preparations, harboring both pro- choring villi. Experiments were performed with placentae between the 6th and liferative CTBs and differentiated EVTs (SI Appendix,Fig.S1E 8th wk. (A) IHC analysis of Notch1 in CCTs after treatment of floating explant and F). Gestation- and differentiation-dependent down-regulation cultures with Notch1-IgG1 or ctrl-IgG1. Sections were stained with Notch1 anti- of Notch1 was also observed in isolated vCTBs/CCTs and EVTs, bodies (brown color) and hematoxylin (blue) to mark nuclear DNA. Representative cellcolumnsofeach40explants(derived from four different placentae) analyzed which were immunopurified with EGFR and HLA-G antibodies, E SI Appendix B are shown. Stippled line demarcates the cell column from the underlying villous respectively (Fig. 1 and ,Fig.S1 ). The decline of stroma (VS). (Scale bars, 50 μm.) (B) Western blot showing down-regulation of the Notch1 toward the end of the first trimester could be associated full-length Notch1 receptor (transmembrane intracellular domain, 120 kDa) in the with the rise in oxygen levels at the time when the placental– presence of Notch1-blocking antibody or control. A representative example maternal circulation and hemotrophic nutrition of the embryo is (10 pooled explants per treatment and placenta, n = 4 placentae analyzed) is established (2). In agreement with that assumption, Notch1 ex- shown. (C) Representative pictures of collagen I-attached anchoring villi (arrows) pression and activity of a canonical Notch reporter increased in after incubation with Notch1-blocking antibody. Explants (a total of 86 explants CTBs under hypoxic conditions (SI Appendix,Fig.S1G and H). 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 Notch1 Promotes Cell Column Stability in Anchoring Villi and Survival of (n = 5). *P < 0.05. (E) Representative images (Scale bar, 50 μm.) showing cyto- Its Progenitors. For manipulation of Notch1 activity in first trimester keratin 18 neoepitope (K18-ne) or p53 IF in Notch1-IgG1–treated floating explant villous explant cultures, a specific Notch1-blocking antibody cultures. CC, cell column; VS, villous stroma. (F) Box plots depict median and + + (Notch1-IgG1), inhibiting ADAM-mediated cleavage (25), was interquartile range (IQR) values of K18-ne or p53 CCTs after Notch1- or ctrl-IgG1 used (Fig. 2). Features of the particular antibody were extensively treatment of each of the 90 explants (n = 5placentae).(G)Westernblotand pretested in different trophoblast cell models (SI Appendix,Fig.S2). quantification of cleaved caspase-3 expression after Notch1 inhibition. For each As previously mentioned (26), treatment with EDTA provoked treatment a total of 40 explants (10 pooled explants per placenta, n = 4) were time-dependent accumulation of Notch1 intracellular domain analyzed. Mean values ± SD (n = 4) normalized to GAPDH are shown. *P < 0.05. (N1ICD) (SI Appendix,Fig.S2A), its nuclear recruitment (SI Ap- pendix,Fig.S2B), and induction of the canonical Notch1 target and B). Upon seeding onto collagen I, Notch1-IgG decreased HES1 (SI Appendix,Fig.S2C). Incubation with the Notch1-blocking 1 antibody diminished EDTA-stimulated generation of N1ICD (SI numbers of anchoring villi per explant, suggesting that either de Appendix,Fig.S2D–F), as well as expression of the full-length novo formation and/or stability of preexisting cell columns are C D Notch1 receptor upon long-term treatment (SI Appendix,Fig.S2G). affected (Fig. 2 and ). Indeed, Western blotting and immu- nofluorescence showed that Notch1 inhibition increased expres- Moreover, Notch1-IgG1 dose-dependently decreased luciferase ac- tivity of the canonical Notch reporter and impaired EDTA-stimu- sion of the apoptotic markers cleaved caspase-3, cytokeratin 18 lated HES1 expression (SI Appendix,Fig.S2H and I). Treatment of neoepitope, and p53 in protein lysates and proximal CCTs of villous explants with the blocking antibody reduced Notch1 signals villous explants (Fig. 2 E–G). Induction of active caspase-3 was in CCTs and diminished total Notch1 protein expression (Fig. 2 A also detected upon siRNA-mediated gene silencing of Notch1 in

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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-ΔRAM. (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.

these cultures (SI Appendix,Fig.S3A). Moreover, Notch1-IgG1 and maintained expression of hepatocyte growth factor activator + treatment increased apoptosis in Notch1 clusters of vCTBs of inhibitor type 1 (HAI-1), a marker of proliferative vCTBs and 6th wk placentae and in cultivated primary CTBs (SI Appendix, CCTs (Fig. 3F and SI Appendix,Fig.S5). Fig. S3 B–D). In addition, treatment of CTBs with camptothecin (CPT) induced apoptosis and expression of Notch1 (SI Appendix, Notch1 Expression in Villous CTBs Induces Proliferation and Inhibits Cell Fig. S3E). Interestingly, cleaved caspase-3 decreased when Notch1 Fusion. To further investigate the role of Notch1 in the placental BIOLOGY levels were highest, suggesting that the receptor could, at least epithelium, N1ICD was overexpressed in vCTBs isolated from 9th- DEVELOPMENTAL partly, counteract CPT-induced apoptosis. Notch1 was shown to to 10th-wk placentae (Fig. 4). Compared with 7th to 8th wk of promote survival by inhibiting degradation of X-linked inhibitor of gestation, Notch1 was largely absent from these cells (SI Appendix, apoptosis (XIAP) (27). Stability of the latter, however, was not Figs. S4F and S6). Similar to its effects in CCTs, N1ICD increased affected upon silencing of Notch1 in CTBs (SI Appendix,Fig.S3F). proliferation and cyclin expression in vCTBs, although the effects were more pronounced (Fig. 4 A–C and SI Appendix,Fig.S7A and Notch1 Maintains Purified Cell Column Progenitors and Inhibits Their B). Again, N1ICD-ΔRAM neither changed expression of cell cycle Differentiation into Extravillous Trophoblasts. To analyze the role of markers nor proliferation. Additionally, N1ICD suppressed in vitro Notch1 in purified progenitors, the two different proliferative CTB syncytialization and differentiation-dependent induction of CGβ,but subtypes, CCT and vCTB, were isolated from first trimester pla- maintained HAI-1 and E-cadherin expression in vCTBs (Fig. 4 D–F). centae using optimized purification protocols. Upon cultivation on fibronectin, vCTBs underwent cell fusion as indicated by the in- Ectopic Notch1 Induces an Extravillous Trophoblast Progenitor-Specific duction of human chorionic gonadotrophin β (CGβ) and suppres- Gene Signature in Fusogenic Precursors. As a prerequisite for the sion of the CTB marker E-cadherin (SI Appendix,Fig.S4A, C,and analysis of Notch1 in trophoblast stemness, putative marker proteins E). The EVT-specific gene HLA-G was not induced upon in vitro of trophoblast self-renewal were studied in placental sections and differentiation of these cells. In contrast, CCTs seeded on fibro- purified progenitor cell types (Fig. 5). Whereas the transcription nectin up-regulated the EVT markers HLA-G and α1 factors p63 and TEA domain family member 4 (TEAD4) were (ITGA1), but not CGβ (SI Appendix,Fig.S4B–D). In both pro- predominantly detected in vCTBs, VE-cadherin and myc were genitor subtypes, Notch1 was down-regulated upon in vitro differ- mainly expressed in proliferative CCTs (Fig. 5 A–C). Over- entiation (SI Appendix,Fig.S4D–F). Subsequently, CCTs were expression of N1ICD in vCTB purified from 9th- to 10th-wk pla- isolated from 9th- to 10th-wk placentae, yielding maximal numbers centae, lacking Notch1, induced CCT-specific myc and VE-cadherin of these cells, and transfected with N1ICD and a mutant variant expression (Fig. 5 D–F and SI Appendix,Fig.S8). In contrast, (N1ICD-ΔRAM), lacking the RBPJκ-associated module (RAM) ΔNp63 and TEAD4, controlling self-renewal of vCTBs and murine for high-affinity binding to the particular transcription factor (Fig. trophoblast stem cells (28, 29), respectively, were suppressed 3A). After transfection, N1ICD was exclusively detected in nuclei of (Fig. 5 D and F and SI Appendix,Fig.S8). In addition, N1ICD CCTs, whereas N1ICD-ΔRAM localized to both cytoplasm and increased expression of IFN regulatory factor 6 (IRF6), a negative nuclei of cells (Fig. 3B). Overexpression of N1ICD supressed regulator of p63 (30) (Fig. 5 D and F and SI Appendix,Fig.S8). cleaved caspase-3, but increased cyclin A protein, cyclin D1 mRNA, N1ICD binds to the myc and IRF6 gene in CTBs. Notch1 was recently shown as well as proliferation of CCTs, measured by EdU-labeling (Fig. 3 to stimulate myc transcription in T-cell acute lymphoblastic leukemia C–E). On the contrary, N1ICD-ΔRAM did not provoke these ef- cells by activating RBPJκ bound to cognate sequences in the prox- fects. Moreover, N1ICD expression prevented differentiation of imal promoter (−97 bp) and in the Notch-dependent myc enhancer CCTs into EVTs, because it inhibited up-regulation of HLA-G (NDME) located 1.4 Mb 3′ of the gene (31, 32). Analyses of the myc

