A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine in Flies, Fish, and Mice.

Dopamine (DA) is a central monoamine neurotransmitter involved in many physiological and pathological processes. A longstanding yet largely unmet goal is to measure DA changes reliably and specifically with high spatiotemporal precision, particularly in animals executing complex behaviors. Here, we report the development of genetically encoded GPCR-activation-based-DA (GRABDA) sensors that enable these measurements. In response to extracellular DA, GRABDA sensors exhibit large fluorescence increases (ΔF/F0 ∼90%) with subcellular resolution, subsecond kinetics, nanomolar to submicromolar affinities, and excellent molecular specificity. GRABDA sensors can resolve a single-electrical-stimulus-evoked DA release in mouse brain slices and detect endogenous DA release in living flies, fish, and mice. In freely behaving mice, GRABDA sensors readily report optogenetically elicited nigrostriatal DA release and depict dynamic mesoaccumbens DA signaling during Pavlovian conditioning or during sexual behaviors. Thus, GRABDA sensors enable spatiotemporally precise measurements of DA dynamics in a variety of model organisms while exhibiting complex behaviors.

Systematic genome editing of the genes on zebrafish Chromosome 1 by CRISPR/Cas9.

Genome editing by the well-established CRISPR/Cas9 technology has greatly facilitated our understanding of many biological processes. However, a complete whole-genome knockout for any species or model organism has rarely been achieved. Here, we performed a systematic knockout of all the genes (1333) on Chromosome 1 in zebrafish, successfully mutated 1029 genes, and generated 1039 germline-transmissible alleles corresponding to 636 genes. Meanwhile, by high-throughput bioinformatics analysis, we found that sequence features play pivotal roles in effective gRNA targeting at specific genes of interest, while the success rate of gene targeting positively correlates with GC content of the target sites. Moreover, we found that nearly one-fourth of all mutants are related to human diseases, and several representative CRISPR/Cas9-generated mutants are described here. Furthermore, we tried to identify the underlying mechanisms leading to distinct phenotypes between genetic mutants and antisense morpholino-mediated knockdown embryos. Altogether, this work has generated the first chromosome-wide collection of zebrafish genetic mutants by the CRISPR/Cas9 technology, which will serve as a valuable resource for the community, and our bioinformatics analysis also provides some useful guidance to design gene-specific gRNAs for successful gene editing.

Heat resilience in embryonic zebrafish revealed using an in vivo stress granule reporter.

Although the regulation of stress granules has become an intensely studied topic, current investigations of stress granule assembly, disassembly and dynamics are mainly performed in cultured cells. Here, we report the establishment of a stress granule reporter to facilitate the real-time study of stress granules in vivo Using CRISPR/Cas9, we fused a green fluorescence protein (GFP) to endogenous G3BP1 in zebrafish. The GFP-G3BP1 reporter faithfully and robustly responded to heat stress in zebrafish embryos and larvae. The induction of stress granules varied by brain regions under the same stress condition, with the midbrain cells showing the highest efficiency and dynamics. Furthermore, pre-conditioning using lower heat stress significantly limited stress granule formation during subsequent higher heat stress. More interestingly, stress granule formation was much more robust in zebrafish embryos than in larvae and coincided with significantly elevated levels of phosphorylated eIF2α and enhanced heat resilience. Therefore, these findings have generated new insights into stress response in zebrafish during early development and demonstrated that the GFP-G3BP1 knock-in zebrafish could be a valuable tool for the investigation of stress granule biology.This article has an associated First Person interview with the first author of the paper.

Near-Infrared Voltage Nanosensors Enable Real-Time Imaging of Neuronal Activities in Mice and Zebrafish.

Optical voltage sensors with the ability to monitor neuronal activities are invaluable tools for studying information processing of the brain. However, the current genetically encoded voltage indicators usually require high-power visible light for excitation and are limited to genetically addressable model animals. Here, we report a near-infrared (NIR)-excited nongenetic voltage nanosensor that achieves stable recording of neuronal membrane potential in intact animals. The nanosensor is composed of a Förster resonance energy transfer (FRET) pair, the outer membrane-anchored upconversion nanoparticle (UCNP), and the membrane-embedded dipicrylamine (DPA). The negative charge of DPA allows membrane potential fluctuation to affect the distance between the DPA and UCNP, therefore changing the FRET efficiency. Consequently, the emission intensity of the nanosensor can report the membrane potential. Using the nanosensor, we monitor not only electrically evoked changes in the membrane potential of cultured cells but also sensory responses of neurons in intact zebrafish and brain state-modulated subthreshold activities of cortical neurons in intact mice.

Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. iNap sensors enabled quantification of cytosolic and mitochondrial NADPH pools that are controlled by cytosolic NAD+ kinase levels and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We found that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase and AMP kinase. Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response in vivo. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live cells and gaining new insights into cell metabolism.

Lysophosphatidic acid acts as a nutrient-derived developmental cue to regulate early hematopoiesis.

Primitive hematopoiesis occurs in the yolk sac blood islands during vertebrate embryogenesis, where abundant phosphatidylcholines (PC) are available as important nutrients for the developing embryo. However, whether these phospholipids also generate developmental cues to promote hematopoiesis is largely unknown. Here, we show that lysophosphatidic acid (LPA), a signaling molecule derived from PC, regulated hemangioblast formation and primitive hematopoiesis. Pharmacological and genetic blockage of LPA receptor 1 (LPAR1) or autotoxin (ATX), a secretory lysophospholipase that catalyzes LPA production, inhibited hematopoietic differentiation of mouse embryonic stem cells and impaired the formation of hemangioblasts. Mechanistic experiments revealed that the regulatory effect of ATX-LPA signaling was mediated by PI3K/Akt-Smad pathway. Furthermore, during in vivo embryogenesis in zebrafish, LPA functioned as a developmental cue for hemangioblast formation and primitive hematopoiesis. Taken together, we identified LPA as an important nutrient-derived developmental cue for primitive hematopoiesis as well as a novel mechanism of hemangioblast regulation.

[The structure and function of habenula].

The habenula (Hb) is an evolutionarily conserved diencephalic structure in vertebrates. It is considered as an emotion center and plays critical roles in regulating diverse types of emotion-related behaviors, including anxiety, fear, reward, depression, and nicotine withdrawal. On the one hand, action selection- and emotion-relevant inputs are transferred to the Hb through the basal ganglia and limbic system, respectively. At the same time, sensory inputs of multiple modalities also converge on the Hb. Among them, the visual input of the Hb from the retina ganglion cells ‒ thalamus pathway has been found to play a critical role in light-preference behavior of zebrafish. On the other hand, the Hb projects to two main neuromodulatory systems, the dopaminergic system and the serotoninergic system. As the Hb receives both internal emotion inputs and external sensory inputs and regulates the function of neuromodulatory systems, its functions are quite diverse and complex. In this review, we summarize the progress in both the structure and connection of the Hb and propose future study direction.

Amperometric Monitoring of Sensory-Evoked Dopamine Release in Awake Larval Zebrafish.

Dopamine plays crucial roles in a broad spectrum of brain functions, and neural circuit mechanisms underlying dopaminergic regulation have been intensively studied in the past decade. As larval zebrafish have relatively simple and highly conserved dopaminergic systems, it can serve as an ideal vertebrate animal model to tackle this issue at a whole-brain scale. For this purpose, it is important to develop methods for monitoring endogenous dopamine release in intact larval zebrafish. Here, we developed a real-time method to monitor dopamine release at high spatiotemporal resolution in the brain of awake larval zebrafish using carbon fiber microelectrodes. As an example for application, we combined this method with genetic tools and in vivo calcium imaging and found that food extract can activate pretectal dopaminergic neurons, which in turn release dopamine at the visual center through their projection, providing a dopaminergic circuit mechanism for olfactory modulation of visual functions. Thus, our study demonstrates, for the first time, the utility of carbon fiber microelectrodes for monitoring sensory-evoked dopamine release in the brain of an awake small organism. SIGNIFICANCE STATEMENT: With carbon fiber microelectrodes, we have succeeded in monitoring sensory-evoked dopamine release in the brain of an awake small organism for the first time. By elucidating the circuitry origin of the dopamine release, we illustrated the potential application of this method in dissection of the neural circuitry mechanisms underlying dopaminergic neuromodulation.

Ildr1b is essential for semicircular canal development, migration of the posterior lateral line primordium and hearing ability in zebrafish: implications for a role in the recessive hearing impairment DFNB42.

