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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

ascl1b-expressing cells give rise exclusively to endocrine cells of both dorsal and ventral bud. a–e', h–i' Genetic lineage tracing using the Cre-loxP system.a Schematic representation of the genetic lineage tracing experiments. The transgenic line Tg(ascl1b:eGFP-creERT2) was crossed with the Tg(ubi:loxP-eGFP-loxP-mCherry) line, abbreviated Tg(ubi:Switch), or with the Tg(ubi:loxP-AmCyan-loxP-ZsYellow) line, abbreviated Tg(ubi:CSY), treated with 4-hydroxytamoxifen (4OHT) at 11, 12, 13, 14, and 15 hpf (b–e') or at 13, 14, and 17 dpf (h–i') and fixed for analysis at the indicated times. Black triangles in a represent loxP sites. b–e' Immunodetection of CRE-mediated rec markers (ZsYellow or mCherry, red) and Isl1 (b–b"), Ins (c–c"), Gcg (d–d"), or Nkx6.1 (e, e') in 4OHT-treated embryos (green). The dotted white line delimits the pancreas (e'). Yellow arrows point to cells co-expressing rec marker (ZsYellow or mCherry) and the respective hormones (Ins or Gcg). f–g' Short-term lineage tracing: immunodetection of GFP and the Ins and Gcg hormones in 5-dpf Tg(ascl1b:eGFP-creERT2) embryos treated from 3 to 5 dpf with DMSO (f, f') or with the Notch signaling inhibitor, LY411575 (g, g'). Yellow arrows in g' point to GFP+/Ins+/Gcg+ secondary endocrine cells found in the IPDs. h–i' Immunodetection at 20 dpf of the CRE-mediated rec marker mCherry together with Ins and Gcg in 4OHT-treated larvae. All views are ventral with the anterior part to the left and represent z-plane confocal images (b–d") or confocal projection images (e–i'). Scale bars = 20 μm
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Fig3: ascl1b-expressing cells give rise exclusively to endocrine cells of both dorsal and ventral bud. a–e', h–i' Genetic lineage tracing using the Cre-loxP system.a Schematic representation of the genetic lineage tracing experiments. The transgenic line Tg(ascl1b:eGFP-creERT2) was crossed with the Tg(ubi:loxP-eGFP-loxP-mCherry) line, abbreviated Tg(ubi:Switch), or with the Tg(ubi:loxP-AmCyan-loxP-ZsYellow) line, abbreviated Tg(ubi:CSY), treated with 4-hydroxytamoxifen (4OHT) at 11, 12, 13, 14, and 15 hpf (b–e') or at 13, 14, and 17 dpf (h–i') and fixed for analysis at the indicated times. Black triangles in a represent loxP sites. b–e' Immunodetection of CRE-mediated rec markers (ZsYellow or mCherry, red) and Isl1 (b–b"), Ins (c–c"), Gcg (d–d"), or Nkx6.1 (e, e') in 4OHT-treated embryos (green). The dotted white line delimits the pancreas (e'). Yellow arrows point to cells co-expressing rec marker (ZsYellow or mCherry) and the respective hormones (Ins or Gcg). f–g' Short-term lineage tracing: immunodetection of GFP and the Ins and Gcg hormones in 5-dpf Tg(ascl1b:eGFP-creERT2) embryos treated from 3 to 5 dpf with DMSO (f, f') or with the Notch signaling inhibitor, LY411575 (g, g'). Yellow arrows in g' point to GFP+/Ins+/Gcg+ secondary endocrine cells found in the IPDs. h–i' Immunodetection at 20 dpf of the CRE-mediated rec marker mCherry together with Ins and Gcg in 4OHT-treated larvae. All views are ventral with the anterior part to the left and represent z-plane confocal images (b–d") or confocal projection images (e–i'). Scale bars = 20 μm

