Exposure to stress during early life may alter the developmental trajectory of an animal by a mechanism known as adaptive plasticity. For example, to enhance reproductive success in an adverse environment, it is known that animals accelerate their growth during development. However, these short-term fitness benefits are often associated with reduced longevity, a phenomenon known as the growth rate-lifespan trade -off. In humans, early life stress exposure compromises health later in life and increases disease susceptibility. Glucocorticoids (GCs) are major stress hormones implicated in these processes. This Review discusses the evidence for GC-mediated adaptive plasticity in development, leading to allostatic overload in later life. We focus on GC-induced effects on brain structure and function, including neurogenesis; highlight the need for longitudinal studies; and discuss approaches to identify molecular mechanisms mediating GC-induced alteration of the brain developmental trajectory leading to adult dysfunctions. Further understanding of how stress and GC exposure can alter developmental trajectories at the molecular and cellular level is of critical importance to reduce the burden of mental and physical ill health across the life course.

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.

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.

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.

Early life stress (ELS) exposure alters stress susceptibility in later life and affects vulnerability to stress-related disorders, but how ELS changes the long-lasting responsiveness of the stress system is not well understood. Zebrafish provides an opportunity to study conserved mechanisms underlying the development and function of the stress response that is regulated largely by the neuroendocrine hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis, with glucocorticoids (GC) as the final effector. In this study, we established a method to chronically elevate endogenous GC levels during early life in larval zebrafish. To this end, we employed an optogenetic actuator, beggiatoa photoactivated adenylyl cyclase, specifically expressed in the interrenal cells of zebrafish and demonstrate that its chronic activation leads to hypercortisolaemia and dampens the acute-stress evoked cortisol levels, across a variety of stressor modalities during early life. This blunting of stress-response was conserved in ontogeny at a later developmental stage. Furthermore, we observe a strong reduction of proopiomelanocortin (pomc)-expression in the pituitary as well as upregulation of fkbp5 gene expression. Going forward, we propose that this model can be leveraged to tease apart the mechanisms underlying developmental programming of the HPA/I axis by early-life GC exposure and its implications for vulnerability and resilience to stress in adulthood.

Glucocorticoid resistance is commonly observed in depression, and has been linked to reduced expression and/or function of the glucocorticoid receptor (NR3C1 in human, hereafter referred to as GR). Previous studies have shown that GR-mutant zebrafish exhibit behavioural abnormalities that are indicative of an affective disorder, suggesting that GR plays a role in brain function. We compared the brain methylomes and brain transcriptomes of adult wild-type and GR-mutant zebrafish, and identified 249 differentially methylated regions (DMRs) that are regulated by GR. These include a cluster of CpG sites within the first intron of fkbp5, the gene encoding the glucocorticoid-inducible heat shock protein co-chaperone Fkbp5. RNA-sequencing analysis revealed that genes associated with chaperone-mediated protein folding, the regulation of circadian rhythm and the regulation of metabolism are particularly sensitive to loss of GR function. In addition, we identified subsets of genes exhibiting GR-regulated transcription that are known to regulate behaviour, and are linked to unipolar depression and anxiety. Taken together, our results identify key biological processes and novel molecular mechanisms through which the GR is likely to mediate responses to stress in the adult zebrafish brain, and they provide further support for the zebrafish GR mutant as a model for the study of affective disorders.

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.

Organisms respond to stressors through a coordinated set of physiological and behavioural responses. Zebrafish provides an opportunity to study conserved mechanisms underlying the stress-response that is regulated largely by the neuroendocrine Hypothalamus-Pituitary-Adrenal/Interrenal (HPA) axis, with glucocorticoids (GC) as the final effector. In this study, we evaluated the effect of chronically active GC signalling in early life on the baseline and stress evoked GC(cortisol) levels in larval zebrafish. To this end, we employed an optogenetic actuator, Beggiatoa photoactivated adenylyl cyclase, expressed in the interrenal cells of zebrafish and demonstrate that its chronic activation leads to hypercortisolaemia and dampens the acute-stress evoked cortisol levels, across a variety of stressor modalities during early life. This blunting of stress-response, a phenotype reported by many studies to be observed in human subjects exposed to early-life trauma, was conserved in ontogeny at a later developmental stage. Furthermore, we observe a strong reduction of proopiomelanocortin (POMC)-expressing cells in the pituitary as well as global upregulation of FKBP5 gene expression, impinging on the negative feedback regulation elicited by elevated cortisol levels. Going forward, we propose that this model can be leveraged to tease apart the mechanisms underlying developmental programming of HPA axis by early-life stress and its implications for vulnerability and resilience to stress in adulthood.

