The AKT1-FOXO4 axis reciprocally regulates hemochorial placentation.

Hemochorial placentation involves the differentiation of invasive trophoblast cells, specialized cells that possess the capacity to exit the placenta and invade into the uterus where they restructure the vasculature. Invasive trophoblast cells arise from a well-defined compartment within the placenta, referred to as the junctional zone in rat and the extravillous trophoblast cell column in human. In this study, we investigated roles for AKT1, a serine/threonine kinase, in placental development using a genome-edited/loss-of-function rat model. Disruption of AKT1 resulted in placental, fetal and postnatal growth restriction. Forkhead box O4 (Foxo4), which encodes a transcription factor and known AKT substrate, was abundantly expressed in the junctional zone and in invasive trophoblast cells of the rat placentation site. Foxo4 gene disruption using genome editing resulted in placentomegaly, including an enlarged junctional zone. AKT1 and FOXO4 regulate the expression of many of the same transcripts expressed by trophoblast cells, but in opposite directions. In summary, we have identified AKT1 and FOXO4 as part of a regulatory network that reciprocally controls critical indices of hemochorial placenta development.

CITED2 is a conserved regulator of the uterine-placental interface.

Establishment of the hemochorial uterine-placental interface requires exodus of trophoblast cells from the placenta and their transformative actions on the uterus, which represent processes critical for a successful pregnancy, but are poorly understood. We examined the involvement of CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2) in rat and human trophoblast cell development. The rat and human exhibit deep hemochorial placentation. CITED2 was distinctively expressed in the junctional zone (JZ) and invasive trophoblast cells of the rat. Homozygous Cited2 gene deletion resulted in placental and fetal growth restriction. Small Cited2 null placentas were characterized by disruptions in the JZ, delays in intrauterine trophoblast cell invasion, and compromised plasticity. In the human placentation site, CITED2 was uniquely expressed in the extravillous trophoblast (EVT) cell column and importantly contributed to the development of the EVT cell lineage. We conclude that CITED2 is a conserved regulator of deep hemochorial placentation.

Intersection of regulatory pathways controlling hemostasis and hemochorial placentation.

Hemochorial placentation is characterized by the development of trophoblast cells specialized to interact with the uterine vascular bed. We utilized trophoblast stem (TS) cell and mutant rat models to investigate regulatory mechanisms controlling trophoblast cell development. TS cell differentiation was characterized by acquisition of transcript signatures indicative of an endothelial cell-like phenotype, which was highlighted by the expression of anticoagulation factors including tissue factor pathway inhibitor (TFPI). TFPI localized to invasive endovascular trophoblast cells of the rat placentation site. Disruption of TFPI in rat TS cells interfered with development of the endothelial cell-like endovascular trophoblast cell phenotype. Similarly, TFPI was expressed in human invasive/extravillous trophoblast (EVT) cells situated within first-trimester human placental tissues and following differentiation of human TS cells. TFPI was required for human TS cell differentiation to EVT cells. We next investigated the physiological relevance of TFPI at the placentation site. Genome-edited global TFPI loss-of-function rat models revealed critical roles for TFPI in embryonic development, resulting in homogeneous midgestation lethality prohibiting analysis of the role of TFPI as a regulator of the late-gestation wave of intrauterine trophoblast cell invasion. In vivo trophoblast-specific TFPI knockdown was compatible with pregnancy but had profound effects at the uterine-placental interface, including restriction of the depth of intrauterine trophoblast cell invasion while leading to the accumulation of natural killer cells and increased fibrin deposition. Collectively, the experimentation implicates TFPI as a conserved regulator of invasive/EVT cell development, uterine spiral artery remodeling, and hemostasis at the maternal-fetal interface.

ASCL2 reciprocally controls key trophoblast lineage decisions during hemochorial placenta development.

