Examination of the swimming capabilities of fish is increasingly being considered as an effective method for determining sublethal toxicity. Acute toxicant exposure is known to cause decreases in swim performance in fish but less is known about how developmental exposure can cause persistent effects that hinder swimming. In addition, little is known about how triglyceride levels fluctuate during fish swimming upon both acute and developmental exposure to toxicant. In this thesis, two studies, one acute and one developmental, were carried out using two different toxicants in order to address these issues.<p> In order to examine acute effects, adult zebrafish (Danio rerio) were exposed to ethanol vehicle or increasing concentrations of 2,4-dinitrophenol (DNP), a mitochondrial electron transport chain uncoupler, for a 24 h period. Following exposure, fish were placed in a swim tunnel for critical swimming speed (Ucrit) determination and swim motion analysis. Whole body triglyceride levels were then determined. Ucrit was decreased in a concentration dependent manner in both the 6 mg/L and 12 mg/L DNP exposure groups, with 6 mg/L DNP being considered sublethal and 12 mg/L approaching the LC50. A decrease in tail beat frequency was observed and is likely the main cause for the decrease in Ucrit in the DNP exposure groups. Triglyceride levels were elevated in a concentration dependent manner in the DNP exposure groups. This increase in triglyceride stores may be due to a behavioral adaption limiting swimming capabilities or due to a direct toxic action of DNP on lipid catabolism.<p> The second study examined whether developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure would cause persistent toxic effects. Zebrafish embryos were exposed to dimethyl sulfoxide control or increasing concentrations of TCDD between 2-4 days post fertilization (dpf). At 5 dpf, cytochrome P450 1A (CYP1A) activity was determined. Fish were raised to 90 dpf with mortalities and deformities being recorded at 5 dpf, 10 dpf, and 90 dpf. At 90 dpf, fish were placed in swim tunnel and Ucrit , swimming motion, and aerobic scope (oxygen consumption rate during exercise minus oxygen consumption rate during rest) were determined. Following swimming, some fish were used for whole body triglyceride analysis while others were used for histological examination. Ucrit was shown to be decreased in the two highest sublethal TCDD exposure groups (0.1 and 1 ng/L) but not in the lowest TCDD exposure group (0.01 ng/L). The exact cause of the decrease in Ucrit is not known, but may be linked to the observed decrease in dorsal aorta diameter, an inability to mobilize triglyceride stores, behavioral adaptations limiting swimming, decreased body length, or a combination of these factors. This TCDD related defect in swimming ability is not due to any increases in gross deformity or mortality rates, nor does it appear that CYP1A induction is required to mediate the toxic effects. Thus, it appears that examination of swim performance may serve as an effective measure of both sublethal acute and developmental toxicities.

<p>The teleost zebrafish (danio rerio) has a highly elevated regenerative capacity compared to mammals, with the ability to quickly and correctly regenerate complex organs such as the fin and the heart following amputation. Studies in other highly regenerative systems suggest that regenerative capacity is directly related to the homeostatic demands of a given tissue, such as high basal levels of cell turnover or the ability to modify tissue size in response to homeostatic changes. However, it is not known if this relationship is present in vertebrate tissues with blastema-based regeneration. To test this idea, we investigated whether markers associated with regeneration are expressed in uninjured zebrafish tissues, and if treatments that block regeneration also lead to homeostatic defects over long periods.</p><p>We found that regenerative capacity is generally required for homeostasis in the fin, as multiple genetic treatments that block regeneration also led to a degenerative loss of distal fin tissue in uninjured animals. In addition, we found that there is extensive cell turnover in the distal fin tissues, accompanied by expression of critical effectors of blastemal regeneration. Both cell proliferation and gene expression were sensitive to changes in Fgf signaling, a factor that is critical for fin regeneration.</p><p>In the heart, we found that although there is little cell turnover in uninjured adult animals, the zebrafish heart can undergo rapid, dramatic cardiogenesis in response to animal growth. These growth conditions induce cardiomyocyte hyperplasia similar to regeneration, and induce gene expression changes in the epicardium, a tissue that is critical for cardiac regeneration. We find that the epicardium continually contributes cells to the uninjured heart, even in the absence of cardiac growth. If this contribution is prevented via a long-term block of Fgf signals, scarring can result, indicating that continual activity of epicardium derived cells (EPDCs) is critical for cardiac homeostasis. We have generated reagents that allow us to visualize EPDCs, and find that they contribute cardiac fibroblasts and perivascular cells during rapid cardiac growth. Uncovering the fate of EPDCs during cardiac homeostasis and regeneration will allow us to better understand their function, and may lead to the development of regenerative therapies for human cardiovascular diseases.</p> / Dissertation

