Browsing Theses and Dissertations by Subject "Zebrafish"
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Characterization of a novel component of Wnt signaling pathway using zebrafish as a model organism.Wnt signaling plays important role in many aspects of embryogenesis such as cell proliferation, cell fate specification, cell polarity and organogenesis(Clevers 2006, van Amerongen and Nusse 2009). Wnt ligands have been shown to activate several intra-cellular signaling cascades, including the canonical or Wnt/-catenin dependent pathway and the non-canonical or -catenin independent pathway. Dishevelled (Dvl) occupies a key position at crossroads of all branches of Wnt signaling cascade. To understand, how Dishevelled (Dvl) may channel signaling into the downstream branches, we sought to identify novel effectors for Dishevelled (Dvl) using a yeast-two hybrid screen. In this study, we used the PDZ domain of Dishevelled (Dvl) as a bait and from this screen, we identified a new binding protein of Dishevelled (Dvl)-termed as Custos. To characterize the functional role of Custos in Wnt signaling pathway, we used mammalian cell culture and zebrafish as a model vertebrate organism. We confirmed the interaction between Custos and Dvl using co-immunoprecipation and GST pull-down. Custos also interacted with -catenin in vivo and this interaction was positively regulated by Wnt stimulation. Immunofluorescence experiments in mammalian cells showed that Custos co-localizes with the nuclear envelope marker, lamin and inhibits translocation of -catenin to the nucleus. In zebrafish embryos, Custos is a maternal gene and expressed throughout development. Spatial in situ hybridization studies showed that Custos was expressed in the dorsal region of the embryo at early stages and in the nervous system in zebrafish at 24hpf. To delineate the biological role of Custos during embryogenesis, we conducted a gain of function and loss of function studies. Overexpression of exogenous Custos and morpholino knockdown of Custos revealed that Custos is critical for embryonic patterning. Knockout of Custos in zebrafish revealed that Custos delays embryonic development and exhibits defects in pigmentation suggesting a plausible role in neural crest development. Taken together, our studies demonstrate that Custos is a novel component of canonical Wnt signaling and required for -catenin translocation into the nucleus and important for embryonic patterning.
Elucidating the Role of the Daam Proteins in Zebrafish Embryonic DevelopmentWnt signaling is an evolutionarily conserved pathway that is essential for the development of the metazoan embryo. Wnt signaling controls essential developmental processes including cell fate, cell polarity, dorsal-ventral patterning and tissue movement. Misregulated Wnt signaling can have disastrous effects on the developing human embryo, leading to potentially fatal congenital malformations including anencephaly and spina bifida. In addition to embryonic development, misregulated Wnt signaling has been implicated in human pathologies including colon and breast cancers and skeletal malformations. Wnt signaling is divided into two main pathway branches, canonical or beta-catenin dependent, and non-canonical, sometimes referred to as the planar cell polarity (PCP) pathway. The former branch activates the transcription of the downstream target genes leading to the patterning the dorsal-ventral axis of the developing embryo, whilst the latter has no downstream transcriptional targets but rather acts upon the cytoskeleton to control cell and tissue polarity and movement. Wnt signaling bifurcates into these two branches at the level of the protein Dishevelled (Dvl). The Dishevelled-associated activator of morphogenesis 1 (Daam1) protein was identified via a yeast-two hybrid screen using Dvl as bait. Daam1 interacts directly with Dvl and mediates activation of the small GTPase Rho, a key player in non-canonical Wnt signaling necessary for proper gastrulation in the Xenopus (frog) embryo. In addition to Daam1, vertebrates possess a second Daam, Daam2, originally identified via an in silico screen in humans. Similar to Daam1, frog Daam2 participates in non-canonical Wnt signaling, contributing to proper formation of the embryonic neural tube. However, conflicting opinions on the function of Daam2 have led to discrepancies regarding its position in Wnt signaling and function in development. Daam1/2 have not been extensively investigated at the genetic level, therefore, I employed the genetic model zebrafish (Danio rerio) to further clarify their role in Wnt signaling. Using techniques such as the latest gene-editing system CRISPR/Cas9 and other well-established molecular methods including in situ hybridization, RT-PCR and knockdown using morpholino oligonucleotides, I sought to further establish the role of the Daam protein family in vertebrate embryonic development. Together, my results indicate that the zebrafish Daam1a/b and Daam2 behave similarly to Daam1 and Daam2 in frog, respectively, by participating in the non- canonical Wnt signaling pathway and mediating morphology in the developing zebrafish embryo.
