endocrine pancreas development and regeneration ...jimmy masjkur,1 steven w. poser,1 polyxeni...

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 andAndreas Androutsellis-Theotokis1,5,6

Endocrine Pancreas Development andRegeneration: Noncanonical IdeasFrom Neural Stem Cell BiologyDiabetes 2016;65:314–330 | DOI: 10.2337/db15-1099

Loss of insulin-producing pancreatic islet b-cells is ahallmark of type 1 diabetes. Several experimental para-digms demonstrate that these cells can, in principle, beregenerated from multiple endogenous sources usingsignaling pathways that are also used during pancreasdevelopment. A thorough understanding of these path-ways will provide improved opportunities for therapeuticintervention. It is now appreciated that signaling path-ways should not be seen as “on” or “off” but that thedegree of activity may result in wildly different cellularoutcomes. In addition to the degree of operation of asignaling pathway, noncanonical branches also play im-portant roles. Thus, a pathway, once considered as “off”or “low” may actually be highly operational but may beusing noncanonical branches. Such branches are onlynow revealing themselves as new tools to assay themare being generated. A formidable source of noncanon-ical signal transduction concepts is neural stem cellsbecause these cells appear to have acquired unusualsignaling interpretations to allow them to maintain theirunique dual properties (self-renewal and multipotency).We discuss how such findings from the neural field canprovide a blueprint for the identification of new molec-ular mechanisms regulating pancreatic biology, with afocus on Notch, Hes/Hey, and hedgehog pathways.

To the neuroscientist, the pancreas can seem like ahighly plastic organ whose cells are able to undergo vastchanges in the context of homeostatic control and

regeneration (1–4). Such plasticity in the adult brain isexhibited only by endogenous neural stem cell (NSC)populations. How NSCs manage to maintain this plas-ticity is an intensely studied question that has led to theidentification of unusual (noncanonical) signal trans-duction pathways that help them manage the cellularproperties that define them as stem cells, namely theability to self-renew and the potential to differentiateinto mature cell types. Part of this noncanonical ma-chinery is a novel branch of the Notch signaling path-way that has pronounced consequences in vitro and invivo (5). It is logical to wonder whether other tissuesalso use these signaling pathways.

Plasticity in the pancreas is demonstrated by multipleexamples of trans- and dedifferentiation among variouscell types reported; these have been experimentallyinduced, whether by pharmacological or genetic manipu-lation or by the implementation of damage and regener-ation models (1,6–12). An impressive amount of workhas been done identifying the molecular mechanisms thatcontrol this plasticity. The patterns that emerge indicatethat a variety of signaling molecules and transcriptionfactors act during development to both suppress and in-duce fates as well as to maintain proper function of theadult cell types, including the Notch and hedgehog path-ways and a number of transcription factors, includingpancreatic and duodenal homeobox 1 (Pdx1), neurogenin3 (Ngn3), and the hairy and enhancer of split-1 (Hes/Hey)family (1,13–42).

1Department of Internal Medicine III, Technische Universität Dresden, Dresden,Germany2First Department of Pediatrics, University of Athens Medical School and AghiaSophia Children’s Hospital, Athens, Greece3Lieber Institute for Brain Development, Baltimore, MD4Diabetes Research Group, Division of Diabetes & Nutritional Sciences, King’sCollege London, London, U.K.5Center for Regenerative Therapies Dresden, Dresden, Germany6Department of Stem Cell Biology, Centre for Biomolecular Sciences, Divisionof Cancer and Stem Cells, School of Medicine, University of Nottingham,Nottingham, U.K.

Corresponding author: Andreas Androutsellis-Theotokis, [email protected].

Received 6 August 2015 and accepted 2 November 2015.

J.M. and S.W.P. contributed equally to this work.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

314 Diabetes Volume 65, February 2016

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A binary view of signal transduction (where a partic-ular pathway is either on or off) seems to be incompleteas emerging evidence points toward several exampleswhere low activity of a particular pathway (e.g., Notchand hedgehog) leads to vastly different developmental andcellular outcomes. But a simply quantitative distinctionbetween low and high activity of a signaling pathwaymay also not be sufficient. A cell that is exhibiting lowactivity of a canonical pathway may actually also exhibithigh activity in noncanonical pathways that are only nowbecoming revealed. For example, whereas canonical Notch(caNotch) signaling is often assessed by the amount ofHes1 expression (a caNotch pathway target gene), it ispossible that the cell exhibits high expression of, forexample, Hes3, an indirect, noncanonical target ofNotch (noncaNotch) with distinct outcomes in NSCs(43). Low activity of a signaling pathway, as assessed bymeasurements of its canonical functions, does not pre-clude that other branches of the same pathway are nothighly active. Noncanonical branches may be unidenti-fied simply because we have not yet designed assays forthem.

NSCs are a treasure trove of noncanonical functions,perhaps because they are required to fit multiple proper-ties into their signal transduction machinery (e.g., theirself-renewal and cell fate–decision abilities), forcing themto come up with unique molecular solutions. Some ofthese have already proven important in pancreatic func-tion. We will focus on lessons learned about noncaNotchand hedgehog signaling in NSCs as a paradigm for theirpotential roles in b-cell development and function. First,we discuss specific aspects of pancreatic development onwhich light can be shed by our emerging understanding ofnoncanonical signaling in NSCs. We do not aim to providea comprehensive review of the developmental processesof the pancreas because this has been meticulously doneelsewhere (1–4).

OVERVIEW OF PANCREAS DEVELOPMENT ANDINVOLVEMENT OF KEY PATHWAYS

In this section we present a brief overview of specificaspects in pancreas development (Fig. 1). We focus only onparticular pathways that are of direct relevance to the con-cepts discussed here. At approximately mouse embryonicday 8.5 (E8.5), a prepancreatic region is specified in the gutendoderm (44). For pancreatic specification to occur andpancreatic markers to be induced, sonic hedgehog (Shh)expression must be suppressed from the presumptivepancreatic endoderm (31,33,34). Subsequently, duringearly pancreatic development (;E9.5; primary transitionphase), the pancreatic bud forms, which contains precur-sor cells (multipotent pancreatic precursors [MPCs]) thatare able to generate differentiated cells from all three ma-jor lineages of the adult pancreas (endocrine, duct, andacinar). These pancreatic progenitors express Pdx1, Sox9,and Hes1, a direct transcriptional target of caNotch sig-naling with multiple developmental roles (25,29,45,46).

