A mouse model of antepartum stillbirth.

BACKGROUND: Many stillbirths of normally formed fetuses in the third trimester could be prevented via delivery if reliable means to anticipate this outcome existed. However, because the etiology of these stillbirths is often unexplained and although the underlying mechanism is presumed to be hypoxia from placental insufficiency, the placentas often appear normal on histopathological examination. Gestational age is a risk factor for antepartum stillbirth, with a rapid rise in stillbirth rates after 40 weeks' gestation. We speculate that a common mechanism may explain antepartum stillbirth in both the late-term and postterm periods. Mice also show increasing rates of stillbirth when pregnancy is artificially prolonged. The model therefore affords an opportunity to characterize events that precede stillbirth. OBJECTIVE: The objective of the study was to prolong gestation in mice and monitor fetal and placental growth and cardiovascular changes. STUDY DESIGN: From embryonic day 15.5 to embryonic day 18.5, pregnant CD-1 mice received daily progesterone injections to prolong pregnancy by an additional 24 hour period (to embryonic day 19.5). To characterize fetal and placental development, experimental assays were performed throughout late gestation (embryonic day 15.5 to embryonic day 19.5), including postnatal day 1 pups as controls. In addition to collecting fetal and placental weights, we monitored fetal blood flow using Doppler ultrasound and examined the fetoplacental arterial vascular geometry using microcomputed tomography. Evidence of hypoxic organ injury in the fetus was assessed using magnetic resonance imaging and pimonidazole immunohistochemistry. RESULTS: At embryonic day 19.5, mean fetal weights were reduced by 14% compared with control postnatal day 1 pups. Ultrasound biomicroscopy showed that fetal heart rate and umbilical artery flow continued to increase at embryonic day 19.5. Despite this, the embryonic day 19.5 fetuses had significant pimonidazole staining in both brain and liver tissue, indicating fetal hypoxia. Placental weights at embryonic day 19.5 were 21% lower than at term (embryonic day 18.5). Microcomputed tomography showed no change in quantitative morphology of the fetoplacental arterial vasculature between embryonic day 18.5 and embryonic day 19.5. CONCLUSION: Prolongation of pregnancy renders the murine fetus vulnerable to significant growth restriction and hypoxia because of differential loss of placental mass rather than any compromise in fetoplacental blood flow. Our data are consistent with a hypoxic mechanism of antepartum fetal death in human term and postterm pregnancy and validates the inability of umbilical artery Doppler to safely monitor such fetuses. New tests of placental function are needed to identify the late-term fetus at risk of hypoxia to intervene by delivery to avoid antepartum stillbirth.

Quantification of Gestational Changes in the Uteroplacental Vascular Tree Reveals Vessel Specific Hemodynamic Roles During Pregnancy in Mice.

The purpose of this study was to establish the time course and hemodynamic significance of de novo formed and enlarged uteroplacental arteries during pregnancy. Using x-ray microcomputed tomography (n = 4-7 placentas from 2-4 dams/gestational group), uteroplacental arterial vascular dimensions were measured at individual implantation sites. Dimensions and topology were used to compute total and vessel-specific resistances and cross-sectional areas. Diameter enlargement of the uterine artery (+55% by Embryonic Day 5.5 [E5.5]) and preplacental radial arteries (+30% by E8.5) was significant only in early gestation. Formation of spiral arteries (E9.5-E11.5), maternal canals, and canal branches (E11.5-E13.5) during midgestation was followed by enlargement of these vessels such that, from E9.5 to E17.5 (near term), spiral artery resistance dropped 9-fold, and canal resistance became negligible. A 12-fold increase in terminal vessel cross-sectional area was nearly sufficient to offset known increases in flow so that blood velocity entering the exchange region was predicted to increase by only 2-fold. The calculated 47% decrease in total resistance downstream of the uterine artery, determined from vascular geometry, was in accord with prior uterine blood flow data in vivo and was due to enlarging spiral artery diameters. Interestingly, radial artery resistance was unchanged after E9.5 so that radial arteries accounted for 91% of resistance and pressure drop in the uteroplacental arterial network by E17.5. These findings led us to propose functional roles for the three morphologically defined vessel types: radial arteries to reduce pressure, spiral artery enlargement to increase flow with gestation, and maternal canal elaboration and enlargement to maintain low exit velocities into the exchange region.

