Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development

Abstract

The transcription factor neurogenin 3 (Neurog3 or Ngn3) controls islet cell fate specification in multipotent pancreatic progenitor cells in the mouse embryo. However, our knowledge of the genetic programs implemented by Ngn3, which control generic and islet subtype-specific properties, is still fragmentary. Gene expression profiling in isolated Ngn3-positive progenitor cells resulted in the identification of the uncharacterized winged helix transcription factor Rfx6. Rfx6 is initially expressed broadly in the gut endoderm, notably in Pdx1-positive cells in the developing pancreatic buds, and then becomes progressively restricted to the endocrine lineage, suggesting a dual function in both endoderm development and islet cell differentiation. Rfx6 is found in postmitotic islet progenitor cells in the embryo and is maintained in all developing and adult islet cell types. Rfx6 is dependent on Ngn3 and acts upstream of or in parallel with NeuroD, Pax4 and Arx transcription factors during islet cell differentiation. In zebrafish, the Rfx6 ortholog is similarly found in progenitors and hormone expressing cells of the islet lineage. Loss-of-function studies in zebrafish revealed that rfx6 is required for the differentiation of glucagon-, ghrelin- and somatostatin-expressing cells, which, in the absence of rfx6, are blocked at the progenitor stage. By contrast, beta cells, whose number is only slightly reduced, were no longer clustered in a compact islet. These data unveil Rfx6 as a novel regulator of islet cell development.

Fig. 1.

Expression of Rfx6 in the gut endoderm and in the developing pancreatic buds in the mouse embryo. (A) Wholemount in situ hybridization (ISH) on an E9.0 mouse embryo (lateral view) with an Rfx6 antisense probe (purple). (B) Transversal section of embryo shown in A at the level of the black arrows. (C-E,G,H) Double immunofluorescence for Rfx6 (green) and Pdx1 (red) on cryosections of E9.5 (C-E), and E10.5 (G) embryos and Rfx6 and Ptf1a (red) at E10.5 (H). (F) ISH for Rfx6 followed by immunohistochemistry for Pdx1. Nuclei are stained with DAPI (blue) in E, G and H. Rfx6 is found in the gut endoderm (blue arrows in A,B) and in the developing pancreatic regions in Pdx1-positive progenitor cells at E9.5 (C-E). At E10.5, Rfx6 is excluded from Pdx1- (F,G) and Ptf1a- (H) positive pancreatic endodermal progenitor cells and restricted to differentiating endocrine cells. All sections are sagittal, B is transverse. In E and G arrows point to Pdx1/Rfx6 double-positive cells. dp, dorsal pancreas; duo, duodenum; nt, neural tube; st, stomach; vp, ventral pancreas.

Fig. 2.

Ngn3-dependent expression of Rfx6 in the mouse islet lineage. (A-F) Double immunofluorescence showing partial overlapping expression of Ngn3 (red) and Rfx6 (green) at E10.5 (A-C) and E15.5 (D-F). Yellow, green and red arrows point to double-labelled, single Rfx6-positive and single Ngn3-positive cells, respectively. Nuclei are stained with DAPI. (G,H) Loss of Rfx6 expression (in situ hybridization in blue) in an Ngn3-deficient pancreas (H) compared with a wild-type pancreas (G), demonstrating that Rfx6 is restricted to the islet lineage at E15.5.

Fig. 3.

Overlapping expression of Rfx6 and other islet transcription factors in the pancreas of mouse embryos. (A-F) In situ hybridization (blue) experiments on adjacent cryosections showing overlapping expression of Rfx6 with NeuroD, Insm1 and Rfx3. All experiments were performed on E15.5 pancreata. Magnifications: 20× in A,B; 40× in C-F.

Fig. 4.

Rfx6 expression is unaffected in the pancreas of Pax4-Arx- and NeuroD-deficient mouse embryos. (A-H) In situ hybridization (blue) for Rfx6 and Ngn3 on adjacent pancreas cryosections of wild-type (A,B), NeuroD- (C,D), Arx- (E,F) and Pax4- (G,H) deficient embryos at E14.5.

Fig. 5.

Rfx6 expression in mature islet cells in the mouse. (A-G) Double immunofluorescence revealing nuclear expression of Rfx6 (green) in hormone-expressing cells (red) in developing alpha (A,B) and beta (C) cells, as well as in mature alpha (D), beta (E), delta (F) and PP (G) cells in adult islets of Langerhans. Gcg, glucagon; Ins, insulin; PP, pancreatic polypeptide; Som, somatostatin. Arrows point to double-positive cells; insets show magnifications.

Fig. 6.

Zebrafish rfx6 is expressed in pancreatic endocrine progenitors as well as in mature endocrine cells. (A) Lateral view of fluorescent wholemount in situ hybridization (WISH) performed on stage 19S embryos with an rfx6 antisense probe (arrowhead). (B) Ventral view of WISH with an rfx6 probe on 24 hpf embryos. (C-N) Lateral views of the pancreas area from embryos analyzed by double-fluorescent WISH, anterior to the left, dorsal to the top. (C) The expression domains of isl1 and sox4b are distinct. (D-F) Relative location of the hormone-expressing cells in relation to the neurod-, isl1- and sox4b-expressing cells. Hormone expression was detected by WISH using a cocktail of insulin, glucagon, somatostatin and ghrelin probes. The diagram shows the respective locations of sox4b-, isl1-, neurod-, and hormone-positive cells within the pancreatic area in a lateral view at 24 hpf. (G-I) At 18S, rfx6 and sox4b expression domains completely overlap, whereas at 24 hpf and 30 hpf sox4b is maintained only in the ventral part of rfx6 domain. (J-L) The expression pattern of rfx6 completely overlaps the pancreatic neurod expression pattern. (M,N) The isl1-expressing cells are located in the dorsal part of the rfx6 expression domain at 24 hpf and 30 hpf.

Fig. 7.

Impaired endocrine cell differentiation, accumulation of islet progenitor cells and failure of insulin-expressing cells clustering in rfx6 morphants in the zebrafish. (A-X) Ventral views of the pancreas area from embryos analysed by WISH, anterior to the left. (A-L) Hormone expression in the control and rfx6 morphants at 30 hpf. (M-X) Pancreatic expression of isl1, sox4b and neuroD at 24 hpf and arx at 30 hpf in the control and rfx6 morphants. (Y-ZII) Lateral views of the pancreas area from 30 hpf embryos analysed by double-fluorescent WISH for sox4b and isl1, anterior to the left, dorsal to the top. Quantifications (right side of figure) represent the number of positive cells per embryo for controls and morphants. Asterisks (*) indicate that the difference between cell number in control and morphants is statistically significant by Student's t-test (P<0.001). Scale bar: 14 μm in A-L; 18 μm in M-X.