Synthesis and Structure of Novel Phenothiazine Derivatives, and Compound Prioritization via In Silico Target Search and Screening for Cytotoxic and Cholinesterase Modulatory Activities in Liver Cancer Cells and In Vivo in Zebrafish.

Phenothiazines (PTZ) are antipsychotics known to modulate a variety of neurotransmitter activities that include dopaminergic and cholinergic signaling and have been identified as potential anticancer agents in vitro. However, it is important to also test whether a highly cytotoxic, repurposed, or novel PTZ has low toxicity and neuromodulatory activity in vivo using vertebrate model organisms, such as zebrafish. In this study, we synthesized novel phenothiazines and screened them in vitro in liver cancer and in vivo in zebrafish embryos/larvae. The syntheses of several intermediate PTZ 10-yl acyl chlorides were followed by elemental analysis and determination of 1H NMR and 13C NMR mass (ESI+) spectra of a large number of novel PTZ 10-carboxamides. Cytotoxicities of 28 PTZ derivatives (1-28) screened against Hep3B and SkHep1 liver cancer cell lines revealed five intermediate and five novel leads along with trifluoperazine (TFP), prochlorperazine (PCP), and perphenazine, which are relatively more cytotoxic than the basic PTZ core. Overall, the derivatives were more cytotoxic to Hep3B than SkHep1 cells. Moreover, in silico target screening identified cholinesterases as some of the commonest targets of the screened phenothiazines. Interestingly, molecular docking studies with acetylcholinesterase (AChE) and butyrylcholinesterase proteins showed that the most cytotoxic compounds 1, 3, PCP, and TFP behaved similar to Huprin W in their amino acid interactions with the AChE protein. The highly cytotoxic intermediate PTZ derivative 1 exhibited a relatively lower toxicity profile than those of 2 and 3 during the zebrafish development. It also modulated in vivo the cholinesterase activity in a dose-dependent manner while significantly increasing the total cholinesterase activity and/or ACHE mRNA levels, independent of the liver cancer cell type. Our screen also identified novel phenothiazines, i.e., 8 and 10, with significant cytotoxic and cholinesterase modulatory effects in liver cancer cells; yet both compounds had low levels of toxicity in zebrafish. Moreover, they modulated the cholinesterase activity or expression of ACHE in a cancer cell line-specific manner, and compound 10 significantly inhibited the cholinesterase activity in zebrafish. Accordingly, using a successful combination of in silico, in vitro, and in vivo approaches, we identified several lead anticancer and cholinesterase modulatory PTZ derivatives for future research.

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Droplet Microarray Based Screening Identifies Proteins for Maintaining Pluripotency of hiPSCs.

Human induced pluripotent stem cells (hiPSCs) are crucial for disease modeling, drug discovery, and personalized medicine. Animal-derived materials hinderapplications of hiPSCs in medical fields. Thus, novel and well-defined substrate coatings capable of maintaining hiPSC pluripotency are important for advancing biomedical applications of hiPSCs. Here a miniaturized droplet microarray (DMA) platform to investigate 11 well-defined proteins, their 55 binary and 165 ternary combinations for their ability to maintainpluripotency of hiPSCs when applied as a surface coating, is used. Using this screening approach, ten protein group coatings are identified, which promote significantly higher NANOG expression of hiPSCs in comparison with Matrigel coating. With two of the identified coatings, long-term pluripotency maintenance of hiPSCs and subsequent differentiation into three germ layers are achieved. Compared with conventional high-throughput screening (HTS) in 96-well plates, the DMA platform uses only 83 µL of protein solution (0.83 µg total protein) and only ≈2.8 × 105 cells, decreasing the amount of proteins and cells ≈860 and 25-fold, respectively. The identified proteins will be essential for research and applications using hiPSCs, while the DMA platform demonstrates great potential for miniaturized HTS of scarce cells or expensive materials such as recombinant proteins.

A chemical probe for BAG1 targets androgen receptor-positive prostate cancer through oxidative stress signaling pathway.

