Browsing by Subject "Zebrafish"
Now showing 1 - 16 of 16
Results Per Page
Sort Options
Item A MOLECULAR ANALYSIS OF PROTEIN TRAFFICKING IN THE VERTEBRATE RETINA: IMPLICATIONS FOR INTRAFLAGELLAR TRANSPORT AND DISEASE(2010-07-14) Krock, Bryan L.Vertebrate photoreceptors are highly specialized sensory neurons that utilize a modified cilium known as the outer segment to detect light. Proper trafficking of proteins to the outer segment is essential for photoreceptor function and survival and defects in this process lead to retinal disease. In this dissertation I focus on two aspects of protein trafficking, intracellular vesicular trafficking in photoreceptors and retinal pigmented epithelial (RPE) cells and how it relates to the human disease choroideremia (CHM), and the trafficking of proteins through the photoreceptor cilium. The human retinal degenerative disease choroideremia (CHM) is caused by mutation of the Rab escort protein-1 (REP1) gene, which is required for proper intracellular vesicular trafficking. However, it was unclear whether photoreceptor degeneration in this disease is cell-autonomous, due to defective opsin transport within the photoreceptor, or is noncell-autonomous and a secondary consequence of defective RPE. Utilizing the technique of blastomere transplantation and a zebrafish line with a mutation in the rep1 gene, I show that photoreceptor degeneration in CHM is noncell-autonomous and is caused by defective RPE. The molecular machinery responsible for protein trafficking through the photoreceptor cilium remained unclear for a long time. Recent studies found Intraflagellar Transport (IFT) is the process that mediates cilia formation and transport of proteins through a cilium, and further analyses showed IFT is important for trafficking proteins to the outer segment. However, many details about how IFT works in photoreceptors remained unclear. By analyzing zebrafish harboring a null mutation in the ift57 gene, I show that Ift57 is only required for efficient IFT, and that the Ift57 protein plays a role in the ATP-dependent dissociation of kinesin II from the IFT particle. Lastly, I investigate the role of retrograde IFT in photoreceptors, a process that had yet to be investigated. By utilizing antisense morpholino oligonucleotides to inhibit expression of cytoplasmic dynein-2 (the molecular motor that mediates retrograde IFT) , I show that retrograde IFT is required for outer segment extension and the recycling of IFT proteins.Item Bisphenol A, and three related alkylphenols exert rapid estrogenic actions on zebrafish (Danio rerio) oocytes to maintain meiotic arrest(2014-12) Fitzgerald, Amanda Crane; Thomas, P. (Peter)Bisphenol A (BPA) is an alkylphenol compound used in plastic manufacturing, which enters the aquatic environment through wastewater treatment plants and landfill leachates. Exposure of fish to BPA results in developmental defects, decreased hatching, and increased occurrence of the egg yolk precursor protein vitellogenin in male fish. BPA is an estrogenic like compound (xenoestrogen) that can bind and activate the nuclear estrogen receptors, ERα and ERβ, causing changes in gene transcription (genomic mechanism). However, the ability of BPA to activate rapid estrogen signaling and to disrupt nongenomic physiological responses to estrogens is not known. One important nongenomic estrogen action in fish is to maintain meiotic arrest of oocytes and to prevent precocious oocyte maturation through activation of the transmembrane G-protein coupled estrogen receptor (Gper). Binding of estrogens to Gper on zebrafish oocytes results in rapid activation of epidermal growth factor receptor (Egfr) signaling and subsequent Mapk3/1 activation. We show here that BPA and three structurally-related chemicals, tetrachlorobisphenol A, tetrabromobisphenol A and nonylphenol, can mimic estrogen by activating this mechanism of meiotic arrest through Gper in zebrafish (Danio rerio). BPA bound to zebrafish Gper and inhibited spontaneous oocyte maturation (OM) of denuded oocytes. Treatment of oocytes with Actinomycin D did not block the effects of BPA, suggesting that this inhibition of OM is through a nongenomic mechanism. Incubation of oocytes with a selective GPER antibody and the specific GPER antagonist G-15 blocked the effects of BPA on OM, further suggesting that BPA inhibition of OM is through its interaction with the receptor. Various inhibitors of the EGFR pathway were utilized to determine if the inhibition of OM by BPA is mediated through this mechanism. BPA activation of the Egfr pathway resulted in Mapk3/1 (also known as Erk 1/2) phosphorylation. The results show that BPA disrupts oocyte maturation through a novel mechanism involving activation of a Gper/Egfr/Mapk3/1 pathway with potential adverse impacts on reproductive success.Item Chemical Genetic Studies of Tumor Suppressors in the Developing Zebrafish(2010-11-02T18:19:46Z) Verduzco, Daniel; Amatruda, James