Search results
Found 12032 matches for
Gestational stress induces the unfolded protein response, resulting in heart defects.
Congenital heart disease (CHD) is an enigma. It is the most common human birth defect and yet, even with the application of modern genetic and genomic technologies, only a minority of cases can be explained genetically. This is because environmental stressors also cause CHD. Here we propose a plausible non-genetic mechanism for induction of CHD by environmental stressors. We show that exposure of mouse embryos to short-term gestational hypoxia induces the most common types of heart defect. This is mediated by the rapid induction of the unfolded protein response (UPR), which profoundly reduces FGF signaling in cardiac progenitor cells of the second heart field. Thus, UPR activation during human pregnancy might be a common cause of CHD. Our findings have far-reaching consequences because the UPR is activated by a myriad of environmental or pathophysiological conditions. Ultimately, our discovery could lead to preventative strategies to reduce the incidence of human CHD.
The onset of circulation triggers a metabolic switch required for endothelial to hematopoietic transition.
Hematopoietic stem cells (HSCs) emerge during development from the vascular wall of the main embryonic arteries. The onset of circulation triggers several processes that provide critical external factors for HSC generation. Nevertheless, it is not fully understood how and when the onset of circulation affects HSC emergence. Here we show that in Ncx1-/- mouse embryos devoid of circulation the HSC lineage develops until the phenotypic pro-HSC stage. However, these cells reside in an abnormal microenvironment, fail to activate the hematopoietic program downstream of Runx1, and are functionally impaired. Single-cell transcriptomics shows that during the endothelial-to-hematopoietic transition, Ncx1-/- cells fail to undergo a glycolysis to oxidative phosphorylation metabolic switch present in wild-type cells. Interestingly, experimental activation of glycolysis results in decreased intraembryonic hematopoiesis. Our results suggest that the onset of circulation triggers metabolic changes that allow HSC generation to proceed.
Functional analysis of a gene-edited mouse model to gain insights into the disease mechanisms of a titin missense variant.
Titin truncating variants are a well-established cause of cardiomyopathy; however, the role of titin missense variants is less well understood. Here we describe the generation of a mouse model to investigate the underlying disease mechanism of a previously reported titin A178D missense variant identified in a family with non-compaction and dilated cardiomyopathy. Heterozygous and homozygous mice carrying the titin A178D missense variant were characterised in vivo by echocardiography. Heterozygous mice had no detectable phenotype at any time point investigated (up to 1 year). By contrast, homozygous mice developed dilated cardiomyopathy from 3 months. Chronic adrenergic stimulation aggravated the phenotype. Targeted transcript profiling revealed induction of the foetal gene programme and hypertrophic signalling pathways in homozygous mice, and these were confirmed at the protein level. Unsupervised proteomics identified downregulation of telethonin and four-and-a-half LIM domain 2, as well as the upregulation of heat shock proteins and myeloid leukaemia factor 1. Loss of telethonin from the cardiac Z-disc was accompanied by proteasomal degradation; however, unfolded telethonin accumulated in the cytoplasm, leading to a proteo-toxic response in the mice.We show that the titin A178D missense variant is pathogenic in homozygous mice, resulting in cardiomyopathy. We also provide evidence of the disease mechanism: because the titin A178D variant abolishes binding of telethonin, this leads to its abnormal cytoplasmic accumulation. Subsequent degradation of telethonin by the proteasome results in proteasomal overload, and activation of a proteo-toxic response. The latter appears to be a driving factor for the cardiomyopathy observed in the mouse model.
Heterozygous loss of WBP11 function causes multiple congenital defects in humans and mice.
