The what,where, when and how of Wnt/β-catenin signaling in pancreas development

The Wnt/β-catenin signaling pathway, conserved across the animal kingdom, is critical for the development of numerous tissues. Several recent studies have focused on the roles that this pathway plays at different stages of pancreatic organogenesis, including specification, proliferation, differentiation and function. Whereas, during early endoderm development, inhibition of the pathway is required for pancreatic specification, subsequent growth and differentiation of the fetal organ depends on the pathway being active. This appears especially true for exocrine acinar cells, the specification and differentiation of which also depend on β-catenin function. Whether the pathway plays an important role in development or function of endocrine islet cells, including insulin-producing β-cells, remains controversial. This question is particularly important in light of recent studies that implicate a downstream component of the pathway, TCF7L2, in human β-cell function. This review will cover recent work on Wnt/β-catenin signaling in pancreas development, emphasizing those points of controversy that most urgently require further investigation.     

The what, where, when and how of Wnt/β-catenin signaling in pancreas development

The Wnt/β-catenin signaling pathway, conserved across the animal kingdom, is critical for the development of numerous tissues. Several recent studies have focused on the roles that this pathway plays at different stages of pancreatic organogenesis, including specification, proliferation, differentiation and function. Whereas, during early endoderm development, inhibition of the pathway is required for pancreatic specification, subsequent growth and differentiation of the fetal organ depends on the pathway being active. This appears especially true for exocrine acinar cells, the specification and differentiation of which also depend on β-catenin function. Whether the pathway plays an important role in development or function of endocrine islet cells, including insulin-producing β-cells, remains controversial. This question is particularly important in light of recent studies that implicate a downstream component of the pathway, TCF7L2, in human β-cell function. This review will cover recent work on Wnt/β-catenin signaling in pancreas development, emphasizing those points of controversy that most urgently require further investigation.     

Pancreatic mesenchyme regulates epithelial organogenesis throughout development

The developing pancreatic epithelium gives rise to all endocrine and exocrine cells of the mature organ. During organogenesis, the epithelial cells receive essential signals from the overlying mesenchyme. Previous studies, focusing on ex vivo tissue explants or complete knockout mice, have identified an important role for the mesenchyme in regulating the expansion of progenitor cells in the early pancreas epithelium. However, due to the lack of genetic tools directing expression specifically to the mesenchyme, the potential roles of this supporting tissue in vivo, especially in guiding later stages of pancreas organogenesis, have not been elucidated. We employed transgenic tools and fetal surgical techniques to ablate mesenchyme via Cre-mediated mesenchymal expression of Diphtheria Toxin (DT) at the onset of pancreas formation, and at later developmental stages via in utero injection of DT into transgenic mice expressing the Diphtheria Toxin receptor (DTR) in this tissue. Our results demonstrate that mesenchymal cells regulate pancreatic growth and branching at both early and late developmental stages by supporting proliferation of precursors and differentiated cells, respectively. Interestingly, while cell differentiation was not affected, the expansion of both the endocrine and exocrine compartments was equally impaired. To further elucidate signals required for mesenchymal cell function, we eliminated β-catenin signaling and determined that it is a critical pathway in regulating mesenchyme survival and growth. Our study presents the first in vivo evidence that the embryonic mesenchyme provides critical signals to the epithelium throughout pancreas organogenesis. The findings are novel and relevant as they indicate a critical role for the mesenchyme during late expansion of endocrine and exocrine compartments. In addition, our results provide a molecular mechanism for mesenchymal expansion and survival by identifying β-catenin signaling as an essential mediator of this process. These results have implications for developing strategies to expand pancreas progenitors and β-cells for clinical transplantation.     

