Alexandra Depince’s research while affiliated with Fish Physiology and Genomics Institute and other places

Chaperone-Mediated Autophagy (CMA) is a major pathway of lysosomal proteolysis involved in numerous cellular processes, and whose dysfunction is associated to several pathologies. Initially studied in mammals and birds, recent findings have identified CMA in fish, reshaping our understanding of its evolution across metazoans. Given the exciting perspectives this finding offered, we have now developed the required tools to investigate and functionally asses that CMA function in a powerful fish genetic model: the zebrafish (Danio rerio). After adapting and validating a fluorescent reporter (KFERQ-Dendra2; previously used to track CMA in mammalian cells) in zebrafish primary embryonic cells, we first demonstrated CMA functionality in this fish species. Then, we developed a transgenic zebrafish line expressing the KFERQ-Dendra2 CMA reporter, enabling the real-time tracking of CMA activity in vivo. This model revealed heterogeneous CMA responses within tissues, highlighting the zebrafish as a valuable model for investigating tissue-specific and cell-scale variations in CMA. Moreover, a novel role for CMA has been uncovered, acting as a gatekeeper of sperm cell proteostasis, thereby playing a crucial role in the production of active and high-quality spermatozoa. Overall, these findings emphasize the zebrafish as a pivotal model for advancing our comprehension of the fundamental mechanisms underlying CMA.

Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis critical for cellular homeostasis and metabolism, and whose defects have been associated with several human pathologies. While CMA has been well described in mammals, functional evidence has only recently been documented in fish, opening up new perspectives to tackle this function under a novel angle. Now we propose to explore CMA functions in the rainbow trout (RT, Oncorhynchus mykiss), a fish species recognized as a model organism of glucose intolerance and characterized by the presence of two paralogs of the CMA-limiting factor Lamp2A (lysosomal associated membrane protein 2A). To this end, we validated a fluorescent reporter (KFERQ-PA-mCherry1) previously used to track functional CMA in mammalian cells, in an RT hepatoma-derived cell line (RTH-149). We found that incubation of cells with high-glucose levels (HG, 25 mM) induced translocation of the CMA reporter to lysosomes and/or late endosomes in a KFERQ- and Lamp2A-dependent manner, as well as reduced its half-life compared to the control (5 mM), thus demonstrating increased CMA flux. Furthermore, we observed that activation of CMA upon HG exposure was mediated by generation of mitochondrial reactive oxygen species, and involving the antioxidant transcription factor Nfe2l2/Nrf2 (nfe2 like bZIP transcription factor 2). Finally, we demonstrated that CMA plays an important protective role against HG-induced stress, primarily mediated by one of the two RT Lamp2As. Together, our results provide unequivocal evidence for CMA activity existence in RT and highlight both the role and regulation of CMA during glucose-related metabolic disorders.Abbreviations: AREs: antioxidant response elements; CHC: α-cyano -4-hydroxycinnamic acid; Chr: chromosome; CMA: chaperone-mediated autophagy; CT: control; DMF: dimethyl fumarate; Emi: endosomal microautophagy; HG: high-glucose; HMOX1: heme oxygenase 1; H2O2: hydrogen peroxide; KFERQ: lysine-phenylalanine-glutamate-arginine-glutamine; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MCC: Manders' correlation coefficient; Manders' correlation coefficient Mo: morpholino oligonucleotide; NAC: N-acetyl cysteine; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; PA-mCherry: photoactivable mCherry; PCC: Pearson's correlation coefficient; ROS: reactive oxygen species; RT: rainbow trout; siRNAs: small interfering RNAs; SOD: superoxide dismutase; Tsg101: tumor susceptibility 101; TTFA: 2-thenoyltrifluoroacetone; WGD: whole-genome duplication.

