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The neural crest (NC) is a multipotent embryonic cell type derived from the ectoderm during neurulation giving rise to a variety of cell lineages such as neurons, glia and pigment cells. Most genes associated with the correct initiation, differentiation and migration of the neural crest have been found through reverse genetics. Similarities between neural crest development and some features of cancer progression are remarkable. For instance, it has been suggested that some cancer types recapitulate NC processes in an unregulated manner such as epithelial-mesenchymal transition or active cell migration throughout the body to form distant metastases. However, to date very little is known about initiators and drivers that direct neural crest cell migration to specific target sites. The Medaka mutant hirame represents an interesting melanocyte specific migration defect on the yolk sac caused by a loss of functional Yes-associated protein (YAP). Medaka hirame mutants were initially studied for their profound changes in body morphology. Genomic mapping identified the causal mutation as a nonsense point mutation within the first WW domain in the Yes-associated protein 1 (YAP1), causing translation of a dysfunctional YAP protein. YAP is a downstream transcriptional co-activator of the recently discovered and evolutionarily conserved Hippo pathway. Alterations within Hippo signalling are linked to cell survival, proliferation and abnormal tissue overgrowth. We demonstrate that hirame melanocyte precursors (melanoblasts) are initially present in normal abundance, but show an early migration defect with a lack of melanoblasts on the yolk sac, and corresponding accumulation in the lateral parts of the body. Subsequently, we observe an overall decline in differentiated melanocyte numbers during late stage embryogenesis. We designed an overexpression cassette linking enhanced GFP to either wild type or a mutated activated version of YAP and present evidence that it can efficiently rescue the melanocyte defect after injection of mRNA into one-cell stage embryos. Furthermore, analysis of the yolk sac anatomy via transmission electron microscopy indicates that a fraction of yolk membrane cells undergo apoptosis and we propose that this may contribute to the establishment of altered environmental cues leading to abnormal melanoblast migration onto the yolk sac. Injection of yap mRNA directly into the yolk sac however, failed to rescue melanoblast patterning. To advance our study, we isolated and characterised a 3.6 kb Medaka dopachrome tautomerase (Dct) promoter fragment, and used it to drive expression of enhanced green fluorescent protein (eGFP) in vivo. We generated germline transgenics with this construct that showed lineage-specific expression of eGFP within early migrating melanoblasts, a phenotype that is maintained in differentiated melanocytes throughout embryogenesis. In addition, using this promoter we overexpressed our egfp-yap fusion cassette and established transgenic lines to assess the cell autonomy of YAP within the melanocyte lineage. However, no fluorescent signal could be detected in the latter transgenics, necessitating future experimentation to properly characterise these lines. Finally, we analysed a range of neural crest markers to examine the extent of the neural crest defects in hirame mutants. In addition to the melanocyte phenotype, we identified a dramatic reduction in xanthophore numbers, although early leucophore development appears unaffected. We also observed a decreased number of dorsal root ganglia in the peripheral nervous system as well as smaller and partly ectopic cranial neural crest ganglia populations within the epibranchial arches. The characterisation of a novel Medaka melanocyte specific promoter as well as additional novel NC markers will be widely applicable and useful to the wider Medaka research community as a tool for the study of neural crest related mechanisms during development.

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The specificity of a medaka vitellogenin (Vtg) enzyme-linked immunosorbent assay (ELISA) kit was tested on adult hybrid tilapia to determine if vitellogenin could be successfully measured in tilapia using this method. Adult hybrid (Orechromis niloticus x Oreochromis mossambicus x Oreochromis aureus) tilapia were injected with 17â-estradiol (E2) at 5 ìg/g body weight (b.w.) in two separate experiments and exposed to 2.5 ìg/g b.w. E2 through aquaria water in a third experiment. All fish were exposed to solvent controls (50/50 PBS/ethanol, or 59/41 PBS/ ethanol) proportional to the volume of estradiol solvent used. Experiment 1 was terminated due to a decrease in fish health and unexpected mortalities. Experiment 2 was continued though there was again a decrease in fish health and mortalities before experiment was completed. According to one-way ANOVA, Experiment 2 treatment groups were significantly different (P<0.05); however, Tukey's pairwise comparisons showed no differences between treatment groups. Experiment 3 results showed statistical differences between some treatment groups (P<0.001). However, maximum vitellogenin levels reached 1250 ng/mL Vtg, which were much lower than induced vitellogenin levels from literature (0.002 mg/mL to 6 mg/mL). Typical induction trends should be as follows: male control<female control< induced male<induced female; however, these trends were not observed in this study. For these reasons, the medaka vitellogenin ELISA kit was determined not specific to tilapia vitellogenin. This research was performed