Browsing Theses and Dissertations by Subjects
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Artificial Chromosome Transgenesis Reveals Long-Distance Negative Regulation of ragl in ZebrafishDespite the essential roles played by the recombination activating genes (ragl and rag2) during V(D)J recombination, the mechanisms that restrict their expression to lymphoid cells are undefined. Using a novel approach to achieve artificial chromosome transgenesis in zebrafish, we demonstrate that distal regulatory elements are critical to suppress ragl expression in inappropriate tissues. In contrast to smaller reporter gene constructs, 125 and 75 kb artificial chromosomes containing the zebrafish rag genomic locus directed GFP expression in a pattern reflective of endogenous rag 1. Mapping experiments identified a positive element 5' of ragl that enhances GFP expression in both lymphoid and non-lymphoid tissues and a negative element 5' of ra g l that specifically suppresses GFP expression in the skeletal muscle. Our transgenic zebrafish also express GFP in olfactory neurons which we show represent an authentic ra g l expression site in zebrafish.
Characterization o f Zebrafish Mutant m erlot as a Non-Mammalian Vertebrate Model for Congenital Anemia Due to Protein 4.1 DeficiencyThe zebrafish mutant merlot (mot) is characterized by onset o f a severe anemia at 96 hours post fertilization. We performed whole mount RNA in situ hybridization and showed that the process o f primitive erythropoiesis is not interrupted in the mot embryos. Blood analysis demonstrated that mot suffers from a severe congenital hemolytic anemia. Using the TU N E L assay, we detected apoptotic erythroid progenitors in the kidneys. We performed electron microscopic analysis and detected membrane abnormalities and a loss o f the cortical membrane organization in the mot cells. We used positional cloning techniques w ith a candidate gene approach to demonstrate that mot encodes the erythroid specific isoform o f protein 4.1R, a critical component o f the red blood cell membrane skeleton. Sequence analysis o f 4.IR cD N A detected nonsense point mutations in both alleles o f mot resulting in premature stop codons. We performed linkage analysis and transgenic rescue experiments to provide further confirmation that the molecular defect in the protein 4 .1R is the underlying cause o f anemic phenotype in mot fish. This study presents the zebrafish mutant merlot as the first characterized non-mammalian vertebrate model o f congenital anemia due to a defect in protein 4.1R integrity.
Developmental and Behavioral Analyses of clarin-2: A Novel Somatosensory Neuron Subtype-Enriched GeneThe trigeminal ganglion (TG) is a somatosensory organ that relays stimuli in the head to the hindbrain and spinal cord, and it comprises multiple subtypes of sensory neurons that respond to different somatosensory stimuli and establish distinct neuronal circuits. The Trpa1b subtype of TG sensory neurons (TGSNs) are responsible for sensing noxious chemicals, but the molecular cues that specify the development of this neuronal subtype remain poorly understood. Zebrafish were previously established as a robust model for studying the development of TGSNs due to its small size, translucency, and robust somatosensory behaviors. A previous microarray study in zebrafish found a novel four transmembrane-domain protein, clarin-2, to be enriched in Trpa1b-expressing cells. Nothing is known about the function of clarin-2, but a close homolog, clarin-1, is one of the causative genes for Usher Syndrome Type 3, a disorder characterized by progressive hearing and vision loss. We hypothesize that clarin-2 may play a role in the development and sensory function of TGSNs. To test this hypothesis, we examined the expression of clarin-2 within the TG during development and used clarin-2 knockout (KO) fish to study the genesis and neurite outgrowth of Trpa1b TGSNs. We found that clarin-2 is indeed enriched in a subset of TGSNs but is not required for the morphogenesis of the TG or the specification of nociceptive sensory neurons. Furthermore, axon projections from Trpa1b neurons were normal in clarin-2 KO fish, compared to control siblings. To test whether clarin-2 is required for the function of TGSNs, we tested somatosensory behaviors in larval zebrafish, including chemo-, thermo-, and mechanosensation. Behavioral analyses showed that clarin-2 is not required for the ability of Trpa1b neurons to detect the chemical irritant mustard oil. Additionally, the detection of heat or vibration was not affected in clarin-2 KO fish. Together, these results suggest that although clarin-2 is enriched in a subset of TGSNs, it is not required for the general morphogenesis of TGSNs or for somatosensation.
Functional analysis of a hematopoietic-specific gene identified using high-throughput tissue-specific cDNA screening in zebrafishZebrafish hematopoiesis begins in an intra-embryonic tissue called the intermediate cell mass (ICM), which specifically expresses the erythroid-specific transcription factor GATA-1. Using a cDNA library constructed from embryonic zebrafish GATA-1- positive cells, we identified a prothymosin alpha-like gene, ProTaL-1. RNA whole mount in situ hybridization showed that ProT~-1 was specifically expressed in the ICM. Microinjection of a construct encoding ProTa.L-1 fused to the green fluorescent protein revealed nuclear localization of ProTa.L-1. Oye~-expression of ProTaL-1 mRNA increased gata-1 expression, whereas knockdown ofProTaL-1 using an antisense, morpholino oligonucleotide resulted in a specific decrease of gata-1 expression in whole embryos. Fluorescence activated cell sorting analy~is of green fluorescent proteinlabeled GATA-I-positive cells from transgenic zebrafish indicated that ProTa.L-1 acted primarily to increase the total number· of ~mbryonic hematopoietic cells. s·ince prothymosin alpha is conserved among species, ProTa.L-1 may play a role in promoting , ' proliferation ofhematopoietic-ptogenitor cells in,higher vertebrates.