Imaging Maturation in Single Adult β-Cells using Fluorescent Reporters for Ins1, Pdx1, MafA and MafB Promoter Activities

Abstract

The functional maturation and dedifferentiation of β-cells is central to diabetes pathogenesis and to β-cell replacement therapy. ‘Snapshots’ of adult β-cells show variable levels of insulin promoter activity, insulin protein and insulin secreting function. We have shown, using a dual fluorescent Pdx1/Ins1 reporter, that this apparent heterogeneity in part reflects a dynamic process of maturation (i.e. gaining insulin promoter activity over time). Gene expression profiling revealed that immature β-cells had increased expression of multiple islet hormones and genes involved in development, proliferation and apoptosis, including MafB. Mature β-cells were enriched in genes that maintain the differentiated β-cell phenotype, including MafA. A follow-up study demonstrated novel roles for Musashi in the control of insulin expression and β-cell proliferation. A factorial screen uncovered that activin A and its antagonist follistatin reciprocally control maturity of adult β-cells. Here, we report the development of new tools that allow for simultaneous analysis of MafA and MafB promoter activities, using fluorescent proteins targeted to the plasma membrane. We found that MafA/MafB promoter-reporters showed differential activity between single β-cells and quantified the dynamics of these gene expression transitions. Using these tools, we have developed ultra-high-content screening platforms to further decode the level of maturity of single adult β-cells in various culture conditions and health/diabetic states. Collectively, these experiments contribute to the understanding of heterogeneity, maturation and putative plasticity of adult pancreatic β-cells. Efforts to harness these immature cells and manipulate their maturation might be one avenue for replacing or repopulating lost/nonfunctional β-cells in the diabetic pancreas.