10 results on '"Law WD"'
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2. Evaluating the mouse neural precursor line, SN4741, as a suitable proxy for midbrain dopaminergic neurons.
- Author
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Boyd RJ, McClymont SA, Barrientos NB, Hook PW, Law WD, Rose RJ, Waite EL, Rathinavelu J, Avramopoulos D, and McCallion AS
- Subjects
- Mice, Humans, Animals, Mesencephalon metabolism, Cell Line, Cell Differentiation, Chromatin metabolism, Dopaminergic Neurons metabolism, Parkinson Disease genetics, Parkinson Disease metabolism
- Abstract
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., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
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3. Establishment of an eHAP1 human haploid cell line hybrid reference genome assembled from short and long reads.
- Author
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Law WD, Warren RL, and McCallion AS
- Subjects
- Genomic Structural Variation, Humans, Hybrid Cells, Nanopore Sequencing, Reference Standards, Cell Line, Genome, Human, Haploidy
- Abstract
Haploid cell lines are a valuable research tool with broad applicability for genetic assays. As such the fully haploid human cell line, eHAP1, has been used in a wide array of studies. However, the absence of a corresponding reference genome sequence for this cell line has limited the potential for more widespread applications to experiments dependent on available sequence, like capture-clone methodologies. We generated ~15× coverage Nanopore long reads from ten GridION flowcells and utilized this data to assemble a de novo draft genome using minimap and miniasm and subsequently polished using Racon. This assembly was further polished using previously generated, low-coverage, Illumina short reads with Pilon and ntEdit. This resulted in a hybrid eHAP1 assembly with >90% complete BUSCO scores. We further assessed the eHAP1 long read data for structural variants using Sniffles and identify a variety of rearrangements, including a previously established Philadelphia translocation. Finally, we demonstrate how some of these variants overlap open chromatin regions, potentially impacting regulatory regions. By integrating both long and short reads, we generated a high-quality reference assembly for eHAP1 cells. The union of long and short reads demonstrates the utility in combining sequencing platforms to generate a high-quality reference genome de novo solely from low coverage data. We expect the resulting eHAP1 genome assembly to provide a useful resource to enable novel experimental applications in this important model cell line., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests or personal relationships that could influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
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4. Parkinson-Associated SNCA Enhancer Variants Revealed by Open Chromatin in Mouse Dopamine Neurons.
- Author
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McClymont SA, Hook PW, Soto AI, Reed X, Law WD, Kerans SJ, Waite EL, Briceno NJ, Thole JF, Heckman MG, Diehl NN, Wszolek ZK, Moore CD, Zhu H, Akiyama JA, Dickel DE, Visel A, Pennacchio LA, Ross OA, Beer MA, and McCallion AS
- Subjects
- Adult, Aged, Aged, 80 and over, Alleles, Animals, Disease Models, Animal, Female, Genotype, Humans, Introns genetics, Male, Mice, Mice, Transgenic, Middle Aged, Pregnancy, Zebrafish, Chromatin genetics, Dopaminergic Neurons pathology, Enhancer Elements, Genetic genetics, Genetic Predisposition to Disease genetics, Parkinson Disease genetics, alpha-Synuclein genetics
- Abstract
The progressive loss of midbrain (MB) dopaminergic (DA) neurons defines the motor features of Parkinson disease (PD), and modulation of risk by common variants in PD has been well established through genome-wide association studies (GWASs). We acquired open chromatin signatures of purified embryonic mouse MB DA neurons because we anticipated that a fraction of PD-associated genetic variation might mediate the variants' effects within this neuronal population. Correlation with >2,300 putative enhancers assayed in mice revealed enrichment for MB cis-regulatory elements (CREs), and these data were reinforced by transgenic analyses of six additional sequences in zebrafish and mice. One CRE, within intron 4 of the familial PD gene SNCA, directed reporter expression in catecholaminergic neurons from transgenic mice and zebrafish. Sequencing of this CRE in 986 individuals with PD and 992 controls revealed two common variants associated with elevated PD risk. To assess potential mechanisms of action, we screened >16,000 proteins for DNA binding capacity and identified a subset whose binding is impacted by these enhancer variants. Additional genotyping across the SNCA locus identified a single PD-associated haplotype, containing the minor alleles of both of the aforementioned PD-risk variants. Our work posits a model for how common variation at SNCA might modulate PD risk and highlights the value of cell-context-dependent guided searches for functional non-coding variation., (Copyright © 2018 American Society of Human Genetics. All rights reserved.)
- Published
- 2018
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5. A genome-wide assessment of conserved SNP alleles reveals a panel of regulatory SNPs relevant to the peripheral nerve.