Invasive trophoblast cells are critical to spiral artery remodeling in hemochorial placentation. Insufficient trophoblast cell invasion and vascular remodeling can lead to pregnancy disorders including preeclampsia, preterm birth, and intrauterine growth restriction. Previous studies in mice identified achaete-scute homolog 2 (ASCL2) as essential to extraembryonic development. We hypothesized that ASCL2 is a critical and conserved regulator of invasive trophoblast cell lineage development. In contrast to the mouse, the rat possesses deep intrauterine trophoblast cell invasion and spiral artery remodeling similar to human placentation. In this study, we investigated invasive/extravillous trophoblast (EVT) cell differentiation using human trophoblast stem (TS) cells and a loss-of-function mutant Ascl2 rat model. ASCL2 transcripts are expressed in the EVT column and junctional zone, which represent tissue sources of invasive trophoblast progenitor cells within human and rat placentation sites, respectively. Differentiation of human TS cells into EVT cells resulted in significant up-regulation of ASCL2 and several other transcripts indicative of EVT cell differentiation. Disruption of ASCL2 impaired EVT cell differentiation, as indicated by cell morphology and transcript profiles. RNA sequencing analysis of ASCL2-deficient trophoblast cells identified both down-regulation of EVT cell-associated transcripts and up-regulation of syncytiotrophoblast-associated transcripts, indicative of dual activating and repressing functions. ASCL2 deficiency in the rat impacted placental morphogenesis, resulting in junctional zone dysgenesis and failed intrauterine trophoblast cell invasion. ASCL2 acts as a critical and conserved regulator of invasive trophoblast cell lineage development and a modulator of the syncytiotrophoblast lineage.

Elf5-centered transcription factor hub controls trophoblast stem cell self-renewal and differentiation through stoichiometry-sensitive shifts in target gene networks.

Elf5 is a transcription factor with pivotal roles in the trophoblast compartment, where it reinforces a trophoblast stem cell (TSC)-specific transcriptional circuit. However, Elf5 is also present in differentiating trophoblast cells that have ceased to express other TSC genes such as Cdx2 and Eomes. In the present study, we aimed to elucidate the context-dependent role of Elf5 at the interface between TSC self-renewal and the onset of differentiation. We demonstrate that precise levels of Elf5 are critical for normal expansion of the TSC compartment and embryonic survival, as Elf5 overexpression triggers precocious trophoblast differentiation. Through integration of protein interactome, transcriptome, and genome-wide chromatin immunoprecipitation data, we reveal that this abundance-dependent function is mediated through a shift in preferred Elf5-binding partners; in TSCs, Elf5 interaction with Eomes recruits Tfap2c to triply occupied sites at TSC-specific genes, driving their expression. In contrast, the Elf5 and Tfap2c interaction becomes predominant as their protein levels increase. This triggers binding to double- and single-occupancy sites that harbor the cognate Tfap2c motif, causing activation of the associated differentiation-promoting genes. These data place Elf5 at the center of a stoichiometry-sensitive transcriptional network, where it acts as a molecular switch governing the balance between TSC proliferation and differentiation.

Similarities and differences in placental development between humans and cynomolgus monkeys.

Background: The placenta is an extraembryonic organ, which is essential to maintain a normal pregnancy. However, placental development in humans is poorly understood because of technical and ethical reasons. Methods: We analyzed the anatomical localization of each trophoblastic subtype in the cynomolgus monkey placenta by immunohistochemistry in the early second trimester. Histological differences among the mouse, cynomolgus monkey, and human placenta were compared. The PubMed database was used to search for studies on placentation in rodents and primates. Main findings: The anatomical structures and subtypes of the placenta in cynomolgus monkeys are highly similar to those in humans, with the exception of fewer interstitial extravillous trophoblasts in cynomolgus monkeys. Conclusion: The cynomolgus monkey appears to be a good animal model to investigate human placentation.

Trophoblast-Specific Conditional Atg7 Knockout Mice Develop Gestational Hypertension.