Haider et al. PNAS | Published online November 14, 2016 | E7713 Downloaded by guest on September 29, 2021 Fig. 4. N1ICD increases proliferation and inhibits cell fusion of purified vCTBs. FLAG-tagged N1ICD or N1ICD-ΔRAM 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.

5′ flanking region (−5kb)usingTransfacPatch1.0identifiedtwo characterized. Derivation of a cell line with trophoblast stem cell putative RBPJκ binding sites, the previously described element at properties from single blastomers of eight-cell embryos suggested −97 bp (32), and another putative cognate sequence at −3.060 bp. that the trophectoderm cell fate could be initiated at very early stages ChIP revealed that N1ICD interacted with the motif at −3.060 bp of pregnancy (43). In addition, trophoblast stem-like cells, expressing upon overexpression in vCTBs as well as with the distal NDME used Cdx2 and p63, were derived from BMP-induced hESCs generating as a control (Fig. 5G and SI Appendix,Fig.S9). As shown (31), both EVTs and STBs (42). However, it remains controversial N1ICD specifically bound to the c1 element in that region (SI Ap- whether bipotential CTBs are maintained during the first trimester pendix,Fig.S9). Moreover, ChIP indicated that IRF6 is also a direct of pregnancy (4). Whereas FGF4 was claimed to redirect fusogenic target of N1ICD. The latter bound to two different RBPJκ cognate CTBs toward EVT differentiation (44), others showed that EVT sequences at −3.6 kb and −2.4 kb in the IRF6 promoter (Fig. 5H and precursors isolated from 6th- to 10th-wk placentae were unable to + SI Appendix,Fig.S9), previously delineated in keratinocytes (33). syncytialize, but spontaneously formed 20% HLA-G cells after Notch1 intracellular domain represses p63 through IRF6 in vCTBs. To ana- 5 d in culture (16). The latter study suggested that distinct tro- lyze whether Notch-dependent down-regulation of ΔNp63 requires phoblast progenitors, committed to syncytialization and EVT IRF6, vCTBs were transfected with N1ICD and siRNA against formation, respectively, have developed in first trimester placen- IRF6. Silencing of the latter increased ΔNp63 levels, indicating a tae. The present study confirms this assumption. Using sequential direct role in N1ICD-induced repression of p63 (Fig. 5I). trypsin digestions of early placental tissues, removal of STB frag- ments by gradient centrifugation and immune depletion of dif- Discussion ferentiated EVTs, CTB progenitor subtypes with high purity were Failures in trophoblast differentiation and function in a variety of obtained. Upon seeding onto fibronectin, vCTBs fused into STBs, pregnancy disorders strongly warrant a better understanding of the but did not induce the EVT markers HLA-G or ITGA1. In con- molecular pathways controlling human placental development. trast, CCTs differentiated into EVTs lacking STB-specific CGβ However, experimental studies are hampered by ethical constraints expression. Notably, formation of STBs and EVTs occurred under to obtain tissues from early human gestation as well as difficulties in identical culture conditions and on the same matrix, suggesting establishing self-renewing human trophoblast stem and progenitor that differentiation is mainly driven by the intrinsic molecular cells from trophoblast isolates (34–36). As a consequence, alter- program of vCTBs and CCTs. Because isolation of the two distinct native models, such as bone morphogenetic protein (BMP)-treated progenitors is based on the consecutive digestions with different human ESCs (hESCs), have been developed, allowing in vitro trypsin concentrations, some marginal cross-contamination cannot formation of the trophoblast lineage (37, 38). Despite concerns be avoided (SI Appendix, Fig. S4C). However, the present protocol regarding specificity of trophectoderm induction with BMP (37, 39, will allow for further genome-wide gene expression analyses of 40), recent investigations using optimized culture conditions sug- these CTB subtypes. Thereby, unique surface markers, suitable for gested that early stages of human trophoblast development could additional purification steps, could be identified. be mimicked in these cells (41, 42). Critical regulators controlling trophoblast stemness and lineage Nevertheless, in vivo localization of human trophoblast stem determination have been vastly studied in developing mice and progenitor cells and their specific features remain poorly (45–48). However, data on key factors controlling human placental

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Fig. 5. Expression pattern of markers of self-renewal and their regulation by N1ICD and IRF6. Representative pictures of first trimester placentae (A and B) showing coimmunofluorescence of TEAD4, ΔNp63, 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 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 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-ΔRAM 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 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.