- Author
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Law WD, Fogarty EA, Vester A, and Antonellis A
- Subjects
- Animals, Gene Expression Regulation genetics, Humans, Mice, Motor Neurons metabolism, Muscle Cells metabolism, Peripheral Nerves cytology, SOXE Transcription Factors metabolism, Schwann Cells metabolism, Tubulin metabolism, Alleles, Genomics, Peripheral Nerves metabolism, Polymorphism, Single Nucleotide
- Abstract
Background: Identifying functional non-coding variation is critical for defining the genetic contributions to human disease. While single-nucleotide polymorphisms (SNPs) within cis-acting transcriptional regulatory elements have been implicated in disease pathogenesis, not all cell types have been assessed and functional validations have been limited. In particular, the cells of the peripheral nervous system have been excluded from genome-wide efforts to link non-coding SNPs to altered gene function. Addressing this gap is essential for defining the genetic architecture of diseases that affect the peripheral nerve. We developed a computational pipeline to identify SNPs that affect regulatory function (rSNPs) and evaluated our predictions on a set of 144 regions in Schwann cells, motor neurons, and muscle cells., Results: We identified 28 regions that display regulatory activity in at least one cell type and 13 SNPs that affect regulatory function. We then tailored our pipeline to one peripheral nerve cell type by incorporating SOX10 ChIP-Seq data; SOX10 is essential for Schwann cells. We prioritized 22 putative SOX10 response elements harboring a SNP and rapidly validated two rSNPs. We then selected one of these elements for further characterization to assess the biological relevance of our approach. Deletion of the element from the genome of cultured Schwann cells-followed by differential gene expression studies-revealed Tubb2b as a candidate target gene. Studying the enhancer in developing mouse embryos revealed activity in SOX10-positive cells including the dorsal root ganglia and melanoblasts., Conclusions: Our efforts provide insight into the utility of employing strict conservation for rSNP discovery. This strategy, combined with functional analyses, can yield candidate target genes. In support of this, our efforts suggest that investigating the role of Tubb2b in SOX10-positive cells may reveal novel biology within these cell populations.
- Published
- 2018
- Full Text
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6. Single-Cell RNA-Seq of Mouse Dopaminergic Neurons Informs Candidate Gene Selection for Sporadic Parkinson Disease.
- Author
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Hook PW, McClymont SA, Cannon GH, Law WD, Morton AJ, Goff LA, and McCallion AS
- Subjects
- Animals, Cell Separation, Gene Regulatory Networks, Genetic Loci, Genetic Markers, Genome-Wide Association Study, Mice, Knockout, Substantia Nigra pathology, Dopaminergic Neurons metabolism, Genetic Association Studies, Parkinson Disease genetics, Parkinson Disease pathology, Sequence Analysis, RNA, Single-Cell Analysis methods
- Abstract
Genetic variation modulating risk of sporadic Parkinson disease (PD) has been primarily explored through genome-wide association studies (GWASs). However, like many other common genetic diseases, the impacted genes remain largely unknown. Here, we used single-cell RNA-seq to characterize dopaminergic (DA) neuron populations in the mouse brain at embryonic and early postnatal time points. These data facilitated unbiased identification of DA neuron subpopulations through their unique transcriptional profiles, including a postnatal neuroblast population and substantia nigra (SN) DA neurons. We use these population-specific data to develop a scoring system to prioritize candidate genes in all 49 GWAS intervals implicated in PD risk, including genes with known PD associations and many with extensive supporting literature. As proof of principle, we confirm that the nigrostriatal pathway is compromised in Cplx1-null mice. Ultimately, this systematic approach establishes biologically pertinent candidates and testable hypotheses for sporadic PD, informing a new era of PD genetic research., (Copyright © 2018 American Society of Human Genetics. All rights reserved.)
- Published
- 2018
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7. Stringent comparative sequence analysis reveals SOX10 as a putative inhibitor of glial cell differentiation.
