Understanding microRNA (miRNA) functions has been hampered by major difficulties in identifying their biological target(s). Currently, the main limitation is the lack of a suitable strategy to identify biologically relevant targets among a high number of putative targets. Here we provide a proof of concept of successful de novo (i.e. without prior knowledge of its identity) miRNA phenotypic target (i.e. target whose de-repression contributes to the phenotypic outcomes) identification from RNA-seq data. Using the medaka mir-202 knock-out (KO) model in which inactivation leads to a major organism-level reproductive phenotype, including reduced egg production, we introduced novel criteria including limited fold-change in KO and low interindividual variability in gene expression to reduce the list of 2853 putative targets to a short list of 5. We selected tead3b, a member of the evolutionarily-conserved Hippo pathway, known to regulate ovarian functions, due to its remarkably strong and evolutionarily conserved binding affinity for miR-202-5p. Deleting the miR-202-5p binding site in the 3' UTR of tead3b, but not of other Hippo pathway members sav1 and vgll4b, triggered a reduced egg production phenotype. This is one of the few successful examples of de novo functional assignment of a miRNA phenotypic target in vivo in vertebrates.

The expansion of the neocortex, a hallmark of mammalian evolution1,2, was accompanied by an increase in cerebellar neuron numbers3. However, little is known about the evolution of the cellular programmes underlying the development of the cerebellum in mammals. In this study we generated single-nucleus RNA-sequencing data for around 400,000 cells to trace the development of the cerebellum from early neurogenesis to adulthood in human, mouse and the marsupial opossum. We established a consensus classification of the cellular diversity in the developing mammalian cerebellum and validated it by spatial mapping in the fetal human cerebellum. Our cross-species analyses revealed largely conserved developmental dynamics of cell-type generation, except for Purkinje cells, for which we observed an expansion of early-born subtypes in the human lineage. Global transcriptome profiles, conserved cell-state markers and gene-expression trajectories across neuronal differentiation show that cerebellar cell-type-defining programmes have been overall preserved for at least 160 million years. However, we also identified many orthologous genes that gained or lost expression in cerebellar neural cell types in one of the species or evolved new expression trajectories during neuronal differentiation, indicating widespread gene repurposing at the cell-type level. In sum, our study unveils shared and lineage-specific gene-expression programmes governing the development of cerebellar cells and expands our understanding of mammalian brain evolution.

The Percidae family comprises many fish species of major importance for aquaculture and fisheries. Based on three new chromosome-scale assemblies in Perca fluviatilis , Perca schrenkii and Sander vitreus along with additional percid fish reference genomes, we provide an evolutionary and comparative genomic analysis of their sex-determination systems. We explored the fate of a duplicated anti-Mullerian hormone receptor type-2 gene ( amhr2bY ), previously suggested to be the master sex determining (MSD) gene in P. flavescens . Phylogenetically related and structurally similar a mhr2 duplications ( amhr2b ) were found in P. schrenkii and Sander lucioperca , potentially dating this duplication event to their last common ancestor around 19-27 Mya. In P. fluviatilis and S. vitreus , this amhr2b duplicate has been lost while it was subject to amplification in S. lucioperca . Analyses of the amhr2b locus in P. schrenkii suggest that this duplication could be also male-specific as it is in P. flavescens . In P. fluviatilis , a relatively small (100 kb) non-recombinant sex-determining region (SDR) was characterized on chromosome-18 using population-genomics approaches. This SDR is characterized by many male-specific single-nucleotide variants (SNVs) and no large duplication/insertion event, suggesting that P. fluviatilis has a male heterogametic sex determination system (XX/XY), generated by allelic diversification. This SDR contains six annotated genes, including three ( c18h1orf198 , hsdl1 , tbc1d32 ) with higher expression in testis than ovary. Together, our results provide a new example of the highly dynamic sex chromosome turnover in teleosts and provide new genomic resources for Percidae, including sex-genotyping tools for all three known Perca species.

Spermatozoa are the cells that are most commonly used for cryopreservation of valuable genetic resources in aquaculture. It is known that fish spermatozoa transmit to the embryo not only their genetic but also their epigenetic profile, especially DNA methylation. Therefore, any alteration of the DNA methylation profile in spermatozoa induces the risk of transmitting epigenetic alterations to the offspring. The aim of this study was to assess the effect of cryopreservation on DNA methylation in rainbow trout spermatozoa. To trigger variable cellular response after freezing–thawing, spermatozoa from mature males were cryopreserved with dimethyl sulfoxide, methanol or glycerol as cryoprotectant. We observed that dimethyl sulfoxide was the best to preserve thawed spermatozoa functions. Methanol only slightly preserved all the cellular parameters, while glycerol failed to protect motility and fertilization ability. The consequences on DNA methylation were assessed using Reduced Representation Bisulfite Sequencing (RRBS). Sperm cryopreservation did not thoroughly impact DNA methylation, although 335–564 differentially methylated cytosines were characterized depending on the cryoprotectant. Very few of them were shared between cryoprotectants, and no correlation with the extent of cellular damage was found. Our study showed that DNA methylation was only slightly altered after sperm cryopreservation, and this may render further analysis of the risk for the progeny very challenging.

