Limits...
Progenitor potential of nkx6.1-expressing cells throughout zebrafish life and during beta cell regeneration.

Ghaye AP, Bergemann D, Tarifeño-Saldivia E, Flasse LC, Von Berg V, Peers B, Voz ML, Manfroid I - BMC Biol. (2015)

Bottom Line: These two genes are initially co-expressed in the pancreatic primordium and their domains segregate, not as a result of mutual repression, but through the opposite effects of Notch signaling, maintaining nkx6.1 expression while repressing ascl1b in progenitors.In contrast to the mouse, pancreatic progenitor markers nkx6.1 and pdx1 continue to be expressed in adult ductal cells, a subset of which we show are still able to proliferate and undergo ductal and endocrine differentiation, providing robust evidence of the existence of pancreatic progenitor/stem cells in the adult zebrafish.Further characterization of these cells will open up new perspectives for anti-diabetic therapies.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Zebrafish Development and Disease Models (ZDDM), GIGA-Research, (Sart-Tilman) University of Liège, Avenue de l'Hôpital 1, B34, 4000, Liège, Belgium. aurelieghaye@outlook.be.

ABSTRACT

Background: In contrast to mammals, the zebrafish has the remarkable capacity to regenerate its pancreatic beta cells very efficiently. Understanding the mechanisms of regeneration in the zebrafish and the differences with mammals will be fundamental to discovering molecules able to stimulate the regeneration process in mammals. To identify the pancreatic cells able to give rise to new beta cells in the zebrafish, we generated new transgenic lines allowing the tracing of multipotent pancreatic progenitors and endocrine precursors.

Results: Using novel bacterial artificial chromosome transgenic nkx6.1 and ascl1b reporter lines, we established that nkx6.1-positive cells give rise to all the pancreatic cell types and ascl1b-positive cells give rise to all the endocrine cell types in the zebrafish embryo. These two genes are initially co-expressed in the pancreatic primordium and their domains segregate, not as a result of mutual repression, but through the opposite effects of Notch signaling, maintaining nkx6.1 expression while repressing ascl1b in progenitors. In the adult zebrafish, nkx6.1 expression persists exclusively in the ductal tree at the tip of which its expression coincides with Notch active signaling in centroacinar/terminal end duct cells. Tracing these cells reveals that they are able to differentiate into other ductal cells and into insulin-expressing cells in normal (non-diabetic) animals. This capacity of ductal cells to generate endocrine cells is supported by the detection of ascl1b in the nkx6.1:GFP ductal cell transcriptome. This transcriptome also reveals, besides actors of the Notch and Wnt pathways, several novel markers such as id2a. Finally, we show that beta cell ablation in the adult zebrafish triggers proliferation of ductal cells and their differentiation into insulin-expressing cells.

Conclusions: We have shown that, in the zebrafish embryo, nkx6.1+ cells are bona fide multipotent pancreatic progenitors, while ascl1b+ cells represent committed endocrine precursors. In contrast to the mouse, pancreatic progenitor markers nkx6.1 and pdx1 continue to be expressed in adult ductal cells, a subset of which we show are still able to proliferate and undergo ductal and endocrine differentiation, providing robust evidence of the existence of pancreatic progenitor/stem cells in the adult zebrafish. Our findings support the hypothesis that nkx6.1+ pancreatic progenitors contribute to beta cell regeneration. Further characterization of these cells will open up new perspectives for anti-diabetic therapies.

No MeSH data available.


