With the US Environment Protection Agency reducing requests for (and funding of) mammalian studies alongside the proposed elimination of requests by 2035, there is an urgent need for fully validated New Approach Methods (NAMs) to fill the resultant gap for safety assessment of agrochemicals. One promising NAM for assessing the potential for human prenatal developmental toxicity potential is the Zebrafish Embryo Developmental Toxicity Assessment, a bioassay that has been used by the pharmaceutical industry for more than a decade in early-stage drug safety assessment. Despite its promise, little data has been generated to assess the validity of ZEDTA for assessing Developmental and Reproductive Toxicity of new agrochemical products. Addressing this knowledge gap, we tested 67 compounds (insecticides, herbicides and fungicides) spanning multiple different chemical groupings and mechanisms of action. ZEDTA assay results were compared with the European Chemicals Agency (ECHA) Classification and Labelling (C&L) for mammalian hazard classification and with publicly available data to determine the ZEDTA’s translation power. Overall, the ZEDTA assay had an effective detection capability of 65 % for sensitivity and 64 % for specificity as compared with the ECHA-C&L classification and publicly available data. Comparing the ZEDTA data there were both strengths and weaknesses in alignments for across the different chemical classes and chemical mechanisms of action. Overall, the data generated, show the performance of the ZEDTA assay was comparable with other bioassays highlighted as alternatives for mammalian assessment and holds good promise as a NAM for screening agrochemical prenatal developmental toxicity during new product human safety assessment.

Early life stress (ELS) is one of the strongest risk factors for developing psychiatric disorders in humans. As conserved key stress hormones of vertebrates, glucocorticoids (GCs) are thought to play an important role in mediating the effects of ELS exposure in shaping adult phenotypes. In this process, early exposure to high level of GCs may induce molecular changes that alter developmental trajectory of an animal and primes differential adult responses. However, comprehensive characterization of identities of molecules that are targeted by developmental GC exposure is currently lacking. In our study, we describe lifelong molecular consequences of high level of developmental GC exposure using an optogenetic zebrafish model. First, we developed a new double-hit stress model using zebrafish by combining exposure to a high endogenous GC level during development and acute adulthood stress exposure. Our results establish that similar to ELS-exposed humans and rodents, developmental GC exposed zebrafish model shows altered behavior and stress hypersensitivity in adulthood. Second, we generated time-series gene expression profiles of the brains in larvae, in adult, and upon stress exposure to identify molecular alterations induced by high developmental GC exposure at different developmental stages. Third, we identify a set of GC-primed genes that show altered expression upon acute stress exposure only in animals exposed to a high developmental GC. Interestingly, our datasets of GC primed genes are enriched in risk factors identified for human psychiatric disorders. Lastly, we identify potential epigenetic regulatory elements and associated post-transcriptional modifications following high developmental GC exposure. Thus, we present a translationally relevant zebrafish model for studying stress hypersensitivity and alteration of behavior induced by exposure to elevated GC levels during development. Our study provides comprehensive datasets delineating potential molecular targets underlying the impact of developmental high GC exposure on adult responses. ### Competing Interest Statement SR holds a patent, European patent number 2928288 and US patent number 10,080,355: A novel inducible model of stress. The remaining authors declare no known competing interests.

Early life stress (ELS) is a major risk factor for developing psychiatric disorders, with glucocorticoids (GCs) implicated in mediating its effects in shaping adult phenotypes. In this process, exposure to high levels of developmental GC (hdGC) is thought to induce molecular changes that prime differential adult responses. However, identities of molecules targeted by hdGC exposure are not completely known. Here, we describe lifelong molecular consequences of hdGC exposure using a newly developed zebrafish double-hit stress model, which shows altered behaviors and stress hypersensitivity in adulthood. We identify a set of primed genes displaying altered expression only upon acute stress in hdGC-exposed adult fish brains. Interestingly, this gene set is enriched in risk factors for psychiatric disorders in humans. Lastly, we identify altered epigenetic regulatory elements following hdGC exposure. Thus, our study provides comprehensive datasets delineating potential molecular targets mediating the impact of hdGC exposure on adult responses.

Exposure to excess glucocorticoid (GC) during early development is implicated in adult dysfunctions. Reduced adult hippocampal neurogenesis is a well-known consequence of exposure to early life stress or elevated GC, however the effects on neurogenesis during development and effects on other brain regions are not well understood. Using an optogenetic zebrafish model, here we analyse the effects of GC exposure on neurogenesis during development in the whole brain. We identify that the hypothalamus is a highly GC-sensitive region where elevated GC causes precocious development. This is followed by failed maturation and early decline accompanied by impaired feeding, growth, and survival. In GC-exposed animals, the developmental trajectory of hypothalamic progenitor cells is strikingly altered, potentially mediated by direct regulation of transcription factors such as rx3 by GC. Our data provide cellular and molecular level insight into GC-induced alteration of the hypothalamic developmental trajectory, a process crucial for health across the life-course.

