Journal articles
Cook A, Beckmann H, Azap R, Ryu S (2023). Acute Stress Modulates Social Approach and Social Maintenance in Adult Zebrafish. eNeuro, 10(9), eneuro.0491-eneu22.2023.
Herget U, Ryu S, De Marco RJ (2023). Altered glucocorticoid reactivity and behavioral phenotype in rx3-/- larval zebrafish. Frontiers in Endocrinology, 14
Ayash S, Lingner T, Ramisch A, Ryu S, Kalisch R, Schmitt U, Müller MB (2023). Fear circuit–based neurobehavioral signatures mirror resilience to chronic social stress in mouse.
Proceedings of the National Academy of Sciences,
120(17).
Abstract:
Fear circuit–based neurobehavioral signatures mirror resilience to chronic social stress in mouse
. Consistent evidence from human data points to successful threat–safety discrimination and responsiveness to extinction of fear memories as key characteristics of resilient individuals. To promote valid cross-species approaches for the identification of resilience mechanisms, we establish a translationally informed mouse model enabling the stratification of mice into three phenotypic subgroups following chronic social defeat stress, based on their individual ability for threat–safety discrimination and conditioned learning: the
. Discriminating-avoiders
. characterized by successful social threat–safety discrimination and extinction of social aversive memories; the
. Indiscriminate-avoiders
. showing aversive response generalization and resistance to extinction, in line with findings on susceptible individuals; and the
. Non-avoiders
. displaying impaired aversive conditioned learning. To explore the neurobiological mechanisms underlying the stratification, we perform transcriptome analysis within three key target regions of the fear circuitry. We identify subgroup-specific differentially expressed genes and gene networks underlying the behavioral phenotypes, i.e. the individual ability to show threat–safety discrimination and respond to extinction training. Our approach provides a translationally informed template with which to characterize the behavioral, molecular, and circuit bases of resilience in mice.
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Abstract.
Jacobs EAK, Ryu S (2023). Larval zebrafish as a model for studying individual variability in translational neuroscience research. Frontiers in Behavioral Neuroscience, 17
Eugenin von Bernhardi J, Biechl D, Miek L, Herget U, Ryu S, Wullimann MF (2022). A versatile transcription factor: Multiple roles of orthopedia a (otpa) beyond its restricted localization in dopaminergic systems of developing and adult zebrafish (Danio rerio) brains.
J Comp Neurol,
530(14), 2537-2561.
Abstract:
A versatile transcription factor: Multiple roles of orthopedia a (otpa) beyond its restricted localization in dopaminergic systems of developing and adult zebrafish (Danio rerio) brains.
Many transcription factors boost neural development and differentiation in specific directions and serve for identifying similar or homologous structures across species. The expression of Orthopedia (Otp) is critical for the development of certain cell groups along the vertebrate neuraxis, for example, the medial amygdala or hypothalamic neurosecretory neurons. Therefore, the primary focus of the present study is the distribution of Orthopedia a (Otpa) in the larval and adult zebrafish (Danio rerio) brain. Since Otpa is also critical for the development of zebrafish basal diencephalic dopaminergic cells, colocalization of Otpa with the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) is studied. Cellular colocalization of Otpa and dopamine is only seen in magnocellular neurons of the periventricular posterior tubercular nucleus and in the posterior tuberal nucleus. Otpa-positive cells occur in many additional structures along the zebrafish neuraxis, from the secondary prosencephalon down to the hindbrain. Furthermore, Otpa expression is studied in shh-GFP and islet1-GFP transgenic zebrafish. Otpa-positive cells only express shh in dopaminergic magnocellular periventricular posterior tubercular cells, and only colocalize with islet1-GFP in the ventral zone and prerecess caudal periventricular hypothalamic zone and the perilemniscal nucleus. The scarcity of cellular colocalization of Otpa in islet1-GFP cells indicates that the Shh-islet1 neurogenetic pathway is not active in most Otpa-expressing domains. Our analysis reveals detailed correspondences between mouse and zebrafish forebrain territories including the zebrafish intermediate nucleus of the ventral telencephalon and the mouse medial amygdala. The zebrafish preoptic Otpa-positive domain represents the neuropeptidergic supraopto-paraventricular region of all tetrapods. Otpa domains in the zebrafish basal plate hypothalamus suggest that the ventral periventricular hypothalamic zone corresponds to the otp-expressing basal hypothalamic tuberal field in the mouse. Furthermore, the mouse otp domain in the mammillary hypothalamus compares partly to our Otpa-positive domain in the prerecess caudal periventricular hypothalamic zone (Hc-a).
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Author URL.
Albert L, Nagpal J, Steinchen W, Zhang L, Werel L, Djokovic N, Ruzic D, Hoffarth M, Xu J, Kaspareit J, et al (2022). Bistable Photoswitch Allows in Vivo Control of Hematopoiesis.
ACS CENTRAL SCIENCE,
8(1), 57-66.
Author URL.
Gemmer A, Mirkes K, Anneser L, Eilers T, Kibat C, Mathuru A, Ryu S, Schuman E (2022). Oxytocin receptors influence the development and maintenance of social behavior in zebrafish (Danio rerio).
Sci Rep,
12(1).
Abstract:
Oxytocin receptors influence the development and maintenance of social behavior in zebrafish (Danio rerio).
Zebrafish are highly social teleost fish and an excellent model to study social behavior. The neuropeptide Oxytocin is associated different social behaviors as well as disorders resulting in social impairment like autism spectrum disorder. However, how Oxytocin receptor signaling affects the development and expression kinetics of social behavior is not known. In this study we investigated the role of the two oxytocin receptors, Oxtr and Oxtrl, in the development and maintenance of social preference and shoaling behavior in 2- to 8-week-old zebrafish. Using CRISPR/Cas9 mediated oxtr and oxtrl knock-out fish, we found that the development of social preference is accelerated if one of the Oxytocin receptors is knocked-out and that the knock-out fish reach significantly higher levels of social preference. Moreover, oxtr-/- fish showed impairments in the maintenance of social preference. Social isolation prior to testing led to impaired maintenance of social preference in both wild-type and oxtr and oxtrl knock-out fish. Knocking-out either of the Oxytocin receptors also led to increased group spacing and reduced polarization in a 20-fish shoal at 8 weeks post fertilization, but not at 4. These results show that the development and maintenance of social behavior is influenced by the Oxytocin receptors and that the effects are not just pro- or antisocial, but dependent on both the age and social context of the fish.
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Author URL.
Anneser L, Gemmer A, Eilers T, Alcantara IC, Loos A-Y, Ryu S, Schuman EM (2022). The neuropeptide Pth2 modulates social behavior and anxiety in zebrafish. iScience, 25(3), 103868-103868.
Eachus H, Ryu S, Placzek M, Wood J (2022). Zebrafish as a model to investigate the CRH axis and interactions with DISC1. Current Opinion in Endocrine and Metabolic Research, 26
Gemmer A, Mirkes K, Anneser L, Eilers T, Kibat C, Mathuru A, Ryu S, Schuman E (2021). Oxytocin receptors influence the development and maintenance of social behavior in zebrafish (<i>Danio rerio</i>).
Abstract:
Oxytocin receptors influence the development and maintenance of social behavior in zebrafish (Danio rerio)
AbstractZebrafish are highly social teleost fish and an excellent model to study social behavior. The neuropeptide Oxytocin is associated different social behaviors as well as disorders resulting in social impairment like autism spectrum disorder. However, how Oxytocin receptor signaling affects the development and expression kinetics of social behavior is not known. In this study we investigated the role of the two oxytocin receptors, Oxtr and Oxtrl, in the development and maintenance of social preference and shoaling behavior in 2- to 8-week-old zebrafish. Using CRISPR/Cas9 mediated oxtr and oxtrl knock-out fish, we found that the development of social preference is accelerated if one of the Oxytocin receptors is knocked-out and that the knock-out fish reach significantly higher levels of social preference. Moreover, oxtr-/- fish showed impairments in the maintenance of social preference. Social isolation prior to testing led to impaired maintenance of social preference in both wild-type and oxtr and oxtrl knock-out fish. Knocking-out one of the Oxytocin receptors also led to increased group spacing and reduced polarization in a 20-fish shoal at 8 weeks post fertilization, but not at 4. These results show that the development and maintenance of social behavior is influenced by the Oxytocin receptors and that the effects are not just pro- or antisocial, but dependent on both the age and social context of the fish.
Abstract.
Ayash S, Lingner T, Ryu S, Kalisch R, Schmitt U, Müller M (2021). Reconceptualising Resilience Within a Translational Framework is Supported by Unique and Brain-Region Specific Transcriptional Signatures in Mice. Biological Psychiatry, 89(9).
Eachus H, Choi M-K, Ryu S (2021). The Effects of Early Life Stress on the Brain and Behaviour: Insights from Zebrafish Models.
Front Cell Dev Biol,
9Abstract:
The Effects of Early Life Stress on the Brain and Behaviour: Insights from Zebrafish Models.