development are scarce. Descriptive analyses in first trimester expressed in vCTBs. However, the stemness markers myc, mark- tissues and trophoblast cell models have been performed to gain edly decreasing during the first trimester of pregnancy (52), and insights into the expression patterns and epigenetic profiles of some VE-cadherin, were mainly detected in CCTs. Besides its classical of these genes (37, 40, 48, 49). However, few of them have been role in endothelial cells, VE-cadherin also specifies a transient, functionally examined by using choriocarcinoma cells as surrogate hematopoetic stem cell population in the developing liver (53). for human primary trophoblasts (28, 50). To extend our present In anchoring villi VE-cadherin specifically localizes to the knowledge, we herein analyzed expression of key regulators in the intermediate region of the cell column containing proliferative two purified CTB progenitor subtypes and performed functional CCT (Fig. 5B), as also previously shown (54). Its expression partly analyses in these cells. Immunofluorescence and quantitative PCR overlaps with Notch1, exclusively found in proximal EVT pro- (qPCR) indicated that TEAD4, a crucial transcription factor in genitors. Cdx2, the central transcriptional activator factor in murine trophectoderm specification and trophoblast progenitor murine trophectoderm development (55), has been detected in self-renewal (29, 51), is also present in human vCTBs but largely vCTBs of early placental tissues and rapidly declines toward the + absent from proximal Notch1 CCTs. Akin to that finding, p63, as- end of the first trimester (42, 48, 49). The present study also de- sociated with the CTB progenitor phenotype (28), was predominantly tected Cdx2 in vCTBs of 6th- wk placentae (SI Appendix,Fig.

Haider et al. PNAS | Published online November 14, 2016 | E7715 Downloaded by guest on September 29, 2021 S1D). However, the factor was absent from both vCTBs and CCTs of increasing oxygen levels could be necessary for on-going EVT of distal villi located at the basal side of the placenta. Instead, formation during the period of spiral artery remodelling. CCTs in this region were positive for Notch1, a key regulator of Notch signaling in stem cells is highly complex and exerts dif- stem cell niches and cell lineage determination (23). Based on ferent effects in tissues and organs (23, 62). Accordingly, Notch + these expression patterns, we hypothesize that putative Cdx2 was shown to promote or inhibit self-renewal, differentiation, and stem cell-like trophoblasts disappear in distally developing regions survival, depending on the specific cellular context. Previous of the placenta, at a time when extensive formation of villi, an- analyses of human placental tissues suggested that Notch sig- choring to the maternal decidua, takes place. Instead, committed naling could be predominantly associated with trophoblast pro- progenitors for cell fusion and EVT differentiation arise, each genitor cell function (20). Although Notch2 has its main role in expressing a unique set of self-renewal genes. invasive trophoblasts (19, 24), down-regulation of HES1 and Development of stable anchoring villi is a prerequisite for a RBPJκ reporter activity during EVT formation indicated that continuous supply with EVTs invading and remodelling maternal canonical Notch signaling is associated with undifferentiated uterine tissues. However, mechanisms controlling establishment trophoblasts (20, 21). Herein, functional analyses of Notch1 in and maintenance of cell columns, replenishing the pool of out- different primary trophoblast cell models corroborate this evi- growing trophoblasts, have not been elucidated. Herein, we iden- dence. Blocking or silencing of Notch1 increased apoptosis in tified Notch1 as a critical regulator of these processes. Notch1 was villous explant cultures and isolated CTBs. In contrast, over- detected in proliferative/mitotic CCTs of the proximal cell column, expression of N1ICD enhanced proliferation and cyclin expres- thereby defining the EVT progenitor cell niche. Despite its short sion but suppressed cleaved caspase-3 in purified vCTBs or half-life of ∼1.8 h (56), nuclear N1ICD was occasionally observed in CCTs. Notch-dependent survival operates through differ- proximal CCTs, suggesting activation of Notch1 cleavage. However, ent mechanisms involving mammalian target of rapamycin – cyclin A and p-Histone H3 were also expressed in neighboring (mTOR) Akt-mediated suppression of p53 or activation of in- − + Notch1 CCTs, suggesting that Notch1 precursors could give rise hibitors of apoptosis such as XIAP (27, 63). Expression of the to committed transient amplifying (TA)-like cells. The latter further latter was not affected by Notch1 in trophoblasts. However, in- + develop into distal HLA-G CCTs progressing toward EVTs. TA hibition of the receptor induced accumulation of nuclear p53. + κ cells, derived by asymmetric cell division of self-renewing Notch1 Notably, silencing of RBPJ in villous explant cultures did not stem cells, have been previously described in other epithelial tissues provoke apoptosis of CCTs (21), suggesting that N1ICD-mediated survival could be an RBPJκ-independent function, as recently (57). In analogy, we assume that Notch1 could control homeostasis κ of placental anchoring villi by adapting rates of progenitor self- shown (64). Different than Notch1, disruption of RBPJ weakly renewal, TA-like cell formation, and EVT differentiation. increased proliferation in explant cultures and, as a consequence EVT formation (21). Several effects could account for this dis- Similar to Cdx2 (49), CTB-specific Notch1 expression markedly κ decreases during the early weeks of pregnancy (SI Appendix,Fig.S1 crepancy. Abolishing RBPJ not only impairs Notch1 but also E and F) and is absent from second trimester trophoblasts (19), activities of Notch3 and Notch4, which are abundantly expressed in CTBs (19, 20). Besides binding to RBPJκ, N1ICD interacts indicating that the number of progenitor cells diminishes at the with a range of different transcription factors (65), of which time of oxygen transition. By binding of NICD to HIF-1α,Notch HIF1α,NFκB, GABPA, and ZNF143 are also detected in our signaling synergizes with hypoxia to keep cells in an undif- + previously published microarray data (66) of EGFR CTBs ferentiated state (58, 59). Hence, we speculated that a low- (accessible at Gene Expression Omnibus, GDS3523). Moreover, oxygen environment could support mRNA expression and protein RBPJκ has Notch-independent roles in different cell types, stability of Notch1, as shown in other cell types (59–61), and mostly as suppressor of gene transcription (67). thereby maintain the proliferative capacity of CCT precursors. In- Notch1 effectively inhibits differentiation in many cellular sys- deed, the present data indicate that hypoxia increases Notch1 as tems, thereby preserving stemness (62). In agreement with that well as activity of a canonical Notch reporter. After establishment – fact, N1ICD maintained expression of genes associated with un- of the placental maternal circulation, declining Notch1 levels might differentiated vCTBs and CCTs, E-cadherin and HAI-1, and affect survival and/or proliferation of these cells, thereby slowing suppressed STB formation of fusogenic precursors as well as down continuous growth of cell columns and EVT differentiation. differentiation-dependent induction of CGβ.Moreover,N1ICD + On the other hand, Notch1 was detected in EVT progenitors also inhibited differentiation of CCTs into HLA-G EVTs. Im- of 12th-wk placentae, at the time when the receptor was completely + munofluorescence of 5th- to 7th-wk placental tissues revealed that absent from vCTBs. Maintenance of Notch1 CCTs in the presence 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 extra- villous 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 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- Fig. 6. Model system depicting the presumptive role of Notch1 in human trophoblast development. N1ICD programs vCTBs into CCTs, expressing the lyzed for markers of vCTB or CCT stemness, defined in the present stemness markers myc and VE-cadherin and prevents EVT differentiation by study. Of note, N1ICD suppressed vCTB-specific p63 and TEAD4 maintaining proliferation and survival of these cells. N1ICD also suppresses expression but induced the CCT markers VE-cadherin and myc. TEAD4 and p63, the latter by inducing its repressor IRF6, thereby alleviating Similar to other cells (31), the latter could be a direct target of self-renewal and cell fusion of vCTBs. active Notch1 in CTBs. ChIP revealed that N1ICD binds to the

