Diverse subtypes and developmental origins of trophoblast giant cellsin the mouse placenta
David G. Simmons a, Amanda L. Fortier b, James C. Cross a,⁎
a Department of Biochemistry and Molecular Biology, University of Calgary, Faculty of Medicine, HSC Room 2279, 3330 Hospital Drive, N.W.,Calgary, Alberta, Canada T2N 4N1
b Department of Human Genetics, Montreal Children,s Hospital Research Institute, Montreal, Quebec, Canada H3Z 2Z3
Received for publication 28 September 2006; revised 19 December 2006; accepted 4 January 2007Available online 10 January 2007
Abstract
Trophoblast giant cells (TGCs) are the first terminally differentiated subtype to form in the trophoblast cell lineage in rodents. In addition tomediating implantation, they are the main endocrine cells of the placenta, producing several hormones which regulate the maternal endocrine andimmune systems and promote maternal blood flow to the implantation site. Generally considered a homogeneous population, TGCs have beenidentified by their expression of genes encoding placental lactogen 1 or proliferin. In the present study, we have identified a number of TGCsubtypes, based on morphology and molecular criteria and demonstrated a previously underappreciated diversity of TGCs. In addition to TGCsthat surround the implantation site and form the interface with the maternal deciduas, we demonstrate at least three other unique TGC subtypes:spiral artery-associated TGCs, maternal blood canal-associated TGCs and a TGC within the sinusoidal spaces of the labyrinth layer of theplacenta. All four TGC subtypes could be identified based on the expression patterns of four genes: Pl1, Pl2, Plf (encoded by genes of theprolactin/prolactin-like protein/placental lactogen gene locus), and Ctsq (from a placental-specific cathepsin gene locus). Each of these subtypeswas detected in differentiated trophoblast stem cell cultures and can be differentially regulated; treatment with retinoic acid induces Pl1/Plf +
The placenta is composed of a variety of differentiatedepithelial cell types (trophoblast) each having specializedfunctions during pregnancy. The trophoblast lineage is bestunderstood in mice and is derived from the trophectoderm,which forms at the blastocyst stage of development. Thetrophectoderm forms an outer shell of cells surrounding theinner cell mass. After implantation, trophectoderm cells not indirect contact with the inner cell mass, the mural trophectoderm,stop dividing and differentiate to form trophoblast giant cells(TGCs) which line the implantation chamber, anastomosing toform a diffuse network of blood sinuses for the early transportand exchange of nutrients and endocrine signals (Bevilacqua
and Abrahamsohn, 1988). In contrast, the trophectodermimmediately overlying the inner cell mass, the polar trophecto-derm, continues to proliferate and gives rise to all the remainingtrophoblast cell types of the placenta (Cross et al., 1994),including spongiotrophoblast, glycogen trophoblast cells,several labyrinth trophoblast cell types, and a later wave ofTGCs (called ‘secondary’ to distinguish them from the initial‘primary’ group) (Simmons and Cross, 2005). Secondary TGCsare thought to derive from the differentiation of ectoplacentalcone precursors, since cultured trophoblast stem cells first passthrough a progenitor phase characterized by the expression ofMash2 and Tpbpa (markers of ectoplacental cone) beforeexpressing TGC-specific genes (Carney et al., 1993; Scott et al.,2000).
TGCs are large, highly polyploid cells that form through theprocess of endoreduplication (Gardner and Davies, 1993;MacAuley et al., 1998). They are inherently invasive and
phagocytic and first mediate the invasion of the implantedconceptus into the maternal decidua (Zybina et al., 2000). Inaddition, they produce paracrine and endocrine factors includingangiogenic factors (Lee et al., 1988; Carney et al., 1993; Vosset al., 2000), vasodilators (Yotsumoto et al., 1998; Gagioti et al.,2000), and anticoagulants (Weiler-Guettler et al., 1996),presumably to promote blood flow to the implantation site,luteotrophic and lactogenic hormones to influence maternalresponses to pregnancy (Linzer and Fisher, 1999; Cross et al.,2002), and interferon that affects the differentiation of decidualstromal cells (Bany and Cross, 2006). Terminally differentiatedTGCs express several members of the prolactin/placentallactogen gene family (Wiemers et al., 2003), including placentallactogen 1 (Pl1/Csh1), placental lactogen 2 (Pl2/Csh2), andproliferin (Plf) (Lee et al., 1988; Faria et al., 1990, 1991;Yamaguchi et al., 1992, 1994; Carney et al., 1993; Hamlin et al.,1994), genes widely used as markers of TGCs. In addition,TGCs also express members of the cathepsin gene family thatare clustered on mouse chromosome 13 (Hemberger et al., 2000;Ishida et al., 2004) which, like the prolactin/placental lactogengene locus (Wiemers et al., 2003), represents a rodent-specificexpansion of genes that appear to be exclusively expressed in theplacenta (Deussing et al., 2002; Sol-Church et al., 2002).