Hypertensive disorder of pregnancy (HDP) is a serious pregnancy complication that is life threatening to both the mother and fetus. Understanding HDP pathophysiology is important for developing medical treatments. This study demonstrates the involvement of autophagy deficiency in adverse maternal and fetal outcomes using trophoblast-specific autophagy related (Atg)7, an autophagy-related protein, knockout mice. Atg7 conditional knockout (cKO) placentas were significantly smaller than controls in the spongiotrophoblast layer but not the labyrinth layer, which significantly elevated blood pressure in dams. A marker of autophagy deficiency, sequestosome 1/p62, was accumulated in giant trophoblast cells and in the spongiotrophoblast layer, accompanying increased apoptosis. However, neither proteinuria in dams nor fetal growth restriction was observed. Regarding trophoblast function, the number of trophoblasts migrating into the maternal decidua was significantly reduced, and the wall/lumen ratio of the spiral arteries was significantly increased in cKO placentas, suggesting shallow trophoblast invasion and inadequate vascular remodeling. The relative expression of placental growth factor mRNA was significantly decreased in cKO placentas compared with the control, likely causing poor placentation; however, other factors were unchanged in cKO placentas. This is the first report of autophagy deficiency leading to impaired placentation complicated by maternal HDP attributable to trophoblast dysfunction, and it suggests that placental autophagy is required for normal placentation.

Lvrn expression is not critical for mouse placentation.

Preeclampsia is a systemic disease caused by abnormal placentation that affects both mother and fetus. It was reported that Laeverin (LVRN, also known as Aminopeptidase Q) was up-regulated in the placenta of preeclamptic patients. However, physiological and pathological functions of LVRN remained to be unknown. Here we characterized Lvrn function during placentation in mice. RT-PCR showed that Lvrn is expressed in both fetus and placenta during embryogenesis, and several adult tissues. When we overexpressed Lvrn in a placenta-specific manner using lentiviral vectors, we did not see any defects in both placentae and fetuses. The mice carrying Lvrn overexpressing placentas did not show any preeclampsia-like symptoms such as maternal high blood pressure and fetal growth restriction. We next ablated Lvrn by CRISPR/Cas9-mediated genome editing to see physiological function. In Lvrn ablated mice, maternal blood pressure during pregnancy was not affected, and both placentas and fetuses grew normally. Collectively, these results suggest that, LVRN is irrelevant to preeclampsia and dispensable for normal placentation and embryonic development in mice.

Lentiviral Vector-Mediated Complementation Restored Fetal Viability but Not Placental Hyperplasia in Plac1-Deficient Mice.

The X-linked Plac1 gene is maternally expressed in trophoblast cells during placentation, and its disruption causes placental hyperplasia and intrauterine growth restriction. In contrast, Plac1 is also reported to be one of the upregulated genes in the hyperplastic placenta generated by nuclear transfer. However, the effect of overexpressed Plac1 on placental formation and function remained unaddressed. We complemented the Plac1 knockout placental dysfunction by lentiviral vector-mediated, placenta-specific Plac1 transgene expression. Whereas fetal development and the morphology of maternal blood sinuses in the labyrinth zone improved, placental hyperplasia remained, with an expanded the junctional zone that migrated and encroached into the labyrinth zone. Further experiments revealed that wild-type placenta with transgenically expressed Plac1 resulted in placental hyperplasia without the encroaching of the junctional zone. Our findings suggest that Plac1 is involved in trophoblast cell proliferation, differentiation, and migration. Its proper expression is required for normal placentation and fetal development.

CRISPR/Cas9-mediated mutation revealed cytoplasmic tail is dispensable for IZUMO1 function and male fertility.

