Development of fish embryonic stem cell lines as tools for basic and applied studies of interest in aquaculture
Living organisms are constantly exposed to cyclical variations, whether daily, monthly or yearly, in environmental factors such as light and temperature. These external fluctuations are the result of terrestrial rotational and translational movements generated by the earth's spinning on its own axis and around the sun. Daily and seasonal variations in light duration (photocycles) affect all living organisms and give rise to a multitude of physiological and behavioural adaptations of a rhythmic nature that favour their survival. With the exception of some organisms that live in the deep ocean or in caves, these cyclical variations of light affect all living beings, making biological rhythms strongly rooted and phylogenetically conserved in most organisms from unicellular to mammals. Therefore, the study of biological rhythms and all their components (synchronising factors or input, molecular clock, rhythmic manifestations or output) is essential to understand the mechanisms of synchronisation, internal time measurement, response and anticipation to predictable environmental variations in living organisms, with circadian rhythms (with a period of around 24 h) being the ones that have aroused most interest in recent decades. As indicated above, light is one of the most studied synchronising environmental factors, determining many of the rhythmic physiological responses observed at the tissue and cellular level. However, light has effects related not only to its time-varying component (photoperiod), but also to variations in intensity and spectral quality. This is particularly important in marine aquatic environments where the water column acts as a colour filter, resulting in non-uniform light penetration as a function of depth and water characteristics (e.g., turbidity). This differential penetration of the electromagnetic wavelengths of visible light in the ocean, and its variation with depth, results in blue and green light having greater penetration of the water column in the open ocean and green light in coastal waters. In fish, as in other vertebrates, the photoperiod and the light spectrum affect important processes such as development, growth, feeding, metabolism or reproduction, both in wild and cultured animals. However, in aquaculture practice, fish are subjected to very specific environmental conditions (light, temperature, culture density, etc.), which in most cases are not optimised and do not correspond to the conditions they experience in their natural environment. This leads to physiological imbalances and deficiencies that compromise animal welfare, affect development, growth, feeding and reproduction, resulting in a deficit in production, with considerable damage to the economic profitability of the aquaculture sector. For this reason, numerous experimental studies have been carried out to gain a deeper understanding of these biological processes of productive interest and to provide a response to these dysfunctions. In this sense, laboratory studies have highlighted the experimental complexity and ethical issues associated with the use of live animals in research. Consequently, there is a need to generate new in vitro tools to complement studies previously carried out in vivo or ex vivo. These in vitro tools can be very useful for both basic and applied studies or for optimising and refining experimental protocols, thus reducing the costs and number of animals used in research, as well as the harm they suffer during sampling and subsequent sacrifice, in compliance with the 3Rs rule. Among these new in vitro tools aimed at simplifying analysis and animal experimentation, the establishment of undifferentiated embryonic stem cell lines is presented as an excellent opportunity to address studies related to growing disciplines such as Developmental Biology, Chronobiology and Environmental Physiology. As a prerequisite, these cell lines must be properly established and characterised in order to obtain reliable and reproducible results. However, for reasons of clinical interest these in vitro cellular tools have been developed mainly in mammals and in freshwater model fish such as zebrafish, with very few embryonic cell lines developed in marine fish. The main objective of this doctoral thesis is to develop of monoclonal embryonic stem cell lines of seabream, Sparus aurata, as a tool to study and characterise the different components of the circadian system (synchronising factors or input, molecular clock, overt rhythms or output) during the development of this species. To achieve this goal, we set out to identify the presence of a molecular clock in these cells, determine their daily rhythms and their synchronisation by light (photoperiod and light spectrum). We also aimed to establish the relationship of this molecular clock with the presence and expression of visual and non-visual photopigments (input) in these embryonic cells and to characterise the output of this clock by studying the daily variations and rhythms in the expression of genes associated with cell proliferation, apoptosis, DNA repair and stress in response to photoperiod and/or light spectrum. In Chapter 1, entitled "Establishment and characterisation of single cell-derived