Event schedule

Hiroyuki Takeda Graduate School of Science, University of Tokyo, Japan
Department of Biological Sciences

The structure and epigenetics of the pluripotent genome in medaka fish

The epigenome of pluripotent cells ensures active transcription of pluripotency-related genes and repression of developmental genes. We generated epigenomic profiles of pluripotent cells which include base-resolution DNA methylomes, histone modification and chromatin accessibility maps using the medaka (Japanese killifish, Oryzias latipes), a vertebrate model suitable for genome science. Native pluripotent cells from medaka blastula embryos were used in rhis study. We first addressed the nucleosome organization and the contribution of DNA sequences to nucleosome positioning in DNA hypomethylated domains (HMDs) using a supervised machine learning algorithm, k-mer SVM. We found that HMDs specifically possess accessible nucleosome organization with longer linkers and identified the strong link between nucleosome positioning and specific DNA sequences at gene promoters in HMDs. Surprisingly, the sequence preference of the nucleosome and linker in HMDs is opposite from that reported previously. We then investigated the dynamics of three-dimensional genome organization during zygotic genome activation (ZGA) using Hi-C in medaka embryos. As reported in Drosophila and mouse, we found that higher-order structures such as compartments are established during ZGA in medaka, independent of transcription. The chromatin accessibility heterogeneity emerges almost simultaneously with compartmentalization. These results suggest that the reprogramming of chromatin folding in early embryos is conserved. Related references 1) Qu W, Hashimoto S, Shimada A, Nakatani Y, Ichikawa K, Saito TL, Ogoshi K, Matsushima K, Suzuki Y, Sugano S, Takeda H, and Morishita S. Genome-wide genetic variations are highly correlated with proximal DNA methylation patterns. Genome Res. 22:1419-25, 2012. 2) Nakamura R, Tsukahara T, Qu W, Ichikawa K, Otsuka T, Ogoshi K, Saito TL, Matsushima K, Sugano S, Hashimoto S, Suzuki Y, Morishita S, and Takeda H. Large hypomethylated domains serve as strong repressive machinery for key developmental genes in vertebrates. Development 141: 2568-80, 2014. 3) Nakamura R, Uno A, Kumagai M, Morishita S, and Takeda H. Hypomethylated domain-enriched DNA motifs prepattern the accessible nucleosome organization in teleosts. Epigenetics Chromatin 10: 44-56, 2017. 4) Ichikawa K, Tomioka S, Suzuki S, Nakamura R, Doi K, Yoshimura J, Kumagai M, Inoue Y, Uchida Y, Irie N, Takeda H and Morishita S. Centromere evolution and CpG methylation during vertebrate speciation. Nat. commun. 8: 1833, 2017. 5) Napo K. M. Cheung, Ryohei Nakamura, Ayako Uno, Masahiko Kumagai, Hiroto S. Fukushima, Shinichi Morishita and Hiroyuki Takeda, Unlinking the methylome pattern from nucleotide sequence, revealed by large-scale in vivo genome engineering and methylome editing in medaka fish. PLoS Genet., 13(12):e1007123, 2017.

Kristin Tessmar Raible MFPL/ University of Vienna, Austria
Platform Rhythms of Life

More than meets the eye: Vertebrate non-visual photoreceptors

The eyes are not the only sites that can perceive light. Light perception by cells in the inner brain of vertebrates, independent of eyes and pineal organs, was already discovered more than 100 years ago. The responsible encephalic photoreceptors have been thought to be specialized cells, similar to the photoreceptors present in the eye and pineal. Consistently, the expression of several opsins has been described at places harboring such deep brain photoreceptors, and hence these opsins were independently claimed to mediate non-visual light responses, such as seasonality. During recent years, an impressive number of non-visual opsins was identified and shown to be in principle able to function as light receptors.
In order to obtain a better understanding of this puzzling complexity of light perception in teleosts, we investigate several ‘non-visual’ Opsins on a functional level. Our particular focus is on TMT/Encephalopsin group, since these Opsins exhibit a particularly slow sequence evolution and some members are conserved across all vertebrate phyla. We have recently shown that the expression of several members of this group is enriched in medaka and zebrafish inter- and motorneurons. Using molecular and behavioral assays we can show that tmt1b in medakafish modulates neurohormone transcript levels and different behaviors in a light-dependent manner. Different types of ‘non-visual’ Opsins have been proposed to convey the light information controlling light-dependent seasonal breeding response, as it is present in medakafish. Null-alleles of medaka tmt1b do not impact on the fish’s gonadal status.

Alexander Aulehla EMBL Heidelberg, Germany

The role of signaling oscillations during embryonic mesoderm pattering

In our group, we study origin and function of signaling oscillations during embryonic development. In vertebrate embryos, oscillatory signalling activities of the Notch [and in mouse embryos also of the Wnt, and Fgf pathways] have been identified during periodic mesoderm segmentation and are part of the somite segmentation clock. Using the mouse model, we have combined quantitative real-time imaging, novel in vitro assays and perturbation approaches to reveal principles and function of these collective signalling oscillations. I will present our recent findings indicating that the relative timing between Notch and Wnt signalling oscillations are critical for mouse embryo mesoderm patterning. To establish medaka as a powerful complementary model to quantitatively study signalling dynamics in vivo, we have initiated experiments to generate segmentation clock real-time reporter medaka knock-in lines; goals, strategies and recent results will be discussed.

Takashi Yoshimura Nagoya University, Japan
Institute of Transformative Bio-Molecules

Medaka as a model to understand the underlying mechanism of vertebrate seasonal adaptation

The appropriate timing of various seasonal processes, such as reproduction, migration and hibernation, is crucial to the survival of animals living in temperate regions. Although this phenomenon attracts great interest, its underlying mechanisms are not well understood. By using non-model organisms that have highly sophisticated seasonal responses, we have uncovered the universality and diversity in the signal transduction pathway regulating seasonal rhythms in vertebrates. Although humans are not typically considered seasonal animals, some evidence suggests that seasonal variation in physiology and behavior also exists in humans. For example, the wavelength settings for the unique yellow hue are shifted to shorter wavelengths in summer compared with those in winter. Seasonal affective disorder patients, experiencing recurrent winter episodes of depressed mood, overeating and hypersomnia, show electroretinogram changes in winter, with lower sensitivity compared with healthy subjects. These observations highlight the potential importance of the retinal in seasonality, but the molecular basis of these seasonal changes remains unknown. We have recently discovered dynamic plasticity in phototransduction regulates seasonal changes in color perception in Japanese medaka fish (Oryzias latipes), an excellent model for studying seasonal adaptation. I will discuss how we can better understand human seasonal rhythms using unique animal models.

Hideaki Takeuichi Okayama University, Japan
Graduate School of Natural Science and Technology

Individual recognition in medaka fish

Previously, we demonstrated that medaka females recognize familiar males following prior visual exposure, and social familiarity influences female mating receptivity. Medaka females can discriminate familiar males from unfamiliar mates and prefer to mate with the former (Okuyama et al., Science, 2014). Here, we found that medaka use faces for individual recognition. Females can discriminate between two male faces and two objects, but upside-down of the faces made it more difficult to discriminate them. When discriminating between two non-face objects, upside-down did not affect it. Thus faces may be special for fish, just as humans (Wang and Takeuchi, elife, 2017). This is the first study that shows the face inversion effect in animals other than mammals.

Kiyoshi Naruse National Institute for Basic Biology, Japan
Lab of Bioresources

Evolution of the sex chromosome and sex-determining genes in Oryzias fish

Sex chromosomes harbor a primary sex-determining signal that triggers sexual development of the organism. In mammals, Sry is the dominant male-determining gene located on the Y chromosome, and has evolved from the neural gene Sox3 on the X chromosome probably through a regulatory mutation. Medaka fishes in the genus Oryzias have different sex chromosomes with different systems (XY and ZW), providing ideal conditions for investigating the mechanisms that lead to the rapid turnover of sex chromosomes. So far, different sex-determining genes, Dmy and GsdfY, have been isolated from the Oryzias species, demonstrating that turnover of sex chromosomes is associated with the substitution of master sex-determining genes. Recently, we identified Sox3 as a novel sex-determining gene on the XY sex chromosomes in the marine medaka Oryzias dancena/melastgma by positional cloning. Sex reversed phenotypes in transgenic fish and loss-of-function mutants of the Y chromosomal Sox3 allele all point to its critical role in sex determination, suggesting that the neo-Y chromosome of O. dancena arose by co-option of Sox3. Furthermore, we found the Sox3 gene also on the XY sex chromosomes in distantly related Oryzias species, O. marmoratus and O. profundicola. Fine mapping and association analysis identified the Y chromosome-specific 430-bp insertion at the Sox3 locus, which appeared to be involved in its male determination function. The Sox3-dependent sex determination system in Oryzias species is polyphyletic, and the Y-specific insertion has not been found in O. dancena, suggesting that Sox3 has evolved as the sex-determining gene independently in different lineages of Oryzias. These results suggest that Sox3 might have acquired the novel male-determining function repeatedly and independently during vertebrate evolution.

