No abstract

Developing systems to identify the cell type-specific functions regulated by genes linked to type 2 diabetes (T2D) risk could transform our understanding of the genetic basis of this disease. However, in vivo systems for efficiently discovering T2D risk gene functions relevant to human cells are currently lacking. Here we describe powerful interdisciplinary approaches combining Drosophila genetics and physiology with human islet biology to address this fundamental gap in diabetes research. We identify Drosophila orthologs of T2D-risk genes that regulate insulin output. With human islets, we perform genetic studies and identify cognate human T2D-risk genes that regulate human beta cell function. Loss of BCL11A, a transcriptional regulator, in primary human islet cells leads to enhanced insulin secretion. Gene expression profiling reveals BCL11A-dependent regulation of multiple genes involved in insulin exocytosis. Thus, genetic and physiological systems described here advance the capacity to identify cell-specific T2D risk gene functions.

Gene targeting studies in primary human islets could advance our understanding of mechanisms driving diabetes pathogenesis. Here, we demonstrate successful genome editing in primary human islets using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). CRISPR-based targeting efficiently mutated protein-coding exons, resulting in acute loss of islet β-cell regulators, like the transcription factor PDX1 and the KATPchannel subunit KIR6.2, accompanied by impaired β-cell regulation and function. CRISPR targeting of non-coding DNA harboring type 2 diabetes (T2D) risk variants revealed changes inABCC8,SIX2andSIX3expression, and impaired β-cell function, thereby linking regulatory elements in these target genes to T2D genetic susceptibility. Advances here establish a paradigm for genetic studies in human islet cells, and reveal regulatory and genetic mechanisms linking non-coding variants to human diabetes risk.

The physiological functions of many vital tissues and organs continue to mature after birth, but the genetic mechanisms governing this postnatal maturation remain an unsolved mystery. Human pancreatic β cells produce and secrete insulin in response to physiological cues like glucose, and these hallmark functions improve in the years after birth. This coincides with expression of the transcription factors SIX2 and SIX3, whose functions in native human β cells remain unknown. Here, we show that shRNA-mediated SIX2 or SIX3 suppression in human pancreatic adult islets impairs insulin secretion. However, transcriptome studies revealed that SIX2 and SIX3 regulate distinct targets. Loss of SIX2 markedly impaired expression of genes governing β-cell insulin processing and output, glucose sensing, and electrophysiology, while SIX3 loss led to inappropriate expression of genes normally expressed in fetal β cells, adult α cells, and other non-β cells. Chromatin accessibility studies identified genes directly regulated by SIX2. Moreover, β cells from diabetic humans with impaired insulin secretion also had reduced SIX2 transcript levels. Revealing how SIX2 and SIX3 govern functional maturation and maintain developmental fate in native human β cells should advance β-cell replacement and other therapeutic strategies for diabetes.

Reliance on rodents for understanding pancreatic genetics, developmental biology and islet function could limit progress in developing interventions for human diseases like diabetes mellitus.Similarities of pancreas morphology and function, and pancreatic disease modeling in pigs suggest that porcine and human pancreas developmental biology may have useful homologies.However, little is known about pig pancreas development. To fill this knowledge gap, we investigated fetal and neonatal pig pancreas at multiple, crucial developmental stages using modern experimental approaches. Purification of islet β-, α-and d-cells via flow cytometry followed by high-throughput transcriptome analysis (RNA-Seq) provided comprehensive gene expression profiles. Morphometric analysis revealed dynamic developmental endocrine cell allocation and islet architectural similarities between pig and human islets. Gene expression analysis identified cell-and stage-specific expression in sorted pig β-and α-cells, and revealed that pig and human β-cells and α-cells shared characteristic molecular and developmental features not observed in mouse β-cells or α-cells. Over 150 causal or candidate 'diabetes risk' genes identified by human studies had dynamic developmental expression in pig β-and α-cells. Our analysis also unveiled scores of signaling pathways linked to native islet β-cell functional maturation. Thus, the findings, conceptual advances, and resources detailed here show how pig pancreatic islet studies complement or surpass other systems for understanding the developmental programs that generate functional β-and a-cells, and that are relevant to human diseases like diabetes mellitus.