Defects in placental syncytiotrophoblast cells are a common cause of developmental heart disease.

Placental abnormalities have been sporadically implicated as a source of developmental heart defects. Yet it remains unknown how often the placenta is at the root of congenital heart defects (CHDs), and what the cellular mechanisms are that underpin this connection. Here, we selected three mouse mutant lines, Atp11a, Smg9 and Ssr2, that presented with placental and heart defects in a recent phenotyping screen, resulting in embryonic lethality. To dissect phenotype causality, we generated embryo- and trophoblast-specific conditional knockouts for each of these lines. This was facilitated by the establishment of a new transgenic mouse, Sox2-Flp, that enables the efficient generation of trophoblast-specific conditional knockouts. We demonstrate a strictly trophoblast-driven cause of the CHD and embryonic lethality in one of the three lines (Atp11a) and a significant contribution of the placenta to the embryonic phenotypes in another line (Smg9). Importantly, our data reveal defects in the maternal blood-facing syncytiotrophoblast layer as a shared pathology in placentally induced CHD models. This study highlights the placenta as a significant source of developmental heart disorders, insights that will transform our understanding of the vast number of unexplained congenital heart defects.

Salt sensitivity of volume and blood pressure in a mouse with globally reduced ENaC γ-subunit expression.

The epithelial Na+ channel (ENaC) promotes the absorption of Na+ in the aldosterone-sensitive distal nephron, colon, and respiratory epithelia. Deletion of genes encoding subunits of ENaC results in early postnatal mortality. Here, we present the initial characterization of a mouse with dramatically suppressed expression of the ENaC γ-subunit. We used this hypomorphic (γmt) allele to explore the importance of this subunit in homeostasis of electrolytes and body fluid volume. At baseline, γ-subunit expression in γmt/mt mice was markedly suppressed in the kidney and lung, whereas electrolytes resembled those of littermate controls. Aldosterone levels in γmt/mt mice exceeded those seen in littermate controls. Quantitative magnetic resonance measurement of body composition revealed similar baseline body water, lean tissue mass, and fat tissue mass in γmt/mt mice and controls. γmt/mt mice exhibited a more rapid decline in body water and lean tissue mass in response to a low-Na+ diet than the controls. Replacement of drinking water with 2% saline selectively and transiently increased body water and lean tissue mass in γmt/mt mice relative to the controls. Lower blood pressures were variably observed in γmt/mt mice on a high-salt diet compared with the controls. γmt/mt also exhibited reduced diurnal blood pressure variation, a "nondipping" phenotype, on a high-Na+ diet. Although ENaC in the renal tubules and colon works to prevent extracellular fluid volume depletion, our observations suggest that ENaC in other tissues may participate in regulating extracellular fluid volume and blood pressure.NEW & NOTEWORTHY A mouse with globally suppressed expression of the epithelial Na+ channel γ-subunit showed enhanced sensitivity to dietary salt, including a transient increase in total body fluid, reduced blood pressure, and reduced diurnal blood pressure variation when given a dietary NaCl challenge. These results point to a role for the epithelial Na+ channel in regulating body fluid and blood pressure beyond classical transepithelial Na+ transport mechanisms.

Advancing human health in the decade ahead: pregnancy as a key window for discovery: A Burroughs Wellcome Fund Pregnancy Think Tank.

Recent revolutionary advances at the intersection of medicine, omics, data sciences, computing, epidemiology, and related technologies inspire us to ponder their impact on health. Their potential impact is particularly germane to the biology of pregnancy and perinatal medicine, where limited improvement in health outcomes for women and children has remained a global challenge. We assembled a group of experts to establish a Pregnancy Think Tank to discuss a broad spectrum of major gestational disorders and adverse pregnancy outcomes that affect maternal-infant lifelong health and should serve as targets for leveraging the many recent advances. This report reflects avenues for future effects that hold great potential in 3 major areas: developmental genomics, including the application of methodologies designed to bridge genotypes, physiology, and diseases, addressing vexing questions in early human development; gestational physiology, from immune tolerance to growth and the timing of parturition; and personalized and population medicine, focusing on amalgamating health record data and deep phenotypes to create broad knowledge that can be integrated into healthcare systems and drive discovery to address pregnancy-related disease and promote general health. We propose a series of questions reflecting development, systems biology, diseases, clinical approaches and tools, and population health, and a call for scientific action. Clearly, transdisciplinary science must advance and accelerate to address adverse pregnancy outcomes. Disciplines not traditionally involved in the reproductive sciences, such as computer science, engineering, mathematics, and pharmacology, should be engaged at the study design phase to optimize the information gathered and to identify and further evaluate potentially actionable therapeutic targets. Information sources should include noninvasive personalized sensors and monitors, alongside instructive "liquid biopsies" for noninvasive pregnancy assessment. Future research should also address the diversity of human cohorts in terms of geography, racial and ethnic distributions, and social and health disparities. Modern technologies, for both data-gathering and data-analyzing, make this possible at a scale that was previously unachievable. Finally, the psychosocial and economic environment in which pregnancy takes place must be considered to promote the health and wellness of communities worldwide.

