Using the self-fertilizing mangrove killifish, we characterized two mutants, shorttail (stl) and balltail (btl). These mutants showed abnormalities in the posterior notochord and muscle development. Taking advantage of a highly inbred isogenic strain of the species, we rapidly identified the mutated genes, noto and msgn1 in the stl and btl mutants, respectively, using a single lane of RNA sequencing without the need of a reference genome or genetic mapping techniques. Next, we confirmed a conserved morphant phenotype in medaka and demonstrate a crucial role of noto and msgn1 in cell sorting between the axial and paraxial part of the tail mesoderm. This novel system could substantially accelerate future small-scale forward-genetic screening and identification of mutations. Therefore, the mangrove killifish could be used as a complementary system alongside existing models for future molecular genetic studies.

The Arabian killifish, Aphanius dispar, is a small tropical teleost fish living in wide range of habitats in sea water and fresh water in the Middle East. Here, we report extraordinary fluorescent pigment cells in the Arabian killifish embryo. These cells appear brown in transmitted light, yellowish white in reflected light, and as strong fluorescence in GFP and RFP filters. TEM and confocal microscopy analyses show the fluorescence emanates from leucosome-like pigment organelles. The cells express the gene encoding GTP cyclohydrolase (gch), a marker for leucophores and xanthophore. Gene knockdown and knockout of gch using morpholino or CRISPR-Cas9 induced loss of fluorescence in these embryos, indicating a crucial role of the enzyme and the associated pterine biosynthesis pathway in the generation of the fluorescence. We concluded that these cells are a highly fluorescent subtype of leucophores and have named them as fluoroleucophores.

Background: Reactive oxygen species (ROS) arise as a result from, and are essential in, numerous cellular processes. ROS, however, are highly reactive and if left unneutralised by endogenous antioxidant systems, can result in extensive cellular damage and/or pathogenesis. In addition, exposure to a wide range of environmental stressors can also result in surplus ROS production leading to oxidative stress (OS) and downstream tissue toxicity. Objectives: Our aim was to produce a stable transgenic zebrafish line, unrestricted by tissue-specific gene regulation, which was capable of providing a whole organismal, real-time read-out of tissue-specific OS following exposure to a wide range of OS-inducing environmental contaminants and conditions. This model could, therefore, serve as a sensitive and specific mechanistic in vivo biomarker for all environmental conditions that result in OS. Methods: to achieve this aim, we exploited the pivotal role of the electrophile response element (EpRE) as a globally-acting master regulator of the cellular response to OS. To test tissue specificity and quantitative capacity, we selected a range of chemical contaminants known to induce OS in specific organs or tissues, and assessed dose-responsiveness in each using microscopic measures of mCherry fluorescence intensity. Results: We produced the first stable transgenic zebrafish line Tg (3EpRE:hsp70:mCherry) with high sensitivity for the detection of cellular RedOx imbalances, in vivo in near-real time. We applied this new model to quantify OS after exposure to a range of environmental conditions with high resolution and provided quantification both of compound- and tissue-specific ROS-induced toxicity. Discussion: Our model has an extremely diverse range of potential applications not only for biomonitoring of toxicants in aqueous environments, but also in biomedicine for identifying ROS-mediated mechanisms involved in the progression of a number of important human diseases, including cancer.

The yolk syncytial layer (YSL) in the zebrafish embryo is a multinucleated syncytium essential for embryo development, but the molecular mechanisms underlying YSL formation remain largely unknown. Here we show that zebrafish solute carrier family 3 member 2 (Slc3a2) is expressed specifically in the YSL and that slc3a2 knockdown causes severe YSL defects including clustering of the yolk syncytial nuclei and enhanced cell fusion, accompanied by disruption of microtubule networks. Expression of a constitutively active RhoA mimics the YSL phenotypes caused by slc3a2 knockdown, whereas attenuation of RhoA or ROCK activity rescues the slc3a2-knockdown phenotypes. Furthermore, slc3a2 knockdown significantly reduces tyrosine phosphorylation of c-Src, and overexpression of a constitutively active Src restores the slc3a2-knockdown phenotypes. Our data demonstrate a signaling pathway regulating YSL formation in which Slc3a2 inhibits the RhoA/ROCK pathway via phosphorylation of c-Src to modulate YSL microtubule dynamics. This work illuminates processes at a very early stage of zebrafish embryogenesis and more generally informs the mechanism of cell dynamics during syncytium formation.

Brominated flame retardants are known to disrupt thyroid hormone (TH) homeostasis in several vertebrate species, but the molecular mechanisms underlying this process and their effects on TH-sensitive tissues during the stages of early development are not well characterised. In this study, we exposed zebrafish (Danio rerio) embryo-larvae to 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and tetrabromobisphenol a (TBBPA) via the water for 96 h from fertilisation and assessed for lethality, effects on development and on the expression of a suite of genes in the hypothalamic-pituitary-thyroid (HPT) axis via both real time quantitative PCR (qRT-PCR) on whole body extracts and whole mount in situ hybridisation (WISH) to identify tissue targets. The 96-h lethal median concentration (96h-LC50) for TBBPA was 0.9 μM and mortality was preceded by retardation of development (smaller animals) and morphological deformities including, oedemas in the pericardial region and tail, small heads, swollen yolk sac extension. Exposure to BDE-47 did not affect zebrafish embryo-larvae survival at any of the concentrations tested (1 – 100 μM) but caused yolk sac and craniofacial deformities, a curved spine and shorter tail at the highest exposure concentration. TBBPA exposure resulted in higher levels of mRNAs for genes encoding deiodinases (dio1), transport proteins (ttr), the thyroid follicle synthesis protein paired box 8 (pax8) and glucuronidation enzymes (ugt1ab) and lower levels of dio3b mRNAs in whole body extracts, with responses varying with developmental stage. BDE-47 exposure resulted in higher levels of thrb, dio1, dio2, pax8 and ugt1ab mRNAs and lower levels of ttr mRNAs in whole body extracts. TBBPA and BDE-47 therefore appear to disrupt the TH system at multiple levels, increasing TH conjugation and clearance, disrupting thyroid follicle development and altering TH transport. Compensatory responses in TH production/ metabolism by deiodinases were also evident. WISH analyses further revealed that both TBBPA and BDE-47 caused tissue-specific changes in thyroid receptor and deiodinase enzyme expression, with the brain, liver, pronephric ducts and craniofacial tissues appearing particularly responsive to altered TH signalling. Given the important role of TRs in mediating the actions of THs during key developmental processes and deiodinases in the control of peripheral TH levels, these transcriptional alterations may have implications for TH sensitive target genes involved in brain and skeletal development. These findings further highlight the potential vulnerability of the thyroid system to disruption by BFRs during early developmental windows.

