Unraveling the transcriptomic landscape of eye migration and visual adaptations during flatfish metamorphosis.

Flatfish undergo a remarkable metamorphosis from symmetrical pelagic larvae to fully asymmetrical benthic juveniles. The most distinctive features of this transformation is the migration of one eye. The molecular role of thyroid hormone in the metamorphosis process in flatfishes is well established. However, the regulatory network that facilitates eye movement remains enigmatic. This paper presents a morphological investigation of the metamorphic process in turbot eyes, using advanced imaging techniques and a global view of gene expression. The study covers migrant and non-migrant eyes and aims to identify the genes that are active during ocular migration. Our transcriptomic analysis shows a significant up-regulation of immune-related genes. The analysis of eye-specific genes reveals distinct patterns during the metamorphic process. Myosin is highlighted in the non-migrant eye, while ependymin is highlighted in the migrant eye, possibly involved in optic nerve regeneration. Furthermore, a potential association between the alx3 gene and cranial restructuring has been identified. Additionally, it confirmed simultaneous adaptation to low light in both eyes, as described by changes in opsins expression during the metamorphic process. The study also revealed that ocular migration activates systems asynchronously in both eyes, providing insight into multifaceted reorganization processes during metamorphosis of flatfish.

Genome-wide chromatin accessibility and gene expression profiling during flatfish metamorphosis.

Metamorphosis is a widely studied post-embryonic process in which many tissues undergo dramatic modifications to adapt to the new adult lifestyle. Flatfishes represent a good example of metamorphosis in teleost fishes. During metamorphosis of flatfish, organ regression and neoformation occur, with one of the most notable changes being the migration of one of the eyes to the other side of the body. In order to create a useful and reliable tool to advance the molecular study of metamorphosis in flatfish, we generated a chromatin accessible atlas as well as gene expression profile during four developmental stages ranging from a phylotypic to a post-metamorphic stage. We identified 29,019 differentially accessible chromatin regions and 3,253 differentially expressed genes. We found stage-specific regulatory regions and gene expression profiles, supporting the quality of the results. Our work provides strongly reproducible data for further studies to elucidate the regulatory elements that ensure successful metamorphosis in flatfish species.

Brains in Metamorphosis: Temporal Transcriptome Dynamics in Hatchery-Reared Flatfishes.

Metamorphosis is a captivating process of change during which the morphology of the larva is completely reshaped to face the new challenges of adult life. In the case of fish, this process initiated in the brain has traditionally been considered to be a critical rearing point and despite the pioneering molecular work carried out in other flatfishes, the underlying molecular basis is still relatively poorly characterized. Turbot brain transcriptome of three developmental stages (pre-metamorphic, climax of metamorphosis and post-metamorphic) were analyzed to study the gene expression dynamics throughout the metamorphic process. A total of 1570 genes were differentially expressed in the three developmental stages and we found a specific pattern of gene expression at each stage. Unexpectedly, at the climax stage of metamorphosis, we found highly expressed genes related to the immune response, while the biological pathway enrichment analysis in pre-metamorphic and post-metamorphic were related to cell differentiation and oxygen carrier activity, respectively. In addition, our results confirm the importance of thyroid stimulating hormone, increasing its expression during metamorphosis. Based on our findings, we assume that immune system activation during the climax of metamorphosis stage could be related to processes of larval tissue inflammation, resorption and replacement, as occurs in other vertebrates.

Dynamic changes in DNA methylation during seahorse (Hippocampus reidi) postnatal development and settlement.

