Final Report Summary - FISHMED (Fishing for Medicines and their targets using Zebrafish models of human diseases)
Executive Summary:
The International Institute of Molecular and Cell Biology (IIMCB) is one of the best biological research institutions in Poland. IIMCB’s strategic objective is to achieve the quality of research and innovative activities of leading research entities worldwide. To achieve this level of excellence and increase our innovative potential, we introduced a new research model: zebrafish. The FishMed Centre, supported by the European Union and Ministry of Science and Higher Education, is composed of a Zebrafish Core Facility and research groups that use zebrafish in innovative projects that study the molecular mechanisms of disease. This ambitious undertaking would not have been possible without support from excellent European partners who shared with us their expertise and resources. The EU and national funding have been used to finance the following: the employment of scientists, technicians, and managers; the purchase of state-of-the-art equipment; exchange visits between IIMCB researchers and their European partners; and participation in and organization of various events, including those that focus on innovation and technology transfer.
One of the key indicators of the success of FishMed has been the ability to compete internationally to recruit experienced researchers and establish a new laboratory that is headed by Dr. Cecilia L. Winata from Singapore. Her group’s main project is dedicated to elucidating the gene regulatory networks of zebrafish heart development using genomic methods. Based on the collaborative agreement, this laboratory has a secondary affiliation with the Max Planck Institute for Heart and Lung Research in Bad Nauheim. This has allowed Dr. Winata and her staff to use the expertise and resources of this well-known center of heart research, including zebrafish research.
IIMCB is well equipped, but FishMed funds allowed us to purchase several additional items, including the first LightSheet Z.1 microscope (SPIM) in Poland and systems for behavioral analyses of zebrafish larvae and adult fish. The SPIM is heavily used by IIMCB staff and external collaborators and is already yielding data, some of which are presented in the first FishMed publications.
The International FishMed Conference on Zebrafish Research was organized by IIMCB to bring together scientists from the field, share recent advances in research on zebrafish, and present the results of the FishMed project to an international audience. The meeting was convened with very high scientific and professional standards and very well received by the speakers and meeting participants from many countries around the world.
In addition to research, we are strongly engaged in popularizing fish as a model for research among current and future scientists. The educational campaign Be Healthy as a Fish targets children and their parents and includes workshops, a short movie, and a book. This action was prepared by public relations specialists who are involved in the FishMed project with strong support from our scientific team and fish facility staff.
The successful implementation of all plans of the FishMed project allows us to be highly optimistic that all of its goals have been achieved or even exceeded. What makes us especially satisfied is that not only IIMCB researchers, but also scientists from other Polish institutions, have benefited from our zebrafish core facility and equipment and the expertise of our staff. Some people from these institutions started new projects using our collection of wildtype, mutant, and transgenic zebrafish lines. The increased demand for zebrafish in Poland convinced us to expand our facility, and we look forward to supporting the zebrafish research of scientists at other institutions at the national and international level.
Let us finish with a statement from experts who were appointed by the European Commission to evaluate IIMCB in the context of FishMed: “FishMed project has been an outstanding success. In the relatively short period since initiation of the project, the zebrafish has been successfully integrated into IIMCB, strong collaborations and international networks have been made, new research projects have been initiated, excellent core facilities have been established or expanded, talented personnel have been recruited to IIMCB, research productivity and grant income has increased, and international visibility of the IIMCB, and Polish research more generally, has been enhanced. FishMed project has given the IIMCB the potential to become a successful internationally competitive hub for research involving zebrafish as a model organism.”
Project Context and Objectives:
The International Institute of Molecular and Cell Biology (IIMCB) is one of the best biological research institutions in Poland. IIMCB’s strategic objective is to achieve the quality of research and innovative activities of leading research entities in the world. To achieve this level of excellence and increase our innovative potential, we introduced a new research model: zebrafish. The FishMed Center, supported by the European Union and Ministry of Science and Higher Education, is composed of a Zebrafish Core Facility and research groups that use zebrafish in innovative projects that study the molecular mechanisms of disease. The EU and national funding have been used to finance the following: the employment of scientists, technicians, and managers; the purchase of state-of-the-art equipment; exchange visits between IIMCB researchers and their European partners; and participation in and organization of various events, including those that focus on innovation and technology transfer.
Objectives
• Twinning of seven IIMCB groups with excellent European zebrafish centers to develop innovative potential using fish models.
• Development of a Zebrafish Core Facility and establishment of a new research group headed by a leader who is selected through an open international competition.
• Acquisition and upgrading of research equipment for a Zebrafish Core Facility and new zebrafish research laboratory.
• Reinforcement of IIMCB innovative potential with the Bio&Technology Innovations Platform (BioTech-IP).
• Construction of an interactive visibility platform to popularize the FishMed Center and research with zebrafish models among scientific and non-scientific communities, including promotion of the project’s innovative results.
The FishMed Center is a consortium of eight groups from IIMCB and six European institutions, including the Max Planck Institute for Heart and Lung Research (MPI-HLR) as a strategic partner. The twinning partners were chosen based on their expertise in research using zebrafish models, excellent publication records, and compatibility with the scientific interests of the FishMed Center groups at IIMCB. European partners share with us their zebrafish models and expertise related to fish research. Twinning allows smooth passage from the initial phase of accommodating a new experimental model to quickly focusing on cutting-edge research that is likely to lead to innovations.
Partners
• Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany, represented by Thomas Braun and Dr. Didier Stainier.
• Austrian Institute of Science and Technology, Klosterneuburg, Austria, represented by Carl-Philipp Heisenberg.
• Department of Physiology, Development, and Neuroscience, Cambridge University, United Kingdom, represented by William Harris.
• MRC Centre for Development and Biomedical Genetics, University of Sheffield, United Kingdom, represented by Oliver Bandmann.
• Department of Biochemistry, University of Geneva, Switzerland, represented by Marcos Gonzalez-Gaitan.
• Department of Molecular Cell Biology, Institute of Biology, Leiden University, The Netherlands, represented by Ewa Snaar-Jagalska.
Project Results:
Matthias Bochtler, Laboratory of Structural Biology
Ten eleven translocation (TET) proteins are alpha-keto-glutarate-dependent dioxygenases that can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5cC). In mammals, TET activity is required for demethylating the paternal pronucleus in the zygote, the mesenchymal-to-epithelial transition in organogenesis, and (partly) demethylation in the germline. 5hmC, the main and first product of the TET-mediated oxidation of 5mC, may also be an epigenomic signal itself, correlating with and likely contributing to cellular differentiation. TET proteins also play a role in the hematopoietic system. Chromosomal aberrations or mutations that affect individual TET genes do not suffice as drivers of leukemias, but such changes are found in up to 25% of human leukemias (depending on the type). In mice, the combined loss of TET genes in the hematopoietic system causes either B-cell (TET1 and TET2 deletions) or myeloid (TET2 or TET3 deletions) malignancies.
Using a combination of TALEN and CRISPR gene-targeting technologies, we generated zebrafish lines that are individually deficient in the TET1, TET2, and TET3 genes. This work was done in collaboration with Olov Andersson (Karolinska Institute, Stockholm). In parallel, we also generated antibodies against bacterially made fragments of the three TET proteins for immunohistochemistry. In agreement with reports that appeared in the literature while this work was in progress, we found that homozygous mutants of single TET genes have at most very mild phenotypes. However, the combined of loss of TET2 and TET3 is incompatible with complete development. The development of fish arrests during the larval period. Our original plans to study leukemias in the fish required modifications in light of the literature that appeared during the project. We plan to rescue TET2 and TET3 double mutants with a floxed TET2 or TET3 allele so that the acute ablation of TET activity is possible in a Cre-driver line-dependent manner. We seek to study the role of TET proteins after the larval stage, overcoming the developmental arrest of TET2 and TET3 double homozygotes. In parallel, we will attempt transplantation experiments, grafting TET2-/- and TET3-/- tissue onto wildtype fish. We are in the process of crossing reporter lines with lines that harbor mutations in the TET genes.
Janusz Bujnicki, Laboratory of Bioinformatics and Protein Engineering
The work of the Bujnicki group has focused on the development and use of computational tools for RNA 3D structure modeling and the modeling of protein-RNA interactions. Numerous software tools were developed with partial support from the FishMed project, which covered the work of Dr. Wayne Dawson and Prof. Bujnicki’s supervision of M.Sc. students who are involved in the project. A major piece of software that was developed based on FishMed support is SimRNA, a computational method of modeling the 3D structures of RNA molecules and RNA-RNA complexes (available at http://genesilico.pl/software/stand-alone/simrna as a standalone tool and at http://genesilico.pl/SimRNAweb as a web server). SimRNA uses a coarse-grained representation, relies on the Monte Carlo method for sampling the conformational space, and employs a statistical potential to approximate the energy and identify conformations that correspond to biologically relevant structures. SimRNA can fold RNA molecules using only sequence information. On established test sequences, it recapitulates the secondary structure with high accuracy, including the correct prediction of pseudoknots. To model complex 3D structures, it can use additional restraints that are derived from experimental or computational analyses, including information about secondary structures and/or long-range contacts. SimRNA can also be used to analyze conformational landscapes and identify potential alternative structures.
The support of FishMed included the conceptual development and testing of tRNAmodpred (a computational method for predicting posttranscriptional modifications in tRNAs; available at http://genesilico.pl/trnamodpred/) and NPDock (a web server for protein-nucleic acid docking; available at http://genesilico.pl/NPDock) and the preparation of articles that described these tools.
Additionally, a number of minor software tools were developed, including software for processing data that are generated by other computational methods.
Agnieszka Chacinska, Laboratory of Mitochondrial Biogenesis
Mitochondria constitute life-essential organelles with multiple cellular functions. They serve as energy and biosynthetic centers, participate in cell stress responses, and are involved in cell death execution. Failure in mitochondrial function is associated with numerous human pathologies. With no cure, the severity of symptoms and disease progression can often lead to death before adolescence. The fact that the treatment of mitochondrial disorders is mostly directed at mitigating the symptoms reflects our relatively poor understanding of the biology of mitochondrial disorders. We seed to find the ways in which faulty mitochondrial biogenesis impinges on the development of a vertebrate organism using Danio rerio as a model.
To meet the goal of our project, we first adapted a variety of basic biochemical skills to the in vivo model. For example, a protocol for protein isolation was developed and optimized that allows the isolation of proteins from zebrafish embryos and larvae at different stages of development. This protocol was subsequently used to study protein levels and their diversity at various stages of zebrafish development. Additionally and specifically to our research interest, we succeeded in enriching mitochondrion protein extracts by isolating zebrafish mitochondria. Taking advantage of a transgenic line with fluorescently labeled mitochondria, we characterized mitochondrial networks at various stages of development and in a plethora of tissues. We accomplished this by using cutting-edge light sheet fluorescent microscopy (LSFM).
We are interested in the universally conserved mitochondrial import assembly (MIA) pathway that is responsible for the import of mitochondrial proteins and well known for its dependence on redox reactions and cross-talk with respiratory chain complexes and complexes that are responsible for maintaining mitochondrial architecture. This MIA pathway is responsible for the biogenesis of cysteine-rich mitochondrial intermembrane space proteins, the majority of which are involved directly or indirectly in the biogenesis of respiratory complexes. Importantly, respiratory complex deficiencies underlie a large proportion of mitochondrial diseases.