E7716 | www.pnas.org/cgi/doi/10.1073/pnas.1612335113 Haider et al. Downloaded by guest on September 29, 2021 3′ NDME of the myc gene, previously shown to promote gene SI Appendix, Table S1. Nuclei were stained with 1 μg/mL DAPI. Tissue were PNAS PLUS expression (31), as well as to a newly identified RBPJκ motif analyzed by fluorescence microscopy (Olympus BX50, CellP software) and present at −3 kb in the proximal promoter region. In contrast to digitally photographed. myc, N1ICD was recently shown to indirectly suppress p63. Immunofluorescence of Cultured Cells. Cells were fixed with 3.7% (wt/vol) N1ICD binds to the IRF6 gene in keratinocytes and activates its paraformaldehyde (10 min), treated with 0.1% Triton X-100 (5 min) and expression (33). In turn, IRF6 promotes proteasomal degradation incubated with primary antibodies overnight at 4 °C (listed in SI Appendix, of p63 (30). An analogous mechanism could regulate p63 in Table S1). Subsequently, cells were washed and incubated for 1 h with 2 μg/mL vCTBs, because IRF6 rapidly increased after N1ICD expression. of secondary antibodies (listed in SI Appendix,TableS1) and nuclei were Also, ChIP analyses showed that N1ICD interacts with two RBPJκ stained with DAPI. Slides were analyzed by fluorescence microscopy (Olympus cognate sequences, recently identified in the proximal IRF6 pro- BX50, CellP software) and digitally photographed. moter region (33). Moreover, silencing of IRF6 in vCTBs im- paired N1ICD-dependent suppression of ΔNp63. Compared with Isolation and Cultivation of Placental Primary CTBs. CTBs were isolated by adapted Δ enzymatic dispersion and Percoll density gradient centrifugation [10–70% (vol/vol); p63 mRNA, loss of Np63 protein was more pronounced in the = – D F SI Appendix GE Healthcare] of pooled first trimester placentae (n 3 6 per isolation) as presence of ectopic N1ICD (Fig. 5 and and ,Fig. described (68, 69) and plated (45 min) in culture medium (DMEM/Ham’sF-12, S8), suggesting that its stability could be affected. Additionally, 10% (vol/vol) FCS, 0.05 mg/mL gentamicin, 0.5 μg/mL fungizone; Gibco) allowing Notch1-mediated inhibition of p63 could diminish numbers of for adherence of contaminating stromal cells. Nonadherent trophoblasts were + self-renewing TEAD4 vCTBs and, as a consequence, cell fusion. collected and CTBs were either seeded in culture medium onto fibronectin- Unlike myc, p63, or IRF6, N1ICD-dependent induction of VE- coated (20 μg/mL; Millipore) dishes (2.5 × 105 cells per square centimeter) or cadherin was only noticed upon long-term expression, indicating further purified using positive selection with EGFR-phycoerythrin (PE) or HLA-G- that it might not be a direct Notch1 target. Notably, immunoflu- PE antibodies (listed in SI Appendix,TableS1) and anti-PE MicroBeads (MACS ’ orescence revealed that the particular adhesion molecule specifi- Miltenyi Biotec) according to the manufacturer s instructions. The contamina- tion with stromal cells was routinely tested by IF with antibodies detecting + cally localizes to proliferative CCTs in the intermediate region of cytokeratin 7 (trophoblast cells) and vimentin (nontrophoblast cells). Vimentin B + + the cell column (Fig. 5 ) (12). Hence, N1ICD-induced VE-cad- cells were fewer than 1%. The purity of isolated EGFR and HLA-G CTB pop- herin expression might suggest that cell column progenitors have ulations was verified using qPCR and Western blotting. further developed into TA-like cells. Finally, we speculate that To verify specificity of the Notch1 blocking, CTBs were allowed to attach for 2 h, Notch1-dependent expression of myc and VE-cadherin could be washed with prewarmed PBS, and preincubated with culture medium containing − sufficient to establish EVT progenitors from Notch1 CTB pre- either 0.5 μg/mL ctrl-IgG1 (ctrl-IgG1 ABIN376845; Antibodies-Online) or 0.5 μg/mL cursors. On the other hand, suppression of cell fusion and down- Notch1-IgG1 (Notch1-IgG1, Genentech) antibodies for 1 h. Subsequently, 5 mM regulation of p63/TEAD4 by N1ICD could negatively affect vCTB EDTA was added for another 30 min, inducing receptor cleavage/generation of self-renewal and thereby trigger CCT formation and EVT differ- N1ICD. For long-term Notch1 inhibition, CTBs were seeded for 2 h and antibodies entiation. Along those lines, silencing of p63 in JEG-3 chorio- 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 con- carcinoma cells was shown to increase migration, a characteristic taining either DMSO (ctrl) or 1 μMCPTfor1,2,4,6,and24h.Toverifywhether feature of invasive EVTs (28). the Notch1 could inhibit apoptosis via XIAP stabilization, CTBs were pre- In summary, the present study shows that Notch1 is a critical incubated with a mixture of four different siRNAs against Notch1 (si-Notch1; BIOLOGY regulator of EVT development in the human placenta. Based on its L-007771–00-0005, ON-TARGETplus SMARTpools, Dharmacon-Thermo Fisher Sci-

