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BACKGROUND: The ferroxidase zyklopen (Zp) has been implicated in the placental transfer of iron to the fetus. However, the evidence for this is largely circumstantial. OBJECTIVES: This study aimed to determine whether Zp is essential for placental iron transfer. METHODS: A model was established using 8- to 12-wk-old pregnant C57BL/6 mice on standard rodent chow in which Zp was knocked out in the fetus and fetal components of the placenta. Zp was also disrupted in the entire placenta using global Zp knockout mice. Inductively coupled plasma MS was used to measure total fetal iron, an indicator of the amount of iron transferred by the placenta to the fetus, at embryonic day 18.5 of gestation. Iron transporter expression in the placenta was measured by Western blotting, and the expression of Hamp1, the gene encoding the iron regulatory hormone hepcidin, was determined in fetal liver by real-time PCR. RESULTS: There was no change in the amount of iron transferred to the fetus when Zp was disrupted in either the fetal component of the placenta or the entire placenta. No compensatory changes in the expression of the iron transport proteins transferrin receptor 1 or ferroportin were observed, nor was there any change in fetal liver Hamp1 mRNA. Hephl1, the gene encoding Zp, was expressed mainly in the maternal decidua of the placenta and not in the nutrient-transporting syncytiotrophoblast. Disruption of Zp in the whole placenta resulted in a 26% increase in placental size (P

Objective Sulfation is an essential physiological process that regulates the function of a wide array of molecules involved in brain development. We have previously shown expression levels for the sulfate transporter Slc13a4 to be elevated during postnatal development, and that sulfate accumulation in the brains of Slc13a4+/− mice is reduced, suggesting a role for this transporter during this critical window of brain development. In order to understand the pathways regulated by cellular sulfation within the brain, we performed a bulk RNA-sequencing analysis of the forebrain of postnatal day 20 (P20) Slc13a4 heterozygous mice and wild-type litter mate controls. Data description We performed an RNA transcriptomic based sequencing screen on the whole forebrain from Slc13a4+/− and Slc13a4+/+mice at P20. Differential expression analysis revealed 90 differentially regulated genes in the forebrain of Slc13a4+/− mice (a p-value of 0.1 was considered as significant). Of these, 55 were upregulated, and 35 were downregulated in the forebrain of heterozygous mice. Moreover, when we stratified further with a ± 1.2 fold-change, we observed 38 upregulated, and 16 downregulated genes in the forebrain of heterozygous mice. This resource provides a useful tool to interrogate which pathways may require elevated sulfate levels to drive normal postnatal development of the brain.

Sulfate is a key anion required for a range of physiological functions within the brain. These include sulfonation of extracellular proteoglycans to facilitate local growth factor binding and to regulate the shape of morphogen gradients during development. We have previously shown that mice lacking one allele of the sulfate transporter Slc13a4 exhibit reduced sulfate transport into the brain, deficits in social behaviour, reduced performance in learning and memory tasks, and abnormal neurogenesis within the ventricular/subventricular zone lining the lateral ventricles. However, whether these mice have deficits in hippocampal neurogenesis was not addressed. Here, we demonstrate that adult Slc13a4+/− mice have increased neurogenesis within the subgranular zone (SGZ) of the hippocampal dentate gyrus, with elevated numbers of neural progenitor cells and intermediate progenitors. In contrast, by 12 months of age there were reduced numbers of neural stem cells in the SGZ of heterozygous mice. Importantly, we did not observe any changes in proliferation when we isolated and cultured progenitors in vitro in neurosphere assays, suggestive of a cell-extrinsic requirement for sulfate in regulating hippocampal neurogenesis. Collectively, these data demonstrate a requirement for sulfate transport during postnatal brain development to ensure normal adult hippocampal neurogenesis., Summary Statement: Heterozygosity for the brain sulfate transporter Slc13a4 during postnatal development results in life-long abnormal hippocampal neurogenesis.

Sulfate is essential for healthy fetal growth and development. Cysteine dioxygenase type 1 (CDO1) plays an important role in the catabolism of cysteine to sulfate. Cdo1 knockout mice exhibit severe and lethal fetal phenotypes but the involvement of CDO1 gene variants in human development is unknown. We searched the NCBI and Ensembl gene databases and identified four alternatively spliced CDO1 coding mRNA transcripts, as well as 148 validated CDO1 gene variants, including 138 missense, 6 nonsense, 1 frameshift, 1 in-frame deletion, and 2 splice site variants. In silico analyses predicted 68 of the missense variants to be deleterious to CDO1 protein structure and function. We examined the relative abundance of the four CDO1 coding mRNA transcripts in human term placentas using qRT-PCR. CDO1 mRNA variant 2 was the most abundant transcript, with intermediate levels of variant 4 and lower levels of variants 1 and 3. Using in situ hybridization, we localised CDO1 mRNA expression to the syncytiotrophoblast layer of human term placenta. To investigate the regulation of CDO1 gene expression, we analysed the transcriptional activity of the human CDO1 5′-flanking region in the JEG-3 placental cell line using luciferase reporter assays. Transcriptional activities were identified in the regions −5 to −269 and − 269 to −1200 nucleotides upstream of the CDO1 transcription initiation site. Mutational analyses of a single nucleotide polymorphism -289C > G that is common in the general population (allele frequency = 0.37) and a putative transcription factor binding motif (CCAAT enhancer binding protein beta) did not alter transcriptional activity of the CDO1 5′-flanking region. Collectively, this study provides an overview and analysis of human CDO1 for future investigations of this gene in human health., Highlights • CDO1 mRNA expressed in syncytiotrophoblast layer of the human term placenta. • CDO1 variant 2 mRNA is the most abundant CDO1 transcript. • 148 validated CDO1 gene variants. • Basal promoter activity contained within 1200 nt of the CDO1 5′-flanking region.

The major milestones in mouse placental development are well described, but our understanding is limited to how the placenta can adapt to damage or changes in the environment. By using stereology and expression of cell cycle markers, we found that the placenta grows under normal conditions not just by hyperplasia of trophoblast cells but also through extensive polyploidy and cell hypertrophy. In response to feeding a low protein diet to mothers prior to and during pregnancy, to mimic chronic malnutrition, we found that this normal program was altered and that it was influenced by the sex of the conceptus. Male fetuses showed intrauterine growth restriction (IUGR) by embryonic day (E) 18.5, just before term, whereas female fetuses showed IUGR as early as E16.5. This difference was correlated with differences in the size of the labyrinth layer of the placenta, the site of nutrient and gas exchange. Functional changes were implied based on up-regulation of nutrient transporter genes. The junctional zone was also affected, with a reduction in both glycogen trophoblast and spongiotrophoblast cells. These changes were associated with increased expression of Phlda2 and reduced expression of Egfr. Polyploidy, which results from endoreduplication, is a normal feature of trophoblast giant cells (TGC) but also spongiotrophoblast cells. Ploidy was increased in sinusoidal-TGCs and spongiotrophoblast cells, but not parietal-TGCs, in low protein placentas. These results indicate that the placenta undergoes a range of changes in development and function in response to poor maternal diet, many of which we interpret are aimed at mitigating the impacts on fetal and maternal health.