ENU-induced mutation in the DNA-binding domain of KLF3 reveals important roles for KLF3 in cardiovascular development and function in mice.

KLF3 is a Krüppel family zinc finger transcription factor with widespread tissue expression and no previously known role in heart development. In a screen for dominant mutations affecting cardiovascular function in N-ethyl-N-nitrosourea (ENU) mutagenized mice, we identified a missense mutation in the Klf3 gene that caused aortic valvular stenosis and partially penetrant perinatal lethality in heterozygotes. All homozygotes died as embryos. In the first of three zinc fingers, a point mutation changed a highly conserved histidine at amino acid 275 to arginine (Klf3(H275R) ). This change impaired binding of the mutant protein to KLF3's canonical DNA binding sequence. Heterozygous Klf3(H275R) mutants that died as neonates had marked biventricular cardiac hypertrophy with diminished cardiac chambers. Adult survivors exhibited hypotension, cardiac hypertrophy with enlarged cardiac chambers, and aortic valvular stenosis. A dominant negative effect on protein function was inferred by the similarity in phenotype between heterozygous Klf3(H275R) mutants and homozygous Klf3 null mice. However, the existence of divergent traits suggested the involvement of additional interactions. We conclude that KLF3 plays diverse and important roles in cardiovascular development and function in mice, and that amino acid 275 is critical for normal KLF3 protein function. Future exploration of the KLF3 pathway provides a new avenue for investigating causative factors contributing to cardiovascular disorders in humans.

Site-specific increases in utero- and fetoplacental arterial vascular resistance in eNOS-deficient mice due to impaired arterial enlargement.

The sites of elevated vascular resistance that impede placental perfusion in pathological pregnancies are unknown. In the current study, we identified these sites in a knockout mouse model (eNOS(-/-)) with reduced uterine (-55%) and umbilical (-29%) artery blood flows caused by endothelial nitric oxide synthase deficiency. Uteroplacental and fetoplacental arterial vascular trees of pregnant mice near term were imaged using x-ray microcomputed tomography (n = 5-10 placentas from 3-5 dams/group). The resulting three-dimensional images were analyzed to assess vessel geometry and vascular resistance. In control and eNOS(-/-) trees, ∼90% of total uteroplacental vascular resistance was located in the radial arteries. Changes in eNOS(-/-) vessel geometry, including 30% reductions in uterine, radial, and spiral artery diameters, were calculated to increase arterial resistance downstream of the uterine artery by 2.3-fold, predicting a 57% decrease in uterine blood flow. Despite large reductions in eNOS(-/-) spiral arteries (-55% by volume) and maternal canals (-67% by volume), these vessels were relatively minor contributors to resistance. In the eNOS(-/-) fetoplacental tree, the number of arterioles (50-75 µm diameter) increased by 26%. Nevertheless, calculated resistance rose by 19%, predominantly because arteries near the periphery of the tree selectively exhibited a 7%-9% diameter reduction. We conclude that previously observed decreases in uterine and umbilical blood flows in eNOS(-/-) pregnancies are associated with markedly divergent structural changes in the uteroplacental versus fetoplacental circulations. Results showed the radial arteries were critical determinants of uteroplacental resistance in mice and therefore warrant greater attention in future studies in pathological human pregnancies.

Effects of genes and environment on the fetoplacental arterial microcirculation in mice revealed by micro-computed tomography imaging.

The fetoplacental arterial tree is critical for efficient distribution of arterial blood to capillaries throughout the placental exchange region; yet, little is known about the factors and mechanisms that control its development. Advances in micro-CT imaging and analysis, and available mutant mouse strains, are facilitating rapid progress. Indeed, micro-CT studies show that genetic differences between the CD1 and C57Bl/6 mouse strains, and between Gcm1 heterozygotes and wild-type littermates alter the developmental trajectory of the fetoplacental arterial tree as do environmental factors including maternal exposure to toxins in cigarette smoke and malarial infection. Relative to other vascular beds, the fetoplacental arterial tree is particularly tractable because veins can more easily be excluded when infusing the contrast agent and because of the placenta's small size, which means that the whole organ can be imaged (maintaining connectivity) and that the tree is simpler (fewer branching generations). Despite these differences, measured parameters were found to be similar to arterial trees in other adult rodent organs. Thus, micro-CT analysis provides a means for advancing of our understanding of the mechanisms controlling development of the fetoplacental arterial tree. Results will likely have relevance to other arterial vasculatures as well.

