Publications by authors named "Christopher Dooley"

27 Publications

  • Page 1 of 1

Evolution of the potassium channel gene Kcnj13 underlies colour pattern diversification in Danio fish.

Nat Commun 2020 12 4;11(1):6230. Epub 2020 Dec 4.

Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076, Tübingen, Germany.

The genetic basis of morphological variation provides a major topic in evolutionary developmental biology. Fish of the genus Danio display colour patterns ranging from horizontal stripes, to vertical bars or spots. Stripe formation in zebrafish, Danio rerio, is a self-organizing process based on cell-contact mediated interactions between three types of chromatophores with a leading role of iridophores. Here we investigate genes known to regulate chromatophore interactions in zebrafish that might have evolved to produce a pattern of vertical bars in its sibling species, Danio aesculapii. Mutant D. aesculapii indicate a lower complexity in chromatophore interactions and a minor role of iridophores in patterning. Reciprocal hemizygosity tests identify the potassium channel gene obelix/Kcnj13 as evolved between the two species. Complementation tests suggest evolutionary change through divergence in Kcnj13 function in two additional Danio species. Thus, our results point towards repeated and independent evolution of this gene during colour pattern diversification.
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http://dx.doi.org/10.1038/s41467-020-20021-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7718271PMC
December 2020

NRAS melanoma tumor formation is reduced by p38-MAPK14 activation in zebrafish models and NRAS-mutated human melanoma cells.

Pigment Cell Melanoma Res 2021 03 24;34(2):150-162. Epub 2020 Sep 24.

University Hospital Zurich, University of Zurich, Zurich, Switzerland.

Oncogenic BRAF and NRAS mutations drive human melanoma initiation. We used transgenic zebrafish to model NRAS-mutant melanoma, and the rapid tumor onset allowed us to study candidate tumor suppressors. We identified P38α-MAPK14 as a potential tumor suppressor in The Cancer Genome Atlas melanoma cohort of NRAS-mutant melanomas, and overexpression significantly increased the time to tumor onset in transgenic zebrafish with NRAS-driven melanoma. Pharmacological activation of P38α-MAPK14 using anisomycin reduced in vitro viability of melanoma cultures, which we confirmed by stable overexpression of p38α. We observed that the viability of MEK inhibitor resistant melanoma cells could be reduced by combined treatment of anisomycin and MEK inhibition. Our study demonstrates that activating the p38α-MAPK14 pathway in the presence of oncogenic NRAS abrogates melanoma in vitro and in vivo. SIGNIFICANCE: The significance of our study is in the accountability of NRAS mutations in melanoma. We demonstrate here that activation of p38α-MAPK14 pathway can abrogate NRAS-mutant melanoma which is contrary to the previously published role of p38α-MAPK14 pathway in BRAF mutant melanoma. These results implicate that BRAF and NRAS-mutant melanoma may not be identical biologically. We also demonstrate the translational benefit of our study by using a small molecule compound-anisomycin (already in use for other diseases in clinical trials) to activate p38α-MAPK14 pathway.
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http://dx.doi.org/10.1111/pcmr.12925DOI Listing
March 2021

PRL3-DDX21 Transcriptional Control of Endolysosomal Genes Restricts Melanocyte Stem Cell Differentiation.

Dev Cell 2020 08 10;54(3):317-332.e9. Epub 2020 Jul 10.

MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh EH4 2XU, UK; Cancer Research UK Edinburgh Centre, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK. Electronic address:

Melanocytes, replenished throughout life by melanocyte stem cells (MSCs), play a critical role in pigmentation and melanoma. Here, we reveal a function for the metastasis-associated phosphatase of regenerating liver 3 (PRL3) in MSC regeneration. We show that PRL3 binds to the RNA helicase DDX21, thereby restricting productive transcription by RNAPII at master transcription factor (MITF)-regulated endolysosomal vesicle genes. In zebrafish, this mechanism controls premature melanoblast expansion and differentiation from MSCs. In melanoma patients, restricted transcription of this endolysosomal vesicle pathway is a hallmark of PRL3-high melanomas. Our work presents the conceptual advance that PRL3-mediated control of transcriptional elongation is a differentiation checkpoint mechanism for activated MSCs and has clinical relevance for the activity of PRL3 in regenerating tissue and cancer.
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http://dx.doi.org/10.1016/j.devcel.2020.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435699PMC
August 2020

Kinematic and Biomechanical Response of Post-Mortem Human Subjects Under Various Pre-Impact Postures to High-Rate Vertical Loading Conditions.

