Student Hamad Bin Khalifa University, Ad Dawhah, Qatar
Abstract: Homozygous mutations in RFX6 lead to neonatal diabetes accompanied by a hypoplastic pancreas, whereas heterozygous mutations cause maturity-onset diabetes of the young (MODY). Recent studies have shown RFX6 variants linkage with type 2 diabetes. Despite RFX6's known function in islet development, its specific role in diabetes pathogenesis remains unclear. Here, we aimed to understand the mechanisms underlying the impairment of pancreatic islet development and subsequent hypoplasia due to loss-of-function mutations in RFX6. We examined RFX6 expression during hESC differentiation into pancreatic islets and analyzed a single-cell RNA-sequencing dataset to identify RFX6-specific cell populations during islet development. Human iPSC lines lacking RFX6 were generated using CRISPR/Cas9 where various approaches used to study the impact during development. Our immunostaining analysis revealed robust RFX6 expression in PDX1+ cells during posterior foregut (PF) stage. However, at pancreatic progenitor (PP) stage, RFX6 did not exhibit co-expression with PDX1 and NKX6.1, whereas it co-localized with NEUROG3 and NKX2.2 during endocrine progenitor (EP) stage. Single-cell analysis demonstrated elevated RFX6 expression in endocrine clusters across various developmental stages. The absence of RFX6 led to a significant decrease in PDX1 expression at the PF stage but did not impact PPs co-expressing PDX1 and NKX6.1. RNA sequencing unveiled downregulation of crucial genes for pancreatic endocrine differentiation, insulin secretion, and ion transport due to RFX6 deficiency. Moreover, RFX6 deficiency led to smaller islet organoids formation via increased cellular apoptosis, attributed to reduced Catalase (CAT) expression, suggesting a protective role for RFX6 in pancreatic islets. Overexpressing RFX6 reversed defective phenotypes in PPs, EPs, and islets These results indicate that the pancreatic hypoplasia and reduced islet cell formation linked to RFX6 mutations is not due to alterations in PDX1+/NKX6.1+ PPs. Instead, they result from cellular apoptosis, decreased CAT expression, and a downregulation in the expression of pancreatic endocrine genes. These insights may pave the way for potential therapeutic strategies, such as enhancing catalase levels, to mitigate diabetes related to RFX6 defects.
Funding Source: This work was funded by grants from Qatar Biomedical Research Institute (QBRI) (Grant No. QBRI-HSCI Project 1). The first author, Noura Aldous, is a PhD student with a scholarship funded from QRDI (GSRA9-L-1-0511-22008).