Trophoblast-specific reduction of VEGFA alters placental gene expression and maternal cardiovascular function in mice.

Given the angiogenic function of vascular endothelial growth factor A (VEGFA), the function of its expression by trophoblast in the avascular placental junctional zone is unknown. In mice, cells from the trophoblast-specific protein alpha (Tpbpa) lineage populate this zone and, in late gestation, some of these cells invade the decidual layer. To diminish Vegfa expression in Tpbpa cells, we crossed Vegfa(flox/flox) females with males carrying Tpbpa-Cre. For single deletion (sd) of Vegfa in Tpbpa cells in 100% of conceptuses (SD100 pregnancies, sd conceptuses) we crossed homozygous lines. For double deletion (dd) of both Vegfa alleles in 50% of the conceptuses (DD50 pregnancies, 50% dd conceptuses and 50% no deletion [nd]), we crossed homozygous Vegfa(flox/flox) females with males heterozygous for Tpbpa-Cre and homozygous for Vegfa(flox/flox). Controls were Vegfa(flox/flox) females bred to wild-type males (V-CTRL pregnancies). In SD100 pregnancies, maternal plasma immunoreactive VEGFA significantly increased and arterial blood pressure decreased, whereas fetal body weight and placental Flt1, sFlt1, and Prl3b1 mRNA were unchanged. In DD50, maternal immunoreactive VEGFA and arterial pressures were unaltered, but both dd and nd conceptuses exhibited significantly increased embryonic lethality, altered expression of Flt1, sFlt1, and Prl3b1 mRNA in the decidual layer, and decreased fetal body weight relative to V-CTRL. Maternal cardiac output significantly increased in proportion to dd conceptuses in the pregnancy. In DD50, results are consistent with altered maternal function beginning in early gestation and adversely impacting both conceptus genotypes. We conclude that maternal function is influenced by Vegfa expression in trophoblast cells at the maternal-fetal interface, likely via an endocrine mechanism.

Priming of hypoxia-inducible factor by neuronal nitric oxide synthase is essential for adaptive responses to severe anemia.

Cells sense and respond to changes in oxygen concentration through gene regulatory processes that are fundamental to survival. Surprisingly, little is known about how anemia affects hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular responses to acute hypoxia, we defined the effects of NOS deficiency in acute anemia. In contrast to endothelial NOS or inducible NOS deficiency, neuronal NOS (nNOS)(-/-) mice demonstrated increased mortality during anemia. Unlike wild-type (WT) animals, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS(-/-) mice. At the cellular level, anemia increased expression of HIF-1α protein and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK1) in the brain of WT, but not nNOS(-/-) mice, despite comparable reductions in tissue PO(2). Paradoxically, nNOS(-/-) mice survived longer during hypoxia, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. Real-time imaging of transgenic animals expressing a reporter HIF-α(ODD)-luciferase chimeric protein confirmed that nNOS was essential for anemia-mediated increases in HIF-α protein stability in vivo. S-nitrosylation effects the functional interaction between HIF and pVHL. We found that anemia led to nNOS-dependent S-nitrosylation of pVHL in vivo and, of interest, led to decreased expression of GSNO reductase. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo and provide essential mechanistic insight into the differences between anemia and hypoxia.

Chronic carbon monoxide inhalation during pregnancy augments uterine artery blood flow and uteroplacental vascular growth in mice.