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.
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Anneser L, Gemmer A, Eilers T, Alcantara IC, Loos A-Y, Ryu S, Schuman EM (2021). The neuropeptide Pth2 modulates social behavior and anxiety in zebrafish.
Abstract:
The neuropeptide Pth2 modulates social behavior and anxiety in zebrafish
SummaryAnimal behavior is strongly context-dependent and behavioral performance is often modulated by internal state. In particular, different social contexts can alter anxiety levels and modulate social behavior. The vertebrate-specific neuropeptide parathyroid hormone 2 (pth2) is directly regulated by the presence or absence of conspecifics in zebrafish. As its cognate receptor, the parathyroid hormone 2 receptor (pth2r), is widely expressed across the brain, we tested fish lacking the functional Pth2 peptide in several anxiety-related and social paradigms. Rodents lacking PTH2 display increased anxiety-related behavior. Here we show that the propensity to react to sudden stimuli with an escape response is increased in pth2-/- zebrafish, consistent with elevated anxiety. While overall social preference for conspecifics is maintained in pth2-/- fish until the early juvenile stage, we found that both social preference and shoaling are altered later in development. The data presented suggest that the neuropeptide Pth2 modulates several conserved behavioral features, and may thus enable the animal to react appropriately in different social contexts.
Abstract.
Ayash S, Lingner T, Ryu S, Kalisch R, Schmitt U, Müller MB (2020). Reconceptualising resilience within a translational framework is supported by unique and brain-region specific transcriptional signatures in mice.
Abstract:
Reconceptualising resilience within a translational framework is supported by unique and brain-region specific transcriptional signatures in mice
ABSTRACTChronic social defeat (CSD) in mice has been increasingly employed in experimental resilience research. Particularly, the degree of CSD-induced social avoidance is used to classify animals into resilient (socially non-avoidant) versus susceptible (avoidant). In-spired by human data pointing to threat-safety discrimination and responsiveness to extinction training of aversive memories as characteristics of resilient individuals, we here describe a translationally informed stratification which identified three phenotypic subgroups of mice following CSD: the Discriminating-avoiders, characterised by successful social threat-safety discrimination and successful extinction of social avoidance; the Indis-criminate-avoiders, showing aversive response generalisation, and the Non-avoiders (absence of social avoidance) displaying impaired conditioned learning. Furthermore, and supporting the biological validity of our approach, we uncovered subgroup-specific transcriptional signatures in classical fear conditioning and anxiety-related brain regions. Our reconceptualisation of resilience in mice refines the currently used dichotomous classification and contributes to advancing future translational approaches.
Abstract.
Anneser L, Alcantara IC, Gemmer A, Mirkes K, Ryu S, Schuman EM (2020). The neuropeptide Pth2 dynamically senses others via mechanosensation. Nature, 588(7839), 653-657.
Castillo-Ramírez LA, Ryu S, De Marco RJ (2019). Active behaviour during early development shapes glucocorticoid reactivity.
Sci Rep,
9(1).
Abstract:
Active behaviour during early development shapes glucocorticoid reactivity.
Glucocorticoids are the final effectors of the stress axis, with numerous targets in the central nervous system and the periphery. They are essential for adaptation, yet currently it is unclear how early life events program the glucocorticoid response to stress. Here we provide evidence that involuntary swimming at early developmental stages can reconfigure the cortisol response to homotypic and heterotypic stress in larval zebrafish (Danio rerio), also reducing startle reactivity and increasing spontaneous activity as well as energy efficiency during active behaviour. Collectively, these data identify a role of the genetically malleable zebrafish for linking early life stress with glucocorticoid function in later life.
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Nagpal J, Herget U, Choi MK, Ryu S (2019). Anatomy, development, and plasticity of the neurosecretory hypothalamus in zebrafish.
Cell Tissue Res,
375(1), 5-22.
Abstract:
Anatomy, development, and plasticity of the neurosecretory hypothalamus in zebrafish.
The paraventricular nucleus (PVN) of the hypothalamus harbors diverse neurosecretory cells with critical physiological roles for the homeostasis. Decades of research in rodents have provided a large amount of information on the anatomy, development, and function of this important hypothalamic nucleus. However, since the hypothalamus lies deep within the brain in mammals and is difficult to access, many questions regarding development and plasticity of this nucleus still remain. In particular, how different environmental conditions, including stress exposure, shape the development of this important nucleus has been difficult to address in animals that develop in utero. To address these open questions, the transparent larval zebrafish with its rapid external development and excellent genetic toolbox offers exciting opportunities. In this review, we summarize recent information on the anatomy and development of the neurosecretory preoptic area (NPO), which represents a similar structure to the mammalian PVN in zebrafish. We will then review recent studies on the development of different cell types in the neurosecretory hypothalamus both in mouse and in fish. Lastly, we discuss stress-induced plasticity of the PVN mainly discussing the data obtained in rodents, but pointing out tools and approaches available in zebrafish for future studies. This review serves as a primer for the currently available information relevant for studying the development and plasticity of this important brain region using zebrafish.
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Langebeck-Jensen K, Shahar OD, Schuman EM, Langer JD, Ryu S (2019). Larval Zebrafish Proteome Regulation in Response to an Environmental Challenge.
Proteomics,
19(14).
Abstract:
Larval Zebrafish Proteome Regulation in Response to an Environmental Challenge.
Adaptation to the environment during development influences the life-long survival of an animal. While brain-wide proteomic changes are expected to underlie such experience-driven physiological and behavioral flexibility, a comprehensive overview of the nature and extent of the proteomic regulation following an environmental challenge during development is currently lacking. In this study, the brain proteome of larval zebrafish is identified and it is determined how it is altered by an exposure to a natural and physical environmental challenge, namely prolonged exposure to strong water currents. A comprehensive larval zebrafish brain proteome is presented here. Furthermore, 57 proteins that are regulated by the exposure to an environmental challenge are identified, which cover multiple functions including neuronal plasticity, the stress response, axonal growth and guidance, spatial learning, and energy metabolism. These represent candidate proteins that may play crucial roles for the adaption to an environmental challenge during development.
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Herzog DP, Beckmann H, Lieb K, Ryu S, Müller MB (2018). Understanding and Predicting Antidepressant Response: Using Animal Models to Move Toward Precision Psychiatry.
Front Psychiatry,
9Abstract:
Understanding and Predicting Antidepressant Response: Using Animal Models to Move Toward Precision Psychiatry.
There are two important gaps of knowledge in depression treatment, namely the lack of biomarkers predicting response to antidepressants and the limited knowledge of the molecular mechanisms underlying clinical improvement. However, individually tailored treatment strategies and individualized prescription are greatly needed given the huge socio-economic burden of depression, the latency until clinical improvement can be observed and the response variability to a particular compound. Still, individual patient-level antidepressant treatment outcomes are highly unpredictable. In contrast to other therapeutic areas and despite tremendous efforts during the past years, the genomics era so far has failed to provide biological or genetic predictors of clinical utility for routine use in depression treatment. Specifically, we suggest to (1) shift the focus from the group patterns to individual outcomes, (2) use dimensional classifications such as Research Domain Criteria, and (3) envision better planning and improved connections between pre-clinical and clinical studies within translational research units. In contrast to studies in patients, animal models enable both searches for peripheral biosignatures predicting treatment response and in depth-analyses of the neurobiological pathways shaping individual antidepressant response in the brain. While there is a considerable number of animal models available aiming at mimicking disease-like conditions such as those seen in depressive disorder, only a limited number of preclinical or truly translational investigations is dedicated to the issue of heterogeneity seen in response to antidepressant treatment. In this mini-review, we provide an overview on the current state of knowledge and propose a framework for successful translational studies into antidepressant treatment response.
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Beretta CA, Dross N, Guglielmi L, Bankhead P, Soulika M, Gutierrez-Triana JA, Paolini A, Poggi L, Falk J, Ryu S, et al (2017). Early Commissural Diencephalic Neurons Control Habenular Axon Extension and Targeting.
Curr Biol,
27(2), 270-278.
Abstract:
Early Commissural Diencephalic Neurons Control Habenular Axon Extension and Targeting.
Most neuronal populations form on both the left and right sides of the brain. Their efferent axons appear to grow synchronously along similar pathways on each side, although the neurons or their environment often differ between the two hemispheres [1-4]. How this coordination is controlled has received little attention. Frequently, neurons establish interhemispheric connections, which can function to integrate information between brain hemispheres (e.g. [5]). Such commissures form very early, suggesting their potential developmental role in coordinating ipsilateral axon navigation during embryonic development [4]. To address the temporal-spatial control of bilateral axon growth, we applied long-term time-lapse imaging to visualize the formation of the conserved left-right asymmetric habenular neural circuit in the developing zebrafish embryo [6]. Although habenular neurons are born at different times across brain hemispheres [7], we found that elongation of habenular axons occurs synchronously. The initiation of axon extension is not controlled within the habenular network itself but through an early developing proximal diencephalic network. The commissural neurons of this network influence habenular axons both ipsilaterally and contralaterally. Their unilateral absence impairs commissure formation and coordinated habenular axon elongation and causes their subsequent arrest on both sides of the brain. Thus, habenular neural circuit formation depends on a second intersecting commissural network, which facilitates the exchange of information between hemispheres required for ipsilaterally projecting habenular axons. This mechanism of network formation may well apply to other circuits, and has only remained undiscovered due to technical limitations.
