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Ly6e expression is restricted to syncytiotrophoblast cells of the mouse placenta
Placenta 34 (2013) 831e835
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Short communication
Ly6e expression is restricted to syncytiotrophoblast cells of the mouse placenta M. Hughes a, B.V. Natale a, D.G. Simmons b, D.R.C. Natale a, * a b
Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 29 May 2013
In the present study, we characterized the expression of lymphocyte antigen 6, locus E (Ly6e) in mouse placental trophoblast. We identified Ly6e mRNA expression in trophoblast stem (TS) cells by a gene expression screen. In vivo, Ly6e was first detectable by mRNA in situ hybridization in the chorion beginning at E8.5 with spatial expression similar to Syncytin a (Syna). At later stages of gestation, Ly6e was restricted to syncytiotrophoblast in the labyrinth. Northern blot confirmed that Ly6e was expressed in both undifferentiated and differentiated TS cell cultures but that its expression increased with differentiation. FACS analysis confirmed these results and allowed us to isolate LY6Eþ cells, which we found to express Syna at a much higher level than did LY6Ee cells. Our findings suggest that LY6E is expressed in differentiated syncytiotrophoblast and may also be useful as an early marker, expressed in progenitors of this cell-type. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Syncytiotrophoblast Mouse Placenta Labyrinth Ly6E
1. Introduction The labyrinth layer of the mouse placenta begins to form around mid-gestation and develops into an extensive network of maternal blood spaces and fetal capillaries that function as the primary site of nutrient and gas exchange between the mother and the growing fetus [1,2]. We have previously reported that the organization of the labyrinth is patterned much earlier in development, at E8.5 in the chorion, prior to extensive branching morphogenesis [3]. In that study, we noted that precursors of the syncytiotrophoblast layers I and II (SynT-I and SynT-II) express the genes Syna and Gcm1/Cebpa/ Synb, respectively. While much is known about labyrinth development and many genes and signaling pathways have been shown to be important for this process [4,5], there have been few reports of layer-specific gene expression for SynT-I and -II. To our knowledge, there is currently no molecular marker to facilitate the early identification and isolation of syncytiotrophoblast precursors other than the genes described above. LY6E is an extracellular, membrane-associated protein that has been shown to identify immature thymocytes and has been implicated as a cell surface receptor in this context [6]. Interestingly, Ly6e homozygous null embryos die in utero at around E15.5 due to an
apparent dilated cardiomyopathy, however the authors also suggested that the embryonic lethality could be secondary to a developmental phenotype, as Ly6e was not found to be significantly expressed in the developing heart [7]. These results suggest that the gene may have other important functions in embryonic development. In the present study, we describe the expression of Ly6e in the mouse placenta. We show that its expression is restricted to a subset of trophoblast cells in the chorion and later, syncytiotrophoblast in the labyrinth. Furthermore, our data suggest that this gene may be useful as a marker to identify early differentiating SynT-I cells. 2. Methods 2.1. Animals and TS cell culture Placentas were dissected at embryonic day (E) 7.5, 8.5 and 15.5 of gestation from pregnant CD-1 females and fixed in 4% paraformaldehyde overnight, embedded in paraffin and sectioned for analysis by histology and in situ hybridization as previously described [3,8,9]. All mouse work was conducted in accordance with University of Calgary guidelines for use of animal models in research. For in vitro studies, trophoblast stem (TS) cells were cultured as previously described [8]. Briefly, proliferating, undifferentiated stem cells were maintained in culture in TS medium in the presence of fibroblast growth factor (FGF) 4 and embryonic fibroblast conditioned medium (EFCM). For differentiation, both FGF4 and EFCM were removed and the cells were then cultured in TS medium alone. 2.2. RNA isolation, Northern blotting and qRT-PCR
* Corresponding author. HSC rm 2263, University of Calgary, 3330 Hospital Rd NW, Calgary, AB, Canada T2N4N1. Tel.: þ1 403 210 6640. E-mail addresses: [email protected], [email protected] (D.R.C. Natale). 0143-4004/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.placenta.2013.05.011