Differentiation of TGCs is often considered a “defaultpathway” as factors which promote trophoblast stem cell self-renewal such as FGF, Nodal, Activin, and TGFβ activelysuppress TGC formation and withdrawal of these factorsresults in rapid differentiation into TGCs (Tanaka et al., 1998;Erlebacher et al., 2004; Guzman-Ayala et al., 2004; Hugheset al., 2004). Despite this, numerous exogenous factors suchas retinoic acid (RA) (Yan et al., 2001; Hemberger et al.,2004), diethylstilbestrol (DES) (Tremblay et al., 2001),parathyroid hormone-related protein (PTHrP) (El-Hashashet al., 2005), and nerve growth factor (NGF) (Kanai-Azumaet al., 1997) have all been shown to promote TGC formation.Whether these paracrine or endocrine factors are required invivo is unclear. The Hand1 transcription factor is required forTGC differentiation as Hand1 deficient conceptuses diebetween embryonic day (E) 7.5 and 8.5 due to a block inTGC formation (Riley et al., 1998; Scott et al., 2000). Hand1appears to be required for both primary and secondary TGCdifferentiation since there are fewer trophoblast cells liningthe implantation site and the ones that are present are smallerthan normal and have reduced expression of TGC-specificgenes (Scott et al., 2000). In vitro, Hand1 mutant trophoblastcells are also less invasive (Hemberger et al., 2004).
Recent observations indicate that TGCs may not be ahomogeneous population. For example, detailed studies of theuterine vasculature during gestation identified Plf-positive/Pl1-negative trophoblast cells which have replaced the maternalendothelial cells in the lumen of the spiral arteries (Adamsonet al., 2002; Cross et al., 2002; Hemberger et al., 2003).Interestingly, the endovascular trophoblast cells express only Plfand not Pl1, even at a time when Pl1 is still strongly expressed inTGCs at the barrier between the decidua and ectoplacental cone,indicating that there may be more than one type of TGC. Infurther support of this idea, there have been several reports of
cells that express Pl2, as well as several cathepsin genes, withinthe labyrinth layer of the placenta during the second half ofgestation (Campbell et al., 1989; Deb et al., 1991; Dai et al.,2000; Sahgal et al., 2000; Lee et al., 2003; Ishida et al., 2004;Simmons and Cross, 2005). These cells appear to be differentthan the TGCs lining the implantation site and invading spiralarteries because, while they express Pl2, they never express Pl1or Plf. Together these observations suggest the existence ofseveral TGCs subtypes within the murine placenta. In the courseof further studying the mouse placenta in detail, we have indeedcharacterized new subtypes of TGCs. Based on characteristiccell morphologies, ploidy analysis, localization, and differentialexpression of genes (Pl1, Pl2, Plf, and Ctsq), we havedistinguished four TGC subtypes both in vivo and in vitro.Using cell lineage tracing, we have found that the TGC subtypeshave different developmental origins.
Materials and methods
Animals
For wild-type conceptuses, CD1 mice (Charles River) were crossed andpregnant females dissected at E8.5 through E18.5 (noon of the day of vaginalplug is designated E0.5). For cell lineage tracing studies, Tpbpa-Cre-GFPfemale mice (Fortier et al., submitted for publication) were crossed with maleZ/AP mice (Lobe et al., 1999) and pregnant females were dissected at E14.5.Briefly, Tpbpa-Cre-GFP mice were constructed by placing a Cre recombinase-IRES-EGFP cassette downstream of 5.4 kb of the Tpbpa promoter (Calzonetti etal., 1995). The construct was then used to generate transgenic mice viapronuclear injection. Three lines were generated which produced similartemporal and spatial expression of Cre-EGFP in accordance with endogenousTpbpa. Line 5 was used for our studies because it displayed the earliest onsetof expression (∼E8.0) which coincides with the onset of endogenous expression(Calzonetti et al., 1995). Expression at this time is restricted to the tip of theectoplacental cone but later is detectable throughout the spongiotrophoblast. Crerecombinase activity was also detectable as early as E8 (Fortier et al., submittedfor publication). All animals were housed under normal light conditions (12 hlight/12 h dark) with free access to food and water. All animal procedures werecarried out in accordance with the University of Calgary Animal CareCommittee.
Cell culture
Trophoblast stem (TS) cells (tgRs26 provided by Tilo Kunath and JanetRossant) were grown as previously described (Tanaka et al., 1998; Hughes et al.,2004) at 37 °C under 95% air and 5% CO2. TS cell medium contained 20% fetalbovine serum (CanSera), 1 mM sodium pyruvate (Invitrogen), 50 μg/mlpenicillin/streptomycin, 5.5×10−5 M β-mercaptoethanol (Invitrogen), 25 ng/mlbasic fibroblast growth factor (Sigma), and 1 μg/ml heparin (Fisher Scientific) inRPMI 1640 (Invitrogen), with 70% of the medium being preconditioned byincubating on embryonic fibroblasts for 48 h. Differentiating medium consistedof the TS medium but without basic fibroblast growth factor, heparin, orembryonic fibroblast preconditioning. Hand1 heterozygous and homozygousmutant TS cells were cultured under the same conditions as tgRs26 TS cells andtheir generation has been previously described (Hemberger et al., 2004). In someexperiments, retinoic acid (RA, Sigma) was dissolved in ethanol and added toTS medium to a final concentration of 5 μM (RA), ethanol only was used as acontrol.
RNA isolation and northern blot analysis
Total RNA was collected from ectoplacental cone plus chorion tissues atE8.5 and E9.5 and placentas from E10.5 through E18.5 by homogenization inTrizol Reagent (Invitrogen), following the manufacturer's instructions. Total
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RNA from TS cell cultures was isolated using one QIAshredder and RNeasycolumn (Qiagen) per well of a six-well plate following the manufacturer'sinstructions. Ten micrograms of total RNA was separated on a 1.1%formaldehyde agarose gel, blotted onto GeneScreen nylon membrane (PerkinElmer), and UV cross-linked. Random-primed DNA labeling of cDNA probesfor Pl1, Pl2, Plf, and Ctsq was carried out with 25 μCi 32P-dCTP and isolatedon Sephadex G-50 columns (AmershamBiosciences). Hybridizations were doneat 60 °C overnight in hybridization buffer as previously described (Church andGilbert, 1984). Signals were detected by exposure to BioMax MR film (Kodak)at −80 °C.