Notch signaling is critical in the maintenance of pancre-atic progenitors (24,35,47,48). Specifically, loss of functionof the Notch ligand Delta-like 1 (Dll1) results in prematureendocrine differentiation and depletion of the pancreaticprogenitors (24,35).

Conversely, overexpression of the Notch intracellulardomain prevents differentiation and traps cells in aprogenitor state (48). This appears to be due to caNotchsignaling because similar phenotypes involving pancreaticgrowth arrest are observed with genetic mouse modelsdeficient in recombining binding protein suppressor ofhairless (RBPJ-k), a transcriptional mediator of caNotchsignaling (14) or Hes1 (24,35,46,49). Apart from Hes1,Notch signaling also regulates Sox9, a transcription factorthat, like Hes1, has multiple developmental roles in manytissues (2,46,50–54). The molecular mechanisms thatcontrol Hes1 are very important for transcriptional con-trol and the responsiveness of pancreatic progenitors tomitogenic support from the surrounding mesenchyme. Inone example, Hes1 opposes expression of p57, which nor-mally induces cell cycle exit (55). Notch activity is neces-sary for the cells to respond to fibroblast growth factor(FGF) 10, a mitogen expressed by the mesenchyme at;E9.5–11.5 (56). In fact, inhibition of Notch cleavageby a g-secretase inhibitor renders cells nonresponsive tothe proliferative effects of FGF10 (57–59).

Later in development, these progenitors become morerestricted in their differentiation potential and separateinto two cell types: unipotent acinar progenitor “tip cells”and bipotent “trunk epithelial” cells that can generateendocrine and duct cells. High caNotch signaling, asassessed by the involvement of RBPJ-k and Hes1, pro-motes the trunk fate (47,48,60–64). High caNotch signal-ing in trunk cells activates Hes1 and Sox9 (a repressor andan activator of Ngn3, respectively). On the one hand,Hes1 activity apparently dominates this fight, leading tolow Ngn3 levels and, subsequently, specification to theductal fate. Low caNotch activity, on the other hand,leads to induction of only Sox9, leading to high Ngn3levels and the acquisition of the endocrine fate (3,65).The interpretation of high versus low Notch activityis an important and recurring theme throughout thisperspective.

In this article, we discuss data acquired from differentdevelopmental stages of the endocrine pancreas and alsofrom the exocrine pancreas; these observations point towardnovel regulatory signaling networks that may be of signif-icant interest to pancreas biology. However, additionalfuture work will be necessary to fully elucidate at whichdevelopmental stages these networks may be operationaland with what precise consequences.

NSCs POINT US TOWARD NONCANONICALSIGNALING PATHWAYS THAT ALSO CONTROLPANCREATIC ISLET CELLS

In this section, we present work from the NSC field dem-onstrating alternative interpretations of common signal

diabetes.diabetesjournals.org Masjkur and Associates 315

transduction pathways, and in subsequent sections, weaddress and speculate how these pathways may con-tribute to the explanation of open questions in pancre-atic biology.

Special Cellular Requirements Force Special SignalTransduction InterpretationsNSCs appear to have acquired an inventive mechanism tofit both their self-renewal and multipotency properties

Figure 1—A simplified diagram of pancreatic development. In early pancreatic development, pancreatic progenitor cells coexpress Pdx1,Sox9, and Hes1. Low caNotch activity promotes the tip cell fate, which later generates acinar cells. In contrast, high caNotch activitypromotes the trunk cell fate, which coexpresses Sox9 and Hes1. In trunk cells, high caNotch activity promotes the generation of ductalcells that maintain Sox9 and Hes1 coexpression. In contrast, low caNotch activity generates the endocrine precursor fate. These cells loseSox9 and Hes1 expression and induce the expression of Ngn3. They are able to generate all cell types of the pancreatic islet, includingb-cells, which maintain low expression of Sox9 and Hes1. Adapted from Shih et al. (2).

316 Neural Stem Cell Clues for Pancreas Biology Diabetes Volume 65, February 2016

into the existing signal transduction machinery. Forexample, they have allocated some of these differentproperties to different phosphorylation sites of the samemolecule, such as those on signal transducer and activatorof transcription 3 (STAT3). STAT3 has two phosphor-ylation sites, one on the tyrosine residue (STAT3-Tyr) at amino acid position 705 (numbering is for themouse protein) and one on the serine residue at position727 (STAT3-Ser) (66). On the one hand, tyrosine phos-phorylation leads to the induction of differentiation,ending their stem cell state (67–69). Serine phosphory-lation, on the other hand, is important for their growthand, therefore, self-renewal (5). In stark contrast, manyother cell types that are only capable of self-renewaland therefore do not have issues of such choices (e.g.,astrocytes), use both phosphorylations for growthand survival (with, typically, the tyrosine site beingmuch more important than the serine site). In NSCs, anoncanonical branch of the Notch signaling pathway is amajor activator of STAT3-Ser phosphorylation (5).

Notch Signaling in Neural DevelopmentNotch signaling is a major regulator in brain develop-ment and the regulation of NSCs. The Notch signalingpathway is involved in a variety of biological process-es, including cell fate specification of many different

tissues, stem cell and progenitor growth, self-renewal,and differentiation; these functions are of direct conse-quence to development, regeneration, and cancer (14,50,60).The Notch family of genes consists of plasma membrane–spanning receptors that, upon activation by ligands (alsomembrane spanning) from adjacent cells, undergo a se-ries of proteolytic cleavages that release the intracellulardomain of the receptor into the cytoplasm. This subse-quently translocates to the nucleus where it interactswith other proteins such as the transcriptional regulatorRBPJ-k (14). A well-studied target gene in many tissuesis the transcription factor Hes1, a member of the Hes/Hey gene family of basic helix-loop-helix (bHLH) tran-scriptional repressors (50). The involvement of RBPJ-kand the induction of Hes1 are often used as indicators ofNotch activation. RBPJ-k and Hes1 involvement typicallydenote the operation of caNotch signaling (70–72) (Fig. 2),although RBPJ-k–independent functions of Notchhave also been reported (but also Notch-independentRBPJ-k functions).