<p>Vertebrates like urodele and teleost have an enhanced capacity for regeneration, when compared to mammals. Recently, the teleost zebrafish (Danio rerio) has become a popular model for studying regenerative events, due to the ability to regenerate multiple organs such as the fin and the heart, and the diverse genetic approaches available for functional studies. In my thesis studies, I have used the zebrafish caudal fin as a model system to understand molecular and cellular mechanism of appendage regeneration. </p><p>Pharmacological and genetic studies have revealed that Fgf signaling is important for appendage regeneration. To dissect the mechanism of Fgfs during zebrafish fin regeneration, lab colleagues and I have generated and utilized transgenic animals in which Fgf signaling can be experimentally increased or decreased. Through these transgenic studies, I found that position-dependent Fgf signaling directs regenerative growth and blastemal proliferation. Proximally-amputated fin regenerates grow at higher rates than the distally-amputated, owing to position-dependent amounts of Fgf activity. Further studies using new transgenics have provided an understanding of mechanisms by which Fgfs influence epidermal regulation of the blastema. Loss- and gain-of-function studies of Fgfs reveal that Fgf signaling both positively and negatively regulated shh expression in the epidermis to maintain blastemal function.</p><p>During the fin regeneration process, pigmentation pattern is re-established as along with bone structures and connective tissues. While the lineage of the blastema is not precisely clear, pigment cells in the fin regenerates are thought to be derived from melanocyte stem cells. Therefore, melanocyte regeneration is an informative system to understand the mechanism underlying regulation of adult stem cells during regeneration. As part of my thesis studies, we generated transgenic animals in which ectopic Ras expression can be experimentally induced. Transgenic studies, combined with pharmacological approaches, have revealed that Ras controls self-renewal of melanocyte stem cells during fin pigment regeneration.</p> / Dissertation

<p>Background: Silver nanoparticles (AgNPs) act as antimicrobials by releasing monovalent silver (Ag+) and are increasingly used in consumer products, thus elevating exposures in human and environmental populations. Materials and Methods: We evaluated Ag+ in a standard model of neuronal cell replication and differentiation, and then determined whether there were similar effects of the ion in vivo using zebrafish. Next, we compared Ag+ and AgNP exposures in the same two models and incorporated the effects of particle coating, size and composition. Conclusions: This work is the first to show that both Ag+ and AgNPs are developmental neurotoxicants in vitro and in vivo. Moreover, although both the soluble ion and the particles impair measures of neurodevelopment, the outcomes and underlying mechanisms of each toxicant are often wholly distinct. Superimposed on the dichotomies between Ag+ and AgNP exposures are clear effects of particle coating, size and composition that will necessitate evaluation of individual AgNP types when considering potential environmental and human health effects. The results presented here provide hazard identification that can help isolate the models and endpoints necessary for developing a risk assessment framework for the growing use of AgNPs.</p> / Dissertation

