ZebraReg-a novel platform for discovering regulators of cardiac regeneration using zebrafish.

Cardiovascular disease is the leading cause of death worldwide with myocardial infarction being the most prevalent. Currently, no cure is available to either prevent or revert the massive death of cardiomyocytes that occurs after a myocardial infarction. Adult mammalian hearts display a limited regeneration capacity, but it is insufficient to allow complete myocardial recovery. In contrast, the injured zebrafish heart muscle regenerates efficiently through robust proliferation of pre-existing myocardial cells. Thus, zebrafish allows its exploitation for studying the genetic programs behind cardiac regeneration, which may be present, albeit dormant, in the adult human heart. To this end, we have established ZebraReg, a novel and versatile automated platform for studying heart regeneration kinetics after the specific ablation of cardiomyocytes in zebrafish larvae. In combination with automated heart imaging, the platform can be integrated with genetic or pharmacological approaches and used for medium-throughput screening of presumed modulators of heart regeneration. We demonstrate the versatility of the platform by identifying both anti- and pro-regenerative effects of genes and drugs. In conclusion, we present a tool which may be utilised to streamline the process of target validation of novel gene regulators of regeneration, and the discovery of new drug therapies to regenerate the heart after myocardial infarction.

Interlaboratory Study on Zebrafish in Toxicology: Systematic Evaluation of the Application of Zebrafish in Toxicology's (SEAZIT's) Evaluation of Developmental Toxicity.

Embryonic zebrafish represent a useful test system to screen substances for their ability to perturb development. The exposure scenarios, endpoints captured, and data analysis vary among the laboratories who conduct screening. A lack of harmonization impedes the comparison of the substance potency and toxicity outcomes across laboratories and may hinder the broader adoption of this model for regulatory use. The Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative was developed to investigate the sources of variability in toxicity testing. This initiative involved an interlaboratory study to determine whether experimental parameters altered the developmental toxicity of a set of 42 substances (3 tested in duplicate) in three diverse laboratories. An initial dose-range-finding study using in-house protocols was followed by a definitive study using four experimental conditions: chorion-on and chorion-off using both static and static renewal exposures. We observed reasonable agreement across the three laboratories as 33 of 42 test substances (78.6%) had the same activity call. However, the differences in potency seen using variable in-house protocols emphasizes the importance of harmonization of the exposure variables under evaluation in the second phase of this study. The outcome of the Def will facilitate future practical discussions on harmonization within the zebrafish research community.

Morphometric analysis of developing zebrafish embryos allows predicting teratogenicity modes of action in higher vertebrates.

The early identification of teratogens in humans and animals is mandatory for drug discovery and development. Zebrafish has emerged as an alternative model to traditional preclinical models for predicting teratogenicity and other potential chemical-induced toxicity hazards. To prove its predictivity, we exposed zebrafish embryos from 0 to 96 h post fertilization to a battery of 31 compounds classified as teratogens or non-teratogens in mammals. The teratogenicity score was based on the measurement of 16 phenotypical parameters, namely heart edema, pigmentation, body length, eye size, yolk size, yolk sac edema, otic vesicle defects, otoliths defects, body axis defects, developmental delay, tail bending, scoliosis, lateral fins absence, hatching ratio, lower jaw malformations and tissue necrosis. Among the 31 compounds, 20 were detected as teratogens and 11 as non-teratogens, resulting in 94.44 % sensitivity, 90.91 % specificity and 87.10 % accuracy compared to rodents. These percentages decreased slightly when referred to humans, with 87.50 % sensitivity, 81.82 % specificity and 74.19 % accuracy, but allowed an increase in the prediction levels reported by rodents for the same compounds. Positive compounds showed a high correlation among teratogenic parameters, pointing out at general developmental delay as major cause to explain the physiological/morphological malformations. A more detailed analysis based on deviations from main trends revealed potential specific modes of action for some compounds such as retinoic acid, DEAB, ochratoxin A, haloperidol, warfarin, valproic acid, acetaminophen, dasatinib, imatinib, dexamethasone, 6-aminonicotinamide and bisphenol A. The high degree of predictivity and the possibility of applying mechanistic approaches makes zebrafish a powerful model for screening teratogenicity.

ZeOncoTest: Refining and Automating the Zebrafish Xenograft Model for Drug Discovery in Cancer.

The xenograft of human cancer cells in model animals is a powerful tool for understanding tumor progression and metastatic potential. Mice represent a validated host, but their use is limited by the elevated experimental costs and low throughput. To overcome these restrictions, zebrafish larvae might represent a valuable alternative. Their small size and transparency allow the tracking of transplanted cells. Therefore, tumor growth and early steps of metastasis, which are difficult to evaluate in mice, can be addressed. In spite of its advantages, the use of this model has been hindered by lack of experimental homogeneity and validation. Considering these facts, the aim of our work was to standardize, automate, and validate a zebrafish larvae xenograft assay with increased translatability and higher drug screening throughput. The ZeOncoTest reliability is based on the optimization of different experimental parameters, such as cell labeling, injection site, automated individual sample image acquisition, and analysis. This workflow implementation finally allows a higher precision and experimental throughput increase, when compared to previous reports. The approach was validated with the breast cancer cell line MDA-MB-231, the colorectal cancer cells HCT116, and the prostate cancer cells PC3; and known drugs, respectively RKI-1447, Docetaxel, and Mitoxantrone. The results recapitulate growth and invasion for all tested tumor cells, along with expected efficacy of the compounds. Finally, the methodology has proven useful for understanding specific drugs mode of action. The insights gained bring a step further for zebrafish larvae xenografts to enter the regulated preclinical drug discovery path.

Comparison of Zebrafish Larvae and hiPSC Cardiomyocytes for Predicting Drug-Induced Cardiotoxicity in Humans.

Cardiovascular drug toxicity is responsible for 17% of drug withdrawals in clinical phases, half of post-marketed drug withdrawals and remains an important adverse effect of several marketed drugs. Early assessment of drug-induced cardiovascular toxicity is mandatory and typically done in cellular systems and mammals. Current in vitro screening methods allow high-throughput but are biologically reductionist. The use of mammal models, which allow a better translatability for predicting clinical outputs, is low-throughput, highly expensive, and ethically controversial. Given the analogies between the human and the zebrafish cardiovascular systems, we propose the use of zebrafish larvae during early drug discovery phases as a balanced model between biological translatability and screening throughput for addressing potential liabilities. To this end, we have developed a high-throughput screening platform that enables fully automatized in vivo image acquisition and analysis to extract a plethora of relevant cardiovascular parameters: heart rate, arrhythmia, AV blockage, ejection fraction, and blood flow, among others. We have used this platform to address the predictive power of zebrafish larvae for detecting potential cardiovascular liabilities in humans. We tested a chemical library of 92 compounds with known clinical cardiotoxicity profiles. The cross-comparison with clinical data and data acquired from human induced pluripotent stem cell cardiomyocytes calcium imaging showed that zebrafish larvae allow a more reliable prediction of cardiotoxicity than cellular systems. Interestingly, our analysis with zebrafish yields similar predictive performance as previous validation meta-studies performed with dogs, the standard regulatory preclinical model for predicting cardiotoxic liabilities prior to clinical phases.

ZeGlobalTox: An Innovative Approach to Address Organ Drug Toxicity Using Zebrafish.

Toxicity is one of the major attrition causes during the drug development process. In that line, cardio-, neuro-, and hepatotoxicities are among the main reasons behind the retirement of drugs in clinical phases and post market withdrawal. Zebrafish exploitation in high-throughput drug screening is becoming an important tool to assess the toxicity and efficacy of novel drugs. This animal model has, from early developmental stages, fully functional organs from a physiological point of view. Thus, drug-induced organ-toxicity can be detected in larval stages, allowing a high predictive power on possible human drug-induced liabilities. Hence, zebrafish can bridge the gap between preclinical in vitro safety assays and rodent models in a fast and cost-effective manner. ZeGlobalTox is an innovative assay that sequentially integrates in vivo cardio-, neuro-, and hepatotoxicity assessment in the same animal, thus impacting strongly in the 3Rs principles. It Reduces, by up to a third, the number of animals required to assess toxicity in those organs. It Refines the drug toxicity evaluation through novel physiological parameters. Finally, it might allow the Replacement of classical species, such as rodents and larger mammals, thanks to its high predictivity (Specificity: 89%, Sensitivity: 68% and Accuracy: 78%).