restricted expression in the proximal region of developing cell col- entific) or nontargeting siRNA (si-ctrl, D-001810-10-20) overnight and sub- DEVELOPMENTAL umns, functional studies in primary trophoblast models have been sequently treated with DMSO (−)or1μMCPT(+)for24h. conducted, indicating a crucial role of the receptor in CCT pro- Isolation and Cultivation of Purified Primary vCTBs and CCTs. CCT and vCTB cell liferation, survival, and differentiation. Notch1 induced markers of − populations were isolated by two consecutive digestion steps followed by Percoll EVT progenitors in Notch1 vCTBs, but suppressed genes controlling density gradient centrifugation. Precisely, placental tissue (6th–8th and 9th–10th wk vCTB self-renewal (Fig. 6). Further studies are required to delineate of gestation, n = 3–5 per isolation) was minced into small pieces (1–3 mm). For Notch ligands involved in this process as well as the mechanism(s) isolation of CCTs, the first digestion was performed with 0.125% trypsin (Gibco) + + initiating Notch1 expression in the developing placenta. 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 Materials and Methods (PAA Laboratories) and cells were filtered through a 100-μm cell strainer (BD Tissue Collection. Placental tissues (6th–12th wk of gestation) were obtained 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 from elective pregnancy terminations. Utilization of tissues and all experi- and processed as described above. Digestion solutions containing either CCTs or mental procedures were approved by the Medical University of Vienna vCTBs were each layered on top of a Percoll gradient [10–70% (vol/vol)] and cells ethics boards. Written informed consent was obtained from all subjects. between 35 and 50% of the Percoll layer were collected. Contaminating RBCs were removed by incubating cells with erythrocyte lysis buffer Immunohistochemistry of Paraffin-Embedded Tissue. Serial sections of paraffin- (155 mM NH Cl, 10 mM KHCO , 0.1 mM EDTA, pH 7.3) for 5 min at room embedded villous explants were analyzed by immunohistochemistry (IHC) using 4 3 temperature and subsequently washed with 1× HBSS. Contaminating Dako EnVision+ System-HRP (DAKO, K4011) as instructed by the manufacturer. stromal cells were depleted from CCT preparations as mentioned above. Sections were incubated with primary antibodies (listed in SI Appendix,Table Finally, differentiated CCTs/EVTs were removed from proliferative CCTs or S1) overnight at 4 °C and nuclei were counterstained with hematoxylin vCTBs, using HLA-G-PE antibodies (Exbio) and anti-PE MicroBeads (MACS (Merck). Sections were digitally photographed using an Olympus BX50 mi- Miltenyi Biotec) as instructed by the manufacturer. CCT and vCTB cells croscope and CellP software. 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. Immunofluorescence of Paraffin-Embedded Tissue. Placental tissue was fixed in CCT and vCTB cells were harvested after 24 h (undifferentiated cells) and be- 7.5% (wt/vol) formaldehyde and embedded in paraffin. To analyze Notch1 tween 72 and 120 h (differentiated cells). Cells were either fixed for IF analyses or localization in different regions of placental villi (stem villus, intermediate vil- snap frozen for qPCR and Western blotting. lus, end villus), tissues were stained with Alcian blue solution and carefully placed in embedding cassettes. Serial sections (3 μm) of paraffin-embedded Notch1 Plasmids and CTB Transfection. Full-length human N1ICD (residues 1762– placental tissue or villous explant cultures were analyzed by immunofluores- 2556) and a N1ICD-ΔRAM domain (residues 1877–2556) were a generous gift of cence (IF) as described elsewhere (20). Briefly, sections were deparaffinized in C. O. Joe, Department of Biological Sciences, Korea Advanced Institute of Sci- Xylol and rehydrated. Antigen retrieval was performed using 1× PT module ence and Technology, Daejeon, South Korea. Notch1 variants are cloned into buffer 1 (Thermo Scientific) for 35 min at 93 °C using a KOS microwave his- pFLAG-CMV-2 (Sigma) as described elsewhere (70). Empty pFLAG-CMV-2 served tostation (Milestone). Sections were incubated with primary antibodies (listed as negative control (mock-CTRL). Isolated CCTs and vCTBs were transfected in SI Appendix,TableS1) overnight at 4 °C. Afterward, slides were washed with the 4D-Nucleofector (program EO-100, Lonza) using the AMAXA SG Cell three times and incubated for 1 h with secondary antibodies (2 μg/mL) listed in Line Kit according to the manufacturer’s instructions. Subsequently, cells were

Haider et al. PNAS | Published online November 14, 2016 | E7717 Downloaded by guest on September 29, 2021 seeded at a density of 3.5 × 105 cells per square centimeter. Transfection with a PVDF (Amersham) membranes and incubated overnight at 4 °C with primary pmaxGFP (Lonza) revealed an average transfection efficiency of 44 ± 18%. antibodies (SI Appendix,TableS1). Subsequently, filters were washed and Transfected trophoblasts were incubated up to 96 h at 37 °C. To evaluate lo- incubated for 1 h with HRP-conjugated secondary antibodies (SI Appendix, calization of transfected N1ICD and N1ICD-ΔRAM, IF was performed as de- Table S1). Signals were developed using ECL Prime Detection Kit (GE Health- scribed above. For down-regulation of IRF6 in N1ICD-overexpressing CTBs, care) and visualized with FluorChemQ Imaging System (Alpha Innotech). siRNA-mediated IRF6 gene silencing was performed using a mixture of four Quantification was performed using ImageJ software. 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- qPCR. RNA isolation (PeqGold Trifast, PeqLab) and reverse transcription Thermo Fisher Scientific). Notch1-transfected cells were seeded in the presence (RevertAid H Minus Reverse Transcriptase, Fermentas, EP0451) were per- of si-ctrl or si-IRF6 and incubated for 24 h. formed as indicated by the manufacturers. Villous explants were homoge- nized with the Precellys 24 (PeqLab) before RNA isolation. qPCR analyses (in Proliferation Assays. Purified CCTs and vCTBs were transfected with mock-CTRL, duplicates) were performed using the 7500 Fast Real-time PCR System (Ap- Δ N1ICD, or N1ICD- RAM, seeded onto fibronectin-coated 48-well dishes, and plied Biosystems, ABI) as described (73). The following TaqMan Gene Ex- μ ′ incubated for 24 h. Afterward, 10 M5-ethynyl-2-deoxyuridine (EdU) pression Assays were used: NOTCH1 (ABI, Hs01062014_m1), CCND1 (ABI, (EdU-Click 488, BaseClick) was added for 4 h. Subsequently, cells were fixed Hs00277039_m1), TEAD4 (ABI, Hs01125032_m1), p63 (ABI, Hs00978340_m1), and EdU was detected according to the manufacturer’s instructions. Nuclei MYC (ABI, Hs00153408_m1), VE-cadherin (ABI, Hs00901465_m1), and HES1 were stained with DAPI (Roche). Cells were digitally photographed (seven + (ABI, Hs00172878_m1). A total of 1 μL cDNA, 0.5 μL primers, 5 μL innuMIX pictures per condition) using the EVOS FL Color Imaging System and EdU qPCR MasterMix Probe (Biometra), and 0.2 μL ROX Reference Dye (Invi- nuclei were counted using Adobe Photoshop CS5. trogen) were used per sample. Signals (ΔCt) were normalized to TATA-box binding protein (TBP) (ABI, 4333769F). Relative expression levels were de- Notch1 Inhibition in First Trimester Villous Explant Cultures. Villous explant termined by using values of controls as a calibrator (ΔΔCt). 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 ChIP. For ChIP analyses, SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling, efficiency to Notch1-blocking antibodies, villous explants were treated with 9003) was used as mentioned by the manufacturer. Briefly, vCTBs were isolated, Δ either 0.5 μg/mL ctrl-IgG1 or 0.5 μg/mL Notch1-IgG1 for 1 h. The Notch1- transfected with N1ICD or N1ICD- RAM, seeded onto fibronectin-coated six- blocking antibody, a generous gift of C. W. Siebel, Genentech, San Francisco, well dishes, and incubated for 20 h. Subsequently, cells were fixed for 45 min binds and stabilizes the negative regulatory region of the receptor, thereby with 2 mM disuccinimidyl glutarate (DSG) and 1% formaldehyde (15 min). inhibiting conformational changes, ADAM-mediated cleavage, and N1ICD- After nuclei preparation, chromatin digestion, and sonication, purified chro- dependent activation of canonical signaling (25). Subsequently, 5 mM EDTA matin lysates were incubated either with Notch1 antibody (1 μg, Cell Signaling, was added for up to 60 min and explants were homogenized to isolate 3608), normal rabbit IgG (negative control) (1 μg; Cell Signaling, 2729) or His- protein lysates. For outgrowth analyses, floating explant pairs were sup- tone H3 XP rabbit mAb (positive control) (1 μg; Cell Signaling, 4620) overnight