RNA was isolated from TS cell cultures (3 independent replicate experiments) by lysis in Qiagen RLT buffer and RNA purified using RNeasy microspin columns as
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outlined by the manufacturer (Qiagen, USA). Northern blotting was conducted using 10 mg of total RNA per lane separated on a 1% agarose gel by electrophoresis, and transferred to membrane as previously described [8]. Membranes were probed with P32-labeled cDNA probes for Essrb, Gcm1, Syna, Tpbpa, Prl3d1 and Rn18s ribosomal RNA as previously published [8]. For Ly6e, a cDNA probe was generated using PCR and the following primers: Forward 50 -TATCCCACTACTGGGCCTTG, Reverse 50 TATCGGGGTTGGTCTTTCAG. mRNA expression was assessed by quantitative reverse transcription (RT)-PCR using the SYBR green method as previously described [10]. Briefly, 1 mg of total RNA was reverse transcribed using the Quantitect Reverse Transcription Kit for SYBR green quantitative PCR (#205311, Qiagen). PCR reactions were then prepared using the Quantitect SYBR green PCR kit (#204143, Qiagen), according to the manufacturers’ instructions, and thermocycling was conducted on an MJ Research, DNA Engine, Opticon2 thermocycler. Primer sequences for Gcm1, Syna and Gapdh are published [10]. Primers for Ly6e, Tpbpa and Ctsq were ordered from Qiagen (Quantitect Primer Assays, QT02280642, QT01749181 and QT00119707, respectively). Quantitative PCR reactions were conducted in triplicate on cDNA representing three independent experimental replicates for each gene. Gapdh was used as a reference gene and data was compiled and analyzed for significant changes in gene expression using the Relative Expression Software Tool (REST) [11]. Significance was determined by T-test, p < 0.01. 2.3. RNA in situ hybridization RNA in situ hybridization was conducted on 7 mm sections of mouse placenta tissue as previously described [3,8,12]. Riboprobes for Gcm1, Syna, Tpbpa and Dlx3 are previously published [3], while a probe for Ly6e was transcribed from a PCR template which was amplified using the following primers, which incorporated T3 and T7 RNA polymerase-specific recognition sequences in the forward and reverse primers, respectively. The primer sequences were: forward/T3: 50 -AATTAACCCTCACTAAAGGGCCATCAATTACCTGCCCCTA reverse/T7: 50 -TAATACGACTCACTATAGGGC TTCACTGTGCTGGCTGTGT. 2.4. Immunostaining and fluorescence activated cell sorting (FACS) Immunostaining to identify the expression of LY6E in placenta was conducted on tissue sections. Sections were de-paraffinized and rehydrated followed by antigen retrieval in Buffer C (Electron Microscopy Sciences, USA) using a 2100-Retriever (Electron Microscopy Science, USA). Sections were then treated with 3% H202 to quench endogenous peroxidase, washed in TBS and blocked for 1 h at room temperature in 1x TBS, 5% rabbit serum, 0.1% BSA. Sections were incubated at 4 C overnight in anti-LY6E antibody (Everest, UK), diluted 1:100 in block solution. For negative controls, slides were incubated in fresh block without primary antibody. Sections were washed in TBS and incubated for 1 h at room temperature in biotinylated rabbit anti-goat secondary antibody (Vector Labs, USA) diluted 1:300, followed by washing and incubation in Extravidin-Peroxidase (Sigma, USA) diluted 1:400 in TBS, 0.1% BSA. Antibody binding was visualized using Vector Nova Red according to the manufacturer’s protocol (Vector Labs, USA). Following counterstaining in hematoxylin (Gills #2, Sigma, USA), sections were dehydrated and mounted in xylene-based mounting medium. In order to confirm the presence of multi-nucleated cells in trophoblast stem cell cultures, cells were grown on glass coverslips. At the desired time cells were fixed for 15 min at room temperature in 2% PFA, 1x PBS followed by incubation in rhodaminephalloidin (1:100; Molecular Probes, Invitrogen, USA). Cells were counterstained in Hoechst 33342 (1:1000; Molecular Probes, Invitrogen, USA) and mounted on slides in Fluorescent Mounting Medium (Dako, USA). LY6Eþ trophoblast cells were identified and isolated from trophoblast stem cell cultures by FACS. Briefly, TS cell cultures were trypsinized at 70e80% confluency, washed and resuspended in 1 mL of 1x PBS containing anti-LY6E antibody [6] (a generous gift from Professor Richard Boyd, Monash University, Melbourne, AU). After incubation at 4 C for 20e30 min, cells were washed gently and incubated in FITC-conjugated goat anti-rat secondary antibody (Jackson Laboratories, USA) for 20e30 min followed by washing, resuspending in 1x PBS and FACS at the University of Calgary Flow Cytometry Facility (Calgary, Canada).