The genetic causes of multiple congenital anomalies are incompletely understood. Here, we report novel heterozygous predicted loss-of-function (LoF) and predicted damaging missense variants in the WW domain binding protein 11 (WBP11) gene in seven unrelated families with a variety of overlapping congenital malformations, including cardiac, vertebral, tracheo-esophageal, renal and limb defects. WBP11 encodes a component of the spliceosome with the ability to activate pre-messenger RNA splicing. We generated a Wbp11 null allele in mouse using CRISPR-Cas9 targeting. Wbp11 homozygous null embryos die prior to E8.5, indicating that Wbp11 is essential for development. Fewer Wbp11 heterozygous null mice are found than expected due to embryonic and postnatal death. Importantly, Wbp11 heterozygous null mice are small and exhibit defects in axial skeleton, kidneys and esophagus, similar to the affected individuals, supporting the role of WBP11 haploinsufficiency in the development of congenital malformations in humans. LoF WBP11 variants should be considered as a possible cause of VACTERL association as well as isolated Klippel-Feil syndrome, renal agenesis or esophageal atresia.
Better communication between experts is needed to solve the environmental origins of birth defects.
More than 6% of babies are born with a structural or functional defect, and many of these need special care and treatment to survive and thrive. Such defects can be inherited, arise through exposure to altered conditions or compounds in the womb, or result from a combination of genetic and environmental factors. Since the 1940s, animal experiments and epidemiological studies have identified many environmental factors that can cause particular birth defects. More recently, advances in genomics have allowed a simple genetic diagnosis in ∼ 30% of birth defects. However, the cause of the remainder is a mystery. I believe that a key limiter to successful identification of new environmental factors is that clinicians, epidemiologists and developmental biologists all approach the topic from different angles. I propose that better communication between such experts will further increase our understanding of the environmental causes of birth defects, and potentially reduce their global burden.
Analysis of Placental Arteriovenous Formation Reveals New Insights Into Embryos With Congenital Heart Defects.
The placental vasculature provides the developing embryo with a circulation to deliver nutrients and dispose of waste products. However, in the mouse, the vascular components of the chorio-allantoic placenta have been largely unexplored due to a lack of well-validated molecular markers. This is required to study how these blood vessels form in development and how they are impacted by embryonic or maternal defects. Here, we employed marker analysis to characterize the arterial/arteriole and venous/venule endothelial cells (ECs) during normal mouse placental development. We reveal that placental ECs are potentially unique compared with their embryonic counterparts. We assessed embryonic markers of arterial ECs, venous ECs, and their capillary counterparts-arteriole and venule ECs. Major findings were that the arterial tree exclusively expressed Dll4, and venous vascular tree could be distinguished from the arterial tree by Endomucin (EMCN) expression levels. The relationship between the placenta and developing heart is particularly interesting. These two organs form at the same stages of embryogenesis and are well known to affect each other's growth trajectories. However, although there are many mouse models of heart defects, these are not routinely assessed for placental defects. Using these new placental vascular markers, we reveal that mouse embryos from one model of heart defects, caused by maternal iron deficiency, also have defects in the formation of the placental arterial, but not the venous, vascular tree. Defects to the embryonic cardiovascular system can therefore have a significant impact on blood flow delivery and expansion of the placental arterial tree.
Functional genomics and gene-environment interaction highlight the complexity of congenital heart disease caused by Notch pathway variants.
Congenital heart disease (CHD) is the most common birth defect and brings with it significant mortality and morbidity. The application of exome and genome sequencing has greatly improved the rate of genetic diagnosis for CHD but the cause in the majority of cases remains uncertain. It is clear that genetics, as well as environmental influences, play roles in the aetiology of CHD. Here we address both these aspects of causation with respect to the Notch signalling pathway. In our CHD cohort, variants in core Notch pathway genes account for 20% of those that cause disease, a rate that did not increase with the inclusion of genes of the broader Notch pathway and its regulators. This is reinforced by case-control burden analysis where variants in Notch pathway genes are enriched in CHD patients. This enrichment is due to variation in NOTCH1. Functional analysis of some novel missense NOTCH1 and DLL4 variants in cultured cells demonstrate reduced signalling activity, allowing variant reclassification. Although loss-of-function variants in DLL4 are known to cause Adams-Oliver syndrome, this is the first report of a hypomorphic DLL4 allele as a cause of isolated CHD. Finally, we demonstrate a gene-environment interaction in mouse embryos between Notch1 heterozygosity and low oxygen- or anti-arrhythmic drug-induced gestational hypoxia, resulting in an increased incidence of heart defects. This implies that exposure to environmental insults such as hypoxia could explain variable expressivity and penetrance of observed CHD in families carrying Notch pathway variants.
