In vertebrate embryonic development, correct specification of tissue fates along the dorsoventral (D/V) axis is known to require the secreted signaling ligand Wnt8. Wnt8 signaling promotes ventral fates and antagonizes the expansion of the dorsal domain known as the organizer. Maintenance of the organizer is critical for proper development as this tissue is known to produce inhibitors of Wnt and BMP (Bone Morphogenetic Protein) family ligands; BMPs are also known to play a major role in promoting ventral fates. In order to understand how Wnt8 antagonizes the organizer, we analyzed the epistatic relationship between Wnt8 and the transcriptional repressors Vent and Vox using zebrafish as a model organism. We found that Wnt8/β-catenin signaling directly regulates the transcriptional levels of vent and vox so that they can repress the transcription of dorsal genes on the ventral side of the embryo. To understand the contribution of Wnt8 towards ventral fate specification, we carefully analyzed its relationship with BMP signaling during gastrula stages. We found that bmp expression in the mesoderm is under the control of Wnt8 at mid-gastrulation and that regulation of bmp explains many of the ventral defects observed in wnt8 mutants. Antagonism of the expression of organizer-derived BMP inhibitors by Wnt8 also indirectly allows timely BMP signaling. Analysis of wnt8; bmp double mutants revealed an early unsuspected function of BMP in the antagonism of the organizer. Further, we uncovered a mechanism through which regulation of vent, vox and a related-gene ved expression by both Wnt8 and BMP antagonizes dorsal/axial mesoderm identity to preserve the integrity of ventral/non-axial tissues. In summary, we have revealed some of the mechanisms of Wnt8 function in D/V mesoderm patterning: it restricts the organizer domain by regulating vent and vox, it allows BMP induced differentiation through its inhibition of BMP antagonists derived from the organizer and it co-regulates vent, vox, and ved with BMP signaling to allow maintenance of the non-axial domain.

Adult neurogenesis is a conserved feature of the central nervous system across the animal kingdom. This process takes place in restricted neurogenic niches of the brain, where active populations of adult stem/progenitor cells are capable of producing newborn neurons. The niche is tightly controlled by intrinsic signals within the microenvironment and from stimuli arising from the external world, which together determine the cellular behaviour of the niche and neuronal output. Currently, our understanding of the biological properties of adult neurogenesis rests mainly on two niches of the vertebrate forebrain. To broaden our view of the diversity of this trait comparative models and new niches must be explored. Here, I have taken advantage of the robust neurogenic capacity of the adult zebrafish brain to examine differences in forebrain and sensory neurogenic niches in regards to cytoarchitectural organization, neurogenic plasticity, and regulation. Five principle findings emerge: (1) up to six morphologically distinct cell types compose forebrain and sensory niches, and are devoid of ependymal cells; (2) heterogeniety in the phenotype of the stem/progenitor cell exists across niches; some having radial glial characteristics; (3) active populations of proliferating stem/progenitor cells reside within primary sensory structures of the adult brain, forming a “sensory neurogenic niche”; different from other models of adult neurogenesis; (4) changes in the social environment induce neurogenic plasticity in sensory niches more readily than integrative niches of the forebrain, and occur independently of cortisol levels; (5) modality-specific stimulation influences stages of adult neurogenesis exclusively in corresponding primary sensory niches as a result of sensory-dependent neurogenic plasticity. Additionally, I have shown that Fibroblast Growth Factor signalling may not be involved in maintaining cell proliferation in sensory niches. These studies showcase the diverse properties of forebrain and sensory neurogenic niches and provide a new perspective concerning the functional role of adult neurogenesis.