Stapp Car Crash J 2019 Nov;63:235-266

Emory University, Atlanta, GA.

Limited data exist on the injury tolerance and biomechanical response of humans to high-rate, under-body blast (UBB) loading conditions that are commonly seen in current military operations, and there are no data examining the influence of occupant posture on response. Additionally, no anthropomorphic test device (ATD) currently exists that can properly assess the response of humans to high-rate UBB loading. Therefore, the purpose of this research was to examine the response of post-mortem human surrogates (PMHS) in various seated postures to high-rate, vertical loading representative of those conditions seen in theater. In total, six PMHS tests were conducted using loading pulses applied directly to the pelvis and feet of the PMHS: three in an acute posture (foot, knee, and pelvis angles of 75°, 75°, and 36°, respectively), and three in an obtuse posture (15° reclined torso, and foot, knee, and pelvis angles of 105°, 105°, and 49.5°, respectively). Tests were conducted with a seat velocity pulse that peaked at ~4 m/s with a 30-40 ms time to peak velocity (TTP) and a floor velocity that peaked at 6.9-8.0 m/s (2-2.75 ms TTP). Posture condition had no influence on skeletal injuries sustained, but did result in altered leg kinematics, with leg entrapment under the seat occurring in the acute posture, and significant forward leg rotations occurring in the obtuse posture. These data will be used to validate a prototype ATD meant for use in high-rate UBB loading scenarios.
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November 2019

The gene regulatory basis of genetic compensation during neural crest induction.

PLoS Genet 2019 06 14;15(6):e1008213. Epub 2019 Jun 14.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom.

The neural crest (NC) is a vertebrate-specific cell type that contributes to a wide range of different tissues across all three germ layers. The gene regulatory network (GRN) responsible for the formation of neural crest is conserved across vertebrates. Central to the induction of the NC GRN are AP-2 and SoxE transcription factors. NC induction robustness is ensured through the ability of some of these transcription factors to compensate loss of function of gene family members. However the gene regulatory events underlying compensation are poorly understood. We have used gene knockout and RNA sequencing strategies to dissect NC induction and compensation in zebrafish. We genetically ablate the NC using double mutants of tfap2a;tfap2c or remove specific subsets of the NC with sox10 and mitfa knockouts and characterise genome-wide gene expression levels across multiple time points. We find that compensation through a single wild-type allele of tfap2c is capable of maintaining early NC induction and differentiation in the absence of tfap2a function, but many target genes have abnormal expression levels and therefore show sensitivity to the reduced tfap2 dosage. This separation of morphological and molecular phenotypes identifies a core set of genes required for early NC development. We also identify the 15 somites stage as the peak of the molecular phenotype which strongly diminishes at 24 hpf even as the morphological phenotype becomes more apparent. Using gene knockouts, we associate previously uncharacterised genes with pigment cell development and establish a role for maternal Hippo signalling in melanocyte differentiation. This work extends and refines the NC GRN while also uncovering the transcriptional basis of genetic compensation via paralogues.
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http://dx.doi.org/10.1371/journal.pgen.1008213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594659PMC
June 2019

Dicer1 is required for pigment cell and craniofacial development in zebrafish.

Biochim Biophys Acta Gene Regul Mech 2019 04 3;1862(4):472-485. Epub 2019 Mar 3.