The noncaNotch signaling branch that leads to STAT3-Ser phosphorylation also involves a number of otherintracellular signaling components (e.g., phosphoinositide3-kinase [PI3K], Akt, mammalian target of rapamycin[mTOR], etc.) as well as the transcription factor Hes3, amember of the Hes/Hey gene family like Hes1 and Hes5

Figure 2—caNotch and noncaNotch signaling branches control NSC self-renewal and differentiation. Notch receptor activation in NSCscan lead to canonical signaling induction that involves the release of the intracellular domain of Notch receptors into the cytoplasm, itsassociation with RBPJ-k and other proteins, and the induction of the transcription of genes, including Hes1. Hes1 promotes differentiationto the glial fate by activating the transcription of genes specific to the glial lineage. In addition, Hes1 can function in the cytoplasm where itmediates the interaction between JAK and STAT3, leading to STAT3-Tyr phosphorylation, which also promotes glial differentiation byactivating the transcription of genes specific to the glial lineage. Notch receptor activation can also lead to the activation of a noncanonicalsignaling branch that involves the sequential activation of PI3K, Akt, mTOR, and STAT3-Ser phosphorylation. This branch leads to in-duction of Hes3, an indirect target of Notch and a member of the same gene family as Hes1. Hes3 promotes the expression of Shh, amorphogen in neural development and mitogen of NSCs. This noncanonical branch promotes the survival of NSCs, and activation in vitroand in vivo increases NSC number. JAK activity inhibits the induction of Hes3 expression (through mechanisms that are not well un-derstood), demonstrating that the canonical and noncanonical pathways may compete for dominance.


(5,54,73). Therefore, Hes3 is also downstream of Notchsignaling but is not a direct transcriptional target like Hes1and Hes5; instead, Notch receptor activation leads to Hes3induction via Akt, mTOR, and STAT3-Ser phosphorylation.Notch ligands that lead to the induction of Hes3 expressiongreatly increase NSC numbers in vitro and in vivo (5,74).

These two Notch signaling branches (ca/Hes1 andnonca/Hes3) have opposing functions in NSCs. Hes1induces differentiation to the glial fate by directly activat-ing prodifferentiation genes and indirectly by promotingthe interaction of Janus kinase (JAK) with STAT3, whichfurther promotes gliogenic differentiation (66–68,75). Thisinvolves the phosphorylation of STAT3 by JAK on thetyrosine residue. Hes3, in contrast, is induced by severalfactors that lead to STAT3-Ser phosphorylation in theabsence of STAT3-Tyr phosphorylation (5).

Therefore, the Hes/Hey family of transcription factorshas multiple functions in the nucleus and cytoplasm,including transcriptional repression, passive repression(through protein-protein interactions), and the mediationof signal transduction. Elucidating these highly complexfunctions for all members of this gene family is likely tocontribute to our detailed understanding of a number ofprocesses where these genes are involved. Because Hes/Hey proteins interact with each other, typically leading tothe repression of their transcriptional activity, studyingthe expression patterns, subcellular localization, andfunction of all family members together in differentbiological systems may be useful.

Hes3 itself is a functional mediator of the non-canonical action of Notch signaling because NSCs fromadult Hes3-null mice fail to respond to Notch activa-tion that normally promotes survival (76). They alsofail to respond to insulin, another stimulator of Akt,mTOR, STAT3-Ser, and Hes3 (Fig. 3A). Hes3 also pro-motes Shh expression, which is a mitogen for NSCs (5).Therefore, Hes3 is an indirect target of Notch and rep-resents the operation of a noncanonical branch of Notchthat involves a number of intracellular signaling com-ponents, including STAT3 when it is phosphorylatedon the serine residue. In other words, this noncanonicalbranch of Notch is an input to the STAT3-Ser/Hes3 sig-naling pathway.

A Generalizable, Noncanonical Molecular Pathway?Deleting Hes3 in mice that already lack the Hes/Heygene family members Hes1 and Hes5 leads to precociousdifferentiation of the NSC population during braindevelopment (77). Aspects of this pathway are not lim-ited to NSCs but can be found in a number of plastic celltypes, including putative cancer stem cells from glioblas-toma multiforme patients and chromaffin progenitorcells from the bovine adrenal medulla (78,79). STAT3-Ser has been shown to drive carcinogenesis in models ofprostate cancer (80). Hes3 expression in vivo has beenimplicated as an indicator of the efficacy of a g-secretaseinhibitor (that blocks Notch signaling) in models of

breast cancer (81). Recent studies have implicatedHes3 (still largely in a correlative manner) in differentreprogramming paradigms (mouse embryonic fibroblaststo induced pluripotent cells and direct adult to inducedNSCs) by demonstrating Hes3 expression regulation dur-ing reprogramming and by correlating Hes3 transductionof adult cells with successful reprogramming to inducedNSCs (82,83).

These observations challenge the question of howwidespread the operation of this pathway may be indifferent cell types in the body, including in those of thepancreas. After all, STAT3-Ser phosphorylation is down-stream of several pathways known to promote b-cellproliferation in various developmental stages of the pan-creas, including FGF signaling (84,85), which has beenshown to intercept Notch signaling components and toregulate cell proliferation and self-renewal in both NSCand pancreatic systems (58,86), several cell surface re-ceptors, Akt, and mTOR. It is possible that Hes3 may bea mediator of some of the functions of these molecules.This would also raise the possibility that, being a bHLHfactor, Hes3 may interfere with the actions of otherbHLH factors of importance to pancreatic developmentand function such as Ngn3 (5,73,87,88). In another par-allel between brain and pancreas, Ngn3 has importantfunctions in the development of the brain (includinghippocampal and hypothalamic neurons and glial pro-genitors), islet cell specification, and b-cell regeneration(18,89–91).

Early data from mice lacking Hes3 corroborate some ofthese ideas because these mice exhibit phenotypes ofdirect relevance to pancreatic function; specifically,whereas otherwise apparently normal, these mice exhibita more pronounced loss of pancreatic b-cells, develop di-abetes faster (43), and show impaired regeneration (asassessed by b-cell marker expression) after streptozotocin(STZ)-induced pancreatic damage (Fig. 3B–D). Further,after damage they fail to induce Ngn3 expression in theexocrine pancreas compartment 5 months after partialSTZ damage compared with wild-type controls (92). Thesedata invite further studies on the possible developmentalroles of Hes3 and its regulators and mediators.