Monocarboxylate transporters (MCTs) have been shown to be important in regulating metabolism during hypoxia in mammals. However, the role of MCTs in hypoxic survival in lower vertebrates is currently unclear. The goal of this study was to investigate the coordination of MCTs along with other metabolic proteins during hypoxia. Therefore, we subjected zebrafish (Danio rerio) to 1.5 mg L-1 O2 over 48 and 96-hr and measured tissue-specific transcriptional changes of MCTs (1, 2 and 4), lactate dehydrogenase A (LDHa), citrate synthase (CS), and other metabolic proteins using real-time RT-PCR. There were no changes in mRNA in muscle at 48 and 96-hr. When data from both time points were pooled in brain, a significant increase was found in MCT4 (+102%) and LDHa (+28%) mRNA indicating a preference towards glycolysis. In gills, there were increases in LDHa at 48-hr (+101%) and MCT1 (+24%) mRNA from pooled data suggesting that both anaerobic and aerobic metabolism is being utilized. Heart had the greatest changes in transcriptional levels compared to other tissues. At 48-hr, increases were found in MCT1 (+117%), MCT4 (+86%), LDHa (+197%), and pooled data showed an increase in CS (+18%) mRNA. These results indicate that the influx and efflux of lactate are both employed as strategies in cardiac tissue during hypoxia. This study has shown that fish utilize tissue-specific regulation of MCTs along with other metabolic genes during hypoxia. / Thesis (Master, Biology) -- Queen's University, 2009-08-24 13:44:06.114

No description available.

Selenium (Se) is an essential trace element to most living organisms, however when compared to other ingested essential trace elements Se has the lowest margin of safety between essential and toxic concentrations. Oviparous vertebrates, especially fishes, are highly susceptible to dietary Se toxicity. Greater incidences of deformities and/or mortalities have been observed in F1 generation larval fishes whose parents were exposed to excess dietary Se in the form of selenomethionine (SeMet), however little information is available on effects of chronic dietary SeMet exposure to adult fish and persistent effects of in ovo SeMet exposure to F1 generation fish. This thesis investigated effects of chronic dietary exposure of excess Se in the form of SeMet on swimming performance (Ucrit), oxygen consumption (MO2), stored energy (triglycerides and glycogen), and the physiological stress response (cortisol production) in adult zebrafish (Danio rerio), as well as immediate (incidence of deformities and mortality) and persistent (e.g. changes in Ucrit, MO2, bioenergetics, the physiological stress response and reproduction) effects of in ovo exposure to SeMet in F1 generation zebrafish. In addition, the study investigated potential underlying mechanisms of SeMet-induced developmental toxicities in early life stages of zebrafish using embryo microinjection. Two separate dietary SeMet exposure studies in adult zebrafish and two in ovo SeMet maternal transfer studies in F1 generation zebrafish were conducted. The first dietary or in ovo exposure study explored effects of excess SeMet exposure on adult zebrafish or the entire life cycle of F1 generation zebrafish. The second study investigated mechanisms of observed SeMet-induced effects on adult or F1 generation zebrafish. In the first feeding study, a significant reduction in Ucrit and greater accumulation of stored energy were observed in the excess dietary SeMet exposed groups when compared to the Se-sufficient dietary control group. The second feeding study showed a greater metabolic rate, and impaired aerobic energy metabolism and triglyceride homeostasis in adult fish fed excess dietary SeMet, which was associated with a reduction in swimming performance and accumulation of triglycerides. Embryos collected from adult zebrafish in both dietary SeMet exposure studies were used to investigate effects of in ovo SeMet exposure on the entire life cycle of F1 generation fish. The first study showed a greater incidence of mortality, an increasing trend for deformities in F1 generation larval zebrafish, and reduced Ucrit in F1 generation adult fish exposed to excess SeMet via in ovo maternal transfer. However, concentrations of stored energy, cortisol and reproduction were unaltered. The second study found that impaired aerobic performance might have been responsible for the reduction in Ucrit of F1 generation adult zebrafish exposed to excess SeMet. Since there is a high variability in Se deposition among eggs via natural maternal transfer, SeMet embryo microinjection was adopted to mimic maternal transfer and to investigate potential mechanisms of SeMet-induced developmental toxicities in early life stages of zebrafish. Greater gene expression of oxidant-inducible transcription factors and impairment in gene expression of an enzyme involved in methionine catabolism were observed in early life stages of zebrafish exposed to excess SeMet via in ovo microinjection. The research presented in this thesis suggests that environmentally relevant dietary SeMet exposure can alter physiological responses in adult fishes and reduce survivability of F1generation fishes, which could impact fitness and recruitment of wild fishes inhabiting Se-contaminated aquatic ecosystems. In addition, the study suggests that SeMet-induced developmental toxicities in early life stages of fishes might be related to oxidative stress or impaired methylation, or a combination of these mechanisms.

