Ancient vertebrate dermal armor evolved from trunk neural crest.

Bone is an evolutionary novelty of vertebrates, likely to have first emerged as part of ancestral dermal armor that consisted of osteogenic and odontogenic components. Whether these early vertebrate structures arose from mesoderm or neural crest cells has been a matter of considerable debate. To examine the developmental origin of the bony part of the dermal armor, we have performed in vivo lineage tracing in the sterlet sturgeon, a representative of nonteleost ray-finned fish that has retained an extensive postcranial dermal skeleton. The results definitively show that sterlet trunk neural crest cells give rise to osteoblasts of the scutes. Transcriptional profiling further reveals neural crest gene signature in sterlet scutes as well as bichir scales. Finally, histological and microCT analyses of ray-finned fish dermal armor show that their scales and scutes are formed by bone, dentin, and hypermineralized covering tissues, in various combinations, that resemble those of the first armored vertebrates. Taken together, our results support a primitive skeletogenic role for the neural crest along the entire body axis, that was later progressively restricted to the cranial region during vertebrate evolution. Thus, the neural crest was a crucial evolutionary innovation driving the origin and diversification of dermal armor along the entire body axis.

Genome-wide comparative methylation analysis reveals the fate of germ stem cells after surrogate production in teleost.

BACKGROUND: Surrogate production by germline stem cell transplantation is a powerful method to produce donor-derived gametes via a host, a practice known as surrogacy. The gametes produced by surrogates are often analysed on the basis of their morphology and species-specific genotyping, which enables conclusion to be drawn about the donor's characteristics. However, in-depth information, such as data on epigenetic changes, is rarely acquired. Germ cells develop in close contact with supporting somatic cells during gametogenesis in vertebrates, and we hypothesize that the recipient's gonadal environment may cause epigenetic changes in produced gametes and progeny. Here, we extensively characterize the DNA methylome of donor-derived sperm and their intergenerational effects in both inter- and intraspecific surrogates. RESULTS: We found more than 3000 differentially methylated regions in both the sperm and progeny derived from inter- and intraspecific surrogates. Hypermethylation in the promoter regions of the protocadherin gamma gene in the intraspecific surrogates was found to be associated with germline transmission. On the contrary, gene expression level and the embryonic development of the offspring remained unaffected. We also discovered MAPK/p53 pathway disruption in interspecific surrogates due to promoter hypermethylation and identified that the inefficient removal of meiotic-arrested endogenous germ cells in hybrid gonads led to the production of infertile spermatozoa. CONCLUSIONS: Donor-derived sperm and progeny from inter- and intraspecific surrogates were more globally hypermethylated than those of the donors. The observed changes in DNA methylation marks in the surrogates had no significant phenotypic effects in the offspring.

Evolutionary conservation of maternal RNA localization in fishes and amphibians revealed by TOMO-Seq.

Asymmetrical localization of biomolecules inside the egg, results in uneven cell division and establishment of many biological processes, cell types and the body plan. However, our knowledge about evolutionary conservation of localized transcripts is still limited to a few models. Our goal was to compare localization profiles along the animal-vegetal axis of mature eggs from four vertebrate models, two amphibians (Xenopus laevis, Ambystoma mexicanum) and two fishes (Acipenser ruthenus, Danio rerio) using the spatial expression method called TOMO-Seq. We revealed that RNAs of many known important transcripts such as germ layer determinants, germ plasm factors and members of key signalling pathways, are localized in completely different profiles among the models. It was also observed that there was a poor correlation between the vegetally localized transcripts but a relatively good correlation between the animally localized transcripts. These findings indicate that the regulation of embryonic development within the animal kingdom is highly diverse and cannot be deduced based on a single model.

Clonal gametogenesis is triggered by intrinsic stimuli in the hybrid's germ cells but is dependent on sex differentiation†.

