A Sox9/Fgf feed-forward loop maintains pancreatic organ identity

Abstract

All mature pancreatic cell types arise from organ-specific multipotent progenitor cells. Although previous studies have identified cell-intrinsic and -extrinsic cues for progenitor cell expansion, it is unclear how these cues are integrated within the niche of the developing organ. Here, we present genetic evidence in mice that the transcription factor Sox9 forms the centerpiece of a gene regulatory network that is crucial for proper organ growth and maintenance of organ identity. We show that pancreatic progenitor-specific ablation of Sox9 during early pancreas development causes pancreas-to-liver cell fate conversion. Sox9 deficiency results in cell-autonomous loss of the fibroblast growth factor receptor (Fgfr) 2b, which is required for transducing mesenchymal Fgf10 signals. Likewise, Fgf10 is required to maintain expression of Sox9 and Fgfr2 in epithelial progenitors, showing that Sox9, Fgfr2 and Fgf10 form a feed-forward expression loop in the early pancreatic organ niche. Mirroring Sox9 deficiency, perturbation of Fgfr signaling in pancreatic explants or genetic inactivation of Fgf10 also result in hepatic cell fate conversion. Combined with previous findings that Fgfr2b or Fgf10 are necessary for pancreatic progenitor cell proliferation, our results demonstrate that organ fate commitment and progenitor cell expansion are coordinately controlled by the activity of a Sox9/Fgf10/Fgfr2b feed-forward loop in the pancreatic niche. This self-promoting Sox9/Fgf10/Fgfr2b loop may regulate cell identity and organ size in a broad spectrum of developmental and regenerative contexts.

Fig. 1.

Sox9-deleted pancreatic progenitors undergo hepatic fate conversion. (A,B) mRNA expression profiling (n=4) (A) and qRT-PCR (n=6) (B) of hepatic mRNAs in E12.5 Sox9^fl/fl; Pdx1-Cre versus control Sox9^fl/fl dorsal pancreas. (C-H′) Immunodetection of AFP or albumin (Alb) reveals almost complete absence in E10.5 (C,C′) or E11.5 (E,E′,G,G′) control or E10.5 Sox9-deleted (D,D′) pancreata, but abundant expression in E11.5 Sox9^fl/fl; Pdx1-Cre dorsal (F,H), but not ventral (F′,H′), pancreas. Arrowheads in E indicate two rare AFP⁺ cells in control dorsal pancreas. (I) AFP⁺ or Alb⁺ cells as a percentage of total pancreatic cells in E11.5 Sox9^fl/fl; Pdx1-Cre (n=5, AFP or 3, Alb) and control Sox9^fl/fl (n=3) dorsal pancreata. (J,K) Phosphohistone H3 (pHH3) staining in Pdx1⁺ progenitors and AFP⁺ cells of dorsal pancreas (J) or AFP⁺ hepatoblasts in liver (K) of E11.5 Sox9^fl/fl; Pdx1-Cre embryos. (L-M′) AFP⁺ cells are observed in dorsal (M) and ventral (M′) pancreas of Sox9^fl/fl; Ptf1a-Cre but not control (L,L′) embryos at E12.5. Transf., transferrin; G-6-P, glucose-6-phosphatase; dp, dorsal pancreas; vp, ventral pancreas; duo, duodenum; li, liver; FDR, false discovery rate. Error bars represent s.e.m.; ^*P<0.05; ^***P<0.001. Scale bars: 20 μm in C-H′,J,K; 50 μm in L-M′.

Fig. 2.

Sox9 maintains Fgfr2b expression in pancreatic progenitors. (A,B) mRNA expression profiling (n=3) (A) and qRT-PCR (n=6) (B) show Fgfr2/Fgfr2b and Fgfr4 downregulation in E12.5 Sox9^fl/fl; Pdx1-Cre versus control Sox9^fl/fl dorsal pancreas. (C-F′) Nuclear Fgfr2 in E10.5 control dorsal (C,C’) and ventral (E,E’) progenitors is extinguished by Sox9 ablation (D,D’,F,F’). dp, dorsal pancreas; vp, ventral pancreas; duo, duodenum; FDR, false discovery rate. Error bars represent s.e.m.; ^**P<0.01; ^***P<0.001. Scale bars: 20 μm.

Fig. 3.

Sox9 and Fgfr2b expression coincide in pancreatic progenitors. (Aa-Bd) Nuclear Fgfr2 expression in E9.0 dorsal pancreatic endoderm is closely correlated with that of Sox9 (arrows) (Aa-Ad); concordantly, neither Sox9 nor Fgfr2 is detectable in ventral pancreatic endoderm (Ba-Bd). (Ca-Dd) Nuclear Fgfr2 expression persists in pancreatic progenitors of dorsal (Ca-Cd) and ventral (Da-Dd) pancreas at E10.5. (Ea-Hd) Between E10.5-E12.5, Fgfr2 is redistributed from nucleus to membrane: at E11.5, Fgfr2 is localized to the membrane and nucleus (Ea-Fd), and by E12.5 is wholly membranous (Ga-Hd). (Ia-Jc) At E15.5, membranous Fgfr2 expression is restricted to the Sox9⁺ progenitor cords of the forming ductal tree. Scale bars: 20 μm.

