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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.


Expression of known pancreatic markers in the transcriptome of adult nkx6.1:eGFP cells. Expression values are expressed as normalized counts. High expression of known ductal genes (orange box) is detected while acinar (green box) and endocrine (red box) markers display low abundance, with the exception of pdx1 and ascl1b (see text)
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Fig10: Expression of known pancreatic markers in the transcriptome of adult nkx6.1:eGFP cells. Expression values are expressed as normalized counts. High expression of known ductal genes (orange box) is detected while acinar (green box) and endocrine (red box) markers display low abundance, with the exception of pdx1 and ascl1b (see text)

Mentions: To get a comprehensive characterization of pancreatic nkx6.1+ ductal cells in adult zebrafish, we determined their transcriptome landscape. Ductal GFP+ cells were isolated from dissected pancreases of adult Tg(nkx6.1:eGFP) fish (with ~95 % purity) and used in RNA-seq experiments. The expression of 15,888 genes could be detected in the ductal transcriptome (complete data available at [38]), in which many genes already known to be expressed in pancreatic ducts in either zebrafish or mammals were found at high expression level, such as sox9b, hnf1ba, onecut1/hnf6, cftr, cdh17, ca2, and ctgfa in addition to nkx6.1 (Fig. 10). We also detected expression of fgfr4 and sdc4, recently proposed as novel ductal markers in the murine embryonic pancreas [39]. In contrast to these ductal genes, the acinar markers ptf1a and rbpjl, the pan-endocrine markers pax6b and isl1, and the lineage specific genes mnx1 and arx were either not detected or detected at extremely low levels in the nkx6.1:eGFP+ ductal cells transcriptome (Fig. 10), underscoring the accuracy of our fluorescence-activated cell sorting (FACS) cell preparations. In contrast to mouse or human adults in which the embryonic pancreatic progenitor marker Pdx1 is not expressed in the pancreatic ducts in normal condition, nkx6.1:eGFP+ duct cells of healthy zebrafish display a robust expression of pdx1. Comparison of the duct transcriptome with those of pancreatic acinar and endocrine cells (manuscript in preparation) highlighted 3,684 genes with preferential expression in duct cells. Among them, 293 duct-specific genes were identified with strong enrichment (≥16-fold) and low expression in the other pancreatic cells (Additional file 8: Table S1), in which we find sox9b, onecut1/hnf6, cdh17, ctgfa, and nkx6.1, corroborating their status as duct-specific markers. Various components of the Notch signaling pathway could also be identified, namely notch2, and different Hairy and enhancer of split-related genes (her6, her9, and her15.1), confirming that a subpopulation of nkx6.1+ cells (the CACs) experiences Notch activity. In addition to genes involved in the Notch signaling cascade, our analyses also identified novel duct-specific markers such as id2a, encoding for a HLH transcription factor, which we also detected co-expressed with sox9b by WISH in 3-dpf larvae (Additional file 9: Fig. S8), and several components of the Wnt pathways such as the Wnt and SFRP ligands sfrp5, sfrp3/frzb, and wnt7bb (Additional file 8: Table S1). At a lower expression level, we could also detect the Wnt receptor fzd7a, which was the only Wnt receptor significantly expressed in the adult pancreas (565 ± 106 normalized counts with 68-fold enrichment in the ducts). These observations suggest that Wnt signaling may play an important role in adult ducts.Fig. 10


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)

Expression of known pancreatic markers in the transcriptome of adult nkx6.1:eGFP cells. Expression values are expressed as normalized counts. High expression of known ductal genes (orange box) is detected while acinar (green box) and endocrine (red box) markers display low abundance, with the exception of pdx1 and ascl1b (see text)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig10: Expression of known pancreatic markers in the transcriptome of adult nkx6.1:eGFP cells. Expression values are expressed as normalized counts. High expression of known ductal genes (orange box) is detected while acinar (green box) and endocrine (red box) markers display low abundance, with the exception of pdx1 and ascl1b (see text)
Mentions: To get a comprehensive characterization of pancreatic nkx6.1+ ductal cells in adult zebrafish, we determined their transcriptome landscape. Ductal GFP+ cells were isolated from dissected pancreases of adult Tg(nkx6.1:eGFP) fish (with ~95 % purity) and used in RNA-seq experiments. The expression of 15,888 genes could be detected in the ductal transcriptome (complete data available at [38]), in which many genes already known to be expressed in pancreatic ducts in either zebrafish or mammals were found at high expression level, such as sox9b, hnf1ba, onecut1/hnf6, cftr, cdh17, ca2, and ctgfa in addition to nkx6.1 (Fig. 10). We also detected expression of fgfr4 and sdc4, recently proposed as novel ductal markers in the murine embryonic pancreas [39]. In contrast to these ductal genes, the acinar markers ptf1a and rbpjl, the pan-endocrine markers pax6b and isl1, and the lineage specific genes mnx1 and arx were either not detected or detected at extremely low levels in the nkx6.1:eGFP+ ductal cells transcriptome (Fig. 10), underscoring the accuracy of our fluorescence-activated cell sorting (FACS) cell preparations. In contrast to mouse or human adults in which the embryonic pancreatic progenitor marker Pdx1 is not expressed in the pancreatic ducts in normal condition, nkx6.1:eGFP+ duct cells of healthy zebrafish display a robust expression of pdx1. Comparison of the duct transcriptome with those of pancreatic acinar and endocrine cells (manuscript in preparation) highlighted 3,684 genes with preferential expression in duct cells. Among them, 293 duct-specific genes were identified with strong enrichment (≥16-fold) and low expression in the other pancreatic cells (Additional file 8: Table S1), in which we find sox9b, onecut1/hnf6, cdh17, ctgfa, and nkx6.1, corroborating their status as duct-specific markers. Various components of the Notch signaling pathway could also be identified, namely notch2, and different Hairy and enhancer of split-related genes (her6, her9, and her15.1), confirming that a subpopulation of nkx6.1+ cells (the CACs) experiences Notch activity. In addition to genes involved in the Notch signaling cascade, our analyses also identified novel duct-specific markers such as id2a, encoding for a HLH transcription factor, which we also detected co-expressed with sox9b by WISH in 3-dpf larvae (Additional file 9: Fig. S8), and several components of the Wnt pathways such as the Wnt and SFRP ligands sfrp5, sfrp3/frzb, and wnt7bb (Additional file 8: Table S1). At a lower expression level, we could also detect the Wnt receptor fzd7a, which was the only Wnt receptor significantly expressed in the adult pancreas (565 ± 106 normalized counts with 68-fold enrichment in the ducts). These observations suggest that Wnt signaling may play an important role in adult ducts.Fig. 10

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.