Loss of Rearranged L-Myc Fusion (RLF) results in defects in heart development in the mouse.

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.

Normothermic ex vivo perfusion provides superior organ preservation and enables viability assessment of hearts from DCD donors.

The shortage of donors in cardiac transplantation may be alleviated by the use of allografts from donation after circulatory death (DCD) donors. We have previously shown that hearts exposed to 30 min warm ischemic time and then flushed with Celsior supplemented with agents that activate ischemic postconditioning pathways, show complete recovery on a blood-perfused ex vivo working heart apparatus. In this study, these findings were assessed in a porcine orthotopic heart transplant model. DCD hearts were preserved with either normothermic ex vivo perfusion (NEVP) using a clinically approved device, or with standard cold storage (CS) for 4 h. Orthotopic transplantation into recipient animals was subsequently undertaken. Five of six hearts preserved with NEVP demonstrated favorable lactate profiles during NEVP and all five could be weaned off cardiopulmonary bypass posttransplant, compared with 0 of 3 hearts preserved with CS (p < 0.05, Fisher's exact test). In conclusion, DCD hearts flushed with supplemented Celsior solution and preserved with NEVP display viability before and after transplantation. Viability studies of human DCD hearts using NEVP are warranted.

Increasing the tolerance of DCD hearts to warm ischemia by pharmacological postconditioning.

Donation after circulatory death (DCD) offers a potential additional source of cardiac allografts. We used a porcine asphyxia model to evaluate viability of DCD hearts subjected to warm ischemic times (WIT) of 20­40 min prior to flushing with Celsior (C) solution. We then assessed potential benefits of supplementing C with erythropoietin, glyceryl trinitrate and zoniporide (Cs), a combination that we have shown previously to activate ischemic postconditioning pathways. Hearts flushed with C/Cs were assessed for functional, biochemical and metabolic recovery on an ex vivo working heart apparatus. Hearts exposed to 20-min WIT showed full recovery of functional and metabolic profiles compared with control hearts (no WIT). Hearts subjected to 30- or 40-min WIT prior to C solution showed partial and no recovery, respectively. Hearts exposed to 30-min WIT and Cs solution displayed complete recovery, while hearts exposed to 40-min WIT and Cs solution demonstrated partial recovery. We conclude that DCD hearts flushed with C solution demonstrate complete recovery up to 20-min WIT after which there is rapid loss of viability. Cs extends the limit of WIT tolerability to 30 min. DCD hearts with ≤30-min WIT may be suitable for transplantation and warrant assessment in a transplant model.

QTL mapping of complex binary traits in an advanced intercross line.

An advanced intercross line (AIL) is an easier and more cost-effective approach compared to recombinant inbred lines for fine mapping of quantitative trait loci (QTL) identified by F(2) designs. In an AIL, a complex binary trait can be mapped through analysis of either continuously distributed proxy traits for the liability of the binary trait or the liability itself, the latter presenting the greater statistical challenge. In another work, we successfully applied both approaches in an AIL to fine map previously identified QTL underlying anatomical parameters of the cardiac inter-atrial septum including patent foramen ovale. Here, we describe the statistical methods that we used to analyse complex binary traits in our AIL design. This is achieved using a likelihood-based method, with the expectation-maximisation algorithm allowing use of standard logistic regression methods for model fitting.

Micrometeorites from the transantarctic mountains.

We report the discovery of large accumulations of micrometeorites on the Myr-old, glacially eroded granitic summits of several isolated nunataks in the Victoria Land Transantarctic Mountains. The number (>3,500) of large (>400 mum and up to 2 mm in size) melted and unmelted particles is orders of magnitudes greater than other Antarctic collections. Flux estimates, bedrock exposure ages and the presence of approximately 0.8-Myr-old microtektites suggest that extraterrestrial dust collection occurred over the last 1 Myr, taking up to 500 kyr to accumulate based on 2 investigated find sites. The size distribution and frequency by type of cosmic spherules in the >200-mum size fraction collected at Frontier Mountain (investigated in detail in this report) are similar to those of the most representative known micrometeorite populations (e.g., South Pole Water Well). This and the identification of unusual types in terms of composition (i.e., chondritic micrometeorites and spherulitic aggregates similar to the approximately 480-kyr-old ones recently found in Antarctic ice cores) and size suggest that the Transantarctic Mountain micrometeorites constitute a unique and essentially unbiased collection that greatly extends the micrometeorite inventory and provides material for studies on micrometeorite fluxes over the recent ( approximately 1 Myr) geological past.