Yukiko Ogino Kyushu University, Japan
Faculty of Agriculture

Diversification of androgen receptor function underlies secondary sex characteristics development of teleost fishes

Gene duplication is a dominant driving force of evolution. Steroid hormone receptor gene family is thought to have arisen from gene duplication. However, the molecular events which produce new protein functions after the genome duplication have not been fully understood. Teleost fishes present a good model to investigate an accurate evolutionary history of protein function after whole genome duplication, because the teleost-specific whole genome duplication (TSGD) 350 million years ago resulted in a variety of duplicated genes that exist in modern fishes. We focused on the androgen receptor (AR) gene, since two different subtype genes, ARα and ARβ, were generated in the TSGD. ARβ has retained the ancestral function, whereas the ARα evolved as a hyperactive form of AR in the teleost lineage. Results of our combined functional and 3 dimensional analyses of medaka (Oryzias latipes) ARs identified the substitutions that led to changes in protein structure and function between medaka ARα and ARβ. One substitution located within the helices 10/11 of ligand binding domain is sufficient for generating higher transactivation of ARα. This amino acid change affects the stability of ligand binding by modifying the hydrogen bonds between ligand and AR. To further address the functional differences between ARα and ARβ in vivo, we isolated the nonsense mutants of ARα and ARβ from the medaka tilling library. ARβ have predominant function for external masculine phenotypes, and ARα might participate the development of traits for male-male competition. Our findings would provide an historical explanation for the retention of the duplicated AR copies in euteleost genome.

References: 1. Y. Ogino et al., (2016) Mol. Biol. Evol. 33, 228-244. 2. Y. Ogino et al., (2014) Endocrinology 155, 449-462. 3. Y. Ogino et al., (2011) Evol. Dev. 13, 315-325. 4. Y. Ogino et al., (2009) Endocrinology 150, 5415-5427.

Saori Yokoi Hokkaido University, Japan
Faculty of Pharmaceutical Sciences

Analysis of molecular basis underlying decision making according to social familiarity

Some social animals can recognize socially-familiarized conspecific individuals (social recognition) and familiarity affects their behavior, which may be important for social adaptation. Disorders in human brain function for this system are assumed to cause mental illnesses, such as autism, and great attention has recently been paid to the underlying neural/molecular mechanisms of these disorders. Oxytocin (OT) is considered to be involved in social recognition and social behavior in rodents and humans. For example, social familiarization (repeated encounters) decreases approach behaviors of male wild-type mice toward unfamiliar females, but not OT KO males. Thus, the OT KO males are thought to have defects in social recognition (social amnesia). In this study, we investigated social behaviors of individual medaka fish, which is a model organism commonly used for molecular genetics, toward group-reared fish (defined as familiar fish) in the same tank and toward unfamiliar conspecifics reared in a different tank. Wild-type males exhibit mating behaviors, irrespective of social familiarity. In contrast, we found that social familiarity strongly affects male social behaviors (e.g., courtship, aggressive, and mate-guarding behaviors) in isotocin-related gene mutants. Isotocin (IT) is a fish homologue of OT. This result suggested that IT-related gene mutants lost social motivation toward conspecifics. In some fish, imprinting affects the social preferences of the juvenile fish based on the traits of their parents that care for them during the early development period. Next, to examine whether or not imprinting mediates social recognition of mutants for IT mutants, we investigated whether social familiarization (rearing in the same tank only as adults) could recover the lost of social motivation in the mutants toward unfamiliar fish.

Minoru Tanaka Nagoya University, Japan
Graduate School of Science

Germ Cells - More than Gametogenesis

Germ cells are the only cells indispensible for producing the next generation. But it is generally recognized that germ cells are passive cells that can develop into only gametes. Actually the gametogenesis is strictly regulated by the surrounded somatic cells. However, our studies have begun to reveal the positive ability that germ cells possess. The germ cells do not only have intrinsic mechanism that determines their fate cell-autonomously, but also have the ability to change their environment. Our recent results indicate that germ cells have an intrinsic power to feminize the gonad and body, which can be against a power of masculinization by male sex determination gene, dmrt1bY. Analysis of controlling the germ cell number also suggests that regulation of germline stem cells is related to the size of gonad. I would like to summarize the potential ability of germ cells that were found during the course of our studies.

Christoph Winkler National University of Singapore, Singapore
Department of Biological Sciences

Transcriptome profiling of osteoblasts in a medaka osteoporosis model identifies novel mediators of bone repair

Bone is a highly dynamic organ that is constantly remodeled to maintain its high quality. During remodeling, old bone matrix is degraded by bone-resorbing osteoclasts and instantly replaced by new matrix from bone-forming osteoblasts. To prevent diseases such as osteoporosis, remodeling must be tightly coordinated to balance osteoblast and osteoclast activity. To date, many aspects that control bone homeostasis and osteoblast-osteoclast coupling remain unclear. To address this, we have chosen the Japanese medaka (Oryzias latipes) as a powerful animal model to mimic aspects of human bone diseases. Previously, we generated transgenic medaka where excessive bone resorption and osteoporosis-like lesions can be triggered by inducible expression of the osteoclast-inducing factor ‘Receptor Activator of Nuclear Factor kappa Ligand’ (RANKL). Using live imaging, we could demonstrate that osteoblast progenitor cells get activated and recruited to the osteoporotic lesion sites, where they differentiate and efficiently repair and remineralize damaged bone matrix. To identify factors involved in osteoblast activation during bone repair, we performed transcriptome profiling of osteoblast progenitors and mature osteoblasts that were purified from osteoporotic fish by Fluorescence Activated Cell Sorting (FACS). We characterized a set of transcripts that are up-regulated in osteoblast progenitors immediately after ectopic osteoclasts had started to induce bone lesions. Among these transcripts, we identified a novel calcium sensing receptor CaSR, which is expressed in osteoblasts. CRISPR-mediated genome editing in medaka revealed that deficient CaSR signaling affects osteogenesis, as well as interferes with the recruitment of osteoblast progenitors to osteoporotic lesions sites. In conclusion, our study revealed a set of novel transcripts up-regulated during osteoporotic bone repair and identified essential roles for CaSR in osteogenesis and bone homeostasis.

Ronald B. Walter Texas State University, USA
Chemistry & Biochemistry

The Genetic Response to Fluorescent Light Exposure Within Internal and External Organs Is Conserved Among Vertebrates (Danio rerio, Oryzias latipes, and Mus musculus)

We have previously shown 4,100 K or “cool white” fluorescent light (FL) exposure induces cellular stress, inflammation and immune responses in the skin of several varied biomedical fish models including; platyfish (Xiphophorus maculatus), medaka, (Oryzias latipes) and zebrafish (Danio rerio)1.
Here we present RNA-seq results that establish similar patterns of modulated gene expression within two internal organs, brain and liver, of zebrafish and medaka exposed to 35 kJ/m2 FL. In addition, FL light induced modulated gene expression within skin, brain and liver of both fish models is compared to similarly FL exposed hairless mice (Mus musculus).
In zebrafish and medaka, each of the three organs tested showed up-modulation of unique gene sets that are expected to activate the Acute Phase Response (APR) signaling pathway, leading to increased inflammation and innate immune response. Our pathway and gene clustering analyses suggest this response is due to increased cellular oxidative stress that promotes induction of the primary up-stream regulator, tumor necrosis factor (TNF). Exposure of hairless mice to FL also serves to induce both inflammation and an immune response that mimics that observed in the fish models for both skin and brain. However, the FL induced genetic response in liver, although consistent and up-modulated in medaka and zebrafish, exhibits a suppressed response in mouse liver, but in the same pathways.
Overall, the sharing of genetic responses to light among divergent diurnal fishes, and a nocturnal mammal, suggests the genetic response to light is likely hard-wired deep within the vertebrate genome, and may extend through the vertebrate classes, perhaps even to humans.

