CRISPR screening in human trophoblast stem cells reveals both shared and distinct aspects of human and mouse placental development.

The placenta serves as the interface between the mother and fetus, facilitating the exchange of gases and nutrients between their separate blood circulation systems. Trophoblasts in the placenta play a central role in this process. Our current understanding of mammalian trophoblast development relies largely on mouse models. However, given the diversification of mammalian placentas, findings from the mouse placenta cannot be readily extrapolated to other mammalian species, including humans. To fill this knowledge gap, we performed CRISPR knockout screening in human trophoblast stem cells (hTSCs). We targeted genes essential for mouse placental development and identified more than 100 genes as critical regulators in both human hTSCs and mouse placentas. Among them, we further characterized in detail two transcription factors, DLX3 and GCM1, and revealed their essential roles in hTSC differentiation. Moreover, a gene function-based comparison between human and mouse trophoblast subtypes suggests that their relationship may differ significantly from previous assumptions based on tissue localization or cellular function. Notably, our data reveal that hTSCs may not be analogous to mouse TSCs or the extraembryonic ectoderm (ExE) in which in vivo TSCs reside. Instead, hTSCs may be analogous to progenitor cells in the mouse ectoplacental cone and chorion. This finding is consistent with the absence of ExE-like structures during human placental development. Our data not only deepen our understanding of human trophoblast development but also facilitate cross-species comparison of mammalian placentas.

Why study human embryo development?

Understanding the processes and mechanisms underlying early human embryo development has become an increasingly active and important area of research. It has potential for insights into important clinical issues such as early pregnancy loss, origins of congenital anomalies and developmental origins of adult disease, as well as fundamental insights into human biology. Improved culture systems for preimplantation embryos, combined with the new tools of single cell genomics and live imaging, are providing new insights into the similarities and differences between human and mouse development. However, access to human embryo material is still restricted and extended culture of early embryos has regulatory and ethical concerns. Stem cell-derived models of different phases of human development can potentially overcome these limitations and provide a scalable source of material to explore the early postimplantation stages of human development. To date, such models are clearly incomplete replicas of normal development but future technological improvements can be envisaged. The ethical and regulatory environment for such studies remains to be fully resolved.

Transforming growth factor-ß signaling governs the differentiation program of extravillous trophoblasts in the developing human placenta.

Abnormal placentation has been noticed in a variety of pregnancy complications such as miscarriage, early-onset preeclampsia, and fetal growth restriction. Defects in the developmental program of extravillous trophoblasts (EVTs), migrating from placental anchoring villi into the maternal decidua and its vessels, is thought to be an underlying cause. Yet, key regulatory mechanisms controlling commitment and differentiation of the invasive trophoblast lineage remain largely elusive. Herein, comparative gene expression analyses of HLA-G-purified EVTs, isolated from donor-matched placenta, decidua, and trophoblast organoids (TB-ORGs), revealed biological processes and signaling pathways governing EVT development. In particular, bioinformatics analyses and manipulations in different versatile trophoblast cell models unraveled transforming growth factor-ß (TGF-ß) signaling as a crucial pathway driving differentiation of placental EVTs into decidual EVTs, the latter showing enrichment of a secretory gene signature. Removal of Wingless signaling and subsequent activation of the TGF-ß pathway were required for the formation of human leukocyte antigen-G+ (HLA-G+) EVTs in TB-ORGs that resemble in situ EVTs at the level of global gene expression. Accordingly, TGF-ß-treated EVTs secreted enzymes, such as DAO and PAPPA2, which were predominantly expressed by decidual EVTs. Their genes were controlled by EVT-specific induction and genomic binding of the TGF-ß downstream effector SMAD3. In summary, TGF-ß signaling plays a key role in human placental development governing the differentiation program of EVTs.

Embryonic statistical analyses reveal 2 growth phenotypes in mouse models of Down syndrome.

