Final Report Summary - ZF-HEALTH (Zebrafish Regulomics for Human Health)
Executive Summary:
"ZF-HEALTH - Zebrafish Regulomics for Human Health" was a Large-scale Integrating Project funded by the European Commission as part of its Seventh Framework Programme (EC Grant Agreement HEALTH-F4-2010-242048). The project started on July 1, 2010 and ran over a period of five and a half years.
ZF-HEALTH built on technologies and concepts developed in the preceding ZF-MODELS project. It utilised these advances for the high-throughput phenotyping of regulatory genes relevant for human disease, by behavioural assays, 3D / 4D imaging and expression profiling (by high-throughput sequencing). It further characterised regulatory elements of such genes by a combination of bioinformatics and transgenics. Small-molecule screening for mutant rescue or disease-relevant processes identified candidate drugs and provided insights into gene function. Our increasing knowledge on the regulators and their interactions with regulatory targets is thereby integrated with knowledge at cellular and organismic level. By capitalising on the virtues of the zebrafish system, this systems biology approach to the regulome allowed to gain unique knowledge complementing ongoing work in mammalian systems, providing important new stimuli for biomedical research.
The research effort was divided in five workpackages covering:
• Generation and distribution of mutants for potential human disease genes. We used TILLING technology to identify 43,098 knock-out alleles in 16,096 genes (61% of all protein-coding zebrafish genes). This is the first time most of the genes of a vertebrate were knocked out. We established a European Zebrafish Resoure Center which has distributed 386 of these alleles to European and international researchers for detailed phenotypic analysis. We generated additional targeted knock-outs by nuclease technologies and started inbreeding of wildtype zebrafish lines to provide better defined lines to researchers.
• Phenotyping of mutants. We carried out initial morphological and behavioral phenotyping for 2,569 genes, identifying 203 abnormal phenotypes, and released the data online. We further performed transcriptional profiling of 200 mutants and behavioral phenotyping of 45 mutants. 4D digital reconstructions were performed of developing embryos of wildtype zebrafish as well as several mutants. 30 scientific articles on mutant phenotypes were published.
• Characterisation of enhancer elements of human disease genes. We developed new bioinformatic approaches for the detection of highly conserved non-coding elements (HCNEs) and produced new genome-wide predictions of genomic regulatory blocks. Three sister species of zebrafish, Danio albolineatus and nigrofasciatus as well as the common carp, were sequenced in order to improve these predictions. A genome browser, ANCORA, was set up showing the results of this work. Many enhancers relevant for development and disease (e.g. for diabetes) were analyzed in transgenic zebrafish.
• Gene expression mapping in the brain. The interactive atlas zebrafishbrain.org established in the ZF-MODELS project and targeted both at students and at advanced researchers, has been greatly expanded with tutorials describing specific structures. Several hundred images of embryos and larvae were generated at cellular resolution with fluorescent labelling of specific cells, cell types or expression driven by specific enhancers, and many of them integrated into the atlas. 10 mutants were also analyzed. The web-based software pipeline ViBE-Z (Virtual brain explorer for zebrafish) was developed to allow researchers to compare large numbers of gene expression domains and map them to anatomical structures at high resolution.
• Small molecule screening. Existing and newly synthesized small-molecule libraries were shared by researchers to perform a total of 11 screens including screens relevant for human diseases such as attention deficit with hyperactivity disorder, epithelial cancer and polycystic kidney disease, and for modulators of ear morphogenesis. Several articles have already been published on compounds identified in these screens.
In total, the project resulted in 158 peer-reviewed articles, 67 doctoral and Masters theses and over 500 presentations and other dissemination activities.
Project Context and Objectives:
In recent years, the zebrafish has emerged as a new vertebrate model organism for biomedical research which offers a unique combination of traits: a short generation time, small size and efficient breeding procedures make it the best choice among vertebrates for forward genetic screening and small-molecule screens, including toxicology, while the transparent embryo and larva offers unique opportunities for imaging of cell movement and gene expression in a developing organism.
Much of the pioneering work on the zebrafish was performed in Europe. The Zebrafish Genome Project led by the Sanger Institute (GRL) produced a genome sequence. Since 2004, the Integrated Project “ZF-MODELS - Zebrafish Models for Human Development and Disease" was funded by the European Commission, strengthening the leading position of European zebrafish.
The ZF-HEALTH project built on several recent advances in the zebrafish field, many of them by ZF-MODELS participants:
• Knock-out of genes by the TILLING method in which target sequences are amplified and sequenced out of a library of mutagenised fish (KNAW, TUD, GRL).
• Zinc finger nuclease induced knock-out based on synthetic proteins that consist of sequence-specific zinc finger domains fused to a bacterial endonuclease.
• High-throughput assays for behavioral research, both for innate (motor, sensory functions) and “evolved” (emotion, motivation, learning) behaviors.
• The breakthrough 4D imaging method, digital scanned laser light sheet microscopy (DSLM), which allows to follow the movement of every cell in the transparent zebrafish embryo (KIT, CNRS).
• The discovery that vertebrate genomes are functionally structured in genomic regulatory blocks (GRBs) which contain long-range enhancers around a developmentally regulated target gene, surrounded by unrelated “bystander” genes (URA).
• High-throughput methods for small-molecule screens that allow to assess drug efficacy and toxicity, in the context of a whole organism, by arraying embryos on a microtiter plate and performing robotic imaging.
The project aimed to utilise all these advances for high-throughput phenotyping of at least a thousand regulatory genes relevant for human disease, by behavioral assays, 3D / 4D imaging and expression profiling. A phenotyping effort of this scale was never undertaken before in any vertebrate organism. We further characterized regulatory elements of such genes by a combination of bioinformatics and transgenics. An online atlas links high resolution gene expression patterns to neuroanatomical structures and will contribute to understanding behavioral phenotypes. Small-molecule screening for mutant rescue or disease-relevant processes will identify candidate drugs and provide insights into gene function.
Our main scientific focus was on development and disease in the areas that generate the largest fraction of health related costs in Europe:
• Development, behavioral, and neurological diseases,
• eye and visual processing,
• tissue regeneration and repair, including regeneration in the brain,
• diabetes and obesity, as well as infectious disease and cancer.
Project Results:
The ZF-HEALTH project comprised five scientific work packages, as well as work packages focusing on dissemination, training and management. Below are the main results by work package:
WP1 – Generation and distribution of mutants for potential human disease genes
The first work package focused on TILLING, a method to obtain knock-out mutations in specific genes by generating a library of mutagenizing animals, and then sequencing the loci of interest until the desired mutation is found. During the project TILLING technology was greatly improved. Rather than screening for mutations in specific amplicons only, the Wellcome Trust Sanger Center (GRL) used high-throughput sequencing of the entire exome (i.e. all translated sequences) of mutagenised fish, identifying knock-out mutations genome-wide. In support of the TILLING project new libraries of mutagenised fish were generated by GRL, the Hubrecht Institute (KNAW) and Technical University of Dresden (TUD). By this method 43,098 knock-out alleles were identified in 16,096 genes (61% of all protein-coding zebrafish genes), far exceeding the original goal of 1,000 genes. All alleles generated were archived as frozen sperm samples and are being submitted to the newly established European Zebrafish Resource Center (EZRC) of Karlsruhe Institute of Technology (KIT) as well as to the Zebrafish International Resource Center (ZIRC, Eugene, Oregon). 32,000 alleles have already been delivered.
Complementing the genome-wide knock-out project, we also focused on technologies that generated targeted knock-outs in genes of special interest with nuclease reagents. At the start of the project, Zinc Finger Nuclease (ZFN) was already available, but this was superseded in the course of the project first by TALEN, and then by CRISPR-Cas9 nucleases. The latter are most efficient and use an RNA strand for guidance which can be generated in vitro, eliminating the need for molecular cloning steps. Four partners initially established pipelines for the Zinc Finger Nuclease (ZFN) technology for generating additional knock-outs. Three of the partners later converted their mutagenesis pipelines to use TALEN technology, while KIT established a TALEN pipeline from the outset. By the end of the project several partners had adopted the even more efficient CRISPR-Cas9 technology. In total, at least 41 alleles were generated by these technologies.