Tissue preparation and in situ hybridization
Implantation sites (E9.0) and placentas (E14.5) were dissected in coldphosphate buffered saline (PBS) and fixed overnight in 4% paraformaldehyde(PFA) in PBS at 4 °C. After 3× rinsing in PBS, tissues went through gradedsucrose solutions (10% in PBS and 25% in PBS) before being embedded in OCT(Tissue Tek). Ten micron sections were cut on a cryostat (Leica), mounted onSuper Frost Plus slides (VWR), and stored at −80 °C. For in situ hybridization,sections were re-hydrated in PBS, post-fixed in 4% PFA for 10 min, treated withproteinase K (15 μg/ml for 5 min at room temperature for E9.0 implantation sitesand 30 μg/ml for 10 min at room temperature for E14.5 placentas), acetylated for10 min (acetic anhydride, 0.25%; Sigma), and hybridized with digoxigenin-labeled probes overnight at 65 °C. Digoxigenin labeling was done according tothe manufacturers instructions (Roche). Hybridization buffer contained 1× salts(200 mM sodium choride, 13 mM tris, 5 mM sodium phosphate monobasic,5 mM sodium phosphate dibasic, 5 mM EDTA), 50% formamide, 10% dextransulfate, 1 mg/ml yeast tRNA (Roche), 1× Denhardt's (1% w/v bovine serumalbumin, 1% w/v Ficoll, 1% w/v polyvinylpyrrolidone), and DIG-labeled probe(final dilution of 1:2000 from reaction with 1 μg template DNA). Two 65 °Cpost-hybridization washes were carried out (1× SSC, 50% formamide, 0.1%tween-20) followed by two RTwashes in 1× MABT (150 mM sodium chloride,100 mM maleic acid, 0.1% tween-20, pH 7.5), and 30 min RNAse treatment(400 mM sodium chloride, 10 mM tris pH7.5, 5 mM EDTA, 20 μg/ml RNAseA). Sections were blocked in 1× MABT, 2% blocking reagent (Roche), 20%heat inactivated goat serum for 1 h, and incubated overnight in block with anti-DIG antibody (Roche) at a 1:2500 dilution. After four 20 min washes in 1×MABT, slides were rinsed in 1× NTMT (100 mMNaCl, 50 mMMgCl, 100 mMtris pH 9.5, 0.1% tween-20) and incubated in NBT/BCIP in NTMTaccording tothe manufacturer's instructions (Promega). Slides were counterstained withnuclear fast red, dehydrated and cleared in xylene, and mounted in cytosealmounting medium (VWR).
For double in situ experiments on TS cell cultures, the same protocol wascarried out with minor modifications. Cells were fixed in 4% PFA for 15 min andwere not proteinase K treated. Also, probes were labeled with fluorescein as wellas digoxigenin according to the manufacturer's instructions and added to thehybridization mix. After the digoxigenin probe was detected with NBT/BCIP,the enzyme was inactivated with heating to 65 °C for 30 min in 1× MABT,followed by 30 min incubation in 0.1 M glycine pH 2.2, blocked for 1 h inblocking solution and incubated overnight at 4 °C with anti-fluorescein antibody(Roche, 1:2500). Fluorescein probes were detected by incubation with INT/BCIP (Roche) until a brown precipitate formed. Reactions were stopped in PBS,counterstained with nuclear fast red, and mounted under 50% glycerol/PBS formicroscopy.
β-Galactosidase and alkaline phosphatase staining
β-Galactosidase and alkaline phosphatase staining was carried out aspreviously described (Lobe et al., 1999). Briefly, placentas were fixed in 2%PFA/0.2% glutaraldehyde for 4 h with rocking (bisected after the first hour) andprocessed through a series of sucrose gradients and embedded in OCT (TissueTek). Ten microgram cryosections were then stained for either β-galactosidaseor alkaline phosphatase activity using NBT/BCIP (Roche) as a substrate forβ-galactosidase or INT/BCIP (Roche) as a substrate for human alkalinephosphatase as previously described (Lobe et al., 1999). For double staining onthe same section, β-galactosidase staining was done first because detection ofhuman alkaline phosphatase activity driven from the transgene required a 30 minheat inactivation of endogenous alkaline phosphatase activity, and this step
inactivates β-galactosidase activity. Counterstaining was done with nuclear fastred and mounting was done using 50% glycerol/PBS.
Nuclear area and estimation of DNA content
Fluorescent in situ hybridizations were done on 10 μm cryosections of E14.5placentas with probes for Plf and Pl2 as described above with the followingmodifications. Detection of the digoxigenin labeled probes was done withhorseradish peroxidase conjugated anti-DIG antibodies (Roche, 1:2500)followed by amplification with fluorescein or TMR conjugated tyramide(Perkin Elmer). Nuclei were counterstained with DAPI. Nuclear area and DNAcontent of Plf + or Pl2+ TGCs were estimated using OpenLab software(Improvision). As a control (representing 2C nuclei), Plf −/Pl2− endothelial cellnuclei or syncytiotrophoblast nuclei were also quantified. Statistical significancewas evaluated using a one-way ANOVA.