Related in: MedlinePlus

In the adult pancreas, Notch-responsive CACs give rise to other ductal and endocrine cells and have the capacity to replicate. Immunodetection on sections through the pancreas of adult Tg(Tp1:VenusPest); Tg(Tp1:H2BmCherry).a Comparison of Venus (green) and H2BmCherry (red) labeling showing a small duct containing H2BmCherry+ cells that have lost Venus. b Comparison of H2BmCherry (red) with the ductal marker 2F11 (green) showing some 2F11+ cells within a small duct co-expressing the stable H2BmCherry marker (yellow arrow). c Weak H2BmCherry labeling near the extremity of a ductular structure (terminal or intercalated duct) (yellow arrows); a CAC (intense H2BmCherry) at the tip of the terminal duct is indicated by an asterisk. d and d' (close-up) Some H2BmCherry+ cells, devoid of Venus (Notch off) co-express the beta cell marker Ins (white) (yellow arrows). Sections acquired in the head of the pancreas, at the level of the main endocrine islet. e–f' Detection of 5-ethynyl-2′-deoxyuridine (EdU) (red), Venus (green), and proliferating cell nuclear antigen (PCNA) (white) in Tg(Tp1:VenusPest) adult fish injected with EdU. The pancreas was analyzed 20 hours (1 day post injection) (e, e') and 5 days (f, f') after EdU injection
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4556004&req=5

Fig8: In the adult pancreas, Notch-responsive CACs give rise to other ductal and endocrine cells and have the capacity to replicate. Immunodetection on sections through the pancreas of adult Tg(Tp1:VenusPest); Tg(Tp1:H2BmCherry).a Comparison of Venus (green) and H2BmCherry (red) labeling showing a small duct containing H2BmCherry+ cells that have lost Venus. b Comparison of H2BmCherry (red) with the ductal marker 2F11 (green) showing some 2F11+ cells within a small duct co-expressing the stable H2BmCherry marker (yellow arrow). c Weak H2BmCherry labeling near the extremity of a ductular structure (terminal or intercalated duct) (yellow arrows); a CAC (intense H2BmCherry) at the tip of the terminal duct is indicated by an asterisk. d and d' (close-up) Some H2BmCherry+ cells, devoid of Venus (Notch off) co-express the beta cell marker Ins (white) (yellow arrows). Sections acquired in the head of the pancreas, at the level of the main endocrine islet. e–f' Detection of 5-ethynyl-2′-deoxyuridine (EdU) (red), Venus (green), and proliferating cell nuclear antigen (PCNA) (white) in Tg(Tp1:VenusPest) adult fish injected with EdU. The pancreas was analyzed 20 hours (1 day post injection) (e, e') and 5 days (f, f') after EdU injection

Mentions: We then wanted to characterize nkx6.1 expression in the pancreas of adult zebrafish. Immunodetection on paraffin sections through the pancreas of Tg(nkx6.1:eGFP) fish that were 6 to 9 months old revealed that nkx6.1:eGFP expression persists in adult zebrafish (Fig. 7a). Comparison of endogenous Nkx6.1 protein and GFP shows that, in the adult too, Tg(nkx6.1:eGFP) recapitulates the pattern of Nkx6.1 expression (data not shown). nkx6.1:eGFP expression is confined to the ducts and to isolated cells scattered throughout the exocrine tissue and was not detected in beta cells (Fig. 7a, a') or acinar cells. These nkx6.1:eGFP+ cells dispersed within the exocrine pancreas exhibit long cellular extensions characteristic of CACs (inset in Fig. 7a) [12]. In the adult zebrafish pancreas, as in mammals, the CACs can also be identified by Notch signaling activity [12]. Thus, to confirm the expression of nkx6.1 in CACs, we used the Notch reporter line Tg(Tp1:VenusPest) [16], in which the destabilized Venus fluorescent protein (VenusPest) highlights cells harboring ongoing Notch activity, and which labels the CACs, as expected (Fig. 7b and inset). All Venus+ cells in the pancreas were found exclusively in the ductal system, and more particularly in the CACs and not within the ductular structures (Additional file 6: Fig. S6). Comparison of Venus with Nkx6.1 confirmed that Nkx6.1 is indeed expressed in all Venus+ CACs (Fig. 7c, c'). In contrast, while ducts also display nkx6.1 expression as revealed with either nkx6.1:eGFP (Fig. 7a, a') or with the endogenous Nkx6.1 protein (Fig. 7d), they are devoid of Notch ongoing activity (white arrows pointing at Venus– ducts in Fig. 7b, d; see also Fig. 8a and Additional file 6: Fig. S6).Fig. 7


Progenitor potential of nkx6.1-expressing cells throughout zebrafish life and during beta cell regeneration.