Adult neurogenesis is a conserved feature of the central nervous system across the animal kingdom. This process takes place in restricted neurogenic niches of the brain, where active populations of adult stem/progenitor cells are capable of producing newborn neurons. The niche is tightly controlled by intrinsic signals within the microenvironment and from stimuli arising from the external world, which together determine the cellular behaviour of the niche and neuronal output. Currently, our understanding of the biological properties of adult neurogenesis rests mainly on two niches of the vertebrate forebrain. To broaden our view of the diversity of this trait comparative models and new niches must be explored. Here, I have taken advantage of the robust neurogenic capacity of the adult zebrafish brain to examine differences in forebrain and sensory neurogenic niches in regards to cytoarchitectural organization, neurogenic plasticity, and regulation. Five principle findings emerge: (1) up to six morphologically distinct cell types compose forebrain and sensory niches, and are devoid of ependymal cells; (2) heterogeniety in the phenotype of the stem/progenitor cell exists across niches; some having radial glial characteristics; (3) active populations of proliferating stem/progenitor cells reside within primary sensory structures of the adult brain, forming a “sensory neurogenic niche”; different from other models of adult neurogenesis; (4) changes in the social environment induce neurogenic plasticity in sensory niches more readily than integrative niches of the forebrain, and occur independently of cortisol levels; (5) modality-specific stimulation influences stages of adult neurogenesis exclusively in corresponding primary sensory niches as a result of sensory-dependent neurogenic plasticity. Additionally, I have shown that Fibroblast Growth Factor signalling may not be involved in maintaining cell proliferation in sensory niches. These studies showcase the diverse properties of forebrain and sensory neurogenic niches and provide a new perspective concerning the functional role of adult neurogenesis.

The ontogeny of carbon dioxide (CO2) sensing in zebrafish (Danio rerio) has not been studied. In this thesis, CO2-mediated increases in heart rate were used to gauge the capacity of zebrafish larvae to sense CO2. CO2 is thought to be sensed through neuroepithelial cells (NECs), which are homologous to mammalian carotid body glomus cells. Owing to its role in facilitating intracellular acidification during exposure to hypercapnia, it was hypothesized that carbonic anhydrase (CA) is involved in CO2 sensing, and that inhibition of CA would blunt the downstream responses. The cardiac response to hypercapnia (0.75% CO2) was reduced in fish exposed to acetazolamide, a CA inhibitor, and in fish experiencing CA knockdown. Based on pharmacological evidence using β-adrenergic receptor (ß-AR) antagonists, and confirmed by β1AR gene knockdown, the efferent limb of the reflex tachycardia accompanying hypercapnia is probably mediated by sympathetic adrenergic neurons interacting with cardiac β1 receptors.