Disruptions to either sulfate supply or sulfation enzymes can affect brain development and have long-lasting effects on brain function, yet our understanding of the molecular mechanisms governing this are incomplete. Perineuronal nets (PNNs) are highly sulfated, specialized extracellular matrix structures that regulate the maturation of synaptic connections and neuronal plasticity. We have previously shown that mice heterozygous for the brain sulfate transporter Slc13a4 have abnormal social interactions, memory, exploratory behaviors, stress and anxiety of postnatal origin, pointing to potential deficits in PNN biology, and implicate SLC13A4 as a critical factor required for regulating normal synaptic connectivity and function. Here, we sought to investigate aberrant PNN formation as a potential mechanism contributing to the functional deficits displayed by Slc13a4+/- mice. Following social interactions, we reveal reduced neuronal activation in the somatosensory cortex of Slc13a4+/- mice, and altered inhibitory and excitatory postsynaptic currents. In line with this, we found a reduction in parvalbumin-expressing neurons decorated with PNNs, as well as reduced expression of markers for PNN maturation. Finally, we reveal that postnatal administration of N-acetylcysteine prevented PNN abnormalities from manifesting in Slc13a4+/- adult animals. Collectively, these data highlight a central role for postnatal SLC13A4 in normal PNN formation, circuit function and subsequent animal behavior.

Background A male fetus is suggested to be more susceptible to in utero and birth complications. This may be due in part to altered morphology or function of the XY placenta. We hypothesised that sexual dimorphism begins at the blastocyst stage with sex differences in the progenitor trophectoderm (TE) and its derived trophoblast lineages, as these cells populate the majority of cell types within the placenta. We investigated sex-specific differences in cell allocation in the pre-implantation embryo and further characterised growth and gene expression of the placental compartments from the early stages of the definitive placenta through to late gestation. Methods Naturally mated Sprague Dawley dams were used to collect blastocysts at embryonic day (E) 5 to characterise cell allocation; total, TE, and inner cell mass (ICM), and differentiation to downstream trophoblast cell types. Placental tissues were collected at E13, E15, and E20 to characterise volumes of placental compartments, and sex-specific gene expression profiles. Results Pre-implantation embryos showed no sex differences in cell allocation (total, TE and ICM) or early trophoblast differentiation, assessed by outgrowth area, number and ploidy of trophoblasts and P-TGCs, and expression of markers of trophoblast stem cell state or differentiation. Whilst no changes in placental structures were found in the immature E13 placenta, the definitive E15 placenta from female fetuses had reduced labyrinthine volume, fetal and maternal blood space volume, as well as fetal blood space surface area, when compared to placentas from males. No differences between the sexes in labyrinth trophoblast volume or interhaemal membrane thickness were found. By E20 these sex-specific placental differences were no longer present, but female fetuses weighed less than their male counterparts. Coupled with expression profiles from E13 and E15 placental samples may suggest a developmental delay in placental differentiation. Conclusions Although there were no overt differences in blastocyst cell number or early placental development, reduced growth of the female labyrinth in mid gestation is likely to contribute to lower fetal weight in females at E20. These data suggest sex differences in fetal growth trajectories are due at least in part, to differences in placenta growth. Electronic supplementary material The online version of this article (doi:10.1186/s13293-017-0138-6) contains supplementary material, which is available to authorized users.

The development of pathologies during pregnancy, including pre-eclampsia, hypertension and fetal growth restriction (FGR), often originates from poor functioning of the placenta. In vivo models of maternal stressors, such as nutrient deficiency, and placental insufficiency often focus on inadequate growth of the fetus and placenta in late gestation. These studies rarely investigate the origins of poor placental formation in early gestation, including those affecting the pre-implantation embryo and/or the uterine environment. The current study characterises the impact on blastocyst, uterine and placental outcomes in a rat model of periconceptional alcohol exposure, in which 12.5% ethanol is administered in a liquid diet from 4 days before until 4 days after conception. We show female-specific effects on trophoblast differentiation, embryo-uterine communication, and formation of the placental vasculature, resulting in markedly reduced placental volume at embryonic day 15. Both sexes exhibited reduced trophectoderm pluripotency and global hypermethylation, suggestive of inappropriate epigenetic reprogramming. Furthermore, evidence of reduced placental nutrient exchange and reduced pre-implantation maternal plasma choline levels offers significant mechanistic insight into the origins of FGR in this model.

Background Sulfate availability is crucial for the sulfonation of brain extracellular matrix constituents, membrane phospholipids, neurosteroids, and neurotransmitters. Observations from humans and mouse models suggest dysregulated sulfate levels may be associated with neurodevelopmental disorders, such as autism. However, the cellular mechanisms governing sulfate homeostasis within the developing or adult brain are not fully understood. Methods We utilized a mouse model with a conditional allele for the sulfate transporter Slc13a4, and a battery of behavioral tests, to assess the effects of disrupted sulfate transport on maternal behaviors, social interactions, memory, olfaction, exploratory behavior, anxiety, stress, and metabolism. Immunohistochemistry examined neurogenesis within the stem cells niches. Findings The sulfate transporter Slc13a4 plays a critical role in postnatal brain development. Slc13a4 haploinsufficiency results in significant behavioral phenotypes in adult mice, notably impairments in social interaction and long-term memory, as well as increased neurogenesis in the subventricular stem cell niche. Conditional gene deletion shows these phenotypes have a developmental origin, and that full biallelic expression of Slc13a4 is required only in postnatal development. Furthermore, administration of N-acetylcysteine (NAC) within postnatal window P14-P30 prevents the onset of phenotypes in adult Slc13a4+/− mice. Interpretation Slc13a4 haploinsufficient mice highlight a requirement for adequate sulfate supply in postnatal development for the maturation of important social interaction and memory pathways. With evidence suggesting dysregulated sulfate biology may be a feature of some neurodevelopmental disorders, the utility of sulfate levels as a biomarker of disease and NAC administration as an early preventative measure should be further explored.

The complement system, typically associated with innate immunity, is emerging as a key controller of nonimmune systems including in development, with recent studies linking complement mutations with neurodevelopmental disease. A key effector of the complement response is the activation fragment C5a, which, through its receptor C5aR1, is a potent driver of inflammation. Surprisingly, C5aR1 is also expressed during early mammalian embryogenesis; however, no clearly defined function is ascribed to C5aR1 in development. Here we demonstrate polarized expression of C5aR1 on the apical surface of mouse embryonic neural progenitor cellsin vivoand on human embryonic stem cell-derived neural progenitors. We also show that signaling of endogenous C5a during mouse embryogenesis drives proliferation of neural progenitor cells within the ventricular zone and is required for normal brain histogenesis. C5aR1 signaling in neural progenitors was dependent on atypical protein kinase C ζ, a mediator of stem cell polarity, with C5aR1 inhibition reducing proliferation and symmetric division of apical neural progenitors in human and mouse models. C5aR1 signaling was shown to promote the maintenance of cell polarity, with exogenous C5a increasing the retention of polarized rosette architecture in human neural progenitors after physical or chemical disruption. Transient inhibition of C5aR1 during neurogenesis in developing mice led to behavioral abnormalities in both sexes and MRI-detected brain microstructural alterations, in studied males, demonstrating a requirement of C5aR1 signaling for appropriate brain development. This study thus identifies a functional role for C5a–C5aR1 signaling in mammalian neurogenesis and provides mechanistic insight into recently identified complement gene mutations and brain disorders.SIGNIFICANCE STATEMENTThe complement system, traditionally known as a controller of innate immunity, now stands as a multifaceted signaling family with a broad range of physiological actions. These include roles in the brain, where complement activation is associated with diseases, including epilepsy and schizophrenia. This study has explored complement regulation of neurogenesis, identifying a novel relationship between the complement activation peptide C5a and the neural progenitor proliferation underpinning formation of the mammalian brain. C5a was identified as a regulator of cell polarity, with inhibition of C5a receptors during embryogenesis leading to abnormal brain development and behavioral deficits. This work demonstrates mechanisms through which dysregulation of complement causes developmental disease and highlights the potential risk of complement inhibition for therapeutic purposes in pregnancy.