3. Results and discussion 3.1. Ly6e expression is restricted to the chorion and labyrinth layers of the mouse placenta We first identified Ly6e expression in mouse trophoblast cells through a microarray experiment conducted on cultured trophoblast stem cells in vitro. To our knowledge this is the first report of Ly6e expression in the mouse placenta and therefore, to further investigate both timing and localization of expression, we conducted mRNA in situ hybridization on mouse placenta tissue
sections. We began our analysis at embryonic day (E) 7.5, where we observed Ly6e expressed in cells within the mesometrial uterine stroma but not in trophoblast of the developing placenta (data not shown). We detected placental Ly6e in the cells of the chorionic ectoderm beginning at approximately E8.5, after chorio-allantoic attachment had occurred (Fig. 1A). Ly6e was restricted to trophoblast cells of the chorion, in close approximation to the ectoplacental cone, a pattern reminiscent of Syna expression [3]. To further investigate the association between Ly6e and Syna, and to more clearly define the trophoblast cell type in which Ly6e is expressed, we expanded our analysis by in situ hybridization to include the expression of Syna, Gcm1, and Tpbpa at E8.5. The expression of these genes, at this time, identifies precursors of the syncytiotrophoblast layers I, II and spongiotrophoblast respectively [3,13]. Gcm1, which is expressed in precursors of syncytiotrophoblast layer II cells, did not appear to overlap with Ly6e and localized to clusters of trophoblast cells of the chorion that were undergoing the first stages of chorio-allantoic branching morphogenesis [3,14,15] (Fig. 1B). Similarly, Tpbpa expression was in trophoblast of the EPC and exclusive of Ly6e-expressing cells (Fig. 1D). Syna, on the other hand, was also detected in trophoblast of the chorion in a pattern that overlapped with Ly6e (Fig. 1C), suggesting that Ly6e expression may be specific to precursors of the SynT-I layer of syncytiotrophoblast. To confirm the presence of Ly6e later in gestation, we compared expression of Ly6e with Gcm1 and Syna, markers of syncytiotrophoblast [3] and Dlx3, a gene previously described as a marker of labyrinthine trophoblast [3,16] (Fig. 1E, F, G, H respectively). At E15.5, placental Ly6e was detected exclusively in the labyrinth and specifically, in syncytiotrophoblast. Sinusoidal giant cells and fetal endothelial cells appeared devoid of Ly6e expression. In contrast, Dlx3 was more generally expressed in the labyrinth although this was expected as it has not been described as SynT-specific [16]. While clearly syncytiotrophoblast-specific, Ly6e appeared more broadly expressed at E15.5 than Gcm1 or Syna. Since both Gcm1 and Syna are downregulated in the labyrinth beginning at E14.5, this is not surprising [3,14,15]. However, it is difficult to determine from the in situ hybridizations whether the broad expression of Ly6e is the result of a maintained expression in the whole of the syncytiotrophoblast layer-1 population, or whether the result of an expanded expression to include syncytiotrophoblast layer-II. From immunostaining results, it appears that both scenarios may be correct as we were able to detect protein at E15.5 in Syn-T, in general (Fig. 1E*). Interestingly, both Ly6e mRNA and protein were also expressed in a layer of trophoblast cells along the chorione mesodermal interface at the edge of the labyrinth, closest to the developing fetus. The cells in this layer are small, tightly associated and columnar in shape and appear to be undifferentiated compared to other known labyrinthine trophoblast subtypes (Fig. 1E**). 3.2. Expression of Ly6e identifies syncytiotrophoblast-like cells in trophoblast stem cell cultures in vitro Staining patterns from in situ hybridization and immunohistochemistry can be difficult to absolutely define in paraffin sections so to further investigate the relationship between LY6E and syncytiotrophoblast differentiation, we utilized the in vitro trophoblast stem (TS) cell model [8,17]. Compared to other markers that we have previously used to identify TS cells and differentiated trophoblast subtypes [3,8], Ly6e mRNA and protein were detectable in both undifferentiated and differentiated TS cell cultures and increased in level of expression as trophoblast cells differentiated (Fig. 2A, representative of 3 replicate experiments). Of note, the pattern of mRNA expression as detected by northern blot is not similar to any other gene that we have previously studied in that it
M. Hughes et al. / Placenta 34 (2013) 831e835
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Fig. 1. Ly6E is expressed in the mouse placenta in the chorion at E8.5 and in the labyrinth later in gestation. At E8.5 (A, B, C, D), ly6E mRNA is detectable by in situ hybridization in the upper cells of the chorion (A). Comparatively, gcm1 is expressed in trophoblast cells at points in the chorion that are beginning branching morphogenesis (B) while the pattern of syncytin-A expression appears to overlap with that of ly6E (C, serial section to A and B). Expression of tpbpa localizes to the ectoplacental cone and does not overlap with ly6E (D). At E15.5 (E, F, G, H), ly6E mRNA is detectable only within the labyrinth of the placenta and appears to be expressed primarily in syncytiotrophoblast (E). Insets E* and E** show high magnification (1000) photomicrographs of Ly6E protein expressed in syncytiotrophoblast (E*, arrowhead) and also in tightly packed, columnar trophoblast lining blood spaces at the bottom edge of the developing labyrinth (E**, arrowhead). For comparison, syncytin-A (F) and to a lesser extent, gcm1 (G) appeared expressed in a subset of ly6E-positive cells. Dlx3 (H), a “labyrinth-specific” gene had a broad expression pattern, similar to ly6E although it was more punctate in appearance. Scale bar ¼ 100 mm.
is detectable in proliferating TS cells, appears to decrease in expression after two days of differentiation and then is upregulated again after four days of differentiation. Based on our in vivo expression analysis, we hypothesized that “early” expression of LY6E might identify trophoblast cells that
have the potential to differentiate into syncytiotrophoblast. In order to test this hypothesis, we first isolated LY6Eþ and e cells from differentiating TS cell cultures (3 days of differentiation). We then queried both groups of cells by qRT-PCR for the expression of a panel of genes including Ly6e, as well as Gcm1, Syna, and Ctsq, all
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Fig. 2. Ly6E expression in TS cell cultures. Ly6E mRNA and protein were detectable in trophoblast stem cells in culture by northern blot (A, left panel) and FACS analysis (A, right panel), respectively. In both cases, while expression was detectable in undifferentiated cells, it increased with differentiation, matching the expression patterns of SynA, Tpbpa and Prl3d1 at 4 and 6 days of differentiation. When measured by FACS, LY6E-expressing cells increased from approximately 28% in undifferentiated cultures to 40% after three days of differentiation (representative results of 3 experimental replicates shown in B; y-axis indicates cells counted, x-axis indicates fluorescence intensity, M1 indicates LY6Eþ cells). When Ly6Eþ cells were isolated from differentiating cultures and analyzed by qRT-PCR relative to Gapdh expression (B), Ly6Eþ trophoblast displayed significantly higher levels of ly6E and syncytin-A expression while showing significantly lower levels of tpbpa (*, T-test; p < 0.01). Furthermore, when Ly6Eþ trophoblast were isolated from undifferentiated cultures and re-plated for an additional 6 days, they formed large, multinucleated cells (C, left panel, arrowheads). That the cells were multinucleated was confirmed by nuclear staining in coordination with F-actin to mark cell borders (C, left panel, arrowhead. blue ¼ nuclei, red ¼ cell borders). By comparison, Ly6E cells remained proliferative and undifferentiated (C, right panel).