A large meteoritic event over Antarctica ca. 430 ka ago inferred from chondritic spherules from the Sør Rondane Mountains.

Large airbursts, the most frequent hazardous impact events, are estimated to occur orders of magnitude more frequently than crater-forming impacts. However, finding traces of these events is impeded by the difficulty of identifying them in the recent geological record. Here, we describe condensation spherules found on top of Walnumfjellet in the Sør Rondane Mountains, Antarctica. Affinities with similar spherules found in EPICA Dome C and Dome Fuji ice cores suggest that these particles were produced during a single-asteroid impact ca. 430 thousand years (ka) ago. The lack of a confirmed crater on the Antarctic ice sheet and geochemical and 18O-poor oxygen isotope signatures allow us to hypothesize that the impact particles result from a touchdown event, in which a projectile vapor jet interacts with the Antarctic ice sheet. Numerical models support a touchdown scenario. This study has implications for the identification and inventory of large cosmic events on Earth.

Schizophrenia-related endophenotypes in heterozygous neuregulin-1 'knockout' mice.

Neuregulin-1 (NRG1) has been shown to play a role in glutamatergic neurotransmission and is a risk gene for schizophrenia, in which there is evidence for hypoglutamatergic function. Sensitivity to the behavioural effects of the psychotomimetic N-methyl-D-aspartate receptor antagonists MK-801 and phencyclidine (PCP) was examined in mutant mice with heterozygous deletion of NRG1. Social behaviour (sociability, social novelty preference and dyadic interaction), together with exploratory activity, was assessed following acute or subchronic administration of MK-801 (0.1 and 0.2 mg/kg) or PCP (5 mg/kg). In untreated NRG1 mutants, levels of glutamate, N-acetylaspartate and GABA were determined using high-performance liquid chromatography and regional brain volumes were assessed using magnetic resonance imaging at 7T. NRG1 mutants, particularly males, displayed decreased responsivity to the locomotor-activating effects of acute PCP. Subchronic MK-801 and PCP disrupted sociability and social novelty preference in mutants and wildtypes and reversed the increase in both exploratory activity and social dominance-related behaviours observed in vehicle-treated mutants. No phenotypic differences were demonstrated in N-acetylaspartate, glutamate or GABA levels. The total ventricular and olfactory bulb volume was decreased in mutants. These data indicate a subtle role for NRG1 in modulating several schizophrenia-relevant processes including the effects of psychotomimetic N-methyl-D-aspartate receptor antagonists.

The small muscle-specific protein Csl modifies cell shape and promotes myocyte fusion in an insulin-like growth factor 1-dependent manner.

We have isolated a murine cDNA encoding a 9-kD protein, Chisel (Csl), in a screen for transcriptional targets of the cardiac homeodomain factor Nkx2-5. Csl transcripts were detected in atria and ventricles of the heart and in all skeletal muscles and smooth muscles of the stomach and pulmonary veins. Csl protein was distributed throughout the cytoplasm in fetal muscles, although costameric and M-line localization to the muscle cytoskeleton became obvious after further maturation. Targeted disruption of Csl showed no overt muscle phenotype. However, ectopic expression in C2C12 myoblasts induced formation of lamellipodia in which Csl protein became tethered to membrane ruffles. Migration of these cells was retarded in a monolayer wound repair assay. Csl-expressing myoblasts differentiated and fused normally, although in the presence of insulin-like growth factor (IGF)-1 they showed dramatically enhanced fusion, leading to formation of large dysmorphogenic "myosacs." The activities of transcription factors nuclear factor of activated T cells (NFAT) and myocyte enhancer-binding factor (MEF)2, were also enhanced in an IGF-1 signaling-dependent manner. The dynamic cytoskeletal localization of Csl and its dominant effects on cell shape and behavior and transcription factor activity suggest that Csl plays a role in the regulatory network through which muscle cells coordinate their structural and functional states during growth, adaptation, and repair.

Outgassed Water on Mars: Constraints from Melt Inclusions in SNC Meteorites.

