TRANSCRIPTIONAL CONTROL OF HUMAN PANCREATIC ISLET DEVELOPMENT: DEFINING THE FUNCTIONS OF RFX6 AND RFX3 IN ENDOCRINE CELL SPECIFICATION AND MATURATION

Abstract

The development of human pancreatic islets is orchestrated by complex transcriptional programs that regulate endocrine cell specification, maturation, and functional competence. Members of the regulatory factor X (RFX) family, RFX6 and RFX3, are key transcription factors implicated in islet development, yet their specific roles in human pancreatic differentiation and diabetes pathogenesis remain poorly understood. Homozygous mutations in RFX6 cause neonatal diabetes with pancreatic hypoplasia, whereas heterozygous mutations are linked to MODY and type 2 diabetes, highlighting its clinical relevance. In contrast, the role of RFX3 in human pancreatic development has not been explored. In this study, we investigated the functions of RFX6 and RFX3 using human pluripotent stem cell-derived pancreatic islets, CRISPR/Cas9-mediated knockout lines, overexpression approaches, and multi-omics analyses including bulk and single-cell RNA sequencing. RFX6 expression was first detected in posterior foregut (PF) in PDX1+ cells, yet, in pancreatic progenitors (PPs), it did not co-localize with PDX1+/NKX6.1+ cells. Instead, RFX6 co-localised with NEUROG3, NKX2.2 and islet hormones in the endocrine progenitors (EPs) and islets. Single-cell analysis confirmed RFX6’s enrichment in endocrine clusters throughout differentiation. Loss of RFX6 led to downregulation of key genes involved in pancreatic endocrine differentiation, insulin secretion, and ion transport, increased apoptosis linked to reduced catalase (CAT) expression, and formation of smaller islet organoids. RFX6 overexpression rescued defective phenotypes in PPs, EPs, and islets. RFX3 was highly expressed across PPs, EPs, and islets. RFX3 knockout disrupted endocrine gene regulation, reduced hormone-secreting cell numbers, impaired β-cell function, and increased the abundance of enterochromaffin cells (ECs). Loss of RFX3 also caused smaller organoids, elevated thioredoxin-interacting protein (TXNIP) levels, and increased apoptosis. Overexpression of RFX3 rescued dysregulated gene expression at progenitor stages. Collectively, these findings demonstrate that RFX6 and RFX3 play complementary but distinct roles in human pancreatic islet development: RFX6 is critical for endocrine gene regulation and cell survival, whereas RFX3 regulates endocrine specification, suppresses EC fate, and promotes β-cell function. This work provides mechanistic insights into transcriptional control of human islet development and has implications for understanding diabetes pathogenesis and improving stem cell-based β-cell replacement strategies.

Date of Award	2026
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences