S. Lee Adamson

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.

A Gja1 missense mutation in a mouse model of oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is an autosomal dominant disorder characterized by pleiotropic developmental anomalies of the limbs, teeth, face and eyes that was shown recently to be caused by mutations in the gap junction protein alpha 1 gene (GJA1), encoding connexin 43 (Cx43). In the course of performing an N-ethyl-N-nitrosourea mutagenesis screen, we identified a dominant mouse mutation that exhibits many classic symptoms of ODDD, including syndactyly, enamel hypoplasia, craniofacial anomalies and cardiac dysfunction. Positional cloning revealed that these mice carry a point mutation in Gja1 leading to the substitution of a highly conserved amino acid (G60S) in Cx43. In vivo and in vitro studies revealed that the mutant Cx43 protein acts in a dominant-negative fashion to disrupt gap junction assembly and function. In addition to the classic features of ODDD, these mutant mice also showed decreased bone mass and mechanical strength, as well as altered hematopoietic stem cell and progenitor populations. Thus, these mice represent an experimental model with which to explore the clinical manifestations of ODDD and to evaluate potential intervention strategies.

Effect of placental embolization on the umbilical arterial velocity waveform in fetal sheep.

Absent diastolic velocity and reversed diastolic velocity have been observed in the umbilical artery of growth-retarded human fetuses by means of Doppler ultrasonography. Because the physiologic mechanism is unknown, the purpose of this study was to determine whether such waveform changes could be induced in sheep fetuses by embolization of the resistance vessels of the placenta. Seven chronically catheterized sheep fetuses were instrumented with Doppler crystals mounted on one umbilical artery. The placenta was progressively embolized from the fetal side with plastic microspheres. In all cases the umbilical arterial waveform showed a progression from normal to zero diastolic velocity with embolization. In six of seven animals diastolic velocity eventually reversed in direction before fetal death. We conclude that occlusion of placental arteries and arterioles in fetal sheep recreates the waveform changes observed in severely growth-retarded human fetuses.

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 ∼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.

Determinants of adverse perinatal outcome in high-risk women with abnormal uterine artery Doppler images

OBJECTIVE The purpose of this study was to evaluate the prognostic role of placental ultrasound imaging at 19-23 weeks of gestation in clinically high-risk women with abnormal uterine artery Doppler (UTAD). STUDY DESIGN Placentas of 60 women with abnormal UTAD were examined at 19-23 weeks of gestation for shape and texture abnormalities. Findings were correlated with clinical outcomes (preterm delivery at <32 weeks of gestation; birth weight <10th percentile [small for gestational age]; preeclampsia/hemolysis, elevated liver enzymes, low platelets; early-onset intrauterine growth restriction with abnormal umbilical artery Doppler; and intrauterine fetal death) and maternal serum screening data. Placental disease was reviewed by 2 perinatal pathologists. RESULTS Women with abnormal placental shape at 19-23 weeks of gestation (n = 28) had higher odds of intrauterine fetal death (odds ratio, 4.5; 95% CI, 1.3-15.6), delivery at <32 weeks of gestation (odds ratio, 4.7; 95% CI, 1.6-14.1]), and intrauterine growth restriction (odds ratio, 4.7; 95% CI, 1.4-15.1]) than did the women with a normal placental shape. Thirty-two of 41 placentas (74%) weighed <10th percentile, and 36 of 43 placentas (83%) had ischemic-thrombotic pathologic condition. There was no association between abnormal placental shape at 19-23 weeks of gestation and placental weight, but 5 of 6 placentas that were <10 cm long were <10th percentile for weight at delivery. There was a poor correlation between measures of ultrasound texture at 19-23 weeks of gestation and the presence of specific lesions at delivery. CONCLUSION Combined abnormal UTAD and placental dysmorphologic condition before fetal viability identifies a subset of women who are at risk of adverse outcomes. Placental size is critical in the determination of the outcome in this situation because of the very high prevalence of destructive lesions, although present methods of placental imaging have significant limitations.

Arterial pressure, vascular input impedance, and resistance as determinants of pulsatile blood flow in the umbilical artery

The flow pulsatility index, the ratio of flow pulse amplitude to mean flow over the cardiac cycle, has been used to quantify pulsatility of blood flow in the umbilical artery. In experiments with fetal sheep, we showed that the flow pulsatility index in the umbilical artery is accurately estimated by the ratio of total umbilico-placental vascular resistance (mean arterial pressure divided by mean umbilical flow) divided by fundamental impedance (umbilical vascular impedance at the heart rate frequency) times the pulsatility index (pulse/mean) of the arterial pressure that drives flow through this bed. The pulsatility index of arterial pressure is primarily determined by upstream factors (e.g. heart rate) whereas fundamental impedance depends primarily on the radius and viscoelastic wall properties of the umbilical artery. An increase in resistance in the microcirculation and/or veins causes proportional changes in the flow pulsatility index because these sites have little influence on fundamental impedance. However, an increase in resistance in the highly vasoactive umbilical arteries has offsetting effects on impedance and resistance; consequently, flow pulsatility changes little even when arterial vasoconstriction markedly reduces mean flow. We conclude that when arterial pressure pulsatility is stable, a change in the flow pulsatility index provides a useful indication of a change in resistance in the microcirculation and/or veins but will not reliably detect a resistance change in the artery.