embryonic stem cell lines from the gilthead seabream, Sparus aurata", we have developed and characterised two embryonic stem cell lines derived from fertilised oocytes of seabream (Sparus aurata) at the morula stage, named SAEC-A3 and SAEC-H7. We have characterised their growth and potential in terms of embryoid body formation, differentiation and transfection capacity. In addition, we have analysed the evolution of cell proliferation under different photoperiods, revealing significant daily oscillations in the proliferative activity of these embryonic cells, which are endogenous as they are maintained under constant dark conditions. In Chapter 2, entitled "A functional light-entrainable molecular clock is revealed in gilthead seabream (Sparus aurata) from early developmental stages using an embryonic stem cell line", we examined whether SAEC-H7 cells have a molecular clock and investigated its regulation by light. Thus, in this study we have characterised the variation and daily rhythms in the expression of the main components of the molecular clock (clock, bmal1, cryptochrome1a, period1, period2 and period3) under controlled light-dark cycles and constant conditions (constant light, constant dark), as well as the response to nocturnal light pulses in this cell line. This study has allowed us to demonstrate that SAEC-H7 cells contain a molecular clock capable of synchronising with the daily light-dark cycles and that these rhythms have an endogenous character, since the oscillations of some genes of the molecular clock are maintained under constant conditions for a certain period of time. In addition, the molecular clock of SAEC-H7 cells exhibits other properties of functional clocks, such as the ability to resynchronise to inverted photocycles and the transcriptional activation of light-inducible genes such as cry1a and per2 during a nocturnal light pulse. In Chapter 3, entitled "Photoperiod and light spectrum modulate daily rhythms and expression of genes involved in cell proliferation, DNA repair, apoptosis and oxidative stress in a seabream embryonic stem cell line", we analysed the effects of white (LDW), blue (LDB), green (LDG), blue/green (LDBG) and red (LDR) light, as well as constant dark (DD) conditions on gene expression and daily rhythms of cellular markers of proliferation (pcna), DNA repair (cry5), apoptosis (bcl2, bax) and cellular and oxidative stress (hsp70, prdx2) in SAEC-H7 cells using RT-qPCR and cosinor analyses. The results obtained revealed the best performance of the cells under blue light conditions (LDB), in which all the genes analysed showed their highest expression levels and the most robust daily variations and rhythms. In blue light, the expression levels of cell proliferation (pcna), DNA repair (cry5), anti-apoptotic (bcl2) and oxidative stress (prdx2) markers peaked at the day-night transition, while pro-apoptotic (bax) and cellular stress (hsp70) markers showed their highest expression at the night-day transition, evidencing the strong synchronisation of transcription of key genes involved in the cell cycle under this light regime. The persistence of significant pcna, cry5, hsp70 and prdx2 rhythms after three days in constant darkness revealed the endogenous and circadian nature of these rhythms, and their control by the molecular clock. Finally, in Chapter 4, entitled "Light sensing in fish embryonic cells: A circadian analysis of visual and non-visual opsin expression using a seabream embryonic stem cell line", we aimed to identify the specific repertoire of visual and non-visual opsins present in the seabream embryonic stem cell line SAEC-H7 that could represent the input pathways to its circadian system and mediate light-entrained cellular responses, as well as to investigate their daily expression patterns and their endogenous nature. This study has allowed us to determine that SAEC-H7 cells express five visual opsins (sws1, sws2a, sws2b, rh2.4 and lws) and eleven non-visual opsins (opn3, opn4m1, opn4m2, opn4m3, opn5, opn6a, opn7a, tmt1a, tmt1b, tmt2a and tmt2b), ensuring sensitivity to all the wavelengths present in white light, but also beyond the visual spectrum. Most of these opsins show daily variations and/or rhythms in their expression levels, with a peak at the beginning of the day, and some of them show robust daily circadian rhythms in constant darkness, suggesting that they are under clock-control. These findings highlight the ability of embryonic fish cells to sense light and maintain synchronised rhythmic responses to light from the earliest stages of segmentation (morula). These basic studies have provided valuable information on the functioning of the circadian system in embryonic cells of seabream (Sparus aurata), which may be of great interest for understanding the effects of environmental factors such as light (and its cycles and spectrum), on processes of a markedly rhythmic nature such as cell differentiation, development, growth, metabolism, stress, feeding, reproduction, as well as on other processes of interest in the aquaculture of this species. This information may also be of great relevance for further studies on developmental biology, environmental physiology and chronobiology of other fish species of basic and aquaculture interest.
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