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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.


The bacterial artificial chromosome reporter line Tg(nkx6.1:eGFP) mirrors the expression of the endogenous nkx6.1 gene. Immunodetection of endogenous Nkx6.1 (red) and GFP (green) in Tg(nkx6.1:eGFP) embryos of the indicated stages. Green arrows point to Nkx6.1–/GFP+ cells and red arrows to Nkx6.1+/GFP- cells. All views are either lateral (b, b', c, and c') or ventral (a, a', d, d', f, and f') with the anterior part to the left. They represent either z-plane confocal images (b, d, e) or confocal projection images (a, c, f). Scale bars = 30 μm. EPD extra-pancreatic duct, IPD intra-pancreatic duct, i islet
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Fig1: The bacterial artificial chromosome reporter line Tg(nkx6.1:eGFP) mirrors the expression of the endogenous nkx6.1 gene. Immunodetection of endogenous Nkx6.1 (red) and GFP (green) in Tg(nkx6.1:eGFP) embryos of the indicated stages. Green arrows point to Nkx6.1–/GFP+ cells and red arrows to Nkx6.1+/GFP- cells. All views are either lateral (b, b', c, and c') or ventral (a, a', d, d', f, and f') with the anterior part to the left. They represent either z-plane confocal images (b, d, e) or confocal projection images (a, c, f). Scale bars = 30 μm. EPD extra-pancreatic duct, IPD intra-pancreatic duct, i islet

Mentions: To label the nkx6.1-expressing cells, we generated a transgenic line driving the expression of the enhanced green fluorescent protein (eGFP) under the control of nkx6.1 regulatory regions. We engineered a BAC spanning from 55 kb upstream to 95 kb downstream of the nkx6.1 gene and inserted the eGFP coding regions into exon 1, replacing the beginning of the nkx6.1 open reading frame (Additional file 1: Fig. S1A). This BAC reporter construct was introduced into the zebrafish genome using the Tol2 transposon system [24, 25] and the stable transgenic line Tg(nkx6.1:eGFP) obtained showed expression of green fluorescent protein (GFP) in the nervous system and in the pancreas, which mirrors the endogenous Nkx6.1 protein expression (Additional file 1: Fig. S1B). Detailed comparison of the localization of these two proteins in the pancreas during development confirmed that GFP is indeed co-expressed with Nkx6.1 (Fig. 1). Indeed, together with the endogenous Nkx6.1 protein [18], GFP is expressed at the base of the endocrine islet at 24 and 30 hpf (Fig. 1b, c), in the ventral bud at 38 and 48 hpf (Fig. 1d, e), and in IPDs and EPDs at 4 days post fertilization (dpf) (Fig. 1f, f'). In contrast, at earlier stages, GFP was detected in a subset of Nkx6.1+ cells, probably due to the delay of GFP expression compared to Nkx6.1. Indeed, at 17 hpf, about 75 % of the Nkx6.1+ cells showed detectable GFP expression (Fig. 1a, a') and at 14 hpf, this proportion dropped even further to about 25–30 % (data not shown). Conversely, a few hours after the onset of nkx6.1 gene expression, some GFP+/Nkx6.1– cells were also detected (green arrows, Fig. 1b'–e'). This GFP labeling is not the result of an ectopic expression of the gfp transcript, as double fluorescent whole-mount in situ hybridization (WISH) showed that the gfp transcripts are present in the same pancreatic domain as nkx6.1 transcripts (data not shown) and importantly, like nkx6.1, gfp transcripts were not found in hormone-expressing cells (Additional file 1: Fig. S1C–C'', D–D''). Hence, prolonged GFP detection is rather due to the well-known high stability of GFP (±24 h half-life [26]), which persists in cells where Nkx6.1 protein is no longer found. This is nicely illustrated at 30 hpf, where strong GFP expression is detected at the base of the forming islet where Nkx6.1+ pancreatic progenitors are located, while weak GFP labeling is found dorsally within the islet, where differentiated endocrine cells, devoid of Nkx6.1, are clustered (Fig. 1c). As expected, this prolonged GFP detection will gradually fade away, finally to disappear completely in the differentiated endocrine cells (Fig. 1f).Fig. 1