In parallel with mutant production, KIT established its European Zebrafish Resource Center and with the help of GRL, established sperm freezing and IVF protocols based on those at use at GRL, carrier identification by allele-specific PCR (KASPar assay) and standard procedures for accepting and distributing a large number of fish lines. The European Zebrafish Resource Center uses space in two different buildings, several hundred meters apart, which helps to keep the core of the resource center free from contamination. All fish to be imported are kept in a Quarantine Room and only their offspring is transferred as disinfected eggs to the Core Fish Room, which is located in a different building. Fish health is monitored by a veterinarian by regularly examining sentinel fish. An internal laboratory database as well as an easily searchable public website and web store were developed and standard operating procedures for processing orders were established. KIT maintains and distributes mutant and transgenic zebrafish lines and also gives zebrafish researchers access to screening services and technologies such as imaging and high-throughput sequencing. Requested lines are shipped as disinfected embryos. Besides the the lines from GRL, it maintains and distributes the entire stock collection of the Nüsslein-Volhard laboratory (MPG), comprising over 2,000 published mutations, as frozen sperm samples, as well as over 100 mutant and transgenic lines from other sources. It closely collaborates with ZIRC to give European researchers cost-effective access to lines available there, since the cost of trans-atlantic shipping is a major impediment to using these lines. It also collaborates with the central model organism database for the zebrafish, ZFIN, which provides a repository for genetic and phenotypic data with “order this” links to the EZRC, and with the Institute of Molecular and Cell Biology (IMCB, Singapore), which provided transgenic lines.
All lines and information on the EZRC are available through its public website: see Fig. 1 and 2. Since the website launch in November 2013 KIT has shipped a total of 558 zebrafish lines, 386 of them GRL mutants, to recipients within and outside the ZF-HEALTH consortium.
Two new tasks were proposed by the Consortium in 2015: inbreeding of zebrafish wildtype lines and generation of additional Cre driver lines for conditional knock-out of genes in neural development by knock-in.
Inbreeding was added to the ZF-HEALTH work programme order to generate zebrafish lines with better defined genetic background then the outbred wildtype lines previously available. It is being performed both at KIT (where 10 full-sib pairs are grown up per generation for 5 wildtype lines) and at ZF BioLabs SL (ZFB, 15 full-sib pairs per generation for 1 line), 65 full-sib pairs in total. KIT has currently reached generation 3, and ZFB generation 5. It is planned to continue the inbreeding effort after the end of the ZF-HEALTH project (with institutional funding) and to make the lines developed at KIT as well as ZFB available through the European Zebrafish Resource Center at KIT.
Generation of Cre driver lines by knock-in into genes of interest, which will be novel and versatile tools for studying neural development, was begun and is still underway at TUD.
WP2 – Phenotyping
Systematic phenotyping, i.e. determing and analyzing the effects of mutations, is a step of central importance in order to make use of the thousands of zebrafish mutants as disease models or to analyze developmental pathways. GRL established a pipeline for initial morphological and behavioural phenotyping (for embryonic and larval phenotypes visible through a stereomicroscope and touch response up to 5 days post fertilization). This was carried out at an unprecedented scale, for over 4,000 TILLING mutations in 2,569 genes, identifying 203 (8 % ) abnormal phenotypes, far more than envisioned. The data can be found on the ZMP website set up by GRL at http://www.sanger.ac.uk/Projects/D_rerio/zmp/ and are being submitted to the ZFIN database. This effort also resulted in a Nature article (Kettleborough et al., 2013). For an example of a phenotype see Fig. 3. Likewise 30 nuclease-induced mutants were phenotyped at several partners institutions. 2 abnormal phenotypes were identified, which is line with this number of phenotypes expected from the phenotyping of TILLING mutants.
Since the start of the project GRL has further performed over 200 expression profiling experiments on on mutant embryos as well as wild-type embryos of a range of developmental. The first results are available online through the ZMP website.
KIT established the photomotor response assay (PMR) for shelf screening of knock-out mutants from GRL, and carried out a pilot screen. This assay originally developed for chemical screening by the laboratory of Randall Peterson (Boston) which promises to give insight into all the major pathways of neural transmission. While this work will be continued, our emphasis has shifted away from shelf screening at the EZRC resource center to a more distributed effort to conduct in-depth phenotyping of mutants directly at the participating labs. This was a decision made in order to make the most productive use of our limited resources since recovery of knock-out mutations from GRL involved considerably more effort than we expected. The success of this approach is reflected by the fact that KIT sent out 386 Sanger mutants for phenotyping by specialized laboratories (WP1), far more than anticipated, and by at least 30 articles that have already been published by Consortium Members on the phenotyping of mutants and the analysis of pathways involved.
The behavioural phenotyping facilities of CNRS and University of Zurich (UZH) were already established in the first reporting period and have performed detailed analysis of a total of 45 mutants, more than originally planned.
In depth phenotyping was performed by several labs. Since the start of the project CNRS studied the Nodal pathway mutant zoep, INSERM studied the RNA-binding protein mutants, qkA and qkB, King’s College London (KCL) the function of foxg1a and dkk1, University of Leiden (UL) the immune-related mutants myd88 and cxcr3, and TUD a zebrafish model for Parkinson’s disease, MPG analysed the role of vasna and vasnb in retinotectal axon guidance, University of Sheffiled (USFD) phenotyped several mutants related to angiogenesis and diseases, Univesity of Cologne (UCO) continued worked on genes involved in skin and bone development, and University of Padua (UNIPD) characterized the role of tcf7l2 in pancreatic development and studied models of pancreatic adenocarcinoma and collagen VI myopathy.
To date 30 articles resulting from the in-depth phenotyping and molecular analysis of mutants have been published by the Consortium, greatly exceeding our original goal of at least 5 publications from analysis of at least 10 selected mutants. Several more are underway. An article on the role of inflammation as the first step towards brain regeneration in zebrafish, was published in Science (Kyritsis et al. 2012).
WP3 – Characterisation of enhancer elements of human disease genes
Our partners University of Sydney (USYD) and Imperial College London (IMPERIAL, who took over the tasks originally assigned to Uni Research AS, Bergen due to a move of the principal investigator) have defined regulatory regions based on several criteria: highly conserved non-coding elements; estimates of synteny span around target genes; and epigenetic marks (histone modification).
IMPERIAL made several updates to its custom genome browser, ANCORA, in which HCNEs, synteny blocks, and relevant epigenetic marks can be assessed for any region of the human genome (http://ancora.genereg.net Fig. 4), and released a software package for studying large sets of promoters. IMPERIAL also explored the relationship of GRB turnover and gene function and identified novel target genes for human disease-associated regulatory variants in the zebrafish, using schizophrenia for a pilot study.
UL and IMPERIAL furthermore sequenced and annotated the common carp genome, which was found to be tetraploid and extremely condensed. Two other sister species, Danio nigrofasciatus and Danio albolineatus were sequenced at moderate coverage by GRL and the University of Freiburg (ALU-FR). These sequences together with the recently available grass carp genome were utilized to dramatically increase the number of known zebrafish CNEs.
In three experiments with a total of over injected 2,000 founders the University of Birmingham (BHAM) analysed human HCNEs associated with type 2 diabetes, with pancreatic agenesis and with the estrogen-receptor related gene gamma alpha (errga).
Moreover USYD and BHAM analysed the enhancers of tp53, miR-9-1, miR-9-2, miR-9-3, Pou3f3, Foxg1 and Sox2 by ChIP-Seq and related methods. ALU-FR used ChIP-Seq to characterize binding sites relevant for zygotic gene activation at the mid-blastula transition. INSERM studied enhancers of Myosin VIIA. UNIPD generated reporter lines for Wnt signaling, hypoxia signaling, the Hippo pathway, thyroid development, the Stat3 and Notch patchways and used reporters to analyse activation of the hypoxia pathways by glucocorticoids. BHAM validated bidirectionally transcribed human enhancers in transgenic zebrafish embryos.
Five papers were published on expression patterns of HCNEs in transgenic zebrafish lines. An article on promoter structure was published in Nature (Haberle et al., 2014).
WP4 – Gene expression mapping in the brain
To understand the function of genes that give behavioral phenotypes when mutated, one needs a detailed description of neuroanatomical gene expression domains as well as of the circuitry of the brain. The overall aims of this work package were to establish a framework to link high resolution 3D gene expression pattern to neuroanatomical structures, and to contribute to resolving the connectivity of circuits in the developing brain with an emphasis on limbic system circuits and monoaminergic circuits. This main tools for this were the ViBE-Z software pipeline and the interactive atlas, zebrafishbrain.org.
ALU-FR developed the Virtual Brain explorer (ViBE-Z), a novel software pipeline that automatically maps gene expression data with cellular resolution to a 3D standard larval zebrafish brain at day 2 – 6. A web interface is publicly at http://vibez.informatik.uni-freiburg.de/. ViBE-Z uses fluorescently stained cell nuclei for image registration and automatically detects 14 predefined anatomical landmarks for aligning new data with the reference brain. This makes it feasible to search for expression in defined anatomical parts and to compare the domains of expression of genes of interest (Fig. 5 and 6).
