Investigation of zebrafish tetraspanin cd63

• Main Author: Trikić, Michael Zivojin
• Published: University of Sheffield 2009
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505348

Tetraspanin CD63 is highly conserved, found in a diverse range of species and has been implicated in a multitude of biological phenomena. Although its precise function is unknown, evidence suggests it has a role in intracellular trafficking. A homolog of CD63 is present in zebrafish of which little is known. The aim of this work was to investigate zebrafish cd63. This thesis first describes validation of zebrafish as a model for the study of CD63, and second investigates cd63 function in this system. Zebrafish cd63 was found to be 45% identical to human CD63 with conserved lysosomal targeting motif and glycosylation sites. To further investigate the characteristics of the cd63 protein a CD63-GFP fusion protein was made and introduced into tissue culture lines as well as live embryos. As predicted from its sequence the construct had an intracellular localisation and appears to be glycosylated. Using In Situ Hybridisation (ISH) and RT-PCR first zygotic expression was identified in pre-polster, later to be found in the notochord and the hatching gland. After validation morpholinos were used to knock down cd63 causing morphological disruption and defective hatching. Assay for release of hatching enzymes revealed that morphant embryos had reduced ability to cleave substrate leading to the working hypothesis that enzyme production was faulty, release defective or a combination of these factors. 'ISH revealed that cd63 was absent in one-eyed pinhead mutants revealing that cd63 is Nodaliy regulated. Study of established markers in morphant embryos provided evidence that cd63 was not involved in specification of the hatching gland, but is required for correct function. A bioinformatic approach was used to identify potential partners of cd63 andúan . experimental methodology proposed for the investigation of these molecules. This was tested for tetraspanin cd82 showing that it has similar spatial and temporal expression to cd63.