Development of a new in vitro cellular tool for the study of cnidarian phenotypic plasticity

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Cnidarian longevity and regeneration

Cnidarians are amongst the longest living animals, and in some cases immortality has been hypothesized. Nevertheless, the range of life span shows a high plasticity within cnidarians. Some polyps of hydrozoans live only a few days (e.g. Campanularia flexuosa; (Strehler & Crowell 1961)), whereas the deep-sea corals Gerardia sp. and Leiopathes sp. are longest lived, 2742 and 4265 years, respectively (Roark et al. 2009). In addition, the hydrozoan species Turritopsis dorhnii (previously known as T. nutricula), is considered immortal, by reverse development of sexually mature medusa into clonal polyps (Piraino et al. 1996).
Most living organisms get old, in a process known as aging. Aging represents one of the biological processes from which we still lack a full answer, mainly due to the diversity of aging across the tree of life. Attempts have been made to define the mechanisms leading to aging, and (López-Otín et al. 2013) categorized nine main molecular and cellular events occurring in cells aging (cf Fig.1.5). These mechanisms are involved at different levels of cellular complexity (from nucleus to the cell tissue). In the nucleus, aging process includes epigenetic alterations (DNA methylation, chromatin remodelling and transcriptional alterations), genomic instability (genomic damages) and telomere attrition (shortening of nucleoprotein structures located at the extremities of the chromosomes). At the organelle level, aging is due to the mitochondria dysfunction (reducing energy source and overproducing reactive oxygen species). At the cellular level, aging is provoked by cellular senescence (the arrest of the cell cycle, i.e. leading to cell death), stem cell exhaustion (depletion of undifferentiated cells capable of differentiation into specific cell types ensuring cell renewal) and loss of proteostasis (accumulation of protein degradation and misfolding of proteins). Finally, aging is the result of altered intercellular communication (perturbation of cell signalling, which leads to failures in the response to inflammation) (López-Otín et al. 2013).
Conversely, in cnidarians, the longevity has been suggested to be explained by some specific strategies overcoming the aging processes. Specifically, mechanisms leading longevity in cnidarians could be linked to the presence, in adult organisms, of stem cells with high and continuous cellular renewal potential. This propriety could then be responsible of two cnidarian specificities: asexual reproduction (responsible of clonal and colonial growth) and tissue regeneration (the process by which animals regrow lost body parts or entire organisms from small body fragments).
From all the above-mentioned mechanisms in cnidarians, regeneration is the most studied. Regeneration can happen in two ways, (1) morphallaxis, i.e. regeneration by reorganization and differentiation of pre-existing cells and (2) epimorphosis, which involves cell proliferation, i.e. cell growth and division leading to an increase in the number of cells (Li et al. 2015). For a long time, the high regenerative capacity of cnidarians was attributed to the presence of stem cells, meaning that regeneration was only done by morphallaxis. In Cnidarians, stem cells were first identified in the hydrozoan genus Hydractinia and later on Hydra (Weismann, 1883). Both groups possess stem cells in the interstitial space of epithelial cells, commonly referred as i-cells (Frank et al. 2009). The totipotency associated to i-cells is responsible for the high regenerative capacity of Hydra, which is able to regenerate two entirely new individuals from head and foot, the head will regrow a foot, and the foot will regrow a head (reviewed in (Holstein et al. 2003b)). Recognized stem cell genes (e.g. piwi, vasa, PL 10) have been used to identify stemness in cnidarian tissues (e.g. Seipel et al. 2003, Siebert et al. 2014) . For instance, piwi is present in the all developmental stages of the hydrozoan Podocoryne carnea with higher expression in the egg and medusa (Seipel et al. 2013) and vasa, piwi and PL 10 have been identified in the epiderm and gastroderm of gastrozooids of the hydrozoan species Nanomia bijuga (Siebert et al. 2014). Nevertheless, recent studies have also shown that other cnidarian species (e.g. the anthozoan Nematostella vectensis) are not able to regenerate without differentiated cell proliferation (Passamaneck & Martindale 2012, DuBuc et al. 2014), showing that regeneration by epimorphosis is also present in cnidarians.
Although regeneration has greatly contributed to our understanding on the mechanisms of longevity in cnidarians, others studies on aging processes could give us a new insight into the extreme life span of cnidarians. One of them is the dynamics of the telomere maintenance in cnidarians. One of the aging mechanisms is the telomere shortening, prevented by the presence of a telomerase. The telomerase is only expressed in germ cells, stem cells and tumours and its activity is responsible for the synthesis of new telomere repeats. In cnidarians, telomerase activity has also been identified (Traut et al. 2007, Ojimi et al. 2009, Zielke & Bodnar 2010); however telomere dynamics over time and their relation with events leading to cell cycle arrest and apoptosis has still to be determined. A relevant way to study telomere and telomerase activity related with cnidarian longevity, growth and stress response could be by the development of in vitro cnidarian culture cells and the analysis of the chromosome dynamics during cell divisions and time. Cell cultures would allow the study of telomerase activity within different types of cells, dividing cells and to determine extension of life span in culture.