Immunoglobulin-like domain containing receptor 1 (ILDR1) is a poorly characterized gene that was first identified in lymphoma cells. Recently, ILDR1 has been found to be responsible for autosomal recessive hearing impairment DFNB42. Patients with ILDR1 mutations cause bilateral non-progressive moderate-to-profound sensorineural hearing impairment. However, the etiology and mechanism of ILDR1-related hearing loss remains to be elucidated. In order to uncover the pathology of DFNB42 deafness, we used the morpholino injection technique to establish an ildr1b-morphant zebrafish model. Ildr1b-morphant zebrafish displayed defective hearing and imbalanced swimming, and developmental delays were seen in the semicircular canals of the inner ear. The gene expression profile and real-time PCR revealed down-regulation of atp1b2b (encoding Na(+)/K(+) transporting, beta 2b polypeptide) in ildr1b-morphant zebrafish. We found that injection of atp1b2b mRNA into ildr1b-knockdown zebrafish could rescue the phenotype of developmental delay of the semicircular canals. Moreover, ildr1b-morphant zebrafish had reduced numbers of lateral line neuromasts due to the disruption of lateral line primordium migration. In situ hybridization showed the involvement of attenuated FGF signaling and the chemokine receptor 4b (cxcr4b) and chemokine receptor 7b (cxcr7b) in posterior lateral line primordium of ildr1b-morphant zebrafish. We concluded that Ildr1b is crucial for the development of the inner ear and the lateral line system. This study provides the first evidence for the mechanism of Ildr1b on hearing in vivo and sheds light on the pathology of DFNB42.

ß-Arrestin1 regulates the morphology and dynamics of microglia in zebrafish in vivo.

Microglia are the primary immune cells in the central nervous system. Microglia typically exist in a 'resting' state in the healthy brain, with ramified processes dynamically exploring the surrounding microenvironment. They become 'activated' under pathological conditions with marked changes in morphology. However, the regulation of their morphology dynamics remains poorly understood. Here, using in vivo time-lapse imaging and three-dimensional morphology analysis of microglia in intact zebrafish larvae, we found that ß-arrestin1, a multifunctional protein involved in various signal transductions, cell-autonomously regulated the microglial morphology. Knockdown of ß-arrestin1 increased the volume size and process number of microglia but reduced the deformation speed in the resting state. Meanwhile, ß-arrestin1 down-regulation led to a high frequency of phagocytic behaviour of microglia. These defects were partially rescued by over-expressing human ß-arrestin1 in microglia. Our study indicated that microglial dynamics in the resting state can be regulated cell-autonomously by ß-arrestin1 signalling.

Integrative analysis of circadian transcriptome and metabolic network reveals the role of de novo purine synthesis in circadian control of cell cycle.

Metabolism is the major output of the circadian clock in many organisms. We developed a computational method to integrate both circadian gene expression and metabolic network. Applying this method to zebrafish circadian transcriptome, we have identified large clusters of metabolic genes containing mostly genes in purine and pyrimidine metabolism in the metabolic network showing similar circadian phases. Our metabolomics analysis found that the level of inosine 5'-monophosphate (IMP), an intermediate metabolite in de novo purine synthesis, showed significant circadian oscillation in larval zebrafish. We focused on IMP dehydrogenase (impdh), a rate-limiting enzyme in de novo purine synthesis, with three circadian oscillating gene homologs: impdh1a, impdh1b and impdh2. Functional analysis revealed that impdh2 contributes to the daily rhythm of S phase in the cell cycle while impdh1a contributes to ocular development and pigment synthesis. The three zebrafish homologs of impdh are likely regulated by different circadian transcription factors. We propose that the circadian regulation of de novo purine synthesis that supplies crucial building blocks for DNA replication is an important mechanism conferring circadian rhythmicity on the cell cycle. Our method is widely applicable to study the impact of circadian transcriptome on metabolism in complex organisms.

[Establishment of an anesthesia model induced by etomidate in larval zebrafish].

Despite the wide application of general anesthetic drugs in clinic, it is still unclear how these drugs induce the state of general anesthesia. Larval zebrafish has emerged as an ideal model for dissecting the mechanism of neural systems due to the conserved and simple brain structure. In the present study, we established an anesthesia model from behavioral to electrophysiological levels using larval zebrafish for the first time. Bath application of etomidate, as a kind of intravenous anesthetic drugs, suppressed the spontaneous locomotion of zebrafish in a concentration-dependent manner. Consistently, in vivo fictive motor patterns of spinal motoneurons recorded extracellularly were significantly inhibited as well. Furthermore, using in vivo extracellular recording and whole-cell recording, we found that etomidate application suppressed local field potentials (LFP) of the brain and blocked visually evoked responses of optic tectal neurons. The study indicates that larval zebrafish can serve as an ideal vertebrate animal model for studying neural mechanisms underlying general anesthesia.