Results
Expression of genes from the prolactin/placental lactogen andcathepsin gene families can distinguish TGC subtypes
In the course of using various molecular markers tocharacterize the different trophoblast cell subtypes within themouse placenta, we focused on four that emerged as importantones for distinguishing different cell phenotypes (Pl1, Pl2, Plf,and Ctsq) (Fig. 1A). Plf expression was detected early ingestation, beginning at E6.5, in TGCs that line the wall ofthe implantation site separating the ectoplacental cone fromthe maternal decidua, herein referred to as “parietal TGCs” orP-TGCs (Fig. 1B, data not shown). Later in gestation, Plfexpression was also detected in the trophoblast cells lining thespiral arteries (SpA-TGCs) and canal spaces (C-TGCs) (Figs.1F, J, N), which result from the coalescence of the spiral arteries(Fig. 1A). Plf expression was not detectable within the labyrinthlayer of the placenta (Fig. 1R) although scattered staining couldbe seen in the spongiotrophoblast layer (Fig. 1F). Similar to Plf,Pl1 expression could be detected early in gestation in P-TGCs(Fig. 1C) and was absent from the labyrinth (Fig. 1S). However,unlike Plf, Pl1 expression was not detected in SpA-TGCs (Fig.1K) or C-TGCs (Fig. 1O) and expression was not maintained inP-TGCs past E10.5 (Fig. 2). Pl2 expression was not detecteduntil ∼E9.5 and was observed in P-TGCs and spongiotropho-blast (Figs. 1D and 2, data not shown). Pl2 expression was notdetected in SpA-TGCs (Fig. 1L). In the mature placenta, Pl2expression was detected in P-TGCs, spongiotrophoblast (Fig.1H), C-TGCs (Fig. 1L), and cells within the labyrinth layer (Fig.1T). Pl2 expression within the labyrinth was restricted to themononuclear trophoblast cells which line the maternal sinusoids(Fig. 1T) (Simmons and Cross, 2005). Ctsq was expressedexclusively in these Pl2+ mononuclear trophoblast cells liningthe maternal sinusoids (Figs. 1E, I, M, Q, U), cells we will referto as sinusoidal TGCs (S-TGCs). Ctsq expression was notdetectable before ∼E11.5 (Figs. 1E and 2) but was evident untilthe end of gestation (Fig. 2). Based on these observations, fourdistinct TGC subtypes could be identified using gene expres-sion patterns and spatial location; Pl1 and Ctsq exclusivelymarked P-TGCs and S-TGCs, respectively. SpA-TGCs wereidentified by expression of Plf alone and C-TGCs by expressionof both Plf and Pl2, but not Pl1 or Ctsq.
Fig. 1. Expression of genes from the prolactin and cathepsin gene families define subsets of trophoblast giant cells. (A) Diagram depicting the structures and cell typesof the murine placenta. (B–U) In situ hybridization for Pl1 (B, F, J, N, R), Pl2 (C, G, K, O, S), Plf (D, H, L, P, T), and Ctsq (E, I, M, Q, U) on E9.0 implantation sites(B–E) and E14.5 placentas (F–U). TGCs lining the implantation site, separating the maternal decidua from the ectoplacental cone (and later the spongiotrophoblastlayer), P-TGCs (B-E), express Pl1, Plf, and Pl2 (after E9.5, not shown), but not Ctsq. Spiral artery-associated TGCs, SpA-TGCs, express Plf exclusively. Maternalcanal-associated TGCs, C-TGCs, express both Pl2 and Plf but do not express Pl1 or Ctsq. TGCs within the labyrinth layer, S-TGC, express both Pl2 and Ctsq but notPl1 or Plf. C—canal lumen, SpA—spiral artery lumen, *—SpA-TGC, black arrow head—C-TGC, black arrows—S-TGC.
Fig. 2. Temporal expression of trophoblast giant cell subtype markers. Northernblot analysis was performed using probes for Pl1, Pl2, Plf, and Ctsq and RNAfrom isolated ectoplacental cone/chorion tissue (E8.5–9.5) and placentas(E10.5–18.5).
Fig. 3. Trophoblast giant cell subtypes are polyploid. (A) SpA-TGCs, C-TGCs,and STGCs have significantly larger nuclei at E14.5 than endothelial orsyncytiotrophoblast cells (p<0.001). P-TGCs have significantly larger nucleithan SpA-TGC, C-TGCs, STGCs, and endothelial or syncytiotrophoblast cells(p<0.001). (B) SpA-TGCs, C-TGCs, and S-TGCs have significantly higherDNA content at E14.5 than endothelial or syncytiotrophoblast cells (p<0.001).P-TGCs have significantly higher DNA content than SpA-TGC, C-TGCs, S-TGCs, and endothelial or syncytiotrophoblast cells (p<0.001). DAPI intensitymeasured in arbitrary units (AU). (C) Representative florescent in situhybridization of Plf (green) or Pl2 (red), indicating Plf (+) P-TGC, Plf (+)SpA-TGC, Pl2 (+) C-TGC and Pl2 (+) S-TGC. Scale bar=0.1 mm.
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All TGC subtypes are mononuclear but polyploid
TGCs were first defined as large cells with high ploidy as aresult of endoreduplication (Gardner et al., 1973; Ilgren, 1983;Zybina and Zybina, 1996). Therefore, nuclear size and relativeDNA content of the different TGC subtypes were measured andcompared with diploid control cells (endothelial and syncytio-trophoblast cells of the labyrinth) in sections of E14.5 placenta.Fluorescent in situ hybridization for Plf and Pl2 was used toidentify each TGC subtype based on a positive signal andspatial location, and DAPI staining was used to visualize theirnuclear size and estimate DNA content (Fig. 3C). The nucleararea of all TGC subtypes was significantly larger than that ofdiploid controls (Fig. 3A, p<0.001). In addition, all TGCsubtypes had significantly higher estimated DNA content thandiploid control cells (Fig. 3B, p<0.001). Notably, P-TGCs hadlarger nuclei and a higher DNA content than the other threeTGC subtypes (Figs. 3A, B, p<0.001).
