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Aims/hypothesis: Homozygous mutations in RFX6 lead to neonatal diabetes accompanied by a hypoplastic pancreas, whereas heterozygous mutations cause MODY. Recent studies have also shown RFX6 variants to be linked 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., Methods: We examined regulatory factor X6 (RFX6) expression during human embryonic stem cell (hESC) differentiation into pancreatic islets and re-analysed a single-cell RNA-seq dataset to identify RFX6-specific cell populations during islet development. Furthermore, induced pluripotent stem cell (iPSC) lines lacking RFX6 were generated using CRISPR/Cas9. Various approaches were then employed to explore the consequences of RFX6 loss across different developmental stages. Subsequently, we evaluated transcriptional changes resulting from RFX6 loss through RNA-seq of pancreatic progenitors (PPs) and endocrine progenitors (EPs)., Results: RFX6 expression was detected in PDX1 + cells in the hESC-derived posterior foregut (PF). However, in the PPs, RFX6 did not co-localise with pancreatic and duodenal homeobox 1 (PDX1) or NK homeobox 1 (NKX6.1) but instead co-localised with neurogenin 3, NK2 homeobox 2 and islet hormones in the EPs and islets. Single-cell analysis revealed high RFX6 expression levels in endocrine clusters across various hESC-derived pancreatic differentiation stages. Upon differentiating iPSCs lacking RFX6 into pancreatic islets, a significant decrease in PDX1 expression at the PF stage was observed, although this did not affect PPs co-expressing PDX1 and NKX6.1. RNA-seq analysis showed the downregulation of essential genes involved in pancreatic endocrine differentiation, insulin secretion and ion transport due to RFX6 deficiency. Furthermore, RFX6 deficiency resulted in the formation of smaller islet organoids due to increased cellular apoptosis, linked to reduced catalase expression, implying a protective role for RFX6. Overexpression of RFX6 reversed defective phenotypes in RFX6-knockout PPs, EPs and islets., Conclusions/interpretation: These findings suggest that pancreatic hypoplasia and reduced islet cell formation associated with RFX6 mutations are not due to alterations in PDX1 + /NKX6.1 + PPs but instead result from cellular apoptosis and downregulation of pancreatic endocrine genes., Data Availability: RNA-seq datasets have been deposited in the Zenodo repository with accession link (DOI: https://doi.org/10.5281/zenodo.10656891 )., Competing Interests: Acknowledgements: We would like to thank N. R. Dunn (A*STAR, Singapore) for providing the HA-RFX6 tagged H9 hESC lines (RFX6HA/HA H9-hESCs). Furthermore, we thank the Genomic Core members at QBRI for their assistance with technical support in RNA-seq. Data availability: RNA-seq datasets have been deposited in the Zenodo repository with accession link (DOI: https://doi.org/10.5281/zenodo.10656891 ). Funding: Open Access funding provided by the Qatar National Library. This work was funded by grants from Qatar Biomedical Research Institute (QBRI) (Grant no. QBRI-HSCI Project 1). NA is a PhD student with a scholarship funded by QRDI (GSRA9-L-1-0511-22008). Authors’ relationships and activities: SH is a co-founder and shareholder of Sequantrix GmbH and has research funding from Novo Nordisk and Askbio. The authors declare that there are no other relationships or activities that might bias, or be perceived to bias, their work. Contribution statement: NA performed most of the experiments and analysed the data. AKE and BM performed experiments and analysed the data. SI and SH analysed the sequencing data. EMA conceived and designed the study, supervised the project, analysed and interpreted the data, and wrote the manuscript. All authors critically reviewed the article and approved the final version of the manuscript. EMA is the guarantor of this work., (© 2024. The Author(s).)

The pathogenesis of diabetes involves complex changes in the expression profiles of mRNA and non-coding RNAs within pancreatic islet cells. Recent progress in induced pluripotent stem cell (iPSC) technology have allowed the modeling of diabetes-associated genes. Our recent study using FOXA2-deficient human iPSC models has highlighted an essential role for FOXA2 in the development of human pancreas. Here, we aimed to provide further insights on the role of microRNAs (miRNAs) by studying the miRNA-mRNA regulatory networks in iPSC-derived islets lacking the FOXA2 gene. Consistent with our previous findings, the absence of FOXA2 significantly downregulated the expression of islet hormones, INS, and GCG, alongside other key developmental genes in pancreatic islets. Concordantly, RNA-Seq analysis showed significant downregulation of genes related to pancreatic development and upregulation of genes associated with nervous system development and lipid metabolic pathways. Furthermore, the absence of FOXA2 in iPSC-derived pancreatic islets resulted in significant alterations in miRNA expression, with 61 miRNAs upregulated and 99 downregulated. The upregulated miRNAs targeted crucial genes involved in diabetes and pancreatic islet cell development. In contrary, the absence of FOXA2 in islets showed a network of downregulated miRNAs targeting genes related to nervous system development and lipid metabolism. These findings highlight the impact of FOXA2 absence on pancreatic islet development and suggesting intricate miRNA-mRNA regulatory networks affecting pancreatic islet cell development., (© 2024. The Author(s).)

Recently, we reported that forkhead box A2 (FOXA2) is required for the development of human pancreatic α- and β-cells. However, whether miRNAs play a role in regulating pancreatic genes during pancreatic development in the absence of FOXA2 expression is largely unknown. Here, we aimed to capture the dysregulated miRNAs and to identify their pancreatic-specific gene targets in pancreatic progenitors (PPs) derived from wild-type induced pluripotent stem cells (WT-iPSCs) and from iPSCs lacking FOXA2 (FOXA2 -/- iPSCs). To identify differentially expressed miRNAs (DEmiRs), and genes (DEGs), two different FOXA2 -/- iPSC lines were differentiated into PPs. FOXA2 -/- PPs showed a significant reduction in the expression of the main PP transcription factors (TFs) in comparison to WT-PPs. RNA sequencing analysis demonstrated significant reduction in the mRNA expression of genes involved in the development and function of exocrine and endocrine pancreas. Furthermore, miRNA profiling identified 107 downregulated and 111 upregulated DEmiRs in FOXA2 -/- PPs compared to WT-PPs. Target prediction analysis between DEmiRs and DEGs identified 92 upregulated miRNAs, predicted to target 1498 downregulated genes in FOXA2 -/- PPs. Several important pancreatic TFs essential for pancreatic development were targeted by multiple DEmiRs. Selected DEmiRs and DEGs were further validated using RT-qPCR. Our findings revealed that FOXA2 expression is crucial for pancreatic development through regulating the expression of pancreatic endocrine and exocrine genes targeted by a set of miRNAs at the pancreatic progenitor stage. These data provide novel insights of the effect of FOXA2 deficiency on miRNA-mRNA regulatory networks controlling pancreatic development and differentiation., (© 2023. The Author(s).)