End-tidal breath carbon monoxide (CO) is abnormally low in women with preeclampsia (PE), while women smoking during pregnancy have shown an increase in CO levels and a 33% lower incidence of PE. This effect may be, in part, due to lowered sFLT1 plasma levels in smokers, and perhaps low-level CO inhalation can attenuate the development of PE in high-risk women. Our previous work showed maternal chronic CO exposure (<300 ppm) throughout gestation had no maternal or fetal deleterious effects in mice. Our current study evaluated the uteroplacental vascular effects in CD-1 maternal mice that inhaled CO (250 ppm) both chronically, gestation day (GD) 0.5 to 18.5, and acutely, 2.5 h on each of GD 10.5 and 14.5. We demonstrated, using microultrasound measurements of blood velocity and microcomputed tomography imaging of the uteroplacental vasculature, that chronic maternal exposure to CO doubled uterine artery blood flow and augmented uteroplacental vascular diameters and branching. This finding may be of benefit to women with PE, as they exhibit uteroplacental vascular compromise. The ratio of VEGF protein to its FLT1 receptor was increased in the placenta, suggesting a shift to a more angiogenic state; however, maternal circulating levels of VEGF, sFLT1, and their ratio were not significantly changed. Doppler blood velocities in the maternal uterine artery and fetal umbilical artery and vein were unaltered. This study provides in vivo evidence that chronic inhalation of 250 ppm CO throughout gestation augments uterine blood flow and uteroplacental vascular growth, changes that may protect against the subsequent development of preeclampsia.

Translational analysis of mouse and human placental protein and mRNA reveals distinct molecular pathologies in human preeclampsia.

Preeclampsia (PE) adversely impacts ~5% of pregnancies. Despite extensive research, no consistent biomarkers or cures have emerged, suggesting that different molecular mechanisms may cause clinically similar disease. To address this, we undertook a proteomics study with three main goals: (1) to identify a panel of cell surface markers that distinguish the trophoblast and endothelial cells of the placenta in the mouse; (2) to translate this marker set to human via the Human Protein Atlas database; and (3) to utilize the validated human trophoblast markers to identify subgroups of human preeclampsia. To achieve these goals, plasma membrane proteins at the blood tissue interfaces were extracted from placentas using intravascular silica-bead perfusion, and then identified using shotgun proteomics. We identified 1181 plasma membrane proteins, of which 171 were enriched at the maternal blood-trophoblast interface and 192 at the fetal endothelial interface with a 70% conservation of expression in humans. Three distinct molecular subgroups of human preeclampsia were identified in existing human microarray data by using expression patterns of trophoblast-enriched proteins. Analysis of all misexpressed genes revealed divergent dysfunctions including angiogenesis (subgroup 1), MAPK signaling (subgroup 2), and hormone biosynthesis and metabolism (subgroup 3). Subgroup 2 lacked expected changes in known preeclampsia markers (sFLT1, sENG) and uniquely overexpressed GNA12. In an independent set of 40 banked placental specimens, GNA12 was overexpressed during preeclampsia when co-incident with chronic hypertension. In the current study we used a novel translational analysis to integrate mouse and human trophoblast protein expression with human microarray data. This strategy identified distinct molecular pathologies in human preeclampsia. We conclude that clinically similar preeclampsia patients exhibit divergent placental gene expression profiles thus implicating divergent molecular mechanisms in the origins of this disease.

A novel Phex mutation in a new mouse model of hypophosphatemic rickets.

X-linked hypophosphatemic rickets (XLH) is a dominantly inherited disease characterized by renal phosphate wasting, aberrant vitamin D metabolism, and defective bone mineralization. It is known that XLH in humans and in certain mouse models is caused by inactivating mutations in PHEX/Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). By a genome-wide N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a dominant mouse mutation that exhibits the classic clinical manifestations of XLH, including growth retardation, skeletal abnormalities (rickets/osteomalacia), hypophosphatemia, and increased serum alkaline phosphatase (ALP) levels. Mapping and sequencing revealed that these mice carry a point mutation in exon 14 of the Phex gene that introduces a stop codon at amino acid 496 of the coding sequence (Phex(Jrt) also published as Phex(K496X) [Ichikawa et al., 2012]). Fgf23 mRNA expression as well as that of osteocalcin, bone sialoprotein, and matrix extracellular phosphoglycoprotein was upregulated in male mutant long bone, but that of sclerostin was unaffected. Although Phex mRNA is expressed in bone from mutant hemizygous male mice (Phex(Jrt)/Y mice), no Phex protein was detected in immunoblots of femoral bone protein. Stromal cultures from mutant bone marrow were indistinguishable from those of wild-type mice with respect to differentiation and mineralization. The ability of Phex(Jrt)/Y osteoblasts to mineralize and the altered expression levels of matrix proteins compared with the well-studied Hyp mice makes it a unique model with which to further explore the clinical manifestations of XLH and its link to FGF23 as well as to evaluate potential new therapeutic strategies.