Correction to: Phases and Mechanisms of Embryonic Cardiomyocyte Proliferation and Ventricular Wall Morphogenesis.

The original version of this article unfortunately contained a mistake. In reviewing the phenotype associated with Mapk14 (p38alpha MAPK) mutation as evaluated by Adams et al. (2000) using tetraploid aggregation chimeric embryos, the authors mistakenly stated that rescue of embryo lethality was short-lived and that embryos died two days later of non-placenta-related causes. In fact, as reported by Adams et al. (2000), when the placental defect of global null embryos was rescued, p38alpha(-/-) embryos developed to term and were normal in appearance. The authors apologize for the error.

Klf14 is an imprinted transcription factor that regulates placental growth.

INTRODUCTION: Imprinted genes are preferentially expressed from one parentally inherited allele, and many are crucial to the regulation of placental function and fetal growth. Murine Krüppel-like factor 14 (Klf14) is a maternally expressed imprinted transcription factor that is a component of the Mest imprinted gene cluster on mouse chromosome 6. We sought to determine if loss of Klf14 expression alters the course of normal mouse extraembryonic development. We also used high-throughput RNA sequencing (RNAseq) to identify a set of differentially expressed genes (DEGs) in placentas with loss of Klf14. METHODS: We generated a Klf14 knockout (Klf14null) mouse using recombineering and transgenic approaches. To identify DEGs in the mouse placenta we compared mRNA transcriptomes derived from 17.5dpc Klf14matKO and wild-type littermate placentas by RNAseq. Candidate DEGs were confirmed with quantitative reverse transcription PCR (qPCR) on an independent cohort of male and female gestational age matched Klf14matKO placentas. RESULTS: We found that 17.5dpc placentas inheriting a maternal null allele (Klf14matKO) had a modest overgrowth phenotype and a near complete ablation of Klf14 expression. However, there was no effect on fetal growth. We identified 20 DEGs differentially expressed in Klf14matKO placentas by RNAseq, and subsequently validated five that are highly upregulated (Begain, Col26a1, Fbln5, Gdf10, and Nell1) by qPCR. The most enriched functional gene-networks included those classified as regulating cellular development and metabolism. CONCLUSION: These results suggest that loss of the maternal Klf14 locus in the mouse placenta acts results in changes in gene expression patterns that modulate placental growth.

Phases and Mechanisms of Embryonic Cardiomyocyte Proliferation and Ventricular Wall Morphogenesis.

If viewed as a movie, heart morphogenesis appears to unfold in a continuous and seamless manner. At the mechanistic level, however, a series of discreet and separable processes sequentially underlie heart development. This is evident in examining the expansion of the ventricular wall, which accounts for most of the contractile force of each heartbeat. Ventricular wall expansion is driven by cardiomyocyte proliferation coupled with a morphogenetic program that causes wall thickening rather than lengthening. Although most studies of these processes have focused on heart-intrinsic processes, it is increasingly clear that extracardiac events influence or even direct heart morphogenesis. In this review, we specifically consider mechanisms responsible for coordinating cardiomyocyte proliferation and ventricular wall expansion in mammalian development, relying primarily on studies from mouse development where a wealth of molecular and genetic data have been accumulated.

PLIN2 Is Essential for Trophoblastic Lipid Droplet Accumulation and Cell Survival During Hypoxia.

