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

Glial cells expressing visual cycle genes are vital for photoreceptor survival in the zebrafish pineal gland

Photoreceptors in the vertebrate eye are dependent on the retinal pigmented epithelium for a variety of functions including retinal re-isomerization and waste disposal. The light-sensitive pineal gland of fish, birds, and amphibians is evolutionarily related to the eye but lacks a pigmented epithelium. Thus, it is unclear how these functions are performed. Here, we ask whether a subpopulation of zebrafish pineal cells, which express glial markers and visual cycle genes, is involved in maintaining photoreceptors. Selective ablation of these cells leads to a loss of pineal photoreceptors. Moreover, these cells internalize exorhodopsin that is secreted by pineal rod-like photoreceptors, and in turn release CD63-positive extracellular vesicles (EVs) that are taken up by pdgfrb-positive phagocytic cells in the forebrain meninges. These results identify a subpopulation of glial cells that is critical for pineal photoreceptor survival and indicate the existence of cells in the forebrain meninges that receive EVs released by these pineal cells and potentially function in waste disposal.Ministry of Education (MOE)National Research Foundation (NRF)This work was funded by a joint grant from the Israel Science Foundation and National Research Foundation of Singapore (6276/17 and NRF2017‐NRF‐ISF002‐2676) to YG and SJ, and a Tier 1 Grant from the Singapore Ministry of Education (RG34/20) to SJ

Visual recognition of social signals by a tectothalamic neural circuit

Social affiliation emerges from individual-level behavioural rules that are driven by conspecific signals1,2,3,4,5. Long-distance attraction and short-distance repulsion, for example, are rules that jointly set a preferred interanimal distance in swarms6,7,8. However, little is known about their perceptual mechanisms and executive neural circuits3. Here we trace the neuronal response to self-like biological motion9,10, a visual trigger for affiliation in developing zebrafish2,11. Unbiased activity mapping and targeted volumetric two-photon calcium imaging revealed 21 activity hotspots distributed throughout the brain as well as clustered biological-motion-tuned neurons in a multimodal, socially activated nucleus of the dorsal thalamus. Individual dorsal thalamus neurons encode local acceleration of visual stimuli mimicking typical fish kinetics but are insensitive to global or continuous motion. Electron microscopic reconstruction of dorsal thalamus neurons revealed synaptic input from the optic tectum and projections into hypothalamic areas with conserved social function12,13,14. Ablation of the optic tectum or dorsal thalamus selectively disrupted social attraction without affecting short-distance repulsion. This tectothalamic pathway thus serves visual recognition of conspecifics, and dissociates neuronal control of attraction from repulsion during social affiliation, revealing a circuit underpinning collective behaviour.publishe

Glial support of pineal photoreceptors in zebrafish

This project investigates a glial cell population in the zebrafish pineal gland, defined by the expression of agrp2.    The files here show confocal stacks of the pineal of Tg(aanat2:exorhodopsin-mcherry, agrp2:eGFP) larvae. Immunolabelling was used to enhance mCherry signal. The images can be opened with FIJI.</p

Agouti-Related Protein 2 Is a New Player in the Teleost Stress Response System

Agouti-related protein (AgRP) is a hypothalamic regulator of food consumption in mammals. However, AgRP has also been detected in circulation, but a possible endocrine role has not been examined. Zebrafish possess two agrp genes: hypothalamically expressed agrp1, considered functionally equivalent to the single mammalian agrp, and agrp2, which is expressed in pre-optic neurons and uncharacterized pineal gland cells and whose function is not well understood. By ablation of AgRP1 -expressing neurons and knockout of the agrp1 gene, we show that AgRP1 stimulates food consumption in the zebrafish larvae. Single-cell sequencing of pineal agrp2-expressing cells revealed molecular resemblance to retinal-pigment epithelium cells, and anatomic analysis shows that these cells secrete peptides, possibly into the cerebrospinal fluid. Additionally, based on AgRP2 peptide localization and gene knockout analysis, we demonstrate that pre-optic AgRP2 is a neuroendocrine regulator of the stress axis that reduces cortisol secretion. We therefore suggest that the ancestral role of AgRP was functionally partitioned in zebrafish by the two AgRPs, with AgRP1 centrally regulating food consumption and AgRP2 acting as a neuroendocrine factor regulating the stress axis

Clock-controlled rhythmic gene expression is disrupted in ΔCLK-expressing pineal glands.