F.The zebrafish is a powerful disease model system. Its distinct advantages allow it to be used for high-throughput genetic and chemical screens with high translational potential. One mutant isolated from a screen for cell cycle deficiencies is the zebrafish cdc25a mutant standstill. This mutant suffers from a G2/M arrest due to activation of the checkpoint protein ATM such that ATM knockdown or inhibition rescues the cell cycle. Additional knockdown studies reveal that the unique zebrafish cdc25 gene cdc25d is responsible for cell cycle progression in the absence of functional Cdc25a or ATM. Assays to measure genomic stress reveal that ATM is activated in the absence of DNA damage. Activation of ATM in the absence of DNA damage is a unique occurrence, but further studies seeking regulators of ATM reveal that ATM is constitutively active at a basal level. This basal ATM activity is sufficient to attenuate cell cycle rate. Other studies involving the use of zebrafish to study cancer involve a screen using peptoids. The peptoid is a class of compound that is modular, easy and inexpensive to synthesize, and stable in vivo. Initial tests reveal that peptoids are able to enter the zebrafish embryo, remain stable, and exert biological functions. Screening of 3,744 compounds for peptoids that specifically affect zebrafish mutant for p53 reveal a group of 4 structurally similar peptoids. Further studies of these peptoids reveal that they are able to perform their function on mouse embryonic fibroblasts in vitro. Finally, I sought the role of the DNA damage marker H2AX in early zebrafish development. While studying the DNA damage response in cdc25a mutants, I discovered that H2AX is present and cyclic in pre-midblastula transition embryos. This phosphorylation is necessary for synchronization and relies on the ability of ATM to phosphorylate H2AX on Ser-139 such that inhibition of ATM or ablation of the phosphorylation site leads to desynchronization and improper development.Item Evaluation on combinative toxicology of sodium perchlorate and sodium arsenate using zebrafish Danio rerio as a model(Texas Tech University, 2006-05) Liu, Fujun; Wang, Jia-Sheng; Theodorakis, Christopher W.; Cobb, George P.; Hooper, Michael J.; Collie, Nathan L.Both perchlorate and arsenic are environmental contaminants, and their co-occurrence has been found in contaminated water. Perchlorate is a definitive thyroid disruptor, and thus affects antioxidant defense system. Arsenate is a pro-oxidant and cause oxidative stress. Arsenic is hypothesized as a thyroid disruptor. Ultimately, both chemicals cause damage on macromolecules. Theoretically, two chemicals have an additive interaction in thyrotoxicity and damage on macromolecules. To test this hypothesis, zebrafish Danio rerio were exposed to sodium arsenate (SA) and sodium perchlorate (SP) individually or in mixture in acute, sub-chronic, and chronic tests. The 96-h LC50s of SA and SP were 272 and 1,365 mg/L, respectively, at larvae stage and 74 and 3,118 mg/L, respectively at juvenile stage. In the chronic toxicity test, we employed some endpoints to evaluate antioxidant (glutathione, GSH), thyrotoxicity (thyroid histopathology), and DNA damage (strand breaks, oxidative bases, and apoptosis). In addition, the toxicokinetics (uptake, accumulation, and depuration) of both chemicals and their mixture was examined. Fish were exposed to SP (10 and 100 mg/L), SA (1 and 10 mg/L), and the mixture—SP+SA (10+1 and 100 +10 mg/L) for up to 90 days. At day 10, 30, 60, and 90, fish were sampled and processed for endpoint assay. In the thyrotoxicity, the sensitivity of the following histopathological indicators for indicating thyroid perturbations by these two chemicals is, in descending order: epithelial cell height>percent of colloid area/follicle area>colloid area/follicular cell height >hyperplasia>angiogenesis>colloid area>follicle area. Models were applied to characterize the toxicokinetics of two chemicals. Two chemicals were taken up and depurated rapidly. Both chemicals reduced the uptake but enhanced the depuration of the other chemical from the zebrafish in mixture. Total and reduced GSH and the GSH status index were significantly altered by high concentrations of SA, SP, or their mixture. Damage to lipid and DNA was not as sensitive as expected. Two chemical displayed additive interactions in terms of some endpoints tested. In summary, this dissertation project explored the mechanism of these two chemicals and their interactions. This dissertation provides toxicological information for these two chemicals and clarifies their joint actions at different levels mechanistically.Item Evaluation on combinative toxicology of sodium perchlorate and sodium arsenate using zebrafish Danio rerio as a model(2006-05) Liu, Fujun; Wang, Jia-Sheng; Theodorakis, Christopher W.; Hooper, Michael J.; Cobb, George P.; Collie, Nathan L.Both perchlorate and arsenic are environmental