The specific functions and essentiality of type II vitellogenin (Vtg2) in early zebrafish development were investigated in this study. A vtg2-mutant zebrafish line was produced and effects of genomic disturbance were observed in F2 females and F3 offspring. No change in vtg2 transcript has been detected, however, Vtg2 abundance in F2 female liver was 5×, and in 1 hpf F3 vtg2-mutant embryos was 3.8× less than Wt (p < 0.05). Fecundity was unaffected while fertilization rate was more than halved in F2 vtg2-mutant females (p < 0.05). Hatching rate was significantly higher in F3 vtg2-mutant embryos in comparison to Wt embryos. Survival rate declined drastically to 29% and 18% at 24 hpf and 20 dpf, respectively, in F3 vtg2-mutant embryos. The introduced mutation caused vitelline membrane deficiencies, significant mortalities at early embryonic stages, and morphological abnormalities in the surviving F3 vtg2-mutant larvae. Overrepresentation of histones, zona pellucida proteins, lectins, and protein degradation related proteins in F3 vtg2-mutant embryos provide evidence to impaired mechanisms involved in vitellin membrane formation. Overall findings imply a potential function of Vtg2 in acquisition of vitellin membrane integrity, among other reproductive functions, and therefore, its essentiality in early zebrafish embryo development.

Replacing fishmeal with alternative protein sources and improving new ingredients diets with feed additives are major objectives in aquaculture. The aim of this study was to evaluate benefits for rainbow trout (Oncorhynchus mykiss) of supplementing a fishmeal-free diet, composed of processed animal proteins (dehydrated poultry protein, hydrolyzed feather meal, poultry and pork blood meal), with yeast extract. Juvenile rainbow trout (initial weight 37 ± 2 g) were fed either with a control diet (19% fishmeal) or with a diet based on terrestrial animal by-products (17%) supplemented or not with 3% of yeast extract. Effects of the diets were evaluated in a 4-week digestibility trial and a 12-week growth experiment. Fish health was investigated by measuring plasma immune markers and performing histological study of the gut. Underlying molecular responses were investigated using unbiased transcriptomic analysis of the liver and distal intestine. Results indicated that supplementing with 3% yeast extract did not influence nutrient digestibility substantially. Nevertheless, fish fed the yeast supplemented fishmeal-free diet grew more than those fed the non-supplemented processed animal protein diet (final body weight of 206.7 ± 14.2 g vs 182.3 ± 13.5 g) but less than their counterparts fed the fishmeal diet (230.5 ± 7.6 g). Plasma and structural parameters indicated no exacerbated immune response or signs of intestinal inflammation in fish fed the fishmeal-free diets. However, plasma total immunoglobulin M levels and intestinal villi were significantly higher in fish fed the diet supplemented with yeast extract. The transcriptomic analysis revealed that the diets influenced immune, inflammatory, pathogen fighting and coagulation gene-related expressions. These results suggest that the dietary inclusion of yeast can enhance a fishmeal-free diet by improving rainbow trout performances and potentially their robustness.

ABSTRACT 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.

AbstractIn France, rainbow trout (Oncorhynchus mykiss) farming traditionally used flow-through systems, which raised concerns about environmental impacts, including limited freshwater availability, and the use of ingredients from intensive agriculture and fishing. To address the growing demand for sustainable food products, there is an increasing interest in organic aquaculture. In this study, we employed an attributional life cycle assessment (LCA) to analyse the environmental impacts of rainbow trout production. We simulated conventional and organic production practices in a hypothetical fish farm to evaluate the differences in environmental impacts at the farm level. The potential impacts were calculated using a product-based functional unit (one tonne of trout) under the two production scenarios and were also expressed using a surface-based functional unit (m2y). Our life cycle impact assessment revealed that organic farming significantly reduced environmental impacts per tonne of trout in seven out of the nine selected impact categories. Notably, freshwater ecotoxicity exhibited the greatest difference, with organic systems showing a 55% decrease. The only exceptions were freshwater eutrophication and water dependence, where organic production led to higher impacts per tonne of trout. In conventional farming, emissions amounted to 14 kg of P eq./tonne, whereas in organic farming, the emissions were slightly higher (15 kg of P eq./tonne). For water dependence, one tonne of trout production in the conventional system mobilized 128 103m3vs. 185 103m3in the organic system. The environmental benefits of organic production were even more marked when using a surface-based functional unit (m2y). We demonstrated the benefits of organic trout production from an environmental perspective. However, our findings highlight the caution needed when interpreting LCA comparisons of such production systems that can be highly influenced by methodological choices such as the functional unit used.

The vertebrate brain emerged more than ~500 million years ago in common evolutionary ancestors. To systematically trace its cellular and molecular origins, we established a spatially resolved cell type atlas of the entire brain of the sea lamprey—a jawless species whose phylogenetic position affords the reconstruction of ancestral vertebrate traits—based on extensive single-cell RNA-seq and in situ sequencing data. Comparisons of this atlas to neural data from the mouse and other jawed vertebrates unveiled various shared features that enabled the reconstruction of cell types, tissue structures and gene expression programs of the ancestral vertebrate brain. However, our analyses also revealed key tissues and cell types that arose later in evolution. For example, the ancestral brain was probably devoid of cerebellar cell types and oligodendrocytes (myelinating cells); our data suggest that the latter emerged from astrocyte-like evolutionary precursors in the jawed vertebrate lineage. Altogether, our work illuminates the cellular and molecular architecture of the ancestral vertebrate brain and provides a foundation for exploring its diversification during evolution.