INTRODUCTION: Most living marine organisms have a biphasic life cycle dependent on metamorphosis and settlement. These critical life-history events mean that a developmentally competent larva undergoes a range of coordinated morphological and physiological changes that are in synchrony with the ecological transition from a pelagic to a benthonic lifestyle. Therefore, transition from a pelagic to a benthonic habitat requires multiple adaptations, however, the underlying mechanisms regulating this process still remains unclear. Epigenetic regulation and specifically DNA methylation, has been suggested to be particularly important for organisms to adapt to new environments. Seahorses (Family Syngnathidae, Genus Hippocampus) are a fascinating group of fish, distinguished by their unique anatomical features, reproductive strategy and behavior. They are unique among vertebrate species due to their "male pregnancy", where males nourish developing embryos and larvae in a brood pouch until hatching and parturition occurs. After birth, free-swimming offspring are pelagic and subsequently they change into a demersal lifestyle. Therefore, to begin to address the question whether epigenetic processes could be involved in the transition from a planktonic to a benthonic lifestyle observed in seahorses, we studied global DNA methylation profiles in a tropical seahorse species (Hippocampus reidi) during postnatal development and settlement. RESULTS: We performed methylation-sensitive amplified polymorphism (MSAP) along with quantitative expression analysis for genes suggested to be involved in the methylation machinery at six age groups: 1, 5, 10, 20, 30 and 40 days after male's pouch release (DAR). Results revealed that the H. reidi genome has a significantly different DNA methylation profile during postnatal development and settlement on demersal habitats. Moreover, gene expression analysis showed up- and down-regulation of specific DNA methyltransferases (DNMTs) encoding genes. CONCLUSION: Our data show that the differences in the DNA methylation patterns seen among developmental stages and during the transition from a pelagic to a benthonic lifestyle suggest a potential for epigenetic regulation of gene expression (through DNA methylation) in this species. Therefore, epigenetic mechanisms could be necessary for seahorse settlement. Nevertheless, if these epigenetic mechanisms come from internal or if they are initiated via external environmental cues should be further investigated.

Author Correction: Functional analysis of new human Bardet-Biedl syndrome loci specific variants in the zebrafish model.

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

Functional analysis of new human Bardet-Biedl syndrome loci specific variants in the zebrafish model.

The multiple genetic approaches available for molecular diagnosis of human diseases have made possible to identify an increasing number of pathogenic genetic changes, particularly with the advent of next generation sequencing (NGS) technologies. However, the main challenge lies in the interpretation of their functional impact, which has resulted in the widespread use of animal models. We describe here the functional modelling of seven BBS loci variants, most of them novel, in zebrafish embryos to validate their in silico prediction of pathogenicity. We show that target knockdown (KD) of known BBS (BBS1, BB5 or BBS6) loci leads to developmental defects commonly associated with ciliopathies, as previously described. These KD pleiotropic phenotypes were rescued by co-injecting human wild type (WT) loci sequence but not with the equivalent mutated mRNAs, providing evidence of the pathogenic effect of these BBS changes. Furthermore, direct assessment of cilia located in Kupffer's vesicle (KV) showed a reduction of ciliary length associated with all the studied variants, thus confirming a deleterious effect. Taken together, our results seem to prove the pathogenicity of the already classified and unclassified new BBS variants, as well as highlight the usefulness of zebrafish as an animal model for in vivo assays in human ciliopathies.

Loss-of-function mutations in the melanocortin 1 receptor cause disruption of dorso-ventral countershading in teleost fish.

The melanocortin 1 receptor (MC1R) is the central melanocortin receptor involved in vertebrate pigmentation. Mutations in this gene cause variations in coat coloration in amniotes. Additionally, in mammals MC1R is the main receptor for agouti-signaling protein (ASIP), making it the critical receptor for the establishment of dorsal-ventral countershading. In fish, Mc1r is also involved in pigmentation, but it has been almost exclusively studied in relation to melanosome dispersion activity and as a putative genetic factor involved in dark/light adaptation. However, its role as the crucial component for the Asip1-dependent control of dorsal-ventral pigmentation remains unexplored. Using CRISPR/Cas9, we created mc1r homozygous knockout zebrafish and found that loss-of-function of mc1r causes a reduction of countershading and a general paling of the animals. We find ectopic development of melanophores and xanthophores, accompanied by a decrease in iridophore numbers in the ventral region of mc1r mutants. We also reveal subtle differences in the role of mc1r in repressing pigment cell development between the skin and scale niches in ventral regions.