BAG1 is a family of polypeptides with a conserved C-terminal BAG domain that functions as a nucleotide exchange factor for the molecular chaperone HSP70. BAG1 proteins also control several signaling processes including proteostasis, apoptosis, and transcription. The largest isoform, BAG1L, controls the activity of the androgen receptor (AR) and is upregulated in prostate cancer. Here, we show that BAG1L regulates AR dynamics in the nucleus and its ablation attenuates AR target gene expression especially those involved in oxidative stress and metabolism. We show that a small molecule, A4B17, that targets the BAG domain downregulates AR target genes similar to a complete BAG1L knockout and upregulates the expression of oxidative stress-induced genes involved in cell death. Furthermore, A4B17 outperformed the clinically approved antagonist enzalutamide in inhibiting cell proliferation and prostate tumor development in a mouse xenograft model. BAG1 inhibitors therefore offer unique opportunities for antagonizing AR action and prostate cancer growth.

In Vitro and In Vivo Toxicity Evaluation of Natural Products with Potential Applications as Biopesticides.

The use of natural products in agriculture as pesticides has been strongly advocated. However, it is necessary to assess their toxicity to ensure their safe use. In the present study, mammalian cell lines and fish models of the zebrafish (Danio rerio) and medaka (Oryzias latipes) have been used to investigate the toxic effects of ten natural products which have potential applications as biopesticides. The fungal metabolites cavoxin, epi-epoformin, papyracillic acid, seiridin and sphaeropsidone, together with the plant compounds inuloxins A and C and ungeremine, showed no toxic effects in mammalian cells and zebrafish embryos. Conversely, cyclopaldic and α-costic acids, produced by Seiridium cupressi and Dittrichia viscosa, respectively, caused significant mortality in zebrafish and medaka embryos as a result of yolk coagulation. However, both compounds showed little effect in zebrafish or mammalian cell lines in culture, thus highlighting the importance of the fish embryotoxicity test in the assessment of environmental impact. Given the embryotoxicity of α-costic acid and cyclopaldic acid, their use as biopesticides is not recommended. Further ecotoxicological studies are needed to evaluate the potential applications of the other compounds.

Miniaturized Drug Sensitivity and Resistance Test on Patient-Derived Cells Using Droplet-Microarray.

Testing the sensitivity of patient-derived tumor cells ex vivo can potentially help determining the appropriate treatment for each patient and spot the development of resistance to a given therapy. The number of cells obtainable from a biopsy is, however, often insufficient for performing ex vivo tests in conventional microtiter plates. Here, we introduce a novel Droplet-Microarray platform based on a hydrophilic-superhydrophobic patterned surface that enables screenings using only 100 cells and 30 picomoles of a drug per individual nanoliter-sized droplet. We demonstrate that the dose-response of as few as 100 primary patient-derived chronic lymphocytic leukemia (CLL) cells to anticancer compounds on the Droplet-Microarray platform resembles the dose-response obtained in 384-well plates requiring 20,000 tumor cells per experiment. The extremely miniaturized Droplet-Microarray platform thus carries great potential for ex vivo drug sensitivity and resistance tests on patient-derived tumor cells and potentially for implementing such tests in medical practice of precision medicine.

Chronic hyperammonemia causes a hypoglutamatergic and hyperGABAergic metabolic state associated with neurobehavioral abnormalities in zebrafish larvae.

Chronic hyperammonemia is a common condition affecting individuals with inherited urea cycle disorders resulting in progressive cognitive impairment and behavioral abnormalities. Altered neurotransmission has been proposed as major source of neuronal dysfunction during chronic hyperammonemia, but the molecular pathomechanism has remained incompletely understood. Here we show that chronic exposure to ammonium acetate induces locomotor dysfunction and abnormal feeding behavior in zebrafish larvae, indicative for an impairment of higher brain functions. Biochemically, chronically elevated ammonium concentrations cause enhanced activity of glutamate decarboxylase isoforms GAD1 and GAD2 with increased formation of GABA and concomitant depletion of glutamate, ultimately leading to a dysfunctional hypoglutamatergic and hyperGABAergic metabolic state. Moreover, elevated GABA concentrations are accompanied by increased expression of GABAA receptor subunits alpha-1, gamma-2 and delta, supporting the notion of an increased GABA tone in chronic hyperammonemia. Propionate oxidation as major anaplerotic reaction sufficiently compensates for the transamination-dependent withdrawal of 2-oxoglutarate, thereby preventing bioenergetic dysfunction under chronic hyperammonemic conditions. Thus, our study extends the hypothesis of alterations in the glutamatergic and GABAergic system being an important pathophysiological factor causing neurobehavioral impairment in chronic hyperammonemia. Given that zebrafish larvae have already been successfully used for high-throughput identification of novel compounds to treat inherited neurological diseases, the reported zebrafish model should be considered an important tool for systematic drug screening targeting altered glutamatergic and GABAergic metabolism under chronic hyperammonemic conditions in the future.