University College London (UCL) continued development of its interactive atlas of the zebrafish brain, http://zebrafishbrain.org begun in the ZF-MODELS FP6 project (Fig. 7). The atlas features 3D images as well as expert-written tutorials that describe specific strutures of the brain, and is as unique and highly valuable tool both for teaching and for collaborative research. 8 tutorials on zebrafish homologs of limbic system structures where added, bringing the total number of tutorials to 25. UCL further developed mosaic single cell labelling methods based on CRISPR/Cas9 genome editing to study the limbic system. UCL also examined gene expression in 28 HCNE derived lines and 65 enhancer lines (including lines generated by USYD in work package 3), and incorporated and annotated part of them in zebrafishbrain.org.
KCL and CNRS established 3D maps of the telencephalon from day 1 to 30 of development, and CNRS analysed the “optic recess region” as a new identified morphogenetic entity.
Finally, both UCL and CNRS established pipelines for characterizing in detail the neuroanatomy of mutant lines, and analyzed over 10 such lines.
WP5 – Chemical screening
The screening of small-molecule libraries containing chemicals with potential pharmacological effects is particularly efficient in the zebrafish, since the zebrafish embryos can be exposed to the chemicals and analysed in microtiter plates, at high throughput, while giving insight into the effecs on an entire vertebrate organism that would not be possible with the commonly used cell culture assays.
Two publicly available small-molecule libraries (FDA approved drugs and ICCB) were acquired by KIT, and in the first reporting period aliquots were distributed to 18 European zebrafish laboratories inside and outside the ZF-HEALTH consortium during the reporting period. The combinatorial chemistry platform of KIT, Complat, generated two additional libraries one of which was tested in the zebrafish.
Several partner laboratories developed novel screening technologies. KIT developed an assay for glucocorticoid signalling, UL optimized a high throughput screening system for cancer and tuberculosis, BHAM developed a transgenic biosensor for screening of kidney development and function, and ZFB optimized hormone-induced spawning.
A total of eleven screens were performed, more than anticipated: for hypoxia signaling (USFD), nicotine analogs in the developing zebrafish embryo (BHAM), mitochondrial dysfunction (ZFB), granulocytic inflammatory response (KIT), attention deficit with hyperactivity disorder (CNRS), epithelial carcinogenesis (UCO), neural tube development (KIT), activation of glucocorticoid signalling (KIT), rescue of epidermal defects in zebrafish hai1a mutants relevant for cancer (UCO) and enhancers and suppressors of the pkd2/cup mutation relevant for polycystic kidney disease (USFD), and for modulators of versican expression in the zebrafish ear (USFD).
Several partners reported the confirmation and further analysis of hits resulting from the screens, UL on compounds that inhibit tuberculosis, USFD on a highly potent HIF activating compound, UCO on potentially anti-carcinogenic compounds, and KIT on anti-inflammatory compounds.
5 articles were published or submitted describing our first results from the analysis of pathways in development and disease, using small compounds identified by chemical screening. A notable result is the suppression of the behavioural phenotype of an autism model by estrogen-related compounds. Other publications concerned tuberculosis drugs and the modulation of inflammatory processes.
Potential Impact:
As stated in the FP7 work programme:
“The overall objective of the Health Theme is to improve the health of European citizens and increase the competitiveness and boost the innovative capacity of European health-related industries and businesses, while addressing global health issues including emerging epidemics. Emphasis will be put on translational research (translation of basic discoveries into clinical applications including scientific validation of experimental results), the development and validation of new therapies, strategies for health, promotion and prevention, child health and healthy ageing, diagnostic tools and medical technologies, as well as sustainable and efficient healthcare systems.”
Our project addressed these objectives as follows.
• It helps to improve the health of European citizens as all of its deliverables are of value for clinical medicine: mutants and phenotypic data for use as disease models, insight into the structural and molecular basis of human behaviour, a functional characterisation of enhancer elements that will help to interpret data on human genetic variability, and validated hits from small-molecule screening for drug candidates.
• In the U.S.A. the importance of zebrafish as a model organism was recognised as early as the last decade. Since that time zebrafish research has been continuously funded by the NIH at far higher levels than in Europe, whether by national funding agencies or the EU. Against this background, our project was of great strategic importance to increase the competitiveness and boost the innovative capacity of European health-related industries and businesses.
• In order to accomplish this we strongly focused on translational research. One SME (ZF Biolabs) participated in the project to help with this focus. Many project partners also have collaborations with SMEs or larger pharmaceutical companies.
Moreover the work programme states:
“The data generated by genomics and proteomics projects will continue to support the European research excellence in related fields by increasing our understanding of key biological processes and provide the foundation for more extensive functional studies concerning genetic variation, the way that genes interact with each other in health and disease, and to speed the search of genes that may underlie diseases in human. This in turn would aid the design of drugs and treatments, including individualised treatments. For example, large-scale functional genomics studies in multi- cellular model organisms will generate new knowledge on human gene function in health and diseases and potential model systems for drug screening.”
Our project directly addressed each of these expected impacts:
• We have substantially increased our understanding of the genetic roots of fundamental biological processes including brain development, visual processing, and behaviour. The zebrafish brain is a uniquely accessible and tractable model for such purposes, which we will leverage by creating a comprehensive atlas of gene expression in development and by studying the effect of knock-out mutations in hundreds of regulatory genes on the brain. There is a growing appreciation that the results of such testing in the zebrafish are directly applicable to mammalian brains, and hence to clinicians. We have further characterised the state of neural and muscle stem cells and aspects of the immune response, areas where the zebrafish has particular strengths as a model organism.
• The genome-wide analysis of genomic regulatory blocks (GRBs) containing long-range enhancers and their target genes will provide a unique new view of the large-scale organisation of vertebrate genomes. This information, mapped to the human genome assembly, will help substantially in understanding human genetic variation and provide a foundation for future targeted searches for non-coding mutations associated with human disease. The identification of such mutations will eventually open the door to an individualised medicine. Our work is integrated with international efforts in human genomics such as the HapMap and ENCODE projects wherever possible. The experimental validation of enhancers in the zebrafish initially focused on regions implicated diseases such as type II diabetes (a condition with a high prevalence in the European population). In future work, building on the methods developed in our project, they will be extended to other common diseases.
• We have systematically assessed the phenotypic effects of (a) targeted mutations over 2,000 genes including transcriptional regulators and homologs of genes suspected in human disease, and (b) disease-relevant mutations identified in previous forward-genetic approaches. A substantial fraction of these are candidates for human disease genes, adding to our knowledge on their function in health and diseases, and providing a shortcut to targeted searches for such genes in the human.
• Many of the mutations are suitable model systems for drug screening. Several project partners have investedsubstantially in the development of robotic technologies for small-molecule screening in the zebrafish. They have refined their technologies and conducted pilot studies within the proposed project, using chemical libraries generated and / or centrally distributed by the consortium. These screens have provided insight into biological processes in themselves, but also served as a proof of principle for the feasibility of large-scale commercial drug and toxicology screens in the zebrafish to be undertaken by the pharmaceutical industry. An SME (small or medium enterprise) end user of our technologies and libraries, the biotechnology company ZFB, was part of our consortium.
In summary, we believe that our project has provided valuable inputs for reaching the objectives of the Seventh Framework Programme, and strongly support European research excellence in the area of model organisms for biomedical research. It has fulfilled our expectation of continuing the success of the ZF-MODELS FP6 project which for the past five years has been a focal point of European zebrafish research, and which has helped to overcome its previous fragmentation by fostering international collaboration, increased the visibility of zebrafish models by its dissemination effort, and mobilised resources far beyond those funded by European Commission.
We disseminate the results obtained during the ZF-HEALTH project to different groups of stakeholders – the biological research community, pharmaceutical companies and clinicians, as well as policy makers and the general public – by different means suited to the particular group, as follows:
To the research community:
1) Through direct release of high-throughput data by public databases linked to the ZF-HEALTH website, as well as specialist databases set up by the Consortium. Mutant and transgenic zebrafish lines and plasmids containing zebrafish sequences are made available by the European zebrafish stock centre that we established. For details see “Management of high-throughput data and biomaterials” below.
2) Publication of new results – including novel tools and technologies – in international scientific journals. 154 articles were produced (far more than the 50 we anticipated), including several in highly ranked journals such as Nature and Science. All these publications acknowledge support by the EC as specified by the Grant Agreements.
3) Presentations at major European and other international conferences. Over 400 such presentations were made over the duration of the project.
