PAX6 maintains β cell identity by repressing genes of alternative islet cell types
Avital Swisa, … , Ruth Ashery-Padan, Yuval Dor
Avital Swisa, … , Ruth Ashery-Padan, Yuval Dor
Published January 3, 2017; First published December 12, 2016
Citation Information: J Clin Invest. 2017;127(1):230-243. https://doi.org/10.1172/JCI88015.
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Research Article Endocrinology Genetics

PAX6 maintains β cell identity by repressing genes of alternative islet cell types

Abstract

Type 2 diabetes is thought to involve a compromised β cell differentiation state, but the mechanisms underlying this dysfunction remain unclear. Here, we report a key role for the TF PAX6 in the maintenance of adult β cell identity and function. PAX6 was downregulated in β cells of diabetic db/db mice and in WT mice treated with an insulin receptor antagonist, revealing metabolic control of expression. Deletion of Pax6 in β cells of adult mice led to lethal hyperglycemia and ketosis that were attributed to loss of β cell function and expansion of α cells. Lineage-tracing, transcriptome, and chromatin analyses showed that PAX6 is a direct activator of β cell genes, thus maintaining mature β cell function and identity. In parallel, we found that PAX6 binds promoters and enhancers to repress alternative islet cell genes including ghrelin, glucagon, and somatostatin. Chromatin analysis and shRNA-mediated gene suppression experiments indicated a similar function of PAX6 in human β cells. We conclude that reduced expression of PAX6 in metabolically stressed β cells may contribute to β cell failure and α cell dysfunction in diabetes.

Authors

Avital Swisa, Dana Avrahami, Noa Eden, Jia Zhang, Eseye Feleke, Tehila Dahan, Yamit Cohen-Tayar, Miri Stolovich-Rain, Klaus H. Kaestner, Benjamin Glaser, Ruth Ashery-Padan, Yuval Dor

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Figure 3

Lineage tracing of Pax6-deleted β cells.

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Lineage tracing of Pax6-deleted β cells. (A) Loss of insulin in Pax6-del...
(A) Loss of insulin in Pax6-deleted β cells, labeled with YFP, after tamoxifen injection. Immunostaining for YFP (green), insulin (red), and DNA (blue). Note that most remaining insulin+ cells at 6 months were YFP– (i.e., had probably escaped deletion of Pax6). Mice were injected with tamoxifen at 2 months of age and sacrificed 1 week or 6 months later as indicated. Original magnification, ×400. (B) Quantification of YFP area versus total islet area in Pax6fl/fl mice (No Cre), MIP-CreER Pax6fl/fl Rosa26-YFP mice without tamoxifen (No TM), and MIP-CreER Pax6fl/fl Rosa26-YFP mice 1 week after tamoxifen injection (βPAX6). n = 3 mice in each group. (C) FACS analysis of sorted YFP+ β cells 3 weeks after tamoxifen injection. βPAX6 cells show a low side scatter (SSC) signal, probably indicating degranulation of β cells. The control sample was from a Pax6fl/fl (flox/flox) mouse. Analysis was performed on 4 samples of each genotype. FSC-A, forward scatter. (D) Pancreatic sections stained for chromogranin A (CHGA, red), YFP (green), and insulin (blue). Chromogranin was retained following long-term deletion of Pax6, suggesting retention of islet cell identity. Original magnification, ×400. (E) Representative electron micrographs of pancreatic sections from a βPAX6 mouse at the age of 3 months, 2 months after tamoxifen injection. Top left: normal insulin granules in WT islet (green arrow); top right: atypical cell type with dark granules (red arrows); bottom left: another atypical cell (red arrows); bottom right: β cell with empty granules (yellow arrow). Samples from 3 flox/flox and 3 βPAX6 mice were imaged by electron microscopy. Original magnification, ×3,000.