The dysfunction of tau protein has been implicated in a number of neurodegenerative diseases, including Alzheimer’s disease (AD) and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). In these diseases, the tau protein is aberrantly hyperphosphorylated and aggregated to form neuropathological deposits in the cell body of neurons. Evidence from genetics studies has shown a linkage between tau mutations and autosomal dominantly inherited FTD. In Chapter one, our current understanding of the mechanisms of tauopathies is summarized. In addition, multiple animal models for mechanistic studies of tauopathies are reviewed. In this thesis, endogenous tau genes in zebrafish were identified and investigated in an attempt to establish zebrafish as an animal model for study of tauopathies. Paper 1 describes the identification of two genes, mapta and maptb in zebrafish that represent duplicates of an ancestral tau orthologue. It examines their complex alternative mRNA splicing patterns and their patterns of expression during embryogenesis. Paper 2 (thesis chapter in the form of a manuscript) describes how we might use zebrafish as an animal model to investigate tau function. Two antibodies that detect Mapta and Maptb specifically are described. In addition, we establish that inhibition of Maptb translation causes an impairment of axonogenesis during zebrafish embryogenesis. / Thesis (M.Sc.) -- University of Adelaide, School of Molecular and Biomedical Science, 2010

Thesis (Ph. D.)--Case Western Reserve University, 2008. / [School of Medicine] Department of Genetics. Includes bibliographical references.

Myelination drastically speeds up action potential propagation along axons, which is fundamental for the correct function of neuronal circuits. However, axon-oligodendrocyte interactions regulating the onset of myelin formation remain unclear. I sought to determine how reticulospinal axons control myelination, as they are the first myelinated in the zebrafish spinal cord. I genetically manipulated zebrafish in order to either remove such axons from a region of the spinal cord, or to increase their number, and characterized oligodendrocyte-lineage cells following this axonal loss- or gain-of-function. In kinesin-binding protein (kbp) mutants, reticulospinal hindbrain neurons start axonogenesis but axons fail to grow along the entire spinal cord as in wildtype, providing an axon-deficient posterior spinal cord and an intact anterior region. I found that early stages of oligodendrocyte development, such as the specification of oligodendrocyte precursors, their distribution and migration were not affected in the posterior spinal cord of these mutants. However, both the proliferation and the survival of late precursors were impaired, resulting in a significant reduction of mature oligodendrocytes in the posterior region of mutants at the onset of myelination. Since the anterior spinal cord of mutants is indistinguishable from wildtype, these results demonstrate that reticulospinal axons provide a mitogenic and a survival signal to a subset of developing OPCs, enabling their differentiation and lineage progression. I then found that the absence of reticulospinal axons did not affect the timing of oligodendrocyte differentiation, which matured on time, suggesting that this follows an intrinsic timer, as previous studies suggested. Oligodendrocytes also did not myelinate incorrect axonal targets, but instead adapted to the reduced axonal surface by elaborating fewer myelin sheaths. Additionally, oligodendrocytes made shorter sheaths, and also incorrectly ensheathed neuron somas in the mutant spinal cord, suggesting that either kbp function or a precise amount of axonal surface are required to prevent ectopic myelination of somas and to promote the longitudinal growth of myelin sheaths. In wildtype animals, the two reticulospinal Mauthner axons are the very first myelinated in the spinal cord. In animals where Notch1a function is temporarily abrogated or hoxb1 genes are temporarily upregulated, supernumerary Mauthner neurons are generated. I found that these extra axons are robustly myelinated, with no impairment of myelination of adjacent axons. Surprisingly, the number of oligodendrocytes was not altered, but I found that each individual oligodendrocyte elaborated more myelin sheaths, whose total length was also longer than in wildtypes. Additionally, dorsal oligodendrocytes, which normally myelinate only small-calibre dorsal axons, readily extended processes ventrally to myelinate the supernumerary large-calibre Mauthner axons, in addition to small-calibre axons. These results suggest that oligodendrocytes are plastic and are not destined to myelinate a particular type of axon, and conversely, that axonal signals that induce myelination are similar for different axons. The long-standing observation that oligodendrocytes tend to myelinate either few large axons or many small axons thus reflects local interactions of oligodendrocyte processes with the nearby axons, rather than different subtypes of oligodendrocytes specified by an intrinsic programme of differentiation. Collectively, this work shows that axons extensively influence both oligodendrocyte lineage progression and oligodendrocyte myelinating potential in vivo.