Pancreatic agenesis and altered m6A methylation in the pancreas of PDX1-mutant cynomolgus macaques

Abstract

As an essential transcriptional activator, PDX1 plays a crucial role in pancreatic development and β-cell function. Mutations in the PDX1 gene may lead to type 4 maturity-onset diabetes of the young (MODY4) and neonatal diabetes mellitus. However, the precise mechanisms underlying MODY4 remain elusive due to the paucity of clinical samples and pronounced differences in pancreatic architecture and genomic composition between humans and existing animal models. In this study, three PDX1-mutant cynomolgus macaques were generated using CRISPR/Cas9 technology, all of which succumbed shortly postpartum, exhibiting pancreatic agenesis. Notably, one tri-allelic PDX1-mutant cynomolgus macaque (designated as M4) developed a pancreas, whereas the two mono-allelic PDX1-mutant cynomolgus macaques displayed no anatomical evidence of pancreatic formation. RNA sequencing of the M4 pancreas revealed substantial molecular changes in both endocrine and exocrine functions, indicating developmental delay and PDX1 haploinsufficiency. A marked change in m6A methylation was identified in the M4 pancreas, confirmed through cultured PDX1-mutant islet organoids. Notably, overexpression of the m6A modulator METTL3 restored function in heterozygous PDX1-mutant islet organoids. This study highlights a novel role of m6A methylation modification in the progression of MODY4 and provides valuable molecular insights for preclinical research.

Keywords: Cynomolgus macaques; M6A methylation modification; MODY4; PDX1.

Figure 1

PDX1-mutant cynomolgus macaques exhibited pancreatic agenesis A: Schematic of PDX1-sgRNAs in cynomolgus monkeys. Blue line: Transactivation domain region; Red arrow: sgRNA. B: Summary of embryo transplantation and PDX1-mutant fetuses in cynomolgus monkeys. C, D: Macroscopic appearance of viscus and pancreas in PDX1-mutant newborns M1, M2 (C), M4, and WT monkeys (D) after delivery. E: H&E-stained sections of M4 and WT pancreas. Blue rectangle: Pancreatic islet; Black rectangle: Pancreatic duct; Red arrow: Inflammatory cell infiltration. F: Representative immunofluorescence images of PDX1, INSULIN, and GLUCAGON staining of WT and M4 pancreas. G: PDX1 protein sequences of M4 monkey with 6 bp deletion of PDX1-mutant alleles indicating loss of two amino acids (in red) compared with WT monkeys.

Figure 2

RNA sequencing analysis of pancreatic tissues in WT and M4 monkeys A: Volcano plot of most significant up-regulated and down-regulated genes in M4 pancreas compared to WT pancreas. Data points in red, blue, and black represent up-regulated, down-regulated, and not significantly differentiated gene expression, respectively. B: Genome (KEGG) pathway enrichment analysis of common DEGs. C: Heatmap of DEGs relative to Alpha, Beta, Delta, and PP cells in pancreatic islets between WT and M4 monkeys. D: Bar plots of representative genes involved in specific β-cell differentiation. E: Heatmap of expression levels of genes related to ductal cells. F: Heatmap of expression levels of genes related to acinar cells. G: Bar plots of PDX1, NEUROG3, and MAFA expression in WT and M4 pancreas. H: Schematic of dynamic changes in PDX1, NEUROG3, and MAFA expression in developing embryos of mice and humans ( Zhu et al., 2017). Development stage of M4 pancreas (red arrow) before W6 compared to humans. E1–E13: Mouse embryonic days 1 to 13, P1–P7: Mouse postnatal days 1 to 7, W0–W12: Gestational weeks 1 to 12.

Figure 3

PDX1 haploinsufficiency in PDX1-mutant islet organoids and cynomolgus macaques A: RT-qPCR analysis of endocrine markers in WT and M4 pancreas. B: RT-qPCR analysis of Nanog, NKX6.1, INS, and PDX1 expression on days 11 and 30 during hiPSC-derived β-cell differentiation ( n=3). C: Bright field of WT, PDX1 ^+/- , and PDX1 ^-/- pancreatic islet-like organoid differentiation on day 11. Scale bar: 500 μm. D: RT-qPCR analysis of NKX6.1 and PDX1 expression in mutant organoids on day 11 of differentiation ( n=3) and RT-qPCR analysis of INS expression on day 30 of organoid differentiation ( n=3). E: Representative immunofluorescence images of C-peptide, INSULIN (INS), GLUCAGON (GCG), and somatostatin (SST) staining of PDX1 ^+/+, PDX1 ^+/-, and PDX1 ^-/- islet organoids on day 30. Scale bar: 100 μm. F: Representative ultrastructure of insulin granules in β cells of mutant organoids, Scale bar: 500 nm. G: GSIS analysis of PDX1 ^+/+, PDX1 ^+/-, and PDX1 ^-/- organoid responses to 2 mmol/L and 16.8 mmol/L glucose stimulation, respectively ( n=5).

Figure 4

Changes in m6A methylation in PDX1-mutant cynomolgus macaques and restoration of PDX1 haploinsufficiency in pancreatic islet organoids by METTL3 A: Heatmap of expression of genes related to m6A modulators in M4 pancreas. B: RT-qPCR analysis of m6A modulator-target gene expression in M4 pancreas. C: RT-qPCR analysis of METTL3 expression in PDX1 ^+/+, PDX1 ^+/-, and PDX1 ^-/- hiPSCs during β-cell differentiation ( n=3). D: Western blot analysis of METTL3 protein levels in PDX1 ^+/+, PDX1 ^+/-, and PDX ^-/- pancreatic islet organoids. E: Representative immunofluorescence images of METTL3 staining of PDX1 ^+/+, PDX1 ^+/-, and PDX1 ^-/ islet organoids on day 30. Scale bar: 100 μm. F: Bright field of WT, PDX1 ^+/- , and PDX1 ^-/- pancreatic islet-like organoid differentiation on day 11. Scale bar: 500 μm. G, H: GSIS analysis of PDX1 ^+/- (G) and PDX1 ^-/- (H) in response to 2 mmol/L and 16.8 mmol/L glucose, respectively, after treatment with METTL3 ( n=5). I: Model depicting effect of decreased expression of m6A modulators in PDX1-mutant animal and cell models (prepared by Figdraw): Heterozygous deletion of PDX1 resulted in reduced PDX1 mRNA and altered m6A modification, which, in turn, reduced mRNA levels of genes related to β-cell differentiation and proliferation, including PDX1, IGF1R, and MAFA. Finally, PDX1-mutants exhibited PDX1-haploinsufficiency, which could be restored by m6A modulator METTL3.

Figure 5

Off-target analysis of fetuses with Cas9-mediated editing at PDX1 locus by whole-genome sequencing A: Summary of potential off-target site analysis. B: Image of detected off-target site by IGV. C: Sanger sequencing of off-target site in WT and M4 monkeys. D: mRNA expression levels of off-target site gene in WT and M4 monkeys by RT-qPCR.