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Biechl D, Tietje K, Ryu S, Grothe B, Gerlach G, Wullimann MF (2017). Identification of accessory olfactory system and medial amygdala in the zebrafish.
Sci Rep,
7Abstract:
Identification of accessory olfactory system and medial amygdala in the zebrafish.
Zebrafish larvae imprint on visual and olfactory cues of their kin on day 5 and 6 postfertilization, respectively. Only imprinted (but not non-imprinted) larvae show strongly activated crypt (and some microvillous) cells demonstrated by pERK levels after subsequent exposure to kin odor. Here, we investigate the olfactory bulb of zebrafish larvae for activated neurons located at the sole glomerulus mdG2 which receives crypt cell input. Imprinted larvae show a significantly increased activation of olfactory bulb cells compared to non-imprinted larvae after exposure to kin odor. Surprisingly, pERK activated Orthopedia-positive cell numbers in the intermediate ventral telencephalic nucleus were higher in non-imprinted, kin odor stimulated larvae compared to control and to kin-odor stimulated imprinted larvae and control. Moreover, DiI tracing experiments in adult zebrafish show a neuronal circuit from crypt/microvillous olfactory sensory neurons via dorsomedial olfactory bulb and intermediate ventral telencephalic nucleus (thus, arguably the teleostean medial amygdala) to tuberal hypothalamus, demonstrating for the first time an accessory olfactory system in teleosts.
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Ryu S, De Marco RJ (2017). Performance on innate behaviour during early development as a function of stress level.
Sci Rep,
7(1).
Abstract:
Performance on innate behaviour during early development as a function of stress level.
What is the relationship between the level of acute stress and performance on innate behaviour? the diversity of innate behaviours and lack of sufficient data gathered under the same experimental conditions leave this question unresolved. While evidence points to an inverted-U shaped relationship between the level of acute stress and various measures of learning and memory function, it is unknown the extent to which such a non-linear function applies to performance on innate behaviour, which develops without example or practice under natural circumstances. The fundamental prediction of this view is that moderate stress levels will improve performance, while higher levels will not. Testing this proposition has been difficult because it entails an overall effect that must be invariant to the nature of the stressor, the behaviour under scrutiny and the stimulus that drives it. Here, we report new experimental results showing that developing zebrafish (Danio rerio) under moderate but not higher levels of stress improved their performance on instinctive activities driven by visual, hydrodynamic and thermal inputs. Our findings reveal, for the first time, the existence of an inverted-U shaped performance function according to stress level during early development in a series of innate behaviours.
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Herget U, Gutierrez-Triana JA, Salazar Thula O, Knerr B, Ryu S (2017). Single-Cell Reconstruction of Oxytocinergic Neurons Reveals Separate Hypophysiotropic and Encephalotropic Subtypes in Larval Zebrafish.
eNeuro,
4(1).
Abstract:
Single-Cell Reconstruction of Oxytocinergic Neurons Reveals Separate Hypophysiotropic and Encephalotropic Subtypes in Larval Zebrafish.
Oxytocin regulates a diverse set of processes including stress, analgesia, metabolism, and social behavior. How such diverse functions are mediated by a single hormonal system is not well understood. Different functions of oxytocin could be mediated by distinct cell groups, yet it is currently unknown whether different oxytocinergic cell types exist that specifically mediate peripheral neuroendocrine or various central neuromodulatory processes via dedicated pathways. Using the Brainbow technique to map the morphology and projections of individual oxytocinergic cells in the larval zebrafish brain, we report here the existence of two main types of oxytocinergic cells: those that innervate the pituitary and those that innervate diverse brain regions. Similar to the situation in the adult rat and the adult midshipman, but in contrast to the situation in the adult trout, these two cell types are mutually exclusive and can be distinguished based on morphological and anatomical criteria. Further, our results reveal that complex oxytocinergic innervation patterns are already established in the larval zebrafish brain.
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De Marco RJ, Thiemann T, Groneberg AH, Herget U, Ryu S (2016). Optogenetically enhanced pituitary corticotroph cell activity post-stress onset causes rapid organizing effects on behaviour.
Nat Commun,
7Abstract:
Optogenetically enhanced pituitary corticotroph cell activity post-stress onset causes rapid organizing effects on behaviour.
The anterior pituitary is the major link between nervous and hormonal systems, which allow the brain to generate adequate and flexible behaviour. Here, we address its role in mediating behavioural adjustments that aid in coping with acutely threatening environments. For this we combine optogenetic manipulation of pituitary corticotroph cells in larval zebrafish with newly developed assays for measuring goal-directed actions in very short timescales. Our results reveal modulatory actions of corticotroph cell activity on locomotion, avoidance behaviours and stimulus responsiveness directly after the onset of stress. Altogether, the findings uncover the significance of endocrine pituitary cells for rapidly optimizing behaviour in local antagonistic environments.
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Vom Berg-Maurer CM, Trivedi CA, Bollmann JH, De Marco RJ, Ryu S (2016). The Severity of Acute Stress is Represented by Increased Synchronous Activity and Recruitment of Hypothalamic CRH Neurons.
J Neurosci,
36(11), 3350-3362.
Abstract:
The Severity of Acute Stress is Represented by Increased Synchronous Activity and Recruitment of Hypothalamic CRH Neurons.
UNLABELLED: the hypothalamo-pituitary-adrenocortical (HPA) axis regulates stress physiology and behavior. To achieve an optimally tuned adaptive response, it is critical that the magnitude of the stress response matches the severity of the threat. Corticotropin-releasing hormone (CRH) released from the paraventricular nucleus of the hypothalamus is a major regulator of the HPA axis. However, how CRH-producing neurons in an intact animal respond to different stressor intensities is currently not known. Using two-photon calcium imaging on intact larval zebrafish, we recorded the activity of CRH cells, while the larvae were exposed to stressors of varying intensity. By combining behavioral and physiological measures, we first determined how sudden alterations in environmental conditions lead to different levels of stress axis activation. Then, we measured changes in the frequency and amplitude of Ca(2+) transients in individual CRH neurons in response to such stressors. The response magnitude of individual CRH cells covaried with stressor intensity. Furthermore, stressors caused the recruitment of previously inactive CRH neurons in an intensity-dependent manner, thus increasing the pool of responsive CRH cells. Strikingly, stressor-induced activity appeared highly synchronized among CRH neurons, and also across hemispheres. Thus, the stressor strength-dependent output of CRH neurons emerges by a dual mechanism that involves both the increased activity of individual cells and the recruitment of a larger pool of responsive cells. The synchronicity of CRH neurons within and across hemispheres ensures that the overall output of the HPA axis matches the severity of the threat. SIGNIFICANCE STATEMENT: Stressors trigger adaptive responses in the body that are essential for survival. How the brain responds to acute stressors of varying intensity in an intact animal, however, is not well understood. We address this question using two-photon Ca(2+) imaging in larval zebrafish with transgenically labeled corticotropin-releasing hormone (CRH) cells, which represent a major regulator of the stress axis. We show that stressor strength-dependent responses of CRH neurons emerge via an intensity-dependent increase in the activity of individual CRH cells, and by an increase in the pool of responsive CRH cells at the population level. Furthermore, we report striking synchronicity among CRH neurons even across hemispheres, which suggests tight intrahypothalamic and interhypothalamic coordination. Thus, our work reveals how CRH neurons respond to different levels of acute stress in vivo.
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Gutierrez-Triana JA, Mateo JL, Ibberson D, Ryu S, Wittbrodt J (2016). iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins.
Development,
143(22), 4272-4278.
Abstract:
iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins.
DNA adenine methyltransferase identification (DamID) has emerged as an alternative method to profile protein-DNA interactions; however, critical issues limit its widespread applicability. Here, we present iDamIDseq, a protocol that improves specificity and sensitivity by inverting the steps DpnI-DpnII and adding steps that involve a phosphatase and exonuclease. To determine genome-wide protein-DNA interactions efficiently, we present the analysis tool iDEAR (iDamIDseq Enrichment Analysis with R). The combination of DamID and iDEAR permits the establishment of consistent profiles for transcription factors, even in transient assays, as we exemplify using the small teleost medaka (Oryzias latipes). We report that the bacterial Dam-coding sequence induces aberrant splicing when it is used with different promoters to drive tissue-specific expression. Here, we present an optimization of the sequence to avoid this problem. This and our other improvements will allow researchers to use DamID effectively in any organism, in a general or targeted manner.
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Herget U, Ryu S (2015). Coexpression analysis of nine neuropeptides in the neurosecretory preoptic area of larval zebrafish.
Front Neuroanat,
9Abstract:
Coexpression analysis of nine neuropeptides in the neurosecretory preoptic area of larval zebrafish.
The paraventricular nucleus (PVN) of the hypothalamus in mammals coordinates neuroendocrine, autonomic and behavioral responses pivotal for homeostasis and the stress response. A large amount of studies in rodents has documented that the PVN contains diverse neuronal cell types which can be identified by the expression of distinct secretory neuropeptides. Interestingly, PVN cell types often coexpress multiple neuropeptides whose relative coexpression levels are subject to environment-induced plasticity. Due to their small size and transparency, zebrafish larvae offer the possibility to comprehensively study the development and plasticity of the PVN in large groups of intact animals, yet important anatomical information about the larval zebrafish PVN-homologous region has been missing. Therefore we recently defined the location and borders of the larval neurosecretory preoptic area (NPO) as the PVN-homologous region in larval zebrafish based on transcription factor expression and cell type clustering. To identify distinct cell types present in the larval NPO, we also generated a comprehensive 3D map of 9 zebrafish homologs of typical neuropeptides found in the mammalian PVN (arginine vasopressin (AVP), corticotropin-releasing hormone (CRH), proenkephalin a (penka)/b (penkb), neurotensin (NTS), oxytocin (OXT), vasoactive intestinal peptide (VIP), cholecystokinin (CCK), and somatostatin (SST)). Here we extend this chemoarchitectural map to include the degrees of coexpression of two neuropeptides in the same cell by performing systematic pairwise comparisons. Our results allowed the subclassification of NPO cell types, and differences in variability of coexpression profiles suggest potential targets of biochemical plasticity. Thus, this work provides an important basis for the analysis of the development, function, and plasticity of the primary neuroendocrine brain region in larval zebrafish.
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Gutierrez-Triana JA, Herget U, Castillo-Ramirez LA, Lutz M, Yeh C-M, De Marco RJ, Ryu S (2015). Manipulation of Interrenal Cell Function in Developing Zebrafish Using Genetically Targeted Ablation and an Optogenetic Tool.
Endocrinology,
156(9), 3394-3401.
Abstract:
Manipulation of Interrenal Cell Function in Developing Zebrafish Using Genetically Targeted Ablation and an Optogenetic Tool.
Zebrafish offer an opportunity to study conserved mechanisms underlying the ontogeny and physiology of the hypothalamic-pituitary-adrenal/interrenal axis. As the final effector of the hypothalamic-pituitary-adrenal/interrenal axis, glucocorticoids exert both rapid and long-term regulatory functions. To elucidate their specific effects in zebrafish, transgenic approaches are necessary to complement pharmacological studies. Here, we report a robust approach to specifically manipulate endogenous concentrations of cortisol by targeting heterologous proteins to interrenal cells using a promoter element of the steroidogenic acute regulatory protein. To test this approach, we first used this regulatory region to generate a transgenic line expressing the bacterial nitroreductase protein, which allows conditional targeted ablation of interrenal cells. We demonstrate that this line can be used to specifically ablate interrenal cells, drastically reducing both basal and stress-induced cortisol concentrations. Next, we coupled this regulatory region to an optogenetic actuator, Beggiatoa photoactivated adenylyl cyclase, to increase endogenous cortisol concentrations in a blue light-dependent manner. Thus, our approach allows specific manipulations of steroidogenic interrenal cell activity for studying the effects of both hypo- and hypercortisolemia in zebrafish.
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Groneberg AH, Herget U, Ryu S, De Marco RJ (2015). Positive taxis and sustained responsiveness to water motions in larval zebrafish.
Front Neural Circuits,
9Abstract:
Positive taxis and sustained responsiveness to water motions in larval zebrafish.
Larval zebrafish (Danio rerio) have become favored subjects for studying the neural bases of behavior. Here, we report a highly stereotyped response of zebrafish larvae to hydrodynamic stimuli. It involves positive taxis, motion damping and sustained responsiveness to flows derived from local, non-stressful water motions. The response depends on the lateral line and has a high sensitivity to stimulus frequency and strength, sensory background and rearing conditions--also encompassing increased threshold levels of response to parallel input. The results show that zebrafish larvae can use near-field detection to locate sources of minute water motions, and offer a unique handle for analyses of hydrodynamic sensing, sensory responsiveness and arousal with accurate control of stimulus properties.
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Gutierrez-Triana J, Herget U, Lichtner P, Castillo-Ramírez LA, Ryu S (2014). A vertebrate-conserved cis -regulatory module for targeted expression in the main hypothalamic regulatory region for the stress response. BMC Developmental Biology, 14(1).
Gutierrez-Triana JA, Herget U, Lichtner P, Castillo-Ramírez LA, Ryu S (2014). A vertebrate-conserved cis-regulatory module for targeted expression in the main hypothalamic regulatory region for the stress response.
BMC Dev Biol,
14Abstract:
A vertebrate-conserved cis-regulatory module for targeted expression in the main hypothalamic regulatory region for the stress response.
BACKGROUND: the homeodomain transcription factor orthopedia (Otp) is an evolutionarily conserved regulator of neuronal fates. In vertebrates, Otp is necessary for the proper development of different regions of the brain and is required in the diencephalon to specify several hypothalamic cell types, including the cells that control the stress response. To understand how this widely expressed transcription factor accomplishes hypothalamus-specific functions, we performed a comprehensive screening of otp cis-regulatory regions in zebrafish. RESULTS: Here, we report the identification of an evolutionarily conserved vertebrate enhancer module with activity in a restricted area of the forebrain, which includes the region of the hypothalamus that controls the stress response. This region includes neurosecretory cells producing Corticotropin-releasing hormone (Crh), Oxytocin (Oxt) and Arginine vasopressin (Avp), which are key components of the stress axis. Lastly, expression of the bacterial nitroreductase gene under this specific enhancer allowed pharmacological attenuation of the stress response in zebrafish larvae. CONCLUSION: Vertebrates share many cellular and molecular components of the stress response and our work identified a striking conservation at the cis-regulatory level of a key hypothalamic developmental gene. In addition, this enhancer provides a useful tool to manipulate and visualize stress-regulatory hypothalamic cells in vivo with the long-term goal of understanding the ontogeny of the stress axis in vertebrates.
Abstract.
Author URL.
Preuss SJ, Trivedi CA, vom Berg-Maurer CM, Ryu S, Bollmann JH (2014). Classification of object size in retinotectal microcircuits.
Curr Biol,
24(20), 2376-2385.
Abstract:
Classification of object size in retinotectal microcircuits.
BACKGROUND: a principal task of the visual system is to detect and classify moving objects in the visual environment. Information about the size of an object is critical for selecting appropriate behavioral responses. Object size is encoded in retinal ganglion cell (RGC) activity. Little is known, however, about how inputs from the multitude of RGC subtypes are distributed to higher visual centers and how information is combined from these feature-selective inputs. RESULTS: Here we show that in the zebrafish optic tectum, prey- or predator-like moving targets evoke activity in distinct groups of RGC fibers dependent on target size, demonstrating a retinal origin of tectal size classification. Small-size-selective retinal inputs are relatively more frequent in the most superficial layer of the tectal neuropil, whereas large-size-selective inputs predominate in deeper layers. Monostratified superficial interneurons (SINs) process large-size- and small-size-selective signals dependent on their dendritic target layer, consistent with the retinal input organization. Further downstream, small- and large-sized objects are encoded in population activity of separate sets of tectal neurons. CONCLUSIONS: Ethologically relevant size classes are preferentially processed in different layers of the tectal neuropil. The tectum categorizes visual targets on the basis of retinally computed size information, suggesting a critical role in visually guided response selection.
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Author URL.
Gasser C, Taiber S, Yeh C-M, Wittig CH, Hegemann P, Ryu S, Wunder F, Möglich A (2014). Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase.
Proc Natl Acad Sci U S A,
111(24), 8803-8808.
Abstract:
Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase.
Sensory photoreceptors elicit vital physiological adaptations in response to incident light. As light-regulated actuators, photoreceptors underpin optogenetics, which denotes the noninvasive, reversible, and spatiotemporally precise perturbation by light of living cells and organisms. of particular versatility, naturally occurring photoactivated adenylate cyclases promote the synthesis of the second messenger cAMP under blue light. Here, we have engineered a light-activated phosphodiesterase (LAPD) with complementary light sensitivity and catalytic activity by recombining the photosensor module of Deinococcus radiodurans bacterial phytochrome with the effector module of Homo sapiens phosphodiesterase 2A. Upon red-light absorption, LAPD up-regulates hydrolysis of cAMP and cGMP by up to sixfold, whereas far-red light can be used to down-regulate activity. LAPD also mediates light-activated cAMP and cGMP hydrolysis in eukaryotic cell cultures and in zebrafish embryos; crucially, the biliverdin chromophore of LAPD is available endogenously and does not need to be provided exogenously. LAPD thus establishes a new optogenetic modality that permits light control over diverse cAMP/cGMP-mediated physiological processes. Because red light penetrates tissue more deeply than light of shorter wavelengths, LAPD appears particularly attractive for studies in living organisms.