contaminants, and their co-occurrence has been found in contaminated water. Perchlorate is a definitive thyroid disruptor, and thus affects antioxidant defense system. Arsenate is a pro-oxidant and cause oxidative stress. Arsenic is hypothesized as a thyroid disruptor. Ultimately, both chemicals cause damage on macromolecules. Theoretically, two chemicals have an additive interaction in thyrotoxicity and damage on macromolecules. To test this hypothesis, zebrafish Danio rerio were exposed to sodium arsenate (SA) and sodium perchlorate (SP) individually or in mixture in acute, sub-chronic, and chronic tests. The 96-h LC50s of SA and SP were 272 and 1,365 mg/L, respectively, at larvae stage and 74 and 3,118 mg/L, respectively at juvenile stage. In the chronic toxicity test, we employed some endpoints to evaluate antioxidant (glutathione, GSH), thyrotoxicity (thyroid histopathology), and DNA damage (strand breaks, oxidative bases, and apoptosis). In addition, the toxicokinetics (uptake, accumulation, and depuration) of both chemicals and their mixture was examined. Fish were exposed to SP (10 and 100 mg/L), SA (1 and 10 mg/L), and the mixture—SP+SA (10+1 and 100 +10 mg/L) for up to 90 days. At day 10, 30, 60, and 90, fish were sampled and processed for endpoint assay. In the thyrotoxicity, the sensitivity of the following histopathological indicators for indicating thyroid perturbations by these two chemicals is, in descending order: epithelial cell height>percent of colloid area/follicle area>colloid area/follicular cell height >hyperplasia>angiogenesis>colloid area>follicle area. Models were applied to characterize the toxicokinetics of two chemicals. Two chemicals were taken up and depurated rapidly. Both chemicals reduced the uptake but enhanced the depuration of the other chemical from the zebrafish in mixture. Total and reduced GSH and the GSH status index were significantly altered by high concentrations of SA, SP, or their mixture. Damage to lipid and DNA was not as sensitive as expected. Two chemical displayed additive interactions in terms of some endpoints tested. In summary, this dissertation project explored the mechanism of these two chemicals and their interactions. This dissertation provides toxicological information for these two chemicals and clarifies their joint actions at different levels mechanistically.Item Examination of the role of different classes of progesterone receptors in oocyte maturation in zebrafish, Danio rerio(2007-12) Harris, Caleb Michael, 1979-; Thomas, P. (Peter)The overall goal of this thesis was to investigate the involvement of three potential progestin receptors in the oocyte maturation of a single model species, zebrafish. Potential mediators of oocyte maturation in lower vertebrates include the nuclear intracellular progesterone receptor (nPR) and two membrane bound progestin receptors, a seven-transmembrane progestin receptor (mPR) and a single transmembrane progestin receptor (PGRMC1). Inhibitory G-protein involvement was inspected as well as ligand activation, relative binding of various ligands and expression patterns of membrane receptors. Intracellular inhibitory G (Gi)-protein signal pathways were shown to be necessary for the steroid induction of oocyte maturation by injecting competent oocytes with pertussis toxin. Various native and synthetic steroid ligands were investigated for their ability to promote germinal vesicle breakdown (GVBD). Classical nPR antagonists were potent inducers of GVBD. Ligand effectiveness of various steroids in promoting GVBD in vitro was correlated to the steroids' relative binding affinity for recombinant zebrafish mPR[Greek small letter alpha] The expression of PGRMC1 was investigated and shown not to be correlated with any oocyte developmental stage. Gonadotropin in vitro incubation resulted in a modest but insignificant increase in PGRMC1 mRNA expression levels. Furthermore, abrogating PGRMC1 expression with antisense morpholino oligonucleotide injections did not significantly impair gonadotropin induction of oocyte maturational competence. This research supports the involvement of the seven transmembrane mPR receptor in the oocyte maturation of zebrafish. Though the involvement of nPR and PGRMC1 is not supported, some trends in data require further investigation as to potential involvement of PGRMC1 in oocyte function.Item The hedgehog pathway in retinal development and disease(2011-08) Bibliowicz, Jonathan; Gross, Jeffrey Martin; FISCHER, JANICE A.; MORGAN, JENNIFER; THOMPSON, WESLEY; VOKES, STEVEN A.The Hedgehog receptor Patched (Ptch) is a well-studied tumor suppressor. Mutations in Ptch have been linked to mis-regulation of stem cell proliferation and tumorigenesis in numerous contexts. To study the role of Ptch function during retinal development and homeostasis, I analyzed retinal growth and patterning in the embryonic and post-embryonic (juvenile) zebrafish ptc2 mutant line. ptc2 deficiency