Background: Encompassing about half of the 60,000 species of vertebrates, fish display the greatest diversity of sex determination mechanisms among metazoans. As such that phylum offers a unique playground to study the impressive variety of gonadal morphogenetic strategies, ranging from gonochorism, with either genetic or environmental sex determination, to unisexuality, with either simultaneous or consecutive hermaphroditism. Summary: From the two main types of gonads, the ovaries embrace the important role to produce the larger and non-motile gametes, which is the basis for the development of a future organism. The production of the egg cells is complex and involves the formation of follicular cells, which are necessary for the maturation of the oocytes and the production of feminine hormones. In this vein, our review focuses on the development of ovaries in fish with special emphasis on the germ cells, including those that transition from one sex to the other as part of their life cycle and those that are capable of transitioning to the opposite sex depending on environmental cues. Key messages: Clearly, establishing an individual as either a female or a male is not accomplished by the sole development of two types of gonads. In most cases, that dichotomy, be it final or transient, is accompanied by coordinated transformations across the entire organism, leading to changes in the physiological sex as a whole. These coordinated transformations require both molecular and neuroendocrine networks, but also anatomical and behavioural adjustments. Remarkably, fish managed to tame the ins and outs of sex reversal mechanisms to take the most advantages of changing sex as adaptive strategies in some situations.

Autophagy is a pleiotropic and evolutionarily conserved process in eukaryotes that encompasses different types of mechanisms by which cells deliver cytoplasmic constituents to the lysosome for degradation. Interestingly, in mammals, two different and specialized autophagic pathways, (i) the chaperone-mediated autophagy (CMA) and (ii) the endosomal microautophagy (eMI), both rely on the use of the same cytosolic chaperone HSPA8 (also known as HSC70) for targeting specific substrates to the lysosome. However, this is not true for all organisms, and differences exist between species with respect to the coexistence of these two autophagic routes. In this paper, we present an in-depth analysis of the evolutionary history of the main components of CMA and eMI and discuss how the observed discrepancies between species may contribute to improving our knowledge of these two functions and their interplays.

To date, more than 20 different vertebrate master sex-determining genes have been identified on different sex chromosomes of mammals, birds, frogs and fish. Interestingly, six of these genes are transcription factors ( Dmrt1 - or Sox3 - related) and 13 others belong to the TGF-β signalling pathway ( Amh , Amhr2 , Bmpr1b , Gsdf and Gdf6 ). This pattern suggests that only a limited group of factors/signalling pathways are prone to become top regulators again and again. Although being clearly a subordinate member of the sex-regulatory network in mammals, the TGF-β signalling pathway made it to the top recurrently and independently. Facing this rolling wave of TGF-β signalling pathways, this review will decipher how the TGF-β signalling pathways cope with the canonical sex gene regulatory network and challenge the current evolutionary concepts accounting for the diversity of sex-determining mechanisms. This article is part of the theme issue ‘Challenging the paradigm in sex chromosome evolution: empirical and theoretical insights with a focus on vertebrates (Part I)’.

Concepts of evolutionary biology suggest that morphological change may occur by rare punctual, but rather large changes, or by more steady and gradual transformations. It can therefore be asked whether genetic changes underlying morphological, physiological, and/or behavioral innovations during evolution occur in a punctual manner, whereby a single mutational event has prominent phenotypic consequences, or if many consecutive alterations in the DNA over longer time periods lead to phenotypic divergence. In the marine teleost, sablefish (Anoplopoma fimbria), complementary genomic and genetic studies led to the identification of a sex locus on the Y Chromosome. Further characterization of this locus resulted in identification of the transforming growth factor (tgfbr1a) gene, gonadal soma-derived factor (gsdf), as the main candidate for fulfilling the master sex determining (MSD) function. The presence of different X and Y Chromosome copies of this gene indicated that the male heterogametic (XY) system of sex determination in sablefish arose by allelic diversification. The gsdfY gene has a spatio-temporal expression profile characteristic of a male MSD gene. We provide experimental evidence demonstrating a pivotal role of a transposable element (TE) for the divergent function of gsdfY By insertion within the gsdfY promoter region, this TE generated allelic diversification by bringing cis-regulatory modules that led to transcriptional rewiring and thus creation of a new MSD gene. This points out for the first time in the scenario of MSD gene evolution by allelic diversification, a single, punctual molecular event in the appearance of a new trigger for male development.