1) Boswell, M., Boswell, W., Lu, Y., Savage, M, et. al., (2017). The Transcriptional Response of Skin to Fluorescent Light Exposure in Viviparous (Xiphophorus) and Oviparous (Danio, Oryzias) Fishes. Comp. Biochem. & Physiology, https://doi.org/10.1016/j.cbpc.2017.10.003

Jochen Wittbrodt COS Heidelberg, Germany

Library of life: the matrix. Medaka to tackle the genetics of individuality

We are all concerned with what makes us special and what is the basis of our individuality. What is the basis for the difference of individuals of the same species and how do nature (genetics) and nurture (environment) contribute? Individual variation is the key parameter in the interplay of intrinsic and extrinsic factors that contribute to states of health and disease, and must be understood if we aim at translating findings between laboratory models and ultimately to the human context. The central issue is to distinguish between phenotypes that are "determined" by widely shared features of genomes, those influenced by individual variation, and stochastic events.In the past ten years we have systematically explored routes to tackle these questions in a model system. One challenge is based in the fact that individuals only represent a very limited resource for systematic studies. Here we would rather need genetically identical groups representing the individuals that in their entirety represent the variably of a population. Strikingly, complex vertebrate model systems had been bred deliberately to reduce variation as a source of noise and to thus ensure experimental reproducibility. Here medaka with the fully established repertoire of laboratory approaches on the one hand and with its rich natural resources provides a unique opportunity. With its high inter-individual variability on the one hand and its resistance to inbreeding on the other, medaka represents the ideal model system to systematically address the genetics of individuality and to delineate the respective genetic and environmental contributions to individuality. Over the last ten years we have identified an unstructured natural population the we have converted into 111 fully sequenced inbred lines reflecting the phenotypic features of the original population. We currently carry out an in-depth phenotyping of these lines at scales ranging from organismal to molecular phenotypes, using cost efficient approaches that can be highly replicated. The project has put a particular focus onto the heart where we integrate information across the cardiovascular systems of vertebrates, especially the wide range of phenotypes observed in humans. I will present an overview of our attempts to serve as an introduction to the topic that will be further deepened by the presentations of Tom Fitzpatrick and Felix Loosli.

Manfred Schartl Biozentrum Universität Würzburg, Germany
Physiological Chemistry

How the Medaka came to Europe

An overview will be given on the history of medaka research in Europe from its very beginning in the eighties of the last century. Initial motivations to select medaka as laboratory model, obstacles and selected examples that mark the triumphal procession of medaka to become the truely complementary and even useful alternative to the zebrafish will be highlighted.

Joaquín Letelier Center for Integrative Biology, Chile

cis-regulatory logic of Shh expression reveals common history of unpaired and paired fins

Despite their evolutionary, developmental, and functional importance the origin of vertebrate paired appendages remains a mystery. Classical ideas center on three hypotheses: that paired fins relate to gill arches, are derived from a fin fold, or that median fins evolved first and paired fins arose by co-option of ancient genetic patterning modules. Importantly, the third hypothesis makes specific predictions about the function and phylogenetic history of cis-regulatory elements (CREs) involved in appendage patterning. Here we show that a major enhancer for Sonic hedgehog (Shh, a key diffusible morphogen essential for proper growth and patterning of limbs and paired fins) is deeply shared between paired and unpaired fins in fish and with the limbs of mice. In mice, a single CRE termed ZRS is solely responsible for coordinating Shh expression in limbs. We use transgenic assays in zebrafish and mouse to trace the functional equivalence of the ZRS across gnathostome phylogeny and its likely absence in agnathans. CRISPR/Cas9- mediated deletion of the medaka core ZRS sequence, inspection of shh epigenetic landscape and in vivo enhancer assays reveal the existence of ZRS shadow enhancers in both teleost and human genomes. Deletion of both ZRS and shadow ZRS abolish shh expression and completely truncate pectoral fin formation. Strikingly, deletion of the medaka ZRS, an experiment expected to only affect the paired fins, results in an almost complete ablation of the dorsal fin. This finding indicates that a ZRS-Shh regulatory module is shared by paired and medial fins, and that paired fins likely emerged by the co-option of developmental programs established in the median fins of stem gnathostomes. Later on, this critical ZRS- driven Shh function was reinforced in pectoral fin development with the recruitment of shadow enhancers, conferring additional robustness.

Satoshi Ansai National Institute for Basic Biology, Japan
Laboratory of Bioresources

The molecular genetic basis of diversified sexually dimorphic traits in Oryzias species endemic to Sulawesi, Indonesia

Sexual dimorphism is prevalent, but often differs remarkably between closely related species. However, we know little about which genetic changes can actually contribute to diversification of such sexually dimorphic traits. Sulawesi endemic Adrianichthyidae species (commonly referred to as medaka) serves an excellent model system because their sexual dimorphic body colorations are significantly diversified in closely-related species. As a first step, we generated a de novo long-read genome assembly of Oryzias celebensis as a reference assembly for the Sulawesi species. This assembly was anchored to 18 chromosomes by a linkage analysis and was annotated using RNA-seq data from adult and embryonic tissues. Using this genomic information, we have studied the molecular mechanisms underlying red coloration in pectoral fins, a characteristic feature of O. woworae males. Quantitative trait loci (QTL) mapping in a F2 intercross between a male of O. woworae and a female of O. celebensis revealed that an autosomal locus controls the red pigmentation. Subsequent RNA-seq analysis showed that csf1 gene is a strong candidate responsible for the red fins in 18 deferentially-expressed genes in the QTL region. This was supported by loss-of-function analysis of the csf1 gene by CRISPR/Cas system, which caused a defect in xanthophores. Semi-quantitive allele specific expression analysis indicates cis-regulatory mutations would cause higher expression in pectoral fins of the males. Further genomic analysis are underway to identify the cis-regulatory mutation(s) responsible for responsible for the red fin traits.

Haobin Zhao Central China Normal University, China
School of Life Sciences

The Proteins Interacting with Prmt5 in Medaka (Oryzias latipes)

Prmt5 plays important roles in regulation of gene expression, RNA processing, cell growth and differentiation, signal transduction and germ cell development, etc in mammal. Prmt5 of medaka had been reported. However, the proteins interacting with Prmt5 in medaka are in dim. In this sudy, medaka Prmt5 was used as a bait to fish the interacting proteins in the library by yeast two-hybrid technology. Positive colonies were obtained by mating yeast cells of Y187 containing pGBKT7-prmt5 and AH109 containing cDNA library on a plate with the dificient medium of SD/-His-Leu-Trp-Ade and by X-α-gal staining. Eight proteins were confirmed interacting with Prmt5 from 69 preliminary positive colonies after four passages, X-α-gal analysis, backcross test and sequencing. The proteins interacting with Prmt5 are methylosome protein 50 (Mep50), apolipoprotein A-I-like （Apo-AI）, PR domain containing protein 1a with zinc fingers (Prdm1a), Prdm1b, Tim-like protein (Tim-l), phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (Paics), NADH dehydrogenase subunit 4 (ND4) and sciellin (Scl). The identification of the proteins interacting with Prmt5 is helpful for study and understanding of Prmt5 function in fish.

Baubak Bajoghli University Hospital Tübingen, Germany
Department of Immunology, Oncology - Labor for Translational Oncology

Long search for the "Holy Grail" in the evolution of adaptive immune system: Identification of lymph node equivalent structures in medaka fish

In the past two decades, we have witnessed a renaissance in the study of the evolution of the adaptive immune system in vertebrates. In particular, these studies have expanded our knowledge of the evolution of thymopoietic tissues, where T-lymphocytes develop. Furthermore, dendritic cells (DCs), which primarily serve as a bridge between the innate and adaptive immune systems were identified in teleost fishes. Overall, these studies demonstrate that the cellular components of the adaptive immune system are evolutionarily conserved between vertebrates. However, secondary lymphoid organs (SLOs), which spatially organising the interaction between lymphocytes and antigen-loaded DCs, are not well conserved across vertebrate groups. For example, lower vertebrates lack lymph node, a vital SLO in mammals. The great mystery is therefore where the adaptive immune response takes place in lower vertebrates. To address this fundamental question, we used medaka fish as a model system. In a comprehensive study, we discovered specific sites, where T-lymphocytes and DCs traffic and interact akin to mammalian lymph nodes. We found that these sites are distributed throughout the body facilitating a rapid adaptive immune response to local infections. Overall, our work provides a new evolutionary framework of the adaptive immune response in teleost fishes.

