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ItemInvestigating The Mechanisms Underlying Metamorphosis in The Chordate Ciona Robusta(Georgia Institute of Technology, 2024-04-29) Johnson, ChristopherIn our study, we investigate the multifaceted roles of papillae in tunicate larvae, pivotal for sensory perception, adhesion, and metamorphosis regulation, particularly in the model organism Ciona. Through molecular marker identification and CRISPR/Cas9-mediated mutagenesis, we delineate the intricate cellular diversity within papillae, elucidating the regulatory networks orchestrated by key transcription factors and signaling pathways. Concurrently, we explore the evolutionary divergence in the expression patterns of Myomaker (Mymk), a fusogenic factor crucial for myoblast fusion and muscle multinucleation, between vertebrates and tunicates. By analyzing cisregulatory sequences of Mymk, we unveil the underlying mechanisms driving the differential spatiotemporal expression patterns in these organisms. Our findings not only deepen our understanding of tunicate development but also provide insights into the evolutionary history of myoblast fusion regulation across chordates.
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ItemChordate-specific gene regulatory network of neuron development in Ciona.(Georgia Institute of Technology, 2023-12-12) Kim, KwantaeIn this research, I investigated the complex gene regulatory networks underlying neurogenesis, taking advantage of the unique neurons of the Ciona model system. I revealed that Fgf signaling is crucial for the neurogenesis of Bipolar Tail Neurons (BTNs) by controlling the expression of Neurogenin, the fate-determining transcription factor in these neurons. Then I also characterized multiple effector genes functioning in the development of BTNs. Additionally, I determined the vital role of the Pax3/7 transcription factor in the neural plate border to induce the neural tube closure. Finally, I demonstrated how the Pax3/7 also orchestrates an intricate gene regulatory network upstream of multiple transcription factors and functional effectors during the neurogenesis of Descending Decussating Neurons (ddNs). I found that the majority of this network’s regulatory branches are shared with other neurons in Ciona or even other organisms including vertebrates. Moreover, I revealed the role of key putative effector genes during the neurogenesis of ddNs. These findings will provide profound insights into developmental mechanisms in the central nervous system of chordates.
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ItemA preliminary investigation into the role of Sipa1l in bipolar tail neurons of Ciona robusta(Georgia Institute of Technology, 2021-05) Shearer, Tanner ReidBipolar tail neurons (BTNs) in Ciona robusta offer a unique chance to assess regulatory networks that give rise to cell behaviors during development. Like most cells within the embryo of Ciona robusta, BTNs develop following highly stereotyped yet dynamic programs. Neurogenin, a transcription factor, was found to be necessary and sufficient for BTN specification, and SIPA1l was observed to be downstream of Neurogenin activation. The present study sought to elucidate the role of SIPA1l in BTN development, and hypothesized that it was required for collective cell migration of BTNs. The gene encoding SIPA1l, Sipa1l, was reconstructed prior to analyzing its domains and its relatedness to the human orthologs Sipa1l1, Sipa1l2, and Sipa1l3. It was found that this gene in Ciona closely resembles its human counterparts in both the domains present in the protein it encodes, as well as, its amino acid sequence alignment. Sipa1l was then targeted using CRISPR/Cas9 in an attempted knock out condition wherein mixed results were obtained that are limited in interpretation. The fluorescent protein used to assess BTN development only labeled anterior BTNs and thus collective cell migration could not be assessed. Moreover, it was observed that Sipa1l did not affect anterior BTN migration or morphology but did appear to affect axonal outgrowth in select embryos. In all, the results of this investigation provide insights into the role of Sipa1l in BTN development and serve as a preliminary study that will prove useful to future researchers seeking to understand BTN development.
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ItemSingle-cell RNA Sequencing Analysis and Experimental Designs to Investigate the Role of Nitric Oxide Signaling in Progenitor Cell Survival in Ciona(Georgia Institute of Technology, 2021-05) Gurgis, Alexandra MarieThe tunicate Ciona is a marine chordate whose biphasic life cycle, relatively simple transcriptome, and ability to be genetically manipulated via electroporation makes it an ideal model for studying phenomena of neurodevelopment and regeneration. During the metamorphosis of larvae to adults, nearly the entire central nervous system is eliminated and rebuilt, with the notable exception of the “Neck”, a compartment of quiescent neural progenitor cells. The mechanism by which these cells are spared from the wave of programmed cell death that occurs around them is not currently understood, but could reveal important principles of cell cycle regulation. In this work, I re-examine single-cell RNA sequencing data to further characterize differential gene expression in the Neck, with a particular emphasis on the nitric oxide signaling pathway as a potential suppressor of apoptosis. I also provide experimental constructs for future investigations of genes I believe to be relevant to understanding Neck cell survival, including designs for in situ hybridization probes and GFP reporters to verify gene expression in vivo, sgRNA primers for CRISPR/Cas9 knockouts, and peakshift primers to verify the efficacy of sgRNAs.