Retinoic acid signaling within pancreatic endocrine progenitors regulates mouse and human β cell specification

Abstract

Retinoic acid (RA) signaling is essential for multiple developmental processes, including appropriate pancreas formation from the foregut endoderm. RA is also required to generate pancreatic progenitors from human pluripotent stem cells. However, the role of RA signaling during endocrine specification has not been fully explored. In this study, we demonstrate that the disruption of RA signaling within the NEUROG3-expressing endocrine progenitor population impairs mouse β cell differentiation and induces ectopic expression of crucial δ cell genes, including somatostatin. In addition, the inhibition of the RA pathway in hESC-derived pancreatic progenitors downstream of NEUROG3 induction impairs insulin expression. We further determine that RA-mediated regulation of endocrine cell differentiation occurs through Wnt pathway components. Together, these data demonstrate the importance of RA signaling in endocrine specification and identify conserved mechanisms by which RA signaling directs pancreatic endocrine cell fate.

Keywords: Diabetes; Pancreas development; Retinoic acid signaling; Wnt signaling; β cell differentiation; δ cell specification.

Fig. 1.

Endocrine-specific RA inhibition disrupts β cell development by E16.5 and increases Sst transcript expression. (A) Representative immunofluorescence images of E16.5 INS, SST, GCG and GHRL in RARdn^flox/flox; Neurog3:cre mutants. (B) Quantification of A (n=4 control, n=5 mutant; statistical analysis was completed for multiple t-tests using the Holm–Sidak method to correct for multiple comparisons; *Padj<0.05 is significant). (C) TUNEL+INS staining at E16.5. DNase1(⁺) sample is a positive control (n=3). (D) β cell proliferation reported as a percentage of proliferating versus non-proliferating β cells (n=3, statistical analysis was performed using an unpaired parametric Student's t-test; P>0.05, not significant). (E) RT-qPCR gene expression analysis of E16.5 whole pancreata from RARdn^flox/flox; Neurog3:cre mutants (n=3, statistical analysis was completed by the Benjamini, Krieger and Yekutieli two-stage step method to correct for multiple comparisons using Student's t-tests; *Q<0.05, **Q<0.005 are significant). (F) Quantification of the percentage of cells expressing Sst RNA but not SST protein (relative to all Sst RNA⁺ cells) in Neurog3:cre alone or RARdn^flox/flox; Neurog3:cre mutants at E16.5 (n=3, statistical analysis was completed by an unpaired parametric Student's t-test; *P<0.05 is significant). Representative images can be seen in Fig. 1G. (G) Dual RNA-protein visualization using RNAscope and immunofluorescence analyses (n=3). White arrows indicate Sst RNA⁺ cells, SST protein^–;INS protein^– cells; yellow arrows indicate Sst RNA⁺ cells, SST protein^–;INS protein⁺ cells. All n values represent biological replicates. Data are mean±s.d. ns, not significant.

Fig. 2.

Postnatal blood glucose homeostasis is disrupted in RA mutants. (A) Gene expression analysis by RT-qPCR of P2 whole pancreata from RARdn^flox/flox; Neurog3:cre mutants (n=3, statistical analysis was completed using the Benjamini, Krieger and Yekutieli two-stage step method to correct for multiple comparisons among multiple Student's t-tests; *Q<0.05 are significant). (B) Quantification of hormones in P2 pancreases (n=5, statistical analysis was completed for multiple Student's t-tests using the Holm–Sidak method to correct for multiple comparisons; *Padj<0.05 is significant). (C) Representative images of immunofluorescence at P2 for INS and SST. Scale bar applies to all panels. (D) Blood glucose at P2, reported in mg/dl. Statistical analysis was performed using an unpaired parametric Student's t-test method (***P<0.0005 is significant). (E) Glucose tolerance tests between 4 and 5 months in male RARdn^fl/fl; Neurog3:cre mutants (n=4). (F) Area under the curve of the intraperitoneal glucose tolerance test (IPGTT) in E (statistical analysis was peformed using an unpaired parametric Student's t-test method; **P<0.005 is significant). All n values represent biological replicates. Data are mean±s.d.

Fig. 3.

Human β cell differentiation requires RA signaling after posterior foregut formation. (A) Summary of human β cell differentiations with RA addition in red. Highlighted area (yellow) indicates when exogenous RA was removed from the media and RA inhibitor (RAi) was added. (B) RT-qPCR gene expression analysis of NEUROG3 expression, normalized to TBP. (C) Percentage of PDX1⁺/NKX6.1⁺ cells at stage 5 (ST5) of the differentiation with and without RAi treatment (n=3 per treatment, statistical analysis was performed using an unpaired parametric Student's t-test method; P>0.05 not significant). (D-F) RT-qPCR gene expression analysis of INS, GCG and SST expression at day 28, normalized to TBP for total RNA and CHGB for endocrine cells to control for differentiation efficiency (n=3 per treatment, statistical analysis was performed using a paired parametric Student's t-test method; *P<0.05 is significant). (G-I) Percentage of cells positive for C-peptide, glucagon and somatostatin by flow cytometric analysis (n=3 per treatment, statistical analysis was performed using an unpaired parametric Student's t-test method; P>0.05, not significant). All n values represent biological replicates. ns, not significant. Data are mean±s.d.

Fig. 4.

RA signaling inhibits the Wnt pathway to promote β cell specification and inhibit the δ cell transcriptional program. (A) Volcano plot of differentially expressed genes from whole-transcriptome analysis by RNA-seq of RARdn^flox/flox; Neurog3:cre mutants (n=3 biological replicates). (B) A selection of genes from A with Log₂ fold change and P-adjusted value (Padj≤0.05 is significant). (C) A selection of upregulated δ cell genes compared with Ins1/2 (Crhr2, Padj=0.053). (D) Significantly upregulated GO terms via PANTHER analysis of genes reported in A and Table S1. (E) Significantly enriched Wnt signaling components (GO:0017147). (F) Model demonstrating that during endocrine differentiation, RA signaling represses Wnt to promote β cell differentiation and repress δ-cell genes.