AbstractChronic or severe stress during development increases psychiatric disease susceptibility. One putative mechanism involves priming of stress-sensitive gene networks by early life Glucocorticoids (GCs) exposure, which modifies responses to stress later in life. However, brain-wide GC-primed molecular alterations are currently unknown. Here, we used an optogenetic zebrafish model to increase endogenous GCs during development and identified comprehensive transcriptomic alterations in the whole brain of GC-exposed fish across the life course. When exposed to acute stress in adulthood, developmental GC-exposed animals show highly exaggerated and pervasive brain-wide transcriptional changes which include gene sets associated with axon development, neuronal signaling, and epigenetic modulators. Strikingly, these altered gene sets showed an overrepresentation of human psychiatric disorder risk genes. We identify in developmental GC-exposed individuals: 1) altered biological processes, 2) hitherto uncharacterized novel GC-primed genes, 3) altered adult behaviors, and 4) subsets of primed genes and putative transcriptional regulators associated with changes in adult social behavior including oxytocin andmyt1la. Thus, our study establishes a new translationally relevant zebrafish model and suggests potential molecular mechanisms by which developmental GC-exposure exerts its life-long impact.

Chronic or severe stress during development increases risk of adult diseases in humans. One putative mechanism involves priming of stress-sensitive gene networks by early life Glucocorticoids (GCs) exposure, which modifies responses to stress later in life. However, brain-wide GC-primed molecular alterations are poorly understood. Here, we used an optogenetic zebrafish model to increase endogenous GCs during development and identified comprehensive transcriptomic alterations in the whole brain of GC-exposed fish across the life course. When exposed to acute stress in adulthood, developmental GC-exposed animals show highly exaggerated and pervasive brain-wide transcriptional changes which include gene sets associated with axon development, neuronal signaling, and epigenetic modulators. Strikingly, these altered gene sets showed an overrepresentation of human psychiatric disorder risk genes. We identify in developmental GC-exposed individuals: 1) altered biological processes, 2) hitherto uncharacterized novel GC-primed genes, 3) altered adult behaviors, and 4) subsets of primed genes and putative transcriptional regulators associated with changes in adult social behavior including oxytocin and myt1la. Thus, our study establishes a new translationally relevant zebrafish model and provides molecular targets through which developmental GC-exposure may exert its life-long impact. Significance statement Chronic or severe stress during development has far-reaching consequences in shaping adult phenotypes and increasing psychiatric disease susceptibility in humans. Glucocorticoids (GCs) as conserved main stress hormones of vertebrates, play a pivotal in this process by “priming” certain genes for altered response in adulthood upon stress re-exposure. However, identities of GC-primed genes are only partially known. In our study, we examined brain-wide molecular consequences of developmental GC exposure using an optogenetic zebrafish model and identified altered biological processes, behaviors, and a comprehensive list of GC-primed genes. Interestingly, the GC-primed gene sets in zebrafish showed an overrepresentation of genes linked to human psychiatric disorders. We propose that GC-primed genes identified here may shape adult responses of early life stress-exposed individuals.

The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts as part of the process for validating potential new drug targets. Traditional approaches (e.g., genetically-modified mice) are optimal for assessing small numbers of genes, but less practical when multiple genes are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation, by providing more confidence in target relevance and facilitating gene selection for definitive loss of function studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes ( API5 , HSPB7 , and LMO2 ) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including changes in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2 , there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports further investigation of the potential roles of API5 , HSPB7 , and LMO2 in human cardiovascular disease. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy.

The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. In this respect, large-scale patient DNA sequencing studies have become an invaluable strategy for identifying potential genetic contributing factors. The complex aetiology of heart failure, however, also means that in vivo models are vital to understand the links between genetic perturbations and functional impacts. Traditional approaches (e.g. genetically-modified mice) are optimal for assessing small numbers of proposed target genes, but less practical when multiple targets are identified. The zebrafish, in contrast, offers great potential for higher throughput in vivo gene functional assessment to aid target prioritisation and support definitive studies undertaken in mice. Here we used whole-exome sequencing and bioinformatics on human patient data to identify 3 genes ( API5, HSPB7 , and LMO2 ) suggestively associated with heart failure that were also predicted to play a broader role in disease aetiology. The role of these genes in cardiovascular system development and function was then further investigated using in vivo CRISPR/Cas9-mediated gene mutation analysis in zebrafish. We observed multiple impacts in F0 knockout zebrafish embryos (crispants) following effective somatic mutation, including reductions in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2 , there was also a significant impact on cardiovascular function as well as an expected reduction in erythropoiesis. The data generated from both the human in silico and zebrafish in vivo assessments undertaken supports roles for API5, HSPB7 , and LMO2 in human cardiovascular disease and identifies them as potential drug targets for further investigation. The data presented also supports the use of human in silico genetic variant analysis, in combination with zebrafish crispant phenotyping, as a powerful approach for assessing gene function as part of an integrated multi-level drug target validation strategy. ### Competing Interest Statement SR, MB, AW and EM are employees of AstraZeneca PLC. There are no other potential conflicts of interest.