Soojin Ryu University Medical School, Johannes Gutenberg University Mainz, Germany
German Resilience Center

Characterization of the endocrine and behavioural stress responses of medaka larvae

When confronted with a stressor animals modify their physiology and behavior in order to cope with the threat. The stressors can be of diverse type but they typically culminate in the activation of the Hypothalami-Pituitary-adrenal (HPA) axis whose final output is the pleiotropic stress hormone glucocorticoids (called cortisol in fish). We are interested in using medaka larvae to study the molecular changes caused by stress exposure during development. As a first step, we have begun to characterise the endocrine and behavioural stress response in larval medaka. Our initial aims are 1) to identify different stressors that elevate cortisol in larval medaka, 2) identify behavioural responses to different stressors, and 3) characterise the ontogeny of the stress response during development.

Narges Aghaallaei University Hospital Tübingen, Germany
Abteilung Innere Medizin II, Labor für Translationale Onkologie

Basal lamina in the base of intestinal furrow is an inductive site for the mucosal adaptive immune response in medaka fish

Several organized lymphoid structures provide a niche that facilitates the initiation of adaptive immune response through stimulation of T-cells by professional antigen-presenting cells (APCs), in particular, dendritic cells (DCs). Gut-associated lymphoid tissues (GALTs) are the most ancient secondary lymphoid tissues in vertebrates, although their organization and cellular composition vary across vertebrate groups. Peyer’s patches (PPs) and mesenteric lymph nodes (MLNs) are the main initiation sites of the GALTs in mammals. However these organized structures are missing in teleost fishes. It is not yet known where DCs migrate to interact with T-cells within the teleost gut. Hence, the organization of teleost GALT or an equivalent tissue is still not fully understood. We report here that young naïve T-cells after leaving the thymus migrate predominantly in the basal lamina in the base of the medaka intestinal furrow, where they traffic along the intestine and interact with antigen-loaded DCs both during homeostasis and in response to mucosal inflammation. Additionally we found that the epithelial cells in the medaka intestinal furrow produce Ccl25/Ccr9, a chemokine/chemokine receptor axis that is required for the recruitment of T-cells. Our findings suggest that the intestinal furrow provides an environment for T-lymphocyte and APC interaction during homeostasis and in response to inflammation. This indicate that the onset of adaptive immune response is spatially well-organized and the lack of lymphoid structures in teleost fish is compensated by recruitment of cellular components of the adaptive immune system in the basal lamina of intestinal furrow to perform their functions.

Alexander Froschauer Technische Universität Dresden, Germany
Institut für Zoologie

Suppressed recombination of the homomorphic Y chromosome in the medaka

The suppression of meiotic recombination is a key feature of sex chromosomes. After the formation of a sex chromosome pair the lack of recombination keeps the identity of the chromosome pair (WZ or XY) and paves the way for morphological differentiation, accumulation of sex specific genes or degeneration. The relatively young Y chromosome of the medaka Oryzias latipes arose by a chromosomal insertion with the dmrt1by gene (aka DMY) driving male sex determination. This Y-specific region has a size of 250 kb and is closely linked to phenotypic markers that are described as early as 1921. The region with suppressed recombination was estimated to be roughly 3.5 Mb in size. When we analyzed the recombination frequency of the 39 Mb LG1 chromosomes in different strains of the medaka, we found a suppression of recombination over at least 16 Mb of the X-chromosome, deduced from phenotypic and PCR-markers mapped in the ensemble database. This recombination block between a transgene (EGFP fluorescence) and the leucophore free locus leads to a genetic distance of only 0.4 cM per 16 Mb and is dependent on the sex of the individual and possibly the origin of the sex chromosomes analyzed. This plasticity reflects the young state of evolution of the chromosomes. Our current analyses compare the physical and genetic recombination frequencies on autosomes and different sex chromosomes to elucidate possible mechanisms of the suppression of recombination. As outlook we discuss the artificial evolution of the medaka sex chromosomes to generate a testable model for evolutionary hypotheses.

Lazaro Centanin COS Heidelberg, Germany

Fixed Developmental Timing Revealed by Trans-Species Transplantation

Jana F. Fuhrmann, Lazaro Centanin.

The temporal requirement to go through embryogenesis is stereotypic within a given specie but largely variable among animals, where the very same organ can display different developmental timing even in related species. Considering teleost fish, a Zebrafish embryo develops faster than a Medaka embryo. Here we use Medaka/Zebrafish chimeras to asses if developmental timing relies mainly on genetic information or if alternatively, is an orchestrated process that responds to instructions from the host. Zebrafish and Medaka blastocysts do not mix to each other when put together in a developing blastula. We have exploited this feature to develop exogenous Zebrafish retinae in medaka host and vice versa, and used it to tackle developmental timing using distinctive events: lens recruitment, onset of pigmentation and neurogenesis. Our results indicate that the genetic timing of retinogenesis is maintained when the organ develops in an exogenous environment, suggesting an autonomous mode of organ formation. I will discuss this findings in the context of community cell effects and self-organising models.

Tonomori Deguchi

Drug discovery screening for lymphatic vessel related diseases using medaka

Evaluation of drug efficacy at the individual-animal-level is essential for advancing to pipeline compounds in all drug development. However, individual experiments take enormous amounts of time and labor and are a major task of drug discovery research. Therefore, we have developed drug discovery screening technology for lymph vessel related diseases using medaka with the aim of constructing a high-throughput screening system at the ultimate individual level capable of screening for drug discovery. First, in order to analyze the lymphatic vessel morphology optically at high speed, we developed a medaka that expresses green fluorescent protein in lymphatic vessels using recombinant DNA technology. In addition, we also developed a special multi well plate for observing medaka, which can observe medaka embryos or fry aligned in the same direction in all wells. Then, we treated the eggs of the lymphatic vessels visualized medaka in the above-mentioned well plate with inhibitor compounds, the images were acquired with an imaging analyzer, and the form change of the lymphatic vessels was examined to evaluate their efficacy. As a result of evaluating the efficacy of about 500 compounds of the existing inhibitor set, death was observed in 126 compounds and teratogenicity was confirmed in 12 compounds. Blood flow abnormality could also be confirmed with 8 compounds. Drug discovery screening for lymphatic-related diseases using medaka, which we have been addressing for years, has become more and more realistic. In the future, we will aim for completion of high-throughput drug discovery screening technology for lymph vessel related diseases using medaka by advancing automation of medaka egg dispensing and automatic analysis of image data.

Yasuhiro Kamei National Institute for Basic Biology, Japan
Spectrography and Bioimaging Facility

Biological and Optical improvement of IR laser-mediated gene induction microscope system

IR laser-mediated gene induction microscope system: IR-LEGO, is a promising technique for developmental biology and biothermology, because the system enables single-cell level local heating. To date, we applied the system to single-cell/local gene induction in many species including medaka and zebrafish. Recently, we utilized the system for a study of heat in biology, especially temperature distribution and dynamics inside cells (we termed this feild as biothermology). Now, we are improving the system to be convenient the operation of IR-LEGO and to be match for biothermological experiment. Although temperature measurement is one of key technology for the IR-LEGO and biothermology, in 2017 we established temperature measurement method in vivo by using fluorescent proteins, gTEMP (Genetically encoded fluorescent thermometer). This FPs realize retiometric temperature measurement in cells without harmful effect. Moreover, we are also improving other points of view. In the meeting, we will explain the outline of improvement of the system in three categories, molecular and microscopic; gTEMP, biological; heat shock factors, and optical; adaptive optics.

Tomohiro Ueno Kyoto University, Japan
Graduate School of Medicine

in vivo MR microcopy of disease models in Medaka

Magnetic Resonance Imaging (MRI) is a non-invasive imaging technique, which is suitable for differentiating soft tissues of internal organs. Thus, MRI is an essential diagnostic tool in a clinical situation. To investigate various human disease models in small fish, we accomplished in vivo MR microscopy by developing a 14.1 T ultra-high field MRI and a hypothermic anesthesia procedure. In the in vivo MR microscopy, we can exclude a water pool and achieve high spatial resolution enough for the small body size of the fish. We created a non-alcoholic fatty liver disease model in medaka and followed its individual disease progression. We quantitatively evaluated the steatosis level by calculating the MRI-estimated proton density fat-fraction (MRI-PDFF), which estimated triglyceride in liver tissue. The MRI-PDFF results agreed with a histological analysis. Moreover, we applied the in vivo MR microscopy to investigate early indication of disease in p53 knockout medaka. We will also discuss results of the time series analysis of the p53 knockout medaka.

