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1.  The cone-dominant retina and the inner ear of zebrafish express the ortholog of CLRN1, the causative gene of human Usher syndrome type 3A 
Gene expression patterns : GEP  2013;13(8):473-481.
Clarin-1 (CLRN1) is the causative gene in Usher Syndrome type 3A, an autosomal recessive disorder characterized by progressive vision and hearing loss. CLRN1 encodes Clarin-1, a glycoprotein with homology to the tetraspanin family of proteins. Previous cell culture studies suggest that Clarin-1 localizes to the plasma membrane and interacts with the cytoskeleton. Mouse models demonstrate a role for the protein in mechanosensory hair bundle integrity, but the function of Clarin-1 in hearing remains unclear. Even less is known of its role in vision, because the Clrn1 knockout mouse does not exhibit a retinal phenotype and expression studies in murine retinas have provided conflicting results. Here, we describe cloning and expression analysis of the zebrafish clrn1 gene, and report protein localization of Clarin-1 in auditory and visual cells from embryonic through adult stages. We detect clrn1 transcripts as early as 24 hours post-fertilization, and expression is maintained through adulthood. In situ hybridization experiments show clrn1 transcripts enriched in mechanosensory hair cells and supporting cells of the inner ear and lateral line organ, photoreceptors, and cells of the inner retina. In mechanosensory hair cells, Clarin-1 is polarized to the apical cell body and the synapses. In the retina, Clarin-1 localizes to lateral cell contacts between photoreceptors and is associated with the outer limiting membrane and subapical processes emanating from Müller glial cells. We also find Clarin-1 protein in the outer plexiform, inner nuclear and ganglion cell layers of the retina. Given the importance of Clarin-1 function in the human retina, it is imperative to find an animal model with a comparable requirement. Our data provide a foundation for exploring the role of Clarin-1 in retinal cell function and survival in a diurnal, cone-dominant species.
doi:10.1016/j.gep.2013.09.001
PMCID: PMC3888827  PMID: 24045267
2.  Zebrafish models in translational research: tipping the scales toward advancements in human health 
Disease Models & Mechanisms  2014;7(7):739-743.
Advances in genomics and next-generation sequencing have provided clinical researchers with unprecedented opportunities to understand the molecular basis of human genetic disorders. This abundance of information places new requirements on traditional disease models, which have the potential to be used to confirm newly identified pathogenic mutations and test the efficacy of emerging therapies. The unique attributes of zebrafish are being increasingly leveraged to create functional disease models, facilitate drug discovery, and provide critical scientific bases for the development of new clinical tools for the diagnosis and treatment of human disease. In this short review and the accompanying poster, we highlight a few illustrative examples of the applications of the zebrafish model to the study of human health and disease.
doi:10.1242/dmm.015545
PMCID: PMC4073263  PMID: 24973743
Usher syndrome; Cancer; Individualized medicine; Muscular dystrophy; Tuberculosis
3.  Complexes of Usher proteins preassemble at the endoplasmic reticulum and are required for trafficking and ER homeostasis 
Disease Models & Mechanisms  2014;7(5):547-559.
Usher syndrome (USH), the leading cause of hereditary combined hearing and vision loss, is characterized by sensorineural deafness and progressive retinal degeneration. Mutations in several different genes produce USH, but the proximal cause of sensory cell death remains mysterious. We adapted a proximity ligation assay to analyze associations among three of the USH proteins, Cdh23, Harmonin and Myo7aa, and the microtubule-based transporter Ift88 in zebrafish inner ear mechanosensory hair cells. We found that the proteins are in close enough proximity to form complexes and that these complexes preassemble at the endoplasmic reticulum (ER). Defects in any one of the three USH proteins disrupt formation and trafficking of the complex and result in diminished levels of the other proteins, generalized trafficking defects and ER stress that triggers apoptosis. ER stress, thus, contributes to sensory hair cell loss and provides a new target to explore for protective therapies for USH.
