Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma.

The mesothelium lines body cavities and surrounds internal organs, widely contributing to homeostasis and regeneration. Mesothelium disruptions cause visceral anomalies and mesothelioma tumors. Nonetheless, the embryonic emergence of mesothelia remains incompletely understood. Here, we track mesothelial origins in the lateral plate mesoderm (LPM) using zebrafish. Single-cell transcriptomics uncovers a post-gastrulation gene expression signature centered on hand2 in distinct LPM progenitor cells. We map mesothelial progenitors to lateral-most, hand2-expressing LPM and confirm conservation in mouse. Time-lapse imaging of zebrafish hand2 reporter embryos captures mesothelium formation including pericardium, visceral, and parietal peritoneum. We find primordial germ cells migrate with the forming mesothelium as ventral migration boundary. Functionally, hand2 loss disrupts mesothelium formation with reduced progenitor cells and perturbed migration. In mouse and human mesothelioma, we document expression of LPM-associated transcription factors including Hand2, suggesting re-initiation of a developmental program. Our data connects mesothelium development to Hand2, expanding our understanding of mesothelial pathologies

Comparative Analysis of Locomotor Behavior and Descending Motor System Anatomy of Larval Zebrafish and Giant Danio

A major challenge for comparative biology is understanding what aspects of an animal’s locomotor repertoire represent general features of motor organization, versus specialized adaptations for its anatomy and ecological niche. In this thesis I investigate the Giant Danio larvae (Devario aequipinnatus) as a potential model for comparative studies with Zebrafish, a well-established animal model in neuroscience. To this end, I study the locomotor behavior of both species and how its differences are reflected in the underlying neural circuit structure. Initially, I compare the anatomy of the descending pathways controlling locomotion in Giant Danio to Zebrafish using retrograde labelling of reticulospinal neurons. I see a striking resemblance of the circuit in both species, with a roughly similar organization and the general division and number of cell clusters being very well conserved. Following, I compare visually guided behaviours in Giant Danio to different Zebrafish strains. Giant Danio show a stronger optomotor response than Zebrafish. The optomotor response of Giant Danio first appear around 4 days post fertilization and can be consistently and reliably evoked. During optomotor tracking Giant Danio show shorter interbout intervals and are able to track motion at higher speeds than Zebrafish. I also observe that the higher manoeuvrability of Giant Danio is also reflected during prey capture. Interestingly, Zebrafish strains derived from more recently wild-caught fish show more robust optomotor behaviour, closer to Giant Danio. Lastly, I demonstrate the suitability of using Giant Danio in a head-restrained preparation with a 3D virtual reality environment. Combined with the potential for comparative approaches with Zebrafish, the faster development, larger neurons, and the rich behavioural repertoire of Giant Danio make it a promising model for neuroscience

Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma

The mesothelium lines body cavities and surrounds internal organs, widely contributing to homeostasis and regeneration. Mesothelium disruptions cause visceral anomalies and mesothelioma tumors. Nonetheless, the embryonic emergence of mesothelia remains incompletely understood. Here, we track mesothelial origins in the lateral plate mesoderm (LPM) using zebrafish. Single-cell transcriptomics uncovers a post-gastrulation gene expression signature centered on hand2 in distinct LPM progenitor cells. We map mesothelial progenitors to lateral-most, hand2-expressing LPM and confirm conservation in mouse. Time-lapse imaging of zebrafish hand2 reporter embryos captures mesothelium formation including pericardium, visceral, and parietal peritoneum. We find primordial germ cells migrate with the forming mesothelium as ventral migration boundary. Functionally, hand2 loss disrupts mesothelium formation with reduced progenitor cells and perturbed migration. In mouse and human mesothelioma, we document expression of LPM-associated transcription factors including Hand2, suggesting re-initiation of a developmental program. Our data connects mesothelium development to Hand2, expanding our understanding of mesothelial pathologies

Machine learning-based automated segmentation with a feedback loop for 3D synchrotron micro-CT

