Review ArticlePax4 and Arx Represent Crucial Regulators of the Developmentof the Endocrine Pancreas
Monica Courtney123 Tamara Rabe34 Patrick Collombat1235 and Ahmed Mansouri3456
1 Universite de Nice-Sophia Antipolis Laboratoire de Genetique du Developpement Normal et Pathologique 06108 Nice France2 Inserm U1091 IBV Diabetes Genetics Team 06108 Nice France3 JDRF 26 Broadway NY 10004 USA4Department of Molecular Cell Biology Max-Planck Institute for Biophysical Chemsitry Am Fassberg 11 37077 Goettingen Germany5 Genome and Stem Cell Center GENKOK Erciyes University Kayseri Turkey6Department of Clinical Neurophysiology University of Goettingen Robert-Koch-Straszlige 40 37075 Goettingen Germany
Correspondence should be addressed to Patrick Collombat collombatunicefr and Ahmed Mansouri amansougwdgde
Received 10 April 2014 Accepted 12 May 2014 Published 29 May 2014
Academic Editor Hui-Qi Qu
Copyright copy 2014 Monica Courtney et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
The development of the endocrine pancreas is under the control of highly orchestrated cross-interacting transcription factorsPancreas genesis is initiated by the emergence of a Pdx1Ptf1a marked territory at the foregutmidgut junction A small fractionof pancreatic fated cells activates the expression of the bHLH transcription factor Ngn3 triggering the endocrine cell programthus giving rise to beta- alpha- delta- PP- and epsilon-cells producing insulin glucagon somatostatin pancreatic polypeptideand ghrelin respectively Two transcription factors Pax4 and Arx play a crucial role in differential endocrine cell subtypespecification They were shown to be necessary and sufficient to endow endocrine progenitors with either a beta- or alpha-celldestiny Interestingly whereas the forced expression of Arx in beta-cells-converts these into cells exhibiting alpha- and PP-cellcharacteristics the sole expression of Pax4 in alpha-cells promotes alpha-cell neogenesis and the acquisition of beta-cell featuresthe resulting beta-like cells being capable of counteracting chemically induced diabetes Gaining new insights into the molecularmechanisms controlling Pax4 and Arx expression in the endocrine pancreas may therefore pave new avenues for the therapy ofdiabetes
1 Introduction
The pancreas is composed of two distinct functional cellcompartments The major part comprises the exocrine tissueconsisting of acinar cells secreting digestive enzymes andan intricate ductal system required for the transport of thedigestive juice to the duodenum The endocrine compart-ment is found scattered throughout the exocrine tissue andis organized into aggregates of hormone-producing cellsforming functional units called islets of Langerhans [1 2]The mouse insulin-producing beta-cells are located in thecore of the islets whereas the remaining cells expressing thehormones glucagon (alpha-cells) somatostatin (delta-cells)pancreatic polypeptide (PP-cells) and ghrelin (epsilon-cells)are detected in islet periphery [1 3]
Insulin producing beta-cells are required to maintainglucose homeostasis Indeed their loss or malfunction even-tually results in diabetes Although medication aiming atcontrolling glycemia has improved glycemic control is oftendifficult to achieve leading to side effects such as hypo-glycemic episodes Therefore it is of fundamental interestto develop alternative therapeutic approaches such as stemcell replacement aiming at replenishing the beta-cell massTowards this goal understanding the molecular mechanismscontrolling beta-cell genesis represents a key step
During embryonic development pancreas formation ischaracterized by the appearance of a ventral and a dorsalbud at the foregutmidgut junction The pancreatic cellterritory is labeled by the expression of two transcriptionfactors Pdx1 and Ptf1a Lineage tracing studies revealed that
Hindawi Publishing CorporationNew Journal of ScienceVolume 2014 Article ID 981569 6 pageshttpdxdoiorg1011552014981569
2 New Journal of Science
Pdx1- and Ptf1a-expressing cells contribute to the develop-ment of all pancreatic derivatives [1 3ndash6] Subsequentlythe pancreatic epithelium proliferates and only a minor cellpopulation becomes fated to the endocrine compartmentsuch cells being characterized by the expression of the tran-scription factor Ngn3 Ngn3 is necessary for the formationof endocrine hormone-expressing cells as its absence resultsin the loss of islet cell genesis [7 8] At E125 the dorsal andventral pancreatic primordia fuse and at around E135 duringthe so-called secondary transition [2] pancreaticmultipotentprogenitor cells which are marked by the expression ofPdx1 Ptf1a Cpa1 and c-myc are detected in the tip of thebranching epithelium [9] At early stages of developmentthese multipotent progenitors have the propensity to give riseto Ngn3+ progenitor acinar and ductal cell lineages How-ever after E14 Cpa1-positive cells lose their multipotencyand are able to generate only exocrine cells [9] Endocrineprecursors initiate the expression of various transcriptionfactors and differentiate to give rise to pancreatic hormone-producing cells Among these transcription factors Pax4 andArx were shown to play a central role in the allocation ofendocrine progenitors towards the insulin producing beta-cell and glucagon producing alpha-cell fates respectively [110 11] Herein wewill discuss in detail our studies concerningthe function of Pax4 and Arx in endocrine cell subtypespecification and their possible future implication in thedevelopment of therapeutic approaches aiming at treatingdiabetes
2 Expression of Pax4 and Arx duringPancreas Development
Pax4 is amember of the paired-box containing family of tran-scription factors [12] Together with Pax6 it plays a crucialrole in endocrine pancreas morphogenesis [13 14] Duringpancreas development Pax4 is first detected in the pancreaticepithelium at E95 [15] At later stages and in adults Pax4expression is restricted to beta-cells [15] Interestingly astudy using the Pax4 promoter to drive Notch expression inthe endocrine pancreas in combination with lineage tracingrevealed that Pax4-expressing cells may represent specifiedendocrine progenitors contributing equally to all four isletsubtypes [16] Several immunohistochemical studies clearlyconfirm that Pax4 is also found in mature beta-cells [17 18]
The Aristaless-related homeobox-encoding gene Arx isexpressed in the central nervous system in skeletal musclesand in the endocrine pancreas [11 19 20] It is first detectedat E95 in the pancreatic epithelium and was found to markbeta- and alpha-precursor cells prior to becoming confinedto alpha- and PP-cells in adults [11] During pancreas genesiswe have previously shown that Pax4 and Arx are present inthe same proendocrine cell [11] The molecular mechanismsleading to the segregation of Arx- and Pax4-expressing cellsare not known However recent findings have demonstratedthat in committed beta-cells the repression of Arx is achievedthroughmethylation of theArx locus [21] Accordingly in theabsence of Dnmt1 gene activity Arx expression is reactivatedin beta-cells converting them into alpha-cells Another studyfurther sustains these findings showing that the targeted
inactivation of Dnmt3a in beta-cells is able to convert theseinto alpha-cells in anArx-dependent manner [22] Moreoverit has been suggested that the repression of Arx gene activityrequired to maintain the beta-cell fate could be achievedthrough Nkx22 interaction with the methylated Arx pro-moter [22] Along the same lines we have demonstratedthat beta-cell commitment is mediated by cross-inhibitoryinteractions between Arx and Pax4 ([1 10 11] and see below)
3 The Role of Pax4 and Arx in Endocrine CellSubtype Specification
The transcription factor Ngn3 which is expressed in allendocrine progenitors is necessary for the formation ofthe endocrine pancreas [7 8] Subsequently a concertedaction of various transcription factors is required for thespecification differentiation and survival of the differenthormone-producing endocrine cell types We have focusedour studies on the role of Pax4 andArx in these processesTheexpression pattern of Pax4 and Arx during pancreas genesisalready indicated that these two factors might promote thespecification of endocrine cells The analysis of Pax4 andArx loss- and gain-of-function mutant mice sustained thisnotion Indeed in the absence of Pax4 function mutant micesuffer fromhyperglycemia and die 2 days postpartum Mutantislets are morphologically normal but the core insulin-producing cells are lost Instead beta-cells appear to bereplaced by a proportional increase in glucagon-labeled cellsIn addition somatostatin-expressing cells are missing [15]This finding suggested that Pax4 not only is required for thespecification of beta- and delta-cells but also may repressthe alpha-cell lineage [15] Hence in the absence of Pax4endocrine progenitors are shunted towards an alpha-cellfate at the expense of a beta-delta-cell destiny Arx mutantmice die two days postpartum and suffer from hypoglycemia[11] Interestingly in these mutant animals a phenotypeopposite to that observed in Pax4-deficient animals is notedIndeed we found that in mutant Arx pancreata alpha-cellsare depleted whereas the beta- and delta-cell contents areproportionally increased Besides inArx-deficient pancreataPax4 transcription appeared upregulated clearly providingevidence of a cross-regulation between the expression ofArx and Pax4 in pancreatic endocrine cell fate specification([1 11] and see below) We concluded that in the absence ofArx activity endocrine progenitor cells are allocated to a beta-delta-cell destiny whereas Pax4-deficiency is accompaniedwith a favoring of an alpha-cell fate at the expense ofbeta-delta-cell lineage [10 11] In addition further studiesuncovered a reciprocal cross-inhibitory interaction betweenArx and Pax4 Indeed combining Gel Mobility Shift Assay(EMSA) and Chromatin ImmunoPrecipitation (ChIP) weestablished that Pax4 could specifically bind to a regionlocalized at the 31015840end of the Arx gene and efficiently repressits transcription Similar experiments using E14 pancreasindicated an interaction of Arx with a 200-bp DNA fragmentpresent within the Pax4 enhancer It appears therefore thatArx and Pax4 directly interact with the respective promoterand repress each otherrsquos transcription [10]
New Journal of Science 3
Ngn3
Pax4 Arx
Pax4Arx
Pax4 X
Pdx1
glucagonα
insulin somatostatin
Exocrine
Pax6ISL1NeuroD1Nkx22
120573-cells 120572-cells120575-cells
120573120575 120572
Figure 1 Cross-inhibitory interaction between Pax4 and Arxpromotes endocrine cell subtype allocation Pancreatic progenitorsexpressing the transcription factor Pdx1 become fated to endocrineand exocrine compartmentsThe endocrine cell program is initiatedby the activation of the b-HLH transcription factorNgn3 Endocrineprogenitors are allocated in a first round of cross-inhibitory interac-tions between Pax4 and Arx to a beta-delta-cell fate or an alpha-cell destiny respectively Similarly a second round of repressiveinteraction between Pax4 and an unknown factor X will promotethe differentiation into beta- and delta-cells respectively
Immunohistochemical analysis revealed that pancreataderived from Pax4Arx double mutant animals display anincreased number of somatostatin-expressing delta-cellswhile alpha- and beta-cells are missing This unexpectedfinding revealed a secondary role for Pax4 in beta-cellgenesis Accordingly our data demonstrate that Pax4 firstpromotes endocrine precursors towards a beta-delta-cell fateand subsequently towards a beta-cell lineage These dataare consistent with a model where an additional factor (X)inducing the delta-cell fate is necessary to repress Pax4activity possibly through an analogous reciprocal interactionmechanism as shown for Pax4 andArx [10] (Figure 1) Futuregenomic analysis may reveal the identity of such a factor
Another transcription factor involved in beta-cell differ-entiation is Nkx22 Indeed the loss of Nkx22 gene activityprovokes the appearance of immature beta-cells in mutantpancreata [23] Of note is the observed upregulation ofArx transcription in the pancreas of these mutant animalssuggesting a possible interaction between these two factors[24ndash26] We have generated ArxNkx22 compound mutantmice The analysis of double mutant pancreata revealed thatNkx22 might act to sustain the transcriptional networkin which Pax4 and Arx are involved to endow endocrineprecursors with beta- and alpha-cell destinies respec-tively [25] This finding is consistent with the notion thatthe concerted action of Pax4 and Nkx22 is required toantagonize Arx activity in differentiating beta-cells [25 26]
Thus our results clearly establish Pax4 and Arx as majorplayers acting in the allocation to the different endocrine
CAG IRESGFP ArxPax4 LacZ PA
Cre-recombinase
ArxPax4 LacZ
(1)
(2) Glu-PromCre-recom
(4)
(3)
L
CAG IRESArxPax4 LacZL
L
Figure 2 Pax4 and Arx are necessary and sufficient to promote thebeta- and alpha-cell fateidentity respectively Schematic describingthe strategy used to conditionally force the expression of Pax4 orArx in the pancreatic endoderm in endocrine cells or in alpha- orbeta cells depending on the promoter used to drive the expressionof the Cre recombinase (1) Transgenic mice carrying a constructallowing the conditional activation of Pax4 or Arx expressionunder the control of beta-actinCMV (promoterEnhancer) displayubiquitous green fluorescent protein (GFP) expression LoxP (L)sites (black bars) flanking GFP coding sequences and a stop codonallowing the activation of Pax4 or Arx only following the crossingof these mice with transgenic mice expressing the Cre recombinaseunder the control of pancreatic promoters (2) (Table 1) Doubletransgenic mice carrying the constructs (1) and (2) will undergoCre-induced homologous recombination to express Pax4 or Arx inconjunctionwith themarker LacZ ((3) and (4)) allowing the tracingof Pax4-expressing cells
Table 1
Pdx1-promoter Expression in pancreatic endodermPax6-promoter Expression in all endocrine cellsPdx1-CreERT2 Inducible by tamoxifen in beta-cellsGlu-Cre Expression in alpha-cellsRIP-Cre Expression in beta-cells
cell fates Hence manipulating the expression of Pax4and Arx may pave new avenues to derive beta-cells fromstemprogenitor cells to develop novel strategies for thetreatment of diabetes
