Abstract
An Investigation of the Molecular Mechanisms Regulating Zebrafish Hindbrain Morphogenesis
The central aim of my thesis research was to advance our understanding of hindbrain morphogenesis, more specifically hindbrain ventricle formation, cerebellar development, and neuronal migration. The embryonic brain begins as a simple tube, the lumen of which forms the brain ventricles, a highly conserved system of cavities with important functions. The first part of my thesis describes my work focused on investigating the roles of zebrafish Zic1 and Zic4 in dorsal neural tube development. I hypothesized that Zic1 and Zic4 proteins function in hindbrain ventricle formation. Using morpholino knockdown and a battery of molecular markers, I provide evidence that zebrafish Zic1 and Zic4 function cooperatively to promote normal hindbrain ventricle morphogenesis. On the basis of morphological and molecular characterization, I showed that in Zic1 and/or Zic4 morphants the ventricle does not open properly due in part to a reduction in dorsal hindbrain progenitor cell proliferation, and I determined that Zic1 and Zic4 function is required for the development of the dorsal roof plate and dorsal rhombomere boundary signaling centers. I showed that roof plate formation and patterning from rhombomere boundaries are critical components of ventricle development. My findings suggest that fundamental Zic functions are likely conserved throughout vertebrates, and aspects of zebrafish Zic1 and Zic4 gene function may inform future studies of DWM. In the second part of my thesis, I explored the molecular mechanisms underlying early cerebellum development in zebrafish by addressing the role of the cMet signaling in this process. In the mouse, cMet signaling has been shown to regulate cerebellum development. Abnormalities in cerebellar structure have been reported in some autistic patients, and altered regulation of human cMET expression has been implicated in autism. I showed that zebrafish cmet is expressed in the cerebellar primordium and the hgf ligand genes expressed in surrounding tissues. Morpholino knockdown of either cMet or its Hgf ligands leads to reduction of the cerebellum, as a consequence of reduced proliferation, disrupted cell type specification of VZ progenitors, and thus differentiation of VZ-derived cell types, and impaired granule cell development. I provide further evidence that cMet signaling regulates the migration of a subpopulation of hindbrain neurons.
Development of the Voltage-Gated Sodium and Potassium Currents Underlying Excitability in Zebrafish Skeletal Muscle
Excitable cells display dynamically regulated changes in the properties of ion currents during development. These changes are crucial for the proper maturation of cellular excitability, and therefore have the potential to affect more sophisticated functions, including neural circuits, movements, and behaviors. Zebrafish skeletal muscle is an excellent model for studying the development of ion channels and their contributions to excitability. They possess distinguishable populations of red and white muscle fibers, whose biological functions are well understood. The main objectives of this thesis were (1) to characterize the development of muscle excitability by examining properties of voltage-gated sodium and potassium currents expressed in embryonic and larval zebrafish during the first week of development and (2) to elucidate some of the mechanisms by which ion current development might be controlled, beginning with activity-dependent and phosphorylation-dependent mechanisms. These objectives were approached using whole-cell electrophysiological techniques to examine the voltage-dependent and kinetic properties of voltage-gated sodium and potassium currents in intact zebrafish skeletal muscle preparations. Mutant sofa potato zebrafish, which lack functional nicotinic acetylcholine receptors, were then utilized to determine whether synaptic activity at the neuromuscular junction is required for proper ion current development. Finally, protein kinases were activated pharmacologically to determine whether they were able to modulate ion currents during development. The results revealed that properties of ion currents undergo a developmental progression, including increased current density, accelerated kinetics, and shifts in voltage dependence; these developments correlated well with the maturation of muscle action potentials and the movements and behaviors they mediate. Sofa potato mutants were found to be deficient in certain aspects of ion current development, but other aspects appeared to be unaffected by a lack of synaptic activity. Protein kinase A demonstrated the ability to drastically reduce potassium current density; however, the effects of protein kinase A were similar at all developmental stages. Overall, these findings provide novel insight into the roles played by voltage-gated currents during the development of excitability in zebrafish skeletal muscle, and expand the rapidly growing body of knowledge about ion channel function in general.
Development and Maturation of the Ocular Lens
The zebrafish (Danio rerio) provides a rare opportunity to observe and quantify in real time the microscopic development of the living eye, one of the most exquisite functional systems in biology. Development of the vertebrate ocular lens begins with an epithelial placode in the cephalic surface ectoderm that normally produces epidermal or neural cells that scatter light and are not refractile or transparent. Cells of the developing lens must undergo exquisite specializations to achieve the high index of refraction, glass-like transparency, and almost perfect symmetry required for a functional element in the optical pathway. The zebrafish has unique differences from the mammal in regard to early lens development and crystallin protein expression that may relate to the aquatic environment in which it lives. Rather than forming a hollow, fluid-filled lens vesicle that pinches off from the surface ectoderm, the developing zebrafish lens forms as a solid mass of cells that delaminate to separate the lens from the cornea. This dissertation will present the first systematic, high-resolution, quantitative, real-time, three-dimensional description of lens development in a living vertebrate from the placode stage at 16 h postfertilization through 4 days postfertilization. Live-cell imaging will establish a fate map for the lens placode and provide a direct and progressive view of the establishment of primary fibers, an anterior epithelium, and secondary fibers during development of the symmetric, transparent lens. Immunohistochemistry will demonstrate primary fiber cell differentiation, anterior epithelial cell differentiation, and progressive delamination of the lens mass from the surface ectoderm that does not involve apoptosis as a primary mechanism. Quasielastic laser light scattering and proteomics will demonstrate similarities in biophysical properties and protein expression between zebrafish lens and cornea. Size exclusion chromatography and shotgun proteomics will characterize protein expression in the zebrafish lens during maturation and aging to identify novel crystallins and demonstrate the importance of alpha crystallin in the aging lens. On the basis of these results, eye organogenesis and lens cell differentiation appear to be well conserved among vertebrates despite the lack of a lens vesicle stage in zebrafish.
Differential Functions of Follicle-Stimulating Hormone and Luteinizing Hormone in Zebrafish Ovary
The pituitary gonadotropins (GTHs), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) are the key hormones controlling vertebrate reproduction. Although the two GTHs have been characterized in numerous teleost species, our understanding of their biological functions remains rather limited. This is largely due to the lack of pure form of homologous GTHs and inadequate understanding of gonadal physiology in most species studied as well as species variation of hormone actions. This study aims at systematically investigating the functional roles of FSH and LH in the ovary using zebrafish as the model. Zebrafish is becoming more and more popular as a model of reproductive and developmental studies due to several advantages. First, although its body size is small, its ovary is relatively large and available all the year around. Second, zebrafish spawns everyday and its development is fast. Last but not least, its bioinformatics information is tremendous compared to other fish models. Previously, our laboratory had established two stable Chinese hamster ovary cell lines expressing recombinant zebrafish FSH (zfFSH) and zebrafish LH (zfLH). However, the production yields are very low. Therefore, this study tried to adopt the yeast Pichia pastoris as another bioreactor to produce recombinant zfFSH and zfLH. Two different forms of expression vectors for a native form and a fusion form carrying a His-tag, respectively, were constructed for each hormone. Their bioactivities were monitored and confirmed by receptor-based reporter gene assays as well as ovarian fragment incubation. As expected, the native form exhibited much higher activities than the fusion form. At the same time, functional studies were carried out to examine and compare bioactivities of the Chinese hamster ovary–derived zfFSH and zfLH in zebrafish ovary, which is the major part of the present project. The following aspects were covered to investigate the actions of zfFSH and zfLH: steroidogenesis and folliculogenesis. We investigated the effects of zfFSH and zfLH on steroidogenesis by examining the regulation of aromatase by these two hormones. Aromatase catalyzes the conversion of androgens into estrogens during steroidogenesis. Both recombinant zebrafish GTHs stimulated the aromatase expression during short-term treatment (8 h) in ovarian fragment culture, with zfFSH much more potent than zfLH. However, zfFSH continued to exhibit powerful effect on aromatase expression after 24 h treatment, whereas zfLH had little effect at all. The stimulatory effect of zfFSH on aromatase expression was time, dose, and stage dependent and was also confirmed by in vivo study. Further, it was zfFSH, not zfLH, that significantly stimulated StAR protein expression during short-term treatment. StAR protein is critical to steroidogenesis by facilitating the movement of cholesterol across the mitochondrial membrane. Both recombinant zebrafish GTHs stimulated activin bA expression but slightly suppressed activin bB expression. During short-term treatment, zfFSH and zfLH exhibited similar stimulatory effects on activin bA expression; the effect of zfLH became more prominent after 24 h treatment, whereas zfFSH had little effect. zfLH was found to be able to induce GVBD in zebrafish, as demonstrated in other fish species. However, our preliminary data showed that zfFSH was also involved in this process. To our knowledge, this is the first time to demonstrate that homologous FSH induces GVBD in teleosts. Although much more work needs to be done to elucidate the functional roles of FSH and LH in fish reproduction, the preset study provides a relatively comprehensive study for us to understand the potential roles of FSH and LH during ovarian development in fish, especially the importance of FSH.
Effects of the Aromatase Inhibitor Fadrozole on Gene Expression in the Zebrafish Brain
Aromatase is responsible for the conversion of androgens to estrogens in the brain and gonadal tissues. Teleosts have been characterized as having high levels of brain estrogen biosynthesis. Little is known about the effects of estrogens on brain function in teleosts. The main objective of this study was to assess the effects of fadrozole, a powerful aromatase inhibitor, on gene expression in the zebrafish brain. A fadrozole exposure leads to a decrease in estrogens and an increase in androgens. In the telencephalon, 235 genes were identified by Affymetrix GeneChip analysis as being differentially regulated. Real-time reverse transcription–polymerase chain reaction and in situ hybridization were used to validate the data obtained from the microarrays. Collectively, the results provide a better understanding of the effects of aromatase inhibitors on gene expression and also shed light on the underlying effects of sex hormone variation and their importance in the brain of teleosts.