In our research, we have utilized several approaches to disrupt the MIA pathway. Specifically, we targeted Mia40 protein, a mitochondrial intermembrane space oxidoreductase, the activity of which determines proper functioning of the MIA pathway. We first used a well-established antisense technique that exploits morpholinos to achieve temporal protein knockdown. In collaboration with the laboratory of our twinning partner Prof. Dider Stainier, we acquired and successfully adapted the TALEN technique to obtain genetic mutants of Mia40. According to our bioinformatic analysis that was performed with the help of the Sangers Institute during the “Working with Zebrafish Genome Resources” workshop that was held in July 2013 in Barcelona during the 8th European Zebrafish Meeting, we confirmed that there are two zebrafish paralogues of the mia40 gene (paralogues 001 and 201). We showed that both paralogues can rescue the lethal phenotype of mia40-depleted yeast S. cerevisiae, proving that they are functional homologues and that the MIA pathway is evolutionarily conserved. We successfully used the TALEN technique to target the genetic loci of both paralogues separately, establishing novel zebrafish mutant lines. These mutants carry frameshift mutations that lead to a premature stop codon and shorter protein products. We used these novel lines to discover that homozygous mutations in paralogue 001 lead to death before the mid-larval stage. In contrast, the homozygous mia40 201 line survives to become fertile adults. The observed discrepancy between these two mia40 paralogues can be explained by differences in their levels of expression. In fact, our quantitative PCR analysis revealed that mia40 paralogue 001 is the predominant transcript during early development, between 24 hours post-fertilization (hpf) and 120 hpf. The facts that (i) the MIA pathway is conserved in evolution and (ii) its dysfunction results in life termination before the juvenile stage make this zebrafish mutant a good model to study the ways in which defects in mitochondrial biogenesis and thus mitochondrial function limit the life and development of vertebrates. We are currently investigating these mutants to find answers to remaining questions in the field of mitochondrion-related pathologies. To accomplish this, we are performing transcriptomic profiling to select the processes, pathways, and organs that are most affected in mutants vs. wildtype siblings. This led us to study glucose levels in our mutants. We found that mia40 001 homozygous mutants have significantly lower glucose levels. Using these novel mutant lines in the transgenic background with fluorescently labeled mitochondria, we observed abnormal mitochondrial structures in skeletal muscles. We also observed prominent cytosolic inclusions that stain positive for green fluorescent protein in muscle cells. These observations were mutant-specific and confirmed in blind experiments. We are currently pursuing electron microscopy analysis of mitochondrial structures and cytoplasmic inclusions in skeletal muscles in a non-transgenic background and focusing on understanding their origin.
Jacek Jaworski, Laboratory of Molecular and Cellular Neurobiology
The major aim of the Laboratory of Molecular and Cellular Neurobiology (LMCN) within the FishMed grant was to develop a new zebrafish model to study mTOR kinase function in neurons in vivo and unravel the mechanisms by which it regulates neuronal differentiation and function. The LMNC also seeks to expand the available models of tuberous sclerosis complex, a mTOR kinase-related disease with severe neurological deficits. To achieve this, available zebrafish mutant strains were used, one of which has depleted mTOR protein, and the other has excess mTOR activity (by disrupting the upstream inhibitor of mTOR, TSC2). Additionally, several new lines have been generated to gain better insights into the molecular biology of mTOR in the fish brain in vivo. These include CRISPER technology-based mutants of Tsc1a, Tsc1b, and Rptor. Through collaborations with twining partners (Harris lab, Stanier lab), we obtained access to additional fish strains that allow live-imaging studies of neuronal development and circuitry formation and CRISPER and CRISPERi technology. These strains and technologies have started to provide coherent information about mTOR kinase function and its molecular pathways in neurons.
By the end of the FishMed project, the LMCN, with technical support from the laboratory of Prof. William Harris, fully characterized cellular characteristics in wildtype and mutant zebrafish retina using cryosections and whole-mount preparations combined with SPIM microscopy. These analyses revealed substantial changes in survival,, migration, differentiation, and neural network formation between the analyzed phenotypes. Additionally, the LMCN performed a thorough analysis of locomotor activity in mutant zebrafish using Zebrabox (Viewpoint). Special attention was paid to epilepsy-like behaviors in TSC mutant fish to clarify whether Tsc gene-deficient fish can be used to prescreen antiepileptic drugs in the future. Protocols for xenograft transplants of stem cells and in vitro cultures of fish retinal neurons were also established. An additional line of research focused on cellular functions and regulation of the Arc gene, which is dysregulated in TSC and the translation and degradation of which depend on mTOR and GSK3 kinases, respectively.
Two potential developments stem from FishMed funds that are allocated to the LMCN that can be used for further applications and medication-oriented studies. First, approaches toward reliable studies of epilepsy-related phenotypes have been established and will be used in the future for potential anti-epileptic drug testing. Second, the LMCN described for the first time a decrease in the caliber of the optic nerve in Tsc2 mutant fish (Switon et al., in preparation), a phenomenon that was recently introduced as a potential biomarker of TSC. This mutant fish phenotype can be further exploited in the search for new TSC treatments.
Jacek Kuznicki, Laboratory of Neurodegeneration
The aim of this project was to investigate possible alterations in calcium homeostasis in a pink1-/- zebrafish model of Parkinson’s disease (PD). We hypothesized that the inhibition of mitochondrial calcium influx resists mitochondrial dysfunction, leading to viable dopaminergic neurons and amelioration of PD progression. The primary approach was to alter multiple mitochondrial Ca2+ influx mechanisms in pink1-/- zebrafish and evaluate the resulting molecular and cellular changes with regard to PD pathology. To accomplish this task, we used a morpholino-based knockdown strategy to silence mcu and vdac1 (i.e. channels that are responsible for calcium influx into mitochondria).
Our results showed that alterations of Ca2+ sequestration through the knockdown of mcu but not vdac1 led to the restoration of mitochondrial respiration and thereby contributed to viable dopaminergic neurons in pink1-/- zebrafish. Subsequent gene expression studies revealed specific upregulation of the mcu regulator micu1 in pink1Y431* mutant zebrafish larvae, and inactivation of micu1 also resulted in the rescue of dopaminergic neurons. The functional consequences of PINK1 deficiency and modified MCU activity were confirmed using a dynamic in silico model of Ca2+-triggered mitochondrial activity. Live imaging using light sheet microscopy was employed to study alterations in basal calcium levels in wildtype and pink1-/- zebrafish tagged with the genetically encoded calcium indicator -GCaMP5. Data analyses were performed with Bitplane Imaris software, which is capable of 3D tracking of single regions in time lapses. pink1-/- is more sensitive to heat shock, giving a faster response in the form of a higher frequency of Ca2+ spikes in brain regions in response to a temperature rise from 27°C to 37°C. Moreover, live imaging studies revealed a 20% increase in CCCP-induced cytosolic Ca2+ efflux in area postrema neurons in pink1-/- zebrafish compared with wildtype zebrafish. Our data suggest that there are alterations in mitochondrial calcium homeostasis in pink1-/- zebrafish, and the modulation of MCU-mediated mitochondrial calcium entry may be a possible neuroprotective strategy in PINK1 mutant PD.
Marta Miaczynska, Laboratory of Cell Biology
The goal of this project was to investigate the role of endocytic proteins in signaling and transcriptional regulation in zebrafish development. As a starting point, unbiased RNAi screens in mammalian cells revealed candidate endocytic proteins that affect transcription in several signaling cascades, including the Wnt and NF-kappaB pathways. These candidate proteins have been further studied with regard to their roles in zebrafish development, in parallel with molecular studies that are performed in cultured mammalian cells.
In the first line of investigation, Tollip adaptor protein was identified as a novel negative regulator of canonical Wnt signaling in mammalian cells. We found that the depletion of Tollip potentiated the activity of the beta-catenin/TCF-dependent transcriptional reporter, whereas its overproduction inhibited reporter activity and the expression of Wnt target genes. These effects occurred independently from dynamin-mediated endocytosis but required the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracted the activation of beta-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibited the pathway after the stage of beta-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive beta-catenin activity.
Having identified the molecular mechanisms of Tollip’s action in vitro in cultured mammalian cells, we further sought to validate its role in Wnt signaling in an in vivo model (i.e. zebrafish) in collaboration with our partner, Prof. Gonzalez-Gaitan. The canonical Wnt pathway is crucial for body patterning during early vertebrate development. Therefore, we tested the effects of tollip overexpression or its morpholino-mediated depletion in zebrafish embryos. The resulting phenotypes were generally reminiscent of those of canonical Wnt signaling mutants. Specifically, morphological defects that were caused by tollip overexpression led to a reduction of the posterior body, the disappearance of somite boundaries, and partial fusion of the eyes that were manifested at 48 hpf. Analyses of the expression of mesoderm-organizing genes, such as goosecoid (gsc) and notochord (ntl), during gastrulation showed that their expression patterns were slightly expanded compared with uninjected embryos. In turn, general developmental effects (at 48 hpf) that were caused by tollip knockdown with various morpholinos included a slight reduction of the size of the posterior body and head, dorsal curvature, smaller eyes, yolk sac extension deficiency, and in some cases heart edema. To evaluate whether some of these phenotypes may be related to a potential inhibitory role of Tollip in Wnt signaling, we attempted to rescue them by reducing the levels of beta-catenin2. After beta-catenin2 knockdown with morpholinos, the embryos displayed weaker pigmentation, an abnormal structure of the head, and a curly tail. Double knockdown resulted in a return to a normal phenotype, suggesting that zebrafish tollip may be involved in Wnt signaling during embryonic development. These results were published in 2015 (Torun et al, PLoS One).
Importantly, studies on the role of tollip in zebrafish development and physiology are continuing, particularly with the ongoing generation of genome-edited zebrafish lines that carry complete knockout of the tollip gene or deletion of its CUE domain, thanks to the efforts of ZF-screens.
In the second line of investigation, components of Endosomal Sorting Complexes Required for Transport (ESCRT) complexes have been found to inhibit the NF-kappaB pathway in mammalian cells. Specifically, we depleted all subunits of the ESCRT complex that control receptor degradation and found that four components (Tsg101, Vps28, UBAP1, and CHMP4B) were essential to restrict constitutive NF-kappaB signaling. Without cytokine stimulation, the depletion of these proteins potently activated both canonical and noncanonical NF-kappaB signaling and induced the NF-kappaB transcriptional response in cultured human cells. These effects depended on cytokine receptors, such as the lymphotoxin beta receptor and tumor necrosis factor receptor (TNFR1). Upon ESCRT depletion, both receptors were concentrated on endosomes and signaled from this compartment. In the case of the lymphotoxin beta receptor, endosomal accumulation induced its oligomerization and signal transduction via TRAF2/3 proteins in the absence of ligands.
In parallel, again in collaboration with our partner Prof. Gonzalez-Gaitan, we investigated whether the effects of depletion of ESCRT subunits on NF-kappaB signaling were evolutionarily conserved. To this end, we analyzed the expression of NF-kappaB target genes in zebrafish embryos that were injected with morpholinos that targeted fish orthologues of Tsg101, Vps28, UBAP1, or CHMP4B (encoded by two genes, chmp4ba and chmp4bb). We measured the expression of fish NF-kappaB target genes, such as nfkbiaa, il1b, and nfkb2, by RT-PCR in whole embryo lysates. A significant increase in nfkb2 expression was detected in vps28 morphants, and a significant increase in il1b was detected in ubap1 and chmp4ba/chmp4bb morphants. We also found significantly higher abundance of nfkbiaa mRNA by in situ hybridization in vps28, ubap1, and chmp4ba/chmp4bb morphants. In all cases, higher nfkbiaa expression was detectable in the same tissues, with enrichment in the pronephros, arguing for the specificity of the observed phenotypes. Cumulatively, these results in fish embryos suggest that ESCRT components inhibit constitutive NF-kappaB signaling also in lower vertebrates and during embryonic development. While performing this work, we initiated an additional collaboration with an established zebrafish researcher, Dr. Maximilian Fürthauer, from the Institut de Biologie de Valrose, University of Nice – Sophia Antipolis, in France. Dr. Fürthauer and his co-worker helped perform the in situ hybridization analyses. The complete study, including the data that were obtained in mammalian cells and in zebrafish embryos, was published in 2016 (Maminska et al., Science Signaling).
Cecilia L. Winata, Laboratory of Zebrafish Developmental Genomics, Max Planck/IIMCB Research Group
Our lab’s research seeks to determine the mechanism of gene regulation during heart development through the application of next-generation sequencing (NGS) to profile the binding sites of key cardiac transcription factors (TFs) and epigenetic markers. We optimized a protocol for isolating pure populations of cardiomyocyte cells from embryos. Preliminary transcriptome profiling by NGS of these cells confirmed their correct identity and high purity. Two heart mutant lines with cardiomyocyte-specific green fluorescent protein expression have been generated through extensive crossing and genotyping. Using the NGS-based RNA-seq method, we generated transcriptome profiles of duplicate samples of cardiomyocytes from wildtype and mutants at 24, 48, and 72 hpf. These datasets are currently undergoing bioinformatics analysis and experimental validation for preparation of a manuscript. We have optimized a ChIP-seq protocol based on the conventional method using antibodies. Currently, we are collecting cardiomyocyte samples from 24, 48, and 72 hpf embryos for a ChIP-seq experiment that is planned at the end of May 2016. At the same time, we are in the process of generating transgenic lines that express fusion-tagged heart TFs using CRISPR technology as an alternative to the conventional ChIP method, which is still being optimized. We have obtained a working sgRNA and designed a construct for homology-directed repair.