BACKGROUND: Down syndrome is associated with several comorbidities, including intellectual disability, growth restriction, and congenital heart defects. The prevalence of Down syndrome-associated comorbidities is highly variable, and intellectual disability, although fully penetrant, ranges from mild to severe. Understanding the basis of this interindividual variability might identify predictive biomarkers of in utero and postnatal outcomes that could be used as endpoints to test the efficacy of future therapeutic interventions. OBJECTIVE: The main objective of this study was to examine if antenatal interindividual variability exists in mouse models of Down syndrome and whether applying statistical approaches to clinically relevant measurements (ie, the weights of the embryo, placenta, and brain) could define cutoffs that discriminate between subgroups of trisomic embryos. STUDY DESIGN: Three commonly used mouse models of Down syndrome (Dp(16)1/Yey, Ts65Dn, and Ts1Cje) and a new model (Ts66Yah) were used in this study. Trisomic and euploid littermate embryos were used from each model with total numbers of 102 for Ts66Yah, 118 for Dp(16)1/Yey, 92 for Ts65Dn, and 126 for Ts1Cje. Placental, embryonic, and brain weights and volumes at embryonic day 18.5 were compared between genotypes in each model. K-mean clustering analysis was applied to embryonic and brain weights to identify severity classes in trisomic embryos, and brain and placental volumetric measurements were compared between genotypes and classes for each strain. In addition, Ts66Yah embryos were examined for malformations because embryonic phenotypes have never been examined in this model. RESULTS: Reduced body and brain weights were present in Ts66Yah, Dp(16)1/Yey, and Ts65Dn embyos. Cluster analysis identified 2 severity classes in trisomic embryos-mild and severe-in all 4 models that were distinguishable using a putative embryonic weight cutoff of <0.5 standard deviation below the mean. Ts66Yah trisomic embryos develop congenital anomalies that are also found in humans with Down syndrome, including congenital heart defects and renal pelvis dilation. CONCLUSION: Statistical approaches applied to clinically relevant measurements revealed 2 classes of phenotypic severity in trisomic mouse models of Down syndrome. Analysis of severely affected trisomic animals may facilitate the identification of biomarkers and endpoints that can be used to prenatally predict outcomes and the efficacy of treatments.

A placenta-on-a-chip model to determine the regulation of FKBPL and galectin-3 in preeclampsia.

Preeclampsia is a pregnancy-specific cardiovascular disorder, involving significant maternal endothelial dysfunction. Although inappropriate placentation due to aberrant angiogenesis, inflammation and shallow trophoblast invasion are the root causes of preeclampsia, pathogenic mechanisms are poorly understood, particularly in early pregnancy. Here, we first confirm the abnormal expression of important vascular and inflammatory proteins, FK506-binding protein-like (FKBPL) and galectin-3 (Gal-3), in human plasma and placental tissues from women with preeclampsia and normotensive controls. We then employ a three-dimensional microfluidic placental model incorporating human umbilical vein endothelial cells (HUVECs) and a first trimester trophoblast cell line (ACH-3P) to investigate FKBPL and Gal-3 signaling in inflammatory conditions. In human samples, both circulating (n = 17 controls; n = 30 preeclampsia) and placental (n ≥ 6) FKBPL and Gal-3 levels were increased in preeclampsia compared to controls (plasma: FKBPL, p < 0.0001; Gal-3, p < 0.01; placenta: FKBPL, p < 0.05; Gal-3, p < 0.01), indicative of vascular dysfunction in preeclampsia. In our placenta-on-a-chip model, we show that endothelial cells are critical for trophoblast-mediated migration and that trophoblasts effectively remodel endothelial vascular networks. Inflammatory cytokine tumour necrosis factor-α (10 ng/mL) modulates both FKBPL and Gal-3 signaling in conjunction with trophoblast migration and impairs vascular network formation (p < 0.005). Our placenta-on-a-chip recapitulates aspects of inappropriate placental development and vascular dysfunction in preeclampsia.

Comprehensive Analysis of Placental DNA Methylation Changes and Fetal Birth Weight in Pigs.