doi:10.1242/dmm.014068
PMCID: PMC4007406  PMID: 24626987
Harmonin; Cadherin23; Ift88; Myo7aa; Usher syndrome; Hair cell; Trafficking; ER stress; Zebrafish
4.  The zebrafish reference genome sequence and its relationship to the human genome 
Howe, Kerstin | Clark, Matthew D. | Torroja, Carlos F. | Torrance, James | Berthelot, Camille | Muffato, Matthieu | Collins, John E. | Humphray, Sean | McLaren, Karen | Matthews, Lucy | McLaren, Stuart | Sealy, Ian | Caccamo, Mario | Churcher, Carol | Scott, Carol | Barrett, Jeffrey C. | Koch, Romke | Rauch, Gerd-Jörg | White, Simon | Chow, William | Kilian, Britt | Quintais, Leonor T. | Guerra-Assunção, José A. | Zhou, Yi | Gu, Yong | Yen, Jennifer | Vogel, Jan-Hinnerk | Eyre, Tina | Redmond, Seth | Banerjee, Ruby | Chi, Jianxiang | Fu, Beiyuan | Langley, Elizabeth | Maguire, Sean F. | Laird, Gavin K. | Lloyd, David | Kenyon, Emma | Donaldson, Sarah | Sehra, Harminder | Almeida-King, Jeff | Loveland, Jane | Trevanion, Stephen | Jones, Matt | Quail, Mike | Willey, Dave | Hunt, Adrienne | Burton, John | Sims, Sarah | McLay, Kirsten | Plumb, Bob | Davis, Joy | Clee, Chris | Oliver, Karen | Clark, Richard | Riddle, Clare | Eliott, David | Threadgold, Glen | Harden, Glenn | Ware, Darren | Mortimer, Beverly | Kerry, Giselle | Heath, Paul | Phillimore, Benjamin | Tracey, Alan | Corby, Nicole | Dunn, Matthew | Johnson, Christopher | Wood, Jonathan | Clark, Susan | Pelan, Sarah | Griffiths, Guy | Smith, Michelle | Glithero, Rebecca | Howden, Philip | Barker, Nicholas | Stevens, Christopher | Harley, Joanna | Holt, Karen | Panagiotidis, Georgios | Lovell, Jamieson | Beasley, Helen | Henderson, Carl | Gordon, Daria | Auger, Katherine | Wright, Deborah | Collins, Joanna | Raisen, Claire | Dyer, Lauren | Leung, Kenric | Robertson, Lauren | Ambridge, Kirsty | Leongamornlert, Daniel | McGuire, Sarah | Gilderthorp, Ruth | Griffiths, Coline | Manthravadi, Deepa | Nichol, Sarah | Barker, Gary | Whitehead, Siobhan | Kay, Michael | Brown, Jacqueline | Murnane, Clare | Gray, Emma | Humphries, Matthew | Sycamore, Neil | Barker, Darren | Saunders, David | Wallis, Justene | Babbage, Anne | Hammond, Sian | Mashreghi-Mohammadi, Maryam | Barr, Lucy | Martin, Sancha | Wray, Paul | Ellington, Andrew | Matthews, Nicholas | Ellwood, Matthew | Woodmansey, Rebecca | Clark, Graham | Cooper, James | Tromans, Anthony | Grafham, Darren | Skuce, Carl | Pandian, Richard | Andrews, Robert | Harrison, Elliot | Kimberley, Andrew | Garnett, Jane | Fosker, Nigel | Hall, Rebekah | Garner, Patrick | Kelly, Daniel | Bird, Christine | Palmer, Sophie | Gehring, Ines | Berger, Andrea | Dooley, Christopher M. | Ersan-Ürün, Zübeyde | Eser, Cigdem | Geiger, Horst | Geisler, Maria | Karotki, Lena | Kirn, Anette | Konantz, Judith | Konantz, Martina | Oberländer, Martina | Rudolph-Geiger, Silke | Teucke, Mathias | Osoegawa, Kazutoyo | Zhu, Baoli | Rapp, Amanda | Widaa, Sara | Langford, Cordelia | Yang, Fengtang | Carter, Nigel P. | Harrow, Jennifer | Ning, Zemin | Herrero, Javier | Searle, Steve M. J. | Enright, Anton | Geisler, Robert | Plasterk, Ronald H. A. | Lee, Charles | Westerfield, Monte | de Jong, Pieter J. | Zon, Leonard I. | Postlethwait, John H. | Nüsslein-Volhard, Christiane | Hubbard, Tim J. P. | Crollius, Hugues Roest | Rogers, Jane | Stemple, Derek L.
Nature  2013;496(7446):498-503.
