Photocatalytic manipulation of Ca2+ signaling for regulating cellular and animal behaviors via MOF-enabled H2O2 generation.

The in situ generation of H2O2 in cells in response to external stimulation has exceptional advantages in modulating intracellular Ca2+ dynamics, including high controllability and biological safety, but has been rarely explored. Here, we develop photocatalyst-based metal-organic frameworks (DCSA-MOFs) to modulate Ca2+ responses in cells, multicellular spheroids, and organs. By virtue of the efficient photocatalytic oxygen reduction to H2O2 without sacrificial agents, photoexcited DCSA-MOFs can rapidly trigger Ca2+ outflow from the endoplasmic reticulum with single-cell precision in a repeatable and controllable manner, enabling the propagation of intercellular Ca2+ waves (ICW) over long distances in two-dimensional and three-dimensional cell cultures. After photoexcitation, ICWs induced by DCSA-MOFs can activate neural activities in the optical tectum of tadpoles and thighs of spinal frogs, eliciting the corresponding motor behaviors. Our study offers a versatile optical nongenetic modulation technique that enables remote, repeatable, and controlled manipulation of cellular and animal behaviors.

ß-Catenin and SOX2 Interaction Regulate Visual Experience-Dependent Cell Homeostasis in the Developing Xenopus Thalamus.

In the vertebrate brain, sensory experience plays a crucial role in shaping thalamocortical connections for visual processing. However, it is still not clear how visual experience influences tissue homeostasis and neurogenesis in the developing thalamus. Here, we reported that the majority of SOX2-positive cells in the thalamus are differentiated neurons that receive visual inputs as early as stage 47 Xenopus. Visual deprivation (VD) for 2 days shifts the neurogenic balance toward proliferation at the expense of differentiation, which is accompanied by a reduction in nuclear-accumulated ß-catenin in SOX2-positive neurons. The knockdown of ß-catenin decreases the expression of SOX2 and increases the number of progenitor cells. Coimmunoprecipitation studies reveal the evolutionary conservation of strong interactions between ß-catenin and SOX2. These findings indicate that ß-catenin interacts with SOX2 to maintain homeostatic neurogenesis during thalamus development.

Epigenetic regulation of GABAergic differentiation in the developing brain.

In the vertebrate brain, GABAergic cell development and neurotransmission are important for the establishment of neural circuits. Various intrinsic and extrinsic factors have been identified to affect GABAergic neurogenesis. However, little is known about the epigenetic control of GABAergic differentiation in the developing brain. Here, we report that the number of GABAergic neurons dynamically changes during the early tectal development in the Xenopus brain. The percentage of GABAergic neurons is relatively unchanged during the early stages from stage 40 to 46 but significantly decreased from stage 46 to 48 tadpoles. Interestingly, the histone acetylation of H3K9 is developmentally decreased from stage 42 to 48 (about 3.5 days). Chronic application of valproate acid (VPA), a broad-spectrum histone deacetylase (HDAC) inhibitor, at stage 46 for 48 h increases the acetylation of H3K9 and the number of GABAergic cells in the optic tectum. VPA treatment also reduces apoptotic cells. Electrophysiological recordings show that a VPA induces an increase in the frequency of mIPSCs and no changes in the amplitude. Behavioral studies reveal that VPA decreases swimming activity and visually guided avoidance behavior. These findings extend our understanding of histone modification in the GABAergic differentiation and neurotransmission during early brain development.

Cell landscape of larval and adult Xenopus laevis at single-cell resolution.

The rapid development of high-throughput single-cell RNA sequencing technology offers a good opportunity to dissect cell heterogeneity of animals. A large number of organism-wide single-cell atlases have been constructed for vertebrates such as Homo sapiens, Macaca fascicularis, Mus musculus and Danio rerio. However, an intermediate taxon that links mammals to vertebrates of more ancient origin is still lacking. Here, we construct the first Xenopus cell landscape to date, including larval and adult organs. Common cell lineage-specific transcription factors have been identified in vertebrates, including fish, amphibians and mammals. The comparison of larval and adult erythrocytes identifies stage-specific hemoglobin subtypes, as well as a common type of cluster containing both larval and adult hemoglobin, mainly at NF59. In addition, cell lineages originating from all three layers exhibits both antigen processing and presentation during metamorphosis, indicating a common regulatory mechanism during metamorphosis. Overall, our study provides a large-scale resource for research on Xenopus metamorphosis and adult organs.

Development of advanced oil/water separation technologies to enhance the effectiveness of mechanical oil recovery operations at sea: Potential and challenges.