Toshiya Nishimura Nagoya University, Japan
Biological Science

Germline sex determination by nanos3, a component of germ plasm, in medaka

Germ cells are the common origin cells of sperm and eggs. In many animals, germ plasm, in which germline determinants are contained, is accumulated in oocytes, and cells which inherit the germ plasm during early embryogenesis develop as primordial germ cells (PGC). Nanos is one of the components of germ plasm and evolutionally conserved from flies to mammals. In medaka, nanos3 mRNA and NANOS3 protein are maternally provided to the embryos and expressed in PGCs during embryonic development. Here, we generated nanos3 loss-of-function mutants by TALEN and examined the function of nanos3 in medaka. nanos3 homozygous (–/–) zygotic mutants derived from nanos3 heterozygous mothers formed germ cells. However, after hatching, the number of germ cells decreased dramatically, leading to infertility in both nanos3–/– males and females. Surprisingly, germ cells in nanos3–/– females reduced the expression level of FOXL3, a gene involved in germline sex determination, and initiate spermatogenesis. Therefore, in addition to the maintenance of germ cells, zygotic nanos3 have a novel role involved in germline sex determination, possibly by regulating foxl3 in medaka.

Felix Loosli Karlsruhe Institute of Technology (KIT), Germany
Institute of Toxicology and Genetics (ITG)

A panel of medaka inbred lines: a resource to study the genetics of individuality

Individuals of a natural population share the same set of genes. However, the polymorphic nature of the gene pool results in genetic individuality with variation in many key phenotypic traits. Statistical approaches, such as genome wide association studies (GWAS) are applied to unravel these complex genetic traits. This requires large numbers of individuals to achieve meaningful statistics and eventually functional tests to prove causality. It is therefore important to establish suitable model systems. We have used medaka fish (Oryzias latipes), an economical, fecund vertebrate with a high tolerance to inbreeding to establish a panel of more than a hundred inbred lines from a wild population. By that we have captured and fixed a large diversity of wild genotypes as near isogenic lines. We will use this panel for GWAS to study the underlying genetics of a select number of complex traits. To this aim we are now establishing assays to screen these lines for phenotypic variation. These assays are designed to measure quantitative differences of a given trait, to allow genome wide association studies with the ultimate aim to identify causative QTLs. I will discuss examples of how we assay specific traits relating to organ development and function, as well as behaviour.

Juan Ramon Martinez-Morales Centro Andaluz de Biología del Desarrollo (CSIC/UPO), Spain
Gene regulation and morphogenesis

Analysis of YAP/TAZ-dependent transcriptional response during early morphogenesis in teleost embryos.

The Hippo signaling pathway is a genetic regulatory cascade that controls tissue homeostasis and organ size by coordinating the expression of proliferation, differentiation and apoptotic genes during development. The main constituents of this pathway are two effector proteins: YAP and its paralog TAZ (WWTR1). When YAP or TAZ are active, they get translocated into the cell nucleus and regulate the expression of their target genes interacting physically with TEAD, among other transcription factors. Here we show that Yap family’s paralogs composition is variable among vertebrates. Whereas many of the vertebrate branches maintained functional copies of both Yap and Taz (such as in tetrapods, spotted gar or zebrafish), Taz is not present in Acanthomorpha, the largest group of teleost fishes (e.g. cod, tilapia, platyfish, stickleback, or medakafish). In contrast, a second copy of Yap1, here referred as yap1b, appears conserved in their genomes. This closer paralog encodes for a protein with a divergent c-terminal transactivation domain. Interestingly, comparative analysis of zebrafish and medaka mutants shows that the mutation of yap1 in medaka (generated by CRISPR-Cas) results in a strong morphogenetic phenotype early during development, which is similar to the simultaneous mutation of yap1 and taz in zebrafish. We use iDamIDseq to identify the potential targets of these transcriptional regulators during gastrulation in medaka embryos. Our results, reinforced after carrying out a transcriptomic approach, indicate that the binding profile of yap1b is limited to a subset of the yap1 TEAD-dependent binding sites. Furthermore, the abundance of genes involved in cell migration in both datasets as well as the evidence from ISH and functional assays suggest the potential role of YAP as a regulatory driver of morphogenesis at early developmental stages.

Didier Stainier MPI Bad Nauheim, Germany

Genetic compensation is triggered by mutant mRNA degradation

In response to deleterious mutations, genetic compensation by transcriptional upregulation of related gene(s) (also known as transcriptional adaptation) has been reported in numerous systems; however, how such a response is activated is unknown. We developed and analyzed several models of transcriptional adaptation in zebrafish and mouse and observed a correlation between the degree of mutant mRNA decay and the transcriptional upregulation of related gene(s). To assess the role of the mutant mRNA in triggering transcriptional adaptation, we generated alleles that fail to transcribe the mutated gene and found that they do not exhibit this response. Moreover, genetic inactivation, silencing or chemical inhibition of the nonsense mediated decay factor Upf1 can also lead to loss of transcriptional adaptation. These and other results presented at the meeting identify a new role for the mRNA surveillance machinery in buffering against mutations by triggering the transcriptional upregulation of related genes. In addition, these results will help design mutant alleles with minimal transcriptional adaptation-derived compensation.

Andrea Pauli Vienna Biocenter, Austria
Research Institute of Molecular Pathology

Small proteins with big roles: Bouncer is necessary and sufficient for species-specific fertilization

Fertilization is fundamental for sexual reproduction, yet its molecular mechanisms are poorly understood. Here, we identify an oocyte-expressed Ly6/uPAR protein, which we call Bouncer, as a crucial fertilization factor in zebrafish. We show that membrane-bound Bouncer mediates sperm-egg binding and is thus essential for sperm entry into the egg. Remarkably, Bouncer is not only required for sperm-egg interaction, but also sufficient to allow cross-species fertilization between zebrafish and medaka, two fish species that diverged over 150 million years ago. Our study thus identifies Bouncer as a key determinant of species-specific fertilization in fish. Bouncer’s closest homolog in tetrapods is restricted to the male gonad in internally fertilizing vertebrates, suggesting that our findings in fish have relevance to human biology.

Thomas Thumberger Heidelberg University, Germany
COS

Acute and inducible knockdown of GFP-tagged proteins using a nanobody-destruction box fusion

I will present our running approaches using CRISPR strategies in medaka. The focus will be on their efficiency to produce mutants, targeted insertions, precise knock-ins, and the most accurate way to unambiguously identify founder fish.

Tomas Fitzgerald EMBL - EBI, United Kingdom
Birney Research

Whole Genome Analysis of the Inbred Medaka Kiyosu Panel

Over the last six years dedicated work has led to the establishment of the inbred medaka Kiyosu panel. Randomly selected mating pairs originating from an outbred wild population in Kiyosu Japan were used to start the inbreeding scheme. After 9 rounds of single brother-sister mating, 111 inbred lines remained from 83 different original wild founding breeding pairs. Thus 28 lines are “sib lines” to another line, a feature we aim to exploit in our statistical analysis. We have completed whole genome sequencing across the entire panel and called homozygous and heterozygous SNPs, INDEL’s and CNV’s in each line against the improved PacBio derived medaka reference. Amazingly over 75% of the lines are greater than 80% homozygous providing a truly unique model organism resource. We present initial analysis of Kiyosu panel genomes focussing on global sequence characteristics in and around detected variation. For QTL mapping, whole organism phenotypes are unlikely to provide specific loci from panel phenotyping alone, here we aim to boost statistical power using carefully designed F2 cross strategies. We have developed an extensive simulation and statistical power assessment of the genetic and environmental effects we might encounter, this simulation scheme handles polygenic phenotypes, multi-trait phenotypes and can simulate the results of phenotyping the Kiyosu panel and expected phenotyping of crosses. We will present simulation results showing a predicted improvement in loci mapping when following specific F2 cross experiment design strategies.