Mentions: Cell fate experiments were performed with Cre/loxP-based lineage tracing approaches by crossing the Tg(ascl1b:eGFP-2A-creERT2) with Cre-responder transgenic lines, either Tg(ubi:loxP-AmCyan-loxP-ZsYellow), termed Tg(ubi:CSY) [27], or Tg(ubi:loxP-eGFP-LoxP-mCherry), termed Tg(ubi:Switch) [28] (Fig. 3a). The double-transgenic embryos were treated five times with 4-hydroxytamoxifen (4OHT) from 11 to 15 hpf, the period when ascl1b expression reaches its maximal level [15], and the embryos were analyzed at 48 or 72 hpf. With these five 4OHT treatments, many ascl1b-expressing cells have undergone CRE recombination while no recombination was detected in the treated single-transgenic embryos used as control (data not shown). The CRE-mediated recombination (rec) marker (standing for either ZsYellow or mCherry) analyzed in double-transgenic embryos at 48 hpf was detected in 38 ± 4.3 % of Isl1+ cells (n = 5), indicating that ascl1b+ cells give rise to the endocrine cells of the dorsal bud (Fig. 3b–b"). In a similar way, the rec marker was detected in 58 ± 7.1 % of the Ins+ cells (n = 9) (Fig. 3c–c") and in 59 ± 3.7 % of the Gcg+ cells (n = 9) (Fig. 3d–d"). In contrast, the rec marker, clearly visible in the endocrine islet, was not detected at 72 hpf in the ductal cells, labeled by Nkx6.1, nor in the acinar cells, which surround them (Fig. 3e, e'), indicating that ascl1b+ cells cannot give rise to exocrine cells. Finally, we determined whether the ascl1b+ cells give rise to the secondary islets emerging from IPDs, either artificially induced by inhibiting the Notch pathway or naturally occurring in 20-dpf larvae. As the combined treatment of LY411575 with 4OHT was lethal, we performed short-term lineage tracing based on GFP expression (instead of Cre/loxP-based lineage tracing analyses). As shown in Fig. 3f, g, LY411575 treatment from 3 to 5 days of the Tg(ascl1b:eGFP-2A-creERT2) larvae led to the appearance of GFP cells all along the IPDs, most of these cells being also positive for glucagon or insulin hormones (yellow arrows, Fig. 3g, g'), indicating that ascl1b+ cells give rise to induced secondary islets. To trace the naturally occurring endocrine cells, we treated Tg(ascl1b:eGFP-2A-creERT2); Tg(ubi:Switch) larvae with 4OHT at 13, 14, and 17 dpf and analyzed the larvae at 20 dpf. The rec marker was detected within the principal islet (Fig. 3h, h') as well as in secondary islets (Fig. 3i, i') confirming that ascl1b+ cells can give rise to secondary islets.Fig. 3


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)

ascl1b-expressing cells give rise exclusively to endocrine cells of both dorsal and ventral bud. a–e', h–i' Genetic lineage tracing using the Cre-loxP system.a Schematic representation of the genetic lineage tracing experiments. The transgenic line Tg(ascl1b:eGFP-creERT2) was crossed with the Tg(ubi:loxP-eGFP-loxP-mCherry) line, abbreviated Tg(ubi:Switch), or with the Tg(ubi:loxP-AmCyan-loxP-ZsYellow) line, abbreviated Tg(ubi:CSY), treated with 4-hydroxytamoxifen (4OHT) at 11, 12, 13, 14, and 15 hpf (b–e') or at 13, 14, and 17 dpf (h–i') and fixed for analysis at the indicated times. Black triangles in a represent loxP sites. b–e' Immunodetection of CRE-mediated rec markers (ZsYellow or mCherry, red) and Isl1 (b–b"), Ins (c–c"), Gcg (d–d"), or Nkx6.1 (e, e') in 4OHT-treated embryos (green). The dotted white line delimits the pancreas (e'). Yellow arrows point to cells co-expressing rec marker (ZsYellow or mCherry) and the respective hormones (Ins or Gcg). f–g' Short-term lineage tracing: immunodetection of GFP and the Ins and Gcg hormones in 5-dpf Tg(ascl1b:eGFP-creERT2) embryos treated from 3 to 5 dpf with DMSO (f, f') or with the Notch signaling inhibitor, LY411575 (g, g'). Yellow arrows in g' point to GFP+/Ins+/Gcg+ secondary endocrine cells found in the IPDs. h–i' Immunodetection at 20 dpf of the CRE-mediated rec marker mCherry together with Ins and Gcg in 4OHT-treated larvae. All views are ventral with the anterior part to the left and represent z-plane confocal images (b–d") or confocal projection images (e–i'). Scale bars = 20 μm
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4556004&req=5

