Report of the 4th European Zebrafish Principal Investigator Meeting

Introduction

The European Zebrafish Principal Investigator Meeting (Fig.1) started in the afternoon of March 15 with an Opening Keynote Lecture given by Marcos Gonzalez-Gaitan (University of Geneva, CH), where he discussed a novel mechanism that allows signalling endosomes to be targeted to one of the daughter cells during asymmetric division. Marcos showed that the asymmetry is already established in the central spindle by the kinesin Klp10A and its antagonist Patronin, which then polarize the direction of endosome motility.

OPEN IN VIEWER

FIG. 1.

The logo of the 4th European Zebrafish Principal Investigator Meeting.

The Scientific Committee composed by Claudia Linker (KCL, United Kingdom), Corinne Houart (KCL, United Kingdom), Carl-Philipp Heisenberg (IST, AU), Laure Bally-Cuif (Institute of Neurobiology Alfred Fessard, FR), Miguel Godinho-Ferreira (IGC, PT), Nadia Mercader (Universität Bern, CH), Robert Kelsh (University of Bath, United Kingdom), Caroline Brennan (Queen Mary University of London, United Kingdom), and Claire Wyart (ICM, FR) selected 63 abstracts for oral presentations. These abstracts were presented in the seven different sessions that composed the meeting, namely Development and Organogenesis (chaired by Elke Ober, DanStem, DK), Disease Models (chaired by Betina Schmid, DZNE, DE), Cell biology and Cell migration (chaired by Lila Solnica-Krezel, Washington University School of Medicine, United States), Stem cells and Regeneration (chaired by Catherina Becker, University of Edinburgh, United Kingdom), Neurosciences (chaired by Michael Brand, TU Dresden, DE), Emerging technologies (chaired by Andy Oates, Crick Institute, United Kingdom), and Behavior (chaired by Ruben Portugues, Max Planck Institute for Neurobiology, DE). The remaining 45 submitted abstracts were invited for poster presentation and were discussed in two different poster sessions that took place after dinner.

In the morning of March 19, Giles Laurent (Max Planck Institute for Brain Research, DE) ended the meeting with a Closing Keynote Lecture, where he discussed the evolution of sleep within vertebrates. There is this accepted idea that all animals sleep and yet the brain's electrophysiological features of sleep (i.e., slow-wave [SW] and rapid eye movement [REM] activities) have only been characterized in birds and mammals. From the recordings obtained in the brain of a lizard, the small Australian dragon Pogona vitticeps, it is clear that SW and REM sleep patterns exist in these reptiles, thus allowing the conclusion that these sleep dynamics evolved much earlier in vertebrate evolution, at least with the emergence of amniotes.

Development and Organogenesis

The first session opened with Didier Stainier (Germany) talking about the role of central nervous system (CNS) progenitors in the regulation of vascular patterning outside the CNS.

Corinne Houart (United Kingdom) followed with the question on how FoxG1 regulates the shift between proliferation and neurogenesis in the telencephalon. She investigated the possible functional interaction between this transcription factor and ASPM, a protein causing microcephaly when mutated in humans. Zebrafish mutants engineered with TALENs and CRISPR-Cas9 technology show smaller telencephala. These mutants also showed ectopic wnt8b expression and premature neurogenesis reminiscent of the foxg1 loss-of-function phenotype. Nevertheless, FoxG1 overexpression did not rescue aspm mutants and therefore the link between these two genes is being investigated.

Still in the head region, Cristina Pujades (Spain) wanted to understand how are rhombomere boundaries maintained so sharply during development despite cells being moving and dividing? Her laboratory is testing the idea that perhaps these boundaries have some elastic properties conferred by actomyosin cables. Their genomic approach raised the evo–devo hypothesis that fast developers such as zebrafish and Astyanax may need actomyosin cables, whereas slower developers such as medaka may not need them for rhombomere boundary formation.

Then, we heard Daniela Panáková (Germany) who told us about the role of actomyosin tension in heart chamber formation. Her group is investigating the role of the Wnt/PCP pathway in actomyosin organization and its impact on mechanosensation by the cardiomyocytes.