Why Did We Miss It?The lack of an obvious phenotype in the Hes3-null mice(88) and the absence of Hes3 probes in some DNA micro-arrays may have hindered research focus on this genebecause these have often favored other Hes/Hey genefamily members that are commonly used to assay caNotchsignaling. An additional difficulty in studying the functionof this gene arises from its regulation at the molecularlevel. Hes3 exists in two isoforms generated by distinctpromoters (87). The coding region in the Hes3-null mousehas been replaced by the lacZ gene (knock-in), and thiswas placed under the control of the “a” promoter, whichproduces the full-length, DNA-binding isoform. There-fore, despite the widespread use of this mouse line, it is


Figure 3—Damage and regeneration paradigms reveal Hes3-related phenotypes. A: Hes3-null (Hes3–/–) mice have no obvious phenotype,and adult NSC (aNSC) cultures can be established from the subventricular zone lining the lateral ventricles of the brain. However, whereaswild-type (wt) aNSCs respond to activation of the STAT3-Ser/Hes3 signaling axis (induced by addition of Notch ligands or insulin to theculture medium) by vast increases in cell number, Hes3–/– mice are largely nonresponsive. These observations suggest Hes3 roles in thecontext of simulated regeneration. B: Similarly, whereas blood glucose levels and gross pancreas morphology are normal in Hes3–/– mice,when mice are challenged through STZ treatments (low-dose STZ, daily injections for 5 days), Hes3–/– mice suffer a greater loss of b-cellscompared with wt controls. These results suggest a role of Hes3 in the protection of pancreatic islets during toxic insults. C: Mice subjected


limited because expression information is provided on onlyone of the two isoforms. Being passive repressors (i.e., re-pressors of transcription factor activity by protein-proteininteractions), the “b” isoform may be hugely important,even though it does not have DNA binding capability. Arole of the b isoform in regeneration may be supported bywork in the developing brain demonstrating that the b iso-form is preferentially expressed in neural progenitors of thedeveloping brain (87).

DO “ALTERNATIVE” HES/HEY GENES OPERATE INPANCREATIC CELLS?

Early reports demonstrated the operation of the STAT3-Ser/Hes3 signaling axis in adult pancreatic cells in vitroand in vivo (43). It may be worth speculating how thispathway may intercept other established signaling path-ways involved in pancreas development and function, in-cluding the mitogenic FGF pathway, Notch (canonical viaHes1, noncanonical via Hes3), and the hedgehog pathway.These testable hypotheses may provide a useful blueprintfor the integration of multiple signaling pathways in pan-creas biology.

Early Clues for the Involvement of Alternative Hes/HeyGenes in Pancreas BiologyEarly experiments with genetically engineered mice sug-gested that complex interactions among members of theHes/Hey gene family profoundly affect pancreatic devel-opment. In Hes1-null mice, ectopic pancreas formation isobserved in areas of the developing primitive stomach,duodenum, and bile duct (93). Hes1 inactivation inducedthe miss-expression of pancreas-specific transcription fac-tor 1a (Ptf1a), a bHLH that is important in the formationof the exocrine pancreas and the spatial organization of theendocrine pancreas in these regions (94). Using Hes1-nullmice that were crossed with a Cre/loxP reporter mouse,allowing the tracing of Ptf1a cells, it was demonstratedthat ectopic Ptf1a-expressing cells had acquired multipo-tent pancreatic progenitor properties in the Hes1-nullmouse that differentiated into cells of the pancreatic line-age, including insulin-producing cells with characteristicnuclear Pdx1 expression. A model where Hes1 suppressesPtf1a was thus suggested as a means of restricting pan-creas formation in the appropriate location. However,Hes1 expression is much broader than the pattern ofectopic pancreas formation in the Hes1-null mice, sug-gesting that additional mechanisms could contribute to

Ptf1a control or the control of other genes helping tolimit ectopic pancreas generation. Hes3 and Hes5, geneswith established roles in neural development and spec-ification (5,74,82,83,95,96), are also expressed in thedeveloping stomach and small intestine, respectively(24,97), so they could be candidates as cocontrollers ofpancreas organogenesis. Another interesting member ofthe Hes/Hey gene family is Hes6, a proneural gene im-plicated in glioma progression (98) and prostate canceraggression, possibly by opposing the hedgehog signalinginhibitor (via Gli1, at least) suppressor of fused (99),with roles in pancreatic islet cells where it opposesHes1 expression, thus promoting the mature b-cell phe-notype (100).

NSC Biology Suggests the Involvement ofNoncanonical Signaling PathwaysSpecifically for Hes3, however, the data from NSC biologymay argue against a compensatory role for Hes1 deletion.This may suggest that other Hes/Hey genes or bHLH genescompensate for Hes1 loss. Assessing the roles of thesesignaling pathways in pancreatic development and adultpancreatic cells will be highly valuable. Putting togetherobservations from the brain and the pancreas, one maysuggest a molecular interaction decreasing its stabilityand inhibiting promoter activity (21,101), leading tolargely mutually exclusive Hes1 and Ngn3 expressionpatterns.

This is based on a number of observations: 1) isolatedadult pancreatic islets from Hes3-null mice have increasedHes1 mRNA levels (43); 2) whereas Hes1 opposes Ngn3expression (21), Hes3 seems to be required for the in-creased Ngn3 expression in the exocrine pancreas afterSTZ-induced damage (92); and 3) the pancreatic pheno-types in the Hes1- and Hes3-null mice are very different(Hes1-null: hindered development [24]; Hes3-null: noobvious phenotype in undamaged animals [88]).

Hes3, therefore, may allow for regeneration-inducedmechanisms to promote the induction of Ngn3 expression(Fig. 3D). How Hes6 may intercept this network is not yetwell understood; however, as we discuss in a later section,Hes6 may activate components of the Shh pathway whilesuppressing Hes1 expression.

caNotch Levels Differentially Regulate the BalanceBetween Sox9 and Hes1In trunk cells, caNotch signaling is an important regulatorof Sox9 and Hes1 (102). The levels of activation of Sox9

to the STZ regimen described can be placed under normal conditions for months to assess their regeneration potential. After 5 months, wtmice regenerate most cells of the pancreatic islet, although their ability to regulate blood glucose levels may still be compromised. Incontrast, Hes3–/– mice are less efficient at regenerating the number of b-cells, suggesting a role of Hes3 in the regeneration of the pan-creatic islet after damage. D: Examples of islets after the experimental procedure described in C. Experimental details for panels B-D:low-dose STZ treatment was performed as previously described (43), but the mice were allowed to survive for 5 months after the lastinjection (n = 6 mice per each of the 4 groups). Immunolabeling images were acquired after 4% paraformaldehyde fixation and sectioning;counting was from 30 islet sections per group. The percentage of Pdx1+ cells per islet (i.e., per number of DAPI+ nuclei in the islet) is asfollows: wt: 79.216 10.9; wt-STZ: 35.86 12.3; Hes32/2: 76.26 13.7; Hes32/2-STZ: 23.76 20.9. A t-test comparison between the wt-STZand Hes32/2-STZ values showed a significant difference (P < 0.05).


and Hes1 determine whether Ngn3 will be subsequentlyinduced (leading to endocrine commitment) or sup-pressed (leading to ductal commitment). These levelsappear to be regulated by highly complex interactionsamong Sox9, Hes1, and Ngn3 (Sox9 induces Hes1 andNgn3 expression; Hes1 opposes Ngn3 expression) as wellas the levels of caNotch signaling (high caNotch leadsto the induction of both Sox9 and Hes1; low caNotchsignaling leads to the preferential induction of Sox9)(Fig. 4).