Trophoblast hypoxia and injury, key components of placental dysfunction, are associated with fetal growth restriction and other complications of pregnancy. Accumulation of lipid droplets has been found in hypoxic nonplacental cells. Unique to pregnancy, lipid accumulation in the placenta might perturb lipid transport to the fetus. We tested the hypothesis that hypoxia leads to accumulation of lipid droplets in human trophoblasts and that trophoblastic PLIN proteins play a key role in this process. We found that hypoxia promotes the accumulation of lipid droplets in primary human trophoblasts. A similar accretion of lipid droplets was found in placental villi in vivo from pregnancies complicated by fetal growth restriction. In both situations, these changes were associated with an increased level of cellular triglycerides. Exposure of trophoblasts to hypoxia led to reduced fatty acid efflux and oxidation with no change in fatty acid uptake or synthesis. We further found that hypoxia markedly stimulated PLIN2 mRNA synthesis and protein expression, which colocalized to lipid droplets. Knockdown of PLIN2, but not PLIN3, enhanced trophoblast apoptotic death, and overexpression of PLIN2 promoted cell viability. Collectively, our data indicate that hypoxia enhances trophoblastic lipid retention in the form of lipid droplets and that PLIN2 plays a key role in this process and in trophoblast defense against apoptotic death. These findings also imply that this protective mechanism may lead to diminished trafficking of lipids to the developing fetus.

Ligand-Dependent Corepressor (LCoR) Is a Rexinoid-Inhibited Peroxisome Proliferator-Activated Receptor γ-Retinoid X Receptor α Coactivator.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is an essential regulator of placental development. To gain deeper insights into placental PPARγ signaling, we dissected its regulation of the Muc1 promoter. We find that, unlike prototypic target activation by heterodimeric receptors, which is either stimulated by or refractory to retinoid X receptor (RXR) ligands (rexinoids), the induction of Muc1 by liganded PPARγ requires RXRα but is inhibited by rexinoids. We demonstrate that this inhibition is mediated by the activation function 2 (AF2) domain of RXRα and that Muc1 activation entails altered AF2 structures of both PPARγ and RXRα. This unique regulation of Muc1 reflects specific coactivation of PPARγ-RXRα heterodimers by the transcription cofactor ligand-dependent corepressor (LCoR), corroborated by significant downregulation of Muc1 in Lcor-null placentas. LCoR interacts with PPARγ and RXRα in a synergistic fashion via adjacent noncanonical protein motifs, and the AF2 domain of ligand-bound RXRα inhibits this interaction. We further identify the transcription factor Krüppel-like factor 6 (KLF6) as a critical regulator of placental development and a component of Muc1 regulation in cooperation with PPARγ, RXRα, and LCoR. Combined, these studies reveal new principles and players in nuclear receptor function in general and placental PPARγ signaling in particular.

Deep intronic mis-splicing mutation in JAK3 gene underlies T-B+NK- severe combined immunodeficiency phenotype.

Severe combined immune deficiency (SCID) is a group of genetically heterogeneous diseases caused by an early block in T cell differentiation and present with life threatening infections, often within the first year of life. Janus kinase (JAK)3 gene mutations have been found to cause autosomal recessive T-B+ SCID phenotype. In this study we describe three patients with a novel deep intronic mis-splicing mutation in JAK3 as a cause of T-B+NK- SCID highlighting the need for careful evaluation of intronic regulatory elements of known genes associated with clearly defined clinical phenotypes. We present the cases and discuss the current literature.

Esrrb-Cre excises loxP-flanked alleles in early four-cell embryos.

Among transgenic mice with ubiquitous Cre recombinase activity, all strains to date excise loxP-flanked (floxed) alleles either at or before the zygote stage or at nondescript stages of development. This manuscript describes a new mouse strain, in which Cre recombinase, integrated into the Esrrb locus, efficiently excises floxed alleles in pre-implantation embryos at the onset of the four-cell stage. By enabling inactivation of genes only after the embryo has undergone two cleavages, this strain should facilitate in vivo studies of genes with essential gametic or zygotic functions. In addition, this study describes a new, highly pluripotent hybrid C57BL/6J x 129S1/SvImJ mouse embryonic stem cell line, HYB12, in which this knockin and additional targeted alleles have been generated.