4 Pax4 and Arx As Cell Identity Determinantsin the Endocrine Pancreas
Based on the aforementioned results we queried whetherPax4 and Arx were not only necessary but also sufficient todrive endocrine progenitors towards a beta- and alpha-cellfate respectively Thus we took advantage of the site-specificCre recombinase system to force the expression of eitherPax4 or Arx in endocrine cells during development andin adult animals As shown in Figure 2 using differenttransgenic mouse lines (see Table 1) we were able to forcethe expression of Arx or Pax4 in the pancreatic endoderm inendocrine cells and in alpha- or in beta-cells In addition inthe double transgenic mice the activation of beta-galactosidase expression is induced serving as markerto label and follow those cells that are forced to express Pax4or Arx (Figure 2)
4 New Journal of Science
Remarkably the analysis of transgenic mice allowingthe forced expression of Arx in Pdx1-labelled pancreaticprecursors in Pax6-expressing endocrine cells or even inmature beta-cells (using inducible Pdx1-Cre mice) revealeda strong hyperglycemia leading to precocious death [27]Accordingly immunohistochemical endocrine marker anal-yses and quantification of islet cells clearly demonstratedthat in all transgenic mouse lines a consistent increase inthe number of cells with alpha- and PP-cell characteristicsoccurred In contrast the number of insulin-producing cellswas dramatically reduced [27] These data indicate that thesole forced expression of Arx in pancreatic cells at differentdevelopmental and adult stages is sufficient to induce changesin islet cell destiny [27] It is interesting to note that themisexpression of Arx in mature beta-cells is able to convertthese into cells displaying alpha- and PP-cell features Thustowards the goal of generating beta-cells fromendocrine cellswe also forced the expression of the transcription factor Pax4not only in the pancreatic endoderm but also in endocrinecells at different stages of endocrine cell development [17]Using a similar strategy as described for Arx the condi-tional misexpression of Pax4 during development revealedits role in promoting the beta-cell fate [17] Importantlya conversion of alpha-cells into beta-like cells was notedthrough the forced expression of Pax4 in alpha-cells Indeedtransgenic mice expressing Pax4 in alpha-cells displayedan age-dependent increase of the beta-cell mass leadingto hypertrophic islets provoked by hyperplasia of insulin-expressing cells Lineage tracing indicated that these beta-like cells were converted from alpha-cells in which Pax4 wasmisexpressed [17] The increase in islet size was intriguingand pointed to permanent regeneration of the alpha-cell poolIn fact this was consistent with the idea that compromisingthe glucagon signaling pathway is associated with alpha-cellregeneration (Figure 3) Indeed mice lacking the glucagonreceptor prohormone convertase 2 or glucagon gene-derivedpeptides display alpha-cell hyperplasia [28ndash30] Accordinglyglucagon supplementation in Pax4 misexpressing mice wasable to diminish the dramatic increase in islet size [17]Thus in Pax4 transgenic mice glucagon signaling alter-ation contributes to alpha-cell regeneration these cells beingconverted into beta-like cells upon Pax4 misexpression Ofnote was the replenishment of insulin producing beta-cellsobserved in Pax4 misexpressing mice following chemicallyinduced diabetes Sixty days after the initiation of beta-cell ablation islets exhibited a normal content of insulin-producing cells that were derived from converted alpha-cellsexpressing Pax4 Altogether these results indicate that theforced expression of Pax4 in alpha-cells is able to induce theirneogenesis and conversion into functional beta-like cellsThese processes were found to depend on the reexpression ofthe proendocrine gene Ngn3 in the ductal lining supportingthe notion of the existence of precursor cells in ducts [17 31ndash37]
The misexpression of Pax4 in alpha-cells was achievedusing transgenic mice expressing the Cre recombinase underthe control of the glucagon promoter (see Figure 2) Accord-ingly in such a strategy Pax4 expression was induced innewly developed alpha-cells [17] Therefore it is reasonable
Compromisedglucagon signaling
Forced expression
Signal
Duct
Conversion
EMT
neogenesis
reactivationof Ngn3
into 120573-cells 120573-cells
120572-cells 120572-cellof Pax4 in 120572-cells
Figure 3 The forced expression of Pax4 in alpha-cells inducestheir conversion to beta-like cells The use of the site-specificrecombination (Figure 2) as well as the tetracycline inducible systemto control the forced expression of Pax4 in alpha-cells was foundto induce their conversion into functional beta-like cells and topromote a concomitant induction of alpha-like cell regenerationThe shortage in glucagon provoked by the conversion of alpha-cells into beta-cells appeared to contribute to the mobilization ofduct-lining precursor cells that reactivate Ngn3 and undergo EMT(epithelial mesenchymal transition) prior to adopting an alpha-likecell identity Such cells are yet again turned into beta-like cells uponPax4 misexpression and should it be maintained
to query whether glucagon-producing alpha-cells residingin the islets of adult mice and being exposed to epigeneticenvironment are also capable of undergoing such conversionupon Pax4 misexpression Hence taking advantage of theinducible tetracycline system Pax4 expression was activatedin alpha-cells of adult mice at various ages and for dif-ferent periods of time Pax4 expression in alpha-cells wastriggered and controlled for a definite time period by thesupplementation of doxycycline in the drinking water [38]Remarkably using this system not only could adult alpha-cells be converted into beta-like cells but also a continuouscycle of alpha-cell neogenesis was observed It is interestingto note that this process was age-independent Transgenicanimals could survive several cycles of chemically-inducedbeta-cell ablation and regenerate multiple times their wholebeta-like cell mass [38] Lineage tracing experiments usingthe site-specific Cre recombinase system clearly establishedthat (a) newly generated beta-like cells were converted fromalpha-cells and (b) a cycle of alpha-cell neogenesis wasinduced such cells originating from duct-lining cells thatreactivated Ngn3 expression and underwent EMT (epithelialmesenchymal transition) [38] Indeed in areas adjacent toducts mesenchymal-like structures were observed wherethe expression of mesenchymal markers such as nestinvimentin snail2 and Sox11 was activated [38 39] Takentogether these findings indicate that the alpha-cell pool inthe endocrine pancreas has the propensity to regenerate andmay therefore represent a valuable source to give rise tonew beta-cells [36] Therefore testing the ability of PAX4
New Journal of Science 5
to convert human alpha-cells into functional beta-cells isof fundamental interest for the development of therapeuticapproaches to treat diabetes
As reported above during the course of developmentPax4 and Arx undergo a reciprocal cross-inhibitory interac-tion allowing the differential specification towards the beta-and alpha-cell fates respectively [1 10] Accordingly theconversion of alpha-cells into beta-like cells mediated bythe forced activation of Pax4 expression may also be due tothe concomitantly suppressed Arx gene activity in glucagon-producing cells It is therefore reasonable to assume thatthe downregulation of Arx gene function in adult glucagon-producing cells may promote their transdifferentiation intobeta-cellsThis has recently been demonstrated by the condi-tional inactivation of Arx in adult alpha-cells which resultedin the appearance of newly generated insulin-producing beta-cells [40] Moreover this alpha-to-beta-like cell transdiffer-entiation induced process triggering a cycle of alpha-cellneogenesis as has been noted in Pax4 misexpressing mice[38ndash40] Remarkably the conditional double knockout ofArxand Pax4 in adult alpha-cells still efficiently promotes thetransdifferentiation of glucagon-producing cells into beta-like cells that could counter chemically induced diabetes[40] These findings are consistent with the idea that Arxfunctional inactivation is the main driving force in achievingthe reprograming of alpha-cells into beta-cells [40] There-fore novel strategies allowing the downregulation of Arxexpression or of its downstream targets in alpha-cells couldopen new avenues to develop innovative approaches forgenerating beta-cells to treat diabetes The identification ofArx target genes andor interacting factors may representan important step in reaching this goal In addition futurestudies are also required to determine whether human alpha-cells are able to transdifferentiate into beta-like cells uponPax4 forced expression or by targeting Arx inactivationFurther exploration of the molecular interactions mecha-nisms controlling endocrine cell subtype specification inthe endocrine pancreas may also uncover novel candidatemolecules that allow the establishment of strategies to directlyprovoke alpha- to beta-cell reprogramming in vivo
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
M Courtney and T Rabe contributed equally
Acknowledgments
This work was supported by the Juvenile Diabetes Researchfoundation (17-2011-16 2-2010-567 26-2008-639 and 17-2013-426) the INSERM AVENIR program the INSERMthe European Research Council (StG-2011-281265) theFMR (DRC20091217179) the ANRBMBF (2009 GENO 1050101KU0906) the ldquoInvestments for the Futurerdquo LABEX SIG-NALIFE (ANR-11-LABX-0028-01) the Max-Planck SocietyClub Isatis Mr and Mrs Dorato the Fondation Generale de
Sante and the Foundation Schlumberger pour lrsquoEducation etla Recherche
References
[1] P Collombat J Hecksher-Sorensen P Serup and A MansourildquoSpecifying pancreatic endocrine cell fatesrdquo Mechanisms ofDevelopment vol 123 no 7 pp 501ndash512 2006
[2] R L Pictet W R Clark R H Williams and W J Rutter ldquoAnultrastructural analysis of the developing embryonic pancreasrdquoDevelopmental Biology vol 29 no 4 pp 436ndash467 1972
[3] F C Pan and C Wright ldquoPancreas organogenesis from bud toplexus to glandrdquo Developmental Dynamics vol 240 no 3 pp530ndash565 2011
[4] G K Gittes ldquoDevelopmental biology of the pancreas a compre-hensive reviewrdquoDevelopmental Biology vol 326 no 1 pp 4ndash352009
[5] G Gu J R Brown and D A Melton ldquoDirect lineage tracingreveals the ontogeny of pancreatic cell fates during mouseembryogenesisrdquoMechanisms of Development vol 120 no 1 pp35ndash43 2003
[6] Y Kawaguchi B Cooper M GannonM Ray R J MacDonaldand C V E Wright ldquoThe role of the transcriptional regulatorPtf1a in converting intestinal to pancreatic progenitorsrdquoNatureGenetics vol 32 no 1 pp 128ndash134 2002
[7] G Gradwohl A Dierich M LeMeur and F Guillemot ldquoneuro-genin3 is required for the development of the four endocrine celllineages of the pancreasrdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 4 pp 1607ndash1611 2000
[8] V M Schwitzgebel D W Scheel J R Conners et al ldquoExpres-sion of neurogenin3 reveals an islet cell precursor populationin the pancreasrdquo Development vol 127 no 16 pp 3533ndash35422000
[9] Q Zhou A C Law J Rajagopal W J Anderson P A Grayand D A Melton ldquoA multipotent progenitor domain guidespancreatic organogenesisrdquoDevelopmental Cell vol 13 no 1 pp103ndash114 2007
[10] P Collombat J Hecksher-Sorensen V Broccoli et alldquoThe simultaneous loss of Arx and Pax4 genes promotes asomatostatin-producing cell fate specification at the expense ofthe 120572- and 120573-cell lineages in the mouse endocrine pancreasrdquoDevelopment vol 132 no 13 pp 2969ndash2980 2005
[11] P Collombat AMansouri J Hecksher-Sorensen et al ldquoOppos-ing actions of Arx and Pax4 in endocrine pancreas develop-mentrdquo Genes amp Development vol 17 no 20 pp 2591ndash26032003
[12] A Mansouri M Hallonet and P Gruss ldquoPax genes and theirroles in cell differentiation and developmentrdquo Current Opinionin Cell Biology vol 8 no 6 pp 851ndash857 1996
[13] A Mansouri G Goudreau and P Gruss ldquoPax genes and theirrole in organogenesisrdquoCancer Research vol 59 no 7 pp 1707Sndash1710S 1999
[14] A Mansouri L St-Onge and P Gruss ldquoRole of Pax genesin endoderm-derived organsrdquo Trends in Endocrinology ampMetabolism vol 10 no 4 pp 164ndash167 1999
[15] B Sosa-Pineda K Chowdhury M Torres G Oliver and PGruss ldquoThe Pax4 gene is essential for differentiation of insulin-producing 120573 cells in themammalian pancreasrdquoNature vol 386no 6623 pp 399ndash402 1997
6 New Journal of Science
[16] A L Greenwood S Li K Jones and D A Melton ldquoNotchsignaling reveals developmental plasticity of Pax4+ pancreaticendocrine progenitors and shunts them to a duct faterdquoMecha-nisms of Development vol 124 no 2 pp 97ndash107 2007
[17] P Collombat X Xu P Ravassard et al ldquoThe ectopic expressionof Pax4 in the mouse pancreas converts progenitor cells into 120572and subsequently120573 cellsrdquoCell vol 138 no 3 pp 449ndash462 2009
[18] J Lu P L Herrera C Carreira et al ldquo120572 cell-specific Men1ablation triggers the transdifferentiation of glucagon-expressingcells and insulinoma developmentrdquo Gastroenterology vol 138no 5 pp 1954ndash1965 2010
[19] S Biressi G Messina P Collombat et al ldquoThe homeobox geneArx is a novel positive regulator of embryonic myogenesisrdquo CellDeath and Differentiation vol 15 no 1 pp 94ndash104 2008
[20] E Colombo P Collombat G Colasante et al ldquoInactivation ofArx the murine ortholog of the X-linked lissencephaly withambiguous genitalia gene leads to severe disorganization of theventral telencephalon with impaired neuronal migration anddifferentiationrdquo The Journal of Neuroscience vol 27 no 17 pp4786ndash4798 2007
[21] S Dhawan S Georgia S-I Tschen G Fan and A BhushanldquoPancreatic 120573 cell identity is maintained by DNA methylation-mediated repression of Arxrdquo Developmental Cell vol 20 no 4pp 419ndash429 2011
[22] J B Papizan R A Singer S-I Tschen et al ldquoNkx22 repressorcomplex regulates islet120573-cell specification and prevents 120573-to-120572-cell reprogrammingrdquo Genes amp Development vol 25 no 21 pp2291ndash2305 2011
[23] L Sussel J Kalamaras D J Hartigan-OrsquoConnor et al ldquoMicelacking the homeodomain transcription factor Nkx22 havediabetes due to arrested differentiation of pancreatic 120573 cellsrdquoDevelopment vol 125 no 12 pp 2213ndash2221 1998