More specifically, to demonstrate the dose-dependenteffects of Notch activity, whole embryonic (E12.5)pancreas explants were cultured under different concen-trations of a g-secretase inhibitor to oppose Notch re-ceptor activation, and it was demonstrated that Sox9transcription requires lower levels of Notch activitythan Hes1 transcription (51). These data are supportedby reports that the expression of Ngn3 and Hes1 is mu-tually exclusive in the developing pancreas (27,35), as isthe expression of Sox9 and Ngn3 at the endocrine pro-genitor stage (46,103–105). Furthermore, Sox9 occupancyregions on the Ngn3 gene promoter have been identified(104,106). In addition, Sox9 may also regulate Ngn3 via

Pdx1 (107,108). (In a seeming paradox, Sox9 also inducesHes1 expression [46], perhaps as a means of limiting itsaction on Ngn3.)

In support of a role of caNotch signaling opposingNgn3 expression, mice deficient in the Notch receptorfamily ligand Dll1 and RBPJ-k exhibit reduced Hes1 ex-pression (35). In concert with these observations, Hes1inhibits the expression of Ngn3 (15), Hes1-null mice ex-hibit increased Ngn3 activity (24), and Hes1 inhibitioninduces the redifferentiation of expanded human pancre-atic b-cell–derived cells (109). Identifying new regulatorsof Hes1 and Sox9 may improve our understanding ofpancreas development.

Possible Involvement of noncaNotch BranchesIn contrast to the caNotch signaling pathway, thenoncaNotch branch involves the indirect (likely via Akt,mTOR, and STAT3-Ser phosphorylation, as shown inNSCs [5]) induction of Hes3, which then suppresses Hes1transcription (the mechanism for this is not yet known),potentially releasing Ngn3 from Hes1-induced inhibition.The contribution of such a putative mechanism in devel-opment is not yet demonstrated because no detailed

Figure 4—caNotch and noncaNotch branches regulating the expression of key pancreatic genes. In this diagram, we fuse data fromdifferent pancreatic cellular systems (pancreatic progenitors, mature b-cells) to create a generalized molecular model of how caNotch andnoncaNotch branches may interact; we suggest that this model can be used as a blueprint to address key open questions in the regulationof pancreas development. caNotch signaling (involving RBPJ-k) can lead to two established cellular outcomes: high caNotch activity intrunk cells leads to the induction of Sox9 and Hes1. Sox9 is an activator of Ngn3, and Hes1 is a repressor of Ngn3. Overall, this leads toloss of Ngn3 expression and the acquisition of the ductal cell fate. In contrast, low caNotch activity leads to the induction of only Sox9 (andnot Hes1), leading to Ngn3 expression and the acquisition of the endocrine precursor fate. Hes3 may intercept this regulatory system givenits roles in adult b-cell lines, in the damaged and regenerating pancreas, in Ngn3 induction in vivo, and in opposing Hes1 expression inpancreatic islets. Hes3, possibly under the control of upstream regulators as described in Fig. 2, may contribute to the suppression of Hes1under low caNotch conditions, ensuring that only Sox9 will be induced (and not Hes1), leading to Ngn3 induction and subsequentendocrine fate specification. Hes6 may also play a role in inhibiting Hes1 in this system.


studies have been performed, although in the adultHes3-null mouse, no gross morphological phenotypesare obvious (43,88). Perhaps, this mechanism is partic-ularly active during regeneration because Hes3-null miceexhibit increased damage and reduced regeneration ofb-cells after STZ damage (43). A technical difficulty inseparating caNotch from noncaNotch effects is that, atleast in NSC cultures, both pathway branches are sensi-tive to g-secretase inhibition (5).

Regardless, the possibility that noncaNotch is alsoinvolved in pancreas development and function promptsthe question of whether low Notch is really simply lowNotch or actually a different equilibrium between caNotch(Hes1-mediated) and noncaNotch (e.g., Hes3-mediated)activity. But is there evidence, even circumstantial, in linewith this possibility?

Integrating FGF10 SignalingThe MPC mitogen FGF10 (56) could provide an entrypoint to Hes3; in fact, it may provide a Notch receptor–independent means of activating both Hes1 (a caNotchtarget) and Hes3 (a noncaNotch target). We speculatethat, in a manner similar to high versus low Notch signaling

(46), high versus low FGF10 signaling may also prefer-entially stimulate the Hes1 (59) versus the Hes3 Notchsignaling branch.

During early pancreatic development, FGF10, derivedfrom mesenchymal cells, leads to Sox9 induction via itsreceptor, Fgfr2b. Sox9 promotes the expression ofFgfr2b, helping to maintain the receptivity of MPCs toFGF10 (53). Pdx1 also promotes Fgfr2b expression, andthis may be mediated by Sox9, because Sox9 and Fgfr2bexpression are lost simultaneously in Pdx1-deficientpancreata. Therefore, Sox9 is regulated by both theNotch and FGF10 pathways. How these apparently dis-parate pathways may regulate the same gene is notwell understood.

The STAT3-Ser/Hes3 signaling axis may possiblycontribute because it provides a molecular mechanismthat connects the Notch signaling pathway with classicmitogenic intracellular signals (e.g., Akt, mTOR), leadingto the induction of Hes3 or still unidentified mediators.Whether this molecular mechanism is operational inMPCs is not yet reported (Fig. 5).