All TGC subtypes appear during differentiation of trophoblaststem cell cultures
To investigate whether all four TGC subtypes appear duringdifferentiation of trophoblast stem cell (TS) cultures, we firstanalyzed the expression of Plf, Pl1, Pl2, and Ctsq by northernblotting. Expression of all four genes was undetectable in stemcells and during early differentiation. Strong expression wasdetected by day 6 of differentiation (Fig. 4A). Based on in vivoexpression patterns, Pl1 and Ctsq expression indicated thepresence of P-TGCs and S-TGCs, respectively. In situhybridization confirmed Pl1 as well as Ctsq expressing TGCsin day 6 differentiated cultures (Figs. 4B, D, E). In situhybridizations with single probes for Pl1, Pl2, Plf, or Ctsqnever showed staining in more than ∼40% of TGCs, alsosuggesting that TGCs are heterogeneous in differentiating TScell cultures. In addition, Ctsq expressing TGCs were lessabundant in day 6 differentiated cultures than TGCs expressingPl1, Pl2, or Plf. To determine whether SpA-TGC or C-TGCwas present in the differentiating TS cell cultures, co-localization was required. Dual color in situ hybridizationidentified TGCs which were Plf +/Pl− (Fig. 4D), Plf +/Pl2−
(Fig. 4C), and Pl2+/Plf + (Fig. 4C). Together these observationsindicated that SpA-TGCs and C-TGCs are most likely present
in differentiating TS cell cultures as well, although triplelabeling would be required to definitively identify them.
Hand1 regulates the formation of all TGC subtypes
Previous studies have indicated a critical role for the bHLHtranscription factor Hand1 during TGC differentiation (Riley etal., 1998; Scott et al., 2000; Hughes et al., 2004). However, theanalysis to date has been limited to what we now define as P-TGCs because Hand1 mutants die before the time that SpA-TGCs, C-TGCs, and S-TGCs develop. In situ hybridizationdemonstrated that Hand1 was expressed in all four TGCsubtypes (Figs. 5C–F). The one exception is that Hand1expression was not detected in SpA-TGCs that had invadedfurthest up into the spiral arteries, cells that do still express Plf,implying that expression is lost by the distal end of their invasionat least at E14.5 (Figs. 5A, B). To explore the functionalsignificance of Hand1, we assessed the expression of Plf, Pl1,Pl2, and Ctsq in Hand1 mutant TS cells. Northern blot analysisof differentiating Hand1−/− TS cells showed reduced expression
Fig. 4. Trophoblast giant cell subtypes appear in differentiating TS cell cultures.(A) Northern blot analysis of Pl1, Pl2, Plf, and Ctsq mRNA expression in TScells over 6 days of culture in the presence (proliferating) or absence(differentiating) of FGF, heparin, and fibroblast conditioned medium (CM).(B) In situ hybridization of TGC subtypes in differentiating TS cell cultures (day6 of differentiation in the absence of FGF/CM). Double in situ hybridizationsindicate Pl1+ TGCs (B, D), Pl2+ TGCs (B, C), Plf+ TGCs (C, D), Pl1/Pl2+TGCs (B), Pl1/Plf+ TGCs (C), and Pl2/Plf+ TGCs (D). Single in situhybridization indicates the expression of Ctsq+ TGCs (E). Scale bar=0.1 mm.
of all TGC marker genes compared to control (Hand1+/−) cells(Fig. 5G), indicating that loss ofHand1 impairs the formation ofall TGCs regardless of subtype. In addition, the expressionpatterns for the trophoblast stem cell markers Esrrb and Eomes(Fig. 5G) were not altered in differentiating Hand1 mutant TScells when compared with control.
Retinoic acid has differential effects on TGC subtypedifferentiation
Retinoic acid (RA) has been shown to promote differentia-tion of TS cells into Pl1-expressing TGCs (Yan et al., 2001;Hemberger et al., 2004). We examined the expression of otherTGC marker genes and, strikingly, found that RA did not induceall genes uniformly. Instead, RA-induced Pl1 and Plf only(indicative of P-TGCs), indicating that S-TGCs and C-TGCswere not present in these cultures (Fig. 6). As expected, RAtreatment induced TGC formation even under proliferatingconditions (in the presence of FGF4 and feeder cell conditionedmedium). Interestingly, under differentiating culture conditions
(in the absence of FGF4 and feeder cell conditioned medium),exposure to RA induced Pl1 and Plf expression but suppressedthe expression of Pl2 and Ctsq (Fig. 6). Together, these datasuggested that P-TGCs are preferentially induced by RA butthat S-TGCs are suppressed.
Not all TGC subtypes arise from Tpbpa+ precursors
It has been hypothesized that differentiation of secondaryTGCs involves intermediate steps, with cells passing through aprogenitor phase characterized by the expression of Mash2 andTpbpa before terminal differentiation (Carney et al., 1993;Scott et al., 2000) and that these precursors reside within theectoplacental cone (and later the spongiotrophoblast layer). Todetermine if all TGC subtypes originate from Tpbpa+
precursors, we took advantage of a transgenic mouse drivingCre recombinase under the control of the Tpbpa promoter. Bycrossing female Tpbpa-Cre mice with male Z/AP dual reportermice (Lobe et al., 1999), Cre induced the removal of the LacZgene encoding β-galactosidase, and therefore the subsequentactivation of human alkaline phosphatase (hAP), in all cellsthat had previously or continued to express Tpbpa. Stainingfor hAP activity therefore identified cells which originatedfrom Tpbpa+ precursors and β-galactosidase activity indicatesthose cells which did not (Fig. 7A). Examination of E14.5placentas indicated that none of the S-TGCs in the labyrinthlayer expressed hAP (Figs. 7B, F, J, N and 8). In contrast,virtually all SpA-TGCs observed were positive for hAP (Figs.7D, H, L, P and 8). Interestingly, the origins of P-TGCs (Figs.7C, G, K, O) and C-TGCs (Figs. 7E, I, M, Q) were mixed,with approximately half of each subtype originating fromTpbpa+ precursors (Fig. 8). Based on these data, TGCsubtypes had different developmental origins. In addition,TGCs of the same subtype could also have differentdevelopmental origins.