Evaluation of Zebrafish Novel Immune-Type Receptor 9
Natural killer (NK) cells are lymphocytes of the innate immune system that express several cell surface receptors, including both activating and inhibitory forms. The natural killer cell receptors (NKRs) distinguish neoplastic or virally infected cells from normal host cells and regulate cytotoxic function. NKRs of different mammalian species differ dramatically and are the products of different multigene families. Because of the recent and rapid evolution of these receptors, it has been difficult to identify orthologs of NKRs in nonmammalian vertebrate species. In this regard, a multigene family of novel immune-type receptors (NITRs) has been identified in bony fish species that are structurally similar to mammalian immunoglobulin-type NKRs. In zebrafish, 14 NITR gene families have been identified, of which Nitr9 is the only activating receptor. In an effort to better understand the role of Nitr9 in zebrafish immunity, it was necessary to develop tools and methods to enable identification, purification, characterization, and function of Nitr9-expressing cells. In Chapter 2, the generation of two anti-Nitr9 monoclonal antibodies (mAbs) that were utilized to determine the Nitr9 protein expression profile in vivo is described. Immunofluorescence and flow cytometric analysis of cells transiently transfected with nitr9 demonstrated that the two mAbs can detect Nitr9-expressing cells. However, due to low levels of expression it was difficult to identify Nitr9-expressing cells in vivo and to subsequently purify the cells. Thus, the goal of the studies presented in Chapter 3 was to determine a method to boost Nitr9 expression in vivo and facilitate the identification and purification of Nitr9-expressing cells with the two mAbs. Preliminary results suggest that nitr9 gene expression is higher in the intestine of rag1-deficient zebrafish, which lack a functional adaptive immune response, compared to rag1-expressing zebrafish. This may provide a potential model to identify Nitr9-expressing cells.
Functional Identification and Initial Characterization of a Fish Coreceptor Involved in Aversive Signaling
Chemoreception plays an important role in predator–prey interactions and feeding dynamics. While the chemoreception of attractant or pleasant-tasting compounds has been well studied, aversive chemoreceptive signaling has been difficult to investigate behaviorally in an ecological context because these interactions are species and context specific, and deterrent compounds vary among prey. Therefore, little is known about the molecular mechanism(s) used in detection of aversive compounds. Using the coral reef system, this thesis explores on a molecular level the deterrent mechanism underlying detection by fish predators of an aversive compound, to gain a greater understanding of predator–prey interactions in this community. Like other organisms that are sessile or slow moving, marine sponges have special mechanisms for defense from predation, commonly containing aversive-tasting compounds that defend these organisms from predation. To this end, we sought to identify and characterize a fish chemoreceptor that detects one or more of these compounds. A behavioral assay demonstrated that many sponge compounds that are known to be deterrent to coral reef predator fish are also deterrent to zebrafish, a freshwater fish whose genome is well characterized. Two of these groups of deterrent triterpene glycosides, formoside and a mixture of ectyoplasides A and B, caused electrophysiological changes in Xenopus oocytes expressing an entire zebrafish cDNA library, β 2 AR, and the ion channel CFTR. Utilizing this electrophysiological bioassay, we fractionated the zebrafish cDNA library and isolated a single cDNA clone encoding RL-TGR, a novel coreceptor involved in the signaling of triterpene glycosides. This coreceptor appears to be structurally and functionally related to receptor-activity-modifying proteins, a family of coreceptors that physically associate with and modify the activity of G-protein-coupled receptors (GPCRs). Structurally, this protein is predicted to have a single-pass transmembrane domain, a short intracellular domain, and a long extracellular domain. Expression in Xenopus oocytes showed that it responds specifically to triterpene glycosides and no other compound tested in a receptor-mediated manner. Additionally, RL-TGR requires coexpression of a GPCR to enable signaling in oocytes, and both of these receptors may be components of a larger signaling complex, as suggested by immunoblotting evidence. Immunoblotting from expressing Xenopus oocyte membranes demonstrated that this protein is membrane associated. A 40 bp portion of the gene is conserved across multiple fish species, but is not found in any other organism with a published genome, suggesting that expression of this receptor is limited to fish species. Therefore, this fish gene may have coevolved with organisms that produce triterpene glycoside defensive compounds, which include sponges, echinoderms, and vascular plants. This work suggests that aversive compounds may be detected by RL-TGR and related proteins in fish. The use of a GPCR and receptor-activity-modifying protein–like coreceptor complex as a detector of deterrent compounds is a clever mechanism in which to perceive potentially harmful compounds. Instead of necessitating expression of a specific bona fide receptor (with the ability to both bind ligand and transduce signals) for each possible compound an organism might need to detect in its lifetime, an organism would only require expression of a limited number of GPCRs and a suite of coreceptors, which can combine in numerous combinations to specifically and efficiently detect a vast number of deterrent compounds, protecting these organism from potentially harmful compounds. This interdisciplinary work crosses the boundaries of behavioral neuroscience, chemical ecology, and molecular biology, and unites fields that rarely overlap. The discovery of RL-TGR is significant not only because it defines a new chemoreceptor–ligand pair in a field where few of these interactions are known, but also because the gene encoding RL-TGR is the first identified that encodes a coreceptor that responds to a chemical defense. This finding may lead the way for the identification of many other receptors that mediate chemical defense signaling in both marine and terrestrial environments, as this protein has the potential to represent the first of an entire family of coreceptors that respond to aversive compounds. The further study of RL-TGR and any related coreceptors will deepen our understanding of the molecular mechanisms of chemical defense compounds and their effects on predator–prey interactions.
Mechanism and Evolution of Mammalian Hedgehog Signaling
The Hedgehog (Hh) signaling pathway is an evolutionarily conserved cascade important for embryonic development and postnatal regeneration. Mutations in components of the Hh pathway in humans result in a number of congenital birth defects, such as holoprosencephaly, as well as numerous cancers. In the fruit fly Drosophila melanogaster, transduction of the Hh signal from the positive membrane effector Smoothened (Smo) to the Ci/Gli transcription factors is mediated by a large cytoplasmic complex. This complex is scaffolded by the kinesin Costal-2, and contains the key regulator Fused (Fu), a putative serine-threonine kinase, and Suppressor of Fused (Sufu), a regulator of Ci/Gli proteins. The role of Fu and Sufu in mammalian Hh signaling and development are less well characterized. In this dissertation, we show that mouse Fu is dispensable for mammalian Hh signaling, but instead participates in construction of the central pair apparatus of motile cilia. Strikingly, zebrafish Fu participates in both motile ciliogenesis and Hh signaling. We link Fu and a Costal-2 ortholog, Kif27 in mouse and Kif7 in zebrafish, to motile ciliogenesis, and advance hypotheses as to how these proteins evolved functions in diverse processes. We also show that mouse Sufu plays an evolutionarily conserved role in promoting the stabilization of Gli2 and Gli3. In part, Sufu opposes the activity of Spop, an adaptor protein for E3 ubiquitin ligases. The increased Hh pathway activity observed in Sufu mutant mice and embryonic fibroblasts can be attributed in part to elevated Gli1 activity. Finally, we investigate the trafficking of mouse Smo to the primary cilium, an organelle required for its activity in Hh transduction. We demonstrate that translocation of Smo to the cilium is necessary but not sufficient for pathway activation, as some classes of Smo antagonists can stimulate movement to the cilium. This result suggests a multistep model for Smo activation in mammalian Hh transduction. Taken together, the data presented in this dissertation shed light on the evolution of the Hh pathway from invertebrates to mammals, and provide a deeper mechanistic understanding of mammalian Hh transduction that will be useful in the future design of rational therapies.
Molecular Pathways in Zebrafish Vascular Development
Proper formation of a functional vascular system is absolutely required for embryonic development and is thought to occur in two distinct phases. In the first phase, blood vessel precursor cells (angioblasts) are generated and migrate to their final location in the embryo by responding to environmental cues. Once at their final location, perivascular support cells are recruited to the nascent vessel and the vessels are remodeled into a system of larger and smaller caliber. In this thesis, I have contributed to our understanding of both of these phases using the zebrafish as a model. Angioblasts that will form the intersegmental vessels (ISVs) in the trunk sprout dorsally from the dorsal aorta at 18 h of development, following the somite boundaries and generating the resulting characteristic chevron shape. In the out of bounds genetic mutant containing molecular lesions in plexin d1 (plxnd1) receptor, ISV angioblasts sprout precociously from the dorsal aorta, and do not respect the somite boundaries. Studies performed in mouse embryos have suggested that Semaphorin 3E (Sema3E) acts as the predominant ligand for PlxnD1 on ISV angioblasts. However, here I show that knockdown of sema3e in zebrafish embryos does not phenocopy the out of bounds mutant, but in fact results in delayed ISV angioblast sprouting and that Sema3e acts both autonomously and nonautonomously on angioblasts. Further, I demonstrate that Sema3e acts not through plxnd1, but likely through plxnb2, to control the timing of ISV angioblast sprouting. Thus, I performed an in situ screen of all previously uncharacterized semaphorins and plexins to determine those that might be involved in controlling ISV angioblast migration and identified sema6bb and sema6dc as potential candidates to control ISV migration. For the second phase of vascular development, I have demonstrated that hedgehog signaling controls embryonic vascular stability by investigating the igu fo10a hedgehog mutant, containing molecular lesions in Daz-interacting protein (Dzip1) and characterized by progressive hemorrhage and lack of perivascular support cell recruitment. Further, I have demonstrated that hemorrhage in the igu fo10a mutant results from a loss of angpt1 expression in cranial mesenchyme and that modulation of angpt1, or its antagonist angpt2, rescues hemorrhage.