A comprehensive view of genome-wide genetic and epigenetic regulatory networks that is generated from this study will provide novel and invaluable insights into heart development, which will be an important step toward a better understanding of the mechanism of congenital heart disease.
Maciej Zylicz, Department of Molecular Biology
A number of mutations in the TP53 gene are overrepresented in samples that are isolated from human tumors, suggesting that they provide a competitive advantage for tumor cells. Many of them belong to the class of “gain-of-function” (GOF) mutations, in which the original function of p53 is lost and the mutated protein acquires new functions.
During the period covered by this report, numerous cell lines have been prepared, based on established cell lines of lung and breast cancer origin, with both epithelial and mesenchymal characteristics. These cell lines are characterized by inducible silencing of the endogenous p53 protein and concurrent expression of the mutated variant of p53, carrying one of the mutations being subject to investigation within this project. Additionally, the cells stably express a red fluorescent protein that enables tracking the cells in vivo following injections into fish embryos.
The cells were tested for tightness of the repression system and its inducibility, and then their migratory and invasive phenotype was characterized in vitro using several methods. In scratch assays and experiments that employed an improved version of this assay, several mutations in p53 resulted in greater motility of the cells, with the magnitude of the increase differing with the mutation. Subsequently, the invasive capability of the cells was investigated by analyzing the invasion of (or migration through) the collagen gel or matrigel. Finally, the invasion of the gel in a 3D culture model (gel-embedded microsphere model) was investigated and quantified. These phenotypic studies confirmed that the migratory and invasive potential of the cells is modified by the presence of the GOF mutants of p53 protein.
In an attempt to determine the molecular mechanisms that are responsible for the increase in invasive potential following the induction of mutated p53, an extensive set of proteins, including many EMT markers, was analyzed by Western blot. Additionally, the expression levels of several chemokines that are involved in cancer invasion and metastasis and their receptors were analyzed by qPCR in cells that expressed the mutated p53s. A subset of the samples was also analyzed by NGS to profile the transcriptome. In summary, the data accumulated to date have not allowed definitive conclusions about the molecular mechanisms of increased invasiveness in cancer cells that express mutated p53.
Zebrafish embryos were employed in the project as a host for xenotransplants of human cancer cells that were labeled with red fluorescent protein and genetically modified with regard to p53 expression. The cells were microinjected into the common cardinal vein of 2 days post-fertilization (dpf) kdrl:EGFP or fli:EGFP embryos with green fluorescent vasculature. On the following days, the extravasation of the cells, the formation of primary metastases, and subsequent local tissue infiltration were observed in the embryos. Because of the high variance of the phenotype, a substantial average change in a large population is required to obtain statistically significant results. Such results have been obtained for some cell line/mutation combinations, and more experimentation is currently underway to be able to accept or reject the hypothesis.
Another line of research in the project is the involvement of gain-of-function p53 mutants in the regulation of angiogenesis. Solid tumors need to stimulate the growth of new blood vessels that deliver nutrients and oxygen because diffusion is insufficient when tumors exceed 2 mm in diameter. The most important cytokine that induces the proliferation and migration of endothelial cells is vascular endothelial growth factor A (VEGF-A). This protein has multiple isoforms, both proangiogenic and antiangiogenic, and their activity is determined by alternative splicing of the last exon. The studies on GOF p53 mutants are ongoing. After the initial phase of experiments in mammalian cell cultures, we now have moved to an animal model, the zebrafish embryo.
Zebrafish Core Facility
The state-of-the-art Zebrafish Core Facility (ZCF) was established in 2012. During the course of the FishMed project, the facility grew, and this growth was noted on many levels. The animal house has expanded from six 5-shelf racks (total capacity of 300 tanks, with 3.5 L volume each) to 12 racks that hold 670 tanks (3.5 L volume each). Further expansion is scheduled for July 2016. Such growth both during and after the FishMed project was possible because of funds that were secured by the ZCF and researchers. The collection of fish that are kept at IIMCB has increased from 28 fish that represent nine lines in 2012 to over 10,000 animals that represent nearly 100 lines that are being kept in 2016. Notably, new mutants have been created by FishMed participants, and they are being used and will be used to develop new models that are available to IIMCB and the general scientific community.
FishMed funds allowed the purchase of specialized equipment (e.g. systems for micromanipulations, behavioral analysis, and imaging) that meet most of the needs of internal and external users. At the beginning of the FishMed project, only eight experienced postdoctoral researchers and three research technicians from IIMCB benefited from the use of the ZCF. Soon thereafter, external users joined. The numbers of both internal and external users have continuously increased. By the end of 2015, the facility was used by over 60 researchers.
FishMed funds also allowed visits and participation in courses/workshops and other meetings that resulted in mastering skills and obtaining new knowledge and contracts. This allowed the development of additional services that are now being provided by the ZCF, including a health screening program, sperm banking, assistance in planning and performing behavioral analysis, and introduction to and advanced courses on the zebrafish model (e.g. husbandry, health, and various techniques used in research projects).
The ZCF staff has been active in promoting the zebrafish model by lecturing on a number of courses for students and established researchers who work with animal models. Thanks to this activity, increasingly more researchers and teachers at various locations in Poland have decided to work with the zebrafish model.
Technology transfer and innovations
In response to the growing innovative potential of IIMCB, the Bio&Technology Innovations Platform (BioTech-IP) was created. It identifies, protects, and commercializes inventions that have market potential. In this scope, BioTech-IP cooperates with technology transfer experts. They assisted in the creation of an IIMCB-owned company (also called a special purpose vehicle [SPV]) that is dedicated to supporting the Institute in technology transfer and commercialization. This SPV company, BioTech-IP Sp. Ltd, is designed to acquire and manage the shares of start-up companies that are established for the commercial exploitation of intellectual property. In the course of the project, BioTech-IP worked on 11 patents and patent applications. The main results are the following:
• Organization of a total of 11 science-business networking brunches attended by 250 participants, during which 40 projects with strong applied potential were presented.
• Providing professional training to a total of approximately 1,250 PhD students and scientists on soft skills, project management, patenting, IPR management, and commercialization strategies.
• Creation of IIMCB-owned company (also called an SPV) that is dedicated to supporting IIMCB in technology transfer and commercialization. An amendment to the Polish Higher Education Act obliges universities and research institutes to establish an independent company (third-party) to support them in efficient technology transfer to industry. According to the Act, SPVs are designed to acquire and manage the shares of start-up companies that are established for the commercial exploitation of intellectual property that comes from a given university or scientific institution. Despite imprecise documents concerning implementation of the Act and legal uncertainty about the formal procedures of SPV creation, IIMCB successfully obtained the required permissions from the Ministry of Science and Higher Education and from the Head of the Polish Academy of Sciences and registered the company, called BioTech-IP Sp. Ltd. This was one of the first SPVs that was created in a scientific institution in Warsaw, with a duly completed business plan that consisted of market and customer analyses, an organizational structure, proposed products, a services portfolio, and a financial forecast.
• Purchasing access to GlobalData business database. Offering technologies to potential investors or industrial partners prompted the need to acquire professional feasibility studies, which are based on more comprehensive information than can be found in internet search engines. Market search revealed that the products that are offered by ThomsonReuters and GlobalData could be helpful in assessing the commercial potential of inventions from the biotechnology, medicine, or medical device fields. However, comparison surveys indicated that the GlobalData-Healthcare product that comprises two units (Pharma eTrack for drugs and Medical for medical devices and diagnostics) fits the TTO profile much better.
• Organization of meeting between scientists and representatives of GlaxoSmithKline (GSK) to coordinate the program called Discovery Partnerships with Academia (DPAc; http://dpac.gsk.com/). The DPAc team was looking for partners in early discovery projects where there is a clear therapeutic hypothesis and an understanding of the target. For suitable projects, GSK offered grant money to be paid when milestones are reached and financial rewards that are shared through royalty payments. During the meeting that was organized by BioTech-IP at IIMCB in November 2014, 14 projects were presented to GSK, from which one was invited to join GSK’s Discovery Fast Track Challenge competition.
• Cooperation with Over Group, an industrial partner that is interested in the commercial application of lytic enzyme technology. The enzyme that was patented by IIMCB has the ability to kill antibiotic-resistant Golden Staph, which is considered one of the most dangerous bacteria in the world. The possible commercial exploitation of the enzyme covered the disinfection of human skin, wounds, and surfaces (in hospitals) and animal protection against the aforementioned bacteria. Feasibility studies indicated that introduction of the enzyme into cattle breeding should be relatively cost effective for the company and profitable. Technology was presented to the following companies: Siveele (siveele.com) Hypred (hypred.com) Ecolab (ecolab.com) BioWet Drwalew S.A. (biowet-drwalew.pl) and Over Group. After negotiations with Over Group, the company introduced the IIMCB technology into their R&D program, with the aim of developing innovative cow udder hygiene products to protect animals against mastitis.
• BioTech-IP also concentrated its efforts on supporting the creation of the first technology-based start-up company, aiming to commercialize inventions concerning restriction enzymes that are capable of the sequence-specific cutting of RNA strands. BioTech-IP supports scientists and IIMCB with regard to business, taxes, and legal issues to make the creation of companies feasible. Moreover, BioTech-IP presented this opportunity to three investment funds: WinQbator, IP-Hub, and Impera Alfa. Although Winqbator pushed to sign the deal, it had to withdraw from negotiations because of internal problems. IIMCB technology and the idea of a start-up company have been presented to two other investment funds: Impera Alfa and IP-hub, which expressed interest. Negotiations with both investors are ongoing. In parallel, BioTech-IP identified a project manager who is experienced in the Polish market and will join the scientific team and supervise the start-up business operations.
• Thanks to BioTech-IP’s support in securing funds for patent protection, it was possible to seek protection for three patents that were granted in Europe and other parts of the world:
- A method of peptide hydrolysis, peptidase, the composition for use as a bacteriostatic and bactericidal agent, a kit and the uses of the active form of LytM from S. aureus or derivatives thereof (EP 2 699 254 B1)
- Sequence-specific engineered ribonuclease H and the method for determining the sequence preference of DNA-RNA hybrid binding proteins (EP 2 718 430)
- dsRNA endoribonucleases (EP 2 718 431)
Potential Impact:
IIMCB’s strategic objective was to achieve the quality of research and innovative activities of leading research entities in the world. To achieve this level of excellence and increase our innovative potential, we successfully established a new research model at IIMCB: zebrafish (Danio rerio). This fish is an excellent organism to study various aspects of disease because it is fully transparent until adulthood. Therefore, development, pathologies, and the effects of treatments can be easily monitored in vivo. Moreover, potential drugs can be added to water to easily test their effects on behavior, anatomy, development, and signaling in high-throughput screens. Using zebrafish models of human diseases, we can study them not only at the molecular and cellular levels but also at the level of live vertebrate organisms, the metabolism of which is very similar to that of mammals.
General impact, including the socioeconomic impact and wider societal implications
Society-oriented research
Research projects that are developed under FishMed focus on human diseases that cause persistent problems in society, such as Parkinson’s disease, neurodegenerative disorders, and metabolic diseases. During the course of these projects, mainly basic biological processes that lead to the development of diseases were studied, but the availability of zebrafish as a research model opened a range of opportunities for collaborations with hospitals and clinics. One of the best examples is the research project, “Identification of genes controlling brain development through genomic analysis of patients with microcephaly,” coordinated by the Institute of Mother and Child (Warsaw, Poland) and involving the Baylor College of Medicine (Houston, Texas, USA) and Laboratory of Zebrafish Developmental Genomics (IIMCB).