Mechanical oil recovery (i.e., booming and skimming) is the most common tool for oil spill response. The recovered fluid generated from skimming processes may contain a considerable proportion of water (10 % ~ 70 %). As a result of regulatory prohibition on the discharge of contaminated waters at sea, vessels and/or storage barges must make frequent trips to shore for oil-water waste disposal. This practice can be time- consuming thus reduces the overall efficiency and capacity of oil recovery. One potential solution is on-site oil-water separation and disposal of water fraction at sea. However, currently available decanting processes may have limited oil/water separation capabilities, especially in the presence of oil-water emulsion, which is inevitable in mechanical oil recovery. The decanted water may not meet the discharge standards and cause severe ecotoxicological impacts. This paper therefore comprehensively reviews the principles and progress in oil/water separation, demulsification, and on-site treatment technologies, investigates their applicability on decanting at sea, and discusses the ecotoxicity of decanted water in the marine environment. The outputs provide the fundamental and practical knowledge on decanting and help enhance response effectiveness and consequently reducing the environmental impacts of oil spills.

Evidence That ITPR2-Mediated Intracellular Calcium Release in Oligodendrocytes Regulates the Development of Carbonic Anhydrase II + Type I/II Oligodendrocytes and the Sizes of Myelin Fibers.

Myelination of neuronal axons in the central nervous system (CNS) by oligodendrocytes (OLs) enables rapid saltatory conductance and axonal integrity, which are crucial for normal brain functioning. Previous studies suggested that different subtypes of oligodendrocytes in the CNS form different types of myelin determined by the diameter of axons in the unit. However, the molecular mechanisms underlying the developmental association of different types of oligodendrocytes with different fiber sizes remain elusive. In the present study, we present the evidence that the intracellular Ca2+ release channel associated receptor (Itpr2) contributes to this developmental process. During early development, Itpr2 is selectively up-regulated in oligodendrocytes coinciding with the initiation of myelination. Functional analyses in both conventional and conditional Itpr2 mutant mice revealed that Itpr2 deficiency causes a developmental delay of OL differentiation, resulting in an increased percentage of CAII+ type I/II OLs which prefer to myelinate small-diameter axons in the CNS. The increased percentage of small caliber myelinated axons leads to an abnormal compound action potentials (CAP) in the optic nerves. Together, these findings revealed a previously unrecognized role for Itpr2-mediated calcium signaling in regulating the development of different types of oligodendrocytes.

Electrophysiological Recording for Study of Xenopus Retinotectal Circuitry.

The innervation of the optic tectum of Xenopus by retinal ganglion cells controls visual information processing and behavioral output. Several indicators can be used to evaluate the functional inputs/outputs of tectal neurons, such as spontaneous activity, visually evoked currents, temporal receptive fields, and spatial receptive fields. Analysis of multiple functional properties in the same neurons allows increased understanding of mechanisms underlying visual system function and plasticity. Patch-clamp recordings combined with gene expression or morpholino-mediated knockdown techniques have been especially powerful in the study of specific genes during development and circuit function. The protocol described here provides instructions for performing in vivo electrophysiological recordings from individual tectal neurons to study retinotectal circuitry in the developing Xenopus tectum.

Wnt/ß-Catenin Signaling in Neural Stem Cell Homeostasis and Neurological Diseases.

Neural stem/progenitor cells (NSCs) maintain the ability of self-renewal and differentiation and compose the complex nervous system. Wnt signaling is thought to control the balance of NSC proliferation and differentiation via the transcriptional coactivator ß-catenin during brain development and adult tissue homeostasis. Disruption of Wnt signaling may result in developmental defects and neurological diseases. Here, we summarize recent findings of the roles of Wnt/ß-catenin signaling components in NSC homeostasis for the regulation of functional brain circuits. We also suggest that the potential role of Wnt/ß-catenin signaling might lead to new therapeutic strategies for neurological diseases, including, but not limited to, spinal cord injury, Alzheimer's disease, Parkinson's disease, and depression.

Xenopus in revealing developmental toxicity and modeling human diseases.

The Xenopus model offers many advantages for investigation of the molecular, cellular, and behavioral mechanisms underlying embryo development. Moreover, Xenopus oocytes and embryos have been extensively used to study developmental toxicity and human diseases in response to various environmental chemicals. This review first summarizes recent advances in using Xenopus as a vertebrate model to study distinct types of tissue/organ development following exposure to environmental toxicants, chemical reagents, and pharmaceutical drugs. Then, the successful use of Xenopus as a model for diseases, including fetal alcohol spectrum disorders, autism, epilepsy, and cardiovascular disease, is reviewed. The potential application of Xenopus in genetic and chemical screening to protect against embryo deficits induced by chemical toxicants and related diseases is also discussed.

d-Glucuronolactone attenuates para-xylene-induced defects in neuronal development and plasticity in Xenopus tectum in vivo.