Zebrafish, Danio rerio, have become an important model for developmental studies and have several advantages over other model systems. These advantages include (1) the easy accessibility of zebrafish embryos for direct observation of their development and (2) their suitability for systematic mutagenesis studies for the identification of genes regulating the development of various tissues and organs, including the skeletal system. Recently, it has been reported that glutamate receptors are expressed in many types of bone cells and regulate bone physiological functions. In the present study, we have examined the effects of a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist¡Xdextromethorphan¡Xon the development of the axial skeleton in zebrafish embryos by using calcein stain. Our results revealed that dextromethorphan significantly attenuates the formation of the axial skeleton and that it is inhibited on pretreatment with glutamate. Moreover, immunohistochemical analysis revealed protein level expression of the NMDA subunit NR1 in the axial region of zebrafish. Our results also indicate that attenuation of NMDA receptor activity-induced change in the axial skeleton may be related to heat-shock protein and extracellular signal-regulated kinase (ERK) signalings. In conclusion, we suggest that the NMDA receptor plays an important role in the development of the axial skeleton. However, further studies are required on the cellular mechanisms of glutamate regulated bone formation.

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.

The inner ear is a complex sensory organ of interconnected chambers, each with a sensory epithelium comprised of hair cells and support cells for detection of sound and motion. This dissertation focuses on the development and regulation of sensory epithelia in zebrafish and utilizes loss of function, gain of function and laser ablation techniques. Hair cells and support cells develop from an equivalence group specified by proneural genes encoding bHLH transcription factors. The vertebrate Atoh1 bHLH transciption factor is a potential candidate for this role. However, data in mouse has led some researchers to conclude it does not have a proneural activity, but, rather, is involved in later stages of hair cell differentiation. In addition, the factors regulating Atoh1 are mostly unknown. We address these issues in zebrafish and show that the zebrafish homologs atoh1a and atoh1b are required during two developmental phases, first in the preotic placode and later in the otic vesicle. They interact with the Notch pathway and are necessary and sufficient for specification of sensory epithelia. Our data confirm atoh1 genes have proneural function. We also go on to show Atoh1 works in a complex network of factors, Pax2/5/8, Sox2, Fgf and Notch. Misexpression of atoh1 alters axial patterning and leads to expanded sensory epithelia, which is enhanced by misexpression of either fgf8 or sox2. Lastly, we examine the role of sox2 in sensory epithelia development and regeneration. Sox2 has been implicated in maintainence of pluripotent stem cells as well as cell differentiation. In the inner ear, Sox2 is initially expressed in the prosensory domain and is required for its formation. Eventually, Sox2 is downregulated in hair cells and maintained in support cells; however, its later role has not been determined. We show that in the zebrafish inner ear, sox2 is expressed after sensory epithelium development has begun and, like in mouse, expression is down regulated in hair cells and maintained in support cells. Our data demonstrate a role for sox2 in maintenance of hair cells and in transdifferentation of support cells into hair cells after laser ablation. Additionally, sox2 is regulated by Aoth1a/1b, Fgf, and Notch.

The vertebrate inner ear is a complex structure responsible for hearing and balance. The inner ear houses sensory epithelia composed of mechanosensory hair cells and non-sensory support cells. Hair cells synapse with neurons of the VIIIth cranial ganglion, the statoacoustic ganglion (SAG), and transmit sensory information to the hindbrain. This dissertation focuses on the development and regulation of both sensory and neuronal cell populations. The sensory epithelium is established by the basic helixloop- helix transcription factor Atoh1. Misexpression of atoh1a in zebrafish results in induction of ectopic sensory epithelia albeit in limited regions of the inner ear. We show that sensory competence of the inner ear can be enhanced by co-activation of fgf8/3 or sox2, genes that normally act in concert with atoh1a. The developing sensory epithelia express several factors that regulate differentiation and maintenance of hair cells. We show that pax5 is differentially expressed in the anterior utricular macula (sensory epithelium). Knockdown of pax5 function results in utricular hair cell death and subsequent loss of vestibular (balance) but not auditory (hearing) defects. SAG neurons are formed normally in these embryos but show disorganized dendrites in the utricle following loss of hair cells. Lastly, we examine the development of SAG. SAG precursors (neuroblasts) are formed in the floor of the ear by another basic helix-loophelix transcription factor neurogenin1 (neurog1). We show that Fgf emanating from the utricular macula specifies neuroblasts, that later delaminate from the otic floor and undergo a phase of proliferation. Neuroblasts then differentiate into bipolar neurons that extend processes to hair cells and targets in the hindbrain. We show evidence that differentiating neurons express fgf5 and regulate further development of the SAG. As more differentiated neurons accumulate, increasing level of Fgf terminates the phase of neuroblast specification. Later on, elevated Fgf stabilizes the transit-amplifying phase and inhibits terminal differentiation. Thus, Fgf signaling regulates SAG development at various stages to ensure that proper number of neurons is generated.

Vertebrates have evolved a unique way to sense their environment: placodallyderived sense organs. These sensory structures emerge from a crescent-shaped domain, the preplacodal domain, which surrounds the anterior neural plate and generates the paired sense organs as well as the cranial ganglia. For decades, embryologists have attempted to determine the tissue interactions required for induction of various placodal tissues. More recently, technological advances have allowed investigators to ask probing questions about the molecular nature of placodal development. In this dissertation I largely focus on development of the otic placode. I utilize loss-of-function techniques available in the zebrafish model system to demonstrate that two members of the fibroblast growth factors family of secreted ligands, Fgf3 and Fgf8, are redundantly required for otic placode induction. I go on to show that these factors are expressed in periotic tissues from the beginning of gastrulation. These findings are consistent with a model where Fgf3 and Fgf8 signal to preotic tissue to induce otic-specific gene expression. This model does not address other potential inducers in otic induction. A study using chick explant cultures suggests that a member of the Wnt family of secreted ligands also has a role in otic induction. I therefore test the relative roles of Wnt and Fgf in otic placode induction. The results demonstrate that Wnt functions primarily to correctly position the Fgf expression domain and that it is these Fgf factors which are directly received by future otic cells. Lastly, I examine the function of the muscle segment homeobox (msx) gene family expressed in the preplacodal domain. This study demonstrates that Msx proteins refine the boundary between the preplacodal domain and the neural plate. Further, msx genes function in the differentiation and survival of posterior placodal tissues (including the otic field), neural crest and dorsal neural cell types. Loss of Msx function results in precocious cell death and morphogenesis defects which may reflect perturbed BMP signaling.

Cryopreservation of gametes provides a promising method to preserve fish genetic materials, which offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful cryopreservation of spermatozoa of about 200 fish species has been achieved, systematic studies on cryopreservation of fish oocytes have only recently been undertaken. The objective of the present studies was to use zebrafish as a model system to develop a cryopreservation protocol for fish oocytes and to develop reliable viability assessment methods for monitoring zebrafish oocyte viability both before and after cryopreservation. A simple and rapid enzymatic method for zebrafish oocytes isolation was developed and the investigations on cryopreservation of zebrafish oocytes using improved controlled slow cooling and vitrification were carried out. Oocyte viability following cryopreservation was investigated by ATP assay, oocyte viability molecular signature (OVMS) and cryomicroscopic observation in addition to staining methods. The optimum conditions for oocyte enzymatic separation were identified as 0.4mg/ml collagenase or 1.6mg/ml hyaluronidase treatment for 10min at 22ºC and this method can be used for oocytes at all stages. The use of sodium free medium (KCl buffer), fast warming and 4-step removal of cryoprotectants in an improved controlled slow cooling protocol significantly enhanced oocyte viability (67.5 ± 1.7%) when compared with a previous study (16.3 ± 2.3%) in this laboratory. Mixtures of cryoprotectants (methanol, Me2SO and propylene glycol), stepwise addition and removal of cryoprotectants in combination of a new vitrification system (CVA65 vitrification system) were used in vitrification studies. Oocyte survivals after vitrification assessed by trypan blue staining were relatively high (76.5 ± 6.3%) shortly after warming in KCl buffer. Furthermore, the result of ATP assay showed that ATP levels in oocytes decreased significantly after cryopreservation indicating the bioenergetic systems of oocytes were damaged. Cryomicroscopic observations demonstrated that Intracellular ice formation (IIF) is the main factor causing injuries during cryopreservation of zebrafish oocytes. The results provided by the present study will assist successful protocol design for cryopreservation of fish oocytes in the future.