Estrogens are well-known to regulate development of sexual dimorphism of the brain; however, their role in embryonic brain development prior to sex-differentiation is unclear. Using estrogen biosensor zebrafish models, we found that estrogen activity in the embryonic brain occurs from early neurogenesis specifically in a type of glia in the olfactory bulb (OB), which we name estrogen-responsive olfactory bulb (EROB) cells. In response to estrogen, EROB cells overlay the outermost layer of the OB and interact tightly with olfactory sensory neurons at the olfactory glomeruli. Inhibiting estrogen activity using an estrogen receptor antagonist, ICI182,780 (ICI), and/or EROB cell ablation impedes olfactory glomerular development, including the topological organisation of olfactory glomeruli and inhibitory synaptogenesis in the OB. Furthermore, activation of estrogen signalling inhibits both intrinsic and olfaction-dependent neuronal activity in the OB, whereas ICI or EROB cell ablation results in the opposite effect on neuronal excitability. Altering the estrogen signalling disrupts olfaction-mediated behaviour in later larval stage. We propose that estrogens act on glia to regulate development of OB circuits, thereby modulating the local excitability in the OB and olfaction-mediated behaviour.

Spring viraemia of carp virus (SVCV) is an OIE listed, notifiable disease affecting carp aquaculture. Disease outbreaks can lead to mass mortality events which poses significant economic threats to farmers and fishery owners. However, SVCV research has been limited due to the lack of full-genome sequences available for isolates from all genogroups, as well as limited information on SVCV pathogenicity. This study performed full-genome sequencing on several SVCV isolates from each of the recognised genogroups. Phylogenetic analysis of these sequences confirmed that isolates fall into 4 genogroups Ia-Id, which had been previously reported in literature. Interestingly, there was an additional sequence complementary to the grass carp thymosin beta-a gene in the 3’ non-coding region of the G gene of a few of the Asian strains (Ia) that could influence the virus replication in that host. A conventional RT-PCR assay was designed to study the prevalence and distribution of the unusual additional sequence. Sequence alignments were used to design a new SVCV-specific TaqMan RT-qPCR assay based on the polymerase (L) gene. Testing of this assay on representative isolates from genogroups Ia-Id showed it was highly specific; detecting all SVCV isolates tested at low virus loads and did not cross-react with other closely related Spriviviruses. An in vitro investigation into differences in the innate immune response to isolates from different genogroups revealed that the Ia genogroup may be able to evade the immune response to promote their own replication whereas isolates from the Ib genogroup induced a strong anti-viral response that could play a role in reducing their pathogenicity. This study has also made good progress towards the development of a reverse genetics reporter system for SVCV and VHSV, incorporating green fluorescent protein (GFP) as a reporter gene. This information can be used going forward to develop a complete reverse genetics system for SVCV in which viable virus is generated from DNA, and then through genetic mutation allow us to learn more about the mechanisms involved in SVCV pathogenicity.

Using the self-fertilizing mangrove killifish, we characterized two mutants, shorttail (stl) and balltail (btl). These mutants showed abnormalities in the posterior notochord and muscle development. Taking advantage of a highly inbred isogenic strain of the species, we rapidly identified the mutated genes, noto and msgn1 in the stl and btl mutants, respectively, using a single lane of RNA sequencing without the need of a reference genome or genetic mapping techniques. Next, we confirmed a conserved morphant phenotype in medaka and demonstrate a crucial role of noto and msgn1 in cell sorting between the axial and paraxial part of the tail mesoderm. This novel system could substantially accelerate future small-scale forward-genetic screening and identification of mutations. Therefore, the mangrove killifish could be used as a complementary system alongside existing models for future molecular genetic studies.

Effluents from wastewater treatment works (WwTW) exhibit both temporal and spatial variation in oestrogenicity, however few studies have attempted to quantify how this variation affects biological responses in fish. Here we used an oestrogen-responsive green fluorescent protein (ERE-GFP) transgenic zebrafish (Danio rerio) to quantify oestrogenic activity and health effects for exposure to three different WwTW effluents. Endpoints measured included survival/hatching rate, GFP induction (measured in target tissues or gfp mRNA induction in whole embryos) and vtg mRNA induction in whole embryos. Exposure to one of the study effluents (at 100%), resulted in some mortality, and exposure to all three effluents (at 50% and 100%) caused decreases in hatching rates. Higher levels of vtg mRNA corresponded with higher levels of steroidal oestrogens in the different effluents, with lowest-observed-effect concentrations (LOECs) between 31 ng/L and 39 ng/L oestradiol equivalents (EEQs). Tissue patterns of GFP expression for all three WwTWs effluents reflected the known targets for steroidal oestrogens and for some other oestrogenic chemicals likely present in those effluents (i.e. nonylphenol or bisphenolic compounds). GFP induction was similarly responsive to vtg mRNA induction (a well-established biomarker for oestrogen exposure). We thus demonstrate the ERE-GFP transgenic zebrafish as an effective model for monitoring the oestrogenic potency and health effects for exposure to complex mixtures of chemicals contained within WwTW effluents.

The Arabian killifish, Aphanius dispar, is a small tropical teleost fish living in wide range of habitats in sea water and fresh water in the Middle East. Here, we report extraordinary fluorescent pigment cells in the Arabian killifish embryo. These cells appear brown in transmitted light, yellowish white in reflected light, and as strong fluorescence in GFP and RFP filters. TEM and confocal microscopy analyses show the fluorescence emanates from leucosome-like pigment organelles. The cells express the gene encoding GTP cyclohydrolase (gch), a marker for leucophores and xanthophore. Gene knockdown and knockout of gch using morpholino or CRISPR-Cas9 induced loss of fluorescence in these embryos, indicating a crucial role of the enzyme and the associated pterine biosynthesis pathway in the generation of the fluorescence. We concluded that these cells are a highly fluorescent subtype of leucophores and have named them as fluoroleucophores.