Functional analysis of tcf21 and tbx20 in zebrafishIn response to cardiac cell death from an injury, zebrafish, as opposed to mammals, are able to regenerate new heart cells without significant scar tissue. Heart attacks, a leading cause of death in the United States, leave behind substantial scar tissue that weakens the heart and leads to a greater chance of a repeated cardiac event. Many genes and major molecular pathways are highly conserved from fish all the way to humans; thus, understanding how the regenerative process works in zebrafish may provide insight into potential therapies for heart attacks in humans. However, we must first understand how heart regeneration occurs in zebrafish at the molecular level. From the time of injury to a zebrafish heart through the completion of regeneration, we want to build a regulatory network showing which genes are up- or down-regulated and how they are interconnected. Transcription factors, such as tcf21 and tbx20, bind to regulatory elements of DNA and can either upregulate or downregulate nearby genes. To build this gene regulatory network, scientists use a technique called ChIP-seq that can determine where in the genome these transcription factors bind. Nearby genes are potential targets of their regulation, and we can validate these enhancers by testing differences in expression using a fluorescent protein reporter construct. ChIP-seq requires high quality antibodies capable of specifically recognizing the transcription factor of interest. These are rarely available. Because each different antibody that is used requires validation and optimization for ChIP-seq, it is not easy to scale up the collection of data for different transcription factors. One way to get around these problems is to express a tagged version of the transcription factor. The tag is recognizable by the same antibody; however, expressing the tagged transcription factor in this manner almost inevitably results in higher than normal levels of expression, leading to false positives in the ChIP-seq data. Using CRISPR/Cas9 technology to target and modify specific sequences in the genome, we developed a novel method to add an epitope tag to these transcription factors at their endogenous loci. This allows us to run ChIP-seq experiments with the transcription factor at physiological levels of expression. We can also use the same antibody to eliminate repeated validation and optimization steps. We have successfully tagged two genes that may be involved in heart regeneration, tcf21 and tbx20. tcf21 is expressed in the developing epicardium and is required for the proper development of the branchial arches. tbx20 is expressed in the cardiomyocytes and is required for the proper development of the heart, and it has also been shown to be upregulated in response to injury in the zebrafish. With tbx20, we have performed a successful ChIP-seq experiment and have tested several promising target genes. It is difficult to test if either tcf21 or tbx20 is required for regeneration, as both of these genes are essential for development and mutants do not survive. The solution to this problem is to engineer the gene so that it can be turned off at a specific time and in a specific cell type. A common method of this in the mouse model utilizes the Cre/loxP system: two loxP sites flank a required segment of a gene, and the introduction of the enzyme Cre deletes the DNA between them. Until CRISPRs this was not feasible in zebrafish, which lacked an efficient method of targeted modification in the genome. We adapted our method for integrating epitope tags to add the two loxP sites in the genome. We have made and tested a fully conditional mutant for tbx20, and we have put in the first of the two loxP sites for tcf21.
Study of the Functional Role of ATP1A3A in the Vertebrate Nervous SystemNa+, K+ ATPases are a group of transmembrane-bound pumps found in all animal cell types. The primary functions of Na+, K+ ATPases are to maintain electrochemical gradients across cell membranes by actively transporting Na+ and K+ ions between intracellular and extracellular spaces using ATP hydrolysis. In vertebrates, Na+, K+ ATPases come in a variety of different isoforms that are expressed in a variety of tissues. Specifically, the α3 isoform, encoded by gene ATP1A3A in humans, has been shown to be expressed in neurons. Mutations in ATP1A3A have been linked to rapid-onset dystonia-parkinsonism and alternating hemiplegia of childhood in humans and has also been shown to cause motor deficits, neuronal excitability, and perinatal death in various animal models. Our lab has generated a mutant in zebrafish (Danio rerio) containing a gene trap that prematurely stops transcription of the ATP1A3A homolog, atp1a3a. We found that larvae homozygous for the gene trap mutation do not survive past 10 days post fertilization. Further analysis revealed that homozygous mutants show vision deficits. We attempted to rescue these phenotypes by expressing atp1a3a in neurons exclusively. However, no rescue of the larval death or vision abnormality was observed, suggesting that atp1a3a presence in cells other than neurons may be critical for survival and proper visual function of the animal.