Cre/loxP-Based Enhancer Trap Improvement For The Organogenesis And Regeneration Of Liver And Pancreatic ? Cell

Liver and pancreatic ? cell are two major internal organs that originate from the endodermal germ layer.The liver serves a broad range of metabolic functions,including glucose homeostasis;lipid homeostasis and ketone bodies production;metabolism of amino acids;detoxification of a variety of different compounds and maintenance of physiological metabolite and protein concentrations in the blood.Most of the liver functions are carried out by the hepatocytes(about 70-75% of hepatic cells),which together with cholangiocytes(10-15 % of hepatic cells)constitute the hepatic parenchyma.The pancreatic beta cell consists of two major functional entities that exert exocrine and endocrine activities,respectively.The exocrine cells produce and secrete various digestive enzymes and they constitute more than 95 % of the total pancreatic cell mass.The four different endocrine cell types are each responsible for the production of one pancreatic hormone: glucagon is made in the ?-cells,insulin in the ?-cells,somatostatin in the ?-cells and pancreatic polypeptide in the PP-cells.Because the liver is essential for viability and livers suitable for transplantation are in short supply,there is intense interest in generating hepatocytes from progenitor cells in the liver as well as from exogenous sources(e.g.,transdifferentiation from other tissues or de novo differentiation from embryonic stem cells [ESCs]).Understanding the factors required for regeneration of pancreatic endocrine cells,especially the insulin-producing ? cell,the only cell type in the body that is responsible for down-regulation of glucose homeostasis will help development therapeutic strategies for millions of diabetics.Liver and pancreatic ? cell from zebrafish and mammals show striking similarities in the molecular control of development,share cellular and subcellular architecture,and have a conserved physiological function.All of these criteria validate zebrafish as a relevant model for studying basic mechanisms of organogenesis and regeneration of liver and pancreatic ? cell.Both adult liver and pancreatic ? cell tissues have the capacity to regenerate during a lifetime or after injury by proliferation of remaining cells,differentiation from multipotent precursor populations,or conversion from another mature cell type.From studies in rodents,there is evidence for all of these mechanisms contributing to homeostasis and response to injury in the liver and pancreatic beta cell,depending on the experimental manipulation and method of analysis.However,how many kinds of precursors that have the potential to contribute to homeostasis and regeneration,what is the nature of these precursors and which factors rule the conversion process remain largely unknown.Search the precursors that contribute to the homeostasis and regeneration and the critical genes that control these processes is an intriguing field of great medical significance.Currently there is no documented strategies that directly visualize the unknown specific genes enriched in pancreatic ? cell and liver.In this study we take advantages of the transgenic zebrafish that express fluorescent protein in liver and pancreatic beta cell,combined with enhancer trap,to screen the genes that play critical roles in pancreatic ? cell and liver physiological functions during development and regeneration.We constructed two double transgenic line,Tg(fabp10:loxP-CFPNTR-loxPDsRed;10×UAS:Cre,cryaa:Venus)and Tg(ins:loxP-CFPNTR-loxP-DsRed;10×UAS: Cre,cryaa:Venus)to mark hepatocyte and pancreatic ? cell.Nitroreductase(NTR)fused with the fluorescent protein CFP is used to catalyze the reduction of the innocuous prodrug metrodinazole(Mtz),thereby producing a cytotoxic product that induces death of the cell containing this transgene.We found both the hepatocytes and pancreatic ? cells were completely ablated under the treatment of 10 mM MtZ,and both show striking recovery at 48 hours respectively after withdraw of the MtZ.By injecting a number of one-cell stage embryos with GAL4 containing transposable Tol2 construct,we creat about 1000 founders carrying enhacer trap.In double transgenetic lines Tg(fabp10:loxP-CFPNTR-loxP-DsRed;10×UAS: Cre,cryaa: Venus)and Tg(ins:loxPCFPNTR-loxP-DsRed;10×UAS:Cre,cryaa:Venus),only under the drive of transcription factor GAL4,can the UAS initiate the expression of Cre.We cross those enhancer trap founders with double transgenetic lines Tg(fabp10:loxP-CFPNTR-loxP-DsRed;10× UAS:Cre,cryaa:Venus)and Tg(ins:loxP-CFPNTR-loxP-DsRed;10× UAS: Cre,cryaa: Venus)respectively,and observe the color shift from blue to red and select the lines with potential liver and pancreatic ? cell-specific genomic insertion.From over 1000 screens,we obtain some lines with red fluorescence marked hepatocytes or beta cells,and their genomic DNA was submitted to reverse PCR for position cloning.All the candidate genes in the vicinity of the reporter insertion was confirmed by in situ hybridization as to whether they are expressed in hepatocyte or not.Meanwhile,taking advantage of these transgenic design,which involves the conversion of the prodrug Mtz into a cytotoxic DNA cross-linking agent under the bacterial nitroreductase(NTR)enzyme,we managed to achieve the targeted ablation of hepatocyte and pancreatic beta cells and these lines was also screened by color change during regeneration.Based on this Cre/loxP based enhancer trap,we identified some tissuespecific genes expressing in liver and ? cell during development as well as regeneration.Of note,pilot enhancer traps captured transiently and weakly expressed genes such as rab3 da and ensab with higher efficiencies than traditional enhancer trap systems.In conclusion,through permanent genetic labeling by Cre/loxP,this improved Tol2-mediated enhancer trap system provides a promising method to identify transiently or weakly expressed,but potentially important,genes during development and regeneration.