ENU-induced mutations reveal quaking functions in embryogenesis and postnatal myelination

Since the discovery of the mouse quaking viable allele (qkv) in 1964, researchers have attempted to elucidate the function of quaking in postnatal brain myelination. More recently, a critical role for quaking in embryogenesis was revealed, but little has been determined on how the loss of quaking function leads to embryonic death. In the work presented here, we report that the defect in the embryonic lethal qkk2 allele is a T to A trans version in the KH domain, and that homozygous qkk2 mutant embryos die from cardiovascular defects. Antibodies against the vascular endothelial marker PECAM-1 reveal that qkk2 homozygous mutants completely lack the yolk sac vascular network present in wild-type embryos. Hearts of qkk2/qkk2 embryos lack the looping morphogenesis of their wild-type littennates, but Nkx2.5, a gene that functions in cardiac looping, is expressed in a wild-type pattern. Cardiac myocytes in mutant embryos also express α-sarcomeric actin protein and whole hearts in organ culture were found to beat at wild-type rates. Real-time quantitative RT-PCR reveals that cardiac developmental markers are expressed at normal levels in qkk2 mutant hearts but that expression of the vascularization gene, Vegf, is increased two-fold compared to wild-type. In addition, the cell cycle genes Ccnd1, Cdkn1a and Cdkn1c have abnormal expression compared to wild-type embryonic hearts. The neural associated genes Shh, Foxa2, Fgf8, and Pax6 as well as the notochord marker T, are all expressed in wild-type patterns in qkk2 embryos at the time of death. However, adult carriers of the qkk2 allele are significantly more susceptible to chemically induced seizures with the convulsant drug pentylenetetrazole than wild-type animals. In an effort to further dissect the function of quaking, we performed a single generation ENU mutagenesis screen to create new alleles. We report here, the generation of the qke5 allele which, unlike the previous induced alleles of quaking, is homozygous viable. The qke5 phenotype consists of postnatal quaking and seizures, which progresses to severe ataxia and early death. Ultrastructural analysis reveals an almost complete lack of myelin compared to both wild-type and qkk2/qkk2 brains. Protein expression analysis in the postnatal brain shows that like qkk2/qkk2, qke5/qke5 mice have low QKI-5 and no QKI-6 and QKI-7 proteins in developing oligodendrocytes. However, oligodendrocytes of both qke5 and qkv homozygous mice express a full range of developmental marker genes indicating that quaking may function relatively late in oligodendrocyte development. We determined by electroencephalogram recordings, that qke5/qke5 surface cortex activity is abnormal and that seizures result in electrical activity characteristic of a decrimental response. Together these results reveal a previously unsuspected and critical role for quaking in cardiovascular development as well as a more important role in postnatal brain myelination then had been determined from 40 years of studies utilizing the qkv allele

Respiratory Syncytial Virus In Connecticut: Predictors Of Seasonal Epidemic Timing

Introduction: Respiratory syncytial virus (RSV) is a primary cause of hospitalizations in children worldwide. Prophylaxis (Palivizumab) can be given to infants who are at high-risk of a severe RSV infection, but the timing of seasonal RSV epidemics need to be known in order to administer prophylaxis at the appropriate time. Methods: This study used data from the Connecticut State Inpatient Database to identify RSV hospitalizations based on ICD-9 diagnosis codes. A harmonic regression analysis was used to evaluate RSV epidemic timing at the county level; subsequently, a hierarchical model was fit to assess RSV epidemic timing at the ZIP code level. Finally, a linear regression model was used to investigate demographic characteristics that were predictive of RSV epidemic timing. Results: 9,740 hospitalizations coded as RSV occurred among children less than 2 years old in Connecticut between July 1, 1997 and June 30, 2013. The seasonal RSV epidemic in the earliest county (Fairfield County) peaked 2.55 weeks earlier than the latest county (Tolland County). The earliest ZIP code had a seasonal RSV epidemic that peaked 4.64 weeks earlier than the latest ZIP code. Demographic characteristics that were significantly associated with ZIP code level RSV peak-timing included population density of childrenblack. Conclusions: Seasonal RSV epidemics in Connecticut occurred earlier in areas that were more urban, had higher population density, and larger black populations. These findings could be used to better time the administration of prophylaxis to high-risk infants

Eurasian watermilfoil fitness loss and invasion potential following desiccation during simulated overland transport

Abstract Vegetative reproduction promotes human-mediated dispersal of aquatic invasive plants as fragments "hitchhike" between water bodies on boats and trailers. However, desiccation of plant fragments may also reduce fitness, decreasing the likelihood of fragment survival as transport distances increase. Current inter-lake invasive species spread models do not directly consider fitness loss due to desiccation and mechanical damage of the transport pathway. Here, we estimate survival as a function of desiccation exposure for Eurasian watermilfoil (Myriophyllum spicatum). Following desiccation treatments, we monitored survival and root formation of individual fragments and assessed the differences between treatments. Highest survival rates occurred for short (< one hour) air exposures and coiled fragments with root production for the coiled treatment occurring in less than two weeks, irrespective of fragment length. In contrast, fragments that experienced desiccation for more than 24 hours had little risk of surviving. Our results emphasize the threat posed by same-day overland movements of boats from invaded to uninvaded waterways, and provide managers with a surveillance radius to inform delimitation surveys arising from the discovery of a new invasion

Alkylation damage causes MMR-dependent chromosomal instability in vertebrate embryos

SN1-type alkylating agents, like N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), are potent mutagens. Exposure to alkylating agents gives rise to O6-alkylguanine, a modified base that is recognized by DNA mismatch repair (MMR) proteins but is not repairable, resulting in replication fork stalling and cell death. We used a somatic mutation detection assay to study the in vivo effects of alkylation damage on lethality and mutation frequency in developing zebrafish embryos. Consistent with the damage-sensing role of the MMR system, mutant embryos lacking the MMR enzyme MSH6 displayed lower lethality than wild-type embryos after exposure to ENU and MNU. In line with this, alkylation-induced somatic mutation frequencies were found to be higher in wild-type embryos than in the msh6 loss-of-function mutants. These mutations were found to be chromosomal aberrations that may be caused by chromosomal breaks that arise from stalled replication forks. As these chromosomal breaks arise at replication, they are not expected to be repaired by non-homologous end joining. Indeed, Ku70 loss-of-function mutants were found to be equally sensitive to ENU as wild-type embryos. Taken together, our results suggest that in vivo alkylation damage results in chromosomal instability and cell death due to aberrantly processed MMR-induced stalled replication forks

Expressed sequence tag analysis of adult human optic nerve for NEIBank: Identification of cell type and tissue markers

<p>Abstract</p> <p>Background</p> <p>The optic nerve is a pure white matter central nervous system (CNS) tract with an isolated blood supply, and is widely used in physiological studies of white matter response to various insults. We examined the gene expression profile of human optic nerve (ON) and, through the NEIBANK online resource, to provide a resource of sequenced verified cDNA clones. An un-normalized cDNA library was constructed from pooled human ON tissues and was used in expressed sequence tag (EST) analysis. Location of an abundant oligodendrocyte marker was examined by immunofluorescence. Quantitative real time polymerase chain reaction (qRT-PCR) and Western analysis were used to compare levels of expression for key calcium channel protein genes and protein product in primate and rodent ON.</p> <p>Results</p> <p>Our analyses revealed a profile similar in many respects to other white matter related tissues, but significantly different from previously available ON cDNA libraries. The previous libraries were found to include specific markers for other eye tissues, suggesting contamination. Immune/inflammatory markers were abundant in the new ON library. The oligodendrocyte marker QKI was abundant at the EST level. Immunofluorescence revealed that this protein is a useful oligodendrocyte cell-type marker in rodent and primate ONs. L-type calcium channel EST abundance was found to be particularly low. A qRT-PCR-based comparative mammalian species analysis reveals that L-type calcium channel expression levels are significantly lower in primate than in rodent ON, which may help account for the class-specific difference in responsiveness to calcium channel blocking agents. Several known eye disease genes are abundantly expressed in ON. Many genes associated with normal axonal function, mRNAs associated with axonal transport, inflammation and neuroprotection are observed.</p> <p>Conclusion</p> <p>We conclude that the new cDNA library is a faithful representation of human ON and EST data provide an initial overview of gene expression patterns in this tissue. The data provide clues for tissue-specific and species-specific properties of human ON that will help in design of therapeutic models.</p

