Dear Editor,

Sulfate is an obligate nutrient for numerous cellular and metabolic processes important for fetal development1. Fetal tissues express sulfotransferases which conjugate sulfate (sulfonate) to molecules such as steroids and hormones, leading to their inactivation2. In addition, sulfonation of glycosaminoglycans is important for development of some tissues, as several growth factor gradients and ligand-receptor interactions are dependent on these sulfonated extracellular constituents3. Not surprisingly therefore, reduced sulfonation capacity has been linked to disorders of skeletal, eye, vascular and craniofacial development, demonstrating a critical requirement for sulfate in these tissues4. Despite a fundamental role for sulfate during development, the fetus appears to have a limited capacity to generate sulfate from sulfur-containing amino acids, and is thought to be reliant on maternal sulfate provision5. In fact, maternal serum sulfate levels increase two-fold during pregnancy, and provide a potential reservoir for fetal consumption6,7. The elevated sulfate levels are due to increased sulfate reabsorption in the maternal kidneys, mediated by increased expression of the SLC13A1 sulfate transporter7. The importance of maintaining high maternal sulfate levels in pregnancy is highlighted by the hyposulfatemia seen in pregnant Slc13a1−/− mice, which causes fetal hyposulfatemia and late gestational fetal loss.

Despite its importance in fetal development, relatively little is known about how sulfate traverses the placenta. The sulfate transporter SLC13A4 is expressed predominantly in the placenta, where it is proposed to mediate sulfate supply to the fetus8. Previously we examined the spatial localization of all 10 sulfate transporters in human9 and mouse placenta8 and identified Slc13a4 as the most abundant sulfate transporter localized to the transporting syncytiotrophoblasts. In the current study we describe the critical requirement of placental Slc13a4 activity for normal fetal development in mice.

We generated Slc13a4 knockout mice using ES cells sourced from the European Conditional Mouse Mutant Program (EUCOMM) to characterize the role of Slc13a4 in placental sulfate transport during fetal development. The targeted Slc13a4 allele (Slc13a4tm1a(EUCOMM)Wtsi) contains a “knockout first” (KOF) targeting cassette (Supplementary information, Figure S1A), which splices to a LacZ trapping element after exon 2, generating a null allele (Figure 1H, Supplementary information, Figure S1A). Crosses between Slc13a4+/KOF mice yielded fewer pups per litter (n = 4) compared with litters from Slc13a4+/KOF × Slc13a4+/+ matings (n = 8), and no Slc13a4KOF/KOF pups were found at birth, indicating loss of Slc13a4 is embryonic lethal (Figure 1A). While loss of Slc13a4 did not result in generalized growth restriction, Slc13a4KOF/KOF embryos displayed a variety of developmental abnormalities grossly detectable as early as embryonic day (E)12.5 (Supplementary information, Figure S2A), which became more severe at E14.5 (Supplementary information, Figure S2A) and E16.5 (Figures 1C and Supplementary information, Figure S2A), with fetal death occurring by E18.5. The most striking phenotypes at E16.5 were pale skin, subcutaneous oedema, craniofacial malformations, vascular hemorrhaging and skeletal defects (Figure 1C). We measured unidirectional maternal-fetal transfer of 35S-sulfate as fetal accumulation of radioisotope at E12.5 after injection into the maternal circulation. At E12.5 35S-sulfate per gram of placenta was significantly lower in Slc13a4KOF/KOF embryos compared with Slc13a4+/KOF and Slc13a4+/+ littermate controls (P < 0.001; Figure 1B, Supplementary information, Figure S3A). Additionally we measured elemental sulfur levels (Supplementary information, Figure S3C); Slc13a4KOF/KOF embryos exhibited an ∼50% reduction in elemental sulfur compared with Slc13a4+/KOF and Slc13a4+/+ littermate controls, in line with a reduced sulfate supply to Slc13a4KOF/KOF embryos.