NDRG1 deficiency attenuates fetal growth and the intrauterine response to hypoxic injury.

Intrauterine mammalian development depends on the preservation of placental function. The expression of the protein N-myc downstream-regulated gene 1 (NDRG1) is increased in placentas of human pregnancies affected by fetal growth restriction and in hypoxic primary human trophoblasts, where NDRG1 attenuates cell injury. We sought to assess the function of placental NDRG1 in vivo and tested the hypothesis that NDRG1 deficiency in the mouse embryo impairs placental function and consequently intrauterine growth. We found that Ndrg1 knock-out embryos were growth restricted in comparison to wild-type or heterozygous counterparts. Furthermore, hypoxia reduced the survival of female, but not male, knock-out embryos. Ndrg1 deletion caused significant alterations in placental gene expression, with a marked reduction in transcription of several lipoproteins in the placental labyrinth. These transcriptional changes were associated with reduced fetal:maternal serum cholesterol ratio exclusively in hypoxic female embryos. Collectively, our findings indicate that NDRG1 promotes fetal growth and regulates the metabolic response to intrauterine hypoxic injury in a sexually dichotomous manner.

Cell-specific PPARγ deficiency establishes anti-inflammatory and anti-fibrogenic properties for this nuclear receptor in non-parenchymal liver cells.

BACKGROUND & AIMS: PPARγ plays an essential role in the transcriptional regulation of genes involved in lipid and glucose metabolism, insulin sensitivity, and inflammation. We recently demonstrated that PPARγ plays a causative role in hepatocyte lipid deposition, contributing to the pathogenesis of hepatic steatosis. In this study, we investigated the role of PPARγ in the inflammatory and fibrogenic response of the liver. METHODS: Heterozygous floxed/null Cre/LoxP mice with targeted deletion of PPARγ in either hepatocytes (Alb-Cre), macrophages (LysM-Cre) or hepatic stellate cells (HSCs) (aP2-Cre) were submitted to carbon tetrachloride (CCl4) liver injury. Further analyses were performed in precision-cut liver slices (PCLS) and primary cultures of hepatocytes, macrophages, and HSCs. RESULTS: LysM-Cre mice displayed an exacerbated response to chronic CCl4 injury and showed higher necroinflammatory injury, lipid peroxidation, inflammatory infiltrate, cleaved-caspase-3 and caspase 3/7 activity, and COX-2, TNF-α, CXCL2, and IL-1ß expression than Alb-Cre and control mice. The deleterious effects of PPARγ disruption in liver macrophages were confirmed in an acute model of CCl4 injury as well as in PCLS incubated with LPS. Moreover, LysM-Cre mice showed an aggravated fibrogenic response to CCl4, as revealed by more prominent Sirius Red and Masson's trichrome staining, elevated hydroxyproline content and induced α-SMA and TIMP-1 expression. Importantly, aP2-Cre mice with specific disruption of PPARγ in HSCs, as confirmed by immunocytochemical analysis of individual liver cells, also showed exacerbated liver damage and fibrogenic response to CCl4. CONCLUSIONS: These data unveil anti-inflammatory and anti-fibrogenic roles for PPARγ in non-parenchymal liver cells.

Placental PPARγ regulates spatiotemporally diverse genes and a unique metabolic network.

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is essential for placental development. For insights into its functions in the placenta, we screened for PPARγ-regulated genes by integrating expression profiles of Pparg-null and Rxra-null placentas with those of WT and Pparg-null trophoblast stem cells differentiated in the presence or absence of a PPARγ agonist. Intersection of these paradigms identified high-probability PPARγ target genes. A few of these genes were previously reported as PPARγ targets in other tissues, but most are new in the context of either PPARγ or placental biology. Transcriptional profiling demonstrated a widespread role for the coactivator NCOA6/AIB3, but not MED1/PBP, in PPARγ-dependent placental gene expression. Spatial and temporal expression analyses revealed that PPARγ impacts genes in diverse trophoblast lineages and during different stages of differentiation. We further validated the Ldhb gene, which encodes the H isoform of lactate dehydrogenase, as a robust PPARγ target in trophoblasts, and propose a hypothetical model that integrates it with a network of PPARγ-regulated genes into a novel pathway of placental fuel metabolism. These findings offer insights not only into the placental functions of PPARγ, but also into unique, previously unsuspected biosynthetic functions of trophoblasts.