of which are expressed in different labyrinthine trophoblast subtypes [3,18]. Finally, we included Tpbpa as an indicator of cells differentiating into spongiotrophoblast and TGCs [18]. LY6Eþ cells preferentially expressed Syna versus all of the other genes examined (Fig. 2B, p < 0.01). In addition, they expressed
statistically significant lower levels of Tpbpa compared to LY6Ee cells (approximately 30%, Fig. 2B, p < 0.01). Surprisingly, both groups expressed Gcm1 and Ctsq at similar levels, which are markers of SynT-II and sinusoidal trophoblast giant cells respectively. This result could be unique to TS cells in culture.
M. Hughes et al. / Placenta 34 (2013) 831e835
Alternatively, it might suggest that while Ly6e is preferentially expressed in SynT-I cells early in placental development, it may be expressed in both layers of syncytiotrophoblast later in gestation as observed in our in vivo analysis. However, as we did not observe Ly6e expression in sinusoidal TGCs in vivo, the detection of Ctsq in LY6Eþ cells in vitro may also result from the difference in sensitivity of the qRT-PCR assay versus mRNA in situ hybridization and the ability to detect lower levels of expression. Finally, to determine whether LY6Eþ cells within proliferating TS cell cultures differentiate into syncytiotrophoblast, we used FACS to isolate LY6Eþ and e cells from proliferating TS cell cultures and replated them, maintaining them in FGF4 to prevent differentiation. While LY6E- cells proliferated and appeared to remain undifferentiated, over the following six days, LY6Eþ cells underwent differentiation, forming large, multi-nucleated cells (Fig. 2C and inset) even in the presence of FGF4. When subjected to cell counts (4 fields of view from 3 replicate experiments), 71% of LY6Eþ cells appeared multi-nucleated while 29% appeared to have an enlarged, single nucleus more typical of TGCs. For comparison, in LY6E cultures, while no multi-nucleated cells were observed, 20% resembled TGCs. The frequency of SynT under normal TS cell differentiation is approximately 3.2%. Based on our in vivo and in vitro analyses, we have determined that LY6E is expressed in trophoblast cells of the developing labyrinth. We propose that it is expressed preferentially in precursors of SynT-I cells during early chorio-allantoic placental development at E8.5, making it, to our knowledge, the only other gene other than Syna a unique marker of these cells. Later in gestation, LY6E expression persists and becomes more broadly expressed, although it appears to remain restricted to syncytiotrophoblast. Acknowledgments The authors wish to thank Professor Richard Boyd for the generous gift of anti-LY6E monoclonal antibody.
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References [1] Cross JC, Simmons DG, Watson ED. Chorioallantoic morphogenesis and formation of the placental villous tree. Ann N Y Acad Sci 2003;995:84e93. [2] Muntener M, Hsu YC. Development of trophoblast and placenta of the mouse. A reinvestigation with regard to the in vitro culture of mouse trophoblast and placenta. Acta Anat (Basel) 1977;98:241e52. [3] Simmons DG, et al. Early patterning of the chorion leads to the trilaminar trophoblast cell structure in the placental labyrinth. Development 2008;135: 2083e91. [4] Rossant J, Cross JC. Placental development: lessons from mouse mutants. Nat Rev Genet 2001;2:538e48. [5] Watson ED, Cross JC. Development of structures and transport functions in the mouse placenta. Physiology (Bethesda) 2005;20:180e93. [6] Classon BJ, Boyd RL. Thymic-shared antigen-1 (TSA-1). A lymphostromal cell membrane Ly-6 superfamily molecule with a putative role in cellular adhesion. Dev Immunol 1998;6:149e56. [7] Zammit DJ, et al. Essential role for the lymphostromal plasma membrane Ly-6 superfamily molecule thymic shared antigen 1 in development of the embryonic adrenal gland. Mol Cell Biol 2002;22:946e52. [8] Natale DR, Hemberger M, Hughes M, Cross JC. Activin promotes differentiation of cultured mouse trophoblast stem cells towards a labyrinth cell fate. Dev Biol 2009;335:120e31. http://dx.doi.org/10.1016/j.ydbio.2009.08.022. PII: S0012-1606(09)01144-0. [9] Natale DR, Starovic M, Cross JC. Phenotypic analysis of the mouse placenta. Methods Mol Med 2006;121:275e93. [10] Watson ED, et al. Cell-cell adhesion defects in Mrj mutant trophoblast cells are associated with failure to pattern the chorion during early placental development. Dev Dyn 2011. http://dx.doi.org/10.1002/dvdy.22755. [11] Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucl Acids Res 2002;30:e36. [12] Simmons DG, Rawn SM, Davies A, Hughes M, Cross JC. Spatial and temporal expression of the 23 murine prolactin/placental lactogen-related genes is not associated with their position in the locus. BMC Genom 2008;9:352. [13] Simmons DG, Fortier AL, Cross JC. Diverse subtypes and developmental origins of trophoblast giant cells in the mouse placenta. Dev Biol 2007;304:567e78. [14] Anson-Cartwright L, et al. The glial cells missing-1 protein is essential for branching morphogenesis in the chorioallantoic placenta. Nat Genet 2000;25:311e4. [15] Basyuk E, et al. The murine Gcm1 gene is expressed in a subset of placental trophoblast cells. Dev Dyn 1999;214:303e11. [16] Morasso MI, Grinberg A, Robinson G, Sargent TD, Mahon KA. Placental failure in mice lacking the homeobox gene Dlx3. Proc Natl Acad Sci U S A 1999;96:162e7. [17] Tanaka S, Kunath T, Hadjantonakis AK, Nagy A, Rossant J. Promotion of trophoblast stem cell proliferation by FGF4. Science 1998;282:2072e5. [18] Simmons DG, Cross JC. Determinants of trophoblast lineage and cell subtype specification in the mouse placenta. Dev Biol 2005;284:12e24.
