Nuclear Receptors and development of marine invertebrates

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The Adverse Outcome Pathway approach in invertebrate EDC research: linking molecular pathways of disruption to biological effects

A survey of the research progress regarding the issue of endocrine disruption in invertebrates has been recently published and, sadly, the results indicate that in the last 20 years the research has not advanced as vigorously as expected (Ford and Le Blanc, 2020). However, the survey also pointed out novel directions and research approaches that could nowadays be promising, one of which is the application of the Adverse Outcome Pathway (AOP) concept (Ford and Le Blanc, 2020; Allen et al., 2014). The AOP is defined as the sequence of events that from the exposure of an individual to a chemical, lead to its adverse effect at the individual or population level (OECD, 2012). Each AOP begins with a molecular initiating event (MIE) in which a chemical interacts with a biological target leading to a first cellular response which induces a sequential series of higher order effects at the organ and the organism level to produce an adverse biological outcome (Fig. 6 from Ankley et al., 2010). The first three steps described in Fig. 6 denote the parameters that define a toxicity pathway, while the last two steps are indicative of the effects on individuals that could lead to loss of population sustainability (Ankley et al., 2010; NRC, 2007). Accordingly, the application of the AOP approach could unravel plausible mechanisms of disruption by invertebrate endocrine-disrupting chemicals along with their effects on individuals that could lead to declines of natural populations (Ford and Le Blanc, 2020).

Marine invertebrate larvae can shed light on invertebrate endocrine disruption

Marine invertebrate larvae and early life stages have long been used as experimental models in various disciplines and not exclusively related to environmental issues (Ortega and Olivares-Bañuelos, 2020; Wilson-Sanders, 2011). Nowadays, larval development of a number of marine invertebrates from distinct phyla is also exploited by standard embryotoxicity tests designed by the International Organization for Standardization – ISO (www.iso.org) and ASTM international (www.astm.org). The main reason is that marine invertebrate larvae meet almost all the characteristics of a good experimental model organisms: they are unexpensive, easy to obtain from adult specimens, they develop fast, they are easy to grow in laboratory conditions, their exploitation for research purposes is not affected by ethical restrictions and the collection of ripening adult specimens from the environment does not generally risks to depauperate natural populations (Lewis et al., 2012; Love, 2009; Ségalat, 2007). Moreover, marine invertebrate larvae are proportionally more sensitive to environmental pollutants and stressors, including EDCs, with respect to their adult stages (Przeslawski, Byrne and Mellin, 2015). Regarding the particular context of endocrine disruption research, marine invertebrate larvae have already been pointed out in previous paragraphs to be the most suitable model for the characterisation of the invertebrate neuro-endocrine system, due to the variety of body plans and morphologies characterising adult stages (Tessmar-Raible, 2007). In addition to that, embryonic and post-embryonic development are the most dynamic periods of NR activity and signalling in vertebrates, carrying out a variety of distinct morphogenetic and developmental processed that are also required, at least in part, in the development of all metazoans, including marine invertebrates (i.e., neurogenesis, lipid metabolism, growth) (Chung and Cooney, 2003; Erwin, 1993). Finally, the fast larval morphogenesis and early developmental transitions of marine invertebrates could serve as potent endpoints of adverse effects of EDCs in the context of the AOP application and establish neuro-endocrine related biomarkers specific to invertebrates (Ford and Le Blanc, 2020; Carrier, Reitzel and Heyland, 2018).

M. galloprovincialis larval development as a model in invertebrate endocrine disruption research

Throughout invertebrates, the morphogenetic processes occurring during larval development are mainly dedicated to equipping the larvae with structures and systems able to provide protection from external stressors as well as converting external stimuli from the environment into appropriate responses (Carrier, Reitzel and Heyland, 2018¸ Gosling, 2015). In M. galloprovincialis, the larval development spans over all the developmental stages from the trochophore to the D-Veliger stage and is characterised by two main morphogenetic processes: shell biogenesis and neurogenesis (Giribet et al., 2020; Gosling, 2015). The process of shell biogenesis enables larvae to protect themselves from external insults while the formation of the nervous system permits to convert external stimuli from the environment in appropriate responses for the regulation of larval behaviour and development (Yurchenko et al., 2019; Marin, 2012).

Larval shell biogenesis in M. galloprovincialis

The growing of a shell is the distinctive character of all conchipheran molluscs, including M. galloprovincialis, on which they rely for protection from external stressors. In addition, the different stages of shell development in molluscs constitute the main diagnostic element of the out-course of their larval development which depends on the growth and shape of the larval shell as well as its ability to enclose the velum (Fig. 2). The larval shell is composed of an organic and inorganic layer (Wanninger and Wollesen, 2018). The organic layer is called organic matrix while the inorganic layer is constituted by the deposition of calcium carbonate (CaCO3). Surprisingly, the ontogeny of the larval shell formation has been described first in M. galloprovincialis (Kniprath, 1980). Shell biogenesis starts with the secretion of the organic matrix operated by a group of ectodermic cells that take the name of shell field (Fig. 3 A) (Kniprath, 1981; Kniprath, 1980).

