Petrone, L; (2016) Circadian clock and light input system in the sea urchin larva. Doctoral thesis , UCL (University College London).

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Abstract

A circadian clock is an endogenous time-keeping mechanism that synchronizes several biological processes with local environment. In metazoans the circadian system is driven by a regulatory network of so called ―clock‖ genes interconnected in transcriptional-translational feedback loops that generate rhythmicity at mRNA and protein level. Sea urchin and its molecular tools can facilitate the comprehension of the evolution of the time-keeping mechanism in bilaterians. For this purpose we identified and analysed the expression of orthologous clock genes in the sea urchin larvae. Genome survey identifies almost all canonical clock genes known in protostomes and deuterostomes, with exception of period, indicating that the last common ancestor of all bilaterians already had a complex clock toolkit. Quantitative gene expression data reveal that the circadian clock begins to oscillate consistently in the free-living larva. Sp_vcry and sp_tim mRNA cycle in both light/dark (LD) and free running (DD) conditions; several other genes consistently show oscillation in LD condition only; while, neither sp_clock, nor sp_bmal have rhythmic expression. Interestingly, in-situ hybridization of key sea urchin clock genes together with cell markers (e.g. serotonin) suggest the presence of two types of light perceiving cells in the apical region of the larva: serotoninergic cells expressing sp_dcry and no-serotoninergic cells expressing sp_opsin3.2. Furthermore, functional analysis was performed to discern linkages in the regulatory network of clock genes. In larvae entrained to light/dark cycles, knockdown of sp_dcry induces arrythmicity in the expression of itself, reduction of amplitude of oscillation in sp_vcry, and reduction of amplitude of oscillation and lower levels of expression in sp_tim. Knockdown of sp_opsin3.2 reduces levels of expression of sp_hlf; and sp_vcry knockdown induces arrythmicity in sp_tim. Importantly, our study highlights differences in the architecture and gene regulation of the sea urchin larval circadian clock compared to other metazoan clocks.

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