Given current risks of pollutant exposures in aquatic environments, there is a growing need to generate reliable computational risk assessment methods to establish how adverse outcomes can be produced across exposure organisms. The adaptive stress response is widely targeted by pollutants of concern and includes transcription factors including nuclear factor (erythroid- derived 2)-like 2 (Nrf2), hypoxia inducible factor (HIF-1α), heat shock factor (HSF1), nuclear factor kappa-light-chain-enhancer of activated -B cells (NFkB), metal transcription factor 1 (MTF1), the aryl hydrocarbon receptor (AhR) and tumor protein P53 (P53). While these TFs are known to be activated by distinct inducers, less is understood about the regulatory links between factors, particularly at the transcription factor (TF) DNA-binding level.
In this thesis, a gene regulatory network (GRN) of adaptive-stress response factors that are key targets of chemical toxicity was constructed based on experimental evidence from mammalian cell-lines. The GRN was modeled using boolean logic and this identified a number of response outcomes that could be attributed to the activation of pathways including antioxidant defence processes and glucose metabolism. The GRN model illustrated that the activation of Nrf2, HIF-1α, AhR, MTF1 and HSF1 led to the same adverse outcomes, suggesting canalisation in stress response pathways.
The ability to use GRNs across different species is widely supported by the identification of TF binding sites (TFBS) within target genes. To assess the efficiency of using the mammalian GRN across teleost fish species, a comprehensive analysis of validated binding sites for the AhR, MTF1, HIF-1α and Nrf2 was conducted across fish-species in comparison to the mammalian consensus binding sequence. This showed variations in binding site composition across validated TFBS for HIF-1α and Nrf2 in fish compared to the mammalian consensus, preventing the identification of the functional sequences for these factors using traditional methods. To establish if such changes affected the efficiency to predict positive downstream target genes for Nrf2 and HIF-1α in mammals and across teleost fish species, random
1
forest classification models were used to compare the efficiency of multiple positional weight matrices (PWM) motifs of TFBS for Nrf2 and HIF-1α. Whilst the result from this analysis identified discrepancies in the ability to predict target genes based on the mammalian motif file used, mammalian motifs were able to predict target genes across fish species. Validated binding sites in fish species were then aligned to generate PWM motifs and sites were predicted across shared target genes hsp70 and hmox1 using both fish based and mammalian based models. This showed that whilst there was some overlap in identified sites across species, fish-specific motifs identified unique sites from mammalian models.
To validate the GRN, gene-expression responses across exposures traditionally associated with activating distinct adaptive stress response factors were collated across the literature. This showed support for some of the key responses identified in the model. Chemical exposure studies were then undertaken in vivo in embryo-larval zebrafish (2 and 4 dpf) to identify potential connectivity between the TFs NFkB, MTF1 and HIF-1α with Nrf2, a key factor in the adaptive stress-response and a regulator of antioxidant response processes. The inducer of Nrf2, tert-butylhydroquinone (tBHQ), was used to determine if there was a change in transcriptional output of mtf1, hif1a and nfkb1 over time and with exposure concentration. This showed a significant difference in expression for nfkb1 and alterations in expression of mtf1 over prolonged exposure scenarios. In addition, the developmental expression of nrf2a, mtf1, hif1α and nfkb1 from 2 hpf to 96 hpf showed differences between transcript levels with hif1α and nfkb1 having the highest levels of expression compared to nrf2a and mtf1.
Overall, the research presented in this thesis provides a novel approach to assess the initiation of adaptive stress-response factors from molecular interactions. The research goes some way in establishing the feedback loops and connections between NFkB, MTF1, Nrf2, AhR, HIF-1α, HSF1 and P53. In doing so, the model generated in this thesis provides a novel approach of establishing outcomes under toxicant exposures.

It is now well established globally that oestrogens are a major environmental contaminant in WwTW effluent discharging into receiving waterways. Even in developed countries despite advances in wastewater treatment and a general reduction in the incidence of gross point source pollution, effluent from most WwTWs contain detectable estrogenic activity; around a third of English rivers currently are thought to contain levels of oestrogenic chemicals sufficient to cause some endocrine disruption in fish. These disruptions in fish present themselves in a
number of different ways including a sex ratio biased in favour of females, the presence intersex individuals; delayed sexual maturation; and alterations in reproductive behaviour. This illustrate the need for a deeper understanding on how oestrogenic chemicals act within the body to affect wildlife health.

Using a transgenic oestrogen response element (ERE), green fluorescent protein (GFP) zebrafish model, this thesis work set out to determine tissue targets and the potential for health impacts of environmental oestrogens and their mixtures within effluents from WwTWs and how these responses may vary both seasonally and for exposures for different developmental life stages. This was measured through a
combination of biological endpoints including mortality/hatching rates, length, weight and condition factor (in adults), vitellogenin (vtg) induction (mRNA) in whole body embryos and in the livers of adults, and also through GFP quantification (measured by fluorescence microscopy on target tissues, Western blotting for whole body GFP induction and GFP mRNA induction).

The body of thesis work also sought to further establish how fluorescence responses in the transgenic zebrafish model compared in terms of sensitivity for the detection of oestrogens with vitellogenin (VTG) induction, a well-established biomarker for oestrogen exposure in fish. The ontogeny of endogenous GFP expression in individual fish was also followed from early life stages through the period of sexual differentiation using fluorescent microscopy, to determine if endogenous GFP expression could be used as a sex marker in our zebrafish
model.

In the WwTW effluent studies, differences in GFP tissue patterning (and of other biological endpoints such as vtg induction) were found to be reflective of the varying concentrations of steroidal oestrogens found in the different wastewaters, (both temporally and between treatment works), and they also illustrated the considerable dynamic nature of the oestrogenic potencies of the WwTW effluents. For chronic effluent exposures, biological responses to both the oestrogenic effluent and the synthetic oestrogen EE2 were differed between the sexes particularly in terms of overall growth and sex differentiation parameters.

