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Generation of conditional Cited2 null alleles.
Cited2 is a transcriptional co-factor that is widely expressed in both embryonic and extraembryonic cells during early development. It is essential for embryonic development with Cited2 null embryos showing abnormal development of organs including heart, neural tube, adrenal glands, and placenta (both in trophoblast derivatives and invading fetal vasculature), as well as having defects in the establishment of the left-right body axis. We report the generation of two conditional null alleles allowing Cre-recombinase-mediated somatic cell gene inactivation. Mice heterozygous or homozygous for these alleles are viable and fertile. Crossing conditional mutants with CMV-Cre transgenic mice produces an embryonic-lethal phenotype in the offspring indistinguishable from germline null mutants. We also demonstrate that conditional deletion results in lacZ expression under the control of the Cited2 promoter. These alleles are therefore useful genetic tools for dissecting the functions of Cited2 in the formation of different organs and patterning of the developing embryo. genesis
Axial skeletal defects caused by mutation in the spondylocostal dysplasia/pudgy gene Dll3 are associated with disruption of the segmentation clock within the presomitic mesoderm.
A loss-of-function mutation in the mouse delta-like3 (Dll3) gene has been generated following gene targeting, and results in severe axial skeletal defects. These defects, which consist of highly disorganised vertebrae and costal defects, are similar to those associated with the Dll3-dependent pudgy mutant in mouse and with spondylocostal dysplasia (MIM 277300) in humans. This study demonstrates that Dll3(neo) and Dll3(pu) are functionally equivalent alleles with respect to the skeletal dysplasia, and we suggest that the three human DLL3 mutations associated with spondylocostal dysplasia are also functionally equivalent to the Dll3(neo) null allele. Our phenotypic analysis of Dll3(neo)/Dll3(neo) mutants shows that the developmental origins of the skeletal defects lie in delayed and irregular somite formation, which results in the perturbation of anteroposterior somite polarity. As the expression of Lfng, Hes1, Hes5 and Hey1 is disrupted in the presomitic mesoderm, we suggest that the somitic aberrations are founded in the disruption of the segmentation clock that intrinsically oscillates within presomitic mesoderm.
Diverse requirements for Notch signalling in mammals.
The Notch signalling pathway has a central role in a wide variety of developmental processes and it is not therefore surprising that mutations in components of this pathway can cause dramatic human genetic disorders. One developmental process in which the Notch pathway is involved at multiple levels is somitogenesis, the mechanism by which the embryo is divided into segments that ultimately form structures such as the axial skeleton and skeletal muscle of the trunk. We are investigating the human genetic disorder spondylocostal dysplasia (SCD), which is a group of malsegmentation syndromes that occur when this process is disrupted. Mutations in the Notch ligand DELTA-LIKE 3 (DLL3) are responsible for cases of autosomal recessive SCD type I (SCDO1), and we are using information derived from these mutations to study the structure of the DLL3 protein. To aid in elucidation of the underlying developmental defect in SCDO1, we have generated a mouse model by targeted deletion of the Dll3 gene (Dunwoodie et al., 2002). These mice show segmentation defects similar to those seen in SCDO1. In addition, these mice have a distinct set of neural defects that may be useful in future neurological assessment of affected individuals. Finally, since not all cases of SCD are due to mutation of DLL3, we are investigating various genes to find other candidates involved in this genetic disease.
Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2.
The yeast UBC9 gene encodes a protein with homology to the E2 ubiquitin-conjugating enzymes that mediate the attachment of ubiquitin to substrate proteins [1]. Depletion of Ubc9p arrests cells in G2 or early M phase and stabilizes B-type cyclins [1]. p18(Ubc9), the Xenopus homolog of Ubc9p, associates specifically with p88(RanGAP1) and p340(RanBP2) [2]. Ran-binding protein 2 (p340(RanBP2)) is a nuclear pore protein [3] [4], and p88(RanGAP1) is a modified form of RanGAP1, a GTPase-activating protein for the small GTPase Ran [2]. It has recently been shown that mammalian RanGAP1 can be conjugated with SUMO-1, a small ubiquitin-related modifier [5-7], and that SUMO-1 conjugation promotes RanGAP1's interaction with RanBP2 [2,5,6]. Here we show that p18(Ubc9) acts as an E2-like enzyme for SUMO-1 conjugation, but not for ubiquitin conjugation. This suggests that the SUMO-1 conjugation pathway is biochemically similar to the ubiquitin conjugation pathway but uses a distinct set of enzymes and regulatory mechanisms. We also show that p18(Ubc9) interacts specifically with the internal repeat domain of RanBP2, which is a substrate for SUMO-1 conjugation in Xenopus egg extracts.
Thylacine 1 is expressed segmentally within the paraxial mesoderm of the Xenopus embryo and interacts with the Notch pathway.
The presomitic mesoderm of vertebrates undergoes a process of segmentation in which cell-cell interactions mediated by the Notch family of receptors and their associated ligands are involved. The vertebrate homologues of Drosophila &Dgr ; are expressed in a dynamic, segmental pattern within the presomitic mesoderm, and alterations in the function of these genes leads to a perturbed pattern of somite segmentation. In this study we have characterised Thylacine 1 which encodes a basic helix-loop-helix class transcription activator. Expression of Thylacine is restricted to the presomitic mesoderm, localising to the anterior half of several somitomeres in register with domains of X-Delta-2 expression. Ectopic expression of Thylacine in embryos causes segmentation defects similar to those seen in embryos in which Notch signalling is altered, and these embryos also show severe disruption in the expression patterns of the marker genes X-Delta-2 and X-ESR5 within the presomitic mesoderm. Finally, the expression of Thylacine is altered in embryos when Notch signalling is perturbed. These observations suggest strongly that Thylacine 1 has a role in the segmentation pathway of the Xenopus embryo, by interacting with the Notch signalling pathway.
Subdivision of the cardiac Nkx2.5 expression domain into myogenic and nonmyogenic compartments.
Nkx2.5 is expressed in the cardiogenic mesoderm of avian, mouse, and amphibian embryos. To understand how various cardiac fates within this domain are apportioned, we fate mapped the mesodermal XNkx2.5 domain of neural tube stage Xenopus embryos. The lateral portions of the XNkx2.5 expression domain in the neural tube stage embryo (stage 22) form the dorsal mesocardium and roof of the pericardial cavity while the intervening ventral region closes to form the myocardial tube. XNkx2.5 expression is maintained throughout the period of heart tube morphogenesis and differentiation of myocardial, mesocardial, and pericardial tissues. A series of microsurgical experiments showed that myocardial differentiation in the lateral portion of the field is suppressed during normal development by signals from the prospective myocardium and by tissues located more dorsally in the embryo, in particular the neural tube. These signals combine to block myogenesis downstream of XNkx2.5 and at or above the level of contractile protein gene expression. We propose that the entire XNkx2.5/heart field is transiently specified as cardiomyogenic. Suppression of this program redirects lateral cells to adopt dorsal mesocardial and dorsal pericardial fates and subdivides the field into distinct myogenic and nonmyogenic compartments.
Old wares and new: five decades of investigation of somitogenesis in Xenopus laevis.
Somites are regular repeated structures formed in pairs on either side of the anterior-posterior axis of developing vertebrate embryos which give rise to all skeletal muscle of the body, the axial skeleton, the tendons and the dorsal dermis. Beginning in the middle of last century, somite formation has been extensively studied in the South African clawed frog (Xenopus laevis) using traditional embryological techniques. Recently, modern molecular methods have been applied to this system, producing substantial insights into the underlying molecular mechanisms driving these morphological events. In this review I discuss these new results in the context of the early embryological observations, looking at all levels of the process of somite formation, from the initial prepatterning of the presomitic mesoderm to the morphomechanical events required for the separation of each somite from the precursor tissue.
Complex SUMO-1 regulation of cardiac transcription factor Nkx2-5.
Reversible post-translational protein modifications such as SUMOylation add complexity to cardiac transcriptional regulation. The homeodomain transcription factor Nkx2-5/Csx is essential for heart specification and morphogenesis. It has been previously suggested that SUMOylation of lysine 51 (K51) of Nkx2-5 is essential for its DNA binding and transcriptional activation. Here, we confirm that SUMOylation strongly enhances Nkx2-5 transcriptional activity and that residue K51 of Nkx2-5 is a SUMOylation target. However, in a range of cultured cell lines we find that a point mutation of K51 to arginine (K51R) does not affect Nkx2-5 activity or DNA binding, suggesting the existence of additional Nkx2-5 SUMOylated residues. Using biochemical assays, we demonstrate that Nkx2-5 is SUMOylated on at least one additional site, and this is the predominant site in cardiac cells. The second site is either non-canonical or a "shifting" site, as mutation of predicted consensus sites and indeed every individual lysine in the context of the K51R mutation failed to impair Nkx2-5 transcriptional synergism with SUMO, or its nuclear localization and DNA binding. We also observe SUMOylation of Nkx2-5 cofactors, which may be critical to Nkx2-5 regulation. Our data reveal highly complex regulatory mechanisms driven by SUMOylation to modulate Nkx2-5 activity.
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.