Release of corticotropin-releasing hormone (CRH) from CRH neurons activates the hypothalamo–pituitary–adrenal (HPA) axis, one of the main physiological stress response systems. Complex feedback loops operate in the HPA axis and understanding the neurobiological mechanisms regulating CRH neurons is of great importance in the context of stress disorders. In this article, we review how in vivo studies in zebrafish have advanced knowledge of the neurobiology of CRH neurons. Disrupted-in-schizophrenia 1 (DISC1) mutant zebrafish have blunted stress responses and can be used to model human stress disorders. We propose that DISC1 influences the development and functioning of CRH neurons as a mechanism linking DISC1 to psychiatric disorders.

The early life period represents a window of increased vulnerability to stress, during which exposure can lead to long-lasting effects on brain structure and function. This stress-induced developmental programming may contribute to the behavioural changes observed in mental illness. In recent decades, rodent studies have significantly advanced our understanding of how early life stress (ELS) affects brain development and behaviour. These studies reveal that ELS has long-term consequences on the brain such as impairment of adult hippocampal neurogenesis, altering learning and memory. Despite such advances, several key questions remain inadequately answered, including a comprehensive overview of brain regions and molecular pathways that are altered by ELS and how ELS-induced molecular changes ultimately lead to behavioural changes in adulthood. The zebrafish represents a novel ELS model, with the potential to contribute to answering some of these questions. The zebrafish offers some important advantages such as the ability to non-invasively modulate stress hormone levels in a whole animal and to visualise whole brain activity in freely behaving animals. This review discusses the current status of the zebrafish ELS field and its potential as a new ELS model.

The Glucocorticoid Receptor (GR) co-ordinates metabolic and behavioural responses to stressors. We hypothesised that GR influences behaviour by modulating specific epigenetic and transcriptional processes in the brain. Using the zebrafish as a model organism, the brain methylomes of wild-type and gr s357 mutant adults were analysed and GR-sensitive, differentially methylated regions (GR-DMRs) were identified. Two genes with GR-DMRs exhibited distinct methylation and transcriptional sensitivities to GR: the widely expressed direct GR target fkbp5 and neuron-specific aplp1 . In larvae, neural activity is required for GR-mediated transcription of aplp1, but not for that of fkbp5 . GR regulates metabotropic glutamate receptor gene expression, the activities of which also modulated aplp1 expression, implicating synaptic neurotransmission as an effector of GR function upstream of aplp1 . Our results identify two distinct routes of GR-regulated transcription in the brain, including a pathway through which GR couples endocrine signalling to synaptic activity-regulated transcription by modulating metabotropic glutamate receptor expression.

The spatial and temporal expression of steroidogenic genes in zebrafish has not been fully characterised. Because zebrafish are increasingly employed in endocrine and stress research, a better characterisation of steroidogenic pathways is required to target specific steps in the biosynthetic pathways. In the present study, we have systematically defined the temporal and spatial expression of steroidogenic enzymes involved in glucocorticoid biosynthesis (cyp21a2, cyp11c1, cyp11a1, cyp11a2, cyp17a1, cyp17a2, hsd3b1, hsd3b2), as well as the mitochondrial electron-providing ferredoxin co-factors (fdx1, fdx1b), during zebrafish development. Our studies showed an early expression of all these genes during embryogenesis. In larvae, expression of cyp11a2, cyp11c1, cyp17a2, cyp21a2, hsd3b1 and fdx1b can be detected in the interrenal gland, which is the zebrafish counterpart of the mammalian adrenal gland, whereas the fdx1 transcript is mainly found in the digestive system. Gene expression studies using quantitative reverse transcriptase-PCR and whole-mount in situ hybridisation in the adult zebrafish brain revealed a wide expression of these genes throughout the encephalon, including neurogenic regions. Using ultra-high-performance liquid chromatography tandem mass spectrometry, we were able to demonstrate the presence of the glucocorticoid cortisol in the adult zebrafish brain. Moreover, we demonstrate de novo biosynthesis of cortisol and the neurosteroid tetrahydrodeoxycorticosterone in the adult zebrafish brain from radiolabelled pregnenolone. Taken together, the present study comprises a comprehensive characterisation of the steroidogenic genes and the fdx co-factors facilitating glucocorticoid biosynthesis in zebrafish. Furthermore, we provide additional evidence of de novo neurosteroid biosynthesising in the brain of adult zebrafish facilitated by enzymes involved in glucocorticoid biosynthesis. Our study provides a valuable source for establishing the zebrafish as a translational model with respect to understanding the roles of the genes for glucocorticoid biosynthesis and fdx co-factors during embryonic development and stress, as well as in brain homeostasis and function.