Next, Jiandong Liu (United States) continued the subject of heart chamber formation with the role of cardiac contraction. Primary cilia were highlighted as mediators of fluid flow to stimulate Notch expression. As trabeculation needs cardiac contraction and in stopped hearts mutants, there is no Notch signaling, and it is thought that contraction promotes trabeculation by notch1b/efnb2a epistasis.

We then moved on to the eye field and heard Florencia Cavodeassi (Spain) who is investigating the nasotemporal patterning of the optic vesicle. Two transcription factors, Foxg1 and Foxd1, by exerting mutual repression, are important for dorsal and ventral fates, respectively. She showed that the dorsal FGF signaling promotes foxg1 expression, whereas ventral Hh signaling promotes foxd1 expression. However, the role of Shh in promoting foxd1 expression is only required in the presence of FGF activity.

Mary Mullins (United States) then talked about adult phenomics in zebrafish. By performing reverse genetics, they carried out a broad-based phenotypic analysis of late larval and adult stages. They already analyzed 600 genes and have confirmed numerous late phenotypes. They aim to report back on novel mutants with maternal effects, male sterility, juvenile lethality, and adult abnormal morphology.

Cecilia Winata (Poland) talked about maternal mRNAs that are translationally regulated by progressive cytoplasmic polyadenylation until the mid-blastula transition (MBT) stage. She showed that this process is crucial for surviving the MBT.

Nadine Vastenhouw (Germany) continued with the same subject and told us that the competition between histones and transcription factors regulates the onset of transcription around MBT. They experimentally showed that an excess of the four core histones in the embryo before MBT represses transcription by competing with transcription factors for binding to DNA.

The last speaker in this session was Roland Dosch (Germany) with the zebrafish germ plasm organizer bucky ball and its functional conservation with Drosophila oskar. The Dosch laboratory showed the functional conservation between these germ plasm organizers despite their lack of sequence homology. These two functionally similar proteins in distant species are characterized for having a fast evolving and intrinsically disordered amino acid sequence.

Disease Models

The second session was the longest session and presented a broad range of disease-related zebrafish models.

DNA replication proteins are not degraded once the cell has entered mitosis and even persist in postmitotic cells, suggesting that they might exert additional functions. Melanie Phillipp (Germany) told us what she thinks replication proteins do when cells do not divide.

Telomeres shorten with age and they are considered molecular timekeepers, which determine cellular life spans. Telomerase is known to be active in stem cells and cancer cells. However, it is currently unclear, if there is also a correlation between telomere length shortening and disease. Miguel Godinho Ferreira's group (Portugal) observed that tert mutant zebrafish, lacking functional telomerase, show premature aging and also display an earlier onset of cancer development compared to wild type. Strikingly, they demonstrated that tert mutant cells exert a dominant damaging effect on wild-type cells in transplantation experiments, strongly suggesting that tissue-specific telomere length is not only a limiting local but also likely a systemic factor for age-related tissue degeneration and disease.

The notochord is a scaffold for growing vertebral bones. Using live imaging and genetic manipulations, Michel Bagnat's group (United States) showed that the vacuoles in notochord cells are necessary to evenly distribute compressive forces of growing vertebrae. Fragmented vacuoles as found in spaetzle mutants lead to kinks of the axis as bones grow. They determined the spaetzle mutation to be in a gene encoding kinase. Most likely, loss of phosphorylation of a vacuole membrane trafficking regulator is responsible for vacuole fragmentation in spaetzle. His study offers mechanistic insight in the etiology of scoliosis.

Yury Miller (United States) developed a zebrafish model for hypertriglyceridemia, which is a risk factor for cardiovascular disease. Zebrafish apolipoprotein C2 (apoc2) mutants show chylomicronemia, severe hypertriglyceridemia, and lipid-laden macrophages, resembling early events in atherosclerosis development in humans. His model promises to be a good tool to investigate the pathogenesis of hypertriglyceridemia-associated diseases.