The SNC (shergottite-nakhlite-chassignite) meteorites, thought to be igneous rocks from Mars, contain melt inclusions trapped at depth in early-formed crystals. Determination of the pre-eruptive water contents of SNC parental magmas from calculations of the solidification histories of these amphibole-bearing inclusions indicates that martian magmas commonly contained 1.4 percent water by weight. When combined with an estimate of the volume of igneous materials on Mars, this information suggests that the total amount of water outgassed since 3.9 billion years ago corresponds to global depths on the order of 200 meters. This value is significantly higher than previous geochemical estimates but lower than estimates based on erosion by floods. These results imply a wetter Mars interior than has been previously thought and support suggestions of significant outgassing before formation of a stable crust or heterogeneous accretion of a veneer of cometary matter.

Analysis of cardiac stem cell self-renewal dynamics in serum-free medium by single cell lineage tracking.

Cardiac colony forming unit-fibroblasts (cCFU-F) are a population of stromal cells residing within the SCA1+/PDGFRα+/CD31- fraction of adult mouse hearts, and which have functional characteristics akin to bone marrow mesenchymal stem cells. We hypothesise that they participate in cardiac homeostasis and repair through their actions as lineage progenitors and paracrine signaling hubs. However, cCFU-F are rare and there are no specific markers for these cells, making them challenging to study. cCFU can self-renew in vitro, although the common use of serum has made it difficult to identify cytokines that maintain lineage identity and self-renewal ability. Cell heterogeneity is an additional confounder as cCFU-F cultures are metastable. Here, we address these limitations by identifying serum-free medium (SFM) for growth, and by using cCFU-F isolated from PdgfraGFP/+ mice to record fate outcomes, morphology and PDGFRα expression for hundreds of single cells over time. We show that SFM supplemented with basic fibroblast growth factor, transforming growth factor-ß and platelet-derived growth factor, enhanced cCFU-F colony formation and long-term self-renewal, while maintaining cCFU-F potency. cCFU-F cultured in SFM maintained a higher proportion of PDGFRα+ cells, a marker of self-renewing cCFU-F, by increasing Pdgfra-GFP+ divisions and reducing the probability of spontaneous myofibroblast differentiation.

Altered motor activity, exploration and anxiety in heterozygous neuregulin 1 mutant mice: implications for understanding schizophrenia.

Human genetic studies have shown that neuregulin 1 (NRG1) is a potential susceptibility gene for schizophrenia. Nrg1 influences various neurodevelopmental processes, which are potentially related to schizophrenia. The neurodevelopmental theory of schizophrenia suggests that interactions between genetic and environmental factors are responsible for biochemical alterations leading to schizophrenia. To investigate these interactions and to match experimental design with the pathophysiology of schizophrenia, we applied a comprehensive behavioural phenotyping strategy for motor activity, exploration and anxiety in a heterozygous Nrg1 transmembrane domain mutant mouse model (Nrg1 HET) using different housing conditions and age groups. We observed a locomotion- and exploration-related hyperactive phenotype in Nrg1 HETs. Increased age had a locomotion- and exploration-inhibiting effect, which was significantly attenuated in mutant mice. Environmental enrichment (EE) had a stimulating influence on locomotion and exploration. The impact of EE was more pronounced in Nrg1 hypomorphs. Our study also showed a moderate task-specific anxiolytic-like phenotype for Nrg1 HETs, which was influenced by external factors. The behavioural phenotype detected in heterozygous Nrg1 mutant mice is not specific to schizophrenia per se, but the increased sensitivity of mutant mice to exogenous factors is consistent with the pathophysiology of schizophrenia and the neurodevelopmental theory. Our findings reinforce the importance of carefully controlling experimental designs for external factors and of comprehensive, integrative phenotyping strategies. Thus, Nrg1 HETs may, in combination with other genetic and drug models, help to clarify pathophysiological mechanisms behind schizophrenia.

Cardiac septal and valvular dysmorphogenesis in mice heterozygous for mutations in the homeobox gene Nkx2-5.