Integrated genomics of susceptibility to alkylator-induced leukemia in mice

<p>Abstract</p> <p>Background</p> <p>Therapy-related acute myeloid leukemia (t-AML) is a secondary, generally incurable, malignancy attributable to chemotherapy exposure. Although there is a genetic component to t-AML susceptibility in mice, the relevant loci and the mechanism(s) by which they contribute to t-AML are largely unknown. An improved understanding of susceptibility factors and the biological processes in which they act may lead to the development of t-AML prevention strategies.</p> <p>Results</p> <p>In this work we applied an integrated genomics strategy in inbred strains of mice to find novel factors that might contribute to susceptibility. We found that the pre-exposure transcriptional state of hematopoietic stem/progenitor cells predicts susceptibility status. More than 900 genes were differentially expressed between susceptible and resistant strains and were highly enriched in the apoptotic program, but it remained unclear which genes, if any, contribute directly to t-AML susceptibility. To address this issue, we integrated gene expression data with genetic information, including single nucleotide polymorphisms (SNPs) and DNA copy number variants (CNVs), to identify genetic networks underlying t-AML susceptibility. The 30 t-AML susceptibility networks we found are robust: they were validated in independent, previously published expression data, and different analytical methods converge on them. Further, the networks are enriched in genes involved in cell cycle and DNA repair (pathways not discovered in traditional differential expression analysis), suggesting that these processes contribute to t-AML susceptibility. Within these networks, the putative regulators (e.g., <it>Parp2</it>, <it>Casp9</it>, <it>Polr1b</it>) are the most likely to have a non-redundant role in the pathogenesis of t-AML. While identifying these networks, we found that current CNVR and SNP-based haplotype maps in mice represented distinct sources of genetic variation contributing to expression variation, implying that mapping studies utilizing either source alone will have reduced sensitivity.</p> <p>Conclusion</p> <p>The identification and prioritization of genes and networks not previously implicated in t-AML generates novel hypotheses on the biology and treatment of this disease that will be the focus of future research.</p

Systematic generation of in vivo G protein-coupled receptor mutants in the rat

G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies

Quaking Regulates Hnrnpa1 Expression through Its 3′ UTR in Oligodendrocyte Precursor Cells

In mice, Quaking (Qk) is required for myelin formation; in humans, it has been associated with psychiatric disease. QK regulates the stability, subcellular localization, and alternative splicing of several myelin-related transcripts, yet little is known about how QK governs these activities. Here, we show that QK enhances Hnrnpa1 mRNA stability by binding a conserved 3′ UTR sequence with high affinity and specificity. A single nucleotide mutation in the binding site eliminates QK-dependent regulation, as does reduction of QK by RNAi. Analysis of exon expression across the transcriptome reveals that QK and hnRNP A1 regulate an overlapping subset of transcripts. Thus, a simple interpretation is that QK regulates a large set of oligodendrocyte precursor genes indirectly by increasing the intracellular concentration of hnRNP A1. Together, the data show that hnRNP A1 is an important QK target that contributes to its control of myelin gene expression

Comparative genetic analysis: the utility of mouse genetic systems for studying human monogenic disease

One of the long-term goals of mutagenesis programs in the mouse has been to generate mutant lines to facilitate the functional study of every mammalian gene. With a combination of complementary genetic approaches and advances in technology, this aim is slowly becoming a reality. One of the most important features of this strategy is the ability to identify and compare a number of mutations in the same gene, an allelic series. With the advent of gene-driven screening of mutant archives, the search for a specific series of interest is now a practical option. This review focuses on the analysis of multiple mutations from chemical mutagenesis projects in a wide variety of genes and the valuable functional information that has been obtained from these studies. Although gene knockouts and transgenics will continue to be an important resource to ascertain gene function, with a significant proportion of human diseases caused by point mutations, identifying an allelic series is becoming an equally efficient route to generating clinically relevant and functionally important mouse models