Discussion
While a few studies have previously described heterogeneityin gene expression within TGCs (Weiler-Guettler et al., 1996;Hunter et al., 1999; Hemberger et al., 2000; Ma and Linzer,2000), most have generally regarded TGCs as a homogeneoussingle cell population that lines the implantation site. Thesecells, what we now call parietal TGCs (P-TGCs), form thebarrier between the maternal decidua and the ectoplacental coneand later the spongiotrophoblast layer. We have demonstratedthe existence of additional TGC subtypes within the matureplacenta. The four different TGC subtypes we have describedare all postmitotic and polyploid, but can be distinguished bydifferential location, gene expression, cell lineage origins, andregulation of differentiation (Fig. 9). Endovascular trophoblastcells have previously been implied based on the expression ofPlf (Adamson et al., 2002; Hemberger et al., 2003), but wehave expanded this to now show that these cells are indeedpolyploid. Pl2 expressing cells have been identified in themouse and rat labyrinth layer, but they have not previously beenconfirmed to be TGCs (Campbell et al., 1989; Deb et al., 1991;
Fig. 5. Hand1 mRNA is expressed in all trophoblast giant cell subtypes and regulates trophoblast giant cell differentiation. Hand1 expression can be detected in allfour trophoblast giant cell subtypes (C–F). Hand1 expression is detected in SpA-TGCs proximal to the placenta (C) but appears absent from more distal SpA-TGCs(A) which still express Plf (B). Hand1 mutant TS cells show reduced expression of marker genes for all TGC subtypes in vitro, but do not have altered expression ofthe TS cell markers Err2 and Eomes (G). C—canal lumen, SpA—spiral artery lumen. Scale bar=0.1 mm.
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Dai et al., 2000; Sahgal et al., 2000; Lee et al., 2003; Ishida etal., 2004; Simmons and Cross, 2005), though a recent studyshowed that the mononuclear trophoblast cells in the labyrinthare indeed polyploid (Coan et al., 2005). We have brought thesedifferent observations together and added significant newdetails. With this knowledge, and the availability of newmarkers, mouse mutants with placental phenotypes can now bedescribed in greater detail. More importantly, the discovery ofseveral new TGC subtypes, some with overlapping patterns ofhormone gene expression, raises significant questions about thefunctions of these cells.
Fig. 6. Formation of trophoblast giant cell subtypes is differentially regulated by retintrophoblast giant cell formation in TS cell cultures, even in the presence of FGF andtrophoblast giant cell subtypes are uniformly up-regulated (compare with marker prTGCs. * Indicates TS cell morphology and arrowhead indicates TGC morphology.
The TGC subtypes that we have identified have distinctpatterns of gene expression, although only two have exclu-sive markers. Pl1 is unique to P-TGCs and Ctsq is unique toS-TGCs. For the other subtypes, we have not yet identified cellsubtype-specific genes. In addition, some genes are expressed inmultiple subtypes. For example, Plf is expressed in SpA-TGCs,P-TGCs, and C-TGCs while Pl2 is expressed in P-TGCs,C-TGCs, and S-TGCs. Distinct patterns of gene expression notonly serve to identify TGC subtypes, but also indicate differentprograms of differentiation. Previous studies have shown thatPl1 expression precedes Pl2 in P-TGCs (Carney et al., 1993).
oic acid. Northern blot analysis demonstrates that retinoic acid (RA) can induceembryonic fibroblast conditioned medium (CM). However, not all markers of
ofile of control) indicating preferential induction of P-TGCs and possibly SpA-Scale bar=0.1 mm.
Fig. 7. Not all trophoblast giant cells arise from Tpbpa+ precursors. Female mice carrying a transgene containing a Cre recombinase-IRES-EGFP cassette under thecontrol of the Tpbpa promoter were crossed with male Z/AP reporter mice to trace the fate of ectoplacental cone/spongiotrophoblast precursors. Placentas were stainedat E14.5 for either β-galactosidase (blue) and/or human alkaline phosphatase (brown). Almost all SpA-TGCs arise from Tpbpa (+) precursors (D, H, L, P) while noneof the S-TGCs do (B, F, J, N). P-TGC (C, G, K, O) and C-TGCs (E, I, M, Q) appear to arise from either Tpbpa (+) precursors or Tpbpa (−) precursors.
However, Pl1 expression is not detectable in the developinglabyrinth layer, and therefore Pl2 expression in S-TGCsappears without first going through a transient Pl1 expressingstage. In addition, SpA-TGCs turn off Pl1 expression beforeP-TGCs and, unlike P-TGCs, never express Pl2. Theseobservations indicate a unique differentiation program foreach TGC subtype.