Tamoxifen administration in pregnant mice can be deleterious to both mother and embryo.
Since it was introduced 20 years ago, tamoxifen-inducible genetic recombination in vivo has become a standard tool in many fields. This technique has great utility, allowing precise temporal and spatial gene recombination mediated by expression of a Cre recombinase-oestrogen receptor hormone binding domain fusion protein. It is frequently used in developmental biology, either for accurate spatio-temporal gene deletion or for lineage-labelling. Administration of high doses of tamoxifen can rapidly induce abortion in pregnant mice but this can be partially overcome by progesterone co-administration. However, administration of tamoxifen to pregnant mice early in pregnancy may have potentially lethal effects on the mother independently of abortion, and can also severely perturb embryonic development. Despite this, only a few published studies mention this fact in passing, and standard parameters for successful or unsuccessful use of tamoxifen in pregnant mice have not been reported. Therefore, in the interests of providing a framework for more humane animal research, we describe our experiences of tamoxifen administration during early gestation in mice. These observations should assist the design of future studies in accordance with the principles of the three Rs (Replacement, Reduction and Refinement of Animals in Research).
Gene-environment interaction impacts on heart development and embryo survival.
Congenital heart disease (CHD) is the most common type of birth defect. In recent years, research has focussed on identifying the genetic causes of CHD. However, only a minority of CHD cases can be attributed to single gene mutations. In addition, studies have identified different environmental stressors that promote CHD, but the additive effect of genetic susceptibility and environmental factors is poorly understood. In this context, we have investigated the effects of short-term gestational hypoxia on mouse embryos genetically predisposed to heart defects. Exposure of mouse embryos heterozygous for Tbx1 or Fgfr1/Fgfr2 to hypoxia in utero increased the incidence and severity of heart defects while Nkx2-5+/- embryos died within 2 days of hypoxic exposure. We identified the molecular consequences of the interaction between Nkx2-5 and short-term gestational hypoxia, which suggest that reduced Nkx2-5 expression and a prolonged hypoxia-inducible factor 1α response together precipitate embryo death. Our study provides insight into the causes of embryo loss and variable penetrance of monogenic CHD, and raises the possibility that cases of foetal death and CHD in humans could be caused by similar gene-environment interactions.
NAD Deficiency, Congenital Malformations, and Niacin Supplementation.
BACKGROUND: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS: We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. RESULTS: Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS: Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).
Kousseff syndrome: a causally heterogeneous disorder.
The existence of Kousseff syndrome as a distinct entity has been thrown into doubt by a recent study conducted on the family originally reported by Kousseff. In all cases where chromosome 22q11.2 FISH testing has been undertaken, including the original sibship, a chromosome 22q11.2-microdeletion has been identified. We report two cases of sacral myelomeningocele associated with a conotruncal cardiac anomaly or "Kousseff syndrome." The first case, a 4-year-old girl, had a sacral myelomeningocele, tetralogy of Fallot, microcephaly, hydrocephalus, hypoplasia of the corpus callosum and mild-moderate developmental delay. Chromosome 22q11.2 FISH was normal and the facial phenotype was not that of velocardiofacial syndrome. Sequencing of the entire coding region of CITED2 did not reveal a mutation. The second case, a male infant, was found to have a 22q11.2-microdeletion. These cases confirm Kousseff syndrome to be a causally heterogeneous disorder.
Sequence of a cDNA encoding chicken high-mobility-group protein-2.