in zebrafish results in an expansion of the stem/progenitor population of the ciliary marginal zone (CMZ), as well as ectopic proliferation within the neural retina at juvenile stages. ptc2-/- mutants also possess vitreo-retinal abnormalities that appear to be embryonic in origin. These phenotypes are similar to the ocular abnormalities previously reported in human patients suffering from Basal Cell Naevus Syndrome (BCNS), a disorder that has been linked to mutations in the human PTCH gene (the orthologue of the zebrafish ptc2), and point to the utility of the ptc2 mutant line as a model for the study of BCNS-related ocular pathologies. In addition, peripheral retinal dysplasias that include ectopic neuronal clusters and disrupted lamination were observed at later, juvenile stages. It has been previously proposed that retinal over-proliferation might contribute to retinal dysplasias observed in the post-natal Ptch1 /- mice (an established model for BCNS); however, this potential relationship has yet to be established experimentally. I demonstrated that a population of ectopically proliferating cells give rise to the ectopic neuronal clusters in the INL of ptc2-/- mutants and established ectopic proliferation as the likely cellular underpinning of retinal dysplasia in juvenile ptc2-/- mutants.Item Inhibitor of differentiation 2A influences growth and differentiation of the developing vertebrate retina upstream of the notch signaling pathway(2012-08) Uribe, Rosa Anna; Gross, Jeffrey Martin; Vokes, Steven A.; Eberhart, Johann; Stein, David; Agarwala, SeemaInhibitor of differentiation (Id) family helix-loop-helix proteins regulate the proliferation, survival and differentiation of numerous cell types during development, however their function during retinal development has not been analyzed. Using loss-of-function and overexpression assays in zebrafish, I demonstrate that Id2a levels modulate retinoblast cell cycle kinetics and thereby influence neuron and glia formation in the retina. Id2a-deficient retinas possess increased numbers of cells occupying S phase, at the expense of mitotic cells, and kinetic analyses demonstrate that Id2a is required for S-phase progression and/or the transition from S to M phase. Id2a-dependent defects in retinoblast proliferation lead to microphthalmia and to an absence of nearly all differentiated inner and outer nuclear layer cell types. Overexpression of id2a has the opposite effect on retinoblast cell cycle kinetics: id2a-overexpressing retinoblasts progress from S to M phase more rapidly and they undergo mitosis more frequently, which results in macrophthalmia. Mosaic analyses reveal that Id2a function in facilitating both cell cycle progression and neuronal differentiation in the retina is non-cell-autonomous, suggesting that Id2a functions upstream of the extrinsic pathways that regulate retinogenesis. In an effort to identify which extrinsic pathways function downstream of Id2a, I discovered that Id2a function is necessary and sufficient to limit Notch pathway activity during retinogenesis. Id2a-deficient retinae possess elevated levels of Notch pathway component gene expression, while retinae overexpressing id2a possess reduced expression of Notch pathway component genes. Attenuation of Notch signaling activity by DAPT or by morpholino knockdown of Notch1a is sufficient to rescue both the proliferative and differentiation defects in Id2a-deficient retinae. In addition to regulating Notch pathway activity, through an RNA-Seq and differential gene expression analysis of Id2a-deficient retinae, I identified a number of additional intrinsic and extrinsic regulatory pathway components whose expression is regulated by Id2a. These data highlight the integral role played by Id2a in the gene regulatory network governing the transition from retinoblast proliferation to terminal differentiation during vertebrate retinogenesis.Item Integrin alphα5/fibronectin1 and focal adhesion kinase are required for lens fiber morphogenesis in zebrafish(2010-08) Hayes, Julie Marie; Gross, Jeffrey Martin; Wallingford, John B.Fibronectin (fn) and integrin α5 (itgα5) are both key players in cell adhesion and intracellar signaling, however the specific in vivo role of these proteins has never been analyzed in the vertebrate lens. The results presented here indicate that Fn1 and Itgα5 proteins are essential for the proper development of the lens. The loss of Fn1 protein in the zebrafish embryo results in distinct adhesion defects, defects in lens fiber morphogenesis, and cataracts. These results were phenocopied in zebrafish itga5 mutants, thereby indicating an essential role for Fn1 and Itgα5 during lens development. Furthermore, embryos with reduced levels of ptk2.1 (focal adhesion kinase – FAK) also phenocopied the defective fn1 and itgα5 lens, suggesting that FAK is a major player in the intracellular signaling mediated by Fn1/Itgα5 interactions in the lens.Item Multiple