Die Entwicklung von Synchrotronlichtquellen der dritten Generation hat die Grundlage für die Untersuchung der 3D-Struktur opaker Proben mit einer Auflösung im Mikrometerbereich und höher geschaffen. Dies führte zur Entwicklung der Röntgen-Synchrotron-Mikro-Computertomographie, welche die Schaffung von Bildgebungseinrichtungen zur Untersuchung von Proben verschiedenster Art förderte, z.B. von Modellorganismen, um die Physiologie komplexer lebender Systeme besser zu verstehen. Die Entwicklung moderner Steuerungssysteme und Robotik ermöglichte die vollständige Automatisierung der Röntgenbildgebungsexperimente und die Kalibrierung der Parameter des Versuchsaufbaus während des Betriebs. Die Weiterentwicklung der digitalen Detektorsysteme führte zu Verbesserungen der Auflösung, des Dynamikbereichs, der Empfindlichkeit und anderer wesentlicher Eigenschaften. Diese Verbesserungen führten zu einer beträchtlichen Steigerung des Durchsatzes des Bildgebungsprozesses, aber auf der anderen Seite begannen die Experimente eine wesentlich größere Datenmenge von bis zu Dutzenden von Terabyte zu generieren, welche anschließend manuell verarbeitet wurden. Somit ebneten diese technischen Fortschritte den Weg für die Durchführung effizienterer Hochdurchsatzexperimente zur Untersuchung einer großen Anzahl von Proben, welche Datensätze von besserer Qualität produzierten. In der wissenschaftlichen Gemeinschaft besteht daher ein hoher Bedarf an einem effizienten, automatisierten Workflow für die Röntgendatenanalyse, welcher eine solche Datenlast bewältigen und wertvolle Erkenntnisse für die Fachexperten liefern kann. Die bestehenden Lösungen für einen solchen Workflow sind nicht direkt auf Hochdurchsatzexperimente anwendbar, da sie für Ad-hoc-Szenarien im Bereich der medizinischen Bildgebung entwickelt wurden. Daher sind sie nicht für Hochdurchsatzdatenströme optimiert und auch nicht in der Lage, die hierarchische Beschaffenheit von Proben zu nutzen. Die wichtigsten Beiträge der vorliegenden Arbeit sind ein neuer automatisierter Analyse-Workflow, der für die effiziente Verarbeitung heterogener Röntgendatensätze hierarchischer Natur geeignet ist. Der entwickelte Workflow basiert auf verbesserten Methoden zur Datenvorverarbeitung, Registrierung, Lokalisierung und Segmentierung. Jede Phase eines Arbeitsablaufs, die eine Trainingsphase beinhaltet, kann automatisch feinabgestimmt werden, um die besten Hyperparameter für den spezifischen Datensatz zu finden. Für die Analyse von Faserstrukturen in Proben wurde eine neue, hochgradig parallelisierbare 3D-Orientierungsanalysemethode entwickelt, die auf einem neuartigen Konzept der emittierenden Strahlen basiert und eine präzisere morphologische Analyse ermöglicht. Alle entwickelten Methoden wurden gründlich an synthetischen Datensätzen validiert, um ihre Anwendbarkeit unter verschiedenen Abbildungsbedingungen quantitativ zu bewerten. Es wurde gezeigt, dass der Workflow in der Lage ist, eine Reihe von Datensätzen ähnlicher Art zu verarbeiten. Darüber hinaus werden die effizienten CPU/GPU-Implementierungen des entwickelten Workflows und der Methoden vorgestellt und der Gemeinschaft als Module für die Sprache Python zur Verfügung gestellt. Der entwickelte automatisierte Analyse-Workflow wurde erfolgreich für Mikro-CT-Datensätze angewandt, die in Hochdurchsatzröntgenexperimenten im Bereich der Entwicklungsbiologie und Materialwissenschaft gewonnen wurden. Insbesondere wurde dieser Arbeitsablauf für die Analyse der Medaka-Fisch-Datensätze angewandt, was eine automatisierte Segmentierung und anschließende morphologische Analyse von Gehirn, Leber, Kopfnephronen und Herz ermöglichte. Darüber hinaus wurde die entwickelte Methode der 3D-Orientierungsanalyse bei der morphologischen Analyse von Polymergerüst-Datensätzen eingesetzt, um einen Herstellungsprozess in Richtung wünschenswerter Eigenschaften zu lenken

How microplastics and adsorbed pollutants affect zebrafish development?