Neuroprotection Mechanisms in an 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Model of Neuronal Degeneration in the Zebrafish
Parkinson's disease is one of the most common neurodegenerative diseases afflicting the elderly. I sought to exploit the zebrafish as a research tool to understand the molecular mechanisms underlying this specific neuronal degeneration. As such, I utilized 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine to induce a severe parkinsonian phenotype in embryonic zebrafish as detected by tyrosine hydroxylase RNA and protein expression. I then applied chemical or genetic perturbations to determine whether I could prevent this neuronal loss from occurring. I observed that inhibition of calpain, a calcium-sensitive protease, and increased antioxidant activity, induced by exposure to superoxide dismutase and catalase enzymes, was sufficient to protect against neuronal loss. Finally, I used acridine orange to assay whether 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was indeed inducing cell death. I observed a statistically significant increase in dying cells in the retina, likely dopaminergic interplexiform cells of the inner nuclear layer; this loss was attenuated by the above neuroprotective strategies.
Regulation of Retinoic Acid in Early Zebrafish Development
Retinoic acid (RA) is an important developmental signaling molecule responsible for the patterning of multiple vertebrate tissues. RA is also a potent teratogen, causing multiorgan birth defects in humans. Endogenous RA levels must therefore be tightly controlled in the developing embryo. On the basis of a microarray approach to identify genes that function as negative feedback regulators of RA signaling, I screened for genes expressed in early somite-stage embryos that respond oppositely to treatment with RA versus RA antagonists, and validated them by RNA in situ hybridization. To study the in vivo RA-related phenotype at early developmental stages, I designed and tested assays on two zebrafish mutants, cyp26a1 mutant giraffe and aldh1a2 mutant neckless, both having aberrant endogenous RA level. Using these assays and the specific marker for RA-dependent developmental processes, I characterize the cyp26a1 mutant in detail to study the function of Cyp26a1 on zebrafish early development. It turned out that cyp26a1 mutant has only limited developmental defects in vivo, suggesting that other regulating mechanisms must play important roles. Focusing on genes known to be involved in RA metabolism, I determined that a dehydrogenase and reducatse short-chain protein family member, dhrs3a, is both RA dependent and strongly RA inducible. Dhrs3a is known to catalyze the dehydrogenation of the RA precursor retinaldehyde to vitamin A; however, its developmental function has not been demonstrated. Using morpholino knockdown and mRNA overexpression, I demonstrated that Dhrs3a functions as an RA feedback inhibitor with primary effects on RA-dependent events in the central nervous system. On the basis of the finding of dhrs3a function and our validated microarray results, as well as results from other labs, I proposed a feedback regulation model on how the accuracy of RA function is achieved. In this model, the RA was regulated by both the positive and negative feedback circuits through multiple RA responsive genes, and forming a regulatory network from multiple levels.
Specificity of Smad Signaling in Hematopoiesis
The BMP signaling pathway has been shown to be important for dorso-ventral patterning and hematopoiesis in the embryo, and proposed to be important for the regulation of adult hematopoiesis. Defects in BMP signaling components are embryonic lethal, complicating the analysis of BMP functions in the adult, and in determining the individual roles of these molecules. Activation of the BMP receptors by ligand binding activates Smads 1, 5, and 8. In this study the effect of knockdown of BMP signaling was tested by manipulating Smad1 and Smad5 in embryos and adults. Loss-of-function approaches were used in the zebrafish model system, in which embryos are not dependent on extraembryonic tissue, to distinguish the functions of Smad1 and Smad5 in embryogenesis. I used Smad1 and Smad5 morpholinos, and the Smad5 mutant, piggytail dty40, to study the effect of gene knockdown. I show that Smad1 knockdown causes a unique phenotype. Smad1 morphants have defects in brain, heart, gut, and hematopoietic system, including an increase in erythrocytes and lack of mature macrophages. This is unlike the Smad5 mutants that have a dorsalized phenotype, and are anemic with macrophage normal numbers. Loss of either Smad, in the embryo, causes a lack of definitive hematopoiesis. I show that Smad1 is able to substitute for Smad5 during embryogenesis, whereas the converse is not true. In the adult, I show that BMP signaling continues to be important for hematopoiesis. Using heterozygote Smad5 mutants, I show that adult fish with the stronger mutant Smad5 allele, somitabun dtc24, are anemic. Smad5 mutant fish also respond to hemolytic anemia with altered kinetics compared to wild type. This indicates that Smad function is relevant to adult hematopoiesis, and identifies specific functions in steady state and stress response erythropoiesis. I have shown that Smad1 and Smad5 have different functions during embryogenesis, in particular, during hematopoiesis. BMP signaling is known to function at multiple places and times during development. How this pathway is able to control different biological functions is only partially understood. This study has shown one way in which this diversity is initiated at the signaling level through differential activation of Smad1 and Smad5.
Structural and Biochemical Studies of TIGAR and ZnuA
Activation of the p53 tumor suppressor by cellular stress leads to variable responses ranging from growth inhibition to apoptosis. TIGAR is a novel p53-inducible gene that inhibits glycolysis by reducing cellular levels of fructose-2,6-bisphosphate, an activator of glycolysis and inhibitor of gluconeogenesis. Here we describe structural and biochemical studies of TIGAR from Danio rerio. The overall structure forms a histidine phosphatase fold with a phosphate molecule coordinated to the catalytic histidine residue and a second phosphate molecule in a position not observed in other phosphatases. The recombinant human and zebrafish enzymes hydrolyze fructose-2,6-bisphosphate as well as fructose-1,6-bisphosphate, but not fructose 6-phosphate in vitro. The TIGAR active site is open and positively charged, consistent with its enzymatic function as bisphosphatase. The closest related structures are the bacterial broad specificity phosphatase PhoE and the fructose-2,6-bisphosphatase domain of the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The structural comparison shows that TIGAR combines an accessible active site as observed in PhoE with a charged substrate-binding pocket as seen in the fructose-2,6-bisphosphatase domain of the bifunctional enzyme.
Yolk Extension Ontogenesis: A Novel Evolutionary and Developmental Module of the Teleostean Phylotypic Period
Embryonic developmental modules have been theorized to provide a mechanistic basis for speciation. This thesis describes how the yolk extension in cypriniform fishes represents a novel evolutionary and developmental module of the teleostean phylotypic period. The yolk extension is an evolutionary module: it is a heritable cladistic trait characteristic of the order Cypriniformes, and is present in sister order Characiformes, and a more basal order Anguilliformes. Moreover, the yolk extension satisfies six criteria proposed by Rudy Raff to define developmental modularity. These are that it (1) has a physical location, (2) undergoes developmental transformation, (3) undergoes evolutionary transformation enabling comparison to a proposed homology, (4) forms autonomously using specific genes, (5) shows connectivity to other modules, and (6) is comprised of a hierarchy of components that may be part of a larger hierarchy. The yolk extension is a cylindrical tube extending posteriorly from a spherical or elliptical yolk ball. It undergoes heterochronic changes in development, shown by comparing its ontogeny in zebrafish, Danio rerio (Cypriniformes), with other cypriniform species. A comparative literature analysis of 461 species, using illustrations depicting the functionally defined vertebrate phylotypic period, spanning the tailbud to the pharyngula stages, demonstrates that the module has undergone an evolutionary transformation. Its formation is homologous to the process of tubulation, the process of enclosing the body axis in nested germ layers as the embryo elongates from a sphere to a rod. Examination of zebrafish mutant literature showed that yolk extension development has a genetic basis. Experimental manipulation of zebrafish embryos showed that it forms autonomously from trunk straightening, while it is mechanically connected to dorsal tissues. The module is part of the hierarchy of tubulation. The yolk extension ontogenesis module is a nested hierarchy of three morphogenetic compartments: the innermost yolk cell, the mesendodermal mantle, and the outermost embryonic integument. Experimental evidence suggests that the contractile embryonic integument reshapes the cohesive, viscoelastic yolk mass, forming the yolk extension. Overall, the yolk extension, as an innovative evolutionary and developmental module of the teleostean phylotypic period, will be useful as a model system in which to frame a plethora of experimental inquiries.
Zebrafish Embryos Exposed to Alcohol Undergo Abnormal Development of Motor Neurons and Muscle Fibers
Children with fetal alcohol spectrum disorder have significantly delayed motor skills, and deficiencies in reflex development. The reasons underlying these motor deficits are not fully understood. The aim of this thesis was to investigate the effect of embryonic exposure to ethanol (EtOH) on motor neuron and muscle fiber morphology and physiology in zebrafish. We observed that EtOH-exposed fish took longer to hatch and exhibited fewer swimming bouts in response to touch. Immunolabeling of motor neurons indicated that EtOH-exposed fish had significantly higher rates of motor neuron axon defects. Examination of muscle fiber morphology revealed that EtOH exposure resulted in significantly smaller muscle fibers. Miniature endplate current recordings from muscle fibers revealed that event amplitudes, rise times, half widths, frequencies, and decay times were affected by EtOH exposure. These findings indicate that motor neurons and muscle fibers of zebrafish are affected by embryonic EtOH exposure, which may be related to deficits in locomotion.
