Functional characteristics of the endogenous clock machinery in krill
University of Padova (UP), Charité Universitätsmedizin Berlin (CUB), Alfred Wegener Institute for Polar and Marine Research (AWI), Australian Antarctic Division (AAD)
Key objectives of this work package are (i) the definition of the molecular components and mechanism of krill’s endogenous circadian clock; (ii) its plasticity in the context of different seasons; and (iii) the identification and characterization of physiological processes that are regulated by krill’s circadian clock, again with respect to different seasons. Ad (i): To define critical clock genes of krill, the Costa lab (UP) – the first who identified a clock gene (Escry) in E. superba  – follows two complementary approaches: It is currently characterizing two other clock genes, clock and an ARNT like gene by using a classic “gene candidate approach”. Additional clock genes will be identified by high-throughput approaches, such as pyro sequencing cDNA libraries. The Costa lab has already pyrosequenced a normalized cDNA library, obtained from head, abdomen and thoracopods. Once the critical clock genes are identified, the Kramer lab (CUB) – in close collaboration with the Costa lab – will characterize the functional interconnections of krill circadian components to characterize the mechanism of krill’s circadian clock. For example, it will be tested using model cell lines (e.g., Drosophila derived cell lines) via luciferase reporter-based transactivation assays , whether in krill (as it is the case in other animal clocks) E-box enhancers serve as critical clock gene promoter elements that are activated by positive clock elements (Clock and ARNT like) and are repressed by negative elements (such as Per).
In addition, based on the critical role of post-translational mechanism that is described for all circadian clocks known to date (ranging from bacteria to humans), the Kramer lab will also test the hypothesis that phosphorylation of clock proteins are of essential importance for Krill’s circadian clock. To this end, the role of CK1 (important for Drosophila and mammalian circadian dynamics) will be tested with biochemical (e.g. mass spectrometry of putative targets, such as PER; ) and pharmacological approaches (e.g. by using CK1 inhibitors; see e.g. ). Ad (ii): To test whether circadian clock dynamics is governed by seasonal influences, the Kramer lab (in collaboration with AWI) will analyze transcript levels of canonical clock genes around the circadian time  under different light:dark (LD) cycles throughout the simulated seasonal course of photoperiod. The group at AAD will provide the specifically entrained samples. Comparison of circadian characteristics (e.g. amplitude, period, phase relation) among clock genes at different stages throughout the simulated seasonal course of photoperiod will provide the possibility to identify critical changes with photoperiod. Ad (iii): To obtain insights into the molecular mechanisms which allow krill endogenous clocks to interpret environmental signaling and modulate physiology and behavior accordingly, the Costa lab will characterize the krill transcriptome around the circadian time, under different light conditions (e.g. seasonal light spectrum, light-dark cycles) and temperature profiles. Again, the group at AAD will provide the specifically entrained samples. Sequences generated by the Costa group and all the available E. superba sequences [21,22,23] from public databases have been recently assembled to create the first krill microarray platform, named Krill 1.1, with a total of 32.217 different probes. 8x60K microarrays (Agilent technology) have been produced, which allow the analysis of eight different samples on single slides. Using krill 1.1 and/or 1.2 Agilent platforms we want to define gene expression signatures of specimens collected in nature at different time points during the day over a complete 24-hour cycle in order to characterize the circadian transcriptome (samples are already available, ). Moreover, the collaboration between these groups aims to define the transcriptional signatures of different developmental stages in E. superba producing stage-specific 3′-UTR cDNA libraries. In this way we will identify larval expressed transcripts and obtain the first transcriptional signature of larval development in the Antarctic krill (estimating the 3’-EST frequency at each stage).