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)

The bacterial artificial chromosome reporter line Tg(nkx6.1:eGFP) mirrors the expression of the endogenous nkx6.1 gene. Immunodetection of endogenous Nkx6.1 (red) and GFP (green) in Tg(nkx6.1:eGFP) embryos of the indicated stages. Green arrows point to Nkx6.1–/GFP+ cells and red arrows to Nkx6.1+/GFP- cells. All views are either lateral (b, b', c, and c') or ventral (a, a', d, d', f, and f') with the anterior part to the left. They represent either z-plane confocal images (b, d, e) or confocal projection images (a, c, f). Scale bars = 30 μm. EPD extra-pancreatic duct, IPD intra-pancreatic duct, i islet
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: The bacterial artificial chromosome reporter line Tg(nkx6.1:eGFP) mirrors the expression of the endogenous nkx6.1 gene. Immunodetection of endogenous Nkx6.1 (red) and GFP (green) in Tg(nkx6.1:eGFP) embryos of the indicated stages. Green arrows point to Nkx6.1–/GFP+ cells and red arrows to Nkx6.1+/GFP- cells. All views are either lateral (b, b', c, and c') or ventral (a, a', d, d', f, and f') with the anterior part to the left. They represent either z-plane confocal images (b, d, e) or confocal projection images (a, c, f). Scale bars = 30 μm. EPD extra-pancreatic duct, IPD intra-pancreatic duct, i islet
Mentions: To label the nkx6.1-expressing cells, we generated a transgenic line driving the expression of the enhanced green fluorescent protein (eGFP) under the control of nkx6.1 regulatory regions. We engineered a BAC spanning from 55 kb upstream to 95 kb downstream of the nkx6.1 gene and inserted the eGFP coding regions into exon 1, replacing the beginning of the nkx6.1 open reading frame (Additional file 1: Fig. S1A). This BAC reporter construct was introduced into the zebrafish genome using the Tol2 transposon system [24, 25] and the stable transgenic line Tg(nkx6.1:eGFP) obtained showed expression of green fluorescent protein (GFP) in the nervous system and in the pancreas, which mirrors the endogenous Nkx6.1 protein expression (Additional file 1: Fig. S1B). Detailed comparison of the localization of these two proteins in the pancreas during development confirmed that GFP is indeed co-expressed with Nkx6.1 (Fig. 1). Indeed, together with the endogenous Nkx6.1 protein [18], GFP is expressed at the base of the endocrine islet at 24 and 30 hpf (Fig. 1b, c), in the ventral bud at 38 and 48 hpf (Fig. 1d, e), and in IPDs and EPDs at 4 days post fertilization (dpf) (Fig. 1f, f'). In contrast, at earlier stages, GFP was detected in a subset of Nkx6.1+ cells, probably due to the delay of GFP expression compared to Nkx6.1. Indeed, at 17 hpf, about 75 % of the Nkx6.1+ cells showed detectable GFP expression (Fig. 1a, a') and at 14 hpf, this proportion dropped even further to about 25–30 % (data not shown). Conversely, a few hours after the onset of nkx6.1 gene expression, some GFP+/Nkx6.1– cells were also detected (green arrows, Fig. 1b'–e'). This GFP labeling is not the result of an ectopic expression of the gfp transcript, as double fluorescent whole-mount in situ hybridization (WISH) showed that the gfp transcripts are present in the same pancreatic domain as nkx6.1 transcripts (data not shown) and importantly, like nkx6.1, gfp transcripts were not found in hormone-expressing cells (Additional file 1: Fig. S1C–C'', D–D''). Hence, prolonged GFP detection is rather due to the well-known high stability of GFP (±24 h half-life [26]), which persists in cells where Nkx6.1 protein is no longer found. This is nicely illustrated at 30 hpf, where strong GFP expression is detected at the base of the forming islet where Nkx6.1+ pancreatic progenitors are located, while weak GFP labeling is found dorsally within the islet, where differentiated endocrine cells, devoid of Nkx6.1, are clustered (Fig. 1c). As expected, this prolonged GFP detection will gradually fade away, finally to disappear completely in the differentiated endocrine cells (Fig. 1f).Fig. 1

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.