Zebrafish have become a popular organism for the study of vertebrate gene function1,2. 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 disease3–5. 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 genes6, 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.
doi:10.1038/nature12111
PMCID: PMC3703927  PMID: 23594743
5.  The zebrafish anatomy and stage ontologies: representing the anatomy and development of Danio rerio 
Background
The Zebrafish Anatomy Ontology (ZFA) is an OBO Foundry ontology that is used in conjunction with the Zebrafish Stage Ontology (ZFS) to describe the gross and cellular anatomy and development of the zebrafish, Danio rerio, from single cell zygote to adult. The zebrafish model organism database (ZFIN) uses the ZFA and ZFS to annotate phenotype and gene expression data from the primary literature and from contributed data sets.
Results
The ZFA models anatomy and development with a subclass hierarchy, a partonomy, and a developmental hierarchy and with relationships to the ZFS that define the stages during which each anatomical entity exists. The ZFA and ZFS are developed utilizing OBO Foundry principles to ensure orthogonality, accessibility, and interoperability. The ZFA has 2860 classes representing a diversity of anatomical structures from different anatomical systems and from different stages of development.
Conclusions
The ZFA describes zebrafish anatomy and development semantically for the purposes of annotating gene expression and anatomical phenotypes. The ontology and the data have been used by other resources to perform cross-species queries of gene expression and phenotype data, providing insights into genetic relationships, morphological evolution, and models of human disease.
doi:10.1186/2041-1480-5-12
PMCID: PMC3944782  PMID: 24568621
6.  Construction and accessibility of a cross-species phenotype ontology along with gene annotations for biomedical research 
F1000Research  2014;2:30.
Phenotype analyses, e.g. investigating metabolic processes, tissue formation, or organism behavior, are an important element of most biological and medical research activities. Biomedical researchers are making increased use of ontological standards and methods to capture the results of such analyses, with one focus being the comparison and analysis of phenotype information between species.
We have generated a cross-species phenotype ontology for human, mouse and zebrafish that contains classes from the Human Phenotype Ontology, Mammalian Phenotype Ontology, and generated classes for zebrafish phenotypes. We also provide up-to-date annotation data connecting human genes to phenotype classes from the generated ontology. We have included the data generation pipeline into our continuous integration system ensuring stable and up-to-date releases.
This article describes the data generation process and is intended to help interested researchers access both the phenotype annotation data and the associated cross-species phenotype ontology. The resource described here can be used in sophisticated semantic similarity and gene set enrichment analyses for phenotype data across species. The stable releases of this resource can be obtained from http://purl.obolibrary.org/obo/hp/uberpheno/.
doi:10.12688/f1000research.2-30.v2
PMCID: PMC3799545  PMID: 24358873
8.  The vertebrate taxonomy ontology: a framework for reasoning across model organism and species phenotypes 
Background
A hierarchical taxonomy of organisms is a prerequisite for semantic integration of biodiversity data. Ideally, there would be a single, expansive, authoritative taxonomy that includes extinct and extant taxa, information on synonyms and common names, and monophyletic supraspecific taxa that reflect our current understanding of phylogenetic relationships.
Description
As a step towards development of such a resource, and to enable large-scale integration of phenotypic data across vertebrates, we created the Vertebrate Taxonomy Ontology (VTO), a semantically defined taxonomic resource derived from the integration of existing taxonomic compilations, and freely distributed under a Creative Commons Zero (CC0) public domain waiver. The VTO includes both extant and extinct vertebrates and currently contains 106,947 taxonomic terms, 22 taxonomic ranks, 104,736 synonyms, and 162,400 cross-references to other taxonomic resources. Key challenges in constructing the VTO included (1) extracting and merging names, synonyms, and identifiers from heterogeneous sources; (2) structuring hierarchies of terms based on evolutionary relationships and the principle of monophyly; and (3) automating this process as much as possible to accommodate updates in source taxonomies.
Conclusions
The VTO is the primary source of taxonomic information used by the Phenoscape Knowledgebase (http://phenoscape.org/), which integrates genetic and evolutionary phenotype data across both model and non-model vertebrates. The VTO is useful for inferring phenotypic changes on the vertebrate tree of life, which enables queries for candidate genes for various episodes in vertebrate evolution.
doi:10.1186/2041-1480-4-34
PMCID: PMC4177199  PMID: 24267744
Data integration; Evolutionary biology; Paleontology; Taxonomic rank
9.  The Human Phenotype Ontology project: linking molecular biology and disease through phenotype data 
Nucleic Acids Research  2013;42(Database issue):D966-D974.