Instituto de Biología Molecular y Celular de Rosario (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) - Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Ocampo y Esmeralda, S2000EZP Rosario, Argentina. Electronic address:

The multidomain RNase III endoribonuclease DICER is required for the generation of most functional microRNAs (miRNAs). Loss of Dicer affects developmental processes at different levels. Here, we characterized the zebrafish Dicer1 mutant, dicer1, which has a single point mutation induced by N-ethyl-N-nitrosourea mutagenesis. Heterozygous dicer1 developed normally, being phenotypically indistinguishable from wild-type siblings. Homozygous dicer1 mutants display smaller eyes, abnormal craniofacial development and aberrant pigmentation. Reduced numbers of both iridophores and melanocytes were observed in the head and ventral trunk of dicer1 homozygotes; the effect on melanocytes was stronger and detectable earlier in development. The expression of microphthalmia-associated transcription factor a (mitfa), the master gene for melanocytes differentiation, was enhanced in dicer1-depleted fish. Similarly, the expression of SRY-box containing gene 10 (sox10), required for mitfa activation, was higher in mutants than in wild types. In silico and in vivo analyses of either sox10 or mitfa 3'UTRs revealed conserved potential miRNA binding sites likely involved in the post-transcriptional regulation of both genes. Based on these findings, we propose that dicer1 participates in the gene regulatory network governing zebrafish melanocyte differentiation by controlling the expression of mitfa and sox10.
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http://dx.doi.org/10.1016/j.bbagrm.2019.02.005DOI Listing
April 2019

Similitude assessment method for comparing PMHS response data from impact loading across multiple test devices.

J Biomech 2018 04 16;72:258-261. Epub 2018 Mar 16.

Johns Hopkins University, Applied Physics Laboratory, 11100 Johns Hopkins Rd., Laurel, MD 20723, United States. Electronic address:

Biological tissue testing is inherently susceptible to the wide range of variability specimen to specimen. A primary resource for encapsulating this range of variability is the biofidelity response corridor or BRC. In the field of injury biomechanics, BRCs are often used for development and validation of both physical, such as anthropomorphic test devices, and computational models. For the purpose of generating corridors, post-mortem human surrogates were tested across a range of loading conditions relevant to under-body blast events. To sufficiently cover the wide range of input conditions, a relatively small number of tests were performed across a large spread of conditions. The high volume of required testing called for leveraging the capabilities of multiple impact test facilities, all with slight variations in test devices. A method for assessing similitude of responses between test devices was created as a metric for inclusion of a response in the resulting BRC. The goal of this method was to supply a statistically sound, objective method to assess the similitude of an individual response against a set of responses to ensure that the BRC created from the set was affected primarily by biological variability, not anomalies or differences stemming from test devices.
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http://dx.doi.org/10.1016/j.jbiomech.2018.03.010DOI Listing
April 2018

A high-resolution mRNA expression time course of embryonic development in zebrafish.

Elife 2017 11 16;6. Epub 2017 Nov 16.

Wellcome Trust Sanger Institute, Hinxton, United Kingdom.

We have produced an mRNA expression time course of zebrafish development across 18 time points from 1 cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3' end transcript counting method we characterise temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. In addition, the data reveal new genes and transcripts, differential use of exons and previously unidentified 3' ends across development, new primary microRNAs and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data can be browsed and downloaded at Expression Atlas and Ensembl.
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http://dx.doi.org/10.7554/eLife.30860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5690287PMC
November 2017

Genetic Screen for Postembryonic Development in the Zebrafish (): Dominant Mutations Affecting Adult Form.

Genetics 2017 10 23;207(2):609-623. Epub 2017 Aug 23.

Department of Orthopedic Research, Boston Children's Hospital, Massachusetts 02115