Interspecific hybridization may trigger the transition from sexual reproduction to asexuality, but mechanistic reasons for such a change in a hybrid's reproduction are poorly understood. Gametogenesis of many asexual hybrids involves a stage of premeiotic endoreplication (PMER), when gonial cells duplicate chromosomes and subsequent meiotic divisions involve bivalents between identical copies, leading to production of clonal gametes. Here, we investigated the triggers of PMER and whether its induction is linked to intrinsic stimuli within a hybrid's gonial cells or whether it is regulated by the surrounding gonadal tissue. We investigated gametogenesis in the Cobitis taenia hybrid complex, which involves sexually reproducing species (Cobitis elongatoides and C. taenia) as well as their hybrids, where females reproduce clonally via PMER while males are sterile. We transplanted spermatogonial stem cells (SSCs) from C. elongatoides and triploid hybrid males into embryos of sexual species and of asexual hybrid females, respectively, and observed their development in an allospecific gonadal environment. Sexual SSCs underwent regular meiosis and produced normally reduced gametes when transplanted into clonal females. On the other hand, the hybrid's SSCs lead to sterility when transplanted into sexual males but maintained their ability to undergo asexual development (PMER) and production of clonal eggs, when transplanted into sexual females. This suggests that asexual gametogenesis is under complex control when somatic gonadal tissue indirectly affects the execution of asexual development by determining the sexual differentiation of stem cells and once such cells develop to female phenotypes, hybrid germ cells trigger the PMER from their intrinsic signals.

Transcriptome and Proteome Analyses Reveal Stage-Specific DNA Damage Response in Embryos of Sturgeon (Acipenser ruthenus).

DNA damage during early life stages may have a negative effect on embryo development, inducing mortality and malformations that have long-lasting effects during adult life. Therefore, in the current study, we analyzed the effect of DNA damage induced by genotoxicants (camptothecin (CPT) and olaparib) at different stages of embryo development. The survival, DNA fragmentation, transcriptome, and proteome of the endangered sturgeon Acipenser ruthenus were analyzed. Sturgeons are non-model fish species that can provide new insights into the DNA damage response and embryo development. The transcriptomic and proteomic patterns changed significantly after exposure to genotoxicants in a stage-dependent manner. The results of this study indicate a correlation between phenotype formation and changes in transcriptomic and proteomic profiles. CPT and olaparib downregulated oxidative phosphorylation and metabolic pathways, and upregulated pathways involved in nucleotide excision repair, base excision repair, and homologous recombination. We observed the upregulated expression of zona pellucida sperm-binding proteins in all treatment groups, as well as the upregulation of several glycolytic enzymes. The analysis of gene expression revealed several markers of DNA damage response and adaptive stress response, which could be applied in toxicological studies on fish embryos. This study is the first complex analysis of the DNA damage response in endangered sturgeons.

Brief communication: rapid elimination of egg stickiness using sodium hypochlorite in wels catfish Silurus glanis.

The wels catfish Silurus glanis is valuable fish for aquaculture. Its production relies mainly on artificial reproduction. One of the crucial steps determining success of the reproduction is elimination of egg stickiness after fertilization. To date, the catfish egg de-adhesion is usually carried out using proteolytic enzymes. Here, we prove a novel method based on oxidation of the egg surface by means of sodium hypochlorite. An effect of different exposure times and concentrations on the egg adhesiveness and damage was tested in the first trial. The selected concentration of sodium hypochlorite 0.3 mg · l-1 with exposure time 40 s was used for comparison with the conventical de-adhesion method using alcalase treatment. The fertilization and hatching rates reached very satisfactory outcome in both treatments (98.3 ± 0.7% vs 97.5 ± 2.2% and 86.6 ± 8.3% vs 91.3 ± 8.5% in alcalase- and sodium hypochlorite-treated embryos, respectively) without any statistical differences. Thus, the de-adhesion method using sodium hypochlorite can be recommended as a suitable method for wels catfish eggs. The method is simple, cheap, very fast, and the treated eggs are disinfected.

Dead-end (dnd) protein in fish-a review.