The Human Phenotype Ontology (HPO) project, available at http://www.human-phenotype-ontology.org, provides a structured, comprehensive and well-defined set of 10,088 classes (terms) describing human phenotypic abnormalities and 13,326 subclass relations between the HPO classes. In addition we have developed logical definitions for 46% of all HPO classes using terms from ontologies for anatomy, cell types, function, embryology, pathology and other domains. This allows interoperability with several resources, especially those containing phenotype information on model organisms such as mouse and zebrafish. Here we describe the updated HPO database, which provides annotations of 7,278 human hereditary syndromes listed in OMIM, Orphanet and DECIPHER to classes of the HPO. Various meta-attributes such as frequency, references and negations are associated with each annotation. Several large-scale projects worldwide utilize the HPO for describing phenotype information in their datasets. We have therefore generated equivalence mappings to other phenotype vocabularies such as LDDB, Orphanet, MedDRA, UMLS and phenoDB, allowing integration of existing datasets and interoperability with multiple biomedical resources. We have created various ways to access the HPO database content using flat files, a MySQL database, and Web-based tools. All data and documentation on the HPO project can be found online.
doi:10.1093/nar/gkt1026
PMCID: PMC3965098  PMID: 24217912
10.  500,000 fish phenotypes: The new informatics landscape for evolutionary and developmental biology of the vertebrate skeleton 
Summary
The rich phenotypic diversity that characterizes the vertebrate skeleton results from evolutionary changes in regulation of genes that drive development. Although relatively little is known about the genes that underlie the skeletal variation among fish species, significant knowledge of genetics and development is available for zebrafish. Because developmental processes are highly conserved, this knowledge can be leveraged for understanding the evolution of skeletal diversity. We developed the Phenoscape Knowledgebase (KB; http://kb.phenoscape.org) to yield testable hypotheses of candidate genes involved in skeletal evolution. We developed a community anatomy ontology for fishes and ontology-based methods to represent complex free-text character descriptions of species in a computable format. With these tools, we populated the KB with comparative morphological data from the literature on over 2,500 teleost fishes (mainly Ostariophysi) resulting in over 500,000 taxon phenotype annotations. The KB integrates these data with similarly structured phenotype data from zebrafish genes (http://zfin.org). Using ontology-based reasoning, candidate genes can be inferred for the phenotypes that vary across taxa, thereby uniting genetic and phenotypic data to formulate evo-devo hypotheses. The morphological data in the KB can be browsed, sorted, and aggregated in ways that provide unprecedented possibilities for data mining and discovery.
doi:10.1111/j.1439-0426.2012.01985.x
PMCID: PMC3377363  PMID: 22736877
11.  PhenoDigm: analyzing curated annotations to associate animal models with human diseases 
The ultimate goal of studying model organisms is to translate what is learned into useful knowledge about normal human biology and disease to facilitate treatment and early screening for diseases. Recent advances in genomic technologies allow for rapid generation of models with a range of targeted genotypes as well as their characterization by high-throughput phenotyping. As an abundance of phenotype data become available, only systematic analysis will facilitate valid conclusions to be drawn from these data and transferred to human diseases. Owing to the volume of data, automated methods are preferable, allowing for a reliable analysis of the data and providing evidence about possible gene–disease associations.
Here, we propose Phenotype comparisons for DIsease Genes and Models (PhenoDigm), as an automated method to provide evidence about gene–disease associations by analysing phenotype information. PhenoDigm integrates data from a variety of model organisms and, at the same time, uses several intermediate scoring methods to identify only strongly data-supported gene candidates for human genetic diseases. We show results of an automated evaluation as well as selected manually assessed examples that support the validity of PhenoDigm. Furthermore, we provide guidance on how to browse the data with PhenoDigm’s web interface and illustrate its usefulness in supporting research.
Database URL: http://www.sanger.ac.uk/resources/databases/phenodigm
doi:10.1093/database/bat025
PMCID: PMC3649640  PMID: 23660285
12.  InterMOD: integrated data and tools for the unification of model organism research 
Scientific Reports  2013;3:1802.