A presença de microplásticos no ecossistema aquático é, nos dias de hoje, uma realidade extremamente preocupante que acarreta sérios riscos para o meio ambiente e para a saúde pública. A capacidade dos microplásticos de adsorverem poluentes orgânicos, como é o caso do benzo[α]pireno (BaP), levanta preocupações adicionais, pois cria uma nova rota de entrada de compostos tóxicos na cadeia alimentar. No entanto, o conhecimento atual sobre o impacto dos microplásticos, inalterados e/ou contaminados, nos organismos aquáticos é ainda insuficiente e requer estudos adicionais. O trabalho desenvolvido no âmbito desta tese pretende assim fornecer novos dados sobre os efeitos biológicos dos microplásticos utilizando como modelo o peixe-zebra (Danio rerio). Pequenos teleósteos, como é o caso do peixe-zebra, oferecem vantagens significativas relativamente aos modelos animais clássicos e são atualmente utilizados como organismos de primeira linha para avaliar os riscos ambientais associados aos compostos tóxicos presentes nos meios aquáticos. Estudos toxicológicos requerem o uso de materiais inertes e condições controladas, todavia, nenhum dos sistemas atualmente comercializados é adequado para avaliar o efeito tóxico dos microplásticos. Estes sistemas contêm componentes feitos de polímeros plásticos que podem libertar partículas plásticas micrométricas, lixiviar os constituintes químicos ou adsorver os compostos químicos em estudo. O sistema de aquários autônomo ZEB316 foi desenvolvido no âmbito deste trabalho com o objetivo de suprimir esta necessidade e de facultar uma solução económica e fácil de implementar que permita a realização de estudos toxicológicos de última geração. Este sistema é construído com materiais inertes e resistentes à corrosão e proporciona boas condições de alojamento através de um sistema eficiente de recirculação e filtragem da água. A avaliação dos parâmetros da água e do desempenho do crescimento dos peixes mostrou que o sistema ZEB316 oferece condições de alojamento comparáveis à dos sistemas comercialmente disponíveis. A maioria dos resultados obtidos no âmbito desta dissertação provêm da análise de imagens de microscopia de campo claro e/ou fluorescência. Embora o uso de imagens de microscopia seja comum na maioria dos laboratórios e permita obter uma quantidade considerável de informação, a análise destas imagens é geralmente um processo moroso, em parte devido à falta de ferramentas automáticas/semiautomáticas dedicadas que permitam a sua análise. Nesse sentido, desenvolvemos diversas macros para o software ImageJ, com o objetivo de reduzir o erro associado à análise pelo usuário, aumentando assim a reprodutibilidade dos dados e a padronização das metodologias experimentais. Uma vez que o foco deste trabalho está centrado no estudo da osteotoxicidade, é de salientar o desenvolvimento de um conjunto de macros específicas para esse fim e denominadas ZFBONE. Este conjunto de ferramentas permite aos usuários avaliar, a partir de imagens 2D, parâmetros morfométricos de várias estruturas ósseas (por exemplo, opérculo, raios e escamas da barbatana caudal), mas também a extensão e a intensidade das colorações específicas do osso. Além disso, outras macros foram desenvolvidas para outros fins, por exemplo, para analisar os vários parâmetros morfométricos relativos aos embriões de peixe ou para avaliar cortes histológicos. Uma vez desenvolvidos os meios e técnicas necessários para a execução dos ensaios toxicológicos, e subsequente análise dos resultados, procedeu-se à realização das várias experiências que visam compreender efeito biológico dos microplásticos e contaminates no peixe-zebra. Assim, larvas de peixe-zebra foram produzidas e mantidas no sistema previamente descrito, ZEB316, e expostas cronicamente, durante o seu desenvolvimento, a partículas de polietileno de 20- 27 μm, inalteradas (MP) ou contaminadas com benzo[α]pireno (MP-BaP). Estas partículas foram adicionadas à dieta dos peixes através de uma suplementação a 1% m/m. Apesar de não se ter registado qualquer alteração ao nível dos parâmetros morfológicos aos 30 dias pós-fertilização (dpf), a presença de MP e MP-BaP acabou por ter um efeito negativo no crescimento dos espécimes aos 90 e 360 dpf. A fecundidade relativa, a morfologia do ovo/embrião e a área do vitelo também sofreram um impacto negativo em peixes-zebra alimentados com MP-BaP. No que respeita ao estado geral do esqueleto, os peixes expostos a dietas experimentais contendo MP e MP-BaP sofreram um aumento significativo na incidência de deformações esqueléticas aos 30 dpf quando comparados com peixes alimentados com a dieta controlo, bem como um desenvolvimento anómalo da barbatana caudal e escamas, e uma diminuição da qualidade do osso aos 90 dpf. Um comprometimento da formação óssea intergeracional foi também observado na prole de espécimes expostos a MP ou MP-BaP, que se refletiu numa redução do osso opercular dos descendentes aos 6 dpf. Além de um claro efeito no desenvolvimento ósseo, a análise histológica do intestino revelou ainda um número reduzido de células caliciformes em peixes alimentados com dieta MP-BaP, um claro sinal de inflamação intestinal. Finalmente, a exposição das larvas a MP-BaP levou a um aumento da expressão de genes associados à via de resposta do BaP, ao mesmo tempo que impactou