Dead end (dnd) is a germ plasm-specific maternal RNA discovered in zebrafish and then in other vertebrates. Dnd protein is essential for migration and motility of primordial germ cells (PGCs), only cells destined to transfer genetic information to offspring. PGCs arise far from somatic cells of developing gonads and they must migrate to their site of function. Migration of PGCs follows complex path by various developing tissues as their disruption impacts on the fertility. Recently, it has been found that dnd is not required for survival of PGCs and dnd-deficient zebrafish PGCs transdifferentiate into the somatic cells. In fish, targeting dnd causes removal of PGCs that ultimately affects sex differentiation. Sterility in various fish species can be achieved by knockdown or knockout of dnd. In our review, we have discussed dnd as a germ cell-specific molecular marker in fish, its interaction with miRNAs, and its use in aquaculture and fish conservation.

Ancient Sturgeons Possess Effective DNA Repair Mechanisms: Influence of Model Genotoxicants on Embryo Development of Sterlet, Acipenser ruthenus.

DNA damage caused by exogenous or endogenous factors is a common challenge for developing fish embryos. DNA damage repair (DDR) pathways help organisms minimize adverse effects of DNA alterations. In terms of DNA repair mechanisms, sturgeons represent a particularly interesting model due to their exceptional genome plasticity. Sterlet (Acipenser ruthenus) is a relatively small species of sturgeon. The goal of this study was to assess the sensitivity of sterlet embryos to model genotoxicants (camptothecin, etoposide, and benzo[a]pyrene), and to assess DDR responses. We assessed the effects of genotoxicants on embryo survival, hatching rate, DNA fragmentation, gene expression, and phosphorylation of H2AX and ATM kinase. Exposure of sterlet embryos to 1 µM benzo[a]pyrene induced low levels of DNA damage accompanied by ATM phosphorylation and xpc gene expression. Conversely, 20 µM etoposide exposure induced DNA damage without activation of known DDR pathways. Effects of 10 nM camptothecin on embryo development were stage-specific, with early stages, before gastrulation, being most sensitive. Overall, this study provides foundational information for future investigation of sterlet DDR pathways.

Preservation of female genetic resources of common carp through oogonial stem cell manipulation.

Several experiments were conducted in order to develop an optimal protocol for slow-rate freezing (-1 °C/min) and short-term storage (-80 or 4 °C) of common carp ovarian tissue fragments with an emphasis on oogonial stem cells (OSCs). Dimethyl sulfoxide (Me2SO) with concentration of 1.5 M was identified as the best cryoprotectant in comparison to propylene glycol and methanol. When comparing supplementation of sugars (glucose, trehalose, sucrose) in different concentrations (0.1, 0.3, 0.5 M), glucose and trehalose in 0.3 M were identified as optimal. Short-term storage options for ovarian tissue pieces at -80 °C and 4 °C were tested as alternatives to cryopreservation and storage in liquid nitrogen. The presence of OSCs was confirmed by immunocytochemistry and viability after storage was determined by the trypan blue exclusion test. This study identified the optimal protocol for OSC cryopreservation using slow rate freezing resulting in ∼65% viability. The frozen/thawed OSCs were labelled by PKH-26 and transplanted into goldfish recipients. The success of the transplantation was confirmed by presence of fluorescent cells in the recipient gonad and later on by RT-PCR with carp dnd1 specific primers. The results of this study can facilitate long-term preservation of common carp germplasm which can be recovered in a surrogate recipient through interspecific germ cell transplantation.

The killifish germline regulates longevity and somatic repair in a sex-specific manner.

Classical evolutionary theories propose tradeoffs among reproduction, damage repair and lifespan. However, the specific role of the germline in shaping vertebrate aging remains largely unknown. In this study, we used the turquoise killifish (Nothobranchius furzeri) to genetically arrest germline development at discrete stages and examine how different modes of infertility impact life history. We first constructed a comprehensive single-cell gonadal atlas, providing cell-type-specific markers for downstream phenotypic analysis. We show here that germline depletion-but not arresting germline differentiation-enhances damage repair in female killifish. Conversely, germline-depleted males instead showed an extension in lifespan and rejuvenated metabolic functions. Through further transcriptomic analysis, we highlight enrichment of pro-longevity pathways and genes in germline-depleted male killifish and demonstrate functional conservation of how these factors may regulate longevity in germline-depleted Caenorhabditis elegans. Our results, therefore, demonstrate that different germline manipulation paradigms can yield pronounced sexually dimorphic phenotypes, implying alternative responses to classical evolutionary tradeoffs.