The larval development of M. galloprovincialis and invertebrate endocrine disruption

In the context of invertebrate endocrine disruption research, the larval development of the mediterranean mussel could serve as a potent model. In addition to the powerful tool that shell biogenesis in itself already constitutes in screening the potential toxicity of environmental pollutants, evidence suggests that the morphogenetic process is at least in part controlled by the larval neuroendocrine signalling operated by the larval neurons and neurotransmitters that arise during early trochophore development (Liu et al., 2020, 2018). What is more, NR signalling supposedly plays a role in regulating these morphogenetic processes in molluscs (Miglioli et al., 2021). It follows that the main morphogenetic processes happening during larval development and their regulation in M. galloprovincialis are potential endocrine disruption targets that could serve as diagnostic endpoints to assess the endocrine effect of environmental pollutants in invertebrates as well as their specific mechanisms and essential information to unravel the functioning and regulatory elements of invertebrate neuro-endocrine systems and of Nuclear Receptors.

Prevention of the effects of 9-cis-Retinoic Acid and TBT on shell formation by co-incubation with UVI3003

In order to assess if the possible effects induced by 9-cis-Retinoic Acid and TBT on larval development might be due to MgRXR ligand-activation, we tested if pharmacological blockage of RXR activity by the full-antagonist UVI3003 could prevent the responses to the bona fide ligand and to TBT alone. The rescue experiment was thus performed by co-exposing fertilized eggs to the highest non-effective concentration of UVI3003 (5μM) and either 9-cis-Retinoic Acid or TBT at concentrations able to induce shell malformations (10nM) chosen on the basis of experiments described in previous paragraphs. Experiments were carried out in 48-wells plates. The negative control added with the vehicle (0.02% and 0.01% DMSO for 9-cis-Retinoic Acid and TBT rescues respectively) and the three positive controls added with 5 μM UVI3003 and 10 nM of 9-cis-Retinoic Acid or TBT, were run in parallel. At 48 hpf, larvae were fixed in 4% PFA and imaged with a Zeiss Axio Imager A2 (Zeiss, France). The effect of the treatments and success of the rescue on global larval development were evaluated by scoring shell malformations and measuring shell lengths in larvae from 3 independent parental pairs and in at least 50 larvae for each parental pair as described in previous paragraphs.

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Isolation and Characterisation of Mytilus galloprovincialis RXR

Thirty-four canonical NR proteins were identified in M. galloprovincialis genome. Putative NRs were verified upon the presence of a single conserved DNA and ligand binding domains. The protein IDs for each MgNR are given in Table 1. Phylogenetic analysis was performed using the amino acid sequences of the DBD and LBD of the 34 MgNRs and the maximum likelihood tree is shown in Fig. 2. M. galloprovincialis NRs include representatives of all the seven NR subfamilies investigated and the preliminary phylogenetic analysis indicated that most of the canonical MgNRs fit in the current nomenclature and classification, with the only exception of the representatives of the bivalve specific NR1P subfamily, previously described in the pacific oyster C. gigas, and the subfamily NR1J described in molluscs, insects, and nematodes (Vogeler et al., 2014; Kaur et al., 2015). The preliminary phylogeny of MgNRs segregated NR1s in a monophyletic group and the subfamilies NR2-NR7/8 in a second major clade, similarly to what was previously described for the NR complements of C. gigas, B. glabrata and L. gigantea (Vogeler et al., 2014; Kaur et al., 2015). Twenty-three of the 34 MgNRs are members of subfamily NR1, with 4 of them belonging to the protostome NR1J group, 2 to the mollusc NR1CDEF group and 8 of them clustering with the bivalve NR1P group (Vogeler et al., 2014; Kaur et al., 2015). Leaving the invertebrate NR1J and molluscan NR1P subgroups aside, M. galloprovincialis possesses single orthologs for all the MgNR1s traditional subgroups, confirming and corroborating data previously obtained in the pacific oyster, apart from the NR1C (PPAR) sub-group, for which three MgNRs were found. Seven MgNRs belong to subfamily 2 (NR2), with a single NR in each subgroup except for NR2E, for which three MgNRs clustering with D. melanogaster TLL, H. sapiens TLX and H. sapiens PNR / D. melanogaster DHR51 were found. Two MgNRs fitted in subfamily 3 (NR3) with high support values with respect to human ER (NR3A) and human/fruit fly ERR (NR3B), whereas no MgNRs clustered in NR3C. Only one MgNR clustered with subfamilies NR4 and NR5. With regards to NR5, the only MgNR5 belonged to the subgroup NR5B, while none clustered with NR5A. Surprisingly, M. galloprovincialis NR complement includes a representative of sub-group NR6A previously believed to be lost in bivalve molluscs, which showed high support values to both the fruit fly and human orthologous sequences (Vogeler et al., 2014; Kaur et al., 2015). M. galloprovincialis NR complement also includes a representative of the newly discovered subfamily NR7/8 (Huang et al., 2018).