Countershading in zebrafish results from an Asip1 controlled dorsoventral gradient of pigment cell differentiation.

Dorso-ventral (DV) countershading is a highly-conserved pigmentary adaptation in vertebrates. In mammals, spatially regulated expression of agouti-signaling protein (ASIP) generates the difference in shading by driving a switch between the production of chemically-distinct melanins in melanocytes in dorsal and ventral regions. In contrast, fish countershading seemed to result from a patterned DV distribution of differently-coloured cell-types (chromatophores). Despite the cellular differences in the basis for counter-shading, previous observations suggested that Agouti signaling likely played a role in this patterning process in fish. To test the hypotheses that Agouti regulated counter-shading in fish, and that this depended upon spatial regulation of the numbers of each chromatophore type, we engineered asip1 homozygous knockout mutant zebrafish. We show that loss-of-function asip1 mutants lose DV countershading, and that this results from changed numbers of multiple pigment cell-types in the skin and on scales. Our findings identify asip1 as key in the establishment of DV countershading in fish, but show that the cellular mechanism for translating a conserved signaling gradient into a conserved pigmentary phenotype has been radically altered in the course of evolution.

Stress, Glucocorticoids and Bone: A Review From Mammals and Fish.

Glucocorticoids (GCs) are the final effector products of a neuroendocrine HPA/HPI axis governing energy balance and stress response in vertebrates. From a physiological point of view, basal GC levels are essential for intermediary metabolism and participate in the development and homeostasis of a wide range of body tissues, including the skeleton. Numerous mammalian studies have demonstrated that GC hormones exert a positive role during bone modeling and remodeling as they promote osteoblastogenesis to maintain the bone architecture. Although the pharmacological effect of the so-called stress hormones has been widely reported, the role of endogenous GCs on bone mineral metabolism as result of the endocrine stress response has been largely overlooked across vertebrates. In addition, stress responses are variable depending on the stressor (e.g., starvation, predation, and environmental change), life cycle events (e.g., migration and aging), and differ among vertebrate lineages, which react differently according to their biological, social and cognitive complexity (e.g., mineral demands, physical, and psychological stress). This review intends to summarize the endogenous GCs action on bone metabolism of mammals and fish under a variety of challenging circumstances. Particular emphasis will be given to the regulatory loop between GCs and the parathyroid hormone (PTH) family peptides, and other key regulators of mineral homeostasis and bone remodeling in vertebrates.

PTH Reloaded: A New Evolutionary Perspective.

The parathyroid hormone (PTH) family is a group of structurally-related secreted peptides involved in bone mineral homeostasis and multitude of developmental processes in vertebrates. These peptides mediate actions through PTH receptors (PTHRs), which belong to the transmembrane G protein-coupled receptor group. To date, genes encoding for PTH and PTHR have only been identified in chordates, suggesting that this signaling pathway may be an evolutionary innovation of our phylum. In vertebrates, we found up to six PTH and three PTHR different paralogs, varying in number between mammals and teleost fishes due to the different rounds of whole-genome duplication and specific gene losses suffered between the two groups of animals. The diversification of the PTH gene family has been accompanied by both functional divergence and convergence, making sometimes difficult the comparison between PTH peptides of teleosts and mammals. Here, we review the roles of all Pth peptides in fishes, and based on the evolutionary history of PTH paralogs, we propose a new and simple nomenclature from PTH1 to PTH4. Moreover, the recent characterization of the Pth4 in zebrafish allows us to consider the prominent role of the brain-to-bone signaling pathway in the regulation of bone development and homeostasis. Finally, comparison between PTH peptides of fish and mammals allows us to discuss an evolutionary model for PTH functions related to bone mineral balance during the vertebrate transition from an aquatic to a terrestrial environment.

Targeted Pth4-expressing cell ablation impairs skeletal mineralization in zebrafish.

