Periodic Reporting for period 1 - ZNEOPSIN_II (The role of novel opsins in non-visual light detection in the zebrafish brain)
Periodo di rendicontazione: 2018-09-01 al 2020-08-31
Vision has been typically seen as the major response to light, however, non-visual light detection modulates diverse physiological responses, including circadian rhythms and sleep. In mammals, both visual and non-visual photoreception is ocular. By contrast, non-mammalian vertebrates possess a wide range of anatomically diverse photoreceptive sites. This fact is especially evident in the zebrafish, where isolated organs can be directly entrained by light. This is true even in early embryonic stages, before the differentiation of any light detecting structures, and in zebrafish cell lines. This general light sensitivity implies that cells and tissues contain the essential photopigments required for light detection. A wide variety of non-visual pigments have been identified in vertebrates, all of which appear to use a basic opsin/vitamin A-based photopigment biochemistry. Moreover, a recent work led the discovery of 4 novel nonvisual opsin classes (opn6, opn7, opn8 and opn9), consisting of 10 unique genes designated novopsins. Expression analysis showed that novopsins are highly expressed in adult zebrafish brain, and they are localized to brain areas previously shown to be light-sensitive. However, a functional link between a specific opsin and its function is still missing and more work is required to unravel the photosensory role of the newly discovered non-visual pigments on zebrafish brain function, behaviour and sleep. The project will be focused on brain, which include an important behavioural aspect, increasing our knowledge on the regulation of specific behaviours by non-visual light detection. Apart from locomotor activity, this includes sleep, where zebrafish has emerged as an important model organism and represents a fundamental research field of considerable importance to human being. The overall aims of the project are outlined below:
Aim 1: Examine the developmental expression pattern of 10 novopsin genes in wild type zebrafish and select candidate photopigments according to their expression pattern to obtain specific mutants.
Aim 2: Determine the consequences of opsin mutations on entrainment of the circadian clock and rhythmic neural activity.
Aim 3: Explore the consequences of deletion on zebrafish locomotor activity and sleep.
Aim 1: Examine the developmental expression pattern of 10 novopsin genes in wild type zebrafish and select candidate photopigments according to their expression pattern to obtain specific mutants.
Aim 2: Determine the consequences of opsin mutations on entrainment of the circadian clock and rhythmic neural activity.
Aim 3: Explore the consequences of deletion on zebrafish locomotor activity and sleep.
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”.
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”.
The impact of the action has been disrupted due to COVID-19 restrictions. Most of the lab work programmed for the last year of the project could not be accomplished. However, the researcher has reinforced her skills and abilities, and competences acquired will be apply in Southern European aquaculture industry, opening new lines of research in the field of non-visual photoreception as light is widely used in aquaculture practices to modulate physiology and behaviour in fish species. Moreover, the researcher has taken advantage of the highly dynamic academic atmosphere at UCL, attending to different events organised. Importantly, the knowledge acquired in zebrafish care and husbandry will allow the researcher to design a zebrafish facility. The mobility aspect has also been very important for the fellow. In addition, the researcher has been involved in managing the project budget and in driving it efficiently, providing invaluable experience for the future in project management. The collaboration between UK and Spain regarding research on circadian clocks have been reinforced, and future research stays are already in preparation. Finally, several valuable tools have been generated within the host institution that will be suitable for future studies.