Awareness of research on animals
Through the creation of discussion forums for the Polish scientific community on the usage of zebrafish models for studies of human diseases as an alternative to mammalian models, FishMed contributed to greater awareness of research on animals. IIMCB has successfully introduced the concept of zebrafish research to the local scientific community and became a local source of information on zebrafish as a research model. As a result, new scientific collaborations with researchers based in Warsaw and other Polish research centers have been established, including the Nencki Institute of Experimental Biology, Warsaw, Medical University of Warsaw, University of Warsaw, Warsaw University of Life Sciences, University of Warmia and Mazury, Olsztyn, Institute of High Pressure Physics, Warsaw, and Poznan University of Medical Sciences.
Ethical issues
All of the research activities at IIMCB are carried out in compliance with fundamental ethical principles and relevant national and international legislation. Following the recent change in national regulations, IIMCB has:
• appointed an Animal Welfare Advisory Team who supervises the wellbeing of animals that are used for scientific or educational purposes
• provided a full-time employment position for a veterinary physician
• obliged all relevant personnel to become familiar with relevant legislation and participate in compulsory training that is imposed by this legislation
Being aware of the importance of ethical issues, IIMCB organized in cooperation with the PolLASA Association “Combined training for persons responsible for planning and conducting procedures and experiments that kill laboratory animals and supplementary training for laboratory animal caretakers.” The training was particularly directed to foreigners as all lectures were translated into English. All participants received required by law certificates entitling to work with laboratory animals. As a rule, all researchers who work with zebrafish undergo relevant training and receive certification that is required by law. Whenever necessary, researchers apply for approval from the Local Ethics Committee for Animal Experiments to use zebrafish for scientific or educational purposes.
Science education
FishMed has had a great impact on society through promotional and educational activities that are focused on the wider public and especially the younger generation who, inspired by our activities, may pursue scientific careers in academia, biotechnology, and the pharmaceutical industry. We involved students and school children through workshops, science festivals, art competitions, info/open days, book fairs, and job fairs. The project generated numerous science education materials, such as a book, a movie, bookmarks, stencils, crossword puzzles, and educational boards. Thus, the project generated output that can be used by policymakers in the field of education, training and youth.
Engaging with civil society and policymakers
We cooperated with the Polish association Supporting People with Inflammatory Bowel Disease “J-elita.” We also approached local authorities, such as the Marshal and Office of the Mazowieckie Voivodeship, Regional Council of the Mazowieckie Voivodeship, and city of Warsaw. They were invited to participate in all major events, such as the kick-off conference, Heart of Europe Zebrafish Meeting, and International FishMed Conference on Zebrafish Research. The representatives of local authorities and funding agencies participated in discussions on the “Evaluation report with guidelines for further development” that was prepared by experts who were appointed by the European Commission. They appreciated the success of FishMed and declared support in further IIMCB development. The city of Warsaw is going to promote IIMCB as an excellent research center and will get involved in organizing international conferences that are planned after the project is completed.
Contribution to the economic and social development of the region
Because of the enhanced research capacity of the FishMed project (i.e. creation of ZCF, employment of experienced researchers, technicians, and managers, and the purchase of state-of-the-art equipment), IIMCB has contributed to the economic and social development of the city of Warsaw and Mazovia province. The successful introduction of a new animal model brought new knowledge and expertise and enhanced the research capacity of the region. Acting as a zebrafish reference center, we share our experience and best practices with others who are interested in adopting this model locally and regionally. Most of the researchers who are employed under FishMed have successfully applied for funding to continue their research at IIMCB. Moreover, by doing so, they created new jobs for researchers, technicians, and support personnel.
Media and communication to general public
To communicate information about FishMed to the general public, IIMCB’s professional public relations office used the following tools: press releases, media briefings, TV coverage, radio coverage, brochures, posters, flyers, films, coverage in specialist/general press, coverage in the national/international press, website, and events that target the general public. To illustrate the results of these activities, we measured the number of IIMCB webpage visits starting from 1 year before FishMed was initiated and the number of appearances of information about IIMCB in the media during the same time period. The number of IIMCB webpage visits increased from 95,910 in 2012 to 114,070 in 2015. The number of appearances of information about IIMCB in the media increased from 3 in 2012 to 34 in 2015.
Innovations and technology transfer
Through BioTech-IP, IIMCB’s Technology Transfer Office, around 1,500 PhD students and scientists at the Biocentre Ochota campus obtained access to professional information that is tailored to their specific needs with regard to commercializing the R&D results of their work. Some of them used these skills in their careers and started business activities. BioTech-IP also helped initiate contacts between scientists and business partners, which led to joint projects and collaborations. Eventually, BioTech-IP developed unique skills to deal with commercialization processes at different stages, which made it possible to create BioTech-IP Innovation, a company that was established to acquire and manage the shares of spin-off companies based on IIMCB’s intellectual property. FishMed successes in innovation and technology transfer will have a positive impact on the economy and regional development in the long-term. IIMCB already has human resources and all of the necessary tools to identify potential targets and support researchers in their commercialization.
Exploitation of results
Bujnicki lab
Description of exploitable foreground: Computer software
We developed a computational method for modeling the 3D structures of RNA molecules and RNA-RNA complexes, called SimRNA. It can computationally simulate the folding of RNA structures if provided with RNA sequence information. It can be used to study the influence of mutations in RNA on the 3D structural stability of RNA. The program is available for free for academic users at http://genesilico.pl/SimRNA/ and may be commercially licensed for industry. The market potential for this type of product is currently unclear. Based on discussions with potential collaborators from industry, a new version of SimRNA that can model RNA-ligand interactions (to be possibly developed in the future) could be useful for commercial applications. The current version is applicable mostly for academic research.
Chacinska lab
Description of exploitable foreground: Novel model and optimized protocol
Mitochondria constitute life-essential organelles with multiple cellular functions. They serve as energy and biosynthetic centers, participate in cell stress responses, and are involved in cell death execution. Failure in mitochondrial function is associated with numerous human pathologies. With no cure, the severity of symptoms and disease progression can often lead to death before adolescence. The fact that the treatment of mitochondrial disorders is mostly directed at mitigating the symptoms reflects our relatively poor understanding of the biology of mitochondrial disorders. Our successful collaboration with the laboratory of Prof. Didier Stainier at the Max Planck Institute for Heart and Lung Disease in Bad Nauheim yielded a novel model with mutations in one of the key proteins that is responsible for mitochondrial biogenesis. We aim to use this zebrafish mutant to identify and understand specific processes in the context of a multicellular organism that are initiated by mitochondrial deficiencies and lead to pathology and eventually death. We also plan to discover putative compensatory mechanisms and defense responses and their role in pathologies that are caused by mitochondrial dysfunction. Our project has great potential to provide novel insights into mitochondrion-related pathologies.
Our optimized protocol for protein extraction in conjunction with the selection of antibodies that specifically detect mitochondrial proteins initiated collaborative research with the laboratory of Prof. Oliver Bandmann in Sheffield, United Kingdom, on the analysis of mitochondrial proteins in zebrafish models of neurodegenerative diseases.
Jaworski lab
Description of exploitable foreground: Analysis of locomotor/epileptic-like activity of TSC mutant zebrafish and analysis of optic nerve caliber of TSC mutant zebrafish.
Understanding mTOR activity-related diseases is of great importance to society. Changes in mTOR activity likely contribute to such neuropathologies as brain tumors, epilepsy, autism spectrum disorders, schizophrenia, addiction, and several neurodegenerative disorders. However, to date, animal models are restricted to either fruit flies or mice and therefore not convenient for large compound/genetic screens with the use of vertebrates. New observations within FishMed established that fish that lack Tsc may be a new model for such screens. Both epileptic-like behaviors and a decreased caliber of the optic nerve in TSC model fish can be exploited by both researchers and collaborating companies to search for new compounds or signaling pathways to be targeted in TSC and epilepsy.
Kuznicki lab
Description of exploitable foreground: Novel research direction
Parkinson's disease (PD) is a neurodegenerative disorder that affects the elderly and remains incurable. Mitochondrial dysfunction has been postulated as a possible etiological factor behind PD. However, the relationship between mitochondrial dysfunction (mitochondrial calcium overload) and dopaminergic cell loss is still unclear. The study aims to reveal the factors that are involved in calcium overload in mitochondria and gain insights into the association between mitochondrial calcium overload and dopaminergic neuron loss in PD.
Miaczynska lab
Description of exploitable foreground: Novel research direction
Based on FishMed results, a new research direction has been developed: studies on the molecular mechanisms that are related to processes in which Tollip protein may be involved at the level of the organism (zebrafish). Studies will concentrate on, among others, the potential contribution of Tollip to the clearance of protein aggregates in a model of Gaucher's disease.
Winata lab
Description of exploitable foreground: Computer software
We developed a computational tool for RNA editing discovery based on NGS called REDiscover. This software is fast (DNAseq & RNAseq processed simultaneously, multithreading), flexible (non-standard experimental designs, multiple inputs [BAM]), reliable, and simple. REDiscover can be used to study RNA editing detection during development. The program is available for free at https://github.com/lpryszcz/REDiscover#rediscover.
Zylicz lab
Description of exploitable foreground: Validation of the zebrafish xenotransplantation model for use in research concerning p53 mutation-induced tumor invasiveness.
This technique has been used by Leiden University, The Netherlands. A transgenic zebrafish line has been prepared that carries a deletion in the tp53 gene. The fish have been prepared for a research project that investigates the mechanisms of oncogenic activity of gain-of-function mutations in p53. Such research requires a p53 null background. Planned activities include the creation of a zebrafish model of one of the human metabolic diseases that has neurological manifestations.
Main dissemination activities
During the course of the project, IIMCB established contacts with non-scientific communities, such as patient organizations, local authorities, and the general public. We have organized special events for selected groups from outside academia and developed an information campaign that is directed at the general public, through which we informed society about the advancements and successes of FishMed.
a. Patient organizations (e.g. workshop for children during the Days of Education organized by the Polish association Supporting People with Inflammatory Bowel Disease “J-elita”
b. Local authorities (e.g. visit by the Marshal of the Mazowieckie Voivodeship to IIMCB and ZCF; participation of representatives of local authorities in the meeting with evaluators appointed by the European Commission and representatives of the Regional Council of the Mazowieckie Voivodeship, city of Warsaw, Office of the Marshal of the Mazowieckie Voivodeship)
c. General public (e.g. Be Healthy as a Fish campaign that comprises a book, a movie, and workshops). A total of 636 primary school children participated in 32 workshops. Around 2300 people received the book, and nearly 1700 people watched the movie. Info days were provided for talented youth, supported by the Polish Children’s Fund, and for students from the Warsaw University of Life Sciences and University of Agriculture in Kraków.
Creation of a discussion forum for the Polish scientific community on the use of zebrafish models for studies of human diseases as an alternative research organism to mammalian models:
a. Organization of 25 open seminars delivered by specialists in zebrafish research
b. Organization of 3 FishMed open report sessions
c. Organization of 2 international conferences devoted to zebrafish in biomedical research: Heart of Europe Zebrafish Meeting and International FishMed Conference on Zebrafish Research
Along with the visibility platform actions, the FishMed results were disseminated to the scientific community via open access publications in internationally recognized journals and lectures and posters that were presented at various scientific events worldwide.
Measures were taken to increase the likelihood of the market uptake of project results, such as identifying and collaborating with potential users and identifying potential partners and sources of financing for commercialization. These activities were undertaken by BioTech-IP, IIMCB’s Technology Transfer Office. In the course of the project, BioTech-IP supported scientists in the initiation of contacts between scientists and business partners, which led to joint projects and collaborations. BioTech-IP also developed unique skills to deal with commercialization processes at different stages, which made it possible to create BioTech-IP Innovation, a company that was established to acquire and manage the shares of spin-off companies based on IIMCB’s intellectual property.
All of these actions have increased the scientific recognition of IIMCB as a research center using zebrafish as a research model and opened new possibilities for inspiring scientific partnerships and collaborations as well as the commercialization of project results in the future.
