Browsing by Subject "zebrafish"
Now showing 1 - 6 of 6
Results Per Page
Sort Options
Item Digitizing Vertebrate Brain Development with Ultrashort Pulse Microscopy(2014-12-01) Gibbs, Holly CEmbryonic development is a process of unparalleled dynamism. The only constant in development is change itself- change in size, change in shape, change in gene expression- all driving the formation of an organism from a single cell. While the complexity of these changes is fascinating, it creates quite a challenge for modern imaging systems aimed at visualizing developmental processes. The embryonic brain is subdivided by unique patterns of gene expression prior to morphogenesis, but the mechanisms linking these phenomena remain poorly understood in part due to the lack of imaging techniques that can capture dynamic molecular, cellular, and tissue-scale dynamics simultaneously in 3-D. We have established the use of Ultrashort Pulse Microscopy, a platform that utilizes the high peak power and short coherence length of broadband ultrashort pulses to simultaneously generate two-photon excited fluorescence and high-resolution optical coherence signals, and 3-D image registration for integrated imaging and analysis of morphogenesis, genetic cell lineage reporter dynamics, and gene expression domains during embryonic brain patterning in the vertebrate model Danio rerio (zebrafish). Using these tools, we characterized wnt1 lineage dynamics during midbrain-hindbrain boundary (MHB) formation and found differences in the refinement of gene expression domains along the dorsoventral axis at the boundary where wnt1 and fgf8a form an interface. We then examined these dynamics in fgf8a loss of function and showed that initiation of the morphogenetic program at the boundary does not require fgf8a, but that anteriorly expanded hindbrain ventricle opening and failure to restrict the posterior limit of wnt1 expression at the dorsal MHB results in a cerebellar to tectal transformation and termination of constriction morphogenesis.Item Inner Ear Sensory Epithelia Development and Regulation in Zebrafish(2011-10-21) Sweet, Elly MaeThe inner ear is a complex sensory organ of interconnected chambers, each with a sensory epithelium comprised of hair cells and support cells for detection of sound and motion. This dissertation focuses on the development and regulation of sensory epithelia in zebrafish and utilizes loss of function, gain of function and laser ablation techniques. Hair cells and support cells develop from an equivalence group specified by proneural genes encoding bHLH transcription factors. The vertebrate Atoh1 bHLH transciption factor is a potential candidate for this role. However, data in mouse has led some researchers to conclude it does not have a proneural activity, but, rather, is involved in later stages of hair cell differentiation. In addition, the factors regulating Atoh1 are mostly unknown. We address these issues in zebrafish and show that the zebrafish homologs atoh1a and atoh1b are required during two developmental phases, first in the preotic placode and later in the otic vesicle. They interact with the Notch pathway and are necessary and sufficient for specification of sensory epithelia. Our data confirm atoh1 genes have proneural function. We also go on to show Atoh1 works in a complex network of factors, Pax2/5/8, Sox2, Fgf and Notch. Misexpression of atoh1 alters axial patterning and leads to expanded sensory epithelia, which is enhanced by misexpression of either fgf8 or sox2. Lastly, we examine the role of sox2 in sensory epithelia development and regeneration. Sox2 has been implicated in maintainence of pluripotent stem cells as well as cell differentiation. In the inner ear, Sox2 is initially expressed in the prosensory domain and is required for its formation. Eventually, Sox2 is downregulated in hair cells and maintained in support cells; however, its later role has not been determined. We show that in the zebrafish inner ear, sox2 is expressed after sensory epithelium development has begun and, like in mouse, expression is down regulated in hair cells and maintained in support cells. Our data demonstrate a role for sox2 in maintenance of hair cells and in transdifferentation of support cells into hair cells after laser ablation. Additionally, sox2 is regulated by Aoth1a/1b, Fgf, and Notch.Item Mechanisms of Wnt8 function in zebrafish mesoderm patterning(Texas A&M University, 2006-08-16) Ramel, Marie-ChristineIn vertebrate embryonic development, correct specification of tissue fates along the dorsoventral (D/V) axis is known to require the secreted signaling ligand Wnt8. Wnt8 signaling promotes ventral fates and antagonizes the expansion of the dorsal