The killifish germline regulates longevity and somatic repair in a sex-specific manner.

Classical evolutionary theories propose tradeoffs between reproduction, damage repair, and lifespan. However, the specific role of the germline in shaping vertebrate aging remains largely unknown. Here, we use the turquoise killifish ( N. furzeri ) to genetically arrest germline development at discrete stages, and examine how different modes of infertility impact life-history. We first construct a comprehensive single-cell gonadal atlas, providing cell-type-specific markers for downstream phenotypic analysis. Next, we show that germline depletion - but not arresting germline differentiation - enhances damage repair in female killifish. Conversely, germline-depleted males instead showed an extension in lifespan and rejuvenated metabolic functions. Through further transcriptomic analysis, we highlight enrichment of pro-longevity pathways and genes in germline-depleted male killifish and demonstrate functional conservation of how these factors may regulate longevity in germline-depleted C. elegans . Our results therefore demonstrate that different germline manipulation paradigms can yield pronounced sexually dimorphic phenotypes, implying alternative responses to classical evolutionary tradeoffs.

A scalable and tunable platform for functional interrogation of peptide hormones in fish.

Pituitary hormones play a central role in shaping vertebrate life history events, including growth, reproduction, metabolism, and aging. The regulation of these traits often requires precise control of hormone levels across diverse timescales. However, fine tuning circulating hormones in-vivo has traditionally been experimentally challenging. Here, using the naturally short-lived turquoise killifish (N. furzeri), we describe a high-throughput platform that combines loss- and gain-of-function of peptide hormones. Mutation of three primary pituitary hormones, growth hormone (gh1), follicle stimulating hormone (fshb), and thyroid stimulating hormone (tshb), alters somatic growth and reproduction. Thus, suggesting that while the killifish undergoes extremely rapid growth and maturity, it still relies on vertebrate-conserved genetic networks. As the next stage, we developed a gain-of-function vector system in which a hormone is tagged using a self-cleavable fluorescent reporter, and ectopically expressed in-vivo through intramuscular electroporation. Following a single electroporation, phenotypes, such as reproduction, are stably rescued for several months. Notably, we demonstrate the versatility of this approach by using multiplexing, dose-dependent, and doxycycline-inducible systems to achieve tunable and reversible expression. In summary, this method is relatively high-throughput, and facilitates large-scale interrogation of life-history strategies in fish. Ultimately, this approach could be adapted for modifying aquaculture species and exploring pro-longevity interventions.

In humans and other vertebrates, a pea-size gland at the base of the brain called the pituitary gland, produces many hormones that regulate how individuals grow, reproduce, and age. Three of the most prominent hormones are known as the growth hormone, the follicle-stimulating hormone, and the thyroid-stimulating hormone. It is important that the body precisely controls the levels of these hormones throughout an individual's life. One way researchers can investigate how hormones and other molecules work is to artificially alter the levels of the molecules in living animals. However, this has proved to be technically challenging and time-consuming for pituitary gland hormones. Moses et al. studied the growth hormone, follicle-stimulating hormone, and thyroid-stimulating hormone in the turquoise killifish, a small fish that grows and matures more rapidly than any other vertebrate research model. The experiments revealed that mutant fish lacking one of the three primary pituitary hormones were smaller, took longer to reach maturity, or were completely sterile. This suggests these three hormones play a similar role in killifish as they do in other vertebrates. The team then developed a new experimental platform to precisely control the levels of the three hormones in killifish. Genes encoding individual hormones were expressed in the muscles of the mutant fish, effectively making the muscles a 'factory' for producing that hormone. Treating mutant fish this way once was enough to restore growth and to fully return reproduction to normal levels for several months. Moses et al. also demonstrated that it is possible to use this platform to express more than one hormone gene at a time and to use drugs to switch hormone production on and off in a reversible manner. For example, this reversible approach made it possible to effectively adjust fertility levels. The new platform developed in this work could be adapted for modifying a variety of traits in animals to explore how they impact health and longevity. In the future, it may also have other applications, such as optimizing how farmed fish grow and reproduce and regulating hormone levels in human patients with hormone imbalances.