Figure 1
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Loss of placental Slc13a4 results in multiple developmental defects and embryonic lethality. (A) Litter sizes at P0.5 for Slc13a4+/KOF females mated with Slc13a4+/KOF males (n = 5 litters, 20 mice) were significantly smaller than for Slc13a4+/+ females (n = 5 litters, 39 mice), **P < 0.01; students t test (B) Placental transfer of 35S-sulfate was significantly less in Slc13a4KOF/KOF embryos compared with Slc13a4+/KOF and Slc13a4+/+ littermate controls (4 litters at E12.5: KOF/KOF n = 9; +/KOF n = 12; +/+ n = 8. Error bars indicate ± SEM, * P < 0.01, **P < 0.001; One-Way ANOVA with Tukey's test (post hoc) for multiple comparison. (C) Gross morphology of embryos at E16.5. Severe phenotypes were observed in Slc13a4KOF/KOF embryos, such as eye defects, oedema, pale appearance, and vascular hemorrhaging in the thoracic region. (D) E16.5 skeletons were stained with alcian blue for cartilage and alizarin red for mineralized bone. Slc13a4KOF/KOF embryos have multiple skeletal defects in the axial skeleton as well as the craniofacial region (left panel). Slc13a4+/KOF embryos (middle panel) exhibit an intermediate phenotype with reduced mineralization in areas of the axial skeleton (arrows) and regions of the skull (arrowhead) and have a disorganized rib cage. Scale bar, 1.0 mm, n = 4 for each genotype. (E) Wholemount ENDOMUCIN immunofluorescence on E14.5 embryo back skin revealed thickened vessels and a decrease in complexity of the vascular network in Slc13a4KOF/KOF embryos. Scale bar, 20 μm, n = 4 for each genotype. (F) Slc13a4KOF/KOF E14.5 embryos exhibit iris coloboma (left panel, arrowhead) and a thickened lens (arrow), scale bar, 10 μm, n = 4 for each genotype. (G) Slc13a4KOF/KOF embryos have craniofacial defects and cleft palate at E14.5, Scale bar, 100 μm. PS - palatal shelves, n = 4 for each genotype. (H) Schematic for generating the Slc13a4Flx allele from the Slc13a4KOF targeting allele (using FLPeR mice), and the subsequent generation of the Slc13a4− allele (using Sox2-Cre mice) (I) Loss of Slc13a4 (either Slc13a4KOF/KOF or Slc13a4−/− alleles) in both placenta and embryo results in embryonic lethality. (J) Loss of Slc13a4 in the embryo, but not the placenta (through epiblast-specific Sox2-Cre deletion of the Slc13a4Flx allele) results in normal embryonic development (See also Supplementary information, Figure S6). (K) Fetal accumulation of 35S-sulfate was not significantly different in Slc13a4−/−TgSox2Cre (n = 7) embryos compared with Slc13a4−/+TgSox2Cre (n = 3), Slc13a4−/Flx (n = 5) and Slc13a4Flx/+(n = 6) littermate controls. Total of three litters at E12.5, bars ± SEM, P = 0.8400, One-Way ANOVA with Tukey's test (posthoc) for multiple comparisons. See also Supplementary information, Figure S3. (L) Rescue of previously observed phenotypes in Slc13a4−/−TgSox2Cre embryos at E16.5 due to the retention of an Slc13a4−/Flx placenta (Scale bar, 1.0 mm) (M) Whole skeletal preparations of Slc13a4−/−TgSox2Cre embryos at E16.5 demonstrate the rescue of skeletal development in Slc13a4−/− embryos with an Slc13a4−/Flx placenta (Scale bar, 1.0 mm) (N) Slc13a4−/− mice from Slc13a4Flx/Flx X Slc13a4−/+TgSox2Cre crosses survive to adulthood and are indistinguishable from Slc13a4−/Flx littermate controls (See Supplementary information, Table S2 and Figure S1).

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Mutations in genes required for intracellular sulfonation have been extensively studied and linked to developmental phenotypes in mice and humans10. For example, the sulfonation of ECM components such as heparin sulfate proteoglycans is critical for growth factor binding and signalling. Mutations within specific heparin sulfotransferases cause defects in eye morphogenesis11, skeletal and craniofacial development11 or affect vascular patterning in vivo12, depending on the organ or tissue the sulfotransferase is endogenously expressed. By contrast, the phenotypes present in Slc13a4KOF/KOF embryos affect multiple tissues, including the skeleton (Figure 1D), vasculature (Figure 1E), eye (iris coloboma and increased lens thickness) (Figure 1F) and palate (Figure 1G), all of which have been individually linked to sulfotransferases or sulfate transporter genes (Supplementary information, Table S1). As the phenotype of Slc13a4KOF/KOF embryos encompasses those of multiple sulfotransferase mutants, it is therefore likely the reflection of a global reduction in sulfate supply to the embryo via the placenta. Importantly, while the skeletons of Slc13a4KOF/KOF embryos displayed a total lack of calcification (alizarin red staining), Slc13a4+/KOF skeletons had an intermediate phenotype with reduced calcification compared with wildtype controls (Figure 1D, middle panel), suggesting that haploinsufficiency of Slc13a4 is also sufficient to perturb or delay fetal skeletal maturation.

