SLC20a1/PiT-1 is required for chorioallantoic placental morphogenesis

in Vascular Biology
Authors:
Ana Correia-Branco Mother Infant Research Institute, Tufts Medical Center, Boston, Massachusetts, USA

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Ariel Mei Mother Infant Research Institute, Tufts Medical Center, Boston, Massachusetts, USA

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Sreehari Pillai Mother Infant Research Institute, Tufts Medical Center, Boston, Massachusetts, USA

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Nirmala Jayaraman Mother Infant Research Institute, Tufts Medical Center, Boston, Massachusetts, USA

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Radhika Sharma Mother Infant Research Institute, Tufts Medical Center, Boston, Massachusetts, USA

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Alison G Paquette University of Washington, Department of Pediatrics, Seattle, Washington, USA

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Naveen K Neradugomma Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, Washington, USA

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Ciara Benson Department of Bioengineering, University of Washington, Seattle, Washington, USA

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Nicholas W Chavkin Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia, USA

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Qingcheng Mao Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, Washington, USA

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Mary C Wallingford Mother Infant Research Institute, Tufts Medical Center, Boston, Massachusetts, USA

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Correspondence should be addressed to M C Wallingford: marycwallingford@gmail.com

*(A Correia-Branco and A Mei contributed equally to this work)

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The placenta mediates the transport of nutrients, such as inorganic phosphate (Pi), between the maternal and fetal circulatory systems. The placenta itself also requires high levels of nutrient uptake as it develops to provide critical support for fetal development. This study aimed to determine placental Pi transport mechanisms using in vitro and in vivo models. We observed that Pi (P33) uptake in BeWo cells is sodium dependent and that SLC20A1/Slc20a1 is the most highly expressed placental sodium-dependent transporter in mouse (microarray), human cell line (RT-PCR) and term placenta (RNA-seq), supporting that normal growth and maintenance of the mouse and human placenta requires SLC20A1/Slc20a1. Slc20a1 wild-type (Slc20a1+/+ ) and knockout (Slc20a1–/– ) mice were produced through timed intercrosses and displayed yolk sac angiogenesis failure as expected at E10.5. E9.5 tissues were analyzed to test whether placental morphogenesis requires Slc20a1. At E9.5, the developing placenta was reduced in size in Slc20a1–/– . Multiple structural abnormalities were also observed in the Slc20a1–/– chorioallantois. We determined that monocarboxylate transporter 1 protein (MCT1+) cells were reduced in developing Slc20a1–/– placenta, confirming that Slc20a1 loss reduced trophoblast syncytiotrophoblast 1 (SynT-I) coverage. Next, we examined the cell type-specific Slc20a1 expression and SynT molecular pathways in silico and identified Notch/Wnt as a pathway of interest that regulates trophoblast differentiation. We further observed that specific trophoblast lineages express Notch/Wnt genes that associate with endothelial cell tip-and-stalk cell markers. In conclusion, our findings support that Slc20a1 mediates the symport of Pi into SynT cells, providing critical support for their differentiation and angiogenic mimicry function at the developing maternalfetal interface.

Abstract

The placenta mediates the transport of nutrients, such as inorganic phosphate (Pi), between the maternal and fetal circulatory systems. The placenta itself also requires high levels of nutrient uptake as it develops to provide critical support for fetal development. This study aimed to determine placental Pi transport mechanisms using in vitro and in vivo models. We observed that Pi (P33) uptake in BeWo cells is sodium dependent and that SLC20A1/Slc20a1 is the most highly expressed placental sodium-dependent transporter in mouse (microarray), human cell line (RT-PCR) and term placenta (RNA-seq), supporting that normal growth and maintenance of the mouse and human placenta requires SLC20A1/Slc20a1. Slc20a1 wild-type (Slc20a1+/+) and knockout (Slc20a1–/–) mice were produced through timed intercrosses and displayed yolk sac angiogenesis failure as expected at E10.5. E9.5 tissues were analyzed to test whether placental morphogenesis requires Slc20a1. At E9.5, the developing placenta was reduced in size in Slc20a1–/–. Multiple structural abnormalities were also observed in the Slc20a1–/–chorioallantois. We determined that monocarboxylate transporter 1 protein (MCT1+) cells were reduced in developing Slc20a1–/–placenta, confirming that Slc20a1 loss reduced trophoblast syncytiotrophoblast 1 (SynT-I) coverage. Next, we examined the cell type-specific Slc20a1 expression and SynT molecular pathways in silico and identified Notch/Wnt as a pathway of interest that regulates trophoblast differentiation. We further observed that specific trophoblast lineages express Notch/Wnt genes that associate with endothelial cell tip-and-stalk cell markers. In conclusion, our findings support that Slc20a1 mediates the symport of Pi into SynT cells, providing critical support for their differentiation and angiogenic mimicry function at the developing maternalfetal interface.

Introduction

Phosphorus is an essential nutrient that serves an essential role in cellular energetics, growth, and bone development (1, 2). It circulates in the bloodstream in the form of inorganic phosphate (Pi). Phosphate symport occurs against a 100-fold concentration gradient via transmembrane proteins, such as the type III sodium-dependent phosphate transporters SLC20A1 (PiT-1) and SLC20A2 (PiT-2). A loss of phosphate homeostasis poses significant challenges, including phosphate wasting, refeeding syndrome, ectopic calcification, chronic kidney disease, and bone mineralization disorders. Phosphate homeostasis is regulated by parathyroid hormone (PTH), vitamin D (VD), and fibroblast growth factor 23 (FGF23) in concert with calcium (3). FGF23 signaling downregulates phosphate transporter gene expression and inhibits the production of VD and PTH (3).

By birth, a human embryo accretes ~15–30 g of phosphorus (4, 5). Maternal–fetal phosphate transport mechanisms are incompletely understood at the molecular level. Phosphate transport flux has been shown to occur against a concentration gradient in hemochorial placentas, which are found in rodents and humans (6, 7). Placental perfusion studies support the fact that placental transport is sodium dependent modulated by PTH and pH, and at least two sodium molecules are exchanged for each phosphorus molecule (8, 9). SLC20A1 and SLC20A2are modulated by PTH, pH, and sodium availability (8, 9). Clinically, altered expression of placental SLC20A1 and SLC20A2 has been associated with preeclampsia, a gestational hypertension disorder caused in part by placental abnormalities (10). Specific fetal developmental requirements for both Slc20a1 and Slc20a2 have been elucidated through the use of genetic mouse models. Slc20a1 loss results in early embryonic lethality and Slc20a2 loss results in subviability (11, 12, 13). Unraveling the expression profiles of SLC20A1/Slc20a1 between human and mouse placenta is critical to understand the relevance of Slc20a1 to human placenta normal vascular development and organ health. Further investigation is required to understand the homeostatic interplay between maternal phosphate homeostasis and placental phosphate transport during pregnancy. In this study, we aimed to assess the SLC20A1/Slc20a1 expression profiles in vitro and in vivo by using in human BeWo placenta cells, human term placenta, and mouse placenta. Indeed, we confirmed that SLC20A1/Slc20a1 was the most highly expressed sodium-dependent transporter.

In this study, we identified a novel requirement for Slc20a1 in chorioallantoic morphogenesis, which is critical for placental vascular development. Briefly, the chorioallantois forms at ~E8.5 after the allantois attaches and spreads out along the fetal side of the chorionic epithelium (extraembryonic ectoderm), resulting in the fusion of the allantois and chorion and development of the chorioallantois which then develops into the labyrinth. Syncytiotrophoblast II (SynT-II) cells begin to differentiate and branch, followed by syncytiotrophoblast I (SynT-I) and sinusoidal trophoblast giant cells (sTGC) cells. Together, SynT-I, SynT-II, and sTGC cells form the trilaminar trophoblast layer of the mouse maternal–fetal interface in the fully developed labyrinth of the mouse placenta. The differentiation of SynT-II cells is driven by Wnt signaling (14) which is required for activation of the terminal SynT-II cell marker Gcm1 (15, 16). Multiple members of the Wnt signaling pathway are required for normal labyrinth morphogenesis, including Wnt7b (17), R-Spondin3 (18), Bcl9 (16), Frizzled 5 (Fzd5) (19), and Wnt2 (20). Crosstalk between WNT and Notch signaling pathways also plays a pivotal role in placenta development (21, 22). Notch signaling, along with pro-angiogenic factors such as vasuclar endothelial growth factor (VEGF), balances tip and stalk cell formation and regulates both tip cell migration and stalk cell proliferation (23, 24, 25, 26). VEGF and expression of delta-like ligand 4 (DLL4) and DLL4-mediated activation of Notch signaling in adjacent cells further promote stalk cell proliferation behind the tip cell (23, 24, 25, 26).

Together, the available data supports a novel model by which Slc20a1 interacts with the Notch–WNT pathway to support SynT differentiation and angiogenic mimicry. This knowledge has the potential to deepen our understanding of critical human placental vascular development signaling pathways that may promote villi angiogenesis and protect against placental vascular dysfunction in some cases of obstetrical complications, such as preterm birth, preeclampsia, and fetal growth restriction (FGR).

Materials and methods

Animal research

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was reviewed and approved by the Tufts Medical Center IACUC (Boston, MA, USA) (protocol #2017-145). Slc20a1 wild-type (Slc20a1+/+) C57Bl/6 mice were purchased from Jackson Laboratories and from Taconic Labs (Hudson, NY, USA); Slc20a1-knockout (Slc20a1–/) mice were purchased from the Jackson Laboratories (ID #B6.Cg-Slc20a1<tm1.2Cmg>/J). Slc20a1-heterozygous (Slc20a1+/–) mice were intercrossed to generate wild-type, heterozygous haploinsufficient, and knockout embryonic tissues. At least three animals/genotype were analyzed for each experiment. Mice were maintained in our animal facility at 22–24°C under a constant day–night rhythm and given food and water ad libitum. The timing of embryonic development was determined by the presence of a vaginal plug the morning after mating, with noon designated as E0.5. At the time of embryonic collection, female mice were euthanized by cervical spine fracture dislocation, and embryos and extraembryonic tissues were collected by mechanical dissection. Yolk sac (YS) was taken for genotyping.

Genotyping

Genotyping was performed using polymerase chain reaction (PCR) and agarose gel-based electrophoresis in TAE (Tris-acetate-EDTA) buffer, as previously described (Wallingford et al. 2016 (13)). Hot Start Green Master Mix 2× (Promega, M5122) was used according to the manufacturer’s directions. Primers were purchased from IDT and included exon 4 forward (Ex4F): 5’-TCTCCGCTCTTTCTGGAT-3’, exon 2 forward (Ex2F): 5’-CTCATCCTGGGCTTCATCAT-3’, and intron 4 reverse (int4R3): 5’-TTCCTTCCTGAATGCCCTCT-3’, as previously described by Festing et al. (2019) (27). 10 µM mass of each primer was added to ~50 ng mass of DNA in each sample. Positive and negative internal controls were included in all PCR experiments. The PCR thermal cycler program included an initial denaturation step of 95°C for 2 min, 40 PCR cycles (95°C for 30 s, 55°C for 45 s, and 72°C for 45 s), followed by a final extension at 72°C for 5 min. Gels were run in a 2% agarose gel at approximately 105 V for 50 min. DirectLoad PCR 100 bp Low Ladder (Sigma–Aldrich, D3687-1VL) was used to estimate amplicon sizes and SYBR Safe (Invitrogen, S33102) was used to observe dsDNA amplicons. Expected amplicon sizes included Slc20a1+/+: ~268 bp, Slc20a1+/+ and Slc20a1–/: ~504 bp (Supplementary Fig. 1E and F, see section on supplementary materials given at the end of this article) (27). Gel images were taken on an FluorChem M gel imaging system with On-board Digital Darkroom and AlphaView software.

BeWo fusion

Cell fusion was quantified as in Aghababaei et al. 2015 (28). Briefly, BeWo cells were cultured and treated with forskolin (5 µM) to induce cell differentiation, and fused cell proportions were calculated as the ratio of the number of nuclei in multinucleated cellular aggregates. Five images were acquired across the midline of each well. An aggregate was defined as ≥5 DAPI-stained nuclei surrounded by continuous E-cadherin (CDH1) membrane staining.

P33 symport assays

Phosphate symport/uptake studies were performed as previously described (29). Briefly, SMCs were incubated in Earle's buffered salt solution (EBSS) containing 0.1, 0.25, and 0.5 mM phosphate and 5 μCi/mL H333PO4 (PerkinElmer Life Science, Inc.). Radioactivity was measured by liquid scintillation. Sodium-dependent phosphate uptake was determined by subtracting the uptake levels measured in EBSS containing choline chloride from uptake levels measured in EBSS containing sodium chloride. Uptake values were normalized to cellular protein content.

Quantitative RT-PCR (hSMC and BeWo)

We examined the Slc20a1 expression and tested the phosphate transport competency of fused trophoblast BeWo cells as a model for human syncytiotrophoblasts and human smooth muscle cell (hSMC) cultures as positive controls. Cells were lysed and stored at −80°C. Total RNA was extracted using the RNeasy Mini Kit (Qiagen, 74104) and DNaseI (Qiagen, 1023460). cDNA synthesis was performed with 1000 ng RNA. Omniscript RT Kit (Qiagen, 205113) was used with both random hexamers (Qiagen, 79236) according to the manufacturer's instructions. TaqMan probes were conjugated with a fluorochrome reporter (FAM) tag at the 5’-end and, an MGB quencher at the 3’-end was used to assess Slc20a1 expression levels. Amplification and detection were carried out in 96-well optical plates on an ABI Prism 7000 Sequence Detection System (Applied Biosystems), with TaqMan Universal PCR 2× master mix (Life Technologies, 4305719) in a final volume of 25 µL per reaction. Each reaction (in triplicate) was carried out at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. Results were analyzed with the manufacturer's software, SDS 1.1 (Applied Biosystems). The gene mRNA expression was normalized to the housekeeping gene 18S and normalized to the NT control using the quantitative method (2–ΔΔCT). Taqman Assays included Slc20a2 (Life Technologies, Mm00660204_mH, FAM-MGB) and 18S ribosomal RNA control reagents (Life Technologies, cat. no. 4308329).

Quantification of human and mouse placenta gene expression

We quantified the bulk expression of sodium-dependent phosphate transporters in human and mouse placentas by microarray and RNA-seq analyses, respectively.

Human data

Human placental expression data were derived from previously published (30) available RNA-sequencing data (SRP094910). This included 200 samples from nonpathological, term pregnancies enriched for large and small for gestational age infants (30). Raw .fastq files were collected from DBGap (31) (database of genotypes and phenotypes), and transcript abundances were estimated using the quantification program Kallisto (32) and condensed to Ensembl Gene IDs using TXImport (33), producing count per million values which were then scaled (log CPM). For in silico analysis of single-nucleus RNA sequencing, single-nucleus RNA-sequencing datasets were derived by Marsh and Bleloch (34). The datasets were manually input into the STRING database to identify interacting protein networks (35).

Mouse data

Transcriptomic data were generated from placentas from wild-type female FVB mice aged 7–10 weeks that were mated with male mice of the same age. Pregnancy progression was monitored by visual inspection and body weight increase. Placentas were collected on E10.5, E15.5, and E19.5. Detailed description of the study design and tissue processing was previously described (36), and the data are publicly available on the Gene Expression Omnibus (37) as GSE41438. Raw data were summarized at the transcript level using robust multi-array average method using the Bioconductor ‘oligo’ package (38, 39) and normalized using quantile normalization within the LIMMA Bioconductor package. For genes which mapped to multiple probes, probe level expression values were averaged to generate gene-level expression values as previously described (40). All analyses were performed in R (Version 3.5.1).

qPCR (YS and placenta)

YS and placenta tissue RNA were analyzed across pregnancy and compared to control tissues (heart, kidney, and lung). RNA was extracted using the RNeasy Mini Kit according to the manufacturer’s directions (Qiagen, 74106). cDNA was made with 1000 µg of total RNA per sample using the Omniscript Reverse Transcriptase kit (Qiagen, 205113). TaqMan probes conjugated with an FAM tag at the 5’-end and an MGB quencher at the 3’-end or custom probes and SYBR green were used to assess expression levels (41). Amplification and detection were carried out in 96-well optical plates on an ABI Prism 7000 Sequence Detection System (Applied Biosystems), with TaqMan Universal PCR 2× master mix (Life Technologies, 4305719) in a final volume of 20 µL per reaction. Each reaction was carried out at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. Results were analyzed with the manufacturer’s software, SDS 1.1 (Applied Biosystems). Gene mRNA expression was normalized to the housekeeping gene 18S (Life Technologies, cat. no. 4308329) using the quantitative method (2–ΔΔCT, where ΔΔCT = (CTgene – CT18S)treated – (CTgene – CT18S)control).

The following probes and Life Technologies Taqman Assays were used to determine the gene expression: Slc17A1 (Mm00436577_m1); Slc17a4 (Mm00621610_m1); Slc17a7 (Mm00812886_m1); Slc34a1 (forward: 5’-GAGCCCTTCACAAGACTCATCAT-‘3 and reverse: 5’-CGGCAATGCTGGTGATCA-‘3); Slc34a2 (forward: 5’-TCGTCAGCATGGTTGCTTCT-‘3 and reverse: 5’-TGTTAGCGCCCATGATGATG-‘3); Slc34a3 (forward: 5’-CCTCAGCTCTGCTTTCCAGCTA-‘3 and reverse: 5’-AGCACCACATTGTCCTTGAAAA-‘3); Slc20a1 (forward: 5’-TTCCTTGTTCGTGCGTTCATC-‘3 and reverse: 5’-AATTGGTAAAGCTCGTAAGCCATT-‘3, probe: 5’-CCGTAAGGCAGATCC-‘3), and Slc20a2 (Mm00660204_mH).

Histology

Samples were dissected and fixed in 4% paraformaldehyde (PFA)/PBS overnight at 4°C. After whole-mount images were taken, the tissue was dehydrated in 100% ethanol, treated with xylene, and embedded in paraffin wax at 60°C. Sections were cut at 7 μm on a Leica RM2135 microtome, dried, and baked at 60°C for 1 h prior to further processing. Immunofluorescence was performed, as previously described (42). Hematoxylin and eosin (H&E) staining was performed as previously described (43, 44). RNAScope was performed using Multiplex Fluorescent v2 system (ACD; 323100), and the standard RNAScope protocol was used according to the manufacturer’s instructions. The following probe was used: mmSlc20a1 (ACD; Mm-Slc20a1-O1). The following antibodies and antibody concentrations were used: anti-E-cadherin (CDH1) (Abcam, ab53033); anti-pH3 (Santa Cruz, sc-374669); anti-monocarboxylate transporter 1 protein (MCT1) (Abclonal, A3013); donkey anti-rabbit Dylight 549 7.5 μg/mL (Jackson ImmunoResearch, 711-505-152); donkey anti-goat Dylight 7.5 μg/mL (Jackson ImmunoResearch, 488 705-485-147); donkey anti-mouse Alexa Fluor 488 7.5 μg/mL (Jackson ImmunoResearch, 715-545-151); and donkey anti-rabbit IgG 15 μg/mL (Jackson ImmunoResearch, 711-545-152). Immunofluorescence was followed by DAPI dilactate for nuclear counterstain (Life Technologies, D3571) and mounted with ProLong Gold Antifade Media (Life Technologies, P36930).

Quantification of histological features

All stained and mounted sections were imaged on a Nikon E800 Upright Microscope Colibri. One section/genotype was imaged on a Keyence Plan Apochromat 40× (BZ-PA40, Keyence Corporation, Osaka, Japan). All histology quantifications were performed blind using ImageJ (45). The ImageJ cell counter plugin was used to assist with cell counting. The ImageJ ROI and watershed plugins were used for MCT1 quantification. Total area analysis tool and cell counter plugin were used to assist with labyrinth MCT1+ cell counting. The area under the curve was calculated using the trapezoidal formula. Remaining quantifications were performed by applying area or freehand line measurement ImageJ cell plugin.

Statistical analysis

At least three independent samples per genotype were analyzed for all experimental data. The following statistical tests were used to analyze the quantitative data (GraphPad Prism version 8.0.0). For comparison of two groups, a P-value was determined by a two-tailed Student's t-test with unequal variance. For comparison of three or more groups, one-way ANOVA was used followed by Tukey's post hoc test when appropriate to compare means between groups.

Results

Slc20a1 is poised to regulate placental phosphate transport

The type III sodium-dependent phosphate transporter Slc20a1 plays key roles in vascular smooth muscle cells (vSMCs) where it serves a crucial role in cellular phosphate symport, but the function of phosphate transporters in the placenta has not yet been fully elucidated. Published data indicated that phosphate transport across the discoid hemochorial placenta (that of mouse, rat, and human) is sodium dependent. We examined the Slc20a1 expression and tested the phosphate transport competency of fused trophoblast BeWo cells as a model for human syncytiotrophoblasts. Slc20a1 is as highly expressed in BeWo as in hSMC, and Slc20a1 levels are increased in response to increased levels of high extracellular Pi (HP) but are not altered between normal phosphate (NP) and HP conditions (Fig. 1A). Fused forskolin-treated BeWo cells were used as a model of syncytiotrophoblast (Fig. 1B, C, D and E). Uptake of radiolabeled P33 revealed active, sodium-dependent phosphate transport compared to choline-treated controls (Fig. 1E). Bulk expression of sodium-dependent phosphate transporters in human (Fig. 2A) and mouse (Fig. 2B) placentas by microarray and RNAseq analyses, respectively, determined that the most highly expressed phosphate transporters included Slc20a1, Slc20a2, and Slc34a2 in both mouse and human placenta. The expression of all three genes increases over time in mouse placenta (Fig. 2B). SLC20A1/Slc20a1 was the most highly expressed sodium-dependent transporter in both species (P < 0.05, ANOVA with Tukey test).

Figure 1
Figure 1

Phosphate (Pi) symport in BeWo trophoblasts is active and sodium dependent. Slc20a1 mRNA qPCR shows that Slc20a1 is as highly expressed in BeWo as in hSMCs, and Slc20a1 levels are increased in response to increased levels of extracellular Pi (HP vs water); however, Slc20a1 levels were not altered between NP normal (NP) and high (HP) extracellular phosphate levels (NP vs HP) (A). Fused BeWo cells were used to model syncytiotrophoblast (B–D). BeWo cell fusion was induced with 5 μM forskolin in DMSO (B–D). The extent of syncytialization was determined by counting the number of syncytia (cells with ≥5 nuclei) in forskolin and DMSO control-treated wells. Three wells per group and five images taken across the diameter of each well were analyzed, confirming effective forskolin-induced fusion (D). Treatment with radiolabeled P33 and sodium or choline demonstrated active, sodium-dependent transport supporting active and sodium-dependent transport (E). *P < 0.05, by Student's t-test. Forsk, forskolin; HP, high phosphate; hSMC, human smooth muscle cells; NP, normal-phosphate.

Citation: Vascular Biology 5, 1; 10.1530/VB-22-0018

Figure 2
Figure 2

The sodium-dependent Pi transporters SLC20A1, SLC20A2, and SLC34A2 are highly expressed in the placenta. SLC20A1, SLC20A2, and SLC34A2 are the most highly expressed sodium-dependent phosphate transporters in human placenta (n = 200) as quantified through RNA-sequencing data (A). Slc20a1, Slc20a2, and Slc34a2 are also the most highly expressed sodium-dependent phosphate transporters in mouse placenta (n = 15) as quantified through microarray data (B). The expression of these three transporters increases across gestational time in the mouse placenta.

Citation: Vascular Biology 5, 1; 10.1530/VB-22-0018

Slc20a1, Slc20a2, and Slc34a2 are expressed in the YS and placenta

Expression levels of type I (Slc17a1, Slc17a4, and Slc17a7), type II (Slc34a1, Slc34a2, and Slc34a3), and type III (Slc20a1 and Slc20a2) in the placenta and YS were examined across pregnancy and compared to control tissues (heart, kidney, and lung), in which each gene is known to serve a required function (Fig. 3A, B, C, D, E, F, G and H). Slc17a1 expression peaked in E11.5 YS and was highly variable between biological replicates (Fig. 3A). Modest levels of Slc17a4 (Fig. 3B) and Slc34a3 (Fig. 3F) were detected in the developing YS that were far below the expression levels observed in control tissues in which they have known biological activity. Slc20a1 (Fig. 3G), Slc20a2 (Fig. 3H), and Slc34a2 (Fig. 3E) are the most highly expressed sodium-dependent phosphate transporters in mouse YS and placenta. The expression of Slc20a2 increased across gestational time in the placenta, whereas Slc20a1 (Fig. 3G) and Slc34a2 (Fig. 3E) plateaued between E9.5 and E10.5.

Figure 3
Figure 3

The sodium-dependent Pi transporters Slc20a1, Slc20a2, and Slc34a2 expression in mouse yolk sac and placenta increases throughout pregnancy. Slc20a1, Slc20a2, and Slc34a2 are the most highly expressed sodium-dependent phosphate transporters in mouse yolk sac and placenta (n = 3) as quantified through qPCR data. The expression of these three transporters increases across gestational time in the placenta, whereas Slc20a1 and Slc34a2 expression peaks between E9.5 and E10.5 and Slc20a2 increases across gestational time in the yolk sac. H, heart; K, kidney; L, lung.

Citation: Vascular Biology 5, 1; 10.1530/VB-22-0018

Slc20a1 loss at E9.5 results in reduced placental development and growth

Previous studies identified that Slc20a1-knockout (Slc20a1–/– ) embryos display embryonic lethality and gross embryonic vascular abnormalities at E10.5 (27), but the placenta phenotypes remained undetermined. In order to address this knowledge gap, we generated heterozygous Slc20a1+/– intercrosses and compared Slc20a1+/+ and Slc20a1–/– placenta tissues. Whole-mount brightfield imaging was not able to detect gross placental width at E9.5 alterations between Slc20a1–/ and Slc20a1+/+(Supplementary Fig. 1A, B, C, and D). Paraffin-embedded tissue sections and H&E staining were then used to evaluate tissue morphology (Fig. 4A, B, C, and D). At E9.5, the developing placenta contains the ectoplacental cone (EPC) and the chorioallantois, which develops into the labyrinth through branching morphogenesis. At E9.5, the EPC and chorioallantois presented normal anatomy in the Slc20a1+/+control tissue (Fig. 4A and C). However, despite the dissected Slc20a1–/–placenta displaying a normal size, the Slc20a1–/–tissue sections revealed a defect in the chorioallantois/developing labyrinth (Fig. 4B and D). This was further supported by a semiquantitative analysis of the developing Slc20a1–/ labyrinth (height and area), which confirmed reduced growth and impaired development (Fig. 4E and F). Slc20a1 RNA expression was surveyed in Slc20a1+/+ and Slc20a1–/ (data not shown) developing placenta and embryo with RNAscope in situ hybridization (ISH) methods. We found that Slc20a1 RNA is expressed in the placenta, chorioallantois and YS compartments of the Slc20a1+/+ (data not shown). We further confirmed that Slc20a1 RNA is not expressed in the placenta, chorioallantois, or YS compartments of the Slc20a1–/– (not shown).

Figure 4
Figure 4

Developing placenta displays reduced development and growth with Slc20a1 loss at E9.5. Developing placenta, constituted of an ectoplacental cone (EPC) and developing labyrinth, was closely examined at E9.5. H&E staining was used to evaluate placental development phenotype (A–D). At E9.5, the Slc20a1+/+ placenta showed normal vascular development with typical EPC and labyrinth with proper morphogenesis (A, C). However, despite Slc20a1–/–EPC showing normal development and size, the Slc20a1–/–developing placenta showed reduced development due to early labyrinth development failure in the chorioallantoic branching step (B, D). We performed longitudinal (yellow arrow in C and D) and area measurements of the chorion and placenta, and the placenta displayed impaired development with Slc20a1 loss (E, F; P ≤ 0.05). Ch/al, chorioallantois; Dec, decidua; P, developing placenta; YS, yolk sac.

Citation: Vascular Biology 5, 1; 10.1530/VB-22-0018

Deletion of Slc20a1 results in reduced trophoblast SynT-I coverage

To identify the cause of chorioallantois morphogenesis failure in the Slc20a1–/–placenta, we first examined the cellular proliferation of E9.5 Slc20a1+/+ and Slc20a1–/ by analyzing pH3+ cell nucleus immunofluorescence (Fig. 5A, B, C, D, E, F, G, H and I). We detected similar levels of pH3+ cells in the chorion and placenta of both Slc20a1+/+ (Fig. 5A, B, C, G, H, and I) and Slc20a1–/–(Fig. 5D, E, F, G and I), indicating that Slc20a1 loss does not disrupt early placental cellular proliferation.

Figure 5
Figure 5

Slc20a1 loss at E9.5 preserves early placental cellular proliferation. Cell proliferation rates of chorion and placenta were determined by pH3+ cell nuclei (red) by immunofluorescence (A–F). Proliferating cells were detected for Slc20a1+/+ (A–C) chorion (B) and placenta (C), as well as Slc20a1–/– (D–F) chorion (E) and placenta (F). Cell proliferation rates were quantified for chorion (H) and placenta (I). No differences in cell proliferation rates were observed with Slc20a1 loss. Scale bar: 100 µm. Ch, chorion; Dec, decidua; P, developing placenta; YS, yolk sac.

Citation: Vascular Biology 5, 1; 10.1530/VB-22-0018

Next, we hypothesize that, although normal rates of proliferation were observed, the differentiation of chorioallantois cells may be disrupted with Slc20a1–/– loss (Fig. 4E and F). Trophoblasts with invasive properties utilize the glucose and the glycolytic pathway for energy and export lactate from SynT-I cells through the MCT1 (46), which can thus be used as a SynT-I specific cell marker. To examine whether trophoblast SynT-I coverage was reduced with Slc20a1 loss, we analyzed MCT1 positivity by immunofluorescence (Fig. 6A, B, C, D, E, F, G, H and I). Overall, total MCT1 was not altered, but MCT1+ cells were reduced in Slc20a1–/–labyrinth (Fig. 6H and I), confirming that Slc20a1 loss reduces trophoblast SynT-I coverage in Slc20a1–/ labyrinth. Despite the normal localization of MCT1 protein, the location and cellular morphology of MCT1+ SynT-I cells further suggested that they failed to migrate toward a high concentration of glucose in maternal blood. Morphologically, SynT-I cellular projections were absent, and we observed the presence of spherical cells, as opposed to elongated cells. This interchanging of cell morphogenesis represents normal angiogenic endothelial cell mimicry. Lastly, we observed that the height of the labyrinth was reduced in Slc20a1–/– (Fig. 6D, E and F).

Figure 6
Figure 6

Slc20a1 loss at E9.5 results in reduced trophoblast SynT-I coverage. SynT-I coverage in the labyrinth was determined by MCT1+ cell (green) immunofluorescence (A–F). Immunofluorescence analysis reveals that Slc20a1 loss reduces trophoblast SynT-I coverage in Slc20a1–/– labyrinth, shown by reduced MCT1+ cells (DF, I). Scale bars: 100 µm. *P < 0.05, by Student's t-test. Ch/al, chorioallantois; Dec, decidua; P, developing placenta; YS, yolk sac.

Citation: Vascular Biology 5, 1; 10.1530/VB-22-0018

Slc20a1–/ embryos die by E10.5 (27), and this time frame corresponds with that of YS malfunction mortalities (47). To test whether the reduction in MCT1+ cells observed in Slc20a1–/– placenta was tissue specific or observed in the YS as well, we analyzed the membrane localization of MCT1 by immunofluorescence (Supplementary Fig. 2A, B, C, and D). No alteration of MCT1 localization in the apical membrane and basal membrane of the YS visceral endoder layer was observed with Slc20a1 loss (Supplementary Fig. 2). This indicates that the reduction of Mct1+ cells in the extraembryonic tissue of Slc20a1–/– is syncytiotrophoblast specific.

Slc20a1 is expressed in SynT-II trophoblast precursor cells

Slc20a1 at E9.5 was examined in silico through the use of single-nucleus RNA-sequencing data published recently by Marsh and Bleloch (34). The results supported the fact that Slc20a1 was detected in blood cells as well as SynT-II trophoblast precursors (Table 1).

Table 1

List of transcripts detected for cluster cell type nuclei.

Gene Cluster cells Cell type
WNT pathway
Slc20a1 Blood cells Synt-II P
Rspo3 Endothelial
Bcl9l Trophoblast SynT-I
Gcm1 Trophoblast SynT-II
Gsk3b Decidual stroma
Axin1 Trophoblast LaTP2
Apc Trophoblast S-TGC
Ctnnb1 Trophoblast SynT-II
Dvl1 Trophoblast S-TGC
Tle1 Fetal mesenchyme
Lef1 Blood cells
Wnt5b1 Fetal mesenchyme
Wnt5b Fetal mesenchyme
Tip and stalk pathway
Cbfa2t3 Blood cells
Flt1 Trophoblast GC
Hes7 Trophoblast SynT-II
Itga6 Endothelial SynT-II
Jag2 Trophoblast SynT-I
Lamc1 Trophoblast GC
Notch1 Endothelial
Notch2 Fetal mesenchyme JZP1
Notch21 Trophoblast SpT P
Notch22 Trophoblast S-TGC P
Notch23 Trophoblast S-TGC
Notch24 Trophoblast
Notch25 Blood cells
Notch26 Fetal mesenchyme
Notch3 Fetal mesenchyme
Plxnd1 Endothelial
Sox13 Endothelial
Sox131 Decidual stroma
Sox4 Fetal mesenchyme
Sox5 Fetal mesenchyme
Sox51 Fetal mesenchyme
Sox52 Fetal mesenchyme
Sox53 Fetal mesenchyme
Sox6 Endothelial
Sox61 Endothelial
Sox62 Fetal mesenchyme
Sox63 Fetal mesenchyme
Tie1 Endothelial
Tie11 Blood cells
Tie12 Endothelial
Tie13 Endothelial
Tie14 Endothelial
Vegfa Decidual stroma SynT-II

GC, glycogen cells; JZP1, junctional zone precursors 1; LaTP2, labyrinth trophoblast progenitor 2; SpT P, spongiotrophoblast precursors; S-TGC, sinusoidal trophoblast giant cells; S-TGC P, sinusoidal trophoblast giant precursor cells; SynT-II P, syncytiotrophoblast II precursors.

To determine whether Slc20a1 is required for angiogenic mimicry, we first hypothesized that SynT-II cells (which are present at the tip of the initial branching labyrinth) and SynT-II precursor cells are syncytiotrophoblast equivalents of the endothelial tip and stalk cells. To test this idea, marker genes associated with endothelial tip and stalk cells, including Notch pathway genes, Tie1, Tie2, and Sox17, were analyzed in the complete Marsch single-nucleus RNA-sequencing dataset, as well as trophoblast-specific clusters. Thirty-three transcripts were detected (Table 1). Both Notch1 and Tie1 were specific to endothelial cells as expected. Tie17 was also detected in endothelial cells. Notch25, Tie12, and Tie13 were detected in the blood cells and Notch2, Notch 3, and Notch 26 were detected in the fetal mesenchyme. The putative tip cell markers Dll4 and Sox17 were not detected. Trophoblast clusters expressed the stalk cell marker Jag2 (junctional zone precursor 1) as well as Notch21 (spongiotrophoblast precursors), Notch22 (sinusoidal trophoblast giant precursor cells, S-TGC P), Noch23 (S-TGC), and Notch24 (SynT-I). Interestingly enough, SynT-II cells mimicked tip cells as they expressed both Integrin 6a (Itga6) and VEGF (Vegfa), and adjacent endothelial and fetal mesenchymal cells expressed Notch1, Notch2, Notch3, and Notch26, respectively (Table 1). This indicates that Slc20a1 may be required to support angiogenic mimicry, and supports the hypothesis that SynT-II cells. Further, SynT-II precursor cells may function as syncytiotrophoblast equivalents of the endothelial tip and stalk cells.

Wnt genes have been shown to drive SynT-II differentiation (48), and knockout mice with null expression of several genes, including Wnt7b, R-Spondin3, Bcl9Fzd5, Fzd5, and Wnt2, have phenotypes reminiscent of the Slc20a1–/– placenta. Furthermore, a crosstalk between WNT and Notch signaling pathways has been previously described to play pivotal roles in placenta development (14, 21, 22). To determine whether Slc20a1 may directly regulate Wnt signaling, Wnt genes were examined in the Marsh dataset (34). Neither Wnt7b, Bcl9Fzd5, Fzd5, or Wnt2 was detected. Twelve transcripts were detected (Table 1). Wnt5b, Wnt5b1, andTLE/Groucho (Tle1) transcripts were all detected in the fetal mesenchyme nuclei. R-Spondin3 (Rspo3) transcript was detected in the endothelial nuclei. LEF (Lef1) was detected in blood cells. GSK3B (Gsk3b) transcript was detected in decidual stroma nuclei. B-catenin (Ctnnb1), Bcl9l, Gcm1, Axin1, Apc, and Dvl1 transcripts were detected on trophoblast nuclei; more specifically, B-catenin (Ctnnb1) and Gcm1 were detected on SynT-II nuclei, Bcl9l was detected on SynT-I nuclei, Axin1 was detected on labyrinth trophoblast progenitor 2 (LaTP2) nuclei, and Apc and Dvl1 were detected on TGC nuclei. This suggests that SynT-II and fetal mesenchyme development is directly regulated by Slc20a1-dependent Wnt signaling pathway. Moreover, B-catenin/BCL9 has been previously described to be involved in the signaling of GCM1/syncytin pathway and regulation of the fusion of human choriocarcinoma cells (49); thus we here propose that in the mouse placenta Slc20a1 regulates tip SynT-II cell fusion by the action of B-catenin/Bcl9/Gcm1 (Fig. 7A).

Figure 7
Figure 7

Proposed model of Slc20a1 and tip and stalk cell differentiation. Slc20a1 regulates Notch–WNT pathway in tip and stalk cell selection (A). Stalk SynT-II precursor cells express Slc20a1, which signals the tip SynT-II cell angiogenic differentiation and fusion by regulation of Notch–WNT pathway. Tip SynT-II cells respond to VEGF through Integrin 6a (Itga6) and undergoes angiogenesis. VEGF may activate Notch proteins in stalk neighboring cells, such as endothelial cells and fetal mesenchyme cells. Tip SynT-II cell activates a signal transduction pathway that regulates cell fusion by activation of WNT signaling, by activation of B-catenin/Gcm1 with recruitment of co-activator Bcl9 from adjacent SynT-I cells, resulting in SynT-II cell fusion. The stalk fetal mesenchyme cells maintain an ON and OFF state of canonical WNT signaling, with Wnt5 activating WNT target genes and TLE/GrouchoWNT repressor silencing the WNT target genes. Notch is activated by WNT pathway in stalk endothelial and fetal mesenchyme cells. Impaired Pi transfer with Slc20a1 loss in Slc20a1–/–- placenta results in defects in the development of SynT-I and disorganization and of the labyrinth compartment (B). This originates insufficient chorioallantois and placental vascular and morphogenesis, observed by reduced placental measurements (area and weight), reduced yolk sac development, and failed embryo development. Bcl9, B-cell lymphoma 9; Gcm1, glial cells missing 1; SynT-I, syncytiotrophoblast layer I; SynT-II, syncytiotrophoblast layer II; TLE, transducin-like enhancer of split; VEGF, vascular endothelial growth factor; WNT, wingless/integrated.

Citation: Vascular Biology 5, 1; 10.1530/VB-22-0018

Discussion

SLC20A1/Slc20a1 was the most highly expressed sodium-dependent transporter in both human and mouse placenta. Slc20a1 expression plateaus between E9.5 and E10.5 in the placenta and YS. Loss of Slc20a1 results in reduced size and arrested development of the placenta at E9.5. This data support the fact that Slc20a1 is required for normal placental development. We determined that MCT1+ cells were reduced in the Slc20a1–/–- labyrinth but not in the YS. These results support that Slc20a1 loss reduces trophoblast SynT-I coverage in Slc20a1–/ labyrinth compartment, indicating insufficient chorioallantois and disrupted placental vascular and morphogenesis (Fig. 7B). Furthermore, the BeWo trophoblast model confirmed cellular competency for active, sodium-dependent phosphate symport as well as extracellular matrix calcification that increased with syncytialization. BeWo syncytialized cells represent an apt model for transporter studies, with 77% of the transporter RNA expression studied showing similarity with primary human trophoblast cells (50). Herein, we confirmed that SLC20A1/Slc20a1 was the most highly expressed sodium-dependent transporter in BeWo cells, human term placenta, and mouse placenta. Taken together, our findings support the fact that normal growth and maintenance of the placenta require SLC20A1/Slc20a1. We now propose the working hypothesis that placental phosphate transport supports the energy requirements for placental vascular development, enabling the development of sufficient maternalfetal interface surface area and minimizing turbulent flow that together promote adequate blood flow, organ health, and maternalfetal transport of nutrients, including phosphorus.

With respect to the required developmental roles, Slc20a1 and Slc34a2 are early embryonic lethal and Slc20a2-null mice are subviable and display FGR (27, 44, 51, 52). Further details on unique vs overlapping expression domains of these transporters may identify distinct roles or cell-type specific activities for these transporters. Slc20a1 knockdown in VSMCs decreases Pi uptake and mineralization (53). Global Slc20a1 loss in the mouse results in a YS angiogenesis defect early in development, which may or may not relate to endothelial tip and stalk cell biology (47). Notably, we found that the Slc20a1–/–- placenta is reduced in size and early labyrinth development is impaired, further supporting the role of Slc20a2 in extraembryonic vascular development. Moreover, we determined that MCT1, a marker of SynT-I, was reduced in Slc20a1–/–labyrinth. As such, Slc20a1 loss reduces trophoblast SynT-I coverage in Slc20a1–/ labyrinth compartment and arrests chorioallantois morphogenesis (Fig. 7B).

Next, we investigated how Slc20a1 may promote the morphogenesis of the chorioallantois and labyrinth in a novel hypothetical model which tests SynT-II and SynT-II precursor cells as equivalents to endothelial tip and stalk cells within the paradigm of placental vascular development at the specialized maternal–fetal interface. In order to test this, we examined the cell type-specific expression of Slc20a1 and endothelial tip/stalk Notch/Wnt pathway genes in silico. SynT-II cells mimicked tip cell gene expression profiles, with the expression of Itg6a, and VEGF, whereas the adjacent SynT-II precursor cells mimicked stalk cells, as they expressed Slc20a1, Notch1, Notch2, Notch3, and Notch26, respectively (Table 1) (Fig. 7A). Wnt signaling pathway genes and B-catenin and Gcm1 were detected on SynT-II nuclei, and Bcl9l was detected on SynT-I and nuclei. We herein propose that in the mouse placenta, Slc20a1 regulates tip SynT-II cell fusion by the action of B-catenin/Bcl9/Gcm1 (Fig. 7A).

Syncytiotrophoblasts can perform endothelial-like functions, including vasculogenic mimicry and angiogenic mimicry. At E9.5, SynT-II cells are, with respect to physical location, at the tip of the initial branching labyrinth (54). It remains unknown, however, whether GCM1+ SynT-II cells actively lead the remaining cells or are passively pushed forward (48). In this study, we proposed and tested the novel idea that SynT-II cells and SynT-II precursors serve analogous functions to the endothelial tip and stalk cells, respectively. Indeed, SynT-II cells mimicked tip cells, with the expression of Itg6a and VEGF, whereas the adjacent SynT-II precursor cells, endothelial cells, and fetal mesenchymal cells mimicked stalk cells, as they expressed Slc20a1, Notch1, Notch2, Notch3, and Notch26, respectively. As such, we propose that Slc20a1 in stalk SynT-II precursor cells is the driver of tip SynT-II cell mobilization, activating VEGF and Integrin 6α. This will activate Notjch signaling and promote the proliferation of the adjacent stalk cells, endothelial cells, and fetal mesenchyme cells (Fig. 7A).

Trophoblasts with invasive properties utilize the glucose and the glycolytic pathway for energy. This results in a buildup of lactate which is exported from SynT-I cells by MCT1 and from SynT-II cells by MCT4 (46). Moreover, MCT1 and MCT4 presented a polarized localization on the maternal side and fetal side of the syncytiotrophoblast layers, respectively (46). Localization of MCT1 and MCT4 in the cell membrane is critical for their lactate transport function. In SiHa tumor cells, MCT1 is essential for glucose sensing and directed invasion toward glucose sources, such as maternal blood within spiral arteries (55). In endometrial cancer cells, MCT1 protein abundance correlates with poor cancer prognosis, presumably due to increased invasiveness (56). Increased use of the glycolytic pathway is recognized as a key characteristic of malignant cells, and it results in abundant lactic acid production (57). A low threshold of either insufficient glucose uptake or lactic acid removal results in cell death of invasive cells which are not close enough to the vascular supply (55). Invasive cells prevent cellular acidosis by increasing proton efflux via the upregulation of pH regulators such as proton pumps, sodium–proton exchangers, and/or MCT1/MCT4 (57). Together, the in vivo invasion phenotype and the morphology of MCT1+ SynT-I cells support the fact that Slc20a1 is required for glucose sensing, glucose signal transduction, or lactate removal. Future studies will be needed to determine which step of this molecular pathway requires Slc20a1, and whether it functions at the cell membrane, in the cytoplasm, or in the mitochondria as well as whether this process is phosphate dependent or phosphate independent. We propose that Slc20a1 is needed for the symport of Pi that supports mitochondrial function enabling the mitochondria to be able to respond to the chemotaxic glucose signal and will test this hypothesis in future work.

Metabolism is becoming increasingly recognized for regulating aspects of embryonic development (58). In line with this, our results supported that syncytiotrophoblast differentiation is disrupted in the absence of Slc20a1. Endogenous and exogenous lactate have been shown to drive cell differentiation toward the extraembryonic endoderm lineage (58). In-depth, cell type-specific gene expression assays are needed to examine this question and determine whether Slc20a1 is required for normal differentiation of syncytiotrophoblast lineages. There is evidence that Slc20a1 directly regulates Wnt signaling, which is the key regulatory pathway which drives SynT-II differentiation (48). Furthermore, studies involving the deletion or inhibition of various components of the Wnt signaling pathway have shown angiogenic and vascular defects in the placenta (59). Notably, loss of Wnt7b (17), R-Spondin3 (18), Bcl9l (16), Fzd5 (19), and/or Wnt2 (20) results in reduced Gcm1+ cells and insufficient labyrinthine morphogenesis (48). In somatotroph adenomas, Slc20a1 levels are correlated positively with tumor size, invasive behavior, and tumor recurrence (60). Furthermore, Slc20a1 knockdown repressed cell invasion and increased the expression of Wnt pathway members Wif1 and secreted frizzled-related protein 4, supporting that Slc20a1 is associated with the activation of the Wnt/B-catenin signaling pathway (60). We observed that B-catenin and Gcm1 were detected in SynT-II nuclei, whereas Bcl9l and Slc20a1 were detected in SynT-I nuclei. Previous studies describe B-catenin/BCL9 to be involved in the signaling of GCM1/syncytin pathway and regulation of the fusion of human choriocarcinoma cells (49). As such, we herein propose that in the mouse placenta Slc20a1 regulates tip SynT-II cell fusion by the action of B-catenin/Bcl9/Gcm1 (Fig. 7A).

Overall, our studies indicate that Slc20a1 is a predominant placental phosphate transporter, and our data support the fact that Slc20a1 is critical for normal morphogenesis of the hemochorial placenta. Furthermore, we present a new working model to test in future work in which Slc20a1 in SynT-II precursors supports: (1) the development of SynT-II trophoblasts in the labyrinth and (2) metabolic and angiogenic mimicry properties of SynT-II trophoblasts to promote the development of the hemochorial placenta.