plemented with 0.5 μg/mL ctrl-IgG1 or 0.5 μg/mL Notch1-IgG1 and kept at 4 °C. Immunoprecipitated chromatin was captured with ChIP-Grade Protein overnight in explant culture medium (DMEM/Ham’s F-12, 0.05 mg/mL gen- G magnetic beads and eluted. Purified DNA was assessed by semi-qPCR using tamicin), respectively. Subsequently, explants were seeded onto rat tail col- Taq Polymerase (Fermentas, EP0402). PCR conditions were as follows: 5 min at lagen I (attachment for 4 h) and subsequently covered with medium 96 °C (initial denaturation); 45 s at 95 °C, 45 s at 58 °C, 45 s at 72 °C (35 cycles), containing either 0.5 μg/mL ctrl-IgG1 or 0.5 μg/mL Notch1-IgG1. After another and 5 min at 72 °C (final extension). MYC PROM primers were designed to span 24 h, explants were digitally photographed and differences in anchorage the region harboring a putative RBPJκ DNA binding motif (CTGCGGGAA) and outgrowth were evaluated using the EVOS FL Color Imaging System. For identified by Transfac Patch 1.0 at −3.060 bp, which differs by one nucleotide long-term antibody- or siRNA-mediated Notch1 inhibition, explants (7th–8th from the consensus sequence CTGTGGGAA. MYC NDME-c1, MYC NDME-c2, wk of gestation) were incubated for 48 h in culture medium supplemented IRF6 PROM-1, and IRF6 PROM-2 primers were published elsewhere (31, 33). μ with 0.5 g/mL Notch1-IgG1 or Notch1 siRNA. Next, explants were either The following primer sequences were used: MYC PROM (229 bp): forward fixed with 7.5% (wt/vol) formaldehyde and embedded in paraffin or ho- 5′-ATGAGGTCAAGCTGGACCTAC-3′ and reverse 5′-TGACGGTGTCTGATCACTTA-3′; mogenized for Western blotting as described below. MYC NDME-c1 (200 bp): forward 5′-GCTGCCACATGCTGATGAAC-3′ and reverse 5′-CCAGGTAGGGGCATTACGTC-3′; MYC NDME-c2 (174 bp): forward 5′-GAG- Cultivation and Luciferase Reporter Assay in Trophoblastic SGHPL-5 Cells and Primary GCCCCCATTCATTACCC-3′ and reverse 5′-GCAGTTCTTCCTACGCTGGT-3′;IRF6 CTBs. SGHPL-5 cells were cultivated in DMEM/Ham’s F-12 supplemented with 10% PROM-1: forward 5′-ACCCTCCCAGCTTGAGTTTT-3′ and reverse 5′-AAACCCCA- (vol/vol) FCS and 0.05 mg/mL gentamicin at standard cell culture conditions. GTGGCATACAAG-3′ (142 bp); IRF6 PROM-2: forward 5′-TCAATGGAGGGCAA- Notch1 cleavage was induced by adding 5 mM EDTA for up to 30 min. Inhibition AATGAT-3′ and reverse 5′-ACGCCTCATCTGCTTGATCT-3′ (162 bp); human of Notch1 cleavage was performed with ctrl-IgG1 or Notch1-IgG1 as mentioned RPL30 (control) (Cell Signaling, 7014). PCR products were analyzed on above. For detection of canonical Notch activity subconfluent SGHPL-5 cells were 1.5% (wt/vol) agarose gels containing Midori green (Nippon Genetics, MG04) cotransfected with 2 μg/mL of a luciferase reporter containing four RBPJκ binding and digitally photographed under UV. Relative occupancy of N1ICD or N1ICD- sites (mutant or wild-type plasmids) and 0.5 μg/mL pCMV–β-galactosidase (CMV- ΔRAM was normalized to normal rabbit IgG. βGal; normalization control) using Lipofectamine 2000 (Invitrogen) as recently described (20). Primary CTBs isolated from pooled 6th-to 8th-wk placentae were Statistical Analyses. Gaussian distribution and equality of variances were exam- transfected with reporter plasmids using the AMAXA system as mentioned above. ined with Kolmogorov–Smirnov and Levene tests, respectively. Statistical analysis After 6 h, medium was changed. SGHPL-5 cells were incubated either with DAPT, of data between two means was performed with Student’s t test or Mann– ctrl-IgG1,orNotch1-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/ Whitney u test using SPSS 14. Comparisons of multiple groups were evaluated – 50 μM CoCl . Protein lysates were prepared after an additional 24 h. Luciferase with one-way ANOVA and appropriate post hoc tests or Kruskal Wallis tests 2 – ’ activity and β-galactosidase activity were determined as previously published (72). 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 experi- ments were conducted in duplicates and replicated at least three times. Western Blotting. Whole-cell lysates were prepared using standard protocols as recently described (72). Villous explants were additionally homogenized for 20 s ACKNOWLEDGMENTS. We thank G. S. Whitley (St. George’s University of at 3,640 × g with a Precellys 24 (PeqLab). Nuclear extracts were isolated using London) for providing SGHPL-5 cells and C. Siebel (Genentech) for providing the NE-PER nuclear and cytoplasmic protein extraction kit (Thermo Scientific). the Notch1 blocking antibody. This study was supported by the Austrian Protein extracts were separated on SDS/PAA gels, transferred onto Hybond-P Science Fund (Grant P-28417-B30).

