Browsing by Author "Heil, Alicia"

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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.