For my investigation into endogenous GFP expression, I found varying levels of fluorescence, throughout the various life stages of our zebrafish to 30dpf. There were four different patterns of GFP expression across the different body tissues recorded, based on the tissues expressing GFP and the timing and duration of that expression. Gonadal sex (measured via histology at 60dph) however was associated only with sex for one of the recorded GFP expression patterns (GFP expression in the liver at 30dph – corresponding to the female phenotype).

Overall, the results from the studies presented in this thesis illustrate the potential for differing health effects of both individual exogenous oestrogens and for a single WwTW effluent over time/season and for effluents from different WwTW. The work also highlights the importance in the timing (developmental stage) of exposure on the biological effects seen. The ability of ERE-GFP transgenic zebrafish model to allow for the analysis of individual tissues, such as the heart or brain, through non-destructive fluorescence microscopy, further demonstrates its significant
advantages for screening and testing for environmental oestrogens.

Whilst these results suggest that GFP induction is less sensitive than VTG
induction per se, related (in part) to the greater magnitude of response for VTG induction, there was a good positive correlation between the two variables illustrating further the utility of the GFP induction as a bio-monitor to detect for the presence of exogenous oestrogens contained in real world effluents. These findings further highlight the importance of integrating biomonitoring approaches to complement analytical chemistry techniques in the monitoring of oestrogenic chemicals in wastewater effluents.

Differences, in the levels of endogenous GFP expression across the different body tissues during early life in our zebrafish model, and prior to gonadal differentiation, are likely a reflection of the varying functional roles oestrogen receptors perform during development (i.e. they are under spatial-temporal control) and are not thought to provide an accurate marker for the determination of sex in the zebrafish
model but could be utilised as a phenotypic marker for identifying females at 30dpf.

With a view towards the future, additional steps now need to be taken in order to further corroborate these findings and to maximise the potential of the transgenic ERE-GFP zebrafish model system. These include the need for identifying the molecular mechanisms of oestrogenic chemicals and of oestrogen responsive genes and pathways, particularly in the responsive target tissues identified in this thesis, to better inform on adverse health outcomes.

Ultimately, the results of this thesis further highlight how the development and application of transgenic fish models can offer huge potential for more integrative health effects assessments, allowing for an improved understanding of the risks posed by oestrogenic chemicals, and for tailoring future approaches to ERA strategies, specifically in respect of AOP frameworks.

Background: Reactive oxygen species (ROS) arise as a result from, and are essential in, numerous cellular processes. ROS, however, are highly reactive and if left unneutralised by endogenous antioxidant systems, can result in extensive cellular damage and/or pathogenesis. In addition, exposure to a wide range of environmental stressors can also result in surplus ROS production leading to oxidative stress (OS) and downstream tissue toxicity. Objectives: Our aim was to produce a stable transgenic zebrafish line, unrestricted by tissue-specific gene regulation, which was capable of providing a whole organismal, real-time read-out of tissue-specific OS following exposure to a wide range of OS-inducing environmental contaminants and conditions. This model could, therefore, serve as a sensitive and specific mechanistic in vivo biomarker for all environmental conditions that result in OS. Methods: to achieve this aim, we exploited the pivotal role of the electrophile response element (EpRE) as a globally-acting master regulator of the cellular response to OS. To test tissue specificity and quantitative capacity, we selected a range of chemical contaminants known to induce OS in specific organs or tissues, and assessed dose-responsiveness in each using microscopic measures of mCherry fluorescence intensity. Results: We produced the first stable transgenic zebrafish line Tg (3EpRE:hsp70:mCherry) with high sensitivity for the detection of cellular RedOx imbalances, in vivo in near-real time. We applied this new model to quantify OS after exposure to a range of environmental conditions with high resolution and provided quantification both of compound- and tissue-specific ROS-induced toxicity. Discussion: Our model has an extremely diverse range of potential applications not only for biomonitoring of toxicants in aqueous environments, but also in biomedicine for identifying ROS-mediated mechanisms involved in the progression of a number of important human diseases, including cancer.

Given current risks of pollutant exposures in aquatic environments, there is a growing need to generate reliable computational risk assessment methods to establish how adverse outcomes can be produced across exposure organisms. The adaptive stress response is widely targeted by pollutants of concern and includes transcription factors including nuclear factor (erythroid- derived 2)-like 2 (Nrf2), hypoxia inducible factor (HIF-1α), heat shock factor (HSF1), nuclear factor kappa-light-chain-enhancer of activated -B cells (NFkB), metal transcription factor 1 (MTF1), the aryl hydrocarbon receptor (AhR) and tumor protein P53 (P53). While these TFs are known to be activated by distinct inducers, less is understood about the regulatory links between factors, particularly at the transcription factor (TF) DNA-binding level.
In this thesis, a gene regulatory network (GRN) of adaptive-stress response factors that are key targets of chemical toxicity was constructed based on experimental evidence from mammalian cell-lines. The GRN was modeled using boolean logic and this identified a number of response outcomes that could be attributed to the activation of pathways including antioxidant defence processes and glucose metabolism. The GRN model illustrated that the activation of Nrf2, HIF-1α, AhR, MTF1 and HSF1 led to the same adverse outcomes, suggesting canalisation in stress response pathways.
The ability to use GRNs across different species is widely supported by the identification of TF binding sites (TFBS) within target genes. To assess the efficiency of using the mammalian GRN across teleost fish species, a comprehensive analysis of validated binding sites for the AhR, MTF1, HIF-1α and Nrf2 was conducted across fish-species in comparison to the mammalian consensus binding sequence. This showed variations in binding site composition across validated TFBS for HIF-1α and Nrf2 in fish compared to the mammalian consensus, preventing the identification of the functional sequences for these factors using traditional methods. To establish if such changes affected the efficiency to predict positive downstream target genes for Nrf2 and HIF-1α in mammals and across teleost fish species, random
1
forest classification models were used to compare the efficiency of multiple positional weight matrices (PWM) motifs of TFBS for Nrf2 and HIF-1α. Whilst the result from this analysis identified discrepancies in the ability to predict target genes based on the mammalian motif file used, mammalian motifs were able to predict target genes across fish species. Validated binding sites in fish species were then aligned to generate PWM motifs and sites were predicted across shared target genes hsp70 and hmox1 using both fish based and mammalian based models. This showed that whilst there was some overlap in identified sites across species, fish-specific motifs identified unique sites from mammalian models.
To validate the GRN, gene-expression responses across exposures traditionally associated with activating distinct adaptive stress response factors were collated across the literature. This showed support for some of the key responses identified in the model. Chemical exposure studies were then undertaken in vivo in embryo-larval zebrafish (2 and 4 dpf) to identify potential connectivity between the TFs NFkB, MTF1 and HIF-1α with Nrf2, a key factor in the adaptive stress-response and a regulator of antioxidant response processes. The inducer of Nrf2, tert-butylhydroquinone (tBHQ), was used to determine if there was a change in transcriptional output of mtf1, hif1a and nfkb1 over time and with exposure concentration. This showed a significant difference in expression for nfkb1 and alterations in expression of mtf1 over prolonged exposure scenarios. In addition, the developmental expression of nrf2a, mtf1, hif1α and nfkb1 from 2 hpf to 96 hpf showed differences between transcript levels with hif1α and nfkb1 having the highest levels of expression compared to nrf2a and mtf1.
Overall, the research presented in this thesis provides a novel approach to assess the initiation of adaptive stress-response factors from molecular interactions. The research goes some way in establishing the feedback loops and connections between NFkB, MTF1, Nrf2, AhR, HIF-1α, HSF1 and P53. In doing so, the model generated in this thesis provides a novel approach of establishing outcomes under toxicant exposures.

