Genetic and Neurological Deficiencies in the Visual System of mct8 Mutant Zebrafish.

Thyroid hormones (THs; T3 and T4) enter cells using specific transporters and regulate development and metabolism. Mutation in the TH transporter monocarboxylate transporter 8 (MCT8, SLC16A2) is associated with brain hypothyroidism and neurological impairment. We established mct8 mutant (mct8-/-) zebrafish as a model for MCT8 deficiency, which causes endocrinological, neurological, and behavioral alterations. Here, we profiled the transcriptome of mct8-/- larvae. Among hundreds of differentially expressed genes, the expression of a cluster of vision-related genes was distinct. Specifically, the expression of the opsin 1 medium wave sensitive 2 (opn1mw2) decreased in two mct8 mutants: mct8-/- and mct8-25bp-/- larvae, and under pharmacological inhibition of TH production. Optokinetic reflex (OKR) assays showed a reduction in the number of conjugated eye movements, and live imaging of genetically encoded Ca2+ indicator revealed altered neuronal activity in the pretectum area of mct8-25bp-/- larvae. These results imply that MCT8 and THs regulate the development of the visual system and suggest a mechanism to the deficiencies observed in the visual system of MCT8-deficiency patients.

A Zebrafish Model for a Rare Genetic Disease Reveals a Conserved Role for FBXL3 in the Circadian Clock System.

The circadian clock, which drives a wide range of bodily rhythms in synchrony with the day-night cycle, is based on a molecular oscillator that ticks with a period of approximately 24 h. Timed proteasomal degradation of clock components is central to the fine-tuning of the oscillator's period. FBXL3 is a protein that functions as a substrate-recognition factor in the E3 ubiquitin ligase complex, and was originally shown in mice to mediate degradation of CRY proteins and thus contribute to the mammalian circadian clock mechanism. By exome sequencing, we have identified a FBXL3 mutation in patients with syndromic developmental delay accompanied by morphological abnormalities and intellectual disability, albeit with a normal sleep pattern. We have investigated the function of FBXL3 in the zebrafish, an excellent model to study both vertebrate development and circadian clock function and, like humans, a diurnal species. Loss of fbxl3a function in zebrafish led to disruption of circadian rhythms of promoter activity and mRNA expression as well as locomotor activity and sleep-wake cycles. However, unlike humans, no morphological effects were evident. These findings point to an evolutionary conserved role for FBXL3 in the circadian clock system across vertebrates and to the acquisition of developmental roles in humans.

Resource: A multi-species multi-timepoint transcriptome database and webpage for the pineal gland and retina.

The website and database https://snengs.nichd.nih.gov provides RNA sequencing data from multi-species analysis of the pineal glands from zebrafish (Danio rerio), chicken (White Leghorn), rat (Rattus novegicus), mouse (Mus musculus), rhesus macaque (Macaca mulatta), and human (Homo sapiens); in most cases, retinal data are also included along with results of the analysis of a mixture of RNA from tissues. Studies cover day and night conditions; in addition, a time series over multiple hours, a developmental time series and pharmacological experiments on rats are included. The data have been uniformly re-processed using the latest methods and assemblies to allow for comparisons between experiments and to reduce processing differences. The website presents search functionality, graphical representations, Excel tables, and track hubs of all data for detailed visualization in the UCSC Genome Browser. As more data are collected from investigators and improved genomes become available in the future, the website will be updated. This database is in the public domain and elements can be reproduced by citing the URL and this report. This effort makes the results of 21st century transcriptome profiling widely available in a user-friendly format that is expected to broadly influence pineal research.

Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior.

The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior.

Altered behavioral performance and live imaging of circuit-specific neural deficiencies in a zebrafish model for psychomotor retardation.