4) The open access part of the ZF HEALTH website, which provides links to online resources and publications produced by the project and throughout the project, announced invitations to participate.
5) Organisation of 3 special interest symposia and 5 workshops targeted at junior researchers. The symposia were titled “Zebrafish Models in Translational Medicine” (Courcelle-sur-Yvette, 2013, coinciding with the 4th Project meeting), “Towards an encyclopedia of DNA elements in zebrafish” (London, 2014) “The Zebrafish: A Vertebrate Model Organism for Biological and Biomedical Investigations" (Gif-sur-Yvette, 2015, coinciding with the 5th workshop). The workshops were titled “Cutting edge technologies in biomedical research” (Karlsruhe, 2011), “Genomics and high throughput sequencing technologies with the zebrafish model” (Cambridge, 2012), “Automation methods for zebrafish research” (Leiden, 2013), “Imaging of Neural Development in Zebrafish” (Karlsruhe, 2014), and “ZF-HEALTH Resources for in Silico Experimentation: VIBE-Z – BioEmergences – MecaGen” (Gif-sur-Yvette, 2015).
6) Incorporation in research training. 67 students obtained their Masters or doctoral degree within the project, results were included in the masters and doctoral research training programmes of each of the partners reaching at least 500 research students over the duration of the project and 5 workshops were organised.
To companies and clinicians:
1) Through articles on new scientific developments, tools and technologies for industrial and medical journals.
2) presentations at major medical and industrial conferences (biotechnological and pharmaceutical),
3) participitation of junior researchers from industry and hospitals in our 5 workshops,
4) collaborations and staff exchanges with companies and hospitals,
5) licensing of patented data, tools and technologies (following the end of the project).
A total of 490 collaborations with industry and medical researchers were reported by Consortium members – for a detailed listing see the annual Dissemination Reports.
To policy makers and the general public:
1) Through a press release (issued at the occasion of the opening of the European Zebrafish Resource Center)
2) popular-science articles for newspapers, popular-science magazines, publications targeted at policy makers on the national and European level, and industrial journals, covering our tools and results (we expect at least 10 such articles throughout the duration of the project),
3) the open-access part of the ZF-HEALTH website which provides a non-specialist introduction to the project's background, objectives and results,
4) a two-page flyer describing the project, for use by the Consortium and the Commission,
5) presentations of the laboratories involved in the ZF-MODELS Project at open days, local and
national science fairs.
These efforts were aided by utilising contacts and expertise from an FP7 COST action, the European Network on Fish Biomedical Models (EUFishBioMed), which represents over 300 zebrafish laboratories.
Project website and flyer
As soon as a Grant Agreement for ZF-HEALTH was concluded with the European Commission, the Scientific Coordinator and Management Support Team set up the project website (http://www.zf-health.org) that provides:
• an overview of the ZF-HEALTH partnership and its research activities
• a section providing background information, project news and announcements (e.g. invitations to participate in our workshops)
• links to a network of public databases (e.g. ZFIN and Ensembl, as well as specialist databases set up by the Consortium) that provide direct access to the high-throughput
data produced in our project.
• a password-protected section for project partners and EC services for the internal
• exchange of data and knowledge, including an internal project news and download section.
The website was regularly updated throughout the project
The Scientific Coordinator and Management Support Team have furthermore prepared a two page- flyer describing the project, its objectives, its research activities, the partnership, its website and online resources, for use by the Consortium and the European Commission. This flyer was kept up to date and was also distributed at the bi-annual International Zebrafish Meetings (in the USA), European Zebrafish Meetings, and European Zebrafish PI Meetings, throughout the duration of the project.
Press releases
The Scientific Coordinator and Management Support Team have issued a press releases on behalf of the partners on the occasion of the opening of the European Zebrafish Resource Center (ERZC) of KIT.
Management of high-throughput data and biomaterials
We aim for openness in our research. High-throughput data and biomaterials produced in our project were therefore released continuously through a network of public databases linked to the ZF-HEALTH website, and through the newly established European zebrafish stock centre. Below is an overview of the high-throughput data and materials produced in our project, and their release:
WP1
Molecular data on mutations through ZFIN, ENSEMBL, and the Zebrafish Mutation Project (ZMP) database set up by GRL, immediately.
WP2
Results of high-throughput phenotyping through ZFIN (using the PATO ontology) and ZMP, within 6 months after phenotyping.
Digital reconstructions from 3D / 4D imaging through the Bioemergences website (still under construction).
Transcriptional profiling results through ZMP, immediately.
Standardised protocols through the ZF-HEALTH website, immediately.
In-depth analysis of phenotypes and pathways through scientific papers, after publication.
WP3
Comparative data on enhancers in human, zebrafish and other species via the ANCORA genome browser of IMPERIAL, immediately.
WP4
Images of expression patterns of transgenic lines in the brain through the zebrafishbrain.org to be established by UCL and ALU-FR, immediately.
WP5
Confirmed hits from chemical screening Scientific papers, licensing to the pharmaceutical industry, after publication.
The following schedule applies to the distribution of biomaterials:
Mutants generated by the TILLING method are made available immediately to all interested parties, through the newly established European Zebrafish Resource Center of KIT.
• Mutants generated by targeted nuclease methods (in which the generating partner may have invested a significant amount of effort) are made available 12 months after their initial phenotyping data are released or after an initial publication, whatever comes first, but may be made available earlier if the generating partner desires.
• Transgenic lines are made available 12 months after images of gene expression are released or after an initial publication, whatever comes first, but may be made be available earlier if the generating partner desires.
•
Patenting and exploitatiom
We intend to distribute the results, tools and technologies generated in our project to all interested parties. However, we also aim to maximise their commercial exploitation. Therefore appropriate measures ensuring the protection of intellectual property rights (IPRs) were taken in order to guarantee possible commercialisation of these results, tools and technologies.
The partners have extensive experience with IPR issues and commited themselves to protecting all new knowledge identified as having economic potential either through patenting or through treating the knowledge as “commercial in confidence”. This protection was done through the technology transfer office of the partner who has generated the knowledge.
While the great majority of our results are in the area of basic research and were therefore not considered to be of direct commercial value, two patents were submitted in the course of the project:
“Spaink, H.P. and Dirks, R.P.: HIGH THROUGHPUT METHOD AND SYSTEM FOR IN VIVO SCREENING”
“Karlsruher Institut für Technologie: Karlsruher Institut für Technologie: MICROSCOPE WITH AT LEAST ONE ILLUMINATING BEAM IN THE FORM OF A LIGHT SHEET”
Our commercial partner, ZFB, further reported the following exploitable foreground:
“Improvement of method of hormonal induction to obtain, in a short period of time, many embryos of zebrafish with high fertility and viability. Furthermore, with the hormonal treatment, ovulation and spermiation is reached on predetermined time and the fertilization and incubation can be done under aseptic conditions, both things are very difficult to obtain using the natural spawning traditional method.”
For details please see section B.1 and B.2 below.
Material Transfer Agreement
Mutants and transgenic lines of zebrafish are made available to parties outside of the ZF- HEALTH Consortium only if the receiving party signs a Material Transfer Agreement (MTA), in line with standard practices in biomedical research. We expect that most mutants and transgenic lines will be distributed through the European stock centre currently being established by KIT-G, which will keep track of the signed MTAs. However, in case such lines are directly distributed by other project partners, the issuing partner will be responsible for obtaining a signed MTA and sending a copy to the Coordinator.
The MTA was drawn up at the start of the project by the Scientific Coordinator and Management Support Team and put in place by the General Assembly. It contain minimal restrictions and was modeleted on the MTA previously used by GRL for its knock-out mutations as well as the MTA of the Zebrafish International Resource Center (ZIRC). Briefly, it specified that:
1) The lines will be used for academic and non-commercial research purposes only and will not be sold or bred for sale.
2) They will not be further distributed to others without written permission from the original provider.
3) The recipient is free to publish results from the lines, but is required must acknowledge the origin of the lines from a member of the our consortium, as well as the EZRC in such publications.
In the case of commercial entities requesting access to mutant or transgenic zebrafish lines, this access will be granted under terms and conditions agreed upon in a collaboration agreement that will be negotiated on an individual basis.
List of Websites:
Public website: http://zf-health.org
Scientific coordinator's address:
Dr. Robert Geisler
Karlsruhe Institute of Technology (KIT)
Institute of Toxicology and Genetics
Hermann-von-Helmholtz-Platz 1
Building 439, Room 310
76344 Eggenstein-Leopoldshafen, Germany
Phone: +49 721 608-24664
Fax: +49 721 608-23354
E-mail: robert.geisler@kit.edu
"ZF-HEALTH - Zebrafish Regulomics for Human Health" was a Large-scale Integrating Project funded by the European Commission as part of its Seventh Framework Programme (EC Grant Agreement HEALTH-F4-2010-242048). The project started on July 1, 2010 and ran over a period of five and a half years.
