Identifcation of pathways that regulate circadian rhythms using a larval zebrafsh small molecule screen

The circadian clock ensures that behavioral and physiological processes occur at appropriate times during the 24-hour day/night cycle, and is regulated at both the cellular and organismal levels. To identify pathways acting on intact animals, we performed a small molecule screen using a luminescent reporter of molecular circadian rhythms in zebrafish larvae. We identified both known and novel pathways that affect circadian period, amplitude and phase. Several drugs identified in the screen did not affect circadian rhythms in cultured cells derived from luminescent reporter embryos or in established zebrafish and mammalian cell lines, suggesting they act via mechanisms absent in cell culture. Strikingly, using drugs that promote or inhibit inflammation, as well as a mutant that lacks microglia, we found that inflammatory state affects circadian amplitude. These results demonstrate a benefit of performing drug screens using intact animals and provide novel targets for treating circadian rhythm disorders.

Introduction

Circadian rhythms help ensure that physiological processes and behaviors occur at appropriate times during the 24-hour day/night cycle. These rhythms are generated and sustained at the cellular level by a transcriptional-translational negative-feedback loop that cycles with a period of approximately 24 hours, and are entrained by environmental cues such as light, food availability and temperature1. Molecular circadian oscillations in cells in different tissues and brain regions cycle with distinct phases, suggesting that non-cell autonomous mechanisms transmit circadian information throughout an animal1. While the suprachiasmatic nucleus (SCN) acts as a central circadian pacemaker to regulate circadian rhythms in mammals, it is unclear how the SCN transmits circadian information and if additional non-cell autonomous mechanisms exist. It is also unclear whether some aspects of the vertebrate circadian clock, which has primarily been studied using nocturnal rodents, differ from those in diurnal vertebrates such as humans. Thus, despite extensive research, mechanisms that regulate circadian rhythms remain incompletely understood.

Attempts to identify these mechanisms have primarily used two approaches. Genetic screens in model organisms have identified many components of the molecular circadian clock1. However, this approach may fail to detect genes that regulate aspects of the clock that do not affect the phenotype studied, or genes that have functionally redundant paralogs. Genetic screens are also difficult to perform using vertebrate animals. More recently, luminescent reporters have been used in cell culture to screen for cell autonomous factors that regulate the mammalian molecular circadian oscillator2,3,4,5,6,7,8. However, this approach lacks in vivo relevance and will not detect mechanisms that act non-cell autonomously or do not operate in the cell types used. Thus, alternative approaches could reveal novel mechanisms that regulate the circadian clock.

Most small molecule screens use in vitro or cell culture assays to identify drugs that bind a specific target or affect a specific process. However, these screens do not recreate the complex environment of whole animals and likely fail to identify some mechanisms that regulate the process under study. To overcome these limitations, we and others have used intact zebrafish as a vertebrate model system for small molecule screens9. This approach combines the in vivo relevance of whole-animal assays with moderate-throughput, low-cost drug screening. It also exploits several features of zebrafish larvae, including a relatively simple yet conserved vertebrate brain that lacks a mature blood-brain-barrier10, a small size that allows for screening in multi-well plates, and optical transparency that facilitates the use of luminescent reporters. Importantly, for the purposes of circadian research, the zebrafish molecular circadian oscillator closely resembles that of mammals11.

Here we describe a screen for small molecules that affect molecular circadian rhythms using a luminescent reporter in zebrafish larvae. We also monitor behavioral circadian rhythms using an assay that we previously used to identify drugs that regulate larval zebrafish locomotor behaviors12. We show that small molecules targeting pathways known to affect the circadian clock induce the expected circadian phenotypes in intact zebrafish. We also identify drugs that implicate novel pathways in regulating circadian rhythms in vivo that are absent in cultured cells. Finally, we show that inflammatory state affects circadian amplitude using both drugs and xpr1b mutant zebrafish, which lack microglia. These results reveal an unexpected role for the immune system in regulating the circadian clock.

Results

A screen for small molecules that affect molecular circadian rhythms in zebrafish larvae

A previous study described transgenic zebrafish in which the promoter for the period3 gene regulates expression of firefly luciferase (Tg(per3:luc)), and showed that this line accurately reports molecular circadian rhythms in zebrafish larvae13. To test whether this line could be used to screen for small molecules that affect molecular circadian rhythms, we asked whether compounds that affect the circadian clock in cell culture induce similar effects in zebrafish larvae. We entrained Tg(per3:luc) larvae in 14:10 hour light:dark (LD) conditions for 6 days at 22 °C13. We then placed individual larvae into each well of a 96-well plate, added small molecules or DMSO vehicle control to each well, and monitored luminescence for 72 hours in constant darkness (DD) (Fig. 1A). To validate our assay, we first tested a drug that targets a pathway known to affect circadian period length. Pharmacological inhibition of casein kinase 1 (CK1) increases period length in mammalian cell culture3,5,14, rodents5,15 and zebrafish5,15,16, similar to some ck1 mutant animals17,18,19,20. We tested a compound, A002195858, that inhibits CK1 in vitro (IC50 = 23 nM) and dose-dependently increases period length in mammalian cells (Fig. S2F), and found that it also dose-dependently increases period length in our larval zebrafish assay (Fig. 1B). We also found that the Src kinase inhibitor SU-665621 dose-dependently increases circadian amplitude in our assay (Fig. 1C). These results indicate that Tg(per3:luc) larvae can be used to report drug-induced changes in molecular circadian rhythms, and that phenotypes observed in mammalian cells can also be observed in zebrafish larvae.