The temporal and spatial patterning that underlies morphogenetic events is controlled by gene regulatory networks (GRNs). These operate through a combinatorial code of DNA – binding transcription factor proteins, and non – coding DNA sequences (cis-regulatory elements, or CREs), that specifically bind transcription factors and regulate nearby genes. By comparatively studying the development of different species, we can illuminate lineage – specific changes in gene regulation that account for morphological evolution. The central nervous system of vertebrates is composed of diverse neural cells that undergo highly coordinated programs of specialization, migration and differentiation during development. Approximately 20% of neurons in the cerebral cortex are GABAergic inhibitory interneurons, which release the neurotransmitter gamma-aminobutyric acid (GABA). Diseases such as autism, schizophrenia and epilepsy are associated with defects in GABAergic interneuron function. Several members of the distal-less homeobox (Dlx) transcription factor family are implicated in a GRN underlying early GABAergic interneuron development in the forebrain. I examined the role played by orthologous dlx genes in the development of GABAergic interneurons in the zebrafish forebrain. I found that when ascl1a transcription factor is down-regulated through the micro-injection of translation – blocking morpholino oligonucleotides, Dlx gene transcription is decreased in the diencephalon, but not the telencephalon. Similarly, gad1a transcription is also decreased in this region for these morphants. As gad1a encodes an enzyme necessary for the production of GABA, these genes are implicated in a cascade underlying GABAergic interneuron development in the diencephalon.

Many studies have reported individual differences in resting metabolic rate (RMR), the energetic cost of self-maintenance. Differences among individuals in the energetic cost of self-maintenance may influence life-history decisions and hence, fitness. In this study, we examined potential causes of intra-specific variation in RMR in zebrafish, Danio rerio. First, the repeatability of RMR was determined to check whether a single measure was reflective of future physiological performance. As predicted, RMR was repeatable over a period of three weeks. However, none of stress-coping style, baseline cortisol levels, metabolically-active organ (gill, heart, intestine and liver) mass, aggression or activity levels were correlated with RMR, i.e. none of these factors were significant contributors to individual variation in RMR. These results imply that other factors must be sought to explain the inter-individual variation in RMR observed in zebrafish.

These studies present a series of novel roles for glucocorticoid signaling in the developing zebrafish embryo. The best-characterized roles of cortisol, the primary circulating corticosteroid in teleost fish, are known to occur by the activation of the glucocorticoid receptor (GR) in the post-hatch animal to mobilize energy reserves for response and recovery from stressful situations. For the first time, evidence is presented that GR and cortisol are key developmental regulators in the pre-hatch zebrafish embryo and that glucocorticoid signaling modulates multiple critical developmental pathways and affects embryogenesis in diverse ways. Prior to these experiments, very little was known regarding the developmental role of glucocorticoids in lower vertebrates. In mammalian models, there has been extensive study of the action of these steroids in late-stage organ maturation, and they have a variety of clinical and biomedical applications. However, in fish, there was a relative dearth of information regarding the basic dynamics and potential functional roles of cortisol and GR in embryogenesis. Zebrafish are a popular model for developmental study, with optically transparent embryos that allow for reliable observation. Additionally, the zebrafish genome is fully sequenced and extensively annotated, and a variety of molecular biology techniques are well-established in the existing literature. The zebrafish is also now recognized as an advantageous model for endocrine and stress axis studies, as it expresses a single GR gene, unique among teleosts but comparable to mammals. Preliminary studies published in the literature described cortisol and GR as deposited in the zebrafish embryo prior to fertilization, and showed their expression declining prior to hatch, then rising significantly as larvae approach the stage of first feeding. This dynamic expression of both ligand and receptor during embryogenesis, combined with knowledge from mammalian models, led to the hypothesis that glucocorticoid signaling may be functionally relevant in zebrafish development. A variety of techniques were used to examine the roles of cortisol and GR in the zebrafish embryo. Morpholino oligonucleotides were injected into one-cell embryos to block GR protein translation, allowing for the identification of GR-responsive developmental events and putative GR target genes. High-density microarray analysis of GR morphants presented numerous novel genes and pathways that are modulated by glucocorticoid signaling in the embryo. The ability to microinject molecules into a newly-fertilized zygote also allowed for other manipulations, including the addition of exogenous cortisol or the use of a cortisol-specific antibody to sequester maternally deposited cortisol. These studies provided the first evidence regarding the functional importance of the maternal cortisol deposition in the zebrafish oocyte prior to fertilization. The detailed temporal and spatial expression of GR mRNA and protein in the developing embryo has been characterized for the first time. GR expression is widespread, especially in developing mesoderm, and de novo GR transcription in the zebrafish embryo begins within 12 hours post fertilization. Lack of GR protein expression in the developing zebrafish embryo causes reduced growth, delayed somitogenesis, altered myogenesis, and severely reduces post-hatch survival. Additionally, GR modulates the expression of bone morphogenetic proteins, a family of morphogens that are involved in major developmental events including dorsoventral patterning, somitogenesis, myogenesis, and organogenesis. Reduction in GR protein content in the developing embryo is also linked to other major developmental processes including neurogenesis, eye formation, cellular adhesion, and development and function of the hypothalamic-pituitary-interrenal (HPI) axis. Cortisol in the early embryo, which is contributed entirely by maternal deposition prior to fertilization, is an important regulator of cardiogenesis and development of the HPI axis. Modulation of cortisol content in the early embryo causes an impairment of the post-hatch response to a physical stressor, as larvae exposed to increased cortisol during embryogenesis displayed an inability to increase heart rate in response to an acute physical stress, and did not display the classical increase in cortisol that follows a stressor challenge. Embryos that experience lowered glucocorticoid signaling in development tend to have a heightened post-hatch response to stress, further supporting the conclusion that HPI axis development is regulated by glucocorticoid signaling. These studies have identified key cardiogenic and HPI axis genes that are GR-responsive, providing mechanistic explanations for these phenotypic changes. Together these findings indicate that maternal deposition of cortisol in the embryo can pattern the post-hatch larva and has definitive impacts on performance as the offspring begin locomotion and approach feeding stages. In total, these studies demonstrate that glucocorticoid signaling is critically important to zebrafish embryogenesis, defining novel roles that are completely independent of the classical vertebrate stress response. These functions have significant effects on diverse developmental pathways and processes, and with the potential applicability of the zebrafish model to studies in higher vertebrates, may have important biomedical applications.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 55-69).