Effect of Mitochondrial Iron on Manganese Superoxide Dismutase
Manganese (Mn)-containing superoxide dismutase enzymes (Sod2p) play a critical role in guarding aerobic cells against oxidative stress. A key step in the activation of Sod2p is the insertion of the Mn cofactor into the active site of the enzyme. Mn is preferentially bound to Sod2p under normal conditions even though the concentration of iron (Fe) in mitochondria is several orders of magnitude higher than Mn. Previous studies in Saccharomyces cerevisiae revealed cases in which Fe misincorporates in yeast Sod2p rather than Mn, leading to the inactivation of this enzyme. It was proposed that mitochondria accumulate at least two pools of Fe: a SOD2-inert pool, which predominates under normal conditions, and a SOD2-reactive pool, which accumulates under certain cases of mitochondrial Fe overload such as in yeast mutants defective in Fe–sulfur (Fe-S) cluster formation (grx5 and ssq1) or in cells lacking the mitochondrial transporter Mtm1p (mtm1), whose function is not known. In Chapter 2 of this thesis, we begin to probe the pathway and mechanism for Fe inactivation of SOD2. We demonstrate that the mitochondrial carrier family members Mrs3p/Mrs4p, previously shown to transport Fe needed for heme and Fe-S cluster biogenesis, are also involved in delivering SOD2-reactive Fe to the mitochondria. We also demonstrate that the Fe in the SOD2-reactive form is indeed very reactive and appears to bind to the active site of yeast Sod2p more rapidly or with higher affinity than Mn. Finally, in this chapter, we studied the effects of Fe overload in mammalian cells on SOD2 activity and find that, similar to yeast, not all forms of mitochondrial Fe that accumulate due to disruptions in Fe homeostasis are reactive with SOD2. In Chapter 3, more detailed analyses of the origins and specificity of SOD2-reactive Fe were carried out. We found that specific disruptions in Fe homeostasis will similarly trigger misincorporation of Fe into bacterial MnSOD expressed in yeast mitochondria. The switch in cofactor selection was only observed with MnSOD and not with a closely related bacterial FeSOD. By XANES and EXAFS analyses, we find that misincorporation of Fe into yeast Sod2p does not correlate with significant changes in the average oxidation state or coordination chemistry of bulk mitochondrial Fe. Instead, small changes in mitochondrial Fe are likely to promote Fe–SOD2 interactions. We also reveal that Isu proteins, scaffolds for Fe-S clusters assembly, are elevated in all the yeast mutants that accumulate SOD2-reactive Fe. We further demonstrate that these Isu proteins are in fact one important source of mitochondrial Fe that is reactive toward Sod2p. Although disruptions in yeast Mtm1p lead to accumulation of SOD2-reactive Fe, the precise function of Mtm1p or its orthologs in higher eukaryotes has remained elusive. In Chapter 4, we identify SLC25A39 as function orthologs of yeast Mtm1p in vertebrates, including zebrafish, mouse, and human. The genes of vertebrates can complement the various phenotypes of yeast mtm1 D mutants. Moreover, our collaborative studies have suggested a role for vertebrate SLC25A39 in Fe homeostasis possibly linked to heme or Fe-S biogenesis. Overall, these studies provide new insight into the underlying mechanism of Fe misincorporation to SOD2 within mitochondria as well as reveal the functional orthologs of yeast Mtm1p in vertebrates, including human.
Ontogeny of the Cypriniform Pharyngeal Jaw Apparatus and Its Associated Musculature
The pharyngeal jaw apparatus is an assemblage of bones and muscles found within the head of fishes. While the oral jaws are used for prey capture in fishes, the pharyngeal jaws are used for prey processing. There is a basic form of the pharyngeal jaw apparatus found among all fishes; however, some fish groups possess features of the pharyngeal jaw apparatus that are specialized for the demands of their diet. One such group are the Cypriniformes. Cypriniformes is one of the world's largest clades of freshwater fishes. They are widely distributed in North America, Europe, Africa, and Asia, but are absent from Australia and South America. The clade is united by several characters, including the presence of a kinethmoid, a lack of teeth on the oral jaws, and an enlarged fifth ceratobranchial that opposes a pad on the basioccipital of the skull. Using developmental and histological methods, I investigated the development of the cypriniform pharyngeal jaw apparatus, a morphological novelty for this group of fishes, using the zebrafish model system. Results indicate that the zebrafish's enlarged fifth ceratobranchial is the largest ceratobranchial very early on in development. Additionally, the levator posterior, which mediates movement of this bone, is also larger than the other levators through ontogeny, beginning very early on in development. Lastly, a unique pattern of bone and muscle development was observed, where the levators externi developed before the appearance of the bony elements on which they attached. In contrast, the levators interni developed at approximately the same time as the bony elements on which they attach. This study is one of the first to provide insight into how the pharyngeal jaw apparatus as a whole develops.
Analysis of the Molecular Mechanism of Autophagosome Formation in Yeast and Zebrafish Models
Autophagy is a conserved intracellular degradative pathway induced by various stress or developmental signals in eukaryotes, and its malfunction contributes to a variety of diseases. During autophagy, cargos such as cytosolic proteins, damaged organelles, and invasive pathogens are engulfed into double-membrane autophagosomes, and transported to and degraded in the lysosome/vacuole. Over 30 ATG (autophagy-related) genes have been identified in the budding yeast Saccharomyces cerevisiae. To understand the molecular mechanism controlling membrane delivery during autophagy, I studied protein interactions involving Atg9, the only known transmembrane protein required for autophagosome formation. In yeast, Atg9 cycles between peripheral sites and the phagophore assembly site (PAS), suggesting its role in supplying membrane for autophagosome nucleation and expansion. Through a yeast two-hybrid screen aimed to find interaction partners of Atg9, I identified Atg11, a component involved in autophagic cargo recognition. Subsequently, I demonstrated that Atg11 mediates Atg9 movement to the PAS along the actin cytoskeleton. Thus, my model suggests that the anterograde transport of Atg9 to the PAS mediated by Atg11 may serve as a membrane shuttle for autophagosome biogenesis. Further, I characterized the self-interaction of Atg9 and generated an Atg9 mutant defective in this interaction. This mutation results in abnormal autophagy, due to altered phagophore formation as well as inefficient membrane delivery to the PAS. On the basis of my analyses, I propose a model suggesting dual functions for the Atg9 complex: by reversibly binding to another Atg9 molecule, Atg9 can both promote lipid transport from the membrane origins, and help assemble an intact phagophore membrane. I also extended my analysis on autophagy in the zebrafish model, which represents a unique system to study autophagy due to its rapid embryonic development and technical advantages in high-throughput drug screens. I identified two zebrafish Atg8 (an autophagosome marker protein) homologs, lc3 and gabarap, and generated two transgenic zebrafish lines expressing GFP-tagged versions of the corresponding proteins. I observed a high level of autophagy activity in zebrafish embryos, which can be further upregulated by the TOR inhibitor rapamycin or the calpain inhibitor calpeptin. Thus, I established a convenient zebrafish tool to assay autophagic activity during embryogenesis in vivo.
Automated Methods to Infer Ancient Homology and Synteny
Establishing homologous (evolutionary) relationships among a set of genes allows us to hypothesize about their histories: how are they related, how have they changed over time, and are those changes the source of novel features? Likewise, aggregating related genes into larger, structurally conserved regions of the genome allows us to infer the evolutionary history of the genome itself: how have the chromosomes changed in number, gene content, and gene order over time? Establishing homology between genes is important for the construction of human disease models in other organisms, such as the zebrafish, by identifying and manipulating the zebrafish copies of genes involved in the human disease. To make such inferences, researchers compare the genomes of extant species. However, the dynamic nature of genomes, in gene content and chromosomal architecture, presents a major technical challenge to correctly identify homologous genes. This thesis presents a system to infer ancient homology between genes that takes into account a major but previously overlooked source of architectural change in genomes: whole-genome duplication. Additionally, the system integrates genomic conservation of synteny (gene order on chromosomes), providing a new source of evidence in homology assignment that complements existing methods. This work applied these algorithms to several genomes to infer the evolutionary history of genes, gene families, and chromosomes in several case studies and to study several unique architectural features of postduplication genomes, such as Ohnologs gone missing.
Cranial Sensory Ganglia Formation: The Role of N-Cadherin
The cranial sensory ganglia (CSG) conduct sensory information from the periphery to the central nervous system. The proper development of these ganglia is critical to an animal's ability to regulate its internal environment and interact with the external world. It is the goal of this dissertation to provide data that will aid in the understanding of the development of these ganglia and their respective circuits. Here, the zebrafish is exploited as a model animal because it is optically translucent, develops rapidly, and is amenable to genetic manipulation, all of which allow observation of developmental processes in an intact, living animal. The transgenic fish, tg (p2xr3.2:eGFP), allows imaging of the CSG neurons throughout development via detection of fluorescent protein expressed specifically within these neurons. Using this and other transgenic fish, aim one characterizes the developmental timing and innervation patterns of the CSG and their respective nerves in the zebrafish. On the basis of this characterization, aims two and three then examine the role of the cell adhesion protein, N-cadherin (cdh2), during the development of these circuits. cdh2 is expressed within these CSG neurons during circuit formation; however, the role of cadherins within these neurons at this time is not understood. By crossing the cdh2 mutant, glass onion (glo) with transgenic fish, aim two describes the effects of ubiquitous cdh2 disruption on the development of the CSG and their respective nerves. In addition to the clustering and axon fasciculation defects previously reported, severe path-finding errors in the facial, glossopharyngeal, and vagus afferents are described. Although these path-finding errors were found to be specific to the afferent components of the cranial nerves, embryos lacking cdh2 function have gross morphological defects that could cause the axons disruptions. To clarify that cdh2 is necessary specifically within the CSG neurons for proper circuit formation, aim three reports on studies in which dominant negative cdh2 constructs are selectively targeted to these neurons. When cdh2 function is disrupted exclusively within these ganglionic neurons, the effects on the afferents recapitulate those described with ubiquitous cdh2 knockout. This indicates that cdh2 functions within CSG neurons to affect critical aspects of afferent wiring.