roles for the zebrafish transcriptional activator SBF/Staf(2009-05-15) Halbig, Kari MicheleEukaryotic transcriptional activators stimulate transcription of genes otherwise expressed at low levels. The typical activator operates by binding to specific sites on DNA with its activating region contacting the multiprotein machinery that directs transcription. SBF/Staf is a transcriptional activator that binds to the SPH element found in the promoters of genes for snRNAs and genes that code for mRNAs. SBF/Staf binds to SPH through a reiterated zinc finger DNA binding domain and also contains two distinct activation domains, one for snRNA genes and one for mRNA genes. To test the role of SBF/Staf in vivo, morpholino antisense oligos were used to knock down SBF/Staf expression in zebrafish. A high percentage of developing zebrafish embryos exhibited abnormalities. Co-injection of a synthetic mRNA construct rescued the morpholino-induced knockdown. Furthermore, both the mRNA and snRNA activation domains have significant roles in the function of SBF/Staf because when each domain was removed separately, partial rescue of the knockdown phenotype was obtained. When both domains were removed, no rescue of the phenotype was observed. Unexpectedly, knockdown of SBF/Staf expression in zebrafish embryos caused an increase in steady-state levels of all endogenous mRNAs tested, as well as transcripts produced from co-injected U6 maxigenes. However, quantitative RT-PCR analysis showed a relatively smaller increase in the steady-state levels of several mRNAs from genes that contain a SPH element in their promoters. In zebrafish U6 genes, the SPH element is in the unique location of being next to the TATA box, instead of ~220 bp upstream of the start site as in mammals. To determine the significance of the proximally-located SPH element for transcription of the zebrafish U6 snRNA gene, the SPH element was mutated. Transcription of a zebrafish U6 maxigene was reduced to 20.6% in transfected ZF4 cells and 26.8% in injected embryos, compared to that of the U6 maxigene with a normal promoter. This work indicates a more global role of SBF/Staf in mRNA gene transcription, instead of only activating the transcription of snRNA and a few mRNA genes, leading to an increased importance of the role of SBF/Staf in transcriptional control.Item A Mutation in Alk6b Causes Impaired Germ Cell Differentation and Testicular Germ Cell Tumors in Zebrafish(2010-11-02T18:19:23Z) Neumann, Joanie; Amatruda, James F.Germ cell tumors (GCTs) affect infants, children and young adults and are increasing in incidence worldwide. GCTs arise from pluripotent germ cells and can exhibit differentiated and undifferentiated histologies, which vary in their malignant potential and response to treatment. The pathways that determine tumor cell differentiation are not known, impeding the development of new therapies. Thus, the treatment of GCTs has remained static since the introduction 30 years ago of cisplatin which, while effective, causes severe side effects including hearing loss, infertility and kidney damage. We identified a zebrafish mutant line with a high incidence of GCT during a forward genetic screen to identify cancer susceptibility loci. Homozygous adult males develop tumors consisting of undifferentiated spermatogonia by 4 months of age while heterozygous males develop tumors around 7 to 9 months of age. We used interval haplotype analysis and high-resolution recombinational mapping to localize the mutation to a 0.82 cM interval on zebrafish chromosome 10. We identified a premature termination codon in Alk6b (Activin Receptor-like Kinase 6b) in the mutant animals. Alk6b is a member of the TGF-beta/BMP superfamily of receptors. BMP signaling has diverse roles including regulation of cell proliferation, differentiation, embryonic development, germ cell specification and gonadogenesis. Misregulation of the BMP signaling pathway has been implicated in various human cancers. In agreement with a critical role for Alk6b in controlling germ cell differentiation, we find evidence of impaired BMP signal transduction in the zebrafish GCTs, as well as evidence of alterations in the expression level of BMP target genes. We have also examined BMP signaling in a series of 40 clinically-annotated human GCTs of diverse histologic subtypes. In agreement with the predictions made from our zebrafish model, we find that undifferentiated GCTs such as dysgerminomas lack BMP signaling activity, whereas signaling is maintained in the differentiated subtype of Yolk Sac Tumors. These results confirm the relevance of the zebrafish model for understanding germ cell tumorigenesis, and will foster the development of improved, targeted therapy of human GCTs.Item Reproductive and developmental toxicity of highly energetic compounds in zebrafish (Danio rerio)(Texas Tech University, 2006-08) Mukhi, Sandeep; Patino, Reynaldo; Smith, Ernest E.; Carr, James