Xylene and its derivatives are known to be neurotoxic to the central nervous system of animals. Our previous work has shown that para-xylene (PX) can cause an increase in apoptotic cells and abnormal avoidance behavior in Xenopus laevis. However, the mechanism underlying the impact of PX on neuronal structural and functional plasticity is less clear. Here, we examined the effects of PX on neuronal development and plasticity in the developing optic tectum. We found that HuC/D-positive neurons were more vulnerable than SOX2-positive progenitor cells or BLBP-positive radial glial cells after exposure to PX at 1 mM for 48 h. The further measurement of postsynaptic receptors and synaptic vesicle proteins showed that the expression levels of GluA1 and GluA2, but not Rab3a and SNAP25, were significantly decreased in the tectal brain. In vivo time-lapse images and electrophysiological recordings showed that PX exposure resulted in significant deficits in neuronal structure, particularly in the total dendritic branch length (TDBL), and visual stimulation-induced excitatory compound synaptic currents (eCSCs) without altering neurotransmitter release probability. Strikingly, coexposure to d-glucuronolactone (GA) and PX rescued the structural and functional deficits caused by PX exposure alone. Furthermore, we found that visual experience-induced structural, functional and behavioral plasticity was blocked by PX exposure, which was also rescued by the simultaneous administration of GA and PX . Thus, our findings indicate that PX is neurotoxic to brain development and plasticity and that GA may be considered a promising candidate to treat PX-induced defects in neural circuits.

Excitatory synaptic dysfunction cell-autonomously decreases inhibitory inputs and disrupts structural and functional plasticity.

Functional circuit assembly is thought to require coordinated development of excitation and inhibition, but whether they are co-regulated cell-autonomously remains unclear. We investigate effects of decreased glutamatergic synaptic input on inhibitory synapses by expressing AMPAR subunit, GluA1 and GluA2, C-terminal peptides (GluA1CTP and GluA2CTP) in developing Xenopus tectal neurons. GluACTPs decrease excitatory synaptic inputs and cell-autonomously decreases inhibitory synaptic inputs in excitatory and inhibitory neurons. Visually evoked excitatory and inhibitory currents decrease proportionately, maintaining excitation/inhibition. GluACTPs affect dendrite structure and visual experience-dependent structural plasticity differently in excitatory and inhibitory neurons. Deficits in excitatory and inhibitory synaptic transmission and experience-dependent plasticity manifest in altered visual receptive field properties. Both visual avoidance behavior and learning-induced behavioral plasticity are impaired, suggesting that maintaining excitation/inhibition alone is insufficient to preserve circuit function. We demonstrate that excitatory synaptic dysfunction in individual neurons cell-autonomously decreases inhibitory inputs and disrupts neuronal and circuit plasticity, information processing and learning.

Role of the visual experience-dependent nascent proteome in neuronal plasticity.

Experience-dependent synaptic plasticity refines brain circuits during development. To identify novel protein synthesis-dependent mechanisms contributing to experience-dependent plasticity, we conducted a quantitative proteomic screen of the nascent proteome in response to visual experience in Xenopus optic tectum using bio-orthogonal metabolic labeling (BONCAT). We identified 83 differentially synthesized candidate plasticity proteins (CPPs). The CPPs form strongly interconnected networks and are annotated to a variety of biological functions, including RNA splicing, protein translation, and chromatin remodeling. Functional analysis of select CPPs revealed the requirement for eukaryotic initiation factor three subunit A (eIF3A), fused in sarcoma (FUS), and ribosomal protein s17 (RPS17) in experience-dependent structural plasticity in tectal neurons and behavioral plasticity in tadpoles. These results demonstrate that the nascent proteome is dynamic in response to visual experience and that de novo synthesis of machinery that regulates RNA splicing and protein translation is required for experience-dependent plasticity.

Visual experience dependent regulation of neuronal structure and function by histone deacetylase 1 in developing Xenopus tectum in vivo.