Remodeling of insulin producing β-cells during Xenopus laevis metamorphosis

Insulin-producing β-cells are present as single cells or in small clusters distributed throughout the pancreas of the Xenopus laevis tadpole. During metamorphic climax when the exocrine pancreas dedifferentiates to progenitor cells, the β-cells undergo two changes. Insulin mRNA is down regulated at the beginning of metamorphic climax (NF62) and reexpressed again near the end of climax. Secondly, the β-cells aggregate to form islets. During climax the increase in insulin cluster size is not caused by cell proliferation or by acinar-to-β-cell transdifferentiation, but rather is due to the aggregation of pre-existing β-cells. The total number of β-cells does not change during the 8 days of climax. Thyroid hormone (TH) induction of premetamorphic tadpoles causes an increase in islet size while prolonged treatment of tadpoles with the goitrogen methimazole inhibits this increase. Expression of a dominant negative form of the thyroid hormone receptor (TRDN) driven by the elastase promoter not only protects the exocrine pancreas of a transgenic tadpole from TH-induced dedifferentiation but also prevents aggregation of β-cells at climax. These transgenic tadpoles do however undergo normal loss and resynthesis of insulin mRNA at the same stage as controls. In contrast transgenic tadpoles with the same TRDN transgene driven by an insulin promoter do not undergo down regulation of insulin mRNA, but do aggregate β-cells to form islets like controls. These results demonstrate that TH controls the remodeling of β-cells through cell-cell interaction with dedifferentiating acinar cells and a cell autonomous program that temporarily shuts off the insulin gene.     

DTA-mediated targeted ablation revealed differential interdependence of endocrine cell lineages in early development of zebrafish pancreas

Cell lineage analysis is critical in understanding the relationship between progenitors and differentiated cells as well as the mechanism underlying the process of differentiation. In order to study the zebrafish endocrine pancreas cell lineage, transgenic expression of diphtheria toxin gene A chain (DTA) under two cell type-specific promoters derived from the insulin (ins) and somatostatin2 (sst2) genes was used to ablate the two types of endocrine cells: insulin-producing β-cells and somatostatin-producing δ-cells, respectively. We found that ablation of β-cells resulted in a reduction of not only β-cells but also glucagon-producing α-cells; in contrast, δ-cells were largely unaffected. Ablation of δ-cells led to reduction of all three types of endocrine cells: α-, β-, and δ. Interestingly, α-cells were more profoundly affected in both β- and δ-cell ablations and were frequently reduced together with β- and δ-cells. By taking advantage of Tg(ins:gfp) and Tg(sst2:gfp) lines, we also monitored the changes of different types of endocrine cells in vivo after ablation and found that both β- and δ-cell populations significantly recovered by 3 dpf after their ablation and it seemed that δ-cells had a better capability of recovery than β-cells. Thus, our current observations indicated differential interdependence of these three cell lineages. The development of zebrafish α-cells, but not δ-cells, is dependent on β-cells, while the development of both α- and β-cells is dependent on δ-cells. In contrast, the development of δ-cells was independent of β-cells.     

Genetic inducible fate mapping in larval zebrafish reveals origins of adult insulin-producing β-cells

The Notch-signaling pathway is known to be fundamental in controlling pancreas differentiation. We now report on using Cre-based fate mapping to indelibly label pancreatic Notch-responsive cells (PNCs) at larval stages and follow their fate in the adult pancreas. We show that the PNCs represent a population of progenitors that can differentiate to multiple lineages, including adult ductal cells, centroacinar cells (CACs) and endocrine cells. These endocrine cells include the insulin-producing β-cells. CACs are a functional component of the exocrine pancreas; however, our fate-mapping results indicate that CACs are more closely related to endocrine cells by lineage as they share a common progenitor. The majority of the exocrine pancreas consists of the secretory acinar cells; however, we only detect a very limited contribution of PNCs to acinar cells. To explain this observation we re-examined early events in pancreas formation. The pancreatic anlage that gives rise to the exocrine pancreas is located in the ventral gut endoderm (called the ventral bud). Ptf1a is a gene required for exocrine pancreas development and is first expressed as the ventral bud forms. We used transgenic marker lines to observe both the domain of cells expressing ptf1a and cells responding to Notch signaling. We do not detect any overlap in expression and demonstrate that the ventral bud consists of two cell populations: a ptf1-expressing domain and a Notch-responsive progenitor core. As pancreas organogenesis continues, the ventral bud derived PNCs align along the duct, remain multipotent and later in development differentiate to form secondary islets, ducts and CACs.     