[Progress in the study of the blood-brain barrier].

Blood-brain barrier (BBB) precisely controls the material exchange between the blood and brain tissue, and plays a critical role in the maintenance of brain microenvironment homeostasis. Brain microvascular endothelial cells connect tightly with each other and intertwine with surrounding pericytes and astrocytes to form the BBB. These cells regulate the development and function of the BBB through expressing tight and adherens junction proteins, transporters, and relevant signal molecules. Neurons and microglia can also regulate the function of BBB in physiological and pathological conditions. Recent studies indicate that the occurrence and progress of various neurological diseases are accompanied with structural and functional impairment of the BBB. Therefore, elucidation of the mechanisms underlying BBB development and function will further benefit our understanding of neurovascular interaction and provide an important theoretical basis for the treatment of neurological diseases. In this review, we briefly summarize the progress of BBB research.

Haemodynamics-driven developmental pruning of brain vasculature in zebrafish.

The brain blood vasculature consists of a highly ramified vessel network that is tailored to meet its physiological functions. How the brain vasculature is formed has long been fascinating biologists. Here we report that the developing vasculature in the zebrafish midbrain undergoes not only angiogenesis but also extensive vessel pruning, which is driven by changes in blood flow. This pruning process shapes the initial exuberant interconnected meshwork into a simplified architecture. Using in vivo long-term serial confocal imaging of the same zebrafish larvae during 1.5-7.5 d post-fertilization, we found that the early formed midbrain vasculature consisted of many vessel loops and higher order segments. Vessel pruning occurred preferentially at loop-forming segments via a process mainly involving lateral migration of endothelial cells (ECs) from pruned to unpruned segments rather than EC apoptosis, leading to gradual reduction in the vasculature complexity with development. Compared to unpruned ones, pruned segments exhibited a low and variable blood flow, which further decreased irreversibly prior to the onset of pruning. Local blockade of blood flow with micro-bead obstruction led to vessel pruning, whereas increasing blood flow by noradrenergic elevation of heartbeat impeded the pruning process. Furthermore, the occurrence of vessel pruning could be largely predicted by haemodynamics-based numerical simulation of vasculature refinement. Thus, changes of blood flow drive vessel pruning via lateral migration of ECs, leading to the simplification of the vasculature and possibly efficient routing of blood flow in the developing brain.

X-ray Radiation-Controlled NO-Release for On-Demand Depth-Independent Hypoxic Radiosensitization.

Multifunctional stimuli-responsive nanotheranostic systems are highly desirable for realizing simultaneous biomedical imaging and on-demand therapy with minimized adverse effects. Herein, we present the construction of an intelligent X-ray-controlled NO-releasing upconversion nanotheranostic system (termed as PEG-USMSs-SNO) by engineering UCNPs with S-nitrosothiol (R-SNO)-grafted mesoporous silica. The PEG-USMSs-SNO is designed to respond sensitively to X-ray radiation for breaking down the S-N bond of SNO to release NO, which leads to X-ray dose-controlled NO release for on-demand hypoxic radiosensitization besides upconversion luminescent imaging through UCNPs in vitro and in vivo. Thanks to the high live-body permeability of X-ray, our developed PEG-USMSs-SNO may provide a new technique for achieving depth-independent controlled NO release and positioned radiotherapy enhancement against deep-seated solid tumors.

Ultrasensitive nanosensors based on upconversion nanoparticles for selective hypoxia imaging in vivo upon near-infrared excitation.

Hypoxia is a distinct feature of malignant solid tumors, which is a possible causative factor for the serious resistance to chemo- and radiotherapy or the development of invasion and metastasis. The exploration of nanosensors with the capabilities like the accurate diagnosis of hypoxic level will be helpful to estimate the malignant degree of tumor and subsequently implement more effective personalized treatment. Here, we report the design and synthesis of nanosensors that can selectively and reversibly detect the level of hypoxia both in vitro and in vivo. The designed nanosensor is composed of two main moieties: oxygen indicator [Ru(dpp)3](2+)Cl2 for detection of hypoxia and upconversion nanoparticles for offering the excitation light of [Ru(dpp)3](2+)Cl2 by upconversion process under 980 nm exposure. The results show that the nanosensors can reversibly become quenched or luminescent under hyperoxic or hypoxic conditions, respectively. Compared with free [Ru(dpp)3](2+)Cl2, the designed nanosensors exhibit enhanced sensitivity for the detection of oxygen in hypoxic regions. More attractively, the nanosensors can image hypoxic regions with high penetration depth because the absorption and emission wavelength are within the NIR and far-red region, respectively. Most importantly, nanosensors display a high selectivity for detection of relevant oxygen changes in cells and zebrafish.