The results of our study have not only added new celltypes, but have indicated a need to revise the past model ofhow the trophoblast cell lineage develops into the variousdifferentiated cell types. Previously, the P-TGC populationwas thought to arise both from the mural trophectoderm andfrom the secondary differentiation of precursors in the
ectoplacental cone and later the spongiotrophoblast layer(Rossant and Ofer, 1977; Johnson and Rossant, 1981; Carneyet al., 1993). Our results have indicated, however, that ∼50%of P-TGCs in the midgestation placenta are derived fromTpbpa− cells. There are only ∼50 mural trophectoderm cellsthat could form the primary wave of P-TGCs and yet there areseveral hundred P-TGCs lining the implantation site by E8.5(Scott et al., 2000). As a result, there must be an additionalsource of secondary P-TGCs in addition to Tpbpa+ precursorsof the ectoplacental cone, perhaps even from outside theectoplacental cone/spongiotrophoblast. The caveat with thestrict interpretation of the cell numbers is that when using Cre-mediated transgene activation, there may be some delay
Fig. 8. Different trophoblast giant cell subtypes have different developmentalorigins. Quantification of trophoblast giant cell subtypes originating fromTpbpa (+) (hPLAP positive—brown) versus Tpbpa (−) (β-galactosidasepositive—blue) precursors. Error bars=±S.E.M.
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between the onset of Cre expression and detection of the hAPactivity (Nagy, 2000). We previously addressed this possibilityby looking at whether there are cells in the spongiotrophoblastlayer that express Tpbpa mRNA but which do not also expresshPLAP, but we found a perfect congruence (A. Fortier et al.,unpublished). We have also found that the Cre transgenebegins expression and has associated recombinase activity asearly as E8.5. Therefore, if there is a delay, it probably is notsignificant and though may account for some unlabeled cells,it is unlikely to account for the hundreds of P-TGCs thatcannot be accounted for by mural trophectoderm precursorsalone.
The potential additional source of P-TGCs is either the directdifferentiation of trophoblast stem cells into P-TGCs ordifferentiation from a previously uncharacterized precursorpopulation in the ectoplacental cone that is devoid of Tpbpaexpression. Previous studies have shown that retinoic acidtreatment of TS cell cultures can induce TGC differentiation(based on morphology and Pl1 expression) without firstexpressing Tpbpa/4311 (Yan et al., 2001), clearly suggestingan alternative pathway of TGC differentiation. Our observationsshow that the type of TGC produced by RA treatment isspecifically the P-TGC subtype. Tpbpa expression at E8.5,when expression is initiated, does not encompass the chorion oreven the whole of the ectoplacental cone, particularly at the baseof the ectoplacental cone nearest the chorion. Therefore, it ispossible that P-TGC precursors reside in this area.
The origin of the other TGC subtypes is specific to eachsubtype. In contrast to P-TGCs, virtually all of the SpA-TGCswe observed expressed hPLAP, indicating their origin fromTpbpa+ precursors. This makes some sense as the ectoplacentalcone is oriented to grow towards the incoming spiral arteries. Incontrast, none of the S-TGCs was positive for hPLAPindicating an alternative developmental origin. As Tpbpaexpression does not encompass the whole of the ectoplacentalcone, particularly at the base closest to the chorion, it seemslikely that S-TGC cells arise from precursors either within theTpbpa− area of the ectoplacental cone or from choriontrophoblast directly. The different origins of SpA-TGCs andS-TGCs are not surprising considering the spatial localizationof the two subtypes in the mature placenta. C-TGCs havemixed developmental origins, with approximately half arisingfrom Tpbpa+ precursors, similar to the findings with P-TGCs.Interestingly, hPLAP+ C-TGCs cells are intermixed with β-
galactosidase+ C-TGCs cells along the entire length of thecanals, with no clear demarcations.
The ability to culture trophoblast stem (TS) cells has beeninvaluable for the study of trophoblast differentiation and TGCformation in particular. In this system, FGF, heparin, andembryonic fibroblast conditioned medium is required tomaintain proliferating conditions and promote TS cell self-renewal (Tanaka et al., 1998). Given the obvious utility of thissystem for the study of TGC differentiation, we used markersfor Plf, Pl1, Pl2, and Ctsq which can distinguish TGC subtypesin vivo to probe northern blots of differentiating TS cell cul-tures. All four markers were induced by day 6 of differentiation,indicating the presence of multiple TGC subtypes within theculture system.
TGC formation relies on the interplay of numerous cellintrinsic factors to guide proper differentiation, most notablybasic helix–loop–helix (bHLH) transcription factors and theirinteracting partners including Hand1, Mash2, Alf1, Itf2, Id1/2,and I-mfa (Cross et al., 1995; Jen et al., 1997; Kraut et al.,1998; Riley et al., 1998; Scott et al., 2000; Hughes et al., 2004).Hand1 in particular is essential for proper TGC differentiationas Hand1 deficient conceptuses die by E7.5 due to a block inTGC formation (Riley et al., 1998; Scott et al., 2000). Resultsof the current study indicated that Hand1 is expressed in allfour TGC subtypes and also that it regulates the differentiationof all TGC subtypes. Expression of all the various TGCsubtype markers was significantly impaired in differentiatingHand1 mutant TS cell cultures (Fig. 5G). The ability of thecells to begin to differentiate in general was not affected inthese cells as indicated by the correct downregulation of thetrophoblast stem cell markers Esrrb and Eomes. DifferentiatedHand1 mutant TS cells have previously been shown to containsignificantly fewer invasive TGCs, demonstrating an intrinsicdeficiency in TGC formation (Hemberger et al., 2004). In vivo,Hand1 is required both for differentiation of mural trophecto-derm into TGCs and to promote secondary TGC differentiation(Riley et al., 1998; Scott et al., 2000). It would be of obviousinterest to see whether other cell intrinsic and paracrine factorsknown to regulate P-TGC differentiation are also required fordifferentiation of other TGC subtypes. Retinoic acid treatmentdid not induce Pl2 or Ctsq expression, and in fact suppressedthe induction of these markers under differentiating conditions,indicating that retinoic acid promotes P-TGCs (and possiblySpA-TGCs also) but not C-TGCs or S-TGCs. These differ-ential effects imply that while there are some common features,the different TGC subtypes are also regulated by their ownmechanisms.
