314 Diabetes Volume 65, February 2016 Jimmy Masjkur,1 Steven W. Poser,1 Polyxeni Nikolakopoulou,1 George Chrousos,2 Ronald D. McKay,3 Stefan R. Bornstein,1 Peter M. Jones,4 and Andreas Androutsellis-Theotokis1,5,6 Endocrine Pancreas Development and Regeneration: Noncanonical Ideas From Neural Stem Cell Biology Diabetes 2016;65:314–330 | DOI: 10.2337/db15-1099 Loss of insulin-producing pancreatic islet b-cells is a regeneration (1–4). Such plasticity in the adult brain is hallmark of type 1 diabetes. Several experimental para- exhibited only by endogenous neural stem cell (NSC) digms demonstrate that these cells can, in principle, be populations. How NSCs manage to maintain this plas- regenerated from multiple endogenous sources using ticity is an intensely studied question that has led to the signaling pathways that are also used during pancreas identification of unusual (noncanonical) signal trans- development. A thorough understanding of these path- duction pathways that help them manage the cellular ways will provide improved opportunities for therapeutic properties that define them as stem cells, namely the intervention. It is now appreciated that signaling path- ability to self-renew and the potential to differentiate “ ” “ ” ways should not be seen as on or off but that the into mature cell types. Part of this noncanonical ma- degree of activity may result in wildly different cellular chinery is a novel branch of the Notch signaling path- outcomes. In addition to the degree of operation of a way that has pronounced consequences in vitro and in signaling pathway, noncanonical branches also play im- vivo (5). It is logical to wonder whether other tissues portant roles. Thus, a pathway, once considered as “off” or “low” may actually be highly operational but may be also use these signaling pathways. using noncanonical branches. Such branches are only Plasticity in the pancreas is demonstrated by multiple now revealing themselves as new tools to assay them examples of trans- and dedifferentiation among various are being generated. A formidable source of noncanon- cell types reported; these have been experimentally PERSPECTIVES IN DIABETES ical signal transduction concepts is neural stem cells induced, whether by pharmacological or genetic manipu- because these cells appear to have acquired unusual lation or by the implementation of damage and regener- signaling interpretations to allow them to maintain their ation models (1,6–12). An impressive amount of work unique dual properties (self-renewal and multipotency). has been done identifying the molecular mechanisms that We discuss how such findings from the neural field can control this plasticity. The patterns that emerge indicate provide a blueprint for the identification of new molec- that a variety of signaling molecules and transcription ular mechanisms regulating pancreatic biology, with a factors act during development to both suppress and in- focus on Notch, Hes/Hey, and hedgehog pathways. duce fates as well as to maintain proper function of the adult cell types, including the Notch and hedgehog path- ways and a number of transcription factors, including To the neuroscientist, the pancreas can seem like a pancreatic and duodenal homeobox 1 (Pdx1), neurogenin highlyplasticorganwhosecellsareabletoundergovast 3 (Ngn3), and the hairy and enhancer of split-1 (Hes/Hey) changes in the context of homeostatic control and family (1,13–42). 1Department of Internal Medicine III, Technische Universität Dresden, Dresden, Corresponding author: Andreas Androutsellis-Theotokis, andreas.theotokis@ Germany uniklinikum-dresden.de. 2 First Department of Pediatrics, University of Athens Medical School and Aghia Received 6 August 2015 and accepted 2 November 2015. Sophia Children’s Hospital, Athens, Greece J.M. and S.W.P. contributed equally to this work. 3Lieber Institute for Brain Development, Baltimore, MD 4Diabetes Research Group, Division of Diabetes & Nutritional Sciences, King’s © 2016 by the American Diabetes Association. Readers may use this article as College London, London, U.K. long as the work is properly cited, the use is educational and not for profit, and 5Center for Regenerative Therapies Dresden, Dresden, Germany the work is not altered. 