A.; Anderson, Todd A.; Lee, Vaughan H.There has been growing concern in recent years about a variety of toxicants, some of which are highly energetic in nature, that can affect human and ecosystem health. Highly energetic compounds such as perchlorate and hexahydro-1,3,5-trinitro-1,3,5-triazine (known as RDX) have been reported in various environments worldwide. The ecotoxicological impacts of these chemicals are relatively not fully understood. The overall objective of this study was to access the toxicological effects of these two chemicals on reproduction, development and other activities in a model aquatic organism, the zebrafish (Danio rerio). Perchlorate is a known thyroid-disrupting chemical. Exposure to environmental relevant concentrations of perchlorate caused several pathological alterations in thyroid follicles of zebrafish, which were used to establish novel and sensitive biomarkers of perchlorate exposure (Chapter 1). Namely, angiogenesis and ¡®colloidal T4 ring¡¯ intensity were far more sensitive indicators of thyroid disruption than previous available biomarkers. The developmental toxicity of perchlorate was studied in larval zebrafish (Chapter 2). Exposure to perchlorate at concentrations that inhibited thyroid function affected growth (length) and the sex ratio of exposed populations. Treatment with perchlorate skewed the sex ratio towards females, whereas co-treatment with thyroxine (T4) skewed the sex ratio towards males. This is the first report for teleost fishes indicating that thyroid hormone is involved in the process of gonadal sex determination and differentiation. An effect of perchlorate on the reproductive performance of zebrafish was also observed in a long-term exposure study (Chapter 3). After 16 weeks of exposure to perchlorate, a decline was observed in the level of T4, but not triiodothyronine, in whole-body homogenates of mothers and their embryos. Perchlorate also impaired fecundity (packed-egg volume), although egg diameters were increased and fertilization and hatching rates were unaffected. Analyses of the embryos also indicated impaired growth of jaw-forming cartilages (Meckel¡¯s and ceratohyal). Relatively little information is available concerning the lethal and sublethal effects of RDX in teleosts. The acute toxicity of RDX was determined for larval zebrafish (Chapter 4). The median lethal concentration of RDX was estimated (96-h LC50, 23-26 ppm), and effects on the surviving fish such as vertebral column deformities and behavioral abnormalities were also documented. The chronic toxicity and bioconcentration potential of RDX were subsequently determined in adult zebrafish (Chapter 5). Long-term exposure to RDX caused severe mortality at a concentration of 9.6 ppm. The bioconcentration factor for RDX was low but slightly increased with time of exposure, from ¡Ü1 at 4 and 8 weeks of exposure to >2 at 12 weeks. The RDX metabolite, MNX, was also found in whole-body extracts. Both RDX and MNX were undetectable in whole-body extracts 2 weeks after the fish were transferred to clean water. Lastly, the effects of RDX at sublethal concentrations on reproduction and egg quality were determined (Chapter 6). Exposure to RDX at the relatively low concentration of 0.5 ppm caused a short-lived increase in fecundity within 2 weeks of the onset of exposures; whereas no effects were noted at the higher concentration tested, 3.2 ppm. Egg fertilization rates and embryo hatching rates were unaffected by parental exposures to RDX. It is concluded that exposure to environmental perchlorate and RDX can potentially impact the health of teleosts, an important component of the aquatic biota.Item Reproductive and developmental toxicity of highly energetic compounds in zebrafish (Danio rerio)(2006-08) Mukhi, Sandeep; Patino, Reynaldo; Anderson, Todd A.; Carr, James A.; Lee, Vaughan H.; Smith, Ernest E.There has been growing concern in recent years about a variety of toxicants, some of which are highly energetic in nature, that can affect human and ecosystem health. Highly energetic compounds such as perchlorate and hexahydro-1,3,5-trinitro-1,3,5-triazine (known as RDX) have been reported in various environments worldwide. The ecotoxicological impacts of these chemicals are relatively not fully understood. The overall objective of this study was to access the toxicological effects of these two chemicals on reproduction, development and other activities in a model aquatic organism, the zebrafish (Danio rerio). Perchlorate is a known thyroid-disrupting chemical. Exposure to environmental relevant concentrations of perchlorate caused several pathological alterations in thyroid follicles of zebrafish, which were used to establish novel and sensitive biomarkers of perchlorate exposure (Chapter 1). Namely, angiogenesis and ¡®colloidal T4 ring¡¯ intensity were far more sensitive indicators of thyroid disruption than previous available biomarkers. The developmental toxicity of