Enhancement of zebrafish sperm production via a large body-sized surrogate with germ cell transplantation.

Zebrafish (Danio rerio) is a commonly-used vertebrate model species for many research areas. However, its low milt volume limits effective cryopreservation of sperm from a single individual and often precludes dividing a single semen sample to conduct multiple downstream procedures such as genomic DNA/RNA extraction and in-vitro fertilization. Here, we apply germ stem cell transplantation to increase zebrafish sperm production in a closely related larger species from the same subfamily, giant danio Devario aequipinnatus. The endogenous germ cell of the host is depleted by dead-end morpholino antisense oligonucleotide. Histology of the sterile gonad and quantitative PCR of gonadal tissue reveals all sterile giant danio develop the male phenotype. Spermatogonial cells of Tg(ddx4:egfp) transgenic zebrafish are transplanted into sterile giant danio larvae, and 22% of recipients (germline chimera) produce donor-derived sperm at sexual maturation. The germline chimera produce approximately three-fold the volume of sperm and 10-fold the spermatozoon concentration of the donor. The donor-derived sperm is functional and gives rise to viable progeny upon fertilization of donor oocytes. We show that the issue of low milt volume can be effectively addressed by employing a larger surrogate parent.

Vitrification of the ovarian tissue in sturgeons.

The aim of this study was to test whether vitrification of sterlet Acipenser ruthenus and Russian sturgeon Acipenser gueldenstaedtii ovarian tissue through needle-immersed vitrification (NIV) is an efficient strategy for the preservation of oogonia (OOG) in order to supplement the current conservation efforts for these endangered fish species. Histological analyses of the gonads displayed that the ovaries of both species were immature and contained predominantly OOG and primary oocytes. The germline origin of these cells was verified by localization of the vasa protein through immunocytochemistry. NIV protocol was optimized by testing different equilibration (ES) and vitrification solutions (VS) containing various concentrations of dimethyl sulfoxide (Me2SO), propylene glycol (PG) or methanol (MeOH). In sterlet, the highest average viability (55.7 ± 11.5%) was obtained by using a combination of 1.5 M PG and 1.5 M Me2SO in the ES, and 1.5 M MeOH and 5.5 M Me2SO in the VS. In Russian sturgeon, the highest average viability (49.4 ± 17.1%) was obtained by using a combination of 1.5 M MeOH and 1.5 M Me2SO in the ES, and 3 M PG and 3 M Me2SO in the VS. To test whether vitrified/warmed OOG are functional, we have conducted an intra-specific transplantation assay to verify whether transplanted sterlet OOG will colonize the gonads of recipient fish. Fluorescently labelled cells were detected within recipient gonads at 2 and 3 months post-fertilization (mpf). Colonization rates of vitrified/warmed OOG (70% at 2 mpf and 61% at 3 mpf) were similar to those of fresh OOG (80% at 2 mpf and 70% at 3 mpf). This study has demonstrated that vitrification of ovarian tissue is an effective method for the preservation of OOG, and that the vitrified/warmed cells are functional and are able to colonize recipient gonads after transplantation similarly to the fresh cells. Since the vitrification procedure displayed in this study is simple and does not require complex and expensive laboratory equipment, it can be readily applied in field conditions, and therefore it can be invaluable for the conservation efforts of the critically endangered sturgeon species. However, care needs to be taken that despite the research conducted so far, donor-derived progeny was not yet obtained in sturgeons.