Heterozygous mutations in the cardiac homeobox gene, NKX2-5, underlie familial cases of atrial septal defect (ASD) with severe atrioventricular conduction block. In this study, mice heterozygous for Nkx2-5-null alleles were assessed for analogous defects. Although ASD occurred only rarely, atrial septal dysmorphogenesis was evident as increased frequencies of patent foramen ovale and septal aneurysm, and decreased length of the septum primum flap valve. These parameters were compounded by genetic background effects, and in the 129/Sv strain, septal dysmorphogenesis bordered on ASD in 17% of Nkx2-5 heterozygotes. In a proportion of neonatal heterozygotes, as well as in adults with ASD, we found that the size of the foramen ovale was significantly enlarged and altered in shape, potentially exposing the normally thin septum primum to excessive hemodynamic forces. Therefore, defective morphogenesis of the septum secundum may be one contributing factor in the generation of patent foramen ovale, septal aneurysm, and certain ASDs. Mild prolongation of P-R interval in females and an increased frequency of stenotic bicuspid aortic valves were also features of the Nkx2-5 heterozygous phenotype. Our data demonstrate that the complex effects of Nkx2-5 haploinsufficiency in mice are weaker but convergent with those in humans. As in the mouse, the phenotype of human NKX2-5 mutations may be modulated by interacting alleles.

Uncertainty of the thyroid dose conversion factor for inhalation intakes of 131I and its parametric uncertainty.

Inhalation exposures of 131I may occur in the physical form of a gas as well as a particulate. The physical characteristics pertaining to these different types of releases influence the intake and subsequent dose to an exposed individual. The thyroid dose received is influenced by the route through which 131I enters the body and its subsequent clearance, absorption and movement throughout the body. The radioactive iodine taken up in the gas-exchange tissues is cleared to other tissues or absorbed into the bloodstream of the individual and transferred to other organs. Iodine in the circulatory system is then taken up by the thyroid gland with resulting dose to that tissue. The magnitude of and uncertainty in the thyroid dose is important to the assessment of individuals exposed to airborne releases of radioiodine. Age- and gender-specific modelling parameters have resulted in significant differences between gas uptake, particulate deposition and inhalation dose conversion factors for each age and gender group. Inhalation dose conversion factors and their inherent uncertainty are markedly affected by the type of iodine intake. These differences are expected due to the modelling of particulate deposition versus uptake of gas in the respiratory tract. Inhalation dose estimates via iodine gases are very similar and separate classifications may not be necessarily based on this assessment.

Quantifying intrinsic and extrinsic control of single-cell fates in cancer and stem/progenitor cell pedigrees with competing risks analysis.

The molecular control of cell fate and behaviour is a central theme in biology. Inherent heterogeneity within cell populations requires that control of cell fate is studied at the single-cell level. Time-lapse imaging and single-cell tracking are powerful technologies for acquiring cell lifetime data, allowing quantification of how cell-intrinsic and extrinsic factors control single-cell fates over time. However, cell lifetime data contain complex features. Competing cell fates, censoring, and the possible inter-dependence of competing fates, currently present challenges to modelling cell lifetime data. Thus far such features are largely ignored, resulting in loss of data and introducing a source of bias. Here we show that competing risks and concordance statistics, previously applied to clinical data and the study of genetic influences on life events in twins, respectively, can be used to quantify intrinsic and extrinsic control of single-cell fates. Using these statistics we demonstrate that 1) breast cancer cell fate after chemotherapy is dependent on p53 genotype; 2) granulocyte macrophage progenitors and their differentiated progeny have concordant fates; and 3) cytokines promote self-renewal of cardiac mesenchymal stem cells by symmetric divisions. Therefore, competing risks and concordance statistics provide a robust and unbiased approach for evaluating hypotheses at the single-cell level.

Two CCAAT boxes in a novel inverted repeat motif are required for Hlx homeobox gene expression.

Hlx is a homeobox transcription factor gene required for normal intestinal and hepatic growth in development. We previously found high sequence identity and 17 conserved consensus cis-regulatory/transcription factor binding elements in the mouse and human Hlx 5' regions. A 594 bp sequence in the Hlx 5' region possessing the same activity in driving luciferase expression as larger Hlx 5' sequences had three segments each necessary but not sufficient for luciferase expression in NIH 3T3 cells (which express Hlx). Nine of the conserved putative regulatory elements are positioned within these segments, including two CCAAT boxes on opposite strands within a conserved 44 bp inverted repeat sequence. To test the hypothesis that these elements are required for promoter activity, we compared the reporter expression activity of segments containing mutations of these elements with activity of the parent Hlx promoter sequence. We found that mutation of either CCAAT box or a conserved AP-2 site resulted in a significant decrease in promoter activity. Restoration of the inverted repeat with complementary mutations of both CCAAT boxes did not restore activity. Further, mutation of other portions of the inverted repeat did not affect promoter activity. Mutation of other elements had no effect on promoter activity.

