Search

Simple Summary: Ring chromosomes (RCs) are formed when chromosome ends fuse to form a circular structure. RCs are seen in <10% of patients with blood cancers (i.e., leukemias, lymphomas, and myelomas), and are associated with poor disease outcomes. We sought to evaluate how frequently RCs arise in patients diagnosed with these cancers and determine if there are any associated genetic variants, regions, or chromosomal abnormalities. Most patients with RCs have mutations in the TP53 gene and gross chromosomal rearrangements, particularly among chromosomes 5, 7, 11, and 17. Most of these patients also possess marker chromosomes with genetic material of unknown origin. The majority of RCs that resemble identifiable chromosomes, as well as chromosomes lacking gross rearrangements, were seen in patients without TP53 mutations. Our data suggest that mechanisms underlying RC generation may arise differently in patient groups with the presence or absence of functional TP53, a potentially important distinction for clinical decision making. Ring chromosomes (RC) are present in <10% of patients with hematological malignancies and are associated with poor prognosis. Until now, only small cohorts of patients with hematological neoplasms and concomitant RCs have been cytogenetically characterized. Here, we performed a conventional chromosome analysis on metaphase spreads from >13,000 patients diagnosed with hematological malignancies at the Johns Hopkins University Hospital and identified 98 patients with RCs—90 with myeloid malignancies and 8 with lymphoid malignancies. We also performed a targeted Next-Generation Sequencing (NGS) assay, using a panel of 642 cancer genes, to identify whether these patients harbor relevant pathogenic variants. Cytogenetic analyses revealed that RCs and marker chromosomes of unknown origin are concurrently present in most patients by karyotyping, and 93% of patients with NGS data have complex karyotypes. A total of 72% of these individuals have pathogenic mutations in TP53, most of whom also possess cytogenetic abnormalities resulting in the loss of 17p, including the loss of TP53. All patients with a detected RC and without complex karyotypes also lack TP53 mutations but have pathogenic mutations in TET2. Further, 70% of RCs that map to a known chromosome are detected in individuals without TP53 mutations. Our data suggest that RCs in hematological malignancies may arise through different mechanisms, but ultimately promote widespread chromosomal instability. [ABSTRACT FROM AUTHOR]

To overcome the ethical and technical limitations of in vivo human disease models, the broader scientific community frequently employs model organism-derived cell lines to investigate disease mechanisms, pathways, and therapeutic strategies. Despite the widespread use of certain in vitro models, many still lack contemporary genomic analysis supporting their use as a proxy for the affected human cells and tissues. Consequently, it is imperative to determine how accurately and effectively any proposed biological surrogate may reflect the biological processes it is assumed to model. One such cellular surrogate of human disease is the established mouse neural precursor cell line, SN4741, which has been used to elucidate mechanisms of neurotoxicity in Parkinson disease for over 25 years. Here, we are using a combination of classic and contemporary genomic techniques – karyotyping, RT-qPCR, single cell RNA-seq, bulk RNA-seq, and ATAC-seq – to characterize the transcriptional landscape, chromatin landscape, and genomic architecture of this cell line, and evaluate its suitability as a proxy for midbrain dopaminergic neurons in the study of Parkinson disease. We find that SN4741 cells possess an unstable triploidy and consistently exhibits low expression of dopaminergic neuron markers across assays, even when the cell line is shifted to the non-permissive temperature that drives differentiation. The transcriptional signatures of SN4741 cells suggest that they are maintained in an undifferentiated state at the permissive temperature and differentiate into immature neurons at the non-permissive temperature; however, they may not be dopaminergic neuron precursors, as previously suggested. Additionally, the chromatin landscapes of SN4741 cells, in both the differentiated and undifferentiated states, are not concordant with the open chromatin profiles of ex vivo, mouse E15.5 forebrain- or midbrain-derived dopaminergic neurons. Overall, our data suggest that SN4741 cells may reflect early aspects of neuronal differentiation but are likely not a suitable proxy for dopaminergic neurons as previously thought. The implications of this study extend broadly, illuminating the need for robust biological and genomic rationale underpinning the use of in vitro models of molecular processes. [ABSTRACT FROM AUTHOR]