Large-scale forward genetic screens have been instrumental for identifying genes that regulate development, homeostasis, and regeneration, as well as the mechanisms of disease. The zebrafish, , is an established genetic and developmental model used in genetic screens to uncover genes necessary for early development. However, the regulation of postembryonic development has received less attention as these screens are more labor intensive and require extensive resources. The lack of systematic interrogation of late development leaves large aspects of the genetic regulation of adult form and physiology unresolved. To understand the genetic control of postembryonic development, we performed a dominant screen for phenotypes affecting the adult zebrafish. In our screen, we identified 72 adult viable mutants showing changes in the shape of the skeleton as well as defects in pigmentation. For efficient mapping of these mutants and mutation identification, we devised a new mapping strategy based on identification of mutant-specific haplotypes. Using this method in combination with a candidate gene approach, we were able to identify linked mutations for 22 out of 25 mutants analyzed. Broadly, our mutational analysis suggests that there are key genes and pathways associated with late development. Many of these pathways are shared with humans and are affected in various disease conditions, suggesting constraint in the genetic pathways that can lead to change in adult form. Taken together, these results show that dominant screens are a feasible and productive means to identify mutations that can further our understanding of gene function during postembryonic development and in disease.
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http://dx.doi.org/10.1534/genetics.117.300187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5629327PMC
October 2017

KDM2A integrates DNA and histone modification signals through a CXXC/PHD module and direct interaction with HP1.

Nucleic Acids Res 2017 02;45(3):1114-1129

MRC Clinical Sciences Centre (CSC), Du Cane Road, London, UK.

Functional genomic elements are marked by characteristic DNA and histone modification signatures. How combinatorial chromatin modification states are recognized by epigenetic reader proteins and how this is linked to their biological function is largely unknown. Here we provide a detailed molecular analysis of chromatin recognition by the lysine demethylase KDM2A. Using biochemical approaches we identify a nucleosome interaction module within KDM2A consisting of a CXXC type zinc finger, a PHD domain and a newly identified Heterochromatin Protein 1 (HP1) interaction motif that mediates direct binding between KDM2A and HP1. This nucleosome interaction module enables KDM2A to decode nucleosomal H3K9me3 modification in addition to CpG methylation signals. The multivalent engagement with DNA and HP1 results in a nucleosome binding circuit in which KDM2A can be recruited to H3K9me3-modified chromatin through HP1, and HP1 can be recruited to unmodified chromatin by KDM2A. A KDM2A mutant deficient in HP1-binding is inactive in an in vivo overexpression assay in zebrafish embryos demonstrating that the HP1 interaction is essential for KDM2A function. Our results reveal a complex regulation of chromatin binding for both KDM2A and HP1 that is modulated by DNA- and H3K9-methylation, and suggest a direct role for KDM2A in chromatin silencing.
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http://dx.doi.org/10.1093/nar/gkw979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388433PMC
February 2017

Evaluation of WIAMan Technology Demonstrator Biofidelity Relative to Sub-Injurious PMHS Response in Simulated Under-body Blast Events.

Stapp Car Crash J 2016 11;60:199-246

Johns Hopkins University Applied Physics Laboratory.

Three laboratory simulated sub-injurious under-body blast (UBB) test conditions were conducted with whole-body Post Mortem Human Surrogates (PMHS) and the Warrior Assessment Injury Manikin (WIAMan) Technology Demonstrator (TD) to establish and assess UBB biofidelity of the WIAMan TD. Test conditions included a rigid floor and rigid seat with independently varied pulses. On the floor, peak velocities of 4 m/s and 6 m/s were applied with a 5 ms time to peak (TTP). The seat peak velocity was 4 m/s with varied TTP of 5 and 10 ms. Tests were conducted with and without personal protective equipment (PPE). PMHS response data was compiled into preliminary biofidelity response corridors (BRCs), which served as evaluation metrics for the WIAMan TD. Each WIAMan TD response was evaluated against the PMHS preliminary BRC for the loading and unloading phase of the signal time history using Correlation Analysis (CORA) software to assign a numerical score between 0 and 1. A weighted average of all responses was calculated to determine body region and whole body biofidelity scores for each test condition. The WIAMan TD received UBB biofidelity scores of 0.62 in Condition A, 0.59 in Condition B, and 0.63 in Condition C, putting it in the fair category (0.44-0.65). Body region responses with scores below a rating of good (0.65-0.84) indicate potential focus areas for the next generation of the WIAMan design.
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November 2016

Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish.