[24] C S Chao Z L Loomis J E Lee and L Sussel ldquoGenetic identi-fication of a novel NeuroD1 function in the early differentiationof islet 120572 PP and 120576 cellsrdquo Developmental Biology vol 312 no 2pp 523ndash532 2007
[25] S Kordowich P Collombat A Mansouri and P Serup ldquoArxand Nkx22 compound deficiency redirects pancreatic alpha-and beta-cell differentiation to a somatostatinghrelin co-expressing cell lineagerdquo BMC Developmental Biology vol 11article 52 2011
[26] T L Mastracci C Wilcox L Arnes et al ldquoNkx22 and Arxgenetically interact to regulate pancreatic endocrine cell devel-opment and endocrine hormone expressionrdquo DevelopmentalBiology vol 359 no 1 pp 1ndash11 2011
[27] P Collombat J Hecksher-Sorensen J Krull et al ldquoEmbryonicendocrine pancreas and mature 120573 cells acquire 120572 and PP cellphenotypes upon Arx misexpressionrdquo The Journal of ClinicalInvestigation vol 117 no 4 pp 961ndash970 2007
[28] M Furuta H Yano A Zhou et al ldquoDefective prohormoneprocessing and altered pancreatic islet morphology in micelacking active SPC2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 94 no 13 pp 6646ndash6651 1997
[29] RWGelling XQDuD SDichmann et al ldquoLower blood glu-cose hyperglucagonemia and pancreatic 120572 cell hyperplasia inglucagon receptor knockout micerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no3 pp 1438ndash1443 2003
[30] Y Hayashi M Yamamoto H Mizoguchi et al ldquoMice deficientfor glucagon gene-derived peptides display normoglycemia
and hyperplasia of islet 120572-cells but not of intestinal L-cellsrdquoMolecular Endocrinology vol 23 no 12 pp 1990ndash1999 2009
[31] P Collombat and A Mansouri ldquoPax4 transdifferentiatesglucagon-secreting 120572 cells to insulin-secreting 120573 endocrinepancreatic cellsrdquo MedecineSciences vol 25 no 8-9 pp 763ndash765 2009
[32] P Collombat and A Mansouri ldquoTurning on the 120573-cell identityin the pancreasrdquo Cell Cycle vol 8 no 21 pp 3450ndash3451 2009
[33] YDor andDAMelton ldquoFacultative endocrine progenitor cellsin the adult pancreasrdquo Cell vol 132 no 2 pp 183ndash184 2008
[34] J F Habener and V Stanojevic ldquo120572-cell role in 120573-cell generationand regenerationrdquo Islets vol 4 no 3 pp 188ndash198 2012
[35] Z Liu and J F Habener ldquoAlpha cells beget beta cellsrdquo Cell vol138 no 3 pp 424ndash426 2009
[36] A Mansouri ldquoDevelopment and regeneration in the endocrinepancreasrdquo ISRN Endocrinology vol 2012 Article ID 640956 12pages 2012
[37] X Xu J DrsquoHoker G Stange et al ldquo120573 cells can be generated fromendogenous progenitors in injured adult mouse pancreasrdquo Cellvol 132 no 2 pp 197ndash207 2008
[38] K Al-Hasani A Pfeifer M Courtney et al ldquoAdult duct-liningcells can reprogram into 120573-like cells able to counter repeatedcycles of toxin-induced diabetesrdquo Developmental Cell vol 26no 1 pp 86ndash100 2013
[39] A Pfeifer M Courtney N Ben-Othman et al et al ldquoInductionof multiple cycles of pancreatic 120573-cell replacementrdquo Cell Cyclevol 12 no 20 pp 3243ndash3244 2013
[40] M Courtney E Gjernes N Druelle et al ldquoThe inactivationof Arx in pancreatic 120572-cells triggers their neogenesis andconversion into functional 120573-like cellsrdquo PLoS Genetics vol 9no 10 article e1003934 2013
Pdx1- and Ptf1a-expressing cells contribute to the develop-ment of all pancreatic derivatives [1 3ndash6] Subsequentlythe pancreatic epithelium proliferates and only a minor cellpopulation becomes fated to the endocrine compartmentsuch cells being characterized by the expression of the tran-scription factor Ngn3 Ngn3 is necessary for the formationof endocrine hormone-expressing cells as its absence resultsin the loss of islet cell genesis [7 8] At E125 the dorsal andventral pancreatic primordia fuse and at around E135 duringthe so-called secondary transition [2] pancreaticmultipotentprogenitor cells which are marked by the expression ofPdx1 Ptf1a Cpa1 and c-myc are detected in the tip of thebranching epithelium [9] At early stages of developmentthese multipotent progenitors have the propensity to give riseto Ngn3+ progenitor acinar and ductal cell lineages How-ever after E14 Cpa1-positive cells lose their multipotencyand are able to generate only exocrine cells [9] Endocrineprecursors initiate the expression of various transcriptionfactors and differentiate to give rise to pancreatic hormone-producing cells Among these transcription factors Pax4 andArx were shown to play a central role in the allocation ofendocrine progenitors towards the insulin producing beta-cell and glucagon producing alpha-cell fates respectively [110 11] Herein wewill discuss in detail our studies concerningthe function of Pax4 and Arx in endocrine cell subtypespecification and their possible future implication in thedevelopment of therapeutic approaches aiming at treatingdiabetes
2 Expression of Pax4 and Arx duringPancreas Development
Pax4 is amember of the paired-box containing family of tran-scription factors [12] Together with Pax6 it plays a crucialrole in endocrine pancreas morphogenesis [13 14] Duringpancreas development Pax4 is first detected in the pancreaticepithelium at E95 [15] At later stages and in adults Pax4expression is restricted to beta-cells [15] Interestingly astudy using the Pax4 promoter to drive Notch expression inthe endocrine pancreas in combination with lineage tracingrevealed that Pax4-expressing cells may represent specifiedendocrine progenitors contributing equally to all four isletsubtypes [16] Several immunohistochemical studies clearlyconfirm that Pax4 is also found in mature beta-cells [17 18]
The Aristaless-related homeobox-encoding gene Arx isexpressed in the central nervous system in skeletal musclesand in the endocrine pancreas [11 19 20] It is first detectedat E95 in the pancreatic epithelium and was found to markbeta- and alpha-precursor cells prior to becoming confinedto alpha- and PP-cells in adults [11] During pancreas genesiswe have previously shown that Pax4 and Arx are present inthe same proendocrine cell [11] The molecular mechanismsleading to the segregation of Arx- and Pax4-expressing cellsare not known However recent findings have demonstratedthat in committed beta-cells the repression of Arx is achievedthroughmethylation of theArx locus [21] Accordingly in theabsence of Dnmt1 gene activity Arx expression is reactivatedin beta-cells converting them into alpha-cells Another studyfurther sustains these findings showing that the targeted
inactivation of Dnmt3a in beta-cells is able to convert theseinto alpha-cells in anArx-dependent manner [22] Moreoverit has been suggested that the repression of Arx gene activityrequired to maintain the beta-cell fate could be achievedthrough Nkx22 interaction with the methylated Arx pro-moter [22] Along the same lines we have demonstratedthat beta-cell commitment is mediated by cross-inhibitoryinteractions between Arx and Pax4 ([1 10 11] and see below)
3 The Role of Pax4 and Arx in Endocrine CellSubtype Specification
The transcription factor Ngn3 which is expressed in allendocrine progenitors is necessary for the formation ofthe endocrine pancreas [7 8] Subsequently a concertedaction of various transcription factors is required for thespecification differentiation and survival of the differenthormone-producing endocrine cell types We have focusedour studies on the role of Pax4 andArx in these processesTheexpression pattern of Pax4 and Arx during pancreas genesisalready indicated that these two factors might promote thespecification of endocrine cells The analysis of Pax4 andArx loss- and gain-of-function mutant mice sustained thisnotion Indeed in the absence of Pax4 function mutant micesuffer fromhyperglycemia and die 2 days postpartum Mutantislets are morphologically normal but the core insulin-producing cells are lost Instead beta-cells appear to bereplaced by a proportional increase in glucagon-labeled cellsIn addition somatostatin-expressing cells are missing [15]This finding suggested that Pax4 not only is required for thespecification of beta- and delta-cells but also may repressthe alpha-cell lineage [15] Hence in the absence of Pax4endocrine progenitors are shunted towards an alpha-cellfate at the expense of a beta-delta-cell destiny Arx mutantmice die two days postpartum and suffer from hypoglycemia[11] Interestingly in these mutant animals a phenotypeopposite to that observed in Pax4-deficient animals is notedIndeed we found that in mutant Arx pancreata alpha-cellsare depleted whereas the beta- and delta-cell contents areproportionally increased Besides inArx-deficient pancreataPax4 transcription appeared upregulated clearly providingevidence of a cross-regulation between the expression ofArx and Pax4 in pancreatic endocrine cell fate specification([1 11] and see below) We concluded that in the absence ofArx activity endocrine progenitor cells are allocated to a beta-delta-cell destiny whereas Pax4-deficiency is accompaniedwith a favoring of an alpha-cell fate at the expense ofbeta-delta-cell lineage [10 11] In addition further studiesuncovered a reciprocal cross-inhibitory interaction betweenArx and Pax4 Indeed combining Gel Mobility Shift Assay(EMSA) and Chromatin ImmunoPrecipitation (ChIP) weestablished that Pax4 could specifically bind to a regionlocalized at the 31015840end of the Arx gene and efficiently repressits transcription Similar experiments using E14 pancreasindicated an interaction of Arx with a 200-bp DNA fragmentpresent within the Pax4 enhancer It appears therefore thatArx and Pax4 directly interact with the respective promoterand repress each otherrsquos transcription [10]
New Journal of Science 3
Ngn3
Pax4 Arx
Pax4Arx
Pax4 X
Pdx1
glucagonα
insulin somatostatin
Exocrine
Pax6ISL1NeuroD1Nkx22
120573-cells 120572-cells120575-cells
120573120575 120572
Figure 1 Cross-inhibitory interaction between Pax4 and Arxpromotes endocrine cell subtype allocation Pancreatic progenitorsexpressing the transcription factor Pdx1 become fated to endocrineand exocrine compartmentsThe endocrine cell program is initiatedby the activation of the b-HLH transcription factorNgn3 Endocrineprogenitors are allocated in a first round of cross-inhibitory interac-tions between Pax4 and Arx to a beta-delta-cell fate or an alpha-cell destiny respectively Similarly a second round of repressiveinteraction between Pax4 and an unknown factor X will promotethe differentiation into beta- and delta-cells respectively
Immunohistochemical analysis revealed that pancreataderived from Pax4Arx double mutant animals display anincreased number of somatostatin-expressing delta-cellswhile alpha- and beta-cells are missing This unexpectedfinding revealed a secondary role for Pax4 in beta-cellgenesis Accordingly our data demonstrate that Pax4 firstpromotes endocrine precursors towards a beta-delta-cell fateand subsequently towards a beta-cell lineage These dataare consistent with a model where an additional factor (X)inducing the delta-cell fate is necessary to repress Pax4activity possibly through an analogous reciprocal interactionmechanism as shown for Pax4 andArx [10] (Figure 1) Futuregenomic analysis may reveal the identity of such a factor
Another transcription factor involved in beta-cell differ-entiation is Nkx22 Indeed the loss of Nkx22 gene activityprovokes the appearance of immature beta-cells in mutantpancreata [23] Of note is the observed upregulation ofArx transcription in the pancreas of these mutant animalssuggesting a possible interaction between these two factors[24ndash26] We have generated ArxNkx22 compound mutantmice The analysis of double mutant pancreata revealed thatNkx22 might act to sustain the transcriptional networkin which Pax4 and Arx are involved to endow endocrineprecursors with beta- and alpha-cell destinies respec-tively [25] This finding is consistent with the notion thatthe concerted action of Pax4 and Nkx22 is required toantagonize Arx activity in differentiating beta-cells [25 26]
Thus our results clearly establish Pax4 and Arx as majorplayers acting in the allocation to the different endocrine
CAG IRESGFP ArxPax4 LacZ PA
Cre-recombinase
ArxPax4 LacZ
(1)
(2) Glu-PromCre-recom
(4)
(3)
L
CAG IRESArxPax4 LacZL
L
Figure 2 Pax4 and Arx are necessary and sufficient to promote thebeta- and alpha-cell fateidentity respectively Schematic describingthe strategy used to conditionally force the expression of Pax4 orArx in the pancreatic endoderm in endocrine cells or in alpha- orbeta cells depending on the promoter used to drive the expressionof the Cre recombinase (1) Transgenic mice carrying a constructallowing the conditional activation of Pax4 or Arx expressionunder the control of beta-actinCMV (promoterEnhancer) displayubiquitous green fluorescent protein (GFP) expression LoxP (L)sites (black bars) flanking GFP coding sequences and a stop codonallowing the activation of Pax4 or Arx only following the crossingof these mice with transgenic mice expressing the Cre recombinaseunder the control of pancreatic promoters (2) (Table 1) Doubletransgenic mice carrying the constructs (1) and (2) will undergoCre-induced homologous recombination to express Pax4 or Arx inconjunctionwith themarker LacZ ((3) and (4)) allowing the tracingof Pax4-expressing cells
Table 1
Pdx1-promoter Expression in pancreatic endodermPax6-promoter Expression in all endocrine cellsPdx1-CreERT2 Inducible by tamoxifen in beta-cellsGlu-Cre Expression in alpha-cellsRIP-Cre Expression in beta-cells
cell fates Hence manipulating the expression of Pax4and Arx may pave new avenues to derive beta-cells fromstemprogenitor cells to develop novel strategies for thetreatment of diabetes
4 Pax4 and Arx As Cell Identity Determinantsin the Endocrine Pancreas
Based on the aforementioned results we queried whetherPax4 and Arx were not only necessary but also sufficient todrive endocrine progenitors towards a beta- and alpha-cellfate respectively Thus we took advantage of the site-specificCre recombinase system to force the expression of eitherPax4 or Arx in endocrine cells during development andin adult animals As shown in Figure 2 using differenttransgenic mouse lines (see Table 1) we were able to forcethe expression of Arx or Pax4 in the pancreatic endoderm inendocrine cells and in alpha- or in beta-cells In addition inthe double transgenic mice the activation of beta-galactosidase expression is induced serving as markerto label and follow those cells that are forced to express Pax4or Arx (Figure 2)