ZF-HEALTH built on technologies and concepts developed in the preceding ZF-MODELS project. It utilised these advances for the high-throughput phenotyping of regulatory genes relevant for human disease, by behavioural assays, 3D / 4D imaging and expression profiling (by high-throughput sequencing). It further characterised regulatory elements of such genes by a combination of bioinformatics and transgenics. Small-molecule screening for mutant rescue or disease-relevant processes identified candidate drugs and provided insights into gene function. Our increasing knowledge on the regulators and their interactions with regulatory targets is thereby integrated with knowledge at cellular and organismic level. By capitalising on the virtues of the zebrafish system, this systems biology approach to the regulome allowed to gain unique knowledge complementing ongoing work in mammalian systems, providing important new stimuli for biomedical research.
The research effort was divided in five workpackages covering:
• Generation and distribution of mutants for potential human disease genes. We used TILLING technology to identify 43,098 knock-out alleles in 16,096 genes (61% of all protein-coding zebrafish genes). This is the first time most of the genes of a vertebrate were knocked out. We established a European Zebrafish Resoure Center which has distributed 386 of these alleles to European and international researchers for detailed phenotypic analysis. We generated additional targeted knock-outs by nuclease technologies and started inbreeding of wildtype zebrafish lines to provide better defined lines to researchers.
• Phenotyping of mutants. We carried out initial morphological and behavioral phenotyping for 2,569 genes, identifying 203 abnormal phenotypes, and released the data online. We further performed transcriptional profiling of 200 mutants and behavioral phenotyping of 45 mutants. 4D digital reconstructions were performed of developing embryos of wildtype zebrafish as well as several mutants. 30 scientific articles on mutant phenotypes were published.
• Characterisation of enhancer elements of human disease genes. We developed new bioinformatic approaches for the detection of highly conserved non-coding elements (HCNEs) and produced new genome-wide predictions of genomic regulatory blocks. Three sister species of zebrafish, Danio albolineatus and nigrofasciatus as well as the common carp, were sequenced in order to improve these predictions. A genome browser, ANCORA, was set up showing the results of this work. Many enhancers relevant for development and disease (e.g. for diabetes) were analyzed in transgenic zebrafish.
• Gene expression mapping in the brain. The interactive atlas zebrafishbrain.org established in the ZF-MODELS project and targeted both at students and at advanced researchers, has been greatly expanded with tutorials describing specific structures. Several hundred images of embryos and larvae were generated at cellular resolution with fluorescent labelling of specific cells, cell types or expression driven by specific enhancers, and many of them integrated into the atlas. 10 mutants were also analyzed. The web-based software pipeline ViBE-Z (Virtual brain explorer for zebrafish) was developed to allow researchers to compare large numbers of gene expression domains and map them to anatomical structures at high resolution.
• Small molecule screening. Existing and newly synthesized small-molecule libraries were shared by researchers to perform a total of 11 screens including screens relevant for human diseases such as attention deficit with hyperactivity disorder, epithelial cancer and polycystic kidney disease, and for modulators of ear morphogenesis. Several articles have already been published on compounds identified in these screens.
In total, the project resulted in 158 peer-reviewed articles, 67 doctoral and Masters theses and over 500 presentations and other dissemination activities.
Project Context and Objectives:
In recent years, the zebrafish has emerged as a new vertebrate model organism for biomedical research which offers a unique combination of traits: a short generation time, small size and efficient breeding procedures make it the best choice among vertebrates for forward genetic screening and small-molecule screens, including toxicology, while the transparent embryo and larva offers unique opportunities for imaging of cell movement and gene expression in a developing organism.
Much of the pioneering work on the zebrafish was performed in Europe. The Zebrafish Genome Project led by the Sanger Institute (GRL) produced a genome sequence. Since 2004, the Integrated Project “ZF-MODELS - Zebrafish Models for Human Development and Disease" was funded by the European Commission, strengthening the leading position of European zebrafish.
The ZF-HEALTH project built on several recent advances in the zebrafish field, many of them by ZF-MODELS participants:
• Knock-out of genes by the TILLING method in which target sequences are amplified and sequenced out of a library of mutagenised fish (KNAW, TUD, GRL).
• Zinc finger nuclease induced knock-out based on synthetic proteins that consist of sequence-specific zinc finger domains fused to a bacterial endonuclease.
• High-throughput assays for behavioral research, both for innate (motor, sensory functions) and “evolved” (emotion, motivation, learning) behaviors.
• The breakthrough 4D imaging method, digital scanned laser light sheet microscopy (DSLM), which allows to follow the movement of every cell in the transparent zebrafish embryo (KIT, CNRS).
• The discovery that vertebrate genomes are functionally structured in genomic regulatory blocks (GRBs) which contain long-range enhancers around a developmentally regulated target gene, surrounded by unrelated “bystander” genes (URA).
• High-throughput methods for small-molecule screens that allow to assess drug efficacy and toxicity, in the context of a whole organism, by arraying embryos on a microtiter plate and performing robotic imaging.
The project aimed to utilise all these advances for high-throughput phenotyping of at least a thousand regulatory genes relevant for human disease, by behavioral assays, 3D / 4D imaging and expression profiling. A phenotyping effort of this scale was never undertaken before in any vertebrate organism. We further characterized regulatory elements of such genes by a combination of bioinformatics and transgenics. An online atlas links high resolution gene expression patterns to neuroanatomical structures and will contribute to understanding behavioral phenotypes. Small-molecule screening for mutant rescue or disease-relevant processes will identify candidate drugs and provide insights into gene function.
Our main scientific focus was on development and disease in the areas that generate the largest fraction of health related costs in Europe:
• Development, behavioral, and neurological diseases,
• eye and visual processing,
• tissue regeneration and repair, including regeneration in the brain,
• diabetes and obesity, as well as infectious disease and cancer.
Project Results:
The ZF-HEALTH project comprised five scientific work packages, as well as work packages focusing on dissemination, training and management. Below are the main results by work package:
WP1 – Generation and distribution of mutants for potential human disease genes
The first work package focused on TILLING, a method to obtain knock-out mutations in specific genes by generating a library of mutagenizing animals, and then sequencing the loci of interest until the desired mutation is found. During the project TILLING technology was greatly improved. Rather than screening for mutations in specific amplicons only, the Wellcome Trust Sanger Center (GRL) used high-throughput sequencing of the entire exome (i.e. all translated sequences) of mutagenised fish, identifying knock-out mutations genome-wide. In support of the TILLING project new libraries of mutagenised fish were generated by GRL, the Hubrecht Institute (KNAW) and Technical University of Dresden (TUD). By this method 43,098 knock-out alleles were identified in 16,096 genes (61% of all protein-coding zebrafish genes), far exceeding the original goal of 1,000 genes. All alleles generated were archived as frozen sperm samples and are being submitted to the newly established European Zebrafish Resource Center (EZRC) of Karlsruhe Institute of Technology (KIT) as well as to the Zebrafish International Resource Center (ZIRC, Eugene, Oregon). 32,000 alleles have already been delivered.
Complementing the genome-wide knock-out project, we also focused on technologies that generated targeted knock-outs in genes of special interest with nuclease reagents. At the start of the project, Zinc Finger Nuclease (ZFN) was already available, but this was superseded in the course of the project first by TALEN, and then by CRISPR-Cas9 nucleases. The latter are most efficient and use an RNA strand for guidance which can be generated in vitro, eliminating the need for molecular cloning steps. Four partners initially established pipelines for the Zinc Finger Nuclease (ZFN) technology for generating additional knock-outs. Three of the partners later converted their mutagenesis pipelines to use TALEN technology, while KIT established a TALEN pipeline from the outset. By the end of the project several partners had adopted the even more efficient CRISPR-Cas9 technology. In total, at least 41 alleles were generated by these technologies.
