Regeneration of the pancreas in adult zebrafish

Abstract

Objective: Regenerating organs in diverse biological systems have provided clues to processes that can be harnessed to repair damaged tissue. Adult mammalian beta-cells have a limited capacity to regenerate, resulting in diabetes and lifelong reliance on insulin. Zebrafish have been used as a model for the regeneration of many organs. We demonstrate the regeneration of adult zebrafish pancreatic beta-cells. This nonmammalian model can be used to define pathways for islet-cell regeneration in humans.

Research design and methods: Adult transgenic zebrafish were injected with a single high dose of streptozotocin or metronidazole and anesthetized at 3, 7, or 14 days or pancreatectomized. Blood glucose measurements were determined and gut sections were analyzed using specific endocrine, exocrine, and duct cell markers as well as markers for dividing cells.

Results: Zebrafish recovered rapidly without the need for insulin injections, and normoglycemia was attained within 2 weeks. Although few proliferating cells were present in vehicles, ablation caused islet destruction and a striking increase of proliferating cells, some of which were Pdx1 positive. Dividing cells were primarily associated with affected islets and ducts but, with the exception of surgical partial pancreatectomy, were not extensively beta-cells.

Conclusions: The ability of the zebrafish to regenerate a functional pancreas using chemical, genetic, and surgical approaches enabled us to identify patterns of cell proliferation in islets and ducts. Further study of the origin and contribution of proliferating cells in reestablishing islet function could provide strategies for treating human diseases.

FIG. 1

β-cell destruction by STZ. A: GFP fluorescence of zebrafish pancreatic explants after 3 days in culture (100×). B: Intact zebrafish: lateral views of right side without skin (50×). Top panel: GFP fluorescence in the main pancreas (rostral) and auxiliary islets (green) of vehicle-injected zebrafish. Bottom panel: STZ-injected zebrafish killed after 3 days. C: Vehicle-injected, killed InsGFP zebrafish. Main pancreas was imaged after in situ treatment with propidium iodide (propidium iodide: red fluorescence). D: STZ-injected zebrafish pancreas + propidium iodide after 3 days. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2

Blood glucose levels return to normal after 2 weeks. Fasted blood glucose readings were recorded from cardiac blood in STZ-treated (black bars) versus vehicle-treated (white bars) zebrafish at 3, 7, and 14 days from 10 (5 male/5 female) STZ- and 6 (3 male/3 female) vehicle-injected InsGFP 1-year-old siblings. A two-fold reduction in blood glucose was observed 1 week after destruction of the β-cells. Gray bars: blood glucose readings from fasted InsNTR 1 year olds 3, 7, or 14 days after MET treatment. IP, intraperitoneal.

FIG. 3

Regeneration of zebrafish islets after STZ treatment. A: Hematoxylin and eosin (H&E) staining of paraffin sections at 3, 7, and 14 days. Vehicle and 14 day STZ: 200× magnification; 3 day and 7 day: 400× magnification. Arrows: islets. Arrowheads: blood vessels. B: STZ Ins/PCNA: insulin antibodies (visualized with red fluorescent secondary antibodies) mark β-cells. PCNA⁺ dividing cells are green. Arrows identify islets. Arrowheads: ducts; vehicle: numerous dividing PCNA⁺ cells are located at the base of intestinal villi. A few non–insulin expressing dividing cells are scattered throughout the islet and surrounding exocrine pancreas (400× magnification); 3 day STZ: a mantle of PCNA-positive cells surrounds affected islets (400×); 7 day STZ: dividing cells are located in and around ducts (400× magnification); inset (200× magnification): CK18 (red) labeling of ducts. Insulin⁺ cells are green; 14 day STZ: 200× magnification. Large islets with scattered dividing cells have appearance similar to vehicle-injected zebrafish. Dividing cells surround insulin-negative areas, similar to the PCNA expression observed after 3 days. C: STZ glucagon/GFP; vehicle (400× magnification): β-cells (green) and α-cells (red). Arrow: islet; 3 day STZ: glucagon⁺ cells within islet remnant (400×); 7 day STZ: glucagon labels GFP-negative islet attached to duct (200×). Inset (400×): glucagon (red) outlines islets ± β-cells (green); 14 day STZ: ductal hyperplasia (200×) with glucagon staining (red). Inset (600×): the ductal epithelium is continuous with the islet (confocal image). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4

Regeneration of islets after metronidazole treatment. 200× magnification. i = islets, int = intestine. Each column contains serial sections except one: 14 day InsNTR, Glu/PCNA. A: Vehicle InsNTR. DIC: differential interference contrast image indicating location of pancreatic islets and ducts. Ins/PCNA: sparse, dividing PCNA⁺ cells (green) in endocrine islets (red, insulin). Arrow: dividing β-cell. Glu/PCNA: glucagon⁺ cells (red) primarily at the perimeter of the islet. Pdx1/CK18: Pdx1⁺ cells located in CK18⁺ (green) ductal epithelium (arrowhead and inset). Pdx1/PCNA: nondividing Pdx1⁺ cells throughout the islet and ducts (arrowhead and inset). Arrow: dividing cell. B: Three day InsNTR. Ins/PCNA: β-cells are absent. Dividing cells (green) occupy the islet. Extra-islet cells are also dividing. Glu/PCNA: glucagon⁺ cells (red) are not dividing. Centrally located α-cells. Pdx1/CK18: Pdx1⁺ (red) cells depicted (inset) in islets and numerous ducts (arrowheads). Pdx1/PCNA: many Pdx1⁺ cells (red) are also dividing (arrows and insert). Many dividing Pdx1⁺ cells (inset) are located in ducts (arrowhead). C: Fourteen day InsNTR. Ins/PCNA: regenerated β-cells in islet (red) occasionally divide (arrow). Glu/PCNA: dividing cells (green) located in and around regenerated islet (α-cells are red). Pdx1/CK18: ducts and vascular epithelium (CK18: green) and Pdx1⁺ cells (red). Pdx1/PCNA: Pdx1 (red) labels islets, ducts (arrowhead), and occasionally intestine (green). Arrows: dividing Pdx1⁺ cells. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5

Regeneration after pancreatectomy. A and B: Right side of intact, living zebrafish before (sham, A) and 14 days after (Ptx, B) surgical removal of the GFP⁺ pancreas (red outline). The tip of the forceps used to remove the pancreas is visible (100× magnification). C: Paraffin section of sham-operated pancreas with few PCNA⁺ dividing cells (PCNA, red) except in the intestine (Int). β-Cells are green (arrow; 200× magnification). D: Many red PCNA⁺ dividing cells in ducts (arrowhead) and in nuclei of regenerating β-cells (yellow; 200× magnification). (A high-quality digital representation of this figure is available on the online issue.)