The recently accepted phenomenon of adult neurogenesis is important for basic biological research and, potentially, can have major implications for the treatment of age-related cognitive decline and disease. Investigation into the mechanisms of adult neurogenesis and its ability to replenish brain circuits with new functional neurons requires whole animal models.  Zebrafish, a diurnal vertebrate, has robust cell proliferation in several neurogenic niches, including the cerebellum and dorsal telencephalon, the latter bearing homology to mammalian hippocampus. Because zebrafish demonstrate rapid regeneration in all tissues, including successful repair following brain traumas, they are promising as a model for designing therapies for human brain traumas or stroke. Their long lifespan and gradual aging also makes them an interesting model for the role of neurogenesis in counteracting human neurodegenerative disorders of aging. In different models, it has been found that cell proliferation in adult brain can be significantly affected by behavioral and environmental factors. Among those is nutrition, impacting adult neurogenesis through the amount of caloric intake, meal frequency, and meal content. The study presented here addressed the effects of nutritional factors on adult neurogenesis in a zebrafish model of premature aging due to excessive caloric food intake since early development. Fish were exposed to fasting, different diets and feeding schedules, with the rate of cell proliferation documented in two largest neurogenic niches of the zebrafish brain, the cerebellum and dorsal telencephalon. Here we show that, under normal conditions, fish with premature aging demonstrate dramatic decline in adult neurogenesis in both niches, when compared to age-matched control. The present findings establish an effect of nutrition on neurogenesis in the cerebellum and dorsal telencephalon of adult zebrafish. Zebrafish maintained on HFD, subjected to fasting, or fed only in the evenings showed significant changes in neurogenesis in two distinct neurogenic niches from that of control fish. Remarkably, the two brain regions under investigation displayed partially different responses to nutrition related factors. This was reflected in the cerebellar niche in which neurogenesis was significantly increased by 24h fast/24h refeed, high fat diet, and evening feeding conditions.  Neurogenesis of the cerebellum was significantly decreased in 24h fast, 42h fast/refeed conditions.  In the dorsal telencephalon, neurogenesis was significantly amplified by high protein, and similar to the cerebellum, high fat diet and evening feeding conditions.  In contrast, neurogenesis of the dorsal telencephalon was significantly attenuated only in the 72h fasting condition. This study provides evidence that nutrition plays important role in the modulation of adult neurogenesis in zebrafish, and presence of niche-specific responses to nutritional factors. This further suggests that zebrafish can serve as a model for studying the effects of specific diets, metabolic factors and drugs that affect metabolism in search for prophylactic and therapeutic measures for age-related cognitive decline or neurodegenerative disorders.