The mechanisms and treatment of psychomotor retardation, which includes motor and cognitive impairment, are indefinite. The Allan-Herndon-Dudley syndrome (AHDS) is an X-linked psychomotor retardation characterized by delayed development, severe intellectual disability, muscle hypotonia, and spastic paraplegia, in combination with disturbed thyroid hormone (TH) parameters. AHDS has been associated with mutations in the monocarboxylate transporter 8 (mct8/slc16a2) gene, which is a TH transporter. In order to determine the pathophysiological mechanisms of AHDS, MCT8 knockout mice were intensively studied. Although these mice faithfully replicated the abnormal serum TH levels, they failed to exhibit the neurological and behavioral symptoms of AHDS patients. Here, we generated an mct8 mutant (mct8-/-) zebrafish using zinc-finger nuclease (ZFN)-mediated targeted gene editing system. The elimination of MCT8 decreased the expression levels of TH receptors; however, it did not affect the expression of other TH-related genes. Similar to human patients, mct8-/- larvae exhibited neurological and behavioral deficiencies. High-throughput behavioral assays demonstrated that mct8-/- larvae exhibited reduced locomotor activity, altered response to external light and dark transitions and an increase in sleep time. These deficiencies in behavioral performance were associated with altered expression of myelin-related genes and neuron-specific deficiencies in circuit formation. Time-lapse imaging of single-axon arbors and synapses in live mct8-/- larvae revealed a reduction in filopodia dynamics and axon branching in sensory neurons and decreased synaptic density in motor neurons. These phenotypes enable assessment of the therapeutic potential of three TH analogs that can enter the cells in the absence of MCT8. The TH analogs restored the myelin and axon outgrowth deficiencies in mct8-/- larvae. These findings suggest a mechanism by which MCT8 regulates neural circuit assembly, ultimately mediating sensory and motor control of behavioral performance. We also propose that the administration of TH analogs early during embryo development can specifically reduce neurological damage in AHDS patients.

Therapeutic Genome Editing and its Potential Enhancement through CRISPR Guide RNA and Cas9 Modifications.

Genome editing with engineered nucleases is a rapidly growing field thanks to transformative technologies that allow researchers to precisely alter genomes for numerous applications including basic research, biotechnology, and human gene therapy. The genome editing process relies on creating a site-specific DNA double-strand break (DSB) by engineered nucleases and then allowing the cell's repair machinery to repair the break such that precise changes are made to the DNA sequence. The recent development of CRISPR-Cas systems as easily accessible and programmable tools for genome editing accelerates the progress towards using genome editing as a new approach to human therapeutics. Here we review how genome editing using engineered nucleases works and how using different genome editing outcomes can be used as a tool set for treating human diseases. We then review the major challenges of therapeutic genome editing and we discuss how its potential enhancement through CRISPR guide RNA and Cas9 protein modifications could resolve some of these challenges.

The light-induced transcriptome of the zebrafish pineal gland reveals complex regulation of the circadian clockwork by light.

Light constitutes a primary signal whereby endogenous circadian clocks are synchronized ('entrained') with the day/night cycle. The molecular mechanisms underlying this vital process are known to require gene activation, yet are incompletely understood. Here, the light-induced transcriptome in the zebrafish central clock organ, the pineal gland, was characterized by messenger RNA (mRNA) sequencing (mRNA-seq) and microarray analyses, resulting in the identification of multiple light-induced mRNAs. Interestingly, a considerable portion of the molecular clock (14 genes) is light-induced in the pineal gland. Four of these genes, encoding the transcription factors dec1, reverbb1, e4bp4-5 and e4bp4-6, differentially affected clock- and light-regulated promoter activation, suggesting that light-input is conveyed to the core clock machinery via diverse mechanisms. Moreover, we show that dec1, as well as the core clock gene per2, is essential for light-entrainment of rhythmic locomotor activity in zebrafish larvae. Additionally, we used microRNA (miRNA) sequencing (miR-seq) and identified pineal-enhanced and light-induced miRNAs. One such miRNA, miR-183, is shown to downregulate e4bp4-6 mRNA through a 3'UTR target site, and importantly, to regulate the rhythmic mRNA levels of aanat2, the key enzyme in melatonin synthesis. Together, this genome-wide approach and functional characterization of light-induced factors indicate a multi-level regulation of the circadian clockwork by light.

Systematic identification of rhythmic genes reveals camk1gb as a new element in the circadian clockwork.

A wide variety of biochemical, physiological, and molecular processes are known to have daily rhythms driven by an endogenous circadian clock. While extensive research has greatly improved our understanding of the molecular mechanisms that constitute the circadian clock, the links between this clock and dependent processes have remained elusive. To address this gap in our knowledge, we have used RNA sequencing (RNA-seq) and DNA microarrays to systematically identify clock-controlled genes in the zebrafish pineal gland. In addition to a comprehensive view of the expression pattern of known clock components within this master clock tissue, this approach has revealed novel potential elements of the circadian timing system. We have implicated one rhythmically expressed gene, camk1gb, in connecting the clock with downstream physiology of the pineal gland. Remarkably, knockdown of camk1gb disrupts locomotor activity in the whole larva, even though it is predominantly expressed within the pineal gland. Therefore, it appears that camk1gb plays a role in linking the pineal master clock with the periphery.

