Fish Physiology and Genomics Institute

ABSTRACT Different adipose tissues (AT) have been described, including subcutaneous and visceral tissues (SCAT and VAT). They display different morphological structures, physiological and metabolic functions. Imaging adipocytes in the whole AT was not feasible because of the large adipocyte sizes and the lipid-full content of the droplets that increased the refractive index. Tissue clearing is then required mainly through a delipidation step, which induces also a tissue shrinkage. Our aim was to image in 3D freshly extracted adipocytes and compare them to those within their tissues. Trout ATs were stained with 5DTAF (extracellular matrix) and Nile Red (lipids). After clearing with Histodenz, 3D images were obtained using a confocal microscope, and adipocytes were segmented and measured. In situ, major differences in adipocyte size and shape were observed between the VAT and SCAT. Ex situ, only the size mattered because all cells were round outside their tissues. This method can be applied to other species, such as mice. In situ, adipocyte sphericity was even higher in the SCAT from a Swiss and a C57Bl6. This approach demonstrates that 3D adipocyte imaging with lipid labeling enables accurate morphological characterization, provides insights into depot-specific structural features, and supports optimization of cell isolation protocols

Abstract Interindividual epigenetic variability, particularly in DNA methylation, is now recognized as a significant contributor to phenotypic diversity in humans and mammals. These epivariable regions, which make up a small fraction of the genome, are strongly influenced by genetic factors and environmental factors, especially during early development. In this context, epigenetic variability of DNA methylation has been proposed as an adaptive force involved in various environmental responses. In fish and other vertebrates, environmental factors are known to influence the health, performance and welfare, likely through the alteration of the epigenetic landscape. However, whether interindividual epigenetic variability may contribute to the phenotypic plasticity of fishes is unknown. Here we provide a first description of the rainbow trout methylome variability using a whole-genome bisulfite sequencing approach in an isogenic line to minimize genetic variation. Variable methylation regions were identified in both liver and hypothalamus tissues of 12 replicate fishes and were found enriched at gene regulatory elements, such as promoters and first introns. Gene Ontology analysis revealed functional clusters related to cellular development, neural communication, metabolic balance, and immune response. Interestingly, some variably methylated regions are found at the same genomic loci in both tissues and showed a strong intraindividual correlation in methylation levels, suggesting establishment during early embryogenesis. Overall, our work demonstrates the existence of interindividual epigenetic variability in rainbow trout and provides valuable insights into the regulatory function of DNA methylation variation that is likely involved in developmental and physiological processes.

Abstract It has been empirically established that genome mixing between divergent species can trigger meiotic aberrations, ultimately leading to the emergence of asexual reproduction through the production of unreduced gametes in various metazoan lineages. Yet, it remains poorly understood how such asexual hybrids cope with co-inherited differences in sex determination systems, diverged regulatory networks, and chromosomal incompatibilities— especially in the context of increased ploidy. Addressing these questions requires high-quality, chromosome-level reference genomes of the parental species involved in hybrid formation. Here, we present the first chromosome-level genome assemblies for three hybridizing Cobitis species ( C. elongatoides , C. taenia , and C. tanaitica ), providing a comprehensive framework to investigate the genetic and cytogenetic basis of hybrid sterility and the transition to asexuality. By integrating genome scaffolding, male/female pooled sequencing, and molecular cytogenetics, we uncover extensive structural variation among homologous chromosomes of the three species, despite their overall syntenic conservation. Population-level Pool-Seq analyses further revealed that each species possesses a distinct, non-homologous sex chromosome, highlighting sex chromosome turnover even among recently diverged lineages. These assemblies enabled the design of chromosome-specific painting probes, which we applied to meiotic metaphase I spreads of diploid hybrids. This approach revealed striking differences in the pairing success of orthologous chromosomes, with some (e.g., Ch01B) frequently forming bivalents, while others (e.g., Ch01A, Ch05, Ch20) failed to do so and remained unpaired. Our results demonstrate that chromosome-specific features, shaped by structural evolution and sex-linked divergence, contribute unequally to hybrid meiotic failure. Together, this work provides a high-resolution genomic and cytogenetic framework to understand how interspecific hybridization gives rise to clonality, and how the architecture of inherited parental genomes shapes the success or breakdown of meiosis in hybrid vertebrates.