In parallel with mutant production, KIT established its European Zebrafish Resource Center and with the help of GRL, established sperm freezing and IVF protocols based on those at use at GRL, carrier identification by allele-specific PCR (KASPar assay) and standard procedures for accepting and distributing a large number of fish lines. The European Zebrafish Resource Center uses space in two different buildings, several hundred meters apart, which helps to keep the core of the resource center free from contamination. All fish to be imported are kept in a Quarantine Room and only their offspring is transferred as disinfected eggs to the Core Fish Room, which is located in a different building. Fish health is monitored by a veterinarian by regularly examining sentinel fish. An internal laboratory database as well as an easily searchable public website and web store were developed and standard operating procedures for processing orders were established. KIT maintains and distributes mutant and transgenic zebrafish lines and also gives zebrafish researchers access to screening services and technologies such as imaging and high-throughput sequencing. Requested lines are shipped as disinfected embryos. Besides the the lines from GRL, it maintains and distributes the entire stock collection of the Nüsslein-Volhard laboratory (MPG), comprising over 2,000 published mutations, as frozen sperm samples, as well as over 100 mutant and transgenic lines from other sources. It closely collaborates with ZIRC to give European researchers cost-effective access to lines available there, since the cost of trans-atlantic shipping is a major impediment to using these lines. It also collaborates with the central model organism database for the zebrafish, ZFIN, which provides a repository for genetic and phenotypic data with “order this” links to the EZRC, and with the Institute of Molecular and Cell Biology (IMCB, Singapore), which provided transgenic lines.
All lines and information on the EZRC are available through its public website: see Fig. 1 and 2. Since the website launch in November 2013 KIT has shipped a total of 558 zebrafish lines, 386 of them GRL mutants, to recipients within and outside the ZF-HEALTH consortium.
Two new tasks were proposed by the Consortium in 2015: inbreeding of zebrafish wildtype lines and generation of additional Cre driver lines for conditional knock-out of genes in neural development by knock-in.
Inbreeding was added to the ZF-HEALTH work programme order to generate zebrafish lines with better defined genetic background then the outbred wildtype lines previously available. It is being performed both at KIT (where 10 full-sib pairs are grown up per generation for 5 wildtype lines) and at ZF BioLabs SL (ZFB, 15 full-sib pairs per generation for 1 line), 65 full-sib pairs in total. KIT has currently reached generation 3, and ZFB generation 5. It is planned to continue the inbreeding effort after the end of the ZF-HEALTH project (with institutional funding) and to make the lines developed at KIT as well as ZFB available through the European Zebrafish Resource Center at KIT.
Generation of Cre driver lines by knock-in into genes of interest, which will be novel and versatile tools for studying neural development, was begun and is still underway at TUD.
WP2 – Phenotyping
Systematic phenotyping, i.e. determing and analyzing the effects of mutations, is a step of central importance in order to make use of the thousands of zebrafish mutants as disease models or to analyze developmental pathways. GRL established a pipeline for initial morphological and behavioural phenotyping (for embryonic and larval phenotypes visible through a stereomicroscope and touch response up to 5 days post fertilization). This was carried out at an unprecedented scale, for over 4,000 TILLING mutations in 2,569 genes, identifying 203 (8 % ) abnormal phenotypes, far more than envisioned. The data can be found on the ZMP website set up by GRL at http://www.sanger.ac.uk/Projects/D_rerio/zmp/ and are being submitted to the ZFIN database. This effort also resulted in a Nature article (Kettleborough et al., 2013). For an example of a phenotype see Fig. 3. Likewise 30 nuclease-induced mutants were phenotyped at several partners institutions. 2 abnormal phenotypes were identified, which is line with this number of phenotypes expected from the phenotyping of TILLING mutants.
Since the start of the project GRL has further performed over 200 expression profiling experiments on on mutant embryos as well as wild-type embryos of a range of developmental. The first results are available online through the ZMP website.
KIT established the photomotor response assay (PMR) for shelf screening of knock-out mutants from GRL, and carried out a pilot screen. This assay originally developed for chemical screening by the laboratory of Randall Peterson (Boston) which promises to give insight into all the major pathways of neural transmission. While this work will be continued, our emphasis has shifted away from shelf screening at the EZRC resource center to a more distributed effort to conduct in-depth phenotyping of mutants directly at the participating labs. This was a decision made in order to make the most productive use of our limited resources since recovery of knock-out mutations from GRL involved considerably more effort than we expected. The success of this approach is reflected by the fact that KIT sent out 386 Sanger mutants for phenotyping by specialized laboratories (WP1), far more than anticipated, and by at least 30 articles that have already been published by Consortium Members on the phenotyping of mutants and the analysis of pathways involved.
The behavioural phenotyping facilities of CNRS and University of Zurich (UZH) were already established in the first reporting period and have performed detailed analysis of a total of 45 mutants, more than originally planned.
In depth phenotyping was performed by several labs. Since the start of the project CNRS studied the Nodal pathway mutant zoep, INSERM studied the RNA-binding protein mutants, qkA and qkB, King’s College London (KCL) the function of foxg1a and dkk1, University of Leiden (UL) the immune-related mutants myd88 and cxcr3, and TUD a zebrafish model for Parkinson’s disease, MPG analysed the role of vasna and vasnb in retinotectal axon guidance, University of Sheffiled (USFD) phenotyped several mutants related to angiogenesis and diseases, Univesity of Cologne (UCO) continued worked on genes involved in skin and bone development, and University of Padua (UNIPD) characterized the role of tcf7l2 in pancreatic development and studied models of pancreatic adenocarcinoma and collagen VI myopathy.
To date 30 articles resulting from the in-depth phenotyping and molecular analysis of mutants have been published by the Consortium, greatly exceeding our original goal of at least 5 publications from analysis of at least 10 selected mutants. Several more are underway. An article on the role of inflammation as the first step towards brain regeneration in zebrafish, was published in Science (Kyritsis et al. 2012).
WP3 – Characterisation of enhancer elements of human disease genes
Our partners University of Sydney (USYD) and Imperial College London (IMPERIAL, who took over the tasks originally assigned to Uni Research AS, Bergen due to a move of the principal investigator) have defined regulatory regions based on several criteria: highly conserved non-coding elements; estimates of synteny span around target genes; and epigenetic marks (histone modification).
IMPERIAL made several updates to its custom genome browser, ANCORA, in which HCNEs, synteny blocks, and relevant epigenetic marks can be assessed for any region of the human genome (http://ancora.genereg.net Fig. 4), and released a software package for studying large sets of promoters. IMPERIAL also explored the relationship of GRB turnover and gene function and identified novel target genes for human disease-associated regulatory variants in the zebrafish, using schizophrenia for a pilot study.
UL and IMPERIAL furthermore sequenced and annotated the common carp genome, which was found to be tetraploid and extremely condensed. Two other sister species, Danio nigrofasciatus and Danio albolineatus were sequenced at moderate coverage by GRL and the University of Freiburg (ALU-FR). These sequences together with the recently available grass carp genome were utilized to dramatically increase the number of known zebrafish CNEs.
In three experiments with a total of over injected 2,000 founders the University of Birmingham (BHAM) analysed human HCNEs associated with type 2 diabetes, with pancreatic agenesis and with the estrogen-receptor related gene gamma alpha (errga).
Moreover USYD and BHAM analysed the enhancers of tp53, miR-9-1, miR-9-2, miR-9-3, Pou3f3, Foxg1 and Sox2 by ChIP-Seq and related methods. ALU-FR used ChIP-Seq to characterize binding sites relevant for zygotic gene activation at the mid-blastula transition. INSERM studied enhancers of Myosin VIIA. UNIPD generated reporter lines for Wnt signaling, hypoxia signaling, the Hippo pathway, thyroid development, the Stat3 and Notch patchways and used reporters to analyse activation of the hypoxia pathways by glucocorticoids. BHAM validated bidirectionally transcribed human enhancers in transgenic zebrafish embryos.
Five papers were published on expression patterns of HCNEs in transgenic zebrafish lines. An article on promoter structure was published in Nature (Haberle et al., 2014).
WP4 – Gene expression mapping in the brain
To understand the function of genes that give behavioral phenotypes when mutated, one needs a detailed description of neuroanatomical gene expression domains as well as of the circuitry of the brain. The overall aims of this work package were to establish a framework to link high resolution 3D gene expression pattern to neuroanatomical structures, and to contribute to resolving the connectivity of circuits in the developing brain with an emphasis on limbic system circuits and monoaminergic circuits. This main tools for this were the ViBE-Z software pipeline and the interactive atlas, zebrafishbrain.org.
ALU-FR developed the Virtual Brain explorer (ViBE-Z), a novel software pipeline that automatically maps gene expression data with cellular resolution to a 3D standard larval zebrafish brain at day 2 – 6. A web interface is publicly at http://vibez.informatik.uni-freiburg.de/. ViBE-Z uses fluorescently stained cell nuclei for image registration and automatically detects 14 predefined anatomical landmarks for aligning new data with the reference brain. This makes it feasible to search for expression in defined anatomical parts and to compare the domains of expression of genes of interest (Fig. 5 and 6).