Main dissemination activities planned after the project is completed
• Publication of at least 11 peer-reviewed articles
• Lectures and posters at international events
• Organization of scientific events related to zebrafish research
• New research projects and grants in collaboration with FishMed partners
• Continuation of the Be Healthy as a Fish campaign
List of Websites:
Website address:
fishmed.iimcb.gov.pl
Contact details:
Dr Urszula Bialek-Wyrzykowska
International Institute of Molecular and Cell Biology
4 Ks. Trojdena St.
02-109 Warsaw, Poland
Tel. +48 22 59 70 769
Fax +48 22 59 70 715
ulawyrzykowska@iimcb.gov.pl
The International Institute of Molecular and Cell Biology (IIMCB) is one of the best biological research institutions in Poland. IIMCB’s strategic objective is to achieve the quality of research and innovative activities of leading research entities worldwide. To achieve this level of excellence and increase our innovative potential, we introduced a new research model: zebrafish. The FishMed Centre, supported by the European Union and Ministry of Science and Higher Education, is composed of a Zebrafish Core Facility and research groups that use zebrafish in innovative projects that study the molecular mechanisms of disease. This ambitious undertaking would not have been possible without support from excellent European partners who shared with us their expertise and resources. The EU and national funding have been used to finance the following: the employment of scientists, technicians, and managers; the purchase of state-of-the-art equipment; exchange visits between IIMCB researchers and their European partners; and participation in and organization of various events, including those that focus on innovation and technology transfer.
One of the key indicators of the success of FishMed has been the ability to compete internationally to recruit experienced researchers and establish a new laboratory that is headed by Dr. Cecilia L. Winata from Singapore. Her group’s main project is dedicated to elucidating the gene regulatory networks of zebrafish heart development using genomic methods. Based on the collaborative agreement, this laboratory has a secondary affiliation with the Max Planck Institute for Heart and Lung Research in Bad Nauheim. This has allowed Dr. Winata and her staff to use the expertise and resources of this well-known center of heart research, including zebrafish research.
IIMCB is well equipped, but FishMed funds allowed us to purchase several additional items, including the first LightSheet Z.1 microscope (SPIM) in Poland and systems for behavioral analyses of zebrafish larvae and adult fish. The SPIM is heavily used by IIMCB staff and external collaborators and is already yielding data, some of which are presented in the first FishMed publications.
The International FishMed Conference on Zebrafish Research was organized by IIMCB to bring together scientists from the field, share recent advances in research on zebrafish, and present the results of the FishMed project to an international audience. The meeting was convened with very high scientific and professional standards and very well received by the speakers and meeting participants from many countries around the world.
In addition to research, we are strongly engaged in popularizing fish as a model for research among current and future scientists. The educational campaign Be Healthy as a Fish targets children and their parents and includes workshops, a short movie, and a book. This action was prepared by public relations specialists who are involved in the FishMed project with strong support from our scientific team and fish facility staff.
The successful implementation of all plans of the FishMed project allows us to be highly optimistic that all of its goals have been achieved or even exceeded. What makes us especially satisfied is that not only IIMCB researchers, but also scientists from other Polish institutions, have benefited from our zebrafish core facility and equipment and the expertise of our staff. Some people from these institutions started new projects using our collection of wildtype, mutant, and transgenic zebrafish lines. The increased demand for zebrafish in Poland convinced us to expand our facility, and we look forward to supporting the zebrafish research of scientists at other institutions at the national and international level.
Let us finish with a statement from experts who were appointed by the European Commission to evaluate IIMCB in the context of FishMed: “FishMed project has been an outstanding success. In the relatively short period since initiation of the project, the zebrafish has been successfully integrated into IIMCB, strong collaborations and international networks have been made, new research projects have been initiated, excellent core facilities have been established or expanded, talented personnel have been recruited to IIMCB, research productivity and grant income has increased, and international visibility of the IIMCB, and Polish research more generally, has been enhanced. FishMed project has given the IIMCB the potential to become a successful internationally competitive hub for research involving zebrafish as a model organism.”
Project Context and Objectives:
The International Institute of Molecular and Cell Biology (IIMCB) is one of the best biological research institutions in Poland. IIMCB’s strategic objective is to achieve the quality of research and innovative activities of leading research entities in the world. To achieve this level of excellence and increase our innovative potential, we introduced a new research model: zebrafish. The FishMed Center, supported by the European Union and Ministry of Science and Higher Education, is composed of a Zebrafish Core Facility and research groups that use zebrafish in innovative projects that study the molecular mechanisms of disease. The EU and national funding have been used to finance the following: the employment of scientists, technicians, and managers; the purchase of state-of-the-art equipment; exchange visits between IIMCB researchers and their European partners; and participation in and organization of various events, including those that focus on innovation and technology transfer.
Objectives
• Twinning of seven IIMCB groups with excellent European zebrafish centers to develop innovative potential using fish models.
• Development of a Zebrafish Core Facility and establishment of a new research group headed by a leader who is selected through an open international competition.
• Acquisition and upgrading of research equipment for a Zebrafish Core Facility and new zebrafish research laboratory.
• Reinforcement of IIMCB innovative potential with the Bio&Technology Innovations Platform (BioTech-IP).
• Construction of an interactive visibility platform to popularize the FishMed Center and research with zebrafish models among scientific and non-scientific communities, including promotion of the project’s innovative results.
The FishMed Center is a consortium of eight groups from IIMCB and six European institutions, including the Max Planck Institute for Heart and Lung Research (MPI-HLR) as a strategic partner. The twinning partners were chosen based on their expertise in research using zebrafish models, excellent publication records, and compatibility with the scientific interests of the FishMed Center groups at IIMCB. European partners share with us their zebrafish models and expertise related to fish research. Twinning allows smooth passage from the initial phase of accommodating a new experimental model to quickly focusing on cutting-edge research that is likely to lead to innovations.
Partners
• Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany, represented by Thomas Braun and Dr. Didier Stainier.
• Austrian Institute of Science and Technology, Klosterneuburg, Austria, represented by Carl-Philipp Heisenberg.
• Department of Physiology, Development, and Neuroscience, Cambridge University, United Kingdom, represented by William Harris.
• MRC Centre for Development and Biomedical Genetics, University of Sheffield, United Kingdom, represented by Oliver Bandmann.
• Department of Biochemistry, University of Geneva, Switzerland, represented by Marcos Gonzalez-Gaitan.
• Department of Molecular Cell Biology, Institute of Biology, Leiden University, The Netherlands, represented by Ewa Snaar-Jagalska.
Project Results:
Matthias Bochtler, Laboratory of Structural Biology
Ten eleven translocation (TET) proteins are alpha-keto-glutarate-dependent dioxygenases that can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5cC). In mammals, TET activity is required for demethylating the paternal pronucleus in the zygote, the mesenchymal-to-epithelial transition in organogenesis, and (partly) demethylation in the germline. 5hmC, the main and first product of the TET-mediated oxidation of 5mC, may also be an epigenomic signal itself, correlating with and likely contributing to cellular differentiation. TET proteins also play a role in the hematopoietic system. Chromosomal aberrations or mutations that affect individual TET genes do not suffice as drivers of leukemias, but such changes are found in up to 25% of human leukemias (depending on the type). In mice, the combined loss of TET genes in the hematopoietic system causes either B-cell (TET1 and TET2 deletions) or myeloid (TET2 or TET3 deletions) malignancies.
Using a combination of TALEN and CRISPR gene-targeting technologies, we generated zebrafish lines that are individually deficient in the TET1, TET2, and TET3 genes. This work was done in collaboration with Olov Andersson (Karolinska Institute, Stockholm). In parallel, we also generated antibodies against bacterially made fragments of the three TET proteins for immunohistochemistry. In agreement with reports that appeared in the literature while this work was in progress, we found that homozygous mutants of single TET genes have at most very mild phenotypes. However, the combined of loss of TET2 and TET3 is incompatible with complete development. The development of fish arrests during the larval period. Our original plans to study leukemias in the fish required modifications in light of the literature that appeared during the project. We plan to rescue TET2 and TET3 double mutants with a floxed TET2 or TET3 allele so that the acute ablation of TET activity is possible in a Cre-driver line-dependent manner. We seek to study the role of TET proteins after the larval stage, overcoming the developmental arrest of TET2 and TET3 double homozygotes. In parallel, we will attempt transplantation experiments, grafting TET2-/- and TET3-/- tissue onto wildtype fish. We are in the process of crossing reporter lines with lines that harbor mutations in the TET genes.
Janusz Bujnicki, Laboratory of Bioinformatics and Protein Engineering
The work of the Bujnicki group has focused on the development and use of computational tools for RNA 3D structure modeling and the modeling of protein-RNA interactions. Numerous software tools were developed with partial support from the FishMed project, which covered the work of Dr. Wayne Dawson and Prof. Bujnicki’s supervision of M.Sc. students who are involved in the project. A major piece of software that was developed based on FishMed support is SimRNA, a computational method of modeling the 3D structures of RNA molecules and RNA-RNA complexes (available at http://genesilico.pl/software/stand-alone/simrna as a standalone tool and at http://genesilico.pl/SimRNAweb as a web server). SimRNA uses a coarse-grained representation, relies on the Monte Carlo method for sampling the conformational space, and employs a statistical potential to approximate the energy and identify conformations that correspond to biologically relevant structures. SimRNA can fold RNA molecules using only sequence information. On established test sequences, it recapitulates the secondary structure with high accuracy, including the correct prediction of pseudoknots. To model complex 3D structures, it can use additional restraints that are derived from experimental or computational analyses, including information about secondary structures and/or long-range contacts. SimRNA can also be used to analyze conformational landscapes and identify potential alternative structures.
The support of FishMed included the conceptual development and testing of tRNAmodpred (a computational method for predicting posttranscriptional modifications in tRNAs; available at http://genesilico.pl/trnamodpred/) and NPDock (a web server for protein-nucleic acid docking; available at http://genesilico.pl/NPDock) and the preparation of articles that described these tools.
Additionally, a number of minor software tools were developed, including software for processing data that are generated by other computational methods.
Agnieszka Chacinska, Laboratory of Mitochondrial Biogenesis
Mitochondria constitute life-essential organelles with multiple cellular functions. They serve as energy and biosynthetic centers, participate in cell stress responses, and are involved in cell death execution. Failure in mitochondrial function is associated with numerous human pathologies. With no cure, the severity of symptoms and disease progression can often lead to death before adolescence. The fact that the treatment of mitochondrial disorders is mostly directed at mitigating the symptoms reflects our relatively poor understanding of the biology of mitochondrial disorders. We seed to find the ways in which faulty mitochondrial biogenesis impinges on the development of a vertebrate organism using Danio rerio as a model.
To meet the goal of our project, we first adapted a variety of basic biochemical skills to the in vivo model. For example, a protocol for protein isolation was developed and optimized that allows the isolation of proteins from zebrafish embryos and larvae at different stages of development. This protocol was subsequently used to study protein levels and their diversity at various stages of zebrafish development. Additionally and specifically to our research interest, we succeeded in enriching mitochondrion protein extracts by isolating zebrafish mitochondria. Taking advantage of a transgenic line with fluorescently labeled mitochondria, we characterized mitochondrial networks at various stages of development and in a plethora of tissues. We accomplished this by using cutting-edge light sheet fluorescent microscopy (LSFM).
We are interested in the universally conserved mitochondrial import assembly (MIA) pathway that is responsible for the import of mitochondrial proteins and well known for its dependence on redox reactions and cross-talk with respiratory chain complexes and complexes that are responsible for maintaining mitochondrial architecture. This MIA pathway is responsible for the biogenesis of cysteine-rich mitochondrial intermembrane space proteins, the majority of which are involved directly or indirectly in the biogenesis of respiratory complexes. Importantly, respiratory complex deficiencies underlie a large proportion of mitochondrial diseases.
