Nuclear receptor signaling and endocrine disruption in a mollusk: the power of functional experiments – MusMod4EDC

To achieve these objectives, the work program is subdivided into four distinct, but interconnected, tasks: - Task 1: Precise spatio-temporal mapping of the expression of nuclear receptors in mussel embryos using an RNA-seq approach, qPCR, immunocytochemistry and in situ markings by “hybridization chain reaction”. - Task 2: Characterization of phenotypes induced by exposure to endocrine disruptors. - Task 3: Identification of nuclear receptor target genes by differential RNA-seq analysis followed by in cellulo functional analysis. - Task 4: Assessment of the developmental functions of nuclear receptors and their involvement in the response to endocrine disruptors.

Task 1: The analysis of the 58 nuclear receptors (46 complete sequences and 12 partial sequences) identified in the genome of M. galloprovincialis by RNA-seq, fluorescent in situ hybridization and qPCR was carried out by partners 1 (Dumollard) and 2 (Schubert) and published in the journal Phil. Trans. B (doi:10.1098/rstb/379/1898). In addition, partners 1 and 2 carried out the complete developmental transcriptome of M. galloprovincialis (between 0 and 72 hours after fertilization) and immunocytochemistry protocols coupled with multiplexed fluorescent in situ markings were developed which allow detailed analysis of the ontogeny of the different embryonic tissues of the mussel. This transcriptome and these new protocols were published in the journal Development (doi:10.1242/dev.202256). This task is now complete. Task 2: Larval mussel cultures in the presence of the endocrine disruptor TBT and sRNA/sDNA extractions were carried out. We established that TBT affects larval shell development as well as neural and muscular development. These effects of TBT can be prevented by prior inhibition of the nuclear receptor RXR, suggesting that RXR is the main mediator of the deleterious effects of TBT on the mussel larva. We plan RNA-seq and ATAC-seq sequencing of embryos cultured in the presence of TBT (with or without RXR inhibitor) for spring 2024. This analysis will allow us to find the gene networks affected by TBT which depend on the RXR activity. Task 3: The phenotypes induced by the RXR agonist called 9 cis-retinoic acid (9cisRA) were characterized. These phenotypes closely resemble TBT-induced phenotypes and are rescued by prior inhibition of RXR, showing once again that TBT exerts its deleterious action on mussel embryos by activating RXR. In addition, in cellulo tests to establish the binding capacities of nuclear receptors were carried out by the ANR post-doc of partner 1 (Miglioli, Dumollard lab) in the laboratory of partner 3 (Santos lab) during a 3-month mission (summer 2023). These analyzes demonstrated that 9cisRA and TBT can directly bind the RXR receptor. Additionally, eight other nuclear receptors were screened for binding to TBT and 9cisRA, and we found that, while 9cisRA can only bind RXR at physiological doses, environmental doses of TBT can activate RXR and NR4A receptors. (HR38). We have therefore identified a new target of TBT (NR4A) in the mussel which could also participate in the toxic mode of action of this endocrine disruptor in this marine invertebrate organism.

Task 1: We established the first comprehensive developmental transcriptome of the Mediterranean mussel (M. galloprovincialis) (published in Development, doi:10.1242/dev.202256). Furthermore, we established the complete mapping of the developmental expression of nuclear receptors during the development of the Mediterranean mussel (M. galloprovincialis) (Phil. Trans. B, doi:10.1098/rstb/379/1898). Task 2: We have established that TBT affects mussel development by binding the nuclear receptor RXR (publication in preparation). Task 4: We will begin this task in winter 2024 by developing transgenesis techniques in the mussel. This task also requires the finalization of task 2 to find target genes of nuclear receptors and to clone the regulatory regions of these target genes.

1. Miglioli A, Tredez M, Boosten M, Sant C, Carvalho JE, Dru P, Canesi L, Schubert M, Dumollard R (2024) The Mediterranean mussel, Mytilus galloprovincialis, a novel model for developmental studies in mollusks. Development doi: 10.1242/dev.202256.
2. Miglioli A, Fonseca A, Besnardeau L, Canesi L, Schubert M, Dumollard R (2024) First characterization of the nuclear receptor superfamily in the Mediterranean mussel Mytilus galloprovincialis: developmental expression dynamics and potential susceptibility to environmental chemicals. Philosophical Transactions of the Royal Society B doi: 10.1098/rstb/379/1898

3. Dumollard R, Miglioli A (2023) Toxicologie développementale sur les larves de moules : apport des méthodes d’immunohistochimie, d’hybridation in situ et d’imagerie pour l’étude fine des tissus, Revue Française d'Histotechnologie 35, 45-72.

Pervasive endocrine disrupting chemical (EDC) pollution is both a healthcare challenge and a threat to biodiversity. The impact of EDC pollution on humans, and other vertebrates, has been extensively studied, and numerous pathologies triggered by EDC exposure at different stages of life have been discovered. In contrast, apart from insects, very little is known about invertebrate endocrine systems, and virtually nothing is known about the toxicity of EDCs in invertebrates. Given that EDCs are found in plastics, personal care products and industrial commodities, vast amounts are released into the ocean by river outlets and wastewater influx, making EDC pollution also a growing problem for marine environments. Amongst invertebrates, mollusks are particularly sensitive to EDC exposure, which induces hermaphroditism in both gastropods and bivalves and causes phenotypes respectively referred to as imposex and intersex. The impact of EDCs is being addressed in ecotoxicology studies, which are generally based on either physiological or biochemical assays, but not on in vivo functional experiments, which has so far prevented the identification of the molecular modes of action of EDCs in marine invertebrates. There are, thus, currently massive gaps of knowledge in our understanding of EDC toxicology in marine invertebrates.
Nuclear receptors (NRs) are ligand-activated transcription factors crucial for regulating the vertebrate endocrine system, and their deregulation, by the binding of EDCs, is one of the leading causes of endocrine disruption in vertebrates. Given the importance of NR signaling in the vertebrate endocrine system, several standardized test systems used to detect endocrine disruption are based on measuring vertebrate NR activity. However, in invertebrates, virtually nothing is known about NR function and the roles of NR-dependent signaling in EDC toxicity. What has been shown is that the presence of a vertebrate NR homolog in an invertebrate is not automatically synonymous with the presence of a vertebrate-like signaling system downstream of this invertebrate NR. We are, thus, currently lacking even a basic understanding of the roles of NRs in endocrine disruption of invertebrates.
This project will remedy this shortcoming, as it will establish the planktonic embryos of the mussel M. galloprovincialis as a laboratory model system for studying NR functions and the roles they play in the toxicity of EDCs. More specifically, we propose to test the hypothesis that NR signaling mediates EDC-induced effects during development of the mussel M. galloprovincialis.
The project is divided into four tasks that will (1) yield detailed information on the expression of NRs during mussel development, both spatiotemporally and quantitatively, (2) compile a detailed characterization of EDC effects on mussel embryos and a list of molecular markers for EDC responses in mussels, (3) determine how genes affected by EDC treatments are regulated by NRs and (4) establish the roles played by NRs during mussel development as well as their contribution to endocrine disruption in mussels. Altogether, by creating novel techniques and resources for M. galloprovincialis, our proposed work will be the first to define NR functions and activities during mussel development and to demonstrate, in vivo, how EDCs modify these functions and activities, hence establishing the planktonic embryos of M. galloprovincialis as a potent study system bridging developmental biology and functional ecotoxicology. The molecular markers for EDC responses that we will identify in the course of this project, for example, will be extremely useful for marine monitoring programs and ecotoxicological surveys to detect endocrine disruption. Given that mussels are a sentinel species used around the world to survey marine environments, international monitoring programs, such as Mussel Watch, will thus benefit greatly from this project.