University College London (UCL) continued development of its interactive atlas of the zebrafish brain, http://zebrafishbrain.org begun in the ZF-MODELS FP6 project (Fig. 7). The atlas features 3D images as well as expert-written tutorials that describe specific strutures of the brain, and is as unique and highly valuable tool both for teaching and for collaborative research. 8 tutorials on zebrafish homologs of limbic system structures where added, bringing the total number of tutorials to 25. UCL further developed mosaic single cell labelling methods based on CRISPR/Cas9 genome editing to study the limbic system. UCL also examined gene expression in 28 HCNE derived lines and 65 enhancer lines (including lines generated by USYD in work package 3), and incorporated and annotated part of them in zebrafishbrain.org.
KCL and CNRS established 3D maps of the telencephalon from day 1 to 30 of development, and CNRS analysed the “optic recess region” as a new identified morphogenetic entity.
Finally, both UCL and CNRS established pipelines for characterizing in detail the neuroanatomy of mutant lines, and analyzed over 10 such lines.
WP5 – Chemical screening
The screening of small-molecule libraries containing chemicals with potential pharmacological effects is particularly efficient in the zebrafish, since the zebrafish embryos can be exposed to the chemicals and analysed in microtiter plates, at high throughput, while giving insight into the effecs on an entire vertebrate organism that would not be possible with the commonly used cell culture assays.
Two publicly available small-molecule libraries (FDA approved drugs and ICCB) were acquired by KIT, and in the first reporting period aliquots were distributed to 18 European zebrafish laboratories inside and outside the ZF-HEALTH consortium during the reporting period. The combinatorial chemistry platform of KIT, Complat, generated two additional libraries one of which was tested in the zebrafish.
Several partner laboratories developed novel screening technologies. KIT developed an assay for glucocorticoid signalling, UL optimized a high throughput screening system for cancer and tuberculosis, BHAM developed a transgenic biosensor for screening of kidney development and function, and ZFB optimized hormone-induced spawning.
A total of eleven screens were performed, more than anticipated: for hypoxia signaling (USFD), nicotine analogs in the developing zebrafish embryo (BHAM), mitochondrial dysfunction (ZFB), granulocytic inflammatory response (KIT), attention deficit with hyperactivity disorder (CNRS), epithelial carcinogenesis (UCO), neural tube development (KIT), activation of glucocorticoid signalling (KIT), rescue of epidermal defects in zebrafish hai1a mutants relevant for cancer (UCO) and enhancers and suppressors of the pkd2/cup mutation relevant for polycystic kidney disease (USFD), and for modulators of versican expression in the zebrafish ear (USFD).
Several partners reported the confirmation and further analysis of hits resulting from the screens, UL on compounds that inhibit tuberculosis, USFD on a highly potent HIF activating compound, UCO on potentially anti-carcinogenic compounds, and KIT on anti-inflammatory compounds.
5 articles were published or submitted describing our first results from the analysis of pathways in development and disease, using small compounds identified by chemical screening. A notable result is the suppression of the behavioural phenotype of an autism model by estrogen-related compounds. Other publications concerned tuberculosis drugs and the modulation of inflammatory processes.
Potential Impact:
As stated in the FP7 work programme:
“The overall objective of the Health Theme is to improve the health of European citizens and increase the competitiveness and boost the innovative capacity of European health-related industries and businesses, while addressing global health issues including emerging epidemics. Emphasis will be put on translational research (translation of basic discoveries into clinical applications including scientific validation of experimental results), the development and validation of new therapies, strategies for health, promotion and prevention, child health and healthy ageing, diagnostic tools and medical technologies, as well as sustainable and efficient healthcare systems.”
Our project addressed these objectives as follows.
• It helps to improve the health of European citizens as all of its deliverables are of value for clinical medicine: mutants and phenotypic data for use as disease models, insight into the structural and molecular basis of human behaviour, a functional characterisation of enhancer elements that will help to interpret data on human genetic variability, and validated hits from small-molecule screening for drug candidates.
• In the U.S.A. the importance of zebrafish as a model organism was recognised as early as the last decade. Since that time zebrafish research has been continuously funded by the NIH at far higher levels than in Europe, whether by national funding agencies or the EU. Against this background, our project was of great strategic importance to increase the competitiveness and boost the innovative capacity of European health-related industries and businesses.
• In order to accomplish this we strongly focused on translational research. One SME (ZF Biolabs) participated in the project to help with this focus. Many project partners also have collaborations with SMEs or larger pharmaceutical companies.
Moreover the work programme states:
“The data generated by genomics and proteomics projects will continue to support the European research excellence in related fields by increasing our understanding of key biological processes and provide the foundation for more extensive functional studies concerning genetic variation, the way that genes interact with each other in health and disease, and to speed the search of genes that may underlie diseases in human. This in turn would aid the design of drugs and treatments, including individualised treatments. For example, large-scale functional genomics studies in multi- cellular model organisms will generate new knowledge on human gene function in health and diseases and potential model systems for drug screening.”
Our project directly addressed each of these expected impacts:
• We have substantially increased our understanding of the genetic roots of fundamental biological processes including brain development, visual processing, and behaviour. The zebrafish brain is a uniquely accessible and tractable model for such purposes, which we will leverage by creating a comprehensive atlas of gene expression in development and by studying the effect of knock-out mutations in hundreds of regulatory genes on the brain. There is a growing appreciation that the results of such testing in the zebrafish are directly applicable to mammalian brains, and hence to clinicians. We have further characterised the state of neural and muscle stem cells and aspects of the immune response, areas where the zebrafish has particular strengths as a model organism.
• The genome-wide analysis of genomic regulatory blocks (GRBs) containing long-range enhancers and their target genes will provide a unique new view of the large-scale organisation of vertebrate genomes. This information, mapped to the human genome assembly, will help substantially in understanding human genetic variation and provide a foundation for future targeted searches for non-coding mutations associated with human disease. The identification of such mutations will eventually open the door to an individualised medicine. Our work is integrated with international efforts in human genomics such as the HapMap and ENCODE projects wherever possible. The experimental validation of enhancers in the zebrafish initially focused on regions implicated diseases such as type II diabetes (a condition with a high prevalence in the European population). In future work, building on the methods developed in our project, they will be extended to other common diseases.
• We have systematically assessed the phenotypic effects of (a) targeted mutations over 2,000 genes including transcriptional regulators and homologs of genes suspected in human disease, and (b) disease-relevant mutations identified in previous forward-genetic approaches. A substantial fraction of these are candidates for human disease genes, adding to our knowledge on their function in health and diseases, and providing a shortcut to targeted searches for such genes in the human.
• Many of the mutations are suitable model systems for drug screening. Several project partners have investedsubstantially in the development of robotic technologies for small-molecule screening in the zebrafish. They have refined their technologies and conducted pilot studies within the proposed project, using chemical libraries generated and / or centrally distributed by the consortium. These screens have provided insight into biological processes in themselves, but also served as a proof of principle for the feasibility of large-scale commercial drug and toxicology screens in the zebrafish to be undertaken by the pharmaceutical industry. An SME (small or medium enterprise) end user of our technologies and libraries, the biotechnology company ZFB, was part of our consortium.
In summary, we believe that our project has provided valuable inputs for reaching the objectives of the Seventh Framework Programme, and strongly support European research excellence in the area of model organisms for biomedical research. It has fulfilled our expectation of continuing the success of the ZF-MODELS FP6 project which for the past five years has been a focal point of European zebrafish research, and which has helped to overcome its previous fragmentation by fostering international collaboration, increased the visibility of zebrafish models by its dissemination effort, and mobilised resources far beyond those funded by European Commission.
We disseminate the results obtained during the ZF-HEALTH project to different groups of stakeholders – the biological research community, pharmaceutical companies and clinicians, as well as policy makers and the general public – by different means suited to the particular group, as follows:
To the research community:
1) Through direct release of high-throughput data by public databases linked to the ZF-HEALTH website, as well as specialist databases set up by the Consortium. Mutant and transgenic zebrafish lines and plasmids containing zebrafish sequences are made available by the European zebrafish stock centre that we established. For details see “Management of high-throughput data and biomaterials” below.
2) Publication of new results – including novel tools and technologies – in international scientific journals. 154 articles were produced (far more than the 50 we anticipated), including several in highly ranked journals such as Nature and Science. All these publications acknowledge support by the EC as specified by the Grant Agreements.
3) Presentations at major European and other international conferences. Over 400 such presentations were made over the duration of the project.