Model organisms are widely used for understanding basic biology, and have significantly contributed to the study of human disease. In recent years, genomic analysis has provided extensive evidence of widespread conservation of gene sequence and function amongst eukaryotes, allowing insights from model organisms to help decipher gene function in a wider range of species. The InterMOD consortium is developing an infrastructure based around the InterMine data warehouse system to integrate genomic and functional data from a number of key model organisms, leading the way to improved cross-species research. So far including budding yeast, nematode worm, fruit fly, zebrafish, rat and mouse, the project has set up data warehouses, synchronized data models, and created analysis tools and links between data from different species. The project unites a number of major model organism databases, improving both the consistency and accessibility of comparative research, to the benefit of the wider scientific community.
doi:10.1038/srep01802
PMCID: PMC3647165  PMID: 23652793
13.  MouseFinder: candidate disease genes from mouse phenotype data 
Human Mutation  2012;33(5):858-866.
Mouse phenotype data represents a valuable resource for the identification of disease-associated genes, especially where the molecular basis is unknown and there is no clue to the candidate gene’s function, pathway involvement or expression pattern. However, until recently these data have not been systematically used due to difficulties in mapping between clinical features observed in humans and mouse phenotype annotations. Here, we describe a semantic approach to solve this problem and demonstrate highly significant recall of known disease-gene associations and orthology relationships. A web application (MouseFinder; www.mousemodels.org) has been developed to allow users to search the results of our whole-phenome comparison of human and mouse. We demonstrate its use in identifying ARTN as a strong candidate gene within the 1p34.1-p32 mapped locus for a hereditary form of ptosis.
doi:10.1002/humu.22051
PMCID: PMC3327758  PMID: 22331800
phenotype; candidate disease genes; model organism; mouse
14.  Phenotypic overlap in the contribution of individual genes to CNV pathogenicity revealed by cross-species computational analysis of single-gene mutations in humans, mice and zebrafish 
Disease Models & Mechanisms  2012;6(2):358-372.
SUMMARY
Numerous disease syndromes are associated with regions of copy number variation (CNV) in the human genome and, in most cases, the pathogenicity of the CNV is thought to be related to altered dosage of the genes contained within the affected segment. However, establishing the contribution of individual genes to the overall pathogenicity of CNV syndromes is difficult and often relies on the identification of potential candidates through manual searches of the literature and online resources. We describe here the development of a computational framework to comprehensively search phenotypic information from model organisms and single-gene human hereditary disorders, and thus speed the interpretation of the complex phenotypes of CNV disorders. There are currently more than 5000 human genes about which nothing is known phenotypically but for which detailed phenotypic information for the mouse and/or zebrafish orthologs is available. Here, we present an ontology-based approach to identify similarities between human disease manifestations and the mutational phenotypes in characterized model organism genes; this approach can therefore be used even in cases where there is little or no information about the function of the human genes. We applied this algorithm to detect candidate genes for 27 recurrent CNV disorders and identified 802 gene-phenotype associations, approximately half of which involved genes that were previously reported to be associated with individual phenotypic features and half of which were novel candidates. A total of 431 associations were made solely on the basis of model organism phenotype data. Additionally, we observed a striking, statistically significant tendency for individual disease phenotypes to be associated with multiple genes located within a single CNV region, a phenomenon that we denote as pheno-clustering. Many of the clusters also display statistically significant similarities in protein function or vicinity within the protein-protein interaction network. Our results provide a basis for understanding previously un-interpretable genotype-phenotype correlations in pathogenic CNVs and for mobilizing the large amount of model organism phenotype data to provide insights into human genetic disorders.
doi:10.1242/dmm.010322
PMCID: PMC3597018  PMID: 23104991
15.  Construction and accessibility of a cross-species phenotype ontology along with gene annotations for biomedical research 
F1000Research  2013;2:30.
Phenotype analyses, e.g. investigating metabolic processes, tissue formation, or organism behavior, are an important element of most biological and medical research activities. Biomedical researchers are making increased use of ontological standards and methods to capture the results of such analyses, with one focus being the comparison and analysis of phenotype information between species.
We have generated a cross-species phenotype ontology for human, mouse and zebra fish that contains zebrafish phenotypes. We also provide up-to-date annotation data connecting human genes to phenotype classes from the generated ontology. We have included the data generation pipeline into our continuous integration system ensuring stable and up-to-date releases.
This article describes the data generation process and is intended to help interested researchers access both the phenotype annotation data and the associated cross-species phenotype ontology. The resource described here can be used in sophisticated semantic similarity and gene set enrichment analyses for phenotype data across species. The stable releases of this resource can be obtained from http://purl.obolibrary.org/obo/hp/uberpheno/.
doi:10.12688/f1000research.2-30.v1
PMCID: PMC3799545  PMID: 24358873
16.  A Unified Anatomy Ontology of the Vertebrate Skeletal System 
PLoS ONE  2012;7(12):e51070.