High-Throughput Screening of Cell Transfection Enhancers Using Miniaturized Droplet Microarrays.

DNA delivery is a powerful research tool for biological research and clinical therapies. However, many nonviral transfection reagents have relatively low transfection efficiency. It is hypothesized that by treating cells with small molecules, the transfection efficiency can be improved. However, in order to identify such transfection-enhancing molecules, thousands of molecules must be tested. Current high-throughput screening (HTS) technologies based on microtiter plates are not suitable for such screenings due to the prohibitively high costs of reagents and operation. Here, the use of the droplet microarray (DMA) platform to screen 774 FDA-approved drugs with CHO-K1, Jurkat and HEK293T cells is reported. The volume of individual aqueous compartments is 20 nL, requiring 0.84 mL of cell suspension and 200 pmoles of each drug (total 0.02 moles) to perform the screening. Thus, the requirement for cells and reagents is 2500 times less than that for the same experiment performed in 384-well plates. The results reveal the potential of the DMA platform as a more cost-effective and less labor-intensive approach to HTS. Furthermore, an increase (approximately two- to fivefold) in transfection efficiency is achieved by treating cells with some molecules. This study clearly demonstrates the potential of the DMA platform for miniaturization of biochemical and cellular HTS.

Silica Nanoparticles Provoke Cell Death Independent of p53 and BAX in Human Colon Cancer Cells.

Several in vitro studies have suggested that silica nanoparticles (NPs) might induce adverse effects in gut cells. Here, we used the human colon cancer epithelial cell line HCT116 to study the potential cytotoxic effects of ingested silica NPs in the presence or absence of serum. Furthermore, we evaluated different physico-chemical parameters important for the assessment of nanoparticle safety, including primary particle size (12, 70, 200, and 500 nm) and surface modification (-NH2 and -COOH). Silica NPs triggered cytotoxicity, as evidenced by reduced metabolism and enhanced membrane leakage. Automated microscopy revealed that the silica NPs promoted apoptosis and necrosis proportional to the administered specific surface area dose. Cytotoxicity of silica NPs was suppressed by increasing amount of serum and surface modification. Furthermore, inhibition of caspases partially prevented silica NP-induced cytotoxicity. In order to investigate the role of specific cell death pathways in more detail, we used isogenic derivatives of HCT116 cells which lack the pro-apoptotic proteins p53 or BAX. In contrast to the anticancer drug cisplatin, silica NPs induced cell death independent of the p53-BAX axis. In conclusion, silica NPs initiated cell death in colon cancer cells dependent on the specific surface area and presence of serum. Further studies in vivo are warranted to address potential cytotoxic actions in the gut epithelium. The unintended toxicity of silica NPs as observed here could also be beneficial. As loss of p53 in colon cancer cells contributes to resistance against anticancer drugs, and thus to reoccurrence of colon cancer, targeted delivery of silica NPs could be envisioned to also deplete p53 deficient tumor cells.

