Browsing by Subject "zebrafish"

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    Defining a model for anterior neural tube closure in the developing zebrafish embryo
    (2018-08) Heil, Alicia
    The neural tube is the precursor to the brain and spinal cord and forms through a process called neurulation. Neurulation is a conserved process among vertebrates and begins with a flat epithelium called the neural plate that folds into a closed tube-like structure. When this folding is disrupted, the neural tube fails to close and results in a neural tube defect (NTD). Previous work in our laboratory found that zebrafish embryos with reduced Nodal signaling had open anterior neural tubes. This finding led to the proposal of a broad model for anterior neurulation in zebrafish. The model begins with Nodal signaling inducing anterior mesendodermal/mesodermal tissues. These tissues then signal to the overlying neuroectoderm to promote cell adhesion in the developing anterior neural tube. Finally, this leads to a closed anterior neural tube. For our first step in our model, we hypothesized that the role for Nodal signaling in neurulation is through mesendoderm/mesoderm induction. In support of this hypothesis we found Nodal signaling is required for the development of a closed anterior neural tube through mid to late blastula stages. This temporal requirement aligns well with the timing for Nodal induction of anterior mesendodermal/mesodermal tissues. Further testing for mesendodermal/mesodermal tissue presence in zebrafish embryos found no single mesendodermal/mesodermal tissue was required for neural tube closure. Our findings support a model in which an overall amount of mesendodermal/mesodermal tissues must be present for neural tube closure, rather than a single tissue. In the second step of our model, we hypothesized that the mesendodermal/mesodermal tissues signal to the overlying neuroectoderm to form a closed neural tube. Further, these signals were thought to act downstream of Nodal signaling to induce or maintain mesendoderm/mesoderm and the neuroectoderm. Using RNAseq to compare Nodal deficient zebrafish embryos with closed and open neural tube phenotypes, we identified several signaling pathways that may have a role in zebrafish anterior neurulation. Our RNAseq data suggested that FGF signaling was reduced in embryos with an open neural tube phenotype. Initial tests using an FGF signaling inhibitor supported our data and the inhibitor was able to induce an open neural tube phenotype in wildtype embryos. In addition, we hypothesized that adherens junction proteins would be reduced in embryos with open neural tubes compared to embryos with closed neural tubes. To test this, several adherens junction proteins were compared between embryos with open and closed neural tubes. This study indicates adherens junctions proteins are still present at relatively similar levels in embryos with open neural tubes compared to those with closed neural tubes. Further studies are needed to determine if adherens junction proteins are localized at the membrane of neural tube cells in embryos with an open neural tube phenotype. To better test our RNAseq data, embryos were examined for effects of FGF signaling and canonical Wnt signaling on anterior neurulation. For FGF signaling, we hypothesized that FGF signaling is required for anterior neurulation and has a similar role to Nodal signaling in neurulation. The FGF signaling pathway was required through the onset of gastrulation for a closed neural tube, and the FGF deficient embryos had a correlation between neural tube closure and mesodermal tissue presence. Embryos deficient in FGF signaling only had mesodermal tissues missing, rather than both mesodermal and mesendodermal tissues found in Nodal deficient embryos. Additionally, we hypothesized canonical Wnt signaling is required for anterior neurulation. To test this, embryos were exposed to LiCl to increase canonical Wnt signaling, as our RNAseq data suggested canonical Wnt signaling was over expressed in embryos with open neural tube phenotypes compared to embryos with closed neural tubes. Our data suggests increased canonical Wnt signaling does not induce NTD in zebrafish embryos.