IZUMO1 is a protein found in the head of spermatozoa that has been identified as essential for sperm-egg fusion. Its binding partner in the egg has been discovered (JUNO); however, the roles of several domains within IZUMO1 remain unexplored. One such domain is the C-terminus, which undergoes major phosphorylation changes in the cytoplasmic portion of the protein during rat epididymal transit. However, the cytoplasmic tail of IZUMO1 in many species is highly variable, ranging from 55 to one amino acid. Therefore, to understand the role of the cytoplasmic tail of IZUMO1 in mouse, we utilised the gene manipulation system of CRISPR/Cas9 to generate a point mutation resulting in a premature stop codon, producing mice with truncated IZUMO1. Mice without the cytoplasmic tail of IZUMO1 showed normal fertility but decreased the amount of protein, indicating that whilst this region is important for the expression level of IZUMO1, it is dispensable for fertilisation in the mouse.

Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes.

The recombinant clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas system has opened a new era for mammalian genome editing. Here, we constructed pX330 plasmids expressing humanized Cas9 (hCas9) and single guide RNAs (sgRNAs) against mouse genes and validated them both in vitro and in vivo. When we randomly chose 291 target sequences within protein coding regions of 73 genes, an average number of off-target candidates (exact match 13 nucleotides from 3' target and NGG) found by Bowtie software was 9.2 ± 21.0 (~1.8 times more than the estimated value, 5.2). We next validated their activity by observing green fluorescence reconstituted by homology dependent repair (HDR) of an EGFP expression cassette in HEK293T cells. Of the pX330 plasmids tested, 81.8% (238/291) were found to be functional in vitro. We finally injected the validated pX330 plasmids into mouse zygotes in its circular form against 32 genes (including two genes previously tested) and obtained mutant mice at a 52.9 ± 22.3% (100/196) mutation frequency. Among the pups carrying mutations on the autosomes, 43.6% (47/96) carried the mutations in both alleles. When off-target candidate sites were examined in 63 mutant mice, 0.8% (3/382) were mutated. We conclude that our method provides a simple, efficient, and cost-effective way for mammalian gene editing that is applicable for large scale mutagenesis in mammals.

The Dual-Pseudotyped Lentiviral Vector with VSV-G and Sendai Virus HN Enhances Infection Efficiency through the Synergistic Effect of the Envelope Proteins.

A gene delivery system utilizing lentiviral vectors (LVs) requires high transduction efficiency for successful application in human gene therapy. Pseudotyping allows viral tropism to be expanded, widening the usage of LVs. While vesicular stomatitis virus G (VSV-G) single-pseudotyped LVs are commonly used, dual-pseudotyping is less frequently employed because of its increased complexity. In this study, we examined the potential of phenotypically mixed heterologous dual-pseudotyped LVs with VSV-G and Sendai virus hemagglutinin-neuraminidase (SeV-HN) glycoproteins, termed V/HN-LV. Our findings demonstrated the significantly improved transduction efficiency of V/HN-LV in various cell lines of mice, cynomolgus monkeys, and humans compared with LV pseudotyped with VSV-G alone. Notably, V/HN-LV showed higher transduction efficiency in human cells, including hematopoietic stem cells. The efficient incorporation of wild-type SeV-HN into V/HN-LV depended on VSV-G. SeV-HN removed sialic acid from VSV-G, and the desialylation of VSV-G increased V/HN-LV infectivity. Furthermore, V/HN-LV acquired the ability to recognize sialic acid, particularly N-acetylneuraminic acid on the host cell, enhancing LV infectivity. Overall, VSV-G and SeV-HN synergistically improve LV transduction efficiency and broaden its tropism, indicating their potential use in gene delivery.

Null mutation of exocyst complex component 3-like does not affect vascular development in mice.