The solute linked carrier 13A4 gene (SLC13A4) is abundantly expressed in the human and mouse placenta where it is proposed to transport nutrient sulfate to the fetus. In mice, targeted disruption of placental Slc13a4 leads to severe and lethal fetal phenotypes, however the involvement of SLC13A4 in human development is unknown. A search of the NCBI and Ensembl gene databases identified two alternatively spliced SLC13A4 mRNA transcripts and 98 SLC13A4 gene variants, including 85 missense, 4 splice site, 5 frameshift and 2 nonsense variants, as well as 2 in-frame deletions. We examined the relative abundance of the two SLC13A4 mRNA transcripts and then compared the sulfate transport function and plasma membrane expression of both isoforms as well as 6 sequence variants that predict disrupted SLC13A4 protein structure and function. SLC13A4 mRNA variant 1 has three additional nucleotides CAG compared to SLC13A4 mRNA variant 2 as a result of alternative splicing at the 5'-end of exon 6. Using qRT-PCR, we show a 4-fold higher abundance of SLC13A4 mRNA variant 1 compared to variant 2 in term human placentas and cultured BeWo and JEG-3 cell lines. The corresponding SLC13A4 protein isoforms 1 and 2 were found to have similar sulfate uptake activity and apical membrane expression in cultured MDCK cells. In addition, sulfate uptake into MDCK cells was similar between SLC13A4 isoform 1 and four missense variants N300S, F310C, E360Q and I570V, whereas V513M and frameshift variant L72Sfs led to partial (≈75% decrease) and complete loss-of-function, respectively. Localisation of these variants in MDCK cells showed N300S, E360Q, V513M and I570V expression on the apical plasma membrane, L72Sfs intracellularly and F310C on both apical and basolateral membranes. Our finding of partial and complete loss-of-function variants warrants further studies of the potential involvement of SLC13A4 in fetal pathophysiology.

Background and Purpose Subtypes of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of cation channels are widely expressed on nerves and smooth muscle cells in many organ systems, where they serve to regulate membrane excitability. Here we have assessed whether HCN channel inhibitors alter the function of airway smooth muscle or the neurons that regulate airway smooth muscle tone. Experimental Approach The effects of the HCN channel inhibitors ZD7288, zatebradine and Cs+ were assessed on agonist and nerve stimulation-evoked changes in guinea pig airway smooth muscle tone using tracheal strips in vitro, an innervated tracheal tube preparation ex vivo or in anaesthetized mechanically ventilated guinea pigs in vivo. HCN channel expression in airway nerves was assessed using immunohistochemistry, PCR and in situ hybridization. Key Results HCN channel inhibition did not alter airway smooth muscle reactivity in vitro to exogenously administered smooth muscle spasmogens, but significantly potentiated smooth muscle contraction evoked by the sensory nerve stimulant capsaicin and electrical field stimulation of parasympathetic cholinergic postganglionic neurons. Sensory nerve hyperresponsiveness was also evident in in vivo following HCN channel blockade. Cs+, but not ZD7288, potentiated preganglionic nerve-dependent airway contractions and over time induced autorhythmic preganglionic nerve activity, which was not mimicked by inhibitors of potassium channels. HCN channel expression was most evident in vagal sensory ganglia and airway nerve fibres. Conclusions and Implications HCN channel inhibitors had a previously unrecognized effect on the neural regulation of airway smooth muscle tone, which may have implications for some patients receiving HCN channel inhibitors for therapeutic purposes.

Nutrient sulfate has numerous roles in mammalian physiology and is essential for healthy fetal growth and development. The fetus has limited capacity to generate sulfate and relies on sulfate supplied from the maternal circulation via placental sulfate transporters. The placenta also has a high sulfate requirement for numerous molecular and cellular functions, including sulfate conjugation (sulfonation) to estrogen and thyroid hormone which leads to their inactivation. Accordingly, the ratio of sulfonated (inactive) to unconjugated (active) hormones modulates endocrine function in fetal, placental and maternal tissues. During pregnancy, there is a marked increase in the expression of genes involved in transport and generation of sulfate in the mouse placenta, in line with increasing fetal and placental demands for sulfate. The maternal circulation also provides a vital reservoir of sulfate for the placenta and fetus, with maternal circulating sulfate levels increasing by 2-fold from mid-gestation. However, despite evidence from animal studies showing the requirement of maternal sulfate supply for placental and fetal physiology, there are no routine clinical measurements of sulfate or consideration of dietary sulfate intake in pregnant women. This is also relevant to certain xenobiotics or pharmacological drugs which when taken by the mother use significant quantities of circulating sulfate for detoxification and clearance, and thereby have the potential to decrease sulfonation capacity in the placenta and fetus. This article will review the physiological adaptations of the placenta for maintaining sulfate homeostasis in the fetus and placenta, with a focus on pathophysiological outcomes in animal models of disturbed sulfate homeostasis.

Exposure of the embryo or fetus to perturbations in utero can result in intrauterine growth restriction, a primary risk factor for the development of adult disease. However, despite similar exposures, males and females often have altered disease susceptibility or progression from different stages of life. Fetal growth is largely mediated by the placenta, which, like the fetus is genetically XX or XY. The placenta and its associated trophoblast lineages originate from the trophectoderm (TE) of the early embryo. Rodent models (rat, mouse, spiny mouse), have been used extensively to examine placenta development and these have demonstrated the growth trajectory of the placenta in females is generally slower compared to males, and also shows altered adaptive responses to stressful environments. These placental adaptations are likely to depend on the type of stressor, duration, severity and the window of exposure during development. Here we describe the divergent developmental pathways between the male and female placenta contributing to altered differentiation of the TE derived trophoblast subtypes, placental growth, and formation of the placental architecture. Our focus is primarily genetic or environmental pertur bations in rodent models which show altered placental responsiveness between sexes. We suggest that perturbations during early placental development may have greater impact on viability and growth of the female fetus whilst those occurring later in gestation may preferentially affect the male fetus. This may be of great relevance to human pregnancies which result from assisted reproductive technologies or complications such as pre-eclampsia and diabetes.

Normal placental function is essential for optimal fetal growth. Transport of glucose from mother to fetus is critical for fetal nutrient demands and can be stored in the placenta as glycogen. However, the function of this glycogen deposition remains a matter of debate: It could be a source of fuel for the placenta itself or a storage reservoir for later use by the fetus in times of need. While the significance of placental glycogen remains elusive, mounting evidence indicates that altered glycogen metabolism and/or deposition accompanies many pregnancy complications that adversely affect fetal development. This review will summarize histological, biochemical and molecular evidence that glycogen accumulates in a) placentas from a variety of experimental rodent models of perturbed pregnancy, including maternal alcohol exposure, glucocorticoid exposure, dietary deficiencies and hypoxia and b) placentas from human pregnancies with complications including preeclampsia, gestational diabetes mellitus and intrauterine growth restriction (IUGR). These pregnancies typically result in altered fetal growth, developmental abnormalities and/or disease outcomes in offspring. Collectively, this evidence suggests that changes in placental glycogen deposition is a common feature of pregnancy complications, particularly those associated with altered fetal growth.