Glutaminase (GLS), which converts glutamine to glutamate, plays a key role In cancer cell metabolism, growth, and proliferation. GLS is being explored as a cancer therapeutic target, but whether GLS inhibitors affect cancer cell-autonomous growth or the host microenvironment or have off-target effects is unknown. Here, we report that loss of one copy of Gls blunted tumor progression in an immune-competent MYC-mediated mouse model of hepatocellular carcinoma. Compared with results in untreated animals with MYC-induced hepatocellular carcinoma, administration of the GLS-specific inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) prolonged survival without any apparent toxicities. BPTES also inhibited growth of a MYC-dependent human B cell lymphoma cell line (P493) by blocking DNA replication, leading to cell death and fragmentation. In mice harboring P493 tumor xenografts, BPTES treatment inhibited tumor cell growth; however, P493 xenografts expressing a BPTES-resistant GLS mutant (GLS-K325A) or overexpressing GLS were not affected by BPTES treatment. Moreover, a customized Vivo-Morpholino that targets human GLS mRNA markedly inhibited P493 xenograft growth without affecting mouse Gls expression. Conversely, a Vivo-Morpholino directed at mouse Gls had no antitumor activity in vivo. Collectively, our studies demonstrate that GLS is required for tumorigenesis and support small molecule and genetic inhibition of GLS as potential approaches for targeting the tumor cell-autonomous dependence on GLS for cancer therapy., Introduction Cancer cells show profound metabolic changes, driven by oncogenes, such as RAS, PI3K, and MYC, or loss of tumor suppressors, such as p53, VHL, and LKB1 (1-5). Aerobic glycolysis [...]

The mutational diversity and phenotypic consequences of the RET (MIM 164761) tyrosine kinase in Hirschsprung disease (HSCR [MIM 142623]) was examined to explore the actual allelic diversity of a gene which impacts the phenotypic diversity and non-Mendelian features of a complex disease. The results demonstrated that both rare and common mutations, coding and noncoding, within the same gene contribute to HSCR in unique and specific ways that lead to recognizable genotype-phenotype associations.

Although the differentiation of ES cells to cardiomyocytes has been firmly established, the extent to which corresponding cardiac precursor cells can contribute to other cardiac populations remains unclear. To determine the molecular and cellular characteristics of cardiac-fated populations derived from mouse ES (mES) cells, we isolated cardiac progenitor cells (CPCs) from differentiating mES cell cultures by using a reporter cell line that expresses GFP under the control of a cardiac-specific enhancer element of Nkx2-5, a transcription factor expressed early in cardiac development. This ES cell--derived CPC population initially expressed genetic markers of both stem cells and mesoderm, while differentiated CPCs displayed markers of 3 distinct cell lineages (cardiomyocytes, vascular smooth muscle cells, and endothelial cells)--Flk1 (also known as Kdr), c-Kit, and Nkx2-5, but not Brachyury--and subsequently expressed Isl1. Clonally derived CPCs also demonstrated this multipotent phenotype. By transcription profiling of CPCs, we found that mES cell--derived CPCs displayed a transcriptional signature that paralleled in vivo cardiac development. Additionally, these studies suggested the involvement of genes that we believe were previously unknown to play a role in cardiac development. Taken together, our data demonstrate that ES cell--derived CPCs comprise a multipotent precursor population capable of populating multiple cardiac lineages and suggest that ES cell differentiation is a valid model for studying development of multiple cardiac-fated tissues., Introduction The heart is the first organ to function in the vertebrate embryo. In mice, the cardiac mesoderm forms a structure called the cardiac crescent at approximately 7.5 dpc; at [...]

RecQ DNA helicases resolve Rad-51-mediated recombination and suppress aberrant homologous recombination. RecQ gene loss is associated with cancer susceptibility and increased mitotic recombination. We have developed an in vivo assay based on a zebrafish pigment mutant for suppression of RecQ activity, and demonstrate that zebrafish RecQ genes have conserved function in suppressing mitotic recombination.

Author(s): Eileen Sproat Emison [1, 4]; Andrew S. McCallion [1, 4]; Carl S. Kashuk [1]; Richard T. Bush [1]; Elizabeth Grice [1]; Shin Lin [1]; Matthew E. Portnoy [2]; David [...]