Abstract.
Author URL.
Herget U, Wolf A, Wullimann MF, Ryu S (2014). Molecular neuroanatomy and chemoarchitecture of the neurosecretory preoptic-hypothalamic area in zebrafish larvae.
J Comp Neurol,
522(7), 1542-1564.
Abstract:
Molecular neuroanatomy and chemoarchitecture of the neurosecretory preoptic-hypothalamic area in zebrafish larvae.
The paraventricular nucleus (PVN) in mammals is the main hypothalamic nucleus controlling hormone release in the pituitary and plays pivotal roles in homeostasis. While the location of a PVN-homologous region has been described in adult fish as the neurosecretory preoptic area (NPO), this region has not been clearly defined in larval zebrafish due to the difficulty in defining cytoarchitectonic nuclear boundaries in the larval brain. Here we identify the precise location of the larval zebrafish NPO using conserved transcription factor and neuropeptide gene expressions. Our results identify the dorsal half of the preoptic-hypothalamic orthopedia a (otpa) domain as the larval NPO and the homologous region to the mammalian PVN. Further, by reconstructing the locations of cells producing zebrafish neuropeptides found in the mammalian PVN (CCK, CRH, ENK, NTS, SS, VIP, OXT, AVP), we provide the first 3D arrangement map of NPO neuropeptides in the larval zebrafish brain. Our results show striking conservation of transcription factor expression (otp, arx, dlx5a, isl1) in and around the NPO/PVN together with neuropeptide expression within it. Finally, we describe the exact anatomical location of cells producing Oxt and Avp in the adult zebrafish. Thus, our results identify the definitive borders and extent of the PVN homologous region in larval zebrafish and serve as an important basis for cross-species comparisons of PVN/NPO structure and function.
Abstract.
Author URL.
Herget U, Wolf A, Wullimann MF, Ryu S (2014). Molecular neuroanatomy and chemoarchitecture of the neurosecretory preoptic‐hypothalamic area in zebrafish larvae. The Journal of Comparative Neurology, 522(13), 3139-3139.
De Marco RJ, Groneberg AH, Yeh C-M, Treviño M, Ryu S (2014). The behavior of larval zebrafish reveals stressor-mediated anorexia during early vertebrate development.
Front Behav Neurosci,
8Abstract:
The behavior of larval zebrafish reveals stressor-mediated anorexia during early vertebrate development.
The relationship between stress and food consumption has been well documented in adults but less so in developing vertebrates. Here we demonstrate that an encounter with a stressor can suppress food consumption in larval zebrafish. Furthermore, we provide indication that food intake suppression cannot be accounted for by changes in locomotion, oxygen consumption and visual responses, as they remain unaffected after exposure to a potent stressor. We also show that feeding reoccurs when basal levels of cortisol (stress hormone in humans and teleosts) are re-established. The results present evidence that the onset of stress can switch off the drive for feeding very early in vertebrate development, and add a novel endpoint for analyses of metabolic and behavioral disorders in an organism suitable for high-throughput genetics and non-invasive brain imaging.
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Author URL.
Yeh C-M, Glöck M, Ryu S (2013). An optimized whole-body cortisol quantification method for assessing stress levels in larval zebrafish.
PLoS One,
8(11).
Abstract:
An optimized whole-body cortisol quantification method for assessing stress levels in larval zebrafish.
Glucocorticoids serve important regulatory functions for many physiological processes and are critical mediators of the stress response. The stress response is a set of bodily processes aimed at counteracting a state of threatened homeostasis. Proper stress response is critical for the survival of an animal, however prolonged or abnormal stress response can be detrimental and is implicated in a number of human diseases such as depression and metabolic diseases. To dissect the underlying mechanism of this complex and important response, the zebrafish, Danio rerio offer important advantages such as ease of genetic manipulations and high-throughput behavioral analyses. However, there is a paucity of suitable methods to measure stress level in larval zebrafish. Therefore, an efficient low-cost method to monitor stress hormone levels will greatly facilitate stress research in zebrafish larvae. In this study, we optimized sample collection as well as cortisol extraction methods and developed a home-made ELISA protocol for measuring whole-body cortisol level in zebrafish larvae. Further, using our customized protocols, we characterized the response of larval zebrafish to a variety of stressors. This assay, developed for efficient cortisol quantification, will be useful for systematic and large-scale stress analyses in larval zebrafish.
Abstract.
Author URL.
De Marco RJ, Groneberg AH, Yeh C-M, Castillo Ramírez LA, Ryu S (2013). Optogenetic elevation of endogenous glucocorticoid level in larval zebrafish.
Front Neural Circuits,
7Abstract:
Optogenetic elevation of endogenous glucocorticoid level in larval zebrafish.
The stress response is a suite of physiological and behavioral processes that help to maintain or reestablish homeostasis. Central to the stress response is the hypothalamic-pituitary-adrenal (HPA) axis, as it releases crucial hormones in response to stress. Glucocorticoids (GCs) are the final effector hormones of the HPA axis, and exert a variety of actions under both basal and stress conditions. Despite their far-reaching importance for health, specific GC effects have been difficult to pin-down due to a lack of methods for selectively manipulating endogenous GC levels. Hence, in order to study stress-induced GC effects, we developed a novel optogenetic approach to selectively manipulate the rise of GCs triggered by stress. Using this approach, we could induce both transient hypercortisolic states and persistent forms of hypercortisolaemia in freely behaving larval zebrafish. Our results also established that transient hypercortisolism leads to enhanced locomotion shortly after stressor exposure. Altogether, we present a highly specific method for manipulating the gain of the stress axis with high temporal accuracy, altering endocrine and behavioral responses to stress as well as basal GC levels. Our study offers a powerful tool for the analysis of rapid (non-genomic) and delayed (genomic) GC effects on brain function and behavior, feedbacks within the stress axis and developmental programming by GCs.
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Author URL.
Wolf A, Ryu S (2013). Specification of posterior hypothalamic neurons requires coordinated activities of Fezf2, Otp, Sim1a and Foxb1.2.
Development,
140(8), 1762-1773.
Abstract:
Specification of posterior hypothalamic neurons requires coordinated activities of Fezf2, Otp, Sim1a and Foxb1.2.
The hypothalamus is a key integrative center in the brain that consists of diverse cell types required for a variety of functions including homeostasis, reproduction, stress response, social and cognitive behavior. Despite our knowledge of several transcription factors crucial for hypothalamic development, it is not known how the wide diversity of neuron types in the hypothalamus is produced. In particular, almost nothing is known about the mechanisms that specify neurons in the posteriormost part of the hypothalamus, the mammillary area. Here, we investigated the specification of two distinct neuron types in the mammillary area that produce the hypothalamic hormones Vasoactive intestinal peptide (Vip) and Urotensin 1 (Uts1). We show that Vip- and Uts1-positive neurons develop in distinct domains in the mammillary area defined by the differential expression of the transcription factors Fezf2, Otp, Sim1a and Foxb1.2. Coordinated activities of these factors are crucial for the establishment of the mammillary area subdomains and the specification of Vip- and Uts1-positive neurons. In addition, Fezf2 is important for early development of the posterior hypothalamus. Thus, our study provides the first molecular anatomical map of the posterior hypothalamus in zebrafish and identifies, for the first time, molecular requirements underlying the specification of distinct posterior hypothalamic neuron types.
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Author URL.
Beretta CA, Dross N, Guiterrez-Triana JA, Ryu S, Carl M (2012). Habenula circuit development: past, present, and future.
Front Neurosci,
6Abstract:
Habenula circuit development: past, present, and future.
The habenular neural circuit is attracting increasing attention from researchers in fields as diverse as neuroscience, medicine, behavior, development, and evolution. Recent studies have revealed that this part of the limbic system in the dorsal diencephalon is involved in reward, addiction, and other behaviors and its impairment is associated with various neurological conditions and diseases. Since the initial description of the dorsal diencephalic conduction system (DDC) with the habenulae in its center at the end of the nineteenth century, increasingly sophisticated techniques have resolved much of its anatomy and have shown that these pathways relay information from different parts of the forebrain to the tegmentum, midbrain, and hindbrain. The first part of this review gives a brief historical overview on how the improving experimental approaches have allowed the stepwise uncovering much of the architecture of the habenula circuit as we know it today. Our brain distributes tasks differentially between left and right and it has become a paradigm that this functional lateralization is a universal feature of vertebrates. Moreover, task dependent differential brain activities have been linked to anatomical differences across the left-right axis in humans. A good way to further explore this fundamental issue will be to study the functional consequences of subtle changes in neural network formation, which requires that we fully understand DDC system development. As the habenular circuit is evolutionarily highly conserved, researchers have the option to perform such difficult experiments in more experimentally amenable vertebrate systems. Indeed, research in the last decade has shown that the zebrafish is well suited for the study of DDC system development and the phenomenon of functional lateralization. We will critically discuss the advantages of the zebrafish model, available techniques, and others that are needed to fully understand habenular circuit development.