Contents lists available at ScienceDirect
Placenta journal homepage: www.elsevier.com/locate/placenta
Short communication
Ly6e expression is restricted to syncytiotrophoblast cells of the mouse placenta M. Hughes a, B.V. Natale a, D.G. Simmons b, D.R.C. Natale a, * a b
Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 29 May 2013
In the present study, we characterized the expression of lymphocyte antigen 6, locus E (Ly6e) in mouse placental trophoblast. We identified Ly6e mRNA expression in trophoblast stem (TS) cells by a gene expression screen. In vivo, Ly6e was first detectable by mRNA in situ hybridization in the chorion beginning at E8.5 with spatial expression similar to Syncytin a (Syna). At later stages of gestation, Ly6e was restricted to syncytiotrophoblast in the labyrinth. Northern blot confirmed that Ly6e was expressed in both undifferentiated and differentiated TS cell cultures but that its expression increased with differentiation. FACS analysis confirmed these results and allowed us to isolate LY6Eþ cells, which we found to express Syna at a much higher level than did LY6Ee cells. Our findings suggest that LY6E is expressed in differentiated syncytiotrophoblast and may also be useful as an early marker, expressed in progenitors of this cell-type. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Syncytiotrophoblast Mouse Placenta Labyrinth Ly6E
1. Introduction The labyrinth layer of the mouse placenta begins to form around mid-gestation and develops into an extensive network of maternal blood spaces and fetal capillaries that function as the primary site of nutrient and gas exchange between the mother and the growing fetus [1,2]. We have previously reported that the organization of the labyrinth is patterned much earlier in development, at E8.5 in the chorion, prior to extensive branching morphogenesis [3]. In that study, we noted that precursors of the syncytiotrophoblast layers I and II (SynT-I and SynT-II) express the genes Syna and Gcm1/Cebpa/ Synb, respectively. While much is known about labyrinth development and many genes and signaling pathways have been shown to be important for this process [4,5], there have been few reports of layer-specific gene expression for SynT-I and -II. To our knowledge, there is currently no molecular marker to facilitate the early identification and isolation of syncytiotrophoblast precursors other than the genes described above. LY6E is an extracellular, membrane-associated protein that has been shown to identify immature thymocytes and has been implicated as a cell surface receptor in this context [6]. Interestingly, Ly6e homozygous null embryos die in utero at around E15.5 due to an
apparent dilated cardiomyopathy, however the authors also suggested that the embryonic lethality could be secondary to a developmental phenotype, as Ly6e was not found to be significantly expressed in the developing heart [7]. These results suggest that the gene may have other important functions in embryonic development. In the present study, we describe the expression of Ly6e in the mouse placenta. We show that its expression is restricted to a subset of trophoblast cells in the chorion and later, syncytiotrophoblast in the labyrinth. Furthermore, our data suggest that this gene may be useful as a marker to identify early differentiating SynT-I cells. 2. Methods 2.1. Animals and TS cell culture Placentas were dissected at embryonic day (E) 7.5, 8.5 and 15.5 of gestation from pregnant CD-1 females and fixed in 4% paraformaldehyde overnight, embedded in paraffin and sectioned for analysis by histology and in situ hybridization as previously described [3,8,9]. All mouse work was conducted in accordance with University of Calgary guidelines for use of animal models in research. For in vitro studies, trophoblast stem (TS) cells were cultured as previously described [8]. Briefly, proliferating, undifferentiated stem cells were maintained in culture in TS medium in the presence of fibroblast growth factor (FGF) 4 and embryonic fibroblast conditioned medium (EFCM). For differentiation, both FGF4 and EFCM were removed and the cells were then cultured in TS medium alone. 2.2. RNA isolation, Northern blotting and qRT-PCR