We provide an overview of recent progress in elucidating the epigenetic mechanisms through which the impacts of psychosocial stressors become biologically embedded, thereby influencing physiology and modulating disease susceptibility. We focus on the stress-induced epigenetic and other regulatory changes to biological pathways that are associated with altered hypothalamo-pituitary-adrenal (HPA) axis activity and proinflammatory signaling. Topics with increasing salience, with respect to the causal pathways linking neuroendocrine stress to elevated risk of chronic disease, are how psychosocial stressors, such as low socioeconomic status, act as inducers of chronic inflammation, and how the glucocorticoid resistance that results from such chronic stress impairs the antiinflammatory functions of glucocorticoids. Altered levels of HPA axis activity, inflammation, and their sensitivity to exogenous glucocorticoids are some of the characteristics of major depressive disorder and posttraumatic stress disorder, and we review current knowledge of the roles of epigenetic changes induced by psychosocial stress in the underlying pathobiology of these disorders. Glucocorticoid resistance is also a characteristic feature of endocrine diseases such as Cushing's disease and Chrousos syndrome, and we consider the pathobiological mechanisms that are at work in these disorders. Finally, we discuss the role of chronic stress in promoting epigenetic age acceleration.

Psychiatric disorders arise due to an interplay of genetic and environmental factors, including stress. Studies in rodents have shown that mutants for Disrupted-In-Schizophrenia-1 (DISC1), a well-accepted genetic risk factor for mental illness, display abnormal behaviours in response to stress, but the mechanisms through which DISC1 affects stress responses remain poorly understood. Using two lines of zebrafish homozygous mutant for disc1, we investigated behaviour and functioning of the hypothalamic-pituitary-interrenal (HPI) axis, the fish equivalent of the hypothalamic-pituitary-adrenal (HPA) axis. Here, we show that the role of DISC1 in stress responses is evolutionarily conserved and that DISC1 is essential for normal functioning of the HPI axis. Adult zebrafish homozygous mutant for disc1 show aberrant behavioural responses to stress. Our studies reveal that in the embryo, disc1 is expressed in neural progenitor cells of the hypothalamus, a conserved region of the vertebrate brain that centrally controls responses to environmental stressors. In disc1 mutant embryos, proliferating rx3-hypothalamic progenitors are not maintained normally and neuronal differentiation is compromised: rx3-derived ff1b+neurons, implicated in anxiety-related behaviours, and corticotrophin releasing hormone (crh) neurons, key regulators of the stress axis, develop abnormally, and rx3-derived pomc+neurons are disorganised. Abnormal hypothalamic development is associated with dysfunctional behavioural and neuroendocrine stress responses. In contrast to wild type siblings, disc1 mutant larvae show altered crh levels, fail to upregulate cortisol levels when under stress and do not modulate shoal cohesion, indicative of abnormal social behaviour. These data indicate that disc1 is essential for normal development of the hypothalamus and for the correct functioning of the HPA/HPI axis.

Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator–like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic–pituitary–interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

In the developing brain, growth and differentiation are intimately linked. Here, we show that in the zebrafish embryo, the homeodomain transcription factor Rx3 coordinates these processes to build the tuberal/anterior hypothalamus. Analysis of rx3 chk mutant/rx3 morphant fish and EdU pulse-chase studies reveal that rx3 is required to select tuberal/anterior hypothalamic progenitors and to orchestrate their anisotropic growth. In the absence of Rx3 function, progenitors accumulate in the third ventricular wall, die or are inappropriately specified, the shh(+) anterior recess does not form, and its resident pomc(+), ff1b(+) and otpb(+) Th1(+) cells fail to differentiate. Manipulation of Shh signalling shows that Shh coordinates progenitor cell selection and behaviour by acting as an on-off switch for rx3. Together, our studies show that Shh and Rx3 govern formation of a distinct progenitor domain that elaborates patterning through its anisotropic growth and differentiation.