negativamente na expressão de genes envolvidos no estresse oxidativo. Os resultados obtidos indicaram um maior comprometimento do desenvolvimento ósseo no peixe-zebra quando sujeito a uma dieta contendo microplásticos contaminados com BaP (MP-BaP), por comparação com a dieta contendo microplásticos não contaminados (MP). Outros autores demostraram também que a presença de BaP afeta a formação das vértebras e o desenvolvimento generalizado do esqueleto, no entanto os mecanismos envolvidos nestes fenómenos permanecem pouco estudados. Desta forma, realizámos ensaios adicionais in vivo com o objetivo de avaliar os efeitos osteotóxicos do BaP durante o desenvolvimento e regeneração óssea em peixe-zebra. A exposição aguda de larvas de peixe-zebra entre os 3 e os 6 dpf ao BaP levou a uma redução do tamanho do osso opercular e uma diminuição quantitativa de células positivas para osteocalcina, indicando um efeito composto na maturação dos osteoblastos. Por sua vez, quando se trata de uma exposição crônica das larvas de peixe-zebra ao BaP, entre os 3 e os 30 dpf, verificou-se que o desenvolvimento do esqueleto axial é afetado, aumentando a incidência e gravidade das deformações esqueléticas. Em peixes jovens adultos, observou-se ainda que a exposição ao BaP afetou não só a mineralização dos raios da barbatana caudal e escamas recém-formados, como prejudicou também o seu padrão morfológico, fenómenos que têm por base uma remodelação óssea desequilibrada. Relativamente às análises de expressão genética de vários marcadores verificou-se que o BaP induziu a ativação das vias xenobióticas e metabólicas e impactou negativamente na formação e organização da matriz extracelular. Curiosamente, a exposição ao BaP regulou positivamente os marcadores de inflamação nas larvas e aumentou o recrutamento de neutrófilos. Uma interação direta entre neutrófilos e a matriz extracelular óssea, ou células responsáveis pela formação do osso, foi observada in vivo, o que sugere um papel dos neutrófilos nos mecanismos subjacentes à osteotoxicidade do BaP. Globalmente, os resultados obtidos no âmbito deste trabalho sugerem que a exposição crónica a microplásticos inalterados e/ou contaminados não prejudica apenas o crescimento dos peixes, mas também o seu desempenho a nível reprodutivo, saúde geral do seu esqueleto, e compromete a descendência por meio de efeitos intergeracionais. Além disso, este trabalho fornece novos dados sobre os principais mecanismos celulares e moleculares envolvidos na osteotoxicidade do BaP e aborda o possível papel dos neutrófilos na redução da resposta óssea inflamatória.The presence of microplastics in the aquatic ecosystem represents a major issue for the environment and human health. The capacity of organic pollutants such as benzo[α]pyrene (BaP) to adsorb onto microplastic particles raises additional concerns, as it creates a new route for toxic compounds to enter the food web. Current knowledge on the impact of pristine and/or contaminated microplastics on aquatic organisms remains insufficient, and this work aims at providing new insights by evaluating their biological effects in zebrafish (Danio rerio). The ZEB316, a standalone housing system built with inert materials, and a comprehensive set of ImageJ semi-automatic tools were first developed and optimized to perform state-of-the-art toxicological studies and obtain meaningful data from morphometric analysis of brightfield/ fluorescence images. Zebrafish larvae were exposed throughout their development to polyethylene microplastics, pristine or spiked with BaP, supplemented in the fish diet. While exposure up to 30 days post-fertilization only increased the incidence of skeletal deformities, long-term exposure to pristine/contaminated microplastics not only jeopardized fish growth, reproduction performance, and skeletal health, but it also caused an intergenerational effect. To further study the mechanisms underlying BaP osteotoxicity, several bone-related in vivo assays were used to evaluate the effects of waterborne exposure to BaP during bone development and regeneration. BaP inhibited osteoblast maturation and ECM mineralization and stimulated osteoclast activity, thus affecting bone remodeling. Transgenic and transcriptomic approaches suggested that besides the activation of xenobiotic and metabolic pathways, which may negatively impact extracellular matrix formation and organization, BaP activates inflammatory mechanisms that recruit neutrophils, which affect both osteoblast and osteoclast activity, possibly through a direct interaction of the neutrophils with the bone matrix. This work provides novel data on the effects of microplastics exposure during zebrafish development, in particular its osteotoxic effects, and gives new insights into the cellular and molecular players involved in BaP osteotoxicity.Marco Tarasco was supported by the Portuguese Foundation for Science and Technology (FCT) through the PhD grant SFRH/BD/128634/2017 and COVID/BD/151848/2021 and by NEUBIAS-COST STSM program as part of action CA15124. This work was funded by FCT through projects UID/Multi/04326/2019, UID/00350/2020, UIDB/04326/2020, UID/MAR/00350/2013 and from the operational programs MAR2020 and COMPETE 2020 through projects OSTEOMAR MAR-02.01.01-FEAMP-0057 and EMBRC.PT ALG-01-0145-FEDER-022121