Skeletal development and mineralization are essential processes driven by the coordinated action of neural signals, circulating molecules and local factors. Our previous studies revealed that the novel neuropeptide Pth4, synthesized by hypothalamic cells, was involved in bone metabolism via phosphate regulation in adult zebrafish. Here, we investigate the role of pth4 during skeletal development using single-cell resolution, two-photon laser ablation of Pth4:eGFP-expressing cells and confocal imaging in vivo. Using a stable transgenic Pth4:eGFP zebrafish line, we identify Pth4:eGFP-expressing cells as post-mitotic neurons. After targeted ablation of eGFP-expressing cells in the hypothalamus, the experimental larvae exhibited impaired mineralization of the craniofacial bones whereas cartilage development was normal. In addition to a decrease in pth4 transcript levels, we noted altered expression of phex and entpd5, genes associated with phosphate homeostasis and mineralization, as well as a delay in the expression of osteoblast differentiation markers such as sp7 and sparc. Taken together, these results suggest that Pth4-expressing hypothalamic neurons participate in the regulation of bone metabolism, possibly through regulating phosphate balance during zebrafish development.

Promoter architecture and transcriptional regulation of musculoskeletal embryonic nuclear protein 1b (mustn1b) gene in zebrafish.

BACKGROUND: Mustn1 is a specific musculoskeletal protein that plays a critical role in myogenesis and chondrogenesis in vertebrates. Whole-mount in situ hybridization revealed that mustn1b mRNAs are specifically expressed in skeletal and cardiac muscles in Zebrafish embryos. However, the precise function and the regulatory elements required for its muscle-specific expression are largely unknown. RESULTS: The purpose of this study was to explore and uncover the target genomic regions that regulate mustn1b gene expression by in vivo functional characterization of the mustn1b promoter. We report here stable expression analyses of eGFP from fluorescent transgenic reporter Zebrafish line containing a 0.8kb_mustn1b-Tol2-eGFP construct. eGFP expression was specifically found in the skeletal and cardiac muscle tissues. We show that reporter Zebrafish lines generated replicate the endogenous mustn1b expression pattern in early Zebrafish embryos. Specific site directed-mutagenesis analysis revealed that promoter activity resides in two annotated genomic regulatory regions, each one corresponding to a specific functional transcription factor binding site. CONCLUSIONS: Our data indicate that mustn1b is specifically expressed in skeletal and cardiac muscle tissues and its muscle specificity is controlled by the 0.2-kb promoter and flanking sequences and in vivo regulated by the action of two sequence-specific families of transcription factors. Developmental Dynamics 246:992-1000, 2017. © 2017 Wiley Periodicals, Inc.

Fish pigmentation and the melanocortin system.

The melanocortin system is a complex neuroendocrine signaling mechanism involved in numerous physiological processes in vertebrates, including pigmentation, steroidogenesis and metabolic control. This review focuses at one of its most fascinating function in fish, its regulatory role in the control of pigmentation, in which the melanocortin 1 receptor (Mc1r), its agonist α-melanocyte stimulating hormone (α-Msh), and the endogenous antagonist agouti signaling protein (Asip1) are the main players. Functional control of Mc1r, which is highly expressed in fish skin and whose activation stimulates melanin production and melanosome dispersion in fish melanophores, is considered a key mechanism for vertebrate pigment phenotypes. The α-Msh peptide, the most documented Mc1r agonist involved in pigmentation, is produced in the pituitary gland, activating melanin synthesis by binding to Mc1r in fish melanophores. Finally, Asip1 is the putative factor for establishing the evolutionarily conserved dorso-ventral pigment pattern found across vertebrates. However, we are just starting to understand how other melanocortin system components are acting in this complex regulatory network.

Pth4, an ancient parathyroid hormone lost in eutherian mammals, reveals a new brain-to-bone signaling pathway.