Expansion of the fetoplacental vasculature in late gestation is strain dependent in mice.

How the fetoplacental arterial tree grows and expands during late gestational development is largely unknown. In this study, we quantified changes in arterial branching in the fetal exchange region of the mouse placenta during late gestation, when capillarization increases rapidly. We studied two commonly used mouse strains, CD1 and C57Bl/6 (B6), at embryonic days (E)13.5, 15.5, and 17.5. B6 mice differ from CD1 mice by exhibiting a blunted fetal weight gain in late gestation. We found that B6 capillarization and interhemal membrane thinning were reduced and placental hypoxia-inducible factor-1α and VEGF-A expression were higher than CD1 near term. Automated vascular segmentation of microcomputed tomography data sets revealed that the number of arterial vessels ≥50 µm remained constant during late gestation in both strains, despite large increases in downstream capillary volume quantified by stereology (+65% in B6 mice and +200% in CD1 mice). Arterial diameters expanded in both strains from E13.5 to E15.5; however, diameters continued to expand to E17.5 in B6 mice only. The diameter scaling coefficient at branch sites was near optimal (-3.0) and remained constant in CD1 mice, whereas it decreased, becoming abnormal, in B6 mice at term (-3.5 ± 0.2). Based on arterial tree geometry, resistance remained constant throughout late gestation (∼0.45 mmHg·s·µl(-1)) in CD1 mice, whereas it decreased by 50% in late gestation in B6 mice. Quantification of the fetoplacental vasculature revealed significant strain-dependent differences in arterial and capillary expansion in late gestation. In both strains, enlargement of the fetoplacental arterial tree occurred primarily by increased arterial diameters with no change in segment numbers in late gestation.

The Sweet Pee model for Sglt2 mutation.

Inhibiting renal glucose transport is a potential pharmacologic approach to treat diabetes. The renal tubular sodium-glucose transporter 2 (SGLT2) reabsorbs approximately 90% of the filtered glucose load. An animal model with sglt2 dysfunction could provide information regarding the potential long-term safety and efficacy of SGLT2 inhibitors, which are currently under clinical investigation. Here, we describe Sweet Pee, a mouse model that carries a nonsense mutation in the Slc5a2 gene, which results in the loss of sglt2 protein function. The phenotype of Sweet Pee mutants was remarkably similar to patients with mutations in the Scl5a2 gene. The Sweet Pee mutants had improved glucose tolerance, higher urinary excretion of calcium and magnesium, and growth retardation. Renal physiologic studies demonstrated a prominent distal osmotic diuresis without enhanced natriuresis. Sweet Pee mutants did not exhibit increased KIM-1 or NGAL, markers of acute tubular injury. After induction of diabetes, Sweet Pee mice had better overall glycemic control than wild-type control mice, but had a higher risk for infection and an increased mortality rate (70% in homozygous mutants versus 10% in controls at 20 weeks). In summary, the Sweet Pee model allows study of the long-term benefits and risks associated with inhibition of SGLT2 for the management of diabetes. Our model suggests that inhibiting SGLT2 may improve glucose control but may confer increased risks for infection, malnutrition, volume contraction, and mortality.