Tissue Targets, Molecular Mechanisms and Health Effects of Bisphenolic Chemicals in Zebrafish

Bisphenol A (BPA) is a chemical incorporated in plastics and resins used for food and beverage containers that has been shown to have estrogenic activity. The fact that BPA possess this activity should not be surprising as it was originally explored for use as a pharmaceutical estrogen. Exposure to BPA has been associated with adverse reproductive and developmental effects in wildlife and laboratory animal models. There are also associations between exposure in humans and adverse health effects, although some of these findings are controversial. The mechanism(s) of action of BPA are well researched, however there is no definitive explanation for the frequently reported discrepancies between in vitro and in vivo studies. Metabolic activation of BPA in vivo has been suggested as a possible reason for this discrepancy in estrogenic potency. As public awareness of the possible health effects of BPA increases manufacturers have increasingly started to use replacement chemicals as monomers in materials that can be labelled as BPA-free. However there is still little information on the estrogenic potency of these structurally similar bisphenol chemicals or how they may affect health outcomes, as observed with BPA. The studies conducted in this thesis therefore aimed to investigate the tissue targets, molecular mechanisms and health effects of BPA, its related chemicals Bisphenol S (BPS), Bisphenol F (BPF) and Bisphenol AF (BPAF) and the BPA metabolite 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP). For this work, a novel ERE transgenic (ERE-TG) zebrafish, that expresses green fluorescent protein (GFP) in response to activation of ERE was employed. These fish can be applied to identify body targets of environmental estrogens in real time with high sensitivity and specificity. BPA, BPF, BPS and BPAF were shown to all preferentially target the heart in ERE-TG zebrafish and GFP induction occurred first in the heart out of the different responding body tissues. The response to BPA was shown to be dependent on the classical estrogen receptor (ER) signalling pathway. However concentrations necessary to induce this response varied for the different bisphenols, with the rank order of potency of BPAF>BPA=BPF>BPS. Bioconcentration factors of the bisphenols were 4.5, 17.8, 5.3 and 0.067 for exposures to 1000 µg BPA/l, 1000 µg BPF/l, 100 µg BPAF/l and 50000 µg BPS/l respectively. These data indicate bioavailability is an important consideration in the differing estrogenic potencies of the different bisphenols. The toxicities of the different bisphenols on early life stage zebrafish followed a similar rank potency order as for the estrogenic activity (BPAF>BPA>BPF>BPS). Specific morphological abnormalities were observed for the different bisphenolic chemical treatments in the toxicity assessments, possibly suggesting that they may act through different ways in inducing their toxic effects. It is recognised that the toxicities for the bisphenolic chemicals were observed at concentrations several orders of magnitude higher than those measured in most aquatic environments and thus the threat they pose to wildlife health might be considered as relatively low, except in circumstances where short but high exposures may occur from accidental release into the environment. The BPA metabolite MBP was found to be up to 1000-fold more potent than the parent compound as an estrogen in ERE-TG fish. The heart was a key target tissue for MBP, as observed for the other bisphenolic compounds. The atrioventricular valves and bulbus arteriosus were identified as the primary targets within the heart. MBP was not measured in zebrafish embryos exposed to BPA and whether this is produced as a metabolite in zebrafish is still not known. Morpholino knockdown of specific ER subtypes indicated that esr1 is a major pathway for the estrogenic response to BPA in the heart during early life stages of zebrafish. Video capture and analysis was used to assess the cardiovascular health of zebrafish exposed to BPA and it was found that at very high exposure concentrations (2500 µg/l) BPA could induce an unstable atrial:ventricular beat ration in 5 dpf larvae and reduced heart beat rate in 14 dpf. In the final study of this thesis transcriptomic profiling was conducted on hearts extracted from 96 hpf ERE-TG zebrafish larvae exposed to BPA. The findings demonstrated that BPA, at an exposure concentration of 150 µg/l caused a down-regulation of a number of genes associated with ion transport and cell-to-cell communication, functions that are essential in maintaining a regular and consistent heart rate. These effect mechanisms may help to explain the effects on the heart seen at the higher BPA exposure concentrations in the previous chapter, although this would need more extensive work to draw any such associations with good confidence Overall, the findings presented in this thesis have provided a body of evidence to show that all of the bisphenolic chemicals tested possess estrogenic activity and as such have the potential for health effects in wildlife and also to humans. It is also the case however that currently in most ambient environments concentrations of these bisphenolic chemicals are far below those that could induce adverse health outcomes. The work in this thesis re-enforces the importance of understanding metabolic activation of chemicals in vivo. It furthermore illustrates the power of transgenic fish and an integrated approach for gaining greater insight into potential health effects of chemicals.NER