Does dietary exposure to 17α-ethinylestradiol alter biomarkers related with endocrine disruption and oxidative stress in the adult triploid of Danio rerio?

This study was conducted to investigate a comprehensive effect of 17α-ethinylestradiol (EE2) in zebrafish (Danio rerio) with the emphasis on endocrine disruption, oxidative stress and detoxification processes at different levels. Adult male triploid zebrafish were exposed to EE2 administered in feed at two concentrations - 10 and 1000 µg/kg for six weeks. The estrogenic potential of EE2 was evaluated using an analysis of vitellogenin, gene expression focused on reproductive disorders and gonad histological examination. The alterations in antioxidant and detoxification status were assessed using analyses of enzyme activities and changes in transcriptional levels of selected genes. The most significant changes were observed especially in fish exposed to a high concentration of EE2 (i.e., 1000 µg/kg). Such high concentration caused extensive mortality (25 %) mainly in the second half of the experiment followed by a highly significant decrease in the length and body weight. Similarly, highly significant induction of vitellogenin level and vtg1 mRNA expression (about 43,000-fold compared to the control) as well as a significant downregulation of gonad aromatase expression (cyp19a1a) and histological changes in testicular tissue were confirmed in this group. In the group exposed to environmentally relevant concentration of EE2 (i.e., 10 µg/kg), no significant differences in vitellogenin were observed, although all fish were positive in the detection of vitellogenin compared to control, where only 40 % of individuals were positive. In addition, the high concentration of EE2 resulted in significant alterations in most monitored antioxidant and detoxifying enzymes with the exception of catalase, followed by strongly significant upregulation in mRNA expression of gsr, gpx1a, cat and cyp1a genes. Furthermore, a significant decrease in the glutathione reductase activity was recorded in fish exposed to 10 µg EE2/kg. To our knowledge, this is the first study which reports the effects of subchronic per oral exposure to EE2 in adult triploid zebrafish.

Optimization of in vitro culture conditions of common carp germ cells for purpose of surrogate production.

Common carp (Cyprinus carpio) is the fourth most-produced fish species in aquaculture and frequently used model species with significant effort invested in development of biotechnological applications. In present study, we attempted to establish an in vitro germ cell culture condition for short term cell culture, which could facilitate further applications such as surrogacy or gene manipulation. Basal media and different types of feeder cells were investigated to optimize carp germ cell culture condition to favor maintenance of mitotic proliferation. Results indicated that germ cells cultured with hESC media and RTG2 cell line as feeder possessed significantly higher proliferation and survival rate compared to that cultured with StemPro media and Sertoli cell line as feeder. In addition, we compared two dissection strategies to compare risk of cell culture contamination and body cavity was open from dorsal part or from ventral part. As a result, carp open from the dorsal side can minimize the risk of contamination. In summary, this is the first study to optimize the cultivation of germ cells in common carp. This opens up new opportunities for the application of specific techniques in the breeding of those species with high commercial value and frequent use as a model fish. Results obtained in this study are important for implementation of new strategies in common carp breeding, conservation of genetic resources, restoration of lines or development of clonal and isogenic carp lines.

Partial fads2 Gene Knockout Diverts LC-PUFA Biosynthesis via an Alternative Δ8 Pathway with an Impact on the Reproduction of Female Zebrafish (Danio rerio).