4) The open access part of the ZF HEALTH website, which provides links to online resources and publications produced by the project and throughout the project, announced invitations to participate.
5) Organisation of 3 special interest symposia and 5 workshops targeted at junior researchers. The symposia were titled “Zebrafish Models in Translational Medicine” (Courcelle-sur-Yvette, 2013, coinciding with the 4th Project meeting), “Towards an encyclopedia of DNA elements in zebrafish” (London, 2014) “The Zebrafish: A Vertebrate Model Organism for Biological and Biomedical Investigations" (Gif-sur-Yvette, 2015, coinciding with the 5th workshop). The workshops were titled “Cutting edge technologies in biomedical research” (Karlsruhe, 2011), “Genomics and high throughput sequencing technologies with the zebrafish model” (Cambridge, 2012), “Automation methods for zebrafish research” (Leiden, 2013), “Imaging of Neural Development in Zebrafish” (Karlsruhe, 2014), and “ZF-HEALTH Resources for in Silico Experimentation: VIBE-Z – BioEmergences – MecaGen” (Gif-sur-Yvette, 2015).
6) Incorporation in research training. 67 students obtained their Masters or doctoral degree within the project, results were included in the masters and doctoral research training programmes of each of the partners reaching at least 500 research students over the duration of the project and 5 workshops were organised.
To companies and clinicians:
1) Through articles on new scientific developments, tools and technologies for industrial and medical journals.
2) presentations at major medical and industrial conferences (biotechnological and pharmaceutical),
3) participitation of junior researchers from industry and hospitals in our 5 workshops,
4) collaborations and staff exchanges with companies and hospitals,
5) licensing of patented data, tools and technologies (following the end of the project).
A total of 490 collaborations with industry and medical researchers were reported by Consortium members – for a detailed listing see the annual Dissemination Reports.
To policy makers and the general public:
1) Through a press release (issued at the occasion of the opening of the European Zebrafish Resource Center)
2) popular-science articles for newspapers, popular-science magazines, publications targeted at policy makers on the national and European level, and industrial journals, covering our tools and results (we expect at least 10 such articles throughout the duration of the project),
3) the open-access part of the ZF-HEALTH website which provides a non-specialist introduction to the project's background, objectives and results,
4) a two-page flyer describing the project, for use by the Consortium and the Commission,
5) presentations of the laboratories involved in the ZF-MODELS Project at open days, local and
national science fairs.
These efforts were aided by utilising contacts and expertise from an FP7 COST action, the European Network on Fish Biomedical Models (EUFishBioMed), which represents over 300 zebrafish laboratories.
Project website and flyer
As soon as a Grant Agreement for ZF-HEALTH was concluded with the European Commission, the Scientific Coordinator and Management Support Team set up the project website (http://www.zf-health.org) that provides:
• an overview of the ZF-HEALTH partnership and its research activities
• a section providing background information, project news and announcements (e.g. invitations to participate in our workshops)
• links to a network of public databases (e.g. ZFIN and Ensembl, as well as specialist databases set up by the Consortium) that provide direct access to the high-throughput
data produced in our project.
• a password-protected section for project partners and EC services for the internal
• exchange of data and knowledge, including an internal project news and download section.
The website was regularly updated throughout the project
The Scientific Coordinator and Management Support Team have furthermore prepared a two page- flyer describing the project, its objectives, its research activities, the partnership, its website and online resources, for use by the Consortium and the European Commission. This flyer was kept up to date and was also distributed at the bi-annual International Zebrafish Meetings (in the USA), European Zebrafish Meetings, and European Zebrafish PI Meetings, throughout the duration of the project.
Press releases
The Scientific Coordinator and Management Support Team have issued a press releases on behalf of the partners on the occasion of the opening of the European Zebrafish Resource Center (ERZC) of KIT.
Management of high-throughput data and biomaterials
We aim for openness in our research. High-throughput data and biomaterials produced in our project were therefore released continuously through a network of public databases linked to the ZF-HEALTH website, and through the newly established European zebrafish stock centre. Below is an overview of the high-throughput data and materials produced in our project, and their release:
WP1
Molecular data on mutations through ZFIN, ENSEMBL, and the Zebrafish Mutation Project (ZMP) database set up by GRL, immediately.
WP2
Results of high-throughput phenotyping through ZFIN (using the PATO ontology) and ZMP, within 6 months after phenotyping.
Digital reconstructions from 3D / 4D imaging through the Bioemergences website (still under construction).
Transcriptional profiling results through ZMP, immediately.
Standardised protocols through the ZF-HEALTH website, immediately.
In-depth analysis of phenotypes and pathways through scientific papers, after publication.
WP3
Comparative data on enhancers in human, zebrafish and other species via the ANCORA genome browser of IMPERIAL, immediately.
WP4
Images of expression patterns of transgenic lines in the brain through the zebrafishbrain.org to be established by UCL and ALU-FR, immediately.
WP5
Confirmed hits from chemical screening Scientific papers, licensing to the pharmaceutical industry, after publication.
The following schedule applies to the distribution of biomaterials:
Mutants generated by the TILLING method are made available immediately to all interested parties, through the newly established European Zebrafish Resource Center of KIT.
• Mutants generated by targeted nuclease methods (in which the generating partner may have invested a significant amount of effort) are made available 12 months after their initial phenotyping data are released or after an initial publication, whatever comes first, but may be made available earlier if the generating partner desires.
• Transgenic lines are made available 12 months after images of gene expression are released or after an initial publication, whatever comes first, but may be made be available earlier if the generating partner desires.
•
Patenting and exploitatiom
We intend to distribute the results, tools and technologies generated in our project to all interested parties. However, we also aim to maximise their commercial exploitation. Therefore appropriate measures ensuring the protection of intellectual property rights (IPRs) were taken in order to guarantee possible commercialisation of these results, tools and technologies.
The partners have extensive experience with IPR issues and commited themselves to protecting all new knowledge identified as having economic potential either through patenting or through treating the knowledge as “commercial in confidence”. This protection was done through the technology transfer office of the partner who has generated the knowledge.
While the great majority of our results are in the area of basic research and were therefore not considered to be of direct commercial value, two patents were submitted in the course of the project:
“Spaink, H.P. and Dirks, R.P.: HIGH THROUGHPUT METHOD AND SYSTEM FOR IN VIVO SCREENING”
“Karlsruher Institut für Technologie: Karlsruher Institut für Technologie: MICROSCOPE WITH AT LEAST ONE ILLUMINATING BEAM IN THE FORM OF A LIGHT SHEET”
Our commercial partner, ZFB, further reported the following exploitable foreground:
“Improvement of method of hormonal induction to obtain, in a short period of time, many embryos of zebrafish with high fertility and viability. Furthermore, with the hormonal treatment, ovulation and spermiation is reached on predetermined time and the fertilization and incubation can be done under aseptic conditions, both things are very difficult to obtain using the natural spawning traditional method.”
For details please see section B.1 and B.2 below.
Material Transfer Agreement
Mutants and transgenic lines of zebrafish are made available to parties outside of the ZF- HEALTH Consortium only if the receiving party signs a Material Transfer Agreement (MTA), in line with standard practices in biomedical research. We expect that most mutants and transgenic lines will be distributed through the European stock centre currently being established by KIT-G, which will keep track of the signed MTAs. However, in case such lines are directly distributed by other project partners, the issuing partner will be responsible for obtaining a signed MTA and sending a copy to the Coordinator.
The MTA was drawn up at the start of the project by the Scientific Coordinator and Management Support Team and put in place by the General Assembly. It contain minimal restrictions and was modeleted on the MTA previously used by GRL for its knock-out mutations as well as the MTA of the Zebrafish International Resource Center (ZIRC). Briefly, it specified that:
1) The lines will be used for academic and non-commercial research purposes only and will not be sold or bred for sale.
2) They will not be further distributed to others without written permission from the original provider.
3) The recipient is free to publish results from the lines, but is required must acknowledge the origin of the lines from a member of the our consortium, as well as the EZRC in such publications.
In the case of commercial entities requesting access to mutant or transgenic zebrafish lines, this access will be granted under terms and conditions agreed upon in a collaboration agreement that will be negotiated on an individual basis.
List of Websites:
Public website: http://zf-health.org
Scientific coordinator's address:
Dr. Robert Geisler
Karlsruhe Institute of Technology (KIT)
Institute of Toxicology and Genetics
Hermann-von-Helmholtz-Platz 1
Building 439, Room 310
76344 Eggenstein-Leopoldshafen, Germany
Phone: +49 721 608-24664
Fax: +49 721 608-23354
E-mail: robert.geisler@kit.edu