* Corresponding author. HSC rm 2263, University of Calgary, 3330 Hospital Rd NW, Calgary, AB, Canada T2N4N1. Tel.: þ1 403 210 6640. E-mail addresses: [email protected], [email protected] (D.R.C. Natale). 0143-4004/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.placenta.2013.05.011
RNA was isolated from TS cell cultures (3 independent replicate experiments) by lysis in Qiagen RLT buffer and RNA purified using RNeasy microspin columns as
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M. Hughes et al. / Placenta 34 (2013) 831e835
outlined by the manufacturer (Qiagen, USA). Northern blotting was conducted using 10 mg of total RNA per lane separated on a 1% agarose gel by electrophoresis, and transferred to membrane as previously described [8]. Membranes were probed with P32-labeled cDNA probes for Essrb, Gcm1, Syna, Tpbpa, Prl3d1 and Rn18s ribosomal RNA as previously published [8]. For Ly6e, a cDNA probe was generated using PCR and the following primers: Forward 50 -TATCCCACTACTGGGCCTTG, Reverse 50 TATCGGGGTTGGTCTTTCAG. mRNA expression was assessed by quantitative reverse transcription (RT)-PCR using the SYBR green method as previously described [10]. Briefly, 1 mg of total RNA was reverse transcribed using the Quantitect Reverse Transcription Kit for SYBR green quantitative PCR (#205311, Qiagen). PCR reactions were then prepared using the Quantitect SYBR green PCR kit (#204143, Qiagen), according to the manufacturers’ instructions, and thermocycling was conducted on an MJ Research, DNA Engine, Opticon2 thermocycler. Primer sequences for Gcm1, Syna and Gapdh are published [10]. Primers for Ly6e, Tpbpa and Ctsq were ordered from Qiagen (Quantitect Primer Assays, QT02280642, QT01749181 and QT00119707, respectively). Quantitative PCR reactions were conducted in triplicate on cDNA representing three independent experimental replicates for each gene. Gapdh was used as a reference gene and data was compiled and analyzed for significant changes in gene expression using the Relative Expression Software Tool (REST) [11]. Significance was determined by T-test, p < 0.01. 2.3. RNA in situ hybridization RNA in situ hybridization was conducted on 7 mm sections of mouse placenta tissue as previously described [3,8,12]. Riboprobes for Gcm1, Syna, Tpbpa and Dlx3 are previously published [3], while a probe for Ly6e was transcribed from a PCR template which was amplified using the following primers, which incorporated T3 and T7 RNA polymerase-specific recognition sequences in the forward and reverse primers, respectively. The primer sequences were: forward/T3: 50 -AATTAACCCTCACTAAAGGGCCATCAATTACCTGCCCCTA reverse/T7: 50 -TAATACGACTCACTATAGGGC TTCACTGTGCTGGCTGTGT. 2.4. Immunostaining and fluorescence activated cell sorting (FACS) Immunostaining to identify the expression of LY6E in placenta was conducted on tissue sections. Sections were de-paraffinized and rehydrated followed by antigen retrieval in Buffer C (Electron Microscopy Sciences, USA) using a 2100-Retriever (Electron Microscopy Science, USA). Sections were then treated with 3% H202 to quench endogenous peroxidase, washed in TBS and blocked for 1 h at room temperature in 1x TBS, 5% rabbit serum, 0.1% BSA. Sections were incubated at 4 C overnight in anti-LY6E antibody (Everest, UK), diluted 1:100 in block solution. For negative controls, slides were incubated in fresh block without primary antibody. Sections were washed in TBS and incubated for 1 h at room temperature in biotinylated rabbit anti-goat secondary antibody (Vector Labs, USA) diluted 1:300, followed by washing and incubation in Extravidin-Peroxidase (Sigma, USA) diluted 1:400 in TBS, 0.1% BSA. Antibody binding was visualized using Vector Nova Red according to the manufacturer’s protocol (Vector Labs, USA). Following counterstaining in hematoxylin (Gills #2, Sigma, USA), sections were dehydrated and mounted in xylene-based mounting medium. In order to confirm the presence of multi-nucleated cells in trophoblast stem cell cultures, cells were grown on glass coverslips. At the desired time cells were fixed for 15 min at room temperature in 2% PFA, 1x PBS followed by incubation in rhodaminephalloidin (1:100; Molecular Probes, Invitrogen, USA). Cells were counterstained in Hoechst 33342 (1:1000; Molecular Probes, Invitrogen, USA) and mounted on slides in Fluorescent Mounting Medium (Dako, USA). LY6Eþ trophoblast cells were identified and isolated from trophoblast stem cell cultures by FACS. Briefly, TS cell cultures were trypsinized at 70e80% confluency, washed and resuspended in 1 mL of 1x PBS containing anti-LY6E antibody [6] (a generous gift from Professor Richard Boyd, Monash University, Melbourne, AU). After incubation at 4 C for 20e30 min, cells were washed gently and incubated in FITC-conjugated goat anti-rat secondary antibody (Jackson Laboratories, USA) for 20e30 min followed by washing, resuspending in 1x PBS and FACS at the University of Calgary Flow Cytometry Facility (Calgary, Canada).