Estrogen plays fundamental roles in a range of developmental processes and exposure to estrogen mimicking chemicals has been associated with various adverse health effects in both wildlife and human populations. Estrogenic chemicals are found commonly as mixtures in the environment and can have additive effects, however risk analysis is typically conducted for single-chemicals with little, or no, consideration given for an animal's exposure history. Here we developed a transgenic zebrafish with a photoconvertable fluorophore (Kaede, green to red on UV light exposure) in a skin pigment-free mutant element (ERE)-Kaede-Casper model and applied it to quantify tissue-specific fluorescence biosensor responses for combinations of estrogen exposures during early life using fluorescence microscopy and image analysis. We identify windows of tissue-specific sensitivity to ethinylestradiol (EE2) for exposure during early-life (0-5 dpf) and illustrate that exposure to estrogen (EE2) during 0-48 hpf enhances responsiveness (sensitivity) to different environmental estrogens (EE2, genistein and bisphenol A) for subsequent exposures during development. Our findings illustrate the importance of an organism's stage of development and estrogen exposure history for assessments on, and possible health risks associated with, estrogen exposure.

Environmental exposure to Bisphenol a (BPA) has been associated with a range of adverse health effects, including on the cardiovascular system in humans. Lack of agreement on its mechanism(s) of action likely stem from comparisons between in vivo and in vitro test systems and potential multiple effects pathways. In rodents, in vivo, metabolic activation of BPA produces 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which is reported to be up to 1000 times more potent as an estrogen than BPA. We investigated the estrogenic effects and estrogen receptor signaling pathway(s) of BPA and MBP following early life exposure using a transgenic, estrogen responsive (ERE-TG) zebrafish and a targeted morpholino approach to knockdown the three fish estrogen receptor (ER) subtypes. The functional consequences of BPA exposure on the cardiovascular system of zebrafish larvae were also examined. The heart atrioventricular valves and the bulbus arteriosus were primary target tissues for both BPA and MBP in the ERE-TG zebrafish, and MBP was approximately 1000-fold more potent than BPA as an estrogen in these tissues. Estrogen receptor knockdown with morpholinos indicated that the estrogenic responses in the heart for both BPA and MBP were mediated via an estrogen receptor 1 (esr1) dependent pathway. At the highest BPA concentration tested (2500 μg/L), alterations in the atrial:ventricular beat ratio indicated a functional impact on the heart of 5 days post fertilization (dpf) larvae, and there was also a significantly reduced heart rate in these larvae at 14 dpf. Our findings indicate that some of the reported adverse effects on heart function associated with BPA exposure (in mammals) may act through an estrogenic mechanism, but that fish are unlikely to be susceptible to adverse effects on heart development for environmentally relevant exposures.

Although Astyanax mexicanus surface fish regenerate their hearts after injury, their Pachón cave-dwelling counterparts cannot and, instead, form a permanent fibrotic scar, similar to the human heart. Myocardial proliferation peaks at similar levels in both surface fish and Pachón 1 week after injury. However, in Pachón, this peak coincides with a strong scarring and immune response, and ultimately, cavefish cardiomyocytes fail to replace the scar. We identified lrrc10 to be upregulated in surface fish compared with Pachón after injury. Similar to cavefish, knockout of lrrc10 in zebrafish impairs heart regeneration without affecting wound cardiomyocyte proliferation. Furthermore, using quantitative trait locus (QTL) analysis, we have linked the degree of heart regeneration to three loci in the genome, identifying candidate genes fundamental to the difference between scarring and regeneration. Our study provides evidence that successful heart regeneration entails a delicate interplay between cardiomyocyte proliferation and scarring.

© 2017 American Chemical Society. Bisphenol a (BPA), a chemical incorporated into plastics and resins, has estrogenic activity and is associated with adverse health effects in humans and wildlife. Similarly structured BPA analogues are widely used but far less is known about their potential toxicity or estrogenic activity in vivo. We undertook the first comprehensive analysis on the toxicity and teratogenic effects of the bisphenols BPA, BPS, BPF, and BPAF in zebrafish embryo-larvae and an assessment on their estrogenic mechanisms in an estrogen-responsive transgenic fish Tg(ERE:Gal4ff)(UAS:GFP). The rank order for toxicity was BPAF > BPA > BPF > BPS. Developmental deformities for larval exposures included cardiac edema, spinal malformation, and craniofacial deformities and there were distinct differences in the effects and potencies between the different bisphenol chemicals. These effects, however, occurred only at concentrations between 1.0 and 200 mg/L which exceed those in most environments. All bisphenol compounds induced estrogenic responses in Tg(ERE:Gal4ff)(UAS:GFP) zebrafish that were inhibited by coexposure with ICI 182780, demonstrating an estrogen receptor dependent mechanism. Target tissues included the heart, liver, somite muscle, fins, and corpuscles of Stannius. The rank order for estrogenicity was BPAF > BPA = BPF > BPS. Bioconcentration factors were 4.5, 17.8, 5.3, and 0.067 for exposure concentrations of 1.0, 1.0, 0.10, and 50 mg/L for BPA, BPF, BPAF, and BPS, respectively. We thus show that these BPA alternatives induce similar toxic and estrogenic effects to BPA and that BPAF is more potent than BPA, further highlighting health concerns regarding the use of BPA alternatives.