Analysis of a gene regulatory cascade mediating circadian rhythm in zebrafish.

In the study of circadian rhythms, it has been a puzzle how a limited number of circadian clock genes can control diverse aspects of physiology. Here we investigate circadian gene expression genome-wide using larval zebrafish as a model system. We made use of a spatial gene expression atlas to investigate the expression of circadian genes in various tissues and cell types. Comparison of genome-wide circadian gene expression data between zebrafish and mouse revealed a nearly anti-phase relationship and allowed us to detect novel evolutionarily conserved circadian genes in vertebrates. We identified three groups of zebrafish genes with distinct responses to light entrainment: fast light-induced genes, slow light-induced genes, and dark-induced genes. Our computational analysis of the circadian gene regulatory network revealed several transcription factors (TFs) involved in diverse aspects of circadian physiology through transcriptional cascade. Of these, microphthalmia-associated transcription factor a (mitfa), a dark-induced TF, mediates a circadian rhythm of melanin synthesis, which may be involved in zebrafish's adaptation to daily light cycling. Our study describes a systematic method to discover previously unidentified TFs involved in circadian physiology in complex organisms.

Real-time in vivo quantitative monitoring of drug release by dual-mode magnetic resonance and upconverted luminescence imaging.

Insufficient or excess drug doses, due to unknown actual drug concentrations at the focus, are one of the main causes of chemotherapy failure for cancers. In this regard, the real-time monitoring of the release of anticancer drugs from nanoparticle drug delivery systems is of crucial importance, but it remains a critical and unsolved challenge. Herein, we report the proposal and development of a novel concept of real-time monitoring of NIR-triggered drug release in vitro and in vivo by using simultaneous upconverted luminescence (UCL) and magnetic resonance (MR) imaging. Such a monitoring strategy features the high sensitivity of UCL and the high-resolution, noninvasiveness, and tissue-depth-independence of MR imaging. The dual-mode real-time and quantitative monitoring of drug release can be applied to determine online the drug concentrations in vivo in the tissue regions of interest and, therefore, to avoid insufficient or excess drug dosings.

Improvement in Tol2 transposon for efficient large-cargo capacity transgene applications in cultured cells and zebrafish ( Danio rerio).

Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes. However, their activity markedly diminishes with payloads exceeding 11 kb. Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs, improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics, metabolic engineering, and transgenic animal production. In this study, we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer ( QBI SP163, ST) and enhanced the nuclear targeting ability using the nuclear localization protein H2B (SHT). The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures (H1299), comparable to the well-established super PiggyBac system. Furthermore, mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads (8 kb, 14 kb, and 24 kb) into zebrafish ( Danio rerio). This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.

Brain topology improved spiking neural network for efficient reinforcement learning of continuous control.

The brain topology highly reflects the complex cognitive functions of the biological brain after million-years of evolution. Learning from these biological topologies is a smarter and easier way to achieve brain-like intelligence with features of efficiency, robustness, and flexibility. Here we proposed a brain topology-improved spiking neural network (BT-SNN) for efficient reinforcement learning. First, hundreds of biological topologies are generated and selected as subsets of the Allen mouse brain topology with the help of the Tanimoto hierarchical clustering algorithm, which has been widely used in analyzing key features of the brain connectome. Second, a few biological constraints are used to filter out three key topology candidates, including but not limited to the proportion of node functions (e.g., sensation, memory, and motor types) and network sparsity. Third, the network topology is integrated with the hybrid numerical solver-improved leaky-integrated and fire neurons. Fourth, the algorithm is then tuned with an evolutionary algorithm named adaptive random search instead of backpropagation to guide synaptic modifications without affecting raw key features of the topology. Fifth, under the test of four animal-survival-like RL tasks (i.e., dynamic controlling in Mujoco), the BT-SNN can achieve higher scores than not only counterpart SNN using random topology but also some classical ANNs (i.e., long-short-term memory and multi-layer perception). This result indicates that the research effort of incorporating biological topology and evolutionary learning rules has much in store for the future.