Delay in development and behavioural abnormalities in the absence of p53 in zebrafish.

p53 is well-known for its tumour-suppressive activity. However, in the past decade it became clear that p53 is also involved in other processes including stem cell proliferation, differentiation and animal development. To investigate the role of p53 in early embryonic development, we targeted p53 by CRISPR/Cas9 to make a p53 knock-out zebrafish (Danio rerio). Our data show developmental and behavioural effects in p53-deficient zebrafish embryos and larvae. Specifically, we found that early development of zebrafish was clearly delayed in the absence of p53. However, after 1 day (1 dpf), the p53-deficient embryos appeared to recover, as evidenced by a similar level of pigmentation at 26 hpf, similar size of the eye at 4 dpf and only a minor difference in body size at 4 dpf compared to p53 wild-type siblings. The recovery of development after 1 dpf in p53-deficient embryos could be due to a compensatory mechanism involving other p53 family members. p63 and p73 were found over-expressed with respect to wild-type siblings. However, despite this adaptation, the hatching time remained delayed in p53-/- zebrafish. In addition to differences in development, p53-null zebrafish embryos also showed differences in behaviour. We observed an overall reduced activity and a reduced travel distance under non-stressed conditions and after exposing the larvae to vibration. We also observed a longer latency until the larvae started to move after touching with a needle. Overall, these data indicate that p53 is involved in early development and locomotion activities.

DIY Automated Feeding and Motion Recording System for the Analysis of Fish Behavior.

Fish species such as medaka or zebrafish are widely used as animal models to study physiology, disease development, and treatment efficacy. They are also used to study the rapidly growing field of behavior research, such as social interactions, anxiety, and the influence of environmental factors. Here we describe an automated experimental setup allowing the recording of general locomotor activity in combination with a food-on-demand system. It can simply be built with some basic electronic knowledge. Our setup enables the recording of locomotor and feeding activity of several fish for long-term studies, excluding disturbing external influences. A description of the automated recording system is given, as well as examples of recordings to illustrate its applicability for the study of fish behavior. The construction manual and operation instructions can be downloaded for free.

The HMG box transcription factors Sox1a and Sox1b specify a new class of glycinergic interneuron in the spinal cord of zebrafish embryos.

Specification of neurons in the spinal cord relies on extrinsic and intrinsic signals, which in turn are interpreted by expression of transcription factors. V2 interneurons develop from the ventral aspects of the spinal cord. We report here a novel neuronal V2 subtype, named V2s, in zebrafish embryos. Formation of these neurons depends on the transcription factors sox1a and sox1b. They develop from common gata2a- and gata3-dependent precursors co-expressing markers of V2b and V2s interneurons. Chemical blockage of Notch signalling causes a decrease in V2s and an increase in V2b cells. Our results are consistent with the existence of at least two types of precursor arranged in a hierarchical manner in the V2 domain. V2s neurons grow long ipsilateral descending axonal projections with a short branch at the ventral midline. They acquire a glycinergic neurotransmitter type during the second day of development. Unilateral ablation of V2s interneurons causes a delay in touch-provoked escape behaviour, suggesting that V2s interneurons are involved in fast motor responses.

An automated screening method for detecting compounds with goitrogenic activity using transgenic zebrafish embryos.

The knowledge on environmentally relevant chemicals that may interfere with thyroid signaling is scarce. Here, we present a method for the screening of goitrogens, compounds that disrupt the thyroid gland function, based on the automatic orientation of zebrafish in a glass capillary and a subsequent imaging of reporter gene fluorescence in the thyroid gland of embryos of the transgenic zebrafish line tg(tg:mCherry). The tg(tg:mCherry) reporter gene indicates a compensatory upregulation of thyroglobulin, the thyroid hormone precursor, in response to inhibition of thyroid hormone synthesis. Fish embryos were exposed to a negative control compound (3,4-dichloroaniline), or a concentration series of known goitrogenic compounds (resorcinol, methimazole, potassium perchlorate, 6-propyl-2-thiouracil, ethylenethiourea, phloroglucinol, pyrazole) with maximum exposure concentration selected based on mortality and/or solubility. Exposure to 3,4-dichloroaniline decreased the fluorescence signal. All goitrogenic compounds exhibited clear concentration-dependent inductions of reporter fluorescence 1.4 to 2.6 fold above control levels. Concentration-response modelling was used to calculate goitrogenic potencies based on EC50 values. The new automated method offers an efficient screening approach for goitrogenic activity.

Zebrafish: A Pharmacogenetic Model for Anesthesia.