Mg2+ regulates many physiological processes and signalling pathways. However, little is known about the mechanisms underlying the organismal balance of Mg2+. Capitalizing on a set of newly generated mouse models, we provide an integrated mechanistic model of the regulation of organismal Mg2+ balance during prenatal development and in adult mice by the ion channel TRPM6. We show that TRPM6 activity in the placenta and yolk sac is essential for embryonic development. In adult mice, TRPM6 is required in the intestine to maintain organismal Mg2+ balance, but is dispensable in the kidney. Trpm6 inactivation in adult mice leads to a shortened lifespan, growth deficit and metabolic alterations indicative of impaired energy balance. Dietary Mg2+ supplementation not only rescues all phenotypes displayed by Trpm6-deficient adult mice, but also may extend the lifespan of wildtype mice. Hence, maintenance of organismal Mg2+ balance by TRPM6 is crucial for prenatal development and survival to adulthood. DOI: http://dx.doi.org/10.7554/eLife.20914.001, eLife digest A balanced diet contains a variety of minerals such as magnesium ions, which are required for many chemical reactions in our body. A shortage of magnesium ions is linked to many diseases and is thought to be especially harmful to babies in the womb and shortly after birth. Magnesium ion deficiency is widespread in human populations and in the US is thought to affect up to 68% of people. Despite its prominent role in human health, our understanding of how the body maintains the right balance of magnesium ions remains extremely vague. Magnesium ions can enter and leave a cell by passing through specific types of proteins that form channels in the membrane surrounding the cell. There are thought to be around ten types of these magnesium ion channels in human cells, but we do not know what roles any of them perform in the body. One such channel called TRPM6 may be particularly important because mutations in the gene that encodes this channel can cause magnesium ion deficiency in human infants. However, the loss of TRPM6 in mice disrupts how mouse embryos develop, suggesting that our current view on the role that TRPM6 plays in regulating the magnesium ion balance in humans may be too simplistic. To address this question, Chubanov et al. studied mice with mutations that disrupted the production of TRPM6 in specific tissues only. The experiments show that TRPM6 primarily operates in the placenta and intestine to regulate the balance of magnesium ions in the body. Further experiments show that the loss of TRPM6 in adult mice leads to reduced lifespan, growth defects and poor health by disrupting important biochemical reactions. Supplying the mutant mice with magnesium ion supplements improved their health and could extend lifespans of normal animals. The findings of Chubanov et al. demonstrate that TRPM6 plays a crucial role in regulating the levels of magnesium ions in mice before birth and into adulthood. The next step is to carry out large-scale experiments to investigate the effects of altering the levels of magnesium ions in human diets. DOI: http://dx.doi.org/10.7554/eLife.20914.002

Maternal alcohol consumption is common prior to pregnancy recognition and in the rat results in altered placental development and fetal growth restriction. To assess the effect of ethanol (EtOH) exposure on the differentiation of trophoblast stem (TS) cells, mouse TS lines were differentiated in vitro for 6 days in 0%, 0.2% or 1% EtOH. This reduced both trophoblast survival and expression of labyrinth and junctional zone trophoblast subtype-specific genes. This suggests that fetal growth restriction and altered placental development associated with maternal alcohol consumption in the periconceptional period could be mediated in part by direct effects on trophoblast development.

Recently we reported that Rearranged L-Myc Fusion, RLF, acts as an epigenetic modifier maintaining low levels of DNA methylation at CpG island shores and enhancers across the genome. Here we focus on the phenotype of Rlf null mutant mice generated via an ENU mutagenesis screen, to identify genes required for epigenetic regulation. RLF is expressed in a range of fetal mouse tissues, including the fetal heart. Comprehensive timed-mating studies are consistent with our previously reported findings that Rlf homozygous mutant mice rarely survive to adulthood, with the majority dying shortly after birth. Histological analysis of two independent Rlf ENU mutant lines at E11.5-E14.5 showed heart defects resembling those present in humans with Left Ventricular Non-Compaction (LVNC). In situ hybridisation analysis localized expression of Rlf to the endocardium and epicardium of embryonic and postnatal hearts, and transiently to cardiomyocytes during heart looping and early chamber formation stages. RNA-seq analysis of Rlf mutant hearts highlighted defective NOTCH pathway signalling, recently describe as one cause of LVNC. This study provides the first evidence that RLF is required for normal heart development in the mouse. The heart morphological defects present at high penetrance in Rlf mutants are consistent with features of LVNC in humans, and molecular analysis identified attenuated JAGGED 1 expression and NOTCH signalling as likely contributors to these defects. Our study highlights the importance of RLF-dependent epigenetic modifications to DNA for maintaining correct gene regulatory network and intercellular signalling interactions during heart chamber and septal development. Further investigations are needed to define the biochemical role of RLF in the developing heart, and whether RLF mutations are a cause of heart defects in humans.

Insights into the anatomical organization of complex neural circuits provide important information about function, and thus tools that facilitate neuroanatomical studies have proved invaluable in neuroscience. Advances in molecular cloning have allowed the production of novel recombinant neuroinvasive viruses for use in transynaptic neural tracing studies. However, the vast majority of these viruses have motility in the retrograde direction only, therefore limiting their use to studies of synaptic input circuitry. Here we describe the construction of an EGFP reporting herpes simplex virus, strain H129, which preferentially moves along synaptically connected neurons in the anterograde direction. In vitro and in vivo characterization studies confirm that the HSV-1 H129-EGFP retains comparable replication and neuroinvasiveness as the wildtype H129 virus. As a proof of principle we confirm anterograde movement of the H129-EGFP along polysynaptic pathways by inoculating the upper airways and tracking time-dependent EGFP expression in previously described ascending sensory pathways. These data confirm a genomic locus for recombining HSV-1 H129 such that normal viral function and replication is maintained. Novel viral recombinants such as HSV-1 H129-EGFP will be useful tools for delineating the central organization of peripheral sensory pathways as well as the synaptic outputs from central neuronal populations.

Trophoblast giant cells (TGCs) are the first terminally differentiated subtype to form in the trophoblast cell lineage in rodents. In addition to mediating implantation, they are the main endocrine cells of the placenta, producing several hormones which regulate the maternal endocrine and immune systems and promote maternal blood flow to the implantation site. Generally considered a homogeneous population, TGCs have been identified by their expression of genes encoding placental lactogen 1 or proliferin. In the present study, we have identified a number of TGC subtypes, based on morphology and molecular criteria and demonstrated a previously underappreciated diversity of TGCs. In addition to TGCs that surround the implantation site and form the interface with the maternal deciduas, we demonstrate at least three other unique TGC subtypes: spiral artery-associated TGCs, maternal blood canal-associated TGCs and a TGC within the sinusoidal spaces of the labyrinth layer of the placenta. All four TGC subtypes could be identified based on the expression patterns of four genes: Pl1, Pl2, Plf (encoded by genes of the prolactin/prolactin-like protein/placental lactogen gene locus), and Ctsq (from a placental-specific cathepsin gene locus). Each of these subtypes was detected in differentiated trophoblast stem cell cultures and can be differentially regulated; treatment with retinoic acid induces Pl1/Plf+ TGCs preferentially. Furthermore, cell lineage tracing studies indicated unique origins for different TGC subtypes, in contrast with previous suggestions that secondary TGCs all arise from Tpbpa+ ectoplacental cone precursors.

The ubiquitously expressed zinc finger transcription factors Sp1 and Sp3 play critical roles in embryonic development. Sp1 knockout mice die around embryonic day 10.5. Mice lacking Sp3 are postnatal lethal. Mice heterozygous for either Sp1 or Sp3 are apparently normal, although slightly smaller. Here, we show that compound heterozygosity of Sp1 and Sp3 results in embryonic lethality accompanied by a spectrum of developmental abnormalities, including growth retardation, morphological alterations of the lung, impaired ossification, anemia, and placental defects. Anemia in Sp1/Sp3 compound heterozygous mutant embryos is associated with impaired maturation of erythrocytes. Analyses of the placenta revealed a markedly reduced spongiotrophoblast layer and a severe disorganization of the labyrinth layer in Sp1/Sp3 compound heterozygous as well as in Sp3-deficient mutant embryos. Our findings demonstrate that a threshold of Sp1 and Sp3 activity is required for normal embryonic development, suggesting that Sp1 and Sp3 act cooperatively to regulate downstream targets.