Environmental adaptation

Cnidarians, as mention above, have a worldwide distribution inhabiting temperate, tropical, deep and surface waters. Colonization of different ecosystems implied adaptation to local environmental conditions, and more recently, anthropogenic threats. One of the ways through which cnidarians adapted was the establishment of mutualistic symbiosis with unicellular phototrophs, an association estimated to have been established 225 million years ago (Rosen 2000).
Some cnidarians live in association with a photosynthetic dinoflagellate from the genus Symbiodinium spp., and form one of the most studied symbioses in the marine realm – cnidarian-dinoflagellate symbiosis. Symbiodinium are unicellular algae that can be found as free-living species or associated with other unicellular organisms (e.g. dinoflagellate, foraminifera) but also cnidarians, molluscs (e.g. giant clams) and sponges (Trench 1993).
The symbiosis between Symbiodinium and Anthozoa – the class of cnidarians comprising sea anemones and corals – is one of the most ecologically significant symbioses, due to its role in the formation of tropical coral reefs, and the great biodiversity herein present (Dubinsky 1990). It is a mutualistic endosymbiotic intracellular association, where the symbiont (Symbiodinium) is located intracellularly in the gastrodermal cells of the host cnidarian, enveloped by a symbiosome membrane that separates the symbiont from the host cytoplasm (Fig. 1.6).

Our cnidarian study model: the sea anemone Anemonia viridis

The snakelocks sea anemone Anemonia viridis (Forskål, 1775) (class Anthozoa) (Fig. 1.10) was adopted as our model species.
It is a temperate species found abundant in shallow waters (up to 20 m) of the Mediterranean, Eastern Atlantic, English Channel and North Sea. It is a non-calcifying symbiotic cnidarian that leaves in association with the dinoflagellate Symbiodinium temperate A clade (Casado-Amezúa et al. 2014). Reproduction in this species is done asexually (by fission) or sexually, and the symbionts are transmitted vertically.
The use of A. viridis as model species presents several advantages: (1) long and non-retractable tentacles gives access to large amounts of tissue; (2) A. viridis has a high tentacle regenerative capacity, (3) facility to obtain aposymbiotic (deprived of symbionts) individuals in laboratory, (4) easy dissociation into epiderm, gastroderm and symbionts, and (5) the absence of a calcium carbonate skeleton favours tissue dissociation. Besides, in the last years, numbers of studies on A. viridis have increased knowledge on its physiology (Furla et al. 1998, 2000, Laurent et al. 2014), biochemistry (e.g. Richier et al. 2003), genetics (e.g. Ganot et al. 2011), cellular biology (Barnay-Verdier et al. 2013) and stress response (Richier et al. 2006, Moya et al. 2012, Suggett et al. 2012, Jarrold et al. 2013, Borell et al. 2014, Horwitz et al. 2015). Evidence from these studies suggests an extended plasticity of A. viridis, at different levels of biological complexity (from whole organism to the cell), as mechanism enabling its success under environmental changes and then justifying the use of A. viridis as a model for our studies.

Impact of ocean acidification on marine organisms

Marine organisms, from algae to fishes, show different sensitivities to OA, and studies predict that there will be winners and losers under future elevated CO₂ conditions (Table 1; Ries et al. 2009, Fabricius et al. 2011). Therefore, it is correct to assume that OA will shape marine communities. Indeed, OA may negatively impact a wide range of species across various phyla, especially calcifying species from plankton to corals. Conversely, some non-calcifying species are able to cope with OA, in natural or experimental conditions, with potential benefits on productivity (Doney et al. 2009, Fabricius et al. 2011). However, most of the studies on the effect of OA in marine organisms have been focused on the ecological impact rather than the mechanisms that trigger the OA response. In marine biota unicellular eukaryotes and cnidarians are two major representative groups for studies of OA impact on calcifying and non-calcifying organisms. Table was built from meta-analysis of Kroeker et al. 2013 and supplemented with data for sea anemones from independent studies (Suggett et al. 2012, Towanda & Thuesen, 2012, Jarrold et al. 2013). n.d. – non-determined by insufficient number of studies; no effect – no significant changes; – decrease; + increase (simplified from Kroeker et al. 2013).