Nkx6.1 and nkx6.2 regulate α- and β-cell formation in zebrafish by acting on pancreatic endocrine progenitor cells

In mice, the Nkx6 genes are crucial to α- and β-cell differentiation, but the molecular mechanisms by which they regulate pancreatic subtype specification remain elusive. Here it is shown that in zebrafish, nkx6.1 and nkx6.2 are co-expressed at early stages in the first pancreatic endocrine progenitors, but that their expression domains gradually segregate into different layers, nkx6.1 being expressed ventrally with respect to the forming islet while nkx6.2 is expressed mainly in β-cells. Knockdown of nkx6.2 or nkx6.1 expression leads to nearly complete loss of α-cells but has no effect on β-, δ-, or ε-cells. In contrast, nkx6.1/nkx6.2 double knockdown leads additionally to a drastic reduction of β-cells. Synergy between the effects of nkx6.1 and nkx6.2 knockdown on both β- and α-cell differentiation suggests that nkx6.1 and nkx6.2 have the same biological activity, the required total nkx6 threshold being higher for α-cell than for β-cell differentiation. Finally, we demonstrate that the nkx6 act on the establishment of the pancreatic endocrine progenitor pool whose size is correlated with the total nkx6 expression level. On the basis of our data, we propose a model in which nkx6.1 and nkx6.2, by allowing the establishment of the endocrine progenitor pool, control α- and β-cell differentiation.     

Antagonistic interactions of hedgehog, Bmp and retinoic acid signals control zebrafish endocrine pancreas development

Pancreatic organogenesis is promoted or restricted by different signaling pathways. In amniotes, inhibition of hedgehog (Hh) activity in the early embryonic endoderm is a prerequisite for pancreatic specification. However, in zebrafish, loss of Hh signaling leads to a severe reduction of β-cells, leading to some ambiguity as to the role of Hh during pancreas development and whether its function has completely diverged between species. Here, we have employed genetic and pharmacological manipulations to temporally delineate the role of Hh in zebrafish endocrine pancreas development and investigate its relationship with the Bmp and retinoic acid (RA) signaling pathways. We found that Hh is required at the start of gastrulation for the medial migration and differentiation of pdx1-expressing pancreatic progenitors at later stages. This early positive role of Hh promotes β-cell lineage differentiation by restricting the repressive effects of Bmp. Inhibition of Bmp signaling in the early gastrula leads to increased β-cell numbers and partially rescued β-cell formation in Hh-deficient embryos. By the end of gastrulation, Hh switches to a negative role by antagonizing RA-mediated specification of the endocrine pancreas, but continues to promote differentiation of exocrine progenitors. We show that RA downregulates the Hh signaling components ptc1 and smo in endodermal explants, indicating a possible molecular mechanism for blocking axial mesoderm-derived Hh ligands from the prepancreatic endoderm during the specification stage. These results identify multiple sequential roles for Hh in pancreas development and highlight an unexpected antagonistic relationship between Hh and other signaling pathways to control pancreatic specification and differentiation.     

Sox9+ ductal cells are multipotent progenitors throughout development but do not produce new endocrine cells in the normal or injured adult pancreas

One major unresolved question in the field of pancreas biology is whether ductal cells have the ability to generate insulin-producing β-cells. Conclusive examination of this question has been limited by the lack of appropriate tools to efficiently and specifically label ductal cells in vivo. We generated Sox9CreER(T2) mice, which, during adulthood, allow for labeling of an average of 70% of pancreatic ductal cells, including terminal duct/centroacinar cells. Fate-mapping studies of the Sox9(+) domain revealed endocrine and acinar cell neogenesis from Sox9(+) cells throughout embryogenesis. Very small numbers of non-β endocrine cells continue to arise from Sox9(+) cells in early postnatal life, but no endocrine or acinar cell neogenesis from Sox9(+) cells occurs during adulthood. In the adult pancreas, pancreatic injury by partial duct ligation (PDL) has been suggested to induce β-cell regeneration from a transient Ngn3(+) endocrine progenitor cell population. Here, we identify ductal cells as a cell of origin for PDL-induced Ngn3(+) cells, but fail to observe β-cell neogenesis from duct-derived cells. Therefore, although PDL leads to activation of Ngn3 expression in ducts, PDL does not induce appropriate cues to allow for completion of the entire β-cell neogenesis program. In conclusion, although endocrine cells arise from the Sox9(+) ductal domain throughout embryogenesis and the early postnatal period, Sox9(+) ductal cells of the adult pancreas no longer give rise to endocrine cells under both normal conditions and in response to PDL.     