Preeclampsia (PE) is a pregnancy-specific disorder characterized by hypertension and proteinuria after 20 wk gestation. Abnormal extravillous trophoblast (EVT) invasion and remodeling of uterine spiral arterioles is thought to contribute to PE development. Interleukin-11 (IL11) impedes human EVT invasion in vitro and is elevated in PE decidua in women. We demonstrate that IL11 administered to mice causes development of PE features. Immunohistochemistry shows IL11 compromises trophoblast invasion, spiral artery remodeling, and placentation, leading to increased systolic blood pressure (SBP), proteinuria, and intrauterine growth restriction, although nonpregnant mice were unaffected. Real-time PCR array analysis identified pregnancy-associated plasma protein A2 (PAPPA2), associated with PE in women, as an IL11 regulated target. IL11 increased PAPPA2 serum and placental tissue levels in mice. In vitro, IL11 compromised primary human EVT invasion, whereas siRNA knockdown of PAPPA2 alleviated the effect. Genes regulating uterine natural killer (uNK) recruitment and differentiation were down-regulated and uNK cells were reduced after IL11 treatment in mice. IL11 withdrawal in mice at onset of PE features reduced SBP and proteinuria to control levels and alleviated placental labyrinth defects. In women, placental IL11 immunostaining levels increased in PE pregnancies and in serum collected from women before development of early-onset PE, shown by ELISA. These results indicate that elevated IL11 levels result in physiological changes at the maternal-fetal interface, contribute to abnormal placentation, and lead to the development of PE. Targeting placental IL11 may provide a new treatment option for PE.

Sensory nerves innervating the mucosa of the airways monitor the local environment for the presence of irritant stimuli and, when activated, provide input to the nucleus of the solitary tract (Sol) and paratrigeminal nucleus (Pa5) in the medulla to drive a variety of protective behaviors. Accompanying these behaviors are perceivable sensations that, particularly for stimuli in the proximal end of the airways, can be discrete and localizable. Airway sensations likely reflect the ascending airway sensory circuitry relayed via the Sol and Pa5, which terminates broadly throughout the CNS. However, the relative contribution of the Sol and Pa5 to these ascending pathways is not known. In the present study, we developed and characterized a novel conditional anterograde transneuronal viral tracing system based on the H129 strain of herpes simplex virus 1 and used this system in rats along with conventional neuroanatomical tracing with cholera toxin B to identify subcircuits in the brainstem and forebrain that are in receipt of relayed airway sensory inputs via the Sol and Pa5. We show that both the Pa5 and proximal airways disproportionately receive afferent terminals arising from the jugular (rather than nodose) vagal ganglia and the output of the Pa5 is predominately directed toward the ventrobasal thalamus. We propose the existence of a somatosensory-like pathway from the proximal airways involving jugular ganglia afferents, the Pa5, and the somatosensory thalamus and suggest that this pathway forms the anatomical framework for sensations arising from the proximal airway mucosa.

A specialized subtype of trophoblast giant cells (TGCs) line the torturous sinusoids of the murine placental labyrinth, and can be distinguished from most other TGCs by the expression of Ctsq. We generated a transgenic mouse line expressing Cre recombinase from the Ctsq promoter. Crosses with Cre-inducible tdTomato reporter mice indicated Cre activity was restricted to the sinusoidal TGCs of the labyrinth, as well as the recently characterized channel TGCs. When crossed with Cre-inducible DTA transgenic mice, ablation of sinusoidal TGCs was achieved in double transgenic embryos, resulting in fetal growth restriction by E16.5, and embryonic lethality by term.

Nutrient sulfate is important for fetal development. The fetus has a limited capacity to generate sulfate and relies on maternal sulfate supplied via the placenta. The gestational age when fetal sulfate generation begins is unknown but would require cysteine dioxygenase (CDO1) which mediates a major step of sulfate production from cysteine. We investigated the ontogeny of Cdo1 mRNA expression in mouse fetal and placental tissues, which showed increasing levels from embryonic day 10.5 and was localised to the decidua and several fetal tissues including nasal cavities and brain. These findings suggest a role for Cdo1 in sulfate generation from mid-gestation.

Endometrial receptivity for embryo implantation in the rat is a transient state occurring on day 5 of pregnancy or pseudopregnancy and is controlled by estrogen and progesterone. To identify genes potentially involved in receptivity, a uterine cDNA library was screened. An interesting pattern for Cystatin C (Cst3) expression was discovered with a peak in abundance just prior to embryo implantation (day 4 of pregnancy) followed by a significant drop the following day when implantation is initiated. Histology localized Cst3 mRNA and CST3 protein to the glandular epithelium on day 4 of pregnancy suggesting that it is secreted into the uterine lumen at this time. In ovariectomized rats endometrial Cst3 mRNA levels decreased within 3 h of treatment with estradiol; this effect was inhibited by the anti-estrogen, ICI 182, 780. The data suggest that the endometrial expression of the cysteine protease inhibitor, Cst3, is modulated by estrogen during the peri-implantation period. (c) 2006 Elsevier Inc. All rights reserved.

Cells of the trophoblast lineage make up the epithelial compartment of the placenta, and their rapid development is essential for the establishment and maintenance of pregnancy. A diverse array of specialized trophoblast subtypes form throughout gestation and are responsible for mediating implantation, as well as promotion of blood to the implantation site, changes in maternal physiology, and nutrient and gas exchange between the fetal and maternal blood supplies. Within the last decade, targeted mutations in mice and the study of trophoblast stem cells in vitro have contributed greatly to our understanding of trophoblast lineage development. Here, we review recent insights into the molecular pathways regulating trophoblast lineage segregation, stem cell maintenance, and subtype differentiation.

During implantation in rodents, attachment and invasion of embryonic trophoblast is accompanied by decidualization of the adjacent endometrial stroma. Decidualization can be initiated only when the endometrium is receptive, and this occurs for a short period in pregnancy. The molecular mechanisms underlying this phenomenon remain unclear. In the current study, using differential display and northern blot analysis, we found that steady-state levels of mRNA for vitamin D3upregulated protein 1 (Vdup1) were significantly higher in ‘refractory’ and ‘delayed’ endometrium compared with ‘receptive’ endometrium or endometrium undergoing artificially induced decidualization. Conversely, thioredoxin (Txn), a ubiquitously expressed cellular redox regulator known to promote growth and proliferation, was found to have elevated transcript levels within the decidualizing endometrium. VDUP1 has previously been shown to bind TXN and inhibit its action. In an inverse, but cooperative, relationship, these molecules have been implicated in regulating cell growth and proliferation in a number of tissues and during transformation to cancer. TheVdup1mRNA is localized to the uterine stroma in the nonreceptive endometrium, the site of increasedTxnmRNA levels during decidualization. In addition,Vdup1mRNA levels are inversely regulated by progesterone and estrogen; prolonged progesterone exposure stimulates an increase inVdup1mRNA levels whereas estrogen decreasesVdup1transcript levels. Together, these results suggest a novel mechanism by which suppression of the decidual response in the nonreceptive endometrium may occur.

The placenta is a highly specialized organ whose primary function is to promote the exchange of nutrients and oxygen between maternal and fetal blood, essential for survival and growth of the baby. The surface area for nutrient transport is a highly convoluted villous structure that forms by branching morphogenesis. In mice, this process begins after embryonic day 8.5, following attachment of allantoic mesoderm to the chorion, and continues through the end of gestation. Gene targeting studies in mice have identified a large number of genes that are essential for chorioallantoic development to give rise to the layer of the placenta called the labyrinth. Collectively, these studies reveal that a number of signaling pathways regulate four distinct phases of labyrinth development: chorioallantoic attachment (involving VCAM1 and its receptor alpha4 integrin, Bmp5/7, and Wnt7b, as well as the cochaperone Mrj), initiation of branching (involving the Gcm1 transcription factor to select sites of branch initiation), extension of villous branching (involving FGF, EGF, and HGF/Met signaling, through the Grb2/Sos1/Mek1/p38alpha MAPK pathway), followed by vascularization of the villous tree. The restricted expression and/or action of the signaling components indicate that a series of intercellular interactions regulate chorioallantoic development.