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/VB-22-0018.

Declaration of interest

The authors have no financial relationships, patents, or copyrights to declare. No payment or services from a third party were received for any aspect of the submitted work.

Funding

This work did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.

Acknowledgements

This work was supported in part by National Institutes of Health K99HD090198 (M. Wallingford). Ongoing support is from the National Institutes of Health R00HD090198 (M. Wallingford); American Heart Association 19CDA34660038 (M. Wallingford); National Institutes of Health DA032507 (Q. Mao); and National Institutes of Health 1K99HD096112-01 (A. Paquette). The authors would like to thank Cecilia Giachelli (University of Washington, Seattle) for support and guidance as well as Kimberly Johansson (Washington University, St Louis) and Hunter Wallingford for discussion.

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  • Figure 1

    Phosphate (Pi) symport in BeWo trophoblasts is active and sodium dependent. Slc20a1 mRNA qPCR shows that Slc20a1 is as highly expressed in BeWo as in hSMCs, and Slc20a1 levels are increased in response to increased levels of extracellular Pi (HP vs water); however, Slc20a1 levels were not altered between NP normal (NP) and high (HP) extracellular phosphate levels (NP vs HP) (A). Fused BeWo cells were used to model syncytiotrophoblast (B–D). BeWo cell fusion was induced with 5 μM forskolin in DMSO (B–D). The extent of syncytialization was determined by counting the number of syncytia (cells with ≥5 nuclei) in forskolin and DMSO control-treated wells. Three wells per group and five images taken across the diameter of each well were analyzed, confirming effective forskolin-induced fusion (D). Treatment with radiolabeled P33 and sodium or choline demonstrated active, sodium-dependent transport supporting active and sodium-dependent transport (E). *P < 0.05, by Student's t-test. Forsk, forskolin; HP, high phosphate; hSMC, human smooth muscle cells; NP, normal-phosphate.

  • Figure 2

    The sodium-dependent Pi transporters SLC20A1, SLC20A2, and SLC34A2 are highly expressed in the placenta. SLC20A1, SLC20A2, and SLC34A2 are the most highly expressed sodium-dependent phosphate transporters in human placenta (n = 200) as quantified through RNA-sequencing data (A). Slc20a1, Slc20a2, and Slc34a2 are also the most highly expressed sodium-dependent phosphate transporters in mouse placenta (n = 15) as quantified through microarray data (B). The expression of these three transporters increases across gestational time in the mouse placenta.

  • Figure 3

    The sodium-dependent Pi transporters Slc20a1, Slc20a2, and Slc34a2 expression in mouse yolk sac and placenta increases throughout pregnancy. Slc20a1, Slc20a2, and Slc34a2 are the most highly expressed sodium-dependent phosphate transporters in mouse yolk sac and placenta (n = 3) as quantified through qPCR data. The expression of these three transporters increases across gestational time in the placenta, whereas Slc20a1 and Slc34a2 expression peaks between E9.5 and E10.5 and Slc20a2 increases across gestational time in the yolk sac. H, heart; K, kidney; L, lung.

  • Figure 4

    Developing placenta displays reduced development and growth with Slc20a1 loss at E9.5. Developing placenta, constituted of an ectoplacental cone (EPC) and developing labyrinth, was closely examined at E9.5. H&E staining was used to evaluate placental development phenotype (A–D). At E9.5, the Slc20a1+/+ placenta showed normal vascular development with typical EPC and labyrinth with proper morphogenesis (A, C). However, despite Slc20a1–/–EPC showing normal development and size, the Slc20a1–/–developing placenta showed reduced development due to early labyrinth development failure in the chorioallantoic branching step (B, D). We performed longitudinal (yellow arrow in C and D) and area measurements of the chorion and placenta, and the placenta displayed impaired development with Slc20a1 loss (E, F; P ≤ 0.05). Ch/al, chorioallantois; Dec, decidua; P, developing placenta; YS, yolk sac.

  • Figure 5

    Slc20a1 loss at E9.5 preserves early placental cellular proliferation. Cell proliferation rates of chorion and placenta were determined by pH3+ cell nuclei (red) by immunofluorescence (A–F). Proliferating cells were detected for Slc20a1+/+ (A–C) chorion (B) and placenta (C), as well as Slc20a1–/– (D–F) chorion (E) and placenta (F). Cell proliferation rates were quantified for chorion (H) and placenta (I). No differences in cell proliferation rates were observed with Slc20a1 loss. Scale bar: 100 µm. Ch, chorion; Dec, decidua; P, developing placenta; YS, yolk sac.

  • Figure 6

    Slc20a1 loss at E9.5 results in reduced trophoblast SynT-I coverage. SynT-I coverage in the labyrinth was determined by MCT1+ cell (green) immunofluorescence (A–F). Immunofluorescence analysis reveals that Slc20a1 loss reduces trophoblast SynT-I coverage in Slc20a1–/– labyrinth, shown by reduced MCT1+ cells (DF, I). Scale bars: 100 µm. *P < 0.05, by Student's t-test. Ch/al, chorioallantois; Dec, decidua; P, developing placenta; YS, yolk sac.

  • Figure 7

    Proposed model of Slc20a1 and tip and stalk cell differentiation. Slc20a1 regulates Notch–WNT pathway in tip and stalk cell selection (A). Stalk SynT-II precursor cells express Slc20a1, which signals the tip SynT-II cell angiogenic differentiation and fusion by regulation of Notch–WNT pathway. Tip SynT-II cells respond to VEGF through Integrin 6a (Itga6) and undergoes angiogenesis. VEGF may activate Notch proteins in stalk neighboring cells, such as endothelial cells and fetal mesenchyme cells. Tip SynT-II cell activates a signal transduction pathway that regulates cell fusion by activation of WNT signaling, by activation of B-catenin/Gcm1 with recruitment of co-activator Bcl9 from adjacent SynT-I cells, resulting in SynT-II cell fusion. The stalk fetal mesenchyme cells maintain an ON and OFF state of canonical WNT signaling, with Wnt5 activating WNT target genes and TLE/GrouchoWNT repressor silencing the WNT target genes. Notch is activated by WNT pathway in stalk endothelial and fetal mesenchyme cells. Impaired Pi transfer with Slc20a1 loss in Slc20a1–/–- placenta results in defects in the development of SynT-I and disorganization and of the labyrinth compartment (B). This originates insufficient chorioallantois and placental vascular and morphogenesis, observed by reduced placental measurements (area and weight), reduced yolk sac development, and failed embryo development. Bcl9, B-cell lymphoma 9; Gcm1, glial cells missing 1; SynT-I, syncytiotrophoblast layer I; SynT-II, syncytiotrophoblast layer II; TLE, transducin-like enhancer of split; VEGF, vascular endothelial growth factor; WNT, wingless/integrated.

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