The zebrafish (Danio rerio) genome contains a single gene fads2 encoding a desaturase (FADS2) with both Δ6 and Δ5 activities, the key player in the endogenous biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs), which serve essential functions as membrane components, sources of energy and signaling molecules. LC-PUFAs include the precursors of eicosanoids and are thus predicted to be indispensable molecules for reproductive health in virtually all vertebrates. In mice, an amniotic vertebrate, fads2 deletion mutants, both males and females, have been confirmed to be sterile. In anamniotic vertebrates, such as fish, there is still no information available on the reproductive (in)ability of fads2 mutants, although zebrafish have become an increasingly important model of lipid metabolism, including some aspects of the generation of germ cells and early embryonic development. In the present study, we apply the CRISPR/Cas9 genome editing system to induce mutations in the zebrafish genome and create crispants displaying a degree of fads2 gene editing within the range of 50-80%. Focusing on adult G0 crispant females, we investigated the LC-PUFA profiles of eggs. Our data suggest an impaired pathway of the LC-PUFA biosynthesis of the ω6 and ω3 series in the first-rate limiting steps of the conversion of linoleic acid (LA) into γ-linolenic acid (GLA), and α-linolenic acid (ALA) into stearidonic acid (SDA), respectively, finally resulting in bad-quality eggs. Our data suggest the existence of an alternative Δ8 pathway, which bypasses the first endogenous LC-PUFA biosynthetic step in zebrafish in vivo, and suggest that the zebrafish bifunctional FADS2 enzyme is actually a trifunctional Δ6/Δ5/Δ8 desaturase.

Efficient CRISPR Mutagenesis in Sturgeon Demonstrates Its Utility in Large, Slow-Maturing Vertebrates.

In the last decade, the CRISPR/Cas9 bacterial virus defense system has been adapted as a user-friendly, efficient, and precise method for targeted mutagenesis in eukaryotes. Though CRISPR/Cas9 has proven effective in a diverse range of organisms, it is still most often used to create mutant lines in lab-reared genetic model systems. However, one major advantage of CRISPR/Cas9 mutagenesis over previous gene targeting approaches is that its high efficiency allows the immediate generation of near-null mosaic mutants. This feature could potentially allow genotype to be linked to phenotype in organisms with life histories that preclude the establishment of purebred genetic lines; a group that includes the vast majority of vertebrate species. Of particular interest to scholars of early vertebrate evolution are several long-lived and slow-maturing fishes that diverged from two dominant modern lineages, teleosts and tetrapods, in the Ordovician, or before. These early-diverging or "basal" vertebrates include the jawless cyclostomes, cartilaginous fishes, and various non-teleost ray-finned fishes. In addition to occupying critical phylogenetic positions, these groups possess combinations of derived and ancestral features not seen in conventional model vertebrates, and thus provide an opportunity for understanding the genetic bases of such traits. Here we report successful use of CRISPR/Cas9 mutagenesis in one such non-teleost fish, sterlet Acipenser ruthenus, a small species of sturgeon. We introduced mutations into the genes Tyrosinase, which is needed for melanin production, and Sonic hedgehog, a pleiotropic developmental regulator with diverse roles in early embryonic patterning and organogenesis. We observed disruption of both loci and the production of consistent phenotypes, including both near-null mutants' various hypomorphs. Based on these results, and previous work in lamprey and amphibians, we discuss how CRISPR/Cas9 F0 mutagenesis may be successfully adapted to other long-lived, slow-maturing aquatic vertebrates and identify the ease of obtaining and injecting eggs and/or zygotes as the main challenges.

Use of all-male triploid population of zebrafish (Danio rerio) for evaluation of 17α-ethinylestradiol effects.

OBJECTIVES: We tested the toxicity of ethinylestradiol, a semisynthetic estrogen used in oral contraceptives, on all-male triploid zebrafish using commercial feeds and three different doses concentrations. We aimed to determine whether ethinylestradiol peroral administration resulted in vitellogenin production and whether all-male triploid zebrafish could serve as a model species for xenoestrogen testing. METHODS: The actual concentrations of 17α-ethinylestradiol were 0.0035 (low); 0.0315 (medium) and 0.365 (high) µg/g. Positive control represented commercial feeds containing 0.0465 µg/g of ß-estradiol. The experiment lasted 8 weeks. RESULTS: Our results indicate that 17α-ethinylestradiol consumption does induce vitellogenin production in triploid zebrafish. CONCLUSIONS: The simple presence of vitellogenin is a definite symptom indicative of the potential for such changes due to the action of estrogenic substances. As such, this experiment has shown that the use of all-male triploid zebrafish populations, rather than the mixed-sex populations of other species previously used, could serve as a suitable alternative model population for controlled testing of the effects of xenoestrogens on fish.