General anesthetics are small molecules that interact with and effect the function of many different proteins to promote loss of consciousness, amnesia, and sometimes, analgesia. Owing to the complexity of this state transition and the transient nature of these drug/protein interactions, anesthetics can be difficult to study. The zebrafish is an emerging model for the discovery of both new genes required for the response to and side effects of anesthesia. Here we discuss the tools available to manipulate the zebrafish genome, including both genetic screens and genome engineering approaches. Additionally, there are various robust behavior assays available to study anesthetic and other drug responses. These assays are available for single-gene study or high throughput for genetic or drug discovery. Finally, we present a case study of using propofol as an anesthetic in the zebrafish. These techniques and protocols make the zebrafish a powerful model to study anesthetic mechanisms and drug discovery.

Microscopy-based high-throughput assays enable multi-parametric analysis to assess adverse effects of nanomaterials in various cell lines.

Manufactured nanomaterials (MNMs) selected from a library of over 120 different MNMs with varied compositions, sizes, and surface coatings were tested by four different laboratories for toxicity by high-throughput/-content (HT/C) techniques. The selected particles comprise 14 MNMs composed of CeO2, Ag, TiO2, ZnO and SiO2 with different coatings and surface characteristics at varying concentrations. The MNMs were tested in different mammalian cell lines at concentrations between 0.5 and 250 µg/mL to link physical-chemical properties to multiple adverse effects. The cell lines are derived from relevant organs such as liver, lung, colon and the immune system. Endpoints such as viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied. Soluble MNMs, Ag and ZnO, were toxic in all cell types. TiO2 and SiO2 MNMs also triggered toxicity in some, but not all, cell types and the cell type-specific effects were influenced by the specific coating and surface modification. CeO2 MNMs were nearly ineffective in our test systems. Differentiated liver cells appear to be most sensitive to MNMs, Whereas most of the investigated MNMs showed no acute toxicity, it became clear that some show adverse effects dependent on the assay and cell line. Hence, it is advised that future nanosafety studies utilise a multi-parametric approach such as HT/C screening to avoid missing signs of toxicity. Furthermore, some of the cell type-specific effects should be followed up in more detail and might also provide an incentive to address potential adverse effects in vivo in the relevant organ.

Evaluation of the Droplet-Microarray Platform for High-Throughput Screening of Suspension Cells.

Phenotypic cell-based high-throughput screenings play a central role in drug discovery and toxicology. The main tendency in cell screenings is the increase of the throughput and decrease of reaction volume in order to accelerate the experiments, reduce the costs, and enable screenings of rare cells. Conventionally, cell-based assays are performed in microtiter plates, which exist in 96- to 1536-wells formats and cannot be further miniaturized. In addition, performing screenings of suspension cells is associated with risk of losing cell content during the staining procedures and incompatibility with high-content microscopy. Here, we evaluate the Droplet-Microarray screening platform for culturing, screening, and imaging of suspension cells. We demonstrate pipetting-free cell seeding and proliferation of cells in individual droplets of 3-80 nL in volume. We developed a methodology to perform parallel treatment, staining, and fixation of suspension cells in individual droplets. Automated imaging of live suspension cells directly in the droplets combined with algorithms for pattern recognition for image analysis is demonstrated. We evaluated the developed methodology by performing a dose-response study with antineoplastic drugs. We believe that the DMA screening platform carries great potential to be adopted for broad spectrum of screenings of suspension cells.

Automated phenotype pattern recognition of zebrafish for high-throughput screening.

Over the last years, the zebrafish (Danio rerio) has become a key model organism in genetic and chemical screenings. A growing number of experiments and an expanding interest in zebrafish research makes it increasingly essential to automatize the distribution of embryos and larvae into standard microtiter plates or other sample holders for screening, often according to phenotypical features. Until now, such sorting processes have been carried out by manually handling the larvae and manual feature detection. Here, a prototype platform for image acquisition together with a classification software is presented. Zebrafish embryos and larvae and their features such as pigmentation are detected automatically from the image. Zebrafish of 4 different phenotypes can be classified through pattern recognition at 72 h post fertilization (hpf), allowing the software to classify an embryo into 2 distinct phenotypic classes: wild-type versus variant. The zebrafish phenotypes are classified with an accuracy of 79-99% without any user interaction. A description of the prototype platform and of the algorithms for image processing and pattern recognition is presented.