due to the fact that tilapia are farmed worldwide, and to increase research with these fish, in order to better understand their response to xenobiotic chemicals, easier methods are desired for measuring their vitellogenin. Vitellogenin induction from exposure to the chemotherapeutics: oxytetracycline, Romet-30®, and copper sulfate was evaluate using Japanese medaka (Oryzias latipes). Medaka were exposed to E2 (0.0552 ìg/L) in aquaria water as a positive control. Medaka were fed oxytetracycline medicated feed at 0.0174 g, 0.0348 g, and 0.087 g, and Romet-30® medicated feed at 0.0261 g, 0.0522 g, and 0.1305 g daily for 11 days and 5 days as recommended. Medaka were exposed to copper sulfate at 1.85 mg/L for one hour (for 1 day, 7 consecutive days, and every three to five days for a total of 7 doses). Vitellogenin levels were measured using a sandwich ELISA. As expected, Vtg levels were induced from exposure to E2 (P<0.01); however, exposure to the chemotherapeutic compounds did not induce elevated Vtg levels. Alternately, copper sulfate exposure for one hour for 1 day did show a significant decrease in vitellogenin (P<0.01). Biological variation accounted for large portions of the variation within treatment groups in medaka vitellogenin in fish exposed to chemotherapeutics (as high as 41.01%). From this research we suggest that these chemicals do not have estrogenic properties capable of inducing vitellogenin in medaka. / Master of Science

Orientador: Rafael Henrique Nóbrega / Resumo: A decisão se a gônada indiferenciada irá se desenvolver em um testículo ou um ovário é uma etapa crítica na diferenciação sexual dos vertebrados. Este processo consiste em uma complexa cascata gênica que culmina na diferenciação das células somáticas presente na gônada indiferenciada. Neste sentido, o hormônio anti-Mülleriano é um importante efetor nesta decisão. Estudos com o peixe medaka mutante hotei, no qual o receptor do Amh tipo 2 (amhrII) é mutado, mostram elevado índice gonadossomático, e reversão sexual de macho para fêmea. Com base nestas informações, acredita-se que o Amh possa regular o número de células germinativas e diretamente ou indiretamente este fato pode influenciar na decisão da gônada bipotencial em se tornar um testículo ou ovário. Sendo assim, o objetivo do presente trabalho foi de se avaliar a sinalização do Amh durante a diferenciação sexual no medaka. Para tanto, estudos de perda de função gênica foram empregados, no qual o gene amh foi deletado através do sistema CRISPR/Cas9. Também foi avaliada a expressão dos genes que atuam na janela de diferenciação sexual por RT-qPCR. Inicialmente, identificamos dois tipos de mutação nos animais heterozigotos (amh (-/+) da F1. A mutação mais interessante foi a deleção de 759 pares de base entre os exons 6 e 7 que compreende parte dos domínios Amh_N e TGF-β. Na geração F2, os mutantes homozigotos amh (-/-) machos XY apresentaram 100% de reversão sexual para fêmeas fenotípicas. Além disso os animais amh (-/-) ap... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The decision whether the undifferentiated gonad will develop in a testicle or an ovary is a critical step in the sexual differentiation of vertebrates. This process consists of a complex genetic cascade that culminates in the differentiation of the somatic cells present in the undifferentiated gonad. In this context, the anti-Müllerian hormone is an important effector in this decision. Studies with the medaka hotei fish, in which the Amh type 2 receptor (amhrII) is mutated, show high gonadosomatic index, and sexual reversion from male to female. Based on this information, it is postulated that the Amh regulates the number of germ cells, and directly or indirectly this fact may influence the decision of the bipotential gonad to become a testicle or ovary. Therefore, the objective of the present study was to evaluate the signaling of Amh during sexual differentiation in medaka. For that, studies of loss of gene function were employed, in which the amh gene was deleted through the CRISPR / Cas9 system. We also evaluated the expression of genes that act in the window of sexual differentiation by RT-qPCR. We initially identified two types of mutation in F1 heterozygous (amh -/+) animals. The most interesting mutation was the deletion of 759 base pairs between exons 6 and 7 which comprises part of the Amh_N and TGF-β domains. In the F2 generation, the homozygous amh (-/-) mutants XY males presented 100% of sexual reversion. In addition, the amh (-/-) animals presented gonadal hyper... (Complete abstract click electronic access below) / Doutor

On August 29, 2005 Hurricane Katrina struck the Gulf Coast and storm surges breached levees flooding much of New Orleans, Louisiana. One month after the storm, sediment was collected and toxicity was tested using Japanese medaka (Oryzias latipes) embryos. Sediments with the highest contaminant levels showed the highest embryonic mortality and most delayed development. However, no sediment caused an increased mutant frequency. When the most contaminated site was resampled in February, 2006 contaminant levels and toxicity decreased. During toxicity testing, approximately 20% of embryos incubated with sediment from one of these sites died and turned red. A red bacterium was isolated that is Gram-negative, cocco-baccilus, non-motile, and most similar to Hahella chejuensis based on genetic and metabolic tests. This bacterium caused 100% infection at 108 bacterial cells per ml and variable infection at lower doses. This study was the first to examine biological effects of exposure to post-Hurricane Katrina sediments.

