Browsing by Subject "Nodal signaling"

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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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    Differentiation and patterning of cells originating in the zebrafish neuroectoderm
    (2013-05) Lund, Caroline E.
    Vertebrate nervous system development requires a complex series of events to transform a flat neuroepithelium into complex structures containing specialized cell types. The anterior neuroectoderm gives rise to the brain and is the origin for some of the first neurons that differentiate. It is also the origin of the cranial neural crest cells that form craniofacial features. This thesis focuses on the embryonic development of two tissues that arise from the zebrafish anterior neuroectoderm, the epithalamus, a region of the dorsal forebrain, and the mandible, the lower jaw. Early in development, the flat neural plate folds into a neural tube. The pineal gland, an organ involved with circadian rhythms, begins as two precursor domains at the lateral edges of the neural plate that converge into a single tissue when the neural tube closes. The pineal gland, along with the parapineal gland and habenula nuclei, form the epithalamus in the dorsal forebrain. In embryos with open neural tubes, the left and the right sides of the pineal and surrounding epithalamus are widely spaced. I found that despite this displacement, pineal cell types differentiate normally and initiate their rhythmic function. Conversely left-right asymmetry in the epithalamus was lost; both sides exhibited left-sided characteristics. Further, this loss of asymmetry in the epithalamus was correlated to severity in neural tube defects. Embryos with left isomerism had significantly wider pineal anlage domains than those with normal or reversed asymmetry. Cranial neural crest cells from the dorsal neural tube migrate to form craniofacial structures, including the cartilaginous precursor to the mandible, Meckel's cartilage. The bigtime (bti) mutant exhibits reductions in mandibular development. I found that although cranial neural crest cells localize normally to the lower jaw region in these mutants, they fail to differentiate into functional chondrocytes that secrete a sufficient amount of collagenous extracellular matrix.
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    Temporal and Spatial Requirement of the Nodal induced head mesendoderm in neurulation AND An inexpensive, efficient method for regular egg collection from a zebrafish in a recirculating system
    (2013-07) Gonsar, Ngawang Youdon
    Chapter 2 Zebrafish in our laboratory are usually bred by removing the fish from the recirculating aquatic system and placing them into 1-2 L spawning tanks. These spawning tanks consist of a bottom reservoir, a lid, and an insert that fits in closely into the bottom reservoir. When the fish breed, the eggs fall through holes of the insert and into the reservoir, thus preventing them from being cannibalized. Because fish in these spawning tanks are not fed and do not get fresh water, they are bred only once a week. During a period where we had high demand for embryos, we instead tried breeding the fish for multiple consecutive days on the recirculating system. Fish were placed into the spawning insert as usual, but the insert was placed into the home tank instead of into the bottom reservoir. We found that there was no significant difference in the number of fertilized eggs produced between the spawning tank and home tank breeding methods. Further, the fish in the home tanks regularly produced fertile embryos over a 28-day time course, with the highest number of eggs per pair produced by the tank with only one pair of adult fish. This method is time-saving as fish bred in home tanks only require to be set up once. It is also an effective way to collect embryos over long periods from the same pair or group of fish and to more easily obtain embryos from stocks with low spawning frequency. Chapter 3 The neural tube is the precursor to the brain and spinal cord. Failure of neural tube closure in humans is one of the most common causes of birth defects. Zebrafish with a decrease in Nodal signaling have a phenotype that is analogous to the fatal human birth defect anencephaly, which is caused by an open anterior neural tube. Previous work in our laboratory has found that Nodal signaling acts through the induction of the head mesendoderm and anterior mesoderm, which underlie the anterior neural tube. Using a pharmacological approach, we determined that Nodal signaling is required up to the late blastula stage of 4.3 hpf for a closed neural tube. This falls within the developmental period when Nodal signaling is most active in mesendoderm and mesoderm induction, supporting the model that Nodal acts through the induction of these tissues. We also found there was a strong correlation between the presence of multiple anterior mesendodermal and mesoderm tissues and neural tube closure. However, no individual tissue was required for neural tube closure. Our finding identifies a specific time window of when Nodal is required for the process of neurulation. This time occurs before the neuroectoderm starts to form, suggesting that Nodal and anterior mesendoderm/mesoderm act very early in the process of neurulation. Further, the finding that multiple mesendodermal/mesodermal tissues are involved suggests that wide region of tissue helps promote closure of the adjacent neural tube.