Mind bomb 1 is required for pancreatic β-cell formation

Abstract

During early pancreatic development, Notch signaling represses differentiation of endocrine cells and promotes proliferation of Nkx6-1(+)Ptf1a(+) multipotent progenitor cells (MPCs). Later, antagonistic interactions between Nkx6 transcription factors and Ptf1a function to segregate MPCs into distal Nkx6-1(-)Ptf1a(+) acinar progenitors and proximal Nkx6-1(+)Ptf1a(-) duct and β-cell progenitors. Distal cells are initially multipotent, but evolve into unipotent, acinar cell progenitors. Conversely, proximal cells are bipotent and give rise to duct cells and late-born endocrine cells, including the insulin producing β-cells. However, signals that regulate proximodistal (P-D) patterning and thus formation of β-cell progenitors are unknown. Here we show that Mind bomb 1 (Mib1) is required for correct P-D patterning of the developing pancreas and β-cell formation. We found that endoderm-specific inactivation of Mib1 caused a loss of Nkx6-1(+)Ptf1a(-) and Hnf1β(+) cells and a corresponding loss of Neurog3(+) endocrine progenitors and β-cells. An accompanying increase in Nkx6-1(-)Ptf1a(+) and amylase(+) cells, occupying the proximal domain, suggests that proximal cells adopt a distal fate in the absence of Mib1 activity. Impeding Notch-mediated transcriptional activation by conditional expression of dominant negative Mastermind-like 1 (Maml1) resulted in a similarly distorted P-D patterning and suppressed β-cell formation, as did conditional inactivation of the Notch target gene Hes1. Our results reveal iterative use of Notch in pancreatic development to ensure correct P-D patterning and adequate β-cell formation.

Fig. 1.

β-Cell formation requires active Notch signaling. (A and B) Optical sections of E18.5 wild-type (A) and Mib1^ΔEdd (B) pancreata stained for insulin, glucagon, and Cdh1. Note complete loss of insulin⁺ cells in Mib1^ΔEdd embryos. (C and D) Optical sections of wild-type (C) and Mib1^ΔEdd (D) embryos stained for Nkx6-1, Ptf1a, and Cdh1 as indicated. Note loss of Cdh1⁺Nkx6-1⁻Ptf1a⁻ duct cells (arrowheads in C) in Mib1^ΔEdd embryos. (E, F, I, J, M, and N) Optical sections of E15.5 pancreata from Mib1^ΔEdd (F), R26^dnMaml1 (J), and Hes1^ΔEdd (N) embryos and controls (E, I, and M) stained for insulin, glucagon, and Cdh1 as indicated. (Scale bars, 50 μm.) (G, K, and O) Insulin⁺ area in micrometers squared is shown relative to total pancreatic epithelial area in millimeters squared. (H, L, and P) qPCR analyses of Glucagon (Gcg), Insulin1 (Ins1), Somatostatin (Sst), and Pancreatic Polypeptide (Ppy) expression at E15.5 in Mib1^ΔEdd (H), R26^dnMaml1 (L), and Hes1^ΔEdd (P) pancreata compared with controls. Data are presented as mean + SD. *P < 0.05, **P < 0.01.

Fig. 2.

Neurog3-expressing precursor cells are lost in Mib1^ΔEdd embryos. (A, B, E, F, I, and J) confocal optical sections of E15.5 pancreata from Mib1^ΔEdd (B), R26^dnMaml1 (F), and Hes1^ΔEdd (J) embryos and controls (A, E, and I) stained for Neurog3, Muc1, and Cdh1 as indicated. (Scale bar, 50 μm.) (C, G, and K) Number of Neurog3⁺ cells is presented relative to total pancreatic epithelial area in mm². (D, H, and L) qPCR data for Neurogenin 3 (Neurog3) expression at e15.5 in Mib1^ΔEdd (D), R26^dnMaml1 (H), and Hes1^ΔEdd (L) pancreata compared with controls. Data are presented as mean + SD. *P < 0.05, **P < 0.01.

Fig. 3.

Nkx6-1⁺Ptf1a⁻ precursor cells are lost from the central pancreatic epithelium. (A, B, D, E, G, and H) Confocal optical sections of E15.5 pancreata from Mib1^ΔEdd (B), R26^dnMaml1 (E), and Hes1^ΔEdd (H) embryos and controls (A, D, and G) stained for Nkx6-1 and Ptf1a as indicated. (Scale bar, 50 μm.) (C, F, I) Number of Nkx6-1⁺, Ptf1a⁺, and Nkx6-1⁺Ptf1a⁺cells is presented relative to total pancreatic epithelial area in millimeters squared. Data are presented as mean + SD. *P < 0.05, **P < 0.01.

Fig. 4.

Nkx6-1⁺Ptf1a⁻ precursor cells are reduced at E12.5. (A, B, D, E, G, and H) Confocal optical sections of E12.5 pancreata from Mib1^ΔEdd (B), R26^dnMaml1 (E), and Hes1^ΔEdd (H) embryos and controls (A, D, and G) stained for Nkx6-1 and Ptf1a as indicated. (Scale bar, 50 μm.) (C, F, and I) Number of Nkx6-1⁺, Ptf1a⁺, and Nkx6-1⁺Ptf1a⁺cells is presented relative to total pancreatic epithelial area in millimeters squared. Data are presented as mean + SD. *P < 0.05, **P < 0.01.