There are clues suggesting complex molecular mecha-nisms by which FGF10 may be connected to caNotch and

Figure 5—FGF10 may intercept both caNotch and noncaNotch. FGF10 can induce classic mitogenic signal transduction pathways,including PI3K, Akt, and mTOR, but it can also activate JAK-STAT signaling. We speculate that the choice may be influenced by thelevels of receptor activation. The PI3K branch may lead, via STAT3-Ser, to a number of events that lead to the induction of Ngn3: Hes3induction, which may then suppress Hes1, and Sox9 induction. In contrast, the JAK-STAT branch may lead to Hes1 induction, whichopposes Ngn3 transcription. Sox9 also induces Hes1, and this, we speculate, may be a molecular mechanism to limit the degree ofNgn3 activation via Sox9. Sox9, possibly via Pdx1, positively regulates the expression of Fgfr2b and maintains receptiveness toFGF10.


noncaNotch signaling in MPCs: genetic perturbations ofFGF10 or its receptor Fgfr2b show that FGF10 signalingleads to hyperplasia, the maintenance of the immaturecell state (high Ptf1a, low Pdx1, low Nkx6.1 state),expression of Hes1, low Dll1, and low Ngn3 (58,59). Atfirst sight, these data may argue against the involvementof Hes3 downstream of FGF10 signaling because Hes3activation may be expected to result in low Hes1 andhigh Pdx1, at least based on data from mouse insulinomacell lines and the adult pancreata from Hes3-null mice (43).However, FGF10 signaling can also activate the JAK-STATpathway, which is a strong inhibitor of Hes3 as well as aninducer of Hes1 in other immature cell types (110). Sim-ilarly, in NSC cultures, the cytokine ciliary neurotrophicfactor can be converted from a prodifferentiation signalsuppressing Hes3 transcription to a mitogen promotingHes3 transcription simply by cotreatment with a JAK in-hibitor (78). These observations raise the intriguing possi-bility that FGF10 may, perhaps under different levelsof activation or cellular and developmental contexts, in-tercept caNotch or noncaNotch signaling with complexdownstream results.

Neural/Pancreas Progenitor Cell Equivalence?Identifying the cell types in the developing pancreas thatmay use aspects of the STAT3-Ser/Hes3 signaling axis willrequire substantial experimentation. Issues to resolveinclude identifying which cell type expresses Hes3 (or afunctional equivalent) and in which cell types STAT3 isphosphorylated on the serine residue and not on thetyrosine residue. As noted earlier, an integrated elucida-tion of the function of all members of the Hes/Hey genefamily may be required.

Few detailed studies on Hes3 expression during devel-opment exist (24,73,88,93). Detailed analyses of adulttissues have demonstrated Hes3 expression in putativeNSCs; such observations during development may be dif-ficult to make by low-magnification inspection (e.g.,whole-embryo immunostaining or in situ hybridization)because Hes3+ cells may be few and Hes3 expression canbe low. Therefore, future studies may shed first light onHes3 expression at different developmental stages of thepancreas.

Is it possible then that this system is operational inbipotent trunk cells? One may speculate that in thedecision between the endocrine and ductal commitment,Hes3 could help bias a progenitor to take the endocrineroute by opposing Hes1 expression. There is no evidence,to our knowledge, that Hes3 regulates Sox9 itself.

It may be interesting to study whether tip cellsexpress Hes3, because they exhibit low caNotch signal-ing and because adult Hes3-null mice exhibit pheno-types in acinar cells (the inability to induce Ngn3 afterpartial STZ damage [43]). Tip cells express Sox9 (111),but there is currently no evidence that this may be me-diated by Hes3; Sox9 induction may be a consequence oflow caNotch signaling.

Obvious developmental deficits in Hes3-null mice arenot known, although detailed studies are still lacking. Thisdoes not mean that Hes3 does not have developmentalroles, because other factors (possibly Hes6, for example)may functionally compensate for the loss of Hes3 in thesegenetic mouse models. However, the current data pointtoward a clearer role for Hes3 in differentiated, insulin-producing cells: In MIN6 cells, Hes3 overexpressioninduces Pdx1 expression, and Hes3 RNA interferenceopposes growth, insulin expression, and insulin sensitiv-ity. In line with the operation of the STAT3-Ser/Hes3signaling axis (which in NSCs is opposed by JAK activity),these cells can be efficiently cultured in conditions thatsuppress JAK-STAT signaling, thereby supporting STAT3-Ser phosphorylation and Hes3 expression (43). These re-sults are mirrored by observations reported in adult micewhere antibody- and PCR-based techniques demonstrateHes3 expression in islets; immunofluorescence experi-ments also suggest the expression of Hes3 in adult humanislets. Further, Hes3–knock-in reporter mice confirm Hes3promoter activity in adult mouse islets and demonstratepromoter activation after STZ-induced damage. The func-tional significance of pancreatic Hes3 is suggested by ob-servations in the adult Hes3-null mice showing increasedsensitivity to STZ damage and impaired regeneration, al-though no stark phenotypes are obvious in the uninjuredadult Hes3-null mice.

Therefore, the STAT3-Ser/Hes3 signaling axis maynot be a molecular mechanism that determines a par-ticular progenitor cell type but a signaling module thatis used by a variety of plastic cell types in the pancreas,both during development and in the adult. Modelingthe operation of this signaling pathway in culture mayprovide new opportunities in basic science and drugdiscovery (discussed later).

WHO CONTROLS Shh?

Shh plays important roles in pancreatic developmentand function, but the mechanisms that regulate itsexpression are not fully understood. Similar to what wediscussed for Notch signaling and possibly also for FGFsignaling, Shh signaling shows dosage-dependent effects,intercepts both caNotch and noncaNotch signaling pathwaybranches (e.g., Hes5 and Hes3) (5,112), and NSC biologymay contribute to our understanding of Shh regulation inthe pancreas.

Shh Signaling in Neural and Pancreatic DevelopmentIn neural development, different levels of Shh result indifferent cellular outcomes, with low Shh levels moreprone to promoting self-renewal/proliferation and highShh more prone to morphogenetic/cell specificationdecisions (112). The dosage-dependent Shh effects maybe linked to the dosage-dependent Notch effects. Infact, there is direct evidence that Shh intercepts bothcaNotch (e.g., Hes5) and noncaNotch signaling (e.g.,Hes3) (5,112).