Rapid embryogenesis, together with genetic similarities with mammals, and the desire to reduce mammalian testing, are major incentives for using the zebrafish model in chemical screening and testing. Transgenic zebrafish, engineered for identifying target gene expression through expression of fluorophores, have considerable potential for both high-content and high-throughput testing of chemicals for endocrine activity. Here we generated an estrogen responsive transgenic zebrafish model in a pigment-free "Casper" phenotype, facilitating identification of target tissues and quantification of these responses in whole intact fish. Using the ERE-GFP-Casper model we show chemical type and concentration dependence for green fluorescent protein (GFP) induction and both spatial and temporal responses for different environmental estrogens tested. We also developed a semiautomated (ArrayScan) imaging and image analysis system that we applied to quantify whole body fluorescence responses for a range of different estrogenic chemicals in the new transgenic zebrafish model. The zebrafish model developed provides a sensitive and highly integrative system for identifying estrogenic chemicals, their target tissues and effect concentrations for exposures in real time and across different life stages. It thus has application for chemical screening to better direct health effects analysis of environmental estrogens and for investigating the functional roles of estrogens in vertebrates.

Some nanoparticles (NPs) may induce adverse health effects in exposed organisms, but to date the evidence for this in wildlife is very limited. Silver nanoparticles (AgNPs) can be toxic to aquatic organisms, including fish, at concentrations relevant for some environmental exposures. We applied whole mount in-situ hybridisation (WISH) in zebrafish embryos and larvae for a suite of genes involved with detoxifying processes and oxidative stress, including metallothionein (mt2), glutathionine S-transferase pi (gstp), glutathionine S-transferase mu (gstm1), haem oxygenase (hmox1) and ferritin heavy chain 1 (fth1) to identify potential target tissues and effect mechanisms of AgNPs compared with a bulk counterpart and ionic silver (AgNO3). AgNPs caused upregulation in the expression of mt2, gstp and gstm1 and down regulation of expression of both hmox1 and fth1 and there were both life stage and tissue-specific responses. Responding tissues included olfactory bulbs, lateral line neuromasts and ionocytes in the skin with the potential for effects on olfaction, behaviour and maintenance of ion balance. Silver ions induced similar gene responses and affected the same target tissues as AgNPs. AgNPs invoked levels of target gene responses more similar to silver treatments compared to coated AgNPs indicating the responses seen were due to released silver ions. In the Nrf2 zebrafish mutant, expression of mt2 (24 hpf) and gstp (3 dpf) were either non-detectable or were at lower levels compared with wild type zebrafish for exposures to AgNPs, indicating that these gene responses are controlled through the Nrf2-Keap pathway.

Bone morphogenetic proteins (Bmp) are major players in the formation of the vertebrate body plan due to their crucial role in patterning of the dorsal-ventral (DV) axis. Despite the highly conserved nature of Bmp signalling in vertebrates, the consequences of changing this pathway can be species-specific. Here, we report that Bmp plays an important role in epiboly, yolk syncytial layer (YSL) movements, and anterior-posterior (AP) axis formation in embryos of the self-fertilizing mangrove killifish, Kryptolebias marmoratus. Stage and dose specific exposures of embryos to the Bmp inhibitor dorsomorphin (DM) produced three distinctive morphologies, with the most extreme condition creating the splitbody phenotype, characterised by an extremely short AP axis where the neural tube, somites, and notochord were bilaterally split. In addition, parts of caudal neural tissues were separated from the main body and formed cell islands in the posterior region of the embryo. This splitbody phenotype, which has not been reported in other animals, shows that modification of Bmp may lead to significantly different consequences during development in other vertebrate species.

Manufactured metal (oxide) nanoparticles are entering the aquatic environment with little understanding on their potential health impacts for exposed organisms. Adopting an integrative approach, we investigated effects of particle size and coating on biological responses for two of the most commonly used metal (oxide) nanoscale particles, silver (Ag) and titanium dioxide (TiO₂) in zebrafish embryos. Titanium dioxide nanoparticles (nominally, 4 nm, 10 nm, 30 nm and 134 nm) had little or no toxicity on the endpoints measured. Ag both in nano form (10 nm and 35 nm) and its larger counterpart (600-1600 nm) induced dose-dependent lethality and morphological defects, occurring predominantly during gastrula stage. of the silver material tested 10 nm nanoparticles appeared to be the most toxic. Coating Ag nanoparticles with citrate or fulvic acid decreased toxicity significantly. In situ hybridisation analysis identified the yolk syncytial layer (YSL) as a target tissue for Ag-nano toxicity where there was a significant induction of the heavy metal stress response gene, metallothionein 2 (Mt2) at sub-lethal exposures. Coherent Anti-stroke Raman Scattering (CARS) microscopy provided no evidence for silver particles crossing the chorionic membrane in exposed embryos. Collectively, our data suggest that silver ions play a major role in the toxicity of Ag nanoparticles.

Background: Various shapes of gastropod shells have evolved ever since the Cambrian. Although theoretical analyses of morphogenesis exist, the molecular basis of shell development remains unclear. We compared expression patterns of the decapentaplegic (dpp) gene in the shell gland and mantle tissues at various developmental stages between coiled-shell and non-coiled-shell gastropods.Results: We analyzed the expression patterns of dpp for the two limpets Patella vulgata and Nipponacmea fuscoviridis, and for the dextral wild-type and sinistral mutant lineage of the pond snail Lymnaea stagnalis. The limpets had symmetric expression patterns of dpp throughout ontogeny, whereas in the pond snail, the results indicated asymmetric and mirror image patterns between the dextral and sinistral lineages.Conclusion: We hypothesize that Dpp induces mantle expansion, and the presence of a left/right asymmetric gradient of the Dpp protein causes the formation of a coiled shell. Our results provide a molecular explanation for shell, coiling including new insights into expression patterns in post-embryonic development, which should aid in understanding how various shell shapes are formed and have evolved in the gastropods. © 2012 Shimizu et al.; licensee BioMed Central Ltd.