4 New Journal of Science
Remarkably the analysis of transgenic mice allowingthe forced expression of Arx in Pdx1-labelled pancreaticprecursors in Pax6-expressing endocrine cells or even inmature beta-cells (using inducible Pdx1-Cre mice) revealeda strong hyperglycemia leading to precocious death [27]Accordingly immunohistochemical endocrine marker anal-yses and quantification of islet cells clearly demonstratedthat in all transgenic mouse lines a consistent increase inthe number of cells with alpha- and PP-cell characteristicsoccurred In contrast the number of insulin-producing cellswas dramatically reduced [27] These data indicate that thesole forced expression of Arx in pancreatic cells at differentdevelopmental and adult stages is sufficient to induce changesin islet cell destiny [27] It is interesting to note that themisexpression of Arx in mature beta-cells is able to convertthese into cells displaying alpha- and PP-cell features Thustowards the goal of generating beta-cells fromendocrine cellswe also forced the expression of the transcription factor Pax4not only in the pancreatic endoderm but also in endocrinecells at different stages of endocrine cell development [17]Using a similar strategy as described for Arx the condi-tional misexpression of Pax4 during development revealedits role in promoting the beta-cell fate [17] Importantlya conversion of alpha-cells into beta-like cells was notedthrough the forced expression of Pax4 in alpha-cells Indeedtransgenic mice expressing Pax4 in alpha-cells displayedan age-dependent increase of the beta-cell mass leadingto hypertrophic islets provoked by hyperplasia of insulin-expressing cells Lineage tracing indicated that these beta-like cells were converted from alpha-cells in which Pax4 wasmisexpressed [17] The increase in islet size was intriguingand pointed to permanent regeneration of the alpha-cell poolIn fact this was consistent with the idea that compromisingthe glucagon signaling pathway is associated with alpha-cellregeneration (Figure 3) Indeed mice lacking the glucagonreceptor prohormone convertase 2 or glucagon gene-derivedpeptides display alpha-cell hyperplasia [28ndash30] Accordinglyglucagon supplementation in Pax4 misexpressing mice wasable to diminish the dramatic increase in islet size [17]Thus in Pax4 transgenic mice glucagon signaling alter-ation contributes to alpha-cell regeneration these cells beingconverted into beta-like cells upon Pax4 misexpression Ofnote was the replenishment of insulin producing beta-cellsobserved in Pax4 misexpressing mice following chemicallyinduced diabetes Sixty days after the initiation of beta-cell ablation islets exhibited a normal content of insulin-producing cells that were derived from converted alpha-cellsexpressing Pax4 Altogether these results indicate that theforced expression of Pax4 in alpha-cells is able to induce theirneogenesis and conversion into functional beta-like cellsThese processes were found to depend on the reexpression ofthe proendocrine gene Ngn3 in the ductal lining supportingthe notion of the existence of precursor cells in ducts [17 31ndash37]
The misexpression of Pax4 in alpha-cells was achievedusing transgenic mice expressing the Cre recombinase underthe control of the glucagon promoter (see Figure 2) Accord-ingly in such a strategy Pax4 expression was induced innewly developed alpha-cells [17] Therefore it is reasonable
Compromisedglucagon signaling
Forced expression
Signal
Duct
Conversion
EMT
neogenesis
reactivationof Ngn3
into 120573-cells 120573-cells
120572-cells 120572-cellof Pax4 in 120572-cells
Figure 3 The forced expression of Pax4 in alpha-cells inducestheir conversion to beta-like cells The use of the site-specificrecombination (Figure 2) as well as the tetracycline inducible systemto control the forced expression of Pax4 in alpha-cells was foundto induce their conversion into functional beta-like cells and topromote a concomitant induction of alpha-like cell regenerationThe shortage in glucagon provoked by the conversion of alpha-cells into beta-cells appeared to contribute to the mobilization ofduct-lining precursor cells that reactivate Ngn3 and undergo EMT(epithelial mesenchymal transition) prior to adopting an alpha-likecell identity Such cells are yet again turned into beta-like cells uponPax4 misexpression and should it be maintained
to query whether glucagon-producing alpha-cells residingin the islets of adult mice and being exposed to epigeneticenvironment are also capable of undergoing such conversionupon Pax4 misexpression Hence taking advantage of theinducible tetracycline system Pax4 expression was activatedin alpha-cells of adult mice at various ages and for dif-ferent periods of time Pax4 expression in alpha-cells wastriggered and controlled for a definite time period by thesupplementation of doxycycline in the drinking water [38]Remarkably using this system not only could adult alpha-cells be converted into beta-like cells but also a continuouscycle of alpha-cell neogenesis was observed It is interestingto note that this process was age-independent Transgenicanimals could survive several cycles of chemically-inducedbeta-cell ablation and regenerate multiple times their wholebeta-like cell mass [38] Lineage tracing experiments usingthe site-specific Cre recombinase system clearly establishedthat (a) newly generated beta-like cells were converted fromalpha-cells and (b) a cycle of alpha-cell neogenesis wasinduced such cells originating from duct-lining cells thatreactivated Ngn3 expression and underwent EMT (epithelialmesenchymal transition) [38] Indeed in areas adjacent toducts mesenchymal-like structures were observed wherethe expression of mesenchymal markers such as nestinvimentin snail2 and Sox11 was activated [38 39] Takentogether these findings indicate that the alpha-cell pool inthe endocrine pancreas has the propensity to regenerate andmay therefore represent a valuable source to give rise tonew beta-cells [36] Therefore testing the ability of PAX4
New Journal of Science 5
to convert human alpha-cells into functional beta-cells isof fundamental interest for the development of therapeuticapproaches to treat diabetes
As reported above during the course of developmentPax4 and Arx undergo a reciprocal cross-inhibitory interac-tion allowing the differential specification towards the beta-and alpha-cell fates respectively [1 10] Accordingly theconversion of alpha-cells into beta-like cells mediated bythe forced activation of Pax4 expression may also be due tothe concomitantly suppressed Arx gene activity in glucagon-producing cells It is therefore reasonable to assume thatthe downregulation of Arx gene function in adult glucagon-producing cells may promote their transdifferentiation intobeta-cellsThis has recently been demonstrated by the condi-tional inactivation of Arx in adult alpha-cells which resultedin the appearance of newly generated insulin-producing beta-cells [40] Moreover this alpha-to-beta-like cell transdiffer-entiation induced process triggering a cycle of alpha-cellneogenesis as has been noted in Pax4 misexpressing mice[38ndash40] Remarkably the conditional double knockout ofArxand Pax4 in adult alpha-cells still efficiently promotes thetransdifferentiation of glucagon-producing cells into beta-like cells that could counter chemically induced diabetes[40] These findings are consistent with the idea that Arxfunctional inactivation is the main driving force in achievingthe reprograming of alpha-cells into beta-cells [40] There-fore novel strategies allowing the downregulation of Arxexpression or of its downstream targets in alpha-cells couldopen new avenues to develop innovative approaches forgenerating beta-cells to treat diabetes The identification ofArx target genes andor interacting factors may representan important step in reaching this goal In addition futurestudies are also required to determine whether human alpha-cells are able to transdifferentiate into beta-like cells uponPax4 forced expression or by targeting Arx inactivationFurther exploration of the molecular interactions mecha-nisms controlling endocrine cell subtype specification inthe endocrine pancreas may also uncover novel candidatemolecules that allow the establishment of strategies to directlyprovoke alpha- to beta-cell reprogramming in vivo
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
M Courtney and T Rabe contributed equally
Acknowledgments
This work was supported by the Juvenile Diabetes Researchfoundation (17-2011-16 2-2010-567 26-2008-639 and 17-2013-426) the INSERM AVENIR program the INSERMthe European Research Council (StG-2011-281265) theFMR (DRC20091217179) the ANRBMBF (2009 GENO 1050101KU0906) the ldquoInvestments for the Futurerdquo LABEX SIG-NALIFE (ANR-11-LABX-0028-01) the Max-Planck SocietyClub Isatis Mr and Mrs Dorato the Fondation Generale de
Sante and the Foundation Schlumberger pour lrsquoEducation etla Recherche
References
[1] P Collombat J Hecksher-Sorensen P Serup and A MansourildquoSpecifying pancreatic endocrine cell fatesrdquo Mechanisms ofDevelopment vol 123 no 7 pp 501ndash512 2006
[2] R L Pictet W R Clark R H Williams and W J Rutter ldquoAnultrastructural analysis of the developing embryonic pancreasrdquoDevelopmental Biology vol 29 no 4 pp 436ndash467 1972
[3] F C Pan and C Wright ldquoPancreas organogenesis from bud toplexus to glandrdquo Developmental Dynamics vol 240 no 3 pp530ndash565 2011
[4] G K Gittes ldquoDevelopmental biology of the pancreas a compre-hensive reviewrdquoDevelopmental Biology vol 326 no 1 pp 4ndash352009
[5] G Gu J R Brown and D A Melton ldquoDirect lineage tracingreveals the ontogeny of pancreatic cell fates during mouseembryogenesisrdquoMechanisms of Development vol 120 no 1 pp35ndash43 2003
[6] Y Kawaguchi B Cooper M GannonM Ray R J MacDonaldand C V E Wright ldquoThe role of the transcriptional regulatorPtf1a in converting intestinal to pancreatic progenitorsrdquoNatureGenetics vol 32 no 1 pp 128ndash134 2002
[7] G Gradwohl A Dierich M LeMeur and F Guillemot ldquoneuro-genin3 is required for the development of the four endocrine celllineages of the pancreasrdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 4 pp 1607ndash1611 2000
[8] V M Schwitzgebel D W Scheel J R Conners et al ldquoExpres-sion of neurogenin3 reveals an islet cell precursor populationin the pancreasrdquo Development vol 127 no 16 pp 3533ndash35422000
[9] Q Zhou A C Law J Rajagopal W J Anderson P A Grayand D A Melton ldquoA multipotent progenitor domain guidespancreatic organogenesisrdquoDevelopmental Cell vol 13 no 1 pp103ndash114 2007
[10] P Collombat J Hecksher-Sorensen V Broccoli et alldquoThe simultaneous loss of Arx and Pax4 genes promotes asomatostatin-producing cell fate specification at the expense ofthe 120572- and 120573-cell lineages in the mouse endocrine pancreasrdquoDevelopment vol 132 no 13 pp 2969ndash2980 2005
[11] P Collombat AMansouri J Hecksher-Sorensen et al ldquoOppos-ing actions of Arx and Pax4 in endocrine pancreas develop-mentrdquo Genes amp Development vol 17 no 20 pp 2591ndash26032003
[12] A Mansouri M Hallonet and P Gruss ldquoPax genes and theirroles in cell differentiation and developmentrdquo Current Opinionin Cell Biology vol 8 no 6 pp 851ndash857 1996
[13] A Mansouri G Goudreau and P Gruss ldquoPax genes and theirrole in organogenesisrdquoCancer Research vol 59 no 7 pp 1707Sndash1710S 1999
[14] A Mansouri L St-Onge and P Gruss ldquoRole of Pax genesin endoderm-derived organsrdquo Trends in Endocrinology ampMetabolism vol 10 no 4 pp 164ndash167 1999
[15] B Sosa-Pineda K Chowdhury M Torres G Oliver and PGruss ldquoThe Pax4 gene is essential for differentiation of insulin-producing 120573 cells in themammalian pancreasrdquoNature vol 386no 6623 pp 399ndash402 1997
6 New Journal of Science
[16] A L Greenwood S Li K Jones and D A Melton ldquoNotchsignaling reveals developmental plasticity of Pax4+ pancreaticendocrine progenitors and shunts them to a duct faterdquoMecha-nisms of Development vol 124 no 2 pp 97ndash107 2007
[17] P Collombat X Xu P Ravassard et al ldquoThe ectopic expressionof Pax4 in the mouse pancreas converts progenitor cells into 120572and subsequently120573 cellsrdquoCell vol 138 no 3 pp 449ndash462 2009
[18] J Lu P L Herrera C Carreira et al ldquo120572 cell-specific Men1ablation triggers the transdifferentiation of glucagon-expressingcells and insulinoma developmentrdquo Gastroenterology vol 138no 5 pp 1954ndash1965 2010
[19] S Biressi G Messina P Collombat et al ldquoThe homeobox geneArx is a novel positive regulator of embryonic myogenesisrdquo CellDeath and Differentiation vol 15 no 1 pp 94ndash104 2008
[20] E Colombo P Collombat G Colasante et al ldquoInactivation ofArx the murine ortholog of the X-linked lissencephaly withambiguous genitalia gene leads to severe disorganization of theventral telencephalon with impaired neuronal migration anddifferentiationrdquo The Journal of Neuroscience vol 27 no 17 pp4786ndash4798 2007
[21] S Dhawan S Georgia S-I Tschen G Fan and A BhushanldquoPancreatic 120573 cell identity is maintained by DNA methylation-mediated repression of Arxrdquo Developmental Cell vol 20 no 4pp 419ndash429 2011
[22] J B Papizan R A Singer S-I Tschen et al ldquoNkx22 repressorcomplex regulates islet120573-cell specification and prevents 120573-to-120572-cell reprogrammingrdquo Genes amp Development vol 25 no 21 pp2291ndash2305 2011
[23] L Sussel J Kalamaras D J Hartigan-OrsquoConnor et al ldquoMicelacking the homeodomain transcription factor Nkx22 havediabetes due to arrested differentiation of pancreatic 120573 cellsrdquoDevelopment vol 125 no 12 pp 2213ndash2221 1998
[24] C S Chao Z L Loomis J E Lee and L Sussel ldquoGenetic identi-fication of a novel NeuroD1 function in the early differentiationof islet 120572 PP and 120576 cellsrdquo Developmental Biology vol 312 no 2pp 523ndash532 2007
[25] S Kordowich P Collombat A Mansouri and P Serup ldquoArxand Nkx22 compound deficiency redirects pancreatic alpha-and beta-cell differentiation to a somatostatinghrelin co-expressing cell lineagerdquo BMC Developmental Biology vol 11article 52 2011