Vessel tortuousity and reduced vascularization in the fetoplacental arterial tree after maternal exposure to polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and the main toxicants found in cigarettes. Women are often exposed to PAHs before pregnancy, typically via prepregnancy smoking. To determine how prepregnancy exposure affects the fetoplacental vasculature of the placenta, we exposed female mice to PAHs before conception, perfused the fetoplacental arterial trees with X-ray contrast agent, and imaged the vasculature ex vivo by microcomputed tomography (micro-CT) at embryonic day 15.5. Automated vascular segmentation and flow calculations revealed that in control trees, <40 chorionic plate vessels (diameter>180 µm) gave rise to ∼1,300 intraplacental arteries (50-180 µm), predicting an arterial vascular resistance of 0.37±0.04 mmHg·s·µl(-1). PAH exposure increased vessel curvature of chorionic plate vessels and significantly increased the tortuousity ratio of the tree. Intraplacental arteries were reduced by 17%, primarily due to a 27% decrease in the number of arteriole-sized (50-100 µm) vessels. There were no changes in the number of chorionic vessels, the depth or span of the tree, the diameter scaling coefficient, or the segment length-to-diameter ratio. PAH exposure resulted in a tree with a similar size and dichotomous branching structure, but one that was comparatively sparse so that arterial vascular resistance was increased by 30%. Assuming the same pressure gradient, blood flow would be 19% lower. Low flow may contribute to the 23% reduction observed in fetal weight. New insights into the specific effects of PAH exposure on a developing arterial tree were achieved using micro-CT imaging and automated vascular segmentation analysis.

Decidual PTEN expression is required for trophoblast invasion in the mouse.

Trophoblast invasion likely depends on complex cross talk between the fetal and maternal tissues and may involve the modulation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling activity in maternal decidual cells. In this report, we studied implantation in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) mice, which lack the PI3K signaling antagonist gene Pten in myometrial and stromal/decidual cells. Primiparous Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) mice were found to be subfertile because of increased fetal mortality at e11.5. Histopathological analyses revealed a failure of decidual regression in these mice, accompanied by reduced or absent invasion of fetal trophoblast glycogen cells and giant cells, abnormal development of the placental labyrinth, and frequent apparent intrauterine fetal growth restriction. Unexpectedly, the loss of phosphate and tensin homolog deleted on chromosome 10 (PTEN) expression in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) decidual cells was not accompanied by a detectable increase in AKT phosphorylation or altered expression or activation of PI3K/AKT downstream effectors such as mammalian target of rapamycin or glycogen synthase kinase-3ß. Terminal deoxynucleotidyl transferase-mediated nick end labeling and bromodeoxyuridine incorporation analyses attributed to the lack of decidual regression mainly to decreased apoptosis in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) decidual cells, rather than to increased proliferation. Remodeling of the maternal vasculature was delayed in Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) uteri at e11.5, as evidenced by persistence of vascular smooth muscle and decreased infiltration of uterine natural killer cells. In addition, thickening of the myometrium and disorganization of the muscle fibers were observed before and throughout gestation. Almost all Pten(tm1Hwu/tm1Hwu);Amhr2(tm3(cre)Bhr/+) mice failed to carry a second litter to term, apparently attributable to endometrial hyperplasia and uterine infections. Together, these data demonstrate novel roles of PTEN in the mammalian uterus and its requirement for proper trophoblast invasion and decidual regression.

Comparative systems biology of human and mouse as a tool to guide the modeling of human placental pathology.

Placental abnormalities are associated with two of the most common and serious complications of human pregnancy, maternal preeclampsia (PE) and fetal intrauterine growth restriction (IUGR), each disorder affecting approximately 5% of all pregnancies. An important question for the use of the mouse as a model for studying human disease is the degree of functional conservation of genetic control pathways from human to mouse. The human and mouse placenta show structural similarities, but there have been no systematic attempts to assess their molecular similarities or differences. We collected protein and mRNA expression data through shot-gun proteomics and microarray expression analysis of the highly vascular exchange region, microdissected from the human and mouse near-term placenta. Over 7000 ortholog genes were detected with 70% co-expressed in both species. Close to 90% agreement was found between our human proteomic results and 1649 genes assayed by immunohistochemistry for expression in the human placenta in the Human Protein Atlas. Interestingly, over 80% of genes known to cause placental phenotypes in mouse are co-expressed in human. Several of these phenotype-associated proteins form a tight protein-protein interaction network involving 15 known and 34 novel candidate proteins also likely important in placental structure and/or function. The entire data are available as a web-accessible database to guide the informed development of mouse models to study human disease.

Endothelial nitric oxide synthase gene expression during murine embryogenesis: commencement of expression in the embryo occurs with the establishment of a unidirectional circulatory system.