Retinoic Acid Promotes the Generation of Pancreatic Endocrine Progenitor Cells and Their Further Differentiation into β-Cells

The identification of secreted factors that can selectively stimulate the generation of insulin producing β-cells from stem and/or progenitor cells represent a significant step in the development of stem cell-based β-cell replacement therapy. By elucidating the molecular mechanisms that regulate the generation of β-cells during normal pancreatic development such putative factors may be identified. In the mouse, β-cells increase markedly in numbers from embryonic day (e) 14.5 and onwards, but the extra-cellular signal(s) that promotes the selective generation of β-cells at these stages remains to be identified. Here we show that the retinoic acid (RA) synthesizing enzyme Raldh1 is expressed in developing mouse and human pancreas at stages when β-cells are generated. We also provide evidence that RA induces the generation of Ngn3⁺ endocrine progenitor cells and stimulates their further differentiation into β-cells by activating a program of cell differentiation that recapitulates the normal temporal program of β-cell differentiation.     

Recent progress in studies of factors that elicit pancreatic β-cell expansion

The loss of or decreased functional pancreatic β-cell is a major cause of type 1 and type 2 diabetes. Previous studies have shown that adult β-cells can maintain their ability for a low level of turnover through replication and neogenesis. Thus, a strategy to prevent and treat diabetes would be to enhance the ability of β-cells to increase the mass of functional β-cells. Consequently, much effort has been devoted to identify factors that can effectively induce β-cell expansion. This review focuses on recent reports on small molecules and protein factors that have been shown to promote β-cell expansion.     

Role of adenosine signalling and metabolism in β-cell regeneration

Glucose homeostasis, which is controlled by the endocrine cells of the pancreas, is disrupted in both type I and type II diabetes. Deficiency in the number of insulin-producing β cells – a primary cause of type I diabetes and a secondary contributor of type II diabetes – leads to hyperglycemia and hence an increase in the need for insulin. Although diabetes can be controlled with insulin injections, a curative approach is needed. A potential approach to curing diabetes involves regenerating the β-cell mass, e.g. by increasing β-cell proliferation, survival, neogenesis or transdifferentiation. The nucleoside adenosine and its cognate nucleotide ATP have long been known to affect insulin secretion, but have more recently been shown to increase β-cell proliferation during homeostatic control and regeneration of the β-cell mass. Adenosine is also known to have anti-inflammatory properties, and agonism of adenosine receptors can promote the survival of β-cells in an inflammatory microenvironment. In this review, both intracellular and extracellular mechanisms of adenosine and ATP are discussed in terms of their established and putative effects on β-cell regeneration.     

Dnmt1 activity is dispensable in δ-cells but is essential for α-cell homeostasis

In addition to β-cells, pancreatic islets contain α- and δ-cells, which respectively produce glucagon and somatostatin. The reprogramming of these two endocrine cell types into insulin producers, as observed after a massive β-cell ablation in mice, may help restoring a functional β-cell mass in type 1 diabetes. Yet, the spontaneous α-to-β and δ-to-β conversion processes are relatively inefficient in adult animals and the underlying epigenetic mechanisms remain unclear. Several studies indicate that the conserved chromatin modifiers DNA methyltransferase 1 (Dnmt1) and Enhancer of zeste homolog 2 (Ezh2) are important for pancreas development and restrict islet cell plasticity. Here, to investigate the role of these two enzymes in α- and δ-cell development and fate maintenance, we genetically inactivated them in each of these two cell types. We found that loss of Dnmt1 does not enhance the conversion of α- or δ-cells toward a β-like fate. In addition, while Dnmt1 was dispensable for the development of these two cell types, we noticed a gradual loss of α-, but not δ-cells in adult mice. Finally, we found that Ezh2 inactivation does not enhance α-cell plasticity, and, contrary to what is observed in β-cells, does not impair α-cell proliferation. Our results indicate that both Dnmt1 and Ezh2 play distinct roles in the different islet cell types.     

Notch/Rbp-j signaling prevents premature endocrine and ductal cell differentiation in the pancreas

To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas.