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

Abstract

BackgroundIn 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.ResultsUsing 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0179-4) contains supplementary material, which is available to authorized users.

Centroacinar cells: At the center of pancreas regeneration

“…However, the regenerative response of the mammalian pancreas can vary depending on the injury model (Criscimanna et al, 2011; Kopp et al, 2011; Lee et al, 2006; Li et al, 2010; Nagasao et al, 2005; Xu et al, 2008), making uncovering mechanisms of regeneration in those animals difficult. On the other hand, as we will discuss here, the zebrafish pancreas displays a remarkable capacity for regeneration (Delaspre et al, 2015; Ghaye et al, 2015; Moss et al, 2009). A detailed understanding of how the fish pancreas regenerates is thus the critical first step towards achieving endogenous β-cell replacement therapy in humans.…”

“…Blocking Notch activity leads to precocious differentiation of CACs into NeuroD-expressing endocrine precursors (Dalgin and Prince, 2015; Parsons et al, 2009). Importantly, larval zebrafish CACs express several major transcriptional regulators of endocrine differentiation such as Sox9b, Nkx6.1, and Nkx2.2a (Delaspre et al, 2015; Ghaye et al, 2015; Manfroid et al, 2012; Pauls et al, 2007) (Figure 3C–D). Inducible genetic lineage tracing of CACs at 5 dpf using the Notch-responsive tp1 promoter has demonstrated that larval CACs give rise to ductal and endocrine cells in the larval and adult pancreas (Wang et al, 2011).…”

“…Two independent groups utilized the NTR/MTZ system to specifically ablate >99% of β cells in the adult (Delaspre et al, 2015; Ghaye et al, 2015). Following ablation, single β cells and β-cell clusters not associated with islets appear along ducts and near CACs, suggesting that β-cell neogenesis is one mechanism by which the pancreas regenerates (Delaspre et al, 2015).…”

“…Following ablation, single β cells and β-cell clusters not associated with islets appear along ducts and near CACs, suggesting that β-cell neogenesis is one mechanism by which the pancreas regenerates (Delaspre et al, 2015). CACs proliferate (Delaspre et al, 2015; Ghaye et al, 2015) and respond dynamically during regeneration by moving closer and even into recovering islets (Delaspre et al, 2015). Using either the Notch-responsive tp1 or the nkx6.1 promoter driving GFP expression, each group observed GFP perdurance in cells beginning to express Insulin, suggesting that CACs themselves contribute to β-cell regeneration (Delaspre et al, 2015; Ghaye et al, 2015).…”

“…Recently, these cells have been revealed to be specialized ductal cells called centroacinar cells (CACs) (Delaspre et al, 2015), which also serve as progenitors to regenerate β cells in the adult zebrafish (Delaspre et al, 2015; Ghaye et al, 2015). CACs are a cell type common to all vertebrates and have several defining characteristics: they are a ductal cell type positioned at the center of acini, with unique cell morphology, active Notch signaling, and expression of the endocrine differentiation regulator Sox9 (Kopp et al, 2011; Manfroid et al, 2012; Seymour, 2014; Seymour et al, 2008; Shih et al, 2012).…”