Thomas Dickmeis (Germany) examined how glucocorticoids regulate diurnal transcription and metabolism. Using a zebrafish glucocorticoid deficiency model, he detected several deregulated metabolic pathways. Interestingly, nonrhythmic dexamethasone treatment could restore most of the diurnal transcriptome. This interesting finding could be explained by the presence of a combination of E-box and glucocorticoid response elements enriched in rescued genes, indicating that glucocorticoids act as permissive cues for the diurnal rhythm.

Dravet syndrome (DS) is a severe epilepsy syndrome leading to 70% seizure freedom in DS children. Peter de Witte (Belgium) made use of the zebrafish scn1Lab mutant, which recapitulates DS, to find new antiepileptic drugs for future, specific DS therapeutics.

Anna-Pavlina Haramis (Netherlands) addressed the role of the tumor supressor LKB1 in autophagy. lkb1 mutants have impaired autophagy, which results in a shortened life span when larvae are challenged with yolk depletion. Both lack of MPK activation and deregulation of PI3K signaling contribute to this autophagy defect. This observation suggests that autophagy inhibition might have therapeutic benefits in LKB1-mutated cancers.

Humans with mutations in the essential myosin light chain (ELC) show a high incidence of sudden cardiac death. David Hassel (Germany) used the zebrafish lazy susan mutant, which lacks the highly conserved ELC phosphorylation site S195, to investigate the role of ELC in cardiomyopathy. Heterozygous adult lazy susan mutants, although unremarkable under normal conditions, die of sudden heart death after being subjected to a physically demanding swimming test. ELC phosphorylation appeared to be essential to adapt heart function to physical stress. Motility analysis in vitro revealed a possible underlying mechanism such as ELC phosphorylation that critically modulates the binding of myosin to actin.

Robert Wheeler (United States) presented a zebrafish swim bladder injection model to study the mechanism of dissemination of Candida albicans. He observed that hyphae and yeast forms are both necessary for dissemination. Hyphae can penetrate the epithelial layer of the swim bladder and yeast is good at dispersal. Neutrophils and macrophages are recruited to the site of infection. C. albicans can survive inside of phagocytes, and active transport in macrophages appears to be one way for C. albicans to get into the bloodstream.

Astrid van der Sar (Netherlands) addressed how mycobacteria can cross the blood–brain barrier in a zebrafish model of bacterial meningitis.

Yi Feng (United Kingdom) previously showed that the innate immune cells provide trophic factors to preneoplastic cells (PNCs) at early tumor-initiating stages. Using a reporter line, she now shows that NFκB signaling in PNCs is involved in neutrophil recruitment toward PNCs. NFκB activating factors remain to be identified in future studies.

Human–zebrafish xenotransplantation models promise to be an alternative to mouse xenograft models for personalized medicine. In this study, Rita Fior (Portugal) showed that inter- and intratumor heterogeneity of human colorectal cancer cells can be detected in zebrafish xenograft models. Most importantly, different responses were found on treatment with two common chemotherapies, FOLFOX and FOLFIRI. This indicates that indeed such zebrafish xenograft models can be used to instruct clinical therapy decisions.

Marina Mione (Germany) developed a zebrafish model that shows both benign lesions and invasive brain tumors, which appear similar to mesenchymal glioblastoma. She used this model to investigate which factors determine benign versus malignant tumor formation and identified a 8-gene YAP signaling signature defining malignant glioblastoma not only in zebrafish but also in human patient samples. An active form of YAP was able to induce aggressive tumors by its own in zebrafish, suggesting that YAP activation is a key factor in malignant tumor formation.

Cell Migration and Cell Biology

A variety of topics were presented from tissue repair and polarity to molecular and cellular interactions controlling cell movement and the generation of forces in tissues. Most of the talks in this session highlighted the importance of high-resolution in vivo imaging coupled to quantification methods of cell movement and shape to better understand dynamic processes.

The powerful combination of mathematical modeling and transplant experiments allowed Carl-Philip Heisenberg (Austria) to put in evidence the role of different tissue layers in the generation of forces driving the initiation of epiboly.