3. Results and discussion 3.1. Ly6e expression is restricted to the chorion and labyrinth layers of the mouse placenta We first identified Ly6e expression in mouse trophoblast cells through a microarray experiment conducted on cultured trophoblast stem cells in vitro. To our knowledge this is the first report of Ly6e expression in the mouse placenta and therefore, to further investigate both timing and localization of expression, we conducted mRNA in situ hybridization on mouse placenta tissue
sections. We began our analysis at embryonic day (E) 7.5, where we observed Ly6e expressed in cells within the mesometrial uterine stroma but not in trophoblast of the developing placenta (data not shown). We detected placental Ly6e in the cells of the chorionic ectoderm beginning at approximately E8.5, after chorio-allantoic attachment had occurred (Fig. 1A). Ly6e was restricted to trophoblast cells of the chorion, in close approximation to the ectoplacental cone, a pattern reminiscent of Syna expression [3]. To further investigate the association between Ly6e and Syna, and to more clearly define the trophoblast cell type in which Ly6e is expressed, we expanded our analysis by in situ hybridization to include the expression of Syna, Gcm1, and Tpbpa at E8.5. The expression of these genes, at this time, identifies precursors of the syncytiotrophoblast layers I, II and spongiotrophoblast respectively [3,13]. Gcm1, which is expressed in precursors of syncytiotrophoblast layer II cells, did not appear to overlap with Ly6e and localized to clusters of trophoblast cells of the chorion that were undergoing the first stages of chorio-allantoic branching morphogenesis [3,14,15] (Fig. 1B). Similarly, Tpbpa expression was in trophoblast of the EPC and exclusive of Ly6e-expressing cells (Fig. 1D). Syna, on the other hand, was also detected in trophoblast of the chorion in a pattern that overlapped with Ly6e (Fig. 1C), suggesting that Ly6e expression may be specific to precursors of the SynT-I layer of syncytiotrophoblast. To confirm the presence of Ly6e later in gestation, we compared expression of Ly6e with Gcm1 and Syna, markers of syncytiotrophoblast [3] and Dlx3, a gene previously described as a marker of labyrinthine trophoblast [3,16] (Fig. 1E, F, G, H respectively). At E15.5, placental Ly6e was detected exclusively in the labyrinth and specifically, in syncytiotrophoblast. Sinusoidal giant cells and fetal endothelial cells appeared devoid of Ly6e expression. In contrast, Dlx3 was more generally expressed in the labyrinth although this was expected as it has not been described as SynT-specific [16]. While clearly syncytiotrophoblast-specific, Ly6e appeared more broadly expressed at E15.5 than Gcm1 or Syna. Since both Gcm1 and Syna are downregulated in the labyrinth beginning at E14.5, this is not surprising [3,14,15]. However, it is difficult to determine from the in situ hybridizations whether the broad expression of Ly6e is the result of a maintained expression in the whole of the syncytiotrophoblast layer-1 population, or whether the result of an expanded expression to include syncytiotrophoblast layer-II. From immunostaining results, it appears that both scenarios may be correct as we were able to detect protein at E15.5 in Syn-T, in general (Fig. 1E*). Interestingly, both Ly6e mRNA and protein were also expressed in a layer of trophoblast cells along the chorione mesodermal interface at the edge of the labyrinth, closest to the developing fetus. The cells in this layer are small, tightly associated and columnar in shape and appear to be undifferentiated compared to other known labyrinthine trophoblast subtypes (Fig. 1E**). 3.2. Expression of Ly6e identifies syncytiotrophoblast-like cells in trophoblast stem cell cultures in vitro Staining patterns from in situ hybridization and immunohistochemistry can be difficult to absolutely define in paraffin sections so to further investigate the relationship between LY6E and syncytiotrophoblast differentiation, we utilized the in vitro trophoblast stem (TS) cell model [8,17]. Compared to other markers that we have previously used to identify TS cells and differentiated trophoblast subtypes [3,8], Ly6e mRNA and protein were detectable in both undifferentiated and differentiated TS cell cultures and increased in level of expression as trophoblast cells differentiated (Fig. 2A, representative of 3 replicate experiments). Of note, the pattern of mRNA expression as detected by northern blot is not similar to any other gene that we have previously studied in that it
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Fig. 1. Ly6E is expressed in the mouse placenta in the chorion at E8.5 and in the labyrinth later in gestation. At E8.5 (A, B, C, D), ly6E mRNA is detectable by in situ hybridization in the upper cells of the chorion (A). Comparatively, gcm1 is expressed in trophoblast cells at points in the chorion that are beginning branching morphogenesis (B) while the pattern of syncytin-A expression appears to overlap with that of ly6E (C, serial section to A and B). Expression of tpbpa localizes to the ectoplacental cone and does not overlap with ly6E (D). At E15.5 (E, F, G, H), ly6E mRNA is detectable only within the labyrinth of the placenta and appears to be expressed primarily in syncytiotrophoblast (E). Insets E* and E** show high magnification (1000) photomicrographs of Ly6E protein expressed in syncytiotrophoblast (E*, arrowhead) and also in tightly packed, columnar trophoblast lining blood spaces at the bottom edge of the developing labyrinth (E**, arrowhead). For comparison, syncytin-A (F) and to a lesser extent, gcm1 (G) appeared expressed in a subset of ly6E-positive cells. Dlx3 (H), a “labyrinth-specific” gene had a broad expression pattern, similar to ly6E although it was more punctate in appearance. Scale bar ¼ 100 mm.