While the placenta expresses a multitude of transporters and channels to facilitate the supply of nutrients such as ions, amino acids, fatty acids, minerals, and glucose to the fetus, studies of gene knockout mice often overlook the contribution of placental supply when investigating the role of individual transporters in fetal development and adult disease. Importantly, the development of placentas from Slc13a4 null embryos appeared normal throughout gestation (Supplementary information, Figure S4). This is an important observation as defects in placental development are known to cause fetal growth restriction13; we do not see a generalized growth restriction in Slc13a4KOF/KOF or Slc13a4−/− embryos. Normal development of Slc13a4KOF/KOF placentas therefore suggests that the tissue-specific embryonic phenotypes are the result of reduced placental SLC13A4 transport activity rather than generalized placental insufficiency and growth restriction as a result of aberrant placental development.

To confirm that the developmental phenotypes and late gestational lethality observed in Slc13a4 null embryos are due to loss of placental Slc13a4 activity, and not due to embryonal tissue-specific requirements for Slc13a4, we generated an Slc13a4 conditional knockout allele by mating Slc13a4+/KOF mice with FLPeR-expressing mice to produce a conditional Slc13a4 “floxed” allele (Slc13a4+/Flx; Figure 1H, Supplementary information, Figure S1A). The conditional allele, when deleted globally through the maternal inheritance of the Sox2-Cre transgene (Tg)14, produced the same phenotypes as seen with the Slc13a4KOF/KOF. Furthermore, crosses of resultant Slc13a4+/− mice also yielded Slc13a4−/− embryos with the same phenotypes as Slc13a4KOF/KOF embryos (Supplementary information, Figure S2B). We next mated homozygous floxed Slc13a4 female mice with male Sox2-Cre deleter mice (Slc13a4−/+TgSox2Cre male X Slc13a4Flx/Flx female) in order to delete Slc13a4 exon 3 within the embryo, while retaining placental Slc13a4 expression; in Slc13a4−/−TgSox2Cre embryos (Figure 1I, J, Supplementary information, Figure S5 and S6) Sox2-driven Cre recombinase expression/activity is restricted to the embryonic epiblast, but is absent from extra-embryonic tissues such as the syncytiotrophoblast where Slc13a4 is endogenously expressed. Litters from Slc13a4−/+TgSox2Cre × Slc13a4Flx/Flx crosses were analyzed on the day of birth (P0.5) and all the resultant pups were genotyped at postnatal day (P)8. Slc13a4−/−TgSox2Cre pups were present within each litter examined from these crosses (Supplementary information, Table S2). At E12.5 we measured unidirectional maternal-fetal transfer of 35S-sulfate in litters from Slc13a4−/+TgSox2Cre x Slc13a4Flx/Flx crosses. There was no significant difference in fetal accumulation of 35S-sulfate per gram of placenta between Slc13a4−/−TgSox2Cre, Slc13a4−/+TgSox2Cre, Slc13a4−/Flx and Slc13a4+/Flx littermate controls (P = 0.840; Figure 1K, Supplementary information, Figure S3B). Slc13a4−/−TgSox2Cre mice from these crosses were indistinguishable from heterozygous littermate controls and analysis of Slc13a4−/−TgSox2Cre embryos at E16.5 demonstrated the rescue of all previously observed phenotypes when Slc13a4 expression was maintained in the placenta (Figure 1L, M). Importantly Slc13a4−/−TgSox2Cre mice survive through to adulthood (Figure 1N). The rescue of severe and lethal embryonic defects in Slc13a4−/−TgSox2Cre embryos when Slc13a4 expression is retained in the placenta, but not the embryo itself, demonstrates the dependence of the fetus on access to circulating maternal sulfate stores via the placenta for normal development. These findings also confirm that the fetus cannot produce enough sulfate from the metabolism of sulfur-containing amino acids to sustain development. To date the role of placental sulfate supply in the development of fetal tissues with a high demand for sulfate metabolism and sulfonation reactions has not been widely considered. The current study highlights a placental contribution to sulfate-dependent fetal abnormalities, and warrants further investigation of placental sulfate transport in human gestation.