The skeleton is of fundamental importance in research in comparative vertebrate morphology, paleontology, biomechanics, developmental biology, and systematics. Motivated by research questions that require computational access to and comparative reasoning across the diverse skeletal phenotypes of vertebrates, we developed a module of anatomical concepts for the skeletal system, the Vertebrate Skeletal Anatomy Ontology (VSAO), to accommodate and unify the existing skeletal terminologies for the species-specific (mouse, the frog Xenopus, zebrafish) and multispecies (teleost, amphibian) vertebrate anatomy ontologies. Previous differences between these terminologies prevented even simple queries across databases pertaining to vertebrate morphology. This module of upper-level and specific skeletal terms currently includes 223 defined terms and 179 synonyms that integrate skeletal cells, tissues, biological processes, organs (skeletal elements such as bones and cartilages), and subdivisions of the skeletal system. The VSAO is designed to integrate with other ontologies, including the Common Anatomy Reference Ontology (CARO), Gene Ontology (GO), Uberon, and Cell Ontology (CL), and it is freely available to the community to be updated with additional terms required for research. Its structure accommodates anatomical variation among vertebrate species in development, structure, and composition. Annotation of diverse vertebrate phenotypes with this ontology will enable novel inquiries across the full spectrum of phenotypic diversity.
doi:10.1371/journal.pone.0051070
PMCID: PMC3519498  PMID: 23251424
17.  ZFIN, the Zebrafish Model Organism Database: increased support for mutants and transgenics 
Nucleic Acids Research  2012;41(Database issue):D854-D860.
ZFIN, the Zebrafish Model Organism Database (http://zfin.org), is the central resource for zebrafish genetic, genomic, phenotypic and developmental data. ZFIN curators manually curate and integrate comprehensive data involving zebrafish genes, mutants, transgenics, phenotypes, genotypes, gene expressions, morpholinos, antibodies, anatomical structures and publications. Integrated views of these data, as well as data gathered through collaborations and data exchanges, are provided through a wide selection of web-based search forms. Among the vertebrate model organisms, zebrafish are uniquely well suited for rapid and targeted generation of mutant lines. The recent rapid production of mutants and transgenic zebrafish is making management of data associated with these resources particularly important to the research community. Here, we describe recent enhancements to ZFIN aimed at improving our support for mutant and transgenic lines, including (i) enhanced mutant/transgenic search functionality; (ii) more expressive phenotype curation methods; (iii) new downloads files and archival data access; (iv) incorporation of new data loads from laboratories undertaking large-scale generation of mutant or transgenic lines and (v) new GBrowse tracks for transgenic insertions, genes with antibodies and morpholinos.
doi:10.1093/nar/gks938
PMCID: PMC3531097  PMID: 23074187
18.  Dcc Regulates Asymmetric Outgrowth of Forebrain Neurons in Zebrafish 
PLoS ONE  2012;7(5):e36516.
The guidance receptor DCC (deleted in colorectal cancer) ortholog UNC-40 regulates neuronal asymmetry development in Caenorhabditis elegans, but it is not known whether DCC plays a role in the specification of neuronal polarity in vertebrates. To examine the roles of DCC in neuronal asymmetry regulation in vertebrates, we studied zebrafish anterior dorsal telencephalon (ADt) neuronal axons. We generated transgenic zebrafish animals expressing the photo-convertible fluorescent protein Kaede in ADt neurons and then photo-converted Kaede to label specifically the ADt neuron axons. We found that ADt axons normally project ventrally. Knock down of Dcc function by injecting antisense morpholino oligonucleotides caused the ADt neurons to project axons dorsally. To examine the axon projection pattern of individual ADt neurons, we labeled single ADt neurons using a forebrain-specific promoter to drive fluorescent protein expression. We found that individual ADt neurons projected axons dorsally or formed multiple processes after morpholino knock down of Dcc function. We further found that knock down of the Dcc ligand, Netrin1, also caused ADt neurons to project axons dorsally. Knockdown of Neogenin1, a guidance receptor closely related to Dcc, enhanced the formation of aberrant dorsal axons in embryos injected with Dcc morpholino. These experiments provide the first evidence that Dcc regulates polarized axon initiation and asymmetric outgrowth of forebrain neurons in vertebrates.
doi:10.1371/journal.pone.0036516
PMCID: PMC3351449  PMID: 22606267
19.  Harmonin (Ush1c) is required in zebrafish Müller glial cells for photoreceptor synaptic development and function 
Disease Models & Mechanisms  2011;4(6):786-800.