is detectable in proliferating TS cells, appears to decrease in expression after two days of differentiation and then is upregulated again after four days of differentiation. Based on our in vivo expression analysis, we hypothesized that “early” expression of LY6E might identify trophoblast cells that
have the potential to differentiate into syncytiotrophoblast. In order to test this hypothesis, we first isolated LY6Eþ and e cells from differentiating TS cell cultures (3 days of differentiation). We then queried both groups of cells by qRT-PCR for the expression of a panel of genes including Ly6e, as well as Gcm1, Syna, and Ctsq, all
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Fig. 2. Ly6E expression in TS cell cultures. Ly6E mRNA and protein were detectable in trophoblast stem cells in culture by northern blot (A, left panel) and FACS analysis (A, right panel), respectively. In both cases, while expression was detectable in undifferentiated cells, it increased with differentiation, matching the expression patterns of SynA, Tpbpa and Prl3d1 at 4 and 6 days of differentiation. When measured by FACS, LY6E-expressing cells increased from approximately 28% in undifferentiated cultures to 40% after three days of differentiation (representative results of 3 experimental replicates shown in B; y-axis indicates cells counted, x-axis indicates fluorescence intensity, M1 indicates LY6Eþ cells). When Ly6Eþ cells were isolated from differentiating cultures and analyzed by qRT-PCR relative to Gapdh expression (B), Ly6Eþ trophoblast displayed significantly higher levels of ly6E and syncytin-A expression while showing significantly lower levels of tpbpa (*, T-test; p < 0.01). Furthermore, when Ly6Eþ trophoblast were isolated from undifferentiated cultures and re-plated for an additional 6 days, they formed large, multinucleated cells (C, left panel, arrowheads). That the cells were multinucleated was confirmed by nuclear staining in coordination with F-actin to mark cell borders (C, left panel, arrowhead. blue ¼ nuclei, red ¼ cell borders). By comparison, Ly6E cells remained proliferative and undifferentiated (C, right panel).
of which are expressed in different labyrinthine trophoblast subtypes [3,18]. Finally, we included Tpbpa as an indicator of cells differentiating into spongiotrophoblast and TGCs [18]. LY6Eþ cells preferentially expressed Syna versus all of the other genes examined (Fig. 2B, p < 0.01). In addition, they expressed
statistically significant lower levels of Tpbpa compared to LY6Ee cells (approximately 30%, Fig. 2B, p < 0.01). Surprisingly, both groups expressed Gcm1 and Ctsq at similar levels, which are markers of SynT-II and sinusoidal trophoblast giant cells respectively. This result could be unique to TS cells in culture.
M. Hughes et al. / Placenta 34 (2013) 831e835
Alternatively, it might suggest that while Ly6e is preferentially expressed in SynT-I cells early in placental development, it may be expressed in both layers of syncytiotrophoblast later in gestation as observed in our in vivo analysis. However, as we did not observe Ly6e expression in sinusoidal TGCs in vivo, the detection of Ctsq in LY6Eþ cells in vitro may also result from the difference in sensitivity of the qRT-PCR assay versus mRNA in situ hybridization and the ability to detect lower levels of expression. Finally, to determine whether LY6Eþ cells within proliferating TS cell cultures differentiate into syncytiotrophoblast, we used FACS to isolate LY6Eþ and e cells from proliferating TS cell cultures and replated them, maintaining them in FGF4 to prevent differentiation. While LY6E- cells proliferated and appeared to remain undifferentiated, over the following six days, LY6Eþ cells underwent differentiation, forming large, multi-nucleated cells (Fig. 2C and inset) even in the presence of FGF4. When subjected to cell counts (4 fields of view from 3 replicate experiments), 71% of LY6Eþ cells appeared multi-nucleated while 29% appeared to have an enlarged, single nucleus more typical of TGCs. For comparison, in LY6E cultures, while no multi-nucleated cells were observed, 20% resembled TGCs. The frequency of SynT under normal TS cell differentiation is approximately 3.2%. Based on our in vivo and in vitro analyses, we have determined that LY6E is expressed in trophoblast cells of the developing labyrinth. We propose that it is expressed preferentially in precursors of SynT-I cells during early chorio-allantoic placental development at E8.5, making it, to our knowledge, the only other gene other than Syna a unique marker of these cells. Later in gestation, LY6E expression persists and becomes more broadly expressed, although it appears to remain restricted to syncytiotrophoblast. Acknowledgments The authors wish to thank Professor Richard Boyd for the generous gift of anti-LY6E monoclonal antibody.
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