Hedgehog signaling in pancreas epithelium regulates embryonic organ formation and adult beta-cell function

Abstract

Objective: Current studies indicate that Hedgehog (Hh) signaling must be excluded during early stages of pancreas formation. However, conflicting evidence suggests that Hh signaling may be active later during pancreas formation and that it is required for insulin production and secretion in cultured beta-cell lines. The objective of this study was to address these discrepancies by assessing the in vivo role of epithelial Hh signaling in the pancreas.

Research design and methods: To identify Hh-active cells in the developing and adult pancreas epithelium, we characterized transgenic reporter Patched1-LacZ mice. To determine the requirement for epithelial Hh signaling in the pancreas, we eliminated an essential Hh signaling component, Smoothened (Smo), in the pancreatic epithelium, and assessed pancreatic development and adult beta-cell physiology phenotypes.

Results: Characterization of Patched1-LacZ reporter mice revealed low-level LacZ expression in pancreatic epithelial cells throughout development until birth, when LacZ activity increases in intensity specifically in endocrine and ductal cells. In the absence of Hh signaling, Smo-deficient mice have delayed pancreas formation leading to a temporary reduction in pancreatic epithelium and beta-cell numbers. Although beta-cell numbers recover by birth, adult Smo-deficient mice display glucose intolerance, increased insulin sensitivity, and reduced total insulin production.

Conclusions: These data show that Hh signaling functions early during pancreas morphogenesis to regulate epithelial and beta-cell expansion and to modulate glucose metabolism by regulating insulin production in adult mice.

FIG. 1.

Hedgehog-active cells reside in the developing and adult pancreas. Pancreatic tissue was isolated from either control mice at e10.5 (A), e12.5 (E), e15.5 (I), e17.5 (M), postnatal day 0 (P0) (Q), and 2 weeks (U), or Patched1-LacZ (Ptch1-LacZ) transgenic mice at e10.5 (B–D), e12.5 (F–H), e15.5 (J–L), e17.5 (N–P), P0 (R–T), 2 weeks (V–X), and 4 weeks (Y–Z'). β-Gal activity was marked by blue staining pattern highlighted by arrowheads and/or arrows. Arrowheads = Ptch1-LacZ staining in the epithelium; arrows = Ptch1-LacZ staining in the mesenchyme. Scale bars in A and B, E and F, I and J, and M and N are equal to 50 μm, while scale bars in Q and R, and U and V, are equal to 100 μm. Lastly, the scale bar in Y is equal to 200 μm. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Pdx1-Cre^early;Smo^flox/null mice have downregulated Smo expression and Hh signaling. Smoothened (green) and insulin (red) costaining in control (A, C, and E) and Pdx1-Cre^early;Smo^flox/null (B, D, and F) P0 islets. Scale bars are equal to 10 μm. G: Smoothened gene expression by Sybr green real-time PCR is downregulated in islets isolated from Pdx1-Cre^early;Smo^flox/null mice vs. control islets (n = 3; ***P < 0.005). Expression of Hh target genes Gli1 (H) and Patched1 (I) is reduced compared with controls (n = 4; ***P < 0.005). P values were determined by Student t test. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Early pancreas formation in Pdx1-Cre^early;Smo^flox/null mice is perturbed. Sections stained with Pdx1 (green), Nkx6.1 (red), and DAPI (blue) staining in e12.5 pancreata show disrupted pancreatic branching in control (A) vs. Pdx1-Cre^early;Smo^flox/null (B) mice. Scale bars are equal to 100 μm. (Arrowheads in A and B indicate branching tips.) C: Total pancreatic epithelial area is reduced in Pdx1-Cre^early;Smo^flox/null mice at e12.5 but normalized by e15.5. (n = 4 for e12.5 samples; **P < 0.02; n = 6 for e15.5 samples). D: Recovery of total pancreatic epithelial area at e15.5 is due to increased total epithelial cell proliferation, as measured by phospho-histone H3 (n = 5; ***P < 0.005). E: The apoptotic rate in e15.5 total pancreatic epithelium is not changed in Pdx1-Cre^early;Smo^flox/null mice (n = 3). P values were determined by Student t test. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Pdx1-Cre^early;Smo^flox/null mice have delayed β-cell formation. Insulin (green) and glucagon (red) staining in e15.5 pancreata show an approximate equivalent number of α-cells (140 α-cells in control vs. 130 α-cells in mutant samples) but a reduced number of β-cells (144 β-cells in control vs. 95 β-cells in mutant samples) between control (A) and Pdx1-Cre^early;Smo^flox/null (B) mice. Neurogenin3 (Ngn3) (green) and Nkx6.1 (red) staining in e15.5 control (C) and Pdx1-Cre^early;Smo^flox/null (D) pancreata. Scale bars in A–D are equal to 100 μm. Quantification of endocrine areas at e15.5 and P0 show that while glucagon areas are normal, insulin area is reduced at e15.5 (E) but normalizes by P0 (F) in Pdx1-Cre^early;Smo^flox/null mice (n = 3 for e15.5 samples; **P < 0.02; n = 4 for P0 samples). G: Proliferation measured by phospho-histone H3 does not account for β-cell recovery at e15.5 or P0 (n = 4; *P < 0.05). While analysis of Ngn3 (green)/Nkx6.1 (red) positive cells in control (C) and Pdx1-Cre^early;Smo^flox/null (D) mice show no significant change in the number of total Ngn3 positive cells (H), a 71% increase in the number of Ngn3/Nkx6.1 positive progenitor β-cells (I) is observed at e15.5 (n = 6; **P < 0.02). P values were determined by Student t test. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Adult histology in Pdx1-Cre^early;Smo^flox/null mice is normal. Hematoxylin and eosin staining in control (A) and Pdx1-Cre^early;Smo^flox/null (B) mice show normal histology. Immunostaining for pancreatic markers amylase (red) and mucin (green) (C and D), glucagon (red) and insulin (green) (E and F), pancreatic polypeptide (red) and somatostatin (green) (G and H), and insulin (red), Pdx1 (green), and DAPI (blue) (I and J) show no apparent defects. Staining performed on 3-month-old pancreata. Scale bars in A–D are equal to 200 μm, while scale bars in E–H are equal to 100 μm. Lastly, scale bars in I–J are equal to 50 μm. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

Pdx1-Cre^early;Smo^flox/null mice are glucose intolerant and produce and secrete less insulin. A: Pdx1-Cre^early;Smo^flox/null mice fasted overnight and challenged with 2 mg/kg of sterile glucose by intraperitoneal injection show glucose intolerance phenotypes at 3 months of age (n = 13; ***P < 5E⁻⁵, two-way ANOVA, a measure of statistical significance for whole datasets). B: Pdx1-Cre^early;Smo^flox/null mice fasted overnight and challenged with 1 unit/kg of sterile insulin by intraperitoneal injection are more insulin sensitive than control mice (n = 5; ***P < 5E⁻⁵, two-way ANOVA). C: Sybr Green real-time PCR shows that while Pdx1 expression is not changed, Insulin expression is downregulated by nearly 40% in adult islets (n = 3; *P < 0.05). D: Insulin content from total pancreata is reduced by nearly 40% (n = 4; **P < 0.02). In vitro insulin secretion assays from isolated islets under high glucose (300 mg/dl) conditions vs. low glucose (30 mg/dl) conditions in the absence (E) (n = 3; ***P < 0.005, **P < 0.02) or presence (F) of 40 mmol/l potassium chloride (n = 3; *P < 0.05). G: Quantification of β-cell mass shows increased β-cell mass in Pdx1-Cre^early;Smo^flox/null mice (n = 4; *P < 0.05). Unless otherwise indicated, P values were determined by Student t test.