perchlorate was studied in larval zebrafish (Chapter 2). Exposure to perchlorate at concentrations that inhibited thyroid function affected growth (length) and the sex ratio of exposed populations. Treatment with perchlorate skewed the sex ratio towards females, whereas co-treatment with thyroxine (T4) skewed the sex ratio towards males. This is the first report for teleost fishes indicating that thyroid hormone is involved in the process of gonadal sex determination and differentiation. An effect of perchlorate on the reproductive performance of zebrafish was also observed in a long-term exposure study (Chapter 3). After 16 weeks of exposure to perchlorate, a decline was observed in the level of T4, but not triiodothyronine, in whole-body homogenates of mothers and their embryos. Perchlorate also impaired fecundity (packed-egg volume), although egg diameters were increased and fertilization and hatching rates were unaffected. Analyses of the embryos also indicated impaired growth of jaw-forming cartilages (Meckel¡¯s and ceratohyal). Relatively little information is available concerning the lethal and sublethal effects of RDX in teleosts. The acute toxicity of RDX was determined for larval zebrafish (Chapter 4). The median lethal concentration of RDX was estimated (96-h LC50, 23-26 ppm), and effects on the surviving fish such as vertebral column deformities and behavioral abnormalities were also documented. The chronic toxicity and bioconcentration potential of RDX were subsequently determined in adult zebrafish (Chapter 5). Long-term exposure to RDX caused severe mortality at a concentration of 9.6 ppm. The bioconcentration factor for RDX was low but slightly increased with time of exposure, from ¡Ü1 at 4 and 8 weeks of exposure to >2 at 12 weeks. The RDX metabolite, MNX, was also found in whole-body extracts. Both RDX and MNX were undetectable in whole-body extracts 2 weeks after the fish were transferred to clean water. Lastly, the effects of RDX at sublethal concentrations on reproduction and egg quality were determined (Chapter 6). Exposure to RDX at the relatively low concentration of 0.5 ppm caused a short-lived increase in fecundity within 2 weeks of the onset of exposures; whereas no effects were noted at the higher concentration tested, 3.2 ppm. Egg fertilization rates and embryo hatching rates were unaffected by parental exposures to RDX. It is concluded that exposure to environmental perchlorate and RDX can potentially impact the health of teleosts, an important component of the aquatic biota.Item Roles for zebrafish trpm7 in growth, skeletogenesis, kidney function and physiological ion homeostasis(2009-12) Elizondo, Michael Reuben; Parichy, David M.; Wallingford, John B.; Fischer, Janice A.; Paull, Tanya T.; Shankland, MartyDevelopment of the adult form requires coordinated growth and patterning of multiple traits in response to local gene activity as well as global endocrine and physiological effectors. In recent years the zebrafish has been utilized as a favorable animal model as a step towards dissecting and better understanding these postembryonic developmental processes. One of the more powerful methods utilized in zebrafish has been the identification of new gene functions through the use of mutant screens. The nutria mutant was recovered from one such screen to identify postembryonic defects in pigment pattern, growth and metamorphosis. These mutants exhibited a pigment cell defect, touch unresponsiveness and severe growth retardation. Here I will discuss my work towards dissecting the underlying developmental processes governing the phenotypic changes in nutria mutants. I characterize gross alterations in skeletal development in nutria mutants that lead to accelerated endochondral ossification but delayed intramembranous ossification. I show that the nutria phenotype results from mutations in trpm7, which encodes a transient receptor potential (TRP) family member that functions as both a cation channel and a kinase. I find trpm7 expression in the fish-specific, ion homeostasis-regulating gland known as the corpuscles of Stannius (CS), and in the mesonephric kidney. I show that mutants also develop kidney stones. Together these results suggest a role for trpm7 activity in regulation of physiological ion homeostasis. Next I confirm that role by identifying late-embryonic and early larval defects in the CS and the kidney, two organs that regulate physiological ion homeostasis. I demonstrate the early larval detection of kidney stones in trpm7 mutants and show that their appearance is presaged by decreased levels of total calcium and magnesium. Furthermore I establish a link between trpm7 function in the CS and stanniocalcin1 (stc1), a potent molecular regulator of calcium homeostasis. Finally, using transgenic overexpression and morpholino-oligonucleotide knockdown, I demonstrate that stc1 modulates calcium and magnesium levels in trpm7 mutant and wild-type