Data collection in support of the Endocrine Disruption (ED) assessment for non-target vertebrates

Disclaimer: The present document has been produced and adopted by the bodies identified above as author(s). This task has been carried out exclusively by the author(s) in the context of a contract between the European Food Safety Authority and the author(s), awarded following a tender procedure. The present document is published complying with the transparency principle to which the Authority is subject. It may not be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.To harmonise vertebrate OECD Test Guidelines for endocrine disruption testing between mammalian and non‐mammalian test species, additional Estrogen, Androgen, Thyroid and Steroidogenesis (EATS) modality endpoints in non‐mammalian models need to be assessed. These would mean for example the addition of hormonal measurements in fish, birds and amphibians. Furthermore, a better reporting of gross pathology findings for birds would also be considered advantageous for the assessment of endocrine disrupting properties.To facilitate adoption of additional measures, guidance on how to perform, report and evaluate these new endpoints is required. In this report, a variety of methods including a systematic evidence map, an extensive literature review and a survey of ecotoxicology laboratories were adopted to collect data on the topic. The systematic evidence map uncovered a range of methods for measuring sex and thyroid hormones in fish, birds and amphibians, although methods for measuring sex‐hormones in fish, were by far, the most frequently encountered in the literature and laboratory survey. However, there are still considerable gaps in knowledge for: optimum sample timing for hormonal measurement (diurnal, developmental stage, etc.), issues with inherent variability, low sample volume (plasma/serum), test species selection, possible impacts of housing/diet/stress. The extensive literature review revealed that although gross pathology and histopathology have been used to investigate the effects of endocrine disrupting chemicals in birds, there are no standardised methods for assessment or interpretation, although relative weight of endocrine organs is frequently used as a gross pathology metric. Reports of how histopathology was assessed varied considerably. From the survey, few contract laboratories are experienced in conducting the avian test guideline and these types of pathology techniques.Recommendations for future work with non‐mammalian taxa include: investigating the optimal time (or timings) for measuring hormones, developing non‐invasive hormone measuring techniques, gaining knowledge of baseline/control hormonal data, and further developing guidance on conducting and assessing gross pathology and histopathology in birds

