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Reverse genetics analysis of biological functions of zinc transporters Znt1 (Slc30a1) & Zip10 (Slc39a10) in zebrafish

Student thesis: Doctoral ThesisDoctor of Philosophy

Zinc is an essential micronutrient indispensable for all forms of life, and disturbances in its homeostasis predispose the body to zinc imbalance-related diseases in man and animals. Mechanisms exist that enable organisms to regulate this metal element, which include proteins that control zinc entry into the cell, chelation whilst in the cell, and also extrusion out of the cell. ZnT1 (SLC30A1) is a zinc exporter originally cloned in mammalian cells but its homologue has also been identified in piscine species. In the present study, we have used genetics and molecular approaches in a model vertebrate system (the zebrafish Danio rerio) to elucidate the nutritional and functional importance of zinc and its transporters in the regulation of some crucial biological processes in the body. The Znt1 mutant of zebrafish strain sa0014 was created by the Sanger Centre using TILLING technology. The mutant fish carries a premature stop codon in the znt1 (slc30a1) gene resulting in a protein that is forty (40) amino acids shorter than the wild- type. A colony of this strain was generated and the mutation studied for its effects on ability of embryos to regulate Zn2+ ions needed for biological processes, such as growth and development, gene expression, zinc acquisition and extrusion, and also its effects on extracellular-regulated kinases (ERK1/2). Investigations were also conducted on embryos with znt1 gene knockdown, through deployment of antisense morpholino- modified oligonucleotides (MO) in embryos of wild-type background, and in all of the studies it was shown that zebrafish embryos with Znt1 deficiencies display subtle but interesting phenotype, often having disturbances in zinc regulation and cell signalling when compared to their wild-type counterparts. Functional studies in adult fish revealed that znt1 mutation affects zinc absorption and transporter regulation as well as diurnal breeding pattern, which was plausibly linked to dysregulation of circadian rhythm- controlled genes and ERK signalling. In addition, investigation was also conducted on the function of Zip10 in embryos and it was found that the zip10 gene is essential in early development through its involvement in epithelial-mesenchymal transition (EMT) and cell migration. This gene was also shown to be important in hatching of zebrafish embryos through its involvement in the proper development and functioning of hatching gland tissue. Interestingly, knockdown of the zip10 gene by MO reduced the expression of hatching gland markers as well as the abundance of Zn2+ ions in the hatching gland cells (HGC) resulting in delayed hatching whereas znt1 knockdown produced the opposite effects. The relevance of all these studies in health and diseases of man and animals are discussed.
Original languageEnglish
Awarding Institution
Award date1 Dec 2013

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