Although sex determination is a fundamental process in vertebrate development, it is very plastic. Diverse genes became major sex determinants in teleost fishes. Deciphering how individual sex-determining genes orchestrate sex determination can reveal new actors in sexual development. Here, we demonstrate that the Y-chromosomal copy of the TGF-β family member gdf6 (gdf6Y) in Nothobranchius furzeri, an emerging model organism in aging research, gained the function of the male sex determinant through allelic diversification while retaining the skeletal developmental function shared with the X-chromosomal gdf6 allele (gdf6X). Concerning sex determination, gdf6Y is expressed by somatic supporting cells of the developing testes. There it induces the male sex in a germ cell-independent manner in contrast to sex determination in zebrafish and the medaka. Looking for downstream effectors of Gdf6Y, we identified besides TGF-β signaling modulators, especially the inhibitor of DNA binding genes id1/2/3, the mRNA decay activator zfp36l2 as a new GDF6 signaling target.

ABSTRACTTeleost fish represent one of the largest and most diverse clades of vertebrates, which makes them great models in various research areas such as ecology and evolution. Recent sequencing endeavors provided high-quality genomes for species covering the main fish evolutionary lineages, opening up large-scale comparative genomics studies. However, transcriptomic data across fish species and organs are heterogenous and have not been integrated with newly sequenced genomes making gene expression quantification and comparative analyses particularly challenging. Thus, resources integrating genomic and transcriptomic data across fish species and organs are still lacking. Here, we present FEVER, a web-based resource allowing evolutionary transcriptomics across species and tissues. First, based on query genes FEVER reconstructs gene trees providing orthologous and paralogous relationships as well as their evolutionary dynamics across 13 species covering the major fish lineages, and 4 model species as evolutionary outgroups. Second, it provides unbiased gene expression across 11 tissues using up-to-date fish genomes. Finally, genomic and transcriptomic data are combined together allowing the exploration of gene expression evolution following speciation and duplication events. FEVER is freely accessible athttps://fever.sk8.inrae.fr/.

Functions of vitellogenins have been in the limelight of fish reproductive physiology research for decades. The Vtg system of acanthomorph teleosts consists of two complete forms of Vtgs (VtgAa and VtgAb) and an incomplete form, VtgC. Insufficient uptake and processing of Vtgs and their yolk proteins lead to inadequate oocyte hydration ensuing failure in acquisition of egg buoyancy and early developmental deficiencies. This review presents a summary of our studies on utilization of multiple Vtgs in species with different egg buoyancy characteristics, as examples. Studies of moronids revealed limited degradation of all three forms of lipovitellin heavy chain derived from their three respective forms of Vtg, by which they contribute to the free amino acid pool driving oocyte hydration during oocyte maturation. In later studies, CRISPR/Cas9 was employed to invalidate zebrafish type I, type II and type III Vtgs, which are orthologs of acanthamorph VtgAa, VtgAb and VtgC, respectively. Results revealed type I Vtg to have essential developmental and nutritional functions in both late embryos and larvae. Genomic disturbance of type II Vtg led to high mortalities during the first 24 h of embryonic development. Despite being a minor form of Vtg in zebrafish and most other species, type III Vtg was also found to contribute essentially to the developmental potential of zebrafish zygotes and early embryos. Apart from severe effects on progeny survival, these studies also disclosed previously unreported regulatory effects of Vtgs on fecundity and fertility, and on embryo hatching. We recently utilized parallel reactions monitoring based liquid chromatography tandem mass spectrometry to assess the processing and utilization of lipovitellins derived from different forms of Vtg in Atlantic halibut and European plaice. Results showed the Lv heavy chain of VtgAa (LvHAa) to be consumed during oocyte maturation and the Lv light chain of VtgAb (LvLAb) to be utilized specifically during late larval stages, while all remaining YPs (LvLAa, LvHAb, LvHC, and LvLC) were utilized during or after hatching up until first feeding in halibut. In plaice, all YPs except LvHAa, which similarly to halibut supports oocyte maturation, are utilized from late embryo to late larval development up until first feeding. The collective findings from these studies affirm substantial disparity in modes of utilization of different types of Vtgs among fish species with various egg buoyancy characteristics, and they reveal previously unknown regulatory functions of Vtgs in maintenance of reproductive assets such as maternal fecundity and fertility, and in embryonic hatching. Despite the progress that has been made over the past two decades by examining multiple Vtgs and their functions, a higher complexity of these systems with much greater diversity between species in modes of Vtg utilization is now evident. Further research is needed to reveal novel ways each species has evolved to utilize these complex multiple Vtg systems and to discover unifying principles for this evolution in fishes of diverse lineages, habitats and life history characteristics.