Study Of β-cell Regeneration Mechanism In Embryonic Zebrafish Pancreas

Maintenance of blood glucose levels are important to normal organism functions.They are regulated by the hormone producing pancreatic endocrine cells of the islets.The isletscompriseinsulin-expressingβ-cells,somatostatin-expressingδ-cells,glucagon-expressingα-cells and ghrelin-expressingε-cells.β-cell loss or failure causes diabetes,a health issue suffering by an increasing proportion of the world’s population.Type 1 diabetic patients can possibly be cured by restoration ofβ-cell mass.For treatment of insulin-dependent diabetes,islet transplant is proven to be a better option to insulin therapy for adult patients.Transplantation of islets requires at least a single donor.Thus transplant rejection and availability of islets are critical,calling for endogenous sources forβ-cells.Researches on normal endocrine pancreas development and regeneration ofβ-cell are providing more and more leads to efficient restoration of functionalβ-cells.Extensive researches have been published focusing onβ-cell regeneration.Animal models are established to study cellular origins and regulators ofβ-cell regeneration for the diabetes cure.Using these models,several endogenous sources likeα-cells andδ-cells have been revealed to contribute to restoration of the lostβ-cell mass.These studies showed endocrine cells maintained plasticity to some degree while rising from the common ancient progenitor and committing to different lineages.Comparing to the knowledge of several origins ofβ-cell neogenesis,little is known about the mechanism ofβ-cell regeneration.Here,we employed a zebrafishβ-cell ablation and regeneration model to investigateβ-cell neogenesis in the first few days after a near-totalβ-cell loss.Regeneration ofβ-cells first occurred within 7 hours post treatment.neurod,pdx1,mnx1and nkx2.2a are among the key regulators of zebrafishβ-cell development.In Tg(neuroD:GFP)and Tg(ins:CFP-NTR)double transgenic embryos,100%of the newβ-cells were GFP and CFP double positive,indicatingβ-cell regeneration happened within the neurod positive endocrine pool.By Tg(pdx1:GFP),Tg(mnx1:GFP)and Tg(nkx2.2a:GFP),we were able to illustrate pdx1,mnx1,nkx2.2a expression inβ-cell regeneration.Statistics showed only 89.2%of the newβ-cell are pdx1~+,54.11%were mnx1~+,while 56.67%were nkx2.2a~+.We used triple transgenic embryos with Tg(pdx1:GFP;mnx1:GFP;ins:CFP-NTR)andTg(pdx1:GFP;nkx2.2a:GFP;ins:CFP-NTR)to further observeβ-cell regeneration.Data turned out that all regeneratedβ-cell were GFP~+.However,in Tg(mnx1:GFP;nkx2.2a:GFP;ins:CFP-NTR)triple transgenic embryos,only 61.9%of the regeneratedβ-cells were GFP~+.Together,neurod,pdx1,mnx1and nkx2.2a all participated in zebrafishβ-cell regeneration.Differential expression pattern of these developmental regulators may suggest variant routes to theβ-cell regeneration.Using Cre/loxP-based lineage tracing,we showed that intra-pancreatic duct cells resisted to give rise to regeneratingβ-cell.Given that transdifferentiation ofα-cell andδ-cell can regenerateβ-cell,here we have provided further molecular evidences that the regeneratingβ-cells originate from multiple cellular origins.