BACKGROUND: Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish) and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38) in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff), contains three copies of oestrogen response elements (3ERE) that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS) elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein). RESULTS: the response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1-2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2), the synthetic oestrogen 17α- ethinyloestradiol (EE2), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures). For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish). We also demonstrate that our construct works in medaka, another model fish test species, suggesting the transient assay is applicable for testing oestrogenic chemicals in fish generally. CONCLUSION: Our results indicate that the transient expression assay system can be used as a rapid integrated testing system for environmental oestrogens and to detect the oestrogenic target sites in developing fish embryos.

The self-fertilizing mangrove killifish, Kryptolebias marmoratus, is an upcoming model species for a range of biological disciplines. To further establish this model in the field of developmental biology, we examined several techniques for embryonic manipulation and for imaging that can be used in an array of experimental designs. These methodological approaches can be divided into two categories: handling of embryos with and without their chorionic membrane. Embryos still enclosed in their chorion can be manipulated using an agarose bed or a methyl cellulose system, holding them in place and allowing their rotation to more specific angles and positions. Using these methods, we demonstrate microinjection of embryos and monitoring of fluorescent yolk syncytial nuclei (YSN) using both stereo and compound microscopes. For higher magnification imaging using compound microscopes as well as time-lapse analyses, embryos were dechorionated and embedded in low-melting-point agarose. To demonstrate this embedding technique, we further examined fluorescent YSN and also analyzed the yolk surface of K. marmoratus embryos. The latter was observed to provide an excellent imaging platform for study of the behavior and morphology of cells during embryonic development, for various types of cells. Our data demonstrate that K. marmoratus is an excellent model species for research in developmental biology, as methodological approaches for the manipulation and imaging of embryos are efficient and readily available.

The yolk syncytial layer (YSL) in the zebrafish embryo is a multinucleated syncytium essential for embryo development, but the molecular mechanisms underlying YSL formation remain largely unknown. Here we show that zebrafish solute carrier family 3 member 2 (Slc3a2) is expressed specifically in the YSL and that slc3a2 knockdown causes severe YSL defects including clustering of the yolk syncytial nuclei and enhanced cell fusion, accompanied by disruption of microtubule networks. Expression of a constitutively active RhoA mimics the YSL phenotypes caused by slc3a2 knockdown, whereas attenuation of RhoA or ROCK activity rescues the slc3a2-knockdown phenotypes. Furthermore, slc3a2 knockdown significantly reduces tyrosine phosphorylation of c-Src, and overexpression of a constitutively active Src restores the slc3a2-knockdown phenotypes. Our data demonstrate a signaling pathway regulating YSL formation in which Slc3a2 inhibits the RhoA/ROCK pathway via phosphorylation of c-Src to modulate YSL microtubule dynamics. This work illuminates processes at a very early stage of zebrafish embryogenesis and more generally informs the mechanism of cell dynamics during syncytium formation.

Developing embryos of the self-fertilising mangrove killifish Kryptolebias marmoratus. PTU-treated embryo (left) lacking melanin pigmentation and a normal embryo (right), 10 days post-fertilisation. Due to its ability to self-fertilise, this species proves to be a highly useful embryological model with unique genetics. From Mourabit et al. Developmental Dynamics 240:1694-1704, 2011.

The mangrove killifish, Kryptolebias marmoratus, is a self-fertilizing vertebrate offering vast potential as a model species in many biological disciplines. Previous studies have defined developmental stages but lacked visual representations of the various embryonic structures. We offer detailed photographic images of K. marmoratus development with revised descriptions. An improved dechorionation method was developed to provide high resolution photographs, in addition to a microinjection technique enabling cell marking in the yolk syncytial layer. Embryos were also treated with PTU (1-phenyl 2-thiourea), an inhibitor of melanogenesis, to provide optical transparency revealing internal structures in late stages of development. Chemical exposures (PTU and retinoic acid) demonstrated that K. marmoratus embryos were sensitive to chemicals, illustrating further their usefulness in developmental biology studies. Our data suggest that K. marmoratus embryos are easily used and manipulated, supporting the use of this hermaphroditic vertebrate as a strong comparative model system in embryology, evolution, genetics, environmental and medical biology.

Angiogenesis plays an important role in vertebrate development and tumor growth. In this process, gicerin, which is known as a kind of cell adhesion molecule, has recently been reported to play an important role but its in vivo function is still unclear in developing vasculature. To address this issue, we used gain-of-function and loss-of-function analyses of gicerin in zebrafish. In the gain of function experiments using enforced expression of various domains of gicerin constructs, extracellular domain induced angiogenic sprouting defects, most notably in the intersegmental vessels, whereas the cytoplasmic domain of gicerin did not affect angiogenic sprouting. Moreover, morpholino-mediated knockdown of gicerin in embryos resulted in angiogenic sprouting defects in intersegmental vessels. Mechanistically, the angiogenic function of gicerin was found to be genetically linked to VEGF signaling in the knock-down experiments using vegf-a mRNA, VEGFR inhibitor and gicerin morpholino. In addition to the physiological angiogenesis during development, gicerin morphants efficiently blocked the tumor angiogenesis in zebrafish. Thus, knock-down of gicerin might have an important implication in controlling tumor angiogenesis.

Due to their large yolk size, salmonid embryos take a longer time for epiboly movements and germ ring closure compared with most other teleost species. Here we analyzed the germ ring closure, tail bud formation and development of the notochord and somites in rainbow trout using live embryo imaging and in situ hybridization with the rt-ntl probe. Rt-ntl is expressed in the germ ring (blastula, gastrula and somitogenesis stage), notochord, tail bud and somites (somitogenesis stage). When epiboly covers half the yolk, a tail bud-like structure is formed and somitogenesis starts. By the time epiboly is completed, the yolk covered and the germ ring closed, the embryo has already reached the 20 somite stage. Therefore, the timing of germ ring closure and tail bud formation is reversed in trout embryos compared with zebrafish and other small model fish embryos (heterochrony). Based on this result, we re-examined the definition of tail bud formation.