An automated and high-throughput Photomotor Response platform for chemical screens.

The zebrafish (Danio rerio) is a well-established vertebrate model organism. Its embryos are used extensively in biology and medicine to perform chemical screens to identify drug candidates or to evaluate teratogenicity and embryotoxicity of substances. Behavioral readouts are increasingly used to assess the effects of compounds on the nervous system. Early stage zebrafish show characteristic behavioral features at stages between 30 and 42 hours post fertilization (hpf) when exposed to a short and bright light flash. This so-called Photomotor Response (PMR) is a reaction of the nervous system of the fish and can be used as a marker in screenings for neuroactive chemicals. To probe a broad and diverse chemical space, many different substances have to be tested and repeated observations are necessary to warrant statistical significance of the results. Although PMR-based chemical screens must use a large number of specimens, there is no sophisticated, automated high-throughput platform available which ensures minimal human intervention. Here we report a PMR platform that was developed by combining an improved automatic sample handling with a remotely controllable microscope setup and an image analysis pipeline. Using infrared illumination during automatic sample preparation, we were able to eliminate excess amounts of visible light that could potentially alter the response results. A remotely controlled microscope setup allows us to screen entire 96-well microtiter plates without human presence that could disturb the embryos. The development of custom video analysis software, including single egg detection, enables us to detect variance among treated specimens and extract easy to interpret numerical values representing the PMR motion. By testing several neuroactive compounds we validated the workflow that can be used to analyze more than one thousand zebrafish eggs on a single 96-well plate.

Differential responsiveness of distinct retinal domains to Atoh7.

During vertebrate eye development retinal progenitor cells (RPCs) differentiate into all neural cell types of the retina. Retinal ganglion cells (RGCs) represent the first cell type to be generated. For their development, Atoh7, a basic Helix Loop Helix (bHLH) transcription factor is crucial. Atoh7 loss of function results in a massive reduction or even a total loss of RGCs. However, inconsistent results have been obtained in atoh7 gain of function experiments with respect to ganglion cell genesis, implying that the effect of Atoh7 is likely to be dependent on the competence state of the RPC. In this study we addressed the differential susceptibilities of early RPCs to Atoh7 in vivo, using medaka. Unexpectedly, we observed a largely normal development of the dorsal retina, although atoh7 was precociously expressed. However, the development of the retina close to the optic nerve head (part of the ventral retina) was disturbed severely. Photoreceptors were largely absent and the Müller glia cell number was reduced significantly. The majority of cells in this domain were ganglion cells and the abnormal development of this area affected the closure of the optic fissure resulting in coloboma.

Genomic and phenotypic characterization of a wild medaka population: towards the establishment of an isogenic population genetic resource in fish.

Oryzias latipes (medaka) has been established as a vertebrate genetic model for more than a century and recently has been rediscovered outside its native Japan. The power of new sequencing methods now makes it possible to reinvigorate medaka genetics, in particular by establishing a near-isogenic panel derived from a single wild population. Here we characterize the genomes of wild medaka catches obtained from a single Southern Japanese population in Kiyosu as a precursor for the establishment of a near-isogenic panel of wild lines. The population is free of significant detrimental population structure and has advantageous linkage disequilibrium properties suitable for the establishment of the proposed panel. Analysis of morphometric traits in five representative inbred strains suggests phenotypic mapping will be feasible in the panel. In addition, high-throughput genome sequencing of these medaka strains confirms their evolutionary relationships on lines of geographic separation and provides further evidence that there has been little significant interbreeding between the Southern and Northern medaka population since the Southern/Northern population split. The sequence data suggest that the Southern Japanese medaka existed as a larger older population that went through a relatively recent bottleneck approximately 10,000 years ago. In addition, we detect patterns of recent positive selection in the Southern population. These data indicate that the genetic structure of the Kiyosu medaka samples is suitable for the establishment of a vertebrate near-isogenic panel and therefore inbreeding of 200 lines based on this population has commenced. Progress of this project can be tracked at http://www.ebi.ac.uk/birney-srv/medaka-ref-panel.