BMC Genomics 2016 Mar 24;17:259. Epub 2016 Mar 24.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

Background: The CRISPR/Cas9 system is a prokaryotic immune system that infers resistance to foreign genetic material and is a sort of 'adaptive immunity'. It has been adapted to enable high throughput genome editing and has revolutionised the generation of targeted mutations.

Results: We have developed a scalable analysis pipeline to identify CRISPR/Cas9 induced mutations in hundreds of samples using next generation sequencing (NGS) of amplicons. We have used this system to investigate the best way to screen mosaic Zebrafish founder individuals for germline transmission of induced mutations. Screening sperm samples from potential founders provides much better information on germline transmission rates and crucially the sequence of the particular insertions/deletions (indels) that will be transmitted. This enables us to combine screening with archiving to create a library of cryopreserved samples carrying known mutations. It also allows us to design efficient genotyping assays, making identifying F1 carriers straightforward.

Conclusions: The methods described will streamline the production of large numbers of knockout alleles in selected genes for phenotypic analysis, complementing existing efforts using random mutagenesis.
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http://dx.doi.org/10.1186/s12864-016-2563-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806435PMC
March 2016

High-throughput and quantitative genome-wide messenger RNA sequencing for molecular phenotyping.

BMC Genomics 2015 Aug 5;16:578. Epub 2015 Aug 5.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK.

Background: We present a genome-wide messenger RNA (mRNA) sequencing technique that converts small amounts of RNA from many samples into molecular phenotypes. It encompasses all steps from sample preparation to sequence analysis and is applicable to baseline profiling or perturbation measurements.

Results: Multiplex sequencing of transcript 3' ends identifies differential transcript abundance independent of gene annotation. We show that increasing biological replicate number while maintaining the total amount of sequencing identifies more differentially abundant transcripts.

Conclusions: This method can be implemented on polyadenylated RNA from any organism with an annotated reference genome and in any laboratory with access to Illumina sequencing.
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http://dx.doi.org/10.1186/s12864-015-1788-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524448PMC
August 2015

Leucophore identity is more gold than silver.

Pigment Cell Melanoma Res 2015 Mar 9;28(2):131. Epub 2015 Feb 9.

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http://dx.doi.org/10.1111/pcmr.12349DOI Listing
March 2015

Medical marijuana in Connecticut.

Conn Med 2014 Apr;78(4):237-9

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April 2014

Of white tigers and solute carriers.

Pigment Cell Melanoma Res 2013 Nov 23;26(6):787-9. Epub 2013 Sep 23.

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http://dx.doi.org/10.1111/pcmr.12163DOI Listing
November 2013

Comparison of the efficacy and safety of low molecular weight heparins for venous thromboembolism prophylaxis in medically ill patients.

Curr Med Res Opin 2014 Mar 19;30(3):367-80. Epub 2013 Nov 19.

Hartford Hospital, Pharmacy , Hartford, CT , USA.

Objective: To conduct a systematic review and mixed-treatment comparison (MTC) meta-analysis to compare the efficacy and safety of low molecular weight heparins (LMWHs) for venous thromboembolism (VTE) prophylaxis in hospitalized medically ill patients. As a secondary objective we compared all therapies within the network to each other.

Methods: We conducted a systematic literature search for randomized trials that evaluated pharmacologic VTE prophylaxis in hospitalized medically ill patients. We conducted a traditional meta-analysis for all pairwise comparisons using a random effects model, reporting relative risks (RRs) and 95% confidence intervals for each outcome. To determine the relative efficacy and safety of included therapies we conducted a MTC meta-analysis using a Bayesian framework, reporting odds ratios (OR) and 95% credible intervals.

Results: Twenty trials met inclusion criteria. Enoxaparin, dalteparin, nadroparin and certoparin were the LMWHs evaluated although none in direct comparative trials. Upon MTC, the relative efficacy of all LMWHs was similar in preventing mortality and VTE as well as in the odds of major and minor bleeding. Dalteparin was not included in the network to evaluate deep vein thrombosis (DVT) and pulmonary embolism (PE) due to lack of reported data and the remaining LMWHs were found to be similar in relative efficacy in preventing these outcomes.