MyoD protein expression in Xenopus embryos closely follows a mesoderm induction-dependent amplification of MyoD transcription and is synchronous across the future somite axis.

The MyoD-related genes code for key regulators of skeletal muscle commitment and differentiation. In this study, expression of MyoD protein has been examined during Xenopus development. Protein is first detected in presumptive mesoderm at early gastrulation, directly following a dramatic increase in MyoD transcription that occurs in response to mesoderm induction. The pattern of expression resembles the muscle fate map at this time. Protein accumulates synchronously along the future somite axis, with no evidence of a spatial regulation which would explain the anterior/posterior wave of myogenic differentiation that follows MyoD expression. During gastrulation, the highest levels of MyoD are in cells next to the developing notochord, suggesting a role for the notochord in induction or maintenance of MyoD expression. After muscle differentiation, MyoD protein is degraded with a half-life of several hours, leading to very low expression in mature somites. These studies support a role for MyoD in induction of muscle mesoderm, but also point to the multi-layered regulation of these events.

Expression of NK-2 class homeobox gene Nkx2-6 in foregut endoderm and heart.

NK-2 class homeobox genes are candidate patterning and lineage regulators in diverse organisms. We report here the embryonic expression pattern of murine member, Nkx2-6. In keeping with its vertebrate relatives, Nkx2-6 was transcribed in ventrolateral embryonic structures. Expression was first detected at E8.0 in endodermal walls of the foregut pocket, tissue destined to become pharyngeal floor. From E8.0-10.5, transcripts were concentrated in pharyngeal pouches and juxtaposed arch ectoderm and mesoderm, as well as in more caudal gut segments. Expression was also seen at opposite poles of the developing heart from E8-8.5 in posterior myocardial progenitors, then sinus venosa and dorsal pericardium, and from E9.5 in outflow tract myocardium.

musculin: a murine basic helix-loop-helix transcription factor gene expressed in embryonic skeletal muscle.

We describe the embryonic expression of musculin, a new murine member of the bHLH family of transcription factors. Musculin protein is closely related to human ABF-1, which is expressed in activated B cells, and to epicardin/capsulin/Pod-1, which is expressed in branchial myoblasts, visceral and urogenital mesoderm and epicardium. In situ hybridisation revealed musculin expression in embryos was largely restricted to the embryonic skeletal muscle lineage. While all skeletal muscles expressed the gene, only a subset of myocytes within each muscle were positive, indicating molecular heterogeneity within fetal muscle.

DAN is a secreted glycoprotein related to Xenopus cerberus.

We report that DAN, a potential cell cycle regulator and tumour suppressor, is a secreted glycoprotein related to Xenopus cerberus. DAN, cerberus, its mouse relative Cer-1/cer-l/Cerberus-like/Cerr1, and the recently described factor DRM/Gremlin, appear to be members of the cystine knot superfamily, which includes TGFbetas and BMPs. Like cerberus and mCer-1, DAN-induced cement glands as well as markers of anterior neural tissue and endoderm in Xenopus animal cap assays, features of BMP signalling blockade. During mouse embryogenesis, Dan was expressed from E8.5 in cranial mesenchyme and somites, then later in limb and facial mesenchyme. The pattern in somites was highly dynamic, with transcripts initially localized to the caudal half of the nascent epithelial somite, then, after maturation, to sclerotomal cells adjacent to the neural tube. Dan was also expressed in the developing myotome. The expression domains include sites in which BMP inhibition is known to be important for development. Thus, DAN appears to be a secreted factor belonging to the cystine knot superfamily, and one of a growing number of antagonists acting to modulate BMP signalling during development.

Otomycosis due to coccidioidomycosis.

The course and treatment of two patients with otomycosis due to Coccidioides immitis, believed to be the first such cases reported, are described. Both infections appeared due to reactivation of hematogenously disseminated foci. Local and systemic chemotherapy plus surgery resulted in remission, and host immune response also appears to be an important factor. One patient, with systemic lupus erythematosus, required more extensive surgery, more chemotherapy, and reduction in steroid dose to arrest the disease. A combined surgical and chemotherapeutic approach appears necessary when otomycosis is due to invasive fungi such as C immitis.