Mutations in blind cavefish target the light-regulated circadian clock gene, period 2

Rosa Maria Ceinos,Silvia Fuselli,Elena Frigato,Pietro Negrini,Nadine Fröhlich,Nicholas S Foulkes,Cristina Pagano,Cristiano Bertolucci,Nicola Cavallari,Daniela Vallone

doi:10.1038/s41598-018-27080-2

Abstract

Light represents the principal signal driving circadian clock entrainment. However, how light influences the evolution of the clock remains poorly understood. The cavefish Phreatichthys andruzzii represents a fascinating model to explore how evolution under extreme aphotic conditions shapes the circadian clock, since in this species the clock is unresponsive to light. We have previously demonstrated that loss-of-function mutations targeting non-visual opsins contribute in part to this blind clock phenotype. Here, we have compared orthologs of two core clock genes that play a key role in photic entrainment, cry1a and per2, in both zebrafish and P. andruzzii. We encountered aberrantly spliced variants for the P. andruzzii per2 transcript. The most abundant transcript encodes a truncated protein lacking the C-terminal Cry binding domain and incorporating an intronic, transposon-derived coding sequence. We demonstrate that the transposon insertion leads to a predominantly cytoplasmic localization of the cavefish Per2 protein in contrast to the zebrafish ortholog which is distributed in both the nucleus and cytoplasm. Thus, it seems that during evolution in complete darkness, the photic entrainment pathway of the circadian clock has been subject to mutation at multiple levels, extending from opsin photoreceptors to nuclear effectors.

Highlights

The circadian clock plays a central role in timing most behavioural and physiological functions
We reported that the expression of both these genes in P. andruzzii shows arrhythmic expression upon exposure to a light-dark cycle[13], compared to the robust oscillation observed with the zebrafish orthologs
We confirm the lack of light responsiveness of these clock genes upon acute exposure to light in the P. andruzzii CF1 cell line compared with the robust light driven induction observed in zebrafish PAC-2 cells (Fig. 1a,b and Table S1)

Summary

Introduction

The circadian clock plays a central role in timing most behavioural and physiological functions. Transient transcriptional induction of a subset of core clock genes in response to light exposure appears to serve as a key step in entrainment of the clock by the day-night cycle. In zebrafish cells the per[2] and cry1a genes are strongly induced upon direct exposure to light via the effect of D-box enhancer promoter elements[7,8]. As well as serving to periodically down-regulate Clock/Bmal-dependent transactivation within the core clock mechanism, Per proteins appear to exhibit more complex functions with reports of both positive and negative regulation of other transcription factor systems[9]. The subcellular localization of many clock proteins and, in particular, the timing of their entry into the nucleus represent key properties, ensuring characteristic delays in the progression of the core feedback loop and ensuring that this timing mechanism generates rhythms with a circadian period. Differential splicing of the pa per[2] transcript leads to insertion of transposon-derived sequences and a premature termination codon, as well as an alteration of the subcellular localization of the predicted protein

Methods

Results

Conclusion