[26] T L Mastracci C Wilcox L Arnes et al ldquoNkx22 and Arxgenetically interact to regulate pancreatic endocrine cell devel-opment and endocrine hormone expressionrdquo DevelopmentalBiology vol 359 no 1 pp 1ndash11 2011
[27] P Collombat J Hecksher-Sorensen J Krull et al ldquoEmbryonicendocrine pancreas and mature 120573 cells acquire 120572 and PP cellphenotypes upon Arx misexpressionrdquo The Journal of ClinicalInvestigation vol 117 no 4 pp 961ndash970 2007
[28] M Furuta H Yano A Zhou et al ldquoDefective prohormoneprocessing and altered pancreatic islet morphology in micelacking active SPC2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 94 no 13 pp 6646ndash6651 1997
[29] RWGelling XQDuD SDichmann et al ldquoLower blood glu-cose hyperglucagonemia and pancreatic 120572 cell hyperplasia inglucagon receptor knockout micerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no3 pp 1438ndash1443 2003
[30] Y Hayashi M Yamamoto H Mizoguchi et al ldquoMice deficientfor glucagon gene-derived peptides display normoglycemia
and hyperplasia of islet 120572-cells but not of intestinal L-cellsrdquoMolecular Endocrinology vol 23 no 12 pp 1990ndash1999 2009
[31] P Collombat and A Mansouri ldquoPax4 transdifferentiatesglucagon-secreting 120572 cells to insulin-secreting 120573 endocrinepancreatic cellsrdquo MedecineSciences vol 25 no 8-9 pp 763ndash765 2009
[32] P Collombat and A Mansouri ldquoTurning on the 120573-cell identityin the pancreasrdquo Cell Cycle vol 8 no 21 pp 3450ndash3451 2009
[33] YDor andDAMelton ldquoFacultative endocrine progenitor cellsin the adult pancreasrdquo Cell vol 132 no 2 pp 183ndash184 2008
[34] J F Habener and V Stanojevic ldquo120572-cell role in 120573-cell generationand regenerationrdquo Islets vol 4 no 3 pp 188ndash198 2012
[35] Z Liu and J F Habener ldquoAlpha cells beget beta cellsrdquo Cell vol138 no 3 pp 424ndash426 2009
[36] A Mansouri ldquoDevelopment and regeneration in the endocrinepancreasrdquo ISRN Endocrinology vol 2012 Article ID 640956 12pages 2012
[37] X Xu J DrsquoHoker G Stange et al ldquo120573 cells can be generated fromendogenous progenitors in injured adult mouse pancreasrdquo Cellvol 132 no 2 pp 197ndash207 2008
[38] K Al-Hasani A Pfeifer M Courtney et al ldquoAdult duct-liningcells can reprogram into 120573-like cells able to counter repeatedcycles of toxin-induced diabetesrdquo Developmental Cell vol 26no 1 pp 86ndash100 2013
[39] A Pfeifer M Courtney N Ben-Othman et al et al ldquoInductionof multiple cycles of pancreatic 120573-cell replacementrdquo Cell Cyclevol 12 no 20 pp 3243ndash3244 2013
[40] M Courtney E Gjernes N Druelle et al ldquoThe inactivationof Arx in pancreatic 120572-cells triggers their neogenesis andconversion into functional 120573-like cellsrdquo PLoS Genetics vol 9no 10 article e1003934 2013
Figure 1 Cross-inhibitory interaction between Pax4 and Arxpromotes endocrine cell subtype allocation Pancreatic progenitorsexpressing the transcription factor Pdx1 become fated to endocrineand exocrine compartmentsThe endocrine cell program is initiatedby the activation of the b-HLH transcription factorNgn3 Endocrineprogenitors are allocated in a first round of cross-inhibitory interac-tions between Pax4 and Arx to a beta-delta-cell fate or an alpha-cell destiny respectively Similarly a second round of repressiveinteraction between Pax4 and an unknown factor X will promotethe differentiation into beta- and delta-cells respectively
Immunohistochemical analysis revealed that pancreataderived from Pax4Arx double mutant animals display anincreased number of somatostatin-expressing delta-cellswhile alpha- and beta-cells are missing This unexpectedfinding revealed a secondary role for Pax4 in beta-cellgenesis Accordingly our data demonstrate that Pax4 firstpromotes endocrine precursors towards a beta-delta-cell fateand subsequently towards a beta-cell lineage These dataare consistent with a model where an additional factor (X)inducing the delta-cell fate is necessary to repress Pax4activity possibly through an analogous reciprocal interactionmechanism as shown for Pax4 andArx [10] (Figure 1) Futuregenomic analysis may reveal the identity of such a factor
Another transcription factor involved in beta-cell differ-entiation is Nkx22 Indeed the loss of Nkx22 gene activityprovokes the appearance of immature beta-cells in mutantpancreata [23] Of note is the observed upregulation ofArx transcription in the pancreas of these mutant animalssuggesting a possible interaction between these two factors[24ndash26] We have generated ArxNkx22 compound mutantmice The analysis of double mutant pancreata revealed thatNkx22 might act to sustain the transcriptional networkin which Pax4 and Arx are involved to endow endocrineprecursors with beta- and alpha-cell destinies respec-tively [25] This finding is consistent with the notion thatthe concerted action of Pax4 and Nkx22 is required toantagonize Arx activity in differentiating beta-cells [25 26]
Thus our results clearly establish Pax4 and Arx as majorplayers acting in the allocation to the different endocrine
CAG IRESGFP ArxPax4 LacZ PA
Cre-recombinase
ArxPax4 LacZ
(1)
(2) Glu-PromCre-recom
(4)
(3)
L
CAG IRESArxPax4 LacZL
L
Figure 2 Pax4 and Arx are necessary and sufficient to promote thebeta- and alpha-cell fateidentity respectively Schematic describingthe strategy used to conditionally force the expression of Pax4 orArx in the pancreatic endoderm in endocrine cells or in alpha- orbeta cells depending on the promoter used to drive the expressionof the Cre recombinase (1) Transgenic mice carrying a constructallowing the conditional activation of Pax4 or Arx expressionunder the control of beta-actinCMV (promoterEnhancer) displayubiquitous green fluorescent protein (GFP) expression LoxP (L)sites (black bars) flanking GFP coding sequences and a stop codonallowing the activation of Pax4 or Arx only following the crossingof these mice with transgenic mice expressing the Cre recombinaseunder the control of pancreatic promoters (2) (Table 1) Doubletransgenic mice carrying the constructs (1) and (2) will undergoCre-induced homologous recombination to express Pax4 or Arx inconjunctionwith themarker LacZ ((3) and (4)) allowing the tracingof Pax4-expressing cells
Table 1
Pdx1-promoter Expression in pancreatic endodermPax6-promoter Expression in all endocrine cellsPdx1-CreERT2 Inducible by tamoxifen in beta-cellsGlu-Cre Expression in alpha-cellsRIP-Cre Expression in beta-cells
cell fates Hence manipulating the expression of Pax4and Arx may pave new avenues to derive beta-cells fromstemprogenitor cells to develop novel strategies for thetreatment of diabetes
4 Pax4 and Arx As Cell Identity Determinantsin the Endocrine Pancreas
Based on the aforementioned results we queried whetherPax4 and Arx were not only necessary but also sufficient todrive endocrine progenitors towards a beta- and alpha-cellfate respectively Thus we took advantage of the site-specificCre recombinase system to force the expression of eitherPax4 or Arx in endocrine cells during development andin adult animals As shown in Figure 2 using differenttransgenic mouse lines (see Table 1) we were able to forcethe expression of Arx or Pax4 in the pancreatic endoderm inendocrine cells and in alpha- or in beta-cells In addition inthe double transgenic mice the activation of beta-galactosidase expression is induced serving as markerto label and follow those cells that are forced to express Pax4or Arx (Figure 2)
4 New Journal of Science
Remarkably the analysis of transgenic mice allowingthe forced expression of Arx in Pdx1-labelled pancreaticprecursors in Pax6-expressing endocrine cells or even inmature beta-cells (using inducible Pdx1-Cre mice) revealeda strong hyperglycemia leading to precocious death [27]Accordingly immunohistochemical endocrine marker anal-yses and quantification of islet cells clearly demonstratedthat in all transgenic mouse lines a consistent increase inthe number of cells with alpha- and PP-cell characteristicsoccurred In contrast the number of insulin-producing cellswas dramatically reduced [27] These data indicate that thesole forced expression of Arx in pancreatic cells at differentdevelopmental and adult stages is sufficient to induce changesin islet cell destiny [27] It is interesting to note that themisexpression of Arx in mature beta-cells is able to convertthese into cells displaying alpha- and PP-cell features Thustowards the goal of generating beta-cells fromendocrine cellswe also forced the expression of the transcription factor Pax4not only in the pancreatic endoderm but also in endocrinecells at different stages of endocrine cell development [17]Using a similar strategy as described for Arx the condi-tional misexpression of Pax4 during development revealedits role in promoting the beta-cell fate [17] Importantlya conversion of alpha-cells into beta-like cells was notedthrough the forced expression of Pax4 in alpha-cells Indeedtransgenic mice expressing Pax4 in alpha-cells displayedan age-dependent increase of the beta-cell mass leadingto hypertrophic islets provoked by hyperplasia of insulin-expressing cells Lineage tracing indicated that these beta-like cells were converted from alpha-cells in which Pax4 wasmisexpressed [17] The increase in islet size was intriguingand pointed to permanent regeneration of the alpha-cell poolIn fact this was consistent with the idea that compromisingthe glucagon signaling pathway is associated with alpha-cellregeneration (Figure 3) Indeed mice lacking the glucagonreceptor prohormone convertase 2 or glucagon gene-derivedpeptides display alpha-cell hyperplasia [28ndash30] Accordinglyglucagon supplementation in Pax4 misexpressing mice wasable to diminish the dramatic increase in islet size [17]Thus in Pax4 transgenic mice glucagon signaling alter-ation contributes to alpha-cell regeneration these cells beingconverted into beta-like cells upon Pax4 misexpression Ofnote was the replenishment of insulin producing beta-cellsobserved in Pax4 misexpressing mice following chemicallyinduced diabetes Sixty days after the initiation of beta-cell ablation islets exhibited a normal content of insulin-producing cells that were derived from converted alpha-cellsexpressing Pax4 Altogether these results indicate that theforced expression of Pax4 in alpha-cells is able to induce theirneogenesis and conversion into functional beta-like cellsThese processes were found to depend on the reexpression ofthe proendocrine gene Ngn3 in the ductal lining supportingthe notion of the existence of precursor cells in ducts [17 31ndash37]
The misexpression of Pax4 in alpha-cells was achievedusing transgenic mice expressing the Cre recombinase underthe control of the glucagon promoter (see Figure 2) Accord-ingly in such a strategy Pax4 expression was induced innewly developed alpha-cells [17] Therefore it is reasonable
Compromisedglucagon signaling
Forced expression
Signal
Duct
Conversion
EMT
neogenesis
reactivationof Ngn3
into 120573-cells 120573-cells
120572-cells 120572-cellof Pax4 in 120572-cells
Figure 3 The forced expression of Pax4 in alpha-cells inducestheir conversion to beta-like cells The use of the site-specificrecombination (Figure 2) as well as the tetracycline inducible systemto control the forced expression of Pax4 in alpha-cells was foundto induce their conversion into functional beta-like cells and topromote a concomitant induction of alpha-like cell regenerationThe shortage in glucagon provoked by the conversion of alpha-cells into beta-cells appeared to contribute to the mobilization ofduct-lining precursor cells that reactivate Ngn3 and undergo EMT(epithelial mesenchymal transition) prior to adopting an alpha-likecell identity Such cells are yet again turned into beta-like cells uponPax4 misexpression and should it be maintained
to query whether glucagon-producing alpha-cells residingin the islets of adult mice and being exposed to epigeneticenvironment are also capable of undergoing such conversionupon Pax4 misexpression Hence taking advantage of theinducible tetracycline system Pax4 expression was activatedin alpha-cells of adult mice at various ages and for dif-ferent periods of time Pax4 expression in alpha-cells wastriggered and controlled for a definite time period by thesupplementation of doxycycline in the drinking water [38]Remarkably using this system not only could adult alpha-cells be converted into beta-like cells but also a continuouscycle of alpha-cell neogenesis was observed It is interestingto note that this process was age-independent Transgenicanimals could survive several cycles of chemically-inducedbeta-cell ablation and regenerate multiple times their wholebeta-like cell mass [38] Lineage tracing experiments usingthe site-specific Cre recombinase system clearly establishedthat (a) newly generated beta-like cells were converted fromalpha-cells and (b) a cycle of alpha-cell neogenesis wasinduced such cells originating from duct-lining cells thatreactivated Ngn3 expression and underwent EMT (epithelialmesenchymal transition) [38] Indeed in areas adjacent toducts mesenchymal-like structures were observed wherethe expression of mesenchymal markers such as nestinvimentin snail2 and Sox11 was activated [38 39] Takentogether these findings indicate that the alpha-cell pool inthe endocrine pancreas has the propensity to regenerate andmay therefore represent a valuable source to give rise tonew beta-cells [36] Therefore testing the ability of PAX4
New Journal of Science 5
to convert human alpha-cells into functional beta-cells isof fundamental interest for the development of therapeuticapproaches to treat diabetes
