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The differentiation of stem cells into insulin-secreting cells in vitro provides a promising cell replacement therapy for diabetes.Despite numerous trail-and-error attempts,the glucose-responsive,insulin-secreting cells generated in vitro are not mature cells and must be transplanted into living mice to gain full competence.Because mammalian pancreatic islets are embedded within the non-transparent pancreas and are difficult to visualize,little is known about the mechanisms of the final maturation of cells in vivo.Here,we have generated a transgenic zebrafish line,ins:Rcamp 1.07,in which the function of every cell in an embryo can be resolved in vivo by a novel two-photon light-sheet microscope.Based on the kinetics of glucose-stimulated Ca2+responses and the expression of MafA,we revealed the sequential maturation of two spatially distinct pools of cells during the hatching period(48-72 hours post-fertilization,hpf).From 48 hpf to 56 hpf,locally synthesized glucose(~3 mM)initiates glucose responsiveness exclusively in the cells in the mantle of the islet.After 56 hpf,islet vascularization enables the efficient delivery of glucose to the islet core to allow internal cells to gain function.The increased supply of glucose to cells via circulation(~8 mM)also drives all glucose-responsive cells towards full maturity.We further demonstrate that the calcineurin/NFAT signalling pathway is the master regulator downstream of glucose,and the activation of this pathway fully rescues the defects of MafA expression and cell function caused by endogenous glucose deprivation.Given the conserved cell development between zebrafish and mammalian species,our findings may shed light on how to improve the functional competence of cells generated from stem cells in vitro.