Concise review: adipocyte origins: weighing the possibilities.

Adipose tissue is the primary energy reservoir in the body and an important endocrine organ that plays roles in energy homeostasis, feeding, insulin sensitivity, and inflammation. While it was tacitly assumed that fat in different anatomical locations had a common origin and homogenous function, it is now clear that regional differences exist in adipose tissue characteristics and function. This is exemplified by the link between increased deep abdominal or visceral fat, but not peripheral adipose tissue and the metabolic disturbances associated with obesity. Regional differences in fat function are due in large part to distinct adipocyte populations that comprise the different fat depots. Evidence accrued primarily in the last decade indicates that the distinct adipocyte populations are generated by a number of processes during and after development. These include the production of adipocytes from different germ cell layers, the formation of distinct preadipocyte populations from mesenchymal progenitors of mesodermal origin, and the production of adipocytes from hematopoietic stem cells from the bone marrow. This review will examine each of these process and their relevance to normal adipose tissue formation and contribution to obesity-related diseases.

Role for PPARγ in obesity-induced hepatic steatosis as determined by hepatocyte- and macrophage-specific conditional knockouts.

Peroxisome proliferator-activated receptor (PPAR) γ is a nuclear receptor central to glucose and lipid homeostasis. PPARγ role in nonalcoholic fatty liver disease is controversial because PPARγ overexpression is a general property of steatotic livers, but its activation by thiazolidinediones reduces hepatic steatosis. Here, we investigated hepatic PPARγ function by using Cre-loxP technology to generate hepatocyte (PPARγ(Δhep))- and macrophage (PPARγ(Δmac))-specific PPARγ-knockout mice. Targeted deletion of PPARγ in hepatocytes, and to a lesser extent in macrophages, protected mice against high-fat diet-induced hepatic steatosis. Down-regulated expression of genes involved in lipogenesis (SCD1, SREBP-1c, and ACC), lipid transport (CD36/FAT, L-FABP, and MTP), and ß-oxidation (PPARα and ACO) was observed in PPARγ(Δhep) mice. Moreover, PPARγ(Δhep) mice showed improved glucose tolerance and reduced PEPCK expression without changes in Pcx, Fbp1, and G6Pc expression and CREB and JNK phosphorylation. In precision-cut liver slices (PCLSs) and hepatocytes, rosiglitazone either alone or in combination with oleic acid increased triglyceride accumulation, an effect that was blocked by the PPARγ antagonist biphenol A diglycidyl ether (BADGE). PCLSs and hepatocytes from PPARγ(Δhep) mice showed blunted responses to rosiglitazone and oleic acid, whereas the response to these compounds remained intact in PCLSs from PPARγ(Δmac) mice. Collectively, these findings establish PPARγ expression in hepatocytes as a prosteatotic factor in fatty liver disease.

STAT6 transcription factor is a facilitator of the nuclear receptor PPARγ-regulated gene expression in macrophages and dendritic cells.

Peroxisome proliferator-activated receptor γ (PPARγ) is a lipid-activated transcription factor regulating lipid metabolism and inflammatory response in macrophages and dendritic cells (DCs). These immune cells exposed to distinct inflammatory milieu show cell type specification as a result of altered gene expression. We demonstrate here a mechanism how inflammatory molecules modulate PPARγ signaling in distinct subsets of cells. Proinflammatory molecules inhibited whereas interleukin-4 (IL-4) stimulated PPARγ activity in macrophages and DCs. Furthermore, IL-4 signaling augmented PPARγ activity through an interaction between PPARγ and signal transducer and activators of transcription 6 (STAT6) on promoters of PPARγ target genes, including FABP4. Thus, STAT6 acts as a facilitating factor for PPARγ by promoting DNA binding and consequently increasing the number of regulated genes and the magnitude of responses. This interaction, underpinning cell type-specific responses, represents a unique way of controlling nuclear receptor signaling by inflammatory molecules in immune cells.

Peroxisome proliferator-activated receptor-gamma abrogates Smad-dependent collagen stimulation by targeting the p300 transcriptional coactivator.