Abstract Background Transcriptional regulatory elements are central to development and interspecific phenotypic variation. Current regulatory element prediction tools rely heavily upon conservation for prediction of putative elements. Recent in vitro observations from the ENCODE project combined with in vivo analyses at the zebrafish phox2b locus suggests that a significant fraction of regulatory elements may fall below commonly applied metrics of conservation. We propose to explore these observations in vivo at the human PHOX2B locus, and also evaluate the potential evidence for genome-wide applicability of these observations through a novel analysis of extant data. Results Transposon-based transgenic analysis utilizing a tiling path proximal to human PHOX2B in zebrafish recapitulates the observations at the zebrafish phox2b locus of both conserved and non-conserved regulatory elements. Analysis of human sequences conserved with previously identified zebrafish phox2b regulatory elements demonstrates that the orthologous sequences exhibit overlapping regulatory control. Additionally, analysis of non-conserved sequences scattered over 135 kb 5' to PHOX2B, provides evidence of non-conserved regulatory elements positively biased with close proximity to the gene. Furthermore, we provide a novel analysis of data from the ENCODE project, finding a non-uniform distribution of regulatory elements consistent with our in vivo observations at PHOX2B. These observations remain largely unchanged when one accounts for the sequence repeat content of the assayed intervals, when the intervals are sub-classified by biological role (developmental versus non-developmental), or by gene density (gene desert versus non-gene desert). Conclusion While regulatory elements frequently display evidence of evolutionary conservation, a fraction appears to be undetected by current metrics of conservation. In vivo observations at the PHOX2B locus, supported by our analyses of in vitro data from the ENCODE project, suggest that the risk of excluding non-conserved sequences in a search for regulatory elements may decrease as distance from the gene increases. Our data combined with the ENCODE data suggests that this may represent a genome wide trend.

Calcium channel subunits, including CACNA1C, have been associated with multiple psychiatric disorders. Specifically, genome wide association studies (GWAS) have repeatedly identified the single nucleotide polymorphism (SNP) rs1006737 in intron 3 of CACNA1C to be strongly associated with schizophrenia and bipolar disorder. Here, we show that rs1006737 marks a quantitative trait locus for CACNA1C transcript levels. We test 16 SNPs in high linkage disequilibrium with rs1007637 and find one, rs4765905, consistently showing allele-dependent regulatory function in reporter assays. We find allele-specific protein binding for 13 SNPs including rs4765905. Using protein microarrays, we identify several proteins binding ≥3 SNPs, but not control sequences, suggesting possible functional interactions and combinatorial haplotype effects. Finally, using circular chromatin conformation capture, we show interaction of the disease-associated region including the 16 SNPs with the CACNA1C promoter and other potential regulatory regions. Our results elucidate the pathogenic relevance of one of the best-supported risk loci for schizophrenia and bipolar disorder. [ABSTRACT FROM AUTHOR]

Most variants implicated in common human disease by genome-wide association studies (GWAS) lie in noncoding sequence intervals. Despite the suggestion that regulatory element disruption represents a common theme, identifying causal risk variants within implicated genomic regions remains a major challenge. Here we present a new sequence-based computational method to predict the effect of regulatory variation, using a classifier (gkm-SVM) that encodes cell type-specific regulatory sequence vocabularies. The induced change in the gkm-SVM score, deltaSVM, quantifies the effect of variants. We show that deltaSVM accurately predicts the impact of SNPs on DNase I sensitivity in their native genomic contexts and accurately predicts the results of dense mutagenesis of several enhancers in reporter assays. Previously validated GWAS SNPs yield large deltaSVM scores, and we predict new risk-conferring SNPs for several autoimmune diseases. Thus, deltaSVM provides a powerful computational approach to systematically identify functional regulatory variants. [ABSTRACT FROM AUTHOR]