Reducing the variability in nuclear transfer outcome requires a better understanding of its cellular and epigenetic determinants, in order to ensure safer fish regeneration from cryobanked somatic material. In this work, clones from goldfish were obtained using cryopreserved fin cells as donor and non-enucleated oocytes as recipients. We showed that the high variability of clones survival was not correlated to spawn quality. Clones were then characterized for their first cleavages pattern in relation to their developmental fate up to hatching. The first cell cycle duration was increased in clones with abnormal first cleavage, and symmetric first two cleavages increased clone probability to reach later on 24 h- and hatching-stages. At 24 h-stage, 24% of the clones were diploids and from donor genetic origin only. However, ploidy and genetic origin did not determine clones morphological quality. DNA methylation reprogramming in the promoter region of pou2, nanog, and notail marker genes was highly variable, but clones with the nicest morphologies displayed the best DNA methylation reprogramming. To conclude, non-enucleated oocytes did allow authentic clones production. The first two cell cycles were a critical determinant of the clone ability to reach hatching-stage, and DNA methylation reprogramming significantly influenced clones morphological quality.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Highly differentiated mature spermatozoa carry not only genetic but also epigenetic information that is to be transmitted to the embryo. DNA methylation is one epigenetic actor associated with sperm nucleus compaction, gene silencing, and prepatterning of embryonic gene expression. Therefore, the stability of this mark toward reproductive biotechnologies is a major issue in animal production. The present work explored the impact of hormonal induction of spermiation and sperm cryopreservation in two cyprinids, the goldfish (Carassius auratus) and the zebrafish (Danio rerio), using LUminometric Methylation Assay (LUMA). We showed that while goldfish hormonal treatment did increase sperm production, it did not alter global DNA methylation of spermatozoa. Different sperm samples repeatedly collected from the same males for 2 months also showed the same global DNA methylation level. Similarly, global DNA methylation was not affected after cryopreservation of goldfish spermatozoa with methanol, whereas less efficient cryoprotectants (dimethylsulfoxide and 1,2‐propanediol) decreased DNA methylation. In contrast, cryopreservation of zebrafish spermatozoa with methanol induced a slight, but significant, increase in global DNA methylation. In the less compact nuclei, that is, goldfish fin somatic cells, cryopreservation did not change global DNA methylation regardless of the choice of cryoprotectant. To conclude, global DNA methylation is a robust parameter with respect to biotechnologies such as hormonal induction of spermiation and sperm cryopreservation, but it can be altered when the best sperm manipulation conditions are not met.

Nuclear transfer consists in injecting a somatic nucleus carrying valuable genetic information into a recipient oocyte to sire a diploid offspring which bears the genome of interest. It requires that the oocyte (maternal) DNA is removed. In fish, because enucleation is difficult to achieve, non-enucleated oocytes are often used and disappearance of the maternal DNA was reported in some clones. The present work explores which cellular events explain spontaneous erasure of maternal DNA, as mastering this phenomenon would circumvent the painstaking procedure of fish oocyte enucleation. The fate of the somatic and maternal DNA during oocyte activation and first cell cycle was studied using DNA labeling and immunofluorescence in goldfish clones. Maternal DNA was always found as an intact metaphase within the oocyte, and polar body extrusion was minimally affected after oocyte activation. During the first cell cycle, only 40% of the clones displayed symmetric cleavage, and these symmetric clones contributed to 80% of those surviving at hatching. Maternal DNA was often fragmented and located under the cleavage furrow. The somatic DNA was organized either into a normal mitotic spindle or abnormal multinuclear spindle. Scenarios matching the DNA behavior and the embryo fate are proposed.