Deciphering the Secret of Sarcomere Assembly and Diseases Using the Zebrafish Model System: Regulation of Myofibrillogenesis by smyd1b and Its Partners
Myofibrillogenesis is a process of precise assembly of sarcomeric proteins into the highly organized sarcomeres, which are essential for muscle cell differentiation and function. Myofibrillogenesis requires proper folding and assembly of newly synthesized sarcomeric proteins. Mutations of the sarcomeric proteins are known to cause skeletal and cardiac muscle diseases. smyd1b is a skeletal and cardiac-muscle-specific gene that encodes two alternatively spliced isoforms, smyd1b_tv1 and smyd1b_tv2. Knockdown of smyd1b (tv1 and tv2) expression resulted in zebrafish larvae without locomotion and heart contraction. Thick filament assembly was significantly disrupted in smyd1b knockdown embryos. Yeast Two-Hybrid study showed that Smyd1 associates with another muscle-specific protein—skNAC; however, skNAC function in muscle cells is unknown. To expand the understanding of smyd1b function and study the working mechanism, I further characterized the function of Smyd1b and its partners, including skNAC and Hsp90a1, during muscle development, and carried out mechanistic studies using zebrafish as a model system. Our findings show that (1), in addition to the thick filament, smyd1b plays an important role in the assembly of thin and titin filaments, as well as Z-line and M-line. (2) Knockdown of smyd1b has no effect for heart tube formation; however, it disrupts the myofibril assembly of the cardiac muscle that causes the heart defect. (3) Smyd1b_tv1, but not Smyd1b_tv2, can be localized on the M-line of sarcomeres. (4) Ser225 on Smyd1b_tv1, which is a potential phosphorylation site, is important for the M-line localization of Smyd1b_tv1. (5) Knockdown of smyd1b causes upregulation of hsp90a1 and unc45b gene expression. (6) hsp90a1 plays an important role for myofibril assembly. (7) Knockdown of smyd1b or hsp90a1 causes the reduction of myosin protein accumulation. (8) Smyd1b_tv1, but not Smyd1b_tv2, associates with the myosin chaperones Hsp90a1 and Unc45b. (9) sknac is required for the thick and thin filament assembly. (10) Knockdown of sknac causes the reduction of myosin protein accumulation. These studies provide us an in-depth characterization of smyd1b and its partners' function and expands the mechanistic understanding of how smyd1b fulfils its vital role in myofibrillogenesis. Most importantly, this study provides new insights to help us understand the complex process of myofibrillogenesis and sarcomere diseases.
Derivation and Characterization of Zebrafish Embryonic Germ Cell Cultures and the Effects of Kit Ligand a
A stem-cell-mediated gene targeting approach is currently not available for zebrafish. To address this problem our lab has developed methods for the culture of pluripotent stem cells derived from early stage zebrafish embryos. Embryonic stem (ES) cell cultures were derived from blastula-stage zebrafish embryos, and primary cultures of pluripotent embryonic germ (EG) cells were initiated from primordial germ cells (PGCs). To be useful for gene-targeting studies, optimization of the culture conditions is required to maintain the stem cells in a pluripotent condition for multiple passages. Also, methods must be established to introduce targeting plasmids into the cells by homologous recombination, and selection strategies are needed to isolate colonies of cells that have undergone the targeting event. In this study, I have developed methods to initiate multiple-passage EG cell cultures derived from zebrafish PGCs. A transgenic approach involving drug selection was used to isolate homogeneous populations of PGCs for cell culture. Optimization of the PGC culture conditions using recombinant zebrafish growth factors demonstrated that Kit ligand a (Kitlga) and Kit ligand b and stromal-cell-derived factor 1a and 1b promoted the in vitro growth of the PGCs. Although PGCs do not express kit receptor in vivo, in culture the cells expressed the receptor and responded to recombinant zebrafish Kitlga added to the medium. After several days in the presence of these factors, the PGC cultures began to exhibit in vitro characteristics of pluripotent EG cells. Kitlga was especially important for promoting expression of pluripotency markers, including nanos, alkaline phosphatase, and SSEA-1. The EG cells also began to express endogenous kitlga and inhibition of its expression or function with antisensense morpholinos or MAP kinase inhibitors demonstrated that the factor was important for maintaining pluripotency. In a separate project, gene targeting methods were established for zebrafish ES cell cultures. The results of these studies demonstrated that the ES cells are able to incorporate plasmid in a targeted fashion by homologous recombination. Two selection strategies were used to isolate ES cell colonies that contained targeted plasmid insertions in either the no tail or myostatin I gene.
Function and Biosynthetic Regulation of Retinoic Acid in Intestinal Development and Homeostasis
It has been hypothesized for almost 100 years that retinoids could play a role in regulating the differentiation and proliferation of epithelial tissues, including the intestine. Some of those original questions still remain as studies to date have been unable to clearly identify the role retinoic acid signaling plays in the intestine. Recent work has also raised questions about how the important process of retinoic acid biosynthesis is regulated. This dissertation presents data that clarify some of mechanisms that regulate retinoic acid biosynthesis in the intestine as well as identify important functions of retinoic acid signaling in intestinal development and homeostasis. The data presented herein identify a requirement for axin1 in intestinal differentiation during zebrafish development. In this novel role axin1 acts in a Wnt-independent manner to positively regulate expression of rdh1l, a retinol dehydrogenase. Further, we present data that suggest that axin1 is able to promote expression of rdh1l by regulating the stability of the transcriptional corepressor Ctbp1. In this dissertation we utilize the apc mcr mutant as a model of retinoic acid deficiency to show that, contrary to the currently accepted model, upregulation of aldh1a2 (raldh2) can be an indication of retinoic acid deficiency rather than an excess. We also provide direct evidence that retinol dehydrogenases are required in the intestine for retinoic acid production and signaling. Further, the data presented here provide genetic evidence that retinoic acid regulates expression of aldh1a2 (raldh2) through an indirect negative regulatory loop, and implicates the known stem cell marker pou5f1 (oct4) as an important positive regulator of aldh1a2 (raldh2) expression. In addition, the data presented in this dissertation clearly identify the essential role of retinoic acid signaling in establishing intestinal progenitor cell number during development. These findings also clearly identify an important role for retinoic acid in maintaining intestinal homeostasis by regulating epithelial differentiation and proliferation in the adult intestine. Together, the data presented in this dissertation establish retinoic acid as an essential regulator of intestinal development and homeostasis, and identify two pathways that are important for regulating the biosynthesis of retinoic acid in the intestine.
Genetic and In Vivo Comparative Analyses of Gli Function in Vertebrates
The Hedgehog (Hh) signaling pathway is a cardinal regulator of a diverse set of developmental processes in amniotes. In mammals, three members of the Gli family of transcription factors serve as the intracellular mediators of Hh signaling. Gli2 and Gli3 have the potential to function as either activators (Gli2/3A) or repressors (Gli2/3R) of Hh target gene expression, whereas Gli1 functions only as an activator. Interestingly, genetic analyses of Gli function in the mouse have revealed that the specific combination of Gli genes, and GliA and GliR functions, required for normal development differs for each tissue type. In addition, analyses of gli function in zebrafish have provided evidence that the developmental requirements for different members of the Gli family have been differentially distributed in zebrafish and mouse. As a means of better understanding the mechanisms underlying tissue- and species-specific requirements for Gli function in vertebrates, a combination of genetic approaches was utilized, including targeted gene replacement and temporal conditional gene removal in mice. By conditionally removing Gli3 from the limb mesenchyme in different loss-of-function and gain-of-function Gli mutant backgrounds, we provide evidence for a model of anterior–posterior limb patterning that integrates the duration and dose of total Gli2 and Gli3 exposure with specific spatial requirements for GliA and GliR functions. By expressing zebrafish gli1 and gli2a from the endogenous mouse Gli2 locus, we provide evidence for differences in zebrafish and mouse Gli1 and Gli2 protein functions that could account for the majority of the differences in Gli gene requirements observed in these two vertebrate species. Our preliminary data are consistent with a model in which the distribution of requirements for Gli1 or Gli2 orthologs in different tissues in zebrafish and mouse reflects the requirement for differing strengths of GliA function, such that mouse Gli2A and zebrafish Gli1A are the stronger GliAs.
Genetic and Molecular Studies of Mammalian Heart Development
The genetic and molecular basis of mammalian cardiac development was studied in two model systems: German shepherd dogs (GSDs) afflicted with inherited juvenile ventricular arrhythmias and the dark-like (dal) mutant mouse, which I identify as a new model for studying myocardial development. GSDs with inherited juvenile ventricular arrhythmias and sudden cardiac death are an established model for studying cardiac ventricular arrhythmias. The inheritance of this disease in GSDs is consistent with a major gene that allows a dog to be affected and modifiers that influence severity. Genome-wide linkage analysis was performed to identify the loci responsible, but no strong evidence of linkage was detected. As altered calcium homeostasis has been observed in the hearts of these dogs, I examined cardiac expression levels of genes implicated in calcium handling. A significant inverse correlation was observed between the levels of ATP2A2 RNA and its protein product, SERCA2, and severity of ventricular arrhythmias, but no mutations were found in the ATP2A2 coding sequence. SERCA2 is a calcium pump located on the sarcoplasmic reticulum that sequesters cytosolic calcium back to the sarcoplasmic reticulum. My work indicates that SERCA2 is a molecular determinant of severity of ventricular arrhythmias in GSDs and suggests that at least one of the causative genes acts upstream of ATP2A2 transcription. Mice homozygous for the dal mutation are small and have darkly pigmented fur. A spectrum of previously unrecognized developmental cardiac abnormalities was observed in dal mutant embryos, including a thickening of ventricular walls, atrial and ventricular septal defects, and enlarged endocardial cushions. No significant differences were detected in the proliferation status of mutant and control embryonic ventricles, but dal mutant embryonic cardiomyocytes were significantly larger than those of controls. This indicated that the thickened ventricles of dal mutant embryos result from developmental cardiac hypertrophy. Functional consequences associated with hypertrophy included early afterdepolarizations and significant irregularities in the Ca2+ transient interval. Positional cloning identified a 4 base pair deletion in the peptidase D (Pepd) gene, and morpholino knockdown of Pepd expression in zebrafish confirmed that Pepd loss of function disrupts normal myocardial development, pigmentation, and body size. Pepd encodes prolidase, an enzyme involved in collagen metabolism. I hypothesized that loss of prolidase function in dal mutant embryos causes cardiac hypertrophy by disrupting the extracellular matrix and integrin signaling. Accordingly, I demonstrated reduced expression of integrin transducers and disrupted cytoskeletal organization in dal mutant embryonic hearts. My work provides a novel understanding of the genetic and molecular basis of developmental cardiac hypertrophy and has implications for the mechanisms that control the normal postnatal transition from proliferative to hypertrophic cardiomyocyte growth. These studies provide important insights into the genetic and molecular mechanisms underlying developmental cardiac disorders and will provide useful insights into understanding the pathogenesis of similar human heart diseases.