Birth weight is a complex multifactorial trait relevant to health states and disease risks in later life. The placenta is essential for proper fetal growth and facilitates gas, nutrient, and waste exchange between the mother and developing fetus. How changes in placental DNA methylation affect fetal birth weight remains to be fully elucidated. In this study, we used whole-genome bisulfite sequencing and RNA sequencing to reveal a global map of DNA methylation and gene expression changes between the placentas of highest birth weight and lowest birth weight piglets in the same litters. The transcriptome analysis identified 1682 differential expressed genes and revealed key transcriptional properties in distinct placentas. We also identified key transcription factors that may drive the differences in DNA methylome patterns between placentas. The decrease in DNA methylation level in the promoter was associated with the transcriptional activation of genes associated with angiogenesis, extracellular matrix remodeling, and transmembrane transport. Our results revealed the regulatory role of DNA methylation in gene transcription activity leading to the differences in placental morphological structures and birth weights of piglets. These results could provide novel clues to clarify the underlying regulatory mechanisms of placental development and fetal growth.

Human Placenta and Evolving Insights into Pathological Changes of Preeclampsia: A Comprehensive Review of the Last Decade.

The placenta, the foremost and multifaceted organ in fetal and maternal biology, is pivotal in facilitating optimal intrauterine fetal development. Remarkably, despite its paramount significance, the placenta remains enigmatic, meriting greater comprehension given its central influence on the health trajectories of both the fetus and the mother. Preeclampsia (PE) and intrauterine fetal growth restriction (IUGR), prevailing disorders of pregnancy, stem from compromised placental development. PE, characterized by heightened mortality and morbidity risks, afflicts 5-7% of global pregnancies, its etiology shrouded in ambiguity. Pertinent pathogenic hallmarks of PE encompass inadequate restructuring of uteroplacental spiral arteries, placental ischemia, and elevated levels of vascular endothelial growth factor receptor-1 (VEGFR-1), also recognized as soluble FMS-like tyrosine kinase-1 (sFlt-1). During gestation, the placental derivation of sFlt-1 accentuates its role as an inhibitory receptor binding to VEGF-A and placental growth factor (PlGF), curtailing target cell accessibility. This review expounds upon the placenta's defining cellular component of the trophoblast, elucidates the intricacies of PE pathogenesis, underscores the pivotal contribution of sFlt-1 to maternal pathology and fetal safeguarding, and surveys recent therapeutic strides witnessed in the past decade.

FoxO1 Deficiency Enhances Cell Proliferation and Survival Under Normoglycemia and Promotes Angiogenesis Under Hyperglycemia in the Placenta.

FoxO1 is an important transcriptional factor that regulates cell survival and metabolism in many tissues. Deleting FoxO1 results in embryonic death due to failure of chorioallantoic fusion at E8.5; however, its role in placental development during mid-late gestation is unclear. In both human patients with gestational diabetes and pregnant mice with hyperglycemia, placental FoxO1 expression was significantly increased. Using FoxO1+/- mice, the effects of FoxO1 haploinsufficiency on placental development under normoglycemia and hyperglycemia were investigated. With FoxO1 haploinsufficiency, the term placental weight increased under both normal and hyperglycemic conditions. Under normoglycemia, this weight change was associated with a general enlargement of the labyrinth, along with increased cell proliferation, decreased cell apoptosis, and decreased expression of p21, p27, Casp3, Casp8, and Rip3. However, under hyperglycemia, the placental weight change was associated with increased fetal blood space, VEGFA overexpression, and expression changes of the angiogenic markers, Eng and Tsp1. In conclusion, FoxO1 plays a role in regulating cell proliferation, cell survival, or angiogenesis, depending on blood glucose levels, during placenta development.

Placental Mammals Acquired Functional Sequences in NRK for Regulating the CK2-PTEN-AKT Pathway and Placental Cell Proliferation.