For successful implantation, the embryo must develop to the blastocyst stage and the endometrium must attain a state that is receptive to the implanting blastocyst. In rodents, the timing, duration, and hormonal regulation of this receptive state has been well defined. However, the molecular cascade of events involved in the onset of the receptive phase remains unclear. In the present study, we sought to identify genes involved in the onset of the receptivity using the technique of suppressive subtraction hybridization. Herein we report the isolation, cloning, and characterization of a novel gene, uterine sensitization-associated gene-1 (UASG-1), that is preferentially expressed within the maximally sensitized/receptive rat endometrium. USAG-1 mRNA encodes a putative protein of 206 amino acids that contains a possible N-terminal secretion signal and a C-terminal cystine knotlike motif. Northern blot analysis revealed that induction of USAG-1 mRNA was restricted to the Day 5 pregnant or pseudopregnant uterus. In situ hybridization experiments demonstrated that this induction was restricted to the uterine glandular epithelial cells. Given the remarkably tight restriction of its expression, USAG-1 may be involved in the onset of endometrial receptivity for implantation/sensitization for the decidual cell reaction.

Introduction Retinoic acid (RA) signaling is a well known regulator of trophoblast differentiation and placental development, and maternal decidual cells are recognized as the source of much of this RA. We explored possible trophoblast-derived sources of RA by examining the expression of RA synthesis enzymes in the developing mouse placenta, as well as addressed potential sites of RA action by examining the ontogeny of gene expression for other RA metabolizing and receptor genes. Furthermore, we investigated the effects of endogenous RA production on trophoblast differentiation. Methods Placental tissues were examined by in situ hybridization and assayed for RARE-LacZ transgene activity to locate sites of RAR signaling. Trophoblast stem cell cultures were differentiated in the presence of ALDH1 inhibitors (DEAB and citral), and expression of labyrinth ( Syna , Ctsq ) and junctional zone ( Tpbpa , Prl7b1 , Prl7a2 ) marker genes were analyzed by qRT-PCR. Results We show Aldh1a3 is strongly expressed in a subset of ectoplacental cone cells and in glycogen trophoblast cells of the definitive murine placenta. Most trophoblast subtypes of the placenta express RA receptor combinations that would enable them to respond to RA signaling. Furthermore, expression of junctional zone markers decrease in differentiating trophoblast cultures when endogenous ALDH1 enzymes are inhibited. Discussion Aldh1a3 is a novel marker for glycogen trophoblast cells and their precursors and may play a role in the differentiation of junctional zone cell types via production of a local source of RA.

Introduction The renin angiotensin system (RAS) is an important mediator of placental development. However, a comprehensive expression profile for 8 key components of the placental RAS throughout murine gestation has not been performed. Furthermore, maternal hypoxia induces dysregulation of RAS expression in fetal tissues but the effects on the murine placental RAS are less well known. Methods Placentas were collected from male and female CD1 mouse fetuses at seven gestational ages for qPCR analysis of Agt , Ren1 , Atp6ap2 , Ace , Ace2 , Agtr1a , Agtr2 and Mas1 . mRNA localisation of Agtr1 and Mas1 and protein localisation of ACE and ACE2 was determined at E18.5. To determine the effects of maternal hypoxia on the placental RAS, mice were housed in 12% oxygen from E14.5–E18.5 and placentas examined at E18.5. Results All RAS genes were expressed in the placenta throughout pregnancy and expression varied with fetal sex and age. Agtr1 was expressed within the labyrinth while Mas1 was expressed within the intraplacental yolk sac. ACE and ACE2 were localised to both labyrinth and junctional zones. In response to maternal hypoxia the expression of Agt , Ace and Ace2 was decreased but expression of Agtr1a was increased. Ace and Agtr1a mRNA levels were affected to a greater extent in females compared to males. Discussion Collectively, the location within the placenta as well as the expression profiles identified, support a role for the placental RAS in labyrinth development. The placental RAS is disturbed by maternal hypoxia in a sexually dimorphic manner and may contribute to impairment of placental vascular development.

The chorioallantoic placenta mediates the bidirectional exchange of nutrients, wastes, and gases between mother and baby, performs essential endocrine activities, and participates in the modulation of the maternal immune and vascular systems. It is therefore not surprising that perturbations in placental formation or function may lead to a myriad of pregnancy complications, endangering maternal health and fetal development. The following chapter provides an overview of the structure and postimplantation morphogenesis of the definitive murine chorioallantoic placenta, including consideration of cell types and lineage. Furthermore, contemporary reagents and strategies for the analyses of placental phenotypes are highlighted.

Nonradioactive in situ hybridization is a simple technique with a large number of steps that can be customized to the varying needs of individual researchers. It permits the localization of one mRNA or simultaneously two mRNAs in tissue sections from the peri-implantation uterus and developing placenta. By varying the detection methods, use of the protocols presented here results in either colorimetric or fluorescent localization of the mRNA(s) of interest, yielding useful data concerning the precise localization of gene expression at the maternal–conceptus interface.

This chapter outlines the morphology of the mouse placenta during development and identifies characteristic features that can be observed on histological cross-sections.

Endometrial receptivity for implantation and sensitization for decidualization in rodents is a transient state under the control of the ovarian steroids estrogen and progesterone. It is unclear, however, what molecular events mediate the onset of uterine receptivity. Messenger RNA differential display was performed on endometrial RNA from ovariectomized rats differentially sensitized for decidualization. Maximally sensitized uteri were at the equivalent of Day 5 of pseudopregnancy, and temporally nonsensitized uteri at Day 4 or 6; hormonally nonsensitized uteri were from animals on Day 5 treated with low or high doses of estradiol on Day 4. A cDNA with endometrial expression restricted to maximally sensitized uteri was isolated, cloned, and sequenced. The cDNA matched the sequence for glucose-regulated protein 78 (GRP78), a heat shock 70-related protein that resides in the lumen of the endoplasmic reticulum (ER) and has roles in several cellular processes including multimeric protein assembly, the degradation of proteins, and the storage and regulation of ER luminal calcium. Northern blot analysis indicated a dramatic increase in GRP78 mRNA levels restricted to the sensitized, Day 5 endometrium, suggesting a role in the onset of the sensitized phase. In situ hybridization and immunohistochemistry experiments localized the up-regulation of GRP78 within the receptive endometrium to the glandular epithelium.

The complement system is involved in a range of diverse developmental processes, including cell survival, growth, differentiation, and regeneration. However, little is known about the role of complement in embryogenesis. In this study, we demonstrate a novel role for the canonical complement 5a receptor (C5aR) in the development of the mammalian neural tube under conditions of maternal dietary folic acid deficiency. Specifically, we found C5aR and C5 to be expressed throughout the period of neurulation in wild-type mice and localized the expression to the cephalic regions of the developing neural tube. C5aR was also found to be expressed in the neuroepithelium of early human embryos. Ablation of the C5ar1 gene or the administration of a specific C5aR peptide antagonist to folic acid–deficient pregnant mice resulted in a high prevalence of severe anterior neural tube defect-associated congenital malformations. These findings provide a new and compelling insight into the role of the complement system during mammalian embryonic development.