domain known as the organizer. Maintenance of the organizer is critical for proper development as this tissue is known to produce inhibitors of Wnt and BMP (Bone Morphogenetic Protein) family ligands; BMPs are also known to play a major role in promoting ventral fates. In order to understand how Wnt8 antagonizes the organizer, we analyzed the epistatic relationship between Wnt8 and the transcriptional repressors Vent and Vox using zebrafish as a model organism. We found that Wnt8/β-catenin signaling directly regulates the transcriptional levels of vent and vox so that they can repress the transcription of dorsal genes on the ventral side of the embryo. To understand the contribution of Wnt8 towards ventral fate specification, we carefully analyzed its relationship with BMP signaling during gastrula stages. We found that bmp expression in the mesoderm is under the control of Wnt8 at mid-gastrulation and that regulation of bmp explains many of the ventral defects observed in wnt8 mutants. Antagonism of the expression of organizer-derived BMP inhibitors by Wnt8 also indirectly allows timely BMP signaling. Analysis of wnt8; bmp double mutants revealed an early unsuspected function of BMP in the antagonism of the organizer. Further, we uncovered a mechanism through which regulation of vent, vox and a related-gene ved expression by both Wnt8 and BMP antagonizes dorsal/axial mesoderm identity to preserve the integrity of ventral/non-axial tissues. In summary, we have revealed some of the mechanisms of Wnt8 function in D/V mesoderm patterning: it restricts the organizer domain by regulating vent and vox, it allows BMP induced differentiation through its inhibition of BMP antagonists derived from the organizer and it co-regulates vent, vox, and ved with BMP signaling to allow maintenance of the non-axial domain.Item Molecular Mechanisms of Wnt8a Regulation: Insights Into Vertebrate Mesoderm Development and Patterning(2012-07-16) Narayanan, AnandVertebrate wnt8a occupies a position at a crossroads linking anteroposterior and dorsoventral axis patterning. While functional aspects of wnt8a are beginning to be understood, the regulation of wnt8a expression and its relationship to mesoderm induction and maintenance pathways are unclear. Three inputs that control wnt8a expression in the zebrafish embryonic margin have been identified: the Brachyury-related T-box transcription factors No tail a (Ntla) and No tail b (Ntlb, previously called Bra) and the maternal zinc-finger transcription factor Zbtb4 (previously called Kzp) are known direct regulators, and Nodal signaling is genetically upstream of wnt8a expression. The transcriptional mechanisms by which the wnt8a locus integrates these diverse temporal inputs are not yet known. We have generated zebrafish transgenic for a modified genomic PAC clone that expresses EGFP from the wnt8a locus. The EGFP reporter transgene is expressed in a pattern nearly identical to wnt8a, including maternal deposition, expression in the ventrolateral mesoderm and in the yolk syncytial layer. Using this transgenic line, we identified two phases of wnt8a transcriptional regulation in zebrafish: phase I comprises Nodal-dependent activation during early gastrulation and phase II comprises No tail (Ntl)-dependent regulation from mid to late gastrula stages onwards. These phases mirror the transition from Nodal-dependent mesoderm induction to Ntl-dependent mesoderm maintenance. To further understand how the wnt8a locus integrates these signals to achieve its transcriptional output, we analyzed upstream cis-regulatory regions through transgenic reporter assays. We identified three promoters in the bicistronic wnt8a locus, two of which drive expression of the upstream coding region (wnt8a.1). We identified two regulatory regions, proximal and distal: the proximal regulatory region contains a mesodermal enhancer with potential binding sites for FoxH1 and Ntl that is required for both the Nodal and Ntl responses. Phase I expression requires Nodal signaling through the mesoderm enhancer in combination with the distal regulatory region, which bears a Zbtb4 consensus binding site. Phase II expression requires Ntl regulation of the mesoderm enhancer in the context of the proximal regulatory region. The distal regulatory region negatively impacts phase II expression driven by the proximal regulatory region, indicating a complex relationship of regulatory elements.Item Neurosensory Development in the Zebrafish Inner Ear(2012-02-14) Vemaraju, ShrutiThe vertebrate inner ear is a complex structure responsible for hearing and balance. The inner ear houses sensory epithelia composed of mechanosensory hair cells and non-sensory support cells. Hair cells synapse with neurons of the VIIIth cranial ganglion, the statoacoustic ganglion (SAG), and transmit sensory information to the hindbrain. This dissertation focuses on the development and regulation of both sensory and neuronal cell populations. The sensory epithelium is established by the basic helixloop- helix transcription factor Atoh1. Misexpression of atoh1a in zebrafish results in induction of ectopic sensory epithelia albeit in limited regions of the inner ear. We show that sensory competence of the inner ear can be enhanced by co-activation of fgf8/3 or sox2, genes that normally act in concert with atoh1a. The developing sensory epithelia express several factors that regulate differentiation and maintenance of hair cells. We show that pax5 is differentially expressed in the anterior utricular macula (sensory epithelium). Knockdown of pax5 function results in utricular hair cell death and subsequent loss of vestibular (balance) but not auditory (hearing) defects. SAG neurons are formed normally in these embryos but show disorganized dendrites in the utricle following loss of hair cells. Lastly, we examine the development of SAG. SAG precursors (neuroblasts) are formed in the floor of the ear by another basic helix-loophelix transcription factor neurogenin1 (neurog1). We show that Fgf emanating from the utricular macula specifies neuroblasts, that later delaminate from the otic floor and undergo a phase of proliferation. Neuroblasts then differentiate into bipolar neurons that extend processes to hair cells and targets in the hindbrain. We show evidence that differentiating neurons express fgf5 and regulate further development of the SAG. As more differentiated neurons accumulate, increasing level of Fgf terminates the phase of neuroblast specification. Later on, elevated Fgf stabilizes the transit-amplifying phase and inhibits terminal differentiation. Thus, Fgf signaling regulates SAG development at various stages to ensure that proper number of neurons is generated.Item Patterns in Teleost Photoreceptor Organization: A Characterization of Basal Body Positioning in Zebrafish Photoreceptors and Variations in Swordtail Photoreceptor Mosaics(2014-04-17) Ramsey, MichelleVertebrate vision is enabled by light-sensitive photoreceptors arranged in a plane in the retina. This study investigates two aspects of this arrangement: 1) positioning of basal bodies within photoreceptors, and 2) positioning of photoreceptors themselves. First, the planar cell polarity of basal bodies, and therefore cilia, is often critical for proper cilia function and is controlled by the planar cell polarity (PCP) pathway. Cilia planar positioning in vertebrate photoreceptors, however, has not been characterized. Because zebrafish photoreceptors form an organized, well-characterized mosaic, they are an ideal system to address photoreceptor basal body positioning. Second, swordtail fish are frequently studied to investigate visually-mediated social behaviors such as mate choice and how these influence evolution. However, less is known about the morphology of their photoreceptor mosaic and how this mosaic influences behavior. Therefore, characterization of the swordtail photoreceptor mosaic is an important step in understanding this relationship between physiology and behavior. In this study, immunohistology is used to characterize cryosectioned flatmounted retinas from zebrafish and swordtails with various genetic, behavioral, and environmental backgrounds. The results of this study reveal that in adult zebrafish retinas, the basal bodies of red-, green-, and blue-sensitive cone photoreceptors localize asymmetrically on the cell edge nearest the optic nerve. In contrast, no patterning is in the basal bodies of ultraviolet-sensitive cones, of rod photoreceptors, or of larval cones. Both rod loss and UV-light addition do not affect cone basal body patterning. Darkness during development leads to bimodality of basal bodies. These results suggest that, after the transition to the adult mosaic, a cellular mechanism involving cell-cell contact, consistent with the PCP pathway, regulates photoreceptor basal body positioning. The results of this study also reveal that the swordtails Xiphophorus malinche, Xiphophorus birchmanni, and their hybrids exhibit an organized square mosaic, although some variations in this pattern exist, including between males and females. As square mosaics have been correlated with sensitivity to changes in light polarization, this warrants future studies in swordtail polarization vision, which may play an important role in visually-mediated behavior. Also, changes in the photoreceptor mosaic might have explanatory power for changes in visually-mediated behavior.