Zebrafish as a model for monocarboxyl transporter 8-deficiency.

Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor retardation characterized by neurological impairment and abnormal thyroid hormone (TH) levels. Mutations in the TH transporter, monocarboxylate transporter 8 (MCT8), are associated with AHDS. MCT8 knock-out mice exhibit impaired TH levels; however, they lack neurological defects. Here, the zebrafish mct8 gene and promoter were isolated, and mct8 promoter-driven transgenic lines were used to show that, similar to humans, mct8 is primarily expressed in the nervous and vascular systems. Morpholino-based knockdown and rescue experiments revealed that MCT8 is strictly required for neural development in the brain and spinal cord. This study shows that MCT8 is a crucial regulator during embryonic development and establishes the first vertebrate model for MCT8 deficiency that exhibits a neurological phenotype.

Sleep-wake regulation and hypocretin-melatonin interaction in zebrafish.

In mammals, hypocretin/orexin (HCRT) neuropeptides are important sleep-wake regulators and HCRT deficiency causes narcolepsy. In addition to fragmented wakefulness, narcoleptic mammals also display sleep fragmentation, a less understood phenotype recapitulated in the zebrafish HCRT receptor mutant (hcrtr-/-). We therefore used zebrafish to study the potential mediators of HCRT-mediated sleep consolidation. Similar to mammals, zebrafish HCRT neurons express vesicular glutamate transporters indicating conservation of the excitatory phenotype. Visualization of the entire HCRT circuit in zebrafish stably expressing hcrt:EGFP revealed parallels with established mammalian HCRT neuroanatomy, including projections to the pineal gland, where hcrtr mRNA is expressed. As pineal-produced melatonin is a major sleep-inducing hormone in zebrafish, we further studied how the HCRT and melatonin systems interact functionally. mRNA level of arylalkylamine-N-acetyltransferase (AANAT2), a key enzyme of melatonin synthesis, is reduced in hcrtr-/- pineal gland during the night. Moreover, HCRT perfusion of cultured zebrafish pineal glands induces melatonin release. Together these data indicate that HCRT can modulate melatonin production at night. Furthermore, hcrtr-/- fish are hypersensitive to melatonin, but not other hypnotic compounds. Subthreshold doses of melatonin increased the amount of sleep and consolidated sleep in hcrtr-/- fish, but not in the wild-type siblings. These results demonstrate the existence of a functional HCRT neurons-pineal gland circuit able to modulate melatonin production and sleep consolidation.

Chemical Modification of Guide RNAs for Improved CRISPR Activity in CD34+ Human Hematopoietic Stem and Progenitor Cells.

Human CD34+ hematopoietic stem and progenitor cells (HSPCs) have the unique ability to repopulate the entire hematopoietic system and thus are at the center of diverse, therapeutically relevant studies. The recent development of the CRISPR/Cas9 tool made the powerful research technique of genome editing highly accessible. Our previous studies demonstrated that high editing efficiency is reached when the CRISPR/Cas9 is introduced to CD34+ HSPCs as a ribonucleoprotein (RNP) complex with chemically modified guide RNAs (gRNAs). The current protocol details a quick 4-day procedure for ex vivo genome editing in CD34+ HSPCs by RNP complexes that are targeted to a specific locus by either a single gRNA (sgRNA) or a 2-part gRNA. The delivery of RNP complexes is performed by electroporation in the presence of a nonspecific, ssDNA electroporation enhancer, which highly improves editing efficiency under the described conditions. This approach is simple and effective with the potential to accelerate many biotechnological and therapeutic applications of the CRISPR/Cas9 technology.

Neural Alterations and Hyperactivity of the Hypothalamic-Pituitary-Thyroid Axis in Oatp1c1 Deficiency.