1. Hamilton WJ, Boyd JD (1960) Development of the human placenta in the first three 4. Aplin JD (2010) Developmental cell biology of human villous trophoblast: current months of gestation. J Anat 94:297–328. research problems. Int J Dev Biol 54(2–3):323–329. 2. Burton GJ, Jauniaux E, Charnock-Jones DS (2010) The influence of the intrauterine 5. Pijnenborg R, Dixon G, Robertson WB, Brosens I (1980) Trophoblastic invasion of environment on human placental development. Int J Dev Biol 54(2–3):303–312. human decidua from 8 to 18 weeks of pregnancy. Placenta 1(1):3–19. 3. Evain-Brion D, Malassine A (2003) Human placenta as an endocrine organ. Growth 6. Red-Horse K, et al. (2004) Trophoblast differentiation during embryo implantation Horm IGF Res 13(Suppl A):S34–S37. and formation of the maternal-fetal interface. J Clin Invest 114(6):744–754.

E7718 | www.pnas.org/cgi/doi/10.1073/pnas.1612335113 Haider et al. Downloaded by guest on September 29, 2021 7. Hustin J, Jauniaux E, Schaaps JP (1990) Histological study of the materno-embryonic 42. Horii M, et al. (2016) Human pluripotent stem cells as a model of trophoblast dif- PNAS PLUS interface in spontaneous abortion. Placenta 11(6):477–486. ferentiation in both normal development and disease. Proc Natl Acad Sci USA 113(27): 8. Pijnenborg R, et al. (1991) Placental bed spiral arteries in the hypertensive disorders of E3882–E3891. pregnancy. Br J Obstet Gynaecol 98(7):648–655. 43. Zdravkovic T, et al. (2015) Human stem cells from single blastomeres reveal pathways 9. Pijnenborg R, Vercruysse L, Hanssens M (2006) The uterine spiral arteries in human of embryonic or trophoblast fate specification. Development 142(23):4010–4025. pregnancy: Facts and controversies. Placenta 27(9–10):939–958. 44. Baczyk D, et al. (2006) Bi-potential behaviour of cytotrophoblasts in first trimester 10. Romero R, Kusanovic JP, Chaiworapongsa T, Hassan SS (2011) Placental bed disorders chorionic villi. Placenta 27(4–5):367–374. in preterm labor, preterm PROM, spontaneous abortion and abruptio placentae. Best 45. Cockburn K, Rossant J (2010) Making the blastocyst: Lessons from the mouse. J Clin Pract Res Clin Obstet Gynaecol 25(3):313–327. Invest 120(4):995–1003. 11. Moffett-King A (2002) Natural killer cells and pregnancy. Nat Rev Immunol 2(9): 46. Latos PA, Hemberger M (2014) Review: The transcriptional and signalling networks of 656–663. mouse trophoblast stem cells. Placenta 35(Suppl):S81–S85. 12. Zhou Y, Damsky CH, Fisher SJ (1997) Preeclampsia is associated with failure of human 47. Rossant J, Cross JC (2001) Placental development: Lessons from mouse mutants. Nat cytotrophoblasts to mimic a vascular adhesion phenotype. One cause of defective Rev Genet 2(7):538–548. endovascular invasion in this syndrome? J Clin Invest 99(9):2152–2164. 48. Knott JG, Paul S (2014) Transcriptional regulators of the trophoblast lineage in 13. Zhou Y, et al. (2013) Reversal of gene dysregulation in cultured cytotrophoblasts mammals with hemochorial placentation. Reproduction 148(6):R121–R136. reveals possible causes of preeclampsia. J Clin Invest 123(7):2862–2872. 49. Hemberger M, Udayashankar R, Tesar P, Moore H, Burton GJ (2010) ELF5-enforced 14. Genbacev O, et al. (2011) Establishment of human trophoblast progenitor cell lines transcriptional networks define an epigenetically regulated trophoblast stem cell – from the chorion. Stem Cells 29(9):1427 1436. compartment in the human placenta. Hum Mol Genet 19(12):2456–2467. 15. Genbacev O, et al. (2016) Integrin α4-positive human trophoblast progenitors: Func- 50. Renaud SJ, et al. (2015) OVO-like 1 regulates progenitor cell fate in human tropho- – tional characterization and transcriptional regulation. Hum Reprod 31(6):1300 1314. blast development. Proc Natl Acad Sci USA 112(45):E6175–E6184. 16. James JL, Stone PR, Chamley LW (2007) The isolation and characterization of a pop- 51. Yagi R, et al. (2007) Transcription factor TEAD4 specifies the trophectoderm lineage ulation of extravillous trophoblast progenitors from first trimester human placenta. at the beginning of mammalian development. Development 134(21):3827–3836. – Hum Reprod 22(8):2111 2119. 52. Pfeifer-Ohlsson S, et al. (1984) Spatial and temporal pattern of cellular myc oncogene 17. Knöfler M, Vasicek R, Schreiber M (2001) Key regulatory transcription factors involved expression in developing human placenta: Implications for embryonic cell pro- – in placental trophoblast development: A review. Placenta 22(Suppl A):S83 S92. liferation. Cell 38(2):585–596. 18. Knöfler M, Pollheimer J (2012) IFPA Award in Placentology lecture: Molecular regu- 53. Oberlin E, et al. (2010) VE-cadherin expression allows identification of a new class of – lation of human trophoblast invasion. Placenta 33(Suppl):S55 S62. hematopoietic stem cells within human embryonic liver. Blood 116(22):4444–4455. 19. Hunkapiller NM, et al. (2011) A role for Notch signaling in trophoblast endovascular 54. Zhou Y, et al. (1997) Human cytotrophoblasts adopt a vascular phenotype as they – invasion and in the pathogenesis of pre-eclampsia. Development 138(14):2987 2998. differentiate. A strategy for successful endovascular invasion? J Clin Invest 99(9): 20. Haider S, et al. (2014) Notch signaling plays a critical role in motility and differenti- 2139–2151. – ation of human first-trimester cytotrophoblasts. Endocrinology 155(1):263 274. 55. Strumpf D, et al. (2005) Cdx2 is required for correct cell fate specification and dif- 21. Velicky P, et al. (2014) Notch-dependent RBPJκ inhibits proliferation of human cyto- ferentiation of trophectoderm in the mouse blastocyst. Development 132(9): trophoblasts and their differentiation into extravillous trophoblasts. Mol Hum Reprod 2093–2102. 20(8):756–766. 56. Hein K, et al. (2015) Site-specific methylation of Notch1 controls the amplitude and 22. Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: Unfolding the duration of the Notch1 response. Sci Signal 8(369):ra30. activation mechanism. Cell 137(2):216–233. 57. Perdigoto CN, Bardin AJ (2013) Sending the right signal: Notch and stem cells. 23. Wang Z, Li Y, Banerjee S, Sarkar FH (2009) Emerging role of Notch in stem cells and Biochim Biophys Acta 1830(2):2307–2322. cancer. Cancer Lett 279(1):8–12. 58. Keith B, Simon MC (2007) Hypoxia-inducible factors, stem cells, and cancer. Cell 24. Plessl K, Haider S, Fiala C, Pollheimer J, Knöfler M (2015) Expression pattern and 129(3):465–472. function of Notch2 in different subtypes of first trimester cytotrophoblast. Placenta 59. Gustafsson MV, et al. (2005) Hypoxia requires notch signaling to maintain the un- 36(4):365–371. differentiated cell state. Dev Cell 9(5):617–628. 25. Wu Y, et al. (2010) Therapeutic antibody targeting of individual Notch receptors. 60. Hiyama A, et al. (2011) Hypoxia activates the notch signaling pathway in cells of the Nature 464(7291):1052–1057.