6Department of Stem Cell Biology, Centre for Biomolecular Sciences, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham, Nottingham, U.K. diabetes.diabetesjournals.org Masjkur and Associates 315 A binary view of signal transduction (where a partic- Notch signaling is critical in the maintenance of pancre- ular pathway is either on or off) seems to be incomplete atic progenitors (24,35,47,48). Specifically, loss of function as emerging evidence points toward several examples of the Notch ligand Delta-like 1 (Dll1) results in premature where low activity of a particular pathway (e.g., Notch endocrine differentiation and depletion of the pancreatic and hedgehog) leads to vastly different developmental and progenitors (24,35). cellular outcomes. But a simply quantitative distinction Conversely, overexpression of the Notch intracellular between low and high activity of a signaling pathway domain prevents differentiation and traps cells in a may also not be sufficient. A cell that is exhibiting low progenitor state (48). This appears to be due to caNotch activity of a canonical pathway may actually also exhibit signaling because similar phenotypes involving pancreatic high activity in noncanonical pathways that are only now growth arrest are observed with genetic mouse models becoming revealed. For example, whereas canonical Notch deficient in recombining binding protein suppressor of (caNotch) signaling is often assessed by the amount of hairless (RBPJ-k), a transcriptional mediator of caNotch Hes1 expression (a caNotch pathway target gene), it is signaling (14) or Hes1 (24,35,46,49). Apart from Hes1, possible that the cell exhibits high expression of, for Notch signaling also regulates Sox9, a transcription factor example, Hes3, an indirect, noncanonical target of that, like Hes1, has multiple developmental roles in many Notch (noncaNotch) with distinct outcomes in NSCs tissues (2,46,50–54). The molecular mechanisms that (43). Low activity of a signaling pathway, as assessed by control Hes1 are very important for transcriptional con- measurements of its canonical functions, does not pre- trol and the responsiveness of pancreatic progenitors to cludethatotherbranchesofthesamepathwayarenot mitogenic support from the surrounding mesenchyme. In highly active. Noncanonicalbranchesmaybeunidenti- one example, Hes1 opposes expression of p57, which nor- fiedsimplybecausewehavenotyetdesignedassaysfor mally induces cell cycle exit (55). Notch activity is neces- them. sary for the cells to respond to fibroblast growth factor NSCs are a treasure trove of noncanonical functions, (FGF) 10, a mitogen expressed by the mesenchyme at perhaps because they are required to fit multiple proper- ;E9.5–11.5 (56). In fact, inhibition of Notch cleavage ties into their signal transduction machinery (e.g., their by a g-secretase inhibitor renders cells nonresponsive to self-renewal and cell fate–decision abilities), forcing them the proliferative effects of FGF10 (57–59). to come up with unique molecular solutions. Some of Later in development, these progenitors become more these have already proven important in pancreatic func- restricted in their differentiation potential and separate tion. We will focus on lessons learned about noncaNotch into two cell types: unipotent acinar progenitor “tip cells” and hedgehog signaling in NSCs as a paradigm for their and bipotent “trunk epithelial” cells that can generate potential roles in b-cell development and function. First, endocrine and duct cells. High caNotch signaling, as we discuss specific aspects of pancreatic development on assessed by the involvement of RBPJ-k and Hes1, pro- which light can be shed by our emerging understanding of motes the trunk fate (47,48,60–64). High caNotch signal- noncanonical signaling in NSCs. We do not aim to provide ing in trunk cells activates Hes1 and Sox9 (a repressor and a comprehensive review of the developmental processes an activator of Ngn3, respectively). On the one hand, of the pancreas because this has been meticulously done Hes1 activity apparently dominates this fight, leading to elsewhere (1–4). low Ngn3 levels and, subsequently, specification to the ductal fate. Low caNotch activity, on the other hand, OVERVIEW OF PANCREAS DEVELOPMENT AND leads to induction of only Sox9, leading to high Ngn3 INVOLVEMENT OF KEY PATHWAYS levels and the acquisition of the endocrine fate (3,65). In this section we present a brief overview of specific The interpretation of high versus low Notch activity aspects in pancreas development (Fig. 1). We focus only on is an important and recurring theme throughout this particular pathways that are of direct relevance to the con- perspective. cepts discussed here. At approximately mouse embryonic In this article, we discuss data acquired from different day 8.5 (E8.5), a prepancreatic region is specified in the gut developmental stages of the endocrine pancreas and also endoderm (44). For pancreatic specification to occur and from the exocrine pancreas; these observations point toward pancreatic markers to be induced, sonic hedgehog (Shh) novel regulatory signaling networks that may be of signif- expression must be suppressed from the presumptive icant interest to pancreas biology. However, additional pancreatic endoderm (31,33,34). Subsequently,