Nemaline myopathy is a subtype of congenital myopathy that is clinically characterized by muscle weakness and early hypotonia of variable severity. Pathologically, nemaline myopathy is characterized by the presence of nemaline rods that stain purple in modified Gӧmӧri trichrome dye in patient biopsies under a microscope. Affected individuals experience skeletal muscle weakness and feeding difficulties, but most individuals will also experience respiratory muscle weakness that is disproportional to the weakness in skeletal muscles. Currently, 6 different subtypes of nemaline myopathy have been identified, each caused by mutations in ACTA1, NEB, TPM2, TPM3, TNNT1, KBTBD13, CFL2, KLHL40, KLHL41, or LMOD3, which are genes that encode either thin filament proteins or Kelch-like proteins. Of these genes, mutations in NEB and ACTA1 account for the majority of nemaline myopathy cases. Due to the genetic heterogeneity of nemaline myopathy, it is imperative to discover therapeutic targets and treatments that can universally treat nemaline myopathy patients. Preliminary data from our lab has demonstrated that proteasome complexes are downregulated in nemaline myopathy patients. Further, proteasomal activators improved motor function in neb zebrafish models, demonstrating the potential for proteasome activators to be therapeutics for nemaline myopathy patients. To extend these studies, the effect of proteasome activators, betulinic acid and Rolipram, was evaluated on the motor function in neb zebrafish models. However, in our experimental trials with betulinic acid and Rolipram, no positive effect on motor function in neb zebrafish was observed. In order to confirm our findings for both betulinic acid and Rolipram, additional trials will need to be conducted. / 2019-10-31T00:00:00Z

Diabetes is a metabolic disease characterized by the loss of functional pancreatic beta cells. The incidence of diabetes has risen rapidly in recent decades, which has been attributed at least partially to alterations in host-associated microbial communities, or microbiota. It is hypothesized that the loss of important microbial functions from the microbiota of affected host populations plays a role in the mechanism of disease onset. Because the immune system also plays a causative role in diabetes progression, and it is well documented that immune cell development and function are regulated by the microbiota, most diabetes microbiota research has focused on the immune system. However, microbial regulation is also required for the development of many other important tissues, including stimulating differentiation and proliferation. We therefore explored the possibility that the microbiota plays a role in host beta cell development. Using the larval zebrafish as a model, we discovered that sterile or germ free (GF) larvae have a depleted beta cell mass compared to their siblings raised in the presence of bacteria and other microbes. This dissertation describes the discovery and characterization of a rare and novel bacterial gene, whose protein product is sufficient to rescue this beta cell developmental defect in the GF larvae. Importantly, these findings suggest a possible role for the microbiota in preventing or prolonging the eventual onset of diabetes through induction of robust beta cell development. Furthermore, the loss of rare bacterial products such as the one described herein could help to explain why low diversity microbial communities are correlated with diabetes.