La règlementation européenne requiert l’évaluation de la reprotoxicité des ingrédients cosmétiques sans avoir recours aux tests sur les organismes définis par la directive européenne sur les animaux utilisés à des fins scientifiques. Pour cribler par exclusion des substances chimiques, l’industrie cosmétique a besoin de développer une méthode alternative à l’expérimentation animale prédictive et spécifique à l’identification d’agents tératogènes (substances entraînant au cours du développement embryonnaire et de manière définitive des malformations physiques et/ou fonctionnelles). Pour cela, le poisson zèbre, le poisson médaka et l’amphibien xénope aux stades embryonnaires ont été évalués sur une liste de 43 substances de référence. Le médaka a été sélectionné pour la fiabilité de son approvisionnement, la robustesse de ses stades embryonnaires lors des manipulations, ainsi que pour les performances du test l’utilisant. De plus, ce test permet de détecter les agents tératogènes les plus puissants, près de la moitié des 26 substances tératogènes de notre liste. Son taux de spécificité est fixé à 100% pour identifier correctement les 17 substances négatives dont l’absence d’effets tératogènes chez l’homme ou un organisme modèle mammifère, est avérée. Néanmoins, les performances de ce test pourraient être améliorées par son automatisation et par l’intégration de la quantification de nouveaux paramètres pour l’identification de malformations fonctionnelles. Enfin, pour prédire l’innocuité d’une substance chimique chez l’Homme, le test prédictif de tératogénicité utilisant les embryons de médaka doit être intégré dans une stratégie globale d’évaluation de la tératogénicité. / European legislation requires the assessment of reproductive toxicity of cosmetic ingredients without using tests on the organisms defined by the European directive on animals used for scientific purposes. To screen by excluding chemicals, cosmetics industry needs to develop an alternative method to animal testing, which needs to be predictive and specific in identifying teratogen agents (during embryonic development substances causing definitive physical and / or functional defects). For this, zebrafish, medaka fish and Xenopus amphibian at embryonic stages were assessed on a list of 43 reference substances. Medaka has been selected to the reliability of its supply, the robustness of its embryonic stages during handling, as well as for performance of its test. In addition, this test can detect the most potent teratogen agents, nearly half of the 26 teratogens of our list. Its specificity rate is set at 100% for correctly identifying 17 negative substances for which the absence of teratogenic effects is proved in humans or mammalian model organism. However, the performance of this test may be improved by its automation and integration of new quantification parameters for the identification of functional defects. Finally, to predict the safety of a chemical in humans, the predictive teratogenicity test using medaka embryos must be integrated into a comprehensive strategy for assessing the teratogenicity.

The oil sands, located in north-eastern Alberta, are one of the largest deposits of oil worldwide. Because the Alberta Environmental Protection and Enhancement Act prohibits the release of oil sands process-affected material into the environment, industry is storing vast quantities of tailings on mine lease sites. The oil sands industry is currently accumulating tailings waste at a rate of >105 m3/day, for which reclamation strategies are being investigated. Naphthenic acids (NAs) have been identified as the most toxic component of oil sands tailings as they are considered acutely toxic to a variety of biota, and are therefore a target contaminant for tailings pond reclamation strategies. Current literature based on Microtox® assays (marine bacteria Vibrio fischeri) suggests that lower molecular weight NAs are more toxic than higher molecular weight NAs. The following thesis involves the utilization of NA fractions and their relative toxicities to determine if NA toxicity is related to NA molecular weight. A previous study generated an oil sands-derived naphthenic acid extract (NAE), which was fractionated by distillation at stepped temperatures, yielding five fractions with increasing median molecular weights (Daltons). In the present study, the same extract and five fractions were utilized. To expand on the earlier characterization which involved a low resolution electrospray ionization mass spectrometry (ESI-MS), the whole extract and five fractions were analysed using electrospray ionization high-resolution mass spectrometry (ESI-HRMS) and synchronous fluorescence spectroscopy (SFS). Mean molecular weights were generated for each fraction, and an increase in molecular weight with increasing fraction number was confirmed. Respective mean Daltons and relative proportions for each fraction are as follows: 237 and 11.9 % (fraction 1), 240 and 32.3% (fraction 2), 257 and 33.4% (fraction 3), 308 and 16.8% (fraction 