Background and Purpose Functional brain imaging using genetically encoded Ca2+ sensors in larval zebrafish is being developed for studying seizures and epilepsy as a more ethical alternative to rodent models. Despite this, few data have been generated on pharmacological mechanisms of action other than GABA(A) antagonism. Assessing larval responsiveness across multiple mechanisms is vital to test the translational power of this approach, as well as assessing its validity for detecting unwanted drug-induced seizures and testing antiepileptic drug efficacy. Experimental Approach Using light-sheet imaging, we systematically analysed the responsiveness of 4 days post fertilisation (dpf; which are not considered protected under European animal experiment legislation) transgenic larval zebrafish to treatment with 57 compounds spanning more than 12 drug classes with a link to seizure generation in mammals, alongside eight compounds with no such link. Key Results We show 4dpf zebrafish are responsive to a wide range of mechanisms implicated in seizure generation, with cerebellar circuitry activated regardless of the initiating pharmacology. Analysis of functional connectivity revealed compounds targeting cholinergic and monoaminergic reuptake, in particular, showed phenotypic consistency broadly mapping onto what is known about neurotransmitter-specific circuitry in the larval zebrafish brain. Many seizure-associated compounds also exhibited altered whole brain functional connectivity compared with controls. Conclusions and Implications This work represents a significant step forward in understanding the translational power of 4dpf larval zebrafish for use in neuropharmacological studies and for studying the events driving transition from small-scale pharmacological activation of local circuits, to the large network-wide abnormal synchronous activity associated with seizures.

The clinical heterogeneity of heart failure has challenged our understanding of the underlying genetic mechanisms of this disease. To gain further insights into this complex pathophysiology we combined human rare variant analysis and in vivo CRISPR/Cas9-mediated mutant phenotyping in zebrafish to identify and investigate the role of 3 genes. Whole-exome sequencing of patients identified API5, HSPB7, and LMO2 as causally associated with heart failure and these genes were further investigated, alongside the positive control gata5, using CRISPR/Cas9-mediated multi-locus in vivo mutation in zebrafish. Following effective somatic mutation, we observed multiple impacts on cardiovascular development and function in F0 embryos including reductions in ventricle size, pericardial oedema, and chamber malformation. In the case of lmo2, there was also a significant impact on heart function. Our analysis suggests novel functions for API5, HSPB7, and LMO2 in human cardiovascular disease and identifies them as potential drug targets. Our data also supports in vivo CRISPR/Cas9-mediated multi-locus gene mutation analysis in F0 zebrafish as a rapid and effective primary screen for assessing gene function, as part of an integrated multi-level drug target validation strategy. Summary Statement Human rare variant analysis and CRISPR/Cas9-generated mutant phenotyping in zebrafish identifies genes as potential drug targets in cardiovascular disease.

The zebrafish (Danio rerio) embryo is currently explored as an alternative for developmental toxicity testing. As maternal metabolism is lacking in this model, knowledge of the disposition of xenobiotics during zebrafish organogenesis is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this study was to assess cytochrome P450 (CYP) activity in zebrafish embryos and larvae until 14 d post-fertilization (dpf) by using a non-specific CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR) and a CYP1-specific substrate, i.e., 7-ethoxyresorufin (ER). Moreover, the constitutive mRNA expression of CYP1A, CYP1B1, CYP1C1, CYP1C2, CYP2K6, CYP3A65, CYP3C1, phase II enzymes uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) and sulfotransferase 1st1 (SULT1ST1), and an ATP-binding cassette (ABC) drug transporter, i.e., abcb4, was assessed during zebrafish development until 32 dpf by means of quantitative PCR (qPCR). The present study showed that trancripts and/or the activity of these proteins involved in disposition of xenobiotics are generally low to undetectable before 72 h post-fertilization (hpf), which has to be taken into account in teratogenicity testing. Full capacity appears to be reached by the end of organogenesis (i.e., 120 hpf), although CYP1—except CYP1A—and SULT1ST1 were shown to be already mature in early embryonic development.