Rfx6 directs islet formation and insulin production in mice and humans

Abstract

Insulin from the beta-cells of the pancreatic islets of Langerhans controls energy homeostasis in vertebrates, and its deficiency causes diabetes mellitus. During embryonic development, the transcription factor neurogenin 3 (Neurog3) initiates the differentiation of the beta-cells and other islet cell types from pancreatic endoderm, but the genetic program that subsequently completes this differentiation remains incompletely understood. Here we show that the transcription factor Rfx6 directs islet cell differentiation downstream of Neurog3. Mice lacking Rfx6 failed to generate any of the normal islet cell types except for pancreatic-polypeptide-producing cells. In human infants with a similar autosomal recessive syndrome of neonatal diabetes, genetic mapping and subsequent sequencing identified mutations in the human RFX6 gene. These studies demonstrate a unique position for Rfx6 in the hierarchy of factors that coordinate pancreatic islet development in both mice and humans. Rfx6 could prove useful in efforts to generate beta-cells for patients with diabetes.

Figure 1. Expression of Rfx6 in mice and human tissues

In a, the mRNA for Rfx genes 1-6 were amplified by RT-PCR from RNA isolated from the pancreas and brain of mouse embryos at e17.5 and from NIH3T3 fibroblasts. In b, levels of RFX6 mRNA were determined by real-time PCR of RNA from whole pancreas of human foetuses at the ages shown. n= 5 samples per foetal age group. *p= 0.017, weeks 8-10 vs. 19-21, by two-tailed Student’s t test. In c, mRNA for RFX6 and control gene Cyclophilin A (PPIA) genes were amplified by RT-PCR from RNA isolated from the human adult tissues shown. In d, levels of Rfx3 and Rfx6 mRNA were determined by real-time RT-PCR (TaqMan) of RNA isolated from the pancreata of wildtype and Neurog3^-/- mouse embryos at e17.5 values and expressed relative to the level of Gusb. n= 3 samples per group. **p = 0.0025, wildtype vs. mutant, by two-tailed Student’s t test.

Figure 2. Expression of Rfx6 in mice

Immunofluorescence staining was performed for Rfx6 (red) in mouse embryos. In a - c, at e9, Rfx6 staining overlaps with Foxa2 in the gut epithelium (including foregut, FG) and nascent dorsal pancreatic bud (DP), but Foxa2 is expressed alone in the liver bud (Li) and extraembryonic endoderm (EE). Separate colour channels are shown for red (a and d) and green (b and e). In d - f, costaining was performed with Pdx1 (green) in gut (duodenum, Du), dorsal pancreas (DP) and ventral pancreas (VP) at e10. In g, e15.5 pancreas was costained for Neurogenin3 (green). Costaining nuclei appear yellow. In h, e18.5 pancreas was costained for insulin (green). In i, adult pancreas was costained for insulin (green). Higher resolution photomicrographs from additional dates with additional markers can be found in Supplementary Figs. 1-7. Scale bars, 25 μm.

Figure 3. Targeting of the Rfx6 gene in mice

In panel a-d, lineage tracing was performed on Rfx6 ^+/+/R26R (left in a and c) or Rfx6 ^+/eGFPcre/R26R (b and d, and right in a and c) mice at e10.5 (a, b) and e12.5 (c, d) by staining for β-galactosidase activity with Xgal (blue). Panel b shows a close-up view of the animal on the right in panel a, and panel d shows a close-up view of the animal on the right in panel c. In panel e, an Rfx6^{eGFPcre/eGFPcre} pup at p2 is shown on the right, with a wildtype liter mate on the left. In panel f, the dissected abdominal viscera are shown for wildtype (left) and Rfx6^{eGFPcre/eGFPcre} (right) pups at p0.5. Additional photographs of the lineage tracing and mutant animals can be found in Supplementary Fig. 10. Li, liver; Du, duodenum; GB, gall bladder; VD, vitelline duct; Th, thymus; Tr, trachea; Oe, oesophagus; Lu, lung; St, stomach; DP, dorsal pancreas; VP, ventral pancreas; Hg, hindgut.

Figure 4. Expression patterns of islet markers in wildtype and Rfx6^{eGFPcre/eGFPcre} mice at e17.5

On pancreas sections from e17.5 embryos with the genotypes shown at the left, immunofluorescence costaining was performed for ChromograninA (ChromoA, red, a - f, green k, l), Synaptophysin (Syn, red, g, h), insulin (green, a, b), glucagon (green, c, d), somatostatin (Sst, green, e, f), ghrelin (green g, h), Nkx6.1 (red in i - l), and pancreatic polypeptide (Ppy, green, k, l). Quantification of cells expressing Ppy and Nkx6.1 is shown in Supplementary Table S3. Scale bars, 25 μm.

Figure 5. Function of the human Rfx6 protein

In a and c, DNA binding of the human in vitro-translated proteins shown above each lane to the double-stranded, radiolabeled oligonucleotide HBV X-box probe was tested by electromobility shift assay (EMSA). In a, combined proteins were co-translated, and probe bound by the heterodimer partners has a mobility between that of the two homodimers. Truncated proteins Rfx3T1 and Rfx3T2 have the first 119 and 160 amino-terminal amino acids removed respectively, but retain the DNA-binding and dimerization domains. In vitro-translated luciferase is included as a negative control. A close-up view of a longer gel is shown in Supplementary Fig. S10a. In b, mouse pancreatic ductal mPac L20 cells were co-transfected with a DNA plasmid containing the reporter constructs shown and another expressing the RFX cDNAs shown, luciferase reporter expression was assayed for each combination. *p = 0.0026 vs. “no cDNA”, 0.0024 vs. Rfx3 alone, and 0.011 vs. Rfx6 alone by two-tailed Student’s t test. In c, a schematic shows the proposed interactions, either direct or indirect, of several transcription factors during pancreas development. In d, increasing amounts of the in vitro-translated human Rfx6 wild type and R181Q and S217P mutant proteins were assayed for binding to the X-box DNA probe. Efficiency of mutant protein production is demonstrated in Supplementary Fig. S10b. In e, mutations found in patients are indicated on a map of the RFX6 gene. All mutations were homozygous except for proband 3.