backgrounds. Together these analyses establish postembryonic roles for trpm7 function in growth, skeletogenesis, kidney function, and physiological ion homeostasis.Item The Role of Fgf and Its Downstream Effectors in Otic and Epibranchial Development in Zebrafish(2012-10-19) Padanad, MaheshIn vertebrates, the otic placode forms inner ear and epibranchial placodes produce sensory ganglia within branchial clefts. Fibroblast growth factor (FGF) family of protein ligands from the surrounding tissues are responsible for otic and epibranchial placode induction. Members of pax2/5/8 family of transcription factors function as mediators during otic induction. To understand the temporal and spatial requirements of Fgf and their interaction with pax2/8 for otic induction, we used heat shock inducible transgenic lines of zebrafish to misexpress fgf3/8 and pax2a/8 under the control of hsp70 promoter. Loss of function studies were done to examine the functions of pax2/8 genes in regulating otic and epibranchial development. We show that global transient activation of hs:fgf3 or hs:fgf8 at mid-late gastrula stages (7-8 hpf) severely impairs otic induction, in part by disrupting formation of the principal signaling centers in the hindbrain. Additionally, mosaic studies show that high-level misexpression blocks otic fate cell-autonomously, whereas low to moderate levels promote otic development. At later stages high-level Fgf misexpression, both globally and locally does not inhibit otic fate, but rather causes a dramatic expansion of endogenous otic domains. Misexpression of hs:pax2a or hs:pax8 also expands endogenous otic domains but is not sufficient to bypass the requirement for Fgf signaling. Co-misexpression of Fgf with pax2a or pax8 leads to production of ectopic otic tissue in a broad range of cranial ectoderm. These data show that changes in timing, distribution and level of Fgf signaling and its downstream effectors influences otic induction. We show that otic and epibranchial placodes are induced at different times and by distinct mechanisms. Initially, Fgf from surrounding tissues induces otic expression of pax8 and sox3, which cooperate synergistically to establish otic fate. Subsequently, pax8 along with pax2a/pax2b downregulate foxi1 expression in otic cells, which is necessary for further otic development. Additionally, pax2/8 activate otic expression of fgf24, which induces epibranchial expression of sox3. Blocking functions of fgf24 or sox3 causes severe epibranchial deficiencies but has little effect on otic development. These results support the model whereby the otic placode forms first and induces epibranchial placodes through pax2/8-dependent Fgf24 signaling.Item Toxicity of cadmium quantum dots compared to cadmium and zinc ions in zebrafish (Danio rerio) and water flea (Daphnia pulex)(2013-05) Tang, SongRecent advances in the ability to manufacture and manipulate materials at the nanoscale have led to increased production and use of many types of nanomaterials in diverse areas. Nanocrystaline semiconductors (Quantum dots; QDs) are small, long-lived fluorescent nanocrystals composed of a core of semiconductor material (e.g. cadmium selenide, zinc sulfide) and shells or dopants of other elements. Often, the continuously increased uses of QDs come increased appearance of in the aqueous environments, and then can be toxic to aquatic organisms and poses significant ecological risks. However, the toxicological impacts of heavy metal constituted QDs on aquatic species are largely unknown, especially at the molecular level. Moreover, it is crucial to determine whether the toxicity of metallic QDs is quantitative or mechanistically different than that of soluble metal components. The aim of this study was to compare the toxicities of ionic cadmium (Cd) and zinc (Zn) and Cd- and Zn-containing QDs at both in vitro via zebrafish liver cells (ZFL) and in vivo through Daphnia pulex. Our results revealed that (1) ionic Cd2+ was more toxic than Zn2+, and the general trend of toxicity of QDs was determined to be CdTe > CdSe/ZnS or InP/ZnS; (2) smaller QDs showed greater toxicity than larger QDs; (3) both Cd2+ and CdTe QDs exposure led to an accumulation of Cd, an increased formation of intracellular reactive oxygen species (ROS), and an induction of DNA strand breaks; (4) CdTe QDs exposure induced expression patterns of metal response, stress defense, and DNA repair genes in a manner similar to that of Cd2+ exposure, while CdSe/ZnS or InP/ZnS QDs altered gene expression in a manner very different from that of the corresponding Cd or Zn salts; and (5) nucleotide excision repair (NER) repair capacity were inhibited with Cd2+ but not with CdTe. The adverse cellular effects caused by acute exposure of QDs might be mediated through differing mechanisms than those resulting from Cd2+ toxicity, and studying the effects of the ionic form may be not enough to explain QD toxicities in aquatic organisms.