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Author URL.
Gabriel JP, Trivedi CA, Maurer CM, Ryu S, Bollmann JH (2012). Layer-specific targeting of direction-selective neurons in the zebrafish optic tectum.
Neuron,
76(6), 1147-1160.
Abstract:
Layer-specific targeting of direction-selective neurons in the zebrafish optic tectum.
Direction selectivity (DS) is an important neuronal property in the visual system, but how DS is generated beyond the retina remains controversial. Here, we report a close correspondence between the preferred direction (PD) and the morphology of DS cells in the optic tectum. Ca(2+) imaging in cells expressing the genetically encoded Ca(2+) indicator GCaMP3 and multiphoton-targeted patch-clamp recordings allowed us to compare structure and function in single neurons. The arbors of differently tuned cell types showed stereotypic differences in shape and laminar profile within the tectal neuropil. Excitatory synaptic inputs were directionally tuned and matched the PD of spike output in these cells, while inhibitory inputs were selective for nonpreferred directions. Functional Ca(2+) imaging in afferent axons showed a matching laminar distribution of DS presynaptic activity. Hence, different directions are represented in different layers, which suggests a simple mechanism for how tectal neurons acquire directional tuning in a nascent circuit.
Abstract.
Author URL.
Tay TL, Ronneberger O, Ryu S, Nitschke R, Driever W (2011). Comprehensive catecholaminergic projectome analysis reveals single-neuron integration of zebrafish ascending and descending dopaminergic systems.
Nat Commun,
2Abstract:
Comprehensive catecholaminergic projectome analysis reveals single-neuron integration of zebrafish ascending and descending dopaminergic systems.
Essential components of animal behaviour are modulated by dopaminergic (DA) and noradrenergic circuitry. In this study, we reveal at cellular resolution the complete set of projections ('projectome') of every single type of DA and noradrenergio neurons in the central nervous system of zebrafish larvae. The most extensive DA projections are established by posterior tubercular otp-dependent neurons, with individual somata integrating the ascending DA system, the descending diencephalospinal, as well as the endohypothalamic circuitry. These findings suggest a major role in the modulation of physiology and behaviour for otp-dependent DA neurons, which correlate with the mammalian A11 group. We further identified an endogenous subpallial DA system that not only provides most of the local DA projections, but also connects to the ventral diencephalon. The catecholaminergic projectome map provides a framework to understand the evolution and function of these neuromodulatory systems.
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Author URL.
Kastenhuber E, Kratochwil CF, Ryu S, Schweitzer J, Driever W (2010). Genetic dissection of dopaminergic and noradrenergic contributions to catecholaminergic tracts in early larval zebrafish.
J Comp Neurol,
518(4), 439-458.
Abstract:
Genetic dissection of dopaminergic and noradrenergic contributions to catecholaminergic tracts in early larval zebrafish.
The catecholamines dopamine and noradrenaline provide some of the major neuromodulatory systems with far-ranging projections in the brain and spinal cord of vertebrates. However, development of these complex systems is only partially understood. Zebrafish provide an excellent model for genetic analysis of neuronal specification and axonal projections in vertebrates. Here, we analyze the ontogeny of the catecholaminergic projections in zebrafish embryos and larvae up to the fifth day of development and establish the basic scaffold of catecholaminergic connectivity. The earliest dopaminergic diencephalospinal projections do not navigate along the zebrafish primary neuron axonal scaffold but establish their own tracts at defined ventrolateral positions. By using genetic tools, we study quantitative and qualitative contributions of noradrenergic and defined dopaminergic groups to the catecholaminergic scaffold. Suppression of Tfap2a activity allows us to eliminate noradrenergic contributions, and depletion of Otp activity deletes mammalian A11-like Otp-dependent ventral diencephalic dopaminergic groups. This analysis reveals a predominant contribution of Otp-dependent dopaminergic neurons to diencephalospinal as well as hypothalamic catecholaminergic tracts. In contrast, noradrenergic projections make only a minor contribution to hindbrain and spinal catecholaminergic tracts. Furthermore, we can demonstrate that, in zebrafish larvae, ascending catecholaminergic projections to the telencephalon are generated exclusively by Otp-dependent diencephalic dopaminergic neurons as well as by hindbrain noradrenergic groups. Our data reveal the Otp-dependent A11-type dopaminergic neurons as the by far most prominent dopaminergic system in larval zebrafish. These findings are consistent with a hypothesis that Otp-dependent dopaminergic neurons establish the major modulatory system for somatomotor and somatosensory circuits in larval fish.
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Author URL.
Löhr H, Ryu S, Driever W (2009). Zebrafish diencephalic A11-related dopaminergic neurons share a conserved transcriptional network with neuroendocrine cell lineages.
Development,
136(6), 1007-1017.
Abstract:
Zebrafish diencephalic A11-related dopaminergic neurons share a conserved transcriptional network with neuroendocrine cell lineages.
Vertebrate dopaminergic neurons develop in distinct neural territories to constitute one of the major neuromodulatory systems. We have identified a zebrafish mutation in the bHLH-PAS family member arnt2, based on a strong reduction in cell number of specific dopaminergic neuron groups in the hypothalamus and posterior tuberculum. Knockdown of sim1 causes a dopaminergic phenotype similar to arnt2 mutants, suggesting that Sim1 acts as a binding partner of Arnt2, similar to their role in hypothalamic neuroendocrine cell specification. sim1, arnt2 and otp are co-expressed in dopaminergic neurons, and combined overexpression of Sim1 and Otp leads to formation of supernumerary dopaminergic neurons in the ventral diencephalon. Arnt2, Sim1 and Otp thus are core components of a conserved transcriptional network, which specifies neuroendocrine as well as A11-related dopaminergic neurons in the fish hypothalamus and posterior tuberculum. Our data suggest a common evolutionary origin of specific hypothalamic neuroendocrine and dopaminergic systems.
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Author URL.
Ryu S, Mahler J, Acampora D, Holzschuh J, Erhardt S, Omodei D, Simeone A, Driever W (2008). Orthopedia Homeodomain Protein is Essential for Diencephalic Dopaminergic Neuron Development. Current Biology, 18(4).
Meng S, Ryu S, Zhao B, Zhang D-Q, Driever W, McMahon DG (2008). Targeting retinal dopaminergic neurons in tyrosine hydroxylase-driven green fluorescent protein transgenic zebrafish.
Mol Vis,
14, 2475-2483.
Abstract:
Targeting retinal dopaminergic neurons in tyrosine hydroxylase-driven green fluorescent protein transgenic zebrafish.
PURPOSE: Dopamine plays key roles in a variety of basic functions in the central nervous system. To study developmental and functional roles of dopaminergic cells in zebrafish, we have generated a transgenic line of zebrafish expressing green fluorescent protein (GFP) under the control of the tyrosine hydroxylase (th1) promoter. METHODS: a 12 kb gene fragment that contains the th1 promoter was isolated and ligated to the MmGFP coding sequence, linearized, microinjected into 1-2 cell stage embryos and the founders crossed with wild-type fish to screen for transgenic lines. Tg(-12th:MmGFP) embryos were visualized under fluorescence microscopy for GFP expression during development. Confocal microscopy was used to visualize GFP-labeled cells in the living whole mount retina and immunostained vertical sections of adult zebrafish retina. Single-cell reverse transcription polymerase chain reaction (RT-PCR) was performed on individual GFP+ cells collected from dispersed retinal cell cultures for th1 and dopamine transporter (dat). Loose-patch recordings of spike activity of GFP+ neurons were made in isolated whole mount retinas. RESULTS: th1 promoter-driven GFP exhibited robust expression in the brain and retina during zebrafish development. In juvenile and adult fish retinas, GFP was expressed in cells located in the inner nuclear layer. Immunocytochemistry with antibodies for GFP and TH showed that 29+/-2% of GFP-labeled cells also expressed TH. Two subpopulations of GFP-labeled cells were identified by fluorescent microscopy: bright GFP-expressing cells and dim GFP-expressing cells. Seminested single-cell RT-PCR showed that 71% of dim GFP-expressing cells expressed both th and dat mRNA. Loose-patch voltage-clamp recording from dim GFP-labeled cells in retinal whole mounts revealed that many of these dopaminergic neurons are spontaneously active in darkness. CONCLUSIONS: Although this Tg(-12th:MmGFP) line is not a completely specific reporter for dopaminergic neurons, using relative GFP intensity we are able to enrich for the selection of retinal dopaminergic cells in vitro and in situ in molecular and electrophysiological experiments. This transgenic line provides a useful tool for studying retinal dopaminergic cells in the zebrafish.