Juan Ramon Martinez-Morales Centro Andaluz de Biología del Desarrollo (CSIC/UPO), Spain
Gene regulation and morphogenesis

Evolutionary emergence of the rac3b/rfng/sgca regulatory cluster refined mechanisms for hindbrain boundaries formation in zebrafish

Developmental programs often rely on parallel morphogenetic mechanisms that guarantee precise tissue architecture. While redundancy constitutes an obvious selective advantage, little is known on how novel morphogenetic mechanisms emerge during evolution. The vertebrate hindbrain is subdivided along the antero-posterior axis in 7 rhombomeres emerging during development through a finely tuned TFs code. In zebrafish, rhombomeric boundaries behave as an elastic barrier, preventing cell intermingling between adjacent compartments. Here, we identify the role of the small-GTPase Rac3b in actomyosin cable assembly at hindbrain boundaries. We show that the novel rac3b/rfng/sgca regulatory cluster, which is specifically expressed at the boundaries, emerged in the Ostariophysi superorder by a chromosomal rearrangement that generated new cis-regulatory interactions. By combining 4C-seq, ATAC-seq, transgenesis, and CRISPR-induced deletions, we characterized this regulatory domain, identifying cis-regulatory elements active specifically at the hindbrain boundaries. Our results suggest that the capacity of boundaries to act as an elastic mesh for segregating rhombomeric cells evolved by co-option of critical genes to a novel regulatory block, refining the mechanisms for hindbrain segmentation.

Jochen Gehrig ACQUIFER, Germany
Digital Biomedical Imaging Systems AG

High content screening by ACQUIFER - Automated microscopy for whole organism screening applications

ACQUIFER is a division of DITABIS, Digital Biomedical Imaging Systems AG, Freiburger Str. 3, 75179 Pforzheim, Germany Phenotypic screening is increasingly employed in biomedical and pharmaceutical research to address scientific questions in the context of whole organism model systems, such as the zebrafish embryo. However, the complexity of a fully developed body imposes methodological challenges in high content screening, demanding novel technical solutions that are compatible with large scale automated imaging and scoring of cell- or tissue-specific and overall phenotypes. At ACQUIFER, we have developed the Imaging Machine, a versatile and flexible high content screening platform following a unique optomechanical design, optimized for non-adherent and motion-sensitive specimen. Its sample centred approach, including a static stage in combination with moving optics and integrated environmental control, provides optimal in-vivo imaging conditions and renders it ideal for image-based screening or parallel long-term observation of biological specimen such as small model organisms. Here, we will give an overview of imaging hardware, discuss workflow requirements for zebrafish screening and highlight example projects. Please refer to our website at www.acquifer.de or call us at +49 (6221) 435 2035 for more detailed information on ACQUIFER products.

Omar Hammouda University Heidelberg, Germany
Center of Organismal Studies Heidelberg

A Change of Heart: Medaka as a model for Human Cardio-Vascular Diseases & GWAS

Cardiovascular diseases (CVD) cover a wide spectrum of disorders involving the heart or blood vessels such as congenital heart diseases, arrhythmias and many more. Together CVD are the leading cause of death globally. Increasing number of Genome Wide Association Studies (GWAS) are being performed, thousands of single nucleotide polymorphisms (SNPs) are being identified and associated to diseases in humans. However, about 93% of identified SNPs lie within non-coding regions of our genome, highlighting the necessity to explore and understand the role of these regions in disease development and progression. Moreover, genetic and experimental limitations in human GWAS urge us to use animal models to fully exploit the advantages of GWAS. My aim is to break down the complex regulatory pathways involved in the development of CVD using medaka (Oryzias latipes) as a model for functional validation of human CVD-associated genes/SNPs. While also introducing it as a robust model for GWAS to identify novel CVD-associated SNPs. Medaka offers many technical advantages such as transparent body and a high tolerance to inbreeding; which will aid in the identification, validation & characterization of novel coding/regulatory mutations leading to CVD-related phenotypes. As a first step, using our newly developed high-throughput heart rate screening protocol, I functionally validate various novel CVD-associated gene hits identified in human GWAS in vivo in medaka embryos. Using CRIPSR/Cas9 targeting complementary human SNP-containing gene regions in medaka, I observe significant changes in heart rate, heart morphology or arrhythmias already in the injected generation. The high isogenicity of medaka inbred lines, ease of the experimental setup & analysis in addition to the rapid gain of results, all pave the way for medaka as an exceptional model for high throughput functional gene validations.

Malte Wachsmuth LUXENDO, Fluorescence Microscopy Business Unit
Bruker Nano Surfaces Division

Luxendo Light-sheet Microscopy: Seeing Life from a Different Angle

Light-sheet microscopy has become the state of the art methodology to address a wide variety of biological questions. Key features of this technique are the extremely minimized phototoxicity, the high-speed image acquisition, and the large imaging depth. This allows long-term imaging of delicate samples in a volumetric manner. Fast subcellular processes and interactions can be observed in the comprehensive context of an organoid, organ, or entire organism. Different samples require different conditions, such that there are also various approaches how to image different samples with light-sheet microscopy. Being a company that is dedicated 100% to light-sheet microscopy, LUXENDO decided to reflect this fact by implementing specialized setups without losing general applicability for each of them. Here, we will introduce the basic concepts of light-sheet microscopy, followed by different implementations. To highlight the advantages suited for specific samples, we will focus on our two recently introduced products: the multiple-view selective plane illumination microscope (MuVi SPIM) and the inverted view selective plane illumination microscope (InVi SPIM). The MuVi-SPIM is a horizontal setup, that is designed to image large volumes very fast. The 4-fold geometry with its two-sided illumination combined with the two-sided detection allows optimal signal detection from anywhere in the sample without the need for rotation. Dedicated to live imaging, the InVi-SPIM is a microscope that is optimized for long-term 3D fluorescence imaging of living specimens. Its maximized photon efficiency, and short illumination times enable long-term imaging under ideal environmental conditions. The optical performance combined with the fast acquisition speed makes the InVi-SPIM perfectly suited for in toto imaging of a large variety of specimens, especially if they are sensitive and need precisely controlled conditions.

Clara Becker Universität Heidelberg, Germany
Centre for Organismal Studies

Growth control in the retinal stem cell niche of medaka

All species with life-long growth are facing similar challenges. Organs have to scale in size, while proliferation of stem and progenitor cells has to be tightly controlled to prevent aberrant growth of tissue. The teleost medaka (Oryzias latipes) is one of those species, and after completion of embryogenesis the retina continues to grow. Retinal stem cells (RSCs), located in the niche at the ciliary marginal zone, divide life-long, ultimately adding new neurons to the growing organ. Since the function of the eye depends on its shape, the activity of stem and progenitor cells is tightly coordinated to establish the proper cell type composition and number. My project combines advanced genetics and targeted modulation of signaling pathways in RSCs to address mechanisms of growth control in the RSC niche. I found that the activation of the insulin signaling pathway in RSCs is sufficient to stimulate massive proliferation. Here, the retina not only grows in radial size but also in thickness of the individual layers. The proliferation rate of stem and progenitor cells is a key determinant not only for eye size but also for its physiological properties. Strikingly we could only achieve this by the physiological signal, but not by oncogenic triggers of proliferation so far. Addressing this aspect, we found that RSCs are under surveillance of mononuclear phagocytic cells. The stem cells express a chemokine ligand attracting mononuclear phagocytic cells expressing the corresponding chemokine receptor. These cells form a network, maintained life-long, in close proximity to the RSC niche. Preliminary data point to an active role of immune cells in protecting and honing the RSC niche.

Anabela Bensimon-Brito Max Planck Institute for Heart and Lung Research, Germany
Dept. III – Developmental Genetics

Zebrafish heart valve regeneration: a model for valve recellularization

Heart valve diseases pose a major threat to human health worldwide, ultimately leading to heart failure and death. The majority of diseased valves are not repairable, and the only possible therapy relies on surgical replacement using mechanical implants or biological scaffolds. Recently, the clinical application of decellularized valves has shown promising results. However, complete recellularization of the implanted valves is still a challenge, which leads to extracellular matrix destabilization, and ultimately valve leaflet degradation. Therefore, identification of cellular and molecular factors promoting efficient recellularization of the implanted valve is crucial to ensure growth potential, repair and effective response to cardiac function demands. Zebrafish are known for their unusual ability to regenerate multiple organs and tissues. Here we show that, upon atrio-ventricular (AV) valve interstitial cell ablation using the Nitroreductase/Metronidazole (NTR/Mtz) system, adult zebrafish are capable of fully regenerating their cardiac valves. Taking advantage of multiple transgenic lines and detailed confocal imaging, we characterized the key stages of valve regeneration. We focused on the positive role of the inflammatory response, cell cycle re-entry, re-differentiation and reconstruction of lost tissue. Moreover, by combining a high-resolution approach of Laser Capture Microdissection with RNAseq, we established a transcriptomic profile of the regenerating valve and surrounding tissues. This dataset allowed us to identify potential pro-regenerative factors driving valve recellularization. Using overexpression transgenic lines and loss-of-function strategies, we are analyzing the role of chemokines and other secreted factors in promoting cell recruitment during valve regeneration. Overall we expect to bring new insights into the field of cardiac valve regeneration, with a particular focus on the molecular factors promoting valvular recellularization.