<p>(A) Experimental procedure for transcriptome analysis. Adult fish were kept under DD and pineal glands were sampled at 12 time points (indicated by arrows) throughout two daily cycles. Black and gray bars correspond to subjective night and day, respectively. (B) The mRNA-seq analysis resulted in the identification of 29 circadian genes in the pineal gland of Tg(<i>aanat2</i>:EGFP-ΔCLK) fish compared with 290 circadian genes in the pineal gland of Tg(<i>aanat2</i>:EGFP) control fish.</p

Generation of Tg(<i>aanat2</i>:EGFP-ΔCLK) fish.

<p>(A) Schematic representation of the transgenic construct used for the generation of Tg(<i>aanat2</i>:EGFP-ΔCLK) fish. The <i>pT2-aanat2</i>:<i>EGFP-2A-Δclocka-5×MYC</i> plasmid consists of two arms from the Tol2 transposable element (black), <i>aanat2</i> regulatory regions (gray), EGFP coding sequence (CDS; green), 2A peptide sequence (orange), ΔCLK CDS (blue), 5×Myc tags (purple) and SV40 poly(A) signal (yellow). (B and B') EGFP expression is restricted to the pineal gland of Tg(<i>aanat2</i>:EGFP-ΔCLK) larvae. Dorsal views of the head region of 7-day post-fertilization (dpf) larvae, anterior to the top; confocal z-stack projection (B) and a single confocal plane (B'). (C) Immunostaining with anti-Myc antibody confirms that ΔCLK is specifically expressed in the pineal gland of Tg(<i>aanat2</i>:EGFP-ΔCLK) larvae. Dorsal view of the head region of a 5-dpf larva, anterior to the top; confocal z-stack projection.</p

Circadian rhythms of locomotor activity under DD and DimDim, but not under LL or LD cycles, are affected by blocking the pineal clock.

<p>Analysis of locomotor activity of 6–8 dpf Tg(<i>aanat2</i>:EGFP-ΔCLK) larvae (ΔCLK) and control larvae under various lighting conditions. A–D, left chart: The average distance moved (cm/10 min) is plotted on the y-axis and circadian time (CT) is plotted on the x-axis; error bars stand for SE (<i>n</i> = 24); black, white and diagonally lined bars represent dark, light and dim light, respectively. A–D, right chart: Distribution of the G-factors (see 'Fourier analysis' in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006445#pgen.1006445.s013" target="_blank">S1 Text</a>) of Tg(<i>aanat2</i>:EGFP-ΔCLK) and control larvae; the median G-factor value for each group is indicated (red lines). (A) Circadian rhythms of locomotor activity under DD, after entrainment by 5 LD cycles, are affected by blocking the pineal clock; a significant difference in the distribution of G-factors was found between Tg(<i>aanat2</i>:EGFP-ΔCLK) and control larvae (<i>p</i><0.0001, Kolmogorov-Smirnov test). (B) Circadian rhythms of locomotor activity under DimDim, after entrainment by 3 LD cycles and 2 light-dim light (LDim) cycles, are affected by blocking the pineal clock; a significant difference in the distribution of the G-factors was found between Tg(<i>aanat2</i>:EGFP-ΔCLK) and control larvae (<i>p</i><0.0001, Kolmogorov-Smirnov test). (C) Circadian rhythms of locomotor activity under LL, after entrainment by 5 LD cycles, are NOT affected by blocking the pineal clock. (D) Circadian rhythms of locomotor activity under LD cycles are NOT affected by blocking the pineal clock.</p

Circadian rhythms of <i>per1b</i> promoter activity in whole larvae are not affected by the pineal ΔCLK mutation.

<p>(A) Mean bioluminescence of Tg[<i>aanat2</i>:EGFP-ΔCLK;(−3.1)<i>per1b</i>::luc] larvae (ΔCLK; green trace; <i>n</i> = 23) and Tg(−3.1)<i>per1b</i>::luc larvae (control; black trace; <i>n</i> = 55), starting from 5 dpf for two daily cycles under DD. Circadian time (CT) is plotted on the x-axis. Gray and black bars represent subjective day and subjective night, respectively. Error bars represent SD. (B) Distribution of the G-factors (a representation of the extent to which the frequency of the luciferase activity pattern corresponds to a 24-hr period; see 'Fourier analysis' in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006445#pgen.1006445.s013" target="_blank">S1 Text</a>) of ΔCLK and control larvae. The median G-factor value for each group is indicated (red lines).</p