There are several members of the high-mobility-group (HMG) of DNA-binding proteins, including HMG-1, HMG-2, HMG-14 and HMG-17 [Johns: The HMG Chromosomal Proteins. Academic Press, London, 1982]. We report here sequences encoding the chicken HMG-2 protein of 207 amino acids (aa). This assignment is made on the basis of available data which indicate 89% homology of the chicken aa sequence to porcine HMG-2. This compares with 78-81% homology to the HMG-1 proteins of rat, hamster, human, porcine, and bovine origin.
A Screening Approach to Identify Clinically Actionable Variants Causing Congenital Heart Disease in Exome Data.
BACKGROUND: Congenital heart disease (CHD)-structural abnormalities of the heart that arise during embryonic development-is the most common inborn malformation, affecting ≤1% of the population. However, currently, only a minority of cases can be explained by genetic abnormalities. The goal of this study was to identify disease-causal genetic variants in 30 families affected by CHD. METHODS: Whole-exome sequencing was performed with the DNA of multiple family members. We utilized a 2-tiered whole-exome variant screening and interpretation procedure. First, we manually curated a high-confidence list of 90 genes known to cause CHD in humans, identified predicted damaging variants in genes on this list, and rated their pathogenicity using American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. RESULTS: In 3 families (10%), we found pathogenic variants in known CHD genes TBX5, TFAP2B, and PTPN11, explaining the cardiac lesions. Second, exomes were comprehensively analyzed to identify additional predicted damaging variants that segregate with disease in CHD candidate genes. In 10 additional families (33%), likely disease-causal variants were uncovered in PBX1, CNOT1, ZFP36L2, TEK, USP34, UPF2, KDM5A, KMT2C, TIE1, TEAD2, and FLT4. CONCLUSIONS: The pathogenesis of CHD could be explained using our high-confidence CHD gene list for variant filtering in a subset of cases. Furthermore, our unbiased screening procedure of family exomes implicates additional genes and variants in the pathogenesis of CHD, which suggest themselves for functional validation. This 2-tiered approach provides a means of (1) identifying clinically actionable variants and (2) identifying additional disease-causal genes, both of which are essential for improving the molecular diagnosis of CHD.
Environmental Risk Factors for Congenital Heart Disease.
Congenital heart disease (CHD) has many forms and a wide range of causes. Clinically, it is important to understand the causes. This allows estimation of recurrence rate, guides treatment options, and may also be used to formulate public health advice to reduce the population prevalence of CHD. The recent advent of sophisticated genetic and genomic methods has led to the identification of more than 100 genes associated with CHD. However, despite these great strides, to date only one-third of CHD cases have been shown to have a simple genetic cause. This is because CHD can also be caused by oligogenic factors, environmental factors, and/or gene-environment interaction. Although solid evidence for environmental causes of CHD have been available for almost 80 years, it is only very recently that the molecular mechanisms for these risk factors have begun to be investigated. In this review, we describe the most important environmental CHD risk factors, and what is known about how they cause CHD.
Insights into the Role of a Cardiomyopathy-Causing Genetic Variant in ACTN2.
Pathogenic variants in ACTN2, coding for alpha-actinin 2, are known to be rare causes of Hypertrophic Cardiomyopathy. However, little is known about the underlying disease mechanisms. Adult heterozygous mice carrying the Actn2 p.Met228Thr variant were phenotyped by echocardiography. For homozygous mice, viable E15.5 embryonic hearts were analysed by High Resolution Episcopic Microscopy and wholemount staining, complemented by unbiased proteomics, qPCR and Western blotting. Heterozygous Actn2 p.Met228Thr mice have no overt phenotype. Only mature males show molecular parameters indicative of cardiomyopathy. By contrast, the variant is embryonically lethal in the homozygous setting and E15.5 hearts show multiple morphological abnormalities. Molecular analyses, including unbiased proteomics, identified quantitative abnormalities in sarcomeric parameters, cell-cycle defects and mitochondrial dysfunction. The mutant alpha-actinin protein is found to be destabilised, associated with increased activity of the ubiquitin-proteasomal system. This missense variant in alpha-actinin renders the protein less stable. In response, the ubiquitin-proteasomal system is activated; a mechanism that has been implicated in cardiomyopathies previously. In parallel, a lack of functional alpha-actinin is thought to cause energetic defects through mitochondrial dysfunction. This seems, together with cell-cycle defects, the likely cause of the death of the embryos. The defects also have wide-ranging morphological consequences.
In Vivo Two-Photon Microscopy Reveals Sensory-Evoked Serotonin (5-HT) Release in Adult Mammalian Neocortex.
The recent development of genetically encoded fluorescent neurotransmitter biosensors has opened the door to recording serotonin (5-hydroxytryptamine, 5-HT) signaling dynamics with high temporal and spatial resolution in vivo. While this represents a significant step forward for serotonin research, the utility of available 5-HT biosensors remains to be fully established under diverse in vivo conditions. Here, we used two-photon microscopy in awake mice to examine the effectiveness of specific 5-HT biosensors for monitoring 5-HT dynamics in somatosensory cortex. Initial experiments found that whisker stimulation evoked a striking change in 5-HT biosensor signal. However, similar changes were observed in controls expressing green fluorescent protein, suggesting a potential hemodynamic artifact. Subsequent use of a second control fluorophore with emission peaks separated from the 5-HT biosensor revealed a reproducible, stimulus-locked increase in 5-HT signal. Our data highlight the promise of 5-HT biosensors for in vivo application, provided measurements are carried out with appropriate optical controls.
A neuronal mechanism controlling the choice between feeding and sexual behaviors in Drosophila.
Animals must express the appropriate behavior that meets their most pressing physiological needs and their environmental context. However, it is currently unclear how alternative behavioral options are evaluated and appropriate actions are prioritized. Here, we describe how fruit flies choose between feeding and courtship; two behaviors necessary for survival and reproduction. We show that sex- and food-deprived male flies prioritize feeding over courtship initiation, and manipulation of food quality or the animal's internal state fine-tunes this decision. We identify the tyramine signaling pathway as an essential mediator of this decision. Tyramine biosynthesis is regulated by the fly's nutritional state and acts as a satiety signal, favoring courtship over feeding. Tyramine inhibits a subset of feeding-promoting tyramine receptor (TyrR)-expressing neurons and activates P1 neurons, a known command center for courtship. Conversely, the perception of a nutritious food source activates TyrR neurons and inhibits P1 neurons. Therefore, TyrR and P1 neurons are oppositely modulated by starvation, via tyramine levels, and food availability. We propose that antagonistic co-regulation of neurons controlling alternative actions is key to prioritizing competing drives in a context- dependent manner.
Differential coding of absolute and relative aversive value in the Drosophila brain.
Animals use prior experience to assign absolute (good or bad) and relative (better or worse) value to new experience. These learned values guide appropriate later decision making. Even though our understanding of how the valuation system computes absolute value is relatively advanced, the mechanistic underpinnings of relative valuation are unclear. Here, we uncover mechanisms of absolute and relative aversive valuation in Drosophila. Three types of punishment-sensitive dopaminergic neurons (DANs) respond differently to electric shock intensity. During learning, these punishment-sensitive DANs drive intensity-scaled plasticity at their respective mushroom body output neuron (MBON) connections to code absolute aversive value. In contrast, by comparing the absolute value of current and previous aversive experiences, the MBON-DAN network can code relative aversive value by using specific punishment-sensitive DANs and recruiting a specific subtype of reward-coding DANs. Behavioral and physiological experiments revealed that a specific subtype of reward-coding DAN assigns a "better than" value to the lesser of the two aversive experiences. This study therefore highlights how appetitive-aversive system interactions within the MB network can code and compare sequential aversive experiences to learn relative aversive value.