Histone deacetylase 1 (HDAC1) is thought to play pivotal roles in neurogenesis and neurodegeneration. However, the role of HDAC1 in neuronal growth and structural plasticity in the developing brain in vivo remains unclear. Here, we show that in the optic tectum of Xenopus laevis, HDAC1 knockdown dramatically decreased the frequency of AMPAR-mediated synaptic currents and increased the frequency of GABAAR-mediated currents, whereas HDAC1 overexpression significantly decreased the frequency of GABAAR-mediated synaptic currents. Both HDAC1 knockdown and overexpression adversely affected dendritic arbor growth and visual experience-dependent structural plasticity. Furthermore, HDAC1 knockdown decreased BDNF expression via a mechanism that involves acetylation of specific histone H4 residues at lysine K5. In particular, the deficits in dendritic growth and visually guided avoidance behavior in HDAC1-knockdown tadpoles could be rescued by acute tectal infusion of BDNF. These results establish a relationship between HDAC1 expression, histone H4 modification and BDNF signaling in the visual-experience dependent regulation of dendritic growth, structural plasticity and function in intact animals in vivo. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 947-962, 2017.

HDAC3 But not HDAC2 Mediates Visual Experience-Dependent Radial Glia Proliferation in the Developing Xenopus Tectum.

Radial glial cells (RGs) are one of the important progenitor cells that can differentiate into neurons or glia to form functional neural circuits in the developing central nervous system (CNS). Histone deacetylases (HDACs) has been associated with visual activity dependent changes in BrdU-positive progenitor cells in the developing brain. We previously have shown that HDAC1 is involved in the experience-dependent proliferation of RGs. However, it is less clear whether two other members of class I HDACs, HDAC2 and HDAC3, are involved in the regulation of radial glia proliferation. Here, we reported that HDAC2 and HDAC3 expression were developmentally regulated in tectal cells, especially in the ventricular layer of the BLBP-positive RGs. Pharmacological blockade using an inhibitor of class I HDACs, MS-275, decreased the number of BrdU-positive dividing progenitor cells. Specific knockdown of HDAC3 but not HDAC2 decreased the number of BrdU- and BLBP-labeled cells, suggesting that the proliferation of radial glia was selectively mediated by HDAC3. Visual deprivation induced selective augmentation of histone H4 acetylation at lysine 16 in BLBP-positive cells. Furthermore, the visual deprivation-induced increase in BrdU-positive cells was partially blocked by HDAC3 downregulation but not by HDAC2 knockdown at stage 49 tadpoles. These data revealed a specific role of HDAC3 in experience-dependent radial glia proliferation during the development of Xenopus tectum.

Increased apoptosis and abnormal visual behavior by histone modifications with exposure to para-xylene in developing Xenopus.

Xylene and its derivatives are raw materials widely used in industry and known to be toxic to animals. However, the mechanism underlying the neurotoxicity of para-xylene (PX) to the central nervous system (CNS) in vivo is less clear. Here, we exposed Xenopus laevis tadpoles to sub-lethal concentrations of PX during the critical period of brain development to determine the effects of PX on Xenopus development and visual behavior. We found that the abnormality rate was significantly increased with exposure to increasing concentrations of PX. In particular, the number of apoptotic cells in the optic tectum was dramatically increased with exposure to PX at 2mM. Long-term PX exposure also resulted in significant deficits in visually guided avoidance behavior. Strikingly, co-incubation with PX and d-glucuronolactone (GA) decreased the number of apoptotic cells and rescued the avoidance behavior. Furthermore, we found that the acetylation of H4K12 (H4K12ac) and the dimethylation of H3K9 (H3K9me2) in the optic tectum were significantly increased in PX-treated animals, and these effects were suppressed by GA treatment. In particular, the increase in apoptotic cells in PX-treated brains was also inhibited by GA treatment. These effects indicate that epigenetic regulation plays a key role in PX-induced apoptosis and animal behavior. In an effort to characterize the neurotoxic effects of PX on brain development and behavior, these results suggest that the neurotoxicity of PX requires further evaluation regarding the safety of commercial and industrial uses.

Experience-Dependent Bimodal Plasticity of Inhibitory Neurons in Early Development.

Inhibitory neurons are heterogeneous in the mature brain. It is unclear when and how inhibitory neurons express distinct structural and functional profiles. Using in vivo time-lapse imaging of tectal neuron structure and visually evoked Ca(2+) responses in tadpoles, we found that inhibitory neurons cluster into two groups with opposite valence of plasticity after 4 hr of dark and visual stimulation. Half decreased dendritic arbor size and Ca(2+) responses after dark and increased them after visual stimulation, matching plasticity in excitatory neurons. Half increased dendrite arbor size and Ca(2+) responses following dark and decreased them after stimulation. At the circuit level, visually evoked excitatory and inhibitory synaptic inputs were potentiated by visual experience and E/I remained constant. Our results indicate that developing inhibitory neurons fall into distinct functional groups with opposite experience-dependent plasticity and as such, are well positioned to foster experience-dependent synaptic plasticity and maintain circuit stability during labile periods of circuit development.