Limitations: Traditional meta-analysis was not possible for many drug comparisons made within the MTC. Heterogeneity was observed in several of the traditional meta-analyses although this may be an inherent limitation of the studied population. Overall rarity of events contributed to imprecise estimates demonstrated by the wide confidence intervals.

Conclusions: Enoxaparin, dalteparin, nadroparin and certoparin are similar in relative efficacy for the prevention of mortality and VTE and in the odds of major or minor bleeding while enoxaparin, nadroparin and certoparin are similar in relative efficacy for the prevention of PE and DVT in hospitalized medical patients.
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http://dx.doi.org/10.1185/03007995.2013.837818DOI Listing
March 2014

Multi-allelic phenotyping--a systematic approach for the simultaneous analysis of multiple induced mutations.

Methods 2013 Aug 23;62(3):197-206. Epub 2013 Apr 23.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

The zebrafish mutation project (ZMP) aims to generate a loss of function allele for every protein-coding gene, but importantly to also characterise the phenotypes of these alleles during the first five days of development. Such a large-scale screen requires a systematic approach both to identifying phenotypes, and also to linking those phenotypes to specific mutations. This phenotyping pipeline simultaneously assesses the consequences of multiple alleles in a two-step process. First, mutations that do not produce a visible phenotype during the first five days of development are identified, while a second round of phenotyping focuses on detailed analysis of those alleles that are suspected to cause a phenotype. Allele-specific PCR single nucleotide polymorphism (SNP) assays are used to genotype F2 parents and individual F3 fry for mutations known to be present in the F1 founder. With this method specific phenotypes can be linked to induced mutations. In addition a method is described for cryopreserving sperm samples of mutagenised males and their subsequent use for in vitro fertilisation to generate F2 families for phenotyping. Ultimately this approach will lead to the functional annotation of the zebrafish genome, which will deepen our understanding of gene function in development and disease.
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http://dx.doi.org/10.1016/j.ymeth.2013.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3770900PMC
August 2013

The zebrafish reference genome sequence and its relationship to the human genome.

Nature 2013 Apr 17;496(7446):498-503. Epub 2013 Apr 17.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.
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http://dx.doi.org/10.1038/nature12111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3703927PMC
April 2013

A systematic genome-wide analysis of zebrafish protein-coding gene function.

Nature 2013 Apr 17;496(7446):494-7. Epub 2013 Apr 17.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes, this number falls considerably short of the more than 22,000 mouse protein-coding genes. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning, insertional mutagenesis, antisense morpholino oligonucleotides, targeted re-sequencing, and zinc finger and TAL endonucleases have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes. Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence, high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.
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http://dx.doi.org/10.1038/nature11992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3743023PMC
April 2013

Mutations in c10orf11, a melanocyte-differentiation gene, cause autosomal-recessive albinism.

Am J Hum Genet 2013 Mar 7;92(3):415-21. Epub 2013 Feb 7.

Applied Human Molecular Genetics, Kennedy Center, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.

Autosomal-recessive albinism is a hypopigmentation disorder with a broad phenotypic range. A substantial fraction of individuals with albinism remain genetically unresolved, and it has been hypothesized that more genes are to be identified. By using homozygosity mapping of an inbred Faroese family, we identified a 3.5 Mb homozygous region (10q22.2-q22.3) on chromosome 10. The region contains five protein-coding genes, and sequencing of one of these, C10orf11, revealed a nonsense mutation that segregated with the disease and showed a recessive inheritance pattern. Investigation of additional albinism-affected individuals from the Faroe Islands revealed that five out of eight unrelated affected persons had the nonsense mutation in C10orf11. Screening of a cohort of autosomal-recessive-albinism-affected individuals residing in Denmark showed a homozygous 1 bp duplication in C10orf11 in an individual originating from Lithuania. Immunohistochemistry showed localization of C10orf11 in melanoblasts and melanocytes in human fetal tissue, but no localization was seen in retinal pigment epithelial cells. Knockdown of the zebrafish (Danio rerio) homolog with the use of morpholinos resulted in substantially decreased pigmentation and a reduction of the apparent number of pigmented melanocytes. The morphant phenotype was rescued by wild-type C10orf11, but not by mutant C10orf11. In conclusion, we have identified a melanocyte-differentiation gene, C10orf11, which when mutated causes autosomal-recessive albinism in humans.
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http://dx.doi.org/10.1016/j.ajhg.2013.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591853PMC
March 2013

On the embryonic origin of adult melanophores: the role of ErbB and Kit signalling in establishing melanophore stem cells in zebrafish.