We recently demonstrated that the gene encoding the RNA binding motif protein 24 (RBM24) is expressed during mouse cardiogenesis, and determined the developmental requirement for its zebrafish homologs Rbm24a and Rbm24b during cardiac development. We demonstrate here that both Rbm24a and Rbm24b are also required for normal somite and craniofacial development. Diminution of rbm24a or rbm24b gene products by morpholino knockdown resulted in significant disruption of somite formation. Detailed in situ hybridization-based analyses of a spectrum of somitogenesis-associated transcripts revealed reduced expression of the cyclic muscle pattering genes dlc and dld encoding Notch ligands, as well as their respective target genes her7, her1. By contrast expression of the Notch receptors notch1a and notch3 appears unchanged. Some RBM-family members have been implicated in pre-mRNA processing. Analysis of affected Notch-pathway mRNAs in rbm24a and rbm24b morpholino-injected embryos revealed aberrant transcript fragments of dlc and dld, but not her1 or her7, suggesting the reduction in transcription levels of Notch pathway components may result from aberrant processing of its ligands. These data imply a previously unknown requirement for Rbm24a and Rbm24b in somite and craniofacial development. Although we anticipate the influence of disrupting RBM24 homologs likely extends beyond the Notch pathway, our results suggest their perturbation may directly, or indirectly, compromise post-transcriptional processing, exemplified by imprecise processing of dlc and dld. [ABSTRACT FROM AUTHOR]

Vertebrate sensory organs originate from both cranial neural crest cells (CNCCs) and placodes. Previously, we have shown that the olfactory placode (OP) forms from a large field of cells extending caudally to the premigratory neural crest domain, and that OPs form through cell movements and not cell division. Concurrent with OP formation, CNCCs migrate rostrally to populate the frontal mass. However, little is known about the interactions between CNCCs and the placodes that form the olfactory sensory system. Previous reports suggest that the OP can generate cell types more typical of neural crest lineages such as neuroendocrine cells and glia, thus marking the OP as an unusual sensory placode. One possible explanation for this exception is that the neural crest origin of glia and neurons has been overlooked due to the intimate association of these two fields during migration. Using molecular markers and live imaging, we followed the development of OP precursors and of dorsally migrating CNCCs in zebrafish embryos. We generated a six4b:mCherry line (OP precursors) that, with a sox10:EGFP line (CNCCs), was used to follow cell migration. Our analyses showed that CNCCs associate with and eventually surround the forming OP with limited cell mixing occurring during this process. Developmental Dynamics 241:1143-1154, 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: The ERBB3 gene is essential for the proper development of the neural crest (NC) and its derivative populations such as Schwann cells. As with all cell fate decisions, transcriptional regulatory control plays a significant role in the progressive restriction and specification of NC derived lineages during development. However, little is known about the sequences mediating transcriptional regulation of ERBB3 or the factors that bind them. Results: In this study we identified three transcriptional enhancers at the ERBB3 locus and evaluated their regulatory potential in vitro in NC-derived cell types and in vivo in transgenic zebrafish. One enhancer, termed ERBB3•MCS6, which lies within the first intron of ERBB3, directs the highest reporter expression in vitro and also demonstrates epigenetic marks consistent with enhancer activity. We identify a consensus SOX10 binding site within ERBB3•MCS6 and demonstrate, in vitro, its necessity and sufficiency for the activity of this enhancer. Additionally, we demonstrate that transcription from the endogenous Erbb3 locus is dependent on Sox10. Further we demonstrate in vitro that Sox10 physically interacts with that ERBB3•MCS6. Consistent with its in vitro activity, we also show that ERBB3•MCS6 drives reporter expression in NC cells and a subset of its derivative lineages in vivo in zebrafish in a manner consistent with erbb3b expression. We also demonstrate, using morpholino analysis, that Sox10 is necessary for ERBB3•MCS6 expression in vivo in zebrafish. Conclusions: Taken collectively, our data suggest that ERBB3 may be directly regulated by SOX10, and that this control may in part be facilitated by ERBB3•MCS6. [ABSTRACT FROM AUTHOR]

Background: Myelin protein zero (MPZ) is a critical structural component of myelin in the peripheral nervous system. The MPZ gene is regulated, in part, by the transcription factors SOX10 and EGR2. Mutations in MPZ, SOX10, and EGR2 have been implicated in demyelinating peripheral neuropathies, suggesting that components of this transcriptional network are candidates for harboring disease-causing mutations (or otherwise functional variants) that affect MPZ expression. Methodology: We utilized a combination of multi-species sequence comparisons, transcription factor-binding site predictions, targeted human DNA re-sequencing, and in vitro and in vivo enhancer assays to study human non-coding MPZ variants. Principal Findings: Our efforts revealed a variant within the first intron of MPZ that resides within a previously described SOX10 binding site is associated with decreased enhancer activity, and alters binding of nuclear proteins. Additionally, the genomic segment harboring this variant directs tissue-relevant reporter gene expression in zebrafish. Conclusions: This is the first reported MPZ variant within a cis-acting transcriptional regulatory element. While we were unable to implicate this variant in disease onset, our data suggests that similar non-coding sequences should be screened for mutations in patients with neurological disease. Furthermore, our multi-faceted approach for examining the functional significance of non-coding variants can be readily generalized to study other loci important for myelin structure and function. [ABSTRACT FROM AUTHOR]

Transcriptional regulation of gene expression plays a significant role in establishing the diversity of human cell types and biological functions from a common set of genes. The components of regulatory control in the human genome include cis-acting elements that act across immense genomic distances to influence the spatial and temporal distribution of gene expression. Here we review the established categories of distant-acting regulatory elements, discussing the classical and contemporary evidence of their regulatory potential and clinical importance. Current efforts to identify regulatory sequences throughout the genome and elucidate their biological significance depend heavily on advances in sequence conservation-based analyses and on increasingly large-scale efforts applying transgenic technologies in model organisms. We discuss the advantages and limitations of sequence conservation as a predictor of regulatory function and present complementary emerging technologies now being applied to annotate regulatory elements in vertebrate genomes. [ABSTRACT FROM AUTHOR]

Abstract: Zebrafish transgenesis is a powerful and increasingly common strategy to assay vertebrate transcriptional regulatory control. Several challenges remain, however, to the broader application of this technique; they include increasing the rate with which transgenes can be analyzed and maximizing the informational value of the data generated. Presently, many rely on the injection of individual constructs and the analysis of resulting reporter expression in mosaic G0 embryos. Here, we contrast these approaches, examining whether injecting pooled transgene constructs can increase the efficiency with which regulatory sequences can be assayed, restricting analysis to the offspring of germ line transmitting transgenic zebrafish in an effort to reduce potential subjectivity. We selected a 64kb interval encompassing the human ASCL1 locus as our model interval and report the analysis of 9 highly conserved putative enhancers therein. We identified 32 transgene-positive zebrafish, transmitting one or more independent constructs displaying ASCL1-like regulatory control. Through examination of embryos harboring one or more transgenes, we demonstrate that five of the nine sequences account for the observed control and describe their likely roles in ASCL1 regulation. These data demonstrate the utility of this approach and its potential for further adaptation and higher throughput application. [Copyright &y& Elsevier]

Sox10 is a dynamically regulated transcription factor gene that is essential for the development of neural crest-derived and oligodendroglial populations. Developmental genes often require multiple regulatory sequences that integrate discrete and overlapping functions to coordinate their expression. To identify Sox10 cis-regulatory elements, we integrated multiple model systems, including cell-based screens and transposon-mediated transgensis in zebrafish, to scrutinize mammalian conserved, noncoding genomic segments at the mouse Sox10 locus. We demonstrate that eight of 11 Sox10 genomic elements direct reporter gene expression in transgenic zebrafish similar to patterns observed in transgenic mice, despite an absence of observable sequence conservation between mice and zebrafish. Multiple segments direct expression in overlapping populations of neural crest derivatives and glial cells, ranging from pan-Sox10 and pan-neural crest regulatory control to the modulation of expression in subpopulations of Sox10-expressing cells, including developing melanocytes and Schwann cells. Several sequences demonstrate overlapping spatial control, yet direct expression in incompletely overlapping developmental intervals. We were able to partially explain neural crest expression patterns by the presence of head to head SoxE family binding sites within two of the elements. Moreover, we were able to use this transcription factor binding site signature to identify the corresponding zebrafish enhancers in the absence of overall sequence homology. We demonstrate the utility of zebrafish transgenesis as a high-fidelity surrogate in the dissection of mammalian gene regulation, especially those with dynamically controlled developmental expression. [ABSTRACT FROM AUTHOR]

Remarkably, although cardiac disease accounts for the largest proportion of adult mortality and morbidity in the industrialized world, the genetic programs controlling early cardiogenesis are largely incompletely understood. To better understand this process, we set out to identify genes whose expression is enriched within early cardiac fated populations, obtaining the transcriptional signatures of mouse embryonic stem cells (mESCs) at defined intervals during their differentiation along a cardiac path. We compared the RNA profiles of cardiac precursors cells (CPCs) with time-matched non-CPCs and undifferentiated mESCs, using a transgenic mESC line harboring an Nkx2-5 cardiac-specific regulatory sequence driving green fluorescent protein (GFP) to facilitate selection of CPCs. We identify 176 transcripts that are significantly elevated in their abundance within CPCs compared with other assayed populations, predicting that they will likely play a role in cardiogenesis. Of note, approximately 24% (43/176) of the cardiogenic candidate transcripts have known roles in cardiac function or development. Importantly, we evaluated the biological relevance of a significant subset 31/133 (23%) of the remaining candidate genes by in situ hybridization at multiple time points during development (embryonic day, E7.5-9.5) and report that all were expressed in key cardiac structures during cardiogenesis. Furthermore 9/31, of which many were previously uncharacterized, were detected as early as the formation of the cardiac crescent. These data demonstrate the potential power of integrating genomic approaches with mESC differentiation to illuminate developmental processes, and provides a valuable resource that may be mined to further elucidate the genetic programs underlying cardiogenesis. [ABSTRACT FROM AUTHOR]

The myelin-associated oligodendrocytic basic protein (MOBP) family constitutes the third most abundant protein in CNS myelin. The mouse Mobp gene comprises eight exons. Mobp pre-mRNA processing gives rise to at least seven Mobp splice variants which are expressed solely in the oligodendrocyte. The predicted proteins all, with one exception, share a 68 residue amino terminus, encoded by exon 3. The carboxyl termini differ in length, giving rise to the diverse array of the protein isoforms. Like myelin basic protein, MOBP is present in the major dense line of CNS myelin suggesting a role in the compaction or stabilization of myelin. However, Mobp homozygous null mice display no overt clinical phenotype and no defect in the process of myelination. MOBP can induce experimental allergic encephalomyelitis in mice and has been proposed to have a role in the pathogenesis of multiple sclerosis. Despite 10 years of rigorous study, the normal physiological function of MOBP remains unknown. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Genetic studies of Hirschsprung disease, a common congenital malformation, have identified eight genes with mutations that can be associated with this condition. Mutations at individual loci are, however, neither necessary nor sufficient to cause clinical disease. We conducted a genome-wide association study in 43 Mennonite family trios using 2,083 microsatellites and single-nucleotide polymorphisms and a new multipoint linkage disequilibrium method that searches for association arising from common ancestry. We identified susceptibility loci at 10q11, 13q22 and 16q23; the gene at 13q22 is EDNRB, encoding a G proteincoupled receptor (GPCR) and the gene at 10qll is RET, encoding a receptor tyrosine kinase (RTK). Statistically significant joint transmission of RET and EDNRB alleles in affected individuals and non-complementation of aganglionosis in mouse intercrosses between Ret null and the Ednrb hypomorphic piebald allele are suggestive of epistasis between EDNRB and RET. Thus, genetic interaction between mutations in RET and EDNRB is an underlying mechanism for this complex disorder. [ABSTRACT FROM AUTHOR]

Rats harboring the agu mutation have altered behavior and brain pathology resembling human Parkinsonian syndromes; notably, they have a movement disorder and age-progressive dysfunction and death of neurons in the midbrain (substantia nigra pars compacta) that use dopamine as a neurotransmitter. We present evidence that this phenotype is due to a mutation in the rat protein kinase Cγ (in rat, Prkcg; in mouse, Prkcc; in human, PRKCG) gene, which generates a premature stop codon, drastically reducing the level of synthesis of the catalytic domain of the brain-specific protein kinase Cγ protein. [ABSTRACT FROM AUTHOR]

Trisomy for human chromosome 21 (Hsa21) results in Down syndrome (DS), one of the most genetically complex conditions compatible with human survival. Assessment of the physiological consequences of dosage-driven overexpression of individual Hsa21 genes during early embryogenesis and the resulting contributions to DS pathology in mammals are not tractable in a systematic way. A recent study looked at loss-of-function of a subset of Caenorhabditis elegans orthologs of Hsa21 genes and identified ten candidates with behavioral phenotypes, but the equivalent over-expression experiment has not been done. We turned to zebrafish as a developmental model and, using a number of surrogate phenotypes, we screened Hsa21 genes for effects on early embyrogenesis. We prepared a library of 164 cDNAs of conserved protein coding genes, injected mRNA into early embryos and evaluated up to 5 days post-fertilization (dpf). Twenty-four genes produced a gross morphological phenotype, 11 of which could be reproduced reliably. Seven of these gave a phenotype consistent with down regulation of the sonic hedgehog (Shh) pathway; two showed defects indicative of defective neural crest migration; one resulted consistently in pericardial edema; and one was embryonic lethal. Combinatorial injections of multiple Hsa21 genes revealed both additive and compensatory effects, supporting the notion that complex genetic relationships underlie end phenotypes of trisomy that produce DS. Together, our data suggest that this system is useful in the genetic dissection of dosage-sensitive gene effects on early development and can inform the contribution of both individual loci and their combinatorial effects to phenotypes relevant to the etiopathology of DS. [ABSTRACT FROM AUTHOR]

Pyridostigmine bromide (PB) is an FDA-approved drug for the treatment of myasthenia gravis and a prophylactic pre-treatment for organophosphate nerve agent poisoning. Current methods for evaluating nerve agent treatments include enzymatic studies and mammalian models. Rapid whole animal screening tools for assessing the effects of nerve agent pre-treatment and post-exposure drugs represent an underdeveloped area of research. We used zebrafish as a model for acute and chronic developmental exposure to PB and two related carbamate acetylcholinesterase (AChE) inhibitors, neostigmine bromide (NB) and physostigmine (PS). Lethal doses and gross morphological phenotypes resulting from exposure to sub-lethal doses of these compounds were determined. Quantitative analyses of motility impairment and AChE enzyme inhibition were used to determine optimal dosing conditions for evaluation of the effects of carbamate exposures on neuronal development; ~ 50% impairment of response to startle stimuli and > 50% inhibition of AChE activity were observed at 80 mM PB, 20 mM NB and 0.1 mM PS. PB induced stunted somite length, but no other phenotypic effects were observed. In contrast, NB and PS induced more severe phenotypic morphological defects than PB as well as neurite outgrowth mislocalization. Additionally, NB induced mislocalization of nicotinic acetylcholine receptors, resulting in impaired synapse formation. Taken together, these data suggest that altered patterns of neuronal connectivity contribute to the developmental neurotoxicity of carbamates and demonstrate the utility of the zebrafish model for distinguishing subtle structure-based differential effects of AChE inhibitors, which include nerve agents, pesticides and drugs. [ABSTRACT FROM AUTHOR]

Glutaminase (GLS), which converts glutamine to glutamate, plays a key role in cancer cell metabolism, growth, and proliferation. GLS is being explored as a cancer therapeutic target, but whether GLS inhibitors affect cancer cell--autonomous growth or the host microenvironment or have off-target effects is unknown. Here, we report that loss of one copy of Gls blunted tumor progression in an immune-competent MYC-mediated mouse model of hepatocellular carcinoma. Compared with results in untreated animals with MYC-induced hepatocellular carcinoma, administration of the GLS-specific inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) prolonged survival without any apparent toxicities. BPTES also inhibited growth of a MYC-dependent human B cell lymphoma cell line (P493) by blocking DNA replication, leading to cell death and fragmentation. In mice harboring P493 tumor xenografts, BPTES treatment inhibited tumor cell growth; however, P493 xenografts expressing a BPTES-resistant GLS mutant (GLS-K325A) or overexpressing GLS were not affected by BPTES treatment. Moreover, a customized Vivo-Morpholino that targets human GLS mRNA markedly inhibited P493 xenograft growth without affecting mouse Gls expression. Conversely, a Vivo-Morpholino directed at mouse Gls had no antitumor activity in vivo. Collectively, our studies demonstrate that GLS is required for tumorigenesis and support small molecule and genetic inhibition of GLS as potential approaches for targeting the tumor cell--autonomous dependence on GLS for cancer therapy. [ABSTRACT FROM AUTHOR]