As reported above during the course of developmentPax4 and Arx undergo a reciprocal cross-inhibitory interac-tion allowing the differential specification towards the beta-and alpha-cell fates respectively [1 10] Accordingly theconversion of alpha-cells into beta-like cells mediated bythe forced activation of Pax4 expression may also be due tothe concomitantly suppressed Arx gene activity in glucagon-producing cells It is therefore reasonable to assume thatthe downregulation of Arx gene function in adult glucagon-producing cells may promote their transdifferentiation intobeta-cellsThis has recently been demonstrated by the condi-tional inactivation of Arx in adult alpha-cells which resultedin the appearance of newly generated insulin-producing beta-cells [40] Moreover this alpha-to-beta-like cell transdiffer-entiation induced process triggering a cycle of alpha-cellneogenesis as has been noted in Pax4 misexpressing mice[38ndash40] Remarkably the conditional double knockout ofArxand Pax4 in adult alpha-cells still efficiently promotes thetransdifferentiation of glucagon-producing cells into beta-like cells that could counter chemically induced diabetes[40] These findings are consistent with the idea that Arxfunctional inactivation is the main driving force in achievingthe reprograming of alpha-cells into beta-cells [40] There-fore novel strategies allowing the downregulation of Arxexpression or of its downstream targets in alpha-cells couldopen new avenues to develop innovative approaches forgenerating beta-cells to treat diabetes The identification ofArx target genes andor interacting factors may representan important step in reaching this goal In addition futurestudies are also required to determine whether human alpha-cells are able to transdifferentiate into beta-like cells uponPax4 forced expression or by targeting Arx inactivationFurther exploration of the molecular interactions mecha-nisms controlling endocrine cell subtype specification inthe endocrine pancreas may also uncover novel candidatemolecules that allow the establishment of strategies to directlyprovoke alpha- to beta-cell reprogramming in vivo
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
M Courtney and T Rabe contributed equally
Acknowledgments
This work was supported by the Juvenile Diabetes Researchfoundation (17-2011-16 2-2010-567 26-2008-639 and 17-2013-426) the INSERM AVENIR program the INSERMthe European Research Council (StG-2011-281265) theFMR (DRC20091217179) the ANRBMBF (2009 GENO 1050101KU0906) the ldquoInvestments for the Futurerdquo LABEX SIG-NALIFE (ANR-11-LABX-0028-01) the Max-Planck SocietyClub Isatis Mr and Mrs Dorato the Fondation Generale de
Sante and the Foundation Schlumberger pour lrsquoEducation etla Recherche
References
[1] P Collombat J Hecksher-Sorensen P Serup and A MansourildquoSpecifying pancreatic endocrine cell fatesrdquo Mechanisms ofDevelopment vol 123 no 7 pp 501ndash512 2006
[2] R L Pictet W R Clark R H Williams and W J Rutter ldquoAnultrastructural analysis of the developing embryonic pancreasrdquoDevelopmental Biology vol 29 no 4 pp 436ndash467 1972
[3] F C Pan and C Wright ldquoPancreas organogenesis from bud toplexus to glandrdquo Developmental Dynamics vol 240 no 3 pp530ndash565 2011
[4] G K Gittes ldquoDevelopmental biology of the pancreas a compre-hensive reviewrdquoDevelopmental Biology vol 326 no 1 pp 4ndash352009
[5] G Gu J R Brown and D A Melton ldquoDirect lineage tracingreveals the ontogeny of pancreatic cell fates during mouseembryogenesisrdquoMechanisms of Development vol 120 no 1 pp35ndash43 2003
[6] Y Kawaguchi B Cooper M GannonM Ray R J MacDonaldand C V E Wright ldquoThe role of the transcriptional regulatorPtf1a in converting intestinal to pancreatic progenitorsrdquoNatureGenetics vol 32 no 1 pp 128ndash134 2002
[7] G Gradwohl A Dierich M LeMeur and F Guillemot ldquoneuro-genin3 is required for the development of the four endocrine celllineages of the pancreasrdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 4 pp 1607ndash1611 2000
[8] V M Schwitzgebel D W Scheel J R Conners et al ldquoExpres-sion of neurogenin3 reveals an islet cell precursor populationin the pancreasrdquo Development vol 127 no 16 pp 3533ndash35422000
[9] Q Zhou A C Law J Rajagopal W J Anderson P A Grayand D A Melton ldquoA multipotent progenitor domain guidespancreatic organogenesisrdquoDevelopmental Cell vol 13 no 1 pp103ndash114 2007
[10] P Collombat J Hecksher-Sorensen V Broccoli et alldquoThe simultaneous loss of Arx and Pax4 genes promotes asomatostatin-producing cell fate specification at the expense ofthe 120572- and 120573-cell lineages in the mouse endocrine pancreasrdquoDevelopment vol 132 no 13 pp 2969ndash2980 2005
[11] P Collombat AMansouri J Hecksher-Sorensen et al ldquoOppos-ing actions of Arx and Pax4 in endocrine pancreas develop-mentrdquo Genes amp Development vol 17 no 20 pp 2591ndash26032003
[12] A Mansouri M Hallonet and P Gruss ldquoPax genes and theirroles in cell differentiation and developmentrdquo Current Opinionin Cell Biology vol 8 no 6 pp 851ndash857 1996
[13] A Mansouri G Goudreau and P Gruss ldquoPax genes and theirrole in organogenesisrdquoCancer Research vol 59 no 7 pp 1707Sndash1710S 1999
[14] A Mansouri L St-Onge and P Gruss ldquoRole of Pax genesin endoderm-derived organsrdquo Trends in Endocrinology ampMetabolism vol 10 no 4 pp 164ndash167 1999
[15] B Sosa-Pineda K Chowdhury M Torres G Oliver and PGruss ldquoThe Pax4 gene is essential for differentiation of insulin-producing 120573 cells in themammalian pancreasrdquoNature vol 386no 6623 pp 399ndash402 1997
6 New Journal of Science
[16] A L Greenwood S Li K Jones and D A Melton ldquoNotchsignaling reveals developmental plasticity of Pax4+ pancreaticendocrine progenitors and shunts them to a duct faterdquoMecha-nisms of Development vol 124 no 2 pp 97ndash107 2007
[17] P Collombat X Xu P Ravassard et al ldquoThe ectopic expressionof Pax4 in the mouse pancreas converts progenitor cells into 120572and subsequently120573 cellsrdquoCell vol 138 no 3 pp 449ndash462 2009
[18] J Lu P L Herrera C Carreira et al ldquo120572 cell-specific Men1ablation triggers the transdifferentiation of glucagon-expressingcells and insulinoma developmentrdquo Gastroenterology vol 138no 5 pp 1954ndash1965 2010
[19] S Biressi G Messina P Collombat et al ldquoThe homeobox geneArx is a novel positive regulator of embryonic myogenesisrdquo CellDeath and Differentiation vol 15 no 1 pp 94ndash104 2008
[20] E Colombo P Collombat G Colasante et al ldquoInactivation ofArx the murine ortholog of the X-linked lissencephaly withambiguous genitalia gene leads to severe disorganization of theventral telencephalon with impaired neuronal migration anddifferentiationrdquo The Journal of Neuroscience vol 27 no 17 pp4786ndash4798 2007
[21] S Dhawan S Georgia S-I Tschen G Fan and A BhushanldquoPancreatic 120573 cell identity is maintained by DNA methylation-mediated repression of Arxrdquo Developmental Cell vol 20 no 4pp 419ndash429 2011
[22] J B Papizan R A Singer S-I Tschen et al ldquoNkx22 repressorcomplex regulates islet120573-cell specification and prevents 120573-to-120572-cell reprogrammingrdquo Genes amp Development vol 25 no 21 pp2291ndash2305 2011
[23] L Sussel J Kalamaras D J Hartigan-OrsquoConnor et al ldquoMicelacking the homeodomain transcription factor Nkx22 havediabetes due to arrested differentiation of pancreatic 120573 cellsrdquoDevelopment vol 125 no 12 pp 2213ndash2221 1998
[24] C S Chao Z L Loomis J E Lee and L Sussel ldquoGenetic identi-fication of a novel NeuroD1 function in the early differentiationof islet 120572 PP and 120576 cellsrdquo Developmental Biology vol 312 no 2pp 523ndash532 2007
[25] S Kordowich P Collombat A Mansouri and P Serup ldquoArxand Nkx22 compound deficiency redirects pancreatic alpha-and beta-cell differentiation to a somatostatinghrelin co-expressing cell lineagerdquo BMC Developmental Biology vol 11article 52 2011
[26] T L Mastracci C Wilcox L Arnes et al ldquoNkx22 and Arxgenetically interact to regulate pancreatic endocrine cell devel-opment and endocrine hormone expressionrdquo DevelopmentalBiology vol 359 no 1 pp 1ndash11 2011
[27] P Collombat J Hecksher-Sorensen J Krull et al ldquoEmbryonicendocrine pancreas and mature 120573 cells acquire 120572 and PP cellphenotypes upon Arx misexpressionrdquo The Journal of ClinicalInvestigation vol 117 no 4 pp 961ndash970 2007
[28] M Furuta H Yano A Zhou et al ldquoDefective prohormoneprocessing and altered pancreatic islet morphology in micelacking active SPC2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 94 no 13 pp 6646ndash6651 1997
[29] RWGelling XQDuD SDichmann et al ldquoLower blood glu-cose hyperglucagonemia and pancreatic 120572 cell hyperplasia inglucagon receptor knockout micerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no3 pp 1438ndash1443 2003
[30] Y Hayashi M Yamamoto H Mizoguchi et al ldquoMice deficientfor glucagon gene-derived peptides display normoglycemia
and hyperplasia of islet 120572-cells but not of intestinal L-cellsrdquoMolecular Endocrinology vol 23 no 12 pp 1990ndash1999 2009
[31] P Collombat and A Mansouri ldquoPax4 transdifferentiatesglucagon-secreting 120572 cells to insulin-secreting 120573 endocrinepancreatic cellsrdquo MedecineSciences vol 25 no 8-9 pp 763ndash765 2009
[32] P Collombat and A Mansouri ldquoTurning on the 120573-cell identityin the pancreasrdquo Cell Cycle vol 8 no 21 pp 3450ndash3451 2009
[33] YDor andDAMelton ldquoFacultative endocrine progenitor cellsin the adult pancreasrdquo Cell vol 132 no 2 pp 183ndash184 2008
[34] J F Habener and V Stanojevic ldquo120572-cell role in 120573-cell generationand regenerationrdquo Islets vol 4 no 3 pp 188ndash198 2012
[35] Z Liu and J F Habener ldquoAlpha cells beget beta cellsrdquo Cell vol138 no 3 pp 424ndash426 2009
[36] A Mansouri ldquoDevelopment and regeneration in the endocrinepancreasrdquo ISRN Endocrinology vol 2012 Article ID 640956 12pages 2012
[37] X Xu J DrsquoHoker G Stange et al ldquo120573 cells can be generated fromendogenous progenitors in injured adult mouse pancreasrdquo Cellvol 132 no 2 pp 197ndash207 2008
[38] K Al-Hasani A Pfeifer M Courtney et al ldquoAdult duct-liningcells can reprogram into 120573-like cells able to counter repeatedcycles of toxin-induced diabetesrdquo Developmental Cell vol 26no 1 pp 86ndash100 2013
[39] A Pfeifer M Courtney N Ben-Othman et al et al ldquoInductionof multiple cycles of pancreatic 120573-cell replacementrdquo Cell Cyclevol 12 no 20 pp 3243ndash3244 2013
[40] M Courtney E Gjernes N Druelle et al ldquoThe inactivationof Arx in pancreatic 120572-cells triggers their neogenesis andconversion into functional 120573-like cellsrdquo PLoS Genetics vol 9no 10 article e1003934 2013
Remarkably the analysis of transgenic mice allowingthe forced expression of Arx in Pdx1-labelled pancreaticprecursors in Pax6-expressing endocrine cells or even inmature beta-cells (using inducible Pdx1-Cre mice) revealeda strong hyperglycemia leading to precocious death [27]Accordingly immunohistochemical endocrine marker anal-yses and quantification of islet cells clearly demonstratedthat in all transgenic mouse lines a consistent increase inthe number of cells with alpha- and PP-cell characteristicsoccurred In contrast the number of insulin-producing cellswas dramatically reduced [27] These data indicate that thesole forced expression of Arx in pancreatic cells at differentdevelopmental and adult stages is sufficient to induce changesin islet cell destiny [27] It is interesting to note that themisexpression of Arx in mature beta-cells is able to convertthese into cells displaying alpha- and PP-cell features Thustowards the goal of generating beta-cells fromendocrine cellswe also forced the expression of the transcription factor Pax4not only in the pancreatic endoderm but also in endocrinecells at different stages of endocrine cell development [17]Using a similar strategy as described for Arx the condi-tional misexpression of Pax4 during development revealedits role in promoting the beta-cell fate [17] Importantlya conversion of alpha-cells into beta-like cells was notedthrough the forced expression of Pax4 in alpha-cells Indeedtransgenic mice expressing Pax4 in alpha-cells displayedan age-dependent increase of the beta-cell mass leadingto hypertrophic islets provoked by hyperplasia of insulin-expressing cells Lineage tracing indicated that these beta-like cells were converted from alpha-cells in which Pax4 wasmisexpressed [17] The increase in islet size was intriguingand pointed to permanent regeneration of the alpha-cell poolIn fact this was consistent with the idea that compromisingthe glucagon signaling pathway is associated with alpha-cellregeneration (Figure 3) Indeed mice lacking the glucagonreceptor prohormone convertase 2 or glucagon gene-derivedpeptides display alpha-cell hyperplasia [28ndash30] Accordinglyglucagon supplementation in Pax4 misexpressing mice wasable to diminish the dramatic increase in islet size [17]Thus in Pax4 transgenic mice glucagon signaling alter-ation contributes to alpha-cell regeneration these cells beingconverted into beta-like cells upon Pax4 misexpression Ofnote was the replenishment of insulin producing beta-cellsobserved in Pax4 misexpressing mice following chemicallyinduced diabetes Sixty days after the initiation of beta-cell ablation islets exhibited a normal content of insulin-producing cells that were derived from converted alpha-cellsexpressing Pax4 Altogether these results indicate that theforced expression of Pax4 in alpha-cells is able to induce theirneogenesis and conversion into functional beta-like cellsThese processes were found to depend on the reexpression ofthe proendocrine gene Ngn3 in the ductal lining supportingthe notion of the existence of precursor cells in ducts [17 31ndash37]
The misexpression of Pax4 in alpha-cells was achievedusing transgenic mice expressing the Cre recombinase underthe control of the glucagon promoter (see Figure 2) Accord-ingly in such a strategy Pax4 expression was induced innewly developed alpha-cells [17] Therefore it is reasonable
Compromisedglucagon signaling
Forced expression
Signal
Duct
Conversion
EMT
neogenesis
reactivationof Ngn3
into 120573-cells 120573-cells
120572-cells 120572-cellof Pax4 in 120572-cells
Figure 3 The forced expression of Pax4 in alpha-cells inducestheir conversion to beta-like cells The use of the site-specificrecombination (Figure 2) as well as the tetracycline inducible systemto control the forced expression of Pax4 in alpha-cells was foundto induce their conversion into functional beta-like cells and topromote a concomitant induction of alpha-like cell regenerationThe shortage in glucagon provoked by the conversion of alpha-cells into beta-cells appeared to contribute to the mobilization ofduct-lining precursor cells that reactivate Ngn3 and undergo EMT(epithelial mesenchymal transition) prior to adopting an alpha-likecell identity Such cells are yet again turned into beta-like cells uponPax4 misexpression and should it be maintained