4), and 355 and 5.6% (fraction 5). When chemical analyses of fractions were compared, it was determined that structures contributing to increased molecular weight included increased cyclic structures (up to 7-ring structures), aromaticity (mono- and diaromatics), nitrogen, sulfur, and oxygen heteroatoms, and dihydroxy/dicarboxy compounds. In addition, characterization data suggested the presence of NAs exhibiting estrogenic structures. Following chemical characterization, NA fractions were subject to embryo/larval bioassays using two fish species: Oryzias latipes (Japanese medaka) and Pimephales promelas (fathead minnow). Endpoints evaluated were mortality, time to hatch, hatch length, and abnormalities. Results suggest that relative NA fraction toxicity is not related to molecular weight, as no trend relating mean Dalton weight to toxicity was observed for any endpoint in both species. Acute toxicity data indicated differences between fractions as high as 2-fold, although results were species-dependent. Fraction 1 displayed the lowest potency (highest LC50) for both Japanese medaka (0.291 mM) and fathead minnow (0.159 mM). Fractions 3 and 2 for Japanese medaka (0.149 and 0.157 mM, respectively), and fractions 5 and 2 for fathead minnow (0.061 and 0.080 mM, respectively) displayed the greatest potencies for mortality (lowest LC50). When fraction LC50s for Japanese medaka were compared to the whole NAE (0.143 mM), the mid molecular weight fractions (fractions 2 and 3) appeared most similar to the whole NA. . In terms of relative toxicity and proportion, constituents in the mid molecular range fractions (2 and 3) likely represent greater risk compared to other fractions, and further chemical and toxicological characterization of constituents within these fractions is warranted particularly for long-chained, monocarboxylic acids, with low aromaticity. Japanese medaka and fathead minnow varied in their sensitivity and their relative response to different fractions. In general, fathead minnow were more sensitive than Japanese medaka based on lower estimates of LC50 and threshold (growth) values in addition to the presence of developmental abnormalities (predominately yolk sac edema) associated with a few of the fractions. Compared to differences in toxicity between fractions for a given species (>2-fold for fathead minnow), there was more variability between species for a given fraction (> 3-fold for fraction 5). Also, the relative toxicity of fractions as indicated in the present study is contrary to the results generated using Vibrio fischeri for the same fractions. Thus, there is a need for multi- endpoint and species toxicity evaluations to assess the efficacy of remediation and reclamation options for reducing toxicity of oil sands tailings.

The American eel (Anguilla rostrata) has suffered a serious population decline in Lake Ontario since the early 1980s due to a decline in recruitment of juveniles migrating from the Sargasso Sea. This has resulted in the closure of the Lake Ontario fishery in 2004 and its listing as endangered under the Ontario Species at Risk act in June of 2008 in Ontario. Due to their complex life cycle, little is known about eels once they leave their freshwater habitats and migrate to the Sargasso Sea to reproduce. Ocean conditions, habitat destruction, disease, reduced lipid content, over-fishing, physical barriers (hydroelectric dams), and chemical contamination are all possible reasons for recruitment decline and may be acting cumulatively. Maternally derived dioxin-like contaminants (DLCs) accumulated during the growth phase of eels in Lake Ontario are toxic to fish embryos, and embryotoxicity is expressed as a series of malformations known as blue sac disease (BSD). I assessed whether these toxicants are in high enough concentrations in sexually maturing, eels to be embryotoxic to their offspring, as assessed by using Japanese medaka (Oryzias latipes), a surrogate species. Medaka embryos were first injected with 2,3,7,8-tetracholordibenzo-p-dioxin (TCDD) to establish their sensitivity to this test chemical expressed as an 11-day EC50 of 3.79 pg/mg, for the induction of BSD. Medaka embryos were injected with eel extracts and their response compared to the TCDD toxicity curve to assess whether extracts caused developmental problems and to estimate the relative concentration of DLCs. Eel extracts from all collection sites caused no dioxin-like toxicity to Japanese medaka embryos. However, significantly higher toxicity at 10 eeq relative to triolein was found for all extracts with no differences among sites, suggesting the presence of non-dioxin-like toxicants. The low level of maternal tissue contamination by DLCs implied by this bioassay is mirrored in chemical monitoring of persistent organic pollutants in Lake Ontario fish. If correct, the low levels of toxicity of extracts to embryos could contribute to the observed increase of eels entering L. Ontario from 2003 to 2008. / Thesis (Master, Biology) -- Queen's University, 2010-09-01 09:31:19.466