Ocean acidification and carbon-concentrating mechanisms

Microalgae are the most important primary producers in the ocean. They absorb inorganic carbon and fix CO2 into organic compounds through Rubisco (ribulose bisphosphate carboxylase/oxygenase), an enzyme with affinity not only to CO2 but also to O2. To overcome this limitation they possess carbon-concentrating mechanisms (CCM) that efficiently exploit HCO3- pool (largest oceanic DIC pool) in the seawater, and convert it in CO2 in order to increase the concentration of available for the active site of the Rubisco (Giordano et al. 2005). An additional component of CCM is carbonic anhydrase (CA) an enzyme responsible for the acceleration of the rather slow inter-conversion between carbon species.
Symbiotic cnidarians have inside their host cells dinoflagellates from the genus Symbiodinium. While, free living Symbiodinium have direct access to DIC from the seawater, once inside host cells, several membranes form a barrier to the diffusion of DIC. One of the crucial contributions of the animal host to the symbiont is then the provision of DIC allowing the process of photosynthesis to proceed. There are two possible sources of DIC to the symbiont, (1) animal host‟s metabolism, i.e. respiration and (2) seawater. Animal host respiration rates are generally lower in respect to the net photosynthetic rates (Furla et al. 2005), indicating the presence of an external source of inorganic carbon. In addition, Symbiodinium Rubisco has low affinities to CO2 therefore efficient allocation of CO2 to the site of fixation is crucial (Rowan et al. 1996). CO2 diffuses passively through host membranes but the low concentration in the seawater makes it insufficient to optimal photosynthesis, making HCO3- the major DIC source for carbon fixation. However, host lipid membranes are impermeable to HCO3- and therefore it must be actively transported through host membranes.
Similarly to microalgae, symbiotic cnidarians possess CCM that efficiently exploits HCO3-pool in the seawater and increases the concentration of CO2 reaching Rubisco (Allemand et al. 1998). Bicarbonate is actively transported by the presence of an H+-ATPase pump located in the epidermal layer, which by the secretion of H+ leads to the acidification of the external medium, and consequent protonation of HCO3- to carbonic acid (H2CO3) (Furla et al. 2000, Bertucci et al. 2010). A membrane-bond carbonic anhydrase (CA) is responsible for the reversible conversion of HCO3- to CO2, which can then freely diffuse into host cells cytoplasm. Once in the cytoplasm, CO2 is trapped and converted to HCO3- by another CA, in agreement to intracellular pH (Venn et al. 2009). The transport of HCO3- to the gastrodermal cells may involve the presence of bicarbonate active transporters (Zoccola et al. 2015). Once HCO3- finally reaches the gastrodermal cells, a CA located close to the symbiont membrane protonates HCO3- into CO2, and this is made available to photosynthesis (Fig. 2.4) (Furla et al. 2000a).

Table of contents :

TABLE OF CONTENTS
LIST OF FIGURES
LIST OF PERSONAL PUBLICATIONS AND COMMUNICATIONS
LIST OF ABBREVIATIONS
Preamble
1. Chapter 1 – General introduction
1.1 The Phylum Cnidaria
1.1.1 Biodiversity
1.1.2 Ecological importance
1.1.3 General anatomy
1.1.4 Cellular anatomy
1.2 Physiological properties of cnidarians
1.2.1 Diversity of natural fluorescence
1.2.2 Cnidarian longevity and regeneration
1.2.3 Environmental adaptation
1.3 Our cnidarian study model: the sea anemone Anemonia viridis
1.4 Thesis objectives
2. Chapter 2 – In vivo studies of phenotypic plasticity
2.1 Introduction
2.1.1 Stress response: general context
2.1.2 Ocean acidification
2.2 Impact of ocean acidification on marine organisms
2.2.1 Impact of ocean acidification on calcification
2.2.2 Ocean acidification and carbon-concentrating mechanisms
2.3 Role of phenotypic plasticity in the response to OA
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Supplementary materials
2.4 Impact of multiple stressors in cnidarians
Material and Methods
Results
Discussion
2.5 Conclusions
3. Chapter 3 – Development of a new in vitro cellular tool for the study of cnidarian phenotypic plasticity
3.1 Cnidarian cellular phenotypic plasticity
3.2 Vertebrate cell culture
3.2.1 Origin and contribution of vertebrate cell cultures
3.2.2 Diversity of vertebrate cell culture methodologies
3.3 Invertebrate cell cultures
3.3.1 Terrestrial cell cultures
3.3.2 Marine cell cultures
3.4 Cnidarian cell culture development
3.4.1 State of the art
3.4.2 Anemonia viridis cell culture establishment
3.5 Characterization and validation of cnidarian primary cell culture
Introduction
Materials and Methods
Results
Discussion
References
Supplementary materials
3.6 Further research on diversification of cell culture
3.6.1 In vitro primary cell culture assays from separated monolayers: epiderm vs. gastroderm .
3.6.2 Hanging drop culture assays for isolation and cultivation of cnidarian pluripotent cells .
3.7 Conclusions
4. Chapter 4 – General conclusions and perspectives
5. References

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