Table of contents :

Preface
Abstract
Résumé
List of Figures and Tables
Introduction
Part 1. Potential impacts of EDCs on marine invertebrates: facts and controversies
1. Endocrine Disrupting Chemicals (EDCs): summary of consensus statements
2. Endocrine disruption: what is known
2.1 The definition of “endocrine disruption” is largely vertebrate-centric
2.2 The basics of endocrine disruption: lessons from vertebrates
2.3 EDCs mimic hormones and compete for the binding to Nuclear Receptors
3. EDCs pollution and Marine Invertebrates
3.1 EDCs in the aquatic environment
3.2 Effects of EDCs on marine invertebrates
3.3 Canonical Endocrine Disruption in marine invertebrates: facts and controversies
3.4 Invertebrate Molecular Disruption (IMD): Invertebrate Nuclear Receptors respond to EDCs
3.5 The Adverse Outcome Pathway approach in invertebrate EDC research: linking molecular pathways of disruption to biological effects
3.6 Marine invertebrate larvae can shed light on invertebrate endocrine disruption
4. References
Part 2. Nuclear Receptors and development of marine invertebrates
Part 3. The larval development of Mytilus galloprovincialis: an experimental model for invertebrate endocrine disruption studies
1. Mytilus galloprovincialis (Lamarck, 1819)
1.1 Characteristics and distribution
1.2 The life cycle of Mytilus galloprovincialis
2. Mytilus galloprovincialis larval development as a model in invertebrate endocrine disruption research
2.1 The larval development of Mytilus galloprovincialis
2.1.1 Larval shell biogenesis in M. galloprovincialis
2.1.2 Larval neurogenesis
2.2 Mytilus galloprovincialis larvae as model organism
2.3 The larval development of M. galloprovincialis and invertebrate endocrine disruption
3. References
Aim of the thesis
Chapter 1. The process of larval shell biogenesis in Mytilus galloprovincialis
Context of the study
Characterization of the main steps in first shell formation in Mytilus galloprovincialis: possible role of tyrosinase
Final remarks
Chapter 2. Neuroendocrine disrupting effects of EDCs in developing larvae of Mytilus galloprovincialis
Context of the study
Case study n°1: Bisphenol-A interferes with first shell formation and development of the serotoninergic system in early larval stages of Mytilus galloprovincialis
Case study n°2: Tetrabromobisphenol-A acts a neurodevelopmental disruptor in early larval stages of Mytilus galloprovincialis
Final remarks
Chapter 3. Implications of NR disruption in developing larvae of Mytilus galloprovincialis: a case study with RXR
Abstract
1. Introduction
2. Material and Methods
2.1 Identification of putative nuclear receptors in the genome of Mytilus galloprovincialis
2.2 MgRXR Sequence Analysis
2.3 Mussels, gamete collection and fertilization
2.4 Experimental conditions and exposure
2.5 Effect of 9-cis-Retinoic Acid, TBT and UVI3003 on larval growth at 48 hpf
2.6 Effect of 9-cis-Retinoic Acid, TBT and UVI3003 on shell formation
2.7 Prevention of the effects of 9-cis-Retinoic Acid and TBT on shell formation by co-incubation with UVI3003
2.8 In situ Hybridisation
2.9 Immunocytochemistry
2.10 Statistical Analyses
3. Results
3.1 Isolation and characterisation of Mytilus galloprovincialis RXR
3.1.1 The Nuclear Receptors of M. galloprovincialis
3.1.2 Sequence analysis of MgRXR
3.2 Effect of 9-cis-RA on larva shell biogenesis
3.2.1 Effect of 9-cis-RA on development of D-veligers at 48 hpf
3.2.2 Effect of increasing concentrations of 9-cis-RA on larval shell formation from 24 to 48 hpf
3.3 Effect of TBT on larva shell biogenesis
3.3.1 Effect of TBT on development of D-veligers at 48 hpf
3.3.2 Effect of increasing concentrations of TBT on larval shell formation from 24 to 48 hpf
3.4 Effect of UVI3003 on larval development of M. galloprovincialis
3.5 9-cis-RA and TBT phenotype rescue expriment at 48 hpf by co-incubation with UVI3003
3.6 Effect of 9-cis-RA and TBT on the expression pattern of MgRXR
3.7 Effect of 9-cis-RA and TBT on the expression pattern of Tyrosinase as a marker of shell development
3.8 Effect of 9-cis-RA and TBT on the dopaminergic system
3.9 Effect of 9-cis-RA and TBT on the number of 5-HTir cells at 48 hpf
3.10 In search of possible MgRXR heterodimers involved in M. galloprovincialis larval development
4. Discussion
5. Conclusions
6. References
General Discussion
1. Shell biogenesis in M. galloprovincialis larvae is regulated by the neuroendocrine system
2. BPA and TBBPA are neuroendocrine disruptors in M. galloprovincialis larvae
3. Nuclear Receptor as possible target of EDCs in early development of M. galloprovincialis
4. RXR initiates the neuroendocrine AOP of TBT in M. galloprovincialis larval development
5. References
Concluding Remarks and Future Perspectives
List of oral communications and published articles
Oral communications
Published Articles
Annex 1
Annex 2

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