Regulation of bone development, growth, and remodeling traditionally has been thought to depend on endocrine and autocrine/paracrine modulators. Recently, however, brain-derived signals have emerged as key regulators of bone metabolism, although their mechanisms of action have been poorly understood. We reveal the existence of an ancient parathyroid hormone (Pth)4 in zebrafish that was secondarily lost in the eutherian mammals' lineage, including humans, and that is specifically expressed in neurons of the hypothalamus and appears to be a central neural regulator of bone development and mineral homeostasis. Transgenic fish lines enabled mapping of axonal projections leading from the hypothalamus to the brainstem and spinal cord. Targeted laser ablation demonstrated an essential role for of pth4-expressing neurons in larval bone mineralization. Moreover, we show that Runx2 is a direct regulator of pth4 expression and that Pth4 can activate cAMP signaling mediated by Pth receptors. Finally, gain-of-function experiments show that Pth4 can alter calcium/phosphorus levels and affect expression of genes involved in phosphate homeostasis. Based on our discovery and characterization of Pth4, we propose a model for evolution of bone homeostasis in the context of the vertebrate transition from an aquatic to a terrestrial lifestyle.-Suarez-Bregua, P., Torres-Nuñez, E., Saxena, A., Guerreiro, P., Braasch, I., Prober, D. A., Moran, P., Cerda-Reverter, J. M., Du, S. J., Adrio, F., Power, D. M., Canario, A. V. M., Postlethwait, J. H., Bronner, M E., Cañestro, C., Rotllant, J. Pth4, an ancient parathyroid hormone lost in eutherian mammals, reveals a new brain-to-bone signaling pathway.

Matricellular protein SPARC/osteonectin expression is regulated by DNA methylation in its core promoter region.

BACKGROUND: SPARC/osteonectin is an evolutionarily conserved matricellular protein that modulates cell-matrix interaction and cell function. In all vertebrates, SPARC is dynamically expressed during embryogenesis. However, the precise function of SPARC and the regulatory elements required for its expression in particular during early embryogenesis are largely unknown. RESULTS: The present study was undertaken to explore the molecular mechanisms that regulate sparc gene expression by in vivo functional characterization of the sparc promoter and identification of possible putative regulatory elements that govern basal promoter activity. We report here transient expression analyses of eGFP expression from transgenic zebrafish containing a Sparc-iTol2-eGFP-BAC and/or 7.25 kb-sparc-Tol2-eGFP constructs. eGFP expression was specifically found in the notochord, otic vesicle, fin fold, intermediate cell mass, and olfactory placode of BAC and Tol2 transposon vectors injected embryos. Deletion analysis revealed that promoter activity resides in the unique 5'-untranslated intronic region. Computer-based analysis revealed a putative CpG island immediately proximal to the translation start site within the intron sequence. Global inhibition of methylation with 5-Aza-2-deoxycytidine promoted sparc expression in association with decreasing CpG methylation. CONCLUSIONS: Taken together, these data identify a contributory role for DNA methylation in regulating sparc expression in zebrafish embryogenesis.

Flow cytometric analysis to evaluate physiological alterations in herbicide-exposed Chlamydomonas moewusii cells.

Investigation of herbicide toxicology in non-target aquatic primary producers such as microalgae is of great importance from an ecological point of view. In order to study the toxicity of the widely used herbicide paraquat on freshwater green microalga Chlamydomonas moewusii, physiological changes associated with 96 h-exposures to this pollutant were monitored using flow cytometry (FCM) technique. Intracellular reactive oxygen species concentration, cytoplasmic membrane potential, metabolic activity and cell protein content were monitored to evaluate the toxicological impact of paraquat on algal physiology. Results showed that herbicide paraquat induced oxidative stress in C. moewusii cells, as it indicated the increase of both superoxide anion and hydrogen peroxide levels observed in non-chlorotic cells of cultures exposed to increasing herbicide concentrations. Furthermore, a progressive increase in the percentage of depolarised cells and a decrease in the metabolic activity level were observed in response to paraquat when non-chlorotic cells were analysed. Chlorotic cells were probably non-viable cells, based on the cytoplasmic membrane depolarisation, its metabolically non-active state and its drastically reduced protein content. In view of the obtained results, we have concluded that a range of significant physiological alterations, detected by flow cytometry, occur when C. moewusii, an ubiquitous microalga in freshwater environments, is challenged with environmentally relevant concentrations of the herbicide paraquat.