How retinal lamination is achieved was explored by Caren Norden (Germany). She showed that the efficient retinal ganglion translocation depends on basal process attachment and an intact microtubule cytoskeleton. However, cells can fall back in a secondary mode of migration making retinal ganglion cell (RGC) translocation a very robust phenomenon as it sets the stage for all later lamination phenomena.

The formation of secondary islets in the pancreas was shown to be a very active process involving single-cell migration, cell recognition, and clustering through highly dynamic filopodia extensions; the PI3K activity was shown to be fundamental in this process (Robin Kimmel, Austria).

The stereotypic choreography of endothelial cell behaviors and the molecular mechanisms of dynamic cell shape changes taking place in the establishment of new blood vessel connections was explored in vivo by Heinz-Georg Belting (Switzerland). Quantitative in vivo imaging and single-cell ablation during neural crest migration in the cranial and trunk region unveiled different cellular mechanisms underlying collective cell migration as shown by Claudia Linker (United Kingdom).

Maximilian Fürthauer (France) presented data establishing a new functional link between Notch signaling and cell polarity in the zebrafish neural tube.

Moreover, the Notch signaling pathway is regulated by Plasmolipin through the endocytic machinery during gut morphogenesis concomitantly controlling cell polarity and the fate of lysosome-rich enterocytes (Fernando Martin-Belmonte, Spain).

The importance of cellular interactions for tissue repair was highlighted by direct observation of macrophage activity at the site of muscle fiber tearing (Uwe Strähle, Germany). Macrophages recognize and clear lipid and protein accumulation at the site of damage, allowing membrane repair. He concluded that membrane repair involves both cell intrinsic and macrophage/cell interactions.

Stem Cells and Regeneration

In the first part of the session, the speakers did a great job in demonstrating how zebrafish can be used as a model for regeneration. The zebrafish possess an incredible capacity to regenerate tissue upon injury, including the heart, liver, fins, brain, and spinal cord.

Michell Reimer (Germany) showed that oligodendrocyte precursors could differentiate into mature oligodendrocytes after spinal cord injury in adults. Axonal, neuronal, and oligodendroglial regeneration leads to functional recovery. Similar traits were shown by Lieve Moons (Belgium) in a zebrafish model of optic nerve crush, where retina ganglion cells (RGCs) completely regenerate the optic nerve within 20 days postinjury. Interestingly, this regeneration potential was seen to decrease with age, in part, due to an attenuation of the intrinsic growth capacity of RGCs and to a senescent cellular environment.

Dimitris Beis (Greece) used an experimental setup with the nitroreductase enzyme coupled with a Gal4/UAS system to show that both embryonic and adult cardiac valves regenerate. This regenerative capacity was mediated by Notch signaling. Jeroen den Hertog (Netherlands) showed some ongoing work on how Ptena/b and Shp2a/b is required for fin regeneration, but not for normal fin outgrowth.

The remaining talks focused on tissue stem cells. Laure Bally-Cuif (France) showed by lineage tracing that neural stem cells (NSCs) build up the adult pallium (dorsal telencephalon) via a pile-up process. The neural cells arising earlier do not mix with neural cells that arise later, and so, they end up in different strata. Some of the NSCs behind this growth process are continuously neurogenic throughout life and present typical characteristics of stem cells as follows: they are quiescent, self-renew, and are multipotent. Robert Kelsh (United Kingdom) identified mutations in a gene expressed in the endothelium that induces neural crest-derived progenitor cells to differentiate prematurely into pigment cells and postulated that an endothelial-secreted factor maintains pigment cell stem cells in an undifferentiated state.

Hematopoietic stem cells (HSCs) are responsible for making blood throughout an organism's life. Ana Cvejic (United Kingdom) applied single-cell sequencing to kidney marrow CD41-positive population and showed a very elegant analysis of the expression data that allowed her to computationally define a hierarchy of progressively differentiated stem and progenitor cell types arising from the HSCs. How these stem cells are made in the embryo was addressed by Rui Monteiro (United Kingdom). He showed that the embryonic precursors of HSCs, the hemogenic endothelium, are specified via a crosstalk mechanism between Notch and autocrine and paracrine TGFβ signaling. A second paracrine input was also required later for the formation of HSCs from the hemogenic endothelium.