The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologs across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: 1) Mouse NRK is localized at the plasma membrane via the CNH domain, where the CIR inhibits CK2. 2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN and 3) leads to the inhibition of AKT signaling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2-PTEN-AKT pathway and placental cell proliferation.

The transposable element-derived transcript of LIN28B has a placental origin and is not specific to tumours.

Transposable elements (TEs) are genetic elements that have evolved as crucial regulators of human development and cancer, functioning as both genes and regulatory elements. When TEs become dysregulated in cancer cells, they can serve as alternate promoters to activate oncogenes, a process known as onco-exaptation. This study aimed to explore the expression and epigenetic regulation of onco-exaptation events in early human developmental tissues. We discovered co-expression of some TEs and oncogenes in human embryonic stem cells and first trimester and term placental tissues. Previous studies identified onco-exaptation events in various cancer types, including an AluJb SINE element-LIN28B interaction in lung cancer cells, and showed that the TE-derived LIN28B transcript is associated with poor patient prognosis in hepatocellular carcinoma. This study further characterized the AluJb-LIN28B transcript and confirmed that its expression is restricted to the placenta. Targeted DNA methylation analysis revealed differential methylation of the two LIN28B promoters between placenta and healthy somatic tissues, indicating that some TE-oncogene interactions are not cancer-specific but arise from the epigenetic reactivation of developmental TE-derived regulatory events. In conclusion, our findings provide evidence that some TE-oncogene interactions are not limited to cancer and may originate from the epigenetic reactivation of TE-derived regulatory events that are involved in early development. These insights broaden our understanding of the role of TEs in gene regulation and suggest the potential importance of targeting TEs in cancer therapy beyond their conventional use as cancer-specific markers.

Decidual derived exosomal miR-99a-5p targets Ppp2r5a to inhibit trophoblast invasion in response to CeO2NPs exposure.

BACKGROUND: The biological effects of cerium dioxide nanoparticles (CeO2NPs), a novel material in the biomedical field, have attracted widespread attention. Our previous study confirmed that exposure to CeO2NPs during pregnancy led to abnormal trophoblast invasion during early placental development, thereby impairing placental development. The potential mechanisms may be related to low-quality decidualization triggered by CeO2NPs exposure, such as an imbalance in trophoblast invasion regulators secreted by decidual cells. However, the intermediate link mediating the "dialogue" between decidual cells and trophoblasts during this process remains unclear. As an important connection between cells, exosomes participate in the "dialogue" between endometrial cells and trophoblasts. Exosomes transfer bioactive microRNA into target cells, which can target and regulate the level of mRNA in target cells. RESULTS: Here, we constructed a mice primary uterine stromal cell-induced decidualization model in vitro, and detected the effect of CeO2NPs exposure on the expression of decidual-derived exosomal miRNAs by high-throughput sequencing. Bioinformatics analysis and dual-luciferase reporter assays were performed to identify target genes of the screened key miRNAs in regulating trophoblast invasion. Finally, the role of the screened miRNAs and their target genes in regulating trophoblast (HTR-8/SVneo cells) invasion was confirmed. The results showed that CeO2NPs exposure inhibited trophoblast invasion by promoting miR-99a-5p expression in decidual-derived exosomes, and Ppp2r5a is a potential target gene for miR-99a-5p to inhibit trophoblast invasion. CONCLUSIONS: This study revealed the molecular mechanism by which CeO2NPs exposure inhibits trophoblast invasion from the perspective of decidual derived exosomal miRNAs. These results will provide an experimental basis for screening potential therapeutic targets for the negative biological effects of CeO2NPs exposure and new ideas for studying the mechanism of damage to trophoblast cells at the decidual-foetal interface by harmful environmental or occupational factors.

Modelling human placental villous development: designing cultures that reflect anatomy.