Background: The thyroid hormones (THs) triiodothyronine (T3) and thyroxine (T4) are crucial regulators of brain development and function. Cell-specific transporter proteins facilitate TH uptake and efflux across the cell membrane, and insufficient TH transport causes hypothyroidism and mental retardation. Mutations in the TH transporters monocarboxylate transporter 8 (MCT8, SLC16A2) and the organic anion-transporting polypeptide 1C1 (OATP1C1, SLCO1C1) are associated with the psychomotor retardation Allan-Herndon-Dudley syndrome and juvenile neurodegeneration, respectively. Methods: To understand the mechanisms and test potential treatments for the recently discovered OATP1C1 deficiency, we established an oatp1c1 mutant (oatp1c1-/-) zebrafish. Results:oatp1c1 is expressed in endothelial cells, neurons, and astrocytes in zebrafish. The activity of the hypothalamic-pituitary-thyroid axis and behavioral locomotor activity increased in oatp1c1-/- larvae. Neuropathological analysis revealed structural alteration in radial glial cells and shorter neuronal axons in oatp1c1-/- larvae and adults. Notably, oatp1c1-/- and oatp1c1-/-Xmct8-/- adults exhibit an enlarged thyroid gland (goiter). Pharmacological assays showed that TH analogs, but not THs, can reduce the size and improve the color of the thyroid gland in adult mutant zebrafish. Conclusion: These results establish a vertebrate model for OATP1C1 deficiency that demonstrates endocrinological, neurological, and behavioral alterations mimicking findings observed in an OATP1C1-deficient patient. Further, the curative effect of TH analogs in the oatp1c1-/- zebrafish model may provide a lead toward a treatment modality in human patients.

Sleep-Dependent Structural Synaptic Plasticity of Inhibitory Synapses in the Dendrites of Hypocretin/Orexin Neurons.

Sleep is tightly regulated by the circadian clock and homeostatic mechanisms. Although the sleep/wake cycle is known to be associated with structural and physiological synaptic changes that benefit the brain, the function of sleep is still debated. The hypothalamic hypocretin/orexin (Hcrt) neurons regulate various functions including feeding, reward, sleep, and wake. Continuous imaging of single neuronal circuits in live animals is vital to understanding the role of sleep in regulating synaptic dynamics, and the transparency of the zebrafish model enables time-lapse imaging of single synapses during both day and night. Here, we use the gephyrin (Gphnb) protein, a central inhibitory synapse organizer, as a fluorescent post-synaptic marker of inhibitory synapses. Double labeling showed that Gphnb-tagRFP and collybistin-EGFP clusters co-localized in dendritic inhibitory synapses. Using a transgenic hcrt:Gphnb-EGFP zebrafish, we showed that the number of inhibitory synapses in the dendrites of Hcrt neurons was increased during development. To determine the effect of sleep on the inhibitory synapses, we performed two-photon live imaging of Gphnb-EGFP in Hcrt neurons during day and night, under light/dark and constant light and dark conditions, and following sleep deprivation (SD). We found that synapse number increased during the night under light/dark conditions but that these changes were eliminated under constant light or dark conditions. SD reduced synapse number during the night, and the number increased during post-deprivation daytime sleep rebound. These results suggest that rhythmic structural plasticity of inhibitory synapses in Hcrt dendrites is independent of the circadian clock and is modulated by consolidated wake and sleep.

Pharmacological treatment and BBB-targeted genetic therapy for MCT8-dependent hypomyelination in zebrafish.

Hypomyelination is a key symptom of Allan-Herndon-Dudley syndrome (AHDS), a psychomotor retardation associated with mutations in the thyroid-hormone (TH) transporter MCT8 (monocarboxylate transporter 8). AHDS is characterized by severe intellectual deficiency, neuromuscular impairment and brain hypothyroidism. In order to understand the mechanism for TH-dependent hypomyelination, we developed an mct8 mutant (mct8-/-) zebrafish model. The quantification of genetic markers for oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes revealed reduced differentiation of OPCs into oligodendrocytes in mct8-/- larvae and adults. Live imaging of single glial cells showed that the number of oligodendrocytes and the length of their extensions are reduced, and the number of peripheral Schwann cells is increased, in mct8-/- larvae compared with wild type. Pharmacological analysis showed that TH analogs and clemastine partially rescued the hypomyelination in the CNS of mct8-/- larvae. Intriguingly, triiodothyronine (T3) treatment rescued hypomyelination in mct8-/- embryos before the maturation of the blood-brain barrier (BBB), but did not affect hypomyelination in older larvae. Thus, we expressed Mct8-tagRFP in the endothelial cells of the vascular system and showed that even relatively weak mosaic expression completely rescued hypomyelination in mct8-/- larvae. These results suggest potential pharmacological treatments and BBB-targeted gene therapy that can enhance myelination in AHDS and possibly in other TH-dependent brain disorders.

Casein kinase 1δ activity: a key element in the zebrafish circadian timing system.