SUMMARY
Usher syndrome is the most prevalent cause of hereditary deaf-blindness, characterized by congenital sensorineural hearing impairment and progressive photoreceptor degeneration beginning in childhood or adolescence. Diagnosis and management of this disease are complex, and the molecular changes underlying sensory cell impairment remain poorly understood. Here we characterize two zebrafish models for a severe form of Usher syndrome, Usher syndrome type 1C (USH1C): one model is a mutant with a newly identified ush1c nonsense mutation, and the other is a morpholino knockdown of ush1c. Both have defects in hearing, balance and visual function from the first week of life. Histological analyses reveal specific defects in sensory cell structure that are consistent with these behavioral phenotypes and could implicate Müller glia in the retinal pathology of Usher syndrome. This study shows that visual defects associated with loss of ush1c function in zebrafish can be detected from the onset of vision, and thus could be applicable to early diagnosis for USH1C patients.
doi:10.1242/dmm.006429
PMCID: PMC3209648  PMID: 21757509
20.  Notch signaling regulates endocrine cell specification in the zebrafish anterior pituitary 
Developmental biology  2008;319(2):248-257.
The vertebrate pituitary gland is a key endocrine control organ that contains six distinct hormone secreting cell types. In this study, we analyzed the role of direct cell-to-cell Delta-Notch signaling in zebrafish anterior pituitary cell type specification. We demonstrate that initial formation of the anterior pituitary placode is independent of Notch signaling. Later however, loss of Notch signaling in mind bomb (mib) mutant embryos or by DAPT treatment leads to increased numbers of lactotropes and loss of corticotropes in the anterior pars distalis (APD), increased number of thyrotropes and loss of somatotrope cell types in the posterior pars distalis (PPD), and fewer melanotropes in the posterior region of the adenohypophysis, the pars intermedia (PI). Conversely, Notch gain of function leads to the opposite result, loss of lactotrope and thyrotrope cell specification, and an increased number of corticotropes, melanotropes, and gonadotropes in the pituitary. Our results suggest that Notch acts on placodal cells, presumably as a permissive signal, to regulate progenitor cell specification to hormone secreting cell types. We propose that Notch mediated lateral inhibition regulates the relative numbers of specified hormone cell types in the three pituitary subdomains.
doi:10.1016/j.ydbio.2008.04.019
PMCID: PMC3178411  PMID: 18534570
adenohypophysis; cell differentiation; cranial; DAPT; Delta; hormone; Notch; organogenesis; patterning; placode
21.  Zebrafish sp7:EGFP: a transgenic for studying otic vesicle formation, skeletogenesis, and bone regeneration 
Genesis (New York, N.Y. : 2000)  2010;48(8):505-511.
Summary
We report the expression pattern and construction of a transgenic zebrafish line for a transcription factor involved in otic vesicle formation and skeletogenesis. The zinc finger transcription factor sp7 (formerly called osterix) is reported as a marker of osteoblasts. Using bacterial artificial chromosome (BAC)-mediated transgenesis, we generated a zebrafish transgenic line for studying skeletal development, Tg(sp7:EGFP)b1212. Using a zebrafish BAC, EGFP was introduced downstream of the regulatory regions of sp7 and injected into 1 cell-stage embryos. In this transgenic line, GFP expression reproduces endogenous sp7 gene expression in the otic placode and vesicle, and in forming skeletal structures. GFP-positive cells were also detected in adult fish, and were found associated with regenerating fin rays post-amputation. This line provides an essential tool for the further study of zebrafish otic vesicle formation and the development and regeneration of the skeleton.