The use of in vitro tools to study trophoblast differentiation and function is essential to improve understanding of normal and abnormal placental development. The relative accessibility of human placentae enables the use of primary trophoblasts and placental explants in a range of in vitro systems. Recent advances in stem cell models, three-dimensional organoid cultures, and organ-on-a-chip systems have further shed light on the complex microenvironment and cell-cell crosstalk involved in placental development. However, understanding each model's strengths and limitations, and which in vivo aspects of human placentation in vitro data acquired does, or does not, accurately reflect, is key to interpret findings appropriately. To help researchers use and design anatomically accurate culture models, this review both outlines our current understanding of placental development, and critically considers the range of established and emerging culture models used to study this, with a focus on those derived from primary tissue.

WNT and NOTCH signaling in human trophoblast development and differentiation.

Correct development of the human placenta and its differentiated epithelial cells, syncytial trophoblasts (STBs) and extravillous trophoblasts (EVTs), is crucial for a successful pregnancy outcome. STBs develop by cell fusion of mononuclear cytotrophoblasts (CTBs) in placental floating villi, whereas migratory EVTs originate from specialized villi anchoring to the maternal decidua. Defects in trophoblast differentiation have been associated with severe pregnancy disorders such as early-onset preeclampsia and fetal growth restriction. However, the evolutionary pathways underlying normal and adverse placentation are poorly understood. Herein, we discuss Wingless (WNT) and NOTCH signaling, two pathways that play pivotal roles in human placenta and trophoblast development. Whereas WNT is necessary for expansion of trophoblast progenitors and stem cells, NOTCH1 is required for proliferation and survival of EVT precursors. Differentiation of the latter is orchestrated by a switch in NOTCH receptor expression as well as by changes in WNT ligands and their downstream effectors.

Placental Morphology and Morphometry: Is It a Prerequisite for Future Pathological Investigations?

Successful pregnancy is dependent on implantation, nutrient and gas exchange, as well as fetal protection from the immunologic attack. Placental pathologies and preterm delivery closely correlate with the size and shape of the placenta. Additionally, normal vaginal microbiota is disturbed during viral insults such as SARS-CoV-2 and HIV, with consequent placental anomalies. This chapter focuses on placental development, morphology, and pathology while also investigating placental bed structure and function. Placental anomalies with regard to HIV-1 and SARS-CoV-2 infection and placental morphometric image analysis and its relevance for verification of placental pathology are explored. Since image analysis remains optional for routine diagnostic purposes, authentication of placental appraisal warrants the use of measurable predefined definitions. Immunohistochemical analyses of placental morphology and angiogenic, epithelial, and apoptotic mechanisms facilitate research into etiopathogenetic pathways involved in placental anomalies with a focus on discovering novel diagnostic foci. Thus, image analyses as an adjunct to complement pathological investigations are recommended.

Integrated Analysis of DNA Methylation and Gene Expression in Porcine Placental Development.

Proper placental development is crucial for the conceptus to grow and survive, because the placenta is responsible for transporting nutrients and oxygen from the pregnant female to the developing fetus. However, the processes of placental morphogenesis and fold formation remain to be fully elucidated. In this study, we used whole-genome bisulfite sequencing and RNA sequencing to produce a global map of DNA methylation and gene expression changes in placentas from Tibetan pig fetuses 21, 28, and 35 days post-coitus. Substantial changes in morphology and histological structures at the uterine-placental interface were revealed via hematoxylin-eosin staining. Transcriptome analysis identified 3959 differentially expressed genes (DEGs) and revealed the key transcriptional properties in three stages. The DNA methylation level in the gene promoter was negatively correlated with gene expression. We identified a set of differentially methylated regions associated with placental developmental genes and transcription factors. The decrease in DNA methylation level in the promoter was associated with the transcriptional activation of 699 DEGs that were functionally enriched in cell adhesion and migration, extracellular matrix remodeling, and angiogenesis. Our analysis provides a valuable resource for understanding the mechanisms of DNA methylation in placental development. The methylation status of different genomic regions plays a key role in establishing transcriptional patterns from placental morphogenesis to fold formation.

Protective role of IL33 signaling in negative pregnancy outcomes associated with lipopolysaccharide exposure.

