Proper implementation of the project has been deeply affected by the COVID-19 situation (difficulty in completing research, exploitation and dissemination).
Aim 1: Relative expression of the ten novopsins was analysed by Real Time Quantitative PCR during early development in wild type zebrafish. All the novel opsins were expressed in zebrafish embryo/larvae, being the onset between the second- and fourth-day post fertilisation. Interestingly, rhythmic expression was found in opn6b, opn7a, 7b, 7c and opn9. Times of the day that showed the highest novopsins expression were selected to perform in situ hybridisation (ISH). Fluorescent riboprobes were synthesised but unfortunately, due to the COVID-19 lockdowns and restrictions ISH were not fully completed and the precise cellular expression pattern of the novel opsins within the zebrafish larvae brain could not be reported. Candidate photopigments were selected according to their pattern and level of expression to obtain specific mutants (opn6b, opn7a, 7b, 7c and opn9). RNA guides were designed to perform CRISPR/Cas and obtain specific mutants. However, training on the injector was not allowed during the last year of the project, and injections of the guides were not feasible. For that reason, some available novopsin mutants from the Sanger Institute were selected. Individual fish was genotyped for the mutation and heterozygous were kept to perform experiments on clock entrainment and behaviour (Aim 2 and Aim 3).
Aim 2: To determine the consequences of opsin mutations on entrainment of the circadian clock and rhythmic neural activity at the molecular level we took advantage of the per3-luciferase line already present in the host lab. However, transgenic lines were too old and before any experiment, new per3-luciferase lines were generated. Luminescence rhythms were tested and confirmed in a Packard Top Count Scintillation Counter until day 6 post fertilisation. Unfortunately, assays for molecular clock function were not accomplished as work in the lab was very restricted during the last year of the project. In the same way, training was not allowed so calcium imaging could not be carried out to determine neuronal activity.
Aim 3: In collaboration with Jason Rihel´s lab, behaviour (activity and sleep) was monitored in zebrafish novopsin mutants. Heterozygous fish were crossed, and embryos were maintained under 14:10 LD cycles until day 3 post fertilisation. On that date, individual healthy larvae were transferred to 96-well plates and the plates were placed into a ViewPoint ZebraBox chamber. The plates were observed by a video camera connected to a computer with video tracking software until day 6-8 post fertilisation. Behavioural “fingerprints” of each mutant line have been obtained. Apparently, no differences between homozygous, heterozygous and wild type fish were relevant in relation to locomotor activity and sleep/wake cycles in the mutants analysed so far. However, activity and statistical analyses are still in process.
The plan for exploitation and dissemination of results has been largely affected by the pandemic and COVID-19 restrictions. Measures to exploit and disseminate the action results were mainly planned during the last year of the project. However, national lockdowns and COVID-19 restrictions made the work in the lab very difficult. Currently, the results of ZNEOPSIN_II will be disseminated as follow:
- International meetings: European Zebrafish Meetings, European Biological Rhythms Society or Gordon Research Conferences.
- Submission of papers for international peer-review scientific journals: it is planned that ZNEOPSIN_II will yield at least 2 publications: “Developmental expression profiles of novel opsins in zebrafish embryo” and “Behavioural fingerprints of selected novopsin mutants in zebrafish”.
