A developmental lineage-based gene co-expression network for mouse pancreatic ß-cells reveals a role for Zfp800 in pancreas development.

To gain a deeper understanding of pancreatic ß-cell development, we used iterative weighted gene correlation network analysis to calculate a gene co-expression network (GCN) from 11 temporally and genetically defined murine cell populations. The GCN, which contained 91 distinct modules, was then used to gain three new biological insights. First, we found that the clustered protocadherin genes are differentially expressed during pancreas development. Pcdhγ genes are preferentially expressed in pancreatic endoderm, Pcdhß genes in nascent islets, and Pcdhα genes in mature ß-cells. Second, after extracting sub-networks of transcriptional regulators for each developmental stage, we identified 81 zinc finger protein (ZFP) genes that are preferentially expressed during endocrine specification and ß-cell maturation. Third, we used the GCN to select three ZFPs for further analysis by CRISPR mutagenesis of mice. Zfp800 null mice exhibited early postnatal lethality, and at E18.5 their pancreata exhibited a reduced number of pancreatic endocrine cells, alterations in exocrine cell morphology, and marked changes in expression of genes involved in protein translation, hormone secretion and developmental pathways in the pancreas. Together, our results suggest that developmentally oriented GCNs have utility for gaining new insights into gene regulation during organogenesis.

Chronic ß-Cell Depolarization Impairs ß-Cell Identity by Disrupting a Network of Ca2+-Regulated Genes.

We used mice lacking Abcc8, a key component of the ß-cell KATP-channel, to analyze the effects of a sustained elevation in the intracellular Ca2+ concentration ([Ca2+]i) on ß-cell identity and gene expression. Lineage tracing analysis revealed the conversion of ß-cells lacking Abcc8 into pancreatic polypeptide cells but not to α- or δ-cells. RNA-sequencing analysis of FACS-purified Abcc8-/- ß-cells confirmed an increase in Ppy gene expression and revealed altered expression of more than 4,200 genes, many of which are involved in Ca2+ signaling, the maintenance of ß-cell identity, and cell adhesion. The expression of S100a6 and S100a4, two highly upregulated genes, is closely correlated with membrane depolarization, suggesting their use as markers for an increase in [Ca2+]i Moreover, a bioinformatics analysis predicts that many of the dysregulated genes are regulated by common transcription factors, one of which, Ascl1, was confirmed to be directly controlled by Ca2+ influx in ß-cells. Interestingly, among the upregulated genes is Aldh1a3, a putative marker of ß-cell dedifferentiation, and other genes associated with ß-cell failure. Taken together, our results suggest that chronically elevated ß-cell [Ca2+]i in Abcc8-/- islets contributes to the alteration of ß-cell identity, islet cell numbers and morphology, and gene expression by disrupting a network of Ca2+-regulated genes.

Pancreatic Inflammation Redirects Acinar to ß Cell Reprogramming.

Using a transgenic mouse model to express MafA, Pdx1, and Neurog3 (3TF) in a pancreatic acinar cell- and doxycycline-dependent manner, we discovered that the outcome of transcription factor-mediated acinar to ß-like cellular reprogramming is dependent on both the magnitude of 3TF expression and on reprogramming-induced inflammation. Overly robust 3TF expression causes acinar cell necrosis, resulting in marked inflammation and acinar-to-ductal metaplasia. Generation of new ß-like cells requires limiting reprogramming-induced inflammation, either by reducing 3TF expression or by eliminating macrophages. The new ß-like cells were able to reverse streptozotocin-induced diabetes 6 days after inducing 3TF expression but failed to sustain their function after removal of the reprogramming factors.