Michelle Collins Max Planck Institute for Heart and Lung Research, Germany
Developmental Genetics

Pitx2c orchestrates embryonic axis extension via mesendodermal cell migration and oriented cell division

Pitx2c, a homeodomain transcription factor, is classically known for its left-right patterning role. However, an early wave of pitx2 expression occurs at the onset of gastrulation in several species, indicating a possible earlier role that remains relatively unexplored. Here, we show that in zebrafish, maternal-zygotic (MZ) pitx2c mutants exhibit a shortened body axis indicative of convergence and extension (CE) defects. Live imaging reveals that MZpitx2c mutants display less persistent mesendodermal migration and randomly oriented cell divisions, which contribute to ineffective CE movements. Transplant experiments indicate that Pitx2c functions cell non-autonomously to regulate these cell behaviors by modulating cell shape and protrusive activity. Using transcriptomic analyses and candidate gene approaches, we identify transcriptional changes in components of the chemokine-ECM-integrin dependent mesendodermal migration network. Together, our results define pathways downstream of Pitx2 that are required during early embryogenesis, and reveal novel functions for Pitx2 as a regulator of morphogenesis.

Alexander Cook Universitätsmedizin der Johannes Gutenberg-Universität Mainz, Germany
Deutsches Resilienz Zentrum, DRZ

Developing a zebrafish model to identify novel molecular resilience mechanisms

Resilience is a dynamic and active process that ensures a trajectory of stable mental health during and after a traumatizing event or a prolonged period of stress, or a relatively rapid, successful recovery. Elucidating and understanding the underlying mechanisms behind this phenomenon is important to combat the negative trajectories non-resilient individuals’ experience. The zebrafish represents a newly established vertebrate animal model in stress research with a high degree of conservation of the stress response system, rendering it an attractive model system to discover molecules contributing to stress resilience. Both acute and chronic stressor paradigms have been reported for zebrafish. However, as of yet, there is no established resilience model, thus, our aim is to develop the first resilience model in zebrafish. Our first objective is to develop a chronic, ecologically relevant stressor in the form of a predator cue and subsequently detect behavioral alterations via multiple behavioral assays. Here we report the development of a high-throughput stressor delivery system suitable for simultaneous chronic stress exposure of a large number of adult zebrafish. The system consists of small monitors fixed above single-housing tanks displaying a ‘looming dot” of defined contrast and angular velocity, resembling an approaching predator. To robustly detect the effect of chronic stress on this behavior, we developed an “open tank” assay, comprising of a large novel environment, offering both a confined deep space and an open exposed space. An individuals’ choice between these two compartments is a measure of stress-induced alteration in exploratory behavior. We discuss the details of this behavioral paradigm and present results on how acute and chronic predator stress exposures alter adult zebrafish behavior in this assay.

Chaitanya Dingare Institute of Cell Biology and Neuroscience, Germany
Dept. of Developmental Biology of Vertebrates

The Hippo Pathway effector Taz is required for the formation of the Micropyle in Zebrafish

In some aquatic species such as teleost fishes, the process of fertilization is external and is mediated by a funnel-shaped opening on the vitelline membrane of the oocyte, called ‘Micropyle’. The micropyle constitutes a unique sperm entry point at the animal pole of the oocyte. It is left behind by a highly specialized follicular cell, the micropylar cell (MC) that is thought to block the formation of the vitelline membrane at the point of contact with the oocyte. Hardly anything is known on how only one cell of the follicular cell layer is first selected and then specified to become such a specialized structure. We recently uncovered that the hippo pathway effector Taz (encoded by the wwtr1 gene) plays a major role in this process. wwtr1 homozygous mutant females are infertile. We could show that this is due to a failure to specify a micropylar cell and, as a consequence, a micropyle. Although it has been clearly shown that the oocyte polarity plays an important role in the specification of the MC, we demonstrate that oocyte polarity is not affected in MZwwtr1 females. We further identified Taz as the first bona fide marker of the MC, and could show that Taz is restricted very early during oogenesis to only one cell, which later forms the functional MC. Finally we uncover cell properties of the MC as potential downstream effectors of Taz in the specification process. Altogether our results identify Taz as the first potential Master regulator of the micropylar cell fate, thus making also an important contribution to understanding fertility in teleost fishes.

Mohamed A. El-Brolosy MPI for heart and lung research, Germany
Developmental genetics

The mRNA surveillance machinery controls transcriptional adaptation to mutations

In response to deleterious mutations, genetic compensation by transcriptional upregulation of related gene(s) (also known as transcriptional adaptation) has been reported in numerous systems; however, how such a response is activated is unknown. We developed and analyzed several models of transcriptional adaptation in zebrafish and mouse and observed a correlation between mutant mRNA decay and transcriptional upregulation of related gene(s). To assess the role of the mutant mRNA in triggering transcriptional adaptation, we generated alleles that fail to transcribe the mutated gene and found that they do not exhibit this response. Moreover, genetic inactivation, silencing or chemical inhibition of the nonsense mediated decay factor Upf1 can also lead to loss of transcriptional adaptation. These results identify a new role for the mRNA surveillance machinery in buffering against mutations by triggering the transcriptional upregulation of related genes. In addition, these results will help design mutant alleles with minimal transcriptional adaptation-derived compensation.

Jakob Gierten Heidelberg University, Germany
COS

Genome-wide Association Study of Cardiac Phenotypes in Medaka Inbred Strains

Objectives: Congenital heart disease (CHD) is one of the most common human birth defects. However, only a minor fraction of CHD cases could be linked to genetic variants/mutations. The aim is to dissect genetic determinants of cardiac traits using medaka inbred strains. For a proof of concept genome-wide association study (GWAS) we focused on heart rate (HR) and its dependence on temperature. Methods: A panel of 100 medaka inbred strains with fully sequenced genomes was used as a mapping population (Loosli, Naruse, Birney, Wittbrodt; unpublished). The panel was profiled applying automated microscopy to score HR of medaka embryos in a temperature range of 21-35 °C. To resolve genetic elements controlling HR, we leveraged two medaka inbred strains with extreme phenotypes: Mop with fast HR and HdrR as reference with slow HR, which were subjected to F2 segregation analysis combining phenotyping with whole-genome sequencing (WGS). Results: WGS of the inbred panel revealed high levels of homozygosity within strains and millions of segregating SNPs across strains. Phenotyping a subset of the panel revealed substantial differences in HRs (max. 24%) and a high broad-sense heritability (H2 = 0.72). The F2 segregation analysis using Mop (fast HR) and HdrR (slow HR) demonstrated an intermediate phenotype in F1-hybrids and a phenotypic distribution in F2 spanning the parental phenotypic interval. To link phenotypic measurements to variants, WGS is currently performed in the F2 population. Conclusion: We present a genome-wide approach to dissect genetic determinants of human disease-relevant phenotypes in a medaka genomics resource. A segregation analysis, focused on HR, demonstrated a mode of complex phenotype inheritance. Strategies to detect and validate novel causative genetic elements are currently being established. We expect that downstream evaluation of candidate variants will indicate novel loci relevant for heart development, function and disease susceptibility.