Mechanism Underlying the Maturation of AMPA Receptors in Zebrafish
Glutamate AMPA receptors (AMPARs) are major excitatory receptors in the vertebrate central nervous system. In many biological systems there are changes in the properties of AMPARs during development that are essential for providing an increase in efficiency of information transfer between neurons and a refinement of motor coordination and sensory perception and cognition. It is not surprising that improper development or loss of function of AMPARs can lead to many neurological disorders such as epilepsy and amyotrophic lateral sclerosis. Thus, determining the mechanisms by which AMPARs mature is of particular importance. The objectives of my thesis were to characterize the developmental changes in AMPAR-mediated currents in zebrafish Mauthner cells and to determine the mechanisms underlying any changes. The major findings reported in this thesis are that (1) there are developmental changes in the properties of AMPAR currents as the Mauthner cell matures; (2) the mechanism underlying these changes is a switch in the composition of AMPAR subtypes; and (3) PKCg is necessary for the developmental switch in AMPAR subtypes from slow receptors to fast receptors. These findings provide valuable insights into the mechanism underlying the development of AMPARs. In addition, they provide the first instance of a signaling link (PKCg) required for the developmental subunit switch and the developmental speeding of AMPAR kinetics.
Microtechnology for Spatial and Temporal Control of Gene Expression in Developing Embryos and Cells
Much of the biology in multicellular development depends upon the exchange of messages between cells: by exchanging chemical or mechanical information, cells influence the state of other cells near them and act in a coordinated fashion. Many tissue patterning events such as migration, proliferation, development, and differentiation that occur in vivo occur in relation to changes in local microenvironment of a cell. The ability to modify two-dimensional specially shaped areas of the local microenvironment with controlled delivery of genes and proteins in patterned shapes allows more biologically relevant manipulations of a complete system's development. Such control or hacking tools are limited at this time, and our technology provides a biological tool to study more of such developmental mechanisms. The work presented in this thesis focuses on the design, fabrication, and application of microfabricated interfaces for the patterned delivery of foreign molecules and gene constructs into developing embryos and cells. Low-voltage microfluidic valves and pumps were designed to deliver multiple compounds in pixel style resolution into growing embryos. Other systems were used to draw two-dimensional patterns of tracer molecules, DNA, and mRNA into the yolk and cells of zebrafish embryos (Danio rerio) at different stages of development. The successful delivery of two-dimensional patterns of trypan blue (normal dye), texas red (fluorescent dye), pCS2eGFP DNA, and GFP-mRNA in both chorionated and dechorionated embryos was also demonstrated. Both DNA and mRNA were expressed in the desired patterns subsequent to delivery. Briefly, 10-mm-wide platinum electrodes were microfabricated into desired patterns and passivated with silicone elastomer. Square pulses of 10–20 V (0.20–0.40 kV/cm), 50–100 ms pulse width were sufficient to create transient pores and introduce compounds from the late blastula period (3 hours postfertilization [hpf]) to early pharyngula period (24 hpf) embryos. Using 24 hpf dechorionated embryos, we achieved a high survival rate of 91.3% and 89%, and a delivery rate of 38% and 50% for GFP-DNA and GFP-mRNA, respectively. We believe that these simple techniques offer the unique advantages of introducing foreign compounds at local sites and in specific patterns unlike any other microsystem techniques and provide new tools to aid advanced studies in cellular development and morphogenesis.
The Bone Marrow Vascular-Stem Cell Niche: Identification, Characterization, and Function
The bone marrow vascular niche (BMVN), comprised of sinusoidal endothelial cells (SECs), is a structurally dynamic vascular network that provides a platform for survival and differentiation of hematopoietic stem and progenitor cells (HSPCs). In these studies, the angiogenic signature and molecular determinants for regeneration of SECs after myelosuppression are identified. During regional regeneration of the BM after myelosuppression, VEGFR2 activation promotes reassembly of regressed SECs, reconstituting HSPCs. VEGFR2 and VEGFR3 expression is restricted to BM vasculature, demarcating a continuous network of VEGFR2 + VEGFR3 + Sca1-SECs and VEGFR3-Sca1 + arterioles. We found that chemotherapy (5 FU) and sublethal irradiation (650 rad) induce minor SEC regression, while lethal irradiation (950 rad) results in severe regression of SECs, requiring BM transplantation for their regeneration. Conditional deletion of VEGFR2 in adult mice blocks regeneration of SECs in sublethally irradiated animals, preventing hematopoietic reconstitution. Inhibition of VEGFR2 signaling in lethally irradiated wild-type mice rescued with BM transplantation severely impairs SEC reconstruction, preventing engraftment and reconstitution of HSPCs. Therefore, regeneration of VEGFR2 + VEGFR3 + Sca1-SECs via VEGFR2 signaling is essential for engraftment HSPCs and restoration of hematopoiesis. In addition, we identify for the first time expression of a novel extracellular matrix localized angiogenic factor, angiomodulin, in the BMVN and show that angiomodulin is upregulated during hemangiogenic recovery after myelosuppression. We sought to study angiomodulin further and found that angiomodulin expression is increased in the vasculature of human tumors as compared to normal tissue. We developed an angiomodulin knockin reporter mouse wherein we demonstrate that angiomodulin is expressed in the vasculature of developing embryos and adult organs and during physiological and pathological angiogenesis. In addition, angiomodulin plays a significant role in the development of embryonic zebrafish vasculature via a genetic interaction with the VEGF-A pathway. These results demonstrate that the vascular-specific marker angiomodulin modulates vascular remodeling by temporizing the proangiogenic effects of VEGF-A during embryogenesis. Collectively, these data suggest that the BMVN is comprised of geographically heterogeneous, hemangiogenic compartments and that induction of neoangiogenesis, perhaps via novel targeting strategies, may accelerate reconstitution of hematopoiesis in patients treated with myeloablative agents.
The Roles of Laminin-Alpha1 and Muscle Contractions in Zebrafish Axon Guidance
During development, axons need to make specific connections with their targets to form a functional nervous system. Axons are guided toward these targets by responding to a variety of guidance cues, which are integrated into behavioral responses by the motile tip of the growth cone. The simple scaffold of axon tracts in the zebrafish provides and ideal model to study how guidance cues collaborate to guide axon in vivo. In this thesis, I use these pathways to investigate how various guidance cues influence growth cone behaviors to affect path-finding decisions. In Chapter 2, I show that an extracellular matrix molecule, laminin-a1, affects the direction of axon growth at choice points, axon extension, cell polarity, and cell survival. In Chapter 3, I provide evidence that muscle contractions help guide overlying sensory axons in the direction of growth and the interaxonal behaviors. Collectively, this work shows the requirements for specific cues in the guidance of a variety of axons throughout the zebrafish embryo, as well as a novel in vivo mechanism of axon guidance.
Unplugged/Muscle-Specific Receptor Kinase Signaling Coordinates Pre- and Postsynaptic Development at the Neuromuscular Junction
Development of the neuromuscular junction is divided into two stages: an early stage when acetylcholine receptors (AChRs) accumulate into prepatterned clusters at the center of muscle, and a later stage when the nerve contacts the muscle and induces neuromuscular synapses. At the later stage, nerve-derived agrin acts through unplugged/muscle-specific receptor kinase (MuSK) to promote synapse formation. At the early stage, unplugged/MuSK induces AChR prepatterning independently of agrin and the nerve. This has raised the question how unplugged/MuSK is activated at the early stage. I have taken a genetic approach in zebrafish to identify a hitherto unknown ligand for unplugged/MuSK. I show that morpholino-mediated knockdown of wnt11r results in pre- and postsynaptic defects at the early stage identical to those in unplugged mutants. I also show that wnt11r and unplugged/MuSK interact genetically and biochemically and that co-overexpression of wnt11r and unplugged/MuSK increases AChR prepatterning. Further, I demonstrate that noncanonical disheveled signaling in muscle is required for unplugged/MuSK function. I propose that wnt ligands activate unplugged/MuSK to organize a central muscle zone to which the growth cones and AChR prepatterns are restricted through a mechanism reminiscent of the planar cell polarity pathway. Using heat-shock inducible transgenes, I further demonstrate that the central muscle zone is dispensable for the formation of neuromuscular synapses, but essential for growth cone guidance. Thus, I propose that wnt-unplugged/MuSK polarizes a region in middle of muscle accessible for motor nerve, thereby determining the synaptic sites. Finally, using the same transgenes, I show that despite its early expression in muscles, unplugged/MuSK activity is first required just before the appearance of AChR prepattern to simultaneously induce AChR accumulation and guide motor axons. I also demonstrate that ubiquitous expression of unplugged/MuSK throughout the muscle membrane results in wild-type-like synaptogenesis. I propose that additional factor(s) restrict unplugged/MuSK signaling to the muscle center. On the basis of these studies, I propose that unplugged/MuSK signaling employs distinct pathways during different stages to ensure the formation of precise synapses within the appropriate target field.
Cortical Granule Exocytosis, Cytokinesis, and Germ Plasm Segregation in Danio rerio
The earliest events in embryonic development are driven and regulated by maternal products already present in the unfertilized egg. In zebrafish (Danio rerio), such maternally driven processes include cortical granule exocytosis, cytokinesis, and germ plasm segregation. A recessive maternal-effect mutation that affects these three processes, in the gene aura, was identified in a zebrafish gynogenesis-based screen. To identify and better understand normal aura function, I have characterized the aura mutant phenotype and determined the molecular identity of this gene. aura affects several events during early development that may be dependent on cytoskeletal rearrangements and it encodes MID1ip1. Cell biology, genetic, and other experimental approaches were used to characterize the aura/MID1ip1 gene function. Together, these studies will contribute to understanding the function and regulation of aura/MID1ip1 during cortical granule exocytosis, cytokinesis, and germ plasm segregation. In this study, I have contributed to the lab's investigation in the relation between the invariant cleavage pattern of the early zebrafish embryo and cortical cues by determining the localization of dynein and dynactin with respect to the centrosome and the spindle. Additionally, I have tested for defects in spindle alignment in several maternal-effect mutants that affect cell shape. These observations, together with experiments by others in the laboratory, reinforce previous findings in other organisms that cortical cues directing spindle orientation are transduced by dynein and dynactin to position the spindle and, consequently, determine the plane of cell cleavage.