doi:10.1002/dvg.20639
PMCID: PMC2926247  PMID: 20506187
bone; Danio rerio; gene transfer techniques; otic vesicle; regeneration; sp7
23.  Emx3 Is Required for the Differentiation of Dorsal Telencephalic Neurons 
emx3 is first expressed in prospective telencephalic cells at the anterior border of the zebrafish neural plate. Knockdown of Emx3 function by morpholino reduces the expression of markers specific to dorsal telencephalon, and impairs axon tract formation. Rescue of both early and late markers requires low-level expression of emx3 at the one- or two-somite stage. Higher emx3 expression levels cause dorsal telencephalic markers to expand ventrally, which points to a possible role of emx3 in specifying dorsal telencephalon and a potential new function for Wnt/beta-catenin pathway activation. In contrast to mice, where Emx2 plays a major role in dorsal telencephalic development, knockdown of zebrafish Emx2 apparently does not affect telencephalic development. Similarly, Emx1 knockdown has little effect. Previously, emx3 was thought to be fish-specific. However, we found all three emx orthologs in Xenopus tropicalis and opossum (Monodelphis domestica) genomes, indicating that emx3 was present in an ancestral tetrapod genome.
doi:10.1002/dvdy.22031
PMCID: PMC2975037  PMID: 19650145
Danio rerio; cell specification; empty spiracles; forebrain; pallium; homeobox; patterning; prosencephalon; transcription factor
24.  ZFIN: enhancements and updates to the zebrafish model organism database 
Nucleic Acids Research  2010;39(Database issue):D822-D829.
ZFIN, the Zebrafish Model Organism Database, http://zfin.org, serves as the central repository and web-based resource for zebrafish genetic, genomic, phenotypic and developmental data. ZFIN manually curates comprehensive data for zebrafish genes, phenotypes, genotypes, gene expression, antibodies, anatomical structures and publications. A wide-ranging collection of web-based search forms and tools facilitates access to integrated views of these data promoting analysis and scientific discovery. Data represented in ZFIN are derived from three primary sources: curation of zebrafish publications, individual research laboratories and collaborations with bioinformatics organizations. Data formats include text, images and graphical representations. ZFIN is a dynamic resource with data added daily as part of our ongoing curation process. Software updates are frequent. Here, we describe recent additions to ZFIN including (i) enhanced access to images, (ii) genomic features, (iii) genome browser, (iv) transcripts, (v) antibodies and (vi) a community wiki for protocols and antibodies.
doi:10.1093/nar/gkq1077
PMCID: PMC3013679  PMID: 21036866
25.  Evolutionary Characters, Phenotypes and Ontologies: Curating Data from the Systematic Biology Literature 
PLoS ONE  2010;5(5):e10708.
Background
The wealth of phenotypic descriptions documented in the published articles, monographs, and dissertations of phylogenetic systematics is traditionally reported in a free-text format, and it is therefore largely inaccessible for linkage to biological databases for genetics, development, and phenotypes, and difficult to manage for large-scale integrative work. The Phenoscape project aims to represent these complex and detailed descriptions with rich and formal semantics that are amenable to computation and integration with phenotype data from other fields of biology. This entails reconceptualizing the traditional free-text characters into the computable Entity-Quality (EQ) formalism using ontologies.
Methodology/Principal Findings
We used ontologies and the EQ formalism to curate a collection of 47 phylogenetic studies on ostariophysan fishes (including catfishes, characins, minnows, knifefishes) and their relatives with the goal of integrating these complex phenotype descriptions with information from an existing model organism database (zebrafish, http://zfin.org). We developed a curation workflow for the collection of character, taxonomic and specimen data from these publications. A total of 4,617 phenotypic characters (10,512 states) for 3,449 taxa, primarily species, were curated into EQ formalism (for a total of 12,861 EQ statements) using anatomical and taxonomic terms from teleost-specific ontologies (Teleost Anatomy Ontology and Teleost Taxonomy Ontology) in combination with terms from a quality ontology (Phenotype and Trait Ontology). Standards and guidelines for consistently and accurately representing phenotypes were developed in response to the challenges that were evident from two annotation experiments and from feedback from curators.
Conclusions/Significance
The challenges we encountered and many of the curation standards and methods for improving consistency that we developed are generally applicable to any effort to represent phenotypes using ontologies. This is because an ontological representation of the detailed variations in phenotype, whether between mutant or wildtype, among individual humans, or across the diversity of species, requires a process by which a precise combination of terms from domain ontologies are selected and organized according to logical relations. The efficiencies that we have developed in this process will be useful for any attempt to annotate complex phenotypic descriptions using ontologies. We also discuss some ramifications of EQ representation for the domain of systematics.
doi:10.1371/journal.pone.0010708
PMCID: PMC2873956  PMID: 20505755

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