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Author URL.
Filippi A, Dürr K, Ryu S, Willaredt M, Holzschuh J, Driever W (2007). Expression and function of nr4a2, lmx1b, and pitx3 in zebrafish dopaminergic and noradrenergic neuronal development.
BMC Dev Biol,
7Abstract:
Expression and function of nr4a2, lmx1b, and pitx3 in zebrafish dopaminergic and noradrenergic neuronal development.
BACKGROUND: Dopaminergic neurons form in diverse areas of the vertebrate di- and mesencephalon to constitute several major neuromodulatory systems. While much is known about mammalian mesencephalic dopaminergic neuron development, little is known about the specification of the diencephalic dopaminergic groups. The transcription factors Pitx3 and Lmx1b play an important role in mammalian mesencephalic dopaminergic specification, and Nurr1/Nr4a2 has been shown to contribute to specification of the dopaminergic neurotransmitter phenotype. We use zebrafish to analyze potentially evolutionarily conserved roles of these transcription factors in a vertebrate brain that lacks a mesencephalic dopaminergic system, but has an ascending dopaminergic system in the ventral diencephalon. RESULTS: We use a combination of fluorescent in situ hybridization and immunohistochemistry to determine whether nr4a2, lmx1b, and pitx3 genes are expressed in mature dopaminergic neurons or in potential precursor populations. We identify a second nr4a2 paralogue, nr4a2a, and find it co-expressed with Tyrosine hydroxylase in preoptic, pretectal and retinal amacrine dopaminergic neurons, while nr4a2b is only expressed in preoptic and retinal dopaminergic neurons. Both zebrafish nr4a2 paralogues are not expressed in ventral diencephalic dopaminergic neurons with ascending projections. Combined morpholino antisense oligo mediated knock-down of both nr4a2a and nr4a2b transcripts reveals that all zebrafish dopaminergic neurons expressing nr4a2a depend on Nr4a2 activity for tyrosine hydroxylase and dopamine transporter expression. Zebrafish lmx1b.1 is expressed in noradrenergic neurons of the locus coeruleus and medulla oblongata, but knock-down reveals that it is specifically required for tyrosine hydroxylase expression only in the medulla oblongata area postrema noradrenergic neurons. Both lmx1b genes and pitx3 are not expressed in dopaminergic neurons, but in a diencephalic territory that might contain precursor cells for ventral diencephalic dopaminergic neurons. Upon morpholino knock-down of both lmx1b paralogues, the number of neurons in diencephalic dopaminergic clusters with ascending projections appears specifically reduced. Thus lmx1b paralogues may contribute to the generation of diencephalic dopaminergic precursors. Conversely, knock-down of pitx3 does not specifically affect any diencephalic DA cluster. CONCLUSION: Our data indicate a conserved evolutionary role of Nr4a2 proteins in specification of the neurotransmitter phenotype, albeit it appears to be only one of several regulatory modules of dopaminergic differentiation, as most ventral diencephalic dopaminergic neurons do not express nr4a2 genes in zebrafish. For zebrafish lmx1b genes, which are not expressed in mature dopaminergic neurons, our data suggest a role in diencephalic precursor populations contributing to the ascending dopaminergic systems. A di-mesencephalic longitudinal domain of lmx1b expression may be the basis for the expansion and posterior shift of ventral di-/mesencephalic dopaminergic populations with ascending projections during evolution.
Abstract.
Author URL.
Ryu S, Mahler J, Acampora D, Holzschuh J, Erhardt S, Omodei D, Simeone A, Driever W (2007). Orthopedia homeodomain protein is essential for diencephalic dopaminergic neuron development.
Curr Biol,
17(10), 873-880.
Abstract:
Orthopedia homeodomain protein is essential for diencephalic dopaminergic neuron development.
Neurons that produce dopamine as a neurotransmitter constitute a heterogeneous group involved in the control of various behaviors and physiology. In mammals, dopaminergic neurons are found in distinct clusters mainly located in the ventral midbrain and the caudal forebrain [1]. Although much is known about midbrain dopaminergic neurons, development of diencephalic dopaminergic neurons is poorly understood. Here we demonstrate that Orthopedia (Otp) homeodomain protein is essential for the development of specific subsets of diencephalic dopaminergic neurons. Zebrafish embryos lacking Otp activity are devoid of dopaminergic neurons in the hypothalamus and the posterior tuberculum. Similarly, Otp-/- mouse [2, 3] embryos lack diencephalic dopaminergic neurons of the A11 group, which constitutes the diencephalospinal dopaminergic system. In both systems, Otp is expressed in the affected dopaminergic neurons as well as in potential precursor populations, and it might contribute to dopaminergic cell specification and differentiation. In fish, overexpression of Otp can induce ectopic tyrosine hydroxylase and dopamine transporter expression, indicating that Otp can specify aspects of dopaminergic identity. Thus, Otp is one of the few known transcription factors that can determine aspects of the dopaminergic phenotype and the first known factor to control the development of the diencephalospinal dopaminergic system.
Abstract.
Author URL.
Brockschmidt A, Todt U, Ryu S, Hoischen A, Landwehr C, Birnbaum S, Frenck W, Radlwimmer B, Lichter P, Engels H, et al (2007). Severe mental retardation with breathing abnormalities (Pitt-Hopkins syndrome) is caused by haploinsufficiency of the neuronal bHLH transcription factor TCF4.
Hum Mol Genet,
16(12), 1488-1494.
Abstract:
Severe mental retardation with breathing abnormalities (Pitt-Hopkins syndrome) is caused by haploinsufficiency of the neuronal bHLH transcription factor TCF4.
Pitt-Hopkins syndrome (PHS) is a rare syndromic mental disorder, which is mainly characterized by severe motor and mental retardation including absent language development, a characteristic facial gestalt and episodes of hyperventilation. We report on a female patient with PHS showing severe mental retardation with absent speech, pronounced muscular hypotonia, ataxia, distinctive facial features, such as a coarse face, a broad nasal bridge and a wide mouth, and hyperventilation attacks. In this patient, genomic profiling by array-based comparative genomic hybridization and fluorescence in situ hybridization studies detected and confirmed a de novo 0.5 Mb deletion in 18q21.2 containing a single gene, the basic helix-loop-helix transcription factor TCF4. cDNA and genomic analyses in the patient and her parents demonstrated TCF4 haploinsufficiency as the underlying cause of the disease. Analysis of the embryonal expression pattern of the Danio rerio ortholog, tcf4, by whole-mount in situ hybridization showed a highly specific expression domain in the pallium of the telencephalon during late somitogenesis, when the patterning of the zebrafish brain is advanced and neural differentiation commences. Later expression domains were restricted to several regions in the central nervous system, including continued expression in the pallium of the telencephalon, and starting expression in the diencephalon (thalamus, ventral thalamus and posterior tuberculum), the midbrain tegmentum, the hindbrain and the branchial arches. This expression pattern correlates with the clinical phenotype. Our results show that haploinsufficiency of TCF4 causes PHS and suggest that D. rerio is a valuable model to study the molecular pathogenesis of PHS and the role of TCF4 in brain development.
Abstract.
Author URL.
Dürr K, Holzschuh J, Filippi A, Ettl A-K, Ryu S, Shepherd IT, Driever W (2006). Differential roles of transcriptional mediator complex subunits Crsp34/Med27, Crsp150/Med14 and Trap100/Med24 during zebrafish retinal development.
Genetics,
174(2), 693-705.
Abstract:
Differential roles of transcriptional mediator complex subunits Crsp34/Med27, Crsp150/Med14 and Trap100/Med24 during zebrafish retinal development.
The transcriptional mediator complex has emerged as an important component of transcriptional regulation, yet it is largely unknown whether its subunits have differential functions in development. We demonstrate that the zebrafish mutation m885 disrupts a subunit of the mediator complex, Crsp34/Med27. To explore the role of the mediator in the control of retinal differentiation, we employed two additional mutations disrupting the mediator subunits Trap100/Med24 and Crsp150/Med14. Our analysis shows that loss of Crsp34/Med27 decreases amacrine cell number, but increases the number of rod photoreceptor cells. In contrast, loss of Trap100/Med24 decreases rod photoreceptor cells. Loss of Crsp150/Med14, on the other hand, only slightly reduces dopaminergic amacrine cells, which are absent from both crsp34(m885) and trap100(lessen) mutant embryos. Our data provide evidence for differential requirements for Crsp34/Med27 in developmental processes. In addition, our data point to divergent functions of the mediator subunits Crsp34/Med27, Trap100/Med24, and Crsp150/Med14 and, thus, suggest that subunit composition of the mediator contributes to the control of differentiation in the vertebrate CNS.
Abstract.
Author URL.
Ryu S, Holzschuh J, Mahler J, Driever W (2006). Genetic analysis of dopaminergic system development in zebrafish.