Endothelial Nitric Oxide Synthase Deficiency Reduces Uterine Blood Flow, Spiral Artery Elongation, and Placental Oxygenation in Pregnant Mice

Preeclampsia is associated with impaired uteroplacental adaptations during pregnancy and abnormalities in the endothelial NO synthase (eNOS)-NO pathway, but whether eNOS deficiency plays a causal role is unknown. Thus, the objective of the current study was to determine the role of eNOS in the mother and/or conceptus in uteroplacental changes during pregnancy using eNOS knockout mice. We quantified uterine artery blood flow using microultrasound, visualized the uteroplacental vasculature using vascular corrosion casts, and used pimonidazole and hypoxia-inducible factor 1&agr; immunohistochemistry as markers of hypoxia in the placentas of eNOS knockout mice versus the background strain, C57Bl/6J (wild type). We found that increases in uteroplacental blood flow, uterine artery diameter, and spiral artery length were reduced, and markers of placental hypoxia in the junctional zone were elevated in late gestation in eNOS knockout mice. Both maternal and conceptus genotypes contributed to changes in uterine artery diameter and flow. Despite placental hypoxia, placental soluble fms-like tyrosine kinase 1 and tumor necrosis factor-&agr; mRNA, and in maternal plasma, soluble fms-like tyrosine kinase 1 were not elevated in eNOS knockout mice. Thus, our results show that both eNOS in the mother and the conceptus contribute to uteroplacental vascular changes and increased uterine arterial blood flow in normal pregnancy.

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

BackgroundNon-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.MethodsWe 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.ResultsGestational 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%.ConclusionMicro-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.

Micro-ultrasound for preclinical imaging

Over the past decade, non-invasive preclinical imaging has emerged as an important tool to facilitate biomedical discovery. Not only have the markets for these tools accelerated, but the numbers of peer-reviewed papers in which imaging end points and biomarkers have been used have grown dramatically. High frequency ‘micro-ultrasound’ has steadily evolved in the post-genomic era as a rapid, comparatively inexpensive imaging tool for studying normal development and models of human disease in small animals. One of the fundamental barriers to this development was the technological hurdle associated with high-frequency array transducers. Recently, new approaches have enabled the upper limits of linear and phased arrays to be pushed from about 20 to over 50 MHz enabling a broad range of new applications. The innovations leading to the new transducer technology and scanner architecture are reviewed. Applications of preclinical micro-ultrasound are explored for developmental biology, cancer, and cardiovascular disease. With respect to the future, the latest developments in high-frequency ultrasound imaging are described.

Fetal growth restriction triggered by polycyclic aromatic hydrocarbons is associated with altered placental vasculature and AhR-dependent changes in cell death

Maternal cigarette smoking is considered an important risk factor associated with fetal intrauterine growth restriction (IUGR). Polycyclic aromatic hydrocarbons (PAHs) are well-known constituents of cigarette smoke, and the effects of acute exposure to these chemicals at different gestational stages have been well established in a variety of laboratory animals. In addition, many PAHs are known ligands of the aryl hydrocarbon receptor (AhR), a cellular xenobiotic sensor responsible for activating the metabolic machinery. In this study, we have applied a chronic, low-dose regimen of PAH exposure to C57Bl/6 female mice before conception. This treatment caused IUGR in day 15.5 post coitum (d15.5) fetuses and yielded abnormalities in the placental vasculature, resulting in significantly reduced arterial surface area and volume of the fetal arterial vasculature of the placenta. However, examination of the small vasculature within the placental labyrinth of PAH-exposed dams revealed extensive branching and enlargement of these vessels, indicating a possible compensatory mechanism. These alterations in vascularization were accompanied by reduced placental cell death rates, increased expression levels of antiapoptotic Xiap, and decreased expression of proapoptotic Bax, cleaved poly(ADP-ribose) polymerase-1, and active caspase-3. AhR-deficient fetuses were rescued from PAH-induced growth restriction and exhibited no changes in the labyrinthine cell death rate. The results of this investigation suggest that chronic exposure to PAHs is a contributing factor to the development of IUGR in human smokers and that the AhR pathway is involved.