Evolutionary origins of apoB mRNA editing: Catalysis by a cytidine deaminase that has acquired a novel RNA-binding motif at its active site

AbstractThe site-specific C to U editing of apolipoprotein B100 (apoB100) mRNA requires a 27 kDa protein (p27) with homology to cytidine deaminase. Here, we show that p27 is a zinc-containing deaminase, which operates catalytically like the E. coli enzyme that acts on monomeric substrate. In contrast with the bacterial enzyme that does not bind RNA, p27 interacts with its polymeric apoB m RNA substrate at AU sequences adjacent to the editing site. This interaction is necessary for editing. RNA binding is mediated through amino acid residues involved in zinc coordination, in proton shuttling, and in forming the αβα structure that encompasses the active site. However, certain mutations that inactivate the enzyme do not affect RNA binding. Thus, RNA binding does not require a catalytically active site. The acquisition of polymeric substrate binding provides a route for the evolution of this editing enzyme from one that acts on monomeric substrates

Structure of the DNA-bound T-box domain of human TBX1, a transcription factor associated with the DiGeorge syndrome

n/

Analysis of the asymmetrically expressed Ablim1 locus reveals existence of a lateral plate Nodal-independent left sided signal and an early, left-right independent role for nodal flow

BACKGROUND: Vertebrates show clear asymmetry in left-right (L-R) patterning of their organs and associated vasculature. During mammalian development a cilia driven leftwards flow of liquid leads to the left-sided expression of Nodal, which in turn activates asymmetric expression of the transcription factor Pitx2. While Pitx2 asymmetry drives many aspects of asymmetric morphogenesis, it is clear from published data that additional asymmetrically expressed loci must exist. RESULTS: A L-R expression screen identified the cytoskeletally-associated gene, actin binding lim protein 1 (Ablim1), as asymmetrically expressed in both the node and left lateral plate mesoderm (LPM). LPM expression closely mirrors that of Nodal. Significantly, Ablim1 LPM asymmetry was detected in the absence of detectable Nodal. In the node, Ablim1 was initially expressed symmetrically across the entire structure, resolving to give a peri-nodal ring at the headfold stage in a flow and Pkd2-dependent manner. The peri-nodal ring of Ablim1 expression became asymmetric by the mid-headfold stage, showing stronger right than left-sided expression. Node asymmetry became more apparent as development proceeded; expression retreated in an anticlockwise direction, disappearing first from the left anterior node. Indeed, at early somite stages Ablim1 shows a unique asymmetric expression pattern, in the left lateral plate and to the right side of the node. CONCLUSION: Left LPM Ablim1 is expressed in the absence of detectable LPM Nodal, clearly revealing existence of a Pitx2 and Nodal-independent left-sided signal in mammals. At the node, a previously unrecognised action of early nodal flow and Pkd2 activity, within the pit of the node, influences gene expression in a symmetric manner. Subsequent Ablim1 expression in the peri-nodal ring reveals a very early indication of L-R asymmetry. Ablim1 expression analysis at the node acts as an indicator of nodal flow. Together these results make Ablim1 a candidate for controlling aspects of L-R identity and patterning

Identification of cardiac malformations in mice lacking Ptdsr using a novel high-throughput magnetic resonance imaging technique

BACKGROUND: Congenital heart defects are the leading non-infectious cause of death in children. Genetic studies in the mouse have been crucial to uncover new genes and signaling pathways associated with heart development and congenital heart disease. The identification of murine models of congenital cardiac malformations in high-throughput mutagenesis screens and in gene-targeted models is hindered by the opacity of the mouse embryo. RESULTS: We developed and optimized a novel method for high-throughput multi-embryo magnetic resonance imaging (MRI). Using this approach we identified cardiac malformations in phosphatidylserine receptor (Ptdsr) deficient embryos. These included ventricular septal defects, double-outlet right ventricle, and hypoplasia of the pulmonary artery and thymus. These results indicate that Ptdsr plays a key role in cardiac development. CONCLUSIONS: Our novel multi-embryo MRI technique enables high-throughput identification of murine models for human congenital cardiopulmonary malformations at high spatial resolution. The technique can be easily adapted for mouse mutagenesis screens and, thus provides an important new tool for identifying new mouse models for human congenital heart diseases

Role of the Transcription Factor Sox4 in Insulin Secretion and Impaired Glucose Tolerance

OBJECTIVES— To identify, map, clone, and functionally validate a novel mouse model for impaired glucose tolerance and insulin secretion

Epiblastic Cited2 deficiency results in cardiac phenotypic heterogeneity and provides a mechanism for haploinsufficiency

AIMS: Deletion of the transcription factor Cited2 causes penetrant and phenotypically heterogenous cardiovascular and laterality defects and adrenal agenesis. Heterozygous human CITED2 mutation is associated with congenital heart disease, suggesting haploinsufficiency. Cited2 functions partly via a Nodal-->Pitx2c pathway controlling left-right patterning. In this present study we investigated the primary site of Cited2 function and mechanisms of haploinsufficiency. METHODS AND RESULTS: A Cited2 conditional allele enabled its deletion in particular cell lineages in mouse development. A lacZ reporter cassette allowed indication of deletion. Congenic Cited2 heterozygous mice were used to investigate haploinsufficiency. Embryos were examined by magnetic resonance imaging, by sectioning and by quantitative real-time polymerase chain reaction (qRT-PCR). Epiblast-specific deletion of Cited2 using Sox2Cre recapitulated penetrant and phenotypically heterogenous cardiovascular and laterality defects. Neural crest-specific deletion using Wnt1Cre affected cranial ganglia but not cardiac development. Mesodermal deletion with Mesp1Cre resulted in low penetrance of septal defect. Mesodermal deletion with T-Cre resulted in adrenal agenesis, but infrequent cardiac septal and laterality defects. beta-Galatactosidase staining and qRT-PCR demonstrated the efficiency and location of Cited2 deletion. Murine Cited2 heterozygosity is itself associated with cardiac malformation, with three of 45 embryos showing ventricular septal defect. Cited2 gene expression in E13.5 hearts was reduced 2.13-fold in Cited2(+/-) compared with wild-type (P = 2.62 x 10(-6)). The Cited2 target gene Pitx2c was reduced 1.5-fold in Cited2(+/-) (P = 0.038) hearts compared with wild-type, and reduced 4.9-fold in Cited2(-/-) hearts (P = 0.00031). Pitx2c levels were reduced two-fold (P = 0.009) in Cited2(+/-) embryos, in comparison with wild-type. Cited2 and Pitx2c expression were strongly correlated in wild-type and Cited2(+/-) hearts (Pearson rank correlation = 0.68, P = 0.0009). Cited2 expression was reduced 7474-fold in Sox2Cre deleted hearts compared with controls (P = 0.00017) and Pitx2c was reduced 3.1-fold (P = 0.013). Deletion of Cited2 with Mesp1Cre resulted in a 130-fold reduction in cardiac Cited2 expression compared with control (P = 0.0002), but Pitx2c expression was not affected. CONCLUSION: These results indicate that phenotypically heterogenous and penetrant cardiac malformations in Cited2 deficiency arise from a primary requirement in epiblast derivatives for left-right patterning, with a secondary cell-autonomous role in the mesoderm. Cardiac malformation associated with Cited2 haploinsufficiency may occur by reducing expression of key Cited2 targets such as Pitx2c

A mutation in the mitochondrial fission gene Dnm1l leads to cardiomyopathy

Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM). However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l) gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease

Genetic Variation in VEGF Does Not Contribute Significantly to the Risk of Congenital Cardiovascular Malformation

Several previous studies have investigated the role of common promoter variants in the vascular endothelial growth factor (VEGF) gene in causing congenital cardiovascular malformation (CVM). However, results have been discrepant between studies and no study to date has comprehensively characterised variation throughout the gene. We genotyped 771 CVM cases, of whom 595 had the outflow tract malformation Tetralogy of Fallot (TOF), and carried out TDT and case-control analyses using haplotype-tagging SNPs in VEGF. We carried out a meta-analysis of previous case-control or family-based studies that had typed VEGF promoter SNPs, which included an additional 570 CVM cases. To identify rare variants potentially causative of CVM, we carried out mutation screening in all VEGF exons and splice sites in 93 TOF cases. There was no significant effect of any VEGF haplotype-tagging SNP on the risk of CVM in our analyses of 771 probands. When the results of this and all previous studies were combined, there was no significant effect of the VEGF promoter SNPs rs699947 (OR 1.05 [95% CI 0.95–1.17]); rs1570360 (OR 1.17 [95% CI 0.99–1.26]); and rs2010963 (OR 1.04 [95% CI 0.93–1.16]) on the risk of CVM in 1341 cases. Mutation screening of 93 TOF cases revealed no VEGF coding sequence variants and no changes at splice consensus sequences. Genetic variation in VEGF appears to play a small role, if any, in outflow tract CVM susceptibility

Pitx2 confers left morphological, molecular, and functional identity to the sinus venosus myocardium

AIMS: The sinus venous myocardium, comprising the sinoatrial node (SAN) and sinus horns (SH), is a region subject to congenital malformations and cardiac arrhythmias. It differentiates from symmetric bilateral mesenchymal precursors, but morphological, molecular, and functional left/right differences are progressively established through development. The role of the laterality gene Pitx2 in this process is unknown. We aimed to elucidate the molecular events driving left/right patterning in the sinus venosus (SV) myocardium by using a myocardial Pitx2 knockout mouse. METHODS AND RESULTS: We generated a myocardial specific Pitx2 knockout model (cTP mice). cTP embryos present several features of Pitx2 null, including right atrial isomerism with bilateral SANs and symmetric atrial entrance of the systemic veins. By in situ hybridization and optical mapping analysis, we compared throughout development the molecular and functional properties of the SV myocardium in wt and mutant embryos. We observed that Pitx2 prevents the expansion of the left-SAN primordium at the onset of its differentiation into myocardium; Pitx2 promotes expansion of the left SH through development; Pitx2 dose-dependently represses the autorhythmic properties of the left SV myocardium at mid-gestation (E14.5); Pitx2 modulates late foetal gene expression at the left SH-derived superior caval vein. CONCLUSION: Pitx2 drives left/right patterning of the SV myocardium through multiple developmental steps. Overall, Pitx2 plays a crucial functional role by negatively modulating a nodal-type programme in the left SV myocardium