to query whether glucagon-producing alpha-cells residingin the islets of adult mice and being exposed to epigeneticenvironment are also capable of undergoing such conversionupon Pax4 misexpression Hence taking advantage of theinducible tetracycline system Pax4 expression was activatedin alpha-cells of adult mice at various ages and for dif-ferent periods of time Pax4 expression in alpha-cells wastriggered and controlled for a definite time period by thesupplementation of doxycycline in the drinking water [38]Remarkably using this system not only could adult alpha-cells be converted into beta-like cells but also a continuouscycle of alpha-cell neogenesis was observed It is interestingto note that this process was age-independent Transgenicanimals could survive several cycles of chemically-inducedbeta-cell ablation and regenerate multiple times their wholebeta-like cell mass [38] Lineage tracing experiments usingthe site-specific Cre recombinase system clearly establishedthat (a) newly generated beta-like cells were converted fromalpha-cells and (b) a cycle of alpha-cell neogenesis wasinduced such cells originating from duct-lining cells thatreactivated Ngn3 expression and underwent EMT (epithelialmesenchymal transition) [38] Indeed in areas adjacent toducts mesenchymal-like structures were observed wherethe expression of mesenchymal markers such as nestinvimentin snail2 and Sox11 was activated [38 39] Takentogether these findings indicate that the alpha-cell pool inthe endocrine pancreas has the propensity to regenerate andmay therefore represent a valuable source to give rise tonew beta-cells [36] Therefore testing the ability of PAX4
New Journal of Science 5
to convert human alpha-cells into functional beta-cells isof fundamental interest for the development of therapeuticapproaches to treat diabetes
As reported above during the course of developmentPax4 and Arx undergo a reciprocal cross-inhibitory interac-tion allowing the differential specification towards the beta-and alpha-cell fates respectively [1 10] Accordingly theconversion of alpha-cells into beta-like cells mediated bythe forced activation of Pax4 expression may also be due tothe concomitantly suppressed Arx gene activity in glucagon-producing cells It is therefore reasonable to assume thatthe downregulation of Arx gene function in adult glucagon-producing cells may promote their transdifferentiation intobeta-cellsThis has recently been demonstrated by the condi-tional inactivation of Arx in adult alpha-cells which resultedin the appearance of newly generated insulin-producing beta-cells [40] Moreover this alpha-to-beta-like cell transdiffer-entiation induced process triggering a cycle of alpha-cellneogenesis as has been noted in Pax4 misexpressing mice[38ndash40] Remarkably the conditional double knockout ofArxand Pax4 in adult alpha-cells still efficiently promotes thetransdifferentiation of glucagon-producing cells into beta-like cells that could counter chemically induced diabetes[40] These findings are consistent with the idea that Arxfunctional inactivation is the main driving force in achievingthe reprograming of alpha-cells into beta-cells [40] There-fore novel strategies allowing the downregulation of Arxexpression or of its downstream targets in alpha-cells couldopen new avenues to develop innovative approaches forgenerating beta-cells to treat diabetes The identification ofArx target genes andor interacting factors may representan important step in reaching this goal In addition futurestudies are also required to determine whether human alpha-cells are able to transdifferentiate into beta-like cells uponPax4 forced expression or by targeting Arx inactivationFurther exploration of the molecular interactions mecha-nisms controlling endocrine cell subtype specification inthe endocrine pancreas may also uncover novel candidatemolecules that allow the establishment of strategies to directlyprovoke alpha- to beta-cell reprogramming in vivo
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
M Courtney and T Rabe contributed equally
Acknowledgments
This work was supported by the Juvenile Diabetes Researchfoundation (17-2011-16 2-2010-567 26-2008-639 and 17-2013-426) the INSERM AVENIR program the INSERMthe European Research Council (StG-2011-281265) theFMR (DRC20091217179) the ANRBMBF (2009 GENO 1050101KU0906) the ldquoInvestments for the Futurerdquo LABEX SIG-NALIFE (ANR-11-LABX-0028-01) the Max-Planck SocietyClub Isatis Mr and Mrs Dorato the Fondation Generale de
Sante and the Foundation Schlumberger pour lrsquoEducation etla Recherche
References
[1] P Collombat J Hecksher-Sorensen P Serup and A MansourildquoSpecifying pancreatic endocrine cell fatesrdquo Mechanisms ofDevelopment vol 123 no 7 pp 501ndash512 2006
[2] R L Pictet W R Clark R H Williams and W J Rutter ldquoAnultrastructural analysis of the developing embryonic pancreasrdquoDevelopmental Biology vol 29 no 4 pp 436ndash467 1972
[3] F C Pan and C Wright ldquoPancreas organogenesis from bud toplexus to glandrdquo Developmental Dynamics vol 240 no 3 pp530ndash565 2011
[4] G K Gittes ldquoDevelopmental biology of the pancreas a compre-hensive reviewrdquoDevelopmental Biology vol 326 no 1 pp 4ndash352009
[5] G Gu J R Brown and D A Melton ldquoDirect lineage tracingreveals the ontogeny of pancreatic cell fates during mouseembryogenesisrdquoMechanisms of Development vol 120 no 1 pp35ndash43 2003
[6] Y Kawaguchi B Cooper M GannonM Ray R J MacDonaldand C V E Wright ldquoThe role of the transcriptional regulatorPtf1a in converting intestinal to pancreatic progenitorsrdquoNatureGenetics vol 32 no 1 pp 128ndash134 2002
[7] G Gradwohl A Dierich M LeMeur and F Guillemot ldquoneuro-genin3 is required for the development of the four endocrine celllineages of the pancreasrdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 4 pp 1607ndash1611 2000
[8] V M Schwitzgebel D W Scheel J R Conners et al ldquoExpres-sion of neurogenin3 reveals an islet cell precursor populationin the pancreasrdquo Development vol 127 no 16 pp 3533ndash35422000
[9] Q Zhou A C Law J Rajagopal W J Anderson P A Grayand D A Melton ldquoA multipotent progenitor domain guidespancreatic organogenesisrdquoDevelopmental Cell vol 13 no 1 pp103ndash114 2007
[10] P Collombat J Hecksher-Sorensen V Broccoli et alldquoThe simultaneous loss of Arx and Pax4 genes promotes asomatostatin-producing cell fate specification at the expense ofthe 120572- and 120573-cell lineages in the mouse endocrine pancreasrdquoDevelopment vol 132 no 13 pp 2969ndash2980 2005
[11] P Collombat AMansouri J Hecksher-Sorensen et al ldquoOppos-ing actions of Arx and Pax4 in endocrine pancreas develop-mentrdquo Genes amp Development vol 17 no 20 pp 2591ndash26032003
[12] A Mansouri M Hallonet and P Gruss ldquoPax genes and theirroles in cell differentiation and developmentrdquo Current Opinionin Cell Biology vol 8 no 6 pp 851ndash857 1996
[13] A Mansouri G Goudreau and P Gruss ldquoPax genes and theirrole in organogenesisrdquoCancer Research vol 59 no 7 pp 1707Sndash1710S 1999
[14] A Mansouri L St-Onge and P Gruss ldquoRole of Pax genesin endoderm-derived organsrdquo Trends in Endocrinology ampMetabolism vol 10 no 4 pp 164ndash167 1999
[15] B Sosa-Pineda K Chowdhury M Torres G Oliver and PGruss ldquoThe Pax4 gene is essential for differentiation of insulin-producing 120573 cells in themammalian pancreasrdquoNature vol 386no 6623 pp 399ndash402 1997
6 New Journal of Science
[16] A L Greenwood S Li K Jones and D A Melton ldquoNotchsignaling reveals developmental plasticity of Pax4+ pancreaticendocrine progenitors and shunts them to a duct faterdquoMecha-nisms of Development vol 124 no 2 pp 97ndash107 2007
[17] P Collombat X Xu P Ravassard et al ldquoThe ectopic expressionof Pax4 in the mouse pancreas converts progenitor cells into 120572and subsequently120573 cellsrdquoCell vol 138 no 3 pp 449ndash462 2009
[18] J Lu P L Herrera C Carreira et al ldquo120572 cell-specific Men1ablation triggers the transdifferentiation of glucagon-expressingcells and insulinoma developmentrdquo Gastroenterology vol 138no 5 pp 1954ndash1965 2010
[19] S Biressi G Messina P Collombat et al ldquoThe homeobox geneArx is a novel positive regulator of embryonic myogenesisrdquo CellDeath and Differentiation vol 15 no 1 pp 94ndash104 2008
[20] E Colombo P Collombat G Colasante et al ldquoInactivation ofArx the murine ortholog of the X-linked lissencephaly withambiguous genitalia gene leads to severe disorganization of theventral telencephalon with impaired neuronal migration anddifferentiationrdquo The Journal of Neuroscience vol 27 no 17 pp4786ndash4798 2007
[21] S Dhawan S Georgia S-I Tschen G Fan and A BhushanldquoPancreatic 120573 cell identity is maintained by DNA methylation-mediated repression of Arxrdquo Developmental Cell vol 20 no 4pp 419ndash429 2011
[22] J B Papizan R A Singer S-I Tschen et al ldquoNkx22 repressorcomplex regulates islet120573-cell specification and prevents 120573-to-120572-cell reprogrammingrdquo Genes amp Development vol 25 no 21 pp2291ndash2305 2011
[23] L Sussel J Kalamaras D J Hartigan-OrsquoConnor et al ldquoMicelacking the homeodomain transcription factor Nkx22 havediabetes due to arrested differentiation of pancreatic 120573 cellsrdquoDevelopment vol 125 no 12 pp 2213ndash2221 1998
[24] C S Chao Z L Loomis J E Lee and L Sussel ldquoGenetic identi-fication of a novel NeuroD1 function in the early differentiationof islet 120572 PP and 120576 cellsrdquo Developmental Biology vol 312 no 2pp 523ndash532 2007
[25] S Kordowich P Collombat A Mansouri and P Serup ldquoArxand Nkx22 compound deficiency redirects pancreatic alpha-and beta-cell differentiation to a somatostatinghrelin co-expressing cell lineagerdquo BMC Developmental Biology vol 11article 52 2011
[26] T L Mastracci C Wilcox L Arnes et al ldquoNkx22 and Arxgenetically interact to regulate pancreatic endocrine cell devel-opment and endocrine hormone expressionrdquo DevelopmentalBiology vol 359 no 1 pp 1ndash11 2011
[27] P Collombat J Hecksher-Sorensen J Krull et al ldquoEmbryonicendocrine pancreas and mature 120573 cells acquire 120572 and PP cellphenotypes upon Arx misexpressionrdquo The Journal of ClinicalInvestigation vol 117 no 4 pp 961ndash970 2007
[28] M Furuta H Yano A Zhou et al ldquoDefective prohormoneprocessing and altered pancreatic islet morphology in micelacking active SPC2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 94 no 13 pp 6646ndash6651 1997
[29] RWGelling XQDuD SDichmann et al ldquoLower blood glu-cose hyperglucagonemia and pancreatic 120572 cell hyperplasia inglucagon receptor knockout micerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no3 pp 1438ndash1443 2003
[30] Y Hayashi M Yamamoto H Mizoguchi et al ldquoMice deficientfor glucagon gene-derived peptides display normoglycemia
and hyperplasia of islet 120572-cells but not of intestinal L-cellsrdquoMolecular Endocrinology vol 23 no 12 pp 1990ndash1999 2009
[31] P Collombat and A Mansouri ldquoPax4 transdifferentiatesglucagon-secreting 120572 cells to insulin-secreting 120573 endocrinepancreatic cellsrdquo MedecineSciences vol 25 no 8-9 pp 763ndash765 2009
[32] P Collombat and A Mansouri ldquoTurning on the 120573-cell identityin the pancreasrdquo Cell Cycle vol 8 no 21 pp 3450ndash3451 2009
[33] YDor andDAMelton ldquoFacultative endocrine progenitor cellsin the adult pancreasrdquo Cell vol 132 no 2 pp 183ndash184 2008
[34] J F Habener and V Stanojevic ldquo120572-cell role in 120573-cell generationand regenerationrdquo Islets vol 4 no 3 pp 188ndash198 2012
[35] Z Liu and J F Habener ldquoAlpha cells beget beta cellsrdquo Cell vol138 no 3 pp 424ndash426 2009
[36] A Mansouri ldquoDevelopment and regeneration in the endocrinepancreasrdquo ISRN Endocrinology vol 2012 Article ID 640956 12pages 2012
[37] X Xu J DrsquoHoker G Stange et al ldquo120573 cells can be generated fromendogenous progenitors in injured adult mouse pancreasrdquo Cellvol 132 no 2 pp 197ndash207 2008
[38] K Al-Hasani A Pfeifer M Courtney et al ldquoAdult duct-liningcells can reprogram into 120573-like cells able to counter repeatedcycles of toxin-induced diabetesrdquo Developmental Cell vol 26no 1 pp 86ndash100 2013
[39] A Pfeifer M Courtney N Ben-Othman et al et al ldquoInductionof multiple cycles of pancreatic 120573-cell replacementrdquo Cell Cyclevol 12 no 20 pp 3243ndash3244 2013
[40] M Courtney E Gjernes N Druelle et al ldquoThe inactivationof Arx in pancreatic 120572-cells triggers their neogenesis andconversion into functional 120573-like cellsrdquo PLoS Genetics vol 9no 10 article e1003934 2013
to convert human alpha-cells into functional beta-cells isof fundamental interest for the development of therapeuticapproaches to treat diabetes
As reported above during the course of developmentPax4 and Arx undergo a reciprocal cross-inhibitory interac-tion allowing the differential specification towards the beta-and alpha-cell fates respectively [1 10] Accordingly theconversion of alpha-cells into beta-like cells mediated bythe forced activation of Pax4 expression may also be due tothe concomitantly suppressed Arx gene activity in glucagon-producing cells It is therefore reasonable to assume thatthe downregulation of Arx gene function in adult glucagon-producing cells may promote their transdifferentiation intobeta-cellsThis has recently been demonstrated by the condi-tional inactivation of Arx in adult alpha-cells which resultedin the appearance of newly generated insulin-producing beta-cells [40] Moreover this alpha-to-beta-like cell transdiffer-entiation induced process triggering a cycle of alpha-cellneogenesis as has been noted in Pax4 misexpressing mice[38ndash40] Remarkably the conditional double knockout ofArxand Pax4 in adult alpha-cells still efficiently promotes thetransdifferentiation of glucagon-producing cells into beta-like cells that could counter chemically induced diabetes[40] These findings are consistent with the idea that Arxfunctional inactivation is the main driving force in achievingthe reprograming of alpha-cells into beta-cells [40] There-fore novel strategies allowing the downregulation of Arxexpression or of its downstream targets in alpha-cells couldopen new avenues to develop innovative approaches forgenerating beta-cells to treat diabetes The identification ofArx target genes andor interacting factors may representan important step in reaching this goal In addition futurestudies are also required to determine whether human alpha-cells are able to transdifferentiate into beta-like cells uponPax4 forced expression or by targeting Arx inactivationFurther exploration of the molecular interactions mecha-nisms controlling endocrine cell subtype specification inthe endocrine pancreas may also uncover novel candidatemolecules that allow the establishment of strategies to directlyprovoke alpha- to beta-cell reprogramming in vivo
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Authorsrsquo Contribution
M Courtney and T Rabe contributed equally
Acknowledgments
This work was supported by the Juvenile Diabetes Researchfoundation (17-2011-16 2-2010-567 26-2008-639 and 17-2013-426) the INSERM AVENIR program the INSERMthe European Research Council (StG-2011-281265) theFMR (DRC20091217179) the ANRBMBF (2009 GENO 1050101KU0906) the ldquoInvestments for the Futurerdquo LABEX SIG-NALIFE (ANR-11-LABX-0028-01) the Max-Planck SocietyClub Isatis Mr and Mrs Dorato the Fondation Generale de
Sante and the Foundation Schlumberger pour lrsquoEducation etla Recherche
References
[1] P Collombat J Hecksher-Sorensen P Serup and A MansourildquoSpecifying pancreatic endocrine cell fatesrdquo Mechanisms ofDevelopment vol 123 no 7 pp 501ndash512 2006
[2] R L Pictet W R Clark R H Williams and W J Rutter ldquoAnultrastructural analysis of the developing embryonic pancreasrdquoDevelopmental Biology vol 29 no 4 pp 436ndash467 1972
[3] F C Pan and C Wright ldquoPancreas organogenesis from bud toplexus to glandrdquo Developmental Dynamics vol 240 no 3 pp530ndash565 2011
[4] G K Gittes ldquoDevelopmental biology of the pancreas a compre-hensive reviewrdquoDevelopmental Biology vol 326 no 1 pp 4ndash352009
[5] G Gu J R Brown and D A Melton ldquoDirect lineage tracingreveals the ontogeny of pancreatic cell fates during mouseembryogenesisrdquoMechanisms of Development vol 120 no 1 pp35ndash43 2003
[6] Y Kawaguchi B Cooper M GannonM Ray R J MacDonaldand C V E Wright ldquoThe role of the transcriptional regulatorPtf1a in converting intestinal to pancreatic progenitorsrdquoNatureGenetics vol 32 no 1 pp 128ndash134 2002
[7] G Gradwohl A Dierich M LeMeur and F Guillemot ldquoneuro-genin3 is required for the development of the four endocrine celllineages of the pancreasrdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 4 pp 1607ndash1611 2000
[8] V M Schwitzgebel D W Scheel J R Conners et al ldquoExpres-sion of neurogenin3 reveals an islet cell precursor populationin the pancreasrdquo Development vol 127 no 16 pp 3533ndash35422000
[9] Q Zhou A C Law J Rajagopal W J Anderson P A Grayand D A Melton ldquoA multipotent progenitor domain guidespancreatic organogenesisrdquoDevelopmental Cell vol 13 no 1 pp103ndash114 2007
[10] P Collombat J Hecksher-Sorensen V Broccoli et alldquoThe simultaneous loss of Arx and Pax4 genes promotes asomatostatin-producing cell fate specification at the expense ofthe 120572- and 120573-cell lineages in the mouse endocrine pancreasrdquoDevelopment vol 132 no 13 pp 2969ndash2980 2005
[11] P Collombat AMansouri J Hecksher-Sorensen et al ldquoOppos-ing actions of Arx and Pax4 in endocrine pancreas develop-mentrdquo Genes amp Development vol 17 no 20 pp 2591ndash26032003
[12] A Mansouri M Hallonet and P Gruss ldquoPax genes and theirroles in cell differentiation and developmentrdquo Current Opinionin Cell Biology vol 8 no 6 pp 851ndash857 1996
[13] A Mansouri G Goudreau and P Gruss ldquoPax genes and theirrole in organogenesisrdquoCancer Research vol 59 no 7 pp 1707Sndash1710S 1999
[14] A Mansouri L St-Onge and P Gruss ldquoRole of Pax genesin endoderm-derived organsrdquo Trends in Endocrinology ampMetabolism vol 10 no 4 pp 164ndash167 1999
[15] B Sosa-Pineda K Chowdhury M Torres G Oliver and PGruss ldquoThe Pax4 gene is essential for differentiation of insulin-producing 120573 cells in themammalian pancreasrdquoNature vol 386no 6623 pp 399ndash402 1997
6 New Journal of Science
[16] A L Greenwood S Li K Jones and D A Melton ldquoNotchsignaling reveals developmental plasticity of Pax4+ pancreaticendocrine progenitors and shunts them to a duct faterdquoMecha-nisms of Development vol 124 no 2 pp 97ndash107 2007
[17] P Collombat X Xu P Ravassard et al ldquoThe ectopic expressionof Pax4 in the mouse pancreas converts progenitor cells into 120572and subsequently120573 cellsrdquoCell vol 138 no 3 pp 449ndash462 2009
[18] J Lu P L Herrera C Carreira et al ldquo120572 cell-specific Men1ablation triggers the transdifferentiation of glucagon-expressingcells and insulinoma developmentrdquo Gastroenterology vol 138no 5 pp 1954ndash1965 2010
[19] S Biressi G Messina P Collombat et al ldquoThe homeobox geneArx is a novel positive regulator of embryonic myogenesisrdquo CellDeath and Differentiation vol 15 no 1 pp 94ndash104 2008
[20] E Colombo P Collombat G Colasante et al ldquoInactivation ofArx the murine ortholog of the X-linked lissencephaly withambiguous genitalia gene leads to severe disorganization of theventral telencephalon with impaired neuronal migration anddifferentiationrdquo The Journal of Neuroscience vol 27 no 17 pp4786ndash4798 2007
[21] S Dhawan S Georgia S-I Tschen G Fan and A BhushanldquoPancreatic 120573 cell identity is maintained by DNA methylation-mediated repression of Arxrdquo Developmental Cell vol 20 no 4pp 419ndash429 2011
[22] J B Papizan R A Singer S-I Tschen et al ldquoNkx22 repressorcomplex regulates islet120573-cell specification and prevents 120573-to-120572-cell reprogrammingrdquo Genes amp Development vol 25 no 21 pp2291ndash2305 2011
[23] L Sussel J Kalamaras D J Hartigan-OrsquoConnor et al ldquoMicelacking the homeodomain transcription factor Nkx22 havediabetes due to arrested differentiation of pancreatic 120573 cellsrdquoDevelopment vol 125 no 12 pp 2213ndash2221 1998
[24] C S Chao Z L Loomis J E Lee and L Sussel ldquoGenetic identi-fication of a novel NeuroD1 function in the early differentiationof islet 120572 PP and 120576 cellsrdquo Developmental Biology vol 312 no 2pp 523ndash532 2007
[25] S Kordowich P Collombat A Mansouri and P Serup ldquoArxand Nkx22 compound deficiency redirects pancreatic alpha-and beta-cell differentiation to a somatostatinghrelin co-expressing cell lineagerdquo BMC Developmental Biology vol 11article 52 2011
[26] T L Mastracci C Wilcox L Arnes et al ldquoNkx22 and Arxgenetically interact to regulate pancreatic endocrine cell devel-opment and endocrine hormone expressionrdquo DevelopmentalBiology vol 359 no 1 pp 1ndash11 2011
[27] P Collombat J Hecksher-Sorensen J Krull et al ldquoEmbryonicendocrine pancreas and mature 120573 cells acquire 120572 and PP cellphenotypes upon Arx misexpressionrdquo The Journal of ClinicalInvestigation vol 117 no 4 pp 961ndash970 2007
[28] M Furuta H Yano A Zhou et al ldquoDefective prohormoneprocessing and altered pancreatic islet morphology in micelacking active SPC2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 94 no 13 pp 6646ndash6651 1997
[29] RWGelling XQDuD SDichmann et al ldquoLower blood glu-cose hyperglucagonemia and pancreatic 120572 cell hyperplasia inglucagon receptor knockout micerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no3 pp 1438ndash1443 2003
[30] Y Hayashi M Yamamoto H Mizoguchi et al ldquoMice deficientfor glucagon gene-derived peptides display normoglycemia
and hyperplasia of islet 120572-cells but not of intestinal L-cellsrdquoMolecular Endocrinology vol 23 no 12 pp 1990ndash1999 2009
[31] P Collombat and A Mansouri ldquoPax4 transdifferentiatesglucagon-secreting 120572 cells to insulin-secreting 120573 endocrinepancreatic cellsrdquo MedecineSciences vol 25 no 8-9 pp 763ndash765 2009
[32] P Collombat and A Mansouri ldquoTurning on the 120573-cell identityin the pancreasrdquo Cell Cycle vol 8 no 21 pp 3450ndash3451 2009
[33] YDor andDAMelton ldquoFacultative endocrine progenitor cellsin the adult pancreasrdquo Cell vol 132 no 2 pp 183ndash184 2008
[34] J F Habener and V Stanojevic ldquo120572-cell role in 120573-cell generationand regenerationrdquo Islets vol 4 no 3 pp 188ndash198 2012
[35] Z Liu and J F Habener ldquoAlpha cells beget beta cellsrdquo Cell vol138 no 3 pp 424ndash426 2009
[36] A Mansouri ldquoDevelopment and regeneration in the endocrinepancreasrdquo ISRN Endocrinology vol 2012 Article ID 640956 12pages 2012
[37] X Xu J DrsquoHoker G Stange et al ldquo120573 cells can be generated fromendogenous progenitors in injured adult mouse pancreasrdquo Cellvol 132 no 2 pp 197ndash207 2008
[38] K Al-Hasani A Pfeifer M Courtney et al ldquoAdult duct-liningcells can reprogram into 120573-like cells able to counter repeatedcycles of toxin-induced diabetesrdquo Developmental Cell vol 26no 1 pp 86ndash100 2013
[39] A Pfeifer M Courtney N Ben-Othman et al et al ldquoInductionof multiple cycles of pancreatic 120573-cell replacementrdquo Cell Cyclevol 12 no 20 pp 3243ndash3244 2013
[40] M Courtney E Gjernes N Druelle et al ldquoThe inactivationof Arx in pancreatic 120572-cells triggers their neogenesis andconversion into functional 120573-like cellsrdquo PLoS Genetics vol 9no 10 article e1003934 2013
[16] A L Greenwood S Li K Jones and D A Melton ldquoNotchsignaling reveals developmental plasticity of Pax4+ pancreaticendocrine progenitors and shunts them to a duct faterdquoMecha-nisms of Development vol 124 no 2 pp 97ndash107 2007
[17] P Collombat X Xu P Ravassard et al ldquoThe ectopic expressionof Pax4 in the mouse pancreas converts progenitor cells into 120572and subsequently120573 cellsrdquoCell vol 138 no 3 pp 449ndash462 2009
[18] J Lu P L Herrera C Carreira et al ldquo120572 cell-specific Men1ablation triggers the transdifferentiation of glucagon-expressingcells and insulinoma developmentrdquo Gastroenterology vol 138no 5 pp 1954ndash1965 2010
[19] S Biressi G Messina P Collombat et al ldquoThe homeobox geneArx is a novel positive regulator of embryonic myogenesisrdquo CellDeath and Differentiation vol 15 no 1 pp 94ndash104 2008
[20] E Colombo P Collombat G Colasante et al ldquoInactivation ofArx the murine ortholog of the X-linked lissencephaly withambiguous genitalia gene leads to severe disorganization of theventral telencephalon with impaired neuronal migration anddifferentiationrdquo The Journal of Neuroscience vol 27 no 17 pp4786ndash4798 2007
[21] S Dhawan S Georgia S-I Tschen G Fan and A BhushanldquoPancreatic 120573 cell identity is maintained by DNA methylation-mediated repression of Arxrdquo Developmental Cell vol 20 no 4pp 419ndash429 2011
[22] J B Papizan R A Singer S-I Tschen et al ldquoNkx22 repressorcomplex regulates islet120573-cell specification and prevents 120573-to-120572-cell reprogrammingrdquo Genes amp Development vol 25 no 21 pp2291ndash2305 2011
[23] L Sussel J Kalamaras D J Hartigan-OrsquoConnor et al ldquoMicelacking the homeodomain transcription factor Nkx22 havediabetes due to arrested differentiation of pancreatic 120573 cellsrdquoDevelopment vol 125 no 12 pp 2213ndash2221 1998
[24] C S Chao Z L Loomis J E Lee and L Sussel ldquoGenetic identi-fication of a novel NeuroD1 function in the early differentiationof islet 120572 PP and 120576 cellsrdquo Developmental Biology vol 312 no 2pp 523ndash532 2007
[25] S Kordowich P Collombat A Mansouri and P Serup ldquoArxand Nkx22 compound deficiency redirects pancreatic alpha-and beta-cell differentiation to a somatostatinghrelin co-expressing cell lineagerdquo BMC Developmental Biology vol 11article 52 2011
[26] T L Mastracci C Wilcox L Arnes et al ldquoNkx22 and Arxgenetically interact to regulate pancreatic endocrine cell devel-opment and endocrine hormone expressionrdquo DevelopmentalBiology vol 359 no 1 pp 1ndash11 2011
[27] P Collombat J Hecksher-Sorensen J Krull et al ldquoEmbryonicendocrine pancreas and mature 120573 cells acquire 120572 and PP cellphenotypes upon Arx misexpressionrdquo The Journal of ClinicalInvestigation vol 117 no 4 pp 961ndash970 2007
[28] M Furuta H Yano A Zhou et al ldquoDefective prohormoneprocessing and altered pancreatic islet morphology in micelacking active SPC2rdquo Proceedings of the National Academy ofSciences of the United States of America vol 94 no 13 pp 6646ndash6651 1997
[29] RWGelling XQDuD SDichmann et al ldquoLower blood glu-cose hyperglucagonemia and pancreatic 120572 cell hyperplasia inglucagon receptor knockout micerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no3 pp 1438ndash1443 2003
[30] Y Hayashi M Yamamoto H Mizoguchi et al ldquoMice deficientfor glucagon gene-derived peptides display normoglycemia
and hyperplasia of islet 120572-cells but not of intestinal L-cellsrdquoMolecular Endocrinology vol 23 no 12 pp 1990ndash1999 2009
[31] P Collombat and A Mansouri ldquoPax4 transdifferentiatesglucagon-secreting 120572 cells to insulin-secreting 120573 endocrinepancreatic cellsrdquo MedecineSciences vol 25 no 8-9 pp 763ndash765 2009
[32] P Collombat and A Mansouri ldquoTurning on the 120573-cell identityin the pancreasrdquo Cell Cycle vol 8 no 21 pp 3450ndash3451 2009
[33] YDor andDAMelton ldquoFacultative endocrine progenitor cellsin the adult pancreasrdquo Cell vol 132 no 2 pp 183ndash184 2008
[34] J F Habener and V Stanojevic ldquo120572-cell role in 120573-cell generationand regenerationrdquo Islets vol 4 no 3 pp 188ndash198 2012
[35] Z Liu and J F Habener ldquoAlpha cells beget beta cellsrdquo Cell vol138 no 3 pp 424ndash426 2009
[36] A Mansouri ldquoDevelopment and regeneration in the endocrinepancreasrdquo ISRN Endocrinology vol 2012 Article ID 640956 12pages 2012
[37] X Xu J DrsquoHoker G Stange et al ldquo120573 cells can be generated fromendogenous progenitors in injured adult mouse pancreasrdquo Cellvol 132 no 2 pp 197ndash207 2008
[38] K Al-Hasani A Pfeifer M Courtney et al ldquoAdult duct-liningcells can reprogram into 120573-like cells able to counter repeatedcycles of toxin-induced diabetesrdquo Developmental Cell vol 26no 1 pp 86ndash100 2013
[39] A Pfeifer M Courtney N Ben-Othman et al et al ldquoInductionof multiple cycles of pancreatic 120573-cell replacementrdquo Cell Cyclevol 12 no 20 pp 3243ndash3244 2013
[40] M Courtney E Gjernes N Druelle et al ldquoThe inactivationof Arx in pancreatic 120572-cells triggers their neogenesis andconversion into functional 120573-like cellsrdquo PLoS Genetics vol 9no 10 article e1003934 2013