In vertebrates, even-skipped related (evx) homeodomain transcription factor encoding genes are expressed in the posterior region during embryonic development, and overexpression experiments have revealed roles in tail development in fish and frogs. Here we analysed the molecular mechanisms of posterior neural development and axis formation regulated by eve1. We show that eve1 is involved in establishing trunk and tail neural ectoderm by two independent mechanisms: First, eve1 posteriorises neural ectoderm via induction of aldh1a2, which encodes an enzyme that synthesises retinoic acid; second, eve1 is involved in neural induction in the posterior ectoderm by attenuating Bmp expression. Further, eve1 can restore trunk neural tube formation in the organiser-deficient ichabod-/- mutant. We conclude that eve1 is crucial for the organisation of the antero-posterior and dorso-ventral axis in the gastrula ectoderm, and furthermore has trunk and tail promoting activity.

The normal embryonic development of the tomato clownfish Amphiprion frenatus was analysed
using live imaging and by in situ hybridization for detection of mesodermal and neurectodermal
development. Both morphology of live embryos and tissue-specific staining revealed significant
differences in the gross developmental programme of A. frenatus compared with better-known
teleost fish models, in particular, initiation of somitogenesis before complete epiboly, initiation of
narrowing of the neurectoderm (neurulation) before somitogenesis, relatively early pigmentation
of melanophores at the 10–15 somite stage and a distinctive pattern of melanophore distribution.
These results suggest evolutionary adaptability of the teleost developmental programme. The ease
of obtaining eggs, in vitro culture of the embryo, in situ staining analyses and these reported
characteristics make A. frenatus a potentially important model marine fish species for studying
embryonic development, physiology, ecology and evolution.

Fibroblast growth factors (FGFs) are members of a family of some 30 secreted proteins important in the regulation of cellular proliferation, migration, differentiation and survival. Here we report the identification of a novel modulator of FGF signal transduction, sef, isolated from a zebrafish embryo library through an in situ hybridization screen. The sef gene encodes a transmembrane protein, and belongs to the synexpression group that includes some of the fgf genes. Sef expression is positively regulated by FGF, and ectopic expression of sef in zebrafish or Xenopus laevis embryos specifically inhibits FGF signalling. In co-immunoprecipitation assays, the intracellular domain of Sef interacts with FGF receptors, FGFR1 and FGFR2. Injection of antisense sef morpholino oligos mimicked the phenotypes observed by ectopic fgf8 expression, suggesting that Sef is required to limit FGF signalling during development.

Random screening for tissue specific genes in zebrafish by in situ hybridization led us to isolate a gene which showed highly restricted expression in the developing eyes and midbrain at somitogenesis stages. This gene was very similar to mouse and human mab21l2. The characteristic expression pattern of mab21l2 facilitates a detailed description of the morphogenesis of the eyes and midbrain in the zebrafish. In the eye field, mab21l2 expression illustrates the transformation of the eye field to form two separate eyes in the anterior neural plate. Mab21l2 staining in the cyclopic mutants, cyc and oep, exhibited incomplete splitting of the eye primodium. In the midbrain, mab21l2 is expressed in the tectum, and its expression follows the expansion of the tectal region. In mutants affecting the mid-hindbrain boundary (MHB), mab21l2 expression is affected differentially. In the noi/pax2.1 mutant, mab21l2 is down-regulated and the size of the tectum remains small, whereas in the ace/fgf8 mutant, mab21l2 expression persists although the shape of the tectum is altered.

Bone morphogenetic proteins (BMP) are members of the TGFbeta superfamily of secreted factors with important regulatory functions during embryogenesis. We have isolated the zebrafish gene, nma, that encodes a protein with high sequence similarity to human NMA and Xenopus Bambi. It is also similar to TGFbeta type I serine/theronine kinase receptors in the extracellular ligand-binding domain but lacks a cytoplasmic kinase domain. During development, nma expression is similar to that of bmp2b and bmp4, and analysis in the dorsalized and ventralized zebrafish mutants swirl and chordino indicates that nma is regulated by BMP signaling. Overexpression of nma during zebrafish and Xenopus development resulted in phenotypes that appear to be based on inhibition of BMP signaling. Biochemically, NMA can associate with TGFbeta type II receptors and bind to TGFbeta ligand. We propose that nma is a BMP-regulated gene whose function is to attenuate BMP signaling during development through interactions with type II receptors and ligands.

The product of the Wilms tumor suppressor gene, WT1, is thought to be a tissue specific transcription factor regulating cell growth and differentiation. To elucidate the function of WT1 in cellular differentiation, we examined the changes in the level of WT1 expression during retinoic acid induced-differentiation of embryonal carcinoma F9 cells into parietal endoderm cells. We found that, in response to retinoic acid addition, the expression of WT1 increased significantly after 12--24 h of incubation, then decreased and finally disappeared after 4 days, by which time most of the cells had differentiated into primitive endoderm cells. To examine the significance of these changes in WT1 expression, we established cell lines constitutively expressing one of the WT1 splicing variants. These cell lines showed a phenotype very similar to parental F9 cells in the absence of retinoic acid. However, in the presence of retinoic acid, they failed to differentiate into primitive endoderm cells and underwent apoptotic death 36 h after the addition of retinoic acid. These results suggest that downregulation of WT1 expression is necessary for normal differentiation of F9 cells into parietal endoderm cells.

WT1, the Wilms tumor-suppressor gene, maps to the human chromosomal region 11p13 and encodes a transcriptional repressor, WT1, implicated in controlling normal urogenital development. Microinjection of the WT1 cDNA into quiescent cells or cells in early to mid G1 phase blocked serum-induced cell cycle progression into S phase. The activity of WT1 varied significantly depending on the presence or absence of an alternatively spliced region located upstream of the zinc finger domain. The inhibitory activity of WT1 was abrogated by the overexpression of cyclin E/CDK2 as well as cyclin D1/CDK4. Furthermore, both CDK4- and CDK2-associated kinase activities were downregulated in cells overexpressing WT1, whereas the levels of CDK4, CDK2, and cyclin D1 expression were unchanged. These findings suggest that inhibition of the activity of cyclin/CDK complexes may be involved in mediating the WT1-induced cell cycle block.