- Author
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Gopinath C, Law WD, Rodríguez-Molina JF, Prasad AB, Song L, Crawford GE, Mullikin JC, Svaren J, and Antonellis A
- Subjects
- Base Sequence, Consensus Sequence, Conserved Sequence, Exons, Genomics methods, High-Throughput Nucleotide Sequencing, Promoter Regions, Genetic, Regulatory Elements, Transcriptional, Response Elements, SOXE Transcription Factors chemistry, SOXE Transcription Factors genetics, Schwann Cells metabolism, Cell Differentiation genetics, Neuroglia cytology, Neuroglia metabolism, SOXE Transcription Factors metabolism
- Abstract
Background: The transcription factor SOX10 is essential for all stages of Schwann cell development including myelination. SOX10 cooperates with other transcription factors to activate the expression of key myelin genes in Schwann cells and is therefore a context-dependent, pro-myelination transcription factor. As such, the identification of genes regulated by SOX10 will provide insight into Schwann cell biology and related diseases. While genome-wide studies have successfully revealed SOX10 target genes, these efforts mainly focused on myelinating stages of Schwann cell development. We propose that less-biased approaches will reveal novel functions of SOX10 outside of myelination., Results: We developed a stringent, computational-based screen for genome-wide identification of SOX10 response elements. Experimental validation of a pilot set of predicted binding sites in multiple systems revealed that SOX10 directly regulates a previously unreported alternative promoter at SOX6, which encodes a transcription factor that inhibits glial cell differentiation. We further explored the utility of our computational approach by combining it with DNase-seq analysis in cultured Schwann cells and previously published SOX10 ChIP-seq data from rat sciatic nerve. Remarkably, this analysis enriched for genomic segments that map to loci involved in the negative regulation of gliogenesis including SOX5, SOX6, NOTCH1, HMGA2, HES1, MYCN, ID4, and ID2. Functional studies in Schwann cells revealed that: (1) all eight loci are expressed prior to myelination and down-regulated subsequent to myelination; (2) seven of the eight loci harbor validated SOX10 binding sites; and (3) seven of the eight loci are down-regulated upon repressing SOX10 function., Conclusions: Our computational strategy revealed a putative novel function for SOX10 in Schwann cells, which suggests a model where SOX10 activates the expression of genes that inhibit myelination during non-myelinating stages of Schwann cell development. Importantly, the computational and functional datasets we present here will be valuable for the study of transcriptional regulation, SOX protein function, and glial cell biology.
- Published
- 2016
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8. SOX10 regulates an alternative promoter at the Charcot-Marie-Tooth disease locus MTMR2.
- Author
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Fogarty EA, Brewer MH, Rodriguez-Molina JF, Law WD, Ma KH, Steinberg NM, Svaren J, and Antonellis A
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- Animals, Charcot-Marie-Tooth Disease physiopathology, Gene Expression Regulation, HeLa Cells, Humans, Mice, Motor Neurons metabolism, Motor Neurons pathology, Mutation, Myelin Sheath genetics, Peripheral Nerves growth & development, Peripheral Nerves metabolism, Peripheral Nerves pathology, Promoter Regions, Genetic, Protein Tyrosine Phosphatases, Non-Receptor genetics, Rats, Schwann Cells metabolism, Schwann Cells pathology, Charcot-Marie-Tooth Disease genetics, Protein Tyrosine Phosphatases, Non-Receptor biosynthesis, Regulatory Elements, Transcriptional genetics, SOXE Transcription Factors genetics
- Abstract
Schwann cells are the myelinating glia of the peripheral nervous system and dysfunction of these cells causes motor and sensory peripheral neuropathy. The transcription factor SOX10 is critical for Schwann cell development and maintenance, and many SOX10 target genes encode proteins required for Schwann cell function. Loss-of-function mutations in the gene encoding myotubularin-related protein 2 (MTMR2) cause Charcot-Marie-Tooth disease type 4B1 (CMT4B1), a severe demyelinating peripheral neuropathy characterized by myelin outfoldings along peripheral nerves. Previous reports indicate that MTMR2 is ubiquitously expressed making it unclear how loss of this gene causes a Schwann cell-specific phenotype. To address this, we performed computational and functional analyses at MTMR2 to identify transcriptional regulatory elements important for Schwann cell expression. Through these efforts, we identified an alternative, SOX10-responsive promoter at MTMR2 that displays strong regulatory activity in immortalized rat Schwann (S16) cells. This promoter directs transcription of a previously unidentified MTMR2 transcript that is enriched in mouse Schwann cells compared to immortalized mouse motor neurons (MN-1), and is predicted to encode an N-terminally truncated protein isoform. The expression of the endogenous transcript is induced in a heterologous cell line by ectopically expressing SOX10, and is nearly ablated in Schwann cells by impairing SOX10 function. Intriguingly, overexpressing the two MTMR2 protein isoforms in HeLa cells revealed that both localize to nuclear puncta and the shorter isoform displays higher nuclear localization compared to the longer isoform. Combined, our data warrant further investigation of the truncated MTMR2 protein isoform in Schwann cells and in CMT4B1 pathogenesis., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2016
- Full Text
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9. Tead1 regulates the expression of Peripheral Myelin Protein 22 during Schwann cell development.