Plasticity within the Auditory Systems of Fishes

Fishes inhabit incredibly cacophonous environments and experience functional, morphological, and transcriptional auditory system plasticity in reproductive state-dependent and auditory experiential contexts. In contrast to the comprehensive study of acoustic overexposure and functional reproductive condition-dependent plasticity within the auditory periphery, the mechanisms underlying acoustic experience-mediated central nervous system plasticity in fishes are generally poorly characterized. Recent research has highlighted neurochemical and transcriptional flexibility within the central nervous systems of fishes in response to prolonged exposure to music. However, the contributions of the acoustic characteristics of musical stimulation to central nervous system plasticity remain unclear. To evaluate the contributions of sound stimulus frequency to brain plasticity, I employed a targeted transcriptional analysis of neuroplasticity-associated genes within the brain of zebrafish (Danio rerio) exposed to 100 Hz and 800 Hz continuous pure tones at a sound pressure level of 140 dB (re 1 μPa) for 1-week intervals across a 4-week period. The transcription of genes involved in mediating connective plasticity fluctuated as a function of duration and frequency of sound exposure, while cellular proliferation did not show variation with sound treatment; suggesting prolonged tonal stimulation may facilitate connective plasticity within the zebrafish brain. These results provide evidence of central nervous system plasticity in response to pure tone exposure and implicate sound-induced behaviour and multisensory inputs in the mediation of sound-induced transcriptional flexibility within the zebrafish brain. Collectively, this thesis highlights the complexity of auditory system plasticity and emphasizes the value of investigating acoustic experience-mediated nervous system plasticity beyond the auditory periphery in fishes

Comparison of electrophysiological auditory measures in fishes

© Springer International Publishing Switzerland 2016. Sounds provide fishes with important information used to mediate behaviors such as predator avoidance, prey detection, and social communication. How we measure auditory capabilities in fishes, therefore, has crucial implications for interpreting how individual species use acoustic information in their natural habitat. Recent analyses have highlighted differences between behavioral and electrophysiologically determined hearing thresholds, but less is known about how physiological measures at different auditory processing levels compare within a single species. Here we provide one of the first comparisons of auditory threshold curves determined by different recording methods in a single fish species, the soniferous Hawaiian sergeant fish Abudefduf abdominalis, and review past studies on representative fish species with tuning curves determined by different methods. The Hawaiian sergeant is a colonial benthic-spawning damselfish (Pomacentridae) that produces low-frequency, low-intensity sounds associated with reproductive and agonistic behaviors. We compared saccular potentials, auditory evoked potentials (AEP), and single neuron recordings from acoustic nuclei of the hindbrain and midbrain torus semicircularis. We found that hearing thresholds were lowest at low frequencies (~75–300 Hz) for all methods, which matches the spectral components of sounds produced by this species. However, thresholds at best frequency determined via single cell recordings were ~15–25 dB lower than those measured by AEP and saccular potential techniques. While none of these physiological techniques gives us a true measure of the auditory “perceptual” abilities of a naturally behaving fish, this study highlights that different methodologies can reveal similar detectable range of frequencies for a given species, but absolute hearing sensitivity may vary considerably

Structure-function properties of the gastrodigestive and hepatic systems of zebrafish (Danio rerio)

While they lack mammal-specific organs, zebrafish provide a high degree of resemblance in their genetic profile, molecular mechanisms and organ physiology to humans and have been established as an excellent complementary platform to rodents. However, their use in gastroenterology and hepatology is under-utilised, conceivably due to a lack of digestive system ultrastructural details as most anatomical studies were performed by light and fluorescence imaging. This thesis provides detailed insights into the structure and function of the zebrafish digestive system, particularly the liver. Multimodal bio-imaging approaches were developed in order to investigate the hepatic ultrastructure and function. Using a protocol that renders samples compatible with multiple imaging platforms, we produce a detailed map of the zebrafish gastrodigestive system from organ to subcellular levels. Findings were compared with the rodent/human counterparts and while some differences exist between the zebrafish and the rodent/human hepatic parenchymal cells and biliary system organisations, many similarities, at the sub/cellular levels, were also demonstrated. Using advances in genetics and a protocol that retains endogenous fluorescence within zebrafish at the same time as ultrastructure for electron microscopy, we further investigated key hepatic functional properties (e.g. macromolecular transport routes) by performing albumin injections and studying the liver macrophages. While we demonstrated similarities in the albumin uptake pathway and in the morphology of liver macrophages in zebrafish, we reveal that zebrafish liver macrophages lack of phagocytic function (a key aspect in rodents and human), which may limit their use in hepatic-immune diseases studies. Altogether, our studies provide new insights and novel protocols for the analysis of the zebrafish liver and lay a foundation to further evaluate uptake routes for gastro-digestive research and drug delivery in various diseases