Continuing on the subject of extracellular signaling, Steffen Scholpp (Germany) presented a mode of propagation of Wnt signaling via cytonemes extending from the cell of origin to the signal-receiving cells. A kinome-wide screen led to the discovery that the Wnt/PCP pathway regulates Wnt cytoneme formation and thus paracrine Wnt/beta-catenin signaling. Finally, Brant Weinstein (United States) presented his most recent work on whole-genome methylation analysis to investigate how DNA methylation regulates gene expression in HSCs. They found that loss of methylation in the cmyb gene promoter led to the progressive loss of cmyb expression in HSCs and thus to failure to differentiate into more restricted hematopoietic lineages such as macrophages, erythrocytes, and T cells.

Neurosciences

The fifth session spanned a range of topics in zebrafish neuroscience, including nervous system development, circuit function, and behavior, as well as exciting new technologies (and even model organisms).

Filippo del Bene (France) started the session by presenting some of his work on development of the retinotectal projection: he showed that Reelin, a large extracellular matrix protein, acts cell nonautonomously to direct correct lamination of retinal axonal arbors in the optic tectum. Tim Czopka (Germany) presented tools and approaches to study myelination of spinal cord axons during larval development as well as during repair following injury.

Three talks followed looking at neural network physiology. First, Emre Yaksi (Norway) presented his work examining population activity in the habenula and its correlation with distinct neuronal clusters in the pallium and olfactory bulb. Then, Tom Baden (Germany) discussed approaches to examine retinal physiology, including the properties of bipolar and ganglion cells, which he has successfully applied in the mouse and plans to translate to zebrafish. Third, Konstantinos Ampatzis (Sweden) discussed his findings using an adult zebrafish spinal cord preparation, which support the progressive recruitment of three populations of motoneurons by corresponding groups of V2a interneurons, as swim speed increases.

Bringing a slight change of direction, Benjamin Judkewitz (Germany) argued the case for studying brain function in Danionella, a tiny cyprinid closed related to Danio. Stephan Neuhauss (Switzerland) opted not to review the Coen Brother's back catalogue but instead showed diverse roles for glutamate transporters at cone-bipolar and cone-horizontal cell synapses.

An exciting technological highlight of the session was presented by Drew Robson and Jennifer Li (United States) who have developed a microscope combining motion cancellation and fast optical sectioning to image neural activity at cellular resolution in freely behaving zebrafish larvae.

Gil Levowitz (Israel) presented his work on the role of hypothalamic oxytocin neurons in the development of social behavior in juvenile zebrafish. Finally, the session was brought to a close by Claire Wyart (France) revealing data on how cerebrospinal fluid contacting neurons integrate sensory information and modulate locomotor programmes at the temporal resolution of single swim bouts.

Emerging Technologies

In zebrafish, and due to an additional genome duplication event, around 30% of the genes have paralogs given a human ortholog. Ozien Konu (Turkey) talked about a computational tool (CompariZome) that allows the comparison of zebrafish transcriptomes across multiple contexts to assess the possible neo- or subfunctionalization of the duplicated genes.

How can we unravel information of individual neurons in vivo in a tightly packed neural cluster? Periklis Pantazis (Switzerland) convinced us that a newly introduced methodology, confined primed conversion of green-to-red photoconvertible proteins such as Dendra2, allows us to do so. In this method, they use low-power, dual-wavelength (488 and 730 nm) illumination combined with a crossed-beam setup in a conventional confocal microscope to successfully label individual neurons in vivo from trigeminal ganglia, tectal cells, and motor neurons.

Reinhard W. Koester (Germany) described the use of ATTAC, Apoptosis Through Targeted Activation of Caspase, as a powerful cell-type-specific ablation tool. He showed that this system can remove about 90% of the targeted cell type and in combination with the already available nitroreductase allows targeting of two-cell populations independently.