J Neural Transm Suppl(70), 61-66.
Abstract:
Genetic analysis of dopaminergic system development in zebrafish.
Zebrafish have become an important model organism to study the genetic control of vertebrate nervous system development. Here, we present an overview on the formation of dopaminergic neuronal groups in zebrafish and compare the positions of DA neurons in fish and mammals using the neuromere model of the vertebrate brain. Based on mutant analysis, we evaluate the role of several signaling pathways in catecholaminergic neuron specification. We further discuss the prospect of identifying novel genes involved in dopaminergic development through forward genetics mutagenesis screens.
Abstract.
Author URL.
Ryu S, Driever W (2006). Minichromosome maintenance proteins as markers for proliferation zones during embryogenesis.
Cell Cycle,
5(11), 1140-1142.
Abstract:
Minichromosome maintenance proteins as markers for proliferation zones during embryogenesis.
Regulation of cell proliferation is of fundamental importance for growth and prevention of cancer. An obligatory step in the regulation of cell proliferation is the control of the initiation of DNA synthesis. The minichromosome maintenance (MCM) proteins are essential DNA replication factors crucial for initiating DNA synthesis once every cell cycle. Recent studies show that the level of MCM proteins is stringently regulated to correlate with cell proliferation and carcinogenesis. Here we discuss recent data, which highlights the usefulness of minichromosome maintenance (MCM) gene expression for detecting proliferating cells as well as zones containing proliferative/stem cells during embryogenesis in a whole organism.
Abstract.
Author URL.
Kastenhuber E, Ryu S, Driever W (2006). [P240]: Guidance of dopaminergic axonal projections in zebrafish. International Journal of Developmental Neuroscience, 24(8), 598-598.
Chapouton P, Adolf B, Leucht C, Tannhäuser B, Ryu S, Driever W, Bally-Cuif L (2006). her5 expression reveals a pool of neural stem cells in the adult zebrafish midbrain.
Development,
133(21), 4293-4303.
Abstract:
her5 expression reveals a pool of neural stem cells in the adult zebrafish midbrain.
Current models of vertebrate adult neural stem cells are largely restricted to the rodent forebrain. To extract the general mechanisms of neural stem cell biology, we sought to identify new adult stem cell populations, in other model systems and/or brain areas. The teleost zebrafish appears to be an ideal system, as cell proliferation in the adult zebrafish brain is found in many more niches than in the mammalian brain. As a starting point towards identifying stem cell populations in this system, we used an embryonic neural stem cell marker, the E(spl) bHLH transcription factor Her5. We demonstrate that her5 expression is not restricted to embryonic neural progenitors, but also defines in the adult zebrafish brain a new proliferation zone at the junction between the mid- and hindbrain. We show that adult her5-expressing cells proliferate slowly, self-renew and express neural stem cell markers. Finally, using in vivo lineage tracing in her5:gfp transgenic animals, we demonstrate that the her5-positive population is multipotent, giving rise in situ to differentiated neurons and glia that populate the basal midbrain. Our findings conclusively identify a new population of adult neural stem cells, as well as their fate and their endogenous environment, in the intact vertebrate brain. This cell population, located outside the forebrain, provides a powerful model to assess the general mechanisms of vertebrate neural stem cell biology. In addition, the first transcription factor characteristic of this cell population, Her5, points to the E(Spl) as a promising family of candidate adult neural stem cell regulators.
Abstract.
Author URL.
Ryu S, Holzschuh J, Erhardt S, Ettl A-K, Driever W (2005). Depletion of minichromosome maintenance protein 5 in the zebrafish retina causes cell-cycle defect and apoptosis.
Proc Natl Acad Sci U S A,
102(51), 18467-18472.
Abstract:
Depletion of minichromosome maintenance protein 5 in the zebrafish retina causes cell-cycle defect and apoptosis.
In multicellular organisms, the control of genome duplication and cell division must be tightly coordinated. Essential roles of the minichromosome maintenance (MCM) proteins for genome duplication have been well established. However, no genetic model has been available to address the function of MCM proteins in the context of vertebrate organogenesis. Here, we present positional cloning of a zebrafish mcm5 mutation and characterization of its retina phenotype. In the retina, mcm5 expression correlates closely with the pattern of cell proliferation. By the third day of development, mcm5 is down-regulated in differentiated cells but is maintained in regions containing retinal stem cells. We demonstrate that a gradual depletion of maternally derived MCM5 protein leads to a prolonged S phase, cell-cycle-exit failure, apoptosis, and reduction in cell number in mcm5(m850) mutant embryos. Interestingly, by the third day of development, increased apoptosis is detectable only in the retina, tectum, and hindbrain but not in other late-proliferating tissues, suggesting that different tissues may employ distinct cellular programs in responding to the depletion of MCM5.
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Holzschuh J, Ryu S, Aberger F, Driever W (2001). Dopamine transporter expression distinguishes dopaminergic neurons from other catecholaminergic neurons in the developing zebrafish embryo.
Mech Dev,
101(1-2), 237-243.
Abstract:
Dopamine transporter expression distinguishes dopaminergic neurons from other catecholaminergic neurons in the developing zebrafish embryo.
To characterize the formation of the dopaminergic system in the developing zebrafish CNS, we cloned cDNAs encoding tyrosine hydroxylase (th), an enzyme in dopamine synthesis, and the dopamine transporter (dat), a membrane transport protein which terminates dopamine action by re-uptake. Dopaminergic neurons are first detected between 18 and 19 h post-fertilization in a cluster of cells in the ventral diencephalon. Subsequently, th and dat detection identifies dopaminergic neurons in the olfactory bulb, the pretectum, the retina and the locus coeruleus. Neurons expressing th but not dat are adrenergic or noradrenergic, and are found in the locus coeruleus, the medulla, the likely analog of the carotid body, and precursors of the enteric and sympathetic nervous system.
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Ryu S, Tjian R (1999). Purification of transcription cofactor complex CRSP.
Proc Natl Acad Sci U S A,
96(13), 7137-7142.
Abstract:
Purification of transcription cofactor complex CRSP.
Transcription of protein coding genes in metazoans involves the concerted action of enhancer binding proteins and the RNA polymerase II apparatus. The cross talk between these two classes of transcription factors is mediated by an elaborate set of cofactor complexes. For the activation of transcription by the promoter specificity protein 1 (Sp1), TATA binding protein-associated factors in the TFIID complex originally were identified as necessary coactivators, but the identity of additional cofactors required for activated transcription was unknown. Recently, we have reported the isolation and properties of a cofactor complex, CRSP (cofactor required for Sp1), which functions in conjunction with the TATA binding protein-associated factors to promote efficient activation of transcription by Sp1. CRSP contains unique subunits as well as polypeptides that are shared with other cofactor complexes. Here, we report a detailed purification protocol for the isolation of CRSP from human HeLa cells. Our purification strategy takes advantage of the ability of CRSP to bind Ni2+-nitrilotriacetic acid-agarose resin as well as other conventional chromatographic resins. We also describe a streamlined purification protocol that allows a more rapid and efficient means to isolate active CRSP.
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Author URL.
Ryu S, Zhou S, Ladurner AG, Tjian R (1999). The transcriptional cofactor complex CRSP is required for activity of the enhancer-binding protein Sp1.
Nature,
397(6718), 446-450.
Abstract:
The transcriptional cofactor complex CRSP is required for activity of the enhancer-binding protein Sp1.
Activation of gene transcription in metazoans is a multistep process that is triggered by factors that recognize transcriptional enhancer sites in DNA. These factors work with co-activators to direct transcriptional initiation by the RNA polymerase II apparatus. One class of co-activator, the TAF(II) subunits of transcription factor TFIID, can serve as targets of activators and as proteins that recognize core promoter sequences necessary for transcription initiation. Transcriptional activation by enhancer-binding factors such as Sp1 requires TFIID, but the identity of other necessary cofactors has remained unknown. Here we describe a new human factor, CRSP, that is required together with the TAF(II)s for transcriptional activation by Sp1. Purification of CRSP identifies a complex of approximate relative molecular mass 700,000 (M(r) approximately 700K) that contains nine subunits with M(r) values ranging from 33K to 200K. Cloning of genes encoding CRSP subunits reveals that CRSP33 is a homologue of the yeast mediator subunit Med7, whereas CRSP150 contains a domain conserved in yeast mediator subunit Rgr1. CRSP p200 is identical to the nuclear hormone-receptor co-activator subunit TRIP2/PBP. CRSPs 34, 77 and 130 are new proteins, but the amino terminus of CRSP70 is homologous to elongation factor TFIIS. Immunodepletion studies confirm that these subunits have an essential cofactor function. The presence of common subunits in distinct cofactor complexes suggests a combinatorial mechanism of co-activator assembly during transcriptional activation.
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Näär AM, Ryu S, Tjian R (1998). Cofactor requirements for transcriptional activation by Sp1.
Cold Spring Harb Symp Quant Biol,
63, 189-199.
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