- Author
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Lopez-Anido C, Poitelon Y, Gopinath C, Moran JJ, Ma KH, Law WD, Antonellis A, Feltri ML, and Svaren J
- Subjects
- Animals, Charcot-Marie-Tooth Disease pathology, DNA Copy Number Variations genetics, DNA-Binding Proteins biosynthesis, Disease Models, Animal, Early Growth Response Protein 2 biosynthesis, Gene Expression Regulation genetics, Humans, Mice, Myelin Sheath genetics, Myelin Sheath pathology, Neurogenesis genetics, Peripheral Nervous System Diseases pathology, Phenotype, Schwann Cells metabolism, Schwann Cells pathology, TEA Domain Transcription Factors, Transcription Factors biosynthesis, Charcot-Marie-Tooth Disease genetics, DNA-Binding Proteins genetics, Early Growth Response Protein 2 genetics, Myelin Proteins genetics, Peripheral Nervous System Diseases genetics, SOXE Transcription Factors genetics, Transcription Factors genetics
- Abstract
Schwann cells are myelinating glia in the peripheral nervous system that form the myelin sheath. A major cause of peripheral neuropathy is a copy number variant involving the Peripheral Myelin Protein 22 (PMP22) gene, which is located within a 1.4-Mb duplication on chromosome 17 associated with the most common form of Charcot-Marie-Tooth Disease (CMT1A). Rodent models of CMT1A have been used to show that reducing Pmp22 overexpression mitigates several aspects of a CMT1A-related phenotype. Mechanistic studies of Pmp22 regulation identified enhancers regulated by the Sox10 (SRY sex determining region Y-box 10) and Egr2/Krox20 (Early growth response protein 2) transcription factors in myelinated nerves. However, relatively little is known regarding how other transcription factors induce Pmp22 expression during Schwann cell development and myelination. Here, we examined Pmp22 enhancers as a function of cell type-specificity, nerve injury and development. While Pmp22 enhancers marked by active histone modifications were lost or remodeled after injury, we found that these enhancers were permissive in early development prior to Pmp22 upregulation. Pmp22 enhancers contain binding motifs for TEA domain (Tead) transcription factors of the Hippo signaling pathway. We discovered that Tead1 and co-activators Yap and Taz are required for Pmp22 expression, as well as for the expression of Egr2 Tead1 directly binds Pmp22 and Egr2 enhancers early in development and Tead1 binding is induced during myelination, correlating with Pmp22 expression. The data identify Tead1 as a novel regulator of Pmp22 expression during development in concert with Sox10 and Egr2., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2016
- Full Text
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10. Differentiation and behavior of human neural progenitors on micropatterned substrates and in the developing retina.
- Author
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Blong CC, Jeon CJ, Yeo JY, Ye EA, Oh J, Callahan JM, Law WD, Mallapragada SK, and Sakaguchi DS
- Subjects
- Cell Culture Techniques, Cell Movement physiology, Cell Proliferation, Cells, Cultured, Collagen chemistry, Collagen pharmacology, Culture Media chemistry, Humans, Laminin chemistry, Laminin pharmacology, Neurogenesis physiology, Neurons cytology, Neurons physiology, Polystyrenes chemistry, Polystyrenes pharmacology, Retina cytology, Retina surgery, Spheroids, Cellular cytology, Spheroids, Cellular physiology, Stem Cells cytology, Cell Differentiation physiology, Culture Media pharmacology, Graft Survival physiology, Retina growth & development, Stem Cell Transplantation methods, Stem Cells physiology
- Abstract
In this study we investigated the differentiation of human neural progenitor cells (hNPCs) in vitro to evaluate their differentiation potential and in vivo to explore their viability and behavior following transplantation. Progenitors were maintained as neurospheres in media containing basic fibroblast growth factor and epidermal growth factor. Micropatterned polystyrene substrates were fabricated and coated with ECL (entactin, collagen, and laminin) to provide physical and chemical guidance during the differentiation of the hNPCs. The hNPCs growing on the micropatterned substrates showed no differences in proliferation or differentiation potential compared with those hNPCs growing on the nonpatterned substrates. However, hNPCs cultured on the micropatterned substrates were aligned in the direction of the micropattern compared with those cells growing on the nonpatterned substrates. Furthermore, hNPC migration was directed in alignment with the micropatterned substrates. Transplantation of the hNPCs into the developing retina was used to evaluate their behavior in vivo. Cells displayed extensive survival, differentiation, and morphological integration following xenotransplant into the retina, even in the absence of immunosuppression. Taken together, our results show that these multipotent hNPCs are a neurogenic progenitor population that can be maintained in culture for extended periods. Although the micropatterned substrates have no major effect on the proliferation or differentiation of the hNPCs, they clearly promoted alignment and directed neurite outgrowth along the pattern as well as directing migration of the cells. These approaches may provide important strategies to guide the growth and differentiation of NPCs in vitro and in vivo., ((c) 2009 Wiley-Liss, Inc.)
- Published
- 2010
- Full Text
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