The existence of numerous TGC subtypes with unique geneexpression patterns and spatial localization begs the question,what is the functional significance of having multiple TGCsubtypes? One could speculate that the specific milieu ofhormones and growth factors produced by each TGC subtypewould have particular functions intimately tied to their locationwithin the placenta. For example, SpA-TGCs are known tosecrete factors which regulate blood flow to the implantationsite such as vasodilators and angiogenic factors. Proliferin(Plf), an angiogenic factor known to stimulate endothelial cell
Fig. 9. Summary of the trophoblast cell lineage and origins of different TGC subtypes.
migration (Jackson et al., 1994) and proliferin-related protein(Plfr), an anti-angiogenic factor (Jackson et al., 1994), are bothexpressed in SpA-TGCs. The combination of hormones andcytokines secreted by P-TGCs may preferentially targetdecidual cells or infiltrating leukocytes such as natural killercells. Prlpa is expressed predominantly by P-TGCs (Ma andLinzer, 2000) and has been shown to modulate uterine naturalkiller cells (Muller et al., 1999; Ain et al., 2003). S-TGCs,located within the labyrinth layer closest to the fetalcirculation, are well situated to facilitate delivery of hormonesand/or cytokines into the fetal circulation or to producemolecules which effectively clear, or modulate the bioactivity,of hormones before they enter the fetal circulation. This couldpotentially be an important function for Ctsq, a proteaseuniquely expressed in S-TGC, as it has been hypothesized tocleave members of the prolactin-like protein family such asPl2 (Ishida et al., 2004). Cleavage of prolactin, itself anangiogenic factor, by cathepsin D for example, results in ashorter fragment with anti-angiogenic activity (Struman et al.,1999; Lkhider et al., 2004; Piwnica et al., 2004). The potentialcleavage of other members of the prolactin family locus, ofwhich there are at least 23 known to be expressed within theplacenta (Wiemers et al., 2003), possibly by placentallyexpressed cathepsins, may prove to be a critical regulatorysystem within the placenta to augment hormone bioactivitybefore entering the fetal circulation or re-entering the maternalcirculation.
Some genes have multiple sites of expression, such as Pl2and Plf. However, the biological action of these gene productslikely depends on the local environment they are secreted into,with different TGC subtypes using the same molecule todifferent ends. For example, Plf can stimulate angiogenesis andendothelial cell migration (Jackson et al., 1994), a functionrequired in the immediate vicinity of SpA-TGCs. But Plfsecretion from P-TGCs may be critical for stimulating uterinecell proliferation (Nelson et al., 1995), something which occurs
widely within the decidua bordering P-TGCs. Likewise, Pl2 isexpressed in multiple TGC subtypes and is known to actthrough the prolactin receptor to regulate maternal adaptationsto pregnancy such as mammary gland development and corpusluteum maintenance (Soares, 2004) or to bind receptors in fetalliver, pancreas, kidney, and intestine influencing fetal develop-ment (Freemark et al., 1993). TGC subtype-specific expressionmay determine which compartment, fetal or maternal, istargeted. Pl2 is expressed early in gestation from P-TGCs,when maternal Pl2 serum levels are high and in the second halfof gestation from S-TGCs when Pl2 first appears in the fetalcirculation (Kishi et al., 1991).
In conclusion, the present study demonstrates a previouslyunderappreciated level of TGC sub-specialization and providesa panel of markers which allows for the identification of at leastfour TGC subtypes. In addition, cell lineage tracing studiesshow that TGC subtypes can have different developmentalorigins, and even TGCs of the same subtype can have more thanone developmental origin. There are likely more subtypes to beidenified as a number of genes expressed in P-TGCs, forexample, are not uniformly expressed within this population,including Mrj (Hunter et al., 1999), Thrombomodulin (Weiler-Guettler et al., 1996), and members of the Cathepsin family(Hemberger et al., 2000). In addition, Ma and Linzer describePrlpa expression in secondary TGCs but not in primaryTGCs, adding a marker distinguishing TGCs from these twodifferent trophectoderm lineages (Ma and Linzer, 2000). Studiesinvestigating trophoblast differentiation or mutant mice withplacental phenotypes have often relied on the expression of justa few TGC markers, particularly Pl1, Pl2, or Plf, as a read outof TGC formation and function. In light of our findings, we nowknow that expression of these specific genes does not reflect thewhole of the TGC population. Clearly, future studies of tro-phoblast differentiation or gene targeted mice with placentalphenotypes should account for the diversity of trophoblastsubtypes.
577D.G. Simmons et al. / Developmental Biology 304 (2007) 567–578
Acknowledgments
The authors would like to thank David Natale for his technicalassistance and valuable discussions and Martha Hughes andColleen Geary for excellent technical assistance. D.S. wassupported by fellowships from the Lalor Foundation and theAlberta Heritage Foundation for Medical Research (AHFMR).The work was supported by operating grants from the CanadianInstitutes of Health Research (CIHR) (to J.C.C.). J.C.C. is aCIHR Investigator and an AHFMR Scientist.
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