Exocyst is an octameric protein complex implicated in exocytosis. The exocyst complex is highly conserved among mammalian species, but the physiological function of each subunit in exocyst remains unclear. Previously, we identified exocyst complex component 3-like (Exoc3l) as a gene abundantly expressed in embryonic endothelial cells and implicated in the process of angiogenesis in human umbilical cord endothelial cells. Here, to reveal the physiological roles of Exoc3l during development, we generated Exoc3l knockout (KO) mice by genome editing with CRISPR/Cas9. Exoc3l KO mice were viable and showed no significant phenotype in embryonic angiogenesis or postnatal retinal angiogenesis. Exoc3l KO mice also showed no significant alteration in cholesterol homeostasis or insulin secretion, although several reports suggest an association of Exoc3l with these processes. Despite the implied roles, Exoc3l KO mice exhibited no apparent phenotype in vascular development, cholesterol homeostasis, or insulin secretion.

Generation of Transgenic Cynomolgus Monkeys Overexpressing the Gene for Amyloid-ß Precursor Protein.

Alzheimer's disease (AD) is the most common cause of dementia and understanding its pathogenesis should lead to improved therapeutic and diagnostic methods. Although several groups have developed transgenic mouse models overexpressing the human amyloid-ß precursor protein (APP) gene with AD mutations, with and without presenilin mutations, as well as APP gene knock-in mouse models, these animals display amyloid pathology but do not show neurofibrillary tangles or neuronal loss. This presumably is due to differences between the etiology of the aged-related human disease and the mouse models. Here we report the generation of two transgenic cynomolgus monkeys overexpressing the human gene for APP with Swedish, Artic, and Iberian mutations, and demonstrated expression of gene tagged green fluorescent protein marker in the placenta, amnion, hair follicles, and peripheral blood. We believe that these nonhuman primate models will be very useful to study the pathogenesis of dementia and AD. However, generated Tg monkeys still have some limitations. We employed the CAG promoter, which will promote gene expression in a non-tissue specific manner. Moreover, we used transgenic models but not knock-in models. Thus, the inserted transgene destroys endogenous gene(s) and may affect the phenotype(s). Nevertheless, it will be of great interest to determine whether these Tg monkeys will develop tauopathy and neurodegeneration similar to human AD.

Ex vivo Trophoblast-specific Genetic Manipulation Using Lentiviral Delivery.

In this protocol report, we describe a lentiviral gene delivery technique for genetic modification of the rat trophoblast cell lineage. Lentiviral packaged gene constructs can be efficiently and specifically delivered to the trophoblast cell lineage of the blastocyst. The consequences of 'gain-of-function' and 'loss-of-function' blastocyst manipulations can be evaluated with in vitro outgrowth assays or following transfer to pseudopregnant rats.

Proton Pump Inhibitors Decrease Soluble fms-Like Tyrosine Kinase-1 and Soluble Endoglin Secretion, Decrease Hypertension, and Rescue Endothelial Dysfunction.

Preeclampsia is a severe complication of pregnancy. Antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin are secreted in excess from the placenta, causing hypertension, endothelial dysfunction, and multiorgan injury. Oxidative stress and vascular inflammation exacerbate the endothelial injury. A drug that can block these pathophysiological steps would be an attractive treatment option. Proton pump inhibitors (PPIs) are safe in pregnancy where they are prescribed for gastric reflux. We performed functional studies on primary human tissues and animal models to examine the effects of PPIs on sFlt-1 and soluble endoglin secretion, vessel dilatation, blood pressure, and endothelial dysfunction. PPIs decreased sFlt-1 and soluble endoglin secretion from trophoblast, placental explants from preeclamptic pregnancies, and endothelial cells. They also mitigated tumor necrosis factor-α-induced endothelial dysfunction: PPIs blocked endothelial vascular cell adhesion molecule-1 expression, leukocyte adhesion to endothelium, and disruption of endothelial tube formation. PPIs decreased endothelin-1 secretion and enhanced endothelial cell migration. Interestingly, the PPI esomeprazole vasodilated maternal blood vessels from normal pregnancies and cases of preterm preeclampsia, but its vasodilatory effects were lost when the vessels were denuded of their endothelium. Esomeprazole decreased blood pressure in a transgenic mouse model where human sFlt-1 was overexpressed in placenta. PPIs upregulated endogenous antioxidant defenses and decreased cytokine secretion from placental tissue and endothelial cells. We have found that PPIs decrease sFlt-1 and soluble endoglin secretion and endothelial dysfunction, dilate blood vessels, decrease blood pressure, and have antioxidant and anti-inflammatory properties. They have therapeutic potential for preeclampsia and other diseases where endothelial dysfunction is involved.

CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice.

Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals. Here we show that large deletions with two sgRNAs as well as dsDNA-mediated point mutations are efficient in mouse embryonic stem cells (ESCs). The dsDNA-mediated gene knockins are also feasible in ESCs. Finally, we generated chimeric mice with biallelic mutant ESCs for a lethal gene, Dnajb13, and analyzed their phenotypes. Not only was the lethal phenotype of hydrocephalus suppressed, but we also found that Dnajb13 is required for sperm cilia formation. The combination of biallelic genome editing in ESCs and subsequent chimeric analysis provides a useful tool for rapid gene function analysis in the whole organism.

The effects of chemically synthesized saposin C on glucosylceramide-ß-glucosidase.

OBJECTIVE: Saposin C (SAP-C) is an essential activator of glucosylceramide (GlcCer)-ß-glucosidase (GCase), the enzyme deficient in Gaucher's disease. In this study, we investigated the effects of chemically synthesized SAP-Cs (synthetic SAP-Cs) on GCase. METHODS: Enzymatic assays and western blot analyses were carried out to evaluate the effects of two kinds of synthetic SAP-Cs, a non-glycosylated form and a N-glycosylated form bearing a complex type nonasaccharide, on GCase with respect to its activation, stabilization, and protection. Imiglucerase (Cerezyme) was used as the GCase. To mimic physiological conditions, GCase activity was assayed in the presence of 4-nitrobenzo-2-oxa-1,3-diazole-labeled GlcCer-containing liposomes composed of bis(monoacylglycero)phosphate, l-α-phosphatidylcholine, and cholesterol. RESULTS: GCase activities increased depending on the concentration of synthetic SAP-Cs. SAP-Cs at a concentration of 1µM increased GCase activities significantly, by 14- to 22-fold (non-glycosylated SAP-C: 22.9±0.16; nona-glycosylated SAP-C: 14.9±0.19; without SAP-C: 1.05±0.035pmol/h/ng GCase). These values equaled or surpassed previously published values obtained using recombinant non-glycosylated SAP-C. Both synthetic SAP-Cs were as effective as bovine serum albumin in stabilizing GCase at 37°C. Western blot analysis revealed that synthetic SAP-Cs specifically protected GCase from cathepsin D digestion. CONCLUSIONS: The results demonstrate that these novel, chemically synthesized SAP-Cs function as activators, stabilizers, and protectors of GCase, suggesting their utility in enzyme replacement therapy in patients with Gaucher's disease.

Sperm calcineurin inhibition prevents mouse fertility with implications for male contraceptive.

Calcineurin inhibitors, such as cyclosporine A and FK506, are used as immunosuppressant drugs, but their adverse effects on male reproductive function remain unclear. The testis expresses somatic calcineurin and a sperm-specific isoform that contains a catalytic subunit (PPP3CC) and a regulatory subunit (PPP3R2). We demonstrate herein that male mice lacking Ppp3cc or Ppp3r2 genes (knockout mice) are infertile, with reduced sperm motility owing to an inflexible midpiece. Treatment of mice with cyclosporine A or FK506 creates phenocopies of the sperm motility and morphological defects. These defects appear within 4 to 5 days of treatment, which indicates that sperm-specific calcineurin confers midpiece flexibility during epididymal transit. Male mouse fertility recovered a week after we discontinued treatment. Because human spermatozoa contain PPP3CC and PPP3R2 as a form of calcineurin, inhibition of this sperm-specific calcineurin may lead to the development of a reversible male contraceptive that would target spermatozoa in the epididymis.