Fig. 4.

Mosaic deletion reveals a cell-autonomous requirement for Sox9 in maintenance of Fgfr2 expression. (A-A″,C-C″) Nuclear Fgfr2 is expressed throughout Sox9⁺ pancreatic progenitors in E11.5 (A-A”) and E12.5 (C-C”) control embryos. (B-B″,D-D″) In Sox9^fl/fl; Ptf1a-Cre mice, Fgfr2 expression is lost in Sox9-deleted progenitors (B-B”,D-D”; white arrowheads) but is maintained in unrecombined Sox9⁺ cells (B-B”,D-D”; yellow arrowheads). The broken line outlines the pancreatic epithelium. dp, dorsal pancreas. Scale bars: 20 μm.

Fig. 5.

Loss of Fgfr2 and hepatic fate conversion in Pdx1-deficient pancreas is Sox9 dependent. (A-D) While control E10.5 dorsal pancreatic progenitors show robust nuclear Fgfr2 expression (A,A′), in Pdx1-deficient dorsal progenitors, Fgfr2 is downregulated in a pattern following that of Sox9 (B-Bb″; arrows indicate Sox9⁺ Fgfr2⁺ cells), consistent with AFP expression in Sox9-deficient progenitors in E11.5 Pdx1^–/– (D), compared with wild-type (WT) (C) mice. Immunofluorescence staining for the truncated Pdx1 protein in Pdx1^–/– dorsal pancreas is shown in Ba and Bb as a reference. (E) AFP⁺ cells as a percentage of total pancreatic cells (E) in E11.5 Pdx1^–/– and wild-type (WT) (n=4) dorsal pancreata. dp, dorsal pancreas; duo, duodenum; li, liver. Error bars represent s.e.m.; ^**P<0.01. Scale bars: 20 μm.

Fig. 6.

Perturbed Fgfr signaling in gut explants induces hepatic fate conversion in dorsal pancreas. (A) Gut region microdissected from E9.5 Sox9-eGFP embryos for explanting. (Ba-Bd) In culture, the eGFP⁺ dorsal pancreatic endoderm forms a dorsal pancreas (dp) over 96 hours (h). (C) Explants were cultured in 10 μM SU5402 or DMSO vehicle control for nested 72-hour windows. (D-Dd,F-Fd,H-Hd,J-Jd) Between day (d)0 and d5 in vitro, the untreated dorsal pancreas expands, branches and differentiates, as in vivo. (E-Ed,G-Gd) Inhibition of Fgf signaling from d0 to d3 results in downregulation of Sox9 (E-Ed) and Pdx1 (G-Gd), and induction of AFP expression (E-Ed,G-Gd). (I-Id,K-Kd) While AFP⁺ cells are also apparent with SU5402 treatment at d1-d4 (I-Id), they are not detected when Fgf signaling is blocked at d2-d5 (K-Kd). For clarity, only eGFP and AFP are shown in z-stacks (D’,E’,F’,G’,H’,I’,J’,K’). (L) AFP⁺ cells as a percentage of total dorsal pancreas cells in gut explants treated with 10 μM SU5402 or DMSO vehicle (CTL) for nested 72-hour windows (n=3); error bars represent s.e.m.; ^*P<0.05; ^**P<0.01. dpe, dorsal pancreatic endoderm; A, anterior; P, posterior. Scale bars: 250 μm in A-Bd; 50 μm in D-Kd.

Fig. 7.

Fgf10 maintains pancreatic fate through Fgfr2b and Sox9. (Aa-Bd) Sox9 and Fgfr2 are coordinately downregulated (arrows indicate Sox9⁺ Fgfr2⁺ cells) in the E9.5 dorsal pancreatic endoderm of Fgf10-deficient (Ba-Bd) compared with wild-type (WT) (Aa-Ad) mice. (C-E) AFP is expressed in E11.5 dorsal pancreata of Fgf10^+/– (D) or Fgf10^–/– (E) mice compared with wild-type pancreas (C), concordant with Sox9 downregulation (E). (F) AFP⁺ cells as a percentage of total pancreatic cells in E11.5 wild-type, Fgf10^+/– and Fgf10^–/– (n=3) dorsal pancreata; error bars represent s.e.m.; ^*P<0.05. dp, dorsal pancreas; li, liver. Scale bars: 20 μm in Aa-Bd; 50 μm in C-E. (G) Mesenchymal Fgf10 signaling, transduced via Fgfr2b on pancreatic progenitors, maintains their Sox9 and, thus, Fgfr2b expression, to maintain Fgf10 receptivity. This Sox9/Fgfr2b/Fgf10 feed-forward loop promotes growth and maintains pancreatic fate in pancreatic progenitors.