Hand1 is a basic helix-loop-helix transcription factor that is essential for development of the placenta, yolk sac and heart during mouse development. While Hand1 is essential for trophoblast giant cell (TGC) differentiation, its potential heterodimer partners are not co-expressed in TGCs. To test the hypothesis that Hand1 functions as homodimer, we generated knock-in mice in which the Hand1 gene was altered to encode a tethered homodimer (TH). Some Hand1(TH/-) conceptuses in which the only form of Hand1 is Hand1(TH) are viable and fertile, indicating that homodimer Hand1 is sufficient for mouse survival. ~2/3 of Hand1(TH/-) and all Hand1(TH/TH) mice died in utero and displayed severe placental defects and variable cardial and cranial-facial abnormalities, indicating a dosage-dependent effect of Hand1(TH). Meanwhile, expression of the Hand1(TH) protein did not have negative effects on viability or fertility in all Hand1(TH/+) mice. These data imply that Hand1 homodimer plays a dominant role during development and its expression dosage is critical for survival, whereas Hand1 heterodimers can be either dispensable or play a regulatory role to modulate the activity of Hand1 homodimer in vivo.

The human solute linked carrier (SLC) 13A4 gene is primarily expressed in the placenta where it is proposed to mediate the transport of nutrient sulfate from mother to fetus. The molecular mechanisms involved in the regulation of SLC13A4 expression remain unknown. To investigate the regulation of SLC13A4 gene expression, we analysed the transcriptional activity of the human SLC13A4 5'-flanking region in the JEG-3 placental cell line using luciferase reporter assays. Basal transcriptional activity was identified in the region -57 to -192 nucleotides upstream of the SLC13A4 transcription initiation site. Mutational analysis of the minimal promoter region identified Nuclear factor Y (NFY), Specificity protein 1 (SP1) and Kruppel like factor 7 (KLF7) motifs which conferred positive transcriptional activity, as well as Zinc finger protein of the cerebellum 2 (ZIC2) and helix-loop-helix protein 1 (HEN1) motifs that repressed transcription. The conserved NFY, SP1, KLF7, ZIC2 and HEN1 motifs in the SLC13A4 promoter of placental species but not in non-placental species, suggests a potential role for these putative transcriptional factor binding motifs in the physiological control of SLC13A4 mRNA expression.

The age-associated decline in growth hormone (GH) secretion may be a consequence of the reduction in the number of GH-releasing hormone (GHRH) positive neurones. However, it remains unclear whether an alteration in the number or distribution of somatostatin (SST) neurones contributes to this change. In the present study, we characterised the role of SST in modulating the change in pulsatile GH secretion in male C57Bl/6J mice throughout puberty and into early adulthood. We assessed pulsatile GH secretion in mice at 4, 8 and 16 weeks of age. These ages correspond to early pubertal, early adulthood and adulthood, respectively. We show an elevation in peak, total and pulsatile GH secretion coinciding with periods of rapid linear growth. Using in situ hybridisation and morphometric methods, we mapped the distribution of Sst mRNA expression within the mouse brain relative to this change in pulsatile GH secretion. The results obtained show that altered pulsatile GH secretion in male mice from 4-16 weeks of age does not coincide with a significant change in the number of Sst mRNA expressing neurones or an abundance of Sst mRNA expression throughout the arcuate nucleus (ARC) and periventricular nucleus (PeV). Rather, we observed a progressive decline in Sst mRNA expressing neurones within subnuclei of the paraventricular nucleus at this time. We conclude that structural changes in Sst expression within the PeV and ARC may not reflect the observed decline in pulsatile GH secretion in mice from puberty into early adulthood.

G protein-coupled receptors (GPCRs) are critical for cardiovascular physiology. Cardiac cells express >100 nonchemosensory GPCRs, indicating that important physiological and potential therapeutic targets remain to be discovered. Moreover, there is a growing appreciation that members of the large, distinct taste and odorant GPCR families have specific functions in tissues beyond the oronasal cavity, including in the brain, gastrointestinal tract and respiratory system. To date, these chemosensory GPCRs have not been systematically studied in the heart. We performed RT-qPCR taste receptor screens in rodent and human heart tissues that revealed discrete subsets of type 2 taste receptors (TAS2/Tas2) as well as Tas1r1 and Tas1r3 (comprising the umami receptor) are expressed. These taste GPCRs are present in cultured cardiac myocytes and fibroblasts, and by in situ hybridization can be visualized across the myocardium in isolated cardiac cells. Tas1r1 gene-targeted mice (Tas1r1(Cre)/Rosa26(tdRFP)) strikingly recapitulated these data. In vivo taste receptor expression levels were developmentally regulated in the postnatal period. Intriguingly, several Tas2rs were upregulated in cultured rat myocytes and in mouse heart in vivo following starvation. The discovery of taste GPCRs in the heart opens an exciting new field of cardiac research. We predict that these taste receptors may function as nutrient sensors in the heart.

Placenta formation during pregnancy requires chorioallantoic branching morphogenesis that involves establishing an amplifying feedback loop between Frizzled5 and Gcm1 to regulate branching initiation and trophoblast differentiation., Chorioallantoic branching morphogenesis is a key milestone during placental development, creating the large surface area for nutrient and gas exchange, and is therefore critical for the success of term pregnancy. Several Wnt pathway molecules have been shown to regulate placental development. However, it remains largely unknown how Wnt-Frizzled (Fzd) signaling spatiotemporally interacts with other essential regulators, ensuring chorionic branching morphogenesis and angiogenesis during placental development. Employing global and trophoblast-specific Fzd5-null and Gcm1-deficient mouse models, combining trophoblast stem cell lines and tetraploid aggregation assay, we demonstrate here that an amplifying signaling loop between Gcm1 and Fzd5 is essential for normal initiation of branching in the chorionic plate. While Gcm1 upregulates Fzd5 specifically at sites where branching initiates in the basal chorion, this elevated Fzd5 expression via nuclear β-catenin signaling in turn maintains expression of Gcm1. Moreover, we show that Fzd5-mediated signaling induces the disassociation of cell junctions for branching initiation via downregulating ZO-1, claudin 4, and claudin 7 expressions in trophoblast cells at the base of the chorion. In addition, Fzd5-mediated signaling is also important for upregulation of Vegf expression in chorion trophoblast cells. Finally, we demonstrate that Fzd5-Gcm1 signaling cascade is operative during human trophoblast differentiation. These data indicate that Gcm1 and Fzd5 function in an evolutionary conserved positive feedback loop that regulates trophoblast differentiation and sites of chorionic branching morphogenesis., Author Summary Abnormal placental development during pregnancy is associated with conditions such as preeclampsia, intrauterine growth restriction, and even fetal death in humans. Here we focus on the earliest steps of placenta formation, which involves the development of the labyrinthine layer, a specialized epithelium that sits between the maternal blood and fetal blood vessels and facilitates the exchange of nutrients, gases, and wastes between the mother and fetus. Pivotal to the development of a functional labyrinth layer are the processes of folding and branching of a flat sheet of trophoblast cells (originally the outer layer of the blastocyst), and of trophoblast cell differentiation. Here, we show in mice that Frizzled5, a receptor component of the Wnt signaling pathway, and Gcm1, an important transcription factor for labyrinth development, form a positive feedback loop that directs normal placental development. We find that Gcm1 up-regulates Fzd5 specifically at branching sites and that elevated Fzd5 expression in turn maintains expression of Gcm1. Moreover, Fzd5-mediated signaling is required for the disassociation of cell junctions and for the up-regulation of Vegf expression in trophoblast cells. Finally, with implications for human disease, we demonstrate that the FZD5-GCM1 signaling cascade operates in primary cultures of human trophoblasts undergoing differentiation.

Maternal exposure to increased synthetic glucocorticoids (GC) during pregnancy is known to disturb fetal development and increase the risk of long-term disease. Maternal exposure to elevated levels of natural GC is likely to be common yet is relatively understudied. The placenta plays an important role in regulating fetal exposure to maternal GC but is itself vulnerable to maternal insults. This study uses a mouse model of maternal corticosterone (Cort) exposure to investigate its effects on the developing placenta. Mice were treated with Cort (33 μg/kg·h) for 60 h starting at embryonic d 12.5 (E12.5) before collection of placentas at E14.5 and E17.5. Although Cort exposure did not affect fetal size, placentas of male fetuses were larger at E17.5 in association with changes in placental Igf2. This increase in size was associated with an increase in placental thickness and an increase in placental junctional zone volume. Placentas from female fetuses were of normal size and had no changes in growth factor mRNA levels. The expression of the protective enzyme 11β-hydroxysteroid dehydrogenase type 2 was increased at E14.5 but was decreased in males at E17.5. In contrast, the expression of Nr3c1 (which encodes the GC receptor) was increased during the Cort exposure and remained elevated at E17.5 in the placentas of male fetuses. Our study has shown that maternal Cort exposure infers a sex-specific alteration to normal placental growth and growth factor expression, thus further adding to our understanding of the mechanisms of male dominance of programmed disease.

Sulfate is important for mammalian growth and development. During pregnancy, maternal circulating sulfate levels increase by 2-fold, enhancing sulfate availability to the fetus. We used quantitative real-time PCR to determine sulfate transporter mRNA levels during mouse gestation in three tissues: kidney and ileum, to identify transporters involved in sulfate absorption and maintaining high maternal circulating sulfate level; and placenta, to build a model of directional sulfate transport from mother to fetus. In the kidney, Slc13a1 and Slc26a1 were the most abundant sulfate transporter mRNAs, which increased by ’2fold at E4.5 or E6.5, whereas lower levels of Slc26a2, Slc26a6, and Slc26a7 mRNA increased by ’3- to 6-fold from E4.5. Ileal sulfate transporter mRNA levels were not increased in gestation, but slight decreases (by ’30–40%) were found for Slc26a3 and Slc26a6. In placentae, Slc13a4 and Slc26a2 mRNAs were most abundant, with levels increasing from E10.5 and peaking (’8fold) from E14.5 to E18.5, whereas Slc26a1 increased by ’3-fold at E18.5. The spatial expression of placental mRNAs was determined by in situ hybridization showing Slc13a4 and Slc26a6 in yolk sac, Slc26a1 in spongiotrophoblasts, and Slc13a4, Slc26a2, Slc26a3, and Slc26a7 in the labyrinthine layer. Within the labyrinth, cell-specific staining revealed Slc13a4 expression in syncytiotrophoblast-II (SynT-II) and Slc26a2 in SynT-I. Together, these data show kidney Slc13a1 and Slc26a1 and placental Slc13a4 and Slc26a2 to be the most abundant sulfate transporter mRNAs in mouse gestation, which likely play important physiological roles in maintaining high maternal serum sulfate levels during pregnancy and mediating sulfate supply to the fetus. placental transport, pregnancy, sulfate transporter

Early placental development in mice involves patterning of the chorion into distinct layers, though little is understood regarding the interactions that regulate its organization. Here we demonstrate that keratin aggregates found in Mrj(-/-) chorionic trophoblast cells are associated with abnormal cell morphology, collapse of the actin cytoskeleton, E-cadherin and β-catenin misexpression and extracellular matrix (ECM) disorganization. Accordingly, Mrj(-/-) trophoblast cells in vitro are nonadherent and display erratic migratory behavior. These cells also fail to differentiate into syncytiotrophoblast cells since Rhox4b expression, a marker of syncytiotrophoblast progenitors, was maintained and Gcm1, Synb, and Syna expression failed to increase. This differentiation defect was not solely attributable to E-cadherin misexpression or ECM disorganization. However, plating Mrj-deficient cells on exogenous laminin-511 normalized their cell behavior. Lastly, we show that Mrj(-/-) chorions at embryonic day 8.5 have expanded Rhox4b expression domains and do not form normal layers of gene expression suggesting that chorion patterning requires Mrj.

Normal placental development and function is essential for fetal growth of eutherian mammals. Mutational studies have shown that numerous growth factors are required for placental development and differentiation of placental lineages. Here, using a gene-trap mutant mouse line, Crim1(KST264), we show that Crim1 is essential for murine placental development. Crim1 is a developmentally expressed, trans-membrane regulator of growth factor activity. Crim1(KST264/KST264) mutant placentae displayed hypoplasia from 13.5 dpc, and altered structure from 15.5 dpc, including alterations in cell number in both the junctional and labyrinth zones. Using the reporter gene from the Crim1(KST264) allele, we found that Crim1 is expressed in multiple cell types of the placenta, including strong expression in the spongiotrophoblast cells of the junctional zone. In the junctional zone of Crim1(KST264/KST264) placentae, there was an increase in the glycogen trophoblast cells adjacent to the spongiotrophoblast cells. In the labyrinth zone, we found a decrease in the density of sinusoidal-trophoblast giant cells. Our findings show that Crim1 is required for placental development, and is necessary for the proper differentiation of sinusoidal-trophoblast giant cells and glycogen trophoblast cells.

Objectives Maternal glucocorticoid (GC) exposure during pregnancy can alter fetal development and program the onset of disease in adult offspring. The placenta helps protect the fetus from excess GC exposure but is itself susceptible to maternal insults and may be involved in sex dependant regulation of fetal programming. This study aimed to investigate the effects of maternal GC exposure on the developing placenta. Study design and main outcome measures Pregnant mice were treated with dexamethasone (DEX-1 μg/kg/h) or saline (SAL) for 60 h via minipump beginning at E12.5. Placentas were collected at E14.5 and E17.5 and the expression of growth factors and placental transporters examined by real-time PCR and/or Western blot. Histological analysis was performed to assess for morphological changes. Results At E14.5, DEX exposed male and female fetuses had a lower weight compared to SAL animals but placental weight was lower in females only. Hsd11b2 and Vegfa gene expression was increased and MAPK1 protein expression decreased in the placentas of females only. At E17.5 placental and fetal body weights were similar and differences in MAPK were no longer present although HSD11B2 protein was elevated in placentas of DEX females. Levels of glucose or amino acid transporters were unaffected. Conclusions Results suggest sex specific responses to maternal GCs within the placenta. Decreased levels of MAPK protein in placentas of female fetuses suggest alterations in the MAPK pathway may contribute to the lower placental weights in this sex. This may contribute towards sex specific fetal programming of adult disease.

The mammalian placenta represents the interface between maternal and embryonic tissues and provides nutrients and gas exchange during embryo growth. Recently, keratin intermediate filament proteins were found to regulate embryo growth upstream of the mammalian target of rapamycin pathway through glucose transporter relocalization and to contribute to yolk sac vasculogenesis through altered bone morphogenetic protein 4 signaling. Whether keratins have vital functions in extraembryonic tissues is not well understood. Here, we report that keratins are essential for placental function. In the absence of keratins, we find hyperoxia in the decidual tissue directly adjacent to the placenta, because of an increased maternal vasculature. Hyperoxia causes impaired vasculogenesis through defective hypoxia-inducible factor 1α and vascular endothelial growth factor signaling, resulting in invagination defects of fetal blood vessels into the chorion. In turn, the reduced labyrinth, together with impaired gas exchange between maternal and embryonic blood, led to increased hypoxia in keratin-deficient embryos. We provide evidence that keratin-positive trophoblast secretion of prolactin-like protein a (Prlpa) and placental growth factor (PlGF) during decidualization are altered in the absence of keratins, leading to increased infiltration of uterine natural killer cells into placental vicinity and increased vascularization of the maternal decidua. Our findings suggest that keratin mutations might mediate conditions leading to early pregnancy loss due to hyperoxia in the decidua.