Generation and Circuit Development of Zebrafish Retinal Horizontal Cells
The construction of functional neuronal circuits requires proper coordination of many developmental processes. Neurons must be generated in the proper number and migrate to their mature locations within the nervous system. Upon completion and sometimes during neuronal migration, neurons must elaborate complex dendritic and axonal arbors and then form precise synaptic connections in the background of a plethora of possible synaptic partners. Glial cells must also establish specialized associations with a number of neuronal structures, including synapses and axons. To create a detailed understanding of how a neuron progresses through all of these stages of development, I have studied a single class of neuron, the retinal horizontal cell (HC), from the stage of cell generation to the establishment of its synaptic circuits and associations with astroglia. HCs are a key component of the circuitry of the outer retina of vertebrates and they function to modulate information transfer from photoreceptors. I examined the genesis of HCs using in vivo multiphoton time-lapse microscopy in the zebrafish retina and discovered a novel precursor cell that is dedicated to the generation of HCs. In contrast to the classical germinal cell, the horizontal precursor cell is unattached to the epithelial surfaces, and instead migrates freely and divides near the final laminar location of mature HCs. I then determined what cellular changes take place in order for postmitotic HCs to establish their specific connectivity with rod and cone photoreceptors. In the zebrafish retina, HCs form well-defined circuits with specific subsets of photoreceptors. I determined how HC dendritic contacts were formed with only a specific subset of photoreceptors by performing in vivo time-lapse microscopy experiments in which a single HC was labeled in the background of all of its proper presynaptic partners. During the course of development, HC dendritic tips formed contacts with both proper and improper photoreceptor presynaptic terminals. However, improper contacts were removed while tips contacting the preferred photoreceptors were preferentially maintained. Thus, the final patterns of connections between HCs and photoreceptors are shaped by a process of refinement. After establishing the events that take place during HC-photoreceptor circuit development, I examined whether Müller glia (MG) influence the development of these circuits. I found that synaptic contacts between HCs and photoreceptors are present before MG processes ensheath the nascent synapses. I also demonstrated that contact with MG did not influence the stability of newly formed HC dendritic tips. Further, preventing the interaction of MG processes with photoreceptor pedicles and HC dendritic tips by targeted MG ablation did not cause the photoreceptor contact with HCs to disassemble. Thus, unlike in other parts of the nervous system, glial contact is not necessary to ensure the stability of newly formed synapses. Together, my findings provide an in vivo view of the cellular mechanisms utilized to generate, localize, and establish the circuitry of a single class of interneuron in the nervous system. My experiments also reveal a novel mechanism of cell generation and a contrasting view of the role of glial cells in stabilizing newly formed circuits.
Genetic Analysis of Cardiac Patterning in Zebrafish
The zebrafish system provides a powerful model to investigate the cellular mechanisms regulating cardiac patterning. This study addresses three different aspects of cardiovascular development in zebrafish and seeks to further elucidate the genetic requirements for vertebrate cardiac patterning. A limitation of the zebrafish system as a model for cardiac disease is the lack of quantitative means to assess cardiac function. In Chapter 2, I describe the zebrafish cardiac analysis system, which enables observation and quantification of cardiac function. An additional advantage of the zebrafish cardiac analysis system is its ability to facilitate high-throughput screening of small molecules or mutations affecting cardiac function. One facet of cardiac patterning is the determination of cardiac laterality. In Chapter 3, I characterize a previously unknown function for Fu in regulating left–right patterning by modulating cilia biogenesis in the Kupffer's vesicle. This role for Fu is independent from its previously described activity downstream of the Hedgehog signaling pathway in zebrafish, demonstrating that Fu function has diverged from Drosophila to vertebrates. Cardiac function requires specification and differentiation of not only the atrial and ventricular chambers but the atrioventricular (AV) boundary as well. Cardiomyocytes exhibit different conducting properties at the AV boundary than in the rest of the heart, and cardiac valve formation requires intricate signaling between the myocardium and the endocardium located at the AV boundary. In the last part of this study (Chapter 4), I characterize and clone the leo1 mutant, which displays cardiac and neural crest phenotypes. I further demonstrate that Leo1 is a conserved member of the PAF1 complex but is not required for all PAF1 complex-mediated transcription. Analysis of leo1 mutants indicates that Leo1 is required for differentiation of the AV boundary and of neural crest cells into pigmentation cells and cartilage. Overall, my study has expanded our understanding of the genetic mechanisms underlying cardiac patterning in zebrafish.
In Vivo Promoter Analysis in Zebrafish of the Fugu rubripes NMDA Receptor Subunit 1 Gene
A 5 kb fragment of the pufferfish Fugu rubripes NR1 promoter spanning nucleotides −2729 to +2343 was shown to be sufficient to drive expression of the NR1 gene in zebrafish. I performed a cross-species sequence comparison of the 5 kb Fugu NR1 promoter with homologous pufferfish (Tetraodon nigroviridis ), zebrafish (Danio rerio), and stickleback (Gasterosteus aculeatus) genomes and identified five noncoding evolutionary conserved regions (ECRs) containing cis-elements that serve as putative binding sites for 11 different trans-acting factors. I performed 5′ serial deletions of the 5 kb NR1 fragment based on the location of the ECRs and subsequently injected the truncated promoter constructs into fertilized zebrafish embryos to assess the activity of the deletion constructs. Analysis of the NR1 promoter requires an investigation of development in an intact organism. Thus, the main aim of my thesis was to establish a transgenic line of zebrafish expressing the 5 kb Fugu NR1 promoter. The transgenic data validated results obtained by in vitro methods regarding the activation of the NR1, which occurs via promoter de-repression, and the regulation of the NR1 gene, whereby tissue-specific trans-acting factors act on its cis-elements and determine its expression. The results suggest the presence of a putative fish NR1 gene control region that spans nucleotides −1360 to −194 and contains ECRs 1–3. The results also demonstrate that the minimal cis-promoter spans nucleotides −149 to +131 and contains ECR4 and ECR5.
Regulation, Function, and Evolution of T2 RNases
T2 RNases have been identified in numerous organisms from plants to animals and even microorganisms. The distribution of this family in almost every organism suggests that it may have an important biological function that has being conserved through evolution. In plants, two different subfamilies are defined. S-RNases are involved in pollen rejection during self-incompatible interactions, whereas S-like RNases are a more diverse group, with not clear function. Although expression studies suggest that S-like RNases are involved in many stress responses, including defense against pests and nutrient starvation, and in developmental processes such as senescence, functional studies addressing their biological role are still lacking. In an attempt to fill this gap in knowledge we initiated an analysis of RNS1, a RNase T2 enzyme from Arabidopsis thaliana. We showed that RNS1 transcript and protein are induced during mechanical wounding of the plant and by treatment with the hormone abscisic acid (ABA). We found that ABA is part of the RNS1 wounding response pathway; yet, in the absence of ABA, the RNS1 transcript is still induced. Thus, RNS1 defines a novel wound-response pathway, independent of known wounding signals such as oligogalacturonides, jasmonates, and ethylene. The unusual regulation of RNS1 by novel ABA-dependent and ABA-independent wounding response pathways suggests a unique, yet undefined, function. To further study the function of T2 RNases, we extended our work to other organisms. We found that petunia nectar is rich in RNase activities, and we identified four T2 RNases in Petunia hybrida. Two of these RNases are similar to S-like RNases, whereas the other two contain characteristics similar to both S- and S-like RNases. The latter two (RNase Phy3 and RNase Phy4) also show patterns of regulation consistent with those of nectarins, suggesting that they may have a role in petunia nectar defense. Although expression analyses can provide clues to understand function of RNases, it was clear that neither of these potential defense roles would be the one selected to keep this family in almost all organisms. Thus, we carried out phylogenetic analyses in search of conservation patterns that could provide more information about this elusive biological role. To this end we characterized RNase T2 proteins from animals (zebrafish) and plants (rice) and identified RNase T2 genes from a variety of species with fully sequenced genomes. We identified two T2 RNase genes in the Danio rerio (zebrafish) genome. Patterns of regulation for these RNases suggest a possible housekeeping function. Evolutionary analysis of these enzymes, along with the emergence of the RNase A family, suggests that many of the stress-related functions preformed by T2 RNases in plants are carried out by the RNase A family in vertebrates; yet, retention of at least one T2 RNase suggests an essential function exists. Expression analysis of eight T2 RNases from Oryza sativa (rice) and phylogenetic analysis of plant T2 RNases present in other fully sequenced plant genomes to led us to conclude that plant S-like RNases are divided in two classes, with RNases in Class I showing signs of rapid evolution and a possible function in stress responses (defense and nutrient deficiency), whereas Class II RNases are expressed ubiquitously and phylogenic conservation suggests a possible housekeeping role. This housekeeping role may be conserved for RNase T2 proteins in animals, whereas Class I functions are carried out by RNase A proteins in vertebrates.
Regulation of Atonal/Atonal Homolog by Distal-Less/DLX Family Members
Animals perceive their environment through information gathered with a diverse set of sensory organs. These specialized structures transmit information about the temperature, humidity, and chemical compounds present in the air and substrate, and pressure waves in the air (sound and motion detection). In Drosophila, the primary organ for sensing sound is Johnston's Organ (JO), a chordotonal organ present in the second segment of the antenna. Despite the importance of hearing as a sensory modality and the role JO plays in it, very little is known about its development. This work begins to elucidate the earliest known events in the specification of JO, namely, the activation of the proneural gene atonal (ato) in the antennal imaginal disc. Later, during pupation, the ato-expressing cells will differentiate into JO. This thesis will start with a brief review of general sense organ biology, followed by a detailed discussion of what is known about the regulation of proneural genes, with particular focus on known enhancers. The second chapter will briefly review proneural regulation and then discuss the identification of several tissue specific enhancers of ato and the regulation of one of the antennal enhancers by the genes distal-less, homothorax, and extradenticle, three key determinants of antennal identity. The third chapter will focus on an intriguing similarity between the regulation of ato in the Drosophila antenna and the regulation of ato homolog 1 in the developing zebrafish inner ear. The fourth chapter will discuss the significance of this research, examine unresolved questions from the field at large, pose some questions that are logical extensions of this work, and suggest experiments that would usefully address these queries.
The Morphological Diversity of Trigeminal Sensory Neurons in the Developing Zebrafish
The vertebrate trigeminal sensory ganglion is responsible for the detection of chemical, mechanical, and thermal stimuli. Individual neurons within this ganglion extend a peripheral axon into the skin to detect cues from the environment. Meanwhile, a central axon projects to the hindbrain to relay this information. These neurons can be divided into large-diameter neurons capable of mechanical detection and small-diameter neurons capable of noxious stimuli and innocuous temperature detection. Considerable research has been focused on discerning patterns in morphology and axon organization among these different functional subsets. However, little is known about how these patterns are established during early development. My thesis work examined the development of peripheral axon arbors expressing the noxious chemical receptor, Trpa1b. Generation of a trpa1b reporter revealed that these arbors are large and more branched than previously described isl1SS sensory arbors. Further, unlike isl1SS, peripheral axons commonly invade into the other side of the head. Despite this contralateral coverage, transplantation experiments indicate that trpa1b arbor territories are still limited by mutual repulsion from neighboring arbors. Interestingly, time-lapse measurements of developing arbors indicate that trpa1b axons may arborize a greater territory by traveling through the skin faster. This remains true even when peripheral axons grow in the absence of neighboring neurons, suggesting that these speed differences are independent of mutual repulsion. Collectively, these studies show that while mutual repulsion serves as a general inhibitory cue, differential outgrowth speeds contribute to the larger territories attributed to trpa1b arbors. Further, my studies also demonstrate the usefulness of functional receptor markers in revealing early and unique patterns in peripheral morphology and receptive field coverage.
The Role of FoxN4 in Regulating Interneuron Specification During Development
This study seeks to understand the development of neurons constituting the vertebrate spinal locomotory network, which is responsible for the intrinsic generation of rhythmic motor activities. Interneurons (INs) of the V2 class of ventral spinal INs in chick and mouse embryos are subdivided into V2a and V2b. The V2 subclasses are of opposite neurotransmitter phenotypes, with the V2a identified as excitatory glutamatergic INs and the V2b being inhibitory GABAergic INs. Their specification during development has been attributed to expression of the transcription factor Foxn4. However, recent overexpression experiments in the chick gave contradicting results as to whether Foxn4 is sufficient to induce V2b INs and if it is also able to suppress the V2a fate. I utilized the much simpler model of embryonic zebrafish with fewer neurons to validate the role of foxn4. I first characterized expression of different foxn4 transcripts at different developmental stages in the zebrafish and confirmed expression of an embryonic-specific transcript and found that all transcripts are downregulated in the adult fish. I also determined the cellular identity of V2b INs in the zebrafish, as double in situ hybridization and immunohistochemistry confirmed these to be GABAergic ventral INs. In an attempt to alter the V2a and V2b populations, I either overexpressed foxn4 mRNA or knocked down its translation using a translation blocking antisense morpholino oligonucleotide. My results indicated no change in V2a or V2b IN numbers when foxn4 was knocked down, but there was a switch from V2a to V2b when foxn4 was overexpressed. Future experiments could hopefully address if altered neural populations are able to form functional neural circuits as this may provide insights to the development and restoration of altered spinal cord motor networks.
The Xylosyltranserase Isoforms: Their Role in Proteoglycan Biosynthesis and Development
The transfer of xylose to specific serine residues in the core protein of proteoglycans is the rate-limiting and the initiating step in the proteoglycan biosynthesis pathway. This important step is catalyzed by xylosyltransferase (Xylt). Two Xylt isoforms have been identified in vertebrates, Xylt1 and Xylt2; however, enzymatic activity has not been defined for Xylt2. We have cloned the full-length murine Xylt2 and made use of Xylt-deficient Chinese hamster ovary cells to express and characterize the activity of Xylt2. We found Xylt2 is capable of modifying both chondroitin sulfate and heparan sulfate proteoglycans, thereby effectively rescuing the mutant Chinese hamster ovary cell phenotype. By generating N-terminal truncation mutants and a Xylt1–Xylt2 N-terminal hybrid, we found a potential regulator region in Xylt2 for aa 33–78, which distinguishes it from Xylt1. Our work has established that Xylt2 is an active transferase that appears to nonspecifically and nonpreferentially modify both chondroitin sulfate and heparan sulfate proteoglycans, and as well may impart some regulation at the initiating step of proteoglycan biosynthesis. We have also exploited the zebrafish as a model to explore the developmental functions of Xylt and address directly the roles of proteoglycans during early vertebrate development. Morpholino knockdown did not reveal a requirement for zebrafish xylt2; by contrast, xylt1 is required for normal development of the zebrafish embryo. Xylt1-deficient embryos have reduced proteoglycan synthesis and show defects in a variety of developing structures, including fins, somites, and the nervous system, reminiscent of phenotypes associated with altered hedgehog (Hh) signaling. In the hindbrain of Xylt1-deficient embryos we find reduced Hh signaling and concomitant alterations in dorsoventral patterning, as well as reduced proliferation. By contrast, spinal cord patterning is relatively normal, but changes in somite patterning reflect elevated Hh signaling. We conclude that proteoglycans, generated by Xylts, are necessary for proper patterning of the nervous system and somites, in part through modulation of Hh signaling, and that signaling regulation via proteoglycans varies with axial level.
Two Germ-Cell-Driven Site-Specific Recombination Systems for the Genetic Containment of Transgenic Fish
According to a 2002 study by the National Research Council, the greatest science-based concern facing animal biotechnology is the ecological and environmental impact from the escape or release of transgenic animals. Transgenic fish and insects were of high concern given their ability to escape, disperse, and become feral. Current physical and biological methods of containment for domestically raised fish, such as net pens/sea cages and triploidy-induced sterility, are inadequate for the 100% containment required of transgenic fish. Recently, genetic containment approaches that utilize germ-cell-driven site-specific recombinases, such as the Cre/loxP system, to excise transgenes from the germline have been described. However, the paradox of these approaches is that while the transgenic gene of interest was removed, the recombinase genes themselves, which are also transgenes, were not excised. We designed and tested a unique genetic containment approach that utilized two germ-cell-driven site-specific recombination systems that excised not only the transgenic gene of interest, but also the recombinase transgenes themselves, leaving behind two loxP footprints in the germline of transgenic fish. Our approach utilized two lines of transgenic zebrafish, a male line and a female line. In the male line, a germ-cell-specific promoter drives flpe recombinase, flanked by loxP sites. In the female line, the gene of interest and its promoter are flanked by FRT sites and situated between the germ-cell-specific promoter and the cre recombinase coding sequence such that cre expression is blocked. This entire construct is also flanked by loxP sites. We theorized that when these male and female lines were crossed, the progeny would express FLPe in germ cells and trigger excision of the FRT-flanked gene of interest. This, in turn, would bring the cre recombinase gene downstream of the germ-cell-specific promoter and its expression would cause excision of the loxP-flanked male line construct as well as its own self-excision from the germ cells. Using this approach, it was anticipated that if these fish were outcrossed to wild-type fish, the gametes would not carry any coding transgene and that only two loxP sites would be transmitted to the F 2 generation. flpe RNA microinjections into embryos containing the female line construct produced a high proportion of EGFP-progeny, were found to express cre transcript in gonads, and produced Cre-mediated excision events in sperm samples. However, when flpe transcript was provided endogenously in the form of a cross with a transgenic male line construct fish, we did not observe excision of the floxed EGFP cassette in F 1 progeny as we did in the flpe RNA microinjections. When F 1 progeny were outcrossed to wild-type fish, we observed a 1:1 ratio of EGFP+ to EGFP− embryos, suggesting that germline excision by FLPe had not been achieved. A PCR screen on F 2 progeny revealed only two out of 136 fish that were positive for a Cre-mediated excision event. The low efficiency of germline transgene excision could have been affected by several factors, including premature self-excision of Cre such that not all floxed sequences were removed, multiple insertions of the female line construct, and poor Cre recombination efficiency related to low temperature.
Uptake of Trace Metals in Aquatic Organisms: A Stable Isotopes Experiment
Aquatic organisms are constantly exposed to metals via dissolved and solid phases. The accumulation levels of trace metals and their toxicological effects are known to be highly dependent on concentrations of major naturally occurring cations (i.e., Ca2+, H+, Mg2+, and Na+). Moreover, other metals present in water may affect accumulation processes of individual metals. Nevertheless, the number of studies investigating the influence of water chemistry on metal uptake or accumulation at environmentally realistic concentrations is rather limited. This research work focuses on the study of trace metals uptake processes in three aquatic organisms (green algae, Pseudokirchneriella subcapitata; water flea, Daphnia magna; and zebrafish, Danio rerio) during a simultaneous exposure to Cd, Cu, Ni, Pb, and Zn at environmentally relevant exposure levels. The study has also explored and applied the possibilities of the stable isotopes technique in combination with ICP-MS as an analytical tool for the investigation of trace metal accumulation. The results showed that water is the dominant route of metal uptake by D. magna compared to food. Concentrations of Ca2+ and Na+ ions, and pH of the medium clearly affected the uptake of all metals except Cu in D. magna. Different from Daphnia responses were observed in zebrafish, showing larger variations in trace metal accumulation in gills compared to the whole body. Coexposure to other metals had an important effect on uptake of individual metals in both Daphnia and zebrafish. The observed metal–metal interactions were metal and species specific and may have important consequences for the risk assessments of metals, which are currently largely based on single-metal approaches. This study is a starting point and has mainly illustrated and confirmed the large potential of the stable isotope technique, which revealed a number of effects and interactions previously not described in literature.