Distribution of neurosensory progenitor pools during inner ear morphogenesis unveiled by cell lineage reconstruction.

Reconstructing the lineage of cells is central to understanding how the wide diversity of cell types develops. Here, we provide the neurosensory lineage reconstruction of a complex sensory organ, the inner ear, by imaging zebrafish embryos in vivo over an extended timespan, combining cell tracing and cell fate marker expression over time. We deliver the first dynamic map of early neuronal and sensory progenitor pools in the whole otic vesicle. It highlights the remodeling of the neuronal progenitor domain upon neuroblast delamination, and reveals that the order and place of neuroblasts' delamination from the otic epithelium prefigure their position within the SAG. Sensory and non-sensory domains harbor different proliferative activity contributing distinctly to the overall growth of the structure. Therefore, the otic vesicle case exemplifies a generic morphogenetic process where spatial and temporal cues regulate cell fate and functional organization of the rudiment of the definitive organ.

The Order and Place of Neuronal Differentiation Establish the Topography of Sensory Projections and the Entry Points within the Hindbrain.

Establishing topographical maps of the external world is an important but still poorly understood feature of the vertebrate sensory system. To study the selective innervation of hindbrain regions by sensory afferents in the zebrafish embryo, we mapped the fine-grained topographical representation of sensory projections at the central level by specific photoconversion of sensory neurons. Sensory ganglia located anteriorly project more medially than do ganglia located posteriorly, and this relates to the order of sensory ganglion differentiation. By single-plane illumination microscopy (SPIM) in vivo imaging, we show that (1) the sequence of arrival of cranial ganglion inputs predicts the topography of central projections, and (2) delaminated neuroblasts differentiate in close contact with the neural tube, and they never loose contact with the neural ectoderm. Afferent entrance points are established by plasma membrane interactions between primary differentiated peripheral sensory neurons and neural tube border cells with the cooperation of neural crest cells. These first contacts remain during ensuing morphological growth to establish pioneer axons. Neural crest cells and repulsive slit1/robo2 signals then guide axons from later-differentiating neurons toward the neural tube. Thus, this study proposes a new model by which the topographical representation of cranial sensory ganglia is established by entrance order, with the entry points determined by cell contact between the sensory ganglion cell bodies and the hindbrain.

Cell lineage analysis reveals three different progenitor pools for neurosensory elements in the otic vesicle.

In the inner ear, sensory versus neuronal specification is achieved through few well-defined bHLH transcription factors. However, the molecular mechanisms regulating the generation of the appropriate cell type in the correct place and at the correct time are not completely understood yet. Various studies have shown that hair cell- and neuron-specifying genes partially overlap in the otic territory, suggesting that mutual interactions among these bHLH factors could direct the generation of the two cell types from a common neurosensory progenitor. Although there is little evidence for a clonal relationship between macular hair cells and sensory neurons, the existence of a single progenitor able to give both sensory and neuronal cell types remains an open question. Here, we identified a population of common neurosensory progenitors in the zebrafish inner ear and studied the proneural requirement for cell fate decision within this population. Expression analysis reveals that proneural genes for hair cells and neurons overlap within the posteromedial otic epithelium. Combined results from single-cell lineage and functional studies on neurog1 and neuroD1 further demonstrate the following: (1) in the anterior region of the ear, neuronal and sensory lineages have already segregated at the onset of proneural gene expression and are committed to a given fate very early; (2) in contrast, the posteromedial part of the ear harbors a population of common progenitors giving both neurons and hair cells until late stages; and finally (3) neuroD1 is required within this pool of bipotent progenitors to generate the hair cell fate.