Blood vessels restrain pancreas branching, differentiation and growth

Abstract

How organ size and form are controlled during development is a major question in biology. Blood vessels have been shown to be essential for early development of the liver and pancreas, and are fundamental to normal and pathological tissue growth. Here, we report that, surprisingly, non-nutritional signals from blood vessels act to restrain pancreas growth. Elimination of endothelial cells increases the size of embryonic pancreatic buds. Conversely, VEGF-induced hypervascularization decreases pancreas size. The growth phenotype results from vascular restriction of pancreatic tip cell formation, lateral branching and differentiation of the pancreatic epithelium into endocrine and acinar cells. The effects are seen both in vivo and ex vivo, indicating a perfusion-independent mechanism. Thus, the vasculature controls pancreas morphogenesis and growth by reducing branching and differentiation of primitive epithelial cells.

Fig. 1.

The vasculature restricts pancreas growth. (A) Hypervascularization of pancreata overexpressing VEGF. Whole-mount staining for Pecam (green) and Pdx1 (red) in pancreata from an E13.5 Pdx1-tTA; TET-VEGF embryo and a control littermate. Scale bar: 100 μm. (B) Smaller pancreas in newborn mice (postnatal day 1) overexpressing VEGF. Dotted lines mark the pancreas. Among control animals, top image is from a Pdx1-tTA animal and bottom image is from a TET-VEGF animal. All images are from the same litter. (C) Reduced pancreas weight in newborn mice overexpressing VEGF. n describes the number of mice analyzed. Graphs show cumulative data from three litters. In each litter, wild-type pancreas weight was normalized to 1, to correct for inter-litter differences. Data are mean±s.e.m. (D) Elimination of endothelial cells upon treatment of pancreatic explants with VEGFR2 inhibitor. Pancreatic buds from E12.5 wild-type embryos were cultured for 3 days in the presence or absence of VEGFR2i. In the lower panels, mice contained a Flk1-LacZ reporter allele. X-gal stains blood vessels in such mice. Scale bars: 200 μm (top); 80 μm (bottom). (E) Larger size of E12.5 pancreatic buds cultured for 3 days in the presence of VEGFR2i. Images show confocal z-stacks of explants in whole-mount stained for Pdx1. Shown are representative images from three individual explants. Yellow numbers describe the area stained for Pdx1. Scale bar: 200 μm. (F) Quantification of explant size after treatment with VEGFR2i, as in E. n describes the total number of explants analyzed. Graphs show cumulative data from ten experiments using ten independent litters. Data are mean±s.e.m.

Fig. 2.

Reduced branching, tip cell formation and endocrine specification in pancreata from E12.5 embryos overexpressing VEGF. (A) Whole-mount immunostaining for Muc1 (red) and Ngn3 (green), showing reduced branching and fewer Ngn3+ cells in VEGF-expressing pancreata. (B) Whole-mount immunostaining for Pdx1 (green), Pecam (red) and Cpa1 (blue), showing elongated unbranched tubes and fewer tip cells in VEGF-expressing pancreata. Scale bars: 200 μm. Images are z-stacks of serial confocal sections.

Fig. 3.

Reduced branching and endocrine differentiation in explanted Pdx1-tTA; TET-VEGF pancreatic buds, suggesting perfusion-independent effects of hypervascularization. (A) Whole-mount immunostaining for Pdx1 (green), Muc1 (red) and Ngn3 (blue) in E10.5 buds cultured for 3 days. (B) Whole-mount immunostaining for insulin (green), Muc1 (red) and Ngn3 (blue) in E12.5 buds cultured for 2 days. Note smaller size, reduced branching and reduced numbers of β-cells in VEGF-expressing pancreata. Scale bars: 200 μm.

Fig. 4.

Excessive formation of tips and acinar differentiation upon ablation of endothelial cells in explanted pancreatic buds. E12.5 wild-type buds were cultured with or without VEGFR2i and stained in whole mount. (A) Staining for Muc1 after culturing for 3 days. (B) Staining for Ptf1a after culturing for 2 days. Upper panels, z-stacks of 11 confocal sections. Bottom panels, individual confocal sections. (C) Top: staining for amylase after culturing for 3 days. Bottom: quantification of amylase-stained area in explants, normalized to the Pdx1-stained area. n denotes the number of individual explants analyzed. Data are mean±s.e.m. Bottom panels in A and C show higher magnification images of the areas marked in yellow in the upper panels. Scale bars: 200 μm. (D) Western blot showing increased amylase expression in avascular buds cultured as in C. Each lane represents a pool of three explants. The intensity of the amylase band relative to β-actin was increased 2.2-fold in the avascular samples. (E) Fold change in the mRNA levels of selected acinar cells upon vascular ablation. Data are extracted from microarray data (see Tables S1-4 and Fig. S9 in the supplementary material).

Fig. 5.

Accelerated formation of endocrine progenitor cells in avascular buds. E9.5 wild type pancreatic buds were cultured for 6 days in the presence or absence of VEGFR2i. (A) Whole-mount immunostaining for Pdx1 (red), Pecam (green) and Ngn3 (white). Each image is a z-stack of one whole individual pancreas. Insets show a higher magnification of Ngn3-stained cells. Scale bar: 100 μm. (B) qRT-PCR for Ngn3, normalized to Pdx1 expression. Results shown are the combined data from six independent litters. n represents the total number of buds in each group. Data are mean±s.e.m.

Fig. 6.

Evidence for involvement of Notch pathway in endothelial signaling to the epithelium. A γ-secretase inhibitor (GSI) partially rescues the branching and endocrine differentiation defects of Pdx1-tTA; TET-VEGF explants. GSI was added to E12.5 transgenic explants for 15 or 24 hours to document the patterns of Ngn3 and Muc1, respectively. Scale bars: 100 μm.

Fig. 7.

Model. Perfusion-independent signals from blood vessels favor self-renewal of trunk pancreatic epithelium. This comes at the expense of forming Ngn3⁺ endocrine progenitors and multipotent tip cells. Consequently, blood vessels restrain the formation of lateral branches, acinar and endocrine differentiation. The net result is a paradoxical restriction of organ size by endothelial cells. We propose that the vasculature acts by modulating Notch-mediated lateral signaling in the epithelium, which favors the coupled formation of endocrine progenitors and tip cells. See text for details.