Myelin is essential for normal nervous system conduction as well as providing metabolic support for the ensheathed axon and has been implicated to influence axon calibre (diameter of the axon body) growth. In demyelinating diseases, the disruption of these functions causes axon degeneration resulting in neurological impairment. The neurons that are myelinated in the CNS and the axon-oligodendrocyte (axon- OL) interactions that might regulate axon calibre and myelination during myelinated axon formation are still mostly unknown, preventing a deeper understanding of CNS development and repair. This doctoral thesis identifies a specific subset of interneurons that are myelinated and investigates the axon-oligodendrocyte interactions during axon calibre growth and initial myelination. In the zebrafish spinal cord, Commisural Primary Ascending interneurons (CoPA), Circumferential Descending interneurons (CiD) and reticulospinal neurons are amongst the first to be myelinated, whereas Commisural Bifurcating Longitudinal interneurons (CoBL) and Circumferential Ascending interneuron (CiA) are not myelinated during early developmental stages. Of the myelinated neurons, axon calibre of reticulo spinal neurons is increased in time with myelin ensheathment, while the axon calibre of CoPA and CiD interneurons is not increased with the onset of myelination. In order to investigate whether there might be a causative relationship between axon calibre increase and myelin ensheathment, the majority of oligodendrocytes were eliminated by olig2 morpholino knockdown. In the absence of oligodendrocytes, the axon calibre of reticulospinal neurons was normal, demonstrating that axon calibre growth is independent of axon-OL interactions and myelin ensheathment. In order to further investigate which aspects of myelinated axon formation might be regulated by axon-OL interactions, axonal activity was reduced through inhibition of synaptic vesicle release by global expression of Tetanus-toxin (TetTx). TetTx treated zebrafish showed a 40% decrease of myelinated axons in the spinal cord. Interestingly, only 10% of this reduction was caused by a decrease in oligodendrocyte number in the spinal cord. Single cell analysis of individual oligodendrocytes revealed a 30% reduction of myelin sheaths per oligodendrocyte in TetTx treated animals, indicating a positive correlation between synaptic vesicle release and the extent of myelination. Timelapse analysis of the myelinating behaviour of individual oligodendrocytes revealed that the decrease in myelin sheaths per cell in the absence of synaptic vesicle release results from a reduction in the initial formation of sheaths rather than an increased retraction of myelin sheaths. Furthermore, individual myelin sheaths formed by the same oligodendrocyte exhibit a dynamic range of different growth rates in control animals, which was reduced to a more uniform, slow growth of myelin sheaths in the absence of synaptic vesicle release. This suggests that local axon-OL interactions can regulate the dynamic myelin sheath growth through synaptic vesicle release. The analyses in this doctoral thesis identifies a subset of the neurons that are myelinated during the onset of myelination in the zebrafish spinal cord, demonstrates that axon caliber growth of these neurons is independent of myelin ensheathment and that axon-OL interactions mediated by synaptic vesicle release can regulate the extent of myelination and influence the dynamic myelinating behavior of oligodendrocytes in vivo. These findings begin to elucidate the axon-OL interactions underlying myelinated axon formation during CNS development, from which future studies might derive neuro-regenerative treatments for demyelinating diseases.

Upon amputation, zebrafish innately regenerate lost or damaged bone by precisely positioning injury-induced, lineage-restricted osteoblast progenitors (pObs). While substantial progress has been made in identifying the cellular and molecular mechanisms underlying this fascinating process, the cell-specific function of these pathways is poorly understood. Understanding how molecular signals initiate osteoblast dedifferentiation, balance progenitor renewal and re-differentiation, and control bone shape during regeneration are of paramount importance for developing human therapies. We show that fin amputation induces a Wnt/β-catenin-dependent epithelial to mesenchymal transformation (EMT) of osteoblasts to generate proliferative Runx2+ pObs. Localized Wnt/β-catenin signaling maintains this progenitor population towards the distal tip of the regenerative blastema. As they become proximally displaced, pObs upregulate sp7 and subsequently mature into re-epithelialized Runx2-/sp7+ osteoblasts that extend pre-existing bone. Autocrine Bone Morphogenetic Protein (BMP) signaling promotes osteoblast differentiation by activating sp7 expression and counters Wnt by inducing Dickkopf-related Wnt antagonists. As such, opposing activities of Wnt and BMP coordinate the simultaneous demand for growth and differentiation during bone regeneration. Previous studies have implicated Hedgehog/Smoothened (Hh/Smo) signaling in controlling the re-establishment of stereotypically branched bony rays during fin regeneration. Using a photoconvertible patched2 reporter, we resolve active Hh/Smo output to a narrow distal regenerate zone comprising pObs and neighboring migratory basal epidermal cells. Hh/Smo activity is driven by epidermal Sonic hedgehog a (Shha) rather than Ob-derived Indian hedgehog a (Ihha), which instead uses non-canonical signaling to support bone maturation. Using high-resolution imaging and BMS-833923, a uniquely effective Smo inhibitor, we show that Shha/Smo promotes branching by escorting pObs into split groups that mirror transiently divided clusters of Shha-expressing epidermis. Epidermal cellular protrusions directly contact pObs only where an otherwise occluding basement membrane remains incompletely assembled. These intimate interactions progressively generate physically separated pOb pools that then regenerate independently to collectively re-form a now branched bone. Our studies elucidate a signaling network model that provides a conceptual framework to understand innate bone repair and regeneration mechanisms and rationally design regenerative therapeutics. This dissertation includes previously published co-authored material. / 10000-01-01

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