Ghaye AP, Bergemann D, Tarifeño-Saldivia E, Flasse LC, Von Berg V, Peers B, Voz ML, Manfroid I - BMC Biol. (2015)

In the adult pancreas, Notch-responsive CACs give rise to other ductal and endocrine cells and have the capacity to replicate. Immunodetection on sections through the pancreas of adult Tg(Tp1:VenusPest); Tg(Tp1:H2BmCherry).a Comparison of Venus (green) and H2BmCherry (red) labeling showing a small duct containing H2BmCherry+ cells that have lost Venus. b Comparison of H2BmCherry (red) with the ductal marker 2F11 (green) showing some 2F11+ cells within a small duct co-expressing the stable H2BmCherry marker (yellow arrow). c Weak H2BmCherry labeling near the extremity of a ductular structure (terminal or intercalated duct) (yellow arrows); a CAC (intense H2BmCherry) at the tip of the terminal duct is indicated by an asterisk. d and d' (close-up) Some H2BmCherry+ cells, devoid of Venus (Notch off) co-express the beta cell marker Ins (white) (yellow arrows). Sections acquired in the head of the pancreas, at the level of the main endocrine islet. e–f' Detection of 5-ethynyl-2′-deoxyuridine (EdU) (red), Venus (green), and proliferating cell nuclear antigen (PCNA) (white) in Tg(Tp1:VenusPest) adult fish injected with EdU. The pancreas was analyzed 20 hours (1 day post injection) (e, e') and 5 days (f, f') after EdU injection
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4556004&req=5

Fig8: In the adult pancreas, Notch-responsive CACs give rise to other ductal and endocrine cells and have the capacity to replicate. Immunodetection on sections through the pancreas of adult Tg(Tp1:VenusPest); Tg(Tp1:H2BmCherry).a Comparison of Venus (green) and H2BmCherry (red) labeling showing a small duct containing H2BmCherry+ cells that have lost Venus. b Comparison of H2BmCherry (red) with the ductal marker 2F11 (green) showing some 2F11+ cells within a small duct co-expressing the stable H2BmCherry marker (yellow arrow). c Weak H2BmCherry labeling near the extremity of a ductular structure (terminal or intercalated duct) (yellow arrows); a CAC (intense H2BmCherry) at the tip of the terminal duct is indicated by an asterisk. d and d' (close-up) Some H2BmCherry+ cells, devoid of Venus (Notch off) co-express the beta cell marker Ins (white) (yellow arrows). Sections acquired in the head of the pancreas, at the level of the main endocrine islet. e–f' Detection of 5-ethynyl-2′-deoxyuridine (EdU) (red), Venus (green), and proliferating cell nuclear antigen (PCNA) (white) in Tg(Tp1:VenusPest) adult fish injected with EdU. The pancreas was analyzed 20 hours (1 day post injection) (e, e') and 5 days (f, f') after EdU injection
Mentions: We then wanted to characterize nkx6.1 expression in the pancreas of adult zebrafish. Immunodetection on paraffin sections through the pancreas of Tg(nkx6.1:eGFP) fish that were 6 to 9 months old revealed that nkx6.1:eGFP expression persists in adult zebrafish (Fig. 7a). Comparison of endogenous Nkx6.1 protein and GFP shows that, in the adult too, Tg(nkx6.1:eGFP) recapitulates the pattern of Nkx6.1 expression (data not shown). nkx6.1:eGFP expression is confined to the ducts and to isolated cells scattered throughout the exocrine tissue and was not detected in beta cells (Fig. 7a, a') or acinar cells. These nkx6.1:eGFP+ cells dispersed within the exocrine pancreas exhibit long cellular extensions characteristic of CACs (inset in Fig. 7a) [12]. In the adult zebrafish pancreas, as in mammals, the CACs can also be identified by Notch signaling activity [12]. Thus, to confirm the expression of nkx6.1 in CACs, we used the Notch reporter line Tg(Tp1:VenusPest) [16], in which the destabilized Venus fluorescent protein (VenusPest) highlights cells harboring ongoing Notch activity, and which labels the CACs, as expected (Fig. 7b and inset). All Venus+ cells in the pancreas were found exclusively in the ductal system, and more particularly in the CACs and not within the ductular structures (Additional file 6: Fig. S6). Comparison of Venus with Nkx6.1 confirmed that Nkx6.1 is indeed expressed in all Venus+ CACs (Fig. 7c, c'). In contrast, while ducts also display nkx6.1 expression as revealed with either nkx6.1:eGFP (Fig. 7a, a') or with the endogenous Nkx6.1 protein (Fig. 7d), they are devoid of Notch ongoing activity (white arrows pointing at Venus– ducts in Fig. 7b, d; see also Fig. 8a and Additional file 6: Fig. S6).Fig. 7