Patrick Müller (Germany) described PyFRAP—a free, open-source, cross-platform software that enables in vivo FRAP measurements in complex geometries thus overcoming previous challenges in FRAP analysis.

Finally, Karuna Sampath (United Kingdom) reminded us that morphogens are required for many developmental processes, but how morphogen gradients are formed in vivo remains unclear. She showed that what concerns the nodal gradient: diffusivity, ligand clearance via degradation and extracellular interactions collectively shapes the gradient and correlates with its signaling range.

Behavior

This short session started with Rui Oliveira (Portugal) arguing in favor of zebrafish as a useful model organism to study social cognition. Zebrafish prefer to spend time in the proximity of other zebrafish rather than alone, and when fighting, they progress through four stages: display, circle, bite, and chase. The winner of a fight will continue to win, and similarly, the loser will continue to lose. They are also social observers and will display various levels of social attention: they will regularly face and attend toward two other interacting fish, will do so less to noninteracting fish, and will not attend to an empty chamber.

Gonzalo de Polavieja (Portugal) presented a phenomenological mathematical model that describes collective behavior in 6–24 dpf larvae. This simple model can describe whether a fish will turn left or right, where there are N or M other fish, respectively. This depends on whether the fish is in a “social mode” or not. If the fish is not social, it will choose left or right randomly. If the fish is social, however, it will pick one other larva at random and turn toward it. So, the probability of turning to the group of N fish is simply given by N/(N + M). It is interesting how this intricate behavior can be understood solely as the interaction between two fish.

Will Norton (United Kingdom) showed data that characterize a nitric oxide synthase mutant and show how it exhibits a marked decrease in aggression, as measured both by dyadic fights and mirror interactions. The mutant also shows an increase in anxiety. In the past, nitric oxide has been shown to be involved in the neuroendocrine system, playing an important role in the hypothalamic–pituitary–adrenal axis. The mutant shows a reduction of dopamine and 5-HT signaling, although pharmacological rescue of the latter was sufficient to rescue the anxiety phenotype.

The final talk of the session saw Michael Orger (Portugal) present a detailed characterization of the locomotor repertoire of zebrafish larvae. The first step involves online high-speed tracking that allows the measurement of many behavioral parameters. Using a novel method, these are then clustered to reveal how all swims fall into 1 of 11 categories, all distinct, which together tile kinematic parameter space. Certain stimuli will preferentially elicit particular locomotor patterns, many of which have already been described in the literature, but which are unified within the same comprehensive framework.

Concluding Remarks

The 4th European Zebrafish Principal Investigator Meeting (EZPM) in Lisbon was a continuation of a highly successful series of meetings on European ground that was initiated in 2010 in the Italian city of Padova by the European Network EuFishBioMed. ¹ This first meeting presented a vibrant atmosphere of discussion and scientific exchange. ² The 4th EZPM showed clearly that zebrafish research has obtained a prominent role in the acquisition of new knowledge uncovering the mechanisms of life and disease and this with increasing momentum driven by the unmatched combination of the experimental virtues of the zebrafish model. In the pioneering days, the zebrafish has been heralded to become the fruit fly with fins and a backbone. The presentations of the 4th EZPM clearly showed we have carried this forecast into effect.

For a research community, strategy comprises not only scientific issues but also entails funding opportunities and regulatory topics. These political and legal frameworks are determined by European authorities and thus cannot be solved in the United States or anywhere else outside of Europe. The 4th EZPM underscored that strategic meetings are required in Europe. However, the driving force of the EZPM should be science and science is international. So, we were most pleased that the adjective “European” in the name of the meeting (intended to reflect the location of the meeting but not the country of origin of its participants) did not prevent colleagues from all over the world to join us. The 4th EZPM provided the right mixture of mind-stimulating scientific talks and strategic discussions at the highest international level hosted in beautiful Lisbon. We are all looking forward to the fifth EZPM to be held in Trento in Northern Italy in 2018.