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Regeneration of the pancreas in adult zebrafish.

Moss JB, Koustubhan P, Greenman M, Parsons MJ, Walter I, Moss LG - Diabetes (2009)

Bottom Line: Dividing cells were primarily associated with affected islets and ducts but, with the exception of surgical partial pancreatectomy, were not extensively beta-cells.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.

View Article: PubMed Central - PubMed

Affiliation: Sarah W. Stedman Nutrition and Metabolism Center, Duke University Medical Center, Durham, North Carolina, USA. jennifer.b.moss@duke.edu

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.

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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.)
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Figure 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.)

Mentions: A histological study of adult zebrafish islets was used to evaluate the surprising return to normoglycemia without the need for insulin administration. Excised gut fragments including the proximal intestine and attached fluorescent main pancreas were fixed and embedded in paraffin. Figure 3 compares stained and immunofluorescence-hybridized sections of vehicles with STZ-injected zebrafish at 3, 7, and 14 days. Hematoxylin and eosin staining showed the main islet was frequently absent and smaller islets were invested with lymphocytic infiltrates and/or necrotic cells after 3 days (Fig. 3A). The islet perimeter was often discontinuous or aberrant (Fig. 3B and C). Weak insulin (Fig. 3B) or GFP (Fig. 3C) staining after 3 days confirmed a loss of β-cells while dividing PCNA-positive cells appeared at the islet perimeter, whereas vehicle-injected zebrafish contained dividing PCNA-positive cells primarily in the intestine (Fig. 3B). Insulin-positive cells were not dividing. Glucagon staining indicated a disruption of islet geometry after 3 days (Fig. 3C). After 7 days, small insulin-positive islets were rarely found. We occasionally observed GFP-positive β-cells within ductal epithelium (Fig. 3B, inset), though dividing cells were not insulin positive. Glucagon marked the perimeter of GFP-positive islets (Fig. 3C, inset) and outlined adjacent structures that appeared to emanate from ducts, visible after 7 days. After 2 weeks, numerous islets were present, and unlike controls, glucagon-positive cells were prominent in ductal epithelium (Fig. 3A and C). Larger islets contained scattered PCNA-positive cells comparable to vehicle-injected zebrafish after 14 days (Fig. 3B).


Regeneration of the pancreas in adult zebrafish.

Moss JB, Koustubhan P, Greenman M, Parsons MJ, Walter I, Moss LG - Diabetes (2009)

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.)
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Related In: Results  -  Collection

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Figure 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.)
Mentions: A histological study of adult zebrafish islets was used to evaluate the surprising return to normoglycemia without the need for insulin administration. Excised gut fragments including the proximal intestine and attached fluorescent main pancreas were fixed and embedded in paraffin. Figure 3 compares stained and immunofluorescence-hybridized sections of vehicles with STZ-injected zebrafish at 3, 7, and 14 days. Hematoxylin and eosin staining showed the main islet was frequently absent and smaller islets were invested with lymphocytic infiltrates and/or necrotic cells after 3 days (Fig. 3A). The islet perimeter was often discontinuous or aberrant (Fig. 3B and C). Weak insulin (Fig. 3B) or GFP (Fig. 3C) staining after 3 days confirmed a loss of β-cells while dividing PCNA-positive cells appeared at the islet perimeter, whereas vehicle-injected zebrafish contained dividing PCNA-positive cells primarily in the intestine (Fig. 3B). Insulin-positive cells were not dividing. Glucagon staining indicated a disruption of islet geometry after 3 days (Fig. 3C). After 7 days, small insulin-positive islets were rarely found. We occasionally observed GFP-positive β-cells within ductal epithelium (Fig. 3B, inset), though dividing cells were not insulin positive. Glucagon marked the perimeter of GFP-positive islets (Fig. 3C, inset) and outlined adjacent structures that appeared to emanate from ducts, visible after 7 days. After 2 weeks, numerous islets were present, and unlike controls, glucagon-positive cells were prominent in ductal epithelium (Fig. 3A and C). Larger islets contained scattered PCNA-positive cells comparable to vehicle-injected zebrafish after 14 days (Fig. 3B).

Bottom Line: Dividing cells were primarily associated with affected islets and ducts but, with the exception of surgical partial pancreatectomy, were not extensively beta-cells.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.

View Article: PubMed Central - PubMed

Affiliation: Sarah W. Stedman Nutrition and Metabolism Center, Duke University Medical Center, Durham, North Carolina, USA. jennifer.b.moss@duke.edu

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.

Show MeSH
Related in: MedlinePlus