To elucidate the role of endothelial NO synthase (eNOS)-derived NO during mammalian embryogenesis, we assessed the expression of the eNOS gene during development. Using transgenic eNOS promoter/reporter mice (with beta-galactosidase and green fluorescent protein reporters), in situ cRNA hybridization, and immunohistochemistry to assess transcription, steady-state mRNA levels, and protein expression, respectively, we noted that eNOS expression in the developing cardiovascular system was highly restricted to endothelial cells of medium- and large-sized arteries and the endocardium. The onset of transcription of the native eNOS gene and reporters coincided with the establishment of robust, unidirectional blood flow at embryonic day 9.5, as assessed by Doppler ultrasound biomicroscopy. Interestingly, reporter transgene expression and native eNOS mRNA were also observed in discrete regions of the developing skeletal musculature and the apical ectodermal ridge of developing limbs, suggesting a role for eNOS-derived NO in limb development. In vitro studies of promoter/reporter constructs indicated that similar eNOS promoter regions operate in both embryonic skeletal muscle and vascular endothelial cells. In summary, transcriptional activity of the eNOS gene in the murine circulatory system occurred following the establishment of embryonic blood flow. Thus, the eNOS gene is a late-onset gene in endothelial ontogeny.

Baf60c is essential for function of BAF chromatin remodelling complexes in heart development.

Tissue-specific transcription factors regulate several important aspects of embryonic development. They must function in the context of DNA assembled into the higher-order structure of chromatin. Enzymatic complexes such as the Swi/Snf-like BAF complexes remodel chromatin to allow the transcriptional machinery access to gene regulatory elements. Here we show that Smarcd3, encoding Baf60c, a subunit of the BAF complexes, is expressed specifically in the heart and somites in the early mouse embryo. Smarcd3 silencing by RNA interference in mouse embryos derived from embryonic stem cells causes defects in heart morphogenesis that reflect impaired expansion of the anterior/secondary heart field, and also results in abnormal cardiac and skeletal muscle differentiation. An intermediate reduction in Smarcd3 expression leads to defects in outflow tract remodelling reminiscent of human congenital heart defects. Baf60c overexpressed in cell culture can mediate interactions between cardiac transcription factors and the BAF complex ATPase Brg1, thereby potentiating the activation of target genes. These results reveal tissue-specific and dose-dependent roles for Baf60c in recruiting BAF chromatin remodelling complexes to heart-specific enhancers, providing a novel mechanism to ensure transcriptional regulation during organogenesis.

Developmental regulation of cardiovascular function is dependent on both genotype and environment.

Adverse developmental environments can increase the risk of adult cardiovascular disease, but not all individuals are affected, suggesting the importance of genotype. Genetically distinct mouse strains allow the genetic dissection of complex traits; however, they have not been used to evaluate the developmental origins of adult cardiovascular disease. Our objective was to determine the effect of prenatal nutrient restriction (R) on adult cardiovascular function in A/J (AJ) and C57BL/6J (B6) mice and whether a postnatal high-fat (HF) diet exacerbates these effects. Pregnant AJ and B6 mice underwent a 30% R or ad libitum diet, and their offspring underwent a HF or control diet. Hypertension (+17 mmHg; P<0.001) was observed in B6R mice at 9 wk, and their arterial pressure tended to remain high at 25 wk (+13 mmHg; not significant). In AJR mice, the normal decrement in arterial pressure over this age range in this strain was abolished. Heart rate prematurely increased in B6R and decreased in AJR (all; P<0.05) mice from 9 to 25 wk. There was no effect of postnatal HF diet on these relationships. The Tei index (from a 26-wk microultrasound) was increased in both AJR and B6R mice (all; P<0.05), suggesting an improved global myocardial performance. Neither R nor HF alone changed diastolic (ratio of E wave to A wave) or systolic (%fractional shortening) function in either strain; however, R and HE combined improved diastolic function in B6 (P<0.05) but not in AJ mice. Therefore, there are strain-dependent alterations in adult cardiovascular function in response to prenatal nutrient restriction. Unexpectedly, a postnatal HF diet did not exacerbate the effects of prenatal nutrient restriction on postnatal cardiovascular outcomes.

Early gene expression and morphogenesis of the murine chorioallantoic placenta in vivo and in vitro.

BACKGROUND: In mice the exchange of oxygen and nutrients between mother and fetus occurs in the chorioallantoic placenta where fetal capillaries come in close proximity with maternal blood perfusing trophoblast-lined sinusoids. Despite its critical importance, quantitative in vivo gene expression over the initial stages of chorioallantoic placental development has not been described, nor are there in vitro systems recapitulating the critical syncytiotrophoblast differentiation step in its formation. Here we describe molecular events that occur during the onset of chorioallantoic morphogenesis in mice in vivo, and in placental explant and whole conceptus cultures in vitro. RESULTS: Chorioallantoic morphogenesis began immediately following allantoic fusion with the chorion in vivo, and was associated with significant upregulation of syncytiotrophoblast associated mRNA (Gcm1 and Syncytin A). However mouse placentas with chorioallantoic point attachment cultured with the allantois or as whole conceptuses did not upregulate Gcm1 and/or Syncytin A, suggesting that syncytiotrophoblast differentiation did not occur in vitro. Failure of morphogenesis appeared to be due to failure to sustain in vitro the chorionic trophoblast cells from which the syncytiotrophoblast cells are derived. In vitro culture conditions did support the upregulation of ectoplacental cone marker Tpbpalpha, maintenance of giant cell marker Pl1, and maintenance of Fgfr2 expression; all of which mimicked in vivo events observed over this developmental interval. CONCLUSIONS: We conclude that chorionic trophoblast maintenance and the early events that occur in vivo between chorioallantoic point attachment and primary villous formation are dependent on undefined intrauterine factors that were not present in the in vitro culture system. Nevertheless, in vitro culture conditions were appropriate to reproduce in vivo expression levels of Fgfr2, Pl1, and Tpbpalpha in placental explants.

In vivo quantification of embryonic and placental growth during gestation in mice using micro-ultrasound.

BACKGROUND: Non-invasive micro-ultrasound was evaluated as a method to quantify intrauterine growth phenotypes in mice. Improved methods are required to accelerate research using genetically-altered mice to investigate the interactive roles of genes and environments on embryonic and placental growth. We determined (1) feasible age ranges for measuring specific variables, (2) normative growth curves, (3) accuracy of ultrasound measurements in comparison with light microscopy, and (4) weight prediction equations using regression analysis for CD-1 mice and evaluated their accuracy when applied to other mouse strains. METHODS: We used 30-40 MHz ultrasound to quantify embryonic and placental morphometry in isoflurane-anesthetized pregnant CD-1 mice from embryonic day 7.5 (E7.5) to E18.5 (full-term), and for C57Bl/6J, B6CBAF1, and hIGFBP1 pregnant transgenic mice at E17.5. RESULTS: Gestational sac dimension provided the earliest measure of conceptus size. Sac dimension derived using regression analysis increased from 0.84 mm at E7.5 to 6.44 mm at E11.5 when it was discontinued. The earliest measurement of embryo size was crown-rump length (CRL) which increased from 1.88 mm at E8.5 to 16.22 mm at E16.5 after which it exceeded the field of view. From E10.5 to E18.5 (full term), progressive increases were observed in embryonic biparietal diameter (BPD) (0.79 mm to 7.55 mm at E18.5), abdominal circumference (AC) (4.91 mm to 26.56 mm), and eye lens diameter (0.20 mm to 0.93 mm). Ossified femur length was measureable from E15.5 (1.06 mm) and increased linearly to 2.23 mm at E18.5. In contrast, placental diameter (PD) and placental thickness (PT) increased from E10.5 to E14.5 then remained constant to term in accord with placental weight. Ultrasound and light microscopy measurements agreed with no significant bias and a discrepancy of less than 25%. Regression equations predicting gestational age from individual variables, and embryonic weight (BW) from CRL, BPD, and AC were obtained. The prediction equation BW = -0.757 + 0.0453 (CRL) + 0.0334 (AC) derived from CD-1 data predicted embryonic weights at E17.5 in three other strains of mice with a mean discrepancy of less than 16%. CONCLUSION: Micro-ultrasound provides a feasible tool for in vivo morphometric quantification of embryonic and placental growth parameters in mice and for estimation of embryonic gestational age and/or body weight in utero.