Early pancreatic development and pancreatic pro-genitor maintenance requires suppression of Shh andactivation of caNotch signaling (involving RBPJ-k andHes1). At ;E9.5, the Shh receptor Ptch1 is still notexpressed in the pancreatic epithelium (31). However,it appears ;1 day later, albeit at low amounts; afterbirth, Ptch1 expression increases and is widespread inthe islets and ducts (113). Therefore, although hedgehogsignaling must be suppressed for pancreatic specifica-tion, it is possible that it is required for optimal devel-opment starting soon after specification, even if atrelatively low levels. Indeed, genetic deletion of theShh mediator Smo in the pancreas epithelium inducesdelayed expansion of the early pancreatic epithelium anddelayed b-cell mass development (113). Several otherstudies demonstrate a role of hedgehog signaling in thegrowth of the pancreas (31,33,34,114–118). These seem-ingly conflicting studies may be consolidated by takinginto account the different mechanisms of hedgehog sig-naling modulation, such as ligand overexpression or Smodeletion. It also suggests that hedgehog signaling opposesthe establishment of pancreas organ boundaries in theforegut while subsequently promoting the expansion ofearly pancreatic epithelium starting at ;E12.5 (113).Adding to the context-dependent complexity of hedgehogsignaling, the actions of this pathway may oppose prolif-eration of pancreatic epithelial cells at around midgesta-tion. We will address the possibility that noncanonicalsignaling pathways may mediate some of the pleiotropiceffects of Shh.

Effects of Shh on Pancreatic FunctionIn adult b-cells, conclusions on the roles of Shh arecomplex, partly because of the different biological sys-tems (cell lines in vitro, in vitro vs. in vivo) used and thedifferent experimental approaches used to modulateShh (pharmacological or genetic manipulation of differ-ent components of the Shh pathway). However, despitedifferences, these studies show important roles of Shhon adult b-cells. b-Cell lines, the Shh signaling small-molecule inhibitor cyclopamine, and ectopic expressionof Shh were used to show that hedgehog signaling pro-motes insulin secretion and content and insulin pro-moter activity (119,120). In a similar experimentalsystem, Shh was also demonstrated to provide protectiveproperties when cells were stressed by the addition ofproinflammatory cytokines (121).

In vivo, a somewhat different story arises on the roleof Shh on adult b-cells. A study that used a differentapproach to activate hedgehog signaling (using an activeversion of Gli2, a mediator of hedgehog signaling inb-cells devoid of primary cilia that normally negativelyregulate hedgehog signaling [122]) showed that increasedShh signaling correlated with increased expression of theprecursor markers Hes1 and Sox9, both direct targets thatare normally excluded from b-cells (123–126). It shouldbe noted that Gli1 and Gli2 overexpression leads to Hes1

induction in several cellular systems, providing a Notch-independent mechanism of inducing Hes1 (126). There-fore, hedgehog signaling in this in vivo system may beperturbed such that it preferentially leads to Hes1 induc-tion at the expense of other target genes, suppressingPdx1 expression. Overall, therefore, there are strong in-dications that hedgehog signaling may both promote andsuppress Pdx1 expression. We propose a model, fusingdata from both the NSC and pancreas fields to consolidatethese observations.

As mentioned above, Shh mediates the nuclear locali-zation of Pdx1 in cultured insulinoma cells (120) (Fig. 6A).Hes3 overexpression also induces Pdx1 expression andnuclear localization in insulinoma cells, and chromatinimmunoprecipitation on-chip data suggest the directregulation of the Pdx1 promoter as a putative mecha-nism (Fig. 6B) (43,127). In NSCs, Hes3 overexpressionpromotes Shh expression (Fig. 6C) (5). A hedgehog sig-naling branch may oppose Pdx1 expression through in-creased Hes1 expression in cells that overexpress Gli2and lack primary cilia (122) (Fig. 6D). Put together, theseobservations suggest the possible (and testable) involve-ment of noncaNotch signaling components in the regu-lation of Shh and Pdx1 (Fig. 7).

These varied observations raise a number of questions:

� Could Hes3 be both an inducer of Shh signaling activityas well as a compensator for the lack of Shh duringearly pancreatic development and perhaps also duringregeneration?

� If Gli2 does not induce Pdx1 expression, is there an-other Gli member that does (128)?

� Are there Gli factors that preferentially lead to Hes1and others to Pdx1 expression?

� How important is the inhibitory effect of primary ciliain Pdx1 regulation?

� Given the positive role of Sox9 in the formation ofprimary cilia (51) and the negative role of primary ciliain the mediation of hedgehog signaling (122), how sig-nificant might this Sox9/hedgehog signaling cross talkbe in terms of development and function?

� How much of the inhibitory effect of the hedgehogbranch on Pdx1 is mediated by Hes1?

CAN WE MODEL DIFFERENT SIGNALING STATESIN CULTURE?

Answering these questions will require a more thoroughunderstanding of the many states that pancreatic cells canadopt during development and disease. It would be a greatadvantage to researchers if these different cellular/signaling states could be modeled in vitro, contributingto our search for alternative growth pathways that mayeventually lead to new therapies.

Again, there are valuable lessons from NSC biologythat can be applied to b-cell research. MIN6 cells are acommonly used transformed mouse insulinoma cell line


(129). Despite the caveats with transformed cell lines,they have proven a valuable tool to study evoked insulinrelease. Still, these cells exhibit low nuclear Pdx1 expres-sion, relative to b-cells in vivo, suggesting that they donot model the in vivo state of b-cells perfectly. They alsoexhibit Hes1 expression, which is normally absent fromb-cells. They are typically cultured in serum-containingmedium, a common strategy to provide ample yet un-defined factors required for cell maintenance and pro-liferation. NSC biology reveals that serum, a potent JAK/STAT pathway activator, suppresses Hes3 expressionand, even more, nuclear Hes3 localization (5,43). Thismight explain why common insulinoma cell line cultureconditions maintain the cells in a low Hes3, low Pdx1,

high Hes1 state that does not model the in vivo state ofadult b-cells.

We recently showed that indeed MIN6 cells can becultured in defined (serum-free conditions) even in thepresence of a JAK inhibitor, which suppresses STAT3-Tyr phosphorylation (43). Hes3 is expressed under theseconditions, and a polyclonal antibody against Hes3 dem-onstrates nuclear localization. Nuclear Pdx1 expressionincidence also increases, providing potential access toits gene targets and representing more accurately b-cellsin vivo. Pdx1 expression is regulated by Hes3, becauseoverexpression of Hes3 in these cells invariably leads tohigh expression of nuclear Pdx1 (43). The Hes3-Pdx1 re-lation may be direct because Hes3 has been demonstrated

Figure 6—Shh and mediators of caNotch and noncaNotch signaling intercept to regulate cell outcome. A: In insulinoma cell lines, Shhoverexpression promotes Pdx1 expression and nuclear localization. B: In insulinoma cell lines, Hes3 overexpression promotes Pdx1expression and nuclear localization; the mechanism may involve the activation of promoter regions of the Pdx1 gene. C: In primary fetalrodent NSC cultures, Hes3 overexpression promotes Shh expression; the mechanism is not yet elucidated. D: In genetically engineeredmice, pancreatic cells that have increased Gli2 expression and lack primary (1ary) cilia (which normally oppose aspects of hedgehogsignaling) exhibit increased Hes1 expression and decreased Pdx1 expression.


to bind to Pdx1 gene promoter regions in MIN6 cells(127). Hes3 RNA interference opposes cell growthand insulin release (43). Switching the same cells be-tween culture conditions induces reversible changesshowcasing the bidirectional ability of the cells to pro-mote their growth via distinct signaling pathways.Such approaches may assist in experimental studiesby locking b-cells in culture in particular signalingstates representing specific aspects of their develop-ment and function.