Zebrafish have become a popular model for studies of the circadian timing mechanism. Taking advantage of its rapid development of a functional circadian clock and the availability of light-entrainable clock-containing cell lines, much knowledge has been gained about the circadian clock system in this species. However, the post-translational modifications of clock proteins, and in particular the phosphorylation of PER proteins by Casein kinase I delta and epsilon (CK1δ and CK1ε), have so far not been examined in the zebrafish. Using pharmacological inhibitors for CK1δ and CK1ε, a pan-CK1δ/ε inhibitor PF-670462, and a CK1ε -selective inhibitor PF-4800567, we show that CK1δ activity is crucial for the functioning of the circadian timing mechanism of zebrafish, while CK1ε plays a minor role. The CK1δ/ε inhibitor disrupted circadian rhythms of promoter activity in the circadian clock-containing zebrafish cell line, PAC-2, while the CK1ε inhibitor had no effect. Zebrafish larvae that were exposed to the CK1δ/ε inhibitor showed no rhythms of locomotor activity while the CK1ε inhibitor had only a minor effect on locomotor activity. Moreover, the addition of the CK1δ/ε inhibitor disrupted rhythms of aanat2 mRNA expression in the pineal gland. The pineal gland is considered to act as a central clock organ in fish, delivering a rhythmic hormonal signal, melatonin, which is regulated by AANAT2 enzymatic activity. Therefore, CK1δ plays a key role in the circadian timing system of the zebrafish. Furthermore, the effect of CK1δ inhibition on rhythmic locomotor activity may reflect its effect on the function of the central clock in the pineal gland as well as its regulation of peripheral clocks.

Multiple PAR and E4BP4 bZIP transcription factors in zebrafish: diverse spatial and temporal expression patterns.

Circadian rhythms of physiology and behavior are generated by an autonomous circadian oscillator that is synchronized daily with the environment, mainly by light input. The PAR subfamily of transcriptional activators and the related E4BP4 repressor belonging to the basic leucine zipper (bZIP) family are clock-controlled genes that are suggested to mediate downstream circadian clock processes and to feedback onto the core oscillator. Here, the authors report the characterization of these genes in the zebrafish, an increasingly important model in the field of chronobiology. Five novel PAR and six novel e4bp4 zebrafish homolog genes were identified using bioinformatic tools and their coding sequences were cloned. Based on their evolutionary relationships, these genes were annotated as ztef2, zhlf1 and zhlf2, zdbp1 and zdbp2, and ze4bp4-1 to -6. The spatial and temporal mRNA expression pattern of each of these factors was characterized in zebrafish embryos in the context of a functional circadian clock and regulation by light. Nine of the factors exhibited augmented and rhythmic expression in the pineal gland, a central clock organ in zebrafish. Moreover, these genes were found to be regulated, to variable extents, by the circadian clock and/or by light. Differential expression patterns of multiple paralogs in zebrafish suggest multiple roles for these factors within the vertebrate circadian clock. This study, in the genetically accessible zebrafish model, lays the foundation for further research regarding the involvement and specific roles of PAR and E4BP4 transcription factors in the vertebrate circadian clock mechanism.

Spectral sensitivity of melatonin suppression in the zebrafish pineal gland.

The pineal gland of the zebrafish (Danio rerio) is a clock-containing photoreceptive organ. Superfused pineal glands kept in darkness display rhythmic melatonin production that lasts for days, with high melatonin levels during the night and low levels during the day. Nocturnal light, however, evokes an acute suppression of melatonin synthesis in the photoreceptor cells. Towards characterizing zebrafish pineal photopigment that is involved in the acute melatonin suppression we have measured the spectral sensitivity of melatonin-suppression response in superfused pineal glands. The effect of 2 h light exposure of seven wavelengths (lambdaavg 408, 460, 512, 560, 608, 660 and 697+/-10-15 nm) at multiple irradiances (10(7)-10(14) photons/cm2/s) was determined, and an action spectrum was plotted. The resultant action spectrum provides evidence for the involvement of multiple photopigments in melatonin suppression. The most efficient melatonin-suppression response was achieved by exposure to light of around 512 nm; however, another peak of lower irradiance sensitivity was observed in the middle to long wavelengths. Opsins-specific RT-PCR analysis confirmed the expression of exo-rhodopsin and visual red-sensitive opsin in the pineal gland, while other zebrafish visual opsins as well as VA and VAL opsins were not detected. Dartnall monograms for exo-rhodopsin and visual red-sensitive opsin account for most but not all of the spectral sensitivity features. Therefore, additional pineal photopigments may contribute to the melatonin-suppression response in the pineal gland.