Development 2013 Mar 30;140(5):1003-13. Epub 2013 Jan 30.

Max-Planck-Institut für Entwicklungsbiologie, Spemannstr 35, 72076 Tübingen, Germany.

Pigment cells in vertebrates are derived from the neural crest (NC), a pluripotent and migratory embryonic cell population. In fishes, larval melanophores develop during embryogenesis directly from NC cells migrating along dorsolateral and ventromedial paths. The embryonic origin of the melanophores that emerge during juvenile development in the skin to contribute to the striking colour patterns of adult fishes remains elusive. We have identified a small set of melanophore progenitor cells (MPs) in the zebrafish (Danio rerio, Cyprinidae) that is established within the first 2 days of embryonic development in close association with the segmentally reiterated dorsal root ganglia (DRGs). Lineage analysis and 4D in vivo imaging indicate that progeny of these embryonic MPs spread segmentally, giving rise to the melanophores that create the adult melanophore stripes. Upon depletion of larval melanophores by morpholino knockdown of Mitfa, the embryonic MPs are prematurely activated; their progeny migrate along the spinal nerves restoring the larval pattern and giving rise to postembryonic MPs associated with the spinal nerves. Mutational or chemical inhibition of ErbB receptors blocks all early NC migration along the ventromedial path, causing a loss of DRGs and embryonic MPs. We show that the sparse like (slk) mutant lacks larval and metamorphic melanophores and identify kit ligand a (kitlga) as the underlying gene. Our data suggest that kitlga is required for the establishment or survival of embryonic MPs. We propose a model in which DRGs provide a niche for the stem cells of adult melanophores.
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http://dx.doi.org/10.1242/dev.087007DOI Listing
March 2013

Slc45a2 and V-ATPase are regulators of melanosomal pH homeostasis in zebrafish, providing a mechanism for human pigment evolution and disease.

Pigment Cell Melanoma Res 2013 Mar 20;26(2):205-17. Epub 2012 Dec 20.

Max Planck Institute for Developmental Biology, Tübingen, Germany.

We present here the positional cloning of the Danio rerio albino mutant and show that the affected gene encodes Slc45a2. The human orthologous gene has previously been shown to be involved in human skin color variation, and mutations therein have been implicated in the disease OCA4. Through ultrastructural analysis of the melanosomes in albino alleles as well as the tyrosinase-deficient mutant sandy, we add new insights into the role of Slc45a2 in the production of melanin. To gain further understanding of the role of Slc45a2 and its possible interactions with other proteins involved in melanization, we further analyzed the role of the V-ATPase as a melanosomal acidifier. We show that it is possible to rescue the melanization potential of the albino melanosomes through genetic and chemical inhibition of V-ATPase, thereby increasing internal melanosome pH.
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http://dx.doi.org/10.1111/pcmr.12053DOI Listing
March 2013

Select non-coding RNA in blood components provide novel clinically accessible biological surrogates for improved identification of traumatic brain injury in OEF/OIF Veterans.

Am J Neurodegener Dis 2012 24;1(1):88-98. Epub 2012 Apr 24.

Department of Neurology, Mount Sinai School of Medicine, New York, NY 10029, USA.