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    Dopaminergic signaling in the spinal cord suppresses locomotion in larval zebrafish development
    (2024-03) Walters, Deborah, L
    The significance of dopamine (DA) and its multifaceted role as a neurotransmitter in the central nervous system has undergone extensive investigation. The research focus of my project centers on dopamine’s role in modulating spinal locomotor circuits in larvae zebrafish. Previous research from our lab showed that larval zebrafish swimming patterns change during development from long episodes durations at 3 days post fertilization (dpf) to short episode durations at 4 dpf and coincides with gross to fine motor control. Dopamine receptor D4 signaling in the spinal cord is necessary in facilitating this switch, likely by modulating dopamine signaling and regulating the activity of motor neurons involved in generating locomotor patterns. We demonstrated that antagonism of D4R signaling starting at 3 dpf prevents the switch from long to short episode durations, while D4R antagonism at 4 dpf reverses the switch from short to long episode durations. We hypothesized that 3 dpf larvae possess sufficient dopaminergic receptors in the spinal cord to bind to DA, enabling the advancement of the developmental switch from immature, long swim patterns to a mature state resembling 4 dpf larvae by exposing larvae at 3 dpf to exogenous DA. To test this, we used transgenic zebrafish that expressed Channelrhodopsin (ChR) in glutamatergic neurons within the spinal cord, allowing for the activation of these neurons using blue-light stimulation. Fictive swimming was measured using peripheral nerve recordings in different conditions, of a baseline (t0), treatment of dopamine (t1), and washout (saline) (t2). Control (untreated) preparations exhibited no significant changes between conditions, indicating that repeated optogenetic stimulation by itself did not induce notable changes in locomotor activity. Dopamine application significantly decreased the number of bursts and episode duration during optogenetic stimulation locomotor activity without affecting number of episodes, burst duration, or inter-burst intervals. These results suggest that exogenous DA affected swim patterns in 3 dpf larvae to resemble their 5 dpf counterparts, indicating a sufficient expression level of dopamine receptors in spinal locomotor networks of 3 dpf larvae to prematurely advance the developmental switch. These results could elucidate how neurodegenerative and motor disorders develop and progress, and shed light on the mechanisms underlying spinal cord injury. These findings could potentially inform translational medical approaches creating novel therapeutic interventions for treating neurodegenerative diseases.
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    Laser Nanowarming: A platform technology for ultra-rapid rewarming of cryopreserved zebrafish embryos
    (2019-06) KHOSLA, KANAV
    This work describes the development of a platform technology called Laser Nanowarming that has enabled the cryopreservation of Zebrafish embryos for the first time. By injecting propylene glycol (PG) and biocompatible gold nanorods (GNR) followed by rapid cooling (90,000 °C/min), embryos were cryogenically stabilized to liquid nitrogen temperatures. Since the effective concentration of PG inside the embryos is approximately 2M, the embryos require rapid rewarming, which was achieved by using a 1064nm powerful millisecond laser pulse that can generate rates up to 14 million °C/min. We leverage biocompatible and photonic GNR that can create rapid and uniform warming throughout the embryo and overcome the damage induced by ice crystallization. We have since adapted this technology to demonstrate successful outcomes in Human Dermal Fibroblasts (HDF) cells as well as Coral larvae (F. Scutaria) and continue to use it to enable the cryopreservation of Pancreatic Islets, Drosophila Embryos, Shrimp Nauplii and other fish embryos. Our future work is geared towards improving the long-term survival rate of biological specimens as well as developing efficient high throughput methods. If successful, this technology can transform the way germplasm are banked and create a huge impact in the fields of species conservation, biomedical research and aquaculture.
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    Revealing Novel Skin Biology Using Protein-Trap Gene-Break Transposon Mutagenesis Technology In The Larval Zebrafish Model
    (2016-12) Westcot, Stephanie
    Abstract Although skin disorders affect as much as a third of the population at any given time, available treatments are limited. Because a more comprehensive understanding of skin development mechanisms can spur the identification of new treatment targets and techniques, we developed the Zebrafish Integument Project (ZIP), an expression-driven platform for identifying new skin genes and new, revertible phenotypes in the vertebrate model Danio rerio (zebrafish). In vivo selection for skin-specific expression of gene-break transposon (GBT) mutant lines identified eleven new, revertible GBT alleles of genes involved in skin development. Eight of those genes had been described in an integumentary context to varying degrees: fras1, grip1, hmcn1, msxc, col4a4, ahnak, capn12, and nrg2a. Three others—arhgef25b, fkbp10b, and megf6a—emerged as novel skin genes. Embryos homozygous for a GBT insertion in neuregulin 2a (nrg2a) revealed a novel requirement for a Neuregulin 2a (Nrg2a) – ErbB2/3 – AKT signaling pathway governing ridge cell morphogenesis and apicobasal organization during median fin fold (MFF) morphogenesis. In nrg2a mutant larvae, the basal keratinocytes that comprise the apical MFF (ridge cells) displayed reduced pAKT levels as well as reduced apical domains and exaggerated basolateral domains. Those defects prevented proper ridge cell elongation into a flattened epithelial morphology, resulting in thickened MFF edges. Additionally, morpholino knockdown of epithelial polarity regulator and tumor suppressor lgl2 ameliorated the nrg2a mutant phenotype. Identifying Lgl2 as an antagonist of Nrg2a – ErbB signaling revealed a significantly earlier role for Lgl2 during epidermal morphogenesis than has been described to date. Furthermore, our findings demonstrated that ridge cells’ squamous flattening morphogenesis drives apical MFF development. We therefore propose MFF ridge cells as a new model for investigating the regulation of cell polarity and cellular morphogenesis with regard to their roles as crucial mechanisms for epithelial morphogenesis generally, and for flattening morphogenesis in particular.