Bottom Line: These two genes are initially co-expressed in the pancreatic primordium and their domains segregate, not as a result of mutual repression, but through the opposite effects of Notch signaling, maintaining nkx6.1 expression while repressing ascl1b in progenitors.In contrast to the mouse, pancreatic progenitor markers nkx6.1 and pdx1 continue to be expressed in adult ductal cells, a subset of which we show are still able to proliferate and undergo ductal and endocrine differentiation, providing robust evidence of the existence of pancreatic progenitor/stem cells in the adult zebrafish.Further characterization of these cells will open up new perspectives for anti-diabetic therapies.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Zebrafish Development and Disease Models (ZDDM), GIGA-Research, (Sart-Tilman) University of Liège, Avenue de l'Hôpital 1, B34, 4000, Liège, Belgium. aurelieghaye@outlook.be.

ABSTRACT

Background: In contrast to mammals, the zebrafish has the remarkable capacity to regenerate its pancreatic beta cells very efficiently. Understanding the mechanisms of regeneration in the zebrafish and the differences with mammals will be fundamental to discovering molecules able to stimulate the regeneration process in mammals. To identify the pancreatic cells able to give rise to new beta cells in the zebrafish, we generated new transgenic lines allowing the tracing of multipotent pancreatic progenitors and endocrine precursors.

Results: Using novel bacterial artificial chromosome transgenic nkx6.1 and ascl1b reporter lines, we established that nkx6.1-positive cells give rise to all the pancreatic cell types and ascl1b-positive cells give rise to all the endocrine cell types in the zebrafish embryo. These two genes are initially co-expressed in the pancreatic primordium and their domains segregate, not as a result of mutual repression, but through the opposite effects of Notch signaling, maintaining nkx6.1 expression while repressing ascl1b in progenitors. In the adult zebrafish, nkx6.1 expression persists exclusively in the ductal tree at the tip of which its expression coincides with Notch active signaling in centroacinar/terminal end duct cells. Tracing these cells reveals that they are able to differentiate into other ductal cells and into insulin-expressing cells in normal (non-diabetic) animals. This capacity of ductal cells to generate endocrine cells is supported by the detection of ascl1b in the nkx6.1:GFP ductal cell transcriptome. This transcriptome also reveals, besides actors of the Notch and Wnt pathways, several novel markers such as id2a. Finally, we show that beta cell ablation in the adult zebrafish triggers proliferation of ductal cells and their differentiation into insulin-expressing cells.

Conclusions: We have shown that, in the zebrafish embryo, nkx6.1+ cells are bona fide multipotent pancreatic progenitors, while ascl1b+ cells represent committed endocrine precursors. In contrast to the mouse, pancreatic progenitor markers nkx6.1 and pdx1 continue to be expressed in adult ductal cells, a subset of which we show are still able to proliferate and undergo ductal and endocrine differentiation, providing robust evidence of the existence of pancreatic progenitor/stem cells in the adult zebrafish. Our findings support the hypothesis that nkx6.1+ pancreatic progenitors contribute to beta cell regeneration. Further characterization of these cells will open up new perspectives for anti-diabetic therapies.

No MeSH data available.


Related in: MedlinePlus