Ligands of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) abrogate the stimulation of collagen gene transcription induced by transforming growth factor-beta (TGF-beta). Here, we delineate the mechanisms underlying this important novel physiological function for PPAR-gamma in connective tissue homeostasis. First, we demonstrated that antagonistic regulation of TGF-beta activity by PPAR-gamma ligands involves cellular PPAR-gamma, since 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)) failed to block TGF-beta-induced responses in either primary cultures of PPAR-gamma-null murine embryonic fibroblasts, or in normal human skin fibroblasts with RNAi-mediated knockdown of PPAR-gamma. Next, we examined the molecular basis underlying the abrogation of TGF-beta signaling by PPAR-gamma in normal human fibroblasts in culture. The results demonstrated that Smad-dependent transcriptional responses were blocked by PPAR-gamma without preventing Smad2/3 activation. In contrast, the interaction between activated Smad2/3 and the transcriptional coactivator and histone acetyltransferase p300 induced by TGF-beta, and the accumulation of p300 on consensus Smad-binding DNA sequences and histone H4 hyperacetylation at the COL1A2 locus, were all prevented by PPAR-gamma. Wild-type p300, but not a mutant form of p300 lacking functional histone acetyltransferase, was able to restore TGF-beta-induced stimulation of COL1A2 in the presence of PPAR-gamma ligands. Collectively, these results indicate that PPAR-gamma blocked Smad-mediated transcriptional responses by preventing p300 recruitment and histone H4 hyperacetylation, resulting in the inhibition of TGF-beta-induced collagen gene expression. Pharmacological activation of PPAR-gamma thus may represent a novel therapeutic approach to target p300-dependent TGF-beta profibrotic responses such as stimulation of collagen gene expression.

PPAR Signaling in Placental Development and Function.

With the major attention to the pivotal roles of PPARs in diverse aspects of energy metabolism, the essential functions of PPARgamma and PPARbeta/delta in placental development came as a surprise and were often considered a nuisance en route to their genetic analysis. However, these findings provided an opportune entrée into placental biology. Genetic and pharmacological studies, primarily of knockout animal models and cell culture, uncovered networks of PPARgamma and PPARdelta, their heterodimeric RXR partners, associated transcriptional coactivators, and target genes, that regulate various aspects of placental development and function. These studies furnish both specific information about trophoblasts and the placenta and potential hints about the functions of PPARs in other tissues and cell types. They reveal that the remarkable versatility of PPARs extends beyond the orchestration of metabolism to the regulation of cellular differentiation, tissue development, and trophoblast-specific functions. This information and its implications are the subject of this review.

Deficiency of PPARbeta/delta in the epidermis results in defective cutaneous permeability barrier homeostasis and increased inflammation.

In cultured human keratinocytes or murine epidermis, peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) (NR1C2) activators (1) stimulate keratinocyte differentiation; (2) decrease keratinocyte proliferation; (3) accelerate permeability barrier repair; (4) increase epidermal lipid synthesis; and (5) reduce cutaneous inflammation. Since these results suggest that PPARbeta/delta could play an important role in cutaneous homeostasis, we assessed here the skin phenotype of mice deficient in PPARbeta/delta. Gross cutaneous abnormalities were not evident, and both stratum corneum (SC) skin hydration and surface pH were normal. However, the epidermis was thickened and proliferating cell nuclear antigen (PCNA) staining was increased, indicating increased cell proliferation. No change in apoptosis was observed but the expression of differentiation markers, such as filaggrin, involucrin, and loricrin, was slightly increased in PPARbeta/delta(-/-) mice. Although basal permeability barrier function was normal, PPARbeta/delta knockout (KO) mice show a significant delay in barrier recovery rates following acute barrier disruption by either acetone treatment or tape-stripping. Delayed barrier recovery correlated with decreased production and secretion of lamellar bodies (LBs), and with reduced numbers of extracellular lamellar membranes in the SC. Finally, PPARbeta/delta KO mice displayed increased inflammation in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Together, these results further demonstrate that PPARbeta/delta in the epidermis: (1) is required for permeability barrier homeostasis; (2) regulates keratinocyte proliferation; and (3) modulates cutaneous inflammation.