intervertebral disc: Implications in degenerative disc disease. Arthritis Rheum 63(5): BIOLOGY 26. Rand MD, et al. (2000) Calcium depletion dissociates and activates heterodimeric 1355–1364.

notch receptors. Mol Cell Biol 20(5):1825–1835. DEVELOPMENTAL 61. Wang Z, et al. (2012) Notch signaling pathway regulates proliferation and differen- 27. Rosati E, et al. (2009) Constitutively activated Notch signaling is involved in survival tiation of immortalized Müller cells under hypoxic conditions in vitro. Neuroscience and apoptosis resistance of B-CLL cells. Blood 113(4):856–865. 214:171–180. 28. Li Y, Moretto-Zita M, Leon-Garcia S, Parast MM (2014) p63 inhibits extravillous tro- 62. Koch U, Lehal R, Radtke F (2013) Stem cells living with a Notch. Development 140(4): phoblast migration and maintains cells in a cytotrophoblast stem cell-like state. Am J 689–704. Pathol 184(12):3332–3343. 63. Mungamuri SK, Yang X, Thor AD, Somasundaram K (2006) Survival signaling by 29. Saha B, Home P, Gunewardena S, Paul S (2016) Transcriptional activity of TEAD4 Notch1: Mammalian target of rapamycin (mTOR)-dependent inhibition of p53. ensures self-renewal of trophoblast progenitors during post-implantation placental – development. FASEB J 30(Suppl 1):585.3 (abstr). Cancer Res 66(9):4715 4724. 30. Moretti F, et al. (2010) A regulatory feedback loop involving p63 and IRF6 links the 64. Perumalsamy LR, Nagala M, Banerjee P, Sarin A (2009) A hierarchical cascade acti- pathogenesis of 2 genetically different human ectodermal dysplasias. J Clin Invest vated by non-canonical Notch signaling and the mTOR-Rictor complex regulates – 120(5):1570–1577. neglect-induced death in mammalian cells. Cell Death Differ 16(6):879 889. 31. Yashiro-Ohtani Y, et al. (2014) Long-range enhancer activity determines Myc sensi- 65. Wang H, et al. (2011) Genome-wide analysis reveals conserved and divergent features tivity to Notch inhibitors in T cell leukemia. Proc Natl Acad Sci USA 111(46): of Notch1/RBPJ binding in human and murine T-lymphoblastic leukemia cells. Proc – E4946–E4953. Natl Acad Sci USA 108(36):14908 14913. 32. Weng AP, et al. (2006) c-Myc is an important direct target of Notch1 in T-cell acute 66. Bilban M, et al. (2009) Identification of novel trophoblast invasion-related genes: lymphoblastic leukemia/lymphoma. Genes Dev 20(15):2096–2109. Heme oxygenase-1 controls motility via peroxisome proliferator-activated receptor – 33. Restivo G, et al. (2011) IRF6 is a mediator of Notch pro-differentiation and tumour gamma. Endocrinology 150(2):1000 1013. suppressive function in keratinocytes. EMBO J 30(22):4571–4585. 67. Sanalkumar R, Dhanesh SB, James J (2010) Non-canonical activation of Notch sig- – 34. James JL, Srinivasan S, Alexander M, Chamley LW (2014) Can we fix it? Evaluating the naling/target genes in vertebrates. Cell Mol Life Sci 67(17):2957 2968. potential of placental stem cells for the treatment of pregnancy disorders. Placenta 68. Biadasiewicz K, et al. (2014) Extravillous trophoblast-associated ADAM12 exerts pro- 35(2):77–84. invasive properties, including induction of integrin beta 1-mediated cellular spread- 35. Roberts RM, Fisher SJ (2011) Trophoblast stem cells. Biol Reprod 84(3):412–421. ing. Biol Reprod 90(5):101. 36. Soares MJ, Vivian JL (2016) Tipping the balance toward trophoblast development. 69. Tarrade A, et al. (2001) Characterization of human villous and extravillous tropho- Proc Natl Acad Sci USA 113(19):5144–5146. blasts isolated from first trimester placenta. Lab Invest 81(9):1199–1211. 37. Li Y, Parast MM (2014) BMP4 regulation of human trophoblast development. Int J Dev 70. Kim SB, et al. (2007) Activated Notch1 interacts with p53 to inhibit its phosphorylation Biol 58(2–4):239–246. and transactivation. Cell Death Differ 14(5):982–991. 38. Xu RH, et al. (2002) BMP4 initiates human embryonic stem cell differentiation to 71. Bauer S, et al. (2004) Tumor necrosis factor-alpha inhibits trophoblast migration trophoblast. Nat Biotechnol 20(12):1261–1264. through elevation of plasminogen activator inhibitor-1 in first-trimester villous ex- 39. Golos TG, Giakoumopoulos M, Gerami-Naini B (2013) Review: Trophoblast differen- plant cultures. J Clin Endocrinol Metab 89(2):812–822. tiation from human embryonic stem cells. Placenta 34(Suppl):S56–S61. 72. Sonderegger S, et al. (2010) Wingless (Wnt)-3A induces trophoblast migration and 40. Lee CQ, et al. (2016) What Is trophoblast? A combination of criteria define human matrix metalloproteinase-2 secretion through canonical Wnt signaling and protein first-trimester trophoblast. Stem Cell Rep 6(2):257–272. kinase B/AKT activation. Endocrinology 151(1):211–220. 41. Yabe S, et al. (2016) Comparison of syncytiotrophoblast generated from human 73. Saleh L, Otti GR, Fiala C, Pollheimer J, Knöfler M (2011) Evaluation of human first embryonic stem cells and from term . Proc Natl Acad Sci USA 113(19): trimester decidual and telomerase-transformed endometrial stromal cells as model E2598–E2607. systems of in vitro decidualization. Reprod Biol Endocrinol 9:155.

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