In our research, we have utilized several approaches to disrupt the MIA pathway. Specifically, we targeted Mia40 protein, a mitochondrial intermembrane space oxidoreductase, the activity of which determines proper functioning of the MIA pathway. We first used a well-established antisense technique that exploits morpholinos to achieve temporal protein knockdown. In collaboration with the laboratory of our twinning partner Prof. Dider Stainier, we acquired and successfully adapted the TALEN technique to obtain genetic mutants of Mia40. According to our bioinformatic analysis that was performed with the help of the Sangers Institute during the “Working with Zebrafish Genome Resources” workshop that was held in July 2013 in Barcelona during the 8th European Zebrafish Meeting, we confirmed that there are two zebrafish paralogues of the mia40 gene (paralogues 001 and 201). We showed that both paralogues can rescue the lethal phenotype of mia40-depleted yeast S. cerevisiae, proving that they are functional homologues and that the MIA pathway is evolutionarily conserved. We successfully used the TALEN technique to target the genetic loci of both paralogues separately, establishing novel zebrafish mutant lines. These mutants carry frameshift mutations that lead to a premature stop codon and shorter protein products. We used these novel lines to discover that homozygous mutations in paralogue 001 lead to death before the mid-larval stage. In contrast, the homozygous mia40 201 line survives to become fertile adults. The observed discrepancy between these two mia40 paralogues can be explained by differences in their levels of expression. In fact, our quantitative PCR analysis revealed that mia40 paralogue 001 is the predominant transcript during early development, between 24 hours post-fertilization (hpf) and 120 hpf. The facts that (i) the MIA pathway is conserved in evolution and (ii) its dysfunction results in life termination before the juvenile stage make this zebrafish mutant a good model to study the ways in which defects in mitochondrial biogenesis and thus mitochondrial function limit the life and development of vertebrates. We are currently investigating these mutants to find answers to remaining questions in the field of mitochondrion-related pathologies. To accomplish this, we are performing transcriptomic profiling to select the processes, pathways, and organs that are most affected in mutants vs. wildtype siblings. This led us to study glucose levels in our mutants. We found that mia40 001 homozygous mutants have significantly lower glucose levels. Using these novel mutant lines in the transgenic background with fluorescently labeled mitochondria, we observed abnormal mitochondrial structures in skeletal muscles. We also observed prominent cytosolic inclusions that stain positive for green fluorescent protein in muscle cells. These observations were mutant-specific and confirmed in blind experiments. We are currently pursuing electron microscopy analysis of mitochondrial structures and cytoplasmic inclusions in skeletal muscles in a non-transgenic background and focusing on understanding their origin.
Jacek Jaworski, Laboratory of Molecular and Cellular Neurobiology
The major aim of the Laboratory of Molecular and Cellular Neurobiology (LMCN) within the FishMed grant was to develop a new zebrafish model to study mTOR kinase function in neurons in vivo and unravel the mechanisms by which it regulates neuronal differentiation and function. The LMNC also seeks to expand the available models of tuberous sclerosis complex, a mTOR kinase-related disease with severe neurological deficits. To achieve this, available zebrafish mutant strains were used, one of which has depleted mTOR protein, and the other has excess mTOR activity (by disrupting the upstream inhibitor of mTOR, TSC2). Additionally, several new lines have been generated to gain better insights into the molecular biology of mTOR in the fish brain in vivo. These include CRISPER technology-based mutants of Tsc1a, Tsc1b, and Rptor. Through collaborations with twining partners (Harris lab, Stanier lab), we obtained access to additional fish strains that allow live-imaging studies of neuronal development and circuitry formation and CRISPER and CRISPERi technology. These strains and technologies have started to provide coherent information about mTOR kinase function and its molecular pathways in neurons.
By the end of the FishMed project, the LMCN, with technical support from the laboratory of Prof. William Harris, fully characterized cellular characteristics in wildtype and mutant zebrafish retina using cryosections and whole-mount preparations combined with SPIM microscopy. These analyses revealed substantial changes in survival,, migration, differentiation, and neural network formation between the analyzed phenotypes. Additionally, the LMCN performed a thorough analysis of locomotor activity in mutant zebrafish using Zebrabox (Viewpoint). Special attention was paid to epilepsy-like behaviors in TSC mutant fish to clarify whether Tsc gene-deficient fish can be used to prescreen antiepileptic drugs in the future. Protocols for xenograft transplants of stem cells and in vitro cultures of fish retinal neurons were also established. An additional line of research focused on cellular functions and regulation of the Arc gene, which is dysregulated in TSC and the translation and degradation of which depend on mTOR and GSK3 kinases, respectively.
Two potential developments stem from FishMed funds that are allocated to the LMCN that can be used for further applications and medication-oriented studies. First, approaches toward reliable studies of epilepsy-related phenotypes have been established and will be used in the future for potential anti-epileptic drug testing. Second, the LMCN described for the first time a decrease in the caliber of the optic nerve in Tsc2 mutant fish (Switon et al., in preparation), a phenomenon that was recently introduced as a potential biomarker of TSC. This mutant fish phenotype can be further exploited in the search for new TSC treatments.
Jacek Kuznicki, Laboratory of Neurodegeneration
The aim of this project was to investigate possible alterations in calcium homeostasis in a pink1-/- zebrafish model of Parkinson’s disease (PD). We hypothesized that the inhibition of mitochondrial calcium influx resists mitochondrial dysfunction, leading to viable dopaminergic neurons and amelioration of PD progression. The primary approach was to alter multiple mitochondrial Ca2+ influx mechanisms in pink1-/- zebrafish and evaluate the resulting molecular and cellular changes with regard to PD pathology. To accomplish this task, we used a morpholino-based knockdown strategy to silence mcu and vdac1 (i.e. channels that are responsible for calcium influx into mitochondria).
Our results showed that alterations of Ca2+ sequestration through the knockdown of mcu but not vdac1 led to the restoration of mitochondrial respiration and thereby contributed to viable dopaminergic neurons in pink1-/- zebrafish. Subsequent gene expression studies revealed specific upregulation of the mcu regulator micu1 in pink1Y431* mutant zebrafish larvae, and inactivation of micu1 also resulted in the rescue of dopaminergic neurons. The functional consequences of PINK1 deficiency and modified MCU activity were confirmed using a dynamic in silico model of Ca2+-triggered mitochondrial activity. Live imaging using light sheet microscopy was employed to study alterations in basal calcium levels in wildtype and pink1-/- zebrafish tagged with the genetically encoded calcium indicator -GCaMP5. Data analyses were performed with Bitplane Imaris software, which is capable of 3D tracking of single regions in time lapses. pink1-/- is more sensitive to heat shock, giving a faster response in the form of a higher frequency of Ca2+ spikes in brain regions in response to a temperature rise from 27°C to 37°C. Moreover, live imaging studies revealed a 20% increase in CCCP-induced cytosolic Ca2+ efflux in area postrema neurons in pink1-/- zebrafish compared with wildtype zebrafish. Our data suggest that there are alterations in mitochondrial calcium homeostasis in pink1-/- zebrafish, and the modulation of MCU-mediated mitochondrial calcium entry may be a possible neuroprotective strategy in PINK1 mutant PD.
Marta Miaczynska, Laboratory of Cell Biology
The goal of this project was to investigate the role of endocytic proteins in signaling and transcriptional regulation in zebrafish development. As a starting point, unbiased RNAi screens in mammalian cells revealed candidate endocytic proteins that affect transcription in several signaling cascades, including the Wnt and NF-kappaB pathways. These candidate proteins have been further studied with regard to their roles in zebrafish development, in parallel with molecular studies that are performed in cultured mammalian cells.
In the first line of investigation, Tollip adaptor protein was identified as a novel negative regulator of canonical Wnt signaling in mammalian cells. We found that the depletion of Tollip potentiated the activity of the beta-catenin/TCF-dependent transcriptional reporter, whereas its overproduction inhibited reporter activity and the expression of Wnt target genes. These effects occurred independently from dynamin-mediated endocytosis but required the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracted the activation of beta-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibited the pathway after the stage of beta-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive beta-catenin activity.
Having identified the molecular mechanisms of Tollip’s action in vitro in cultured mammalian cells, we further sought to validate its role in Wnt signaling in an in vivo model (i.e. zebrafish) in collaboration with our partner, Prof. Gonzalez-Gaitan. The canonical Wnt pathway is crucial for body patterning during early vertebrate development. Therefore, we tested the effects of tollip overexpression or its morpholino-mediated depletion in zebrafish embryos. The resulting phenotypes were generally reminiscent of those of canonical Wnt signaling mutants. Specifically, morphological defects that were caused by tollip overexpression led to a reduction of the posterior body, the disappearance of somite boundaries, and partial fusion of the eyes that were manifested at 48 hpf. Analyses of the expression of mesoderm-organizing genes, such as goosecoid (gsc) and notochord (ntl), during gastrulation showed that their expression patterns were slightly expanded compared with uninjected embryos. In turn, general developmental effects (at 48 hpf) that were caused by tollip knockdown with various morpholinos included a slight reduction of the size of the posterior body and head, dorsal curvature, smaller eyes, yolk sac extension deficiency, and in some cases heart edema. To evaluate whether some of these phenotypes may be related to a potential inhibitory role of Tollip in Wnt signaling, we attempted to rescue them by reducing the levels of beta-catenin2. After beta-catenin2 knockdown with morpholinos, the embryos displayed weaker pigmentation, an abnormal structure of the head, and a curly tail. Double knockdown resulted in a return to a normal phenotype, suggesting that zebrafish tollip may be involved in Wnt signaling during embryonic development. These results were published in 2015 (Torun et al, PLoS One).
Importantly, studies on the role of tollip in zebrafish development and physiology are continuing, particularly with the ongoing generation of genome-edited zebrafish lines that carry complete knockout of the tollip gene or deletion of its CUE domain, thanks to the efforts of ZF-screens.
In the second line of investigation, components of Endosomal Sorting Complexes Required for Transport (ESCRT) complexes have been found to inhibit the NF-kappaB pathway in mammalian cells. Specifically, we depleted all subunits of the ESCRT complex that control receptor degradation and found that four components (Tsg101, Vps28, UBAP1, and CHMP4B) were essential to restrict constitutive NF-kappaB signaling. Without cytokine stimulation, the depletion of these proteins potently activated both canonical and noncanonical NF-kappaB signaling and induced the NF-kappaB transcriptional response in cultured human cells. These effects depended on cytokine receptors, such as the lymphotoxin beta receptor and tumor necrosis factor receptor (TNFR1). Upon ESCRT depletion, both receptors were concentrated on endosomes and signaled from this compartment. In the case of the lymphotoxin beta receptor, endosomal accumulation induced its oligomerization and signal transduction via TRAF2/3 proteins in the absence of ligands.
In parallel, again in collaboration with our partner Prof. Gonzalez-Gaitan, we investigated whether the effects of depletion of ESCRT subunits on NF-kappaB signaling were evolutionarily conserved. To this end, we analyzed the expression of NF-kappaB target genes in zebrafish embryos that were injected with morpholinos that targeted fish orthologues of Tsg101, Vps28, UBAP1, or CHMP4B (encoded by two genes, chmp4ba and chmp4bb). We measured the expression of fish NF-kappaB target genes, such as nfkbiaa, il1b, and nfkb2, by RT-PCR in whole embryo lysates. A significant increase in nfkb2 expression was detected in vps28 morphants, and a significant increase in il1b was detected in ubap1 and chmp4ba/chmp4bb morphants. We also found significantly higher abundance of nfkbiaa mRNA by in situ hybridization in vps28, ubap1, and chmp4ba/chmp4bb morphants. In all cases, higher nfkbiaa expression was detectable in the same tissues, with enrichment in the pronephros, arguing for the specificity of the observed phenotypes. Cumulatively, these results in fish embryos suggest that ESCRT components inhibit constitutive NF-kappaB signaling also in lower vertebrates and during embryonic development. While performing this work, we initiated an additional collaboration with an established zebrafish researcher, Dr. Maximilian Fürthauer, from the Institut de Biologie de Valrose, University of Nice – Sophia Antipolis, in France. Dr. Fürthauer and his co-worker helped perform the in situ hybridization analyses. The complete study, including the data that were obtained in mammalian cells and in zebrafish embryos, was published in 2016 (Maminska et al., Science Signaling).
Cecilia L. Winata, Laboratory of Zebrafish Developmental Genomics, Max Planck/IIMCB Research Group
Our lab’s research seeks to determine the mechanism of gene regulation during heart development through the application of next-generation sequencing (NGS) to profile the binding sites of key cardiac transcription factors (TFs) and epigenetic markers. We optimized a protocol for isolating pure populations of cardiomyocyte cells from embryos. Preliminary transcriptome profiling by NGS of these cells confirmed their correct identity and high purity. Two heart mutant lines with cardiomyocyte-specific green fluorescent protein expression have been generated through extensive crossing and genotyping. Using the NGS-based RNA-seq method, we generated transcriptome profiles of duplicate samples of cardiomyocytes from wildtype and mutants at 24, 48, and 72 hpf. These datasets are currently undergoing bioinformatics analysis and experimental validation for preparation of a manuscript. We have optimized a ChIP-seq protocol based on the conventional method using antibodies. Currently, we are collecting cardiomyocyte samples from 24, 48, and 72 hpf embryos for a ChIP-seq experiment that is planned at the end of May 2016. At the same time, we are in the process of generating transgenic lines that express fusion-tagged heart TFs using CRISPR technology as an alternative to the conventional ChIP method, which is still being optimized. We have obtained a working sgRNA and designed a construct for homology-directed repair.
A comprehensive view of genome-wide genetic and epigenetic regulatory networks that is generated from this study will provide novel and invaluable insights into heart development, which will be an important step toward a better understanding of the mechanism of congenital heart disease.
Maciej Zylicz, Department of Molecular Biology
A number of mutations in the TP53 gene are overrepresented in samples that are isolated from human tumors, suggesting that they provide a competitive advantage for tumor cells. Many of them belong to the class of “gain-of-function” (GOF) mutations, in which the original function of p53 is lost and the mutated protein acquires new functions.
During the period covered by this report, numerous cell lines have been prepared, based on established cell lines of lung and breast cancer origin, with both epithelial and mesenchymal characteristics. These cell lines are characterized by inducible silencing of the endogenous p53 protein and concurrent expression of the mutated variant of p53, carrying one of the mutations being subject to investigation within this project. Additionally, the cells stably express a red fluorescent protein that enables tracking the cells in vivo following injections into fish embryos.
The cells were tested for tightness of the repression system and its inducibility, and then their migratory and invasive phenotype was characterized in vitro using several methods. In scratch assays and experiments that employed an improved version of this assay, several mutations in p53 resulted in greater motility of the cells, with the magnitude of the increase differing with the mutation. Subsequently, the invasive capability of the cells was investigated by analyzing the invasion of (or migration through) the collagen gel or matrigel. Finally, the invasion of the gel in a 3D culture model (gel-embedded microsphere model) was investigated and quantified. These phenotypic studies confirmed that the migratory and invasive potential of the cells is modified by the presence of the GOF mutants of p53 protein.
In an attempt to determine the molecular mechanisms that are responsible for the increase in invasive potential following the induction of mutated p53, an extensive set of proteins, including many EMT markers, was analyzed by Western blot. Additionally, the expression levels of several chemokines that are involved in cancer invasion and metastasis and their receptors were analyzed by qPCR in cells that expressed the mutated p53s. A subset of the samples was also analyzed by NGS to profile the transcriptome. In summary, the data accumulated to date have not allowed definitive conclusions about the molecular mechanisms of increased invasiveness in cancer cells that express mutated p53.
Zebrafish embryos were employed in the project as a host for xenotransplants of human cancer cells that were labeled with red fluorescent protein and genetically modified with regard to p53 expression. The cells were microinjected into the common cardinal vein of 2 days post-fertilization (dpf) kdrl:EGFP or fli:EGFP embryos with green fluorescent vasculature. On the following days, the extravasation of the cells, the formation of primary metastases, and subsequent local tissue infiltration were observed in the embryos. Because of the high variance of the phenotype, a substantial average change in a large population is required to obtain statistically significant results. Such results have been obtained for some cell line/mutation combinations, and more experimentation is currently underway to be able to accept or reject the hypothesis.
Another line of research in the project is the involvement of gain-of-function p53 mutants in the regulation of angiogenesis. Solid tumors need to stimulate the growth of new blood vessels that deliver nutrients and oxygen because diffusion is insufficient when tumors exceed 2 mm in diameter. The most important cytokine that induces the proliferation and migration of endothelial cells is vascular endothelial growth factor A (VEGF-A). This protein has multiple isoforms, both proangiogenic and antiangiogenic, and their activity is determined by alternative splicing of the last exon. The studies on GOF p53 mutants are ongoing. After the initial phase of experiments in mammalian cell cultures, we now have moved to an animal model, the zebrafish embryo.
Zebrafish Core Facility
The state-of-the-art Zebrafish Core Facility (ZCF) was established in 2012. During the course of the FishMed project, the facility grew, and this growth was noted on many levels. The animal house has expanded from six 5-shelf racks (total capacity of 300 tanks, with 3.5 L volume each) to 12 racks that hold 670 tanks (3.5 L volume each). Further expansion is scheduled for July 2016. Such growth both during and after the FishMed project was possible because of funds that were secured by the ZCF and researchers. The collection of fish that are kept at IIMCB has increased from 28 fish that represent nine lines in 2012 to over 10,000 animals that represent nearly 100 lines that are being kept in 2016. Notably, new mutants have been created by FishMed participants, and they are being used and will be used to develop new models that are available to IIMCB and the general scientific community.
FishMed funds allowed the purchase of specialized equipment (e.g. systems for micromanipulations, behavioral analysis, and imaging) that meet most of the needs of internal and external users. At the beginning of the FishMed project, only eight experienced postdoctoral researchers and three research technicians from IIMCB benefited from the use of the ZCF. Soon thereafter, external users joined. The numbers of both internal and external users have continuously increased. By the end of 2015, the facility was used by over 60 researchers.
FishMed funds also allowed visits and participation in courses/workshops and other meetings that resulted in mastering skills and obtaining new knowledge and contracts. This allowed the development of additional services that are now being provided by the ZCF, including a health screening program, sperm banking, assistance in planning and performing behavioral analysis, and introduction to and advanced courses on the zebrafish model (e.g. husbandry, health, and various techniques used in research projects).
The ZCF staff has been active in promoting the zebrafish model by lecturing on a number of courses for students and established researchers who work with animal models. Thanks to this activity, increasingly more researchers and teachers at various locations in Poland have decided to work with the zebrafish model.
Technology transfer and innovations
In response to the growing innovative potential of IIMCB, the Bio&Technology Innovations Platform (BioTech-IP) was created. It identifies, protects, and commercializes inventions that have market potential. In this scope, BioTech-IP cooperates with technology transfer experts. They assisted in the creation of an IIMCB-owned company (also called a special purpose vehicle [SPV]) that is dedicated to supporting the Institute in technology transfer and commercialization. This SPV company, BioTech-IP Sp. Ltd, is designed to acquire and manage the shares of start-up companies that are established for the commercial exploitation of intellectual property. In the course of the project, BioTech-IP worked on 11 patents and patent applications. The main results are the following:
• Organization of a total of 11 science-business networking brunches attended by 250 participants, during which 40 projects with strong applied potential were presented.
• Providing professional training to a total of approximately 1,250 PhD students and scientists on soft skills, project management, patenting, IPR management, and commercialization strategies.
• Creation of IIMCB-owned company (also called an SPV) that is dedicated to supporting IIMCB in technology transfer and commercialization. An amendment to the Polish Higher Education Act obliges universities and research institutes to establish an independent company (third-party) to support them in efficient technology transfer to industry. According to the Act, SPVs are designed to acquire and manage the shares of start-up companies that are established for the commercial exploitation of intellectual property that comes from a given university or scientific institution. Despite imprecise documents concerning implementation of the Act and legal uncertainty about the formal procedures of SPV creation, IIMCB successfully obtained the required permissions from the Ministry of Science and Higher Education and from the Head of the Polish Academy of Sciences and registered the company, called BioTech-IP Sp. Ltd. This was one of the first SPVs that was created in a scientific institution in Warsaw, with a duly completed business plan that consisted of market and customer analyses, an organizational structure, proposed products, a services portfolio, and a financial forecast.
• Purchasing access to GlobalData business database. Offering technologies to potential investors or industrial partners prompted the need to acquire professional feasibility studies, which are based on more comprehensive information than can be found in internet search engines. Market search revealed that the products that are offered by ThomsonReuters and GlobalData could be helpful in assessing the commercial potential of inventions from the biotechnology, medicine, or medical device fields. However, comparison surveys indicated that the GlobalData-Healthcare product that comprises two units (Pharma eTrack for drugs and Medical for medical devices and diagnostics) fits the TTO profile much better.
• Organization of meeting between scientists and representatives of GlaxoSmithKline (GSK) to coordinate the program called Discovery Partnerships with Academia (DPAc; http://dpac.gsk.com/). The DPAc team was looking for partners in early discovery projects where there is a clear therapeutic hypothesis and an understanding of the target. For suitable projects, GSK offered grant money to be paid when milestones are reached and financial rewards that are shared through royalty payments. During the meeting that was organized by BioTech-IP at IIMCB in November 2014, 14 projects were presented to GSK, from which one was invited to join GSK’s Discovery Fast Track Challenge competition.
• Cooperation with Over Group, an industrial partner that is interested in the commercial application of lytic enzyme technology. The enzyme that was patented by IIMCB has the ability to kill antibiotic-resistant Golden Staph, which is considered one of the most dangerous bacteria in the world. The possible commercial exploitation of the enzyme covered the disinfection of human skin, wounds, and surfaces (in hospitals) and animal protection against the aforementioned bacteria. Feasibility studies indicated that introduction of the enzyme into cattle breeding should be relatively cost effective for the company and profitable. Technology was presented to the following companies: Siveele (siveele.com) Hypred (hypred.com) Ecolab (ecolab.com) BioWet Drwalew S.A. (biowet-drwalew.pl) and Over Group. After negotiations with Over Group, the company introduced the IIMCB technology into their R&D program, with the aim of developing innovative cow udder hygiene products to protect animals against mastitis.
• BioTech-IP also concentrated its efforts on supporting the creation of the first technology-based start-up company, aiming to commercialize inventions concerning restriction enzymes that are capable of the sequence-specific cutting of RNA strands. BioTech-IP supports scientists and IIMCB with regard to business, taxes, and legal issues to make the creation of companies feasible. Moreover, BioTech-IP presented this opportunity to three investment funds: WinQbator, IP-Hub, and Impera Alfa. Although Winqbator pushed to sign the deal, it had to withdraw from negotiations because of internal problems. IIMCB technology and the idea of a start-up company have been presented to two other investment funds: Impera Alfa and IP-hub, which expressed interest. Negotiations with both investors are ongoing. In parallel, BioTech-IP identified a project manager who is experienced in the Polish market and will join the scientific team and supervise the start-up business operations.
• Thanks to BioTech-IP’s support in securing funds for patent protection, it was possible to seek protection for three patents that were granted in Europe and other parts of the world:
- A method of peptide hydrolysis, peptidase, the composition for use as a bacteriostatic and bactericidal agent, a kit and the uses of the active form of LytM from S. aureus or derivatives thereof (EP 2 699 254 B1)
- Sequence-specific engineered ribonuclease H and the method for determining the sequence preference of DNA-RNA hybrid binding proteins (EP 2 718 430)
- dsRNA endoribonucleases (EP 2 718 431)
Potential Impact:
IIMCB’s strategic objective was to achieve the quality of research and innovative activities of leading research entities in the world. To achieve this level of excellence and increase our innovative potential, we successfully established a new research model at IIMCB: zebrafish (Danio rerio). This fish is an excellent organism to study various aspects of disease because it is fully transparent until adulthood. Therefore, development, pathologies, and the effects of treatments can be easily monitored in vivo. Moreover, potential drugs can be added to water to easily test their effects on behavior, anatomy, development, and signaling in high-throughput screens. Using zebrafish models of human diseases, we can study them not only at the molecular and cellular levels but also at the level of live vertebrate organisms, the metabolism of which is very similar to that of mammals.
General impact, including the socioeconomic impact and wider societal implications
Society-oriented research
Research projects that are developed under FishMed focus on human diseases that cause persistent problems in society, such as Parkinson’s disease, neurodegenerative disorders, and metabolic diseases. During the course of these projects, mainly basic biological processes that lead to the development of diseases were studied, but the availability of zebrafish as a research model opened a range of opportunities for collaborations with hospitals and clinics. One of the best examples is the research project, “Identification of genes controlling brain development through genomic analysis of patients with microcephaly,” coordinated by the Institute of Mother and Child (Warsaw, Poland) and involving the Baylor College of Medicine (Houston, Texas, USA) and Laboratory of Zebrafish Developmental Genomics (IIMCB).
Awareness of research on animals
Through the creation of discussion forums for the Polish scientific community on the usage of zebrafish models for studies of human diseases as an alternative to mammalian models, FishMed contributed to greater awareness of research on animals. IIMCB has successfully introduced the concept of zebrafish research to the local scientific community and became a local source of information on zebrafish as a research model. As a result, new scientific collaborations with researchers based in Warsaw and other Polish research centers have been established, including the Nencki Institute of Experimental Biology, Warsaw, Medical University of Warsaw, University of Warsaw, Warsaw University of Life Sciences, University of Warmia and Mazury, Olsztyn, Institute of High Pressure Physics, Warsaw, and Poznan University of Medical Sciences.
Ethical issues
All of the research activities at IIMCB are carried out in compliance with fundamental ethical principles and relevant national and international legislation. Following the recent change in national regulations, IIMCB has:
• appointed an Animal Welfare Advisory Team who supervises the wellbeing of animals that are used for scientific or educational purposes
• provided a full-time employment position for a veterinary physician
• obliged all relevant personnel to become familiar with relevant legislation and participate in compulsory training that is imposed by this legislation
Being aware of the importance of ethical issues, IIMCB organized in cooperation with the PolLASA Association “Combined training for persons responsible for planning and conducting procedures and experiments that kill laboratory animals and supplementary training for laboratory animal caretakers.” The training was particularly directed to foreigners as all lectures were translated into English. All participants received required by law certificates entitling to work with laboratory animals. As a rule, all researchers who work with zebrafish undergo relevant training and receive certification that is required by law. Whenever necessary, researchers apply for approval from the Local Ethics Committee for Animal Experiments to use zebrafish for scientific or educational purposes.
Science education
FishMed has had a great impact on society through promotional and educational activities that are focused on the wider public and especially the younger generation who, inspired by our activities, may pursue scientific careers in academia, biotechnology, and the pharmaceutical industry. We involved students and school children through workshops, science festivals, art competitions, info/open days, book fairs, and job fairs. The project generated numerous science education materials, such as a book, a movie, bookmarks, stencils, crossword puzzles, and educational boards. Thus, the project generated output that can be used by policymakers in the field of education, training and youth.
Engaging with civil society and policymakers
We cooperated with the Polish association Supporting People with Inflammatory Bowel Disease “J-elita.” We also approached local authorities, such as the Marshal and Office of the Mazowieckie Voivodeship, Regional Council of the Mazowieckie Voivodeship, and city of Warsaw. They were invited to participate in all major events, such as the kick-off conference, Heart of Europe Zebrafish Meeting, and International FishMed Conference on Zebrafish Research. The representatives of local authorities and funding agencies participated in discussions on the “Evaluation report with guidelines for further development” that was prepared by experts who were appointed by the European Commission. They appreciated the success of FishMed and declared support in further IIMCB development. The city of Warsaw is going to promote IIMCB as an excellent research center and will get involved in organizing international conferences that are planned after the project is completed.
Contribution to the economic and social development of the region
Because of the enhanced research capacity of the FishMed project (i.e. creation of ZCF, employment of experienced researchers, technicians, and managers, and the purchase of state-of-the-art equipment), IIMCB has contributed to the economic and social development of the city of Warsaw and Mazovia province. The successful introduction of a new animal model brought new knowledge and expertise and enhanced the research capacity of the region. Acting as a zebrafish reference center, we share our experience and best practices with others who are interested in adopting this model locally and regionally. Most of the researchers who are employed under FishMed have successfully applied for funding to continue their research at IIMCB. Moreover, by doing so, they created new jobs for researchers, technicians, and support personnel.
Media and communication to general public
To communicate information about FishMed to the general public, IIMCB’s professional public relations office used the following tools: press releases, media briefings, TV coverage, radio coverage, brochures, posters, flyers, films, coverage in specialist/general press, coverage in the national/international press, website, and events that target the general public. To illustrate the results of these activities, we measured the number of IIMCB webpage visits starting from 1 year before FishMed was initiated and the number of appearances of information about IIMCB in the media during the same time period. The number of IIMCB webpage visits increased from 95,910 in 2012 to 114,070 in 2015. The number of appearances of information about IIMCB in the media increased from 3 in 2012 to 34 in 2015.
Innovations and technology transfer
Through BioTech-IP, IIMCB’s Technology Transfer Office, around 1,500 PhD students and scientists at the Biocentre Ochota campus obtained access to professional information that is tailored to their specific needs with regard to commercializing the R&D results of their work. Some of them used these skills in their careers and started business activities. BioTech-IP also helped initiate contacts between scientists and business partners, which led to joint projects and collaborations. Eventually, BioTech-IP developed unique skills to deal with commercialization processes at different stages, which made it possible to create BioTech-IP Innovation, a company that was established to acquire and manage the shares of spin-off companies based on IIMCB’s intellectual property. FishMed successes in innovation and technology transfer will have a positive impact on the economy and regional development in the long-term. IIMCB already has human resources and all of the necessary tools to identify potential targets and support researchers in their commercialization.
Exploitation of results
Bujnicki lab
Description of exploitable foreground: Computer software
We developed a computational method for modeling the 3D structures of RNA molecules and RNA-RNA complexes, called SimRNA. It can computationally simulate the folding of RNA structures if provided with RNA sequence information. It can be used to study the influence of mutations in RNA on the 3D structural stability of RNA. The program is available for free for academic users at http://genesilico.pl/SimRNA/ and may be commercially licensed for industry. The market potential for this type of product is currently unclear. Based on discussions with potential collaborators from industry, a new version of SimRNA that can model RNA-ligand interactions (to be possibly developed in the future) could be useful for commercial applications. The current version is applicable mostly for academic research.
Chacinska lab
Description of exploitable foreground: Novel model and optimized protocol
Mitochondria constitute life-essential organelles with multiple cellular functions. They serve as energy and biosynthetic centers, participate in cell stress responses, and are involved in cell death execution. Failure in mitochondrial function is associated with numerous human pathologies. With no cure, the severity of symptoms and disease progression can often lead to death before adolescence. The fact that the treatment of mitochondrial disorders is mostly directed at mitigating the symptoms reflects our relatively poor understanding of the biology of mitochondrial disorders. Our successful collaboration with the laboratory of Prof. Didier Stainier at the Max Planck Institute for Heart and Lung Disease in Bad Nauheim yielded a novel model with mutations in one of the key proteins that is responsible for mitochondrial biogenesis. We aim to use this zebrafish mutant to identify and understand specific processes in the context of a multicellular organism that are initiated by mitochondrial deficiencies and lead to pathology and eventually death. We also plan to discover putative compensatory mechanisms and defense responses and their role in pathologies that are caused by mitochondrial dysfunction. Our project has great potential to provide novel insights into mitochondrion-related pathologies.
Our optimized protocol for protein extraction in conjunction with the selection of antibodies that specifically detect mitochondrial proteins initiated collaborative research with the laboratory of Prof. Oliver Bandmann in Sheffield, United Kingdom, on the analysis of mitochondrial proteins in zebrafish models of neurodegenerative diseases.
Jaworski lab
Description of exploitable foreground: Analysis of locomotor/epileptic-like activity of TSC mutant zebrafish and analysis of optic nerve caliber of TSC mutant zebrafish.
Understanding mTOR activity-related diseases is of great importance to society. Changes in mTOR activity likely contribute to such neuropathologies as brain tumors, epilepsy, autism spectrum disorders, schizophrenia, addiction, and several neurodegenerative disorders. However, to date, animal models are restricted to either fruit flies or mice and therefore not convenient for large compound/genetic screens with the use of vertebrates. New observations within FishMed established that fish that lack Tsc may be a new model for such screens. Both epileptic-like behaviors and a decreased caliber of the optic nerve in TSC model fish can be exploited by both researchers and collaborating companies to search for new compounds or signaling pathways to be targeted in TSC and epilepsy.
Kuznicki lab
Description of exploitable foreground: Novel research direction
Parkinson's disease (PD) is a neurodegenerative disorder that affects the elderly and remains incurable. Mitochondrial dysfunction has been postulated as a possible etiological factor behind PD. However, the relationship between mitochondrial dysfunction (mitochondrial calcium overload) and dopaminergic cell loss is still unclear. The study aims to reveal the factors that are involved in calcium overload in mitochondria and gain insights into the association between mitochondrial calcium overload and dopaminergic neuron loss in PD.
Miaczynska lab
Description of exploitable foreground: Novel research direction
Based on FishMed results, a new research direction has been developed: studies on the molecular mechanisms that are related to processes in which Tollip protein may be involved at the level of the organism (zebrafish). Studies will concentrate on, among others, the potential contribution of Tollip to the clearance of protein aggregates in a model of Gaucher's disease.
Winata lab
Description of exploitable foreground: Computer software
We developed a computational tool for RNA editing discovery based on NGS called REDiscover. This software is fast (DNAseq & RNAseq processed simultaneously, multithreading), flexible (non-standard experimental designs, multiple inputs [BAM]), reliable, and simple. REDiscover can be used to study RNA editing detection during development. The program is available for free at https://github.com/lpryszcz/REDiscover#rediscover.
Zylicz lab
Description of exploitable foreground: Validation of the zebrafish xenotransplantation model for use in research concerning p53 mutation-induced tumor invasiveness.
This technique has been used by Leiden University, The Netherlands. A transgenic zebrafish line has been prepared that carries a deletion in the tp53 gene. The fish have been prepared for a research project that investigates the mechanisms of oncogenic activity of gain-of-function mutations in p53. Such research requires a p53 null background. Planned activities include the creation of a zebrafish model of one of the human metabolic diseases that has neurological manifestations.
Main dissemination activities
During the course of the project, IIMCB established contacts with non-scientific communities, such as patient organizations, local authorities, and the general public. We have organized special events for selected groups from outside academia and developed an information campaign that is directed at the general public, through which we informed society about the advancements and successes of FishMed.
a. Patient organizations (e.g. workshop for children during the Days of Education organized by the Polish association Supporting People with Inflammatory Bowel Disease “J-elita”
b. Local authorities (e.g. visit by the Marshal of the Mazowieckie Voivodeship to IIMCB and ZCF; participation of representatives of local authorities in the meeting with evaluators appointed by the European Commission and representatives of the Regional Council of the Mazowieckie Voivodeship, city of Warsaw, Office of the Marshal of the Mazowieckie Voivodeship)
c. General public (e.g. Be Healthy as a Fish campaign that comprises a book, a movie, and workshops). A total of 636 primary school children participated in 32 workshops. Around 2300 people received the book, and nearly 1700 people watched the movie. Info days were provided for talented youth, supported by the Polish Children’s Fund, and for students from the Warsaw University of Life Sciences and University of Agriculture in Kraków.
Creation of a discussion forum for the Polish scientific community on the use of zebrafish models for studies of human diseases as an alternative research organism to mammalian models:
a. Organization of 25 open seminars delivered by specialists in zebrafish research
b. Organization of 3 FishMed open report sessions
c. Organization of 2 international conferences devoted to zebrafish in biomedical research: Heart of Europe Zebrafish Meeting and International FishMed Conference on Zebrafish Research
Along with the visibility platform actions, the FishMed results were disseminated to the scientific community via open access publications in internationally recognized journals and lectures and posters that were presented at various scientific events worldwide.
Measures were taken to increase the likelihood of the market uptake of project results, such as identifying and collaborating with potential users and identifying potential partners and sources of financing for commercialization. These activities were undertaken by BioTech-IP, IIMCB’s Technology Transfer Office. In the course of the project, BioTech-IP supported scientists in the initiation of contacts between scientists and business partners, which led to joint projects and collaborations. BioTech-IP also developed unique skills to deal with commercialization processes at different stages, which made it possible to create BioTech-IP Innovation, a company that was established to acquire and manage the shares of spin-off companies based on IIMCB’s intellectual property.
All of these actions have increased the scientific recognition of IIMCB as a research center using zebrafish as a research model and opened new possibilities for inspiring scientific partnerships and collaborations as well as the commercialization of project results in the future.
Main dissemination activities planned after the project is completed
• Publication of at least 11 peer-reviewed articles
• Lectures and posters at international events
• Organization of scientific events related to zebrafish research
• New research projects and grants in collaboration with FishMed partners
• Continuation of the Be Healthy as a Fish campaign
List of Websites:
Website address:
fishmed.iimcb.gov.pl
Contact details:
Dr Urszula Bialek-Wyrzykowska
International Institute of Molecular and Cell Biology
4 Ks. Trojdena St.
02-109 Warsaw, Poland
Tel. +48 22 59 70 769
Fax +48 22 59 70 715
ulawyrzykowska@iimcb.gov.pl