Interleukin 33 (IL33) signaling has been implicated in the establishment and maintenance of pregnancy and in pregnancy disorders. The goal of this project was to evaluate the role of IL33 signaling in rat pregnancy. The rat possesses hemochorial placentation with deep intrauterine trophoblast invasion; features also characteristic of human placentation. We generated and characterized a germline mutant rat model for IL33 using CRISPR/Cas9 genome editing. IL33 deficient rats exhibited deficits in lung responses to an inflammatory stimulus (Sephadex G-200) and to estrogen-induced uterine eosinophilia. Female rats deficient in IL33 were fertile and exhibited pregnancy outcomes (gestation length and litter size) similar to wild-type rats. Placental weight was adversely affected by the disruption of IL33 signaling. A difference in pregnancy-dependent adaptations to lipopolysaccharide (LPS) exposure was observed between wild-type and IL33 deficient pregnancies. Pregnancy in wild-type rats treated with LPS did not differ significantly from pregnancy in vehicle-treated wild-type rats. In contrast, LPS treatment decreased fetal survival rate, fetal and placental weights, and increased fetal growth restriction in IL33 deficient rats. In summary, a new rat model for investigating IL33 signaling has been established. IL33 signaling participates in the regulation of placental development and protection against LPS-induced fetal and placental growth restriction.

The unexplored role of sedentary time and physical activity in glucose and lipid metabolism-related placental mRNAs in pregnant women who are obese: the DALI lifestyle randomised controlled trial.

OBJECTIVE: We aimed to explore: (i) the association of sedentary time (ST) and physical activity (PA) during pregnancy with the placental expression of genes related to glucose and lipid metabolism in pregnant women who are obese; (ii) maternal metabolic factors mediating changes in these placental transcripts; and (iii) cord blood markers related to the mRNAs mediating neonatal adiposity. DESIGN: Multicentre randomised controlled trial. SETTING: Hospitals in nine European countries. POPULATION: A cohort of 112 pregnant women with placental tissue. METHODS: Both ST and moderate-to-vigorous PA (MVPA) levels were measured objectively using accelerometry at three time periods during pregnancy. MAIN OUTCOME MEASURES: Placental mRNAs (FATP2, FATP3, FABP4, GLUT1 and PPAR-γ) were measured with NanoString technology. Maternal and fetal metabolic markers and neonatal adiposity were assessed. RESULTS: Longer periods of ST, especially in early to middle pregnancy, was associated with lower placental FATP2 and FATP3 expression (P < 0.05), whereas MVPA at baseline was inversely associated with GLUT1 mRNA (P = 0.02). Although placental FATP2 and FATP3 expression were regulated by the insulin-glucose axis (P < 0.05), no maternal metabolic marker mediated the association of ST/MVPA with placental mRNAs (P > 0.05). Additionally, placental FATP2 expression was inversely associated with cord blood triglycerides and free fatty acids (FFAs; P < 0.01). No cord blood marker mediated neonatal adiposity except for cord blood leptin, which mediated the effects of PPAR-γ on neonatal sum of skinfolds (P < 0.05). CONCLUSIONS: In early to middle pregnancy, ST is associated with the expression of placental genes linked to lipid transport. PA is hardly related to transporter mRNAs. Strategies aimed at reducing sedentary behaviour during pregnancy could modulate placental gene expression, which may help to prevent unfavourable fetal and maternal pregnancy outcomes. TWEETABLE ABSTRACT: Reducing sedentary behaviour in pregnancy might modulate placental expression of genes related to lipid metabolism in women who are obese.

Mapping the expression of transient receptor potential channels across murine placental development.

