Benato, Francesca (2008) Analisi in vivo dello sviluppo del pancreas di zebrafish. [Tesi di dottorato]
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The vertebrate pancreas is an endoderm-derived organ composed by an endocrine portion with cells secreting hormones, such as insulin, glucagon somatostatin, pancreatic polipeptide and ghrelin in the blood flow, and an exocrine one that releases digestive enzimes in the gut lumen.
One of the first decisions in the pancreatic differentiation is between endocrine and exocrine cell fates. The transcription factor ptf1a is an essential gene involved in the exocrine differentiation. In these years zebrafish has been widely employed as a model to study genes involved in endoderm and endoderm-derived organs specification and differentiation. This study is possible because genes and molecular mechanism implicated in embryonic development are highly conserved between this animal model and higher vertebrates. Despite several works done in the last years, the knowledge of molecular mechanisms underlying pancreatic development and differentiation in zebrafish is incomplete and many factors involved in this process are still unknown.
During these three years I produced a zebrafish transgenic line in which the GFP is directed in the exocrine pancreas by 5,5 kbp of ptf1a upstream regulatory region. This work gave us a new useful "tool" to clarify pancreatic organogenesis and to understand how ptf1a itself is regulated. The same ptf1a promotorial region was cloned upstream the DsRed gene with a new system called Tol2 that is able to increase the transgene integration rate in the zebrafish germinal cells genome. This strategy allowed us to obtain a transgenic zebrafish line that can be crossed to many transgenic lines where GFP is under the control of different tissue-specific promoters.
In order to determine what is the minimum ptf1a promoter region necessary to drive GFP expression in the zebrafish exocrine pancreas, 2,6 kbp of that region were also cloned. In this case I observed a ubiquitous GFP expression. With the aim to better clarify the genetics of pancreatic development in zebrafish I undertook a large-scale genetic screen, which took place at the Max Planck Institute in Tuebingen (Germany), using zebrafish larvae obtained from lines mutagenized with the chimical ENU. During that period I screened for the presence of defects in the insulin expression, detected by in situ hybridization; more than 1200 families were analyzed. One of the mutants identified, in which insulin was absent, was characterized and mapped, in collaboration with Prof. Dirk Meyer. Candidate gene analysis detected a new mutation in the raldh2 (ald1a2) gene, causing a phenotipe similar to the zebrafish mutant neckless. During that period, the astrocytes marker gfap was also used, to detect astroglial cell defects in the screened embryos. For two out of nine mutant families with defects in gfap expression I determined, in collaboration with Prof. Robert Geisler, the linkage group bearing the mutation responsible of the observed phenotype.
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