This study was designed to identify clinically accessible molecular biomarkers of mild traumatic brain injury (mTBI) that could be used to help identify returning Operation Iraqi Freedom (OIF) and Operation Enduring Freedom (OEF) Veterans who are suffering from the effects of mTBI. While analyzing the expression profile of small non-coding RNAs in peripheral blood mononuclear cells (PBMCs) from an OEF/OIF veteran study cohort using a high throughput array chip platform, we identified 18 candidate small non-coding RNA biomarkers that are differentially regulated in PBMCs of mTBI compared to non-TBI control cases. Independent quantitative real-time polymerase chain reaction assays confirmed that 13 of these candidate small RNA biomarker species are, indeed, significantly down-regulated in PBMCs of mTBI compared to non-TBI control veteran cases. Based on unsupervised clustering analysis, we identified a 3-biomarker panel which was most able to distinguish mTBI from non-TBI control veteran cases with high accuracy, selectivity and specificity. The majority of mTBI cases in our biomarker study were co-morbid with Post-Traumatic Stress Disorder (PTSD), and thus our non-TBI control cases were selected to match PTSD diagnoses. Therefore, our identified panel of 3 small RNA biomarkers likely represents a biological index selective for mTBI. Outcomes from our studies suggest that additional applications of the clinically accessible small non-coding RNA biomarkers to current diagnostic criteria may lead to improved mTBI detection and more sensitive outcome measures for clinical trials. Future studies exploring the physiological relevance of mTBI biomarkers will also provide a better understanding of the biological mechanisms underlying mTBI and insights into novel therapeutic targets for mTBI.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3560446PMC
February 2013

Differences in immune response may explain lower survival among older men with pneumonia.

Crit Care Med 2009 May;37(5):1655-62

Department of Critical Care Medicine, CRISMA Laboratory, University of Pittsburgh, Pittsburgh, PA, USA.

Objective: Lower life expectancy in men is generally attributed to higher likelihood of risky behavior and because men develop chronic conditions earlier. If sex-related differences in survival are independent of preinfection chronic health and health behavior, it would suggest that survival differences may occur because of sex differences in quality of care and biological response to infection, and these differences may contribute to sex differences in life expectancy. We assessed if sex-related survival difference following community-acquired pneumonia (CAP) is due to differences in clinical characteristics, quality of care, or immune response.

Design, Setting, And Subjects: Prospective observational cohort of 2183 subjects with CAP.

Measurements And Main Results: Mean age was 64.9 years. Men were more likely to smoke and had more comorbidity compared with women. At emergency department presentation, men had different biomarker patterns, as evidenced by higher inflammation (tumor necrosis factor, interleukin [IL]-6, and IL-10) and fibrinolysis (d-dimer), and lower coagulation biomarkers (antithrombin-III and factor IX) (p < 0.05). Small differences in favor of men were seen in care quality, including antibiotic timing and compliance with American Thoracic Society guidelines. Men had lower survival at 30, 90, and 365 days. The higher 1-year mortality was not attenuated when adjusted for differences in demographics, smoking, resuscitation, insurance, and vaccination status, comorbidity, hospital characteristics, and illness severity (unadjusted hazard ratio [HR] = 1.35, p = 0.003; and adjusted HR = 1.29, p = 0.004). HR was no longer statistically significant when additionally adjusted for differences in emergency department concentrations of tumor necrosis factor, IL-6, IL-10, d-dimer, antithrombin-III, and factor IX (adjusted HR = 1.27, p = 0.17). Patterns of biomarkers observed in men were associated with worse survival for 1 year.

Conclusions: Lower survival among men following CAP was not explained by differences in chronic diseases, health behaviors, and quality of care. Patterns of inflammatory, coagulation, and fibrinolysis biomarkers among men may explain reduced short-term and long-term survival.
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http://dx.doi.org/10.1097/CCM.0b013e31819da853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2760065PMC
May 2009

Large-scale mapping of mutations affecting zebrafish development.

BMC Genomics 2007 Jan 9;8:11. Epub 2007 Jan 9.

Department 3--Genetics, Max-Planck-Institut für Entwicklungsbiologie, Spemannstr, 35/III, 72076 Tübingen, Germany.

Background: Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers.

Results: We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM.

Conclusion: By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations.
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http://dx.doi.org/10.1186/1471-2164-8-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1781435PMC
January 2007
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