Transient receptor potential (TRP) channels play prominent roles in ion homeostasis by their ability to control cation influx. Mouse placentation is governed by the processes of trophoblast proliferation, invasion, differentiation, and fusion, all of which require calcium signaling. Although certain TRP channels have been shown to contribute to maternal-fetal transport of magnesium and calcium, a role for TRP channels in specific trophoblast functions has been disregarded. Using qRT-PCR and in situ hybridisation, the spatio-temporal expression pattern of TRP channels in the mouse placenta across gestation (E10.5-E18.5) was assessed. Prominent expression was observed for Trpv2, Trpm6, and Trpm7. Calcium microfluorimetry in primary trophoblast cells isolated at E14.5 of gestation further revealed the functional activity of TRPV2 and TRPM7. Finally, comparing TRP channels expression in mouse trophoblast stem cells (mTSCs) and mouse embryonic stem cells (mESC) confirmed the specific expression of TRPV2 during placental development. Moreover, TRP channel expression was similar in mTSCs compared to primary trophoblasts and validate mTSC as a model to study TRP channels in placental development. Collectivity, our results identify a specific spatio-temporal TRP channel expression pattern in trophoblasts, suggesting a possible involvement in regulating the process of placentation.

Paternal knockout of Slc38a4/SNAT4 causes placental hypoplasia associated with intrauterine growth restriction in mice.

The placenta is critical in mammalian embryonic development because the embryo's supply of nutrients, including amino acids, depends solely on mother-to-embryo transport through it. However, the molecular mechanisms underlying this amino acid supply are poorly understood. In this study, we focused on system A amino acid transporters Slc38a1/SNAT1, Slc38a2/SNAT2, and Slc38a4/SNAT4, which carry neutral, short-side-chain amino acids, to determine their involvement in placental or embryonic development. A triple-target CRISPR screen identified Slc38a4/SNAT4 as the critical amino acid transporter for placental development in mice. We established mouse lines from the CRISPR founders with large deletions in Slc38a4 and found that, consistent with the imprinted paternal expression of Slc38a4/SNAT4 in the placenta, paternal knockout (KO) but not maternal KO of Slc38a4/SNAT4 caused placental hypoplasia associated with reduced fetal weight. Immunostaining revealed that SNAT4 was widely expressed in differentiating cytotrophoblasts and maturing trophoblasts at the maternal-fetal interface. A blood metabolome analysis revealed that amino acid concentrations were globally reduced in Slc38a4/SNAT4 mutant embryos. These results indicated that SNAT4-mediated amino acid transport in mice plays a major role in placental and embryonic development. Given that expression of Slc38a4 in the placenta is conserved in other species, our Slc38a4/SNAT4 mutant mice could be a promising model for the analysis of placental defects leading to intrauterine growth restriction in mammals.

High-resolution contrast-enhanced microCT reveals the true three-dimensional morphology of the murine placenta.

Genetic engineering of the mouse genome identified many genes that are essential for embryogenesis. Remarkably, the prevalence of concomitant placental defects in embryonic lethal mutants is highly underestimated and indicates the importance of detailed placental analysis when phenotyping new individual gene knockouts. Here we introduce high-resolution contrast-enhanced microfocus computed tomography (CE-CT) as a nondestructive, high-throughput technique to evaluate the 3D placental morphology. Using a contrast agent, zirconium-substituted Keggin polyoxometalate (Zr-POM), the soft tissue of the placenta (i.e., different layers and cell types and its vasculature) was imaged with a resolution of 3.5 µm voxel size. This approach allowed us to visualize and study early and late stages of placental development. Moreover, CE-CT provides a method to precisely quantify placental parameters (i.e., volumes, volume fraction, ratio of different placental layers, and volumes of specific cell populations) that are crucial for statistical comparison studies. The CE-CT assessment of the 3D morphology of the placentas was validated (i) by comparison with standard histological studies; (ii) by evaluating placentas from 2 different mouse strains, 129S6 and C57BL/6J mice; and (iii) by confirming the placental phenotype of mice lacking phosphoinositol 3-kinase (PI3K)-p110α. Finally, the Zr-POM-based CE-CT allowed for inspection of the vasculature structure in the entire placenta, as well as detecting placental defects in pathologies characterized by embryonic resorption and placental fusion. Taken together, Zr-POM-based CE-CT offers a quantitative 3D methodology to investigate placental development or pathologies.