HDAC1 regulates the proliferation of radial glial cells in the developing Xenopus tectum.

In the developing central nervous system (CNS), progenitor cells differentiate into progeny to form functional neural circuits. Radial glial cells (RGs) are a transient progenitor cell type that is present during neurogenesis. It is thought that a combination of neural trophic factors, neurotransmitters and electrical activity regulates the proliferation and differentiation of RGs. However, it is less clear how epigenetic modulation changes RG proliferation. We sought to explore the effect of histone deacetylase (HDAC) activity on the proliferation of RGs in the visual optic tectum of Xenopus laevis. We found that the number of BrdU-labeled precursor cells along the ventricular layer of the tectum decrease developmentally from stage 46 to stage 49. The co-labeling of BrdU-positive cells with brain lipid-binding protein (BLBP), a radial glia marker, showed that the majority of BrdU-labeled cells along the tectal midline are RGs. BLBP-positive cells are also developmentally decreased with the maturation of the brain. Furthermore, HDAC1 expression is developmentally down-regulated in tectal cells, especially in the ventricular layer of the tectum. Pharmacological blockade of HDACs using Trichostatin A (TSA) or Valproic acid (VPA) decreased the number of BrdU-positive, BLBP-positive and co-labeling cells. Specific knockdown of HDAC1 by a morpholino (HDAC1-MO) decreased the number of BrdU- and BLBP-labeled cells and increased the acetylation level of histone H4 at lysine 12 (H4K12). The visual deprivation-induced increase in BrdU- and BLBP-positive cells was blocked by HDAC1 knockdown at stage 49 tadpoles. These data demonstrate that HDAC1 regulates radial glia cell proliferation in the developing optical tectum of Xenopus laevis.

Subcellular Localization of Class I Histone Deacetylases in the Developing Xenopus tectum.

Histone deacetylases (HDACs) are thought to localize in the nucleus to regulate gene transcription and play pivotal roles in neurogenesis, apoptosis, and plasticity. However, the subcellular distribution of class I HDACs in the developing brain remains unclear. Here, we show that HDAC1 and HDAC2 are located in both the mitochondria and the nucleus in the Xenopus laevis stage 34 tectum and are mainly restricted to the nucleus following further brain development. HDAC3 is widely present in the mitochondria, nucleus, and cytoplasm during early tectal development and is mainly distributed in the nucleus in stage 45 tectum. In contrast, HDAC8 is broadly located in the mitochondria, nucleus, and cytoplasm during tectal development. These data demonstrate that HDAC1, HDAC2, and HDAC3 are transiently localized in the mitochondria and that the subcellular distribution of class I HDACs in the Xenopus tectum is heterogeneous. Furthermore, we observed that spherical mitochondria accumulate in the cytoplasm at earlier stages, whereas elongated mitochondria are evenly distributed in the tectum at later stages. The activity of histone acetylation (H4K12) remains low in mitochondria during tectal development. Pharmacological blockades of HDACs using a broad spectrum HDAC inhibitor of Trichostatin A (TSA) or specific class I HDAC inhibitors of MS-275 and MGCD0103 decrease the number of mitochondria in the tectum at stage 34. These findings highlight a link between the subcellular distribution of class I HDACs and mitochondrial dynamics in the developing optic tectum of Xenopus laevis.

Acute synthesis of CPEB is required for plasticity of visual avoidance behavior in Xenopus.

Neural plasticity requires protein synthesis, but the identity of newly synthesized proteins generated in response to plasticity-inducing stimuli remains unclear. We used in vivo bio-orthogonal noncanonical amino acid tagging (BONCAT) with the methionine analog azidohomoalanine (AHA) combined with the multidimensional protein identification technique (MudPIT) to identify proteins that are synthesized in the tadpole brain over 24 hr. We induced conditioning-dependent plasticity of visual avoidance behavior, which required N-methyl-D-aspartate (NMDA) and Ca(2+)-permeable α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, αCaMKII, and rapid protein synthesis. Combining BONCAT with western blots revealed that proteins including αCaMKII, MEK1, CPEB, and GAD65 are synthesized during conditioning. Acute synthesis of CPEB during conditioning is required for behavioral plasticity as well as conditioning-induced synaptic and structural plasticity in the tectal circuit. We outline a signaling pathway that regulates protein-synthesis-dependent behavioral plasticity in intact animals, identify newly synthesized proteins induced by visual experience, and demonstrate a requirement for acute synthesis of CPEB in plasticity.