Fig3: ascl1b-expressing cells give rise exclusively to endocrine cells of both dorsal and ventral bud. a–e', h–i' Genetic lineage tracing using the Cre-loxP system.a Schematic representation of the genetic lineage tracing experiments. The transgenic line Tg(ascl1b:eGFP-creERT2) was crossed with the Tg(ubi:loxP-eGFP-loxP-mCherry) line, abbreviated Tg(ubi:Switch), or with the Tg(ubi:loxP-AmCyan-loxP-ZsYellow) line, abbreviated Tg(ubi:CSY), treated with 4-hydroxytamoxifen (4OHT) at 11, 12, 13, 14, and 15 hpf (b–e') or at 13, 14, and 17 dpf (h–i') and fixed for analysis at the indicated times. Black triangles in a represent loxP sites. b–e' Immunodetection of CRE-mediated rec markers (ZsYellow or mCherry, red) and Isl1 (b–b"), Ins (c–c"), Gcg (d–d"), or Nkx6.1 (e, e') in 4OHT-treated embryos (green). The dotted white line delimits the pancreas (e'). Yellow arrows point to cells co-expressing rec marker (ZsYellow or mCherry) and the respective hormones (Ins or Gcg). f–g' Short-term lineage tracing: immunodetection of GFP and the Ins and Gcg hormones in 5-dpf Tg(ascl1b:eGFP-creERT2) embryos treated from 3 to 5 dpf with DMSO (f, f') or with the Notch signaling inhibitor, LY411575 (g, g'). Yellow arrows in g' point to GFP+/Ins+/Gcg+ secondary endocrine cells found in the IPDs. h–i' Immunodetection at 20 dpf of the CRE-mediated rec marker mCherry together with Ins and Gcg in 4OHT-treated larvae. All views are ventral with the anterior part to the left and represent z-plane confocal images (b–d") or confocal projection images (e–i'). Scale bars = 20 μm
Mentions: Cell fate experiments were performed with Cre/loxP-based lineage tracing approaches by crossing the Tg(ascl1b:eGFP-2A-creERT2) with Cre-responder transgenic lines, either Tg(ubi:loxP-AmCyan-loxP-ZsYellow), termed Tg(ubi:CSY) [27], or Tg(ubi:loxP-eGFP-LoxP-mCherry), termed Tg(ubi:Switch) [28] (Fig. 3a). The double-transgenic embryos were treated five times with 4-hydroxytamoxifen (4OHT) from 11 to 15 hpf, the period when ascl1b expression reaches its maximal level [15], and the embryos were analyzed at 48 or 72 hpf. With these five 4OHT treatments, many ascl1b-expressing cells have undergone CRE recombination while no recombination was detected in the treated single-transgenic embryos used as control (data not shown). The CRE-mediated recombination (rec) marker (standing for either ZsYellow or mCherry) analyzed in double-transgenic embryos at 48 hpf was detected in 38 ± 4.3 % of Isl1+ cells (n = 5), indicating that ascl1b+ cells give rise to the endocrine cells of the dorsal bud (Fig. 3b–b"). In a similar way, the rec marker was detected in 58 ± 7.1 % of the Ins+ cells (n = 9) (Fig. 3c–c") and in 59 ± 3.7 % of the Gcg+ cells (n = 9) (Fig. 3d–d"). In contrast, the rec marker, clearly visible in the endocrine islet, was not detected at 72 hpf in the ductal cells, labeled by Nkx6.1, nor in the acinar cells, which surround them (Fig. 3e, e'), indicating that ascl1b+ cells cannot give rise to exocrine cells. Finally, we determined whether the ascl1b+ cells give rise to the secondary islets emerging from IPDs, either artificially induced by inhibiting the Notch pathway or naturally occurring in 20-dpf larvae. As the combined treatment of LY411575 with 4OHT was lethal, we performed short-term lineage tracing based on GFP expression (instead of Cre/loxP-based lineage tracing analyses). As shown in Fig. 3f, g, LY411575 treatment from 3 to 5 days of the Tg(ascl1b:eGFP-2A-creERT2) larvae led to the appearance of GFP cells all along the IPDs, most of these cells being also positive for glucagon or insulin hormones (yellow arrows, Fig. 3g, g'), indicating that ascl1b+ cells give rise to induced secondary islets. To trace the naturally occurring endocrine cells, we treated Tg(ascl1b:eGFP-2A-creERT2); Tg(ubi:Switch) larvae with 4OHT at 13, 14, and 17 dpf and analyzed the larvae at 20 dpf. The rec marker was detected within the principal islet (Fig. 3h, h') as well as in secondary islets (Fig. 3i, i') confirming that ascl1b+ cells can give rise to secondary islets.Fig. 3

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