Alexander A. Goloborodko Göttingen Center for Molecular Biosciences (GZMB), Germany
Developmental Biochemistry

Molecular Mechanisms of Germ Plasm Localization during Early Zebrafish Embryogenesis

The zebrafish germline is specified early during embryogenesis by inherited maternal RNAs and proteins called germ plasm. Only those cells containing germ plasm will become part of the germline, whereas other cells will commit to somatic cell fates. Therefore, proper localization of germ plasm is crucial for germ cell specification. To investigate germ plasm localization, we use the Bucky ball (Buc) gene discovered in our lab as a molecular proxy. Buc is indispensable for germ plasm aggregation in zebrafish. Moreover, Buc is the first protein inducing the formation of primordial germ cells in vivo. Fascinatingly, Buc protein mirrors germ plasm localization during all stages of zebrafish embryogenesis and oogenesis. For example, Buc is restricted to the cleavage furrows at the 4-cell stage, which will constitute the entire germline of the embryo. Interestingly, I found that a motif of 47 of 639 amino acids is necessary and sufficient for Buc localization. However, it was recently shown that the localization signal of the Buc homologue in Xenopus contains prion domains, which are responsible for protein aggregation (Boke et al., Cell, 2016). Nonetheless , I could show that Buc localization is independent of protein aggregation via prion domains. Furthermore, I found that Buc is recruited to cell-cell junctions, since it co-localizes with the adherens junction protein ZO-1 and hemidesmosomal protein integrin-alfa-5. Further co-localization experiments will determine the subtype of cell-cell junction, which anchors germ plasm to the cleavage furrow. In addition, I plan to use the localization domain as a bait to isolate the biochemical interaction network controlling Buc positioning in the early embryo. Taken together my experiments suggest that Buc acts as a scaffold recruiting germ plasm proteins to four cell-cell junctions, which then separate the germline from the soma during zebrafish embryogenesis.

Christian Helker Max Planck Institute for Heart and Lung Research, Germany
Department of Developmental Genetics

In vivo secretome-wide and chemical screening to identify novel regulators of pancreatic β-cell function

Diabetes Mellitus is foreseen to be the 7th leading cause of death in 2030, warranting a high demand to identify new therapeutics. Type 2 Diabetes, which affects 90% of diabetes patients, initiates with insulin resistance of the target tissue but only manifests in individuals where the β-cells cannot meet the higher demand for Insulin. Several in vitro drug screens have been carried out to identify signaling pathways inducing β-cell expansion and functionality. However, the drugs identified often fail in the following in vivo studies due to the xenobiotic defense mechanism of the organism. To monitor β-cell functionality in vivo, we established two zebrafish transgenic lines using the firefly luciferase as a sensitive and quantitative readout of pdx1 and insulin promoter activity. To specifically identify conserved and clinically relevant proteins, we screened a library of the human secretome (1700 cDNAs) as well as 7000 chemical compounds including FDA approved drugs. So far, our in vivo strategy has identified 29 human proteins that induce the insulin promoter and 7 small molecules that induce the pdx1 promoter. We further validated several of the small molecules and found that the hit compounds indeed induce β-cell function in primary human tissue samples as well as in a stem cell based human β-cell differentiation protocol. Thus, by using our in vivo high throughput screening strategy, we have been able to identify potential modulators of the activity of the pdx1 and insulin promoters in vivo. We speculate that some of these proteins and compounds will provide a beneficial effect when tested in diabetic patients.

Jens Kroll Heidelberg University, Germany
Vascular Biology

Loss of erythropoietin aggravates hyperglycemia induced renal damage and alters embryonic renal development via induction of apoptosis in zebrafish

Ravindra Peravali Karlsruhe Institute of Technology, Germany
Institute of Toxicology and Genetics

From Morphology to Behavior: Phenotyping Medaka Inbred Lines

With advances in genotyping and cost-effective sequencing technologies, Genome-wide association (GWA) studies have emerged as approaches to study the genetics of natural variation and gene-environment interaction. GWA studies are particularly useful when inbred lines are available (as once they are genotyped, these lines can be phenotyped multiple times) and also with the availability of automated image acquisition and analysis systems for rapid phenotyping. The teleost fish Medaka (Oryzias latipes) is an ideal model organism for these kinds of studies because of the presence of still free living wild populations in Japan and East Asia, the ability to generate highly polymorphic inbred lines and the availability of the complete genome sequence. We present the development of automated and intelligent screening platforms for high-throughput acquisition of morphometric and behavioral data. We characterize gross morphological features of different southern and northern inbred Medaka lines and evaluate the level of phenotypic variation between these lines. Furthermore, we show results of a quantitative assessment of fundamental behavioral traits such as locomotion and feeding in larval Medaka. Mathematical algorithms developed for morphometry and behavioral analysis will be discussed. The aim of this work is to identify a variety of phenotypic traits derived from morphology and behavior in order to assist in the investigation of the genetic basis for polymorphism in Medaka.

Ali Seleit COS, Germany
Animal Physiology

Neural stem cells induce the formation of their physical niche during organogenesis

Most organs rely on stem cells to maintain homeostasis during post-embryonic life. Typically, stem cells of independent lineages work coordinately within mature organs to ensure proper ratios of cell types. Little is known, however, on how these different stem cells locate to forming organs during development. Here we show that neuromasts of the posterior lateral line in medaka are composed of two independent life-long lineages with different embryonic origins. Clonal analysis and 4D imaging revealed a hierarchical organisation with instructing and responding roles: an inner, neural lineage induces the formation of an outer, border cell lineage (nBC) from the skin epithelium. Our results demonstrate that the neural lineage is necessary and sufficient to generate nBCs highlighting self-organisation principles at the level of the entire embryo. We hypothesise that induction of surrounding tissues plays a major role during the establishment of vertebrate stem cell niches.

Erika Tsingos Heidelberg University, Germany
Centre for Organismal Studies (COS)

Neural stem cells coordinate post-embryonic morphogenesis in the eye of medaka

How do anatomically and functionally distinct tissues coordinate to direct growth and shape in complex organs?
Teleost fish grow throughout life, providing an excellent system to address this question. The eye consists of many anatomically distinct tissues; i.e. cells in one tissue do not contribute new cells to other tissues in the organ. Thus, the tissues grow independently, and yet their growth rates must be precisely tuned to maintain the 3D shape crucial for visual function.
In medaka (Oryzias latipes), the neural retina (NR) and retinal pigmented epithelium (RPE) contain independent stem cells in a ring-shaped domain. In both tissues, cell size is constant, cell death is negligible, and there is no cell rearrangement. Therefore, the critical parameter that needs to be tuned to adjust tissue growth rate to organ growth rate is the proliferation of the tissue stem cells.
We explore conceptual modes of growth and shape-giving in the NR and RPE in an agent-based computational model. Using experimental lineage tracing in medaka, we validate the model predictions and introduce step-wise increments in complexity to explain the experimental findings. The combination of computational modelling and experiments uncovers that - despite the identical topology of NR and RPE - stem cells in the NR drive growth and control shape of the organ, whereas RPE stem cells follow external instructive signals.
Our work highlights how a minimal target node for evolution – the proliferation of neuroretinal stem cells – can be exploited to adapt complex organ morphogenesis in a vertebrate system.

Carina Beatrice Vibe EMBL Heidelberg, Germany
Developmental Biology Unit

Visualizing signaling oscillations during embryonic patterning in the Medaka model

During development, cells are required to interpret dynamic signals and coordinate a precise response in space and time. Our lab is interested in how embryonic patterning can be encoded in the dynamic properties of such signals. To explore the function of signaling dynamics in development, we study somitogenesis in vertebrate embryos. The timing of somitogenesis is controlled by a “segmentation clock” that periodically activates a system of oscillating genes in the presomitic mesoderm (PSM). It is known that these dynamic signals are important for somite formation, but how cells interpret such signals is an open question. While somitogenesis itself is a conserved process in vertebrates, the oscillatory signaling pathways involved vary between different species. For example, in the Mouse PSM this includes the Notch, Wnt and FGF pathways, while in the Zebrafish PSM there is only evidence for the involvement of the Notch pathway, notably the her genes. To complement existing models of vertebrate somitogenesis, we study signaling dynamics in the Japanese Medaka (Oryzias Latipes). Using CRISPR/Cas9, we aim to knock in reporters in the endogenous locus of molecular players in the Notch, Wnt and FGF pathways, followed by imaging of signaling oscillations in real time. I will present recent progress in the development and imaging of these dynamic reporters. Following this approach, we hope to discover which pathways are involved in the Medaka segmentation clock, and how general the principles of dynamic signal encoding are across vertebrate species. Ultimately, these reporter lines can be used not only to characterize the Medaka segmentation clock, but also to correlate signaling dynamics with spatial patterns through genetic and chemical perturbations.