It is possible that Shh and Hes3 signaling have similaroutcomes to cells at different developmental stages orsignaling states. Despite the similar effects of hedgehogand Hes3 signaling manipulation, the effects of hedgehogsignaling were reported using cell culture conditions thatplace cells in a signaling state that is nonresponsive toHes3 manipulation. Specifically, the effects of hedgehogsignaling modulation were seen in commonly used serum-containing culture systems where Hes3 is excluded from

the nucleus and Hes3 RNA interference does not signif-icantly affect cell growth. In contrast, Hes3 manipulationhas powerful roles in serum-free, defined culture condi-tions, where Hes3 is allowed in the nucleus. These resultsgive a glimpse to strategies that may allow locking cells indifferent states to allow their study. Whether Shh manip-ulation affects cells in defined conditions is not yet known.It will be important to define cell states using distinctculture conditions and reevaluate the effects of theperturbation of canonical and noncanonical signalingpathway components.

CONCLUSIONS

The protection and regeneration of insulin-producingpancreatic islet b-cells of the endocrine pancreas is a ma-jor focus of diabetes research (3,4). Compared with otheradult tissues, the endocrine pancreas exhibits consider-able plasticity in that many stimuli, including age, nutri-tion, pregnancy, insulin sensitivity, excessive caloricintake, and various paradigms of damage, can affect

Figure 7—Notch and Shh signaling pathways intercept to regulate the expression of key pancreatic transcription factors. In this diagram,we fuse together observations from different cell types to create a generalized model of how Shh and Notch signaling may intercept inpreviously unexplored ways. Hes3 directly regulates Pdx1 expression in MIN6 cells. Shh also directly does this, suggesting that in these cellsystems Shh and Hes3 share common functions. In NSCs, Hes3 overexpression leads to Shh expression induction, suggesting that inaddition to the parallel functions of Shh and Hes3, Hes3 may also be a regulator of Shh. However, this possibility is yet unexplored in thepancreatic system, to our knowledge. It is not unlikely, however, because another member of the Hes/Hey family of genes, Hes6, doesregulate Shh signaling in prostate cancer cells, opposes Hes1 action in culture, and promotes Pdx1 expression. In insulinoma cell linecultures, Shh pathway activation increases insulin secretion and content and promotes cell survival after inflammatory cytokine–inducedstress. Hes3 has similar roles in MIN6 cells. A particular branch of the Shh signaling pathway, however, suggests more complex roles ofhedgehog signaling. In a genetic mouse model where Gli2, a transcriptional mediator of hedgehog signaling, is overexpressed and wherecells lack primary (1ary) cilia (which normally contribute to the suppression of hedgehog signaling), increased expression of the precursormarkers Hes1 and Sox9 was observed. This apparent discrepancy may be explained by the fact that Gli2 promotes Hes1 expression,demonstrating how Shh signaling intercepts with caNotch signaling. Hes1, in turn, suppresses Pdx1 expression; for example, Hes1-nullmice exhibit ectopic Pdx1 expression and pancreas formation. Hes6 opposes Hes1 expression. Taken together, Notch, hedgehog, andHes/Hey signaling intercept in canonical and noncanonical ways to regulate the development and function of the pancreas. This complexsignaling network offers many opportunities for manipulation and study and argues for an extensive elucidation of additional aspects of thisnetwork, including a better understanding of other Hes/Hey genes and Gli factors in this process.


b-cell proliferation and alter b-cell mass, at least in ex-perimental rodent models. Understanding the signaltransduction pathways behind these phenomena will pro-vide potentially new therapeutic avenues. Clues are pro-vided by reports of experimental interventions thatmanipulate b-cell mass using treatments with knowndownstream signaling pathways. These include hypergly-cemia, incretins, Nodal (a TGF-b family member), vascularendothelial growth factor, Wnt pathway activators, andg-aminobutyric acid, for example (4). Additional clues areprovided by reports pointing out the importance of tran-scription factors in the specification of the b-cell fate,including Pdx1, MafA, and Ngn3 (130).

It is important to elucidate in detail how thesesignaling pathways regulate their downstream transcrip-tion factors to provide a deeper understanding of pancre-atic development and function and to give more precisedirection to drug discovery programs. Recent evidencesuggests that the view that a signaling pathway is on/offor high/low may be too simplistic because it does notaccount for the operation of alternative (noncanonical)branches of these pathways that may be highly activewhile canonical branches may be suppressed. In anexample of cross-disciplinary approach, the NSC field mayprovide a template to evaluate the involvement of suchnoncanonical signaling pathway branches in pancreaticdevelopment and function, providing new ideas that mayhelp understand and manipulate better the plasticity ofthis organ.

Funding. This work was partly supported by the Helmholtz AllianceImaging and Curing Environmental Metabolic Diseases (ICEMED), through theHelmholtz Association Initiative and Networking Fund grant 051_40001, DeutscheForschungsgemeinschaft grant SFB 655 “Cells Into Tissues” Project A24, andDeutsche Forschungsgemeinschaft Clinical Research Unit grant KFO 252.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. J.M. and P.N. collected, assembled, analyzed,and interpreted the data and revised the manuscript. S.W.P. collected,assembled, analyzed, and interpreted the data; revised the manuscript; andprovided a critical revision for important intellectual content. G.C., R.D.M., andS.R.B. analyzed and interpreted the data, revised the manuscript, and provided acritical revision for important intellectual content. P.M.J. analyzed and interpretedthe data, wrote the manuscript, revised the manuscript, and provided a criticalrevision for important intellectual content. A.A.-T. conceived the topic, drafted thearticle, wrote the manuscript, and interpreted and analyzed data. All authorsapproved the final manuscript. A.A.-T. is the guarantor of this work and, as such,had full access to all the data in the study and takes responsibility for the integrityof the data and the accuracy of the data analysis.

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